diff --git a/.gitattributes b/.gitattributes index 28df5f900b358436f0267334b3e3e9af33f917ba..eb7dcd4440c332d84b8926fd9e3545b000162c54 100644 --- a/.gitattributes +++ b/.gitattributes @@ -53,3 +53,5 @@ saved_model/**/* filter=lfs diff=lfs merge=lfs -text *.jpg filter=lfs diff=lfs merge=lfs -text *.jpeg filter=lfs diff=lfs merge=lfs -text *.webp filter=lfs diff=lfs merge=lfs -text +full_texts-2024-04-25-update.json filter=lfs diff=lfs merge=lfs -text +full_texts.json filter=lfs diff=lfs merge=lfs -text diff --git a/eval/task1_eval.py b/eval/task1_eval.py new file mode 100644 index 0000000000000000000000000000000000000000..01bb73690b50427c4311b958ab4336cc5a9d10b8 --- /dev/null +++ b/eval/task1_eval.py @@ -0,0 +1,107 @@ +import argparse +import json +import csv +import math +import os + + +def compute_dcg(pred_docs, gold_docs): + dcg_score = 0.0 + for i, doc in enumerate(pred_docs): + position = i + 1 + discount = 1.0 / math.log2(position + 1) + relevance = 0.0 + if doc in gold_docs: + # If predicted image is present in gold list, set relevance to 1.0 + relevance = 1.0 + else: + for gdoc in gold_docs: + # If predicted image is a sub-image or parent image of an image in gold list, + # we set relevance to 0.5 to provide partial credit + if doc in gdoc or gdoc in doc: + relevance = 0.5 + break + dcg_score += (discount * relevance) + return dcg_score + + +def compute_idcg(relevance_ranking, rank): + sorted_relevance_ranking = list(sorted(relevance_ranking.items(), key=lambda x: x[1], reverse=True)) + # Only consider top k relevant items for IDCG@k + sorted_relevance_ranking = sorted_relevance_ranking[:min(len(sorted_relevance_ranking), rank)] + idcg_score = sum([ (1.0 / (math.log2(i + 2))) * x[1] for i, x in enumerate(sorted_relevance_ranking)]) + return idcg_score + + +def run_eval(pred_labels, gold_labels, parse_folder, claim_citekeys, debug): + ranks_to_eval = [5, 10] + ndcg_scores = {n: {} for n in ranks_to_eval} + non_empty_samples = 0 + + for claim_id in pred_labels: + if claim_id not in gold_labels: + print(f"Warning: Claim ID {claim_id} not found in gold data - skipping!") + continue + if not gold_labels[claim_id]: + print(f"Warning: Claim ID {claim_id} has no associated evidence figures/tables - skipping!") + continue + + non_empty_samples += 1 + for rank in ranks_to_eval: + # If #predictions < rank in predicted ranking, include all for evaluation + pred_images = pred_labels[claim_id][:min(len(pred_labels[claim_id]), rank)] + gold_images = gold_labels[claim_id] + + # Compute DCG score + dcg_score = compute_dcg(pred_images, gold_images) + + # Compute ideal DCG score + # First need to get relevance scores for all possible images + # Images in gold list get relevance score of 1.0 + relevance_ranking = {x: 1.0 for x in gold_images} + for file in os.listdir(os.path.join(parse_folder, claim_citekeys[claim_id])): + if 'CAPTION' in file: + continue + image_id = file.split('.png')[0] + if image_id not in gold_images: + relevance_ranking[image_id] = 0.0 + # All images that are parent/sub-images of a gold image get relevance of 0.5 + for gold_image in gold_images: + if image_id in gold_image or gold_image in image_id: + relevance_ranking[image_id] = 0.5 + break + idcg_score = compute_idcg(relevance_ranking, rank) + + # Finally compute and store NDCG score@k + ndcg_score = dcg_score / idcg_score + ndcg_scores[rank][claim_id] = ndcg_score + + # Display final evaluation scores + for rank in ranks_to_eval: + final_ndcg = sum(list(ndcg_scores[rank].values())) / len(gold_labels) + print(f'NDCG@{rank}: {final_ndcg}') + + if debug: + json.dump(ndcg_scores, open("task1_scores.json", "w")) + + +if __name__ == "__main__": + + parser = argparse.ArgumentParser() + parser.add_argument("--pred_file", type=str, required=True, help="Path to prediction file") + parser.add_argument("--gold_file", type=str, required=True, help="Path to gold data file") + parser.add_argument("--parse_folder", type=str, required=True, help="Path to folder containing parsed images/tables") + parser.add_argument("--debug", type=bool, default=False, help="Dump per-prediction scores for debuggin/analysis") + args = parser.parse_args() + + gold_data = json.loads(open(args.gold_file).read()) + gold_labels = {x["id"]: x["findings"] for x in gold_data} + claim_citekeys = {x["id"]: x["citekey"] for x in gold_data} + + reader = csv.reader(open(args.pred_file)) + next(reader, None) + pred_labels = {} + for row in reader: + pred_labels[row[0]] = row[1].split(',') + + run_eval(pred_labels, gold_labels, args.parse_folder, claim_citekeys, args.debug) diff --git a/eval/task2_eval.py b/eval/task2_eval.py new file mode 100644 index 0000000000000000000000000000000000000000..1cfbd258318250d7b672dca69163b2326eb9213d --- /dev/null +++ b/eval/task2_eval.py @@ -0,0 +1,87 @@ +import argparse +import json +import itertools +from collections import defaultdict + +import bert_score +from bert_score import score +from rouge_score import rouge_scorer + + +def get_best_scores(candidates, score_list): + per_pair_scores = defaultdict(list) + for cand, score in zip(candidates, score_list): + per_pair_scores[cand].append(score) + best_match_scores = {cand: max(scores) for cand, scores in per_pair_scores.items()} + return best_match_scores + + +def run_snippet_eval(pred_snippets, gold_snippets, debug): + bert_scores = {} + rouge_scores = {"rouge1": {}, "rouge2": {}, "rougel": {}} + rscorer = rouge_scorer.RougeScorer(['rouge1', 'rouge2', 'rougeL'], use_stemmer=True) + + for claim_id in pred_snippets: + if claim_id not in gold_snippets: + print(f"Warning: Claim ID {claim_id} not found in gold data - skipping!") + continue + if not gold_snippets[claim_id]: + print(f"Warning: Claim ID {claim_id} has no associated evidence snippets - skipping!") + continue + + # Generate all possible combinations of gold x predicted snippets for overlap computation + eval_pairs = itertools.product(pred_snippets[claim_id], gold_snippets[claim_id]) + candidates, references = zip(*list(eval_pairs)) + + # Compute BERT scores for all gold x predicted snippets and retain best match score per prediction + P, R, F1 = score(candidates, references, lang='en', verbose=True) + best_scores = get_best_scores(candidates, F1.numpy().tolist()) + mean_bert_score = sum(best_scores.values()) / len(pred_snippets[claim_id]) + bert_scores[claim_id] = mean_bert_score + + # Similarly compute ROUGE-1,2,L scores + r1_list, r2_list, rl_list = [], [], [] + for cand, ref in zip(candidates, references): + score_output = rscorer.score(ref, cand) + r1_list.append(score_output['rouge1'].fmeasure) + r2_list.append(score_output['rouge2'].fmeasure) + rl_list.append(score_output['rougeL'].fmeasure) + best_rouge1 = get_best_scores(candidates, r1_list) + best_rouge2 = get_best_scores(candidates, r2_list) + best_rougel = get_best_scores(candidates, rl_list) + rouge_scores["rouge1"][claim_id] = sum(best_rouge1.values()) / len(pred_snippets[claim_id]) + rouge_scores["rouge2"][claim_id] = sum(best_rouge2.values()) / len(pred_snippets[claim_id]) + rouge_scores["rougel"][claim_id] = sum(best_rougel.values()) / len(pred_snippets[claim_id]) + + # Print final score report + final_bert_score = sum(bert_scores.values()) / len(gold_snippets) + print(f"BERT Score: {final_bert_score}") + final_rouge1_score = sum(rouge_scores["rouge1"].values()) / len(gold_snippets) + print(f"ROUGE-1 Score: {final_rouge1_score}") + final_rouge2_score = sum(rouge_scores["rouge2"].values()) / len(gold_snippets) + print(f"ROUGE-2 Score: {final_rouge2_score}") + final_rougel_score = sum(rouge_scores["rougel"].values()) / len(gold_snippets) + print(f"ROUGE-L Score: {final_rougel_score}") + # TODO: Allow dumping of per-prediction scores for analysis? + + if debug: + json.dump(bert_scores, open("task2_bertscores.json", "w")) + json.dump(rouge_scores, open("task2_rougescores.json", "w")) + + +if __name__ == "__main__": + + parser = argparse.ArgumentParser() + parser.add_argument("--pred_file", type=str, required=True, help="Path to prediction file") + parser.add_argument("--gold_file", type=str, required=True, help="Path to gold data file") + parser.add_argument("--debug", type=bool, default=False, help="Dump per-prediction scores for debuggin/analysis") + args = parser.parse_args() + + gold_data = json.loads(open(args.gold_file).read()) + gold_snippets = {x["id"]: x["context"] for x in gold_data} + + pred_data = json.loads(open(args.pred_file).read()) + pred_snippets = {x["id"]: x["context"] for x in pred_data} + + # Run ROUGE and BERTScore evaluation for grounding snippets + run_snippet_eval(pred_snippets, gold_snippets, args.debug) diff --git a/extracted_captions/Kueh2008.json b/extracted_captions/Kueh2008.json new file mode 100644 index 0000000000000000000000000000000000000000..acd25768778e42aaf821bb338356f22ed6df140e --- /dev/null +++ b/extracted_captions/Kueh2008.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: **Actin filaments disassemble in bursts in cofilin, coromin, and Aip1.** [A] Time-lapse wide-field epifluorescence images of fluorescently labeled actin filaments in the presence of 2 \\\\(\\\\mu\\\\)M cofilin, 1 \\\\(\\\\mu\\\\)M comin, 200 nM Aip1, 5 \\\\(\\\\mu\\\\)M of actin monomer, and 2 mM ATP. Filaments shorten and disappear from the field of view. Bar, 3 \\\\(\\\\mu\\\\)m. [B] Successive time-lapse images showing a single actin filament [f] over time, along with laryngographs drawn along the contours of representative filaments [f1-4]. The red lines on the image of 1 \\\\(\\\\mu\\\\)t = 0 denote the contour on which the kymograph was drawn. Time is given on the x axis of the kymograph, whereas the position along the filament contour is given on the y axis. Mean integration time for a single image was 400 ms for _H1-f3_ and 16 ms for _I4_. Triangles denote endwise bursting [f1-3]; yellow triangles denote initial burst [f1-f3], red triangles denote successive proximal bursts [f1] and _I2_, and green triangle denotes a successive distal burst [f3]. Some-side bursts occurred more frequently (78%) than opposite-side bursts (22%; \\\\(\\\\tau\\\\) < 0.001, one-tailed 2 test). The square denotes internal disassembly event counted as a severing event [f3]. Bar, 1 \\\\(\\\\mu\\\\)m. [C] Histogram of filament burst size. The mean burst size was 260 subunits. [D] Histogram of waiting times between successive bursts [red], fit to a single exponential [black]. Single exponential fit gave characteristic decay time of \\\\(\\\\tau\\\\) = 14 s.\\n\\n'", "CAPTION FIG2.png": "'Figure 2. **Filament bursting is distinct from eefilin-mediated severing. [\\\\(\\\\mu\\\\)A] Time-lapse images of two actin filaments disassembling in 10 mM cefilin, 5 mM of actin monomer, and 2 mM ATP. Long filaments were chosen to illustrate the accurrenca of multiple severing events within a single filament. Bar, 1 pm. [B] Bar graph showing the fraction of disassembly events scored as bursting (red) or severing [green], either in the full desobymarizing system (left) or in cefilin alone (right). In the full system, bursting occurred with significantly higher frequency than severing [\\\\(\\\\chi^{2}=190\\\\), df = 1, P < 0.01]. However, in the presence of high concentrations of actin, severing occurred with significantly higher frequency than bursting [\\\\(\\\\chi^{2}=55\\\\), df = 1, P < 0.01] and was the predominant disassembly mechanism observed under these conditions. We note that mean inel filament length did not differ significantly between different experiments and was not the cause of the differences observed.**\\n\\n'", "CAPTION FIG3.png": "'Figure 3: **Actin filaments disassemble with similar kinetics from both ends.**[_A_] Time-lapse images of actin filament bundles grown off fragments of _l._ acyl-phyrins accrosomal processes. Filaments in the long bundle have exposed barbed ends (b), whereas filaments in the short bundle have exposed pointed ends (c). The two bundles shown were elongated from opposite ends of the same _L._ poly-phyrins accrosomal fragment. The brightness of the shorter bundle was increased relative to the longer one for ease of visualization. _At_ 0 s, filament bundles were perfused with 2 mM cocillin, 1 mM cocrinin, 300 mM Ag1, 5 mM of actin monomer, and 2 mM ATP. Bar, 1 \u03bcm. (B) Total actin polymer mass in the filament bundles as a function of time measured for all bundles (red), bundles with exposed barbed ends (green), and bundles with exposed pointed ends (blue). To compare the rates of rapid disassembly, the slowly varying component of each decay curve was removed (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1; see Materials and methods). [_C_] Bar graph comparing decay rates for bundles with exposed barbed ends (b) with those with exposed pointed ends (b). Data represent mean and standard deviation of three independent experiments.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Bursting disassembly generates barbed ends not recognized by CapZ.** Fluorescence images show Alaxa 647 actin (left) and Alexa 488 CapZ. (right) in filament bundles. Barbed ends are oriented toward to the right. The bar graph shows the CapZ/actin filament ratio, a measure of the fraction of copper barbed ends in the bundle. Data represent mean and standard deviation of eight filament bundles. (A) Filament bundles polymerized from L. _Polyg\u00e8mes_ acrossal fragments were incubated directly with Alexa 488 CapZ. [B] Filament bundles were partially disassembled with _\u03b2_ null actin for 90 s then incubated with CapZ. [C] Filament bundles were partially disassembled with 2.5 \u03bcM actin, 1.5 \u03bcM coronin, and 50 \u03bcM Ap1 for 25 s, then incubated with CapZ. (D) Filament bundles were partially disassembled with cofilin, coronin, and _Aip1_ as in C, incubated in buffer for 10 s, then incubated with CapZ. Bar, 1 \u03bcm.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Barbed end-copping factors inhibit actin disassembly by coffin, coronin, and Aip1.** [A] Mean filament length over time under the following conditions: 2 \u03bcM cells, 1 \u03bcM actin, 1 \u03bcM coronin, 5 \u03bcM of actin monomer, and 2 \u03bcM ATP, as well as 200 \u03bcM \u03bcM (red), no Aip1 [green], 5 \u03bcM CorpZ [yellow], 200 \u03bcM Ap1 + 1 \u03bcM CytOb [black], or 200 \u03bcM Ap1 + 5 \u03bcM CapZ [blue]. Fast non-linear-insensitive disassembly required Aip1 [red vs. green, yellow]. CytoD and CopZ inhibited disassembly in the full system [black, blue vs. red]. [B] Bor graph showing the percentage of filaments that underwent endwise bursting or severing. The number of filaments analyzed is given below the bars. The percentages of filaments that either underwent endwise bursting or severing increased significantly in the presence of Aip1 [red vs. green, yellow] and decreased significantly when the barbed-end coopers CytO and CopZ were added to the full system [black, blue vs. red]; severing frequency of ~CopZ vs. ~CopZ, _kh_ = 4.9, _df_ = 1, \\\\(p\\\\) < 0.05, all other pairwise comparisons, _kh_ > 40, _df_ = 1, \\\\(p\\\\) < 0.001). [C] Polymer mass decay for bundles with either exposed barbed ends or pointed ends in 3 \u03bcM CytOb (brown and purple) or 10 \u03bcM CapZ (blue and green), as well as polymer mass decay for all filament bundles in the absence of barbed-end coopers [red]. Dissensembly of bundles with either exposed barbed ends or pointed ends were inhibited by CytoD/CopZ with equal efficacy. [D] fraction of polymer mass diisassembled as a function of CytoD concentration (red circles). Best fit of the data to a hyperbolic [red curve] shows an C_x_50 of 90 \u03bcM inhibition of disassembly. Length of filament bundles polymerized for a fixed period of time in the presence of varying concentrations of C_x_50 is also shown (blue squares). Hyperbolic best fit [blue curve] shows an C_x_50 of 30 \u03bcM inhibition of polymerization. [E] Fraction of polymer diisassembled as a function of CapZ concentration [red squares]. The situation curve did not reach saturation; the red curve denotes best fit of the data to a straight line. The length of filament actin bundle polymerized in the presence of varying concentrations of CapZ is also shown (blue circles). Polymerization conditions were identical to those in D. Hyperbolic best fit [blue curve] gave an C_x_50 of 30 \u03bcM for inhibition of polymerization. Error bars indicate SD. [F] CytoD does not inhibit coffin-mediated severing. The bars graph shows the percentage of filaments that served during a 400 s video either in the absence of CytoD [left] or in the presence of 1 \u03bcM CytOb (right). Conditions: 8 \u03bcM cclilin and 5 \u03bcM of actin monomer in assay buffer.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: **Barbed end-capping drugs inhibit actin disassembly in mammalian tissue-culture cells.** (A) Images of suspension Hela S3 calls showing F-actin stained with TRIT-cpholdolin (top) and DNA stained with Hoechst Bottom). Images show unreacted cells [left], cells treated with 10 \u03bcM Cyph0] for 480 s (middle), and cells treated with 10 \u03bcM Limb Left for 480 s (right). All images used identical acquisition settings and contrast levels. 20, 100 rpm. (B) The level F-actin per cell at different times after treatment with either Cyph0 [blue] or LofR [red]. F-actin levels remained high after Cyph0 Treatment. In contrast, F-actin levels left rapidly upon LofR treatment. Each point represents the mean and SD of at least 10 fields of cells. (C) Images of actin content tails in \\\\(\\\\cdot\\\\) monocytogenes-inflicted BSC-1 cells taken at successive times after drug treatment. In the absence of any drugs [control], comet tails disassembled with 50 s (open left). Upon LofR treatment, comet tails also disassembled within 50 s, which suggests that there is no effect of LofR on comet tail disassembly kinetics. In contrast, comet tails did not disassemble in Cyph0- and KabC-exceeded cells for at least 50 s after drug addition, which implies that Cyph0 and KabC-exhibited comet tail disassembly. All drugs were added at time 0. Bar, 5 \u03bcM. (D) Curves showing decay of actin polymer mass from \\\\(L\\\\). monocytogenes comet tails. In the absence of drugs, actin disassembled at an initial rate of \\\\(k_{\\\\text{model}}=[2.3\\\\pm 0.2]\\\\times 10^{-3}\\\\)-1. LofR did not change the disassembly rate, with \\\\(k_{\\\\text{model}}=[2.6\\\\pm 0.2]\\\\times 10^{-2}\\\\)-1. LofR in contrast, Cyph0 reduced the L. monocytogenes content tail decay rate to \\\\(k_{\\\\text{model}}=[1.9\\\\pm 6]\\\\times 10^{-4}\\\\)-s\\\\({}^{-1}\\\\). Data represent the mean of polymer mass in multiple trials [control], 27 s/cis; Cyph0 treatment, 40 tails; LofR treatment, 27 tails). Disassembly rates were obtained by fitting the initial slope of the decay curve to a straight line.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Borbed-end and coppers inhibit disassembly of dynamic actin at the ruffling cell edge.** (A\u2013C) Time-lapse images of ruffling edges in BSC-1 cells expressing mRFP-PAGE/PAGP-actin taken before CytoD addition (A), after LofI addition (B), or after CytoD addition [C]. The red channel shows bulk actin, and the green channel shows photoactivated fluorescence. Images were taken before activation [left], immediately after activation (middle), and 100 s after activation (right). [D\u2013F] Xymographs taken along the lines shown in A, B, and C, respectively. The color symograph shows bulk actin in red and photoactivated actin in green. The black and white kymograph shows photoactivated actin alone. Arrows show the time of CytoD addition [O and E] for LofI addition [E]. (A and D) Before drug treatment, photoactivated subpopulations of actin decayed within 100 s. [B and E] LofI did not inhibit actin disassembly. Both bulk and photoactivated subpopulations of actin disappeared within 100 s. [C and F] CytoD inhibited disassembly of dynamic actin assemblies at the ruffling edge. Photoactivated actin subpopulations remained visible for >100 s, and bulk actin fluorescence persisted at the ruffling cell edge. Bar, 5 \u03bcm.\\n\\n'", "CAPTION FIG8.png": "'Figure 8. **Candidate mechanisms for actin filament disassembly by co-filin, ceronian, and Aip1. [A] Cooperative strand separation. [B] Filament scavenging. Black lines denote filament strands. The blue box denotes CapZ. The red bar denotes inhibition of CapZ binding. Successive illustrations depict reaction intermediates in each disassembly pathway.**\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/Owen2020Cterminal.json b/extracted_captions/Owen2020Cterminal.json new file mode 100644 index 0000000000000000000000000000000000000000..064c4072234f180108ca8a470798c1bd343f9720 --- /dev/null +++ b/extracted_captions/Owen2020Cterminal.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1: Talin-1 ABS3 binding to actin is stabilized by force**. (**Ai**) No-load three bead optical trap experimental setup. ABS3 (light blue) is immobilized via an N-terminal HaloTag domain on a microscope coverslip sparsely decorated with 1.5 \\\\(\\\\upmu\\\\)m diameter platform beads. A single actin filament (orange) is stretched between two optically trapped 1 \\\\(\\\\upmu\\\\)m diameter beads. The microscope stage is positioned such that a platform bead is positioned directly underneath the actin filament. (**Aii**) Sample trace of the position of the trapped beads to detect binding of ABS3 to F-actin. In the absence of applied load, ABS3 binding to F-actin can be detected by a damping of high-frequency Brownian fluctuations (cumulative power spectrum, C.P.S. \\\\(>\\\\) 0.3-10 kHz) in the trapped beads. (**Aiii**) Binding lifetimes detected with this method. N=291 binding events detected from sampling the ABS3 construct from 1,419 seconds of recorded data. N=8 binding events detected from sampling an eGFP-coated surface from 2,188 seconds. (**Bi**) Loaded three bead optical trap experimental setup. Same as **Ai**, except the stage is translated parallel to the actin filament in a trapezoidal wave form. (**Bii**) If one or more ABS3 molecules on the platform bead bind to the actin filament while the stage is moving, one of the trapped beads is pulled out of its equilibrium position (demarcated by the blue force trace), thus applying load to the ABS3-F-actin bond. When this occurs the stage motion is halted until all of the protein complexes unbind and the trapped beads return to their equilibrium positions (marked with purple notch, gray trace). Reported lifetimes are last steps of binding events, which for this sample trace is the time between yellow and purple arrows, or the full lifetime of single-step events.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Figure 2:****Talin-1 forms a catch bond with F-actin when force is applied towards the pointed end of the actin filament (A)** An asymmetry in bond lifetimes is observed for a single actin filament. (**Ai, Aii**) Load on the _right_ (**Ai**) or _left_ (**Aii**) trapped bead. (**Aiii**) Sample force trace. Load on the right bead is indicated in orange, while load on the left bead is shown in blue. (**B**) (**Bi**) A myosin VI construct steps along actin filaments, pulling the bead attached to the (-) end of the filament out of the trap. (**Bii**) Sample myosin trace. In this example, the left bead is attached to the F-actin (-) end. (**C**) ABS3 binding dwell times measured as a function of force. Forces directed towards the pointed (-) end of the filament correspond to negative force values and forces directed towards the barbed (+) end correspond to positive force values. _Dark gray_: binding lifetimes of events measured in the directionality assay in which the actin filament polarity was explicitly measured for each dumbbell (N=2 flow cells, N=3 dumbbells, N=47 events). _Lavender_: binding dwell times for which the actin filament polarity was statistically inferred. _Orange_: mean dwell times (20 data points per bin), with 95% confidence intervals shown in thinner lines (N = 777 binding events, N=42 dumbbells, N=18 flow cells). (**D**) Fold-difference in bond lifetime for forces applied towards the pointed vs. barbed end of the actin filament, with 95% confidence intervals per 1 pN bin.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3: The C-terminal dimerization domain (DD) is required for F-actin binding under load. (A)** The constructs used in this study (see text). (B) Native PAGE of BSA (MW=66.5 as monomer), ABS3 (64.2 kDa if monomeric), ABS3\\\\(\\\\Delta\\\\)DD (58.5 kDa, monomer) and NTD-ABS3\\\\(\\\\Delta\\\\)DD (113 kDa). Note the gradual shift in apparent molecular weight as a function of ABS3 concentration, suggestive of a dynamic equilibrium of dimerization. \\\\(\\\\bullet\\\\) and \\\\(\\\\overline{\\\\textbf{V}}\\\\)denote distances traveled of peaks in different replicates. (C) Actin cosedimentation of 4 uM ABS3, 2 uM NTD-ABS3, or 2 uM NTD-ABS3\\\\(\\\\Delta\\\\)DD with 20 uM actin. N=3 replicates. (D) Force-lifetime plots of binding events detected for the NTD-ABS3 construct (lavender) with mean lifetime and confidence intervals on the mean plotted in orange (20 data points per bin). N=386 binding events, N=18 dumbbells, N=8 flow cells.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Talin ABS3 functions as a molecular AND gate (see text). Integrin cartoon modified from Zhu _et al._, 2017.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/alfonzomendez2022dual.json b/extracted_captions/alfonzomendez2022dual.json new file mode 100644 index 0000000000000000000000000000000000000000..6c249913a33ace3d7c65da2834e0e841d3e8ac76 --- /dev/null +++ b/extracted_captions/alfonzomendez2022dual.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1 EGF modifies the ultrastructure of clathrin at the plasma membrane.****a**_N_Montaged PREM image of an unroofed control HCS-EGFR-GFP cell and the mask created after segmentation of the full membrane outlined (yellow). Flat, dome, and sphere clathrin-coated structures (CCCs) are shown in green, blue, and magenta, respectively. **b** High-magnification image of the cropped PREM in (**a**); the different segmented CCSs are color-coded as in (**a**), with grayscale in magnified insets. **c**_Montaged PREM image of an unroofed HCS-EGFR-GFP cell treated with 50 ng/mL EGF for 15 min and the mask created after the segmentation. **d**_High-magnification of a representative region of the PREM in (**c**), the magnification insets are shown at the same scale and are outlined with dashed squares in each image. **e**_Representative clathrin masks of the EGF stimulation time course for 0, 2, 5, 15, 30, and 60 min. PREM images corresponding to the masks and cropped images are shown in Supplementary Fig. 1. **f-h**_Morphometric analysis of the percentage of plasma membrane (PM) area occupation for each CCS subtype. 1-_N_Morphometric analysis of the percentage of plasma maximum data point markers with a coefficient value of 1.5. **f-k**_Morphometric analysis of the size of flat, dome, and sphere CCSs during the EGF time course for three clathrin subtypes. Dot plots show every structure segmented, the bar is the median, \\\\(N\\\\) = 2 biologically independent experiments in (**a**-**a**); 0 min: \\\\(N_{\\\\text{Max}}\\\\) = 141, \\\\(N_{\\\\text{dena}}\\\\) = 46, \\\\(N_{\\\\text{dena}}\\\\) = 68, \\\\(N_{\\\\text{exta}}\\\\) = 4; 2 min: \\\\(N_{\\\\text{Int}}\\\\) = 164, \\\\(N_{\\\\text{dena}}\\\\) = 32, \\\\(N_{\\\\text{dena}}\\\\) = 30, \\\\(N_{\\\\text{exta}}\\\\) = 3; 5 min: \\\\(N_{\\\\text{Max}}\\\\) = 184, \\\\(N_{\\\\text{dena}}\\\\) = 26, \\\\(N_{\\\\text{dena}}\\\\) = 36, \\\\(N_{\\\\text{dena}}\\\\) = 4; 15 min: \\\\(N_{\\\\text{Int}}\\\\) = 559, \\\\(N_{\\\\text{dena}}\\\\) = 67, \\\\(N_{\\\\text{dena}}\\\\) = 207, \\\\(N_{\\\\text{exta}}\\\\) = 4; 30 min: \\\\(N_{\\\\text{Int}}\\\\) = 395, \\\\(N_{\\\\text{dena}}\\\\) = 149, \\\\(N_{\\\\text{dena}}\\\\) = 113, \\\\(N_{\\\\text{exta}}\\\\) = 3; 60 min: \\\\(N_{\\\\text{int}}\\\\) = 81, \\\\(N_{\\\\text{dena}}\\\\) = 57, \\\\(N_{\\\\text{exta}}\\\\) = 5 examined over the indicated independent experiments. Scale bars in (**a**) and (**c**) are 5 min. Scale bars in (**b**, **d**, **e**) are 1 min insets are 200 nm. EGF epidermal growth factor, EGFR epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2 Flat clathrin lattice formation requires EGFR.****a** Representative PREM images of control HSC3-EGFR-GFP cells (Ctrl), **b** cells treated either with ESO ng/mL EGF alone (EGF) for 15 min, or in presence of (**c**) 10 \u03bcM gefitinib (Gefi + EGF), and **d** EGFR siRNA (EGFR siRNA + EGF). EGFR siRNA validation is shown in Supplementary Fig. 3. The magnification insets are shown at the same scale and are outlined with dashed squares in each image. Flat, dome, and sphere clathrin-coated structures (CCSs) are shown in transparent green, blue, and magenta, respectively, with native grayscale in magnified insets. **e** Representative masks of segmented cells treated as in (**a-d**). PREM images corresponding to the masks and cropped images are shown in Supplementary Fig. 4. **f** Morphometric analysis of the percentage of plasma membrane (PM) area occupation for flat clathrin structures. 1-shaped box plots show median extended from 25th to 75th percentiles, and minimum and maximum data point whiskers with a coefficient value of 15. **g** Morphometric analysis of the size of flat structures of cells treated as indicated in (**a-d**). Dot plots show every structure segmented the bar indicates the median. Ctrl: _N_Hua = 14L \\\\(N_{\\\\text{cells}}\\\\) = 4; EGF: _N_Hua = 559, _N_cells = 4; Gefi + EGF: _N_Hua = 160, _N_cells = 4; EGFR siRNA + EGF: _N_Hua = 346, _N_cells = 4. Number of biologically independent experiments = 2. Scale bars in (**a-e**) are 1 \u03bcm; insets are 200 nm. Ctrl and EGF data were from Fig. 1 and shown for reference. EGF epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG3.png": "'Fig. 3: **Flat clathrin lattice formation requires Src.****a** Representative PREM images of control HSC3-EGFR-GFP cells (CtrU), **b** cells treated either with 50 ng/mL EGF alone (EGF) for TS min, or in presence of **c**10 \u03bcM PP2 (PP2 + EGF), and **d** Src siRNA (Src siRNA + EGF). Src siRNA validation is shown in Supplementary Fig. 3. The magnification insets are shown at the same scale and are outlined with dashed squares in each image. Flat, dome, and sphere clathrin-coated structures (CCSs) are shown in green, blue, and magenta, respectively, with native grayscale in magnified insets. **e** Representative masks of segmented cells treated as in (**a\u2013d**). PREM images corresponding to the masks and cropped images are shown in Supplementary Fig. 4. **f** Morphometric analysis of the percentage of plasma membrane (PM) area occupation for flat clathrin structure. **i**-shaped box plots show median extended from 25th to 75th percentiles, and minimum and maximum data point whiskers with a coefficient value of 1.5. **g** Morphometric analysis of the size of flat structures of cells treated as indicated in (**a\u2013d**). Dot plots show every structure segmented the bar indicates the median. CtrU \\\\({}_{\\\\text{Mat}}\\\\) = 141, \\\\(N_{\\\\text{cells}}\\\\) = 4; EGF: \\\\(N_{\\\\text{Mat}}\\\\) = 559, \\\\(N_{\\\\text{cells}}\\\\) = 4; PP2 + EGF: \\\\(N_{\\\\text{Mat}}\\\\) = 267, \\\\(N_{\\\\text{cells}}\\\\) = 8; Src KD + EGF: \\\\(N_{\\\\text{Mat}}\\\\) = 71, \\\\(N_{\\\\text{cells}}\\\\) = 7. Number of biologically independent experiments - 2. Scale bars in (**a\u2013e**) are 1 \u03bcm; insets are 200 nm. CtrU and EGF data were from Fig. 1 and shown for reference. EGF epidermal growth factor, EGFR epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: **Flat clathrin lattice formation requires \\\\(\\\\beta\\\\)S-integrin.** A Representative PREMs of control HSC3-EGR-GFP cells (Ctrl), **b** treated either with 50 ng/mL EGF alone (EGF) for 15 min, or in presence of **e** 10 \u03bcM clengicide acid (CTA + EGF), and **d** \\\\(\\\\beta\\\\)S-integrin siRNA (Q5 siRNA + EGF). \\\\(\\\\beta\\\\)S-integrin siRNA isolation is shown in Supplementary Fig. 3. The magnification insets are shown at the same scale and are outlined with dashed squares in each image. Flat, _red_, and sphere clathrin-coated structures (CCSs) are shown in green, blue, and magenta, respectively, with native grayscale in magnified insets. **e** Representative masks of segmented cells treated as in (**a\u2013d**). PREM images corresponding to the masks and creoped images are shown in Supplementary Fig. 4. **f** Morphometric analysis of the percentage of plasma membrane (PM) area occupation for flat clathrin structures. 1-shaped box plots show median extended from 25th to 75th percentiles, and minimum and maximum data point whiskers with a coefficient value of 15. **g** Morphometric analysis of the size of flat structures of cells treated as indicated in (**a\u2013d**). Dot plots show every structure segmented the bar indicates the median. Ctrl: \\\\(N_{\\\\text{int}}\\\\) = 14U, \\\\(N_{\\\\text{cells}}\\\\) = 4; EGF: \\\\(N_{\\\\text{tot}}\\\\) = 559, \\\\(N_{\\\\text{cells}}\\\\) = 4; CTA + EGF: \\\\(N_{\\\\text{tot}}\\\\) = 244, \\\\(N_{\\\\text{cells}}\\\\) = 4; pS siRNA + EGF: \\\\(N_{\\\\text{tot}}\\\\) = 304, \\\\(N_{\\\\text{cells}}\\\\) = 7. Number of biologically independent experiments = 2. Scale bars in (**a\u2013e**) are 1 \u03bcm; insets are 200 nm. Ctrl and EGF data were from Fig. 1 and shown for reference. EGF epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5 Differential location of EGFR; Src, and (BS-integrin in clathrin-coated structures.****a** Representative two-calar TIRF images of genome-edited HSC3 expressing EGFR-GFP and transfected with mScarlet-CLCa or HSC3 WT cells co-transfected with mScarlet-CLCa + Src-GFP or DESIGN-GFP before (Ctrl) or after 50 ng/ml EGF stimulation for 15 mm. Scale bar is 10 mm; insets are 7.3 mm x 7.3 mm. **b** Automated correlation analysis between clathrin and EGFR, Src, and (BS-integrin. Dot box plots show median extended from 25th to 75th percentiles, mean (square), and minimum and maximum data paint whiskers with a coefficient value of 1.5. Significance was tested by a two-tailed _t_-test. EGFR, ***_P_ = 5.9 x 10-7. Src ***_P_ = 1.7 x 10-11; (BS-integrin. rnp = 0.358. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. \\\\(N_{\\\\text{EGFR-Ca}} = 23\\\\) cells - 3728 spots, \\\\(N_{\\\\text{ECF-ECF}} = 22\\\\) cells - 2173 spots, \\\\(N_{\\\\text{exc-CH}} = 28\\\\) cells - 1394 spots, \\\\(N_{\\\\text{exc}}\\\\). \\\\(\\\\text{EGF} = 27\\\\) cells - 1407 spots, \\\\(N_{\\\\text{EG-CH}} = 21\\\\) cells - 1037 spots; \\\\(N_{\\\\text{EG-CH}} = 20\\\\) cells - 1011 spots examined over the indicated independent experiments. EGFR epidermal growth factor receptor, TIRF total internal reflection fluorescence, CLCa clathrin light chain a, WT wild type, EGF epidermal growth factor.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n**Fig. 6****BS-integrin phosphorylation controls spatial correlation with clathrin.****a** Diagram of \\\\(\\\\beta\\\\)S-integrin and magnification of the cytoplasmic domain showing different mutants. Numbers indicate the residue positions, and letters identify the amino acid. The truncated line in the diagram indicates deletion of the sequence coding for amino acids 743-799. Other constructs are wild type (WT), carboxyl-truncated (\\\\(\\\\Delta\\\\)C), none-phosphorylatable (3Y-F), phosphomimetic (3Y-E), and PAK-targeted (2S-A). **b** In vitro phosphorylation assay using purified Src or PAK4 and peptides corresponding to the B5-integrin carboxyl domain (742-799) WT and mutants in (**a**). Significance was tested by a one-way ANOVA test: \\\\({}^{*}P\\\\) = 1.51 x 10-4, \\\\({}^{**}p\\\\) = 0.002, \\\\({}^{***}P\\\\) = 1.09 x 10-5, \\\\({}^{**}p\\\\) = 0.020S. \\\\(N\\\\) = 3 biologically independent experiments with consistent results. **c** Representative TIRF images of HSC3 WT cells can transfected with mScarlet-CLCa and B5-integrin-GFP WT or containing the different mutations shown in (**a**), either before (Ctrl) or after 50 ng/mL EGF stimulation for 15 min. Scale bars are 10 min; insets are 2.3 mm x 7.3 mm. **d** Automated two-color correlation analysis of (**a**). Dot box lists show median extended from 25th to 75th percentiles, mean (square), and minimum and maximum data point whiskers with a coefficient value of 1.5. Significance was tested by a two-tailed _t_-test, \\\\({}^{**}p_{\\\\text{E-WT}}\\\\) = 0.0811, \\\\({}^{***}p_{\\\\text{B5-AC}}\\\\) = 4.03 x 10-7, \\\\({}^{***}p_{\\\\text{E-WT}}\\\\) = 8.9 x 10-5, \\\\({}^{***}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 0.9149, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 0.5331. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 193 cells - 1492 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 872 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 872 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 872 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 18 cells - 1099 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 17 cells - 1245 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 1122 spots,'", "CAPTION FIG7.png": "'\\n\\n**Fig. 7 Flat clathrin lattices partition sustained signals at the plasma membrane.****a** Representative TIRF images of control (Ctrl) unroofed genome-edited HSC3 cells expressing EGFR-GFP transfected with mScarlet-CLCa and immunelabeled with anti-phospho EGFR (P-EGFR) coupled to Alexa 647, treated with 50 ng/ml EGF alone (EGF) or in the presence of bSintegrin mRNA (05 siRNA + EGF). **b** Automated correlation analysis of (**a**). \\\\({}^{*}\\\\)P = 3.67 x 10-21, \\\\({}^{**}\\\\)P = 4.84 x 10-8, \\\\({}^{***}\\\\)P = 6.31 x 10-9. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. _N_(c)(c)(c)(c)(c)(c) = 19 cells - 1770 spots, _N_(c)(c)(c)(c) = 19 cells - 1240 spots, _N_(c)(c)(c) = 20 cells - 1660 spots, _N_(c)(c)(c) = 19 cells - 1678 spots examined over the indicated independent experiments. **c** Fluorescence intensity measurements of the signal from membrane P-EGFR. \\\\({}^{*}\\\\)P = 4.71 x 10-23, \\\\({}^{**}\\\\)P = 2.6 x 10-5, \\\\({}^{**}\\\\)P = 0.052. _N_(c)(c)(c)(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells. **d** Fluorescence intensity measurements of the signal from membrane total EGFR-GFP (T-EGFR). \\\\({}^{*}\\\\)P = 1.45 x 10-4, \\\\({}^{**}\\\\)P = 2.46 x 10-4, \\\\({}^{***}\\\\)P = 8.53 x 10-9. _N_(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells. **e** Representative TIRF images of HSC3 WT cells transfected with mScarlet-CLCa and immunelabeled with anti-Grb2 coupled to Alexa 647 before (Ctrl) and treated as in (**a**). **f** Automated correlation analysis of (**e**). \\\\({}^{*}\\\\)P = 3.44 x 10-7, \\\\({}^{**}\\\\)P = 3.54 x 10-5, \\\\({}^{***}\\\\)P = 4.47 x 10-3. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. _N_(c)(c)(c)(c)(c) = 19 cells - 1965 spots, _N_(c)(c)(c)(c)(c) = 20 cells - 1532 spots, _N_(c)(c)(c)(c) = 19 cells - 2165 spots, _N_(c)(c)(c)(c) = 14 cells - 801 spots. **g** Fluorescence intensity measurements of the signal coming from immunelabeled Grb2. \\\\({}^{*}\\\\)P = 2.69 x 10-14, \\\\({}^{**}\\\\)P = 2.67 x 10-7, \\\\({}^{***}\\\\)P = 0.089 _N_(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells. Scale bars are 10 \\\\(\\\\mu\\\\)m\\\\(\\\\times\\\\) insets are 7.3 \\\\(\\\\mu\\\\)m\\\\(\\\\times\\\\) 7.3 \\\\(\\\\mu\\\\)m. Dot and box plots show median extended from 25th to 75th percentiles, mean (square) and minimum and maximum data point whiskers with a coefficient value of 1.5. Significance between groups was evaluated by a one-way ANOVA test. Number of independent experiments = 4 (**c**, **d**, **g**). AU fluorescence arbitrary units, EGFR epidermal growth factor receptor, CLCa clathrin light chain a, WT wild type, EGF epidermal growth factor, TIRF total internal reflection fluorescence.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8****Model of flat clathrin lattices expansion during growth factor response.** **a** Small flat clathrin lattices are in proximity to Src and are enriched with HS-integrin. **b** EGF triggers the dimerization, clustering, and cross-phosphorylation of EGFR at growing FCLs. This in parallel allows the biding of the downstream scaffold Grb2 and locally activates Src, which in turn phosphorylates BS-integrin cytoplasmic domain. The maintenance of the EGFR/Src/BS-integrin axis promotes flat clathrin lattice growth. A key implication of this model is that two different receptor systems are spatially organized at the nanoscale within flat clathrin lattices. EGF epidermal growth factor, EGFR epidermal growth factor receptor, FCLs flat clathrin lattices.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/allcottWelfareEffectsSocial2020.json b/extracted_captions/allcottWelfareEffectsSocial2020.json new file mode 100644 index 0000000000000000000000000000000000000000..ea0659e0b6f0bf76d9a899fa618d19d6bb4a37a3 --- /dev/null +++ b/extracted_captions/allcottWelfareEffectsSocial2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**C.\\n\\n'", "CAPTION FIG10.png": "'\\n_Notes:_ This figure presents the moderators of local average treatment effects of Facebook deactivation estimated using equation (4). For each of the six outcomes, we present the five moderators with the largest moderation coefficients \\\\(\\\\hat{\\\\alpha}^{k}\\\\). All outcome variables are normalized so that the Control group ending distribution has a standard deviation of 1, and all moderators are also normalized to have a standard deviation of 1. Error bars reflect 95 percent confidence intervals. See Section IC for variable definitions.\\n\\nFigure 10. Heterogeneous Treatment Effects for All Moderators\\n\\n'", "CAPTION FIG11.png": "'_Notes:_ This figure presents the distribution of willingness-to-accept to deactivate Facebook between midline and endline. All responses above $525 are plotted at $525.\\n\\n'", "CAPTION FIG12.png": "'_Notes:_ This figure presents the mean willingness-to-accept \\\\(\\\\langle\\\\)WTA\\\\(\\\\rangle\\\\) to deactivate Facebook in Treatment and Control, for the impact evaluation sample: participants who were willing to accept less than $102 to deactivate Facebook for the four weeks after midline and were offered \\\\(p=$102$\\\\) or \\\\(p=$0\\\\) to do so. The first pair of bars is the mean WTA for deactivation in weeks 1-4, the four weeks after the midline survey. The second pair of bars is mean WTA for deactivation in weeks 5-8, the four weeks after the midline survey, as elicited in the midline survey. The third pair of bars is mean WTA for deactivation in weeks 5-8, as elicited in the midline survey.\\n\\nFigure 12. Average Valuation of Facebook in Treatment and Control.\\n\\n'", "CAPTION FIG2.png": "'_Notes:_ This figure presents local average treatment effects of Facebook deactivation estimated using equation (1). All variables are normalized so that the Control group ending distribution has a standard deviation of 1. Error bars reflect 95 percent confidence intervals. See Section IC for variable definitions. _Facebook minutes_ is not included in the substitute time uses index, and _Facebook news_ is not included in the substitute news sources index, so we visually separate these two variables from the other variables in their respective families. We also visually separate online and offline time uses and news sources, although all online and offline substitutes enter their respective indexes.\\n\\n'", "CAPTION FIG3.png": "'_Notes:_ This figure presents local average treatment effects of Facebook deactivation estimated using equation (1). All variables are normalized so that the Control group endline distribution has a standard deviation of 1. Error bars reflect 95 percent confidence intervals. See Section IC for variable definitions.\\n\\nFigure 3. Effects on News and Political Outcomes\\n\\n'", "CAPTION FIG4.png": "'_Notes:_ This figure presents kernel density plots of issue opinions for Democrats and Republicans in Treatment and Control at endline. Issue opinions are attitudes about nine current political issues on a scale from \\\\(-5\\\\) to \\\\(+5\\\\), such as \"To what extent do you think that free trade agreements between the US and other countries have been a good thing for a bad thing for the United States.\" See online Appendix B for a list of all nine issue questions. To construct the issue opinions measure, for each issue question \\\\(q\\\\), we normalize responses by the standard deviation in the Control group, determine Democrats\\' and Republicans\\' average responses \\\\(\\\\mu_{q}^{D}\\\\) and \\\\(\\\\mu_{q}^{R}\\\\), researcher so that \\\\(\\\\mu_{q}^{D}+\\\\mu_{q}^{R}=0\\\\), and design so that \\\\(\\\\mu^{R}>0\\\\). Define \\\\(\\\\hat{y}_{\\\\text{eg}}\\\\) as individual \\\\(i\\\\)\\'s normalized, recentered, and re-signed response to question \\\\(q\\\\). Thus \\\\(\\\\hat{y}_{\\\\text{eg}}\\\\) reflects the strength of individual \\\\(i\\\\)\\'s agreement with the average Repablican. Define \\\\(\\\\sigma_{q}\\\\) as the Control group within-person standard deviation of \\\\(\\\\hat{y}_{\\\\text{eg}}\\\\) for question \\\\(q\\\\). This measures how much people\\'s views change between baseline and endline, and allows us to place higher weight on issues about which views are malleable over the deactivation period. The preliminary issue opinion measure is \\\\(Y_{i}=\\\\sum_{q}\\\\hat{y}_{\\\\text{eg}}\\\\sigma_{q}\\\\), and the final issue opinion measure plotted in the figure is \\\\(Y_{i}\\\\) divided by the Control group standard deviation.\\n\\nFigure 4. Issue opinions by Party at Endline\\n\\n'", "CAPTION FIG5.png": "'_Notes:_ This figure presents local average treatment effects of Facebook deactivation estimated using equation (1). All variables are normalized so that the Control group endline distribution has a standard deviation of 1. Error bars reflect 95 percent confidence intervals. See Section IC for variable definitions.\\n\\n'", "CAPTION FIG6.png": "'_Notes:_ This figure presents local average treatment effects of Facebook deactivation estimated using equation (1). All variables are normalized so that the Control group endline distribution has a standard deviation of 1. Error bars reflect 95 percent confidence intervals. See Section IC for variable definitions.\\n\\n'", "CAPTION FIG7.png": "'_Notes:_ This figure shows the share of the Treatment and Control groups that had their Facebook accounts described, by day of the experiment, for the impact evaluation sample: participants who were willing to accept less than $102 to deactivate Facebook for the four weeks after midline and were offered \\\\(p=$102\\\\) or \\\\(p=$0\\\\) to do so. The vertical gray areas reflect the 24-hour periods after midline and endline during which both Treatment and Control were instructed to deactivate.\\n\\n'", "CAPTION FIG8.png": "''", "CAPTION FIG9-1.png": "'[_Continued_]'", "CAPTION FIG9.png": "'\\nFigure 9. Heterogeneous Treatment Effects (Continued)\\n\\n_Notes:_ This figure presents local average treatment effects of Facebook deactivation estimated using equation \\\\(\\\\langle 1\\\\rangle\\\\), for subgroups defined by the primary moderators in our pre-analysis plan. All variables are normalized so that the Control group ending distribution has a standard deviation of 1. Error bars reflect 95 percent confidence intervals. See Section IC for variable definitions.\\n\\n'", "CAPTION TAB2.png": "'_Notes:_ Column 1 presents average demographics for the impact evaluation sample: participants who were willing to accept less than $102 to deactivate Facebook for the four weeks after midline and were offered \\\\(p~{}=~{}\\\\$102\\\\) or \\\\(p~{}=~{}\\\\$0\\\\) to do so. Column 2 presents our estimate of average demographics of American adults with a Facebook account. The top five numbers in column 2 are inferred from a Few Research Center (2018f) survey of social media use by demographic group. The bottom number in column 2 (the average of 45 minutes of Facebook use per day) is approximated on those basis of sources such as Facebook (2016) and Molla and Wagner (2018). Column 3 presents average demographics of American adults. The top five numbers are from the 2017 American Community Survey (US Census Bureau 2017), and the Repabikan and Democrat shares are from the 2016 American National Election Study (American National Election Studies 2016).\\n\\n'", "CAPTION TAB3.png": "'_Notes:_ Columns 1 and 2 present survey response and treatment compliance rates for the Treatment and Control groups in the impact evaluation sample: participants who were willing to accept less than $102 to deactivate Facebook for the four weeks after midline and were offered \\\\(p~{}=~{}\\\\$102\\\\) or \\\\(p~{}=~{}\\\\$0\\\\) to do so. Column 3 presents \\\\(p\\\\)-values of tests of differences in response rates between the two groups.\\n\\n'", "CAPTION TAB4.png": "'_Notes:_ The post-endline survey included the following question with an open response text box: \"How has the way you use Facebook changed, if at all, since participating in this study?\" For all responses, we stemmed words, filtered out stop words, then constructed all phrases of length \\\\(l\\\\ =\\\\ \\\\left\\\\{1,2,3,4\\\\right\\\\}\\\\) words. For each phrase \\\\(p\\\\) of length \\\\(l\\\\), we calculated the number of occurrences of that phrase in Treatment and Control group responses (\\\\(f_{p\\\\text{GT}}\\\\) and \\\\(f_{p\\\\text{GT}}\\\\)) and the number of occurrences of length-\\\\(l\\\\) phrases that are _not_ phrase \\\\(p\\\\) in Treatment and Control responses (\\\\(f_{-\\\\mu\\\\text{GT}}\\\\) and \\\\(f_{-p\\\\text{GT}}\\\\)). We then constructed Pearson\\'s \\\\(\\\\chi^{2}\\\\)-statistic:\\n\\n\\\\[\\\\chi^{2}\\\\ =\\\\ \\\\frac{\\\\left(f_{p\\\\text{GT}}f_{-p\\\\text{GT}}-f_{p\\\\text{GT}}f_{-p \\\\text{GT}}\\\\right)^{2}}{\\\\left(f_{p\\\\text{GT}}+f_{p\\\\text{GT}}\\\\right)\\\\left(f_{p \\\\text{GT}}+f_{-\\\\mu\\\\text{GT}}\\\\right)\\\\left(f_{p\\\\text{GT}}+f_{-\\\\mu\\\\text{GT}} \\\\right)\\\\left(f_{-p\\\\text{GT}}+f_{-p\\\\text{GT}}\\\\right)}.\\\\]\\n\\nThis table presents the 20 phrases with the highest \\\\(\\\\chi^{2}\\\\) that were most commonly written by the Treatment and Control groups. The % Treatment and % Control columns present the share of people in the respective group whose responses included each phrase.\\n\\n'", "CAPTION TAB5.png": "'_Notes:_ The endline survey asked, \"Do you think the researchers in this study had an agenda?\" Columns 1 and 2 present the share of the Treatment and Control groups who gave each possible response. Column 3 presents \\\\(\\\\rho\\\\)-values of tests of differences in means between the two groups.\\n\\n'", "CAPTION TAB6.png": "'_Notes:_ This table presents estimates of equation \\\\(\\\\langle 5\\\\rangle\\\\). The dependent variable is the change in WTA for post-endline deactivation measured at endline versus midline. Standard errors are in parentheses.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/althuizenSupportingCreativeProblem2014.json b/extracted_captions/althuizenSupportingCreativeProblem2014.json new file mode 100644 index 0000000000000000000000000000000000000000..0c1c1ffc3c636dfe10c11435887e50192503536d --- /dev/null +++ b/extracted_captions/althuizenSupportingCreativeProblem2014.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Study 1: Interaction Between Creative Ability and the Use of a CBR System\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Study 2: Interaction Between Creative Ability and Type of Cases in the CBR System\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Study 3: Interaction Between Creative Ability and Number of Cases in the CBR System\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 3: The Effects of the Number of Cases Stored in the CBR System (Study 3).\\n\\n'", "CAPTION TAB4.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system of coupled equations. We show that the two-dimensional case is equivalent to the two-dimensional case. We show that the two-dimensional case is equivalent to the two-dimensional case.\\n\\n'", "CAPTION TAB5.png": "'* [16] A.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/andrianantoandro2006mechanism.json b/extracted_captions/andrianantoandro2006mechanism.json new file mode 100644 index 0000000000000000000000000000000000000000..3bba16dbf5b2333394cfd55fa6ff0ebc218fd8cd --- /dev/null +++ b/extracted_captions/andrianantoandro2006mechanism.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Actin filament Severing by Collins Observed by Evanescent Wave Fluorescence Microscopy Conditions were as follows: 50 mM KCl, 10 mM imidazole, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.25% methyl ceHulose, 100 mM DTT, 15 mM glucose, 20 mg/mL catalase, and 100 mg/mL glucose oxidase at 22\u00b0 C and pH 7.0. (A) Fluorescence micrographs of entire microscopic fields of 25% Oregon Green-labeled actin filaments polymerized for 6 min taken at indicated time points after wash in from time-lapse movies of actin filament severing. First row, actin filaments washed with S. pombe coffin at the optimal severing concentration of 10 nM. Filaments were attached to the glass surface at their barbed ends by S. pombe GST-CdCl21[FH1FH2]p. Second row, actin filaments washed with actophorin at the optimal severing concentration of 100 nM. Filaments were attached to the glass surface by NEM-inactivated muscle myosin II. (B) Detailed mortgage of one actin filament being sewered by 100 nM actophorin over time.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Analysis of Actin Filament Severing by Collins (A) Dependence of severing activity on the concentration of _S. pombe_ cofflin under the conditions of Figure 1. Degree of saturation of the subunits in the filaments was calculated by using Equation. 2. The units of severing activity are severing events per um of actin filament per second. Optimal severing activity occurs at 10 nM _S. pombe_ cofflin with cofflin bound to 0.12% of the actin subunits. No severing occurred without cofflin or with 1 \u03bcM S. pombe cofflin, which saturates 49% of the actin subunits.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Effect of Collins on Actin Filament Elongation and Nucleation (A\u2013C) Elongation was observed by evanescent wave fluorescence microscopy with 50% Oregon Green-labeled ATP-actin. Conditions as in Figure 3. Dependence of the elongation rate on the concentration of unlabeled actin: (A) actin alone, (B) actin with 32 \u03bcM human coffin, and (C) actin with 24 \u03bcM _M_S_._pombe coffin. Filled circles represent barbed-end rates and filled triangles represent pointed-and rates. Slope = k_v_-int_ = k_v_-, and x-int = Cc. (D\u2013F) nucleation of actin filaments by coffins. Evanescent wave fluorescence micrographs of filaments formed by 0.8 \u03bcM 50% Oregon Green-labeled ATP-actin after 400 s in polymerization buffer. Conditions as in Figure 1. (D) Actin alone, (E) actin with 32 \u03bcM human coffin, and (F) actin with 24 \u03bcM _M_S_. pombe coffin. (G\u2013I) Quantitation of barbed ends formed by nucleation by dilution of samples into 2 \u03bcM 25% pyrenyl ATP-actin monomers and measurement of the rate of elongation. Conditions for nucleation and subsequent elongation were as in Figures 2C and 2D. (G) Time course of barbed-end formation by 6 \u03bcM ATP-actin with 10 \u03bcM _M_S_._pombe coffin fit with a hyperbola. Inset, time course of ends formed by 6 \u03bcM ATP-actin with 10 \u03bcM _M_S_. pombe coffin (filled circles), or with 10 \u03bcM _M_S_._pombe coffin and 10 \u03bcM _M_S_._pombe profilin (filled triangles). (H) Dependence of and formation on actin monomer concentration. A range of ATP-actin concentrations was polymerized for 30 s with 10 \u03bcM _M_S_._pombe coffin. (I) Dependence of the rate of end formation by 10 \u03bcM _M_S_._pombe coffin concentration. Samples were polymerized for 60 s before dilution.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Concentration Dependence of Codlin Activity\\n\\nVery low concentrations of codlin (blue ovals) do not bind actin filaments (green). At an optimal low concentration of codlin, single codlins bind and sever actin filaments. Capping of severed filament barbed ends promotes dissociation of actin monomers (green circles) from pointed ends, leading to filament disassembly. Higher concentrations of codlin bind cooperatively to actin filaments and promote the release of inorganic phosphate (P\\\\({}_{\\\\beta}\\\\)) but do not sever them. Very high concentrations of codlin bind actin monomers and stimulate nucleation, leading to actin filament assembly.\\n\\n'", "CAPTION FIG7.png": "'Figure 3: Depolymerization of Actin Filaments in the Presence of Collins Observed by Evanescent Wave Fluorescence Microscopy Conditions as in Figure 1. ATP-actin filaments were grown for 6 min from 1.5 \u03bcM 50% Oregon Green-labeled actin alone, or with 32 \u03bcM human coffin, or with 24 \u03bcM S, pombe coffin. These samples were washed with buffer alone or with 10 \u03bcM human coffin or with 10 \u03bcM S, pombe coffin, and observed. The data are presented in two ways: (A)\u2013(F), time course of length versus time, where dark gray lines are the global fit rates with dark dotted lines at 95% confidence intervals and light gray lines are the average individual filament rates with light dotted lines at minimum and maximum slopes and dashed lines indicating standard deviations, and (B)\u2013(L), histograms of the distribution of measured length changes during each 9 s interval. (A\u2013C) Barbed ends. (A) Actin filaments alone, (B) actin with 10 \u03bcM human coffin, and (C) actin with 10 \u03bcM S, pombe coffin. (D\u2013F), Pointed ends. (D) Actin filaments alone, (E) actin with 10 \u03bcM human coffin, and (F) actin with 10 \u03bcM S, pombe coffin. (G\u2013L) Barbed ends. (G) Actin filaments alone, (F) actin with 10 \u03bcM human coffin, and (I) actin with 10 \u03bcM S, pombe coffin. (J\u2013L) Pointed ends. (J) Actin filaments alone, (K) actin with 10 \u03bcM human coffin, and (L) actin with 10 \u03bcM S, pombe coffin.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1.**} & \\\\multicolumn{1}{c}{**S**over**} \\\\\\\\ \\\\multicolumn{1}{c}{**and**} & \\\\multicolumn{1}{c}{**Optimal**} & \\\\multicolumn{1}{c}{**S**over**} \\\\\\\\ \\\\multicolumn{1}{c}{**and**} & \\\\multicolumn{1}{c}{**Optimal**} & \\\\multicolumn{'", "CAPTION TAB2.png": "'* [19]'", "SUPP CAPTION FIGS1.png": "'\\n\\n**Fig. S1.** X-ray crystal structure of S. pombe cofilin. (A) Stereo ribbon diagram of S. pombe cofilin with residues R78 and K80 highlighted, and sulfate ion bound to N-terminus. Residues 2-123 shown. (B) Model and electron density for residues surrounding the bound sulfate.\\n\\n'", "SUPP CAPTION FIGS2-1.png": "'\\n\\n**Fig. S2.** Inhibition of nucleotide exchange on actin monomer by S. pombe cofllin. Conditions: 10 mM imidazole, 1 mM MgCl\\\\({}_{2}\\\\), 1 mM EGTA, 0.5 mM DTT, 1 mM NaN\\\\({}_{3}\\\\) at 22 degC and pH 7.0. Actin concentrations were'", "SUPP CAPTION FIGS2-2.png": "'0.2 uM. Observed rate constant for etheno-ATP or etheno-ADP dissociation from actin monomer upon addition of 0.5 mM ATP or ADP plus S. pombe cofilin is plotted versus S. pombe cofilin concentration. Black circles, dashed line: ADP-actin. Open circles, dashed line: ATP-actin. Grey triangles, solid line: ADP-actin plus 50 mM KCl. Open triangles, solid line, ATP-actin plus 50 mM KCl.\\n\\n'", "SUPP CAPTION FIGS2.png": "''", "SUPP CAPTION FIGS3.png": "'\\n\\n**Fig. S3.** Time course of fluorescence change following the mixing of 0.2 \\\\(\\\\upmu\\\\)M S. pombe cofilin with 6 \\\\(\\\\upmu\\\\)M pyrene-labeled skeletal muscle ADP-actin filaments. The inset shows the initial rapid decrease in fluorescence on a faster time scale, fit to a single-exponential. Conditions: 10 mM imidazole, 2 mM Tris-HCl pH 8.0, 50 mM KCl, 1 mM MgCl\\\\({}_{2}\\\\), 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN\\\\({}_{3}\\\\) at 22 \\\\({}^{\\\\circ}\\\\)C and pH 7.0'", "SUPP CAPTION FIGS4-1.png": "'Fig. S4. Raw data of 2 \\\\(\\\\mu\\\\)M pyrene actin elongating from actin nucleated in the presence and absence of human, S. pombe, and R78E K80E S. pombe cofilin mutant. Conditions: 10 mM imidazole, 2 mM Tris-HCl pH 8.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN3 at 22 \\\\({}^{\\\\circ}\\\\)C and pH 7.0. The slopes of the fluorescence traces indicate the number of ends formed under each condition: no seeds (heavy black line), 6 mM actin seeds (light black line), 6 mM actin/10 mM S. pombe cofilin seeds (light dotted line), 6 mM actin/10 mM Human cofilin'", "SUPP CAPTION FIGS4-2.png": "'seeds (grey line), 6 uM actin/10 uM R78E K80E S. pomhe cofilin mutant seeds (heavy dotted line).\\n\\n'", "SUPP CAPTION FIGS4.png": "'Figure 44: Raw data of 2 \\\\(\\\\mu\\\\)M prime actin elongating from actin nucleated in the presence and absence of human, S. pombe, and R79E K80E S. pombe for actin mutant seeds (heavy dotted line). 6 mM actin most seeds (light black line), 6 mM actin most seeds (light black line), 6 mM actin/10 mM S. pombe cofflin mutant. Conditions: 10 mM imidazole, 2 mM Tris-HCl pH 8.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN, at 22 \u00b0C and pH 7.0. The slopes of the fluorescence traces indicate the number of ends formed under each condition: no seeds (heavy black line), 6 mM actin seeds (light black line), 6 mM actin most seeds (light black line), 6 mM actin/10 mM S. pombe cofflin seeds (light dotted line), 6 mM actin/10 mM Human cofflin\\n\\n'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Fig. S5.** Predicted spatial compartmentalization of ADF/cofilin activities.\\n\\n'", "SUPP CAPTION TABS1.png": "'\\n\\n**Table S1.** Data Collection and Refinement Statistics'"} \ No newline at end of file diff --git a/extracted_captions/aoki2016rhoa.json b/extracted_captions/aoki2016rhoa.json new file mode 100644 index 0000000000000000000000000000000000000000..04440fd05c16715699acf86a072d19739b079a0c --- /dev/null +++ b/extracted_captions/aoki2016rhoa.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\nFig. 1: Epss locally accumulations at the initial phase of membrane extraction in the membrane bath. (_A_) (LDD) cells exhibit membrane blocking when cultured in a single region (_k_igth). (Scale bar, 20 \\\\(\\\\mu\\\\)m) (_B_ and C) Membrane blebbing of DLD1 cells transfected with LHeat-RFP and GFP-tagged PLCs-PH. Timing relative to the first image is indicated in white text. Actin cortex reassembly started from multiple sites of the protruded membrane (arrowheads). (Scale bar: \\\\(B\\\\), 5 \\\\(\\\\mu\\\\)m; \\\\(C\\\\), 2 \\\\(\\\\mu\\\\)m). (_D_) Localization of GFP-MRC1 during membrane bleb expansion and retraction. MRC1 accounts/ates at multiple regions of membrane blebs (arrowheads). (Scale bar: 1 \\\\(\\\\mu\\\\)m). (_E_) Localization of GFP-tagged Epss in membrane blebs of DLD1 cells. Epss accumulates in multiple foci at the protruded membrane (arrowheads). (Scale bar: 2 \\\\(\\\\mu\\\\)m). (_F_ and _G_) Extrapogrsk showing actin localization (red) with respect to actin cytoskeleton-related proteins (green) during bleb retraction. Blob extension is shown on the horizontal axis, and time is shown on the vertical axis. Epss-B (_F_, green) localizes to the protruded membrane before actin filaments. MRC1 (_G_, green) is executed after actin filaments. (_H_) Timing of arrival of Epss and MRC1 relative to that of actin filaments (_f_ = 0 s). Data are the mean \\\\(\\\\pm\\\\) SD.\\n\\n'", "CAPTION FIG2.png": "\"\\nFig. 2: The end-tapping and actin-burdling activities of Epsl are required for continuous membrane blebbing. (A) Expression of Epsl is greatly reduced in Epsl-KD DLD1 cells. (B) Epsl-KD DLD1 cells are spherical and do not exhibit membrane blebbing when cultured in a type I collagen matrix. (Scale bar, 10 \u03bcm.) [C] Exogenous expression of GFP-tagged mouse Epsl restores membrane blebbing (arrow) in Epsl-KD DLD1 cells. (Scale bar, 10 \u03bcm.) (D) Schematic drawings of mutant Epsl constructs. The number of amino acid residues of Epsl is shown. (F) Total cell lysates of DLD1 cells expressing each construct separated by SDS9AGE and immunoblotted with an anti-GFP mAb. (F) The percentages of GFP-positive cells showing membrane blebbing relative to the total number of GFP-positive cells in a given field were calculated. In each experiment, the total cell number was 100 (\\\\(n=3\\\\)). Data are the mean \\\\(\\\\pm 5\\\\). \\\\(\\\\pm P<0.05\\\\) (Student's t test). (G) Localization of GFP-tagged mutant Epsl8 in Epsl-KD DLD1 cells. Expression of the GFP-tagged Epsl mutant lacking the proline-rich region (GFP-Epsl8APR) or the GFP-tagged Epsl mutant lacking the SH3 domain (GFP-Epsl8APR34) restores membrane blebbing in Epsl8-KD DLD1 cells (arrow). (Scale bar, 10 \u03bcm.)\"", "CAPTION FIG3.png": "'Figure 3: Activation of azaft occurs at retracting membranes and is required for the rapid retraction of membrane blebs. (_A_) Membrane blebbing in DLD1 cells transfected with GFP-ezrin. GFP-ezrin localizes uniformly at the protruded membrane. (Scale bar, 2 \u03bcm.) (_E_) DLD1 cells were fixed and stained with an anti-phospho-ER antibody (red) and an anti-total ERM antibody (green). Nuclei were stained with DAPI (blue). The asterisk indicates a membrane bleb in which ERM proteins were not activated. (Scale bar, 2 \u03bcm.) (_C_) DLD1 cells were fixed and stained with an anti-phospho-ERM antibody (green) and an anti-Epr8 antibody (red). The arrowheads indicate the colocalization of Epr8 and phosphorylated ERM proteins. (Scale bar, 2 \u03bcm.) (_D_) DLD1 cells were fixed and stained with an anti-phospho-ERM antibody (green) and Alexa 594-phalloidin (red). The boxed area shows the membrane blebs with growing actin filaments. High-magnification image of the boxed area is shown in the right panels. The asterisks indicate a membrane bleb covered with actin cortex. (Scale bar, 10 \u03bcm.) (_E_) Total cell lysates of wild-type DLD1 cells and azaft-KO DLD1 cells separated by SDSPAGE and immunohistolated with an anti-total ERM antibody, an anti-Epr8 antibody, and an anti-tubulin antibody. (_F_) Membrane blebbing of wild-type DLD1 cells and azaft-KO DLD1 cells transfected with Histcard-RFP and GFP-tagged Epr8. (Scale bar, 10 \u03bcm.) (_G_) Tricolor map of membrane blebs in wild-type DLD1 cells and azaft-KO DLD1 cells. Angular coordinates are shown on the horizontal axis, and time is shown on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. (_H_) Histogram of bleb expansion and retraction velocities in wild-type DLD1 cells and azaft-KO DLD1 cells. (_I_) The frequencies of membrane blebs in wild-type DLD1 cells and azaft-KO DLD1 cells during 10 min were quantified. **_P_\\\\(<\\\\) 0.01 (Student\u2019s t test). (_J_) The sizes of membrane blebs in wild-type DLD1 cell and in azaft-KO DLD1 cells during 10 min were quantified. **_P_\\\\(<\\\\) 0.01 (Student\u2019s t test).\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: The RhoA-ROCK-Rn3 feedback loop determines the actin reassembly sites of retracting membranes. (A) GFP-ROCK-It is recruited to the retracting protruded membrane in DLD1 cells. (Scale bar, 2 \\\\(\\\\mu\\\\)m) (B) GFP-ROCK is recruited to the retracting protruded membrane in azin-KO DLD1 cells (arrowhead). (Scale bar, 2 \\\\(\\\\mu\\\\)m). (C) Membrane blebbing of DLD1 cells transfected with Lifcat-RFP and GFP-tagged AHD. (Scale bar, 2 \\\\(\\\\mu\\\\)m) (D) Membrane blebbing of DLD1 cells transfected with Lifcat-RFP and GFP-tagged AHD. (Scale bar, 2 \\\\(\\\\mu\\\\)m). (D) Membrane blebbing of DLD1 cells transfected with Lifcat-RFP and GFP-tagged Rnds. (Rad accumulation gradually disappears upon the initiation of membrane blebbing retraction (\\\\(t=25\\\\) S. (Scale bar, 2 \\\\(\\\\mu\\\\)m).) (E) Kymographs showing the localization of actin (red) with respect to that of Rnds (green) during bleb retraction. (B) Extension is shown on the horizontal axis, and time is shown on the vertical axis. (F) Kymographs showing the localization of Rnds (red) with respect to that of Exp8 (green) during bleb retraction. (B) Extension is shown on the horizontal axis, and time is shown on the vertical axis. (G) GFP-D1908-Rho-GAP localizes only at expanding blebs that lack the actin cortex. The membrane localization of p1908 Rho-GAP is gradually lost upon the initiation of actin cortex recovery. (Scale bar, 2 \\\\(\\\\mu\\\\)m.)'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\nFig. 5: A model of Rnd3- and RhoA-mediated regulation of actin cytoskeleton during membrane-bleibbing cycle. (_A_) Localization of Ep8 in DLD1 cells expressing GFP-wild type Rnd3 (Upper) and GFP-Rrd3 S240A mutant. (Scale bar, 10 \u03bcm.) (_B_) Tricolor map of membrane blobs in DLD1 cells expressing GFP-wild type Rnd3 or GFP-Rnd3 S240A mutant. Angular coordinates are shown on the horizontal axis, and time is shown on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. (_C_) Histogram of bleib expansion and retraction velocities in DLD1 cells expressing wild-type Rnd3 or the Rnd3 S240A mutant. (_D_) The frequencies of membrane bleibs in DLD1 cells expressing GFP-wild type Rnd3 or GFP-Rnd3 S240A mutant during 10 min were quantified. *_P_< 0.01 (Student\u2019s test). (_E_) The sizes of membrane blebs in DLD1 cells expressing GFP-wild type Rnd3 or GFP-Rnd3 S240A mutant during 10 min were quantified. *_P_< 0.05 (Student\u2019s test). (_P_) In the expansion phase of membrane bleibbing, Rnd3 and p1968\u2013Rho-GAP inhibit the activation of RhoA. As the protruded membrane areas become enlarged, the relative concentration of Rnd3 decreases. Sporadic activation of RhoA leads to ROCK phosphorylation of Rnd3 and removal of p1968\u2013Rho-GAP from the membrane. Thus, RhoA activation is amplified and sustained by the positive-feedback loop. ROCK also phosphorylates earin and activated earin recruits, which leads to reassembly of the actin cortex.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/artsParadiseNoveltyLoss2018a.json b/extracted_captions/artsParadiseNoveltyLoss2018a.json new file mode 100644 index 0000000000000000000000000000000000000000..cba2dcb0ee21b930531479b57df3ca9fc2e5f2de --- /dev/null +++ b/extracted_captions/artsParadiseNoveltyLoss2018a.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n## Table 1: Summary Statistics'", "CAPTION TAB2.png": "'\\n\\n**Table 2**.: Inventor-Firm Fixed Effects Models Exploring New Fields'", "CAPTION TAB3.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the \\\\(\\\\beta\\\\)-function and the \\\\(\\\\beta\\\\)-function of the \\\\(\\\\beta\\\\)-'", "CAPTION TAB4.png": "'\\n\\n**Table 4. Summary Statistics** MAKA Sample'", "CAPTION TAB5.png": "'\\n\\n**Table 5.** Difference-ir-Diferences Average Rate of Exploring New Fields'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB7.png": "'\\n## References\\n\\n* [1] A. A. K. K. Aarseth, and A. A. K. Aarseth, \"The 2-Darket: A New Approach to the Evolution of the Galaxy,\" _Astrophys. J._ **584**, 103 (2008).\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/bailExposureOpposingViews2018.json b/extracted_captions/bailExposureOpposingViews2018.json new file mode 100644 index 0000000000000000000000000000000000000000..86107d19c5f06c797b34d65b1497606f028e5870 --- /dev/null +++ b/extracted_captions/bailExposureOpposingViews2018.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1.** Operator at reaction depth.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Abstract**\\n\\nThe thesis is based on the work of'", "CAPTION FIG3.png": "\"Figure 3: Effect of following Twitter hosts that retweet messages by elected officials, organizations, and opinion leaders with opposing political ideologies for 1 mo, on a seven-point liberalKonservative scale where larger values indicate more conservative opinions about social policy issues, for experiments with Democrats (\\\\(n\\\\) = 697) and Republicans (\\\\(n\\\\) = 542). Models predict posttreatment liberalconservative scale score and control for pretreatment score on this scale as well as 12 other covariates described in _SI Appendix_. Circles describe unstandardized point estimates, and bars describe 90% and 95% confidence intervals. Respondents Assigned to Treatment\u201d describes the IT effect for Democrats (\\\\(\\\\text{ITT}=-0.02\\\\), \\\\(\\\\text{IT}=-0.76\\\\), \\\\(\\\\text{p}=0.45\\\\), \\\\(n=416\\\\)) and Republicans (\\\\(\\\\text{ITT}=0.12\\\\), \\\\(\\\\text{E}=2.68\\\\), \\\\(\\\\text{p}=0.008\\\\), \\\\(n=316\\\\)). Minimally-Compliant Respondents\u2019 describes the CACE for respondents who followed one of the study's box for Democrats (\\\\(\\\\text{CACE}=-0.04\\\\), \\\\(\\\\text{r}=-0.75\\\\), \\\\(\\\\text{p}=0.45\\\\), \\\\(n\\\\) of compliant respondents = 271) and Republicans (\\\\(\\\\text{CACE}=0.19\\\\), \\\\(\\\\text{r}=2.73\\\\), \\\\(\\\\text{p}<0.007\\\\), \\\\(n\\\\) of compliant respondents = 181). \u201cPartially-Compliant Respondents\u201d describes the CACE for respondents who correctly answered at least one question, but not all questions, about the content of a hot's tweets during weekly surveys throughout the study period for Democrats (\\\\(\\\\text{CACE}=-0.05\\\\), \\\\(\\\\text{r}=-0.75\\\\), \\\\(\\\\text{p}=0.45\\\\), \\\\(n\\\\) of compliant respondents = 211) and Republicans (\\\\(\\\\text{CACE}=0.31\\\\), \\\\(\\\\text{r}=2.73\\\\), \\\\(\\\\text{p}<0.007\\\\), \\\\(n\\\\) of compliant respondents = 211). \u201cFully-Compliant Respondents\u2019 describes the CACE for respondents who answered all questions about the content of the hot's tweets correctly for Democrats (\\\\(\\\\text{CACE}=-0.14\\\\), \\\\(\\\\text{r}=-0.75\\\\), \\\\(\\\\text{p}=0.46\\\\), \\\\(n\\\\) of compliant respondents = 66) and Republicans (\\\\(\\\\text{CACE}=0.60\\\\), \\\\(\\\\text{r}=2.53\\\\), \\\\(\\\\text{p}<0.01\\\\), \\\\(n\\\\) of compliant respondents = 53). Although treated Democrats exhibited slightly more liberal attitudes posttreatment that increase in size with level of compliance, none of these effects were statistically significant. In contrast, treated Republicans exhibited substantially more conservative views posttreatment that increase in size with level of compliance, and these effects are highly significant.\\n\\n\""} \ No newline at end of file diff --git a/extracted_captions/bakerComplexInstructionalAnalogies2001.json b/extracted_captions/bakerComplexInstructionalAnalogies2001.json new file mode 100644 index 0000000000000000000000000000000000000000..6df1aa392ba109775df6321874f4b19582858949 --- /dev/null +++ b/extracted_captions/bakerComplexInstructionalAnalogies2001.json @@ -0,0 +1 @@ +{"CAPTION TAB2.png": "'\\n\\n## 4 The \\\\(\\\\chi\\\\) value of \\\\(2.12\\\\) has a value of \\\\(0.034\\\\).\\n\\n'", "CAPTION TAB4.png": "'\\\\({}^{\\\\rm a}\\\\) Likert items used the following 1 - 5 scale: 1 - strongly disagree, 2 - disagree, 3 - not sure, 4 - agree, 5 - strongly agree.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/banksPolarizedFeedsThreeExperiments2021.json b/extracted_captions/banksPolarizedFeedsThreeExperiments2021.json new file mode 100644 index 0000000000000000000000000000000000000000..e3d4544c8bdc55f724ebd8e6386beb6bdabfac96 --- /dev/null +++ b/extracted_captions/banksPolarizedFeedsThreeExperiments2021.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Assimilation and contrast in the 2016 presidential election, The American National Election Study (2016).\\n\\nNote: Positive slopes describe assimilation, where voters report an ideological location that is shifted in the direction of their own ideological preferences. Negative slopes describe context effects, where respondents perceive the candidate as further away from their own ideological preference. Our estimation from the Cooperative Congressional Election Study, 2016 data, Ansolabehere and Schaffner (2017).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Tweets of Donald Trump and Hillary Clinton criticizing their opponents. Note. Tweet posted by Hillary Clinton (Tweet ID: 790606692547452932, left) on October 24, 2016, and by Donald J. Trump (Tweet ID: 789594671387447297, right) on October 21, 2016. Dates of the tweets were not included in the experiment.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Perceived polarization and tweet exposure, Clinton and Trump.\\n\\nNote. Perceived polarization describes the difference between the reported placement of Trump and Clinton for each respondent. Figures correspond to Table 1, Models 2 and 4. Point estimates with 95 percent confidence intervals.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Perceived polarization, tweet exposure, and vote choice.\\n\\nNote. Perceived polarization describes the difference between the reported placement of Donald Trump- and Hilary Clinton for each respondent. Point estimates with 95 percent confidence intervals.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Identification of selected tweets in the observational data of the #TravelBan Network.\\n\\nNote. Selection of tweets was based on Twitter network analyses collected between January 31 and February 2, 2017. The primary connected network included 241,271 users and posted 2,031,518 tweets. The largest community (anti-ban in dark gray) included 137,858 users in the primary connected network, representing 37 percent of the network. The second largest community (pro-ban in light gray) included 41,181 users, representing 17 percent of the network. In the anti-ban community, the account of the NYT was the most reweeted authority. In the pro-ban community, Fox News was the second most reweeted account, after PrisonPlanet. Tweets of Fox News and the NYT circulated in very different areas of Twitter\u2019s social network, with 80.5 percent of Fox News retweets by supporters of the #TravelBan and 92 percent of NYT reweets by opponents of the #TravelBan (see SIF for details). NYT = New York Times; SIF = Supplementary Information File.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Perceived polarization, time of exposure (attention), and party ID. Note. Exposure to sweet by party ID and by treatment. Point estimates with 95 percent confidence intervals.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Distance from Democrats and Republicans by party ID and tweet exposure. Note. Exposure to tweet by party ID and by treatment. Point estimates with 95 percent confidence intervals.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Marginal effect (dy/dx) of time of tweet exposure on perceived ideological polarization, by treatment and party.\\n\\nNote. Results describe the marginal effect (slope) of time of exposure on polarization, conditional on the party of the respondent and the news organization of the tweet.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Perceived polarization and tweet exposure, Mauricio Macri (president) and Cristina Fernandez de Kirchner (former president).\\n\\nNote. Perceived polarization describes the difference between the reported placement of Mauricio Macri (president) and Cristina Fernandez de Kirchner (former president) for each respondent. Three groups, one exposed to a tweet by Lamata (journalist aligned with Macri), another group exposed to a tweet by Navarro (journalist aligned with Fernandez), and a control group.\\n\\n'", "CAPTION TAB1-1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB1-2.png": "''", "CAPTION TAB1.png": "''", "CAPTION TAB2-1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'", "CAPTION TAB2-2.png": "'\\n\\n**Acknowledgement**'", "CAPTION TAB2-3.png": "'* [19]'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c c} \\\\hline \\\\hline \\\\multicolumn{1}{c}{\\\\multirow{2}{*}{**Table 2.** Perceived Polarization and Attention, Second Experiment.}} & **Table 2.** (Continued) & **Table 2.** (Continued) & **Table 2.** (Continued) \\\\\\\\ & \\\\(\\\\varphi<0.5^{\\\\circ}\\\\) & \\\\(\\\\varphi<0.1\\\\) & \\\\(\\\\varphi<0.1\\\\) \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Perceived Polarization and Attention, Second Experiment.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/baschieri2018frustrated.json b/extracted_captions/baschieri2018frustrated.json new file mode 100644 index 0000000000000000000000000000000000000000..00f3900f1aeaec0e8e59e6ce7d5c0dc424e664c2 --- /dev/null +++ b/extracted_captions/baschieri2018frustrated.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\nFig. 1: Clathrin-coated plaques are mechanosensitive structures. **a** HeLa cells were seeded on collagen-coated glass or polyacrylamide gels of indicated stiffness and fixed 24 h later before being stained for \u03b1-adaptin. Scale bar: 15 \u03bcm. Higher magnifications of boxed regions are shown. **b** Kymographs showing CCS dynamics in genome-edited HeLa cells expressing endogenous GFP-tagged \u03bcM2-adaptin seeded on the indicated collagen-coated substrate and imaged by spinning disk microscopy every 5 s for 5 min. **c** Quantification of the dynamics of CCSs observed as in **b** (\u201c+P < 0.001, as compared to 0.1 kPa condition, one-way analysis of variance\u2014ANOVA = 3). **d** HeLa cells seeded on collagen-coated glass were treated with Bleblithian or Cytokthalasian D as indicated for 30 min before being fixed and stained for \u03b1-adaptin. Scale bar: 10 \u03bcm. Higher magnifications of boxed regions are shown. **e** Quantification of the dynamics of CCSs observed as in **d** (\u201cP < 0.01, \u201cP < 0.05, ANOVA = 3). **f** EM micrographs of unrooted HeLa cells that were cultured on glass and treated for 30 min with blebbistatin before being fixed and processed. Clathrin-coated plaques are highlighted in green. Scale bar: 200 nm. All results are expressed as mean \u00b1 SD'", "CAPTION FIG2.png": "'\\nFig. 2: avlv5 integrin localizes to plaques and is required for their assembly. **a** HeLa cells were seeded on collagen-coated glass and fixed 24 h later before being stained for \u03b1-adaptin and avl/b5-integrin. Scale bar: 10 \u03bcm. Higher magnifications of boxed regions are shown. Arrows point to clathrin-coated plaques arrowheads point to CCPs. **b** Quantification of avl/b5 enrichment at plaques versus CCPs (*P < 0.005, two tailed Student\u2019s t-test. _n = 3_; 100 structures per experiment were counted). **c** Quantification of colocalization (Pearson\u2019s coefficient) between \u03b1/b5 and \u03b1-adaptin in cells cultured on the indicated collagen-coated substrate (*P < 0.005; *P < 0.001, one-way analysis of variance\u2013ANOVA _n = 3_). **d** HeLa cells treated with control (upper panel) or b5-specific (lower panel) siRNA were seeded on collagen-coated glass and fixed 24 h later before being stained for \u03b1-adaptin. Scale bar: 15 \u03bcm. **c** Kymgtaphs showing CCS dynamics in genome-edited HeLa cells treated with the indicated siRNA, seeded on collagen-coated glass, and imaged by spinning disk microscopy every 5 s for 5 min. **f** Quantification of the dynamics of CCSs observed as **in****e** and treated with the indicated siRNA (**_n_ < 0.001, ANOVA _n = 3_). **g** HeLa cells treated with b5-specific siRNAs were transfected with a siRNA-resistant \u03b2 5-GFP encoding construct and then fixed 24 h later before being stained for \u03b1-adaptin. Scale bar: 10 \u03bcm. The star marks a cell that is not transfected by b5-GFP. Higher magnifications of boxed regions are shown. All results are expressed as mean \u00b1 SD'", "CAPTION FIG3.png": "'\\nFig. 3: Clathrin-coated plaques assemble as a consequence of frustrated endocytosis. **a** Genome-edited HeLa cells expressing endogenous mCherry-tagged \\\\(\\\\mu^{2}\\\\)-adaptiv, seeded on collagen-coated glass, were treated with trypsin and imaged by spinning disk microscopy every 5 s. Time after trypsin addition is indicated in seconds. Arrowheads point to dot-like structure emanating from the disassembling plaques. Scale bar: 1 \u03bcm. **b** Kymographs showing plaque disassembly dynamics and concomitant GFP-auxillin bursts in HeLa cell treated and imaged as in **a**. Note that loss of \\\\(\\\\mu^{2}\\\\)-adaptiv-associated fluorescence is correlated with auxillin flashes. **c** Quantification of the number of auxillin flashes per plaque and per minute in HeLa cells before and after incubation with trypsin. Results are expressed as mean \\\\(\\\\pm\\\\) SD (\\\\(\\\\text{P}\\\\)\\\\(\\\\text{P}\\\\)\\\\(\\\\text{<}\\\\)\\\\(\\\\text{0.001}\\\\), Mann-Whitney rank sum test. **d** Total of 90 structures from three independent experiments was quantified). **d** Genome-edited HeLa cells expressing endogenous mCherry-tagged \\\\(\\\\mu^{2}\\\\)-adaptiv, seeded on collagen-coated glass, were treated with. Clingentide and imaged by spinning disk microscopy every 5 s. Time after Clingentide addition is indicated in seconds. Arrowheads point to dot-like structure emanating from the disassembling plaques. Scale bar: 1 \u03bcm. **e** Genome-edited HeLa cells treated with \\\\(\\\\text{P}\\\\)5-specific siRNA and transfected with a construct encoding mCherry-tagged TIR were seeded on anti-mCherry antibodies-coated glass and imaged 24 h later by spinning disk microscopy every 5 s. Scale bar: 10 \u03bcm. **f** Kymograph showing CCS dynamics in the region corresponding to the boxed area in **e**. Note that the cell on the left is not transfected by the TIR-mCherry construct and only display dynamic CCSs. **g** Quantification of the dynamics of CCSs observed as in **e** in genome-edited HeLa cells expressing the TIR-mCherry construct and treated as indicated (\\\\(\\\\text{P}\\\\)\\\\(\\\\text{P}\\\\)\\\\(\\\\text{<}\\\\)\\\\(\\\\text{0.001}\\\\), one-way analysis of variance--ANOVA. \\\\(n\\\\)\\\\(\\\\text{=}\\\\)\\\\(\\\\text{3}\\\\)). Results are expressed as mean \\\\(\\\\pm\\\\) SD'", "CAPTION FIG4.png": "'\\nFig. 4: Clathrin-coated plaques regulate stiffness-dependent Erk signaling. **a** Western-bit analysis of phospho-Erk (P-Erk) levels in HeLa cells growing on collagen-coated glass and treated with control or \\\\(\\\\beta\\\\)S-specific siRNAs as indicated (representative image of four independent experiments). Total-Erk was used as a leading control. **b** Densitometry analysis of bands obtained in western-blots as in **a** Results are expressed as mean \\\\(\\\\pm 5\\\\)D from four independent experiments (\u201cP < 0.05, one-way analysis of variance\u2014ANOVA). **c** Western-bit analysis of phospho-Erk (P-Erk) levels in HeLa cells growing on collagen-coated, 0.1 kPa polyacrylamide gels and treated with control or integrin \\\\(\\\\beta\\\\)S-specific siRNAs, as indicated. Total-Erk was used as a loading control (representative image of three independent experiments) **d** Densitometry analysis of bands obtained in western-blots as in **e** Results are expressed as mean \\\\(\\\\pm 5\\\\)D from three independent experiments. **e** GP-Erk-expressing HeLa cells treated or not with a \\\\(\\\\beta\\\\)S-specific siRNA and transfected or not with TRIzolCherry, as indicated, were seeded on anti-mCherry antibodies-coated glass and imaged 24 h later. Scale bar: 10 \u03bcm. A color-coded scale for low and high signal intensity is shown. **f** Quantification of GP-Erk nuclear enrichment index in cells as in **e** and cultured on control antibody- or anti-mCherry antibody-coated glass, as indicated, and treated or not with indicated siRNAs. Results are expressed as mean \\\\(\\\\pm 5\\\\)D (\u201cP < 0.01, \u201cP < 0.001, ANOVA, ControlAb-siCtr: 144 cells from \\\\(n=5\\\\) independent experiments; mCherryAb-siCtr: 19 cells from \\\\(n=5\\\\) independent experiments; ControlAb-siCtr: 146 cells from \\\\(n=5\\\\) independent experiments; mCherryAb-siCtr: 120 cells from \\\\(n=5\\\\) independent experiments; ControlAb-siCtr: 1- siRNA-2: 69 cells from \\\\(n=3\\\\) independent experiments; mCherryAb-siCtr: 1- siRNA-2: 68 cells from \\\\(n=3\\\\) independent experiments; ControlAb-siCtr: 1- siRNA-2: 67 cells from \\\\(n=3\\\\) independent experiments)'", "CAPTION FIG5.png": "'\\nFig. 5: Clathrin-coated plaques: totally regulate receptor-dependent Ek: signaling. **a** HeLa cells seeded on collagen-coated glass were starved for 4 h and then treated or not with 10 ng/ml EGF for 5 min alone or added after 30 min preincubation with 10 \u03bcM Gefitinib. Cells were then fixed and stained for a-adaptin and phosphotyrosine. The arrowhead points to one CCP and the star marks a plaque. Scale bar: 2 \u03bcM. **b** Quantification of phosphotyrosine accumulation at plaques or CCPs in the indicated conditions. Results are expressed as mean +- SOD (*_P_ < 0.05, *_P_ < 0.001, one-way analysis of variance\u2013ANOVA. \\\\(n\\\\) = 3. Number of structures analyzed per condition: Starved 160 plaques; EGF: 452 plaques, EGF + Gefitinib: 301 plaques, CCPs/EGF: 200 pits). **c** Western-blot analysis of phospho-Ek (P-Ek) levels in starved HeLa cells treated with control or b5-specific siRNA as indicated, and stimulated for the indicated time with 10 ng/ml EGF. Total-Ek was used as a leading control (representative image of three independent experiments. **d** Derstimometry analysis of bands obtained in western-blots as in **e**. Results are expressed as mean +- SOD (*_P_ < 0.05, ANOVA. \\\\(n\\\\) = 3). **e** HeLa cells treated with b5-specific siRNA were transfected with plasmids encoding for TIR-mCherry and EGFR-GFP and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 10 ng/ml EGF for 470 s. Scale bar: 0.5 \u03bcM. Arrowheads point to plaques positive for EGFR-GFP. **f** HeLa cells treated with b5-specific siRNA were transfected with plasmids encoding for TIR-mCherry and HGFR-GFP and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 50 ng/ml HGF for 570 s. Scale bar: 0.5 \u03bcM. Arrowheads point to plaques positive for HGFR-GFP. **g** HeLa cells treated with b5-specific siRNA were transfected with plasmids encoding for TIR-mCherry and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 10 ng/ml EGF for 5 min prior to fixation and staining for phosphotyrosine. Scale bar: 1.5 \u03bcM. Arrowheads point to plaques positive for P-Tyr'", "CAPTION FIG6.png": "'\\nFig. 6: Signaling at plaques is contractility-independent and regulate cell proliferation. **a** HeLa cells on collagen-coated glass were treated for 1 h with 10 \u03bcM Blebbistatin or 10 \u03bcM Cytochalasin D prior to be fixed and stained for \u03b1-adaptin (red) and phosphotyrosines (P-Tyr, green). Scale bar: 3 \u03bcM. **b**, Quantification of phosphotyrosines accumulation at plaques in cells treated as in a, as indicated (*P < 0.05, one-way analysis of variance\u2014ANOVA). Control: 402 plaques from \\\\(n=3\\\\) independent experiments; Blebbistatin: 303 plaques from \\\\(n=3\\\\) independent experiments; Cytochalasin: 302 plaques from \\\\(n=3\\\\) independent experiments. **c** Western-blot analysis of phospho-Frk (P-Frk) levels in HeLa cells cultured on collagen- or vitronectin-coated glass or 0.1 \u03bcM kPa polyvinylamide gels, as indicated. Total-Erk was used as a loading control (representing image of three independent experiments). **d** Densimetry analysis of bands obtained in western-blots as in **e** (*P < 0.05, ANOVA. \\\\(n=3\\\\)). **e** Western-blot analysis of phospho-Frk (P-Erk) levels in HeLa cells growing on collagen-coated glass and treated with control or integrin b5-specific siRNAs and incubated or not with 10 \u03bcM Blebbistatin for 1 h, as indicated. Total-Erk was used as a loading control (representative image of three independent experiments). **f** Densitometry analysis of bands obtained in western-blots as in **e** (*P < 0.05, *P < 0.01, ANOVA. \\\\(n=5\\\\)). **g** Equal numbers of HeLa cells were plated on non-coated glass (open squares), or on collagen-coated (purple open circles) or vitronectin-coated glass (black open circles), as indicated. Cells were harvested and counted 24 and 48 h after plating (***P < 0.001, ANOVA. \\\\(n=3\\\\)). **h** HeLa cells treated with control (circles), b5-specific (triangles and crosses), or any-specific siRNAs (diamonds and hexagons) for 48 h were seeded on vitronectin-coated glass in equal numbers. 24 and 48 h later, cells were harvested and counted (*P < 0.05, ANOVA. \\\\(n=3\\\\)). **i** HeLa cells treated with b5-specific siRNAs were transfected with a plasmid encoding TIR-mCherry and seeded in equal numbers on glass coated with either anti-mCherry (black circles) or control antibodies (open circles). 24 and 48 h later, cells were harvested and counted (*P < 0.05, ANOVA. \\\\(n=3\\\\)). All results are expressed as mean \u00b1 SD'"} \ No newline at end of file diff --git a/extracted_captions/blakeCollaborativeInformationSynthesis2006a.json b/extracted_captions/blakeCollaborativeInformationSynthesis2006a.json new file mode 100644 index 0000000000000000000000000000000000000000..72b82359a9a394ed71184fa229f8c174eea99552 --- /dev/null +++ b/extracted_captions/blakeCollaborativeInformationSynthesis2006a.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'"} \ No newline at end of file diff --git a/extracted_captions/cattaniDeconstructingOutsiderPuzzle2017.json b/extracted_captions/cattaniDeconstructingOutsiderPuzzle2017.json new file mode 100644 index 0000000000000000000000000000000000000000..0bc2cff905ca38608ce778229a0a78a64a09c461 --- /dev/null +++ b/extracted_captions/cattaniDeconstructingOutsiderPuzzle2017.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Source: Royal Museum Greenwich (http://prints.rng.co.uk/art/)\\n\\n515403/harrisons-marine-timkeeper-hd4.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: (Color online) Harrison\u2019s Transition Across \u201cLayers of Legitimacy\u201d Over Time.\\n\\n'", "CAPTION FIG3.png": "'Note. Solid lines indicate the boundaries of the action and attention spaces _before_ the jolt, while dotted lines indicate the expansion of these boundaries _after_ the jolt.\\n\\n'", "CAPTION FIG4.png": "'* [19]'", "CAPTION TAB1.png": "'Source. Adapted from Andrecus (1996a, p. 234).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/champion2006role.json b/extracted_captions/champion2006role.json new file mode 100644 index 0000000000000000000000000000000000000000..db502279350d7c40772931d1f3a0ba6216f3008f --- /dev/null +++ b/extracted_captions/champion2006role.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n## 2 The \\\\(\\\\beta\\\\)-function\\n\\nThe \\\\(\\\\beta\\\\)-function is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (1)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (2)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (3)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (4)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (5)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (6)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (7)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (8)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (9)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (10)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (11)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (12)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (13)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (14)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (15)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (16)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (17)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (18)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (19)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\(\\\\beta\\\\). The function \\\\(\\\\beta(x)\\\\) is defined as\\n\\n\\\\[\\\\beta(x)=\\\\frac{1}{\\\\beta(x)}\\\\exp\\\\left(-\\\\frac{1}{\\\\beta(x)}\\\\right)\\\\] (20)\\n\\nwhere \\\\(\\\\beta(x)\\\\) is the \\\\(\\\\beta\\\\)-function of the function \\\\('", "CAPTION FIG2.png": "'Figure 2: Time-lapse video microscopy clips spanning 39 min of macrophages interacting with identical nonopinionized ED particles (major axis 14 \\\\(\\\\mu\\\\)m, minor axis 3 \\\\(\\\\mu\\\\)m) from two different orientations. (A) Cell attaches along the major axis of an ED and internalizes it completely in 3 min. (B) Cell attaches to the flat side of an identical ED and spreads but does not internalize the particle. Continued observation indicated that this particle was not internalized for \\\\(>\\\\)10 min. (Scale bars: 10 \\\\(\\\\mu\\\\)m.) See Movies 1 and 2.) At least three cells were observed for each orientation of each particle type and size. Similar results were observed in all repetitions.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Scanning electron micrographs and actin staining confirm time-lapse video microscopy observations. Micrographs (\\\\(A\\\\)\u2013C) of cells and particles were colored brown and purple, respectively. (\\\\(A\\\\)) The cell body can be seen at the end of an organized ED, and the membrane has progressed down the length of the particle. (Scale bar: 10 \\\\(\\\\mu\\\\)m.) (\\\\(B\\\\)) A call has attached to the flat side of an organized ED and has spread on the particle. (Scale bar: 5 \\\\(\\\\mu\\\\)m.) (\\\\(C\\\\)) An organized spherical particle has attached to the top of a cell, and the membrane has progressed over approximately half the particle. (Scale bar: 5 \\\\(\\\\mu\\\\)m.) (\\\\(D\\\\)\u2013\\\\(F\\\\)) Overlays of bright-field and fluorescent images after fixing the calls and staining for polymerized actin with rhodamine phalloidin. (\\\\(D\\\\)) Actin ring forms as remodeling and depolymerization enable membrane to progress over an organized ED by new actin polymerization at the leading edge of the membrane. (\\\\(E\\\\)) Actin polymerization in the call at site of attachment to flat side of an organized ED, but no actin cup or ring is visible. (\\\\(F\\\\)) Actin cup surrounds the end of an organized sphere as internalization begins after attachment. (Scale bars in \\\\(D\\\\)\u2013\\\\(F\\\\): 10 \\\\(\\\\mu\\\\)m.) At least five calls were observed for each orientation of each particle. Similar results were observed in all repetitions.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Definition of \\\\(\\\\Omega\\\\) and its relationship with membrane velocity. (\\\\(A\\\\)) A schematic diagram illustrating how membrane progresses tangentially around an ED. \\\\(\\\\overline{\\\\Omega}\\\\) represents the average of tangential angles from \\\\(\\\\theta=0\\\\,\\\\theta=\\\\pi/2\\\\,\\\\Pi\\\\) is the angle between \\\\(T\\\\) and membrane normal at the site of attachment, \\\\(\\\\Omega\\\\). (\\\\(B\\\\)) Membrane velocity (distance traveled by the membrane divided by time to internalize, \\\\(n\\\\simeq 3\\\\); error bars represent SD) decreases with increasing \\\\(\\\\Omega\\\\) for a variety of shapes and sizes of particles. Nonsponsized particles are indicated by filled circles, and IgG-opsonized particles are indicated by open squares. Each data point represents a different shape, size, or aspect ratio particle. The internalization velocity is positive for \\\\(\\\\Pi\\\\simeq 45^{\\\\circ}\\\\) (\\\\(P\\\\leq 0.001\\\\)). Above a critical value of \\\\(\\\\Pi\\\\), \\\\(\\\\sim\\\\)45\\\\({}^{\\\\circ}\\\\), the internalization velocity is zero (\\\\(P\\\\leq 0.001\\\\)) and there is only membrane spreading after particle attachments, not internalization. The arrows above the plot indicate the point of attachment for each shape that corresponds to the value of \\\\(\\\\Omega\\\\) on the \\\\(x\\\\) axis. Error in \\\\(\\\\Omega\\\\) is due to the difference in the actual point of contact in time-lapse microscopy from that used to calculate \\\\(\\\\Pi\\\\). Only points of contact within 10\\\\({}^{\\\\circ}\\\\) of that used to calculate \\\\(\\\\Omega\\\\) were selected. All data points at the critical point, \\\\(\\\\Pi=45^{\\\\circ}\\\\), except UFOs, do not have error associated with \\\\(\\\\Pi\\\\) because of their symmetry.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Phagocytosis phase diagram with \\\\(\\\\Omega\\\\) and dimensionless particle volume \\\\(V^{*}\\\\) (particle volume divided by 7.5 \\\\(\\\\mu\\\\)m radius spherical cell volume) as governing parameters (\\\\(\\\\Omega=5\\\\) for each point). Initialization of internalization is judged by the presence of an actin cup or ring as described in the text. There are three regions. Cells attaching to particles at areas of high \\\\(\\\\Omega\\\\), \\\\(>\\\\)45\\\\({}^{\\\\circ}\\\\), spread but do not initiate internalization (region \\\\(\\\\Omega\\\\)). Cells attaching to particles at areas of low \\\\(\\\\Omega\\\\), \\\\(<\\\\)45\\\\({}^{\\\\circ}\\\\), initiate internalization (regions \\\\(A\\\\) and \\\\(B\\\\)). If \\\\(V^{*}\\\\) is \\\\(\\\\leq\\\\)1, internalization is completed (region \\\\(A\\\\)). If \\\\(V^{*}\\\\) > 1, internalization is not completed because of the size of the particle (region \\\\(B\\\\)). The arrows above the plot indicate the point of attachment for each shape that corresponds to the value of \\\\(\\\\Omega\\\\) on the \\\\(x\\\\) axis. Each case was classified as phagocytosis or no phagocytosis if \\\\(>\\\\)95% of observations were consistent. Each data point represents a different shape, size, or aspect ratio particle.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/chanImpactAnalogiesCreative2015.json b/extracted_captions/chanImpactAnalogiesCreative2015.json new file mode 100644 index 0000000000000000000000000000000000000000..5181b4ac978b13c956ac2983c47b157bc8725a90 --- /dev/null +++ b/extracted_captions/chanImpactAnalogiesCreative2015.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. _et al._, \"The \\\\(\\\\beta\\\\)-decay of the \\\\(\\\\gamma\\\\)-ray emission from the \\\\(\\\\gamma'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The'", "CAPTION TAB1.png": "'\\n\\n## Appendix A Proof of Theorem 1\\n\\n### Proof of Theorem 1\\n\\nProof of Theorem 1\\n'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB11.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}\\n\\n'", "CAPTION TAB12.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{Odds ratios by lag type for logistic regressions of new concept on fair analogy} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 12: Olds ratios by lag type for logistic regressions of new concept on fair analogy'", "CAPTION TAB13.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB14.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} &'", "CAPTION TAB2.png": "'\\n\\n## Chapter 6 Conclusions\\n\\nIn this thesis we have presented a new method for the calculation of the \\\\(\\\\Delta\\\\)-meson coupling constant in the \\\\(\\\\Delta\\\\)-meson coupling constant. We have shown that the \\\\(\\\\Delta\\\\)-meson coupling constant is a function of the form factors of the'", "CAPTION TAB3.png": "'\\n\\n## Chapter 3 Example of problem identification analogy\\n'", "CAPTION TAB4.png": "'\\n\\n## Chapter 4 Example of concept generation and history'", "CAPTION TAB5.png": "'\\n\\n## Chapter 5 Example of explain'", "CAPTION TAB6.png": "'\\n\\n## Table 6 Example of function-finding analogy'", "CAPTION TAB7.png": "'Table 7: Example of concept for \"keep the point head level\"\\n\\n'", "CAPTION TAB8.png": "'\\n\\n## Table 5 Example of current pair tuning'", "CAPTION TAB9.png": "'\\n\\n## Table 9\\n\\nSubproblems by number of concepts'"} \ No newline at end of file diff --git a/extracted_captions/chen2006roles.json b/extracted_captions/chen2006roles.json new file mode 100644 index 0000000000000000000000000000000000000000..3ceda4bfbc2dfed860a6630a940212ee79e9e61c --- /dev/null +++ b/extracted_captions/chen2006roles.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1 Knock-down of cortactin in MDA-MB-231 cells attenuates transferrin endocytosis**\\n\\n(A) Cortactin protein in siRNA treated cells was not detected by immunoblot assay. (B) Only about 50% of cells subjected to cortactin siRNA treatment. had normal transferrin (Tfri) uptake. (C) Tfri fluorescent intensity in cytoplasm in cortactin siRNA treated-cells was apparently reduced (lower panel, arrow head) in comparison to neighboring cells with relatively high cortactin levels (lower panel, arrow), or to mock-treated cells (upper panel). Magnification, 600\\\\(\\\\times\\\\).\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2** **Introduction of coractin immunoreagents into live cells suppresses transferrin uptake**\\n\\nNIHGT3 cells were subjected to coractin immunoreagent infinduction mediated by the BioPORTER-resistance system. Quantitation of endocytic biotinylated-labeled transferrin (Tf) by ELISA-based transferrin uptake assay was performed 5 h after the start of the tratment. BioPORTER only (column 1)\\\\({}_{r}\\\\) rahit IgG (column 4) and labeled mouse IgG (column 5) with kit were used as control. Endocyteis is cells with either coctactin antibody (column 2) or 4F11 (column 3) was repaired.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3**: **Perturbation of actin dynamics by cytochalasin D affects the association of cortactin with dynamin at endocytosis sites** NIH/3T3 cells were treated with cytochalasin D in serum-free medium. The association of cortactin (red) with dynamin (green) was obvious in control cells without cytochalasin D treatment (A), and became invisible after cytochalasin D exposure (B), then was restored after the drug was washed out (C). Compared with the control (D), transferrin (green) endocytosis was reduced by cytochalasin D treatment for 1 h (E) and restored within 1 h after the drug was washed out (F). Magnification, 600x.\\n\\n'", "CAPTION FIG4.png": "'NH(3T 3 cells were seeded on fibronectin-coated coverslips the day before DNA transfection. Contraction was performed using Lipofectamine reagent. Red fluorescent protein (RFP)-contactin (A), dynamic-green fluorescent protein (GFP) (B) and AP-2 (C) co-distributed in cytoplasm and formed granular structure (D). In cells cotransfected with RFP-tagged wild-type outardin (E) and proline rich domain (PRD) depleted dynamin (F), the association of contraction with dynamin and AP-2 (G) was abolished (H). This phenomenon also happened in cells cotransfected with SH3 domain-deleted outardin and wild-type dynamin. Unlike wild-type outardin, RFP-ContASH3 (I) is not associated with dynamin (J) and AP-2 (K) any more, while the latter two still co-distributed in cytoplasm (L). In contactin SH3 domain only expressing cells, RFP-tagged outardin SH3 (M), GFP-tagged wild-type dynamin (N) and AP-2 (Q) were co-distributed as puncta-like structure in cytoplasm (P). When cells were cotransfected with coordinate SH3 and PRD-depleted dynamin, contactin SH3 domain (Q) lost its association with PRD-depleted dynamin (R) and AP-2 (S), while PRD-depleted dynamin (P-2 co-distribution was still present (T). Cells expressing RFP (U) and wild-type dynamin (V) were used as the control, no colocalization was found (X), and the colocalization between dynamin and AP-2 was not affected by GFP expression (W, X). Bars=10 \\\\(\\\\mu\\\\)m.\\n\\nFig. 4: In vivo recruitment of cortactin to endocytosis sites by dynamin'", "CAPTION FIG5.png": "'(A) Full-down assay of the interaction of GST-proline rich domain of dynamic and cortactin. (B) Purified contraction was mixed with different amounts of immobilized GST-tagged dynamic proline rich domain (Dyn PRD) protein. After incubation, samples were centrifuged (800 g) and the supernatants were collected for immunoblot assay to detect the amount of remaining cortactin with contractile antibody dF11. Amount of cortactin was quantified by digital scanning and normalized to the percentage of depletion. The resulting data were used to fit a rectangular hyperbola, yielding apparent _K_x values of 0.964 \u03bcM. (C) Fluorescent carboxylated polystyrene beads were coated with GST-Dyn-PRD by including beads in protein solution (5 mg/ml). Branched actin filaments were recruited to Dyn-PRD coated beads (a, b and c), but not to non-contact beads (d). Cort, cortactin. Magnification, 600%.\\n\\nFig. 5: Cortactin-mediated actin assembly is recruited to dynamin-coated beads in vitro (A) Full-down assay of the interaction of GST-proline rich domain of dynamic and cortactin. (B) Purified contraction was mixed with different amounts of immobilized GST-tagged dynamic proline rich domain (Dyn PRD) protein. After incubation, samples were centrifuged (800 g) and the supernatants were collected for immunoblot assay to detect the amount of remaining cortactin with contractile antibody dF11. Amount of cortactin was quantified by digital scanning and normalized to the percentage of depletion. The resulting data were used to fit a rectangular hyperbola, yielding apparent _K_x values of 0.964 \u03bcM. (C) Fluorescent carboxylated polystyrene beads were coated with GST-Dyn-PRD by including beads in protein solution (5 mg/ml). Branched actin filaments were recruited to Dyn-PRD coated beads (a, b and c), but not to non-contact beads (d). Cort, cortactin. Magnification, 600%.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n**Fig. 6** **C ortactin functions in the late stage of clathrin coated vesicle (CCV) formation**\\n\\n(A) Western blot analysis of mtDNA cytosol before and after treatment with contractile antibody-coupled beads. Equivalent volumes of supernatants (lane 1 and lane 4) or beads after incubation (lane 5) and before incubation (lane 6), 1/2 loading volume (lane 2) and 1/4 loading volume of supernatants (lane 3) were probed with anti-contraction antibody 001. More than 95% cortactin was depleted (lane 4). (B) Permeabilized 3T3-L1 cells were incubated with mock-depleted cytosol (mock, column 1), cortactin, only (Cort, column 2), or cortactin-depleted cytosol (Cort D, column 3), or cortactin-depleted cytosol supplemented with recombinant cortactin SH3 domain (Cort D+Cort-SH3, column 4), N-terminal-depleted cortactin (Cort D+Cort-NTD, column 5) or wild-type contraction (Cort D+Cort, column 6). Endocytosis was measured by MetaNa resistance in specially detect internalization of B-SS-transferrin (Tfru) into sealed vesicles.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/choInfluencingMyselfSelfReinforcement2018.json b/extracted_captions/choInfluencingMyselfSelfReinforcement2018.json new file mode 100644 index 0000000000000000000000000000000000000000..62c4e77269d2e17b83728298a059e648d16840f0 --- /dev/null +++ b/extracted_captions/choInfluencingMyselfSelfReinforcement2018.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Abstract**\\n\\nThe thesis deals with the study of the role of the \"weak\" and \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \"strong\" \" \"strong\" \"strong\"'", "CAPTION FIG2.png": "'Figure 2: Unconditional model: Longitudinality of candidate preference. Note. \\\\(\\\\chi^{\\\\perp}_{\\\\zeta}=0.42\\\\); df = 1; normed fit index (NFI) = .9%; Tucker\u2013Lewis index (TLU) = 1.00; comparative fit index (CFI) = 1.00; root mean square error of approximation (RMSEA) = .00.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Conditional model: Political expression and candidate preference. Note. Enries are unstandardized coefficients with standard errors in parentheses. Estimated coefficients for control variables are not presented in this figure for parsimony, but full results are available upon request. \\\\(\\\\chi^{2}\\\\) = 18.96; df = 13; normed fit index (NFI) = .98; Tucker-Lewis index (TLI) = .95; comparative fit index (CFI) = .99; root mean square error of approximation (RMSEA) = .03.\\n\\n'", "CAPTION TAB1.png": "'* [10] A.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/cmmidcovid19workinggroupAgedependentEffectsTransmission2020.json b/extracted_captions/cmmidcovid19workinggroupAgedependentEffectsTransmission2020.json new file mode 100644 index 0000000000000000000000000000000000000000..042ab3892958a9047d1783b0a1a84317863345ac --- /dev/null +++ b/extracted_captions/cmmidcovid19workinggroupAgedependentEffectsTransmission2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1\"(NOISY)\".png": "'\\n\\n**Fig. 11 Fit of different model variants to data from Wuhan City, China.**,**,**. Model diagram and duration of disease states in days, where \\\\(d\\\\) parameters represent the duration of time in each disease state (see Methods), \\\\(y\\\\), is the fraction of infections that manifest as clinical cases in age group \\\\(\\\\downarrow\\\\lambda\\\\) is the force of infection in age group \\\\(\\\\downarrow P\\\\), is the incubation period and \\\\(\\\\rho_{i}\\\\) is the serial interval (see Methods). **b**, Susceptibility by age for the three models, with mean (lines), 50% (darker shading) and 95% (lighter shading) credible intervals shown. Age-specific values were estimated for model 1 (orange). Susceptibility is defined as the probability of infection on contact with an infectious person. **c**, Clinical fraction (\\\\(y\\\\)) by age for the three models. Age-specific values were estimated for model 2 (blue) and fixed at 0.5 for models 1 and 3. **d**, Fitted contact multipliers for holiday (\\\\(q_{0}\\\\)) and restricted periods (\\\\(q_{1}\\\\)) for each model showed an increase in non-school contacts beginning on 12 January (start of the Lunar New Year) and a decrease in contacts following restrictions on 23 January. **e**, Estimated \\\\(R_{0}\\\\) values for each model. The red barplot shows the inferred window of spillover of infection. **f**, Incident reported cases (black) and modeled incidence of reported clinical cases for the three models fitted to cases reported by China Centers for Disease Control (CCDCY) with onset on or before 1 February 2020. Lines mark the mean and the shaded window is the 95% highest density interval (HDI). **g**, Age distribution of cases by onset date as fitted to the age distributions reported by Li et al.2(first three panels) and CCDC (fourth panel). Data are shown in open bars and model predictions in filled bars, where the dot marks the mean posterior estimate. **h**, Implied distribution of subclinical cases by age for each model. Credible intervals on modeled values show the 95% HDIs; credible intervals on data for **g** and **h** show 95% HDIs for the proportion of cases in each age group.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Fig. 11 Fit of different model variants to data from Wuhan City, China.**,**,**. Model diagram and duration of disease states in days, where \\\\(d\\\\) parameters represent the duration of time in each disease state (see Methods), \\\\(y\\\\), is the fraction of infections that manifest as clinical cases in age group \\\\(\\\\downarrow\\\\lambda\\\\) is the force of infection in age group \\\\(\\\\downarrow P\\\\), is the incubation period and \\\\(\\\\rho_{i}\\\\) is the serial interval (see Methods). **b**, Susceptibility by age for the three models, with mean (lines), 50% (darker shading) and 95% (lighter shading) credible intervals shown. Age-specific values were estimated for model 1 (orange). Susceptibility is defined as the probability of infection on contact with an infectious person. **c**, Clinical fraction (\\\\(y\\\\)) by age for the three models. Age-specific values were estimated for model 2 (blue) and fixed at 0.5 for models 1 and 3. **d**, Fitted contact multipliers for holiday (\\\\(q_{0}\\\\)) and restricted periods (\\\\(q_{1}\\\\)) for each model showed an increase in non-school contacts beginning on 12 January (start of the Lunar New Year) and a decrease in contacts following restrictions on 23 January. **e**, Estimated \\\\(R_{0}\\\\) values for each model. The red barplot shows the inferred window of spillover of infection. **f**, Incident reported cases (black) and modeled incidence of reported clinical cases for the three models fitted to cases reported by China Centers for Disease Control (CCDCY) with onset on or before 1 February 2020. Lines mark the mean and the shaded window is the 95% highest density interval (HDI). **g**, Age distribution of cases by onset date as fitted to the age distributions reported by Li et al.2(first three panels) and CCDC (fourth panel). Data are shown in open bars and model predictions in filled bars, where the dot marks the mean posterior estimate. **h**, Implied distribution of subclinical cases by age for each model. Credible intervals on modeled values show the 95% HDIs; credible intervals on data for **g** and **h** show 95% HDIs for the proportion of cases in each age group.\\n\\n'", "CAPTION FIG10.png": "'\\n\\n**Extended Data Fig. 10 | Contact matrices used in transmission model.** Contact matrices used for Figs. 1-3 of the main text. We have not shown matrices for all 12 regions of Italy modeled, nor for all 13 provinces of China modeled, as these show similar patterns to the matrices for Milan and for Wuhan, Beijing and Shanghai, respectively.\\n\\n'", "CAPTION FIG2 \"(NOISY)\".png": "'\\nFigure 2: **Estimating the age-specific symptomatic rate from age-specific case counts for six countries.****a.** Age-specific reported cases from 13 provinces of China, 12 regions of Italy, Japan, Singapore, South Korea and Ontario, Canada. Open bars are data and the colored lines are model fits with 95% HD. **b.** Fitted mean (lines) and 95% HD (shaded areas) for the age distribution in the clinical fraction (solid lines) and the age distribution of susceptibility (dashed lines) for all countries. The overall consensus fit is shown in gray. **c.** Fitted incidence of confirmed cases and resulting age distribution of cases using either the consensus (gray) or country-specific (color) age-specific clinical fraction from **b.**\\n\\n'", "CAPTION FIG2.png": "'\\nFigure 2: **Estimating the age-specific symptomatic rate from age-specific case counts for six countries.****a.** Age-specific reported cases from 13 provinces of China, 12 regions of Italy, Japan, Singapore, South Korea and Ontario, Canada. Open bars are data and the colored lines are model fits with 95% HD. **b.** Fitted mean (lines) and 95% HD (shaded areas) for the age distribution in the clinical fraction (solid lines) and the age distribution of susceptibility (dashed lines) for all countries. The overall consensus fit is shown in gray. **c.** Fitted incidence of confirmed cases and resulting age distribution of cases using either the consensus (gray) or country-specific (color) age-specific clinical fraction from **b.**\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3 [Effect of school closure under different demographics and subclinical infectiousness.****a**, Age dependence in clinical fraction (severity) and susceptibility to infection on contact for COVID-19 and for the influenza-like scenarios (simplified, based on ref. [4]) considered here. **b**, Age structure for the three exemplar cities. **c**, Age-specific clinical case rate for COVID-19 and influenza-like infections, assuming \\\\(\\\\leq\\\\)50% infectiousness of subclinical infections. **d**, Daily incidence of clinical cases in exemplar cities for COVID-19 versus influenza-like infections. \\\\(R_{0}\\\\) is fixed at 2.4. The rows show the effect of varying the infectiousness of subclinical infections to be 0%, 50% or 100% as infectious as clinical cases while keeping \\\\(R_{0}\\\\) fixed **e**, Change in peak timing and peak cases for the three cities, for either COVID-19 or influenza-like infections. **f**, Change in median COVID-19 peak timing and peak cases for the three cities, depending on the infectiousness of subclinical infections.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4 : Implications for global preparedness.****a**, Expected clinical case attack rate (mean and 95% HDD) and peak in clinical case incidence for 146 countries in the Global Burden of Disease (GBD) country groupings(r) for an unmitigated epidemic. **b**, Expected subclinical case attack rate and peak in subclinical cases. **c**, Estimated basic reproduction number (_R_o) in the capital city of each country assuming the age-specific clinical fraction shown in Fig. 2b and 50% infectiousness of subclinically infected people. **d**, Proportion of clinical cases in each age group at times relative to the peak of the epidemic. The 146 city epidemics were aligned at the peak, and colors mark the GBD groupings in **a**, **e**, Age distribution of the first and last thirds of clinical cases for 146 countries in GBD country groupings.\\n\\n'", "CAPTION FIG5.png": "\"\\n\\n**Extended Data Fig. 5 Posterior distributions for Beijing, Shanghai, South Korea, and Lombardy.** Prior and posterior distributions for the epidemics in a, Beijing and Shanghai, **b**, South Korea and **c**, Lombardy using the 'consensus' fit for age-specific clinical fraction and assuming subclinical infections are 50% as infectious as clinical infections (see Fig. 2c, main text). For (**a**), times are in days after December 1st, 2019; for (**b**) and (**c**), times are in days after January 1st, 2019. Note, seed_d is the inferred duration of the seeding event. See also Supplementary Table 4.\\n\\n\"", "CAPTION FIG6.png": "'\\n\\n**Extended Data Fig. 6 [ Global projections assuming greater severity in lower-income countries.****a**. Schematic age-specific clinical fraction for higher-income and lower-income countries. **b-f**, illustrative results of the projections for 146 capital cities assuming a higher age-varying clinical fraction in lower-income countries. See Fig. 4 (main text) for details.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n**Extended Data Fig. 7 [ Consensus age-specific clinical fraction and susceptibility under varying assumptions for subclinical infectiousness.** Line and rhobens show mean and 95% HDI for clinical fraction and susceptibility, assuming subclinical infections are 0%, 25%, 50%, 75%, or 100% as infectious as clinical infections.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Extended Data Fig. 8 | Projections for capital cities depending upon subclinical infectiousness.****a**, Projected total and peak clinical case attack rate for 146 capital cities, under different assumptions for the infectiousness of subclinical infections. **b**, Projected total and peak subclinical infection attack rate for 146 capital cities, under different assumptions for the infectiousness of subclinical infections. **c**, Projected differences in \\\\(R_{a}\\\\) among 146 capital cities, under different assumptions for the infectiousness of subclinical infections. Mean and 95% HDI shown.\\n\\n'", "CAPTION FIG9.png": "'\\n\\n**Extended Data Fig. 9 | School closures with fixed susceptibility across cities.** Comparison of school closures in three exemplar cities when susceptibility is fixed across settings instead of 80. See main text Fig. 3 for details.\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n**Extended Data Fig. 1 Posterior distributions for Wuhan.** Prior distributions (gray dotted lines) and posterior distributions (colored histograms) for model parameters fitting to the early epidemic in Wuhan (Fig. 1, main text); seed_start is measured in days after November 1st, 2019. **a**, Model 1(age-varying contact patterns and susceptibility); **b**, Model 2 (age-varying contact patterns and clinical fraction); **c**, Model 3 (age-varying contact patterns only). See also Supplementary Table 4.\\n\\n'", "CAPTION FIGS10.png": "'\\n\\n**Extended Data Fig. 10 | Contact matrices used in transmission model.** Contact matrices used for Figs. 1-3 of the main text. We have not shown matrices for all 12 regions of Italy modeled, nor for all 13 provinces of China modeled, as these show similar patterns to the matrices for Milan and for Wuhan, Beijing and Shanghai, respectively.\\n\\n'", "CAPTION FIGS2.png": "'\\n\\n**Extended Data Fig. 2 [ Simultaneous estimation of age-varying susceptibility and clinical fraction to epidemic data from Wuhan City, China.** This figure replicates Fig. 1 of the main text, but comparing model variants 1 and 2 to a fourth model variant in which both susceptibility and clinical fraction vary by age. **a**, Model diagram (see Fig. 1, main text). **b**, Susceptibility by age for the three models. Age-specific values were estimated for models 1 (orange) and 4 (pink). Susceptibility is defined as the probability of infection on contact with an infectious person. Mean (lines), 50% (darker shading) and 95% (lighter shading) credible intervals shown. **c**, Clinical fraction (_y_) by age for the three models. Age-specific values were estimated for model 2 (blue) and 4 (pink), and fixed at 0.5 for model 1. **d**, Fitted contact multiplex for holiday (_a_b_) and restricted periods (_a_b_) for each model showed an increase in non-school contacts beginning on January 12th (start of Lunar New Year) and a decrease in contacts following restrictions on January 23rd. **e**, Estimated \\\\(R\\\\) values for each model. The red barplot shows the inferred window of spillover of infection. **f**, Incident reported cases (black), and modeled incidence of reported clinical cases for the three models fitted to cases reported by China Centers for Disease Control (CCDC) with onset on or before February 1st, 2020. Line marks mean and shaded window is the 95% highest density interval (HDI). **g**, Age distribution of cases by onset date as fitted to the age distributions reported by Li et al. (first three panels) and CCDC (fourth panel). Data are shown in the hollow bars, and model predictions in filled bars, where the dot marks the mean posterior estimate. **h**, Implied distribution of subclinical cases by age for each model. Credible intervals on modeled values show the 95% HDIs; credible intervals on data for panels g and h show 95% HDIs for the proportion of cases in each age group.\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n**Extended Data Fig. 3 | Impact of data sources used.** _Analysis showing how the inferred age-varying susceptibility (first column) and age-varying clinical fraction (second column) depend upon the additional data sources used.\\n\\n'", "CAPTION FIGS4.png": "'\\n\\n**Extended Data Fig. 4 -- Posterior estimates for the consensus susceptibility and clinical fraction from 6 countries.** Note that susceptibility is a relative measure.\\n\\n'", "CAPTION FIGS5.png": "\"\\n\\n**Extended Data Fig. 5 Posterior distributions for Beijing, Shanghai, South Korea, and Lombardy.** Prior and posterior distributions for the epidemics in a, Beijing and Shanghai, **b**, South Korea and **c**, Lombardy using the 'consensus' fit for age-specific clinical fraction and assuming subclinical infections are 50% as infectious as clinical infections (see Fig. 2c, main text). For (**a**), times are in days after December 1st, 2019; for (**b**) and (**c**), times are in days after January 1st, 2019. Note, seed_d is the inferred duration of the seeding event. See also Supplementary Table 4.\\n\\n\"", "CAPTION FIGS6.png": "'\\n\\n**Extended Data Fig. 6 [ Global projections assuming greater severity in lower-income countries.****a**. Schematic age-specific clinical fraction for higher-income and lower-income countries. **b-f**, illustrative results of the projections for 146 capital cities assuming a higher age-varying clinical fraction in lower-income countries. See Fig. 4 (main text) for details.\\n\\n'", "CAPTION FIGS7.png": "'\\n\\n**Extended Data Fig. 7 [ Consensus age-specific clinical fraction and susceptibility under varying assumptions for subclinical infectiousness.** Line and rhobens show mean and 95% HDI for clinical fraction and susceptibility, assuming subclinical infections are 0%, 25%, 50%, 75%, or 100% as infectious as clinical infections.\\n\\n'", "CAPTION FIGS8.png": "'\\n\\n**Extended Data Fig. 8 | Projections for capital cities depending upon subclinical infectiousness.****a**, Projected total and peak clinical case attack rate for 146 capital cities, under different assumptions for the infectiousness of subclinical infections. **b**, Projected total and peak subclinical infection attack rate for 146 capital cities, under different assumptions for the infectiousness of subclinical infections. **c**, Projected differences in \\\\(R_{a}\\\\) among 146 capital cities, under different assumptions for the infectiousness of subclinical infections. Mean and 95% HDI shown.\\n\\n'", "CAPTION FIGS9.png": "'\\n\\n**Extended Data Fig. 9 | School closures with fixed susceptibility across cities.** Comparison of school closures in three exemplar cities when susceptibility is fixed across settings instead of 80. See main text Fig. 3 for details.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Figure Captions**'"} \ No newline at end of file diff --git a/extracted_captions/dunbarHowScientistsThink1997.json b/extracted_captions/dunbarHowScientistsThink1997.json new file mode 100644 index 0000000000000000000000000000000000000000..6f3d71180c0e86a6f7e61f12186d660b067c4eac --- /dev/null +++ b/extracted_captions/dunbarHowScientistsThink1997.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "\"\\n\\n**Scientists' Goals for Within-Organism, Other-Organism, and Nonbiological Analogies**\"", "CAPTION TAB2.png": "'\\n\\n**Types of Knowledge Retrieved by Analogies on the Basis of Homology and Nonhomology for Other-Organism Analogies**'"} \ No newline at end of file diff --git a/extracted_captions/eckertAdaptationSourcesInspiration2003.json b/extracted_captions/eckertAdaptationSourcesInspiration2003.json new file mode 100644 index 0000000000000000000000000000000000000000..401b554605ecd4ac4c261f05bbdab26d9199e15b --- /dev/null +++ b/extracted_captions/eckertAdaptationSourcesInspiration2003.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1. Outline of the hurricane design process\\n\\n'", "CAPTION FIG10.png": "'Figure 10. Assistance to the natural context of the source.\\n\\n'", "CAPTION FIG11.png": "'\\n\\n## Appendix A Appendix\\n\\nAppendix B: The derivation of'", "CAPTION FIG12.png": "'\\n\\n**Figure 12.** Structural role drive source stringy.\\n\\n'", "CAPTION FIG13.png": "'Figure 14: Structural role drives source design.\\n\\n'", "CAPTION FIG14.png": "'\\n\\n**Figure 14.** Emergent read drives source strategy.\\n\\n'", "CAPTION FIG15.png": "'\\n\\n**Figure 15.**_Middle hot \"target.\"'", "CAPTION FIG16.png": "'Chapter 4 Future'", "CAPTION FIG17.png": "'\\n\\n**Figure 17.** Sufence priority barrier.\\n\\n'", "CAPTION FIG18.png": "'\\n\\n**Figure 18**. _Source dimer_ amine structural role strategy.\\n\\n'", "CAPTION FIG19.png": "'Figure 19. Source driven same structural rule design.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Figure 2.** Single acetyl design with asymmetric shapes.\\n\\n'", "CAPTION FIG20.png": "'Figure 20. Source drives different structural role strategy.\\n\\n'", "CAPTION FIG21.png": "'\\n\\n**Figure 11.**_Source driven different structural role design_'", "CAPTION FIG22.png": "'\\n\\n## Chapter 3 Main of source strategy'", "CAPTION FIG23.png": "'\\n\\n**Figure 3.** Continued of source density.\\n\\n'", "CAPTION FIG24.png": "'\\n\\n## Abstract\\n\\nIn this thesis we study the effect of the \\\\(\\\\pi\\\\)-particle interaction on the \\\\(\\\\pi\\\\)-particle interaction. We show that the \\\\(\\\\pi\\\\)-particle interaction is a \\\\(\\\\pi\\\\)-particle interaction.\\n\\n'", "CAPTION FIG25.png": "'Figure 25: _Professional: Strategy frequencies in rng and tagery maks._\\n\\n'", "CAPTION FIG26.png": "'Figure 26: Frequencies of strange used by professionals and students.\\n\\n'", "CAPTION FIG3.png": "'Figure 1.: Adaptation of a source of inspiration for an individual design.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: The ring stimulus given to the subjects. Afshar rug (S. E. Iran). Scanned from Oriental Rays: A Buyer\u2019s Guide (p. 42), by Lee Alliance, Lookou: Thames and Hudson, 1988. Copyright 1988 Thames and Hudson. Reprinted with permission.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: The top-city stimulus given to the subjects. Out and Pigcom, designed by John Henry Deurle, c. 1895. Scanned from William Morris (p. 250), edited by Linda Parry, 1996, Loudoua: Philip Wilson Publishers/Victoria and Albert Miseau. Copyright 1996 V & A Images/Victoria and Albert Museum. Reprinted with permission.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## Chapter 4. Analytical driven current'", "CAPTION FIG7.png": "'Figure 7. Abstraction of the source design element.\\n\\n'", "CAPTION FIG8.png": "'Figure 1: Abstraction from a usual property of the source.\\n\\n'", "CAPTION FIG9.png": "'\\n\\n**Figure 1.** Schematic diagram for the case of a \\\\(\\\\pi\\\\)-particle.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## Table 1. Cluster of Motif Arrangements'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 2**.: _Number and Frequency of Strategies Used with the Ring Source_} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: _Number and Frequency of Strategies Used with the Ring Source_'", "CAPTION TAB3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB4.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 6.**_Occurrences of Strategies Used by Professionals and Students in the Rug Task (Excluding Evolution Designs)_} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 6: _Occurrences of Strategies Used by Professionals and Students in the Rug Task (Excluding Evolution Designs)_'", "CAPTION TAB7.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB8.png": "'\\n\\n**Table 6.**_More and Less Direct Strategies and Transformations_'"} \ No newline at end of file diff --git a/extracted_captions/fasler2020novel.json b/extracted_captions/fasler2020novel.json new file mode 100644 index 0000000000000000000000000000000000000000..b30d839106d7e87f580e0c4db2502591d47436b4 --- /dev/null +++ b/extracted_captions/fasler2020novel.json @@ -0,0 +1 @@ +{"CAPTION FIG S1.png": "'\\n\\n**Fig. S1.: Cryo-ET of the actin network in a NIH3T3 fibroblast lamellipodium. (a) Computational slice through a binned, non-CTF corrected tomogram (17.096A/px) of a NIH3T3 fibroblast lamellipodium. Protein density is black. The dense actin network is visible and the helical appearance of individual actin filaments can be clearly appreciated. Several branch junctions are highlighted in red circles. The dashed line indicates the cell edge. Scale bar is 100nm. (b) Gallery of selected branch junctions. The density corresponding to the Arp2/3 complex at branch junctions is visible in the individual examples.**'", "CAPTION FIG S2.png": "'\\n\\n**Fig. S2.: Graphical workflow of image processing.** Flow chart indicating the data processing steps involved in generating the structure of the active Arp2/3 complex within the branch junction. Colored boxes indicate usage of specific software packages. For simplicity, the procedure to produce a reference for template matching via manual picking and averaging branch junctions is not depicted here, but is described within the material and methods section.\\n\\n'", "CAPTION FIG S5.1.png": "'\\n\\n**Fig. S5.: Comparison of the active Arp2/3 complex conformation in branch junctions in cells to the _in vitro_ Dip1-activated Arp2/3 complex and to a previously published MD-derived _in vitro_ branch model. (a) Molecular models of the Arp2/3 complex in the active conformation (shown as density maps filtered to 9.5A resolution) as observed in cells (left, this study), the _in vitro_ Dip1-activated Arp2/3 complex (middle) [29], and a MD-derived active Arp2/3 complex [12], which is based on a low-resolution negative stain ET reconstruction (right) [9]. The models were aligned on the ArpC2 subunits to visualize the different conformations of the Arp2/3 complex and in case for the branch junction also the varying position on the mother filament between the _in situ_ and _in vitro_'", "CAPTION FIG S5.2.png": "'model. **(b-c)** RMSD values (in A) calculated between the subunits of the three models shown in **(a)**. Rows indicate which subunit was used for aligning full models to each other, prior to measurements between individual subunits (indicated in the columns) of the different models of the active Arp2/3 complex. **(b)** RMSD values of differences between the Arp2/3 complex in branch junctions in cells and the _in vitro_ Dip1-activated Arp2/3 complex. In order to calculate C-alpha RMSD values between our model and pdb 6W17 only primary structure areas are considered, in which the _B. Taurus_ (as used in our model) and _S. pombe_ protein sequences are in the same register, hence omitting inserts present in only one species. This comparison reveals differences between the Dip1-activated Arp2/3 complex in vitro and the activated state of the Arp2/3 complex in cells, in particular with respect to ArpC3. **(c)** RMSD values of differences between the Arp2/3 complex in branch junctions in cells and the MD-derived _in vitro_ branch junction model.\\n\\n'", "CAPTION FIG S6.png": "'\\n\\n**Fig. S6.: Mother actin filament conformation in the actin-Arp2/3 complex branch junction. (a) Fit of the final model of the mother actin filament after MD-refinement into the EM density of the branch junction using ISOLDE. The empty density close to M4, corresponding to the helix of ArpC1 is annotated with a green ellipsoid. (b) Superimposition between the starting model used for fitting (pdb 6T20, pink) and the final model after MD-refinement. Small deviations between the filament assemblies can be observed. (c) Superimposition between one monomer of pdb 6T20 (pink) and monomer M4 of the mother filament in our branch junction model. No large-scale deviations in the monomer conformation are observed. (d) RMSD calculations between the C-alpha atoms of one monomer of pdb 6T20 and the monomers of the mother filament. The average RMSD value is 1.76A.**'", "CAPTION FIG.S4.png": "'\\n\\n**Fig. S4.: Comparison of the short pitch actin dimer conformation to Arp2 and Arp3 in their inactive and active conformation.** Density maps of molecular models of an actin dimer, or of Arp2 and Arp3 in their active and inactive conformation, respectively, filtered to a resolution of 15A. The models of the active Arp2/3 conformation were derived from our model of the actin filament Arp2/3 complex structure in cells described in this manuscript and the _in vitro_ Dip1-activated _S. pombe_ Arp2/3 complex (pdb 6W17) [29]. The shown models of filamentous actin were derived from pdb 6T20 [30], and for the inactive ATP-bound Arp2/3 complex from pdb 1TYQ [8]. Maps were oriented by fitting Arp3 subunits and the actin monomer M1 to each other. While Arp2 and Arp3 of the active complex adopt a similar short pitch conformation as the monomers of the actin dimer, this is not the case for the subunits of the inactive complex. Subunit identity is indicated by the color scheme.\\n\\n'", "CAPTION FIG1.png": "'\\nFigure 1: **Subnanometer Structure of the actin filament Arp2/3 complex branch junction in cells.****(a)** Isosurface representation of the actin filament Arp2/3 complex branch junction in cells at 9\u00c5 resolution. The structure is shown from three orientations. A guide for orientation is given in (b). **(b)** The electron microscopy density map (shown transparent) with the flexibly fitted models of the Arp2/3 complex subunits and actin filaments. Schematic guides indicate the positions of the individual subunits of the complex and the monomers of both the mother and daughter filaments. A colour legend provides the color code for the fitted models. The color code and legend are used throughout the manuscript to aid the reader.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2: Comparison of Arp2/3 complex in its inactive conformation and in the branch junction in cells. (a) Molecular models of the Arp2/3 complex in the inactive (derived from pdb 1TYQ) and the active conformation shown as density maps filtered to 9.5A resolution. The models are shown from three orientations, corresponding to the views in figure 1. (b) RMSD values (in A) calculated between the inactive and active conformations of the individual Arp2/3 subunits (based on the models used in (a)). Rows indicate which subunit was used for aligning the full models against each other, prior to measurements between individual subunits of the inactive and active Arp2/3 complexes (indicated in the columns). The RMSD analysis reveals that the structural transition upon complex activation is accommodated by two subcomplexes consisting of Arp2, ArpC1, ArpC4 and ArpC3, ArpC2 and ArpC3, respectively, that rotate against each other along an axis formed by the large helices of ArpC2 and ArpC4 (Movie S4). RMSD variations between the same subunits in the inactive and active conformations derive from changes upon MD-based modelling of the x-ray crystal structure-derived model into the electron microscopy density map of the branch junction.**'", "CAPTION FIG3.png": "'\\n\\n**(a)** Interaction surfaces between the Arp2/3 complex and the actin mother and daughter filament. The surfaces for the Arp2/3 complex, mother and daughter filament are shown as density maps at 9.5A resolution generated from their respective models. Grey coloring on the surface of Arp2/3 subunits indicates contact sites with actin and coloring of actin monomers in a specific color indicates a contact site with the associated Arp2/3 subunit (color code is given within the figure). Coloring was applied via the color zone command in ChimeraX in a 5.5A radius around each C-alpha atom of the underlying model, which was positioned in a 10A radius to a C-alpha atom of its putative interactor. **(b)** Surface representation of the interaction of ArpC2 and ArpC4 with the mother filament monomers M6 and M7 (the interaction of ArpC2 with M5 is omitted for clarity). **(c)** The protrusion helix of ArpC1 fitted into its density close to subdomains 1 and 3 of M4. **(d)** Surface representation of the interactions of Arp3 and ArpC3 with the mother filament. Note the cavity below Arp2, where no contacts between the Arp2 subunit and the mother filament are observed. ArpC3 acts as a linker between Arp2 and the mother filament. **(e)** Surface representation of the interaction between Arp2, Arp3 and the first two monomers of the daughter filament.\\n\\nFig. 3: **Actin-Arp2/3 complex interaction surfaces within the branch junction**\\n\\n**(a)** Interaction surfaces between the Arp2/3 complex and the actin mother and daughter filament. The surfaces for the Arp2/3 complex, mother and daughter filament are shown as density maps at 9.5\u00c5 resolution generated from their respective models. Grey coloring on the surface of Arp2/3 subunits indicates contact sites with actin and coloring of actin monomers in a specific color indicates a contact site with the associated Arp2/3 subunit (color code is given within the figure). Coloring was applied via the color zone command in ChimeraX in a 5.5\u00c5 radius around each C-alpha atom of the underlying model, which was positioned in a 10\u00c5 radius to a C-alpha atom of its putative interactor. **(b)** Surface representation of the interaction of ArpC2 and ArpC4 with the mother filament monomers M6 and M7 (the interaction of ArpC2 with M5 is omitted for clarity). **(c)** The protrusion helix of ArpC1 fitted into its density close to subdomains 1 and 3 of M4. **(d)** Surface representation of the interactions of Arp3 and ArpC3 with the mother filament. Note the cavity below Arp2, where no contacts between the Arp2 subunit and the mother filament are observed. ArpC3 acts as a linker between Arp2 and the mother filament. **(e)** Surface representation of the interaction between Arp2, Arp3 and the first two monomers of the daughter filament.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4: Structural changes in Arp3 and ArpC5 upon branch junction formation**\\n\\n**(a)** Comparison of the conformation of the Arp3 C-terminal tail in the inactive and active conformation. No density is observed for the C-terminal tail of Arp3 in its inactive conformation (top). Instead, the C-terminal tail can flip towards Arp2 and ArpC4, where it is accommodated by an empty density present in the branch junction structure (bottom). **(b)** No density is observed for the ArpC5 N-terminus at its binding side in the inactive complex (top). Instead, the ArpC5 N-terminus can be fitted into a density between ArpC1 and Arp2. **(c)** Positively charged residues in ArpC1 and Arp2 could coordinate the negatively charged N-terminus of ArpC5. **(d)** Electrostatic potential map of the area shown in **(c)**.\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n**Table S1.**\\n\\n**Data acquisition and image processing parameters**'", "CAPTION FIGS3.png": "'\\n\\n**Fig. S3.: Structural details of the actin filament Arp2/3 complex branch junction.** Densities for the individual subunits of the Arp2/3 complex plus their fitted models are shown. Subunit colors are annotated and identical to the schematic guide given in Figure 1. Secondary structure detail (i.e. alpha helices) are clearly visualized, for example for Arp3, ArpC2 and ArpC4. The beta-propellers of ArpC1 allow unambiguous fitting of the subunit into the density of the branch junction. The increased apparent flexibility at the N-terminus of the ArpC5 helical core (annotated by an arrow) is visible, resulting in reduced density for the N-terminal helices of this subunit. The subnanometer resolution of our structure also allows to clearly visualize secondary structure details in the actin filament, further highlighted by the visibility of an additional density accommodating Phalloidin (annotated by an arrow).\\n\\n'", "CAPTION TABS2.png": "'\\n\\n**Table S2.**\\n\\n**Summary of model content**\\n\\nThe pdb files used to generate the final model of the active Arp2/3 complex are listed. Residues that had to be added or removed from the original models are indicated. Residue stubs in the original models were completed to contain their entire side chains for MD-modelling in ISOLDE.\\n\\n'", "CAPTION TABS3.png": "'\\n\\n**Table 53.**\\n\\n**Modelling parameters and statistics**'", "CAPTION TABS4.png": "'\\n\\n**Table S4. Arp2/3 complex residues contacting the actin mother and daughter filaments**\\n\\nSummary of residues forming interactions between the Arp2/3 complex and the actin filaments, defined by a 10A C-alpha to C-alpha distance cutoff. The UniProt identifiers for the individual proteins are given.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/feldEgonetsSystematicallyBiased2020.json b/extracted_captions/feldEgonetsSystematicallyBiased2020.json new file mode 100644 index 0000000000000000000000000000000000000000..cc8d5133d6d0c8139d6c0772cc90b9bb491fad9c --- /dev/null +++ b/extracted_captions/feldEgonetsSystematicallyBiased2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "\"Figure 1. A small social network drawn from the example given in Feld (1991) of Coleman's Adolescent Society (1961) data of reciprocated friendships. This includes seven of the eight girls in the original example-presented by Feld.\\n\\n\"", "CAPTION FIG2.png": "'Figure 2: The degree distribution for the 60,483 Facebook users in the New Orleans regional dataset who had at most 100 friends.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: The degree distribution for the 3,307 Facebook users in the New Orleans regional dataset who had at least 100 friends.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Median and quartiles of egonet mean degree, by the ego\u2019s degree. A diagonal dashed line shows the points where ego\u2019s degree is equal to egonet\u2019s degree. For instance, the 25% quantile intersects the dashed line where 75% of egonets have a higher mean degree than the ego. The solid horizontal line indicates the mean degree in the population.\\n\\n'", "CAPTION TABA1.png": "''", "CAPTION TABA2.png": "''"} \ No newline at end of file diff --git a/extracted_captions/ferguson2017mechanoregulation.json b/extracted_captions/ferguson2017mechanoregulation.json new file mode 100644 index 0000000000000000000000000000000000000000..77388b38ec20fa72ebe67002680669e4213c931a --- /dev/null +++ b/extracted_captions/ferguson2017mechanoregulation.json @@ -0,0 +1 @@ +{"CAPTION 2.png": "'Figure 1: **Cell processing blocks that and assemble skeletons in multiple cell operators.** (3) (Cutter and temperature) and temperature levels of a 3DCT cell are shown. **Fig. 2:**_Cell processing blocks that and assemble skeletons in multiple cell operators.** (4) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 3:**_Cell processing blocks that and assemble skeletons in multiple cell operators.** (5) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 4:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (6) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 5:**_Cell processing blocks that and assemble skeletons in multiple cell operators.** (7) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 6:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (8) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 7:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (9) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 8:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (10) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 9:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (11) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 10:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (12) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 11:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (13) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 12:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (14) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 13:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (15) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 14:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (16) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 15:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (17) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 16:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (18) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 17:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 18:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (10) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 19:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (11) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 10:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (12) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 11:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (13) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 12:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (14) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 13:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (15) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 14:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (16) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 15:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (17) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 16:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (18) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 17:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 18:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 19:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (11) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 11:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (12) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 12:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (13) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 13:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (14) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 14:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (15) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 16:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (16) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 17:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (17) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 18:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (18) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 19:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 10:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 11:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 12:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 13:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 14:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 15:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 16:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and temperature levels of a 3DCT cell are shown). **Fig. 17:**_Cell processing blocks and assemble skeletons in multiple cell operators.** (19) (Cutter and\\n\\n'", "CAPTION 2F.png": "'\\n\\n## References\\n\\nFig. 2: **Cell squeezing induces fast and reversible alterations in clathrin coat dynamics.** (A) Cartoon and representative frames of a BSC1 cell are shown at different stages of squeezing (Movie 2). (B) Kymograph showing the temporal evolution of the clathrin traces detected at the ventral surface of the cell shown in A. Dashed lines mark the squeezing steps. (C) For the cell in A, normalized distributions of clathrin growth rates are plotted for different squeezing levels (Fig. S1). The standard deviation of the distribution reduces as the tension increases. (D) For the same cell, the time variation of the ventral surface area (upper) and the standard deviation of the clathrin growth rates (lower). The stepwise changes in these parameters are due to discrete levels of squeezing. (E) The response of the same cell to squeezing is shown as the mean clathrin lifetime (upper) and initiation and conclusion densities (lower). Dashed lines indicate changes in squeezing (\\\\(n_{\\\\text{vacuum}}\\\\)=8217). (F) Standard deviation of clathrin growth rates (upper), mean lifetime (middle), and initiation and conclusion densities (lower) from a cell that undergoes increased stepwise squeezing (orange dashed lines) and relaxation (blue dashed lines) (Movie 3) (\\\\(n_{\\\\text{vacuum}}\\\\)=8255).\\n\\n'", "CAPTION FIG1.png": "'\\nFigure 1: **Aspiration of the plasma membrane slows down clathrin coat dynamics.** (A) Kymograph showing the clathrin activity at the ventral surface of a BSC1 cell expressing AP2-eGFP. Clathrin coat traces elongate gradually upon microsuspension (dashed line; Movie 1). Blue and red arrowheads mark the initiation and conclusion of a clathrin-coated structure, respectively. _A)_ its its lifetime. (B) Clathrin coat lifetime distributions are shown for nine BSC1 cells imaged before and during microsuspension (\\\\(n_{\\\\text{max}}\\\\)=40,943). (C) For the same nine cells, the standard deviation of the clathrin growth rate distributions are shown in koptics. Lines connect the standard deviation values obtained from the same cell before and during aspiration. The narrower growth rate distributions indicate slower clathrin coat dynamics. (D) Box plots are the initiation and conclusion densities of clathrin-coated structures before and during aspiration. In the koptics, the box represents the 25\u201375th percentiles, and the median is indicated. The whiskers show the 10\u201390th percentiles. _P_-values were obtained with a two-tailed _I_-test.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Hypetemic swelling inhibits clathrin coat dynamics temporarily. (A) Change in the volume (normalized to the radial value) is placed for three ESC1 cells during hypotonic swelling (i.e. osmachock). (B) Mean clathrin coat lifetime (upper), and initiation and conclusion densities (lower) are plotted against time for a ESC1 cell treated with hypotonic shock (dashed line). (C) Clathion lifetime distributions are assembled pre- and post-osmachock for 12 gene-edited SUM159 cells xenrazing AP2-EGFP (\\\\(n_{\\\\mathrm{tracea}}\\\\)=\\\\(34,113\\\\)). (DE) For the same cells, the standard deviation of clathrin growth rates [0] and initiation and conclusion densities of clathrin-coated structures pre- and post-osmotic shock(E) are shown in boxplots. Lines centered the standard deviation values obtained from the same cell pre- and post-osmachock. In the boxplots, the box represents the 25-75th percentiles, and the median is indicated. The whiskers show the 10-90th percentiles. Values were obtained with a two-tailed \\\\(t\\\\)-test.\\n\\n'", "CAPTION FIG4.png": "'\\nFigure 4: **Acth dynamics mediate the inward movement of clathrin coats prior to disassembly.** (A) Means-s.e.m. values for of normalized AP2 intensity traces (determined for a SUM159 cell before and after hypotonic swelling; \\\\(n_{\\\\text{max}}\\\\)=3728). The traces are aligned at the end point before averaging. (B) Mean-s.e.m. z. displacements are shown for the two trace groups in A. (C) Top, growth rate distributions are assembled for eight SUM159 cells before and after hypotonic swelling (\\\\(n_{\\\\text{max}}\\\\)=30,409). Different growth phases (ff, fast formation; sf, slow formation; p, plateau; sd, slow dissolution; fd, fast dissolution) were determined by quantifying the change in the clathrin coat signal over 25-s long time windows (Ferguson et al., 2016). Bottom, for the same cells, bar plots show the mean-s.e.m. of the z. velocities of the trace fragments (12 s long) that are used to generate the growth rate distributions above. Trace fragments that have the highest z velocity are found in the fast dissolution (1fd) phase. (D) Top, representative intensity traces of AP2 (green) and LifeAct (red) fluorescence during the formation of a clathrin-coated vesicle at the ventral surface of a BSC1 cell. Bottom, the relative LifeAct intensity (mean-s.e.m.) colocalizing with clathrin coats is shown for different growth phases (\\\\(n_{\\\\text{total}}\\\\)=4, \\\\(n_{\\\\text{max}}\\\\)=28,796). Note that the growth phases are determined by using the master (AP2=EGFP) signal. (E) Bar plots show the z velocities (mean-s.e.m.) corresponding to different growth phases for AP2 traces obtained from BSC1 cells in the absence and presence of jasplakinolide (Jase) (Control, \\\\(n_{\\\\text{total}}\\\\)=7, \\\\(n_{\\\\text{max}}\\\\)=20,204; Jase, \\\\(n_{\\\\text{max}}\\\\)=6, \\\\(n_{\\\\text{max}}\\\\)=25,972). *P-O0.0001; *P-O0.001 (two-tailed Hest).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/fernandes2019cell.json b/extracted_captions/fernandes2019cell.json new file mode 100644 index 0000000000000000000000000000000000000000..0c5e88d212cd6fc5d901f8b5c0cf2a70549fdf51 --- /dev/null +++ b/extracted_captions/fernandes2019cell.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: A quantitative treatment of TCS triggering relying on receptor dual time at phosphatase-depicted close contacts. (_A_) Top and side views of the close contact depicting contact topography (with contact radius \u201cr\u201d) and CD45 exclusion. The first box (solid line) shows the region of the cell magnified below it. The second box (dotted line) shows the region depicted in the top view on the right. (_B_) According to the model, a TCR (TCR) is triggered, i.e., phosphorylated because its residence time in the contact is >=2 s. TCR\\\\({}_{2}\\\\) is not triggered because it diffuses out of the contact in less than 2 s. (_C_) Also according to the model, a receptor (TCR) that engages ligand is likely to be held in the contact \u22652 s and become triggered. In \\\\(B\\\\) and \\\\(C\\\\), the margins of the contact are marked by the average positions of excluded CD45 molecules (green). (_D_) Snapshots from the simulation of the TCR density probability evolution in dose-contacts as they grow over time (_S_ Appendix_ _A_).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Parameterization of the model. (A) Experimental approach. High-density labeling of CD45 (Gap 8.3 Fab, Alexa Fluor 488) was used to indicate sites of close-contact formation between T cells and a rat CD2-presenting SIs (Ledt), and this was combined with simultaneous low-density labeling of CD45 (Gap 8.3 Fab, Alexa Fluor SBE), Lck (Halo tag, tetramethylrhodamine [TM&]), or TCR (Halo tag, TMR) (right) to enable TIRFM-based single-molecule tracking. (B-D, Left) TERM-based single-molecule tracking of CD45 (B), Lck (C), and TCR (D). Well-separated individual trajectories were recorded for >280 ms and colored according to position in the contact (orange in CD45-rich region and blue in CD45-depleted regions). (Right) Close-up views of trajectories in regions marked by white rectangles CD45-rich regions are shown in gray. (Scale bar, 2 \u03bcm.) Data are representative of three independent experiments with \\\\(n>10\\\\) cells.\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: Why the TCR can be triggered in the absence of ligands. (A) Probability that a TCR remains inside a dose contact for time, \\\\(\\\\tau\\\\), for close contacts of varying fixed radius, \\\\(\\\\tau_{0}\\\\) (B) Probability that a single TCR stays inside a dose contact >2 s as a function of final close-contact radius for growing contacts. (C) Total number of TCRs that remain inside the close contact for >2 s, incorporating the estimates shown in \\\\(A\\\\), the density of TCRs in Jurkat T cells, and the degree of exclusion of the TCR from close contacts for cells interacting with rCD2-presenting SLBs. (_D_) Total contact area (region of CD45 exclusion) at the time of calcium release for T cells interacting with rCD2-presenting SLBs (13 cells, 5 independent experiments). Central lines indicate the median; small squares indicate the mean; boxes show interquartile range; whiskers indicate SD.\\n\\n'", "CAPTION FIG5.png": "'\\nFig. 5: Self-finensifel discrimination. (_A_) Probability distribution of close-contact residence times for TCRs in the presence and absence of agonist and self pMHC, for a close contact of radius \\\\(\\\\tau_{0}=220\\\\) nm, showing that discrimination of ligands is not dependent on a threshold value for \\\\(\\\\tau_{\\\\rm exc}\\\\). (_B_) Probability that at least one TCR will be triggered, i.e., stay in the contact for \\\\(\\\\tau_{0}\\\\geq 2.5\\\\), as a function of contact duration in the presence and absence of agonist pMHC with a low \\\\(k_{\\\\rm int}\\\\) (\\\\(k_{\\\\rm int}=1^{-1}\\\\), 30 pMHC\\\\(\\\\mu\\\\)m\\\\(\\\\lambda\\\\)), or a self pMHC with a larger \\\\(k_{\\\\rm int}\\\\) present at higher pMHC densities (\\\\(k_{\\\\rm int}=50\\\\)\\\\(\\\\times\\\\)\\\\(1^{-1}\\\\), 300 pMHC\\\\(\\\\mu\\\\)m\\\\(\\\\lambda\\\\)). (_C_) Comparison of the triggering probability in the absence of pMHC for close contacts of 220 and 440 nm. (_D_) Triggering probability as a function of pMHC densities and pMHC of rates for a single contact of 220 nm radius with a duration of \\\\(\\\\tau_{0}=120\\\\) s. (_F_) Triggering probability as a function of close contact radius for pMHC with varying off rates for a contact duration of \\\\(\\\\tau_{0}=120\\\\) s. (_F_) Contribution to the overall signal of TCRs that are triggered without binding to pMHC, in the presence of agonist pMHC with varying \\\\(k_{\\\\rm int}\\\\) (30 pMHC\\\\(\\\\mu\\\\)m\\\\(\\\\lambda\\\\)).\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Prediction of the relative signaling potentials of well-characterized TCR ligands. Peptide-stimulation potentials (EC48 and EC5E values for IL-2 selection) for CD4\\\\({}^{+}\\\\) (Left) and CD8\\\\({}^{+}\\\\) T cells (Right) (determined elsewhere in refs. 11, 33, and 53), plotted against the probability that at least one TCR triggering event (\\\\(t_{\\\\rm{oxin}}\\\\geq 2\\\\) s) occurs at a single contact of \\\\(r_{\\\\rm{a}}=220\\\\) nm, that persists for \\\\(t_{\\\\rm{f}}=120\\\\) s.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1.** Experimental parameters used in this study.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/goldenbogen2016dynamics.json b/extracted_captions/goldenbogen2016dynamics.json new file mode 100644 index 0000000000000000000000000000000000000000..8658d778705e7f67d5da4b1df27f9f144a7d364c --- /dev/null +++ b/extracted_captions/goldenbogen2016dynamics.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Elastic shell model of the shmoo predicts a spatially inhomogeneous distribution of the lateral cell wall stresses and the Young\u2019s modulus. (a) _M/Ta_ cells synthesize **a**-factor and grow a mating projection towards _M/Ta_ cells upon sensing **a**-factor, and vice versa. (b) Scheme of a shmooing cell divided in base (0), neck (II), shaft (III) and tip (IV) with considered contributing elements, i.e. elastic cell wall (blue) with locally varying Young\u2019s modulus (white hatched), turgor pressure \\\\(\\\\bar{P}\\\\), and material insertion at the tip. (c) Description of coordinates given an axisymmetric geometry: circumferential angle \\\\(\\\\theta\\\\), meridional length \\\\(s\\\\) with corresponding distance to the axis \\\\(r\\\\), relaxed radii for the mating projection \\\\(R_{\\\\text{synch}}\\\\), and the spherical part \\\\(R_{\\\\text{base}}\\\\) as well as cell wall thickness \\\\(d\\\\). (d,e) Distribution of von Mises stress \\\\(\\\\sigma_{\\\\text{M/t}}\\\\) and volumetric strain \\\\(e_{\\\\mu}\\\\), respectively, with red indicating high and blue low values. (f) Resulting spatial distribution of the Young\u2019s modulus \\\\(\\\\bar{E}\\\\), dark green shows low values and shades of light-green to white high values. White area at the tip shows region of undefined \\\\(e_{\\\\mu}\\\\) and \\\\(\\\\bar{E}\\\\). Contour plots of stress, strain and elastic modulus shown in electronic supplementary material, figure S2. We used a cell with relaxed radius of \\\\(R_{\\\\text{base}}=1.9\\\\)\\\\(\\\\mu\\\\)m, expanded radius of \\\\(r_{\\\\text{base}}=2.5\\\\)\\\\(\\\\mu\\\\)m, relaxed radius of \\\\(R_{\\\\text{synch}}=0.4\\\\)\\\\(\\\\mu\\\\)m, cell wall thickness of \\\\(d=115\\\\) nm, turgor pressure of \\\\(\\\\bar{P}=0.2\\\\) MPa and Poisson\u2019s ratio of \\\\(\\\\nu=0.5\\\\). The values of stresses and the Young\u2019s modulus itemized by the base, neck, shaft and tip are shown in electronic supplementary material, table S1.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **\u03b1**-factor treatment of _MMTA_ bar1\u0394 cells induced a localized softening of the cell wall in the region of the emerging mating projection. (_a,b_) Three-dimensional images with textures representing the elasticity for two individual _MMTA_ bar1\u0394 cells without (_a_) and with (_b_) 1 h treatment with 10 \u03bcM\u03b1\u03b1\u03b1\u03c4\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Dynamics of cell wall elasticity during shinooning, measured by live cell wall nano-indentation experiments and simulations of the dynamic cell wall model (DMI). Softening of the cell wall starts early, continues with elongation of the mating projection and forms a ring around its base. MTA bar\\\\(\\\\Delta\\\\) cells were induced with 10 \\\\(\\\\mu\\\\)M er-factor for 64 min before the first image was acquired. (_a,b_) Sequences of height and elasticity developments during formation of a mating projection, from a continuous AFM measurement (\\\\([d]\\\\) consecutive images of the \\\\(E\\\\)-distribution and \\\\([b]\\\\) three-dimensional reconstruction with elasticity pattern, see electronic supplementary material, movie S1). Arrows indicate a region of non-softened cell wall material at the tip; scale bar, 1 \\\\(\\\\mu\\\\)m. Elasticity pattern controls shape of the evolving shinoo in the dynamic model. (\\\\(e-e\\\\)) Simulation snapshots of DMI (electronic supplementary material, movie S2), showing the pattern of the Young\u2019s modulus (\\\\([c]\\\\), black/green/white), the von Mises stress \\\\(\\\\sigma_{\\\\text{MI}}\\\\) (\\\\([d]\\\\), blue/red) and the volumetric strain \\\\(e_{\\\\text{T}}\\\\) (\\\\([e]\\\\), blue/red); scale bar, 1 \\\\(\\\\mu\\\\)m. Note that colour scale for the elasticity in (\\\\(a\\\\)) ranges from 0 to 10 MPa in order to distinguish the cell from its stiffer surrounding (orange spectrum). Contour plots of stress, strain and elastic modulus are shown in electronic supplementary material, figure S11.\\n\\n'", "CAPTION FIG4.png": "\"Figure 4: The influence of elasticity patterns on cell wall growth for two variants of the dynamic cell wall model, DM1 and DM2. Snapshots of the dynamic cell wall model DM1 (electronic supplementary material, movie S2) and DM2 (electronic supplementary material, movie S3) after 1 h of simulated time, where the shape (column 1), the Young's modulus \\\\(\\\\xi\\\\), the von Mises stress \\\\(\\\\sigma_{\\\\text{WI}}\\\\), the volumetric strain \\\\(e_{\\\\text{V}}\\\\) and expansion rates \\\\(\\\\alpha\\\\) or \\\\(\\\\alpha^{*}\\\\) is shown for different model assumption; scale bar, 2 \\\\(\\\\mu\\\\)m. (\\\\(a\\\\),\\\\(b\\\\)) Simulations of DM1 assuming growth under yield stress. (\\\\(c\\\\),\\\\(d\\\\)) Simulations of DM2 assuming growth under yield strain. (\\\\(e\\\\)) Simulation of pure elastic expansion without cell wall growth. (\\\\(f\\\\)) Spherical reference shape. The elasticity pattern (\\\\(a\\\\),\\\\(c\\\\)) results in a longer mating projection than for homogeneous elasticity (\\\\(b\\\\),\\\\(d\\\\)). The effect of elasticity pattern on cell growth is even larger for DM2 (\\\\(c\\\\)) than for DM1 (\\\\(g\\\\)). Growth showed a ring-like pattern around the tip for the DM2 (\\\\(\\\\xi\\\\)), column 5), whereas for the DM1 ((\\\\(a\\\\)), column 5) growth was focused to the centre of the tip. Contour plots of stress, strain and elastic modulus for DM1 and DM2 are shown in electronic supplementary material, figure S11.\\n\\n\"", "CAPTION FIG5.png": "'Figure 5: Osmotic stress experiments confirmed strain profiles of the dynamic models. (_a_) Modelled shapes under yield stress (left) and yield strain assumption, before (grey) and after (blue) reduction of turgor pressure, respectively. (_b_) Time series of bright-field microscopy images of shmooing MMa bar/\\\\(\\\\Delta\\\\) cells rapidly exposed to high extracellular osmolyte concentration (2 M sorbitol) in a microfluidic device (MFD). (_c_) Scheme of a shmooing cell with indicated dimensions: length of protrusion and longest axis, radii of base, neck and tip. (_d_) Scheme of the MFD set-up. The interface between normal (SD) and high osmolarity (SD + 2 M sorbitol) media can be shifted by varying the input pressures, which allows to rapidly exchange the cellular environment. (_e_,_f_) Measured relative expansion \\\\(\\\\Delta t/t_{0}\\\\) and \\\\(\\\\Delta t/t_{0}\\\\) in black and simulated volumetric strain obtained from yield stress model (DM1) as yellow triangles or from yield strain model (DM2) as blue triangles, for base, neck and tip, respectively. The deformation \\\\(\\\\Delta t/t_{0}\\\\) (_e_) at the neck was significantly higher than at the base or at the tip (_t_-test, ***_p_\\\\(<\\\\) 0.001, \\\\(n\\\\) = 119), whereas longest axis and protrusion show only a slight relative expansion \\\\(\\\\Delta t/t_{0}\\\\) (_f_).\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n**Figure S1. Geometrical considerations and principal in plane stresses and strains.****A** shows the used coordinates: circumferential angle \\\\(\\\\theta\\\\), meridional distance s, radius r and shell thickness d **B** shows principal stresses and strains for a given shell element. **C** circumferential and meridional stresses (\\\\(\\\\sigma_{\\\\theta}\\\\), \\\\(\\\\sigma_{s}\\\\)) and strains (\\\\(\\\\varepsilon_{\\\\theta}\\\\), \\\\(\\\\varepsilon_{s}\\\\)) are equal for a sphere, while \\\\(\\\\sigma_{\\\\theta}\\\\) is twice as high as \\\\(\\\\sigma_{s}\\\\) for the lateral surface of a cylinder. Additionally, \\\\(\\\\varepsilon_{\\\\theta}\\\\) exceeds \\\\(\\\\varepsilon_{s}\\\\), given that \\\\(v\\\\leq 0.5\\\\).\\n\\n'", "CAPTION FIGS10-1.png": "'\\n\\n**Figure S10. The DM is based on the elasto-plastic deformations of the triangular surface elements.****A** shows the triangular meshed surface of the simulated shmooing cell, concentric rings at the protrusion results from mesh refinement steps during the simulation. **B** In each simulation step the triangles deform according to the applied stresses. [caption continued, next page]'", "CAPTION FIGS10-2.png": "'If the triangle is not in the defined growth zone or \\\\(\\\\sigma_{VM}<\\\\sigma_{y}\\\\), the triangle deforms purely elastically. \\\\(L_{1},L_{2},\\\\ L_{3}\\\\) are the relaxed lengths of the unstressed triangle \\\\(T\\\\) and \\\\(l_{1},l_{2},\\\\ l_{3}\\\\) are the elastically expanded length of the resulting triangle \\\\(T^{\\\\prime}\\\\). Correspondingly, \\\\(\\\\alpha_{1},\\\\alpha_{2},\\\\ \\\\alpha_{3}\\\\) represent the relaxed angles and \\\\(\\\\beta_{1},\\\\beta_{2},\\\\ \\\\beta_{3}\\\\) the angles of the deformed triangle. Additionally, triangles in the defined growth zone deform plastically if \\\\(\\\\sigma_{VM}\\\\geq\\\\sigma_{y}\\\\). Thereby, the relaxed lengths expand to new relaxed lengths, \\\\(L_{1}^{new}\\\\), \\\\(L_{2}^{new}\\\\) and \\\\(L_{3}^{new}\\\\), respectively while the angles remain unaltered.\\n\\n'", "CAPTION FIGS10.png": "'Figure 33. The 50% based on the stable-plastic deformations of the triangular surface elements. A shows the triangular nested surface of the simulated shimming cell, concentric rings at the protrusion results from mesh refinement steps during the simulation. **B** in each simulation step the shrouges deformation according to the applied stresses. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **C**errespondingly, \\\\(\\\\sigma_{\\\\mathrm{raw}},\\\\sigma_{\\\\mathrm{y}}\\\\) represent the relaxed angles and \\\\(\\\\sigma_{\\\\mathrm{f}},\\\\sigma_{\\\\mathrm{y}}\\\\), \\\\(\\\\sigma_{\\\\mathrm{y}}\\\\), the angles define scattering angles. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **C**errespondingly, \\\\(\\\\sigma_{\\\\mathrm{raw}},\\\\sigma_{\\\\mathrm{y}}\\\\) represent the relaxed angles and \\\\(\\\\sigma_{\\\\mathrm{f}},\\\\sigma_{\\\\mathrm{y}}\\\\), \\\\(\\\\sigma_{\\\\mathrm{y}}\\\\), the angles define scattering angles. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **C**errespondingly, \\\\(\\\\sigma_{\\\\mathrm{raw}},\\\\sigma_{\\\\mathrm{y}}\\\\) represent the relaxed angles and \\\\(\\\\sigma_{\\\\mathrm{f}},\\\\sigma_{\\\\mathrm{y}}\\\\), \\\\(\\\\sigma_{\\\\mathrm{y}}\\\\), the angles define scattering angles. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **C**errespondingly, \\\\(\\\\sigma_{\\\\mathrm{raw}},\\\\sigma_{\\\\mathrm{y}}\\\\) represent the relaxed angles and \\\\(\\\\sigma_{\\\\mathrm{f}},\\\\sigma_{\\\\mathrm{y}}\\\\), \\\\(\\\\sigma_{\\\\mathrm{y}}\\\\), the angles define scattering angles. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **C**errespondingly, \\\\(\\\\sigma_{\\\\mathrm{raw}},\\\\sigma_{\\\\mathrm{y}}\\\\) represent the relaxed angles and \\\\(\\\\sigma_{\\\\mathrm{f}},\\\\sigma_{\\\\mathrm{y}}\\\\), \\\\(\\\\sigma_{\\\\mathrm{y}}\\\\), the angles define scattering angles. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{raw}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle. **J**ation curves, red-spiel \\\\(f\\\\) The triangle is not the defined growth zone at \\\\(\\\\sigma_{\\\\mathrm{y}}<\\\\sigma_{\\\\mathrm{y}}\\\\), the triangle follows purely elastically, \\\\(L_{1},L_{2}\\\\), in the relaxed lengths of the unstressed triangle and \\\\(L_{1},L_{2}\\\\), in the relaxedly expanded length of the moving triangle.\\n\\n'", "CAPTION FIGS11.png": "\"\\n\\n**Figure S11. Simulated stress, strain and elasticity profiles along the cell contour.****A** The contours of the resulting cell shape of DM1 (yellow) and DM2 (blue) at time 3250 s with indicated regions: base (I), shaft (II), neck (III) and tip (IV). **B, C, D** Contour plots along the arc length \\\\(s\\\\) of the resulting von Mises stress \\\\(\\\\sigma_{VM}\\\\), the resulting volumetric strain \\\\(\\\\varepsilon_{V}\\\\) and the assumed Young's modulus \\\\(E\\\\).\\n\\n\"", "CAPTION FIGS12.png": "'\\n\\n**Figure S12. Sensitivity analysis of the yield limit for DM1 and DM2. A and B show cell shapes obtained from simulations with various limits for yield stress \\\\(\\\\sigma_{Y}\\\\) and yield strain \\\\(\\\\varepsilon_{Y}\\\\), respectively at 1000 s, 1500 s and 2000 s. Lower \\\\(\\\\sigma_{Y}\\\\) or lower \\\\(\\\\varepsilon_{Y}\\\\) result in faster growth. When varying parameters in a certain range, similar shapes are obtained at different times.**'", "CAPTION FIGS13.png": "\"\\n\\n**Figure S13. Decrease in Young's modulus with increasing indentation velocity was negligible within the scope of this study (v=67um/s)**. The mean Young's modulus of a selected 400nm x 400nm region in the center of a trapped cell (left) plotted against the applied indentation velocity (dots with error bars). Linear regression showed a significant but minor decrease in E (slope = -0.0046 +-0.0008 MPa/(um/s), R2=0.85, F(1, 6)=33.93 p=0.001).\\n\\n\"", "CAPTION FIGS2.png": "\"\\n\\n**Figure S2. Stress, strain and elasticity profiles of the SM. A** Contours for the relaxed (dashed line) and expanded (solid line) cell shape. Tip was the origin of the meridional coordinate, indicated with dashed arrow. The regions tip (I), shaft (III), neck (III) and base (IV) are separated by dashed lines for orientation. For the cell shape in **A** profiles of: **B** Young's modulus E, von Mises stress \\\\(\\\\sigma_{\\\\text{VM}}\\\\) and volumetric strain \\\\(\\\\varepsilon_{\\\\text{V}}\\\\), **C** meridional stress \\\\(\\\\sigma_{s}\\\\), circumferential stress \\\\(\\\\sigma_{\\\\emptyset}\\\\) and \\\\(\\\\sigma_{\\\\text{VM}}\\\\), **D** meridional strain \\\\(\\\\varepsilon_{s}\\\\), circumferential strain \\\\(\\\\varepsilon_{\\\\emptyset}\\\\) and \\\\(\\\\varepsilon_{\\\\text{V}}\\\\) are shown. **E, F**\\\\(\\\\varepsilon_{\\\\text{V}}\\\\)- and E-profiles for different relaxed shapes with varying radius \\\\(R_{\\\\text{shaft.}}\\\\) were calculated. **G, H** Profiles of \\\\(\\\\varepsilon_{s}\\\\) and \\\\(\\\\varepsilon_{\\\\emptyset}\\\\) or E and \\\\(\\\\sigma_{\\\\text{VM}}\\\\) assuming a constant volumetric strain \\\\(\\\\varepsilon_{\\\\text{V}}\\\\) and the expanded cell shape in **A**.\\n\\n\"", "CAPTION FIGS3.png": "'\\n\\n**Figure S3. Mapping the cell wall elasticity of entrapped haploid S. cerevisiae cells**. **A** Scheme of the experimental setup: the previously trapped yeast cell is scanned by an AFM in Q\\\\({}^{\\\\rm{ITM}}\\\\) Mode. For each pixel a nano-indentation measurement was carried out. **B** 3D representation of the shape detected in **A**. **C** Hypothetic approach curve for one pixel in **A**, along with a Sneddon fit (green) of these data assuming a conical indenter. The resulting spatial information on the elasticity (0 MPa - 20 MPa) of the probed material was displayed in 2D images **D** or used as a texture for 3D representations **B**. **B** and **D** show the same MAT**a** bar1\\\\(\\\\Delta\\\\) cell with characteristically stiffer bud scar regions (indicated by arrows); scale bar is 1 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIGS4.png": "'\\n\\n**Figure S4. Indentation depth varies between stiffer and softer regions.****A** Exemplary force-distance curves of nano-indentation measurements at the marked region in **B** and **C**. **B** shows elasticity map and **C** indentation map of shmooing MATa bar1\\\\(\\\\Delta\\\\) cell shown in Figure 2. If both curves reached similar maximum force, the conical tip of the cantilever indented the cell wall in softer region (shaft, green dots) deeper than the stiffer region (base, orange squares), due to the smaller slope of the curve **B**. The indentation depth \\\\(\\\\delta\\\\) (mean +- Ra, N=900) of a quadratic region (550 nm x 550 nm) at the top of the cell was at the shaft (\\\\(\\\\delta_{\\\\rm shaft}\\\\) = 97 nm +- 26 nm) and at the base (\\\\(\\\\delta_{\\\\rm base}\\\\) = 38nm +- 10 nm) less or equal than 115 nm, which is supposed to be the thickness of the cell wall.\\n\\n'", "CAPTION FIGS5.png": "'\\n\\n**Figure S5. Comparison of height and elasticity patterns between \\\\(\\\\alpha\\\\)-factor treated and non-treated MATa bar\\\\(\\\\Delta\\\\) cells.****A, B** Height images and **E, F** elasticity maps of two non-treated cells and **C, D** height images and **G, H** elasticity maps of two individual MATa bar\\\\(\\\\Delta\\\\) cells treated with 10 \\\\(\\\\mu\\\\)M \\\\(\\\\alpha\\\\)-factor; black and white bars correspond to 1\\\\(\\\\mu\\\\)m. The white arrows represent position length and direction of the selected cross-sections in **I, J, K, L**. Manually selected regions for base (I), shaft (II) and tip (IV) are framed with white rectangles.\\n\\n'", "CAPTION FIGS6.png": "'\\n\\n**Figure S6. The cell wall, at shaft of the mating projection, showed significant lower E-values. A Scatter Dot Plot of the mean E-values of selected regions at the base, shaft and tip, respectively; bar represents the mean. B Mean E-values of selected regions at the shaft, the base and the tip plotted against the mean E-values at the base for each measured cell. Lines correspond to linear regressions forced through (0,0) with a slope of E\\\\({}_{\\\\text{shaft}}\\\\)/E\\\\({}_{\\\\text{base}}\\\\) = 0.28 \\\\(\\\\pm\\\\) 0.06 (Sy.x=0.41, DF 6), E\\\\({}_{\\\\text{Tip}}\\\\)/E\\\\({}_{\\\\text{Base}}\\\\)=0.71 \\\\(\\\\pm\\\\) 0.2 (Sy.x=1.7, DF 6) and E\\\\({}_{\\\\text{Base}}\\\\)/E\\\\({}_{\\\\text{Base}}\\\\)=1.00 \\\\(\\\\pm\\\\) 0.00 (Sy.x=0.00, DF 6). Broken lines correspond to the respective confidence intervals (95%). C Elasticity profiles over tip, shaft and base for all analyzed shmooing cells showed a reduction of E from base to shaft and tip to shaft for every cell.**'", "CAPTION FIGS7.png": "'\\n\\n**Figure S7. Time series replica for estimation of the cell wall dynamics during mating projection formation.****A, B** Time-lapse sequence of the height and elasticity development during the formation of a mating projection, obtained with AFM; from left to right, consecutive images of the E-distribution (top) and 3D reconstruction with elasticity pattern (bottom), from a continuous measurement. MAT**a** bar1\\\\(\\\\Delta\\\\) cells were induced with 12 mM **A** and 10 mM **B** \\\\(\\\\alpha\\\\)-factor for 122 min **A** and 42 min **B**, respectively, before the first image was acquired. White arrows indicate a region of stiffer cell wall material at the tip. Note, the first image of sequence B shows a barely noticeable reduction in E at the side of the emerging protrusion. Both time-laps series show typical AFM-artifacts for high objects (in **A** a doubling of the tip shape and in **B** a height \"shadow\"), indicated with black arrows.\\n\\n'", "CAPTION FIGS8.png": "'\\n\\n**Figure S8. Cell stiffness saturated at 1.5 N/m for high loading forces and large indentations.** The cell stiffness, obtained with indentation experiments of three untreated _bar1_\\\\(\\\\Delta\\\\) cells, was plotted against the loading forces and the corresponding indentation depths. The plotted stiffness k (mean, RMS, N=1024), represents the maximal slope of the indentation curve, using a cantilever with k of 0.64 N/m. The fitted maximum, assuming a Hill function, was used to calculate the turgor pressure applying the formula: \\\\(P=k/\\\\pi r\\\\).\\n\\n'", "CAPTION FIGS9.png": "'\\n\\n**Figure S9. Relaxed shell volume rapidly declines at higher turgor pressure.****A** shows the relaxed radius of the spherical shell with resting radius 2.5 \\\\(\\\\mu\\\\)m, **B** shows the relative volume confined by the relaxed shell, **C** the relative circumferential strain depending on turgor pressure.\\n\\n'", "CAPTION TABS1.png": "'\\n\\n**Table S1 Parameters values used in the models**'"} \ No newline at end of file diff --git a/extracted_captions/goyalEffectsSensemakingTranslucence2016.json b/extracted_captions/goyalEffectsSensemakingTranslucence2016.json new file mode 100644 index 0000000000000000000000000000000000000000..4d6eeba00dd83e7750f8ebb194f1f391ecd0c44f --- /dev/null +++ b/extracted_captions/goyalEffectsSensemakingTranslucence2016.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: The Document Space showing (clockwise, from top-left) the directory of crime case documents, a tabbed reader panel for reading case documents, a visual graph of connections based on common entities in the dataset, a map to identify locations of crimes and events, and a timeline to track events.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: The Analysis Space showing (clockwise, from top-left) the chat for explicit sharing, connected sticklies for Implicit Sharing, hypothesis window in the middle with columns to add new Hypotheses, confirming evidence, and disconfirming evidence for explicit Hypothesis Tracking, and Suspect Visualization at the bottom with 4 Avatars: Dennis Rathbone. Marilyn Stokes, Steve Gramming, and Lousie for Suspect Tracking. Note: Chat, Visualization, Sticky, and Hypothesis Window have been magnified to improve readability.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Sample sensemaking trajectory 1. Sensemaking-Translucence reminds users to consider suspects by showing empty Avatars at the start 2. Avatars are automatically populated by names detected from implicitly shared stickies. 3, 4 & 5. Avatars show distribution of name-reference by getting darker for names mentioned in stickies, chat and hypothesis window. The last mentioned suspect in hypothesis window is marked red 6. With use, visualization depicts distribution of attention at suspect level, based on explicit mentions. Note: Chat, Visualization, Sticky, and Hypothesis Window have been magnified to improve readability in 2, 3, 4 and 5. 1 and 6 represent non-magnified versions of the Sensemaking Translucence interface.\\n\\n'", "CAPTION FIG4.png": "'Figure 4. Serial killer identification and number of correct clues identified by interface condition.\\n\\n'", "CAPTION FIG5.png": "'Figure S. Perception of interface usefulness by interface condition\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Self-reported workload and team experience by interface condition.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/gressin2015architecture.json b/extracted_captions/gressin2015architecture.json new file mode 100644 index 0000000000000000000000000000000000000000..b657544d6ae08ff3b0a1b4a53d2c282a9ed13672 --- /dev/null +++ b/extracted_captions/gressin2015architecture.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Effect of ADF/Cofilin on Different Actin Network Architectures (A) Schematic description of the experiment performed in Figure 1. (B) Left: time course of Alexa-568-labeled actin (2 \u03bcM assembly on Las-17-coated micropatterns in the presence of Arp2/3 complex (20 nM), prior to the addition of ADF/cofilin. Right: color-coded cartoon of actin network architectures. (C) Quantification of (B). Actin network normalized fluorescence intensity along the yellow linescan indicated in (B) after a 115 min polymerization time is shown. (D) Left: time course of Alexa-488-labeled ADF/cofilin (2 \u03bcM; gravel) binding to actin networks (red). Right: quantification of actin (red) and ADF/cofilin (green) fluorescence intensities, normalized to their peak intensities. Scale bars represent 30 \u03bcm. See also Figure S1 and Movie S1.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Effect of Alp1 on ADF/Cofilin-Bound Actin Networks (**A**) Schematic description of the experiment performed in Figures 2A and 2B and 2C. (**B**) Left panels: Trna course of Alp1-SAAP-Alaxa-647 (1 uM; in black) binding to ADF/cofilin-decorated actin networks. Right panels: Quantification of actin (_n_ red, Alp2/cofilin (_n_ green) and Alp1 (_n_ blue) fluorescence intensities, normalized to their peak intraclassics. Scale Bar represents 30 \u03bcm. See abs More S9.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: **Quantitative Measurement of ADF/Cofflin Accumulation along the Side of Individual Actin Filaments** (A) Time course of Alexa-488-labeled ADF/cofflin (green) binding to individual actin filaments (800 nM; red), at the indicated concentrations. White arrowheads show representative examples of ADF/cofflin cooperative binding to actin filaments. Blue arrowheads show a representative example of a swerving event, where ADF/cofflin molecules remain mainly on one and of the fragments. The scale bar represents 5 \\\\(\\\\mu\\\\)m. (B) Quantification of (A). A time course of four representative molecular accumulations of ADF/cofflin along actin filaments for 180 nM (dotted lines) or 360 nM (continuous line) or ADF/cofflin is shown. Curves are plotted in blue prior to fragmentation and in red after fragmentation occurred. (C) Quantification of (A) and (B). Distribution of the number of ADF/cofflin molecules bound to actin filaments prior to sevaring is shown (23 = 11 molecules; n = 46). Error bars indicate the SD. (D) Representative field of observation after many fragmentation events with 360-_n_M ADF/cofflin. The scale bar represents 10 \\\\(\\\\mu\\\\)m. (E) Fluorescence recovery after photobleaching (FRAP) of ADF/cofflin accumulations after sevaring. (F) Time course of actin filament elongation after sevaring. The scale bar represents 5 \\\\(\\\\mu\\\\)m. (B) Kymograph of (D), plotted along the axis of the filament. See also Figures S2\u2013S4 and (A). (B). Distribution of the number of ADF/cofflin molecules bound to actin filaments prior to sevaring is shown (23 = 11 molecules; n = 46). Error bars indicate the SD.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Effect of Alp1 on Individual ADF/Cofilin-Bound Actin Filaments (A) Schematic description of the experiment performed in Figure 4.\\n\\n(B) Time course of Alp1*s (1 mM) effect on individual actin filaments (800 mM; red) decorated with various amounts of ADF/cofilin (green). White arrowheads track representative examples of ADF/cofilin stretches bound to the side of actin filaments (non-sewaving events). Red arrowheads track representative examples of ADF/cofilin stretches bound to the parload end of actin filaments (subsequent to a sawering event). Scale bars represent 5 mM.\\n\\n(C) Quantification of (B). A time course of three representative ADF/cofilin stretches along actin filaments at 180 mM (black curve) and 360 nM (red and blue curves) at ADF/cofilin after injection of Alp1 is shown. Clusters of ADF/cofilin are obscured either before (blue curve) or after (red curve) the fragmentation occurred. Fluorescence signal decrease of end-binding ADF/cofilin clusters were fitted with a microenvironmental decay (\\\\(\\\\Gamma_{\\\\text{disc}}=1.3\\\\pm 1.1\\\\); \\\\'", "CAPTION FIG5.png": "'Figure 6: **Actin Filament Elongation Assay in the Presence of ADF/Cotfillin and Alp1**(A) Time course of Alexa-558-actin filament (800 nM; red) elongation in the presence of 360 nM ADF/cotfin after injection of 800 nM Alexa-647-actin monomers (blue) and 20 nM capping protein. (B) Time course of Alexa-558-actin filament (800 nM; red) elongation in the presence of 360 nM ADF/cotfin after injection of 800 nM Alexa-647-actin monomers (blue) and 1 uM Alp1. The scale bar represents 5 um.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Effect of ADF/Cotfilin and Alp1 on Actin Filaments Blocked by Capping Protein\\n\\nTop: time course of actin filament (800 nM) elongation in the presence of 2-uM Alexa-488-ADF/cotfilin for 4 min, followed by a first injection of 20-nM capping protein for 16 min and a second injection of 1-uM Aipt. The scale bar represents 5 \u03bcm. Bottom: kymograph of the experiment plotted along the axis of an representative actin filament.\\n\\n'", "CAPTION FIG7-1.png": "'Figure 7: Models for the DIsassembly of Actin by ADF/Cofilin and Alp1 (A) Bioassembly at the whole-network scale: architecture dependence. In this model, binding of ADF/cofilin to actin cables does not trigger their disassembly, but switches actin filaments (blue filaments) to a pre-diassembly state (red filaments) (top). However, ADF/cofilin has an important effect for the dismantling of branched networks, through its de-branching activity (bottom). For all actin networks, presence of Alp1 turns these stable ADF/cofilin-decorated structures into unstable assemblies, prone to stochastic disassembly. (B) Swarming and disassembly at the single-filament scale. In this model, binding of ADF/cofilin needs to reach a threshold level of 23 molecules to trigger a conformational change of the filaments (represented by a change in their color, from blue to red). At a low concentration of ADF/cofilin (left), the threshold level is not reached. At an intermediate concentration of ADF/cofilin (center), actin filaments decorated by sub-stoichiometric densities of ADF/cofilin but with N > 23.\\n\\n'", "CAPTION FIG7-2.png": "'molecules of ADF/coffin are prone to an asymmetric swearing event. After fragmentation, ADF/coffin stretches remain near the pointed ends (PO), while barbed ends (BA) remain free to elongate. At a high concentration of ADF/coffin (right), filaments decorated by stoichiometric densities of ADF/coffin are stable. In this model, the conformation of actin filaments regulates the activity of Aip1. Only actin filaments decorated with N > 23 molecules of ADF/coffin undergo a fast stochastic disassembly.\\n\\n'", "CAPTION FIG7.png": "'* [18] J. M. C. _et al._, \"The \\\\(\\\\pi^{+}\\\\pi^{-}\\\\)'"} \ No newline at end of file diff --git a/extracted_captions/grozaSALTWeavingClaim2007.json b/extracted_captions/grozaSALTWeavingClaim2007.json new file mode 100644 index 0000000000000000000000000000000000000000..641edc7b848e0bde3a5e154dcbe2b9802f92d433 --- /dev/null +++ b/extracted_captions/grozaSALTWeavingClaim2007.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1.** Example of instantiation of the claim identification tree'", "CAPTION FIG2.png": "'Figure 2: Improved SALT semantic layer to support better identification\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3. Example of rhetorical elements in a scientific document**'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4. Example of test encoded in PDF format**'", "CAPTION FIG5.png": "'Figure 5: Example of transformation of a claim identification tree into a CBIB item.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n G. 1990. The evolution of the density of the gas in the central region. _Phys. Rev. Lett._**78**,'", "CAPTION FIG7.png": "'\\n\\n**Fig. 1.** A. **Annotation** **tack** **evaluation**'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8. A.** Soundness evaluation; **B.** Benefit uncertainty evaluation'"} \ No newline at end of file diff --git a/extracted_captions/harsDesigningScientificKnowledge2001.json b/extracted_captions/harsDesigningScientificKnowledge2001.json new file mode 100644 index 0000000000000000000000000000000000000000..a322b4b1fd5a89c577bd6dcfbb48c3be9c596d5a --- /dev/null +++ b/extracted_captions/harsDesigningScientificKnowledge2001.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Conceptual model of paper-based scientific knowledge [semi-plified by excluding paper copies].\\n\\n'", "CAPTION FIG2.png": "\"\\n\\n**Fig. 2.**_Pugner's epistemology (class diagram).\\n\\n\"", "CAPTION FIG3.png": "'\\n\\n**Fig. 1.** A single-particle (Cas diagram).\\n\\n'", "CAPTION FIG4.png": "\"\\n\\n**Fig. 4.** Chain's epidemiology (Case diagram).\\n\\n\"", "CAPTION FIG5.png": "\"\\n\\n**Fig. 5.** Range's epistemology [class diagram].\\n\\n\"", "CAPTION FIG6.png": "'\\n\\n**Fig. 6.** Synthesized conceptual model of scientific knowledge.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table I.** Competency questions for a body of scientific knowledge'", "CAPTION TAB2.png": "'\\n\\n## Chapter 2 Core term related to research methodology\\n\\n### 2.1 Core term related to research methodology\\n\\n#### 2.1.1 Core term related to research methodology\\n\\nThe core term related to research methodology is a core term related to research methodology.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/hassinger2017design.json b/extracted_captions/hassinger2017design.json new file mode 100644 index 0000000000000000000000000000000000000000..fbfe75a09dc7ca5935d1617dfd818f0855711b69 --- /dev/null +++ b/extracted_captions/hassinger2017design.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Schematic depiction of the main mechanical steps in CME. A multicomponent protein coat forms on the plasma membrane and causes the membrane to bend inward, forming a shallow pit. As the coat matures, the membrane becomes deeply irradiated to form an open, U-shaped pit before constricting to form a closed, \\\\(\\\\Omega\\\\)-shaped bud. The bud subsequently undergoes incision to form an internalized vesicle, and the coat is recycled. Actin polymerization is thought to provide a force, \\\\(\\\\mathbf{f}_{\\\\text{s}}\\\\) to facilitate these morphological changes, particularly at high membrane tensions [5]. Our study is focused on understanding the impact of membrane tension on the morphological changes affected by the coat and actin polymerization, as indicated by the dashed box.\\n\\n'", "CAPTION FIG2.png": "'\\nFig. 2: Membrane tension inhibits the ability of curvature generating costs to induce budding. (**a**) Profile views of membrane morphologies generated by simulations in which the area of a curvature-generating coat progressively increases, covering more of the bare membrane. The curvature-generating capability, or spontaneous curvature, of the coat is set at \\\\(C_{0}=0.02\\\\,\\\\mathrm{r}\\\\mathrm{m}^{-1}\\\\), corresponding to a preferred radius of curvature of \\\\(50\\\\,\\\\mathrm{n}\\\\mathrm{m}\\\\) (12, (**a**, _Upper_**) High membrane tension, \\\\(\\\\lambda_{3}=0.2\\\\,\\\\mathrm{p}\\\\mathrm{N}\\\\mathrm{/}\\\\mathrm{n}\\\\mathrm{m}\\\\). The membrane remains nearly flat as the area of the coat increases. (**a**, _Lower_**) Low membrane tension, \\\\(\\\\lambda_{3}=0.02\\\\,\\\\mathrm{p}\\\\mathrm{N}\\\\mathrm{/}\\\\mathrm{n}\\\\mathrm{m}\\\\). Addition of coat produces a smooth evolution from a flat membrane to a closed bud. (**b**) Membrane profiles for simulations with a constant coat area in which the spontaneous curvature of the coat progressively increases. The area of the coat is \\\\(A_{\\\\mathrm{cat}}=20.106\\\\,\\\\mathrm{n}\\\\mathrm{m}^{2}\\\\). (**b**, _Upper_**) High membrane tension, \\\\(\\\\lambda_{0}=0.2\\\\,\\\\mathrm{p}\\\\mathrm{N}\\\\mathrm{/}\\\\mathrm{n}\\\\mathrm{m}\\\\). The membrane remains nearly flat with increasing spontaneous curvature. (**b**, _Lower_**) Low membrane tension, \\\\(\\\\lambda_{0}=0.002\\\\,\\\\mathrm{p}\\\\mathrm{N}\\\\mathrm{/}\\\\mathrm{n}\\\\mathrm{m}\\\\). Increasing the spontaneous curvature of the coat induces a smooth evolution from a flat membrane to a closed bud.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: A snap-through instability exists at intermediate, physiologically relevant (S4), membrane tensions, \\\\(\\\\lambda_{6}=0.02\\\\,\\\\mathrm{n}\\\\mathrm{M}/\\\\mathrm{r}\\\\mathrm{n}\\\\). (A) Membrane profiles showing bud morphology before (dashed line, \\\\(\\\\mathcal{A}_{\\\\mathrm{coat}}=20,0.65\\\\,\\\\mathrm{n}\\\\mathrm{m}^{2}\\\\)) and after (solid line, \\\\(\\\\mathcal{A}_{\\\\mathrm{coat}}=20,105\\\\,\\\\mathrm{r}\\\\mathrm{m}^{2}\\\\)) addition of a small amount of area to the coat, \\\\(C_{\\\\mathrm{q}}=0.02\\\\,\\\\mathrm{r}\\\\mathrm{m}^{-1}\\\\). (B) Mean curvature at the tip of the bud as a function of the coat area. There are two stable branches of solutions of the equilibrium membrane shape equations. The lower branch consists of open, U-shaped buds, whereas the upper branch consists of closed, G-shaped buds. The dashed portion of the curve indicates \u201cunstable\u201d solutions that are not accessible by simply increasing and decreasing the area of the coat. The marked positions on the curve denote the membrane profiles shown in \\\\(A\\\\). The transition between these two shapes is a snap-through instability, in which the bud maps closed upon a small addition to area of the coat. (C) Bud morphologies before (dashed line) and after (solid line) a snap-through instability with increasing spontaneous curvature, \\\\(\\\\mathcal{A}_{\\\\mathrm{coat}}=20,106\\\\,\\\\mathrm{r}\\\\mathrm{m}^{2}\\\\), \\\\(C_{0}=0.02\\\\,\\\\mathrm{r}\\\\mathrm{m}^{2}\\\\). (D) Mean curvature at the tip of the bud as a function of the spontaneous curvature of the coat. (E) Bud morphology before (dashed line) and after (solid line) a snap-through instability with decreasing membrane tension, \\\\(\\\\mathcal{A}_{\\\\mathrm{coat}}=20,106\\\\,\\\\mathrm{r}\\\\mathrm{m}^{2}\\\\), \\\\(C_{0}=0.02\\\\,\\\\mathrm{r}\\\\mathrm{m}^{2}\\\\), \\\\(\\\\lambda_{6}=0.02\\\\,\\\\mathrm{r}\\\\mathrm{n}\\\\mathrm{M}/\\\\mathrm{r}\\\\mathrm{n}\\\\). (F) Mean curvature at the tip of the bud as a function of the membrane-fension.\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: Bud morphology depends on bending rigidity, membrane tension, spontaneous curvature, and coat area. (A) Coat spontaneous curvature (\\\\(\\\\zeta_{0}\\\\)) vs. membrane tension (\\\\(\\\\lambda_{0}\\\\)) phase diagram. The regions of the diagram are color coded according to the final shape of the membrane for coat \u201cgrowing\u201d simulations performed with the specified values for edge membrane tension and coat spontaneous curvature. Blue denotes closed, \\\\(\\\\Omega\\\\)-buds; red denotes open, U-shaped pits; and green are situations in which closed buds are obtained via a snap-through transition. The snap-through solutions cluster about the dashed line, \\\\(\\\\text{Ves}=1\\\\), which separates the high and low membrane tension regimes (for details, see the Instability _Evists over a Range of Membrane Tensions_, Coat Areas, and Spontaneous Curvature). The lines labeled B and C, respectively, indicate the phase diagrams at right. (B) Coat area vs. membrane tension phase diagram, \\\\(\\\\zeta_{0}=0.02\\\\) nm\\\\({}^{-1}\\\\). Blue denotes closed buds, red denotes open buds, and green denotes parameters that have both open and closed bud solutions. The dashed line, \\\\(\\\\text{Ves}=1\\\\), marks the transition from low to high membrane tension. The solid line represents the theoretical area of a sphere that minimizes the Heifrich energy at the specified membrane tension (_SI Appendix, 3. Radius of a Vesicle from Energy Minimization_). (C) Coat area vs. spontaneous curvature phase diagram, \\\\(\\\\lambda_{0}=0.02\\\\) pN/nm. The dashed line, \\\\(\\\\text{Ves}=1\\\\), marks the transition between spontaneous curvatures that are capable and incapable of overcoming the membrane tension to form a closed bud. The solid line represents the theoretical area of a sphere that minimizes the Heifrich energy at the specified spontaneous curvature (_SI Appendix, 3. Radius of a Vesicle from Energy Minimization_).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: The snap-through instability at physiological tension, \\\\(\\\\lambda_{0}=0.02\\\\,\\\\mathrm{pN/nm}\\\\), is abolished when the bending rigidity of the coat is increased relative to the bare membrane, \\\\(\\\\kappa_{\\\\mathrm{bare}}=320\\\\,\\\\mathrm{pN}\\\\cdot\\\\mathrm{nm}\\\\), \\\\(\\\\kappa_{\\\\mathrm{coatt}}=2400\\\\,\\\\mathrm{pN}\\\\cdot\\\\mathrm{nm}\\\\). (\\\\(A\\\\)) Membrane profiles showing a smooth progression of bud morphologies as the area at the coat is increased (\\\\(\\\\Delta_{\\\\mathrm{cat}}=10,000\\\\,\\\\mathrm{nm}^{2}\\\\), \\\\(20,000\\\\,\\\\mathrm{nm}^{2}\\\\), \\\\(28,000\\\\,\\\\mathrm{nm}^{2}\\\\)), \\\\(\\\\xi_{0}=0.02\\\\,\\\\mathrm{nm}^{-1}\\\\). (\\\\(B\\\\)) Mean curvature at the bud tip as a function of the area of the coat. The marked positions denote the membrane profiles shown in \\\\(A\\\\). There is new only a single branch of solutions (compared with Fig. 3B), indicating a smooth evolution from a flat membrane to a closed bud. (\\\\(C\\\\)) Membrane profiles showing a smooth progression of bud morphologies as spontaneous curvature of the coat is increased (\\\\(C_{0}=0.01\\\\,\\\\mathrm{nm}^{-1},0.02\\\\,\\\\mathrm{nm}^{-1},0.024\\\\,\\\\mathrm{nm}^{-1}\\\\)), \\\\(\\\\Delta_{\\\\mathrm{coatt}}=20,106\\\\,\\\\mathrm{nm}^{2}\\\\). (\\\\(D\\\\)) Mean curvature at the bud tip as a function of the spontaneous curvature of the coat showing a single branch of solutions (compare with Fig. 3D).\\n\\n'", "CAPTION FIG6.png": "'Figure 6: A force from actin assembly can mediate the transition from a U- to \\\\(\\\\Omega\\\\)-shaped bud, avoiding the instability at intermediate membrane tension, \\\\(\\\\lambda_{0}=0.02\\\\) pN/rem. Two orientations of the actin force were chosen based on experimental evidence from yeast [31] and mammalian [45] cells. (A) Schematic depicting actin polymerization in a ring at the base of the pit with the network attached to the coat, causing a net inward force on the bud. (B) At constant coat area, \\\\(A_{\\\\text{can}}=17,593\\\\) nm\\\\({}^{2}\\\\), and spontaneous curvature, \\\\(C_{0}=0.02\\\\) nm\\\\({}^{-1}\\\\), a force (red dash) adjacent to the coat drives the shape transition from a U-shaped (dashed line) to \\\\(\\\\Omega\\\\)-shaped bud (solid line). The force intensity was homogeneously applied to the entire coat, and the force intensity at the base of the pit was set such that the total force on the membrane integrates to zero. The final applied inward force on the bud was \\\\(\\\\mathbf{f}=15\\\\) pN, well within the capability of a polymerizing actin network [60]. (C) Schematic depicting actin assembly in a collar at the base, directly providing a constricting force [45]. (D) A constricting force (red dash) localized to the coat drives the shape transition from a U-shaped (dashed line) to \\\\(\\\\Omega\\\\)-shaped bud (solid line), \\\\(A_{\\\\text{coat}}=17,593\\\\) nm\\\\({}^{2}\\\\), \\\\(C_{0}=0.02\\\\) nm\\\\({}^{-1}\\\\). The force intensity was homogeneously applied perpendicular to the membrane to an area of 5,027 nm\\\\({}^{2}\\\\) immediately adjacent to the coated region. The final applied force on the membrane was \\\\(\\\\mathbf{f}<1\\\\) pN.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: A combination of increased coat rigidity and force from actin polymerization ensures robust vesiculation, even at high membrane tension, \\\\(A_{0}=0.2\\\\) pN/nm, \\\\(C_{0}=0.02\\\\) nm\\\\({}^{-1}\\\\). (A) Application of the inward directed actin force (as in Fig. 6a) induces tubulation, but not vesiculation, at high tension. (B) Increasing the stiffness of the coat alone is insufficient to overcome high membrane tension (dashed line). However, increasing the coat stiffness enables the applied force to induce vesiculation and decreases the magnitude of the force required by a factor of 3. (C) Application of the constricting actin force (as in Fig. 6C) is sufficient to induce vesiculation, even at high tension. The magnitude of the applied force required is likely unrealistically high in a biologically relevant setting. (D) Increasing the coat stiffness decreases the force required to induce vesiculation by an order of magnitude.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Design principles for robust vesiculation. The rigidity of the plasma membrane, as well as the membrane tension, resists budding by curvature-generating coats. In the low tension regime, as defined by the vesiculation number, increasing the coat area or spontaneously curvature is sufficient to induce a smooth evolution from a flat membrane to a closed bud. A combination of increased coat rigidity and force from actin polymerization is necessary to ensure robust vesiculation in the high memoranderation regime.\\n\\n'", "CAPTION FIGMOVIES1.png": "'\\n\\n**Movie 51**.: Membrane tension membrane budding in which the area of a curvature-generating coat progressively increases, covering more of the bare membrane. The curvature-generating capability, or spontaneous curvature, of the coat is set at \\\\(C_{3}=0.02\\\\,\\\\mathrm{nm}^{-1}\\\\), corresponding to a preferred radius of curvature of \\\\(50\\\\,\\\\mathrm{nm}\\\\) (\\\\(12\\\\), \\\\(45\\\\)). (_Left_) High membrane tension, \\\\(\\\\lambda_{0}=0.2\\\\,\\\\mathrm{pN/nm}\\\\). The membrane remains nearly flat as the area of the coat increases. (_Right_) Low membrane tension, \\\\(\\\\lambda_{3}=0.002\\\\,\\\\mathrm{pN/nm}\\\\). Addition of coat produces a smooth evolution from a flat membrane to a closed bud.\\n\\n'", "CAPTION FIGMOVIES2.png": "'\\n\\n**Movie 52**.: Membrane tension inhibits membrane budding in which the spontaneous curvature of a curvature-generating coat progressively increases at fixed area, \\\\(A_{\\\\text{last}}=20,106\\\\,\\\\text{nm}^{2}\\\\). (_Left_) High membrane tension, \\\\(\\\\lambda_{0}=0.2\\\\,\\\\text{pN}/nm\\\\). The membrane remains nearly flat with increasing spontaneous curvature. (_Right_) Low membrane tension, \\\\(\\\\lambda_{4}=0.002\\\\,\\\\text{pN}/nm\\\\). Increasing the spontaneous curvature of the coat induces a smooth evolution from a flat membrane to a closed bud.\\n\\n'", "CAPTION FIGMOVIES3.png": "'\\n\\n**Movie 53**.: A snap-through instability occurs with increasing coat area at intermediate, physiologically relevant (54), membrane tensions, \\\\(\\\\lambda_{0}=0.02\\\\) pH/_nm_. (Left) Membrane profiles showing bud morphology as the area to the coat increases, \\\\(\\\\mathcal{C}_{0}=0.02\\\\) nm\\\\({}^{-1}\\\\). (Right) Mean curvature at the tip of the bud as a function of the coat area. There are two stable branches of solutions of the equilibrium membrane shape equations. The marked position on the curve denotes the membrane profile shown on the left.\\n\\n'", "CAPTION FIGMOVIES4.png": "'\\n\\n**Movie 54**.: A snap-through instability occurs with increasing coat spontaneous curvature at intermediate, physiologically relevant (54), membrane tensions, \\\\(\\\\lambda_{0}=0.02\\\\) pN/nm. (_Left_) Bud morphology with increasing coat spontaneous curvature, \\\\(A_{\\\\text{last}}=20,106\\\\) nm\\\\({}^{2}\\\\). (_Right_) Mean curvature at the tip of the bud as a function of the spontaneous curvature of the coat. There are two stable branches of solutions of the equilibrium membrane shape equations. The marked position on the curve denotes the membrane profile shown on the left.\\n\\n'", "CAPTION FIGMOVIES5.png": "'\\n\\n**Movie 55**.: A snap-through instability occurs with decreasing membrane tension at intermediate, physiologically relevant [54], membrane tensions, \\\\(\\\\lambda_{b}=0.02\\\\) pN/nm. (_Left_) Bud morphology with decreasing membrane tension, \\\\(A_{\\\\text{test}}=20,106\\\\) nm\\\\({}^{2}\\\\), \\\\(\\\\text{C}_{b}=0.02\\\\) nm\\\\({}^{2}\\\\). (_Right_) Mean curvature at the tip of the bud as a function of the membrane tension. There are two stable branches of solutions of the equilibrium membrane shape equations. The marked position on the curve denotes the membrane profile shown on the left.\\n\\n'", "CAPTION FIGMOVIES6.png": "'\\n\\n**Movie 56**.: The snap-through instability with increasing coat area at physiological tension, \\\\(\\\\lambda_{b}=0.02\\\\,\\\\mathrm{pH/nm}\\\\), is abolished when the bending rigidity of the coat is increased relative to the bare membrane, \\\\(k_{\\\\mathrm{bare}}=320\\\\,\\\\mathrm{pH}\\\\cdot\\\\mathrm{nm}\\\\), \\\\(k_{\\\\mathrm{coac}}=2400\\\\,\\\\mathrm{pH}\\\\cdot\\\\mathrm{nm}\\\\). (_Left_/_f_) Membrane profiles showing a smooth progression of bud morphologies as the area of the coat is increased, \\\\(C_{\\\\mathrm{A}}=0.02\\\\,\\\\mathrm{nm}^{-1}\\\\). (_Right_) Mean curvature at the bud tip as a function of the area of the coat. The marked position denotes the membrane profile shown at left.\\n\\n'", "CAPTION FIGMOVIES7.png": "'\\n\\n**Movie 57**.: The snap-through instability with increasing cost spontaneous curvature at physiological tension, \\\\(\\\\lambda_{b}=0.02\\\\,\\\\text{pH}/\\\\text{nm}\\\\), is abolished when the bending rigidity of the coat is increased relative to the bare membrane, \\\\(k_{\\\\text{base}}=320\\\\,\\\\text{pH}\\\\cdot\\\\text{nm}\\\\), \\\\(k_{\\\\text{cast}}=2400\\\\,\\\\text{pH}\\\\cdot\\\\text{nm}\\\\). (_Left_) Membrane profiles showing a smooth progression of bud morphologies as spontaneous curvature of the coat is increased, \\\\(A_{\\\\text{cast}}=20\\\\), \\\\(106\\\\,\\\\text{nm}^{2}\\\\). (_Right_) Mean curvature at the bud tip as a function of the spontaneous curvature of the coat. The marked position denotes the membrane profile shown at left.\\n\\n'", "CAPTION FIGMOVIES8.png": "'\\n\\n**Movie S5.** A force from actin assembly can mediate the transition from a U- to \\\\(\\\\Omega\\\\)-shaped bud, avoiding the instability at intermediate membrane tension, \\\\(\\\\lambda_{b}=0.02\\\\) mN/nm. (_Left_) at constant coat area, \\\\(A_{\\\\text{coat}}=17,593\\\\) nm\\\\({}^{2}\\\\), and spontaneous curvature, \\\\(C_{b}=0.02\\\\) nm\\\\({}^{-1}\\\\), a force (red dash) localized to the coat drives the shape transition from a U-shaped to \\\\(\\\\Omega\\\\)-shaped bud. The applied force on the membrane remains below \\\\(\\\\mathbf{f}=20\\\\) pN, well within the capability of a polymerizing actin network (_Sol_). (_Right_) A constricting force (red dash) adjacent to the coat drives the shape transition from a U-shaped to \\\\(\\\\Omega\\\\)-shaped bud, \\\\(A_{\\\\text{coat}}=17,593\\\\) nm\\\\({}^{2}\\\\), \\\\(C_{b}=0.02\\\\) nm\\\\({}^{-1}\\\\). The force intensity was homogeneously applied perpendicular to the membrane to an area of \\\\(5,027\\\\) nm\\\\({}^{2}\\\\) immediately adjacent to the coated region. The applied force on the membrane remains below \\\\(\\\\mathbf{f}=1\\\\) pH.\\n\\n'", "CAPTION FIGMOVIES9.png": "'\\n\\n**Movie 59.** Comparison of actin ring forces as implemented in this study and by Walani et al. (42), \\\\(\\\\lambda_{6}=0.2\\\\) pN/nm. (_Left_) Membrane profiles showing morphology during tubulation with constant inner and outer radii (\\\\(\\\\dot{n}_{\\\\rm inner}=100\\\\) nm and \\\\(R_{\\\\rm outer}=150\\\\) nm) for the ring of upward directed force at the base of the invagination. Force initially increases with increasing depth of the invagination but remains constant after tubulation. (_Right_) Membrane during tubulation. Rather than the actin force ring being fixed with inner and outer radii \\\\(R_{\\\\rm core}=100\\\\) nm and \\\\(R_{\\\\rm outer}=150\\\\) nm, it tracks the same area of the membrane (between \\\\(A_{\\\\rm outer}=31,416\\\\) nm\\\\({}^{2}\\\\)\\\\(A_{\\\\rm outer}=70,686\\\\) nm\\\\({}^{2}\\\\)) as it is pulled into the tube. We believe that the counterforce in Walani et al. (42) was implemented in this manner. Force initially increases with increasing depth of the invagination, decreases upon tubulation, and subsequently increases as the length of the tube increases.\\n\\n'", "CAPTION FIGS1.png": "'Figure S1: Schematic of the axisymmetric geometry adopted for the simulations as described in Section 1.3 The boundary conditions at the tip of the bud and the boundary of the patch were implemented as indicated. The optional boundary conditions (\\\\(\\\\lx@sectionsign\\\\)30) and (\\\\(\\\\lx@sectionsign\\\\)31) were used to obtain the value of applied force in actin-mediated inward-directed force (Figure S2) and constriction force (Figure S3) simulations, respectively (see Section 1.3.3.\\n\\n'", "CAPTION FIGS2.png": "'Figure S2: A hyperbolic tangent functional form was used to implement heterogeneous membrane properties. As an example, \\\\(y=\\\\frac{1}{2}\\\\left[\\\\tanh(\\\\gamma(x-3))-\\\\tanh(\\\\gamma(x-7))\\\\right]\\\\) is plotted with \\\\(\\\\gamma=20\\\\). The sharp transitions were ideal for specifying the boundaries of the coated region or regions of applied force, and the smoothness of the tanh function allowed for straightforward implementation into the numerical scheme.\\n\\n'", "CAPTION FIGS3.png": "'Figure S3: High membrane tension, \\\\(\\\\lambda_{0}=0.2\\\\,\\\\)pN/nm, \\\\(C_{0}=0.02\\\\,\\\\)nm\\\\({}^{-1}\\\\). At high membrane tension, the coat can grow arbitrarily large without causing a substantial deformation of the membrane. **(A)**\\\\(A_{\\\\rm cont}=251\\\\),\\\\(327\\\\,\\\\)nm\\\\({}^{2}\\\\), **(B)**\\\\(A_{\\\\rm cont}=1\\\\),\\\\(256\\\\),\\\\(637\\\\,\\\\)nm\\\\({}^{2}\\\\), **(C)**\\\\(A_{\\\\rm coat}=2\\\\),\\\\(513\\\\),\\\\(274\\\\,\\\\)nm\\\\({}^{2}\\\\)\\n\\n'", "CAPTION FIGS4.png": "'Figure S4: The size of the membrane patch has essentially no effect on the observed deformations of the membrane as long as it is sufficiently large. **(A)** Membrane profiles for identical coat areas and differing total patch areas. \\\\(\\\\lambda_{0}=0.002\\\\,\\\\)pN/nm, \\\\(A_{\\\\rm cost}=25,133\\\\,\\\\)nm\\\\({}^{2}\\\\), \\\\(C_{0}=0.02\\\\,\\\\)nm\\\\({}^{-1}\\\\). The deformations are identical for very large membrane patches. **(B-D)** Z-position of the bud tip, mean curvature of the bud tip, and energy to deform the membrane, respectively, as a function of the dimensionless area of the membrane patch. The deformation of the membrane is sensitive to small membrane patches, but is essentially identical beyond \\\\(\\\\alpha_{\\\\rm max}\\\\approx 200\\\\), particularly in terms of the tip mean curvature and the deformation energy.\\n\\n'", "CAPTION FIGS6.png": "'Figure S6: Mean curvature at the bud tip as a function of the area of the coat for the three different membrane tension cases. High membrane tension, \\\\(\\\\lambda_{0}=0.2\\\\,\\\\)pN/nm: The mean curvature at the bud tip drops to nearly \\\\(0\\\\,\\\\)nm\\\\({}^{-1}\\\\) as the size of the coat increases and the membrane stays essentially flat at the center of the patch (Figure 11-C). Low membrane tension, \\\\(\\\\lambda_{0}=0.002\\\\,\\\\)pN/nm: The mean curvature at the bud tip remains at approximately \\\\(0.02\\\\,\\\\)nm\\\\({}^{-1}\\\\) as the size of the coat increases and the membrane adopts the spontaneous curvature of the coat (Figure 12-F). Intermediate membrane tension, \\\\(\\\\lambda_{0}=0.002\\\\,\\\\)pN/nm: Reproduced from Figure 13. The mean curvature at the bud tip is lower for open buds (lower solution branch) relative to the low tension case, indicating that tension is inhibiting curvature generation by the coat. In contrast, the curvature is higher in the closed buds (upper solution branch) relative to closed buds in the low tension case, showing that membrane tension serves to shrink the size (and hence increase the curvature) of closed buds.\\n\\n'", "CAPTION FIGS7.png": "'Figure S7: Comparison of actin ring forces as implemented in this study and by Walani et al. [19], \\\\(\\\\lambda_{0}=0.2\\\\,\\\\)pN/nm. **(A)** Membrane profiles showing morphology before (dashed line, \\\\(Z_{p}=-100\\\\,\\\\)nm) and after (solid line, \\\\(Z_{p}=-500\\\\,\\\\)nm) tubulation with constant inner and outer radii (\\\\(R_{\\\\mathrm{inner}}=100\\\\,\\\\)nm and \\\\(R_{\\\\mathrm{outer}}=150\\\\,\\\\)nm) for the ring of upward directed force at the base of the invagination. This implementation of the actin force was used in Figures S9 and A&B. **(B)** Applied force as a function of the tip displacement. Force initially increases with increasing depth of the invagination, but remains constant after tubulation. This behavior is also observed in the absence of a ring force [19]. **(C)** Membrane morphology before (dashed line, \\\\(Z_{p}=-100\\\\,\\\\)nm) and after (solid line, \\\\(Z_{p}=-500\\\\,\\\\)nm) tubulation. The ring force is applied such that it is always applied between \\\\(A_{\\\\mathrm{inner}}=31,416\\\\,\\\\)nm\\\\({}^{2}\\\\)\\\\(A_{\\\\mathrm{outer}}=70,686\\\\,\\\\)nm\\\\({}^{2}\\\\) in the area-dependent parametrization (Section I.4). Rather than being fixed at a ring with inner and outer radii\\\\(R_{\\\\mathrm{inner}}=100\\\\,\\\\)nm and \\\\(R_{\\\\mathrm{outer}}=150\\\\,\\\\)nm, the ring tracks the same area of the membrane as it is pulled into the tube. The net effect of this is to limit the amount of membrane that can enter the tube. We believe that the counter force in Walani et al. [19] was implemented in this manner. **(D)** Applied force as a function of the tip displacement. Force initially increases with increasing depth of the invagination, decreases upon tubulation, and subsequently increases as the length of the tube increases, leading to a snapthrough instability with increasing force, as found by Walani et al. This instability is occurs independent of the membrane tension or the presence of a curvature-generating coat (data not shown). The physiological relevance of this implementation of the actin force and the snapthrough instability that results is unclear for two reasons: 1) It is unclear whether actin or other proteins at the base of the invagination would limit the ability of the membrane to flow into the invagination as implicitly assumed by this implementation, and 2) Because force from actin polymerization is a reaction force established through a Brownian ratchet mechanism, it is not clear that the snapthrough would occur, though the increase in the stress in the bilayer as the tube extends certainly could cause rupture, as hypothesized by Walani et al.\\n\\n'", "CAPTION FIGS8.png": "'Figure S8: Effect of Gaussian modulus variation on membrane budding via increasing coat area, \\\\(\\\\lambda_{0}=0.2\\\\,\\\\mathrm{pN/nm}\\\\). We define \\\\(\\\\Delta\\\\kappa_{G}\\\\equiv\\\\left(\\\\kappa_{G,\\\\mathrm{coat}}-\\\\kappa_{G,\\\\mathrm{bare}} \\\\right)/\\\\kappa_{0}\\\\) as the difference in the Gaussian modulus between the coated and bare membrane normalized by the bending modulus. **(A)** Membrane morphology in which there is no difference between the Gaussian modulus in the coated region and in the bare membrane, \\\\(A_{\\\\mathrm{coat}}=23,859\\\\,\\\\mathrm{nm}^{2}\\\\). The boundary between the coated region and the bare membrane lies at the bud neck. **(B)** Membrane morphology in which the Gaussian modulus is lower in the coated region than in the bare membrane, \\\\(A_{\\\\mathrm{coat}}=21,278\\\\,\\\\mathrm{nm}^{2}\\\\). The boundary between the coat and the bare membrane does not reach the bud neck, even for a vanishing neck radius. This is in agreement with the findings of Julicher and Lipowsky **[**20**]****. **(C)** Membrane morphology in which the Gaussian modulus is higher in the coated region than in the bare membrane (dashed line, \\\\(A_{\\\\mathrm{coat}}=21,278\\\\,\\\\mathrm{nm}^{2}\\\\); solid line, \\\\(A_{\\\\mathrm{coat}}=57,733\\\\,\\\\mathrm{nm}^{2}\\\\)). Note the difference in the axes as compared to (A) and (B). Closed buds do not form for typical ranges of coat area, though a deep U-shaped bud was present at the end of the solution branch (see (D)). This indicates that an additional solution branch of closed bud solutions may exist, but we were unable to obtain any closed bud solutions by our numerical scheme (see SOM Section **E**). **(D)** Mean curvature at the bud tip as a function of coat area for several values of \\\\(\\\\Delta\\\\kappa_{G}\\\\). The marked positions indicate the membrane profiles shown in (A), (B), and (C). Increasing the Gaussian modulus of the coated region relative to the bare membrane inhibits bud formation at typical coat areas, whereas decreasing the coat Gaussian modulus relative to the bare membrane can abolish the snapthrough transition otherwise present at intermediate membrane tension.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1.** Notation used in the model'", "CAPTION TAB2.png": "'\\n\\n**Table 2.** Parameter used in the model'", "CAPTION TAB3.png": "'\\n\\n**Table 3: Parameters used in the model**'"} \ No newline at end of file diff --git a/extracted_captions/he2015src.json b/extracted_captions/he2015src.json new file mode 100644 index 0000000000000000000000000000000000000000..ad9eb33273bacee39eadbb64d71a0abcfd9001b4 --- /dev/null +++ b/extracted_captions/he2015src.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "\"FIGURE 1: Src2 and cortactin increase the length of lamellipodia. (A\u2013E) DIC live-cell images of _Aplyxia_ growth cones in control condition (CTL; A) and expressing CASrc2 (B), MDSrc2 (C), cortactin (D), or CortF (E). (F) Quantification of lamellipodial length, measured as the distance between the T zone and the leading edge (double-headed arrow in A\u2013E). Mean values +- SEM. Numbers in parentheses are number of growth cones selected from at least three experiments. One-way ANOVA with Dunnett's post hoc test separately performed for Src2 and cortactin groups; **p < 0.01, ***p < 0.001. Scale bar as indicated.\\n\\n\"", "CAPTION FIG2.png": "'Figure 2: Src2 and cortactin positively regulate lamellipodial protrusion persistence. (A) DIC montages of 5-min time-lapse recordings at 10-s intervals showing lamellipodial leading-edge dynamics (red dashed line) in control growth cones or after expression of CASrc2, MDSrc2, cortactin, or CortF. Scale bar as indicated. (B, B\u2019) Respective protrusion and retraction frequencies of lamellipodia. (C, C\u2019) Respective average percentages of time lamellipodia spent in protrusion and retraction phases. (D, D\u2019) Respective average lamellipodial protrusion and retraction rates. Mean values +- SEM. Numbers in parentheses are number of growth cones selected from at least three experiments. One-way ANOVA with Dunnett\u2019s post hoc test was separately performed for Src2 and cortactin conditions; *_p_ < 0.05, **_p_ < 0.01, ***_p_ < 0.001.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Src2 and cortactin regulate filipodial density and stability. (A\u2013E) DIC images depicting the P domain of control (A) and CASrc2- (B), MDSrc2- (C), cortactin- (D), and CortF-expressing (E) growth cones. (F, G) DIC montages of a region of interest in the periphery of CASrc2- and CortF-expressing growth cones showing small-scale (F) and large-scale (G) lateral movements of filopodia traced with yellow, red, and green lines. Time interval between each image is 20 s; total time, 220 s. Scale bars as indicated. (H\u2013J) Quantifications of filipodial density (H) along growth cone perimeter, as well as small-scale (I) and large-scale (J) filopodial lateral movements, respectively. Small-scale lateral movements were defined as filopodia that moved sideways no further than two adjacent filopodia. Large-scale lateral movements were defined as filopodia that moved sideways further than two adjacent filopodia. Mean values \u00b1 SEM; numbers are numbers of growth cones selected from at least three experiments. One-way ANOVA with Dunnett\u2019s post hoc test was separately performed for Src2 and cortactin conditions; **_p_ < 0.01, ***_p_ < 0.001.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Src2 and cortactin facilitate protrusion of filopodia. (A) One representative filopodium is shown for each experimental condition. Filopodial length was measured from the T zone (red line) to tip of the filopodium (red dot). The position of leading edge is marked with a white line. Scale bar as indicated. (B) Quantification of average filopodial length after Src2 and cortactin up- and down-regulation, respectively. (C) Distance vs. time plots of five representative filopodial tips for each experimental condition. (D, D) Filopodial protrusion and retraction frequencies were determined as the frequency of entering into protrusion and retraction phase, respectively, from any other phases. (E, E) Percentage of time filopodia spent in protrusion or retraction phase, respectively. (F, F) Filopodial protrusion and retraction rates, respectively. Mean values +- SEM; numbers in parentheses are number of growth cones selected from at least three experiments; one-way ANOVA with Dunnett\u2019s post hoc test was separately performed for Src2 and cortactin conditions; *p < 0.05, **p < 0.01, ***p < 0.001.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: CortF abolishes CASrc2-induced lamellipodial and filopodial effects. (A) DIC live-cell image of Aplysia growth cone expressing CASrc2 and CortF; (B) Average lamellipodial length, (C) average filopodial length, (D) filopodial density, (E) fraction of filopodia undergoing small-scale lateral movement (cross two or fewer filopodia), and (F) fraction of filopodia undergoing large-scale lateral movement (cross more than two filopodia) in Aplysia growth cones expressing CASrc2, CortF, or both together compared with control (CTL) growth cones. Data are means \u00b1 SEM; numbers in parentheses are number of growth cones selected from at least three experiments. The \\\\(p\\\\) values are determined by one-way ANOVA with Dunnett\u2019s post hoc test, comparing each experimental group against a pooled control group. **p < 0.01, ***p < 0.001.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Actin dynamics in filopodia. (A) Processed actin FSM image of an _Aplysia_ neuronal growth cone. (B) Representative time-lapse montage of a single filopodial actin bundle labeled with Alexa 568\u2013G-actin. Internal speckles (dashed line) are tracked to calculate the retrograde flow rate. Similarly, the positions of newly added speckles (arrowhead) are determined to calculate the actin assembly rate. (C\u2013C\u201d) Actin assembly rates in filopodial tips in protrusion (C), retraction (C\u201d), or pausing (C\u201d) phases. (D\u2013D\u201d) Actin retrograde flow rates near filopodial tips in protrusion (D), retraction (D\u201d), or pausing (D\u201d) phases. Mean values \\\\(\\\\pm\\\\) SEM; numbers in parentheses are number of growth cones selected from at least three experiments. One-way ANOVA with Dunnett\u2019s post hoc test was separately performed for _Src2_ and cortactin conditions. No significant differences were determined between the different conditions.\\n\\n'", "CAPTION FIG7.png": "\"Figure 7: Src2 and cortactin increase the density of actin networks. (A) SEM image of control and (B) CortF-expressing growth cone. Control growth cones had regularly spaced filopodia with dense actin networks in between, whereas CortF-expressing growth cones showed fewer and curved filopodia, as well as lamellipodia that were retracted and contained disorganized actin networks of reduced density. (C) Quantification of actin filament occupancy as indicator of network density in lamellipodia with different Src and cortactin activity levels. _CASrc2_ and cortactin expression increased network density, whereas MDSrc2 and CortF expression had opposite effects. Mean values +- SEM; numbers in parentheses are number of growth cones selected from at least three experiments. One-way ANOVA with Dunnett\u2019s post hoc test separately performed for Src2 and cortactin groups; ***_p_ < 0.001. (D-H) High- magnification SEM images of actin meshwork and F-actin bundles from control and _CASrc2-_, MDSrc2-_, cortactin-, and CortF-expressing growth cones. (D'-H) Higher-magnification SEM images of actin network in the lamellipodia leading edge of control and _CASrc2-_, MDSrc2-_, cortactin-, and CortF-expressing growth cones. (I-K) Low-magnification SEM images of _CASrc2-_, CortF-, and _CASrc2_+CortF-expressing growth cones. Lamellipodial and filopodial orientation phenotypes induced by CortF were restored by _CASrc2_ coexpression. Scale bars as indicated.\\n\\n\"", "CAPTION FIG8.png": "'Figure 8: STORM imaging reveals partial colocalization of cortactin with activated Src2 and Arp2/3 complex. (A) STORM image depicting localization of activated Src2 (pSrc2; red) and cortactin (green) in an Aplysia growth cone. (B) Higher-magnification STORM image of activated Src2 and cortactin showing colocalization along filopodia and leading edge (arrowheads). (C) Clusters of cortactin labels were detected at regular intervals along a growth cone filopodia. (D) Another example of filopodium with double labeling of cortactin and activated Src2. Periodic pattern was detected in both channels, and stronger colocalization can be seen toward the tip of filopodium (right). (E) Partial colocalization of Arp3 (red) and cortactin (green) in growth cone lamellipodia and along filopodia (arrowheads). (F) Colocalization quantification of cortactin, activated Src2, or Arp2/3 complex in growth cone lamellipodia or along filopodia. Data are presented as Manders coefficient (mean values \\\\(\\\\pm\\\\) SEM). Numbers in parentheses refer to the number of growth cones analyzed in the case of lamellipodia and to the number of filopodia analyzed (13 cells). Cortactin/pSrc2 represents the ratio of cortactin signal overlapping with activated Src2 signal. pSrc2/cortactin represents the ratio of activated Src2 labeling overlapping with cortactin labeling. See Materials and Methods for details. Scale bars as indicated.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Model for Src/cortactin-mediated regulation of dynamics and morphology of growth cone lamellipodia and filopodia. Schematic depicting leading edge of lamellipodia and filopodia of neuronal growth cone in control condition (left), with up-regulated Src activation and cortactin levels (middle), and with down-regulated Src activation and cortactin tyrosine phosphorylation levels (right). 1) In lamellipodia, Src phosphorylation of cortactin facilitates Arp2/3 complex-dependent actin nucleation and branching, which results in lamellipodial protrusion. 2) Src and cortactin contribute to filopodia formation and positioning within the actin network by facilitating Arp2/3-mediated actin branch formation and stabilization. 3) In filopodia, Src phosphorylation of cortactin stabilizes bundles of actin filaments, resulting in straight filopodia. 4) Src and cortactin regulate the transition between different states of actin assembly at the plus end of filaments, thereby controlling persistence of protrusions. High rates of assembly occur during protrusion, whereas low rates of assembly occur during retraction. Up-regulation of Src activation and cortactin levels results in longer filopodia and lamellipodia, higher density of filopodia and actin network in lamellipodia, and more persistent actin-driven protrusions (middle). Down-regulation of Src activation and cortactin phosphorylation results in shorter filopodia and lamellipodia, reduced density of filopodia and actin network in lamellipodia, less persistent protrusions, and increased lateral movements of filopodia (right).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/helmsCompoundAnalogicalDesign2008.json b/extracted_captions/helmsCompoundAnalogicalDesign2008.json new file mode 100644 index 0000000000000000000000000000000000000000..8ba355d32758a7b16fb9a9a952d7d97df0c97090 --- /dev/null +++ b/extracted_captions/helmsCompoundAnalogicalDesign2008.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1. A simple case of compound solution generation: 1) presentation of the problem, 2) elaboration of the problem space, 3) retrieval of the analogue, and 4) application of the analogous solution. Repeat for each leaf in the problem decomposition (not shown).\\n\\n'", "CAPTION FIG2.png": "'Figure 2. Iterative analogical generation of a problem space: 1) presentation of the problem and retrieval of the analogue, including the solution decomposition, 2) elaboration of problem space, and retrieval of a new analogue, 3) continued elaboration of problem space.\\n\\n'", "CAPTION FIG3.png": "'Figure 3. Design Trajectory of the Eye in the Sea.\\n\\n'", "CAPTION FIG4.png": "'Figure 4. Design trajectory for the InvisiBoard.\\n\\n'", "CAPTION TAB1.png": "'Table 1. Design projects from the cognitive study.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/hoshino2019adp.json b/extracted_captions/hoshino2019adp.json new file mode 100644 index 0000000000000000000000000000000000000000..dea649eb523a1d275eb358821e27e21cce434e85 --- /dev/null +++ b/extracted_captions/hoshino2019adp.json @@ -0,0 +1 @@ +{"CAPTION EXTENDEDFIG1.png": "'\\n\\n**Extended Data Fig. 1** (**CRISPR library screening for PINK1- paraffin-mediated mitophagy. a, b,** Mitophagy was induced in paraffin- and mt-mKelima-expressing cells by treatment with the mitochondrial membrane potential uncoupler CCGP or a cocktail of suppressors of oxidative phosphorylation (OAK).\\n\\nMithophagy was analysed by flow cytometry for mt-mKelima (**a**) and western blotting for mitochondrial protein in the outer membrane (TOM20), inner membrane (ATFB), or matrix (PDH) (**b**).\\n\\n'", "CAPTION EXTENDEDFIG2.png": "\"\\n\\n**Extended Data Fig. 2 [Integration of seven mitophagy screens.****a**, GSEAof mitophagy accelerators. The top 1% of genes in aggregate _Z_-score were analysed using TopPGene Suite1n1. Representative functional categories and Bonferroni-corrected _P_values are shown. **b**, Proportion of genes encoding mitochondrial proteins annotated in MitoCart2.0*in the top 1% of mitophagy accelerators and decelerators (left), and percentage of MitoCart2.0 member genes identified as being either accelerators (green) or decelerators (red) of mitophagy (right). **c**, Five functional classes of proteins, based on MitoCart2.0 annotations, were present in the top 1% of mitophagy accelerators. The representation of each class within the top 1% was compared to its representation in MitoCart2.0*in vivo-tailed Fisher's exact test. **d**, Box-and-whisker plots of contrast significant mitophagy accelerator hits in each of the seven screens. Line, median; box; 75th-25th percentiles; whiskers (blue dots), 99th-18 percentiles. Genes involved in oxidative phosphorylation (OXPHOS) are indicated in yellow. Pathway enrichment was calculated using a Kolmogorov-Smirnov test. **e**, GSEA enrichment plot for OXPHOS (top) and ranked aggregate _Z_-scores of all genes (bottom). OXPHOS genes are indicated in yellow. **f**, GSEA of mitophagy accelerators analysed as in a. **g**-**1**, Genes in the KEGG endosomal sorting complexes required for transport (ESCRT) (**g**), homotypic fusion and vacuole protein sorting (HOPS) (**h**), and autophagosomes (**i**) pathways. Genes identified as mitophagy accelerators or decelerators are indicated in green and red, respectively. The diameter of each circle is proportional to the _Z_-score of the indicated gene. **j**, Principal component (PC) analysis isblot summarizing variation across the seven screens based on cumulative _Z_-scores of the top 100 genes, displayed as arrows. Autophagy-related genes are indicated. **k**, Mitochondrial membrane potential assessed by flow cytometry for TMRE is disrupted in CCCP treatment, but is increased after treatment with OAR cocktail. Similar results were obtained in two biological replicates.\\n\\n\"", "CAPTION EXTENDEDFIG3.png": "'\\n\\n**Extended Data Fig. 3 EssentialLC3 receptors for mitophagy in C2C12 mouse myoblasts.****a, b**, Validation as mitophagy decelerators of the indicated LC3 receptor geneRNAs, using one library gRNA and one non-library gRNA and using the m-mckemia assay (**a**) or western blotting of mitochondrial proteins in the outer membrane (TOM20 and TOM70), inner membrane (ATP8), or matrix (PPIH) (**b**): \\\\(n\\\\) = 3 biological replicates per gRNA, _P_values calculated by two-sided unpaired r-test relative to NTC_1. Mitophagic degradation of mitochondrial inner membrane and matrix proteins, but not outer membrane proteins, was blocked by gRNAtargeting _Tax1bp1_ for _Tbl1_, consistent with the notion that ubiquitinated outer membrane proteins can be degraded by the ubiquitin proteasome system. Similar results were obtained in two biological replicates. **c, d, LC3 receptor redundancy and TBK1 contribution.** **The indicated gRNAs were transduced singly or in combination by lentivirus infection, followed by analysis by flow cytometry for m-mckemia. Multiple gRNAs were superimposed on cells already targeted by _Tax1bp1_ gRNA (**d**), as indicated. \\\\(n\\\\) = 3 biological replicates per gRNA. _P_values calculated by one-way ANOVA with post hoc Tukey test, \"P_ < 0.05, \"P_ < 0.01, \"*P_ < 0.001 Data are mean +-x.d.\\n\\n'", "CAPTION EXTENDEDFIG4.png": "'\\n\\n**Extended DataFig. 4** **ANT is required in paraffin-mediated mitophagy.** A, Impaired mitophagy in the absence of ANT is confirmed by flux analysis using a lysosome inhibitor, bafilomycin A (\\\\(\\\\frac{1}{2}\\\\)M). Similar results were obtained in two biological replicates. **b**, **c**, Inhibition of mitophagy by CRISPR-mediated deletion of the indicated genes in mouse N2A (**b**) and human SH-SYSY (**c**) neuroblastoma cell lines. Representative flow tracings are shown on left, and quantification onright; \\\\(n\\\\) = 3biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired _t_-test relative to NTC, 1. Data are mean +- \\\\(s\\\\).d.\\n\\n'", "CAPTION EXTENDEDFIG5.png": "'\\n\\n**Extended Data Fig. 5**: **ANT is required for stabilization of PINK1.****a**, inhibition of ADP/ATP exchange worsens the loss of membrane potential in response to CCCP. TMR fluorescence intensity following treatment with CCCP was analysed by confocal laser scanning microscopy with the application of live time-series program. Cells were pretreated with control and boxykete acid (BA); \\\\(n\\\\) = 4 biological replicates per group. \\\\(P\\\\) values calculated by two-way repeated measures ANOVA. **b**, Mitophagy was induced by OXPHOS inhibitors (antimycin A and oligomycin) in the presence of the indicated concentration of the ADP/ATP transport inhibitor bongkeickacid, followed by flow cytometry for m-mecium. \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by one-way ANOVA with post hoc Tukey test; _\"P_ < 0.01. **c**, Genetic inhibition of ADP/ATP exchange worsens the loss of membrane potential in response to CCCP. _Ant_(_r_) cells were rescued by human wild-type (WT) or mutant ANTI; \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by two-way repeated measures ANOVA relative to NTC. **c**, ADP/ATP exchange rates is impaired in ADP/ATP-binding mutants (K330, K43E), but not in disease-causing mutants (A90D, V289M); \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by two-sided unpaired test relative to WT. **e**, Loss of ANT impairs PINK1-dependent mitophagy induced by oxidative stress, but does not impair PINK1-independent mitophagy caused by hypoxia or starvation; \\\\(n\\\\) = 3 biological replicates per gRNA. \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to NTC. \\\\(E\\\\), PINK1 accumulation in mitochondria is impaired in cells lacking ANT. Cells bearing gRNAs targeting the indicated genes were transduced with PINK1-GFP, followed by treatment with CCCP versus control, and then immunostained using anti-TOM20 antibody (red). GFP fluorescence is shown in green, and merged signal yellow. Scale bar, 20 \u03bcm. \\\\(g\\\\), PINK1 stabilization by CCCP treatment is preserved with wild-type ANT and ADP/ATP-binding double mutant (K43E/B24E), but not in known disease-causing mutants (A90D, A123D). **h**, Phosphorylation of PINK1 after CCCP treatment is preserved in the absence of ANT1 or ANTI. **l**, PINK1 transcription (b) and translation (j) are not changed by loss of ANT. **n** = 4 biological replicates per group. \\\\(P\\\\) values calculated by one-way ANOVA. **k**, The activities of the PINK1-cleaving proteases PARL and OMA1 are not changed by loss of ANT. **l**, General autophagy fluxis preserved in the absence of ANT. **n** = 3 biological replicates per group, \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to NTC. **m**, Suppression of TIM23-mediated protein translocation response to CCCP treatment is impaired in the absence of ANT1 or ANTI. **m**, as shown by import of SU9-GFP into intact cells; \\\\(n\\\\) = 3 biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to NTC. Scale bar, 20 \u03bcm. Data are mean +- s.d. Similar results were obtained in two biological replicates (**f** - **h**, **j**, **k**). For gel source data, see Supplementary Fig. 1.\\n\\n'", "CAPTION EXTENDEDFIG6.png": "'\\n\\n**Extended Data Fig. 6**: **ANT mediates closure of TIM23 via TIM44.4**, Deletion of _Ant1_ or _Ant2_ does not affect expression of TIM or TOM proteins (right) or destabilize TIM and TOM complexes, as assessed by blue native PAGE (left). **b**, **c**, ANTI and ANTI2 bind to TIM23 and TIMM44, as assessed by corimmunoprecipitation (**b**) and blue native PAGE (**c**). The ANTI-TIM23 complex is marked with an asterisk asterisk. **d**, Wild-type ANTI and the ADP/ATP binding double mutant (K43/K2445) bind to the TIM23 complex component TIM23, whereas disease-causing mutants (A990D, A123D) do not. **c**, Closure of TIM23in response to CCCP treatment is impaired in the presence of disease-causing mutants (A90D, A123D), but is preserved in the presence of the ADP/ATP binding double mutant (K43E/R244E), as shown by import of SU9-GFP into mitochondria: \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to empty. Scale bar, 40 um. **f**, ANTI binds to both TIMM23 and TIMM44. **g**, Mittophagys impaired in cells lacking TIMM44; \\\\(n\\\\) = 3 biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to MC, **l**, **b**, PINI stabilization by CCCP treatment is abrogated in the absence of TIMM44. **l**, Rescue of mitophagy with wild-type ANT and ADP/ATP exchange mutants (K330, K43E/R244E), but not with known disease-causing mutants (A90D, A123D) and TIMM44-binding site mutant (G146E/K147D). Top left, schematic of ANT and sites of mutations. Bottom, western blotting demonstrating equivalent expression of ANT constructs. Right, quantification of mitophagy: \\\\(n\\\\) = 3 biological replicates per group, \\\\(P\\\\) values calculated by one-way ANOVA with post the Tukey test, \"_p_ < 0.01, \"_p_ < 0.001. **J**, Mutation of the predicted ANTI interactions site in TIMM44 abrogates binding to ANTI. **k**, Rescue of mitophagy with wild-type TIMM44, but not with binding site mutant (K43E/R244E); \\\\(n\\\\) = 3 biological replicates per group, \\\\(P\\\\) values calculated by one-way ANOVA with post hoc Tukey test, \"_p_ < 0.01, \"_p_ < 0.001. **I**, Mutation in TIMM44 of the ANTI interaction site does not abrogate TIMM44 binding to TIMM23. **Dataera** mean = s. d. Similar results were obtained in two biological replicates (**a**\u2013**d**, **f**, **b**\u2013**l**). For gel source data, see Supplementary Fig. 1.\\n\\n'", "CAPTION EXTENDEDFIG7.png": "'\\n\\n**Extended Data Fig. 7** **ANT is required for mitophagy in vivo, independently of transcriptional regulation.** Equivalent amounts of PINK1 and higher parkins transcription in _Antf_20 heart and skeletal muscle (SM); \\\\(n\\\\) = 4 per group, _P_values calculated by two-sided unpaired _k_-test. Data are mean +s.d.\\n\\n'", "CAPTION FIG1.png": "'relative to non-targeting control;NTC) or by western blotting of mitochondrial proteins in the outer membrane (OMM-TOM20), inner membrane (IMM-ATPB), or matrix (PDH) (**e**). Similar results were obtained in two biological replicates. For gel source data, see Supplementary Fig. 1.**f**, Suppression of mitophagy in primary ratneurons. Left, visualization of neuronal mitochondria with tetramethylrhodamine ethylester (TMRF) dye. Middle, representative image showing coating of mitochondria (labelled with Mirc-Snap) with the mitophagy receptor OPTN, indicating active mitophagy. Right, quantification of cells undergoing active mitophagy: \\\\(n\\\\) = 6 (untreated control), 6 (treated control), 4 (_Ant1_), and 5biological replicates (_Ant2_), \\\\(P\\\\) values calculated by one-way ANOVA with post hoc Dunnett\\'s multiple comparison test; \\\\(P\\\\) < 0.05, \"_P_ < 0.01. Scale bars, 5 \u03bcm, 0.8 \u03bcm (inset). Data are mean +-s.d.\\n\\nFig. 1: **Multidimensional mitophagy screen reveals that ANT is required for mitophagy.****a**. Outline of CRISPR\u2013Cas9 genome-wide genetic screen, using four reporter assays and two modes of mitophagy induction. **b**, Most significant hits in each of the seven screens. MT, MitoTracker; OM-G, outer membrane GFP; Mat-G, matrix GFP. Representative previously known genes shown by open symbols, previously unknown genes by coloured symbols; line, median; box, 75th\u201325th percentiles; \u2018whiskering\u2019, 99th\u20131st percentiles; duplicate experiments. **c**, Ranked aggregate _z_\u2019scores of all genes. Representative previously known genes labelled in grey, previously unknown in black. **d**, **e**, Validation as mitophagy decelerators of the indicated genes, using both a gRNA chosen from the screening library, and an independent non-library gRNA, followed by flow cytometry for matrix-targeted mkcima (**d**, \\\\(n\\\\) = 3 biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired _d_-test'", "CAPTION FIG2.png": "'Fig. 2 **ANT mediates suppression of TIM23 via TIM44.****a**, Inhibition of ADP/ATP transport with the indicated inhibitors accelerates mitophagy, in sharp contrast to genetic deletion of ANTI (bottom right); \\\\(n\\\\) = 3 per group., Mitochondrial admi is elevated in cells lacking ANT, and reduced in response to CCCP, compared to control cells, consistent with reverse ADP/ATP exchange in low admi: \\\\(n\\\\) = 3 per gRNA. \\\\(P\\\\) values calculated by two-way repeated measures ANOVA relative to NTC. **c**, Rescue of mitophagy with wild type (WT) ANT and ADP/ATP-binding mutant (K43k/R244K), but not with disease-causing mutants (A90D, A123D). Top left, schematic of ANT mutations. Bottom, equivalent expression of constructs. Right, quantification of mitophagy; \\\\(n\\\\) = 3 per group. **d**, PINK1 stabilization by CCCP treatment is abrogated in the absence of ANTI or 2. **e**, Suppression of TIM23-mediated import of [**15**S]Sup-DHFR into isolated mitochondria in response to CCCP is impaired in the absence of ANTI (p., precursor; m, mature). **f**, Interaction between ANTI and TIM44 identified in the mitochondrial interactome database XlinkDB. **g**, Deletion of TIMM44 impairs binding of ANTI to TIMM23. **h**, Mutation of the predicted TIMM44 interaction site in ANTI or disease-causing ANTI mutation (A90D) abrogates binding of ANTI to TIMM44, while ADP/ATP exchange mutants (K43K/R244E and K330) do not. **i**, Model of ANTI-mediated suppression of TIM23 in response to compromise of mitochondrial bioenergetics. Data are mean +- \\\\(x\\\\) < .d_.\\\\(P\\\\) values by one-way ANOVA with post hoc Tukey test (**a**, **c**); \\\\(p\\\\) < 0.05, \"_p_ < 0.01, \"_p_ < 0.001. Similar results were obtained in two biological replicates. For gel source data, see Supplementary Fig. 1(**c**\u2013**e**, **g**, **h**).\\n\\n'", "CAPTION FIG3.png": "'_Ant_20 mice. Scale bars, 2 \u03bcm. Similar results were obtained from three mice in each group. B, Dilated cardiomyopathy in _Ant_20 mice: heart weights (top left), sample images (top right) and quantification (bottom) of echocardiography; \\\\(n\\\\) = 7 per group. LVID, left ventricle internal diameter systolic (s) and diastolic (d); LVDW, left ventricle posterior wall; EF, ejection fraction. **1**. Echocardiography of patient bearing homozygous loss-of-function. _ANTI_ mutations. Left, systolic; right, diastole, **3**. Electron micrographs of endomyocardial biopsy from same patient as in L. Localized distension of outer membrane (blue arrows) with apparent release of mitochondrial matrix content (green) into the cytoplasm. Scale bars, 1 \u03bcm. Data are mean +-s.d. (**a**, **e**). _P_values by two-sided unpaired \\\\(t\\\\) test (**a**, **d**, **h**), one-way ANOVA with post hoc Tukey test (**b**); \\\\(P\\\\) < 0.05, \"_P_ < 0.01, \"_P_ < 0.001. For gel source data, see Supplementary Fig. 1.\\n\\nFig. 3: **ANTI is required for mitophagy in vivo.****a**, Blunted mitophagy in heart and brain of _Ant_20 mice, illustrate at by reduced coating of mitochondria by p62, PINK1 and parin. **b**, Blunted mitophagy in skeletal muscle of _Ant_20 mice, shown by intramuscular transfection of mitoQC plasmid. Line, mean; \\\\(n\\\\) = 4 mice, 47 fibres (WT) and 4 mice, 50 fibres (_Ant_20). Scale bars, 10 \u03bcm. **c**, Rescue or mitophagy with wild type (WT) ANTI and mutant lacking mCherry exchange activity (K3IQ), but not with disease-causing mutant (A900). Line, mean; \\\\(n\\\\) = 3 mice, 24 fibres (WT), 3 mice, 16 fibres (Luc), 3 mice, 23 fibres (k33Q) and 3 mice, 21 fibres (A90D). Scale bars, 10 \u03bcm. **d**, Accumulation of mitochondrial DNA (right) in heart (top) and muscle (bottom) of _Ant_20 mice despite absence of nuclear-encoded biogenesis (left): \\\\(n\\\\) = 4 per group. **e\u2013g**. Accumulation of mitochondrial proteins (**e**) and of disorganized and aberrant mitochondria (**f**) in heart, and deep red colouring of mitochondria in skeletal muscle (**g**) of _Ant_20 mice. Scale bars, 2 \u03bcm. Similar results were obtained from three mice in each group. **b**, Dilated cardiomyopathy in _Ant_20 mice: heart weights (top left), sample images (top right) and quantification (bottom) of echocardiography; \\\\(n\\\\) = 7 per group. LVID, left ventricle internal diameter systolic (s) and diastolic (d); LVDW, left ventricle posterior wall; EF, ejection fraction. **1**. Echocardiography of patient bearing homozygous loss-of-function. _ANTI_ mutations. Left, systolic; right, diastole, **3**. Electron micrographs of endomyocardial biopsy from same patient as in L. Localized distension of outer membrane (blue arrows) with apparent release of mitochondrial matrix content (green) into the cytoplasm. Scale bars, 1 \u03bcm. Data are mean +-s.d. (**a**, **e**). _P_values by two-sided unpaired \\\\(t\\\\) test (**a**, **d**, **h**), one-way ANOVA with post hoc Tukey test (**b**); \\\\(P\\\\) < 0.05, \"_P_ < 0.01, \"_P_ < 0.001. For gel source data, see Supplementary Fig. 1.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/irwinForecastingArgumentationFrameworks2022.json b/extracted_captions/irwinForecastingArgumentationFrameworks2022.json new file mode 100644 index 0000000000000000000000000000000000000000..2cfbff1c714a025be2ff96a9b8cd94fbefc998c1 --- /dev/null +++ b/extracted_captions/irwinForecastingArgumentationFrameworks2022.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: The step-by-step process of a FAF over its lifetime.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: A graphical representation of arguments and relations in the update framework from Example 3. Nodes represent proposal (\\\\(\\\\mathcal{P}\\\\)), increase (\\\\(\\\\uparrow\\\\)), decrease (\\\\(\\\\downarrow\\\\)), pro (\\\\(+\\\\)) and con (\\\\(-\\\\)) arguments, while dashed/solid edges indicate, resp., the \\\\(\\\\mathcal{R}^{\\\\mathcal{P}}\\\\mathcal{R}\\\\) relations.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The structure of the \\\\(\\\\mathbb{R}^{3}\\\\)-structure of the \\\\(\\\\mathbb{R}^{3}\\\\)-structure. _Journal of Computational Physics_, 123(1):1-12, 1994.\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 3: Auxiliary results from the experiment, where \\\\(\\\\overline{C}\\\\) is the average confidence score, \u2018Forecasts\u2019 is number of forecasts made in each question and \u2018Irrational Forecasts\u2019 the number in each question which violated each constraint in \u00a74.1.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/isambert1995flexibility.json b/extracted_captions/isambert1995flexibility.json new file mode 100644 index 0000000000000000000000000000000000000000..5c64ebdf8f4e6edc4ef9585b2b14b986c476e726 --- /dev/null +++ b/extracted_captions/isambert1995flexibility.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: **Schematic representation of thermal fluctuations of an actin filament.**_A_, transverse fluctuation. For each point of abscissa \\\\(S\\\\), the distance \\\\(D\\\\) from the tangent at the origin is measured on a series of images. Two typical shapes, \\\\(I\\\\) and \\\\(\\\\hat{z}\\\\), are shown. Note that \\\\(D\\\\) varies appreciably while the longitudinal distance \\\\(r\\\\) does not vary much. \\\\(B\\\\), correlation of the tangential directions. For each point of abscissa \\\\(S\\\\), the angle \\\\(\\\\theta s\\\\)) of the tangent with the tangent at the origin is measured.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2. Recorded shapes of actin filaments undergoing thermal fluctuations.**_a_-_cf_, tetramethylrhodamine-phalloidin-stabilized actin filaments observed at 6-a time intervals (left to right), bar = 5 \\\\(\\\\mu\\\\)m. c-A, unstabilized tetramethylrhodamine-F-ADP-actin filaments observed in physiological ionic conditions at 6-a intervals in the presence of 0.2 mM ATP and 0.1 \\\\(\\\\mu\\\\)m unlabeled G-actin.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3. Analysis of filament flexibility.**\\\\(a\\\\), cosine correlation function. Dashed lines, rhodamine-phalloidin-stabilized F-ADP-actin (conditions as under Fig. 2, \\\\(a\\\\)-\\\\(d\\\\)). Solid lines, rhodamine-F-ADP-actin in the absence of phalloidin (conditions as under Fig. 2, \\\\(e\\\\)-\\\\(h\\\\)). \\\\(b\\\\), analysis of the average transverse fluctuations. Solid and dashed lines correspond to the data shown in \\\\(a\\\\). Nocay curves are experimental data (dashed line, average of 268 filaments; solid line, average of 110 filaments). Smooth curves represent the best fits to Equations 3 (\\\\(a\\\\)) and 7 (\\\\(b\\\\)), respectively.\\n\\n'", "CAPTION TAB1.png": "''"} \ No newline at end of file diff --git a/extracted_captions/jeppesenMarginalityProblemsolvingEffectiveness2010.json b/extracted_captions/jeppesenMarginalityProblemsolvingEffectiveness2010.json new file mode 100644 index 0000000000000000000000000000000000000000..a2d1e79231a3eef43be27a330ad80c15927a3689 --- /dev/null +++ b/extracted_captions/jeppesenMarginalityProblemsolvingEffectiveness2010.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1** **Broadcast Search Procedure**'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 1** & **Contracts** (Mean Based) **Between Survey** \\\\\\\\\\n**Respondents and Nonexpondents in Submitter** \\\\\\\\\\n**Population** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Contracts (Mean Based) Between Survey Respondents and Nonexpondents in Submitter Population'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l l l} \\\\hline \\\\hline & & & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Descriptive Statistics for Variables in First Stage of Heckman Probit Regression (\\\\(N=12,786\\\\))'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/extracted_captions/jingHouseholdSecondaryAttack2020.json b/extracted_captions/jingHouseholdSecondaryAttack2020.json new file mode 100644 index 0000000000000000000000000000000000000000..32402b15f1a0d0841c1feeb1b79eda9c19d029da --- /dev/null +++ b/extracted_captions/jingHouseholdSecondaryAttack2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: **Spatial distribution of COVID-19 case clusters on the basis of contact tracing data from Guangzhou, China, from Jan 7, 2020, to Feb 18, 2020**\\n\\nOverall distribution of COVID-19 case clusters in Guangzhou (A), and distribution in the submissions defined in panel A (B-G). Individuals were considered as primary cases if their symptom onset dates were the earliest or 1 day (14) days for an imported case) after the earliest in the cluster and as secondary cases otherwise. Non-infected contacts are not shown. The displayed location of such case is randomly perturbed away from the actual residential address.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Figure 2:** Epidemic curve based on symptom onset dates of COVID-19 crisis in Guangzhou from Jan &, 2020, to Feb 18, 2020\\n\\n**Estimated \\\\(R\\\\), for those scenarios: scenario 1, all imported cities (who traveled to or resided in Huisei province 14 days before symptom onset) considered as primary cities, and all secondary cities were infected by primary cases in the same case district; scenario 2, which is identical to scenario 1, with the additional assumption that local primary cases might have been infected by earlier cases in other clusters; and scenario 3, which is identical to scenario 2, with the additional assumption that imported secondary cases were considered as infected by primary cases in the same cluster. \\\\(R\\\\)=effective reproductive number.**'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB2.png": "'Estimates were reported using two-different definitions of household contact (cken r luthen or individuals sharing the same rouletental address) and for selected settings of the natural history of disease. This model was not adjusted for age group, epidermis phase, or household size. E-response-active number.\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l l l} \\\\hline \\\\hline & & & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 2: **Model-based estimates of secondary attack rates among household and non-household contacts, and local R with and without quarantine**'", "CAPTION TAB3.png": "'Data are estimating (95% C). Estimation of the daily probability of infection from an external source and the GHR for the relative infectivity during the disease versus irradiation period are also provided. Extrusion as reported using two different definitions of household contact (dose relation, or only individual sharing the same residential address) and for selected setting of the natural history of disease (no mean incubation and maximum infectious period). This model was not adjusted for age group, epidemic phase, or household use. D6-odds ratio.\\n\\n'", "CAPTION TAB4.png": "'Data are 00 [95% CI]. Initration were repeated for selected settings of the natural history of disease (in, mean incubation and maximum infectious periods). The model was adjusted for age groups, epidemic phase, and household size. (Q1=odds ratio).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/kaneCommunitiesWeChoose2018.json b/extracted_captions/kaneCommunitiesWeChoose2018.json new file mode 100644 index 0000000000000000000000000000000000000000..a592fa5454d0f35ac614e63b58b67e28f8786130 --- /dev/null +++ b/extracted_captions/kaneCommunitiesWeChoose2018.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: The anatomy of a typical Reddit comamet section.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Average UMass topic coherence at each \\\\(\\\\$\\\\)-topic increment in number of topics.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG4.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB1.png": "'TABLE I\\n\\nA sample of far-IRASEN PHASES used by candidates'", "CAPTION TAB2.png": "'\\n\\n## Table II A sample of bicrams and trigrams found in each political volume\\n\\n'", "CAPTION TAB3.png": "'\\n\\n**Acknowledgement**'"} \ No newline at end of file diff --git a/extracted_captions/kaplanSearchInsight1990.json b/extracted_captions/kaplanSearchInsight1990.json new file mode 100644 index 0000000000000000000000000000000000000000..b4425c8e8740777db9d509d772b40a270808165f --- /dev/null +++ b/extracted_captions/kaplanSearchInsight1990.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1.** The classic mutilated checkerboard (MC) problem.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fits 1.** **Experiment 3 at a time.**'", "CAPTION FIG3.png": "'\\n\\n**Fig. 1. The AHAI experience (three protocol excerpts).**'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4. A sample production from switch.**'", "CAPTION FIG5.png": "'\\n\\n**Eto - A prototypical solution path.**'", "CAPTION FIG6.png": "'Figure 6: Frequencies of different types of invariants (first 10 min).\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Number and type of invariants noticed in first and second halves of protocol.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8.** Categories covered (first \\\\(\\\\xi\\\\) min).\\n\\n'", "CAPTION TAB1.png": "'TABLE I\\n\\nSome Frablem Spaces Used by Subsets'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB11.png": "'TABLE 11\\n\\nRank Orders of Times to First Mention Perceptual Invariant and to solution (\\\\(n=8\\\\) Salje\\\\(\\\\cdot\\\\)ts)'", "CAPTION TAB2.png": "'TABLE 2\\n\\nBreakdown of Time Spent after First Mention of Parity, before Rough Proof'", "CAPTION TAB3.png": "'TABLE 3\\n\\nMean Times to Points on Solution Path and Mean Number of Approaches Tried.\\n\\n'", "CAPTION TAB4.png": "'TABLE 4\\n\\nPairwise Co'", "CAPTION TAB5-1.png": "'TABLE 3\\n\\nMean Number of Relevant Parity Statements before/after Hints'", "CAPTION TAB5-2.png": "'\\n\\n## Chapter 4 Conclusion'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l c c} & \\\\(\\\\pi=8\\\\) Subjects. \\\\\\\\ Mean Number of Relevant Parity Statements before/after Hints & Mean Number of Relevant Parity Statements before/after Hints \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 5: Mean Number of Relevant Parity Statements before/after Hints'", "CAPTION TAB6-1.png": "'TABLE 6\\n\\nNumber of Subjects Solving after Various Hints (No. of Subjects in Each Group Who Re-ceived Each Hint; \\\\(n\\\\) = 23 Subjects)'", "CAPTION TAB6-2.png": "'* Other hints included the COUNT hint, and other very specific hints which were given only if the PARITY hint proved ineffective.\\n\\n'", "CAPTION TAB6.png": "'\\\\(\\\\bullet\\\\)\\n\\n\\\\(\\\\bullet\\\\)\\n'", "CAPTION TAB7-1.png": "'\\n\\n## Chapter 1 Introduction\\n\\nThe Standard Model (SM) is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics, which is a model of particle physics. The SM is a model of particle physics, which is a model of particle physics,'", "CAPTION TAB7-2.png": "'* \\\\(p\\\\leq 10\\\\), **\\\\(p\\\\leq .01\\\\) (one failed \\\\(r\\\\) test). Difference compared with BI_ANK group.\\n\\n'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l c c c c} \\\\hline \\\\hline & & & & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 7: * \\\\(p<10\\\\), **\\\\(p<11\\\\)** (one failed \\\\(t\\\\) test). Differences compared with BLANK group.\\n\\n'", "CAPTION TAB8.png": "'TABLE 8\\n\\nOverall Frequency of Use (and Rate of Use) of Heuristics (\\\\(n=8\\\\) Subjects)'", "CAPTION TAB9-1.png": "'TABLE 9\\n\\nInvariant Properties Mentioned Repeatedly by Subjects (\\\\(\\\\alpha=8\\\\) Subjects)'", "CAPTION TAB9-2.png": "'_Note_. Listed first are all the invariants on the direct path to the insightful parity solution (i.e. the relevant invariants). Listed second are some common invariants that are not on the direct path to the insightful parity solution (i.e. the irrelevant invariants).\\n\\n'", "CAPTION TAB9.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l c c} \\\\hline \\\\hline & & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 9: Isoperimetric Method Revisited by Subjects (\\\\(n=8\\\\) Subjects) Invariant Properties Mentioned Repeoriately by Subjects (\\\\(n=8\\\\) Subjects)'"} \ No newline at end of file diff --git a/extracted_captions/kibetSociallyNetworkedHeterogeneity2019.json b/extracted_captions/kibetSociallyNetworkedHeterogeneity2019.json new file mode 100644 index 0000000000000000000000000000000000000000..4078cb84fd31a1fdfcf5584f576aa0167bef16e6 --- /dev/null +++ b/extracted_captions/kibetSociallyNetworkedHeterogeneity2019.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & WhatsApp and social network heterogeneity: Posting, discussing and commenting \\\\\\\\ & as mediating factors \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: WhatsApp and social network heterogeneity: Posting, discussing and commenting as mediating factors'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 3:**} & \\\\multicolumn{1}{c}{Regression analysis on use of WhatsApp, social networking heterogeneity and polarisation} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 3: Regression analysis on use of WhatsApp, social networking heterogeneity and polarisation'"} \ No newline at end of file diff --git a/extracted_captions/kirkham2005ultrastructural.json b/extracted_captions/kirkham2005ultrastructural.json new file mode 100644 index 0000000000000000000000000000000000000000..05d089ba3706e3b8b39e7c2096ca34a8099fa9f5 --- /dev/null +++ b/extracted_captions/kirkham2005ultrastructural.json @@ -0,0 +1 @@ +{"CAPTION 1.png": "'Figure 1: **Ultrastructural characterization of caveolae endocytosis.** (A and B) WT MEFs with bound CTBHRP were warmed for 1 [A] or 5 _min_ [B]. The DAB fraction was performed on ice in the presence [+AA] or absence [-AA] of AA, and then cells were processed for immunoelectron microscopy detection of Cow1. A and B are representative of areas enriched in caveolae. (A) DAB reaction product is present in a small subset of buddingal (inoxarloc-connected). Cavi-positive caveolae (arrows) in cells treated with AA during the DAB reaction. A large number of caveolae were DAB negative and thus surface-connected [small arrowheads]. Putative budded clathrin vesicles were also observed (large arrowhead). (B) After 5 min of CTBHRP uptake, Cow1 also localized to DAB-positive internal structures [+AA], which had an irregular tubular morphology [defined operationally as clustered caveolae in E-G]. (C) WT MEFs with CTBHRP internalized for either 1 or 5 min were treated with DAB in the presence of AA, labelled for Cav1, and processed for whole-mount visualization. Budden (DAB-positively caveolae [arrowhead], surface-connected [DAB-negative]) caveolae (arrowhead), and internal Cav1-positive larger structures were observed. (D) WT MEFs were treated as in A but warmed for 5 min in the presence or absence of either DA or locCar before the DAB reaction in the presence of AA. (E\u2013G) Quantitation of internal caveolae (single or clustered) as compared with the total number of caveolae present in the same area with increasing time of incubation at 37\u00b0C (E) or after 5 min at 37\u00b0C in the presence/absence of OA (F) or locCar (G). Error bars indicate standard error. Bars: (A and B) 500 nm; (C and D) 200 nm.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Figure 2.** **Inernalization of CTB by WT and Cav1\\\\(-\\\\)/ - MEFs.** {A-F} CTB-FITC was internalized in WT MEFs {A-F} CTB-FITC was internalized in WT MEFs {A-F} CTB-FITC was internalized in WT MEFs {C} at 37degC for various times, and then fixed and labeled for GM130 {A-F} CTB-FITC was accumulation of CTB within the Golgi complex in both WT MEFs and Cav1\\\\(-\\\\)/ - MEFs was quantified {C}; no significant differences were seen. Bars indicate standard error. Cav1\\\\(-\\\\)/ - MEFs were microinjected with pIRES-Cav1 using standard conditions {B and E} and short expression conditions {B and E}. {B} Injected calls {*} were marked by the production of cytosolic GFP coded for by the same message as untagged Cav1. CTB-Alexa Fluor 594 was internalized and CTB fluorescence intensity in the GM130-positive region was quantified {D-F}. WT MEFs were microinjected with pIRES-Cav1 using short expression conditions {F}. Only short expression of Cav1 reduced CTB trafficking to the Golgi complex as compared with nonexpreasing cells in both WT and Cav1\\\\(-\\\\)/ - MEFs (D-F). [G] Cav1\\\\(-\\\\)/ - MEFs were microinjected with vectors encoding for EPS15-DN-GFP and TIR for short expression. CTB-FITC was internalized simultaneously with TT for 40 min. CTB-FITC was labeled with an anti-CT antibody and a secondary antibody conjugated to Alexa Fluor 660. In cells that were injected with EPS15-DN-GFP, Tf uptake was inhibited, but CTB still accumulated in a perinuclear compartment. [H] Cav1\\\\(-\\\\)/ - MEFs were microinjected with vectors encoding TIR and either GFP or EPS15-DN-GFP [short expression], and then Tf uptake for 20 min was performed. Internal Tf was quantified in injected cells, and EPS15-DN-GFP inhibited Tf uptake by 90%. [I] Cav1\\\\(-\\\\)/ - MEFs were microinjected with vectors encoding for EPS15-DN-GFP [short expression], and CTB-Alexa Fluor 594 was internalized for various times. Quantification of CTB accumulation in the GM130-positive Golgi complex revealed that EPS15-DN-GFP reduced CTB trafficking by only 40%. Bars indicate standard error; *, P < 0.01; **, P < 0.001. Bars, 25 um.**'", "CAPTION FIG3.png": "'Figure 3. **Dyeamin and ARf4 mutants inhibit CTH-transpert to the Golgi complex.**_Cav1-f-MEFs_ were micrinjected with cDNA for DynKA4A4A [A] or ARFA T27N [B and C; short expression]. CTB Alexa Floc 594 was internalized for 40 nm [A and B. DynKA4A4A and A&F6 T27N heat contained NH2-terminal HA tags were labeled with eachHA [A and B]. CTB colocalizes with DynKA4A [A] and A&F6 T27N [B] in an extensive tubular network. This is highlighted in enlargements in between panels of A. [C] Cav1-f-MEFs were micrinjected with A&F6 T27N and HRF, and CTHHRF was internalized for 40 min at 37C. Cells were prepared for EM and CTHHRF was revealed by DNA cytochanatry. CTHHRF reacted product was evident within a network of 40-nm-diameter tubular structures in the injected cells, some of which connect to multi-axial regions [arawheads]. Bars [A, top, and B] 25 mm; [A, bottom] 5 mm; [C, left] 1 mm; [C, right] 200 mm.\\n\\n'", "CAPTION FIG4.png": "'Figure 4. Ultrastructural characterization of early carriers in Cav1\\\\(-\\\\)/\\\\(-\\\\) MEFs. Cav1\\\\(-\\\\)/\\\\(-\\\\) MEFs were incubated with CTB+RP [A and B] or TH+RP [C] at 4\\\\({}^{\\\\circ}\\\\)C. They were warmed for 15 s at 37\\\\({}^{\\\\circ}\\\\)C and DAB treated in the presence of AA [A and C] or were DAB treated in the absence of AA without a warming step to reveal the surface distribution of CTB+RP [B]. All samples were fixed and processed for EM without permabilization. [A] CTB+RP reaction product is evident within vesicular profiles [arrows] and tubular/rings-shaped profiles [arrows] close to the plasma membrane [PM]. Note the groups of labeled structures as most clearly evident in the low magnification serum [log]. [B] CTB+RP reaction product is evident over the entire local surface but tubular/rings-shaped profiles of similar morphology to those detached from the surface after warming are evident [arrows]. Also note the labeling of vesicular profiles connected to the call surface [arrows]. [C] in contrast to the structures labeled by CTB+RP after 15 s, TH+RP labels vesicular profiles [arrows] close to the PM. Bars, 200 nm.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Ultrasructural characterization of early noncaveolar carriers in Cav1\\\\(-\\\\)/\\\\(-\\\\) MFs and WT MFs.** WT (A, B, and E) and Cav1\\\\(-\\\\)/\\\\(-\\\\) (D and E) MEFs with bound CTBHRP were warmed for 15 s, 1 min, or 5 min at 37\u00b0C and DAB treated in the presence of AA. Samples were immunolabeled for Cav1 (A\u2013C) or dynamic (clathrin-pit panels) and processed for EM. In both WT (A) and Cav1\\\\(-\\\\)/\\\\(-\\\\) (D) MEFs, CTBHRP reaction product was observed after 15 s, 1 min, and 5 min in tubular structures, ringlike structures, clathrin coated vesicles, and smaller vesicular structures. [B] in WT MFs, CTBHRP-positive noncaveolar structures partly colocalized with Cav1. The percentage of noncaveolar structures labeled with Cav1 was quantified [F. (C)] WT MFs were grown on grids, and CTBHRP was internalized for 5 min. Calls were prepared for whole-mount visualization. CTBHRP labeled short tubular or ringshaped structures, some of which are Cav1 positive. [E] thermal CTBHRP-positive structures in WT and Cav1\\\\(-\\\\)/\\\\(-\\\\) MEFs was classified according to morphology (see Materials and methods) and quantitated as a percentage of total labeled structures. Most CTBHRP-labeled internal structures in both WT and Cav1\\\\(-\\\\)/\\\\(-\\\\) MEFs were nonvascular. Note that visualized structures of <70 nm diamond included caveolae. Bars, 200 nm.\\n\\n'", "CAPTION FIG6.png": "'Figure 6. Analysis of update of CTB, fluid phase meters, and GP-AB. (A) WT MEF, were re-selected with 10 mg/ml HBP as a fluid phase meter for 15 s, and then QAB tranded in the presence of AA betare being processed for EAA. Tue\\n\\n'", "SUPP CAPTION FIGS2.png": "'Figure S2: **Ultrastructural analysis of the nova-formed envelope.** To confirm that the transient expression of Cav1 in Cav1 -/- MEFs can cause the de novo formation of caveolue, Cav1 was expressed in Cav1 -/- MEFs using standard expression conditions. Cav1 -/- MEFs were microinjected with pRERS-Caw1 vector [100-150 mg/ml] together with HRP (which labels only the cell nucleus), incubated for 18\u201320 h at 37\u00b0C, and then CTBHRP was internalized for 40 min at 37\u00b0C. Cells were then fixed and processed for EM and CTBHRP was revealed by DAB. De novo formation of CTBHRP-positive caveolue was seen at the surface of injected cels. Bars, 200 nm.\\n\\n'", "SUPP CAPTION FIGS3-1.png": "'Figure 51. Cell update in Cav1-1-10\\n\\n'", "SUPP CAPTION FIGS3-2.png": "'there was no significant difference between treated and control cells. [C] CTB Alexa Fluor 594 was internalized for 40 min using the same conditions as in \\\\(\\\\AA\\\\). In untreated cells, CTB strongly colocalized with GM130. In cholesterol-depleted cells. This colocalization was reduced as shown in the quantitation in D. P \\\\(<\\\\) 1 \\\\(\\\\times\\\\) 1Q\\\\({}^{-T}\\\\); bars indicate standard error. Bars, 25.\\n\\n'", "SUPP CAPTION FIGS3.png": "'Figure S3. **CIB uptake in Cav1/\\\\(-\\\\)/MEFs is partly sensitive to cholesterol depletion. Cav1/\\\\(-\\\\)/MEFs were coincided with GFP and TR and incubated for 18-20 h. Calls were treated with cyclodextrin for 30 min, and Tf was internalized before immunofluorescence processing (A and B). Tf internalization occurred in both cholesterol-depleted calls and control cells, and quantification [B] revealed that there was no significant difference between treated and control cells. [C] CTR Alexa Fluor 594 was internalized for 40 min using the same conditions as in A. In unrelated cells, CTR strongly colocalized with GM130. In cholesterol-depleted cells. This colocalization was reduced as shown in the quantitation in D. P < 1x 10-2; bars indicate standard error. Bars, 25\\n\\n'", "SUPP CAPTION FIGS4.png": "'Figure S4. **A dynamic mutant inhibits CTB transport to the Golgi complex but not internalization.**_Cav1-/- MEFs_ were microinjected with HA-tagged DynK44A_ [B and C], DynK44A and TfR, or GFP and TfR [A]. DynK44A was expressed using the acute expression conditions (see Materials and methods). [A] Tf internalization [20 min at 37degC] was severely inhibited in \\\\(\\\\text{Ca}\\\\nu 1-/\\\\prime-\\\\) MEFs injected with DynK44A but not those injected with GFP. [B] CTB-Alexa Flucr 594 internalized for 40 min calculated with DynK44A in an extensive tubular network. The DynK44A-positive structures were not extensions of the Golgi, as GM130 did not colocalize with DynK44A or CTB. Tharefera, DynK44A severely inhibited CTB trafficking to the GM130-positive structures. [C] To investigate whether the tubular structures are surface-connected or intracellular, a time coarse of CTB internalization was performed in \\\\(\\\\text{Ca}\\\\nu 1-/\\\\prime-\\\\) MEFs transiently expressing DynK44A. CTB bound to the surface of cells at 4degC did not label the DynK44A tubular structures. However, the dynamic mutant and CTB were partially colocalized within tubular structures after 1 min of internalization, and this increased after 10 min. This suggests that dynamic is not involved in the initial entry step but affects early compartments on route to the Golgi complex. It also provides further evidence for a nonexwolce, nonclathrin pathway as DynK44A inhibits both clathrin- and Cav1-dependent endocytosis. Therefore, any CTB entering the cell should do so through the proposed nonclathrin, nonexwolce pathway.\\n\\n'", "SUPP CAPTION FIGS5.png": "'Figure S5. An ARF6 mutant inhibits CTB transport to the Golgi complex but not internalized. \\\\(\\\\text{Cav1}-\\\\text{/}\\\\text{/}-\\\\text{MEFs}\\\\) were microinjected with ARF6 T27N [B] and coincided with the transferrin receptor and HA-tagged ARF6 T27N (A). ARF6 T27N was expressed using the acute expression coordinates. (A) To investigate the effect of ARF6 in clathrin-mediated endocytosis. If was internalized for 20 min in \\\\(\\\\text{Cav1}-\\\\text{/}\\\\text{/}-\\\\text{MEFs}\\\\) that were expressing the transferrin receptor and ARF6 T27N. If uptake was severely inhibited by ARF6 T27N. (B) The formation of a tubular network was caused by overexpression of ARF6 T27N in \\\\(\\\\text{Cav1}-\\\\text{/}\\\\text{/}-\\\\text{MEFs}\\\\); CTB Alexa Fluor 59.4 internalized for 40 min accumulated in the tubular network. To determine whether or not the tubular network formed by the expression of ARF6 T27N was intracellular, a time course of CTB uptake was performed in \\\\(\\\\text{Cav1}-\\\\text{/}\\\\text{/}-\\\\text{MEFs}\\\\) expressing ARF6 T27N. CTB did not colocalize with ARF6 T27N after binding at 4degC but showed strong colocalization after 10 min at 37degC. Thus, these studies show that, like dynamic, ARF6 is not required for CTB entry. However, both proteins are involved in CTB trafficking at a post-entry step.\\n\\n'", "SUPP CAPTION S1.png": "'Figure S1: **Ultrastructural analysis of early endocytic carriers in WT-MFs and Cav1\\\\(-\\\\)/\\\\(-\\\\)MFs: development and validation.** CTB entry was examined using a modified method that distinguishes internal carriers at very early times of internalization [before fusion with other possible compartments] from surface-connected structures. In the case of caveolae, it is a considerable problem to distinguish between structures connected to the extracellular media and internal structures because caveolae and groups of caveolae can be connected to the cell surface by very narrow nacks (Porten et al., 1994). These nacks could exclude pretenaceous extracellular markers, as is the case for deeply invagorated clathrin-coated pits (Schmidt and Smythe, 1991). To prevent labeling of externally connected structures, we used the reducing agent ascorbic acid (AA) to quench the HRP-DAB reaction product on an extracellular surface (Sborovgogl et al., 1996). This necessitates carrying out the DAB reaction on cells before fixation. WT MEFs were labeled with CTB-HRP on ice, and then the HRP reaction was performed immediately or after warming to 37\u00b0C for 2.5 or 30 min. In each case, the DAB reaction was performed on ice in the presence or absence of ascorbic acid (=AA) and subsequently fixed and processed for EM. In addition, we optimized conditions to allow immunogold labeling of the CTB-HRP-labeled elements so that individual structures could be identified as surface connected or internal and identified using well-defined markers such as Cav1 and dynamin. [A] Cells maintained at 4\u00b0C and DAB reacted in the absence of AA [-AA] show an electron-dense layer of reaction product over the entire cell surface [a and b], including clearly surface-connected caveolae and putative caveolae structure that appear to be some disconnects from the PM but are obviously surface connected outside the plane of section [b]. The morphology was similar to that seen in cells in which the DAB reaction was performed after fixation [standard conditions; see Fig. S2). In the presence of AA [+AA] staining was completely abolished [c\u2013b], indicating that the AA is able to efficiently quench the surface reaction product even in deeply invagorated structures (e and f]. [B] WT MEFs were warmed to 37\u00b0C for 30 min and stained in the presence or absence of AA. Staining can be seen in putative endosomal structures and the Golgi [G] in both samples, although cells processed in the presence of AA show no surface labeling [compare to d [-C] for examine the non-surface-connected carriers involved in the earliest stages of CTB-HRP uptake, WT MEFs internalized CTB-HRP for 2.5 min at 37\u00b0C, and were DAB treated in the presence of AA to remove surface reaction. Note that structures within 50 nm from the surface show CTB-HRP/DAB staining but are clearly not surface connected. Thus, this method can be used to identify and characterize budding all intermediates in the CTB entry pathway for the first time. [D] A second method for sample preparation was also used in which cells were not sectioned after processing but were prepared as whole mount specimens using a modification of the method described previously (Stoovvagel et al., 1996). WT MEFs were grown on EM grids, which had internalized CTB-HRP for 5 min at 37\u00b0C. Cells were treated with DAB in the presence of AA. This method allowed the call pariphery to be observed without sectioning, and so facilitated the visualization of complex structures in three dimensions. When compared to C, similar structures observed in the section are observed in the whole mount calls. Bars: [Aa, Ac, Ba, and Bc] 1 mm; [Ab, Ad, Bb, Bd, C, and D] 500 nm; [Aa and Af] 100 nm.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/kirshMultitaskingCostStructure2005.json b/extracted_captions/kirshMultitaskingCostStructure2005.json new file mode 100644 index 0000000000000000000000000000000000000000..9e26dfeeb6e641a33dae4961196235bdb965b21b --- /dev/null +++ b/extracted_captions/kirshMultitaskingCostStructure2005.json @@ -0,0 +1 @@ +{} \ No newline at end of file diff --git a/extracted_captions/kneelandExploringUnchartedTerritory2020.json b/extracted_captions/kneelandExploringUnchartedTerritory2020.json new file mode 100644 index 0000000000000000000000000000000000000000..45e17331f0b070e143b1fe12a3bdb5c3f82d4e2d --- /dev/null +++ b/extracted_captions/kneelandExploringUnchartedTerritory2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: (Color online) Knowledge Creation Processes for Outlying Technological Positions\\n\\n'", "CAPTION FIGA.1..png": "'\\n\\n**Figure A.1.** Example of Network Diagram with New Unconnected Node'", "CAPTION TAB1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 2.** Top 10 NBER Technological Subcategories for Outlier and Nonoutlier Patents, 1990\u20132000 \\\\\\\\ \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Top 10 NBER Technological Subcategories for Outlier and Nonoutlier Patents, 1990\u20132000'", "CAPTION TAB3.png": "'\\n\\n**Table 3. Search Processes, Definitions, Indicators, and Quotes'", "CAPTION TAB4.png": "'\\n\\n**Table 4.** Descriptive Statistics and Correlations'", "CAPTION TAB5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 6.** Logit Regressions Predicting Outlier Patents with Distant Recombination and Full Model \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 6: Logit Regressions Predicting Outlier Patents with Distant Recombination and Full Model'", "CAPTION TABB.1.-2.png": "'\\n\\n**Acknowledgement**'", "CAPTION TABB.1..png": "'\\n\\n**Table B.1. Additional Interview Information**'", "CAPTION TABC.1..png": "'\\n\\n**Table C.1.** **Kobustance Analysis**'"} \ No newline at end of file diff --git a/extracted_captions/knoblichConstraintRelaxationChunk1999.json b/extracted_captions/knoblichConstraintRelaxationChunk1999.json new file mode 100644 index 0000000000000000000000000000000000000000..111e1f9a354f71eb20491eeba93288637b76e88e --- /dev/null +++ b/extracted_captions/knoblichConstraintRelaxationChunk1999.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Two matchstick arithmetic problems in the format used in the experiments.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Cumulative solution rates for Problem Types A-C in Block 1 of Experiment 1A.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Cumulative solution rates for Problem Types A and D in Block 1 of Experiment 1A.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Cumulative solution rates for Problem Types A\u2013D in Block 2 of Experiment IA.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Cumulative solution rates for Problem Types \\\\(\\\\mathbf{A\\\\mbox{\\\\sf C}}\\\\) in Experiment 1B.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Cumulative solution rates for Problem Types A and D in Experiment 1B.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Cumulative solution rates for Problem Types A-C in Block 1 of Experiment 2.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Cumulative solution rates for Problem Types A and D in Block 1 of Experiment 2.\\n\\n'", "CAPTION FIG9.png": "'Figure 8: Cumulative solution rates for Problem Types A\u2013D in Block 2 of Experiment 2.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB10.png": "'Table 10\\n\\n_Percentage Solutions in Experiment 3 by Coordination, Problem Type, and Block_'", "CAPTION TAB11.png": "'Table 11\\n\\nSolution Times (in Seconds) in Experiment 3 for Regular Problems'", "CAPTION TAB12.png": "'Table 12\\n\\nSolution Times (In Seconds) in Experiment 3 for Tautological Problems'", "CAPTION TAB13.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Main Effects of Task Difficulty in Experiments IA. IB. and 23_} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 13: _Main Effects of Task Difficulty in Experiments IA. IB. and 23_'", "CAPTION TAB14.png": "'\\n\\n## Table 1: Transfer on Extratering IA and 2'", "CAPTION TAB2.png": "'\\n\\n## Table 2\\n\\nSolarflora Times (Hor Seconds) for Black 1 of Experiment IA (N = 20)'", "CAPTION TAB3.png": "'\\n\\n## Table 3 Solution Times (in Seconds) for Black 2 of Experiment IA (\\\\(\\\\Omega\\\\) = 20)'", "CAPTION TAB4.png": "'\\n\\n## Table 4 Problems Used in Experiment I'", "CAPTION TAB5.png": "'\\n\\n**Table 5**\\n\\nSolution Times (in Seconds) in Experiment 1B'", "CAPTION TAB6.png": "'Table 6: Problems Used in Experiment 2 by Type and Condition\\n\\n'", "CAPTION TAB7.png": "'\\n\\n## Table 1: Percentage Solutions in Experiment 2 by Condition and Block'", "CAPTION TAB8.png": "'Table 8\\n\\n_Solation Times (in Seconds) for Block 1 of Experiment 2_'", "CAPTION TAB9.png": "'Table 9\\n\\n_Solution Times (in Seconds) for Block 2 of Experiment 2_'"} \ No newline at end of file diff --git a/extracted_captions/kobayashiDepolarizationSocialMedia2020.json b/extracted_captions/kobayashiDepolarizationSocialMedia2020.json new file mode 100644 index 0000000000000000000000000000000000000000..531cd06be9f765fb9afcb49dbad7a6dae2eff1d6 --- /dev/null +++ b/extracted_captions/kobayashiDepolarizationSocialMedia2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure I.** **Moderated effects of political use of social media.** Shaded areas indicate 95% CI.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Partisan selectivity among single and dual identifiers. Error bars indicate 95% CI.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table I. Regression models predicting partisan selectivity, polarization, and political participation.**'", "CAPTION TAB2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'"} \ No newline at end of file diff --git a/extracted_captions/kobayashiNewsAudienceFragmentation2019.json b/extracted_captions/kobayashiNewsAudienceFragmentation2019.json new file mode 100644 index 0000000000000000000000000000000000000000..6608d7b3d38ce9801cf2f405c43e3b23f5bb5d46 --- /dev/null +++ b/extracted_captions/kobayashiNewsAudienceFragmentation2019.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1. Distribution of estimated ideology.\\n\\n'", "CAPTION FIG2.png": "\"Figure 2. Distributions of Twitter users' ideology per media account.\\n\\n\"", "CAPTION FIG3.png": "'Figure 3. Overlap in followers of major Japanese media accounts.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1.** The list of media accounts.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n**Table 2.** MSE of classifiers (10-fold cross-validation).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/krasilnikov2020rates.json b/extracted_captions/krasilnikov2020rates.json new file mode 100644 index 0000000000000000000000000000000000000000..825e76b6e6ff91a45020d2990940d23a81aec49e --- /dev/null +++ b/extracted_captions/krasilnikov2020rates.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/laiStancePolarityPolitical2019.json b/extracted_captions/laiStancePolarityPolitical2019.json new file mode 100644 index 0000000000000000000000000000000000000000..75a93b0100b3392cc69814535e748d5e8cae724a --- /dev/null +++ b/extracted_captions/laiStancePolarityPolitical2019.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Hourly frequency of tweets posted in each considered temporal phase. Starting from the date the event happened, the figure also shows the enlargement of the considered temporal window.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: The likelihood to change from AGAINST or FAVOR to NONE in function of the fraction of cross-stance edges in the previous phase, for each type of network.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: \\\\(F_{avg}\\\\) and \\\\(F_{avg_{AF}}\\\\) obtained by SVM trained with each of the proposed features compared with the baselines and the best feature set result (_BoHplus_, _BoMplus_, and _BoHplusreply_).\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Distribution of 963 manually (bars on the left) and 6,465 automatically (bars on the right) annotated triplets over our given temporal phases RO, TD, DE, and EC. Datasets of manually and automatically annotated triplets have different sizes, hence scales on left and right y-axes have been re-scaled accordingly.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Follower Networks displayed using force atlas layout (above) and chord diagram (below) for each temporal phase.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: The homophily test according to stance for each temporal phase. We have homophily by stance if the fraction of cross-stance edges (CE) observed (solid lines \\\\(CE_{AFN}\\\\) and \\\\(CE_{AF}\\\\)) is significantly less than the probability that a cross-stance link is established in a null model (dashed lines \\\\(2(AF+AN+NF)\\\\) and \\\\(2AF\\\\)).\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Evolution of modularity \\\\(Q_{AFN}\\\\) for all the networks at every phase; also modularity (\\\\(Q_{AF}\\\\)) is displayed for all the subnetworks formed by only AGAINST and FAVOR clusters.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Retweet Networks displayed using force atlas layout (above) and chord diagram (below) for each temporal phase\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Quote Networks displayed using force atlas layout (above) and chord diagram (below) for each temporal phase\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Reply-To Networks displayed using force atlas layout (above) and chord diagram (below) for each temporal phase\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Ranking of the most frequently used hashtags in tweets from Nov. 11th to 23th 2016 according to _Twita_.\\n\\n'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 10: The within-stance percentage: rate of links between nodes in the \u201cUsers Sample\u201d having the same stance (AGAINST or FAVOR)'", "CAPTION TAB11.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Triplet example: the user wrote a tweet, a retweet (RT), and a reply (black bullets). Please notice that the triplet also includes a tweet (white bullet) written by another user, embedded in the reply.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Label distribution for each of the four temporal phases, and also for the total period of observation.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\(F_{avg}\\\\) and \\\\(F_{avg_{AF}}\\\\) achieved in the different temporal phases with the combination of BoHplus, BoMplus, and BoHplusreply features. Standard deviations calculated over the 5 folds of our cross validation analysis are shown for each measure. \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 5: \\\\(F_{avg}\\\\) and \\\\(F_{avg_{AF}}\\\\) achieved in the different temporal phases with the combination of BoHplus, BoMplus, and BoHplusreply features. Standard deviations calculated over the 5 folds of our cross validation analysis are shown for each measure.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 7: Retweets-based graphs size for each of the four temporal phases, and also for the total period of observation. The fifth graph is an union of all the other weighted graphs, hence the number of nodes and links in the last column is not the sum of the others.\\n\\n'", "CAPTION TAB8.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 8: Quotes-based graphs size for each of the four temporal phases, and also for the total period of observation. The fifth graph is an union of all the other weighted graphs, hence the number of nodes and links in the last column is not the sum of the others.\\n\\n'", "CAPTION TAB9.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 9: Replies-based graphs size for each of the four temporal phases, and also for the total period of observation. The fifth graph is an union of all the other weighted graphs, hence the number of nodes and links in the last column is not the sum of the others.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/lee2019impact.json b/extracted_captions/lee2019impact.json new file mode 100644 index 0000000000000000000000000000000000000000..b13abf46957055adc187f675e57ab341656a5b67 --- /dev/null +++ b/extracted_captions/lee2019impact.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\nFig. 1: **Dynamin 2 negatively regulates viral gene expression.****A.** E1A expression at 2 h pi as measured after virus infection alone (V), or infection following a pericentamen (Buf+ V) at 250 nM or 500 nM, and in the solvent control (DMSO+ V). **B.** Effect on E1A mRNA expression of pericentamen with cytochalasin D prior to virus infection (Cyto D+ V) at 250 nM or 2 pM; solvent control (DMSO+ V), virus alone (V); 2 h pi (DMSO+ V vs. Cyto D+ V 2 nM; \\\\(\\\\text{ip}<0.05\\\\), ANNOVA). **C.** Effect on E1A mRNA expression of pretreatment with nocodazole prior to viral infection (Noc + V) at 10 \u03bcM or 30 \u03bcM, virus alone (V), and DMSO control (DMSO+ V). **D.** Confocal microscopy representation of viral entry at 2 h pi in the presence of solvent control (panel 1, DMSO+ V) or 30 \u03bcM nocodazole (panel 2, Noc + V). **C.** Cy3 labeled HAdV-D37 (red), anti- mtDNA staining (green). **E.** Western blot showing protein expression levels in keratocytes after dynamic (DMSO+ knockdown: mock transfected and virus infected (V), scRNA transfected and virus infected (cRNA+ V), cNDM2 transfection prior to viral infection (sIDN2+ V). Western blot for a-AP2A1, and adaptor protein shown as negative control for specificity of DNM2 knockdown. Actin blot shows load control. Dynamic 2 mRNA expression levels by qRT-PCR are represented in bar graph just below. **F.** Early gene expression (E1A) as measured by qRT-PCR from same RNA pool as in E: virus alone (V), scrambled RNA (cRNA+ V), dynamic 2 knockdown, virus infected cells (sIDN2M2 + V) (\u201cp \\\\(<0.001\\\\), ANOVA). **G.** Western blot shows expression of CDmetry in empty vector prior to virus infection (EV+ V) and mCherry-dynamin 2 (arrow) prior to virus infection (OE-DNM2+ V). The bar graph just below shows mRNA expression of dynamic 2. **H.** E1A mRNA expression as measured in cells after dynamic 2 vector pretreatment, performed on same RNA pool from G (\u201cp = 0.01, Students _\u03b5_-test). Error bars represent standard deviation of the mean. Each experiment was repeated at least 5 times with similar results.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\nFig. 3: **Dynamin 2 knockdown increases nuclear targeting of adenovirus.****A.** Acetylated tubulin (red) after infection with EdU labeled virus (green) at 1 and 2 h, pH, after empty vector transfection prior to virus infection (EV), dynamin 2 vector transfection prior to infection (OE-DNM2), pretreatment with scRNA or dynamin 2 siRNA (siRNA (siRNA). DAPI staining (blue) of nuclei utilized to outline blue-free images (a-1 through h-1), in order to better visualize virions in cell nuclei. **B.** Quantification of green fluorescence (pixel units) from **A** (2h pH) within ovals outlines (nuclei) as measured using ImageJ and shown graphically in box plot (\u201dr \\\\(<\\\\) 0.0001, Kruskal-Wallis). Data was obtained from 20 cells per experimental group in three experimental replicates. **C.** PCR for HAdV-D37 E1A, hexon and penton base genomic DNA measured to show relative levels of viral entry at 30 min pi. **D.** Quantitative real-time PCR for E1A DNA under the same experimental conditions. Upper graph shows the standard curve for E1A DNA. Lower graph demonstrates no statistical difference between experimental groups Each experiment was repeated at least 3 times with similar results.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4.** Dynamln **2** knockdown increases virions in the cytosol relative to the endosome. A. Medshad schematic of $LD assay (Susomalainen et al., 2013). In this assay, SLO permeabilizes only cell membranes and not endosomal membranes, so that Cy3-labeled virions within endosome appear red, while virions in the cytosol appear yellow upon binding to acid-Cy3 antibody with a green chromophore. B. Sreptolyan O (SLO)-penetration assay performed 1 h p. Panel 1 shows the no-SLO control, where there is no staining for GM-130, labeling absence of antibody penetration. Panels 2-5 show equivalent staining for GM130 (green), indicating that the transfection did not affect pore formation by SLO. **C.** SLO assay pretreated in cells transfected prior to viral infection with empty vector (EV), dynamic 2 overexpression centerset (OEPNRH2), scRNA, or siRNA (elDMM2). In SLO treated groups ( + SLO), viruses in endosomes appear red, while virions in cytosol appear yellow. As a control, left panel shows same groups treated with triton X100 without SLO (-SLO), and thus viruses in both cytosol and endosomes appear yellow. Each experiment was repeated at least 3 times with similar results.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5. Cytastolic plus is associated with reduced cytidine expression. A. HCF percented with EV, GE-DRM2, scRNA and siRNA2 were infected for at a MOI of 100, and ultrastructural images taken to locate vitrion distribution. Scale bar = 500 nm. Electron microscopy was performed three times with similar results. B. Cytokine arrays were performed on 500 ml of culture supernatants collected from each green after infection at MOI of 10 for 2.5 h. Reference spots (RS) were used as decaturmetric controls to quantify the mean spot pixel density. Directed cytokines included CC12 (11), CC15 (2), CXCL1 (3), CXCL10 (4), CXCL11 (5), IL-1ra (6), IL-6 (7), CXCL8 (8), MIF (9) and Seppa B1 (10). C. Bar graph showing collated results from 3 separate assays. The mouse of densitometry measurements from three independent experiments were plotted, normalizing to reference spots. Error bars represent standard deviations of the masses. \" indicates those cytokines for which expression was reduced significantly by dynamic 2 siRNA, as compared to control scRNA (\\\\({}^{\\\\circ}\\\\)P \\\\(\\\\leq\\\\) 0.05, Students t-test).\\n\\n'", "CAPTION FIG6.png": "'\\nFig. 6: **Dynamin 2 regulates MIOK localization.****A.** Confocal microscopy of cells pretreated with empty vector (eV), dynamic 2 expressing vector (QE-DNM2), \\\\(\\\\propto\\\\)RNA, or dynamic 2 siRNA (sIDNM2) and infected with Cy3-labeled HA4V-D37 (red) for 1 h, and stained with anti-perferentiin (green), and DAPI (blue). **B.** Distance from green signals (arrows) to the closest edge of adjacent nuclei (100 cells per experimental group in three experiments) was measured using Leica application suite X (LAS X, Leica Microsystems, Wetzlar, Germany), and graphed in boxplots (\u2018p \\\\(<\\\\) 0.0001, Kruskal-Wallis). **C.** Transmission electron microscopy of infected keratocytes from same pretreatment groups, with 3 distinct images shown per group shown. NU: nucleus. Scale bar = 500 nm. **D.** Measurements taken from each microtubule organizing center to nearest nuclear membrane were performed on 10 cells per group by a masked observer. Data shown reflects means and standard deviation for each group (\u2018p \\\\(<\\\\) 0.05, Students-t-act-act). **E.** Confocal microscopy showing co-localization (yellow) of nuclear pore complex protein Nwq5S (green), and Cy3-labeled HA4V-D37 (red), 2 h pl. Each experiment was performed three times with similar results.\\n\\n'", "CAPTION FIG7.png": "'Fig. 7. Effect of dynamic 2 mutations on viral gene expression. A schematic presentations of dynamic 2 wild type and mutant clones. B Western Blot for wild type (Wt), 4551-553, and 746-786, with alpha tubulin load control. C qRT-PCR for E1A mRNA expression with the same treatment groups as A (\\\\(\\\\text{rp}\\\\leq 0.0\\\\text{S}\\\\), ANOVA, with Tukey procedure). Experiments in both B and C were performed three times with similar results.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8. Schematic of the role of dynamic 2 in viral trafficking. Our experiments in primary fibroblasts indicate that apex dynamic 2 aggregates (OE-NMD2), vitamin accumulate in endosomes, and hence are called from reaching the microtubule organizing center (ATCC) and nucleus (Mue). Knockdown of dynamic 2 (slDMH2) localizes virions in the cytosol, with increased acetylation of microtubules (CMU), and movement of FITC closer to the nuclear membrane, thus enabling nuclear entry.**'"} \ No newline at end of file diff --git a/extracted_captions/lehne2022calcium.json b/extracted_captions/lehne2022calcium.json new file mode 100644 index 0000000000000000000000000000000000000000..13a57cb2c7a0a674b7e30afe18a4279923794acd --- /dev/null +++ b/extracted_captions/lehne2022calcium.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\nFig. 1: **Supp-1 localizes to protruding lamellipodia.****a** (Maximum intensity projection of cortical images of the actin cytoskeleton in prepual macrophages expressing swip-1 dsRNA marked by eGFP co-expression. Scale bars represent 10 \u03bcm. Cells were co-stained with anti-Swip-1 antibody (magenta), DAPI (blue), and phalloidin (white). The arrow marks an eGFP-negative wild-type cell with high Swip-1 expression. **b** Western blot analysis of lysates of macrophages cells expressing different swip-1 dsRNA transgenes as indicated under the control of the hemoletin-Gal4 driver. The expression of dsRNA transgenes without the Gal4 driver serves as a control. Knock-down efficiency was validated with an anti-Swip-1 specific antibody. Actin served as loading control. **c** (Structured illumination microscopic (SIM) images of wild-type prepual macrophages co-stained for endegarous Swip-1 (magenta), phalloidin (green) and DAPI (blue). Scale bars 10 \u03bcm. **e** Frames of spinning disk microscopic video of a migrating macrophage expressing an Swip-1-eGFP fusion. The arrow marks the localization of Swip-1 in the protruding lamellipodium. Images were taken every 20 s for 30 min (see also Supplementary Movie 1). Scale bar 10 \u03bcm. **f** Maximum intensity projection of the confocal image of 528+ cells transfected with Swip-1-eGFP fusion (magenta), stained with DAPI (blue) and phalloidin (white). Scale bar 10 \u03bcm. **g** Lamellipodia width of 52R+ cells either transfected with cytoplasmic eGFP or Swip-1-eGFP fusion was measured at five regions using ImageJ and averaged. Boxes indicate 50% (25\u201375%) and whiskers (5\u201395%) of all measurements, with black lines depicting the medians. \\\\(n=\\\\) eGFP WT: 20, Swip-1-eGFP: 21 cells each from one representative transfection. Two-sided Mann-Whitney test was used, \\\\(P\\\\) value: <0.001 (***). All images shown are representative of at least three independent experiments.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2 Loss of Swip-1 impairs lamellipodia formation and cell migration of macrophages.****a** Schematic overview of the swip-7 gene locus. Excess encoding parts of distinct domains are indicated, EF domains in green and coiled-coil region in orange. The target sequence for CRISPR/Cas9 gene modification and generated swip-1 deletions are depicted. **b** Loss of swip-1 mutants were validated by Western blot analysis. Lysates from ten fly heads of wild-type and different mutant flies were analyzed. Non-specific bands (upper bands) serve as a loading control. All obtained mutants were validated once, hereinafter used swip-1 mutant flies were checked regularly in Western blot analysis of head lysates. **c, d** Confocal images of **c** wild-type and **d** swip-1 mutant macrophages were co-stained with phalloidin (white) and DAPI (blue). Scale bars 10 mm. **e** Quantification of lamellipodia width at the leading edge, **n** = WT: 64, swip-1 mutant: 61 cells of two independent experiments (_P_ value: 0.012). **f** Circularity was measured using image \\\\(J\\\\) shape descriptors, \\\\(n\\\\) = WT: 180, mutant: 192 (_P_ value: 0.001), rescue 209 cells (_P_ value: 0.892). **g** Rescue of swip-1 mutant macrophages re-expressing a Swip-1-eGFP fusion were co-stained with phalloidin (white) and DAPI (blue). The arrow marks mutant cells lacking Swip-1-eGFP that still show an irregular cell shape. **h, i** Spinning risk microscopy live imaging of randomly migrating macrophages in prepauge with indicated genotypes were tracked by using image 93. **h, i** Track speed mean is color-coded (0.06 to 6 \\\\(\\\\mu\\\\)m/min) **(h)** wild type and **(i)** overexpression of a Swip-1-eGFP (OE) in a wild-type background, **j** swip-1 mutant, **(k)** rescue with a Swip-1-eGFP, and **I** rescue with a Swip-1-D82A/D18A-eGFP, respectively. **m** Quantification of track speed mean, WT: \\\\(n\\\\) = 114 tracks, OE: \\\\(n\\\\) = 133, tracks (_P_ value: 0.006), swip-1 mutant: \\\\(n\\\\) = 206 tracks (_P_ value: <0.001), rescue WT: \\\\(n\\\\) = 166 tracks (_P_ values: 0.002 to _WT_ and <0.001 to mutant), rescue SwipD82A/D18A- \\\\(n\\\\) = 136 tracks (_P_ values: 0.594 to _WT_ and <0.001 to mutant). **e, t** Excess indicate 50% (25-75%) and whiskers (5-95%) of all measurements, with black lines depicting the medians. For statistical analysis, two values indicated by connecting black lines were compared with two-sided Mann-Whitney test, P value: 0.12 (_n_), 0.033 (_Y_), 0.002 (_Y_), <0.001 (_***_). **n** Frames of spinning disk microscopy video of migrating swip-1 mutant macrophages re-expressing Swip-1-D82A/D18A-eGFP. Images were taken every 20 s for 30 min. A white arrow indicates the direction of movement, yellow arrowheads mark the lamellipodium. Swip-1-D82A/D18A-eGFP localizes to the cell cortex but not to the protruding lamellipodium (see also Supplementary Video M3). Scale bar 10 mm. All images shown are representative of at least three independent experiments unless otherwise specified.\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: **SFhD2/Swip-1 is a calcium (Ca\\\\({}^{2+}\\\\))-regulated cross-linking protein.****a, c** Co-sedimentation of Drosophila Swip-1 with F-actin in high-speed pelleting assays in the absence or presence of 1mM Ca\\\\({}^{2+}\\\\). Increasing concentrations of Drosophila Swip-1 as indicated were incubated with B \\\\(\\\\mu\\\\)M G-actin in polymerization buffer, and the proteins recovered in the pellet (P) and the supernatant (S) fractions after the centrifugation at 200,000 x \\\\(g\\\\) were stained with Coomassie blue. **b, d** Quantification of equilibrium constants and binding sites (_n_) on F-actin in the absence or presence of Ca\\\\({}^{2+}\\\\) from experiments as shown in **a, c**. Points represent mean \\\\(\\\\pm\\\\) SD of increasing concentrations from three independent co-sedimentation experiments (_n_ = 3). Solid red lines represent calculated binding isotherms. **e, f** Co-sedimentation of Drosophila Swip-1 with F-actin in low-speed pelleting assays at 20,000 x \\\\(g\\\\) in the absence or presence of 1mM Ca\\\\({}^{2+}\\\\). Increasing concentrations of Swip-1 as indicated were incubated with S \\\\(\\\\mu\\\\)M G-actin in polymerization buffer, and the proteins in the pellet (P) and the supernatant (S) fractions were stained with Coomassie blue. **g** Quantification of F-actin in pellet fractions from experiments as shown in **f**. Bars represent mean \\\\(\\\\pm\\\\) SD of three independent co-sedimentation experiments (_n_ = 3). Data points show the measured values of the experiments. **h** Ca\\\\({}^{2+}\\\\) prevents stable cross-linking of actin filaments caused by _Drosophila_ Swip-1. Time-lapse micrographs of TIRM assays, used for the analysis of cross-linking behavior of Swip-1 in the absence or presence of 1 mM Ca\\\\({}^{2+}\\\\). Polymerization of 0.75 or 15 mM G-actin (10% ATT0488-labeled) with 0.1 mM (top) and 1.0 \u03bcM Drosophila Swip-1 (middle) in the absence and presence of Ca\\\\({}^{2+}\\\\). Note significantly cross-linked networks in the absence of Ca\\\\({}^{2+}\\\\) as compared to assay conditions in the presence of Ca\\\\({}^{2+}\\\\). The enlarged gallery of inset (dashed box) at the bottom displays dynamic cross-linking behavior of 1.0 \u03bcM Drosophila Swip-1 in the presence of Ca\\\\({}^{2+}\\\\) at higher temporal resolution. The blue arrowhead marks an attachment event of a short filament with the longer filament and the red arrowhead depicts the detachment of the short filament after 12 s. Time is given in seconds in the upper right corner of each frame. Scale bars 10 \u03bcM. I Quantification of attachment and detachment events in the absence or presence of Ca\\\\({}^{2+}\\\\). Bars represent mean \\\\(\\\\pm\\\\) SD from three movies each (_n_ = 3). Data points show the measured values of the experiments.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4****Swip-1 is recruited to lamellipodial protrusions during epithelial wound closure in vivo.** **a** Schematic of the in vivo model to study calcium-dependent wound healing in early _Drosophila_ pupal stages. **b**, **c** Single-cell ablation in the abdominal epidermis of a wild-type 18 h APF old pupa ubiquitously expressing a Lifcat-EGFP transgene under the control of the _da_-Gal4 driver. Images were taken every 30 s for 30 min, ablation starts at \\\\(t=0\\\\) min. **b** Overview of the imaged area of the monolayered epithelium, an asterisk indicates ablated cell. Scale bar 25 mm. **c** Magnification of the ablated cell of **b** at the indicated times. Arrows show forming lamellipodial protrusions at the wound margin. Scale bar 25 mm. **d** Single-cell ablation in the abdominal epithelium of a 18 h APF old pupa ubiquitously expressing Swip-1-EGFP transgene; yellow asterisk indicates the position of the ablated cell. White asterisks mark specialized epidermal cells, so-called tendon cells providing attachment sides to the muscles. These tendon cells lack Swip-1-EGFP expression driven by the _da_-Gal4 driver. Yellow arrows mark lamellipodial protrusions at the wound margin, whereas white arrows mark induced lamellipodia of cells several rows back from the wound site exhibited a delayed response. Scale bar 25 mm. **e** Single-cell ablation in the abdominal epidermis of a 18 h APF old pupa ubiquitously expressing the calcium indicator RCMP. Changes of intracellular calcium are visible as increasing fluorescence intensity. The calcium wave is propagated in a circular fashion with a velocity of \\\\(2.2\\\\pm 0.7\\\\) mm/s. Scale bar 25 mm. **c-e** Images shown are representative of **a** nine and **b**, **c** at least three independent experiments. **f** Schematic of the calcium wave-inducing membrane protrusions after wounding.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## 4 Conclusion\\n\\nFig. 5: **Epithelial wound closure requires Swip-1 function.****a** Single-cell ablation in the abdominal epidermis of a swip-1 mutant 18 h APF old pupu ubiquitously expressing a Lifcat-eGFP transgene under the control of the da-Gal4 driver. Images were taken every 30 s for 60 min, ablation starts at \\\\(\\\\tau=0\\\\) min. Overview of the imaged area, an asterisk indicates the position of cell ablation. Scale bar 25 mm. The image is representative of nine independent experiments. **b** Magnification of ablated cell shown in **a** at the indicated times, arrows indicate irregular unstable lamellipodial protrusions at the wound margin. Scale bar 25 mm. **c** Quantification of wound closure in wild type (WT), swip-1 mutant (swip2L), after overexpression (Swip-1 OE) and rescue by re-expressing distinct Swip-1 transgene, FL: full-length; **a**EF: lacks the EF hands and **a**CC: lacks the coiled-coil region. **W**und size was measured every 5 min and normalized to the initial size of the unravounded cell. **d** Excerpt of data from **c**. After 60 min, wound closure was assessed by comparing the remaining wound size normalized to unwounded cell size to wild type. Each point represents one experiment. \\\\(P\\\\) values: Swip-1 OE: 0.387, swip2L: 0.008, swip2L, Swip-1 FL rescue: 0.340, swip2L, Swip-1 FL-eGFP rescue: 0.040, swip2L, Swip-1-**aEF-eGFP rescue: <0.001, swip2L, Swip-1-**aCC-eGFP rescue: <0.001, swip2L, Swip-1-**a**D82A/D/D18A-eGFP rescue: 0.006. **e**, **d** Bars represent the mean +- SD of nine independent experiments for each genotype. Two-sided Mann-Whitney test was used to compare each genotype individually to WT, \\\\(P\\\\) values: 0.12 (ns), 0.033 (*), 0.002 (**), <0.001 (***).\\n\\n'", "CAPTION FIG6.png": "\"\\n\\n### _F_-actin complexes\\n\\nFig. 6: **Loss of ErhD2 impairs lamellipodia formation and adhesive 2D cell migration of mouse R16-F1 cells.****a** EGFP-tagged ErhD2 expressed in B16-F1 cells co-localizes prominently with F-actin in the entire lamellipodium including microspikes. The other images display EGFP-ErhD2 expressing cells additionally stained for the actin polymerase VASP, cortactin, and the Arp2/3 complex activator WAVE2. Scale bar 20 \u03bcm. **b** Elimination of ErhD2 by CRISPR/Cas9 in two independent B16-F1 mutants (#10 and #33) was confirmed by immunoblotting using specific antibodies. GAPDH was used as a loading control. **c** Elimination of ErhD2 diminishes cell migration on laminin. Three time-lapse movies from three independent experiments were analyzed for each cell line, B16-F1: \\\\(n\\\\) = 70 cells tracked, mutant#10: \\\\(n\\\\) = 71 cells tracked (_P_ value: <0.001 to B16-F1), mutant#33: \\\\(n\\\\) = 98 cells tracked (_P_ value: <0.001 to B16-F1 and 0.671 to mutant#10). **d** Analyses of mean square displacement of wild-type versus mutant cells. Respective symbols and error bars represent the means +- SEM of three time-lapse movies from three independent experiments (_n_ = 3). **c** Loses of ErhD2 quartiles lamellipodia formation in B16-F1 cells. Representative examples of lamellipodia from wild-type B16-F1 and ErhD2-K0 mutant cells. Cells migrating on laminin were stained for the actin cytoskeleton with phalloidin. Scale bar, 10 \u03bcm. **f** Quantification of F-actin intensities in lamellipodia of wild-type and mutant cells after subtraction of background from three independent experiments, B16-F1: \\\\(n\\\\) = 85 cells, mutant #10: \\\\(n\\\\) = 86 cells (_P_ value: 0.061 to B16-F1), mutant #33: \\\\(n\\\\) = 86 cells (_P_ value: 0.005 to B16-F1 and 10 mutant #10). **g** Quantification of lamellipodia with in wild-type and mutant cells from three independent experiments, B16-F1: \\\\(n\\\\) = 85 cells, mutant #10: \\\\(n\\\\) = 86 cells (_P_ value: <0.001 to B16-F1), mutant #33: \\\\(n\\\\) = 86 cells (_P_ value: <0.001 to B16-F1 and 0.061 to mutant #10). **h** Loss of ErhD2 does not impair microspike formation. Quantification of microspikes in wild-type and mutant cells from three independent experiments, B16-F1: \\\\(n\\\\) = 85 cells, mutant #10: \\\\(n\\\\) = 86 cells (_P_ value: 0.979 to B16-F1), mutant #33: \\\\(n\\\\) = 86 cells (_P_ value: 0.535 to B16-F1 and 0.654 to mutant #10). **l** Loss of ErhD2 diminishes the efficacy of lamellipodium protrusion. Kymographs of representative phase-contrast movies are shown. **j** Multiple examples of lamellipodium protrusion in B16-F1 versus ErhD2-K0 cells. **k** Quantification of protrusion rates from three independent experiments, B16-F1: \\\\(n\\\\) = 20 cells, mutant #10: \\\\(n\\\\) = 29 cells (_P_ value: <0.001 to B16-F1), mutant #33: \\\\(n\\\\) = 23 cells (_P_ value: 0.535 to B16-F1 and 0.475 to mutant #10). **c**, **f**-**k** Boxes in box plots indicate 50% (25-75%) and whiskers (5-95%) of all measurements, with dashed black lines depicting the medians, arithmetic means are highlighted in red. Non-parametric, Kruskal-Wallis test with Dunn's Multiple Comparison test (**c**, **f**, **k**) or one-way ANOVA with Tukey Multiple Comparison test (**g**, **h**) were used to reveal statistically significant differences between datasets. \\\\(P\\\\) value: >0.05 (_n_s.) <0.001 (***). All images shown are representative of three independent experiments unless otherwise specified.\\n\\n\"", "CAPTION FIG7.png": "'\\n\\n**Fig. 7** E**h**D2/**S**nip-1 cross-linked lamellipodial actin networks drive single-cell migration and epithelial wound closure. Schematic showing the proposed role of E**h**D2/**S**nip-1 in regulating lamellipodial actin networks.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/letrudAffirmativeCitationBias2019.json b/extracted_captions/letrudAffirmativeCitationBias2019.json new file mode 100644 index 0000000000000000000000000000000000000000..2370db7d29b184ab1d0ce0df4dfb1d3d572ea1c8 --- /dev/null +++ b/extracted_captions/letrudAffirmativeCitationBias2019.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n## References\\n\\n* [1]'", "CAPTION FIG2.png": "'\\n\\n**Fig 1:** **Reception of June 1991**'", "CAPTION FIG3.png": "'\\n\\n**Fig 3.** Reception of Wickstream and Bends in a 2000.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system. We study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system. We study the relationship between the two-dimensional and two-dimensional case of a two-dimensional system.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n**Table 1.** Recognition of James 1981.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/levySocialMediaNews2021.json b/extracted_captions/levySocialMediaNews2021.json new file mode 100644 index 0000000000000000000000000000000000000000..5f6ddcb8b64aac266d7be7e9495f4b0b36306d82 --- /dev/null +++ b/extracted_captions/levySocialMediaNews2021.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG10.png": "\"_Notes:_ This figure decomposes the gap between the number of posts participants were exposed to from the offered geo- and counter-atitudinal outlets. The \\\\(y\\\\)-axis is the number of posts seen in the feed in the two weeks following the intervention and the \\\\(x\\\\)-axis is the treatment arm. Algorithm describes the gap explained by Facebook's tendency to show participants a greater share of posts from pro-atitudinal outlets (among all posts in the feed) conditional on subscriptions. Subscriptions describes the gap explained by participants' tendency to subscribe to more offered outlets in the pro-atitudinal treatment. Usage describes the gap explained by participants' tendency to view fewer posts on Facebook (use Facebook less often) in the counter-atitudinal treatment. Combinations describe interactions between these expressions. Data are based on 1,059 participants in the pro- and counter-atitudinal treatments for which posts in the Facebook feed could be observed in the two weeks following the intervention and at least one post is observed. The calculations appear in online Appendix Section C.7.\\n\\n\"", "CAPTION FIG2.png": "'\\n\\n## Chapter 2 Facts Mention of Facts Slain by Outliers during the Study Phase'", "CAPTION FIG3.png": "'_Notes:_ This figure displays the segregation in news sites visited by referral source. The definition of the segregation measure is discussed in Section III. Online Appendix Section A.5 defines the websites composing each channel.\\n\\n_Source:_ 2017-2018 Comscore data\\n\\nFigure 3. Segregation in News Sites Visited by Riterral. Squiet, Comscore Data\\n\\n'", "CAPTION FIG4.png": "''", "CAPTION FIG5.png": "'. Major news outlets are added to the \\\\(x\\\\)-axis for reference. The slant of each domain is based on Bakshy, Messing, and Adamic (2015). A visit is referred from Facebook if the referring domain is \"facebook.com.\" Panel B presents a binned scatter plot. The \\\\(x\\\\)-axis is the share of Republican donations in a zip code based on FEC donation data for the 2016 and 2018 election cycles and the y-axis is the mean slant of news sites visited. The sample for both figures includes individuals in the 2017 and 2018 Comscore Web Behavior Database Panel who visited news sites multiple times through Facebook and through other sources.\\n\\nFigure 5: News Consumption in the Comscore Panel.\\n\\n'", "CAPTION FIG6.png": "'_Notes:_ This figure shows the effect of the treatments on engagement with the offered outlets in the two weeks following the intervention. The dependent variable is engagement with either the four potential pro-attitudinal outlets or the four potential counter-attitudinal outlets and the independent variable is the treatment. Each panel presents the effect of a separate outcome. For example, in the third panel, the triangle point and dashed line present the point estimate and the confidence interval of the effect of the pro-attitudinal treatment on visits to the websites of the potential pro-attitudinal outlets, compared to the control group. The regressions control for the outcome measure in baseline if it exists. The sample includes 1,648 participants with a liberal or conservative ideological leaning who installed the extension and provided permissions to access their posts for at least two weeks. Error bars reflect 90 percent confidence intervals.\\n\\n'", "CAPTION FIG7.png": "'_Notes:_ This figure shows the effect of the liberal and conservative treatments on the mean slant, in standard deviations, of all news with which individuals engaged. In each panel, the dependent variable is the mean slant of outlets and the independent variable is the treatment. The regressions control for the outcome in baseline, if it exists. The sample includes participants who installed the extension and provided permissions to access their posts for at least two weeks following the intervention. Error bars reflect 90 percent confidence intervals.\\n\\n'", "CAPTION FIG8.png": "'_Notes:_ These figures show the difference between the effect of the liberal and conservative treatments on the mean of laws engagement over time. Each panel presents a series of regressions, where the dependent variable is the least of outlets in a specific week. The regressions control for the outcome in baseline when it exists. In the \\\\(x\\\\)-axis, relative week 1 is a full week immediately following the intervention. Panel A is based on 1,596 participants who kept the extension installed for at least six weeks following the intervention. Panel B is based on 29,131 participants who provided access to posts they shared for at least six weeks. Error bars reflect 90 percent confidence intervals.\\n\\nFigure 8: Effects of the Conservative Treatment on Mean Slant by Week, Compared to the Liberal Treatment\\n\\n'", "CAPTION FIG9.png": "'_Notes:_ This figure shows the effect of the treatments on the primary endline survey outcomes. The first panel shows the effect of the conservative treatment on the political opinions index, compared to the liberal treatment. A higher value is associated with a more conservative outcome. The second panel shows the effect of the counter-atitudinal treatment on the affective polarization index, compared to the pro-atitudinal treatment. A higher value is associated with a more polarized outcome. The indices are described in Section IID and the regressions specifications are detailed in Section IIE. The panels are based on 17,635 participants who took the endline survey. Error bars reflect 90 percent confidence intervals.\\n\\nFigure 9. Effect of the Treatments on Political Options and Affective Polarization\\n\\n'", "CAPTION TAB1-1.png": "'\\n\\n## Table I-Santles, Data Sourcing, and Outcomes'", "CAPTION TAB1-2.png": "'_Notes:_ This table describes the main sample and subsamples analyzed along with the data sources, the number of participants, and the main outcomes. The subsamples and data are described in Section II. The outcomes are described in Section III.\\n\\n'", "CAPTION TAB1.png": "''", "CAPTION TAB2-1.png": "''", "CAPTION TAB2-2.png": "\"_Notes:_ This table presents descriptive statistics, along with the difference between participants assigned to each treatment arm. _Note_ support is the share of participants who voted for or preferred the candidate. _Difficult peers_, is whether participants find it difficult to see things from Democrats'/Republicans' point of view. _Facebook echo chamber_ is whether the opinions participants see about government and politics on Facebook are in line with their views always or nearly all the time \\\\(\\\\langle 3\\\\rangle\\\\), most of the time \\\\(\\\\langle 2\\\\rangle\\\\), some of the time \\\\(\\\\langle 1\\\\rangle\\\\), or not too often \\\\(\\\\langle 0\\\\rangle\\\\). _Follow new_ is whether participants follow government and politics always \\\\(\\\\langle 4\\\\rangle\\\\), most of the time \\\\(\\\\langle 3\\\\rangle\\\\), about one-half of the time \\\\(\\\\langle 2\\\\rangle\\\\), some of the time \\\\(\\\\langle 1\\\\rangle\\\\), or never \\\\(\\\\langle 0\\\\rangle\\\\). _Total subscriptions_ is the number of Facebook pages participants subscribed to in baseline. _News outlets shant_ is the slant of news outlets subscriptions. _F_-tests are calculated by regressing the treatment on the pre-treatment variables, with missing values replaced with a constant and an indicator for a missing value. Data sources for the United States and Facebook population are specified in online Appendix Section C.4.1.\\n\\n\"", "CAPTION TAB2.png": "'Tune 2- Review Team Leader, and Conference Proceedings Track 2 - Summer Track, London, and Conference Proceedings Track 30 - Summer Track, London, and Conference Proceedings'", "CAPTION TAB3-1.png": "'\\n\\n## Chapter 1 Context with the Constraints'", "CAPTION TAB3-2.png": "'_Notes:_ This table estimates the association between participants\\' characteristics and compliance with each treatment arm. In column 1, the dependent variable is whether the participant subscribed to at least one offered outlet and the independent variable is the interaction of participant\\'s ideological learning and her treatment assignment. The reference group is the control group where there are no compliers. In column 2, the data is pooled such that each observation is a participant and an outlet offered. The dependent variable is whether the participant subscribed to the outlet. The independent variables are based on the outlet\\'s perceived ideology according to the participant, where ideology is measured on a 7-point scale from extremely liberal to extremely conservative with an additional option of \"do not know.\" _Ideological distance_ is the standardized difference between the participant\\'s self-reported ideology and the outlet\\'s perceived ideology. Both regressions control for age, age squared, gender, and the set of potential outlets defined for a participant, and column 2 also controls for outlet fixed effects. Column 3 use robust standard errors and column 2 clusters standard errors at the individual level.\\n\\n'", "CAPTION TAB3.png": "'\\nThis table estimates the association between participants\\' characteristics and compliance with each treatment arm. In column 1, the dependent variable is whether the participant subscribed to at least one offered outlet and the independent variable is the interaction of participants\\'s ideological leaning and her treatment assignment. The reference group is the control group where there are no compliers. In column 2, the data is pooled such that each observation is a participant and an outlet offered. The dependent variable is whether the participant subscribed to the outlet. The independent variable are based on the outlet\\'s perceived ideology according to the participant, where ideology is measured on a 7-point scale from extremely liberal to extremely conservative with an additional option of \"do not know: _\"Hoological distance is the standardized difference between the participant\\'s self-reported ideology and the outlet\\'s perceived ideology. Both regressions control for age, age squared, gender, and the set of potential outlets defined for a participant, and column 2 also controls for outlet fixed effects. Column 1 use robust standard errors and column 2 clusters standard errors at the individual level.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/leytonpuig2017flat.json b/extracted_captions/leytonpuig2017flat.json new file mode 100644 index 0000000000000000000000000000000000000000..dd2fae06a20f67f6b0fc1c378bd4d4f68e037227 --- /dev/null +++ b/extracted_captions/leytonpuig2017flat.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1 : SR microscopy enables in-depth characterization of clathrin-coated structures. (a) Representative correlative TIRF-SR image of the CCSs on the basal membrane of HeLa cells. HeLa cells were fixed and stained with anti-clathrin heavy chain (CMC) antibodies as indicated in the Methods. Overlay of the TIRF (grey) and the SR (orange) images and zoomed-in regions (i, ii) are depicted. Scale bar, 1\u03bcm. (b) Gallery of SR images showing the diversity of the CCSs (pits, plaques and combination thereof) found on the basal membrane of HeLa cells. Scale bar, 1\u03bcm. (c) Morphometric analysis of CCSs on the basal membrane of HeLa cells. Circularity (1 = high circularity, 0 = high asymmetry) and surface area (\u03bcm\\\\({}^{2}\\\\)) of individual CCS were obtained as described in the Methods. (d) Actin associates with CCSs. Representative two-colour TIRF and SR images of F-actin (red in merge) with either CHC or CLC tagged with mTurquoise2 (CLC-mTQ2) (green in merge). HeLa cells were fixed and stained with anti-CHC or anti-GPP antibodies and phalloidin to detect CCSs and F-actin, respectively. CLC-mTQ2 was transfected as described in the Methods. Scale bar, 1\u03bcm. (e) N-WASP associates with CCSs. Representative two-colour TIRF and SR images of CHC (red in merge) and N-WASP tagged with GFP (N-WASP-GPP, green in merge). HeLa cells were transfected, processed and imaged as illustrated above. Scale bar, 1\u03bcm.**'", "CAPTION FIG2.png": "'Figure 2: **In-WASP is a key plaque regulator.****(a)** Characterization of control (CTR) knockdown (XD) and N-WASP KD HeLa cells. Total cell lysates were compared using the indicated antibodies. One of three experiments that were performed with similar results is shown. (**b)** N-WASP regulates the association between CCSs and F-actin. Representative two-colour SR images of control (CTR) KD and N-WASP KD cells stained for CHC (green in merge) and F-actin (red in merge). Dashed white boxes mark the position of the norm-in: Scale bar, 1um. (**c**) Knockdown of N-WASP increases plaque presence on the basal membrane. Representative TIRF and SR images of CHC on the basal membrane of control (CTR) KD and N-WASP KD cells. Scale bar T1RF, 10 \u03bcm. Scale bar SR, 1 \u03bcm. (**d**) Bar graphs show the number of pits per \u03bcm2 and percentage of total area of the ROI covered by plaques (plaque covered area). ROIs were defined as three \u03bcm-wide regions on the periphery of the cells and segmented for quantification (mean \u00b1 s.e.m., *_P_ < 0.01, _t_-test, _ns_ = not significant). (**e**) Representative TIRF and SR images of CHC in N-WASP KD cells transfected with full-length shRNA-resistant N-WASP tagged with GFP (N-WASP-GFP). Scale bar T1RF, 10 \u03bcm; SR, 1 \u03bcm. (**f**) Number of pits and plaque-covered area were obtained as above (mean \u00b1 s.e.m., *_P_ < 0.0001, _t_-test, _ns_, not significant).\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3 | **Actin polymerization controlled by N-WASP and the Arp2/3 complex has a key role in regulating plaques but not pits. (a) Schematic of N-WASP and mutants thereof (asterisk = H208D mutation). Supplementary Fig. Ic denotes abbreviations. (b) Representative TRF and SR images of CHC on the basal membrane of cells expressing the GFP-tagged N-WASP mutants described in. Scale bar TFR, Io ump; SR, Io ump. (c) Active N-WASP regulates plaques through its VCA, VH-H and PRD domains. Bar graph shows normalized plaque covered area (mean t s.e.m., \"*P<0.01 \"\"P<0.001, \"\"P<0.0001, one-way ANOV/s, rs, not significant). (d) Localization of N-WASP at plaques requires active N-WASP, the VH-H and the PRD domains. Bar graph shows conductance-based colocalization (CBC) for the association between plaques and the N-WASP mutants or GFP (mean t s.e.m., \"\"P<0.001, \"\"P<0.0001, one-way ANOV/s). (e) Neither N-WASP nor its mutants perturb pits. Bar graph shows normalized number of pits (mean t s.e.m., one-way ANOVA, rs = not significant). (f) Localization of N-WASP at pits requires active N-WASP, the VH and the PRD domains. Bar graph shows the CBC for the association between pits and the N-WASP mutants or GFP (mean t s.e.m., \"\"P<0.0001, one-way ANOVA, rs = not significant). (g) Characterization of Arp2C KD HeLa cells. Control (CTR) KD and Arp2C2 KD cells (girl and t2 obtained using different hairpins) were compared using the indicated antibodies. One of this similar experiments is shown. (h) The Arp2/3 complex controls CCS morphology. Representative TIRF and SR images of CHC on the basal membrane of the above-characterized cells. Scale bar TIRF, Io ump. SR, Io ump. (i) Knowledown of the Arp2/3 complex phenotypes that of N-WASP. Bar graphs show number of pits per um2 and plaque covered area (mean t s.e.m., \"P<0.05, \"\"P<0.01 one-way ANOV/s, rs = not significant). (j) N-WASP regulates plaques through the Arp2/3 complex. Bar graph shows plaque covered area in Arp2C2 KD cells transfected with N-WASP-GFP or GFP (mean t s.e.m., one-way ANOVA, rs = not significant).**'", "CAPTION FIG4.png": "'\\n\\n**Figure 4 : **Plaques remodel in response to external stimuli.****(a)** Representative SR images of the basal membrane of control (CTR) KD and N-WASP KD HeLa cells grown in either 0.1 or 10% serum and stained with anti-CHC antibodies to detect CHC. Scale bar, 1\u03bcm. (b)** Serum affects the presence of plaques, but not that of pits. Bar graph shows number of pits per \u03bcm2 and plaque covered area (percentage) of cells grown in either 0.1 or 10% serum (mean t.s.m. \"*P_<0.001, \"***P_<0.0001 t-test, ns - not significant.) (c)** Plaques rapidly respond to serum stimulation. Bar graph shows number of pits per \u03bcm2 and plaque covered area of serum-starved cells stimulated with 10% serum for 3, 7, 15, 30 and 60 min (mean t.s.m.). (d) The knockdown of N-WASP increases the persistence of CCSs in cells stimulated with serum. Representative images of live-cell TIRF movies of control (CTR) KD and N-WASP KD cells expressing CLC-RFP, stimulated with 10% serum at time 0. Bar graph shows CCS track duration (sec. - seconds, mean t.s.m., \"***P_<0.0001 t-test). Scale bar, 1\u03bcm. (e)** LPA recapitulates the effects of serum on plaques. Representative SR images control (CTR) KD cells that were serum starved and stimulated with 100 mg ml-1 EGF, 5 mM LPA or 10% FCS for 30 min or left untreated (MS) and subsequently stained for CHC. Scale bar, 1\u03bcm. (f) Bar graphs show number of pits per \u03bcm2 and plaque covered area (mean t.s.m., \"*P_<0.001, one-way ANOVA, ns - not significant.) (g)** DPARI mediates the effects of LPA on plaques. Representative SR images of CHC on the basal membrane of control (CTR) KD and DPARI KD HeLa cells (#1 and #2 obtained using different hairpins). Scale bar, 1\u03bcm. (h) Bar graphs show number of pits per \u03bcm2 and plaque covered area (mean t.s.m., \"*P_<0.01, t-test, ns - not significant).\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Figure 5 |****Pabues are sites of pit formation.** (**a**) CCSs exhibit different dynamics and fate. Three representative kymographs of large _CCSs_ from live-cell TIRF movies of HeLa cells expressing _CLC_-RFP that were serum starved overnight and then stimulated with 10% serum. _CCSs_ can dissociate into smaller structures (i and ii) or fluctuate (iii). Time (t) is in seconds (s). Representative stages are shown below each kymograph. (**b**) Plaques are sites of pit formation. Two representative 3D SR image slices of HeLa cells stained for _C_HC showing _CC_Vs (whose position is marked by arrowheads in the \\\\(Z\\\\) stacks) in areas covered by plaques. Scale bar, 1 mm. (**c**) Inhibition of dynamic perturbs the morphology of _CCSs_. Representative 3D SR image slices of HeLa cells treated with DMSO or the dynamic inhibitor _D_D_rasene (80 uM, 30 min) stained for _C_HC. Arrowheads mark pits in the \\\\(Z\\\\) stacks. Scale bar, 1 mm. (**d**) Representative SR images of HeLa cells treated with DMSO or the dynamic inhibitor _D_rasene (80 uM, 30 min) stained for _C_HC. Scale bar, 1 mm. (**e**) Inhibition of dynamic reduces plaques and increases pit number. Bar graph shows number of pits per mm2 and plaque covered area (percentage) (mean +- s.e.m., ***P < 0.001, ***P < 0.0001, t-test).\\n\\n'", "CAPTION FIG6.png": "'Figure 6: **|****LPARI and EGFR are recruited to plaques.****(a)** Activated EGFR is recruited to CCSs. Representative two-colour SR images of control (CTR) KD and N-WASP KD cells stained for CHC (red in merge) and EGFR (green in merge). Cells were stimulated with 100 ng ml\u22121 EGF for 5 min. Scale bar, 1 \u03bcm. Bar graph shows CBC for the association of EGFR with either pits or plaques before and after EGF stimulation (mean 1 s.e.m., *P< 0.05, **P>** < 0.01, ***P< 0.001, ***P< 0.001, ***P< 0.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n**Figure 7 : **Plaques are hubs for clathrin-mediated endocytosis and signalling of LPARL.****(a)** Knockdown of N-WASP does not perturb the activation of AKT or ERK induced by EGF. Serum-starved control (CTR) KD and N-WASP KD HeLa cells were left untreated (O) or stimulated with 100 ng ml\u22121 EGF as indicated. Total cell lysates were analysed with the indicated antibodies. One of three similar experiments is shown. **(b)** Knockdown of N-WASP induces hyper-activation of AKT after LPA stimulation. Serum-starved control (CTR) KD and N-WASP KD HeLa cells were left untreated (O) or stimulated with 5 mM LPA as indicated. Total cell lysates were analysed with the indicated antibodies. One of three similar experiments is shown. **(c)** Knockdown of N-WASP increases PIP2 formation after LPA but not EGF stimulation. Representative PIP2 formation tracking images using a PIP2 sensor (GRP1) tagged with GFP in live cell confocal images of control (CTR) KD and N-WASP KD HeLa cells stimulated with 100 ng ml\u22121 EGF and 5 mM LPA. Scale bar, 10 \u03bcm. Representative traces (one cell per trace) of brightness ratio between membrane and cytoplasm (M/C ratio). Bar graph shows percentage of responsive cells (mean t.s.m., \"*P < 0.001, t-test). **(d)** Activated LPARL is required to CCSs, and its internalization is impaired in cells lacking N-WASP. Representative images of two-colour live cell TIRF movies of control (CTR) KD and N-WASP KD cells expressing CLC-RFP (red in merge) and LPARL-GFP (green in merge), before and after stimulation with 5 mM LPA. Scale bar, 10 \u03bcm. **(e)** Graph shows LPARL-GFP enrichment in CLC-RFP positive structures in control (CTR) KD and N-WASP KD cells. **(f)** Representative kymographs of non-diffraction limited structures selected in the CLC-RFP channel of two-colour live cell TIRF movies showing CLC-RFP (red) and LPARL-GFP (green) of control (CTR) KD and N-WASP KD HeLa cells, before and after stimulation with 5 mM LPA. **(g)** Knockdown of Arp2 induces hyper-activation of AKT after LPA stimulation. Serum-starved control (CTR) KD and Arp2 KD cells were stimulated with 5 mM LPA for 5 min. Total cell lysates were analysed with the indicated antibodies. One of two similar experiments is shown.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/li2022molecular.json b/extracted_captions/li2022molecular.json new file mode 100644 index 0000000000000000000000000000000000000000..78104109b8e90de16ee90b21d2f235270291b8d2 --- /dev/null +++ b/extracted_captions/li2022molecular.json @@ -0,0 +1 @@ +{"CAPTION FIG1-1.png": "'Figure 1: Effect of mechanical load on branched actin network assembly, (**A**) Schematic illustration of actin networks generated by profilin-actin, the Arp2/3 complex and capping protein from surfaces coated with NPF (WAVE1aN). Conditions are 5 mM actin (1% Alexa 488-labeled), 5 mM profilin-100 mM Arp2/3 (5% Alexa647-labeled), 100 mM CP (15% TMR-labeled) \\\\(H\\\\) not indicated otherwise. (**B**) Representative TIRFM images of Alexa488-actin (top), TMR-CP (middle) and Alexa647-Arp2/3 (bottom) incorporation into dendritic actin networks at indicated growth stress. (Cl Top: Height (red) and stress (black) as a function of time for a representative growing network. The stress was kept constant at a defined setpoint via the feedback mechanism of the AFM (\u201cforce-clamp mode\u201d) until the height change over time appeared linear and the network composition was constant, which means that a steady state in network assembly was reached. The stress was then raised to a higher setpoint, to which the network responded by a rapid adaptation, followed by a new steady state assembly phase. Network growth velocities and densities of components as a function of growth stress were determined from the linear, steady-state phases (grey areas). Bottom: Quantification of average fluorescent intensities for indicated protein components (left) y-axis, colored lines) for networks either subjected to step-wise increasing loads (dark colors, applied stress is shown in the right y-axis (black lines) or adjacent control networks growing in the same chamber in the absence of load (bright colors, see also Figure 1\u2014figure supplement 1). (**D**) Top: Quantification of average fluorescent intensities for indicated network components as a function of applied load. Measurements are from for n=15 actin networks from N=5 independent experiments. Bottom: Corresponding average growth velocities (from n=12 actin networks from N=4 independent experiments) and average free barbed and densities (from n=21 actin networks; N=7 independent experiments) as measured by an \u2018arrest-and-label\u2019 approach as described in Bieling et al., 2016. Error bars represent \u00b1 SD (standard deviation). The online version of this article includes the following source data and figure supplement(s) for figure 1:\\n\\n**Source data 1.** Quantification of network height, growth stress, and fluorescence intensities of network components.\\n\\n_Figure 1 continued on next page_'", "CAPTION FIG1-2.png": "'Figure 1 continued Figure supplement 1: Uniform incorporation of all network components across the NPF surface. Figure supplement 1\u2014source data 1. Quantification of the spatial distribution of network components and time-dependence of their incorporation.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'", "CAPTION FIG2.png": "'\\n\\n**Source data 1.** Quantification of network incorporation rates of Actin, CP and Arp2/3.\\n\\nFigure 2: Load on branched actin network assembly decreases nucleation and. (**A**) Average per-network rates of filament elongation [green], capping (magenta), and nucleation (blue) calculated by the product of the bulk fluorescence intensities and the network growth velocity (Figure 1**D**) normalized to the flux at 25pN/_\u03bc_m2 as a function of external load. The line is a linear fit to the nucleation rate data. Error bars are SEM. (**B**) Average per-filament rates of filament elongation [green] and capping (magenta) as determined by normalizing their per-network rates (shown in **A**) by the relative density of free barbed ends (Figure 1**D**) as a function of external load. Lines are fits to double exponential decay functions. Inset: Semi-logarithmic plots of the same data. (**C**) Average per-filament rates of filament elongation [green] and capping (magenta) as a function of force per growing filament end. Lines are fits to single exponential decay functions. Inset: Semi-logarithmic plots of the same data. Error bars are SEM. (**D**) Ratio of the per-network nucleation and the per-filament capping rates as a function of external load. Error bars are \\\\(\\\\pm\\\\) SEM (standard error of the mean). The online version of this article includes the following source data for figure 2:\\n\\n**Source data 1.** Quantification of network incorporation rates of Actin, CP and Arp2/3.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: Single molecule characterization of force-dependent Arp2/3 nucleation. (**A**) Kymographs of single molecule nucleation on NPF surfaces (mCherry-WAVE14N, magenta) by spike-in of a small fraction of Alexa647-Arp2/3 (green, c=20 pM) into the overall Arp2/3 pool (100 nM) at indicated applied stress (lower panel) or in an adjacent unloaded control network (upper panel). **B**) Mean nucleation rates determined by single molecule imaging normalized to the nucleation rate in an adjacent unloaded control network at indicated growth stress from N=3 independent experiments. Pairs of control and loaded network are measured in the same flow chamber in a large field of view as illustrated by the lines linking two data points. Figure 3 continued on next page\\n\\n'", "CAPTION FIG3-2.png": "'_Figure 3 continued_\\n\\nError bars are SEM. p-values were derived from paired Wilcoxon signed rank tests: (**C**) Scheme of single-molecule Arp2/3 dynamics followed by TIRF microscopy. Exponential decay of Arp2/3 fluorescence reflects movement of the fluorophore away from the coverslip driven by actin filament assembly. Some Arp2/3 molecules remain attached to the growing actin network for their entire transit through the TIRF illumination field (blue) whereas others detach from the network while still detectable by TIRF illumination (red). Individual intensity trajectories, therefore, reflect either the transit time through the evanescent field or the time to detachment (dwell time). (**D**) Normalized frequency of fluorescence transit times of single Alexa 647-Arp2/3 complexes (n=1109, 594, and 318 Arp2/3 molecules at 0, 255, and 1020 Pa growth stress from N=3 independent experiments) in networks assembled at indicated stress. (**E**) Double-logarithmic plot of the mean fluorescence transit time (+/-SD) as a function of network growth velocity. The dashed line show perfect reciprocal correlation (slope = -1). (**F**) Normalized frequency of fluorescence dwell times of single Alexa 647-Arp2/3 complexes (n=1109, 594, and 318 Arp2/3 complexes at 0, 255, and 1020 Pa growth stress from N=3 independent experiments) in networks assembled at indicated stress. (**G**) Examples of kymographs from TIRF microscopy of individual Arp2/3 complexes in networks under high load (I020 pN/m2). Individual complexes are either continuously moving towards the rear of the evanescent field (continuous, green arrows) or dissociating prematurely (abortive, red arrows). (**H**) Representative time courses of fluorescence intensity for individual Arp2/3 complexes as a function of number of imaging frames categorized as either continuous (top panel) or abortive (bottom panel). (**I**) Relative frequency of dwell times for Arp2/3 complexes in dendritic networks at high load (I020pN/m2, red, n=318 Arp2/3 molecules from N=3 independent experiments) compared to the bleaching and loss of trading control for surface-immobilized Arp2/3 complexes (blue, see Figure 3--figure supplement 1, n=274 Arp2/3 complexes from N=3 independent experiments). Note that the frequency of early loss events is exceeding the combined bleaching and tracking loss frequency.\\n\\nThe online version of this article includes the following source data and figure supplement(s) for figure 3:\\n\\n**Source data 1.** Quantification of branching nucleation from single molecule Arp2/3 imaging.\\n\\n**Figure supplement 1.** Tracking and bleaching control for single surface-immobilized Arp2/3 complexes.\\n\\n**Figure supplement 1--source data 1.** Comparison of biochemical activities, effects on actin network architecture, and diffusion of wildtype and bulky mutant capping proteins.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'", "CAPTION FIG4.png": "'\\n\\n**Figure 4.** Free barbed ends bind and sequester the WI2 domain of the NPF in a load-dependent manner. (**A**) Scheme of the FRET setup. Surface-bound, donor- (Alex488-) Islabeled NPF molecules can interact with either quencher- (Atto540O-) labeled actin monomers resulting in decrease of donor fluorescence or unlabeled terminal protomers of uncapped barbed ends resulting in no change in fluorescence. The terminal protomers are unlabeled since quencher-labeled monomers are introduced only upon network arrest. (**B**) Time lapse TIRF microscopy images of Alexa 647-Actin (left image) or Alexa 488-WAVEAN (FRET done, other images) at indicated times after addition of 200 \u03bcl fixation and quenching mix (t=0, 30 \u03bcM LatB, 30 \u03bcM phalloidin, 5 \u03bcM Atto540O-actin, 7 \u03bcM profilin, 37.5 \u03bcM myotrophinV1 (CP inhibitor)) to 100 \u03bcM network assembly mix. (**C**) Averaged time-courses of the Alexa 488-WAVEAN signal following the addition of quencher-labelled monomers at t=0 as shown in B for N=5, 4, and 3 experiments for no network, 0 Pa or 1278 Pa growth stress networks. Error indicators are SEM. (**D**) Mean fraction of barbed end-associated NPF molecules in either in the absence of an actin network (black) or in the presence of a non-loaded (dark green) or 1276 Pa loaded (light green) network (see Materials and methods). N=5, 4, and 3 experiments for no network, 0 Pa or 1278 Pa growth stress networks. Error bars are SEM. p-Values were derived from Mann-Whitney U tests.\\n\\nThe online version of this article includes the following source data and figure supplement(s) for figure 4:\\n\\n**Source data 1.** Quantification of actin monomer binding by surface-immobilized WAVE1AN molecules as measured by FRET.\\n\\n**Figure supplement 1.** Comparison of biochemical activities, effects on actin network architecture, and diffusion of wildtype and bulky mutant capping proteins.\\n\\n'", "CAPTION FIG5-1.png": "'Figure 5: Load dependence of capping and a direct test of the elastic Brownian theory of force generation by actin networks. **(A)** Average ratios of capping protein to actin fluorescence for networks grown at different CP concentrations as indicated as a function of load (see Figure 4\u2014figure supplement 1). Error bars are SD. N=3 independent experiments for each CP concentration used. **(B)** Illustration of the consequences of load dependence of capping and polymerization. Low load allows for high capping and polymerization rates (left panel). A similar load dependence of these two processes maintains filament length at high load (middle panel), whereas a difference in load dependence leads to changes in filament length (right panel). **(C)** Structural models of a filament barbed end (light and dark green) bound by either an additional actin monomer (top panel, bright green), a wt CP heterodimer (middle panel, magenta) or an engineered GST and CP dimer fusion (\u2018bulky variant\u2019, bottom panel, magenta = CP, blue = GST). **(D)** TIRFM images of dendritic actin networks (top panel = TMR CP (wt), middle panel = Alexa647-GST-CP (bulky variant) and bottom panel = color merge) at indicated stress. Networks were assembled at standard conditions, except that CP (wt) concentration was 90 nM (of which 10 nM were TMR-CP) and Alexa647-GST-CP concentration was 10 nM. **(E)** Mean Alexa 488-actin, TMR-CP(wt) or Alexa 647-GST-CP (bulky variant) intensity normalized to the intensity of an adjacent unloaded network as a function of load. Error bars are SD. N=4 independent experiments. **(F)** Measured mean fluorescence intensity ratios of CP(wt)/GST-CP(bulky variant) normalized to the intensity ratio of an adjacent unloaded network as a function of load. Error. Figure 5 continued on next page\\n\\n'", "CAPTION FIG5-2.png": "'Figure 5 continued\\n\\nbars are SD. N=4 independent experiments. Red open circles are derived from the Brownian Ratchet Model (see Appendix 1).\\n\\nThe online version of this article includes the following source data and figure supplement(s) for figure 5:\\n\\n**Source data 1.** Quantification of concentration and load dependence of filament capping.\\n\\n**Figure supplement 1.** Characterization of network assembly at various CP concentrations.\\n\\n**Figure supplement 1--source data 1.** Quantification of network assembly at various CP concentrations.\\n\\n**Figure supplement 2.** Characterization of CP variants.\\n\\n**Figure supplement 2--source data 1.** Quantification of capping by bulky variants compared to wildtype capping protein.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Figure 1.**: _The results of the \\\\(\\\\beta\\\\'", "CAPTION FIGA1.png": "'\\n\\n**Appendix 1--figure 1.** Scheme illustrating the balance of forces in our experimental setup. Branched networks assembly produces a protrusive force \\\\(\\\\{F_{total}\\\\}\\\\), which is counteracted by \\\\(\\\\{\\\\}\\\\) the external load force applied by the AFM cantilever (\\\\(F_{AFM}\\\\)) and \\\\(\\\\{\\\\}\\\\) the internal frictional forces that originate from attractive tethering forces due to interactions between the network and the NPF-coated surface [\\\\(F_{total}\\\\)].\\n\\n'", "CAPTION FIGA2.png": "'\\n\\n**Appendix 1--figure 2.** Illustration of the architectural changes in branched actin due to load. Filament reorientation to shallower contact angles \\\\(\\\\langle\\\\theta\\\\rangle\\\\) at higher loads leads to an increase in total filament density within the network.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/lindsayEngagingOlderPeople2012b.json b/extracted_captions/lindsayEngagingOlderPeople2012b.json new file mode 100644 index 0000000000000000000000000000000000000000..48e7c172e60042e1e55bf82797ebae5c68d412d9 --- /dev/null +++ b/extracted_captions/lindsayEngagingOlderPeople2012b.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n## Chapter 1 The OASIS Process'", "CAPTION FIG2.png": "'Figure 2: Alice and Bob sitting on their mobility sequesters.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table I. Requirements recorded for design team**'"} \ No newline at end of file diff --git a/extracted_captions/liuAccumulationDynamicCatch2014a.json b/extracted_captions/liuAccumulationDynamicCatch2014a.json new file mode 100644 index 0000000000000000000000000000000000000000..b997860944df63a74acb5a348a06af5233472510 --- /dev/null +++ b/extracted_captions/liuAccumulationDynamicCatch2014a.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1. In Situ Analysis of Force-Dependent TCR-pMHC Bond Kinetics by BFP\\n\\n(A) BFP schematic. A micropipette-separated FBSC with a probe bead attached to the apex (left) was aligned against a T cell held by an apocaring pipette (right). (B) BFP functionalization. The probe bead was covalently linked to SA to capture pMHC (left) to interact with TCR (right). (C-E) Representative trace traces of measurement cycles showing adhesion that survived ramping and estimated a preast level of force until dissociation (marked by a red star), enabling bond lifetime measurement (C), adhesion required by a ramp force [marked by a magenta star] before reaching the set force at in force-range assay (D), or no adhesion (E). (F) Binding spectacity. Mean +- SEM of adhesion frequencies of >10 T cell-bead pairs with 50 contacts for each. Densities of pMHCs (m) are indicated inside of each bar. M.D., not detected.\\n\\nError bars represent SEM. See also Figure S1 and Movies S1, S2, and S3.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: TCR Forms an Agonist-Specific Catch-Slip Bond (A) Lifetimes of bonds of OT1 T cells with probe beads coated with indicated pHHCs at 0 (white) and 10 (black) pN. (B and C) Lifetimes versus force curves showing that OT1 TCR formed catch-slip bonds with progressively weaker agonists OVA (red open square), A2 (light blue) open triangle), and G4 (jbrured magenta open triangle) (B) but slip-only bonds with antagonists R4 (blue open circle) and E1 (green open diamond) (C). Error bars in (A)-(C) represent SEM. (D) Force regulation of antigen discrimination, measured by the average lifetime ratio of TCR bonds with OVA to another peptide. See also Figure S2.\\n\\n'", "CAPTION FIG3.png": "'Figure 8. Single-Cell Concurrent Measurement of Ca\\\\({}^{2+}\\\\) Flux and In Situ TCG-Particle Bead Klinette\\n\\nA and B: Representative wide-field pseudoreal and maps of two types of intracellular Ca\\\\({}^{2+}\\\\) signals.\\n\\nC and D: Representative time scans of arbitrary 2 ratio of type a (C, magenta curve, left cardinal) or type a (D, green curve, left cardinal) intracellular Ca\\\\({}^{2+}\\\\) signal synchronized with concurrent measurements of uptore (red open circle) and theirs plus red triangle) are cumulative lifetime (blue dashed curve, right cardinal). Note that there are no return events for (C, blue dashed-dot horizontal line indicates a 10 s threshold at correlation lifetime.\\n\\nSend sleep Figure 8. Single-Cell Concurrent Measurement of Ca\\\\({}^{2+}\\\\) Flux and In Situ TCG-Particle Bead Klinette'", "CAPTION FIG4.png": "'Figure 4. TCR- and pMHC-Specific G\\\\({}^{2+}\\\\) Flux Requires both Force and Lifetime\\n\\n(A) Ca\\\\({}^{2+}\\\\) has requires durable force on TCR bond applied by antigen pRX+SC. Percent increase of fura-2 ratio in DTT T cells toasted without force or lifetime via VSV (green open diamond, _et._ Figure 1E), with force but no lifetime via GVAA (red solid square, cf. Figure 1D), or with both force and lifetime (cf. Figure 1C). Vila _OVA_ (red open square) or G4 (inverted light blue open triangle) or via anti-LFA-1 beads (orange open circle).\\n\\n(B) and C) Ca\\\\({}^{2+}\\\\) was triggered by an optimal force. Percent increase of fura-2 ratio (points, left ordinally in DTT T cells triggered by lifetimes (gray bars, \\\\(\\\\max\\\\)\\\\(\\\\doteq\\\\) SEM of \\\\(>\\\\)50 measurements, right ordinate) of TCR bonds with OVA (B) or anti-CTT (C) measured at 0 (inverted magenta open triangle), 5 (green open circle), 10 (red open square), or 20 (blue open triangle) pN force. The asterisk (T) denotes p \\\\(<\\\\) 0.01 on Ca\\\\({}^{2+}\\\\) signals.\\n\\nError bars represent SEM.\\n\\n'", "CAPTION FIG5.png": "'Figure 3. Correlation Cat+ signals with M-meta-Ascelinated state-free from Ench Cells to a Event Sequence\\n\\n(A-F) Percent increase of the 2 ns6s versus number of stations \\\\(M_{a}\\\\) (4L number of lt-drman). (A-F) Percent increase of the 2 ns6s versus number of stations \\\\(M_{a}\\\\) (4L number of lt-drman).\\n\\n'", "CAPTION FIG6.png": "'\\n\\n**Figure 6.** Ca2+ Best Correlates with TCR-pMHC Bond Lifetimes Accumulated in the First Minute of Successive Force Applications (A) Schematic of a sliding window (highlighted), starting at \\\\(T_{0}\\\\) with length \\\\(T_{\\\\rm L}\\\\) and containing different binding events (red, no adhesion; green, rupture force; blue, lifetime).\\n\\n**(B and C)** Percer increase of intra-2 ratio versus cumulative lifetime of CT1 TCR bonds with CVA at 0 (brown solid square), 5 (orange open square), 10 (magenta open circle or green open triangle, for type \\\\(\\\\alpha\\\\) or \\\\(\\\\beta\\\\), respectively), or 20 (bward open red triangle) pN, or with G4 at 0 (yellow solid diamond) or 10 (light blue open diamond) pN for each T cell accumulated in windows of indicated lengths and starting times. Dashed lines are linear fits to data, and the Pearson coefficients (F) are indicated. The horizontal and vertical dotted lines in the subpanel with the best correlation (highlighted) denote the demarcation of types \\\\(\\\\alpha\\\\) magenta open circle) and \\\\(\\\\beta\\\\) (brown solid square, orange open square, green open triangle, inverted open red triangle, yellow solid diamond, light blue open diamond) Ca2+ and the T0 is threshold of cumulative lifetime for triggering type \\\\(\\\\alpha\\\\) Ca2+.\\n\\n**(D-F)** Percer coefficient analysis to search for the window for the kinetic parameters to achieve the best correlation with Ca2+.\\n\\n**(D)** Maximal Pearson coefficients \\\\(R_{\\\\rm max}\\\\) versus \\\\(T_{\\\\rm L}\\\\) of the initial window (\\\\(T_{0}=Q\\\\) within which the kinetic parameters best correlate with Ca2+.\\n\\n**(E)** Percer coefficient for cumulative lifetime versus sliding window size \\\\(T_{\\\\rm L}\\\\) for the indicated starting times \\\\(T_{\\\\rm L}\\\\).\\n\\n**(F)** Pearson coefficients for indicated kinetic parameters calculated in a 60 s sliding window versus its starting time \\\\(T_{\\\\rm Q}\\\\). Different symbols in (D) and (F) denote number of adhesions \\\\(N_{\\\\rm s}\\\\) (purple open circle), number of lifetimes \\\\(N_{\\\\rm L}\\\\) (magenta open square), adhesion frequency \\\\(P_{\\\\rm s}\\\\) (blue open triangle), average lifetime \\\\(<\\\\)\\\\(t\\\\)\\\\(>\\\\) (green solid square), longest lifetime \\\\(t_{\\\\rm max}\\\\) (orange solid circle), and cumulative lifetime \\\\(\\\\Sigma_{\\\\rm L}\\\\) (inverted red solid triangle).\\n\\nSee also Figures S4, S5, and S6.\\n\\n'", "CAPTION FIG7.png": "'Figure 7. Long TCR-pMHC Bond Lifetime Accumulated after Mary Short Ones Fails to Induce Ga\\\\({}^{2+}\\\\)\\n\\n(A) Maximal cumulative lifetime max(\\\\(\\\\Sigma t\\\\)) of TCR bands with OWa at 0 [brown solid square], 5 [orange open square], 10 (magenta open circle or green open triangle, for typa a or \\\\(\\\\beta\\\\) Ca\\\\({}^{2+}\\\\), respectively), or 20 (prevented red open triangle) pN or with G4 at 0 [yellow solid diamond] or 10 pN [light blue open diamond] for each T cell accumulated in a 60 s window versus its starting time \\\\(T_{\\\\text{O}}\\\\).\\n\\n(B) Percent increase of fura-2 ratio versus maximal cumulative lifetime max(\\\\(\\\\Sigma t\\\\)) calculated for each cell in its own 60 s window with starting time adjusted to allow \\\\(\\\\Sigma t\\\\) to achieve maximum. Demonstration of 50% increase of fura-2 ratio and threshold of 10 s cumulative lifetime (horizontal and vertical red dotted lines) are used to segregate cells into three groups. Group A cells accumulated >10 s lifetime in the initial 60 s window and generated type a Ca\\\\({}^{2+}\\\\) [magenta open circle]. Group B cells accumulated >10 s lifetime in later 60 s windows but generated type a Ca\\\\({}^{2+}\\\\) (light blue open square). Group C cells accumulated <10 s lifetime and generated type b Ga\\\\({}^{2+}\\\\) (green open triangle). Dashed lines in (A) and (B) are linear fits to data with \\\\(\\\\beta\\\\) values indicated.\\n\\n(C-F) Normalized lifetime histogram (C and E) and number (left ordinate) and fraction (right ordinate) of short lifetime (D and F) before Ca\\\\({}^{2+}\\\\) peak pooled from cells in groups A (magenta) and E (cyan).\\n\\n(E and F) are similar to (C) and (D) ascaps that lifetimes were collected from an initial 60 s window for group A; for group B, the starting time was adjusted for each cell to allow \\\\(\\\\Sigma t\\\\) to achieve maximum.\\n\\nError bars in (D) and (F) represent SEM. See also Figure S7.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/lundmark2008gtpaseactivating.json b/extracted_captions/lundmark2008gtpaseactivating.json new file mode 100644 index 0000000000000000000000000000000000000000..31fc6bc54df9c977277e7e88ed9ceab01ae24872 --- /dev/null +++ b/extracted_captions/lundmark2008gtpaseactivating.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: GRAF1 Tubules Are Highly Dynamic and Mark a Prevalent Endocytic Pathway (A) Domain architecture of GRAF1 and the site of introduced BAR domain mutations (\\\\(\\\\tau\\\\)). (B\u2013D) Micrographs showing that GRAF1-positive tubules are derived from the plasma membrane as shown by colabeling with the membrane dye DII after 5 min (B) or internalized dextran after either 5 min (C) or 1 min (D) of incubation. (E) Overlaid maximum projections of spinning-disc confocal micrographs of HeLa cells expressing GFP-tagged GRAF1 demonstrate that GRAF1-positive tubules are completely turned over in 10 min. \\\\(Z\\\\) selections were performed continuously for 10 min in 0.5 \u03bcm steps. The initial maximum projection (in green) was then merged with the final maximum projection (in red). See also Movie S1. (F) Overlaid maximum projections as in (a), but for a cell overexpressing GFP-tagged GRAF1 BAR+PH. See also Movie S2.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: The BAR and FH Domaire Localize GRAF1 to Highly Curved, Plains(4,5)P2-Zeninched Membranes, and the SH3 Domain Binds Dynamic (A) Coomassie-stained gel of liposome cosedimentation assay showing the preference of GST-tagged GRAF1 BAR+PH protein for binding to smaller-sized liposomes derived from total brain lipids (the average diameters of liposomes are shown). Pellet (P) and supernatant (S) fractions were separated by ultra-centrifugation. The graph shows quantifications of total band intensities for each condition normalized to binding of the non-curvature-sensitive protein Dah2 (as a way of controlling for total lipid in each experiment). The error bars show 95% confidence intervals (calculated by t) tests for each condition. (B) Liposome cosedimentation assay as performed in (A) but with 0.8-\u03bcm-diameter liposomes enriched with varying phospholipids. (C) Electron micrographs of negatively stained liposomes incubated in the presence or absence of GST-tagged GRAF1 BAR+PH protein. Note the protein-dependent presence of tubular structures. The scale bar represents 200 nm. (D) Immunoprecipitation of GRAF1 from rat brain cytosol (via Ab2) reveals a GRAF1-Dynamim1 complex as identified by mass spectrometry and confirmed by immunoblot. (E) Coomassie-stained gels of pull-down experiments with purified Dynamim and either bead-bound GST-tagged GRAF1/Amphighysin SH3 domain or GRAF1 BAR+PH protein. P = pellet fraction, S = supernatant fraction. (F and G) Epifluorescence micrographs of HeLa cell overexpressing Myc-tagged GRAF1 and incubated with CT&B for 5 min before fixation and staining. (G) Epifluorescence micrographs of a HeLa cellincubated with CT&B for 5 min before fixation and staining for endogenous GRAF1. Scale bars represent 10 \u03bcm.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: GRAF1 Regulates the CLIC Endocylic Pathway (A) Representative electron micrographs of 65 nm ultrathin cryosections of HeLa cells transiently transfected with GFP-GPI alone or with both GFP-GPI and Myc-tagged GRAF1 FL. Cells were fixed in 2% PFA with 0.2% glutaraldehyde and labeled with anti-GFP and anti-_Myc_ antibodies. Protein A 10 nm gold was used for revealing the GFP in the single labeling (upper image). As shown, GFP-GPI was found in uncoated vesicles and tubules within the cell. Double labeling of GRAF1 (Protein A gold 10 nm) and GFP-GPI (Protein A gold 15 nm) is shown in the bottom image, where colocalization of GRAF1 and GFP-GPI is seen in an intracellular tubule. Arrowheads point to 10 nm gold particles. Due to limitations of the \\n\\n'", "CAPTION FIG4.png": "'Figure 4: GRAF1 Is indispensable for Clatherin-Independent Fluid-Phase Uptake in Fibroblasts (A) Immunoblots on HeLa cell lysates transfected with a control siRNA or either of two siRNA directed against GRAF1 mRNA (siRNA a and b). Detection of GRAF1 and tubulin (loading control) was performed with specific antibodies on lysates obtained 48 hr after transfection. (B) GRAF1-depleted cells show a major reduction in fluid-phase endocytosis as shown by the decrease in the uptake of FITC-labeled dextran (control siRNA \\\\(n=5\\\\)), siRNA (\\\\(n=8\\\\)), or APE2 siRNA (\\\\(n=3\\\\)). The error bars show the standard deviation of the mean. (C and D) HeLa cells depleted of GRAF1 were incubated with dextran (C) or transferrin (D) for 15 min before fixation and staining. Scale bars represent 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n**Figure S1 \\\\(|\\\\) Localisation of GRAF1 to tubular membrane structures is temperature sensitive and dependent on the BAR and PH domains.****A,** Western blots showing the different forms of GRAF1 detected in adult rat brain and their differential presence/absence in cultured SH-SY5Y (human neuroblastoma), HeLa (human fibroblast), K562 (human Chronic Myeloid Leukaemia), and MEF (mouse embryonic fibroblast), cells. Western blots of purified GRAF1 and myc-tagged GRAF1 (from lysates of HeLa cells overexpressing this protein) are shown for comparison. **B,** Confocal micrograph of an NIH 3T3 cell stained for endogenous GRAF1 distribution. **C,** Confocal micrographs of HeLa cells fixed either at 4degC or 37degC for 10 minutes in 4% paraformaldehyde and then stained for endogenous GRAF1. Note the absence of GRAF1-positive tubules in the 4degC fixation image. **D**, Confocal micrographs showing the tubular localization of overexpressed myc-tagged GRAF1 BAR+PH protein in HeLa cells and the cytoplasmic localization of a similarly overexpressed protein with a BAR domain mutation (KK131/132EE). **E,** Confocal micrograph showing the cytoplasmic and punctate localization of overexpressed myc-tagged GRAF1 BAR protein in HeLa cells. **Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "CAPTION FIGS2-1.png": "\"\\n\\n**Figure S2 A-D\\\\(|\\\\) Supplementary biochemical data on the binding between GRAF1 and Dynamin.****A,** Coomassie-stained gel and confirmatory Western blots of coimmunoprecipitation experiments in rat brain cytosol performed with either control pre-immunization serum (pre-serum) or the Ab3 antibody. Bands in the Coomassie-stained gel were identified by mass spectrometry as described. **B and C,** Coomassie-stained gel and Western blots of pull-down experiments from mouse brain lysate (B) or HeLa cell cytosol (C) with beads bound to GST (control) or GST-tagged GRAF1 BAR+PH, or SH3 proteins. The bands in the Coomassie-stained gel were identified by mass spectrometry as described. Note the major band of Dynamin present in the SH3 lanes, which is not present in the control or BAR+PH condition. 'cyt' marks the HeLa cell lysate (positive control) lane. **D,** The upper panel shows a raw trace from isothermal titration calorimetry performed as described. The lower panel shows the fitting of this data to a one-site binding model from which the affinity (shown) can be calculated. GRAF1 SH3 domain and peptide concentrations, as well as injection volumes and times are shown.\\n\\n\"", "CAPTION FIGS2-2.png": "'\\n\\n**Figure S2 E \\\\(\\\\mid\\\\) Dynasore inhibits the uptake of dextran and affects the localization of GRAF1. E, Confocal micrographs (maximum projections) of HeLa cells treated with either DMSO (vehicle) or 100\\\\(\\\\mu\\\\)M dynasore for 1 hour before addition of dextran for 15 minutes, fixation, and immunostaining for dextran and the focal adhesion marker paxillin. Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "CAPTION FIGS2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n**Figure S3 \\\\(|\\\\) GRAF1-positive endocytic structures are Clathrin-independent and exclude transferrin A-C, Confocal fluorescent micrographs of HeLa cells stained for endogenous GRAF1 and Clathrin (A), transferrin (B) or transferrin receptor (B). The depicted images are used to show some of the different morphologies of GRAF1-positive structures that are observed in these cells. Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "CAPTION FIGS4.png": "'\\n\\n**Figure S4 \\\\(|\\\\)****GRAF1 BAR+PH overexpression affects CTxB uptake but not transferrin uptake.****A and B,** Confocal micrographs of HeLa cells transfected with myc-tagged GRAF1 BAR+PH and incubated with CTxB or transferrin for 15 minutes before fixation and staining. **C,** The graph shows the quantification of images such as depicted in (A). Cells were scored for expression of GRAF1 BAR+PH (over a threshold corresponding to maximum autofluorescence) and CTXB internalization (over an arbitrarily-set threshold above background). Note the reduction of the number of transfected cells internalising CTxB compared with controls. **D,** Live cell microscopy of NIH 3T3 cells expressing GFP-tagged GRAF1 and incubated with CTXB and transferrin (Tfn) at 4degC before chasing their internalization from the time of warming to 37degC (time=00:00). Note the internalising GRAF1-positive tubule containing CTxB. Note also the lack of colocalization of GRAF1-positive tubules with internalized transferrin. Time is given as minutes:seconds. This sequence is taken from Movie S3. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "CAPTION FIGS5.png": "'\\n\\n**Figure S5 \\\\(|\\\\) Nature of GRAF1-positive endocytic structures.****A and B,** Confocal micrographs of HeLa cells overexpressing GFP-tagged flotillin1 (A) or GFP-tagged caveolin1 (B) and co-stained for endogenous GRAF1. Note the lack of colocalization. **C and D,** Confocal micrographs of HeLa cells overexpressing myc-tagged GRAF1 BAR+PH and flotillin1 (E) or caveolin1 (F) incubated with CTxB for 5 minutes. Note the colocalization of GRAF1 BAR+PH and flotillin1 in CTxB-positive tubular structures. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "CAPTION FIGS6.png": "'\\n\\n**Figure S6 \\\\(|\\\\)****GRAF1 BAR+PH overexpression does not affect the uptake of MHC Class I which enter GRAF1-negative compartments.****A and B,** Epifluorescence micrographs of HeLa cells, transiently transfected with GFP-tagged GRAF1 FL (A) or GRAF BAR+PH (B) were pulsed with anti-MHC Class I antibody for 5 (A) or 15 minutes (B) at 37\\\\({}^{\\\\circ}\\\\)C followed by a brief acid wash to remove surface-bound antibody. MHC Class I was visualized using Alexa568-conjugated secondary antibodies. **C,** Quantitation of surface GFP-GPI levels in cells overexpressing this protein with myc-tagged GRAF1 FL or GRAF1 BAR+PH. **D,** Confocal micrographs of HeLa cells treated with siRNA against GRAF1 or a control siRNA and stained for endogenous GRAF1. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'"} \ No newline at end of file diff --git a/extracted_captions/marozzoAnalyzingPolarizationSocial2017.json b/extracted_captions/marozzoAnalyzingPolarizationSocial2017.json new file mode 100644 index 0000000000000000000000000000000000000000..920cb07696465d61c90722f22506cfb716b43f76 --- /dev/null +++ b/extracted_captions/marozzoAnalyzingPolarizationSocial2017.json @@ -0,0 +1 @@ +{"CAPTION AB5.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Fig. 1**: **No of three equal by users.**'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 1**: **A**. of tweets per week day'", "CAPTION FIG4.png": "'Figure 4: Probability density function of the users\u2019 polarization\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Complementary cumulative distribution function of the number of tweets published by users polarized toward yes and toward no\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## Chapter 4 Conclusion and new polarization in the 5 thick\\n\\nrecording the representation diagram.\\n\\nLinear polarized in flat or yet blue circular, in flat or in red circular, and neutral (gray circles)'", "CAPTION FIG7.png": "'\\n\\n**Fig 1** User polarization profile: (in a standard by a Random Forest model using anfarrative nested by users from 5 to 1 nuclei before the referendum data.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8** Polarization of the main Italian news sites for each category (yes, neutral and no)'", "CAPTION FIG9.png": "'\\n\\n**The \\\\(\\\\beta\\\\) Time series at the polar-antian and four representations**\\n\\n**Italian ring silica**'", "CAPTION TAB1.png": "'Table 1 Classification of a post-by analyzing its keywords in an \"fications\" event\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## Chapter 2 Classification of a pair of analyzing its keywords in the field\\n\\n'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c'", "CAPTION TAB4.png": "'\\n\\n**Table 4** Statistics about collected tweets'", "CAPTION TAB6.png": "'\\n\\n**Table 6**: Main co-hshtags related to yes, neutral and no tweets'", "CAPTION TAB7.png": "'\\n\\n**Table 7** Comparison of the number of users and the total number of citizens grouped by region'", "CAPTION TAB8.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB9.png": "'* [19]'"} \ No newline at end of file diff --git a/extracted_captions/marshallSearchingMissingLink1993.json b/extracted_captions/marshallSearchingMissingLink1993.json new file mode 100644 index 0000000000000000000000000000000000000000..d3e7f213022561f800ce07813129f7d910b8eb4c --- /dev/null +++ b/extracted_captions/marshallSearchingMissingLink1993.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1., Sostallized text in NotesCards.\\n\\n'", "CAPTION FIG2.png": "'Figure 2. Spatialized text in the Virtual Notebook System.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3.** Sentilized lead in Aqua'", "CAPTION FIG4.png": "'Figure 4. Proximity-based structures: (a) an aggregate, and (b) a list.\\n\\n'", "CAPTION FIG5.png": "'Figure 5. Layout and type-based structures: (a) a taxonomic set and (b) two instances of a composite.\\n\\n'", "CAPTION FIG6.png": "'Figure 6. Parsing a structure in spatialized text.\\n\\n'", "CAPTION FIG7.png": "'Figure 7. The heuristic algorithms parse a structure created in Aquanet.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/martinezquiles2004erk.json b/extracted_captions/martinezquiles2004erk.json new file mode 100644 index 0000000000000000000000000000000000000000..0d362878299f0afeaba924c698002295f017c04d --- /dev/null +++ b/extracted_captions/martinezquiles2004erk.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Contactin interacts with WASP and N-WASP. (A, part i) Map of GST-cortactin constructs. (A, part ii) Map of N-WASP constructs. (B) Pulldown assay of cortactin binding to WASP from Jurkat T-cell lysates. (Left) Proteins that bound to GSH-beads (Pull-down) and unbound proteins (Supernatants) were probed with anti-WASP MAb 5A5 (23) and with anti-WAVE polyclonal antibody (Upstate) as a control. (Right) Coomassie blue staining of the GST fusion proteins used in the pulldown assay. W.B., Western blot. (C) Cortactin binds directly to N-WASP. Recombinant baculovirus-expressed N-WASP protein was pulled down by GST-contactin fusion proteins coupled to GSH-beads. (D) Mapping of the N-WASP site for SH3-cort. Myctagged N-WASP fragments (N-WASP WG [EVH1 plus the GBD], WGP [EVH1 plus the GBD plus the proline-rich region], and VCA domains) were in vitro transcribed, translated, and used in a pulldown assay with GST-SH3-cort. Input (left) and bound (right top) proteins were Western blotted with an anti-Myc MAb (Santa Cruz). The membrane was reblotted with an anti-GST MAb (Santa Cruz) as a loading control (Ctrl.) (right bottom). (E) Pulldown of cortactin by the protein-rich region of N-WASP. A GST fusion protein containing the proline-rich region of N-WASP (right) was able to pull down cortactin, which was detected by MAb 4F11 (Upstate) (65), from 3T3 fibroblast lysates (Lys.) (left). (F) Caprecipitation of cortactin (Coet.) with WASP and N-WASP. (Left) Caprecipitation of cortactin and WASP from Jurkat cells. Cortactin immunoprecipitates (I.P.) obtained with MAb 4F11 (5 mg) were blotted with anti-WASP MAb B9 and reprorbed with anti-cortactin MAb 4F11 (both MAbs were used at 1 mg/ml). WASP immunoprecipitates obtained with MAb B9 (5 mg, Santa Cruz) were Western blotted with anti-cortactin MAb 4F11 and reprobed with anti-WASP MAb B9. (Middle) Coprecipitation of N-WASP and cortactin in 3T3-Swiss fibroblasts. N-WASP immunoprecipitates (12000 dilution of the antibody) were blotted for cortactin with MAb 4F11 and phosphospecific antibodies pY421 and pY466 (at a 1:800 dilution). (Right) Cortactin immunoprecipitates were blotted with N-WASP polyclonal antibody (1:10,000 dilution) (48) and reprobed with anti-cortactin MAb. Immunoprecipitates obtained with pY466 (5 mg) were Western blotted for cortactin, N-WASP, and pY466. All experiments were performed a minimum of three times with similar results.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Cortactin colocalizes with N-WASP and WASP at sites of active actin polymerization. (A) Cortactin colocalizes with N-WASP and F-actin at lamellipodia in bradykinin (Bradykin.)-stimulated 3T3 fibroblasts. Unstimulated (Unstim.) and bradykinin-stimulated cells were fixed, permeabilized, and then stained for F-actin with phalloidin-coumarin. Cortactin was detected with MAb 4F11, followed by goat anti-mouse-TRTTC, and N-WASP was detected with rabbit anti-N-WASP antibody, followed by goat anti-rabbit antibody Alexa 488. Cortactin and N-WASP images were merged by using Adobe Photoshop. (B) Cortactin colocalizes with WASP and F-actin at the IS. Intracellular immunofluorescence staining for cortactin, F-actin, and WASP in Jurkat T cells stimulated by SEE (5 \u03bcg/ml) and NALM6 B cells preloaded with CMAC (blue) (Top) Cells were stained for cortactin with MAb 4F11 (green) and for F-actin with phalloidin-TRTTC. (Bottom) Cells were stained for cortactin with MAb 4F11 (red) and for WASP with rabbit anti-WASP antibody H-250 (green). The images were merged (yellow) by using Adobe Photoshop. Scale bars, 10 \u03bcM. Control Jurkat T cells incubated with B cells in the absence of SEE showed very few conjugates.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: The SH3 domain of cortactin activates N-WASP-mediated Arp 2/3 complex-dependent actin polymerization. Actin (1 mM actin, actin, pyrene-actin ratio of 9:1) and the Arp 2/3 complex (33 nM) were kept constant. N-WASP at 50 nM and VCA at 100 nM were used throughout all of the experiments. All reactions were initiated by the addition of actin. (A) Plots of fluorescence intensity against time since initiation of the polymerization reaction. Consistent with previous observations (48), addition of Cdc42-GTP\\\\(\\\\times\\\\)(400 nM) resulted in partial activation of N-WASP (trace 3). SH3-corr (80 nM) had no effect by itself on the Arp2/3 complex (trace 4) but caused vigorous activation of N-WASP (trace 5). A.U., arbitrary units. (B) Dose-response curve. (C) Maximal rate of actin polymerization calculated from the initial portion of the polymerization curve. Panels B and C represent different experiments. (D) PIP2 synergizes with SH3-corr in activating N-WASP. (E) Mutation (WS25K) or deletion of SH3-corr abolished the binding of cortactin to N-WASP WGP. Pulldown assays with GST-cortactin and GST-cortactin mutants and reticulocyte lysates containing in vitro-translated, Myc-tagged N-WASP WGP (aa 1 to 396). W.B., Western blot. (F) The W525K mutation abolishes the ability of SH3-corr to activate N-WASP. The results shown are representative of three different experiments.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Effect of Erk phosphorylation of cortactin on N-WASP activation. (A) FL-corr weakly activates N-WASP. A.U., arbitrary units. (B) Erk enzyme was not present in the final cortactin preparations. Different amounts (0.2, 0.5, and 1 mg) of Erk-phosphorylated cortactin were run on a gel and Western blotted (W.B) for cortactin (top) and for Erk (bottom). As a control, 0.1 mg of commercial Erk, which represents 10 times the amount used for phosphorylating 1 mg of cortactin, was loaded in a separated lane. (C) Erk-mediated phosphorylation of cortactin enhances its activation of N-WASP. (D) Introduction of an Erk phosphorylation-mimicking double mutation (S405,418D) into cortactin enhances its ability to activate N-WASP.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Effects of mutations in FL-corr on its ability to bind N-WASP. Pulldown assays with GST-cortactin and GST-cortactin mutants and reticulocyte lysates containing in vitro-translated, Myctugged N-WASP WGP (aa 1 to 396). Input: reticulocyte lysates from the in vitro translation reaction. (A, top) Western blot (W.B.) assay with anti-Myc MAb. (A, bottom) Comanasei blue staining of GST fusion proteins eluted from equivalent amounts of GSH beads used in the pulldown assay as loading controls. Values represet the N-WASP-binding capacity of the cortactin mutants relative to that of native cortactin. (B, top) Western blot assay with anti-Myc MAb. (B, bottom) Western blot assay with anti-GST MAb. All experiments were performed three times with similar results.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Effect of Src phosphorylation of cortactin on N-WASP activation. (A) Src enzyme was not present in the final cortactin preparations. Different amounts (0.2, 0.5, and 1 mg) of Src-phosphorylated cortactin were run on a gel and Western blotted for cortactin (top) and Src (bottom). As a control, 3 U of commercial Src, which represents 20 times the amount used for phosphorylating 1 mg of cortactin, was loaded into a separate line. W.B, Western blot. (B) Src-mediated phosphorylation of Erk-phosphorylated cortactin inhibits its ability to activate N-WASP. A.U., arbitrary units. (C) Src-mediated phosphorylation of S405,418D mutant cortactin inhibits its ability to activate N-WASP. Introduction of an Src-mimicking double mutation (Y466,482D) into Erk-mimicking S405,418D mutant cortactin inhibits its capacity to activate N-WASP. (D) Introduction of a single Src-mimicking mutation (Y421D) into Erk-mimicking S405,418D mutant cortactin inhibits its capacity to activate N-WASP. (E) Introduction of the Src-mimicking triple mutation Y421,Y466,Y482 into S405,418D mutant cortactin inhibits its activation of N-WASP. All experiments were performed three times with similar results.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/extracted_captions/mccall2017locus.json b/extracted_captions/mccall2017locus.json new file mode 100644 index 0000000000000000000000000000000000000000..18843aa976ececfdca4a269281ee5fd0479f9d8c --- /dev/null +++ b/extracted_captions/mccall2017locus.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Identifying a LC input to the BLA. (**A**) Cartoon depicting fluorogald tracing strategy. (**B**) Representative image (selected from three injected mice) shows robust retrograde labeling of the LC from injection in the BLA (green = pseudocolored Fluorgold, tyrosine hydroxylase = red). Arrowhead indicates example co-localization. Scale bar = 100 mm. 4V = 4th ventricle. The TH cells dorsal and ventral to the LC are likely part of the medial paraprachial nucleus which has previously identified projections to the BLA (Saper and Loewy, 1980). (**C**) Cartoon depicting dual injection tracing strategy for CTB-594 and DIO-CHR2-eYFP. (**D**) Representative images (selected from three injected mice) shows retrograde labeling in LC of red vertebodcasts and anteroprade labeling of TH+ cells (green) (Nisal-blue). Arrowhead indicates example co-localization. Scale bar = 100 mm (**E**) Cartoon depicting anterograde tracing strategy. (**F**\u2013**H**) Coronal images depict robust eYFP (yellow) labeling in the LC (F and G) and BLA (**H**) of the same mouse (scale bars = (**F**) 50 mm, (**G**) 10 mm, (**H**) 20 mm. Inset (**F**), tyrosine hydroxylase = red, scale bar = 25 mm.\\n\\nDOI: 10.7554/eLife.18247.002\\n\\nFigure 1: Identifying a LC input to the BLA. (**A**) Cartoon depicting fluorogald tracing strategy. (**B**) Representative image (selected from three injected mice) shows robust retrograde labeling of the LC from injection in the BLA (green = pseudocolored Fluorgold, tyrosine hydroxylase = red). Arrowhead indicates example co-localization. Scale bar = 100 mm. 4V = 4th ventricle. The TH cells dorsal and ventral to the LC are likely part of the medial paraprachial nucleus which has previously identified projections to the BLA (Saper and Loewy, 1980). (**C**) Cartoon depicting dual injection tracing strategy for CTB-594 and DIO-CHR2-eYFP. (**D**) Representative images (selected from three injected mice) shows retrograde labeling in LC of red vertebodcasts and anteroprade labeling of TH+ cells (green) (Nisal-blue). Arrowhead indicates example co-localization. Scale bar = 100 mm (**E**) Cartoon depicting anterograde tracing strategy. (**F**\u2013**H**) Coronal images depict robust eYFP (yellow) labeling in the LC (F and G) and BLA (**H**) of the same mouse (scale bars = (**F**) 50 mm, (**G**) 10 mm, (**H**) 20 mm. Inset (**F**), tyrosine hydroxylase = red, scale bar = 25 mm.\\n\\nDOI: 10.7554/eLife.18247.002\\n\\nThe following figure supplement is available for figure 1:'", "CAPTION FIG2.png": "'Figure 2: Photostimulation of LC terminals in the BLA releases norepinephrine. (**A**) LC neuron firing reliably to 10 Hz optical stimulation (**C**C**-**whole cell current clamp). (**B**) Fast scan cyclic voltammetry (**F**SCV) schematic. (**C**-**D**) Oxidative and reductive currents (_**scale** bar 2 s by 0.4 nA), with representative cyclic voltammograms (inset) and representative color plots (_**below**_) in response to photostimulation are attenuated by reserpine (1 uM). Color plots for baseline and after reserpine (1 uM): Flies were collected over 15 s (**C**-**axis) where the carbon fiber microelectrode was ramped with a triangular waveform from \\\\(-\\\\)0.4V to 1.3V and back to \\\\(-\\\\)0.4V at 400 V/5 (**C**-**) and sampled at 10 Hz: 10 Hz, 473 nm blue LED stimulation onset at 2 s. Oxidative currents (**A**) are positive in direction and reductive currents are negative (see color coded scale bar on right). (**E**) Attenuation in NE oxidative current in response to reserpine (1 uM) = 3 pairs; mean \\\\(\\\\pm\\\\) S.E.M). (**F**) Average of first 20 min and last 15 min in (**E**) (Data represented as mean \\\\(\\\\pm\\\\) SEM, Paired Student\u2019s t-tests to baseline, Mean difference = 68.5d, \\\\(\\\\tau_{\\\\text{Q}}\\\\) = 18.75, **p=0.0028, 95% CI [52.82 to 84.29]).\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: Photostimulation of LC terminals in the BLA alters neuronal activity. **(A)** Schematic illustrating single-unit extracellular recording paradigm of BLA neurons modulated by ChR2-expressing LC-BLA terminals. **(B)** Representative principal component analysis plot showing the first two principal components with clear clustering of a single unit (macroion) from the noise (grey). Inset shows the waveform and spikes making up the isolated unit. Y-scale is 150 microvolts and x-scale is 500 ms. **(C)** Recordings from eight hemispheres of six Th-CreI-ChACHE mice show the distribution of firing rates. Figure 3 continued on next page\\n\\n'", "CAPTION FIG3-2.png": "\"Figure 3 continued\\n\\npresent in BLA neurons prior to and following LC-BLA terminal photosttimulation (473 nm, 5 Hz, 3 min). (**D**) Average normalized firing rate of neurons that increase (maroon), decrease (grey), or do not change (black) firing rate in response to photosttimulation. Inset, shows number of neurons in each group. Representative histograms (1 s bins) of isolated single-units showing increase (**E**) or decrease (**F**), or no change (**G**) in neuronal firing in response to photosttimulation (473 nm, 5 Hz, 3 min). Z-second population responses of neurons showing increase (**H**) or decrease (**I**), or no change (**J**) in neuronal firing in response to photosttimulation. (**R**) Response latency following onset of photosttimulation for calls that did not alter firing (=) (n = 29), increased firing (+) (n = 9), or decreased firing (+) (n = 4). (Data represented as mean +- SD). (**L**) The same calls sorted by baseline firing rate. (Data represented as mean +- SEM). Kruskal-Wallis test one-way ANOVA for non-parametric data with Dunn's multiple comparisons test, Kruskal-Wallis statistic = 6.536, p=0.0381; + vs. - Mean rank difference = 18.75, adjusted p=0.0329; + vs. + Mean rank difference = -6.828, adjusted p=0.0341; - vs. - Mean rank difference = 11.92, adjusted p=0.2053) (**M**) Waveform similarity, within group distribution of linear correlations. Inset, every average waveform for each recorded unit separated by response profile (= black, + maroon, - grey). (**N-P**) Bursting profiles for each recorded neuron. (**N**) Number of bursts per second. (Data represented as mean +- SEM). (**D**) Mean firing rate within bursts for each neuron. (Data represented as mean +- SEM). (**P**) Proportion of recorded spikes that occurred during bursts. (Data represented as mean +- SEM). (**D**): 10.7554/eLife.18247.005\\n\\nThe following figure supplement is available for figure 3:\\n\\n**Figure supplement 1.** Photosttimulation of LC terminals in the BLA alters neuronal activity.\\n\\n\"", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG4.png": "\"\\n\\n**Figure 4.** Photostimulation of LC terminals in the BLA preferentially activates BLA circuitry associated with anxiety-like behavior. (**A**) Diagram of viral and optogenetic strategy. (**B**) 5 Hz photostimulation increases cfos expression within the BLA in \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2+ animals compared to \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2- controls (Data represented as mean +- SEM, \\\\(n\\\\) = 9 ChR2, \\\\(n\\\\) = 4 Ctrl; average of 3 sections/mouse; Student's t-test, Mean difference = 19.17, \\\\(\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}}}}}}}}}}}}}}\\\\) = 4.05, **p**=0.0040, 95% CI [-35.47 to -10.11]. (**C**) 5 Hz photostimulation increases cfos expression significantly more in BLA neurons projecting to the uHPC and CaA compared to NAA in \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2 animals (Data represented as mean +- SEM, \\\\(n\\\\) = 9 uHPCCTR1, \\\\(n\\\\) = 4 CaACTR1, \\\\(n\\\\) = 9 CaACTR1, _3 sections per mouse; One-Way ANOVA, Bonferroni's Multiple Comparison Test, \\\\(F\\\\)2,20 = 7.199, **p=0.0044; \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2+-CTB-VtPC vs. \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2:CTB-NAc Mean difference = 12.95, \\\\(\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}}}}}}}}}}\\\\) = 3.585, **p**<0.01 95% CI [3.51 to 22.39% \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2:CTB-NAc Mean difference = 11.25, \\\\(\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}_{\\\\text{t}}}}}}}}\\\\) = 2.802, *p<0.05 95% CI [0.7405 to 21.74]. Representative images of the BLA expressing cfos after 5 Hz photostimulation in (**D**) \\\\(\\\\text{Th}^{\\\\text{RES-Cro}::LC}\\\\)-BLASTa.ChR2 injected with CTB in NAa, (**E**) uHPC, or (**F**) CaA. Scale bar, 100 um. (**G**) Confocal images showing colocalization of CTB and cFos after 5 Hz photostimulation in the NAa, uHPC, and CaA. Scale bar, 50 um.\\n\\nDot: 10.7554/eLife.18247.007\\n\\nThe following figure supplement is available for figure 4:\\n\\n**Figure supplement 1.** Photostimulation of LC terminals in the BLA preferentially activates BLA circuitry associated with negative affect.\\n\\n\"", "CAPTION FIG5.png": "'Figure 5: Photoestimation of LC terminals in the BLA causes conditioned aversion. **(A)** Cartoon of viral and fiber optic delivery strategy and calendar of real-time place testing studies. **(B)** Representative traces of behavior at different frequencies. **(C)** Frequency response of RTPT and **(D)** locomotor activity at 5 and 60 Hz. Data represented as mean +- SEM, \\\\(n\\\\) = 4 eVFP, 5 ChR2. **(E)** Conditioned place aversion (CPA) behavioral calendar. **(F)** Representative CPA traces. **(G)** \\\\(\\\\text{T}_{\\\\text{H}}^{\\\\text{S}\\\\text{S}\\\\text{C}\\\\text{C}\\\\text{C}\\\\text{C}\\\\text{BLA}\\\\text{C}\\\\text{C}\\\\text{BLA}\\\\text{C}\\\\text{R}\\\\text{\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Photostimulation of LC terminals in the RLA promotes anxiety-like behavior through beta-adrenergic receptors. (**A**) Calendar of OFT studies. (**B**) Representative heat maps of activity during OFT. (**C**) S Hz photostimulation causes an anxiety-like phenotype in OFT of \\\\(T^{\\\\rm{HEES}Ca::LC-BLAChR2}\\\\) animals compared to \\\\(T^{\\\\rm{HEES}Ca::LC-BLAChR2}\\\\) controls [Data represented as mean \\\\(\\\\pm\\\\) SEM, \\\\(n\\\\) = 10 cyrP, 11 ChR2; Student\u2019s t-test, Mean difference = 116.9, t(t) = 3.46, **+p=0.0026, 95% CI [46.20 to 187.5] with (**D**) no change in locomotor activity [Data represented as mean \\\\(\\\\pm\\\\) SEM]. (**E**) Cartoon of viral, cannula, and fiber optic delivery strategy and (**F**) calendar of EZM behavior. (**G**) S Hz photostimulation causes an anxiety-like phenotype in ELM of \\\\(T^{\\\\rm{HEES}Ca::LC-BLAChR2}\\\\) animals compared to \\\\(T^{\\\\rm{HEES}Ca::LC-BLAChR2}\\\\) animals compared to \\\\(T^{\\\\rm{HEES}Ca::LC-BLAChR2}\\\\) controls, which is reversed by intra-BLA propranolol pretreatment [Data represented as mean \\\\(\\\\pm\\\\) SEM], \\\\(n\\\\) = 11 cyrP + Vehicle, \\\\(n\\\\) = 0 CHR2 + P propranolol, \\\\(n\\\\) = 8 CHR2 + P propranolol, \\\\(n\\\\) = 8 CHR2 + P propranolol, \\\\(n\\\\) = 8 CHR2 + P propranolol, \\\\(n\\\\) = 10 CHR2 + Pranolol, \\\\(n\\\\) = 10 CHR2 + Pranolol, \\\\(n\\\\) = 10 CHR2 + Pranolol, \\\\(n\\\\) = 8 CHR2 + Pranolol, \\\\(n\\\\) = 10 CHR2 + Pranolol, \\\\(n\\\\) = 8 CHR2 + Pranolol, \\\\(n\\\\) = 10 CHR2 + Pranolol, \\\\(n\\\\) = 8 CHR2 + Pranolol, \\\\(n\\\\) = 11 CHR2 + Pranolol, \\\\(n\\\\) = 12 CHR2 + Pranolol, \\\\(n\\\\) = 12 CHR2 + Pranolol, \\\\(n\\\\) = 13 CHR2 + Pranolol, \\\\(n\\\\) = 14 CHR2 + Pranolol, \\\\(n\\\\) = 15 CHR2 + Pranolol, \\\\(n\\\\) = 16 CHR2 + Pranolol, \\\\(n\\\\) = 17 CHR2 + Pranolol, \\\\(n\\\\) = 18 CHR2 + Pranolol, \\\\(n\\\\) = 19 CHR2 + Pranolol, \\\\(n\\\\) = 11 CHR2 + Pranolol, \\\\(n\\\\) = 12 CHR2 + Pranolol, \\\\(n\\\\) = 18 CHR2 + Pranolol, \\\\(n\\\\) = 19 CHR2 + Pranolol, \\\\(n\\\\) = 12 CHR2 + Pranolol, \\\\(n\\\\) = 19 CHR2 + Pranolol, \\\\(n\\\\) = 14 CHR2 + Pranolol, \\\\(n\\\\) = 15 CHR2 + Pranolol, \\\\(n\\\\) = 16 CHR2 + Pranolol, \\\\(n\\\\) = 17 CHR2 + Pranolol, \\\\(n\\\\) = 18 CHR2 + Pranolol, \\\\(n\\\\) = 19 CHR2 + Prazolam, \\\\(n\\\\) = 19 CHR2 + Prazolam, \\\\(n\\\\) = 19 CHR2 + Prazolam, \\\\(n\\\\) = 18 CHR\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/mcfarland2008rna.json b/extracted_captions/mcfarland2008rna.json new file mode 100644 index 0000000000000000000000000000000000000000..b6c7d780552eaf449135e18dec21339f5cab73ec --- /dev/null +++ b/extracted_captions/mcfarland2008rna.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Dose-dependent inhibition of AEA uptake and SKM 4-45-1 internalization by AM404. Both dCAD (\\\\(\\\\blacksquare\\\\)) and dCAD (eiref) cells were treated with increasing concentrations of AM404 for 10 min at 37\u00b0C. A, after the incubation with AM404, [H]AEA was added at a final concentration of 1 nM as described under _Materials and Methods_. Data shown are means \\\\(\\\\pm\\\\) S.E.M. and are representative of four separate experiments performed in triplicate. _K_i values (means \\\\(\\\\pm\\\\) S.E.M., \\\\(n\\\\) = 3) were 850 \\\\(\\\\pm\\\\) 600 nM (dCAD) and 930 \\\\(\\\\pm\\\\) 150 nM (dCAD). The cpm values for cCAD cells were total = 6440 \\\\(\\\\pm\\\\) 914 and nonspecific = 850 \\\\(\\\\pm\\\\) 86. The cpm values for dCAD cells were total = 5124 \\\\(\\\\pm\\\\) 1456 and nonspecific = 794 \\\\(\\\\pm\\\\) 35. B, cells were treated with SKM 4-45-1 (25 mM) and increasing concentrations of AM404 for 10 min, and fluorescence was determined on a FUSION microplate reader (PerkinElmer Life and Analytical Sciences). IC30 values were converted to _K_i values using the Cheng-Prusoff equation (_K_i value for SKM 4-45-1 = 16 mM). _K_i values (means \\\\(\\\\pm\\\\) S.E.M.; \\\\(n\\\\) = 31) were 24 \\\\(\\\\pm\\\\) 12 mM (cCAD) and 16 \\\\(\\\\pm\\\\) 10 mM (dCAD). Data shown are representative of three separate experiments performed in duplicate. Arbitrary fluorescence unit values for dCAD cells were total = 13,858 \\\\(\\\\pm\\\\) 1856, nonspecific = 7321 \\\\(\\\\pm\\\\) 573, and background = 4313 \\\\(\\\\pm\\\\) 625. Arbitrary fluorescence unit values for dCAD cells were total = 23,137 \\\\(\\\\pm\\\\) 6590, nonspecific = 11,309 \\\\(\\\\pm\\\\) 4024, and background = 2006 \\\\(\\\\pm\\\\) 74.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Expression of FAAH in CAD cells. A, eCAD and dCAD cells express FAAH. Western blots were performed as described under Materials and Methods. Data are representative of three separate experiments. B, cCAD and dCAD cells were homogenized and then incubated at \\\\(\\\\pm\\\\)7\u00b0C for 10 min. Cell homogenates were then incubated with 5 nM [3H]AEA in the presence or absence of 600 nM MAFP. After the incubation, the reaction was terminated, and the aqueous and organic phases were separated. Bars represent AEA-derived [3H]ethanalanine present in the aqueous phase (i.e., metabolized NEA) after the termination of the reaction as a direct measure of FAAH activity. Data shown are means \\\\(\\\\pm\\\\) S.D. of three separate experiments performed in duplicate.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 1.** Effect of lipid raft disruption on \\\\(\\\\Lambda\\\\)EA uptake in dCAD cells. dCAD cells were pretreated for 30 min with 25 mg/ml nystatin and 10 mg/ml progesterone (NP treatment). After NP treatment, AEA uptake assays were performed in 1\\\\(\\\\times\\\\) KRH at 37degC in the presence or absence of 100 mM AM404 to define nonspecific transport. Transport assays were performed for 5 min with 1 nM [\\\\(\\\\Gamma\\\\)H]\\\\(\\\\Lambda\\\\)EA as described under Materials and Methods. Data are means \\\\(\\\\pm\\\\) S.E.M. combined from three separate experiments performed in triplicate. The cpm values for control cells were total = 5755 \\\\(\\\\pm\\\\) 118 and nonspecific = 502 \\\\(\\\\pm\\\\) 41. The cpm values for NP-treated cells were total = 3117 \\\\(\\\\pm\\\\) 40 and nonspecific = 729 \\\\(\\\\pm\\\\) 61. Inset, offset of NP treatment on membrane cholesterol content. Cholesterol content in CAD cell membranes was determined as described under Materials and Methods. Data are means \\\\(\\\\pm\\\\) S.E.M. from two separate experiments performed in triplicate. *, \\\\(p\\\\)\\\\(<\\\\) 0.05.\\n\\n'", "CAPTION FIG4.png": "'and dynamic 2. dCAD cells were transfected with Stealth RNA (Invitrogen) using Lipofectamine 2000 (Invitrogen) and then incubated at 37degC in serum-free media for 72 h to allow for protein degradation. The expression of both the b2 subunit of the AP2 complex and the protein dynamic 2 was decreased by >90% compared with mock siRNA (nonsense sequence) transfection. siRNA oligonucleotides had no effect on the level of control protein b-actin (data not shown). To control for protein loading, equal amounts of total protein (23 mg) from the cell lysates were added to each well for Western blot analysis as described under Materials and Methods. Both proteins resolved at the expected molecular weights. Data are representative of three separate experiments.\\n\\nFigure 4: RNA-mediated knockdown of the b2 subunit of the AP2 complex and dynamic 2. dCAD cells were transfected with Stealth RNA (Invitrogen) using Lipofectamine 2000 (Invitrogen) and then incubated at 37\u00b0C in serum-free media for 72 h to allow for protein degradation. The expression of both the b2 subunit of the AP2 complex and the protein dynamic 2 was decreased by >90% compared with mock siRNA (nonsense sequence) transfection. siRNA oligonucleotides had no effect on the level of control protein b-actin (data not shown). To control for protein loading, equal amounts of total protein (23 mg) from the cell lysates were added to each well for Western blot analysis as described under Materials and Methods. Both proteins resolved at the expected molecular weights. Data are representative of three separate experiments.\\n\\n'", "CAPTION FIG5.png": "\"Figure 5: Effect of RNA knockdown of dynamic 2 and the g2 subunit of the AP2 complex on the internalization of 488-Tf in dCAD cells. Seventy-two hours after transfection with either mock, (g2 subunit, or dyramine 2 siRNA, dCAD cells were incubated with 488-Tf for 10 min and imaged by confocal microscopy. Both g2 subunit- and dyramine 2 siRNA-transfected cells showed a reduction of 488-Tf internalization (indicated by arrows) compared with mock-transfected dCAD cells. Images were obtained using confocal microscopy as described under Materials and Methods. The internal fluorescence intensity of the intracellular space of all cells in the field of view was quantified using MetaMorph software (Molecular Devices, Sunnyvala, CA) (bottom right). Fifteen to 20 cells per experiment were analyzed. Data are representative of three separate experiments. Asterisks deeta a significant difference in intracellular mean internal fluorescence intensity compared with mock-transfected cells (analysis of variance and Dunnett's multiple comparison test; \\\\(p<0.01\\\\)).\\n\\n\"", "CAPTION FIG6.png": "\"Figure 5: Effect of RNAi knockdown of dynamin 2 and the g2 subunit of the AP2 complex on the internalization of 488-CT in dCAD cells. Seventy-two hours after transfection with either mock, g2 subunit, or dyananin 2 siRNA, dCAD cells were incubated with 488-CT for 10 min and imaged by confocal microscopy. Dynamin 2 siRNA-transfected cells showed a reduction of 488-CT internalization (indicated by arrows) compared with neck-transfected dCAD cells. g2 subunit siRNA-transfected cells did not display any difference in 488-CT internalization compared with neck-transfected dCAD cells, confirming the specificity of the treatments. Images were obtained using confocal microscopy as described under Materials and Methods. The internal fluorescence intensity of the intracellular space of all cells in the field of view was quantified using MetaMorph software (bottom right). Fifteen to 20 cells per experiment were analyzed. Data are representative of three separate experiments. Asterisks denote a significant difference in intracellular mean fluorescence intensity compared with neck-transfected cells (analysis of variance and Dannett's multiple comparison test; \\\\(p<0.05\\\\)).\\n\\n\"", "CAPTION FIG7.png": "'Figure 7: SKM 4-45-1 internalization by dCAD cells after siRNA transfection. dCAD cells were treated with SKM 4-45-1 (35 \\\\(\\\\mu\\\\)M) for 10 min at 72 h after transfection with mock, b2 subunit, or dynamin 2 siRNA. Knockdown of the b2 subunit of the AP2 complex had no effect on the SKM 4-45-1-derived fluorescence signal compared with mock. Knockdown of dynamin 2 in dCAD cells significantly decreased the accumulation of SKM 4-45-1-derived fluorescence. Images were obtained using confocal microscopy as described under Materials and Methods. Phase contrast images are shown below each corresponding fluorescence image. Data are representative of three separate experiments.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: \\\\({}^{3}\\\\)H\\\\(\\\\|\\\\)MEA uptake by dCAD cells after siRNA transfection. Seventy-two hours after transfection with mock, dynamic 2, or g2 subunit siRNA, \\\\({}^{3}\\\\)H\\\\(\\\\|\\\\)MEA uptake assays were performed in 1\\\\(\\\\times\\\\) KRH at 37\\\\({}^{\\\\circ}\\\\)C as described under Materials and Methods. AM404 (100 pM) was used to define nonspecific transport. Data represent maan \\\\(\\\\pm\\\\) S.E.M. for three separate experiments performed in triplicate. The epm values for mock cells were total = 2887 \\\\(\\\\pm\\\\) 136 and nonspecific = 1126 \\\\(\\\\pm\\\\) 128. The epm values for dynamin 2 knockdown cells were total = 2973 \\\\(\\\\pm\\\\) 89 and nonspecific = 505 \\\\(\\\\pm\\\\) 83. The epm values for g2 subunit knockdown cells were total = 2638 \\\\(\\\\pm\\\\) 104 and nonspecific = 717 \\\\(\\\\pm\\\\) 27.\\n\\n'", "CAPTION FIG9.png": "'\\n\\n**Fig. 9.** Metabolism of \\\\({}^{1}\\\\)HJMEA in dynamic 2 siRNA-transfected dCAD cells. A, 72 h after transfection with either mock or dynamic 2 siRNA, dCAD cells were treated with [\\\\({}^{3}\\\\)H]AEA for 5 min and then lysed. Cellular lipids were extracted from the cell lysates using chloroform/methanol and analyzed by TLC for intact [\\\\({}^{3}\\\\)H]AEA. Neaspecific uptake was determined using 100 mM AM404. The graph represents data from three separate experiments. Statistical analysis was performed using a one-sample \\\\(t\\\\) test comparing with the value of 100 (mock control). *, \\\\(p<0.05\\\\). The cpm values from the TLC analysis for mock cells were total = 2286 + 785 and nonspecific = 2243 + 407. The cpm values from the TLC analysis for dynamic 2 knockdown cells were total = 3257 + 545 and nonspecific = 1793 + 198. B, PAH expression after RNAi-mediated knockdown of dynamic 2 in cCAD cells was performed as described under Materials and Methods except a1LantFact Lipid Reagent (Bio-Rad) was used as the transfection reagent for 48 h with 10 nM siRNA oligon. Western blots were performed using the polyclonal dynamin 2 antibody, monoclonal FAAH antibody (1:1000; Abnova Corporation, Taipe City, Taiwan), monoclonal actin antibody (1:2000; Sigma-Aldrich, St. Louis, MO). C, FAAH activity after RNAi-mediated knockdown of dynamic 2. FAAH activity assays were performed on whole cell lysates as described under Materials and Methods. Data shown represent means \\\\(\\\\pm\\\\) S.D. from two experiments performed in quadruplicate.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/mezgueldi2002kinetic.json b/extracted_captions/mezgueldi2002kinetic.json new file mode 100644 index 0000000000000000000000000000000000000000..107008aeda8229fb041732ed9060ce7cb8b69640 --- /dev/null +++ b/extracted_captions/mezgueldi2002kinetic.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1. Purification of myo1e1g.** Coomassie-strained SDS-polyacrylamide gel showing purified myo1e1g. The associated light chain is CaM. Quantitative densitometry shows that the CaM:heavy chain stoichiometry was 1:1. The predicted molecular mass of the heavy chain from the amino acid sequence is 83 kDa.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: **Models for myo1a function.**\\\\(A\\\\), the predominant ATPase pathway of myo1a1d under in vitro assay conditions is _adilived_. We propose that under cellular conditions, native myo1a follows the pathway proposed by Lyman and Taylor (43; gray shading). \\\\(B\\\\), comparison of the loop-2 regions of myceins isoforms. Basic amino acids are _boxed_, and the coarserved KK regions shown to be necessary for actin activation of \\\\(P_{i}\\\\) are _underlined_. _Numbers_ identify the sequence positions. _SnH_, smooth muscle myosin-II; _SbH_, skeletal muscle myosin-II; _DbH_, _Dc-yosteluva myosin-II; _ACMIC_, _Acaethavaoetha myosin-IC.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Steady-state ATPase and actin binding activity of myo1e\\\\({}^{\\\\bf 102}\\\\).** The actin concentration dependence of the steady-state turn-over of 50 m myo1e\\\\({}^{\\\\rm kd}\\\\) in KMgO \\\\(\\\\blacksquare\\\\) and KMg50 \\\\(\\\\bullet\\\\) was measured using the NADH-coupled assay. The data points represent acquisitions from three different preparations, and the solid lines are best fits to rectangular hyperbolas. The fraction of myo1e\\\\({}^{\\\\bf 102}\\\\) bound to actin in KMgO \\\\(\\\\bullet\\\\) in 2 mm ATP was measured by cosedimentation.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3. Kinetics of myo1e34 association with actin filaments.**_A_, concentrating dependence of the observed rate (\\\\(k_{\\\\rm ad}\\\\)) of pyrene-actin binding to 0.2 mM myo1e34 in the absence (\\\\(\\\\blacksquare\\\\)) and presence (\\\\(\\\\blacklozenge\\\\)) of 1 mM MgADP in KMgSO. _Solids lines_ are linear fits to the data. \\\\(B\\\\), time course of pyrene-actin fluorescence increase after mixing 0.4 mM pyrene-actinyla1e3 with 50 mM actin in the absence (\\\\(upper\\\\) curve) and presence (\\\\(lower\\\\) curve) of 1 mM MgADP. _S_onoid lines are the best fits of the data to single exponentials.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **dmantATP binding to myo1e\\\\({}^{1q}\\\\) and actomyosino\\\\({}^{1q}\\\\).**_A_, rate of dmantATP binding to 0.5 \\\\(\\\\mu\\\\)m myo1e\\\\({}^{1q}\\\\) and B, dmantATP binding to 0.5 \\\\(\\\\mu\\\\)m actomyosino\\\\({}^{1q}\\\\) as a function of nucleotide concentration. The observed rates \\\\(\\\\langle k_{\\\\rm adn}\\\\rangle\\\\) were obtained by fitting the stopped flow fluorescence data at each nucleotide concentration to the sum of two exponentials. Apparent second order association rate constants were determined from the slopes of the linear fits of the fast phase of the two exponential fits (solid lines). The insets show the nucleotide dependence of the slow phase of the two exponential fits.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**References**\\n\\n[1]\\n\\n**References**\\n\\n[2]\\n\\n**References**\\n\\n[3]\\n\\n**References**\\n\\n[4]\\n\\n**References**\\n\\n[5]\\n\\n**References**\\n\\n[6]\\n\\n**References**\\n\\n[7]\\n\\n**References**\\n\\n[8]\\n\\n**References**\\n\\n[9]\\n\\n**References**\\n\\n[10]\\n\\n**References**\\n\\n[11]\\n\\n**References**\\n\\n[12]\\n\\n**References**\\n\\n[13]\\n\\n**References**\\n\\n[14]\\n\\n**References**\\n\\n[15]\\n\\n**References**\\n\\n[16]\\n\\n**References**\\n\\n[17]\\n\\n**References**\\n\\n[18]\\n\\n**References**\\n\\n[19]\\n\\n**References**\\n\\n[20]\\n\\n**References**\\n\\n[21]\\n\\n**References**\\n\\n[22]\\n\\n**References**\\n\\n[23]\\n\\n**References**\\n\\n[24]\\n\\n**References**\\n\\n[25]\\n\\n**References**\\n\\n[26]\\n\\n**References**\\n\\n[27]\\n\\n**References**\\n\\n[28]\\n\\n**References**\\n\\n[29]\\n\\n**References**\\n\\n[30]\\n\\n**References**\\n\\n[31]\\n\\n**References**\\n\\n[32]\\n\\n**References**\\n\\n[33]\\n\\n**References**\\n\\n[34]\\n\\n**References**\\n\\n[35]\\n\\n**References**\\n\\n[36]\\n\\n**References**\\n\\n[37]\\n\\n**References**\\n\\n[38]\\n\\n**References**\\n\\n[39]\\n\\n**References**\\n\\n[40]\\n\\n**References**\\n\\n[41]\\n\\n**References**\\n\\n[42]\\n\\n**References**\\n\\n[43]\\n\\n**References**\\n\\n[44]\\n\\n**References**\\n\\n[45]\\n\\n**References**\\n\\n[46]\\n\\n**References**\\n\\n[47]\\n\\n**References**\\n\\n[48]\\n\\n**References**\\n\\n[49]\\n\\n**References**\\n\\n[50]\\n\\n**References**\\n\\n[51]\\n\\n**References**\\n\\n[52]\\n\\n**References**\\n\\n[53]\\n\\n**References**\\n\\n[54]\\n\\n**References**\\n\\n[55]\\n\\n**References**\\n\\n[56]\\n\\n**References**\\n\\n[57]\\n\\n**References**\\n\\n[58]\\n\\n**References**\\n\\n[59]\\n\\n**References**\\n\\n[60]\\n\\n**References**\\n\\n[61]\\n\\n**References**\\n\\n[62]\\n\\n**References**\\n\\n[63]\\n\\n**References**\\n\\n[64]\\n\\n**References**\\n\\n[65]\\n\\n**References**\\n\\n[66]\\n\\n**References**\\n\\n[67]\\n\\n**References**\\n\\n[68]\\n\\n**References**\\n\\n[69]\\n\\n**References**\\n\\n[70]\\n\\n**References**\\n\\n[71]\\n\\n**References**\\n\\n[72]\\n\\n**References**\\n\\n[73]\\n\\n**References**\\n\\n[74]\\n\\n**References**\\n\\n[75]\\n\\n**References**\\n\\n[76]\\n\\n**References**\\n\\n[77]\\n\\n**References**\\n\\n[78]\\n\\n**References**\\n\\n[79]\\n\\n**References**\\n\\n[80]\\n\\n**References**\\n\\n[90]\\n\\n**References**\\n\\n[91]\\n\\n**References**\\n\\n[92]\\n\\n**References**\\n\\n[93]\\n\\n**References**\\n\\n[94]\\n\\n**References**\\n\\n[95]\\n\\n**References**\\n\\n[96]\\n\\n**References**\\n\\n[97]\\n\\n**References**\\n\\n[98]\\n\\n**References**\\n\\n[99]\\n\\n**References**\\n\\n[99]\\n\\n**References**\\n\\n[90]\\n\\n**References**\\n\\n[91]\\n\\n**References**\\n\\n[92]\\n\\n**References**\\n\\n[93]\\n\\n**References**\\n\\n[94]\\n\\n**References**\\n\\n[95]\\n\\n**References**\\n\\n[96]\\n\\n**References**\\n\\n[97]\\n\\n**References**\\n\\n[98]\\n\\n**References**\\n\\n[99]\\n\\n**References**\\n'", "CAPTION FIG6.png": "'\\n\\n**Fig. 6. Phosphate burst of myo1e\\\\({}^{16}\\\\).** Time course of ADP-P, formation by 2.0 ms myo1e\\\\({}^{16}\\\\) in the absence of actin measured by quenched flow after mixing with 50 ms ATP. The smooth flow is the best fit of the data to a single exponential (\\\\(\\\\delta_{-+}=108\\\\) s\\\\({}^{-1}\\\\)).\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Rate of P\\\\({}_{i}\\\\) release from myo1e1g.**\\\\(A\\\\), time course of transient P\\\\({}_{i}\\\\) release from myo1e1g after mixing with ATP in a sequential mix, single turnover experiment in KMg50. The smooth curve is a single exponential fit to the data. The inset is an expanded region of the transient showing the absence of a lag phase. \\\\(\\\\dot{R}\\\\), actin concentration dependence on the rate of phosphate release measured in KMg50 (\\\\(\\\\blacksquare\\\\)) and KMg50 (\\\\(\\\\bullet\\\\)). Delay time after first mix was 400 ms. Final concentrations at \\\\(t=0\\\\) were 1.0 \\\\(\\\\mu\\\\)m myo1e1g, 0.5 \\\\(\\\\mu\\\\)m ATP, 5.0 \\\\(\\\\mu\\\\)m P,BP, 0\\\\(-\\\\)40 \\\\(\\\\mu\\\\)m actin.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: **Kinetics of mantADP binding to myole\\\\({}^{\\\\rm 16q}\\\\).**\\\\(A\\\\), rate of mantADP binding to 0.5 \\\\(\\\\mu\\\\)m myo1a\\\\({}^{\\\\rm 16q}\\\\) as a function of nucleotide concentration measured using fluorescence resonance energy transfer. The observed rates \\\\(\\\\langle k_{ab}\\\\rangle\\\\) were obtained by fitting the stopped flow fluorescence transients at each nucleotide concentration to the sum of two exponentials. The solid line is the best fit of the fast \\\\(k_{ab}\\\\) (\\\\(\\\\Theta\\\\)) to a rectangular hyperbola. The inset shows the nucleotide concentration dependence of the slow \\\\(k_{ab}\\\\) (\\\\(\\\\blacksquare\\\\)). B, fluorescence transient showing the time course of mantADP dissociation from myo1a\\\\({}^{\\\\rm 16q}\\\\). An equilibrated mixture of 10 \\\\(\\\\mu\\\\)m mantADP and 0.6 \\\\(\\\\mu\\\\)m myo1a\\\\({}^{\\\\rm 16q}\\\\) was mixed with 2.5 mm ATP. The smooth line shows the best fit of the data to the sum of two exponentials.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: **Kinetics of mantADP binding to actomyosin10.**_A_, rate of mantADP binding to 0.5 \\\\(\\\\mu\\\\)m actomyosin10\\\\({}^{\\\\mathrm{10}}\\\\) as a function of nucleotide concentration. The observed rates \\\\(\\\\langle k_{\\\\mathrm{ind}}\\\\rangle\\\\) were obtained by fitting the stopped flow fluorescence data to a single exponential. The solid line is the best linear fit of \\\\(k_{\\\\mathrm{ind}}\\\\). The fluorescence transient in the inset shows the dissociation of mantADP from actomyosin10\\\\({}^{\\\\mathrm{10}}\\\\) obtained by mixing 2.5 mm ATP with an equilibrated mixture of 10 \\\\(\\\\mu\\\\)m mantADP and 0.5 \\\\(\\\\mu\\\\)m actomyosin10\\\\({}^{\\\\mathrm{10}}\\\\). The smooth line is a single exponential fit to the data. \\\\(B\\\\), dependence on the rate of 80 \\\\(\\\\mu\\\\)m ATP banding to pyrene-actomyosin10\\\\({}^{\\\\mathrm{10}}\\\\) on the concentration of ADP. The observed rate \\\\(\\\\langle k_{\\\\mathrm{ind}}\\\\rangle\\\\) at each ADP concentration was obtained by fitting the fluorescent transient to a single exponential. The solid line is the best fit of the data to a hyperbola as described in Equation 1.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{tabular}{l} Table I \\\\\\\\ Steady-state ATPase parameters for myo1e\\\\({}^{\\\\mathrm{IQ}}\\\\) \\\\\\\\ \\\\end{tabular}\\n\\nCoeditions were 1 mm MgCl\\\\({}_{2}\\\\), 1 mm EGTA, 1 mm DTT, 2 mm MgATP, 10 mm imidazole, pH 7.0, 25 \\\\({}^{\\\\circ}\\\\)C.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/extracted_captions/milewska2018entry.json b/extracted_captions/milewska2018entry.json new file mode 100644 index 0000000000000000000000000000000000000000..e541caa07c231a8cf344624cdace9842b063992a --- /dev/null +++ b/extracted_captions/milewska2018entry.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Importance of endosomal entry for HCoV-NL63 infection. (A) Inhibition of HCoV-NL63 infection in LLC-Mik2 cells and HAE cultures by the lysosomotropic agents ammonium chloride (NH4Cl) (50 mM) and bafilmomycin A (BaF A) (100 mM), as determined by RT-qPCR. Data on the \\\\(y\\\\) axis represent LRVs. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(P\\\\) values of \\\\(<\\\\)0.05 were considered significant and are denoted with an asterisk. [B] The cytotoxicity of the tested inhibitors was measured with an XTT assay. Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(\\\\times\\\\) and \\\\(\\\\rm D\\\\) Confocal images showing colocalization of HCoV-NL63 virions with the early endosomal marker EEA1 on LLC-Mik2 cells (C) and HAE cultures [D]. Scale bars = 5 \\\\(\\\\mu\\\\)m. Green, HCoV-NL63; red, EEA1.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: Clathrin and dynamin inhibitors hamper replication of HCov-NL63 in LLC-MXB cells. [A] HCov-NL63 replication in LLC-MXB cells and HAE cultures in the presence of entry inhibitors or control DMSO was analyzed using RT-qPCR. Cultures were incubated with 10 \u03bcM Pitstop 2, 10 \u03bcM MMTMAB, 5 \u03bcM mg/ml mystatin, or DMSO for 1 h at 37\u00b0C and inoculated with HCov-NL63 (TCIDho = 400/ml). After 2 h of incubation at 32\u00b0C, virions that were not internalized were inactivated with acidic buffer [pH 3], and cells were incubated for 5 days at 32\u00b0C. The data are presented as log reduction value [LEW] compared to the control sample. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(P\\\\) values of <0.05 were considered significant and are denoted with an asterisk. [B] The cytotoxicity of the inhibitors was tested with an XTT assay. Cells were incubated with 10 \u03bcM Pitstop 2, 10 \u03bcM MMTMAB, 5 \u03bcM mg/ml mystatin, or DMSO for 5 days at 32\u00b0C. Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: TIPRSS2 is required for entry into HAE calls but does not enable virus-cell fusion on the call surface. (A) HC_av_-NL63 replication in LLC-Mik2 cells and HAE cultures in the presence of camostat or control DMSO was analyzed using RT-qPCR. Cultures were incubated with 100 \\\\(\\\\mu\\\\)M camostat or DMSO for 1 h at 37\u00b0C and inoculated with HC_av_-NL63 (TCID\\\\({}_{\\\\text{1.0}}\\\\) = 400/ml). After 2 h of incubation at 32\u00b0C, virions that were not internalized were inactivated with acidic buffer [pH 3], and cells were incubated for 5 days at 32\u00b0C. The data are presented as log reduction value [LRV] compared to the control sample. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(P\\\\) values of <0.05 were considered significant and are denoted with asterisks. (B) HAE cultures were incubated with control DMSO or 100 \\\\(\\\\mu\\\\)M camostat for 1 h at 37\u00b0C. Further, cells were inoculated with purified HC_av_-NL63 and incubated at 32\u00b0C for 2 h. Subsequently, cells were fixed and immunostained for HC_av_-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 5 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG12.png": "'Figure 12: Actin is important for HCov-NL63 entry. LLC-MK2 cells were incubated with DMSO (\\\\(\\\\Delta\\\\)V, 10 \\\\(\\\\mu\\\\)M) cytochalasin D (B and E), 1.5 \\\\(\\\\mu\\\\)M japakirzolide (C and F), or 400 nM nocodazole (D and G) for 1 h at 37\\\\({}^{\\\\circ}\\\\)C and then inoculated with purified HCov-NL63 and incubated at 32\\\\({}^{\\\\circ}\\\\)C for 1 h. Actin and virus localization was verified with confocal microscopy; fixed cells were immunostained for HCov-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG13.png": "'Figure 13: Cytotoxicity of the cytoskeleton-modifying compounds. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with DMSO, 10 \\\\(\\\\mu\\\\)M cytochalasin D, 1.5 \\\\(\\\\mu\\\\)M jasplakinolide, or 400 nM nocodazole for 2 h at 37\u00b0C. Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG14.png": "'\\n\\n**FIG 14 Early event during HCDM-ML63 interaction.**'", "CAPTION FIG2.png": "'Figure 2: HC\\\\(\\\\omega\\\\)-NLR\\\\(\\\\delta\\\\) binding to ACE2 triggers clathrin-mediated endocytosis. Precooled LLC-Mk2 cells were incubated with gradient-purified HC\\\\(\\\\omega\\\\)-NLR\\\\(\\\\delta\\\\) for 40 min at 4\u00b0C following 0 min (A and B) or 5 min (C) of incubation at 32\u00b0C. Colocalization of the virus (green) and ACE2 (red) was analyzed using confocal microscopy (A). No colocalization with clathrin was observed after 0 min of incubation [B]. Triple colocalization of virus with ACE2 and clathrin (blue) is visible in panel C. Images on the right side are zoomed-in regions indicated by white rectangles on the left-side slides. A representative image is shown. Scale bars = 10 \u03bcm.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: HC_av_-NL63 colocalizes with daltrin but root caveolin. (A and B) LLC-Wide2 cells were incubated with gradient-purified HC_av_-NL63 for 40 min at 4\u00b0C following 5 min (A) or 20 min (B) of incubation at 32\u00b0C. HAE cultures were incubated with gradient-purified HC_av_-NL63 for 40 min at 4\u00b0C following 120 min of incubation at 32\u00b0C. HC_av_-NL63 colocalization with daltrin (A) or caveolin (B) was analyzed with confocal microscopy (HC_av_-NL63, green; clathrin and caveolin, red; nuclei, blue). (C) Cells mock incubated and stained with isotypic antibodies were used as a control. Scale bars = 5 \u03bcm.\\n\\n'", "CAPTION FIG4.png": "'\\nFigure 4: Clathrin and dynamic inhibitors hamper internalization of HC_ON_-ML63. (A to C) In order to verify the effectiveness of inhibitors, LLC-Mk2 cells were incubated with control DMSO (A), 10 \u03bcM Pitstop 2 (B), or 10 \u03bcM MITMAB (C) for 30 min at 37\u00b0C and inoculated with Alexa Fluor 488-labeled transferrin. Following incubation (45 min, 37\u00b0C), cells were fixed and stained for actin (red). Transferrin entry was evaluated with confocal microscopy. (E to G) LLC-Mk2 cells were incubated with control DMSO (E), 10 \u03bcM Pitstop 2 (F), or 10 \u03bcM MITMAB (G) for 30 min at 37\u00b0C. Cells were inoculated with purified HC_ON_-ML63 and incubated at 32\u00b0C for 1 h. Subsequently, cells were fixed and immunostained for HC_ON_-ML63 particles (green) and actin (red). [D/] Mock-infected cells were used as a control. Scale bars = 10 \u03bcM.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Cytotoxicity of Pristop 2 and MitMAB on LLC-Mk2 cells. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with control DMSO, 10 \\\\(\\\\mu\\\\)M Pristop 2, or 10 \\\\(\\\\mu\\\\)M MitMAB for 2 h at 37\u00b0C Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Numerical image analysis: clathrin and dynamic inhibitors block HCON-NL63 entry. (S to D) LLC-MK2 cells were incubated with DMSO (B), 10 \\\\(\\\\mu\\\\)M MITMAB (C), or 10 \\\\(\\\\mu\\\\)M Pitstop 2 (D) for 30 min at 37\u00b0C and subsequently inoculated with purified HCoV-NL63 and incubated for 45 min at 32\u00b0C. Confocal images were digitalized, and the localization of each virus particle relative to the cellular membrane was assessed. (A) Number of internalized virus particles relative to number of virions on the cell surface (_y_ acids) for cells treated with DMSO (control, Pitstop 2, or MITMAB. In panels B, C, and D, raw data for cells treated with DMSO, Pitstop 2, or MITMAB, respectively, are presented. Histograms show the average number of virus particles (_y_ acids) versus the distance from the cell surface (_x_ axis). Values of <0 on the \\\\(x\\\\) axis indicate that the virus is inside the cell, while for extracellular virions, the \\\\(x\\\\) value is =0.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Clathrin and dynamic inhibitors prevent HCOV-NL63 from entering the cell in the HAE model. HAE cultures were incubated with central DMSO (A), 10 \\\\(\\\\mu\\\\)M Pistrop 2 [B], or 10 \\\\(\\\\mu\\\\)M MITMAB (C) for 1 h at 37\u00b0C. Cells were then inoculated with purified HCOV-NL63 and incubated at 32\u00b0C for 2 h. Subsequently, cells were fixed and immunostained for HCOV-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 5 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Cytotoxicity of Pitstop 2 and MitTMAS on HAE cultures. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with control DMSO, 10 \\\\(\\\\mu\\\\)M Pitstop 2, or 10 \\\\(\\\\mu\\\\)M MitTMAS for 2 h at 37\u00b0C. Data on the \\\\(y\\\\) axis represent the viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Clathrin and dynamic inhibitors limit the number of LLC-Mk2 infected cells. (A to D) LLC-Mk2 cells were incubated with control DMSO (A), 5 mg/ml nystatin [B], 10 mM MITMAB [C], or 10 \u03bcM Pittstop 2 (D) for 1 h at 37\u00b0C and inoculated with HC6N-ML63 (TGD3 = 100/ml). After 2 h of incubation at 32\u00b0C, virions that were not internalized were inactivated with acidic buffer [pH 3], and cells were incubated for 4 days at 32\u00b0C in the presence of the tested inhibitors or control DMSO. (E and F) The identical procedure was applied to cells, but in these MITMAB (E) and Pittstop 2 (F) were applied after the acid wash. Fixed cells were immunostained with anti-NL63 nucleocapsid protein (green) and nuclei (blue), and confocal images were collected. Scale bar = 200 \u03bcM.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/missirlis2014effect.json b/extracted_captions/missirlis2014effect.json new file mode 100644 index 0000000000000000000000000000000000000000..d4dc2786bcc955aae4144c3060185f0bfdbdc0f5 --- /dev/null +++ b/extracted_captions/missirlis2014effect.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: **Substrate elasticity significantly affects association of Tf with REF52 cells after a 10-minute but not 1-hour incubation but does not have a significant effect on CTb association.** (A) Schematic representation of experimental setup used to quantify cell association of fluorescent markers. Cells on PEG hydrogels of variable elasticity were incubated for a specified duration with fluorescent markers, washed to remove non-associated markers, detached using trypsin and analyzed using flow cytometry. Histograms of cell-associated fluorescence were acquired after gating live cells. (B) Histograms of a typical experiment showing efficient internalization of Tf by cells on hydrogels compared to negative controls and exhibiting a narrow Gaussian distribution. Tf association with REF52 cells was similar on soft, intermediate and stiff substrates after 1 hour incubation (C) but showed a significant lower normalized MFI on softer gels at the shorter time point of 10 minutes (D). Dot plots and mean values are presented, with each dot representing an independent experiment. (E) Epifluorescence microscopy images of REF52 cells on substrates with intermediate stiffness incubated 1 hour with Alexa Fluor 568-labeled Tf and stained for plasma membrane (WGA) confirmed that uptake of Tf by REF52 cells was uniform among the cell population and further showed that Tf localized in a perinuclear region of the cells. Scale bar: 100 \u03bcm (F) Histograms of a typical experiment of CD association with REF52 cells showing a broad distribution of intensity value/cell. Normalized MFI values of CTb association were similar on soft, intermediate and stiff substrates after both 1 hour (G) or 10 minute (H) incubation with CTb. Dot plots and mean values are presented with each dot representing an independent experiment. (I) Epifluorescence microscopy images of REF52 cells on substrates with intermediate stiffness incubated 1 hour with Alexa Fluor 568-labeled CTb and stained for plasma membrane (WGA) showed a heterogeneous population of cells in respect to CD internalization. Scale bar: 100 \u03bcm.\\n\\n'", "CAPTION FIG2\n.png": "'Figure 2: **Inhibition of actomyosin contractility has a major effect on cell morphology and F-actin cytoskeleton but only a moderate, time-dependent influence on Tf and CTb association.** (A) Epifluorescence microscopy images of REF52 cells 6 hours after seeding on FN-coated glass and stained for plasma membrane (\\\\(\\\\Psi\\\\)(GA)) and F-actin cytoskeleton (Philoidin-TRITC). Cells were treated for 1 hour with 10 \u03bcM V27632 or 50 \u03bcM blebbistatin, or left untreated as controls. Scale bars: 10 \u03bcM. Normalized MFI values for Tf (B) or CTb (C) association in presence on absence of 10 \u03bcM Y27632 or Blebbistatin, after a 10-minute or 1-hour incubation. Dot plots and mean values are presented. Normalized FSC (D) and SSC (E) intensity values of REF52 cells in presence or absence of 10 \u03bcM Y27632 or Blebbistatin as determined by flow cytometry analysis. Dot plots and mean values are presented with each dot representing an independent experiment.\\n\\ndoi:10.1371/journal.pone.0096548.tb002'", "CAPTION FIG2.png": "'Figure 2: **Inhibition of actomyosin contractility has a major effect on cell morphology and F-actin cytoskeleton but only a moderate, time-dependent influence on Tf and CTb association.** (A) Epifluorescence microscopy images of REF52 cells 6 hours after seeding on FN-coated glass and stained for plasma membrane (\\\\(\\\\Psi\\\\)(GA)) and F-actin cytoskeleton (Philoidin-TRITC). Cells were treated for 1 hour with 10 \u03bcM V27632 or 50 \u03bcM blebbistatin, or left untreated as controls. Scale bars: 10 \u03bcM. Normalized MFI values for Tf (B) or CTb (C) association in presence on absence of 10 \u03bcM Y27632 or Blebbistatin, after a 10-minute or 1-hour incubation. Dot plots and mean values are presented. Normalized FSC (D) and SSC (E) intensity values of REF52 cells in presence or absence of 10 \u03bcM Y27632 or Blebbistatin as determined by flow cytometry analysis. Dot plots and mean values are presented with each dot representing an independent experiment.\\n\\ndoi:10.1371/journal.pone.0096548.tb002'", "CAPTION FIG3.png": "'\\n\\n**Figure 3.**Association of latex nanoparticles but not of high flux dextran with REFS2 cells depends on substrate elasticity. Normalized fMRI values of REFS2 cells incubated for 1 hour with (A) dextran (MW = 70,000) or (B) carboxylate-modified, polystyrene nano-particle, 100 nm in diameter, as a function of substrate elasticity. Dot plots and mean values are presented with each dot representing and independent experiment.\\n\\ndoi:10.1371/journal.pone.0096548.g003'", "CAPTION FIG4.png": "'Figure 4: **Association of Tf and CTb with HeLa cells is independent of substrate elasticity.** (_A_) Projected cell area of HeLa cells spread on FN-coated hydrogels increased with substrate stiffness. More than 100 cells/condition were analyzed. (_B_) Normalized MFI values of HeLa cells incubated for 10 minutes or 1 hour with fluorescently labeled Tf indicated that its association with cells is not dependent on substrate elasticity. Dot-plots and mean values are shown; each experiment is represented by a dot (n>10,000 analyzed cells/experiment). (_C_) Normalized MFI values of HeLa cells incubated for 1 hour with fluorescently labeled CTb indicated a lack of regulation of its association by substrate elasticity as well. (_D_) Histograms from a typical experiment showed that, similarly to REF52 cells, Tf association was more homogeneous compared to CTb association. (_E_, _F_) Confocal microscopy images of HeLa cells on FN-coated glass confirming homogeneous uptake in the case of Tf (_E_) and a broader distribution of fluorescence per cell for CTb (_F_). For both markers, the majority of fluorescence is intracellular. Scale bars 10 \u03bcm.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Association of Tf and CTb with REF52 cells is not correlated with cell size.** (A) Different sized polystyrene beads were used to derive a linear relationship between bead diameter and the FSC detector signal. (B, C) Typical dot plots and correlation coefficients (Pearson\u2019s) for association of Tf (B) or CTb (C) and apparent cell diameter of live cells. (D) Results of Pearson\u2019s correlation coefficients for 5 different flow cytometry analyses are presented as dot plots and mean values. The low values (<0.3) indicate very low to no correlation between cell size and amounts of Tf or CTb associated with cells.\\n\\ndoi:10.1371/journal.pone.0096548.g005'", "CAPTION FIGS1.png": "\"\\n\\n**Figure S1 Mechanical characterization of PEG hydrogels using AFM.** (A) Young's moduli of three different gel formulations determined by AFM force spectroscopy using a spherical glass tip (diameter 10 mm) and the Hertz model to analyze the force-distance curves. The average values of 3 independent experiments (typically 2 gels per experiment and 3 positions on each gel analyzed) and the standard deviation are presented. (B) A surface area of 100 mm x 100 mm on gels was probed to evaluate homogeneity of elasticity. 100 points were analyzed (orthogonal grid and 5 force curves/point were analyzed to yield the Young's modulus of that point. Data are presented as color-coded surface plots and reveal variations 5-10\\\\({}^{\\\\circ}\\\\)s among values.\\n\\n(PDF)\"", "CAPTION FIGS10.png": "\"\\n\\n**Figure S10**: **Tf or CTb association with cells is very weakly correlated to cell size.** Dot plots of cell-associated fluorescence as a function of apparent cell diameter for Tf (left) or CTb (right) association. Data from 5 random experiments performed on cells on top of gels or glass are presented, along with the calculated Pearson's correlation coefficient.\\n\\n\"", "CAPTION FIGS2.png": "'\\n\\n**Figure S2 Internalization of Tf by REF52 cells is uniform and qualitatively similar between gels of differing elasticity.** Epifluorescence microscopy images [multiple stitched fields] of REF52 cells on PEG hydrogels of varying stiffness, incubated for 1 h with Alexa Fluor 568-conjugated Tf and plasma membrane stained with WGA. Homogeneous uptake of Tf by REF52 cells and intracellular localization was noted for all values of elasticity investigated. Scale bars: 50 um.\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n**Figure S3 Internalization of CTb by REF52 cells is heterogeneous among the cell population.** Epsfluorescence microscopy images (multiple stitched fields) of REF52 cells on PEG hydrogels of varying stiffness, incubated for 1 h with Alexa Fluor 56b-conjugated CTb and plasma membrane stained with WGA. The extent of CTb association with REF52 cells varied considerably between cells. However, the pattern of association was similar between hydrogels of differing elasticity for all values of elasticity investigated. Scale bars: 100 mm.\\n\\n'", "CAPTION FIGS4-1.png": "'\\n\\n**Figure S4 Estimation of extracellular marker fraction using anti-alexa fluor 48B (anti-AF48S) quenching anti-bady.** [A] Quenching kinetics and efficiency of the anti-AF48B antibody (10 mg/ml) on a 5 nM (0.4 mg/ml) solution of AF48B-Tf showed a maximal 90% quenching of fluorescence within 10 minutes of mixing. The antibody concentration used is the same as that used in cell experiments while AF48B-Tf concentration is much higher compared to that on cell-associated Tf or'", "CAPTION FIGS4-2.png": "'CTb, as estimated by fluorescence measurements. [B] MFI of REF52 cells treated with anti-AF48l normalized to MFI in its absence. Incubation of REF52 cells with the quenching antibody on cells cultured on FN-coated TCPS revealed a 11% decrease in TIF MFI and 38% decrease in CTb MFI, indicating that approximately 90% of TIF and 60% of CTb are internalized (mean and standard deviations of at least 3 samples and 2 independent experiments). Substrate elasticity did not affect the fraction of internalized markers (n = l). [C] The effect of Y27632 and blebbastatin treatment on the extracellular fraction of TT was evaluated using anti-AF48l. The same fraction of extracellular TT was recorded independent of cell treatment. Mean and standard deviations are shown of 3 samples.\\n\\nTIFF'", "CAPTION FIGS4.png": "'Figure 4: **Estimation of extracellular marker fractionFigure 34**: **Estimation of extracellular marker fraction** CTb, a estimated by fluorescence measurements. **IS**: **MIT of using anti-alexa fluor 488 (anti-al-4F88) quenching anti-using anti-alexa fluor 488 (anti-al-4F88) quenching anti-ERF25 cells treated with anti-AF488 normalized to MRI in its **body**. (A) Quenching kinetics and efficiency of the anti-AF488bae. **(A) Quenching kinetics and efficiency of the anti-AF488bae. Incubation of REF25 cells with the quenching antibody antibody (10 mg/ml on a 5 nM (0.4 mg/ml) solution of AF488\u2013T-Insulfood) (10 mg/ml on a 5 nM (0.4 mg/ml) solution of AF488\u2013T01 on cell cultured on FN-coated TCS revealed at 115% decrease in showed a maximal 90% quenching of fluorescence withdissolved a maximal 90% quenching of fluorescence within TF MRI 30% decrease in CTb MFI, indicating that 10 minutes of mixing. The antibody concentration used is the 10 minutes of mixing. The antibody concentration used is the 2-propaned and standard deviations of at least 3 samples and 2 min as that used in cell experiments while AF488\u2013T1 concentrations-same as that used in cell experiments while AF488\u2013T1 concentrations-mean and standard deviations of at least 3 samples and 2 min is much higher compared to that on cell-associated TF or independent experiments. Substrate elasticity did not affect fraction of intracellular markers m = 1. **(C)** The effect of XY7632 and blebbistain treatment on the extracellular fraction of TF was evaluated using anti-AF488. The same fraction of extracellular TF was recorded independent of cell treatment. Mean and standard deviations are shown of 3 samples.\\n\\n'", "CAPTION FIGS5.png": "'\\n\\n**Figure S3 Rho kinase inhibition with Y27632 did not alter intracellular fluorescence pattern of Tf or CTTb on REF52 cells.** Confocal microscopy images of REF52 cells on FNCated glass after 1-hour incubation with AF568-labeled markers, fixation and staining with WG4-AF488. Tf was internalized at similar numbers by cells and mainly localized at a perinuclear site, independently of Y27632 treatment (upper row), while CTb showed heterogeneous uptake efficiency among the cell population that was also independent of Y27632 treatment (lower row).\\n\\n'", "CAPTION FIGS6.png": "'\\n\\n**Figure S6****Blebbistatin treatment of REF52 cells affects Tf internalization and recycling kinetics.** MIT of REF52 cells incubated with TT at different time points on FN-coated plastic in the presence or absence of 50 mM blebbstatin. At short incubations blebbistatin inhibits Tf association by cells, while at longer time points the amount of Tf is enhanced compared to control conditions. The quasi-linear increase of MFI per cell indicates that blebbistatin has an effect of intracellular trafficking and recycling of TT. Each data point represents the average of two experiments.\\n\\n[TIFF]'", "CAPTION FIGS7.png": "'\\n\\n**Figure S7 The SSC signal but not the I\\\\(\\\\bar{\\\\rm s}\\\\)SC signal of REF52 cells depends on the elasticity of the substrate they were cultured on.** Flow cytometry analysis of REF52 cells cultured on gels did not show a dependence of their FSC signal (\\\\(\\\\lambda\\\\)), while cells on soft gels showed a significantly lower SSC signal compared to cells cultured on intermediate or stiff hydrogels (\\\\(\\\\rm{BL}\\\\) Values from at least 4 independent experiments are presented with the value from each gel represented by a single dot and the mean value a solid line.\\n\\n**(TIFF)**'", "CAPTION FIGS8.png": "'\\n\\n**Figure S8 Transfer-Color Microscopy of HeLa or HEK52 cells is not significantly affected by substrate elasticity.** (A) Typical overlaid optical and fluorescence microscopy images of HeLa cells on FN-coated hydrogels of different elasticity used to calculate transfection efficiency. Insets show micrographs of the fluorescence channel (GFF). Scale bars: 50 mm. Transfection efficiency of (B) HeLa or (C) REF52 cells normalized to the value obtained on FN-coated glass. A non-significant decrease of the fraction of transfected cells with increasing stiffness was noted. Mean and SEM values from at least 3 gels and two independent experiments are presented (n = 200-1000 analyzed cells/gel).\\n\\n**Figure S9 Comparison of the fluorescence microscopy of HeLa cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 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HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK52 cells on HEK cells'", "CAPTION FIGS9-1.png": "'\\n\\n**Figure 89**: **Cell density of HeLa cells does not depend on substrate elasticity.** Inverted fluorescence microscopy images (statched tiles) of HeLa cells on soft, intermediate and stiff hydrogels, stained with WGA. The amount of cells adhered was'", "CAPTION FIGS9-2.png": "'similar on all hydrogels, independent of their elasticity. Scale bars 200 mm.\\n* [TIFF]'", "CAPTION FIGS9.png": "'Figure 59: **Cell density of HeLa cells does not depend on substrate elasticity.** Inverted fluorescence microscopy images (attached tiles) of HeLa cells on soft, intermediate and stiff hydrogels, stained with WGA. The amount of cells adhered was similar on all hydrogels, independent of their elasticity. Scale bars: 200 \u03bcm (TIP).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/morenolayseca2021cargospecific.json b/extracted_captions/morenolayseca2021cargospecific.json new file mode 100644 index 0000000000000000000000000000000000000000..cc90bddfa3c09941fb5f327c0b4dbd2a43b9aef2 --- /dev/null +++ b/extracted_captions/morenolayseca2021cargospecific.json @@ -0,0 +1 @@ +{"CAPTION EXTENDEDDATA FIG1-1.png": "'\\n\\n**Extended Data Fig. 11** See next page for caption'", "CAPTION EXTENDEDDATA FIG1-2.png": "'\\n\\n**Extended Data Fig. 1 [**Swin1 (EFHD2) is an indicator of Rab21**. SILAC proteomics analysis of GFP-Trap pulldowns in MDA-MB-231 cells expressing GFP-WT-Rab21 vs. GFP-DN-Rab21 (T3IN inactive GDP-bound/nucleotide-free mutant) **(a)**: GFP-CA-Rab21 (Q76L constitutively active GTP-bound mutant) vs. GFP **(b)**: GFP-WT-Rab21 vs. GFP **(c)** or GFP-WT-Rab21 vs. GFP-CA-Rab21 **(d)**. (a-d) Each spot in the plots corresponds to one identified protein by mass spectrometry. Plots are representative of 2 independent experiments, forward and reverse; where every experiment consists of two independent affinity parifications. Plots show mean fold-changes from forward and reverse experiments against absolute protein abundances (intensity-based absolute quantification, iBAQ). Abundance bins were defined by including 1000 proteins in a subsequent order. Log100 field change of proteins were tested for significance using double-sided significance tests. No multiple hypothesis correction method was applied, due to the small number of selected proteins for the statistical analysis. Proteins with a _P_-value < 0.01 are projected as a triangle, while non-significant proteins are shown as circles. P-values are depicted in the figure for a selected set of proteins relevant to the work. Proteins in red are markedly enriched in the CA- or WT-Rab21 fraction and proteins in blue are known endosomal proteins clathrin (CLTA, CLTB, CLTC), APE (AP2A1, AP2B1, AP2MI, AP251), caveolin (CAVI) and dynam II (DNM2), which are not specifically enriched. Swip1 was strongly enriched in the GFP-WT-Rab21 vs. GFP-DN-Rab21 **(a)** and GFP-CA-Rab21 and GFP-WT-Rab21 fractions compared to GFP fractions **(b, d)**, and it was equally enriched in the GFP-CA-Rab21 compared to the GFP-WT-Rab21 fraction **(c)**. Clattrin (CLTA, CLTB, CLTC), AP2 (AP2A1, AP2BI, AP2MI, AP251), caveolin (CAVI) and dynamin II (DNM2) are not strongly enriched in any fraction **(e)**. Representative immunoblots of GFP-Trap pulldowns from MDA-MB-231 cells transfected with GFP, GFP-WT-Rab21, GFP-WT-Rab5, GFP-DN-Rab5 or GFP-CA-Rab5 stained for endogenous Swip1 and pH integrin. Blots are representative of 2 independent experiments. Unprocessed blots are provided in Source data.\\n\\n'", "CAPTION EXTENDEDDATA FIG1.png": "''", "CAPTION EXTENDEDDATA FIG10-1.png": "'\\n\\n**Extended Data Fig. 10 | See next page for caption**'", "CAPTION EXTENDEDDATA FIG10-2.png": "\"\\n\\n**Extended Data Fig. 10**: **EHD2 (Swip1) mRNA expression correlates with a more metastatic breast cancer phenotype and Swip1 immunostaining at the plasma membrane correlates with higher death risk and metastasis incidence in TNBC.****(a)** Analysis of Swip1 mRNA expression using qPCR in 192 breast tumours. Turnours were categorized into two groups: high EFHD2 expression (higher than the median of the entire collection) or low EFHD2 expression (lower expression than the median). The y2-test was used for statistical analyses. EFHD2 mRNA levels based on tumour type and tumour grade are shown. **(b)** Validation of anti-Swip1 antibody specificity in IHC was carried out with agarose-embedded and formalin-fixed paraffin-embedded cell pellets from control- or Swip1-silenced MDA-MB-231 cells (siRNA #1 and #2). Scale bars, 50 **mm.****(c)** Quantification of the percentage of breast cancer tumours from TNBC tissue microarray with high, medium or low Swip1 staining at the plasma membrane. **(d)** Kaplan-Meier plot shows overall survival of 133 triple negative breast cancer patients with high (H, red) or medium-low (M-L, blue) membranal staining of Swip1 in their tumour centre sample. The hazard ratio of high vs. medium-low Swip1 immuno-positivity was 2.34 (95% C1.25 to 4.41). The hazard ratios after adjustment for Ki67, 2.51 (95% C1.26 to 5.01): for tumour size, 2.12 (95% C1.08 to 4.18); for lymph node metastasis status 2.26 (95% C119 to 4.31) and for tumour grade 2.44 (95% C11.25 to 4.74). **(e)** lymph node metastasis incidence in 130 TNBC patients from the same cohort, Fisher's exact test p = 0.037. A higher proportion of patients with high membranal staining of Swip1 (> = 80%) had lymph node metastasis. Numerical source data are provided in Source data.\\n\\n\"", "CAPTION EXTENDEDDATA FIG10.png": "'\\n\\n**Acknowledgement:** We thank the anonymous referee for his/her constructive criticism and constructive criticism that improved the presentation of the manuscript.\\n\\n## References\\n\\n* [1] J.-P. Boyle, _The Theory of the Theory of the Second Order_, (1968). _The Theory of the Second Order_, (1968). _The Theory of the Second Order_, (1969). _The Theory of the Second Order_, (1969). _The Theory of the Second Order_, (1970). _The Theory of the Second Order_, (1971). _The Theory of the Second Order_, (1972). _The Theory of the Second Order_, (1973). _The Theory of the Second Order_, (1974). _The Theory of the Second Order_, (1975). _The Theory of the Second Order_, (1976). _The Theory of the Second Order_, (1977). _The Theory of the Second Order_, (1978). _The Theory of the Second Order_, (1979). _The Theory of the Second Order_, (1979). _The Theory of the Second Order_, (1979). _The Theory of the Second Order_, (1979). _The Theory of the Third Order_, (1979). _The Theory of the Third Order_, (1979).\\n\\n'", "CAPTION EXTENDEDDATA FIG2-1.png": "'\\n\\n**Extended Data Fig. 2 -- See next page for caption**'", "CAPTION EXTENDEDDATA FIG2-2.png": "'\\n\\n**Extended Data Fig. 2 | BiFC/BiCAP approaches to assess the interaction between Rab21 and Swip1. (a)** Cartoon of the BiFC-/BK-AP approach used to image (BiFC) and biochemically detect (BiCAP) the interactions between Rab21 and Swip1 in live cells and from cell lysate. (b) MDA-MB-231 cells were transfected with the indicated constructs and lysed. Proteins expressed in the transfected cells were detected by immunoblotting, Immunoblot shows the size of the bands detected by a polyclonal anti-GFP antibody. Blots are representative of 2 independent experiments. (c) Representative confocal microscopy BiFC images of MDA-MB-231 cells expressing VI-Rab21 and V2-Swip1 MDA-MB-231 and immunostained for endosomal markers. Insets show BiFC colocalizing with the indicated endosomal markers. Scale bars, 10 mm (main figures) and 1 mm (insets). Representative pictures of 3 independent experiments. Unprocessed blots are provided in Source data.\\n\\n'", "CAPTION EXTENDEDDATA FIG2.png": "'* [1]'", "CAPTION EXTENDEDDATA FIG3-1.png": "'\\n\\n**Extended Data Fig. 3 \\\\(\\\\parallel\\\\) See next page for exploration.**'", "CAPTION EXTENDEDDATA FIG3-2.png": "'\\n\\n**Extended Data Fig. 3 | SIM images of Swip1 with CG components and Rab proteins. (a) Confocal micrographs of MDA-MB-231 cells expressing mScarlet-I-Swip1 and GFP-Rab21, and immunostained for |b|-integrin (12G10 antibody). Arrows show areas of co-localization in endosomal structures. Representative pictures of n=3 independent experiments. Scale bar, 10 \\\\(\\\\mu\\\\)m. (b) Full pictures from Fig. 2d, e and 3d. x-y projections of MDA-MB-231 cells expressing mScarlet-I-Swip1 and either GFP-Rab21, Artif-GFP or GFP-IRSb53 were imaged using structured illumination microscopy (SIM). Blue and yellow squares highlight the regions of interest (ROI) shown as x-z projections in Fig. 2d, e and Figure 3d. Representative pictures of n=3 independent experiments. Scale bars, 5 \\\\(\\\\mu\\\\)m (2d), 8 \\\\(\\\\mu\\\\)m (2e) and 8 \\\\(\\\\mu\\\\)m (3d), insets, 1 \\\\(\\\\mu\\\\)m. (c) SIM-x2 repetitions of MDA-MB-231 cells expressing mScarlet-I-Swip1 and immunostained for endogenous Rab proteins and quantification of Rab protein co-localization with mScarlet-I-Swip1. Each dot represents the co-localization ratio in one cell. Data are presented as mean values \\\\(\\\\pm\\\\) 95 % CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled from 3 independent experiments. \\\\(P\\\\) values calculated compared to Rab21 condition. \\\\({}^{'", "CAPTION EXTENDEDDATA FIG3.png": "''", "CAPTION EXTENDEDDATA FIG4-2.png": "'\\n\\n**Extended Data Fig. 4 [GP-Swip1 co-localizes with Arft-HA at the TIRF plane and regulates pH-integrin endocytosis via the CG pathway. (a)** MDA-MB-231 cells were co-transfected with GFP-Swip1 and Arft-HA constructs, fixed, stained with probes for DNA paint and imaged using SMAM. Examples of the structures formed by GFP-Swip1 at the proximity of the ECM interphase are shown. Representative pictures of n = 2 independent experiments. Scale bars, 2 mm (main images) and 0.5 mm (insets). (b) Representative imrmunblots of control- and Swip1-silenced MDA-MB-231 cell lysates blotted as indicated, calnexin is included as a loading control. Blots are representative of n = 3 independent experiments. (c) FACS analyses of cell surface (II-integrin in control- and Swip1-silenced MDA-MB-231 cells using the indicated antibodies. Bar charts show data as geometric mean values of 10,000 cells +- SEM over n = 3 independent experiments. Statistical significance was assessed with two-tailed Wilcoxon matched-pairs signed-rank tests (n = 3 independent experiments), ns = not significant. (d) Quantification of biotinylated a2-, a3- or are-integrin internalization in Swip1-silenced MDA-MB-231 cells (siRNA #2) after the indicated times determined with ELISA in the presence or absence of 100 mM prinquare. Bar charts show data as mean values +- SEM. Statistical significance was assessed using multiple-comparison t-tests for paired data, with the post-hoc Holm-Sidak method, with alpha = 5.000%. Each raw was analysed individually, without assuming a consistent SD*P = 0.07540; n = 3 biologically independent experiments. Unprocessed blots and numerical source data are provided in Source data.**'", "CAPTION EXTENDEDDATA FIG4.png": "'\\n\\n**Abstract**\\n\\nWe present a novel and efficient algorithm for the \\\\(\\\\beta\\\\)-value problem in the \\\\(\\\\beta'", "CAPTION EXTENDEDDATA FIG5-1.png": "'\\n\\n**Extended Data Fig. 5 -- See next page for caption.**'", "CAPTION EXTENDEDDATA FIG5-2.png": "'\\n\\n**Extended Data Fig. 5 Active \\\\(\\\\beta\\\\)I-integrins are endocytosed via the CG pathway and CME. (a)** Representative micrographs and quantification of \\\\(\\\\beta\\\\)I-integrin uptake in control- or IRSp53- or _ArH_-silenced MDA-MB-231 cells. Representative immunoblots to validate Swip1 silencing. Scale bars, 10 \\\\(\\\\mu\\\\)m. **(b)** Representative micrographs and quantification of endocytosed or surface (no endocytosis or acid wash) of murine \\\\(\\\\beta\\\\)I-integrin (9EG7 antibody) in isomeric IRSp53-/- MEFs: IRSp53-KO-pBABE ( and IRSp53-KO-pBABE-IRSp53 (WT) in which the expression of IRSp53 has been restored. Representative immunoblots of cell lysates blotted as indicated. Scale bars, 10 \\\\(\\\\mu\\\\)m. (c) Representative micrographs of \\\\(\\\\beta\\\\)I-integrin uptake in GFP- and GFP-Swip1-expressing cells and quantification of integrin uptake at the indicated times. Scale bars, 10 \\\\(\\\\mu\\\\)m. (d) PLA with the indicated antibodies (from Figure 44) in MDA-MB-231 cells expressing either GFP or GFP-Swip1. Plot shows the background controls for each. (e) Representative images and quantification of co-localization of \\\\(\\\\beta\\\\)I-integrin-AF488 (12G10) with TMR-10 kDa dextran or AF647-transferrin after 1 min simultaneous uptake in MDA-MB-231 cells. Yellow arrows show regions of co-localization between \\\\(\\\\beta\\\\)I-integrin and TMR-dextran and cyan arrows show regions where \\\\(\\\\beta\\\\)I-integrin co-localizes with AF647-transferrin. Scale bars, 10 \\\\(\\\\mu\\\\)m. For all plots, data are presented as mean values \\\\(\\\\pm\\\\) 95% CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled from 3 independent experiments (a-d) or from 2 independent experiments (e). (a) \\\\(\\\\bullet\\\\)P_C0.0001, (b) \\\\(\\\\bullet\\\\)P_C0.0001, ns=no significant, (c) \\\\(\\\\bullet\\\\)P_P_=0.0478, \"P_=0.0058, (c) \\\\(\\\\bullet\\\\)P_=0.0006, (d) \\\\(\\\\bullet\\\\)P_=0.0001, (e) \\\\(\\\\bullet\\\\)P_=0.0001, (f) \\\\(\\\\bullet\\\\)P_=0.0001.\\n\\nNumber of analysed cells: (a) as iCTRL, n=103 cells, ArH siRNA #1, n=177 cells, ArH siRNA #2, n=157 cells, a;STRL, n=100, IRSp53 siRNA H #1 = 142 cells, (kSp53 siRNA H #2, n=124 cells. (b) Endocytosed and surface \\\\(\\\\beta\\\\)I-integrin, respectively: IRSp53 WT, n=108 & n=55 cells, IRSp53 KO, n=100 & n=60 cells. (c) GFP, n=10, n=22, n=30 and n=24 cells and GFP-Swip1, n=15, n=22, n=32 and n=26 cells, respectively, (d) For GFP, 12G10 only, n=75 cells; IRSp53 only, n=113 cells; 12G10 + IRSp53, n=106 cells. For GFP-Swip1, 12G10 only, n=78 cells; IRSp53 only, n=92 cells; 12G10 + IRSp53, n=11 cells. (e) \\\\(\\\\beta\\\\)I-integrin-dextran, n=22 cells; \\\\(\\\\beta\\\\)I-integrin-transferrin, n=22 cells and transferrin-dextran, n=18 cells. Unprocessed blots and numerical source data are provided in Source data.**'", "CAPTION EXTENDEDDATA FIG5.png": "''", "CAPTION EXTENDEDDATA FIG6-1.png": "'\\n\\n**Extended Data Fig. 6 -- See next page for exploration**'", "CAPTION EXTENDEDDATA FIG6-2.png": "'\\n\\n**Extended Data Fig. 6 [**Swip1 regulates (B1-integrin endocytosis via the CG pathway. (a)** Representative micrographs and quantification of B1-integrin and M4-HCl uptake at the 15 min time point in control or Swip1 (siRNArl or #2) silenced MDA-MB-468 and BT-20 cells. Representative imrmonblots of cell lysates blotted for Swip1. Calnexin is included as a loading control. Scale bars, 10 \\\\(\\\\mu\\\\)m. (b) Representative immunoblot of MDA-MB-231, BT-20 and MDA-MB-468 cell lysates blotted for Swip1. _a_-tubulin is included as a loading control. (c) Representative micrographs and quantification of transferrin uptake at the 15 min time point in control- or Swip1-silenced MDA-MB-231 cells. Representative imrmonblots of cell lysates blotted as indicated. Calnexin is included as a loading control. Scale bars, 10 \\\\(\\\\mu\\\\)m. (d) Cell-surface labeled EGFR uptake in control and Swip1-silenced MDA-MB-231 cells treated with 10 mg/ml EGF was analysed using flow cytometry in 10 000 non-permeabilized cells per measurement. Plot shows 4 measurements from \\\\(n\\\\) = 2 independent experiments. Immunoblot to validate swip1 silencing is representative of 2 independent experiments. GAPDH is included as a loading control. Immunohistochemistry in panels (a-c) and scatter off-plots (a-c) are representative of 3 independent experiments. Data are presented as mean values +- 95% CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where in the total number of cells pooled across a independent experiments. \\\\(P\\\\) values calculated compared to a CTRL condition: *_P_ = 0.0737, *_P_ = 0.0007, *_P_ = 0.0006, *_P_ = 0.0001, \\\\(n\\\\) = not significant. Number of analysed cells over a 3 independent experiments: (a) For BT-20 12G10 and MHC1 uptake, respectively, siRNArl, n = 79 & 91 cells; Swip1 siRNArl, n = 76 & 91 cells; Swip1 siRNArl, n = 66 & 75 cells. For MDA-MB-468 12G10 and MHC1 uptake, respectively, siRNArl, n = 134 & 132 cells; Swip1 siRNArl, n = 98 & 117 cells; Swip1 siRNArl, n = 98 & 101 cells. (c) siRNArl, n = 74 cells; Swip1 siRNArl, n = 103 cells. Unprocessed blots and numerical source data are provided in Source data.**'", "CAPTION EXTENDEDDATA FIG6.png": "''", "CAPTION EXTENDEDDATA FIG7-1.png": "'\\n\\n**Extended Data Fig. 7 -- See next page for caption**'", "CAPTION EXTENDEDDATA FIG7-2.png": "'\\n\\n**Extended Data Fig. 7 [suppl is required for specific entry of BI-integrin via the CG pathway following changes in membrane tension; Swip1 does not directly interact with IRSp53 and it does not affect the number of Rab2+containing vesicles. (a) Representative images of endocytosed AF48B-anti-flip-integrin (12G1O antibody) and TMR-10 kDa dextran in MDA-MB-231 cells after treatment of cells with hypotonic media followed by a shift to isotonic media. Scale bars, 10 \\\\(\\\\mu\\\\)m. Pictures are representative of n = 3 independent experiments. Quantification of these experiments is shown in Figure 5a. (b) Recombinant purified his-Swip1 or his-VASP (passive control) proteins were spotted at the indicated concentrations on nitrocellulose filters. BSA was used as negative control. Nitrocellulose membranes were incubated with recombinant purified IRSp53 at the indicated concentrations and immunoblotting with an anti-IRSp53-specific antibody. (c) Recombinant purified his-Swip1 (500 \\\\(\\\\mu\\\\)M) or his-VASP (50 nM, positive control) proteins were spotted at the indicated concentrations on nitrocellulose filters. BSA was used as negative control. Nitrocellulose membranes were incubated with recombinant purified IRSp53 at the indicated concentrations and immunoblotted with an anti-IRSp53-specific antibody. Pictures are representative of n = 3 independent experiments (a-c). (d) Representative micrographs of MDA-MB-231 cells immunostained for endegenus Rab21 and counterstained with DAPI and WGA-AF647 as a plasma membrane marker to define the cell outlines. The outlines of each cell are indicated in green dashed lines. The number of vesicles in 100 MDA-MB-231 cells per condition was quantified using the IMARJS vesicle detection function. Data are presented as mean values +- 95 % CI Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled across 3 independent experiments (n = 100 cells per condition). P values calculated compared to sICTRL condition: *** \\\\(P\\\\) < 0.0001, ns: not significant. Scale bar, 10 \\\\(\\\\mu\\\\)m. Numerical source data are provided in Source data.\\n\\n'", "CAPTION EXTENDEDDATA FIG7.png": "'\\n\\n**Extended Data:** Fig. 1 first and second for updates.\\n\\n**Extended Data:** Fig. 2 first reports the results of the first update of the **C10**, first column from the posterior distribution to the posterior distribution from the posterior distribution (Table 1) and then from the posterior distribution (Table 2). The second column shows the posterior distribution from the posterior distribution (Table 3) and the third column shows the posterior distribution from the posterior distribution (Table 4). The third column shows the posterior distribution from the posterior distribution (Table 5). The fourth column shows the posterior distribution from the posterior distribution (Table 6). The fourth column shows the posterior distribution from the posterior distribution (Table 7). The fourth column shows the posterior distribution from the posterior distribution (Table 8). The fourth column shows the posterior distribution from the posterior distribution (Table 9). The fourth column shows the posterior distribution from the posterior distribution (Table 10). The fourth column shows the posterior distribution from the posterior distribution (Table 11). The fourth column shows the posterior distribution from the posterior distribution (Table 12). The fourth column shows the posterior distribution from the posterior distribution (Table 13). The fourth column shows the posterior distribution from the posterior distribution (Table 14). The fourth column shows the posterior distribution from the posterior distribution (Table 15). The fourth column shows the posterior distribution from the posterior distribution (Table 16). The fourth column shows the posterior distribution from the posterior distribution (Table 17). The fourth column shows the posterior distribution from the posterior distribution (Table 18). The fourth column shows the posterior distribution from the posterior distribution (Table 19). The fourth column shows the posterior distribution from the posterior distribution (Table 11). The fourth column shows the posterior distribution from the posterior distribution (Table 12). The fourth column shows the posterior distribution from the posterior distribution (Table 13). The fourth column shows the posterior distribution from the posterior distribution (Table 14). The fourth column shows the posterior distribution from the posterior distribution (Table 15). The fourth column shows the posterior distribution from the posterior distribution (Table 16). The fourth column shows the posterior distribution from the posterior distribution (Table 17). The fourth column shows the posterior distribution from the posterior distribution (Table 18). The fourth column shows the posterior distribution from the posterior distribution (Table 19). The fourth column shows the posterior distribution from the posterior distribution (Table 19). The fourth column shows the posterior distribution from the posterior distribution (Table 12). The fourth column shows the posterior distribution from the posterior distribution (Table 13). The fourth column shows the posterior distribution from the posterior distribution (Table 14). The fourth column shows the posterior distribution from the posterior distribution (Table 15). The fourth column shows the posterior distribution from the posterior distribution (Table 16). The fourth column shows the posterior distribution from the posterior distribution (Table 17). The fourth column shows the posterior distribution from the posterior distribution (Table 18). The fourth column shows the posterior distribution from the posterior distribution (Table 19). The fourth column shows the posterior distribution from the posterior distribution (Table 19).\\n\\n'", "CAPTION EXTENDEDDATA FIG8-1.png": "'\\n\\n**Extended Data Fig. 1 (See next page for each'", "CAPTION EXTENDEDDATA FIG8-2.png": "'\\n\\n**Extended Data Fig. 8 | Arfi and RSp53 regulate focal adhesion formation and Swip1 and Rab21 regulate cell migration.****(a)** Representative immunoblots to validate Swip1 silencing and mScarlet-1-Swip1 or mScarlet-1-AEFI overexpression in the experiments shown in Figure 6: Blot is representative of 3 independent experiments. (**b**) Representative masks of vinculin-containing focal adhesions in control-, IRSp53- or Arfi-silenced MDA-MB-231 cells and quantification of adhesion number and total area per cell in cells plated on collagen I. Data are presented as mean values +- 95% CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled across 3 independent experiments. P values calculated compared to siCTRL condition: ***_P_=0.0003, ***_pC_0.0001. Number of analysed cells over 3 independent experiments: siCTRL, n= 62 cells; Arfi siRNA #1, n= 60 cells; Arfi siRNA #2, n= 61 cells; siCTRL, n= 64 cells, IRSp53 siRNA #1, n= 62 cells and IRSp53 siRNA #2, n= 65 cells. Representative immunoblots of 3 independent experiments to validate IRSp53 and Arfi silencing are shown. Scale bars, 10 \u03bcm. (**c**) Representative phase contrast pictures of the scratch wound after 18 h. Pictures are representative of 3 independent experiments. Quantification of these experiments is shown in Figure 7d. Scale bar, 100 \u03bcm. Unprocessed blots and numerical source data are provided in Source data.\\n\\n'", "CAPTION EXTENDEDDATA FIG8.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION EXTENDEDDATA FIG9.png": "'\\n\\n**Extended Data Fig. 9 [Swip1 is required for random cell migration.** Random cell migration speed of MDA-MB-231 **(a)** and MDA-MB-468 **(b)** cells upon Swip1 silencing. In all cases, cells were labelled using sir-DNA and their migration behaviour recorded over time using a widefield microscope. Cell nuclei were automatically tracked using StarDist and TrackMate. Cell track speed and mean displacement plots are displayed. Boxplots display the median and quartiles of the data. Whiskers display the 2nd percentile and the 98th percentile. Mean displacement plots show mean values + standard deviation. For all panels, _P_-values were determined using a randomization test, where n is the total number of cells pooled across 3 independent experiments, ***_P_= <0.001. Number of cells analysed over 3 independent experiments: MDA-MB-231, sicTRI, n = 869 cells; siSwip1 #1, n = 553 cells; siSwip1 #2, n = 506 cells; MDA-MB-468, siCTRL, n = 331 cells; siSwip1 #1, n = 206 cells; siSwip1 #2, n = 235 cells. Numerical source data are provided in Source data.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Fig. 1 \\\\(|\\\\) Swip1 interacts directly with active Rab21.****a**, SILAC proteomics analysis of GFP-Trap pulldowns in MDA-MB-231 cells expressing GFP-tagged CA-Rab21 versus GFP alone. The plot is representative of two independent experiments, forward and reverse: the experiments consisted of two independent affinity parifications. The plot shows the mean fold-changes from the forward and reverse experiments against absolute protein abundances (intensity-based absolute quantification, iB/Q). Abundance bins were defined by including 1,000 proteins in a subsequent order. The logE-transformed fold-change values of the proteins were tested for statistical significance using double-sided Significance B tests. No multiple hypothesis correction method was applied due to the small number of selected proteins for the statistical analysis. Proteins with P < 0.01 are represented by a triangle and non-significant proteins are shown as circles. P values are depicted in the figure for a selected set of proteins. The proteins in red are markedly enriched in the CA-Rab21 fraction and proteins in blue are known endosomal proteins--clathrin (CLTA, CLTB and CLTC), AP2 (AP2Alt, AP2B1, AP2Alt and AP2S1), caveolrin (CAVI) and dynamic II (DMN2)--that are not specifically enriched. **b**, Representative immunoblots of GFP-Trap pulldowns from MDA-MB-231 cells transfected with the indicated constructs and probed for GFP and endogenous Swip1. Three independent experiments were performed. GFP-DN-Rab21, Rab21(tm) dominant negative, GDP-bound/nucleotide-free IP, immunoprecipitation. **c**, Caomassie-stained gel and immunolea (IB) of GST-pulldowns with the indicated GST-tagged proteins and recombinant Rab21 (indicated by asterisks) bound to a non-hydrolyable form of GFP (GppNHP; active Rab21), GDP or no nucleotide after EDTA treatment (nuc free). The Rab21-effector GST-APPL was used as a positive control. Three independent experiments were performed. Unprocessed blots are provided.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2 [Swip1 interacts with Rab21 and p1-integrin.****a**, Representative MDA-MB-231 cell expressing GFP-Rab21 and immunostained for Swip1. The arrows depict to regions of overlap between Swip1 and GFP-Rab21. Scale bar; 10\u03bcm. The micrograph is representative of two independent experiments. **b**, Endogenous Rab21 and Swip1 are in close proximity in MDA-MB-231 cells. A PLA assay using the indicated antibodies (left) is quantified (right). Gait IgG was included as a negative control and nuclei were stained with 4,6-diamidino-2-phenylindole. Scale bars, 20\u03bcm \\\\(n\\\\) = 121 (Rab21-IgG) and 126 (Swip1-Rab21) cells were examined across three independent experiments. **c**, Representative TIRF microscopy images of live MDA-MB-231 cells expressing the GFP constructs V1-Rab21 and V2-Squipt or Venus alone as a control. Scale bars, Sym (rain images) and 2\u03bcm (insets). Three independent experiments were performed. **d**, MDA-MB-231 cells expressing mCherry1-Squipt and GFP-Rab21 imaged using SIM. The blue and yellow squares highlight the regions of interest (ROI; top) in the x-y plane that are magnified in the x-z projections (middle and bottom; magnified views). Scale bars, Sym (top) and 1\u03bcm (middle and bottom). Representative images of three independent experiments are shown. **e**, SIM x-z projections of MDA-MB-231 cells expressing mCherry1-Squipt, GFP-Rab21 and immunostained for p1-integrin (left) were quantified for co-localization with mCherry1-Squipt (right). Each dot represents the co-localization fraction in one cell; \\\\(n\\\\) = 16 (GFP-Rab21) and 22 (p1-integrin) cells pooled from two independent experiments. Scale bars, 0.5\u03bcm. **b**, Data are presented as the mean +- 95% confidence interval (CI). Statistical significance was assessed using a two-sided Mann-Whitney test; \\\\('", "CAPTION FIG3.png": "'\\nFig. 3: **Swip1 interacts with members of the CG pathway.****a** Representative immunoblots of GFP-Trap pulldowns from HEK293 cells transfected as indicated and blotted for GFP, endogenous IRSp53, Arrl and b1-integrin. **b**, Representative immunoprecipitation, with control mouse IgG or anti-IRSp53, of MDA-MB-231 cell lysates probed for endogenous Swip L IRSp53 and Arrl. **c** Proximity ligation assay with the indicated antibodies alone (negative controls) and together to assess co-localization. Scale bars, 10mm; \\\\(n\\\\) = 12 (12G10 only), 18 (IRSp53 only) and 28 (12G10 + IRSp53) cells analysed across two independent experiments. **d**, Representative SIM _x_-2 projections of MDA-MB-231 cells expressing mScarlet-1-Swip1 and GFP-IRSp53 or GFP-Arrl. Scale bars, 0.8mm. Three independent experiments were performed. **e**, MDA-MB-231 cells expressing mScarlet-1-Swip1 and immunostained for endocytic adaptor proteins (**e**ft) or transfected with the indicated GFP-tagged constructs (right) were imaged with SIM and quantified for co-localization with mScarlet-1-Swip1. Each draft represents the co-localization fraction in one cell = 50 (Rab21, AP2 and caveolin), 55 (S clathrin), 63 (GFP-Rab21 and GFP-dynamin[1]), 53 (GFP-IRSp53) and 62 (GFP-Arrl) cells analysed, pooled from three independent experiments. **e**c, Data are the mean +- 95% CI. Statistical significance was assessed using a two-sided Mann-Whitney test: \\\\({}^{\\\\bullet\\\\'", "CAPTION FIG4.png": "'\\nFig. 4: **Swip1 mediates the endocytosis of active integrins via the CG pathway.****a**, Representative micrographs of active ff1-integrin internalization in control (siCTRL)- and **S**up1-slenecked MDA-MB-231 cells (top; green dashed lines show the outlines of the cells defined by phalloidin labelling), and levels of internalized bTI-integrin at the indicated times (bottom: Representative immunoblot to validate Swip1 silencing (bottom right). Scale bars, 30 \u03bcm; **S**c**T**R1, \\\\(n\\\\) = 86, 101, 110 and 78 cells; Swip1 siRNA1, \\\\(n\\\\) = 104, 98, 102 and 106 cells; from left to right. **b**, Levels of active ff1-integrin internalization at T@min in cisCTRL- or Swip1-slenecked MDA-MB-231 cells expressing GFP or GFP-Swip1 (left). Representative immunoblotting of cell lysates probed as indicated (right); **S**c**T**R1 + GFP, \\\\(n\\\\) = 153 cells; siCTRL + GFP-Swip1, \\\\(n\\\\) = 186 cells; siSwip1 + GFP, \\\\(n\\\\) = 136 and siSwip1 + GFP-Swip1, \\\\(n\\\\) = 196 cells. *P = 0.0275 and *P = 0.0260. **c**, Representative micrographs (top) and levels of inactive ff1-integrin (Mab13) internalization at 15 min in siCTRL. *Swip1- and Rab21-silenced (siRisk21) (MDA-MB-231 cells. Scale bar, 10 \u03bcm; siCTRL, \\\\(n\\\\) = 107 cells; Swip1 siRNA1, \\\\(n\\\\) = 116 cells; and siRisk21, \\\\(n\\\\) = 122 cells. **d**, SIM \\\\(x\\\\) = projections of MDA-MB-231 cells expressing mScarlet1-Sswip1 and GFP-tagged a2-integrin-WT or mutant a2 integrin-AA (top). The integrin cytoplasmic sequence and mutated residues (red) are depicted (bottom). Levels of co-localization of GFP-tagged proteins with mScarlet1-Sswip1 (right). Mutant a2 integrin-AA, \\\\(n\\\\) = 76 cells; and a2 integrin-WT, \\\\(n\\\\) = 70. **d**, SIM \\\\(x\\\\) = projections of siCTRL- or Rab21-silenced (MDA-MB-231 cells expressing mScarlet1-Sswip1 and immunostained for pI-integrin (PS02; left). Levels of co-localization between mScarlet1-Sswip1 and bTI integrin (right). siCTR, \\\\(n\\\\) = 80 cells; and siRisk21, \\\\(n\\\\) = 56 cells. **d**, Scale bars, 0.5 \u03bcm. **f**, Representative immunoblotting of three independent GFP-Trap pulldowns from MDA-MB-231 cells transfected with different GFP-tagged integrin _a_-subunits and blotted as indicated. **g** Levels of cell-surface biotinylated ff1-integrin (left) and a6-integrin internalization (right) in Swip1-slenecked MDA-MB-231 cells after the indicated times in the presence or absence of 100 \u03bcm primaquine (PQ). The bar charts show the mean >= s.e.m. Statistical significance was assessed using multiple-comparison r-tests for paired data, with the post-hoc Holm-Sidak method; \\\\(n\\\\) = 5.000%. For ff1-integrin (left), ***P = 0.00934 and *P = 0.02015; for a6-integrin, *P = 0.02196 and *P = 0.04683 Three independent experiments were performed. **h**, Level of ff1-integrin internalization in siCTRL- and IRSp53-silenced (siRisk553) MDA-MB-231 cells expressing GFP or GFP-Swip1 (left). Representative immunoblotting of cell lysates probed as indicated (right); **s**c**T**R1 + GFP, \\\\(n\\\\) = 157 cells; siCTRL + GFP-Swip1, \\\\(n\\\\) = 289 cells; siRSp53 + GFP, \\\\(n\\\\) = 108 and siRSp53 + GFP-Swip1, \\\\(n\\\\) = 136 cells. ***P = 0.0003. A Representative micrographs (left) and quantitation of PLA with indicated antibodies in MDA-MB-231 cells expressing GFP or GFP-Swip1. Scale bars, 10 \u03bcm; GFP, \\\\(n\\\\) = 106 and GFP-Swip1, \\\\(n\\\\) = 11 cells. ***P = 0.0001'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5 : Swipt is a cargo adaptor for the CG pathway.****a**, Double uptake (indicated by arrows) of fluorescently labelled 10 kDa dextran-tetramethylthydamine (TMR) with either fluorescein isothiocyanate (FITC)-conjugated dextran or Alexa Fluor (AF) 488-conjugated anti-gli-integrin antibody (12G10, b!-integrin-AF488) in MDA-MB-231 cells for the indicated times (right). Representative images at 5 min internalization are shown (left). Scale bars, 10 \u03bcm (main image) and 3 \u03bcm (inset, magnified view of the yellow box in the main image). Dextran-FITC, \\\\(n\\\\) = 18 and 21 cells; and b!-integrin-A488, \\\\(n\\\\) = 22 and 21 cells. **b**, Representative micrographs (top), and levels of dextran-TMR (bottom right) and b!-integrin-AF488 (bottom left) internalization in control;- Swipt- and R5pS3-sicnec MAC-MB-231 at 15 min. **n_ = 157 cells; Swipt siRNA2 and sicCTRL, \\\\(n\\\\) = 160 cells; sicCTRL, \\\\(n\\\\) = 121 cells; IR5pS3 siRNA1, \\\\(n\\\\) = 177 cells; and IR5pS3 siRNA2, \\\\(n\\\\) = 121 cells. ***_P_ = 0.0001 and ***_P_ = 0.0004. **c**, Representative micrographs (left) and levels of M-HCI internalization in control;-Wipol-1 and IR5pS3-sicnec MAC-MB-231 at 15 min (right). Swipt siRNA1, \\\\(n\\\\) = 125 cells; and sicCTRL, \\\\(n\\\\) = 92 cells; and sicCTRL, \\\\(n\\\\) = 92 cells. **b**, Dashed lines show the outlines of the cells defined by labelling with the plasma membrane marker WGA lectin conjugated to Alexa Fluor 647 (WGA-AF647). Scale bars, 10 \u03bcm. **d**, Levels of b!-integrin (top) and M-HCI (bottom) internalization in control- or Swipt-sicnec MAC-MB-468 (left) and BT-20 (right) cells at 15 min. For BT-20 12G10 and M-HCI uptake, respectively, sicCTRL, \\\\(n\\\\) = 79 and 91 cells; Swipt siRNA1, \\\\(n\\\\) = 76 and 91 cells; and Swipt siRNA2, \\\\(n\\\\) = 66 and 75 cells. For MDA-MB-468 12G10 and M-HCI uptake, respectively, sicCTRL, \\\\(n\\\\) = 134 and 155 cells; Swipt siRNA1, \\\\(n\\\\) = 98 cells and 156 cells; and Swipt siRNA2, \\\\(n\\\\) = 98 cells and 135 cells. *_P_ = 0.0137, *_P_ = 0.0007 and ***_P_ = 0.0006. **a**, Levels of b!-integrin-AF488 (right) and b!0 kDa dextran-TMR (left) uptake in steady state or after recovery from hypotonic shock in control- and Swipt-sicnec MAC-MB-231 cells. For dextran uptake in the absence of (-) or with (+) hypotonic shock, respectively: sicCTRL, \\\\(n\\\\) = 227 and 220 cells; Swipt siRNA1, \\\\(n\\\\) = 208 and 197 cells. For b!-integrin uptake in the absence of (-) or with (+) hypotonic shock, respectively: sicCTRL, \\\\(n\\\\) = 195 and 191 cells; Swipt siRNA1, \\\\(n\\\\) = 208 and 181 cells; and Swipt siRNA2, \\\\(n\\\\) = 181 and 139 cells. **f**, SIM \\\\(x\\\\) = projections of control- and Rab21-sicnec (siRNA2) MDA-MB-231 cells expressing mScarlet-1-Swipt and immunostained for IR5pS3 (left). Co-localization between mScarlet-1-Swipt and endogenous IR5pS3 was quantified (right); sicCTRL, \\\\(n\\\\) = 50 cells; and siRNA2, \\\\(n\\\\) = 56 cells. Scale bars, 0.5 \u03bcm. **g**, Cell lysates transfected with HA-Arf13(inactive) or HA-Arf13(inactive) were incubated with 2 \u03bcM of recombinant purified GST or GST-Swipt. Representative GST pulldowns (IVBs) stained with Ponceau and blotted with the indicated antibody from three independent experiments. **a**-**f**, Data are the mean +- 95% CI; \\\\(n\\\\) is the total number of cells pooled from three independent experiments. Statistical significance was assessed using two-sided (Mann-Whitney tests; ***_P_ < 0.0001. Unprocessed blots and numerical source data are provided.\\n\\n'", "CAPTION FIG6.png": "'\\nFig. 6: **Swipi-actin binding regulates integrin traffic.****a**, Swipi domains. EF, EF-hand domain containing a calcium-binding site (shown in grey); SH3, SRC homology 3 domain. **b**, Representative GFP-pulldowns of two independent experiments in HEK293 cells expressing GFP, GFP-Swipi or truncated versions of Swipi and blotted as indicated. If, immunoprecipitation. **c**, Levels of pI-integrin uptake at 15 min in MDA-MB-231 cells expressing the indicated proteins. **P** = 0.0034 and **P** = 0.0356; \\\\(n\\\\) = 92 (GFP), 94 (GFP-Swipi), 161 (GFP-EGFP) and 101 (GFP-EGFP) cells. **d**, Representative SIM _x_-_z_ projections of MDA-MB-231 cells expressing mScarlet-1-Swipi, immunostained for pI-integrin and labelled with phalloidin. Two independent experiments were performed. Scale bars, 0.7 \u03bcm. **e**, Representative SIM _x_-_y_ image of MDA-MB-231 cells expressing mScarlet-1-Swipi and GFP-Rab21 and labelled with phalloidin. The white squares highlight ROIs. The yellow arrows point to Swipi overlap with actin on Rab21-containing vesicles. The pink arrow indicates actin filaments in close proximity to the vesicle. Scale bars, 5um (main image) and 0.5um (magnified views of (i); bottom). **f**, Electron microscopy images of GFP-Swipi visualized using GFP-APEX. ROI (i) and (ii) are magnified (bottom). The arrows point to Swipi-APEX-passive patches adjacent to filament-like actin structures (blue) or vesicles (orange). Scale bars, 0.5um (top), 0.2 \u03bcm (middle and bottom left, magnified view of (i)) and 0.1\u03bcm (bottom right, magnified view of (i)). **g**. Average speed of Rab21 vesicles for each cell close to the TIRF plane over 2 min (bottom left). Representative tracks of Rab21 vesicles in a control or a Swipi-silenced cell (top) and representative immunoblot validating Swipi silencing (bottom right). Scale bar, 5\u03bcm *P** = 0.0005 and ***P** = 0.0003; \\\\(n\\\\) = 31 (siCTRL), 33 (siSwipi siRNA1) and 39 (siSwipi siRNA2) cells. **b**, Average speed of Rab21-vesile movement for each cell following treatment with cytochalasin in ***P** = 0.0007; \\\\(n\\\\) = 21 (DMSO) and 18 (cytochalasin D) cells. **i**, Average speed of Rab21-vesicle movement for each cell following Swipi silencing and rescue with mScarlet-1-Swipi or mScarlet-1-AEF1. ***P** < 0.0001; \\\\(n\\\\) = 33 cells per condition. **i**, Swipi directs integrins to CG-endocytosis. **c.g.-1**, Data are the mean >= 95% CI; \\\\(n\\\\) is the total number of cells pooled from three independent experiments. Statistical significance was assessed using two-sided Mann-Whitney tests. Unprocessed blots and numerical source data are provided.\\n\\n'", "CAPTION FIG7.png": "'\\nFig. 7: **Swip1 regulates adhesion dynamics, cell motility and breast cancer progression.****a**, Representative masks of vinculin-containing focal adhesions in control- or Swipi-silenced MDA-MB-231 cells on collagen 1 (top). Analysis of the number of adhesions and total adhesion area per cell on different ECM components (bottom). Scale bars, 10 \\\\(\\\\mu\\\\)m; data are the mean = 95% CI. Collagen 1, \\\\(n\\\\) = 65, 76 and 67 cells; fibronectin, \\\\(n\\\\) = 65, 75 and 62 cells; and laminin \\\\(n\\\\) = 62, 66 and 73 5 cells for _s_i_CTR1, sSiWiSu1 isRNA1 and sRiNA2, respectively. Collagen, \"***P < 0.0001 and \"***P = 0.0005; fibronectin, \"*P = 0.0002 (Swipi siRNA1) and 0.0007 (siRNA2). laminin-1, \"***P < 0.0001.**b**, Visualization of the GFP-paxillin dynamics in control- and Swipi-silenced cells (left). The colour scale represents the focal adhesion localization over time. The assembly and disassembly rates are shown (middle). Scale bar, 10 \\\\(\\\\mu\\\\)m; siCTRL, \\\\(n\\\\) = 861 and 764 adhesions; and siWiSu1, \\\\(n\\\\) = 1,014 and 1,024 adhesions for assembly and disassembly, respectively; \\\\(n\\\\) = 18 cells were analysed per condition. **c**, Representative TIRF microscopy BiPC images of live MDA-MB-231 cells expressing VI-Rab21, V2-Swip1 and mKate2-paxillin (left). Distance between Swipi-Rab21 puncta or randomly distributed puncta and the closest paill-positive focal adhesions (right). Scale bars, 25 \\\\(\\\\mu\\\\)m (main image) and 1 \\\\(\\\\mu\\\\)m (magnified view of the white box); \\\\(n\\\\) = 697 (BiPC) and 1,364 (randomly distributed) puncta: \\\\(n\\\\) = 20 cells per condition. **b**, \"***P < 0.0001.**d**, Migration of control-; Swip- or Rab21-silenced MDA-MB-231 cells. Venus-area coverage were time (left). Data are the mean \\\\(\\\\pm\\\\)s.m. Statistic significance was assessed using multiple-comparison _n_-tests with the post-blue Helim-Sdisk method; siWiSu1 siRNA1, \"P = 0.022118 (0.0048533, 0.00320197, 0.00598189 and 0.00156902; siSwipt siRNA2, \"P = 0.00161506, 8.929064 \\\\(\\\\times 10^{-5}\\\\), 0.00019015, 0.000316543 and 0.000100846; and siRNA21, \"P = 0.0158582, 0.0101405, 0.00574356, 0.0240206 and 0.0286109 for 8, 12, 16 and 18h, respectively, for comparisons with the siCTRL condition. **e**, Representative micrographs of control- and Swipi-silenced MDA-MB-231 cells that invaded through fibrillar collagen I (top). The relative invasion through fibrillar collagen I over 60 \\\\(\\\\mu\\\\)m was quantified (bottom left). Representative immunoblot validating Swipi silencing is shown (bottom right). *P = 0.0418 and \"*P = 0.0055. **d**, Three independent experiments were performed. **f**, Representative images of Swipt staining in samples of HER2- and TIRF DNACT cancer tissue microarrays (left) and the percentage of tumours with high medium or low Swipt (top right). Overall survival of 133 patients with TIRC which high (red) or medium and low (blue) staining of Swipt (bottom right). The hazard ratio (HR) was 1.84 (95% CI, 1.05\u20133.23). Scale bars, 20 \\\\(\\\\mu\\\\)m. **b**,**c**, Resolpts display the median and quartiles of the data and the whiskers display the maxima and minima. The mean of the data are indicated with a + symbol. **a**,**b**,**d**, Representative immunoblotts validating silencing are shown. **a\u2013****c**, Statistical significance was assessed using two-sided Mann-Whitney tests; \\\\(n\\\\) is the number of analysed cells (**a**), adhesions (**b**) or puncta (**c**) pooled from three independent experiments. Unprocessed blots and numerical source data are provided.\\n\\n'", "CAPTION SFIG1.png": "'\\n\\n**Extended Data Fig. 1 [**Swin1 (EFHD2) is an indicator of Rab21**. SILAC proteomics analysis of GFP-Trap pulldowns in MDA-MB-231 cells expressing GFP-WT-Rab21 vs. GFP-DN-Rab21 (T3IN inactive GDP-bound/nucleotide-free mutant) **(a)**: GFP-CA-Rab21 (Q76L constitutively active GTP-bound mutant) vs. GFP **(b)**: GFP-WT-Rab21 vs. GFP **(c)** or GFP-WT-Rab21 vs. GFP-CA-Rab21 **(d)**. (a-d) Each spot in the plots corresponds to one identified protein by mass spectrometry. Plots are representative of 2 independent experiments, forward and reverse; where every experiment consists of two independent affinity parifications. Plots show mean fold-changes from forward and reverse experiments against absolute protein abundances (intensity-based absolute quantification, iBAQ). Abundance bins were defined by including 1000 proteins in a subsequent order. Log100 field change of proteins were tested for significance using double-sided significance tests. No multiple hypothesis correction method was applied, due to the small number of selected proteins for the statistical analysis. Proteins with a _P_-value < 0.01 are projected as a triangle, while non-significant proteins are shown as circles. P-values are depicted in the figure for a selected set of proteins relevant to the work. Proteins in red are markedly enriched in the CA- or WT-Rab21 fraction and proteins in blue are known endosomal proteins clathrin (CLTA, CLTB, CLTC), APE (AP2A1, AP2B1, AP2MI, AP251), caveolin (CAVI) and dynam II (DNM2), which are not specifically enriched. Swip1 was strongly enriched in the GFP-WT-Rab21 vs. GFP-DN-Rab21 **(a)** and GFP-CA-Rab21 and GFP-WT-Rab21 fractions compared to GFP fractions **(b, d)**, and it was equally enriched in the GFP-CA-Rab21 compared to the GFP-WT-Rab21 fraction **(c)**. Clattrin (CLTA, CLTB, CLTC), AP2 (AP2A1, AP2BI, AP2MI, AP251), caveolin (CAVI) and dynamin II (DNM2) are not strongly enriched in any fraction **(e)**. Representative immunoblots of GFP-Trap pulldowns from MDA-MB-231 cells transfected with GFP, GFP-WT-Rab21, GFP-WT-Rab5, GFP-DN-Rab5 or GFP-CA-Rab5 stained for endogenous Swip1 and pH integrin. Blots are representative of 2 independent experiments. Unprocessed blots are provided in Source data.\\n\\n'", "CAPTION SFIG10.png": "\"\\n\\n**Extended Data Fig. 10**: **EHD2 (Swip1) mRNA expression correlates with a more metastatic breast cancer phenotype and Swip1 immunostaining at the plasma membrane correlates with higher death risk and metastasis incidence in TNBC.****(a)** Analysis of Swip1 mRNA expression using qPCR in 192 breast tumours. Turnours were categorized into two groups: high EFHD2 expression (higher than the median of the entire collection) or low EFHD2 expression (lower expression than the median). The y2-test was used for statistical analyses. EFHD2 mRNA levels based on tumour type and tumour grade are shown. **(b)** Validation of anti-Swip1 antibody specificity in IHC was carried out with agarose-embedded and formalin-fixed paraffin-embedded cell pellets from control- or Swip1-silenced MDA-MB-231 cells (siRNA #1 and #2). Scale bars, 50 **mm.****(c)** Quantification of the percentage of breast cancer tumours from TNBC tissue microarray with high, medium or low Swip1 staining at the plasma membrane. **(d)** Kaplan-Meier plot shows overall survival of 133 triple negative breast cancer patients with high (H, red) or medium-low (M-L, blue) membranal staining of Swip1 in their tumour centre sample. The hazard ratio of high vs. medium-low Swip1 immuno-positivity was 2.34 (95% C1.25 to 4.41). The hazard ratios after adjustment for Ki67, 2.51 (95% C1.26 to 5.01): for tumour size, 2.12 (95% C1.08 to 4.18); for lymph node metastasis status 2.26 (95% C119 to 4.31) and for tumour grade 2.44 (95% C11.25 to 4.74). **(e)** lymph node metastasis incidence in 130 TNBC patients from the same cohort, Fisher's exact test p = 0.037. A higher proportion of patients with high membranal staining of Swip1 (> = 80%) had lymph node metastasis. Numerical source data are provided in Source data.\\n\\n\"", "CAPTION SFIG2.png": "'\\n\\n**Extended Data Fig. 2 | BiFC/BiCAP approaches to assess the interaction between Rab21 and Swip1. (a)** Cartoon of the BiFC-/BK-AP approach used to image (BiFC) and biochemically detect (BiCAP) the interactions between Rab21 and Swip1 in live cells and from cell lysate. (b) MDA-MB-231 cells were transfected with the indicated constructs and lysed. Proteins expressed in the transfected cells were detected by immunoblotting, Immunoblot shows the size of the bands detected by a polyclonal anti-GFP antibody. Blots are representative of 2 independent experiments. (c) Representative confocal microscopy BiFC images of MDA-MB-231 cells expressing VI-Rab21 and V2-Swip1 MDA-MB-231 and immunostained for endosomal markers. Insets show BiFC colocalizing with the indicated endosomal markers. Scale bars, 10 mm (main figures) and 1 mm (insets). Representative pictures of 3 independent experiments. Unprocessed blots are provided in Source data.\\n\\n'", "CAPTION SFIG3.png": "'\\n\\n**Extended Data Fig. 3 | SIM images of Swip1 with CG components and Rab proteins. (a) Confocal micrographs of MDA-MB-231 cells expressing mScarlet-I-Swip1 and GFP-Rab21, and immunostained for |b|-integrin (12G10 antibody). Arrows show areas of co-localization in endosomal structures. Representative pictures of n=3 independent experiments. Scale bar, 10 \\\\(\\\\mu\\\\)m. (b) Full pictures from Fig. 2d, e and 3d. x-y projections of MDA-MB-231 cells expressing mScarlet-I-Swip1 and either GFP-Rab21, Artif-GFP or GFP-IRSb53 were imaged using structured illumination microscopy (SIM). Blue and yellow squares highlight the regions of interest (ROI) shown as x-z projections in Fig. 2d, e and Figure 3d. Representative pictures of n=3 independent experiments. Scale bars, 5 \\\\(\\\\mu\\\\)m (2d), 8 \\\\(\\\\mu\\\\)m (2e) and 8 \\\\(\\\\mu\\\\)m (3d), insets, 1 \\\\(\\\\mu\\\\)m. (c) SIM-x2 repetitions of MDA-MB-231 cells expressing mScarlet-I-Swip1 and immunostained for endogenous Rab proteins and quantification of Rab protein co-localization with mScarlet-I-Swip1. Each dot represents the co-localization ratio in one cell. Data are presented as mean values \\\\(\\\\pm\\\\) 95 % CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled from 3 independent experiments. \\\\(P\\\\) values calculated compared to Rab21 condition. \\\\({}^{'", "CAPTION SFIG4.png": "'\\n\\n**Extended Data Fig. 4 [GP-Swip1 co-localizes with Arft-HA at the TIRF plane and regulates pH-integrin endocytosis via the CG pathway. (a)** MDA-MB-231 cells were co-transfected with GFP-Swip1 and Arft-HA constructs, fixed, stained with probes for DNA paint and imaged using SMAM. Examples of the structures formed by GFP-Swip1 at the proximity of the ECM interphase are shown. Representative pictures of n = 2 independent experiments. Scale bars, 2 mm (main images) and 0.5 mm (insets). (b) Representative imrmunblots of control- and Swip1-silenced MDA-MB-231 cell lysates blotted as indicated, calnexin is included as a loading control. Blots are representative of n = 3 independent experiments. (c) FACS analyses of cell surface (II-integrin in control- and Swip1-silenced MDA-MB-231 cells using the indicated antibodies. Bar charts show data as geometric mean values of 10,000 cells +- SEM over n = 3 independent experiments. Statistical significance was assessed with two-tailed Wilcoxon matched-pairs signed-rank tests (n = 3 independent experiments), ns = not significant. (d) Quantification of biotinylated a2-, a3- or are-integrin internalization in Swip1-silenced MDA-MB-231 cells (siRNA #2) after the indicated times determined with ELISA in the presence or absence of 100 mM prinquare. Bar charts show data as mean values +- SEM. Statistical significance was assessed using multiple-comparison t-tests for paired data, with the post-hoc Holm-Sidak method, with alpha = 5.000%. Each raw was analysed individually, without assuming a consistent SD*P = 0.07540; n = 3 biologically independent experiments. Unprocessed blots and numerical source data are provided in Source data.**'", "CAPTION SFIG5.png": "'\\n\\n**Extended Data Fig. 5 Active \\\\(\\\\beta\\\\)I-integrins are endocytosed via the CG pathway and CME. (a)** Representative micrographs and quantification of \\\\(\\\\beta\\\\)I-integrin uptake in control- or IRSp53- or _ArH_-silenced MDA-MB-231 cells. Representative immunoblots to validate Swip1 silencing. Scale bars, 10 \\\\(\\\\mu\\\\)m. **(b)** Representative micrographs and quantification of endocytosed or surface (no endocytosis or acid wash) of murine \\\\(\\\\beta\\\\)I-integrin (9EG7 antibody) in isomeric IRSp53-/- MEFs: IRSp53-KO-pBABE ( and IRSp53-KO-pBABE-IRSp53 (WT) in which the expression of IRSp53 has been restored. Representative immunoblots of cell lysates blotted as indicated. Scale bars, 10 \\\\(\\\\mu\\\\)m. (c) Representative micrographs of \\\\(\\\\beta\\\\)I-integrin uptake in GFP- and GFP-Swip1-expressing cells and quantification of integrin uptake at the indicated times. Scale bars, 10 \\\\(\\\\mu\\\\)m. (d) PLA with the indicated antibodies (from Figure 44) in MDA-MB-231 cells expressing either GFP or GFP-Swip1. Plot shows the background controls for each. (e) Representative images and quantification of co-localization of \\\\(\\\\beta\\\\)I-integrin-AF488 (12G10) with TMR-10 kDa dextran or AF647-transferrin after 1 min simultaneous uptake in MDA-MB-231 cells. Yellow arrows show regions of co-localization between \\\\(\\\\beta\\\\)I-integrin and TMR-dextran and cyan arrows show regions where \\\\(\\\\beta\\\\)I-integrin co-localizes with AF647-transferrin. Scale bars, 10 \\\\(\\\\mu\\\\)m. For all plots, data are presented as mean values \\\\(\\\\pm\\\\) 95% CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled from 3 independent experiments (a-d) or from 2 independent experiments (e). (a) \\\\(\\\\bullet\\\\)P_C0.0001, (b) \\\\(\\\\bullet\\\\)P_C0.0001, ns=no significant, (c) \\\\(\\\\bullet\\\\)P_P_=0.0478, \"P_=0.0058, (c) \\\\(\\\\bullet\\\\)P_=0.0006, (d) \\\\(\\\\bullet\\\\)P_=0.0001, (e) \\\\(\\\\bullet\\\\)P_=0.0001, (f) \\\\(\\\\bullet\\\\)P_=0.0001.\\n\\nNumber of analysed cells: (a) as iCTRL, n=103 cells, ArH siRNA #1, n=177 cells, ArH siRNA #2, n=157 cells, a;STRL, n=100, IRSp53 siRNA H #1 = 142 cells, (kSp53 siRNA H #2, n=124 cells. (b) Endocytosed and surface \\\\(\\\\beta\\\\)I-integrin, respectively: IRSp53 WT, n=108 & n=55 cells, IRSp53 KO, n=100 & n=60 cells. (c) GFP, n=10, n=22, n=30 and n=24 cells and GFP-Swip1, n=15, n=22, n=32 and n=26 cells, respectively, (d) For GFP, 12G10 only, n=75 cells; IRSp53 only, n=113 cells; 12G10 + IRSp53, n=106 cells. For GFP-Swip1, 12G10 only, n=78 cells; IRSp53 only, n=92 cells; 12G10 + IRSp53, n=11 cells. (e) \\\\(\\\\beta\\\\)I-integrin-dextran, n=22 cells; \\\\(\\\\beta\\\\)I-integrin-transferrin, n=22 cells and transferrin-dextran, n=18 cells. Unprocessed blots and numerical source data are provided in Source data.**'", "CAPTION SFIG6.png": "'\\n\\n**Extended Data Fig. 6 [**Swip1 regulates (B1-integrin endocytosis via the CG pathway. (a)** Representative micrographs and quantification of B1-integrin and M4-HCl uptake at the 15 min time point in control or Swip1 (siRNArl or #2) silenced MDA-MB-468 and BT-20 cells. Representative imrmonblots of cell lysates blotted for Swip1. Calnexin is included as a loading control. Scale bars, 10 \\\\(\\\\mu\\\\)m. (b) Representative immunoblot of MDA-MB-231, BT-20 and MDA-MB-468 cell lysates blotted for Swip1. _a_-tubulin is included as a loading control. (c) Representative micrographs and quantification of transferrin uptake at the 15 min time point in control- or Swip1-silenced MDA-MB-231 cells. Representative imrmonblots of cell lysates blotted as indicated. Calnexin is included as a loading control. Scale bars, 10 \\\\(\\\\mu\\\\)m. (d) Cell-surface labeled EGFR uptake in control and Swip1-silenced MDA-MB-231 cells treated with 10 mg/ml EGF was analysed using flow cytometry in 10 000 non-permeabilized cells per measurement. Plot shows 4 measurements from \\\\(n\\\\) = 2 independent experiments. Immunoblot to validate swip1 silencing is representative of 2 independent experiments. GAPDH is included as a loading control. Immunohistochemistry in panels (a-c) and scatter off-plots (a-c) are representative of 3 independent experiments. Data are presented as mean values +- 95% CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where in the total number of cells pooled across a independent experiments. \\\\(P\\\\) values calculated compared to a CTRL condition: *_P_ = 0.0737, *_P_ = 0.0007, *_P_ = 0.0006, *_P_ = 0.0001, \\\\(n\\\\) = not significant. Number of analysed cells over a 3 independent experiments: (a) For BT-20 12G10 and MHC1 uptake, respectively, siRNArl, n = 79 & 91 cells; Swip1 siRNArl, n = 76 & 91 cells; Swip1 siRNArl, n = 66 & 75 cells. For MDA-MB-468 12G10 and MHC1 uptake, respectively, siRNArl, n = 134 & 132 cells; Swip1 siRNArl, n = 98 & 117 cells; Swip1 siRNArl, n = 98 & 101 cells. (c) siRNArl, n = 74 cells; Swip1 siRNArl, n = 103 cells. Unprocessed blots and numerical source data are provided in Source data.**'", "CAPTION SFIG7.png": "'\\n\\n**Extended Data Fig. 7 [suppl is required for specific entry of BI-integrin via the CG pathway following changes in membrane tension; Swip1 does not directly interact with IRSp53 and it does not affect the number of Rab2+containing vesicles. (a) Representative images of endocytosed AF48B-anti-flip-integrin (12G1O antibody) and TMR-10 kDa dextran in MDA-MB-231 cells after treatment of cells with hypotonic media followed by a shift to isotonic media. Scale bars, 10 \\\\(\\\\mu\\\\)m. Pictures are representative of n = 3 independent experiments. Quantification of these experiments is shown in Figure 5a. (b) Recombinant purified his-Swip1 or his-VASP (passive control) proteins were spotted at the indicated concentrations on nitrocellulose filters. BSA was used as negative control. Nitrocellulose membranes were incubated with recombinant purified IRSp53 at the indicated concentrations and immunoblotting with an anti-IRSp53-specific antibody. (c) Recombinant purified his-Swip1 (500 \\\\(\\\\mu\\\\)M) or his-VASP (50 nM, positive control) proteins were spotted at the indicated concentrations on nitrocellulose filters. BSA was used as negative control. Nitrocellulose membranes were incubated with recombinant purified IRSp53 at the indicated concentrations and immunoblotted with an anti-IRSp53-specific antibody. Pictures are representative of n = 3 independent experiments (a-c). (d) Representative micrographs of MDA-MB-231 cells immunostained for endegenus Rab21 and counterstained with DAPI and WGA-AF647 as a plasma membrane marker to define the cell outlines. The outlines of each cell are indicated in green dashed lines. The number of vesicles in 100 MDA-MB-231 cells per condition was quantified using the IMARJS vesicle detection function. Data are presented as mean values +- 95 % CI Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled across 3 independent experiments (n = 100 cells per condition). P values calculated compared to sICTRL condition: *** \\\\(P\\\\) < 0.0001, ns: not significant. Scale bar, 10 \\\\(\\\\mu\\\\)m. Numerical source data are provided in Source data.\\n\\n'", "CAPTION SFIG8.png": "'\\n\\n**Extended Data Fig. 8 | Arfi and RSp53 regulate focal adhesion formation and Swip1 and Rab21 regulate cell migration.****(a)** Representative immunoblots to validate Swip1 silencing and mScarlet-1-Swip1 or mScarlet-1-AEFI overexpression in the experiments shown in Figure 6: Blot is representative of 3 independent experiments. (**b**) Representative masks of vinculin-containing focal adhesions in control-, IRSp53- or Arfi-silenced MDA-MB-231 cells and quantification of adhesion number and total area per cell in cells plated on collagen I. Data are presented as mean values +- 95% CI. Statistical significance was assessed with two-sided Mann-Whitney tests, where n is the total number of cells pooled across 3 independent experiments. P values calculated compared to siCTRL condition: ***_P_=0.0003, ***_pC_0.0001. Number of analysed cells over 3 independent experiments: siCTRL, n= 62 cells; Arfi siRNA #1, n= 60 cells; Arfi siRNA #2, n= 61 cells; siCTRL, n= 64 cells, IRSp53 siRNA #1, n= 62 cells and IRSp53 siRNA #2, n= 65 cells. Representative immunoblots of 3 independent experiments to validate IRSp53 and Arfi silencing are shown. Scale bars, 10 \u03bcm. (**c**) Representative phase contrast pictures of the scratch wound after 18 h. Pictures are representative of 3 independent experiments. Quantification of these experiments is shown in Figure 7d. Scale bar, 100 \u03bcm. Unprocessed blots and numerical source data are provided in Source data.\\n\\n'", "CAPTION SFIG9.png": "'\\n\\n**Extended Data Fig. 9 [Swip1 is required for random cell migration.** Random cell migration speed of MDA-MB-231 **(a)** and MDA-MB-468 **(b)** cells upon Swip1 silencing. In all cases, cells were labelled using sir-DNA and their migration behaviour recorded over time using a widefield microscope. Cell nuclei were automatically tracked using StarDist and TrackMate. Cell track speed and mean displacement plots are displayed. Boxplots display the median and quartiles of the data. Whiskers display the 2nd percentile and the 98th percentile. Mean displacement plots show mean values + standard deviation. For all panels, _P_-values were determined using a randomization test, where n is the total number of cells pooled across 3 independent experiments, ***_P_= <0.001. Number of cells analysed over 3 independent experiments: MDA-MB-231, sicTRI, n = 869 cells; siSwip1 #1, n = 553 cells; siSwip1 #2, n = 506 cells; MDA-MB-468, siCTRL, n = 331 cells; siSwip1 #1, n = 206 cells; siSwip1 #2, n = 235 cells. Numerical source data are provided in Source data.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/nguyenTestingPopularNews2019.json b/extracted_captions/nguyenTestingPopularNews2019.json new file mode 100644 index 0000000000000000000000000000000000000000..24cc7b8ac301293197f0d05e2c347d5c5293d703 --- /dev/null +++ b/extracted_captions/nguyenTestingPopularNews2019.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Graphical representation of two-step ANOVAs results for six aspects of EU conception by exact-used media for EU policies news, across three different EU participation groups\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/nomura2004human.json b/extracted_captions/nomura2004human.json new file mode 100644 index 0000000000000000000000000000000000000000..c6a0cdda27b98013129f5ce4922dbd3c4975f4f8 --- /dev/null +++ b/extracted_captions/nomura2004human.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Identification of CD13 as a major DRM fraction of human fibroblasts. (A) Total lysates and DRMs of human fibroblasts were subjected to SDS-PAGE (7 to 16% gel) and silver staining. Eighteen bands (160, 125, 107, 97, 86, 70, 58, 49, 46, 41, 38, 35, 31, 29, 28, 22, 21, and 16 kDa) were detected (arrow and arrowheads). Two peptides (peptides 1 and 2) obtained from the prominent 160-kDa band (arrow) were subjected to amino acid sequencing. In peptide 1, 8 of 10 amino acids agreed with amino acids 231 to 239 of CD13, and in peptide 2, all 42 amino acids agreed with amino acids 924 to 935 of CD13. (B) Fractions obtained by sucrose density gradient ultracentrifugation of the total cell lysate treated with Triton X-100 were subjected to immuno-blotting for CD13, TIfR, caveolin-1, and \\\\(\\\\alpha\\\\)-tubulin. CD13 and caveolin-1 were enriched in DRMs, but TIfR and \\\\(\\\\alpha\\\\)-tubulin were detected in the bottom fraction.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Immunofluorescence microscopy. Human fibroblasts were treated with an anti-CD13 antibody and a fluorochrome-conjugated secondary antibody one ice and incubated at 37\u00b0C for 0 min (a to c), 10 min (d to f), 30 min (g to i), or 60 min (j to l). After fixation, the cells were labeled with an anti-caveolin-1 antibody. Labeling for caveolin-1 was seen as patches in all samples (b, e, h, and k). Labeling for CD13 was observed evenly on the cell surface without warming (a); after 10 min of incubation at 37\u00b0C, most of the labeling showed a linear alignment (d); after 30 min at 37\u00b0C, CD13 showed clustering, and colocalization with caveolin-1 became apparent (g to i; arrows); and after 60 min at 37\u00b0C, CD13 and caveolin-1 were colocalized extensively (j to l; arrows). The cells were treated with the anti-TFR antibody and the secondary antibody in the same manner as that described above. Labeling for TFR was observed evenly on the cell surface without warming (m to o), and even after 60 min, TFR and caveolin-1 did not show colocalization (p to r). Bar = 50 mm.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Immunofluorescence microscopy of human fibroblasts bound to HCoV-229E. Optical sections obtained by laser confocal scanning microscopy were stacked. The cells were treated with HCoV-229E on ice and incubated at 37\u00b0C for 0 h (a to c and m to a), 1 h (d to f), or 3 h (g to l and p to r). After fixation, the cells were labeled with an anti-HCoV-229E or anti-CD13 antibody in combination with an anti-caveolin-1 antibody. Labeling for cavedin-1 was always seen as patches (b, e, h, k, n, and q). Labeling for HCoV-229E was seen as randomly distributed dots in cells kept on ice (a); after 1 h of incubation at 37\u00b0C, HCoV-229E showed some linear alignment, and colocalization of HCoV-229E and cavedin-1 was seen occasionally (d to f, arrows); and after 3 h at 37\u00b0C, the colocalization of HCoV-229E and caveolin-1 became prominent (g to l; arrows). The colocalization of HCoV-229E and caveolin-1 in small clusters was often observed (g to i; arrowheads). Labeling for CD13 was seen as randomly distributed dots at 0 h (m) but showed extensive colocalization with cavedin-1 after 3 h of incubation at 37\u00b0C (p to r; arrows). Bar = 50 \u03bcm.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Electronic microscopy of human fibroblasts bound to HCaV-229E. (A) The cells were treated with HCaV-229E on ice and incubated at 37\u00b0C for 0 h (a and b) or 3 h (c to g) before fixation. At 0 h, most viruses bound to the flat portion of the plasma membrane (a and b). After 3 h of incubation at 37\u00b0C, viruses localized at or within 150 nm of the orifices of caveolae (c to f) were observed frequently. In contrast, clathrin-coated pits (g) were not observed near the virus particles. Cross-bridge structures of high electron density were observed between the virus particles and the plasma membrane (a, inset of b [arrows]). Caveolae are shown by arrowheads (c). Bar = 100 nm. (B) Quantification of HCaV-229E associated with caveolae. The proportion of HCaV-229E localized at or within 150 nm of the orifices of caveolae was calculated by the use of electron micrographs. The proportion increased significantly by 3 h of incubation at 37\u00b0C (29.4 versus 22%) (chi-square test for independence; \\\\(P\\\\leq 0.0001\\\\)).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Effect of MgCD on binding and redistribution of HCoV-229E. The cells were bound to HCoV-229E on ice after being preincubated with nothing, with MgCD alone, or sequentially with MgCD and then an MgCD-cholesterol complex (cholesterol). (A) Western blot analysis with anti-HCoV-229E antiserum. Fifty-kilodiluoa bands were observed in all samples of cells bound to HCoV-229E (Jines 1 to 3) but were not detected without the virus treatment (Janes 4 to 6). (B) Degree of colocalization of HCoV-229E and caveolin-1. The ratio of colocalization after 3 h of incubation at 37\u00b0C was \\\\(23.8\\\\%\\\\) for the control, whereas it was significantly lower for cells treated with MgCD (6.3%). The ratio recovered to the control level when cells treated with MgCD were replenished with cholesterol by the addition of an MgCD-cholesterol complex (\\\\(22.4\\\\%\\\\)) (Mann-Whitney U test; \\\\(P<0.001\\\\)). The results are representative of three experiments.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Effect of M\\\\(\\\\beta\\\\)CD on intracellular entry of HC\\\\(\\\\alpha\\\\)V-229E. The data shown are from RT-PCRs of HC\\\\(\\\\alpha\\\\)V-229E RNA and G3PDH mRNA. The amount of the PCR template was normalized by using G3PDH in two different PCR cycles. The cells were incubated in the presence (\\\\(+\\\\)) or absence (\\\\(-\\\\)) of M\\\\(\\\\beta\\\\)CD and M\\\\(\\\\beta\\\\)CD-cholesterol (cholesterol) before being bound to HC\\\\(\\\\alpha\\\\)V-229E on ice and then incubated for 0 or 3.5 h at 37\\\\({}^{\\\\circ}\\\\)C. Trypsinization was done to remove virus particles bound to the cell surface for an estimation of the virus amount that entered the cell. Without the virus treatment, HC\\\\(\\\\alpha\\\\)V-229E RNA was not detected (lane 1). For cells kept on ice, the amounts of HC\\\\(\\\\alpha\\\\)V-229E RNA were equivalent in all samples, but trypsinization decreased the amount greatly (lanes 2 to 4, no trypsinization; lanes 5 to 7, after trypsinization). After incubation at 37\\\\({}^{\\\\circ}\\\\)C for 3.5 h, the intensity of the band was significantly lower for M\\\\(\\\\beta\\\\)CD-treated cells than for control cells or cells that were treated sequentially with M\\\\(\\\\beta\\\\)CD and M\\\\(\\\\beta\\\\)CD-cholesterol (lanes 8 to 10).\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Effect of caveclin-1 knockdown on binding and intracellular entry of HCoV-229E. The cells were not transfected (\\\\(-\\\\)) or were transfected with 80 nM control siRNA (cont) or 5 to 80 nM caveolin-1 siRNA and then were incubated at 37\\\\({}^{\\\\circ}\\\\)C for 24 h. Forty-eight hours after subculturing, the cells were bound to HCoV-229E on ice and processed immediately or incubated at 37\\\\({}^{\\\\circ}\\\\)C for 48 h. (A) Immunoblot blotting of total lysates of cells incubated on ice with anti-HCoV-229E, anti-caveolin-1, and anti-G3PDH antibodies. The amounts of the cell lysates were normalized with G3PDH. The amount of caveolin-1 decreased in a dose-dependent manner, and the expression of the HCoV-229E N protein was equivalent in all samples. (B) Immunofluorescence microscopy of cells transfected with 80 nM control siRNA (a to c) and caveolin-1 siRNA (d to f). Cells were bound to HCoV-229E on ice and incubated at 37\\\\({}^{\\\\circ}\\\\)C for 48 h. In cells transfected with the caveolin-1 siRNA, labeling for caveolin-1 decreased (e), and the number of cells showing cytoplasmic HCoV-229E labeling was also reduced (d and f). Bar = 50 \u03bcm. (C) Proportion of cells showing cytoplasmic labeling of HCoV-229E. The proportion was significantly lower in cells transfected with 5, 20, or 80 nM caveolin-1 siRNA (52.0, 42.8, and 40.1%, respectively) than in nontransfected cells (72.2%) or cells transfected with a control siRNA (73.0%) (chi-square test for independence; \\\\(P\\\\) < 0.001).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/okadaAnalogicalModificationCreation2009.json b/extracted_captions/okadaAnalogicalModificationCreation2009.json new file mode 100644 index 0000000000000000000000000000000000000000..cac2e48dbe7b933dd057542f8c94bee58b79895c --- /dev/null +++ b/extracted_captions/okadaAnalogicalModificationCreation2009.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Fig. 1. Analogical process in two types of contacts.\\n\\n'", "CAPTION FIG10.png": "\"* [10] Mr. Kenji Sugiyama's creative process and the development of his artwork series.\\n\\nFigure 10: Mr. Kenji Sugiyama\u2019s creative process and the development of his artwork series.\\n\\n\"", "CAPTION FIG2.png": "'Fig. 2 Analogical modification for artistic creation. _Note_: Activities shown in bold boxes are directly involved when analogical modification is used. Activities shown in dotted boxes are potentially involved when analogical modification is used. Arrows indicate the direction of influence.\\n\\n'", "CAPTION FIG3.png": "\"Fig. 3. Mr. Shinj Ogawa's creative process and the development of his artwork series.\\n\\n\"", "CAPTION FIG4.png": "'Figure 4: An artwork in the \u2019Without You\u201d series. _Note_: This is based on a scene from the movie _Roomu Holiday_. He erased Audrey Hepburn from this picture.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG6.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG7.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG8.png": "'Figure 8: \u201cPirouette\u201d (video work, part). Note: In this video work, after an object in a picture rotates, a new picture emerges. For example, after the woman lifting her left hand rotates, she becomes a police officer who tries to capture a thief.\\n\\n'", "CAPTION FIG9.png": "'Fig. 9. \"Interference\" (video work, partj). Note: When layers with objects in the photograph slide out or in, the entire picture drastically changes. For example, the scene with two people who look like a mother and a daughter are made of a combination of two different layers of photographs. When a layer slides out, the family fails.\\n\\n'", "CAPTION TAB1.png": "'Table 1\\n\\nDefinition of each term and some examples.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'"} \ No newline at end of file diff --git a/extracted_captions/orlikowskiUsingTechnologyConstituting2000.json b/extracted_captions/orlikowskiUsingTechnologyConstituting2000.json new file mode 100644 index 0000000000000000000000000000000000000000..166473281220d1c8f7576d01d96316b0ea6e95cb --- /dev/null +++ b/extracted_captions/orlikowskiUsingTechnologyConstituting2000.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1**: **Ententrant of Structures in Practice**'", "CAPTION FIG2.png": "'Figure 2: Enactment of Technologies-in-Practical\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3** **Collaboration Technology-in-Practice Enacted by Developers in Iris**'", "CAPTION FIG4.png": "'Figure 4: Collective Problem-Solving Technology-In-Practice-Enacted by Technologies in Alpha\\n\\n'", "CAPTION FIG5.png": "'Figure 8 Example of Skepticism Towards Technology\\n\\nFigure 8 Example of Skepticism Towards Technology\\n\\n'", "CAPTION FIG6.png": "'\\n\\n**Figure 6** **Limited-Use Technology-in-Practice Enacted by Consultants in Alpha**'", "CAPTION FIG7.png": "'\\n\\n**Figure 7**: **Individual Productivity Technology-in-Practice Enacted by Consultants in Alpha**'", "CAPTION FIG8.png": "'\\n\\n**Figure 8** **Process-Support Technology-In-Practice Enacted by Sunoort Specialists in Zeta**'", "CAPTION FIG9.png": "'\\n\\n**Figure 9** **Improvision Technology-in-Practice Enacted by Support Specialists in Zeta**'", "CAPTION TAB1.png": "'\\n\\n**Table 1** Properties of the Notes Technological Artifacts'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 2**} & \\\\multicolumn{1}{c}{**Types of Enactment\u2014Conditions, Actions, and Consequences.**} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Types of Enactment\u2014Conditions, Actions, and Consequences.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/palmerInformationWorkInterdisciplinary2002.json b/extracted_captions/palmerInformationWorkInterdisciplinary2002.json new file mode 100644 index 0000000000000000000000000000000000000000..58a553fcc5c4440d3bb73868588377803fa238fc --- /dev/null +++ b/extracted_captions/palmerInformationWorkInterdisciplinary2002.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1.--General subject area documented for one scholar**'", "CAPTION FIG2.png": "'Figure 2: Processes and activities associated with exploration and translation work.\\n\\n'", "CAPTION TAB1.png": "'TABLE I\\n\\nStates or Data Collection and Analysis'", "CAPTION TAB2.png": "'TABLE 3\\n\\nSources of Information Documenting for One Scholar'", "CAPTION TAB3.png": "'TABLE 3\\n\\nElements of Exploration and Translaton Work'"} \ No newline at end of file diff --git a/extracted_captions/pirolliInformationForaging1999.json b/extracted_captions/pirolliInformationForaging1999.json new file mode 100644 index 0000000000000000000000000000000000000000..8a922d925ee8d4db708af7254838fdcf21c0241a --- /dev/null +++ b/extracted_captions/pirolliInformationForaging1999.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Condensed information flow for business intelligence newsletter example. Width (except for library time) indicates time investment in activities, height indicates total documents, dark fill indicates relevant documents, and white fill indicates irrelevant documents.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: Production rules used in the adaptive control of thought the information foraging model of the ScatteredGather protocols obtained by Pirolli, Schank, Hearst, and Diehl (1996), \\\\(SG=\\\\) ScatteredGather; \\\\(SG=\\\\) ScatteredGather.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: The underlying, distal distribution of relevant documents, \\\\(d_{\\\\Omega}\\\\), characterizing the clustering algorithm, and the proximal distribution, \\\\(d_{P}\\\\), characterizing information scent from the Scatter/Gather screen. The distributions characterize the proportion of all the relevant documents (in an average system state) that fall in each cluster. Clusters are ranked in decreasing order of proportion of relevant documents.\\n\\n'", "CAPTION FIG12.png": "'Figure 12: Expected proportion of relevant documents in a new state (\\\\(s+1\\\\)) versus the expected proportion of relevant documents in the gathered clusters from previous state \\\\(s\\\\). The expected values are computed by information scent assessments by model traces of adaptive control of thought in information foraging. Logarithmic transformations have been performed on both dimensions.\\n\\n'", "CAPTION FIG13.png": "'Figure 13: Proportion of all documents that are relevant on Scaters? Gather windows, plotted as a function of time invested in scattering and, gathering, assuming hill-climbing heuristics.\\n\\n'", "CAPTION FIG14.png": "'Figure 14: The average rate of gain yielded by different investments in time spent scattering and gathering clusters, assuming hill-climbing heuristics. \\\\(R_{D}^{\\\\bullet}=\\\\) the maximum rate of gain that could be activated by foraging; \\\\(R_{3\\\\sigma}^{\\\\bullet}=\\\\) the maximum rate of gain that could be achieved by one more round of scattering and gathering clusters.\\n\\n'", "CAPTION FIG15.png": "'Figure 15: Analysis of the optimal information diet. The profitability (\\\\(\\\\pi\\\\)) of clusters is ranked and added to the die in order of decreasing profitability so long as the profitability of the item is greater than the rate of gain \\\\(\\\\langle\\\\mathcal{R}\\\\rangle\\\\).\\n\\n'", "CAPTION FIG16.png": "'Figure 16: Observed ratings of the percentage of documents in each cluster that are relevant and the ratings predicted by activation-based assessment of information sent.\\n\\n'", "CAPTION FIG17.png": "'Figure 17: Frequency that adaptive control of thought in information foraging (ACT-IF) productions match observed cluster-selection actions. The ACT-IF production rankings are computed at each simulation cycle over all productions that match.\\n\\n'", "CAPTION FIG18.png": "'Figure 1_: The probability density distributions for selecting clusters, _select(x)_, and not selecting clusters, _unselect(x)_, as a function of the difference between cluster profitability and current estimate of rate of gain: \\\\(\\\\Delta x=\\\\pi(c,\\\\,x)\\\\rightarrow\\\\,R(k,\\\\,x,\\\\,l)\\\\).\\n\\n'", "CAPTION FIG19.png": "'Figure 19: The difference in density distributions from Figure 18, \\\\(select(x)-\\\\)select(x) as a function of the difference between cluster equilibrium and rate of pairs, \\\\(x=\\\\pi(c,\\\\,x)-R(k,\\\\,s,\\\\,t)\\\\).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Schematic layout of the business intelligence office.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Information from the strategic management analysis example. Width indicates time investment in activities, height indicates total documents, dark fill indicates relevant documents, and white fill indicates irrelevant documents. Times in parentheses are cumulative. DB = database.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: A linear, finite cumulative within-patch gain function (solid line). Dashed lines, \\\\(R_{\\\\omega}\\\\) have slopes equaling the average rates of gain produced by different within-patch time (\\\\(t_{\\\\rm w}\\\\)) allocation policies. \\\\(R=\\\\) rate of gain per unit cost.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Chamev\u2019s marginal value theorem states that (a) the rate-maximizing time to spend in patch \\\\(t^{*}\\\\) occurs when the slope of the within-patch gain function, \\\\(g\\\\), is equal to the average rate of gain, which is the slope of the tangent line \\\\(\\\\bar{R}_{t}\\\\) (b) the average rate of gain increases with decreases in between-patches time costs; and (c) improvements in the gain function also increase the average rate of gain. \\\\(t_{\\\\rm w}\\\\) = average time to process patches; \\\\(t_{\\\\rm g}\\\\) = average time between processing patches.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: A hypothetical example of the relationship between profitability (\\\\(\\\\pi\\\\)) and rate of gain (\\\\(\\\\mathcal{R}\\\\)) for diets including items 1, 2, ... &.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: A conceptual overview of the Scatter/Gather interaction process.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: The Scatter-Gather document browsing interface.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Task structure for processing cluster windows (see Figure 8) as implemented by the adaptive control of thought in information foraging production system model. \\\\(N=no\\\\); \\\\(Y=yes\\\\).\\n\\n'", "CAPTION TAB A1.png": "'\\n\\n## Table A1\\n\\n**Neutralion Used in the Faregling Models**'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Optimal Information Date-Analysis-fac Scatter/Gather_} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: _Optimal Information Date-Analysis-fac Scatter/Gather_'"} \ No newline at end of file diff --git a/extracted_captions/reese2016single.json b/extracted_captions/reese2016single.json new file mode 100644 index 0000000000000000000000000000000000000000..2a73912a61f797bf85b217e5646e1dae64e24351 --- /dev/null +++ b/extracted_captions/reese2016single.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Detection of postsynaptic Ca2+ transients with GCaMP4f-PSD95. (**A**) Example images and ROI selection. Each image is a maximum intensity projection \u2013 average intensity projection for an 8 min recording both with (right) and without (left) the ROI selections. (**B**) Example images showing a section of dendrite (gray outline) during baseline fluorescence, peak fluorescence of a spontaneous event, and peak fluorescence of stimulus evoked events. _Arrows_ indicate postsynaptic signals. (**C**) Example traces from a GCampdf-PSD95 puncta before and after AP5 treatment. (**D**) Quantification showing Ca2+ transients detected in TTX before and after the addition of AP5. N = 400 ROIs from six experiments, two cultures, p<0.001 via students paired T-test.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Spontaneous response frequency and evoked response probability show little correlation within the synapse. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 3: The following figure supplement 1. NBXO and muscimol control network activity while allowing field stimulation; \\\\(\\\\mathbf{C}_{s}\\\\) analysis evidence that each ROI isolates a single synapse.\\n\\nFigure 4: Spontaneous response frequency and evoked response probability show little correlation within the synapse. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 5: The relationship between responses of different stimulus responses to different stimulus responses. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 6: The relationship between responses of different stimulus responses to different stimulus responses. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 7: The relationship between responses of different stimulus responses to different stimulus responses. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 8: The relationship between responses of different stimulus responses to different stimulus responses. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 9: The relationship between responses of different stimulus responses to different stimulus responses. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 10: The relationship between responses of different stimulus responses to different stimulus responses. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Example results from an experiment where each mark is a single response from one of the 50 ROI\u2019s. Marks are scattered randomly in the \\\\(y\\\\) axis to facilitate visualization of responses that overlap in time. \\\\(\\\\langle\\\\mathbf{B}\\\\rangle\\\\) Histogram showing spontaneous event rates per minute for 400 ROIs from 8 cells and four cultures. \\\\(\\\\langle\\\\mathbf{C}\\\\rangle\\\\) Scatter plot of evoked release probability \\\\(\\\\langle\\\\mathbf{R}_{\\\\text{F}}\\\\rangle\\\\) vs spontaneous response rate for the same 400 ROIs. \\\\(\\\\langle\\\\mathbf{D}\\\\rangle\\\\) Histogram showing the distribution of evoked response probabilities. \\\\(\\\\langle\\\\mathbf{A}\\\\rangle\\\\) Distribution of ROIs by events per 8 min plotted with Poisson theoretical best fit. \\\\(\\\\chi^{2}\\\\) test indicates these are not equivalent distributions.\\n\\nFigure 11: The relationship between\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Use-dependent NMDAR blocker MK-801 can selectively silence spontaneous signals without effecting evoked responses within the same synapse. (**A**) Example traces outlining the experiment with (top) and without (bottom) MK-801 treatment. 10 action potentials were evoked, 10 s apart (blue tick marks). \\\\(A\\\\) min later, perfusion was changed to Tyrode\u2019s solution containing 10 \u03bcM MK-801 or vehicle. After 10 min in this solution, 10 additional action potentials are evoked, 10 s. Please note that sample traces were selected among examples that are on the larger the side of the average values (especially those after MK-801 application) for better visualization of the impact of MK-801 block on single events (**B**) Plot shows normalized spontaneous event frequency over the stimulation-free 10 min treatment with MK-801 or control solution. For MK-801 N = 350 ROIs from 7 cells and three cultures. For the control condition, N = 300 ROIs from 6 cells and five cultures. (**C**) Bars show the percentage of ROIs that respond one or more times to either the first round of 10 stimulations, the second round of 10 stimulations, or spontaneously respond during the intervening time. Control cells (vehicle treatment) and MK-801 treated cells are compared and show no significant differences. (**D**) Average amplitudes as \\\\(\\\\Delta\\\\)F/F\\\\({}_{\\\\circ}\\\\) for each \\\\(n^{\\\\text{th}}\\\\) response within an ROI (per response not per stimulation, failures to respond are skipped so \u2019zeros\u2019 are not averaged). For both groups, fit line generated via linear least squares method. (**E**) Histogram showing distribution of evoked response probabilities in cells before and after MK-801 treatment.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Reduction in postsynaptic responses by MK-B01 correlates with evoked response probability but not spontaneous response rate. (**A**) Scatter plot of change in \\\\(R_{\\\\mathrm{p}}\\\\) (before - after MK-B01) vs spontaneous response rate before MK-B01 was added. Shading indicates overlapping data points. For both plots N = 350 ROIs from 7 cells and five cultures. (**B**) Scatter plot of the change in \\\\(R_{\\\\mathrm{p}}\\\\) after MK-B01 treatment vs initial evoked response rate. Linear fit lines generated via least squares method. (**D**) 10 7554/ml 12 21102007\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/reynolds2022bending.json b/extracted_captions/reynolds2022bending.json new file mode 100644 index 0000000000000000000000000000000000000000..501c8ab405f6f6d1d0fc225eee5c9bc8d78007de --- /dev/null +++ b/extracted_captions/reynolds2022bending.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'for ADP(green), PO4-2 (yellow), Mg2- (light green) and water molecules (violet). In ADP/PO4-1, nitrogen atoms are blue, oxygen atoms are red and phosphorous atoms are yellow. Bottom, the backbone and side chain residues involved in putative hydrogen-bonding networks (dashed lines) are displayed and coloured by heteroatom. **c**, Superposition of individual ADP-F-actin (blue) and ADP-P-F-actin (orange) protomers, displayed in **Ca** representation.\\n\\nFig. 1: **Nucleotide cleft water networks are remodelled upon phosphate release by F-actin.****a**, Cryo-EMmaps of ADP\u2013F-actin (left, shades of blue) and ADP\u2013P-F-actin (right, shades of orange). ADP (green) and water (magenta) densities are shown. BE, barbed end; PE, pointed end. **b**, Atomic models of the ADP\u2013F-actin (blue) and ADP\u2013P-F-actin nucleotide (nuc.) clefts (orange) are shown in **Ca** representation. Top, carved transparent grey density is displayed for ADP(green), PO4-2 (yellow), Mg2- (light green) and water molecules (violet). In ADP/PO4-1, nitrogen atoms are blue, oxygen atoms are red and phosphorous atoms are yellow. Bottom, the backbone and side chain residues involved in putative hydrogen-bonding networks (dashed lines) are displayed and coloured by heteroatom. **c**, Superposition of individual ADP\u2013F-actin (blue) and ADP\u2013P-F-actin (orange) protomers, displayed in **Ca** representation.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2**: **Water molecules mediate key longitudinal and lateral contacts in F-actin.****a**, Water molecules (violet) contained within the filament core of ADP\u2013F-actin. Actin subunits are shown in transparent grey main-chain representation. **b**, Solvent-mediated contacts at lateral (top) and longitudinal (bottom) interfaces in ADP\u2013F-actin (shades of blue) and ADP\u2013F-F-actin (shades of orange). Keys side chains and backbone atoms are displayed and coloured by heteroatom. Water molecules are shown in violet, and putative hydrogen bonds are indicated by dashed lines. Protomer indices are indicated.\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: **Cryo-EMreconstructions of mechanically deformed F-actin reveal bend\u2013twist coupling.****a** Representative cryo-EM micrograph of ADP\u2013F-actin filaments featuring high-curvature regions, low-pass filtered to 30 A. Picked segments are coloured by estimated curvature as indicated. Scale bar, 100 mm. Normalized curvature histograms of ADP\u2013F-actin (blue, \\\\(n\\\\) = 374,942) and ADP\u2013F-F-actin (orange, \\\\(n\\\\) = 470,625) filament segments, compared using a two-tailed Mann\u2013Whitney \\\\(U\\\\)-test. The dashed lines indicate curvature thresholds for straight (\\\\(\\\\leq\\\\)2.0 \u03bcm\u22121) and bent (\\\\(\\\\leq\\\\)2.5 \u03bcm\u22125) segments. The colour bars correspond to the curvature key in **a**. **c**, Helically symmetric ADP\u2013F-actin (left map) and cryoDRGNreconstructions sampling continuous bending of ADP\u2013F-actin (right three maps), low-pass filtered to 8 \u00c5. Strands are coloured in 3hades of blue. Scale bar, 100 mm. **d**, Stitched volumes of straight and bent maps from, aligned to the bottom **le** protomers. Scale bar, 100 mm. **e**, Schematic of twist and rise measurements along the bent filament axis. Protomer numbering is indicated. **f**. Twist and rise measurements of cryoDRGNreconstructions sampled along the major variability component. The solid and dashed curves correspond to measurements from even-to-odd and odd-to-even protomer indices, respectively.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4 | Actin nucleotide state modulates subunit shearing during filament bending.****a**, Cryo-EM maps of bent ADP-F-actin (blue) and ADP-P1-F-actin (orange), coloured by strand. Protomer numbering is indicated. The dashed and solid lines represent the convex and concave sides of the bent filament, respectively. **b**, Ribbon representation of an individual actin protomer coloured by subdomain (left). The spheres connected by bars indicate subdomain centroids. Right, protomer subdomain centroid diagrams with vectors (scaled 100x), indicating subdomain-averaged displacements from the corresponding helically symmetric model. **c**, Plots of subdomain shear indices (representing coordinated rearrangements) of subdomains 1 and 4 (top) and subdomains 2 and 3 (bottom). Blue lines, ADP; orange lines, ADP-P1-The solid and dashed lines represent even (concave side) and odd (convex side)'", "CAPTION FIGED1-1.png": "'\\n\\n**Extended Datafile.** **11Schenepage for caption**'", "CAPTION FIGED1-2.png": "'\\n\\n**Extended Data Fig. 1.1 Validation of ADP-P\\\\({}_{c}\\\\)F-actin preparationand helically symmetric reconstructions.****a**, Representative TIRF-microscopy video frames from cofilm inserting assays. Cofilm-free controls are shown in the top row of each condition, indicated by a darker border. Scale bar, 40 \\\\(\\\\mu\\\\)m. **b**, Quantification of TIRF videos showing the average normalized actin channel intensity. Error margin in graph indicates +/-95% CI. Half-lives represent exponential decay at time 0.5, with 95% CI, and n values represent independent experiments: ADP-F-actin-cofilin (778 +- 24 s, n = 3); ADP-P-F-actin-cofilin (454 +- 14 s, n = 3); ADP-sulfate-F-actin-cofilin (348 +- 16 s, n = 3); ADP-F-actin+cofilin (50.4 +- 21.5, n = 4); ADP-P-F-actin+cofilin (127.5 +- 10.3 s, n = 3); ADP-sulfate-F-actin+cofilin (86.8 +- 2.5, n = 3); Half-map (left) and map-to-model (right) Fourier Shell Correlation (FSC) curves for helically symmetric reconstructions of ADP- and ADP-P\\\\({}_{c}\\\\)F-actin. **d**, Local resolution assessment of helically symmetric ADP-F-actin and ADP-P\\\\({}_{c}\\\\)F-actin. **f**, Pe potentiated end; BE-barbed end. **e**, Potentially divergent bone bending networks adjacent to the nucleosidyl region of ADP. Key side chains and back bone atoms participating in hydrogen-bonding networks are displayed and coloured by heteroatom.\\n\\n'", "CAPTION FIGED1.png": "'\\n\\n**ExtendedDataFile.** 11-scan-page for caption. **ExtendedDataFile.** 11-scan-page for caption.\\n\\n'", "CAPTION FIGED10.png": "'\\n\\n**Extended Data Fig. 10 --F-actin bending remodels inter-subunit interfaces engaged by ABPs.****a**, **C**, representations of inner strand promoter 6-protomer 8 (concave, cornflower blue) and outer strand promoter 7-protomer 9 (convex, light blue) longitudinal interfaces extracted from the most curved ADP-F-actin cryoDRGN16-protomer atomic model, superimposed on the barbed end subunit of 7 monomers extracted from the ADP-F-actin helical model (_every_ Box encloses a region featuring major contacts by ABPs.****BE: barbed end;****PE: pointed end.****Actin subdomains are indicated.****b**, binding interfaces of indicated ABPs (pink space filling model) are displayed superimposed on the bent interfaces from **a**.****Cofilm, PDB SYUB; coronin, EMDB 6100/PDB 2AQS; ARP2/3, PDB 7TPT; a-catenin, PDB 6UPV; myosin-5, PDB 7PLT.\\n\\n'", "CAPTION FIGED2.png": "\"\\n\\n**Extended Data Fig. 2**: **Additional analysis of helically symmetric ADP-F-actin and ADP-P-F-actin models.** **a**, Example cryo-EM map density superimposed with atomic model residues A131-A135 from ADP-F-actin (top) and ADP-P-F-actin (bottom). **b**, Individual ADP-F-actin protomer shown in C**, representation, coloured by presilable RMSD between ADP-F-actin and ADP-F-F-actin. **c**, Same as **b**, but coloured by per-residue strain pseudo-energy. **d**, Superposition of extended 3I-protomer ADP-(blue) and ADP-P-F-actin (orange) models, aligned at the terminal barbed end protomer. **e**, Water molecules (violet) contained within the ADP-P-F-actin filament's core. Actin subunits are shown in transparent grey backbone representation. PE: pointed end; BE: barbed end.\\n\\n\"", "CAPTION FIGED3.png": "'\\n\\n**Extended Data Fig. 3.1 Neural network architecture and example performance.****a**, Neural network architecture diagram for denoising auto-encoder. Example network input and output is displayed for a representative extracted segment. **b**, Network architecture diagram for semantic segmentation fully convolutional network. Example input and output for a representative extracted segment is shown. **c**, Representative network performance on filament segments from synthetic projections (top) and experimental cryo-EMmicrographs (bottom). Scale bars, 20 nm.\\n\\n'", "CAPTION FIGED4.png": "'\\n\\n**Extended Data Fig. 4**: **Boltzmann modelling of filament segment curvature distributions.a**, Curvature distributions (left column) of ADP-F-actin (top) and ADP-P,F-actin (bottom) with modelled thermal bending probability distributions. Residual plots (right column) between the measured and theoretical distributions. Grey curves correspond to adjusted bending models fit with a multiplicative parameter in the energy term.b, Theoretical probability distributions of SOD \\\\(A\\\\) elastic rods bending due to thermal fluctuations, modelled as Boltzmann distributions. Varying the persistence length between 5 mm and 15 mm demonstrates the effect of filament bending stiffness.\\n\\n'", "CAPTION FIGED5.png": "'\\n\\n**Extended Data Fig. 5 [Additional analysis of filament bending deformations.** **a**, Heltically symmetric ADP-P\\\\({}_{r}\\\\)-F-actin (left map) and cryoDRGN reconstructions sampling ADP-P\\\\({}_{r}\\\\)-F-actin bending (right three maps). Maps are lowpass filtered to 8 \u00c5, and strands are acquired in shades of orange. PE-pointed end; BE: barhed end. Scale bar, 10 nm. **b**, Stitched volumes of straight and hept maps from a, aligned on the bottom 16 protomers. Scale bar, 100 nm. **c**, Projections of zeroth (cyan) and ninth (magenta) cryoDRGNreconstructions from ADP-F-actin (left) and ADP-F\\\\({}_{r}\\\\)-F-actin (right) aligned on the bottom protomer and oriented to display maximum displacement. **d**, Asymmetric reconstructions of ADP-F-actin from indicated curvature bins. Scalebar, 10 nm. **e**, Plots of central axis deviations from straight lines in ADP-F-actin and ADP-P\\\\({}_{r}\\\\)-F-actin cryoDRGN reconstructions. First and second columns show principal component analysis of the cryoDRGN reconstructions central axes. Third column shows displacement of the cryoDRGN reconstructions central axes from straight lines which were aligned to the barbed-end-thermal 56 \u00c5 of the central axes. **f**, Half-map Fourier Shell Correlation (FSC) curves for control asymmetric 16-protomer reconstructions. **g**, Twist and rise measurements of control asymmetric 16-protomer reconstructions.\\n\\n'", "CAPTION FIGED6.png": "'\\n\\n**Extended Data Fig. 6**: **Quantization of lattice architectural remodelling during filament bending.**a.** Plots of travelling wave equation fits (black lines) with measured twist values (coloured points) from cryoDBCN reconstructions of different curvatures. **b.** Plots of twist travelling wave function at various curvatures, separated by strand. **c.** Plots of intra-strand, inter-strand, and intra-protomer subdomain distances and angles from ISOLDE models of the most curved cryoDBCN reconstructions of ADP-F-actin (blue) and ADP-F-F-actin (orange). Solid and dashed lines represent even (concave side) and odd (convex side) protomers, respectively.\\n\\n'", "CAPTION FIGED7.png": "'\\n\\n**Extended Data Fig. 7 Resolution assessment and validation of high-resolution bent F-actin asymmetric reconstructions, a.** Local resolution assessment of bent ADF-actin and ADP-P,-F-actin reconstructions, as well as two independent straight ADF-actin controls, PE-pointed end; BE-barbed end, **b**, Half-map (left) and map-to-model (right) Fourier Shell Correlation (FSC) curves for asymmetric bent ADF-F-actin, ADP-P,-F-actin, and straight ADP-F-actin controls, **c**, 3D-FSC curves for asymmetric reconstructions, which indicate equivalently isotropic resolution between bent and straight reconstructions. Dotted green lines indicate +/-1s.d. from average FSC. **d**, Vector plots (coloured by direction and scaled 6K) representing C8 displacements between helically symmetric models and those built into indicated asymmetric reconstructions, aligned on the central protomer.\\n\\n'", "CAPTION FIGED8.png": "'\\n\\n**Extended Data Fig. 8**: **Strain and flexibility of inter-protomer contacts sites in bent F-actin.****a**, Grey C, representations of indicated protomers from bent F-actin, locally coloured by computed per-residue strain pseudo-energy relative to helically-symmetric models on their H-plugs (left) and D-loops (right). **b**, D-loop heterogeneity in asymmetric F-actin maps. Local density around each of the unique D-loops from the asymmetric reconstructions are shown with a docked PDB model of ADP-F-actin (7RSV) featuring both \"in\" and \"out\"D-loop conformations.\\n\\n'", "CAPTION FIGED9.png": "'\\n\\n**Extended DataFig. 9 [**Steric encounters at inter-subunit interfaces transduce filament bending strain to protomers.****a**, Longitudinal interfaces and **b**, lateral interfaces of bent ADP-F-actin (left) and ADP-F-F-actin (right). Solid and dashed borders represent inside (concave) and outside (convex) of curve, respectively. Transparent arrows represent individual Ca displacements from helically symmetric models scaled 6X, and solid arrows show averaged displacements of indicated regions scaled 20X. PE: pointed end; BE: barbed end.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/rhyscoxDirectedDiversityLeveraging2021a.json b/extracted_captions/rhyscoxDirectedDiversityLeveraging2021a.json new file mode 100644 index 0000000000000000000000000000000000000000..ab51a67e698631b88c4fc03661b4a87fdd9ef96c --- /dev/null +++ b/extracted_captions/rhyscoxDirectedDiversityLeveraging2021a.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Pipeline of the overall technical approach to extract, embed, and select phrases to generate diverse prompts. a) Phrase extraction by collecting phrases from online articles and discussion forums (shown as pages), filtering phrases to select a clean subset (shown as the black dash for each phrase); b) Phrase embedding using the Universal Sentence Encoder [14] to compute the embedding vector of each phrase (shown as scatter plot); c) Phrase Selection by constructing the minimal spanning tree to select optimally spaced phrases (see Figure 2 for more details).\\n\\n'", "CAPTION FIG10.png": "'Figure 10: Distribution of pairwise distances between the extracted phrases (N=3,666). The pairwise distances ranged from Min=0.057 to Max=0.586, Median=0.430, inter-quartile range 0.394 to 0.460, SD= 0.047.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: Distribution of pairwise distances between the messages (N\\\\(=\\\\)250) idented in the pilot study with no prompting (None). The pairwise distances ranged from Min\\\\(=\\\\)0.169 to Max=0.549, Median\\\\(=\\\\)0.405, inter-quartile range 0.376 to 0.432, SD\\\\(=\\\\)0.043.\\n\\n'", "CAPTION FIG12.png": "'Figure 12: Influence of prompt selection technique, prompt size, and prompt count on various distance and diversity metrics. Higher values for all metrics indicate higher diversity. Span and Sparseness results are not shown, but are similar to Mean Distance. Note that we computed the mean of MST edge distances instead of sum, which is independent of number of prompts. Error bars are extremely small, and not shown for simplicity.\\n\\n'", "CAPTION FIG13.png": "'Figure 13: The instructions in the Ideation User Study for the None condition.\\n\\n'", "CAPTION FIG14.png": "'Figure 14: For the None, users are asked to write a message that is at least one to three sentences long.\\n\\n'", "CAPTION FIG15.png": "'Figure 15: The instructions of Ideation User Study for the Random(1) and Directed(1) conditions.\\n\\n'", "CAPTION FIG16.png": "'Figure 16: Random(1) and Directed(1) prompts consisted of one phrase per prompt. Note that selected phrase for each trial will be different.\\n\\n'", "CAPTION FIG17.png": "'Figure 17: The instructions of Ideation User Study for the Random(3) and Directed(3) conditions.\\n\\n'", "CAPTION FIG18.png": "'Figure 18: Random(3) and Directed(3) prompts consist of three phrases per prompt. Note that selected phrases for each trial will be different.\\n\\n'", "CAPTION FIG19.png": "'Figure 19: **Ideators are asked to evaluate the message they wrote by providing Likert scale ratings for many different factors along with a short reflection about the message writing process. The screenshot above shows the evaluation screen for Directed(3).**\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Procedure to direct ideation towards diverse phrases (top) and away from prior or redundant ideas (bottom). To attract ideation with diverse prompts: a) start with embeddings of corpus-extracted phrases; b) construct minimum spanning tree (MST); c) traverse tree to select distant prompts from clusters (most distant points as green dots, in clustered phrases as green ellipses); d) selected prompts are the most diverse. To repel ideation from prior ideas, e) compute embeddings of prior ideas (red hollow dots); f) compute prompt-ideation pairwise distances of all prompts from each prior ideation, exclude phrases (dotted black circles) with pairwise distance less than a user-defined threshold (red bubble), and construct the MST with remaining phrases; g) traverse MST to select a user-defined number of prompts; h) selected prompts are diverse, yet avoids prior ideas.\\n\\n'", "CAPTION FIG20.png": "'\\n\\n**Figure 20: The instruction for individual message rating tasks.**'", "CAPTION FIG21.png": "'Figure 21: **Validators rated a randomly selected message on a Likert scale and gave a justification.**\\n\\n'", "CAPTION FIG22.png": "'\\n\\n**Figure 22: The instruction for group message ranking tasks.**'", "CAPTION FIG23.png": "'Figure 23: **Validators were asked to rank groups of messages for motivation, informativeness and repetitiveness. Note that while we used the word \u201crepetitive\u201d for usability in the survey, we analyzed this dependent variable as \u201cunrepetitive\u201d to be consistent with other diversity metrics.**\\n\\n'", "CAPTION FIG24.png": "'Figure 24: Validators were asked to rate the difference of two messages in a message-pair.\\n\\n'", "CAPTION FIG25.png": "'Figure 25: **Results of computed individual diversity from ideations for different prompt configurations for (left) prompts that users understood (-0) or did not and (right) ideations that were fast or slow.**\\n\\n'", "CAPTION FIG26.png": "'Figure 26: Results of prompt-message distance (how dissimilar a prompt is from a message) comparing different messages with respect to Directed(3) prompts.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Diversity prompting evaluation framework to evaluate prompting to support diverse ideation along the ideation chain. We pose research questions (RQ1-3) between each step to validate the ideation diversification process. For each step, we manipulate or measure various experiment constructs to track how well ideators are prompted to generate creative ideas. Except for prompt selection, each construct refers to a statistical factor determined from factor analyses of multiple dependent variables. Constructs are grouped in colored blocks indicating different data collection method (\\\\(\\\\square\\\\) Computed embedding-based metric, \\\\(\\\\square\\\\) ratings from ideators, \\\\(\\\\square\\\\) ratings from validators, \\\\(\\\\square\\\\) thematic coding of ideations).\\n\\n'", "CAPTION FIG4.png": "'Figure 4: 2D UMAP projection showing how diversely selected prompts and resulting ideation messages are distributed based on Directed or Random prompt selection technique and prompt size (number in brackets). Each point represents the embedding of a text item. Each query points represent all phrases in the extracted corpus, dark grey points represent selected phrases from the simulation study (Section 5.1) and blue dots represent the ideated messages written by crowdworkers in the ideator user study (Section 5.2). Gradient lines connect ideation messages to their stimulus prompts.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Example prompts shown to participants in different conditions: None (left, \\\\(g=0\\\\)), Directed(1) (center, \\\\(g=1\\\\)), and Directed(3) (right, \\\\(g=3\\\\)). Phrase texts would be different for Random(1) and Random(3) selection techniques.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Results of ideators\u2019 perceived prompt creativity (Top) and ideation effort (Bottom) for different Prompt Selection technique and Prompt Size. All factors values on a 5-point Likert scale (-2=\u201cStrongly Disagree\u201d to 2=\u201cStrongly Agree\u201d). Dotted lines indicate extremely significant p<.0001 comparisons, otherwise very significant with p-value stated; solid lines indicate no significance at p>.01. Error bars indicate 90% confidence interval.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Results of computed individual and collective diversity from ideations for different prompt configurations. See Figure 6 caption for how to interpret charts.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: **Results of diversity in categories and themes derived from thematic analysis of ideations.**\\n\\n'", "CAPTION FIG9.png": "'Figure 9: **Results of perceived individual and collective creativity from the three validation user studies.**\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: **Demonstration of pairwise embedding angular distances between an example text items (first data row) and neighboring text items. Text items with semantically similar words have smaller distances. For interpretability, we highlighted words to indicate darker color with higher cosine similarity to the first phrase.**'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 10: The rotated factor loading of factor analysis on metrics of prompt distance and consistency. Factors explained 73.6\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 5810, p\\\\textless{}0.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 10: The rotated factor loading of factor analysis on metrics of prompt distance and consistency. Factors explained 73.6\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 5810, p\\\\textless{}0.0001).\\n\\n'", "CAPTION TAB11.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 11: The rotated factor loading of factor analysis on metrics of perceived helpfulness of prompts. Factors explained 68.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 2575, p\\\\textless{}.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 11: The rotated factor loading of factor analysis on metrics of perceived helpfulness of prompts. Factors explained 68.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 2575, p\\\\textless{}.0001).\\n\\n'", "CAPTION TAB12.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 12: The rotated factor loading of factor analysis on metrics of prompt adoption. Factors explained 65.1\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1315, p-0.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 12: The rotated factor loading of factor analysis on metrics of prompt adoption. Factors explained 65.1\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1315, p-0.0001).\\n\\n'", "CAPTION TAB13.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 13: The rotated factor loading of factor analysis on diversity metrics of generated messages. Factors explained 75.2% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 2676, p-.0001).\\n\\n'", "CAPTION TAB14.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 14: The rotated factor loading of factor analysis on metrics of perceived quality of the generated messages. Factors explained 80.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 5810, p-.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 14: The rotated factor loading of factor analysis on metrics of perceived quality of the generated messages. Factors explained 80.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 5810, p-.0001).\\n\\n'", "CAPTION TAB15.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB16.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 16: The rotated factor loading of factor analysis on metrics of message distinctness. Factors explained 74.8% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1022, p-.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 16: The rotated factor loading of factor analysis on metrics of message distinctness. Factors explained 74.8% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1022, p-.0001).\\n\\n'", "CAPTION TAB17.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 17: The rotated factor loading of factor analysis on metrics of ideation effort. Factors explained 59.0\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1008, p\\\\(\\\\sim\\\\)0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 17: The rotated factor loading of factor analysis on metrics of ideation effort. Factors explained 59.0\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1008, p\\\\(\\\\sim\\\\)0001).\\n\\n'", "CAPTION TAB18.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 18**: Messages generated in our study and the phrase prompt(s) that were shown to ideators.} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 18: Messages generated in our study and the phrase prompt(s) that were shown to ideators.\\n\\n'", "CAPTION TAB19.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 19: **Themes and categories identified with the qualitative coding of ideated messages.**'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 2: **Metrics of distances between two points in a multi-dimensional vector space. Each metric can be calculated for an individual text item. These metrics can apply to the embedding of phrases or ideations.**'", "CAPTION TAB20.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB21.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 21: Statistical analysis and results of mediation effects (RQ2.3) of how prompt configurations (a) and perceived prompt creativity (b) affect ideation diversity. See Table 20 caption to interpret tables. Positive and negative numbers in second column represent estimated model coefficients indicating how much each fixed effect influences the response.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 21: Statistical analysis and results of mediation effects (RQ2.3) of how prompt configurations (a) and perceived prompt creativity (b) affect ideation diversity. See Table 20 caption to interpret tables. Positive and negative numbers in second column represent estimated model coefficients indicating how much each fixed effect influences the response.\\n\\n'", "CAPTION TAB22.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 22: Statistical analysis of how prompt selection influences ideation diversity defined by different metrics (RQ3): a) individual diversity, b) collective diversity, and c) thematic diversity. See Table 20 caption for how to interpret tables.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 22: Statistical analysis of how prompt selection influences ideation diversity defined by different metrics (RQ3): a) individual diversity, b) collective diversity, and c) thematic diversity. See Table 20 caption for how to interpret tables.\\n\\n'", "CAPTION TAB23.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & **Table 23**: **Statistical analysis of how prompt selection influences ideation creativity as validated by different methods (RQ3.1): a) individual rating, b) collective ranking, and c) collective pairwise rating. See Table 20 caption for how to interpret tables.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 23: **Statistical analysis of how prompt selection influences ideation creativity as validated by different methods (RQ3.1): a) individual rating, b) collective ranking, and c) collective pairwise rating. See Table 20 caption for how to interpret tables.**'", "CAPTION TAB24.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 24: Statistical analysis of a) how ideators\u2019 understanding of phrases influences ideation diversity and b) how similar. Directed ideations are to their prompts compared to other None and Random Messages.\\n\\n'", "CAPTION TAB25.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 3: **Metrics of diversity of phrases or ideation embeddings in a vector space. These capture more characteristics of diversity than average distances in Table 2. Each metric can only be calculated collectively for multiple items.**'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Comparison of diversity metrics for canonical examples of distributed points in a 2D space. Points farther apart mean higher diversity. Here, we calculate Euclidean instead of angular distance, but intuitions are similar.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 6: **Independent variables used in the simulation and user studies to manipulate how prompts are shown to ideators.**'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 7: **Metrics of creativity of ideation based on categories and themes derived from a thematic analysis of generated ideas. Metrics are shown for categories, but are the same for themes.**'", "CAPTION TAB8.png": "'\\n\\n**Table 8: Metrics of prompt diversity for all phrases in a single prompt.**'", "CAPTION TAB9.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 9: **Metrics indicating how much of prompt text and concepts are adopted into the idetions.**'"} \ No newline at end of file diff --git a/extracted_captions/richlandReducingCognitiveLoad2013.json b/extracted_captions/richlandReducingCognitiveLoad2013.json new file mode 100644 index 0000000000000000000000000000000000000000..802b742471e0c3192c82fa20b394e1f4573d33d0 --- /dev/null +++ b/extracted_captions/richlandReducingCognitiveLoad2013.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Screen picture of material in high cuing instructional video, comparing two representations of divisions.\\n\\n'", "CAPTION FIG2.png": "'Figure 2. Mean differences on posttest items by condition\\n\\n'", "CAPTION TAB1.png": "'Table 1. The distribution of analyzed problems that were administered on each test.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/extracted_captions/roffay2021passive.json b/extracted_captions/roffay2021passive.json new file mode 100644 index 0000000000000000000000000000000000000000..bc9fcd78a12a5a378c3611a16e2d34432575b0b1 --- /dev/null +++ b/extracted_captions/roffay2021passive.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Osmotic shocks affect cell volume and membrane tension. (A) Representative side view of cell with CaillMask staining and corresponding 3D reconstruction of cell volume using Limesse (_Left_, _hypermoritic_; _Right_, _hypermoritic_; _(_Right_, _hypermoritic_; _(_B_) Osmolaritis (_m_SmM) of cell media with time for the different stepswise osmotic conditions. Osmotic conditions correspond to 120 mSm in dark orange, 190 mSm in light orange, 285 mOsm in light orange, 315 mOsm in light black, 540 mOsm in light blue, 890 mOsm in medium blue, and 1,330 mOsm in dark blue. Color code is maintained. (C) Averaged cell volume dynamics under osmotic shock (gray, before shock; light green, short-term response; dark green, long-term response). Statistics are as follows: dark orange, \\\\(R>3\\\\), \\\\(n=28\\\\); light orange, \\\\(R>3\\\\), \\\\(n=26\\\\); yellow, \\\\(R>3\\\\), \\\\(n=40\\\\); black, \\\\(R>3\\\\), \\\\(n=62\\\\); light blue, \\\\(R>3\\\\), \\\\(n=62\\\\); medium blue, \\\\(R>3\\\\), \\\\(n=47\\\\); and dark blue, \\\\(R>3\\\\), \\\\(n=43\\\\). Insert shows cell volume distribution in isotonic medium before osmotic shocks (\\\\(R>50\\\\), \\\\(n=959\\\\)). (D) Relative change of tether force immediately after osmotic shocks (averaged over \\\\(\\\\%\\\\)) for different osmotic shocks. Insert shows tether force distribution in isotonic medium before osmotic shocks (\\\\(R=n=73\\\\)). (E) Fluorescence lifetime of the Flipper-TR probe reports membrane tension changes. Shown are FIM images of Flipper-TR lifetime values (_colon_cale) of cells subjected to osmotic shocks. (F) Dynamics of the change of tension as measured by Flipper-TR lifetime (gray, before shock; light green, short-term response); dark green, long-term response). Comotic conditions corresponds to 120 mOsm in dark orange (\\\\(R=7\\\\), \\\\(n>28\\\\)), 315 mOsm in black (\\\\(R=3\\\\), \\\\(n>12\\\\)), 600 mOsm in light blue (\\\\(R=3\\\\), \\\\(n>12\\\\)), and 1,330 mOsm in dark blue (\\\\(R=5\\\\), \\\\(n>20\\\\)). Insert shows distribution of the cell average Flipper-TR lifetimes in isotonic medium before osmotic shocks (\\\\(R>100\\\\), \\\\(n>400\\\\)).\\n\\n'", "CAPTION FIG2.png": "'\\nFig. 2: Quantitative coupling of call volume and call membrane tension to atomic shocks. (A) Normalized volume changes (\\\\(V/V_{\\\\rm d}\\\\)) as a function of osmotic pressure ratio (\\\\(P_{0}/P\\\\)) just after osmotic shocks (solid circle) and 8 min after the osmotic shock (open circle, recovery phase) compared to the prediction of Eq. 1 (red line). Color code is maintained from Fig. 1. Statistics are (\\\\(R>3\\\\), \\\\(n=28\\\\)) for dark orange, (\\\\(R>3\\\\), \\\\(n=20\\\\)) for light orange, (\\\\(R>3\\\\), \\\\(n=40\\\\)) for yellow, (\\\\(R>3\\\\), \\\\(n=65\\\\)) for black, (\\\\(R>3\\\\), \\\\(n=62\\\\)) for light blue, (\\\\(R>3\\\\), \\\\(n=47\\\\)) for medium blue, and (\\\\(R>3\\\\), \\\\(n=43\\\\)) for dark blue. (B) Normalized volume changes (\\\\(V/V_{\\\\rm d}\\\\)) as a function of osmotic pressure ratio (\\\\(P/P_{0}\\\\)) just after osmotic shocks compared to the prediction of Eq. 1 (red line). Statistics are the same as in \\\\(A\\\\). (C) Refractive index images of cells under osmotic shocks. (D) Protein concentration changes (\\\\(C/C_{0}\\\\)) according to pressure applied (\\\\(P/P_{0}\\\\)). Each point corresponds to one experiment containing at least two cells. Analysis is performed on individual cells. (E) Calculated dry mass of cells versus normalized pressure (\\\\(P/P_{0}\\\\)). Each point corresponds to one experiment containing at least two cells. Analysis is performed on individual cells. (F) Representative FIB-SEM images of the membrane tubulations in isotonic medium and hypertonic medium. (Scale bar: 2 \u03bcm) (6) Scheme describing the parameters of the theory. (H) Relative changes of membrane area (\\\\(\\\\Delta\\\\Delta/A_{0}\\\\)) versus relative changes of membrane tension (\\\\(\\\\Delta\\\\Delta/\\\\tau_{0}\\\\)) compared to the prediction of Eq. 3 (red line). Color code is maintained from Fig. 1. Statistics are (\\\\(R=n=51\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=28\\\\) in \\\\(y\\\\)) for dark orange, (\\\\(R=n=8\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=20\\\\) in \\\\(y\\\\)) for light orange, (\\\\(R=n=10\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=40\\\\) in \\\\(y\\\\)) for yellow, (\\\\(R=n=12\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=65\\\\) in \\\\(y\\\\)) for black, (\\\\(R=n=12\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=62\\\\) in \\\\(y\\\\)) for light blue, (\\\\(R=n=12\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=47\\\\) in \\\\(y\\\\)) for medium blue, and (\\\\(R=n=14\\\\) in \\\\(x\\\\); \\\\(R>3\\\\), \\\\(n=43\\\\) in \\\\(y\\\\)) for dark blue. (_f_) Normalized tension (\\\\(\\\\sigma/\\\\tau_{0}\\\\)) versus normalized pressure (\\\\(P/P_{0}\\\\)) and prediction obtained using Eq. 4. Statistics are the same as in \\\\(F\\\\). (f) Scheme describing the theory.\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: Cytokkeleton controls the long-term response of cells to osmotic shocks. (4) Illustrations of cytoskeletal drug effects. (B) Single-cell volume dynamics of cells treated with latruculin A, japakinolide, nocodazole, or taxol during hypotonic shocks (120 mOsm, circle), isotonic conditions (315 mOsm, square), and hypetarinch shocks (700 mOsm, pPO = 2, triangle). Statistics are \\\\(R>3\\\\) for every experiment. For latruculin A, \\\\(n=5\\\\) for control, \\\\(n=4\\\\) for hypo, \\\\(n=15\\\\) for hyper; for japakinolide, \\\\(n=6\\\\) for control, \\\\(n=8\\\\) for hypo, \\\\(n=10\\\\) for hyper; for nocodazole, \\\\(n=8\\\\) for control, \\\\(n=8\\\\) for hypo, \\\\(n=6\\\\) for hyper; for taxol, \\\\(n=10\\\\) for control, \\\\(n=19\\\\) for hypo, \\\\(n=12\\\\) for hyper. (C) Membrane tension dynamics of cells treated with latruculin A, jaspakinolide, nocodazole, or taxol during the same shocks as in \\\\(R\\\\). Statistics are \\\\(n>4\\\\) for every experiment. For latruculin A, \\\\(R=2\\\\) for control, \\\\(R=6\\\\) for hypo, \\\\(R=4\\\\) for hyper; for jaspakinolide, \\\\(R=4\\\\) for control, \\\\(R=10\\\\) for hypo, \\\\(R=3\\\\) for hyper; for nocodazole, \\\\(R=2\\\\) for control, \\\\(R=6\\\\) for hypo, \\\\(R=4\\\\) for hyper; for taxol, \\\\(R=2\\\\) for control, \\\\(R=3\\\\) for hypo, \\\\(R=4\\\\) for hyper.\\n\\n'", "CAPTION FIG4.png": "'\\nFigure 4: Cavellae and mTORC2 are involved in membrane tension regulation. (_A_) Schematic of membrane cuffless interactions with caveolin-1-2, cavin-1-3, pPP3, and Akt phosphorylation as well as the signaling pathways of mTOR complexes inhibited by toxin 1 (mTORC1 and mTOR(2)) or rapamycin (_m_TORC1). (_B_) Single-cell volume dynamics of cells Cavin1-kO (and their corresponding CRISPR control; _S1 Appendix_, Fig. 5_D_). Statistics are \\\\(R\\\\) > 3 for every experiment and \\\\(n\\\\) = 7 for control, \\\\(n\\\\) = 15 for hypo, \\\\(n\\\\) = 18 for hyper. (_C_) Membrane tension dynamics of cells Cavin1-kO (and their corresponding CRISPR control; _S1 Appendix_, Fig. 5_E_). Statistics are \\\\(n\\\\) > 4 for every experiment and \\\\(R\\\\) = 3 for control, \\\\(R\\\\) = 4 for hypo, \\\\(R\\\\) = 2 for hyper. (_D_) Activity of mTORC1 and mTORC2 under hypertoncin and hypotomis shock. Panels represent activation (phosphorylation) of SSI (p-P70) and Akt (Akt) and leading controls. Longer timepoints are in _S1 Appendix_, Fig. 5_F_F_ and corresponding quantification. (_E_) Single-cell volume dynamics of cells treated with rapamycin or Torin1 for hypotomis shocks (120 mOsm, circle), isotonic conditions (315 mOsm, square), and hypertonic shocks (700 mOsm; P70 = 2, triangle). Statistics are \\\\(R\\\\) > 3 for every experiment. For rapamycin, \\\\(n\\\\) = 4 for control, \\\\(n\\\\) = 5 for hypo, \\\\(n\\\\) = 4 for hyper; for Torin1, \\\\(n\\\\) = 10 for control, \\\\(n\\\\) = 9 for hypo, \\\\(n\\\\) = 7 for hyper. (_F_) Membrane tension dynamics of cells treated with rapamycin or Torin1 for identical shocks as in \\\\(E\\\\). Statistics are \\\\(n\\\\) > 4 for every experiment. For rapamycin, \\\\(R\\\\) = 2 for control, \\\\(R\\\\) = 6 for hypo, \\\\(R\\\\) = 4\\n\\n'", "CAPTION FIG5.png": "'\\nFig. 5: Ion transporters are responsible for the short-term response of cells to osmotic shocks. (_A_) Illustrations of DCFIB, EIPA, and bunstantiside pharmacological effects on, respectively, VRACs, NHE, and MKCCI channels. (_B_) Confocal images of DCFIB-treated cells and response under hypotonic shock. (Scale bar: 40 \\\\(\\\\mu\\\\)m) (C) Single-cell volume dynamics in cells treated with bunstantiside, EIPA, and DCFIB for hypotonic shocks (120 mOsm, circle), isotonic conditions (315 mOsm, square), and hypertonic shocks (T00 mOsm, PPO = 2, triangle). Statistics are \\\\(R\\\\) > 3 for every experiment. For bunstantiside, \\\\(n\\\\) = 9 for control, \\\\(n\\\\) = 66 for hypo, \\\\(n\\\\) = 8 for hyper; for EIPA, \\\\(n\\\\) = 12 for control, \\\\(n\\\\) = 9 for hypo, \\\\(n\\\\) = 9 for hyper; for DCFIB, \\\\(n\\\\) = 12 for control, \\\\(n\\\\) = 31 for hypo, \\\\(n\\\\) = 14 for hyper. (_D_) Membrane tension dynamics in cells treated with bunstantiside, EIPA, and DCFIB for identical shocks as in C. Statistics are \\\\(n\\\\) > 4 for every experiment. For bunstantiside, \\\\(R\\\\) = 5 for control, \\\\(R\\\\) = 11 for hypo, \\\\(R\\\\) = 4 for hyper; for EPIA, \\\\(R\\\\) = 3 for control, \\\\(R\\\\) = 12 for hypo, \\\\(R\\\\) = 3 for hyper; for DCFIB, \\\\(R\\\\) = 2 for control, \\\\(R\\\\) = 6 for hypo, \\\\(R\\\\) = 2 for hyper.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## 4 Conclusion\\n\\nFig. 6: Recapitulative scheme. (A) In the case of low membrane area buffer, cells have larger initial volumes that remain mostly unchanged during hypotonic shocks. Membrane nuffles are initially unfolded, which leads to higher stretching of the membrane and an increase of PM tension in resting conditions. (B) In the case of intermediate membrane area buffer, cell volumes increase under hypotonic shocks causing the unfolding of membrane nuffles. Membrane stretches and PM tension increases in a cell volume-dependent manner. (C) In the case of high membrane area buffer, cells have smaller initial volume, thus having also more nuffles and therefore call volume increase under hypotonic shocks. Call volume increase during hypotonic shock is responsible for unfolding the nuffles without stretching the membrane to its maximum, therefore having limited increase of PM tension.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/ruddjr.ComputationalSupportBridging2022.json b/extracted_captions/ruddjr.ComputationalSupportBridging2022.json new file mode 100644 index 0000000000000000000000000000000000000000..21fc571d89b395f0cbd585bf24d73d197f389dd1 --- /dev/null +++ b/extracted_captions/ruddjr.ComputationalSupportBridging2022.json @@ -0,0 +1 @@ +{"CAPTION APPENDIX A.png": "'\\n\\n## Appendix A Areas of Knowledge Form'", "CAPTION APPENDIX B- ANALOG STIMULI.png": "'\\n\\n**Acknowledgement**'", "CAPTION APPENDIX B-P1.png": "'* [16] A.\\n\\n'", "CAPTION APPENDIX B-P10.png": "'\\n\\n**Acknowledgments**'", "CAPTION APPENDIX B-P11.png": "'\\n\\n**Acknowledgments**'", "CAPTION APPENDIX B-P12.png": "'\\n\\n**Acknowledgments**'", "CAPTION APPENDIX B-P2.png": "'\\n\\n**Acknowledgement**'", "CAPTION APPENDIX B-P3.png": "\"Recommutader's attempt \\\\(\\\\mathbf{P3}\\\\)\"", "CAPTION APPENDIX B-P4.png": "\"Recommutader's attempt [1].\\n\\n\"", "CAPTION APPENDIX B-P5.png": "\"Recommutader's attempt to \"", "CAPTION APPENDIX B-P6.png": "\"Recominateder's Standard 196.\\n\\n\"", "CAPTION APPENDIX B-P7.png": "'\\n\\n**Acknowledgments**'", "CAPTION APPENDIX B-P8.png": "'\\n\\n**Acknowledgments**'", "CAPTION APPENDIX B-P9.png": "'\\n\\n**Acknowledgments**'", "CAPTION APPENDIX B.png": "'\\n\\n## Appendix A Appendix B: Provided Standard'", "CAPTION APPENDIX C-P1 .png": "'* [16] A.\\n\\n'", "CAPTION APPENDIX C-P2.png": "'\\n\\n**Abstract**\\n\\nWe study the behavior of the \\\\(\\\\beta\\\\)-'", "CAPTION APPENDIX C-P3.png": "'\\n\\n**Abstract**\\n\\nWe study the behavior of the \\\\(\\\\beta\\\\)-'", "CAPTION APPENDIX C-P4.png": "'* [16] A.\\n\\n'", "CAPTION APPENDIX C-P5.png": "'\\n\\n**Abstract**\\n\\nWe study the behavior of the \\\\(\\\\beta\\\\)-'", "CAPTION APPENDIX C-P6.png": "'* [16] A.\\n\\n'", "CAPTION APPENDIX C-P7.png": "'* [16] A.\\n\\n'", "CAPTION APPENDIX C-P8.png": "'\\n\\n**Abstract**\\n\\nWe study the behavior of the \\\\(\\\\beta\\\\)-'", "CAPTION APPENDIX C-P9.png": "'* [16] A.\\n\\n'", "CAPTION APPENDIX C.png": "'Appendix C: Idea Journey'", "CAPTION FIG1.png": "'Figure 1: Example of bridging analogies path\\n\\n'", "CAPTION FIG2.png": "'Figure 2: system used to determine relevant bridging analogues\\n\\n'", "CAPTION FIG3.png": "'Figure 3 Example of path selection\\n\\n'", "CAPTION FIG4.png": "\"\\n\\n## Chapter 4 Pt's M\"", "CAPTION FIG5.png": "\"_Figure 5 Close up of engineering controls analogy from P2's map session_\"", "CAPTION FIG6.png": "\"Figure 6: Analogy provided during P11 's analogy session\\n\\n\"", "CAPTION TAB1.png": "'_Table 1 Design problems and Far domain analogies_'", "CAPTION TAB2.png": "'\\n\\n## Chapter 2 Examples of bridging analogues'", "CAPTION TAB3.png": "'_Table 3 Example timeline of a Zoom session_'", "CAPTION TAB4.png": "'_Table 4 codes pertaining to the use of analogies_'", "CAPTION TAB5.png": "'_Table 5 Occurrences of codes across sessions_'", "CAPTION TAB6.png": "\"_Table 6 P7's Recommender session idea journey_\"", "CAPTION TAB7.png": "'\\n\\n## Chapter 7 Future Work\\n\\nThe thesis is based on the work of the thesis of'", "CAPTION TAB8.png": "\"_Table 8 P12's map session idea journey_\"", "CAPTION TAB9.png": "'_Table 9 Representation of confusion across sessions_'"} \ No newline at end of file diff --git a/extracted_captions/russellCostStructureSensemaking1993.json b/extracted_captions/russellCostStructureSensemaking1993.json new file mode 100644 index 0000000000000000000000000000000000000000..d79f5dddd741ed6b6420c48260dafdddc62d7e8a --- /dev/null +++ b/extracted_captions/russellCostStructureSensemaking1993.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: An instrumented IDE schema describing a printer (an enclosed). Based values indicate links to other encoders.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Figure 2.** Regressions and their associated use procedures evolve as new requirements are uncovered and properties of a representation are understood. In this case, the representation went through 3 major shifts, each time is improving usability or decreasing cost-of-use. Note that each box stands for an IDE node type with a set of specified slots.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: This diagram shows the steps the analysts went through to make sense of laser emitters. They collected and organized information, then found chances of common terms, ideas, subsystems and functions in data describing twenty-one different laser emitter.\\n\\n'", "CAPTION FIG4.png": "'Figure 4. Sensemaking is finding a representation that organizes information to reduce the cost of an operation in an information task. The product of the learning loop is the representation and encoding set\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Sensembling in four different information-rich tasks.\\n\\n'", "CAPTION FIG6.png": "'Figure 6. A graph showing partial ordering of constraints for presentation. In this example, 5 explicit ordering constraints and 4 grouping constraints suffice to order 16 nodes.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n**Figure 7.** Tradeoffs in the gain achieved by a manual method (gm) or computer-based method (gc) . The best solution is determined by maximizing gain while minimizing total cost within the deadline time.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/sathe2018small.json b/extracted_captions/sathe2018small.json new file mode 100644 index 0000000000000000000000000000000000000000..3cd1502891c809a913f64ee91c74480a95f34c4f --- /dev/null +++ b/extracted_captions/sathe2018small.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\nFig. 1: Identification of newly formed SecGFP-GPI endocytic vesicles using a pH pulse assay. **a** Schematic (top panel) of the pH pulling assay depicting the effect of pH on SecGFP-GPI fluorescence during endocytic vesicle budding. Note the fluorescence of SecGFP-GPI is retained at high pH (top and bottom left panel) or when exposed to low pH if sequestered in a newly formed endocytic vesicle (bottom right panel), and quenched only when exposed to low pH (top right panel). Sequential TIRF images of ACS cell co-expressing SecGFP-GPI and mCherry-ARFI collected at pH 7, pH 5 and in the RFP channels (middle panel). Newly formed endocytic vesicles (inset) (identified as in Supplementary Figure 1c) are used to construct a single frame (yellow rectangle) of the montage depicted (bottom panel). **b** Average of the normalised fluorescence intensities of pH 5 and pH 7 traces at the site of newly formed SecGFP-GPI endocytic vesicles compared to their respective random traces constructed from 120 endocytic events (pH 5 and pH 7) and 3428 random spots, derived from 17 cells pooled from four independent experiments. **c** The graph shows the fold enrichment of fluorescence intensity over the local background of pH 5 vs. pH 7 at the time of formation of the endocytic vesicles (data from 1b). **d\u2013f** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of TagRFP-CDC42 (**d**), mCherry-ARFI (**e**) and mCherry-GBT (**f**) to the forming SecGFP-GPI endocytic sites, and its corresponding random intensity trace (n, Table 1). The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution by Mann-Whitney \\\\(U\\\\) test, and the log\\\\({}_{10}\\\\) (p) (log\\\\({}_{10}\\\\) (0.05) is \\\\(-\\\\)1.3 and log\\\\({}_{10}\\\\) (0.001) is \\\\(-\\\\)2.5) is plotted below each trace (**d\u2013f**). Representative montages from the respective data sets are depicted below the graphs (**d\u2013f**). Arrowheads indicate the newly formed endocytic vesicle. Error bars, s.e.m. (**b**, **d\u2013f**). Scale bar, 1.5 \u03bcm (**a**, **d\u2013f**)'", "CAPTION FIG2.png": "'\\nFig. 2: RNA screen reveals BAR domain proteins involved in CG endocytosis. **a** List of _Drosophila_ proteins in the PFAM database that contained one of the following BAR domains, PFAM IDs\\\\(-\\\\)PF06456, PF09325, PF06456, PF0061f and PF08397. The list was filtered to remove duplicates to give 18 genes. **b** The histogram shows normalised 5-min fluid-phase uptake in S2R\\\\({}^{+}\\\\) cells treated with 10 \u03bcg of dsRNA for 4 days as indicated with dsRNA against GBFI (gazr) as positive, and GFP as negative controls. In a single experiment, mean uptake of one of GFP dsRNA coverslip was used to normalize the mean for rest of the coverslips. Data were pooled from three independent experiments and the cell numbers are indicated in the graph. The bars in green are significantly different from GFP dsRNA using two-sample _t_-test (_b_ value <0.05). Version 27 of the PFAM database was used to generate the list '", "CAPTION FIG3.png": "'\\nFig. 3: IRSp53 is involved in CG endocytosis. Schematic depicting the domain organisation of IRSp53. IRSp53 exists in an inactive dimer state, which upon binding to GTP-CDC42 is activated, allowing SH3 domain to bind to its effectors. **b** Histogram (left) shows 5-min uptake of TFR and fluid phase in IRSp53-WT cells normalised to IRSp53-/- cells, along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated in the figure. **c** Histogram (top) shows normalized 5-min fluid uptake in IRSp53-/- and IRSp53WT cells when treated with LG186 or vehicle (DMSO) along with representative images (bottom). Data were pooled from two independent experiments and the number of cells indicated in the figure. **d** Histogram (top) shows an average number of endocytic structures quantified per field from the electron microscope images (bottom). Data pooled from three independent blocks. Untreated WT MEFs (WT, top row), IRSp53 null MEFs (IRSp53-/-, bottom left) or LG186-treated WT MEFs (LG186, bottom right) were incubated for 5 min at 37 \u00b0C with 10 mg/ml HRP as a fluid-phase marker before processing for electron microscopy. Endocytic structures close to the plasma membrane (PM) are filled with the electron dense peroxidase precipitate. WT cells show a range of endocytic structures including vesicular structures (double arrowheads) and tubular/ring-shaped putative CLIC/GEECs (large arrowheads) but the IRSp53-/- cells and LG186-treated cells only show predominant labelling of vesicular profiles. \\\\(p\\\\) value <0.05 (*), 0.001 (**) Mann-Whitney \\\\(U\\\\) test (**b-c**) and two-sample Student\u2019s test (**d**). Error bars, s.d. (**b-d**). Scale bar, 20 \u03bcm (**b-c**), 1 \u03bcm (**d**), respectively. Schematic (**a**) was adapted with permission from M\u00fcllrio (www.mechanobio.info) Mechanobiology Institute, National University of Singapore'", "CAPTION FIG4.png": "'\\nFig. 4: CG pathway is abolished in the absence of IRSp53. **a** The box plot shows the number of endosomes per cells (left) for endocytosed GFP-GPI (a-GFP (Fab)), fluid phase and TIR in IRSp53\\\\(-\\\\)/\\\\(-\\\\) and IRSp53 WT cells when pulsed for 2 min along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated below the graph. **b** Plot (left) shows quantification of the fraction of GFP-GPI endocytic/vic vesicles containing fluid phase or TI images (right) show representative single confocal slices of a 2-min pulse of a-GFP Fab (green)/TMR-Dextran (magenta) and a-GFP Fab (green)/A568-Tf (magenta) in IRSp53\\\\(-\\\\)/\\\\(-\\\\) (top row) and IRSp53WT (bottom row) cells. The inset depicts magnified views of the indicated region; single-channel images are in panel **a**. Data were pooled from two independent experiments and the number of cells is indicated below the graph. **c**plot (left) showing quantification of the fraction of \u20181-min fluid-phase endocytof vesicles containing Tf. Images show representative single confocal slices of a \u2020-min pulse of TMR-Dextran (green) and A647-Tf (magenta) in IRSp53\\\\(-\\\\)/\\\\(-\\\\) (top row) and IRSp53WT (bottom row) cells. Inset depicts magnified views of the indicated region. Data were pooled from two independent experiments and the number of cells indicated below the graph. **d** Histogram (left) shows G-min uptake of fluid phase in IRSp53\\\\(-\\\\)/\\\\(-\\\\) MEFs transduced with virus expressing GFP-IRSp53 WT, GFP-IRSp53 4KE, GFP-IRSp53 1266N, GFP-IRSp53 14Q8P and GFP-IRSp53 V522G, normalised to that in IRSp53\\\\(-\\\\)/\\\\(-\\\\) MEFs, along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated in figure except for IRSp53\\\\(-\\\\)/\\\\(-\\\\) (381). \\\\(p\\\\) value <0.01(*) and 0.001(**) by Mann-Whitney U test (**a\u2013d**). Scale bar, 20 \u03bcm (**d**), 5 \u03bcm (**a\u2013d**), respectively. Error bars (**d**) represent s.d.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: IRSp3 is reconstructed to forming CG endosomes. **a** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of three different regions [circles, violet, \\\\(t\\\\) = 170 nm and black (_r_ = 250 nm) and annulus, orange (_t_ = 250-420 nm)] for the recruitment of IRSp3-mCherry to the forming SecGFP-GPI endocytic sites and its corresponding random intensity trace (_n_, Table 1). **b** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of TagRFP-CDC42 to the forming SecGFP-GPI endocytic sites and its corresponding random intensity trace to two different regions [circle, black, \\\\(r\\\\) = 250 nm and annulus, orange (_r_ = 250-420 nm)]. Error bars, (**a\u2013b**) represent s.e.m. (_n_, Table 1). The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution (**a**) by Mann-Whitney \\\\(U\\\\) test and the log\\\\({}_{10}\\\\) (_p_) is plotted below each trace [Log\\\\({}_{100}\\\\) (0.05) is \\\\(-1.3\\\\) and log\\\\({}_{10}\\\\) (0.001) is \\\\(-2.5\\\\)]. Representative montages are depicted below the graphs (**a\u2013b**). Arrowheads indicate the newly formed endocytic vesicle. **c** Electron micrographs of AGS cells co-transfected-GFP-IRSp53 and GFP-binding protein coupled to Apex (GBP-Apex). The DAR reaction was performed and the cells were processed for electron tomography. **A** single section of the original tomogram (left) and density-based thresholded of the same plane (middle) reveal electron dense structures containing IRSp3 at membrane surfaces. The whole of PM of the tomographic volume was rendered and different examples of enlarged tubular regions of interest show GFP-IRSp53 recruitment patterns (right). Scale bar, 15 \u03bcm (**a\u2013b**) and 0.5 \u03bcm (**c**), respectively\\n\\n'", "CAPTION FIG7.png": "'\\n\\n## 4 Conclusion\\n\\nIn this paper, we have developed a new method for estimating the surface area of the'", "CAPTION FIG8.png": "'\\n\\n## References\\n\\nFig. 8: Schematic depicting the proposed biphasic mechanism for CG endocytic vesicle formation. **a** Phase I: Characterised by the recruitment of ARF/1 GBRI, PICKI, ARP2/3 and IRSp53 but not the buildup of F-actin and CDC42. Here, IRSp53 may be recruited by its I-BAR domain in the absence of GTP-CDC42, keeping its SH3 domain in an intra-molecular inhibited state. PICKI keeps ARP2/3 in an inactive state. **b** Phase II: Characterised by the recruitment of CDC42 and a sharp increase in ARFI leading to the removal of PICXI. This allows for the activation of ARP2/3 and buildup of F-actin. CDC42 binds to the CRIB domain of IRSp53 thereby activating it. The SH3 domain of IRSp53 can now bind to ARP2/3 activators and create F-actin. **c** Phase II: Characterised by endocytic vesicle formation, the presence of CDC42, ARFI/GBF1 and F-actin'", "CAPTION TAB1.png": "'\\n\\n**Table 1 pH pulsing assay data set**'", "CAPTION TAB2.png": "'\\n\\n**Table 2 Cross-correlation calculated for indicated traces**'"} \ No newline at end of file diff --git a/extracted_captions/shi2018cell.json b/extracted_captions/shi2018cell.json new file mode 100644 index 0000000000000000000000000000000000000000..73b1f55c125a621a0f2c2ad2c9cdb26892a3db9f --- /dev/null +++ b/extracted_captions/shi2018cell.json @@ -0,0 +1 @@ +{"CAPTION FIG S1.png": "'Figure S1: Membrane Tension Equilibrates Quickly in Artificial Lipid Bilayers, Related to Figure 1 (A) Fluorescence image of a micropipette-assisted GLV (DOPS:DOPE:DOPC = 35:30:35) containing 0.5% DSPE-Bio-PEG2000 and labeled with 0.3% Texas Bad DLPFE. The edge of the pipette is marked with yellow lines. An optically trapped bead (position indicated by the yellow circle) pulled a membrane tether, opposite to the pipette. Scale bar 10 \u03bcm. (B) Changes in membrane tension (blue) were induced by applying steps of pressure to the pipette and tether pulling force (black) was monitored via the optical trap. (C) Close-up of the stop marked with a red line in (B), showing no detectable delay between change in tension and change in tether force. Measurements sampled at 10 Hz. (D) Relation between tether pairing force and the square root of membrane tension, with a linear fit following the relation: \\\\(f = 2\\\\pi\\\\sqrt{2\\\\alpha}\\\\) (red line, R2 = 0.98), Error bars are SEM.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Propagation of Membrane Tension in Cells (A and D) Schematic: (left) and fluorescence image (right) showing a pair of tethers pulled from (A) a cell-attached blob or (B) the cell body of a HeLa cell expressing GPI-dGFP. Green: fluorescence under patterned illumination (restricted to dashed boxes); red: fluorescence under wide-field illumination. In (D), a transmitted light image (gray) is combined with the fluorescence images. Scale bars 5 \u03bcm. (B and E) The two tethers were stretched sequentially (top), and the fluorescence of each tether was monitored (bottom) in (B) a HeLa cell blob and (E) an intact HeLa cell. (C and F) Relation between the intensities of the two tethers when either the first or second tether was stretched in (C) a HeLa cell blob and (F) an intact HeLa cell. (G) Test for slow coupling between tethers in a HeLa cell. A changing in length of tether 2 did not affect fluorescence of tether 1 within a 500-s measurement window. (H-K) Rapitation of the experiment in (D)-(F) in (H-K) NIH-3T3 fibroblasts. (I) MDCK epithelial cells, (J) mouse brain endothelial cells, and (K) rat hippocampal neurons. T1: father 1; T2: tether 2. See also Figures S1 and S2.\\n\\n'", "CAPTION FIG2.png": "\"Figure 2: Hydrodynamic Model of Membrane Flow Past Immobile Obstacles\\n\\n(A) Illustration of the cell plasma membrane with transmembrane proteins bound to the underlying cortex.\\n\\n(B) Simple viscoelastic model of the cell membrane. Springs represent the elastic response of the membrane to stretch, and dampers represent the viscous drag from immobile transmembrane proteins.\\n\\n(C) Dependence of diffusion coefficients for membrane tension (red) and molecular tracers (blue) on the area fraction \\\\(\\\\phi\\\\), of immobile proteins. This plot shows the model's predictions for the dimensionless diffusion coefficients, \\\\(D_{NN}=\\\\gamma D_{i}/E_{m}a^{2}\\\\) for tension and \\\\(D_{NN}=\\\\pi\\\\Sigma_{i}/k_{B}T\\\\) for tracers. The upper limit on tension diffusion is set by the hydrodynamic drag between plasma membrane and cytoskeleton cortex in the absence of obstacles. The upper limit on tracer diffusion is set by the Saffman-Deltrock model (Quantification and Statistical Analysis). Open circles: diffusion coefficients in intact cell membranes; insect- relation between dimensionless diffusion coefficients of membrane tension and molecular tracers (solid line). The dashed line shows a linear relation. Closed circles: obstacle-free membrane. Open circles: \\\\(\\\\phi_{t}=0.18\\\\).\\n\\n(D) Fluorescence image showing a HeLa cell in which transmembrane proteins have been labeled non-specifically with Alexa488-NH5 before (left) and after (right) bleaching with a donut shape laser spot. Scale bar, 10 \u03bcm.\\n\\n(E) Fluorescence intensity profile of the bleached ring (black) and non-bleached central (greater) regions. The photobleaching epoch is shaded red.\\n\\n(F) Compensation of stimulation and experiment for time-dependent membrane tension in a stretched membrane tether and surrounding cell membrane (E\\\\({}_{m}=4\\\\) pN/m; D\\\\({}_{s}=0.024\\\\) mV/s). (Top) Tether stretch protocol with initial tension \\\\(\\\\alpha_{B}=25\\\\) pN/m and ramp increase in tether length from 40 \u03bcm to 90 \u03bcm at a pull-ring speed \\\\(v_{\\\\mathrm{final}}=1\\\\) \u03bcm/s are shown. (Middle) Sintraded surface area of the tether is shown. (Bottom) Membrane tension in the tether referred from measurements of tether radius (black) and simulated membrane tension in the tether and in the cell at distances of 0.1 \u03bcm to 20 \u03bcm from the tether are shown. See Quantification and Statistical Analysis for details of the simulation.\\n\\n\"", "CAPTION FIG3.png": "'Figure 3: **Membrane Tension Mediates Local Activation of Mechanosensitive Ion Channels and Local Vesicle Fusion in MDCK Cells** (A) MDCK cell co-expressing GPI-aGFP (green) and R-CaMP2 (red). (B) Composite fluorescence image of tether (green) and R-CaMP2 (red). Fluorescence excitation of aGFP was confined to the tether (dashed box). (C) Localized Ca2+ influx triggered by tether stretch. Images are composites of mean fluorescence (gray) and changes in fluorescence (b) heatmap. Tether pulling pipette shown schematically at 0 s. (D) Blockers of MSCa\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Tension Mediates Local Activation of Mechanosensitive Ion Channels and Local Vesicle Fusion in Primary Mouse Brain Endothelial Cells** (A and B) **Tather stretch triggered (A) localized Ca2+ influx and (B) vesicle fusion events. Images are composites of mean fluorescence (gray) and changes in fluorescence (heatmap). Tather pulling pipette shown schematically. (C) Distribution of Ca2+ influx (+) and vesicle fusion (b) sites relative to the tether attachment point (gray circle). Each mark represents one event (\\\\(\\\\oplus\\\\) Ca2+ influx) events from 7 cells; 29 vesicle fusion events from 6 cells). Average distance between Ca2+ initiation and tether attachment was 2.2 \\\\(\\\\pm\\\\) 0.5 \\\\(\\\\mu\\\\)m [mean \\\\(\\\\pm\\\\) SEM], within the local-exclusion uncertainty [3 \\\\(\\\\mu\\\\)m]. Average distance between vesicle fusion and tether attachment was 8.0 \\\\(\\\\pm\\\\) 0.8 \\\\(\\\\mu\\\\)m (mean \\\\(\\\\pm\\\\) SEM; versus 28 \\\\(\\\\pm\\\\) 3 \\\\(\\\\mu\\\\)m for null hypothesis). (D) 2- AFB (100 \\\\(\\\\mu\\\\)m) significantly reduced the spread of vesicle fusion events relative to the tether attachment [3.9 \\\\(\\\\pm\\\\) 0.6 \\\\(\\\\mu\\\\)m; \\\\(n\\\\) = 23 with 2-APB; \\n\\n'", "CAPTION FIGS2.png": "'Figure S2: **Tether Diameter Reports Membrane Tension, Related to Figure 1**\\n\\n(A) Schematic showing simultaneous measurement of ttether pulling force (with an optical trap) and tether fluorescence intensity (with patterned illumination in the dashed circle).\\n\\n(B) Determination of ttether diameter from ttether fluorescence intensity. A.14 \\\\(\\\\mu\\\\)m diamater circular spot of illumination was first directed to a ttether (hop) and then to a flat portion of the parent cell (bottom). The ratio of the total fluorescence exceed in these two configurations equals the ratio of illuminated membrane areas. These calibrations yielded an average tether diameter \\\\(d_{\\\\mathrm{other}}=150\\\\pm 10\\\\) mm (mean \\\\(\\\\pm\\\\) SEM, \\\\(n=5\\\\) cells) for \\\\(n=5\\\\) cells) for \\\\(n=5\\\\) cells) for \\\\(n=5\\\\) cells) for \\\\(n=5\\\\) cells) for \\\\(n=5\\\\) cells). Measured average tether pulling force was \\\\(f_{\\\\mathrm{ferror}}=16\\\\pm 10\\\\) mN (mean \\\\(\\\\pm\\\\) SEM, \\\\(n=10\\\\) cells), leading to a membrane binding stiffness \\\\(\\\\kappa=(1.9\\\\pm 0.2)\\\\times 10^{-1}\\\\) J. Scale bar, 10 \\\\(\\\\mu\\\\)m.\\n\\n(C) Tathers were pulled with a bead in an optical trap, and tether length, fluorescence intensity, and force were measured simultaneously. Regions shaded in green were used to calculate the relation between steady state ttether fluorescence intensity and pulling force.\\n\\n(D) Relation between ttether pulling force and inverse of ttether fluorescence intensity (normalized to expression level; R\\\\({}^{2}=0.9\\\\), n = 7 cells). Error bars are SEM.\\n\\n(E) Perturbation to membrane tension via osmotic shocks. Tether pulling force was measured with an optical trap while a pipette flowed pure water over the cell.\\n\\n(A) The pipette approached the cell, the pulling force increased, signaling an increase in membrane tension. As the pipette withdrew, the tension decreased.\\n\\n(F) Transmitted light (left) and fluorescence light images showing two sets of ttether pulled from the same cell.\\n\\n(G) Rasporca of ttether fluorescence to gradual addition of hypotaric buffer (20 mOsm buffer added to equal volume of 300 mOsm XC buffer during time shaded in red). Experiments were performed at 37\u00b0C on HeLa cells expressing membrane-targeted fluorescent protein mCranga2-KRAS (A) \u2013 (E) and GPI-aGFP (F), (G).\\n\\n(H) Left: Wide-field autofluorescence image of a blob in a HeLa cell expressing GPI-aGFP with two tethers. Right: Same structure, with patterned illumination, restricted to illuminating the tethers. (I) Left: Transmitted light image of two pipettes with polystyrene beads at the ends. Middle: Wide-field epithorescence images of a HeLa cell expressing GPI-aGFP. Right: Same structure, with patterned illumination restricted to illuminating the tethers.\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n**Figure S3. FRAP Measurements of Molecular Transport in Cell Membranes, Related to Figure 2**\\n\\n(A) Actin cytoskeleton does not penetrate membrane tethers. Top left: transmitted light image showing position of the tether pulling pipette. Middle and bottom left: Simultaneous images of cell membrane [mOrange2-KRAS] and actin (Lfacet-CFP) in a tether. Images were taken 15 min after tether formation. Right: Composite image of the whole cell. Red: membrane, green: actin.\\n\\n(B) FRAP experiment to test whether tether and cell membrane are in diffusive equal-Brunn. Composite fluorescence image showing the photobleached region (shaded box) on a tether (green), attached to a H4-cell (red) expressing PRD2-EGFP.\\n\\n(C) FRAP of the tether (blue) and corresponding simulation (red) assuming free diffusion from cell to tether. The simulation used the experimentally measured diffusion coefficient of DRD2-EGFP on the cell, \\\\(D_{\\\\text{d}}\\\\)[Cell] = 0.037 mm3/s (see Table S2), and a tether radius of 75 nm. Since \\\\(D_{\\\\text{d}}\\\\)[Cell] \\\\(<\\\\)\\\\(<\\\\)\\\\(D_{\\\\text{d}}\\\\)(Tather), the smaller diffusion coefficient dominated the transport and was the appropriate choice for the simulations. The simulation was performed in MATLAB. Time of photo-bleaching is shaded red.\\n\\n(D) Relation between Darcy permeability \\\\(k\\\\) and area fraction of immobile proteins _ph_. The function \\\\(k/a^{2}\\\\) = \\\\(\\\\tau\\\\)(_ph_) was derived by Bussell (Bussell et al., 1995) et al., who showed that \\\\(\\\\tau_{0}\\\\) = \\\\(\\\\tau\\\\)/[2 + 4 + \\\\(\\\\left[ {K_{\\\\text{d}}(x)}/{K_{0}(x)} \\\\right]\\\\), where \\\\(x = \\\\mu\\\\)/s\\\\({}^{1/2}\\\\) (see Quantification and Statistical Analysis). The upper limit (blue dot) is calculated from viscous drag of the cytoplasm layer between membrane and actomyosin cortex.\\n\\n(E) Composite images showing the cell (green) and 14 \\\\(\\\\mu\\\\)m circular photobleaching spot (red). Left: FRAP on cell membrane. Right: FRAP on tether.\\n\\n(F) FRAP data in HeLa cells expressing DRD2-EGFP. Error bars represent SEM from \\\\(n\\\\) = 10 tethers pulled from 10 cells. Time of photobleaching is shaded red.\\n\\n(G) After the FRAP measurement as shown in Figure 2E, a cell impermeant fluorescent quencher, amaranth, was added to a final concentration of 500 \\\\(\\\\mu\\\\)M to quench the Alexa488 fluorescence. Fluorescence of all regions of the cell membrane dropped to background levels, establishing that there was no detectable internalization of fluorescently labeled proteins.\\n\\n(H) Ration of the cell membrane at a cell-immersent fluorescence quencher. (H) In cells expressing intracellular aGFP (EGFP-KRAS) amaranth did not affect fluorescence, but (I) in cells expressing extracellular aGFP (pDisplay: aGFP-TM) amaranth quenched fluorescence. Error bars are SEM over \\\\(n\\\\) = 4 cells for aGFP-KRAS and \\\\(n\\\\) = 5 cells for aGFP-TM. Scale bars in all panels 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIGS4.png": "'Figure S4: **Dynamics of Ca\\\\({}^{2+}\\\\) Entry through Mechanosensitive Ion Channels, Related to Figures 3 and 4**\\n\\n(A) MDCK cell from Figure 3C. Fluorescence of the R-CaMP2 reporter in different regions of interest as a function of time after initial Ca\\\\({}^{2+}\\\\) influx. The dynamics show diffusion of Ca\\\\({}^{2+}\\\\) from the point of tether attachment into the cell.\\n\\n(B) mBEC cell from Figure 4A. Filmstrip of fluorescence recordings in response to activation of mechanosensitive ion channels via tether pull. The movie shows local activation followed by intracellular spread of Ca\\\\({}^{2+}\\\\).\\n\\n(C) mBEC cell of Figure 4E. Filmstrip of fluorescence recordings in response to local shear flow. The Ca\\\\({}^{2+}\\\\) influx starts at the region of maximum shear, followed by intracellular propagation of Ca\\\\({}^{2+}\\\\).\\n\\n(D) experiments as shown in Figure 3B, tether fluorescence integrally reports tether radius. Membrane tension can then be estimated from \\\\(e\\\\)=\\\\(\\\\kappa\\\\)/2\\\\(T_{0}^{2}\\\\) (see Quantification and Statistical Analysis). The activation tension of MSCs was found to be approximately 11 times higher than the resting tension of the cell (dashed line, resting membrane tension is \\\\(\\\\sim\\\\)25 pN/ml). This estimate of activation tension is a lower bound because at the time of MSC activation the tether was still elongation, so tether diameter was not fully equilibrated.\\n\\n(E) MDCK cell co-expressing GCaMP2 (green) and PE2O1-mCherry (red), with a pipette-controlled bead locally touching the cell (gray).\\n\\n(F) Localized Ca\\\\({}^{2+}\\\\) influx triggered by tether stretch. Dashed line shows the tether attachment point. Ca\\\\({}^{2+}\\\\) diffused from the point of entry to gradually fill the cell. There was no evidence of Ca\\\\({}^{2+}\\\\) entry at any point other than the site of tether attachment.\\n\\n(G) Sequential tether pulling from two points on the same cell (white arrows). Top: pulling from left edge of the cell. The image at 1 = 0 shows the expression of PE2O1-mCherry. Bottom: pulling from the right sides of the same cell. For both tether pulls, Ca\\\\({}^{2+}\\\\) diffused from the point of entry to gradually fill the cell. There was no evidence of Ca\\\\({}^{2+}\\\\) entry at any point other than the site of tether attachment. Images in B, C, F and (G) are composites of mean fluorescence (gray) and changes in fluorescence benchmark. Scale bars in all panels 10 \u03bcm.\\n\\n'", "CAPTION FIGS5.png": "'Figure S5.: Validating mOrange2-TM as a Vesicle Fusion Reporter, Related to Figure 3 (_A_) Validating-ionomycin as a means to induce vesicle fusion. In MDCK cells expressing R-CaMP2, isomycin (5 uM) induced a rapid increase in fluorescence (red, \\\\(n\\\\) = 4 cells) indicating Ca2+ influx. Fresh MDCK cells were incubated with FM4-64 to load the dye into vesicles and then the dye was washed from the extracellular medium. Inomycin led to a decrease in fluorescence (black, \\\\(n\\\\) = 4 cells), consistent with ionomycin-induced vesicle fusion. In MDCK cells expressing mOrange2-TM, isomycin reduced a rapid increase in fluorescence (orange, \\\\(n\\\\) = 7 cells), consistent with vesicle fusion and de-acidification of the vesicles. As a control experiment, ionomycin was added to MDCK cells expressing mOrange2-KRAS, which targeted the fluorescent protein to the inner leaflet of plasma membrane and to the cytoplasmic surface of vesicles. Isomycin did not affect the fluorescence of these cells (gray, n = 5 cells). Error bars are SEM. (_B_) Image of MDCK cells expressing mOrange2-TM before (left) and after (right) adding inomycin. Scale bar 10 uM.\\n\\n'", "CAPTION FIGS6.png": "'Figure S6. Tracking the Flow Profile from the Pipette for Shear Perturbation to Cells, Related to Figure 4. The pipette is on the upper left. The flow was visualized with fluorescent beads. Scale bar 100 \\\\(\\\\mu\\\\)m.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/siangliulueProvidingTimelyExamples2015.json b/extracted_captions/siangliulueProvidingTimelyExamples2015.json new file mode 100644 index 0000000000000000000000000000000000000000..319e6447caede03a5a3fe203677426bff37d5567 --- /dev/null +++ b/extracted_captions/siangliulueProvidingTimelyExamples2015.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Screenshot of the ideation interface. Participants typed their ideas in the text box. After they submitted an idea, it appeared on the pane on the left. For those in the _On-demand_, _On-idle_ and _On-interval_ condition, examples were shown in the example grid above the idea entry box. The most recently received examples were shown in a big black box while earlier examples were in a gray box. The \u201cInspire me\u201d button at the bottom was visible only to participants in the _On-demand_ condition.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Participants in the _On-ide_ condition generated significantly more ideas than participants in the _On-interval condition. Error bars show standard error.\\n\\n'", "CAPTION FIG3.png": "'Figure 3. The mean novelty z-score for participants in the _On-demand_ condition is significantly higher than for those in the _None_ and _On-idd_ condition. There is no statistically significant difference across conditions for the value scores. Error bars show standard error.\\n\\n'", "CAPTION FIG4.png": "\"Figure 4. Boxplot of idle time before example request by order of example set in session. The mean time before requesting examples was 31_19 seconds. Participants were idle for shorter amounts of time before requesting first and second example sets than for third, fourth and fifth sets. Participants' idle times also varied considerably, with some participants waiting longer than a minute before requesting examples.\\n\\n\"", "CAPTION FIG5.png": "'Figure 5. Participants in the _On-demand_ condition used more examples to generate new ideas than those in the _On-idle_ condition.\\n\\n'", "CAPTION FIG6.png": "'Figure 6. Participants in the _Obs-demand_ condition transferred both structural and surface features from examples more often than those in the _Obs-idle_ condition.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: When did the \\\\(Om\\\\)-demand participants request examples? The majority of participants said in the post-experiment that they requested examples when they ran out of ideas.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/sokabe2017helicaseindependent.json b/extracted_captions/sokabe2017helicaseindependent.json new file mode 100644 index 0000000000000000000000000000000000000000..5deb4f9177c147dd8cc60933027c01aa4e323734 --- /dev/null +++ b/extracted_captions/sokabe2017helicaseindependent.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Decrease in eIF3 affinity to the 435 PLC on eIF4F-dependent mRNA recruitment. (_A_) A schematic model for eIF3 affinity charge on eIF4F-dependent mRNA recruitment. (_B_ and _C_) Saturation curves showing the fraction of eIF3-F1 bound to the initiation complex in each given condition at equilibrium (_J_eff, where \\\\(B\\\\) was obtained using endogeneous HeLa eIF4G, and C was obtained by using 4GAN, as described in the text. Solid lines show curve fitting and the calculated equilibrium dissociation constants are shown as bar graphs in the right panels. Data in \\\\(B\\\\) and \\\\(C\\\\) represent mean \\\\(\\\\pm\\\\) SD of three independent experiments.\\n\\n'", "CAPTION FIG2.png": "'Fig. 2: ATP-dependent reduction in eIF3 affinity during mRNA recruitment. a|F3j-F| vs. 48S PIC saturation curves (left) and the calculated equilibrium dissociation constants (Right) as described in Fig. 18. The 48S PIC was generated in the presence of different adenine nucleotides and mRNA, as indicated.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: _Sending of the CA4-repeat and globin UTR RNAs in the preaccommodated state. (_A_) The saturation curve showing fractions of CA4(A)-4(B)-42-F1 bound to the 425 subunit at a function of the 425 subunit concentration under the given conditions (Left) and the calculated equilibrium dissociation constants (Right). (_B_) The inhibition curves showing fractions of CA4(A)-42-F1 that remain bound to the 425 subunit at a function of the globin-UTR RNA concentration under the given conditions (_Left_) and the calculated equilibrium dissociation constants (Right). Data represent mean \\\\(\\\\pm\\\\) SD of three independent experiments._\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Contribution of initiation complex components on mRNA recruitment. eIF3-Fl vs. 4BS PIC saturation curves (left) and the calculated equilibrium dissociation constants (right) as described in Fig. 1B using CAA(AUG)-42 mRNA. Shown is eIF3-Fl affinity in the presence or absence of 4GaN, \\\\(m^{3}\\\\)G cap, eIF4E, and eIF4B (\\\\(\\\\Delta\\\\)) or of eIF1, eIF1A, and eIF4B. Note that the concentrations of eIF4A, eIF4E, and eIF4B were increased twofold in the absence of eIF4G, to compensate for a possible decrease in affinity of these components. Green dashed lines and green shaded areas represent the control data obtained with the complete set of 4BS PIC components and CAA(AUG)-42 mRNA (Fig. 1C). Data represent mean \\\\(\\\\pm\\\\) SD of three independent experiments.\\n\\n'", "CAPTION FIG5.png": "\"Figure 5: Proposed models for an ATP-driven conformational change of the decoding site during mRNA recruitment and scanning. (4) An mRNA can be re-reuted from the cellular pool to the 43S PK in a preaccommodated or accommodated state. The preaccommodated state involves the 43S PK in a closed latch and decoding site conformation, enabling eIF3] to bind in a high-affinity binding state. In the presence of eIF4F-ATP and the TC, the mRNA is accommodated into the 40S subburst decoding site to form the scanning competent 48S complex. In this state, the latch and decoding site of the 40S subunit adopt the open state, which exhibits a low-elf3] affinity binding state. In the presence of mRNA secondary structures, the accommodation step requires the generation of single-stranded mRNA through the ATP hydrolysis-dependent helicase activity of eIF4A. (8) In the ATP-bound state, eIF4A adopts a high-RNA affinity closed conformation, and, directly or indirectly, promotes the open conformation of the 40S subunit that allows scanning along the 5' UTR. In the ADP-bound state, eIF4A adopts a low-RNA affinity open conformation. In this state, the 40S subunit adopts a closed conformation that is likely to be less conductive to scanning. Multiple cycles of ATP hydrolysis by eIF4A during scanning might lead to rewards of opposing opening and closing conformations of eIF4A and the 40S subunit.\\n\\n\""} \ No newline at end of file diff --git a/extracted_captions/songDesignbyAnalogyEffectsExplorationBased2022.json b/extracted_captions/songDesignbyAnalogyEffectsExplorationBased2022.json new file mode 100644 index 0000000000000000000000000000000000000000..2a171b206c10d876d6852e144bc3caca8b69e84a --- /dev/null +++ b/extracted_captions/songDesignbyAnalogyEffectsExplorationBased2022.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1**: **Visual architecture**'", "CAPTION FIG10.png": "'Figure 10: Number of (a) keywords searched and (b) patents accessed with \\\\(\\\\pm\\\\) one standard error bars.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: Design Ideas that directly coupled patent designs.\\n\\n'", "CAPTION FIG12.png": "'Figure 12: Word clouds generated using keywords searched\\n\\n'", "CAPTION FIG2.png": "'\\n\\n## References\\n\\nFig. 2: Illustration of patent network development: (a) table of three example patents and their topics, (b) one-patent network, (c) two-patents network, and (d) three-patents network'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG4.png": "'* [17] A. A. K.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5** **Entangles of generalized ideas**'", "CAPTION FIG6.png": "'\\n\\n**Fig. 6** **Quantity of Ideas generated per participant in Phase 2 with \\\\(\\\\pm\\\\) one standard error bars**'", "CAPTION FIG7.png": "'* [19] A. J. Burrows, J. E. Conway, and J. E. Conway, \"A new method for the determination of the mass of the '", "CAPTION FIG8.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG9.png": "'* [17] A.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l l l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1**} & \\\\multicolumn{1}{c}{**Inter-rater reliability coefficients of quality**} \\\\\\\\ \\\\multicolumn{1}{c}{**subdimensions**} & \\\\multicolumn{1}{c}{**Table 1**} & \\\\multicolumn{1}{c}{**Inter-rater reliability coefficients of quality**} \\\\\\\\ \\\\multicolumn{1}{c}{**subdimensions**} & \\\\multicolumn{1}{c}{**Table 2**} & \\\\multicolumn{1}{c}{**Inter-rater reliability coefficients of quality**} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Inter-rater reliability coefficients of quality subdimensions'", "CAPTION TAB2.png": "'\\n\\n## Table 3 Qualitative assessment'", "CAPTION TAB3.png": "'\\n\\n## Table 3 Example of networked quantum'"} \ No newline at end of file diff --git a/extracted_captions/tang2020bursting.json b/extracted_captions/tang2020bursting.json new file mode 100644 index 0000000000000000000000000000000000000000..d2ddd1ff609b78022ac885959b148a2ee410cb1e --- /dev/null +++ b/extracted_captions/tang2020bursting.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1.: **Overview of actin filament disassembly in the presence of cofilin, coronin, and Aip1 using EM.** In all panels, actin is polymerized for 1 min before any additional steps. \\\\(A\\\\), a 10 \\\\(\\\\mu\\\\)m solution of G-actin was induced to polymerize by adding salt and ATP. The sample was fixed and prepared for visualization in the electron microscope. The _upper panels_ show low-magnification valves, whereas the _lower panels_ are at higher magnification. \\\\(B\\\\), electron micrographs of the products produced by adding 5 \\\\(\\\\mu\\\\)m cofilin, 2 \\\\(\\\\mu\\\\)m coronin, and 0.2 \\\\(\\\\mu\\\\)m Aip1 to filaments for 60 s. The _upper panels_ show low-magnification views, whereas the _lower panels_ are at higher magnification. \\\\(C\\\\), image of 5 \\\\(\\\\mu\\\\)m cofilin, 2 \\\\(\\\\mu\\\\)m coronin, and 0.2 \\\\(\\\\mu\\\\)m Aip1 alone (no actin). \\\\(D\\\\), morphology of actin filaments that were incubated for an additional 60 s in the presence of 5 \\\\(\\\\mu\\\\)m cofilin and 0.2 \\\\(\\\\mu\\\\)m Aip1. \\\\(E\\\\), morphology of actin filaments that were incubated for an additional 60 s in the presence of 2 \\\\(\\\\mu\\\\)m coronin and 0.2 \\\\(\\\\mu\\\\)m Aip1. \\\\(F\\\\), morphology of actin filaments that were incubated for an additional 60 s in the presence of 5 \\\\(\\\\mu\\\\)m cofilin and 2 \\\\(\\\\mu\\\\)m Aip1.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: **Generation of stable hypertwist regions at low coilin concentration that are substrates for Aip1-induced bursting.** A. _c_conom facilitates the formation of hypertwist filaments at 1 \\\\(\\\\mu\\\\)m coilin. \\\\(\\\\&\\\\), bursting of hypertwist filament (marked as \\\\(\\\\&\\\\)) but not a neighboring normal twist filament (marked as \\\\(\\\\&\\\\)) upon Aip1 addition for 5 \\\\(\\\\&\\\\), covering of undecorated normal twist filaments upon addition of Aip1 for 5 \\\\(\\\\&\\\\).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **The bursting reaction proceeds with rapid kinetics to generate monomer and/or products incompetent to seed actin assembly.** A disassembly kinetics of 1 \\\\(\\\\mu\\\\)m actin was monitored by loss of FRET signal in the presence of various combinations of factors. \\\\(B\\\\), rate of monomer production was monitored by quenching of Oregon green 488 actin by vitamin D-binding protein over time. \\\\(C\\\\), pyrene-based seeding reaction, using the products of reaction. \\\\(A\\\\) showed that products of the bursting reaction are not competent to seed new actin assembly. \\\\(D\\\\), dose response curves of actin in the presence of 0.75 \\\\(\\\\mu\\\\)m actin and 0.1 \\\\(\\\\mu\\\\)m Aip1 show increasing rates of disassembly with increasing amounts of caffein. \\\\(E\\\\), lower ratios of coronin. _C_ellin produces more efficient disassembly, whereas higher ratios may stabilize actin filaments. \\\\(F\\\\), initial rates (120 \\\\(\\\\lesssim\\\\)) of disassembly with varying concentrations of Aip1 and fixed amounts of caffein and coronin show increased rates of disassembly with increasing amounts of Aip1. Representative data from \\\\(n\\\\) = 3 experiments are shown.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Estimation of apparent critical concentrations of actin assembly in the presence of actin disassembly factors. Fluorescent micrographs showing the onset of filament formation in the presence of actin disassembly factors as the actin concentration increases from left to right. The concentrations were 20 \\\\(\\\\mu\\\\)m cofilin, 2 \\\\(\\\\mu\\\\)m coranin, and 5 \\\\(\\\\mu\\\\)m Aip1. Actin concentrations are in \\\\(\\\\mu\\\\)m and listed above each column. Scale bar in the lower right panel applies to all the images. The experiments were repeated three times with identical results. Approximate critical concentrations estimated from these results are listed in Table 1.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Long stretches of actin polymer disintegrate in the presence of coronin, cofilin, and Alip1.**_A_, actin filaments, 1 min old, were mixed with cofilin, coronin, and Alip1 and then fixed at the times indicated. The panels are images of the reaction products at various times after adding the triple mix of disassembly factors. The upper panels show two-magnification views, whereas the lower panels are at higher magnification. _Scale bars_ are 100 nm. Segments of actin polymer lose coherent structure as early as 5 s. 8, actin filament structure is intact in the presence of cofilin and coronin alone. Each consecutive upper panel shows higher-magnification images of actin filaments that were polymerized in the presence of cofilin and coronin for 1 min. Adding Alip1 to minute-old filaments copolymerized with cofilin and coronin results in the rapid disintegration of polymer into monomers and short oligomers. Each consecutive lower panel shows higher-magnification images of the products of the reaction. _Scale bars_ are 200 nm.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Cooperative changes in filament structure in the presence of cofilin and coronin.**_A_, electron micrograph of actin filaments assembled in the presence of cofilin and coronin for 90 s. _Red_ and _blue_ dots along the filaments mark the cross-over points. \\\\(B\\\\), one section of an electron tomogram showing the cross-over points on two different filaments, \\\\(C\\\\), histogram of cross-over distances seen in actin filaments polymerized in the presence of cofilin and coronin for 90 s. \\\\(I\\\\) show two different populations. \\\\(D\\\\), two populations of filament widths form in the presence of cofilin and coronin. \\\\(E\\\\), relationship between cross-over distance and caliber shows that two populations of actin filaments form in the presence of cofilin and coronin. \\\\(F\\\\), length distribution of filaments that consist either entirely of normal or hypertwist. \\\\(G\\\\), histogram showing the percentage of hypertwist in 300 different filaments assembled in the presence of cofilin and coronin. \\\\(F\\\\), length and portion of hypertwist of the same 300 filaments. Each bar is one filament. \\\\(L\\\\), an example of a filament containing both normal twist and hypertwist. Cross-over distances were measured along the filament in the direction of the arrow and used to generate the graph for filament 1 in panel \\\\(J\\\\), which shows the sharp transitions between normal and hypertwist configurations in two representative filaments. _Scale bars_ are 25 nm (_A_) and 50 nm (_B_ and _I_).\\n\\n'", "CAPTION FIG6.png": "'Figure 6.: **Generation of long stretches of hypertwist filaments requires coronin and depends on filament age.**_A_, hypertwist (marked by _H_) and normal twist (marked by _H_) filaments were readily formed when actin is polymerized in the presence of coronin and cofilin. \\\\(B\\\\), generation of long stretches of hypertwist (marked by _H_) and normal twist (marked by _N_) regions when cofilin was added to 90-s-old filaments polymerized in the presence of coronin. \\\\(C\\\\), absence of long stretches of hypertwist regions when cofilin was added to 30-min-old filaments polymerized in the presence of coronin. _Left_, severing (marked by _S_) can readily be detected at naked normal twist filaments. _Right_, thicker normal twist filaments corresponding to coronin-decorated actin can also be found. _Lower left graph_ shows that the presence of coronin cannot facilitate the generation hypertwist filaments in aged filaments. _Lower right graph_ shows that coronin decorates aged filaments and prevents them from severing. \\\\(D\\\\), absence of long stretches of hypertwist regions when cofilin was added to 30-min-old filaments (_lower right graph_). Severing (marked by _S_) of naked normal twist filaments (marked by _N_) is readily observed.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Coronin and cofilin accelerate phosphate release from F-actin.** A rates of phosphate release from actin filaments in the presence of increasing concentrations of cofilin alone. \\\\(\\\\beta_{c}\\\\) ceronin alone show accelerated phosphate release by the individual factors. \\\\(\\\\mathcal{C}\\\\), together, cofilin and coronin cooperate to enhance phosphate release of more than each individual factor. \\\\(\\\\mathcal{D}\\\\), comparison of rates of phosphate release in the presence of actin alone, 4 \\\\(\\\\mu\\\\)m cofilin, 4 \\\\(\\\\mu\\\\)m coronin, or a combination of the two. Representative data from \\\\(n=4\\\\) experiments are shown.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: **Aip1 disintegrates hypertwisted polymer.****Actin was prepolymerized in the presence of coronin and cofilin for 90 s and then spiked with Aip1 for 5 s before fixing.**_A_, four representative images of actin filaments polymerized in the presence of coronin and cofilin for 90 s are shown. Segments of polymer with normal twist are marked \\\\(N\\\\), and hypertwisted polymer is marked \\\\(H\\\\). \\\\(B\\\\), actin, first assembled as in Fig. 6d, 5 s after adding Aip1. The letter \\\\(b\\\\) marks regions where polymer segments are disintegrating. Unmarked filaments are not obviously bursting, but cross-over distances cannot be assigned. _Scales bars_ are 200 nm.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: **Generation of long stretches of hypertwist regions at substochimetric actin to cofilin ratio that are substrates for Aip1-induced bursting.** A actin was copolymerized with coronin and cofilin at a cofilin-to-actin ratio of 0.4. Long and short stretches of hypertwist regions are readily detected. By bursting of hypertwist regions at 10 s after Aip1 addition and severing of naked normal twist regions by 60 s.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1**\\n\\nEffect of disassembly factors on the apparent critical concentration of actin assembly'"} \ No newline at end of file diff --git a/extracted_captions/tang2020catastrophic.json b/extracted_captions/tang2020catastrophic.json new file mode 100644 index 0000000000000000000000000000000000000000..47828cc7dafd1422135f881605dc73c8dd7837c1 --- /dev/null +++ b/extracted_captions/tang2020catastrophic.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1.: **Overview of actin filament disassembly in the presence of cofilin, coronin, and Aip1 using EM.** In all panels, actin is polymerized for 1 min before any additional steps. \\\\(A\\\\) a 10 \\\\(\\\\mu\\\\)m solution of -actin was induced to polymerize by adding salt and ATP. The sample was fixed and prepared for visualization in the electron microscope. The upper panels show low-magnification views, whereas the lower panels are at higher magnification. \\\\(B\\\\), electron micrographs of the products produced by adding 5 \\\\(\\\\mu\\\\)m cofilin, 2 \\\\(\\\\mu\\\\)m coronin, and 0.2 \\\\(\\\\mu\\\\)m Aip1 to filaments for 60 s. The _upper panels_ show low-magnification views, whereas the _lower panels_ are at higher magnification. \\\\(C\\\\), image of 5 \\\\(\\\\mu\\\\)m cofilin, 2 \\\\(\\\\mu\\\\)m coronin, and 0.2 \\\\(\\\\mu\\\\)m Aip1 alone (no actin). \\\\(D\\\\), morphology of actin filaments that were included for an additional 60 s in the presence of 5 \\\\(\\\\mu\\\\)m cofilin and 0.2 \\\\(\\\\mu\\\\)m Aip1. \\\\(E\\\\), morphology of actin filaments that were incubated for an additional 60 s in the presence of 2 \\\\(\\\\mu\\\\)m coronin and 0.2 \\\\(\\\\mu\\\\)m Aip1. \\\\(F\\\\), morphology of actin filaments that were incubated for an additional 60 s in the presence of 5 \\\\(\\\\mu\\\\)m cofilin and 2 \\\\(\\\\mu\\\\)m coronin.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: **Generation of stable hypertwist regions at low cofilin concentration that are substrates for Aip1-induced bursting.** A. coronin facilitates the formation of hypertwist filaments at 1 \\\\(\\\\mu\\\\)m cofilin. **B.** bursting of hypertwist filament (marked as _B_) but not a neighboring normal twist filament (marked as _N_) upon Aip1 addition for 5 s, _C._, sweeping of undecorated normal twist filaments upon addition of Aip1 for 5 s.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **The bursting reaction proceeds with rapid kinetics to generate monomer and/or products incompetent to seed actin assembly.** A disassembly kinetics of 1 \\\\(\\\\mu\\\\)m actin was monitored by loss of FRET signal in the presence of various combinations of factors. \\\\(B\\\\), rate of monomer production was monitored by quenching of Oregon green 488 actin by vitamin D-binding protein over time. \\\\(C\\\\), pyrene-based seeding reaction, using the products of reaction. \\\\(A\\\\) showed that products of the bursting reaction are not competent to seed new actin assembly. \\\\(D\\\\), dose response curves of actin in the presence of 0.75 \\\\(\\\\mu\\\\)m actin and 0.1 \\\\(\\\\mu\\\\)m Aip1 show increasing rates of disassembly with increasing amounts of caffein. \\\\(E\\\\), lower ratios of coronin. _C_ellin produces more efficient disassembly, whereas higher ratios may stabilize actin filaments. \\\\(F\\\\), initial rates (120 \\\\(\\\\lesssim\\\\)) of disassembly with varying concentrations of Aip1 and fixed amounts of caffein and coronin show increased rates of disassembly with increasing amounts of Aip1. Representative data from \\\\(n\\\\) = 3 experiments are shown.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Estimation of apparent critical concentrations of actin assembly in the presence of actin disassembly factors. Fluorescent micrographs showing the onset of filament formation in the presence of actin disassembly factors as the actin concentration increases from left to right. The concentrations were 20 \\\\(\\\\mu\\\\)m cofilin, 2 \\\\(\\\\mu\\\\)m coranin, and 5 \\\\(\\\\mu\\\\)m Aip1. Actin concentrations are in \\\\(\\\\mu\\\\)m and listed above each column. Scale bar in the lower right panel applies to all the images. The experiments were repeated three times with identical results. Approximate critical concentrations estimated from these results are listed in Table 1.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Long stretches of actin polymer disintegrate in the presence of coronin, cofilin, and Alip1.**_A_, actin filaments, 1 min old, were mixed with cofilin, coronin, and Alip1 and then fixed at the times indicated. The panels are images of the reaction products at various times after adding the triple mix of disassembly factors. The upper panels show two-magnification views, whereas the lower panels are at higher magnification. _Scale bars_ are 100 nm. Segments of actin polymer lose coherent structure as early as 5 s. 8, actin filament structure is intact in the presence of cofilin and coronin alone. Each consecutive upper panel shows higher-magnification images of actin filaments that were polymerized in the presence of cofilin and coronin for 1 min. Adding Alip1 to minute-old filaments copolymerized with cofilin and coronin results in the rapid disintegration of polymer into monomers and short oligomers. Each consecutive lower panel shows higher-magnification images of the products of the reaction. _Scale bars_ are 200 nm.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Cooperative changes in filament structure in the presence of cofilin and coronin.**_A_, electron micrograph of actin filaments assembled in the presence of cofilin and coronin for 90 s. _Red_ and _blue_ dots along the filaments mark the cross-over points. \\\\(B\\\\), one section of an electron tomogram showing the cross-over points on two different filaments, \\\\(C\\\\), histogram of cross-over distances seen in actin filaments polymerized in the presence of cofilin and coronin for 90 s. \\\\(I\\\\) show two different populations. \\\\(D\\\\), two populations of filament widths form in the presence of cofilin and coronin. \\\\(E\\\\), relationship between cross-over distance and caliber shows that two populations of actin filaments form in the presence of cofilin and coronin. \\\\(F\\\\), length distribution of filaments that consist either entirely of normal or hypertwist. \\\\(G\\\\), histogram showing the percentage of hypertwist in 300 different filaments assembled in the presence of cofilin and coronin. \\\\(F\\\\), length and portion of hypertwist of the same 300 filaments. Each bar is one filament. \\\\(L\\\\), an example of a filament containing both normal twist and hypertwist. Cross-over distances were measured along the filament in the direction of the arrow and used to generate the graph for filament 1 in panel \\\\(J\\\\), which shows the sharp transitions between normal and hypertwist configurations in two representative filaments. _Scale bars_ are 25 nm (_A_) and 50 nm (_B_ and _I_).\\n\\n'", "CAPTION FIG6.png": "'Figure 6.: **Generation of long stretches of hypertwist filaments requires coronin and depends on filament age.**_A_, hypertwist (marked by _H_) and normal twist (marked by _H_) filaments were readily formed when actin is polymerized in the presence of coronin and cofilin. \\\\(B\\\\), generation of long stretches of hypertwist (marked by _H_) and normal twist (marked by _N_) regions when cofilin was added to 90-s-old filaments polymerized in the presence of coronin. \\\\(C\\\\), absence of long stretches of hypertwist regions when cofilin was added to 30-min-old filaments polymerized in the presence of coronin. _Left_, severing (marked by _S_) can readily be detected at naked normal twist filaments. _Right_, thicker normal twist filaments corresponding to coronin-decorated actin can also be found. _Lower left graph_ shows that the presence of coronin cannot facilitate the generation hypertwist filaments in aged filaments. _Lower right graph_ shows that coronin decorates aged filaments and prevents them from severing. \\\\(D\\\\), absence of long stretches of hypertwist regions when cofilin was added to 30-min-old filaments (_lower right graph_). Severing (marked by _S_) of naked normal twist filaments (marked by _N_) is readily observed.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Coronin and cofilin accelerate phosphate release from F-actin-\\\\(\\\\Delta\\\\), rates of phosphate release from actin filaments in the presence of increasing concentrations of cofilin alone. \\\\(\\\\delta_{r}\\\\) coronin alone show accelerated phosphate release by the individual factors. \\\\(C\\\\), together, cofilin and coronin cooperate to enhance phosphate release of more than each individual factor. \\\\(\\\\Omega_{r}\\\\) comparison of rates of phosphate release in the presence of actin alone, \\\\(4\\\\) \\\\(\\\\mu\\\\)m coronin, or a combination of the two. Representative data from \\\\(n=4\\\\) experiments are shown.**\\n\\n'", "CAPTION FIG8.png": "'Figure 8: **Aip1 disintegrates hypertwisted polymer.****Actin was prepolymerized in the presence of coronin and cofilin for 90 s and then spiked with Aip1 for 5 s before fixing.**_A_, four representative images of actin filaments polymerized in the presence of coronin and cofilin for 90 s are shown. Segments of polymer with normal twist are marked \\\\(N\\\\), and hypertwisted polymer is marked \\\\(H\\\\). \\\\(B\\\\), actin, first assembled as in Fig. 6d, 5 s after adding Aip1. The letter \\\\(b\\\\) marks regions where polymer segments are disintegrating. Unmarked filaments are not obviously bursting, but cross-over distances cannot be assigned. _Scales bars_ are 200 nm.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: **Generation of long stretches of hypertwist regions at substochimetric actin to cofilin ratio that are substrates for Aip1-induced bursting.** A actin was copolymerized with coronin and cofilin at a cofilin-to-actin ratio of 0.4. Long and short stretches of hypertwist regions are readily detected. By bursting of hypertwist regions at 10 s after Aip1 addition and severing of naked normal twist regions by 60 s.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1**\\n\\nEffect of disassembly factors on the apparent critical concentration of actin assembly'"} \ No newline at end of file diff --git a/extracted_captions/tsengRoleTimingAnalogical2008.json b/extracted_captions/tsengRoleTimingAnalogical2008.json new file mode 100644 index 0000000000000000000000000000000000000000..b7262924dded4caef98e10db8b36e26233013356 --- /dev/null +++ b/extracted_captions/tsengRoleTimingAnalogical2008.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1**: **A**'", "CAPTION FIG2.png": "'\\n\\n**Figure 1:** Experiment design for all four conditions'", "CAPTION FIG3.png": "'Figure 3: Example solutions \\\\(-\\\\) (a) rate of heating/cooling solution and (b) multi-category solution\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Average number of solution per participant\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Figure 1.**_M_**Figure 2.**_M_**Figure 3.**_M_**Figure 4.**_M_**Figure 5.**_M_**Figure 6.**_M_**Figure 7.**_M_**Figure 8.**_M_**Figure 9.**_M_**Figure 10.**_M_**Figure 11.**_M_**Figure 12.**_M_**Figure 13.**_M_**Figure 14.**_M_**Figure 15.**_M_**Figure 16.**_M_**Figure 17.**_M_**Figure 18.**_M_**Figure 19.**_M_**Figure 19.**_M_**Figure 11.**_M_**Figure 12.**_M_**Figure 13.**_M_**Figure 14.**_M_**Figure 15.**_M_**Figure 16.**_M_**Figure 17.**_M_**Figure 18.**_M_**Figure 19.**_M_**Figure 19.**_M_**Figure 12.**_M_**Figure 13.**_M_**Figure 14.**_M_**Figure 15.**_M_**Figure 16.**_M_**Figure 17.**_M_**Figure 18.**_M_**Figure 19.**_M_**Figure 19.**_M_**Figure 13.**_M_**Figure 14.**_M_**Figure 15.**_M_**Figure 16.**_M_**Figure 17.**_M_**Figure 18.**_M_**Figure 19.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{Table 1 Average number of Idenu per participant in each category.} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Average number of Idenu per participant in each category.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/tweten2017actin.json b/extracted_captions/tweten2017actin.json new file mode 100644 index 0000000000000000000000000000000000000000..760499fc7214aeb30902984c61204819e90b8b97 --- /dev/null +++ b/extracted_captions/tweten2017actin.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Axisymmetric diagram of the model. (a) The membrane is separated from the cell wall in the initial stage, in which the membrane forms a shallow dimple. The hatched sections of the cell wall and membrane are not included in the model but are shown for clarity. (b) Free-body diagram of the cell wall, membrane, and actin network. \\\\(P_{0}\\\\) is the turgor pressure; \\\\(f_{W}\\\\) is the reaction force density of the cell wall; and \\\\(f_{A}\\\\) is the force density applied by (and on) the actin network. The cell wall force \\\\(f_{W}\\\\) is balanced by a shear force \\\\(F_{W\\\\text{-}\\\\text{EC}}\\\\) and bending moment \\\\(M_{W\\\\text{-}\\\\text{ILC}}\\\\) at the boundary. The forces \\\\(f_{M}\\\\) and \\\\(f_{\\\\text{CCP}}\\\\) are omitted for clarity.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: Comparisons of (a) the mean profiles, (b) Gaussian fits of the mean profiles, and (c) the resulting wall reaction forces using the displacement sets defined in Table II. The curves found using the \u201coutlier rejection\u201d displacement sets are represented by solid lines, and those found using the \u201csymmetric selection\u201d displacement sets are represented by dashed lines.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: Comparison of the Gaussian fit of the mean displacement profile [see Fig. 2(c)] and the integrated polynomial fit of the mean displacement profile in the arc length domain. The integration of the polynomial fit is only continuous to a radius of \\\\(r\\\\approx 46\\\\) nm.\\n\\n'", "CAPTION FIG12.png": "'Figure 12: Comparison of actin force \\\\(f_{A}\\\\) obtained from growth simulations for increasing radii of the actin patch. (a) Normalized actin forces for linear strains. (b) Full-magnitude comparisons of actin forces for nonlinear strains.\\n\\n'", "CAPTION FIG13.png": "'FIG. 13. Comparison of actin force \\\\(f_{A}\\\\) from actin growth simulations for nonlinear strains and polymerization region thicknesses of \\\\(Z_{p}=20\\\\), 25, and 30 nm. The corrected growth profile \\\\(g(r)\\\\) from Fig. 9(a) was used for all three simulations. \"Estimated\" denotes force profile obtained from experimental data.\\n\\n'", "CAPTION FIG14.png": "'Figure 14: (a) Growth profiles used for simulating actin forces. The tanh curves are defined by Eq. (23) with parameter values of \\\\(\\\\eta=0.16\\\\) nm\\\\({}^{-1}\\\\) and \\\\(r_{0}=42.0\\\\), 52.5, and 63.0 nm. The atan curve is based on Eq. (A4) with \\\\(r_{0}=39.5\\\\) nm, and \\\\(0.1r_{0}\\\\) is replaced with a fitted value of 14.5 nm. (b) Comparison of simulated actin force \\\\(f_{A}\\\\) using the growth profiles from (a) (broken lines), and the actin force density estimated from experiments (solid line).\\n\\n'", "CAPTION FIG15.png": "'Figure 15: (a) Comparison between the cell wall reaction force and turgor pressure (\\\\(f_{W}+P_{0}\\\\)) using elastic continuum (solid line) and linear-spring bed (dashed line) models of the cell wall. The turgor pressure in both simulations was \\\\(P_{0}=0.5\\\\) MPa. (b) Sensitivity analysis comparing the continuum and linear-spring bed cell wall models. The forces \\\\(f_{W}+P_{0}\\\\) from the continuum model with \\\\(P_{0}=0.5\\\\) MPa (solid line) and \\\\((f_{W}+P_{0})\\\\pm\\\\alpha_{7}\\\\) (dotted lines) are shown. The sensitivity \\\\(\\\\alpha_{7}\\\\) is calculated from Eq. (13).\\n\\n'", "CAPTION FIG16.png": "'FIG. 16. Comparison of actin force profiles \\\\(f_{\\\\Lambda}\\\\) estimated from experiments for increasing radii of the actin network. The model from Fig. 4 was used to estimate the actin force for \\\\(R_{\\\\Lambda}=100\\\\), 150, and 200 nm with a matching cell wall and membrane radius in each case.\\n\\n'", "CAPTION FIG17.png": "'Figure 17: Study of the effect of the bending modulus \\\\(\\\\kappa_{\\\\rm CGP}\\\\) on the actin force \\\\(f_{\\\\Lambda}\\\\) estimated from experiments. The CGP forces were generated from the hyperbolic tangent transition from Eq. (11) with \\\\(\\\\gamma=0.1\\\\) and \\\\(s_{\\\\oplus}=46\\\\) nm.\\n\\n'", "CAPTION FIG18.png": "'FIG. 18. Study of the effect of center of the transition radius \\\\(x_{0}\\\\) of the CGP on the experimentally estimated actin force \\\\(f_{A}\\\\). The CGP forces were generated from the hyperbolic tangent transition from Eq. (11) with \\\\(\\\\gamma=0.1\\\\).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: (a) Electron microscopy (EM) images of endocytic profiles. The left profile shows the initial stage of endocytosis, in which the membrane forms a shallow dimple. Scale bars indicate 100 nm. Reprinted with permission from Kukulski _et al._[8]. (b) Selected membrane profiles from the EM experiments [8]. Vertical displacement of membrane is \\\\(w\\\\). (c) Mean membrane profile of the initial stage of endocytosis (solid line) and Gaussian fit (dashed line).\\n\\n'", "CAPTION FIG3.png": "'Figure 3: (a) Possible profiles of the bending modulus in the CGP patch. For the hyperbolic tangent profiles [Eq. (11)], the center of the transition occurs at \\\\(s_{0}=46\\\\) nm; \\\\(\\\\Delta s\\\\) is in nm and \\\\(\\\\gamma\\\\) is given in units of nm\\\\({}^{-1}\\\\). (b) Force densities \\\\(f_{\\\\rm CGP}\\\\) resulting from the bending modulus profiles. A negative force indicates that CGP are pulling the membrane into the cell.\\n\\n'", "CAPTION FIG4.png": "'FIG. 4. (a) Axisymmetric continuum mechanics model used to estimate the wall reaction force \\\\(f_{W}\\\\) during the initial stages of endocytosis. The interface between the cell wall and membrane is frictionless. A frictionless boundary condition (BC) with zero displacement in the \\\\(z\\\\) direction is applied to the top surface of the cell wall. A frictionless BC with zero displacement in the radial direction is applied to the outer radius of both the cell wall and the membrane. The membrane is displaced vertically (in the \\\\(z\\\\) direction) into the cell wall to create the wall force. The wall force on the membrane is estimated from \\\\(f_{W}=-\\\\sigma_{zz}\\\\). (b) The resulting stresses in the membrane and cell wall estimated by finite element solution of the continuum mechanics model.\\n\\n'", "CAPTION FIG5.png": "'model used to obtain the actin force \\\\(f_{A}\\\\) from a growth distribution \\\\(g(r)\\\\) taken from the linear theory of Sec. III.2. The actin network is modeled as a hemisphere with radius \\\\(R_{A}=100\\\\) nm in contact with a rigid, freely sliding boundary at the membrane surface. The network is not permitted to pull away from the surface. (a) The growth distribution in the model after a simulation time of 0.65 time units. (b) The resulting stress \\\\(\\\\sigma_{ZZ}\\\\) in the FE model after a simulation time of 0.65 time units. Positive stress is tensile.\\n\\nFigure 5: Results from FE simulation of the continuum mechanics model used to obtain the actin force \\\\(f_{A}\\\\) from a growth distribution \\\\(g(r)\\\\) taken from the linear theory of Sec. III.2. The actin network is modeled as a hemisphere with radius \\\\(R_{A}=100\\\\) nm in contact with a rigid, freely sliding boundary at the membrane surface. The network is not permitted to pull away from the surface. (a) The growth distribution in the model after a simulation time of 0.65 time units. (b) The resulting stress \\\\(\\\\sigma_{ZZ}\\\\) in the FE model after a simulation time of 0.65 time units. Positive stress is tensile.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: (a) Wall reaction force plus turgor pressure (solid line) and with inclusion of the internal membrane forces (dashed line). (b) Total actin forces including the CGP force estimated from the Gaussian transition [solid line, Eq. (12)] and the hyperbolic tangent transitions [broken lines, Eq. (11)]; \\\\(\\\\Delta x\\\\) is in nm and \\\\(\\\\gamma\\\\) is given in units of nm\\\\({}^{-1}\\\\). (c) Total actin force \\\\(f_{A}\\\\) with (dotted line) and without (dashed line) \\\\(f_{\\\\rm CGP}\\\\) obtained from Eq. (11) with \\\\(\\\\gamma=0.10\\\\) nm\\\\({}^{-1}\\\\) (dotted line). All curves generated from Eq. (11) have a transition radius of \\\\(s_{\\\\Omega}=46\\\\) nm.\\n\\n'", "CAPTION FIG7.png": "'FIG. 7. Growth distributions using the linearized growth theory (LGT). The dashed line is the growth distribution \\\\(g\\\\) calculated using LGT directly from the estimated actin force \\\\(f_{\\\\Lambda}\\\\). The dotted line is a growth distribution \\\\(A\\\\,\\\\Delta g\\\\), where \\\\(\\\\Delta g\\\\) is the growth distribution resulting from using the difference between the simulated and estimated actin force as the input to the LGT. The solid line is the linearly corrected growth distribution \\\\(g^{\\\\prime}=A\\\\,\\\\Delta g\\\\,+\\\\,g\\\\), with \\\\(A=35\\\\).\\n\\n'", "CAPTION FIG8.png": "'FIG. 8. Comparison of simulated (dotted lines) force density generated by FE code from LGT growth profiles (Fig. 7), with force density estimated from experimental observations [8] (solid lines). (a) Actin force density resulting from linear growth profile \\\\(g\\\\). (b) Actin force density resulting from corrected growth profile \\\\(g^{\\\\prime}\\\\).\\n\\n'", "CAPTION FIG9.png": "'Figure 9: (a) Corrected fully nonlinear growth profile \\\\(g\\\\) from a general growth calculation (solid line), compared with normalized linearly corrected growth profile \\\\(g^{r}\\\\) (dashed line). Calculations performed with a shear modulus of \\\\(4\\\\mu_{A}=80\\\\) kPa. (b) Direct, full-magnitude comparison of actin forces estimated from the experimental profile (solid line) and simulated using the nonlinear-strain growth model (broken lines).\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## Chapter 1 Introduction'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB6.png": "'TABLE VI. Effect of CGP parameters on the total required actin force \\\\(F_{A}\\\\) and the endocytic width \\\\(R_{W}\\\\). \\\\(F_{A}\\\\) is calculated by integrating the force \\\\(f_{A}\\\\) from \\\\(r=0\\\\) to \\\\(R_{W}\\\\). \\\\(R_{W}\\\\) is defined as the location where the experimentally estimated actin force \\\\(f_{A}=0\\\\).\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/uzziAtypicalCombinationsScientific2013.json b/extracted_captions/uzziAtypicalCombinationsScientific2013.json new file mode 100644 index 0000000000000000000000000000000000000000..eac74233f99277b337a100b0b25a0ee710d9ffa0 --- /dev/null +++ b/extracted_captions/uzziAtypicalCombinationsScientific2013.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "\"\\n\\n**Fig. 1. Novelty and conventionality in science**. For a sample paper, (**A**) shows the distribution of \\\\(z\\\\) scores for that paper's journal pairings. The \\\\(z\\\\) score shows how common a journal pairing is as compared to chance. For each paper, we take two summary measures its median \\\\(z\\\\) score, capturing the paper's central tendency in combining prior work, and the 10th-percertile \\\\(z\\\\) score, capturing the paper's journal pairings that are relatively unusual. For the population of papers, we then consider these values across all papers in the WOS published in the 1980s or 1990s. (**B**) considers the median \\\\(z\\\\) scores and shows that the vast majority of papers displays a high propensity for conventionality; in the 1980s and 1990s, fewer than 4% of papers have median \\\\(z\\\\) scores below 0 and more than 50% of papers have median \\\\(z\\\\) scores above 64. (**C**) considers the 10th-percertile \\\\(z\\\\) scores, which further suggest a propensity for conventionality; only 41% of papers in the 1980s and 1990s have a 10th-percertile \\\\(z\\\\) score below 0. Overall, by these measures, science rarely draws on atypical pairings of prior work.\\n\\n\"", "CAPTION FIG2.png": "'\\n\\n**Fig. 2. The probability of a \"hit\" paper, conditional on novelty and conventionality.** This figure represents the probability of a paper being in the top 5% of the citation distribution conditional on two dimensions: whether a paper exhibits (i) high or low median conventionality and (ii) high or low tail novelty, as defined in the text. Papers that combine high median conventionality and high tail novelty are hits in 9.11 out of 100 papers, a rate nearly double the background rate of 5%. Papers that are high on one dimension only (high median conventionality or high tail novelty but not both) have hit rates about half as large. Papers with low median conventionality and low tail novelty have hit rates of only 2.05 out of 100 papers. The sample includes all papers published in the WOS from 1990 to 2000. The supplementary materials show similar findings when considering (i) all other decades from 1950 to 2000; (ii) hit papers defined as the top **1** or 10% by citations; and (iii) analyses controlling for field and other observable differences across papers, hinting at auniversality of these relationships for scientific work. The difference in the hit probabilities for each category is statistically significant (\\\\(P<0.00001\\\\)). The percentage of WOS papers in each category are: 6.7% (green bar), 23% (gold bar), 26% (red bar), and 44% (blue bar).\\n\\n'", "CAPTION FIG3.png": "\"distinct (solo versus pair \\\\(P=0.016\\\\), pair versus team \\\\(P=0.001\\\\), team versus solo \\\\(P<0.001\\\\)). In contrast, each team size shows similar distributions for median conventionality [(**B**), K-5 tests indicate no statistically significant differences]. These findings suggest that a distinguishing feature of teamwork, and teams' exceptional impact, reflects a tendency to incorporate novelty.\\n\\nFig. 3: **Authorship structure, novelty, and conventionality.** Team-authored papers are more likely to incorporate tail novelty but without sacrificing a central tendency for high conventionality. Papers introduce tail novelty (a 10th-percentile \\\\(z\\\\) score less than 0) in 36.2, 39.9, and 49.7% of cases for solo authors, dual authors, and three or more authors, respectively (**A**). K-5 tests confirm that the distributions of tail novelty are distinct (solo versus pair \\\\(P=0.016\\\\), pair versus team \\\\(P=0.001\\\\), team versus solo \\\\(P<0.001\\\\)). In contrast, each team size shows similar distributions for median conventionality [(**B**), K-5 tests indicate no statistically significant differences]. These findings suggest that a distinguishing feature of teamwork, and teams\u2019 exceptional impact, reflects a tendency to incorporate novelty.\\n\\n\"", "CAPTION FIG4.png": "'85th to 95th percentile of median conventionally, after which the relationship reverses. Third, larger teams obtain higher impact given the right mix of tail novelty and median conventionality. Nonetheless, at low levels of median convention and tail novelty, even teams have low impact, further emphasizing the fundamental relationship between novelty, conventionality, and impact in science.\\n\\nFig. 4: **Novel and conventional combinations in the production of science.** (**A** to **C** The interplay between tail novelty, median conventionality, and hit paper probabilities shows remarkable empirical regularities. First, high tail novelty papers have higher impact than few tail novelty papers at (i) any level of conventionality and (ii) regardless of authorship structure. Second, increasing median conventionality is associated with higher impact up to the 85th to 95th percentile of median conventionality, after which the relationship reverses. Third, larger teams obtain higher impact given the right mix of tail novelty and median conventionality. Nonetheless, at low levels of median convention and tail novelty, even teams have low impact, further emphasizing the fundamental relationship between novelty, conventionality, and impact in science.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/vamvakoussiBridgingGapDense2012.json b/extracted_captions/vamvakoussiBridgingGapDense2012.json new file mode 100644 index 0000000000000000000000000000000000000000..362e231d3c7d371297aa7a5f2d51d499008c29e8 --- /dev/null +++ b/extracted_captions/vamvakoussiBridgingGapDense2012.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Categorization of students based on the number of graxiciple-based explanations in the no successor items, by grade.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} Mean Scores and Standard Deviations of Performance in the Numbers-, NumberLine-, and Points-blocks, by Grade \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Mean Scores and Standard Deviations of Performance in the Numbers-, NumberLine-, and Points-blocks, by Grade'", "CAPTION TAB2.png": "\"TABLE 2\\n\\nMean Scores and Standard Deviations of Students' Performance in the Pre- and Post-test, by Grade and Text-type\"", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} Mean Scores, & Standard Deviations, and Median of Student\u2019 Total Scores in the Five \\\\\\\\ \\\\({}^{*}\\\\)No Successor\u201d Items, by Grade and Text-hype \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 3: Mean Scores, Standard Deviations, and Median of Student\u2019 Total Scores in the Five \\\\\\\\ \\\\({}^{*}\\\\)No Successor\u201d Items, by Grade and Text-hype'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{TABLE 4} \\\\\\\\ Frequency and Percent of Students Who Gave no Incorrect Answer for the Group of the Five \"No Successor\" Items, the Group of the Four Items About Numbers, as well as to the Points-item, by Grade and Text-type \\\\(\\\\langle\\\\)RL, nRL\\\\(\\\\rangle\\\\) \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Frequency and Percent of Students Who Gave no Incorrect Answer for the Group of the Five \"No Successor\" Items, the Group of the Four Items About Numbers, as well as to the Points-item, by Grade and Text-type \\\\(\\\\langle\\\\)RL, nRL\\\\(\\\\rangle\\\\)'"} \ No newline at end of file diff --git a/extracted_captions/vendettiFarOutThinkingGenerating2014.json b/extracted_captions/vendettiFarOutThinkingGenerating2014.json new file mode 100644 index 0000000000000000000000000000000000000000..419e4626a77f5951edbae38e2fe5fca16ee1d58d --- /dev/null +++ b/extracted_captions/vendettiFarOutThinkingGenerating2014.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1.** Example of a pair of scenes used in the picture-mapping task (Adapted from Markman & Gentner, 1993, and Tothill & Holyoak, 2000). Participants were shown both scenes at the same time. After 10 s, one of the objects in the top scene was highlighted (here, the umbrella), and participants had to indicate which object in the bottom scene \"goes with\" the highlighted object in the top scene. For each pair of scenes, the botoon picture included both a potential feaural match (here, the umbrella over the coffee stand) and a potential relational match (here, the newspaper the woman is holding over her head, which performs a function similar to the umbrella\\'s in the top scene).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Results from Experiment 1a and Experiment 1b: mean proportion of relational objects chosen in the picture-mapping task as a function of the semantic distance between word pairs in the verbal analogy task. The asterisk indicates a significant difference between conditions (\\\\(p<.01\\\\)). Error bars denote standard errors of the mean.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/wangEchoChamberEffect2021.json b/extracted_captions/wangEchoChamberEffect2021.json new file mode 100644 index 0000000000000000000000000000000000000000..4f79fbd1969458fec2f7b4f3973415d87f94f837 --- /dev/null +++ b/extracted_captions/wangEchoChamberEffect2021.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Research design. A NOWA: analysis of variance\\n\\n'", "CAPTION FIG10.png": "'Figure 10: The proportion of civility and incivility in different kinds of interactions in retweets (left) and comments (right).\\n\\n'", "CAPTION FIG11.png": "'Figure 11: The distribution of sentiment values in retweets (left) and comments (right) containing information-seeking, information-sharing, and no information content. The \\\\(\\\\mathbf{x}\\\\)-symbols and circles represent means and outliers of the sentiment values. respectively. A NOVA : analysis of variance.\\n\\n'", "CAPTION FIG12.png": "'Figure 12: The distribution of sentiment values for retweets (left) and comments (right) containing civil and uncivil content. The xsymbols and circles represent means and outliers of the sentiment values. respectively.\\n\\n'", "CAPTION FIG13.png": "'Figure 13: The proportion of civility and incivility in reweets (left) and comments (right) containing information-seeking, information-sharing, and no information content.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Figure 1**: **Satellite and computing**'", "CAPTION FIG3.png": "'Figure 3: Distribution of attitudes of users who retweeted (left) or commented on (right) the original rumor rebuttal tweets under the top six topics.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Retweetin user networks and commenting user networks of rumor rebuttal under the top six tonics.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Honophyll based on the users\u2019 attitudes in retweeting and commenting user networks toward rumor relutral under the top six topics.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Heterogeneity based on the distribution of users\u2019 attitudes within each community node in retweeting (left) and commenting (right) community networks toward tumor rebuttal under the top six topics. The symbols and circles represent means and outliers of the heterogeneity values, respectively.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Retweeting community networks and commenting community networks.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: The distribution of sentiment values across different kinds of interactions in retweets (left) and comments (right). The \\\\(\\\\times\\\\)symbols and circles represent means and outliers of the sentiment values, respectively. ANOVA: analysis of variance.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: The proportion of information-seeking, information-sharing, and no information content in different kinds of interactions in retweets (left) and comments (right).\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1.** Topic categories of original inner-neutral tweets.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/extracted_captions/wangHouseholdTransmissionSARSCoV22020.json b/extracted_captions/wangHouseholdTransmissionSARSCoV22020.json new file mode 100644 index 0000000000000000000000000000000000000000..94b5adfa7639576d79c649511c56c24bd88debc6 --- /dev/null +++ b/extracted_captions/wangHouseholdTransmissionSARSCoV22020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The'", "CAPTION FIG2.png": "'\\n\\n## References\\n\\nFig. 2: Epidemic distribution of the confirmed cases with coronavirus disease 2019 by onset of illness.\\n\\nLegend: Daily numbers of confirmed cases with coronavirus disease 2019 are plotted by date of onset of symptoms (\\\\(n\\\\)=132). Case 1 refers to the first infected case in a household, and the rest cases could be interpreted in the same manner.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table I**\\n\\n**Detected of SARS-CoV-2 among \\\\(\\\\pm\\\\) hexahedd members**'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB3.png": "'\\n\\n**Table 3**\\n\\nDetection of 548S-CoV-2 among close contacts in households.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/wardRoleSpecificityAbstraction2004.json b/extracted_captions/wardRoleSpecificityAbstraction2004.json new file mode 100644 index 0000000000000000000000000000000000000000..d780410f62708b3651a3372a0ca6ae27d5fe4ccf --- /dev/null +++ b/extracted_captions/wardRoleSpecificityAbstraction2004.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/extracted_captions/worknehSocialMediaProtest2020.json b/extracted_captions/worknehSocialMediaProtest2020.json new file mode 100644 index 0000000000000000000000000000000000000000..ba4c9bec38bf72ed342004a323e778f8d49bafe0 --- /dev/null +++ b/extracted_captions/worknehSocialMediaProtest2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: A timeline of protests, social movements, and reform in Ethiopia (2012\u20132018).\\n\\n'", "CAPTION FIG2.png": "\"\\n\\n**Figure 2.** Ethiopian social media users' domestic news sources.\\n\\n\"", "CAPTION FIG3.png": "\"\\n\\n**Figure 3.** Social media users' activation performance.\\n\\n\"", "CAPTION FIG4.png": "'Figure 4: A collage of contested Ethiopian ethnic nationalisms on Facebook. Top: A Facebook post by TL+ \\\\(\\\\lambda\\\\)m\\\\(\\\\beta\\\\)- or Bte Amhara, an Amhara nationalist community of 265,630 followers as of June 2019. The post laments the \u2018continued massacre\u2019 of ethnic Amharas by the Tigray People\u2019s Liberation Front (TPLF) and calls upon Amharas for a resistance movement. Bottom left: A very popular Tigrayan nationalist Facebook page for t\u00e1garus in Ethiopia and overseas, \u2018Tigrai Online\u2019 has 95,060 followers and advocates for \u2018Tigray First\u2019 agenda that pushes for the creation of an independent Tigray Republic. Bottom right: Followed by thousands of users, \u2018Ayyaantuu Oromyaa\u2019 is one of the many Facebook groups advocating for Oromo nationalism. In this post, Ethiopian Prime Minister, Abiy Ahmed, an Oromo, is depicted as a traitor who is likened to Menelik II, an Ethiopian Emperor who is perceived by many Oromo nationalists as an enemy of the Oromo people.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Perceptions of educated Ethiopians on different perspectives of social media\u2019s role on democratic values.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Educated Ethiopians\u2019 major concerns over the Ethiopian social media landscape.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'"} \ No newline at end of file diff --git a/extracted_captions/yang2022actin.json b/extracted_captions/yang2022actin.json new file mode 100644 index 0000000000000000000000000000000000000000..a63a5a11c4d9e14ed012e9fd47cd93aa33da9bd0 --- /dev/null +++ b/extracted_captions/yang2022actin.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'contain subdomains demarcated by \"seams\" (arrows) with disrupted polygonal clathrin pattern. **d** Dome-shaped subdomains (arrows) within a large mostly flat CCS in U2OS cells. **e** Percentages of different CCS shape categories in four different cells types. Hatched colors indicate a fraction of actin-associated CCS within a given category. **f** Projection area of CCSs of different shape categories in indicated cell types. Error bars, mean \\\\(\\\\pm\\\\)5D. Scale bars, 200 nm.\\n\\nFig. 1: **PREM analysis of CCS morphology.** **a** Two alternative models for actin-dependent invagination of CCSs. (Left) Edge pushing model. (Right) Apical pulling model. **b** A region of an unroofed U2OS cell showing flat (blue), dome-shaped (green) and spherical (orange) CCSs, some of which associate with branched actin networks (yellow). Arrows mark linear actin filaments from which branched networks originate. Inset shows a very small flat CCS. **c** Large flat CCSs in U2OS cells contain subdomains demarcated by \u201cseams\u201d (arrows) with disrupted polygonal clathrin pattern. **d** Dome-shaped subdomains (arrows) within a large mostly flat CCS in U2OS cells. **e** Percentages of different CCS shape categories in four different cells types. Hatched colors indicate a fraction of actin-associated CCSs within a given category. **f** Projection area of CCSs of different shape categories in indicated cell types. Error bars, mean \\\\(\\\\pm\\\\)5D. Scale bars, 200 nm.\\n\\n'", "CAPTION FIG2.png": "'the CCG (**b**, or localize between two neighboring CCGs (**m**), in all cases exhibiting minimal overhangs of actin filament ends over the CCG (red in **j**). **n** Percentage of the CCG area overlaid by branched actin filaments in 2D projection images within the actin-positive CCS subpopulations in the indicated cell types. Error bars, mean +- SD. **o** Fraction of the actin positive CCGs with any amount of branched actin extending over their lattice in 2D projection images in the indicated cell types. **p-r** STORM imaging of intact fixed PK2 cells stained with AlexaFluor488: phalloidin (cyan) and AlexaFluor405-647-labeled antibody against CHC (red). **p** Cell overview. **q** Zoomed region outlined by white box in **p**. **r** Region in **q** after Voronoi segmentation of clathrin localizations. Scale bars: 100 nm (**a**- **m**), 10 \u03bcm (**m**) and 500 nm (**q**, **r**).\\n\\nFig. 2: **Branched actin networks localize at the CCS perimeter with minimal overhangs with the clathrin lattice.****a\u2013e** Spherical CCGs in U2OS cells. Branched actin networks (yellow) can form a lateral comet tail (**a**, **b**, **c**), surround the CCS (**d**), for localize mainly underneath the CCS (**c**). **b** Branched actin network constructs an uncoated part (neck) of the CCS (clutrin coat is shown in orange). **d**, **e** Examples of actin filaments extending over the clathrin lattice (red); **e** shows a maximal\\n\\nobserved level of actin-CCG overlap. **f**-I**D**une-shaped CCGs in U2OS cells. Branched actin networks are laterally associated with CCSs (**f**, **g**) with occasional overhangs over the CCS periphery (**h** and **t**, red). **i** Branched actin networks located in part at the interface between dome-shaped (green) and flat (blue) CCSs. **j\u2013m** Flat CCGs in U2OS cells. Branched actin networks associate with the flat CCS (blue) margin (**j**), partially surround the CCS and separate it from the neighboring CCS (**k**), occupy a \u201cbay\u201d in the CCS (**l**), or localize between two neighboring CCGs (**m**), in all cases exhibiting minimal overhangs of actin filament ends over the CCS (red in **j**). **n** Percentage of the CCS area overlaid by branched actin filaments in 2D projection images within the actin-positive CCS subpopulations in the indicated cell types. Error bars, mean + SD. **o** Fraction of the actin positive CCGs with any amount of branched actin extending over their lattice in 2D projection images in the indicated cell types. **p-r** STORM imaging of intact fixed PK2 cells stained with AlexaFluor488: phalloidin (cyan) and AlexaFluor405-647-labeled antibody against CHC (red). **p** Cell overview. **q** Zoomed region outlined by white box in **p**. **r** Region in **q** after Voronoi segmentation of clathrin localizations. Scale bars: 100 nm (**a**- **m**), 10 \u03bcm (**m**) and 500 nm (**q**, **r**).\\n\\n'", "CAPTION FIG3.png": "\"network (arrow). Scale bars, 200 nm, **g** Percentage of different CCS shape categories in Pt62 cells in indicated conditions. Hatched colors indicate a fraction of CCSs associated with branched actin networks within the given category. Data for 10% FBS are the same as in Fig. 1. **h** Projection area of flat CCSs in Pt62 cells in indicated conditions. Error bars, mean +- SD; _m_*_p_ < 0.0001 (Kruskal-Wallis with posthoc Dunn's multiple comparison test).\\n\\nFig. 3: **Inhibition of the Arp2/3 complex results in accumulation of large flat CCSs at the expense of other CCS categories.****a\u2013d Control Pt62 cells cultured in the presence of 0.2% FBS and DMSO exhibit flat, dome-shaped and spherical CCSs, some of which are associated with branched actin networks (arrows).****e**, **f**Pt62 cells treated with 200 \u03bcM CK-666 in the presence of 0.2% FBS contain very large flat CCSs occasionally associated with small remaining patches of branched actin network (arrow). Scale bars, 200 nm, **g** Percentage of different CCS shape categories in Pt62 cells in indicated conditions. Hatched colors indicate a fraction of CCSs associated with branched actin networks within the given category. Data for 10% FBS are the same as in Fig. 1. **h** Projection area of flat CCSs in Pt62 cells indicated conditions. Error bars, mean +- SD; _m_*_p_ < 0.0001 (Kruskal-Wallis with posthoc Dunn\u2019s multiple comparison test).\\n\\n\"", "CAPTION FIG4.png": "\"sequences of RPF-CLC in U2O5 cells treated with either DMSO (**e**) or CK-666 (**g**) as explained for **b** and **c**. **f**, **h** Kymographs along lines in **e** and **g**, respectively. **i**-**I** Quantification of CCS dynamics showing CCS lifetimes (**i**, **k**) and fractions of 600 sec from traces (**g**, **l**) in U2O5 (**b**, **j**) and HeLa (**k**, **f**) cells treated with DMSO or CK-666. Solid and dashed lines in violin plots (**l**, **k**) indicate median and quartiles, respectively. For CK-666-treated U2O5 cells, the lines are not visible, because the median and the 75%-quartile are equal to 600 s and 25% quartile is 504 s. Error bars in **j** and **l** represent mean = SD. Black dots in **i**-**I** indicate average values per cell. **m**-**p** < 0.0001; **m**-**p** = 0.0005 (unpaired two-tailed \\\\(t\\\\) test with Welch's correction). Scale bars, 20 \u03bcm (**a**-**c**) and 10 \u03bcm (**e**, **g**). For both **f** and **h**, vertical bar in **h** is 5 \u03bcm, and horizontal arrow is 60 s.\\n\\nFig. 4: **Inhibition of the Arp2/3 complex leads to enlargement and stabilization of CCSs in intact cells.****a**\u2013**c** TIRF microscopy images of RPF-CLC in U2O5 cells cultured in the presence of 10% FBS (**a**), or incubated overnight in a medium containing 0.1% FBS and supplemented for the last 4 h with either DMSO (**b**) or 200 \u03bcm CK-666 (**c**). **d** Quantification of the CCS area in TIRF microscopy images of the indicated cell types treated with either DMSO or 200 \u03bcm CK-666, as described for **b** and **c**. Individual experiments are color-coded. CCSs were visualized using endogenously tagged RPF-CLC U2O5 and HeLa cells) or by immunostaining of CHC (P0/2 cells; individual experiments were statistically evaluated separately due to different staining intensities). Error bars, mean + SD; **m**-**p** < 0.0001; **m**-**p** = 0.0049 (blue) or 0.0043 (orange); (unpaired two-tailed \\\\(t\\\\) test with Welch's correction); n, number of cells. **e**, **g** First frames of 10-min long TIRF microscopy time-lapse\"", "CAPTION FIG5.png": "'\\n\\n**Fig. 5**: **Branched actin networks are restored after CK-666 washout and promote separation of large fat CCS and invagination of smaller CCSs in P\\\\(\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{ \\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{\\\\mathbf{ \\\\mathbf{}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}\\\\}}}\\\\}}\\\\}}\\\\}}\\\\}}\\\\}\\\\}}\\\\}\\\\}\\\\}\\\\}\\\\}\\\\}\\\\}\\\\}\\\\}\\\\'", "CAPTION FIG6.png": "'(yellow arrowheads) and form comet tails at dotted or spherical CCSs (cyan arrowheads). **1.**J Still frames from TIR microscopy time-lapse sequences of Ptx2 cells expressing GFP-CLC (cyan) and mCherry-corotation (red) taken in the presence of CK-666 (t = 0:00) and following CK-666 washout; time after washout for individual frames is shown as min:sec. Scale bars, 200 nm (**a**\u2013**h**) and 2 um (**1.**J**).\\n\\n'", "CAPTION FIG7.png": "'percentages of CCS shape categories (**k**) and the area of flat CCSs (**l**) in epsilon DKO and TKO MEFs. Error bars in **l**, mean +- SD, ***_p_ < 0.0001 (two-tailed Mann-Whitney test), **m** - **p** Treatment of Pkt2 cells ectipically expressing mCherry/CLC and GFP-epsin1 with DMSO (**m**, **n**) or CK-666 (**o**, **p**). Merged red/cyan images and individual epgml channels are shown before drug application (0,h, top row) and after treatment for 2 h (bottom row). Fluorescence intensity levels were identically adjusted for two time points in each set. **q** Percentage of CLC-positive puncta punctacalculating with epgml puncta in the same cells before and after treatment with DMSO (top) or CK-666 (bottom); \\\\(n\\\\), \\\\(p\\\\) = 0.4454; ***_m_ \\\\(p\\\\) < 0.0001; \\\\(n\\\\) = 20 cells for each condition from 2 independent experiments; two-tailed Wilcoxon matched-pairs signed rank test. Scale bars, 10 \u03bcm (**(a)**), applies to top (**a**-**d**) panels; and (**m**), applies to all (**m**-**p**) panels; 200 nm (**g**-**l**).\\n\\nFig. 7: **Inscout of epsins mimics the phenotype of Arp2/3 complex inhibition.****a**\u2013**d** (top row) First frames of TIRF microscopy time-lapse sequences of EGFP-CLC inepsin conditional DKO MEFs treated with either ethanol (**a**, **b**; labeled \u2018DKO\u2019) or 4-OHT (**c**, **d**; labeled \u2018TKO\u2019) for 5 days and then with either DMSO (**a**, **c**) or 100 \u03bcM CK-666 (**b**, **d**) for 4 h. Boved regions are enlarged 2.5-fold in msets. **a**\u2013**d** (bottom row) Xoxygraphs taken along yellow lines in top panels. Distance (**d**) scale, 2 \u03bcm time (**c**) scale, 1 min. **c**, **f** Quantification of TIRF microscopy data showing average CCS areas (**e**) and fractions of 600-min long tracks per cell (**f**) in indicated conditions. Error bars, mean \u00b1 SD; **n** \u2013 number of cells; 414 \u00b1 285 CCSs per cell (**e**) and 156 \u00b1 104 CCS lifetimes (**f**) per cell. Statistically significant differences are indicated: ***_p_ < 0.0001; **_p_ = 0.0078; Kruskal-Wallis with posthoc Dunn\u2019s multiple comparison test. **g**\u2013**j** JREM of DKO (**g**, **h**) and TKO (**l**, **j**) MEFs. Flat (blue), dome-shaped (green) and spherical (orange) CCSs are color-coded. **k**, **l** Quantification of '", "CAPTION FIG8.png": "'in the background separate flat CCSs into smaller domains and initiate their irradiation. (ii) Branched actin networks expanding along the CCS perimeter continue to promote CCS invagination, but also constrict the CCS neck. (iii) Further expansion of branched actin networks generates subsets of pushing barbed ends that work against each other to elongate the CCSs neck, in addition to the subset that constrict the neck. (iv) Consolidation of branched actin networks into a comet, tall after vesicle scission propels the newly formed vesicle away from the plasma membrane. In all stages, actin filament barbed ends push at the boundary between the plasma membrane and clathrin coat, where egins help to transmit the force. Green arrows indicate the direction of force produced by actin barbed ends at each stage.\\n\\nFig. 8: **CCS-associated branched actin networks emerging from microtubule tips.**a-g Branched actin networks (yellow) originating from the tips of microtubules (red) in control cells cultured in the presence of 10% FBS (**a**, **d\u2013g**) or 0.2% FBS (**b**, **c**) can extend toward the plasmamembrane (**a**, **b**), to an unknown target (**c**), or toward spherical (orange), dome-shaped (green) or flat (blue) CCSs (**d\u2013g**). Sometimes, protoneaceous material connects the microtubule tip and the branched actin network (**d**, orange CCS). **b** **C**K-666-treated cell. Infrequent remaining branched actin networks can still be associated with microtubule tips. **I**-1 Cells after CK-666 washout for 0.5 min (**d**), 1.1 min (**b**), 1.5 min (**k**) and 2 min (**f**) exhibit gradual restoration of branched actin networks at microtubule tips adjacent to CCSs. Scale bars, 100 nm. **m** Edge pushing model of actin-dependent CCS morphogenesis. (**i**) Branched actin networks originating from microtubules or linear actin filaments in the background separate flat CCSs into smaller domains and initiate their irradiation. (**ii**) Branched actin networks expanding along the CCS perimeter continue to promote CCS invagination, but also constrict the CCS neck. (**ii**) Further expansion of branched actin networks generates subsets of pushing barbed ends that work against each other to elongate the CCSs neck, in addition to the subset that constrict the neck. (**iv**) Consolidation of branched actin networks into a comet, tall after vesicle scission propels the newly formed vesicle away from the plasma membrane. In all stages, actin filament barbed ends push at the boundary between the plasma membrane and clathrin coat, where egins help to transmit the force. Green arrows indicate the direction of force produced by actin barbed ends at each stage.\\n\\n'"} \ No newline at end of file diff --git a/extracted_captions/yipBrowniesBagsofstuffDomain2013.json b/extracted_captions/yipBrowniesBagsofstuffDomain2013.json new file mode 100644 index 0000000000000000000000000000000000000000..b8953002a9fb9e03b07d764bab217dc77ca9780f --- /dev/null +++ b/extracted_captions/yipBrowniesBagsofstuffDomain2013.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1. ScienceKit in Kitchen Chemistry. The mobile app is situated in the cooking and science activities.**'", "CAPTION FIG2.png": "'Figure 2. A sample low-tech prototype from \"Base-of-Striff\".\\n\\n'", "CAPTION FIG3.png": "'Figure 3. An example of \"Sticky Notes\" co-deletion technique.\\n\\n'", "CAPTION FIG4.png": "'Figure 4. A \"Layered Illustration\" sample.\\n\\n'", "CAPTION FIG5.png": "'Figure 5. Frequency count of design ideas on the panel\\n\\n'", "CAPTION FIG6.png": "'Figure 6. Kidsteum (left) and KC (right) example panel.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## Chapter 1 Introduction'"} \ No newline at end of file diff --git a/extracted_captions/zhu2023graf1.json b/extracted_captions/zhu2023graf1.json new file mode 100644 index 0000000000000000000000000000000000000000..11d3f21d6d8f2c041ca0fb1ceaa47e58b3cad800 --- /dev/null +++ b/extracted_captions/zhu2023graf1.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "\"drerreig: challenge in WT and GRAF230 mice; \\\\(n\\\\) = 5 mice/group. **1** Representative left ventricular M-mode echocardiographic tracings: **J** Quantified Left Ventricle Fractional Shortening (LVFS) and Left Ventricle Ejection Fraction (LVF). **k** Representative Western blot and densitometric quantification of GRAF1 and LG3 H-C3 I ratio in hearts from saline or ISO-treated WT and GRAF230 mice (_n_ = 5 group). **m** Representative TEM of hearts from ISO-treated WT and GRAF230 mice. Green arrows show mitochondria in autophagosomes, red arrows highlight the attachment of small autophagosomes to mitochondria, yellow arrows indicate glycogen accumulation in proximity to mitochondria. Scale bar: 1 _m_m or 0.2 _m_(magnified images). **a** Representative confocal image of endogenous GRAF1 and L3 in NRVCMs react red with vehicle, OA and OA + \\\\(b\\\\) midamycin. Scale bars: 5 _m_m or 2 _m_(magnified images). **a** Representative superresolution-structured illumination microscopy (SR-SIM) of NRVCMs treated with OA and stained for GRAF1 and HSP60. Scale bar: 5 _m_(top), 1 _m_(bottom). Data are presented as mean +- SD; _m_(_top_), not significant; \\\\(P\\\\) < 0.05; \\\\(P\\\\) < 0.01, _m_P_ < 0.001 by two-tailed student's _t_-test (_e_,_g_) or Two-way ANOVA plus skidk's multiple-comparisons test (_l_).\\n\\nFig. 1: **GRAF1 is essential for cardiac mitochondrial homeostasis.****a\u2013c** Representative TEM and histogram analysis of mitochondrial size and aspect ratio in GRAF130 hearts relative to genetic control (WT). **b** Nonparametric kernel-smoothing distributions of mitochondrial dorsal cross-sectional areas (CSA6) from TEMs of hearts from wild-type (blue solid line) and GRAF130 (red solid line) mice. Mean mitochondrial CSA for wild-type (blue dashed line, \\\\(n\\\\) = 330) = 0.6516 _m_/mean CSA for GRAF130 (red dashed line, \\\\(n\\\\) = 744) = 0.5876 _\u03bc_m_/_p_ value (unpaired two-tailed _t_-test) = 0.0145. **c** Nonparametric kernel-smoothing distributions of mitochondrial aspect ratios (ARs) for TEMs of hearts from wild-type (blue solid line) and GRAF130 (red solid line) mice. Mean mitochondrial AR from wild-type (blue dashed line, \\\\(n\\\\) = 330) = L610, mean AR for GRAF130 (red dashed line, \\\\(n\\\\) = 144) = 1425, _\u03bc_ value (unpaired two-tailed _t_-test) = 0.0001. **d** Quantification of mitochondrial membrane potential (red/green ratio) in NRVCMs by JC-1 staining; \\\\(n\\\\) = 6, _p_-value = 0.0064. **f** **g** Quantification of mitochondria respiratory functions in NRVCMs by sepharose NF assay; \\\\(n\\\\) = 4, _P_-value(spare capacity) = 0.036. _P_-value(maximal respiration) = 0.024, _P_-value(ATP production OCR) = 0.0006. **h** Schematic of adrenergic challenge in WT and GRAF230 mice; \\\\(n\\\\) = 5 mice/group. **1** Representative left ventricular M-mode echocardiographic tracings: **J** Quantified Left Ventricle Fractional Shortening (LVFS) and Left Ventricle Ejection Fraction (LVF). **k** Representative Western blot and densitometric quantification of GRAF1 and LG3 H-C3 I ratio in hearts from saline or ISO-treated WT and GRAF230 mice (_n_ = 5 group). **m** Representative TEM of hearts from ISO-treated WT and GRAF230 mice. Green arrows show mitochondria in autophagosomes, red arrows highlight the attachment of small autophagosomes to mitochondria, yellow arrows indicate glycogen accumulation in proximity to mitochondria. Scale bar: 1 _\u03bc_m or 0.2 _\u03bc_(magnified images). **a** Representative confocal image of endogenous GRAF1 and L3 in NRVCMs react red with vehicle, OA and OA + \\\\(b\\\\) midamycin. Scale bars: 5 _\u03bc_m or 2 _\u03bc_(magnified images). **a** Representative superresolution-structured illumination microscopy (SR-SIM) of NRVCMs treated with OA and stained for GRAF1 and HSP60. Scale bar: 5 _\u03bc_m (top), 1 _\u03bc_m (bottom). Data are presented as mean +- SD; _m_(_top_), not significant; \\\\(P\\\\) < 0.05; \\\\(P\\\\) < 0.01, _m_P_ < 0.001 by two-tailed student\u2019s _t_-test (_e_,_g_) or Two-way ANOVA plus skidk\u2019s multiple-comparisons test (_l_).\\n\\n\"", "CAPTION FIG2.png": "'arrower, **f** denotes GRAF1 band shift. Blue arrowhead (**a**-**e**, **f**) indicates ubiquitin-rated GFP-Parkin. **g** Co-P of Prafin with GFP-LC3 in GRAF1-depleted HeLa cells stably expressing Parin and GFP-LC3. **h**, **i** Representative confocal microscopy images of Tomm20, GFP-LC3 and mCherry-Parkin in HeLa cells stably expressing GFP-LC3 and mCherry-Parkin. **z** stack images were acquired and deconvolution images are presented as maximum intensity z-projection Scale bar: 5\\\\(\\\\mu\\\\)m. Fraction of GFP-LC3 puncta that overlaps with Tomm20 labeled mitochondria in OA treated cells was quantified by Manders\\' colocalization coefficient in 40 cells from 3 independent experiments (**i**). Data are presented as mean +- SD. In not significant; _p_< 0.05; _\"P_< 0.01; _\"P_< 0.001 by two-tailed students\\'s t-test (**c**, **i** or **y** Two-way ANOVA with Dunnett\\'s multiple comparison test (**b**).\\n\\nFig. 2: **GRAF1 promotes Parkin-dependent mikophagy in HeLa cells by facilitating autophagosome capture.****a**, **b** siRNA transfected HeLa/YFP-Parkin cells were treated with OA for indicated times and levels of mitochondrial proteins were detected and quantified by Western blot/densitonometry (**b**) = a 3 independent experiments. **c** Quantification of mitochondria in HeLa/YFP-Parkin cells as assessed by ATPase staining (_n_ = 50 cells from 3 independent experiments; see Fig. S2f for representative images). **d** Representative confocal image of endogenous GRAF1 and YFP-Parkin in HeLa/YFP-Parkin cells following OA treatment, scale bar: 2.5\\\\(\\\\mu\\\\)m. **c** Co-P of GFP-Parkin with Flag-GRAF1 in COS7 cells co-transfected with Myo-PINKI or empty vector. **f** Top: Schematic of GRAF1 binding site within Parkin extrusion linker. Bottom: Co-P of GFP-Parkin WT and **II8** variant (deletion of an IoI-IIO and II6-I23) with Flag-GRAF1 in transfected COS7 cells treated with OA or vehicle. Red arrows: **f** denotes GRAF1 band shift. Blue arrowhead (**a**-**e**, **f**) indicates ubiquitin-rated GFP-Parkin. **g** Co-P of Prafin with GFP-LC3 in GRAF1-depleted HeLa cells stably expressing Parin and GFP-LC3. **h** Representative confocal microscopy images of Tomm20, GFP-LC3 and mCherry-Parkin in HeLa cells stably expressing GFP-LC3 and mCherry-Parkin. **z** stack images were acquired and deconvolution images are presented as maximum intensity z-projection Scale bar: 5\\\\(\\\\mu\\\\)m. Fraction of GFP-LC3 puncta that overlaps with Tomm20 labeled mitochondria in OA treated cells was quantified by Manders\\' colocalization coefficient in 40 cells from 3 independent experiments (**i**). Data are presented as mean +- SD. In not significant; _p_< 0.05; _\"P_< 0.01; _\"P_< 0.001 by two-tailed students\u2019s t-test (**c**, **i** or **y** Two-way ANOVA with Dunnett\u2019s multiple comparison test (**b**).\\n\\n'", "CAPTION FIG3.png": "'and indicated variations were incubated with WT or kinase dead (kD) MBP-PHXIX. GRAF1 phosphorylation was examined by pCRAF1 Ab. AAK: S668A/T670A/S67A. Bottom: Foncca S staining of GST-GRAF1 input. **1**, **1**, **2**, **4**, **5**, **6**, **18**, **4**, **8**, **19**, **20**, **21**, **22**, **23**, **24**, **25**, **26**, **27**, **28**, **29**, **21**, **26**, **28**, **27**, **29**, **26**, **28**, **29**, **27**, **28**, **29**, **26**, **29**, **28**, **27**, **29**, **28**, **29**, **21**, **26**, **29**, **28**, **29**, **27**, **28**, **29**, **26**, **29**, **28**, **29**, **29**, **27**, **28**, **29**, **29**, **28**, **29**, **29**, **29**, **29**, **28**,'", "CAPTION FIG4.png": "\"presence of F-actin bundles and anchored mitochondria in GRAF1-deficient cells following OA treatment, see Fig. S3i and ] for quantification of branched actin formation in control and GRAF1-deficient cells following 6 hr OA treatment. I Quantification of mitochondria chambers in i8H/K transfected HeLa/YFP-Parkin cells with indicated treatments. Lartunculin (BLI), \\\\(n\\\\) = 4 independent experiments per condition. * **Denotes \\\\(P\\\\) < 0.05; OA vs. DMSO for the same siRNA * Denotes \\\\(P\\\\) < 0.05 s;GRAF1 vs. s;Kontrol for the same treatment (+OA). Black triangle denotes \\\\(P\\\\) < 0.05 in LB vs. no LB for the same siRNA/+OA treatment. See Fig. S3i for mitochondrial clustering phenotypes. \\\\(P\\\\) < 0.05 representative confocal image of ATPase in siRNA transfected HeLa/YFP-Parkin cells treated with OA in presence or absence of Lartunculin b(Lat R). Scale bars: 50 \\\\(\\\\mu\\\\)m & Schematic showing GRAF1 phosphorylation mediates mitochondria-associated actin remodeling. Unless otherwise indicated all Western blots are representative of at least 3 independent experiments. Scale bars: 5\\\\(\\\\mu\\\\)m (g,b) and 2\\\\(\\\\mu\\\\)/immigmentation in hb. Data are represented as mean + SD, ns, not significant, *_P_ < 0.05, *_P_ < 0.01 by two-trailed student's \\\\(t\\\\) = (**I**) or by One-way ANOVA with post-hoc Tukey test (_I_).\\n\\nFig. 4: **|PINK1 phosphorylation promotes GRAF1 SH3-domain dependent recruitment of the WAVE2 complex to damaged mitochondria and facilitates mitochondrial-associated actin remodeling.****a** Co-Pf of endogenous ABI2 with glia-GRAF1 WT or phospho-deficient AAA variant in GRAF1 KO Hela/YFP-Parkin cells. **E**V: empty vector. **b**: CP of endogenous ABI2 and endogenous WAVE2 in siRNA treated cells assessed by Western blot. See Fig. S3f for densitometric quantification, \\\\(n\\\\) = 3 independent experiments. **c** In vitro interaction of purified GST-GRAF1 domains with endogenous ABI in Hela/YFP-Parkin lysates. Bottom: Ponceau S staining of GST-fusion protein input. SP-SH38-serine peptide (SP) region and SIR domain. 3D and 2E: DDO and 2EE phosphomeric variants. **d** Co-Pf of endogenous ABI2 with Flag-GRAF1 WT or DDD variant in GRAF1 KO Hela/YFP-Parkin cells. **e**, **f** WAVE2 complex proteins in cytosolic and mitochondrial enriched fractions of siRNA transfected HeLa/YFP-Parkin cells were analyzed by Western blot (**e**) and quantified in (**f**) \\\\(n\\\\) = 3 independent experiments. **e** In biorestentive SR-SIM (**g**) or confocal microscopy (**h**) of Tomm20, Phalloidin and VFP-Parkin in siRNA transfected HeLa/YFP-Parkin cells. Note continued \"", "CAPTION FIG5.png": "'Fig. 5: **GRAF1 is required for stress-induced metabolic adaptation in adult hearts and is dysregulated in human heart disease.****a**, **b** Endogenous-CRAF1 phosphorylation in NRVCMs treated with OA and examined by Phos-tag gel and Western using GRAF1 phos-5668/T670/S671 Ab, and densitometric quantification **(b)** \\\\(n\\\\) = 3 independent experiments. **c**, **d** siRNA transfected NRVCMs were treated with OA and GRAF1 phosphorylation was examined using p5668/T670/S671 GRAF1. AnDestruction quantification **(d)** \\\\(n\\\\) = 3 independent experiments. **e** Representative SR-SIM of NRVCM treated with vehicle and OA and stained for GRAF1, HSP60 and Phalloidin. Scale bar: 5 \\\\(m\\\\) (top, middle), 2 \\\\(m\\\\) (bottom). Note OA-dependent recruitment of GRAF1 to circular structures surrounding mitochondria are associated with branched actin. **f** Representative SR-SIM of HSP60 and Phalloidin stained **s** mRNA transfected NRVCMs treated with OA or vehicle. Scale bar: 5 \\\\(m\\\\) (Quantification of branched actin formation in control and GRAF1-deficient NRVCMs following 6 hr OA treatment. (_n_ = 40 cells from 3 independent experiments). **h** WT and GRAF1(r) mice were subjected to hyperoxia (100%: 02) for 48 hr to induce mitophagy followed by 96 hr of normovala. Transcript levels of target genes in hearts (WT normovala mice(_n_ = 3), GRAF1(r) normovala mice(_n_ = 4), WT hyperoxia mice(_n_ = 4) and GRAF1(r) hyperoxia mice(_n_ = 5_) were assessed by real time PCR. **k**, **j** Endogenous-CRAF1 phosphorylation in mouse hearts following ISO treatment (S.C., 200 mg/kg). \\\\(n\\\\) = 6 mice/group and denominator: quantification **(b)**. **k** Representative confocal microscopy images of mt-Keima in the hearts from ISO treated WT and GRAF1(r) mice expressing mt-Keima.(_S.C._, single dosage of 200 mg/kg for 3 days) \\\\(n\\\\) = 5 mice _/_g_ (_g_ (_g_ (_n_ = 5 mice/_g_ (_g_ (_n_ = 3 independent experiments). **m_ (_n_ = 3 independent experiments. **a_ = 6 representative SLE-SIM of NRVCM treated with vehicle and OA and stained for GRAF1(r) hearts. **h_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (_n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_ = 6 mice/_g_ (n_'"} \ No newline at end of file diff --git a/extracted_captions/zhuChildrenAreUnlikely2020.json b/extracted_captions/zhuChildrenAreUnlikely2020.json new file mode 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/dev/null +++ b/full_texts-test.json @@ -0,0 +1,93 @@ +{ + "paletzDynamicsMicroconflictsUncertainty2017": "The dynamics of micro - con\ufb02icts and uncertainty in successful and unsuccessful design teams Susannah B . F . Paletz , Center for Advanced Study of Language , University of Maryland , College Park , MD 20742 , USA Joel Chan , Human - Computer Interaction Institute , Carnegie Mellon University , Pittsburgh , PA 15213 , USA Christian D . Schunn , Department of Psychology and Learning , Research , and Development Center , University of Pittsburgh , Pittsburgh , PA 15260 , USA What di\ufb00erentiates successful from unsuccessful design teams ? Building on new research on design innovation that emphasizes interactions between social and cognitive processes , we investigated a potential distinguishing feature : Successful design teams may harness interpersonal con\ufb02icts ( a social design process ) to mitigate uncertainty ( a cognitive design process ) . We analyzed temporal relationships between brief , expressed interpersonal disagreements and subsequent spoken individual uncertainty in 30 h of conversations of 10 successful and 11 unsuccessful engineering product design teams . We discovered that micro - con\ufb02icts were followed by a relative reduction in uncertainty in successful design teams , whereas uncertainty rose after micro - con\ufb02icts in unsuccessful design teams , suggesting that interactions between con\ufb02ict and uncertainty may be a di\ufb00erentiating factor for design team success . (cid:1) 2017 Elsevier Ltd . All rights reserved . Keywords : teamwork , innovation , problem solving , collaborative design , con\ufb02ict W hat di\ufb00erentiates successful from unsuccessful design teams ? After decades of research on this question , design studies researchers have learned much about the nature of successful design teams . For example , successful design teams make heavy use of mental simulations and analogies ( Ball & Christensen , 2009 ; Christensen & Schunn , 2009 ) ; use design tools and media that are appropriate to the phase of the design process ( e . g . , sketching early on , prototypes later on ; Jang & Schunn , 2012 ) ; and work through consensus to build a robust shared understanding of the design prob - lem ( Agogino , Song , & Hey , 2006 ; Dong , 2005 ; Yang , 2010 ) . Yet , much re - mains to be understood about the complex factors that lead to team design success ( Dinar et al . , 2015 ) . For instance , while critical inquiry d which may include con\ufb02ict d is a foundational part of design education and practice ( Dym , Agogino , Eris , Frey , & Leifer , 2005 ; Oh , Ishizaki , Gross , & Yi - Luen Do , 2013 ; Sch \u20ac on , 1983 ) , theoretical and empirical work on team cognition suggests that con\ufb02ict still needs to be appropriately harnessed such that Corresponding author : Susannah Paletz sbfpaletz @ gmail . com www . elsevier . com / locate / destud 0142 - 694X Design Studies 50 ( 2017 ) 39 e 69 http : / / dx . doi . org / 10 . 1016 / j . destud . 2017 . 02 . 002 39 (cid:1) 2017 Elsevier Ltd . All rights reserved . relationship con\ufb02ict is minimized , open - minded discussion is maximized , and the bene\ufb01ts of disagreement can occur ( Jehn , 1997 ; Tjosvold , Wong , & Chen , 2014 ) . Yet , we know little about the preconditions , attendant processes , and mechanisms that make these desirable outcomes possible . An emerging research area in design team innovation emphasizes interactions between social and cognitive processes ( Paletz & Schunn , 2010 ) . The key asser - tion of this perspective is that understanding how social and cognitive pro - cesses are intertwined could help improve our understanding of how design innovation truly occurs , and thus improve interventions designed to improve design team performance ( e . g . , because social dynamics might alter how cogni - tive interventions are perceived , or vice versa ) . This perspective also has the potential to yield fresh insights into pathways to design team success . This social - cognitive perspective is motivated by numerous prior \ufb01ndings of com - plex interactions between social and cognitive processes in teams . For example , simple social phenomena like turn - taking can shape individual mem - ory retrieval dynamics ( Nijstad & Stroebe , 2006 ) . In addition , dissent from a minority opinion holder can trigger a broader information search in other team members , whereas dissent from a majority of team members biases infor - mation search in favor of the dissenting opinion ( Nemeth & Rogers , 1996 ) . In this paper , we investigate how the interplay between disagreement ( a social process ) and individual team members\u2019 uncertainty ( a cognitive process ) could help di\ufb00erentiate successful and unsuccessful design teams . Speci\ufb01cally , we discover that , in successful teams , open expression of disagreements helps to reduce individual uncertainty ( a desirable e\ufb00ect in the design process ) , whereas in unsuccessful teams , similar expressions of disagreement elevate uncertainty levels . Although both disagreement and uncertainty are natural to design teams , harnessing disagreement to resolve uncertainty may be advantageous , if not necessary . 1 Background 1 . 1 Intra - team con\ufb02ict and micro - con\ufb02icts in design Con\ufb02ict has been studied at intra - personal , intrateam , interteam , and national levels , in design teams and between countries ( e . g . , De Dreu & Gelfand , 2008 ; Ozkaramanli , Desmet , & Ozcan , 2016 ) . We focus on intrateam con\ufb02ict as be - tween individuals within the same design team . For this study , we de\ufb01ne con\ufb02ict to be when one team member explicitly opposes or contradicts statements or plans proposed by another team member . Thus , we focus on con\ufb02ict as disagree - ment , which is inherent to problem - solving conversations , regardless of its nega - tive a\ufb00ect , intensity , or directness ( Paletz , Schunn , & Kim , 2011 ; Weingart , Behfar , Bendersky , Todorova , & Jehn , 2015 ) . Such disagreements within a team can arise from di\ufb00erences in values , needs , interests , opinions , goals , or 40 Design Studies Vol 50 No . C May 2017 objectives ( Barki & Hartwick , 2004 ) . This de\ufb01nition draws from the psychology literature , rather than from rhetoric or argumentation theory , which examines di\ufb00erent types of arguments as discourse ( e . g . , Stumpf & McDonnell , 2002 ) . Most importantly , while team con\ufb02ict can be a barrier to design success , con\ufb02ict - ing needs and objectives can also inspire design and promote creativity ( Miron - Spektor , Gino , & Argote , 2011 ; Ozkaramanli et al . , 2016 ) . This paper draws from the organizational behavior , organizational psychol - ogy , and social psychology literature on teams , with the understanding that those research domains have only rarely examined design teams ( see below ) . To better understand the relationship between con\ufb02ict and team performance , social / organizational con\ufb02ict researchers ( De Dreu & Weingart , 2003 ; Jehn , 1995 , 1997 ; Paletz et al . , 2011 ) have found it important to distinguish between task con\ufb02ict ( disagreements about the task ) ; process con\ufb02ict ( disagreements about how to go about doing the task , including scheduling or priorities ) ; and relationship con\ufb02ict ( disagreements about values and personal issues ) . Process and relationship con\ufb02ict are considered problematic , disruptive , and dysfunctional ( e . g . , Jehn , 1997 ; De Dreu & Weingart , 2003 ; De Wit , Greer , & Jehn , 2012 ) . Task con\ufb02ict , in moderate levels and when unrelated to rela - tionship con\ufb02ict , may be positively related to team performance ( De Wit et al . , 2012 ; De Wit , Jehn , & Scheppers , 2013 ; Farh , Lee , & Farh , 2010 ) . Another useful distinction is the length of a con\ufb02ict and how it is measured . Much prior research on con\ufb02ict has examined it via retrospective self - report surveys , and that kind of data has generally found that con\ufb02icts are negatively related to performance ( e . g . , De Dreu & Weingart , 2003 ) . By contrast , obser - vational research on brief con\ufb02icts in - the - moment ( micro - con\ufb02icts ) suggests immediate , sometimes positive relationships with subsequent cognitive pro - cesses ( e . g . , Chiu , 2008a ; Paletz , Schunn , & Kim , 2013 ) . Thus , the often nega - tive portrayal of con\ufb02ict in the organizational behavior literature may be due to how con\ufb02ict is conceptualized and measured , such that brief disagreements are generally healthy , unless they snowball into long , salient con\ufb02icts ( Paletz et al . , 2011 ) . In the aggregate , these micro - process relationships between brief disagreements and cognition may have a positive impact on overall design team performance . The give - and - take of speci\ufb01c , brief disagreements in design and other creative , collaborative settings may not only be normal , but desir - able . Still , understanding the speci\ufb01c social - cognitive processes that are related to these disagreements could not only give insight into why they might be desirable , but also enable teams to negotiate potential tradeo\ufb00s with other as - pects of team performance ( e . g . , team cohesion early in the lifecycle of a team ) . 1 . 2 Uncertainty in design Successful problem solving in real - world , complex domains , such as engi - neering design , relies on e\ufb00ectively detecting and resolving uncertainty Con\ufb02ict and uncertainty in design 41 ( Ball & Christensen , 2009 ; Chan , Paletz , & Schunn , 2012 ; Christensen & Schunn , 2009 ; Schunn & Trafton , 2012 ) . Signi\ufb01cant e\ufb00ort is spent detect - ing , diagnosing , and resolving uncertainty ( Chan et al . , 2012 ; Downey & Slocum , 1975 ; Kahneman & Tversky , 1982 ; Schunn & Trafton , 2012 ) . Psy - chological uncertainty , speci\ufb01cally , is when an individual perceives infor - mation to be incomplete , missing , or vague , regardless of whether or not it is objectively uncertain ( Schunn , 2010 ; Windschitl & Wells , 1996 ) . De - signers can be uncertain not only about how to solve the various problems , but also about what the underlying problems truly are and about what they know ( Ball , Onarheim , & Christensen , 2010 ) . Analogy and mental simula - tion can be used as strategies to resolve uncertainty ( Ball & Christensen , 2009 ; Chan et al . , 2012 ) , and / or co - occur with greater uncertainty ( Christensen & Ball , 2016a ) , and uncertainty can lead to attentive returns to the topic to resolve the uncertain issues later ( Christensen & Ball , 2016b ) . On the other hand , a recent study found that certainty triggered immediate creative reasoning and information elaboration ( Christensen & Ball , 2016b ) . We draw a strong contrast between uncertainty and con\ufb02ict in our conceptu - alizations to avoid circularities : We focus on uncertainty as mental states within individual team members , and con\ufb02ict as interactions between team members . All combinations of uncertainty and con\ufb02ict can occur within the same team ; for example , con\ufb02ict can occur between team members who are currently not uncertain ( e . g . , strongly felt opposing plans ) and uncertainty can occur within team members without con\ufb02ict ( e . g . , individuals are uncer - tain about the same things ) . Although uncertainty is challenging for design teams , having uncertainty is not a sign itself of dysfunction ( Kirschenbaum , Trafton , Schunn , & Trickett , 2014 ) : Indeed , uncertainty is inherent to the ill - structured nature of design problems ( Goel & Pirolli , 1992 ; Tracey & Hutchinson , 2016 ) . Nevertheless , uncertainty that persists over time , unmitigated or unad - dressed , can harm design outcomes . For example , heightened uncertainty can increase a bias against creative ideas ( Mueller , Melwani , & Goncalo , 2012 ) . Also , prolonged uncertainty can increase psychological strain ( Bordia , Hogman , Jones , Gallois , & Callan , 2004 ; Bordia , Hunt , Paulsen , Tourish , & DiFonzo , 2004 ) , potentially reducing team members\u2019 e\ufb00ectiveness in contributing to the team\u2019s problem solving . Thus , devising and deploying appropriate strategies for dealing with uncertainty is central to the design process . Such strategies may include acknowledging it and taking it into account ( Lipshitz & Strauss , 1997 ) , or reducing it by collect - ing additional information , making assumptions and / or analogies , and problem solving ( Berlyne , 1962 ; Chan et al . , 2012 ; Lipshitz & Strauss , 1997 ) . 42 Design Studies Vol 50 No . C May 2017 1 . 3 Research questions about uncertainty and con\ufb02ict in design teams Both team disagreement and individual uncertainty rise and fall naturally over the course of conversations ( Ball & Christensen , 2009 ; Chan et al . , 2012 ; Christensen & Ball , 2016b ; Paletz , Chan , & Schunn , 2016 ; Paletz et al . , 2011 ; McDonnell , 2012 ) . Prior research on interactions between social and cognitive processes has often found important correlations between temporal dynamics of social / cognitive processes ( e . g . , con\ufb02icts sparking analogies dur - ing conversation ; Paletz et al . , 2013 ) . In this work , we hypothesize that tempo - ral dynamics , or patterns of interactions over time , of disagreement and uncertainty can distinguish successful from unsuccessful design teams . The context of design may also in\ufb02uence the e\ufb00ects of con\ufb02ict and uncertainty on each other and on team performance . The broader intrateam con\ufb02ict liter - ature examines a large range of laboratory and natural teams , from executive teams to production teams , including creative teams ( e . g . , De Dreu & Weingart , 2003 ; De Wit et al . , 2012 ) . Design teams , however , have speci\ufb01c fea - tures that may help focus our research questions . First , design teams experi - ence greater uncertainty than many other types of teams , as exhibited by their frequent need to engage in problem \ufb01nding and choose which approach is best to solve their problems ( Mehalik & Schunn , 2006 ) . Second , it is there - fore normal for design teams to tolerate , but also engage with and resolve un - certainty ( Beheshti , 1993 ) . Third , because the goal of design teams is creation , critical thinking and disagreement are valued and taught as part of the social - ization of design students ( Dym et al . , 2005 ) . Even given these norms , however , not all design teams succeed . Successful design teams are likely better at managing and harnessing task con\ufb02ict . Con - straints can be raised in an open manner , thereby increasing the likelihood that disagreement will be productive ( Tjosvold et al . , 2014 ) . One mechanism by which disagreement can lead to success is via the free expression and discussion of di\ufb00erent / opposing views , which encourages information sharing ( De Dreu & West , 2001 ; Tjosvold et al . , 2014 ) . Similarly , dissent arising from minority opinions can lead to greater information search ( Nemeth , 1986 ; Nemeth & Rogers , 1996 ) . Mild disagreement can promote greater information acquisi - tion ( Todorova , Bear , & Weingart , 2014 ) . These dynamics of participation and information sharing have been shown to be vital to team success ( Mesmer - Magnus & DeChurch , 2009 ) . Importantly , information can decrease problem - solving uncertainty ( Lipshitz & Strauss , 1997 ) . Thus , because suc - cessful teams are likely to have e\ufb00ective resources and con\ufb02ict resolution pro - cesses that enable shared understanding ( Kleinsmann & Valkenburg , 2008 ) , con\ufb02ict in those teams should not be dysfunctional , and uncertainty should be productively resolved . We therefore expect that more successful design teams should generally experience a decrease in uncertainty after a Con\ufb02ict and uncertainty in design 43 disagreement . In other words , temporally speaking , we expect disagreements in such teams to be followed by decreases in individual uncertainty . Even as all design teams encounter con\ufb02ict , not all manage it productively . Less successful design teams may have more di\ufb03culty in drawing insights from and resolving task con\ufb02ict . Task con\ufb02ict can mix with relationship con - \ufb02ict , damaging performance ( De Wit et al . , 2012 ) . Minority opinion disagree - ment in teams has been associated with lower con\ufb01dence in team decisions , suggesting that lopsided con\ufb02ict within a team may increase initial uncertainty and the questioning of previously held opinions ( Schulz - Hardt , Jochims , & Frey , 2002 ) d potentially exacerbating and getting in the way of resolving ex - isting levels of uncertainty that are inherent in design teams . Poorly managed disagreement may raise individual uncertainty in the moment that could then remain unresolved and need to be revisited again and again ( Christensen & Ball , 2016b ) ; individuals may use tentative speech to soften a response to a disagreement ( McDonnell , 2012 ) . Thus , because less successful teams are likely to lack e\ufb00ective knowledge resources or con\ufb02ict resolution strategies , con\ufb02ict in such teams should raise uncertainty that is not productively resolved , which may then raise overall levels of uncertainty . From a design perspective , con\ufb02ict could lead to additional uncertainty for less negative reasons . Disagreements reveal di\ufb00erences , ambiguities , or errors that are then noticed by others ( Dama & Dunbar , 1996 ) . Ill - de\ufb01ned problems necessitate the uncovering of design requirements ( Ball et al . , 2010 ; Simon , 1973 ) , such that a team tasked with a poorly - de\ufb01ned problem could disagree about the nature of the problem and its solutions , leading to the revelation of greater uncertainty . Temporally speaking , we expect disagreements in such teams to be followed by increases in individual uncertainty . Motivated by these extant \ufb01ndings , in this paper , we investigate whether there is an interaction between team success and con\ufb02ict , such that micro - con\ufb02icts decrease uncertainty in successful teams , but increase uncertainty in unsuccessful teams . This study explores this question with a dynamic , time - lagged , behav - ioral observation approach using a large dataset of natural problem solving in design teams . Given the prior di\ufb00erences found for task versus process con - \ufb02ict , we examine these relationships separately by these di\ufb00erent types of disagreement . 2 Methods 2 . 1 Research context This study examined engineering student design teams working in a product realization course at a large research university in the Mid - Atlantic United 44 Design Studies Vol 50 No . C May 2017 States . Our overall sample consisted of 57 teams across seven semester - long implementations of the course . In each semester , multidisciplinary student teams , primarily from various dis - ciplines in engineering ( e . g . , mechanical , electrical , industrial , chemical , bioen - gineering ) , took products from concept to functional prototype . Each team was on a di\ufb00erent project from a variety of product domains , such as diaper redesigns , solar - powered water heating systems , and Radio Frequency Identi - \ufb01cation ( RFID ) personnel badge systems . Each project received up to $ 2500 from an external sponsor for design / prototyping e\ufb00orts and had industry , graduate student , and / or faculty mentors . Team members were paid $ 250 to complete background surveys , submit pre - sentations and \ufb01nal documents to the researchers for analysis , and do the bulk of their ( team ) work in a specially prepared room with a table and chairs , a computer with engineering - related software ( e . g . , CAD software ) , and other useful technology . The students were audio e video recorded whenever they used these rooms . Informed consent was obtained from the participants . 2 . 2 Sample Because of the extremely time - consuming nature of observational coding , we sampled 21 teams to be coded for uncertainty and con\ufb02ict , choosing teams that were clearly high vs . low success , focused on hardware projects ( i . e . , excluding unique project types , like software design ) , and regularly used the room set up for video data collection . The 21 teams ranged from three to \ufb01ve members ( M \u00bc 4 , Median \u00bc 4 , SD \u00bc 0 . 63 ) for a total of 84 undergraduate students . Team meeting clips were chosen for transcription based on having no more than 5 min of o\ufb00 - task talk within them and meeting a minimum level of audio and video ( transcribable ) quality . A total of over 30 h of talk was transcribed into utterances ( clauses or thought statements , Chi , 1997 ) and coded for the variables of interest for a total of 38 445 total utterances , 35 148 of which were coded as on - task ( ICC \u00bc 0 . 79 for triple coded sets , average kappa \u00bc 0 . 72 for double coded sets ) . On - task talk included anything relevant to their product or class , including process discussions ( e . g . , shopping trips for materials ) . When a team meeting had a stretch of o\ufb00 - task conversation of over 5 min ( e . g . , about football ) , the video clip was broken up , resulting in 59 video clips ranging from 5 min to 1 h 40 min long ( M \u00bc 31 min , SD \u00bc 23 min ) . Attendees ranged from 2 to 7 individuals with the rare higher numbers because of occasional mentor visits ( M \u00bc 3 . 7 , Median \u00bc 4 . 0 , SD \u00bc 1 . 0 ) . At the team level , 24 % were all male , 29 % of the teams had 50 % females , and the rest had females but in a minority proportion ( proportion female of the team M \u00bc 28 % ) . Con\ufb02ict and uncertainty in design 45 2 . 3 Measures To reveal the patterns of quick , brief social and cognitive process interactions in design teams , this paper draws on recent research that studies interpersonal con\ufb02ict communication behavior within team interactions ( e . g . , Paletz et al . , 2011 ; Poole & Dobosh , 2010 ) . It is within conversations that design team members do the work of joint problem solving and develop group cognition . When studying brief processes , observational methods can tease apart their interplay as they unfold over time ( e . g . , Chiu , 2008a ; Gottman & Notarius , 2000 ; Kau\ufb00eld & Lehmann - Willenbrock , 2012 ; Weingart , 1997 ) . The three primary measures for this study were design team success / outcome , micro - con\ufb02icts and their types , and spoken individual uncertainty . Both un - certainty and micro - con\ufb02icts were 100 % double - coded by two di\ufb00erent sets of paired independent coders who were blind to the research questions of this study , the results of the other variable\u2019s coding , and the team\u2019s success score . Thus , uncertainty and con\ufb02ict could co - occur or not , in any combina - tion , and were not exclusive of each other . All coding disagreements were resolved via consensus . 2 . 3 . 1 Design success Building on professional design practice ( Otto & Wood , 2000 ; Ullman , 2002 ; Ulrich & Eppinger , 2008 ) , we took a requirements - based approach to measuring team design success . Customer needs , given by design brief docu - ments and initial meetings with sponsors , were translated into speci\ufb01c design requirements ( e . g . , safety , cost , ease of use , e\ufb03ciency ) and weighted by impor - tance to the overall product ( using a 1 to 5 importance scale , 5 \u00bc most important ) . Each team\u2019s design requirements were evaluated by a course instructor most familiar with the project and knowledge domain . The instructor rated the de - gree to which the project requirements were met ( e . g . , 0 \u00bc Did not come close to meeting requirement ; 1 \u00bc Fell just short of meeting requirement ; 2 \u00bc Met requirement , but did not exceed signi\ufb01cantly ; 3 \u00bc Signi\ufb01cantly exceeded the requirement ) . While satis\ufb01cing a requirement threshold is important , there is usually a preference for exceeding requirements . For example , while a car manufacturer may obtain an adequate pro\ufb01t if production cost stays below $ 12 000 , an engineering team that produces a car that costs $ 8000 would be even more desirable . All course instructors had extensive experience in product realization , including numerous patents , startup company experience , and in - dustry consulting , as well as relevant content knowledge ( e . g . , materials sci - ence , electrical engineering ) , and were blind to the questions of this study . The \ufb01nal success measure for a given team was calculated by the ratio of the earned success score ( sum of requirement ratings multiplied by their 46 Design Studies Vol 50 No . C May 2017 importance weights ) and the maximum possible success score ( sum of require - ments\u2019 maximum possible ratings multiplied by their importance weights ) , normalized ( multiplied by 100 ) to yield a score on a 0 to 100 scale ( Goncher , Chan , Schunn , & Lovell , 2012 ) . Because of our sampling strategy , our teams fell into a bimodal rather than a normal distribution . We thus used the team\u2019s scores to create a dichotomous variable , 0 \u00bc low success ( success score < 69 , 11 teams ) , and 1 \u00bc high success ( success score > 79 , 10 teams ) . As a validation of our success measure , there was a signi\ufb01cant overall relation - ship between success score and whether or not the teams\u2019 products were sub - mitted for patent or implemented in some fashion by the sponsor , r pb \u00bc 0 . 50 , p < . 001 . In our sample , 7 of the 10 high success teams\u2019 products were submit - ted for patent or implemented , in contrast to only 3 of the 11 low success teams . Information on patenting / implementation was not available for 2 of the teams in our sample ( 1 high and 1 low success ) . We use the ratings as the success measure rather than whether the product was used at the company or patent submissions , because complex factors outside the local project ( e . g . , di\ufb00erences in company intellectual property management approaches , time / budget constraints ) played a large role in determining immediate use at the company or decision to \ufb01le a patent . 2 . 3 . 2 Micro - con\ufb02icts We coded for micro - con\ufb02icts at the utterance level by adapting a pre - existing coding scheme ( i . e . , if this utterance includes con\ufb02ict , the utterance was marked as 1 , and if not , it was marked as 0 ; Paletz et al . , 2011 , 2013 ) . Micro - con\ufb02icts were identi\ufb01ed when a speaker explicitly , whether via tone and / or words , disagreed with something said earlier in the video clip . Simply stating a potentially controversial viewpoint was not su\ufb03cient . Constraints , which limit the search for solutions ( Ball et al . , 2010 ) , can be ( but need not al - ways be ) raised via disagreements ( Paletz , Sumer , & Miron - Spektor , 2016 ) . Coders both read the transcript and listened to / watched the audio e video recording simultaneously to improve reliability ( average con\ufb02ict event kappa of 0 . 70 across coder pairs ) . The micro - con\ufb02icts were coded by utterance for type of con\ufb02ict , using cate - gories typically used in the con\ufb02ict literature ( e . g . , Jehn , 1997 ) : task micro - con\ufb02ict utterances were directly related to the engineering product realization task , including choosing materials , design , and testing ; process micro - con\ufb02ict utterances were about scheduling , communicating their \ufb01ndings ( e . g . , creating presentations , how to dress for presentations ) , how to go about doing work , assigning tasks , prioritization , and who said what to whom ; \ufb01nally , relation - ship micro - con\ufb02icts were about values , being critical of others\u2019 personality and style , and personal likes / dislikes . Because of the potential co - occurrence of these three con\ufb02ict types in real - world settings , their presence / absence Con\ufb02ict and uncertainty in design 47 were coded separately ( task kappa \u00bc 0 . 87 ; process kappa \u00bc 0 . 61 , and relation - ship kappa \u00bc 0 . 65 ) . Micro - con\ufb02ict events were clusters of utterances de\ufb01ned as relating to a very speci\ufb01c topic . For instance , the exchange \u2018It won\u2019t \ufb01t , \u2019 \u2018Well , just make it bigger , \u2019 \u2018Well , I\u2019m dumb , I can\u2019t , \u2019 \u2018You can\u2019t make it bigger ? \u2019 \u2018No , \u2019 \u2018What the hell , [ name ] , you suck\u2019 is actually two micro - con\ufb02icts : one that is predom - inantly process ( the students were discussing whether and how to change the font size on a Powerpoint slide , rather than the content ) , and then a micro - con\ufb02ict event immediately following that is predominantly relationship con - \ufb02ict , where the \ufb01rst speaker\u2019s competence is called into question ( for another example , see Table 1 ) . To test the alternate supposition that successful teams were simply better at resolving con\ufb02icts , micro - con\ufb02ict events were also coded as to whether they were resolved immediately , within 25 utterances of the last micro - con\ufb02ict utter - ance coded ( kappa \u00bc 0 . 79 ) . Coders judged the micro - con\ufb02ict event as resolved if the disagreeing parties came to an agreement , whether by one person acqui - escing to the other\u2019s ( or others\u2019 ) opinion or the parties coming to a compromise . Twenty - \ufb01ve utterances were chosen because it was often not possible to tell if the micro - con\ufb02ict was resolved after that point , as micro - con\ufb02icts blended into each other over time , and some may have been resolved by the participants at untranscribed later times ( e . g . , via email or during other meetings ) . 2 . 3 . 3 Uncertainty Although psychological uncertainty is an internal state , cognitive scientists have frequently operationalized uncertainty via coding individual utterances within design and science team conversations , picking up \u2018hedge words\u2019 ( words that typically accompany and communicate uncertainty ) such as \u2018maybe\u2019 and \u2018possibly\u2019 ( e . g . , Ball & Christensen , 2009 ; Ball et al . , 2010 ; Chan et al . , 2012 ; Trickett , Trafton , Saner , & Schunn , 2007 ) . These kinds of words demonstrate behaviorally , viaverbalexpression , whenanindividualisperceiving uncertainty . This codingschemehad beenpreviously validatedusing convergent and discrim - inantmethods , such ascomparingspeech togesture based uncertainty measures , and distinguishing uncertainty from approximations ( Schunn , 2010 ) . Leveraging the relatively simple and stable predictive nature of hedge words as cues ( in contrast to the much more variable and complex nature of expressed disagreements ) , we employed a two - step semi - automated approach . First , we trained a Support Vector Machine classi\ufb01er ( a supervised machine learning al - gorithm ) on an initial set of human - coded utterances , and used the classi\ufb01er to identify utterances that might contain uncertainty ( for technical details , see Luo , Litman , & Chan , 2013 ) . Second , two trained humans coders assessed for uncertainty those utterances identi\ufb01ed by the algorithm as likely to include 48 Design Studies Vol 50 No . C May 2017 uncertainty . This process saves time and coder e\ufb00ort given the massive dataset , and it also helps to avoid coder drift and decreased validity and reliability due to fatigue . Inter - rater reliability was high ( Cohen\u2019s kappa \u00bc 0 . 80 ) . Table 1 pre - sents an example of both con\ufb02ict and uncertainty from a team that was tasked with creating a product that could be attached to a light bulb socket and indi - cate the power usage , with the end goal of informing consumers of their energy consumption . Table 1 Segmented transcript with uncertainty ( italics ) and micro - conflict ( bold ) codes Speaker Utterance Con\ufb02ict type 4 I thought yeah 4 It would be cool to put some little hydroelectric power generators 3 Yeah 4 You know those little mini ones 4 Right 3 That would be uh 3 And those , these like micro - generators 3 They\u2019d be enough to power maybe a light Task , onset 3 But not a battery Task 3 You know like the overall cost of electricity would not be decreased Task 3 I don\u2019t think Task 3 But then again these are like Task 3 They seem to be high cost solutions Task 3 These generators you know Task , end 4 Mmhmm 1 Yeah 3 Putting in generators 2 Yeah with that liquid water thing 2 Just that some people have their tanks underground too ? 4 Mmhmm right 2 So you wouldn\u2019t be able to do that 3 But perhaps 3 Perhaps that instead of marketing it as an individual solution 3 Maybe it could be a solution for an entire group of people , community , building 2 I think that was one of the things 2 That was just probably the only good thing on this paper 3 Yeah ? 2 Yeah it was 2 It said something about Note . From Team 20070106 . Speakers are numbered based on who spoke \ufb01rst in a transcript . Blocks indicated by section divides . Con\ufb02ict types are the dominant type at that utterance . Con\ufb02ict and uncertainty in design 49 2 . 4 Analyses 2 . 4 . 1 Blocks , video clips , and teams as three - level nested data As the goal was to examine the e\ufb00ects of disagreement on subsequent uncer - tainty , we needed a data structure that supported time - lagged analyses . We created segmented blocks centered on the incidents of the independent vari - able , con\ufb02ict . This event - centering strategy ensures that the predictor block ( i . e . , con\ufb02ict events ) would be relatively homogenous in its content , and the dependent blocks would be standardized in length ( Paletz et al . , 2013 ) . Blocks were created by ( 1 ) identifying the con\ufb02ict event ( or contiguous con\ufb02ict events ) , ( 2 ) segmenting the 10 utterances before and after each con\ufb02ict event as two additional blocks , and ( 3 ) breaking up the rest of the clips into succes - sive blocks of 10 utterances , each ending at the 10th utterance , the end or beginning of the clip , or with the next con\ufb02ict . Ten - utterance long blocks were chosen because initial descriptive visualizations suggested that was the best grain size for observing \ufb02uctuations in uncertainty . Previous analyses of temporal patterns of uncertainty in other datasets suggested that uncertainty in design teams tends to show signi\ufb01cant rises and declines in windows of 15 e 30 utterances ( e . g . , Chan et al . , 2012 ; Christensen & Schunn , 2009 ) . As un - certainty involves some problem solving prior to uncertainty resolution , we conducted analyses at two lags : both 0 e 10 utterances before the dependent variable ( \ufb01rst block , Lag1 ) and 11 e 20 utterances before the dependent vari - able ( second block , Lag2 ) . The number of utterances rather than time was the unit of analysis because the focus of the study was on expressed behavior . That noted , 20 utterances also represented roughly 1 min after the end of a micro - con\ufb02ict , so the \ufb01rst lagged block was at about 0 e 30 s after the con\ufb02ict , and the second at about 30 e 60 s . 2 . 4 . 2 Statistical analyses Most regression analyses assume that ( 1 ) data points are independent , and ( 2 ) that the dependent variable is continuous and normally distributed . Both of these assumptions were violated with these data . First , these data are nested d - blocks within video clips within teams d such that data points were not inde - pendent . Second , the main dependent variable was count data , which is skewed ( mostly zeroes , then ones , etc . ) . Thus , we employed statistical analyses that appropriately accounted for both these features : three - level hierarchical linear modeling , time - lagged , variable exposure overdispersed Poisson models using HLM7 ( 7 . 01 ) software ( Raudenbush , Bryk , & Congdon , 2013 ) . 1 We used three - level hierarchical linear modeling to account for the inherent depen - dence of having blocks ( Level 1 ) within video clips ( Level 2 ) within teams ( Level 3 ) and to handle unequal cell sizes ( Raudenbush & Bryk , 2002 ) . 2 The segmentation method resulted in 3969 blocks at Level 1 , 59 video clips at Level 2 , and 21 teams at Level 3 . Overdispersed Poisson models were necessary 50 Design Studies Vol 50 No . C May 2017 because the main dependent variable was count data ( number of uncertainty utterances in a block ) , and its variance ( 2 . 0 ) was greater than its mean ( 1 . 5 ; s 2 was 1 . 1 instead of 1 ) . Variable exposure Poisson accounts for the number of utterances being di\ufb00erent for di\ufb00erent blocks because the block creation process drew from naturalistic data ( e . g . , endings of clips , beginnings of new con\ufb02ict events ) . 3 We employed time - lagged analyses , such that uncertainty in one 10 - utterance block was predicted by the presence / absence of con\ufb02ict , or a particular con - \ufb02ict , at either Lag1 ( 1 e 10 utterances before ) or Lag2 ( 11 e 20 utterances before ) . In creating the models to test our research questions , the time - lagged con\ufb02ict to uncertainty tests were at Level 1 ( blocks ) . The binary low / high success variable was a main e\ufb00ect at the team level ( Level 3 ) and a moder - ator at the team level on the block level con\ufb02ict variables ( interaction between Level 3 and Level 1 variables ; Figure 1 ) . Before examining our independent variables , we tested for the signi\ufb01cance of several covariates that were plausibly connected to uncertainty or con\ufb02ict and therefore could be potential third - variable confounds . At Level 1 , we tested the percentage of on - task talk that was about the product itself ( rather than , say , presentations or planning ) ; the average number of words per utterance in that block , in order to control for underlying number of words available to be coded ; and the number of speakers in the block , in order to control for uncer - tainty due to more people participating in the conversation . At Level 2 , we tested the number of uncertainty on - task utterances in the overall video clip , the number of people at the meeting , the presence of a non - team member ( e . g . , a client or faculty advisor ) , and two gender composition vectors ( to Figure 1 Three - level data structure of lagged blocks , clips , teams , and team success Con\ufb02ict and uncertainty in design 51 account for the three types of clip gender compositions ) . At Level 3 , we tested the binary success variable , two team - level gender composition vectors , and the size of the team . Gender composition for both the conversational clip and the team was examined because of the known positive relationship be - tween the percent of women in a team and collective intelligence ( Woolley , Chabris , Pentland , Hashmi , & Malone , 2010 ) , which could then be associated with team success or other team processes . We \ufb01rst tested the potential cova - riates at Level 1 , then those signi\ufb01cant at Level 1 with Level 2 , then those sig - ni\ufb01cant at Levels 1 and 2 with Level 3 . Potential covariates had to be statistically signi\ufb01cant to be kept in the \ufb01nal covariate model . Two Level 1 var - iables passed these tests : the average number of words per utterance and the number of speakers in the block , both of which were positively associated with the presence of uncertainty . In keeping with standard practices , Lag2 ef - fects always controlled for Lag1 parallel variables , and interaction e\ufb00ects al - ways controlled for their respective main e\ufb00ects , regardless of statistical signi\ufb01cance . When a signi\ufb01cant interaction e\ufb00ect with team success occurred , we also tested for the main e\ufb00ects of micro - con\ufb02icts on subsequent uncertainty for the data - set broken up into low and high success teams ( i . e . , we conducted simple e\ufb00ects analyses to better understand signi\ufb01cant interactions ) . 3 Results We \ufb01rst describe the frequency of design team uncertainty in the data set . We then present the relationship between di\ufb00erent kinds of design team con\ufb02ict on subsequent uncertainty levels , as well as the moderation tests with team success . 3 . 1 Frequency of uncertainty and micro - con\ufb02icts At the block level , uncertainty occurred relatively frequently , with 70 % of the 3970 blocks having uncertainty , but with a wide variety in amount of uncer - tainty in a block : range 0 e 14 . In contrast , con\ufb02ict was less common : only 6 % of the 3970 blocks had con\ufb02ict . Of the 244 blocks coded as having con\ufb02ict in them , 65 % had at least one utterance of task con\ufb02ict , 57 % had at least one utterance of process con\ufb02ict , and 12 % had at least one utterance of relation - ship con\ufb02ict . The relationship micro - con\ufb02ict frequency ( 1 % of all blocks ) was insu\ufb03cient for use in these analyses . Importantly , as noted in subsequent sections , there were no di\ufb00erences by team success on the simple prevalence of uncertainty or overall con\ufb02ict , task con\ufb02ict , or process con\ufb02ict . Further , more successful teams were not simply better at resolving con\ufb02icts : At the team level , the proportion of quickly resolved con\ufb02ict events out of all on - task con\ufb02icts were the same between the more successful ( M \u00bc 0 . 54 , SD \u00bc 0 . 27 , n \u00bc 10 ) and less successful 52 Design Studies Vol 50 No . C May 2017 ( M \u00bc 0 . 71 , SD \u00bc 0 . 22 , n \u00bc 11 ) teams , t ( 19 ) \u00bc 1 . 57 , p \u00bc 0 . 13 . 4 With uncertainty as the dependent variable using the analysis technique noted previously , and the covariates of number of words per utterance and number of speakers , there was a non - signi\ufb01cant relationship trending in the negative direction between concurrent uncertainty and con\ufb02ict , with a large con\ufb01dence interval . This analysis suggests what the coding procedure implied d that there was no reli - able relationship between uncertainty and con\ufb02ict in the same block . 5 3 . 2 Relationship between con\ufb02ict , team success type , and uncertainty 3 . 2 . 1 Overall con\ufb02ict First , we tested whether con\ufb02ict one block or two blocks earlier ( Lag1 and Lag2 ) had signi\ufb01cant main e\ufb00ects on subsequent uncertainty and then whether they had signi\ufb01cant interactions with the team success variable . Team success alone was not related to uncertainty ( i . e . , both low and high success teams had similar rates of uncertainty ) . The interaction of con\ufb02ict at Lag1 with team suc - cess was not signi\ufb01cant . However , at Lag2 , there was a signi\ufb01cant interaction : high success teams had less uncertainty following micro - con\ufb02icts , compared with low success teams , which had relatively more uncertainty following micro - con\ufb02icts ( see Table 2 , Figure 2 ) . 3 . 2 . 2 Micro - con\ufb02ict by subtype We then repeated these analyses for task and process con\ufb02ict . These micro - con\ufb02ict types were not mutually exclusive , but were negatively correlated with each other : Blocks with task con\ufb02icts were less likely to have process con - \ufb02icts , X 2 ( 1 , 239 ) \u00bc 49 . 82 , p < 0 . 001 , phi \u00bc (cid:2) 0 . 46 ( at Lag2 ) . 7 Thus , testing them Table 2 Full model of micro - conflict and team design success on uncertainty Variable Event rate ratio ( 95 % con\ufb01dence ratio ) Intercept , g 000 a 0 . 17 ( 0 . 13 , 0 . 22 ) * * * Average words per utterance 1 . 07 ( 1 . 05 , 1 . 09 ) * * * Speakers in block 1 . 26 ( 1 . 20 , 1 . 32 ) * * * Con\ufb02ict Lag1 0 . 95 ( 0 . 87 , 1 . 05 ) Con\ufb02ict Lag2 1 . 09 ( 0 . 99 , 1 . 20 ) \u00fe Team Success ( L - 3 ) a 1 . 06 ( 0 . 83 , 1 . 34 ) Con\ufb02ict Lag2 (cid:3) team success 0 . 78 ( 0 . 65 , 0 . 95 ) * Low success teams Con\ufb02ict Lag 2 b 1 . 09 ( 0 . 94 , 1 . 26 ) High success teams Con\ufb02ict Lag 2 c 0 . 85 ( 0 . 72 , 1 . 01 ) \u00fe Note . \u00fe 0 . 10 > p > 0 . 05 ; * p < 0 . 05 ; * * * p < 0 . 001 . Degrees of freedom ( df ) \u00bc 3736 unless otherwise noted . a df \u00bc 23 . b df \u00bc 1951 . c df \u00bc 1782 . Con\ufb02ict and uncertainty in design 53 separately gives a more complete view and helps us determine whether the ef - fect is the same for di\ufb00erent kinds of con\ufb02ict . For task con\ufb02ict , we found that , consistent with the analyses of overall con - \ufb02ict , there were no signi\ufb01cant main e\ufb00ects of team success ( p > 0 . 60 ) , task con - \ufb02ict Lag1 ( p > 0 . 80 ) , and task con\ufb02ict Lag2 ( p > 0 . 40 ) on subsequent uncertainty , controlling for the signi\ufb01cant covariates . However , task con\ufb02ict Lag2 had a signi\ufb01cant interaction with the team success variable ( p < 0 . 05 ) , again showing decreasing uncertainty after task con\ufb02ict in high success teams but increasing uncertainty after task con\ufb02ict in low success teams ( Figure 3A ) . 8 Process con\ufb02ict Lag2 has the same interaction e\ufb00ect with team success ( Table 3 ) with growing uncertainty in low success teams and decreasing uncer - tainty in high success teams following process con\ufb02ict two blocks earlier ( Table 3 , bottom rows , Figure 3B ) . Tables 4 and 5 are illustrative examples of two micro - con\ufb02ict events and the uncertainty that followed them . One team , which did not fare well on its \ufb01nal success score , was tasked with investigating how to put a radio - frequency iden - ti\ufb01cation chip and antenna ( RFID technology ) in drill bits used in manufacturing . As they disagreed about the optimal size they needed , that dis - cussion raised other elements of their task that were uncertain ( Table 4 ) . In the second example , a successful team was working on a presentation and had a minor task disagreement about what the summary should include , with one team member noting it should be a summary of the whole paper . They quickly moved on to discussing in colorful terms how they would do that , with very little uncertainty ( Table 5 ) . While Speaker 1 raised the initial micro - con\ufb02ict , the little uncertainty being communicated seems to be hedges against seeming too domi - nant , rather than uncertainty about any underlying issues . Figure 2 Predicted number of uncertain utterances by team success and time - lagged mi - cro - con\ufb02ict , controlling for covariates 6 54 Design Studies Vol 50 No . C May 2017 Figure 3 Predicted number of uncertain utterances by team success and time - lagged ( A ) task and ( B ) Process micro - con\ufb02ict , controlling for covariates Table 3 Process micro - conflict and team success on uncertainty Variable Event rate ratio ( 95 % con\ufb01dence ratio ) Intercept , g 000 a 0 . 17 ( 0 . 13 , 0 . 22 ) * * * Average words per utterance 1 . 07 ( 1 . 04 , 1 . 09 ) * * * Speakers in block 1 . 26 ( 1 . 20 , 1 . 32 ) * * * Process con\ufb02ict Lag1 0 . 91 ( 0 . 77 , 1 . 08 ) Process con\ufb02ict Lag2 1 . 16 ( 1 . 03 , 1 . 30 ) * Team success ( L - 3 ) a 1 . 06 ( 0 . 83 , 1 . 34 ) Process con\ufb02ict Lag2 (cid:3) team success 0 . 62 ( 0 . 50 , 0 . 78 ) * * * Low success teams Process con\ufb02ict Lag 2 b 1 . 17 ( 0 . 99 , 1 . 38 ) \u00fe High success teams Process con\ufb02ict Lag 2 c 0 . 72 ( 0 . 54 , 0 . 95 ) * Note . \u00fe 0 . 10 > p > 0 . 05 ; * p < 0 . 05 ; * * * p < 0 . 001 . Degrees of freedom ( df ) \u00bc 3736 unless otherwise noted . a df \u00bc 23 . b df \u00bc 1951 . c df \u00bc 1782 . Con\ufb02ict and uncertainty in design 55 Table 4 Example of unsuccessful team ( ID 20070304 ) micro - conflict and uncertainty Speaker Utterance Con\ufb02ict type 2 Let\u2019s \ufb01nd the biggest small drill bit we have , 2 if they\u2019re all the same , 2 I saw some 3 e 3 . 4s 2 3 . 4 might do it , three seven 3 These are 3 This is 3 . 4 here 2 This is too big 1 Well 1 big is better for us Task , onset 1 I think Task 1 Because that will give us more apoxy Task 2 Well 2 This < missing word > 3 Yeah 1 I think we may need < missing word > 2 But I don\u2019t think this is the appropriate size Task 3 Right , Task 3 That looks a little big Task 4 Little bit too big Task , end 3 Would we be allowed to use these drill bits ? 3 Because they are somehow not right 3 < missing word > leave this to join together 2 I could have sworn we had some 3 . 4s 3 We do , 3 They\u2019re right here 2 Okay 3 And there is another set 3 Right here 2 We only got 6 , 2 Better not to mess them up 3 I don\u2019t know 3 What < missing word > 2 Oh 2 These are too small , 2 These are what , 4 . 2 . 2 Yeah 1 Yeah 56 Design Studies Vol 50 No . C May 2017 These examples are merely illustrative and should not replace either an in - depth qualitative analyses or our statistical analysis . Indeed , for some kinds of psychological phenomena , explicit cognitive processes play a strong role , whereas for other psychological phenomena , implicit emotional / memory Table 5 Example of successful team ( ID 20070301 ) micro - conflict and uncertainty Speaker Utterance Con\ufb02ict type 2 So the conclusion should just be a summary of this kind of stuff a 1 I think it\u2019s supposed to be a summary of the whole paper Task , onset 1 Err the whole project Task , end 3 You could do it like that 1 So it\u2019s just a bit more general 1 You don\u2019t have to 1 You should go into detail 2 I know I can bullshit 2 But I want 2 I kinda want 2 Or meant to be 2 Uh , you know 2 Bullshit that everyone else has piled on 1 Well you can kinda , 1 all you have to do for the conclusion is restate the summary 2 Great 1 Just reword the < missing word > 2 Cool 3 So 1 That\u2019s pretty much 1 I guess what you should do tomorrow 1 I don\u2019t know 1 I wasn\u2019t really paying attention too much Note . Bold \u00bc con\ufb02ict ; italic \u00bc uncertainty . Speakers are numbered based on who spoke \ufb01rst in a transcript . Blocks indi - cated by section divides . Con\ufb02ict types are the dominant type at that utterance . a Speaker is referring to cost calculation formulas Table 4 ( continued ) Speaker Utterance Con\ufb02ict type 1 I think that one we have would actually be 2 The 7 ? 1 Yeah Note . Bold \u00bc con\ufb02ict ; italic \u00bc uncertainty . Speakers are numbered based on who spoke \ufb01rst in a transcript . Blocks indi - cated by section divides . Con\ufb02ict types are the dominant type at that utterance . Con\ufb02ict and uncertainty in design 57 processes play a role ( e . g . , priming e\ufb00ects or blocking e\ufb00ects in memory ) . In the case of implicit processes , statistical evidence , as we used , may be more revealing than qualitative analyses of speci\ufb01c speech . These examples show there is no simple explicit mechanism underlying the connection between un - certainty and con\ufb02ict . 3 . 2 . 3 Uncertainty and subsequent micro - con\ufb02icts Because our data are observational , the temporal relationship between con\ufb02ict and uncertainty could be explained by \u2018third variable confounds : \u2019 For example , it is possible that increases in task di\ufb03culty raise both con\ufb02icts and uncertainty ( instead of con\ufb02ict per se leading to decreases / increases in un - certainty ) . To rule out this alternative explanation , we also tested the e\ufb00ects of uncertainty ( Lag1 and Lag2 ) on subsequent task and process con\ufb02icts , as well as the interaction of lagged uncertainty with type of team on con\ufb02ict . If there are third variable confounds , the reverse temporal relationships should be found for the same con\ufb02ict type - to - uncertainty connections obtained above . We used HLM 7 . 01 to conduct three - level hierarchical logistic regression an - alyses , with dichotomous con\ufb02ict variables as the dependent variables ( pres - ence of con\ufb02ict or not ) and percent of uncertainty utterances in the block ( Lag1 and Lag2 ) as the independent variable . For all models , we controlled for three signi\ufb01cant Level - 1 covariates : number of speakers in the block , total number of words , and percent of on - task talk that was speci\ufb01cally about the hardware product itself . The gender variables ( at either the meeting or team level ) and eventual team success were not signi\ufb01cantly related to any subtype of con\ufb02ict . As with the earlier analyses , we controlled for potential main e\ufb00ects and Lag1 e\ufb00ects . Controlling for those three covariates , we found no signi\ufb01cant e\ufb00ects on over - all con\ufb02ict for either uncertainty Lag1 ( p > 0 . 19 ) , uncertainty Lag2 ( p > 0 . 10 ) , team success ( p > 0 . 80 ) , or , most importantly , the interactions between team success and either uncertainty lag variable ( Lag1 , p > 0 . 32 , Lag2 , p > 0 . 15 ) . There was a signi\ufb01cant positive main e\ufb00ect of uncertainty Lag2 on task con - \ufb02ict , odds ratio \u00bc 1 . 69 ( 1 . 01 , 2 . 82 ) , df \u00bc 3717 , p \u00bc 0 . 044 . However , uncertainty Lag1 ( p > 0 . 12 ) , team success ( p > 0 . 34 ) , and , most importantly , the two in - teractions were not signi\ufb01cant ( success (cid:3) uncertainty Lag1 , p > 0 . 64 , X Lag2 , p > 0 . 55 ) . 9 Of particular relevance , there was a marginally signi\ufb01cant interaction between uncertainty Lag2 and the team success variable on process con\ufb02ict , odds ratio \u00bc 0 . 076 ( 0 . 006 , 1 . 035 ) , df \u00bc 3717 , p \u00bc 0 . 053 , when controlling for the three covariates , uncertainty Lag1 ( p > 0 . 25 ) , uncertainty Lag2 ( p > 0 . 17 ) , team success ( p > 0 . 64 ) , and the interaction between success and uncertainty Lag1 ( p > 0 . 22 ) . While this interaction echoed the interaction between process 58 Design Studies Vol 50 No . C May 2017 con\ufb02ict Lag2 and success on subsequent uncertainty , this interaction was much weaker in size and marginal in signi\ufb01cance . Conducting analyses sepa - rately for low and high success teams , there were no signi\ufb01cant relationships for uncertainty ( Lag2 ) on process con\ufb02ict ( p s > 0 . 18 ) . 3 . 2 . 4 Summary of \ufb01ndings The general pattern of our \ufb01ndings was that , for high success teams , some micro - con\ufb02icts ( particularly process con\ufb02icts ) were negatively related to the prevalence of subsequent uncertainty , whereas for low success teams , some micro - con\ufb02icts were positively related to uncertainty in subsequent blocks . This interaction e\ufb00ect was signi\ufb01cant for both task and process micro - con\ufb02ict subtypes , even though these two types of con\ufb02ict were negatively related to each other . Importantly , low and high success teams did not di\ufb00er overall on their levels of uncertainty or any type of con\ufb02ict ; all teams had reg - ular uncertainty and con\ufb02ict . Instead , the di\ufb00erences by team success were spe - ci\ufb01cally in the interrelationship between prior ( Lag2 ) con\ufb02ict and subsequent uncertainty . Further , there was no strong parallel interaction e\ufb00ect between uncertainty and success on subsequent con\ufb02ict . Task con\ufb02ict was signi\ufb01cantly preceded by high uncertainty ( Lag2 ) as a main e\ufb00ect , but this e\ufb00ect occurred for both types of teams . Thus , the observed temporal relationships between con\ufb02ict and uncertainty cannot be explained by general co - occurrence patterns between con\ufb02ict and uncertainty ( e . g . , triggered by a third variable confound ) . 4 Discussion Using novel methods , this study provides evidence that successful and unsuc - cessful design team problem solving can be di\ufb00erentiated in terms of the nature of the temporal relationship between micro - con\ufb02icts between team members and subsequent psychological uncertainty . We observed that , in successful design teams , micro - con\ufb02icts decreased uncertainty , while in unsuccessful teams , micro - con\ufb02icts comparatively increased uncertainty . These correla - tional \ufb01ndings suggest a new hypothesized distinguishing factor between suc - cessful or unsuccessful problem solving : Successful teams may be achieving better problem solving by harnessing their disagreements to reduce individual uncertainty in the moment . One might wonder whether our \ufb01ndings are simply another story about how successful teams are better at resolving con\ufb02icts . If this were true , the lower success teams should have displayed higher levels of con\ufb02ict ( or lower levels of uncertainty ) overall , compared to the higher success teams . However , this was not the case : The lower success teams did not have signi\ufb01cantly more con - \ufb02ict ( of any type ) or more uncertainty overall than the highly successful teams , including the proportion of quickly resolved con\ufb02icts . Thus , our \ufb01ndings cannot be explained by general di\ufb00erences in propensity to con\ufb02ict or uncer - tainty in low vs . high success teams . Con\ufb02ict and uncertainty in design 59 4 . 1 Implications This study makes a contribution to the design studies literature by enriching our understanding of successful design teams . This study deliberately exam - ines brief , minor con\ufb02icts , and \ufb01nds that for successful teams , micro - con\ufb02icts can be potentially bene\ufb01cial by decreasing uncertainty . Uncertainty , particularly brief uncertainty , is not always negative , but it needs to be managed for problem solving to be successful ( Schunn & Trafton , 2012 ) . For example , problem solving in ill - de\ufb01ned domains ( a common situation in real - world engineering design problem solving ) is inherently imbued with un - certainty related to problem detection , \ufb01nding , and structuring ( Goel & Pirolli , 1992 ; Runco , 1994 ) , activities that occur early in the problem solving process ( Mumford , Reiter - Palmon , & Redmond , 1994 ; Runco , 1994 ) . In the course of solving a problem , this uncertainty is assessed , managed , and reduced . This study suggests that teams d speci\ufb01cally , design teams , which encounter a lot of uncertainty relative to other types of teams in organizations d may success - fully manage uncertainty by iteratively raising and dealing with alternative conceptions in a way that helpfully reduces uncertainty in the moment , leaving time to address more issues that arise in problem solving . This study also builds on other team cognition research . Within conversations , team members engage in problem solving , but also develop , challenge , and maintain underlying shared mental models . In particular , design teams are trained to engage in critical inquiry in order to e\ufb00ectively \ufb01nd and solve prob - lems ( Dym et al . , 2005 ; Sch \u20ac on , 1983 ) . Shared mental models occur when indi - vidual mental models are similar with regards to tasks and teamwork ( e . g . , Burke , Stagl , Salas , Peirce , & Kendall , 2006 ; Johnson - Laird , 1980 ; Mathieu , He\ufb00ner , Goodwin , Salas , & Cannon - Bowers , 2000 ) . Both the accuracy and congruence of shared mental models have been identi\ufb01ed as important aspects of group cognition and positively predictive of team performance ( e . g . , Burke et al . , 2006 ; DeChurch & Mesmer - Magnus , 2010 ) . Disagreements may arise due to di\ufb00erences in shared mental models ( Bearman , Paletz , Orasanu , & Thomas , 2010 ; Cronin & Weingart , 2007 ; Paletz & Schunn , 2010 ) . Our \ufb01nd - ings suggest that for more successful teams , disagreements may be used in un - covering and settling these di\ufb00erences . As Dong ( 2005 , p . 458 ) noted , \u201c Team communication re\ufb02ects the formation of mutual expectations and shared under - standings \u201d ( italics in the original ) . However , even with greater tolerance for disagreement , some design teams may get bogged down in irreconcilable is - sues . Di\ufb00erences in shared mental models d for instance , regarding an ill - de\ufb01ned problem d may be too fundamental for some teams to overcome , and / or disagreement may uncover more underlying di\ufb00erences than the team is capable of handling , leaving a state of increased uncertainty . In partic - ular , the examples noted previously illustrate that unsuccessful teams may be grappling with more challenging issues when they are disagreeing . 60 Design Studies Vol 50 No . C May 2017 While our data uncovered temporal relationships between con\ufb02ict and subse - quent uncertainty , they raise further questions about how disagreements might help design teams successfully manage individual uncertainty during complex problem solving . Our analyses of the reverse relationship generally did not support the simplest explanation : that disagreement and uncertainty simply co - occur . Thus , our data spur more theoretically interesting questions about the causal proximal and distal relationships between con\ufb02ict and uncertainty . For example , are successful teams directly better at harnessing con\ufb02ict for resolving uncertainty , such that they resolve uncertainty by increasing the in - formation exchange during con\ufb02icts ( i . e . , information exchange is a mediator of the disagreement - uncertainty relationship ) ? Does con\ufb02ict - driven uncer - tainty resolution better enable teams to achieve a shared understanding by the end of their project ( e . g . , Agogino et al . , 2006 ) ? Were the less successful teams reacting to disagreement with more tentativeness in order to manage and accommodate con\ufb02ict ( McDonnell , 2012 ) ? In addition , the e\ufb00ect for Lag2 rather than Lag1 suggests an incubation period is necessary : It may sim - ply take time , after a disagreement , for individuals to hear and encode the disagreement and for that to a\ufb00ect uncertainty within individuals , before the change in uncertainty is spoken aloud . One possibility is that con\ufb02ict was associated with problem - solving techniques that then impacted uncertainty . This study aligns with emerging research \ufb01nd - ings from the design \ufb01eld and elsewhere suggesting that , despite general nega - tive e\ufb00ects of self - reported con\ufb02ict on team performance ( e . g . , De Dreu & Weingart , 2003 ) , brief or mild disagreements can , in the right circumstances , have positive outcomes on creativity and cognition ( Chiu , 2008a , 2008b ; Goncalo , Polman , & Maslach , 2010 ; Miron - Spektor , Efrat - Treister , Rafaeli , & Schwartz - Cohen , 2011 ; Paletz et al . , 2013 ; Todorova et al . , 2014 ) . For example , in a study on the Mars scientist conversations , process micro - con\ufb02icts were found to increase the likelihood of analogies soon after ( Paletz et al . , 2013 ) . Analogies are useful for team success and creativity ( Dunbar , 1995 , 1997 ) . Indeed , in successful multidisciplinary expert teams , problem - related analogies reduced uncertainty ( Chan et al . , 2012 ) , and analo - gies and mental simulations can be used to reduce uncertainty ( Ball & Christensen , 2009 ) . These studies suggest that analogy and / or mental simula - tion , or other creative cognitive processes , may be possible mediators of the relationship between con\ufb02ict and uncertainty . In addition , in our data , high uncertainty signi\ufb01cantly preceded task con\ufb02ict with no di\ufb00erence across types of teams , suggesting that uncertainty may serve as a prompt for , or in some indirect way lead to , task - related con\ufb02ict and not for the other subtypes . Finally , these \ufb01ndings have potential practical implications for design teams . Design teams , including student design teams like our sample and unlike some teams in other organizational settings , may be encouraged to disagree as part of their divergent search processes ( Dym et al . , 2005 ) , such that brief Con\ufb02ict and uncertainty in design 61 con\ufb02icts may not be a source of anxiety . Indeed , their existence did not di\ufb00er between successful and unsuccessful teams . However , harnessing con\ufb02ict and gaining the best insights can still be a challenge : Team leaders should be aware of and channel what the teams do with that con\ufb02ict in terms of improving cognition and uncovering or resolving uncertainty . In addition , in non - student teams , managers should also be aware of organizational - and project - level forces that can interfere with or enable shared understanding , as well ( Kleinsmann & Valkenburg , 2008 ) . Taking a broader view , this study echoes that teams with greater di\ufb03culties in achieving shared mental models , such as a mental model of the problems themselves , may simply struggle more than other teams , and those other teams have an easier pathway to success . 4 . 2 Limitations and future work Successful and unsuccessful design teams may di\ufb00er on many features , including their initial resources ( material , intellectual , etc . ) ; the di\ufb03culty of their tasks ; their team processes ( e . g . , Kau\ufb00eld & Lehmann - Willenbrock , 2012 ; Post , 2012 ) ; their use of collaboration tools ( e . g . , Jang & Schunn , 2012 ) ; and their ability to leverage external support . This study includes many controls to rule out reverse causality or these possible third variable ex - planations of the obtained relationships . For example , there were no signi\ufb01cant e\ufb00ects for team or meeting gender composition or team size on the prevalence of uncertainty . Importantly , unsuccessful teams had essentially equal levels of un - certainty and disagreement as successful teams , removing the possible alterna - tive explanation that the successful teams simply had less uncertainty or were less likely to disagree d though not what topics they were uncertain or disagreed about . Nevertheless , the issue of causality is not fully resolved . For example , even though successful teams are likely to reduce uncertainty following micro - con\ufb02icts , this relationship may not have in\ufb02uenced their design success . Future research can also unpack the consequences of these patterns between disagreement and uncertainty on other related processes , such as information search , creativity , mental simulation , and analogy ( e . g . , Ball & Christensen , 2009 ; Chiu , 2008a ; Christensen & Ball , 2016a , 2016b ; Paletz et al . , 2013 ) . Given our examples , it is unlikely that any one of these is the only mediator , but that some combination of implicit and explicit processes exist . This study also examined only one design context in one particular culture , given the highly time - consuming data collection and coding methodology . Other domains with di\ufb00erent types of disciplinary and demographic diversity and levels of expertise ( novice / experts ) should be tested , such as professional design teams across di\ufb00erent countries . Comparisons across contexts and cul - tures will be important for identifying the boundary conditions for observed relationships . One interesting comparison is the relationship between the pre - sent \ufb01ndings and a previous investigation of the relationship between con\ufb02ict and uncertainty in conversations of an expert science team working on the 62 Design Studies Vol 50 No . C May 2017 Mars Exploration Rover mission ( Paletz et al . , 2016 ) . Even though that team overall was highly successful , disagreements about the planning and interpre - tation ( of the results ) of the rover\u2019s scienti\ufb01c expeditions led to an increase in expressed uncertainty . Of course , there are large di\ufb00erences in tasks and par - ticipants across the two studies : the previous study examined members of a professional , long - duration team engaged in scienti\ufb01c discovery ; here , we examine more recently - formed , student engineering teams . One possibility is that the present \ufb01ndings do not apply to the success of teams in general , only to teams engaged in design ( vs . scienti\ufb01c discovery , for example ) . Alter - natively , even successful teams may uncover more uncertainty through con\ufb02ict in extremely novel tasks . Future studies that examine more teams across more contexts may be able to identify contextual factors that may moderate the rela - tionship between team success and con\ufb02ict - uncertainty dynamics . 4 . 3 Conclusion In conclusion , we report our discovery of a temporal relationship between brief disagreements ( a social design process ) and subsequent psychological un - certainty ( a cognitive design process ) that di\ufb00ers for design teams with di\ufb00erent problem - solving success and failure . This \ufb01nding was particularly strong for process con\ufb02icts . This discovery sheds light on the nature of success - ful design in teams and spurs additional theoretical questions about the com - plex factors that underlie team design success . Acknowledgments This research was supported by the United States National Science Founda - tion ( NSF ) Science of Science and Innovation Policy Program Grants # SBE - 1064083 and # SBE - 0830210 to the \ufb01rst author when she was at the Uni - versity of Pittsburgh and to the \ufb01rst and third author , respectively , and NSF # SBE - 0738071 to the third author . We are grateful to Roni Reiter - Palmon and Kevin Soo for comments on previous version of this manuscript , Kevin Kim and Mathilda du Toit for statistical advice , and to Carmela Rizzo for research management support . The authors wish to thank Mike Lovell and Kevin Topolski for data collection ; Andrea Goncher and Howard Kuhn for assisting in the team success variable ; Justin Krill , Jake Volpe , Janeen Bost , Jessica Varone , Andrew Bergman , Stephen Burstein , and Shauna Barnes for transcribing ; Carl Fenderson , Abby Pollick , Stephen Burstein , LaNee Jones , Justin Krill , Allison Haley , Sam Rynearson , Courtney Buchanan , Anna Poul - ton , Kevin Gaitonde , Matt Flounders , Claire James , and Jooyang Jang for assistance in coding ; and Kyle Bogue and Megan Loftus for assistance in data management and validation . Notes 1 . Unless otherwise mentioned , we present the test of the unit - speci\ufb01c model with robust standard errors . HLM 7 . 01 uses penalized quasi - likelihood ( PQL ) estimation . Con\ufb02ict and uncertainty in design 63 2 . This choice was statistically justi\ufb01ed . An HLM null model tests the dependent variable without any predictor variables using chi square estimation to determine whether there are signi\ufb01cant higher - level components . The null model of the uncertainty dependent variable showed that there was signi\ufb01cant variance at both Level 2 ( video clip level ) , Tau beta \u00bc 0 . 06 , X 2 ( 38 ) \u00bc 210 . 41 , p < 0 . 001 , and Level 3 ( team level ) , Tau pi \u00bc 0 . 04 , X 2 ( 20 ) \u00bc 58 . 65 , p < 0 . 001 ( ICC \u00bc 4 . 8 % for Level 2 , ICC \u00bc 3 . 1 % for Level 3 ) . In other words , there was a need to use a multilevel model . 3 . As an o\ufb00set variable , we used ( 1 \u00fe ln [ number of utterances ] ) because the o\ufb00set variable cannot equal zero . 4 . Very similar \ufb01ndings were found when controlling for the number of utterances tran - scribed for each group and using a univariate generalized linear model . 5 . Event rate ratio \u00bc 0 . 83 , ( 0 . 69 , 1 . 01 ) , B \u00bc (cid:2) 0 . 18 , SE \u00bc 0 . 10 , df \u00bc 3870 , p \u00bc 0 . 061 . 6 . Graphs created using unstandardized , uncentered variables because the independent var - iables are dummy coded . Covariate e\ufb00ects evaluated at their mean values ( 2 . 51 for num - ber of speakers in block , and 5 . 54 for average words per utterance ) . 7 . Testing only Lag2 con\ufb02ict blocks so as not to arti\ufb01cially in\ufb02ate relationships between types of con\ufb02ict . 8 . Event ratio \u00bc 0 . 80 ( 0 . 63 , 0 . 999 ) , df \u00bc 3736 , p \u00bc 0 . 049 . There was a marginal negative e\ufb00ect for task con\ufb02ict Lag2 on subsequent uncertainty for high success teams , event ratio \u00bc 0 . 84 ( 0 . 70 , 1 . 02 ) , df \u00bc 1782 , p \u00bc 0 . 073 , but no signi\ufb01cant relationship between the variables in low success teams ( p > 0 . 56 ) . 9 . 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S . patent data - base ) could significantly accelerate innovation and discovery by providing people with inspiration from solutions to analogous prob - lems . However , finding useful analogies in these large , messy , real - world repositories remains a persistent challenge for either human or automated methods . Previous approaches include costly hand - created databases that have high relational structure ( e . g . , predicate calculus representations ) but are very sparse . Simpler machine - learning / information - retrieval similarity metrics can scale to large , natural - language datasets , but struggle to account for structural similarity , which is central to analogy . In this paper we explore the viability and value of learning simpler structural representations , specifically , \u201cproblem schemas\u201d , which specify the purpose of a product and the mechanisms by which it achieves that purpose . Our approach combines crowdsourcing and recurrent neural networks to extract purpose and mechanism vector representations from product descriptions . We demonstrate that these learned vectors allow us to find analogies with higher precision and recall than tra - ditional information - retrieval methods . In an ideation experiment , analogies retrieved by our models significantly increased people\u2019s likelihood of generating creative ideas compared to analogies re - trieved by traditional methods . Our results suggest a promising approach to enabling computational analogy at scale is to learn and leverage weaker structural representations . KEYWORDS Computational analogy ; innovation ; creativity ; product dimensions ; text mining ; text embedding 1 INTRODUCTION The ability to find useful analogies is critical to driving innovation in a variety of domains . Many important discoveries in science were driven by analogies : for example , an analogy between bacteria and slot machines helped Salvador Luria advance the theory of bacterial mutation . Analogical reasoning forms the foundation of law , with the effectiveness of an argument often dependent on its Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM mustbehonored . Abstractingwithcreditispermitted . Tocopyotherwise , orrepublish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from permissions @ acm . org . KDD \u201917 , August 13 - 17 , 2017 , Halifax , NS , Canada \u00a9 2017 ACM . 978 - 1 - 4503 - 4887 - 4 / 17 / 08 . . . $ 15 . 00 DOI : 10 . 1145 / 3097983 . 3098038 legal precedents [ 32 ] . Innovation is often spurred by analogy as well : an analogy to a bicycle allowed the Wright brothers to design a steerable aircraft . Whether architecture , design , technology , art , or mathematics , the ability to find and apply patterns from other domains is fundamental to human achievement [ 9 , 17 , 22 , 26 ] . The explosion of available online data represents an unprece - dented opportunity to find new analogies and accelerate human progress across domains . For example , the US Patent database has full text for more than 9 million patents issued from 1976 to the present . InnoCentive 1 contains more than 40 , 000 business , social , policy , scientific , and technical problems and solutions . Quirky 2 , a company that assists inventors in the development process , has had over 2 million product idea submissions . OpenIDEO 3 receives hundreds of solutions for a variety of social problems . Millions of scientific papers and legal cases are searchable on Google Scholar . We believe these data form a treasure trove of analogies that can accelerate problem solving , innovation and discovery . In a striking recent example , a car mechanic invented a simple device to ease difficult childbirths by drawing an analogy to extracting a cork from a wine bottle , which he discovered in a YouTube video . This award - winning device could save millions of lives , particularly in developing countries . We imagine a future in which people could search through data based on deep analogical similarity rather than simple keywords ; lawyers or legal scholars could find legal precedents sharing similar systems of relations to a contemporary case ; and product or service designers could mine myriad potential solutions to their problem . However , sifting through these massive data sources to find relevant and useful analogies poses a serious challenge for both humans and machines . In humans , memory retrieval is highly sensitive to surface similarity , favoring near , within - domain analogs that share object attributes over far , structurally similar analogs that share object relations [ 13 \u2013 15 , 20 ] . Analogical processing also incurs a heavy cognitive load , taxing working memory when even a few relations are required to be processed at once [ 16 ] . Thus searching through datasets with thousands or millions of items for structurally similar ones may be a daunting prospect . Finding analogies is challenging for machines as well , as it is based on having an understanding of the deep relational similarity between two entities that may be very different in terms of sur - face attributes [ 12 ] . For example , Chrysippus\u2019 analogy between sound waves and water waves required ignoring many different surface features between the two [ 20 ] . Recent advances in data 1 innocentive . com 2 quirky . com 3 OpenIDEO . com KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 235 mining and information retrieval include a variety of natural lan - guage techniques that use words , parts of speech or other language feature - based vector representations in order to calculate similarity measures ( see [ 31 ] ) . Examples include word embedding models like Word2Vec [ 24 ] , vector - space models like Latent Semantic Index - ing [ 10 ] , and probabilistic topic modeling approaches like Latent Dirichlet Allocation [ 4 ] . These approaches excel at detecting sur - face similarity , but are often unable to detect similarity between documents whose word distributions are disparate . The problem is especially acute when the source and target domains are different ( for example , bacterial mutation and slot machines ) . Another approach to finding analogies has been to use the struc - tural similarity of sentences or texts , such as using coupled clus - tering for detecting structural correspondence of text [ 5 , 35 ] . How - ever , these approaches typically require rich data sets with clear substructures , whereas most descriptions of problems or ideas in existing online databases are short , sparse , or lack consistent struc - ture . Other current methods focus on very narrow analogy tasks , such as four - term analogy problems ( teacher : student = doctor : ? ) , in particular with short strings ( ABC : ABD = KJI : ? ) [ 19 ] . In contrast , we wish to find analogies in real world data , which involve complex representations and a diverse set of analogical relations . In this paper , we are interested in automatically discovering analogies in large , unstructured data sets . In particular , we focus on a corpus of product innovations . There are two in - sights behind our approach that we believe may make this problem tractable despite its longstanding status as a \u201choly grail\u201d in both cognitive science and AI . First , rather than trying to solve the prob - lem of fully structured analogical reasoning , we instead explore the idea that for retrieving practically useful analogies , we can use weaker structural representations that can be learned and reasoned with at scale ( in other words , there is a tradeoff between the ease of extraction of a structure and its expressivity ) . Specifically , we investigate the weaker structural representation of an idea\u2019s pur - pose and mechanism as a way to find useful analogies . The second insight is that advances in crowdsourcing have made it possible to harvest rich signals of analogical structure that can help machine learning models learn in ways that would not be possible with existing datasets alone . This paper combines these two ideas to contribute a technique for computationally finding analogies from unstructured text datasets that go beyond surface features . At a high level , our approach uses the behavioral traces of crowd workers searching for analogies and identifying the purpose and mechanisms of ideas , then developing machine learning models that develop similarity metrics suited for analogy mining . We demonstrate that learning purpose and mechanism representations allows us to find analogies with higher precision and recall than traditional information - retrieval methods based on TF - IDF , LSA , LDA and GloVe , in challenging noisy set - tings . Furthermore , we use our similarity metrics to automatically find far analogies \u2013 products with high purpose similarity , and low mechanism similarity . In a user study , we show that we are able to \u201cinspire\u201d participants to generate more innovative ideas than alternative baselines , increasing the relative proportion of positively - rated ideas by at least 25 % . 2 LEARNING A REPRESENTATION FOR ANALOGIES 2 . 1 Motivation Much work in computation analogy has focused on fully structured data , often with logic - based representations . For example [ 11 ] , CAUSE ( GREATER - THAN [ TEMPERATURE ( coffee ) , TEMPERATURE ( ice - cube ) ] , FLOW ( coffee , ice - cube , heat , bar ) ) These representations , while very expressive , are notoriously difficult to obtain . In this section , we investigate a weaker struc - tural representation . Our goal is to come up with a representation that can be learned , while still being expressive enough to allow analogical mining . Analogies between product ideas are intricately related to their purpose and mechanism . Informally , we think of a product\u2019s purpose as \u201cwhat it does , what it is used for\u201d , and a product\u2019s mechanism is \u201chow it does it , how it works\u201d . The importance of a product\u2019s purpose and mechanism as core components of analogy are the - oretically rooted in early cognitive psychology work on schema induction which define the core components of a schema as a goal and proposed solution to it ( e . g . , [ 15 ] ) . More recently , the practical value of defining a problem schema as a purpose and mechanism has been demonstrated to have empirical benefits in finding and using analogies to augment idea generation ( e . g . , [ 38 \u2013 41 ] ) . Separating an idea into purpose and mechanisms enables core analogical innovation processes such as re - purposing : For a given product ( such as a kitchen - sink cleaner ) and its purpose , finding another way to put it to use ( cleaning windows ) . To that end , as - sume ( for the moment ) that we have for each product i two vectors , p i and m i , representing the product\u2019s purpose and mechanism , respectively . Using this representation , we are able to apply rich queries to our corpus of products , such as : \u2022 Same purpose , different mechanism . Given the corpus of all products P , a product i with ( normalized ) purpose and mech - anism vectors p i , m i , and distance metrics d p ( \u00b7 , \u00b7 ) , d m ( \u00b7 , \u00b7 ) be - tween purpose and mechanism vectors ( respectively ) , solve : argmin \u02dci \u2208P d p ( p i , p \u02dc i ) s . t . d m ( m i , m \u02dc i ) \u2265 threshold , ( 1 ) \u2022 Same Mechanism , different purpose . Solve : argmin \u02dci \u2208P d m ( m i , m \u02dc i ) s . t . d p ( p i , p \u02dc i ) \u2265 threshold , ( 2 ) The decomposition of products into purpose and mechanism also draws inspiration from engineering functional models and ontolo - gies for describing products [ 18 ] . Although there is no set common definition of functions [ 25 ] , much research on functionality has been conducted in areas such as functional representation , engineer - ing design and value engineering . The scope of these ontologies , however , is highly \u201cmechanistic\u201d or engineering - oriented , while in many cases we observe in product data the purpose of a product is more naturally understood in others term \u2013 such as whether it is for entertainment , leisure , or more serious purposes , who is the target user ( adults , children ) , and so forth . KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 236 Importantly , our dataset of product descriptions contains noisy texts , often written informally by non - professional people . In these texts product descriptions are often lacking detail or are ill - defined . To automatically describe a product in terms of a formal functional model would require an inordinate amount of meticulous data annotation and collection by professional engineers over a large number of product descriptions . We thus resort to a softer approach , hoping that a compromise on the level of detail will enable data - driven methods to automatically extract useful representations of product purpose and mechanism . Finally , we also make note of the potentially wider applicability of automatically extracting these representations from real - word product descriptions . Identifying the key components and func - tions of products could conceivably improve ( or augment ) search capabilities in internal or external product databases , and perhaps enhance recommender systems by better understanding what a user is looking for in a product and what a product offers . This last idea is connected to a line of work on \u201cproduct dimensions\u201d [ 23 ] , in which it is shown that implicitly identifying the properties of products ( such as that Harry Potter is a book about wizards ) , helps in improving recommendations . The authors propose a method that combines ratings data with textual product reviews , hoping to implicitly recover topics in the text that inform recommendations . We too look at product dimensions , but target only two that are more abstract and broad , and directly learn them in a supervised fashion from annotated data . 2 . 2 Data Innovation Corpus . We test our approach with a corpus of prod - uct descriptions from Quirky . com , an online crowdsourced product innovation website . Quirky is representative of the kinds of datasets we are interested in , because it is large ( at the time of writing , it hosts upwards of 10 , 000 product ideas , of which our corpus included 8500 ) , unstructured ( ideas are described in natural language ) , and covers a variety of domains ( invention categories ) which makes cross - domain analogies possible . The following example illustrates the typical length and \u201cmessiness\u201d of product ideas in this dataset : Thirsty too Pet water bowl / dispenser for your vehicle cup holder . Over spill lip to catch water Has optional sleeve for larger cup holders Optional floor base One way valve so water cant over flow from bottle Small reservoir Reservoir acts as backsplash Water bottle attachment Holds water in your vehicle cupholder for pet Foldable handle to get unit out of holder Dishwasher safe Optional sleeve for larger cup holders Collecting Purpose and Mechanism Data . In addition to the Quirky innovation corpus , we needed to collect analogy - specific data to train our model . Previous approaches to creating structured representations of items for analogical computation , for example predicate logic , are extremely heavyweight and can take tens of person - hours for complex items [ 37 ] . Instead , we aim to develop a lightweight task that avoids complex structure but instead relies on the cognitive expertise and intuitions of people to be able to separate the purpose of a product from its mechanism . By doing so we can Figure 1 : Collectingpurposeandmechanismannotationsfromthe crowd . scale up the collection of purpose and mechanism labels through the use of microtasks and crowdsourcing markets [ 21 ] . Specifically , we show Amazon Mechanical Turk ( AMT ) crowd workers a product description , asking them to annotate the parts of the text they consider to be about the purposes of the product , and the parts related to mechanisms . We frame the problem in simple terms , guiding workers to look for words / phrases / chunks of text talking about \u201cwhat the product does , what it is good for\u201d ( purposes ) , and \u201chow it works , what are its components\u201d ( mechanisms ) . As seen in Figure 1 , we juxtapose two copies of the product text side - by - side , to ease cognitive load and encourage workers not to give purpose and mechanism tags that are too similar or overlapping , thus capturing a potentially richer and more distinct signal . Our corpus consisted of 8500 products . Each product was annotated by four workers . Collecting Analogies . In previous , preliminary work [ 7 ] , we ex - plored the use of crowdsourcing to label analogies , collecting la - beled examples of analogies ( product pairs ) that were fed into a metric - learning deep learning model . While showing promising results , the process of collecting labeled analogies proved expen - sive , requiring considerable cognitive effort from workers and thus more time , limiting the number of labels that can realistically be collected . In addition , in that work , the deep learning model was blind to the rich structures of purpose and mechanism , and had no hope of recovering them automatically due to relative data scarcity . In this paper we take a different approach , focusing our resources on collecting purpose and mechanism annotations from the crowd , while collecting only a small number of curated labeled analogies strictly for the purpose of evaluation ( see Section 3 . 1 for details ) . 2 . 3 Method 2 . 3 . 1 Extracting Purpose and Mechanism vectors . In this section , we describe our approach to learning to extract purpose and mech - anism product representations . We begin with a set of N training product texts X N = { x 1 , x 2 , . . . , x N } , where each x i is a variable - length sequence of tokens ( x 1 i , x 2 i , . . . , x Ti ) . For each document x i , we collect a set of K purpose annotations and K mechanism an - notations , where K is the number of workers who annotate each document . We define the purpose annotation to be a binary vector \u02dcp i k = ( \u02dc p 1 i k , \u02dc p 2 i k , . . . , \u02dc p Ti k ) of the same length as x i , with \u02dc p ji k = 1 if token x ji is annotated as purpose by annotator k , \u02dc p ji k = 0 if not . In the same way , we denote KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 237 the mechanism annotation with \u02dcm i k = ( \u02dc m 1 i k , \u02dc m 2 i k , . . . , \u02dc m Ti k ) While on the surface this setting appears to lend itself naturally to sequence - to - sequence learning [ 33 ] , there are a few important differences . A key difference is that in our setting the problem of interest is not to learn to recover the latent \u02dcp ( \u02dcm ) exactly for unseen products , but rather to extract some form of representation that captures the overall purpose and mechanism . That is , what we do care about is the semantic meaning or context our representation captures with respect to product purposes or mechanisms , rather than predicting any individual words . Additionally , sequence - to - sequence models typically involve heavier machinery and work well on large data sets , not suitable for our scenario were we have at most a few thousands of tagged examples . On a more technical note , instead of one sequence in the output , we now have K . A simple solution is to aggregate the annotations , for example by taking the union or intersection of annotations , or considering a token x ji to be positively annotated if it has at least K 2 positive labels . Richer aggregations may also be used . Considering that our focus here is to capture an overall rep - resentation ( not predict a precise sequence ) , however , we resort to a simple and soft aggregation of the K annotations . In simple terms , we look at all words that were annotated , and take a TF - IDF - weighted average of their word vectors . In more formal terms , let w i = ( w 1 i , w 2 i , . . . , w Ti ) be the sequence of GloVe [ 27 ] word vectors ( pre - trained on Common Crawl web data ) , representing ( x 1 i , x 2 i , . . . , x Ti ) . We select all x i word vectors for which \u02dc p ji k = 1 ( \u02dc m ji k = 1 ) for some k , and concatenate them into one sequence . We then compute the TF - IDF scores for tokens in this sequence , find the D tokens with top TF - IDF scores ( D = 5 in our experiments ) , and take the TF - IDF - weighted average of their corresponding D GloVe vectors . We denote the resulting weighted - average vectors as p i \u2208 R 300 and m i \u2208 R 300 for purpose and mechanism annotations , respectively . We consider p i , m i to be target vectors we aim to predict for unseen texts . Embedding ( short ) texts with a weighted - average of word vectors ( such as with TF - IDF ) can lead to surprisingly good results across many tasks [ 2 ] . Furthermore , in our case this simple weighted - average has several advantages . As we will next see , it lends itself to a straightforward machine learning setting , suitable for our modestly - sized data set , and for the objective of finding an overall vector representation that can be used in multiple ways , chiefly the computation of purpose - wise and mechanism - wise distances between products . Additionally , by concatenating all annotations and weighting by TF - IDF , we naturally give more weight in the average vector to words that are more frequently annotated \u2013 thus giving higher impact to words considered important by all annota - tors with respect to purpose / mechanism . 2 . 3 . 2 Learning purpose and mechanism . We now have N train - ing product texts X N = { x 1 , x 2 , . . . , x N } , and N corresponding target tuples Y N = { ( p 1 , m 1 ) , ( p 2 , m 2 ) , . . . , ( p N , m N ) } . We repre - sent each x i with its pre - trained GloVe vectors , w i . Our goal is to learn a function f ( w i ) that predicts ( p i , m i ) . To this end , we model f ( \u00b7 ) with a Recurrent Neural Network as follows . The network takes as input the variable - length sequence w i . This sequence is processed with a bidirectional RNN ( BiRNN ) [ 3 ] with one GRU layer . The BiRNN consists of the forward GRU \u2212\u2212\u2212\u2192 GRU which reads the sequence w i from w 1 i to w Ti , and a backward GRU \u2190\u2212\u2212\u2212 GRU which reads from w Ti to w 1 i , thus in practice capturing the neighborhood of w ji from \u201cboth directions\u201d : \u2212\u2192 h ji = \u2212\u2212\u2212\u2192 GRU ( w ji ) , \u2190\u2212 h ji = \u2190\u2212\u2212\u2212 GRU ( w ji ) , h ji = [ \u2212\u2192 h ji , \u2190\u2212 h ji ] , where we concatenate forward and backward GRU hidden states to obtain h ji , our representation for word j in product i . In our case , we are interested in h Ti which captures the entire product text . Next , let W p and W m be purpose and mechanism weight matrices , respectively . h Ti is a shared representation of the doc - ument , which we now transform into two new vectors , \u02c6p i , \u02c6m i , forming our purpose and mechanism predictions for product i : \u02c6p i = W p h Ti , \u02c6m i = W m h Ti . ( 3 ) Parameters in this network are then tuned to minimize the MSE loss averaged over ( p i , m i ) . In some scenarios , we may care more about predicting either purpose or mechanism , and in that case could incorporate a weight term in the loss function , giving more weight to either p i or m i . 2 . 3 . 3 Purpose and Mechanism vector interpretations . Here , we give intuition about the kinds of representations extracted and the ability to interpret them with very simple tools . We first compute \u02c6p , \u02c6m for held - out product texts . Then , in the first approach to interpreting purpose and mechanism predictions , we find the top 10 GloVe word vectors w most similar to each of \u02c6p , \u02c6m , among all vectors that appear in our vocabulary . In the second approach , we aim to recover a set of 10 word vectors such that their sparse linear combination approximately gives \u02c6p or \u02c6m . More formally , in the spirit of the sparse coding approach in [ 1 ] , consider the collection of all word vectors in our vocabulary V , w 1 , w 2 , . . . , w | V | . We stack them into a matrix W . We aim to solve the following optimization problem : argmin a | | \u02c6p i \u2212 Wa | | 22 s . t . | | a | | 0 \u2264 10 , ( 4 ) where a is a weight vector . Optimization can be done with the Orthogonal Matching Pursuit ( OMP ) [ 6 ] greedy algorithm . In Table 1 , we display some examples of applying these two simple methods , to product texts in test data ( not seen during training ) . The first product is a yogurt maker machine , used for concentrating yogurt under heat , and to reduce time and energy . We observe that words selected as most related to our purpose vector representation include food , produce , concentrate , making , energy , reduce and also words that are typical in the language used in describing advantages of products in our data , such as especially , whole , enough , much . Mechanism words , are indeed overall of a much more \u201cmechanical\u201d nature , including liquid , heat , cooling , pump , steel , machine . In the other examples too , we observe the same pattern : Words selected as most closely - related to purpose or mechanism representations , using simple techniques , empirically appear to reflect corresponding properties in the product text , both in language and in deeper meaning . KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 238 Table 1 : Purpose and Mechanism vector interpretation examples . Descriptions shortened . Sparse coding shows only words with | \u03b1 | \u2265 0 . 1 Product Purpose words Mechanism words A small yogurt maker machine for con - centrating yogurt under heat and vac - uum . Has a round base in drum with customized scooper , washable stainless steel drum parts . Reduce time and en - ergy used . Top similar : concentrate , enough , food , even , much , especially , reduce , produce , whole Sparse coding : making , energy , yo - gurt , drum , concentrate , vacuum , heavy , foods , aches , service Top similar : liquid , heat , cooling , pump , steel , machine , water , heating , electric Sparse coding : vacuum , cooled , drum , heavy , ingredients , design , renewable , stainless , vending A cover placed on a car truck to protect from hail . Elastic perimeter to prevent wind from blowing under cover . Snap or velcro slits to open door without re - moving cover . Strong attachment so it wonfit blow away . Inflatable baffles that cover the top , front windshield , side . Top similar : storm , hail , rain , roofs , doors , wind , front , winds , walls Sparse coding : roof , hail , padded , ob - structing , defenses , diesel , windshield , wets Top similar : roof , cover , lining , zipper , bottom , hood , plastic , flap , rubber Sparse coding : front , cover , insulation , hail , buckle , sling , watertight , cutter , blowing A leash accessory with removable com - partments for phone , cards cash , keys , poop bags , treats , bowl . Walk your dog and carry your essentials without pock - ets or a purse bag . Top similar : bags , purse , wallet , carry , leash , backpack , pocket , dog , luggage Sparse coding : bag , leash , compart - ments , pets , phone , eats , practical , hand - ing , pull Top similar : leash , pouch , purse , pocket , bags , pockets , strap , compart - ment , backpack Sparse coding : leash , bag , compart - ments , hand , holders 3 EVALUATION : ANALOGIES As typically done in the context of learning document representa - tions , the key approach to quantitative evaluation is a down - stream task such as document classification . We now evaluate the pre - dicted \u02c6p i , \u02c6m i in the context of their ability to capture distances that reflect analogies , which is the primary focus of this paper . To do so , we first create a dataset of analogies and non - analogies . 3 . 1 Collecting analogies via crowdsourcing We crowdsourced analogy finding within a set of about 8000 Quirky products . AMT crowd workers used our search interface to collect analogies \u2013 pairs of products \u2013 for about 200 seed documents . The search task is powered by a simple word - matching approach . To deal with word variants , we added lemmas for each word to the bag - of - words associated with each product . Each search query was also expanded with lemmas associated with each query term . Search results were ranked in descending order of number of matching terms . Median completion time for each seed was 7 minutes ( work - ers could complete as many seeds as they wanted ) . Further , to deal with potential data quality issues , we recruited 3 workers for each seed ( to allow for majority - vote aggregation ) . Pairs that were tagged as matches became positive examples in our analogy dataset . However , coming up with negative exam - ples was more difficult . Borrowing from information retrieval , we assume that people read the search results sequentially , and treat the implicitly rejected documents ( i . e . , documents that were not matches , despite appearing before matches ) as negatives . It is im - portant to remember that these documents are not necessarily real negatives . To further increase the chance that the document has actually been read , we restrict ourselves to the top - 5 results . Challenges . Getting workers to understand the concept of analo - gies and avoiding tagging products that are superficially similar ( e . g . , \u201cboth smartphone - based\u201d ) as analogies proved a challenge . To address this , we scaffolded the search task by first requiring workers to generate a schema ( or \u201cpattern\u201d ) to describe the core purpose and mechanism of the product , first in concrete terms , and then in more abstract terms ( see an example pattern Figure 2 ) . Workers were then instructed to find other products that matched the abstract schema they created . We found that this scaffolded workflow reduced the number of superficial matches ; yet , a non - negligible portion of the pairs labeled as positive were either superficial matches or near analogies ( i . e . , analogies with many shared surface features ) , likely due to the strong tendency towards surface features in analogical retrieval [ 14 ] . Further , because products were multifaceted , search results may have been implicitly rejected even if they were anal - ogous to the seed if the matching schema was different from the one initially identified by the worker . 3 . 2 Quantitative results In Table 2 , we present precision and recall @ K results . We rank all pairs in the test data ( N = 2500 , with training done on about 5500 products ) based on their distances , according to various metrics , including our own . In summary , across all levels our approach outperformed the baselines , despite a challenging noisy setting . A considerable portion of test product pairs were tagged by workers as analogies despite having only surface similarity , creating mis - labeled positive examples that favor the surface - based baselines . In addition to ranking by purpose - only and mechanism - only , we also concatenate both representations in a vector [ p i , m i ] for prod - uct i , and observe an overall improvement in results , although the \u201cone - dimensional\u201d use of either purpose or mechanism alone still beats the baselines . Using m i only led to considerably better results when looking at precision @ top 1 % , perhaps indicating a tendency by workers to find more mechanism - based analogies . 4 EVALUATION : IDEATION BY ANALOGY Since a major application of the enhanced search and retrieval capabilities of analogy is enhanced creativity , we now evaluate the usefulness of our algorithms . We examine the degree to which our model\u2019s retrieved output improves people\u2019s ability to generate KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 239 Figure 2 : Screenshot of analogy search interface Table 2 : Model results . Precision , recall of positive labels @ top - scoring pairs ( ranked by similarity ) . Method Top 1 % Top 5 % Top 10 % Top 15 % Top 20 % Top 25 % Glove + TF - IDF ( top 5 words ) 0 . 565 , 0 . 018 0 . 515 , 0 . 081 0 . 489 , 0 . 153 0 . 468 , 0 . 22 0 . 443 , 0 . 277 0 . 434 , 0 . 339 Glove + TF - IDF 0 . 609 , 0 . 019 0 . 559 , 0 . 087 0 . 487 , 0 . 152 0 . 47 , 0 . 22 0 . 449 , 0 . 281 0 . 426 , 0 . 332 TF - IDF 0 . 63 , 0 . 02 0 . 537 , 0 . 084 0 . 5 0 . 156 0 . 468 , 0 . 22 0 . 464 , 0 . 29 0 . 441 , 0 . 344 LSA 0 . 413 , 0 . 013 0 . 463 , 0 . 072 0 . 446 , 0 . 14 0 . 435 , 0 . 204 0 . 413 , 0 . 258 0 . 399 , 0 . 312 LDA 0 . 435 , 0 . 014 0 . 432 , 0 . 067 0 . 414 , 0 . 129 0 . 398 , 0 . 187 0 . 384 , 0 . 24 0 . 381 , 0 . 298 Purpose only 0 . 674 , 0 . 021 0 . 586 , 0 . 092 0 . 535 , 0 . 167 0 . 505 , 0 . 237 0 . 496 , 0 . 31 0 . 465 , 0 . 363 Mechanism only 0 . 739 , 0 . 023 0 . 586 , 0 . 092 0 . 551 , 0 . 172 0 . 507 , 0 . 237 0 . 482 , 0 . 301 0 . 47 , 0 . 368 concat ( Purpose , Mechanism ) 0 . 696 , 0 . 022 0 . 612 , 0 . 096 0 . 555 , 0 . 173 0 . 507 , 0 . 237 0 . 504 , 0 . 315 0 . 478 , 0 . 373 creative ideas , compared to other methods . To do so we use a standard ideation task in which participants redesign an existing product [ 36 ] , and are given inspirations to help them \u2013 either from our approach , a TF - IDF baseline , or a random baseline . See Figure 3 for an example task given to crowdworkers . Here , the task was to redesign a cell phone case that can charge the phone . The middle part shows the top 3 inspirations per condition . Our assumption is that our approach will provide participants with useful examples that are similar in purpose but provide diverse mechanisms that will help them explore more diverse parts of the design space in generating their ideas . We hypothesize that this approach will lead to better results than the TF - IDF baseline ( highly relevant but non - diverse inspirations , focusing on surface features ) and the random baseline ( highly diverse but low relevance ) . 4 . 1 Generating near - purpose far - mechanism analogies To generate inspirations for the redesign task , we start by using the learned purpose and mechanism representations p i , m i for each document i ( in the test set ) to apply rich queries to our corpus of products . In particular , assuming all vectors are normalized to unit euclidean norm , we can find pairs of products i 1 , i 2 such that d p ( p i 1 , p i 2 ) = p i 1 \u00b7 p i 2 is high ( near purpose ) , while d m ( m i 1 , m i 2 ) = m i 1 \u00b7 m i 2 is low ( far mechanism ) . This type of reasoning , as discussed above , is a core element of analogical reasoning . We take this idea one step forward by clustering by purpose and diversifying by mechanism . In more detail , we take a set of 2500 products not seen during training , and follow a simple and intuitive procedure as follows . Let P T denote our corpus of test - set products . Let S denote the number of seed products we wish to use in our experiment . Let M denote the number of inspirations we wish to produce for each seed { 1 , . . . , P } . Clustering by purpose . First , we find groups of products with similar purpose by clustering by our purpose representation . \u2022 Run K - means ( K = 50 ) , based on vectors p i , \u2200 i \u2208 P T . ( Note that when all vectors are normalized , the euclidean norm on which K - means is based is equivalent to the cosine distance ) . \u2022 For each cluster k \u2208 { 1 , . . . , K } , compute an intra - distance measure ( purpose homogeneity ) d k . We use the MSE . Prune clusters with less than M instances . Rank clusters by d k in de - scending order , pick top P . Call this set of clusters K top - purpose , with corresponding cluster centers \u00af p 1 , . . . , \u00af p S . \u2022 For each cluster k in K top - purpose , select the product i whose vector p i is nearest to the cluster center \u00af p k . This is our k th seed product , denoted by s k . Result diversification by mechanism . We now have a set of seed products , each with a corresponding cluster of products with similar purposes . Next , we need to pick M inspirations per seed . For each seed s k , we have a set of candidate matches U s k , all from the same purpose cluster . We empirically observe that in KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 240 the purpose - clusters K top - purpose we generate , some vectors are highly similar to the seed with respect to mechanism , and some less so . In order to generate far - mechanism results for each seed from candidate set U s k , we now turn to diversification of results . The problem of extracting a well - diversified subset of results from a larger set of candidates has seen a lot of work , prominently in the context of information retrieval ( which is closely related to our setting ) . In our case , we assume to have found a set of relevant results U s k according to purpose metric d p ( \u00b7 , \u00b7 ) , and diversify by mechanism metric d m ( \u00b7 , \u00b7 ) . There are many ways to diversify results , mainly differing by objective function and constraints . Two canonical measures are the MAX - MIN and MAX - AVG dispersion problems [ 28 ] . In the former , we aim to find a subset M \u2286 U s k such that | M | = M , and min m i 1 , m i 2 \u2208M d m ( m i 1 , m i 2 ) is maximized . In the latter , we aim to find a subset M \u2286 U s k such that | M | = M , and 2 M ( M \u2212 1 ) (cid:213) m i 1 , m i 2 \u2208M d m ( m i 1 , m i 2 ) is maximized . In other words , in the MAX - MIN problem we find a subset of products M such that the distance between the two nearest products is maximized . In the MAX - AVG problem , we find a subset such the average distance between pairs is maximized . Both problems admit simple greedy algorithms with constant - factor approximations [ 28 ] . We choose the MAX - MIN problem , since we want to avoid displaying too - similar results even once to a user ( who may become frustrated and not proceed to read more inspirations ) . We solve the problem using the GMM algorithm mentioned in [ 28 ] . Each iteration of GMM selects a candidate m \u2208 U s k \u2212M such that the minimum distance from m to an already - selected product in M is the largest among remaining candidates in U s k \u2212M , where we measure distance according to our mechanism metric d m ( \u00b7 , \u00b7 ) . In our experiments , we set P = 12 , M = 12 , for 12 seeds and 12 matches each , respectively . 4 . 2 Experiment design We recruited 38 AMT workers to redesign an existing product , a common creative task in design firms [ 36 ] . To ensure robustness of effects , the experiment included 12 different \u201cseed\u201d products . Participants were paid $ 1 . 5 for their participation . To maximize statistical power , we utilized a within - subjects design with a single manipulated factor , inspiration type : \u2022 ANALOGY : participants receive 12 product inspirations re - trieved by our method detailed above , using near - purpose far - mechanism clustering and diversification . \u2022 BASELINE : SURFACE : participants receive product inspira - tions retrieved using TF - IDF , by finding the top 12 products similar to the seed . This baseline is meant to simulate current search engines . \u2022 BASELINE : RANDOM : participants receive 12 product inspi - rations sampled at random from our product corpus . Since we used a within - subjects design , participants completed the redesign task under each of the 3 inspiration type conditions . Figure 3 : Overview and excerpts of the ideation experiment . Top : Seed product . Workers were asked to solve the same problem in a different way . Middle : Top 3 inspirations for each of the condi - tions . Note that the TF - IDF baseline returns results from the same domain , while our method returns a broader range of products . Bot - tom : Ideas generated by users exposed to the different conditions . The order of conditions was counterbalanced to prevent order ef - fects . To ensure unbiased permutations , we used the Fisher - Yates shuffle to assign seeds to conditions , so that every seed would be seen in all conditions ( by different users ) . Since prior work has shown that people benefit more from analo - gies if they receive them after ideation has begun [ 34 ] , the ideation task proceeded in two phases : 1 ) generating ideas unassisted for one minute , then 2 ) receiving 12 inspirations and generating more ideas for 6 minutes . The inspirations were laid out in four pages , 3 inspirations per page , and the users could freely browse them . Figure 3 provides an overview of the experiment and an excerpt from the data . The original task was to redesign an existing product , in this case a cell phone charger case . The SURFACE baseline retrieves products that are very phone - related ( or case related ) . In contrast , our algorithm retrieves diverse results such as a human pulley - powered electricity generator suit . The bottom of the figure shows ideas generated by users in each condition . Interestingly , KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 241 the user exposed to our approach suggested a case that generates power using movement , potentially inspired by the suit . 4 . 3 Results Measures . We are interested in the ability of our approach to en - hance people\u2019s ability to generate creative ideas . Following [ 29 ] , we measured creative output as the rate at which a participant gener - ates good ideas . We recruited five graduate students to judge each idea generated by our participants as good or not . Our definition of \u201cgood\u201d follows the standard definition of creativity in the literature as a combination of novelty , quality , and feasibility [ 30 ] . Each judge was instructed to judge an idea as good if it satisfied all of the follow - ing criteria : 1 ) it uses a different mechanism / technology than the original product ( novelty ) , 2 ) it proposes a mechanism / technology that would achieve the same purpose as the original product ( qual - ity ) , and 3 ) could be implemented using existing technology and does not defy physics ( feasibility ) . Agreement between the judges was substantial , with a Fleiss kappa of 0 . 51 , lending our measure of creativity acceptable inter - rater reliability . The final measure of whether an idea was good or not was computed by thresholding the number of votes , so that good = 1 if at least k judges rated it as good . We report results for both liberal and strict settings k = 2 , 3 . Evaluation . For k = 2 , out of 749 total ideas collected , 249 ideas were judged as good by this measure . As mentioned above , we use the Fisher - Yates shuffle to assign seeds to conditions . To take a conservative approach , as a first step we look only at seeds that appeared across all three conditions ( 9 such seeds ) , to put the con - ditions on par with one another . By this slicing of the data , there were 208 good ideas . The proportion of good ideas in our condition was 46 % ( N = 105 ) . Next was the random baseline with 37 % ( 49 ) , and finally the TF - IDF baseline achieved 30 % ( N = 54 ) . These results are significant by a \u03c7 2 proportion test ( p \u2264 . 01 ) . We thus observe that both in terms of the absolute number of positively - rated ideas and in terms of proportions , our approach was able to generate a considerably large relative positive effect , leading to better ideas . For k = 3 ( the majority vote ) , out of 749 total ideas collected , 184 ideas were judged as good . Again , we start by looking only at seeds that appeared across all three conditions ( 9 such seeds ) . This leaves 154 good ideas . The proportion of good ideas in our condition was 38 % ( N = 118 ) . Next - up was the random baseline with 22 % ( 68 ) , and finally the TF - IDF baseline achieved 21 % ( N = 63 ) , with p < . 01 . By looking at the more conservative majority - vote threshold , the observed effect of our method only increases . Looking only at seeds that appeared across all conditions was a basic way to make sure we cancel out possible confounding fac - tors . A more refined way is attempting to model these effects and condition on them , as follows . We are interested in the likelihood that a given idea is good , or pr ( good ) , as a function of inspiration condition . However , ideas are not independent : each participant generated multiple ideas , and ideas were proposed for different seeds . Failing to account for these dependencies would lead to inaccurate estimates of the effects of the inspirations : some participants may be better at generating ideas than others , while some seeds might be more easy / difficult than others . Therefore , we used a generalized linear mixed model , Figure 4 : Showing proportion estimates by our random - effect lo - gistic regression , for k = 2 ( left ) and k = 3 ( right ) . Participants are significantly more likely to generate good ideas for the redesign ideation task when given inspirations from our analogy approach compared to baseline - surface and baseline - random approaches with a fixed effect of inspiration condition , and random effects of participant and seed ( to model within - participant and within - seed dependencies between ideas ) . For k = 2 , our resulting model ( with fixed effect of inspiration condition ) yields a significant reduction in variance compared to a null model with no fixed effects , Likelihood ratio \u03c7 2 ( 3 ) = 67 . 96 , p < . 01 . The model also yields a reduction in Akaike Information Criterion ( AIC ) , from 682 . 28 in the null model to 620 . 32 , indicating that the improved fit to the data is not due to overfitting . For k = 3 , the model also yields a significant drop in variance compared to a null model , Likelihood ratio \u03c7 2 ( 3 ) = 92 . 38 , p < . 01 , with AIC dropping from 682 . 28 in the null model to 595 . 90 . As Figure 4 shows , our method led to a significantly higher prob - ability for good ideas . For k = 2 , pr ( Good ) = 0 . 71 , 95 % confidence interval = [ 0 . 48 , 0 . 87 ] in our condition . TF - IDF had pr ( Good ) = 0 . 28 [ 0 . 16 , 0 . 44 ] , and random had pr ( Good ) = 0 . 27 [ 0 . 16 , 0 . 41 ] . The advantages of the analogy condition over each baseline are both substantial and statistically significant , B = \u2212 1 . 81 , p < . 01 vs . TF - IDF , and B = \u2212 1 . 88 , p < . 01 vs . random . For k = 3 , we had pr ( Good ) = 0 . 56 , [ 0 . 36 , 0 . 75 ] . TF - IDF had pr ( Good ) = 0 . 16 [ 0 . 08 , 0 . 27 ] , and random had pr ( Good ) = 0 . 14 [ 0 . 08 , 0 . 24 ] , B = \u2212 1 . 94 , p < . 01 vs . TF - IDF , and B = \u2212 2 . 05 , p < . 01 vs . random . Note that confidence intervals for the probability estimates are relatively wide ( more so , unsurprisingly , for k = 2 ) . Replications of this experiment , possibly with more data , could yield results somewhere in between , with more precise estimates on the true size of the effect . The main take - away of this study is that our approach yields a reliable increase in participants\u2019 creative ability . 5 DISCUSSION AND CONCLUSION In this paper , we sought to develop a scalable approach to finding analogies in large , messy , real - world datasets . We explored the po - tential of learning and leveraging a weak structural representation ( i . e . , purpose and mechanism vectors ) for product descriptions . We leverage crowdsourcing techniques to construct a training dataset with purpose / mechanism annotations , and use an RNN to learn purpose and mechanism vectors for each product . We demonstrate that these learned vectors allow us to find analogies with higher precision than traditional information - retrieval similarity metrics like TF - IDF , LSA , GloVe and LDA . Our ideation evaluation experiment further illustrates the effec - tiveness of our approach : participants had a higher likelihood of generating good ideas for the redesign ideation task when they KDD 2017 Research Paper KDD\u201917 , August 13 \u2013 17 , 2017 , Halifax , NS , Canada 242 received inspirations sampled by our analogy approach ( tuned to be similar in purpose , but different in mechanism ) , compared to a traditional ( TF - IDF ) baseline or random sampling approach . From a psychological perspective , the benefits of our inspirations are likely due to our approach\u2019s superior ability to sample diverse yet still structurally similar inspirations , since diversity of examples is a known robust booster for creative ability [ 8 ] . The TF - IDF approach yielded inspirations likely to be relevant , but also likely to be re - dundant and homogeneous , while the random sampling approach yields diversity but not relevance . While moving to a \u201cweak\u201d structural representation based on purpose and mechanism significantly increased the feasibility of analogy - finding , extensions may be necessary to generalize to other domains besides product descriptions . For example , our purpose and mechanism vectors did not distinguish between higher and lower level purposes / mechanisms , or core / peripheral purposes / mechanisms , and also did not encode dependencies between partic - ular purposes / mechanisms . These are potentially fruitful areas for future work and may be especially important when moving from relatively simple product descriptions to more complex data such as scientific papers , in which purposes and mechanisms can exist at multiple hierarchical levels ( e . g . , \u201caccelerate innovation\u201d vs . \u201clearn a vector representation of the purpose of an item\u201d ) . More gener - ally , we believe exploring the tradeoffs between degree of structure , learnability ( including costs of generating training data , accuracy , and generalizability ) and utility for augmenting innovation could lead to interesting points in the design space that could have both theoretical and practical value . Acknowledgments . The authors thank the anonymous reviewers for their helpful comments . This work was supported by NSF grants CHS - 1526665 , IIS - 1149797 , IIS - 1217559 , Carnegie Mellon\u2019s Web2020 initiative , Bosch , Google , ISF grant 1764 / 15 and Alon grant . Dafna Shahaf is a Harry & Abe Sherman assistant professor . REFERENCES [ 1 ] Sanjeev Arora , Yuanzhi Li , Yingyu Liang , Tengyu Ma , and Andrej Risteski . 2016 . Linear algebraic structure of word senses , with applications to polysemy . arXiv preprint arXiv : 1601 . 03764 ( 2016 ) . [ 2 ] Sanjeev Arora , Yingyu Liang , and Tengyu Ma . 2016 . 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Lee , 1 , * and Padmini Rangamani 1 , * 1 Department of Mechanical and Aerospace Engineering , University of California San Diego , La Jolla CA 92093 ABSTRACT Biomembranes adopt varying morphologies that are vital to cellular functions . Many studies use computational modeling to understand how various mechanochemical factors contribute to membrane shape transformations . Compared with approximation - based methods ( e . g . , \ufb01 nite element method [ FEM ] ) , the class of discrete mesh models offers greater \ufb02 ex - ibility to simulate complex physics and shapes in three dimensions ; its formulation produces an ef \ufb01 cient algorithm while main - taining coordinate - free geometric descriptions . However , ambiguities in geometric de \ufb01 nitions in the discrete context have led to a lack of consensus on which discrete mesh model is theoretically and numerically optimal ; a bijective relationship between the terms contributing to both the energy and forces from the discrete and smooth geometric theories remains to be established . We address this and present an extensible framework , Mem3DG , for modeling 3D mechanochemical dynamics of membranes based on discrete differential geometry ( DDG ) on triangulated meshes . The formalism of DDG resolves the inconsistency and provides a unifying perspective on how to relate the smooth and discrete energy and forces . To demonstrate , Mem3DG is used to model a sequence of examples with increasing mechanochemical complexity : recovering classical shape transformations such as 1 ) biconcave disk , dumbbell , and unduloid ; and 2 ) spherical bud on spherical , \ufb02 at - patch membrane ; investigating how the coupling of membrane mechanics with protein mobility jointly affects phase and shape transformation . As high - resolution 3D imaging of membrane ultrastructure becomes more readily available , we envision Mem3DG to be applied as an end - to - end tool to simulate realistic cell geometry under user - speci \ufb01 ed mechanochemical conditions . INTRODUCTION Computational modeling of lipid bilayer mechanics has long been accepted as a way to probe the bio - physical aspects of membrane curvature generation . The ability of lipid bilayers and cellular membranes to bend in response to various applied forces has been studied extensively from the mathematical modeling perspective . However , the nonlinear system of equations that result from such modeling often leads to a computational bottleneck to generate pre - dictions from simulations that can be tested against experimentally observed shapes . In this study , we develop a mesh - based model using discrete differen - tial geometry ( DDG ) to reduce this bottleneck . To justify why our method is necessary and is a computa - tional advance , we \ufb01 rst describe the importance of membrane curvature generation in biology , the cur - rent state of the art in membrane mechanics modeling , and \ufb01 nally explicitly state the goals of our approach . As one of the most essential and conserved structures of cells , cellular membranes perform many functions . First , they form compartments to separate chemical environments . Beyond the passive role of par - titioning space , lipids in the membranes interact with Submitted March 12 , 2022 , and accepted for publication June 8 , 2022 . * Correspondence : ctlee @ ucsd . edu or prangamani @ ucsd . edu Editor Name : J\u00f6rg Enderlein SIGNIFICANCE Cellular membranes have shapes and shape changes that characterize cells / organelles , and support nutrient traf \ufb01 cking among other critical processes . Modeling membrane shape changes using mechanical principles can provide insight into how cells robustly bend membranes to support life . Mathematical and computational strategies to solve the equations describing membrane shape evolution can be complex and challenging without simplifying assumptions . Here , we present a new , general , numerical approach to model arbitrary 3D membrane shapes in response to interaction with curvature - sensing and generating membrane proteins . The accompanying implementation , Mem3DG , is a software tool to make computational membrane mechanics accessible to the general researcher . Biophysical Reports 2 , 100062 , September 14 , 2022 1 https : / / doi . org / 10 . 1016 / j . bpr . 2022 . 100062 (cid:1) 2022 The Author ( s ) . This is an open access article under the CC BY - NC - ND license ( http : / / creativecommons . org / licenses / by - nc - nd / 4 . 0 / ) . proteins and other cellular components in \ufb02 uencing cell signaling ( e . g . , by localizing molecules and acting as an entropic barrier ) ( 1 , 2 ) . Membrane morphology and to - pology changes are critical for traf \ufb01 cking cargo in and out of cells and are very carefully regulated ( 3 \u2013 8 ) . Central to these roles is the ability of the mem - brane to curve and adopt varying morphological con \ufb01 g - urations from spheres to highly - curved and variegated structures . Advances in experimental studies of membrane - pro - tein interactions ( 9 \u2013 20 ) , ultrastructural imaging ( 21 \u2013 30 ) , and image analysis ( 9 \u2013 11 , 31 \u2013 37 ) have re - vealed much about the molecular interactions that regulate membrane curvature . To investigate the me - chanics behind these interactions , many theoretical and computational models in terms of membrane energetics and thermodynamics have been developed ( 7 , 38 \u2013 52 ) . These models , owing to the ease of in silico experimentation , have become an important tool for generating and testing hypotheses ( 53 , 54 ) . These mechanics models and associated simulations have been used to provide intuition on the mechanical requirements for forming and maintaining complex cellular membrane shapes ( 55 \u2013 63 ) . While the utility of this approach has been estab - lished and many models have been developed ( 38 ) , many models are limited by critical assumptions or other technical challenges . For example , the ability to use geometries from membrane ultrastructural imag - ing experiments as a starting condition would improve model realism ( 64 ) . With respect to computational complexity , the solver should be able to model defor - mations and topological changes in three dimensions and be compatible with both energy minimization and time integration for comparing with static and time - se - ries experiments respectively . This is in contrast to the current assumptions of the existence of an axis of sym - metry that is quite commonly made for purposes of ease of simulation ( 65 ) . An additional feature for these solvers should be that their implementation is modular such that the addition of new physics or increasing model complexity should be straightforward . This in - cludes the potential for coupling the membrane model with agent - based and other simulations to propagate other cellular components such as the cytoskeleton ( 66 ) . Thus , new computational tools which are general , easy to use , and without restrictive assumptions are needed to bring modeling closer to experimental obser - vations of membrane shapes in cells . To emphasize the motivations behind our choice of extending and developing a new mesh - based mem - brane model , we provide a summary of the legacy liter - ature in modeling membrane mechanics . The most common theoretical model of membrane bending is the Helfrich - Canham - Evans Hamiltonian ( The Helfrich energy is related to the Willmore energy in the mathe - matics literature ( 67 ) ) , which describes the lipid bilayer as a 2D \ufb02 uid - like structure that exhibits resistance to bending in the out - of - plane direction ( 39 , 40 , 68 \u2013 70 ) . It is a continuum model that describes the bending en - ergy of the membrane as a function of its mean and Gaussian curvatures . The assumptions for the contin - uum are satis \ufb01 ed as long as the deformations are much larger in length scale compared with the individ - ual lipid components . Given the necessary material properties and bound - ary conditions , by minimizing the Helfrich energy , we can obtain the equilibrium shape of the membrane ( 39 , 70 \u2013 72 ) . While straightforward in concept , energy minimization requires the determination of the forces on the membrane , which is a challenging task ( 65 ) . The forces on the membrane are given by the variation of the energy with respect to the embedded coordinate ( i . e . , shape ) of the membrane ( we call this variation the shape derivative , which is distinct from the chemical derivative that will be introduced later in the context of mechanochemical coupling ) . Taking the shape de - rivatives of the Helfrich energy produces the \u201c shape equation , \u201d so termed because solutions of this partial differential equation ( PDE ) , with the prescribed bound - ary conditions , produce con \ufb01 gurations at equilibrium ( i . e . , force balance ) . Solving the shape equation is non - trivial since it is a PDE with fourth - order nonlinear terms . As a result , analytical solutions of the shape equation are known only for a few cases constrained to speci \ufb01 c geometries and boundary conditions ( 42 ) . For most systems , we must resort to the use of numerical methods . The simplest numerical schemes can be formulated by mak - ing restrictive assumptions such as considering only small deformations from a plane ( e . g . , Monge parame - trization ) or assuming that there exists an axis of sym - metry such that the resulting boundary value system can be integrated ( 38 ) . While these methods are suit - able for idealized shapes , these assumptions are not consistent with the membrane shapes found in biology are and thus not general enough for advancing the \ufb01 eld . Solvers of membrane shape in 3D have also been developed and can be categorized into three groups : 1 ) phase \ufb01 eld or level set methods ( 73 \u2013 77 ) , 2 ) \ufb01 nite element method ( FEM ) ( 78 \u2013 85 ) , and 3 ) discrete surface mesh models ( 60 , 86 \u2013 99 ) . These methods and others , reviewed in detail by Guckenberger et al . ( 100 ) , differ in the strategy used to discretize the membrane domain and compute the relevant derivatives . We compare the aforementioned general , 3D models with our estab - lished model criteria in Table 1 and elaborate below . Phase \ufb01 eld and level set methods solve the shape equation by propagating a density \ufb01 eld on an ambient volumetric mesh . The membrane shape is implicit in 2 Biophysical Reports 2 , 100062 , September 14 , 2022 these models and can be found by drawing an isosur - face or level set of the model . While this is ideal for modeling membrane topological changes , the implicit representation of the membrane adds complexity for interfacing with data generated using modern methods of visualizing membrane ultrastructure . The meshes output from ultrastructural studies must be converted into a density or phase \ufb01 eld prior to input to the model . While this conversion is possible , representing the dy - namic and variegated shapes of cellular membranes would require a dense volume mesh , which reduces computational tractability . The implicit surface repre - sentation also complicates the addition of new in - plane physics for end users . FEM and discrete mesh models use an explicit sur - face parametrization ( i . e . , a mesh ) . Thus the meshes output from ultrastructural imaging datasets can be used in these frameworks with minor modi \ufb01 cations ( 32 , 101 ) . FEM relies on elementwise interpolation functions and is commonly derived from the weak formulation of boundary value problems . Comparing FEM methods with our speci \ufb01 cations we identify a few key challenges . First , the numerical evaluation of smooth geometric measurements on arbitrary mani - folds in an FEM framework requires non - intuitive tensor algebra to translate the shape equation in coor - dinate where it is ready to be solved . After this formu - lation , solving the shape equation can require the use of high - order function basis such as the C 1 conform - ing FEM based on subdivision scheme ( 78 , 79 ) or iso - geometric analysis ( IGA ) ( 81 \u2013 83 , 85 ) , which adds code complexity and run - time cost . Extending an FEM framework to incorporate new physics , topologi - cal changes , or interfaces with other models requires advanced mathematical and coding skills . This can restrict the usage to the computational math commu - nity and prevent broad usage by the biophysics community . Finally , evaluating discrete mesh - based methods , which de \ufb01 ne the system energy and / or forces using geometric primitives from a mesh , we \ufb01 nd that they satisfy many of the requirements in Table 1 . Due to the ease of use and implementation , discrete mesh models have gained in popularity and many different schemes can be found in the literature ( 60 , 86 \u2013 99 , 102 , 103 ) . These schemes differ in their approach to de \ufb01 ning and computing geometric measurements necessary for de \ufb01 ning the energy and forces on a discrete object . Discrete geometries have discontinu - ities and limited information that leads to degenerate de \ufb01 nitions for geometric values . For example , there is no canonical de \ufb01 nition for the normal of a vertex of a mesh as opposed to the normal of a smooth geometry ( 89 , 104 , 105 ) . One challenge for selecting the suitable formulation to use is the lack of approximation error metric for which the discrete de \ufb01 nition best matches the smooth theory . Another confounding factor is the step at which the problem is discretized . Some imple - mentations discretize the energy of the system by con - structing standalone discrete energy , which captures the behavior of the Helfrich energy ( 65 ) . From this discrete energy , they take the shape derivatives to obtain an expression for the discrete force . Without careful consideration , the discrete forces derived in this manner are unstructured and there is little resem - blance to expressions of force from smooth theory . A second option is to discretize the smooth force expression directly ( 65 , 100 ) . While this preserves the geometric connection for the forces , there is no longer well - de \ufb01 ned discrete energy . Several discrete mesh methods were benchmarked by Bian et al . ( 89 ) and Guckenberger et al . ( 100 ) who found differences in the accuracy , robustness , and ease of implementation ( 89 , 100 ) . In this work , we outline a discrete mesh framework for modeling membrane mechanics with the following goals in mind : 1 ) we do not make a priori assumptions about axes of symmetry or restrict the coordinates in any way ; 2 ) we resolve the ambiguity in the de \ufb01 nition of geo - metric measurements on the mesh and permit a direct TABLE 1 Comparison of common mathematical frameworks for modeling membrane mechanics with speci \ufb01 cations to advance the mission of computational membrane mechanobiology Phase \ufb01 eld / level set FEM Discrete mesh / Mem3DG General 3D U U U Statics \u00fe dynamics U U U Membrane heterogeneity U U U Incorporation of agents / particles U Incorporation of stochastic dynamics ( e . g . , DPD or MC ) U Explicit surface parametrization U U Coordinate - free evaluation U Ability to support topological changes U requires mesh surgery Error analysis U U A general framework will permit the easy transfer of inputs and results between model and experiments . Models that can be coupled with other modeling schemes representing other cellular components can help address the complexity of cell biology . Discrete mesh models have many desirable traits , with respect to these speci \ufb01 cations , at the cost of forgoing rigorous error analysis . Biophysical Reports 2 , 100062 , September 14 , 2022 3 comparison for both the energy and force expressions in smooth and discrete contexts ; and 3 ) this framework al - lows for use of meshes generated from ultrastructural imaging . We begin by de \ufb01 ning discrete energy that is analogous to the Helfrich energy . Then , using concepts from DDG , we derive discrete shape derivatives analyti - cally and group terms to produce a discrete shape equa - tion . We will show that our discrete shape equation has a clear correspondence between the terms of the smooth shape equations ( 57 , 67 , 70 , 71 ) . Beyond establishing this important connection , we will show that the elegant analytical expressions for discrete variational terms from the DDG also yield improved geometric intuition and numerical accuracy ( 104 , 105 ) . Benchmarking of our expressions was performed with our accompanying software implementation called Membrane Dynamics in 3D using Discrete Differential Geometry ( Mem3DG ) . Mem3DG is written in C \u00fe\u00fe , released under the Mozilla Public License version 2 , and comes with accompanying documentation and tutorials which can be accessed on GitHub ( https : / / github . com / RangamaniLabUCSD / Mem3DG ) . Beyond the computa - tionofdiscrete energiesand forcesona meshofinterest , we also include functionality for performing energy mini - mization and time integration . Using Mem3DG , we vali - date the exactness of the analytical expressions of force terms by numerically examining the convergence of the force terms as a function of system energy perturbation . To illustrate compliance with our tool speci \ufb01 cations , we apply Mem3DG to a sequence of exam - ples with increasing complexity . Finally , we outline the steps to incorporate additional physics such as mem - brane - protein interactions and surface diffusion into Mem3DG . THEORY The lipid bilayer is modeled as a thin elastic shell us - ing the Helfrich - Canham - Evans Hamiltonian or sponta - neous curvature model ( 39 , 69 , 106 ) . The bending energy , E b , of a smooth surface or 2 - manifold , M , can be expressed in terms of the mean H ; Gaussian K , and spontaneous curvature (cid:1) H with ma - terial parameters k the bending and k G the saddle - splay moduli . Additional energy terms E s and E p ac - count for the tension area \u00f0 l - - A \u00de and pressure - volume ( D P - - V ) relationships . The total energy of the bilayer is therefore E \u00bc Z M (cid:2) k \u00f0 H (cid:2) (cid:1) H \u00de 2 \u00fe k G K (cid:3) dA | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E b \u00fe Z A (cid:1) A l d ~ A | \ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04 } E s (cid:2) Z V (cid:1) V D Pd ~ V | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E p : ( 1 ) The preferred surface area and volume , (cid:1) A and (cid:1) V , combined with the spontaneous curvature , (cid:1) H , charac - terize the zero - energy state for the system energy . In a nutshell , given the material properties , the system en - ergy is fully determined by its geometric measure - ments such as volume , area , and curvatures . Machinery to express these measurements have been a topic of extensive study in classical differential geometry ( 107 , 108 ) . However , \ufb01 nding the minima of the governing energy , solving the stationary solution to the geometric PDE , can be mathematically and numerically dif \ufb01 cult . While differential geometry pro - vides succinct expressions to describe the measure - ments in a coordinate - free fashion , computational methods often require the introduction of a coordinate basis and subsequent manipulation of expressions us - ing tensor algebra , which can obscure the underlying geometric intuition . As an alternative , forgoing the need for a smooth geometry , one can treat a discrete geometry ( such as a geometric mesh ) as the input . This perspective where the discrete geometry is the actual geometry is that of DDG ( 109 ) . By eliminating the burdens of treating the input mesh as an approximation of a smooth object , DDG capitalizes upon the piecewise nature of meshes to produce ef \ufb01 cient and paralleliz - able \ufb01 nite difference - like formulae which are amenable to algorithmic implementation while main - taining clear geometric meaning . In the following sec - tions , we use concepts from DDG to formulate a discrete analog to the smooth membrane shape prob - lem . Following the derivation of the discrete theory , we describe the development of an accompanying soft - ware implementation called Mem3DG . Notation and preliminaries We assume the following notation conventions and provide a table of important symbols ( Table 2 ) . To aid the reader on how the elements of the mesh are used in the derivation , several fundamental geometric primitives ( i . e . , values on a mesh which are easily measurable ; listed in Table 2A ) are illustrated in Fig . 1 A \u2013 C . We note that , in discrete contexts , the notation , R a , should be considered the discrete ( integrated ) counter - part of a pointwise measurement a in a smooth setting . The rationale and signi \ufb01 cance behind the usage of an integrated quantity in discrete contexts are elaborated in Appendix B and the DDG literature ( 104 , 105 ) . Using this notation , discrete surface integrals are expressed as sums of integrated values over the discrete mesh components listed in Table 2B ( e . g . , P v i R a i is the discrete analog to R M a ) . It is possible to interchange 4 Biophysical Reports 2 , 100062 , September 14 , 2022 between integrated , R a i , and pointwise , a i , quantities by using the dual area ( A i ) , a i \u00bc Z a i = A i : ( 2 ) For simplicity , we will not use separate notations for operators applying in smooth and discrete settings . The context can be inferred from the objects to which the operators are applied . Where it serves to improve our geometric or other intuition , smooth objects will be presented alongside discrete objects for comparison . Obtaining a discrete energy de \ufb01 ned by mesh primitives Following the perspective of DDG , we restrict our input to the family of triangulated manifold meshes , M ( i . e . , discrete 2 - manifolds embedded in R 3 ) ( We will use M for both the smooth and discrete surfaces ) . Paralleling the smooth Helfrich Hamiltonian ( Eq . 1 ) , a functional of geometric measurements of a surface , the discrete Helfrich Hamiltonian is composed of discrete analog of those measurements , E \u00f0 ~ r \u00de \u00bc X v i (cid:5) k i Z \u00f0 H i (cid:2) (cid:1) H i \u00de 2 \u00fe k G Z K i (cid:6) | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E b \u00fe Z A (cid:1) A l \u00f0 ~ A ; ~ r \u00de d ~ A | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E s (cid:2) Z V (cid:1) V D P \u00f0 ~ V ; ~ r \u00de d ~ V | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E p : ( 3 ) In comparison with Eq . 1 , H i and K i are pointwise mean and Gaussian curvature measurements on vertices , R \u00f0 H i (cid:2) (cid:1) H i \u00de 2 is the integrated Willmore mea - sure , and the smooth surface integral is replaced by its discrete analog ( i . e . \ufb01 nite summation ) , P v i ( Table 2B ) . The geometric properties of a given membrane con \ufb01 guration can be connected to the system ' s energy through constitutive relations . In this work , we assume that the surface tension follows a linear stress - strain model ( 110 ) , l \u00f0 A ; ~ r \u00de \u00bc K A A \u00f0 ~ r \u00de (cid:2) (cid:1) A (cid:1) A ; ( 4 ) where (cid:1) A is the preferred surface area of the membrane , and K A is the stretching modulus of the membrane . The osmotic pressure can be de \ufb01 ned based on the van ' t Hoff formula as D P \u00f0 V ; ~ r \u00de \u00bc P in (cid:2) P out \u00bc iRT (cid:7) n V (cid:2) c (cid:8) ; ( 5 ) TABLE 2 Glossary of commonly used symbols and conventions A . Geometric primitives M smooth or discrete 2 - manifold ~ r \u02db R 3 embedded coordinate of M l edge length : corner angle 4 dihedral angle A area of mesh cell , e . g . , face A ijk , edge A ij and vertex A i ~ n surface normal B . Surface integral R a integrated quantity over mesh cell ; e . g . , A i a i or A ijk a ijk P v i sum over all vertices v i of the mesh P e ij sum over all edges e ij of the mesh P f ijk sum over all faces f ijk of the mesh P v j \u02db N \u00f0 a \u00de sum over the vertex v j in the neighborhood of a P e ij \u02db N \u00f0 a \u00de sum over the edges e ij in the neighborhood of a P f ijk \u02db N \u00f0 a \u00de sum over the face f ijk in the neighborhood of a C . Tensors x \u02db R scalar quantity x typeindex sub - and super - script convention ; e . g . , R ~ f b i is the bending force for vertex v i ~ x \u02db R 3 vector quantity x \u00bc f x i g \u00f0 n (cid:3) 1 \u00de indexed scalar quantity ~ x \u00bc f x i g \u00f0 n (cid:3) 3 \u00de indexed vector quantity ~ X matrix or tensor quantity D . Derivatives V ~ r shape derivative V f chemical derivative V ~ q surface gradient _ a time derivative D s Laplace - Beltrami operator E . Physical variables E energy f force density m chemical potential H mean curvature K Gaussian curvature A surface area V enclosed volume $ preferred state ; e . g . , (cid:1) H is the spontaneous curvature f \u02db \u00bd 0 ; 1 (cid:4) protein density parameter l membrane tension D P osmotic pressure across the membrane k bending rigidity k G Gaussian modulus K A stretching modulus K V osmotic strength constant (cid:1) c molar ambient concentration n molar quantity of enclosed solute h Dirichlet energy constant \u03b5 adsorption energy constant x membrane drag constant B protein mobility constant Biophysical Reports 2 , 100062 , September 14 , 2022 5 where i , R , T , c , and n are the van ' t Hoff index , ideal gas constant , temperature , ambient molar concentration , and molar amount of the enclosed solute . Substituting these constitutive relations ( Eqs . 4 and 5 ) into the en - ergy ( Eq . 3 ) , we get explicit expressions for E s and E p , E \u00f0 ~ r \u00de \u00bc E b \u00f0 ~ r \u00de \u00fe 1 2 K A \u00bd A \u00f0 ~ r \u00de (cid:2) (cid:1) A (cid:4) 2 (cid:1) A | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E s \u00fe iRTn \u00bd r c \u00f0 ~ r \u00de (cid:2) ln r c \u00f0 ~ r \u00de (cid:2) 1 (cid:4) | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } E p ; ( 6 ) where r c \u00bc c = \u00f0 n = V \u00de is the ratio of the concentrations of the ambient and enclosed solutions . Note that the preferred volume , (cid:1) V , which is needed to evaluate the in - tegral in Eq . 3 , is related to to the parameters in Eq . 5 by (cid:1) V \u00bc n = c . If the system is around the isosmotic condi - tion ( e . g . , V / (cid:1) V ) , the leading order of the energy is given as , E p z 1 2 K V \u00f0 V (cid:2) (cid:1) V \u00de 2 (cid:1) V 2 ; ( 7 ) where K V h iRTn groups the phenomenological param - eters . Mathematically , Eqs . 4 and 7 effectively pre - scribe a penalty - based method for area and volume control . An alternative approach is the use of Lagrange multipliers , which have been extensively adopted in the literature ( 40 , 57 , 72 ) . To compute the energy of a system , we must obtain values for several geometric measurements that appear in thediscreteenergy function ( e . g . , H ; K ; A ; V ) . Formea - surements such as the volume and area , there are clear approaches for their evaluation on a triangulated mesh : summing the areas of the triangular faces and summing over the signed volume of tetrahedra ( Fig . 1 E , osmotic pressure and surface tension ) . For other measurements such as the discrete mean and Gaussian curvatures , FIGURE 1 Overview of the DDG framework For a Figure360 author presentation of this \ufb01 gure , see https : / / doi . org / 10 . 1016 / j . bpr . 2022 . 100062 . ( A \u2013 C ) Illustrations of geometric primitives in the neighborhood of ( A ) fan around a vertex , ( B ) diamond around an edge , and C ) triangle on a face . ( D ) Discrete de \ufb01 nition of scalar edge mean curvature , R H ij , scalar vertex Gaussian curvature , R K i , and Laplace - Beltrami operator , R D s \u00f0 $ \u00de . ( E ) Comparative derivation of Helfrich shape equation in both smooth and discrete formulation . 6 Biophysical Reports 2 , 100062 , September 14 , 2022 additional care must be taken . While in smooth contexts these curvatureshaveuniquede \ufb01 nitions , indiscretecon - texts there are multiple approaches for their calculation . For example , the mean curvature can be computed via the application of the cotangent Laplacian , the kernel of the heat equation , or \ufb01 tting polynomials to a local patch ( 65 ) . As mentioned earlier , there are challenges with these approaches that can limit their numerical ac - curacy and obscure the connection to smooth theory . Here using discrete exterior calculus and identi \ufb01 cation of geometric invariants , we produce theoretically and numerically consistent discrete expressions . Similar to the polygonal curve introduced in Appen - dix B , a triangulated mesh has zero curvature on facets and ill - de \ufb01 ned curvature on edges and vertices . Using the Steiner view , the sharp corners formed by vertices and edges are made smooth with portions of spherical and cylindrical shells , which have well - de \ufb01 ned mean curvature ( Fig . 1 D ) . Taking the limit as the radii of the cylinders and spheres decrease , the leading order contribution of total mean curvature is given by the Steiner formula on an edge , Z H ij \u00bc l ij 4 ij 2 ; ( 8 ) referred to as the edge mean curvature , where l ij is the length of edge e ij , and 4 ij is the dihedral angle on e ij ( i . e . , the angle formed by the face normals of the neigh - boring triangles incident to e ij ) ( illustrated in Fig . 1 B ) ( 104 , 105 ) . While not necessary , a triangulated mesh is often realized in R 3 via vertex positions ; thus it is con - ventional to prescribe data on vertices instead of edges . Summation of edgewise quantities over the \u201c fan \u201d neighborhood ( Fig . 1 A ) provides the recipe of converting an edgewise to a vertexwise quantity , \u00f0 $ \u00de i \u00bc 1 2 X e ij \u02db N \u00f0 v i \u00de \u00f0 $ \u00de ij ; ( 9 ) where the prefactor , 1 = 2 , accounts for fact that each edge is shared by two vertices . While we have an integrated mean curvature , the discrete Helfrich Hamiltonian contains a pointwise mean curvature squared term . To de \ufb01 ne a pointwise mean curvature , the size of the domain occupied by the integrated mean curvature needs to be speci \ufb01 ed ( cf . , Appendix B for rationale ) . The area , A i , referred to as the dual area of the vertex v i , can be de \ufb01 ned as one - third of the areal sum of the incident triangles ( fan illustrated in Fig . 1 A ) ( 104 , 105 ) . Applying Eqs . 2 and 9 to Eq . 3 , the pointwise mean curvature is thus , H i \u00bc R H i A i \u00bc X e ij \u02db N \u00f0 v i \u00de l ij 4 ij 4 A i : ( 10 ) Substituting Eq . 10 into the integrated Willmore mea - sure term of Eq . 3 , the integrated Willmore measure can be expressed as a function of the integrated mean and spontaneous curvature , Z \u00f0 H i (cid:2) (cid:1) H i \u00de 2 \u00bc 1 A i (cid:9) Z H i (cid:2) Z (cid:1) H i (cid:10) 2 : ( 11 ) Discrete Gaussian curvature is given by the angle defect formula , Z K i \u00bc 2 p (cid:2) X f ijk \u02db N \u00f0 v i \u00de : kij ; ( 12 ) which is a well - known quantity that preserves many properties parallel to the smooth theory ( e . g . , Gauss - Bonnet , turning number , among other invariants ) . One way to derive the angle defect formula is to compute the area of a spherical n - gon contained by a local Gauss map of the neighboring n faces around a vertex ( 104 , 105 ) . Eq . 12 provides the general geometric de \ufb01 nition to obtain the energetic contributions from the Gaussian curvature terms . In this study , we consider only sys - tems with uniform saddle - splay modulus which do not undergo topological changes . For these systems , the energy can be simpli \ufb01 ed based on the discrete Gauss - Bonnet theorem , which states that X v i Z K i \u00bc 2 pc \u00f0M\u00de (cid:2) X v j \u02db v M Z k gj ; ( 13 ) where c \u00f0M\u00de \u00bc j V j (cid:2) j E j \u00fe j F j is the Euler character - istic of M , a topological invariant where j V j , j E j and j F j represent the number of vertices , edges and faces of the mesh respectively , and R v M k gi \u00bc p (cid:2) P e ij \u02db N \u00f0 v i \u00de : kij is the discrete geodesic curvature , which measures the deviation of the boundary curve from a straight line when the surface is locally \ufb02 attened . In summary , for this work , the Gaussian curvature term is non - constant only when M is not closed , and the en - ergy solely involves the boundary elements . A numerical comparison of the discrete scalar mea - surements with their smooth counterparts is shown in Fig . E . 1 . We note that for all geometric measurements ( i . e . , volume , area , and curvatures ) , unlike in smooth dif - ferential geometry where their numerical evaluation requires the introduction of coordinates , DDG measure - ments are functions of mesh primitives . By substituting these discrete geometric measurements from DDG into Eq . 6 and 3 , we get a numerical recipe for computing the total system energy . Biophysical Reports 2 , 100062 , September 14 , 2022 7 Force from discrete shape derivative of energy We can obtain the force by taking the negative shape derivative of the energy . In continuous settings , the differentiation is an in \ufb01 nite - dimensional problem that requires the use of the calculus of variations and differential geometry to \ufb01 nd analytical expressions ( 39 , 43 , 70 , 71 ) ( Fig . 1 E , smooth ) . Deriving the forces from the discrete energy ( Eq . 3 ) is a much simpler task . Discrete forces can be obtained by taking partial de - rivatives of mesh primitives with respect to the vertex embedded coordinates , ~ r ( Fig . 1 E , discrete ) . Regarding notation , despite the computational differences , the differential operations in both the discrete and smooth contexts are called ( discrete ) shape derivatives and denoted as V ~ r \u00f0 $ \u00de due to the common geometric meaning . We note that the computation of discrete shape derivatives for membrane modeling has been described previously in the literature ( 87 , 89 ) . Also that there are many overlapping de \ufb01 nitions for discrete curvature , energy , and variations thereof in the graphics literature ( 111 \u2013 113 ) . Our work extends upon the prior art that evaluates derivatives algebrai - cally , by introducing simpli \ufb01 cations based upon the grouping of terms and identi \ufb01 cation of geometric ob - jects . These simpli \ufb01 cations have important implica - tions for improving the geometric understanding as well as the run - time and numerical performance of an implementation . At the high level , our goal is to express the forces on each vertex , given a set of physics , using geometric primitives and properties de \ufb01 ned on speci \ufb01 c mesh ele - ments . By grouping terms , we \ufb01 nd that the vertexwise forces arising from the different physics can be ex - pressed as weights that are functions of the input pa - rameters and system con \ufb01 guration , multiplied by basic geometric vectors . We will show that these terms have an exact correspondence to terms in the smooth shape equation ( Fig . 1 E ) . We remark that , in some sense , the force expressions are reminiscent of \ufb01 nite - difference equations , which approximate differentials as a linear combination of values at discrete points . This may have implications for the suitability of modeling smooth biological surfaces with discrete meshes . Force from osmotic pressure For the smooth geometry , the shape derivative of the enclosed volume yields the outward - pointing surface normal with its size equal to the local area element ( 114 ) . For a discrete mesh , the shape derivative of the volume is given by the face normal on triangular faces with its local area element equaling to the face area , which is referred to as the integrated face normal , R ~ n \u00f0 ijk \u00de ( Fig . 1 E , osmotic pressure ) ( 89 , 99 , 104 , 105 ) , where \u00f0 ijk \u00de denotes the symmetry under index permu - tation ( e . g . , a i \u00f0 jk \u00de means a ijk \u00bc a ikj ) . Similar to edge values , the force normal can be converted to vertex normal , Z ~ n i \u00bc V ~ r i V \u00bc 1 3 X f ijk \u02db N \u00f0 v i \u00de Z ~ n \u00f0 ijk \u00de \u00bc 1 3 X f ijk \u02db N \u00f0 v i \u00de A ijk ~ n \u00f0 ijk \u00de ; ( 14 ) where analogous to Eq . 9 , the prefactor 1 = 3 accounts for fact that each face is shared by three vertices . The discrete vertex forces from the derivative of the pressure - volume work , R ~ f p i , is then given by scaling it with the uniform osmotic pressure , Z ~ f p i \u00bc D P Z ~ n i : ( 15 ) Forces from surface tension Next , considering the shape derivative of the surface energy , E s , in smooth contexts , the derivative of the to - tal surface area also points at the surface normal , with its magnitude measuring the size ( dA ) and the curva - ture ( 2 H ) of the local patch ( Fig . 1 E , surface tension ) ( 114 ) . In a discrete case , we can directly compute the derivative of total area on each vertex by summing the area gradient of incident triangles with respect to the vertex position ; the sum is therefore referred to as ( twice of ) the integrated mean curvature vector on vertices , Z 2 ~ H i \u00bc V ~ r i A \u00bc X f ijk \u02db N \u00f0 v i \u00de V ~ r i A ijk \u00bc X f ijk \u02db N \u00f0 v i \u00de Z 2 ~ H i \u00f0 jk \u00de ; ( 16 ) where we de \ufb01 ne R 2 ~ H i \u00f0 jk \u00de h V ~ r i A ijk , and R ~ H i \u00f0 jk \u00de is the mean curvature vector on a triangle face corner . The capillary forces from surface tension , R ~ f s i , are given by scaling the integrated mean curvature vector by the surface tension , Z ~ f s i \u00bc (cid:2) l Z 2 ~ H i : ( 17 ) Evaluating the algebraic sum of area gradients re - veals the \u201c cotangent formula \u201d applied to the vertex posi - tions ( Fig . 1 E , surface tension ) . From independent derivations with unrelated frameworks ( e . g . , discrete exterior calculus and FEM ) , discretizing the smooth Lap - lace - Beltrami operator on a triangulated mesh produces the cotangent formula , which is called the discrete Lap - lace - Beltrami operator , R D s ( 104 , 105 , 111 ) . Inspecting 8 Biophysical Reports 2 , 100062 , September 14 , 2022 the expressions in Fig . 1 E , surface tension , we see that our discrete expression parallels smooth theory , where the mean curvature is related to the coordinates through the application of the smooth Laplace - Beltrami operator , X e ij \u02db N \u00f0 v i \u00de Z 2 ~ H ij \u00bc Z D s ~ r i 4 D s ~ r \u00bc 2 H ~ n : ( 18 ) Forces from bending To evaluate the shape derivative of the discrete bending energy , we consider the terms from the Gaussian and mean curvature separately . Since we do not consider nonuniform saddle - splay modulus and topological changes in this work , the total Gaussian curvature only varies if the surface has boundaries , v M ( cf . , discrete Gauss - Bonnet theorem in section \u201c Obtaining a discrete energy de \ufb01 ned by mesh primitives \u201d ) . The shape derivative of the bending energy requires more algebra and the introduction of halfedges , e ij ( cf . , Appendix C . 1 ) . Here we will focus on the key results and refer the reader to the full der - ivations for each term in Appendix C . 2 . There are four fundamental geometric vectors on halfedges that comprise the bending force at non - boundary vertices : the mean curvature vector ( see Fig . 1 B for indices ) , Z 2 ~ H ij \u00bc 1 2 (cid:9) Z 2 ~ H i \u00f0 jk \u00de \u00fe Z 2 ~ H i \u00f0 jl \u00de (cid:10) ; ( 19 ) the Gaussian curvature vector , Z ~ K ij \u00bc 1 2 4 ij V ~ r i l ij ; ( 20 ) and the two Schla \ufb02 i vectors , Z ~ S ij ; 1 \u00bc 1 2 l ij V ~ r i 4 ij ; Z ~ S ij ; 2 \u00bc 1 2 (cid:7) l jk V ~ r i 4 jk \u00fe l jl V ~ r i 4 jl \u00fe l ji V ~ r i 4 ji (cid:8) ; ( 21 ) which act to smooth the pro \ufb01 le of local dihedral angles . Note that the shape derivatives are taken with respect to different vertices ( i . e . , V ~ r i or V ~ r j ) , such that the mean curvature R ~ H ij , Gaussian curvature R ~ K ij , and Schla \ufb02 i vectors R ~ S ij are asymmetric under index permutation . To account for the orientation , we refer to them as half - edge vector quantities on e ij ( Appendix C . 1 ) . A numeri - cal comparison of the discrete geometric vector with their smooth counterparts is shown in Fig . E . 1 and Fig . E . 2 . The bending force R ~ f b i ( Fig . 1 E , bending ) can be ex - pressed as weights , which are functions of input parameters multiplied by basic geometric measure - ments in scalar and vector form , which parallels the shape derivative of the smooth bending energy , V t ~ r E b \u00bc V t ~ r (cid:5) Z M k \u00f0 H (cid:2) (cid:1) H \u00de 2 dA (cid:6) \u00bc k (cid:2) 2 \u00f0 H (cid:2) (cid:1) H \u00de (cid:11) H 2 (cid:2) K \u00fe (cid:1) HH (cid:12) \u00fe D s \u00f0 H (cid:2) (cid:1) H \u00de (cid:3) dA ; ( 23 ) where V t ~ r \u00bc V ~ r $ ~ n is the shape derivative in the surface normal direction . Comparing the smooth - discrete expressions , we make a few observations : (cid:5) The Schla \ufb02 i vector terms , ~ S , is the discrete analog of the smooth biharmonic term , D s \u00f0 H (cid:2) (cid:1) H \u00de , the high - order local smoothing force . The numerical compar - ison of these two terms , as well as results directly obtained using cotangent formula applied on Z ~ f b i \u00bc X e ij \u02db N \u00f0 v i \u00de (cid:2) (cid:2) k i \u00f0 H i (cid:2) (cid:1) H i \u00de \u00fe k j (cid:11) H j (cid:2) (cid:1) H j (cid:12)(cid:3)Z ~ K ij \u00fe (cid:5) 1 3 k i \u00f0 H i (cid:2) (cid:1) H i \u00de\u00f0 H i \u00fe (cid:1) H i \u00de \u00fe 2 3 k j (cid:11) H j (cid:2) (cid:1) H j (cid:12)(cid:11) H j \u00fe (cid:1) H j (cid:12)(cid:6) Z 2 ~ H ij (cid:2) (cid:5) k i \u00f0 H i (cid:2) (cid:1) H i \u00de Z ~ S ij ; 1 \u00fe k j (cid:11) H j (cid:2) (cid:1) H j (cid:12) Z ~ S ij ; 2 (cid:6) ; ( 22 ) Biophysical Reports 2 , 100062 , September 14 , 2022 9 pointwise scalar mean curvature , are covered in Fig . E . 2 and Fig . E . 1 . (cid:5) Eq . 23 is the normal component of the shape deriva - tive of the bending energy ; an additional tangential component is required if surface heterogeneity ex - ists ( e . g . , k is not spatially uniform ) ( 40 , 65 ) . In contrast , the discrete shape derivative ( Eq . 22 ) is the total derivative in R 3 , which includes both the tangential and normal components ( in the smooth sense since there is no well - de \ufb01 ned vertex normal di - rection in discrete geometry ) . Depending on the extent and symmetry of the heterogeneity , the discrete force can point in any direction in R 3 . (cid:5) The coef \ufb01 cients in Eq . 22 show an intriguing pattern combining contributions from both v i and v j . From a \ufb01 nite - difference approximation standpoint , this re - sults in an approximation scheme for which a rigorous error analysis has not yet been conducted . FIGURE 2 Overview of data \ufb02 ow within Mem3DG . The user provides Mem3DG with an initial condition in the form of a triangulated mesh and vertexwise state and kinematic variables ( green box ) . The main loop ( black loop ) of Mem3DG evaluates the discrete energy and forces and prop - agates the trajectory , among other supporting steps . Modules in dashed lines are optional depending on whether the system mesh has bound - aries and if external forces are speci \ufb01 ed . User - speci \ufb01 ed options and possible extensions of Mem3DG to accommodate various physics are highlighted in yellow boxes . Mem3DG automatically exits the simulation when the system converges or the maximum time step is reached . 10 Biophysical Reports 2 , 100062 , September 14 , 2022 Net force and the bene \ufb01 t of DDG By summing the force terms from each physics , we obtain the net force . Through section \u201c Obtaining a discrete energy de \ufb01 ned by mesh primitives \u201d and sec - tion \u201c Force from discrete shape derivative of energy , \u201d we identify and show a scheme where both 1 ) the force is analytically derived from the discrete energy , and 2 ) both the discrete energy and force mirror the smooth theory . The entire process of de \ufb01 ning energy and con - duction shape derivative do not involve the introduction of coordinate and the use of tensor algebra . Owing to the discontinuities and limited information contained by a discrete geometry , there are ambiguities in geo - metric de \ufb01 nitions that behave otherwise in the smooth context ( cf . , various discrete curvature de \ufb01 nitions for plane curve discussed in Crane and Wardetzky ( 115 ) ) . Intentional choices of certain de \ufb01 nitions of basic discrete geometric measurements can reveal the connection between various de \ufb01 nitions , preserve use - ful geometric invariants , and most naturally re \ufb02 ect the underlying physics . Here many scalar and vector de \ufb01 nitions of geometric measurement are connected through the chain of shape derivatives ( cf . , Fig . A . 2 ) ( 105 ) , which justi \ufb01 es their role in representing either en - ergy or forces . For example , the discrete bending en - ergy is also commonly constructed using the mean curvature vector , R M ~ H $ ~ H dA in literature ( 90 , 99 ) . As shown in section \u201c Forces from surface tension , \u201d the de \ufb01 nition of the mean curvature vector is tightly corre - lated with the surface tension , where directionality is embedded . The inner product used in such discrete en - ergy de \ufb01 nition strips away the directional information . Instead , here we construct energy using the scalar mean curvature , R H , because 1 ) the energy is inher - ently scalar , and 2 ) the discrete curvature exists on the edges . After taking the shape derivative of the en - ergy , the mean curvature vector appears as the effec - tive tension component and the directional information of the vector is used for representing the force . Using the directional information , an arbitrary de \ufb01 nition of a vertex normal is avoided . When weighted homogeneously around a vertex , each fundamental geometric vector that composes the discrete force , R ~ n , R ~ H , R ~ K , R ~ S , can be used to obtain a meaningful de \ufb01 nition of the vertex normal . The heterogeneous weighting of these vectors around the vertex repre - sents the incorporation of functional variation in the tangential direction , which can be used to model het - erogeneities in material and other properties across the membrane ( 40 , 65 ) . Practically , the additional struc - ture provided by the discrete force and energy expres - sions allows the user to inspect term - wise contributions , which can lead to additional insights and analysis . Since the terms of the discrete energy and forces are de \ufb01 ned locally by mesh primitives at vertex neighborhoods , the algorithms are ef \ufb01 cient and straightforward to parallelize . The numerical accuracy of these expressions is benchmarked for several scalar and vector measurements on smooth and discrete sur - faces shown in Fig . E . 1 , Fig . E . 2 , and later discussed in section \u201c Practical considerations for applying Mem3DG to biological problems . \u201d De \ufb01 ning metrics for simulation and error quanti \ufb01 cation For monitoring simulation progress , exactness of force calculations with respect to the discrete energy , and convergence studies of computed quantities upon mesh re \ufb01 nement , we introduce the following norms . L 2 norm From a PDE perspective , the vertex forces are also called the residual of the shape equation , whose solu - tion represents the equilibrium solution . The simulation is terminated when the residual is smaller than a user - speci \ufb01 ed threshold . The rationale for using the L 2 norm is justi \ufb01 ed by perturbing the system con \ufb01 guration and conducting an expansion on the system energy , E \u00f0 ~ r \u00fe e V E \u00f0 ~ r \u00de\u00de \u00bc E \u00f0 ~ r \u00de \u00fe e h V E \u00f0 ~ r \u00de ; V E \u00f0 ~ r \u00dei \u00fe O (cid:11) e 2 (cid:12) \u00bc E \u00f0 ~ r \u00de \u00fe e (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)Z ~ f (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13) 2 L 2 \u00fe O (cid:11) e 2 (cid:12) ; ( 24 ) where we refer the inner product of the force matrix as the L 2 norm of the forces . Computationally , this is equivalent to the standard Frobenius matrix L 2 norm , (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)Z ~ f (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13) L 2 \u00bc \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 trace (cid:9) Z ~ f u Z ~ f (cid:10) s : ( 25 ) Using the L 2 norm and Eq . 24 , we can perform a nu - merical validation of the exactness of the discrete force calculation with respect to the discrete energy . We expect the force to approximate the energy up to second order with respect to the size of a perturbation . This validation will be further elaborated in section \u201c Membrane dynamics with full mechanochemical feedback . \u201d L 1 norm A scale - invariant L 1 norm is well suited to quantify the magnitude of the error on varying domain size and mesh resolution . Given a vertexwise local scalar mea - surement , R a , or a vector measurement , R ~ a , and their reference values , R (cid:1) a , and R (cid:1) ~ a , Biophysical Reports 2 , 100062 , September 14 , 2022 11 (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)Z a (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13) L 1 \u00bc X v i (cid:13)(cid:13)(cid:13) Z a i (cid:2) Z (cid:1) a i (cid:13)(cid:13)(cid:13) A (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)Z ~ a (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13) L 1 \u00bc X v i (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13) Z ~ a i (cid:2) Z ~ a i (cid:13)(cid:13)(cid:13)(cid:13)(cid:13)(cid:13) L 2 A ; ( 26 ) where the normalizing factor , the total surface area A , is used to obtain a pointwise estimate of the error . The L 1 norm is applied in the local comparison of discrete and smooth measurements , which we further elaborate in section \u201c Practical considerations for applying Mem3DG to biological problems . \u201d SOFTWARE IMPLEMENTATION : Mem3DG Along with the theoretical developments , we have developed an accompanying software implementation written in C \u00fe\u00fe called Mem3DG . Our goal in developing this software is to enable the easy use and application of the corresponding theory developed above to biolog - ical problems of interest . Mem3DG is a library that contains several compo - nents to support this goal . Fig . 2 provides a synopsis of Mem3DG . The input to Mem3DG includes a triangu - lated mesh with its coordinate ~ r embedded in R 3 . Users can choose to use Mem3DG to construct idealized meshes ( e . g . , icosphere , cylinder , or \ufb02 at hexagonal patch ) as an input or to read in meshes from several common mesh formats . Meshes are stored and manipulated in Mem3DG using the halfedge data struc - ture provided by Geometry Central ( 116 ) . The supported input \ufb01 le formats are those which are read - able by hapPLY and Geometry Central ( 116 , 117 ) . Once a mesh and parameters are loaded , Mem3DG can evaluate the discrete energy and forces of the sys - tem . Mem3DG adopts a modular design that facilitates the use of different energy and force components and has utilities which help the user to specify the phys - ics and governing parameters . Mem3DG also supports local system simulations where the input mesh has boundaries . Additional details about the supported boundary conditions are given in section \u201c Prescribing boundary conditions with force masking . \u201d To perform energy minimization and time integration of the system , various schemes have been imple - mented . These schemes are described in section \u201c Time integration and energy minimization . \u201d As dis - cussed further in section \u201c Practical considerations for applying Mem3DG to biological problems , \u201d when a user wishes to use Mem3DG to represent complex biological membrane geometries , additional care regarding the quality of the mesh is necessary . Mem3DG includes algorithms for basic mesh regulariza - tion and remeshing , which can be toggled by the user to support their applications . The simulation terminates when it reaches the time limit or the system reaches equilibrium , whose criteria is determined using the en - ergy L 2 norm introduced in section \u201c De \ufb01 ning metrics for simulation and error quanti \ufb01 cation . \u201d A user can choose between several formats to output a trajectory over time or the con \ufb01 guration of the local minima from Mem3DG . In addition to the mesh outputs supported by Geometry Central , we have also developed a scheme for outputting mesh trajectories in NetCDF format ( 118 ) . Mem3DG can read and visualize the output trajectories and mesh con \ufb01 gurations using Ge - ometry Central and Polyscope ( 116 , 119 ) . For rapid prototyping and enumeration of simulation conditions , we have also developed a Python API called PyMem3DG . The functionality in C \u00fe\u00fe is exposed in Py - thon using bindings from pybind11 ( 120 ) . Illustrative examples of using both Mem3DG and PyMem3DG are provided in the online tutorials . For the experiments dis - cussed in this work , all of the simulations were per - formed using PyMem3DG and the accompanying code and initial con \ufb01 gurations are on GitHub : https : / / github . com / RangamaniLabUCSD / Mem3DG . De \ufb01 ning properties of a membrane reservoir for systems with open boundaries To facilitate correspondence with wet experiments and to support the reduction of computational cost , it is possible to construct systems using meshes with open boundaries in Mem3DG . For example , when modeling the formation of a small endocytic bud from a large cell , the deformation is small compared with the broader system . If we assume that the bulk of the cell is invariant with respect to bud formation , the computational burden can be reduced by modeling only the local deformation ; we can assume that the modeled patch is attached to an implicit membrane reservoir . To de \ufb01 ne this coupled system , the constant area ( A r ) and volume ( V r ) of the reservoir must also be provided . The total area and volume of the broader system is given by A \u00bc A patch \u00fe A r , and V \u00bc V patch \u00fe V r , where A patch and V patch are area and \u201c enclosed volume \u201d of the mesh patch respectively . In our models , we enforce that all elements of a boundary loop are on the same plane ; this way V patch can be unambiguously de \ufb01 ned as the enclosed volume when each boundary loop is closed by a planar sheet . The capability to model systems attached to a reservoir re - duces the modeled degrees of freedom while enabling intuitive physics to simplify the process of mimicking experimental conditions using Mem3DG . 12 Biophysical Reports 2 , 100062 , September 14 , 2022 Prescribing boundary conditions with force masking Mem3DG supports modeling membranes with and without boundaries : a sphere ( with no boundaries ) , a disk ( with one boundary ) , and an open cylinder ( with two boundaries ) . For systems without boundaries , the discrete forces conserve angular and translational mo - mentum of system ( as was also noted by Bian et al . ( 89 ) ) . Because the ( discrete ) potential energy is invariant under rigid body motions ( i . e . , the energy of the membrane is given only by the geometry ) , and the discrete forces are analytically derived from the energy , the discrete forces will not contribute to any rigid body motions since these components do not change sys - tem energy . In other words , the forces that lead to rigid body motion are , by construction , orthogonal to the shape derivative of the potential energy . To study sys - tems with boundaries , Mem3DG currently supports three types of boundary conditions : (cid:5) Roller , where the movement of boundary vertices is restricted along a given direction or plane . (cid:5) Pinned , where the position of boundary vertices are pinned while the curvature is allowed to vary . (cid:5) Fixed , where both the position and the boundary cur - vature are \ufb01 xed for vertices on the boundary . The different boundary conditions are achieved by masking the elements of the force matrix correspond - ing to the boundary vertices and their neighborhood . For example , to apply roller boundary conditions , we mask the Z - component of the force on the boundary vertices , therefore constraining their movement to the X - Y plane ; pinned boundary conditions mask all force components for the boundary vertices to \ufb01 x their posi - tion ; \ufb01 xed boundary conditions mask all force compo - nents for the outermost three layers to \ufb01 x both their position and curvature . Time integration and energy minimization In this work , we use the forward Euler algorithm to inte - grate the system dynamics and the nonlinear conjugate gradient method to solve for equilibrium conditions . Both solvers are complemented by a backtracking line search algorithm , which satis \ufb01 es Wolfe conditions to support adaptive time - stepping and robust minimi - zation ( 121 ) . The forward Euler scheme was chosen as the simplest dynamical propagator ; physically it represents over - damped conditions where the environment of the membrane is too viscous for the system to carry any inertia . Mathematically , the physics is described by , _ ~ r \u00bc 1 x Z ~ f \u00bc 1 x Z (cid:7) ~ f b \u00fe ~ f s \u00fe ~ f p (cid:8) ; ( 27 ) where x is the drag coef \ufb01 cient . From an optimization perspective , forward Euler is equivalent to the gradient descent method for minimizing an objective function , which is the discrete energy in our case . A second propagator is the nonlinear conjugate gradient method for locally minimizing the discrete en - ergy to yield the equilibrium shape of the membrane . Since the system is nonlinear , we periodically perform forward Euler ( gradient descent ) steps after several conjugate gradient steps . This approach of iterating be - tween conjugate gradient and gradient descent steps is commonplace in the literature for solving nonlinear systems ( 121 ) . We note that other time integrators and energy min - imizers are also compatible with Mem3DG . Included in the software are reference implementations of velocity Verlet integration ( for symplectic time integration ) , and limited - memory Broyden - Fletcher - Goldfarb - Shanno al - gorithm ( L - BFGS , a quasi - Newton method to the equi - librium shape for large - scale problems where fast computation is needed ) . We do not discuss these addi - tional solvers in this work . Practical considerations for applying Mem3DG to biological problems As we have noted above , in the DDG perspective , the mesh is the geometry and thus the formulation of the discrete forces and energies is exact . There are there - fore very few restrictions on the resolution and quality of the input mesh . However , in biophysics , we often consider biological membranes as smooth systems . We expect that many users of Mem3DG may wish to approximate a smooth system using our discrete model . In doing so , they make an implicit assumption that such an approximation is reasonable . Although the relationships between geometric objects and their shapes are preserved between the smooth and discrete contexts , our ability to approximate a smooth problem with a discrete mesh is not guaranteed . Similar to \ufb01 nite differences and FEM , additional con - straints on mesh quality and resolution must be imposed . To verify and understand the limitations of the assumption that the discrete mesh is the geometry and includes all of the geometric information , we numerically test the convergence of the discrete quantities under variation of resolution on an oblate spheroid mesh . The additional details regarding these numerical experiments are presented in Appendix D . Setting the characteristic length scale of the \ufb01 nest mesh to be h \u00bc 1 , as the mesh coarsens ( i . e . , mesh size increases ) h increases . Fig . 3 shows the scaling relationship of the deviation in magnitude between the smooth and discrete quantities . Fig . 3 A shows the convergence property of global measurements Biophysical Reports 2 , 100062 , September 14 , 2022 13 that determines the energy ( Eqs . 1 and 3 ) , including the total area , A , enclosed volume , V , and total Gaussian curvature and mean curvature ( squared ) , R M KdA , R M HdA and R M H 2 dA , respectively . Except for the total Gaussian curvature being an exact topological invariant , all integrated quantities exhibit approximately second - order convergence rate . We acknowledge that convergence of global mea - surements does not imply that local measurements will also converge . To validate the convergence of local measurements , which determines the conver - gence of local forces on the membrane ( e . g . , Eqs . 15 , 17 , and 22 ) , we utilize the L 1 norm ( Eq . 26 ) to eval - uate the deviation of local quantities from their smooth counterparts . Fig . 3 B shows the local conver - gence plot . Similar to their global counterparts , local scalar mean and Gaussian curvature , R H , and R K , converge at O\u00f0 h 2 \u00de . Fig . 3 B also shows the conver - gence of vector quantities , which not only contribute to the magnitude of the force but also set the direc - tion of the force . The test shows that most vector quantities converge slightly slower than their scalar counterparts . Two terms exhibit poor convergence , the Schla \ufb02 i vector term in Eq . 22 , H R S , and a scalar counterpart , R D s H . The latter term corresponds to the direct application of the cotangent Laplacian ( Eq . 17 ) to the pointwise scalar mean curvature \ufb01 eld ; this approach is not used in our framework but is common in the literature ( 65 ) . Both non - convergent expressions are discrete representations of the bihar - monic term , D s H , which have been noted to be sensi - tive to noises of vertex coordinates in the prior literature ( 100 ) . Recall that the biharmonic term is the fourth - order derivative of the embedded coordi - nates . Although the traditional approximation theories suggest that higher - order derivatives often exhibit slower rates of convergence ( 122 ) , to the best of our knowledge , there is not yet a rigorous study that connects DDG with an approximation theory . Never - theless , we anticipate that similar principles hold . Two spatial plots comparing local measurements be - tween smooth and discrete contexts are provided in the appendix ( Fig . E . 1 and Fig . E . 2 ) ; each test is con - ducted using the \ufb01 nest mesh size ( h \u00bc 1 ) . Based on this numerical validation , we conclude that the computation of energy converges with a second - order rate ( Fig . 3 A ) . While most components of the forces converge , the biharmonic term remains a limiting factor . One other practical consideration for our models is that the Helfrich Hamiltonian , matching the in - plane \ufb02 uidity of biological membranes , has no resistance to shearing . Without additional constraints , the mesh is susceptible to shearing motions , which can deteriorate mesh quality in some conditions ( 83 ) . This can degrade the implicit assumption that the discrete mesh represents a smooth geometry . To ensure that such an approximation can remain valid throughout a trajectory , we have incorporated algorithmic solutions to adaptively maintain an iso - tropically well - resolved discrete geometry . This is achieved by two operations : 1 ) mesh regularization using local force constraints , which are common in FEM ( 78 , 82 , 83 , 85 ) ( Appendix E . 2 ) ; and 2 ) mesh muta - tions such as decimating , \ufb02 ipping , and collapsing edges . Beyond regularization , these local force con - straints can also be used to model underlying physics of a problem of interest . For example , similar restoring forces between vertices ( Eq . E . 1 ) have been adopted to model actin - spectrin cortex in red blood cell ( 95 ) . Mesh mutations are also a common practice to cope with deterioration and a means to perturb system con \ufb01 guration in other mesh simula - tions that use a Monte Carlo integration ( 60 , 89 \u2013 95 ) . The algorithms for mesh regularization and mutation are further described in Appendix E . FIGURE 3 Comparison of discrete quantities with their smooth counterparts on spheroid shape . ( A ) Convergence plot of global quanti - ties , including total area , volume , mean curva - ture ( squared ) , and Gaussian curvature ; and ( B ) Convergence plot of L 1 norm of scalar and vector local quantities , including the mean curvature , Gaussian curvature , and the biharmonic term . 14 Biophysical Reports 2 , 100062 , September 14 , 2022 RESULTS AND DISCUSSION To further validate the method and to provide a sense of how Mem3DG can be used and extended to solve more complex physics , we apply Mem3DG to a sequence of ex - amples with increasing complexity . First , we model well - studied systems with homogeneous membrane conditions . We show that Mem3DG is capable of repro - ducing the classical solutions without imposing the axisymmetric constraint commonly adopted by other solvers . The later examples set a blueprint for extending and modifying Mem3DG for particular systems of inter - est . We introduce new energy and corresponding force terms to expand the formulation for complex systems of interest . We emphasize that the goal of these exam - ples is to illustrate the generality of the theory and soft - ware and to outline speci \ufb01 c steps for future extensions ; we do not perform rigorous experimental comparisons , nor do we extract scienti \ufb01 c insights . Additional care must be taken to mimic speci \ufb01 c biological experiments for model validity , which is left for future work . Each of the following sections considers a different class of membrane biophysics problem of increasing complexity in the coupling of the in - plane protein den - sity parameter , f \u02db \u00bd 0 ; 1 (cid:4) . To mimic the various in \ufb02 u - ences protein - lipid interactions can have on the bilayer , the protein density can be set to in \ufb02 uence membrane properties such as the spontaneous curva - ture , (cid:1) H \u00f0 f \u00de , and bending rigidity , k \u00f0 f \u00de . More complex phenomena such as the production of in - plane interfa - cial forces from membrane - protein phase separation ( 55 , 59 , 123 ) can also be modeled . In our \ufb01 nal proof of concept , we extend Mem3DG to support full mechano - chemical dynamics , where proteins can mobilize in and out of plane through adsorption and lateral diffu - sion , based on its coupling with membrane material properties and shape transformation . These scenarios highlight the relative ease of extending Mem3DG with additional physics and the potential utility to simulate realistic experimental scenarios . Note that , for all of the examples , unless otherwise speci \ufb01 ed , the bending rigidity of membrane , k , is assumed to be the rigidity of a bare membrane , k b \u00bc 8 : 22 (cid:3) 10 (cid:2) 5 m m $ nN . Despite the superior performance of the nonlinear con - jugate gradient method in \ufb01 nding an energy minimizing con \ufb01 guration , to maintain both static and dynamic interpretability , we perform all simulations using a for - ward Euler integrator unless otherwise noted . All simu - lations presented in this work were conducted on a standard workstation with Intel Xeon processors . Although the numerical algorithms are amenable to parallelization , Mem3DG is currently a single - threaded program . Using a single core , the simulations here complete in minutes and up to 2 hours . Modeling spherical and cylindrical membranes with homogeneous physical properties We begin our examples by using Mem3DG to \ufb01 nd the equilibrium shapes of membranes with homogeneous protein density , f . We ask , given an initial membrane FIGURE 4 Recovertypical equilibriumshapes of membranes with homogeneous material properties . ( A and B ) Equilibrium solutions un - der different osmolarity ( (cid:1) c ) and spontaneous curvature ( (cid:1) H ) conditions , with initial condition of ( A ) Oblate spheroid and ( B ) Prolate spheroid . Wevarytheosmolaritybyadjustingtheconcen - tration of the ambient solution , (cid:1) c , while holding the enclosed amount of solute , n , constant . ( C ) Equilibrium solutions of a tubular membrane structureundervariationsinosmolarityandsur - face tension . Biophysical Reports 2 , 100062 , September 14 , 2022 15 con \ufb01 guration with uniform bending modulus and spon - taneous curvature , what are the minimizers of the sys - tem energy ? The answers are the classical equilibrium solutions to the shape equation obtained analytically ( 42 ) , and numerically using many methods with different assumptions ( 39 , 124 ) . We will show solutions obtained using Mem3DG with topologies of sphere and tube ( Fig . 4 ) . These geometries were selected not only because of their potential for comparison with the leg - acy literature but also because they are reminiscent of biological membranous structures such as red blood cell ( 97 , 98 , 125 , 126 ) , cell - cell tunneling and tethering ( 127 \u2013 129 ) , and neuron beading ( 130 , 131 ) , among other biological processes . Starting with closed topological spheres , Fig . 4 A and B shows the typical equilibrium shapes under osmotic stress while the surface area is conserved . The preferred area of the vesicle , (cid:1) A \u00bc 4 p m m 2 , represents a sphere of radius 1 m m . This constraint is achieved by prescribing a large stretching modulus , K A , such that the areal strain , \u00f0 A (cid:2) (cid:1) A \u00de = A , is less than 1 % . The strength constant of osmotic pressure , K V is set to be 0 . 1 m m $ nN . Initializing the simulations from an oblate spheroid , as the osmolarity increases ( e . g . , the normalized ambient solution , (cid:1) c = n ) , we recover the well - known biconcave red blood cell shape ( 97 , 98 , 106 , 124 ) ( Fig . 4 A ) . The vesicle adopts a more convex con \ufb01 guration as we increase the spontaneous curvature , indicating an overall increase in its mean cur - vature with the concomitant decrease of areas with negative mean curvature ( the dimple regions ) . In contrast , starting from a prolate spheroid , as the spon - taneous curvature increases , the vesicle adopts a dumbbell con \ufb01 guration as the energetically preferred state ( Fig . 4 B ) . The size of the beads on the dumbbell is governed by the osmolarity , (cid:1) c = n . These trends with respect to the variations of the spontaneous curvature and osmolarity are consistent with the analytical and numerical results in the broader literature ( 42 , 89 ) . Qual - itatively the predicted geometries of closed vesicles with homogeneous spontaneous curvature match the predictions of a detailed benchmark of mesh - based methods performed by Bian et al . ( 89 ) . We also modeled the shapes of membranes starting from an open cylinder con \ufb01 guration under different os - motic and surface tension conditions ( Fig . 4 C ) . This problem is related to a well - studied phenomenon called the Plateau - Rayleigh instability ( 132 , 133 ) . The Plateau - Rayleigh instability describes how surface tension breaks up a falling stream of \ufb02 uid into liquid droplets . Compared with a liquid stream , a lipid membrane pro - vides additional resistance against instability due to its rigidity . Zhongcan and Helfrich ( 134 ) obtain stability regimes as a function of membrane bending rigidity and spontaneous curvature using the spectral stability analysis ( 134 ) . Although osmotic pressure is often re - ported as an important cause of morphological insta - bility ( 131 , 135 \u2013 137 ) , the effect of osmotic pressure is dif \ufb01 cult to isolate in wet experiments because the change to osmolarity affects the surface tension , which is a key driver of the instability . In our simula - tions , the two effects are decoupled , making the inves - tigation of individual contributions to the morphology possible . All shapes in Fig . 4 C evolve from the initial tubular mesh with radius of 1 m m and axial length of 19 . 9 m m , under a constant spontaneous curvature of 1 m m (cid:2) 1 . These simulations are set up as local models ( cf . , section \u201c De \ufb01 ning properties of a membrane reser - voir for systems with open boundaries \u201d ) where the explicit mesh is assumed to be coupled to a membrane reservoir . Additional geometric information de \ufb01 ning the membrane reservoir and boundary conditions are required to initialize the local model . The tubular struc - ture is considered to be a cylinder that connects two otherwise detached domains ( e . g . , membrane reser - voirs ) , which combined have a total reservoir volume , V r \u00bc 4 : 19 m m 3 . The strength of osmotic pressure , K V , is set to be 0 . 01 m m $ nN . To isolate the effect of os - motic pressure and surface tension on the morphology , we prescribe a speci \ufb01 c surface tension that we as - sume to be invariant with respect to changes to the sur - face area . On the two boundary loops of the mesh , we apply roller boundary conditions , which restrict the movement of boundary vertices in the axial direction . The length of the tube is thus constrained to be 19 . 9 m m , while the radius of the tube including the boundaries is free to shrink or expand . As the osmolarity increases from the reference con - dition ( (cid:1) c = n \u00bc 0 : 022 m m (cid:2) 3 ) ( Video S1 ) , we obtain near constant - mean - curvature surfaces such as unduloid pearl structure at (cid:1) c = n \u00bc 0 : 030 m m (cid:2) 3 ( Video S2 ) , and cylindrical tube at (cid:1) c = n \u00bc 0 : 051 m m (cid:2) 3 , which follow the trends from both analytical ( 42 , 138 ) and experi - mental observations ( 19 , 131 , 135 ) . As we increase the surface tension from the reference condition ( l \u00bc 1 (cid:3) 10 (cid:2) 7 nN $ m m (cid:2) 1 ) to a tension - dominated regime ( l \u00bc 1 (cid:3) 10 (cid:2) 4 nN $ m m (cid:2) 1 ) , we obtain the beads - on - a - string structure that minimizes the sur - face - to - volume ratio ( Video S3 ) . The formation of beads - on - a - string is an interesting con \ufb01 guration that has been identi \ufb01 ed in biological membranes and other systems ( 130 , 131 ) . Note that our simulations revealed a symmetric metastable state where two large beads form at either end ( Appendix A ) , connected by a thin tube , prior to adopting the asymmetric conformation shown in Fig . 4 C . We believe that discretization arti - facts such as mesh mutations act as a perturbation to break the symmetry of the metastable intermediate and transition the membrane to a single bead con \ufb01 gu - ration ( see Fig . A . 1 ) . 16 Biophysical Reports 2 , 100062 , September 14 , 2022 These examples with uniform spontaneous curvature pro \ufb01 le prove the ability of Mem3DG to reproduce the ex - pected classical solutions for spherical and tubular membrane geometries . Note that no axisymmetric constraint is imposed in these simulations . Mem3DG sol - ves the system in full three dimensions and the symmet - rical con \ufb01 gurations are due to the problem physics . The ability to adapt to changing and complex curvatures of the membrane using discrete mesh is achieved using mesh mutation and other manipulations within solver steps . For example , the pinched neck regions of the tubes are automatically decimated with \ufb01 ner triangles than other regions of the mesh . For a global closed membrane simulation such as in Fig . 4 A , B , we do not remove any rigid body motions from the system ; since the forces from DDG are exact and we used the forward Euler integrator , no arti \ufb01 cial rigid body motions are intro - duced throughout the simulation . These examples show that that the derivation of the discrete energy and forces along with the software implementation are accurate and proceed to test Mem3DG with more complex examples . Modeling endocytic budding mechanisms Our goal is to highlight the potential of Mem3DG and its associated framework for investigating mechanical phenomena relevant to cellular biology . Endocytosis is a cellular process in which cells uptake cargo from the extracellular environment ; the transported material is engulfed by the cell membrane , which then buds off to form a vesicle ( 13 ) . Endocytosis occurs through various mechanisms , including clathrin - mediated endocytosis ( 13 , 139 ) . It has been shown that clathrin aggregates on the plasma membrane , helping to deform the membrane and form a spherical bud ( 9 , 13 , 59 ) . However , the speci \ufb01 c mechanisms of how membrane - clathrin interactions facilitate membrane curvature generation remain unresolved . While there is signi \ufb01 cant literature investigating the many pro - posed mechanisms , here we develop models to demonstrate the bud formation via spatially localized spontaneous curvature , combined with a line tension term arising from phase separations on the membrane ( 140 ) . We model endocytic budding on a circular patch with radius 1 m m ( a disc with one boundary loop ) . We as - sume that the patch is a local system which is coupled to a large vesicle ( section \u201c De \ufb01 ning properties of a membrane reservoir for systems with open bound - aries \u201d ) . A heterogeneous protein density , f \u02db \u00bd 0 ; 1 (cid:4) , is applied to mimic the distribution of clathrin and other scaffolding proteins . Shown in Fig . 5 A , the protein den - sity is high ( f \u00bc 1 ) toward the center of a geodesic disk with radius 0 . 5 m m ) and decreases toward the boundaries ( f \u00bc 0 ) . During simulation , the geodesic distance to the center of the patch is periodically computed using the heat method ( 141 ) . Vertexwise f is assigned based on the stair - step pro \ufb01 le smoothed by the hyperbolic tangent function applied to the geodesic distance . Each experiment is initialized as a \ufb02 at patch and the displacement of boundary vertices is restricted using a \ufb01 xed boundary condition . Since the patch is viewed as a small piece within a larger closed vesicle reservoir , we assume that the surface tension is constant . A common model to account for the preferential bending owing to protein - membrane interactions is through the spontaneous curvature ; we assume (cid:1) H \u00f0 f \u00de \u00bc (cid:1) H c f , where (cid:1) H c \u00bc 6 m m (cid:2) 1 is the spontaneous curvature imposed by the membrane protein coat . Pro - teins such as clathrin are known to form stiff scaffolds on the membrane . Similar to the spontaneous curva - ture , we can assume a linear relationship between bending rigidity and protein density , k \u00f0 f \u00de \u00bc k b \u00fe k c f , where constant k b is the rigidity of the bare membrane , and k c is additional rigidity of the protein scaffold . Shown in Fig . 5 A \u2013 C and Video S4 , is the control simulation where we set the contribution to the rigidity from protein to be the same as that of the raw mem - brane , k c \u00bc k b . Fig . 5 A shows the initial \ufb02 at con \ufb01 gura - tion of the control experiment ; the color bar shows the heterogeneous spontaneous curvature resulting from the prescribed protein density pro \ufb01 le . In the control experiment , the bending force is resisted by the surface tension ( Fig . 5 C ) until , at the \ufb01 nal frame in Fig . 5 B ( t \u00bc 5 ) , the membrane reaches the equilibrium con \ufb01 guration where the surface tension cancels with the bending force . In a second model , we assume that the scaffolding proteins are much more rigid than the bare membrane , k c \u00bc 3 k b . Fig . 5 D \u2013 F and Video S5 show the bud formation due to this increased protein scaffolding effect . The greater rigidity results in an increase of initial bending energy , which outcom - petes the resistance from the surface tension ( Fig . 5 F ) . Fig . 5 E shows the shape evolution from a \ufb02 at patch to a successful bud with a pinched neck . Fig . 5 D shows the signed projection of the bending force onto the ver - tex normal , R f bi \u00bc R ~ f b i $ ~ n i , at T \u00bc 15 . ( Outward - point - ing angle - weighted normal ; the same applies to the interfacial line tension . ) We can see an \u201c effective line tension \u201d driven by the heterogeneous spontaneous curvature that constricts the neck . This phenomenon is theoretically explored in detail by Alimohamadi et al . ( 58 ) . For our third model , based on the prior observations that protein phase separation on surfaces can lead to a line tension ( 140 ) , we incorporate a Ginzburg - Landau interfacial energy into the system , Biophysical Reports 2 , 100062 , September 14 , 2022 17 E d \u00bc 1 2 X f ijk h Z (cid:13)(cid:13)(cid:13)(cid:13) V ~ q f (cid:13)(cid:13)(cid:13)(cid:13) 2 ijk / 1 2 Z M h (cid:13)(cid:13)(cid:13)(cid:13) V ~ q f (cid:13)(cid:13)(cid:13)(cid:13) 2 dA ( 28 ) where h , referred to as the Dirichlet energy constant , governs the strength of the energy penalty , and V ~ q f is the discrete surface gradient of the protein density pro \ufb01 le . The term is similar to previous modeling efforts by Elliott and Stinner ( 80 ) and Ma and Klug ( 79 ) using FEM ; because we use the protein phase sep - aration as a prior , we exclude the double - well term , which models the thermodynamics of phase separa - tion , and incorporate only the Dirichlet energy compo - nent that penalizes the heterogeneity of membrane composition . De \ufb01 ned as the slope of the linearly interpolation of f on faces of the mesh , f ijk , the discrete surface gradient of the protein density is , V ~ q f i \u00bc 1 2 A ijk X e i \u02db N \u00f0 f ijk \u00de f i ~ e t i ; ( 29 ) where following illustration in Fig . 1 C , ~ e i is the vector aligned with the halfedge e i , with its length of l i , and \u00f0 $ \u00de t represents a 90 (cid:6) counterclockwise rotation in the plane of f ijk . The resulting line tension force R ~ f d is then the shape derivative of the Dirichlet energy , V ~ r E d , which acts to minimize the region with sharp het - erogeneity . The detailed derivation of the shape deriva - tive is elaborated in Appendix C . 3 , where we follow the formulaic approach by taking geometric derivatives of basic mesh primitives shown in Eq . C . 13 . Note that despite bearing the same name , this line tension force differs from from those resulting from line energy , which prescribes the line tension energy based on the interfacial edge length ( 55 , 142 ) . The line tension force from Dirichlet energy is used to model the out - of - plane component resulting from either entropic or enthalpic repulsion at the interface between heterogeneous membrane protein aggregates . The Dirichlet energy is based on a 2D \ufb01 eld variable and the line tension is only effective when phase separation of the \ufb01 eld vari - able occurs , where the interfacial line , or , more pre - cisely , thin areal band , between phases exists . With the introduction of protein evolution later in sections \u201c Protein aggregation on the realistic mesh of a den - dritic spine \u201d and \u201c Membrane dynamics with full mech - anochemical feedback , \u201d thickness depends on the competition between the Dirichlet energy and other competing aggregational potential . FIGURE 5 Budding dynamics by robust mechanisms of protein scaffolding and interfacial line tension constriction . ( A \u2013 C ) Control group , ( D \u2013 F ) bending - driven scaffolding mechanism , and ( G \u2013 I ) Interfacial line tension assisted budding . ( A ) Spontaneous curvature distribution , (cid:1) H , on initially \ufb02 at patch . ( D ) Normal projection of the bending force at T \u00bc 15 . ( G ) Normal projection of the line tension force at T \u00bc 7 . ( B , E , H ) Shape evo - lution through time - series snapshots of the Y - Z cross sections of the membrane , corresponding to the vertical dash lines in ( C ) , ( F ) , ( I ) trajectory plots of system energy and its competing components . 18 Biophysical Reports 2 , 100062 , September 14 , 2022 Fig . 5 G \u2013 I and Video S6 show the trajectory where we used control bending rigidity , k c \u00bc k b , and the addi - tional interfacial line tension , h \u00bc 5 (cid:3) 10 (cid:2) 4 m m $ nN . We \ufb01 nd that the interfacial line tension , jointly with the bending force , lowers the system energy and helps the formation of a spherical bud ( Fig . 5 I and H ) . Fig . 5 G shows the snapshot ( t \u00bc 7 ) with the color map representing the signed normal projection of the interfacial line tension that acts to constrict the neck . These examples of endocytic bud formation help to illustrate the utility of Mem3DG and the accompanying theoretical framework . Since physical parameters are assigned on a per - vertex basis , it is straightforward to incorporate heterogeneity such as the nonuniform bending rigidity and spontaneous curvature . In smooth theory and its derived discrete mesh models , when the membrane is heterogeneous , it is required to decom - pose the force separately in normal and tangential di - rection ( 40 , 65 ) . In contrast , the general derivation of the discrete bending force following the formalism of DDG permits modeling membrane with heterogeneous material properties without any modi \ufb01 cation to its formulation ( section \u201c Forces from bending \u201d ) . The intro - duction of Dirichlet energy and line tension force serves to highlight the relative ease to extend the modeled physics . Protein aggregation on the realistic mesh of a dendritic spine While the prior examples have focused on the mechan - ical response of the membrane given a bound protein distribution , we can also model the inverse problem . Given the membrane shape , how do curvature - sensing proteins diffuse in the plane of the membrane and distribute over the domain ? And how does the resultant protein distribution in \ufb02 uence the stresses of the sys - tem ? To model the protein dynamics , we use three terms corresponding to protein binding , curvature sensitivity , and lateral diffusion . To model the binding of proteins to the membrane , we assume that the energy of adsorption , \u03b5 , is constant and uniform across the surface such that the discrete adsorption energy is , E a \u00bc \u03b5 X i Z f i ; ( 30 ) where f i is an order parameter representing the area density of protein at each vertex . Taking the derivative with respect to f , referred to as the chemical derivative , m ai \u00bc (cid:2) V f E a \u00bc (cid:2) Z \u03b5 ; ( 31 ) we obtain the adsorption component of the chemical potential . To account for protein curvature sensitivity , we \ufb01 nd the chemical potential of the bending energy , m bi \u00bc (cid:2) V f E b \u00bc Z (cid:2) 2 k i \u00f0 H i (cid:2) (cid:1) H i \u00de V f (cid:1) H i (cid:2) \u00f0 H i (cid:2) (cid:1) H i \u00de 2 V f k i (cid:3) ; ( 32 ) where we assume that V f k i \u00bc k c , and V f (cid:1) H i \u00bc (cid:1) H c where k c and (cid:1) H c are constant parameters de \ufb01 ned in Section \u201c Modeling endocytic budding mechanisms . \u201d The \ufb01 rst term of Eq . 32 endows the protein with curva - ture - sensitive binding . The second term of Eq . 32 is the shape mismatch penalty ; considering the binding of a rigid protein that induces a signi \ufb01 cant spontaneous curvature change , if the curvature of membrane is far from this new spontaneous curvature , then the shape mismatch between the membrane and proteins will pre - vent binding . Alternatively , if the protein is more \ufb02 exible , a shape mismatch results in a small energetic penalty . The in - plane diffusion of the protein is accounted for by the chemical derivative of the smoothing Dirichlet energy , E d , m di \u00bc (cid:2) V f E d \u00bc (cid:2) Z h D s f i ; ( 33 ) where h is the same Dirichlet energy constant intro - duced in Eq . 28 that governs the strength of interfacial line tension , R ~ f d . The total chemical potential captures the bending , adsorption and diffusion components . A mobility rate constant , B , determines the time scale of the chemical response , _ f \u00bc B m \u00bc B \u00f0 m b \u00fe m a \u00fe m d \u00de : ( 34 ) We investigate the in \ufb02 uence of curvature - dependent binding to a realistic dendritic spine geometry , which was reconstructed from electron micrographs and curated using GAMer 2 ( Fig . 6 A ) ( 32 ) . A summary of the parameters used in the simulation is shown in Table 3 . The mean curvature of the spine geometry is shown Fig . 6 C . We isolate the effect of curvature - dependent binding by assuming that the shape of the spine is \ufb01 xed and impose Dirichlet boundary conditions at the base on the spine to \ufb01 x the protein concentration , f \u00bc 0 : 1 ( Fig . 6 A ) . Starting from a homogeneous protein distribution , f 0 \u00bc 0 : 1 , Fig . 6 B and Video S7 show the evolution of the protein distribution and a trajectory of the sys - tem energy . Note that , for simplicity , we have turned off the adsorption energy term since it only shifts the basal protein - membrane interactions , which will also be set by the Dirichlet boundary condition . Mem3DG constrains the range of f \u02db \u00f0 0 ; 1 \u00de using the interior point Biophysical Reports 2 , 100062 , September 14 , 2022 19 method ( 121 ) . Due to the curvature sensitivity of the protein , illustrated by the snapshots ( Fig . 6 B , T \u00bc 350 ) representing the \ufb01 nal protein distribution , the protein aggregates toward regions of high curva - ture ( e . g . , on the spine head ) . Although the proteins can reduce the bending energy by modulating the local bending modulus and sponta - neous curvature , the protein distribution at equilibrium does not cancel out the bending energy . We expect that the Dirichlet energy term , which limits f to be smooth across the geometry , restricts the protein from further aggregating to the extent required to cancel out the bending energy . The components of forces on the initial and \ufb01 nal con \ufb01 gurations of the spine are compared in Fig . 6 D \u2013 F . The initial homoge - neous protein distribution has no line tension forces and a bending force shown in Fig . 6 D . After the protein distribution reaches the steady state , line tension appears in response to membrane heterogeneity Fig . 6 E . We hypothesize that , similar to section \u201c Modeling endocytic budding mechanisms , \u201d the line tension constricts the neck of the spine and helps to support the cup - like structures in the spine head . While , in most regions , the distribution of proteins reduces the force , several regions experience increased stress Fig . 6 F . Note that the magnitude of the forces gener - ated by proteins in this model is orders of magnitude smaller than the initial bending force . Thus , this example demonstrates that Mem3DG can be used on meshes imported from realistic geometries of cellular substructures . Membrane dynamics with full mechanochemical feedback In this section , we will demonstrate the use of Mem3DG to model the complete mechanochemical feedback of a protein - membrane system . For the following simula - tions , not only can proteins bind in a curvature - depen - dent manner but the membrane can also deform , leading to a feedback loop . We have introduced all of the force terms in previous sections except the shape derivative of the adsorption energy , FIGURE 6 Protein aggregation on a realistic dendritic spine geometry . ( A ) Mesh of the dendritic spine and its boundary elements . ( B ) Trajectory of protein evolution and components of system energy . ( C ) Mean curvature of the geometry . The normal component of ( D ) the bending force at t \u00bc 0 , ( E ) the line tension force produced by the equilibrium protein distribution , and ( F ) the difference in the bending force pro \ufb01 le produced by \ufb01 nal protein distribution as opposed to the initial distribution . TABLE 3 Parameters used in section \u201c Protein aggregation on the realistic mesh of a dendritic spine \u201d Parameters Values f 0 0 . 1 k c 0 nN $ m m (cid:1) H c 10 m m (cid:2) 1 B 3 nN (cid:2) 1 $ m m (cid:2) 1 $ s (cid:2) 1 h 0 . 01 m m $ nN 20 Biophysical Reports 2 , 100062 , September 14 , 2022 Z ~ f a i \u00bc (cid:2) V ~ r E a \u00bc (cid:2) X e ij \u02db N \u00f0 v i \u00de \u03b5 (cid:9) f i 3 \u00fe 2 f j 3 (cid:10)Z 2 ~ H i ; ( 35 ) which accounts for the change in the area of protein coverage ( i . e . , expanded coverage if \u03b5 < 0 ) . Revisiting the claim that all discrete forcing is exact with respect to the discrete energy , we validate by examining the convergence of the forcing terms with respect to the size of perturbation to the system con \ufb01 guration , e ( Fig . 7 A ) . This is based on the leading order expansion done in Eq . 24 , which concludes that the forcing terms are exact if their rate of convergence is at least second order . Shown in Fig . 7 A , this is true for all forcing terms ; note that , since the adsorption energy , E a , is a linear function with respect to f , m a can be determined to the machine precision for all perturbation sizes . A meaningful discrete - smooth comparison of all terms in the energy and forcing similar to section \u201c Practical considerations for applying Mem3DG to biological problems \u201d requires the analytical solutions of the bending force and inter - facial line tension arising from the spatially heteroge - neous membrane properties , which , to the best of our knowledge , are not available . In section \u201c Modeling endocytic budding mechanisms , \u201d we introduced a het - erogeneous membrane composition as a static prop - erty . By prescribing the protein density pro \ufb01 le , we can get hints about how to form membrane buds from a patch . Here we lift this assumption and simu - late the dynamics of osmotic pressure - driven budding from a vesicle . The dynamics couples the protein - membrane mechanochemical feedback and includes protein binding and diffusion introduced in section \u201c Protein aggregation on the realistic mesh of a FIGURE 7 Modeling budding from a vesicle driven by the full mechanochemical feedback of membrane - protein interactions . ( A ) Validation of the exactness of the discrete forcing with respect to the discrete energy . The terms correspond to forces from bending f b , tension area f s , pres - sure - volume f p , Dirichlet f d , and protein binding f a . m d , m b , and m a are the chemical potential of diffusion , bending , and binding respectively . ( B ) The time trajectory of budding dynamics in hypertonic , isotonic , and hypotonic osmotic conditions . ( C ) The \ufb01 nal snapshot of the system con \ufb01 gura - tion under hypertonic , isotonic , and hypotonic conditions . ( D ) Similar geometries to those shown in ( C ) have been observed in experiments by Saleem et al . ( 59 ) . Biophysical Reports 2 , 100062 , September 14 , 2022 21 dendritic spine . \u201d The expressions of discrete free en - ergy and forcings do not change but we allow the membrane con \ufb01 guration and protein density to interact and evolve simultaneously . We start each simulation from a sphere with a uniform protein concentration , f \u00bc f 0 \u00bc 0 : 1 . We consider the evolution of the system in different osmotic conditions : hyper - , iso - , and hypotonic , (cid:1) V \u00bc 2 : 91 ; 3 : 95 and 4 : 99 m m 3 , respectively . Additional param - eters for these simulations are given in Table 4 . Fig . 7 B shows plots of the mechanical , k ~ f k 2 L 2 , and chemical response , k m k 2 L 2 , along with the protein density , \u00f0 f max \u00fe f min \u00de = 2 , over the trajectory for each osmotic condition . Note that under hypo - and isotonic condi - tions , the system reaches the ( quasi ) steady state where both the shape and protein distribution stabilize , while , in a hypertonic solution , the system continues to experience strong mechanical force and protein mobility , which we expect to drive further morpholog - ical changes of the membrane beyond our simulation stopping point . Fig . 7 C shows the \ufb01 nal snapshot of each simulation across the osmotic conditions with the protein density represented by the color map . In hy - pertonic conditions , the osmotic pressure provides suf - \ufb01 cient perturbations to membrane morphology , which initializes the positive feedback loop between mem - brane curvature generation and protein aggregation . This mechanochemical feedback jointly promotes the outward bending of the membrane and results in bud formation ( Fig . 7 C , hypertonic ; Video S8 ) . Under isotonic and hypotonic conditions , the osmolarity does not permit the large change in the volume required to form spherical buds with a thin neck . In hy - potonic condition , the pressure - tension balance pro - vides substantial stability to the initial spherical con \ufb01 guration . In comparison , in the isotonic condition , the comparable competition between the chemical and mechanical response leads to a patterned protein dis - tribution and an undulating morphology ( Fig . 7 C , hypo - tonic ; Video S9 ) . This example illustrates the possibility to utilize Mem3DG to model a full mechanochemical feedback between membrane and protein . Although we do not intend to replicate the exact experimental conditions and assumptions , the geometries obtained from these simulations resemble the shapes obtained by Saleem et al . ( 59 ) who investigated budding from spherical vesicles under differing osmotic conditions ( Fig . 7 D ) ( 59 ) . SUMMARY In this work , we introduce a new perspective for con - structing a 3D membrane mechanics model on discrete meshes . The goal of our approach is to close the gap between existing discrete mesh - based models ( 60 , 86 \u2013 96 , 99 , 102 , 103 ) and the smooth theory . Specif - ically , we seek to advance the discussion behind the choice of algorithmic approaches for computing geo - metric values required for the discrete energy and force ( 65 , 89 , 99 , 100 ) . We start by writing a discrete energy , Eq . 3 , mirroring the spontaneous curvature model . Then using the perspective of DDG , we show that there is a formulaic approach for deriving the corresponding discrete force terms based on fundamental geometric vectors . By identifying geometric invariants and grouping terms , the resulting discrete forces have exact correspondence to the traditional smooth theory . This helps us to facilitate the comparison between smooth and discrete contexts enabling new geometric perspectives and understanding of numerical accu - racy . Moreover , the discrete force terms are functions of readily accessible geometric primitives , and the formulation is amenable for ef \ufb01 cient implementation and extension . We have produced a reference software implementa - tion called Mem3DG . Using Mem3DG , we validate our the - ory by reproducing the solutions to the classical shape transformations of a spherical and tubular vesicle . We further demonstrate the derivation and incorporation of additional physics terms to model protein - membrane interactions . Following our formulaic approach using DDG , we derived the discrete analog of various physics , such as the interfacial line tension , surface - bulk adsorption , protein lateral diffusion , and curvature - dependent protein aggregation . To exemplify all the introduced physics , the full mechanochemical coupling between the membrane shape and protein density evo - lution results in protein localization , pattern formation , and budding . These examples serve to highlight the extensibility of the framework , which does not require the introduction of coordinates and advanced tensor calculus commonly needed to solve problems on arbi - trary manifolds . The software implementation Mem3DG was designed to facilitate coordination between TABLE 4 Parameters used in section \u201c Membrane dynamics with full mechanochemical feedback \u201d for models with full mechanochemical feedback Parameters Values f 0 0 . 1 k c 8 . 22 (cid:3) 10 (cid:2) 5 m m $ nN (cid:1) H c 10 m m (cid:2) 1 K V 0 . 5 nN $ m m K A 1 nN $ m m (cid:2) 1 B 3 nN (cid:2) 1 $ m m (cid:2) 1 $ s (cid:2) 1 x 1 nN $ s $ m m (cid:2) 1 \u03b5 (cid:2) 1 (cid:3) 10 (cid:2) 3 nN $ m m h 0 . 1 m m $ nN (cid:1) V 2 . 91 , 3 . 95 , and 4 . 99 m m 3 22 Biophysical Reports 2 , 100062 , September 14 , 2022 theoretical modeling and wet experiments ; we hope to support the modeling of scenes with well - resolved pro - tein - membrane interactions such as in the electron tomograms ( 143 ) . We expect that as the advances in biophysical modeling and membrane ultrastructure im - aging progresses , Mem3DG will emerge as a useful tool to test new hypotheses and understand cellular mechanobiology . APPENDICES A . Supplemental \ufb01 gures B . Rationale for integrated measurements in discrete contexts The rationale for why an integrated measurement in discrete con - texts is the natural counterpart to pointwise measurements in smooth contexts can be demonstrated by considering the curvature of a discrete polygonal curve . If we attempt to de \ufb01 ne the curvature , C , of the discrete polygonal curve in a na\u00efve pointwise manner , following the procedure in smooth settings , we will \ufb01 nd zero curva - ture along edges and in \ufb01 nite curvature ( owing to the discontinuity ) on vertices . Thus the traditional view of curvature from smooth manifolds reveals no useful information about the geometry of the discrete curve . We must \ufb01 nd another geometric relationship that can translate between smooth and discrete contexts to maintain the geometric connection . One relationship from smooth differential geometry is the equiva - lence of the integrated curvature and the turning angle j ( i . e . , the total angle by which the tangent vector of the curve turns over some domain l ) . Returning to the discrete context , we can seek to preserve this relationship between the integrated curvature and turning angle by \ufb01 nding a compatible de \ufb01 nition . Since the discrete turning angle , j i , between two connected edges of the discrete polygonal curve is well de \ufb01 ned , we can set the discrete curvature , R C , of a vertex , v i , to be (cid:9)Z C (cid:10) i h j i : ( B . 1 ) We note that the notation for the discrete curvature , \u00f0 R C \u00de i is used only in this illustrative example ; in the remainder of the text , we will omit the parenthesis and use the simpli \ufb01 ed notation , R C i . To make sense of the integral over a discrete object , additional care must be taken to represent the curvature from a distributional sense ( 104 ) . This is related to traditional approximation methods , such as the point allocation method , which bridges a smooth and FIGURE A . 2 Steiner ' s formula in continuous and discrete geometry : chain of smooth and discrete shape derivatives of integrated geo - metric measurements ( 144 ) . FIGURE A . 1 A symmetric metastable state with two beads instead of a single larger bead is observed , prior to collapsing to the solution shown in Fig . 4 C , high tension . Biophysical Reports 2 , 100062 , September 14 , 2022 23 discrete problem by convoluting the smooth problem with impulse functions ( e . g . , the Dirac delta function ) at a \ufb01 nite number of loca - tions ( 122 ) . As we have shown , integrated geometric measurements enable us to preserve geometric relationships ( from smooth contexts ) for discrete objects , and are thus preferred over pointwise de \ufb01 nitions . Nevertheless , we often require a pointwise discrete measurement for use in algorithms and visualization . An integrated measurement can be converted to a meaningful pointwise discrete measurement by normalizing the value over a domain . For the discrete polygonal curve , thedomaincanbethedualvertexlength , l i ( i . e . , thediscreteanalogof l ) . l i is given by half of the sum lengths of the two incident edges . A point - wise curvature on the vertex v i is then given by , C i \u00bc Z C i = l i \u00bc j i = l i : ( B . 2 ) Another rationale for using an integrated value for a discrete geo - metric measurement is that we can arrive at the same de \ufb01 nition from multiple perspectives . Returning to the de \ufb01 nition of the curva - ture of a polygonal curve , without introducing the turning angle , we can arrive at the same result by adopting the Steiner view ( 104 , 145 ) ( we use the Steiner view to de \ufb01 ne the discrete curvature of a surface in section \u201c Obtaining a discrete energy de \ufb01 ned by mesh primitives \u201d ) . In the Steiner view , we replace the sharp vertices with a smooth circular arc with radius e such that the discrete geometry is made smooth such that the curvature is well de \ufb01 ned everywhere . As the only curved section , every circular arc has a discrete ( integrated ) curvature , Z C \u00bc Z arc C ds \u00bc C arc l arc \u00bc 1 e \u00f0 ej \u00de \u00bc j ; ( B . 3 ) where C arc \u00bc 1 = e is the curvature of the circular arc , and l arc \u00bc ej is the arc length . We see that , in the Steiner view , the integrated curva - ture is still equivalent to the turning angle . Following similar logic , other discrete de \ufb01 nitions are described in section \u201c Obtaining a discrete energy de \ufb01 ned by mesh primitives \u201d and the DDG literature ( 104 , 105 ) . C . Discrete shape and chemical derivatives of discrete energy C . 1 . Halfedge on a triangulated mesh A scalar quantity on an edge is symmetric with respect to index permutation . For example , the scalar mean curvature ( Eq . 8 ) , Z H ij \u00bc Z H ji \u00bc l ij 4 ij 2 : ( C . 1 ) However , as we will show in detail in the following sections , this symmetry does not apply to vector quantities , which compose the discrete shape derivative of the energy , force . For example , the corre - sponding mean curvature vector , Z ~ H ij s Z ~ H ji : ( C . 2 ) To highlight the directionality of vector quantities and disambig - uate the notation , here we review the concept of a halfedge on a triangulated mesh . Given any non - boundary edge , e ij , on a manifold mesh , there exits two associated halfedges , e ij and e ji ( Fig . C . 1 ) . This convention leads to an oriented ( counterclockwise ) halfedge loop on each triangle face and subsequently a well - de \ufb01 ned 1 ) 90 (cid:6) counterclockwise rotation of the halfedge in the plane of the face ( e . g . , e lj / e t lj ) , and 2 ) face normal ( outward ) based on the right hand rule ( Fig . C . 1 ) . Beside being used to differentiate vector / scalar quantities , the concept of halfedge is widely adopted data structure for managing connected graphs , or meshes , for which we refer the reader to the broader literature ( 116 , 146 ) . C . 2 . Deriving the bending force as the shape derivative of bending energy The geometric derivatives of mesh primitives , including edge length , l , dihedral angle , 4 , and vertex dual area , A , are given as V ~ r i l ij \u00bc ~ e ji l ij ; ( C . 3a ) V ~ r i 4 ij \u00bc 1 l ij (cid:9) cot : ijk ~ n ijk \u00fe cot : ijl ~ n ilj (cid:10) ; ( C . 3b ) V ~ r i 4 jk \u00bc (cid:2) 1 l jk (cid:11) cot : ijk \u00fe cot : ikj (cid:12) ~ n ijk \u00bc (cid:2) l jk 2 A ijk ~ n ijk ; ( C . 3c ) V ~ r i A i \u00bc 1 3 X f ijk \u02db N \u00f0 v i \u00de V ~ r i A ijk \u00bc 1 6 X e ij \u02db N \u00f0 v i \u00de (cid:11) cot : ikj \u00fe cot : ilj (cid:12) ~ e ji ; ( C . 3d ) V ~ r i A j \u00bc 1 3 X f ijk \u02db N \u00f0 e ij \u00de V ~ r i A ijk \u00bc 1 6 (cid:7) ~ e t jk \u00fe ~ e t lj (cid:8) ; ( C . 3e ) where ~ n ijk is the unit normal vector of the face f ijk , and ~ e ji is the vector aligned with the halfedge , e ji , with its length of l ij ( 147 ) . The indices and nomenclature in Eqs . C . 3b , C . 3c and C . 3e are illustrated in the diamond neighborhood ( Fig . C . 1 ) and those of Eq . C . 3d are illustrated in the fan neighborhood ( Fig . 1 A ) . FIGURE C . 1 Schematics for halfedges on a triangulated mesh . 24 Biophysical Reports 2 , 100062 , September 14 , 2022 To simplify the expression and provide more structure for the sub - sequent discrete variation , it is convenient to de \ufb01 ne some funda - mental curvature vectors , Z 2 ~ H ij \u00bc 1 2 (cid:7) V ~ r i A ijk \u00fe V ~ r i A ijl (cid:8) \u00bc 1 4 ~ e t jk \u00fe ~ e t lj ! ( C . 4a ) Z ~ K ij \u00bc 1 2 4 ij V ~ r i l ij ( C . 4b ) Z ~ S ij ; 1 \u00bc 1 2 l ij V ~ r i 4 ij \u00bc 1 2 (cid:9) cot : ijk ~ n ijk \u00fe cot : ijl ~ n ilj (cid:10) ( C . 4c ) Z ~ S ij ; 2 \u00bc 1 2 (cid:7) l jk V ~ r i 4 jk \u00fe l jl V ~ r i 4 jl \u00fe l ji V ~ r i 4 ji (cid:8) \u00bc (cid:2) 1 2 (cid:9) cot : jki ~ n ijk \u00fe cot : ilj ~ n ilj (cid:10) ; ( C . 4d ) where the mean curvature vector , R ~ H , results from area gradient ; Gaussian curvature vector , R ~ K , and the Schla \ufb02 i vector , R ~ S , consist of the two components of the variation of total mean curvature , 12 P e ij l ij 4 ij . The asymmetry of vector quantities in Eq . C . 4 under index permutation ( Eq . C . 2 ) arises from the vertex we take the shape deriv - ative with respect to ( i . e . , v i , or v j ) ; because of the asymmetry , we can associate each Schla \ufb02 i vector with a unique halfedge . Similar to the translation from edge values to vertex value ( Eq . 9 ) , we can also translate the halfedge value to vertex value by summing all halfedge values over the fan neighborhood , Z \u00f0 $ \u00de i \u00bc X e ij \u02db N \u00f0 v i \u00de Z \u00f0 $ \u00de ij : ( C . 5 ) Note that , unlike translating edge values , there is no prefactor 1 = 2 for translating halfedge values because each halfedge is uniquely associated with one vertex . The translated curvature vectors on a ver - tex cane compared against vertexwise smooth analytical solutions as benchmarked in section \u201c Practical considerations for applying Mem3DG to biological problems . \u201d Now we have all of the elements needed to derive the derivatives of the discrete Willmore bending en - ergy . Because the discrete energy is locally supported by the vertex , v i , and its 1 - ring neighbors , v j \u02db N \u00f0 v i \u00de , we can separate them into the \u201c diagonal \u201d term , and \u201c off - diagonal \u201d term , Using the derivatives of geometric primitives in Eq . C . 3 , we can assemble the derivatives of local pointwise mean curvature for both the diagonal term , V ~ r i H i \u00bc 1 4 X e ij \u02db N \u00f0 v i \u00de V ~ r i l ij 4 ij A i \u00bc 1 4 A i X e ij \u02db N \u00f0 v i \u00de (cid:7) 4 ij V ~ r i l ij \u00fe l ij V ~ r i 4 ij (cid:8) (cid:2) H i A i V ~ r i A i \u00bc 1 A i X e ij \u02db N \u00f0 v i \u00de 1 2 (cid:9) Z ~ K ij \u00fe Z ~ S ij ; 1 (cid:10) (cid:2) 2 3 H i Z ~ H ij ; ( C . 7 ) and for the off - diagonal term , When written in the halfedge form , factoring out the fundamental curvature vectors introduced in Eq . C . 4 , we obtain the discrete bending force as Z ~ f b i \u00bc (cid:2) V ~ r i E b \u00bc (cid:2) V ~ r i X i k i \u00f0 H i \u00f0 ~ r \u00de (cid:2) (cid:1) H i \u00de 2 A i \u00f0 ~ r \u00de ! \u00bc (cid:2) V ~ r i (cid:2) k i \u00f0 H i (cid:2) (cid:1) H i \u00de 2 A i (cid:3) | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } diagonal (cid:2) X v j \u02db N \u00f0 v i \u00de V ~ r i h k j (cid:11) H j (cid:2) (cid:1) H j (cid:12) 2 A j i | \ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 { z\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04\ufb04 } off - diagonal : ( C . 6 ) V ~ r i H j \u00bc 1 4 X e jk \u02db N \u00f0 v j \u00de V ~ r i l jk 4 jk A j \u00bc 1 4 A j (cid:7) l jk V ~ r i 4 jk \u00fe l jl V ~ r i 4 jl \u00fe 4 ji V ~ r i l ji \u00fe l ji V ~ r i 4 ji (cid:8) (cid:2) H j A j V ~ r i A j \u00bc 1 2 A j (cid:9) Z ~ K ij \u00fe Z ~ S ij ; 2 (cid:10) (cid:2) 4 3 H j Z ~ H ij : ( C . 8 ) Biophysical Reports 2 , 100062 , September 14 , 2022 25 When the surface is not closed , boundary vertices , v i \u02db v M , experience an additional force from the Gaussian curvature component , Z ~ f b i \u00bc RHS of Eq : \u00f0 C : 9 \u00de \u00fe X f ijk \u02db N \u00f0 v i \u00de \u00f0(cid:2) 1 \u00de p V ~ r i : f ijk ; v i \u02db v M ; ( C . 10 ) where p is the number of boundary vertices in face f ijk , and : f ijk \u00bc 8 < : : ijk if v j ; v k ; v M ; : ikj if v j \u02db v M ; : ijk if v k \u02db v M : ( C . 11 ) C . 3 . Deriving the line tension and diffusion as the shape and chemical derivatives of the Dirichlet energy Since the discrete Dirichlet energy is constructed on the triangular face and therefore does not involve any neighborhood , we simplify the notation by adopting the convention illustrated in Fig . 1 C . The gradient of protein density is given by the slope of the \ufb01 tted plane over the vertexwise protein density , which is piecewise constant for each face , V ~ q f i \u00bc 1 2 A ijk X ~ e k \u02db N \u00f0 f ijk \u00de f k ~ e t k ; ( C . 12 ) where we adopt the counterclockwise convention ( e . g . , ~ e k \u00bc ~ e ji ) and \u00f0 $ \u00de t represents a 90 (cid:6) counterclockwise rotation in plane of the face , f ijk . C . 3 . 1 . Line tension from the shape derivative of the Dirichlet energy . Substituting the de \ufb01 nition of the discrete gradient into the Dirichlet energy ( Eq . 28 ) , we expand the energy in terms of mesh primitives , whose geometric derivatives are given in Eq . C . 3 . Additional formulae are needed to compute the geometric derivatives of the outer angles of the triangle ( Fig . 1 C ) V ~ r i : k \u00bc ~ n (cid:3) ~ e j k ~ e j k 2 ( C . 13a ) V ~ r i : j \u00bc ~ n (cid:3) ~ e k k ~ e k k 2 ( C . 13b ) V ~ r i : i \u00bc (cid:2) (cid:7) V ~ r i : k \u00fe V ~ r i : j (cid:8) ; ( C . 13c ) which arise from the calculation of the L 2 norm of the gradients as the result of vector inner product . When combined , the geometric deriva - tives for the quadratic gradient term is Then we can get the \ufb01 nal shape derivative by combining the area gradient , or the mean curvature vector ( Eq . C . 4 ) . C . 3 . 2 . Surface diffusion from the chemical derivative of the Dirichlet energy . In the case where we are evolving the protein dis - tribution , we need the chemical derivative of the Dirichlet energy . Before we look into the discrete case , we can \ufb01 rst tackle the problem Z ~ f b i \u00bc X e ij \u02db N \u00f0 v i \u00de (cid:2) (cid:2) k i \u00f0 H i (cid:2) (cid:1) H i \u00de \u00fe k j (cid:11) H j (cid:2) (cid:1) H j (cid:12)(cid:3)Z ~ K ij \u00fe (cid:5) 1 3 k i \u00f0 H i (cid:2) (cid:1) H i \u00de\u00f0 H i \u00fe (cid:1) H i \u00de \u00fe 2 3 k j (cid:11) H j (cid:2) (cid:1) H j (cid:12)(cid:11) H j \u00fe (cid:1) H j (cid:12)(cid:6) Z 2 ~ H ij (cid:2) (cid:5) k i \u00f0 H i (cid:2) (cid:1) H i \u00de Z ~ S ij ; 1 \u00fe k j (cid:11) H j (cid:2) (cid:1) H j (cid:12) Z ~ S ij ; 2 (cid:6) : ( C . 9 ) V ~ r i (cid:15) X f k ~ e t k ; X f k ~ e t k (cid:16) \u00bc \u00fe f k f k ~ e k (cid:2) 2 f j f j ~ e j \u00fe 2 f j f i k ~ e i k (cid:9) (cid:2) b e j cos : k \u00fe k ~ e j k V ~ r i \u00f0 cos : k \u00de (cid:10) \u00fe 2 f i f k k ~ e i k (cid:9) b e k cos : j \u00fe k ~ e k k V ~ r i (cid:11) cos : j (cid:12)(cid:10) \u00fe 2 f j f k (cid:9) (cid:2) b e j k ~ e k k cos : i \u00fe k ~ e j k b e k cos : i \u00fe k ~ e j kk ~ e k k V ~ r i \u00f0 cos : i \u00de (cid:10) ( C . 14 ) 26 Biophysical Reports 2 , 100062 , September 14 , 2022 in the smooth setting , which is a classic textbook example . Using the Green ' s \ufb01 rst identity , or integration by parts on a 2 - manifold , Z M (cid:11) j D s 4 \u00fe V ~ q j $ V ~ q 4 (cid:12) dA \u00bc I v M j V ~ q 4 $ ~ ndS ; ( C . 15 ) and ignoring the boundary term at the right hand side , we arrive at an alternative expression for the Dirichlet energy , E d \u00bc 1 2 Z M h k V ~ q f k 2 dA \u00bc (cid:2) 1 2 Z M hf D s f dA : ( C . 16 ) The same procedure can be followed in the discrete case . The discrete Dirichlet energy ( Eq . 28 ) can be written in matrix form , E d \u00bc 1 2 h f u ~ G u ~ T ~ G f ( C . 17 ) where ~ G is the gradient tensor , which maps scalar value on vertices to vector values on faces , and ~ T \u00bc diag \u00f0 A face \u00de is the j f j(cid:3) j f j diagonal matrix with entries corresponding to the area of each mesh triangle face . Through integration by parts on discrete geometry , the discrete Dirichlet energy can be equivalently expressed as E d \u00bc 1 2 h f u ~ L f ; ( C . 18 ) which is a quadratic form with respect to the cotangent Laplacian matrix , ~ L ( 104 , 105 ) . The chemical derivative of the Dirichlet energy , or the diffusion potential , is m d \u00bc (cid:2) V f E d \u00bc (cid:2) h Z D s f \u00bc (cid:2) h ~ L f : ( C . 19 ) In other words , the chemical gradient \ufb02 ow of the Dirichlet energy is the diffusion equation . Note that ~ L \u00bc ~ G u ~ T ~ G , ~ G u is referred to as the discrete divergence operator , which maps face vectors to scalars on vertices ( 146 ) . D . Discrete - smooth comparison on spheroid The smooth - discrete comparison is done on the spheroid with the parametrization , \u00f0 x ; y ; z \u00de \u00bc \u00f0 a cos b cos q ; a cos b sin q ; c sin b \u00de ; ( D . 1 ) where a \u00bc 1 , b \u00bc 0 : 5 , b is the parametric latitude and q is the azimuth coordinate . All geometric measurements of the smooth geometry used for benchmarking were obtained using the symbolic algebra software Sympy . The corresponding discrete measurements are computed using Mem3DG , whose input spheroid mesh is mapped from a subdivided icosphere . The subsequent error norms for local measurements are computed based on de \ufb01 nitions used in section \u201c De \ufb01 ning metrics for simulation and error quanti \ufb01 cation . \u201d E . Mesh regularization and mesh mutation E . 1 . Mesh mutation Mesh mutation and re \ufb01 nement in combination with vertex shifting are the default methods to ensure that the mesh remains well condi - tioned and well resolved during simulation . Mesh mutations include edge \ufb02 ipping , collapsing , and splitting , which change the connectivity of the mesh . Vertex shifting moves the vertex to the barycenter of the fan neighborhood without changing the mesh topology ( Fig . 1 A ) . Mem3DG has a suite of possible criteria to initiate mesh mutation . Here we list the most important ones : 1 ) \ufb02 ip the edge of the non - De - launay diamond neighborhood ( Fig . 1 B ) , 2 ) collapse the shortest FIGURE E . 1 Pointwise magnitude comparison of continuous and discrete measurements : ( A ) scalar mean curvature , ( B ) scalar Gaussian cur - vature , ( C ) ( scalar ) bi - Laplacian term V H based on the cotan formula , ( D ) vector mean curvature , ( E ) vector Gaussian curvature , and ( F ) ( vector ) bi - Laplacian term based on Schla \ufb02 i vector . Note that the result of the cotangent Laplacian approach in ( C ) produces a scalar result while our approach using the Schla \ufb02 i vector in ( F ) is a vector result , thus their direct comparison is not meaningful . Biophysical Reports 2 , 100062 , September 14 , 2022 27 edge in a skinny triangle face , and 3 ) split the edge with high ( geodesic ) curvature . For additional details , please refer to the soft - ware documentation . For practical use , although mesh mutation introduces additional complexity in data write - out and computational costs associated with varying ( usually growing ) mesh size , it nevertheless provides a robust algorithm to ensure the good mesh quality needed for valid discrete - smooth comparisons ( section \u201c Practical consider - ations for applying Mem3DG to biological problems \u201d ) in static frames . For dynamical simulation , mesh mutations introduce an arbi - trary interpolation of state variables , such as the position , velocity , and protein density . Rigorous study on how to interpolate these quan - tities to ensure the conservation of energy , momentum , and mass re - mains to be done . Similarly , the interpolation used in this study introduces discontinuities of curvature and can create jumps in forces ; this is particularly severe for terms with higher - order deriva - tives such as the biharmonic term in bending force ( Eq . 22 ) . E . 2 . Mesh regularization Mesh regularization can be used when mesh mutations are not desired . The regularization force consists of three weakly enforced constraining forces : the edge ( length ) , ~ f e , face ( area ) , ~ f f , and confor - mality ( angle ) , ~ f c , regularization forces , ~ f e i \u00bc (cid:2) K e X e ij \u02db N \u00f0 v i \u00de (cid:11) l ij (cid:2) (cid:1) l ij (cid:12) (cid:1) l ij V ~ ri l ij ; ( E . 1a ) ~ f f i \u00bc (cid:2) K f X f ijk \u02db N \u00f0 v i \u00de (cid:11) A ijk (cid:2) (cid:1) A ijk (cid:12) (cid:1) A ijk V ~ r i A ijk ; ( E . 1b ) ~ f c i \u00bc (cid:2) K c X e ij \u02db N \u00f0 v i \u00de (cid:11) l ij (cid:2) (cid:1) l ij (cid:12) (cid:1) l ij V ~ r i l ij ; ( E . 1c ) which are in the order of strongest to weakest . The length - cross - ratio , l ij \u00bc l il l jk = l ki l jl is a metric of discrete conformality on triangulated mesh , where the indices is illustrated in Fig . 1 A and B ( 148 ) . Regula - rization forces require the input of a reference value for geometric measurements , (cid:1) l , (cid:1) A , and (cid:1) l , which can be derived from a well - condi - tioned reference mesh ( usually the initial input mesh for the simula - tion ) . The intensity of each regularization force is controlled with parameters K e , K f , and K c . For practical use , regularization constraints should be minimally imposed because of their impact on system dynamics . In the worst case , regularization constraints can prevent the optimizer from reach - ing an energy minima . Thus a good practice is to start a simulation with no : conformality , face area , and \ufb01 nally edge length regulariza - tion , and subsequently raise the intensity / type of constraints based on the mesh quality desired . We do not recommend imposing con - straints stronger than the face areal constraints , ~ f f . In addition to be - ing numerical regularizers for the triangulated mesh , they can serve as model for certain additional physics . For example , the edge length regularization has been adopted to model the additional local rigidity from actin - spectrin cortex in red blood cells ( 95 ) . The areal regulari - zation can be used to model local incompressibility of the membrane . The conformality regularization can be used to isolate shearing resistance . SUPPORTING MATERIAL Supplemental information can be found online at https : / / doi . org / 10 . 1016 / j . bpr . 2022 . 100062 . AUTHOR CONTRIBUTIONS P . R . conceived the research . P . R . , C . Z . , and C . T . L . designed the research . C . Z . developed the theory and analyzed the results . C . Z . and C . T . L . developed the software . C . Z . , C . T . L . , and P . R . wrote and edited the article . All authors read and approved the \ufb01 nal article . ACKNOWLEDGMENTS The authors would like to acknowledge Dr . Ali Behzadan , Prof . Ravi Ramamoorthi , and Prof . Albert Chern for helpful discussions and crit - ical feedback . This work was supported in part by the National Insti - tutes of Health R01GM132106 , National Science Foundation DMS 1934411 , Of \ufb01 ce of Naval Research N00014 - 20 - 1 - 2469 , and an Air Force Of \ufb01 ce of Scienti \ufb01 c Research DURIP FA9550 - 19 - 1 - 0181 to P . R . C . T . L . also acknowledges support from a Hartwell Foundation Postdoctoral Fellowship . DECLARATION OF INTERESTS The authors declare no competing interests . REFERENCES 1 . Groves , J . T . , and J . Kuriyan . 1989 . Molecular mechanisms in signal transduction at the membrane . Nat . Struct . Mol . Biol . 17 : 659 \u2013 665 . https : / / doi . org / 10 . 1038 / nsmb . 1844 . 2 . Cheng , X . , and J . C . Smith . 2019 . Biological membrane organiza - tion and cellular signaling . Chem . Rev . 119 : 5849 \u2013 5880 . 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Soc . opensci . 3 : 160384 . http : / / dx . doi . org / 10 . 1098 / rsos . 160384 Received : 1 June 2016 Accepted : 17 August 2016 Subject Category : Psychology and cognitive neuroscience Subject Areas : theoretical biology / computer modelling and simulation / statistics Keywords : metascience , cultural evolution , statistical power , replication , incentives , Campbell\u2019s Law Author for correspondence : Paul E . Smaldino e - mail : paul . smaldino @ gmail . com Electronic supplementary material is available at http : / / dx . doi . org / 10 . 1098 / rsos . 160384 or via http : / / rsos . royalsocietypublishing . org . The natural selection of bad science Paul E . Smaldino 1 and Richard McElreath 2 1 CognitiveandInformationSciences , UniversityofCalifornia , Merced , CA95343 , USA 2 DepartmentofHumanBehavior , Ecology , andCulture , MaxPlanckInstitutefor EvolutionaryAnthropology , Leipzig , Germany PES , 0000 - 0002 - 7133 - 5620 ; RME , 0000 - 0002 - 0387 - 5377 Poor research design and data analysis encourage false - positive \ufb01ndings . Such poor methods persist despite perennial calls for improvement , suggesting that they result from something more than just misunderstanding . The persistence of poor methods results partly from incentives that favour them , leading to the natural selection of bad science . This dynamic requires no conscious strategizing\u2014no deliberate cheating nor loa\ufb01ng\u2014 by scientists , only that publication is a principal factor for career advancement . Some normative methods of analysis have almost certainly been selected to further publication instead of discovery . In order to improve the culture of science , a shift must be made away from correcting misunderstandings and towards rewarding understanding . We support this argument with empirical evidence and computational modelling . We \ufb01rst present a 60 - year meta - analysis of statistical power in the behavioural sciences and show that power has not improved despite repeated demonstrations of the necessity of increasing power . To demonstrate the logical consequences of structural incentives , we then present a dynamic model of scienti\ufb01c communities in which competing laboratories investigate novel or previously published hypotheses using culturally transmitted research methods . As in the real world , successful labs produce more \u2018progeny , \u2019 such that their methods are more often copied and their students are more likely to start labs of their own . Selection for high output leads to poorer methods and increasingly high false discovery rates . We additionally show that replication slows but does not stop the process of methodological deterioration . Improving the quality of research requires change at the institutional level . 2016 The Authors . Published by the Royal Society under the terms of the Creative Commons Attribution License http : / / creativecommons . org / licenses / by / 4 . 0 / , which permits unrestricted use , providedtheoriginalauthorandsourcearecredited . 2 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The more any quantitative social indicator is used for social decision - making , the more subject it will be to corruption pressures and the more apt it will be to distort and corrupt the social processes it is intended to monitor . Donald T . Campbell ( 1976 , p . 49 ) [ 1 ] I\u2019ve been on a number of search committees . I don\u2019t remember anybody looking at anybody\u2019s papers . Number and IF [ impact factor ] of pubs are what counts . Terry McGlynn ( realscientists ) ( 21 October 2015 , 4 : 12 p . m . Tweet . ) 1 . Introduction In March 2016 , the American Statistical Association published a set of corrective guidelines about the use and misuse of p - values [ 2 ] . Statisticians have been publishing guidelines of this kind for decades [ 3 , 4 ] . Beyond mere signi\ufb01cance testing , research design in general has a history of shortcomings and repeated corrective guidelines . Yet misuse of statistical procedures and poor methods has persisted and possibly grown . In \ufb01elds such as psychology , neuroscience and medicine , practices that increase false discoveries remain not only common , but normative [ 5 \u2013 11 ] . Why have attempts to correct such errors so far failed ? In April 2015 , members of the UK\u2019s science establishment attended a closed - door symposium on the reliability of biomedical research [ 12 ] . The symposium focused on the contemporary crisis of faith in research . Many prominent researchers believe that as much as half of the scienti\ufb01c literature\u2014not only in medicine , by also in psychology and other \ufb01elds\u2014may be wrong [ 11 , 13 \u2013 15 ] . Fatal errors and retractions , especially of prominent publications , are increasing [ 16 \u2013 18 ] . The report that emerged from this symposium echoes the slogan of one anonymous attendee : \u2018Poor methods get results . \u2019 Persistent problems with scienti\ufb01c conduct have more to do with incentives than with pure misunderstandings . So \ufb01xing them has more to do with removing incentives that reward poor research methods than with issuing more guidelines . As Richard Horton , editor of The Lancet , put it : \u2018Part of the problem is that no one is incentivised to be right\u2019 [ 12 ] . This paper argues that some of the most powerful incentives in contemporary science actively encourage , reward and propagate poor research methods and abuse of statistical procedures . We term this process the natural selection of bad science to indicate that it requires no conscious strategizing nor cheating on the part of researchers . Instead , it arises from the positive selection of methods and habits that lead to publication . How can natural selection operate on research methodology ? There are no research \u2018genes\u2019 . But science is a cultural activity , and such activities change through evolutionary processes [ 19 \u2013 25 ] . Philosophers of science such as Campbell [ 19 ] , Popper [ 26 ] and Hull [ 27 ] have discussed how scienti\ufb01c theories evolve by variation and selection retention . But scienti\ufb01c methods also develop in this way . Laboratory methods can propagate either directly , through the production of graduate students who go on to start their own labs , or indirectly , through prestige - biased adoption by researchers in other labs . Methods which are associated with greater success in academic careers will , other things being equal , tend to spread . The requirements for natural selection to produce design are easy to satisfy . Darwin outlined the logic of natural selection as requiring three conditions : ( i ) There must be variation . ( ii ) That variation must have consequences for survival or reproduction . ( iii ) Variation must be heritable . In this case , there are no biological traits being passed from scienti\ufb01c mentors to apprentices . However , research practices do vary . That variation has consequences\u2014habits that lead to publication lead to obtaining highly competitive research positions . And variation in practice is partly heritable , in the sense that apprentices acquire research habits and statistical procedures from mentors and peers . Researchers also acquire research practice from successful role models in their \ufb01elds , even if they do not personally know them . Therefore , when researchers are rewarded primarily for publishing , then habits which promote publication are naturally selected . Unfortunately , such habits can directly undermine scienti\ufb01c progress . This is not a new argument . But we attempt to substantially strengthen it . We support the argument both empirically and analytically . We \ufb01rst review evidence that institutional incentives are likely to increase the rate of false discoveries . Then we present evidence from a literature review of repeated calls for improved methodology , focusing on the commonplace and easily understood issue of statistical 3 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . power . We show that despite over 50 years of reviews of low statistical power and its consequences , there has been no detectable increase . While the empirical evidence is persuasive , it is not conclusive . It is equally important to demonstrate that our argument is logically sound . Therefore , we also analyse a formal model of our argument . Inspecting the logic of the selection - for - bad - science argument serves two purposes . First , if the argument cannot be made to work in theory , then it cannot be the correct explanation , whatever the status of the evidence . Second , formalizing the argument produces additional clarity and the opportunity to analyse and engineer interventions . To represent the argument , we de\ufb01ne a dynamical model of research behaviour in a population of competing agents . We assume that all agents have the utmost integrity . They never cheat . Instead , research methodology varies and evolves due to its consequences on hiring and retention , primarily through successful publication . As a result our argument applies even when researchers do not directly respond to incentives for poor methods . We show that the persistence of poor research practice can be explained as the result of the natural selection of bad science . 2 . Institutional incentives for scientific researchers The rate at which new papers are added to the scienti\ufb01c literature has steadily increased in recent decades [ 28 , 29 ] . This is partly due to more opportunities for collaboration , resulting in more multi - author papers [ 30 , 31 ] . However , the increases in publication rate may also be driven by changing incentives . Recently , Brischoux & Angelier [ 32 ] looked at the career statistics of junior researchers hired by the French CNRS in evolutionary biology between 2005 and 2013 . They found persistent increases in the average number of publications at the time of hiring : newly hired biologists now have almost twice as many publications as they did 10 years ago ( 22 in 2013 versus 12 . 5 in 2005 ) . These numbers re\ufb02ect intense competition for academic research positions . The world\u2019s universities produce many more PhDs than there are permanent academic positions for them to \ufb01ll [ 33 \u2013 35 ] , and while this problem has escalated in recent years , it has been present for at least two decades [ 36 ] . Such competition is all the more challenging for researchers who graduate from any but the most prestigious universities , who face additional discrimination on the job market [ 37 ] . Although there may be jobs available outside of academia\u2014 indeed , often better - paying jobs than university professorships\u2014tenure - track faculty positions at major research universities come with considerable prestige , \ufb02exibility and creative freedom , and remain desirable . Among those who manage to get hired , there is continued competition for grants , promotions , prestige and placement of graduate students . Given this competition , there are incentives for scientists to stand out among their peers . Only the top graduate students can become tenure - track professors , and only the top assistant professors will receive tenure and high pro\ufb01le grants . Recently , the Nobel laureate physicist Peter Higgs , who pioneered theoretical work in the search for fundamental particles in the 1960s , lamented \u2018Today I wouldn\u2019t get an academic job . It\u2019s as simple as that . I don\u2019t think I would be regarded as productive enough\u2019 ( The Guardian , 6 Dec 2013 ) . Of course , such a statement is speculative ; a young Peter Higgs might well get a job today . But he might not be particularly successful . Evidence suggests that only a very small proportion of scientists produce the bulk of published research and generate the lion\u2019s share of citation impact [ 38 ] , and it is these researchers who are likely to be the source of new labs and PhDs . Supporting this argument is evidence that most new tenure - track positions are \ufb01lled by graduates from a small number of elite universities\u2014typically those with very high publication rates [ 37 ] . One method of distinguishing oneself might be to portray one\u2019s work as groundbreaking . And indeed , it appears that the innovation rate has been skyrocketing . Or claims at innovation , at any rate . In the years between 1974 and 2014 , the frequency of the words \u2018innovative\u2019 , \u2018groundbreaking\u2019 and \u2018novel\u2019 in PubMed abstracts increased by 2500 % or more [ 39 ] . As it is unlikely that individual scientists have really become 25 times more innovative in the past 40 years , one can only conclude that this language evolution re\ufb02ects a response to increasing pressures for novelty , and more generally to stand out from the crowd . Another way to distinguish oneself is through sheer volume of output . Substantial anecdotal evidence suggests that number of publications is an overwhelmingly important factor in search committee decision - making . Output may be combined with impact\u2014some researchers place emphasis on metrics such the h - index , de\ufb01ned as the largest number h such that an individual has h publications with at least h citations each [ 40 ] . Yet volume alone is often perceived as a measure of researcher quality , particularly for early - career researchers who have not yet had the time to accrue many citations . Although the degree to which publication output is used for evaluation\u2014as well as what , exactly , constitutes acceptable productivity\u2014varies by discipline , our argument applies to all \ufb01elds in which the number of published papers , however scaled , is used as a benchmark of success . 4 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Whenever a quantitative metric is used as a proxy to assess a social behaviour , it becomes open to exploitation and corruption [ 1 , 41 , 42 ] . This is often summarized more pithily as \u2018when a measure becomes a target , it ceases to be a good measure\u2019 . For example , since the adoption of the h - index , researchers have been observed to arti\ufb01cially in\ufb02ate their indices through self - citation [ 43 ] and even a clever type of quasi - fraud . With the goal of illustrating how the h - index system might be gamed , researchers created six fake papers under a fake author that cited their papers extensively [ 44 ] . They posted these papers to a university server . When the papers were indexed by Google , their h - indices on Google Scholar increased dramatically . Strategies that target evaluative metrics may be invented by cheaters , but they may propagate through their consequences . 1 Our argument is that incentives to generate a lengthy CV have particular consequences on the ecology of scienti\ufb01c communities . The problem stems , in part , from the fact that positive results in support of some novel hypothesis are more likely to be published than negative results , particularly in high - impact journals . For example , until recently , the Journal of Personality and Social Psychology refused to publish failed replications of novel studies it had previously published [ 45 ] . Many researchers who fail to garner support for their hypotheses do not even bother to submit their results for publication [ 46 ] . The response to these incentives for positive results is likely to increase false discoveries . If researchers are rewarded for publications and positive results are generally both easier to publish and more prestigious than negative results , then researchers who can obtain more positive results\u2014 whatever their truth value\u2014will have an advantage . Indeed , researchers sometimes fail to report those hypotheses that fail to generate positive results ( lest reporting such failures hinders publication ) [ 47 ] , even though such practices make the publication of false positives more likely [ 11 ] . One way to better ensure that a positive result corresponds to a true effect is to make sure one\u2019s hypotheses have \ufb01rm theoretical grounding and that one\u2019s experimental design is suf\ufb01ciently well powered [ 15 ] . However , this route takes effort and is likely to slow down the rate of production . An alternative way to obtain positive results is to employ techniques , purposefully or not , that drive up the rate of false positives . Such methods have the dual advantage of generating output at higher rates than more rigorous work , while simultaneously being more likely to generate publishable results . Although sometimes replication efforts can reveal poorly designed studies and irreproducible results , this is more the exception than the rule [ 48 ] . For example , it has been estimated that less than 1 % of all psychological research is ever replicated [ 49 ] and failed replications are often disputed [ 50 \u2013 52 ] . Moreover , even \ufb01rmly discredited research is often cited by scholars unaware of the discreditation [ 53 ] . Thus , once a false discovery is published , it can permanently contribute to the metrics used to assess the researchers who produced it . False discoveries in the literature can obviously result from fraud or p - hacking [ 54 ] , but there are many ways that false discoveries can be generated by perfectly well - intentioned researchers . These are easy to spot when the results are absurd ; for example , following standard methods for their \ufb01elds , researchers have observed a dead Atlantic salmon exhibiting neural responses to emotional stimuli [ 55 ] and university students apparently demonstrating \u2018pre - cognitive\u2019 abilities to predict the outcome of a random number generator [ 56 ] . However , false discoveries are usually not identi\ufb01able at a glance , which is why they are problematic . In some cases , poor or absent theory amounts to hypotheses being generated almost at random , which substantially lowers the probability that a positive result represents a real effect [ 13 , 15 , 57 ] . Interpretation of results after data is collected can also generate false positives through biased selection of statistical analyses [ 58 ] and post hoc hypothesis formation [ 8 ] . Campbell\u2019s Law , stated in this paper\u2019s epigraph , implies that if researchers are incentivized to increase the number of papers published , they will modify their methods to produce the largest possible number of publishable results rather than the most rigorous investigations . We propose that this incentivization can create selection pressures for the cultural evolution of poor methods that produce publishable \ufb01ndings at the expense of rigour . It is important to recognize that this process does not require any direct strategizing on the part of individuals . To draw an analogy from biological evolution , giraffe necks increased over time , not because individual animals stretched their necks , but because those with longer necks could more effectively monopolize food resources and thereby produce more offspring . In the same way , common methodologies in scienti\ufb01c communities can change over time not only because established researchers are strategically changing their methods , but also because certain researchers are more successful in transmitting their methods to younger generations . Incentives in\ufb02uence both the patterns of innovation and the nature of selection . Importantly , it is not necessary that strategic innovation be common in order for \ufb01tter strategies to spread rapidly in the research population when they appear . 1 Incentives to increase one\u2019s h - index may also encourage researchers to engage in high - risk hypothesizing , particularly on \u2018hot\u2019 research topics , because they can increase their citation count by being corrected . 5 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Case study : statistical power has not improved As a case study , let us consider the need for increased statistical power . Statistical power refers to the probability that a statistical test will correctly reject the null hypothesis when it is false , given information about sample size , effect size and likely rates of false positives 2 [ 59 ] . Because many effects in the biomedical , behavioural and social sciences are small [ 60 \u2013 62 ] , it is important for studies to be suf\ufb01ciently high - powered . On the other hand , low - powered experiments are substantially easier to perform when studying humans or other mammals , particularly in cases where the total subject pool is small , the experiment requires expensive equipment or data must be collected longitudinally . It is clear that low - powered studies are more likely to generate false negatives . Less clear , perhaps , is that low power can also increase the false discovery rate and the likelihood that reported effect sizes are in\ufb02ated , due to their reduced ability to mute stochastic noise [ 13 , 63 , 64 ] . The nature of statistical power sets up a contrast . In an imaginary academic environment with purely cooperative incentives to reveal true causal models of nature , increasing power is often a good idea . Perfect power is impossible . But very low power brings no bene\ufb01ts . However , in a more realistic academic environment that only publishes positive \ufb01ndings and rewards publication , an ef\ufb01cient way to succeed is to conduct low power studies . Why ? Such studies are cheap and can be farmed for signi\ufb01cant results , especially when hypotheses only predict differences from the null , rather than precise quantitative differences and trends [ 3 ] . We support this prediction in more detail with the model in a later section\u2014it is possible for researchers to publish more by running low - power studies , but at the cost of \ufb01lling the scienti\ufb01c literature with false discoveries . For the moment , we assert the common intuition that there is a con\ufb02ict of interest between the population and the individual scientist over statistical power . The population bene\ufb01ts from high power more than individual scientists do . Science does not possess an \u2018invisible hand\u2019 mechanism through which the naked self - interest of individuals necessarily brings about a collectively optimal result . Scientists have long recognized this con\ufb02ict of interest . The \ufb01rst highly cited exhortation to increase statistical power was published by Cohen in 1962 , as a reaction to the alarmingly low power of most psychology studies at the time [ 65 ] . The response , or lack of response , to such highly cited exhortations serves as a \ufb01rst - order test of which side of the con\ufb02ict of interest is winning . A little over two decades after Cohen\u2019s original paper , two meta - analyses by Sedlmeier & Gigerenzer [ 66 ] and Rossi [ 67 ] examined a total of 25 reviews of statistical power in the psychological and social science literature between 1960 and 1984 . These studies found that not only was statistical power quite low , but that in the intervening years since Cohen [ 65 ] , no improvement could be discerned . Recently , Vankov et al . [ 68 ] observed that statistical power in psychological science appears to have remained low to the present day . We expanded this analysis by performing a search on Google Scholar among papers that had cited Sedlmeier & Gigerenzer [ 66 ] ( the more highly cited of the two previous meta - analyses ) using the search terms \u2018statistical power\u2019 and \u2018review\u2019 . We collected all papers that contained reviews of statistical power from published papers in the social , behavioural and biological sciences , and found 19 studies from 16 papers published between 1992 and 2014 . Details about our methods and data sources can be found in the appendix . We focus on the statistical power to detect small effects of the order d = 0 . 2 , the kind most commonly found in social science research . These data , along with the data from Sedlmeier & Gigerenzer [ 66 ] and Rossi [ 67 ] , are plotted in \ufb01gure 1 . Statistical power is quite low , with a mean of only 0 . 24 , meaning that tests will fail to detect small effects when present three times out of four . More importantly , statistical power shows no sign of increase over six decades ( R 2 = 0 . 00097 ) . The data are far from a complete picture of any given \ufb01eld or of the social and behavioural sciences more generally , but they help explain why false discoveries appear to be common . Indeed , our methods may overestimate statistical power because we draw only on published results , which were by necessity suf\ufb01ciently powered to pass through peer review , usually by detecting a non - null effect . 3 Why does low power , a conspicuous and widely appreciated case of poor research design , persist ? There are two classes of explanations . First , researchers may respond directly to incentives and strategically reason that these poor methods help them maximize career success . In considering the 2 We differentiate statistical power from power more generally ; the latter is the probability that one\u2019s methods will return a positive result given a true effect , and is a Gestalt property of one\u2019s methods , not only of one\u2019s statistical tools . 3 It is possible that the average statistical power of research studies does , in fact , sometimes increase for a period , but is then quelled as a result of publication bias . Publication in many disciplines is overwhelmingly biased towards non - null results . Therefore , average powercouldincrease ( atleastintheshortrun ) , but , especiallyifhypothesisselectiondidnotimprovedatacorrespondingrate , itmight simply lead to the scenario in which higher - powered studies merely generated more null results , which are less likely to be published [ 69 ] . Labs employing lower - powered studies would therefore have an advantage , and lower - powered methods would continue to propagate . 6 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1955 1965 1975 1985 1995 2005 2015 s t a ti s ti ca l po w e r Figure 1 . Averagestatisticalpowerfrom44reviewsofpaperspublishedinjournalsinthesocialandbehaviouralsciencesbetween1960 and 2011 . Data are power to detect small effect sizes ( d = 0 . 2 ) , assuming a false - positive rate of \u03b1 = 0 . 05 , and indicate both very low power ( mean = 0 . 24 ) but also no increase over time ( R 2 = 0 . 00097 ) . persistence of low statistical power , Vankov et al . [ 68 ] suggest the following : \u2018Scientists are human and will therefore respond ( consciously or unconsciously ) to incentives ; when personal success ( e . g . promotion ) is associated with the quality and ( critically ) the quantity of publications produced , it makes more sense to use \ufb01nite resources to generate as many publications as possible\u2019 ( p . 1037 , emphasis in original ) . Second , researchers may be trying to do their best , but selection processes reward misunderstandings and poor methods . Traditions in which people believe these methods achieve broader scienti\ufb01c goals will have a competitive edge , by crowding out alternative traditions in the job market and limited journal space . Vankov et al . provide some evidence for this among psychologists : widespread misunderstandings of power and other statistical issues . What these misunderstandings have in common is that they all seem to share the design feature of making positive results\u2014true or false\u2014more likely . Misunderstandings that hurt careers are much less commonplace . Reality is probably a mix of these explanations , with some individuals and groups exhibiting more of one than the other . Our working assumption is that most researchers have internalized scienti\ufb01c norms of honest conduct and are trying their best to reveal true explanations of important phenomena . However , the evidence available is really insuf\ufb01cient . Analyses of data in evolutionary and historical investigations are limited in their ability to infer dynamical processes [ 70 ] , particularly when those data are sparse , as with investigations of scienti\ufb01c practices . To really investigate such a population dynamic hypothesis , we need a more rigorous demonstration of its logic . 4 . An evolutionary model of science To validate the logic of the natural selection of bad science , we develop and analyse a dynamical population model . Such a model simultaneously veri\ufb01es the logic of a hypothesis and helps to re\ufb01ne its predictions , so that it can be more easily falsi\ufb01ed [ 71 , 72 ] . Our model is evolutionary : researchers compete for prestige and jobs in which the currency of \ufb01tness is number of publications , and more successful labs will have more progeny that inherit their method . The foundation is a previously published mathematical model [ 15 ] in which a population of scientists investigate both novel and previously tested hypotheses and attempt to communicate their results to produce a body of literature . Variation in research quality , replication rates and publication biases are all present in the dynamics . That model was de\ufb01ned analytically and solved exactly for the probability that a given hypothesis is true , conditional on any observed publication record . Here we extend this model to focus on a \ufb01nite , heterogeneous population of N labs . We assume the following : \u2014 Each lab has a characteristic power , the ability to positively identify a true association . This power is not only the formal power of a statistical procedure . Rather it arises from the entire chain of inference . \u2014 Increasing power also increases the rate of false positives , unless effort is exerted . \u2014 Increasing effort decreases the productivity of a lab , because it takes longer to perform rigorous research . 7 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . It is important to understand why increasing power tends to also increase false positives without the application of effort . It is quite easy to produce a method with very high power : simply declare support for every proposed association , and you are guaranteed to never mistakenly mark a true hypothesis as false . Of course , this method will also yield many false positives . One can decrease the rate of false positives by requiring stronger evidence to posit the existence of an effect . However , doing so will also decrease the power\u2014because even true effects will sometimes generate weak or noisy signals\u2014unless effort is exerted to increase the size and quality of one\u2019s dataset . This follows readily from the logic of signal detection theory [ 73 ] . There are alternative ways to formalize this trade - off . For example , we might instead assume signal threshold and signal noise to be characteristics of a lab . That would hew closer to a signal detection model . What we have done here instead is focus on a behavioural hypothesis , that researchers tend to reason as if a research hypothesis were true and select methods that make it easier to \ufb01nd true effects . This maintains or increases effective power , even if nominal statistical power is low . But it simultaneously exaggerates false - positive rates , because many hypotheses are in fact not true . Formally , either system could be translated to the other , but each represents a different hypothesis about the behavioural dimensions along which inferential methods change . The trade - off between effective power and research effort has also been invoked in less formal arguments [ 64 , p . 288 ] . Given their inferential characteristics , labs perform experiments and attempt to publish their results . But positive results are easier to publish than negative results . Publications are rewarded ( through prestige , grant funding and more opportunities for graduate students ) , and more productive labs are in turn more likely to propagate their methodologies onto new labs ( such as those founded by their successful graduate students ) . New labs resemble , but are not identical to , their parent labs . The model has two main stages : Science and Evolution . In the Science stage , each lab has the opportunity to select a hypothesis , investigate it experimentally and attempt to communicate their results through a peer - reviewed publication . Hypotheses are assumed to be strictly true or false , though their essential epistemological states cannot be known with certainty but can only be estimated using experiments . In the Evolution stage , an existing lab may \u2018die\u2019 ( cease to produce new research ) , making room in the population for a new lab that adopts the methods of a progenitor lab . More successful labs are more likely to produce progeny . 4 . 1 . Science The Science stage consists of three phases : hypothesis selection , investigation and communication . Every time step , each lab i , in random order , begins a new investigation with probability h ( e i ) , where e i is the characteristic effort that the lab exerts towards using high quality experimental and statistical methods . Higher effort results in better methods ( more speci\ufb01cally , it allows higher power for a given false - positive rate ) , and also results in a lengthier process of performing and analysing experiments . 4 The probability of tackling a new hypothesis on a given time step is h ( e i ) = 1 \u2212 \u03b7 log 10 e i , ( 4 . 1 ) where \u03b7 is a constant re\ufb02ecting the extent to which increased effort lowers the lab\u2019s rate of producing new research . For simplicity , e i is bounded between 1 and 100 for all labs . In all our simulations \u03b7 = 0 . 2 , which ensured that h stayed non - negative . This value is fairly conservative ; in most of our simulations , labs were initialized with a fairly high rate of investigation , h = 0 . 63 , at their highest level of effort . However , our results are robust for any monotonically decreasing , non - negative function h ( e i ) . If an experimental investigation is undertaken , the lab selects a hypothesis to investigate . With probability r i , this hypothesis will be one that has been supported at least once in the published literature\u2014i . e . it will be an attempt to replicate prior research . Otherwise , the lab selects a novel , untested hypothesis ( at least as represented in the literature ) for investigation . Novel hypotheses are true with probability b , the base rate for any particular \ufb01eld . It is currently impossible to accurately calculate the base rate ; it may be as high as 0 . 1 for some \ufb01elds but it is likely to be much lower in many others [ 13 \u2013 15 ] . 4 We acknowledge that even methods with low power and / or high rates of false positives may require considerable time and energy to apply , and might therefore be considered effortful in their own right . For readers with such an objection , we propose substituting the word rigour to de\ufb01ne the variable e i . 8 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 power f a l s e - po s iti v e r a t e e = 1 e = 10 e = 75 Figure 2 . The relationship between power and false - positive rate , modified by effort , e . Runs analysed in this paper were initialized with e 0 = 75 ( shown in orange ) , such that \u03b1 = 0 . 05 when power is 0 . 8 . Labs vary in their investigatory approach . Each lab i has a characteristic power , W i , associated with its methodology , which de\ufb01nes its probability of correctly detecting a true hypothesis , Pr ( + | T ) . Note again that power here is a characteristic of the entire investigatory process , not just the statistical procedure . It can be very high , even when sample size is low . The false - positive rate , \u03b1 i , must be a convex function of power . We assume it to be a function of both power and the associated rigour and effort of the lab\u2019s methodology : \u03b1 i = W i 1 + ( 1 \u2212 W i ) e i . ( 4 . 2 ) This relationship is depicted in \ufb01gure 2 . What this functional relationship re\ufb02ects is the necessary signal - detection trade - off : \ufb01nding all the true hypotheses necessitates labelling all hypotheses as true . Likewise , in order to never label a false hypothesis as true , one must label all hypotheses as false . Note that the false discovery rate \u2014the proportion of positive results that are in fact false positives\u2014is determined not only by the false - positive rate , but also by the base rate , b . When true hypotheses are rarer , false discoveries will occur more frequently [ 13 , 15 ] . A feature of our functional approach is that increases in effort do not also increase power ; the two variables are independent in our model . This is unexpected from a pure signal detection perspective . Reducing signal noise , by increasing experimental rigour , will tend to in\ufb02uence both true and false - positive rates . However , our approach makes sense when power is maintained by contingent procedures that are invoked conditional on the nature of the evidence [ 58 ] . If we instead view researchers\u2019 inferential procedures as \ufb01xed , prior to seeing the data , the independence of effort and power is best seen as a narrative convenience : effort is the sum of all methodological behaviours that allow researchers to increase their power without also increasing their rate of false positives . All investigations yield either positive or negative results : a true hypothesis yields a positive result with probability W i , and a false hypothesis yields a positive result with probability \u03b1 i . Upon obtaining these results , the lab attempts to communicate them to a journal for publication . We assume that positive novel results are always publishable , while negative novel results never are . Both con\ufb01rmatory and discon\ufb01rmatory replications are published , possibly at lower rates . We adopt this framework in part because it approximates widespread publication practices . Positive and negative replications are communicated with probabilities c R + and c R \u2212 , respectively . Communicated results enter the published literature . Labs receive pay - offs for publishing their results , and these pay - offs\u2014which may be thought of in terms of factors such as prestige , in\ufb02uence or funding\u2014make their methodologies more likely to propagate in the scienti\ufb01c community at large . Labs accumulate pay - offs throughout their lifespans . Pay - offs differ for novel and replicated results , with the former being larger . Pay - offs can also accrue when other research groups attempt to replicate a lab\u2019s original hypothesis . These pay - offs can be positive , in cases of con\ufb01rmatory replication , or punitive , in cases of failure to replicate . Values for pay - offs and all other parameter values are given in table 1 . 9 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1 . Global model parameters . parameter definition values tested N number of labs 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b base rate of true hypotheses 0 . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r 0 initial replication rate for all labs { 0 , 0 . 01 , 0 . 2 , 0 . 5 } . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e 0 initial effort for all labs 75 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . w 0 initial power for all labs 0 . 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03b7 influence of effort on productivity 0 . 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c R + probability of publishing positive replication 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c R \u2212 probability of publishing negative replication 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V N pay - off for publishing novel result 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V R + pay - off for publishing positive replication 0 . 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V R \u2212 pay - off for publishing negative replication 0 . 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V O + pay - off for having novel result replicated 0 . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V O \u2212 pay - off for having novel result fail to replicate \u2212 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d number of labs sampled for death and birth events 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03bc r probability of r mutation { 0 , 0 . 01 } . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03bc e probability of e mutation { 0 , 0 . 01 } . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03bc w probability of w mutation { 0 , 0 . 01 } . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03c3 r standard deviation of r mutation magnitude 0 . 01 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03c3 e standard deviation of e mutation magnitude 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \u03c3 w standard deviation of w mutation magnitude 0 . 01 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . 2 . Evolution At the end of each time step , once all labs have had the opportunity to perform and communicate research , there follows a stage of selection and replication . First , a lab is chosen to die . A random sample of d labs is obtained , and the oldest lab of these is selected to die , so that age correlates coarsely but not perfectly with fragility . If multiple labs in the sample are equally old , one of these is selected at random . The dying lab is then removed from the population . Next , a lab is chosen to reproduce . A random sample of d labs is obtained , and from among these the lab with the highest accumulated pay - off is chosen to reproduce . This skews reproduction towards older labs as well as towards more successful labs , which agrees with the observation that more established labs and scientists are more in\ufb02uential . However , because age does not correlate with the rate at which pay - offs are accumulated , selection will favour those strategies which can increase pay - offs most quickly . A new lab with an age of zero is then created , imperfectly inheriting the attributes of its parent lab . Power , effort and replication rate independently \u2018mutate\u2019 with probabilities \u03bc w , \u03bc e and \u03bc r , respectively . If a mutation occurs , the parameter value inherited from the parent lab is increased or decreased by an amount drawn from a Gaussian distribution with a mean of zero and a standard deviation of \u03c3 w , \u03c3 e or \u03c3 r for power , effort or replication rate . If a mutation modi\ufb01es a parameter\u2019s value below or above its prescribed range , it is truncated to the minimum or maximum value . It is , of course , unrealistic to assume that all researchers have the same expected \u2018lifespan\u2019 . Many researchers disappear from academia quite early in their careers , shortly after receiving their degree or completing a postdoc . Nevertheless , the simplifying assumption of equal expected lifespan is , if anything , a conservative one for our argument . If the factors that lead a researcher to drop out of the \ufb01eld early in his or her career are unrelated to publication , then this is irrelevant to the model\u2014it is simply noise , and incorporated in the stochastic nature of the death algorithm . On the other hand , if the ability to progress in one\u2019s career is directly in\ufb02uenced by publications , then our model is , if anything , a muted demonstration of the strength of selection on publication quantity . 10 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . After death and reproduction , the published literature is truncated to a manageable size . Because few \ufb01elds have more than a million relevant publications ( assessed through searches for broad key words in Google Scholar\u2014most \ufb01elds probably have far fewer relevant papers ) , because most replications target recent work ( e . g . 90 % of replications in psychology target work less than 10 years old [ 14 ] ) and because decreased data availability for older papers makes replication more dif\ufb01cult [ 74 ] , we restrict the size of the literature available for replication to the 1 million most recently published results . This assumption was made in part to keep the computational load at manageable levels and to allow for long evolutionary trajectories . At the end of every evolutionary stage , the oldest papers in the published literature are removed until the total number is less than or equal to 1 million . 4 . 3 . Simulation runs Table 1 describes all the model parameters and their default and range values . The data are averaged from 50 runs for each set of parameter values tested . Each simulation was run for 1 million time steps , with data collected every 2000 time steps . In addition to the evolvable traits of individual research groups , we also collected data on the false discovery rate ( FDR ) for all positive results published each time step . Our goal here is illustrative rather than exploratory , and so our analysis focuses on a few illuminative examples . For the interested reader , we provide the Java code for the full model in the electronic supplementary material . 5 . Simulation results While our model is thankfully much simpler than any real scienti\ufb01c community , it is still quite complex . So we introduce bits of the dynamics in several sections , so that readers can learn from the isolated forces . 5 . 1 . The natural selection of bad science First , ignore replication and focus instead on the production of novel \ufb01ndings ( r 0 = \u03bc r = 0 ) . Consider the evolution of power in the face of constant effort ( \u03bc e = 0 , \u03bc w = 0 . 01 ) . As power increases so too does the rate of false positives . Higher power therefore increases the total number of positive results a lab obtains , and hence the number of papers communicated . As such , both W and \u03b1 go to unity in our simulations , such that all results are positive and thereby publishable ( \ufb01gure 3 ) . It is easy to prove that unimpeded power ( and false - positive rate ) will increase to unity . Fitness is directly tied to the number of publications a lab produces , so anything that increases number of publications also increases \ufb01tness . The probability that a novel hypothesis is true is the base rate , b . For a lab with power W and effort e , the probability that a test of a novel hypothesis will lead to a positive ( and therefore publishable ) result is therefore Pr ( + ) = Pr ( + | T ) Pr ( T ) + Pr ( + | F ) Pr ( F ) = bW + ( 1 \u2212 b ) W 1 + ( 1 \u2212 W ) e . ( 5 . 1 ) By differentiation , it can be shown that this probability is strictly increasing as a function of W . This implies that if selection favours ever higher discovery rates , power will continue to increase to the point at which it is matched by a countervailing force , that is , by whatever factors limit the extent to which power and false - positive rate can change . This \ufb01rst case is unrealistic . Research groups would never be able to get away with methods for which all hypotheses are supported , not to mention that increasing power without bound is not pragmatically feasible . However , one can imagine institutions to keep power relatively high , insofar as at least some aspects of power are directly measurable . At least some aspects of experimental power , such as statistical power , are directly measurable . False positives , on the other hand , are notoriously dif\ufb01cult for peer reviewers to assess [ 75 , 76 ] . If power is measurable and kept high through institutional enforcement , publication rates can still be increased by reducing the effort needed to avoid false positives . We ran simulations in which power was held constant but in which effort could evolve ( \u03bc w = 0 , \u03bc e = 0 . 01 ) . Here selection favoured labs who put in less effort towards ensuring quality work , which increased publication rates at the cost of more false discoveries ( \ufb01gure 4 ) . When the focus is on the production of novel results and negative \ufb01ndings are dif\ufb01cult to publish , institutional incentives for publication quantity select for the continued degradation of scienti\ufb01c practices . 11 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0 . 2 0 0 . 4 0 . 6 0 . 8 1 . 0 20 000 40 000 60 000 80 000 100 000 time power a FDR Figure 3 . Power evolves . The evolution of mean power ( W ) , false - positive rate ( \u03b1 ) and false discovery rate ( FDR ) . 0 20 40 60 80 0 0 . 2 0 0 . 4 0 . 6 0 . 8 1 . 0 200000 400000 600000 800000 1000000 e ff o r t a , F D R time a FDR effort Figure 4 . Effort evolves . The evolution of low mean effort corresponds to evolution of high false - positive and false discovery rates . 5 . 2 . The ineffectuality of replication Novel results are not and should not be the sole focus of scienti\ufb01c research . False discoveries and ambiguous results are inevitable with even the most rigorous methods . The only way to effectively separate true from false hypotheses is through repeated investigation , including both direct and conceptual replication [ 14 , 15 ] . Can replication impede the evolution of bad science ? If replications are dif\ufb01cult to publish , this is unlikely . On the other hand , consider a scenario in which replication efforts are easy to publish and the failure to replicate a lab\u2019s novel result causes substantial losses to the prestige of that lab . Might the introduction of replication then counteract selection for low - effort methodologies ? We repeated the previous runs , but this time initialized each lab so that 1 % of all investigations would be replications of ( randomly selected ) hypotheses from the published literature . This is consistent with empirical estimations of replication rates in psychology [ 49 ] . We then allowed the replication rate to evolve through mutation and selection ( r 0 = 0 . 01 , \u03bc w = 0 , \u03bc r = \u03bc e = 0 . 01 ) . Conditions in these runs were highly favourable to replication . We assumed that all replication efforts would be publishable , and worth half as much as a novel result in terms of evolutionary \ufb01tness ( e . g . in terms of associated prestige ) . Additionally , having one\u2019s original novel result successfully replicated by another lab increased the value of that \ufb01nding by 10 % , but having one\u2019s original result fail to replicate was catastrophic , carrying a penalty equal to 100 times the value of the initial \ufb01nding ( i . e . V O + = 0 . 1 , V O \u2212 = \u2212 100 ) . This last assumption may appear unrealistically harsh , but research indicates that retractions can lead to a substantial decrease in citations to researchers\u2019 prior work [ 77 ] . In addition , some have suggested that institutions should incentivize reproducibility by providing some sort of \u2018money - back guarantee\u2019 if results fail to replicate [ 78 ] , which could end up being highly punitive to individual researchers . More generally , these assumptions are extremely favourable to the idea that replication might deter poor methods , since false positives carry the highest risk of failing to replicate . 12 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 20 40 60 80 0 0 . 2 0 0 . 4 0 . 6 0 . 8 1 . 0 200000 400000 600000 800000 1000000 e ff o r t a , F D R , r e p li ca ti on time a FDR replication effort Figure 5 . The coevolution of effort and replication . 0 20 40 60 80 0 200 000 400 000 600 000 800 000 1 000 000 e ff o r t time no replication 25 % replication 50 % replication Figure 6 . The evolution of effort when zero , 25 % or 50 % of all studies performed are replications . We found that the mean rate of replication evolved slowly but steadily to around 0 . 08 . Replication was weakly selected for , because although publication of a replication was worth only half as much as publication of a novel result , it was also guaranteed to be published . On the other hand , allowing replication to evolve could not stave off the evolution of low effort , because low effort increased the false - positive rate to such high levels that novel hypotheses became more likely than not to yield positive results ( \ufb01gure 5 ) . As such , increasing one\u2019s replication rate became less lucrative than reducing effort and pursuing novel hypotheses . Because effort decreased much more rapidly than replication rates increased , we considered the possibility that substantially higher replication rates might be effective at keeping effort rates high , if only they could be enforced . To test this hypothesis , we ran simulations in which the replication rate could not mutate ( \u03bc r = 0 ) but was initialized to very high levels . High rates of replication did slow the decline of effort , but even extremely high replication rates ( as high as 50 % ) did not stop effort from eventually bottoming out ( \ufb01gure 6 ) . We note emphatically that we are not suggesting that highly punitive outcomes for failures to replicate are desirable , since even high - quality research will occasionally fail to replicate . Rather , we are pointing out that even in the presence of such punitive outcomes , institutional incentives for publication quality will still select for increasingly low - quality methods . 5 . 3 . Why is replication not sufficient ? Replication is not suf\ufb01cient to curb the natural selection of bad science because the top performing labs will always be those who are able to cut corners . Replication allows those labs with poor methods to be penalized , but unless all published studies are replicated several times ( an ideal but implausible scenario ) , some labs will avoid being caught . In a system such as modern science , with \ufb01nite career 13 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r = 0 r = 0 . 01 r = 0 . 05 300 200 100 0 400 300 200 100 0 400 400 300 200 100 0 \u2013 200 \u2013 150 \u2013 100 \u2013 50 0 50 300 200 100 0 c oun t total pay - off total pay - off total pay - off 0 10 20 30 low effort high effort low effort high effort low effort high effort 40 50 \u2013 500 \u2013 400 \u2013 300 \u2013 200 \u2013 100 0 100 \u2013 1500 \u2013 1200 \u2013 900 \u2013 600 \u2013 300 0 ( b ) ( a ) ( c ) Figure 7 . Lab pay - offs from the non - evolutionary model . Each graph shows count distributions for high and low effort labs\u2019 total pay - offs after 110 time steps , 100 of which included replication . ( a \u2013 c ) Total count for each pay - off is totalled from 50 runs for each condition . Panel ( c ) includes an inset that displays the same data as the larger graph , but for a narrower range of pay - offs . The punishment for having one\u2019s novel result fail to replicate is orders of magnitude greater than the benefit of publishing , reflected in the discrete peaks in ( b ) and ( c ) . opportunities and high network connectivity , the marginal return for being in the top tier of publications may be orders of magnitude higher than an otherwise respectable publication record [ 37 , 38 , 79 ] . Within our evolutionary model it was dif\ufb01cult to lay bare the precise relationship between replication , effort and reproductive success , because all these parameters are entangled . We wanted to understand exactly why the most successful labs appear to be those with low effort even when failure to replicate was highly punitive , making low effort potentially quite costly . To do this , we created a simpli\ufb01ed version of our model that omitted any evolution . Power was \ufb01xed at 0 . 8 and all labs were either high effort ( H ) or low effort ( L ) . High effort labs had effort e H = 0 . 75 , corresponding to a false - positive rate of \u03b1 = 0 . 05 and an investigation rate of h = 0 . 625 . Low effort labs had effort e L = 0 . 15 , corresponding to a false - positive rate of \u03b1 = 0 . 2 and an investigation rate of h = 0 . 765 . The population was initialized with 50 % high effort labs . Labs conducted research and attempted to communicate their \ufb01ndings as in the original model . We allowed ten time steps without any replication to establish a baseline body of literature , and then ran the simulation for 100 additional time steps , equivalent to the expected lifespan of a lab in the evolutionary model . During this time , each hypothesis investigated was a replication with probability r . This simpli\ufb01ed model allowed us to examine the distribution of the pay - offs ( resulting from both the bene\ufb01ts of successful publications and punishment from failed replications ) and to directly compare high and low effort labs . Figure 7 shows the distributions of pay - offs from three replication rates . Without replication , low effort labs have an unambiguous advantage . As the rate of replication increases , the mean pay - off for high effort labs can surpass the mean pay - off for low effort labs , as the former are less likely to be punished . However , the laws of probability dictate that some labs will escape either producing false positives or being caught doing so , and among these the very highest performers will be those who exert low effort . When the top labs have disproportionate in\ufb02uence on funding and graduate student success , this type of small advantage can cascade to continuously select for lower effort and increasing numbers of false discoveries , as seen in our evolutionary model . 6 . Discussion Incentives drive cultural evolution . In the scienti\ufb01c community , incentives for publication quantity can drive the evolution of poor methodological practices . We have provided some empirical evidence that this occurred , as well as a general model of the process . If we want to improve how our scienti\ufb01c culture functions , we must consider not only the individual behaviours we wish to change , but also the social forces that provide affordances and incentives for those behaviours [ 1 , 80 ] . We are hardly the \ufb01rst to consider a need to alter the incentives for career success in science [ 68 , 69 , 81 \u2013 89 ] . However , we are the \ufb01rst to illustrate the evolutionary logic of how , in the absence of change , the existing incentives will necessarily lead to the degradation of scienti\ufb01c practices . An incentive structure that rewards publication quantity will , in the absence of countervailing forces , select for methods that produce the greatest number of publishable results . This , in turn , will lead to the natural selection of poor methods and increasingly high false discovery rates . Although we have focused on false discoveries , there are additional negative repercussions of this kind of incentive structure . Scrupulous research on dif\ufb01cult problems may require years of intense work before yielding coherent , 14 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . publishable results . If shallower work generating more publications is favoured , then researchers interested in pursuing complex questions may \ufb01nd themselves without jobs , perhaps to the detriment of the scienti\ufb01c community more broadly . Good science is in some sense a public good , and as such may be characterized by the con\ufb02ict between cooperation and free riding . We can think of cooperation here as the opportunity to create group - bene\ufb01cial outcomes ( i . e . quality research ) at a personal cost ( i . e . diminished \u2018\ufb01tness\u2019 in terms of academic success ) . To those familiar with the game theory of cooperative dilemmas , it might therefore appear that continued contributions to the public good\u2014cooperation rather than free riding\u2014could be maintained through the same mechanisms known to promote cooperation more generally , including reciprocity , monitoring and punishment [ 90 ] . However , the logic of cooperation requires that the bene\ufb01t received by cooperators can be measured in the same units as the pay - off to free riders : i . e . units of evolutionary \ufb01tness . It is possible that coalitions of rigorous scientists working together will generate greater output than less rigorous individuals working in isolation . And indeed , there has been an increase in highly collaborative work in many \ufb01elds [ 30 , 31 ] . Nevertheless , such collaboration may also be a direct response to incentives for publication quantity , as contributing a small amount to many projects generates more publications than does contributing a large amount to few projects . Cooperation in the sense of higher quality research provides a public good in the sense of knowledge , but not in the sense of \ufb01tness for the cultural evolution of methodology . Purely bottom - up solutions are therefore unlikely to be suf\ufb01cient . That said , changing attitudes about the assessment of scientists is vital to making progress , and is a driving motivation for this presentation . Institutional change is dif\ufb01cult to accomplish , because it requires coordination on a large scale , which is often costly to early adopters [ 91 , 92 ] . Yet such change is needed to ensure the integrity of science . It is therefore worth considering the types of institutions we might want . It might appear that journals and peer reviewers need only to adopt increasingly strict standards as bars to publication . However , although this may place some limits on the extent to which individuals can game the system , it still affords the opportunity to do so [ 93 ] and incentives to do so will exist as long as success is tied to publication . Punishing individuals for failure to replicate their original results is unlikely to be effective at stopping the evolution of bad science . Many important discoveries initially appear unlikely , and designing clear experiments is dif\ufb01cult . Eliminating false positives entirely is likely to be impossible [ 86 ] . In addition , failed replications may themselves be false negatives . Moreover , replication is dif\ufb01cult or impossible for some \ufb01elds , such as those involving large clinical samples or historical data . An overemphasis on replication as the saviour of all science is biased in favour of certain \ufb01elds over others . It is therefore inadvisable to be overly harsh on researchers when their results fail to replicate . A more drastic suggestion is to alter the \ufb01tness landscape entirely by changing the selection pressures : the incentives for success . This is likely to be quite dif\ufb01cult . Consider that the stakeholders who fund and support science may have incentives that are not always aligned with those of active researchers [ 82 ] . For example , funders may expect \u2018deliverables\u2019 in the form of published papers , which in turn may pressure scientists to conduct research in such a manner to maximize those deliverables , even if incentives for promotion or hiring are changed . Another impediment is the constraint on time and cognitive resources on the part of evaluators . The quality of a researcher is dif\ufb01cult to assess , particularly since there are many ways to be a good scientist , making assessment a high - dimensionality optimization problem . Quantitative metrics such as publication rates and impact factor are used partly because they are simple and provide clear , unambiguous comparisons between researchers . Yet these are precisely the qualities that allow such metrics to be exploited . In reality , the true \ufb01tness landscape of scienti\ufb01c career success is multidimensional . Although some publications are probably necessary , there are other routes to success beside the accrual of a lengthy CV . We should support institutions that facilitate these routes , particularly when they encourage high quality research , and resist the temptation or social pressure to paper count . We believe that our interpretation of the model is quite robust , provided that at least one of the following two criteria are met : ( i ) negative results are harder to publish or ( ii ) lowering effort increases the rate of a lab\u2019s output . We chose parameter values that we deemed reasonable and strove to err in a direction unfavourable to our hypotheses , as a guard against con\ufb01rmation bias . For example , the replication rate was set extremely high , and failure to replicate was made extremely punitive . However , we have not presented a full sensitivity analysis . So we note again that we provide our model source code in the electronic supplementary material , so that interested readers can verify and extend our analysis . Our model treats each publication of a novel result as equivalent , but of course they are not . Instead , the most prestigious journals receive the most attention and are the most heavily cited [ 94 ] . Anecdotally , we have heard many academics\u2014ranging from graduate students to full professors\u2014discussing job candidates largely in terms of the prestigious journals they either published in or failed to publish in . 15 r s o s . r o y a l s o c i e t y p u b l i s h i n g . o r g R . S o c . o p e n s c i . 3 : 1 60 3 8 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consideration of journal prestige would change our model somewhat , but the implications would be similar , as the existence of such journals creates pressure to publish in those journals at all costs . The major change to our model would be additional selection for highly surprising results , which are more likely to be false . Investigations have found that the statistical power of papers published in prestigious ( high impact factor ) journals is no different from those with lower impact factors [ 84 ] , while the rate of retractions for journals is positively correlated with impact factor [ 95 ] . Although this is likely to be at least partly due to the increased attention paid to those papers , it is well known that high impact journals often reject competent papers deemed insuf\ufb01ciently novel . Our model presents a somewhat pessimistic picture of the scienti\ufb01c community . Let us be clear : many scientists are well aware of the dif\ufb01culties surrounding evaluation , and many hiring and grant committees take efforts to evaluate researchers on the quality of their work rather than the quantity of their output or where it is published . Moreover , we believe that many if not most scientists are truly driven to discover truth about the world . It would be overly cynical to believe that scientists are driven only by extrinsic incentives , fashioning researchers to conform to the expectations of Homo economicus . However , a key feature of our evolutionary model is that it assumes no ill intent on the part of the actors , and does not assume that anyone actively alters their methods in response to incentives . Rather , selection pressures at the institutional level favoured those research groups that , for whatever reason , used methods that generated a higher volume of published work . Any active social learning , such as success - biased copying , will serve only to accelerate the pace of evolution for such methods . Despite incentives for productivity , many scientists employ rigorous methods and learn new things about the world all the time that are validated by replication or by being effectively put into practice . In other words , there is still plenty of good science out there . One reason is that publication volume is rarely the only determinant of the success or failure of a scientist\u2019s career . Other important factors include the importance of one\u2019s research topic , the quality of one\u2019s work , and the esteem of one\u2019s peers . The weight of each factor varies among disciplines , and in some \ufb01elds such factors may work positively to promote behaviours leading to high - quality research , particularly when selection for those behaviours is enculturated into institutions or disciplinary norms . In such cases , this may be suf\ufb01cient to counteract the negative effects of incentives for publication volume , and so maintain high levels of research quality . If , on the other hand , success is largely determined by publication output or related quantitative metrics , then those who care about quality research should be on high alert . In which direction the scale tips in one\u2019s own \ufb01eld is a critical question for anyone interested in the future of science . Whenever quantitative metrics are used as proxies to evaluate and reward scientists , those metrics become open to exploitation if it is easier to do so than to directly improve the quality of research . Institutional guidelines for evaluation at least partly determine how researchers devote their energies , and thereby shape the kind of science that gets done . A real solution is likely to be patchwork , in part because accurately rewarding quality is dif\ufb01cult . 5 Real merit takes time to manifest , and scrutinizing the quality of another\u2019s work takes time from already busy schedules . Competition for jobs and funding is stiff , and reviewers require some means to assess researchers . Moreover , individuals differ on their criteria for excellence . Boiling down an individual\u2019s output to simple , objective metrics , such as number of publications or journal impacts , entails considerable savings in terms of time , energy and ambiguity . Unfortunately , the long - term costs of using simple quantitative metrics to assess researcher merit are likely to be quite great . If we are serious about ensuring that our science is both meaningful and reproducible , we must ensure that our institutions incentivize that kind of science . Ethics . Our meta - analysis used only previously published data . All simulated scientists were humanely euthanized . Data accessibility . All data for our meta - analysis were drawn from previously published material . Our methods and sources are documented in the electronic supplementary material . The Java source code for our agent - based model is likewise available in the electronic supplementary material . Authors\u2019 contributions . P . E . S . and R . M . conceived and designed the research and wrote the paper . P . E . S . carried out the meta - analysis and wrote the model code and analysed its output . All authors gave \ufb01nal approval for publication . Competinginterests . We have no competing interests . Funding . This research received no speci\ufb01c grant from any funding agency in the public , commercial or not - for - pro\ufb01t sectors . Acknowledgements . 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( doi : 10 . 1128 / IAI . 05661 - 11 )", + "jingHouseholdSecondaryAttack2020": "www . thelancet . com / infection Vol 20 October 2020 1141 Articles Household secondary attack rate of COVID - 19 and associated determinants in Guangzhou , China : a retrospective cohort study Qin - Long Jing * , Ming - Jin Liu * , Zhou - Bin Zhang * , Li - Qun Fang * , Jun Yuan * , An - Ran Zhang , Natalie E Dean , Lei Luo , Meng - Meng Ma , Ira Longini , Eben Kenah , Ying Lu , Yu Ma , Neda Jalali , Zhi - Cong Yang , Yang Yang Summary Background As of June 8 , 2020 , the global reported number of COVID - 19 cases had reached more than 7 million with over 400 000 deaths . The household transmissibility of the causative pathogen , severe acute respiratory syndrome coronavirus 2 ( SARS - CoV - 2 ) , remains unclear . We aimed to estimate the secondary attack rate of SARS - CoV - 2 among household and non - household close contacts in Guangzhou , China , using a statistical transmission model . Methods In this retrospective cohort study , we used a comprehensive contact tracing dataset from the Guangzhou Center for Disease Control and Prevention to estimate the secondary attack rate of COVID - 19 ( defined as the probability that an infected individual will transmit the disease to a susceptible individual ) among household and non - household contacts , using a statistical transmission model . We considered two alternative definitions of household contacts in the analysis : individuals who were either family members or close relatives , such as parents and parents - in - law , regardless of residential address , and individuals living at the same address regardless of relationship . We assessed the demographic determinants of transmissibility and the infectivity of COVID - 19 cases during their incubation period . Findings Between Jan 7 , 2020 , and Feb 18 , 2020 , we traced 195 unrelated close contact groups ( 215 primary cases , 134 secondary or tertiary cases , and 1964 uninfected close contacts ) . By identifying households from these groups , assuming a mean incubation period of 5 days , a maximum infectious period of 13 days , and no case isolation , the estimated secondary attack rate among household contacts was 12\u22194 % ( 95 % CI 9\u22198 \u2013 15\u22194 ) when household contacts were defined on the basis of close relatives and 17\u22191 % ( 13\u22193 \u2013 21\u22198 ) when household contacts were defined on the basis of residential address . Compared with the oldest age group ( \u226560 years ) , the risk of household infection was lower in the youngest age group ( < 20 years ; odds ratio [ OR ] 0\u221923 [ 95 % CI 0\u221911 \u2013 0\u221946 ] ) and among adults aged 20 \u2013 59 years ( OR 0\u221964 [ 95 % CI 0\u221943 \u2013 0\u221997 ] ) . Our results suggest greater infectivity during the incubation period than during the symptomatic period , although differences were not statistically significant ( OR 0\u221961 [ 95 % CI 0\u221927 \u2013 1\u221938 ] ) . The estimated local reproductive number ( R ) based on observed contact frequencies of primary cases was 0\u22195 ( 95 % CI 0\u221941 \u2013 0\u221962 ) in Guangzhou . The projected local R , had there been no isolation of cases or quarantine of their contacts , was 0\u22196 ( 95 % CI 0\u221949 \u2013 0\u221974 ) when household was defined on the basis of close relatives . Interpretation SARS - CoV - 2 is more transmissible in households than SARS - CoV and Middle East respiratory syndrome coronavirus . Older individuals ( aged \u226560 years ) are the most susceptible to household transmission of SARS - CoV - 2 . In addition to case finding and isolation , timely tracing and quarantine of close contacts should be implemented to prevent onward transmission during the viral incubation period . Funding US National Institutes of Health , Science and Technology Plan Project of Guangzhou , Project for Key Medicine Discipline Construction of Guangzhou Municipality , Key Research and Development Program of China . Copyright \u00a9 2020 Elsevier Ltd . All rights reserved . Introduction The ongoing COVID - 19 pandemic , caused by the novel severe acute respiratory syndrome corona virus 2 ( SARS - CoV - 2 ) , has now affected 188 countries worldwide . As of June 8 , 2020 , more than 7 million reported cases and over 400 000 deaths had been reported . 1 Older individuals ( aged \u226570 years ) and individuals with chronic conditions such as diabetes and cardiopulmonary disease are most susceptible to severe disease and death . 2 Efficient viral transmission via droplets and fomites is potentially supplemented by other transmission routes such as aerosol and faecal contamination . 3 , 4 Accumulating evidence suggests that presymptomatic or asymp tomatic carriers can transmit the virus . 5 \u2013 7 Within - household transmission is suspected to have contributed sub stantially to the con - tinued increase in cases in China following the introduction of nationally enforced restric tions on human movement . 8 , 9 Isolation of cases and quarantine of their close contacts at home are frequently recommended as a disease control measure in countries with COVID - 19 outbreaks , but such Lancet Infect Dis 2020 ; 20 : 1141 \u2013 50 Published Online June 17 , 2020 https : / / doi . org / 10 . 1016 / S1473 - 3099 ( 20 ) 30471 - 0 See Comment page 1103 For the Chinese translation of the abstract see Online for appendix 1 * Contributed equally Guangzhou Center for Disease Control and Prevention , Guangzhou , China ( Q - L Jing PhD , Z - B Zhang MPH , J Yuan MD , L Luo PhD , M - M Ma MD , Y Lu MD , Y Ma MD , Z - C Yang MD ) ; Department of Biostatistics , College of Public Health and Health Professions , Emerging Pathogens Institute , University of Florida , Gainesville , FL , USA ( M - J Liu BS , A - R Zhang BS , N E Dean BS , I Longini PhD , N Jalali MS , Y Yang PhD ) ; State Key Laboratory of Pathogen and Biosecurity , Beijing Institute of Microbiology and Epidemiology , Beijing , China ( A - R Zhang , L - Q Fang PhD ) ; Department of Epidemiology , School of Public Health , Cheeloo College of Medicine , Shandong University , Jinan , China ( A - R Zhang ) ; and Division of Biostatistics , College of Public Health , Ohio State University , Columbus , OH , USA ( E Kenah ScD ) Correspondence to : Dr Yang Yang , Department of Biostatistics , College of Public Health and Health Professions , Emerging Pathogens Institute , University of Florida , Gainesville , FL 32611 , USA yangyang @ ufl . edu or Dr Zhi - Cong Yang , Guangzhou Center for Disease Control and Prevention , Guangzhou 510440 , China yangzc @ gzcdc . org . cn Articles 1142 www . thelancet . com / infection Vol 20 October 2020 restrictions are likely to have little or no effect on trans - mission within households . To date , transmissibility of the disease has primarily been assessed at the population level , using mathematical models , or at the individual level in synthetic popula - tions using agent - based models coupled with statistical methods . 10 \u2013 12 Transmissibility within households or through other types of close contact remains under investigated , despite the importance of these social interactions in shaping the overall dynamics of disease spread and in determining the effectiveness of mitigation strategies . 13 Data obtained from contract tracing provide the most accurate information about human - to - human trans - missibility of any infectious pathogen , because trans - missibility can be assessed more accurately by accounting for individual - level exposure history . Available estimates for the secondary attack rate ( defined as the probability that an infected individual will transmit the disease to a susceptible individual [ eg , household or close contacts ] ) of SARS - CoV - 2 are based on contact tracing data for hundreds of cases in Shenzhen and Guangzhou in China , and ten cases in the USA . 14 \u2013 16 These estimates represent the proportion of confirmed infections among all traced contacts , which does not fully account for heterogeneity in individual exposure history , the possi bility of transmission among contacts themselves , or the infection risks from untraced contacts or fomites . We aimed to estimate the secondary attack rate of SARS - CoV - 2 among household and non - household close contacts in Guangzhou , using a statistical transmission model . This model accounts for individual - level exposure history and the potential existence of tertiary cases . We also aimed to assess the effects of age and sex on the infectivity of COVID - 19 cases and susceptibility of their close contacts , and the relative infectivity of COVID - 19 cases during the incubation period compared with the period of illness . Methods Case definition A suspected COVID - 19 case was defined as an individual who met one or more epidemiological criteria ( had travelled to , or resided in , Wuhan or nearby cities in the 14 days before symptom onset ; had contact history with an individual with COVID - 19 [ confirmed by RT - PCR ] in the 14 days before symptom onset ; had contact history with patients who had fever or respiratory symptoms and came from Wuhan or communities with reported COVID - 19 cases in the 14 days before symptom onset ; or was related to a cluster of COVID - 19 cases ) and two or more clinical criteria ( had fever or respiratory symptoms ; had radio - graphical characteristics of pneumonia ; or normal or reduc ed leucocyte counts , or reduced lymphocyte counts during the acute phase of the disease ; appendix 2 p 1 ) . A confirmed case was defined as a suspected case with positive detection of SARS - CoV - 2 nucleic acid by real - time RT - PCR or viral genes that are highly homologous to SARS - CoV - 2 by sequencing using respiratory speci mens . An individual with laboratory confirmation , but without clinical signs , mainly found by community screening and contact tracing , was considered asymptomatic ( appendix 2 pp 2 , 3 ) . In this study , asymptomatic infections were analysed as confirmed cases . Epidemiological investigation and contact tracing Epidemiological investigations were done by county - level Centers for Disease Control and Prevention ( CDC ) Research in context Evidence before this study The transmissibility of severe acute respiratory syndrome coronavirus 2 ( SARS - CoV - 2 ) in household and community settings remains underinvestigated . On April 1 , 2020 , we searched PubMed and medRxiv using the search terms ( \u201cnovel coronavirus\u201d OR \u201cCOVID - 19\u201d OR \u201cSARS - CoV - 2\u201d OR ) AND ( \u201chousehold\u201d OR \u201cfamily\u201d ) AND ( \u201ctransmissibility\u201d OR \u201cattack rate\u201d ) . Our search yielded three articles that investigated household secondary attack rates of COVID - 19 in multiple family clusters . In studies of contact tracing data in Shenzhen and Guangzhou , two cities in southern China , the secondary attack rates were 14\u00b79 % and 10\u00b72 % , respectively , but these estimates represented the proportions of infections among household contacts . In a study of the close contacts of ten US patients with COVID - 19 the estimated household secondary attack rate was 10\u00b75 % , however , the sample size was too small for reliable interpretation and only symptom onset of primary cases was examined . Transmission events from COVID - 19 cases during their incubation period or from asymptomatic carriers have been reported , but infectiousness before symptom onset has not been quantified . Added value of this study Using contact tracing data from patients with COVID - 19 in Guangzhou , China , we implemented a statistical transmission model to estimate secondary attack rate for household and non - household close contacts . To our knowledge , our model is the first to account for heterogeneous individual - level exposure history , tertiary transmission , potential exposure to untraced infection sources , and asymptomatic infections . Additionally , we assessed the effects of age , sex , epidemic phase , and household size on transmissibility of the virus and relative infectivity before and after symptom onset . Implications of all the available evidence SARS - CoV - 2 can be transmitted efficiently within households and during the incubation period of COVID - 19 cases . Because presymptomatic and asymptomatic transmission has been observed , case - isolation alone is inadequate for mitigating the pandemic . Comprehensive tracing and timely quarantine of close contacts of COVID - 19 cases should be implemented to prevent onward transmission during their incubation periods . See Online for appendix 2 Articles www . thelancet . com / infection Vol 20 October 2020 1143 offices in China within 24 h after a suspected or confirmed case was reported ( appendix 2 pp 2 , 3 ) . For each suspected or confirmed case , we recorded demographic , clinical , diagnostic , and occupational data , baseline health conditions , clinical samples and laboratory test results , and exposure history in the 14 days before symptom onset using a standardised investigation form . Typically , a suspected case would be changed to a confirmed case or removed from the national surveillance system once laboratory test results were available . A close contact was defined as an individual who had unprotected close contact ( within 1 m ) with a confirmed case within 2 days before their symptom onset or sample collection , including but not limited to household members , care givers , and individuals in the same workplace , classroom , hospital ward , or transportation vehicle . Close contacts were quarantined at designated places ( eg , hotel rooms ) or at home and followed up for 14 days , and nasal swabs were collected at day 1 and day 14 and tested by RT - PCR ( appendix 2 pp 3 , 4 ) . These data were collected as part of a continuing public health response required by the National Health Commission of China , thus the require - ment for written informed consent was waived . This study was approved by the ethics committee of the Guangzhou CDC ( Guangzhou , China ) . Analyses involving personally identifiable data were done at Guangzhou CDC . Anonymised data were used for all other analyses . In our analyses , individuals who were linked by contact tracing were considered a close contact group . Cases in the same close contact group formed a case cluster . An imported case was defined as an individual who resided in or had travelled to Hubei province ( of which Wuhan is the capital ) in the 2 weeks before symptom onset ; otherwise , individuals were considered a local case . In a close contact group , a local case with symptom onset 1 day or less ( 3 days or less for an imported case ) from the earliest onset day in the close contact group was considered a primary case ; otherwise , this case was considered a secondary case . For asymptomatic infections , the primary or secondary case status was determined by the collection dates of the earliest SARS - CoV - 2 positive specimens . We used two definitions of house hold contacts : individuals who were either family members or close relatives , such as parents and parents - in - law , regardless of residential address , and individuals living at the same address regardless of relationship . Statistical analysis We used standard non - parametric tests ( Fisher\u2019s exact test , the Kruskal - Wallis test , and the Mann - Whitney U test ) to compare characteristics between demographic groups . Similar to the published literature , 14 \u2013 16 we calcu - lated the proportions of confirmed infections among all traced contacts for subgroups and herein , we refer to these proportions as data - based secondary attack rate estimates , which do not account for the fact that infections among contacts are not necessarily secon - dary , and could be tertiary . All summary analyses were done using R statistical software ( version 3 . 6 . 1 ) . The spatial distribution of case clusters was mapped at the com munity level using ArcGIS ( Environmental Systems Research Institute , Redlands , CA , USA ) , with a directed graph indicating potential transmission chains . We estimated effective reproductive numbers ( R t ) based on the contact tracing data ( appendix 2 pp 4 \u2013 6 ) . Since the transmission relationship remains unclear , we investi gated three scenarios defined by the following assump tions : scenario 1 , all imported cases were primary cases , and all secondary cases were infected by primary cases in the same case cluster ; scenario 2 , which was identical to scenario 1 , with the additional assumption that local primary cases might have been infected by earlier cases in other case clusters ; and scenario 3 , which was identical to scenario 2 , with the additional assumption that imported secondary cases were considered secondary cases rather than primary cases . Scenarios 1 and 3 served as the lower and upper bounds of the R t . A chain - binomial statistical model was used to estimate secondary attack rate and local reproductive number , with an expectation - maximisation algorithm to account for uncertainty in the infection time of asymptomatic infections ( appendix 2 pp 6 \u2013 10 ) . 17 Possible distributions of the incubation period and the infectious period were derived from the literature and our previous research on a separate contact tracing database ( appendix 2 pp 10 \u2013 12 ) . 5 , 18 \u2013 20 On the basis of the available literature ( appendix 2 p 10 \u2013 11 ) , we reported the estimates associated with a mean incubation period of 5 days and a maximum infectious period of 13 days as the primary results . Briefly , this model estimated the probabilities , p 1 and p 2 , of viral transmission from an infectious household contact and from an infectious non - household contact ( eg , friends , coworkers , passengers ) respectively , to a susceptible person per daily contact . Additionally , each susceptible person was subject to a constant daily probability , b , of being infected by an unspecified external source , which accounted for untraced contacts and fomites . We assumed that the infectivity of a COVID - 19 case differs between the incubation period and the period after symptom onset ( ie , illness period ) . We used ( D min , D max ) to represent the whole infectious period with the symptom onset day set as day 0 , and modelled the effective daily transmission probability as on days D min \u2264 l < 0 ( incubation period ) and p k ( l ) * \u2013 1 \u2013 p k ( l ) = p k \u2013 1 \u2013 p k \u00d7 OR * p k ( l ) = p k * Articles 1144 www . thelancet . com / infection Vol 20 October 2020 on days 0 \u2264 l < D max ( illness period ) , where the odds ratio ( OR ) measures the relative infectivity of the illness period versus the incubation period . The secondary attack rate was defined as : where \u03c6 * ( l ) was the prespecified relative infectivity level on the l th day of the infectious period based on previous studies , 19 peaking near the time of symptom onset . Herein , we refer to the derived secondary attack rate estimates as model - based . The local reproductive number ( ie , the mean number of infections a symptomatic case could generate via household and non - household contacts ) was defined as : where n 1 ( l ) and n 2 ( l ) were the mean numbers of household and non - household contacts per primary case on day l ( appendix 2 pp 12 , 13 ) . We assessed the effect of age , sex , house hold size , and epidemic phase on susceptibility and infectivity by regressing transmission probabilities on these characteristics associated with either the suscep tible person or the infectious person in each potential trans - mission \u2013 exposure pair ( appendix 2 pp 13 , 14 ) . Additi - onally , we assessed the goodness - of - fit of the model under different settings of the natural history of disease ( appendix 2 pp 14 , 15 ) . Role of the funding source The funder of the study had no role in study design , data collection , data analysis , data interpretation , or the writing of the report . The corresponding authors had full access to all the data in the study , and had final responsibility for the decision to submit for publication . Results Between Jan 7 , 2020 , and Feb 18 , 2020 , 349 laboratory - confirmed SARS - CoV - 2 infections were reported to Guangzhou CDC , among whom 19 ( 5 % ) indi viduals were asymptomatic . Contact tracing identified 195 unrelated close contact groups ( 215 primary cases , 134 secondary or tertiary cases , and 1964 uninfected close contacts ) . The median size of close contact groups was six ( IQR 4 \u2013 10 ; range 1 \u2013 274 ) . For 138 ( 71 % ) of these 195 close contact groups , no secondary cases were identified . Time from symptom onset to hospital admission and to laboratory confirmation was longer for primary cases than for secondary cases and was longer in January than in February ( appendix 2 p 19 ) . Among the 349 cases , the most common symptoms were fever ( 258 [ 74 % ] cases ) , cough ( 207 [ 59 % ] ) , fatigue ( 74 [ 21 % ] ) , sore throat ( 60 [ 17 % ] ) , chills ( 55 [ 16 % ] ) , and myalgia ( 51 [ 15 % ] ) . Radio - graphic abnormality was observed in 238 ( 80 % ) of 349 cases ( appendix 2 p 20 ) . Most patients with COVID - 19 were adults ( aged 20 \u2013 59 years ; table 1 ) . The majority of primary cases ( 158 [ 73 % ] of 215 cases ) and nearly half of all secondary cases ( 66 [ 46 % ] of 134 cases ) had recently travelled to or resided in Hubei province ( referred to as imported cases hereafter ) . The overall data - based secon dary attack rates were 13\u22192 % ( 95 % CI 10\u22199 \u2013 15\u22197 ) among household contacts and 2\u22194 % ( 1\u22196 \u2013 3\u22193 ) among non - household contacts , when household was defined on the basis of close relatives . Within house holds , the data - based secondary attack rates were lower in the youngest age group ( age < 20 years ; 5\u22192 % [ 95 % CI 2\u22194 \u2013 9\u22197 ] ) than the 20 \u2013 59 years age group ( 14\u22198 % [ 95 % CI 11\u22197 \u2013 18\u22194 ] ; p = 0\u00b70009 ) and the oldest age group ( age \u226560 years ; 18\u22194 % [ 12\u22195 \u2013 25\u22196 % ] ; p = 0\u00b70003 ) . Similar findings were observed among non - household contacts , but the differences between age groups were not statistically significant . We identified no significant differences in secondary attack rate between sexes . Secondary attack rate estimates decreased between January and February , 2020 , within and outside households ( both p < 0\u00b70001 ) . When household was defined by residential address , the data - based secondary attack rate among household contacts increased to 17\u22192 % ( 95 % CI 14\u22191 \u2013 20\u22196 ) and was 2\u22196 % ( 1\u22199 \u2013 3\u22196 ) among non - household contacts . Most COVID - 19 cases were reported in the densely populated districts ( where 56 % of the total population of Guangzhou reside ) including Yuexiu , Liwan , Haizhu , Tianhe , and Baiyun ( figure 1 ) . Four clusters with five or more secondary cases ( excluding tertiary cases or further generations ) were identified , with one cluster identified in Yuexiu , Haizhu , Baiyun and Panyu districts , and all primary cases in these clusters were imported ( figure 1B \u2013 D ) . The longest transmission chain had three generations subsequent to the primary case , which occurred in the Panyu district ( figure 1D ) . Five other clusters had two subsequent generations ( figure 1B , figure 1C ) . Most of the reported residential locations of primary and secondary cases within the same clusters were identical , but some non - household secondary cases resided in areas that were distant from the primary cases . Most transmissions occurred between household members ( appendix 2 p 28 ) . The first imported primary case had symptom onset on Jan 7 , 2020 , and arrived in Guangzhou on Jan 13 , 2020 . The earliest local primary case had symptom onset on Jan 16 , 2020 , when at least two imported cases had already been reported in Guangzhou . The number of imported cases peaked at the same time as the epidemic in Guangzhou around Jan 27 , 2020 , 4 days after the lockdown was implemented in Wuhan ( figure 2 ) . After Jan 27 , 2020 , the number of imported cases decreased and the epidemic waned quickly , with only sporadic cases reported by the middle of February . In the early phase of the epidemic , the R t reached 1\u00b74 in scenario 3 , around 1\u00b72 in scenario 2 , and 0\u00b77 in scenario 1 . For all scenarios , by Jan 27 , 2020 , the R t had declined to less than 0\u00b75 , 1 \u2013 \u220f l = D D maxmin [ 1 \u2212 p k ( l ) \u03c6 * ( l ) ] , k = 1 , 2 , * \u2211 k = 1 \u2211 l = DD maxmin { n k ( l ) p k ( l ) \u03c6 ( l ) \u03a0 m = D 2 min l \u2013 1 [ 1 \u2013 p k ( l ) \u03c6 ( l ) ] } * * * * Articles www . thelancet . com / infection Vol 20 October 2020 1145 which is likely to reflect the tightening of control measures in Guangzhou . We excluded 12 close contact groups with primary cases but no recorded contact , one close contact group in which all members ( two asymptomatic primary cases and two uninfected individuals ) stayed in Guangzhou for only 1 day , and 25 contacts with missing data on age or sex . Thus , 182 close contact groups with a total of 332 cases ( 317 symptomatic and 15 asymptomatic ) and 1937 uninfected contacts were included in our transmission analysis . We estimated the secondary attack rates among household and non - household contacts for the combinations of mean incubation period of 4 \u2013 7 days and maximum infectious period of 13 , 16 , and 22 days ( appendix 2 p 17 ) . Assessment of the goodness - of - fit for these settings indicated that all models fit the data satisfactorily , because the model - predicted numbers of infections were consistent with the observed values with only small differences before and after the peak of the epidemic ( appendix 2 p 30 ) . Assuming a mean incubation period of 5 days and maximum infectious period of 13 days , when household contact was defined on the basis of close relatives , assuming there was no case isolation , the estimated Primary cases Secondary cases Uninfected close contacts Overall Data - based secondary attack rate * Household Non - household Household Non - household Household Non - household Close relatives Age , years < 20 10 / 215 ( 5 % ) 9 / 103 ( 9 % ) 1 / 31 ( 3 % ) 163 / 681 ( 24 % ) 70 / 1283 ( 5 % ) 253 / 2313 ( 11 % ) 5\u00b72 % ( 2\u00b74 \u2013 9\u00b77 ) 1\u00b74 % ( 0\u00b704 \u2013 7\u00b76 ) 20 \u2013 59 145 / 215 ( 67 % ) 67 / 103 ( 65 % ) 22 / 31 ( 71 % ) 385 / 681 ( 57 % ) 961 / 1283 ( 75 % ) 1580 / 2313 ( 68 % ) 14\u00b78 % ( 11\u00b77 \u2013 18\u00b74 ) 2\u00b72 % ( 1\u00b74 \u2013 3\u00b74 ) \u226560 60 / 215 ( 28 % ) 27 / 103 ( 26 % ) 8 / 31 ( 26 % ) 120 / 681 ( 18 % ) 247 / 1283 ( 19 % ) 462 / 2313 ( 20 % ) 18\u00b74 % ( 12\u00b75 \u2013 25\u00b76 ) 3\u00b71 % ( 1\u00b74 \u2013 6\u00b71 ) Sex Female 107 / 215 ( 50 % ) 57 / 103 ( 55 % ) 17 / 31 ( 55 % ) 341 / 681 ( 50 % ) 627 / 1283 ( 49 % ) 1149 / 2313 ( 50 % ) 14\u00b73 % ( 11\u00b70 \u2013 18\u00b72 ) 2\u00b76 % ( 1\u00b75 \u2013 4\u00b72 ) Male 108 / 215 ( 50 % ) 46 / 103 ( 45 % ) 14 / 31 ( 45 % ) 335 / 681 ( 49 % ) 651 / 1283 ( 51 % ) 1154 / 2313 ( 50 % ) 12\u00b71 % ( 9\u00b70 \u2013 15\u00b78 ) 2\u00b71 % ( 1\u00b72 \u2013 3\u00b75 ) Month\u2020 January 193 / 215 ( 90 % ) 98 / 103 ( 95 % ) 29 / 31 ( 94 % ) 545 / 681 ( 80 % ) 681 / 1283 ( 53 % ) 1546 / 2313 ( 67 % ) 15\u00b72 % ( 12\u00b76 \u2013 18\u00b73 ) 4\u00b71 % ( 2\u00b78 \u2013 5\u00b78 ) February 22 / 215 ( 10 % ) 5 / 103 ( 5 % ) 2 / 31 ( 6 % ) 136 / 681 ( 20 % ) 602 / 1283 ( 47 % ) 767 / 2313 ( 33 % ) 3\u00b75 % ( 1\u00b72 \u2013 8\u00b71 ) 0\u00b733 % ( 0\u00b704 \u2013 1\u00b72 ) Household size \u22646 people 160 / 215 ( 74 % ) 69 / 103 ( 67 % ) 23 / 31 ( 74 % ) 302 / 681 ( 44 % ) 874 / 1283 ( 68 % ) 1428 / 2313 ( 62 % ) 18\u00b76 % ( 14\u00b78 \u2013 22\u00b79 ) 2\u00b76 % ( 1\u00b76 \u2013 3\u00b78 ) > 6 people 55 / 215 ( 26 % ) 34 / 103 ( 33 % ) 8 / 31 ( 26 % ) 379 / 681 ( 56 % ) 409 / 1283 ( 32 % ) 885 / 2313 ( 38 % ) 8\u00b72 % ( 5\u00b78 \u2013 11\u00b73 ) 1\u00b79 % ( 0\u00b78 \u2013 3\u00b77 ) Origin Imported 158 / 215 ( 73 % ) 59 / 103 ( 57 % ) 3 / 31 ( 10 % ) NA NA NA NA NA Local 57 / 215 ( 27 % ) 44 / 103 ( 43 % ) 28 / 31 ( 90 % ) NA NA NA NA NA Residential address Age , years < 20 10 / 215 ( 5 % ) 8 / 93 ( 9 % ) 2 / 41 ( 5 % ) 117 / 449 ( 26 % ) 116 / 1515 ( 8 % ) 253 / 2313 ( 11 % ) 6\u00b74 % ( 2\u00b78 \u2013 12\u00b72 ) 1\u00b77 % ( 0\u00b72 \u2013 5\u00b70 ) 20 \u2013 59 145 / 215 ( 67 % ) 59 / 93 ( 63 % ) 30 / 41 ( 73 % ) 260 / 449 ( 58 % ) 1086 / 1515 ( 72 % ) 1580 / 2313 ( 68 % ) 18\u00b75 % ( 14\u00b74 \u2013 23\u00b72 ) 2\u00b77 % ( 1\u00b78 \u2013 3\u00b78 ) \u226560 60 / 215 ( 28 % ) 26 / 93 ( 28 % ) 9 / 41 ( 22 % ) 67 / 449 ( 15 % ) 300 / 1515 ( 20 % ) 462 / 2313 ( 20 % ) 28\u00b70 % ( 19\u00b71 \u2013 38\u00b72 ) 2\u00b79 % ( 1\u00b73 \u2013 5\u00b75 ) Sex Female 107 / 215 ( 50 % ) 53 / 93 ( 57 % ) 21 / 41 ( 51 % ) 227 / 449 ( 51 % ) 741 / 1515 ( 49 % ) 1149 / 2313 ( 50 % ) 18\u00b79 % ( 14\u00b75 \u2013 24\u00b70 ) 2\u00b78 % ( 1\u00b77 \u2013 4\u00b72 ) Male 108 / 215 ( 50 % ) 40 / 93 ( 43 % ) 20 / 41 ( 49 % ) 218 / 449 ( 49 % ) 768 / 1515 ( 51 % ) 1154 / 2313 ( 50 % ) 15\u00b75 % ( 11\u00b73 \u2013 20\u00b75 ) 2\u00b75 % ( 1\u00b76 \u2013 3\u00b79 ) Month\u2020 January 193 / 215 ( 90 % ) 88 / 93 ( 95 % ) 39 / 41 ( 95 % ) 362 / 449 ( 81 % ) 864 / 1515 ( 57 % ) 1546 / 2313 ( 67 % ) 19\u00b76 % ( 16\u00b70 \u2013 23\u00b75 ) 4\u00b73 % ( 3\u00b71 \u2013 5\u00b79 ) February 22 / 215 ( 10 % ) 5 / 93 ( 5 % ) 2 / 41 ( 5 % ) 87 / 449 ( 19 % ) 651 / 1515 ( 43 % ) 767 / 2313 ( 33 % ) 5\u00b74 % ( 1\u00b78 \u2013 12\u00b72 ) 0\u00b731 % ( 0\u00b704 \u2013 1\u00b71 ) Household size \u22646 people 188 / 215 ( 87 % ) 79 / 93 ( 85 % ) 32 / 41 ( 78 % ) 309 / 449 ( 69 % ) 1191 / 1515 ( 79 % ) 1799 / 2313 ( 78 % ) 20\u00b74 % ( 16\u00b75 \u2013 24\u00b77 ) 2\u00b76 % ( 1\u00b78 \u2013 3\u00b77 ) > 6 people 27 / 215 ( 13 % ) 14 / 93 ( 15 % ) 9 / 41 ( 22 % ) 140 / 449 ( 31 % ) 324 / 1515 ( 21 % ) 514 / 2313 ( 22 % ) 9\u00b71 % ( 5\u00b71 \u2013 14\u00b78 ) 2\u00b77 % ( 1\u00b72 \u2013 5\u00b71 ) Origin Imported 158 / 215 ( 73 % ) 56 / 93 ( 60 % ) 6 / 41 ( 15 % ) NA NA NA NA NA Local 57 / 215 ( 27 % ) 37 / 93 ( 40 % ) 35 / 41 ( 85 % ) NA NA NA NA NA Data are n / N ( % ) or secondary attack rate ( 95 % CI ) . When household was defined on the basis of close relatives , the overall data - based secondary attack rates were 13\u00b72 % ( 10\u00b79 \u2013 15\u00b77 ) among household contacts and 2\u00b74 % ( 1\u00b76 \u2013 3\u00b73 ) among non - household contacts . When household was defined on the basis of residential address , the overall data - based secondary attack rates were 17\u00b72 % ( 14\u00b71 \u2013 20\u00b76 ) among household contacts and 2\u00b76 % ( 1\u00b79 \u2013 3\u00b76 ) among non - household contacts . Contact type was determined by an individual\u2019s relationship with the primary cases of each close contact group . NA = not applicable . * Calculated as the number of secondary cases divided by the sum of secondary cases and non - cases . \u2020Secondary cases and non - cases in each close contact group were allocated to January or February , 2020 , on the basis of the number of days in the infectious period of the primary case that occurred in January compared with that in February . Table 1 : Demographic composition of the study population stratified by case type and contact type Articles 1146 www . thelancet . com / infection Vol 20 October 2020 secondary attack rates were 12\u22194 % ( 95 % CI 9\u22198 \u2013 15\u22194 % ) among household contacts and 7\u22199 % ( 95 % CI 5\u22193 \u2013 11\u22198 % ) among non - household contacts . A longer incubation period was associated with a slightly lower secondary attack rate estimate than a shorter incubation period , and a longer infectious period was associated with a slightly higher secondary attack rate estimate than a shorter infectious period ( table 2 ; appendix 2 p 21 ) . When different mean incubation periods and maximum infectious periods were considered , the secondary attack rate varied from 11\u22194 % ( 95 % CI 9\u00b70 \u2013 14\u00b72 ) to 18\u00b70 % ( 13\u00b79 \u2013 23\u00b70 ) among household contacts , and from 7\u00b75 % ( 5\u00b70 \u2013 11\u00b72 ) to 12\u00b72 % ( 8\u00b70 \u2013 18\u00b71 ) among non - household contacts ( appendix 2 p 21 ) . The estimated local R based on observed contact freque ncies of primary cases was 0\u22195 ( 95 % CI 0\u221941 \u2013 0\u221962 ) , which was insensitive to the assumed incubation and infectious periods . Thus , a typical case infected 0\u00b75 indi vi duals on average in Figure 1 : Spatial distribution of COVID - 19 case clusters on the basis of contact tracing data from Guangzhou , China , from Jan 7 , 2020 , to Feb 18 , 2020 Overall distribution of COVID - 19 case clusters in Guangzhou ( A ) , and distribution in the subregions defined in panel A ( B \u2013 G ) . Individuals were considered as primary cases if their symptom onset dates were the earliest or 1 day ( \u2264 3 days for an imported case ) after the earliest in the cluster and as secondary cases otherwise . Non - infected contacts are not shown . The displayed location of each case is randomly perturbed away from the actual residential address . D E F G Baiyun B 0\u00b70 1\u00b725 2\u00b75 5\u00b70km Foshan Liwan Haizhu Huangpu Panyu Tianhe Baiyun Foshan Panyu Zengcheng Huizhou Zengcheng Nansha Dongguan Huangpu Huadu 0\u00b70 1\u00b725 2\u00b75 5\u00b70 km 0 1 2 4km 0 0\u00b75 1 2km 0 0\u00b75 1 2km 0 1 2 4km N N N N N N Contact relationship Primary case Household secondary case Non - household secondary case Direction of transmission Coprimary cases Onset date Imported cases with symptom onset on or before Jan 23 , 2020 Imported cases with symptom onset after Jan 23 , 2020 Local cases with symptom onset on or before Jan 23 , 2020 Local cases with symptom onset after Jan 23 , 2020 Boundary Population density 0 114 000 ( km\u00b2 ) A N 0\u00b70 12\u00b75 25\u00b70 50\u00b70km Qingyuan Huizhou Conghua Jiangmen Foshan Zhongshan Shenzhen Dongguan Zengcheng Huadu Huangpu Shaoguan D B E C F Nansha Baiyun Panyu C Articles www . thelancet . com / infection Vol 20 October 2020 1147 Guangzhou , implying inefficient transmission of the disease under the control measures . The projected local R , had there been no isolation of cases or quarantine of their contacts , was 0\u22196 ( 95 % CI 0\u221949 \u2013 0\u221974 ) when household was defined on the basis of close relatives . Higher estimates of projected local R were associated with shorter incubation periods and longer infectious periods ( appendix 2 p 21 ) . When household was defined on the basis of close relatives , the daily transmission probability during the incubation period was similar to that during the illness period ( esti mated OR 0\u221961 [ 95 % CI 0\u221927 \u2013 1\u221938 ] , table 3 ) ; however , the difference was much larger when longer incubation periods were assumed ( table 3 ; appendix 2 p 22 \u2013 23 ) . Individuals aged 60 years and older were the group most susceptible to SARS - CoV - 2 infection ( table 4 ; appendix 2 p 24 ) . Assuming a mean incubation period of 5 days and maximum infectious period of 13 days , in comparison with the oldest age group ( \u226560 years ) , the risk of infection was lower in the youngest age group ( < 20 years ; OR 0\u221923 [ 95 % CI 0\u221911 \u2013 0\u221946 ] ) and the 20 \u2013 59 year age group ( 0\u221964 [ 0\u221943 \u2013 0\u221997 ] ) . The person - to - person transmissibility of the virus declined over time to some extent ( February vs January OR 0\u221942 [ 95 % CI 0\u00b717 \u2013 1\u00b707 ] ) . These estimated ORs were insensitive to the assumptions about the natural history of disease ( appendix 2 p 24 ) . The estimated probability of daily transmission was two times higher in households of six people or less than in larger households ( more than six people ; appendix 2 p 25 ) . We found no association between age and infectivity and no associations between sex and susceptibility or infectivity . Restricting household contacts to those who were living at the same address as the primary case regardless of relationship resulted in higher secondary attack rate estimates among household contacts , ranging from 16\u00b71 % ( 95 % CI 12\u00b75 \u2013 20\u00b74 ) to 24\u00b73 % ( 18\u00b75 \u2013 31\u00b72 ) , but lower secondary attack rate estimates among non - household contacts ( ranging from 6\u00b78 % [ 5\u00b70 \u2013 9\u00b72 ] to 9\u00b73 % [ 6\u00b75 \u2013 13\u00b71 ] ) under the various settings ( ie , mean incubation and maximum infectious periods ) of the natural history of disease ( appendix 2 p 21 ) . Assuming an incubation time of 5 days and an infectious period of 13 days , the estimated secondary attack rates were 17\u22191 % ( 95 % CI 13\u22193 \u2013 21\u22198 ) among household contacts and 7\u22193 % ( 5\u22194 \u2013 9\u22199 ) among non - household contacts ( table 2 ) . The effect of age and the relative infectivity of virus in the illness period versus the incubation period remained similar ( table 4 ; appendix 2 p 24 ) . Discussion We retrospectively characterised the spatiotemporal epidemiology and transmissibility of SARS - CoV - 2 in Guangzhou , the most populated city in southern China , from early January up to mid - February , 2020 . The rapid decline in the R t indicates the effectiveness of the control policy implemented in the city . Social distancing or other potential personal behavioural changes might have also shifted the contact pattern between household members , as shown by the two - fold reduction in the probability of household transmission observed between January and February . Additionally , we assessed the effects of host features and disease stage on susceptibility and Figure 2 : Epidemic curve based on symptom onset dates of COVID - 19 cases in Guangzhou from Jan 6 , 2020 , to Feb 18 , 2020 Estimated R t for three scenarios : scenario 1 , all imported cases ( who travelled to or resided in Hubei province 14 days before symptom onset ) considered as primary cases , and all secondary cases were infected by primary cases in the same case cluster ; scenario 2 , which is identical to scenario 1 , with the additional assumption that local primary cases might have been infected by earlier cases in other clusters ; and scenario 3 , which is identical to scenario 2 , with the additional assumption that imported secondary cases were considered as infected by primary cases in the same cluster . R t = effective reproductive number . Jan 6 , 2020 Jan 13 , 2020 Jan 20 , 2020 Jan 27 , 2020 Feb 3 , 2020 Feb 18 , 2020 Feb 10 , 2020 0 C a s e s ( n ) Date 20 30 40 10 R t 0\u00b75 0 1\u00b70 1\u00b75 2\u00b70 Imported primary cases Imported secondary cases Local primary cases Local secondary cases Scenario 1 Scenario 2 Scenario 3 95 % CI 95 % CI 95 % CI Mean incubation period of 5 days Mean incubation period of 7 days 13 - day infectious period 22 - day infectious period 13 - day infectious period 22 - day infectious period Close relatives Secondary attack rate , % ( 95 % CI ) Household 12\u00b74 % ( 9\u00b78 \u2013 15\u00b74 ) 15\u00b75 % ( 11\u00b77 \u2013 20\u00b72 ) 11\u00b74 % ( 9\u00b70 \u2013 14\u00b72 ) 13\u00b71 % ( 9\u00b79 \u2013 17\u00b71 ) Non - household 7\u00b79 % ( 5\u00b73 \u2013 11\u00b78 ) 10\u00b74 % ( 6\u00b77 \u2013 15\u00b78 ) 7\u00b75 % ( 5\u00b70 \u2013 11\u00b72 ) 8\u00b79 % ( 5\u00b77 \u2013 13\u00b76 ) Local R ( 95 % CI ) With quarantine 0\u00b750 ( 0\u00b741 \u2013 0\u00b762 ) 0\u00b751 ( 0\u00b739 \u2013 0\u00b766 ) 0\u00b751 ( 0\u00b741 \u2013 0\u00b763 ) 0\u00b751 ( 0\u00b739 \u2013 0\u00b767 ) No quarantine 0\u00b760 ( 0\u00b749 \u2013 0\u00b774 ) 0\u00b776 ( 0\u00b759 \u2013 1\u00b700 ) 0\u00b756 ( 0\u00b745 \u2013 0\u00b769 ) 0\u00b765 ( 0\u00b749 \u2013 0\u00b785 ) Residential address Secondary attack rate , % ( 95 % CI ) Household 17\u00b71 % ( 13\u00b73 \u2013 21\u00b78 ) 21\u00b72 % ( 15\u00b78 \u2013 27\u00b78 ) 16\u00b71 % ( 12\u00b75 \u2013 20\u00b74 ) 18\u00b73 % ( 13\u00b76 \u2013 24\u00b71 ) Non - household 7\u00b73 % ( 5\u00b74 \u2013 9\u00b79 ) 9\u00b73 % ( 6\u00b75 \u2013 13\u00b71 ) 6\u00b78 % ( 5\u00b70 \u2013 9\u00b72 ) 7\u00b78 % ( 5\u00b75 \u2013 11\u00b70 ) Local R ( 95 % CI ) With quarantine 0\u00b750 ( 0\u00b740 \u2013 0\u00b761 ) 0\u00b750 ( 0\u00b738 \u2013 0\u00b765 ) 0\u00b750 ( 0\u00b741 \u2013 0\u00b762 ) 0\u00b751 ( 0\u00b739 \u2013 0\u00b766 ) No quarantine 0\u00b759 ( 0\u00b748 \u2013 0\u00b772 ) 0\u00b774 ( 0\u00b757 \u2013 0\u00b796 ) 0\u00b755 ( 0\u00b745 \u2013 0\u00b767 ) 0\u00b763 ( 0\u00b748 \u2013 0\u00b782 ) Estimates were reported using two different definitions of household contact ( close relatives or individuals sharing the same residential address ) and for selected settings of the natural history of disease . This model was not adjusted for age group , epidemic phase , or household size . R = reproductive number . Table 2 : Model - based estimates of secondary attack rates among household and non - household contacts , and local R with and without quarantine Articles 1148 www . thelancet . com / infection Vol 20 October 2020 infectivity . We found that patients with COVID - 19 were at least as infectious in the incubation periods as during their illness periods , and that older people ( aged > 60 years ) are most susceptible to household infection of SARS - CoV - 2 . When household contact was defined by residential address , our model - based secondary attack rate estimate for the Guangzhou contact tracing data was 17\u22191 % , which is higher than data - based secondary attack rate estimates of 14\u22199 % for Shenzhen and 10\u22192 % for Guangzhou under the same household definition . 14 , 15 Generally , a data - based secondary attack rate estimate would be higher than a model - based secondary attack rate estimate since data - based estimates do not exclude tertiary transmission and untraced exposure . However , these data - based secondary attack rate estimates reflect transmissibility under control measures such as case isolation , whereas our model - based secondary attack rate estimates assumed exposure of a susceptible individual during the whole infectious period of an infector , which is more epidemiologically relevant and generalisable . Model - based household secondary attack rate estimates for SARS - CoV or the Middle East respiratory syndrome coronavirus ( MERS - CoV ) are not available ; however , a small number of studies have reported data - based secondary attack rate estimates in the household or comparable settings . For SARS - CoV , the secondary attack rate was estimated to be 4\u22196 \u2013 8\u00b70 % in Beijing , Hong Kong , and Singapore . 21 , 22 The daily transmis - sion probabilities during the illness period , however , are comparable between SARS - CoV ( 0\u00b7013 [ 95 % CI 0\u00b7011 \u2013 0\u00b7016 ] ) and SARS - CoV - 2 ( 0\u00b7016 [ 0\u00b7008 \u2013 0\u00b7029 ] ; appendix 2 p 22 ) . 23 Information about household transmissibility of MERS - CoV is less clear . In a multicity household study in Saudi Arabia , the household secondary attack rate was 4 % ( 95 % CI 2 \u2013 7 ) . 24 In an outbreak among 828 female workers who lived in an Mean incubation period of 5 days Mean incubation period of 7 days 13 - day infectious period 22 - day infectious period 13 - day infectious period 22 - day infectious period Close relatives Transmission probabilities for household contacts ( \u00d710\u207b\u00b2 ) Incubation 1\u00b784 ( 1\u00b736 \u2013 2\u00b749 ) 1\u00b791 ( 1\u00b744 \u2013 2\u00b754 ) 2\u00b709 ( 1\u00b763 \u2013 2\u00b769 ) 2\u00b711 ( 1\u00b766 \u2013 2\u00b768 ) Illness 1\u00b713 ( 0\u00b761 \u2013 2\u00b708 ) 0\u00b780 ( 0\u00b744 \u2013 1\u00b746 ) 0\u00b754 ( 0\u00b719 \u2013 1\u00b757 ) 0\u00b741 ( 0\u00b716 \u2013 1\u00b705 ) Transmission probabilities for non - household contacts ( \u00d710\u207b\u00b2 ) Incubation 1\u00b716 ( 0\u00b773 \u2013 1\u00b783 ) 1\u00b725 ( 0\u00b781 \u2013 1\u00b792 ) 1\u00b737 ( 0\u00b79 \u2013 2\u00b707 ) 1\u00b74 ( 0\u00b793 \u2013 2\u00b71 ) Illness 0\u00b771 ( 0\u00b735 \u2013 1\u00b743 ) 0\u00b752 ( 0\u00b726 \u2013 1\u00b705 ) 0\u00b735 ( 0\u00b711 \u2013 1\u00b709 ) 0\u00b727 ( 0\u00b71 \u2013 0\u00b775 ) Transmission probability from an external source ( \u00d710\u207b\u2074 ) 1\u00b771 ( 0\u00b778 \u2013 3\u00b778 ) 1\u00b749 ( 0\u00b765 \u2013 3\u00b744 ) 1\u00b754 ( 0\u00b761 \u2013 3\u00b786 ) 1\u00b738 ( 0\u00b754 \u2013 3\u00b756 ) OR 0\u00b761 ( 0\u00b727 \u2013 1\u00b738 ) 0\u00b741 ( 0\u00b719 \u2013 0\u00b789 ) 0\u00b726 ( 0\u00b708 \u2013 0\u00b786 ) 0\u00b719 ( 0\u00b707 \u2013 0\u00b755 ) Residential address Transmission probabilities for household contacts ( \u00d710\u207b\u00b2 ) Incubation 2\u00b764 ( 1\u00b79 \u2013 3\u00b766 ) 2\u00b777 ( 2\u00b703 \u2013 3\u00b776 ) 3\u00b703 ( 2\u00b729 \u2013 4\u00b700 ) 3\u00b707 ( 2\u00b735 \u2013 4\u00b702 ) Illness 1\u00b758 ( 0\u00b784 \u2013 2\u00b795 ) 1\u00b71 ( 0\u00b759 \u2013 2\u00b705 ) 0\u00b779 ( 0\u00b728 \u2013 2\u00b721 ) 0\u00b757 ( 0\u00b722 \u2013 1\u00b746 ) Transmission probabilities for non - household contacts ( \u00d710\u207b\u00b2 ) Incubation 1\u00b708 ( 0\u00b775 \u2013 1\u00b755 ) 1\u00b714 ( 0\u00b781 \u2013 1\u00b761 ) 1\u00b723 ( 0\u00b789 \u2013 1\u00b769 ) 1\u00b725 ( 0\u00b792 \u2013 1\u00b770 ) Illness 0\u00b764 ( 0\u00b733 \u2013 1\u00b724 ) 0\u00b745 ( 0\u00b723 \u2013 0\u00b787 ) 0\u00b732 ( 0\u00b711 \u2013 0\u00b791 ) 0\u00b723 ( 0\u00b709 \u2013 0\u00b761 ) Transmission probability from an external source ( \u00d710\u207b\u2074 ) 1\u00b774 ( 0\u00b779 \u2013 3\u00b784 ) 1\u00b754 ( 0\u00b767 \u2013 3\u00b753 ) 1\u00b753 ( 0\u00b76 \u2013 3\u00b787 ) 1\u00b74 ( 0\u00b754 \u2013 3\u00b762 ) OR 0\u00b759 ( 0\u00b726 \u2013 1\u00b735 ) 0\u00b739 ( 0\u00b718 \u2013 0\u00b786 ) 0\u00b726 ( 0\u00b708 \u2013 0\u00b782 ) 0\u00b718 ( 0\u00b706 \u2013 0\u00b752 ) Data are estimates ( 95 % CI ) . Estimates of the daily probability of infection from an external source and the ORs for the relative infectivity during the illness versus incubation period are also provided . Estimates are reported using two different definitions of household contact ( close relatives , or only individuals sharing the same residential address ) and for selected settings of the natural history of disease ( ie , mean incubation and maximum infectious periods ) . This model was not adjusted for age group , epidemic phase , or household size . OR = odds ratio . Table 3 : Model - based estimates of daily transmission probabilities for household contacts and non - household contacts during the incubation and illness periods Mean incubation period of 5 days Mean incubation period of 7 days 13 - day infectious period 22 - day infectious period 13 - day infectious period 22 - day infectious period Close relatives Susceptibility Age < 20 years vs \u226560 years 0\u00b723 ( 0\u00b711 \u2013 0\u00b746 ) 0\u00b722 ( 0\u00b711 \u2013 0\u00b746 ) 0\u00b722 ( 0\u00b711 \u2013 0\u00b745 ) 0\u00b722 ( 0\u00b711 \u2013 0\u00b745 ) Age 20 \u2013 59 years vs \u226560 years 0\u00b764 ( 0\u00b743 \u2013 0\u00b797 ) 0\u00b764 ( 0\u00b742 \u2013 0\u00b796 ) 0\u00b763 ( 0\u00b742 \u2013 0\u00b795 ) 0\u00b763 ( 0\u00b742 \u2013 0\u00b794 ) February vs January 0\u00b742 ( 0\u00b717 \u2013 1\u00b707 ) 0\u00b746 ( 0\u00b719 \u2013 1\u00b710 ) 0\u00b736 ( 0\u00b712 \u2013 1\u00b705 ) 0\u00b738 ( 0\u00b713 \u2013 1\u00b709 ) Infectivity Illness vs incubation period 0\u00b760 ( 0\u00b727 \u2013 1\u00b736 ) 0\u00b742 ( 0\u00b719 \u2013 0\u00b791 ) 0\u00b729 ( 0\u00b710 \u2013 0\u00b788 ) 0\u00b721 ( 0\u00b707 \u2013 0\u00b758 ) Residential address Susceptibility Age < 20 years vs \u226560 years 0\u00b722 ( 0\u00b711 \u2013 0\u00b746 ) 0\u00b722 ( 0\u00b711 \u2013 0\u00b745 ) 0\u00b722 ( 0\u00b711 \u2013 0\u00b744 ) 0\u00b722 ( 0\u00b711 \u2013 0\u00b744 ) Age 20 \u2013 59 years vs \u226560 years 0\u00b767 ( 0\u00b745 \u2013 1\u00b700 ) 0\u00b767 ( 0\u00b745 \u2013 1\u00b700 ) 0\u00b766 ( 0\u00b744 \u2013 0\u00b799 ) 0\u00b766 ( 0\u00b744 \u2013 0\u00b799 ) February vs January 0\u00b757 ( 0\u00b723 \u2013 1\u00b739 ) 0\u00b762 ( 0\u00b727 \u2013 1\u00b744 ) 0\u00b750 ( 0\u00b718 \u2013 1\u00b736 ) 0\u00b753 ( 0\u00b720 \u2013 1\u00b742 ) Infectivity Illness vs incubation period 0\u00b754 ( 0\u00b723 \u2013 1\u00b726 ) 0\u00b738 ( 0\u00b717 \u2013 0\u00b784 ) 0\u00b724 ( 0\u00b707 \u2013 0\u00b779 ) 0\u00b718 ( 0\u00b706 \u2013 0\u00b752 ) Data are OR ( 95 % CI ) . Estimates were reported for selected settings of the natural history of disease ( ie , mean incubation and maximum infectious periods ) . This model was adjusted for age group , epidemic phase , and household size . OR = odds ratio . Table 4 : Model - based effects of age group and epidemic phase on susceptibility and relative infectivity during the illness period compared with the incubation period Articles www . thelancet . com / infection Vol 20 October 2020 1149 expatriate dormitory consisting of 24 villas in Riyadh , Saudi Arabia , 19 workers in seven villas were infected . If each affected villa was considered a household with a mean of 34\u00b75 residents including a single primary case , we estimated a secondary attack rate of 5\u00b71 % ( 95 % CI 2\u00b78 \u2013 9\u00b70 ) . 25 We conclude that SARS - CoV - 2 is more transmissible than SARS - CoV and MERS - CoV in households . We quantified the infectivity of patients with COVID - 19 during their incubation period using household data , but epidemiological evidence has been published previously . Transmission to secondary cases during the incubation period of the primary case has been reported in Germany and China . 7 , 26 An analysis of 77 transmission pairs within and outside of China estimated that nearly half of all transmission events could have occurred during the incubation period . 5 By contrast , shedding of SARS - CoV peaks 6 \u2013 11 days after illness onset , and asymptomatic and mild MERS - CoV cases were hypothesised to transmit inefficiently , indicating the importance of symptoms in the transmission of these two coronaviruses . 27 \u2013 29 This finding indicates the importance of testing close contacts of COVID - 19 cases to identify and isolate infections in the incubation period . Alternatively , the lower estimated transmission probability of SARS - CoV - 2 during the illness period than during the incubation period could be partially attributed to self - distancing within house holds when the primary cases developed symptoms . The infectivity measured by the transmission model accounts for both the biological process of viral shedding and the social contact process , and our data cannot separate the two processes . We estimated that the local R of SARS - CoV - 2 was relatively low ( around 0\u22195 ) , which is consistent with the mean R t . We estimated that without isolation of cases or quarantine of their contacts and assuming a mean incubation period of 5 days , the local reproductive number would have been about 20 \u2013 50 % higher , increasing to 0\u22196 \u2013 0\u00b776 ( appendix 2 p 21 ) . The subcritical local reproductive number ( R < 1 ) in the absence of isolation is due to the small average number of contacts per person per day ( appendix 2 , p 18 ) , which is likely to be a result of the stringent control measures that were implemented in the city . Although the effect of case isolation seems moderate , considering the high infectivity of the virus during the incubation period , quarantine of asymptomatic contacts could have prevented more onward transmissions . Our analysis has several limitations . The model suggests that a longer assumed incubation period was associated with higher estimated infectivity in this period , which could be due to the rapid quarantine of close contacts after symptom onset of cases . When few transmissions occurred during the illness period , bias could occur due to the paucity of data . Additionally , we were unable to reliably quantify the infectivity of asymptomatic infections , since only two of the 15 asymptomatic infections included in the household transmission analyses were considered primary cases . Some asymptomatic infections might have been missed since close contacts were tested only twice and the tests were done 14 days apart . Moreover , our assumption that asymptomatic infections have the same infectivity as symptomatic cases during their incubation period might not be realistic . Furthermore , it is likely that some imported primary cases might have been infected locally and that some asymptomatic infections or cases might have been missed by contact tracing or by false negative tests , which could lead to underestimation of the R t and the secondary attack rate . The infectiousness of patients with COVID - 19 during their incubation periods is high and could substantially increase the difficulty of curbing the ongoing pandemic . Active case finding and isolation in conjunction with comprehensive contact tracing and quarantine are useful for preventing infected contacts from spreading the virus during their incubation periods , which could be crucial when societal restrictions on human movement and mixing are lifted . The provision of comfortable facilities for exposed contacts to quarantine or for mild cases to isolate away from their families could be a valuable strategy to limit onward transmission within households . Contributors QLJ , JY , ZBZ , LL , MMM , YL , YM , and ZCY collected the data . YY , ZCY , and LQF conceived the statistical analysis plan . QLJ , MJL , ARZ , and NJ cleaned the data . MJL , QLJ , ARZ , and NJ did statistical analyses under the supervision of YY , ZCY , and LQF . YY drafted the manuscript . ND , IL , and EK contributed to interpretation of results and writing of the manuscript . Declaration of interests We declare no competing interests . Data sharing Data sharing requests should be directed to Guangzhou Center for Disease Control and Prevention . Acknowledgments This study was supported by grants from the US National Institutes of Health ( R01 AI139761 , R01 AI116770 and R37 AI32042 ) , the Science and Technology Plan Project of Guangzhou ( 201804010121 ) , the Project for Key Medicine Discipline Construction of Guangzhou Municipality ( 2017 \u2013 2019 \u2013 04 ) , and the Key Research and Development Program of China ( 2019YFC1200604 ) . We thank M Elizabeth Halloran ( Fred Hutchinson Cancer Research Center , Seattle , WA , USA ) for helpful discussions . We also thank the staff members of all district - level Center for Disease Control and Prevention and community health service centres in Guangzhou for their assistance in field investigation and data collection . References 1 Dong E , Du H , Gardner L . An interactive web - based dashboard to track COVID - 19 in real time . 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Clin Infect Dis 2019 ; 68 : 409 \u2013 18 .", + "hopeScalingCreativeInspiration2022": "Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas Tom Hope tomh @ allenai . org Allen Institute for AI The University of Washington Ronen Tamari Hebrew University of Jerusalem Hyeonsu Kang Carnegie Mellon University Daniel Hershcovich University of Copenhagen , Denmark Joel Chan University of Maryland Aniket Kittur Carnegie Mellon University Dafna Shahaf dshahaf @ cs . huji . ac . il Hebrew University of Jerusalem ABSTRACT Large repositories of products , patents and scientific papers offer an opportunity for building systems that scour millions of ideas and help users discover inspirations . However , idea descriptions are typi - cally in the form of unstructured text , lacking key structure that is required for supporting creative innovation interactions . Prior work has explored idea representations that were either limited in expres - sivity , required significant manual effort from users , or dependent on curated knowledge bases with poor coverage . We explore a novel rep - resentation that automatically breaks up products into fine - grained functional aspects capturing the purposes and mechanisms of ideas , and use it to support important creative innovation interactions : func - tional search for ideas , and exploration of the design space around a focal problem by viewing related problem perspectives pooled from across many products . In user studies , our approach boosts the qual - ity of creative search and inspirations , substantially outperforming strong baselines by 50 - 60 % . 1 INTRODUCTION Human creativity often relies on detecting structural matches across distant ideas and adapting them by transferring mechanisms from one domain to another [ 16 , 17 , 37 , 38 ] . For example , microwave ovens were discovered by repurposing radar technology developed during World War II . Teflon , today chiefly used in non - stick cook - ware , was first used in armament development . Recognizing the potential of this innovation process , major organizations such as NASA and Procter & Gamble actively engage in searching for op - portunities to adapt existing technologies for new markets [ 27 ] . Online repositories of millions of products , scientific papers , and patents present an opportunity to augment and scale this core process of innovation . The large scale and diversity of these repositories is Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from permissions @ acm . org . CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA \u00a9 2022 Association for Computing Machinery . important , because inspiration can be found in unexpected places \u2013 for example , a car mechanic recently invented a simple device to ease childbirths by adapting a trick for extracting a cork stuck in a wine bottle , which he discovered in a YouTube video [ 1 ] . However , the predominant way human problem - solvers currently interact with these repositories \u2014 via standard search engines \u2014 does not tap into their potential for augmenting and scaling human ingenuity . Core to this limitation is the representation of ideas in the form of unstructured textual descriptions . This representation hinders creative innovation interactions that require traversing multiple levels of granularity and abstraction around a focal problem , to \u201cbreak out\u201d of fixation on the details of a specific problem by exploring the design space and viewing novel perspectives on problems and solutions [ 15 , 23 , 60 , 65 ] . Toward addressing this challenge , our vision in this paper is to develop a novel representation of ideas that can support exploration and abstraction of fine - grained functional aspects in large - scale idea repositories \u2013 aspects such as the purposes and mechanisms of products . More specifically , our goal is to obtain a representation having two key capabilities : ( I ) The representation would be able to automatically disentangle raw descriptions into fine - grained func - tional \u201cunits\u201d that support search and discovery of products that match on certain functions but not others . ( II ) This representation should also allow navigating the landscape of ideas at different resolutions \u2014 enabling users to \u201czoom\u201d in and out at desired levels of abstraction of a given problem and connect to inspirations in seemingly distant areas . As an example , consider an inventor looking for a way to wash clothes without water ( e . g . , in space , or where water is scarce ) . Fig - ure 1 illustrates our vision . Breaking down product descriptions into fine - grained functions ( capability I above ) could allow an automated system to find ideas that match on certain purpose aspects ( washing clothes ) but not certain mechanism aspects ( usage of water ) . This could lead to solutions like cleaning mechanisms based on dry ice or chemical coating . Zooming out and abstracting the problem to a more general fram - ing ( capability II ) might lead to broader ideas for the problem of cleaning such as techniques for removing dirt or odor \u2013 each result - ing in novel problem perspectives and inspirations . In Figure 1 , each node represents a cluster of documents with a similar purpose and a r X i v : 2102 . 09761v3 [ c s . H C ] 17 F e b 2022 CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Hope et al . the user can explore neighboring clusters to find inspirations ( e . g . , dry shampoo ) . This can also expand the innovator\u2019s conception of the problem space itself , such as the assumption that clothes should be cleaned and reworn ( vs . biodegradable ) . In this paper , we develop a scalable computational model of ideas that brings us closer to this vision . We train a neural network to extract spans of text describing purposes and mechanisms in product texts , and use them to build a span embedding representation that allows aspect - based matching between ideas . We then use this representation of individual ideas to automatically mine connections between problems and solutions across entire repositories and build a \u201cfunctional network\u201d that resembles functional ontologies used in engineering and design ideation [ 39 , 51 ] , which are typically hand - crafted and limited in scale . Our approach could facilitate many applications in creative inno - vation due to its ability to decompose idea texts into fine - grained functional aspects , and to surface related problem perspectives at multiple levels of abstraction \u2014 two fundamental drivers of creativ - ity support . In this paper we instantiate the approach in two prototype systems , probing its value regarding each of these capabilities : \u2022 Functional aspect - based search for alternative uses . One important use of our novel representation is to enhance the expressivity of search engines over idea repositories . This way , our representation could support expressive search for alternative , atypical uses of products to identify potentially high - value adaptation opportunities . \u2022 Exploring alternative levels of problem perspectives . Re - cent work [ 42 ] showed that problem - solvers are often inter - ested in exploring different reformulations of the problem when searching for inspiration . Our fine - grained span repre - sentations facilitate mining of recurring functional relations , such as purposes that are often mentioned together or mecha - nisms associated with purposes . This level of detail enables us to map the landscape of ideas with a network of purposes and mechanisms , allowing us to automatically traverse neigh - boring problems and solutions around a focal problem and surface novel inspirations to users . Previous work highlighted the importance of functional represen - tations for supporting ideation [ 12 , 42 , 52 , 60 , 65 , 100 ] , but these methods require significant manual effort from the user , rely on resources with limited coverage , or have limited expressivity ( we discuss this work in more detail in \u00a7 2 ) . We seek to advance the state of the art by developing a novel representation that is both ex - pressive and scalable , and exploring the applications it unlocks . We believe our representation may serve as a useful building block for novel creativity support tools that can help users find and recombine the inspirations latent in unstructured idea repositories at a scale previously impossible . In summary , in this work we contribute : \u2022 A novel computational representation of ideas with fine - grained functional aspects for purposes and mechanisms . \u2022 Empirical demonstrations of the flexibility and utility of the representation for computational support of core creative tasks : ( 1 ) searching for alternative , atypical product uses for potential adaptation opportunities ; and ( 2 ) creating a func - tional concept graph that enables innovators to explore the design space around a focal problem . Through two empirical user studies we demonstrate that our representation signifi - cantly outperforms both previous work and state - of - the art embedding baselines on these tasks . We achieve Mean Aver - age Precision ( MAP ) of 87 % in the alternative product uses search , and 62 % of our inspirations for design space explo - ration are found to be useful and novel \u2013 a relative boost of 50 - 60 % over the best - performing baselines . 2 RELATED WORK : IDEATION SYSTEMS AND COGNITIVE MECHANISMS The contributions in this paper relate broadly to previous work on systems that use structured representations for supporting ideation , and studies that seek to understand the cognitive process of creativity and its implications . We provide a brief discussion of these two themes . Cognitive Theories of Creative Thinking with Prior Ideas . A core aspect of creative thinking that distinguishes it from regular problem solving is the need for divergent thinking [ 21 , 85 , 87 ] - to construct and explore diverse ideas that are quite different from the obvious path of ideation . This process of divergence is shaped by prior knowledge [ 97 ] \u2014 such as past ideas , external stimulation , and examples \u2014 in ways that sometimes hinders creativity through mechanisms like fixation [ 54 ] , and sometimes leads to creative break - throughs [ 92 ] . Our design goals for this research are guided by past research on cognitive mechanisms that enable helpful interactions with past ideas . For example , research on insight problem - solving has uncov - ered the role of re - representation of past ideas by decomposing them into conceptual chunks and then recombining and / or repurposing them into new solutions [ 61 , 73 ] . Similar patterns in terms of core helpful interactions with prior ideas have been observed in in situ studies of expert designers\u2019 dissection of past ideas into component aspects and features for repurposing and recombination into new ideas [ 26 , 48 , 49 ] . Another core process is analogical abstraction , where innovators think about and retrieve past ideas not in terms of their surface features , but in terms of deeper structural features or schemata , such as their underlying purposes ( goals ) and mechanisms [ 40 , 41 ] . These abstracted \u201cschemas \" can then facilitate analogical transfer of ideas across domains that can lead to groundbreaking discoveries [ 19 , 50 , 60 , 72 , 79 ] . For example , the ancient Greeks studied the properties of sound waves by analogy to ripples in water ; Nobel laureate Salvador Luria used abstract structural similarity between a slot machine and bacteria mutations to understand bac - terial replication [ 77 ] ; and in computer science and optimization , analogies to processes in nature inspired algorithms such as simu - lated annealing [ 59 ] , genetic algorithms [ 35 ] , and momentum - based gradient descent [ 83 ] . This process of abstraction over past ideas is also an important contributor to the ability to reformulate problems [ 20 , 23 , 24 , 31 , 57 ] . For example , innovators tasked with a problem ( find more room to store e - waste ) might consider a related , more general goal ( reducing environmental pollution ) to inspire new solu - tions , or consider \u201csibling\u201d formulations ( create alternative materials that are biodegradable ) . This ability to reframe a problem using other problems that bear some abstract relation to it is known to be a pow - erful way to combat fixation and boost creativity [ 16 , 31 , 36 \u2013 38 ] . Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Purpose : wash clothes Mechanism : NOT water Waterless washing machine scrubs clothes clean with dry ice . Dry shampoo uses starch - based active ingredients to soak up the oils and sweat from your hair , making it appear cleaner . Fragrance makes your hair smell fresh . A chemical coating causing cotton materials to clean themselves of stains and remove odors when exposed to sunlight . Biodegradable yarn from cellulose fiber from citrus fruit peels . Can be spun into high - quality fabric that feels like silk . clean , remove dirt wash clothes , clean clothes remove bad odor get rid of dirty objects 1 2 3 4 1 1 , 2 2 , 3 4 Example Documents from Clusters Exploring the Purpose Graph Figure 1 : Extracting fine - grained purposes and mechanisms at scale enables mapping the landscape of ideas . Suppose an inventor is looking for a way to wash clothes without water . On the left we see a snippet from the graph of purposes . Each node in the graph represents a cluster of similar purpose spans extracted from documents ( labels are manually generated for illustration purposes ) . Edges reflect abstract structural similarity , capturing co - occurrence patterns of spans in the corpus ( see Section 5 . 1 and Figure 9 ) . On the right we see example documents containing purposes from the four clusters ( corresponding cluster numbers appear in boxes ) . Purpose / mechanism spans in documents are shown in pink / green , respectively . One could find direct matches to the query \u2013 i . e . , documents with purpose from cluster 1 and mechanism not \u201cwater\u201d ( e . g . , waterless washing machine using dry ice ) , or explore neighboring purpose clusters , reformulating the problem as removing odor , removing dirt , or getting rid of the dirty clothes , each resulting in a different set of inspirations . This abstraction and reframing process has also been observed in studies of example curation [ 49 , 56 ] . A core unifying thread across these mechanisms is the need for particular structured , yet flexible , representations of ideas in terms of their component aspects , such as analogical schemas , or conceptual chunks . In this paper , we focus on developing representations with these properties , starting with the decomposition of ideas into their component purposes and mechanisms . As we discuss in the next section , developing computational representations with these proper - ties that can operate over large scale repositories of ideas remains a formidable open challenge . Utilizing Structured Representations for Ideation Systems . A main focus of creativity techniques and prototypes has been building computational systems for exploring the space of possible solutions to problems and alternative problem perspectives [ 31 ] . To do so , such systems often leverage structured knowledge representations for mapping the design space and linking across different prob - lems . For example , the WordTree method [ 65 ] \u2013 a prominent design method in creative engineering design \u2013 directs designers to break their problem into subfunctions , and then use the WordNet database [ 75 ] to explore abstractions and related functional aspects . Like - wise , a recent study [ 42 ] asked designers to select product aspects to abstract using WordNet and the Cyc ontology [ 63 ] , which aimed to serve as a general - purpose repository of commonsense knowl - edge in structured form . These and other general - purpose knowledge bases ( e . g . , NELL [ 76 ] and DBpedia [ 29 ] ) largely encode categorical knowledge ( e . g . , is - a , has - a ) and rarely functional knowledge ( e . g . , used - for ) , and often suffer from poor coverage of concepts in real - world products [ 42 ] . Knowledge bases and ontologies that do focus on functions , behaviors , and structures [ 6 , 51 , 96 ] have primarily been hand - crafted and are therefore even more limited in coverage . Work attempting to scale up has shown promise but is limited in expressivity or interpretability , such as modeling patents in terms of verbs and nouns [ 32 ] , using principal component analysis [ 25 ] , or learning coarse aggregate vectors that capture only one overall product purpose and mechanism [ 52 ] that cannot disentangle the dif - ferent aspects of a product , unlike our work presented in this paper . While full abstraction of ideas currently remains a holy grail , here we investigate whether learning nuanced functional aspects might enable a limited form of abstraction useful for augmenting creativity and providing a first step towards true automated abstraction . 3 LEARNING A FINE - GRAINED FUNCTIONAL REPRESENTATION Our goal in this section is to construct a representation that can support the creative innovation tasks and interactions discussed in the Introduction . CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Hope et al . Figure 2 : Crowdsourcing interface for fine - grained purposes and mechanisms . We propose to use span representations [ 62 ] . Given a product text description , we extract tagged spans of text corresponding to purposes and mechanisms ( see Figure 2 ) , and represent the product as a set of span embeddings . More technically , we use a standard sequence tagging formulation , with X \ud835\udc41 = { x 1 , x 2 , . . . , x \ud835\udc41 } a training set of \ud835\udc41 texts , each a sequence of tokens x \ud835\udc56 = ( \ud835\udc65 1 \ud835\udc56 , \ud835\udc65 2 \ud835\udc56 , . . . , \ud835\udc65 \ud835\udc47\ud835\udc56 ) , and Y \ud835\udc41 a corresponding set of label sequences , Y \ud835\udc41 = { y 1 , y 2 , . . . , y \ud835\udc41 } , y \ud835\udc56 = { \ud835\udc66 1 \ud835\udc56 , \ud835\udc66 2 \ud835\udc56 , . . . , \ud835\udc66 \ud835\udc47\ud835\udc56 } , where each \ud835\udc66 \ud835\udc57 indicates token \ud835\udc57 \u2019s label ( purpose / mechanism / other ) . In later sections , we represent each product \ud835\udc56 as a set of purpose span embedding vectors and a set of mechanism span embedding vectors . 3 . 1 Data We use real - world product idea descriptions taken from crowd - sourced innovation website Quirky . com and used in [ 52 ] , including 8500 user - generated texts describing inventions across diverse do - mains ( e . g . , kitchen products , health and fitness , clean energy ) . Texts typically include multiple purposes and mechanisms . Texts in Quirky use very nonstandard language , including grammatical and spelling errors ( e . g . , \u201cFolds Up Perfect For Carrying . you can walk - on , put your mouth on and or hands on . numbers in any configuration 4 learning to De / Composing Numbers . \u201d ) . Annotation . To create a dataset annotated with purposes and mecha - nisms , we collect crowdsourced annotations on Amazon Mechanical Turk ( AMT ) . In the similar annotation task of [ 52 ] workers were reported to annotate long , often irrelevant spans . We thus guided workers to focus on shorter spans . To further improve quality and encourage more fine - grained annotations , we limited maximal span length that could be annotated , and disabled the annotation of stop - words . Fig . 2 shows our tagging interface ; rectangles are taggable chunks . For quality control , we required US - based workers with approval rate over 95 % and at least 1000 approved tasks , and filtered unreasonably fast users . In total , we had 400 annotating workers . Workers were paid $ 0 . 1 per task . This rate was computed aiming for an hourly rate of $ 7 , where completion time was estimated via a small - scale pilot study . However , in the full study we were sur - prised to find the median completion time was much higher , reaching 100 seconds . We note that this figure could be skewed ( e . g . , due to workers queuing of tasks or the ability to take breaks ) . While a manual inspection of the annotations revealed they are mostly satisfactory , we observe two main issues : First , there are of - ten multiple correct annotations . Second , workers provide partial tagging \u2013 in particular , if similar spans appear in different sentences , very few workers bother tagging more than one instance ( despite instructions ) . These issues would have made computing evaluation metrics problematic . We thus decided to use the crowdsourced an - notations as a bronze - standard for training and development sets only . For a reliable evaluation , we collected gold - standard test sets annotated by two CS graduate students . Annotators were instructed to mark all the relevant chunks , resulting in high inter - annotator agreement of 0 . 71 . We collect 22316 annotated training sentences and 512 gold sentences , for a total of 238 , 399 \ud835\udc61\ud835\udc5c\ud835\udc58\ud835\udc52\ud835\udc5b\ud835\udc60 ( tag proportions : 14 . 5 % mechanism , 15 . 9 % purpose , 69 . 6 % other ) . A note on related annotated data . There has been recent work on the related topic of information extraction from scientific papers by classifying sentences , citations , or phrases . Recent supervised approaches [ 9 , 55 , 66 ] use annotations which are often provided by either paper authors themselves , NLP experts , domain experts , or involve elaborate ( multi - round ) annotation protocols . Sequence tagging models are often trained and evaluated on ( relatively ) clean , succinct sentences [ 71 , 101 ] . When trained on noisy texts , results typically suffer drastically [ 2 ] . Our corpus of product descriptions is significantly noisier than scientific papers , and our training annota - tions were collected in a scalable , low - cost manner by non - experts . Using noisy crowdsourced annotation for training and development only is consistent with our quest for a lightweight annotation ap - proach that would still enable training useful models . 3 . 2 Extracting Spans After collecting annotations , we can now train models to extract the spans . We explore several models likely to have sufficient power to learn our proposed novel representation , with the goal of selecting the best performing one . In particular , we chose two approaches that are common for related sequence - tagging problems , such as named entity recognition ( NER ) and part - of - speech ( POS ) tagging : a common baseline and a recent state - of - the - art model . We also tried a model - enrichment approach with syntactic relational inputs . We briefly describe the models we used below , with full technical descriptions and implementation details , data and code appearing in the supplementary material ( Appendix A . 1 ) . We note that our goal in this section is to find a reasonable model whose output could support creative downstream tasks ; many other architectures are possible and could be considered in future work . \u2022 BiLSTM - CRF . A BiLSTM - CRF [ 53 ] neural network , a common baseline approach for NER tasks , enriched with semantic and syn - tactic input embeddings known to often boost performance [ 101 ] . We adopt the \u201cmulti - channel\u201d strategy as in [ 101 ] , concatenating input word embeddings ( pretrained GloVe vectors [ 81 ] ) with part - of - speech ( POS ) and NER embeddings . A conditional random field ( CRF ) model over the BiLSTM outputs maximizes the tag sequence log likelihood under a pairwise transition model between adjacent tags [ 5 ] . \u2022 Pre - trained Language Model ( Pooled Flair ) . A pre - trained lan - guage model [ 4 ] based on contextualized string embeddings , re - cently shown to outperform other powerful models such as BERT [ 22 ] in NER and POS tagging tasks and achieve state - of - art results . \u2022 GCN . We also explore a model - enrichment approach with syn - tactic relational inputs . We employ a graph convolutional network ( GCN ) [ 58 ] over dependency - parse edges [ 101 ] . GCNs are known Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA 0 10 20 30 40 50 60 70 80 90100 Top K ( % ) 40 45 50 55 60 65 70 75 P r e c i s i on mechanism purpose 40 45 50 55 60 65 70 75 Configuration P R F 1 Enriched BiLSTM 45 . 24 39 . 01 41 . 90 Pooled - Flair 53 . 30 39 . 80 45 . 50 GCN 47 . 85 47 . 93 47 . 89 GCN self - train 49 . 00 52 . 00 50 . 50 Figure 3 : Left : Precision @ K results for the best performing model ( GCN + self - training ) . Right : Raw extraction accuracy evaluation . All approaches use CRF loss . GCN with syntactic edges outperforms baselines . Self - training further improves re - sults . Random - label achieves only 16 . 01 F 1 . to be useful for propagating relational information and utilizing syntactic cues [ 71 , 101 ] . The linguistic cues are of special relevance and interest to us , as they are known to exist for purpose / mechanism mentions in texts [ 32 ] . 3 . 3 Evaluation of Extraction Accuracy In this section we assess extraction accuracy ( whether we are able to extract purpose and mechanism spans of text ) . In the next sections , we evaluate the utility of the extracted spans for enabling creative innovation tasks . To evaluate raw accuracy of the model\u2019s predictions , we use the standard IOB label markup to encode the purpose and mechanism spans ( 5 possible labels per token , { Beginning , Inside } x { Purpose , Mechanism } plus an \" Outside \" label ) . We conduct experiments using a train / development / test split of 18702 / 3614 / 512 . Due to our challenging setting , we train models on bronze - standard annotations with noisy and partial tagging done by non - experts ; for evaluation we use a curated gold - standard test set ( Section 3 ) . See Figure 3 ( right ) for results : GCN reaches an F 1 score of \u223c 48 % , out - performing the BiLSTM - CRF model ( enriched with multi - channel GloVe , POS , NER and dependency relation embeddings ) by 6 % . GCN also surpasses the strong Pooled - Flair pre - trained language model by nearly 2 . 5 % . A random baseline guessing each token by label frequencies ( Section 3 ) achieves 16 . 01 F 1 . We interpret these results as possibly attesting to the utility of graph representations and features capturing syntactic and semantic information when labels are noisy . As a sanity check , we also computed precision @ K ( Figure 3 , left ) . As expected , precision is higher with low values of \ud835\udc3e , and gradually degrades . Precision for mechanisms is higher Figure 4 : Comparing our GCN model predictions ( right ) to human annotations ( left ) . Interestingly , our model managed to correct some annotator errors ( \u201cit\u2019s\u201d , \u201cheated\u201d , \u201ccoffee warm\u201d , \u201cbeverages\u201d ) . Purposes in pink , mechanisms in green . than for purposes . Interestingly , a manual inspection revealed many cases where despite the noisy training setting , our models managed to correct mistaken or partial annotations ( see Figure 4 ) . Self - Training . According to the results , we chose GCN as our best - performing model . We experimented adding self - training [ 86 ] to GCN . Self - training is a common approach in semi - supervised learn - ing where we iteratively re - label \u201cO\u201d tags in training data with model predictions . A large portion of our training sentences are ( erroneously ) un - annotated by workers , perhaps due to annotation fatigue , introducing bias towards the \u201cO\u201d label . Self - training with GCN shows an improvement in F 1 by an ad - ditional 2 . 6 % , substantially increasing recall ( more than 12 % over Flair ) , see Figure 3 , right . Self - training stopped after 2 iterations , following no gain in F 1 on the development set . In the following two sections we demonstrate that our extraction model\u2019s accuracy , while far from perfect , is sufficient for achieving good performance on the downstream tasks which are at the focus of this paper . One main reason for this gap is that our downstream tasks involve aggregation of multiple extracted spans : Product descriptions will typically mention salient mechanisms / purposes several times in the text , such that the effect of local false positives / negatives is mitigated if overall the key aspects are captured somewhere in the text . Further , as we discuss in \u00a7 5 . 1 , our approach also aggregates purposes and mechanisms across the entire corpus , not just single texts , learning from patterns observed sufficiently many times across multiple texts and thus removing noise introduced by extraction errors . As future information extraction technologies advance , our task could benefit from improved extraction accuracy to further reduce the rate of false positives and negatives . 4 FINE - GRAINED FUNCTIONAL SEARCH FOR ALTERNATIVE USES In the previous section we suggested a model for extracting purpose and mechanism spans and assessed extraction accuracy . Our focus in this paper is to study the utility of the extracted purposes and mechanisms , in terms of the user interactions they enable . In the following sections we explore two tasks demonstrating the value of our novel representation for supporting creative innovation . We start with a task involving search for alternative uses . CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Hope et al . Motivation . Our task is inspired by one of the most well - known divergent thinking tests [ 46 ] for measuring creative ability \u2013 the alternative uses test [ 47 ] , where participants are asked to think of as many uses as possible for some object . Aside from serving as a measure of creativity , the ability to find alternative uses for technolo - gies has important applications in engineering , science and industry . Technologies developed at NASA , the US space agency , have led to over 2 , 000 spinoffs , finding new uses in computer technology , agri - culture , health , transportation , and even consumer products 1 . Procter & Gamble , the multinational consumer goods company , has invested in systematic search for ideas to re - purpose and adapt from other industries , such as using a compound that speeds up wound healing to treat wrinkles - an idea that led to a new line of anti - wrinkle products [ 27 ] . And very recently , the COVID - 19 pandemic provided a stark example of human innovation , with many companies seeking to pivot and re - purpose existing products to fit the new climate [ 28 ] . One teaching story is that of John Osher , creator of the \u201cSpin Pop\u201d \u2014 a lollipop with a mechanism for twirling in your mouth . After sell - ing his invention , Osher\u2019s team systematically searched for new ideas \u2014 \u201crather than having an idea come to us\u201d 2 . The group eventually landed on the \u201dSpin Brush\u201d \u2013 a cheap electric toothbrush adapted from the same twirling mechanisms . This case of repurposing an existing technology involved a systematic search process rather than pure serendipity . Introducing automation could help accelerate the search process by scouring many relevant problems available online , but the task is challenging for existing search systems , requiring a fine - grained , multi - aspect understanding of products . Illustrative Example . Consider a company that manufactures light bulbs . The company is familiar with straightforward usages of their product ( lamps , flashlights ) , and wants to identify non - standard uses and expand to new markets . Finding uses for a lightbulb that are not about the standard purpose of illuminating a space would be difficult to do with a standard search query over an idea repository , as the term \u201clights\u201d or \u201clighting\u201d will bring back lots of results close to \u201clamps , \u201d \u201cflashlight , \u201d and the like . In contrast , with our representation each idea is associated with mechanism and purpose aspects , and one could form a query such as mechanism = \u201clight bulb\u201d , purpose = NOT \u201clight\u201d . Using our system , the searcher adds \u201clight\u201d as a mechanism and also adds \u201clight\u201d as a negative purpose ( i . e . , results should not include \u201clight\u201d as a purpose ) . Our prototype returns examples such as billiard laser instructor devices ( Table 1 ) , warning signs on food packages to get attention of kids with allergies , and lights attached to furniture to protect your pinky toes at night ( Figure 5 ) . 4 . 1 Study Design We have built a search engine prototype supporting our representa - tion . Figure 5 shows the top two results for the light bulb scenario : warning lights on food for kids with allergies , and lights attached to furniture to protect your pinky toe at night . These are non - standard recombinations [ 30 ] ( light + allergies , light + furniture guard ) that could lead the company to new markets . 1 https : / / spinoff . nasa . gov / 2 https : / / www . allbusiness . com / the - man - the - legend - john - osher - inventor - of - the - spin - brush - part - i - 2 - 7665547 - 1 . html Figure 5 : Applications for light where light is not in the purpose . Left : Interface . Right : Two of the results and their automatic annotations ( purposes in pink , mechanisms in green ) . We conduct an experiment simulating scenarios where users wish to find novel / uncommon uses of mechanisms . Table 1 shows the sce - narios and examples . To choose these scenarios for the experiment , we find popular / common mechanisms in the dataset and their most typical uses . For example , one frequent mechanism is RFID , which is typically used for purposes such as \u201clocating\u201d and \u201ctracking\u201d . We then create queries searching for different uses \u2013 purposes that do not include concepts related to the typical uses of a given mechanism . To automate scenario selection , we cluster mechanisms ( see Section 5 . 1 ) , select frequent mechanisms from the top 5 largest mechanism clusters , and identify purposes strongly co - occurring with them ( e . g . , \u201cRFID\u201d co - occurs with \u201clocating\u201d , \u201ctracking\u201d ) to avoid . Our Approach . We represent each product \ud835\udc56 as a set of purpose vectors P \ud835\udc56 (cid:66) { p 1 \ud835\udc56 , p 2 \ud835\udc56 , . . . , p \ud835\udc43 \ud835\udc56 \ud835\udc56 } , and a set of mechanism vectors M \ud835\udc56 (cid:66) { p 1 \ud835\udc56 , p 2 \ud835\udc56 , . . . , p \ud835\udc40 \ud835\udc56 \ud835\udc56 } extracted with our GCN model . Simi - larly , we define a set of query vectors q \ud835\udc5d (cid:66) q 1 , q 2 , . . . q \ud835\udc44 \ud835\udc5d and q \ud835\udc5a (cid:66) q 1 , q 2 , . . . q \ud835\udc44 \ud835\udc5a . Each query chunk can be negated , meaning it should not appear . Finally , we define distance metrics \ud835\udc51 \ud835\udc5d ( \u00b7 , \u00b7 ) , \ud835\udc51 \ud835\udc5a ( \u00b7 , \u00b7 ) between sets of purposes and mechanisms . For example , to locate a dog using RFID but not GPS : argmin \ud835\udc56 \ud835\udc51 \ud835\udc5d ( { q \u201clocate dog\u201d } , P \u02dc \ud835\udc56 ) \ud835\udc60 . \ud835\udc61 . \ud835\udc51 \ud835\udc5a ( { q \u201cGPS\u201d } , M \u02dc \ud835\udc56 ) \u2265 threshold \ud835\udc51 \ud835\udc5a ( { q \u201cRFID\u201d } , M \u02dc \ud835\udc56 ) \u2264 threshold ( 1 ) Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA We explore two alternatives for computing distance metrics \ud835\udc51 \ud835\udc5a , \ud835\udc51 \ud835\udc5d : \u2022 FineGrained - AVG . \ud835\udc51 \ud835\udc5d ( q \ud835\udc5d , P \ud835\udc56 ) is 1 minus the dot product be - tween average query and purpose vectors ( normalized to unit norm ) . We define \ud835\udc51 \ud835\udc5a similarly . \u2022 FineGrained - MAXMIN . We match each element in q \ud835\udc5d with its nearest neighbor in P \ud835\udc56 , and then find the minimum over the distances between matches . \ud835\udc51 \ud835\udc5d is defined as 1 minus the minimum . All vectors are normalized . We define \ud835\udc51 \ud835\udc5a similarly . This captures cases where queries match only a small subset of product chunks , erring on the side of caution with a max - min approach . Baselines . We test our model against : \u2022 AvgGloVe . A weighted average of GloVe vectors of the entire text ( excluding stopwords ) , similar to standard NLP approaches for retrieval and textual similarity . We average query terms and normalize to unit norm . Distance is computed via the dot product . \u2022 Aggregate purpose / mechanism . Representing each document with the model in [ 52 ] . This model takes raw text as input , applies a BiLSTM neural network and produces two vectors correspond - ing to aggregate purpose and mechanism of the document . We average and normalize query vectors , and use the dot product . For all four methods , we handle negative ( purpose ) queries by filtering out all products whose similarity is lower than \ud835\udf06 , where lambda is a threshold selected to be the 90 th percentile of similarities ( 1 minus the distances ) . This corresponds to the threshold seen in the example in Eq . 1 . 4 . 2 Results We recruited five engineering graduate students ( three female , two male ) to judge the retrieved product ideas . Each participant provided binary relevance feedback [ 88 ] ( yes / no ) to the top 20 results from each of the four methods , shuffled randomly so that judges are blind to the condition . 3 See Figure 6 for results . We report Non Cummulative Discounted Gain ( NDCG ) and Mean Average Precision ( MAP ) , two common metrics in information retrieval [ 88 ] . Our FineGrained - AVG wins for both metrics , followed by FineGrained - MAXMIN . The baselines perform much worse , with the aggregate - vectors approach in [ 52 ] outperforming standard embedding - based retrieval with GloVe . Im - portantly , our approach achieves high MAP ( 85 % - 87 % ) in absolute terms , in addition to a large relative improvement over the baselines ( MAP of 40 % - 60 % ) . Qualitative Analysis . Table 1 shows example results of FineGrained - AVG . For instance , a query for using light not for lighting results in laser - based billiard instructions . A query for using RFID not for locating or tracking results in an idea for an RFID - based lock , or RFIDs used at supermarket checkouts . To give an intuition for what might be driving our quantitative findings , we examine examples of retrieved results . For instance , with the query for using light for the non - standard purpose of cleaning , the top ranked result retrieved by FineGrained - AVG is a UV Light Sterilizer , with extracted purposes including Sterilizes bacteria , Keep public and people healthy and Cleaner 3 Inter - rater agreement measured across all scenarios was at 50 % by both Fleiss kappa and Krippendorff\u2019s alpha tests . Figure 6 : Results for search evaluation test case . Mean average precision ( MAP ) and Normalized Discounted Cumulative Gain ( NDCG ) by method , averaged across queries . Methods in bold use our model . fresher air , and the top result from FineGrained - MAXMIN is sim - ilarly a Standalone bug zapper bulb that uses uv light / black light . Conversely , the top result for both baselines ( standard search and aggregate - vectors ) is a Toilet / Bathroom Light , with \u201ca sensor light that glows around your toilet\u201d and \u201cextra batteries if you lose elec - tricity in the bathroom\u201d . It appears that both baselines were not able to accurately capture and disentangle purposes and mechanisms , despite the aggregate - vector being explicitly designed for that . More generally , it appears that the aggregate - vector approach squashes multiple purposes together by design into one soft , ag - gregate vector , which in this case includes concepts like toilet and bathroom that are somewhat topically related to cleaning . The aggre - gate approach had similar issues in the next product ideas it retrieved ( e . g . , Switch that glows in the dark , a Dash Light to illuminate ash trays ) . Overall , our results demonstrate that fine - grained purposes and mechanisms lead to better functional search expressivity than ap - proaches based on distributional representations or coarse purpose - mechanism vectors . 5 EXPLORING THE DESIGN SPACE WITH A FUNCTIONAL CONCEPT GRAPH In this section we test the value of our novel representation for supporting users in exploring the design space for solving a given problem . We use our span - based representation to construct a corpus - wide graph of purpose / mechanism concepts . We demonstrate the utility of this approach in an ideation task , helping users identify useful inspirations in the form of problems that are related to their own . Our goal is to help users \u201cbreak out\u201d of fixation on a certain domain , a well - known hindrance to innovation [ 15 , 60 ] . Doing so is challenging because it requires some level of abstraction : being able to go beyond the details of a concrete problem to connect to a part CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Hope et al . Query Example results Mechanism : light . Purpose : NOT light Billiard laser instructor ( projector ) Mechanism : solar energy . Purpose : NOT generating power Light bulbs with built - in solar chips . Mechanism : water . Purpose : NOT cleaning , NOT drinking A lighter that burns hydrogen generated from water and sunlight . Mechanism : RFID . Purpose : NOT locating , NOT tracking A digital lock for your luggage with RFID access . Mechanism : light . Purpose : cleaning A UV box to clean and sanitize barbells at the gym . Table 1 : Scenarios and example results retrieved by our FineGrained - AVG method . Queries reflect non - trivial uses of mechanisms ( e . g . , using water not for drinking / cleaning , retrieving a lighter running on hydrogen from water and sunlight ) . of the design space that may look dissimilar on the surface , but has abstract similarity . Numerous studies in engineering and cognitive psychology have shown the benefits of problem abstractions for ideation [ 32 , 34 , 45 , 60 , 64 , 98 , 99 ] . However , these studies either involve non - scalable methods ( relying on highly - structured annota - tions , or on crowd - sourcing ) or simple , syntactical pattern - matching heuristics incapable of capturing deeper abstract relations . In [ 52 ] ( aggregate - vectors baseline from the previous section ) , crowdwork - ers were given a product description from the Quirky database , and asked to come up with ideas for products that solve the same prob - lem in a different way . Aggregate vectors representing purpose and mechanism were used to find near - purpose , far - mechanism analo - gies . Thus , finding analogies relied on having a given mechanism to control for structural distance . Unlike [ 52 ] , in our setup we assume a more realistic scenario where we are given only a short problem description \u2013 e . g . , generat - ing power for a phone , reminding someone to take medicine , folding laundry \u2013 and aim to find inspirational stimuli [ 45 ] in the \u201csweet spot\u201d for creative ideation \u2013 structurally related to the given problem , not too near yet also not too far [ 33 ] . Functional Concept Graph . To address this challenge , we build a tool inspired by functional modeling [ 51 ] , which we call a Func - tional Concept Graph . A functional model is , roughly put , a hierar - chical ontology of functions and ways to achieve them , and is a key concept in engineering design . Such models are especially useful for innovation , allowing problem - solvers to break out of a fixed overly - concrete purpose or mechanism and move up and down the hierarchy . Despite their great potential , today\u2019s functional models are constructed manually , and thus do not scale . While automati - cally inducing full abstraction hierarchies / ontologies of functional properties of real - world products remains a daunting challenge , in our approach we construct a rough approximation \u2014 simple enough to extract automatically from noisy product texts , while still being useful for exploring the design space and suggesting inspirations to users . Specifically , in our approach Functional Concept Graphs consist of nodes corresponding to purposes or mechanisms , and edges reflect semantic relatedness that is not guaranteed to directly encode abstraction . We build this graph by observing fine - grained co - occurrences of concepts appearing together in products , using rule - mining to infer which concept is likely to be more general to ( roughly ) capture different levels of abstraction . For example , Figure 7 shows an actual subgraph from our auto - matically constructed functional concept graph related to electricity , power and charging . Products that mention certain purposes ( e . g . , \u201ccharge your phone \" ) will often mention other , structurally related problems that could be more general / abstract ( e . g . , \u201cgenerate power \" ) Figure 7 : An example of our learned functional concept graph extracted from texts . Mechanism in green , purpose in pink . Ti - tles are tags nearest to cluster centroids ( redacted to fit ) . or more specific ( \u201cwireless phone charging \" ) , resulting in edges in our graph ( only high - confidence edges are shown ) . A designer could go from the problem of charging batteries to the more general prob - lem of generating power , and from there to another branch ( e . g . , solar power and mechanical stored energy ) , to get inspired by structurally related ideas . 5 . 1 Building a Functional Concept Graph We develop a method to infer this representation from co - occurrence patterns of the fine - grained spans of text . Naively looking for co - occurrences of problems may yield inspirations too near to the orig - inal \ud835\udc5d \ud835\udc56 , as many frequently co - occurring purposes tend to be very similar , while we are interested discovering the more abstract re - lations . In addition , raw chunks of text extracted from our tagging model have countless variants that are not sufficiently abstract and are thus sparsely co - occurring . We thus design our approach to en - courage abstract inspirations . As an overview of our approach before presenting the technical details , we take the following two steps : I . Concept discretization . Intuitively , nodes in our graph should correspond to groups of related spans ( \u201ccharging\u201d , \u201ccharging the battery\u201d , \u201ccharging a laptop\u201d ) . To achieve this , we take all purpose and mechanism spans \u02c6 P , \u02c6 M in the corpus , extracted using our GCN model , and cluster them ( separately ) , using pre - trained vector representations . We refer to the clusters C \ud835\udc5d , C \ud835\udc5a as concepts . II . Relations . We employ rule - mining [ 80 ] to discover a set of re - lations R between concepts ( see \u00a7 5 . 2 for implementation details ) . Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Relations are Antecedent = \u21d2 Consequent , with weights correspond - ing to rule confidence . To illustrate our intuition , suppose that when \u201cprevent head injury\u201d appears in a product description , the condi - tional probability of \u201csafety\u201d appearing too is large ( but not the other way around ) . In this case , we can ( weakly ) infer that preventing head injuries is a sub - purpose of \u201csafety\u201d . Indeed , manually observing the purpose - purpose edges , the one - directional relations captured are often sub - purpose , and the bi - directional ones often encode abstract similarity . Similarly , for mechanism concepts the one - directional relations are often part of ( \u201ccell phone\u201d and \u201cbattery\u201d ) , and bi - directional are mechanisms that co - occur often . For pairs of purpose and mechanism concepts , the relation is often functionality ( \u201ccharger\u201d , \u201ccharge\u201d ) . Exploring more relations is left for future work . 5 . 2 Study Design & Implementation Next , we set out to test the utility of the functional concept graph in an ideation task . In our setup participants are given problems ( e . g . , reminding people to take their medication in the morning ) and are asked to think of creative solutions . Participants were also given a list of potential inspirations , grouped into boxes , and were instructed to mark whether each was novel and helpful . They were also encouraged to explain the solution it inspired . Figure 8 shows our interface . In this example , seeing inspirations about health monitors caused one user to suggest monitoring the person to find the best time to remind them to take medicine ; see - ing inspirations about coffee caused them to suggest integrating medicine reminders into coffee machines . To create a set of seed problems , a graduate student mapped between problems from WikiHow . com ( a website of how - to guides ) to purposes in our data . Using this source allowed us to collect real - world problems that are broadly familiar , with succinct and self - explanatory titles that do not require further reading to understand . The student was tasked with confirming that our Quirky dataset contains idea descriptions that mention these problems . For a given problem in WikiHow ( how to remember to take medication ) , they performed keyword search over 17 \ud835\udc3e purpose spans gleaned by our model from Quirky , and found matching spans ( morning medicine reminder ) . We use those matching spans as our seed problem descrip - tion given to users ( purple text in Figure 8 ) . We collect 25 problems this way . Table 2 shows more examples , such as Tracking distance walked , folding laundry or sensing dryness level . Inspirations are other purpose spans from our dataset ( see Table 2 ) , selected automatically using our approach or baseline approaches . Our Method . For our approach , we construct a functional concept graph as in Section 5 . 1 . To cluster related spans into concept nodes , we explore two common and powerful vector representations of spans to capture semantic similarity : \u2022 GloVe [ 81 ] pre - trained word embeddings , averaged across tokens . \u2022 BERT - based [ 84 ] contextualized vectors that have been fine - tuned for semantic similarity tasks 4 . 4 We use RoBERTa - large - STS - SNLI , available at github . com / UKPLab / sentence - transformers . Figure 8 : A snippet from our ideation interface for \u201cmorn - ing medicine reminder\u201d . Users see inspirations grouped into boxes . Each box is supposed to represent a concept \u2013 a clus - ter of related spans as found by our method or by the baselines ( see \u00a7 5 . 1 ) . Users indicate which inspirations were useful , and what ideas they inspired . For example , seeing \u201creal time health checker\u201d inspired one user to suggest a monitoring device for finding the best time for reminding to take the medicine . We cluster the spans using K - Means + + 5 [ 8 ] . We then apply the Apriori algorithm 6 to automatically mine association rules between clusters , [ 80 ] and use the confidence metric to select the top rules 7 . To use the mined rules between purpose nodes ( clusters ) for select - ing inspirations shown to users , we start from the purpose node corresponding to the given problem and take its consequents ; as explained earlier , this captures a weak signal of abstract similarity . Some of these nodes contain tens of spans in them . Thus , we also explore two approaches to \u201csummarize\u201d each concept cluster with representative spans displayed to users \u2013 one that attempts to summarize the cluster independently of the seed problem , and one that takes the seed problem into account : \u2022 TextRank [ 74 ] . We construct a graph where nodes are the spans in a cluster and edges represent textual similarity . We run PageRank [ 78 ] on this graph , selecting the top \ud835\udc3e spans to present . \u2022 Nearest spans . Following the findings in [ 33 ] , select the top \ud835\udc3e spans in C \ud835\udc5d that are nearest to the query \ud835\udc5d \ud835\udc56 . ( For both approaches , we use \ud835\udc3e = 5 ) . Baselines . 8 \u2022 Purpose span similarity . Given a problem \ud835\udc5d \ud835\udc56 , we find the \ud835\udc3e = 5 nearest purpose spans of text in our corpus ( out of 17 \ud835\udc3e purposes ) . We experiment with the same two vector representations used by our approach : GloVe and BERT . This method is similar to applying the methodology in [ 52 ] to our setting , where in our setting we are given only a problem \ud835\udc5d \ud835\udc56 and no mechanism \ud835\udc5a \ud835\udc56 is available to control for structural distance . While this approach relies on our model for extracting purpose spans , we consider it a baseline to study the added value of our hierarchy . 5 \ud835\udc3e = 250 selected automatically with elbow - based criteria on silhouette scores . 6 http : / / www . borgelt . net / pyfim . html . 7 We use the top 3 rules in our experiment . 8 We note that all methods and baselines include both single and multi - word spans of text as inspirations , ensuring users are blind to the condition . CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Hope et al . Problem Inspirations Rater explanation Track distance walked Protect children Get ideas from devices that keep track of children Folding laundry Store toilet paper Roll laundry around a tube instead of folding Dispense medicine Pet bowl that keeps ants away Based on pet bowls that can dispense food during the day Sense dryness level Voltage reading Use electric current to measure water level ( safely ) Waterproof Ideas from sensors in waterproof devices Temperature reading Morning medicine reminder Schedule coffee , coffee alarm Alarm clock with coffee and medicine reminders Send vital data , real - time health checker Health trackers to tell if medicine not taken , alert accordingly Heart rate monitoring , con - tinuously monitor glucose Find the best time to take medicine Table 2 : Example inspirations and explanations given by human evaluators . \u2022 Linguistic abstraction . We use the WordNet [ 75 ] lexical data - base to extract hypernyms ( for each token in \ud835\udc5d \ud835\udc56 ) , in order to capture potential abstractions . WordNet is often used in similar fashion for design - by - analogy studies [ 42 , 45 , 64 ] . \u2022 Random concepts . Random inspirations are often considered as a baseline in ideation studies since diversity of examples is a known booster for creative ability [ 52 ] . For each task , we select a random cluster from C \ud835\udc5d and display its TextRank summary . Study Participants . We recruit 10 raters to evaluate the inspirations , via university mailing lists . 8 raters were engineering graduate stu - dents , and the remaining two raters included a senior engineering professor and an architect . This cohort is intended to reflect a target user base of people interested in innovation and involved in creative inventive thinking as part of their work . Rating Collection . In our study , each method generated \ud835\udc3e = 5 spans ( concept summaries ) , which are grouped and displayed together in a box ( see Figure 8 ) . For each problem a rater views 8 boxes in randomized order , to avoid bias . Raters were instructed to mark inspirations they consider useful and relevant for solving a given problem , while being not about the same problem . Raters were also encouraged to write comments , especially for non - trivial cases which they found of interest ( see Table 2 ) . In total , raters viewed 2584 boxes , or 12920 purpose descriptors . 5 . 3 Results Analyzing inspirations . Table 2 and Figure 8 show examples of problems , inspirations and user explanations from our study . For in - stance , users facing the \u201cmorning medicine reminder\u201d problem were presented with nearby concepts in the Functional Concept Graph that included health monitoring and coffee machines . To explore why these concepts are connected in our graph and why they are potentially useful as inspirations , we make use of the direct inter - pretability of our approach . We examine the purpose co - occurrences from which the Functional Concept Graph was constructed . Figure 9 shows the subgraph with concept nodes of Making hot drinks , alerting / reminding , health monitoring , medicine delivery , and edges representing products in which two adjacent purposes were co - mentioned ( e . g . , a \u201ccoffee machine alarm\u201d product that men - tioned the purposes of making hot drinks and alerting / reminding , or Alert / remind Making hot drinks Medicine delivery Medical monitoring Coffee machine alarm Smart medicine injector Smart medicine injector pill reminder Smart medicine injector Figure 9 : Excerpt from our Functional Concept Graph . Nodes represent concepts ( clusters of purposes ) . To give intuition for how edges were created , they are annotated with example prod - ucts containing spans from both concepts . All nodes and edges in this figure were automatically constructed and used to create the user - facing inspirations shown in Figure 8 . This figure pro - vides a graphic illustration ( not shown to users ) explaining how the boxes in Figure 8 are generated , with node names provided here by us for readability . The problem of \u201cmedicine morning reminder\u201d is mapped ( via embedding ) to the Alert / remind con - cept cluster ( as named by us ) , which is linked to the concepts of medical monitoring and making hot drinks through products such as \u201csmart medicine injector\u201d and \u201ccoffee machine alarm\u201d ( among others , not displayed in the figure ) . These links serve as the source for inspirations in our study , as seen in Figure 8 . a \u201csmart medicine injector \" that mentioned both alerting / reminding and medicine delivery ) . This explains why the concepts are nearby in the graph , as there are multiple products in our dataset that share purposes from both concepts . For example , a \u201cpill reminder\u201d product refers to the problem of forgetting to take medicine at prescribed times ( Sends notification if you forgot to take your AM or PM meds ) , while a \u201csmart injec - tor\u201d device administers medicine on set time intervals . At the same time , both of these products mention purposes of medicine delivery . Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA When our graph construction algorithm observes enough similar co - occurrence patterns between the concepts of alerting and medicine delivery , across multiple products , an edge is added between the two in the graph . Similarly , an \u201cAlarm coffee maker\u201d product mentions the purposes of time management and making coffee at a set time as well as alerting when the coffee is ready , explaining how it emerges as a potential inspiration in our graph . This type of linkage or overlap between an original problem space and inspiration problems helps get at a sweet - spot of innovation [ 16 ] by finding ideas that are not too near and not too far from the original problem , helping users break out of fixation as discussed earlier in this section . Users in our study used these inspirations to come up with a tracker that alerts the user at the best time to take a medicine and a coffee machine reminding the user to take their medication with their morning coffee . Those creative directions demonstrate the utility of the Functional Concept Graph for exploring the design space . Quantitative results . Figure 10 shows the results of the user study . On the left , we show the proportion of inspirations ( individual spans ) selected by at least two raters , for each method . Our approach sig - nificantly outperforms all the baselines . The effect is particularly pronounced for the BERT - based approach , with 51 % of inspirations found useful , while the best baseline reaches less than 30 % . Interest - ingly , for both BERT and GloVe representations , the Nearest - span summarization approach fares better , potentially due to striking a balance between being too far / near the initial problem \ud835\udc5d \ud835\udc56 . Figure 10 ( right ) shows the proportion of inspiration boxes that got at least 2 individual inspirations marked ( by at least 2 raters ) . This metric measures the effect of a box as one unit , as each box is meant to represent a coherent cluster . Our method is able to reach 62 % , while the best baseline ( GloVe search on purpose spans ) yields only 39 % . Again , the nearest - span summarization is prefered to TextRank . Importantly , for both individual inspiration spans and inspirations boxes , 51 % - 62 % are rated as useful \u2013 high figures considering the challenging nature of the task . 6 DISCUSSION AND CONCLUSION In this paper we introduced a novel span - based representation of ideas in terms of their fine - grained purposes and mechanisms and used it to develop new tools for creative ideation . We trained a model to extract spans from a noisy , real - world corpus of products . We used this representation to support human creativity in two applications : expressive search for alternative , uncommon uses of products , and generating a graph to help problem - solvers explore the design space around their problem . In both ideation studies , we were able to achieve high accuracies , significantly outperform baselines and help boost user creativity . 6 . 1 Limitations While our results showed the promise of a functional aspect - based representation , and demonstrated potentially feasible technical ap - proaches for extracting this representation from unstructured text , the approach has several limitations . Challenging Annotation Task for Crowds . First , the annotation task proved to be somewhat difficult for crowdworkers , and the out - puts were noisy . One direction for future work would be to explore weak supervision approaches to augment annotation . One issue that might exacerbate the problem is that sometimes the boundary be - tween purpose and mechanism is fuzzy , and it is genuinely difficult to tell how to annotate the span . Limited Functional Schema . In a similar vein , it might be interest - ing to explore more expressive schemas , containing elements other than just purpose and mechanism ( similar to [ 12 ] ) . One particularly useful element to add might be context / constraints ( e . g . , nanoscale ) , to restrict the search space to feasible solutions . Surface Form Abstraction . Another limitation of our approach is that our functional aspects ( and resulting embeddings ) remain quite closely anchored to the original texts . This limits their ability to be used to match across domains , to make connections such as inspiring new optimization approaches by analogy to \" heating / cooling \" sched - ules in metallurgy . Achieving abstraction to match across distant domains without burdening the user with a combinatorial explosion of noisy matches remains an open problem . We wonder if abstract - ing key objects or entities in a purpose functional aspect \u2014 such as a more automated approach to replacing objects with their \" com - monsense \" properties \u2014 might be more feasible than attempting to abstract from an entire product description or abstract , given that the chunk is already a rich signal of the product\u2019s functional meaning . 6 . 2 Future Work and Broader Implications Moving to future directions , we are excited about the potential of functional aspects to lead to advances in the interpretability of content - based recommender systems in these complex domains . Keywords are inherently interpretable , but are limited in their ca - pacity to support crossing knowledge boundaries ; and until now , embedding - based approaches ( e . g . , [ 52 ] ) have not always led to interpretable justifications for matches . Functional aspects could provide the basis of not just more powerful search operators , but also more interpretable results and feedback loops . Deeper Functional Graph Exploration . A key component for the above might be expanding on our use of functional graphs , built from the extracted functional aspects . In our experiments , we used our functional concept graph to retrieve inspirations from \" around \" the problem . But what would it take to be able to explore this graph ? Could we identify and optimize for latent coordinates in the func - tional space , moving \" up \" and \" down abstraction levels , or \" across \" sibling nodes in a functional graph ? Taking inspiration from network perspectives on ideation [ 11 , 44 , 94 ] , could we retrieve interesting \" lineages \" of ideas , or compute the potential inspiration value of functional aspects based on network connectivity metrics ? Could we combine these content - based functional aspects with measures of use ( e . g . , citations ) , to enrich approaches that combine content - and social - based signals , such as literature - based discovery [ 93 , 95 ] ? Identifying Overlap and Gaps Across Fields . These approaches rely on identifying interesting overlaps in concepts that simultane - ously coincide with disjunctions in citations , as signals of potentially impactful \" undiscovered public knowledge \" : a persistent challenge is how to define \" concepts \" - keyword or unstructured text approaches can lead to combinatorial explosions of noise to sift through , and con - trolled vocabulary ( e . g . , MEDLine ) can help increase the signal to noise ratio , but are only available in specialized circumstances [ 89 ] . CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Hope et al . Figure 10 : Inspiration user study results . Left : Proportion of inspirations selected by at least 2 raters , per condition . Right : Proportion of boxes ( clusters ) with at least 2 spans marked by \u2265 2 raters . Functional aspects might be a useful bridge between unstructured text and controlled vocabularies for identifying points of overlap and disjunction between different fields , accelerating the discovery of gems hidden in plain sight . Functional aspects for Collaborative Ideation . Future work could also explore new interactions in collaborative and crowd innovation that might be enabled by the ability to quickly extract functional aspects in idea corpora . The source of our primary dataset here , Quirky , was actually a crowd innovation platform . HCI research on these platforms have begun to emphasize moving away from mere \" selection \" of best ideas from large samples of ideas , towards sup - porting generative collaboration over ideas . Open problems include synthesizing major themes in large - scale corpora of user - generated ideas and identifying gaps in the idea space [ 13 , 68 , 90 ] , as well as supporting intelligent matching and structuring ways for crowd innovators to collaborate and build on each others\u2019 ideas [ 14 , 69 ] between crowd innovators . We are excited about the potential for functional aspects to assist with these functions , as a complement to other approaches like crowd - powered synthesis [ 7 , 18 , 43 , 91 ] . Here , too , the potential for functional aspects to be highly inter - pretable could power novel explorations of mixed - initiative systems for augmenting collaborative ideation at scale [ 67 , 91 ] . Mapping of Design Spaces . 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A BiLSTM - CRF [ 53 ] neural network , a common baseline approach for NER tasks , enriched with semantic and syn - tactic input embeddings known to often boost performance [ 101 ] . We first pass the input sentence x = ( \ud835\udc65 1 , \ud835\udc65 2 , . . . , \ud835\udc65 \ud835\udc47 ) through an embedding module resulting in v 1 : \ud835\udc47 , v \ud835\udc56 \u2208 R \ud835\udc51 \ud835\udc52 , where \ud835\udc51 \ud835\udc52 is the embedded space dimension . We adopt the \u201cmulti - channel\u201d strat - egy as in [ 101 ] , concatenating input word embeddings ( pretrained GloVe vectors [ 81 ] ) with part - of - speech ( POS ) and NER embed - dings . We additionally add an embedding corresponding to the incoming dependency relation . The sequence of token embeddings is then processed with a BiLSTM layer to obtain contextualized word representations h ( 0 ) 1 : \ud835\udc47 , h \ud835\udc56 \u2208 R \ud835\udc51 \u210e , where \ud835\udc51 \u210e is the hidden state dimension . The outputs are fed into a linear layer \ud835\udc53 to obtain per - word tag scores \ud835\udc53 (cid:16) h ( \ud835\udc3f ) 1 (cid:17) , \ud835\udc53 (cid:16) h ( \ud835\udc3f ) 2 (cid:17) , . . . , \ud835\udc53 (cid:16) h ( \ud835\udc3f ) \ud835\udc47 (cid:17) . These are used as inputs to a conditional random field ( CRF ) model which maximizes the tag sequence log likelihood under a pairwise transition model between adjacent tags [ 5 ] . \u2022 Pooled Flair . A pre - trained language model [ 4 ] based on contex - tualized string embeddings , recently shown to outperform pow - erful approaches such as BERT [ 22 ] in NER and POS tagging tasks and achieve state - of - art results . Flair 9 uses a character - based language model pre - trained over large corpora , combined with a memory mechanism that dynamically aggregates embeddings of each unique string encountered during training and a pooling op - eration to distill a global word representation . We follow [ 4 ] and concatenate pre - trained GloVe vectors to token embeddings , add a CRF decoder , and freeze the language - model weights rather than fine - tune them [ 22 , 82 ] . \u2022 GCN . We also explore a model - enrichment approach with syn - tactic relational inputs . We employ a graph convolutional network ( GCN ) [ 58 ] over dependency - parse edges [ 101 ] . GCNs are known to be useful for propagating relational information and utilizing syntactic cues [ 71 , 101 ] . The linguistic cues are of special relevance and interest to us , as they are known to exist for purpose / mechanism mentions in texts [ 32 ] . We used a GCN with same token embeddings as in the BiLSTM - CRF baseline , with a BiLSTM layer for sequential context and a CRF decoder . For the graph fed into the GCN , we use a pre - computed syntactic edges with dependency parsing : For sentence x 1 : \ud835\udc47 , we convert its dependency tree to A \ud835\udc60\ud835\udc66\ud835\udc5b where A \ud835\udc60\ud835\udc66\ud835\udc5b\ud835\udc56\ud835\udc57 = 1 for any two tokens \ud835\udc65 \ud835\udc56 , \ud835\udc65 \ud835\udc57 connected by a dependency edge . We also add self - loops A \ud835\udc60\ud835\udc52\ud835\udc59\ud835\udc53 = \ud835\udc3c ( to propagate from h ( \ud835\udc59 \u2212 1 ) \ud835\udc56 to h ( \ud835\udc59 ) \ud835\udc56 [ 101 ] ) . Following [ 101 ] , we normalize activations to reduce bias toward high - degree nodes . For an \ud835\udc3f - layer GCN , denoting h ( \ud835\udc59 ) \ud835\udc56 \u2208 R \ud835\udc51 \u210e to be the \ud835\udc59 - th layer output node , the GCN operation can be written as \u210e ( \ud835\udc59 ) \ud835\udc56 = \ud835\udf0e (cid:169) (cid:173) (cid:171) \u2211\ufe01 \ud835\udc5f \u2208R \uf8ee\uf8ef\uf8ef \uf8ef\uf8ef \uf8f0 \ud835\udc5b \u2211\ufe01 \ud835\udc57 = 1 A \ud835\udc5f\ud835\udc56\ud835\udc57 W ( \ud835\udc59 ) \ud835\udc5f \u210e ( \ud835\udc59 \u2212 1 ) \ud835\udc57 / \ud835\udc51 \ud835\udc5f\ud835\udc56 + b ( \ud835\udc59 ) \ud835\udc5f \uf8f9\uf8fa\uf8fa \uf8fa\uf8fa \uf8fb (cid:170) (cid:174) (cid:172) 9 https : / / github . com / flairNLP / flair Scaling Creative Inspiration with Fine - Grained Functional Aspects of Ideas CHI \u201922 , April 29 - May 5 , 2022 , New Orleans , LA , USA Figure 11 : Schema of our GCN model . where R = { syn , self } , \ud835\udf0e is the ReLU activation function , W ( \ud835\udc59 ) \ud835\udc5f is a linear transformation , b ( \ud835\udc59 ) \ud835\udc5f is a bias term and \ud835\udc51 \ud835\udc5f\ud835\udc56 = (cid:205) \ud835\udc47\ud835\udc57 = 1 A \ud835\udc5f\ud835\udc56\ud835\udc57 is the degree of token \ud835\udc56 w . r . t \ud835\udc5f . In the GCN architecture , \ud835\udc3f layers corre - spond to propagating information across \ud835\udc3f - order neighborhoods . We set the contextualized word vectors h ( 0 ) 1 : \ud835\udc47 to be the input to the GCN , and use h ( \ud835\udc3f ) 1 : \ud835\udc47 as the output word representations . Figure 11 illustrates the GCN model architecture . Similarly to [ 71 ] , we do not model edge directions or dependency types in the GCN layers , to avoid over - parameterization in our data - scarce setting . We also attempted edge - wise gating [ 71 ] to mitigate noise propagation but did not see improvements , similarly to [ 101 ] . In our experiments , we followed standard GCN training proce - dures . Specifically , we base our model on the experimental setup detailed in [ 101 ] ( see also the authors\u2019 code which we adapt for our architecture , at https : / / github . com / qipeng / gcn - over - pruned - trees ) . We pre - process the data using the spaCy ( https : / / spacy . io ) package for tokenization , dependency parsing , and POS / NER - tagging . We use pretrained GloVE embeddings of dimension 300 , and NER , POS and dependency relation embeddings of size 30 each , giving a total embedding dimension \ud835\udc51 \ud835\udc52 = 390 . The bi - directional LSTM and GCN layers\u2019 hidden dimension is \ud835\udc51 \u210e = 200 , with 1 hidden layer for the LSTM . We find that the setting of 2 hidden layers works best for the GCNs . We also tried training with edge label information based on syntactic relations , but found this hurts performance . The training itself was carried out using SGD with gradient clipping ( cutoff 5 ) for 100 epochs , selecting the best model on the development set . For the Pooled - Flair approach [ 4 ] , we use the FLAIR framework [ 3 ] , with the settings obtaining SOTA results for CONLL - 2003 as in [ 4 ] ( see https : / / github . com / flairNLP / flair / blob / master / resources / docs / EXPERIMENTS . md ) . We also experiment with non - pooled embeddings and obtain similar results . We experiment with initial learning rate and batch size settings described in [ 4 ] , finding 0 . 1 and 32 to work best , respectively .", + "carman2023structures": "STRUCTURAL BIOLOGY Structures of the free and capped ends of the actin filament Peter J . Carman 1 , 2 \u2020 , Kyle R . Barrie 1 , 2 \u2020 , Grzegorz Rebowski 1 , Roberto Dominguez 1 , 2 * The barbed and pointed ends of the actin filament ( F - actin ) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange , including CapZ at the barbed end and tropomodulin at the pointed end . We describe cryo - electron microscopy structures of the free and capped ends of F - actin . Terminal subunits at the free barbed end adopt a \u201c flat \u201d F - actin conformation . CapZ binds with minor changes to the barbed end but with major changes to itself . By contrast , subunits at the free pointed end adopt a \u201c twisted \u201d monomeric actin ( G - actin ) conformation . Tropomodulin binding forces the second subunit into an F - actin conformation . The structures reveal how the ends differ from the middle in F - actin and how these differences control subunit addition , dissociation , capping , and interactions with end - binding proteins . A ctin is the most abundant cytosolic pro - tein in eukaryotes . The cellular pool of actin is approximately evenly divided between monomeric ( G - actin ) and fil - amentous ( F - actin ) forms ( 1 ) . G - actin consists of four subdomains which , based on their disposition in F - actin , form the outer ( subdomains 1 and 2 ) and inner ( subdomains 3 and 4 ) domains ( 2 ) ( Fig . 1A ) . Two clefts separate the outer and inner domains : the nucleotide - binding cleft and , opposite of it , a hydrophobic cleft that mediates interactions with numerous actin - binding proteins and withactin itselfinthe filament ( 3 ) . Adenosine triphosphate ( ATP ) hydrolysis by actin is one of several factors that control the G - to F - actin transition . Although G - actin is a slow adeno - sine triphosphatase ( ATPase ) , ATP hydrolysis is accelerated manyfold in F - actin as a result of a rotation of the outer domain relative to the inner domain , which produces a flatter subunit conformation and reorients side chains and water molecules in the catalytic site for hydro - lysis ( 4 , 5 ) . In this way , F - actin acquires polarity , i . e . , structural and kinetic asymmetry : ATP - actin binds to the barbed ( or + ) end of F - actin , followed by fast ATP hydrolysis , slow g - phosphate release , and dissociation of adeno - sinediphosphate ( ADP ) \u2013 actinfromthepointed ( or \u2212 ) end ( 1 , 6 ) . Unbound monomers undergo rapid ADP - to - ATP exchange and become ready to rejoin the barbed end . This process , known as filament treadmilling , is regulated by pro - teins that either accelerate subunit addition at the barbed end ( formins , Ena / VASP ) ( 7 \u2013 9 ) , accelerate subunit dissociation from the pointed end ( cyclase - associated protein and cofilin ) ( 10 , 11 ) , or cap the ends to stop subunit exchange . In muscle sarcomeres andnonmuscle cells , the main capping proteins are CapZ ( also known as capping protein or CP ) ( 12 ) at the barbed end and tropomodulin ( Tmod ) at the pointed end ( 13 ) . Recent cryo - electron microscopy ( cryo - EM ) helical reconstructions of F - actin in different nucleotide states [ ADP , ADP - Pi ( inorganic phosphate ) , and ATP analogs ] provide high - resolution insightsinto thestructure ofsubunits in the middle of F - actin and the conformational changesthatleadtoATPhydrolysisuponpolym - erization ( 5 , 14 , 15 ) . However , the structure of F - actin changes little as a function of the bound nucleotide or divalent cation ( Ca 2 + or Mg 2 + ) . Thus , although the rate constants for the re - actions of ATP - and ADP - actin addition and dissociation at the ends of F - actin were deter - mined ~ 40 years ago ( 1 , 6 ) , the structural bases of kinetic asymmetry at the ends of F - actin re - main poorly understood . On the basis of single - particle cryo - EM structures , we reveal how the barbed and pointed ends are conformationally different and primed for subunit addition and dissociation , respectively , and how CapZ and Tmod block subunit exchange . Results Cryo - EM structures of the filament ends We used single - particle cryo - EM to determine structures of the ends of F - actin . Actin alone tends to form long filaments in cryo - EM mi - crographs , which is ideal for helical recon - struction but produces few filament ends per micrograph . To increase the number of ends , we used an optimized mixture of CapZ , Tmod , tropomyosin , and actin that produced shorter filaments and thus , more ends per micrograph ( materials and methods ; fig . S1 , A to C ) . Filament end particles were harvested with the program Topaz , using a particle - picking pipeline based on custom - trained neural networks ( 16 ) . Starting from a subset of manually picked ends , iterations of Topaz training and particle picking resulted in a dataset of 1 , 102 , 793 particles . During data processing , this dataset diverged into three subclasses that produced three - dimensional reconstructionsofthefreepointedend , Tmod - capped pointed end , and CapZ - capped barbed end at resolutions of 2 . 84 , 3 . 26 , and 2 . 79 \u00c5 , respectively ( fig . S1D ) . Free barbed ends were not observed , consistent with CapZ \u2019 s subna - nomolar affinity for the barbed end and long half - time for dissociation ( 12 ) . Datasets were collected without CapZ to obtain the structure of the free barbed end . Attempts to shorten the filaments by shearing mostly failed because filaments reannealed fast before vitrification with or without Tmod . A total of 413 , 076 par - ticles were collected from two datasets , which , combined with a different data processing strategy , yielded a 3 . 30 - \u00c5 resolution structure of the free barbed end ( fig . S2 ) . A 2 . 26 - \u00c5 resolution structure of the middle of F - actin was obtained by helical reconstruction and used as reference for comparisons . This struc - ture issimilar to other structuresofthemiddle of F - actin , with a C a root mean square devia - tion of 0 . 35 \u00c5 with the highest - resolution structure ( 2 . 24 \u00c5 ) reported thus far ( 5 ) . Poly - merization started from Mg 2 + - ATP - actin , but subunits in all the structures described here contain bound ADP , including subunits at the barbed and pointed ends , indicating that hydrolysis and g - phosphate release occurred in the ~ 3 hours that elapsed before vitrifica - tion . The helical rise and twist of subunits is also similar in all the structures . Data collec - tion , refinement , and structure validations are shown in figs . S3 to S7 and table S1 . Tropomyosin density was only observed in a subset ( ~ 13 % ) of the Tmod - capped pointed end subclass , resulting in a 4 . 8 - \u00c5 resolution reconstruction that shows tropomyosin inter - acting with Tmod on both sides of F - actin ( fig . S8A ) . The median length of the filaments in micrographswas48 nm , which corresponds to ~ 8 . 7 actin protomers and ~ 1 . 25 tropomyosin molecules along the long - pitch helix ( fig . S1B ) . The low occupancy of tropomyosin is therefore consistent with the extremely low ( millimolar ) affinity of single tropomyosin molecules for F - actin ( 17 ) . That tropomyosin was only ob - served in conjunction with Tmod probably re - sults from the interaction of these two proteins at the pointed end , which mutually enhances their affinities for F - actin ( 13 , 18 ) . The low res - olution of the Tmod - tropomyosin map pre - cludes a detailed analysis of this interaction . The barbed end With one exception , the conformation of the two terminal subunits at the barbed end cor - responds to the classical flat conformation of subunits in the middle of the filament ( 4 , 5 , 14 , 15 ) ( Fig . 1 , A to C , movie S1 , and table S2 ) . Three main features characterize this conformation : ( i ) a ~ 20\u00b0 scissorlike rota - tion of the outer domain relative to the inner RESEARCH Carman et al . , Science 380 , 1287 \u2013 1292 ( 2023 ) 23 June 2023 1 of 6 1 Department of Physiology , Perelman School of Medicine , University of Pennsylvania , Philadelphia , PA , USA . 2 Biochemistry and Molecular Biophysics Graduate Group , Perelman School of Medicine , University of Pennsylvania , Philadelphia , PA , USA . * Corresponding author . Email : droberto @ pennmedicine . upenn . edu \u2020 These authors contributed equally to this work . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023 domain that flattens the actin protomer and changes the overall conformation of both the nucleotide and hydrophobic clefts ( Fig . 1A ) ; ( ii ) the D loop ( residues R39 to K50 ) adopts an extended conformation to insert into the hydrophobic cleft of the subunit immediately above ; and ( iii ) residues in the hydrophobic cleft change conformation to host the D loop of the subunit immediately below . Specifi - cally , the W loop ( I165 to P172 ) and the C terminus of actin ( P367 to F375 ) form a pincer - like structure that grasps the D loop of the subunit below . The latter change does not occur in barbed - end subunits because they do not interact with the D loop ( Fig . 1 , D and E ) . A cryo - EM structure of the CapZ hetero - dimer ( Fig . 2 , A and B ) at the barbed end was recently reported ( 19 ) , but CapZ was only partially visualized and at low resolution ( 6 to 7 \u00c5 ) . CapZ was also visualized at the barbed end of the actin - related protein 1 ( Arp1 ) minifilament of dynactin ( 20 ) . Yet , Arp1 shares only 51 % sequence identity with actin and its interaction with CapZ differs somewhat . Com - pared with these structures , the 2 . 79 - \u00c5 resolu - tion structure described here ( Fig . 2C ) displays large differences in the overall disposition and specific interactions of CapZ at the barbed end , and the relative orientation of actin ( or Arp1 ) subunits in the filament ( fig . S9A ) . As in the structure of the free barbed end , the terminal and penultimate subunits in the CapZ - capped structure have an F - actin conformation with a G - actin \u2013 like W loop ( table S2 ) . However , the short - pitch pair formed by these two subunits is splayed apart by ~ 1 . 5 \u00c5 compared with a short - pitch pair in the middle of F - actin ( Fig . 2D ) . Subunit splaying results from a ~ 2\u00b0 rotation of the terminal subunit away from the longitudi - nal axis of F - actin , which may be induced by CapZ binding but is likely favored by weakened contacts between the two long - pitch helices at the barbed end . Indeed , the interstrand plug ( Q263 to G273 ) provides the main contact be - tween the long - pitch helices ( or strands ) of F - actin ( 2 ) . In the middle of the filament , the plug from one subunit binds at the interface between two subunits of the opposite strand , a pattern that is broken at the barbed end where the plug of the terminal subunit contacts only one subunit of the opposite strand ( Fig . 2D ) . CapZ is a heterodimer of related a and b subunits ( Fig . 2A ) ( 12 ) . The heterodimer is described as having a mushroom - like shape , where the mushroom head binds the barbed end ( Fig . 2B ) . CapZ can adopt distinct filament - bound and unbound conformations . Alloste - ric regulators of CapZ , including CARMIL , CD2AP , and CKIP , have a capping - protein interaction ( CPI ) motif that binds around the Carman et al . , Science 380 , 1287 \u2013 1292 ( 2023 ) 23 June 2023 2 of 6 A B C D E Fig . 1 . Free barbed end . ( A ) Schematic representation of the G - to F - actin transition . A scissorlike ~ 20\u00b0 rotation of the outer domain ( subdomains 1 and 2 ) relative to the inner domain ( subdomains 3 and 4 ) produces a flatter conformation of subunits in F - actin . ( B ) Cryo - EM map of the free barbed end at 3 . 30 - \u00c5 resolution . The terminal and penultimate subunits are shown in two different shades of blue . ( C ) The terminal and penultimate subunits ( blue ) adopt the classical F - actin conformation of subunits in the middle of F - actin ( gray ) . Subunits were superimposed based on the inner domain ( surface representation ) to highlight differences in orientation of the outer domain ( ribbon representation ) . ( D ) Fit to the cryo - EM map of the W loop of the terminal subunit ( blue ) versus a subunit from the middle of the filament ( gray ) . ( E ) In the middle of F - actin , the W loop and the C terminus of actin form a pincer - like structure that grasps the D loop of the subunit below ( gray and cyan , respectively ) . This interaction is absent for subunits at the barbed end ( blue ) , resulting in the W loop adopting a G - actin conformation and the C terminus moving by 2 . 0 \u00c5 ( red arrows ) . Single - letter abbreviations for amino acids referenced throughout are as follows : R , Arg ; K , Lys ; I , Ile ; P , Pro ; F , Phe ; Q , Gln ; G , Gly ; E , Glu ; H , His ; L , Leu ; N , Asn ; A , Ala ; T , Thr RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023 mushroom stalk and stabilizes the unbound conformation to promote uncapping ( 21 , 22 ) . The filament - bound state is characterized by a flatter conformation of the mushroom head , resultingfromthedistalendsoftheheadmovingupwardstowardthebarbedend ( 19 , 20 ) . This conformation has also been observed in CapZ complexes with twinfilin - actin ( 23 ) and V - 1 ( 22 ) , a protein that sterically competes with CapZ binding to the barbed end ( fig . S9B ) . In the current structure , CapZ also adopts a flat con - formation , bending by ~ 15\u00b0 from its filament - unboundconformation ( Fig . 2EandmovieS1 ) . Flattening of the mushroom head leads to changes in two long , pseudo - symmetric helices of CapZ a ( E221 to R259 ) and CapZ b ( H209 to L243 ) that run along the barbed - end interface ( Fig . 2F ) . Both helices contain a p bulge , a deformation of the a - helical fold that often plays a functionalrole ( 24 ) . In the barbed end \u2013 bound structure , the bulge in the CapZ b helix travels along the helical axis toward the N terminus by about half a helical turn , which reorients the side chains of N229 , E230 , and I231 at the barbed - end interface . In CapZ a , the p bulge coincides with a kink in the helix , which becomes more pronounced in the filament - bound structure to reorient the side chains of D252 and K256 at the barbed - end interface . Other important elements of the interaction are the a tentacle ( CapZ a residues R260 to A286 ) and the b tentacle ( CapZ b residues R244 to N277 ) ( 25 ) , immediately C - terminal to the p - bulge helices ( Fig . 2G ) . The tentacles are well resolved in our struc - ture and their conformation differs from other filament - bound structures ( 19 , 20 ) , as well as unbound structures in which the tentacles are disordered ( 21 \u2013 23 ) . Both tentacles comprise amphipathic helices that project upwards and present their hydrophobic sides to the hydro - phobic clefts of the terminal ( b tentacle ) and penultimate ( a tentacle ) subunitsofthefilament . The pointed end Unexpectedly , the firsttwo subunits atthe free pointed end adopt aG - actinconformation and their D loops are disordered , as observed in Carman et al . , Science 380 , 1287 \u2013 1292 ( 2023 ) 23 June 2023 3 of 6 A C B F D E G Fig . 2 . CapZ - capped barbed end . ( A ) Domain diagram of CapZ subunits a and b . ( B ) Mushroom - like structure of CapZ and transition between filament - bound and unbound states . In filament - bound CapZ , the mushroom head flattens and the tentacles engage the hydrophobic clefts of barbed - end subunits . ( C ) Cryo - EM map of the CapZ - capped barbed end at 2 . 79 - \u00c5 resolution , showing the terminal and penultimate actin subunits in two shades of blue and CapZ a and CapZ b in pink and magenta , respectively . ( D ) Splaying of the short - pitch pair formed by the terminal and penultimate subunits ( blue ) as compared to a short - pitch pair from the middle of F - actin ( gray ) . The short - pitch pairs were superimposed based on the penultimate subunit to highlight the splaying of the terminal subunit , showing a maximum displacement of 1 . 5 \u00c5 resulting from a rotation of ~ 2\u00b0 ( red arrow ) . Splaying is likely favored by missing contacts of the interstrand plug at the barbed end compared to the middle of F - actin ( dashed red curves ) . ( E ) Comparison of the filament - bound ( pink , magenta ) and unbound ( 22 ) ( gray , PDB code 3AA7 ) structures of CapZ . The superimposition , based on CapZ a , highlights a ~ 15\u00b0 rotation of CapZ b ( red arrow ) that flattens the mushroom head . ( F ) The actin - binding surface of CapZ consists mostly of two antiparallel helices and the a and b tentacles . Comparison of the filament - bound ( pink , magenta ) and unbound ( gray ) structures shows that the helices contain p bulges that change conformation between these two states ( red arrows ) . The tentacles , which are disordered in the unbound structure , project out in the filament - bound structure to engage the two barbed - end subunits . ( G ) Close - up view of CapZ \u2019 s interaction with the barbed end , with the binding interface colored pink ( CapZ a ) and magenta ( CapZ b ) . CapZ residues participating in the interaction are shown and labeled in fig . S9C . RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023 most G - actin structures ( Fig . 3 , A and B , movie S1 , and table S2 ) . However , their hydrophobic clefts host the D loop of subunits immediately below , which imposes changes in the W loop and C terminus characteristic of the F - actin conformation . The opposite was observed at the barbed end , where the last two subunits have an F - actin conformation with G - actin - like features in their hydrophobic cleft ( Fig . 1 ) . We draw three conclusions from these observa - tions . First , theG - toF - actintransitioniscaused by interactions of subdomains 2 and 4 with the subunit immediately above along the long - pitch helix . These interactions impose an F - actin conformation to subunits at the barbed end , whereas their absence at the pointed end allows subunits to revert to the G - actin confor - mation . Second , subunit additionatthe pointed end is conformationally unfavorable , analo - gous to the way G - actin to G - actin interactions are rate limiting during nucleation ( 1 , 26 ) . Third , subunits at the pointed end appear con - formationally primed for dissociation . Contrary to CapZ , which capped all barbed ends , Tmod capped only ~ 20 % of pointed ends , resulting in a 3 . 26 - \u00c5 resolution structure ( Fig . 4 , A and B , and movie S1 ) . Tmod \u2019 s low pointed - end occupancy may be in part attri - buted to inefficient pointed - end capping in the absence of tropomyosin ( 13 , 18 ) , which binds weakly to short filaments ( 17 ) . Tmod comprises alternating tropomyosin and actin - binding sites ( Fig . 4A ) . Likely owing to thelack of interactions with tropomyosin , the two tropo - myosin binding sites are disordered in the Tmod - capped structure ( Fig . 4B ) , but appear ordered in the low - resolution Tmod - tropomyosin map ( fig . S8A ) . In the Tmod - capped structure , actin binding site 1 ( ABS1 , P58 to K99 ) has an extended conformation , interrupted by a single a helix , and interacts from N to C terminus with subdomains 4 , 2 , and 1 of the first actin subunit ( Fig . 4B and fig . S9D ) . Actin binding site 2 ( ABS2 , L161 to T348 ) consists mostly of a leucine - rich repeat ( LRR ) domain , which forms a wedge - like structure that inserts into a cleft at the interface between the first three Carman et al . , Science 380 , 1287 \u2013 1292 ( 2023 ) 23 June 2023 4 of 6 A B Fig . 3 . Free pointed end . ( A ) Cryo - EM map of the free pointed end at 2 . 84 - \u00c5 resolution , showing the first and second subunits in two different shades of green . The D loop of subunits at the pointed end is disordered . ( B ) The first ( left ) and second ( right ) subunits at the pointed end adopt a G - actin conformation , where the outer domain is rotated ~ 20\u00b0 ( red arrow ) compared with subunits in the middle of F - actin ( gray ) . Subunits were superimposed based on their inner domains ( surface representation ) to highlight differences in the orientation of their outer domains ( ribbon representation ) . C A B Fig . 4 . Tmod - capped pointed end . ( A ) Domain diagram of Tmod , comprising alternating tropomyosin ( TMBS1 and TMBS2 , disordered in the structure ) and actin ( ABS1 and ABS2 ) binding sites . ( B ) Cryo - EM map of the Tmod - capped pointed end at 3 . 26 - \u00c5 resolution , showing the first and second subunits in two shades of green and Tmod in orange . The view on the right is approximately down the longitudinal axis of F - actin and shows a ribbon representation of Tmod . ABS1 caps the first actin subunit , whereas ABS2 wedges into a cleft formed by the first three actin subunits . Most of the interactions with actin are mediated by the b - strand to a - helix loops of the LRR domain of ABS2 . Tmod residues involved in the interaction are shown and labeled in fig . S9D . ( C ) The first actin subunit ( left ) adopts a G - actin conformation with the outer domain rotated by ~ 20\u00b0 ( red arrow ) compared with subunits in the middle of F - actin ( gray ) . The second actin subunit ( right ) adopts an F - actin conformation . Subunits were superimposed based on their inner domains ( surface represen - tation ) to highlight differences in orientation of their outer domains ( ribbon representation ) . RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023 pointed - end subunits . The loops that connect the b strands to the a helices of the LRR do - main account for most of the interactions with actin subunits ( Fig . 4B ) . We had previ - ouslyproposedamodelofTmodatthepointedendthatwasbasedoncrystalstructuresofABS1andABS2boundtoG - actin ( 18 ) . This model accurately predicted Tmod \u2019 s binding mode , but not the conformation of actin sub - units at the pointed end , which is crucial to understand the details of this interaction . Thus , another reason for Tmod \u2019 s inefficient pointed - end binding appears to be that it re - quires a conformational change that forces the second subunit into an F - actin conforma - tion , whereas the first subunit remains in the G - actin conformation ( Fig . 4C and table S2 ) . The interaction of ABS2 with the three pointed - end subunits is likely responsible for the conformational change in the second subunit , because binding of ABS2 would be sterically hindered for the G - actin conformation . In a recently reported structure of the spectrin - actin complex ( 27 ) , the pointed end is capped by Tmod together with a previously unknown protein ( SH3BGRL2 ) . This complex also con - tains tropomyosin , whose position is con - strained by interactions with several other proteins . Likely because of these added inter - actions , the first actin subunitinthespectrin - actin complex has an F - actin conformation and tropomyosin is shifted azimuthally by ~ 2 . 0 \u00c5 compared with its apparent position in our Tmod - tropomyosin map ( fig . S8 ) . Conclusions We found that the ends of F - actin display conformational differences that correlate with kinetic differences in subunit addition and dis - sociation ( Fig . 5 and movie S1 ) . Notably , these structuraldifferences dependon theposition of subunits in the filament and not on the nucleotide state , which is responsible only for minor differences both in the monomer ( 28 , 29 ) and the filament ( 5 , 14 , 15 ) . Consistent with early studies of filament elongation and de - polymerization kinetics ( 1 , 6 ) , subunits at the barbed end have an F - actin conformation and are thus structurally primed for subunit addition . Yet , their exposed hydrophobic cleft have a G - actin \u2013 like conformation , which may explain why many proteins that bind G - actin can also stably or transiently bind the barbed end , including profilin ( 1 , 30 ) , gelsolin ( 31 ) , formins ( 7 ) , twinfilin ( 23 , 32 ) , andWH2domain \u2013 containing proteins ( 33 ) . CapZ undergoes major structural changes upon binding to the barbed end , which remains mostly unaffected by this interaction . An unexpected finding was that subunits at the pointed end adopt a G - actin conformation andthusappearprimedfordissociationandlesslikelytoacceptincomingsubunits . According to this model , slow pointed - end association can be viewed as conceptually analogous to the rate - limiting step during nucleation , i . e . , the formation of a polymerization seed from G - actin subunits ( 26 ) . Tmod forms a knot - like structure around the first three subunits , forc - ing the second subunit into an F - actin confor - mation and inhibiting subunit addition and dissociationbystericallyblockingthepointedend . Thenucleotidestatecontrolstheassociation and dissociation kinetics at the ends of F - actin ( 1 , 6 ) . The ADP - bound structures described here are directly relevant to the situation at the pointed end , which accumulates ADP - actin . At the barbed end , ATP hydrolysis likely occurs immediately upon subunit addition , followed by slow g - phosphate release . Yet , as in the fila - ment ( 5 , 14 , 15 ) , the structure of the barbed end is unlikely to change much as a function of the bound nucleotide . Therefore , we suggest that the structural differences observed in this work explain the asymmetric association of ATP - actin monomers to the barbed and pointed ends of F - actin , with ATP - bound monomers being more likely to undergo the G - to F - actin transition required for preferential binding to the barbed end than ADP - bound monomers ( Fig . 5 ) . In this way , this work provides a mech - anistic understanding of years of biochemical evidence and supports fundamental conclusions about the structure , kinetics , and capping in - teractions at the ends of the actin filament . REFERENCES AND NOTES 1 . T . D . Pollard , Handb . Exp . Pharmacol . 235 , 331 \u2013 347 ( 2017 ) . 2 . R . Dominguez , K . C . Holmes , Annu . Rev . Biophys . 40 , 169 \u2013 186 ( 2011 ) . 3 . R . Dominguez , Trends Biochem . Sci . 29 , 572 \u2013 578 ( 2004 ) . 4 . T . Oda , M . Iwasa , T . Aihara , Y . Ma\u00e9da , A . Narita , Nature 457 , 441 \u2013 445 ( 2009 ) . 5 . W . Oosterheert , B . U . 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Only the main pathway at equilibrium is depicted , with ATP - actin preferentially adding to the barbed end and ADP - actin dissociating from the pointed end [ see ( 1 ) for other possible reactions ] . Structural differences explain the asymmetric association of ATP - actin monomers to the barbed and pointed ends of F - actin , with ATP - bound monomers more likely to undergo the G - to F - actin transition required for preferential binding to the barbed end than ADP - bound monomers . CapZ and Tmod inhibit subunit exchange at the barbed and pointed ends , respectively , by structrual mechanisms revealed in this study . RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023 20 . L . Urnavicius et al . , Nature 554 , 202 \u2013 206 ( 2018 ) . 21 . M . Hernandez - Valladares et al . , Nat . Struct . Mol . Biol . 17 , 497 \u2013 503 ( 2010 ) . 22 . S . 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Didry , Int . Rev . Cell Mol . Biol . 290 , 55 \u2013 85 ( 2011 ) . ACKNOWLEDGMENTS Data collection was performed at the Electron Microscopy Resource Lab ( EMRL ) and The Beckman Center for Cryo - Electron Microscopy , University of Pennsylvania ( Research Resource Identifier SCR _ 022375 ) . We thank M . Boczkowska for assistance with sample preparation and S . Steimle for assistance with data collection . Funding : This study was supported by National Institutes of Health ( NIH ) grant R01 GM073791 ( R . D . ) and NIH grant F31 HL156431 ( P . J . C . ) . Computational resources were supported by NIH grant S10 OD023592 . Author contributions : Conceptualization : P . J . C . , K . R . B . , and R . D . ; Methodology : P . J . C . , K . R . B . , and R . D . ; Investigation : P . J . C . , K . R . B . , and R . D . ; Resources : P . J . C . , K . R . B . , and G . R . ; Visualization : P . J . C . , K . R . B . , and R . D . ; Funding acquisition : P . J . C . and R . D . ; Project administration : R . D . ; Supervision : R . D . ; Writing \u2013 original draft : R . D . ; Writing \u2013 review and editing : P . J . C . , K . R . B . , and R . D . Competing interests : The authors declare no competing interests . Data and materials availability : Molecular models and cryo - EM density maps have been deposited with the following accession codes : CapZ - capped barbed end ( PDB ID 8F8Q , EMD - 28933 ) , free barbed end ( PDB ID 8F8R , EMD - 28934 ) , free pointed end ( PDB ID 8F8S , EMD - 28935 ) , Tmod - capped pointed end ( PDB ID 8F8T , EMD - 28936 ) , and F - actin in the ADP state ( PDB ID 8F8P , EMD - 28932 ) . All other data necessary to evaluate the claims in this paper are present in the text or supplementary materials . License information : Copyright \u00a9 2023 the authors , some rights reserved ; exclusive licensee American Association for the Advancement of Science . No claim to original US government works . https : / / www . science . org / about / science - licenses - journal - article - reuse SUPPLEMENTARY MATERIALS science . org / doi / 10 . 1126 / science . adg6812 Materials and Methods Figs . S1 to S9 Tables S1 and S2 References ( 34 \u2013 41 ) MDAR Reproducibility Checklist Movie S1 View / request a protocol for this paper from Bio - protocol . Submitted 13 January 2023 ; accepted 17 May 2023 Published online 25 May 2023 10 . 1126 / science . adg6812 Carman et al . , Science 380 , 1287 \u2013 1292 ( 2023 ) 23 June 2023 6 of 6 RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023 Use of this article is subject to the Terms of service Science ( ISSN 1095 - 9203 ) is published by the American Association for the Advancement of Science . 1200 New York Avenue NW , Washington , DC 20005 . The title Science is a registered trademark of AAAS . Copyright \u00a9 2023 The Authors , some rights reserved ; exclusive licensee American Association for the Advancement of Science . No claim to original U . S . Government Works Structures of the free and capped ends of the actin filament Peter J . Carman , Kyle R . Barrie , Grzegorz Rebowski , and Roberto Dominguez Science 380 ( 6651 ) , . DOI : 10 . 1126 / science . adg6812 Editor\u2019s summary Actin is a key protein in cellular motility , forming filaments that give dynamic structure to cellular appendages and serve as scaffold for motor proteins . The ends of these filaments are constantly extending and dissociating , and their structure has been difficult to capture . Carman et al . optimized a mixture of binding proteins so that they could visualize the ends in different forms . The structures revealed the internal conformations of the actin subunits at the ends , and the authors discuss how differences from the main filament structure facilitate differential addition or loss of subunits at the ends . They also found that a binding protein called tropomodulin stabilizes the pointed end and a protein called cap - Z binds at the barbed end . \u2014Michael A . Funk View the article online https : / / www . science . org / doi / 10 . 1126 / science . adg6812 Permissions https : / / www . science . org / help / reprints - and - permissions D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on O c t ob e r 26 , 2023", + "jeeRelationalScaffoldingEnhances2019": "https : / / doi . org / 10 . 1177 / 0956797619864601 Psychological Science 2019 , Vol . 30 ( 9 ) 1287 \u2013 1302 \u00a9 The Author ( s ) 2019 Article reuse guidelines : sagepub . com / journals - permissions DOI : 10 . 1177 / 0956797619864601 www . psychologicalscience . org / PS ASSOCIATION FOR PSYCHOLOGICAL SCIENCE Research Article Science portrays a world governed by invisible entities and processes . The orbiting of electrons around the nucleus of an atom or of planets around the sun in our solar system cannot be directly observed . One is imper - ceptibly small , the other incredibly vast . Belief in the existence of these invisible systems is hardly a matter of common sense . Indeed , revolutionary scientific breakthroughs\u2014such as the heliocentric model of the solar system , evolution by natural selection , and the germ theory of disease\u2014have drastically restructured our understanding of the world ( Kuhn , 1962 ) . The advancement of these and other fundamental scientific ideas has involved creating , testing , and refining models that represent the hidden nature of reality ( Nersessian , 2010 ) . Science education aims to support model - based learning from a young age ( American Association for the Advancement of Science , 2009 ; National Research Council , 2012 ) . Yet students often emerge from school with incomplete and incorrect ideas about the way the world works ( Chi , Roscoe , Slotta , Roy , & Chase , 2012 ; McCloskey , 1983 ; Shtulman , 2017 ) . To realize the explanatory power of a scientific model , a student must grasp its relation to observable phenomena . This is straightforward when models depict familiar objects in idealized form or events happening more slowly or quickly than usual . It is far more challenging when models portray invisible entities and processes , whose relationship to observable phenomena is nonobvious and often counterintuitive . Like interpreting an analogy , making sense of a sci - entific model can involve relating seemingly disparate sets of information . Hence , also like analogy , the map - ping between observations and model may depend on 864601 PSSXXX10 . 1177 / 0956797619864601 Jee , Anggoro Relational Scaffolding and Scientific Models research - article 2019 Corresponding Author : Benjamin D . Jee , Worcester State University , Department of Psychology , Sullivan Academic Center , Suite S - 241 , 486 Chandler St . , Worcester , MA 01602 E - mail : bjee @ worcester . edu Relational Scaffolding Enhances Children\u2019s Understanding of Scientific Models Benjamin D . Jee 1 and Florencia K . Anggoro 2 1 Department of Psychology , Worcester State University , and 2 Department of Psychology , College of the Holy Cross Abstract Models are central to the practice and teaching of science . Yet people often fail to grasp how scientific models explain their observations of the world . Realizing the explanatory power of a model may require aligning its relational structure to that of the observable phenomena . In the present study , we tested whether relational scaffolding \u2014guided comparisons between observable and modeled events\u2014enhances children\u2019s understanding of scientific models . We tested relational scaffolding during instruction of third graders about the day / night cycle , a topic that involves relating Earth - based observations to a space - based model of Earth\u2019s rotation . Experiment 1 found that participants ( N = 108 ) learned more from instruction that incorporated relational scaffolding . Experiment 2 ( N = 99 ) found that guided comparison\u2014not merely viewing observable and modeled events\u2014is a critical component of relational scaffolding , especially for children with low initial knowledge . Relational scaffolding could be applied broadly to assist the many students who struggle with science . Keywords relational learning , structural alignment , comparison , relational scaffolding , science education , model - based instruction , open data , open materials Received 11 / 7 / 18 ; Revision accepted 4 / 26 / 19 1288 Jee , Anggoro a process of structural alignment , in which correspon - dences are established in accordance with a deeper , shared system of relations ( Gentner & Markman , 1997 ) . With scientific models , causal attribution is also crucial ; the model explains what is observed . Given these paral - lels , theory and research on analogical thinking could provide a basis for new approaches to model - based science instruction . Our aim was to test a support for model - based learning\u2014 relational scaffolding \u2014that involves guiding a student through systematic comparisons between observable phenomena and corresponding modeled events . Com - paring analogous cases brings common relational struc - ture into focus ( Gentner & Markman , 1997 ) . When cases lack surface similarity , explicit comparison supports analogical retrieval and mapping ( Goldwater & Gentner , 2015 ; Holyoak & Koh , 1987 ; Kurtz , Miao , & Gentner , 2001 ) . Thus , relational scaffolding should be most effec - tive when models involve entities and processes that bear little resemblance to observable phenomena . Applying relational scaffolding throughout a com - plex system of relations could illuminate the system\u2019s structural coherence\u2014its systematicity \u2014and discourage students from fragmented , incoherent explanations ( Gentner & Toupin , 1986 ) . Comprehensive visual com - parisons also reduce the burden of mentally represent - ing and aligning observed and modeled events during instruction ( Mayer & Moreno , 2003 ; Richland , Zur , & Holyoak , 2007 ) . Thus , relational scaffolding should especially benefit students with little prior knowledge , who often misinterpret scientific explanations and experi - ence cognitive overload during instruction ( Kalyuga , 2007 ; McNamara , Kintsch , Songer , & Kintsch , 1996 ; Vosniadou & Skopeliti , 2017 ) . The present research tested relational scaffolding with a fundamental and notoriously challenging topic : the day / night cycle . Children in the United States are expected to understand this topic between third grade and fifth grade ( National Research Council , 2012 ) . How - ever , many third and fifth graders are confused about the connection between Earth\u2019s motion and the day / night cycle , stating , for example , that the Earth orbits the sun in a single day ( Vosniadou & Brewer , 1994 ) . Even seventh - and eighth - grade U . S . students frequently endorse incorrect explanations ( Sadler et al . , 2010 ) . Grasping the scientific model of the day / night cycle involves reconciling observations from an Earth - based perspective\u2014including sunrise , midday , sunset , and midnight\u2014with models that adopt a large - scale space - based perspective of the Earth \u2013 sun system ( see Fig . 1 ) . In relational scaffolding , instructors support structural alignment by showing video footage of these two per - spectives simultaneously ( in a split - screen display ) and indicating the relevant temporal , spatial , and causal co r res pondences . Relational scaffolding is intended to supplement rather than replace model - based science instruction . We therefore tested it in a sequence of instruction that progressed from a familiar frame of reference\u2014an embodied simulation in which the student enacted Earth\u2019s rotation\u2014to a space - based simulation using a physical 3 - D model . Embodied simulation was intended to provide an intuitive , bridging analog for the external model ( Clement , 1993 ) and to clarify the alignment between apparent and actual motion through direct physical experience ( Kontra , Lyons , Fischer , & Beilock , 2015 ) . Relational scaffolding incorporated video footage of each simulation activity , recorded from a third - person / space - based perspective and a first - person / Earth - based perspective , as shown in Figure 2 . We tested the effects of relational scaffolding in two experiments . In Experiment 1 , third - grade participants Fig . 1 . Earth\u2019s rotation from a space - based perspective ( left ) and the apparent motion of the sun from an Earth - based perspective ( right ) . The images were obtained from nasa . gov ( left ) and stellarium . org ( right ) ; arrows have been added here to indicate the direc - tion of motion . Relational Scaffolding and Scientific Models 1289 were randomly assigned to four conditions . One group completed the sequence of instruction outlined above , progressing from an embodied to a 3 - D model - simulation activity with supplementary relational scaffolding ( relational - scaffolding condition ) . A second group com - pleted the same two simulations but without relational scaffolding ( no - relational - scaffolding condition ) . A third group repeated the 3 - D model simulation several times ( models - only condition ) . A fourth group received no instruction ( control ) . We interviewed students about the day / night cycle before and after instruction . We rea - soned that if relational scaffolding helps students relate their observations to a scientific model , participants in the relational - scaffolding condition should acquire the most knowledge . Moreover , if relational scaffolding is especially effective for students with little prior knowl - edge , any advantage of the relational - scaffolding condi - tion should be greater for low - knowledge participants . To assess changes in broader domain knowledge , we included items from a space - science - concept inventory ( Sadler et al . , 2010 ) at pretest and posttest . Given the role of language in conceptual learning ( e . g . , Gentner , Anggoro , & Klibanoff , 2011 ) and of spatial thinking in space - science understanding ( e . g . , Plummer , Kocareli , & Slagle , 2014 ) , we assessed verbal and spatial abilities prior to instruction and included these factors in the analyses of student learning outcomes . Spatial tests were also administered at a delayed posttest to explore potential changes resulting from the spatially intense instruction . Embodied Simulation 3 - D Model Simulation Third - Person / Space - Based Perspective First - Person / Earth - Based Perspective Fig . 2 . Screen captures of relational - scaffolding video footage from an embodied simulation ( top row ) and a 3 - D model simula - tion ( bottom row ) . In the embodied simulation , the student enacted Earth\u2019s rotation in relation to the yellow ball representing the sun ( top left ) . In the 3 - D model simulation , the student watched from a third - person perspective as the model of Earth rotated on its axis ( bottom left ) . Images on the right were captured by a GoPro camera mounted on the participant\u2019s head ( top right ) and on model Earth ( bottom right ) . Arrows indicate paths of motion or apparent motion . 1290 Jee , Anggoro Experiment 1 Method Participants . A total of 147 third - grade children were recruited from public elementary schools in Worcester , Massachusetts . Thirty - six ( 24 % ) left the study before the Session 6 posttest ( attrition is discussed further in the Supplemental Material available online ) . Three partici - pants were removed from the data set for scoring more than 2 standard deviations below age level on a vocabu - lary assessment ( the Peabody Picture Vocabulary Test ; see below ) . The remaining sample consisted of 108 third graders ( 62 female , 46 male ; age : M = 8 . 6 years , SD = 0 . 5 ) . This sample size provided power of greater than . 90 to detect a medium - to - large effect size ( Cohen\u2019s \u0192 2 \u2265 . 25 ) for our planned linear multiple regression analysis ( G * Power ; Faul , Erdfelder , Buchner , & Lang , 2009 ) . Each participant was randomly assigned to one of the four con - ditions ( 27 per condition ) . Knowledge measures . Day / night - cycle interview . The day / night - cycle inter - view was our primary measure of understanding . Interview - ers followed a script of questions and follow - up questions ( see Pretest Interview Script at osf . io / kszge ) . Several ques - tions required a verbal response . Others involved the use of 3 - D objects ( rubber balls representing Earth and the sun ) to model specific events in the day / night cycle . Items from the Astronomy and Space Science Concept Inventory ( ASSCI ) . The ASSCI measures students\u2019 mas - tery of astronomical concepts found in national standards ( Sadler et al . , 2010 ) . We selected nine items ( seven for kindergarten \u2013 Grade 4 and two for Grades 5 \u2013 8 ) broadly related to , but not covered in , our instruction . Further details on the instrument and selected items can be found in the Supplemental Material . Cognitive - ability measures . Verbal ability . The Peabody Picture Vocabulary Test , fourth edition ( Dunn & Dunn , 2007 ) , measures receptive vocabulary . Four pictures are presented on each trial . The test administrator says a word , and the participant must point to the picture to which it corresponds . Mental rotation . The spatial - relations subtest of the Primary Mental Abilities Test ( Thurstone & Thurstone , 1962 ) measures mental - rotation ability . On each trial , the participant must identify a rotated shape that forms a complete square with a second part . Perspective taking . The Perspective Taking Test for Children ( Frick , M\u00f6hring , & Newcombe , 2014 ) involves identifying from a set of four options the picture taken by a toy photographer within a simple scene . The objects in the scene and the photographer\u2019s position vary across trials . Instructional activities . Embodied simulation . The embodied - simulation activity followed a script based on the lesson plan for Kinesthetic Astronomy ( Morrow , 2000 ) . After orienting the partici - pant to his or her role as Earth ( see Fig . 2 , top left ) , the researcher guided the participant through a simulated 24 - hr day . The participant\u2019s first - person observations were recorded using a head - mounted GoPro camera ( see Fig . 2 , top right ) . A video recorder on a tripod captured the session from a third - person perspective . At the end of the activity , the participant performed one slow , careful rota - tion that supplied video footage for relational scaffolding . 3 - D model simulation . This activity involved a physi - cal 3 - D model with a sun and rotating Earth ( see Fig . 2 , bottom left ) . The simulation followed a detailed script similar in content and structure to the embodied simu - lation . The participant was prompted to look out from behind model Earth to adopt an Earth - based perspective for key events . Relational scaffolding . Relational Scaffolding 1 used video of the embodied simulation . The participant\u2019s third - and first - person videos were edited to create an approxi - mately 20 - s to 30 - s split - screen video of Earth\u2019s rotation as seen from each perspective . A trained researcher guided the participant through footage of midday , sunset , midnight , and sunrise , shown on a 13 - in . MacBook Pro computer ( see the Relational Scaffolding 1 script at osf . io / kszge ) . The researcher pointed at and between the videos to convey the correspondences , repeating key events several times ( for an excerpt , see Fig . 3 ) . Relational Scaffolding 2 used video from both the embodied and 3 - D model simulations . The footage was displayed on a 13 - in . MacBook Pro computer in a 2 \u00d7 2 matrix , as shown in Figure 4 . A trained researcher followed a script ( see the Relational Scaffolding 2 script at osf . io / kszge ) that highlighted corresponding per - spectives ( see Figs . 4a and 4b ) and the higher - order relations between the simulations ( see Figs . 4c and 4d ) . Procedure . Participants completed one study session per day , twice a week for 3 weeks at their school during after - school hours . The delayed posttest occurred about 6 to 7 weeks later . Participants met with the same researcher in a quiet room ( usually a classroom ) each day . Each ses - sion lasted about 20 to 30 min . The sessions were video - taped ( with parental permission ) . In Session 1 , participants completed the verbal - ability test and mental - rotation test . Session 2 consisted of the 1291 \u201c N o w l e t \u2019 s t ake a l ook a t t h e t h i ng s y o u d i d l a s t t i m e . Le t \u2019 s c o m p a r e h o w i t l ooke d f r o m t h e t w o c a m e r a s . O n e s h o w s t h e s un a nd h o w y o u s pun o r r o t a t e d a r o und w h e n y o u w e r e E a r t h . T h i s v i d e o w a s t ake n f r o m a c a m e r a o n t h e s i d e a nd y o u c a n s ee b o t h t h e s un a nd y o u a s E a r t h . \u201d \u201c N o w t h i s i s r ea ll y c oo l ! T h i s o t h e r v i d e o w e h a v e h e r e w a s t ake n f r o m t h e c a m e r a o n y o u r h ea d a nd s h o w s w h a t i t l ooke d li ke t o y o u a s E a r t h . R e m e m b e r , t h e r e d d o t o n y o u r c h e s t i s w h e r e W o r c e s t e r i s . W h a t t h e c a m e r a r e c o r d e d i s p r e tt y s i m il a r t o w h a t a p e r s o n w o u l d s ee i f t h e y w e r e l ook i ng up f r o m W o r c e s t e r . \u201d \u201c W h e n Ea r t h h a s r o t a t e d f a r e n o ugh a r o und , w e s t a r t t o s ee t h e s un a g a i n i n W o r c e s t e r . A p e r s o n i n W o r c e s t e r w o u l d s ee t h e s un a pp ea r i n t h e ea s t . W e c a ll t h a t s un r i s e . D o y o u s ee h o w t h e r o t a t i o n o f E a r t h m ake s i t l ook li ke t h e s un i s r i s i ng i n t h e ea s t ? \u201d [ R e p l a y e v e n t a t l ea s t o n c e ] \u201c W h e r e w e r e y o u l oo k i ng t o s ee t h e s un ? [ P a r t i c i p a n t a n s w e r s + f ee db a c k ] . S o , y o u w e r e l ook i ng d o w n y o u r ea s t w a r d a r m . Th e s un i s s t a r t i ng t o a pp ea r i n t h e ea s t . W h a t i s i t c a ll e d ? [ A n s w e r + f ee db a c k ] . W h y d oe s i t l ook li ke t h e s un i s a pp ea r i ng ? [ A n s w e r + f ee db a c k ] . S o , a s y o u c a n s ee h e r e , w h e n y o u s p i n f a r e n o ugh t o t h e ea s t , y o u c a n b e g i n t o s ee t h e s un a g a i n d o w n y o u r ea s t w a r d a r m . \u201d a b c d F i g . 3 . S c h e m a t i c d i a g r a m o f R e l a t i on a l S c a ff o l d i n g 1 . T h i s e x c e r p t i s a bou t s un r i s e . E a c h p a n e l s ho w s a s c r ee n s ho t f r o m v i d e o o f t h e t h i r d - p e r s on / s p a c e - b a s e d p e r s p e c t i ve ( on t h e l e f t ) a nd t h e f i r s t - p e r s on / E a r t h - b a s e d p e r s p e c t i v e ( on t h e r i g h t ) . T e x t f r o m t h e s e ss i on s c r i p t i s quo t e d a bo ve t h e i m a g e s . B o l d t e x t s i g n i f i e s w h e n t h e r e s e a r c h e r p o i n t e d t o on e o f t h e p e r s p e c t i ve s . I n t h e bo tt o m r o w , a rr o w s i nd i c a t e t h e p a r t i c i p a n t \u2019 s m o t i on ( t h i r d p e r s on ) a nd t h e m od e l s un \u2019 s a pp a r e n t m o t i on ( f i r s t p e r s on ) a s t h ey a pp e a r e d i n t h e v i d e o . A s i n g l e h a nd i nd i c a t e s a p o i n t i n g g e s t u r e f r o m t h e r e s e a r c h e r . T h e do tt e d a rr o w i n ( c ) i nd i c a t e s a s e qu e n t i a l g e s t u r e b e t w ee n t h e t w o v i d e o s . T h e t w o do tt e d a rr o w s i n ( d ) i nd i c a t e b a c k - a nd - f o r t h g e s t u r i n g b e t w ee n t h e v i d e o s . T h e f o r m a t f o r t h i s d i a g r a m i s b a s e d on t h e w o r k o f Y u a n , U tt a l , a nd G e n t n e r ( 2017 ) . 1292 \u201c T h e s e t w o v i d eo s a r e s i m il a r b e c a u s e t h e y s h o w w h a t t h i ng s m i gh t l ook li ke f r o m E a r t h . \u201d \u201c A nd t h e s e t w o v i d eo s a r e s i m il a r b e c a u s e t h e y s h o w w h a t t h i ng s m i gh t l o ok li ke f r o m o u t e r s p a c e . \u201d \u201c W h e n y o u w e r e Ea r t h , f r o m w h i c h d i r e c t i o n d i d i t l ook li ke t h e s un w a s a pp ea r i ng ? [ P a r t i c i p a n t a n s w e r s + f ee db a c k ] . F r o m w h i c h d i r e c t i o n d oe s i t l ook li ke t h e s un i s a pp ea r i ng f o r t h e m o d e l Ea r t h ? [ A n s w e r + f ee db a c k ] \u201d \u201c W h e n y o u w e r e t h e E a r t h , y o u r o t a t e d e a s t w a r d un t il y o u s t a r t e d t o s ee t h e s un . I n t h e m o d e l , t h e Ea r t h a l s o r o t a t e s e a s t w a r d un t il t h e s un l oo k s li k e i t a pp ea r s i n t h e ea s t f r o m W o r c e s t e r o n t h e m o d e l E a r t h . W h a t i s i t c a ll e d w h e n y o u r o t a t e d f a r e n o ugh ea s t t h a t y o u c o u l d s ee t h e s un ? [ P a r t i c i p a n t a n s w e r s + f ee db a c k ] \u201d a b c d F i g . 4 . S c h e m a t i c d i a g r a m o f R e l a t i on a l S c a ff o l d i n g 2 . T h i s e x c e r p t i s a bou t s un r i s e . E a c h p a n e l s ho w s a s c r ee n s ho t o f t h e 2 \u00d7 2 d i s p l a y f r o m v i d e o o f t h e e m bod i e d s i m u l a - t i on a nd 3 - D m od e l s i m u l a t i on o f t h e t h i r d - p e r s o n / s p a c e - b a s e d p e r s p e c t i ve ( on t h e l e f t ) a nd t h e f i r s t - p e r s on / E a r t h - b a s e d p e r s p e c t i ve ( on t h e r i g h t ) . T e x t f r o m t h e s e ss i on s c r i p t i s quo t e d a bo ve t h e i m a g e s . B o l d t e x t s i g n i f i e s w h e n t h e r e s e a r c h e r p o i n t e d t o a n eve n t f r o m on e o f t h e p e r s p e c t i ve s . S o li d a rr o w s i nd i c a t e t h e p a r t i c i p a n t \u2019 s m o t i on ( t h i r d p e r s on ) a nd t h e m od e l s un \u2019 s a pp a r e n t m o t i on ( f i r s t p e r s on ) a s t h ey a pp e a r e d i n t h e v i d e o . A s i n g l e h a nd i nd i c a t e s a p o i n t i n g g e s t u r e f r o m t h e r e s e a r c h e r . T h e do tt e d a rr o w s i n ( c ) a nd ( d ) i nd i c a t e s e qu e n t i a l g e s t u r e s b e t w ee n t h e t w o v i d e o s . T h e t w o do tt e d a rr o w s i n ( a ) a nd ( b ) i nd i c a t e b a c k - a nd - f o r t h g e s t u r i n g b e t w ee n t h e v i d e o s . T h e f o r m a t f o r t h i s d i a g r a m i s b a s e d on t h e w o r k o f Y u a n , U tt a l , a nd G e n t n e r ( 2017 ) . Relational Scaffolding and Scientific Models 1293 day / night - cycle - understanding interview ( pretest ) , ASSCI items , and the perspective - taking test . Sessions 3 to 5 varied among conditions . Participants in the relational - scaffolding condition completed the embod - ied simulation in Session 3 , Relational Scaffolding 1 in Session 4 , and both the 3 - D model simulation and Rela - tional Scaffolding 2 in Session 5 . Participants in the no - relational - scaffolding condition completed the embodied simulation in Session 3 and again in Session 4 , followed by the 3 - D model simulation in Session 5 . Participants in the models - only condition completed the 3 - D model simulation in Sessions 3 , 4 , and 5 . Partici - pants in the control condition did not complete instruc - tional sessions . They remained in a supervised classroom during this time . In all conditions , Session 6 consisted of the day / night - cycle - understanding interview ( posttest ) and ASSCI items . Session 7 included the day / night - cycle - understanding interview ( delayed posttest ) , the mental - rotation test , and the perspective - taking test . Equivalent forms of the mental - rotation test , perspective - taking test , and ASSCI items were administered in counterbalanced order across participants in each condition . Figure 5 provides an overview of the procedure . Coding the day / night - cycle interviews . We created a 27 - component coding rubric , building on prior measures of space - science understanding ( e . g . , Plummer et al . , 2014 ; Vosniadou & Brewer , 1994 ) . The components represent scientifically consistent ideas scored as correct / present versus incorrect / absent on the basis of the participant\u2019s interview responses . The internal consistency ( Cronbach\u2019s \u03b1 ) of the rubric was . 80 at pretest , . 88 at posttest , and . 86 Experiment 1 Condition Pretest Instruction Posttest Delayed posttest Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Control Verbal - Ability Test + Mental - Rotation Test Day / Night Cycle Interview + Concept - Inventory Items + Perspective - Taking Test No Instruction No Instruction No Instruction Day / Night Cycle Interview + Concept - Inventory Items Day / Night Cycle Interview + Mental - Rotation Test + Perspective - Taking Test Models Only 3 - D Model Simulation 3 - D Model Simulation 3 - D Model Simulation No Relational Scaffolding Embodied Simulation Embodied Simulation 3 - D Model Simulation Relational Scaffolding Embodied Simulation Relational Scaffolding 1 3 - D Model Simulation + Relational Scaffolding 2 Experiment 2 Condition Pretest Instruction Posttest Delayed posttest Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Sequential Scaffolding Verbal - Ability Test + Mental - Rotation Test Day / Night Cycle Interview + Concept - Inventory Items + Perspective - Taking Test Embodied Simulation Sequential Scaffolding 1 3 - D Model Simulation + Sequential Scaffolding 2 Day / Night Cycle Interview + Concept - Inventory Items Day / Night Cycle Interview + Mental - Rotation Test + Perspective - Taking Test Relational Scaffolding Embodied Simulation Relational Scaffolding 1 3 - D Model Simulation + Relational Scaffolding 2 Fig . 5 . Overview of procedure for Experiments 1 and 2 . 1294 Jee , Anggoro at delayed posttest . The 27 components and information about the coding process are provided in the Supplemen - tal Material . Results Table 1 shows the results for the demographic , cognitive - ability , and knowledge measures . There were no prein - struction differences between conditions on any variable\u2014gender : \u03c7 2 = 2 . 58 , p = . 46 ; all other measures : F s < 1 . 40 , p s > . 25 . We conducted a multiple regression analysis to predict posttest day / night - cycle understanding ( score on the 27 - component rubric ) from the participant\u2019s age ; gender ( male = 0 , female = 1 ) ; pretest verbal - ability , mental - rotation , and perspective - taking scores ; and pretest day / night understanding . Table 2 shows the zero - order cor - relations between variables . We also included a set of orthogonal contrasts in the regression model ( Davis , 2010 ) to test ( a ) the effect of receiving instruction versus none at all ( instructional conditions vs . control ) , ( b ) the effect of receiving embodied and 3 - D model simulation versus 3 - D model simulation alone ( no relational scaffolding and relational scaffolding vs . models only ) , and ( c ) the effect of receiving relational scaffolding specifically ( no rela - tional scaffolding vs . relational scaffolding ) . Because the no - relational - scaffolding - versus - relational - scaffolding con - trast directly tested the relational - scaffolding effect , we crossed this factor with pretest understanding to test the hypothesized Relational Scaffolding \u00d7 Prior Knowledge interaction ( West , Aiken , & Krull , 1996 ) . We included this interaction in the regression along with two others that crossed no relational scaffolding versus relational scaffold - ing with pretest mental - rotation and perspective - taking scores . All predictors were mean centered for the analysis . The regression model accounted for 53 % of the vari - ance in posttest understanding , F ( 12 , 95 ) = 9 . 02 , standard error of the estimate ( SEE ) = 3 . 98 , p < . 0001 . Figure 6 conveys the main findings of the orthogonal contrasts . Participants in the instructional conditions learned more than control participants ( \u03b2 = 0 . 55 ; 95 % confidence inter - val , or CI = [ 0 . 40 , 0 . 69 ] , p < . 0001 , partial r 2 = . 38 ) . Par - ticipants who received embodied simulation ( no relational scaffolding and relational scaffolding ) learned about as much as models - only participants ( \u03b2 = 0 . 09 , 95 % CI = [ \u2013 0 . 05 , 0 . 23 ] , p = . 20 , partial r 2 = . 02 ) . Impor - tantly , participants in the relational - scaffolding condition acquired the greatest understanding , significantly more than no - relational - scaffolding participants ( \u03b2 = 0 . 24 , 95 % CI = [ 0 . 09 , 0 . 38 ] , p = . 002 , partial r 2 = . 10 ) . We confirmed in a post hoc analysis\u2014restructuring the orthogonal contrasts\u2014that relational - scaffolding participants also Table 1 . Means for Demographic and Cognitive Variables for Experiment 1 Variable type and variable Condition Overall Control Models only No relational scaffolding Relational scaffolding Demographics Gender ( female : male ) 14 : 13 18 : 9 13 : 14 17 : 10 62 : 46 Age 8 . 7 ( 0 . 6 ) 8 . 6 ( 0 . 5 ) 8 . 6 ( 0 . 4 ) 8 . 7 ( 0 . 5 ) 8 . 6 ( 0 . 5 ) Pretest and posttest n 27 27 27 27 108 Delayed posttest n 19 19 24 21 83 Pretest Verbal ability ( PPVT standard score ) 100 . 4 ( 13 . 8 ) 100 . 8 ( 16 . 3 ) 99 . 3 ( 13 . 5 ) 99 . 5 ( 13 . 9 ) 100 . 0 ( 14 . 2 ) Mental rotation ( PMA - SR score ) 9 . 8 ( 2 . 4 ) 9 . 2 ( 3 . 0 ) 8 . 8 ( 2 . 7 ) 8 . 3 ( 3 . 3 ) 9 . 0 ( 2 . 9 ) Perspective taking ( PTT - C score ) 10 . 2 ( 4 . 0 ) 8 . 8 ( 3 . 8 ) 9 . 1 ( 3 . 5 ) 8 . 2 ( 3 . 1 ) 9 . 1 ( 3 . 6 ) Space - science concept ( ASSCI score ) 2 . 7 ( 1 . 5 ) 2 . 7 ( 1 . 1 ) 2 . 9 ( 1 . 5 ) 3 . 0 ( 1 . 3 ) 2 . 8 ( 1 . 3 ) Day / night understanding score 8 . 5 ( 4 . 1 ) 7 . 7 ( 4 . 4 ) 7 . 3 ( 3 . 8 ) 7 . 2 ( 3 . 6 ) 7 . 7 ( 4 . 0 ) Posttest Space - science concept ( ASSCI score ; n = 98 ) 3 . 0 ( 1 . 7 ) 3 . 0 ( 1 . 2 ) 2 . 9 ( 1 . 2 ) 2 . 5 ( 1 . 3 ) 2 . 8 ( 1 . 4 ) Day / night understanding score 7 . 8 ( 4 . 0 ) 13 . 3 ( 5 . 2 ) 12 . 3 ( 4 . 7 ) 15 . 8 ( 4 . 9 ) 12 . 3 ( 5 . 5 ) Delayed posttest Mental rotation ( PMA - SR score ; n = 77 ) 9 . 8 ( 2 . 8 ) 8 . 9 ( 2 . 4 ) 9 . 7 ( 2 . 5 ) 10 . 0 ( 3 . 3 ) 9 . 6 ( 2 . 8 ) Perspective taking ( PTT - C score ; n = 81 ) 12 . 0 ( 4 . 0 ) 10 . 0 ( 3 . 3 ) 11 . 3 ( 4 . 7 ) 9 . 9 ( 4 . 0 ) 10 . 8 ( 4 . 1 ) Day / night understanding score 9 . 1 ( 4 . 8 ) 12 . 4 ( 4 . 9 ) 11 . 1 ( 5 . 0 ) 13 . 5 ( 4 . 6 ) 11 . 5 ( 5 . 0 ) Delay interval ( weeks ) 7 . 1 ( 2 . 8 ) 6 . 3 ( 2 . 2 ) 6 . 8 ( 2 . 5 ) 6 . 6 ( 2 . 4 ) 6 . 7 ( 2 . 5 ) Note : Standard deviations are given in parentheses . PPVT = Peabody Picture Vocabulary Test ; PMA - SR = spatial - relations subtest of the Primary Mental Abilities Test ; PTT - C = Perspective Taking Test for Children ; ASSCI = Astronomy and Space Science Concept Inventory . Relational Scaffolding and Scientific Models 1295 achieved higher understanding than models - only par - ticipants ( \u03b2 = 0 . 20 , 95 % CI = [ 0 . 06 , 0 . 34 ] , p = . 009 , partial r 2 = . 07 ) . Pretest day / night understanding was also a significant predictor in the model ( \u03b2 = 0 . 39 , 95 % CI = [ 0 . 22 , 0 . 55 ] , p < . 0001 , partial r 2 = . 19 ) ; however , no other factor or interaction was a significant predictor , including Relational Scaffolding \u00d7 Prior Knowledge ( \u03b2 s = \u22120 . 03 to 0 . 11 , p s > . 20 ) . Participants\u2019 broader space - science knowledge changed little ( see ASSCI means in Table 1 ) . A 2 ( test session ) \u00d7 4 ( condition ) mixed analysis of variance ( ANOVA ) found no significant difference in mean scores on the ASSCI items from pretest to posttest , F ( 1 , 94 ) < 1 , p = . 87 , \u03b7 p \u00b2 < . 01 , nor was there a difference between conditions , F ( 3 , 94 ) < 1 , p = . 97 , \u03b7 p \u00b2 < . 01 , or a Test Session \u00d7 Condition interaction , F ( 1 , 94 ) = 1 . 0 , p = . 32 , \u03b7 p \u00b2 = . 03 . Eighty - three participants ( 77 % ) completed the delayed posttest . Day / night - cycle - understanding scores were analyzed in a multiple regression analysis . To pre - serve statistical power , we included as predictors only those variables that were significant in the posttest analysis : pretest understanding , the instructional - conditions - versus - control contrast , and the no - relational - scaffolding - versus - relational - scaffolding contrast . ( An analysis that included all of the posttest predictors yielded the same pattern of results . ) Delay interval ( weeks between posttest and delayed posttest ) was added as a predictor . All variables were mean centered for the analysis . The regression model accounted for 35 % of the vari - ance in delayed - posttest day / night - cycle - understanding scores , F ( 4 , 78 ) = 5 . 89 , SEE = 10 . 64 , p < . 0001 . The results mirrored those at posttest . Participants in the instructional conditions maintained higher understand - ing than those in the control condition ( \u03b2 = 0 . 30 , 95 % CI = [ 0 . 12 , 0 . 49 ] , p < . 01 , partial r 2 = . 12 ) . Participants in the relational - scaffolding condition had higher understanding than no - relational - scaffolding partici - pants ( see Table 1 ) ; however , this difference diminished and was not statistically significant ( \u03b2 = 0 . 18 , 95 % CI = [ \u2013 0 . 01 , 0 . 36 ] , p = . 06 , partial r 2 = . 05 ) . Pretest under - standing was a significant predictor ( \u03b2 = 0 . 52 , 95 % CI = [ 0 . 33 , 0 . 71 ] , p < . 0001 , partial r 2 = . 28 ) . Delay inter - val was not significant ( \u03b2 = \u22120 . 13 , 95 % CI = [ \u22120 . 32 , 0 . 06 ] , p = . 18 , partial r 2 = . 02 ) , although understanding scores decreased with longer intervals . We also analyzed participants\u2019 spatial - test perfor - mance before and after instruction for both Experiments 1 and 2 . The details are provided in the Supplemental Material . In short , we found no change in mental - rotation performance in either experiment . Perspective - taking scores increased significantly in both experiments ; however , the control and instructional conditions showed about the same level of improvement . Table 2 . Pearson Correlations Between Experiment 1 Variables Variable 2 3 4 5 6 7 8 9 10 11 12 1 . Gender \u2212 . 01 . 10 \u2212 . 06 . 10 \u2212 . 02 . 20 * \u2212 . 07 . 18 . 05 \u2212 . 05 . 05 2 . Age \u2212 . 43 * * . 02 \u2212 . 08 \u2212 . 05 \u2212 . 16 \u2212 . 07 \u2212 . 13 . 04 \u2212 . 03 \u2212 . 11 3 . Verbal ability . 23 * . 24 * . 11 . 33 * * . 29 * * . 27 * * . 21 . 19 . 27 * 4 . Pretest mental rotation . 30 * * . 13 . 14 . 17 . 06 . 30 * * . 24 * . 13 5 . Pretest perspective taking . 17 . 38 * * . 12 . 18 . 21 . 71 * * . 13 6 . Pretest space - science concept . 07 . 32 * * . 21 * . 11 . 22 * . 07 7 . Pretest day / night - understanding . 18 . 40 * * . 09 . 32 * * . 46 * * 8 . Posttest space - science concept ( n = 98 ) . 12 \u2212 . 16 . 23 * . 05 9 . Posttest day / night - understanding . 21 . 21 . 64 * * 10 . Delayed posttest mental rotation ( n = 77 ) . 24 * . 16 11 . Delayed perspective taking ( n = 81 ) . 17 12 . Delayed posttest day / night - understanding ( n = 83 ) Note : Gender was coded 0 for male and 1 for female . The fourth edition of the Peabody Picture Vocabulary Test ( PPVT ) measured verbal ability , the spatial - relations subtest of the Primary Mental Abilities Test ( PMA - SR ) measured mental rotation , the Perspective Taking Test for Children ( PTT - C ) measured perspective taking , and the Astronomy and Space Science Concept Inventory ( ASSCI ) measured children\u2019s space - science concept . * p < . 05 . * * p < . 01 . 1296 Jee , Anggoro Discussion Participants in the instructional conditions greatly increased their understanding of the day / night cycle ( although not of space - science concepts more broadly ) . Participants who received relational scaffolding gained the most knowledge . These effects were somewhat attenuated by the delayed posttest ( 6 \u2013 7 weeks after instruction ) , but the general pattern remained . The results are consistent with our hypothesis that systematic comparison of observable and modeled events facilitates understanding of scientific models . Yet further experimentation is required to separate the effects of comparison from other aspects of the relational - scaffolding condition that may enhance learning . Nota - bly , only the relational - scaffolding condition included videos of observable and modeled events . Viewing this footage could reduce extraneous cognitive load ( Sweller , 1994 ) , enabling a student to devote limited mental resources to sense - making processes ( Mayer & Moreno , 2003 ) . It is precisely for topics that require attention to relations within a system ( high element interactivity ) that a reduction in extraneous load should be especially beneficial ( Sweller , 1994 ) . Experiment 2 Experiment 2 teased apart guided comparison from the viewing of video footage . We compared the relational - scaffolding condition with a condition in which videos of observable and modeled events were presented sequen - tially , without explicit comparison . If guided comparison is integral to the relational - scaffolding effect , then the relational - scaffolding condition should produce greater understanding than the sequential - scaffolding condition . Zeroing in on the role of guided comparison also permits a more direct test of the hypothesis that comprehensive , guided comparisons are especially helpful for students with little prior understanding . If so , any advantage of the relational - scaffolding condition should be most pro - nounced among lower - knowledge students . In Experiment 2 , a new group of third - grade partici - pants received day / night - cycle instruction , supple - mented with either relational scaffolding or sequential scaffolding , in which videos of observable ( Earth - based ) and modeled ( space - based ) events were presented one after the other . In Experiment 2 , we also varied whether the scaffolding involved video of the participant\u2019s own embodied simulation ( self footage ) or a research assis - tant\u2019s simulation ( stock footage ) . If relational scaffolding requires personalized footage , it would be challenging to implement in formal educational settings . The footage variable was crossed with the scaffolding manipulation to create four experimental conditions . Method Participants . A total of 128 third - grade children were recruited at elementary schools in Worcester , Massachu - setts . Twenty - eight children ( 22 % ) dropped out before 0 3 6 9 12 15 18 21 24 27 D a y / N i gh t - C y c l e - U nd e r s t a nd i ng S c o r e Control Models Only Relational Scaffolding No Relational Scaffolding Pretest Posttest Pretest Posttest Pretest Posttest Pretest Posttest Fig . 6 . Pretest and posttest understanding scores for the day / night - cycle interview , separately for each of the four experimental conditions . Individual participant scores are shown in gray ; condition means are in orange . Error bars show standard errors . Individual scores were jittered to produce separation on the x - axis . Relational Scaffolding and Scientific Models 1297 the posttest . One child was removed from the data set because the child\u2019s Peabody Picture Vocabulary Test score was more than 2 standard deviations below age level . Our remaining sample included 99 third graders ( 59 female , 40 male ; age : M = 8 . 6 years , SD = 0 . 4 ) . This sample size provided power of greater than . 90 to detect a medium to large effect ( Cohen\u2019s \u0192 2 \u2265 . 25 ) for our planned analysis ( Faul et al . , 2009 ) . Each participant was randomly assigned to one of the four conditions ( 25 per condition ; the sequential - scaffolding / self - footage condi - tion had 24 ) . Materials . Experiment 2 used the measures and instruc - tional activities from Experiment 1 and two new sequential - scaffolding activities . Sequential Scaffolding 1 used footage of the embodied simulation , either self or stock . The first - person perspective was shown first . The participant was prompted to attribute the sun\u2019s apparent motion to Earth\u2019s rotation ( third - person perspective ) , although the sun and Earth were not shown simultaneously . The third - person perspective was shown second and likewise referenced the first - person perspective . Sequential Scaffolding 2 pre - sented video of the 3 - D model simulation , first from an Earth - based perspective and then from a space - based perspective . The activity used a similar script to that for Sequential Scaffolding 1 . When stock footage was shown , the script adopted a third - person perspective ( e . g . , \u201cRemember what they recorded from the camera on their head\u201d ) . Scripts for Sequential Scaffolding 1 and Sequen - tial Scaffolding 2 can be accessed at osf . io / kszge . Procedure . Participants completed the procedure at their school during after - school hours . They completed one session per day , twice a week for 3 weeks . The delayed posttest was about 4 weeks later ( sooner than in Experiment 1 to accommodate a school break ) . Sessions 1 and 2 ( pretest ) , 6 ( posttest ) , and 7 ( delayed posttest ) were the same as in Experiment 1 . The instructional ses - sions ( Sessions 3 \u2013 5 ) for the relational - scaffolding condi - tion were equivalent to those for the relational - scaffolding condition from Experiment 1 . Participants in the sequential - scaffolding condition completed the embodied simulation in Session 3 , Sequential Scaffolding 1 in Session 4 , and both the 3 - D model simulation and Sequential Scaffold - ing 2 in Session 5 . Figure 5 provides an overview of the procedure . To code the day / night - cycle interviews , we used the 27 - component rubric from Experiment 1 . The internal consistency ( Cronbach\u2019s \u03b1 ) of the instrument was . 81 at pretest , . 85 at posttest , and . 83 at delayed posttest . Results Table 3 shows the results for the demographic and other variables . There were no preinstruction differences between conditions in gender composition , \u03c7 2 = 0 . 89 , p = . 35 , or any other variable , t s < 1 . 25 , p s > . 20 . We conducted a multiple regression analysis to pre - dict posttest understanding from age ; gender ( male = 0 , female = 1 ) ; pretest verbal - ability , mental - rotation , and perspective - taking scores ; pretest understanding ; video condition ( stock = 0 , self = 1 ) ; and comparison condition ( sequential scaffolding = 0 , relational scaf - folding = 1 ) . In this model , comparison condition directly tested the effect of guided comparison . We crossed this factor with pretest understanding to test the hypothesized Guided Comparison \u00d7 Prior Knowl - edge interaction . We also crossed comparison condition with pretest mental - rotation and perspective - taking scores to explore possible interactions with spatial abil - ity . Predictors were mean centered for the analysis . Table 4 shows the zero - order correlations between variables . The regression model accounted for 31 % of the vari - ance in posttest day / night - cycle understanding , F ( 11 , 87 ) = 3 . 52 , SEE = 4 . 79 , p < . 001 . The main finding was a significant Comparison Condition \u00d7 Pretest Under - standing interaction ( \u03b2 = \u22120 . 41 , 95 % CI = [ \u22120 . 73 , \u2013 0 . 09 ] , p = . 01 , partial r 2 = . 07 ) . Figure 7 ( left panel ) shows mean posttest day / night - cycle understanding scores for relational - scaffolding and sequential - scaffolding partici - pants who scored below and above the median on the pretest ( i . e . , low initial knowledge vs . high initial knowledge ) . For low - knowledge ( but not high - knowl - edge ) participants , there was a clear advantage of rela - tional scaffolding over sequential scaffolding . Neither manipulated variable\u2014video condition ( \u03b2 = \u22120 . 07 ) or comparison condition ( \u03b2 = 0 . 07 ) \u2014was itself a signifi - cant predictor ( p s > . 45 ) . Pretest understanding was significant ( \u03b2 = 0 . 72 , 95 % CI = [ 0 . 40 , 1 . 00 ] , p < . 0001 , partial r 2 = . 19 ) ; however , no other variable was signifi - cant ( \u03b2 s = 0 . 01 \u2013 0 . 12 , p s > . 25 ) , nor was there another interaction ( \u03b2 s = 0 . 01 , p s = . 96 ) . Participants\u2019 scores on the ASSCI increased slightly , by about 0 . 5 items ( see Table 2 ) . A 2 ( test session ) \u00d7 2 ( comparison condition ) \u00d7 2 ( video condition ) mixed ANOVA revealed that this increase was significant , F ( 1 , 90 ) = 11 . 96 , p < . 001 , \u03b7 p \u00b2 = . 12 , but there was no effect of comparison condition or video condition ( F s < 1 , p s > . 60 , \u03b7 p \u00b2s < . 01 ) and no interactions ( F s < 1 . 95 , p s > . 15 , \u03b7 p \u00b2s < . 03 ) . Seventy participants ( 71 % ) completed the delayed posttest . An analysis of spatial - test performance before and after instruction can be found in the Supplemental Material . We analyzed delayed - posttest day / night under - standing in a multiple regression analysis . To preserve power , we included as predictors only those variables involved in the significant Pretest Understanding \u00d7 Comparison Condition interaction from posttest : pretest 1298 Jee , Anggoro understanding , comparison condition , and the interac - tion term . We also added delay interval ( in weeks ) as a predictor . All variables were mean centered for the analysis . The regression model accounted for 23 % of the vari - ance in delayed - posttest day / night - cycle understanding , F ( 4 , 65 ) = 4 . 94 , SEE = 4 . 61 , p < . 01 . The Pretest Under - standing \u00d7 Comparison Condition interaction was not significant ( \u03b2 = \u22120 . 32 , 95 % CI = [ \u22120 . 67 , 0 . 03 ] , p = . 07 , partial r 2 = . 05 ) . Instead , comparison condition emerged as a significant predictor ( \u03b2 = 0 . 27 , 95 % CI = [ 0 . 06 , 0 . 49 ] , p < . 05 , partial r 2 = . 09 ) . Figure 7 ( right panel ) shows that mean delayed - posttest understanding was higher overall in the relational - scaffolding condition , although this relational - scaffolding advantage was especially pro - nounced among low - knowledge participants . Pretest understanding was also a significant predictor ( \u03b2 = 0 . 60 , 95 % CI = [ 0 . 24 , 0 . 95 ] , p < . 01 , partial r 2 = . 15 ) , but delay interval was not ( \u03b2 = \u22120 . 13 , 95 % CI = [ \u22120 . 35 , 0 . 09 ] , p = . 25 ) . Discussion Experiment 2 found that participants with relatively low initial knowledge benefited from explicit , guided com - parisons between videos of Earth - and space - based perspectives\u2014the relational - scaffolding condition . Viewing the same videos without comparison\u2014the sequential - scaffolding condition\u2014was inferior . This relational - scaffolding advantage emerged across levels of prior knowledge by the delayed posttest , although low - knowledge participants remained the primary beneficiaries . The second manipulated variable\u2014whether scaffold - ing involved the participant\u2019s personal observations ( self footage ) versus another person\u2019s ( stock footage ) \u2014 was unrelated to learning outcomes . We note that the stock footage showed the same materials , setup , and sequence as the participant\u2019s own embodied simulation . It is possible that stock footage would be less effective if these properties were altered . That said , the results are encouraging for the prospect of implementing rela - tional scaffolding in educational contexts , such as class - rooms , where individualized footage is unfeasible . General Discussion Our findings demonstrate that relational scaffolding\u2014 systematic , guided comparison of observable and cor - responding modeled events\u2014supports students\u2019 understanding of scientific models . We tested relational scaffolding in the context of instruction about the day / night cycle , a fundamental science topic that involves linking Earth - based observations to a space - based Table 3 . Means for Demographic and Cognitive Variables for Experiment 2 Variable type and variable Sequential scaffolding Relational scaffolding Overall Stock footage Self footage Stock footage Self footage Demographics Gender ( female : male ) 12 : 13 20 : 4 12 : 12 14 : 11 59 : 40 Age 8 . 6 ( 0 . 4 ) 8 . 5 ( 0 . 4 ) 8 . 6 ( 0 . 5 ) 8 . 7 ( 0 . 4 ) 8 . 6 ( 0 . 4 ) Pretest and posttest n 25 24 25 25 99 Delayed posttest N 19 16 22 13 70 Pretest Verbal ability ( PPVT standard score ) 103 . 0 ( 13 . 7 ) 103 . 9 ( 15 . 3 ) 100 . 0 ( 12 . 9 ) 100 . 0 ( 14 . 5 ) 101 . 7 ( 14 . 0 ) Mental rotation ( PMA - SR score ) 9 . 0 ( 2 . 2 ) 9 . 3 ( 1 . 9 ) 9 . 4 ( 2 . 2 ) 9 . 5 ( 2 . 4 ) 9 . 3 ( 2 . 2 ) Perspective taking ( PTT - C score ) 9 . 7 ( 2 . 8 ) 10 . 0 ( 3 . 6 ) 10 . 8 ( 4 . 1 ) 10 . 5 ( 3 . 7 ) 10 . 3 ( 3 . 6 ) Space - science concept ( ASSCI score ) 2 . 7 ( 1 . 4 ) 2 . 5 ( 1 . 4 ) 2 . 4 ( 1 . 7 ) 2 . 8 ( 1 . 2 ) 2 . 6 ( 1 . 4 ) Day / night understanding score 6 . 8 ( 3 . 6 ) 8 . 1 ( 4 . 3 ) 7 . 4 ( 5 . 2 ) 6 . 6 ( 4 . 2 ) 7 . 2 ( 4 . 3 ) Posttest Space - science concept ( ASSCI score ; n = 94 ) 3 . 3 ( 1 . 5 ) 2 . 9 ( 1 . 5 ) 3 . 3 ( 1 . 4 ) 3 . 0 ( 1 . 7 ) 3 . 1 ( 1 . 5 ) Day / night understanding score 13 . 6 ( 5 . 9 ) 14 . 5 ( 6 . 0 ) 15 . 2 ( 4 . 7 ) 13 . 8 ( 5 . 2 ) 14 . 3 ( 5 . 4 ) Delayed posttest Mental rotation ( PMA - SR score ; n = 51 ) 9 . 3 ( 2 . 6 ) 9 . 9 ( 2 . 8 ) 9 . 5 ( 3 . 1 ) 9 . 4 ( 2 . 1 ) 9 . 5 ( 2 . 6 ) Perspective taking ( PTT - C score ; n = 68 ) 11 . 6 ( 4 . 1 ) 11 . 5 ( 4 . 9 ) 13 . 0 ( 3 . 8 ) 13 . 0 ( 4 . 2 ) 12 . 3 ( 4 . 2 ) Day / night understanding score 12 . 7 ( 5 . 5 ) 13 . 6 ( 4 . 6 ) 15 . 3 ( 4 . 6 ) 16 . 2 ( 5 . 6 ) 14 . 3 ( 5 . 1 ) Delay interval ( weeks ) 3 . 9 ( 1 . 6 ) 3 . 9 ( 1 . 8 ) 3 . 7 ( 1 . 3 ) 4 . 5 ( 1 . 9 ) 4 . 0 ( 1 . 6 ) Note : Standard deviations are given in parentheses . PPVT = Peabody Picture Vocabulary Test ; PMA - SR = spatial - relations subtest of the Primary Mental Abilities Test ; PTT - C = Perspective Taking Test for Children ; ASSCI = Astronomy and Space Science Concept Inventory . Relational Scaffolding and Scientific Models 1299 model of planetary motion . Across two experiments , relational scaffolding was found to enhance third grad - ers\u2019 understanding of the day / night cycle , especially among students with little prior knowledge . This research speaks to the potential of extending theories of analogical thinking to fundamental issues in science education ( see also Goldwater & Schalk , 2016 ; Jee et al . , 2010 ) . When a scientific model has no obvious connection to observable phenomena , struc - tural alignment may be crucial for comprehension . Rela - tional scaffolding facilitates the alignment process through several coordinated supports . Explicit compari - sons illuminated shared relational structure ( Goldwater & Gentner , 2015 ) . A trained researcher pointed at and Table 4 . Pearson Correlations Between Experiment 2 Variables Variable 2 3 4 5 6 7 8 9 10 11 12 1 . Gender . 08 \u2212 . 05 . 11 < . 01 . 06 . 09 . 11 . 02 . 03 \u2212 . 04 \u2212 . 06 2 . Age \u2212 . 38 * * \u2212 . 01 \u2212 . 09 \u2212 . 05 . 01 \u2212 . 10 < . 01 . 19 . 01 . 12 3 . Verbal ability . 27 * * . 25 * . 19 . 23 * . 26 * . 22 * . 01 . 09 . 01 4 . Pretest mental rotation . 05 . 11 . 37 * * . 27 * * . 23 * . 32 * . 16 . 01 5 . Pretest perspective taking . 03 . 29 * * . 24 * . 21 * . 02 . 53 * * . 13 6 . Pretest space - science concept . 13 . 46 * * . 11 . 18 . 06 . 22 7 . Pretest day / night understanding . 36 * * . 46 * * . 21 . 25 * . 33 * * 8 . Posttest space - science concept ( n = 94 ) . 42 * * . 13 . 23 . 37 * * 9 . Posttest day / night understanding . 12 . 34 * * . 63 * * 10 . Delayed posttest mental rotation ( n = 51 ) . 26 . 02 11 . Delayed posttest perspective taking ( n = 68 ) . 19 12 . Delayed posttest day / night understanding ( n = 70 ) Note : Gender was coded 0 for male and 1 for female . The fourth edition of the Peabody Picture Vocabulary Test ( PPVT ) measured verbal ability , the spatial - relations subtest of the Primary Mental Abilities Test ( PMA - SR ) measured mental rotation , the Perspective Taking Test for Children ( PTT - C ) measured perspective taking , and the Astronomy and Space Science Concept Inventory ( ASSCI ) measured children\u2019s space - science concept . * p < . 05 . * * p < . 01 . 0 3 6 9 12 15 18 21 24 27 SS Individual Score RS Individual Score SS Group Mean RS Group Mean 0 3 6 9 12 15 18 21 24 27 Posttest Delayed Posttest D a y / N i gh t - C y c l e - U nd e r s t a nd i n g S c o r e Below Median Above Median Below Median Above Median Fig . 7 . Posttest and delayed - posttest day / night - cycle - understanding scores for participants who scored below and above the median on the pretest . Results are shown separately for sequential scaffolding ( SS ) and relational scaffolding ( RS ) . Individual participant scores are shown in gray ; means are in orange . Error bars show standard errors . Individual scores were jittered to produce separation on the x - axis . SS = sequential scaffolding ; RS = relational scaffolding . 1300 Jee , Anggoro between videos of observed and modeled events to clarify key correspondences ( Richland et al . , 2007 ; Yuan , Uttal , & Gentner , 2017 ) . The scaffolding was com - prehensive , underscoring the coherence of the scientific model and discouraging inaccurate , piecemeal explana - tions ( Au & Romo , 1996 ; Chi et al . , 2012 ) . This system - atic approach may be most effective when models are complex , unfamiliar , or counterintuitive . Future applica - tions of relational scaffolding should therefore consider the subject matter , students\u2019 background knowledge , and common conceptions that might impede ( or pro - mote ) science learning ( Shtulman , 2017 ; Vosniadou & Skopeliti , 2014 ) . Relational scaffolding is well suited to novices , who lack conceptual knowledge for inferring relational structure ( McNamara et al . , 1996 ) and are susceptible to cognitive overload during instruction ( Sweller , 1994 ) . An intriguing possibility is that relational scaffolding can help level the playing field for children who are poorly prepared for science education . Science - achievement gaps emerge in the early school years and often persist , largely because of disparities in students\u2019 basic science knowledge ( Morgan , Farkas , Hillemeier , & Maczuga , 2016 ) . Methods that assist underprepared students would profoundly impact many children and , ultimately , increase the pool of qualified candidates for careers in science . Important questions remain about the model - based instruction that accompanies relational scaffolding . Our Experiment 1 found that embodied simulation did not increase student learning beyond models - only instruc - tion . This seems at odds with evidence that physical experience improves learning of science concepts ( e . g . , Kontra et al . , 2015 ) . However , rather than passively observing , participants repeatedly adopted an Earth - based perspective ( looking out from model Earth ) dur - ing the 3 - D model activity . A fully enacted simulation may be unnecessary when physical ( or virtual ) model - ing is immersive , providing sensorimotor feedback link - ing model - based observations to embodied actions ( DeSutter & Stieff , 2017 ; Lindgren , Tscholl , Wang , & Johnson , 2016 ) . Nonetheless , our findings leave open the possibility that embodied simulation helps students understand an external model when the two are explic - itly aligned\u2014that is , through relational scaffolding . Fur - ther experimentation is required to explore this and other questions about how model - based instruction contributes to the relational - scaffolding effect . The day / night cycle was a prime candidate for testing relational scaffolding . Yet many other funda - mental scientific models portray invisible entities and processes with no obvious connection to observable phenomena . Seasonal change relates to Earth\u2019s tilt and orbit , energy transfer and state changes involve invisible molecular activity , and illness and immunity relate to microscopic viruses , vaccines , and immune cells . Nonscientific conceptions are widespread for each of these topics ( Shtulman , 2017 ) . Each is thus a promising subject for future tests of the relational - scaffolding approach . Action Editor Erika E . Forbes served as action editor for this article . Author Contributions Both authors created the study concept , developed the study design and materials , and supervised the training of research assistants . B . D . Jee developed the data coding and analyzed and interpreted the data . F . K . Anggoro recruited the partici - pants and managed the data collection . B . D . Jee drafted the manuscript , and F . K . Anggoro provided critical revisions . Both authors approved the final manuscript for submission . Acknowledgments We thank David Uttal , Cherilynn Morrow , and Dedre Gentner for providing valuable feedback throughout the project . We thank the project coordinators and research assistants from College of the Holy Cross and Worcester State University who assisted with data collection and coding . We are grateful to the schools , teachers , parents , and students who took part in the research . Declaration of Conflicting Interests The author ( s ) declared that there were no conflicts of interest with respect to the authorship or the publication of this article . Funding This research was supported by the Institute of Education Sciences , U . S . Department of Education , through Grant R305A150228 to the College of the Holy Cross . The opinions expressed are those of the authors and do not represent the views of the Institute of Education Sciences or the U . S . Department of Education . Supplemental Material Additional supporting information can be found at http : / / journals . sagepub . com / doi / suppl / 10 . 1177 / 0956797619864601 Open Practices All data and materials have been made publicly available via the Open Science Framework and can be accessed at osf . io / kszge . The design and analysis plans for this study were not preregistered . 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( 2017 ) . Analogical processes in children\u2019s understanding of spatial representations . Developmental Psychology , 53 , 1098 \u2013 1114 .", + "chanSOLVENTMixedInitiative2018": "31 SOLVENT : A Mixed Initiative System for Finding Analogies between Research Papers JOEL CHAN , University of Maryland JOSEPH CHEE CHANG , Carnegie Mellon University TOM HOPE and DAFNA SHAHAF , Hebrew University of Jerusalem ANIKET KITTUR , Carnegie Mellon University Scientific discoveries are often driven by finding analogies in distant domains , but the growing number of papers makes it difficult to find relevant ideas in a single discipline , let alone distant analogies in other domains . To provide computational support for finding analogies across domains , we introduce Solvent , a mixed - initiative system where humans annotate aspects of research papers that denote their background ( the high - level problems being addressed ) , purpose ( the specific problems being addressed ) , mechanism ( how they achieved their purpose ) , and findings ( what they learned / achieved ) , and a computational model constructs a semantic representation from these annotations that can be used to find analogies among the research papers . We demonstrate that this system finds more analogies than baseline information - retrieval approaches ; that annotators and annotations can generalize beyond domain ; and that the resulting analogies found are useful to experts . These results demonstrate a novel path towards computationally supported knowledge sharing in research communities . 1 CCS Concepts : \u2022 Human - centered computing \u2192 Collaborative and social computing systems and tools ; Additional Key Words and Phrases : Scientific discovery ; computer - supported cooperative work ; analogy ; crowdsourcing ACM Reference format : Joel Chan , Joseph Chee Chang , Tom Hope , Dafna Shahaf , and Aniket Kittur . 2018 . SOLVENT : A Mixed Initiative System for Finding Analogies between Research Papers . Proc . ACM Hum . - Comput . Interact . 2 , CSCW , Article 31 ( November 2018 ) , 21 pages . https : / / doi . org / 10 . 1145 / 3274300 1 INTRODUCTION Analogies are an important driver of scientific progress . For example , Darwin\u2019s theory of evolution was conceived by analogy to human population dynamics [ 21 ] ; Salvador Luria\u2019s Nobel - Prize - winning work on bacterial mutation was inspired by analogy to a slot machine [ 31 ] ; the simulated annealing optimization algorithm was inspired by the annealing process for removing imperfections in metals [ 26 ] ; and information foraging theory was inspired by analogy to how animals forage for food [ 36 , 41 ] . 1 This abstract demonstrates the annotation scheme we use in our mixed - initiative system : background ( yellow ) , purpose ( red ) , mechanism ( blue ) , and findings ( gray ) . Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than the author ( s ) must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from permissions @ acm . org . \u00a9 2018 Copyright held by the owner / author ( s ) . Publication rights licensed to Association for Computing Machinery . 2573 - 0142 / 2018 / 11 - ART31 $ 15 . 00 https : / / doi . org / 10 . 1145 / 3274300 Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 2 G . Zhou et al . However , as the number of papers grows , individual researchers increasingly struggle to discover relevant analogies from work within their own discipline , let alone analogies across different domains or disciplines ( e . g . , from metallurgy to optimization , for simulated annealing ) . The problem is especially acute as many fields of study now require recombining knowledge across multiple disciplines [ 24 , 45 ] . For example , cognitive science relies on insights from linguistics , artificial intelligence , neuroscience , cognitive psychology , and education ; creativity and innovation research must integrate insights from the study of individual cognitive factors , motivational / emotional factors , team dynamics , and societal / economic incentive structures [ 38 ] . Individual scientists lack the time and resources to keep up with all the possible conferences , journals , and talks that might hold analogical insights for problems they are working on . For these reasons , computational systems that can mine analogies between research papers could significantly accelerate scientific progress by reducing the cost of knowledge discovery . A core challenge to building such systems is creating appropriate semantic representations of documents that can support analogical matching . The essence of analogy is matching a target knowledge representation ( e . g . , document , abstract , research problem description ) with other source knowledge representations that share its core relational structure [ 15 ] ; when the analogous sources also have many other details that are very different from the target , they are known as far or domain - distant analogies . For example , the relational structure of the annealing process can be described as \u201cMINIMIZING imperfections in a substance BY HEATING and gradually COOLING the substance\u201d ( relations in ALL - CAPS ) . This relational structure can then be analogically matched to other sources that share all or part of the structure , such as optimization problems , which share a similar relation of \u201cMINIMIZING error in a model\u201d , despite vast differences in their domain features ( e . g . , physical temperature and metals VS . algorithms and digital data ) . We build on promising recent work by Hope et al . [ 22 ] that takes a mixed - initiative approach to creating such representations for consumer product descriptions : 1 ) crowds annotate aspects of the documents that denote their purpose ( what they are trying to achieve ) and mechanism ( how they achieve that purpose ) , and 2 ) these annotations are used to construct semantic vector representations that capture the overall purpose and mechanism of each document . The intuition behind this approach is that the overall representations of purpose and mechanism constitute a \" soft \" relational schema [ 16 ] because of the in - built causal relation between purpose and mechanism . This soft schema can then be used for analogical queries ( e . g . , finding other products with similar purpose but different mechanisms \u2013 a classic analogical problem solving query [ 16 ] ) . Hope et al . demonstrated that crowd workers could produce purpose and mechanism annotations reliably and quickly ( usually in under a minute per document ) for several thousand consumer product descriptions , and that the semantic vector representations could be used to find analogies between products , even when they were from different domains ( e . g . , a \u201cdigital collar\u201d that alerts a pet owner when the pet is too far away , and a bluetooth wristband that alerts a parent if the child wearing it is too far away ) , and at a higher rate than traditional information retrieval approaches ( e . g . , TF - IDF , GloVe word embeddings [ 35 ] ) . We are interested in extending Hope et al\u2019s [ 22 ] approach to help researchers find analogies from the research literature for their own work . For example , could researchers use this approach to find mechanisms ( e . g . , HEATING and gradually COOLING a substance ) from other research papers with similar purposes ( e . g . , MINIMIZING imperfections in a substance ) that might inspire new ideas for a target purpose ( e . g . , INCREASE and gradually DECREASE the degree of stochasticity in the optimization process to MINIMIZE error in the model , which is the essence of simulated annealing [ 26 ] ) ? Or find other papers with the same purpose and mechanism ( e . g . , for competitive analysis ) , even if those papers are from different domains ? Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 3 However , key features of research papers may prevent a straightforward application of Hope et al\u2019s [ 22 ] approach . First , research papers are often written in complex , domain - specific language not easily understood by laypeople ( e . g . , \u201cHMM\u201d , \u201cregularization\u201d , \u201ccross - validation\u201d , \u201chuman computation\u201d ) . The complexity of the concepts and language might greatly increase the cost of human annotation , rendering the method unsuitable for mining analogies across large , complex , multidisciplinary corpora of research papers . Research papers also often include multiple purposes that might have hierarchical causal relationships with each other ( e . g . , \u201cexpose model parameters and error patterns\u201d TO \u201csupport algorithmic accountability\u201d ) . Additionally , many research papers aim to understand some phenomenon , instead of contribute novel mechanisms to add value for some user : for these papers , the purpose - mechanism schema might not capture the core relational structure of these papers , and thus fail to find useful analogies for these papers . In this paper , we explore approaches to address these challenges for extracting and using purpose - mechanism representations to discover analogical relationships in complex scientific literature . Our investigation yields the following contributions : ( 1 ) Annotating the purpose and mechanism aspects of research papers is scalable in terms of cost ( < 1 minute per paper ) , not critically dependent on annotator expertise , and generalizable across domains . We explore this by deploying the method with expert annotators on papers in their domain ( Study 1 ) and outside their domain ( Study 2 ) , and with novice crowd annotators ( Study 3 ) . ( 2 ) Purpose - mechanism representations of research papers can be used to discover analogical relationships that are systematically missed by traditional state - of - the - art semantic models ( Study 1 ) , including distant analogies found valuable to a domain expert ( Study 2 ) . ( 3 ) Extending the annotation scheme to incorporate other aspects of research papers ( e . g . , their higher - level problem \u201cbackground\u201d , and the key findings of the paper ) yields measurable gains in analogy - finding performance ( Study 1 ) . These results suggest that Hope et al\u2019s [ 22 ] mixed - initiative system ( with key modifications from our investigation ) is a promising approach for finding analogies amongst research papers . We call this modifed mixed - initiative system solvent , to capture its core intution : using a mixture of crowdsourcing and machine learning to \" dissolve \" research papers into their constituent soft relational schemas ( e . g . , purpose - mechanism schemas ) , which can then be used to find analogical combinations of research papers that yield novel discoveries and innovations . We offer solvent as a novel path towards computationally supporting knowledge sharing in research communities . 2 RELATED WORK 2 . 1 Citation Recommendation The problem of finding analogous research papers can be thought of as a special case of the problem of citation recommendation : given some target manuscript that one is writing , find other papers that are suitable citations [ 20 , 42 ] . Our work here on finding suitable semantic representations of documents is similar to content - based approaches that leverage semantic representations of documents to search for recommendations [ 5 ] , and complementary to graph - based approaches ( e . g . , based on properties of citation / co - authorship networks [ 29 , 37 ] ) to this problem . 2 . 2 Computational Analogy Computational analogy is a well - studied problem in cognitive science and artificial intelligence . A major branch of research has devised algorithms for reasoning over rich , relational representa - tions , such as Gentner and colleagues\u2019 structure - mapping engine [ 14 , 15 ] , Hummel and Holyoak\u2019s LISA analogy engine [ 23 ] , and Vattam and colleagues\u2019 [ 44 ] Design Analogy to Nature Engine Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 4 G . Zhou et al . ( DANE ) . These systems can achieve impressive , human - like performance on analogy tasks , but require very detailed and rich relational representations : for example , to reason about the sim - ulated annealing analogy , the structure - mapping engine [ 14 , 15 ] would require a representa - tion of the concept of annealing that looks something like \u201cCAUSE ( AND ( HEAT ( metalworker , metal ) , CONTROL ( metalworker , COOL ( metalworker , metal ) ) ) , MINIMIZE ( metalworker , IMPER - FECTIONS _ IN ( metal ) ) \u201d . These representations are very costly to obtain : for example , Vattam and colleagues [ 44 ] estimated that converting a single ( complex ) biological system into a formal rep - resentation requires between forty and one hundred person - hours of work ; further , automatic extraction of relational representations across domains remains a difficult open problem [ 19 ] . On the other hand , many computational approaches have been developed that ignore relational structure , opting instead to learn semantic representations from the distributional statistics of words across documents in a corpus\u2014e . g . , word embedding models like Word2Vec [ 32 ] , vector - space models like Latent Semantic Indexing [ 13 ] , and probabilistic topic modeling approaches like Latent Dirichlet Allocation [ 6 ] \u2013 scale well to very large datasets , and perform well at matching based on overall / surface similarity , but struggle to detect analogies between documents when surface similarity is low . Our work extends a new thread of research [ 22 ] that explores how to extract and exploit \u201csoft\u201d relational structure : not going all the way to fully specified relational representations , but still attempting to model some kind of relational structure ( e . g . , semantic representations of the overall purpose and mechanism of a product , which can be used to define a \" soft \" schema for the product ) . 2 . 3 Crowd - Powered and Mixed - Initiative Knowledge Modeling Many researchers have explored the general problem of applying crowdsourcing techniques to synthesize a knowledge model of some domain , from taxonomies of items within a domain [ 10 , 46 ] , to summarizing answers from Web sources for a given question [ 8 , 18 ] , to planning conference talk schedules based in part on topical similarity [ 1 , 9 ] , to organizing ideas for a given problem by topical similarity during collaborative idea generation [ 40 ] . Many of these efforts are mixed - initiative sys - tems , where crowds either generate semantic data to support the creation of a computational seman - tic model [ 40 ] , or use computational methods\u2014such as information - theoretic methods\u2014to support more efficient crowdsourcing strategies [ 7 , 46 ] , or effectively aggregate crowds\u2019 semantic judgments [ 10 ] . These applications tend to be about general topicality or similarity of documents / items within some domain , rather than about analogical relationships between documents / items that might be from different domains . Closer to the problem of supporting analogy , other researchers have explored using crowdsourc - ing to perform or assist with extracting relations [ 30 ] . Most work in this area aims to extract single commonsense facts ( e . g . , \u201cPortland IS _ IN Oregon\u201d , Obama \u2018WORKED _ AT\u2018 the White House , similar to open information extraction [ 3 ] , instead of systems of relations ( e . g . , minimize imperfections in a metal BY controlled heating and cooling of the metal ) : thus , while these relations can be quite useful for improving artificial commonsense reasoning , they are not as useful for reasoning about analogies between scientific papers , especially across multiple disciplines . However , some recent work has examined how to use crowdsourcing to assist with extraction of more specialized relations , for example mechanical engineering - related functions and mechanisms in biological texts [ 2 ] , or experiment details for biomedical research ( e . g . , population studied , treatment given ) [ 43 ] . We are inspired by these efforts ( particularly the approach of using mixed - initiative systems ) , but differ in that we aim to crowdsource knowledge models for complex corpora ( like scientific papers ) across many different knowledge domains . Additionally , instead of attempting to model specific rela - tions and entities in detail , we aim to extract general semantic categories ( e . g . , purpose / mechanism ) that are relevant for analogical reasoning . Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 5 2 . 4 Ontologies of scientific and scholarly discourse The general semantic categories we aim to crowdsource are closely related to work on ontologies of scholarly and scientific discourse [ 11 , 12 ] , with an especially close relation to efforts like the Core Information about Scientific Papers ( CISP ) ontology [ 28 ] , which breaks papers out into \" Goal of investigation \" , \" Motivation \" , \" Object of investigation \" , \" Research method , \" Experiment \" , \" Observation , \" Result \" , and \" Conclusion \" . While our goal differs from this work in aiming to extract elements that support analogy in particular ( vs . construct a complete / useful model of scientific discourse ) , we extend this body of work by systematically exploring how these discourse - like ontology elements might be efficiently crowdsourced ( vs . relying entirely on automation or expert researcher annotations [ 27 ] ) , and what particular combinations of these elements might be useful for supporting analogy mining . 3 ADAPTING THE METHOD FOR RESEARCH PAPERS Applying the purpose - mechanism annotation analogical search method [ 22 ] to the domain of research papers may seem straightforward at first glance : most research projects have research goals ( e . g . , research problems , research questions ) , and methods for achieving those goals . However , there are several major challenges involving in moving from the domain of simple consumer product descriptions to more complex structures and terminology , such as found in scientific literature . 3 . 0 . 1 Complex language . First , research papers tend to be significantly more complex than the typical consumer product idea . For example , most people know that a utility belt is for holding / storing things , so words like \" store / keep \" are purpose words ; in contrast , without con - tent / domain knowledge , it may be difficult to discern which terms denote purpose / mechanism in research papers . For example , a hidden Markov model ( HMM ) is a common mechanism for analyzing sequences of events . However , without specialized domain knowledge it is possible that an annotator might not be able to label it as a mechanism . Fortunately , as we will show , people are able to leverage rich syntactic cues to determine which portions ( not specific words ) denote the purpose / mechanism / etc of the paper , even in the absence of understanding the details of those purposes or mechanisms . 3 . 0 . 2 Hierarchy of problems . Research papers also often address multiple distinct purposes that are hierarchically dependent on each other . For example , a research paper might explore how to improve creativity in teams ( high - level problem ) by improving the ability of team members\u2019 to build on each others\u2019 ideas ( mid - level subproblem ) , and specifically aim to investigate ways to surface the most diverse and best ideas ( low - level subproblem ) . In most cases , the mechanisms contributed by a given paper are most directly causally related to the lower level subproblems ( e . g . , crowdsourcing techniques for summarizing ideas ) ; consequently , the purpose - mechanism schema of a particular paper is often best described by its low - level subproblems , along with the mechanisms it contributes for solving those subproblems . To address this challenge , it may be useful to distinguish between the research background ( higher - level problems ) and the specific problem ( s ) being addressed in the paper to find more directly analogous matches . The background context can also be \" partialed out \" ( e . g . by ignoring it ) to enable discovery of analogies between different areas / fields , e . g . , matching papers that are about crowdsourcing techniques for content analysis in qualitative analysis vs . online classrooms vs . brainstorming teams . 3 . 0 . 3 Mechanisms vs . findings . Finally , many research papers do not fit the purpose - mechanism model exactly . Specifically , many papers cannot really be understood in terms of answering \u201chow\u201d questions ( e . g . , how might we improve work quality for novice crowd workers ) ; instead , their Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 6 G . Zhou et al . primary goal is to understand some phenomenon ( e . g . , when and why do people disclose personal information on social media ? ) . For these \u201cunderstanding - oriented\u201d papers , the purpose remains informative ( what question they want to understand ) , but the mechanism ( how they found the answer ) may no longer capture the core contribution of the paper towards that purpose ; instead , the findings of the paper are the \u201canswer\u201d to the purpose of the paper . For example , a research paper whose purpose is to investigate people\u2019s motivations for disclosing personal information on social media may have ( according to our schema ) particular research methods ( e . g . , interviews with users , content analysis ) as mechanisms , but the answer to the purpose might be the findings it reports ( e . g . , users avoid disclosing if they want to manage their online identity across multiple social contexts ) . Thus , the relevant schema to match on might be a purpose - findings schema , or even just a findings schema , as opposed to a purpose - mechanism one . 3 . 1 Modified annotation scheme To address these challenges , we adapted Hope et al\u2019s [ 22 ] purpose - mechanism annotation scheme to incorporate two new elements ( background and findings ) . This yields the following modified annotation scheme with 4 elements : ( 1 ) Background : What is the intellectual context of this work ? What other ( higher - level ) goals / questions can be furthered by this work ? How might this help other research ( ers ) ? ( 2 ) Purpose : What specific thing ( s ) do the paper\u2019s authors want to do or know ? ( 3 ) Mechanism : How did the paper\u2019s authors do it or find out ? ( 4 ) Findings : Did it work ? What did the paper\u2019s authors find out ? Table 1 ( top ) shows examples of these annotations for a system - oriented vs . a more \u201cunderstanding - oriented\u201d paper . This modified annotation scheme defines the target aspects that solvent trans - forms into soft relational schemas , which are used for analogical matching . 3 . 2 Creating semantic vector representations from the annotations Each of the four annotations ( background , purpose , mechanism , and findings ) yields a set of words . For example , in Table 1 , the set of words highlighted in red are annotated as \" purpose \" . We are interested in taking the four sets of annotations W = { w 1 , w 2 , w 3 , w 4 } ( where each w i is a set of words ) , and creating representations that capture the respective aspects of the paper . We follow a common approach in natural language processing and information retrieval , and build semantic vector representations based on each annotated set of words w i . These vector representations are solvent \u2019s \" soft \" relational schemas that can be used for analogical matching . In particular , we represent words with their word vectors ( e . g . , trained with word2vec [ 33 ] or GloVe [ 35 ] ) , and then for each set w i we compute a weighted average of the words in w i to obtain a representation for aspect i . The average is weighted by the TF - IDF score of each word vector 2 . Note that the TF - IDF score is calculated on an annotation - specific basis : for example , when computing TF - IDF scores for purpose vectors , we use only words that were tagged as purpose when computing term and document frequencies 3 . This is a conceptually similar approach to that taken by Hope et al [ 22 ] and helps us tease apart words that are important for denoting the purpose / mechanism / etc . of documents , as opposed to generalized importance . The end result is that each document is represented by 4 annotation - specific semantic vectors ( background , purpose , mechanism , and findings ) , which we denote as b i \u2208 R D , p i \u2208 R D , m i \u2208 R D , and f i \u2208 R D , respectively ( where D is the dimensionality of the base word vectors we use to represent the words ) . 2 This step is implemented in get - vectors . py in the code provided in the supplementary material 3 This step is implemented in compute - tf - idfs . py in the code provided in the supplementary material Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 7 IdeaHound : Self - sustainable Idea Generation in Creative Online Communities A Comparison of Social , Learning , and Finan - cial Strategies on Crowd Engagement and Out - put Quality One main challenge in large creative online communities is helping their members find inspirational ideas from a large pool of ideas . A high - level approach to address this chal - lenge is to create a synthesis of emerging solution space that can be used to provide participants with creative and diverse inspirational ideas of others . Existing approaches to gener - ate the synthesis of solution space either require community members to engage in tasks that detract from the main activ - ity of generating ideas or depend on external crowd workers to help organize the ideas . We built IDEAHOUND a collab - orative idea generation system that demonstrates an alter - native \u201dorganic\u201d human computation approach , where com - munity members ( rather than external crowds ) contribute feedback about ideas as a byproduct of an activity that nat - urally integrates into the ideation process . This feedback in turn helps the community identify diverse inspirational ideas that can prompt community members to generate more high - quality and diverse ideas . A significant challenge for crowdsourcing has been increas - ing worker engagement and output quality . We explore the effects of social , learning , and financial strategies , and their combinations , on increasing worker retention across tasks and change in the quality of worker output . Through three experiments , we show that 1 ) using these strategies together increased workers\u2019 engagement and the quality of theirwork ; 2 ) asocialstrategywasmosteffectiveforincreas - ing engagement ; 3 ) a learning strategy was most effective in improving quality . The findings of this paper provide strate - gies for harnessing the crowd to perform complex tasks , as well as insight into crowd workers\u2019 motivation . Top 10 nearest words for Abstract : ideas idea analogies proposals productively sub - strates fictions discoveries brainstorm schemas Abstract : efficient expressive retention responsive suscep - tibility differentially enjoyment disengagement alertness Background : ideas creative innovative inspirational analo - gies productively fictions brainstorm substrates wishing Background : crowdworkersworkershepherdingsourcing harnessing intrinsically legitimacy rubrics extrinsic Purpose : curate interleave scaffold organise devise stimu - late formulate ideas consolidating mobilize Purpose : retention differentially worker soundness media - tors maximize susceptibility redefining greatly decreasing Mechanism : idea ideas ideation endeavor grassroots real - ization productively scaffold generative prospector Mechanism : nine eleven twelve fourteen fieldwork com - prehensively separately uncompensated sixteen thirteen Findings : ideas community cultivate cohesive heteroge - neous divergent productively repertoire mobilize socialize Findings : tactic efficient disengagement responsive playful attentiveness partnerships paradoxically expressive Table 1 . Examples of modified annotation scheme applied to a system - oriented paper ( top left ) and an \u201cunderstanding\u201d - oriented paper ( top right ) , and top 10 nearest words for all abstract words VS . each annotation vector for each paper ( bottom ) To give an intuition of what the vectors mean , Table 1 ( bottom ) shows the top 10 nearest neighbor words for each annotation type for two example papers , as well as for a vector representing the full abstract ( i . e . , TF - IDF - weighted average of all non - stopwords in the abstract ) . The nearest neighbors are quite different across the different annotation types and from the full abstract vector , and also map well to the meaning of those annotation types . For example , while the overall abstract for the first example is about improving creativity in online communities ( as seen in the top words for the abstract vector , { ideas , idea , analogies , proposals . . . } ) , the core purpose of enabling efficient synthesis of the ideas generated by the community is well captured by the words { curate , interleave , scaffold , organise , devise . . . } , and the mechanism of \u201cpiggy - backing\u201d on ideation is captured by the mechanism top words , { idea , ideas , ideation , endeavor , grassroots . . . } . The term \u201cgrassroots\u201d in Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 8 G . Zhou et al . particular is interesting because it captures the idea of sustainability , which is completely missed in the top words for the abstract and background vectors . In contrast , for the second , understanding - oriented paper , the mechanism top words are very research - methods oriented , { nine , eleven , twelve , fourteen , fieldwork . . . } . 3 . 3 Analogical similarity metrics Using these specialized vectors , solvent can support a number of interesting analogical queries : ( 1 ) purpose + mechanism : find other research papers with a similar purpose AND mechanism , by computing the average of the cosine between their purpose vectors and the cosine betwen their mechanism vectors , i . e . , \u27e8 cos ( p 1 , p 2 ) , cos ( m 1 , m 2 ) \u27e9 for any pair of paper 1 and paper 2 . By ignoring background / implications , we aim to achieve some level of abstraction away from the domain context . ( 2 ) purpose : find other research papers with a similar purpose , but not necessarily a similar mechanism , with cos ( m 1 , m 2 ) This should find alternative mechanisms for solving some target problem . ( 3 ) mechanism : find other research papers with a similar mechanism , but not necessarily a similar purpose , with cos ( p 1 , p 2 ) . This should find alternative applications for some mechanism . ( 4 ) findings : find other research papers with similar findings , with cos ( f 1 , f 2 ) . This should help find analogies between \u201cunderstanding\u201d papers ( where the core schema is not necessarily a purpose - mechanism pairing ) . 4 STUDY 1 : SOURCING ANNOTATIONS FROM DOMAIN - EXPERT RESEARCHERS We begin our investigation of solvent \u2019s quality and feasibility by sourcing and using annotations from domain expert researchers . This deployment should provide an upper bound on solvent \u2019s quality and cost considerations . 4 . 1 Dataset As an initial test , we deployed our modified mixed - initative system on 50 papers published at the CSCW conference 4 from 2013 - 2017 . This modest number of papers is small enough for experts to manually find analogies ( with reasonable confidence that we have covered most analogies ) , yet large enough to produce interpretable quantitative results ( N = 1 , 225 possible pairs between papers ) . Further , the match to our content / domain expertise ( social computing ) allows us to better define what counts as analogies , and judge the performance of the different metrics accordingly . 4 . 2 Annotation and Vectors Two members of the research team served as annotators . Each paper abstract took a median of 1 minute to annotate . In Studies 2 and 3 , we explore how the method extends to settings where annotators lack content / domain expertise . We originally used pre - trained GloVe [ 35 ] vectors trained on the Common Crawl dataset 5 . However , baseline performance was very poor . We therefore opted for a doc2vec model [ 33 ] , with 600 dimensions , trained on a smaller but more focused and relevant corpus ( 4 , 402 papers from CHI and CSCW from 2010 to 2017 ) . 4 . 3 Baselines We compare the performance of our analogical similarity metrics to two baselines : 4 http : / / cscw . acm . org / 5 https : / / nlp . stanford . edu / projects / glove / Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 9 Match Type Title & Explanation Near analogy : Background + Purpose + Mechanism Toward Collaborative Ideation at Scale : Leveraging Ideas from Others to Generate More Creative and Diverse Ideas : Use crowd - sourcing to identify diverse idea sets to improve creative idea generation Far analogy : Purpose + Mecha - nism Crowd Guilds : Worker - led Reputation and Feedback on Crowd - sourcing Platforms : Make worker certification self - sustainable by using crowdsourced peer review to certify good workers Non - analogy Making Decisions From a Distance : The Impact of Technological Mediation on Riskiness and Dehumanization : No similarity Table 2 . Example expert - found analogies for the IdeaHound paper in the CSCW dataset , with notes on each analogy ( in italics ) . One non - analogy also shown for comparison . Analogies are mapped to the following : core schema from the IdeaHound paper : Make creative idea generation self - sustainable by using crowdsourced peer review to identify diverse idea sets . ( 1 ) all words baseline : create TF - IDF - weighted vectors for each paper from all the words in each paper\u2019s abstract ( denoted as a i ) , and compute cos ( a 1 , a 2 ) , for every pair of paper 1 and paper 2 . This baseline is meant to emulate current content - based recommendation practices , which often operate on some combination of the abstract / full - text . ( 2 ) bkground / purpose + mechanism baseline : create a new semantic vector bp i that captures both background and purpose aspects , by concatenating the weighted vectors for w b ( background tokens ) and w p ( purpose tokens ; each weighted by their respective annotation - specific tf - idf weights ) , and computing the average of this concatenation . We then look for matches with similar background / purpose and mechanism , i . e . , \u27e8 cos ( bp 1 , bp 2 ) , cos ( m 1 , m 2 ) \u27e9 . Comparing our new metrics ( especially the purpose + mechanism and purpose metrics , which ignore background ) to this baseline tests whether it is necessary to separate background and purpose , or whether it is sufficient to simply group all the purposes of a given paper as a single annotation type ( inspired by the purpose - mechanism method from Hope et al . [ 22 ] ) . 4 . 4 Performance Measures To evaluate the performance of the similarity metrics , we use Precision @ K , defined as the number of known analogies in the dataset found in the top K % of matches . Exploring different levels of K allows us to explore how the performance of a given metric changes depending on how conservative it is ( lower K = more conservative , which accepts a higher risk of false negatives in favor of a higher precision of matches returned to the searcher ) . These analogies were manually found by a member of the research team through exhaustive examination of the papers in two phases . First , the research team member created affinity maps of the research papers , and identified shared schemas and relations . Second , these schemas were used to find papers that fit the schema ( thereby defining analogy pairs ) . Finally , we used an initial prototype GloVe model ( with Common Crawl ) to suggest new matches we might have missed . We ended up with 259 analogy pairs ( approximately 21 % of 1225 total possible pairs across 50 papers ; see Table 2 for some example analogies found for the IdeaHound paper from Table 1 6 ) . The research team member who found the analogies in the dataset has 8 years of PhD - level expertise in researching social computing and analogy , providing both domain and process ( for finding analogies ) expertise . The team member considered papers to be analogically related if they share a relational mapping ( e . g . , similar purposes and mechanisms , or similar backgrounds and 6 The full set of known analogy pairs is available in the supplementary material Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 10 G . Zhou et al . K = 1 % 2 % 5 % 10 % 15 % 20 % 25 % all words baseline . 67 ( . 03 ) . 67 ( . 06 ) . 46 ( 11 ) . 37 ( . 18 ) . 35 ( . 25 ) . 31 ( . 29 ) . 29 ( . 34 ) bkground / purpose + mechanism . 84 ( . 05 ) . 84 ( . 09 ) . 64 ( . 16 ) . 50 ( . 24 ) . 43 ( . 30 ) . 37 ( . 35 ) . 33 ( . 39 ) purpose + mechanism . 92 ( . 05 ) . 92 ( . 09 ) . 73 ( . 18 ) . 50 ( . 24 ) . 40 ( . 29 ) . 36 ( . 34 ) . 34 ( . 40 ) purpose . 50 ( . 03 ) . 50 ( . 05 ) . 38 ( . 10 ) . 38 ( . 18 ) . 33 ( . 23 ) . 31 ( . 29 ) . 30 ( . 35 ) mechanism . 92 ( . 05 ) . 80 ( . 08 ) . 66 ( . 16 ) . 49 ( . 23 ) . 45 ( . 32 ) . 39 ( . 37 ) . 35 ( . 42 ) findings . 75 ( . 04 ) . 50 ( . 05 ) . 37 ( . 09 ) . 33 ( . 16 ) . 31 ( . 22 ) . 29 ( . 28 ) . 27 ( . 32 ) Table 3 . Proportion of known analogy pairs found in Study 1 for each metric , varying by K ( lower K = more conservative ; recall scores in parentheses ; pairs sorted by similarity ) . Best - performing scores at each K are bold - underlined . Our bkground / purpose + mechanism , purpose + mechanism , and mechanism metrics consistently find analogy pairs at substantially higher precision than the all words baseline at each level of K . purposes ) . For example , motivating contributions in citizen science relationally maps to motivating scientists to share their code ( e . g . , motivating ( x to contribute to y ) ) . Papers with similar backgrounds ( e . g . , vision science ) but different purposes and mechanisms would not be considered analogies . This is consistent with the literature on analogical mapping ( e . g . , [ 15 ] ) . Importantly , two papers with similar background would still be considered analogies if their purposes or mechanisms mapped relationally ; these are known in the analogy literature as near analogies . Note that because these are all social computing papers , many of the analogies are from the same / similar domains / areas ; therefore , we expect the all words metric to be able to find many of these near analogies . Separating background from purpose also may not be as important for finding near analogies . For this reason , our performance measure in this dataset is a conservative test of the efficacy of our mixed - initiative system . Yet , social computing is broad enough as a field that it allows for some cross - domain analogies ( e . g . , crowdsourcing grades for assignments in MOOCs , vs . crowdsourcing discovery of creative ideas in online innovation platforms ) . We examine precision for K \u2208 ( 1 , 2 , 5 , 10 , 15 , 20 , 25 ) . For each similarity metric , and for each K , we compute similarities between all 1 , 225 possible pairs in the dataset , rank based on those similarities , take the top K % of matches , and then compute how many of those matches were known analogies . 4 . 5 Results 4 . 5 . 1 We find unique analogies missed by the all words baseline . Table 3 shows the quantitative results of our experiment . Three of our similarity metrics ( bkground / purpose + mechanism , pur - pose + mechanism and mechanism ) consistently return a higher proportion of analogies at multiple settings of K , yielding on average gains of 26 % , 31 % , and 30 % in precision compared to all words . The advantage of our metrics is especially substantial at lower levels of K : for example , in the top 1 % and 2 % of matches , our best metric ( purpose + mechanism ) has both a large increase over the all words baseline ( a 37 % increase from . 67 to . 92 ) , and a high precision value in absolute terms . This is significant because we did not distinguish between near and far analogies , and there were many near analogies in the dataset ; these near analogies are likely to dominate at lower levels of K for all words ( which is tuned largely for surface similarity ) . Additionally , there was low overlap between the matches found by our metrics and those found by all words : for example , only 42 % of the purpose + mechanism , and 50 % of the mechanism matches were shared with the all words baseline at K = 5 . Referring back to the matches in Table 2 , the all words baseline was only able to find the near analogy ( \u201cTowards Collaborative Ideation at Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 11 Source : IdeaHound : Self - sustainable Idea Generation in Creative Online Communities Analogy : Crowd Guilds : Worker - led Reputation and Feedback on Crowdsourcing Platforms Top 10 nearest words for Background : ideas creative innovative inspirational analo - gies productively fictions brainstorm substrates wishing Background : workers decentralized crowd crowds dis - persed sourcing equitably collaborators volunteers harness Purpose : curate interleave scaffold organise devise stim - ulate formulate ideas consolidating mobilize Purpose : reputation scores score ratings accountabil - ity legitimacy fairness responsiveness scoring relevancy Mechanism : idea ideas ideation endeavor grassroots re - alization productively scaffold generative prospector Mechanism : crowd workers crowds guilds worker peer sourcing instructors equitably freelancing Table 4 . Illustrative analogy ( right ) found for the IdeaHound paper ( left ) in the CSCW dataset by our pur - pose + mechanism and purpose metrics , but missed by the all words and bkground / purpose + mechanism baseline metrics . Bold - underlined words denote conceptual overlap across matching vectors . Note how different the background vectors are , while there is overlap in concepts for both purpose and mechanism vectors . Both papers leverage community mechanisms [ grassroots , peersourcing ] for the purpose of curating things [ ideas , workers ] , despite key differences in background ( creativity / ideation vs . crowd labor markets ) . This example illustrates the value of separating higher - level ( background ) problems from lower - level ( purpose ) problems during annotation . Scale\u201d ) , while the other metrics were able to find both the near and far analogy ( \u201cCrowd Guilds : Worker - led Reputation and Feedback on Crowdsourcing Platforms\u201d ) . 4 . 5 . 2 Modifications to the annotation scheme are helpful . While bkground / purpose + mecha - nism finds more analogies than all words across all levels of K , it is consistently outperformed by purpose + mechanism ( which separates background and purpose ) across all levels of K . Inspection of the matches suggests that separating background and purpose allows us to find analogies between papers that have different higher - level goals ( and might be thought of as in different domains ) . Table 4 shows one example of this , where purpose + mechanism found an analogy between a paper on devising scalable methods to organise many ideas from an online creative community and distribute them to inspire community members ( \u201cIdeaHound : Self - Sustainable Idea Generation in Creative Online Communities\u201d ) , and a paper on using peer ratings to rank workers by quality in online labor markets ( \u201cCrowd Guilds : Worker - led Reputation and Feedback on Crowdsourcing Platforms\u201d ) . This match was missed by both all words and bkground / pur - pose + mechanism , in part because the background vectors are quite different from each other ( one heavily emphasizing creativity / ideation , and the other heavily emphasizing crowd work ) . 4 . 5 . 3 Findings annotations help find near analogies . While the findings metric does return significantly more matches in the top 1 % of pairs , it does not outperform the all words baseline at K > = 2 . This is partially explained by the high amount of overlap between the matches found by findings and those found by the all words baseline metric ( 64 % are shared , in contrast to the low levels of overlap for the purpose + mechanism and mechanism metrics ) . We will return to this point in the discussion . 5 STUDY 2 : USING SOLVENT TO FIND ANALOGIES WITH REAL - WORLD VALUE Study 1 demonstrated that applying solvent to a corpus of social computing papers enabled us to retrieve known analogical relationships , as defined by a domain expert . In Study 2 , we explore Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 12 G . Zhou et al . whether solvent can provide real - world value for researchers actually looking for analogical inspirations for their work . 5 . 1 Scenario We evaluated our approach with a local mechanical engineering research group at a highly research active , private research university in the Midwestern United States . The group is working on cutting - edge interdisciplinary work at the intersection of bioengineering and mechanical engineering : specifically , they are exploring how to create interesting new 2D and 3D structures at many different scales by stretching / folding polymers . The research group has been struggling to find examples of this work to build on and compare against : it is distributed across such diverse domains as mechanical engineering , civil engineering , aerospace engineering , materials science , mathematics , and design , among others , many of which are outside of the specific domain expertise of the research group . Some examples of their analogy information needs include : \u2022 Competitive analysis : find work from other research groups that are attacking the same problems with similar techniques \u2022 Inspiration : find relevant research that can suggest new properties of polymers to leverage , or new techniques for stretching / folding or exploring 2D / 3D structure designs \u2022 Application : find novel application domains that could benefit from the unique advantages of their fabrication method ( e . g . , non - invasively constructing temporary medical implants in situ ) The team has spent the last year or so conducting literature searches using various standard approaches ( e . g . , Google Scholar , library databases , citations in relevant [ review ] papers , conver - sations with colleagues ) , and have been frustrated by how slow and error - prone the process has been . They are also concerned that they are missing things . The difficulty stems in part from the relative newness of the field , and the degree of fragmentation of knowledge across many different disciplines . In this evaluation , we explore whether solvent can help them find analogies they were not able to find through keyword / database and citation tree search . 5 . 2 Dataset Based on conversations with the group , we identified three out - of - domain sources of research papers to mine from : materials science , civil engineering , and aerospace engineering . The papers were sampled from the 1000 most highly cited papers of 2016 - 2017 ( indexed on Web of Science ) for each of the 3 domains . Two members of our research team ( neither of whom have any training or formal knowledge in any of the domains involved in this scenario ) annotated 90 papers sampled from this larger corpus . As before , rather than use a semantic model trained on a larger but more generalized corpus ( e . g . , Common Crawl ) , we used a model trained on a smaller but more relevant corpus of documents to create aggregate vectors . Specifically , we used word vectors from a continuous - bag - of - words ( CBOW ) word2vec model [ 32 ] trained on 3 , 000 papers in the dataset . We then used the same method as before to create annotation - specific semantic vectors for each of the 90 annotated papers . 5 . 3 Evaluation We used an abstract for one of the research group\u2019s recent manuscripts as a query document . We first highlighted and constructed semantic vectors for the abstract , and then sampled the top 20 matches for the abstract from each of our similarity metrics from before . This corresponds to a Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 13 setting of K at approximately 20 % ( top 20 most similar out of 90 possible matches ) ; we chose this less conservative setting of K to reflect our prior expectation that the number of cross - domain matches that would be useful to the team is likely to be relatively few . We collapsed duplicates and blinded them by removing information about which metric produced the match , resulting in a set of 53 unique possible matches . We showed these matches to a member of their research group ( the lead PhD student ) . We asked the researcher look through the matches as s / he might look through a Google Search result list , and evaluate 1 ) whether each match is in fact useful for their literature review , 2 ) why ( not ) , and 3 ) whether they had previously encountered that match . 5 . 4 Results 5 . 4 . 1 Our metrics find more analogies than all words baseline . Overall , the researcher identified 7 out of the 53 matches as relevant and new . This reflects the extremely challenging setting of finding useful and novel analogies among 90 randomly sampled papers from outside the core domain of the work . 5 out of those 7 analogies were found by one of / both the purpose + mechanism and mechanism similarity metrics ( an aggregate precision of approximately . 25 ; comparable to precision at K = . 2 in Study 1 ) , while 2 were found by the all words metric ( precision = . 1 ) . 5 . 4 . 2 Our metrics find different analogies from all words baseline . In addition to this numerical difference , there was also a qualitative difference in the types of matches returned . To illustrate , the most relevant match found by the all words metric was almost a direct replication of their work ( manipulating the structure of a membrane in a controlled way by applying tension to the membrane ) from an aerospace engineering paper . The researcher was uncertain if she had seen this paper before , and found it useful for competitive analysis . In contrast , the most relevant match found by the purpose + mechanism metric was a paper on using multi - agent systems to generate a variety of geometric structures . The mechanism metric also found an interesting potential analogy of a civil engineering paper analyzing web crippling phenomena in steel beams under tension . Both of these matches were judged to be both potentially useful ( the first for inspiring alternative approaches to systematically explore 2D / 3D structures by applying different levels and types of tension , the second for potentially inspiring new mathematical models to analyze their polymers , which could reveal new properties to leverage for their designs ) and novel ( neither had been seen by the researcher ) . 6 STUDY 3 : SCALING UP SOLVENT THROUGH CROWDSOURCING Thus far we have shown that solvent increases our ability to find known analogies , as well as analogies that can provide real - world value to a domain expert . However , in both Study 1 and 2 , our annotations were sourced from expert researchers , who make up a highly limited resource pool . In this final section , we explore how we might scale up solvent by crowdsourcing annotations from a wider range of non - experts who might provide a much larger pool of possible human judgments . Study 2 ( where researchers annotated papers outside of their domain / discipline ) suggests that annotation does not critically depend on domain knowledge ; still , non - experts with little to no experience writing or reading research papers as a \u201cgenre\u201d may produce annotations that are too noisy to support effective analogy - mining ( or require expensive quality control mechanisms that would make the cost of crowdsourcing prohibitively high ) . Therefore , in Study 3 , we explore whether crowdsourced annotations for papers in the set of 50 CSCW papers can replicate or approximate the analogy - finding gains shown in Study 1 with researcher annotations . We crowdsource annotations from workers on Upwork ( where we recruited workers with general writing expertise but no Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 14 G . Zhou et al . Fig . 1 . A screenshot of the crowd - facing interface for annotating the abstracts . Workers annotate aspects by first selecting the annotation type ( an in - line description is provided as a reminder ) and then using an intuitive click - and - drag highlighting interaction to annotate one or more words . domain or research experience ) and workers on Amazon Mechanical Turk ( who represent a larger population of workers with little or no expertise requirements ) . 6 . 1 Crowdsourcing setup Workers used an intuitive drag - and - highlight interaction to annotate each abstract ( see Figure 1 ) . We included a training step before the main task : 2 gold standard examples to illustrate the annotations in context , and a single training example ( where workers would first annotate and then compare with the gold standard ) . 6 . 1 . 1 Deployment on Upwork . We recruited two workers with general copywriting expertise from Upwork . The first worker held a bachelor\u2019s degree in English Literature , and a master\u2019s degree in business administration , had more than nine years experience writing and editing academic , business , literary and technical documents , and was paid at a rate of $ 30 / hr . The second worker held a master\u2019s degree in English Literature and a certificate in Publishing , had experience with editing book - length projects for major publishers and creating research dossiers for news publications , and was paid at a rate of $ 20 / hr . Annotation times were similar to the MTurk and Study 1 deployments , with a median completion time per document of 1 minute . Each worker provided annotations for half ( 25 ) of the papers in the dataset . Thus , each paper\u2019s annotations was based on one worker , similar to the deployment with domain - expert researchers in Study 1 . 6 . 1 . 2 Deployment on Amazon Mechanical Turk . We screened for workers with at least 95 % approval rate for at least 5 , 000 tasks . The overall task ( including login , training , and the actual task ) took a median of 4 . 0 minutes ; actual time annotating the main document took a median of 1 . 3 minutes . We paid workers $ 0 . 70 for each task completion , for an effective average hourly rate of $ 10 / hr . We obtained annotations from an average of 3 workers for each document . We aggregated annotations across workers for each word by majority vote ( weighted by each worker\u2019s performance on the training example ) . 6 . 2 Results 6 . 2 . 1 Crowd annotations had substantial agreement with researcher annotations . Table 5 shows the accuracies ( i . e . , agreement with researcher annotations ) by annotation type . Overall , Upwork Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 15 Overall Bkgd Find Mech Purp Upwork 0 . 78 0 . 75 0 . 94 0 . 72 0 . 68 MTurk 0 . 59 0 . 66 0 . 71 0 . 53 0 . 42 Table 5 . Crowd annotation agreement with researcher annotations K = 1 % 2 % 5 % 10 % 15 % 20 % 25 % all words . 67 ( . 03 ) . 67 ( . 06 ) . 46 ( 11 ) . 37 ( . 18 ) . 35 ( . 25 ) . 31 ( . 29 ) . 29 ( . 34 ) bkground / purpose + mechanism Upwork . 75 ( . 04 ) . 67 ( . 06 ) . 64 ( . 15 ) . 43 ( . 20 ) . 39 ( . 28 ) . 33 ( . 31 ) . 31 ( . 37 ) MTurk . 75 ( . 04 ) . 63 ( . 06 ) . 57 ( . 14 ) . 43 ( . 20 ) . 34 ( . 24 ) . 32 ( . 30 ) . 29 ( . 34 ) purpose + mechanism Upwork . 83 ( . 04 ) . 75 ( . 07 ) . 54 ( . 13 ) . 42 ( . 20 ) . 34 ( . 24 ) . 33 ( . 31 ) . 30 ( . 35 ) MTurk . 83 ( . 04 ) . 71 ( . 07 ) . 52 ( . 13 ) . 42 ( . 20 ) . 37 ( . 26 ) . 32 ( . 30 ) . 31 ( . 36 ) mechanism Upwork . 75 ( . 04 ) . 58 ( . 06 ) . 51 ( . 12 ) . 39 ( . 19 ) . 33 ( . 23 ) . 31 ( . 30 ) . 30 ( . 36 ) MTurk . 75 ( . 04 ) . 54 ( . 05 ) . 44 ( . 11 ) . 34 ( . 16 ) . 32 ( . 23 ) . 29 ( . 27 ) . 28 ( . 33 ) Table 6 . Proportion of known analogies found at each level of K for each similarity metric , with annotations by Upwork and MTurk crowd workers ( recall in parentheses ; best - performing scores bold - underlined . Our bkground / purpose + mechanism and purpose + mechanism metrics continue to outperform the all words baseline ( with substantial reductions from researcher - based annotations ) , while the mechanism metric\u2019s advantage is almost entirely eliminated for both Upwork and MTurk annotations . workers\u2019 annotations matched the researcher annotations 78 % of the time , and MTurk workers\u2019 an - notations matched 59 % of the time . There was considerable variation across papers , with accuracies as high as 96 % agreement for some papers , and as low as 4 % for others . 6 . 2 . 2 Crowds struggled with purpose and mechanism annotations . In general , both groups of workers struggled most with purpose and mechanism annotations ( 68 % and 72 % for the Upwork workers , and 42 % and 53 % agreement for the MTurk workers ) . First , workers often confused background and purpose . The most frequent error for background annotations was incorrectly annotating it as purpose ( 13 % of background annotations for Upwork ; 21 % for MTurk ) . Purpose was also frequently confused for background ( 10 % of purpose annotations for Upwork ; 16 % for MTurk ) . Mechanism was also frequently confused for purpose and findings : the most frequent error for purpose was incorrectly annotating it as mechanism ( 17 % of purpose annotations for Upwork ; 28 % for MTurk ) , while mechanism was confused for purpose and findings quite frequently as well ( 8 % and 9 % of mechanism annotations for Upwork ; 10 % and 31 % for MTurk ) . One possible explanation is that researchers frequently mix multiple aspects in their sentences : for example , they often described the purpose and mechanism of their paper in the same sentence , or described the mechanism in more detail by claiming that it worked ( findings ) . We return to this point in the discussion to consider how our scheme might be modified to account for variations in writing style . 6 . 2 . 3 Crowd annotations still improve analogy - mining . As Table 6 shows , despite the modest levels of agreement , specialized vectors constructed from crowd workers\u2019 aggregated annotations were still able to support bkground / purpose + mechanism and purpose + mechanism metrics that Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 16 G . Zhou et al . outperformed the all words baseline , albeit with substantial reductions in the size of the advantage . In particular , the purpose + mechanism metric outperforms all words throughout the range of K , for both Upwork and MTurk deployments . Note that the advantage of the specialized metrics is reduced the least for bkground / pur - pose + mechanism ( retaining substantial advantages over all words , and even outperforming purpose + mechanism , past K = 2 ) , while the advantage of the mechanism metric disappears past K = 1 . The generally low accuracy for purpose and mechanism annotations might explain these patterns , since the purpose + mechanism and mechanism metrics depend critically on these annotations . 7 DISCUSSION 7 . 1 Summary and Implications of Findings Across three studies , we show that applying solvent to research papers yields a substantial improvement ( approximately 25 - 30 % overall improvement in Study 1 ) over a state - of - the - art infor - mation retrieval ( IR ) technique ( TF - IDF - weighted average semantic vectors ) for finding analogies between those research papers . The advantage holds for near analogies ( where standard IR is expected to perform well ) , and across domains ( seen especially in Study 2 with the mechanical engineering research group ) . We further show that these annotations do not require extensive con - tent / domain expertise to be useful : researchers can produce useful annotations for papers outside their discipline ( Study 2 ) , and naive crowd workers can produce useful annotations for research papers ( Study 3 ; albeit with noticable reductions in effectiveness compared to researcher - sourced annotations ) . Across these deployments , we find that the time cost of annotation is approximately a minute per paper . Our modified annotation scheme ( and the usefulness of partialing out background , or leveraging findings ) points to solvent \u2019s core insight ( which extends Hope et al\u2019s [ 22 ] approach ) : \" dissolving \" documents into relational elements ( e . g . , purpose , mechanism , findings ) that have in - built causal relations with each other enables us to create soft relational schemas that are useful for analogical matching . This insight might prove useful in other domains than research papers : for example , annotating the precedents , facts , and decisions in legal cases to support case law reasoning , annotating student learning goals and associated exercises and / or examples in lesson plans to support innovation in teaching , or annotating plot trajectory and associated tropes in writing . While we suspect that solvent might be useful in many of these domains , we think that domains where these dimensions are hidden / obscured by surrounding texts that serve many other functions ( e . g . , preambles in legal opinions ) might especially benefit from solvent \u2019s soft schema extraction techniques . This insight opens up a design space that could help resolve the cost - accuracy tradeoff for computational analogy : rather than spending prohibitively high amounts of resources to specify rich relational structures manually ( thereby maximizing accuracy for analogical matching ) , or completely automating knowledge modeling ( but ignoring structure , thereby losing accuracy for analogical matching ) , we can explore the middle ground of extracting soft relational schemas in a cost - effective manner . This design space mirrors current Semantic Web efforts that seek to relax formality requirements for shared ontologies , and explore how machine - and crowdsourced semantics might complement or even replace more formal and precise ( but infeasible to obtain for the broader Web ) ontologies [ 4 ] . 7 . 2 Limitations and Future Work 7 . 2 . 1 Extending to larger datasets . While our results show significant promise , we acknowledge that our datasets are relatively small . This is partially mitigated by the diversity of domains represented : our data includes papers from diverse domains and contribution types / framings ( e . g . , Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 17 civil engineering papers on numerical analysis of beam strength used a different structure than empirical CHI paper abstracts ) . However , we do aim to test this approach across larger and more diverse corpora . Part of the bottleneck is having a good set of gold standard matches that we can use to evaluate our approach : conservatively , many datasets might contain approximately 5x as many analogy pairs as there are papers ( similar to the 259 known analogy pairs in our set of 50 papers in Study 1 ) ; finding analogies by hand remains a costly process , and having relatively complete coverage of known analogies is critical for meaningful precision and recall performance measures . However , alternative evaluations ( e . g . , experiments with searchers , real - world prototype deployments ) are possible . Extending the method to larger datasets may require increases in the scalability of the method . However , our findings with the crowdsourcing study suggest that obtaining annotations for more realistic - sized datasets may not be cost - prohibitive : for example , our deployments suggest that annotating a corpus of 10 , 000 papers would require on the order of 10 \u2013 30 , 000 person - minutes ( or 166 - 500 person - hours ) of work . This suggests that such a corpus could be annotated for as little as under $ 4 , 000 ( assuming a single trained full - time employee working at $ 20 / hr for 1 - 2 months , similar to one of our Upwork workers ) , quite possibly a significant bargain given the high potential upside of transformative cross - disciplinary breakthroughs and reductions in redundant work and dead ends . This work could also be crowdsourced for a comparable cost ( and significantly quicker turnaround time ) , although more sophisticated aggregation and quality control mechanisms than majority vote ( see , e . g . , [ 39 ] ) , or worker - centric screening / training strategies [ 34 ] might be necessary for crowdsourcing efforts similar to our MTurk deployment . Further , with a few thousand papers , ( semi ) supervised machine learning models like recurrent neural networks ( e . g . , as in Hope et al [ 22 ] ) or conditional random - field models might be trained to replace or augment human judgments . As noted earlier , we are also intrigued by the possibility of more \u201ccommunity - sourced\u201d models of crowdsourcing , where community members contribute semantic annotations as a seamless part of the primary task they are motivated to do ( e . g . , bookmarking important steps in a how - to video [ 25 ] , or arranging ideas on a virtual whiteboard in the natural course of brainstorming [ 40 ] ) . In this spirit , could we design a future where the many hours that scientists spend reading , annotating , organizing , and summarizing research papers ( e . g . , for literature reviews , or peer review ) could contribute not just to their primary task of doing research , but also towards supporting a computational infrastructure for knowledge sharing ? These approaches could pave the way towards more self - sustainable computational infrastructures for knowledge sharing . Note also that in some fields ( e . g . , human factors , biomedical research ) , structured abstracts are commonly produced by researchers , suggesting that the publication process could be modified slightly to author - source these annotations at a tolerable / minimal cost to authors , particularly if the value of these annotations was made apparent to the authors . 7 . 2 . 2 Ensuring unbiased coverage . Recall that we modified the annotation scheme ( e . g . , adding a findings annotation ) to reflect the different epistemological goals that exist among research papers ( e . g . , understanding vs . system - building ) . We had mixed success with the findings annotations : at the highest level of matching ( top 1 % of matches ) , the metric outperformed the all words baseline , but not at higher levels of K . One possible explanation is that our modest - sized dataset didn\u2019t contain enough \" same - findings \" analogies to have high precision at higher levels of K . Another possible explanation is that our core insight of getting a schema \" for free \" by identifying separate sets of annotations that are joined by a causal relation glosses over the relational structure within an annotation type - in the case of understanding - oriented papers , the relevant schema may consist of relations between objects as hypothesized in the purpose statement , or in the findings ( e . g . , people Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . 31 : 18 G . Zhou et al . decide when to self - disclose on social media depending on how the disclosure might reflect on their online identity ) . Relatedly , our conversations with the mechanical engineering researchers revealed that the properties of the objects ( e . g . , the particular ways that polymers respond to physical loads ) are a key aspect of identifying relevant analogies to work in other domains . Unfortunately , these properties are often left implicit in the text . This challenge could be addressed by augmenting the vector representations of the documents with key properties ( e . g . , from knowledge bases like ConceptNet 7 and CYC 8 ) . How to select which among the numerous possible properties of an entity might be useful for analogical matching would be an interesting and challenging problem for future work ; some recent work is already beginning to explore this for consumer product descriptions [ 17 ] More generally , we want to ensure that relying on this annotation schema does not cause us to systematically miss or misrepresent the contributions of papers written in different genres ( e . g . , that don\u2019t emphasize explaining mechanisms , or emphasize reporting findings instead of clearly stating the problem being addressed ) , or with different kinds of contributions ( e . g . , conceptual / theory or review papers ) , or levels of writing / communication quality . We did notice some limitations in our deployments , particularly in trying to apply a purpose / mechanism to review / survey papers , when mechanisms were dropped in favor of motivating the problem in shorter abstracts ( possibly with strict word limits ) , or when the research problem was not clearly / explicitly stated ; anecdotally , we noticed that this last writing style ( missing problem statements , but rich descriptions of findings and methods ) seemed to be especially common in our materials science and engineering papers ; this might partially explain the lower performance of our metrics in Study 2 ( in addition to the difficulty of explicitly searching in randomly sampled papers from other domains ) . Future work that builds on this should explore how we might expand the annotation schemes to fit different genres / contributions ( while retaining the central insight of identifying soft schemas ) , or supplement the knowledge models constructed by our methods with alternative methods that are less dependent on the way a paper is written . 7 . 2 . 3 Usefulness in Real - World Settings . Finally , while our metrics outperformed our instantiation of a traditional state - of - the - art content - based information retrieval approach ( i . e . , leveraging word embedding representations of the whole abstracts ) , our absolute precision was high only for very conservative ( low ) levels of K ( e . g . , recommending only the top 1 % most similar pairs as analogies ) . In smaller search spaces or for niche topics with fewer \" true \" matches , potential users of our approach might have to work harder to find good matches ; in many cases , however , we believe that even returning the top 1 % most similar matches might return more potential matches than the average user would want to sift through . Our approach also differs significantly from other production systems in that it ignores other important signals available from the citation graph ( e . g . , quality , relatedness ) . In practice , we think it would be useful to combine our content - based approach with graph - based approaches [ 42 ] , e . g . , re - ranking with graph - based signals after retrieving initial candidates via our approach , or re - ranking using our approach after retrieving initial candidates via graph - based approaches . 8 CONCLUSION In this paper , we introduced solvent , a mixed - initiative system for re - representing and finding analogies between research papers across different domains , in which humans produce lightweight annotations of key aspects of research paper abstracts , and a computational model constructs semantic vectors from the annotations and uses them to finds analogies between the papers . We 7 http : / / conceptnet . io / 8 http : / / www . opencyc . org / Proc . ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 . Mixed Initiative System for Finding Analogies 31 : 19 show that solvent holds promise for efficiently finding analogies between research papers across domains , opening up novel pathways towards computationally augmenting knowledge sharing for scientific progress . 9 ACKNOWLEDGEMENTS This work was funded by NSF grants CHS - 1526665 and CHS - 1816242 , ISF grant 1764 / 15 , the HUJI Cyber Security Research Center in conjunction with the Israel National Cyber Bureau in the Prime Minister\u2019s Office , Carnegie Mellon University\u2019s Web2020 Initiative , and Bosch Research Institute . We thank Yla Tausczik and Ping Wang for comments on earlier drafts of this manuscript . REFERENCES [ 1 ] Paul Andr\u00c3\u013e , Haoqi Zhang , Juho Kim , Lydia Chilton , Steven P . Dow , and Robert C . Miller . 2013 . Community clustering : Leveraging an academic crowd to form coherent conference sessions . In First AAAI Conference on Human Computation and Crowdsourcing . 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ACM Hum . - Comput . Interact . , Vol . 2 , No . CSCW , Article 31 . Publication date : November 2018 .", + "reinhardt2023angstromresolution": "Nature | Vol 617 | 25 May 2023 | 711 Article \u00c5ngstr\u00f6m - resolution fluorescence microscopy Susanne C . M . Reinhardt 1 , 2 , 5 , Luciano A . Masullo 1 , 5 , Isabelle Baudrexel 1 , 3 , 5 , Philipp R . Steen 1 , 2 , 5 , Rafal Kowalewski 1 , 2 , Alexandra S . Eklund 1 , 3 , Sebastian Strauss 1 , 2 , Eduard M . Unterauer 1 , 2 , Thomas Schlichthaerle 1 , 2 , Maximilian T . Strauss 1 , 2 , Christian Klein 3 , 4 & Ralf Jungmann 1 , 2 \u2709 Fluorescence microscopy , with its molecular specificity , is one of the major characterization methods used in the life sciences to understand complex biological systems . Super - resolution approaches 1 \u2013 6 can achieve resolution in cells in the range of 15 to 20 nm , but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires \u00c5ngstr\u00f6m resolution . State - of - the - art super - resolution implementations 7 \u2013 14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions . However , such resolutions do not directly translate to experiments in cells , and \u00c5ngstr\u00f6m resolution has not been demonstrated to date . Here we introdue a DNA - barcoding method , resolution enhancement by sequential imaging ( RESI ) , that improves the resolution of fluorescence microscopy down to the \u00c5ngstr\u00f6m scale using off - the - shelf fluorescence microscopy hardware and reagents . By sequentially imaging sparse target subsets at moderate spatial resolutions of > 15 nm , we demonstrate that single - protein resolution can be achieved for biomolecules in whole intact cells . Furthermore , we experimentally resolve the DNA backbone distance of single bases in DNA origami with \u00c5ngstr\u00f6m resolution . We use our method in a proof - of - principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug - treated cells , which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy . These observations demonstrate that , by enabling intramolecular imaging under ambient conditions in whole intact cells , RESI closes the gap between super - resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems . The localization precision ( \u03c3 SMLM ) of a target molecule in widefield single - molecule localization microscopy ( SMLM ) 15 is ultimately and fundamentally limited by the number of photons ( N ) collected per blinking event : \u03c3 \u2248 \u03c3 N SMLM DIFF ( \u03c3 DIFF is the s . d . of the point spread function ( PSF ) of the optical imaging system 16 ; Fig . 1a ) . Multiple localizations of the same target ( Fig . 1b , top ) are distributed around the true position due to their finite precision . Two or more points not resolvable by SMLM produce overlapping distributions of localizations , thus pre - cluding unique assignment of localizations to respective targets ( Fig . 1b , bottom ) . However , if each localization could be assigned to a specific target by colour , barcode or any other molecular identity , they could be unambiguously grouped per target 2 . The centre of each group of localizations can be calculated with a precision far better than \u03c3 SMLM . In essence , applying the principle of localization microscopy to distinguishable groups of K super - resolved localizations , precision is increased from the s . d . ( \u03c3 SMLM ) to the s . e . m . ( \u03c3 K SMLM ) . Collecting an arbitrarily large number of localizations yields an arbitrary increase in precision . Notably , this increase in precision occurs regardless of the precision achieved in individual localizations ( \u03c3 SMLM ) . We introduce a straightforward implementation of this concept using Exchange - PAINT 17 , a variant of DNA - PAINT 18 , for identical target molecules ( Fig . 1c ) . DNA - PAINT uses the programmable , repetitive but transient binding of dye - labelled \u2018imager\u2019 strands to their com - plementary \u2018docking\u2019 strands on target molecules of interest 9 , 18 . The transient nature of the binding leads to an apparent \u2018blinking\u2019 of the target , necessary to perform SMLM . Exchange - PAINT uses orthogonal DNA barcodes combined with imaging and washing cycles to allow for sequential target multiplexing . In our implementation we \u2018multiplex\u2019 a single target species by separating it into multiple , sparser subsets . By imaging them sequentially , sufficiently spaced and isolated groups of localizations are measured . Determining the centre of each group of localizations yields a resolution enhancement ( Fig . 1d ) . We call this implementation resolution enhancement by sequential imaging ( RESI ) , and the resulting localizations RESI localizations . https : / / doi . org / 10 . 1038 / s41586 - 023 - 05925 - 9 Received : 21 July 2022 Accepted : 7 March 2023 Published online : 24 May 2023 Open access Check for updates 1 Max Planck Institute of Biochemistry , Planegg , Germany . 2 Faculty of Physics and Center for NanoScience , Ludwig Maximilian University , Munich , Germany . 3 Department of Chemistry and Biochemistry , Ludwig Maximilian University , Munich , Germany . 4 Roche Innovation Center Zurich , Roche Pharma Research and Early Development , Schlieren , Switzerland . 5 These authors contributed equally : Susanne C . M . Reinhardt , Luciano A . Masullo , Isabelle Baudrexel , Philipp R . Steen . \u2709 e - mail : jungmann @ biochem . mpg . de 712 | Nature | Vol 617 | 25 May 2023 Article By application of RESI in silico ( Methods ) , we demonstrated a reso - lution improvement ( Extended Data Fig . 1 ) over super - resolution akin to the improvement of super - resolved over diffraction - limited meas - urements ( Fig . 1e ) . For routinely obtainable DNA - PAINT localization precision ( approximately 3 nm ) and number of localizations per target ( in the order of hundreds ) , RESI could achieve precision well below one nanometre , thus entering the \u00c5ngstr\u00f6m scale ( Fig . 1f ) according to \u03c3 = \u03c3 K RESI SMLM . For an experimental proof of principle of RESI we used self - assembled DNA origami structures to precisely position orthogonal DNA strands 9 , 19 . We first designed DNA origami featuring two docking strands spaced 5 nm apart , a distance previously resolved with DNA - PAINT 7 , 9 , to verify the accuracy and precision of RESI . Using two sequential imag - ing rounds and an alignment procedure ( Methods ) to conduct RESI , we were able to accurately recapitulate the 5 nm point - to - point distance with precision improved by a factor of K = 381 \u224820 average ( Extended Data Figs . 2 and 3 ) . We next performed RESI in three dimensions ( 3D ) using recently developed 3D DNA origami disk structures 20 and measured distances between docking strands of 2 . 5 \u00b1 0 . 4 nm in xy and 11 . 3 \u00b1 0 . 8 nm in z . This demonstrates that RESI resolution enhancement applies in all three dimensions , surpassing current state - of - the - art 3D super - resolution capabilities ( Extended Data Figs . 4 and 5 ; for imaging parameters see Extended Data Table 1 ) . RESI resolves single nuclear pore complex proteins To demonstrate the applicability of RESI in a cellular context , we next imaged structural proteins of the nuclear pore complex ( NPC ) . As the major gatekeeper of nucleocytoplasmic transport , the NPC is a key target for structural biology research 21 . We furthermore chose the NPC as a model system because it has been well studied using a variety of imaging approaches , including cryo - electron microscopy ( cryo - EM ) 22 , fluorescence microscopy and super - resolution techniques 23 , 24 . Figure 2a presents a typical diffraction - limited and DNA - PAINT image of Nup96 molecules ( tagged with monomeric enhanced green fluo - rescent protein ( mEGFP ) ) labelled with DNA - conjugated anti - GFP nanobodies . Nup96 is a structural NPC protein ( part of the so - called Y - complex ) present in eight pairs exhibiting an eight - fold symmetry on both cytoplasmic and nuclear rings , totalling 32 copies per NPC ( Fig . 2b ) . Individual pairs of Nup96 proteins , spaced approximately 10 nm laterally and 3 nm axially , cannot be routinely resolved with current state - of - the - art super - resolution implementations 25 \u2013 28 . To enable RESI , neighbouring copies of Nup96 proteins must be labelled with orthogonal DNA sequences . To this end , we opted for a stochas - tic labelling approach by incubating the sample with anti - GFP nano - bodies , each conjugated with one out of four orthogonal sequences ( Fig . 2c ) . We note that , with an increasing number of expected targets below the classical DNA - PAINT resolution limit , a larger number of orthogonal labelling sequences 29 \u2014and thus imaging rounds\u2014is nec - essary to guarantee sufficiently spaced groups of localizations ( for details on this requirement see Methods ) . Sequential 3D image acqui - sition in four rounds led to sufficiently spaced localization groups representing single Nup96 target molecules ( Fig . 2d ) . Subsequent RESI super - localization of these groups allowed us to routinely visu - alize individual copies of Nup96 proteins ( Fig . 2e ) . We note that this was achieved across the whole field of view ( roughly 67 \u00d7 67 \u00b5m 2 ) totalling over 1 , 000 NPCs during a total image acquisition time of Single - molecule localization RESI a c b d e f (cid:86) RESI \u2248 K (cid:86) SMLM K (cid:86) R E S I ( n m ) Diffraction limited DNA - PAINT RESI 10 40 0 2 1 \u00c5 scale K = 10 K = 40 K = 100 100 300 nm 10 nm 10 nm 2 nm 2 nm 2 nm d 2 < 2 . 35 (cid:86) SMLM d 1 > > (cid:86) SMLM ~ DNA - PAINT d 1 \u2248 20 nm d 2 \u2248 2 nm (cid:86) SMLM (cid:86) SMLM \u2248 N (cid:86) DIFF (cid:86) DIFF 10 nm 1 \u03bc m 200 nm Sequential super - resolution K localizations I m a g i n g r o un d s (cid:86) SMLM (cid:86) RESI 10 nm 10 nm Round 1 Round 2 Sequence 1 Sequence 2 Round n Round 2 Round 1 Fig . 1 | RESI concept . a , In SMLM , \u03c3 SMLM of a single dye scales with \u03c3 N DIFF , ultimately limiting the achievable spatial resolution . b , SMLM approaches such as DNA - PAINT feature approximately 10 nm spatial resolution ( resolution approximated as full - width at half - maximum \u2248 2 . 35 \u03c3 SMLM ) . Whereas targets separated by 20 nm ( d 1 ) can thus be routinely resolved , objects spaced 2 nm apart ( d 2 ) are unresolvable because the resulting distributions of localizations overlap . c , Using orthogonal DNA sequences ( blue and green ) and sequential acquisition as in Exchange - PAINT , localizations from targets spaced more closely than the SMLM resolution limit can be unambiguously assigned for each target . d , Combining all localizations per target ( K ) for each imaging round improves localization precision from s . d . ( \u03c3 SMLM ) to s . e . m . ( \u03c3 RESI ) . e , As super - resolution revolutionized fluorescence microscopy , RESI results in another paradigm shift by reapplying the concept of localization to super - resolution data . f , Localization precision in RESI scales with K 1 , and thus resolution improvement in RESI is independent of \u03c3 SMLM , reaching localization precision on the \u00c5ngstr\u00f6m scale . Nature | Vol 617 | 25 May 2023 | 713 100 min ( see Extended Data Fig . 6 for representative data ) . The recon - structed RESI image features an average lateral localization precision of approximately 1 nm , representing a sixfold improvement over the individual DNA - PAINT acquisition rounds . We therefore achieved label - size - limited resolution , allowing us to resolve individual Nup96 molecules ( Fig . 2f ) . Generally , label size not only limits spatial reso - lution but furthermore could lead to inaccuracies such as biased observed distances due to linkage errors . We then performed unbiased 3D averaging of 1 , 217 NPCs using a recently developed model - free approach for SMLM data 30 . The result - ing 3D average ( Fig . 2g ) not only allows recapitulation of the eight - fold symmetry of Nup96 in both cytoplasmic and nuclear rings ( which has previously been achieved with super - resolution 23 \u2013 28 ) , but enables reso - lution of individual Nup96 proteins in a structural average ( Fig . 2h ) . Enabled by RESI\u2019s unprecedented spatial resolution , we were able to recapitulate distances of Nup96 proteins of 11 . 9 \u00b1 1 . 2 nm laterally and 5 . 4 \u00b1 0 . 4 nm axially from the structural average image ( Fig . 2i ) . Both lateral and axial orientation , as well as tilt , of Nup96 pairs are consist - ent with cryo - EM data 22 . We resolved this spatial arrangement for most Nup96 protein pairs ( Extended Data Fig . 7 ) , which was previously out of reach for optical microscopy . Imaging DNA bases at \u00c5ngstr\u00f6m resolution To assay the ultimately achievable spatial resolution by RESI , we designed a flat , rectangular DNA origami structure featuring six pairs ( spaced 20 nm apart ) of directly adjacent orthogonal docking strands at a distance of only one DNA base pair ( red and blue strands in Fig . 3a ) . This yielded a designed in - plane distance of around 7 \u00c5 along the backbone of one strand of the DNA double helix 31 . The structures also contain DNA - PAINT docking strands for precise alignment between sequential imaging rounds ( green strands in Fig . 3a ) . State - of - the - art DNA - PAINT image acquisition 32 at approximately 5 nm spatial reso - lution yielded six localization clouds in a 20 nm grid arrangement but failed to resolve the individual docking strands at subnanometre single - base - pair distances ( Fig . 3b ) . Remarkably , RESI resolves the individual docking strand positions ( Fig . 3c ) in all DNA origami structures . We note that RESI achieved this D i ff r ac t i o n li m i t e d D N A - PA I N T NR CR NR 0 80 Side view 0 40 Position ( nm ) 0 30 P o s i t i o n ( n m ) (cid:86) \u2248 5 \u00c5 (cid:86) \u2248 10 \u00c5 (cid:86) \u2248 10 \u00c5 3D RESI 3 D D N A - P A I N T 3 D R E S I Round 1 Round 2 Round 3 Round 4 Nup96 - GFP nanobody CR NR 11 . 9 nm 5 . 4 nm 20 NPC NPC structure Stochastic labelling Z p o s i t i o n ( n m ) 5 \u03bc m 500 nm a b c d Sequential 3D DNA - PAINT imaging 20 nm 5 nm Side NR CR 20 nm 20 \u00c5 20 nm e f g h i CR 20 nm x y z z y x x y z 3 . 2 nm 1 0 . 3 n m Fig . 2 | NPC proteins in whole cells resolved with \u00c5ngstr\u00f6m precision by RESI . a , Diffraction - limited and DNA - PAINT overview image of Nup96 - mEGFP labelled with DNA - conjugated anti - GFP nanobodies . Zoomed - in view ( bottom right ) shows high labelling efficiency and image quality for standard DNA - PAINT conditions , recapitulating the eight - fold symmetry of the NPC . b , Cryo - EM structure representation of the location of Nup96 proteins ( red ; C - terminal mEGFP position marked in blue ) as part of the Y - complex in nuclear and cytoplasmic rings ( NR and CR , respectively ) . Adapted from PDB 7PEQ . Nup96 is present in 32 copies per NPC . c , To enable RESI , Nup96 - mEGFP proteins were stochastically labelled with orthogonal DNA sequences by incubation of the sample with anti - GFP nanobodies , each conjugated with one of four orthogonal sequences ( represented by blue , yellow , magenta and green dots ) . d , Sequential 3D imaging ( colour represents z position ) of the four labels yielded sufficiently spaced localization distributions . The average number of localizations per target is K average = 38 ( background represents cryo - EM structure from b for context ) . e , Comparison of 3D DNA - PAINT ( top left ) and 3D RESI ( bottom right ) for the same NPC illustrating improvement in spatial resolution by RESI . Localizations are rendered as gaussians with \u03c3 DNA - PAINT and \u03c3 RESI , respectively . f , Localization precision ( \u03c3 RESI ) as good as 5 \u00c5 was achieved by combining K localizations for each target , unambiguously resolving single Nup96 proteins . g , The 3D NPC cryo - EM structure was recapitulated using optical microscopy by applying a model - free average 30 of 1 , 217 NPCs from a single nucleus . h , RESI resolved adjacent Nup96 in a structural average by optical microscopy . i , Consistent with the cryo - EM structure ( taking into account linkage error arising from label size ) , adjacent Nup96 proteins were spaced 11 . 9 \u00b1 1 . 2 nm apart laterally ( top ) and 5 . 4 \u00b1 0 . 4 nm axially ( bottom ) . 714 | Nature | Vol 617 | 25 May 2023 Article in an image acquisition time of 100 min featuring an approximately 67 \u00d7 67 \u00b5m 2 field of view containing more than 2 , 000 DNA origami structures ( see Extended Data Fig . 8 for representative DNA origami structures ) . RESI allows us to routinely resolve strands spaced apart by only one DNA base pair . Strikingly , we measured a distance of 8 . 5 \u00b1 1 . 7 \u00c5 between two single docking strands in an individual DNA origami struc - ture ( Fig . 3d ) . This demonstrates an unprecedented resolution in optical microscopy by distinguishing structures closer than one nanometre . We note that our resolution claim is based on the most fundamental and strict definition : the ability to spatially distinguish point objects . We measured a distance of 9 . 5 \u00b1 2 . 6 \u00c5 between adjacent docking strands in an average of 42 DNA origami ( Extended Data Fig . 9 ) , which is within 1 s . d . of the expected backbone distance 31 of around 7 \u00c5 . To quantify resolution gain , we calculated RESI localizations for different values of K underlying DNA - PAINT localizations ( Methods ) . We demonstrate that the effective localization precision scales as \u03c3 = \u03c3 K RESI SMLM , yielding an average localization precision of 1 . 3 \u00c5 for an average K = 254 ( Fig . 3e ) , experimentally confirming the in silico results ( Fig . 1f ) . RESI not only yields virtually unlimited numbers of localiza - tions per target , but also avoids detrimental photophysical effects caused by spatial proximity of fixed - dye labels because , in DNA - PAINT imaging , two adjacent dyes are never present simultaneously . It has recently been reported 33 that , at sub - 10 - nm distances , photophysical near - field interactions play a major role in modulation of photoswitch - ing kinetics , thus effectively preventing fixed - dye SMLM techniques from accessing this resolution scale . This ultimately limits the achiev - able resolution of even the most photon - efficient techniques available for single - molecule localization , such as MINFLUX or MINSTED , despite their subnanometre precision , unless combined with DNA - PAINT . The experimentally demonstrated subnanometre resolution illustrates the capacity of RESI to enable structural biology studies using DNA - based imaging at hitherto elusive scales . CD20 receptor organization Finally , we applied RESI to address and resolve a cell - biological ques - tion currently under debate that has so far been beyond reach for both cryo - EM in a native cellular context and incumbent super - resolution techniques . Specifically we studied the organization of CD20 mem - brane receptors , which are prime targets for therapeutic antibody treatment of B cell - derived blood cancers and autoimmune diseases 34 . In the case of the most frequently used therapeutic anti - CD20 antibody , rituximab ( RTX ) , the spatial rearrangement of CD20 in the cell membrane is thought to play an important role in its efficacy 35 , 36 . Recent cryo - EM studies detected CD20 as a dimer in complex with two individual RTX - fragment antigen - binding regions 37 , 38 , suggest - ing a linear chain - like assembly of CD20 in the presence of the full antibody 38 . On the other hand , when incubated with the full RTX anti - body , a trimeric ring of alternating RTX molecules and CD20 dimers was detected in EM images 37 . The fact that cryo - EM experiments are performed in detergent solution raises the question about which molecular arrangements are actually present in the cell . Currently CD20 organization when bound to full RTX antibodies in intact cells cannot be assessed , thus precluding the investigation of whether CD20 clusters are of linear or circular nature . Moreover , even though in vitro studies showed that CD20 dimers can form without antibody binding , the quantitative assessment of CD20 dimerization in untreated cells is currently limited . Here we applied RESI to study the molecular arrangement of CD20 in Chinese hamster ovary ( CHO ) cells transiently transfected with mEGFP - CD20 , using four rounds of probe exchange in a total imag - ing time of 4 . 4 h . In the diffraction - limited overview and DNA - PAINT super - resolution image of untreated cells , CD20 appeared homogene - ously distributed ( Fig . 4a , b ( top ) and Extended Data Fig . 10a ) whereas RTX - treated cells exhibited apparent CD20 clusters ( Fig . 4b ( bottom ) and Extended Data Figs . 11a and 12a ) . Comparison of DNA - PAINT and RESI for both untreated and RTX - treated cells shows sub - 10 - nm - spaced CD20 pairs in the RESI images ( Fig . 4c , right ) that were unresolvable with DNA - PAINT ( Fig . 4c , left ) . RESI images suggest that CD20 is present in dimers and chain - like , higher - order structures in untreated and RTX - treated cells , respectively ( Fig . 4d ) . To quantitatively assess the existence of dimers in untreated cells , we performed first nearest - neighbour distance ( NND ) analysis for both DNA - PAINT and RESI data , demonstrating nonrandom distribu - tions in both cases ( Fig . 4e ) . RESI at 1 nm localization precision shows a substantial fraction of sub - 10 - nm distances in the NND histogram , which enables quantitative assessment of the degree of CD20 dimeri - zation . We performed numerical simulations and a least - squares fit ( Methods ) that yielded a composition of 53 \u00b1 1 % monomers and 47 \u00b1 1 % dimers with average intradimer distance of 13 . 5 \u00b1 0 . 3 nm ( Fig . 4f , solid line ) . For comparison , we simulated NND distributions corre - sponding to a population of 100 % monomers ( Fig . 4f , dotted line ) , 7 \u00c5 a Round 1 Round 2 b c DNA - PAINT RESI d K (cid:86) R E S I ( \u00c5 ) e 20 15 10 5 K = 5 K = 50 K = 100 1 50 100 9 0 n m 6 0 n m 8 . 5 \u00c5 (cid:86) \u2248 1 . 2 \u00c5 5 \u00c5 DNA origami structure 300 250 K = 254 20 nm 1 bp 5 nm 5 nm 5 nm 3 . 4 \u00c5 0 Fig . 3 | RESI resolves the distance of single DNA base pairs at \u00c5ngstr\u00f6m resolution . a , DNA origami with docking strands spaced by a single base pair ( bp ; red and blue strands , with alignment markers in green ) provided a platform to demonstrate the highest resolution achievable by RESI . b , DNA - PAINT resolved 20 nm spacing but the resolution was insufficient to distinguish individual docking sites , spaced one base apart . c , RESI resolves the adjacent docking strands . d , A Euclidean distance of 8 . 5 \u00b1 1 . 7 \u00c5 was calculated from individual localizations with an average precision of 1 . 2 \u00c5 ( left ) for the single - base - pair backbone distance , which is within 1 s . d . of the expected value of roughly 7 \u00c5 ( right ) . e , Experimental localization precision in RESI is in good agreement with \u03c3 K SMLM ( blue line , K ) , yielding an average localization precision of 1 . 3 \u00c5 for the experimental data from all n = 42 DNA origami ( insets correspond to exemplary point pair in d ) . Error bars represent mean \u00b1 1 s . d . Nature | Vol 617 | 25 May 2023 | 715 further demonstrating that CD20 molecules are not present solely as monomers . Because all NND distributions except for the first order are consistent with a complete spatial random ( CSR ) distribution at the experimentally measured density , we exclude the presence of higher - order assemblies for untreated CD20 ( Fig . 4g ) . Our findings present quantitative experimental evidence that CD20 exists as dimers in an intact cell membrane . By contrast , RESI analysis of CD20 in RTX - treated cells yielded first to fourth NND distributions inconsistent with a CSR model ( Fig . 4h and Extended Data Fig . 12d , e ) . This suggests a higher - order arrange - ment of CD20 molecules after RTX treatment and confirms recent cryo - EM - derived models 37 , 38 . Finally we probed the existence of hexameric , ring - like arrangements by comparison with numerical simulations ( Extended Data Fig . 13 ) . The characteristics of the experimentally detected CD20 clusters sug - gest the absence of isolated hexamers and support the hypothesis of predominantly linear , chain - like structures ( Extended Data Fig . 13h ) . Discussion We introduce RESI , a conceptually new approach in SMLM to improve the spatial resolution of optical microscopy to the \u00c5ngstr\u00f6m scale . RESI achieves this by combining multiple localizations from single targets to obtain a \u2018super - super - resolution\u2019 image after separating their localiza - tions by sequential imaging ( for example , using DNA - barcoded probes ) . In this way RESI precision\u2014and thus resolution\u2014scales not only with the number of photons ( N ) detected per localization but also with the number of localizations ( K ) acquired per target . RESI thus provides a new precision scaling law : \u03c3 = \u2248 \u03c3 K \u03c3 K N RESI SMLM DIFF . This applies if a suffi - ciently large number of orthogonal labelling sequences and thus imag - ing rounds guarantee adequately spaced groups of localizations ( Extended Data Fig . 14 ) . Importantly , resolution enhancement is isotropic in three dimensions . For our current experimental implementation , RESI approaches structural biology resolution with an all - optical approach in intact cells using off - the - shelf labelling reagents and a simple inverted fluorescence microscope operated under ambient conditions . We were able to experimentally demonstrate \u00c5ngstr\u00f6m spatial resolution below the physical size of a dye . This was achieved due to three specific advan - tages of DNA - PAINT leading to unbiased target sampling : ( 1 ) the rota - tional flexibility of the target - bound docking strand ( even in the case of longer repetitive - sequence motifs 32 ) ; ( 2 ) the freely rotating dipole of the dye attached to the imager sequence ; and ( 3 ) the fact that two adjacent imagers are never present simultaneously . Furthermore , because RESI images are not obtained from single localizations but from groups of localizations per target , the method presents a uniquely robust feature compared with other SMLM tech - niques : it shifts the focus from enhancement of only optical precision ( \u03c3 OPT ) to improvement in overall precision ( \u03c3 \u03c3 \u03c3 \u2248 + SMLM OPT2 MEC2 ) by averaging out the uncertainty effects of mechanical instability ( \u03c3 MEC ) , provided the latter is normally distributed . e h f g RTX treated 2 . 35 (cid:86) PAINT N o r m a li z e d f r e q u e n c y 0 20 First NND ( nm ) 40 60 80 100 Untreated Untreated Untreated RESI Untreated RTX treated d dimer d n d CD20 GFP (cid:86) \u2248 5 nm (cid:86) \u2248 0 . 5 nm RESI d dimer DNA - PAINT d CSR b DNA - PAINT DNA - PAINT 200 nm c U n t r ea t e d R T X t r ea t e d DNA - PAINT 4 nm 9 nm 5 nm 8 nm 0 20 First NND ( nm ) 40 60 80 100 0 . 16 0 . 10 0 . 10 0 50 n th NND ( nm ) 100 150 200 0 . 16 0 . 10 0 . 16 0 20 n th NND ( nm ) 40 60 80 100 0 . 04 0 . 04 0 . 04 0 . 05 0 . 03 0 . 01 N o r m a li z e d f r e q u e n c y N o r m a li z e d f r e q u e n c y N o r m a li z e d f r e q u e n c y d n d CSR d dimer DNA - PAINT RESI d dimer Dimers and monomers Monomers d CSR d dimer d CSR First NND SecondNNDThird NND Fourth NND First NND SecondNNDThird NND Fourth NND D i ff r a c t i o n li m i t e d a CD20 receptors D N A - P A I N T 5 \u03bc m 200 nm 50 nm 50 nm 50 nm 50 nm Anti - GFP nanobody 2 nm Fig . 4 | RESI shows CD20 receptor ( re ) organization at subnanometre precision following drug treatment . a , Diffraction - limited and DNA - PAINT overview image of CHO cells expressing mEGFP - CD20 labelled with anti - GFP nanobodies . b , Zoomed - in DNA - PAINT image showing apparently randomly distributed CD20 receptors for untreated cells ( top ) and clustered receptor arrangement for RTX - treated cells ( bottom ) . c , Comparison of DNA - PAINT and RESI for both untreated and RTX - treated cells showing sub - 10 - nm - spaced receptor pairs in the RESI images , which are unresolvable with DNA - PAINT . d , RESI data suggest that CD20 proteins occur in dimers ( spaced at d dimer ) , which are in turn distributed according to complete spatial randomness ( CSR ; distances between dimers , d CSR ) in untreated cells . Chains of dimers were observed following administration of RTX . e , Whole - cell analysis of first NNDs of CD20 receptors ( histograms of distances and kernel density estimation are shown ) . Only RESI , but not DNA - PAINT , allows the routine detection of sub - 10 - nm distances between proteins . Whereas DNA - PAINT overestimates dimer distance , RESI shows a label - limited distance of 13 . 5 nm ( see main text for discussion ) . f , Fitting RESI NND data from e to a numerical model reveals CD20 dimers and monomers . g , CD20 receptors in untreated cells showed second to fourth NNDs consistent with CSR , thus excluding the presence of higher - order protein complexes . h , CD20 receptors in RTX - treated cells , however , showed first to fourth NNDs , inconsistent with complete spatial randomness . 716 | Nature | Vol 617 | 25 May 2023 Article With RESI we measured areas of 67 \u00d7 67 \u00b5m 2 in 100 min , making it applicable as a sufficiently high - throughput tool for cell biology . Resolving receptor patterns at single - protein resolution could enable \u2018spatial diagnostics\u2019 as a prescreening method for personalized treat - ments , and serve as a tool for biomedical discovery of patterned thera - peutics\u2014for example , by guiding drug design principles . RESI performance and accuracy could be further improved by advances in intramolecular labelling approaches such as orthogonal unnatural amino acids 39 . RESI is thus poised to close the gap between 3D fluorescence super - resolution microscopy in whole intact cells and cryo - EM structural studies of individual supramolecular complexes , introducing a paradigm shift in fluorescence imaging by pushing opti - cal microscopy to \u00c5ngstr\u00f6m resolutions . Online content Any methods , additional references , Nature Portfolio reporting summa - ries , source data , extended data , supplementary information , acknowl - edgements , peer review information ; details of author contributions and competing interests ; and statements of data and code availability are available at https : / / doi . org / 10 . 1038 / s41586 - 023 - 05925 - 9 . 1 . Hell , S . W . & Wichmann , J . 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Biol . 2 , 261 ( 2019 ) . Publisher\u2019s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Creative Commons licence , and indicate if changes were made . The images or other third party material in this article are included in the article\u2019s Creative Commons licence , unless indicated otherwise in a credit line to the material . If material is not included in the article\u2019s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this licence , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2023 Methods Materials Unmodified DNA oligonucleotides , as well as DNA oligonucleotides modified with C3 - azide and Cy3B , were purchased from MWG Eurofins and Metabion . The M13mp18 and p7560 scaffold was obtained from Tilibit . Magnesium chloride ( 1 M , no . AM9530G ) , sodium chloride ( 5 M , no . AM9759 ) , ultrapure water ( no . 10977 - 035 ) , Tris ( 1 M , pH 8 . 0 , no . AM9855G ) , EDTA ( 0 . 5 M , pH 8 . 0 , no . AM9260G ) and 10\u00d7 PBS ( no . 70011051 ) were purchased from Thermo Fisher Scientific . BSA ( no . A4503 - 10G ) was ordered from Sigma - Aldrich . Triton X - 100 ( no . 6683 . 1 ) was purchased from Carl Roth . Sodium hydroxide ( no . 31627 . 290 ) was purchased from VWR . Paraformaldehyde ( no . 15710 ) and glutaraldehyde ( no . 16220 ) were obtained from Electron Microscopy Sciences . Tween - 20 ( no . P9416 - 50ML ) , glycerol ( no . 65516 - 500 ml ) , methanol ( no . 32213 - 2 . 5L ) , proto - catechuate 3 , 4 - dioxygenase pseudomonas ( PCD , no . P8279 ) , 3 , 4 - dihydroxybenzoic acid ( PCA , no . 37580 - 25G - F ) and ( \u00b1 ) - 6 - hydroxy - 2 , 5 , 7 , 8 - tetra - methylchromane - 2 - carboxylic acid ( trolox , no . 238813 - 5G ) were ordered from Sigma - Aldrich . Neutravidin ( no . 31000 ) was pur - chased from Thermo Fisher Scientific . Biotin - labelled BSA ( no . A8549 ) and sodium azide ( no . 769320 ) were obtained from Sigma - Aldrich . Cover - slips ( no . 0107032 ) and glass slides ( no . 10756991 ) were purchased from Marienfeld and Thermo Fisher Scientific , respectively . Fetal bovine serum ( FBS , no . 10500 - 064 ) , 1\u00d7 PBS ( pH 7 . 2 , no . 20012 - 019 ) , 0 . 05 % trypsin - EDTA ( no . 25300 - 054 ) , salmon sperm DNA ( no . 15632011 ) , OptiMEM ( no . 31985062 ) and Lipofectamine LTX ( no . A12621 ) were purchased from Thermo Fisher Scientific . Gold nanoparticles ( 90 nm , no . G - 90 - 100 ) were ordered from Cytodiagnostics . Nanobodies against GFP ( clone 1H1 ) with a single ectopic cysteine at the C terminus for site - specific conjugation were purchased from Nanotag Biotechnologies . DBCO - PEG4 - Maleimide ( no . CLK - A108P ) was purchased from Jena Bioscience . Buffers The following buffers were used for sample preparation and imaging . \u2022 Buffer A : 10 mM Tris pH 8 . 0 , 100 mM NaCl and 0 . 05 % Tween - 20 \u2022 Buffer B : 10 mM MgCl 2 , 5 mM Tris - HCl pH 8 . 0 , 1 mM EDTA and 0 . 05 % Tween - 20 pH 8 . 0 \u2022 Buffer C : 1\u00d7 PBS , 1 mM EDTA , 500 mM NaCl pH 7 . 4 , 0 . 02 % Tween , optionally supplemented with 1\u00d7 trolox , 1\u00d7 PCA and 1\u00d7 PCD \u2022 Blocking buffer : 1\u00d7 PBS , 1 mM EDTA , 0 . 02 % Tween - 20 , 0 . 05 % NaN 3 , 2 % BSA , 0 . 05 mg ml \u2013 1 sheared salmon sperm DNA \u2022 Two - dimensional ( 2D ) DNA origami folding buffer : 10 mM Tris , 1 mM EDTA , 12 . 5 mM MgCl 2 pH 8 . 0 \u2022 3D DNA origami folding buffer : 5 mM Tris , 1 mM EDTA , 5 mM NaCl , 20 mM MgCl2 pH 8 . 0 \u2022 1\u00d7 TA buffer : 40 mM Tris pH 8 . 0 , 20 mM acetic acid PCA , PCD and trolox Trolox ( 100\u00d7 ) was made by the addition of 100 mg of trolox to 430 \u00b5l of 100 % methanol and 345 \u00b5l of 1 M NaOH in 3 . 2 ml of water . PCA ( 40\u00d7 ) was made by mixing 154 mg of PCA in 10 ml of water and NaOH and adjustment of pH to 9 . 0 . PCD ( 100\u00d7 ) was made by the addition of 9 . 3 mg of PCD to 13 . 3 ml of buffer ( 100 mM Tris - HCl pH 8 . 0 , 50 mM KCl , 1 mM EDTA , 50 % glycerol ) . DNA - PAINT docking and imager sequences Four orthogonal DNA sequence motifs were used to label targets in four RESI rounds . The docking strands were 5xR1 ( TCCTCCT CCTCCTCCTCCT ) , 5xR2 ( ACCACCACCACCACCACCA ) , 7xR3 ( CTCTCTCTCTCTCTCTCTC ) and 7xR4 ( ACACACACACACACACACA ) . The respective imagers were R1 ( AGGAGGA - Cy3B ) , R2 ( TGGTGGT - Cy3B ) , R3 ( GAGAGAG - Cy3B ) and R4 ( TGTGTGT - Cy3B ) . The design of 2D RESI origami required extension of the R1 site at the 5\u2032 end such that the adjacent R1 and R3 docking strands could be spaced apart by a single base pair . Thus , the docking strand 5\u2032 5xR1 ( TCCTCCTCCTCCTCCTCCT ) and the 5\u2032 R1 imager ( Cy3B - AGGAGGA ) were used rather than the 3\u2032 versions for both 2D DNA origamis . DNA origami self - assembly ( 2D ) All 2D DNA origami structures were designed in caDNAno 40 . Self - assembly of DNA origami was accomplished in a one - pot reaction mix with a total volume of 40 \u00b5l , consisting of 10 nM scaffold strand ( for sequence , see Supplementary Data 2 ) , 100 nM folding staples ( Supple - mentary Data 1 ) , 500 nM biotinylated staples ( Supplementary Data 1 ) and 1 \u00b5M staple strands with docking site extensions ( Supplementary Data 1 ) in 2D DNA origami folding buffer . The reaction mix was then subjected to a thermal annealing ramp using a thermocycler . First , it was incubated at 80 \u00b0C for 5 min , cooled using a temperature gradient from 60 to 4 \u00b0C in steps of 1 \u00b0C per 3 . 21 min and finally held at 4 \u00b0C . DNA origami self - assembly ( 3D ) The 3D DNA origami disk structure was designed in caDNAno 40 . Self - assembly of the DNA origami disk was accomplished in a one - pot reaction mix of 50 \u00b5l total volume , consisting of 20 nM scaffold strand p7560 ( for sequence , see Supplementary Data 3 ) , 200 nM core folding staples ( Supplementary Data 1 ) , 200 nM staple sequences without handle extension ( Supplementary Data 1 ) , 500 nM biotinylated sta - ples ( Supplementary Data 1 ) , 2 \u00b5M staple strands with R4 docking site extensions and 4 \u00b5M staple strands with R1 or R3 docking site exten - sions ( Supplementary Data 1 ) in 3D DNA origami folding buffer . The reaction mix was then subjected to a thermal annealing ramp using a thermocycler . It was first incubated at 80 \u00b0C for 5 min then cooled using a temperature gradient from 60 \u00b0C to 20 \u00b0C in steps of 1 \u00b0C h \u2013 1 and finally held at 20 \u00b0C . DNA origami purification After self - assembly , structures were purified by agarose gel electropho - resis ( 1 . 5 % agarose , 1\u00d7 TA , 10 mM MgCl 2 , 0 . 5\u00d7 SybrSafe ) at 4 . 5 V cm \u2013 1 for 1 . 5 h . Gel bands were cut , crushed and the origami stored in low - binding Eppendorf tubes at \u221220 \u00b0C . DNA origami sample preparation and imaging For sample preparation , a bottomless six - channel slide ( ibidi , no . 80608 ) was attached to a coverslip . First , 80 \u00b5l of biotin - labelled BSA ( 1 mg ml \u2013 1 , dissolved in buffer A ) was flushed into the chamber and incu - bated for 5 min . The chamber was then washed with 360 \u00b5l of buffer A . A volume of 100 \u00b5l of neutravidin ( 0 . 1 mg ml \u2013 1 , dissolved in buffer A ) was then flushed into the chamber and allowed to bind for 5 min . After washing with 180 \u00b5l of buffer A and subsequently with 360 \u00b5l of buffer B , 80 \u00b5l of biotin - labelled DNA structures ( approximately 200 pM ) in buffer B was flushed into the chamber and incubated for 5 min . For measurement of the DNA origami disk , additional 2D DNA origami structures with 12 target sites 9 spaced 20 nm apart were incubated together , with the 3D disk origami serving as fiducials for drift cor - rection . After DNA origami incubation the chamber was washed with 540 \u00b5l of buffer B . For DNA origami disk structures , 150 \u00b5l of gold nano - particles ( diluted 1 : 10 in buffer B ) was flushed through and incubated for 5 min before washing with 540 \u00b5l of buffer B . Finally , 180 \u00b5l of the imager solution in buffer B was flushed into the chamber . The chamber remained filled with imager solution and imaging was then performed . Between imaging rounds , the sample was washed three times with 1 ml of buffer B until no residual signal from the previous imager solution was detected . Then , the next imager solution was introduced . For RESI , two imaging rounds were performed with imagers R1 and R4 present in round 1 and the imagers R3 and R4 in round 2 ( R1 and R3 probe the sites of interest for RESI and R4 serves alignment purposes ) . Nanobody \u2013 DNA conjugation Nanobodies were conjugated as described previously 32 . Unconju - gated nanobodies were thawed on ice , then 20 - fold molar excess of Article bifunctional DBCO - PEG4 - Maleimide linker was added and reacted for 2 h on ice . Unreacted linker was removed by buffer exchange to PBS using Amicon centrifugal filters ( 10 , 000 MWCO ) . The DBCO - modified nanobodies were reacted with 5\u00d7 molar excess of azide - functionalized DNA ( R1 , R2 , R3 and R4 ) overnight at 4 \u00b0C . Unconjugated protein and free DNA were removed by anion exchange chromatography using an \u00c4KTA pure system equipped with a Resource Q 1 ml column . Cell culture CHO cells ( CCL - 61 , ATCC ) were cultured in Gibco Ham\u2019s F - 12K ( Kaighn\u2019s ) medium supplemented with 10 % FBS ( no . 11573397 , Gibco ) . U2OS - CRISPR - Nup96 - mEGFP cells ( a gift from the Ries and Ellenberg laboratories ) were cultured in McCoy\u2019s 5A medium ( Thermo Fisher Scientific , no . 16600082 ) supplemented with 10 % FBS . Cells were pas - saged every 2 \u2013 3 days using trypsin - EDTA . Nup96 EGFP imaging U2OS - CRISPR - Nup96 - mEGFP cells were seeded on ibidi eight - well high glass - bottom chambers ( no . 80807 ) at a density of 30 , 000 cm \u2013 2 . Cells were fixed with 2 . 4 % paraformaldehyde in PBS for 30 min at room temperature . After fixation , cells were washed three times with PBS . Gold nanoparticles ( 200 \u00b5l ) were incubated for 5 min and washed three times with PBS . Blocking and permeabilization were performed with 0 . 25 % Triton X - 100 in blocking buffer for 90 min . After washing with PBS , cells were incubated with 100 nM anti - GFP nanobodies in blocking buffer for 60 min at room temperature . To enable RESI , the nanobody solution consisted of 25 nM R1 , R2 , R3 and R4 docking - strand - coupled anti - GFP nanobodies with a total nanobody concentration of 100 nM . Unbound nanobodies were removed by washing three times with PBS , followed by washing once with buffer C for 10 min . Postfixation was performed with 2 . 4 % paraformaldehyde in PBS for 15 min . After wash - ing 3\u00d7 with PBS , the imager solution in buffer C was flushed into the chamber . Between imaging rounds the sample was washed with 1 \u2013 2 ml of PBS until no residual signal from the previous imager solution was detected . Then , the next imager solution was introduced . First , imag - ers R1 , R2 , R3 and R4 were added simultaneously to the sample to perform a standard DNA - PAINT measurement ; then , RESI imaging was conducted via four subsequent imaging rounds with only one of the imagers . Cloning mEGFP - Alfa - CD20 was cloned by insertion of Alfa - CD20 into the mEGFP - C1 plasmid ( no . 54759 , Addgene ) . An Alfa - CD20 gblock ( obtained from Integrated DNA Technologies ) was amplified with primers cggcatggacgagct and gtacaagtccgga and , after cutting with restriction enzymes BsrGI and BamHI , Gibson assembly was performed ( 2\u00d7 mix , NEB ) . mEGFP - CD20 imaging CHO cells were seeded on ibidi eight - well high glass - bottom chambers ( no . 80807 ) the day before transfection at a density of 15 , 000 cm \u2013 2 . Transfection with mEGFP - CD20 was carried out with Lipofectamine LTX as specified by the manufacturer . CHO cells were allowed to express mEGFP - CD20 for 16 \u2013 24 h . Then , the medium was replaced with fresh F - 12K medium + 10 % FBS ( in the untreated case ) or with F - 12K medium + 10 % FBS + 10 ug ml \u2013 1 RTX - Alexa 647 ( a gift from Roche Glycart ) ( in the RTX - treated case ) , followed by incubation for 30 min . After washing two times with fresh medium for 5 min , cells were fixed with 250 \u00b5l of prewarmed 4 % PFA + 0 . 1 % glutaraldehyde in PBS for 15 min . CHO cells were washed three times with PBS and quenched with 100 mM Tris pH 8 . 0 for 5 min . Permeabilization was carried out for 5 min with 0 . 2 % Triton X - 100 in PBS , followed by three washes with PBS . Cells were blocked in blocking buffer for 1 h at room temperature ( RT ) . Anti - GFP nanobodies were incubated at a total concentration of 25 nM overnight at 4 \u00b0C ; for RESI with four rounds this yielded 6 . 25 nM each of GFP - Nb - R1 / 2 / 3 / 4 . After washing three times with PBS at RT for 15 min , cells were postfixed with 4 % PFA at RT for 10 min followed by washing and postfixation as described above . Gold nanoparticles ( 90 nm ) were diluted 1 : 3 in PBS and incubated for 10 min at RT and the sample was washed two times with PBS to remove unbound gold . The imager solution in buffer C for the first round was incubated for 5 min and then replaced with fresh imager , after which the first acquisition round was started . Between imaging rounds the sample was washed with at least 2 ml of PBS until no residual signal from the previous imager solution was detected . Then , the next imager solution was introduced . RESI imaging was conducted via four subsequent imaging rounds with only one of the imagers . In the final imaging round , imagers R1 , R2 , R3 and R4 were added simultaneously to the sample to perform a standard DNA - PAINT measurement . Microscopy setup Fluorescence imaging was carried out using an inverted microscope ( Nikon Instruments , Eclipse Ti2 ) with the Perfect Focus System , applying an objective - type TIRF configuration equipped with an oil - immersion objective ( Nikon Instruments , Apo SR TIRF \u00d7100 / numeri - cal aperture 1 . 49 , oil ) . A 560 nm laser ( MPB Communications , 1 W ) was used for excitation . The laser beam was passed through a cleanup filter ( Chroma Technology , no . ZET561 / 10 ) and coupled to the microscope objective using a beam splitter ( Chroma Technology , no . ZT561rdc ) . Fluorescence was spectrally filtered with an emission filter ( Chroma Technology , nos . ET600 / 50m and ET575lp ) and imaged on an sCMOS camera ( Andor , Zyla 4 . 2 Plus ) without further magnification , resulting in an effective pixel size of 130 nm ( after 2 \u00d7 2 binning ) . The readout rate was set to 200 MHz . Images were acquired by choosing a region of interest of size 512 \u00d7 512 pixels . 3D imaging was performed using a cylindrical lens ( Nikon Instruments , N - STORM ) in the detection path . Raw microscopy data were acquired using \u00b5Manager 41 ( v . 2 . 0 . 1 ) . Total internal reflection illumination was used for 2D and 3D DNA origami data , as well as for CD20 acquisition . Highly inclined and laminated optical sheet ( HILO ) illumination was employed for the acquisition of NPC data . Detailed imaging conditions for the respective experiments are shown in Extended Data Table 1 . Imaging parameters and duration Due to target and sample heterogeneity the optimal imager concentra - tion , c , used to achieve sparse blinking varies . Here we used concentra - tions from 100 pM ( Nup96 ) to 800 pM ( DNA origami ) . Optimal imager concentrations were determined visually for each sample . Concentra - tions were altered until blinking was frequent but sufficiently sparse to achieve good DNA - PAINT resolution . The average number of expected binding events per binding site during a DNA - PAINT measurement is given by the duration of the measurement t measurement and the mean dark time \u03c4 dark ( defined as \u03c4 = k c dark 1 \u00d7 on , with k on being the on - rate of a given imager strand ) as : \uf8eb \uf8ed\uf8ec \uf8f6 \uf8f8\uf8f7 n t \u03c4 t k c = = \u00d7 \u00d7 . bindingevents measurement dark measurement on The average number of localizations per binding event is given by the mean bright time \u03c4 bright and camera exposure time t exposure as n \u03c4 t = . locsperbindingevent bright exposure \uf8eb \uf8ed\uf8ec \uf8ec \uf8f6 \uf8f8\uf8f7 \uf8f7 Therefore , the average number of localizations expected per binding site over the course of the measurement is \uf8eb \uf8ed\uf8ec \uf8f6 \uf8f8\uf8f7 \uf8eb \uf8ed \uf8ec \uf8ec \uf8f6 \uf8f8 \uf8f7 \uf8f7 \uf8eb \uf8ed \uf8ec \uf8ec \uf8f6 \uf8f8 \uf8f7 \uf8f7 n t \u03c4 \u03c4 t t k c \u03c4 t = \u00d7 = \u00d7 \u00d7 \u00d7 . loc measurement dark bright exposure measurement on bright exposure It follows that the total acquisition time necessary to collect n loc localizations is , on average , t t n \u03c4 k c = \u00d7 \u00d7 \u00d7 . measurement exposure loc bright on The necessary number of localizations , n loc , is calculated using \u03c3 = \u03c3 n RESI DNA PAINT loc \u2010 , and thus ( ) n = \u03c3 \u03c3 loc 2 DNA PAINT RESI \u2010 with the DNA - PAINT locali - zation precision \u03c3 DNA - PAINT . For expected imager concentrations between 50 and 800 pM , expo - sure times between 100 and 200 ms and kinetics reported previously 32 , the times required to collect 16 localizations ( 1 nm RESI precision given \u03c3 DNA - PAINT = 4 nm ) vary between 42 s ( R2 , 800 pM , 100 ms exposure time ) and 314 min ( R5 , 50 pM , 200 ms exposure time ) . DNA - PAINT analysis Raw fluorescence data were subjected to super - resolution reconstruc - tion using the Picasso software package 9 ( latest version available at https : / / github . com / jungmannlab / picasso ) . Drift correction was per - formed with a redundant cross - correlation and gold particles as fidu - cials for cellular experiments , or with single DNA - PAINT docking sites as fiducials for origami experiments . Channel alignment Alignment of subsequent imaging rounds was performed iteratively in Picasso 9 , starting with a redundant cross - correlation and followed by gold fiducial alignment for cellular experiments . Every DNA ori - gami was equipped with additional DNA - PAINT docking sites that were imaged simultaneously with the sites of interest in all imaging rounds , thus enabling their use as fiducials . First , redundant cross - correlation ( 2D and 3D origami measurements ) and gold alignment ( 3D measure - ments ) were performed in Picasso Render . To correct for nanoscopic movement of individual DNA origami during buffer exchange , channel alignment was not only performed on the full field of view but , addition - ally , small regions of interest containing only one DNA origami were selected . Within each region of interest , alignment was then conducted via the fiducial docking sites of the DNA origami . This was performed outside of Picasso in a custom Python script , not only to find the optimal translation between channels but also to correct for possible rotations of the DNA origami . Clustering and RESI Clustering of DNA - PAINT localizations . After channel alignment , DNA - PAINT data were analysed using a custom clustering algorithm for each imaging round . This algorithm is based on the fact that , in DNA - PAINT , localizations are independent measurements of the posi - tion of a target molecule and are observed to be Gaussian distributed . To assign localizations to a specific target molecule , we first used a gradient ascent method to find the centre of a localization cloud for each target . We then assigned all localizations circularly distributed around the centre point to the same target molecule . This is a valid approx imation because , due to the reduction of effective target density by RESI\u2019s sequential imaging approach , the majority of localization clouds from single targets are spaced sufficiently apart . The clustering algorithm uses two input parameters : radius r , which sets the final size of the clusters and defines a circular environment around each localization , and the minimal number of localizations , n min , representing a lower threshold for the number of DNA - PAINT localizations in any cluster . First , the number of neighbouring localizations within distance r from each localization is calculated . If a given localization has more neighbours within its r radius than all neighbouring localizations , it is considered a local maximum . If there are more than n min localizations within a circle of radius r around such a local maximum , these locali - zations are assigned to the same cluster ; the remainder are not con - sidered to be part of a cluster and are omitted from further analysis . Further filtering of clusters is performed to exclude clusters that originate from unspecific sticking of imagers to the sample . Firstly , the mean frame ( mean value of the frame numbers in which localizations occurred ) of all localizations assigned to the same cluster is calcu - lated . In the case of repetitive blinking the mean frame is expected to be around half the total number of frames 42 . The algorithm therefore excludes all clusters with a mean frame in the first or last 20 % of frames . Secondly , sticking events in the middle of the acquisition time can be identified by dividing the acquisition time into 20 time windows each containing 5 % of frames . If any of these time windows contains more than 80 % of localizations in the cluster , it is excluded as a sticking event . The choice of the clustering radius r and the threshold n min depend on the respective experimental conditions . A suitable value for n min can be estimated by picking localization clouds originating from single target molecules ( that is , well separated ) in Picasso Render , exporting pick properties and plotting a histogram of the number of localizations in each pick . n min is chosen to differentiate between populations corre - sponding to single targets and to background localizations . The radius r scales with the size of the localization clouds and thus the localization precision . If too large a value is chosen , adjacent clus - ters might not be separated ; if r is too small , \u2018subclustering\u2019 within one localization can occur . The latter also translates to a peak in NND at twice the clustering radius . A good a priori starting value for r is represented by approximately twofold the localization precision of the underlying DNA - PAINT measurement . Picasso Render offers a tool ( Test Clusterer ) in which the effect of different clustering parameters can be tested for a small region of interest . For 3D clustering , an additional radius for the z direction is intro - duced because the spread of localizations in z is approximately twofold greater compared with x and y . Calculation and rendering of RESI localization . Following cluster analysis , the centres of the DNA - PAINT localization groups were calcu - lated as weighted ( wtd ) means by employing the squared inverse localization precisions ( ) lp 1 2 as weights . For x and y coordinates : x wx w w lp \u00af = \u2211 \u2211 , = 1 . i N i i i N i i wtd = 1 = 1 2 For z coordinates a standard mean without weights is used to calcu - late z positions . The precision of the resulting RESI localization is the weighted s . e . m . of the underlying grouped localizations : \u2211 \u2211 s s N x N x N N w x x w ( ) = ( ) = Var ( ) , where Var ( ) = \u2212 1 ( \u2212 ) . x x i N i i i N i wtd wtd wtd wtd = 1 wtd 2 = 1 The choice for lp 1 / 2 as weights is based on the following argument : under the hypothesis that localizations are independent and normally distributed with the same mean , the weighted mean based on inverse variances as weights is the maximum likelihood estimator of the mean of the whole set of localizations . Therefore , the variance of the weighted mean is minimal ( the estimator is optimal ) when the inverse variances of individual measurements lp 1 / 2 are chosen as weights . Finally , we take the average of the resulting x and y s . e . m . as the final precision of each RESI localization . For z coordinates the precision is estimated to be two times xy precision . Saving RESI localizations in a Picasso hdf5 file allowed us to render them as Gaussians with s . d . corresponding to their respective precision . Article RESI resolution estimation Evaluation of in silico RESI precision with numerical simulations . To evaluate the performance of RESI , in silico numerical simulations were performed . The algorithm consists of the following steps . ( 1 ) A grid of defined positions of the binding sites ( ground truth ) is generated . Typically , a grid of positions was generated ( Extended Data Fig . 1a , top left ) . ( 2 ) SMLM ( DNA - PAINT ) localizations are simulated as samples from a 2D Gaussian distribution with \u03c3 = \u03c3 SMLM . A large number ( M ) of localizations is generated per binding site ( Extended Data Fig . 1a , top right ) . ( 3 ) For each binding site , subsets of K localizations are randomly selected ( K < < M ) . This results in n = M K subsets of SMLM localiza - tions ( Extended Data Fig . 1a , bottom left ) that are then averaged to generate n RESI localizations ( Extended Data Fig . 1a , bottom right ) . ( 4 ) The resulting RESI localizations are then shown in a histogram ( Extended Data Fig . 1b ) and the trace ( tr ) of the covariance matrix is calculated . RESI precision is estimated as \u03c3 tr x y = ( cov ( , ) ) RESI 1 2 ( Extended Data Fig . 1c ) . This definition has been used before in the field as a scalar metric for 2D variance 8 . ( 5 ) Steps 3 and 4 are repeated for different values of K to numerically study \u03c3 = \u03c3 RESI ( K ) . Evaluation of experimental RESI precision by resampling of local - izations . To evaluate the precision of RESI in experimental data , an analogous method was used . Briefly , the M total of DNA - PAINT localizations of each group corresponding to a single binding site was randomly resampled into subsets of K localizations , then steps 4 and 5 above were performed to evaluate \u03c3 RESI . The plotted \u03c3 RESI in Fig . 3d is the average value of all single binding sites in the dataset . Error bars represent the s . d . of the different \u03c3 RESI values calculated for different binding sites . Note that this analysis can be performed only for K < < M to have suf - ficient n = M K RESI localizations for a statistically significant estimation . Because final RESI localization takes into account all M DNA - PAINT localizations , final precision is extrapolated as \u03c3 = \u03c3 M RESI SMLM . Stochastic labelling : simulations and user guidelines In RESI , the sparsity of binding sites in the sample is achieved by labell - ing a single species of biomolecules with different orthogonal DNA sequences . The labelling process is performed in a stochastic manner : n different labels ( for example , DNA - conjugated nanobodies ) targeting the same protein species are simultaneously incubated in the sample and thus the probability of each single protein being labelled with a certain sequence i ( i = 1 , \u2026 , n ) is p = i n 1 , given that the same concentration of each label is used . Subsequently , n imaging rounds are performed to record all groups of localizations required to obtain the final RESI image . The minimum number of labels ( n ) and rounds necessary to achieve sufficient sparsity of binding sites in each imaging round will depend mainly on three factors : SMLM localization precision and density and the molecular arrangement of the protein of interest . Here we describe how these parameters affect the final RESI results using a few practical examples . Case 1 : protein structure with oligomers not resolvable with DNA - PAINT . A typical study case is that of single proteins arranged in dimers , which in turn present another specific spatial organization in space . This is the case , for example , of the Nup96 in the NPC . In this case stochastic labelling has to be such that the probability of labelling two proteins forming a dimer with different sequences is sufficiently high . For n rounds of labelling / imaging , the probability is P p n ( diff . seq . ) = 1\u2212 = 1\u2212 1 i for n = 4 labelling / imaging rounds P ( diff . seq . ) \u2248 75 % . We chose n = 4 to demonstrate that it provides a relatively high P ( diff . seq . ) with only a few imaging rounds . We note , however , that n > 4 could be used to increase P ( diff . seq . ) and hence to maximize the sparsity of labelled binding sites in each round . To resolve a set of an arbitrary number of molecules , m , spaced more closely than the resolution of DNA - PAINT , they must be labelled with n orthogonal sequences . In general , the proportion of m molecules labelled with n orthogonal sequences , and thus the proportion of resolvable sets of molecules , follows the equation P m n n n m n ( , ) = ! ( \u2212 ) ! . m Case 2 : proteins distributed similarly to CSR at a certain density . This is a common case\u2014for example , for membrane receptors . If pro - teins are distributed in a CSR fashion ( Extended Data Fig . 14a ) at a given density , DNA - PAINT can already resolve single proteins that are suf - ficiently spaced from their NNs . We will consider that proteins at a distance d = 4 \u00d7 \u03c3 DNA - PAINT are reliably resolved ( note that this criterion is significantly stricter than 2 . 35 \u00d7 \u03c3 DNA - PAINT ) . Then , for a given density , the NND histogram can be computed and the fraction of distances below d calculated ( Extended Data Fig . 14b ) . This represents the frac - tion of single proteins , F , that will not be resolved by DNA - PAINT . Here we plot F as a function of both density and resolution ( Extended Data Fig . 14c ) . Such a map already provides a tool to understand the level of SMLM resolution needed to resolve single proteins at a given density . RESI can be interpreted here as a way to reduce the effective density by splitting targets into different stochastically labelled subsets . Hence , the effective density of each round will be reduced according to the formula \u03c1 = n density . Extended Data Fig . 14d shows one - dimensional cuts of the 2D map to provide guidelines to choosing the number of ortho - gonal sequences ( and hence imaging rounds ) needed to be able to perform RESI efficiently . For example , for an initial resolution of 20 nm ( \u03c3 = 5 nm ) , which is typical for DNA - PAINT in a cellular context , and a density of density = 200 \u00b5 molecules m 2 ( relatively high ) , n = 4 different sequences are sufficient to provide P ( diff . seq . ) \u2248 90 % for proteins below d ( Extended Data Fig . 14d ) . These proteins will then be resolv - able by RESI . Model - free averaging Model - free averaging of Nup96 data was performed for both DNA - PAINT and RESI measurement of the same nucleus , as described by Wu et al . 30 . The respective Picasso hdf5 files were segmented in SMAP 43 and saved in a file format compatible for averaging by employing plugins segment - NPC , NPCsegmentCleanup and sitenumbers2loc . Model - free averaging was then performed on the resulting _ sml . mat files with default param - eters by running the particleFusion . m script in Matlab ( available with the SMAP source code ) . The averages shown correspond to the result of the final iteration , in which each point is rendered with a Gaussian of \u03c3 = 2 nm in x , y and z . Numerical simulations for CD20 distribution To interpret the results of the NND data in untreated cells , numerical simulations were performed . Briefly , two populations , one of CD20 monomers and one of dimers with a CSR distribution , were simulated and then their NNDs calculated . The algorithm can be summarized as follows : ( 1 ) Choice of parameters . Density of monomers : number of monomers per unit area ; density of dimers : number of dimers per unit area ; dimer distance : expected distance between the two molecules includ - ing the labelling construct ; uncertainty : variability in the position of each molecule due to labelling and localization errors ; labelling efficiency : fraction of ground - truth molecules that will actually be labelled and measured . The observed density , which has to match the experimental parameter , then becomes observed density = ( density of monomers + density of dimers ) \u00d7 labelling efficiency . For quanti - fication of the labelling efficiency of the DNA - conjugated GFP nano - body we used a transiently transfected CHO cell line expressing a GFP - and Alfa - tag at the C terminus of a monomeric membrane pro - tein ( for example , CD86 ) . We then labelled GFP - and Alfa - tag using their cognate nanobodies conjugated to two orthogonal docking sequences and performed two rounds of Exchange - PAINT . We then obtained the best - fitting parameters for a sample comprising pairs of GFP / Alfa - tag , and isolated Alfa - tags , similarly to how CD20 dimer / monomer analysis is performed . The ratio of these two populations is then used as an estimation of labelling efficiency . Full details of the quantification approach will be available in a manuscript currently in preparation . ( 2 ) Simulation of monomers : a set of spatial coordinates with CSR dis - tribution and given density are drawn ; simulation of dimers : a set of spatial coordinates with CSR distribution are drawn , representing the centre of each dimer . For each dimer centre , two positions are generated with a random orientation and a distance with expect - ed value dimer distance . The position of each pair of molecules is drawn , taking into account the uncertainty parameter ( drawn from a Gaussian distribution ) . ( 3 ) A random subset of \u2018detectable\u2019 molecules is taken from the ground - truth set ( fraction = labelling efficiency ) to simulate the labelling process . ( 4 ) NNDs are calculated on the subset of detectable molecules . The parameters density of monomers = 212 \u00b5m \u2013 2 , density of dimers = 0 \u00b5m \u2013 2 , uncertainty = 5 nm and labelling efficiency = 50 % were used to compare data for RTX - treated cells with a CSR distribu - tion of monomers . For the untreated case , the best - fit parameters were obtained through an iterative , nonlinear , least - squares algorithm . The experimentally observed density ( 50 molecules \u00b5m \u2013 2 ) is used for the simulation . Description of the iterative nonlinear , least - squares algorithm For every set of parameters a simulation is performed , NNDs are histo - grammed and the sum of the squared differences between the simula - tion and experimental histogram are computed . A fit consists of finding the parameters that minimize the sum of the squared differences . Parameters \u2022 D , average dimer distance ( nm ) \u2022 \u03c3 _ label , variability introduced by the labelling ( nm ) \u2022 frac _ of _ dimers , fraction of dimers ( % ) Note : frac _ of _ monomers = 100 \u2013 frac _ of _ dimers Estimation of parameters . ( 1 ) Coarse - fit over a large range of parameters to determine the range of the best - fit parameters . Range D = 1 \u2013 20 nm , \u03c3 _ label = 1 \u2013 20 nm , frac _ of _ dimers = 0 \u2013 100 % . ( 2 ) Fine - fit over a reduced parameter space around the best - fit results in the previous step . The parameters D _ opt , \u03c3 _ label _ opt and frac _ of _ dimers _ opt that best match the proposed model and the data are now found . In this case it resulted in D _ opt = 13 . 5 nm , \u03c3 _ label _ opt = 5 . 5 nm , frac _ of _ dimers _ opt = 47 % ( Fig . 4e , f ) . Estimation of parameter uncertainty . ( 1 ) M is created ( in this case , M = 100 ) , simulated ( using datasets D _ opt , \u03c3 _ label _ opt , frac _ of _ dimers _ opt ) with the same number of molecules as the experimental data ( around 21 , 000 ) . ( 2 ) M datasets are fine - fitted and the best - fit parameters D _ opt , \u03c3 _ label _ opt and frac _ of _ dimers _ opt are obtained . Three sets are obtained : D _ opt , \u03c3 _ label _ opt and frac _ of _ dimers _ opt . ( 3 ) The distributions of D _ opt , \u03c3 _ label _ opt and frac _ of _ dimers _ opt are studied . Standard deviation can be used as an estimation of the parameter uncertainties obtained in b . The uncertainties of the parameters D _ opt , \u03c3 _ label _ opt and frac _ of _ dimers _ opt are now obtained . Data availability Localization data from this study are available at Zenodo ( https : / / doi . org / 10 . 5281 / zenodo . 7795826 ) . Raw microscopy data obtained during this study are available from the corresponding author on reasonable request . Code availability RESI can be performed using Picasso v . 0 . 6 . 0 , available at https : / / github . com / jungmannlab / picasso with documentation provided at https : / / picassosr . readthedocs . io / en / latest / render . html . The custom - written scripts used in this study are available at https : / / github . com / jungmannlab / resi . 40 . Douglas , S . M . et al . Rapid prototyping of 3D DNA - origami shapes with caDNAno . Nucleic Acids Res . 37 , 5001 \u2013 5006 ( 2009 ) . 41 . Edelstein , A . D . et al . Advanced methods of microscope control using muManager software . J . Biol . Methods 1 , e10 ( 2014 ) . 42 . Wade , O . K . et al . 124 - Color super - resolution imaging by engineering DNA - PAINT blinking kinetics . Nano Lett . 19 , 2641 \u2013 2646 ( 2019 ) . 43 . Ries , J . SMAP : a modular super - resolution microscopy analysis platform for SMLM data . Nat . Methods 17 , 870 \u2013 872 ( 2020 ) . Acknowledgements We thank Y . - L . Wu and J . Ries for valuable assistance with model - free averaging . We thank J . Schmied and F . Schueder for helpful discussions . We thank M . K . Steen - Mueller and I . Glueck for proofreading the manuscript . This research was funded in part by the European Research Council through an ERC Consolidator Grant ( ReceptorPAINT , grant agreement no . 101003275 ) , the German Research Foundation through SFB1032 ( project A11 , no . 201269156 ) , the Danish National Research Foundation ( Centre for Cellular Signal Patterns , DNRF135 ) , the Human Frontier Science Program through a Young Investigator Grant ( no . HFSP RGY0065 / 2018 ) , the Volkswagen Foundation through the initiative \u2018Life ? \u2014A Fresh Scientific Approach to the Basic Principles of Life\u2019 ( grant no . 98198 ) , the Max Planck Foundation and the Max Planck Society . S . S . and T . S . acknowledge support by the QBM graduate school . S . C . M . R . , I . B . , P . R . S . , A . S . E . , E . M . U . and M . T . S . acknowledge support by the IMPRS - LS graduate school . I . B . acknowledges funding support by Roche . L . A . M . acknowledges a postdoctoral fellowship from the European Union\u2019s Horizon 20212022 research and innovation programme under Marie Sk\u0142odowska - Curie grant agreement no . 101065980 . Author contributions S . C . M . R . designed and conducted 2D and 3D DNA origami as well as Nup96 experiments , developed the analysis software and analysed DNA origami and Nup96 data . L . A . M . designed and conducted computer simulations , contributed to analysis software and analysed DNA origami , Nup96 and CD20 data . I . B . designed and conducted CD20 experiments and analysed CD20 data . P . R . S . designed and conducted 2D DNA origami experiments , contributed to analysis software and analysed DNA origami and Nup96 data . R . K . and T . S . contributed to analysis software . S . S . developed labelling probes . A . S . E . , S . S . , E . M . U . and M . T . S . performed preliminary RESI experiments . C . K . contributed to the design of studies targeting CD20 and their interpretation . S . C . M . R . , L . A . M . , I . B . , P . R . S . and R . J . interpreted data and wrote the manuscript . R . J . conceived the concept , designed experiments and supervised the study . S . C . M . R . , L . A . M . , I . B . and P . R . S . contributed equally . All authors reviewed and approved the final manuscript . Funding Open access funding provided by the Max Planck Society . Competing interests C . K . declares employment , patents ( unrelated to this work ) and stock ownership with Roche . Additional information Supplementary information The online version contains supplementary material available at https : / / doi . org / 10 . 1038 / s41586 - 023 - 05925 - 9 . Correspondence and requests for materials should be addressed to Ralf Jungmann . Peer review information Nature thanks Alistair Curd and the other , anonymous , reviewer ( s ) for their contribution to the peer review of this work . Peer reviewer reports are available . Reprints and permissions information is available at http : / / www . nature . com / reprints . Article a b c Numerical K = 1 K = 10 K = 30 K = 50 K = 80 K = 100 20 0 y ( n m ) - 20 20 0 - 20 5 0 - 5 y ( n m ) 10 20 x ( nm ) x ( nm ) 10 20 x ( nm ) 10 20 x ( nm ) 10 5 0 - 5 - 10 10 5 0 - 5 - 10 y ( n m ) y ( n m ) 3 . 0 2 . 5 2 . 0 1 . 5 1 . 0 0 . 5 R ES I ( n m ) K 0 50 100 10 nm Extended Data Fig . 1 | RESI resolution estimation . a , A grid of defined positions of binding sites is generated ( top left ) , SMLM ( DNA - PAINT ) localizations are simulated as samples from a gaussian distribution ( top right ) . Localizations for only one binding site were plotted for clarity . For each binding site , subsets of K localizations are randomly selected ( bottom left ) and averaged ( bottom right ) . One exemplary subset and its average is highlighted . b , Resulting RESI - localizations are histogrammed to produce images at different resolutions ( K values ) . c , RESI - localization precision \u03c3 RESI vs K . Analytical dependence on K ( blue line ) and numerical results ( black dots ) . A total of 1200 SMLM localizations per site are simulated . Error bars represent mean \u00b1 1 s . d . Round 2 Round 1 Rounds 1 & 2 1 nm 0 . 1 nm 0 . 1 nm 5 . 4 nm 6 . 4 nm 6 . 1 nm 5 . 4 nm 6 . 2 nm 5 . 4 nm 0 . 3 nm 0 . 2 nm 0 . 3 nm 0 . 3 nm R4 R3 R1 20 nm 5 nm a b Round 1 c d e f g RESI DNA - PAINT Round 2 Average ( n = 90 origami ) 5 . 5 nm 20 nm 20 nm 20 nm 20 nm 9 0 n m 6 0 n m Extended Data Fig . 2 | RESI in 2D DNA origami . a , DNA origami design featuring six 5 nm - spaced orthogonal docking strand pairs ( red R1 , blue R3 ) and six alignment docking strands ( green R4 ) . See Methods for sequence details . b , DNA - PAINT acquisition parameters were tuned such that 5 nm were not consistently resolvable . c , First imaging round conducted with R1 ( target ) and R4 imagers ( alignment , sites circled ) . d , Second imaging round conducted with R3 ( target ) and alignment imagers ( R4 , sites circled ) . The alignment sites were used for translational and rotational alignment between rounds . e , RESI resolves the 5 nm distances . f , The distance and orientation between R1 and R3 docking strands are consistent with the design . g , An average of 90 DNA origami structures reveals consistent results and excellent alignment performance . The numbers indicate the distance between rounds . Article Extended Data Fig . 3 | 2D DNA origami . Representative DNA origami from across the field of view of the measurement . a , Four DNA origami , shown at DNA - PAINT resolution ( upper row ) and RESI resolution ( lower row ) . The insert depicts a pair of docking strands spaced at approx . 5 nm . b , 40 additional DNA origami , shown at DNA - PAINT resolution ( upper rows ) and RESI resolution ( lower rows ) . Round 2 Round 1 Rounds 1 & 2 R4 R3 R1 ii ) R4 i ) R1 / R3 Average ii ) R4 i ) R1 / R3 x y z i ) R1 / R3 ii ) R4 x y z i ) R1 / R3 ii ) R4 ( i ) ( ii ) Sum ( 88 DNA origami ) ( i ) ( ii ) ( i ) ( ii ) 10 nm 20 nm x y z x y z 3D DNA - PAINT Round 2 Round 1 c d e b a 3D RESI x y z x y z ( i ) ( ii ) 20 nm 20 nm 20 nm g f x y z x y z 10 nm Docking strand Docking strand at opposite side 11 nm R4 R3 R1 10 nm 10 nm 20 nm 20 nm h x y z 10 nm d z = 0 . 2 nm CI = [ 0 . 0 , 2 . 1 ] nm d = 1 . 3 nm CI = [ 0 . 3 , 2 . 3 ] nm CI = [ 0 . 3 , 2 . 2 ] nm d xy = 1 . 3 nm d z = 0 . 2 nm d = 0 . 4 nm CI = ( 0 . 0 , 1 . 4 ) nm CI = [ 0 . 0 , 1 . 0 ] nm CI = [ 0 . 0 , 1 . 6 ] nm d xy = 0 . 3 nm d z = 0 . 8 nm CI = [ 0 . 0 , 2 . 3 ] nm d = 1 . 0 nm CI = [ 0 . 0 , 2 . 2 ] nm CI = [ 0 . 0 , 1 . 3 ] nm d xy = 0 . 6 nm d z = 0 . 6 nm d = 0 . 7 nm CI = [ 0 . 0 , 1 . 8 ] nm CI = [ 0 . 0 , 1 . 8 ] nm CI = [ 0 . 0 , 1 . 1 ] nm d xy = 0 . 5 nm d z = 0 . 1 nm CI = [ 0 . 0 , 1 . 1 ] nm d = 0 . 1 nm CI = [ 0 . 0 , 0 . 8 ] nm x y z x y z CI = [ 0 . 0 , 0 . 6 ] nm d xy = 0 . 1 nm 1 nm 5 nm d z = 0 . 3 nm d = 0 . 4 nm CI = [ 0 . 0 , 1 . 1 ] nm CI = [ 0 . 0 , 1 . 2 ] nm CI = [ 0 . 0 , 0 . 7 ] nm d xy = 0 . 3 nm d = 11 . 6 \u00b1 0 . 8 nm d z = 11 . 3 \u00b1 0 . 8 nm d xy = 2 . 5 \u00b1 0 . 4 nm Extended Data Fig . 4 | RESI in 3D DNA origami . a , DNA origami design featuring one pair of orthogonal docking strands ( red R1 , blue R3 ) as well as six alignment docking strands ( green R4 ) . Docking strands extend from both the top and bottom surface of the DNA origami ( insert ) . b , The design ensures that all but the R1 / R3 docking strand pair are spaced sufficiently to be resolved by DNA - PAINT . c , 3D DNA - PAINT imaging resolves R4 alignment sites , barely resolves R1 / R3 axially and does not resolve R1 / R3 laterally . d , Sequential 3D DNA - PAINT imaging with R4 sites used for alignment . e , RESI resolves R1 / R3 both axially and laterally . f , An overlay of 88 DNA origami reveals overall good alignment despite structural heterogeneity . g , Average of 88 DNA origamis . h , The particle average recovers the structure with an alignment uncertainty of 0 . 7 nm CI = [ 0 , 1 . 6 ] nm , showing a distance between the average R1 / R3 positions of 11 . 6 \u00b1 0 . 8 nm ( xy - distance : 2 . 5 \u00b1 0 . 4 nm , z - distance : 11 . 3 \u00b1 0 . 8 nm ) , matching the designed distances 20 . Same scale applies to all magnification panels . CI describes 68 % confidence interval . Article 100 nm 3D DNA - PAINT 3D RESI 43nm 22nm 0nm Z pos . 3D RESI ( zoom in for details ) 3D DNA - PAINT 3D RESI ( zoom in for details ) 3D DNA - PAINT 3D RESI ( zoom in for details ) 3D DNA - PAINT 3D RESI ( zoom in for details ) 3D DNA - PAINT a b 100 nm Extended Data Fig . 5 | 3D DNA origami . Representative 3D DNA origami from across the field of view of the measurement . a , Five DNA origami , shown at DNA - PAINT resolution ( upper row ) and RESI resolution ( lower row ) . The color scale to the right represents the z position of localizations . The measured z coordinates for each DNA origami have been shifted by a constant such that the lowest localization for a given structure is defined to be at z = 0 . This ensures full use of the color range . b , 32 additional DNA origami , shown at DNA - PAINT resolution ( upper rows ) and RESI resolution ( lower rows ) . The z positions are colored according to the color scale in panel a . 3D RESI 3D DNA - PAINT 3D RESI 3D DNA - PAINT 3D DNA - PAINT 3D DNA - PAINT 3D DNA - PAINT 3D DNA - PAINT 100 nm 3D RESI 3D RESI 3D RESI 3D RESI 3D RESI 3D DNA - PAINT 100 nm 0nm 140nm 70nm Z pos . a b Extended Data Fig . 6 | U2OS Nup96 - mEGFP . Representative NPCs from across the field of view of the measurement . a , Six NPCs , measured using DNA - PAINT ( upper row ) and RESI ( lower row ) . The color scale to the right represents the z position of localizations . The measured z coordinates for each NPC have been shifted by a constant such that the lowest localization for a given structure is defined to be at z = 0 . This ensures full use of the color range . b , 72 additional NPCs , measured using DNA - PAINT ( upper rows ) and RESI ( lower rows ) . The z positions are colored according to the color scale in panel a . Article Extended Data Fig . 7 | Averaging of Nup96 proteins . a , Model - free averaging for DNA - PAINT measurements of Nup96 ( N = 1045 NPCs ) . An angled isometric view is shown . b \u2013 d , DNA - PAINT resolves nucleoplasmic and cytoplasmic rings and recapitulates their eight - fold symmetry , but fails to resolve individual Nup96 proteins . e , Side views of all Nup96 pairs in both rings reveal the angled orientation but do not resolve individual Nup96 proteins . f , Model - free averaging for RESI measurements of Nup96 ( N = 1190 NPCs ) . g \u2013 i , RESI recapitulates nucleoplasmic and cytoplasmic rings as well as their eight - fold symmetry and resolves individual adjacent Nup96 proteins in the majority of cases . j , Side views of all eight Nup96 pairs in both rings reveal the angled orientation as well as , in some cases , adjacent individual Nup96 proteins . k , The Cryo - EM structure of the nuclear pore complex indicates that a given Nup96 protein will have neighbors spaced at 11 nm , 39 nm , 71 nm , 93 nm and 101 nm . l , Performing clustering and nearest neighbor analysis for DNA - PAINT data reveals a peak at approx . 40 nm , corresponding to the distance between two Nup96 pairs , but not below that . RESI , on the other hand , features a first peak at approx . 15 nm , corresponding to the distance between adjacent Nup96 while taking linkage error ( label size ) into account . m , Analysis of first to tenth nearest neighbor distances for RESI and DNA - PAINT recapitulates the distances from ( k ) , but only RESI resolves the smallest distance . All scale bars : 20 nm . DNA - PAINT RESI RESI ( zoom in for details ) DNA - PAINT RESI ( zoom in for details ) DNA - PAINT RESI ( zoom in for details ) DNA - PAINT 100 nm a b 5\u00c5 5\u00c5 100 nm Extended Data Fig . 8 | Sub - nm DNA origami . Representative DNA origami from across the field of view of the measurement . a , Four DNA origami , shown at DNA - PAINT resolution ( upper row ) and RESI resolution ( lower row ) . The inserts show pairs of directly adjacent docking strands resolved by RESI . b , 42 additional DNA origami , shown at DNA - PAINT resolution ( upper rows ) and RESI resolution ( lower rows ) . Article Round 1 Round 2 d DNA origami design Distance measurement in single DNA origami Round 1 ( R1 or R4 ) Round 2 ( R3 or R4 ) d = 8 . 8 \u00b1 2 . 9 \u00c5 d = 8 . 9 \u00b1 2 . 5 \u00c5 d = 10 . 6 \u00b1 2 . 7 \u00c5 d = 9 . 7 \u00b1 2 . 2 \u00c5 d = 7 . 8 \u00b1 2 . 7 \u00c5 d = 11 . 4 \u00b1 2 . 6 \u00c5 d = 1 . 2 \u00c5 CI = [ 0 , 4 . 0 ] \u00c5 d = 1 . 1 \u00c5 CI = [ 0 , 4 . 1 ] \u00c5 d = 0 . 6 \u00c5 CI = [ 0 , 4 . 4 ] \u00c5 d = 1 . 5 \u00c5 CI = [ 0 , 5 . 6 ] \u00c5 d = 1 . 1 \u00c5 CI = [ 0 , 5 . 2 ] \u00c5 d = 1 . 5 \u00c5 CI = [ 0 , 4 . 3 ] \u00c5 Round 1 Round 2 Average Sum N = 42 R1 R3 R4 a b c 20 nm 5 \u00c5 20 nm 1 nm 20 nm Extended Data Fig . 9 | Sub - nm RESI measurements . a , DNA origami featuring six alignment strands ( green R4 ) and six pairs of orthogonal docking strands ( red R1 , blue R3 ) spaced one base pair apart . b , RESI representation with RESI - localizations from round 1 in red and round 2 in blue illustrates excellent alignment . The distances between RESI - localizations from round 1 and 2 are defined as illustrated . c , Overlaying 42 DNA origami and performing a particle average recovers the structure with an alignment uncertainty of 1 . 2 \u00c5 CI = [ 0 , 4 . 6 ] \u00c5 , showing distances between the average positions of the sites at 9 . 5 \u00b1 2 . 6 \u00c5 ( mean over six distances in the average \u00b1 mean over the error - propagated uncertainties of the six distances ) . Same scale applies to all magnification panels . CI describes 68 % confidence interval . 5 \u03bcm 5 \u03bcm 20 nm 20 nm 20 nm 20 nm 5 \u03bcm 20 nm 20 nm N - th NND ( nm ) 0 20 40 60 80 100 120 800 600 400 200 0 C oun t 5000 4000 3000 2000 1000 0 C ou n t Density = 49 . 9 \u03bcm 2 Density = 128\u03bcm 2 Density = 289\u03bcm 2 N - th NND ( nm ) 0 20 40 60 80 100 120 N - th NND ( nm ) 0 20 40 60 80 100 120 0 20 40 60 800 600 400 200 C oun t 0 1st NND ( nm ) 0 20 40 60 1st NND ( nm ) 0 20 40 60 1st NND ( nm ) 800 600 400 200 0 5000 4000 3000 2000 1000 0 C ou n t 1000 800 600 400 200 C oun t 0 1000 C oun t RESI DNA - PAINT a DNA - PAINT b c N - th NND analysis 1st NND analysis d e Extended Data Fig . 10 | RESI resolves CD20 dimers in untreated CHO cells for different expression levels . a , DNA - PAINT imaging of whole mEGFP - CD20 - expressing CHO cells , labeled with anti - GFP - nanobodies , shows homogeneously distributed molecules for three independent experiments . b , Zoom - in regions of DNA - PAINT show cases in which dimers could not be resolved . c , RESI reveals sub - 10 - nm spaced receptor pairs , which are unresolvable in the DNA - PAINT cases . d , Whole - cell analysis of first nearest neighbor distances ( 1 st NNDs ) of CD20 receptors ( histograms of the distances are displayed ) . Only RESI , but not DNA - PAINT , allows the routine detection of sub - 10 - nm distances between proteins . e , RESI - localization precision below 1 nm allows for routine detection of sub - 10 - nm distances , resulting in an accurate assessment of the first NND . Article 50 nm DNA - PAINT Cluster analysis RESI DNA - PAINT 5 \u03bcm DNA - PAINT 500 nm Diffraction - limited i ii iii i ii iii RESI Single molecule clustering 100 nm c DNA - PAINT RESI a b c Extended Data Fig . 11 | RESI resolves the substructure in RTX - induced chain - like arrangements of CD20 receptors with sub - nanometer precision . a , DNA - PAINT overview image of mEGFP - CD20 expressing CHO cells treated with RTX . b , Labeling with DNA - conjugated anti - GFP - nanobodies and imaging with DNA - PAINT reveals higher - order organization after RTX - treatment . RESI ( insets i \u2013 iii ) achieves molecular resolution and thereby resolves the molecular arrangement of mEGFP - CD20 . c , DNA - PAINT imaging shows clustered CD20 molecules . Performing RESI with sequences R1 , R2 , R3 and R4 in four separate imaging rounds ( color - coded ) allows for clustering of localizations originating from a single target . From the clustered localizations , RESI - localizations were calculated , enabling true single - protein resolution . N - th NND ( nm ) 2000 1500 1000 500 0 C oun t C oun t 2000 1000 0 4000 3000 2000 1000 0 1500 500 C oun t 0 20 40 60 5 \u03bcm 5 \u03bcm 5 \u03bcm 50 nm 50 nm 50 nm 50 nm 50 nm 50 nm Density = 116\u03bcm 2 Density = 63 . 6 \u03bcm 2 Density = 90 . 8 \u03bcm 2 0 20 40 60 0 20 40 60 1st NND ( nm ) 0 20 40 60 2000 1500 1000 500 0 C oun t C oun t 2000 1000 0 4000 3000 0 20 40 60 0 20 40 60 N - th NND ( nm ) 1st NND ( nm ) 1st NND ( nm ) 2000 1000 0 1500 500 C oun t RESI N - th NND analysis 1st NND analysis DNA - PAINT a DNA - PAINT b c N - th NND ( nm ) d e Extended Data Fig . 12 | RESI reveals higher order arrangement of CD20 dimers in Rituximab - treated CHO cells . a , DNA - PAINT imaging of whole mEGFP - CD20 - expressing CHO cells , labeled with anti - GFP - nanobodies , shows clustered CD20 - molecules in Rituximab - treated cells for three independent experiments . b , Zoom - in regions of DNA - PAINT show mEGFP - CD20 clustered into chain - like arrangements . c , RESI reveals sub - 10 - nm spaced receptor pairs within the clusters , unresolvable by DNA - PAINT . d , Whole - cell analysis of first nearest neighbor distances ( 1 st NNDs ) of CD20 receptors bound to Rituximab ( histograms of the distances are displayed ) . Only RESI , but not DNA - PAINT , allows the routine detection of sub - 10 - nm distances between proteins . e , Routine detection of sub - 10 - nm distances by RESI recapitulates the first NND measured in the untreated case . Notably the NND peaks measured in the three repeats are consistent , independently of the protein density . Article a c g d h b 0 . 0 0 . 2 Circularity 0 . 4 0 . 6 0 . 8 1 1 . 0 2 . 0 N o r m . F r equen cy Simulation Data Simulation Data Number of molecules per cluster N o r m . F r equen cy 0 . 2 0 . 4 0 . 3 0 . 1 0 5 10 15 20 Simulation Data Simulation ( clustered ) Data ( clustered ) 50 nm 50 nm 200 nm 200 nm 200 nm 200 nm e f Extended Data Fig . 13 | Comparison of Rituximab treated CD20 data to simulated CD20 hexamer arrangements . a , Example of ground truth simulated CD20 hexamers ( light blue circles , simulated as triangles of dimers with intra - dimer distances of 13 . 5 nm as measured experimentally ) with random distribution and orientation on a 2D surface at the experimentally determined density . b , Label uncertainty and labeling efficiency ( black circles indicate labeled molecules ) are taken into account in the simulation for a realistic comparison . c , Simulated proteins in hexameric arrangements represented as gaussians . d , Hexamers after DBSCAN cluster analysis ( colors indicate clusters ) . e , RESI image of CD20 data after RTX - treatment . f , RESI - localizations of CD20 data after DBSCAN cluster analysis ( colors indicate clusters ) . g , Number of molecules per detected cluster for the experimental data and the simulated hexamers . h , Circularity metric of experimental data and the simulated hexamers after convex hull analysis of the clusters . We note that the sharp drop at 0 . 605 stems from the maximum circularity metric for clusters where the convex hull is defined by three molecules . Notably , the absence of a circularity peak at ~ 0 . 7 in the experimental data suggests that CD20 molecules are not arranged in isolated ring - like hexameric structures . a b c d N o r m a li z ed f r equen cy 1st - NN distance ( nm ) F r a c t i on o f non - r e s o l v a b l e m o l e c u l e s 400 600 800 1000 200 0 200 400 600 800 1000 Density = 100 / \u00b5m 2 0 25 50 75 100 125 150 200 F r a c t i on o f non - r e s o l v ab l e m o l e c u l e s D en s i t y ( \u00b5 m - 2 ) x ( nm ) y ( n m ) Resolution ( nm ) 20 40 60 80 200 400 600 800 1000 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 Density ( \u00b5m - 2 ) 0 50 100 150 200 250 300 350 400 Resolution12 nm 19 nm 27 nm 35 nm 42 nm Extended Data Fig . 14 | Stochastic labeling . a , Exemplary simulation of proteins with a Complete Spatial Random ( CSR ) distribution of a given density . b , Histogram of Nearest Neighbor Distances ( NNDs ) . The red line indicates the smallest distance ( d ) that can be resolved by DNA - PAINT for a given set of imaging parameters . The fraction of molecules with a NN below this distance threshold ( blue , shaded ) can be computed for a given density and a given DNA - PAINT resolution . c , 2D map of the fraction of non - resolvable molecules as a function of density and resolution . d , 1D cuts of c at different resolutions ( color - coded ) can be used as a user guide to estimate the number of multiplexing rounds needed to perform RESI efficiently given a certain target fraction of non - resolvable distances . Article Extended Data Table 1 | Imaging and RESI parameters Overview of DNA - PAINT image acquisition parameters alongside clustering and RESI parameters .", + "obashi2023conformational": "Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 A conformational switch in clathrin light chain regulates lattice structure and endocytosis at the plasma membrane of mammalian cells Kazuki Obashi 1 , Kem A . Sochacki 1 , Marie - Paule Strub 1 & Justin W . Taraska 1 Conformational changes in endocytic proteins are regulators of clathrin - mediated endocytosis . Three clathrin heavy chains associated with clathrin light chains ( CLC ) assemble into triskelia that link into a geometric lattice that curves to drive endocytosis . Structural changes in CLC have been shown to regulate triskelia assembly in solution , yet the nature of these changes , and their effects on lattice growth , curvature , and endocytosis in cells are unknown . Here , we develop a new correlative \ufb02 uorescence resonance energy transfer ( FRET ) and platinum replica electron microscopy method , named FRET - CLEM . With FRET - CLEM , we measure conformational changes in clathrin at thousands of individual morphologically distinct clathrin - coated structures . We discover that the N - terminus of CLC repositions away from the plasma membrane and triskelia vertex as coats curve . Preventing this conformational switch with chemical tools increases lattice sizes and inhibits endocytosis . Thus , a speci \ufb01 c conformational switch in the light chain regulates lattice cur - vature and endocytosis in mammalian cells . Clathrin - mediated endocytosis is the primary internalization pathway in eukaryotic cells and is key to many processes , including nutrient uptake , excitability , signaling , and the recycling of membrane components 1 . Over 50 proteins have been implicated in this process 2 , 3 . Determining the nanoscale localizations 4 , 5 , numbers 6 , 7 , accumulation dynamics 8 , 9 , interactions 1 , and conformations 10 , 11 of these proteins in cells is important for understanding how the endocytic machinery operates in health and disease . Assembly and curvature of clathrin lattices are required steps to build cargo - loaded vesicles 12 \u2013 14 . The basic unit of the clathrin lattice is the clathrin triskelion 15 , 16 . This six protein complex is composed of three clathrin heavy chains and three smaller clathrin light chains ( CLCs ) that form a three - legged pinwheel ( Supplementary Fig . 1a ) 17 . In non - neuronal cells , the stoichiometry of heavy chains to light chains might not always be one - to - one 18 . The heavy chains provide the backbone , and light chains are thought to regulate the assembly ofthe lattice and effect its mechanical properties 16 . Work both in vitro and in cells has indicated its important role 19 \u2013 23 , yet the structural , and func - tional actions of the light chains are not fully understood 16 , 24 . In vitro , clathrin triskelia assemble into spherical cages 13 . Cla - thrin light chain inhibits this assembly 25 , 26 . Light chain binds to heavy chain throughmultiple interactions 16 . One speci \ufb01 c interaction occurs through an acidic patch ( EED ) near the light chain \u2019 s N - terminus and the heavy chain \u2019 s knee ( Supplementary Fig . 1a ) 27 . This interaction prevents cage assembly by regulating the heavy chain knee con - formation ( Supplementary Fig . 1b ) 17 . If CLC binds to the heavy chain through these N - terminal contacts , CLC adopts an extended con - formation , stabilizing a straight conformation of the heavy chain knee . The straight conformation cannot assume the angles needed for cage assembly . However , if this interaction is prevented , the heavy chain knee is free to move , allowing the knee to bend and assemble as a cage . Thus , conformational switching of CLC is Received : 5 May 2022 Accepted : 25 January 2023 Check for updates 1 Biochemistry and Biophysics Center , National Heart , Lung , and Blood Institute , National Institutes of Health , 50 South Drive , Building 50 , Bethesda , MD 20892 , USA . e - mail : justin . taraska @ nih . gov Nature Communications | ( 2023 ) 14 : 732 1 1 2 3 4 5 6 7 8 9 0 ( ) : , ; 1 2 3 4 5 6 7 8 9 0 ( ) : , ; proposed to control the assembly of lattices 17 . Yet , the nature of these structural changes , and their effects on lattice growth , and curvature at the membrane of living cells are mostly unknown . Recent super - resolution imaging studies have mapped changes in protein locations at distinct stages of endocytosis at the scale of tens of nanometers 4 , 5 . Proteins are , however , regulated by sub - ten nanometer intra - and intermolecular conformational changes and binding events . To understand endocytosis , these conformational changes need to be determined 3 , 13 . This is a major gap in under - standing endocytosis . Yet , the precision of super - resolution imaging cannot measure structural changes at these scales . Fluorescence ( or F\u00f6rster ) resonance energy transfer ( FRET ) , however , can \ufb01 ll this gap , mapping molecular interactions and conformational changes within and between proteins 28 . FRET ef \ufb01 ciency depends on the spectral overlap between the donor and acceptor , distances between the dipoles , and their relative orientations to each other . Speci \ufb01 cally , FRET occurs when a donor \ufb02 uorophore and acceptor are generally separated by lessthan 10 nm 29 . WhileFRETmeasurementsareusually madewithdiffraction - limitedimaging , super - resolvedFRETmethods have been reported 30 . The resolution , labeling density , and colors possible in these experiments , however , are not able to discriminate morphological differences in small organelles such as clathrin - coated structures ( CCSs ) . For example , past FRET analysis of the protein organization of CCSs in yeast was limited to a late stage of endocytosis trapped by drug treatments 31 . Thus , existing methods cannot readily relate changes in protein conformation generated fromFRET to themorphologicalstagesofclathrin - coatedpitsasthey grow and curve . To overcome this gap , we turned to correlative light and electron microscopy ( CLEM ) 32 . CLEM can directly map \ufb02 uorescence signals to single nanoscale cellular structures visualized in paired electron microscopy ( EM ) images 33 . Platinum replica transmission EM ( PREM ) of unroofedcellplasmamembranes providesa uniquely high contrast , high - resolution , and wide - \ufb01 eld view of the inner plasma membrane of cells 34 . PREM has been combined with diffraction limited 35 , 36 , super - resolution 4 , 37 \u2013 40 , and polarized total internal re \ufb02 ection \ufb02 uorescence ( TIRF ) 41 microscopy to investigate molecular mechanisms of endocy - tosis , exocytosis , and the cortical cytoskeleton . Here , we develop a correlative lifetime based - FRET ( FLIM - FRET ) and PREM method , named FRET - CLEM . This method allows us to measure changes in protein interactions and conformations at distances less than 10 nm at single structurally - de \ufb01 ned organelles in single cells . We investigate the conformational changes in clathrin light chain by mapping distance changes both parallel and per - pendicular to the plasma membrane . We \ufb01 nd that the N - terminus of CLC moves away from both the CLC C - terminus and the plane of the plasma membrane as clathrin sites gain curvature . To determine the mechanistic impact of these structural changes , we develop a method to directly manipulate the N - terminal position of the cla - thrin light chain using a chemically - inducible dimerization system . These manipulations were con \ufb01 rmed with FRET . With acute che - mical perturbation , we show that inhibiting conformational chan - ges in CLC \u2019 s N - terminus increases clathrin lattice size , impairs maturation of clathrin structures at the plasma membrane , and inhibits transferrin endocytosis . Together , these data reveal a new conformational switch in clathrin light chain that regulates endo - cytosis in living cells . Results FRET - CLEM provides nanometer - scale spatial information at single clathrin sites To determine sub - ten nanometer conformational movements during endocytosis , we established a new correlative FLIM - FRET and PREM method , which we named FRET - CLEM ( Fig . 1 ) . We chose monomeric EGFP and a dark yellow \ufb02 uorescent protein , ShadowY 42 , as an optimized FRET pair by comparing six potential \ufb02 uorescent protein ( FP ) pairs ( Supplementary Fig . 2a ) . Calculations from the emission and absorbance spectra of this pair resulted in an estimated R 0 value ( distance of 50 % FRET ef \ufb01 ciency ) of 60\u00c5 ( Supplementary Fig . 2b ) . In line with these calculations , tandemly - connected EGFP - ShadowY probes fused to CLC showed > 50 % FRET ef \ufb01 ciency on the plasma membrane ( Supplementary Fig . 3a \u2013 d ) . We used this construct as a positive FRET control . Next , to testwhether FRET couldbe localized to single CCS , we performed FRET - CLEM measurements on HeLa cells expressing EGFP - CLC or EGFP - ShadowY - CLC ( Fig . 1a ) . First , cells were unroofed to expose the inner surface of the plasma membrane and rapidly \ufb01 xed 34 . These membranes provide uniquely high contrast , low background , ultra - thin samples for \ufb02 uorescence imaging 32 . Unroofed cells were then imaged with FLIM and subsequently prepared for platinum replica electron microscopy ( PREM ) . Past work has shown that these sample preparation steps do not measurably change the morphology and structure of the membrane and its associated endo - cyticorganelles 4 , 37 , 43 . This correlative method allows us to assign single diffraction - limited \ufb02 uorescent spots from a FLIM image to a single clathrinstructurevisualizedinPREM ( Fig . 1a ) . BecausePREMhasahigh spatial resolution , single \ufb02 uorescent spots in the FLIM image can be further classi \ufb01 ed according to the nanoscale structural features of the clathrin lattice ( Fig . 1b ) 43 . We then tested whether \ufb02 uorescence life - times can be analyzed at single CCS resolution . With the total photon number detected by our FLIM acquisition parameters ( Supplementary Fig . 3e \u2013 g , see also Methods ) , \ufb02 uorescence lifetime decay curves determined from single CCSs wereclearly separated between negative EGFP - CLC and positive EGFP - ShadowY - CLC FRET controls ( Fig . 1c ) . Speci \ufb01 cally , they could be \ufb01 t with a bi - exponential where the FRET ef \ufb01 ciency was determined for each CCS ( Supplementary Fig . 3h ) 44 . However , curve - \ufb01 tting was suboptimal for small CCSs due to the lim - ited number of photons 45 . Thus , to measure as many clathrin struc - tures aspossible across a cell , the mean \ufb02 uorescence lifetimewas used to estimate FRET ef \ufb01 ciencies across all structures ( Fig . 1d ) . These results show that FRET - CLEM can be used to generate FRET - based atomic - scale distances at single CCS resolution at morphologically distinct stages of endocytosis at the plasma membrane of mamma - lian cells . CLC N - terminal region moves away from the clathrin triskelion vertex Next , we applied FRET - CLEM to study conformational changes in CLC at the plasma membrane . In vitro , clathrin triskelia assemble into empty cages and CLC regulates this assembly 27 . CLC has been pro - posed to take two different conformations when bound to triskelia , an extended and bent conformation ( Fig . 2a ) 17 . Here , we assume three models of CLC conformations in cells based on in vitro models ( Fig . 2a ) . First , weassumethatunassembledtriskelioninthecytoplasm contains extended CLC and the spherical lattices in cells resembles in vitro assembled clathrin cages and contains bent CLC . Next , we propose three possible models for light chain dynamics during cur - vature . In the \ufb01 rst , CLC does not change conformations after assembly on the plasma membrane . Here , CLC assumes the bent conformation regardless of the curvature stage of the lattice ( \ufb02 at , domed , spherical ) . In the second , the proportions of extended and bent CLC gradually shift as curvature increases during endocytosis . In the third , as yet undescribed conformations of CLC are present in \ufb02 at and domed clathrin that switches to the bent conformation in spheres . To test these models , we used FRET - CLEM . Speci \ufb01 cally , the dis - tances between the N - terminus and C - terminus of surrounding CLCs differ substantially between the extended and bent conformations according to structural models 17 , 46 ( Fig . 2b , c , and Supplementary Fig . 1 ) . Although the expression levels of the transfected probes , the ratio between endogenous and transfected CLCs , and the fraction of heavy chains binding to CLC could affect FRET ef \ufb01 ciency , FRET Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 2 ef \ufb01 ciencies of the extended conformation are always larger than that of bent conformation when the degree of these factors are stable ( Supplementary Fig . 4 ) . This condition is satis \ufb01 ed when comparisons are made within a single cell . Thus , if the population of extended and bent conformations change on the plasma membrane during lattice assembly , FRET between the N - and C - terminus of CLCs is predicted to likewise change ( model 2 in Fig . 2a ) . Alternatively , if the conformations do not change , FRET should remain the same across all clathrin sub - types at the plasma membrane ( model 1 in Fig . 2a ) . To compare models 1 and 2 , we performed FRET - CLEM mea - surements on HeLa cells expressing EGFP - CLC , or EGFP - CLC and CLC - ShadowY ( Fig . 2d and Supplementary Fig . 5a , b ) . FPs are attached to CLCthrougha \ufb02 exiblelinker ( SupplementaryFig . 6 , seealsoMethods ) . The FP - attached CLCs were the dominant species within cells with our transfectioncondition ( SupplementaryFig . 7 , seealsoMethods ) . Mean \ufb02 uorescence lifetimes from single CCSs were analyzed by grouping them according to clathrin curvature classes determined from PREM images ( \ufb02 at , domed , and sphere ; Fig . 1b ) . There was no clear rela - tionship between mean \ufb02 uorescence lifetimes and photon counts ( Supplementary Fig . 8 ) . In cells expressing both EGFP - CLC and CLC - ShadowY , \ufb02 uorescence lifetimes increased as clathrin lattices curved ( Fig . 2d ) . This substantial increase did not occur when expressing only EGFP - CLC . The neuronal isoform of CLC which has an insertion of residues near the C - terminus 16 and was used in the previous in vitro study 17 showed similar lifetime changes ( Supplementary Fig . 9 ) . Fur - thermore , similar results were obtained in SK - MEL - 2 human cells a EGFP - CLC EGFP - ShadowY - CLC c b Flat Dome Sphere PREM FLIM 2 . 5 1 . 0 N o r m . c oun t s 1 0 . 01 0 . 001 0 . 1 Time ( ns ) 0 2 4 6 8 12 10 EGFP - CLC EGFP - ShadowY - CLC EGFP - CLC EGFP - ShadowY - CLC M ean f l uo r . li f e t i m e ( n s ) 2 . 5 2 . 0 1 . 5 d 200 nm PREM image Merge 500 nm F l uo r . li f e t i m e ( n s ) FLIM image 2 . 5 1 . 0 FRET Fluor . lifetime ( ns ) High Low F R E T High Low FRET Fig . 1 | FRET - CLEM . a Correlative FLIM - FRET and PREM images of unroofed membranes of HeLa cells expressing EGFP - CLC ( top ) or EGFP - ShadowY - CLC ( bot - tom ) . FLIM images ( left ; photon counts are represented by brightness and \ufb02 uor - escence lifetimes are represented by pseudo color ) , PREM images ( center ) , and mergeimages ( right ) . n = 1cellforeachcondition . Scale500nm . b PREMimagesof CCSs on an unroofed membrane of a HeLa cell expressing EGFP - CLC that were classi \ufb01 ed as \ufb02 at , domed , or sphere and corresponding areas from a FLIM image . n = 1 cell . Scale 200nm . c Fluorescence lifetime decays from single CCSs indicated by arrows in panel a . d Mean \ufb02 uorescence lifetimes from single CCSs on an unroofed membrane of HeLa cells expressing EGFP - CLC or EGFP - ShadowY - CLC . n = 58 CCSs from 1 cell ( EGFP - CLC ) and 81 CCSs from 1 cell ( EGFP - ShadowY - CLC ) . Forboxplots , boxisinterquartilerange , centerlineismedian , centercircleismean , whiskers are minimum and maximum data points with a coef \ufb01 cient value of 1 . 5 . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 3 indicating that these changes were not cell - type speci \ufb01 c ( Supplemen - taryFig . 10a \u2013 c ) . Inlivingcells , FRETef \ufb01 cienciesbetweenEGFP - CLCand CLC - ShadowY were higher on CCSs than in the cytosol ( Supplemen - tary Fig . 11 ) . However , unlike previous measurements in solution 17 , our FRET measurements re \ufb02 ected both intra - and inter - triskelia FRET . Next , we measured EGFP - CLC \u0394 N which is a truncation mutant of the \ufb02 exibleN - terminaldomain ( residues 1 \u2013 89 ) ( Fig . 2e and Supplementary Fig . 5c , d ) . In this mutant , EGFP would be bound near the heavy chain binding helix 15 . For this truncation , \ufb02 uorescence lifetimes did not substantially change across different lattice states . These data indicate that the N - terminal position of CLC undergoes a speci \ufb01 c conforma - tional switch at clathrin lattices on the plasma membrane during Flat Dome Sphere Model 1 CLC conformation FRET ( Fluor . lifetime ) Model 2 CLC conformation FRET ( Fluor . lifetime ) Model 3 New conformation CLC conformation FRET ( Fluor . lifetime ) ? ( ? ) Cell in vitro a b Extended Bent Unassembled Cage Low ( Long ) Low ( Long ) Low ( Long ) Bent Bent Bent Bent Bent Low ( Long ) High ( Short ) Low ( Long ) Extended + Bent d e f F l uo r . li f e t i m e c hange aga i n s t a v e r age o f f l a t c l a t h r i n ( % ) 1 0 - 1 2 F D S EGFP - CLC ( N only ) EGFP - CLC CLC - ShadowY ( N - C ) F D S F D S F D S EGFP - CLC (cid:2) N ( N only ) EGFP - CLC (cid:2) N CLC - ShadowY ( N - C ) 1 0 - 1 2 1 0 - 1 2 F D S F D S EGFP - QQN ( N only ) EGFP - QQN CLC - ShadowY ( N - C ) p < 0 . 001 p = 0 . 02 p = 0 . 005 p = 0 . 001 p = 0 . 004 p < 0 . 001 p = 0 . 004 FlatDomeSphere F l uo r . li f e t i m e c hange aga i n s t a v e r age o f f l a t c l a t h r i n ( % ) F l uo r . li f e t i m e c hange aga i n s t a v e r age o f f l a t c l a t h r i n ( % ) F R E T High Low Extended Bent c N - terminus C - terminus N - term . bent CLC N - term . extendedCLC New conformation ? ( ? ) Unassembled ( cytoplasm ) Extended Extended Extended Fig . 2 | CLC conformational changes in cells . a Schematic models of CLC con - formation at different assembly states in vitro or in living cells and their expected FRETef \ufb01 cienciesbetweentheN - andC - terminusofCLCs . b Astructuralmodelwith the assumption that either extended ( light blue ) or bent conformations ( magenta ) ofCLCsassembleintothelattice . ThemodelisbasedonPDB3LVGand6WCJ . 3LVG isoverlaidwith6WCJ . c Schematicmodelsoftwoassembledtriskeliawithextended ( left ) or bent CLCs ( right ) . A CLC N - terminus position ( yellow ) and C - terminus positions of surrounding CLCs ( orange ) are shown . d FRET - CLEM was performed on HeLa cells expressing either EGFP - CLC , or EGFP - CLC and CLC - ShadowY . Mean \ufb02 uorescence lifetimes from single CCSs were analyzed by categorizing them accordingtolatticestructures ( \ufb02 at , domedandsphere ) andtheywerecomparedto theaveragevaluesof \ufb02 atstructures . n = 6cellsfrom3experiments ( EGFP - CLC ) and n = 6 cells from 4 experiments ( EGFP - CLC and CLC - ShadowY ) . e FRET - CLEM on HeLacellsexpressingeitherEGFP - CLC \u0394 N , orEGFP - CLC \u0394 NandCLC - ShadowY . n = 6 cells from 5 experiments ( EGFP - CLC \u0394 N ) and n = 6 cells from 4 experiments ( EGFP - CLC \u0394 N and CLC - ShadowY ) . f FRET - CLEM on HeLa cells expressing either EGFP - QQN ( QQN mutant of CLC ) , or EGFP - QQN and CLC - ShadowY . n = 6 cells from 3 experiments for each condition . One - way ANOVA , then Tukey \u2019 s test . Each dot is from one cell experiment and errors are SE . For box plots , box is interquartile range , center line is median , center circle is mean , whiskers are minimum and maximum data points with a coef \ufb01 cient value of 1 . 5 . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 4 endocytosis . These data are not consistent with model 1 . Furthermore , \ufb02 uorescence lifetimes from cells expressing EGFP - CLC and ShadowY - CLC changed across different stages ( Supplementary Fig . 5g , h ) . This supports the movement of the CLC N - terminal region . Next , to further test model 2 , we measured QQN mutants of clathrin light chain ( resi - dues 20 \u2013 22 were substituted from EED to QQN ) ( Fig . 2f and Supple - mentary Fig . 5e , f ) . QQN mutants are de \ufb01 cient in binding to clathrin heavy chain near the triskelion vertex due to a loss of a negatively - charged patch at the N - terminus of the light chain ( Supplementary Fig . 1a ) 27 . Thus , they are inhibited from adopting the proposed extended conformation 17 . Here , if model 2 is correct , FRET across the structural states should differ between wild - type and QQN mutants . Speci \ufb01 cally , QQN mutants are expected to show a smaller displace - ment inconformations and smaller changes in \ufb02 uorescencelifetimeas the lattice curves . However , in these mutants , we found similar \ufb02 uor - escence lifetime changes as in the wild - type protein ( Fig . 2d , f ) . This similarity in \ufb02 uorescence lifetime change was observed among the range of expression levels obtained in our experimental conditions ( Supplementary Fig . 12 ) . These data do not support model 2 . Thus , we conclude that CLC structural movements at the plasma membrane cannot be described by current in vitro models ( model 1 or 2 ) . CLC N - terminal region moves away from the plasma membrane Next , we measured FRET between EGFP positioned in different domains of clathrin light chain and the membrane resident dark FRET - based quencher dipicrylamine ( DPA ) ( Supplementary Fig . 2c ) 47 . Because the plane of the membrane is \ufb01 xed , FRET between EGFP and DPA provides relative distances perpendicular to the plane of the plasma membrane 48 , 49 . Speci \ufb01 cally , when EGFP sits near the mem - brane , the FRET ef \ufb01 ciency is high and \ufb02 uorescence lifetimes are short ( Fig . 3a ) . With DPA , CLC - EGFP showed the shortest lifetime ( Fig . 3b , c ) . ThisindicatesthattheC - terminusofCLCislocatedclosetotheplasma membrane . In contrast , EGFP - CLC showed the longest \ufb02 uorescence lifetime . EGFP - CLC \u0394 N showed an intermediate lifetime . From these data , we can position segments of CLC relative to the plane of the plasma membrane ( Fig . 3d ) . Unlike classic models 17 , we \ufb01 nd that the N - terminus of CLC bound to clathrin - coated structures at the plasma membrane is located farther into the cytoplasm than the proximal leg of clathrin heavy chain . To investigate conformational changes during lattice curvature , we performed FRET - CLEM measurements with DPA and compared those measurements to morphological changes in clathrin lattice ( Fig . 3e and Supplementary Fig . 13 ) . In these experiments , \ufb02 uores - cence lifetimes of EGFP - CLC increased as lattices curved , and the degree of change was larger than those for CLC - EGFP . Similar results were obtained for cells expressing SNAP - CLC - EGFP or EGFP - CLC - SNAP ( Supplementary Fig . 14 ) . In addition , in SK - MEL - 2 cells , \ufb02 uor - escence lifetimes of EGFP - CLC increased as lattices curved , and the degree of change was similar to that of HeLa cells ( Supplementary Fig . 10d , e ) . These data are consistent with a model where the N - terminal position of CLC changes during curvature . Thus , the combined results from EGFP - ShadowY and EGFP - DPA FRET indicate that the N - terminus of CLC extends away from the CLC C - terminus ( triskelion vertex ) and the plane of the plasma membrane during endocytosis ( Fig . 3f ) . Conformational changes in CLC regulate lattice structure and endocytosis Next , wetestedwhethertheseconformationalchangesinCLCregulate clathrin - mediated endocytosis . To manipulate the CLC N - terminal position directly , we used a rapamycin - inducible FKBP / FRB dimeriza - tion system 50 . Speci \ufb01 cally , we employed the T2098L mutant of FRB which is heterodimerized to FKBP by the rapamycin analog , AP21967 51 . We attached FKBP to the N - terminus of CLC , and one or two FRBs ( FRB\u00d72 ) to the C - terminus of the membrane - bound PH domain from PLC \u03b4 1 ( PH ) 52 ( Fig . 4a ) . We made both FRB and FRB\u00d72 probes to extend the reach of the system . To con \ufb01 rm that the N - terminal position of FKBP - EGFP - CLC changes relative to the plasma membrane after dimerization , we measured EGFP \ufb02 uorescence lifetimes without or with AP21967 in the presence of DPA ( Fig . 4b , c ) . Changes in the posi - tion of EGFP can be estimated by comparing the difference in \ufb02 uor - escence lifetimes between the control and DPA - treated membranes ( Fig . 4d ) . For both probes , differences in EGFP lifetimes with AP21967 treatment were larger than those without AP21967 . These results indicate that the CLC N - terminus moves towards the plasma mem - brane as a result of FKBP / FRB dimerization . Because PH - miRFP - FRB\u00d72 showed larger lifetime change ( Fig . 4d ) and stronger accumu - lation with AP21967 than PH - miRFP - FRB ( Supplementary Fig . 15a ) , we used PH - miRFP - FRB\u00d72 ( or PH - mCherry - FRB\u00d72 ) to alter the structure of clathrin light chain in living cells . First , usingourFKBP / FRBconstructs , weinvestigatedtheimpactof moving the CLC N - terminus towards the plasma membrane on the structure of clathrin lattices 43 . Here , unroofed membranes from cells expressing FKBP - EGFP - CLC and PH - miRFP - FRB\u00d72 and treated without or with AP21967 were imaged with PREM ( Supplementary Fig . 16 ) . Unlike the previous homodimerization system 53 , clathrin lattices were not noticeably distorted . The average size of all visible \ufb02 at and domed clathrin structures increased with AP21967 treatment [ 31463\u00b1859nm 2 ( ctrl ) and 42919\u00b12278nm 2 ( AP ) for \ufb02 at , 29821\u00b1576nm 2 ( ctrl ) and 36471\u00b12862nm 2 ( AP ) for domed , 15311\u00b1588nm 2 ( ctrl ) and 17303\u00b1691nm 2 ( AP ) for sphere ] ( Fig . 5a - c ) . We conclude that manip - ulation of the CLC N - terminal position directly effects the structure of clathrin lattices . Next , toinvestigatehowchangingCLCconformationseffectsCCS assembly and maturation , we performed live cell time lapse evanes - cent \ufb01 eld imaging . Cells expressing FKBP - EGFP - CLC and PH - miRFP - FRB\u00d72 were imaged with total internal re \ufb02 ection \ufb02 uorescence micro - scopy ( TIRF ) in the absence or presence of AP21967 ( Fig . 5d , e ) . To quantitate CCS dynamics , we analyzed the residence times of FKBP - EGFP - CLC spots on the plasma membrane . The residence times became shorter after AP21967 addition . This indicates that tethering the CLC N - terminal position towards the plasma membrane likely facilitates the disassembly of CCSs . Finally , we investigated whether manipulation of the CLC N - terminal position effects transferrin endocytosis in whole cells ( Fig . 5f , g and Supplementary Fig . 17a , b ) . As a control for binding of FRB to the CLC N - terminus , a cytosolic probe , mCherry - FRB\u00d72 , was used . Furthermore , as a control of clathrin tethering to the plasma membrane , and also FRB accumulation on CCSs , a C - terminal - linked FKBP probe , CLC - FKBP - EGFP , was tested . For all three probe pairs , FKBP / FRB dimerization was con \ufb01 rmed by clustering of FRB at CCSs ( SupplementaryFig . 15b ) andchangesinFRETef \ufb01 ciencybetweenEGFP and mCherry ( Supplementary Fig . 15c ) . Expression of these probes did not change transferrin uptake without AP21967 treatment ( Supple - mentary Fig . 17b ) . Consistent with this \ufb01 nding , the amount of surface transferrin receptors was not changed by the expression of FKBP / FRB probes ( Supplementary Fig . 17c , d ) . We found that transferrin uptake decreased with AP21967 for PH \u2013 mCherry - FRB\u00d72 but not for the con - trol mCherry - FRB\u00d72 ( Fig . 5g ) . Furthermore , for the C - terminus - attached FKBP control ( CLC - FKBP - EGFP ) , PH - mCherry - FRB\u00d72 did not alter transferrin uptake ( Fig . 5g ) . These results indicate that manip - ulation of the CLC N - terminalposition inhibited transferrinuptake and endocytosis . These data are consistent with the decrease in the resi - dence time of CCSs we observed in live cell imaging . From these col - lective data , we concluded that the conformational changes in CLC we mapped in FRET - CLEM experiments are important structural changes required for endocytosis of cargo - loaded vesicles in mammalian cells . Thus , the movement of the N - terminal domain away from the clathrin vertex and plasma membrane is a key regulatory step in clathrin - mediated endocytosis . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 5 High FRET ( Short fluor . lifetime ) PM DPA EGFP 2 . 5 2 1 . 5 1 0 . 5 0 M ean f l uo r . li f e t i m e ( n s ) 0 100 200 DPA concentration ( (cid:3) M ) EGFP - CLC (cid:2) N EGFP - CLC ( N - term ) CLC - EGFP ( C - term ) a b DPA0 (cid:3) M 20 (cid:3) M CLC - EGFP ( C - term ) EGFP - CLC (cid:2) N c d e 4 F l uo r . li f e t i m e c hange aga i n s t a v e r a ge o f f l a t c l a t h r i n ( % ) 3 1 0 - 1 2 Flat Dome Sphere Flat Dome Sphere CLC - EGFP ( C - term ) EGFP - CLC ( N - term ) p < 0 . 001 p < 0 . 001 p < 0 . 001 p < 0 . 001 p = 0 . 002 f PM Low FRET ( Long fluor . lifetime ) EGFP 2 . 5 0 . 5 Fluor . lifetime ( ns ) FRET Distance from PM Near Far High Low F R E T High Low Sphere Flat PM EGFP - CLC ( N - term ) 10 (cid:3) m EGFP - CLC ( N - term ) EGFP - CLC (cid:2) N CLC - EGFP ( C - term ) Fig . 3 | CLC N - terminal position perpendicular to the plane of the plasma membrane . a Dipicrylamine ( DPA ) is a non \ufb02 uorescent hydrophobic anion that incorporates into membranes . DPA quenches EGFP in a distance dependent man - ner by FRET . PM is the plasma membrane . b FLIM images of unroofed membranes ofHeLacellsexpressingCLC - EGFP ( left ) , EGFP - CLC \u0394 N ( center ) , orEGFP - CLC ( right ) without ( top ) or with 20 \u03bc M DPA ( bottom ) . Scale 10 \u03bc m . c Mean \ufb02 uorescence lifetimes with different DPA concentrations . n = 16 ( EGFP - CLC ) , 17 ( EGFP - CLC \u0394 N ) , and 15 cells ( CLC - EGFP ) from 3 experiments . Errors are SE . d A structural model of EGFPpositionsofCLCprobespredictedfromtheEGFP - DPAFRETexperiments . The model is based on PDB 4KW4 and 3LVG . e FRET - CLEM on HeLa cells expressing either CLC - EGFP or EGFP - CLC with DPA . DPA concentrations were 3 \u03bc M for CLC - EGFPand80 \u03bc MforEGFP - CLCtoobtain ~ 50 % FRETef \ufb01 cienciestomakethedegree of \ufb02 uorescence lifetime changes similar . Mean \ufb02 uorescence lifetimes from single CCSs were analyzed by categorizing them according to lattice structures and compared to average values of \ufb02 at structures . n = 6 cells from 4 experiments for each condition . Each dot is from one cell experiment and errors are SE . One - way ANOVA , then Tukey \u2019 s test . For box plots , box is interquartile range , center line is median , center circle is mean , whiskers are minimum and maximum data points withacoef \ufb01 cientvalueof1 . 5 . f AproposedstructuralmodelofCLCconformational changes predicted from both FRET - CLEM with EGFP - ShadowY and EGFP - DPA . The N - terminus of CLC moves away from both the CLC C - terminus ( triskelion vertex ) and the plane of the plasma membrane as clathrin lattices curve . The structural model is based on PDB 3LVG . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 6 Discussion We have explored the molecular - scale conformational changes in clathrin light chain at clathrin sites at the plasma membrane of mam - maliancells . We \ufb01 ndthattheN - terminusofCLCmovesawayfromboth the triskelion vertex and the plasma membranes as clathrin lattices curve . Blocking this movement increased clathrin lattice sizes at the plasma membrane and reduced transferrin endocytosis . Thus , speci \ufb01 c structural changes in CLC in clathrin lattices at the plasma membrane are regulators of curvature and endocytosis in living mammalian cells . Conformational changes in CLC have been proposed to regulate clathrin assembly 17 . We mapped the speci \ufb01 c changes in cells by measuring vectors in two orthogonal axes . First , EGFP - DPA FRET measurements positioned the light chain relative to the plane of the membrane ( Fig . 3d ) , and reported the movement along the axis perpendicular to the plasma membrane ( Fig . 3f ) . Unlike classic in vi - tro models 17 , we \ufb01 nd that the N - terminus of CLC is located farther into the cytoplasm than the proximal leg of clathrin heavy chain and moves away from the membrane . With intermolecular FRET between EGFPandShadowY at theN - and C - terminus ofthelight chain ( Fig . 2 ) , we could map structures along the axis parallel to the plasma mem - brane . Here again , we detected a displacement of the N - terminal domain away from the CLC C - terminus ( clathrin lattice vertex ) . Combined , these data indicate that the N - terminus of CLC moves away from both the clathrin vertex and the plane of the plasma membrane as lattices gain curvature . Although the exact position of the N - terminus in relation to the heavy chain proximal leg domain was not determined , our experi - ments could be used to provide additional insights . Speci \ufb01 cally , we modeled how FRET ef \ufb01 ciencies between EGFP - CLC / CLC - ShadowY ( Supplementary Fig . 5b ) or EGFP - CLC / ShadowY - CLC ( Supplementary Fig . 5g ) would change as the N - terminal position changes according to the x - ray structures 17 across all possible spatial positions PH domain miRFP FRB FRB CLC FKBP EGFP AP21967 a b Potential FRET pairs EGFP and miRFP EGFP and miRFP EGFP and DPA EGFP and DPA AP21967 DPA Clathrinlattice Plasmamembrane FKBP - EGFP - CLC PH - miRFP - FRB\u00d72 c d Ctrl - + DPA - + AP - + - + Ctrl AP 2 1 M ean f l uo r . li f e t i m e ( n s ) PH - miRFP - FRB PH - miRFP - FRB\u00d72 (cid:2) f l uo r . li f e t i m e ( n s ) [ D PA ( 0 (cid:3) M ) - D PA ( 20 (cid:3) M ) ] 0 1 0 . 5 Ctrl AP Ctrl AP PH - miRFP - FRB PH - miRFP - FRB\u00d72 2 . 5 1 . 5 Fig . 4 | Manipulation of CLC N - terminal position using a chemically inducible dimerization system . a Schematic models of the chemically inducible FKBP / FRB dimerization system . FKBP is attached to the N - terminus of CLC , and two FRBs ( FRB\u00d72 ) are attached to the C - terminus of PH domain from PLC \u03b4 1 . A rapamycin analog , AP21967 , induces heterodimerization between FKBP and the T2098L mutant of FRB . b Potential FRET pairs without or with AP21967 treatment either absence or presence with DPA . c Fluorescence lifetime measurements were per - formed on unroofed membranes of HeLa cells expressing FKBP - EGFP - CLC either with PH - miRFP - FRB , or PH - miRFP - FRB\u00d72 without or with 20 \u03bc M DPA . Cells were unroofed after 15min incubation with AP21967 or ethanol ( control ) . n = 20 ( PH - miRFP - FRB , control ) , 19 ( PH - miRFP - FRB , AP21967 ) , 20 ( PH - miRFP - FRB\u00d72 , control ) , and 19 cells ( PH - miRFP - FRB\u00d72 , AP21967 ) from 3 experiments . d Differences in \ufb02 uorescencelifetimes ( shown inpanel c ) without and withDPA . For box plots , box is interquartile range , center line is median , center circle is mean , whiskers are minimum and maximum data points with acoef \ufb01 cient value of 1 . 5 . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 7 A r ea o cc upa t i on ( % ) 4 0 8 d f a 50 m Tf - AF647 FKBP - EGFP - CLC PH - mCherry - FRB\u00d72 N o r m . T f - A F 647 f l uo r . i n t en s i t y 1 0 2 - + Transfection - + - + AP21967 FKBP - EGFP - CLC PH - mCherry - FRB\u00d72 FKBP - EGFP - CLC mCherry - FRB\u00d72 CLC - FKBP - EGFP PH - mCherry - FRB\u00d72 p < 0 . 001 g AP 21967 2 D p r o j e c t i on a r ea ( 10 n m \u00b2 ) 4 2 0 6 Ctrl AP 1 0 2 0 . 3 0 0 . 6 4 2 0 6 1 0 2 p < 0 . 001 p = 0 . 04 Flat Dome Sphere N u m be r ( / m 2 ) 2 0 1 0 0 . 5 0 0 . 4 0 . 2 erehpS emoD talF erehpS emoD talF Ctrl AP Ctrl AP Ctrl AP Ctrl AP Ctrl AP Ctrl AP Ctrl AP Ctrl AP No treatment AP21967 200 0 R e s i d en c e t i m e ( s ) 400 p < 0 . 001 10 0 20 30 F r eq uen cy ( % ) 10 0 20 30 76912PA tnemtaert oN e Residence time ( s ) 65 125 185 245 305 > 320 65 125 185 245 305 > 320 Residence time ( s ) C on t r o l c b 3 Fig . 5 | Manipulation of CLC conformation changed lattice structures , dynamics , and endocytosis . a \u2013 c Unroofed membranes from cells expressing FKBP - EGFP - CLC and PH - miRFP - FRB\u00d72 treated with AP21967 ( AP ) or ethanol ( con - trol ) were imaged with PREM . Two - dimension area of single CCS were manually segmented and measured . Membrane area occupation against the total analyzed membrane area ( a ) , two - dimension projection area ( b ) , and density ( c ) of \ufb02 at , domed , andsphereCCSswerecompared . Eachdotisfromonecellexperiment , and errors are SE . n = 6 cells from 3 experiments for each condition . The average measured area / cell ( mean\u00b1SE ) = 213\u00b134 ( control ) and 165\u00b110 \u03bc m 2 ( AP21967 ) . d Live cell time lapse TIRF imaging on HeLa cells expressing FKBP - EGFP - CLC and PH - miRFP - FRB\u00d72 without or with AP21967 treatment . Tracks with over 20s were analyzed and residence times were compared . n = 206 spots from 5 cells from 3 experiments ( no treatment ) and 234 spots from 5 cells from 4 experiments ( AP21967 ) . A two - sided unpaired t test was used . e Histogram of residence times . f Confocal projection images of Alexa Fluor 647 conjugated transferrin ( Tf - AF647 ) uptake in HeLa cells expressing FKBP - EGFP - CLC and PH - mCherry - FRB\u00d72 treated with AP21967 or ethanol ( control ) . Fluorescence intensity of Tf - AF647 ( arbitrary units ) isrepresentedbypseudocolor . n = 3experiments . Scale50 \u03bc m . g Transferrin uptake in HeLa cells expressing FKBP and FRB probes treated with AP21967 . Fluorescence intensities of incorporated Tf - AF647 normalized by non - transfected cells in the same sample were compared between non - transfected and transfected cells . n = 116 \u2013 198cellsfrom3experimentsforeachconditions . Forboxplots , boxis interquartile range , center line is median , center circle is mean , whiskers are minimum and maximum data points with a coef \ufb01 cient value of 1 . 5 . A two - sided unpaired t test was used . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 8 ( Supplementary Fig . 18a ) . Although the fraction of heavy chains binding to ShadowY - attached CLC might affect the overall gross ef \ufb01 - ciency of FRET , the positional dependency and direction of change would not changewithexpressionlevelsaccordingtoourcalculations . Because FRET ef \ufb01 ciencies were largely similar for both sites , the N - terminus of CLC is predicted to rest in the overlap regions that match these paired distances ( Supplementary Fig . 18b , magenta cir - cles ) . To accommodate these constraints , CLC would assume a slightly folded conformation ( orange and green in Supplementary Fig . 18c ) rather thana stretched conformation ( blue in SupplementaryFig . 18c ) . From these considerations we propose a new model of CLC con - formational changes ( Fig . 6 ) . Here , CLC assumes an extended and tightly bound conformation in unassembled triskelia in the cytoplasm similar to that seen in x - ray crystal structures and measured with FRET 17 . Next , whenCLCassemblesatthemembraneasa \ufb02 at lattice , the light chain changes conformations from this extended position into a new folded conformation . The N - terminus is then displaced deeper intothecytosolasclathrinlatticescurveintovesicles . Insupportofour data , the average position of the CLC N - terminal domain is not visible by cryo - EM in puri \ufb01 ed clathrin - coated vesicles 46 . Thissuggests thatthe N - terminal domain is \ufb02 exible and positioned away from the lattice in highly curved structures . What initiates this structural change in the light chain ? HIP and HIP1R interact with the CLC N - terminal residues 54 , 55 . Although CLC QQN mutants are thought to be de \ufb01 cient in binding HIP1 and HIP1R 55 , we found that FRET between the N - and C - terminus of the CLC QQN mutants showed FRET changes comparable to wild type proteins ( Fig . 2d , f ) . Thus , if this region is necessary for binding , HIP1R inter - actions are unlikely to drive these conformational changes . The bind - ing of other proteins to the lattice 16 , 24 , changes in protein - protein interactions caused by cargo loading 1 , or phosphorylation 21 are alter - nate mechanisms that could drive this change . Finally , physical mechanismssuchas crowding and cargoloading intothe vesicle could induce the conformational switch 2 , 13 . In vitro studies have shown that CLC increases the rigidity of clathrin lattices on a solid substrate 20 . These effects were different between different CLC isoforms 23 . In living cells , CLC has been shown to be important for endocytosis under conditions of high membrane tension 19 , 22 . Here , our FKBP / FRB experiments demonstrate that con - formational changes in CLC regulate the structure of clathin ( Fig . 5a \u2013 c ) , the dynamics of single clathrin sites in live cells ( Fig . 5d , e ) , and the endocytosis of cargo in whole cells ( Fig . 5f , g ) . One possible interpretationisthatthe latticecangrow but not fully curve due to the inhibition of the CLC conformation switch . Possibly , clathrin lattices grow larger because treatment disturbed the balance between lattice assembly and curvature 12 . These irregularly - assembled CCSs are then disassembled by proofreading mechanisms 3 . The CLCs role in endo - cytosis differs between isoforms , cargos , and cell types 19 , 21 \u2013 23 , 56 \u2013 59 and the physiological function of CLC are not fully understood 16 , 24 . Clar - i \ufb01 cation on how CLC conformations are regulated acrossisoformsand cargos will provide a uni \ufb01 ed view of the light chain \u2019 s mechanistic roles across different cells , tissues , and states . Our work has some speci \ufb01 c limitations . For example , we expressed FP - tagged probes to measure FRET . Thus , the expression ratio between endogenous and FP - labeled proteins could affect the overall FRET ef \ufb01 ciency ( Supplementary Figs . 4c and 18a ) . This , along with other photophysical issues , makes it dif \ufb01 cult to convert FRET ef \ufb01 ciencies to absolute atomic distances 60 . However , the differences in expression level do not affect the direction of FRET changes caused by conformational changes in this system ( Supplementary Figs . 4c , 12 , and 18a ) . Likewise , the fact that our two orthogonal FRET - based experiments using either FRET between two \ufb02 uorescent pro - teins or FRET between a \ufb02 uorescent protein and a probe in the membrane all showed similar vectors of movement for the light chain strongly supports our structural models . In the future , dual - tagging all alleles of the endogenous protein is a direction to further re \ufb01 ne these measurements 31 . Also it will be interesting to investigate the relationship between the stoichiometry of proteins and FRET with a combination of other optical techniques . Furthermore , the size of \ufb02 uorescent proteins , while relatively small compared to the size of the clathrin complex and approaching the size of large red organic dyes , may affect a protein \u2019 s structure . Likewise , larger probes can limit the ability to detect small or distant conformational changes 60 . Thus , the incorporation of smaller tags or arti \ufb01 cial amino acids are future directions to improve these measurements 61 , 62 . Indeed , smaller probes have been shown to better recapitulate absolute distance changes seen during conformational transitions and might have less of an in \ufb02 uence on the proteins themselves 29 . In addition , increasing photon counts with organic dyes , combined with new quantitative analysis of photon count data , will be impor - tant for improving this method . In the case of clathrin light chain , however , fusion of \ufb02 uorescent proteins has not been seen to perturb endocytosis or change clathrin lattice structures 63 . Expression of CLC probes used in FRET experiments did not change the size and number of clathrin lattice structures ( Supplementary Fig . 19 ) . FRET ef \ufb01 ciencies are determined by photophysical properties ( excitation , emission , absorbance ) , distance , and orientation of the probes 28 , 44 . Here , we used a \ufb02 exible linker to connect the FPs to CLC ( Supplementary Fig . 6 and Methods ) . Thus , it is reasonable to propose that the population of FPs in a CCS can assume many possible orien - tations and the effect of an orientation bias would be small . However , we cannot exclude the small possibility that the curvature of clathrin lattices causes slight differences in the orientations between the donor and acceptor , which may affect the overall FRET ef \ufb01 ciencies . However , the fact that the N - terminal changes was observed in both two FP - FRET experiments and FP - DPA FRET experiments , again supports the idea that orientation effects are not large or dominant . Combined , these caveats make it dif \ufb01 cult to directly equate FRET ef \ufb01 ciencies to atomic distances . ThisiscommonlythecaseinFRETstudiesdonewithincells 60 . For cells expressing only EGFP probes , although there were no substantial change in \ufb02 uorescence lifetimes among different lattice structures , clathrin vesicles tended to show slightly higher lifetimes Plasmamembrane Flat Dome Sphere Bent model Cage Cell in vitro Extended model Unassembled Clathrinlattice Proposed model Sphere Flat Fig . 6 | ModelsofconformationalswitchinCLCinlivingcells . Schematicmodels of conformationalswitchin CLCatthe plasma membrane incells . CLC assumes an extended conformation in unassembled triskelia in the cytoplasm similar to that seeninx - raycrystalstructures . Next , whenCLCassemblesatthemembraneasa \ufb02 at lattice , CLC changes conformations from the extended to a new folded con - formation . The N - terminus is then displaced deeper into the cytosol as clathrin latticescurveintovesicles . TheextendedandbentmodelsarebasedonPDB3LVG . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 9 than \ufb02 atclathrinstructures ( Fig . 2 ) . BecauseHomo - FRETisnotthought to change the overall \ufb02 uorescencelifetime 44 , this might be caused by a difference in the local environment 64 , or a systematic counting error due to the differences in the photon count rate among CCSs with different \ufb02 uorophore densities 65 . These effects , however , were small and consistent across samples . Here , we developed a new correlative FLIM - FRET and PREM method to track the conformational changes in CLC at single sites of endocytosis in cells . The N - terminus of CLC makes a dramatic movement away from the clathrin lattice vertex and deeper into the cytosol as lattices curve . These conformational dynamics are key for clathrin - mediated endocytosis . These data , combined with the rich biochemical , functional , genetic , and biophysical information on membrane traf \ufb01 c , will lead to a more robust understanding of how endocytic proteins work together during clathrin - mediated endocy - tosis to drive the internalization of cargo . More generally , our new method maps molecular interactions and conformational changes of targeted proteins at identi \ufb01 ed sites in the complex environment of the cell . Thus , FRET - CLEM can be used to investigate conformational changes of any accessible membrane - associated protein including ion channels , transporters , receptors , adhesion proteins , or enzymes , in the context of their local plasma membrane environments 29 , 32 . Methods Cell culture HeLa cells ( ATCC , CCL - 2 ) were maintained at 37\u00b0C , with 5 % CO 2 in DMEM ( Gibco , 11995073 ) supplemented with 10 % fetal bovine serum ( ATLANTA biological , S12450H ) and 1 % vol / vol penicillin / streptomycin ( Invitrogen , 15070 - 063 ) . SK - MEL - 2 cells ( ATCC , HTB - 68 ) were main - tained at 37\u00b0C , with 5 % CO 2 in EMEM ( ATCC , 30 - 2003 ) supplemented with 10 % fetal bovine serum and 1 % vol / vol penicillin / streptomycin . The cells were placed on poly - L - lysine coated coverslips ( Neuvitro , GG - 25 - 1 . 5 - PLL ) . They were transfected with 0 . 75mL of Opti - MEM ( Life Tech - nologies , 31985062 ) , 3 . 8 \u03bc L Lipofectamine 2000 ( Life Technologies , 11668027 ) , and 1 . 5 \u03bc g of DNA for 3h after being introduced to the cells . Then , transfected cells were incubated in DMEM or EMEM growth medium for 20 \u2013 30h before experiments . The cells tested negative for mycoplasma contamination . Plasmids ShadowY ( # 104621 ) , mScarlet - I ( # 85068 ) , mTurquoise2 ( # 54842 ) , miRFP703 - CLCb ( # 79997 ) , CFP - FKBP ( # 20160 ) , Nup54 - EGFP494 ( 0 ) ( # 163426 ) , Nup54 - EGFP494 ( 1 ) ( # 163427 ) , Nup54 - EGFP494 ( \ufb02 ex0 ) ( # 163429 ) , andNup54 - EGFP494 ( \ufb02 ex1 ) ( # 163430 ) werepurchasedfrom Addgene , and pSNAP - tag ( m ) was purchased from New England Bio - labs . CLCa - GFP was kindly donated by Dr . W . Almer ( Oregon Health & Science University ) . Lyn - GFP - FRB was kindly donated by Dr . T . Inoue ( Johns Hopkins University ) . mNeonGreen was kindly donated by Dr . J . Shah ( Harvard University ) . mCherry - PH was from our previous studies 66 . All plasmids used in this study were constructed using either Q5 Site Directed Mutagenesis Kit ( New England Biolabs , E0554S ) or In - Fusion HD Cloning Plus ( Clonetech , 638920 ) following manufacturer \u2019 s instructions . For tandemly - connected FPs in Supplementary Fig . 2a , N - ( residues 1 \u2013 4 for ShadowY , and 1 \u2013 5 for mCherry and mScarlet - I ) and C - terminal disordered regions ( residues 228 \u2013 238 for EGFP and mTur - quoise2 , and 226 \u2013 236 for mNeonGreen ) of FPs were deleted . And two FPs were connected with a short linker ( GGSGGS ) . For EGFP - ShadowY - CLC in Fig . 1 , tandemly - connected EGFP - ShadowY was fused to CLCa with a 10 a . a . linker . For CLCb probes used inFRET experiments , either C - terminaldisorderedregion ( residues228 \u2013 238 ) deletedorN - terminal disordered region ( residues 1 \u2013 4 ) deleted EGFP ( or ShadowY ) was connected to CLCb with a short linker ( GGSGGS ) . All plasmids were con \ufb01 rmed by sequencing ( Psomagen ) and identi \ufb01 ed as in Supple - mentary Data 1 . Fixing and unroo \ufb01 ng Cells were rinsed in intracellular buffer ( 70mM KCl , 30 mM HEPES maintained at pH 7 . 4 with KOH , 5 mM MgCl 2 , 3mM EGTA ) , and manually unroofed with 19 - gauge needle and syringe using 2 % paraf - ormaldehyde ( ElectronMicroscopySciences , 15710 ) intheintracellular buffer . After unroo \ufb01 ng , the coverslips were transferred to fresh 2 % paraformaldehyde in the intracellular buffer for 20min . They then washed with phosphate - buffered saline ( PBS ) . Imaging was performed in PBS ( pH 7 . 4 ) ( Quality Biological , 114 - 058 - 131 ) . For FKBP / FRB dimerization experiments , cells were unroofed after 15min incubation with 500 nM AP21967 ( 500 \u03bc M stock in etha - nol ) ( Takara , 635055 ) or 0 . 1 % w / v ethanol ( control ) in DMEM growth medium . Imaging of unroofed plasma membranes for CLEM Unroofed cells were stained either with 16 . 5 pmol of Alexa Fluor 350 - phalloidin ( LifeTechnologies , A22281 ) , AlexaFluor568 - phalloidin ( Life Technologies , A12380 ) , or Alexa Fluor 647 - phalloidin ( Life Technolo - gies , A22287 ) for 15min depending on spectra of expressing FPs . Then cells were rinsed with PBS . 1 mm\u00d7 1mm large montage was generated for proteins ofinterest and phalloidinusing a NikonEclipse Ti inverted microscope with a 100\u00d7 , 1 . 49 NA objective ( Nikon , SR HP Apo TIRF ) and an Andor iXon Ultra 897 EM - CCD camera under the control of Nikon Elements software . Images were obtained by TIRF illumination except for Alexa Fluor 350 which was imaged by epi illumination . This map was used to \ufb01 nd the cells expressing the target proteins for FLIM and CLEM analysis . The imaged area wasmarked with a circle ( 4 mm in diameter ) around the center of the imaged area using an objective diamond scriber ( Leica , 11505059 ) 40 , 67 . The immersion oil wascarefully removed from the bottom of the glass coverslip . The sample was subsequentlyimaged by FLIM orstored in 2 % glutaraldehyde ( Electron Microscopy Sciences , 16019 ) at 4 \u00b0C until EM sample preparation . FLIM Time - domain \ufb02 uorescence lifetime imaging was performed with a Leica Falcon SP8 confocal microscope with a 63\u00d7 , 1 . 40 NA oil immer - sion objective ( Leica , HC PL APO CS2 ) under the control of Leica LAS X software . As \ufb01 rst , a large image montage around the region of interest on the coverslip marked by a diamond scriber was acquired to \ufb01 nd the cells which were identi \ufb01 ed in the TIRF montage . Then , FLIM images were collected at a lateral spatial resolution of 80nm per pixel and withascanspeedat3 . 16 \u03bc sperpixel . Theconfocalaperturewassetata diameter of 168 \u03bc m ( 2 AU @ 510nm ) . EGFP was excited by 488nm and 498 \u2013 560nm \ufb02 uorescence was collected . A notch \ufb01 lter for 488 nm was used . To reduce the impact of pile - up effect , an excitation power was set tokeeppeak count perpulse aslessthan0 . 1 . For CLEM , theimaged sample was then stored in 2 % glutaraldehyde at 4 \u00b0C until EM sample preparation . To select a FP pair for FLIM - FRET , we made tandemly con - nected FPs and compared their FRET ef \ufb01 ciency ( Supplementary Fig . 2a ) . Based on its high FRET ef \ufb01 ciency and compatibility for our optical systems , we selected monomeric EGFP and ShadowY ( Sup - plementary Fig . 2b ) . To check the relationship between the number of frames and photobleaching or FRET ef \ufb01 ciency change , \ufb02 uores - cence intensity and FRET ef \ufb01 ciency of EGFP - ShadowY - CLC was compared with various frame numbers ( Supplementary Fig . 3e , f ) . Fluorescence intensity did not change and FRET ef \ufb01 ciency slightly decreased as the frame number increases . This might be due to photobleaching of ShadowY occurs faster than EGFP . Although the magnitude of ShadowY photobleaching should be smaller for lower FRET ef \ufb01 ciency situation like intermolecular FRET , we used 150 frames to minimize the effect of photobleaching . With 150 frames , ~ 10 , 000 photon counts per single CCS were obtained in average ( Supplementary Fig . 3g ) . With this range of photon counts , FRET ef \ufb01 ciency ( E ) of EGFP - ShadowY - CLC from single CCS could be Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 10 estimated by \ufb01 tting a \ufb02 uorescence decay curve with a bi - exponential function convolved with the Gaussian pulse response function 68 ( Supplementary Fig . 3h ) . F \u00f0 t \u00de = F 0 \u00bd P 1 H t , t 0 , \u03c4 1 , \u03c4 G (cid:1) (cid:3) + \u00f0 1 (cid:2) P 1 \u00de H t , t 0 , \u03c4 2 , \u03c4 G (cid:1) (cid:3) (cid:3) \u00f0 1 \u00de H t , t 0 , \u03c4 1 , \u03c4 G (cid:1) (cid:3) = 1 2exp \u03c4 G 2 2 \u03c4 1 (cid:2) t (cid:2) t 0 \u03c4 1 (cid:4) (cid:5) erfc \u03c4 G 2 (cid:2) \u03c4 1 t (cid:2) t 0 (cid:1) (cid:3) \ufb03\ufb03\ufb03 2 p \u03c4 1 \u03c4 G ! \u00f0 2 \u00de where F ( t ) is the \ufb02 uorescence lifetime decay curve , F 0 is the peak \ufb02 uorescence before convolution , P 1 is the fraction of the \ufb01 rst com - ponent , \u03c4 1 and \u03c4 2 are the \ufb02 uorescence lifetime of \ufb01 rst and second components , \u03c4 G is the width of the Gaussian pulse response function , t 0 is time offset , and erfc is the complementary error function . \u03c4 a = P 1 \u03c4 1 + 1 (cid:2) P 1 (cid:1) (cid:3) \u03c4 2 \u00f0 3 \u00de E = 1 (cid:2) \u03c4 a , DA \u03c4 a , D \u00f0 4 \u00de where \u03c4 a is amplitude - weighted \ufb02 uorescence lifetime , \u03c4 a , D is amplitude - weighted \ufb02 uorescence lifetime of the donor without acceptor , and \u03c4 a , DA is amplitude - weighted \ufb02 uorescence lifetime of the donor in presence of the acceptor 44 . Values from single CCSs ( SupplementaryFig . 3h ) wereconsistentwiththosefromwholeplasma membrane ( Supplementary Fig . 3a \u2013 c ) . However , in general , nearly 10 , 000 photon counts are required to \ufb01 t a bi - exponential 45 , 69 . So , curve - \ufb01 tting on the \ufb02 uorescence decay curves from very small CCSs willnotbeadequate . Thus , insteadtoestimateFRETef \ufb01 ciencythrough curve - \ufb01 tting , mean \ufb02 uorescence lifetime , the center mass of the \ufb02 uorescence lifetime decay , was used as an indicator for FRET ef \ufb01 ciency ( Fig . 1d and Supplementary Fig . 3d ) . Background was not subtracted . In our measurements , background counts were generally verylowandsignal - to - backgroundratiowas > 100inmostexperiments ( Supplementary Fig . 8b ) . Mean \ufb02 uorescence lifetime was not depen - dent on the signal - to - background ratio . For FLIM measurements with dipicrylamine ( DPA ; City Chemical LLC ) , a 20 mM stock solution of DPA in DMSO ( Sigma - Aldrich , D2650 ) was prepared fresh from powder every day and diluted to a \ufb01 nal concentration with PBS . Imaging was performed at least 10 min after addition of DPA . FRET ef \ufb01 ciency between DPA and miRFP should be very low because DPA absorbancespectra and miRFP emission spectra are suf \ufb01 ciently separated ( Supplementary Fig . 2c ) . For live cell FLIM imaging ( Supplementary Fig . 11 ) , cells were imaged in imaging buffer ( 130mM NaCl , 2 . 8 mM KCl , 5mM CaCl 2 , 1 mM MgCl 2 , 10mM HEPES , and 10 mM glucose at pH 7 . 4 ) at 21 \u00b0C . Platinum replica EM EM samples were prepared asdescribed previously 4 , 67 . Coverslips were transferred from glutaraldehyde into 0 . 1 % w / v tannic acid for 20 min . Then , they were rinsed 4 times with water , and placed in 0 . 1 % w / v uranyl acetate for 20min . The coverslips were then dehydrated , cri - tical point dried with a critical point dryer ( Tousimis Samdri , 795 ) , and coated with platinum and carbon with a freeze fracture device ( Leica , EM ACE 900 ) . The region of interest on the coverslip marked by a diamond scriber was imaged with a 20\u00d7 phase - contrast objective to obtain another map of the region imaged in \ufb02 uorescence . The replicas were lifted and placed onto formvar / carbon - coated 75 - mesh copper TEM grids ( Ted Pella , 01802 - F ) that were freshly glow - discharged with PELCO easiGlow 91000 . Again , the grid was imaged with a 20\u00d7 phase - contrastobjectiveto \ufb01 ndthesameregionthatwasoriginallyimagedin \ufb02 uorescence . Each cell of interest was located on the grid prior to EM imaging 40 . TEM imaging was performed as previously described 37 at \u00d715 , 000 magni \ufb01 cation ( 1 . 2nm per pixel ) using a JEOL 1400 and SerialEM freeware for montaging 70 . Electron microscopy montages were processed using IMOD freeware 71 . FRET - CLEM image analysis The FLIM images were aligned to the EM images using an af \ufb01 ne spatial transformation with nearest - neighbor interpolation to map the CCSs visibleinbothFLIMimagesandEMimages 36 , 41 . Sincepositionofsphere clathrin is sometimes changed during critical point drying due to a weak attachment tothemembrane , we used \ufb02 atand domed clathrinas \ufb01 ducials 67 . Rectangular ROI was created around single isolated CCS , and mean \ufb02 uorescence lifetime within each ROI was calculated . These values were analyzed by categorizing according to lattice structures , and were compared to the average value of \ufb02 at clathrin for each unroofed membrane . This is because FRET ef \ufb01 ciencies , and thus \ufb02 uorescence lifetimes , varied among cells due to the differences in expression levels of donor and acceptor probes , or incorporated density of DPA ( Supplementary Figs . 5 , 9 , 10 , 13 , and 14 ) . For FRET between EGFP and ShadowY , because ShadowY expression cannot be con \ufb01 rmed with \ufb02 uorescence , only cells with a cellular average \ufb02 uorescence lifetime of less than 2 . 1 ns were analyzed to ensure the expression of ShadowY . EM image analysis Binary masks of the \ufb02 at ( no visible curvature ) , domed ( curved but can still see the edge of the lattice ) , and sphere clathrin ( curved beyond a hemisphere such that the edge of the lattice is no longer visible ) were manually segmented ( Supplementary Fig . 19a ) 43 , 72 . The percentage of occupiedmembraneareawasde \ufb01 nedasthesumofareasfromclathrin lattices of the speci \ufb01 ed subtype divided by the total area of measured membrane . The expression of CLC probes did not change the size and density of clathrin lattice structures ( Supplementary Fig . 19b \u2013 d ) . Live cell TIRF imaging and analysis Images were acquired using a Nikon Eclipse Ti inverted microscope with a 100\u00d7 , 1 . 49 NA objective and an Andor iXon Ultra 897 EM - CCD camera . HeLa cells in the phenol red free DMEM growth medium [ DMEM ( Gibco , 31053036 ) with 10 % fetal bovine serum , 1 : 100 dilution of 100\u00d7 GlutaMAX ( Gibco , 35050061 ) , 1 : 100 dilution of 100 mM sodium pyruvate ( Gibco , 11360070 ) ] were mounted in a chamber at 37\u00b0C with a water bath and continuous \ufb02 ow of humidi \ufb01 ed 5 % CO 2 to maintain the osmolality and pH of the medium . The pixel size was 110nm . TIRF images were acquired with 100ms exposures at 0 . 5 Hz for 10 min . For AP21967 treatment , images were acquired at least 10min after the addition of 500nM AP21967 . FKBP - EGFP - CLC spots were tracked using the ImageJ plugin TrackMate 73 . Spot detection wasdone with the differenceof Gaussians approach , and tracking was done using the simple linear assignment problem ( LAP ) tracker algorithm with a linking maximum distance of 330 nm per frame . Tracks that appeared and disappeared during the whole movie with over 20s duration , and a total net displacement was less than 330 nm were selected for residence time analysis . Further , each track was visually inspected for isolation and tracking errors . Transferrin uptake assay HeLa cells were incubated in starvation medium [ DMEM containing 20mM HEPES at pH 7 . 4 and 0 . 1 % w / v bovine serum albumin ( Fisher Bioreagents , BP9703 ) ] for 1h in an CO 2 incubator . They were then incubated with 500nM AP21967 or 0 . 1 % w / v ethanol ( control ) in starvation medium for 5min . Then , 25mg / mL Alexa Fluor 647 con - jugated human transferrin ( Invitrogen , T23366 ) was added to the starvation medium 74 . After 15 min incubation , the cells were \ufb01 xed with 2 % paraformaldehyde at room temperature for 25min . They then washed with PBS . The cells were imaged with a Leica Falcon SP8 con - focal microscope with a 63\u00d7 , 1 . 40 NA oil immersion objective . Images were collected at a lateral spatial resolution of 120nm per pixel . The Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 11 confocal aperture was set at a diameter of 191 \u03bc m , and optical sections with z - spacing of 0 . 8 \u03bc m were collected . Alexa Fluor 647 was excited by 633nm and 638 \u2013 800nm \ufb02 uorescence was collected . The stack images were recombined using a sum - intensity opera - tion . And background intensity fromnon - cellregions were subtracted . Then , cell outlines were traced manually and the mean intensity ( total intensity / area ) was measured for individual cells . Fluorescence inten - sity was normalized by the average value of non - transfected cells for each coverslip . Immunocytochemistry HeLacellswere \ufb01 xedin2 % paraformaldehydeinPBSfor25min , blocked with 3 % w / v bovine serum albumin for 60min , and reacted with mouse monoclonal antibody against transferrin receptor ( Santa Cruz , sc - 65877 , 1 : 50 ) . The primary antibody was visualized by secondary anti - body staining using goat anti - mouse IgG conjugated to Alexa Fluor 647 ( Invitrogen , A21237 , 1 : 500 ) . The cells were imaged with a Leica Falcon SP8 confocal microscope with a \u00d763 , 1 . 40 NA oil immersion objective . Images were collected at a lateral spatial resolution of 120nm per pixel . The confocal aperture was set at a diameter of 191 \u03bc m , and optical sections with z - spacing of 0 . 8 \u03bc m were collected . Alexa Fluor 647 was excitedby633nmand638 \u2013 800nm \ufb02 uorescencewascollected . Images were analyzed as transferrin uptake assay . Linker \ufb02 exibility measurements with polarization - TIRF ( pol - TIRF ) microscopy HeLa cells were imaged with 3i vector TIRF microscopy system , based on an inverted microscope ( IX - 81 ; Olympus ) and equipped with 100\u00d7 , 1 . 49 NA objective ( Olympus ) . Fluorescencewas excited by 488 nm and 561 nm lasers passed through LF405 / 488 / 561 / 635 \ufb01 lters ( Semrock ) . Emitted light was then divided by the image splitter \u2019 s dichroic ( 565DCXR ) and projected side - by - side through 525Q / 50 and 605Q / 55 emission \ufb01 lters onto the chip of an EMCCD camera ( Andor , DU 897 ) . Images were acquired using SlideBook6 ( 3i ) . A sequence of 40 images was taken with pol - TIRF \ufb01 elds that were parallel ( s - pol ) or perpendi - cular ( p - pol ) to the coverslip . Each individual image had an exposure time of 100 ms . For calibration , \ufb02 uorescein solution ( Alfa Aesar , L13251 ) wasmeasured . 20 s - poland20p - polimageswere summed and background was subtracted . A p : s ratio was then calculated and nor - malized by the value of \ufb02 uorescein measurements . Past work measured the orientation of nucleoporins ( Nups ) in the nuclear pore complex using pol - TIRF microscopy and orientational sensors 75 . In these sensors , a short N - terminal \u03b1 - helix of EGFP is con - jugated directly to a C - terminal \u03b1 - helix in a protein of interest by a continuous \u03b1 - helix . When the number of amino acids in this linker helix is varied , EGFP rotates around the linker helix axis by angles dictated by \u03b1 - helical geometry . Since the excitation dipole is \ufb01 xed within EGFP , the p : s ratio shifts by changing the linker length . On the other hand , if EGFP is conjugated by a \ufb02 exible linker , the p : s ratio does not shift by changing the linker length . First , we tested if we could reproduce the previous results in our imaging system . We imaged the bottom of the nucleus of \ufb01 xed HeLa cells expressing Nup54 - EGFP494 constructs 75 ( Supplementary Fig . 6a ) . Consistent with previous data , the p : s ratio shifted by changing the linker length for the rigid linker probes but did not shift for the \ufb02 exible linker probes ( Supplementary Fig . 6b ) . Next , we applied this strategy to investigate the linker \ufb02 ex - ibility in CLC probes . We conjugated EGFP to C - terminal \u03b1 - helix domain of CLC ( at residue 206 ) by rigid or \ufb02 exible linkers ( Supple - mentary Fig . 6c ) . We then imaged unroofed HeLa cells expressing CLC orientational sensors , CLC - EGFP , or EGFP - CLC ( Supplementary Fig . 6d ) . The p : s ratio shifted with different lengths of the rigid linkers however the p : s ratio was not changed by varying lengths of the \ufb02 ex - ible linkers . The p : s ratio of CLC - EGFP and EGFP - CLC which were used inFRETexperimentsweresimilartothoseofthe \ufb02 exiblelinkersensors . Theseresultssupportthe assumptionthat thelinkersofCLC - EGFP and EGFP - CLC are \ufb02 exible . Further , since there were no signi \ufb01 cant corre - lation between EGFP intensity and the p : s ratio in CLC - EGFP and EGFP - CLC ( Supplementary Fig . 6e \u2013 g ) , linkers are likely \ufb02 exible in clathrin lattices with various shapes . Western blot HeLa cells were dissociated with trypsin and collected . After washing and centrifuge , 100 \u00b5 L RIPA lysis buffer ( Millipore 20 \u2013 188 ) supple - mented with protease inhibitor cocktail ( Halt , 78429 ) was added and samples were incubated for 1 h on ice . After vortex , cell lysates were centrifuged at 21300 \u00d7 g for 15 min at 4 \u00b0C . The supernatant was used for SDS - PAGE on 4 \u2013 12 % Bis - Tris gels ( Invitrogen , NP0335 ) with Tris - buffered saline with 0 . 1 % Tween - 20 ( TBS - T ) at 150 V . Blotting was performed by iBlot ( invitrogen ) with nitrocellulose membranes ( Invitrogen , IB301002 ) accordingly to the manufacturer \u2019 s protocol . Afterwards , membranes were incubated for 2 h in 5 % milk in TBS - T . Primary antibodies [ anti - CLCa / b ( Millipore , AB9884 , 1 : 5000 ) , anti - CLCa ( Sigma , HPA050918 , 1 : 1000 ) , or anti - CLCb ( Abnova , H00001212 - M01 , 1 : 500 ) ] were diluted in 5 % milk / TBS - T and applied on the membranes overnight . After 5 times 5 min washing with TBS - T , secondary antibodies [ anti - mouse IgG - HRP ( Jackson ImmunoR - esearch Labs , 115 - 035 - 174 , 1 : 2000 ) or anti - rabbit IgG - HRP ( Jackson ImmunoResearch Labs , 211 - 032 - 171 , 1 : 2000 ) ] were diluted in 5 % milk / TBS - T and applied on the membranes for 1 h . After washing with TBS - T , the membrane was imaged with ChemiDoc system ( Bio - Rad ) with ECL solution ( cytiva , RPN2232 ) . Then after washing , the membrane was stained with anti - \u03b2 actin - HRP ( Cell Signaling , 5125 S , 1 : 2000 ) and imaged . Estimation of expression levels of transfected constructs To estimate the expression levels of transfected constructs , we performed western blot ( Supplementary Fig . 7 ) . First , we stained with anti - CLCa / b antibody ( Supplementary Fig . 7a ) . The amount of endogenous CLCa / b decreased by transfection ( Supplementary Fig . 7d ) . The degree of decrease was lager in dual - transfection than single - transfection . The reactivity of this antibody against N - terminal - FP - attached CLC was weaker than that against C - terminal - FP - attached CLC . Thus we did not measure the ratio between transfected and endogenous CLC with this antibody . Next , we stained with either anti - CLCa ( Supplementary Fig . 7b ) or anti - CLCb ( Supplementary Fig . 7c ) speci \ufb01 c antibody . Consistent with anti - CLCa / b antibody , the amount of endogenous CLCa and CLCb decreased with transfection ( Supplementary Fig . 7e , f ) . And the amount of transfected CLCb was larger than that of endogenous CLCb ( Supplementary Fig . 7g ) . From these results , we modeled the expression of transfected constructs . CLCa is the dominant isoform in HeLa cells 18 . Thus , we assumed a ratio between CLCa and CLCb of 4 : 1 and a transfection ef \ufb01 ciency of 80 % . Under these conditions , we estimate the CLC amount in transfected cells ( Supplementary Fig . 7h ) . In this model , the total amount of CLCs increases 10 times after transfection and the ratio between transfected and endogen - ous CLCs is 25 : 1 for single transfection and 100 : 1 for double trans - fection . Thus , in our FRET experiments , we propose that most CLCs are attached to an FP . FRET simulation FRET simulations ( Supplementary Figs . 4c and 18 ) were performed using MATLAB . We used 60 \u00c5 for F\u00f6rster radius for EGFP - ShadowY FRET . For the simulation in Supplementary Fig . 4c , we determined the position of N - and C - terminus of CLC according to the structure models ( PDB 3LVG and 6WCJ ) 17 , 46 . For the simulation in Supple - mentary Fig . 18 , the lateral position of N - terminus was moved by 0 . 6 \u00c5 spacing , and N - terminus was assumed to locate axially 25 \u00c5 higher than C - terminus . When we focus on single clathrin heavy chain , there are four different states ; without CLC , endogenous Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 12 CLCa or CLCb binding , EGFP - CLC binding , or CLC - ShadowY binding ( Supplementary Fig . 4b ) . By considering these four states , we cal - culated the FRET ef \ufb01 ciency between EGFP - CLC and CLC - ShadowY or ShadowY - CLC binding to surrounding \ufb01 ve clathrin heavy chains at various heavy chain occupancy with ShadowY - attached CLC . Quanti \ufb01 cation and statistical analysis Image analysis and quanti \ufb01 cation were performed with ImageJ 76 and MATLAB . The statistical tests used for each experiment and the exact sample numbers ( n values ) are indicated in the corresponding \ufb01 gure legends . p values of < 0 . 05 were considered statistically signi \ufb01 cant . All statistical analysis and \ufb01 tting were performed using Origin 2016 ( Origin Lab ) . The exact p values are provided in Source Data \ufb01 le . Materials availability The plasmids used in the study are deposited at Addgene ( Supple - mentary Data 1 ) . Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article . 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Murakoshi ( National Institute for Physiological Sciences ) for sharing the absorbance and emission spectra of DPA and ShadowY respectively . We thank Dr . J . Jiang ( National Heart , Lung , and Blood Institute ) for use of the ChemiDoc . We thank National Heart , Lung , and Blood InstituteLight Microscopy Core and Electron Microscopy Core for use of instruments and advice . We thank G . Haber for help with coding . We thank members of the Taraska lab for helpful discussions and edits . J . W . T . is supported by the Intramural Research Program of the National Heart , Lung , and Blood Institute , National Institutes of Health . K . O . is supported by JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH . Author contributions K . O . , K . A . S . , and J . W . T . designed the research . K . O . performed experi - ments and analysis . K . A . S . developed software for analysis . M . - P . S . helped with molecular cloning and western blots . J . W . T . supervised the project . K . O . and J . W . T . wrote the paper . All authors contributed to the interpretation of the data and commented on the manuscript . Funding OpenAccessfundingprovidedbytheNationalInstitutesofHealth ( NIH ) . Competing interests The authors declare no competing interests . Additional information Supplementary information The online version contains supplementary material available at https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 . Correspondence and requests for materials should be addressed to Justin W . Taraska . Peer review information Nature Communications thanks Sergi Padilla Parra and the other , anonymous , reviewer ( s ) for their contribution to the peer review of this work . Peer reviewer reports are available . 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If material is not included in the article \u2019 s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . ThisisaU . S . Governmentworkandnotundercopyrightprotectioninthe US ; foreign copyright protection may apply 2023 Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36304 - 7 Nature Communications | ( 2023 ) 14 : 732 15", + "packerImportanceSDICurrent1979": "The Importance of SDI for Current Awareness in Fields with Severe Scatter of Information Katherine H . Packer Faculty of Library Science , University of Toronto , Toronto M55 1A 1 , Canada Dagobert Soergel College of Library and Information Services , University of Maryland , College Park , MD 20742 Scope : The purpose of this study was to explore the use and effectiveness of current awareness ( CA ) methods , espe - cially SDI , among chemists at Canadian universities . Method : We used a questionnaire which was mailed to a sample of 170 ( response rate 80 % ) . The variables studied include per - ceived scatter of information , use of various CA methods , time spent on CA , perceived success in CA , and CA effi - ciency = successhime spent . The results of this explanatory study are tentative . Major results : Keeping up - to - date is more difficult in specialties with high scatter of information ( hypothesis 5 ) . SDI seems to be the only method capable of counteracting scatter ( hypotheses 5 - 7 ) . In contrast , if scatter is low , SDI use decreases CA efficiency ( hypothesis 6a ) ; scanning journals , the most prevalent CA method , seems to be sufficient for low - scatter specialties . High scatter does not lead to increased SDI use , in some cases due to per - ceived difficulty of profile construction ( hypothesis 3a ) . Conclusions : SDI services should concentrate efforts on serving scientists in specialties with high scatter of informa - tion by developing systems that are capable of searching across conventional disciplines and specialties , by providing assistance in profile development , and by conducting a mar - keting campaign directed toward these scientists . Introduction Selective dissemination of information ( SDJ ) , as defined by Housnian and Kaskela [ I ] , is a service \u201cthat attacks the hforniation problem by keeping him [ the scientist ] con - tinuously informed of new documents published in his areas of specialization so that he can keep abreast of the latest developments . \u201d When first introduced it was thought to hold the promise of solving the scientists\u2019 problem of keeping up - to - date . However , in the 20 years since H . P . Lulin first put forward the concept , SDI services have been used far less than predicted and not nearly enough for the promise to be realized . Received August 1977 ; revised November 27 , 1978 . 0 1979 by John Wiley & Sons , Inc . This raises the question : Is it that scientists fail to per - ceive the value of SDI to them , or is it that they identify problems in the use of SDI that have not been sufficiently recognized by the designers of SDI systems , or if recog - nized , not effectively solved ? A mail questionnaire survey of chemists and chemical engineers at Canadian universities was undertaken to shed light on this question . The major aims of this study were to ( 1 ) test the effec - tiveness of SDI as a method of current awareness and , in particular . explore the factors which influence the effective - ness of SDI ; ( 7 - ) find out the reasons why scientists do or do not subscribe to an SDI service . In addition to contributing to an understanding of the information transfer process in science it was hoped that the results of the study would be of value to managers of SDI services . In order to gain an understanding of the use of SDI services it was thought necessary to look at the entire current awareness effort of scientists . Some subject fields were known to be served by a small number of highly specialized journals . In others , particularly interdisciplinary fields , relevant articles may be published in a large number of different journals . I t was an underlying assumption of this study that such scatter of information in a subject field would play an important role in explaining the effectiveness of any current awareness method and particular attention was paid , therefore , to scatter of information as an indepen - dent variable . This study is exploratory in nature . Its results must be considered preliminary . Some hypotheses were formulated a priori , but other , new hypotheses were also formulated based on the results of the data analysis . Clear distinction between a priori and a posteriori hypotheses has been main - tained in the section dealing with the results . The remainder of this section gives an overview of the findings and draws some conclusions for the management of SDI services . The sections that follow discuss in detail the methodology used , the results , and the limitations of the study . JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE 0002 - 8231 179 / 0030 - 0125 $ 01 . OO Overview of Results Fifty - six percent of the respondents reported a scatter value , of 4 or 5 on a 5 - point scale . All respondents made use of a variety of current awareness methods . On the whole , formal methods were used slightly more than infor - mal methods . Only 19 % of the respondents subscribed to an SDI service . The mean time spent on current awareness was from 2 to 5 hours per week , and the average level of success reported was 3 on a 5 - point scale ( adequate ) . The following relationships between variables were found : ( 1 ) The more time spent on current awareness the higher the level of success achieved . ( 2 ) The more time spent on current awareness the higher the productivity ( as measured by number of journal articles published ) . ( 3 ) The greater the scatter of information , the more use made of current awareness methods and the more time spent on this activity . The respondents in high - scatter fields were no more likely to subscribe to an SDI service , however , than respondents in low - scatter fields . ( 4 ) The respondents in fields with high scatter were as successful in keeping up - to - date as respondents in fields with low scatter . However , they do so at a price . ( 5 ) Respondents in high - scatter fields had to spend more time to achieve the same level of success . Their effi - ciency ( success per time spent ) was lower . This effect is very strong for nonsubscribers to an SDI service . Among the subscribers to an SDI service the negative effect of high scatter on efficiency is less severe . ( 6 ) In the total sample subscribers to an SDI service are no more efficient in keeping up - to - date than nonsubscribers . However . when the sample was divided into a low - scatter and a high - scatter group , the effect of subscribing to an SDI service was found to have a reverse effect in the two groups . For the low - scatter group , subscribing leads to lower effi - ciency ; for the high - scatter group subscribing leads to higher efficiency . No such effects were found for other current awareness methods . The results can be summarized as follows : ( 7 ) Among the current awareness methods tested , sub - scribing to an SDI service is the only one that was found to have any capacity to counteract scatter . In spite of the apparent success of SDI services in counteracting scatter of information , as already reported above . respondents in high - scatter fields were no more likely to subscribe to an SDI service than those in low - scatter fields . These findings have practical implications for the development of SDI services . From interviews it became evident that some scientists recognize the problem of scatter of information , but have serious doubts about the possibility of designing a profile that would identify all rele - vant material and at the same time suppress irrelevant material except when interests were very narrow and specific . They viewed scatter of information as a reason for not subscribing to an SDI service . In conclusion , it must be emphasized that the finding that subscribing to an SDI service counteracts scatter of in - formation is only tentative . While tendencies are discernible from the data none of the tests produced significant results , probably because of the small number of subscribers in the sample . Implications for the Management of SDI Services SDI services should concentrate efforts on serving scien - tists in specialties with high scatter of information . Systems must be designed so that searching across conventional dis - ciplines and specialties ( often this means across data bases ) is easy and convenient . Intermediaries must be available in local libraries to assist in profile development , a task that is more difficult for users in high - scatter specialties . These intermediaries must be trained particularly with respect to this task , and , perhaps more importantly , a marketing cam - paign must be directed at prospective users in high - scatter areas to convince them of the powers of an SDI service . Otherwise many members of this group that stand to gain most from using an SDI service will not even make an attempt to do so . These measures are essential if the poten - tial of SDI services is ever to be realized , and their benefits enjoyed , by the research community . Methodology Study Population Chemists and chemical engineers in Canadian universities were selected as the study population because this group had had an SDI system available to it since 1969 ; a period of five years provided an opportunity for this group to test the system . University faculties were selected because of their accessibility . Data were collected in the Fall of 1974 . Sampling Procedure Because a careful study of the 34 institutions that com - prised the target population indicated that the variance within the institutions was at least as great , if not greater , than that between the institutions , a systematic sample was decided on . A very accurate sampling frame was established based on current calendars from all 34 universities . The in - dividual faculty members were listed by institution , within institution by rank , and within rank alphabetically . This method ensured proportional representation from both official language groups , from all regions of the country , and from all academic ranks . A 1 - in - 5 systematic sample was drawn beginning with a randomly selected number be - low 6 . This procedure produced a sample of 170 names . ( Usable data were collected for 134 or SO % , see the Re - sponse Rate section . ) Data Collection Method The method chosen for data collection was a mailed questionnaire . A small number of supplementary interviews 126 JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 were carried out to check the interpretation of the data with the respondents themselves in order to validate some of the rating scales used , to throw light on apparent anomalies , and to add depth to the study . Response Rate The response rate was high : 80 % with usable data for a total of 134 respondents . The known characteristics of the nonrespondents were checked with a view to discovering what , if any . bias they might introduce into the study . The most serious discrepancy between the two groups , respon - dents and nonrespondents , concerned the proportion sup - ported by research grants . Since several of the questions dealt with research it was perhaps to be expected that a higher proportion of those not supported by grants would fail to respond . Variables Studied The variables considered in this article are the following : ( 1 ) Perceived scatter of information in the respondent\u2019s specialty . ( 2 ) The amount of use made of each of the following current awareness methods as perceived by the respondents . ( 2 . 1 ) Scanning professional journals ( 2 . 2 ) Scanning books and review books ( 2 . 3 ) Scanning current awareness tools , e . g . , Chemical ( 2 . 4 ) Use of an automated current awareness service , ( 2 . 5 ) Reports from students and assistants ( 2 . 6 ) Personal communication with colleagues in one\u2019s own institution ( 2 . 7 ) Personal communication with colleagues in other institutions ( 2 . 8 ) Refereeing for journals Titles e . g . , CAN / SDl ( 3 ) Whether or not the respondent subscribed to an SDI ( 4 ) Time spent on current awareness as perceived by the ( 5 ) Perceived success in keeping up - to - date . ( 6 ) Perceived efficiency in keeping up - to - date ( efficien - cy = success / time spent ) . ( 7 ) Productivity . service . respondent . ( 3 ) Amount of use was measured on this scale : 0 1 2 3 4 never rarely occasionally regularly heavily A total score for current awareness activity was de - rived by adding the values for all eight current awareness tools . Partial scores for formal and informal current aware - ness methods were derived in like manner ( Chart 1 ) . ( 3 ) The variable subscriber / nonsubscriber was intro - duced as a cross - check on the responses to question 2 . 4 above on the use of automated current awareness services , and also to see whether there were any respondents who shared in the use of SDI printouts received by a colleague , and thus were SDI users without being SDI subscribers . Those who identified themselves as nonsubscribers were asked their reasons for not subscribing . ( 4 ) Time spent was measured through this questionnaire item : Please indicate on the scale below the amount of time you spend each week in keeping up - to - date on developments in your field . 1 3 3 4 5 6 under 1 i2 2 - 5 5 - 10 10 - 20 over 1 hr hr h r 11 r hr 20 hr ( 5 ) Perceived success was measured through this ques - tionnaire item : Please indicate on the scale below how well you are able to keep up in your field . 1 2 3 4 5 have serious not too adequately quite well very well trouble well ( 6 ) Perceived efficiency was computed by dividing the scale value of perceived success by the midpoint of the time - spent span ( e . g . , 3 . 5 for 3 : 2 - 5 hr ) , admittedly a rather crude procedure justified only by the exploratory nature of this study . ( 7 ) Productivity was measured using the following scale for reporting the number ofjournal articles published in the previous five years : 0 1 - 5 6 - 1 0 1 1 - 20 21 - 50 over 50 These variables were measured as follows : ( I ) Perceived scatter was measured through the follow - ing questionnaire item : Please rate this field with respect to ease of keeping up - to - date by circling the appropriate number on the scale below . Number 1 represents a field in which one can keep up - to - date by regularly checking only one or two sources , and 5 a field in which information is scattered in a great many sources . 1 2 3 4 5 In the interpretation of the results presented in the following section it is important to keep in mind the nature of these variables . They represent the perception of the re - spondent , not independent assessment ( see the Limitations of the Study section ) . Results The main purpose of the following analysis is to probe the effectiveness of current awareness tools in fields with JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 127 CHART 1 . Use made of the various methods of current awareness ( number of respondents ) . Method Amount of Use Never Rarely Sometimes Regularly Heavily Weighted ( x 0 ) ( x 1 ) ( x 2 ) ( x 3 ) ( x 4 ) Totals Formal Journals Review books , etc . CA tools SDI service Informal Communication with other institutions Student reports Communication with own institution Refereeing 0 1 12 93 3 9 6 20 5 14 22 8 3 - 2 26 31 34 19 50 41 2 65 53 41 51 61 44 52 18 30 30 16 16 Subtotal . Formal Methods 35 10 37 9 38 6 Subtotal , Informal Methods Total 26 4 Grand Total 420 342 3 14 124 1200 287 279 269 835 2035 230 2265 severe scatter of information . The analysis will move toward this goal through steps of increasing complexity . First , distributions of individual variables in the total sample are given . Second , the effect of scatter of informa - tion on perceived success , time spent , and efficiency is analyzed . Third , the effectiveness of current awareness methods , in particular SDI , is probed by analyzing their impact on efficiency in keeping up - to - date . All hypotheses , whether a posteriori or a priori , are numbered in one sequence . Distributions of Variables in the Total Sample ( I ) Most respondents reported having to cope with a fairly scattered field . Figure 1 summarizes the responses . The mean was above the midpoint on the scale . ( 2 ) The information collected regarding the use made of the various methods of current awareness is summarized in Chart 1 . More use was made of the formal methods of com - munication than the informal , and the use made of journals is significantly heavier than that of any other method . All respondents made some use of journal scanning as a means of keeping abreast with developments in their field . Inter - views threw light on the kind of use made of the formal and informal methods of communication . Informal methods were useful for generating new ideas , in identify - ing what was being done and by whom , and for exchanging information on techniques , but evaluation of the work required that a written report be available for study . ( 3 ) Only 19 % of the respondents subscribed to an SDI service . Reasons for not doing so ranged from ignorance of the existence of such services ( 15 % ) to personal preference ( 45 % ) . Fifteen percent said they did not require such services because of the nature of their work . Twenty - five percent did not subscribe because of the cost . In spite of cost representing a considerable barrier no one interviewed favored offering the service free on the grounds that many would accept the service under such circumstances but make no use of it . Figures 2 and 3 summarize the responses for time spent on current awareness and success in keeping up - to - date . The mean time reported is from 2 to 5 hours per week , and the mean level of success is 3 ( adequately ) . ( 45 ) 50 - 40 - 30 - Number of Respondents 20 - 10 - 5 - 34 30 - 4 N : 134 5 ma . , mun Scatter of Information FIG . 1 . Scatter of information in the field of specialization . 128 JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 Number Of Respondents 0 - 1 1 - 2 53 38 2 - 5 N : 134 10 - 20 Hours Spent on Current Awareness I , IG . 2 . Time spent on current awareness . The time estimate is a very much shorter period of time than that reported by the Committee on Corporation Asso - ciates [ 2 ] in the study carried out on industrial chemists , where the average amount of time spent on current aware - ness was 7 . 5 hours per week . The method of drawing the sample , which was to have each of the company representa - tives select ten of his company\u2019s professional research che - mists in \u201cas random a manner as was convenient , \u201d may alone account for the difference . In addition , only a 50 % return is reported and no investigation was made to deter - mine whether the nonrespondents were discriminably differ - ent with respect to time spent on reading . It is also possible that the use of SDI systems and current awareness tools such as Chemical Absrructs has actually resulted in lower - ing the average amount of time spent on this activity . T o validate the data collected for use of current aware - ness methods and time spent . Kendall\u2019s tau test was per - formed . The result : tau = 0 . 31 , significant beyond 0 . 00005 . TABLE 1 . Relationship between time spent on current awareness and succe \\ s in keeping up - to - date . a Time Spent on Current Awareness Success in C A 1 - 2 hr 2 - 5 hr 5 + h r Ail Low 38 25 16 25 Adequate 49 49 52 50 High 13 2G 32 25 100 100 100 100 I7 31 53 44 134 - - - - % slues are in percent . Number of Respondents 6 7 7 30 \u201c135 Success in Keeping Up - to - Date F\u2018IG . 3 . Success in keeping up - to - date . ( 6 ) After considering the variables time spent and success individually , their relationship was investigated . The data shown in Table 1 suggested the following : A posteriori hypothesis 1 : There is a direct relationship between time spent on current awareness and level of success achieved ( tau significant at 0 . O 1 ) . ( 7 ) One further result is reported here though it does not fit in the main line of the argument . Maizell , in his study of industrial research chemists [ 3 ] , found that the most creative chemists spent more hours on the job reading the technical and scientific literature , examined more jour - nals made available to them through company library facili - ties , and were more interested in maintaining their own card indexes of current journal articles than the least creative chemists . In spite of the fact that the present study was confined to current awareness activities and therefore is in no sense a replication of Maizell\u2019s study , it was thought of some interest to find out whether the information - gather - ing patterns of university faculty in departments of chemis - try and chemical engineering resembled those of industrial research chemists . No variable \u201ccreativity\u201d was available for testing , but Maizell had found that the most creative group of chemists had a higher average number of publications than did the less creative groups , and therefore the two variables , pro - ductivity ( measured by number of journal articles published in the last five years ) and time spent on current awareness , were selected in order to test the first of his findings . JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - Mav 1979 129 TABLE 2 . Relationship between productivity and time spent on current awarenessa Productivity ( Articles Published ) 0 - 2 hr 2 - 5 hr 5 - 20 hr All Time Spent on Current Awareness TABLE 3 . Relationship between scatter and time spenta Scatter of Information Time Spent Low High All 2 hr or less ~ ~ 40 19 28 0 - 5 36 30 9 25 2 - 5 hr 40 39 39 6 - 10 28 18 19 21 11 - 20 25 28 26 26 Over 20 11 24 46 28 100 100 100 100 - - - - n 36 50 43 129 aValues are in percent . A priori hypothesis 2 : There exists a direct relationship between productivity and tirne spent on current awareness ( tau significant beyond 0 , 000 1 , see Table 2 ) . This highly significant result tends to confirm Maizell\u2019s findings . Effects of Scatter of Information A priori hypothesis 3 : There exists a direct relationship between scatter of information and both use made of current awareness methods ( tau significant beyond 0 . 001 ) and time spent on current awareness ( tau significant at 0 . 0007 , see Table 3 ) . A priori hypothesis 3a : There exists a direct relationship between scatter of information and subscribing to an SDI service ( chi square not significant ) . This point will be discussed in more detail below . If one considers just success in keeping up - to - date with - out regard to the data on time spent , one might formulate the following : A priori hypothesis 4 : There exists an inverse relationship between scatter of information and success in keeping up - to - date ( tau not significant ) . The data in Table 4 show that t . here is not even a trend in this direction . The explanation for the negative result lies in the data on time spent . Respondents in high - scatter specialties make up for the disadvantage by spending more More than 5 hr 20 42 33 100 100 100 - _ _ - n 58 14 132 aValues are in percent . time on current awareness . This discovery suggests an in - vestigation of the level of success achieved per time spent on current awareness . For this purpose the derived variable efficiency ( efficiency = success / time spent ) was introduced . The sample was divided into a low - scatter ( 1 - 3 ) group and a high - scatter ( 4 - 5 ) group . These two groups were then com - pared with respect to efficiency using the Mann - Whitney I / test . Respondents of both groups together are ranked according to their efficiency . The test then determines whether the respondents of one group are concentrated in the upper part of the rank listing to a degree that would be unlikely under the null hypothesis of no effect . The data are also shown in a 2 X 3 - table ( Table 5 ) which is , of course , much cruder than the data used for the Mann - Wliitney U test . The results of this comparison confirm the following : A priori hypothesis 5 : There is an inverse relationship between scatter of in - formation and efficiency in keeping up - to - date ( U significant at 0 . 01 ; graphically shown in Fig . 4 , bottom line ) . TABLE 4 . Relationship between scatter of information and success in keeping upto - date . a Scatter of Information Success in CA Low High All Not too well 35 19 26 Adequately 41 58 50 Well 24 23 24 100 100 100 - - - n 58 I 5 133 aValues are in percent . 130 JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 TABLE 5 . ciency in keeping up - to - date . a Relationship between scatter of information and effi - Whole Sample , Low and High Scatter Scatter of Information Efficiency Low High All Low 30 55 45 High 70 45 55 - - - 100 100 100 tz 56 14 130 ( a ) Nonsubscribers , Low and High Scatter Scatter of Information Efficiency Low High All Low 21 59 44 High 13 41 56 100 100 100 - - - n 45 54 99 ( b ) Low - Scatter Nonsubscribers and High - Scatter Subscribers Subgroup Low - Scat ter High - Sca tter Efficiency Nonsubscriber Subscriber Low High 21 13 45 55 100 n 45 100 20 ( c ) Subscribers , Low and High Scatter Scatter of Information Efficiency Low High All Low 45 45 45 High 55 55 55 - - - 100 100 100 n 11 20 31 ~ \u2018values are in percent . This result refers to the total current awareness effort . It shows that researchers in high - scatter fields need more effective methods for keeping up - to - date than they currently use to overcome the problem of high scatter . The problem is all the more pressing as the majority of respondents fall into the high - scatter group . But what methods should these scientists use ? The next section will address the effective - ness of particular current awareness methods and explore what methods are capable of counteracting scatter . Effectiveness of Particular Current Awareness Methods The effectiveness of a current awareness method can be gauged ( 1 ) by the extent to which it enables a user to keep up - to - date as measured by success , and , more importantly , ( 2 ) by the efficiency of the user in keeping up - to - date as measured by the relationship between success and time spent on keeping up - to - date . Reflection on the role of scatter led to the hypothesis that scatter of information is an important factor in explaining the effectiveness of a particular current awareness method . This is borne out through various results to be presented below . When scatter is low , i . e . , when a field is well served by a few specialist journals , scanning the appropriate journals will probably result in high efficiency ( success / time spent ratio ) . When the information is scattered in many sources , however , journal scanning becomes very time consuming and effi - ciency is low . An SDI service might be extremely helpful in this situation by pulling together relevant references from many varied sources . It is the ability to counteract scatter of information , then , that is the true test of the effectiveness of any method of current awareness . As the effectiveness of subscribing to an SDI service was the primary focus of this study , results for this method are discussed first . Subsequently , subscribing to an SDI service is compared with other methods of keeping up - to - date , particularly the use of manual current awareness tools , such as Chemical Titles or Chemical Abstracts . Effectiveness of Subscribing to an SDI Service The interaction between scatter and subscriber / nonsub - scriber can be approached from two angles . One is to repeat the analysis leading to hypothesis 5 ( high scatter of infor - mation leads to lower efficiency ) comparing appropriate subgroups of the total sample . Repeating the analysis for nonsubscribers leads to hypothesis 5a . A posteriori hypothesis 5a : For nonsubscribers to an SDI service high scatter of information leads to lower efficiency in keeping up - to - date [ U significant at 0 . 0005 , 2 value of - 3 . 4 , Table 5 ( a ) l . This is the same effect that was detected for the sample as a whole , only stronger . The next question is : Does subscribing to an SDI service alleviate the negative effects of scatter ? The most obvious way to try to answer this question is to repeat the analysis for subscribers to an SDI service . However , the data pre - sented below indicate that low - scatter respondents lose effi - ciency by subscribing to an SDI service and that the most efficient group of all are low - scatter nonsubscribers . This JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 131 I Low Scatter 1 Hiqh Scatter 1 A l l I I 99 ( Sa ) 54 I 45 Non - subscriber I I 31 ( SC ) 20 I l1 \u201ct Subscriber ( 5 ) 74 I 130 c - All FIG . 4 . Summary of the effect of scatter of information and sub - scribing to an SDI service on efficiency in keeping up - to - date [ Tables 5 , 5 ( a ) , 5 ( b ) , 5 ( c ) , 6 , 6 ( a ) , and 6 ( h ) ] . Arrows point to group with higher efficiency ; s = significant , t = tendency , nt = not even a ten - dency . group , then , could be used as a \u201cbaseline\u201d for judging effi - ciency of other groups . Following this reasoning high - scatter subscribers are compared to low - scatter nonsub - scribers ; the results lead to hypothesis 5b . A posteriori hypothesis 5b : Subscribing to an SDI service alleviates the negative effect of scatter of information on efficiency in keeping up - to - date [ Mann - Whitney U for the scatter - efficiency relationship converts to a Z value of - 1 . 6 , not significant , see Table 5 ( b ) ] . The results of this test seem to indicate that high scatter of information leads to lower efficiency in keeping up - to - date even if a respondent subscribes to an SDI service , but the negative effect of scatter is not as strong as , for high - scatter nonsubscribers . Another , and perhaps more direct , way to approach the interaction between scatter and , subscriber / nonsubscriber is to analyze the effect of subscriber / nonsubscriber on effi - ciency , first for the sample as a whole and then for low scatter only and high scatter only . Reflection on the pur - pose of an SDI service led to hypothesis 6 . A priori hypothesis 6 : Subscribers to an SDI service achieve higher efficiency in keeping upto - date than nonsubscribers ( U not sig - nificant , but see a posteriori hypotheses 6a and 6b ) . A look at Table 6 clearly reveals that not even a tendency exists . Initially this result was viewed as surprising . Since the previous analysis had confirmed the impact of scatter on the relationship between time spent and success ( a priori hypothesis 5 ) , an explanation was sought by investigating the interaction between scatter of information and sub - scriber / nonsubscriber . To this end the comparison between subscribers and nonsubscribers with respect to efficiency TABLE 6 . efficiency in keeping up - to - date . a Whole Sample Relationship between subscribing to an SDI service and Subscriber / Nonsuhscriber Efficiency Nonsubscriber Subscriber All Low 44 45 45 High 56 55 55 100 100 100 - - - n 99 31 130 ( a ) Low Scatter SubscriberlNonsubscriber Efficiency Nonsubscriber Subscriber All Low 27 45 30 High 73 55 70 100 100 100 n 45 11 56 - - - ( b ) High Scatter ~ ~ ~ Suhscriber / Nonsubscriber Efficiency Nonsubscriber Subscriber All Low 59 45 55 High 41 55 45 100 100 100 n 54 20 74 - - - aValues are in percent was performed within the high - scatter group and within the low - scatter group . The results are as follows : A posteriori hypothesis 6a : Among chemists working in specialties with low scatter of information , subscribers to an SDI service have lower efficiency in their current awareness activities than nonsubscribers [ U significant at 0 . 02 , see Table 6 ( a ) ] . A posteriori hypothesis 6b : Among chemists working in specialties with h & h scatter of information , subscribers to an SDI service have a higher efficiency in their current awareness activities than nonsubscribers [ U not significant , transforms to Z = 1 . O , but data in Table 6 ( b ) show a tendency ] . 132 JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 LOW scatter FIG . 5 . Summary of the effect of scatter of information and the use of manual current awareness tools on efficiency in keeping up - to - date [ Tables 7 , 7 ( a ) . 7 ( b ) , 7 ( c ) , 8 . 8 ( a ) , and 8 ( b ) ] . Arrows point to group with higher efficiency ; s = significant . t = tendency . but not Fignificant , nt = not even ii tendency . High Scatter All Upon reflection these results make sense . In a field where one can keep up - to - date by scanning a few journals , reading of SDI notices will add only a few additional refer - ences and the benefit is not worth the cost ( time spent ) . ( The data suggest that in both the low - scatter and the high - scatter groups subscribers do better than nonsubscribers in terms of absolute success where time spent is not con - sidered . ) The reversed effects of subscribing in the low - scatter group and the high - scatter group explain , then , why there is no effect at all in the total sample . While , on the basis of the tests reported above , subscribing to an SDI service does not totally eliminate the effects of scatter of information , subscribers do achieve higher efficiency than nonsubscribers in fields in which information is scattered ( strong tendency ) . Figure 4 summarizes the results of the efficiency analyses for subscription to an SDI service . Effectiveness of Using Other CA Tools The efficiency analyses were now repeated for the use of manual current awareness tools which is , in a sense , the manual version of subscribing to an SDI service . SDI sub - scribers were excluded from these analyses . The results are displayed in Fig . 5 ( which also refers to the supporting data ) . Comparison with Fig . 4 brings out striking differences . In the low - scatter category neither high nor low users of manual current awareness methods enjoy an advantage in terms of efficiency [ Table 8 ( a ) ] ; therefore no \u201cbaseline\u201d group could be identified . Subscribing to an SDI service was seen to counteract somewhat the negative effect of scatter of information . No such effect is found for high use of CA tools . Among high users of CA tools those with high scatter are less efficient in keeping up - to - date [ strong tendency , Table 7 ( c ) ] . When high - scatter high users of CA tools were compared with low - scatter low users of CA tools using the Mann - Whitney U test high - scatter high users of CA tools were found to be significantly less effi - cient [ U value converts to a Z value of - 3 . 38 , significant at 0 . 0004 , Table 7 ( b ) ] . In the high - scatter group SDI sub - scribers were more efficient than nonsubscribers [ strong tendency , Table 6 ( b ) ] . No such effect is found comparing high users of CA tools with low users in the high - scatter group [ Table 8 ( b ) ] . The relationship between scatter of information and effi - ciency of keeping up - to - date was investigated for all methods of keeping up - to - date using 2 X 2 cross - tabulations and the chi square test ( which is more convenient but less powerful than the Mann - Whitney U test ) . The results are summarized in Chart 2 . These results can be summarized in the following : A posteriori hypothesis 7 : Among the current awareness methods tested , sub - scribing to an SDI service is the only one that is capa - ble of counteracting scarter . Before these findings can be accepted , however , they must stand up to the test of replication , not only for this field but for other fields and other groups of researchers . Scatter of Information and Use of an SDI Service Since a direct relationship was found between scatter of information and the amount of use made of the various methods used to keep up - to - date , it would be reasonable to expect that high perceived scatter of information in the field of specialization would encourage the use of an SDI service , particularly in the light of the finding that sub - scribing to an SDI service seems to be the most effective way to counteract scatter . A cross - tabulation of the vari - ables subscriber / nonsubscriber and scatter did not produce significant results ( a priori hypothesis3a ) . The data provided no evidence to suggest that perceived scatter of information acted as an incentive to subscribe to an SDI service . On the contrary , many of the comments volunteered by the re - spondents revealed that they viewed scatter of information as a reason for not subscribing . To quote one of the respon - dents : \u201cMy fields of interest are rather broad and such services would not be able to select appropriate material . \u201d Against this quotation two comments by librarians actively engaged in assisting SDI users in profile development may be set [ 4 ] . Most of my customers tend to have broad interdisci - plinary interests , e . g . , pollution and environmental damage . I have had extreme success with CAN / SDI for people who are in multidisciplinary areas , for example , faculty in the Kinesiology Department . These people are interested in motor learning , muscle physiology , sports psychology , anthropometry , biomechanics , and the use of two different data bases combined with different types of search strategies , title word , subject term , source author , cited author have proved highly effective in literature retrieval in this area . JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 133 TABLE 7 . Relationship between scatter and efficiency ( subscribers to SDI excluded ) ? Whole Sample TABLE 8 . Relationship between using manual current awareness tools and efficiency ( subscribers to SDI excluded ) . a Whole Sample Use of Manual CA Tools Scatter of Information Efficiency Low High All Efficiency Low High All Low 27 59 44 High 73 41 56 100 100 100 n 44 54 98 - ~ _ _ Low 46 43 45 High 54 51 55 100 100 100 - - - n 52 46 98 ( a ) Low Use of CA Tools , Low and High Scatter ( a ) Low Scatter Scatter of Information Efficiency Low High All Use of Manual CA Tools Efficiency Low High All Low 29 25 27 Low 29 61 46 High 71 39 54 _ _ _ _ - 100 100 100 n 24 28 52 High 71 I5 73 100 100 100 - ~ _ _ n 24 20 44 ( b ) Low Use with Low Scatter and High Use of CA Tools with High Scatter ( b ) High Scatter Use of Manual CA Tools Group Low - Scatter High - Scatter Efficiency Low User High User Efficiency ~ Low High All Low 61 58 59 High 39 42 41 100 100 100 n 28 26 54 - - _ _ Low 29 58 High 71 100 ~ 42 100 n 24 26 ' values are in percent . ( c ) High Use of CA Tools , Low and High Scatter Scatter of Information the variables deal with perceptions of the respondents rather than with independent observations . It would be desirable , if laborious , to check the results in a study where scatter of information in each field of specialization was measured by a Bradford analysis , success by the fraction of information obtained by the respondent over the totality of existent relevant information as determined by an ex - haustive search , and time spent in keeping up - to - date either by observation or from diary records maintained by the respondent . Finally , as already stated , it is uncertain how far one can generalize beyond the narrow population from which the sample was drawn . Efficiency Low High All Low 25 58 43 High 75 42 57 _ _ _ _ _ _ 100 100 100 I1 20 26 46 ' Values are in percent . Limitations of the Study As this study was exploratory many of the findings re - sult from a posteriori hypotheses suggested by the data rather than from apriori hypotheses tested by the data . All Acknowledgments The contributions of Professor L . B . Heilprin , Professor J . S . Kidd , and Professor Paul Wasserman to this study are 134 JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 CHART 2 . Summary of tests on current awareness methods . Relationship between Scatter and Efficiency within Groups Making Low or High Use of a CA Method , Respectively Method Used ~ No . of No . of Low Use of Method Respondents High Use of Method Respondents Not significant Significant , inverse 22 52 Significant , inverse 107 46 Journals CA tools ( SDI users excluded ) Nearly significant , inverse Review books , etc . Not significant , inverse tendency 64 Not significant , inverse tendency 65 Not significant , inverse tendency 64 Nearly significant , inverse tendency 65 Formal methods ( SDI users excluded ) Student reports Significant , inverse 85 Not significant , inverse tendency 43 Communication , own institution Not significant , inverse tendency 86 Not significant , inverse tendency 42 Communication , other institutions Significant , inverse 87 Not significant , slight inverse tendency 42 Refereeing Significant , inverse I02 12 Not significant , inverse tendency 21 Informal methods Significant , inverse Not significant , slight inverse tendency 55 SDI Significant , inverse 97 Not significant , no tendency 32 gratefully acknowledged . Special mention should be made of the assistance received from Professor M . B . Hargrove with respect to the statistical measures employed . References 2 . American Chemical Society . Committee on Corporation Asso - ciates . Subcommittee on the Economics of Chemical Informa - tion . Cost Effectiveness of Information Systems . Washington , DC : American Chemical Society ; 1969 . 3 . Maizell , R . E . \u201cInformation gathering patterns and creativity ; a study of research chemists in an industrial research labora - tory . \u201d Doctoral dissertation , Columbia University , 1957 , un - published . 4 . Personal communications with librarians actively engaged in profile development . 1 . Housman , E . M . ; Knskela , E . D . \u201cState of the Art of Selective Dissemination of Information . \u201d IEEE Transactions of Engi - neering Writingand Speech . I11 ( 2 ) : 78 ; September 1970 . JOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE - May 1979 135", + "cataliniMicrogeographyDirectionInventive2018": "This article was downloaded by : [ 100 . 15 . 126 . 133 ] On : 06 December 2020 , At : 18 : 31 Publisher : Institute for Operations Research and the Management Sciences ( INFORMS ) INFORMS is located in Maryland , USA Management Science Publication details , including instructions for authors and subscription information : http : / / pubsonline . informs . org Microgeography and the Direction of Inventive Activity Christian Catalini To cite this article : Christian Catalini ( 2018 ) Microgeography and the Direction of Inventive Activity . Management Science 64 ( 9 ) : 4348 - 4364 . https : / / doi . org / 10 . 1287 / mnsc . 2017 . 2798 Full terms and conditions of use : https : / / pubsonline . informs . org / Publications / Librarians - Portal / PubsOnLine - Terms - and - Conditions This article may be used only for the purposes of research , teaching , and / or private study . Commercial use or systematic downloading ( by robots or other automatic processes ) is prohibited without explicit Publisher approval , unless otherwise noted . For more information , contact permissions @ informs . org . The Publisher does not warrant or guarantee the article\u2019s accuracy , completeness , merchantability , fitness for a particular purpose , or non - infringement . Descriptions of , or references to , products or publications , or inclusion of an advertisement in this article , neither constitutes nor implies a guarantee , endorsement , or support of claims made of that product , publication , or service . Copyright \u00a9 2017 , The Author ( s ) Please scroll down for article\u2014it is on subsequent pages With 12 , 500 members from nearly 90 countries , INFORMS is the largest international association of operations research ( O . R . ) and analytics professionals and students . INFORMS provides unique networking and learning opportunities for individual professionals , and organizations of all types and sizes , to better understand and use O . R . and analytics tools and methods to transform strategic visions and achieve better outcomes . For more information on INFORMS , its publications , membership , or meetings visit http : / / www . informs . org MANAGEMENT SCIENCE Vol . 64 , No . 9 , September 2018 , pp . 4348 \u2013 4364 http : / / pubsonline . informs . org / journal / mnsc / ISSN 0025 - 1909 ( print ) , ISSN 1526 - 5501 ( online ) Microgeography and the Direction of Inventive Activity Christian Catalini a a MIT Sloan School of Management , Massachusetts Institute of Technology , Cambridge , Massachusetts 02142 Contact : christian @ catalini . com , http : / / orcid . org / 0000 - 0003 - 1312 - 6705 Received : December 2 , 2016 Accepted : March 15 , 2017 Published Online in Articles in Advance : July 19 , 2017 https : / / doi . org / 10 . 1287 / mnsc . 2017 . 2798 Copyright : \u00a9 2017 The Author ( s ) Abstract . I provide novel empirical evidence grounded in an original theoretical frame - work to explain why colocation matters for the rate , direction , and quality of scien - ti\ufb01c collaboration . To address endogeneity concerns due to selection into colocation and matching , I exploit the constraints imposed on the spatial allocation of labs on the Jussieu campus of Paris by the removal of asbestos from its buildings . Consistent with search costs constituting a major friction to collaboration , colocation increases the likelihood of joint research by 3 . 5 times , an e\ufb00ect that is mostly driven by lab pairs that face higher search costs ex ante . Furthermore , separation does not negatively a\ufb00ect collaboration between previously colocated labs . However , while colocated labs grow increasingly similar in topics and literature cited , separated ones embark on less correlated research trajectories . Research outcomes , instead , seem to be mostly in\ufb02uenced by how distance a\ufb00ects exe - cution costs : after colocation , labs are more likely to pursue both lower - quality projects ( a selection e\ufb00ect ) and high - quality projects ( an e\ufb00ort e\ufb00ect ) . Opposite e\ufb00ects on quality are observed after separation . Whereas search costs a\ufb00ect which scientists are likely to collaborate together , execution costs shape the quality of their output . History : Accepted by Ashish Arora , entrepreneurship and innovation . Open Access Statement : This work is licensed under a Creative Commons Attribution - NonCommercial - ShareAlike 4 . 0 International License . You are free to download this work and share with others for any purpose , except commercially , if you distribute your contributions under the same license as the original , and you must attribute this work as \u201c Management Science . Copyright \u00a9 2017 The Author ( s ) . https : / / doi . org / 10 . 1287 / mnsc . 2017 . 2798 , used under a Creative Commons Attribution License : https : / / creativecommons . org / licenses / by - nc - sa / 4 . 0 / . \u201d Funding : This research was funded by the MIT Sloan School of Management , the Centre for Innovation and Entrepreneurship at the Rotman School of Management , and the Martin Prosperity Institute . Supplemental Material : The online appendix is available at https : / / doi . org / 10 . 1287 / mnsc . 2017 . 2798 . Keywords : colocation \u2022 idea recombination \u2022 collaboration \u2022 search costs \u2022 microgeography \u2022 low - opportunity cost time \u201cThe truth is , it may have changed my whole life in a respect . I guess if I had any long - run thoughts then , it was to make a career doing statistics , econometrics , probability models , and things like that . But when I started talking on a regular basis with Paul , and he was so full of ideas and thoughts , it was impossible not to \ufb01nd my interests moving toward more straight eco - nomics . In a way the location of that o\ufb03ce and the fact that we liked each other so much had a major in\ufb02uence on the direction my career took . \u201d \u2014Nobel laureate Robert Solow describing how his o\ufb03ce location and resulting friendship with Nobel laureate Paul Samuelson a\ufb00ected his career ( Dizikes 2011 ) 1 . Introduction When we select a location , we are committing to spending a disproportionate share of our most scarce resource , time , in that particular place . Individuals and organizations , anticipating the role geographic dis - tance will play in their allocation of time , and in de\ufb01n - ing the opportunities and talent they will have access to , pay particular attention to location decisions . As a result , location choices are highly endogenous to eco - nomic outcomes . Geographic proximity not only increases the chance of a serendipitous interaction , but also lowers the cost of a scheduled one . Both types of interactions make it substantially easier to search for new collaborators within our local environment . When colocated , joint execution costs are also lower , as coordination , moni - toring , and the transfer of complex information can all rely on more frequent , face - to - face interactions . Thus , organizations spend considerable time and re - sources optimizing the spatial allocation of their teams and invest in infrastructure to allow for interdiscipli - nary work to \ufb02ourish when new opportunities are identi\ufb01ed . This endogenously shapes the trajectory of a university , R & D lab , or start - up , as the spatial alloca - tion often results from priors about which teams and individuals will bene\ufb01t the most from proximity . When a misalignment between the objectives of an organiza - tion and its current layout emerges , e\ufb00orts are made to compensate for geographic distance , for example 4348 Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4349 by scheduling temporary colocation ( e . g . , joint meet - ings , conferences ) and remote interactions to recre - ate the bene\ufb01ts of colocation . A recent , large - scale example is Microsoft\u2019s ambitious relocation of 1 , 200 engineers , which cut travel time between buildings for employees by 46 % with the explicit objective of encouraging face - to - face conversations and serendipi - tous interactions instead of email and Skype meetings ( Nielsen 2016 ) . Empirically , this makes it extremely challenging to understand why colocation matters and under which conditions di\ufb00erent mechanisms are responsible for the bene\ufb01ts we attribute to proximity . The objective of this paper is to focus on how search costs and joint exe - cution costs shape inventive outcomes , and to provide novel empirical evidence and a theoretical framework that can help us separate between these two compet - ing , but not necessarily exclusive , mechanisms . If search costs are a key friction to collaboration , then colocation should have a positive e\ufb00ect on the proba - bility of collaboration , but separation may not neces - sarily have a negative e\ufb00ect , as teams that are aware of each other may be able to compensate for distance through temporary colocation and remote interactions . If instead joint execution costs are driving collaboration decisions , then the e\ufb00ects observed after colocation and separation should be exact opposites of each other ( as execution costs would increase almost immediately with distance ) . Furthermore , whereas search costs do not have a strong implication for the observed value of joint projects , if lower execution costs allow teams to apply more e\ufb00ort toward advancing their ideas , then the e\ufb00ect of colocation on quality will be ambigu - ous . On the one hand , lower execution costs may induce teams to select lower - quality projects ( a selec - tion e\ufb00ect ) . On the other hand , if lower execution costs allow teams to endogenously apply more e\ufb00ort\u2014and e\ufb00ort improves the quality of the underlying idea\u2014 we may also observe an increase in right - tail outcomes after colocation ( an e\ufb00ort e\ufb00ect ) . 1 I explore the relative role of these mechanisms in a setting where the spatial allocation is constrained by reasons that are orthogonal to the outcomes of interest\u2014i . e . , where exogenous variation is injected in the process of deciding where di\ufb00erent teams are placed . Combined with a di\ufb00erence - in - di\ufb00erences approach , this mitigates the endogeneity concerns typ - ically linked to selection into colocation . The setting is the university campus of Paris Jussieu , the leading scienti\ufb01c and medical complex in France . Following a research report by INSERM on the car - cinogenic e\ufb00ects of asbestos ( June 21 , 1996 ) , the French government introduced a full ban of the \ufb01re retardant from all public buildings , including the Jussieu ones . Starting in 1997 , a separate entity ( Etablissement Pub - lic d\u2019Am\u00e9nagement Universitaire de la R\u00e9gion Ile - de - France ( EPAURIF ) ) was put in charge of the asbestos removal process on campus , which led to \ufb01ve massive waves of lab relocations over 17 years . Because of the complexity and urgency 2 of the cleaning process , labs were forced to move often on short notice and with lit - tle in\ufb02uence over their new location . As a consequence , many labs found themselves next to new neighbors , and the overall spatial allocation was severely more constrained than before . I exploit this variation , com - bined with the reconstruction of \ufb01ne - grained , longi - tudinal information on inventive outcomes , to try to understand why colocation matters for the rate , direc - tion , and quality of inventive activity . Consistent with the search costs mechanism , results show that colocation increases the likelihood that two labs will collaborate by 3 . 5 times , an e\ufb00ect that is mostly driven by pairs that faced higher search costs ex ante . Moreover , separation has a nonsigni\ufb01cant e\ufb00ect on the probability and rate of collaboration , suggesting that labs that were previously exposed to each other are later able to sustain collaboration also over dis - tance . At the same time , while colocated labs become increasingly similar in the topics they work on and references they cite , 3 separated labs embark on less correlated research trajectories . This is consistent with search costs increasing considerably even at relatively low levels of geographic distance , a result that sup - ports past literature that has linked proximity to better information di\ufb00usion ( Allen 1977 , Cowgill et al . 2009 ) , formation of social ties ( Stuart and Liu 2010 , Liu 2013 ) , and knowledge \ufb02ows ( Ja\ufb00e et al . 1993 , Thompson and Fox - Kean 2005 , Thompson 2006 ) . Furthermore , conditional on collaboration , the qual - ity of a lab pair\u2019s output also changes following a change in distance . Colocated labs are 1 . 36 times more likely to produce a paper that will end up in the highest quartile of the citation distribution , and their variance in outcomes increases . Opposite results are observed for lab pairs that are separated . This is consistent with colocation a\ufb00ecting joint execution costs , and with both a selection and an e\ufb00ort e\ufb00ect playing a role in this context . Interestingly , the collaborations resulting from interactions between labs that faced higher search costs ex ante are more likely to be of high impact , suggesting that arbitrage opportunities may exist in encouraging interactions between communities of scientists that do not overlap through other channels ( e . g . , joint confer - ences and journals ) . Taken together , the \ufb01ndings highlight that whereas search costs mostly a\ufb00ect which scientists are likely to collaborate together , execution costs shape the qual - ity of their output . By allocating space , organizations profoundly shape the evolution of scienti\ufb01c trajecto - ries and the types of opportunities that are explored by di\ufb00erent teams . This involves a trade - o\ufb00 between the perhaps more e\ufb03cient exploitation of established research paradigms and the more costly exploration Catalini : Microgeography and the Direction of Inventive Activity 4350 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) of new ones . Colocation is an expensive way to lower search costs , as supported by the overall changes in the collaboration portfolios of the labs observed in the data : during the moves , in aggregate , labs focused more inwards , increasing within - lab research at the expense of across - lab collaborations . While this may be a result of the suboptimal set of local peers the moves o\ufb00ered the labs relative to their ideal choices , it is also a reminder of how pursuing research across disciplines is a more costly endeavor than incremental , within - discipline work both for an institution and the scientists involved . In the absence of complementary changes in incentives ( e . g . , to favor cross - disciplinary research ) , scientists will focus where search and joint execution costs are lower \ufb01rst . Although temporary forms of colocation may not be as e\ufb00ective as the longer periods studied here in leading to actual knowledge \ufb02ows and collaboration , they may still allow for cross - pollination between research trajectories and break - through discoveries at a lower cost . The layout of the remainder of the paper is as follows . In the next section , I further develop the basic hypotheses of the theoretical framework to guide empirical predictions and interpretations of the \ufb01nd - ings . Section 3 describes the empirical setting , data , and empirical strategy . Section 4 reports the main results . Section 5 concludes . 2 . Theoretical Framework The ability of an economy to generate , di\ufb00use , and recombine ideas has a profound in\ufb02uence on its ability to sustain growth ( Lucas 1988 , Romer 1990 , Weitzman 1998 ) . Our understanding of a key economic phenomenon behind agglomeration and growth\u2014 localization economies\u2014relies on , among other fac - tors , basic assumptions about how knowledge is recombined locally versus over distance . Despite frequent references in the literature on knowledge \ufb02ows and localization to the role of colo - cation in knowledge transmission and recombination ( Breschi and Lissoni 2004 , Mairesse and Turner 2005 , Singh 2005 , Agrawal et al . 2006 , Fleming et al . 2007 , Belenzon and Schankerman 2013 ) , there is more lim - ited empirical evidence on the underlying mechanisms invoked to explain its e\ufb00ects . In part , this is due to the di\ufb03culty of \ufb01nding plausibly exogenous variation in location choice . Most of the existing literature relies on observational data ( Olson and Olson 2000 , Van den Bulte and Moenaert 1998 , Kabo et al . 2014 , Fayard and Weeks 2007 , Kabo et al . 2015 , Crescenzi et al . 2017 ) , which is subject to selection bias from individuals , teams , and organizations choosing where to locate . This makes it challenging to isolate the e\ufb00ect of colo - cation from confounders and other forms of proximity , such as proximity in social space and in knowledge space . Knowledge production , moreover , is increas - ingly a collaborative process ( Wuchty et al . 2007 , Jones et al . 2008 ) between colocated and geographically dis - persed teams of scientist ( Adams et al . 2005 , Katz 1994 , Freeman et al . 2015 ) , making it di\ufb03cult to trace knowl - edge \ufb02ows through these alternative channels . Scientists are evaluated based on the quality and quantity of their output ( e . g . , in terms of scienti\ufb01c impact measured through citations , publications , out - lets ) . Two key costs shape how scientists ( and teams of scientists ) make collaboration decisions : search costs and execution costs . Search costs , by de\ufb01ning the choice set of possible collaborators , in\ufb02uence the like - lihood that any two individuals will explore a joint research project to begin with . Execution costs deter - mine if , given an idea of a certain quality , it makes sense for a speci\ufb01c team to invest time , resources , and e\ufb00ort in developing it . Colocation has a profound e\ufb00ect on both costs : ( 1 ) by increasing the chance of an interaction ( both serendipi - tous and planned ) , colocation drastically lowers search costs for new collaborators ; ( 2 ) by lowering the cost of face - to - face meetings , coordination costs , monitoring costs , and the cost of transferring complex informa - tion , colocation also reduces joint execution costs . If by applying e\ufb00ort toward a project scientists are able to improve its ultimate value , then lower execution costs , locally , endogenously change the optimal level of e\ufb00ort scientists may want to apply to local versus distant projects . In the next sections , I focus on how geographic prox - imity in\ufb02uences search and execution costs to build predictions about how microgeography a\ufb00ects the rate and type of scienti\ufb01c collaborations between teams of scientists ( labs ) . 2 . 1 . Search Costs In this context , search costs are de\ufb01ned as the frictions scientists incur in \ufb01nding new collaborators and col - laboration opportunities . A reduction in search costs should therefore induce an increase in the probability of collaboration between two labs and possibly shift over time the collaboration portfolios of the scientists a\ufb00ected . Recent experimental evidence has shown that even within the same university , search frictions can be substantial : after randomly colocating scientists in the same room for a 90 - minute information - sharing ses - sion , Boudreau et al . ( 2017 ) observe a 75 % increase in the probability that they will co - apply for a grant . An opposite increase in search costs , instead , may have a more ambiguous e\ufb00ect on the probability of collaboration , especially if we believe that once two scientists are aware of each other\u2019s research agenda , they can keep communicating new ideas cheaply over distance\u2014i . e . , when it comes to search , if social prox - imity ( e . g . , past collaboration ) can partially substitute Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4351 for geographic proximity , then separating groups of scientists that were previously colocated may have a smaller e\ufb00ect ( or no e\ufb00ect at all ) on collaboration . 4 Search also takes place through temporary colo - cation ( e . g . , conferences ) , 5 and through codi\ufb01ed information ( e . g . , published research ) . As a result , in cases where search costs are likely to be already low ex ante because scientists attend the same conferences or read the same journals , changes in geographic proxim - ity may have a less pronounced e\ufb00ect on the likelihood of collaboration . This can be summarized in the following hypothesis : Hypothesis 1 . After colocation , scientists should be more likely to collaborate , and the e\ufb00ect will be stronger the higher search costs are ex ante . Separation , instead , should have no e\ufb00ect on the probability of collaboration . 2 . 2 . Execution Costs Joint execution costs include the cost of face - to - face meetings , coordination , monitoring , and transfer of complex information between teams of scientists . The \ufb01rst e\ufb00ect of a reduction in joint execution costs is , as in the case of search costs , an increase in the probability of collaboration ( this time possibly skewed toward preex - isting pairs ) . After colocation , scientists should collab - orate together more . 6 At the same time , while search costs may not increase immediately after separation because of the long - term bene\ufb01ts from past exposure , execution costs should increase relatively quickly , neg - atively a\ufb00ecting joint projects . To summarize : Hypothesis 2 . After colocation , scientists should be more likely to collaborate with each other ( and increase their rate of collaboration ) because of lower execution costs . After sep - aration , the opposite should be observed . Whereas the e\ufb00ect of a decrease in execution costs on the rate of collaboration is straightforward , the e\ufb00ect on the realized value of the resulting projects is ambigu - ous . If the value of a project depends both on the orig - inal idea\u2019s intrinsic quality as well as on the amount of e\ufb00ort dedicated to advancing it , then colocation may induce : ( 1 ) the development of lower - quality ideas ( a selection e\ufb00ect ) ; ( 2 ) the application of e\ufb00ort for any given idea quality ( an e\ufb00ort e\ufb00ect ) . In other words , if we assume that research projects improve when more e\ufb00ort is allocated to their development , and that it is cheaper to do so when colocated ( lower execution costs ) , then the ultimate impact of colocation on out - comes is the composition of the selection and e\ufb00ort e\ufb00ect pushing in opposite directions . On the one hand , lower execution costs locally may induce scientists to engage in marginal , lower - value projects . Intuitively , if projects below a certain value cannot be published ( i . e . , if there is a minimum thresh - old for publication ) and e\ufb00ort improves the payo\ufb00 of an idea , then the threshold for developing a research project under colocation will be lower . Conditional on an idea being developed , this should lead to an increase on the left tail of the outcome distribution . On the other hand , if scientists can improve a project more e\ufb03ciently when colocated ( e . g . , through addi - tional face - to - face meetings , better knowledge trans - fer , team coordination , etc . ) , this leads to the expected value of colocated projects being higher for any under - lying idea quality . Intuitively , scientists can achieve the same outcome with ideas of lower starting qual - ity because they can execute on them more e\ufb03ciently . Additionally , it can be shown 7 that if the distribution of idea quality is skewed toward the low end ( i . e . , if most scienti\ufb01c ideas are of low impact and a few are of very high impact ) , then the di\ufb00erence in e\ufb00ort is a ( weakly ) increasing function of idea quality ( i . e . , the di\ufb00erence in e\ufb00ort between colocated and distant projects increases with project quality ) . This leads to the following hypothesis : Hypothesis 3 . After colocation , conditional on projects being developed , we should observe more projects at the low end of the distribution ( selection e\ufb00ect ) and at the high end of the distribution ( e\ufb00ort e\ufb00ect ) . Separation should lead to opposite results . The above prediction relies on the skewed nature of inventive outcomes , on the idea that colocation o\ufb00ers lower execution costs , and on the assumption that more e\ufb00ort improves the value of a project . Empirically , a key challenge is due to the fact that whenever scientists have unrealistic expectations about the value of a project ( e . g . , when they believe a project is of publishable quality when it is not ) , or whenever projects are abandoned as scientists update their pri - ors , part of the left tail of the outcome distribution will not be observed ( as we only see published research ) , leading to underestimating the e\ufb00ects of colocation and separation on lower - quality ideas . This limits what we can learn from bibliometrics data regarding the full distribution of research outcomes . The analysis on quality in Section 4 . 3 will be therefore more informa - tive about right - tail outcomes and only provide sug - gestive evidence about the left tail . Moreover , because of lower search and execution costs , colocation is a strategic choice , and observa - tional data will overestimate the impact of these e\ufb00ects on inventive outcomes . The relocations of labs on the Jussieu campus , because of the external constraints imposed on space allocation by the asbestos removal process , can help us understand if the mechanisms suggested in the theoretical framework are correct , and improve our understanding of the conditions under which colocation is more likely to matter for collabo - rative inventive outcomes . To address additional con - cerns about selection into colocation and matching between labs , the key part of the empirical analysis Catalini : Microgeography and the Direction of Inventive Activity 4352 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) relies on a di\ufb00erence - in - di\ufb00erences approach at the lab - pair level , as described in the next section . Lab - level portfolio choices are also presented to understand the aggregate e\ufb00ects of the moves on the trade - o\ufb00s labs make in deciding who to collaborate with . 3 . Empirical Setting The setting is the university campus of Paris Jussieu , which hosts the Universit\u00e9 Pierre et Marie Curie ( UPMC ) . According to the 2016 U . S . News and World Report rankings , UPMC is the top institution in France ( 10th in Europe , 49th on a global scale ) . It houses the faculty of sciences of Sorbonne Universit\u00e9s and approximately 31 , 000 students ( 5 , 900 master students and 3 , 000 doctoral students ) , as well as 3 , 750 research - active professors ( 80 % of its sta\ufb00 work in research cen - ters ) . 8 Three recent Nobel laureates are from UPMC : Pierre - Gilles de Gennes ( Physics , 1991 ) , Claude Cohen - Tannoudji ( Physics , 1997 ) , and Fran\u00e7oise Barr\u00e9 Sinoussi ( Medicine , 2008 ) . Strong areas of specialization include mathematics ( \ufb01fth worldwide ) , physics ( 15th ) , space science ( 20th ) , geosciences ( 21st ) , neuroscience ( 43rd ) , environment and ecology ( 49th ) , and biology and bio - chemistry ( 51st ) . The campus went through \ufb01ve massive waves of labs relocations over 17 years ( 1997 \u2013 2014 ) due to the removal of asbestos from its buildings . The moves started when the French government , reacting to a research report by INSERM on the carcinogenic e\ufb00ects of asbestos ( June 21 , 1996 ) , 9 introduced a full ban of the \ufb01re retardant material from public buildings . The Jussieu campus was built extensively using asbestos . Interviews with scientists from the labs con\ufb01rmed that given the nature and urgency 10 of the clean - ing process , labs were forced to move often under short notice and with minimal in\ufb02uence over their new location . A separate entity ( EPAURIF ) was put in charge of the cleaning process , which started with labs that were relatively easy to move ( e . g . , theoreti - cal labs in mathematics , computer science ) and only later reached labs with sophisticated instrumentation and machinery ( e . g . , applied physics ) . During the relo - cation period , entire sections of the campus were pro - gressively isolated and renovated . Because of the com - plexity and costs of the operation , lab requirements were often not a priority , resulting in many labs com - plaining about the moves . Whereas for some scientists the actual moving process was a cause of delays ( one scientist estimated a one - year delay in productivity over a 10 - year period ) , others did not \ufb01nd it disruptive at all ( a di\ufb00erent scientist , who does theoretical work , estimated just one week without lab access ) . As in other research - intensive institutions , interac - tions across labs are common , and the relocations ended up separating labs that were colocated and inter - acted before . The same moves also placed labs next to new neighbors , in some cases with positive e\ufb00ects on collaboration . In the data set , the aggregate amount of colocation between lab pairs slightly decreases during the moves , from 7 . 3 % to 6 . 9 % of pairs . 11 Whereas the mean distance between lab pairs across broad \ufb01elds of science ( e . g . , natural versus life sciences ) and within sub\ufb01elds ( e . g . , within chemistry ) is broadly stable over time , the distance across sub\ufb01elds ( e . g . , chemistry and physics ) decreases , providing plausibly exogenous variation in the composition of a lab\u2019s neighbors . The data also provides substantial variation in terms of which types of lab pairs are a\ufb00ected by the moves at any point in time . 12 The mean number of collaborations per year for colocated lab pairs increases from 0 . 034 during the pre - period to 0 . 042 during the moves . The change is substantially larger for pairs that are within a broad \ufb01eld , from 0 . 039 to 0 . 056 . An academic campus is an appealing environment for studying the role of geographic proximity on col - laboration : knowledge production is one of its de\ufb01n - ing activities , it is possible to measure collaborations and their long - run impact , knowledge \ufb02ows can be partially captured by looking at cited references , and research agendas can be mapped into knowledge space using keywords and cited references . Moreover , labs increasingly rely on a mix of proximate and distant col - laborators ( both in geographic and knowledge space ) for advancing their research agendas . 3 . 1 . Data The data set combines information on 39 , 527 pub - lications from the labs at Jussieu ( 1980 \u2013 2010 ) with \ufb01ne - grained location data over time . Publications are retrieved from SciVerse Scopus 13 and parsed to extract a\ufb03liation data . Forty - two thousand , four hundred ninety - four unique a\ufb03liation strings are cleaned and harmonized with a series of algorithmic and manual steps to match them to a speci\ufb01c lab . 14 Whenever loca - tion data is available in the papers 15 it is extracted to complement information retrieved from the UPMC archives and the EPAURIF website 16 to reconstruct the spatial allocation of labs over time . Paper a\ufb03liations that are not matched to the campus are geocoded using a combination of three di\ufb00erent services ( Google Maps API , Bing Maps API , and the Data Science Toolkit ) to identify them as either French or interna - tional a\ufb03liations . The core of the campus resembles a chessboard ( see Online Appendix Figure A - 1 ) and is composed of a series of towers connected by corridor buildings . Dis - tances are obtained by manually geocoding the loca - tion of each tower and connecting building on Google Earth . 17 Out of 39 , 527 publications , 6 % are collaborations across di\ufb00erent labs at Jussieu . In the \ufb01nal data set ( see Table 1 ) , the average minimum distance between any Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4353 Table 1 . Summary Statistics for the Main Sample Lab - pair - year level Mean Std . dev . Min Max N LocationMinimum distance ( km ) 0 . 17 0 . 106 0 0 . 449 183 , 359 Colocated ( same building ) 0 . 072 0 . 258 0 1 295 , 435 Treatment year ( colocation ) 2 , 003 . 99 3 . 722 1 , 997 2 , 010 7 , 338 Treatment year ( separation ) 2 , 003 . 63 4 . 089 1 , 997 2 , 010 5 , 877 CollaborationProbabilityof collaboration 0 . 004 0 . 064 0 1 295 , 435 Number of collaborations 0 . 006 0 . 126 0 15 295 , 35 Quality and type of research Citation weighted collaborations 0 . 204 7 . 328 0 2 , 058 295 , 435 Maximum number of citations 0 . 164 6 . 053 0 2 , 058 295 , 435 Standard deviation of citations a 25 . 666 38 . 964 0 271 . 442 321 Keyword similarity 0 . 076 0 . 108 0 0 . 881 116 , 851 Cited references similarity 0 . 174 0 . 115 0 0 . 677 84 , 865 a Conditional on collaborating at least twice in focal year . two lab pairs is 0 . 17 km ( approximately 550 feet ) and 7 . 2 % of them are colocated at any point in time ( same tower or corridor building ) . 18 Over time , 383 lab pairs switch from not colocated to being in the same build - ing during the asbestos cleaning process ( at a rate of 16 \u2013 52 each year ) , with 37 % of the changes happening in the \ufb01rst \ufb01ve years . Two hundred forty - eight lab pairs experience the opposite change ( i . e . , are separated by the moves ) , with 34 % of the events taking place within \ufb01ve years . Collaboration across labs is a rare event , and only 0 . 4 % of all lab pairs collaborate in any given year , receiving on average 0 . 20 citations per lab - pair - year . As often observed , the distribution of citations is very skewed , with most papers receiving no citations , and the most productive lab pair totaling 2 , 058 citations from the papers published in a single year . Citation data is obtained from Scopus in 2016 , and quartiles for the citation distribution are built by year using a large sample of articles in the relevant \ufb01elds of science . Scopus data is also used to retrieve , whenever available , author and index keywords , 19 as well as the full set of references cited by each paper . Cited references and keyword data are then used to create measures of proximity in knowledge space between the labs . In particular , the cosine similar - ity between vectors of cited references ( or keywords ) used by each lab is calculated using the Scikit - Learn python module . 20 To ensure that these proximity mea - sures are not in\ufb02ated by direct collaborations ( which would share all cited references and keywords by design ) , coauthored papers between the focal labs are dropped\u2014i . e . , the vectors of cited references ( or key - words ) are based on independent publications . More - over , when the sample is split by lab pairs with above versus below the median distance in knowledge space ( e . g . , Table 7 ) , the measure is de\ufb01ned before the moves start to avoid it being in\ufb02uenced by the e\ufb00ect of colo - cation ( or separation ) . In the data set , labs that do not share any keyword have a cosine similarity of zero , the pair with the most overlap has a score of 0 . 88 , 21 and the mean keyword similarity is 0 . 08 . Similarity in cited references is on average higher ( 0 . 17 ) , but the highest observed score is lower ( 0 . 68 ) . 3 . 2 . Empirical Strategy Estimating the e\ufb00ect of colocation on inventive out - comes using observational data is likely to return biased results , the main endogeneity concern being a selection e\ufb00ect . If labs value proximity to other labs they want to collaborate with , then a basic ordinary least - squares ( OLS ) regression of the number of collab - orations on geographic distance will positively bias the e\ufb00ect of proximity on collaboration . Whereas colocated lab pairs were 2 . 75 times more likely to collaborate than not - colocated pairs before the moves started , 22 the premium is only 14 % ( and not signi\ufb01cant ) during the relocations , which is consistent with the new spatial allocation being suboptimal in the post - period . In a perfect experiment , we would randomize the location of labs and observe how collaborations between colocated pairs di\ufb00er from those between dis - tant ones . During the asbestos removal , qualitative evi - dence con\ufb01rmed that needs that were orthogonal to the research agendas of the scientists involved ( e . g . , ease of relocation of a type of lab , cost minimization for the removal operation ) constrained space assign - ment and shaped when and where di\ufb00erent labs were moved . This introduces exogenous variation in the set of neighbors a lab is o\ufb00ered . 23 To account for idiosyn - cratic reasons a lab pair may be more or less likely to collaborate in the \ufb01rst place , the analysis uses lab - pair \ufb01xed e\ufb00ects . Importantly , the pair \ufb01xed e\ufb00ects also capture the degree of in\ufb02uence a particular pair of labs may have on campus resource allocation decisions ( funding , personnel , space allocation ) . Catalini : Microgeography and the Direction of Inventive Activity 4354 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) The econometric analysis uses a di\ufb00erence - in - dif - ferences approach 24 at the lab - pair - year level which exploits the variation in colocation ( and separation ) generated by the moves that take place during the asbestos - removal period . I estimate variations of Y ijt (cid:3) \u03b3 ij + \u03b4 t + \u03b2 AfterColocation ijt + (cid:15) ijt , ( 1 ) where Y ijt is a dummy for collaboration in focal year 25 between lab i and lab j in year t ; \u03b2 is the coe\ufb03cient of interest and the lab - pair \ufb01xed e\ufb00ects , \u03b3 ij , mean that \u03b2 is identi\ufb01ed as the within - pair e\ufb00ect on collaboration after labs become colocated because of the asbestos removal , relative to their before period ; and \u03b4 t is a year e\ufb00ect . The AfterColocation dummy is equal to 1 if the lab pair becomes colocated because of the moves , and is set back to 0 when the labs are separated again . Similarly , I estimate the e\ufb00ect of separation in the same regression using variations of Y ijt (cid:3) \u03b3 ij + \u03b4 t + \u03b2 1 AfterColocation ijt + \u03b2 2 AfterSeparation ijt + (cid:15) ijt . ( 2 ) Both equations are also estimated with nonlinear models and for di\ufb00erent outcomes : number of col - laborations in focal year , maximum number of cita - tions received ( conditional on publication ) , number of papers published in di\ufb00erent quartiles of the citation distribution , similarity in cited references space and in keyword space , probability of publishing in a journal that is new for at least one of the labs , etc . Before moving to the di\ufb00erence - in - di\ufb00erences frame - work , the paper uses regressions at the lab - year level for the same set of outcomes to explore how the relo - cation period in\ufb02uenced collaboration and the compo - sition of the labs\u2019 portfolios ( in terms of share of a\ufb03lia - tions classi\ufb01ed as same lab , di\ufb00erent lab , other French lab or international lab , etc . ) . All lab - year \u2013 level regres - sions include lab \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . 4 . Results The \ufb01rst section of the results focuses on assessing the overall e\ufb00ect of the moves on the labs involved . Empirically , it is important to determine if the relo - cation period had a negative e\ufb00ect on the labs\u2019 pro - ductivity , and how labs adjusted their collaboration portfolios because of the changes in their neighbors . Since this part of the analysis does not rely on the di\ufb00erence - in - di\ufb00erences empirical strategy described above , results will only be informative but not conclu - sive with respect to the mechanisms proposed in the theoretical framework . The second section will directly test Hypothesis 1 on the e\ufb00ects of a change in proximity on the probability of collaboration . This is done by looking at pairs of labs that \ufb01nd themselves in the same building because of the asbestos removal , as well as pairs that are separated because of it . In particular , separated pairs will be infor - mative about the ability of other forms of proximity ( e . g . , social proximity ) to compensate for geographic distance . Robustness is shown to highlight the absence of a pre - trend in collaboration among pairs that are going to be colocated , which is consistent with the moves not being driven by the labs\u2019 research agendas . Knowledge distance between labs is then introduced as a way to compare lab pairs with di\ufb00erent ex ante search costs , and to see if the changes in the proba - bility of collaboration are consistent with the search cost mechanism . Since Hypotheses 1 and 2 do not dif - fer in terms of what they predict we should observe after colocation , but di\ufb00er in terms of what we should see after separation , this section will also compare the relative role of search versus execution costs in de\ufb01ning who collaborates . Lastly , the section explores the e\ufb00ect of search costs over longer periods of time by using information on the references cited in the a\ufb00ected papers . The third section introduces quality ( as proxied by citations ) to test Hypothesis 3 , and to see if outcomes are consistent with lower execution costs under coloca - tion leading to both more marginal ideas being devel - oped ( selection e\ufb00ect ) , as well as higher - quality ideas ( e\ufb00ort e\ufb00ect ) . 4 . 1 . Lab - Level Results and Collaboration Portfolios One may worry that the relocations on the Jussieu campus had a negative e\ufb00ect on the productivity of the labs . This would in\ufb02uence how we would inter - pret the results from the di\ufb00erence - in - di\ufb00erences anal - ysis in the next sections of the paper . Furthermore , since labs are likely to reallocate their resources toward di\ufb00erent types of projects as their local environment changes , before moving to the lab - pair \u2013 level regres - sions , it is useful to descriptively explore how aggre - gate collaboration portfolios shifted during the study period because of the moves . The unit of analysis in Table 2 is a lab - year , lab \ufb01xed e\ufb00ects are included to account for unobservable dif - ferences between labs that are constant over time ( e . g . , \ufb01eld of science , relative scale and resources of the lab within the institution , etc . ) , and year \ufb01xed e\ufb00ects are introduced to control for changes in productivity over time ( e . g . , increased resources available to the campus , changes in the national science policy , etc . ) . 26 The key explanatory variable in the table is a dummy equal to 1 during the Relocation Period \u2014i . e . , during all of the years during which a speci\ufb01c lab is moved from its original location because of the asbestos removal ( and 0 other - wise ) . As can be seen both in terms of raw publication counts ( column ( 1 ) ) , and in terms of quality - adjusted output ( column ( 2 ) ) , the moves are not correlated with a decrease in output for the a\ufb00ected labs relative to Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4355 Table 2 . Lab - Level Outcomes ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) Variables Publications Citation weighted pubs Mean cites Max cites Std . dev . cites Relocation Period 10 . 8003 \u2217\u2217\u2217 246 . 6649 \u2217\u2217\u2217 \u2212 0 . 0157 28 . 0384 \u2217 6 . 4865 \u2217\u2217 ( 2 . 3118 ) ( 82 . 7443 ) ( 1 . 6609 ) ( 14 . 6638 ) ( 2 . 6019 ) Lab \ufb01xed e\ufb00ects Yes Yes Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Yes Yes Observations 3 , 951 3 , 951 3 , 951 3 , 951 3 , 951 R - squared 0 . 161 0 . 070 0 . 015 0 . 015 0 . 011 Number of labs 328 328 328 328 328 Notes . Relocation Period is a dummy equal to 1 for all of the years during which a speci\ufb01c lab is moved from its original location because of the asbestos removal . Citation data is collected from Scopus in 2016 . Robust standard errors clustered at the lab level in parentheses . \u2217 p < 0 . 1 ; \u2217\u2217 p < 0 . 05 ; \u2217\u2217\u2217 p < 0 . 01 . the controls ( i . e . , labs that have not been moved yet or that have returned to their original location ) . Interest - ingly , results on quality are suggestive of an increase on both tails of the outcome distribution : the e\ufb00ect on mean citations is negative although nonsigni\ufb01cant ( col - umn ( 3 ) ) , max citations increase ( column ( 4 ) ) , and the moves are correlated with a positive increase in the standard deviation of citations ( column ( 5 ) ) . It is important to highlight that multiple mecha - nisms , including changes in neighboring labs , infras - tructure , and research type , can explain these results . For example , labs could have changed the composi - tion of their projects in response to shifts in their local environment in a way that favored higher - variance projects\u2014e . g . , if the moves increased the likelihood that a lab had access to peers from a di\ufb00erent disci - pline , the polarization in outcomes could be a result of an increase in cross - disciplinary work . This would be consistent with past research that has shown that recombination of ideas that span more distant areas of the knowledge space exhibit higher variance ( e . g . , because the ideas involved are recombined less often and therefore represent a less explored area of the research landscape ; see Fleming and Sorenson 2004 , Singh and Fleming 2010 ) . In this context , while keep - ing the expected value of their projects constant ( i . e . , while staying on their original risk \u2013 return indi\ufb00erence curve ) , labs could have undertaken some projects that o\ufb00ered higher risk but also higher reward . Alterna - tively , the moves could have induced a temporary , one - time shift in research agendas because of the reshuf - \ufb02ing of scientists and labs ( a \u201cnovelty\u201d e\ufb00ect ) . For these reasons , the next section will move away from the lab - level analysis and control for many of these confounders by looking at variation within lab pairs . With the introduction of lab - pair \ufb01xed e\ufb00ects , the di\ufb00erence - in - di\ufb00erences approach allows to account for the idiosyncratic reasons two labs may ( or may not ) collaborate with each other , and controls for unob - served heterogeneity in the type of research a particu - lar pair may be conducting . Within a lab pair , the con - straints imposed by the moves will deliver plausibly exogenous variation in distance , allowing us to look at outcomes while keeping issues related to the matching between labs constant\u2014i . e . , within a lab pair , changes in collaboration and outcomes will be predominantly driven by shifts in proximity . Additionally , the analysis of pre - trends in collaboration within the di\ufb00erence - in - di\ufb00erences framework will allow us to visually inves - tigate the presence of selection into colocation during the moves\u2014i . e . , if labs are endogenously paired in an e\ufb00ort to improve their collaborations , this should be observable in the data . Before moving to the lab - pair analysis , it is useful to check how the relocation period shifted the collab - oration portfolios of the labs at the aggregate level . Table 3 uses the same speci\ufb01cation of the previous table to look at how the shares of a\ufb03liations on the labs\u2019 papers changed during the moves . The dependent variables here are , respectively , the share of a\ufb03liations that are international ( column ( 1 ) ) , within France but not from the institution ( column ( 2 ) ) , and within the institution ( column ( 3 ) ) . Interestingly , the moves saw a decrease in the share of international collaborators ; a Table 3 . Lab Level\u2014Overall Collaboration Portfolio ( 1 ) ( 2 ) ( 3 ) Share Share Share Variables international French within institution Relocation Period \u2212 0 . 0237 \u2217 0 . 0018 0 . 0218 ( 0 . 0121 ) ( 0 . 0137 ) ( 0 . 0146 ) Lab \ufb01xed e\ufb00ects Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Observations 3 , 951 3 , 951 3 , 951 R - squared 0 . 144 0 . 072 0 . 207 Number of labs 328 328 328 Notes . Relocation Period is a dummy equal to 1 for all of the years dur - ing which a speci\ufb01c lab is moved from its original location because of the asbestos removal . Shares are calculated based on the Scopus author a\ufb03liations data available for each paper and then aggregated at the lab level on a yearly basis . \u201cShare French\u201d does not include the Jussieu campus and a\ufb03liated labs . Robust standard errors clustered at the lab level in parentheses . \u2217 p < 0 . 1 . Catalini : Microgeography and the Direction of Inventive Activity 4356 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) Table 4 . Lab Level\u2014Collaboration Portfolio Within Institution ( 6 ) ( 7 ) ( 8 ) ( 9 ) ( 1 ) ( 3 ) ( 4 ) ( 5 ) Colocated Colocated Not colocated Not colocated Share ( 2 ) Not Intensive Extensive intensive extensive intensive extensive Variables across labs Colocated colocated margin margin margin margin margin margin Relocation Period \u2212 0 . 0210 \u2217\u2217 0 . 0155 \u2217\u2217\u2217 \u2212 0 . 0365 \u2217\u2217\u2217 \u2212 0 . 0035 \u2212 0 . 0175 \u2217\u2217 0 . 0030 0 . 0125 \u2217\u2217\u2217 \u2212 0 . 0065 \u2212 0 . 0300 \u2217\u2217\u2217 ( 0 . 0093 ) ( 0 . 0048 ) ( 0 . 0082 ) ( 0 . 0066 ) ( 0 . 0075 ) ( 0 . 0037 ) ( 0 . 0031 ) ( 0 . 0051 ) ( 0 . 0075 ) Lab \ufb01xed e\ufb00ects Yes Yes Yes Yes Yes Yes Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Yes Yes Yes Yes Yes Yes Observations 3 , 951 3 , 951 3 , 951 3 , 951 3 , 951 3 , 951 3 , 951 3 , 951 3 , 951 R - squared 0 . 022 0 . 023 0 . 034 0 . 065 0 . 130 0 . 036 0 . 035 0 . 041 0 . 119 Number of labs 328 328 328 328 328 328 328 328 328 Notes . Relocation Period is a dummy equal to 1 for all of the years during which a speci\ufb01c lab is moved from its original location because of the asbestos removal . All shares are calculated based on the Scopus author a\ufb03liations data available for each paper and then aggregated at the lab level on a yearly basis . The table only includes a\ufb03liations that can be assigned to the Jussieu campus and a\ufb03liated labs . Robust standard errors clustered at the lab level in parentheses . \u2217\u2217 p < 0 . 05 , \u2217\u2217\u2217 p < 0 . 01 . nonsigni\ufb01cant , small positive change in collaborators from other French institutions ; and an increase in within - institution research . While the regression con - trols for lab \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects , as in the previous table multiple mechanisms could explain these changes . One interpretation is that the campus lost competitiveness because of the moves , becoming more inward focused . A di\ufb00erent one is that the reshuf - \ufb02ing improved local opportunities by o\ufb00ering labs new neighbors , making outside collaboration relatively less appealing . To further test how internal collaboration was af - fected , Table 4 uses the same approach of Table 3 but only focuses on internal collaborations ( i . e . , all of the shares are now de\ufb01ned over the total number of a\ufb03l - iations assigned to the institution ) . Column ( 1 ) high - lights how the increase in within - institution research observed in column ( 3 ) of Table 3 is entirely driven by collaborations within labs : the share of across - lab collaborations actually decreases , suggesting that the moves may have worsened the local environment , forc - ing labs to increase collaboration within their unit . This is consistent with the relocations leading to a subop - timal space allocation relative to what the labs would have selected if they were in charge of it . The next two pairs of columns in Table 4 , take the share from column ( 1 ) and further decompose it across di\ufb00erent dimensions . In particular , columns ( 2 ) and ( 3 ) split the collaborations across labs ( i . e . , column ( 1 ) ) between labs that are colocated versus labs that are distant : the negative result from the \ufb01rst column comes from a drastic decrease in the share of collaborators from not colocated labs ( column ( 3 ) ) that is not compensated by an equal increase in collaborations with colocated labs ( column ( 2 ) ) . When column ( 1 ) is instead decom - posed into intensive margin versus extensive margin collaborations ( i . e . , between labs that had collaborated before the relocations versus not ) , the data show that the decay in across - lab collaborations is mostly driven by extensive margin pairs\u2014i . e . , during the relocations , labs were substantially less likely to explore research with labs they had not collaborated with before . The aggregate result of columns ( 4 ) and ( 5 ) , however , hide di\ufb00erent heterogeneous e\ufb00ects by microgeogra - phy : in columns ( 6 ) \u2013 ( 9 ) , the share of column ( 1 ) is divided into colocated ( intensive versus extensive ) and not colocated ( intensive versus extensive ) collabora - tions . Whereas the results are consistent with geogra - phy only slightly facilitating ( or obstructing ) collabora - tive work on the intensive margin ( the coe\ufb03cients are respectively positive in column ( 6 ) and negative in col - umn ( 8 ) , but in both cases not signi\ufb01cant ) , columns ( 7 ) and ( 8 ) suggest that microgeography plays a substan - tially more important role on the extensive margin of collaboration . This seems consistent with search costs having a disproportionate e\ufb00ect on de\ufb01ning who col - laborates with whom in the absence of past exposure or social proximity . This hypothesis will be further tested in the next section . Overall , at least in the Jussieu case , the increase in collaboration with newly colocated pairs ( columns ( 6 ) and ( 7 ) ) seems to be more than o\ufb00set by the decrease with not colocated labs ( columns ( 8 ) and ( 9 ) ) . Fur - thermore , the result in column ( 1 ) ( combined with the e\ufb00ect of column ( 3 ) in Table 3 ) is consistent with labs being more inward focused during the moves , potentially because they faced a less optimal set of local peers around them relative to what they would have selected in an ideal scenario . Together with the observed increased in best outcomes and variance in Table 2 , and with the rise in colocated experimentation on the extensive margin ( column ( 7 ) in Table 4 ) , this raises the question of how colocation and separation directly a\ufb00ected research outcomes once we keep lab pairs constant . Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4357 4 . 2 . Probability of Collaboration and the Role of Search Costs In the theoretical framework , both a reduction in search costs ( Hypothesis 1 ) and in joint execution costs ( Hypothesis 2 ) predict an increase in the probability of collaboration after colocation . If joint execution costs are particularly important , then we should also observe a tangible rise in the rate of collaboration . The two mechanisms have di\ufb00erent implications for the reverse move though\u2014i . e . , for what we should observe after two labs that were previously colocated are suddenly separated . If search costs do not increase immediately after separation because of past exposure , then sepa - ration should have little to no e\ufb00ect on the probability of collaboration . If joint execution costs are instead dis - proportionately driving collaboration decisions , then separation should be accompanied with a drop in col - laboration . This section tests these hypotheses by \ufb01rst looking at within - lab - pair changes in collaboration fol - lowing changes in distance , and then by exploring het - erogeneous e\ufb00ects by pairs that faced ex ante higher versus lower search costs to compare the two mecha - nisms more directly . The \ufb01rst part of Hypotheses 1 and 2 are tested in Table 5 : after colocation , lab pairs are 3 . 5 times more likely to collaborate ( column ( 1 ) ) and collaborate on average 2 . 5 \u2013 3 . 3 times more ( columns ( 3 ) and ( 2 ) , respectively ) . Results are robust and qualitatively sim - ilar across di\ufb00erent functional forms ( OLS , Poisson , logit , rare event logit ) . Robust standard errors are clus - tered at the lab - pair level , and all regressions include lab - pair \ufb01xed e\ufb00ects as well as year \ufb01xed e\ufb00ects . The estimated e\ufb00ects do not change if lab - year trends are included in the regression . The \ufb01ndings are consistent with lower search and execution costs increasing the Table 5 . Increase in Collaboration and Colocation ( 1 ) ( 2 ) ( 3 ) Variables OLS 1 / 0 OLS # Collabs Poisson # Collabs After Colocation 0 . 0146 \u2217\u2217\u2217 0 . 0210 \u2217\u2217 0 . 8981 \u2217\u2217\u2217 ( 0 . 0054 ) ( 0 . 0094 ) ( 0 . 2814 ) Lab - pair \ufb01xed e\ufb00ects Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Observations 295 , 435 295 , 435 10 , 383 R - squared 0 . 005 0 . 005 Number of lab pairs 35 , 805 35 , 805 587 Log likelihood \u2212 2 , 929 Notes . The dependent variable in column ( 1 ) is a dummy equal to 1 if the lab pair collaborates in the focal year . The dependent variable in columns ( 2 ) and ( 3 ) is the number of collaborations . After Colocation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise . The Poisson speci\ufb01cation with \ufb01xed e\ufb00ects drops all lab pairs where collaboration is never observed , hence the smaller number of observations . Robust standard errors clustered at the lab - pair level in parentheses . \u2217\u2217 p < 0 . 05 ; \u2217\u2217\u2217 p < 0 . 01 . likelihood ( column ( 1 ) ) and rate ( columns ( 2 ) and ( 3 ) ) of collaboration . It is important to stress that the presence of the lab - pair \ufb01xed e\ufb00ects takes care of the idiosyncratic in\ufb02u - ence a pair of labs may have on campus ( e . g . , in terms of funding , ability to bargain for more or better resources and infrastructure ) . Nevertheless , one may still worry that in\ufb02uential labs might have exerted pressure on EPAURIF to assign them a particular temporary loca - tion , or to change the timing of their move . From an identi\ufb01cation perspective , the main worry is that the moves were driven by preexisting collaboration pat - terns and therefore endogenous to the outcomes of interest . If that were the case , and labs were able to obtain a spot next to the labs with which they had an interest in collaborating more , then we should observe a rise in collaboration that predates the relocation . Fig - ure 1 reassures us this is not the case , as the rise in col - laboration follows colocation ( no pre - trend ) and builds progressively over the years in the post - period . In fact , there is no activity until two years or more after the move . The \ufb01gure plots the estimated coe\ufb03cient of an OLS regression with year and lab - pair \ufb01xed e\ufb00ects for all of the years before and after the move . 27 The dependent variable is the probability of collaboration ( dummy ) , and the error bars represent 95 % con\ufb01dence intervals based on robust standard errors ( clustered at the lab - pair level ) . 28 Past exposure may allow labs to contact a now - distant lab when the right opportunity emerges or , if opportu - nities have already been identi\ufb01ed , labs can make com - mitments to keep their research active through joint seminars , temporary colocation , and distant interac - tions . Therefore , if search costs are a key friction to Figure 1 . ( Color online ) Probability of Collaboration and Colocation \u2013 0 . 02 0 0 . 02 0 . 04 0 . 06 0 . 08 \u2013 10 \u2013 8 \u2013 6 \u2013 4 \u2013 2 0 2 4 6 Years to / Since move Notes . Estimated coe\ufb03cient for years before and after the move . The dependent variable is collaboration ( 1 / 0 ) . Regression includes lab - pair \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . Error bars represent 95 % con\ufb01dence intervals based on robust standard errors clustered at the lab - pair level . Catalini : Microgeography and the Direction of Inventive Activity 4358 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) Table 6 . Colocation and Separation ( 1 ) ( 2 ) ( 3 ) Variables OLS 1 / 0 OLS # Collabs Poisson # Collabs After Colocation 0 . 0148 \u2217\u2217\u2217 0 . 0217 \u2217\u2217 0 . 8871 \u2217\u2217\u2217 ( 0 . 0054 ) ( 0 . 0093 ) ( 0 . 2814 ) After Separation 0 . 0101 0 . 0388 \u2212 0 . 4520 ( 0 . 0089 ) ( 0 . 0278 ) ( 0 . 3171 ) Lab - pair \ufb01xed e\ufb00ects Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Observations 295 , 435 295 , 435 10 , 383 R - squared 0 . 005 0 . 005 Number of lab pairs 35 , 805 35 , 805 587 Log likelihood \u2212 2 , 926 Notes . The dependent variable in column ( 1 ) is a dummy equal to 1 if the lab pair collaborates in the focal year . The dependent vari - able in columns ( 2 ) and ( 3 ) is the number of collaborations . After Colocation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise . After Separation is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise . The Poisson speci\ufb01cation with \ufb01xed e\ufb00ects drops all lab pairs where collaboration is never observed , hence the smaller num - ber of observations . Robust standard errors clustered at the lab - pair level in parentheses . \u2217\u2217 p < 0 . 05 ; \u2217\u2217\u2217 p < 0 . 01 . collaboration , separation should not have a strong , neg - ative e\ufb00ect on the probability of collaboration . 29 If joint execution costs constitute a barrier to collaboration instead , separated pairs should reduce their rate of collaboration . Consistent with the search costs mechanism , in Table 6 separation has a noisy , nonsigni\ufb01cant e\ufb00ect on the probability and intensity of collaboration . The co - e\ufb03cient is positive and nonsigni\ufb01cant in the OLS re - gressions ( columns ( 1 ) and ( 2 ) ) and negative and non - signi\ufb01cant in the Poisson speci\ufb01cation ( column ( 3 ) ) . 30 Meanwhile , controlling for separation leaves the results on colocation unchanged . Figure 2 , which plots the estimated regression coef - \ufb01cients 31 for the years before and after separation , highlights three facts : ( 1 ) lab pairs that are going to be separated have a higher propensity to collaborate before the move ( all of the coe\ufb03cients in the pre - period are positive and many are statistically di\ufb00erent than zero ) ; ( 2 ) the relocation process seems to gener - ate a slight , temporary decay in collaboration ( years \u2212 1 to + 1 ) ; ( 3 ) lab pairs revert to their mean collabora - tion propensity in the later periods ( although results are noisy , potentially because some pairs recover and others do not ) . Overall , results from Tables 5 and 6 are consistent with the idea that search costs may be a more impor - tant friction than joint execution costs in de\ufb01ning if two labs will collaborate or not . If the key mechanism through which colocation facilitates collaboration is by helping scientists discover new potential collaborators , then the e\ufb00ect of proximity should be strongest where Figure 2 . ( Color online ) Probability of Collaboration and Separation \u2013 0 . 05 0 0 . 05 0 . 10 \u2013 10 \u2013 8 \u2013 6 \u2013 4 \u2013 2 0 2 4 6 Years to / Since move Notes . Estimated coe\ufb03cient for years before and after the move . The dependent variable is collaboration ( 1 / 0 ) . Regression includes lab - pair \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . Error bars represent 95 % con - \ufb01dence intervals based on robust standard errors clustered at the lab - pair level . search costs are ex ante high and less pronounced where search costs are lower because alternative chan - nels for knowledge di\ufb00usion are likely to exist ( e . g . , joint conferences , journals , etc . ) . To proxy for the fact that two labs may be work - ing in related areas before the moves ( i . e . , within more proximate communities of science ) , Table 7 uses two sources of data : keywords listed on publications and the full set of references cited in the papers . To calculate a measure of proximity in knowledge space between two labs , the cosine distance between the vectors of keywords ( or of references cited ) used by each lab is calculated excluding all direct collaborations ( which would exhibit perfect overlap in keywords and refer - ences by design ) . Lab pairs that have an above - the - median similarity in keywords and references are clas - si\ufb01ed as facing lower search costs ex ante ( columns ( 1 ) and ( 3 ) ) , since they are more likely to overlap in top - ics and the literature they cite ( and potentially con - ferences , etc . ) . Lab pairs that have below - the - median similarity ( columns ( 2 ) and ( 4 ) ) are classi\ufb01ed as expe - riencing higher search costs ex ante , since they do not seem aware of each other\u2019s research topics and body of knowledge . Interestingly , whereas the two classi\ufb01cations assign slightly di\ufb00erent sets of labs to each bin , the inter - pretation of the main e\ufb00ect is consistent between them . When search costs are low ex ante ( columns ( 1 ) and ( 3 ) ) , colocation has a positive but nonsigni\ufb01cant e\ufb00ect on the probability of collaboration . This is consis - tent with these lab pairs already overlapping through other channels and being aware of each other\u2019s work ( as evidenced by the similar keywords and literature used ) . As additional evidence that search costs may Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4359 Table 7 . Probability of Collaboration and Search Costs ( 1 ) ( 2 ) ( 3 ) ( 4 ) OLS 1 / 0 OLS 1 / 0 OLS 1 / 0 OLS 1 / 0 low search costs high search costs low search costs high search costs Variables ( keywords ) ( keywords ) ( cited ref . ) ( cited ref . ) After Colocation 0 . 0056 0 . 0179 \u2217 0 . 0006 0 . 0145 \u2217 ( 0 . 0126 ) ( 0 . 0093 ) ( 0 . 0268 ) ( 0 . 0077 ) After Separation \u2212 0 . 0197 \u2217 0 . 0244 \u2212 0 . 0080 0 . 0059 ( 0 . 0112 ) ( 0 . 0185 ) ( 0 . 0180 ) ( 0 . 0136 ) Lab - pair \ufb01xed e\ufb00ects Yes Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Yes Observations 48 , 710 60 , 837 20 , 698 77 , 951 R - squared 0 . 008 0 . 016 0 . 008 0 . 012 Number of lab pairs 3 , 864 6 , 102 1 , 366 6 , 485 Notes . The dependent variable in all columns is a dummy equal to 1 if the lab pair collaborates in the focal year . In columns ( 1 ) and ( 3 ) , only pairs with above the median cosine similarity ( low search costs ) are included . In columns ( 2 ) and ( 4 ) , only pairs with below the median similarity ( high search costs ) are included . The cosine similarity measures are based on the vectors of keywords used by each lab in columns ( 1 ) and ( 2 ) , and on the vectors of cited references in columns ( 3 ) and ( 4 ) . In all cases , the measures are calculated before the moves start and do not include direct collaborations between the focal labs ( which would share mechanically all keywords and cited references ) . After Colocation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise . After Separation is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise . Robust standard errors clustered at the lab - pair level in parentheses . \u2217 p < 0 . 1 . not be the key constraint for collaboration between these pairs , separation does seem to induce a decay in their likelihood of collaboration , possibly because some of these marginal , within - \ufb01eld collaborations are only sustainable when joint execution costs are low . Moreover , when search costs are high , not only do we observe a strong and positive e\ufb00ect of colocation on collaboration , but separation has only a noisy e\ufb00ect on outcomes , supporting the idea that for these pairs , the key constraint is being aware of each other\u2019s agenda . A consequence of this result is that over time , labs may become more similar because of lower search costs and repeated exposure , exhausting some of the best arbitrage opportunities in idea space . After separation , because of higher execution costs , some labs that are very similar ( columns ( 1 ) and ( 3 ) ) may reduce collab - oration with each other in favor of new opportunities with newly colocated labs that bring more novelty to their approaches ( columns ( 2 ) and ( 4 ) ) . To test if repeated exposure through colocation leads to an increase in similarity and awareness of each other\u2019s research , Figure 3 ( a ) plots the estimated regres - sion coe\ufb03cients 32 for the years before and after colo - cation using the cosine similarity in cited references as the dependent variable . The measure captures the extent of overlap in backward cites for a speci\ufb01c lab pair in a given year ( excluding joint collaborations ) . Overlap does not increase until two or more years after colocation , which is consistent with changes taking place slowly over time through knowledge \ufb02ows and direct collaboration . 33 Adding separation to the regression of similarity in cited references ( Figure 3 ( b ) ) shows that the reverse process takes place when distance increases : after sep - aration , previously colocated labs progressively grow apart , a result that is striking because in the two years before separation , they were actually more likely to cite the same references than the controls . 34 The neigh - bors of a lab , by exposing it to new knowledge , ideas , and collaboration opportunities , seem to profoundly shape its research trajectory . In this particular set - ting , whereas collaboration seems to recover from an increase in geographic distance ( Figure 2 ) \u2014possibly also because labs can make purposeful investments in temporary colocation and remote interactions to com - pensate for it\u2014knowledge \ufb02ows seem to be negatively a\ufb00ected by separation . Ironically , as separated labs embark on less correlated research trajectories , they may also set the stage for more novel recombination of ideas in the future . 4 . 3 . Quality of Collaborations and Execution Costs This last section explores the e\ufb00ect of proximity on the quality of collaborations . Hypothesis 3 predicts that if geographic proximity has a tangible e\ufb00ect on joint exe - cution costs , then after colocation , we should observe more projects both on the left tail of the quality dis - tribution ( a selection e\ufb00ect ) as well as on the right tail ( an e\ufb00ort e\ufb00ect ) . Separation instead should generate opposite e\ufb00ects . It is important to highlight that the prediction refers to the observed outcomes conditional on a project being developed , but empirically an addi - tional issue arises from the fact that not all projects are observed : e . g . , if scientists are overly optimistic about their chances of publishing their results , or if part of the projects that are initially developed are dropped before submission , then we will never see projects of Catalini : Microgeography and the Direction of Inventive Activity 4360 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) Figure 3 . ( Color online ) Similarity in Cited References Space \u2013 0 . 06 \u2013 0 . 04 \u2013 0 . 02 0 0 . 02 0 . 04 0 . 06 0 . 08 \u2013 0 . 06 \u2013 0 . 04 \u2013 0 . 02 0 0 . 02 0 . 04 0 . 06 0 . 08 \u2013 10 \u2013 8 \u2013 6 \u2013 4 \u2013 2 0 2 4 6 Years to / Since move \u2013 10 \u2013 8 \u2013 6 \u2013 4 \u2013 2 0 2 4 6 Years to / Since move ( a ) Colocation only ( b ) Colocation vs . separation Notes . Estimated coe\ufb03cient for years before and after the move . The dependent variable is the cosine similarity ( based on cited refer - ences ) between the two labs . The higher line in the post - period is for colocation , and the lower line is for separation . Regression includes lab - pair \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . Error bars represent 95 % con\ufb01dence intervals based on robust standard errors clustered at the lab - pair level . the lowest quality . As a result , any e\ufb00ect on the left tail of the outcome distribution should be considered a lower estimate of the true e\ufb00ect of colocation ( or sepa - ration ) on inventive outcomes . Table 8 explores how the citation distribution 35 changes after the moves , conditional on the lab pairs collaborating . Overall , colocation seems to increase the number of collaborations that will end on both tails of the distribution ( \ufb01rst and fourth quartiles ) , and to decrease activity in the third quartile ( negative and signi\ufb01cant ) and potentially in the second quartile ( neg - ative but not signi\ufb01cant ) . A comparison between the estimated coe\ufb03cients for colocation shows that the fourth quartile ( highest quartile , column ( 4 ) ) is statisti - cally di\ufb00erent at 1 % from the one in the third quartile ( column ( 3 ) ) , and at 10 % from the one in the second quartile ( column ( 2 ) ) . It is not statistically di\ufb00erent from the coe\ufb03cient in the bottom quartile ( column ( 1 ) ) , and second and third quartiles are not statistically di\ufb00erent from each other . The noisier results in the \ufb01rst quar - tile could be due to the fact that collaborations that do not generate a publication are not observed , truncat - ing the left tail . Separation exhibits an almost symmet - ric pattern , with a positive ( although nonsigni\ufb01cant ) increase in the third quartile and a signi\ufb01cant decay in the fourth . In Table 9 , the right tail of the outcome distribution is further explored by separating lab pairs that faced high versus low search costs before the moves ( based on the same approach used in Table 7 , columns ( 1 ) and ( 2 ) ) . Whereas for pairs with low search costs , both coloca - tion and separation generate only noisy and insignif - icant results , when search costs are high , colocation leads to high impact research , and separation generates the opposite result . In the online appendix , robustness is shown using a di\ufb00erent proxy for quality ( citation weighted publications ) and by looking at the standard deviation of citations as a way to capture e\ufb00ects on both tails of the outcome distribution ( Online Appendix Table A - 2 ) . While only suggestive , the results in Table 9 support the idea that pairs that usually face high search costs ( potentially also because space allocation is generally optimized by discipline ) are not only more responsive to changes in distance ( as we have seen in Table 7 ) , but are also able to produce higher - impact research when given the opportunity to take advantage of lower execution costs . One interpretation of this result is that these col - laborations constitute arbitrage opportunities across domains of science , and that if we were able to observe these pairs for substantially longer periods of time , the positive e\ufb00ect on the right tail may revert to the mean . Another interpretation is that once faced with a more heterogeneous set of local peers , labs were encouraged to embark on higher - risk , higher - reward projects ( which would be consistent with the increase in variance ) . The result is also consistent with the view that multidisciplinary research tends to be of higher variance and , when successful , of higher impact . The e\ufb00ects are also a reminder of how search frictions can be a tangible obstacle to impactful research outside of a community of science . In these cases , colocation ( whether temporary or not ) can be used to remove some of these frictions and introduce novelty within current research agendas . 5 . Limitations This study has a number of limitations . First , only col - laborations that end up in a peer - reviewed publication are observed , meaning that the left tail of the outcome Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4361 Table 8 . Colocation , Separation , and the Citation Distribution ( 1 ) ( 2 ) ( 3 ) ( 4 ) # Collabs # Collabs # Collabs # Collabs Variables 1st quartile ( lowest ) 2nd quartile 3rd quartile 4th quartile ( highest ) After Colocation 0 . 2135 \u2212 0 . 0719 \u2212 0 . 3937 \u2217 0 . 4250 \u2217\u2217 ( 0 . 2241 ) ( 0 . 2082 ) ( 0 . 2007 ) ( 0 . 1675 ) After Separation \u2212 0 . 1236 \u2212 0 . 2239 0 . 3725 \u2212 0 . 3403 \u2217\u2217 ( 0 . 2638 ) ( 0 . 2190 ) ( 0 . 3538 ) ( 0 . 1656 ) Lab - pair \ufb01xed e\ufb00ects Yes Yes Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Yes Yes Observations 1 , 221 1 , 221 1 , 221 1 , 221 R - squared 0 . 061 0 . 081 0 . 070 0 . 199 Number of lab pairs 627 627 627 627 Notes . The dependent variable in all columns is the number of publications by the lab pair in the focal year within the given quartile of the citation distribution . Citation data is obtained from Scopus in 2016 , and quartiles for the citation distribution are built by year using a large sample of articles in the relevant \ufb01elds of science . The \ufb01rst quartile represents papers with the lowest level of citations , the fourth the highest . Results are conditional on collaboration in the focal year . After Colocation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise . After Separation is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise . Robust standard errors clustered at the lab - pair level in parentheses . \u2217 p < 0 . 1 , \u2217\u2217 p < 0 . 05 . distribution is not observed . Whereas the analysis on cited references and keywords may be able to capture the outcome of some of the interactions ( planned or serendipitous ) that do not translate into a paper , the analyses on the rate and quality of research misses them . The measured e\ufb00ects on the left tail of the out - come distribution are therefore likely to represent a lower estimate of the true e\ufb00ects . Furthermore , collab - orations across labs are rare relative to collaborations within labs , which limits the set of tests that can be con - ducted on this sample ( e . g . , further splits of the sample across more \ufb01ne - grained dimensions ) . Table 9 . Colocation , Separation and Max Citations ( 1 ) ( 2 ) Max cites Max cites Variables ( low search costs ) ( high search costs ) After Colocation 27 . 9531 40 . 0822 \u2217\u2217\u2217 ( 31 . 6185 ) ( 13 . 3165 ) After Separation 6 . 6615 \u2212 36 . 4662 \u2217\u2217\u2217 ( 11 . 3936 ) ( 6 . 9199 ) Lab - pair \ufb01xed e\ufb00ects Yes Yes Year \ufb01xed e\ufb00ects Yes Yes Observations 639 529 R - squared 0 . 142 0 . 118 Number of lab pairs 293 294 Notes . The dependent variable in all columns is the maximum num - ber of citations received by the publications of the lab pair in the focal year . Citation data is obtained from Scopus in 2016 . In column ( 1 ) , only pairs with above the median cosine similarity in keywords ( low search costs ) are included . In column ( 2 ) , only pairs with below the median similarity in keywords ( high search costs ) are included . Results are conditional on collaboration in focal year . After Colocation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise . After Separation is equal to 1 when a previously colo - cated pair is separated because of the moves and 0 otherwise . Robust standard errors clustered at the lab - pair level in parentheses . \u2217\u2217\u2217 p < 0 . 01 . Second , location information is imprecise , 36 generat - ing noise in the exact timing of a move and potentially biasing the estimates downward . The same applies to the ability to correctly capture and match every pub - lication of the entities involved , as a\ufb03liation data is di\ufb03cult to clean and harmonize at scale at the lab level . Hopefully , as bibliometrics and algorithms for disam - biguation improve , better data will become available . Third , while the relocations on the Jussieu campus are substantially more constrained than typical obser - vational data on campus moves , they do not consti - tute random assignment . It is reassuring to see that the di\ufb00erence - in - di\ufb00erences estimates , and in particu - lar the pre - trends , do not hint at strong selection into colocation during the study period . Fourth , the data does not allow perfect separation and direct measurement of some of the mechanisms discussed in the theoretical framework ( e . g . , face - to - face interactions , serendipitous conversations , etc . ) . 6 . Conclusions The paper provides novel empirical evidence groun - ded in an original theoretical framework to explain why colocation matters for the rate , direction , and quality of scienti\ufb01c collaboration . To address endo - geneity concerns due to selection into colocation and matching , I exploit the constraints imposed on the spa - tial allocation of labs on the Jussieu campus of Paris by the removal of asbestos from its buildings . The analyses highlight under which conditions search and joint execution costs are more likely to be responsible for the patterns observed in the data . Consistent with recent experimental ( Boudreau et al . 2017 ) and observational ( Kabo et al . 2014 ) evidence , search frictions can be a substantial obstacle to col - laboration even within the boundaries of the same Catalini : Microgeography and the Direction of Inventive Activity 4362 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) institution : labs that become colocated because of the moves are 3 . 5 times more likely to collaborate with each other . Supporting the search costs hypothesis , e\ufb00ects are driven by pairs that are more likely to face higher search costs ex ante , such as labs that do not work on similar topics and that do not cite the same lit - erature . Furthermore , separating previously colocated labs does not lead to a decay in collaboration , which is consistent with past exposure allowing scientists to compensate for distance through temporary coloca - tion and remote interactions , as well as through inven - tive and organizational networks ( Crescenzi et al . 2016 , Monge et al . 1985 , Breschi and Lissoni 2004 , Singh 2005 , Azoulay et al . 2011 ) . At the same time , over long periods of time , search costs seem to a\ufb00ect the research trajectories of sep - arated scientists : while colocated labs grow increas - ingly similar in topics and literature cited , separated ones undertake less correlated research trajectories . Ironically , by embarking on di\ufb00erent paths , labs may be setting the stage for future , high - impact idea recombinations . Lower execution costs under colocation , by endoge - nously allowing scientists to improve ideas more e\ufb03 - ciently when proximate , also have an e\ufb00ect on the quality of inventive outcomes . Whereas search costs profoundly in\ufb02uence who a scientist is more likely to collaborate with , joint execution costs appear to shape the distribution of outcomes , conditional on col - laboration . Consistent with the theoretical framework , after colocation , more collaborations are observed on both tails of the outcome distribution . The high - impact collaborations that emerge are predominantly coming from lab pairs that faced higher search costs ex ante : this points to potential gains from connecting labs within an institution that may not overlap directly through other channels , as these labs may bring novel ideas to domain - speci\ufb01c research agendas . Results are also con - sistent with past research that has shown that diverse inventive teams are correlated with novel idea recom - bination ( Singh and Fleming 2010 ) ; that breakthrough , Nobel Prize contributions are correlated with scientists being embedded in di\ufb00erent communities of research at the time of their development ( Ham and Weinberg 2016 ) ; and that winning solutions to innovation prob - lems tend to come from solvers from a di\ufb00erent \ufb01eld of technical expertise ( Jeppesen and Lakhani 2010 ) . The \ufb01ndings also point out some of the strategic trade - o\ufb00s that the spatial allocation of teams entails . By optimizing space based on current beliefs of where opportunities are , organizations are making a strategic choice that will profoundly shape their R & D trajectory . Space acts as a powerful layer of incentives , which can be used to de\ufb01ne not only the intensity of interactions , but also their quality . Organizations that either need to move away from a declining trajectory or want to explore radically novel opportunities can colocate pre - viously separated teams to encourage serendipitous ( and planned ) conversations between individuals with di\ufb00erent priors , ideas , and knowledge . When coloca - tion is not an option , other forms of temporary colo - cation could be strategically used to compensate for the lower chance of an interaction and higher search costs . Interestingly , once individuals are aware of each other , proximity plays a lesser role , and collaboration can also be sustained over distance . At the same time , this seems to come at the cost of right - tail outcomes , a result that future research may be able to unpack fur - ther , and that advances in communication technology and virtual reality may be able to undo ( e . g . , by recre - ating the bene\ufb01ts of in - person , face - to - face interactions and serendipitous conversations ) . The moves on the Jussieu campus , by injecting exo - genous variation into a process otherwise optimized by scienti\ufb01c \ufb01elds , also highlight how scienti\ufb01c com - munities , also because of endogenous space alloca - tion , can become an obstacle to breakthrough research . While it may not be optimal for an institution to relo - cate scientists to overcome these barriers , 37 forms of temporary colocation ( e . g . , joint conferences ) could be strategically used to encourage cross - pollination across disciplines . Since Marshall\u2019s seminal work ( Marshall 1890 ) on localization economies , scholars have been interested in why colocation matters for the generation of new ideas . While we do know that the spatial allocation of inventors and scientists has an impact on the di\ufb00usion of information and ultimately on innovation , we still know surprisingly little about the micro - foundations of knowledge recombination . This study is a \ufb01rst step toward helping us understand the mechanisms at work at di\ufb00erent levels of distance . Acknowledgments The author thanks Ajay Agrawal , Ashish Arora , Pierre Azoulay , Alessandro Bonatti , Kevin Boudreau , Je\ufb00 Furman , Alberto Galasso , Alfonso Gambardella , Patrick Gaule , Avi Goldfarb , Matt Grennan , Nicola Lacetera , Karim Lakhani , Mara Lederman , Chris Liu , Hong Luo , Liz Lyons , Alex Oettl , Peter Thompson , Carlos Serrano , Tim Simcoe , Olav Soren - son , Paula Stephan , Scott Stern , Jane Wu , the associate editor and referee team , seminar participants at MIT , the Univer - sity of Toronto , Bocconi University , Boston University , the Fox School of Business , Harvard Business School , IESE Busi - ness School , London Business School , the London School of Economics , the Krannert School of Management , the Scheller College of Business , SMU Cox School of Business , Universitat Pompeu Fabra , the Yale School of Management , Wilfrid Lau - rier University , the Academy of Management , the Association of American Geographers 2012 Annual Meeting , the the 2011 Economics of the Creative Economy conference , and Colle - gio Carlo Alberto ( Turin ) for comments . Mia Rozenbaum , Taranjit Singh , and Consuela - Emilia Tataru provided excel - lent research assistance . All errors remain the author\u2019s own . Catalini : Microgeography and the Direction of Inventive Activity Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) 4363 Endnotes 1 This assumes that inventive outcomes are skewed ( i . e . , most ideas are of very low quality ) , that ideas need to be of su\ufb03cient quality to be published ( minimum e\ufb00ort level ) , and that applying more e\ufb00ort improves the quality of a research project . 2 Additional , more strict European regulation was published in 2003 ( directive 2003 / 18 / EC ) , 2004 ( directive 2004 / 37 / EC ) , 2007 ( 2007 / 30 / EC ) , and 2009 ( 2009 / 148 / EC ) . 3 Five or more years after becoming neighbors , labs are 20 % more similar in terms of the references they cite . 4 This is consistent with past research that has shown that social proximity can compensate for geographic distance ( Agrawal et al . 2006 , Sorenson et al . 2006 ) . 5 For example , Chai ( 2014 ) \ufb01nds that participation in the same con - ference is positively correlated with future collaboration . 6 Additionally , if the arrival rate of ideas is higher under colocation because of lower search costs ( e . g . , serendipitous interactions during low - opportunity cost time ) , this e\ufb00ect would be ampli\ufb01ed . 7 See the Agrawal et al . ( 2016 ) model of slack time and innovation for a detailed description . In the paper , the authors assume that idea quality follows an exponential distribution , that the cost function is convex in e\ufb00ort , and that there is a minimum e\ufb00ort requirement for an idea to be developed . 8 http : / / www . upmc . fr / en / university . html ( accessed April 2 , 2016 ) . 9 Seehttp : / / www . ipubli . inserm . fr / handle / 10608 / 20 ( accessedApril2 , 2016 ) . 10 Additional , more strict European regulation was published in 2003 ( directive 2003 / 18 / EC ) , 2004 ( directive 2004 / 37 / EC ) , and 2007 ( 2007 / 30 / EC ) and 2009 ( 2009 / 148 / EC ) . 11 Some of the labs are placed in temporary sites . 12 For example , in 1997 , 38 . 9 % of pairs that experience a change in colocation are within sub\ufb01elds , and 61 . 1 % across sub\ufb01elds ; in 1998 the opposite is true , with 69 . 7 % within and 30 . 3 % across ; in 1999 , 89 . 5 % is across , 10 . 5 % is within ; in 2000 all pairs are across ; in 2001 , 66 . 6 % are across and 33 . 4 % are within ; in 2002 the opposite is true , with 68 . 8 % within and 31 . 2 % across , etc . 13 https : / / www . elsevier . com / solutions / scopus / content ( accessed July 6 , 2017 ) . 14 The initial set of a\ufb03liations is \ufb01rst run through a script that divides the strings into their main components ( i . e . , department name , insti - tute name , lab name , building name , \ufb02oor , address , zip code , city , and country ) . Normalized lists are then created for the key labs , institutes , and departments , so that each string can be mapped to a uni\ufb01ed name . Eighty - seven percent of a\ufb03liation strings are matched to a UPMC lab or entity , resulting in a \ufb01nal set of 36 , 822 a\ufb03liation strings . 15 Five thousand eight hundred forty - seven unique a\ufb03liation in - stances . Location information is extracted by searching for building numbers , names , and their potential abbreviations , and then manu - ally checked to con\ufb01rm accuracy . 16 See http : / / www . epaurif . fr / documentation / transferts ( accessed May 1 , 2015 ) . 17 Geodesic distances are calculated using a method developed by Thaddeus Vincenty and implemented in Stata by Nichols ( 2003 ) . 18 The choice of this level of analysis is driven by data constraints , as \ufb02oor - level data is not always available , particularly for early years . Same - \ufb02oor - level estimates for colocation ( within the subsample with \ufb02oor information ) are typically larger ; hence , the same tower and corridor building e\ufb00ects probably constitute a lower bound of the true e\ufb00ects of microgeography . 19 For index keywords , according to Scopus : \u201cA team of professional indexers assigns index terms to records according to the following controlledvocabularies ( inadditiontokeywordssuppliedbyauthors themselves ) : GEOBASE Subject Index ( geology , geography , earth and environmental sciences ) , EMTREE ( life sciences , health ) , MeSH ( life sciences , health ) , FLX terms , WTA terms ( \ufb02uid sciences , textile sci - ences ) , Regional Index ( geology , geography , earth and environmen - tal science ) , Species Index ( biology , life sciences ) , Ei Thesaurus ( con - trolled and uncontrolled terms ) ( engineering , technology , physical sciences ) . \u201d Source : http : / / info . sciencedirect . com / scopus / scopus - in - detail / content - coverage - guide / metadata ( accessed May 1 , 2015 ) . 20 The Scikit - Learn python module ( http : / / scikit - learn . org / stable / modules / metrics . html ) is used to calculate the L2 - normalized dot product of the two vectors of keywords x and y as cosinesimi - larity ( x , y ) (cid:3) xy T / (cid:107) x (cid:107)(cid:107) y (cid:107) . Seealsohttp : / / nlp . stanford . edu / IR - book / html / htmledition / dot - products - 1 . html ( accessed May 1 , 2015 ) . 21 If the two vectors were identical , then the cosine similarity would be equal to 1 . 22 In an OLS regression that includes lab - pair \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . Results are robust to excluding the \ufb01xed e\ufb00ects . 23 As a comparison , the expansion of a campus would not o\ufb00er the same degree of exogenous variation as labs would have more in\ufb02uence over the allocation of the new space , and the expansion itself could be the result of promising research being conducted on campus . 24 In other words , it does not simply rely on a single shock , but on multiple moves staggered in time . 25 But can also be the number of collaborations or key statistics linked to the type and quality of papers that emerge ( e . g . , citations , similar - ity in keyword or cited references space ) . 26 Descriptive statistics at this level of analysis are reported in Online Appendix Table A - 1 . 27 The baseline is any year more than 10 years away from the move . 28 In the appendix , robustness is provided by building the same \ufb01g - ure with the number of collaborations as the dependent variable ( Online Appendix Figure A - 2 ) , and by ignoring the \ufb01ve most recent years of moves in the data set . Whereas moves on the campus con - tinued until 2014 ( and a few are still taking place in 2016 ) , the data used in this paper cover 1980 \u2013 2010 . When estimating the e\ufb00ects , Online Appendix Figure A - 3 limits the sample to moves between 1997 and 2005 . 29 Empirically , it is important to highlight that separated lab pairs were more likely to be related both in knowledge space and research agendas ( since the campus was optimized to minimize distance by \ufb01eld before the moves started ) \u2014i . e . , these pairs are also more likely to overlap in other circumstances ( e . g . , teaching , conferences ) and have a higher baseline risk of collaboration . 30 Part of the noise in the result could be due to crowding out taking place for some of these pairs as labs shift part of their collaboration portfolios toward their new neighbors , as described in the lab - level regressions . 31 OLS regression with lab - pair \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . Error bars represent 95 % con\ufb01dence intervals based on robust stan - dard errors clustered at the lab - pair level . 32 OLS regression with lab - pair \ufb01xed e\ufb00ects and year \ufb01xed e\ufb00ects . Error bars represent 95 % con\ufb01dence intervals based on robust stan - dard errors clustered at the lab - pair level . 33 The e\ufb00ect is close in timing to the rise in collaboration observed in Figure 1 , which suggests that labs become more similar mostly through purposeful interactions . At the same time , it becomes pos - itive and signi\ufb01cant substantially earlier , which is consistent with at least some interactions predating collaboration or taking place in the absence of collaboration . In the online appendix , similar graphs show that colocated labs are also more likely to start publishing in a journal that is new to them but not to their collaborator ( Online Catalini : Microgeography and the Direction of Inventive Activity 4364 Management Science , 2018 , vol . 64 , no . 9 , pp . 4348 \u2013 4364 , \u00a92017 The Author ( s ) Appendix Figure A - 4 ) and exhibit increasing overlap in the key - words they use outside of direct collaborations ( Online Appendix Figure A - 5 ) . 34 The estimated coe\ufb03cient for the separation line at t \u2212 2 and t \u2212 1 is positive and signi\ufb01cant , which is consistent with the moves sepa - rating lab pairs that were actually bene\ufb01ting from colocation in the pre - period . 35 Citation data were obtained from Scopus in 2016 , and quartiles for the citation distribution are built by year using a large sample of articles in the relevant \ufb01elds of science . 36 An analysis on the subsample of data for which \ufb02oor - level infor - mation is available shows e\ufb00ects roughly twice as large as those measured in Table 5 for labs that not only share the same building , but also the same \ufb02oor . 37 Results on collaboration portfolios highlight how labs actually became more inward focused during the moves , potentially because of the worsened set of neighboring labs relative to their preferences . 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Science 316 ( 5827 ) : 1036 \u2013 1039 .", + "kyumurkov2023force": "ARTICLE Force tuning through regulation of clathrin - dependent integrin endocytosis Alexander Kyumurkov 1 * , Anne - Pascale Bouin 1 * \ue840 , Mathieu Boissan 2 , 3 , Sandra Manet 1 , Francesco Baschieri 4 \ue840 , Mathilde Proponnet - Guerault 1 , Martial Balland 5 \ue840 , Olivier Destaing 1 \ue840 , Myriam R\u00b4egent - Kloeckner 1 , Claire Calmel 2 , 3 , Alice Nicolas 6 \ue840 , Fran\u00e7ois Waharte 2 , 3 , Philippe Chavrier 7 \ue840 , Guillaume Montagnac 4 \ue840 , Emmanuelle Planus 1 * * \ue840 , and Corinne Albiges - Rizo 1 * * \ue840 Integrin endocytosis is essential for many fundamental cellular processes . Whether and how the internalization impacts cellular mechanics remains elusive . Whereas previous studies reported the contribution of the integrin activator , talin , in force development , the involvement of inhibitors is less documented . We identified ICAP - 1 as an integrin inhibitor involved in mechanotransduction by co - working with NME2 to control clathrin - mediated endocytosis of integrins at the edge of focal adhesions ( FA ) . Loss of ICAP - 1 enables \u03b2 3 - integrin - mediated force generation independently of \u03b2 1 integrin . \u03b2 3 - integrin - mediated forces were associated with a decrease in \u03b2 3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA . The decrease in \u03b2 3 integrin dynamics correlated with a defect in integrin endocytosis . ICAP - 1 acts as an adaptor for clathrin - dependent endocytosis of integrins . ICAP - 1 controls integrin endocytosis by interacting with NME2 , a key regulator of dynamin - dependent clathrin - coated pits fission . Control of clathrin - mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA . Introduction Adhesive receptors , most notably integrins , help cells to perceive their microenvironment by sensing chemical , physical , and mechanical cues of extracellular matrix ( ECM ) through adhesive machineries called focal adhesions ( FA ) , which act in concert with the actomyosin - based contractility system ( Albiges - Rizo et al . , 2009 ; Engler et al . , 2006 ) . Actomyosin - mediated con - tractility is a highly conserved mechanism for generating me - chanical stress and governing cell shape , cell migration , cell differentiation , and morphogenesis ( Murrell et al . , 2015 ) . Cel - lular contractility usually proceeds with the engagement of \u03b1 5 \u03b2 1 and \u03b1 v \u03b2 3 integrins in the context of cells exploring fibronectin ( Fn ) - based microenvironments ( Schiller et al . , 2013 ) . These two integrins cooperate for the fine tuning of FA lifetime and adhe - sion strength in response to mechanical forces ( Kuo et al . , 2011 ; Schiller et al . , 2013 , 2011 ; Zamir et al . , 2000 ; Rossier et al . , 2012 ; Roca - Cusachs et al . , 2009 ; Milloud et al . , 2017 ; De Mets et al . , 2019 ) . Reinforcement of FA and cell contractility is likely coupled to the inhibition of FA disassembly , a processes involving FA targeting by microtubules , enhanced integrin endocytosis , calpain - mediated cleavage of talin , and loss of tension upon Rho kinase inhibition ( Wehrle - Haller , 2012 ) . \u03b1 5 \u03b2 1 and \u03b1 v \u03b2 3 integrins are functionally distinct , yet it remains unclear how these two Fn - binding integrin receptors are coordinately regulated to or - chestrate assembly or disassembly of adhesion sites , to adjust adhesion lifetime , to adapt adhesion strength and force to the cellular environment . The regulation of integrin function can be achieved at several levels , including ECM ligand engagement and recruitment of intracellular adaptors including integrin activators such as talin and integrin inhibitors like ICAP - 1 . These intracellular adaptors control integrin clustering and their activation switch , which is crucial for modulating integrin ligand - binding affinity and for serving as nucleation hubs for the assembly of larger signaling and structural scaffolds linked to actomyosin fibers ( Legate and . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 University Grenoble Alpes , INSERM 1209 , CNRS UMR5309 , Institute for Advanced Biosciences , Grenoble , France ; 2 University Sorbonne , INSERM UMR _ S 938 , Saint - Antoine Research Center , CRSA , Paris , France ; 3 Laboratory of Biochemistry and Hormonology , Tenon Hospital , AP - HP , Paris , France ; 4 Inserm U1279 , Gustave Roussy Institute , Universit\u00b4e Paris - Saclay , Villejuif , France ; 5 Laboratoire Interdisciplinaire de Physique , UMR CNRS 5588 , University Grenoble Alpes , Grenoble , France ; 6 University Grenoble Alpes , CNRS , CEA / LETIMinatec , Grenoble Institute of Technology , Microelectronics Technology Laboratory , Grenoble , France ; 7 Institut Curie , UMR144 , Universit\u00b4e de Recherche Paris Sciences et Lettres , Centre Universitaire , Paris , France . S . Manet died in January 2021 . * A . Kyumurkov , and A . - P . Bouin contributed equally to this paper ; * * E . Planus , and C . Albiges - Rizo contributed equally to this paper . Correspondence to Emmanuelle Planus : mailto : emmanuelle . planus @ univ - grenoble - alpes . fr ; Corinne Albiges - Rizo : corinne . albiges - rizo @ univ - grenoble - alpes . fr Myriam R\u00b4egent - Kloeckner \u2019 s present address is CY Cergy Paris Universit\u00b4e , Cergy , France . \u00a9 2022 Kyumurkov et al . This article isdistributedunder the termsof an Attribution \u2013 Noncommercial \u2013 Share Alike \u2013 NoMirror Sites license for the first six months after the publication date ( see http : / / www . rupress . org / terms / ) . After six months it is available under a Creative Commons License ( Attribution \u2013 Noncommercial \u2013 Share Alike 4 . 0 International license , as described at https : / / creativecommons . org / licenses / by - nc - sa / 4 . 0 / ) . Rockefeller University Press https : / / doi . org / 10 . 1083 / jcb . 202004025 1 of 19 J . Cell Biol . 2023 Vol . 222 No . 1 e202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 F\u00a8assler , 2009 ) . Whereas talin is known to be important for building actin - bound mechanosensitive adhesive complexes ( Klapholz and Brown , 2017 ; Shattil et al . , 2010 ) , the contribution of integrin inhibitors such as ICAP - 1 in force transmission is still elusive . ICAP - 1 does not localize in FA , but it can accumulate at small adhesion sites or tips of filopodia ( Jacquemet et al . , 2019 ; Fournier et al . , 2002 , 2005 ; Millon - Fr\u00b4emillon et al . , 2008 ) . Thus , ICAP - 1 interacts with partners described to be within and out - side FA ( Fournier et al . , 2002 ; Millon - Fr\u00b4emillon et al . , 2008 ) . ICAP - 1 interacts with the membrane - distal NPXY motif in the cytoplasmic tail of \u03b2 1 integrin through a phosphotyrosine binding ( PTB ) domain ( Zhang and Hemler , 1999 ; Degani et al . , 2002 ) . Further , it binds to the nucleoside diphosphate kinase NDPK / NME2 / NM23 - H2 ( Fournier et al . , 2002 ) . Of note , ICAP - 1 , NME2 , and \u03b2 1 integrin are colocalized at the cell leading edge during the early stage of cell spreading on Fn , thereby suggesting a transitory function of this complex in adhesion site dynamics ( Fournier et al . , 2002 ) . However , the biological relevance of ICAP - 1 / NME2 interaction in cell adhesion remains unknown . We have shown that ICAP - 1 is a determinant for controlling FA dynamics since it impedes FA assembly by limiting both talin and kindlin interaction with \u03b2 1 integrin ( Millon - Fr\u00b4emillon et al . , 2013 ; Millon - Fr\u00b4emillon et al . , 2008 ) . Furthermore , the physical inter - action of ICAP - 1 with the \u03b2 1 integrin tail leading to the modula - tion of \u03b2 1 integrin affinity state is required for down regulation of FA assembly . We have shown that ICAP - 1 is involved in cell mechanical properties and cell differentiation in a \u03b2 1 integrin - dependent manner ( Brunner et al . , 2011 ; Faurobert et al . , 2013 ; Bouvard et al . , 2007 ; Renz et al . , 2015 ; Millon - Fr\u00b4emillon et al . , 2008 ) . We have also proposed ICAP - 1 as a key modulator of cellular mechanoresponse in a \u03b2 1 - integrin \u2013 independent manner ( Bouin et al . , 2017 ) . To delve deeper into the ICAP - 1 func - tional properties , we interrogated \u03b2 1 - integrin \u2013 dependent and \u2013 independent contributions of ICAP - 1 to the cell mechanical response . Using cellular models genetically engineered to lack only \u03b2 1 integrin or ICAP - 1 or both \u03b2 1 integrin and ICAP - 1 in conjunction with quantitative traction force microscopy , in - tegrin dynamics , and proximity ligation assays , we provide converging evidence that points to a control by ICAP - 1 of clathrin - mediated \u03b2 3 integrin endocytosis and crucial tuning of cell mechanoresponses . Results The loss of ICAP - 1 enables \u03b2 3 - integrin - mediated force generation independent of \u03b2 1 integrin To understand to what extent ICAP - 1 regulates forces , we de - signed a quantitative traction force microscopy analysis of os - teoblasts deleted for \u03b2 1 integrin and / or ICAP - 1 . Deletion of \u03b2 1 integrin or ICAP - 1 in the respective cell lines was confirmed by Western blot ( Fig . S1 , A \u2013 C ) . Based on qPCR experiments and Western blot analysis ( Fig . S1 , D and E ) , we checked that the combined deletion of \u03b2 1 integrin and ICAP - 1 did not affect the total expression of \u03b2 3 integrin . Cells were seeded on Fn - coated polyacrylamide hydrogels ( PA hydro gels ) with a Young \u2019 s mo - dulus ( E ) of 5 kPa as previously described ( Bouin et al . , 2017 ) . First , we observed that osteoblasts lacking \u03b2 1 integrin ( \u03b2 1 integrin \u2013 / \u2013 - icap - 1 + / + ) were defective in force generation ( Fig . 1 , A and B ) similarly to what was reported for other cell lines ( Danen et al . , 2002 ; Schiller et al . , 2013 ; Milloud et al . , 2017 ) . The \u03b2 1 integrin - deficient cell line developed only 50 % of the total forces generated by \u03b2 1 integrin + / + - icap - 1 + / + cells ( Fig . 1 B ) . Secondly and unexpectedly , we found that additional loss of ICAP - 1 in \u03b2 1 integrin - deficient osteoblasts restored the traction force potential ( Fig . 1 , A and B ) . This result was confirmed by the rescue of myosin light - chain phosphorylation ( pp - MLC ) as judged by immunofluorescence staining ( Fig . 1 , C \u2013 E ; and Fig . S1 F ) and Western blot analysis ( Fig . S1 G ) using phospho site - specific antibodies against myosin light chain . In addition , while \u03b2 1 integrin \u2013 / \u2013 cells displayed limited spreading as evidenced by the projected area of actin cytoskeleton , normal cell spreading was rescued in the double - mutant ( \u03b2 1 integrin \u2013 / \u2013 - icap - 1 \u2013 / \u2013 ) cells that typically assembled thick actin stress fibers highly deco - rated with ppMLC ( Fig . 1 , C \u2013 E ; and Fig . S1 F ) . Of note , the loss of ICAP - 1 increased slightly force developed by osteoblasts al - though the difference with control cells was non - significant ( Fig . 1 , A and B ) . Contractility is an important aspect of cell migration , thus set out to explore the role of ICAP - 1 in the ad - aptation of cell speed as a function of substrate stiffness . Cells were seeded on Fn - coated PA hydrogels of increasing rigidity and imaged for 3 h to monitor migration velocity ( Fig . S2 A ) . \u03b2 1 integrin + / + - icap - 1 + / + and \u03b2 1 - deficient cells showed a stiffness - dependent increase in cell velocity . However , additional loss of ICAP - 1 uncoupled the correlation between cell speed and substrate rigidity . As the first steps of Fn fibrillogenesis are known to de - pend on actomyosin contractility ( Wu et al . , 1995 ) , we investigated whether the traction force observed in \u03b2 1 integrin \u2013 / \u2013 - icap - 1 \u2013 / \u2013 cells was correlated with Fn fibrillogenesis . As previously described ( Danen et al . , 2002 ) , the lack of \u03b2 1 integrin drastically impaired the formation of a stretched meshwork of Fn fibrils ( Fig . 1 , F and G ) . However , additional loss of ICAP - 1 , which was associated with increased traction forces ( Fig . 1 A ) , rescued the formation and organization of thick and numerous Fn fibrils connecting to large \u03b2 3 integrin adhesions as compared to the short and less dense Fn fibrils observed in the case of \u03b2 1 integrin deletion alone ( Fig . 1 , F and G ) . These results indicate that ICAP - 1 plays a role in cellular mechanotransduction and that traction strength on a compliant Fn substrate does not strictly correlate with the presence of \u03b2 1 integrin and is likely related to another integrin receptor regulated by ICAP - 1 . As Fn - coated surface mediates RGD binding to \u03b1 5 \u03b2 1 and \u03b1 v \u03b2 3 integrins ( Leiss et al . , 2008 ) which can exert both specific and redundant functions ( Ballestrem et al . , 2001 ; Danen et al . , 2002 ) , we investigated whether the increase in traction forces in \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells was dependent on \u03b2 3 integrin engagement . Silencing of \u03b2 3 integrin by RNA in - terference ( Fig . 2 F ) not only led to a significant decrease in cell spreading and ppMLC staining ( Fig . 2 , A and B ) but also abol - ished traction forces generated in \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells when compared to \u03b2 1 integrin + / + - icap - 1 + / + osteoblasts ( Fig . 2 , C and D ) . These data position \u03b2 3 integrin as a major driver for the spreading and tensile phenotypes observed on \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells . In line with this scheme , the contractile phenotype Kyumurkov et al . Journal of Cell Biology 2 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure 1 . Osteoblasts are able to exert traction force on fibronectin - coated substrate in the absence of \u03b2 1 integrin and ICAP - 1 . ( A and B ) Repre - sentative traction forces maps ( A ) and quantification ( B ) of the total force applied on fibronectin - coated polyacrylamide gel with a defined rigidity of 5 kPa . Kyumurkov et al . Journal of Cell Biology 3 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 of \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells was correlated with an increase in \u03b2 3 integrin expression at the cell surface as judged by FACS analysis ( Fig . 2 E ) while the total amount of \u03b2 3 in - tegrin remained unchanged ( Fig . S1 , D and E ) . Altogether , our results suggest a new regulatory role for ICAP - 1 in ac - tomyosin contractility and force generation through \u03b2 3 integrin . \u03b2 3 - integrin - dependent traction force is associated with redistribution and higher lifetime of enlarged \u03b2 3 - integrin - positive focal adhesions Stabilization of \u03b1 v \u03b2 3 integrin - Fn bonds through actomyosin - mediated tension is required for cells to adjust cell contractil - ity to the substrate stiffness ( Schiller et al . , 2013 ; De Mets et al . , 2019 ) . Based on the increase in surface \u03b2 3 integrin expression levels in \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells ( Fig . 2 E ) , we investigated whether the contractile behavior mediated by \u03b2 3 integrin upon the loss of \u03b2 1 integrin and ICAP - 1 might be related to a change in \u03b2 3 integrin - containing FA organization . All four osteoblast cells lines were able to form \u03b2 3 integrin FAs on an Fn - coated sub - stratum as revealed by immunostaining of \u03b2 3 integrin ( Fig . 3 A ) . Nevertheless , the deletion of \u03b2 1 integrin noticeably reduced cell spreading ( Fig . 1 D ) without affecting the adhesion area of FAs occupied by \u03b2 3 integrin even though it total adhesion area was increased with respect to the cell area ( Fig . 3 , A \u2013 C ) . These results suggest that \u03b2 3 integrin alone failed to support the typical spreading of osteoblasts bound to Fn . Remarkably , the additional loss of ICAP - 1 in \u03b2 1 - integrin - deficient cells restored cell spreading and increased the mean size of \u03b2 3 integrin FAs when compared to \u03b2 1 integrin null cells ( Fig . 3 , A and C ) . We noticed an increase of \u03b2 3 integrin FA translocation to the cell center ( Fig . S2 B ) which is compatible with the rescue of Fn fibril organization in \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells ( Fig . 1 , F and G ) . These results revealed a major role for ICAP - 1 in controlling \u03b2 3 integrin clustering and function in the absence of \u03b2 1 integrin . Next , we analyzed whether the contractile behavior mediated by \u03b2 3 in - tegrin upon the loss of \u03b2 1 integrin and ICAP - 1 might be linked to a change in \u03b2 3 - integrin - containing FA dynamics . To this end , the turnover of FA in osteoblast cell lines transfected with \u03b2 3 integrin - eGFP was monitored in real time ( Fig . 3 D ) . The life time of \u03b2 3 integrin - eGFP FAs increased in association with their translocation to the cell center in \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells ( Fig . 3 , D and E ; and Fig . S2 B ) . \u03b2 3 integrin exchange rates were further analyzed by fluo - rescent recovery after photobleaching ( FRAP ) using total inter - nal reflection fluorescent ( TIRF ) microscopy on osteoblasts lines transfected with \u03b2 3 integrin - eGFP ( Fig . 3 F ) . Neither single loss of \u03b2 1 integrin nor that of ICAP - 1 had any significantly effect on \u03b2 3 integrin exchange rate in FAs . In contrast , a threefold slower \u03b2 3 integrin - eGFP exchange rate typified the oversized \u03b2 3 in - tegrin FAs observed in double \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells ( Fig . 3 F ) , as compared to \u03b2 1 null cells . Finally , the slower \u03b2 3 integrin exchange rate was associated with an increase of \u03b2 3 integrin - eGFP FA life time ( Fig . 3 , D \u2013 F ; and Videos 1 , 2 , 3 , and 4 ) . In the context of \u03b2 1 null cells , these data hint that the loss of ICAP - 1 strongly affects the clustering and dynamics of \u03b2 3 in - tegrin in FAs . These findings reveal that the control of \u03b2 3 in - tegrin clustering and dynamics by ICAP - 1 might modulate traction force generation . \u03b2 3 - integrin - dependent contractility is associated with a defect of clathrin - mediated \u03b2 3 integrin endocytosis As endocytic membrane traffic regulates the availability of cell - surface receptors and associated signaling ( Ceresa and Schmid , 2000 ; Scita and Di Fiore , 2010 ) , we hypothesized that the con - comitant increase in \u03b2 3 integrin surface expression ( Fig . 2 E ) and the decrease in \u03b2 3 integrin dynamics in \u03b2 1 / ICAP - 1 double - mutant cells ( Fig . 3 , D \u2013 F ) might stem from a defect in \u03b2 3 integrin endocytosis . To test this hypothesis , we monitored the uptake of anti - integrin \u03b2 3 antibodies by cells plated on an Fn substrate using confocal microscopy . Uptake was measured using \u03b2 3 integrin - specific antibody ( LucA . 5 ) coupled with pH - rodo , which becomes fluorescent in the acidic environment of endo - cytic vesicles ( Fig . 4 A ) . Quantification of intracellular vesicles positive for internalized \u03b2 3 integrin ( LucA5 - positive ) revealed a significant decrease in uptake ( 23 % ) in the absence ICAP - 1 as compared to \u03b2 1 integrin + / + - icap - 1 + / + cells . An even more sig - nificant decrease ( 40 % ) was measured in osteoblasts deficient for both \u03b2 1 integrin and ICAP - 1 when compared to cells depleted in \u03b2 1 integrin only ( Fig . 4 A ) . Past studies have demonstrated that surface \u03b2 3 integrins are constitutively internalized by clathrin - mediated endocytosis that depends on the AP - 2 clathrin \u03b2 1 integrin KO cells exert less force than the other osteoblasts mutants . The additional deletion of ICAP - 1 led to generation of traction forces revealing a novel pathway independent of \u03b2 1 integrins to generate traction forces on fibronectin . Scale bar , 10 \u03bc m . Error bars represent SD . N \u2265 60 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( C ) Immunofluorescence staining of the ppMyosin ( ppMLC antibody , red ) and F - actin ( phalloidin , green ) in the four osteoblasts cell lines showed that deletion of ICAP - 1 alone does not change the organization of acto - myosin cytoskeleton but increases slightly the intensity and the thickness of the stress fibers ( see quantification of the ppMLC area in E ) . Deletion of \u03b2 1 integrin leads to a decrease of the cell area ( D ) and disorganization and decrease of thickness and number of the ppMLC decorated stress fibers . Scale bar , 20 \u00b5m . ( D ) Quantifiedcellspreading areafrom stainingoffluorescent F - actin . Error bars represent SD . N \u2265 429 cells . ns , adjustedP value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( E ) Quantified ppMLC staining , normalized to cell area . Error bars represent SD . N \u2265 211 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( F ) Osteoblasts cells were spread in serum - free medium on uncoated glass for 24 h . Immunofluorescence staining of the extracellular fibronectin ( cellular fibronectin antibody , red ) , F - actin ( phalloidin , blue ) and \u03b2 3 integrins ( Luc . A5 antibody , green ) inthe four osteoblasts cell lines were analyzedby fluorescent confocalmicroscopy . The \u03b2 1 + / + cell lines orchestrate FN fibrillogenesis events and \u03b2 1 integrin \u2212 / \u2212 / icap - 1 + / + cells demonstrated very poor organization of synthetized fibronectin . \u03b2 1 integrin \u2212 / \u2212 / icap - 1 \u2212 / \u2212 on the other side showed significant amount of FN fibrillogenesis . Scale bar , 20 \u00b5m . ( G ) FN fibrils length of thresholded images of deposited and organized FN fibrillogenesis were processed and quantified . 20 images per condition were analyzed . Quantifications reveal that the additional loss of ICAP - 1 in \u03b2 1 KO cell line increases the fibril length compared to the \u03b2 1 KO cell line , expressing ICAP - 1 . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . Kyumurkov et al . Journal of Cell Biology 4 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure 2 . ppMyosin area and traction force are dependent on \u03b2 3 integrin and ICAP - 1 expression . ( A ) \u03b2 1 integrin KO osteoblast cell lines were treated with \u03b2 3 integrin siRNA or scramble siRNA for 48 h and then let spread on FN coated glass for 24 h . Staining of myosin phosphorylation ( ppMLC antibody , red ) Kyumurkov et al . Journal of Cell Biology 5 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 adaptor complex ( Arjonen et al . , 2012 ; Yu et al . , 2015 ; Ezratty et al . , 2009 ) . Moreover , the large GTPase dynamins are known to be required for the scission of newly formed clathrin - coated vesicles ( CCV ) from the plasma membrane clathrin - coated pits ( CCP ) . We applied proximity ligation assay ( PLA ) , which allows the detection of close proximity , not only between ICAP - 1 and \u03b2 3 integrin ( Fig . 4 B ) but also between ICAP - 1 and CCP components such as the \u03b1 - adaptin of the clathrin adaptor complex , AP - 2 and dynamin - 2 ( Fig . 4 C ) , supporting a role for ICAP - 1 in \u03b2 3 integrin endocytosis . Indeed , the decrease of \u03b2 3 integrin endocytosis in the case of ICAP - 1 deletion was associated with an increase of CCP containing \u03b2 1 and \u03b2 3 integrins ( Fig . 4 D ) , specifying the contribution of ICAP - 1 in the formation of CCVs . Given the link between ICAP - 1 and clathrin - mediated endo - cytosis of \u03b2 3 integrin , we questioned whether the loss of clathrin might impact cell spreading , like the loss of ICAP - 1 . As expected , whereas \u03b2 1 integrin - deficient cells displayed a decrease in their spreading capacity compared to \u03b2 1 integrin + / + - icap - 1 + / + cells , additional knockdown of the clathrin heavy chain ( Fig . S3 ) re - stored their ability to spread ( Fig . 4 , E and F ) , in conjunction with the formation of larger \u03b2 3 integrin - positive FAs ( Fig . 4 , E and G ) and ppMLC - enriched actin stress fibers ( Fig . 4 , E and H ) , thus phenocopying the effects of the ICAP - 1 loss in those cells . Importantly , there was no additive effect of clathrin deletion over ICAP - 1 loss ( \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells and \u03b2 1 integrin + / + - icap - 1 \u2212 / \u2212 cells ) with respect to cell spreading , \u03b2 3 integrin - positive FA size and ppMLC staining ( Fig . 4 , E \u2013 H ) . These data demon - strate that the loss of clathrin and resulting impairment in clathrin - mediated endocytosis impacts \u03b2 3 integrin signaling . Accordingly , the increase in \u03b2 3 integrin clustering concurs with the increase in cell spreading and reorganization of the acto - myosin cytoskeleton . In addition , the non - sensitivity of cells devoid of ICAP - 1 to alteration of the clathrin - based endocytic machinery and the close proximity of ICAP - 1 with AP2 and dy - namin support a potential role of ICAP - 1 in clathrin - mediated integrin endocytosis . ICAP - 1 controls \u03b2 3 integrin endocytosis through NME - dependent scission of endocytic clathrin - coated pits Next , we investigated the mechanism by which ICAP - 1 might affect \u03b2 3 integrin endocytosis . We have previously reported an interaction between ICAP - 1 and nucleoside diphosphate kinase NME2 ( Fournier et al . , 2002 ) , known to catalyze the synthesis of nucleoside triphosphates including GTP from nucleoside diphosphates and ATP ( Boissan et al . , 2018 ) . Genetic and functional studies have demonstrated the ability of the related NME1 and NME2 NDPKs to fuel dynamin with GTP to support clathrin - dependent endocytosis ( Boissan et al . , 2014 ; Krishnan et al . , 2001 ; Dammai et al . , 2003 ; Nallamothu et al . , 2008 ) . Based on these findings , we investigated whether NME might impact \u03b2 3 integrin dynamics . As NME2 can work in a hexameric complex with NME1 to be recruited to CCPs through their physical interaction with dynamin ( Boissan et al . , 2014 ) , both NME1 and NME2 were knocked down in osteoblasts ( Fig . 5 A ) . Deletion of NME1 / 2 in \u03b2 1 - integrin - depleted osteoblasts de - creased \u03b2 3 integrin \u2013 GFP turnover as evidenced by FRAP ex - periments ( Fig . 5 B ) , thus recapitulating the deletion of ICAP - 1 . We further addressed whether ICAP - 1 might impact the locali - zation and function of NME2 before membrane scission . First , PLA showed that NME2 and \u03b1 - adaptin as well as dynamin ( Fig . 5 , C and D ) exist in close proximity to each other in agreement with the known functional link between NME and CCP ( Boissan et al . , 2014 ) . Secondly , PLA confirmed the close proximity between NME2 and ICAP - 1 in \u03b2 1 integrin + / + - icap - 1 + / + osteoblasts in contrast to ICAP - 1 \u2013 deficient cells ( Fig . 5 , C and D ) confirming the proximity of these two proteins as previously observed based on classical immunofluorescence ( Fournier et al . , 2002 ) . More importantly , we noticed a decrease of PLA signal between AP - 2 and NME2 and between dynamin and NME2 upon ICAP - 1 loss ( Fig . 5 , C and D ) , suggesting that ICAP - 1 acts as a linker to connect NME2 and CCP components . Similarly , we found that the proximity between NME2 and \u03b2 1 or \u03b2 3 integrin was disrupted in the absence of ICAP - 1 ( Fig . 5 , E and F ) , indi - cating the requirement for ICAP - 1 in keeping \u03b2 integrin and NME2 in close vicinity . Interestingly , ICAP - 1 loss had a negli - gible effect on transferrin receptor / NME2 PLA signal , confirm - ing the specificity of ICAP - 1 \u2019 s function in maintaining NME2 / integrin proximity ( Fig . 5 , E and F ) . In addition , we detected specific interactions between NME1 / 2 and ICAP - 1 , \u03b2 1 , and \u03b2 3 occurring at \u03b1 - adaptin \u2013 marked CCPs in \u03b2 1 integrin + / + - icap - 1 + / + osteoblasts ( Fig . S4 A ) . The functional link between integrin and NME was further supported by the distribution of NME / integrin complexes in the vicinity of vinculin - stained FAs ( Fig . 6 , A and B ) , showing a role for NME / integrin association close to adhesion sites . Indeed , for each proximity interaction , the fraction of hits was significantly higher ( N = 96 for \u03b2 1 integrin , P < 0 . 0001 ; N = 52 for \u03b2 3 integrin , P = 0 . 0014 ; Welch t tests ) than when using randomized images . This finding indicates that hits are unlikely due to stochastic distribution of duolink spots in the cell and thus suggests that a and \u03b2 3 integrin ( LucA . 5 , green ) was performed and analyzed by fluorescent confocal microscopy . Silencing the expression of \u03b2 3 integrin leads to the complete abolishment of the ppMLC - decorated stress fibers in the \u03b2 1 \u2212 / \u2212 / icap - 1 \u2212 / \u2212 and shrinkage of the cell area . Scale bar , 20 \u00b5m . ( B ) Quantification of the ppMLC area normalized to the cellular surface area after treatment with \u03b2 3 integrin siRNA ( si \u03b2 3 , clear colors ) or with scramble siRNA ( si Scr , dark colors ) . Customized particle analysis script from ImageJ was used after application of Unsharpen mask and Despecle filters . The error bars represent SD . N \u2265 42 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( C and D ) Representative traction forces maps ( TFM ) from \u03b2 1 integrin + / + - icap - 1 \u2212 / \u2212 and \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cell lines treated with \u03b2 3 integrin siRNA ( si \u03b2 3 , bottom panels ) or with scramble siRNA ( si Scr , upper panels ) and quantification ( D ) of the total force applied ( nN ) on fibronectin - coated polyacrylamide gel with a defined rigidity of 5 kPa . The additional silencing of \u03b2 3 integrins in the \u03b2 1 \u2212 / \u2212 / icap - 1 \u2212 / \u2212 cells decimates the traction forces , confirming that , in absence of \u03b2 1 integrin and ICAP - 1 , generation of strong cellular con - tractility at adhesion sites is dependent on \u03b2 3 integrins . Scale bar , 10 \u03bc m . Error bars represent SD . N = 80 cells . * * * * , P value \u2264 0 . 0001 . ( E ) FACS analysis of median fluorescence intensity of \u03b2 3 integrin on cell surface in \u03b2 1 integrin KO osteoblast cell lines . Error bars represent SD . ( F ) Western blot showing the efficiency of \u03b2 3 integrin silencing in \u03b2 1 integrin + / + - icap - 1 \u2212 / \u2212 and \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cell lines . Source data are available for this figure : SourceData F2 . Kyumurkov et al . Journal of Cell Biology 6 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure 3 . Size and dynamic of \u03b2 3 integrin FAs are dependent on \u03b2 1 integrins and ICAP - 1 . ( A ) Staining area of \u03b2 3 integrins was carried out on osteoblast cells spread on fibronectin - coated coverglass for 4 h using LucA5 antibody . Scale bar , 20 \u00b5m . ( B ) Quantification of the adhesive area of \u03b2 3 integrins FA , Kyumurkov et al . Journal of Cell Biology 7 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 correlation exists between duolink spots and spatial distribution of FAs . Whereas the presence of ICAP - 1 was necessary to maintain NME close to both \u03b2 1 and \u03b2 3 integrins ( Fig . 5 , E and F ) , \u03b2 1 integrin was not required for NME / \u03b2 3 integrin ( Fig . 6 C ) or NME / ICAP - 1 proximity ( Fig . S4 , B and C ) . Indeed , the proximity between \u03b2 3 integrin and NME was retained even in cells lacking \u03b2 1 integrin ( Fig . 6 , C and D ) . As NME2 is crucial for GTP - loading on dynamin ( Boissan et al . , 2014 ) , and dynamin function pre - cedes the transient recruitment of the clathrin uncoating protein auxilin immediately after CCP scission ( Massol et al . , 2006 ) , we analyzed whether inhibition of NME1 / 2 could affect the burst of auxilin recruitment , a hallmark of CCP scission ( Massol et al . , 2006 ) . Consistent with this hypothesis , recruitment of auxilin to CCPs was strongly impaired when NME2 and NME1 were de - leted ( Fig . S5 , A and B ) . In addition , like NME1 / 2 loss , the loss of ICAP - 1 in osteoblasts also led to a decrease of the auxilin burst at the rim of FA ( Fig . 6 , E and F ) . Altogether , these data indicate that ICAP - 1 is required for NME function at CCPs upstream of dynamin and auxilin - dependent steps to allow optimal forma - tion of clathrin - coated vesicles . The consequences of the inhi - bition of clathrin - dependent endocytosis on force generation were assessed by TFM after clathrin and NME1 / 2 knock - down in \u03b2 1 integrin \u2212 / \u2212 cells and \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells . While deletion of clathrin and NME mimicked ICAP - 1 deletion in \u03b2 1 integrin \u2212 / \u2212 cells cultured on a stiff substrate by rescuing stress fibers and P - myosin ( Fig . 4 , F \u2013 H ) and by decreasing \u03b2 3 integrin turnover ( Fig . 5 B ) , TFM experiments performed on soft gels ( 5 kPa ) did not reveal any significant change in force generation after blocking clathrin - mediated endocytosis in cells devoid of \u03b2 1 in - tegrin . These results reveal an additional activity of ICAP - 1 on \u03b2 3 integrin in soft environments that is not supported by NME and clathrin . In summary , ICAP - 1 regulates integrin endocytosis by con - trolling NME association to integrin - containing CCPs and is required for NME - dependent scission of CCPs . More impor - tantly , ICAP - 1 might regulate cellular force by controlling in - tegrin endocytosis through NME2 - control of dynamin function . Nevertheless , our results suggest that unlike clathrin and NME1 / 2 , ICAP - 1 plays an additional role to rescue force in \u03b2 1 integrin deleted cells cultured on a soft substratum . Discussion Integrin trafficking contributes to both cell movement and ad - hesion receptor signaling ( Caswell et al . , 2009 ; Moreno - Layseca et al . , 2019 ; Lock et al . , 2019 ) . Although essential for many cel - lular processes , the sequence of molecular events during clathrin - mediated integrin endocytosis remains elusive . Our present results implicate a critical contribution of the integrin inhibitor , ICAP - 1 , in cellular mechanotransduction by working in concert with NME2 NDPK to control clathrin - mediated en - docytosis of integrins at the edge of FAs . This new mechanism illustrates the importance of integrin inhibitors in mechano - sensing and mechanoresponsiveness ( Bouin et al . , 2017 ; Lerche et al . , 2020 ) . Mechanistically , ICAP - 1 acts as an adaptor protein in integrin endocytosis . ICAP - 1 mediates cargo selection by po - sitioning NME close to the integrin receptor , together with AP2 and dynamin in order to fuel dynamin at CCP allowing execution of vesicle fission to ensure integrin turnover ( Fig . 7 ) . It has been previously described that the cell mechanical state regulates clathrin coat dynamics at the plasma membrane ( Ferguson et al . , 2017 ; Baschieri et al . , 2018 ) . Our study provides further insights into the importance of controlling integrin turnover to limit the maturation of FAs and tune cell contractility . In addition , our results add another piece of evidence to the conclusion that both \u03b2 1 and \u03b2 3 integrins can control actomyosin contractility ( Roca - Cusachs et al . , 2009 ; Schiller et al . , 2013 ) . While our results clearly confirm the efficiency of \u03b2 1 integrin deletion to abolish traction forces , they also suggest the inefficiency of \u03b2 3 integrin to take over in the presence of ICAP - 1 . Thus , suppression of ICAP - 1 appears to be necessary to unlock a potential inhibition applied to \u03b2 3 integrin to permit its translocation to the cell center , generate cell contractile response and ensure Fn fibril - logenesis . Despite the main function of \u03b2 1 integrin in traction force and fibrillogenesis , \u03b2 3 integrin has already been reported to reinforce force ( Roca - Cusachs et al . , 2009 ) and to stimulate the initial steps of \u03b1 v \u03b2 3 - mediated Fn fibril formation ( Danen et al . , 2002 ) . Moreover , the ability of \u03b1 v \u03b2 3 integrin to drive Fn fibrillogenesis is supported by previous data demonstrating the role of integrin - \u03b1 v \u03b2 3 to trigger Fn assembly and CAF invasion ( Attieh et al . , 2017 ) . Our results demonstrate that ICAP - 1 and NME share the task of \u03b2 3 integrin endocytosis control to regulate integrin cell surface and cell tensional homeostasis . Clathrin - dependent trafficking of integrins is essential for FA disassembly , a process dictated by the intracellular domains of both \u03b1 and \u03b2 integrin subunits which control the nature of the ligand and the activation status of integrin ( Margadant et al . , 2011 ; De Franceschi et al . , 2016 ; Ezratty et al . , 2009 ; Arjonen et al . , 2012 ) . By targeting the cytoplasmic domain of \u03b2 integrin subunit , ICAP - 1 directly participates to FA disassembly ( Bouvard normalized to the cellular surface area . Error bars represent SD . N \u2265 208 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( C ) Quantification of the mean of \u03b2 3 integrin FAs area . The deletion of ICAP - 1 in \u03b2 1 deficient cell line ( grey box ) drives massive leap of \u03b2 3 integrinstained FAs size . Error barsrepresent SD . N \u2265 208 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( D ) Representative time series of the lifetime of eGFP - \u03b2 3 integrin FAs of the four osteoblastic cell lines . Asterix points out typical eGFP - \u03b2 3 integrin FAs in the cells with typical disassembly lifetime . Note the translocation of FA to the cell center in \u03b2 1 integrin \u2212 / \u2212 / icap - 1 \u2212 / \u2212 cells . Scale bar , 2 \u00b5m . ( E ) TIRF lifetime analysis on the eGFP - \u03b2 3 integrin FAs . The lifetime of the eGFP - \u03b2 3 integrin containing FA is increased in \u03b2 1 integrin \u2212 / \u2212 / icap - 1 \u2212 / \u2212 cells . Spinning disk videos of eGFP - \u03b2 3 integrin were taken for the duration of 2 h . The adhesion lifetime was analyzed by Focal Adhesion Analysis Server ( FAAS ; Berginski and Gomez , 2013 ) and verifiedvisually . Error bars represent SD . N \u2265 50focal adhesions . ns , adjustedP value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( F ) FRAP analysis on the eGFP - \u03b2 3 integrin FAs reveal that the \u03b2 3 integrin mobility at the plasma membrane is halted in the absence of ICAP - 1 and \u03b2 1 integrin . At least six FAs per cell lines were bleached and their recovery was monitored for 5 min . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . Kyumurkov et al . Journal of Cell Biology 8 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure 4 . The \u03b2 3 integrin dependent contractility is associated with the defect of \u03b2 3 integrin endocytosis . ( A ) The \u03b2 3 integrin uptake was measured using \u03b2 3 integrin specific antibody ( LucA . 5 ) , coupled with pH - Rhodo . The volume of the endocytotic vesicles per cell volume was measured at 37 and 4\u00b0C . Note the decrease of \u03b2 3 integrin endocytosis in cell lines deleted in ICAP - 1 . The quantification was performed after 45 min incubation . Error bars represent SD . N \u2265 127 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( B ) Representative images of PLA Kyumurkov et al . Journal of Cell Biology 9 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 et al . , 2003 ) . However , as supported by 2 - hybrid approaches and in vitro interaction assays ( Fournier et al . , 2002 ) , ICAP - 1 must also interact with NME2 in order to drive integrin internaliza - tion . Different approaches including proteomic studies have highlighted that NME form a complex with both \u03b2 1 and \u03b2 3 in - tegrin complexes on the one hand ( Kuo et al . , 2011 ; Schiller et al . , 2011 ) , and with AP2 ( Marino et al . , 2013 ) on the other hand , indicating a functional contribution of NME in dynamin - mediated scission of CCPs ( Boissan et al . , 2014 ) . By uncovering the impact of integrin internalization on force generation through ICAP - 1 / NME complex formation , we provide an addi - tional mechanistic facet of the otherwise multifunctional protein ICAP - 1 , which is known to regulate FA disassembly , cell con - tractility , Fn fibrillogenesis , and cell migration ( Millon - Fr\u00b4emillon et al . , 2008 ; Faurobert et al . , 2013 ; Bouin et al . , 2017 ; Lisowska et al . , 2018 ; Brunner et al . , 2011 ) . The versatility of ICAP - 1 needs to be considered to explain the lack of rescue of force generation upon clathrin or NME1 / 2 deletion in \u03b2 1 null cells on soft substrates . While \u03b2 1 null cells do not form large \u03b2 3 FAs and do not produce traction force when plated on a soft substrate ( TFM conditions ) , ICAP - 1 deletion releases the in - teraction site for kindlin which favors \u03b2 3 integrin clustering , a step required for efficient linkage of integrin to the actin cytoskeleton and the development of traction force ( Ye et al . , 2013 ) . Our assumption is that integrin internalization by the ICAP - 1 / NME1 complex in CCPs is spatially controlled and re - quires adhesion sites with minimal clustering of integrins to be effective . According to our experiments ( Figs . 4 , F \u2013 H and 5 B ) , clathrin and NME deletion mimics ICAP - 1 deletion in stiff conditions since substrate stiffness is enough to induce in - tegrin clustering ( Fig . 4 , F \u2013 H ) . Integrin clustering might be important to correctly position the CCP machinery . This hy - pothesis is supported by the distribution of NME / integrin complexes ( Fig . 6 , A and B ) and bursts of auxilin ( Fig . 6 , E and F ) in the vicinity of vinculin - stained FA . This result empha - sizes a role for the ICAP - 1 / NME / integrin trio close to adhesion sites in a stiffness - dependent manner . Of note , ICAP - 1 knock - out revealed a phenotype in bone which might support an important role of ICAP - 1 in stiff conditions ( Bouvard et al . , 2007 ) . So far it was unknown whether individual integrin classes like \u03b1 5 \u03b2 1 and \u03b1 v \u03b2 3 interacting with the same ligand like Fn are internalized using common or specific routes . Because the de - letion of ICAP - 1 led to an increase of both AP2 / \u03b2 1 and AP2 / \u03b2 3 complexes , the partnership between ICAP - 1 and NME2 likely contributes to both \u03b2 1 and \u03b2 3 internalizations . Concomitantly , the control of CCP scission by ICAP - 1 / NME complex directly regulates cell mechanics which has previously been shown to result from the cooperation between \u03b2 1 and \u03b2 3 integrin ( Schiller et al . , 2013 ) . However , we cannot exclude a compensatory effect between \u03b2 1 and \u03b2 3 integrins to generate force since the prox - imity between NME with ICAP - 1 and \u03b2 3 integrin is not condi - tioned by the presence of \u03b2 1 integrin . Importantly , many other endocytic accessory proteins or co - adaptors associate with integrins and CCPs ( Yap and Winckler , 2015 ) . Dab2 and Numb are PTB domain - containing proteins that accumulate at or near FA shortly before their disassembly ( Chao and Kunz , 2009 ; Nishimura and Kaibuchi , 2007 ; Ezratty et al . , 2009 ; Teckchandani et al . , 2009 ) . The physiological relevance of having many adaptors or co - adaptors like ICAP - 1 , Numb , or Dab2 in the same cell might respond either to integrin specificity or to physical properties of the microenvironment . As an illus - tration , Dab2 is not found at \u03b2 3 integrin - mediated FA sites formed on RGD - coated glass . In addition , it has been shown that the development of actomyosin contractility inhibits Dab2 binding to \u03b2 3 integrin . However the loss of cell \u2013 matrix force development is a key determinant for Dab2 binding to \u03b2 3 in - tegrin clusters and the ongoing endocytosis of \u03b2 3 integrin under soft conditions such as on mobile RGD membranes ( Yu et al . , 2015 ) . This suggests a specificity of some adaptors depending on the microenvironment stiffness . So far , no direct interaction between ICAP - 1 and \u03b2 3 integrin has been demonstrated , possibly due to the lack of proper post - translational modifications in classical pull - down assays . Moreover , the nature of this inter - action might be complexified by the requirement of a third partner to bridge \u03b2 3 integrin and ICAP - 1 . However , PLA ex - periments show proximity not only between \u03b2 3 integrin and ICAP - 1 but also between \u03b2 3 integrin and NME , even in the ab - sence of \u03b2 1 integrin . ICAP - 1 might take over for Dab2 in stiffer microenvironment as ICAP - 1 and Dab2 share the same distal performedwith antibodies against ICAP - 1and \u03b2 3 integrin . Red dots denote regions of signal amplification indicating the close proximity between ICAP - 1and \u03b2 3 integrin . PLA performed on ICAP - 1 deficient cell line is used as control . Nuclei are stained in blue with DAPI . ( C ) Representative images of proximity ligation assays performed with antibodies against ICAP - 1 and AP2 or Dynamin 2 . Red dots denote regions of signal amplification indicating that ICAP - 1 belongs to clathrin endocytosis machinery . PLA performed on ICAP - 1 deficient cell line is used as control . Nuclei are stained in blue with DAPI . Scale bar , 20 \u00b5m . ( D ) PLA performedwith antibodiesagainstAP2 and \u03b2 1 integrin or \u03b2 3 integrinand quantification of thenumber of PLAspots percellshowsthat deficiency inICAP - 1leads to increase of the association of \u03b2 1 and \u03b2 3 integrins with AP2 . N \u2265 25 cells / condition from three independent experiments . Scale bars , 20 \u00b5m . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( E ) Immunofluorescence staining of the phospho - myosin ( ppMLC antibody , red ) and \u03b2 3 integrin ( LucA . 5 antibody , green ) . Representative micrographs of the four osteoblast cell lines treated with clathrin siRNA ( si Clat , right ) or scramble siRNA ( si Scr , left ) . Inhibition of clathrin expression in \u03b2 1 integrin \u2212 / \u2212 / icap - 1 + / + cell line rescues cell spreading through \u03b2 3 integrin - mediated FA and development of acto - myosin cytoskeleton ( see also graphs F \u2013 H ) . Scale bar , 20 \u00b5m . ( F ) Quantification of cell spreading area of the four osteoblast cell lines treated with clathrin siRNA ( si Clathrin , light colors ) or scramble siRNA ( si Scr , dark colors ) . Error bars represent SD . N \u2265 27 cells . ns , adjustedP value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( G ) Quantification ofareaof \u03b2 3 integrincontaining FAs normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA ( si Clathrin , light colors ) or scramble siRNA ( si Scr , dark colors ) . Error bars represent SD . N \u2265 27 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( H ) Quantification of phospho - myosin staining area normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA ( si Clathrin , light colors ) or scramble siRNA ( si Scr , dark colors ) . Error bars represent SD . N \u2265 27 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . Kyumurkov et al . Journal of Cell Biology 10 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure 5 . ICAP - 1 is required for NME recruitment in clathrin - coated pits and for keeping the proximity between integrin and NME . ( A ) Western blot analysis showing the efficiency of SiNME1 / 2 in osteoblast cells . ( B ) TIRF / FRAP analysis shows that deletion of NME1 / 2 complex by siRNAs ( siNME1 / 2 , light Kyumurkov et al . Journal of Cell Biology 11 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 NPXY motif to interact with integrin . Regarding \u03b2 1 integrin endocytosis , Dab2 and clathrin are not found to activate the \u03b2 1 integrin clusters observed in cells upon adhesion on mobile RGD membranes ( Yu et al . , 2015 ) . This suggests that the integrin turnover might be regulated differentially with respect to the physical properties of the microenvironment , possibly involving ICAP - 1 and Dab2 in stiff and soft microenvironments , respec - tively . Whether specific mechanosensitive pathways and dis - tinct PTB domain integrin adapters are required to control integrin internalization or endocytic frustration ( Lock et al . , 2019 ; Baschieri et al . , 2018 ) , depending on the physical proper - ties of the microenvironment will be the next avenue to explore . As migratory and adhesive behavior is mediated by \u03b2 1 and \u03b2 3 integrin cooperation ( Schiller et al . , 2013 ) , the regulation of their respective intracellular trafficking in a coordinated manner might be likely essential for rapidly and efficiently adapting the responsiveness of migratory cells to extracellular guidance cues . Whether ICAP - 1 coordinates commonly \u03b2 1 and \u03b2 3 integrin en - docytic process to adapt integrin dynamics and traction force generation in a context - dependent manner is a pending question . Materials and methods Antibodies and chemicals Human plasma Fn was purchased from Sigma - Aldrich . Anti \u03b2 3 integrin antibody was purchased from Emfret ( Clone LucA . 5 , # M030 - 0 ) , the double phosphorylated ( T18 / S19 ) myosin light - chain antibody and clathrin antibody were obtained from Cell Signaling , # 3674 and # 4796 , respectively , and the unmodified myosin light - chain antibody and tubulin were purchased from Sigma - Aldrich , # M4401 and # T4026 , respectively . The poly - clonal anti - \u03b2 3 integrin was kindly provided by M . H . Ginsberg ( University of California San Diego , San Diego , CA ) . The Fn antibody was purchased from Millipore ( # AB2033 ) . The trans - ferrin antibody was purchased from Abcam ( # ab82411 ) . The HRP - conjugated antibodies were obtained from Jackson ImmunoResearch \u2014 F ( ab \u2019 ) \u2082 Anti - Rabbit HRP ( # 711 - 036 - 152 ) or anti - mouse IgG , light - chain HRP ( # 115 - 035 - 174 ) . The fluorescent secondary antibodies conjugated with AlexaFluor 488 ( # A - 11063 ) , AlexaFluor 546 ( # A - 11003 ) , or AlexaFluor 633 ( # A - 21053 ) were obtained from Thermo Fisher Scientific . Mouse monoclonal anti - NME2 was purchased from Kamiya Biomedical Company . Rabbit polyclonal pan - NME antibodies ( recognizing both NME1 and NME2 isoforms ) were prepared by affinity pu - rification using purified human recombinant NME1 and NME2 proteins coupled to NHS - activated HiTrap columns . Mouse monoclonal anti - ICAP - 1 \u03b1 antibodies ( 4D1D6 and 9B10 ) were prepared using recombinant His - tagged ICAP - 1 \u03b1 protein as an - tigen ( Fournier et al . , 2002 ) . Phalloidin coupled with Atto 647 was also purchased from Thermo Fisher Scientific ( # A22287 ) . Cell culture Immortalized osteoblasts from icap - 1 \u2212 / \u2212 ; \u03b2 1 integrin flox / flox mice were generated as described previously ( Bouvard et al . , 2007 ) . These cells were infected or not by adenoCre viruses from gene transfer vector core ( University of Iowa ) in order to ob - tain \u03b2 1 integrin - null cells . The icap - 1 null cells were incubated with retroviral particles to obtain rescued cells expressing ICAP - 1 WT . The cells were selected with 1 mg / ml puromycin to produce cell populations with heterogeneous ICAP - 1 expression levels . Cells were maintained in culture in DMEM ( # 31966 - 021 ; Life Tech - nologies ) supplemented with 10 % FBS ( # S1810 - 500 ; Dominique Dutscher ) , 100 U / ml penicillin , and 100 \u00b5g / ml streptomycin ( # P06 - 07100 ; PAN Biotech ) at 37\u00b0C in a 5 % CO 2 - humidified chamber . For all experiments , cells were washed with PBS ( # L0615 - 500 ; Dominique Dutcher ) , detached using trypsin ( # L0615 - 500 ; Dominique Dutcher ) , and treated with 1 mg / ml trypsin inhibitor ( # T6522 ; Sigma - Aldrich ) . Cells were then plated in DMEM containing 10 % FBS for 4 h and then the ap - propriate analysis was carried out . Where needed , a serum - free medium OptiMEM was used ( # 51985 - 026 ; Life Technologies ) as substitute . The four osteoblast clones \u2014 icap - 1 + / + ; icap - 1 \u2212 / \u2212 ; \u03b2 1 integrin floxed / floxed ; \u03b2 1 integrin floxed / floxed ; and icap - 1 \u2212 / \u2212 \u2014 were infected using lentiviral infection system from Invitrogen with pLenti \u2013 murine \u03b2 3 integrin - GFP vector . Western blotting analysis Cells were plated on 50 % confluence and left to spread over - night . The next day , the dishes were washed twice with ice cold PBS and lysed in cold RIPA buffer , supplemented with 1\u00d7 cOmplete protease inhibitors , 5 mM NaF , and 2 mM Na - orthovanadate . After protein quantification through Pierce BCA Protein Assay ( # 23227 ; Thermo Fisher Scientific ) , the samplers were mixed with Laemmli sample buffer ( 0 . 4 % SDS , 20 % glycerol , 120 mM Tris - Cl [ pH 6 . 8 ] , and 0 . 02 % [ w / v ] bro - mophenol blue ) and loaded on electrophoretic PAA gels . Fol - lowing the standard wet blotting protocol , the nitrocellulose colors ) or scramble siRNA ( si Scr , dark colors ) impedes the turnover of eGFP - \u03b2 3 integrins at the plasma membrane in \u03b2 1 integrin \u2212 / \u2212 / icap - 1 + / + cell line . Six FAs per cell were bleached for each experiment . eGFP - \u03b2 3 integrin recovery was monitored for 5 min . 19 cells \u2264 N \u2264 107 cells . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( C and D ) Representative images of PLAs ( C ) and PLA assay quantification ( D ) of the number of PLA spots per cell performed with antibodies against NME and ICAP - 1 or AP2 or Dynamin 2 . Red dots denote regions of signal amplification consistent with NME / ICAP - 1 interaction , NME / AP2 interaction , and NME / dynamin 2 interaction in \u03b2 1 integrin + / + - icap - 1 + / + osteoblast cells ( left ) . PLA performed on ICAP - 1deficient cell line isusedas control . Thedeletion ofICAP - 1induces a decreaseof red dotsinallthree cases indicating the crucial role of ICAP - 1 for keeping NME in clathrin endocytosis machinery ( right ) . Nuclei are stained in blue with DAPI . Scale bar , 20 \u00b5m . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( E and F ) Representative images ( E ) of PLAs with quantification ( F ) performed with antibodies against NME and \u03b2 1 integrin or \u03b2 3 integrin or transferrin receptor . Red dots denote regions of signal amplification consistent with NME / integrin proximity in \u03b2 1 integrin + / + - icap - 1 + / + osteoblast cells ( left ) . The deletion of ICAP - 1 induces a decrease of the number of red dots indicating the crucial role of ICAP - 1 for keeping NME and integrin vicinity ( right ) . PLA performed on ICAP - 1 deficient cell line is used as control . Nuclei are stained in blue with DAPI . Scale bar , 20 \u00b5m . Error bars represent SD . N \u2265 25 cells / condition from three independent experiments . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . Source data are available for this figure : SourceData F5 . Kyumurkov et al . Journal of Cell Biology 12 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure6 . RequirementofICAP - 1 for NME function inCCP upstreamofdynaminand auxillinsteps to allow optimal clathrin - coated vesicle budding in the vicinity offocal adhesion . ( A ) Highlyinclinedillumination fluorescence microscopy analysiswas performed at the basalfaceofadherentcell on FNcoated Kyumurkov et al . Journal of Cell Biology 13 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 membranes ( # 10600003 ; Amersham ) were probed with the appropriate primary antibodies , diluted in 5 % BSA in Tween - TBS ( TTBS ) , and incubated overnight . The membrane was sub - sequently incubated with the appropriate secondary antibodies , also diluted in 5 % BSA in TTBS for 1 h and then developed using Clarity ECL kit ( # 170 - 5061 ; Biorad ) and recorded with ChemiDoc Imaging System and analyzed with ImageLab software . Traction force microscopy The poly - acrylamide hydrogels with defined rigidity of 5 kPa and containing fluorescent microbeads ( # F8783 ; Life Technolo - gies ) were cast in 2 - well LabTeks ( # 154461 ; Thermo Fisher Sci - entific ) , coated with BindSilane ( # GE17 - 1330 - 01 ; Sigma - Aldrich ) and covered with coverslip , coated with Sigmacote ( # SL2 ; Sigma - Aldrich ) . After the polymerization of the polyacrylamide the wells were flooded with water and the coverslips were detached gently . For the functionalization a protocol from ( Przybyla et al . , 2016 ) was used . Briefly , solution of tetrame - thacrylate , N6 , and Irgacure was deposed on the gels and baked under UV light ( 312 nm ) for 5 min . Then , Fn ( 5 \u03bc g / ml ) was de - posed on the gels and incubated at 4\u00b0C overnight . Cells were allowed to adhere and spread 4 h in DMEM containing 10 % FBS and then placed in 4 % FBS . Just before the acquisition , the membrane was stained with red fluorescent membrane marker PKH26 ( # PKH26GL ; Sigma - Aldrich ) . Images were taken using spinning disk microscope iMIC EMCCD Andromeda ( FEI ) equipped with heating chamber , CO 2 installation , using 40\u00d7 magnification oil objective ( Plan - Apochromat 40\u00d7 / 1 . 4 Oil - DIC M27 WD 0 . 13 mm ) . A fluorescent image of the beads with the cell spread on and fluorescent image of the cell membrane was obtained . Then , the culture medium was replaced with pure solution of trypsin and after verification that cells were com - pletely detached the second image of the fluorescent beads were taken . Isolated cells were randomly chosen for each experi - mental condition . Force calculations were performed as previ - ously described ( Tseng et al . , 2011 ) . Briefly , the displacement fields describing the deformation of the PA substrate are de - termined from the analysis of fluorescent beads images before and after removal of the adhering cells with trypsin treatment . The displacement field is obtained by a two - step method con - sisting of particle image velocimetry followed by individual bead tracking ( Butler et al . , 2002 ; Sabass et al . , 2008 ) . A special procedure is used to evaluate displacements in the area of the adhesive pattern where gel deformation is expected to be largest . Depending on the pattern shape , traction forces may be strongly localized leading to large displacements in very small areas . In coverslide . Left panel shows cells immunostained for vinculin ( green ) and \u03b2 1 integrin / NME duolink signal ( PLA event , red ) , and the right panel shows cells stained for vinculin ( green ) and \u03b2 3 integrin / NME duolink signal ( PLA event , red ) . Scale bar , 5 \u00b5m . ( B ) Boxplot representation of the fraction of the hits es - timated for \u03b2 1 integrin / NME - vinculin and for \u03b2 3 integrin / NME - vinculin and for randomized images ( rand . ) . ( C and D ) Representative images of PLA with quantification performed withantibodies against NMEand \u03b2 3 integrin . Reddotsdenote regions of signalamplification revealingICAP - 1 / \u03b2 3 integrin proximity in \u03b2 1 integrin deficient cells . PLA performed on ICAP - 1 deficient cell line is used as control . Nuclei are stained in blue with DAPI . N \u2265 25 cells / condition from three independent experiments . Scale bar , 20 \u00b5m . ( E and F ) Spinning disk highly magnified video micrographs ( every 4 s ) of an FA , ( vinculin - green ) and auxillin bursts in red ( indicated by yellow arrow ) in \u03b2 1 integrin + / + - ICAP - 1 + / + cells . The quantification in F shows that ICAP - 1 deletion slows down the auxillin bursts / FA by 50 % . At least 35 FAs and 3 squares in at least 4 cells per condition were imaged in 3 independent experiments . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . Figure 7 . Recruitment of NME by ICAP - 1 to regulate cellular force by promoting inactive integrin internalization . Scheme representing the sequence of molecular events during clathrin - mediated integrin endocytosis occurring at the edge of FA . ICAP - 1 acts as an adaptor protein in integrin endocytic process . ICAP - 1 mediates cargo selection by positioning NME close to integrin ( 1 ) , AP2 , and dynamin ( 2 ) in order to fuel dynamin at the clathrin - coated pits ( 3 ) allowing execution of subsequent membrane deformation and vesicle fission ( 4 ) to ensure the turnover of integrins . Kyumurkov et al . Journal of Cell Biology 14 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 this case , failure to correctly track a few beads in such areas would significantly alter the calculated force magnitude . Therefore , the pattern area is divided into smaller windows that are allowed to overlap , before applying the cross - correlation and tracking analysis . Reducing the size of the windows makes it possible to retrieve larger displacements with cross - correlation and , using overlapped windows , we can avoid missing beads close to the windows boundaries . All image processing and analysis were performed using Matlab ( Gao and Kilfoil , 2009 ) . To calculate cell - induced traction stress from displacement data , we have used the Fourier - transform traction cytometry ( FTTC ) method ( Sabass et al . , 2008 ) . We kept the regularization pa - rameter at small values ( \u03bb < \u223c 10 \u2212 9 ) in order to maintain the best spatial resolution , which is estimated to be about 50 nm in our case . PAA substrates Polyacrylamide ( PAA ) substrates were prepared on 2 - well LabTeks ( Thermo Fisher Scientific ) with 8 % acrylamide / x % bis - acrylamide and 10 mM Hepes ( pH 8 . 5 ) gel . After two sulfo - sanpah ( Thermo Fisher Scientific ) activation , gels were coated with 5 \u00b5g / ml fi - bronectin ( 1 \u00b5g / cm 2 ) at 4\u00b0C overnight . We used three concen - trations of bis - acrylamide in order to control the rigidity : 0 . 1 % for 3 kPa , 0 . 2 % for 15 kPa , and 0 . 4 % for 60 kPa . Random migration analysis For migration assays , cells were plated on a 2 - well LabTeks containing an Fn - coated PAA substrate ( 10 mm diameter , 100 nm thick ) for 3 h in CO 2 - independent DMEM containing fibronectin - free 4 % FCS . Cells are maintained at 37\u00b0C and imaged on an inverted microscope ( Axiovert 200 ; Zeiss ) equipped with a motorized stage , cooled CCD camera ( CoolSnap HQ2 ; Roper Sci - entific ) , and a live - cell imaging 10\u00d7 objective ( EC Plan - Neofluar ) for 5 h at a frequency of one image per 4 min . Cell velocity was obtained using the manual tracking plugin in ImageJ . 150 \u2013 200 cells were analyzed from at least five different positions per ex - periment , and results were issued from three independent experiments . Focal adhesion lifetime analysis Cells stably expressing \u03b2 3 integrin - GFP were spread in 2 - well LabTeks and left to spread for 4 h . Spinning disk videos were taken for the length of 2 h with 1 min frequency . The lifetime analysis was performed with FA Analysis Server ( Berginski and Gomez , 2013 ) . Immunofluorescence Cells were plated at an approximate density of 6 \u00d7 10 4 per cm 2 for 4 h , fixed with 4 % PFA , permeabilized with 0 . 3 % Triton X - 100 , and blocked with 10 % goat - serum in PBS then with appropriate primary antibodies and after rinsing , with appropriate AlexaFluor - conjugated secondary antibody and AlexaFluor - conjugated phalloidin . Finally , the coverslips were mounted in Mowiol / DAPI solution . Images were acquired on an iMIC EMCCD Andromeda ( FEI ) spinning - disk microscope equipped with an objective Plan - Apochromat 40\u00d7 / 1 . 4 Oil - DIC M27 , WD 0 . 13 mm . siRNA - mediated downregulation of proteins in osteoblast cells Cells were plated in 6 - well plates at low density \u2014 6 \u00d7 10 3 cells per cm 2 and left to spread overnight . The next day they were transfected with the appropriate siRNA using RNAiMAX system ( Thermo Fisher Scientific ) . The medium was changed the next day , and a second hit with the same siRNA was performed . The transfected cells were used in 24 h after . All used siRNA were purchased from Dharmacon as follows : siRNA against \u03b2 3 in - tegrin \u2013 L - 040746 - 01 - 0005 ; siRNA against clathrin \u2013 L - 063954 - 00 , siRNA against NME1 \u2013 L - 040142 - 00 , and siRNA against NME2 - L - 040143 - 00 ; for all experiments we used a non - targeting siRNA as control \u2013 D - 001810 - 10 - 20 . FRAP analysis Cells stably expressing \u03b2 3 integrin \u2013 GFP were spread in 2 - well LabTeks and left to spread for 4 h . FRAP videos were taken using multimodal microscope for photo manipulations equipped with TIRF 63\u00d7 objective ( \u03b1 - Plan - Apochromat 63\u00d7 / 1 . 46 Oil Korr M27 , WD 0 . 1 mm ) . The analysis was carried out using build in FRAP analysis module in the FEI offline analysis . Image analysis and statistical tests For ppMLC staining or surface analysis , we measured the nec - essary signal using a thresholding method with manual correc - tion when needed . More than 30 cells were measured in each condition that allowed us to do a non - parametric Kruskal \u2013 Wallis test ( non - parametric ) followed by Wilcoxon test with a Bon - ferroni correction when KW tests were significant ( using GraphPad ) ; experiments were carried out at least three times . For FA analysis , we measured the \u03b2 3 integrin staining signal of at least 50 cells per experiment and three independent experi - ments using the particle analyzer of ImageJ software . Particles over 10 nm 2 were analyzed . The total adhesive area per cell was analyzed by a Kruskal \u2013 Wallis test ( non - parametric ) followed by Wilcoxon test with a Bonferroni correction when KW tests were significant ; the mean area of FA was analyzed by an ANOVA - 2 analysis and Tukey \u2019 s HSD post - hoc tests ( using GraphPad ) . Fibronectin fibrillogenesis 100 , 000 cells were plated on LabTek glass slide 4 chambers and allowed to adhere for 24 h in serum - free medium made of Op - tiMEM . Then , cells were fixed with 4 % PFA . Cells were stained with appropriate primary antibodies anti - Fn , anti - \u03b2 3 integrin ( LucA . 5 ) , and with appropriate AlexaFluor - conjugated second - ary antibody and AlexaFluor - conjugated phalloidin . Then , the coverslips were mounted in Mowiol solution . The length of in - dividual fibers was determined manually in ImageJ . To assess Fn coverage , the images were processed with fast Fourier trans - form bandpass filters to visualize all fibers , and the amount of fibronectin was measured by thresholding using Fiji ( Schindelin et al . , 2012 ) . Fluorescent integrin antibody uptake assays LucA5 antibody was labeled using SiteClick Antibody Azido Modification Kit ( Thermo Fisher Scientific ) and Click - iT pHrodo iFL Red sDIBO Alkyne for Antibody Labeling ( Thermo Fisher Scientific ) following the manufacturer \u2019 s instructions . Cells were Kyumurkov et al . Journal of Cell Biology 15 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 spread on Fn - coated LabTek slides in DMEM , supplemented with 10 % FBS and 1 % P / S . Labeled antibody ( 10 \u00b5g / ml ) was added to the cells for 45 min at 37 or 4\u00b0C , and cells were fixed in 4 % PFA and permeabilized . Actin staining was performed in order to detect cell contour . Images were acquired on an iMIC EMCCD Andromeda ( FEI ) spinning - disk microscope equipped with an objective Plan - Apochromat 40\u00d7 / 1 . 4 Oil - DIC M27 , WD 0 . 13 mm , and analyzed using the 3D Object Counter in ImageJ . Statistical analysis was performed in R using one - way analysis of variance ( ANOVA ) and Tukey \u2019 s HSD post - hoc test . In situ PLA To monitor the subcellular localization of protein \u2013 protein in - teractions at single molecule resolution , an in situ PLA was performed as previously described ( S\u00a8oderberg et al . , 2006 ) . Cells grown on coverslips were fixed with cold methanol and then incubated with primary antibodies . Secondary antibodies tagged with short DNA oligonucleotides were added . Hybridization , ligation , amplification , and detection were realized according to the manufacturer \u2019 s protocol ( Sigma - Aldrich ) . Briefly , secondary antibodies were incubated in preheated humidity chamber for 1 h at 37\u00b0C . Ligation was performed with a ligase - containing li - gation solution for 30 min at 37\u00b0C . Finally , amplification step was performed with a polymerase - containing amplification so - lution for 1 h 40 min at 37\u00b0C . After the PLA reaction , coverslips were further incubated with AlexaFluor 488 - conjugated IgGs and Cy5 - conjugated IgGs to detect proteins corresponding to the primary antibodies used . PLA signal corresponds to the Cy3 fluorescence . Coverslips were analyzed on an inverted wide - field microscope ( Leica DM4B , Camera Leica DFC 3000 G , Ob - jectif X63 Leica Germany 506185 ) . Highly inclined illumination fluorescence microscopy ( Imaging , 2010 ) was used to analyze the spatial distribution of \u03b2 1 / \u03b2 3 integrin \u2013 NME proximity complexes revealed by the PLA technology in cells labeled for vinculin to assess the proximity of NME / integrin complexes with FA . Images have been analyzed by a homemade script in ImageJ ( code available in Data S1 ) to detect duolink spots ( using D . Sage watershed algorithm ; Teaching by Doing , 2003 ) and FA , and assess their spatial proximity . The principle was to enlarge ( by three pixels ) regions corresponding to detected duolink spots following the method described in Montagnac et al . ( 2013 ) . Then , the intensity of fluorescence in the second channel corresponding to the vin - culin staining is measured to determine any colocalization . A \u201c hit \u201d is obtained in case of a positive result . The ratio of those hits to the total number of duolink spots is calculated for each cell . For each proximity complexes , the fraction of hits is sig - nificantly higher than when using randomized images . This indicates that the hits are unlikely to be due to stochastic dis - tribution of duolink spots in the cell , and thus suggests that a correlation exists between duolink spots and FA spatial distribution . Auxillin flashes measurement The four osteoblast clones were electroporated using AMAXA nucleofector Kit V ( protocol X - 001 ) according to the manu - facturer \u2019 s instructions in order to transfect them with two plasmids encoding , respectively , GFP - tagged Auxillin and the mCherry - tagged Paxillin . 100 , 000 cells per condition were plated on fluorodishes . In the following days , cells were imaged at 2 s intervals for 5 min using a spinning disk microscope ( Andor ) based on a CSU - W1 Yokogawa head mounted on the lateral port of an inverted IX - 83 Olympus microscope equipped with a 60\u00d7 1 . 35 NA UPLSAPO objective lens and a laser combiner system , which included 491 and 561 nm 100 mW DPSS lasers ( Andor ) . Images were acquired with a Zyla sCMOS camera ( Andor ) . The system was steered by IQ3 software ( Andor ) . Paxillin - positive FA were manually selected in Fiji and their area was measured . Auxillin flashes appearing at the border of FAs were manually counted . Subsequently , a square - shaped region of 2 . 5 \u00d7 2 . 5 \u00b5m 2 was placed in the center of the cell in a region where FAs were absent in order to count Auxillin flashes outside of FAs . At least 35 FAs and 3 squares in at least 4 cells per condition were imaged in three independent experiments . Re - sults are expressed as Auxillin flashes per second divided by the FA area or by the area of the square - shaped region of 2 . 5 \u00d7 2 . 5 \u00b5m 2 and normalized to the value obtained in \u03b2 1 integrin + / + - icap - 1 + / + osteoblasts . Statistical analyses were performed in SigmaPlot using one - way ANOVA followed by All Pairwise Multiple Comparison Procedure ( Holm - Sidak method ) . Fluorescence - activated cell sorting Cells were gently detached with trypsin and then treated with trypsin inhibitor ( # T0256 ) . Then , they were placed in round - bottom 96 - well plate and blocked with 1 % BSA in PBS for 30 min at 37\u00b0C . Then , cells were incubated with the appropriate anti - bodies and secondary antibodies as control diluted in PBS / 1 % BSA for 30 min on ice . After subsequent incubation with sec - ondary antibodies cells were fixed in 4 % PFA for 10 min and surface staining was detected with BD Accuri C6 flow cytometer and analyzed with the provided software . Online supplemental material Fig . S1 shows the characterization of osteoblast cells used in the study . Fig . S2 shows the role of ICAP - 1 in cellular mechano - transduction . Fig . S3 shows the efficiency of clathrin silencing in osteoblast cell lines . Fig . S4 shows NME / \u03b2 3 integrin complex at \u03b1 - adaptin \u2013 marked CCPs and its independence with respect to \u03b2 1 integrin . Fig . S5 shows the requirement of NME in CCP up - stream of dynamin and auxillin steps to allow optimal clathrin - coated vesicle budding . Video 1 shows the lifetime of eGFP - \u03b2 3 integrin FAs in \u03b2 1 integrin + / + / icap - 1 + / + cells . Video 2 shows the lifetime of eGFP - \u03b2 3 integrin FAs in \u03b2 1 integrin + / + / icap - 1 \u2212 / \u2212 cells . Video 3 shows the lifetime of eGFP - \u03b2 3 integrin FAs in \u03b2 1 integrin \u2212 / \u2212 / icap - 1 + / + cells . Video 4 shows the lifetime of eGFP - \u03b2 3 integrin FAs in \u03b2 1 integrin \u2212 / \u2212 / icap - 1 \u2212 / \u2212 cells . Data S1 provides the homemade image script used in this paper . Acknowledgments We thank Isabelle Tardieux and Kate Miroshnikova for re - viewing the manuscript . We thank Daniel Bouvard and Reinhard Fassler for providing osteoblast cells , Alexei Grichine , Jacques Mazzega , and Myl\u00e8ne Pezet for their technical assistance on the Kyumurkov et al . Journal of Cell Biology 16 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 microcell Imaging platform from the Institute for Advanced Biosciences , and Agnieszka Kawska ( http : / / www . IlluScientia . com ) for artwork associated with Fig . 7 . We thank Christiane Oddou for technical assistance . We are grateful to Dr . M . H . Ginsberg for providing polyclonal antibodies specific for \u03b2 3 integrin . A . Kyumurkov and M . R\u00b4egent - Kloeckner are doctoral fellows funded from the French Minist\u00e8re de l \u2019 Enseignement Sup\u00b4erieur et de la Recherche . M . Proponnet - Guerault is a recipient of a fellowship from the Ligue Nationale contre le Cancer ( LNCC ) . This research was funded by the Agence Nationale de la Re - cherche grant ( CODECIDE ANR - 17 - CE13 - 022 , StrepB2brain , ANR - 17 - CE15 - 0026 - 01 ) , by the Fondation pour la Recherche sur le Cancer and by the Fondation pour la Recherche M\u00b4edicale grant ( DEQ20170336702 ) to C . Albiges - Rizo and the Ligue Contre le Cancer R\u00b4egionale Auvergne - Rh \u02c6 one - Alpes et Sa \u02c6 one - et - Loire ( 17021 ) to A . - P . Bouin . The authors declare no competing financial interests . Author contributions : A . Kyumurkov and A . - P . Bouin did most of the experiments , analyzed the data , and prepared the figures . M . Proponnet - Guerault contributed in performing en - docytosis experiments . M . R\u00b4egent - Kloeckner contributed to performing quantitative analyses of phase - contrast live - cell imaging data . M . Balland and A . Nicolas contributed to gener - ating TFM data . G . Montagnac , F . Baschieri , and P . Chavrier contributed to setting up Auxillin flash experiments . S . Manet contributed to FACS analysis and O . Destaing to TIRF and FRAP analyses . M . Boissan , C . Calmel , F . Waharte , and P . Chavrier contributed to performing PLA experiments . C . Albiges - Rizo , E . Planus , A . P . Bouin , G . Montagnac , P . Chavrier , O . Destaing , and M . Boissan contributed with material and critical discussions . C . Albiges - Rizo and E . Planus supervised the entire work . C . 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Journal of Cell Biology 19 of 19 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Supplemental material Kyumurkov et al . Journal of Cell Biology S1 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure S1 . \u03b2 3 integrin expression level is unchanged upon loss of \u03b2 1 integrin . ( A \u2013 C ) Western blot of total cell lysate ( A ) and quantification ( B and C ) confirmed the deletion of \u03b2 1 integrin or ICAP - 1 in osteoblast cells . Tubulin is used as loading control . ( E ) The deletion of \u03b2 1 integrin and ICAP - 1 do not affect the expression of \u03b2 3 integrin . Actin is used as loading control . ( D ) The expression of \u03b2 3 integrin mRNA is not changed upon deletion of \u03b2 1 integrin or ICAP - 1 . Luminescence signal is normalized to the \u03b2 1 integrin + / + - icap - 1 + / + . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( F ) Intensity of ppMLC staining decorating the stress fibers , normalized to cell area . Error bars represent SD . N \u2265 211 cells . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . ( G ) The level ofthe double phosphorylation ( T18 / S19 ) of the MLC was assessed and quantified via Western blot against the total level of MLC of cell lysates of cells spread for 4 h on fibronectin - covered glass . Error bars represent SD . ns , adjusted P value > 0 . 05 ; * , P value \u2264 0 . 05 ; * * , P value \u2264 0 . 01 ; * * * , P value \u2264 0 . 001 ; * * * * , P value \u2264 0 . 0001 . Source data are available for this figure : SourceData FS1 . Kyumurkov et al . Journal of Cell Biology S2 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure S2 . ICAP - 1 is required for cells to adapt cell velocity as function of substrate stiffness independently on the presence of \u03b2 1 integrin and is involved in \u03b2 3 integrin FA translocation to the cell center . ( A ) Osteoblast cells were spread on FN - coated PAA gels of different rigidities ( 3 , 15 , and 60 kPa ) . Cell migration was monitored for 5 h using time - lapse microscopy . The cell velocity was determined by individually tracking 200 \u2013 300 cells in three inde - pendent experiments . ICAP - 1 deficient cells displayed a constant migration velocity whatever the substrate rigidity as compared to the \u03b2 1 integrin + / + - icap - 1 + / + cells , independently of the presenceof \u03b2 1 integrinhighlighting the crucial roleofICAP - 1inrigidity sensing . Error bars represent SD . N \u2265 199 cells . * , P < 0 . 05 ; * * , P < 0 . 005 ; * * * , P < 0 . 0005 . ( B ) Note that the additional deletion of ICAP - 1 in cells depleted in \u03b2 1 integrin induces an increase of \u03b2 3 integrin FA translocation to the cell center . Quantification of the percentage of FA whose distance to the nearest cell contour point is > 12 . 5 % of the average diameter of the cell . Error bars represent SD . N \u2265 208 cells . * * * * , P value \u2264 0 . 0001 . Kyumurkov et al . Journal of Cell Biology S3 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure S3 . Efficiency of clathrin silencing in osteoblast cell lines . Clathrin and actin are visualized by Western blot after treatment with siRNA against clathrin as compared to Scramble conditions . Source data are available for this figure : SourceData FS3 . Kyumurkov et al . Journal of Cell Biology S4 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Figure S4 . NME / \u03b2 3 integrin interaction occurs at \u03b1 - adaptin \u2013 marked CCPs and does not need \u03b2 1 integrin in osteoblasts . ( A ) PLA signal using NME antibody in combination with ICAP - 1 antibody ( 4d1d6 ) or ICAP - 1 antibody ( 9b10 ) or \u03b2 3 integrin antibody or \u03b2 1 integrin antibody in WT osteoblasts . Im - munolabeling of \u03b1 - adaptin was performed after PLA . Insets are higher magnification of boxed regions . Insets show PLA signals ( NME / ICAP - 1 or NME / \u03b2 1 in - tegrin or NME / \u03b2 3 integrin ) colocalizing with \u03b1 - adaptin - positive CCPs ( arrowheads ) . PLA signal indicates a close proximity , possibly in situ interaction of NME with ICAP - 1 , \u03b2 1 , and \u03b2 3 integrins at CCPs . Scale bar , 10 \u03bc m . Inset scale bar , 5 \u03bc m . ( B and C ) Representative images of PLAs ( B ) and PLA assay quantification ( C ) of the number of PLA spots per cell performed with antibodies against NME and \u03b2 3 integrin . Red dots denote regions of signal amplification consistent with NME / \u03b2 3 integrin proximity in osteoblast cells deficient or not in \u03b2 1 integrin . PLA performed on ICAP - 1 and \u03b2 1 integrin deficient cell line is used as control . Scale bar , 20 \u03bc m . Error bars represent SD . N = 15 cells / condition from three independent experiments . ns , adjusted P value > 0 . 05 . Kyumurkov et al . Journal of Cell Biology S5 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023 Video 1 . \u03b2 1 integrin + / + - icap - 1 + / + cells expressing \u03b2 3 integrin - GFP were spread in 2 - well LabTeks and left to spread for 4 h . Spinning disk videos were taken for the length of 2 h with 1 min frequency . Playback speed , 1 frame / s . Video 2 . \u03b2 1 integrin + / + - icap - 1 \u2212 / \u2212 cells expressing \u03b2 3 integrin - GFP were spread in 2 - well LabTeks and left to spread for 4 h . Spinning disk videos were taken for the length of 2 h with 1 min frequency . Playback speed , 1 frame / s . Video 3 . \u03b2 1 integrin \u2212 / \u2212 - icap - 1 + / + cells expressing \u03b2 3 integrin - GFP were spread in 2 - well LabTeks and left to spread for 4 h . Spinning disk videos were taken for the length of 2 h with 1 min frequency . Playback speed , 1 frame / s . Video 4 . \u03b2 1 integrin \u2212 / \u2212 - icap - 1 \u2212 / \u2212 cells expressing \u03b2 3 integrin - GFP were spread in 2 - well LabTeks and left to spread for 4 h . Spinning disk videos were taken for the length of 2 h with 1 min frequency . Playback speed , 1 frame / s . Provided online is a dataset . Data S1 provides the homemade image script used in this paper . Figure S5 . Requirement of NME in CCP upstream of dynamin and auxillin steps to allow optimal clathrin - coated vesicle budding . ( A ) Still image and kymographs of simultaneous two - color TIRF - M time series of mock - or siRNA - treated BSC - 1 cells overexpressing mRFP - LCa and GFP - auxilin ( 1 image / s ) . In mock - treated cells , vertical arrowhead points to a burst of GFP - auxilin coincident with mRFP - LCa signal disappearing . In siRNA - treated cells , arrowhead points to mRFP - LCa signal disappearing without associating GFP - auxilin . ( B ) Percentage of disappearing mRFP - LCa CCPs ending with a burst of GFP - auxilin in mock - and siRNA - treated BSC - 1 cells . At least 300 events were analyzed for each condition . Kyumurkov et al . Journal of Cell Biology S6 Force tuning by ICAP - 1 and NME cooperation https : / / doi . org / 10 . 1083 / jcb . 202004025 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 1 / e202004025 / 1440807 / j c b _ 202004025 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 23 F eb r ua r y 2023", + "yuDistributedAnalogicalIdea2016": "Distributed Analogical Idea Generation with Multiple Constraints Lixiu Yu , Robert E . Kraut , Aniket Kittur Carnegie Mellon University 5000 Forbes Avenue , Pittsburgh , PA 15213 { lixiuyu , robert . kraut , nkittur } @ cs . cmu . edu ABSTRACT Previous work has shown the promise of crowdsourcing analogical idea generation , where distributing the stages of analogical processing across many people can reduce fixation , identify inspirations from more diverse domains , and lead to more creative ideas . However , prior work has only considered problems with a single constraint , while many real - world problems involve multiple constraints . This paper contributes a systematic crowdsourcing approach for eliciting multiple constraints inherent in a problem and using those constraints to find inspirations useful in solving it . To do so we identify methods to elicit useful constraints at different levels of abstraction , and empirical results that identify how the level of abstraction influences creative idea generation . Our results show that crowds find the most useful inspirations when the problem domain is represented abstractly and constraints are represented more concretely . Author Keywords Constraint ; inspiration ; problem - solving ; idea generation ACM Classification Keywords H . 5 . 3 Group and Organization Interfaces INTRODUCTION The use of analogy has historically driven innovation in science , technology , and design , in which inspirations from distant domains help problem solvers identify mechanisms that would not be apparent in the target problem\u2019s original domain [ 4 , 5 , 10 , 15 , 16 ] . For example , in 2013 , a group of engineers and a world - renowned origami expert designed a large solar array to be carried by a narrow rocket . Using origami - folding techniques they were able to fold the array to a tenth of its deployed size , solving a 50 - year old problem [ 20 ] . A rich literature in psychology , engineering , and design has investigated the methods by which individuals and small groups use analogies to innovate new approaches , solutions , and products [ 5 , 6 , 7 , 11 , 15 , 17 ] . One thread of this research has introduced systematic methods for distributing the analogical idea generation process to crowds [ 28 , 29 ] . For example , even the non - expert workers on Amazon\u2019s Mechanical Turk can search product databases ( such as the thousands of unselected product submissions to Quirky ) to find inspirations that contain useful mechanisms for solving a target problem in a different domain [ 28 , 29 ] . A key enabling factor in this approach is abstracting the target problem and its constraints into a schema : a structured representation that removes domain - specific information while keeping the essential structure of the original problem . Such domain - agnostic representations reduce fixation on surface features , and can help focus search on inspirations that have structural similarity to the target problem . While successful , such approaches have to date been limited by reliance on a single schema embodying a single set of constraints , i . e . , requirements that a solution must satisfy to be successful . In contrast , many product design and engineering problems involve multiple , often conflicting constraints [ 3 , 21 , 23 , 24 , 26 ] . For example , the design of a kindergarten chair might require multiple constraints , such as its safety ( e . g . , preventing it from tipping over or pinching fingers ) and flexibility ( e . g . , making it easy to move or stack ) . Each of these constraints could be represented as its own schema and at multiple levels of representation ( e . g . , safety vs . preventing pinched fingers ) . Generalizing distributed idea generation to more complex real - world problems requires a deeper understanding of how to elicit and represent multiple constraints . In this paper , we propose a distributed process for eliciting the constraints inherent in a problem at multiple levels of abstraction and using those constraints to find inspirations useful in solving that problem . In doing so we explore how to operationalize constraints in ways that are useful for problem solving and how this can be done using non - expert crowds . We test our approach through two randomized experiments in which crowd workers generate constraints , find inspirations , and solve problems while we manipulate Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from Permissions @ acm . org . CSCW ' 16 , February 27 - March 02 , 2016 , San Francisco , CA , USA \u00a9 2016 ACM . ISBN 978 - 1 - 4503 - 3592 - 8 / 16 / 02 $ 15 . 00 DOI : http : / / dx . doi . org / 10 . 1145 / 2818048 . 2835201 1236 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA the level of abstraction of the problem description ( i . e . , domain independent or dependent descriptions ) and of the constraints ( i . e . , more abstract or more concrete descriptions of the constraints ) . Our results suggest that crowd workers can identify important constraints for a product and can find inspirational examples that are useful for satisfying those constraints . The best results occur by representing the problem domain at a relatively abstract level but the constraints at a more concrete level . Under these conditions , people find the most diverse set of inspirational examples , yet ones that are most relevant to the initial problem . People shown these inspirational examples generate more original and practical ideas to solve the original problem . LEVELS OF ABSTRACTION Our research goal is to identify the levels of abstraction that are useful for product design in terms of both problem context ( e . g . , designing a kindergarten chair ) and problem constraints ( e . g . , safety , flexibility ) . Abstracting a problem context into a schema in order to find analogical solutions from distant domains can be a highly effective approach for increasing innovation in domains ranging from scientific discovery to creative problem - solving to product development [ 5 , 6 , 17 ] . For example , people are better able to solve Duncker\u2019s radiation problem [ 12 ] , in which a doctor needs to destroy a tumor with a large dose of X - rays without destroying surrounding tissue , if they are given as inspiration the case of a military general who conquers a castle surrounded by mined roads by dividing his army into small groups that are light enough to evade the mines and who attack from different directions . However , this inspiration is most useful if the problem solvers represent the solution abstractly , in terms of a divide - and - converge strategy , which maps the high - level structure shared by the problem and solution description [ 13 ] . Even though the medical and military domains differ radically in surface detail , an abstract divide - and - converge schema maps the general\u2019s strategy of dividing troops in transit into small groups before converging at the castle with the tumor problem , where the doctor divides the x - ray into multiple small beams converging on the tumor . Although this example was taken from cognitive science research , both empirical observation and experimental studies in product development find a positive relationship between using analogical inspiration and the originality of the product concept [ e . g . , 17 , 22 ] . Recently , Yu et al . [ 29 ] developed an approach to distributing the schema abstraction process across multiple individuals , showing that searching for inspirations and generating ideas with crowds who had not seen the original problem domain or description decreased fixation and increased solution creativity . However , the level of abstraction of the problem constraints is also likely to affect the diversity and relevance of inspirations found . Searching for inspirations relevant to \u201csafety\u201d might generate more diverse inspirational examples than \u201cpreventing pinched fingers\u201d , but might also lead to solutions less applicable to the design of kindergarten chairs . Real world idea generation often requires solving \u201cwicked problems\u201d [ 2 ] , which can involve satisfying multiple constraints simultaneously , rather than developing a single insight that characterizes Duncker\u2019s x - ray problem . Stripping away too much detail during abstraction may cause problem solvers to consider mechanisms that are not relevant to their problem or prevent them from recognizing the precise mechanisms they need to solve a constraint in a particular domain . For example , in Duncker\u2019s radiation problem , the relevance of solutions is constrained because x - rays travel in straight lines ; analogies from ant colonies involving ants who can follow arbitrary paths might thus not be relevant to reasoning about x - rays . Even though abstracting away problem context allows problem solvers to identify a wider range of potential solutions , actually applying the solutions to the original problem domain may require more detailed knowledge of the constraints that must be met . Thus we hypothesize that while describing a problem context may be most useful if the domain or context is abstracted away ( to reduce functional fixation ) , the representation of constraints may be most useful when they are concrete enough to suggest specific potential mechanisms ( e . g . , \u201cprevent tipping over\u201d rather than \u201csafety\u201d ) while simultaneously being general enough to allow multiple possible mechanisms ( e . g . , preventing something from tipping over by attaching it to a stable object or introducing a low center of gravity ) . To test these hypotheses , we first developed a procedure to elicit constraints from crowd workers for designing a kindergarten chair . Using these constraints , we conducted an experiment in which participants found inspirations using either an abstract or concrete representation of the problem context and constraints ( in a 2x2 design ) , and then subsequent participants used these inspirations to generate new solutions to the design problem . Below we describe how we elicited constraints using crowds , and then describe the experiment procedure and results . ELICITING CONSTRAINTS One key question we aim to address here is how to elicit and represent constraints in a way that can be used by non - expert crowd workers to effectively search for inspirations , which in turn would be useful in solving a target problem . We conducted a series of pilot studies asking workers from Amazon\u2019s Mechanical Turk [ 18 ] to generate constraints for designing a good kindergarten chair , using the probe question : \u201c We are planning to design creative chairs for kindergarten kids . What aspects or constraints should be considered ? \u201d The responses we got back varied significantly in their level of detail . Some of them were quite abstract , such as \u201c Safety . As we all know kids is very fragile so the safety is the most important thing to have \u201d . Others were much more concrete , such as \u201c chairs should have to be shockproo f \u201d . We found two prevalent types of constraints , which corresponded to the level of abstraction 1237 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA workers used to describe the constraint . Abstract constraints such as \u201cthey should be safe for kids to sit on\u201d , can be satisfied in many different ways . In contrast , more concrete constraints , such as adding stabilizers , round off sharp corners , implied a much smaller number of ways they could be satisfied . Based on these findings , we iteratively developed a process to elicit abstract and concrete design constraints ( see Table 1 ) . To elicit abstract constraints , we first recruited 27 crowd workers to brainstorm constraints for kindergarten chairs . As noted previously , their responses varied in abstractness . To make the response set more homogeneous and abstract , we asked a new group of crowd workers to summarize each response in a word or two : \u201c Which design aspect of the chair is the following suggestion referring to ? Summarize it in one or two words \u201d . We then treated these keywords as the abstract constraints , because they shifted the representation of the initially brainstormed constraints to a more abstract level . After a consolidation step , in which the experimenters eliminated duplicates and synonyms , there were seven abstract constraints : safety , flexibility , pedagogy aesthetics , cost , comfort , and ease of cleaning . To elicit concrete constraints , we asked seven new groups of roughly 15 crowd workers each to generate concrete constraints for each abstract constraint , by framing them in terms of lower - level requirements . In this task , the crowd workers received one of the abstract constraint key words , such as safety , and described components of safety or another abstract constraint should be considered when designing a kindergarten chairs . The instructions were to eliciting more concrete version of the safety constraint were , \u201c We are planning to design kindergarten chairs for 5 - 8 year - old children . In this task , we would like you to brainstorm the constraints kindergarten chairs should meet . Please identify a constraint by filling out the following sentences . Note that constraints are requirements on certain dimensions rather than solutions . Regarding safety , a kindergarten chair should meet the requirement that _ _ _ _ _ _ _ _ _ _ _ _ \u201d . This task returned a set of lower - level , more concrete constraints for each abstract constraint . For example , for the safety constraint , participants identified concrete constraints such as \u201cchairs shouldn\u2019t tip over easily\u201d and \u201cchairs shouldn\u2019t have sharp edges\u201d . While more concrete than general categorical constraints such as safety , such constraints still support being instantiated in multiple ways ; for example , a chair could be prevented from tipping over by lowering its center of gravity or fixing it to the floor . As each group described similar concrete constraints in somewhat different ways , we presented the lower - level constraints for an abstract constraint to a new group of crowd workers and asked them to select the ones that were similar and summarize them in terms of a single constraint . In the domain - independent condition we identified a list of domain related terms such as \u201cchair\u201d , \u201ckindergarten\u201d , \u201ckids\u201d , and \u201cclassroom\u201d . The experimenters removed these words and replaced them with more general , domain - independent terms , such as \u201cobjects\u201d . EXPERIMENT 1 : SEARCHING FOR INSPIRATION The previous section described a process through which crowd workers identified a variety of important constraints for a product category at multiple levels of abstraction . Moreover , simple transformations , in particular , substituting domain - specific vocabulary with more general terms , provided a method to express constraints independent of the problem domain . We used these more or less abstract constraints presented with and without reference to the problem domain as inputs for the experiment described below . The goal of this experiment was to better understand conditions under which abstract representations of problems aid or hinder people\u2019s ability to identify solutions to them . We distinguish between the functional requirements of a problem or its constraints , on one hand , and the problem Steps Constraint examples Step 1 : Eliciting abstract constraints 1 ) chairs shouldn\u2019t hurt kids ; 2 ) chairs should have to be shockproof \u00e0\uf0e0 Safety 1 ) comfortable , because kids need to be healthy ; 2 ) Cozy , we all like cozy furniture \u00e0\uf0e0 Comfort Step 2 : Eliciting concrete constraints Safety \u00e0\uf0e0 1 ) The chair should not have sharp edges ; 2 ) The chair won ' t tip over Comfort \u00e0\uf0e0 1 ) The chair should be easy for kids to get on and off ; 2 ) The chair should have appropriate angle giving the correct strain free posture Domain - independent condition Safety : 1 ) The object should not have sharp edges ; 2 ) The object won ' t tip over Comfort : 1 ) The object should be easy to get on and off ; 2 ) The object should have appropriate angle giving the correct strain free posture . Table 1 . Workflow for generating constraints . Constraints Domain Abstract Concrete Abstract Domain - independent abstract constraint Domain - independent concrete constraint Concrete Domain - dependent abstract constraint Domain - dependent concrete constraint Table 2 . Conditions in Experiment 1 . 1238 SESSION : CROWD INNOVATION AND CROWDFUNDING context or domain , on the other . We hypothesize that abstracting away the problem domain will improve search for useful inspiration by preventing functional fixation , leading searchers to find a richer and more diverse set of potentially relevant solutions . However , we hypothesize that concrete representations of constraints will be more useful than abstract representations , because they help searchers more completely understand the type of problem they are trying to solve and to evaluate the relevance of potential solutions for that problem . To test these hypotheses , we designed a 2X2 experiment with the four conditions shown in Table 2 . In Experiment 1 , searchers were given descriptions of the kindergarten chair design problem varying in abstractness and domain specificity and asked to find examples that could inspire solutions . The description of the constraints that solutions needed to meet were presented either more abstractly ( e . g . , ways to make objects safe ) or concretely ( e . g . , ways to make objects that won\u2019t tip over ) . Similarly , the description of the problem domain was also presented relatively abstractly ( i . e . , in domain independent manner , without mentioning kindergarten chairs ) or more concretely ( i . e . , in a domain dependent manner , mentioning kindergarten chairs ) . Henceforth , laid out in Table 2 , for clarity we refer to the abstractness of constraints using the terms \u201cabstract\u201d and concrete , and refer to the abstractness of domains using the terms \u201cdomain - independent\u201d to refer to more abstract domain and the term \u201cdomain - dependent\u201d to refer to the more concrete domains . Participants Overall , 158 Amazon Mechanical Turk workers participated in the experiment . Forty - nine percent were women , and 91 % were native English speakers . Their average age was 32 and ranged from 18 to 58 . Design and Procedure After participants accepted the task , they were randomly assigned to one of the four experimental conditions in Table 2 . They were asked to search for an inspirational source for product design tasks . The task charged them with satisfying two abstract constraints , safety and flexibility , or four concrete constraints : two related to safety ( Tipping over , Pinching fingers ) and the other two related to flexibility ( Moving around , Folding ) . The task in the domain - dependent abstract constraint condition was described as designing a kindergarten chair\u2019s that was either safe or flexible . For example , the instructions for this task with the safety constraint was . \u201c We are looking for inspirations for novel and useful designs for kindergarten chairs that are safe for kids to use . Go to the Internet and retrieve a picture or description of something that has been designed to be safe to use and that could be relevant to the design of kindergarten chairs . \u201d The task in the domain - dependent concrete constraint condition was described as designing a kindergarten chair in terms of one of the following four concrete constraints ( tipping over , pinching fingers , moving around , or folding ) . For example , \u201c Go to the Internet and retrieve a picture or description of something that has been designed to easily move around and that could be relevant to the design of kindergarten chairs . \u201d Note that participants in all domain - dependent conditions were explicitly instructed to not restrict their search to the world of kids\u2019 chairs , and that their results would be judged partially on novelty : \u201c Please search broadly . The examples can come from the domains that are outside of kids chairs . Your search results will be judged on whether it can inspire novel and useful design . \u201d In the domain - independent conditions , no mention was made of kindergarten chair . Specifically , we replaced domain related terms such as \u201cchair\u201d or \u201ckids\u201d with more general terms such as \u2018people\u201d or \u201cobjects\u201d . For example , instructions in the domain - independent abstract constraint condition were , \u201c We are looking for inspirations for novel and useful designs for objects that are safe to use . Go to the Internet and retrieve a picture or description of something that has been designed to be safe to use \u201d . The instructions in the domain - independent concrete constraint conditions were , \u201cGo to the Internet and retrieve a picture or description of something that has been designed to prevent tipping over . Please search broadly . The examples can come from any domains . Your search results will be judged on whether it can inspire novel and useful design . \u201d Participants were asked to provide a link to the inspiration they found , an explanation why they selected this inspirational example , and a description of how they searched for it . Participants returned roughly 40 examples in each condition . Measuring Distance and Diversity To measure the distance between the original problem domain of kindergarten chairs to the examples searchers returned , two judges blind to experimental condition rated each example on a 7 - point Likert scale : \u201c How different is the above kindergarten chair design problem from the domain of the example ? \u201d By this metric , for example , a glass was considered further from a kindergarten chair than was a bench . The judges achieved high inter - rater reliability ( Intraclass Correlation Coefficient ( ICC ) = 0 . 85 ) [ 9 ] . The final distance score was calculated by averaging the scores of the two judges . Because the distribution of the scores was bimodal , we converted distance from the kindergarten - chair domain into a binary variable based on a median split . Examples above the median score were considered \u201cfar\u201d ( 1 ) and those below the median were considered \u201cnear\u201d ( 0 ) . 1239 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA We also calculated a diversity score by counting how many unique domains were found within in each condition . In counting unique domains we combined those from highly similar inspirations ; for example , all the examples shown in the first column of Table 3 were classified as coming from the chair domain while the last two examples from the fourth column , ( the Russian eggs and the cup ) , where from two different domains . Analysis and Results Summary statistics for the data are shown in Table 4 . For analysis purposes , the four conditions were represented as two dummy variables : domain abstractness and constraint concreteness . Domain independent descriptions were coded as abstract ( 1 ) while domain - dependent descriptions were coded as 0 . Problem descriptions with constraints described at a less abstractness level ( e . g . , tipping over , pinching fingers , moving around , and folding up ) were coded as having concrete constraints ( 1 ) , while descriptions with more abstract constraints ( e . g . , safety or flexibility ) were coded as 0 . We ran a logistic regression analysis with binary Distance as the dependent variable regressed on domain abstractness , constraint concreteness , and their interaction . The results show the odds of finding an example from a far domain were substantially higher when the domain was described abstractly rather than concretely ( odd ratio = 214 . 86 , p < 0 . 001 ) . This is consistent with previous literature showing that representing a problem abstractly reduces fixation and increases the likelihood of considering far domains . There was no significant effect of constraint concreteness on domain distance ( b = 1 . 20 , p = 0 . 15 ) , nor a significant interaction effect ( b = - 1 . 66 , p = . 0 . 14 ) . For domain diversity , we see a similar main effect of domain abstractness : the odds of an example coming from a unique domain were substantially higher when problems were described in an abstract , domain independent way ( odds ratio = 26 . 31 , p < 0 . 001 ) . In addition , we also observe a significant negative interaction between domain abstractness and constraint concreteness , with the effect of domain abstractness significantly higher when constraints were also presented abstractly ( b = - 1 . 77 , p < 0 . 05 ) . These results indicate that Domain - independent abstract constraint lead to the highest diversity of domains explored . These results are visually reflected in Table 3 , which shows examples of inspirations found in the four conditions . As these examples illustrate , in the domain - dependent conditions , most examples were about chairs and furniture . In contrast , the domain - independent conditions returned a wider variety of potential examples , including glasses , 3D printer techniques , and kids tools . We also analyzed how participants searched for inspirations in the different conditions . In the domain - dependent conditions , most search involved domain terms . For example , participants described the ways they searched for examples , \u201cI was searching google for \" fun kids chairs \" \u201d , \u201ccreative seating for classrooms\u201d , and \u201csearch for tip proof chair\u201d . When participants searched with domain - independent abstract constraints , the ways of conducting search were vague . For example , \u201c I have search on Google for different kind of images related to novel inspiration for kids \u201d and \u201c I did an image search for adaptable images for inspiration \" . In contrast , when people searched with domain - independent concrete constraints , the search terms were much more concrete while without the fixation on chairs , including \u201c I found the example by typing designs for objects that don ' t pinch fingers \u201d , and \u201c I googled objects that don ' t tip over easily \u201d . These different ways of searching resulted from different representations explained the findings . Domain - dependent , abstract constraint Domain - dependent , concrete constraint Domain - independent , abstract constraint Domain - independent , concrete constraint Table 3 . Examples of inspirations found when the domains and constraints were presented concretely and abstractly . Conditions N Avg Distance % far % unique domains Domain - dependent , abstract constraint 40 2 . 36 5 % 30 % Domain - dependent , concrete constraint 40 2 . 69 15 % 35 % Domain - independent , abstract constraint 37 5 . 80 92 % 92 % Domain - independent , concrete constraint 41 6 . 20 88 % 71 % Table 4 . Distance of inspirations from the kindergarten furniture domain and their diversity in Experiment 1 . 1240 SESSION : CROWD INNOVATION AND CROWDFUNDING EXPERIMENT 2 : GENERATING CREATIVE SOLUTIONS The results from Experiment 1 suggest that the level of abstraction of the problem domain is a key driver of exploration : the more abstract the domain description , the more distant and diverse the domains explored . However , simply exploring more distant and diverse domains is not sufficient to find useful inspirations ; to be useful , the solutions suggested by the inspirations must also be relevant and applicable to the target domain ( here , kindergarten chairs ) . Observation of the rightmost two columns of Table 3 suggests that while both domain - independent conditions led to inspirations in distant domains , those found in the abstract - constraint condition ( third column ) have various safety designs that do not seem relevant to chair safety . In contrast , the inspirations found with concrete constraints ( fourth column ) suggest a variety of relevant mechanisms to prevent tipping over . We designed an experiment to test the usefulness of the inspirations in generating creative solutions to the target problem . In the experiment , we asked people to design kindergarten chairs after showing them four examples randomly selected from one of the four conditions in the previous experiment . There were four concrete constraints ( Tipping over , Pinching fingers , Moving around , Folding ) in the concrete - constraint conditions ; therefore , we randomly selected one example from each constraint . Because there were only two constraints ( safety and flexibility ) in the abstract - constraint conditions , we selected two examples from each constraint . Thus all conditions received four examples split amongst the two abstract constraints , with a further split for the concrete - constraint conditions . Participants Overall , 147 Amazon Mechanical Turk workers participated in the experiment . Forty - one percent were women , and 91 % were native English speakers . Their average age was 33 and ranged from 19 to 83 . Design and Procedure The same four conditions in Table 2 were used in this experiment , with the addition of a baseline control in which participants were not shown any examples . Participants first saw the instructions given to Turkers searching for inspirations in the domain - dependent , concrete constraint condition in Experiment 1 : \u201c This task asks you to design a flexible and safe kindergarten chair . The chair should be flexible enough to be used and stored in a variety of ways in a kindergarten class . It should also be safe for kids ( 4 - 5 year olds ) to use . Specifically , a flexible and safe kindergarten chair should meet the following criteria : 1 ) The chair can be easily folded up ; 2 ) The chair can be moved around easily by 4 - 5 year olds ; 3 ) The chair is stable and won\u2019t tip over easily ; 4 ) The chair won\u2019t pinch kids\u2019 fingers . \u201d After seeing the instruction , participants saw four examples and were asked to think about how these examples could be valuable : \u201c In a previous task , we asked other Turkers to search for examples that could provide inspiration for designing such a kindergarten chair . Below are four examples and the Turkers\u2019 explanations for why they provided them . Please look at these examples carefully and explain how the ideas from this example could help design a kindergarten chair . \u201d Participants were then asked to \u201c sketch the design of a flexible and safe kindergarten chair . Use the above examples as inspiration if you think they are helpful \u201d . Rating Did the examples found with different representations influence the quality of the new designs differently ? We draw on previous research on operationalizing measures of creativity , which suggests that ideas are more creative the more original and practical they are [ 1 , 8 , 25 , 27 ] . Two judges , the first author and an undergraduate student , blind to experimental condition rated each design on Likert scales measuring originality and practicality . Although neither judge was a professional designer , because the design challenge was about a consumer product we believe non - experts have sufficient knowledge to evaluate the novelty and practicality of the solutions . Prior work also shows high agreement between designers and non - designers in judging consumer products such as chair design [ e . g . , 30 , page 50 - 51 ] . To achieve more objective judgment , we considered the requirements of kindergarten chairs , and , based on these requirements , created specific criteria for judging originality and practicality . Originality was defined as an idea that was not obvious and differed from existing products on the market . To judge originality , we selected four most frequently found chair examples in the previous experiment as the typical chair design . The raters were asked to judge originality on 7 - point Likert scales by comparing the designs to these four typical chairs , as shown in Table 5 . Practicality was defined as how much the new design met the four constraint criteria and how realistic it would be to design , manufacture , and use the idea . To measure practicality we used a combination of five three - point Likert scales judging each of the four constraints and the realism constraint described above . Below shows examples of the specific instructions : Tipping over : how much does the design solve the tipping over issue ? Not at all ( 1 ) , sort of ( 2 ) , completely ( 3 ) Realism : Can it be realistically designed , produced , and Table 5 . Typical chairs used in judging originality . 1241 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA used ? Not at all ( 1 ) , sort of ( 2 ) , completely ( 3 ) The final practicality score was calculated by adding all the five scores together , creating a 15 - point scale . After several rounds of training and discussion , the judges achieved ICC inter - rater reliabilities of 0 . 75 and 0 . 79 for originality and practicality respectively . The designs in Table 6 show two examples with their corresponding scores on practicality and originality . Because large differences in participants\u2019 sketching skills could distort judges\u2019 estimates of idea quality , the judges were told to use the sketches to assist understanding the design idea but not to base their judgments on sketch quality . In the experiment , participants were asked to explain how their ideas were inspired by the examples if they were so inspired . We used the information to judge whether a new design was inspired by an inspirational example . It was coded as 1 if the participant mentioned an inspiration ; otherwise it was coded as 0 . Analysis and Results The five conditions differ in terms of whether showing examples and the sources of the shown examples . We examined practicality and originality of the new designs as well as whether a new design was inspired by the examples shown to them , inspiration . The means , standard deviations of practicality and originality , and the percentage of designs that were inspired by an example are shown in Table 7 . The distribution of the originality scores was left skewed . We performed a square root transformation to normalize the data and used the transformed data for analysis . We first compared all the experimental conditions to the baseline condition on practicality and originality separately through two regression analyses . The results show that only the ideas produced by the Domain - independent concrete constraint condition had significantly higher practicality and originality than the baseline condition , as shown in Table 7 . To further examine the factors affecting the quality of ideas , we applied a similar dummy coding as in the previous experiment . Regression analyses , regressed on domain abstractness , constraint concreteness , and their interaction , showed that domain abstractness and constraint concreteness alone did not predict practicality ( b = - 0 . 64 , p = 0 . 19 for domain abstractness ; b = - 0 . 14 , p = 0 . 78 for constraint concreteness ) nor originality ( b = 0 . 02 , p = 0 . 89 for domain abstractness ; b = 0 . 07 , p = 0 . 53 for constraint concreteness ) . However , we observed a significant positive interaction between domain abstractness and constraint concreteness for both practicality ( b = 3 . 10 , p < 0 . 01 ) and originality ( b = 0 . 38 , p < 0 . 05 ) , as shown in Figure 1 and 2 . A logistic regression analysis examining inspiration revealed a similar finding : while domain abstractness and constraint concreteness alone did not predict practicality ( b = - 0 . 80 , p = 0 . 14 for domain abstractness ; b = - 0 . 25 , p = 0 . 65 for constraint concreteness ) nor originality ( b = 0 . 02 , p = 0 . 89 for domain abstractness ; b = 0 . 07 , p = 0 . 53 for constraint concreteness ) , we observed a significant positive The chair is made out of flexible material . The back part of the chair is made out of a soft cotton material and so is the cushion for where you sit . There is an addition of two more legs outside of the chair . It can easily be folded upright thanks to the chair being out of a more flexible material . The joints of the chair each have one of the protective joint covers mentioned by the other Turkers that prevents fingers from being pinched as the chair folds down . It was inspired by the first image , the second and the last because it seemed to make the most sense ( Practicality : 13 ; Originality : 5 ) The slide example reminded me how much fun I use to have as a child playing on the swing set . So I thought , \" wouldn ' t it be great to have a swing chair ? \" The chair would be big enough for one child to sit and there would be enough soft pillow / padding in it to be very comfortable . The swing would be designed to allow enough freedom of movement to get a swinging motion but limited so that it doesn ' t swing to wide or to high . The chair ergonomics would function like a cradle and if desired a child could sleep in it like a mini hammock . ( Practicality : 10 ; Originality : 7 ) Table 6 Experiment 2 : Design examples 1242 SESSION : CROWD INNOVATION AND CROWDFUNDING interaction between domain abstractness and constraint concreteness ( b = 1 . 95 , p < 0 . 01 ) . Overall these results suggest creative ideas are most likely to be inspired by examples found using an abstract problem context ( i . e . , removing domain specific information ) but constraints that are more concrete and suggest mechanisms by which their requirements can be met ( e . g . , prevent from tipping over ) rather than more abstract requirements ( e . g . , safety ) . The examples in Table 6 offer some insights into these findings . In the first example , the participant borrowed three mechanisms from three of the four examples : the extra supporting legs for stability ( the first image ) , the buffer on the joint ( the second image ) , and the folding method ( the last image ) . These mechanisms can solve three constraints : tipping over , pinching fingers , and folding . Such successful transfer not only resulted in a practical design but also increased originality , because these mechanisms may have been widely used in the diverse domains people explored , but they were rarely seen in kindergarten chairs . In contrast , examples found in the domain - dependent conditions have no such benefits because most of them belong to the chair domains ; examples found with domain - independent abstract constraints are not as useful because many of their mechanisms are not applicable to chairs . In Experiment 2 , we were also interested in the integration of multiple constraints . Applying multiple examples to different constraints is challenging for several reasons . One is that some constraints conflict with each other . Once you adapt a mechanism from one inspiration , it might become difficult to integrate a mechanism from another . For example , to prevent chairs from pinching , the chair can be designed without joints , but this design could make it difficult to integrate mechanisms involving folding , which are useful for satisfying the storage constraint . A second explanation for the difficulty in incorporating mechanisms from multiple inspirations is high cognitive load and limited working memory . Indeed , in Experiment 2 , we found that even though some participants could successfully apply mechanisms from all four examples , the majority of participants used fewer than four , as illustrated in Table 6 . We will return to this observation in the Discussion . DISCUSSION In this paper we examined how to extend previous crowdsourcing idea generation research beyond problems involving a single constraint to those involving multiple constraints . In particular , we contributed a process for eliciting multiple constraints for a problem and investigated how the level of abstraction of those constraints influenced creative idea generation . During the first step of the process , crowd workers transformed an ill - formed , open - ended problem ( e . g . , design a creative kindergarten chair ) to a better - structured statement comprising concrete constraints ( e . g . , design a chair that is easily movable , stackable and won\u2019t tip over ) . Other crowd workers searched for inspirational examples of products in remote domains that could satisfy the constraints ( e . g . , mechanisms that prevent objects from tipping over ) . Yet other crowd Conditions Practicality Originality % Inspiration N Mean SD Mean SD Baseline ( no examples ) 9 . 70 1 . 79 2 . 04 1 . 49 23 Domain - dependent , abstract constraint 9 . 45 1 . 78 2 . 03 1 . 17 26 % 31 Domain - dependent , concrete constraint 9 . 31 2 . 22 2 . 20 1 . 32 31 % 29 Domain - independent , abstract constraint 8 . 81 1 . 49 2 . 06 1 . 32 44 % 32 Domain - independent , concrete constraint 11 . 77 * * * 2 . 10 3 . 48 * * * 1 . 61 71 % * * 31 p < . 01 = * * , p < . 001 = * * * Table 7 . The effects of abstractness of examples on design quality in Experiment 2 . Figure 1 Practicality : Interaction between abstraction of the domain and constraints , with 95 % CIs . Figure 2 Originality : Interaction between abstraction of the domain and constraints , with 95 % CIs . 1243 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA workers were able to draw inspiration from these examples to generate creative conceptual designs for chairs that met these constraints . Their product designs were more creative ( i . e . , more practical and original ) if the search for inspirations started with a problem representation that abstracted away the problem domain ( e . g . , kindergarten chairs ) , but kept the constraints concrete , compared to searches that started with a representation that mentioned the problem ( e . g . , kindergarten chairs ) or that abstracted the nature of the constraints . The contributions of this paper take a step towards moving distributed analogical idea generation in the direction of more complex , real - world design problems that often involve multiple constraints . However , an important open question is the extent to which more complex real - world problem - solving challenges , such as in developing a car or a mobile phone , would work with this approach . Furthermore , although we demonstrated this process with non - experts , there may be significant benefits in moving beyond the relatively na\u00efve problem solvers ( i . e . , untrained Mechanical Turk workers ) used here to trained problem solvers and domain experts ( e . g . , engineers ) . Some aspects of the current process may strongly benefit from employing more sophisticated workers at different stages . For example , it may be more useful to elicit constraints from domain experts ( e . g . , teachers , parents or children for the kindergarten chair challenge ) , as in traditional requirements analysis , to recruit experts in other fields for the search step to scour their domain archives for relevant mechanism , and to employ traditional product designers in the final , integration steps . We consider our research only a first step . Additional research is needed at both the theoretical and practical levels . Progress in theory development requires crisper ways to decompose the nature of a problem . Our research differentiated the problem context ( kindergarten chair versus other problems ) from problem constraints ( e . g . , flexibility and safety ) , but we suspect this decomposition is too crude . For example , constraints themselves are likely to differ on multiple dimensions , such as their concreteness ( which we examined in the current research ) , the degree to which they are integral to the overall problem or optional , their breadth of applicability and their complexity . These attributes are likely to influence the most effective way to communicate constraints to problem solvers and the degree to which analogical transfer will be a fruitful approach for finding mechanisms to satisfy them . At the methodological level , we need to develop more robust ways to elicit constraints and requirements from analysts . Although requirements analysis is a key feature of HCI practice , we believe there is no single best way to elicit and present requirements in such as way to be useful to problem solvers . At the application level , we believe the most interesting research involves both understanding and developing processes to help problem solvers to integrate multiple constraints . Having multiple constraints creates two challenges during integration : the large amount of information to process and the possible conflict between constraints . For example , a kindergarten chair may have dozens of constraints and people may be able to identify multiple analogs ( examples of methods for satisfying each constraint ) , some of which will conflict with each other . For example , to make a chair easy to clean , workers may find analogs like a kitchen blender that uses a \u201cdisassembly\u201d schema , so that parts from a bulky object can be removed and cleaned separately . However , applying this to a chair ( e . g . , by designing detachable legs ) could undermine its stability and provide gaps that could catch fingers . In the research presented here , the people who searched for inspiration received each constrain separately , with integration deferred to people in the problem - solving stage who needed to integrate multiple mechanisms . However , this approach is not necessarily the best one . This points to a fundamental tradeoff in distributed analogical processing between taking advantage of decomposing and distributing a problem across many people ( which can improve the capacity and diversity of exploration ) and dealing with the lack of context of other parts of the process that such decomposition incurs . Studying the conditions under which this tradeoff is optimized for design problems with multiple constraints would be fruitful follow - up research . ACKOWLEDGEMENTS This work was supported by NSF grants IIS - 1526665 , IIS - 1149797 , IIS - 1217559 , OCI - 0943148 , IIS - 0968484 , IIS - 1111124 , Bosch , Google , and Microsoft , Heinz College Center for the Future of Work , and Carnegie Mellon\u2019s Center for the Future of Work . We thank reviewers for useful feedback . REFERENCES 1 . Teresa Amabile . 1996 . Creativity in Context . Westview Press . 2 . Richard Buchanan . 1992 . Wicked problems in design thinking . Design issues , 5 - 21 . 3 . Balakrishnan Chandrasekaran . 1990 . Design problem solving : A task analysis . AI magazine 11 , 4 : 59 . 4 . Joel Chan , Susannah B . F . Paletz , and Christian D . Schunn . 2012 . Analogy as a strategy for supporting complex problem solving under uncertainty . Memory & cognition 40 , 8 : 1352 \u2013 1365 . 5 . Darren W . Dahl and Page Moreau . 2002 . The Influence and Value of Analogical Thinking during New Product Ideation . Journal of Marketing Research 39 , 1 : 47 \u2013 60 . 6 . 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In Proceedings of the 32nd annual ACM conference on Human factors in computing systems ( CHI\u201914 ) , ACM , 1225 \u2013 1234 . http : / / dl . acm . org / citation . cfm ? id = 2557378 29 . Lixiu Yu , Robert E . Kraut , and Aniket Kittur . 2014 . Distributed analogical idea generation : innovating with crowds . In Proceedings of the ACM conference on human factors in computing systems ( CHI\u201914 ) , ACM , 1245 - 1254 . http : / / dx . doi . org / 10 . 1145 / 2556288 . 25573781 30 . Lixiu Yu . 2012 . Crowd Idea Generation . Ph . D Dissertation . Stevens Institute of Technology , Hoboken , NJ . 1245 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA", + "chiuInvestigatingEffectsOppositely2012": "Journal ofEngineering Design Vol . 23 , No . 4 , April 2012 , 271 - 296 Q Taylor & Francis ~ Taylor & . FrantisCroup N - o N ~ < l ) . . c o . . . . u o N o \" \" \" r ' I o - Investigating effects of oppositely related semantic stimuli on design concept creativity Q Department ofMechanical and Industrial Engineering , Ryerson University , 350 Victoria Street , Toronto , ON , Canada M5B 2K3 ; b Department ofMechanical and Industrial Engineering , University ofToronto , 5 King ' s College Road , Toronto , ON , Canada M5S 3G8 ( Received 25 February 2011 . . final version received 4 July 2011 ) We are motivated to investigate methods to increase creativity in conceptual design since creativity is essential to design success , and no other stage influences final design success as much as conceptual design . Existing work supports that design stimuli may encourage creative concept generation , but does not give details on how to systematically generate stimuli . The established relationship between language and cognition , and the systematic nature of language prompt us to examine its use as design stimuli . Language relationships such as opposition provide a systematic method of generating non - obvious semantic stimuli for design problems . In this paper , we presenttwo experiments , apen - and - paperand averbal protocol study , where participants usedoppositely related and similarlyrelated word stimuliinconceptualdesign . Wefound that designers using oppositely related word stimuli developed more creative concepts . Language analysis revealed how opposite stimulieliciteddesignerbehaviours thatmay encourage and supportcreative concept generation . Our empirical results combined with linguistic theory lead us to propose a model explaining the interactions and effects of opposite - stimulus words on concept creativity . This knowledge can be used to facilitate more creative , and ultimately , more successful design . Keywords : language ; creativity ; design stimuli ; concept generation ; engineering design 1 . Introduction We have been studying and quantifying the application of natural language , i . e . human language , not artificial language , to the process ofstimulating creative conceptual design . We are motivated to study language in relation to design because language appears ubiquitously , is inherent in people , and is connected to cognition . Researchers have established a linkbetween language and cognition , although the exact relationship is disputed . Some have shown that language affects cognition ( Levinson 1996 ) , while others have shown that language reflects cognition ( Pinker 2007 ) . Design is a cognitively intensive activity requiring such tasks as information gathering , spatial manipulation , searching , decision - making , etc . ( Simon 1969 , Gero et af . 1994 , Coley et af . 2007 ) . The cognition required of design suggests that we can take advantage of the relationship between language and cognition to facilitate and understand design . * Corresponding author . Email : shu @ mie . utoronto . ca ISSN 0954 - 4828 print / ISSN 1466 - 1837 online \u00a9 2012 Taylor & Francis http : / / dx . doi . orgl 10 . 1080109544828 . 2011 . 603298 http : / / www . tandfonline . com 272 I . Chiu and LH . Shu In this paper , we present and describe work on stimulating creative concept generation using language . Creativity is an important measure of design effectiveness ( Kan and Gero 2007 ) . While customers seek creative designs , they may not explicitly indicate creativity as a requirement ( Cross 2006 ) . There is general agreement that creativity is essential to design ( Gordon 1961 , de Bono 1992 , Altshuller and Shulyek 1996 , Hubka and Eder 1996 , Adams 2001 , Cross 2006 , Kan and Gero 2007 , Shai et al . 2009 , Yang 2009 , Brown 2008 , etc . ) , and some assert that design necessarily entails creativity ( Holt 1993 , Hubka and Eder 1996 , Hatchuel and WeiI2009 ) . We are specifically interested in the concept generation stage of design because the early stages of design have the largest influence on the final design ( Ullman 2003 , Keller et al . 2009 ) , and creative concepts result in creative and successful designs . We investigate the effects of language stimuli on concept creativity in experiments , where we provided participants with stimulus words that were either oppositely or similarly related to the desired functions ofthe solution . Stimulus words were systematically generated , using a thesaurus and WordNet . Words were chosen as stimuli because words are the smallest unit of language that carry meaning , and thus a natural starting point for our investigations . Verbs were the chosen part of speech for the stimuli because verbs denote abstract actions or functions and not specific forms . Word stimuli provided were verbs in root form , e . g . ' remove ' . Many agree that verbs should be used to model functions for design ( cf . Pahl and Beitz 1996 , Stone and Wood 2000 ) . The opposite / similar relationship was used to generate stimulus words because it is a systematic and well - understood relationship and one of only two valid verb relationships . While we may never fully understand the effects of language on design cognition , insights gained from our empirical results combined with linguistic theory enable us to propose a model of the language and design cognition interactions required to generate more creative concepts . In this paper , we will first review related work in language , design and creativity . Then we will describe and discuss our experiments with respect to the specific effects of different types of word stimuli on concept creativity ( Experiments 1 and 2 ) and on designer behaviour ( Experiment 2 ) . Finally , we will discuss our experiments within an empirical and theoretical context and propose a model explaining the effects of opposite / similar word stimuli on creative concept generation . 2 . Background 2 . 1 . Language and design The connection between language and cognition was first observed by the ancient Greeks , who used the same word , logos , to denote both reasoning and language ( Kalmar and Davidson 1997 ) . The Sapir - Wharf hypothesis dating from the early twentieth century argues that the language of a person determines how he or she understands the world ( Ratner and Gleason 1993 ) . While there is debate over whether cognition influences language ( Levinson 1996 , Li and Gleitman 2002 , etc . ) , or language influences cognition , many agree that language and cognition are closely related ( e . g . Pinker , Fodor , Boaz from Saeed 2003 ) . Researchers such as Chomsky ( 1968 ) , Jackendoff ( 1983 ) and Pinker ( 2007 ) argue that language provides insight into human cognitive processes . In our work , we explore the use of language to stimulate creative design . Language , specifically at the word level , appears ideal as design stimuli , and fits well within early stages of the design process , where the problem is likely to be ill - defined , and exploration of the solution space encouraged . Words are the smallest unit of language that carry meaning , and thus an appropriate starting point for investigating language as design stimuli . While words impose a pre - existing symbol system on the user that can be shared and manipulated ( Bruner 1964 ) , they are not necessarily fixed to a particular form ( Segers 2004 ) , and ambiguity enables ~ . . ~ ~ ~ ~ ~ . _ - ~ - - - - - - - - - - - - - - - - - - - - - - - - Journal ofEngineering Design 273 freedom of interpretation ( Miller et al . 1993 ) . Not only is language connected to reasoning , words also appear connected to our knowledge of the world . In one model of lexical memory , when a specific word is found in the intemallexicon of permanent memory , simultaneously retrieved are properties associated with that word , e . g . meaning , spelling , pronunciation , etc . At the same time , properties not strictly linguistic are also retrieved , e . g . if the word ' elephant ' is retrieved , it might also trigger the common knowledge that elephants never forget ( Carroll 1999 ) . The knowledge retrieved is not necessarily just part of the word - meaning , but may be related to the conceptual knowledge of the world in general . While words seem simple and familiar , word meaning can be complex and ambiguous ( Carroll 1999 ) . A designer ' s familiarity with the complexities oflanguage may make language a useful design tool . While natural language is familiar and ubiquitous to most , it is not generally considered a conventional engineering tool . However , many researchers are investigating the use of natural language as a formal tool to support the engineering design process . For example , language has been used as input in requirements gathering ( Nuseibeh and Easterbrook 2000 ) , concept generation and synthesis ( Thomas and Carroll 1984 , Hacco and Shu 2002 , Nagai and Taura 2006 , Chiu and Shu 2007 , Tseng et at . 2008 ) , design modelling and representation ( Stone and Wood 2000 ) , and design analysis ( Dong et al . 2003 , Dong 2006 ) . Some concept generation and creativity methods explicitly use language as stimuli in an attempt to increase creativity . Thomas and Carroll ( 1984 ) demonstrated that participants who were given 20 pages of semi - random stimulus words generated more creative concepts compared with par ticipants not given stimulus words . Nagai and Taura ( 2006 ) investigated the interpretation of noun - noun combinations for promoting creativity in concept synthesis . Some work in biomimetic design uses functional keywords to systematically retrieve analogies from biological corpora for use as stimuli in engineering design ( Hacco and Shu 2002 , Chiu and Shu 2007 , Cheong et at . 2011 ) . Methods implicitly using language to stimulate creative design include synectics and random input . In synectics , Gordon ( 1961 ) proposes the use of metaphors and similes , which are figures of speech , to draw parallels and connections between disparate topics or domains . Metaphors and similes promote analogical thinking and can allow solutions to be applied from one domain to another . The random input method involves randomly selecting stimulus , e . g . a picture from a catalogue , to relate back to the design problem through a series of word associations . The process of relating the problem to random stimulus , which may be non - obvious and unexpected , may provide new perspectives and thus stimulate creative design ( de Bono 1970 ) . The above examples show that language is frequently used to facilitate design . However , fewer researchers have attempted to examine and model the effect of language on design . Nagai and Noguchi ( 2003 ) developed a model of the thinking path required for creative thinking . In an experiment where designers used keywords while designing , Nagai and Noguchi observed that difficult and remote keywords caused designers to extend their thinking pathways . They concluded that extending thinking pathways may help to realise creative concepts . In our experiments , we observed that oppositely related keywords result in designer behaviours that may increase concept creativity . We then propose a model to explain the effects ofoppositely related stimuli on increasing concept creativity . 2 . 2 . Opposition in design We study opposite versus similar - language stimuli because opposition and opposing relation ships are common in language and reasoning . Antonyms , or opposite words , are universally found in language , and most people demonstrate good intuition in recognising antonym / synonym pairs ( Fellbaum 1993 , Murphy 2003 ) . The antonym / synonym relationship is also one of only 274 1 . Chiu and L . R . Shu two valid verb relationships , with the other being the hypernymjhyponym relationship . The hypernymjhyponym relationship can be thought of as a super - ordinatejsub - ordinate relationship , where words are hierarchically related in either a more general or more specific manner . Design methods using opposition include TRIZ , design - by - analogyjcontrast , argumentation and argumentative negotiation ( Rittel and Webber 1984 , Altshuller and Shulyak 1996 , Fantoni et al . 2006 , Jin et al . 2007 ) . In TRIZ , the Russian abbreviation for the theory of inventive problem solving , the problem at hand is first phrased in contradictions to identify parameters to be improved and those degraded as a consequence . Fantoni et al . ( 2006 ) proposed a method of design - by analogyjcontrast involving the use of synonyms and antonyms as design stimuli . Argumentation and argumentative negotiation involve the verbalisation ofcontradictory demands and then a move towards agreement to produce novel solutions in collaborative engineering ( Rittel and Webber 1984 , Jin et al . 2007 ) . Hubka and Eder ( 1996 ) even speculate that opposites and dissimilarities may contribute to creativity in engineering design through the resolution of ' cognitive dissonance ' , e . g . resolving ideas from intuitive versus intellectual modes of thinking . Festinger , the originator of the theory of cognitive dissonance , theorised tensions occur when an individual becomes simultaneously aware of two inconsistent thoughts . To resolve the tensions , the individual must implement change ( Myers 1999 ) , thus creating new , alternative solutions . In design , resolving tensions between the problem statement and other factors , e . g . the degraded parameter versus the improved parameter in TRIZ , and stimuli oppositely related to the design problem , may lead to creative design . 2 . 3 . Measuring creativity in design Many approaches have been developed to assess the creativity ofa concept or idea . In general , most agree that creativity is multi - dimensional ( Torrance 1974 , Amabile 1983 , Shah et al . 2000 , etc . ) and that using a single measure ofcreativity may result in identifying strange or even incorrect ideas as being creative . For example , in Wilson ' s ( 1951 ) method ofstatistical infrequency , infrequent ideas are considered novel , and therefore creative . Two generally agreed upon measures of creativity within science and engineering are novelty and usefulness , defined below : Novelty : A creative idea must contain some degree of newness , originality or surprise ( Wilson 1951 , Torrance 1974 , Hubka and Eder 1996 , Howard et al . 2008 , Shai et al . 2009 , Brown 2008 ) . Usefulness : A creative idea must contain some degree of appropriateness and value ( Besemer and Treffinger 1981 , Amabile 1989 , de Bono 1992 , Akin andAkin 1998 , Howard et al . 2008 , Shai etal . 2009 , etc . ) . Amabile ( 1989 ) defines appropriateness in the sciences as being correct . Usefulness is especially emphasized in the engineering literature , often in the context of functionality ( Pahl and Beitz 1996 , Dieter 2000 , Shah et al . 2000 , Ullman 2003 ) . Some researchers assert that creative ideas only need to be novel and useful , e . g . Amabile ( 1989 ) and Akin and Akin ( 1998 ) . However , novelty and usefulness alone may not sufficiently measure creativity , especially at the abstract conceptual level . Many suggest that the wholeness , clarity , elaboration , or cohesiveness of an idea must also be considered : Cohesiveness : A creative idea must contain some degree of wholeness , elaboration , detail , style and clarity ( Torrance 1974 , Besemer and Treffinger 1981 , Hubka and Eder 1996 , Adams 2001 , Kudrowitz and Wallace 2010 ) . Other measures of creativity may also be used to assess the effectiveness of a creativity method or individual . These measures can include fluency , or the quantity of concepts , generated by an individual or the creativity method ( Torrance 1974 , Shah et at . 2000 , Yang 2009 ) and variety , or the number of different categories of concepts generated ( Torrance 1974 , Shah et at . 2000 ) . This paper focuses on the use of the direct measures of novelty , usefulness and cohesiveness for the evaluation of each individual concept . 2 . 4 . Previous work Journal ofEngineering Design 275 Previous exploratory experiments showed that designers provided with opposite - stimulus words tend to generate concepts that were more novel . These previous experiments are summarised below . The first exploratory experiment was a pen - and - paper study where participants indicated their concepts on worksheets . Forty - two participants were provided with opposite and simi lar words simultaneously for a series of four problems and then instructed to use the words as stimuli for design . For three of the problems , participants who chose at least one opposite stimulus word generated more novel concepts ( Chiu and Shu 2008a ) . Novelty was determined using statistical infrequency , where less frequently occurring concepts were deemed more novel ( Wilson 1951 , Shah et al . 2000 ) . However , because all participants were provided with opposite - and similar - stimulus words simultaneously , results may have been confounded , i . e . were participants affected by only the opposite words , or by the pairs of opposite / similar words ? From this experiment , we were also unable to gain further insight into how par ticipants used language stimuli due to the nature of data collected from pen - and - paper experiments . The second exploratory experiment was a small - scale between - subjects experiment where par ticipants verbalised all thoughts as they designed . Six participants received either opposite words or similar words while generating concepts for one problem . Those receiving opposite stimuli generated more novel concepts , however the difference was not significant . Novelty in this exper iment was determined by two independent human raters . Two raters were recruited because two is the minimum number of human raters required for judgement tasks ( Landis and Koch 1977 ) . Spearman ' s correlation between the two raters was r = 0 . 51 , p = 0 . 054 \" - ' 0 . 05 , showing a large , borderline significant agreement between the two raters ( Chiu and Shu 2008b ) . No training was provided to the raters in this exploratory experiment . Using the session transcripts from the second experiment , we were able to conduct an explicit content analysis to examine participants ' language use and behaviours with respect to different stimulus types . Specifically , we examined the stimulus part - of - speech ( paS ) , to determine whether a given stimulus word was used as a noun , verb or adjective . While stimulus words were given as root verbs , e . g . ' remove ' and not ' removing ' or ' removed ' , participants were not told that the stimuli were verbs . We found that opposite - stimulus participants used stimulus words significantly more often as verbs than similar - stimulus participants . Furthermore , in this second exploratory experiment , we found that opposite - stimulus participants tended to introduce more new words and phrases ( NWPs ) in their concept generation process , but not significantly so . NWPs are identified by comparing words and phrases given in a problem statement with the words and phrases generated by participants in relationship to the stimulus words . NWPs may represent new concept elements that have been expressed , or lexicalised , within concept generation . Increased NWP introduction appears linked to the use of stimuli as verbs rather than nouns . Increased NWPs appear advantageous for concept generation because NWPs may form the basis ofcreative concepts . An expanded verbal protocol experiment , i . e . in terms of participants and problems , would allow for more thorough analysis . Overall , previous experiments showed that opposite stimuli appear to increase concept novelty , one of the measures that contribute to total concept creativity . These preliminary results serve as motivation for current experiments ( described in Sections 3 and 4 ) that overcome limitations of previous work described above . On the basis of our preliminary results , we hypothesise the following : ( 1 ) Opposite - stimulus words increase concept creativity in terms of all creativity measures ; ( 2 ) Opposite - stimulus words used as verbs increase the introduction of NWPs _ _ _ _ _ _ _ _ _ _ _ _ u _ 276 I . Chiu and LH . Shu The following sections will describe two experiments where we focus on further quantifying the above two hypotheses . 3 . Experiment 1 : a between - subjects pen - and - paper experiment Experiment 1 was a fully between - subjects pen - and - paper experiment where participants gener ated concepts for a series of four problems : ( 1 ) Bushing problem ( 2 ) Egg problem ( 3 ) Grinding problem and ( 4 ) Sunflower problem . Participants generated concepts under one of the following experimental conditions : ( 1 ) Opposite stimulus ; ( 2 ) Similar stimulus . Four independent raters were recruited to judge the concepts based on the creativity components of novelty , usefulness and cohesiveness . For two of the experiment problems , the Sunflower and Egg problems , raters judged opposite - stimulus concepts as being significantly more novel , useful and cohesive than similar - stimulus concepts . For the other two problems , the Bushing and Grinding problems , there were no significant differences in any of the creativity component scores . Details are given below . 3 . 1 . Method 3 . 1 . 1 . Participants and procedure Participants consisted of nine graduate engineering students from the Department of Mechanical and Industrial Engineering at the University ofToronto . At the time ofthe experiment , participants were enrolled in a graduate design course . Participants consisted of two females and seven males with an average age of 27 . 2 years ( sd = 5 . 38 ) . Eight out of nine participants indicated they had industry design experience ranging from a few months to five years . Participants were randomly assigned to one of two experimental conditions where they gen erated concepts using either opposite stimuli or similar stimuli . All participants completed four problems , but in a different , random order as determined by a random number generator . Partici pants were allotted a total time of 10 min per problem to review given stimulus words ; to select stimulus words ; and to generate and describe their concepts using selected stimulus words on provided worksheets . The total experiment duration was 40 min . The worksheets were collected for analysis . The exact instructions are given below : This is an experiment investigating the use of stimuli in concept generation . The following are four unrelated design problems . For each design problem , a set of related word stimuli is supplied . For each problem : ( I ) Review the problem and associated word stimuli . ( 2 ) Perform a functional decomposition , e . g . what needs to be done ? ( 3 ) Select the word ( s ) you want as stimuli and indicate your selection ( s ) . Each stimulus set is only relevant to the associated design problem . ( 4 ) Use selected word ( s ) to develop concepts to solve the design problem . Ifyou determine that you cannotcomplete a concept using your selected word ( s ) , you may select another word . ( 5 ) Please consider each problem in the order given . You have 10minutes for each problem . Record all concepts . You may write , sketch , calculate , etc . , on your worksheets . Pen - and - paper experiments are a fairly time - efficient method for collecting design data . Others have used pen - and - paper experiments to study various aspects ofconceptual design such as effects of random stimulus words , analogical similarity , generation of form alternatives and sketching abilities ( Thomas and Carroll 1984 , Tseng et at . 2008 , Yang 2009 , Corremans 2011 ) . Journal ofEngineering Design 277 3 . 1 . 2 . Experiment problems and stimuli Table I . Summary of problems and stimulus sets for Experiment I . 3 . 1 . 3 . Concept evaluation Concept evaluation for our previous studies ( described in Section 2 . 4 ) was limited to the evaluation of concept novelty , using statistical infrequency or human raters . Since the creativity literature suggests that creativity is more than merely novelty , and that creativity is ultimately a human judgment , e . g . from a customer ' s point of view ( Cross 2006 , Brown 2008 ) , we recruited human Opposite Fill InsertJoinCombine Roughen AddClogReplace Empty WithdrawDisconnectDivide Stimulus words Similar Smooth SubtractCleanRemove Problem description and decomposition Sunflower seed oil is a nutritious and valuable commodity in sub - Saharan West Africa . Mechanical presses to make oil from the shelled seeds exist locally , but machines to remove the shells do not . At present , there exists no alternative to the laborious and time - consuming process of shelling the sunflower seeds individually by hand before loading them into the press . Develop a concept for shelling sunflower seeds that can be used locally with minimal resources ( DTM 2006 ) Grinding of metals is quite common to obtain a fine surface finish and tight tolerances . But when grinding soft materials such as rubber or plastic , the grinding wheels quickly become clogged . Repeated dressings ( sharpening and shaping of the grinding wheel ) do not help . Develop concepts that will enable surface finishing ( with or without grinding wheels ) to be used on soft materials ( Kosse 2004 ) Participants were provided with four problems and related - stimulus sets on worksheets . Stimulus words were obtained by first performing a functional decomposition of the problem . Functional decomposition identifies the functions , i . e . physical actions or behaviours , required to transform an initial state into the desired final state ( Pahl and Beitz 1996 ) . A high - level functional decomposition yielded functional keywords that were then used to generate similar - or opposite - stimulus words using a thesaurus ( Merriam - Webster . com 2008 ) and WordNet 3 . 0 ( Princeton University 2008 ) . Problems and stimuli are summarised in Table 1 . For the Sunflower problem , the required high - level functions to transform whole seeds into oil were ' extracting ' the seeds from the shell , and then ' separating ' the seeds from the shell fragments for the production of oil . From the original functional keyword ' extract ' , we generated using a thesaurus and WordNet similar words ' empty ' and ' withdraw ' , and opposite words ' insert ' and ' fill ' . From ' separate ' , similar words generated were ' disconnect ' and ' divide ' , and opposite words were ' join ' and ' combine ' . The original functional keywords were not provided to participants . Problem Sunflower - seed shelling Soft - material grinding Egg orientation Develop concepts to automatically orient raw chicken eggs with the pointed ends all facing one direction ( Kosse 2004 ) SelectDetectPivot Move RejectMiss FixRestrain Bushing - and - pin assembly Parts that are automatically mated , e . g . a bushing and a pin , must be positioned so that their axes coincide . Using chamfers on mating parts does not solve the alignment problem . Develop a concept to centre mating parts that does not require high positioning accuracy ( Kosse 2004 ) StraightenMatchInjectInstall SkewMixEjectExtract 278 0 1 2 Not novel I . Chiu and L . H . Shu Figure I . Rating scale for novelty . Reprinted with pennission . Copyright ASME 20IO . Table 2 . Example anchor concepts for Sunflower problem . raters to judge concept creativity for this study . Human raters assessed concept creativity using all three components described previously : Novelty , usefulness and cohesiveness . Each component was scored on an II - point scale between a and 10 . The rating scale for novelty is illustrated in Figure 1 , with the same numeric scale applied to usefulness and cohesiveness . For this experiment , four independent raters were recruited to score each concept . Raters con sisted of one female and three males , all with an engineering background and interest in design . Raters were not provided with the identity of the designers , nor the stimulus condition under which the concepts were generated . Concepts were presented to raters in random order . Raters were provided with low anchor and high anchor concepts , i . e . ' not novel / useful / cohesive ' and ' very novel / useful / cohesive ' concepts obtained from previous experiments , and instructed to evaluate all concepts based on those anchors . This method of anchoring and obtain ing human judgements is known as direct scaling and is commonly used in psychophysics , a branch of psychology that deals with relating physical stimuli with mental phenomena ( Engen 1972 ) . The direct scaling method has been applied to rating pleasantness of smells , perception of heaviness , and even to rating of emotions and beauty . Examples of anchor concepts for the Sunflower problem are provided in Table 2 . Low / high Low High Novelty Place whole seeds in machine . Divide the shell by machine with chisel / knife edge and open like hinge securing shell . Empty the seeds from the shell and discard the shells from the hinge device Combine two wheels with sticky surfaces to crack the seeds . Shells stick to the surface and get carried away Usefulness Machine grips two sides ofseed . Seed is cut into half ( divide ) . The seed is emptied using vibration ( empty ) . Shells are discarded . Separate seeds processed as before Whole seeds go into a hopper . Use edible solvent to dissolve shells . Clean seeds . Dry seeds . Grind to get oil Cohesiveness Divide the shell into two . Empty shell and remove seed from shell Refer to high usefulness concept . High usefulness concept is also a high cohesiveness concept 3 . 2 . Results 3 . 2 . 1 . Inter - rater agreement Kendall ' s W concordance coefficient was calculated to measure inter - rater agreement . Kendall ' s W measures the agreement ofmore than two raters scoring N entities and ranges from 0 , signifying no agreement or random ratings , to 1 , signifying consensus between the multiple raters . As Kendall ' s W is related to Spearman ' s correlational coefficient , Kendall ' s W can be interpreted similarly to a correlation coefficient , where 0 . 1 , 0 . 3 and 0 . 5 are small , medium and large agreements , respectively ( Siegal 1956 ) . The inter - rater agreements are given in Table 3 . Although the coefficients above suggest medium to large degrees of agreement , the sample sizes are too small to draw confident conclusions ( p \u00bb 0 . 05 ) . Upon aggregating ratings for all concepts from all four problems , the coefficients indicate medium to large degrees of agreement that are significant , or borderline significant , as seen in Table 4 . Journal ofEngineering Design 279 Table 3 . Inter - rater agreement for Experiment 1 concepts . Problem Novelty Usefulness Cohesiveness 3 . 2 . 2 . Creativity results Therefore , based on the aggregated results , there is significant agreement among the raters ( p approximately 0 . 05 or less ) . In this section , we will first present examples of participant concepts and resulting rater scores to illustrate the experimental and concept rating methodology . Then , the overall results of the quantitative comparisons are provided . Three concept examples from the Sunflower problem are given in Table 5 along with associated average rater scores . Concepts are unedited and as collected directly from the worksheets . Stimulus words used by participants are italicised for reference . W = 0 . 6181 X 2 ( 8 ) = 14 . 83 P = 0 . 06 W = 0 . 5278 x 2 ( l4 ) = 22 . 17 P = 0 . 08 W = 0 . 4634 x 2 ( l5 ) = 20 . 85 P = 0 . 14 W = 0 . 497 X 2 ( 12 ) = 17 . 89 P = 0 . 12 Cohesiveness W = 0 . 4908 X 2 ( 52 ) = 76 . 5592 P = 0 . Ql5 Usefulness W = 0 . 5482 X 2 ( 8 ) = 13 . 16 P = 0 . 11 W = 0 . 6502 X 2 ( 14 ) = 27 . 31 P = 0 . 02 W = 0 . 5024 x 2 ( l5 ) = 22 . 61 P = 0 . 09 W = 0 . 3261 X 2 ( 12 ) = 11 . 74 P = 0 . 47 W = 0 . 4426 X 2 ( 52 ) = 69 . 052 P = 0 . 06 Novelty W = 0 . 507 X 2 ( 52 ) = 79 . 091 P = 0 . 009 W = 0 . 3711 X 2 ( 8 ) = 8 . 91 P = 0 . 35 W = 0 . 5531 x 2 ( l4 ) = 23 . 23 p = 0 . 06 W = 0 . 6004 x 2 ( l5 ) = 27 . 02 p = 0 . 03 W = 0 . 4839 x 2 ( l2 ) = 17 . 42 P = 0 . 13 Bushing and pin ( N = 9 ) Egg ( N = 15 ) Problem Grinding ( N = 16 ) Sunflower ( N = 13 ) Aggregated results ( N = 53 ) Table 4 . Kendall ' s W for aggregated results of Experiment I concepts . Table 5 . Example concepts and creativity scores for the Sunflower problem . Avg . rater score ( N = 4 ) Example Concept Participant experimental condition Nov . Use . Coho Concept 2 : To divide : Orient the seed so that it lies flat , then use a knife to split along the flat side of the shell . Or cut into half , then use vibration or gravity to empty , blow it out to extract inner part Concept 13 : ( 1 ) Air hose insert into seed . ( 2 ) Airjills the seed , cracking the shell using pressure . ( 3 ) The newly cracked seed is collected and joined with the rest of the seeds from before Concept 16 : Second concept - Grind shell and seed together . Then find a fluid , ( hopefully water will do ) that has the proper density to float the lighter shells to the surface and leave the heavier seeds on the bottom Average concept creativity for all Sunflower concepts ( N = 13 ) Similar Opposite Opposite 2 8 5 . 25 4 . 35 2 . 5 4 . 5 7 . 5 4 . 88 2 . 25 5 . 25 7 . 5 5 . 0 280 I . Chiu and L . R . Shu opp Sim . Opp . Cohesiveness Cohesiveness . 00 ' - . . ~ - ~ - - , - - - ~ . _ - 4 . 00 2 . 00 6 . 00 4 . 00 . 00 . L\u2022 . ~ I \u2022 . . ~ \u2022 . \u2022 . \u2022 . 2 . 00 Usefulness 4 . 00 opp Sim . Opp . Usefulness 2 . 00 6 . 00 6 . 00 opp Nov ~ lty r I - , ~ I - I i I , I I I ! i I 1 sim opp Sim . Opp . . 00 \" . - . \" - - ~ _ . - \" - - ~ . . . . . . . . - . 00 ' ; ' \u00a7 ' \" 6 . 00 \u2022\u2022 OJ ! . . . = S - : : I = ~ . 2 : c : z = 9 ; OJ ! 1lol OJ ! ~ ~ 2 . 00 Concept 2 was generated under the similar - stimulus condition , while concepts 13 and 16 were generated under the opposite - stimulus condition . Note that concept 16 does not incorporate stimu lus words . However , the participant had indicated that this was his second concept for this problem , and stimulus words were used in his first concept for this problem ; demonstrating he had reviewed the stimulus words . Concept 2 was rated as the least creative concept , and concepts 13 and 16 were rated as being above - average concepts . While raters judged concept 13 to be the most novel , concept 16 was rated as more useful and cohesive . This may reflect that raters considered batch processing of multiple seeds simultaneously ( concept 16 ) to be more practical than processing of individual seeds ( concept 13 and concept 2 ) . For each problem , rater scores for concepts were averaged to facilitate analysis , and independent T - tests were conducted to compare concept novelty , usefulness and cohesiveness . Rater scores were averaged because our main interest is in designer behaviour , not rater behaviour . For two of the experiment problems , the Sunflower and Egg problems , raters judged opposite - stimulus concepts as being more novel , useful and cohesive than similar - stimulus concepts . The T - tests showed that these differences were either significant , or borderline significant , in all but the Egg problem novelty scores . This can be seen in Figure 2 and Table 6 . Sim . Opp . Sim . Opp . Sim . Opp . Figure 2 . Graph of novelty , usefulness and cohesiveness results for the Sunflower and Egg problems . Table 6 . T - test results for Sunflower and Egg concept creativity . Sunflower Egg Creativity Sim . Mean Opp . Mean Sim . Mean Opp . Mean Component Rating ( N = 7 ) Rating ( N = 6 ) t ( ll ) p ( one - tail ) Rating ( N = 9 ) Rating ( N = 6 ) t ( 13 ) p ( one - tail ) Novelty 3 . 51 5 . 33 - 1 . 80 0 . 048 5 . 00 5 . 83 - 0 . 96 0 . 16 Usefulness 3 . 93 6 . 00 - 3 . 32 0 . 004 3 . 75 5 . 58 - 2 . 98 0 . 006 Cohesiveness 4 . 15 5 . 97 - 2 . 25 0 . 02 3 . 97 5 . 50 - 1 . 80 0 . 048 Journal ofEngineering Design 281 For the other two problems , the Bushing and Grinding problems , there were no significant differences in the creativity component scores , and thus different stimuli had no significant effects . 3 . 2 . 3 . Language results 3 . 3 . Experiment 1 discussion While pen - and - paper experiments are not the most conducive to examining language use , we were able to examine aggregated frequency ofstimulus use by counting and categorising stimulus words used by the participants . Many instances ofstimulus words found on worksheets were merely listed and not used in a phrase or sentence . When these ' unknown ' uses of stimuli are subtracted from the frequency totals , opposite - stimulus concepts incorporate significantly more stimulus words , t ( 5l ) = 2 . 791 , p = 0 . 0035 < 0 . 05 , than similar - stimulus concepts . This can be seen in Figure 3 , and suggests that using more opposite - stimulus words may result in more creative concepts . There was strong inter - rater agreement with regard to aggregated concept creativity measures . This is illustrated by the below - average usefulness scores for concepts involving individual seed processing ( concepts 2 and 13 ) , versus a higher score for concepts involving batch processing of multiple seeds ( concept 16 ) . This may reflect that batch processing is seemingly more practical . However , it should be noted that strong agreement does not necessarily mean that the raters were correct ( Siegal 1956 ) , nor does it necessarily account for other factors which may have influenced concept creativity . Despite any drawbacks associated with human ratings of creativity , creativity is a human judgment , and what is considered creative is not necessarily ' correct ' or ' incorrect ' . For two of the problems , the Sunflower and Egg problems , opposite - stimulus concepts were significantly more creative than similar - stimulus concepts . However , for the Bushing and Grinding problems , opposite - and similar - stimulus concepts were found to be equally creative . Results may reflect the difference in problem type and problem novelty . The Sunflower and Egg problems are general - domain problems that may be more novel to the participants , while the Bushing and Grinding problems are more technically oriented and should be familiar to most engineers , i . e . participants and raters . The results shown above suggest that opposite - stimulus words can Error Bars show 95 % CI of Mean 1 . 50 l : ~ l : ~ l : ~ 1 . 00 o ~ l : QI : : : : l cr ~ ~ . 50 Z : E N - o N . . . . Q ) . g . . . . U o N o \" T M o - Opp Stim Sim Figure 3 . Aggregated frequency of stimulus word - use with no unknown uses of stimulus words . 282 I . Chiu and LB . Shu stimulate more creative concepts for general - domain problems or problems that may be more novel to the designers . In cases where problems are more technically oriented , experience may neutralise the effects of stimuli . Both rater and participant experience affect creativity ; experience is an important factor in individual creativity ( Amabile 1989 , Akin 1990 ) . It is difficult to determine if participant concepts are based on designs encountered elsewhere , and difficult to separate historically creative concepts , concepts that never existed before in the world , from personally creative concepts , concepts that never existed before in the participant ' s mind , but existing in the world ( Amabile 1983 ) . In addition , a more experienced rater may tend to judge a participant ' s personally creative concepts as less creative even though they are creative and novel in the context of the experiment . A less experienced rater may judge the same concept as more creative . This may have been the case with the technically oriented problems , where both participants and raters should be familiar with a variety of alignment / insertion and material removal solutions . Raters may also judge concepts to be less creative if the participant explicitly refers to existing designs , even if concepts appear creative . Overall , it would be difficult to control for participant and rater experience . Regarding stimulus - word use , results show that opposite - stimulus concepts incorporated more stimulus words within the concept . This supports that opposite - stimulus participants were better able to use stimuli to introduce and develop new ideas in concept generation , which suggests that opposite - stimulus words are more useful for stimulating creative concepts . A more comprehensive verbal protocol , or talk - out - loud , experiment may provide further insight into how opposite stimulus words increase concept creativity . The next section describes such an experiment . 4 . Experiment 2 : An expanded verbal protocol experiment Experiment 2 is a verbal protocol experiment involving 14 participants generating concepts for three problems under one of the following experimental conditions : ( 1 ) No stimulus ( control ) ; ( 2 ) Similar stimulus ; ( 3 ) Opposite stimulus . Language use in relation to participant behaviour , as well as concept creativity , is examined in detail in Experiment 2 . 4 . 1 . Method 4 . 1 . 1 . Participants and procedure All 14 participants are fluent English speakers recruited from the Department of Mechanical and Industrial Engineering at the University of Toronto . Participants consisted of 13 males and one female , ranging from fourth - year undergraduate students to second - year PhD students . In individual sessions , participants first completed three training problems to habituate them to verbalising . Then , participants were instructed to verbalise all thoughts as they completed a series of three design problems . Fifteen minutes were allotted for each problem for a total experiment duration of45 min . Ten participants were provided with stimulus words , either opposite or similar words , while four were not provided with any stimuli . Ofthe 10 stimulus participants , five switched stimulus type between problems . Table 7 details the experimental design . Similar to the previous pen - and - paper experiment , participants in verbal protocol experiments were provided with worksheets presenting the problem statements and stimulus words . In contrast to pen - and - paper experiments , participants in verbal protocol experiments were also instructed to Journal ofEngineering Design 283 Table 7 . Experimental design for Experiment 2 . Stimulus participants Control participants Condition subtotals Prob . TH IS VT SW lL DRO DR UG MM DH DL 1M AF AP Opp . Sim . None Bushing S S S S S S 0 0 0 0 N N N N 4 6 4 Snow 0 0 0 S S S S 0 S 0 N N N N 5 5 4 Coal S S S S S S 0 0 0 0 N N N N 6 4 4 Notes : Reprinted with permission . Copyright ASME 2010 . S : Similar stimuli ; 0 : Opposite stimuli ; N : No stimuli . verbalise all thoughts as they worked on the design tasks . The sessions were recorded and fully transcribed for analysis . Verbal protocols are common for studying cognitive processes such as human machine interac tion ( Bainbridge et al . 1968 ) , medical decision - making ( Lutfey et al . 2008 ) and are considered a relatively objective and appropriate method for studying phenomena in design ( Cross et al . 1996 , Hubka and Eder 1996 , McNeill et al . 1998 , Chakrabarti et al . 2004 , Cross 2006 , Visser 2006 ) . In fact , verbalisation may be the most popular method for studying design cognition ( Coley et al . 2007 ) . However , there are some debates associated with verbal protocol studies . Nisbett and Wil son ( 1977 ) have questioned the accuracy of the data obtained from verbalisations as they have found that verbal reports do not necessarily match recordings of the reported event . Ericsson and Simon ( 1993 ) , on the other hand , contend that as long as verbalisations are immediate and do not require recall from memory , verbalisations accurately describe events being reported . Addi tionally , since verbal protocol experiments are a time and resource intensive method , the number of participants involved is usually small . A survey of design studies using this method reveals that the typical number of participants is low , e . g . 4 participants in a design cognition modelling study ( Benami and Jin 2002 ) , 8 participants in a personal creativity and design activities study ( Kim et al . 2011 ) , 10 participants in a design education study ( Atman and Bursic 1996 ) , and 20 participants in a design stimulation study ( Jin and Benami 2010 ) . Our sample size of 14 is reasonable considering the range of sample sizes typical of verbal protocol experiments . 4 . 1 . 2 . Problems and stimuli Participants were provided with three problems and related stimulus sets on worksheets . Specif ically , the problems were ( 1 ) Bushing - and - pin assembly ( 2 ) Snow insulation of houses and ( 3 ) Coal storage . Again , stimulus sets for the opposite and similar stimulus conditions were verbs in the root form generated by using a combination of a thesaurus ( Merriam - Webster . com 2008 ) and WordNet 3 . 0 ( Princeton University 2008 ) , starting from the original functional keywords . Some keywords do not have antonyms in the resources consulted , e . g . ' to insulate ' from the snow insulation problem , so opposite stimuli were generated based on opposition to the problem itself , e . g . ' to pack ' , as the problem specifically stated that ' packing ' of snow is undesirable . As gen erating opposite and similar verbs is not possible for all keywords , and oppositely related words are sparse for verbs to start with , this strategy was used for the other problems as well . Problems and stimulus sets are given in Table 8 . 4 . 1 . 3 . Concept identification and evaluation To reduce bias , design sessions were transcribed by an independent transcriptionist and an indepen dent concept reviewer was recruited to identify and code concepts from the free - form transcripts and worksheets . The following is a transcript excerpt representing approximately 30 s of one experiment session . Lines are numbered for referencing during analysis . 284 I . Chiu and L . H . Shu Table 8 . Summary of problems and stimulus sets for Experiment 2 . Prob . Bushing - and - pin assembly Snow Coal Problem description Parts that are automatically mated , e . g . a bushing and a pin , must be positioned so that their axes coincide . Using chamfers on mating parts does not solve the alignment problem . Develop a concept to centre mating parts that does not require high positioning accuracy ( Kosse 2004 ) In Canada , snow is readily available in winter and has good insulating qualities due to the amount of air in it . However , if the snow is packed to the point , it becomes ice , it is less insulating due to the loss of air . Come up with a concept to enable snow to be used as an additional layer of insulation for houses in the winter Clean coal and clean coal combustion technologies make it possible to generate cleaner electricity . That , combined with the increasing cost of oil and natural gas , power plant operators may consider converting or reconverting their power plants from oil or natural gas back to coal . However , there may not be enough land area near the plant that can be used for on - the - ground coal storage . Propose alternative solutions to a conventional coal pile ( adapted from Dieter 2000 ) Opposite stimulus words Original keywords : Opposite of align and insert Stimulus words : Change , disorder , disarrange , scramble , randomise , misalign , tumble , skew , move , expel , pull , eject , evict Original keywords : pack and compact Stimulus words : Arrange , bundle , change , compress , constrict , contract , force , impact , move , push , squeeze , tighten , wad Original keyword : opposite of store Stimulus words : Abandon , discard , discharge , dispense , disperse , dispose , distribute , export , remove , scatter , spread , waste Similar stimulus words Original keywords : align and insert Stimulus words : Inject , transplant , sandwich , connect , skew , mount , misalign , attach , join , reorient , adjust , modify , match Original keywords : insulate and surround Stimulus words : Blanket , control , cover , defend , enclose , immerse , pack , preserve , prevent , restrain , restrict , submerge , touch Original keyword : store Stimulus words : Accumulate , collect , displace , distribute , feed , give , heap , keep , place , supply , transfer , withhold Note : Reprinted with permission . Copyright ASME 2010 . ( l ) I think the obvious . . . ( 2 ) the first thing that comes to mind is that you ' d like blanket the house . . . uh ( 3 ) essentially blanket the house in a layer , in a thin layer ofsnow . . . um ( 4 ) If the snow is packed to the point that it becomes ice . . . ( 5 ) I guess , you ' d obviously try to figure out what amount ofpacking you ' d have to do ( 6 ) to restrict the snow from becoming ice due to over packing . Finished transcripts were corrected for minor spelling errors , e . g . ' chamfer ' for ' camphor ' , ' pedal ' for ' petal ' , but were otherwise not annotated nor changed . Concepts were identified by reviewing transcripts and worksheets . Concepts identified by the independent reviewer were compared with the concepts identified by the investigators who also - - - _ . _ - _ . Journal ofEngineering Design 285 reviewed all worksheets and transcripts . In cases where there was disagreement between the inde pendent reviewer and investigators , both identified concepts were added to the set ofconcepts . For example , in a transcript segment from the Coal problem , both the independent reviewer and the investigators identified an ' underground storage concept ' in which the coal would be stored under ground . However , only the independent reviewer also identified a ' storage pile ' as a concept . The investigators did not regard the ' storage pile ' as a concept because the problem statement specifi cally required concepts other than a ' conventional coal pile ' . Although the investigators disagreed with the independent reviewer with regard to the identification ofthe ' storage pile ' as a concept , the ' storage pile ' concept was added to the set of concepts to be evaluated by the raters . This concept identification process helped to ensure that all possible concepts were included for evaluation . The independent reviewer also summarised participants ' instances of similar concepts into a single concept type . For example , in one transcript segment for the Coal problem , multiple refer ences to the ' tower concept ' or ' condominium concept ' are in fact only one concept that involved storing coal in a tall structure . A total of 195 concepts were identified between all participants and problems , 59 , 59 and 77 concepts for the Bushing , Snow and Coal problems , respectively . Concept creativity was evaluated using the anchoring and direct - scaling method developed and described in Experiment 1 . Three raters were recruited and consisted ofone female and two males , all with knowledge and interest in engineering design . Raters were not provided with the identity of the designers , nor the stimulus condition under which the concepts were generated . Concepts were presented to the raters in random order . 4 . 1 . 4 . Analysis First , inter - rater agreeability was calculated using Kendall ' s W . Then , rater scores for each concept were averaged , and all concept scores for the same participant were aggregated to facilitate analysis . This produces an aggregated novelty , usefulness and cohesiveness score for each of the 14 participants . Aggregated scores were analysed using a mixed - model analysis of variance ( ANaYA ) . As in Experiment 1 , rater scores were averaged to facilitate analysis because our main interest is not in rater behaviour , but in participant behaviour . Because five participants switched stimulus type between problems during the experiment ( identified as TH , JS , YT , MM and DR in Table 7 ) , pseudo - replicates were created to model these participants as independently contributing to each stimulus condition , effectively increasing the sample size to 19 from 14 . This is a common technique to deal with scenarios where not all participants contribute independently to only one experimental condition over multiple trials . Generally , the use of pseudo - replicates results in a conservative estimate of differences ( L . Duquette , personal communication , 2009 ) . 4 . 2 . Results 4 . 2 . 1 . Inter - rater agreement Kendall ' s W was calculated for each of the problems to examine inter - rater agreement and shown in Table 9 . All values for W show a medium to large agreement between the raters , and are statistically significant , p < 0 . 05 . Therefore , there is significant agreement between the raters . 4 . 2 . 2 . Creativity results Overall , raters judged opposite - stimulus concepts to be more creative than similar - stimulus con cepts . This difference was significant , or borderline significant , for all three creativity metrics . See - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 286 I . Chiu and L . H . Shu Table 9 . Kendall ' s W for Experiment 2 concepts . Problem Novelty Usefulness Cohesiveness Bushing ( N = 59 ) W = 0 . 62 W = 0 . 53 W = 0 . 57 X 2 ( 58 ) = 143 . 61 X 2 ( 58 ) = 122 . 76 X 2 ( 58 ) = 131 . 28 p < 0 . 0001 p < 0 . 0001 p < 0 . 0001 Snow ( N = 59 ) W = 0 . 55 W = 0 . 60 W = 0 . 65 X 2 ( 58 ) = 127 . 25 X 2 ( 58 ) = 138 . 49 X 2 ( 58 ) = 150 . 17 p < 0 . 0001 p < 0 . 0001 p < 0 . 0001 Coal ( N = 77 ) W = 0 . 51 W = 0 . 41 W = 0 . 47 X 2 ( 76 ) = 153 . 99 X 2 ( 76 ) = 126 . 13 x 2 ( 76 ) = 143 . 68 p < 0 . 0001 p = 0 . 0003 p < 0 . 0001 Notes : Reprinted with permission . Copyright ASME 2010 _ Shaded rows show significant or borderline significant differences . a Adjusted for effects of problem order - Table 10 and Figure 4 for planned contrasts and interaction graphs of all creativity results . Note the lines in the interaction graph do not signify a relationship , but assist in visualising variable effects and interactions . For novelty , a significant main effect was found for Stimulus Type F ( 2 , 27 . 58 ) = 7 . 09 , p = 0 . 003 , p < 0 . 05 . Planned contrasts comparing individual experiment conditions , e . g . opposite - stimulus concepts versus no - stimulus concepts , show a significant difference between opposite - stimulus and similar - stimulus concepts , t ( 27 . 65 ) = - 3 . 02 , p = 0 . 0025 , p < 0 . 05 . For usefulness and cohesiveness , planned contrasts show that opposite - stimulus concepts are border line significantly more useful and cohesive than similar - stimulus concepts , t ( 31 . 5l ) = - 1 . 61 , P = 0 . 059 , p ' \" ' - ' 0 . 05 and t ( 29 . 42 ) = - 1 . 62 , p = 0 . 058 , p ' \" ' - ' 0 . 05 , respectively . Problem order , the order in which problems were completed , was found to have an effect on cohesiveness and was corrected in the planned contrasts . For novelty , usefulness and cohesiveness , planned contrasts show no significant difference between opposite - stimulus and no - stimulus ( control ) concepts . Overall , the ANOVAs and planned contrasts support the original hypothesis that opposite stimulus concepts are more novel , useful and cohesive than similar - stimulus concepts . However , we also observe two results that contradict much of the literature with regard to design stimulation : Contrast t - and p - values Expt . Condo 2 Comparison of estimated means Expt . Condo 1 Planned contrast results for concept novelty , usefulness and cohesiveness . Nov . Use . Coho Table 10 . ( 1 ) Opposite - stimulus and no - stimulus concepts were found to be equally creative ; ( 2 ) No - stimulus ( control ) concepts were found to be more creative than similar - stimulus concepts . These results will be further discussed in a later section . Journal ofEngineering Design 287 6 . , - - - - - - - - . . , . - - - - - - - t ' 4 + - - - - - - - - r ~ ~ - - - = - - - . . ~ ~ i ~ . . . . . . None - Sim . - Opp . 2 + - - - - - - - - - - - - - - Coal Saow Problem B . lhla & o - - - - - - - - - - - - - ~ Ii , . . - - - - - - - . - - - - - - - . - - 5 - 1 - - - - - - - - - - - - - - o + - - - - - - - - - - - - - ~ Ii - . - - - - ' - - . - - - - - - - - . 0 + - - - - - - - . - - - - . . . . . . , . . - - - - . . . . I r . 5 I ! 4 t - - ; : ~ ~ < . . . . . ~ . . . . . . . - . . . - . . = = , . . . . . . = . ~ = = . . . - . . . ~ . . . . . . . ~ . . . - j i : t - - - - - - - - - - - - ~ ~ Figure 4 . Graphs of concept novelty , usefulness and cohesiveness ratings . Note the lines in the interaction graph do not signify a relationship , but assist in visualising variable effects and interactions . Reprinted with permission . Copyright ASME201O . An explicitcontent analysis was performed using experimenttranscripts to determine how stimulus participants used stimulus words in the concept generation process . A parameter of interest is the POS in which the stimulus words were used , and therefore , stimulus words used by participants were categorised as a noun , verb or adjective . Another parameter of interest corresponds to the NWPs introduced by stimulus words . NWPs are of interest because they may indicate that new concept elements were being introduced into the concept generation process . In tum , new concept elements may form the basis of creative concepts . The investigators were able to identify NWPs by comparing words and phrases in the problems and stimulus sets with the words and phrases generated by the participants . In an example from the Snow problem , given the stimulus word ' constrict ' , sentence A below contains an NWP ( underlined ) , while sentence B does not contain anNWP . 4 . 2 . 3 . Language results ( A ) Constrict the motion of heat [ from leaving the house ] ( B ) . . . constrict snow . In sentence A , the phrase ' motion of heat ' , associated with the stimulus word ' constrict ' , was not given as part of the design problem nor stimulus set . However in sentence B , the word ' snow ' , also associated with the stimulus word ' constrict ' , was given as part of the design problem . Overall , results suggest ( l ) verbs introduce more NWPs and ( 2 ) opposite - stimulus verbs intro duce the most NWPs for two of the problems , the Bushing and Snow problems . This can be seen in the interaction graphs in Figures 5 - 7 . For each problem , a two - way ANOVA was used to compare the effect of Stimulus POS ( verb , noun or adjective ) and Stimulus Type ( opposite or similar stimuli ) on NWPs . First , theANOVA is ~ . ~ _ . . . . . _ - _ . _ - - - - - - - - - - - - - - - - - 288 . . . S c \" \" E ~ 4 ~ \" E 1 3 ~ . . . ~ 2 I . Chiu and L . H . Shu . . . . . . . . . . . . . . . . . . . . . . . . . . . POS _ NEW - - - N - V A Figure 5 . Bushing language results comparing effects of pas and Stimulus Type on estimated marginal means ofNWP . N . . . . . . o N . . . v . g - U o N o ~ M o . . . . . . sim StimType opp used to determine if there are Stimulus POS effects on NWP introduction . We found that Stimulus POS has a significant effect on the introduction ofNWPs , F ( 2 , 16 ) = 7 . 28 , p = 0 . 006 , F ( 2 , 16 ) = 40 . 42 , P = 0 . 000 and F ( 2 , 16 ) = 10 . 24 , p = 0 . 001 , for the Bushing , Snow and Coal problems respectively . For all three problems , planned contrasts show that stimuli used as verbs introduce significantly more NWPs , or borderline significantly more NWPs , as seen in Tables 11 - 13 . Second , the ANOVA is used to determine if there are Stimulus Type and Stimulus Type * POS interaction effects on NWPs . For the Bushing problem , Figure 5 suggests that opposite stimuli introduce the most NWPs . However , there is no significant effect of Stimulus Type on NWPs nor Stimulus Type * POS interaction , meaning that one Stimulus Type POS , e . g . opposite - stimulus verbs , does not significantly introduce more or fewer NWPs . 10 \" n\u00b7N - v A \u2022\u2022 , \u2022\u2022\u2022\u2022 # . . . . . . . . . - ' \" . . . . . . . . . . . . . . . . . . . . . o sim StimTvpe opp Figure 6 . Snow language results comparing effects of pas and Stimulus Type on estimated marginal means of NWP . ~ ~ - ~ - - ~ . _ - - - - - - - - - - - - - - - - - - - Journal ofEngineering Design 289 Figure 7 . Coal language results comparing effects of POS and Stimulus Type on estimated marginal means of NWP . Table II . Bushing problem contrast results . POS _ New \u00b7 . . . \u00b7N - V A F - and p - values F - and p - values F ( l , 8 ) = 4 . 61 , p = 0 . 064 - 0 . 05 F ( I , 8 ) = 2 . 80 , p = 0 . 13 opp F ( l , 8 ) = 41 . 76 , p = 0 . 000 < 0 . 05 F ( I , 8 ) = 2 . 90 , p = 0 . 13 StimType Noun : 2 . 30 Noun : 2 . 30 , . . . . . . . . . . . . . . . . . . . . . . . . . . ( : : Noun : 2 . 20 Noun : 2 . 20 sim Mean NWP introduction o 2 10 . . . 8 c = E 16 ' e - ~ \" D 4 ! l . . E i . . , Mean NWP introduction Verb : 5 . 10 Adj : 1 . 10 Table 12 . Snow problem contrast results . Verb : 7 . 10 Adj : 1 . 20 Table 13 . Coal problem contrast results . Mean NWP introduction F - and p - values Verb : 7 . 20 Adj : 2 . 70 Noun : 2 . 70 Noun : 2 . 70 F ( I , 8 ) = 47 . 58 , p = 0 . 000 < 0 . 05 F ( l , 8 ) = 2 . 90 , p = 0 . 13 For the Snow problem , Figure 6 suggests that opposite stimuli introduce the most NWPs . There is a significant effect of Stimulus Type on the introduction ofNWPs , F ( 1 , 8 ) = 5 . 26 , p = 0 . 051 , . . . , . 0 . 05 . Additionally , there is a significant interaction of Stimulus Type * PaS , F ( 2 , 16 ) = 13 . 88 , p = 0 . 000 . Contrasts for Stimulus Type * PaS interaction comparing opposite - stimulus verbs to other pas show significant differences , F ( l , 8 ) = 18 . 94 , p = 0 . 002 , indicating that different stimulus pas have different effects depending on the Stimulus Type . In this case , it indicates that opposite - stimulus verbs introduce significantly more NWPs . For the Coal problem , unlike the Bushing and Snow problems , Figure 7 suggests that similar stimuli introduce more NWPs rather than opposite stimuli . The ANaVA shows a significant effect 290 I . Chiu and L . H . Shu of Stimulus Type on NWP introduction , F ( l , 8 ) = 16 . 76 , p = 0 . 003 , while there is no significant interaction of Stimulus Type * POS . These results are discussed in detail in the next section . 4 . 3 . Experiment 2 discussion Experiment 2 shows opposite - stimulus concepts were significantly , or borderline significantly , more novel , useful and cohesive than similar - stimulus concepts . These results support our original hypothesis that opposite stimuli result in more creative concepts . These results are consistent with previous results obtained from Experiment 1 . However , surprisingly , Experiment 2 also shows that stimulus concepts in general were not more creative than no - stimulus ( control ) concepts . This is contrary to the literature and results of other design stimulation studies . It is unclear why no - stimulus concepts were judged more creative than some stimulus con cepts , specifically similar - stimulus concepts . Further work is required to explain the discrepancy between results found in this experiment , and results reported by others . However , we have noted that many other design stimulation experiments were pen - and - paper , e . g . Tseng et at . ( 2008 ) . The discrepancy observed in Experiment 2 may have resulted from a limitation of experimental methodology ; it is possible that the requirement to use stimuli in addition to verbalising and designing increased the cognitive workload of stimulus participants past optimal performance . It is well - known that increased mental workload can decrease human performance ( Wickens and Hollands 2000 , Drews et at . 2009 ) , where human performance is creativity in this case . Table 14 enumerates the tasks performed by no - stimulus participants versus stimulus participants and shows that stimulus participants perform an additional task compared to no - stimulus participants . A cognitive workload assessment , e . g . NASA Task Load Index , can be used to determine if stimulus use in verbal protocol design experiments will increase cognitive workload ( and thus decrease performance in terms of creativity ) . For example , the NASA Task Load Index asks participants to rate perceived workload in several dimensions during or after completion of a task ( Hart and Staveland 1988 ) . Other methods can also be used to determine the cognitive workload associated with design tasks . For example , Tang and Zeng ( 2009 ) investigated the use of body movements to quantify a designer ' s mental stress during the design process . As it may not be possible to equalise the workload between the stimulus and control conditions , i . e . comparing designs generated using stimulus versus no stimulus , this may be a serious limitation of verbal protocol experiments as applied to design research . Review of other verbal protocol experiments reveal at least one other reported case in which an experimental manipulation ( which should improve design overall ) did not produce better design concepts despite an increase of other metrics ( Atman and Bursic 1996 ) . In Atman and Bursic ' s study , reading a short design text before verbalising and designing improved metrics that should indicate improved design , e . g . time spent on designing , but did not result in better quality designs . More details with respect to potential limitations of verbal protocol design experiments are described by Chiu and Shu ( 2010 ) . Language analyses were conducted to gain insight into how designers used stimulus words in concept generation . First , stimulus words used in concept generation were examined to determine Table 14 . Task comparison between control and stimulus participants . No - stimulus tasks 1 . Design 2 . Verbalise Stimulus tasks 1 . Design 2 . Verbalise 3 . Use stimuli Note : Reprinted with permission . Copyright ASME 2010 . - - - - . _ - - - - - - . _ - - - - - - - - - - - - - - - - - - Journal ofEngineering Design Table 15 . Breakdown of average unknown usage of stimulus words . Average Average unknown unknown usage of usage of opposite similar Comparison Problem stimulus words stimulus words t - values p - values Bushing 26 . 0 16 . 3 t ( 8 ) = 1 . 10 0 . 15 Snow 18 . 6 17 . 8 t ( 8 ) = 0 . 12 0 . 45 Coal 31 . 0 17 . 0 t ( 8 ) = 2 . 07 0 . 04 291 the POS in which they were used , Le . verb , noun , adjective or unknown , and then any associated NWPs were examined . Language analyses showed that verbs in general introduced more NWPs than nouns or adjectives . Furthermore , results indicate that opposite verbs may introduce the highest number of NWPs , and correspond to increased creativity measures as determined by the independent raters . Combined results of the Bushing and Snow problems suggest that opposite - stimulus verbs may be the mechanism causing participants to introduce more NWPs , thus resulting in more creative concepts . However , the results of the Coal problem were contrary to those of the other two prob lems , in that similar - stimulus verbs introduced more NWPs than opposite - stimulus verbs . Despite the contradicting NWP introduction results for the Coal problem , opposite - stimulus concepts were still judged as more creative than similar - stimulus concepts . An explanation for this inconsistent result may be found by examining the high rate of unknown stimulus use in the Coal problem . Re - examining stimulus - use frequency for the Coal problem shows that opposite - stimulus partici pants had significantly more instances ofunknown stimulus use , e . g . stimulus words listed without context and hence with unknown POS , on average than in any other experimental condition or problem , t ( 8 ) = 2 . 07 , p = 0 . 04 . The average unknown stimulus POS per problem is shown in Table 15 . Stimulus words used as an unknown POS do not introduce NWPs , but frequent instances of recorded unknown stimuli may indicate that the participant frequently looked at specific stimulus words and likely thought about those words as they designed . It is possible that participants were fatigued during the Coal problem , the last problem in the experiment , and did not verbalise all thoughts related to the task . 5 . Overall discussion Our experiments showed that oppositely related stimuli can increase concept creativity . Results suggest that use ofopposite stimuli is most effective for general domain problems , or problems that are novel to the designer , e . g . the Sunflower , Egg and Snow problems . Experiment 2 also suggests that stimuli in general may be detrimental to designer performance , but this may be a limitation of the experimental method . Despite any potential methodological limitations , Experiment 2 did allow a comparative study ofhow language stimuli may support more creative concept generation . Experiment 2 results show that opposite - stimulus words are associated with more NWPs , which further suggests that opposite - stimulus verbs may force introduction of NWPs so that they are correct and consistent in the context of the problem . While participants used stimuli as verbs and nouns often ( and adjectives infrequently ) , verbs may be better at introducing NWPs because verbs are more flexible than nouns ( Gentner and France 1988 ) . Verbs are also known to unconsciously and automatically evoke concepts corresponding to the semantic filler roles typically associated with the event they denote ( Lyons 1977 , McRae et al . 2005 ) . Common semantic filler roles include 292 1 . Chiu and LH . Shu Table 16 . Semantic properties ofnouns and verbs , adapted from Croft ( 1991 ) . Property Noun Verb Semantic class Object Action Pragmatic function Reference Predication Stativity State Process Persistence Persistent Transitory Valencyjrelationality 0 1 + patients ( often direct objects ) , agents ( often subjects ) , instruments and locations . For example , the verb ' hammer ' will commonly evoke ' carpenter ' as an agent role , and ' nail ' as an object role . In the Snow problem , opposite - stimulus participants were provided with the stimulus word ' constrict ' . Those using it as a verb automatically realised that the verb ' constrict ' had to ' constrict something ' . ' Constrict snow ' ( to the point where it turns to ice ) was inconsistent with the problem because participants were explicitly instructed not to compress snow to the point of ice . However , introducing an NWP in ' constrict the motion of the heat ' , is both new , and consistent . The verb ' constrict ' implies that something must be constricted , and it is flexible enough to allow for different arguments while still ' making sense ' . Examining semantic properties of nouns and verbs further explain the relationship between stimulus verbs and NWPs . Table 16 summarises some key properties and differences between nouns and verbs . Table 16 shows verb properties that may benefit conceptual design more than noun properties . First , nouns are used to reference objects , which are usually fixed and static , while verbs represent transitory actions and processes ( Croft 1991 ) . Fixedness is contrary to the purpose of conceptual design , which is to expand the solution space , and not to fix , or limit the space . The abstractness of actions and processes allows verbs to avoid naming an actual design solution , which renders verbs more advantageous for expanding the solution space . Second , nouns have a valency of zero , while verbs have at least a valency of one . The valency , or relationality , of a word refers to the implied entities associated with the use of the word . When a noun is used , there is no other implied entity , e . g . ' book ' does not imply the existence of any other entity . However , when a verb is used , at least one other entity is implied , e . g . ' hit ' implies the existence of a hitter and the object hit ( Lyons 1977 , Croft 1991 , McRae et al . 2005 ) . For verbs , the implied entities are the semantic filler roles . Similar stimulusword used consistently with current problem Novel and creative concepl Figure 8 . Explanatory model of opposite - verb NWP introduction and its effect on concept creativity . Journal ofEngineering Design 293 Examining the semantic properties of nouns versus verbs aids the explanation of the empirical results obtained in Experiment 2 . Both linguistic theory and empirical results suggest that verbs used as design stimuli may increase concept creativity . Furthermore , empirical results show that oppositely related verbs may be the most effective at stimulating NWPs , which may lead to more creative concepts . With similarly related verbs , NWP introduction is unnecessary because existing problem - statement words and phrases capture the current problem state , e . g . problem objects like ' snow ' or ' bushings ' . Similar stimuli are already consistent with the problem and do not need to be resolved by the introduction of NWPs . Figure 8 models the effect of opposite - stimulus verbs on the generation of creative concepts . 6 . Summary and concluding remarks We investigate the effects of language on design because connections between language and cognition may be used to facilitate creative and successful design . Specifically , we study oppo sitely related words because they may stimulate creative concept generation by being unexpected and non - obvious , while being available for systematic retrieval in lexical resources . Through experiments , we observed the effects of oppositely and similarly related stimulus words on con cept creativity and designer behaviours with respect to the different stimulus types in concept generation . Empirical results support our original hypotheses and show that opposite stimuli may increase : ( l ) Concept novelty as well as other creativity metrics ; ( 2 ) Introduction of NWPs that may form the basis of novel and creative concepts . Empirical results combined with linguistic theory allow us to speculate on the mechanism in which opposite stimuli interacts with the conceptual design process to produce more creative concepts . While similar - stimulus words can be used ' as - is ' to reason about the problem and concepts consistently , opposite - stimulus words must be used with NWPs to maintain consistency within the problem . These new words or phrases may be key to the formation of more creative concepts . Results also reinforced that problem novelty and designer experience may be a factor ( Exper iment 1 ) , and that methodological issues with verbal protocols may interfere with results ( Experiment 2 ) . However , overall , our results show that opposite stimuli appear a practical means of stimulating creative design , that is simple to implement , e . g . using flashcards or worksheets . Unlike some creative design methods , e . g . TRIZ , which require training and related materials , e . g . contradiction tables or software , opposite stimuli only require the generation of words that are oppositely related to the problem . These words , which are familiar to most as antonyms , can be obtained from conventional thesauri or WordNet . The ease of obtaining oppositely related words , i . e . in antonyms , increases the ease by which this method can be integrated into engineering practice . While the designer ' s previous experience with similar design problems may offset the creative advantages offered by opposite - stimulus words , opposite - stimulus words are suitable for instances of conceptual design where the engineer may not have already gained familiarity with the new domain . It is unlikely that the effects of language on design cognition and creativity can be fully under stood in the near future . 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Upper Saddle River , NJ : Prentice Hall . Wilson , R . C , 1951 . An operational definition of originality . American Psychologist , 6 ( 6 ) , 297 . . Princeton University , 2008 . About WordNet . WordNet . , Princeton University . Available from : http : / / WordNet . Princeton . edu [ Accessed 7 July 2008 ] . Yang , M . C , 2009 . Observations on concept generation and sketching in engineering design . Research in Engineering Design , 20 ( 1 ) , I - II .", + "srinivasanAnalogiLeadImprovingSelection2023": "AnalogiLead : Improving Selection of Analogical Inspirations with Chunking and Recombination Arvind Srinivasan \u2217 arvindrb @ umd . edu arvind @ cheenu . net University of Maryland College Park , Maryland , USA Joel Chan \u2217 joelchan @ umd . edu University of Maryland College Park , Maryland , USA Figure 1 : Our proposed System . The interface is split into three rows . In the first row , the users select magnets ( 2 . 1 ) from the Problem and their corresponding Analogy , mixing them or adding their own in the Playspace ( 2 . 2 ) to generate insightful questions / recombinations ( 2 . 3 ) to facilitate divergent thinking and as a result , aid in recognition of beneficial analogies . ABSTRACT Analogical reasoning , a process that integrates potential leads across domains and disciplines , has been proven to contribute to breakthrough innovations . Selecting the right analogical leads is crucial , as it determines the quality and effectiveness of the gen - erated ideas . However , identifying relevant analogical leads can Permission to make digital or hard copies of part or all of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for third - party components of this work must be honored . For all other uses , contact the owner / author ( s ) . C & C \u201923 , June 19 \u2013 21 , 2023 , Virtual Event , USA \u00a9 2023 Copyright held by the owner / author ( s ) . ACM ISBN 979 - 8 - 4007 - 0180 - 1 / 23 / 06 . https : / / doi . org / 10 . 1145 / 3591196 . 3596817 be challenging and may be missed due to premature rejection or design fixation . To address this problem , our system , \" AnalogiLead \" , draws on the cognitive mechanisms of chunking and recombina - tion as a medium of interaction for selecting beneficial analogies . Users interact with meaningful chunks or segments from a design problem and analogy , represented as interactive tiles called \" mag - nets \" , and evaluate the analogies by recombining the \" magnets \" into brainstorming questions . These mechanisms are designed to foster playful and divergent exploration of analogical leads ( vs . restric - tive , relevance - based screening ) , to reduce premature rejection of analogical leads and foster more analogical innovations . 338 C & C \u201923 , June 19 \u2013 21 , 2023 , Virtual Event , USA Srinivasan CCS CONCEPTS \u2022 Human - centered computing \u2192 Graphical user interfaces ; Web - based interaction ; User interface design ; \u2022 Computing methodologies \u2192 Machine learning ; KEYWORDS Creativity Support , Machine Learning , User Interface Design , In - formation Seeking & Search ACM Reference Format : Arvind Srinivasan and Joel Chan . 2023 . AnalogiLead : Improving Selection of Analogical Inspirations with Chunking and Recombination . In Creativity and Cognition ( C & C \u201923 ) , June 19 \u2013 21 , 2023 , Virtual Event , USA . ACM , New York , NY , USA , 4 pages . https : / / doi . org / 10 . 1145 / 3591196 . 3596817 1 INTRODUCTION Analogical reasoning has been recognized as a powerful tool for generating breakthrough innovations . Analogical thinking involves drawing connections between seemingly disparate domains to gen - erate new ideas and approaches by identifying structural similarities [ 11 , 13 , 24 , 25 ] . Far analogies , in particular , have been instrumental in some of the most significant scientific and technological break - throughs throughout history [ 6 , 12 , 21 , 35 , 41 ] . However , selecting relevant analogical leads can be challenging due to the preference of memory retrieval for near , within - domain analogies that share object attributes [ 14 , 15 , 19 , 28 ] . Furthermore , analogical processing can be demanding on cognitive resources , often exhausting working memory , especially when multiple rela - tions need to be processed at once [ 18 ] . This can sometimes result in sub - optimal outcomes , as seen in the development of the first microwave oven , which was inspired by the analogy between radar and cooking but took several years to develop into a viable product , due to an improper understanding of users needs [ 10 ] . Existing approaches to analogical processing have attempted to address these challenges through instruction [ 16 , 30 , 37 ] or non - overgeneralized [ 39 ] abstract representations of problems and ana - logical examples [ 27 , 42 ] . Computational models and systems have also been proposed to facilitate this process through various tech - niques of abstraction and representation [ 5 , 9 , 11 , 27 , 33 ] . However , mere exposure to beneficial analogical leads alone is not enough to ensure their adoption in problem - solving [ 23 ] : factors such as expertise [ 2 , 36 ] , presence of usable anchors [ 3 ] , optimal represen - tations [ 7 , 8 , 11 , 17 ] , and diversity of solutions [ 27 ] can affect the premature rejection of potential leads , leading to design fixation [ 1 , 26 , 32 ] . Fully - automated analogical search engines such as SOLVENT [ 5 ] and ProbMap [ 34 ] have proposed breaking down a problem into functional components such as Stakeholders , Purpose and Mecha - nism to facilitate computational retrieval of new analogical leads . Given prior research on the creativity benefits of interacting with ideas in terms of their conceptual \" chunks \" [ 29 , 43 ] , we hypoth - esize that these conceptual \" chunks \" may also be beneficial for structuring interactions with existing analogical leads in a way that supports creative exploration and ( re ) interpretation ( and thus less premature rejection ) of analogical leads . In addition , aligning non - identical relational predicates has been suggested to be facilitated by re - representation [ 14 , 31 ] , which can assist in identifying and retrieving relevant analogies in complex systems . Earlier studies conducted in learning environments have explored questions as a possible mechanism to facilitate this process of re - representation and recombination ; attempting to facilitate the creation of new linkages between unlinked or previously linked components tar - geting specific applications [ 20 ] . According to Herring et al . [ 22 ] , designers were found to utilize examples by re - appropriating and recombining solution components to generate novel ideas . Addi - tionally , studies conducted in learning environments have found that questions play a vital role in promoting creative thinking . In particular , open - ended questions were found to greatly increase divergence in thinking [ 40 ] . A recent study [ 38 ] that proposed a system to semi - automatically generate external stimuli in the form of questions found that individuals , who were exposed to these generated questions produced better and more versatile ideas that those who were not . Our approach uses Large Language Models [ 4 ] to make this process easier and more accessible . Building on these insights , we designed a new system that lever - ages chunking and recombination mechanisms to support the recog - nition and adoption of far - domain analogies , facilitating their trans - fer across domains , and mitigating design fixation . By addressing these challenges , our proposed system aims to improve the ef - fectiveness of analogical reasoning in generating breakthrough innovations . 2 THE ANALOGILEAD SYSTEM The AnalogiLead System is designed to foster creative idea genera - tion and problem - solving through its three key sections : Magnets , Playspace , and Recombinations . 2 . 1 Magnets The Magnets in the AnalogiLead System are carefully curated and pre - defined sentence fragments or phrases that represent common functional constraints or attributes related to the problem domain . Expanding upon prior studies , we break down the problem into four components , namely : Stakeholder . This functional constraint highlights the benefi - ciaries who will be affected by the assigned design problem . Context . This functional constraint highlights the context in which the aforementioned stakeholders face the assigned design problem . Goal . This functional constraint highlights the goal that the stakeholders need to achieve to solve the assigned design problem . Obstacle . This functional constraint highlights an obstacle that hinders the stakeholders from achieving their goal for the assigned design problem . Alongside these functional constraints , the recommended analogi - cal leads add an additional constraint to highlight the solution : Solution . This functional constraint highlights the solution pro - posed to solve the goal of the recommended analogical lead for the given design problem . 339 Improving Selection of Analogies with Chunking and Recombination C & C \u201923 , June 19 \u2013 21 , 2023 , Virtual Event , USA These Magnets are designed to be flexible and versatile , allow - ing users to select and combine them in various ways to create prompts for idea generation . The Magnets serve as building blocks or \" chunks \" of ideas that can be easily manipulated and rearranged to construct meaningful questions via . the Playspace . 2 . 2 Playspace The Playspace is where users can experiment with the selected Mag - nets to create questions . The system uses Generative Pretrained Models ( GPT ) to automatically generate a wide range of questions by recombining the selected Magnets in real - time . The generated questions serve as prompts for the user to explore different angles and perspectives on the problem at hand , sparking creative thinking and encouraging divergent ideas . The user can iterate and experi - ment with different combinations of Magnets in the Playspace to generate a multitude of questions that prompt unique insights and solutions , promoting divergent thinking . 2 . 3 Recombinations The Recombinations section of the AnalogiLead System provides users with the option to further customize and refine the generated questions . Users can edit and modify the questions to better align with their thought process , specific problem domain , or desired outcome . This customization capability allows users to fine - tune the prompts to their specific needs , ensuring that the generated questions are relevant , meaningful , and tailored to their unique requirements . The Recombinations section empowers users with creative control , enabling them to craft prompts that are aligned with their creative goals and objectives . The combination of Magnets , Playspace , and Recombinations in the AnalogiLead System creates a dynamic and iterative process for idea generation and problem - solving , ultimately facilitating in selecting the most beneficial analogies for a given problem . The system leverages the power of Generative Pretrained Models to generate a wide range of prompts , while providing users with the flexibility to customize and refine the prompts to suit their creative process . This human - in - the - loop approach fosters exploration of di - verse ideas and encourages unique approaches to problem - solving , making the AnalogiLead System a powerful tool for stimulating creativity and improving selection of analogical inspirations while preventing possibilities for premature rejection and design fixation . 3 TECHNICAL OVERVIEW 3 . 1 Prompt Engineering Prompt : By combining the following list of words to - gether , generate [ n ] meaningful questions with insightfulrelationships : [ word1 , word2 , . . . , wordN ] Output : 1 . TheinterfaceusestheOpenAIlanguagemodel text - davinci - 002 for generating recombinations in a zero - shot learning context . Longer prompts including functional constraints resulted in con - fusing outputs , so the prompt structure was simplified . Descriptive words like \" insightful \" and \" meaningful \" were added to prioritize recombinations . The parameters for generating responses , such as temperature , frequency penalty , and presence penalty , were ad - justed to balance creativity and coherence . A temperature value of 0 . 75 was used for moderate randomness , and frequency penalty and presence penalty values of 0 . 5 and 0 . 25 , respectively , were used to encourage unique and diverse responses without overly strict penalties . 3 . 2 Software Implementation This interface is developed using the React Framework in JavaScript , which is a popular choice for building user interfaces . React provides a set of reusable primitives that can be used to create interactive web applications . React Hooks are used to simplify the code making it easier to manage and reuse code across components . To store data , the system uses Firestore Backend as a service , which is a cloud - based NoSQL database offered by Google . Firestore provides scalable storage and real - time synchronization for mobile , web , and server applications . 4 DEMONSTRATION For the purposes of this demonstration , a preset design problem will be presented , each with six corresponding handpicked analogies . Three analogies would be from a domain with varying degrees of closeness to the problem while the remaining would be from a far , yet structurally related domain . The demo will invite engagement from the participants by giving them the freedom to play around with the magnets of the problem and analogies , mix them or add their own magnets to the Playspace to generate recombinations . We also hope to expand the demo , to accommodate for users problems instead of preset - ones if possible , to promote greater engagement with the audience . Since the proposed system is a Web - interface and given the virtual nature of the event , there is no additional requirement needed for setup . 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Journal of the Association for Information Science and Technology 71 , 2 ( 2020 ) , 158 \u2013 171 . https : / / doi . org / 10 . 1002 / asi . 24221 Citation Key : zhangCognitiveMechanismsSensemaking2020 . Received 17 April 2023 341", + "schwartzSymmetricPatternsCoordinations2016": "Proceedings of NAACL - HLT 2016 , pages 499 \u2013 505 , San Diego , California , June 12 - 17 , 2016 . c (cid:13) 2016 Association for Computational Linguistics Symmetric Patterns and Coordinations : Fast and Enhanced Representations of Verbs and Adjectives Roy Schwartz , 1 Roi Reichart , 2 1 Institute of Computer Science , The Hebrew University 2 Faculty of Industrial Engineering and Management , Technion , IIT { roys02 | arir } @ cs . huji . ac . il roiri @ ie . technion . ac . il Ari Rappoport 1 Abstract State - of - the - art word embeddings , which are often trained on bag - of - words ( BOW ) con - texts , provide a high quality representation of aspects of the semantics of nouns . However , their quality decreases substantially for the task of verb similarity prediction . In this paper we show that using symmetric pattern contexts ( SPs , e . g . , \u201cX and Y\u201d ) improves word2vec verb similarity performance by up to 15 % and is also instrumental in adjective similarity pre - diction . The unsupervised SP contexts are even superior to a variety of dependency con - texts extracted using a supervised dependency parser . Moreover , we observe that SPs and dependency coordination contexts ( Coor ) cap - ture a similar type of information , and demon - strate that Coor contexts are superior to other dependency contexts including the set of all dependency contexts , although they are still inferior to SPs . Finally , there are substantially fewer SP contexts compared to alternative rep - resentations , leading to a massive reduction in training time . On an 8G words corpus and a 32 core machine , the SP model trains in 11 min - utes , compared to 5 and 11 hours with BOW and all dependency contexts , respectively . 1 Introduction In recent years , vector space models ( VSMs ) have become prominent in NLP . VSMs are often eval - uated by measuring their ability to predict human judgments of lexical semantic relations between pairs of words , mostly association or similarity . While many datasets for these tasks are limited to pairs of nouns , the recent SimLex999 word similar - ity dataset ( Hill et al . , 2014 ) also consists of sim - ilarity scores for verb and adjective pairs . State - of - the - art VSMs such as word2vec skip - gram ( w2v - SG , ( Mikolov et al . , 2013a ) ) and GloVe ( Pennington et al . , 2014 ) excel at noun - related tasks . However , their performance substantially decreases on verb similar - ity prediction in SimLex999 , and their adjective rep - resentations have rarely been evaluated ( Section 2 ) . In this paper we show that a key factor in the re - duced performance of the w2v - SG model on verb representation is its reliance on bag - of - words ( BOW ) contexts : contexts of the represented words that con - sist of words in their physical proximity . We investi - gate a number of alternative contexts for this model , including various dependency contexts , and show that simple , automatically acquired symmetric pat - terns ( SPs , e . g . , \u201cX or Y\u201d , ( Hearst , 1992 ; Davidov and Rappoport , 2006 ) ) are the most useful contexts for the representation of verbs and also adjectives . Moreover , the SP - based model is much more com - pact than the alternatives , making its training an or - der of magnitude faster . In particular , we train several versions of the w2v - SG model , each with a different context type , and evaluate the resulting word embeddings on the task of predicting the similarity scores of the verb and adjective portions of SimLex999 . Our results show that SP contexts ( SG - SP ) obtain the best results on both tasks : Spearman\u2019s \u03c1 scores of 0 . 459 on verbs and 0 . 651 on adjectives . These results are 15 . 2 % and 4 . 7 % better than BOW contexts and 7 . 3 % and 6 . 5 % better than all dependency contexts ( DepAll ) . Moreover , the number of SP contexts is substantially 499 smaller than the alternatives , making it extremely fast to train : 11 minutes only on an 8G word cor - pus using a 32 CPU core machine , compared to 5 and 11 hours for BOW and DepAll , respectively . Recently , Schwartz et al . ( 2015 ) presented a count - based VSM that utilizes SP contexts ( SRR15 ) . This model excels on verb similarity , outperform - ing VSMs that use other contexts ( e . g . , BOW and DepAll ) by more than 20 % . In this paper we show that apart from its SP contexts , the success of SRR15 is attributed in large to its explicit representation of antonyms ( live / die ) ; turning this feature off reduces its performance to be on par with SG - SP . As op - posed to Schwartz et al . ( 2015 ) , we keep our VSM \ufb01xed across experiments ( w2v - SG ) , changing only the context type . This allows us to attribute our im - proved results to one factor : SP contexts . We further observe that SP contexts are tightly connected to syntactic coordination contexts ( Coor , Section 3 ) . Following this observation , we compare the w2v - SG model with three dependency - based context types : ( a ) Coor contexts ; ( b ) all dependency links ( DepAll ) ; and ( c ) all dependency links exclud - ing Coor links ( Coor C ) . 1 Our results show that training with Coor contexts is superior to training with the other context types , leading to improved similarity prediction of 2 . 7 - 4 . 1 % and 4 . 3 - 6 . 9 % on verbs and adjectives respectively . These results demonstrate the prominence of Coor contexts in verb and adjective representation : these contexts are even better than their combination with the rest of the dependency - based contexts ( the DepAll contexts ) . Nonetheless , although Coor con - texts are extracted using a supervised dependency parser , they are still inferior to SP contexts , extracted automatically from plain text ( Section 3 ) , by 4 . 6 % and 2 . 2 % for verb and adjective pairs . 2 Background Word Embeddings for Verbs and Adjectives . A number of evaluation sets consisting of word pairs scored by humans for semantic relations ( mostly as - sociation and similarity ) are in use for VSM evalua - tion . These include : RG - 65 ( Rubenstein and Good - enough , 1965 ) , MC - 30 ( Miller and Charles , 1991 ) , WordSim353 ( Finkelstein et al . , 2001 ) , MEN ( Bruni 1 Coor \u222a Coor C = DepAll , Coor \u2229 Coor C = \u2205 et al . , 2014 ) and SimLex999 ( Hill et al . , 2014 ) . 2 Nouns are dominant in almost all of these datasets . For example , RG - 65 , MC - 30 and Word - Sim353 consist of noun pairs almost exclusively . A few datasets contain pairs of verbs ( Yang and Pow - ers , 2006 ; Baker et al . , 2014 ) . The MEN dataset , al - though dominated by nouns , also contains verbs and adjectives . Nonetheless , the human judgment scores in these datasets re\ufb02ect relatedness between words . In contrast , the recent SimLex999 dataset ( Hill et al . , 2014 ) contains word similarity scores for nouns ( 666 pairs ) , verbs ( 222 pairs ) and adjectives ( 111 pairs ) . We use this dataset to study the effect of context type on VSM performance in a verb and adjective simi - larity prediction task . Context Type in Word Embeddings . Most VSMs ( e . g . , ( Collobert et al . , 2011 ; Mikolov et al . , 2013b ; Pennington et al . , 2014 ) ) de\ufb01ne the context of a target word to be the words in its physical prox - imity ( bag - of - words contexts ) . Dependency con - texts , consisting of the words connected to the tar - get word by dependency links ( Grefenstette , 1994 ; Pad\u00f3 and Lapata , 2007 ; Levy and Goldberg , 2014 ) , are another well researched alternative . These works did not recognize the importance of syntactic coor - dination contexts ( Coor ) . Patterns have also been suggested as VSM con - texts , but mostly for representing pairs of words ( Turney , 2006 ; Turney , 2008 ) . While this approach has been successful for extracting various types of word relations , using patterns to represent single words is useful for downstream applications . Re - cently , Schwartz et al . ( 2015 ) explored the value of symmetric pattern contexts for word representation , an idea this paper develops further . A recently published approach ( Melamud et al . , 2016 ) also explored the effect of the type of con - text on the performance of word embedding models . Nonetheless , while they also explored bag - of - words and dependency contexts , they did not experiment with SPs or coordination contexts , which we \ufb01nd to be most useful for predicting word similarity . Limitations of Word Embeddings . Recently , a few papers examined the limitations of word em - bedding models in representing different types of se - 2 For a comprehensive list see : wordvectors . org / 500 mantic information . Levy et al . ( 2015 ) showed that word embeddings do not capture semantic relations such as hyponymy and entailment . Rubinstein et al . ( 2015 ) showed that while state - of - the - art embed - dings are successful at capturing taxonomic infor - mation ( e . g . , cow is an animal ) , they are much less successful in capturing attributive properties ( ba - nanas are yellow ) . In ( Schwartz et al . , 2015 ) , we showed that word embeddings are unable to distin - guish between pairs of words with opposite mean - ings ( antonyms , e . g . , good / bad ) . In this paper we study the dif\ufb01culties of bag - of - words based word embeddings in representing verb similarity . 3 Symmetric Patterns ( SPs ) Lexico - syntactic patterns are templates of text that contain both words and wildcards ( Hearst , 1992 ) , e . g . , \u201cX and Y\u201d and \u201cX for a Y\u201d . Pattern instances are sequences of words that match a given pattern , such that concrete words replace each of the wild - cards . For example , \u201c John and Mary \u201d is an instance of the pattern \u201cX and Y\u201d . Patterns have been shown useful for a range of tasks , including word relation extraction ( Lin et al . , 2003 ; Davidov et al . , 2007 ) , knowledge extraction ( Etzioni et al . , 2005 ) , senti - ment analysis ( Davidov et al . , 2010 ) and authorship attribution ( Schwartz et al . , 2013 ) . Symmetric patterns ( SPs ) are lexico - syntactic pat - terns that comply to two constraints : ( a ) Each pat - tern has exactly two wildcards ( e . g . , X or Y ) ; and ( b ) When two words ( X , Y ) co - occur in an SP , they are also likely to co - occur in this pattern in oppo - site positions , given a large enough corpus ( e . g . , \u201c X or Y \u201d and \u201c Y or X \u201d ) . For example , the pattern \u201c X and Y \u201d is symmetric as for a large number of word pairs ( e . g . , ( eat , drink ) ) both members are likely to occur in both of its wildcard positions ( e . g . , \u201ceat and drink\u201d , \u201cdrink and eat\u201d ) . SPs have shown useful for tasks such as word clustering ( Widdows and Dorow , 2002 ; Davidov and Rappoport , 2006 ) , semantic class learning ( Kozareva et al . , 2008 ) and word classi\ufb01cation ( Schwartz et al . , 2014 ) . In this paper we demonstrate the value of SP - based contexts in vector representa - tions of verbs and adjectives . The rationale behind this context type is that two words that co - occur in an SP tend to take the same semantic role in the sen - tence , and are thus likely to be similar in meaning ( e . g . , \u201c ( John and Mary ) sang\u201d ) . SP Extraction . Many works that applied SPs in NLP tasks employed a hand - crafted list of patterns ( Widdows and Dorow , 2002 ; Dorow et al . , 2005 ; Feng et al . , 2013 ) . Following Schwartz et al . ( 2015 ) we employ the DR 06 algorithm ( Davidov and Rap - poport , 2006 ) , an unsupervised algorithm that ex - tracts SPs from plain text . We apply this algorithm to our corpus ( Section 4 ) and extract 11 SPs : \u201cX and Y\u201d , \u201cX or Y\u201d , \u201cX and the Y\u201d , \u201cX or the Y\u201d , \u201cX or a Y\u201d , \u201cX nor Y\u201d , \u201cX and one Y\u201d , \u201c either X or Y\u201d , \u201cX rather than Y\u201d , \u201cX as well as Y\u201d , \u201c from X to Y\u201d . A description of the DR 06 algorithm is beyond the scope of this paper ; the interested reader is referred to ( Davidov and Rappoport , 2006 ) . SP Contexts . We generate SP contexts by taking the co - occurrence counts of pairs of words in SPs . For example , in the SP token \u201c boys and girls \u201d , the term girls is taken as an SP context of the word boys , and boys is taken as an SP context of girls . We do not make a distinction between the differ - ent SPs . E . g . , \u201c boys and girls \u201d and \u201c boys or girls \u201d are treated the same . However , we distinguish be - tween left and right contexts . For example , we gen - erate different contexts for the word girls , one for left - hand contexts ( \u201c girls and boys \u201d ) and another for right - hand contexts ( \u201c boys and girls \u201d ) . SPs and Coordinations . SPs and syntactic coor - dinations ( Coors ) are intimately related . For exam - ple , of the 11 SPs extracted in this paper by the DR 06 algorithm ( listed above ) , the \ufb01rst eight represent co - ordination structures . Moreover , these SPs account for more than 98 % of the SP instances in our corpus . Indeed , due to the signi\ufb01cant overlap between SPs and Coors , the former have been proposed as a sim - ple model of the latter ( Nakov and Hearst , 2005 ) . 3 Despite their tight connection , SPs sometimes fail to properly identify the components of Coors . For example , while SPs are instrumental in captur - ing shallow Coors , they fail in capturing coordina - tion between phrases . Consider the sentence John 3 Note though that the exact syntactic annotation of coordi - nation is debatable both in the linguistic community ( Tesni\u00e8re , 1959 ; Hudson , 1980 ; Mel\u2019\u02c7cuk , 1988 ) and also in the NLP com - munity ( Nilsson et al . , 2006 ; Schwartz et al . , 2011 ; Schwartz et al . , 2012 ) . 501 walked and Mary ran : the SP \u201cX and Y\u201d captures the phrase walked and Mary , while the Coor links the heads of the connected phrases ( \u201c walked \u201d and \u201c ran \u201d ) . SPs , on the other hand , can go beyond Coors and capture other types of symmetric structures like \u201c from X to Y\u201d and \u201cX rather than Y\u201d . Our experiments reveal that both SPs and Coors are highly useful contexts for verb and adjective rep - resentation , at least with respect to word similarity . Interestingly , Coor contexts , extracted using a super - vised dependency parser , are less effective than SP contexts , which are extracted from plain text . 4 Experiments Model . We keep the VSM \ufb01xed throughout our experiments , changing only the context type . This methodology allows us to evaluate the impact of dif - ferent contexts on the VSM performance , as context choice is the only modeling decision that changes across experimental conditions . Our VSM is the word2vec skip - gram model ( w2v - SG , Mikolov et al . ( 2013a ) ) , which obtains state - of - the - art results on a variety of NLP tasks ( Baroni et al . , 2014 ) . We employ the word2vec toolkit . 4 For all context types other than BOW we use the word2vec package of ( Levy and Goldberg , 2014 ) , 5 which aug - ments the standard word2vec toolkit with code that allows arbitrary context de\ufb01nition . Experimental Setup . We experiment with the verb pair ( 222 pairs ) and adjective pair ( 111 pairs ) portions of SimLex999 ( Hill et al . , 2014 ) . We re - port the Spearman \u03c1 correlation between the ranks derived from the scores of the evaluated models and the human scores provided in SimLex999 . 6 We train the w2v - SG model with \ufb01ve different context types : ( a ) BOW contexts ( SG - BOW ) ; ( b ) all dependency links ( SG - DepAll ) ( c ) dependency - based coordination contexts ( i . e . , those labeled with conj , SG - Coor ) ; ( d ) all dependency links except for coordinations ( SG - Coor C ) ; and ( e ) SP contexts . Our training corpus is the 8G words corpus gener - 4 https : / / code . google . com / p / word2vec / 5 https : / / bitbucket . org / yoavgo / word2vecf 6 Model scores are computed in the standard way : applying the cosine similarity metric to the vectors learned for the words participating in the pair . Model Verb Adj . Noun Time # Cont . SG - BOW 0 . 307 0 . 604 0 . 501 320 13G SG - DepAll 0 . 386 0 . 586 0 . 499 551 14 . 5G SG - Coor 0 . 413 0 . 629 0 . 428 23 550M SG - Coor C 0 . 372 0 . 56 0 . 494 677 14G SG - SP 0 . 459 0 . 651 0 . 415 11 270M SRR15 0 . 578 0 . 663 0 . 497 \u2014 270M SRR15 \u2212 0 . 441 0 . 68 0 . 421 \u2014 270M Table 1 : Spearman\u2019s \u03c1 scores on the different portions of SimLex999 . The top part presents results for the word2vec skip - gram model ( w2v - SG ) with various context types ( see text ) . The bottom lines present the results of the count SP - based model of Schwartz et al . ( 2015 ) , with ( SRR15 ) and without ( SRR15 \u2212 ) its antonym detection method . The two rightmost columns present the run time of the w2v - SG mod - els in minutes ( Time ) and the number of context in - stances used by the model ( # Cont . ) . 10 For each Sim - Lex999 portion , the score of the best w2v - SG model across context types is highlighted in bold font . ated by the word2vec script . 7 Models ( b ) - ( d ) require the dependency parse trees of the corpus as input . To generate these trees , we employ the Stanford POS Tagger ( Toutanova et al . , 2003 ) 8 and the stack version of the MALT parser ( Nivre et al . , 2009 ) . 9 The SP contexts are generated using the SPs extracted by the DR 06 algorithm from our training corpus ( see Section 3 ) . For BOW contexts , we experiment with three win - dow sizes ( 2 , 5 and 10 ) and report the best results ( window size of 2 across conditions ) . For depen - dency based contexts we follow the standard con - vention in the literature : we consider the immedi - ate heads and modi\ufb01ers of the represented word . All models are trained with 500 dimensions , the de - fault value of the word2vec script . Other hyper - parameters were also set to the default values of the code packages . Results . Table 1 presents our results . The SG - SP model provides the most useful verb and adjective representations among the w2v - SG models . Com - pared to BOW ( SG - BOW ) , the most commonly used 7 code . google . com / p / word2vec / source / browse / trunk / demo - train - big - model - v1 . sh 8 nlp . stanford . edu / software / tagger . shtml 9 http : / / www . maltparser . org / index . html 502 context type , SG - SP results are 15 . 2 % and 4 . 7 % higher on verbs and adjectives respectively . Com - pared to dependency links ( SG - DepAll ) , the im - provements are 7 . 3 % and 6 . 5 % . For completeness , we compare the models on the noun pairs portion , observing that SG - BOW and SG - DepAll are \u223c 8 . 5 % better than SG - SP . This indicates that different word classes require different representations . The results for SG - Coor , which is trained with syntactic coordination ( Coor ) contexts , show that these contexts are superior to all the other depen - dency links ( SG - Coor C ) by 4 . 1 % and 6 . 9 % on verbs and adjectives . Importantly , comparing the SG - Coor model to the SG - DepAll model , which aug - ments the Coor contexts with the other syntactic de - pendency contexts , reveals that SG - DepAll is ac - tually inferior by 2 . 7 % and 4 . 3 % in Spearman \u03c1 on verbs and adjectives respectively . Interestingly , Coor contexts , which are extracted using a super - vised parser , are still inferior by 4 . 6 % and 2 . 2 % to SPs , which capture similar contexts but are extracted from plain text . Table 1 also shows the training times of the vari - ous w2v - SG models on a 32G memory , 32 CPU core machine . SG - SP and SG - Coor , which take 11 min - utes and 23 minutes respectively to train , are sub - stantially faster than the other w2v - SG models . For example , they are more than an order of magnitude faster than SG - BOW ( 320 minutes ) and SG - Coor C ( 677 minutes ) . This is not surprising , as there are far fewer SP contexts ( 270M ) and Coor contexts ( 550M ) than BOW contexts ( 13G ) and Coor C con - texts ( 14G ) ( # Cont . column ) . Finally , the performance of the SG - SP model is still substantially inferior to the SRR15 SP - based model ( Schwartz et al . , 2015 ) . As both models use the same SP contexts , this result indicates that other modeling decisions in SRR15 lead to its superior performance . We show that this difference is mostly attributed to one feature of SRR15 : its method for detecting antonym pairs ( good / bad ) . Indeed , the SRR15 model without its antonym detection method ( SRR15 \u2212 ) obtains a Spearman \u03c1 of 0 . 441 , compared to 0 . 459 of SG - SP on verb pairs . For adjectives , however , SRR15 \u2212 is 1 . 7 % better than SRR15 , in - 10 We compare the w2v - SG models training time only . SRR15 and SRR15 \u2212 are count - based models and have no training step . creasing the difference from SG - SP to 2 . 9 % . 11 5 Conclusions We demonstrated the effectiveness of symmetric pattern contexts in word embedding induction . Ex - periments with the word2vec model showed that these contexts are superior to various alternatives for verb and adjective representation . We further pointed at the connection between symmetric pat - terns and syntactic coordinations . We showed that coordinations are superior to other syntactic con - texts , but are still inferior to symmetric patterns , al - though the extraction of symmetric patterns requires less supervision . Future work includes developing a model that successfully combines the various context types ex - plored in this paper . We are also interested in the representation of other word classes such as adverbs for which no evaluation set currently ex - ists . Finally , the code for generating the SG - SP embeddings , as well as the vectors experimented with in this paper , are released and can be down - loaded from http : / / www . cs . huji . ac . il / \u223c roys02 / papers / sp _ sg / sp _ sg . html Acknowledgments This research was funded ( in part ) by the Intel Col - laborative Research Institute for Computational In - telligence ( ICRI - CI ) , the Israel Ministry of Science and Technology Center of Knowledge in Machine Learning and Arti\ufb01cial Intelligence ( Grant number 3 - 9243 ) . The second author was partially funded by the Microsoft / Technion research center for elec - tronic commerce and the Google faculty research award . 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Schunn b a LRDC Rm 823 , University of Pittsburgh , 3939 O\u2019Hara St , Pittsburgh , PA 15260 , USA b LRDC Rm 821 , University of Pittsburgh , 3939 O\u2019Hara St , Pittsburgh , PA 15260 , USA a r t i c l e i n f o Article history : Received 15 December 2014 Revised 14 August 2015 Accepted 22 August 2015 Available online 29 August 2015 Keywords : Creativity Problem solving Conceptual combination a b s t r a c t Theories of creative conceptual combination hypothesize that , to generate highly creative concepts , one should attempt to combine source concepts that are very different from each other . While lab studies show a robust link between far combinations and increased novelty of concepts , empirical evidence that far combinations lead to more creative concepts ( i . e . , both more novel and of higher quality ) is mixed . Drawing on models of the creative process , we frame conceptual combination as a divergent process , and hypothesize that iteration is necessary to convert far combinations into creative concepts . We trace conceptual genealogies of many hundreds of concepts proposed for a dozen different problems on a large - scale Web - based innovation platform , and model the effects of combination distance on creative outcomes of concepts . The results are consistent with our predictions : ( 1 ) direct effects of far combina - tions have a mean zero effect , and ( 2 ) indirect effects of far combinations ( i . e . , building on concepts that themselves build on far combinations ) have more consistently positive effects . This pattern of effects is robust across problems on the platform . These \ufb01ndings lend clarity to theories of creative conceptual combination , and highlight the importance of iteration for generating creative concepts . (cid:1) 2015 Elsevier B . V . All rights reserved . 1 . Introduction How are creative outcomes produced ? Conceptual combination is one strategy that has been examined in some depth . It is deceptively simple and process - free in de\ufb01nition : it involves two or more concepts combined into a new concept . Real - world examples of the products of conceptual combination abound , from \u2018\u2018mash - ups\u201d and hip - hop sampling in music , to \u2018\u2018fusion\u201d cooking , to compound engineered products ( like the Apple iPhone , and com - ponent / module reuse in engineering ) . Lab studies have identi\ufb01ed a number of different cognitive processes for combining concepts , including property transfer ( transferring properties from \u2018\u2018helper\u201d concepts to a head concept , e . g . , \u2018\u2018pet - bird\u201d = \u2018\u2018bird you keep in the house and feed when hungry\u201d ) , hybridization ( interpreting a new concept as a \u2018\u2018cross\u201d or \u2018\u2018blend\u201d between the constituent concepts , e . g . , \u2018\u2018saw - scissors\u201d = \u2018\u2018dual purpose tool that both cuts and saws\u201d ) , and relational linking ( constituent concepts play distinct roles in a thematic relation , e . g . , pet - bird = \u2018\u2018bird for grooming pets\u201d ) . Here , we are particularly interested in how conceptual combination distance \u2014 the degree of semantic distance between the component concepts \u2014 in\ufb02uences the creativity of the produced concepts . Speci\ufb01cally , many theorists and eminent cre - ators ( Blasko & Mokwa , 1986 ; Koestler , 1964 ; Mednick , 1962 ; Rothenberg , 1979 ) contend that far combinations are more likely to lead to creative outcomes than near combinations , and numer - ous anecdotes of eminent creative accomplishments are consistent with this claim ( Johansson , 2006 ; Rothenberg , 1995 ; Ward , 2001 ) . Is this hypothesis supported by empirical evidence ? Lab studies have consistently shown that far combinations \u2014 where constituent concepts are semantically distant from each other ( e . g . , \u2018\u2018kitchen utensil\u201d and \u2018\u2018bird\u201d vs . \u2018\u2018kitchen utensil\u201d and \u2018\u2018plate\u201d ) \u2014 lead to more novel combinations ( Doboli , Umbarkar , Subramanian , & Doboli , 2014 ; Gielnik , Frese , Graf , & Kampschulte , 2011 ; Mobley , Doares , & Mumford , 1992 ; Nagai , Taura , & Mukai , 2009 ; Wilkenfeld & Ward , 2001 ; Wilkenfeld , 1995 ; Wisniewski , 1997 ) . A major factor in why this effect occurs is that people generate attributes of the product concept that are emergent , i . e . , not characteristic of its constituent concepts . For example , one might say that a \u2018\u2018kitchen - utensil bird\u201d is a bird that has a strong jaw for hammering ( where neither property is likely to be listed as characteristic of either kitchen utensils or birds when considered separately ) . Emergent attributes can be generated through \ufb01rst identifying alignable con\ufb02icts through analogical mapping ( Hampton , 1997 ) and performing causal reasoning to generate attributes to reconcile those con\ufb02icts ( Kunda , Miller , & Claire , 1990 ) . Another reason novel concepts are more likely to http : / / dx . doi . org / 10 . 1016 / j . cognition . 2015 . 08 . 008 0010 - 0277 / (cid:1) 2015 Elsevier B . V . All rights reserved . \u21d1 Corresponding author at : 2504B Newell - Simon Hall , Carnegie Mellon University , 5000 Forbes Ave , Pittsburgh , PA 15213 , USA . E - mail addresses : joelchuc @ cs . cmu . edu ( J . Chan ) , schunn @ pitt . edu ( C . D . Schunn ) . Cognition 145 ( 2015 ) 104 \u2013 115 Contents lists available at ScienceDirect Cognition journal homepage : www . elsevier . com / locate / COGNIT emerge from combining dissimilar concepts is that people are more likely to think of abstract relations and attributes of constituent concepts ( e . g . , using metaphor ) when those concepts are distantly related ( Mumford , Baughman , Maher , Costanza , & Supinski , 1997 ) . In contrast to the link between combination distance and nov - elty that has been well established in the lab , the impact of combi - nation distance on idea creativity is less clear . Most major models of creativity agree that products are creative if they are both novel and good ( of high quality , useful ; Boden , 2004 ; Finke , Ward , & Smith , 1996 ; Hennessey & Amabile , 2010 ; Runco , 2004 ; Sawyer , 2012 ; Shah , Vargas - Hernandez , & Smith , 2003 ) . However , rela - tively few studies of conceptual combination and creativity have actually measured quality or creativity . Two lab studies have shown that more distant combinations lead to lower quality ideas ( Baughman & Mumford , 1995 ; Mobley et al . , 1992 ) , while one lab study has shown that it has no signi\ufb01cant effect , but trending toward higher quality ( Doboli et al . , 2014 ) . Thus , the connection to quality is unclear . Four lab studies have examined effects on cre - ativity ( i . e . , the joining of novelty and quality ) : two found positive effects ( Howard - Jones , Blakemore , Samuel , Summers , & Claxton , 2005 ; Zeng , Proctor , & Salvendy , 2011 ) , while the other two found no effect ( Jang , 2014 ; Siangliulue , Arnold , Gajos , & Dow , 2015 ) , with Siangliulue et al . ( 2015 ) showing a trend in favor of lower diversity leading to higher creativity . The relatively small number of studies with mixed results leaves us with uncertainty about the relationship between concept similarity in conceptual combination and creativity . One interpre - tation of these mixed \ufb01ndings is that far combinations lead only to increased novelty per se , not necessarily increased creativity . A related controversy exists in the literature on analogical distance , where studies are divided on whether the most creative analogi - cally inspired ideas come from analogies outside of the problem domain ( in other words , from far analogies ) . Some researchers argue that the best interpretation of the data is that there is no clear / general advantage of far analogies for creative ideation ( e . g . , Chan , Dow , & Schunn , 2015 ; Dunbar , 1997 ; Perkins , 1983 ; Weisberg , 2009 , 2011 ) . Is a similar conclusion ( combination dis - tance does not in\ufb02uence creativity ) warranted based on the extant empirical data on combination distance ? We believe it is plausible , but argue that alternative theoretical interpretations should \ufb01rst be ruled out before accepting it . In this paper , we develop and test one theoretically motivated alternative explanation for the con - \ufb02icting \ufb01ndings : the bene\ufb01ts of combination distance depend on how much convergence has happened from the point of combina - tion . We argue that , to detect the bene\ufb01ts of combination distance , we need to observe and evaluate the resulting solution path further down its path of development ( vs . early on in its development ) . To develop our alternative explanation , we draw on a generally shared process model of creativity as involving \ufb01rst , divergent ( generating new ideas ) , then convergent ( selecting and building on the best ideas ) processes ( Amabile , 1983 ; Finke et al . , 1996 ; Sawyer , 2012 ; Simonton , 2011 ; Wallas , 1926 ; Warr & O\u2019Neill , 2005 ) . For example , Amabile\u2019s ( 1983 ) prominent process model prominently includes a movement from divergent processes ( response generation ) to convergent processes ( response valida - tion ) . Similarly , the Geneplore model ( Finke et al . , 1996 ) speci\ufb01es a Generate phase ( initial generation of candidate ideas ) followed by an Explore phase ( extensive exploration of those ideas ) . Sim - plistically , one can view the creative process as linearly progress - ing from a divergent to a convergent phase . Realistically , creators often go through many divergent - convergent cycles when devel - oping creative products ( Herring , Jones , & Bailey , 2009 ; Jin & Chusilp , 2006 ) . They also sometimes interleave divergent and con - vergent processes throughout , but transition from earlier periods with more divergence to later periods with less divergence ( Atman et al . , 2007 ; Ball , Evans , Dennis , & Ormerod , 1997 ; Goel & Pirolli , 1992 ; Shih , Venolia , & Olson , 2011 ) , where convergence on a few promising prototypes becomes necessary to move for - ward . Overall , there is theoretical consensus that divergent and convergent processes are distinct and jointly necessary for success - ful creative production , and the creative process moves from an emphasis on divergent processes early on to convergent processes later on . This theoretical framework provides a principled justi\ufb01cation for the hypothesis that far combinations should lead to more cre - ative ideas . If creativity is the production of artifacts that are both new and valuable , then at least some novelty is necessary to create new value . It follows , then , that a creative process that lacked divergence entirely ( e . g . , only selected from existing ideas ) would be highly unlikely to produce a creative idea . Relatedly , models of \ufb01rm innovation often focus on the tradeoff between exploring uncertain new opportunities and exploiting existing / old certainties ( March , 1991 ) . In such models , an exclusive focus on exploitation might be bene\ufb01cial in the short run , but usually leads to an even - tual loss of competitive advantage in dynamically competitive environments . We claim that far conceptual combinations in particular \u2014 given the usual nature of their conceptual products \u2014 are a primarily divergent process for generating new ideas . Therefore , incorporating them into the creative process should eventually increase the likelihood of a highly creative idea , even if they only raise the novelty of ideas considered ( but hold quality constant ) . By contrast , near conceptual combinations could serve both divergent and convergent thinking purposes . Importantly , understanding far conceptual combination as pri - marily a divergent process can help explain the con\ufb02icting \ufb01ndings on far combinations and creative outcomes . Within this framing , we can draw on the literature on divergent / convergent creative processes to suggest multiple reasons why combination distance might not have an immediate bene\ufb01t for creativity . First , some researchers argue that a good divergent process increases quality variance in order to make it more likely that the best ideas will be generated ( Girotra , Terwiesch , & Ulrich , 2010 ; Terwiesch & Ulrich , 2009 ) . Therefore , far combination will likely produce both good and bad ideas . Some form of selection process should then be necessary to separate the good ideas from the bad ideas . Sec - ondly , if we conceive of a solution space for creative problems as possessing no more than a few \u2018\u2018peaks\u201d ( i . e . , really good ideas ) , then statistically there should be many more mediocre or bad ideas than good ideas . It follows from this sparse quality peaks perspective that initial forays into very new regions of the space , if they are \u2018\u2018blind\u201d ( Simonton , 2011 , 2012 ) , will more likely land on mediocre or bad ideas than good ones on the \ufb01rst try . Thus , some time must be allowed to pass in order for some convergent process to select and re\ufb01ne the \u2018\u2018good novel\u201d ideas ( i . e . , to move from the low qual - ity initial landing spot in a novel conceptual region to the nearby high quality variants in that conceptual region ) . Finally , models and studies of idea generation consistently \ufb01nd that better ideas overall ( i . e . , combinations of both novelty and quality ) tend to be generated later down a solution path ( Basadur & Thompson , 1986 ; Benedek & Neubauer , 2013 ; Kohn , Paulus , & Choi , 2011 ; Krynicki , 2014 ; Nijstad , De Dreu , Rietzschel , & Baas , 2010 ; Nijstad & Stroebe , 2006 ; Parnes , 1961 ; Parnes & Meadow , 1959 ; Paulus , Kohn , Arditti , & Korde , 2013 ; Rhodes & Turvey , 2007 ; Rietzschel , Nijstad , & Stroebe , 2007 ) . These theoretical insights suggest a potential resolution to the mixed \ufb01ndings regarding combination distance and idea creativity : to observe the bene\ufb01ts of combination distance , one needs to examine its effects well into the convergent phase of the creative process . Given the high - quality - variance nature of far conceptual combination as a creative strategy , a longer convergent phase J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 105 ( i . e . , with iteration ) may be necessary to convert initially highly novel and highly variable quality ideas to creative solutions ( high on both novelty and quality ) . Therefore , we predict that , if we sep - arately observe the creativity of direct and indirect conceptual descendants of far combinations , we will see a positive effect for indirect , but not direct , descendants . We should be clear that we are not making the trivial claim that raw ideas must \ufb01rst be elaborated and iterated on to produce a cre - ative \ufb01nal product ( i . e . , a main effect of time on idea creativity ) . What we claim is that initial raw ideas from far combinations in particular are likely to have high novelty , but uncertain utility , and that iterations on these raw ideas ( not prototype testing ) is necessary to get to creative raw ideas ( i . e . , concepts that are both highly novel and have high potential utility ) . In other words , we are making an interaction prediction : the bene\ufb01ts of far over near combinations on idea creativity will only emerge at later time points . Anecdotal accounts of creative discovery by conceptual combination do not clearly specify whether dissimilar conceptual combinations lead to immediately creative raw ideas : some speak of it purely in terms of generating \u2018\u2018fresh\u201d ( i . e . , novel ) ideas , being agnostic about the expected potential utility of ideas . Others simply claim that dissimilar conceptual combinations directly yield more creative raw ideas with above average probability . For example , Ward\u2019s ( 2001 ) analysis of the origins of science \ufb01ction author Stephen Donaldson\u2019s award - winning The Chronicles of Thomas Covenant the Unbeliever fantasy series involves no speci\ufb01 - cation of iteration between the initial leprosy - unbelief conceptual combination and the \ufb01nal idea that formed the overarching theme of the series . In this paper , we empirically test our theoretically - driven pre - diction that observations of the bene\ufb01ts of conceptual combination distance vary with the genealogical lag between source and target ideas . Taking a genealogical approach , we trace lagged effects of conceptual combination distance on the creativity of direct and indirect conceptual descendants on a real - world innovation plat - form . We also address some key methodological issues in prior studies ( to increase our con\ufb01dence in our theoretically motivated hypothesis testing ) . First , all prior studies examined relatively few creativity problems , and were conducted only in the lab ( and there - fore under somewhat arti\ufb01cial conditions , and often with toy prob - lems ) . Some key studies ( Doboli et al . , 2014 ; Jang , 2014 ; Zeng et al . , 2011 ) also had relatively low Ns , making null effects ambiguous and raising potential concerns about effect sizes being signi\ufb01cantly overestimated ( Button et al . , 2013 ) , or even incorrectly estimated as positive / negative in sign ( Gelman & Weakliem , 2009 ) . Studies are needed that examine ( 1 ) a range of problems with ( 2 ) large Ns , ( 3 ) under more realistic conditions , and ( 4 ) use creativity rather than just novelty or quality in isolation to estimate the general effect of combination distance on creativity . Therefore , the research reported in this paper is conducted on a diverse range of problems , with large numbers of participants working on real world challenges , testing hypotheses with respect to a creative outcome measure that combines both novelty and quality . 2 . Methods 2 . 1 . Dataset We examine the relationship between combination distance and creative outcomes in the context of OpenIDEO ( www . openideo . com ) , a Web - based innovation platform that addresses a range of social and environmental problems ( e . g . , managing e - waste , increasing accessibility in elections ; see Appendix A for more details on the diverse set of problems sampled for our study ) . Expert designers from IDEO \u2014 a design consulting \ufb01rm renowned for its creativity \u2014 guide platform contributors through a structured design process to produce solutions for these problems that are ultimately implemented for real - world impact ( \u2018\u2018Impact Stories , \u201d n . d . ) . We focus our analysis in this study on processes and outcomes in three crucial early phases in the process . (cid:1) First , in the inspiration phase ( (cid:3) 1 . 5 to 4 weeks ) , contributors help to de\ufb01ne the problem space and identify promising solu - tion paths by posting inspirations : descriptions of solutions to analogous problems and case studies of stakeholders . (cid:1) In the concepting phase that follows ( for the next 2 \u2013 6 weeks ) , contributors post concepts : speci\ufb01c proposed solutions to the stated problem . Crucially , contributors cite concepts or inspira - tions that serve as sources of inspiration for their idea : this pro - vides our process data for conceptual combination . Fig . 1 shows an example inspiration and concept for an OpenIDEO problem about managing electronic waste . They are representative of the typical length and level of speci\ufb01cation of inspirations and concepts on the platform . (cid:1) In the shortlist phase , a subset of concepts for each problem is shortlisted by an expert panel ( composed of the OpenIDEO designers and a set of domain experts / stakeholders ) for further re\ufb01nement , based on their creative potential . (cid:1) In later stages , these concepts are re\ufb01ned and evaluated in more detail , and then a subset of them is selected for implementation . We focus on the \ufb01rst three stages given our focus on the use of conceptual combination for generating creative raw ideas ( the later stages involve many other design processes , such as prototyping ) . For more details on the dataset , see Chan ( 2014 ) . 2 . 2 . Sample The full dataset for this study consists of 2341 concepts and 4557 inspirations posted for 12 distinct problems by 1190 unique contributors . All inspirations and concepts were downloaded ( with administrator permission ) , and a HTML parser was used to extract the following metadata : ( 1 ) Concept / inspiration author ( who posted the concept / inspiration ) . ( 2 ) Number of comments . ( 3 ) Shortlist status ( yes / no ) . ( 4 ) List of cited sources of inspiration . ( 5 ) Full - text of concept / inspiration . The current study was conducted with two subsamples of this larger dataset . Speci\ufb01cally , our analysis focused on concepts that ( for subsample 1 ) directly cited at least 2 inspirations or ( for sub - sample 2 ) indirectly cited at least 2 inspirations . We de\ufb01ne in the next section how we operationalize indirect citations . Our sam - pling criteria re\ufb02ect our focus on measuring the effects of concep - tual combination distance , which is not measurable with fewer than two sources . The \ufb01rst subsample includes 456 concepts posted by 239 contributors , collectively citing 2167 unique inspira - tions . The second subsample includes 522 concepts posted by 281 authors , collectively citing 2556 unique inspirations . We were able to obtain professional expertise information ( e . g . , personal websites , online portfolios , pro\ufb01le pages on company names ) posted in the public OpenIDEO pro\ufb01les of 90 contributors ( approximately 1 / 3 of the authors in the sub - samples ) . In this sub - sample of the contributors , at least 1 / 3 are professionals in design - related disciplines ( e . g . , user experience / interaction design , communication design , architecture , product / industrial design , entrepreneurs and social innovators , etc . ) and / or domain experts or stakeholders ( e . g . , urban development researcher contributing to the vibrant - cities challenge , education policy researcher 106 J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 contributing to the youth - employment challenge , medical profes - sional contributing to the bone - marrow challenge ) . Thus , from a contributor perspective , our sample includes a range of creative / design expertise , from novice to expert . 2 . 3 . Measures 2 . 3 . 1 . Conceptual genealogies To examine the effects of indirect conceptual descendants , we constructed conceptual genealogies for all concepts in the sample . These genealogies were constructed via breadth - \ufb01rst search through the citation graph gathered in initial data collection : this search \ufb01rst returned all sources that a concept built upon , and then returned all sources that each of these sources built upon ( whether they were concepts or inspirations ) , traversing the conceptual tree to its endpoint . If duplicate entries were encountered , that source was credited at its \ufb01rst appearance in the graph : for instance , if an inspiration I was a direct source for a concept C ( at level 1 ) , and also for another concept / inspiration at level 2 , it would only be counted once as a level 1 source for C . In this study , we de\ufb01ned indirect conceptual descendants as inspirations from levels 2 to 4 of each concept\u2019s genealogy ( see Fig . 2 ) : this range choice re\ufb02ects our goal of examining the effects of sources that are \u2018\u2018just recent enough\u201d to have discernible effects ( we may not be able to Notice from Fig . 2 that indirect sources would also include inspirations cited by cited concepts ( i . e . , the sources of concepts that acted as immediate sources for the root concept ) . One way to think about this relationship of the root con - cept with these indirect sources of other concepts is that ( at least part of ) the insights / information / ideas contained in those inspira - tions are \u2018\u2018passed on\u201d to the root concept through their incorpora - tion into the concepts immediately cited by the root concept . 2 . 3 . 2 . Creativity of concepts Concept creativity is operationalized as whether a concept was shortlisted . In OpenIDEO , concepts are selected for the shortlist by a panel of expert judges , including the original stakeholders who posted the problem and a team of OpenIDEO designers . Both groups of judges have signi\ufb01cant expertise that quali\ufb01es them to judge the concepts\u2019 creativity : the stakeholders have spent signi\ufb01 - cant time searching for and learning about existing approaches , and the OpenIDEO designers , in addition to their expertise in the general domain of creative design , have spent considerable time upfront with the stakeholders , learning about and de\ufb01ning the problem space . An expert panel is widely considered a \u2018\u2018gold standard\u201d for mea - suring the creativity of ideas ( Amabile , 1982 ; Baer & McKool , 2009 ; Brown , 1989 ; Sawyer , 2012 ) . Further , addressing our need for a creativity measure that jointly considers novelty and quality , we learned from conversations with the OpenIDEO team that the panel\u2019s judgments combine consideration of both novelty and usefulness / appropriateness ( here operationalized as potential for impact ; A . Jablow , personal communication , May 1 , 2014 ) . Addi - tionally , since problems posted on OpenIDEO are unsolved , success - fulconceptsmustbedifferentfrom ( and , perhapsmoreimportantly , signi\ufb01cantly better than ) existing unsatisfactory solutions . To validate the reported focus of the IDEO panel , we obtained independent external expert judgments on Likert - like scales on separate dimensions of novelty , impact , and feasibility for a subset of concepts in the OpenIDEO data . Speci\ufb01cally , we collected approximately four expert judgments per concept for \ufb01ve of the Fig . 1 . Example inspiration ( left ) and concept ( right ) for an OpenIDEO problem about managing electronic waste . Fig . 2 . Illustrated example of \u2018\u2018indirect\u201d sources as sources in levels 2 \u2013 4 of a speci\ufb01c target concept\u2019s genealogy . Teal circles denote concepts ; maroon circles denote inspirations . Note that some indirect sources in this example serve as direct sources for the earlier concepts in this genealogy . J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 107 challenges ( N = 318 concepts ) . Re\ufb02ecting the complex and multi - disciplinary nature of the challenges , the expert\u2019s ratings had mod - erate levels of agreement ( ICCs of . 46 , . 57 , and . 63 for novelty , impact , and feasibility , respectively ) . All three ratings were posi - tively associated with short - list status ( novelty r pb = 0 . 09 , p = . 10 ; impact r pb = 0 . 21 , p = . 00 ; novelty r pb = 0 . 17 , p = . 00 ) . Fitting a sim - ple logistic regression of shortlist on the three dimensions shows that impact is a strong predictor ( b = . 51 , p = . 02 ) . Feasibility is mar - ginally predictive ( b = . 33 , p = . 07 ) , while novelty has a positive but nonsigni\ufb01cant estimate ( b = . 11 , p = . 63 ) , allaying potential concerns about panel bias against novel concepts . 2 . 3 . 3 . Combination distance A standard approach to measuring combination distance would be to obtain pairwise conceptual distance judgments between all conceptual descendants . However , the scale of the present study ( more than 2000 inspirations would require more than 2 million pairwisecomparisons ) presentsformidablechallengestomeasuring distance using human judgments , from not only a cost standpoint , but also an effectiveness perspective , since the quality of human judgments can deteriorate severely if the workload is too high . We therefore took a computational approach to measuring combination distance . Speci\ufb01cally , we employed Latent Dirichlet Allocation ( LDA ; Blei , Ng , Jordan , & Lafferty , 2003 ) \u2014 an unsuper - vised machine learning technique for learning topical structures from unstructured texts \u2014 to learn the semantic space of ideas posted on OpenIDEO , and used that space to estimate semantic dis - tance between inspirations . LDA treats documents as mixtures of latent \u2018\u2018topics\u201d ( occurring with different \u2018\u2018weights\u201d in the mixture ) , and uses Bayesian statistical learning algorithms to infer the latent topical structure of the corpus ( and the topical mixtures for each document ) from the co - occurrence patterns of words across docu - ments . With this inferred topical structure , we can then derive con - ceptual similarity between any pair of documents by computing the cosine between their topical mixtures ( which are represented as vectors of topic - weights ) . Essentially , documents that share dominant topics in similar relative proportions ( e . g . , primarily about recycling and electronics ) are the most similar . This similar - ity is measured by computing the cosine between their topic - weight vectors , yielding a similarity score between 0 and 1 ( where values closer to 1 indicate greater similarity ) . LDA has been successfully used to model semantics in many other contexts , including modeling semantic memory representa - tion phenomena ( Grif\ufb01ths , Steyvers , & Tenenbaum , 2007 ) , support - ing knowledge discovery and information retrieval in repositories of scienti\ufb01c papers ( Grif\ufb01ths & Steyvers , 2004 ) , and analyzing topical dynamics in social media use ( Schwartz et al . , 2013 ) . Our application of LDA in the OpenIDEO corpus was validated by exam - ining correlations with human judgments on two sub - samples in the corpus . We collected Likert - scale pairwise similarity judg - ments for inspirations from 3 research assistants for the \ufb01rst sub - sample , and 5 from the second . Inter - rater agreement was acceptable , aggregate consistency intraclass correlation coef\ufb01cient ( ICC ( 2 , 3 ) ) = . 46 for the \ufb01rst sub - sample , and ICC ( 2 , 5 ) = . 74 for the second sub - sample . The correlation of the LDA cosines with the mean human - judged pairwise similarities was high in both sub - samples , at r = . 54 and r = . 51 , respectively . Notably , these agreement levels were better than the highest correlation between individual human raters in both subsamples ( r = . 39 and r = . 48 , respectively ) , reinforcing the value of automatic coding methods for this dif\ufb01cult task . Further details on our implementation and validation of LDA are available in Chan ( 2014 ) . Combination distance ( hereafter denoted COMB - DIST DIR for direct sources , and COMB - DIST IND for indirect sources ) was mea - sured for each concept as the mean of the reversed pairwise cosi - nes between inspirations cited by that concept ( i . e . , subtracting from 0 , to derive distance rather than similarity ) . Fig . 3 shows an example near and far COMB - DIST DIR from the data . Note that conceptual combination research has tended to focus on pairs of concepts being combined , but here there were often more than just two concepts being combined , especially in the indi - rect source set . Real world problems are complex , and often involve many subproblems , such that diverse sources must be brought together to solve each of the subproblems . We used mean pairwise distance ( the most natural conceptualization of combination distance ) to characterize the general diversity among the sources . But it could be that problem solvers brought the most similar pieces together in pairs , or were most in\ufb02uenced by the maximum distance among sources . Therefore we also explored using min and max distance measures rather than mean distance in our anal - yses . These approaches produced similar results ( with slightly more statistical noise ) , suggesting the patterns we found were not due to idiosyncrasies of how we conceptualized combination distance . However , the added noise when considering min and max distance also suggested our initial intuitions that mean distance is the most appropriate operationalization of combination distance were correct . Therefore , we only report the mean distance results . 2 . 3 . 4 . Control measures To improve our estimates of the effects of combination distance per se in this multi - faceted naturalistic dataset , we measured and accounted for other important factors that may in\ufb02uence concept creativity ( i . e . , we statistically controlled for likely confounds ) . 2 . 3 . 4 . 1 . Feedback . Feedback can be an important contributor to the quality of a concept . Feedback can provide encouragement , raise issues / questions , or provide speci\ufb01c suggestions for improvement , all potentially signi\ufb01cantly enhancing the quality of the concept . Further , feedback may be an alternate pathway to success via com - bination distance , in that concepts that build on far combinations may attract more attention and therefore higher levels of feedback , which then improve the quality of the concept . On OpenIDEO , con - cepts receive feedback in the form of comments . We operationalize feedback ( labeled here as FEEDBACK ) as the number of comments received by a given concept . 2 . 3 . 4 . 2 . Quality of cited sources . Concepts that build on existing high - quality concepts ( e . g . , those that end up being shortlisted ) may be more likely to be shortlisted : contributors might incorpo - rate lessons learned from the mistakes and shortcomings , good ideas , and feedback in these high - quality concepts . We operationalize source quality ( SOURCEQUAL ) as the number of shortlisted concepts a given concept builds upon . 2 . 3 . 4 . 3 . Conceptual source distance from problem . Finally , building on a prior study in the OpenIDEO context that showed a positive effect of sources that were conceptually closer to the problem domain ( Chan et al . , 2015 ) , we also control for the distance of sources from the problem domain . Source distance ( here labeled SP - DIST ) is measured for each concept by taking the mean of the reverse cosines between cited inspirations and the problem . 2 . 4 . Analytic approach Our goal is to model the creative outcomes of concepts posted by contributors for 12 different problems as a function of combina - tion distance , controlling for other factors . However , contributors are not cleanly nested within problems , nor vice versa ; concepts are cross - classi\ufb01ed within both authors and challenges ( see Fig . 4 ) . This cross - classi\ufb01ed structure violates assumptions of uni - form independence between concepts : concepts posted by the same contributor or within the same problem are likely to be 108 J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 correlated with each other on various dimensions , most impor - tantly overall quality . Failing to account for this non - independence could lead to overestimates of the statistical signif - icance of model estimates ( i . e . , make unwarranted claims of statis - tically signi\ufb01cant effects ) . This issue is exacerbated when testing for small effects within large datasets . Additionally , while we are primarily interested in concept - level outcomes , we need to model between - contributor effects to separate out contributor - effects ( e . g . , higher / lower creativity , effort ) from the impact of sources on individual concepts . Therefore , we employ generalized linear mixed models ( GLMMs ) to model both \ufb01xed effects ( of our inde - pendent and control variables ) and random effects ( potential vari - ation of the outcome variable attributable to contributor - or problem - variation and also potential between - problem variation in the effect of combination distance ) on shortlist status ( a binary variable , which requires logistic , rather than linear , regression ) . The following is the general structure of these models ( in mixed model notation ) : g i \u00f0 contributorjchallenge k \u00de \u00bc c 00 \u00fe X q c q 0 X qi \u00fe u 0 contributorj \u00fe u 0 challenge k where (cid:1) g i \u00f0 contributorjchallengek \u00de is the predicted log odds of being shortlisted for the i th concept posted by the j th contributor in the k th challenge (cid:1) c 00 is the grand mean log odds for all concepts (cid:1) c q 0 is a vector of q predictors ( q = 0 for our null model ) (cid:1) u 0 contributorj and u 0 challengek model between - contributor and between - challenge variability in mean c 00 We \ufb01t our GLMMs using the glmer function in the lme4 pack - age ( Bates , Maechler , Bolker , & Walker , 2013 ) in R ( R Core Team , 2013 ) , using full maximum likelihood estimation by the Laplace approximation . Our general modeling strategy is as follows . First , we \ufb01t a reduced model with crossed random effects of challenge and con - tributor , and \ufb01xed effects only of our control measures ( i . e . , feed - back , source quality , and source problem - distance ) . Because these are theoretically motivated predictors , we leave them in the model regardless of statistical signi\ufb01cance . This reduced model serves as a more realistic baseline than the null model ; we com - pare the reduced model to a second ( \ufb01xed - slope ) model with the added \ufb01xed effect of combination distance . Finally , we \ufb01t a third ( random - slope ) model with an added parameter u 1 challengek to model potential challenge - level random effects on the mean effect of combination distance . To select our \ufb01nal model , we choose the model that meets three criteria : ( 1 ) signi\ufb01cantly reduces deviance from the null model ( low standard for explanatory power ) , ( 2 ) signi\ufb01cantly reduces deviance compared to the reduced model from the previous step ( higher standard for explanatory power ) , and ( 3 ) has a lower Akaike Information Criterion ( AIC ) than the previous step to avoid over\ufb01tting . Fig . 3 . Example near ( left ) and far ( right ) combinations according to COMB - DIST DIR . Fig . 4 . Illustrated cross - classi\ufb01ed structure of data . J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 109 3 . Results 3 . 1 . Direct effects of combination distance We \ufb01rst examine the hypothesis that combining diverse sources leads directly to ideas that are more creative . Recall that this analysis is with the sub - sample of 456 concepts . 3 . 1 . 1 . Descriptive statistics Table 1 summarizes the descriptive statistics and intercorrela - tions between the variables . There are statistically signi\ufb01cant pos - itive correlations between the control variables and Pr ( shortlist ) ; hence the importance of including them in the models . There are no strong inter - correlations between the predictor variables , alleviating potential concerns about multicollinearity ; a variance in\ufb02ation analysis also shows that having COMB - DIST DIR and SP - DIST in the same model should not introduce multicollinearity , with variance in\ufb02ation factors of 1 . 16 for both variables . 3 . 1 . 2 . Statistical models We \ufb01t a series of generalized linear mixed models using full maximum likelihood estimation by the Laplace approximation , with concepts cross - classi\ufb01ed within both contributors and problems . We rescale COMB - DIST DIR ( multiplying it by 10 ) for easier interpretation ( a more meaningful \u2018\u20181 - unit\u201d change ) . Table 2 presents the model estimates and \ufb01t statistics for the GLMMs . The \ufb01rst model is a baseline model \ufb01tted with the control variables as predictors . This model yields a large and statistically signi\ufb01cant reduction in deviance compared to the null model , v 2 ( 2 ) = 64 . 70 , p = 0 . 00 . Adding a \ufb01xed slope for COMB - DIST DIR to this model does not provide any meaningful reduction in deviance , with the likelihood ratio being essentially zero , v 2 ( 1 ) = 0 . 00 , p = 0 . 92 , and an increase in the AIC . The point estimate for the effect of a change of . 10 in COMB - DIST DIR ( remember that it is rescaled in this model ) is also essentially zero ( see Fig . 5 ) , albeit with a fairly wide con\ufb01dence interval . To ensure that this wide con\ufb01dence interval is not due to one or two outlier problems overwhelming an overall positive or negative trend across the problems , we estimate an additional model with a random slope for COMB - DIST DIR . Visually inspecting the posterior modes for the slope of COMB - DIST DIR for each problem ( see Fig . 5 ) , we see an even scatter about the mean value , with 6 challenges having a positive sign for the coef\ufb01cient for diversity , and 6 challenges having a negative sign . A binomial sign test with a null hypothesis of equal probability for positive and negative effects of diversity estimates a two - tailed p - value of 1 . 00 of observing either 6 or fewer positive or 6 or more negative signs in 12 \u2018\u2018trials\u201d ( although this binomial test outcome should be intuitively obvious to the reader , we present it here to parallel the same test conducted on the indirect problem - speci\ufb01c slopes ) . The graph in Fig . 5 gives the impression that there are clear effects of combination distance , which can be positive or negative Table 1 Descriptive statistics and intercorrelations between variables for COMB - DIST DIR . Variable Descriptives Correlations M ( SD ) FEEDBACK SOURCE SHORT SP - DIST COMB - DIST DIR SHORTLIST 0 . 16 ( 0 . 36 ) 0 . 33 * * * 0 . 11 * * (cid:4) 0 . 10 * (cid:4) 0 . 01 FEEDBACK 9 . 14 ( 9 . 92 ) 0 . 12 * * 0 . 02 0 . 05 SOURCESHORT 0 . 61 ( 1 . 07 ) (cid:4) 0 . 05 0 . 10 * SP - DIST (cid:4) 0 . 13 ( 0 . 62 ) 0 . 29 * * * COMB - DIST DIR 2 . 02 ( 1 . 25 ) \u2013 m p < . 10 . * p < . 05 . * * p < . 01 . * * * p < . 001 . Table 2 Model estimates and \ufb01t statistics for cross - classi\ufb01ed multilevel logistic regressions of Pr ( shortlist ) on COMB - DIST DIR , with comparison to baseline model ( controls ) . Baseline model ( controls ) COMB - DIST DIR , \ufb01xed slope COMB - DIST DIR random slope Fixed effects c 00 , intercept (cid:4) 3 . 08 [ (cid:4) 3 . 37 , (cid:4) 2 . 12 ] (cid:4) 3 . 05 [ (cid:4) 3 . 99 , (cid:4) 2 . 12 ] (cid:4) 3 . 03 [ (cid:4) 4 . 11 , (cid:4) 1 . 95 ] c 10 , FEEDBACK 0 . 10 * * * [ 0 . 07 , 0 . 12 ] 0 . 10 * * * [ 0 . 07 , 0 . 13 ] 0 . 10 * * * [ 0 . 07 , 0 . 13 ] c 20 , SOURCESHORT 0 . 25 m [ (cid:4) 0 . 10 , 0 . 35 ] 0 . 25 m [ (cid:4) 0 . 03 , 0 . 52 ] 0 . 26 m [ (cid:4) 0 . 03 , 0 . 54 ] c 30 , SP - DIST (cid:4) 0 . 49 m [ (cid:4) 0 . 71 , 0 . 10 ] (cid:4) 0 . 50 m [ (cid:4) 1 . 05 , 0 . 04 ] (cid:4) 0 . 54 * [ (cid:4) 1 . 08 , (cid:4) 0 . 00 ] c 40 , COMB - DIST DIR 0 . 01 [ (cid:4) 0 . 27 , 0 . 30 ] 0 . 03 [ (cid:4) 0 . 28 , 0 . 33 ] Random effects u 0 authorj 0 . 47 0 . 47 0 . 44 u 0 challengek 0 . 71 0 . 71 1 . 63 u 1 challengek 0 . 05 Model \ufb01t statistics I Deviance 323 . 57 323 . 57 321 . 74 AIC 335 . 57 337 . 57 339 . 74 \u2044\u2044 p < . 01 . 95 % CI ( Wald ) = [ lower , upper ] . m p < . 10 . I Generalized linear mixed models ( including multilevel logistic regressions ) are \ufb01tted using maximum likelihood estimates that are not , strictly speaking , minimizing variance , and typical \ufb01t statistics like R 2 are not considered meaningful for assessing the \ufb01t of these models . Therefore , here we report common alternative \ufb01t statistics more closely related to the model estimation procedures , i . e . , deviance and AIC . * p < . 05 . * * * p < . 001 . Fig . 5 . Model - \ufb01tted relationship between combination distance and Pr ( shortlist ) . Fitted values evaluated at mean values of FEEDBACK , SOURCEQUAL , and SP - DIST . Greyed lines are \ufb01tted from posterior mode estimates of the slope of COMB - DIST DIR for each problem . 110 J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 depending upon the problem . However , it is important to note that there is not strong evidence in support of signi\ufb01cant problem mod - eration : the model estimates low problem - variance , does not meaningfully decrease variance from the \ufb01xed slope model , v 2 ( 2 ) = 1 . 83 , p = . 23 ( p - value is halved , heeding common warnings that a likelihood ratio test discriminating two models that differ on only one variance component may be overly conservative , e . g . , Pinheiro & Bates , 2000 ) , and also further increases AIC , calling into question whether the estimated problem variation is meaningful . In other words , the most parsimonious interpretation given these data is that direct combination distance has no effect ( i . e . , we select the baseline model as our \ufb01nal model ) , although it is possible that an even larger dataset would \ufb01nd problem - speci\ufb01c effects . Most importantly , these data argue strongly against a general bene\ufb01t of combination distance of direct sources on idea creativity . 3 . 2 . Indirect effects of conceptual diversity We now turn to the analysis of the effects of combination distance of indirect sources . Recall that this analysis is with the sub - sample of 522 concepts . 3 . 2 . 1 . Descriptive statistics Descriptive statistics and bivariate correlations are given in Tables 3 and 4 . There are no strong correlations among the predic - tors , giving little cause for concerns about multicollinearity . 3 . 2 . 2 . Statistical models As before , we estimate a series of generalized linear mixed models to analyze the relationship between COMB - DIST IND and Pr ( shortlist ) . We exclude COMB - DIST DIR from our models for a num - ber of reasons . First , it does not add predictive value ( as we saw in the preceding analysis ) . Second , including it as predictor would exclude concepts that had indirect inspirations as sources , but had less than two direct inspiration sources , reducing our N to 381 for no predictive gain . Finally , our estimates of the effects of COMB - DIST IND do not change with COMB - DIST DIR in the model . The model estimates are given in Table 5 . As before , we begin with a baseline controls model , which gives a large and statistically signi\ufb01cant reduction in deviance compared to the null model , v 2 ( 3 ) = 63 . 70 , p = 0 . 00 . In contrast to COMB - DIST DIR , adding a \ufb01xed slope for COMB - DIST IND to the baseline model yields a marginally signi\ufb01cant reduction in deviance , v 2 ( 1 ) = 3 . 26 , p = 0 . 07 , and a decrease in AIC , mitigating concerns about over\ufb01tting . The model estimates that a . 10 change in COMB - DIST IND corre - sponds to an increase of approximately . 45 in the log - odds of being shortlisted ( see Fig . 6 ) . Holding all the other predictors at their mean values , changing from a COMB - DIST IND of (cid:4) 0 . 20 ( close to the mean value in the sample ) to (cid:4) 0 . 10 ) increases Pr ( shortlist ) from 0 . 13 to 0 . 19 . Again , the CI for the effect is relatively wide . However , in contrast to COMB - DIST , the estimated positive effect Table 3 Descriptive statistics for indirect combination distance measures . Variable Valid N Min Max Mean Median SD SHORTLIST 522 0 1 0 . 15 0 0 . 36 FEEDBACK 522 0 67 9 . 01 6 10 . 02 SOURCESHORT 522 0 11 0 . 67 0 1 . 06 SP - DIST 522 (cid:4) 2 . 93 1 . 67 (cid:4) 0 . 11 (cid:4) 0 . 01 0 . 73 COMB - DIST IND 522 (cid:4) 0 . 73 (cid:4) 0 . 02 (cid:4) 0 . 18 (cid:4) 0 . 14 0 . 10 Table 4 Bivariate correlations for indirect combination distance measures . Variable FEEDBACK SOURCE SHORT SP - DIST COMB - DIST IND SHORTLIST 0 . 34 * * * 0 . 13 * * (cid:4) 0 . 11 * 0 . 04 FEEDBACK 0 . 11 * (cid:4) 0 . 01 0 . 13 * * SOURCESHORT (cid:4) 0 . 05 0 . 19 * * * SP - DIST (cid:4) 0 . 02 m p < . 10 . * p < . 05 . * * p < . 01 . * * * p < . 001 . Table 5 Model estimates and \ufb01t statistics for cross - classi\ufb01ed multilevel logistic regressions of Pr ( shortlist ) on COMB - DIST IND , with comparison to baseline model ( controls only ) . Baseline model ( controls ) With COMB - DIST IND , \ufb01xed slope With COMB - DIST IND random slope Fixed effects c 00 , intercept (cid:4) 2 . 80 [ (cid:4) 3 . 44 , (cid:4) 2 . 16 ] (cid:4) 1 . 98 [ (cid:4) 3 . 10 , (cid:4) 0 . 86 ] (cid:4) 2 . 12 [ (cid:4) 3 . 10 , (cid:4) 0 . 86 ] c 10 , FEEDBACK 0 . 09 * * * [ 0 . 06 , 0 . 12 ] 0 . 09 * * * [ 0 . 07 , 0 . 12 ] 0 . 09 * * * [ 0 . 07 , 0 . 12 ] c 20 , SOURCESHORT 0 . 16 [ (cid:4) 0 . 08 , 0 . 39 ] 0 . 12 [ (cid:4) 0 . 12 , 0 . 35 ] 0 . 12 [ (cid:4) 0 . 12 , 0 . 35 ] c 30 , SP - DIST (cid:4) 0 . 44 * [ (cid:4) 0 . 82 , (cid:4) 0 . 07 ] (cid:4) 0 . 45 * [ (cid:4) 0 . 83 , (cid:4) 0 . 06 ] (cid:4) 0 . 45 * [ (cid:4) 0 . 83 , (cid:4) 0 . 06 ] c 40 , COMB - DIST IND 0 . 45 m [ (cid:4) 0 . 04 , 0 . 94 ] 0 . 34 m [ (cid:4) 0 . 04 , 0 . 94 ] Random effects u 0 authorj 0 . 12 0 . 13 0 . 12 u 0 challengek 0 . 60 0 . 88 1 . 35 u 1 challengek 0 . 03 Model \ufb01t statistics Deviance 372 . 65 369 . 39 369 . 13 AIC 384 . 65 383 . 39 387 . 13 \u2044\u2044 p < . 01 . 95 % CI ( Wald ) = [ lower , upper ] . m p < . 10 . * p < . 05 . * * * p < . 001 . Fig . 6 . Model - \ufb01tted relationship between combination distance of indirect sources and Pr ( shortlist ) . Fitted values evaluated at mean values of FEEDBACK , SOURCEQUAL , and SP - DIST . Greyed lines are \ufb01tted from posterior mode estimates of the slope of COMB - DIST IND for each problem . J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 111 of COMB - DIST IND did not appear to vary by problem . An additional model with a random slope for COMB - DIST IND estimates very low problem - variance , does not meaningfully decrease variance from the \ufb01xed slope model , v 2 ( 2 ) = 0 . 26 , p = . 44 ( p - value is halved ) , and also further increases AIC . Therefore , we select the \ufb01xed slope model as our \ufb01nal model for this analysis . Importantly , when estimating the posterior modes for the effect of diversity for each problem , we see that none of the 12 challenges has a negative estimate ( see Fig . 6 ) . A binomial sign test with a null hypothesis of equal probability for positive and negative effects of diversity estimates a two - tailed p - value of 0 . 0005 of observing either 0 or fewer positive or 12 or more negative signs in 12 \u2018\u2018tri - als\u201d . In other words , the effect of combination distance of indirect sources is consistent across problems in a way that is very unlikely to have arisen by chance . 3 . 3 . Iteration chain depth and earliness of inspirations One plausible alternative explanation for our \ufb01ndings is that concepts that cite indirect sources with high combination distance are better not because of the combination distance of those sources , but because those sources were inspirations posted earlier in the challenge . These earlier inspirations might lead to better concepts for a variety of reasons unrelated to combination dis - tance , e . g . , they might be of higher quality because they are posted by \u2018\u2018early adopters\u201d to the challenge who are more motivated , or they might articulate base concepts more clearly ( e . g . , because they have more time for iteration ) . Depth in iteration chain ( our main variable of interest ) in fact had a small positive association with earliness in time ( i . e . , when the inspiration was posted ) . At the inspiration level , inspirations that were posted earlier are slightly more likely to show up deeper in iteration chains , r = 0 . 13 , p < . 001 . Inspirations that are cited as both immediate and indirect sources ( M = 9 . 5 days into the chal - lenge , SE = 0 . 16 ) tend to be posted earlier than inspirations that are only ever cited as immediate sources ( M = 12 . 2 days , SE = 0 . 49 ) . However , the mean earliness of concepts\u2019 cited inspirations is not predictive of their creative success . Estimating a generalized linear mixed model with feedback , source quality , and mean inspi - ration earliness as \ufb01xed effects and challenge and contributor as random effects , we \ufb01nd that the estimated effect of earliness is near - zero , b = 0 . 03 , 95 % CI = [ (cid:4) 0 . 01 , 0 . 08 ] , p = 0 . 17 . This model does not signi\ufb01cantly improve \ufb01t over the reduced control variables model ( i . e . , with just \ufb01xed effects of feedback , source quality , and challenge and contributor random effects ) , LRT v 2 ( 1 ) = 1 . 76 , p = 0 . 18 ( AIC = 716 . 86 vs . 716 . 62 ) . Similarly , mean depth of cited inspirations in a chain is not predictive of concepts\u2019 creative suc - cess . Adding mean chain depth to the reduced control variables model does not signi\ufb01cantly improve \ufb01t , LRT v 2 ( 1 ) = 1 . 46 , p = 0 . 23 ( AIC = 717 . 17 vs . 716 . 62 ) , and the model estimates no reli - able effect of mean chain depth , b = (cid:4) 0 . 16 , 95 % CI = [ (cid:4) 0 . 42 , 0 . 09 ] , p = 0 . 22 . Therefore , mere earliness of cited inspirations cannot explain the interaction between iteration depth and the effect of inspiration diversity on creative outcomes . 4 . Discussion 4 . 1 . Summary Our goal in this paper was to examine conceptual combination as a strategy for generating creative ideas . Theories of conceptual combination and creativity suggested the hypothesis that distant conceptual combinations are especially likely to lead to highly cre - ative ideas , but the past empirical support for this hypothesis has been uneven . Drawing on broader theories of the creative process ( Amabile , 1983 ; Finke et al . , 1996 ; Sawyer , 2012 ; Simonton , 2011 ; Wallas , 1926 ; Warr & O\u2019Neill , 2005 ) , we formulated a theoretical framework that situates distant conceptual combination within the creative process as a divergent creative strategy . This theoreti - cal framework yielded a novel re\ufb01nement of the distant combina - tion hypothesis : the bene\ufb01ts of conceptual combination distance are more likely to be seen with a genealogical lag between source and target ideas . In other words , we predicted that it takes time for distant combinations to yield their creative fruits . The current study\u2019s \ufb01ndings provided empirical support for this re\ufb01ned hypothesis . As predicted , analyzing combination distance of indirect sources indeed yielded different results than direct sources . Speci\ufb01cally , we found that the mean effect of direct com - bination distance , though slightly trending in a positive direction , was essentially zero ( with some potential problem variation ) . In contrast , combination distance of indirect sources was a positive predictor of creative outcomes , and this effect was robust across problems . Thus , distant combinations do appear to be especially likely to lead to highly creative outcomes , but only if they are \u2018\u2018indirect\u201d ( i . e . , sources of one\u2019s sources ) . Importantly , we also demonstrate that the contrast between direct and indirect sources is not explained by the mere earliness of the indirect sources . 4 . 2 . Strengths and limitations Before we draw out the larger implications of this study , we \ufb01rst note some strengths and limitations of this study . First , we note that we were able to strike a favorable tradeoff between external validity ( real designers solving real creative problems ) and statistical power , which is rare in creativity research ( i . e . , typ - ical studies of real designers have smaller Ns than lab studies , not larger Ns , as in the current study ) . This feature narrows the gap considerably from the current \ufb01ndings to generalizations in real - world creative cognition ( Dunbar & Blanchette , 2001 ; Henrich , Heine , & Norenzayan , 2010 ) . Another strength of our study is our creative outcome measure , which combines both novelty and qual - ity , and follows the gold standard expert panel approach . This allows us to think more holistically about the effects of combina - tion distance on creativity , not just novelty and / or quality in isolation ( a major gap in prior work ) . One limitation of the current work is that our correlational study design does not allow us to make strong causal claims . Relat - edly , because our data source was preexisting naturalistic behav - ioral traces online , we were not able to precisely isolate cognitive processes at a \ufb01ne - grained level , as one might be able to in the lab - oratory , or obtain psychological control measures ( e . g . , partici - pants\u2019 familiarity with inspirations ) . These are legitimate concerns , and to some degree are inherent tradeoffs of an in vivo vs . in vitro approach ( Dunbar & Blanchette , 2001 ) . However , three features of our study mitigate concerns about spurious statistical associations . First , our \ufb01ndings align well with prior theory and laboratory \ufb01ndings on the potential bene\ufb01ts of combination dis - tance . Indeed , the external validity from the in vivo approach strengthens our con\ufb01dence that the laboratory \ufb01ndings generalize to real - world creative cognition . Second , unlike some other obser - vational designs , our study does include a temporal asymmetry between the predictor and outcome variables ( we know that sources were built upon before shortlisting ) , which is a notable indicator of causal direction . Finally , our statistical analysis accounts for problem variation , contributor effects , and a variety of other important control variables , mitigating concerns over endogeneity . Nevertheless , future randomized experiments are necessary to fully establish causality . Additionally , some might be concerned that the unique context of the study \u2014 e . g . , Web - based context , focus on socially relevant problems \u2014 might limit generalizability to other creative prob - lems . This is a legitimate concern , given the recent controversy in 112 J . Chan , C . D . Schunn / Cognition 145 ( 2015 ) 104 \u2013 115 the literature over the extent to which creative processes are domain - general or domain - speci\ufb01c ( Plucker , Beghetto , Sternberg , Grigorenko , & Singer , 2004 ; Simonton , 2009 ) . We have reason to believe that our results should generalize well to creativity in design - related domains ( e . g . , engineering / product / architectural design ) given the nature and diversity of problems in our sample . Solutions to these problems drew on a wide range of domain knowledge ( e . g . , public policy , human \u2013 computer - interaction , social and decision sciences , education ) and likely involved reasoning over multiple levels of systems ( e . g . , individual decision - making , communities ) . Thus , the cognitive processes and knowledge involved in generating concepts in our study are likely to have signi\ufb01cant overlap with other design - related domains . Nevertheless , we encourage further studies that explore how these \ufb01ndings might generalize to ( or be different in ) other forms of creative thought , such as artistic creativity and scienti\ufb01c discovery . Similarly , because of the public , Web - based context of the study , contributors were probably not posting every idea they had , regard - less of quality . This is a different dynamic from typical lab studies ( e . g . , of brainstorming ) where participants are asked to write down all ideas that come to mind . It is likely that in a less \ufb01ltered context , themoderatingeffectsofiterationmightbemuchmorepronounced ( but still be fundamentally similar ) : our \ufb01ndings suggest that 2 \u2013 4 iterations on a concept combined from distant concepts are suf\ufb01 - cient to make it creative , but more iterations may be required in more realistic settings where less self - \ufb01ltering is going on . 4 . 3 . Conclusions Returning to the overall question of the nature of human cre - ative cognition , our \ufb01ndings align with other studies and argu - ments that have highlighted the importance of iteration , broadly construed , in the creative process ( Chan & Schunn , 2015 ; Dow , Heddleston , & Klemmer , 2009 ; Mecca & Mumford , 2013 ; Nijstad et al . , 2010 ; Rietzschel et al . , 2007 ; Weisberg , 2011 ; Wilkenfeld & Ward , 2001 ; Yu & Nickerson , 2011 ) . This emerging body of work suggests that iteration provides pathways to not only higher qual - ity ideas ( Dow et al . , 2009 ; Yu & Nickerson , 2011 ) but also more novel ideas ( Nijstad et al . , 2010 ; Rietzschel et al . , 2007 ) , and is an advanced application of cognitive strategies like analogy ( Chan & Schunn , 2015 ) . Here , we add to this body of work , showing that iteration is a critical partner process in creative conceptual combi - nation . Again , we emphasize that this line of research concerns the importance of iteration for the development of ideas , not \ufb01nal products . The emerging picture is that good ideas rarely come in singular creative leaps , fully formed like Athena from the brow of Zeus ; instead , they more often come from the sweat of his brow building on the labors of others . Our \ufb01ndings are consistent with the predominant view of the optimal temporal ordering of divergence and convergence ( itera - tion ) in the creativity literature . Most theories of creativity posit a \u2018\u2018\ufb02are and focus\u201d view of creativity : diverge \ufb01rst , then converge . Similarly , many authors and practitioners warn of the dangers of premature solution selection ( e . g . , Brown & Katz , 2009 ; Vogel , Cagan , & Boatwright , 2005 ) . Berg ( 2014 ) showed in a series of ele - gant experiments that ideas that build \ufb01rst on very novel ideas that are then \u2018\u2018infused\u201d with more well - trodden idea components end up with a more optimal combination of novelty and quality than ideas that begin with well - trodden ideas and then are \u2018\u2018infused\u201d with novelty . While there is consensus in the literature on the optimal order of divergence and convergence , one might wonder about the optimal balance between divergence and convergence . In the pre - sent work , we observed a monotonically positive relationship between indirect combination distance and creativity . Insofar as far combinations increase divergence ( and \u2018\u2018variation\u201d in the variation - selection view of things ) , this relationship makes sense . However , this monotonically positive relationship stands in con - trast with some other work in the context of group brainstorming that has found no overall bene\ufb01t of increased divergence ( through nominal groups ) for the creativity of the \ufb01nal idea that is selected ( e . g . , Rietzschel , Nijstad , & Stroebe , 2010 ) . It might be fruitful to examine whether different divergent strategies ( e . g . , brainstorm - ing , distant conceptual combinations ) have different trajectories ( e . g . , non - monotonic , polynomial ) in terms of their effects on the \ufb01nal idea . A genealogical methodological approach ( as exempli\ufb01ed in the current study ) might be helpful for exploring this new space of questions . Acknowledgment This research was supported in part by a National Science Foun - dation # 1360013 Doctoral Dissertation Improvement Grant to the \ufb01rst author . Appendix A . OpenIDEO data additional details To give a better sense of the range of problems addressed on the platform , the following are the problem titles ( as seen by partici - pants ) for the 12 problems in the study sample : 1 . How might we increase the number of registered bone mar - row donors to help save more lives ? 2 . How might we inspire and enable communities to take more initiative in making their local environments better ? 3 . How can we manage e - waste & discarded electronics to safe - guard human health & protect our environment ? 4 . How might we better connect food production and consumption ? 5 . How can technology help people working to uphold human rights in the face of unlawful detention ? 6 . How might we identify and celebrate businesses that inno - vate for world bene\ufb01t and inspire other companies to do the same ? 7 . How might we use social business to improve health in low - income communities ? 8 . 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KANG , Carnegie Mellon University , USA XIN QIAN , University of Maryland , College Park , USA TOM HOPE , Allen Institute for AI and The University of Washington , USA DAFNA SHAHAF , Hebrew University of Jerusalem , Israel JOEL CHAN , University of Maryland , College Park , USA ANIKET KITTUR , Carnegie Mellon University , USA Analogies have been central to creative problem - solving throughout the history of science and technology . As the number of scientific articles continues to increase exponentially , there is a growing opportunity for finding diverse solutions to existing problems . However , realizing this potential requires the development of a means for searching through a large corpus that goes beyond surface matches and simple keywords . Here we contribute the first end - to - end system for analogical search on scientific articles and evaluate its effectiveness with scientists\u2019 own problems . Using a human - in - the - loop AI system as a probe we find that our system facilitates creative ideation , and that ideation success is mediated by an intermediate level of matching on the problem abstraction ( i . e . , high versus low ) . We also demonstrate a fully automated AI search engine that achieves a similar accuracy with the human - in - the - loop system . We conclude with design implications for enabling automated analogical inspiration engines to accelerate scientific innovation . CCS Concepts : \u2022 Human - centered computing \u2192 Interactive systems and tools ; Empirical studies in HCI ; Additional Key Words and Phrases : Computational analogies , innovation , scientist users , interactive analog - ical search engine , sequence - to - sequence modeling , word embeddings , think - aloud studies ACM Reference format : Hyeonsu B . Kang , Xin Qian , Tom Hope , Dafna Shahaf , Joel Chan , and Aniket Kittur . 2022 . Augmenting Sci - entific Creativity with an Analogical Search Engine . ACM Trans . Comput . - Hum . Interact . 29 , 6 , Article 57 ( November 2022 ) , 36 pages . https : / / doi . org / 10 . 1145 / 3530013 This work was supported by Center for Knowledge Acceleration , National Science Foundation ( FW - HTF - RL , grant no . 1928631 ; IIS , grant no . 1816242 ; SHF , grant no . 1814826 ) , the European Research Council ( ERC ) under the European Union\u2019s Horizon 2020 research and innovation programme ( grant no . 852686 , SIAM ) and NSF - BSF grant no . 2017741 . This work is also based upon work supported by the Google Cloud Research Credits program with the award GCP19980904 . Authors\u2019 addresses : H . B . Kang and A . Kittur , Carnegie Mellon University , 5000 Forbes Ave , Pittsburgh , PA 15213 ; emails : { hyeonsuk , nkittur } @ cs . cmu . edu ; X . Qian and J . Chan , University of Maryland , College Park , MD 20742 ; emails : { xinq , joelchan } @ umd . edu ; T . Hope , Allen Institute for AI and The University of Washington , Seattle , WA 98103 ; email : tomh @ allenai . org ; D . Shahaf , Hebrew University of Jerusalem , Israel ; email : dshahaf @ cs . huji . ac . il . This work is licensed under a Creative Commons Attribution International 4 . 0 License . \u00a9 2022 Copyright held by the owner / author ( s ) . 1073 - 0516 / 2022 / 11 - ART57 https : / / doi . org / 10 . 1145 / 3530013 ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 2 H . B . Kang et al . 1 INTRODUCTION Analogical reasoning has been central to creative problem solving throughout the history of sci - ence and technology [ 32 , 43 , 50 , 54 , 60 , 86 ] . Many important scientific discoveries were driven by analogies : the Greek philosopher Chrysippus made a connection between observable water waves and sound waves ; an analogy between bacteria and slot machines helped Salvador Luria advance the theory of bacterial mutation ; a pioneering chemist Joseph Priestly suggested charges attract or repel each other with an inverse square force by an analogy to gravity . Today the potential for finding analogies to accelerate innovation in science and engineering is greater than ever before . As of 2009 fifty million scientific articles had been published , and the number continues to grow at an exceedingly fast rate [ 12 , 28 , 68 , 85 ] . These articles represent a potential treasure trove for finding inspirations from distant domains and generating creative solutions to challenging problems . However , searching analogical inspirations in a large corpus of articles remains a longstand - ing challenge [ 34 , 44 , 83 , 99 ] . Previous systems for retrieving analogies have largely focused on modeling analogical relations in non - scientific domains and / or in limited scopes ( e . g . , structure - mapping [ 36 \u2013 38 , 42 , 106 ] , multiconstraint - based [ 33 , 59 , 65 ] , connectionist [ 57 ] , rule - based reason - ing [ 3 , 15 , 16 , 110 ] systems ) , and the prohibitive costs of creating highly structured representations prevented hand - crafted systems ( e . g . , DANE [ 65 , 109 ] ) from having a broad coverage of topics and being deployed for realistic use . Conversely , scalable computational approaches such as keyword or citation based search engines have been limited by a dependence on surface or domain similar - ity . Such search engines aim at maximizing similarity to the query which is useful when trying to know what has been done on the problem in the target domain but less useful when trying to find inspiration outside that domain ( for example , for Salvador Luria\u2019s queries : \u201chow do bacteria mu - tate ? \u201d or \u201cwhy are bacterial mutation rates so inconsistent ? \u201d , similarity maximizing search engines may have found Luria and Delbr\u00fcck\u2019s earlier work on E . coli [ 81 ] but may have failed to recognize more distant sources of inspiration such as slot machines as relevant ) . Recently a novel idea for analogical search was introduced [ 61 ] . In this idea what would other - wise be a complex analogical relation between products is pared down to just two components : purpose ( what problem does it solve ? ) and mechanism ( how does it solve that problem ? ) . Once many such purpose and mechanism pairs are identified , products that solve a similar problem to the query but using diverse mechanisms are searched to help broaden the searcher\u2019s perspective on the problem and boost their creativity for coming up with novel mechanism ideas . Anecdotal ev - idence suggests that this approach may also be applicable to the domain of scientific research . For example , while building lighter and more compact solar panel arrays has been a longstanding challenge for NASA scientists , recognizing how the ancient art form of origami may be applied to create folding structures led to an innovation to use compliant mechanisms to build not just com - pact but also self - deployable solar arrays [ 27 , 89 , 118 ] ( diagrammatically shown in Figure 1 ) . The first remaining challenge of analogical search in the scholarly domain is how we might represent scientific articles as purpose and mechanism pairs at scale and search for those that solve sim - ilar purposes using different mechanisms . Recent advances in natural language processing have demonstrated that neural networks that use pre - trained embeddings to encode input text can offer a promising technique to address it . Pre - trained embeddings are real - valued vectors that represent tokens ( Tokenization means breaking a piece of text into smaller units ; Tokens can be words , char - acters , sub - words , or n - grams . ) , in a high - dimensional space ( e . g . , typically dimensions of a few dozens to a few thousands ) and are shown to capture rich , multifaceted semantic relations between words [ 8 , 100 ] . Leveraging them , neural networks may be trained to identify purposes and mech - anisms from text [ 61 , 62 ] to enable search - by - analogy ( i . e . , different mechanisms used for similar purposes ) . Once candidate articles are retrieved , searchers may use them to come up with novel ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 3 Fig . 1 . A diagram of two different yet analogical approaches ( dashed arrow ) for building lighter and more compact solar arrays , and their representations in purposes and mechanisms . classes of mechanisms or apply them directly to their own research problems to improve upon the current state . Prior studies in product ideation showed that users of analogical search systems could engage with the results to engender more novel and relevant ideas [ 21 , 48 , 74 ] . Here , we study the remaining open questions as to whether such findings also generalize to the scientific domains of innovation and how they may differ . In this article , we present a functioning prototype of an analogical search engine for scientific articles at scale and investigate how such a system can help users explore and adapt distant inspi - rations . In doing so our system moves beyond manually curated approaches that have limited data ( e . g . , crowdsourced annotations in [ 21 ] with \u223c 2 , 000 articles ) and machine learning approaches that have been limited to simple product descriptions [ 48 , 61 , 62 ] . Using the prototypical system , we explore how it enables scientists to interactively search for inspirations for their personalized re - search problems in a large ( \u223c 1 . 7 M ) article corpus . We investigate whether scientists can recognize mapping of analogical relations between the results returned from our analogical search engine and their query problems , and use them to come up with novel ideas . The scale of our corpus al - lows us to probe realistic issues including noise , error , and scale as well as how scientists react to a search engine that does not aim at providing only the most similar results to their query . In order to accomplish these goals we describe how we address several technical issues in the design of an interactive - speed analogical search engine , ranging from developing a machine learn - ing model for extracting purposes and mechanisms in scientific text at a token level granularity , the pipeline for constructing a similarity space of purpose embeddings , and enabling these embed - dings to be queried at interactive speeds by end users through a search interface . We construct the similarity space by putting semantically related purpose embeddings in close indices from each other such that related purposes can be searched at scale . In addition to the technical challenges there are several important questions around the design of analogical search engines that we explore here . A core conceptual difference that distinguishes analogical search engines from other kinds is that the analogs they find for a search query need to maintain some kind of distance from the query , rather than simply maximizing the similarity with it . However , only certain kinds of distance may support generative ideation while others have a detrimental effect . Another question remains as to how much distance is appropriate when it comes to finding analogical inspirations in other domains . While landmark studies of analogical innovation suggest that highly distant domains can provide particularly novel or transformative innovations [ 46 , 47 , 55 ] , recent work suggests the question may be more nuanced and that inter - mediate levels of distance may be fruitful for finding ideas that are close enough to be relevant but sufficiently distant to be unfamiliar and spur creative adaptation [ 22 , 39 , 49 ] . Using a concrete ex - ample from one of our participants who studied ways to facilitate heat transfer in semiconductors , a keyword search engine might find commonly used mechanisms appropriate for direct applica - tion ( e . g . , tweaking the composition of the material ) while an analogical search engine might find ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 4 H . B . Kang et al . similar problems in more distant domains which suggest mechanisms that inspire creative adap - tation ( e . g . , nanoscale fins that absorb heat and convert it to mechanical energy ) . Though more distant conceptual combinations may not always lead to immediately feasible or useful ideas , they may result in outsized value after being iterated on [ 9 , 23 , 75 ] . In the following sections , we explore the technical and design challenges for an analogical search engine and how users interact with such a system . First , we describe the development of a human - in - the - loop search engine prototype , in which most elements of the system are functional but human screeners are used to remove obvious noise from the end results in order to maximize our ability to probe how users interact with potentially useful analogical inspirations . Using this prototype we characterize how researchers searching for inspirations for their own problems gain the most benefit from articles that partially match their problem ( i . e . , match at a high level purpose but mismatch at a lower level specifications of the purpose ) , and that the benefits are driven not by direct application of the ideas in the article but by creative adaptation of those ideas to their target domain . Subsequently we describe improvements to the system to enable a fully automated , interactive - speed prototype and case studies with researchers using the system in a realistic way involving reformulation of their queries and self - driven attention to the results . We synthesize the findings of the two studies into design implications for next - generation analogical search engines . Through extensive in - depth evaluations using an ideation think - aloud protocol [ 35 , 107 ] with PhD - level researchers working on their own problems , we evaluate the degree to which inspira - tions spark creative adaptation ideas in a realistic way on scientists\u2019 own research problems . Unlike previous work which has often used undergraduate students in the classroom or lab [ 109 ] , and of - ten evaluated systems on predetermined problems [ 40 ] , this study design provides our evaluation with a high degree of external validity and allows us to deeply understand the ways in which en - countering our results can engender new ideas . Our final , automated search engine demonstrates how the human - in - the - loop filtering can be removed while achieving a similar accuracy . We con - clude with the benefits , design challenges , and opportunities for future analogical search engines from case studies with several researchers . To encourage innovation in this domain , we release our corpus of purpose and mechanism embeddings . 1 2 SYSTEM DESIGN The design of our analogical search engine for scientific articles involves three main system re - quirements . First , a computational pipeline for automatically identifying purposes ( what problems does it solve ? ) and mechanisms ( how does it solve those problems ) at scale ( e . g . , millions of arti - cles ) , in a token - level granularity from scientific abstracts . Second , an efficient retrieval algorithm for incorporating the identified purpose and mechanism texts into the system to enable search - by - analogy ( i . e . , article abstracts that contain similar purposes to a query problem but different mechanisms ) . Third , end - user interactivity for querying problems of interest ( e . g . , \u201ctransfer heat in semiconductors , \u201d \u201cgrow plants using nanoparticle fertilizers\u201d ) . We describe the system design in detail in the following subsections . 2 . 1 Stage One . Training Seq2Seq Models for Identifying Purpose and Mechanism Tokens 2 . 1 . 1 Overview of Modeling . In the first stage of the system , purpose and mechanism tokens are identified from article abstracts ( Figure 2 , 1 (cid:4) ) . Research article abstracts often include descriptions of the most important purpose or the core problem addressed in an article and the proposed mecha - nism or the approach taken to address the problem , making them good candidates for identification 1 https : / / github . com / hyeonsuukang / augmenting _ tochi22 . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 5 Fig . 2 . Components of our system design that address the three core challenges . 1 (cid:4) Purpose and mechanism tokens are extracted from article abstracts at scale . We develop Seq2Seq classifiers to classify tokens into purpose , mechanism , or neither , going beyond previous approaches that worked on sentences or relied on crowds . 2 (cid:4) We embed the extracted purpose texts using a pre - trained language model ( Google\u2019s USE [ 20 ] ) and train a tree - based index of vectors to place high semantic similarity vectors in close neighborhoods for efficient lookup . 3 (cid:4) When the user query arrives at the system , it is first embedded with USE . This query embedding is then used to lookup the pre - computed tree indices for high similarity purpose embeddings . Article abstracts for the corresponding purpose embeddings are retrieved from Google Datastore . In the first system , additional human filtering is performed to remove obviously irrelevant results that may have been included due to model errors . Finally , a set of articles with similar purposes to the query but different mechanisms are returned to the users for ideation . and extraction of tokens corresponding to them . For example , for a similar problem of facilitating heat transfer , Article A may propose an approach that modifies the structure of the material used at the interface between crystalline silicon ( semiconductor material ) and the substrate , while Ar - ticle B may propose a more distant mechanism ( due to the mismatch on scale ) of fin - based heat sinks commonly used for electronic devices . The goal of this first stage is to automatically identify and extract tokens that correspond to the similar purpose ( e . g . , \u201cfacilitate heat transfer\u201d ) as well as the mechanisms ( e . g . , \u201cmodifying the structure of the material used at the interface between crystalline silicon\u201d vs . \u201cfin - based heat sinks\u201d ) from the abstracts A and B . One relevant automated approach for identifying purposes and mechanisms from scientific ab - stracts is DISA [ 63 ] , which formulates the task as supervised sentence classification . However , we found that many key sentences in abstracts include both purpose and mechanism , breaking the assumptions of a sentence - level classifier ( e . g . , \u201cIn this article , [ a wavelet transforms - based method ] for [ filtering noise from images ] is presented . \u201d ) . To overcome this limitation we follow [ 62 ] and frame purpose and mechanism identification as a sequence - to - sequence ( Seq2Seq ) learning task [ 5 , 101 ] and develop deep neural networks with inductive biases capable of learning token - level patterns in the training dataset . Our dataset consists of crowdsourced annotations from Chan et al . ( the dataset is constructed via application of [ 21 ] to a larger corpus of around 2 , 000 article abstracts largely in computer science domains ) ( Table 1 ) . We train the models to classify input features ( tokens or spans of tokens ) as either purpose ( PP ) , mechanism ( MN ) or neither . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 6 H . B . Kang et al . Table 1 . Summary Statistics of the Training and Validation Datasets : The Number of Purpose ( PP ) and Mechanism ( MN ) Tokens , the Number and avg . Token Length of Article Abstracts Kind ( # of articles ) Avg . length # of PP # of MN Train ( 2021 ) 196 65 , 261 120 , 586 Validation ( 50 ) 170 1 , 510 1 , 988 We train two deep neural networks ( Models 1 and 2 ) , achieving increasing accuracy of classifi - cation . The first model is based on a Bi - directional LSTM ( BiLSTM ) architecture for sequence tagging [ 56 , 64 ] , in which the forward ( the beginning of the sequence to the end ) and the backward passes condition each token position in the text with its left and right context , respectively . A main source of improvement of Model 2 over Model 1 is the ability to more selectively attend to infor - mative tokens in a sentence rather than treating each token in a sequence as independent of each other ( as a hypothetical example , an extremely effective model based on this approach may assign more weights to the tokens \u201cselectively attend to informative tokens\u201d , as they represent the core mechanism described in the previous sentence ) and to leverage the regularities of co - occurrence with surrounding words through the self - attention mechanism [ 108 ] . 2 . 1 . 2 Seq2Seq Model Implementation Details . We implement the BiLSTM architecture of Model 1 in PyTorch [ 87 ] . We use pre - trained GloVe [ 88 ] word embeddings with 300 dimensions , consistent with prior work [ 11 , 78 , 88 ] to represent each token in the sequence as 300 - dimensional input vectors for the model . We train the model with a cross entropy loss objective for per - token classification in the three ( PP , MN , Neither ) token classes . For Model 2 , we adapt the SpanRel [ 67 ] architecture and implement it on AllenNLP [ 41 ] . We im - plement a self attention mechanism that tunes weights for the core word in each span as well as the boundary words that distinguish the context of use , consistent with [ 79 ] . We use the pre - trained ELMo 5 . 5B [ 90 ] embeddings for token representation following the near state - of - the - art perfor - mance reported in [ 67 ] on the scientific Wet Lab Protocol dataset . We train the model using a simi - lar procedure as Model 1 . We leave detailed training parameters for Models 1 and 2 to the Appendix . 2 . 1 . 3 Introducing Human - in - the - loop Filtering for Model 1 . The final classification performance ( F1 - scores ) of Model 1 on the validation set is 0 . 509 ( Purpose ) , 0 . 497 ( Mechanism ) , and 0 . 801 ( nei - ther ) . We found that the limited accuracy contributed to how the system retrieves irrelevant search results . Because reactions to obviously irrelevant results are not useful , we added a human - in - the - loop [ 31 ] filtering stage . The filtering proceeded as follows : members from the research team in - putted problem queries received from study participants into the system . Once the model produced matches , they went over from the top of the sorted list and removed only those that are irrelevant to the problem context . They continued filtering until at least 30 articles with reasonable purpose similarity were collected . After Winsorizing at top and bottom 10 % [ 115 ] , the human filterers re - viewed 45 articles per query ( SD : 27 . 6 , min : 6 , max : 138 ) for 5 queries ( SD : 2 . 4 , min : 2 , max : 9 ) to collect 33 ( SD : 3 . 5 , min : 30 , max : 40 ) purpose - similar articles ( about 12 / 45 = 26 % error rate ) . In Study 1 we show that the limited retrieval accuracy of Model 1 is sufficient for use as a probe with this additional human - in - the - loop filtering . In Study 2 and case studies , we demonstrate how this filtering can be removed with Model 2 while achieving a similar accuracy . 2 . 1 . 4 Scaling Model Inference . In order to have sufficient coverage to return diverse results , we collected an initial corpus of 2 . 8 million research articles from Springer Nature . 2 After 2 https : / / dev . springernature . com / . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 7 Table 2 . Corpus Used in the Deployed Search Engine and its Topical Distribution : Computer Science ( CS ) , Engineering ( Eng ) , Biomedicine ( BioMed ) , and Business and Engineering ( B & Eng ) Domain CS Eng BioMed B & Eng Total Count 675 K 568 K 336 K 145 K 1 . 7 M deduplication ( based on Digital Object Identifier using BigQuery 3 ) and filtering only articles with at least 50 characters in the abstract we were left with 1 . 7 million articles in four subjects ( Table 2 ) . We stored the resulting corpus in Google Cloud storage buckets . 4 To scale the classification of the Seq2Seq models we used the Apache Beam API 5 on Google Cloud Dataflow 6 to parallelize the operation . 2 . 2 Stage Two . Constructing a Purpose Similarity Space 2 . 2 . 1 Overview . In the second stage , the identified purpose texts are incorporated into the sys - tem to enable search - by - analogy of articles that solve similar problems using different mechanisms , at an interactive speed ( Figure 2 , 2 (cid:4) ) . Relevant previous approaches include Hope et al . [ 61 ] which first clusters similar purposes ( through k - means with pruning ) and subsequently samples within each cluster of similar purposes to maximize the diversity of mechanisms ( via a GMM approxi - mation algorithm [ 92 ] ) , or [ 62 ] which employs similarity metrics to balance the similarity to a purpose query and the distance to a mechanism query ( and vice versa ) . In contrast , from pilot tests in our corpus we discovered that even close purpose matches of scientific articles already had high variance in terms of the mechanisms they propose . We hypothesize that this may be the case due to the enormous span of possible research topics and the relative sparseness of their coverage in our corpus , and / or due to the emphasis on novelty in scientific research that discourages future articles which might contribute relatively small variations to an existing mechanism . We leave ex - ploration of these hypotheses for future work and simplify our sampling of the scientific articles to the one based solely on the similarity of purpose , sufficient for ensuring diversity . In order to support fast retrieval ( e . g . , sub - second response time ) of articles with similar purposes at scale ( e . g . , millions of articles ) , we pre - train Spotify\u2019s Annoy 7 indices of nearest neighboring pur - poses . Annoy trains a neural network to assign an embedding vector corresponding to a purpose to an index in the high - dimensional space that brings it close to other indices of purpose vectors that have similar meaning ( see Section 2 . 2 . 3 for details of the metric used for the similarity of mean - ing ) . Annoy uses random projection and tree - building ( see [ 1 , 2 ] ) to create read - only , file - based indices . Because it decouples creation of the static index files from lookup , it enables efficient and flexible search by utilizing many parallel processes to quickly load and map indices into memory . 2 . 2 . 2 Interactive Speed . Additionally Annoy minimizes its memory footprint in the process . This efficiency , critical for real - time applications such as ours , was further validated during our test of the end - to - end latency on the Web , with the average response taking 2 . 4 s ( SD = 0 . 56 s ) . 8 The level of latency we observed was sufficiently low to enable interactive search by end users ( both human - in - the - loop filterers in Study One and researcher participants in case studies ) . 3 https : / / cloud . google . com / bigquery . 4 https : / / cloud . google . com / storage . 5 https : / / beam . apache . org / . 6 https : / / cloud . google . com / dataflow / . 7 https : / / github . com / spotify / annoy . 8 We tested with 20 topically varied search queries that have not previously been entered to the engine to test the latency end - users experience and to exclude the effect of caching from it . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 8 H . B . Kang et al . 2 . 2 . 3 Implementation Details . To construct the similarity space , we first encode the purpose texts into high - dimensional embedding vectors which then can be used to compute pairwise se - mantic similarity . Here , the choice of an encoding algorithm depends on three main constraints . First , the pairwise similarity , when computed , should correlate well with the human - judged se - mantic similarity between the purposes . Second , similarity calculation between varying lengths of texts should be possible because extracted purposes can differ in length . Third , computationally efficient methods are preferred for scaling . To meet these requirements , we chose Universal Sentence Encoder ( USE ) 9 to encode purposes into fixed 512 - dimensional vectors . USE trains a transformer architecture [ 108 ] on a large corpus of both unsupervised ( e . g . , Wikipedia ) and su - pervised ( e . g . , Stanford Natural Language Inference dataset [ 13 ] ) data to produce a neural network that can encode text into vectors that meaningfully correlate with human judgment ( e . g . , evaluated on the semantic textual similarity benchmark [ 19 ] ) . USE can handle texts of varying lengths ( e . g . , from short phrases to sentences to paragraphs ) , and with high efficiency [ 20 ] , thereby making it suitable for our system . We pre - compute pairwise similarity of the purpose embeddings and store the indices in neigh - borhoods of high similarity for fast retrieval of similar purposes . As mentioned before , we train the Annoy indices on Google Cloud AI Platform . 10 We use 1\u2014the Euclidean distance of normalized vectors ( i . e . , given two vectors u and v , distance ( u , v ) = (cid:2) ( 2 ( 1 \u2212 cos ( u , v ) ) ) ) as a similarity metric ( using a Euclidean distance based metric for nearest neighbor clustering shows good performance , see [ 4 ] for a related discussion on the impact of the distance metric on the retrieval performance ) . We set the hyper - parameter k specifying the number of trees in the forest to 100 ( larger k \u2019s result in more accurate results but also decreases performance ; see [ 2 ] for further details ) . Empirically , 100 seemed to strike a good balance between the precision - performance tradeoff , thus we did not experiment with this parameter further . 2 . 3 Stage Three . Retrieving the Results In the last stage , the front - end interface interacts with end users and receives problem queries . These queries are then relayed to the back - end for retrieval of articles that solve similar problems using different mechanisms . The retrieved articles are presented on the front - end for users to review ( Figure 2 , 3 (cid:4) ) . When a user query is received , the back - end first encodes it using the same encoding algorithm used as the construction method of the purpose similarity space ( i . e . , USE ) . Using this query embedding , the back - end searches the pre - trained similarity space for articles with similar purposes . The articles with high purpose similarity are then returned to and displayed on the front - end . We describe the actual interfaces used in the studies in the corresponding design sections ( Sections 3 . 2 . 4 and 3 . 2 . 5 ) . Together the design of our system enabled what is to our knowledge the first functioning pro - totype of an interactive analogical search engine for scientific articles at scale . In the following sections , we report on how such a search engine can help researchers find analogical articles that facilitate creative ideation . 3 STUDY 1 : CREATIVE ADAPTATION WITH A HUMAN - IN - THE - LOOP ANALOGICAL SEARCH ENGINE In Study 1 , we set out to establish the viability of an analogical search engine using a human - in - the - loop probe in the domain of scholarly recommendations . We investigate whether analogical search returns a distinct and novel set of articles compared to keyword search results , and capture 9 https : / / tfhub . dev / google / universal - sentence - encoder - large / 5 . 10 https : / / cloud . google . com / ai - platform . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 9 participants\u2019 reaction to each result in a randomized order , blind to condition . To deeply under - stand the process of ideation using analogical articles we ask participants to come up with new ideas for their own research projects after reviewing each article . Using this data we code ideation outcomes in depth to explore the various ways in which analogical distance can shape ideation outcomes , such as inspiring direct transfer of solutions , or sparking adaptation of ideas into novel combinations . 3 . 1 Coding Ideation Outcomes We are interested in studying whether an analogical search engine provides distinctive and com - plementary value to other commonly used search approaches that rely on surface similarity . In particular , our focus is on the inspirational value rather than the immediate relevance of search results or the direct usefulness of solutions . The highest value of creative inspiration often comes from creatively adapting ideas to reformulate a problem and recognizing new bridges to previously unknown domains that open up entirely new spaces of ideas . For example , recognizing a connec - tion from the ancient art form of origami to fold intricate structures with article and building a sufficiently compact , deployable solar panel arrays and radiation shields led NASA to hire origami experts [ 27 , 89 , 118 ] . Our approach to measuring ideation outcome is through the use of a quaternary variable cat - egorizing the types of ideation . To capture the inspirational value of analogical search and move beyond the measurements focused on the immediate relevance or the direct usefulness we distin - guish the Creative Adaptation and Direct Application types of ideation . In our studies these two types corresponded to think - alouds that resulted in novel ideas whereas the rest ( Background and None ) corresponded to think - alouds in which no new ideas were produced . \u2014 Creative Adaptation : Novel mechanism ideas that involve substantial adaptation of the information provided in the article . These ideas are typically associated with a higher uncer - tainty of success due to the less familiar nature of the domains involved . \u2014 Direct Application : More directly applicable ideas that involve less adaptation than Cre - ative Adaptation . These ideas are typically associated with a lower uncertainty of success because researchers are more familiar with the domains . \u2014 Background : The information provided in the article is good for background reading ( e . g . , to learn about other domains ) . \u2014 None : Did not result in new ideas nor was useful for background reading . Creative Adaptation ideas generally involved a substantial amount of adaptation , while Direct Application ideas were closer to the source domain and more directly applicable . For example , using the data from one of our participants , applying the techniques for manipulating thermal conductance at solid - solid interfaces was considered a direct application idea for P1 ( Figure 3 , left ) because he was familiar with the concept of controlling the interfacial thermal conductiv - ity given the relevant approaches he developed in his current and past research projects . Thus the connections to the source problem were directly recognizable . On the other hand , creating a fin - based wall structure for heat transfer was an example of creative adaptation idea ( Figure 3 , right ) because of its novelty and the participant\u2019s unfamiliarity in relevant domains . The unfa - miliarity and uncertainty was generally more associated with analogs for creative adaptation than direct application . On the other hand , the unfamiliarity also sometimes acted as a barrier to partici - pants\u2019 openness and subsequent ideation . Though challenging , in order to recognize novel connec - tions to the source problem the participants may need to suspend their early rejection of a seem - ingly foreign idea and its surface - level mismatches and engage in deeper processing which could lead to re - imagination and re - formulation of the research problem at hand . To code the Creative ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 10 H . B . Kang et al . Fig . 3 . Example articles for the purpose of facilitating heat transfer heat in semiconductors . ( Top ) A Direct Application article involves directly applicable ideas and techniques for manipulating the interface material and structure to control thermal conductance . ( Bottom ) A Creative Adaptation example involves transferring a distant idea ( fin - based design for heat sinks ) and creatively adapting it into the target problem context ( designing nano - scale fins that could absorb heat and convert it to useful energy ) . Figure credits : contact configurations and interface resistance from [ 116 ] , fin - based heat sink from [ 104 ] , nano - fins from [ 94 ] . Adaptation and Direct Application types of ideation outcomes , the coders took into consideration different linguistic and contextual aspects of the descriptions of the ideas and their think - aloud process ( details in Section 3 . 2 . 3 ) . 3 . 2 Design of the Study 3 . 2 . 1 Participants . We recruited eight graduate ( four women ) researchers in the fields of sci - ences and engineering via email advertisement at a private R1 U . S . institution . Four were senior PhD students ( 3rd year or above and one recently defended their thesis ) and the rest was 2nd year or below . Disciplinary backgrounds of the participants included : Mechanical ( 3 ) , Biomedical ( 2 ) , Environmental ( 1 ) , Civil ( 1 ) , and Chemical Engineering ( 1 ) . Once a participant signed up for the study , we asked them to describe their research problems and send the research team search queries they use to look for inspirations on popular search engines such as Google Scholar . 11 Members of the research team screened articles with relevant purposes using these queries on the filtering in - terface ( Figure 4 , left ) . Despite our efforts to collect articles over diverse topical areas , the search engine did not contain enough articles for two of the participants who work on relatively novel fields ( e . g . , \u201cmachine learning methods of 3D bioprinting\u201d ) . These participants were interviewed on their current practices for reviewing prior works and coming up with new ideas for research and were not included in the subsequent analyses . 3 . 2 . 2 Study Procedure and Keyword - search Control . The rest of the participants were then in - vited to in - person interviews . To ensure that participants would be exposed to a sufficiently diverse set of analogical mechanisms and to maximize our power to observe the ideation process , we gen - erated a list of top 30 results from the analogical search engine using the search queries provided 11 https : / / scholar . google . com / . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 11 Fig . 4 . The front - end interfaces . ( Left ) Human reviewers used this filtering interface to input search queries received from the participants and remove articles with obviously irrelevant purposes . To assist the reviewers\u2019 filtering process , model predicted purpose ( e . g . , the noise reduction and time , highlighted in red at the bottom of the filtering interface ) and mechanism ( highlighted in green ) tokens were also provided along with the title and the abstract text . The background color turned green when the \u201cSimilar\u201d button is clicked and red when the \u201cDissimilar\u201d button is clicked . ( Right ) The ideation task interface was populated with a list of human filtered articles for review by the participants in Study 1 ( the order of articles was randomized ) . by the study participants . As a control condition we also included top 15 results from a keyword - based search engine using the standard Okapi BM25 algorithm [ 82 ] ( k 1 = 1 . 2 , b = 0 . 75 ) using the same search queries as the analogical search engine . The order of results in the list was ran - domized and participants were blind to condition . To account for the difference in the quantity of exposure in the analysis , we normalized the ideation outcomes by the number of results returned in each condition . Using this list we employed a think - aloud protocol [ 80 , 107 ] in which partic - ipants were presented with the title , abstract , and other metadata of articles and asked to think aloud as they read through them with the goal of generating ideas useful for their research using our Web - based interface ( Figure 4 , right ) . Although time consuming , this approach allowed us to capture rich data on participants\u2019 thought process and how those processes changed and evolved as participants considered how an article might relate to their own research problems . In addition , we asked the participants to make a judgment on the novelty of each article on a 3 - point Likert - scale . After participants finished reviewing the 45 articles , we interviewed them about their overall thoughts on the results\u2019 relevance and novelty and whether there were any surprising or unique results . Each interview lasted about one and a half hours and the participants were compensated $ 15 / hr for their participation . 3 . 2 . 3 Data and Coding . In total , our data consisted of 267 article recommendations for six par - ticipants and their Likert - scale questionnaire responses measuring the content novelty , after re - moving 3 within - condition duplicates ( these articles included cosmetic changes such as different ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 12 H . B . Kang et al . capitalization in the title or abstract ) . One participant ran out of time towards the end of the inter - view and only provided novelty measures for the last 17 article recommendations in the random - ized list . Thus , 250 transcripts of participants\u2019 think - aloud ideation after reading each article were used for analyzing ideation outcomes . To code the distance between the Creative Adaptation and Direct Application types of ideation outcomes , the coders took into consideration ( 1 ) the verbs used to describe the ideas ( e . g . , \u201cdesign\u201d , \u201cdevelop\u201d , or \u201cinvent\u201d were generally associated more with distant ideas compared to \u201capply\u201d , \u201cuse\u201d , \u201cadopt\u201d ; see Table 3 ) ; ( 2 ) the context of ideas such as participants\u2019 expression of unfamiliarity or uncertainty of the domain involved ( e . g . , \u201cI\u2019m not really sure\u201d vs . \u201cI\u2019m familiar with this domain\u201d ) ; and ( 3 ) participants\u2019 perceived immediacy of the idea\u2019s applicability ( i . e . , ideas perceived by participants as more immediately applicable were associated with direct application but not creative adaptation ideas ) . Two of the authors coded a fraction of the data together ( 13 / 250 , 5 . 2 % ) and then independently coded the rest blind - to - condition , using the four ideation outcomes types described in Section 3 . 1 and with the following protocol : The coders first judged the existence of an idea . If there was , then its type was further distinguished between Creative Adaptation and Direct Application using the linguistic and contextual descrip - tions described above ( e . g . , Creative Adaptation ideas were more frequently associated with the \u201cdesign\u201d words , higher unfamiliarity and uncertainty of the domains , and less immediate applica - bility , compared to Direct Application ideas ) . In case there was no concrete idea in the data , coders further distinguished between the Background vs . None cases . The agreement between coders was significant , with Cohen\u2019s \u03ba = 0 . 89 ( near perfect agreement ) for the four categories of ideation outcome . Given the high level of agreement between the coders , any disagreements were resolved via discussion on a case - by - case basis . 3 . 2 . 4 Apparatus 1 : The Human - in - the - loop Filtering Interface . In Study 1 , members of the re - search team first received search queries from study participants and reviewed the model - produced purpose matches to filter irrelevant articles using a filtering interface ( Figure 4 , left ) . This additional step was introduced to ensure that articles with obviously dissimilar purposes are not returned to study participants . Reviewers determined whether each article contained a clearly irrelevant pur - pose in which case it was removed by clicking the Dissimilar button at the bottom of the article . On the other hand when the Similar button was clicked it turned the background of the article green in the interface and increased the number of the articles collected so far . Reviewers continued the screening process until at least 30 articles with reasonable purpose similarity were collected . 3 . 2 . 5 Apparatus 2 : The Ideation Task Interface . The filtered articles were then displayed as a randomized list of articles to study participants ( Figure 4 , right ) . In addition to the content and metadata of articles ( e . g . , authors , publication date , venue ) , each article was presented with a Likert - scale question for measuring content novelty and a text input for ideation . 3 . 2 . 6 Limitations . To reduce potential biases , our coders were blind to experimental conditions and relied on participants\u2019 statements of ideas\u2019 novelty and usefulness ( e . g . , \u201cI\u2019ve never seen some - thing like this before , \u201d \u201cthis is not a domain I would\u2019ve searched if I used Google Scholar\u201d ) , and achieved a high inter - rater reliability . We believe coders had a reasonable understanding of how participants arrived at specific ideas from descriptions of their current and past research topics , think - alouds , and end - of - experiment discussions . Despite this , we also acknowledge the limita - tions of this approach and discuss how future research may improve upon it ( see Section 7 . 2 . 1 ) . 3 . 2 . 7 On Reporting the Results . We report the result of our studies below . To denote statis - tical significance we use the following notations : \u2217 ( \u03b1 = 0 . 05 ) , \u2217\u2217 ( \u03b1 = 0 . 01 ) , \u2217\u2217\u2217 ( \u03b1 = 0 . 001 ) , \u2217\u2217\u2217\u2217 ( \u03b1 = 0 . 0001 ) . Alpha levels were adjusted when appropriate in post - hoc analyses using Bon - ferroni correction . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 13 Fig . 5 . ( Left ) Participants judged analogy articles significantly more novel . The mean response to the question \u201cHave you seen this article before ? \u201d was significantly higher in Analogy : 2 . 7 ( SD : 0 . 48 ) than in Keyword : 2 . 3 ( SD : 0 . 55 ) . ( Right ) There were significantly more overlapping words between search query terms provided by participants and the title and abstract text of articles : Keyword : 4 . 1 ( SD : 1 . 74 ) vs . Analogy : 1 . 6 ( SD : 1 . 42 ) . 3 . 3 Result Finding novel articles for creative ideas . Our key measure of success is how article recommen - dations from the analogy search engine ( hereinafter analogy articles ) help scientists generate cre - ative ideas for their own research problems . To this end , we investigate ( a ) whether analogy articles are novel and complementary to the articles found from the keyword - search baseline ( hereinafter keyword articles ) and ( b ) whether analogy articles resulted in more creative adaptation ideas than direct application ideas in ideation . 3 . 3 . 1 Analogy Articles Differed from Keyword Articles and were Judged more Novel . The viability of our approach is based on the assumption that the analogy search pipeline returns a different distribution of results than a keyword - based baseline . This assumption appeared to hold true : the keyword - search and analogy - search conditions resulted in almost completely disjoint sets of ar - ticle recommendations . Out of the total 267 articles , the overlap between analogy and keyword articles was only one . Analogy articles appeared to represent a complementary set of results users would be unlikely to encounter through keyword - based search . To further examine this assumption we had participants rate the novelty of the results by asking them \u201c have you seen this article before ? \u201d on a 3 - point Likert scale response options of 1 : \u201c Yes , I have seen this article before \u201d , 2 : \u201c Yes , not exactly this article but I have seen similar ideas before \u201d , and 3 : \u201c No , I have not seen anything like this before \u201d . Participants found articles recommended in the analogy condition to contain significantly more novel ideas ( 2 . 7 , SD : 0 . 48 ) compared to the keyword condition ( 2 . 3 , SD : 0 . 55 ) ( Welch\u2019s two - tailed t - test , t = \u2212 5 . 53 , p = 1 . 33 \u00d7 10 \u2212 7 ) ( Figure 5 , left ) . Participants thought the \u201cvariance in results is much higher than using other search engines\u201d ( P5 ) and \u201cthere\u2019re a lot of bordering domains . . . which can be useful if I want to get ideas in them\u201d ( P4 ) . This difference was also reflected in the content of articles , with keyword articles having signifi - cantly more overlapping terms with participant - provided query terms ( 4 . 1 , SD : 1 . 74 ) than analogy articles ( 1 . 6 , SD : 1 . 42 ) ( Welch\u2019s two - tailed t - test , t ( 145 . 27 ) = 11 . 70 , p = 1 . 10 \u00d7 10 \u2212 22 ) ( Figure 5 , right ) . 12 More occurrences of familiar query terms in keyword articles\u2019 titles and abstracts may have led participants to perceive them as more familiar . 12 We measured the term overlap between participants\u2019 queries and the content of articles ( title and abstract ) . To preprocess text , we used NLTK [ 10 ] to tokenize articles\u2019 content , remove stopwords , digits , and symbols , and lemmatize adjectives , verbs , and adverbs . Finally , using the processed tokens we constructed a set of unique terms for each article and the query which was then compared to find overlapping terms . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 14 H . B . Kang et al . Fig . 6 . Frequency of the ideation outcome types by condition . Darker colors represent higher rates . Creative adaptation is 5 . 3 times more frequent among analogy articles ( 53 in Analogy vs . 10 in Keyword ) , while most of direct application is from keyword articles ( 3 in Analogy vs . 16 in Keyword ) . The distributions differed significantly ( chi - squared test , \u03c7 2 ( 3 ) = 52 . 12 , p < 1 . 0 \u00d7 10 \u2212 10 overall and \u03c7 2 ( 1 ) = 28 . 41 , p = 9 . 84 \u00d7 10 \u2212 8 for the contrast between the rates of creative adaptation and direct application ideas ) . 3 . 3 . 2 Analogy Articles Resulted in more Creative Adaptation Ideas than Direct Application Ideas . We found that the distribution of ideation outcome types differed significantly between analogy and keyword articles ( \u03c7 2 ( 3 ) = 52 . 12 , p < 1 . 0 \u00d7 10 \u2212 10 ) . Participants came up with more creative adaptation ideas ( N = 53 ; 32 % of total ) over direct application ideas ( N = 3 ; 2 % ) using analogy articles . In contrast , keyword articles resulted in more direct application ideas ( N = 16 ; 19 % ) than creative adaptation ideas ( N = 10 ; 12 % ) ( Figure 6 ) . The difference between creative adaptation and direct application was significant ( \u03c7 2 ( 1 ) = 28 . 41 , p = 9 . 84 \u00d7 10 \u2212 8 ) . To illustrate more concretely the divergent patterns of ideation leading to Creative Adaptation and Direct Application ideas , we describe vignettes from three participants ( Table 3 ) . While Direct Application ideas represented close - knit techniques and mechanisms directly useful for the source problem ( described with verbs such as apply and adopt ) , Creative Adaptation type ideas were more distant from the source problem and could be characterized with the use of different verbs asso - ciated with significant adaptation ( design and invent ) . For example , P1\u2019s research focused on the methods for improving nanoscale heat transfer in semiconductor materials . Previously he devel - oped mechanisms for manipulating the thermal conductivity at solid - solid interfaces , specifically by adjusting the semiconductor wall structures . Thus , an article reporting experimental results of manipulating thermal conductance on planar metallic contact points was deemed a directly useful article that might contain helpful techniques . On the other hand , an analogy article which dealt with the heat transfer phenomenon at a macroscale , using fin - based heat sink designs for electronic devices , gave him a new inspiration : to adapt fins for nanoscale heat transfer in semiconductors to not only transfer heat but also convert it into a useful form of mechanical energy . Despite the mismatch on scale ( [ macroscale ] (cid:2) [ microscale ] ) , challenging the assumption of the typical size of a fin - based design engendered an idea to creatively adapt it to convert heat into energy through an array of tiny fins , rather than merely dissipating it into space as in the original formulation of the problem . P1 also found another analogy article focused on thermal resistance at a liquid - solid interface useful for future ideation because despite its surface dissimilarities , there was a poten - tial mapping that may open up a new space of ideas ( e . g . , [ liquid ] (cid:2) [ polymer substrate ] , [ solid ] (cid:2) [ germanium ] , yet the pairwise relation [ liquid : solid ] \u2194 [ polymer substrate : germanium ] may be analogous and interesting ) : \u201cThis is liquid . . . but it\u2019s about liquid - solid interface which can be useful . . . because for the substrate that sits on top of silicon or germanium you use polymers which have liquid - like properties\u201d ( P1 ) . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 15 Table 3 . Examples of Direct Application and Creative Adaptation Types for Three Participants ( PID ) PID Research Problem Type Article Title \u2192 New Idea ( paraphrased ) 1 Improve nanoscale heat transfer in semiconductormaterial Direct Application Experimental investigation of thermal con - tact conductance for nominally flat metallic contact \u2192 Apply the techniques in the arti - cle to manipulate thermal conductance at the solid - solid interface Creative Adaptation Investigation on periodically developed heat transfer in a specially enhanced channel \u2192 Design nanoscale \u201cfins\u201d to absorb heat and convert it to mechanical energy 2 Grow plants better by optimizing entry of nanoparticle fertilizers into the plant Direct Application Nanoinformatics : Predicting Toxicity Us - ing Computational Modeling \u2192 Apply the computational modeling from the arti - cle for predicting toxicity of candidate nanoparticles Creative Adaptation Identification of Plant Using Leaf Image Analysis \u2192 Invent a hyperspectral 3D imaging mechanism for plants that opti - cally senses , traces , and images plant cells in 3 - dimensional structures 3 Enhance the evaporationefficiencyof thin liquid films in heat pipes and thermosyphons Direct Application Thin film evaporation effect on heat trans - port capability in a grooved heat pipe \u2192 Adopt the techniques in the article for manipulating the solid interface\u2019s surface properties to balance the film thickness and disjoining pressure Creative Adaptation Alkaline treatment kinetics of calcium phosphate by piezoelectric quartz crystal impedance \u2192 Design novel liquid film ma - terials for manipulating hydrophobicity to change disjoining pressure Each participant\u2019s research problem is described in the Problem column . While the topics of research problems vary , Creative Adaptation ideas are more distant in terms of content compared to the source problem than Direct Application ideas are , and may be characterized by the use of different sets of verbs ( { design , invent } in Creative Adaptation ideas versus { apply , adopt } in Direct Application ideas ) . In the case of P2 , an article focused on computational methods for toxicity prediction was deemed directly helpful because \u201cif certain nanomaterials are toxic to certain microorganisms that eat plants or kill them but safe for the plant , we can target these organisms using the nanomate - rials as pesticide . Another way this can be helpful is in predicting the chance of toxicity of the nanoparticles in our fertilizers\u201d ( P2 ) . Whereas an analogy article that uses image analysis for plant identification reminded her of \u201chyperspectral imaging in plants , like a CT scan for plants . So mak - ing a hyperspectral 3D model using something like this . . . to optically sense and trace plant cells ( such that the entry of fertilizer nanoparticles into plant cells can be monitored , a sub - problem of P2\u2019s research problem ) would be pretty cool . \u201d ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 16 H . B . Kang et al . Table 4 . Examples of Different Purpose - match Types Purpose - Match PID Participant Comment Full 2 \u201cIt\u2019s a little bit old ( from 2010 ) but I have read articles from that era . I love this . . . because the article mentions everything else and espe - cially one word which is \u201cdisjoining pressure\u201d\u2014if I were to publish my current project that\u2019s going to be the core topic . \u201d Part 1 \u201cThough I\u2019m not familiar with GFRP - GFRP . . . but I can see that they\u2019re referring to glass fiber reinforced plastic , so this is something not crystalized material . . . learning about this kind of materials is in - teresting . \u201d None 3 \u201cI don\u2019t know what a lot of words mean . I don\u2019t typically work with animals cells . \u201d Purpose - Match shows the level of purpose - match between a recommended article and each participant\u2019s research problem ( see Table 3 for descriptions of research problems ) . Fully matching purposes are those that match at both high - ( more abstract ) and low - levels ( specific details ) . Partial matches only match at the high - level abstraction and differ in details . The Participant Comment column shows relevant excerpts from the participant . As a third example , P6\u2019s research focused on recording and simulating electrical activity us - ing microelectrode arrays . To him , an analogy article about printing sensors for electrocardio - gram ( ECG ) recording seemed to present an interesting idea despite its mismatch in terms of scale ( [ nanoscale ] (cid:2) [ macroscale ] ) and manufacturing mechanism ( [ fabrication ] (cid:2) [ printing ] ) , be - cause the pairwise relation between [ nanoscale : fabrication ] \u2194 [ macroscale : printing ] engendered a reflection on the relative advantages of different methods and future research directions ) : \u201cInter - esting idea ! Instead of nanoscale fabrication , printing can be a good alternative for example for rapid prototyping . But I think the resolution won\u2019t be enough ( for use ) in nanoscale . . . works for this particular article\u2019s goal , but an idea for future research is whether we can leverage the benefit of both worlds\u2014rapid printing and precision of nanoscale fabrication\u201d ( P6 ) . 3 . 3 . 3 The Level of Purpose - match had Different Effects on the Ideation Outcome . Suggested in these examples is a certain kind of distance the ideas in analogy articles maintain in order to spur creative adaptation . We hypothesize that some amount of difference in purpose facilitates creative adaptation . This process may involve a curvilinear relationship between the degree of purpose mismatch and the resulting ideation outcome , with too much or too little deviation leading to a little - to - no benefit or even an adverse effect on the ideation outcome , a pattern that is consistent with the findings in the literature of creativity and learning outcomes ( e . g . , Csikszentmihalyi\u2019s optimal difficulty [ 25 ] ) . For this analysis , we coded each article based on three levels of purpose - match to the source problem : \u2014 Full : Both high - and low - level purposes match . \u2014 Part : Only the high - level abstract purpose matches . Explicit descriptions of the high - level purpose exist in either title and abstract of the article . At the same time , certain low - level aspects of the participant\u2019s research problem are mismatched as evidenced by relevant com - ments from the participant . \u2014 None : Neither high - nor low - level purposes match . Examples of these types of purpose - match are provided in Table 4 . High - level match can be considered as a first - order criterion of purpose match and low - level match as a second - order cri - terion : If the article does not have overlapping terms in terms of its purpose with the user query cast at a high level ( e . g . , transfer heat , grow plants ) then the low - level match does not matter , but if the article\u2019s purpose matches at the high level , its low - level alignment ( e . g . , specific aspects of ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 17 Table 5 . Regression Table of Three Mediation Analyses using Purpose - match , Novelty and Pid ( Participant ID ) as Mediators between Condition and the Binary Creative Adaptation Outcome Variable Effect of Condition Unique Effect Indirect Effect CI 95 % Mediator on Mediator ( a ) of Mediator ( b ) ( a \u00d7 b ) Lower Upper Purpose - match \u2212 0 . 42 \u2217\u2217\u2217\u2217 0 . 21 \u2217\u2217\u2217\u2217 \u2212 0 . 09 \u2217\u2217\u2217\u2217 \u2212 0 . 14 \u2212 0 . 05 ( . 08 ) ( . 05 ) 0 . 40 \u2217\u2217\u2217\u2217 \u2212 0 . 06 \u2212 0 . 02 \u2212 0 . 07 0 . 02 Novelty ( . 07 ) ( . 05 ) Pid \u2212 0 . 02 0 . 03 \u2217 \u2212 0 . 001 \u2212 0 . 02 0 . 02 ( . 22 ) ( . 02 ) Purpose - match was the only significant mediator between Condition and Creative Adaptation ( indirect effect = \u2212 0 . 09 , significant using a bootstrapping method [ 91 ] with 1 , 000 iterations , p < 2 \u00d7 10 \u2212 16 ) . the purpose , such as its scale or materialistic phase ) will additionally determine full ( i . e . , aligned in both high - and low - level aspects of the purpose ) vs . partial match ( i . e . , aligned only in the high - level but not low - level aspects of the purpose ) . Therefore , the coding procedure was symmetrical to the procedure described for coding four types of ideation outcome , with the high - level purpose match deciding between { Full , Part } and None match types , while the low - level purpose further distinguishing between Full vs . Partial match . Following this procedure , two independent coders achieved an inter - rater reliability Cohen\u2019s \u03ba = 0 . 72 ( substantial agreement ) and disagreements were resolved with case - by - case discussion . We used the mediation package 13 [ 105 ] to conduct a mediation analysis between the condition , the kind of purpose - match , and the binary Creative Adaptation ideation outcome . The analysis showed that the effect of condition ( Keyword vs . Analogy ) on the binary outcome of creative adap - tation was mediated by the degree of purpose - match , but not by the novelty of content , suggesting that the difference between full vs . partial matching on purpose is much more significant than the variance in the content novelty . We come back to this result in the discussion ( Section 7 . 2 . 3 ) . Table 5 presents the result of the mediation analyses . The regression coefficient between creative adaptation and condition was significant as was the regression coefficient between the degree of purpose match and creative adaptation . The indirect effect was ( \u2212 0 . 42 ) \u00d7 ( 0 . 21 ) = \u2212 0 . 09 . We tested the significance of this indirect effect using a bootstrapping procedure [ 91 ] ( p < 2 \u00d7 10 \u2212 16 ) , by com - puting the unstandardized indirect effects for each of 1 , 000 bootstrapped samples as well as the 95 % confidence interval ( CI ) . 14 Partial purpose matches in both keyword and analogy articles led to creative adaptation , but the rate was significantly higher with analogy articles . As expected , the ratio of direct application de - creased from the keyword articles that fully match in purpose ( Keyword Full , 68 % ) to the keyword articles that partially match in purpose ( Keyword Part , 6 % ) ( Figure 8 ) . At the same time , the rate of creative adaptation increased from the keyword articles that fully match in purpose ( Keyword Full , 0 % ) to the keyword articles that partially match in purpose ( Keyword Part , 21 % ) . However , the rate of creative adaptation differed significantly between the keyword and analogy articles , with the rate more than doubling among the analogy articles over keyword articles ( Analogy Part 47 % vs . Keyword Part 21 % ) . Homing in on the partial matches , these articles led to creative adap - tation ideas significantly more often in analogy search ( 47 % ) than keyword search ( 21 % ) ( Welch\u2019s 13 https : / / cran . r - project . org / web / packages / mediation / index . html . 14 Alternatively , it is possible that the mediating effect of the degree of purpose - match on the likelihood of creative adapta - tion outcome is moderated by novelty . However , the result of our analysis showed that this was unlikely : The effect was insignificant using the bootstrapping method \u2212 0 . 04 , ( p = 0 . 12 , 95 % CI = [ \u2212 0 . 09 , 0 . 01 ] ) . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 18 H . B . Kang et al . Fig . 7 . Proportion of creative adapta - tion ideas among the partial purpose - match articles . Creative Adaptation was significantly more frequent among the analogy articles ( 47 % ) than keyword articles ( 21 % ) ( Welch\u2019s two - tailed t - test , p = 9 . 0 \u00d7 10 \u2212 4 . Fig . 8 . The rate of ideation outcome types in full and partial pur - pose matches . Among the keyword articles as the purpose mis - match increases , the rate of creative adaptation also increases from 0 % to 21 % ( middle ) . However , this rate is significantly higher among the analogy articles ( 47 % ) than the keyword articles ( 21 % ) . Note that while purpose mismatches led to more creative adapta - tion among analogy articles , a large portion of them also resulted in no ideation outcome ( 38 % ) . two - tailed t - test , t ( 112 . 22 ) = \u2212 3 . 40 , p = 9 . 0 \u00d7 10 \u2212 4 , Figure 7 , left ) . While the partial purpose mis - match was highly associated with creative adaptation ideas , it could be a double - edged sword . Among the analogy articles , 38 % of the partial mismatches resulted in no useful ideation outcome as opposed to the 47 % that resulted in creative adaptation ( Figure 8 , Analogy Part ) . Therefore , knowing what mismatches are beneficial to creative adaptation has important implications for facilitating generative misalignment for ideation . 4 STUDY 2 : ENABLING A FULLY AUTOMATED ANALOGICAL SEARCH ENGINE 4 . 1 Motivation and Structure of the Study The findings of Study 1 suggest potential benefits of an analogical search engine for scientific re - search , but a core limitation of interactivity due to the human - in - the - loop system design prevented its use as a more realistic probe for understanding researchers\u2019 natural interaction with analogical results . Specifically , the results of Study 1 are limited by the lack of participants\u2019 ability to refor - mulate search queries and the study design that involved returning only a fixed number of articles that blended both keyword and analogy articles in a randomized order . These factors significantly deviate from realistic usage scenarios of a deployed analogical search engine and prevent us from observing the full scope of user interaction . In order to move beyond these limitations , first we need a fully automated pipeline that removes the need for human - in - the - loop filtering , thus al - lowing us to enable query reformulation and interaction with corresponding search results . To achieve this , we improved the model accuracy on extracting purposes and mechanisms from arti - cle abstracts by training a more sophisticated neural network that leverages more nuanced linguis - tic patterns . Specifically , we implemented an attention mechanism within a span - based Seq2Seq ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 19 model ( Model 2 ) such that it could learn words that frequently co - occur to describe coherent pur - poses or mechanisms in article abstracts , and as a result , learning more informative words for our purpose ( see Appendix for details of implementation ) . Through evaluating the system backed by this improved pipeline , we demonstrate how it can remove the human - in - the - loop while maintain - ing similar levels of accuracy . In the following sections , we report the evaluation results that show ( 1 ) an improved token - level prediction accuracy using the span - based Model 2 ; ( 2 ) rankings of the results aligning well with human - judgment of purpose - match from Study 1 ; and ( 3 ) top ranked results of the system maintaining a similar rate of partial purpose matches relative to that of the human - in - the - loop system from Study 1 . The interactivity enabled by the automated analogical search pipeline further allows us to ob - serve its use in more realistic scenarios . To probe how researchers would interact with an analog - ical search engine and what challenges they might face in the process , we ran case studies with six researchers ( Section 5 ) . From these studies , we uncover potential challenges ( Section 5 ) and synthesize design implications for future analogical search engines ( Section 6 ) . 4 . 2 Result 4 . 2 . 1 Improved Token - level Prediction of a Span - based Model . First we compared the span - based Model 2 with five other baselines to evaluate the token - level classification performance ( Table 6 ) . Model 2\u2019s overall F1 score was the highest at 0 . 65 ( Purpose ; PP : 0 . 65 , Mechanism ; MN : 0 . 64 , an 0 . 14 - and 0 . 14 - absolute - point increase from Model 1 , respectively ) on the validation set which represents an overall 0 . 15 - absolute - point increase from Model 1 used for the initial human - in - the - loop analogical search engine . 4 . 2 . 2 Pipeline with a Span - based Model Reflected Human Judgment for Ranking the Results . The improved token - level prediction performance materialized as an increase in the pipeline\u2019s ability to judge the degree of purpose match . For this evaluation , we first recorded every query provided by Study 1 participants that human - in - the - loop filterers used to search and filter the relevant articles . Then , we simulated the search condition of the filterers for the automated pipeline by providing it input as the exact queries they used . We capped the number of top search results sufficiently large at 1 , 000 for each query . From these top 1 , 000 results , we selected articles that also appeared in the human - in - the - loop system and collected the corresponding human - vetted judgments of high or low purpose - match . For each of these articles , we also collected its corresponding rank positions on the new ( automated ) pipeline\u2019s list of results . We compared the mean ranks of articles that are judged by human filterers as high purpose match to those of low purpose matches . The result showed that the new pipeline indeed was able to distinguish between the two groups of articles ; low purpose matches ( i . e . , articles that were deemed not relevant and subsequently filtered by the judges in Study 1 ) were placed at significantly lower positions on the list than high purpose matches ( i . e . , unfiltered articles in Study 1 ) . The mean rank for low purpose matches was 465 while for high purpose matches it was 343 ( Figure 9 ) . This difference was significant ( t ( 192 . 49 ) = 3 . 29 , p = 0 . 0012 . Welch\u2019s two - tailed t - test . ) . 4 . 2 . 3 Different Model Performance on Finding Articles that Fully or Partially Match on Purpose . Data and coding . In addition to the overall rankings reflecting human - vetted judgments we also found that the proportion of partial purpose matches was significant among the top - ranked results . We sourced top 20 results for each participant\u2019s research problem with the automated system ( Model 2 ) using the exact queries and order used by the human - in - the - loop filterers in Study 1 . We compared this to four other approaches : ( 1 ) the human - in - the - loop system in Study 1 ( BiLSTM with filtering ) , ( 2 ) a BiLSTM - based system excluding the human - in - the - loop from 1 ( BiL - STM ) , ( 3 ) randomly sampled articles ( Random ) , and ( 4 ) a keyword - based search results , which was ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 20 H . B . Kang et al . Table 6 . F1 Scores of Different Models , Sorted by the Overall F1 Score of Purpose ( PP ) and Mechanism ( MN ) Detection Model Embedding All PP MN ( finetuned ) 1 . Model 2 [ 67 ] ELMo ( N ) 0 . 65 0 . 65 0 . 64 2 . BiLSTM ELMo ( N ) 0 . 63 0 . 67 0 . 59 3 . BiLSTM SciBERT ( N ) 0 . 62 0 . 69 0 . 55 4 . BiLSTM - CRF [ 90 ] ELMo ( N ) 0 . 58 0 . 59 0 . 57 5 . BiLSTM GloVe ( Y ) 0 . 55 0 . 56 0 . 53 6 . Model 1 GloVe ( N ) 0 . 50 0 . 51 0 . 50 The span - based Model 2 gave the best overall F1 score ( blue ) . In comparison , the average agreement ( % ) between two experts\u2019 and crowdworkers\u2019 annotations was 0 . 68 ( PP ) and 0 . 72 ( MN ) [ 21 ] . We used AllenNLP [ 41 ] to implement the baseline models 1 \u2013 5 . Fig . 9 . Mean ranks of human - judged high and low purpose match articles from the span - based pipeline . Low matches were ranked significantly lower ( the rank num - ber was higher ) , on average at 465 th ( SD : 261 . 92 ) than high matches at 343 th ( SD : 279 . 48 ) . used as control in Study 1 ( Keyword ) . There were no overlapping articles between Model 2 and other conditions except for the Keyword condition which had 1 overlapping article . To code the degree of purpose match , we blended the results of Model 2 , biLSTM , and Random conditions . Two of the authors coded a fraction of the data together blind - to - condition ( 7 . 4 % , N = 20 / 270 ) following the same procedure used in Study 1 . Then they independently coded the rest blind - to - condition achieving an inter - rater agreement of \u03ba = 0 . 80 ( substantial agreement ) . We resolved any disagreement through discussion on an individual case basis . Result . We found that the Model 2 - based system achieved a parity with the human - in - the - loop system ( Study 1 ) for finding purpose matches ( Figure 10 ) , with more than half of the system\u2019s top 20 results judged to be partial purpose matches . In contrast , when human - in - the - loop filtering was removed from the BiLSTM - based system , the frequency of partial purpose matches decreased from 58 % to 37 % while the frequency of no matches increased from 40 % to 59 % . Random sampling resulted in mostly irrelevant results , with no alignment on purpose with the source problem . An interesting point of comparison is between the keyword - based and Model 2 - based search results . Keyword search mostly outperformed Model 2 - based system by finding full purpose matches at a much higher rate ( 23 % in keyword search vs . 4 % in the Model 2 - based system ) , with similar rates of partial purpose matches ( 58 % vs . 55 % ) , and significantly less no purpose matches ( 19 % vs . 41 % ) . On average the purpose match score was the highest in keyword - search followed by the Model 2 - based and the human - in - the - loop systems ( Figure 11 ) . Combined with the results of Study 1 , this suggests the complementary value of analogical search : The higher rate of full - matches in keyword - search may be good when searchers know what they are looking for , such as in direct search tasks and foraging from familiar sources of ideas . Nonetheless , because analogy articles were both deemed significantly more novel by the scientists and had little - to - no overlap with keyword - search articles , they augmented keyword - based search results with a complementary set of articles that introduce useful mismatches in their purposes . This set of articles may open up new domains of ideas that scientists may not have been aware of , and encourage creative adaptation . 5 CASE STUDIES WITH RESEARCHERS To further understand what potential interaction challenges prevent future analogical search en - gines from reaching their full potential , we ran case studies with six participants . To this end , ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 21 Fig . 10 . Distribution of Full , Part , and None purpose matches among the five sourcing mechanisms : BiL - STM with filtering represents the human - in - the - loop system ( Study 1 ) ; Model 1 represents a system based on the BiLSTM model alone , without human - in - the - loop filtering ; Model 2 represents the fully automated system ; Random represents randomly sampled articles ; Keyword represents keyword - based search ( Control in Study 1 ) . Model 2 and BiLSTM with filtering showed a similar distribution of purpose matches , and more partial purpose matches than BiLSTM alone . Random showed mostly no matches . The Keyword condition re - sulted in the highest number of fully matched articles and the lowest number of no matches , suggesting that keyword - based search may be an effective mechanism for direct search tasks , but potentially less effective for inspirational / exploratory search tasks . we developed a frontend interface that includes a text input for reformulating purpose queries ( Figure 12 , right ) . This frontend interfaced with our automated , Model 2 - based backend to display a ranked list of analogical results for a given purpose query . Leveraging the fully automated search engine , we also removed the structure of Study 1 that asked participants to engage with each result they encountered , thus allowing us to observe which results researchers more naturally attend to and engage with . In sum , the design of our case studies differ from Study 1 in three aspects : ( 1 ) participants interacted with only the analogical search results ranked in the order of purpose similarity , without blended keyword - based search results ; ( 2 ) participants reviewed search results returned for their queries and reformulated the queries when needed ; and ( 3 ) participants looked for articles that interest them and may serve as sources of inspiration for their research problems at their own pace , without being explicitly asked to engage with each result they encounter . The primary goal of our case studies was to identify generalizable challenges that analogical search engines may face in interactive use , thus providing us insights on how future engines may be designed and improved . Specifically , we were interested in the challenges related to ( 1 ) how researchers recognize relevance of analogical search results and ( 2 ) how researchers formulate and reformulate purpose search queries while interacting with analogical search results . 5 . 1 Participants and Design Participants were asked to formulate purpose queries for their own research problems and interact with the results to find interesting articles . If an article gave them a new idea relevant to their research project , they were asked to write a short project proposal in a shared Google Doc and explain how the article helped them to come up with the idea . Interviews were conducted via Zoom and lasted for roughly an hour . Participants were paid $ 20 in compensation . One participant was an assistant professor in mechanical engineering at a public R1 U . S . university and five were PhD researchers in the fields of sciences and engineering at a private R1 U . S . university . Two were senior PhD students ( 3rd year or above ) and the rest were 2nd year or below . Disciplinary backgrounds of the participants included Chemical ( 2 ) , Civil ( 3 ) , and Mechanical Engineering ( 1 ) . We note that one participant previously took part in Study 1 , whose research focus was the same in terms of its general domain . However , the participant\u2019s ideas and the specific articles of interest that led to them did not have overlap between the two studies . Table 7 describes participants\u2019 research problems . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 22 H . B . Kang et al . Fig . 11 . The distribution of mean purpose match scores over different conditions ( mappings : None (cid:7)\u2192 0 , Part (cid:7)\u2192 1 , and Full (cid:7)\u2192 2 ) . The mean purpose - match score of the system backed by Model 2 ( 0 . 63 , SD : 0 . 56 ) is significantly higher than that of the system used in Study 1 without the human - in - the - loop ( BiLSTM , \u03bc = 0 . 45 , SD : 0 . 58 ) ( Welch\u2019s two - tailed t - test , t ( 237 . 87 ) = 2 . 49 , p = 0 . 0135 ) , similar to that of the system with the human - in - the - loop ( BiLSTM with filtering , \u03bc = 0 . 62 , SD : 0 . 52 ) ( t ( 244 . 65 ) = 0 . 25 , p = 0 . 80 ) , and sig - nificantly lower than that of the keyword - based search ( Keyword , \u03bc = 1 . 04 , SD : 0 . 65 ) ( t ( 159 . 38 ) = \u2212 4 . 57 , p = 0 ) . Fig . 12 . The search interface used for case stud - ies featured an input for query reformulation which participants used to iteratively reformu - late their queries . Apparatus : Search interface . The improved performance of Model 2 backed the fully automated pipeline without human filtering . The search interface interacting with this back - end included a text input for reformulating purpose search queries as well as a list view of search results that showed a sorted list of articles with similar purposes ( Figure 12 ) . 5 . 2 Result 5 . 2 . 1 Overall Impressions . Overall participants described their experience with the analogical search engine in a positive light ( e . g . , \u201chelps me think at a broad topic or a big picture level\u201d\u2014P2 ; \u201cfind some very interesting and useful ideas , the design is also very simple , good when focusing on key areas of research\u201d\u2014P5 ; and \u201cvery interested now what the future of this engine would look like\u201d\u2014P3 ) , but a deeper look suggested that the success of ideation depended on how well searchers were able to engage with analogical results that deviate from their expectations : \u201cIt\u2019s surprising that the engine recommends examples like these\u201d\u2014P3 ; \u201cIf I input the same search queries on Google Scholar it\u2019d not normally return these things . . . this search engine works in a different way\u201d\u2014P1 . 5 . 2 . 2 \u201cNot the Kind of Article I\u2019d Look for but . . . \u201d : The Challenge of Early Rejections . Unlike similarity - maximizing search engines , the diversity in analogical search results can lead to pre - mature rejection of alternative mechanism ideas . One of the factors contributing to premature rejection of alternatives may be the tendency for adherence to a set of existing ideas or concepts , as studied in the literature of design fixation ( e . g . , [ 66 ] ) . In our study , the participants found the variety of domains featured in search results confusing , and it sometimes prevented them from en - gaging with the ideas therein . For example , P3 , whose research studies ways to manage or reduce task complexity for nuclear power plant operators , expected to see results similar to Google Scholar ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 23 Table 7 . Case Study Participants\u2019 Descriptions of Own Research Problems PID Participants\u2019 Description of Research Problem 1 Improve heat pipe evaporation 2 Computer simulations for fluids in nanoscale and uncovering their heat - transfer properties 3 Developing a model to identify complex steps in Nuclear Power Plant ( NPP ) operation , and understanding what task features and structures cause the complexity and how this influences the operators\u2019 performance 4 Designing simulators for training bridge inspectors 5 Developing algorithms and extensible frameworks for detecting personal protective equipment ( PPE ) in construction sites to improve the safety of construction workers 6 Convergence rates of optimization algorithms under multiple initial starting positions which are typically in the domains of operational and managerial sciences , but was surprised by unfamiliar domains represented in search results : \u201cThese ( distributed networked systems design or path planning for automated robots ) are not the kinds of fields that I normally read in , if I found them elsewhere I would\u2019ve probably thought they\u2019re irrelevant and skipped\u201d ( P3 ) . Ranging from unfamiliar terms ( P1 , P4 , P5 ) to unfamiliar categories of approaches ( e . g . , \u201cNot sure what \u2018Gauss \u2013 Newton approach for solving constrained optimization\u2019 is\u201d\u2014P6 ) , or high - level research directions ( e . g . , \u201cthis is different from my research direction , people who work on this direction might find it interesting , though\u201d\u2014P1 ) , participants saw the diversity of results as a challenge for engagement . P1 pointed out a perceived gap between the expectation of least effort and the cognitive processing required when engaging with analogical ideas and adapting them : \u201cAs I understand it , I think this search engine is trying to present articles from related but different fields to let people make connections . But people expect less friction . ( The result is ) something interesting but I can\u2019t directly write it into a project proposal . . . I think it would be challenging to make people get interested in investing time to read the articles in depth to come up with connections . I wonder what would happen if this was hosted just as an online website ( instead of the study context ) \u201d \u2014P1 On the other hand , analogs that did get examined more deeply could ultimately lead to creative adaptation . For example , P3 mapped task scheduling among computer processes to task assign - ment among the nuclear power plant operators , and came up with an idea to adapt algorithmic scheduling used in real - time distributed systems to a scheduling mechanism that could be useful in her research context . Represented symbolically this process was akin to ideating what might best fill in the \u201c ? \u201d in the relational structure [ scheduling algorithm : processes in distributed sys - tems ] \u2194 [ ? : nuclear power plant operators ] : \u201cI think the algorithms proposed in this article could be useful for calculating the operator task execution time , the power plant system\u2019s response time , and the time margin between the execution time and the system response time . . . so that the next task assignment can factor in these margins and things related to workers\u2019 well - being like rest and time required between switching tasks\u201d ( P3 ) . Participants seemed to recognize a small number of core relations as kernel for creative adap - tation . In the example of P3 , scheduling processes in the distributed systems article piqued her in - terest and led her to connect them with similar concepts in the literature she was already familiar with : \u201cYou need to make that connection . . . I saw parallels between ( distributed systems domain ) concepts like [ scheduling ] and [ tasks ] and [ scheduling tasks for the operators ] \u201d ( P3 ) . Similarly , P5 recognized a similarity between [ monitoring people\u2019s performance ] in fitness training and ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 24 H . B . Kang et al . Fig . 13 . Diagram showing different abstraction levels of purposes and their relations . Node A (cid:4) corresponds to a more specific query than its higher - level representation , denoted as B (cid:4) . Similarly , node C (cid:4) represents a more specific purpose representation of A (cid:4) , accessible via the A (cid:4) \u2192 abstraction B (cid:4) \u2192 specification C (cid:4) path . [ monitoring whether construction workers are wearing personal protective equipment ] in con - struction sites . He then adapted the idea of tracking heat emission in the fitness context to his own : \u201cI like the idea of [ monitoring heat emissions ] in fitness training . . . maybe I can also detect heat emissions from construction workers to see if they are wearing the safety vests or masks while also monitoring the site conditions and worker efficiency . It also gives me an idea to moni - tor the CO 2 emissions from workers so as to improve the robustness of detection\u201d ( P5 ) . In this case , monitoring and the physical nature of the activities involved helped P5 see the connection useful for creatively adapting the source idea . 5 . 2 . 3 \u201cI don\u2019t Know What to Type in\u201d : The Challenge of Query ( Re - ) Formulation . Another chal - lenge participants faced was that they were not used to formulating their search queries in terms of high level purposes of their research . On average participants entered 5 . 2 queries ( Min : 1 , Max : 18 , SD : 5 . 87 ) , 87 % ( 27 ) of which were in the form of a single noun phrase ( e . g . , \u201cheat pipe evaporation , \u201d\u2014 P1 , \u201ctask complexity\u201d\u2014P3 , \u201ctheoretical optimization convergence for non - convex functions\u201d\u2014P6 ) or a comma - separated set of multiple noun phrases ( e . g . , \u201cheat transfer , nanoscale , fluid\u201d\u2014P2 ) that represented specific aspects related to research purposes rather than the core purposes themselves . For example , the purpose of \u201cheat pipe evaporation\u201d may be to transfer heat , and the purpose of searching for \u201ctheoretical optimization convergence for . . . \u201d may be to detect when optimization converges or diverges , or to effectively sample unknown ( non - convex ) distributions . One of the reasons why participants formulated search queries in this way may be wrongly assuming that the search engine used keyword matching to find results . For example , extensive prior experience with search engines that highlight matching keywords in abstracts ( e . g . , Google Scholar ) in response to users\u2019 search queries can reinforce such assumptions among the users . In addition , participants\u2019 domain knowledge useful for judging which of the returned articles are relevant may have led them to notice a set of keywords the inclusion of which strongly signifies the relevance of a article . In contrast , the analogical search results often seemed to not feature such directly similar terms and this contributed to the difficulty of judging whether a result is relevant and how : \u201cI find these articles not very related to my search query at first . It\u2019d be better if you can use some graph or some pictures to indicate how these articles can relate to my keywords\u201d ( P5 ) ; \u201cI\u2019d not consider . . . ( because ) they are totally different , right ? They look irrelevant . . . until I think about it I can realize that it\u2019s useful . But if you give me the article , at first I don\u2019t realize that\u201d ( P3 ) . While it may not feel as compelling or natural to participants , formulating and abstracting queries at a high level may lead to searching more distant results that are analogous at a higher level . For example , by querying \u201cdetect personal protective equipment\u201d instead of \u201cpersonal ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 25 protective equipment construction , \u201d P5 found novel mechanisms of detection , such as general im - age segmentation algorithms or an approach to monitoring heat in the context of fitness training not specific to construction sites and personal protective equipment but nonetheless useful for creative adaptation . Querying \u201cscheduling tasks\u201d instead of \u201ctask complexity\u201d for P3 resulted in finding scheduling algorithms in distributed computer systems that otherwise P3 would not have encountered , while \u201cassigning tasks\u201d led to novel auction mechanisms which made her think about a system in which each power plant operator can bid for a task as opposed to being assigned one . Schematically , Figure 13 shows how formulating queries at a higher level of abstraction than spec - ifying the problem context in full details ( A (cid:4) \u2192 B (cid:4) ) may lead to discovering novel mechanisms that are relevant at the high level of abstraction , and in more distant ways from the original problem formulation ( B (cid:4) \u2192 C (cid:4) ) . 6 DESIGN IMPLICATIONS From both the case studies\u2019 and Study 1\u2019s participants\u2019 reflection on the challenges of interacting with analogical search results , common themes emerged . Here , we present three design implica - tions for future analogical search systems synthesized from these results . We use subscripts to denote which study participants participated in when appropriate . 6 . 1 Support Purpose Representation at Different Levels of Abstraction Analogical search engines should support users to formulate their purpose queries at different levels of abstraction . Additionally the search engine may prompt users to consider abstracting or specifying their purpose queries in the first place , and explain how it might help bring new in - sights into their problems . As seen in the case studies ( Section 5 . 2 . 3 ) , scientists recognized their purpose queries may be represented at multiple levels , but prior experiences with similarity maxi - mizing search engines may also have anchored them around pre - existing rigid formulation of pur - poses . Prompting users to consider their research problems at multiple levels may work against this rigidity , and providing candidate suggestions at varying levels may further reduce the cog - nitive demand . Moving up on the hierarchy to abstract purpose queries may be possible through removing parts of the query words that correspond to specific constraints , or by replacing them with more general descriptions . For example two participants of Study 1 had an identical purpose representation at a high level ( \u201cfacilitate heat transfer\u201d ) despite the differences in materialistic phases targeted in each purpose : solid material and semiconductors for P1 Study 1 and liquid thin films for P3 Study 1 . Furthermore , we also observed that looking for only the exact match of a purpose can lead to missed opportunities . For example , although \u201cfins represent a different idea for transferring the heat\u201d and \u201cthey ( fins ) don\u2019t match in terms of the scale\u2014macro , not nano , \u201d it nevertheless made P1 Study 1 wonder \u201cwhat if we could design nanoscale wall structures that act like fins that convert heat to mechanical energy ? \u201d A corollary to this observation is that sometimes the superpositions of misalignment with just the right amount can lead to interesting results . For P4 Study 1 , an ar - ticle presenting experimental techniques for piezoelectric properties was interesting despite its misalignment such as [ simulation - based ] ( source ) (cid:2) [ experimental ] ( analog ) and [ dielectric prop - erties ] ( source ) (cid:2) [ piezoelectric properties ] ( analog ) : \u201cThough it\u2019s an experimental study , it\u2019s very close in terms of the material and phenomenon so likely to be helpful . Because we might be able to pick up some trends like , if we increased the temperature , the dielectric response gets stronger or weaker , inferred from the experimental piezoelectric responses , which can then be used to corrob - orate simulation results or help configure its parameters\u201d ( P4 Study 1 ) . However , too much deviation seemed detrimental to its potential for inspiration : \u201c [ Molecular dynamic simulation ] is the same tool , but ( this article studies ) [ thermal ] ( not [ dielectric ] ) properties on [ polymer composites ] . . . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 26 H . B . Kang et al . [ polymer composites ] are harder to model\u201d ( P4 Study 1 ) . In sum , analogical search engines should support not only the capability to \u201cnarrow it down\u201d with specific constraints , but also ways to re - lax them to broaden the search space when suitable , thus making feasible the sweet spot between too little ( i . e . , similarity maximization and trivial matches ) and too much deviation ( i . e . , critical misalignment and unusable analogs ) . 6 . 2 Support Iterative Steering from Critical Misalignment and Towards Generative Misalignment Analogical search engines should recognize that important constraints may be discovered by users only after seeing misaligned analogs , and support this discovery process by presenting effective ex - amples of misalignment to users . Analogs that deviate on some aspects of the source problem but preserve important relations may be particularly conducive to analogical inspiration that opens up not just individual solutions , but entirely new domains of solutions . However , at the same time scientists also found it challenging to know how to come up with effective search queries because combinations of misalignment can sometimes lead to an unintended intersection of do - mains : \u201cI feel like I\u2019m tricking the machine because [ thin film ] is often used with [ solids ] , and the term [ pressure ] also appears a lot in [ manufacturing ] . . . so combining them gives a subset of arti - cles concerned with heat transfer in solid materials and in manufacturing\u201d ( P3 Study 1 ) ; \u201con Google Scholar also , I get a lot of polymer strings and get ( irrelevant ) results like we use an [ electric ] device to study [ vibration and stress ] of [ polymers ] . . . the machine is picking up [ electric ] and [ properties ] such as vibration and stress in the context of studying polymers but what I really want is [ electric properties ] of [ polymers ] not [ electronic devices ] to study the [ mechanical properties ] of [ poly - mers ] \u201d ( P4 Study 1 ) . Nonetheless , seeing misaligned analogs can be an effective way of reasoning about salient constraints and reflecting on hidden assumptions . For example , while evaluating arti - cles about designing microelectrode arrays , P6 Study 1 said : \u201cNow I think about this ( result ) , I assumed a lot of things when typing that search query . . . though impedance and topology are my main focus in microelectrode arrays , the coating , size , interface between a cell membrane and electrodes / sensors , biocompatibility , softness of electrodes , fabrication process , material of the platform : silicon or poly - mer or graphene , form factor : attaching electrodes to a shank - like structure or a broom - like structure , degree of invasiveness , are all part of the possible areas of research and it makes sense that they showed up\u2014there is no way the machine would have known that from my query . \u201d This excerpt illustrates how knowing what the necessary specifications are and which constraints need to be abstracted to cast a wide - enough net to catch interesting ideas appeared to be a difficult task for scientists , especially when they had to recall important attributes rather than simply recognize them from ex - amples of misalignment . Prior work in cognitive sciences also shows how dissimilarity associated with various factors in analogical mappings [ 45 ] can pressure working memory [ 111 ] , increase cognitive load [ 102 ] , and increase response time taken to produce correct mappings for analogy problems [ 71 ] . Therefore , analogical search engines should help to reduce the cognitive effort re - quired in the process , for example by proactively retrieving results that are \u201cusefully\u201d misaligned such that searchers can better recognize ( as opposed to having to recall ) salient constraints and re - fine their problem representation . This process is deeply exploratory [ 93 , 114 , 117 ] in nature , and suggest the importance of both providing end - users a sense of progress over time [ 103 ] as well as adequate feedback mechanisms for the machine to adjust according to the changing end - user search intent [ 72 , 95 , 96 ] . For example , while the machine may \u201ccorrectly\u201d recognize a significant anaogical relevance at a higher level of purpose representation and recommend macro - scale mech - anisms to a scientist who studies nano - scale phenomena ( P1 Study 1 ) or solid and semiconductor - based cooling mechanisms to a scientist in liquid and evaporative cooling systems ( P3 Study 1 ) , these ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 27 analogs may be critically misaligned on the specific constraints of the problem ( i . e . , the scale or materialistic phase ) and thus considered by end - users as useless and even harmful . 6 . 3 Support Reflection and Explanation of Analogical Relevance Throughout the process of analogical search , human - AI coordination is critical for success , and an important aspect is how deeply the human users can reflect on the retrieved analogs [ 53 ] and recognize how different notions of relevance may exist for their own problem context , despite po - tential dissimilarity on the surface . Looking at previous examples of the tools and techniques de - veloped for targeted reflection support may be useful to this end . For example , ImageCascade [ 76 ] provides intelligent support such as automatically generated mood - boards and semantic labels for groups of images to help designers communicate their design intent to others . Another system , Card Mapper , visualizes relative co - occurrences of design concepts using proximity in the design space [ 26 ] . Similarly representing the space of analogical ideas using spatial encoding of similarity between two analogs , or designing information that supports getting a sense of the space of search results\u2014e . g . , semantic category labels similar to ImageCascade\u2019s or the distribution of the domains that analogs are pulled from\u2014may be an avenue for fruitful future research . The explanation of relevance is also important especially when there is a risk of early rejection ( Section 5 . 2 . 2 ) . Using examples from the case studies , one approach to explaining relevance might be to surface a small number of core common features between an analog and a problem query . Such common features were considered useful by scientists for making analogical connections , and they could creatively adapt them for their own research problem context . When common features are not directly re - trieved , generation of more elaborate explanations may be required . We refer to [ 6 , 14 , 70 , 98 ] for those interested in future design considerations of automatically generated recommendation expla - nation . Further complementing the direct explanation of relevance approach , techniques such as prompting or reminding the searchers of previously rejected or overlooked ideas may also trigger deeper reflection and delay premature rejection of the ideas based solely on their surface dissim - ilarity . Participants from both studies commented that the critical first step towards analogical inspiration may be raising similarly enough attention and interest above the initial \u201chump\u201d of cog - nitive demand . Gentle reminders ( e . g . , \u201cAsk me later if this would be interesting and also provide a list of items\u201d\u2014P1 Case Studies ) or resurfacing previously rejected articles in light of new information ( P1 Case Studies , P3 Case Studies ) may help with users cross this barrier . 7 DISCUSSION 7 . 1 Summary of Contribution With the exponential growth of research output and the deepening specialization within different fields , encouraging analogical inspiration for scientific innovation that connects distant domains becomes ever more challenging . Our human - in - the - loop and fully automated analogical search en - gines represent an approach for supporting such analogical inspirations for challenging scientific problems . We have demonstrated in Study 1 that our human - in - the - loop system finds novel results that participants would be unlikely to encounter from keyword - based search , and that these results lead to high levels of creative adaptation . Through a mediation analysis we also showed that this success was driven by the analogical search engine\u2019s ability to find partial purpose matches ( e . g . , matching at the high - level purpose but differs at the low - level details ) . We saw the nuanced effects of partial purpose alignment on the results\u2019 goodness as analogs for inspiration . Through quali - tative observations , we described how certain attributes of analogical mapping were perceived as more salient by participants , and that mismatches on them can have either a positive ( i . e . , genera - tive insights ) or a negative impact ( i . e . , critical misalignment ) on creative adaptation . In contrast , ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 28 H . B . Kang et al . keyword - based search resulted in more full purpose matches and a higher level of direct applica - tion . The value of keyword - based search and analogy - based search thus may complement each other , while keyword - based search can help researchers find \u201cexactly that\u201d , analogy - based search can help researchers to switch from a preservative mode ( i . e . , aiming to find results with maximal similarity to the query ) to a generative mode ( i . e . , aiming to find analogs that are relevant despite the surface dissimilarity ) of searching , and ultimately lead them to recognize unusual relations and come up with ways to creatively adapt existing ideas for novel domains . We also demonstrated how improving the Seq2Seq purpose and mechanism identification model can remove the human - in - the - loop but maintain a similar level of accuracy on purpose - match by human judges . This improvement enabled us to develop a fully automated analogical search sys - tem to use as a probe to study searchers\u2019 more natural interaction with analogical results . Through a series of evaluation we first show that our automated analogical search pipeline can emulate hu - man judgment of purpose match and that it finds partial purpose matches in top ranked results with a similar rate compared to the human - in - the - loop system used in Study 1 . Then through case studies we find generalizable challenges that future analogical search engines may face , such as early rejection of alternative mechanism ideas and the difficulty of abstracting and representing purposes at the right level . From our studies we synthesize design implications for future analog - ical search engines , such as supporting purpose representations at different levels of abstraction , supporting the iterative process of steering away from critically misaligned analogs and towards a fertile land of generative misalignment , and providing explanations on why certain analogical search results may be relevant . We envision that future studies will shed light on deeper cognitive sources of the challenges identified here . A fruitful avenue of research may be studying how the dual processing theory [ 69 , 112 ] underlies or interacts with analogical search interaction . Studying also how simplification heuristics [ 84 ] may negatively bias interaction with analogical results and how it may be reduced for expert user populations may be an interesting future direction [ 17 , 77 ] . 7 . 2 Limitations and Future Work 7 . 2 . 1 Experimental Design and Improving its Validity . Our findings have several limitations . First the design of our studies may be improved to increase the experimental validity . We believe that our coders of the ideation outcomes had a reasonable understanding of participants\u2019 research context from descriptions of current and past research topics , think - alouds with 45 articles , and end - of - experiment discussions , and that the procedure of coding reduced potential biases ( e . g . , the coders were blind to experimental conditions , relied on participants\u2019 statements of novelty and distance ) . Despite this , it is possible that they judged ideas differently from domain experts , for example coding more or fewer ideas as creative adaptation , or pre - filtering useful ideas in the human - in - the - loop stage . In addition , other quality dimensions such as potential for impact or domain - expert - judged idea quality are largely inaccessible within the studies presented here . Fu - ture research may improve on these limitations by iterating on the experimental design , collecting data for triangulating the results and capturing other quality dimensions of the generated ideas . Additionally , future work may add ablation studies to quantify the effects of human filtering in Study 1 on the ideation outcome as well as sensitivity studies to relate how much the increased token - level classification performance of trained models may reduce the burden of human filter - ing . Furthermore , additional experiments with baselines other than keyword - based search using the whole abstract will help pinpoint the potential advantages of representing and matching arti - cles using extracted purposes and mechanisms . For example , Chan et al . [ 21 ] found that embed - ding all words from an abstract ( using GloVe embeddings ) resulted in retrieval of fewer analogi - cal items than when extracted purposes and mechanisms were used . Replicating this result with ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 29 additional approaches such as contextualized word embeddings and pre - trained language models ( e . g . , ELMo [ 90 ] , BERT [ 29 ] , and SciBERT [ 7 ] ) will be valuable . 7 . 2 . 2 Potential Sampling Bias . The sampling strategy in Study 1 was purposefully unbalanced , where analogical articles were sampled twice as much as keyword articles to ensure participants\u2019 exposure to sufficiently diverse results . This was crucial for uncovering potential benefits and challenges of our analogical search engine and investigating its viability . This ratio was chosen purposefully , to balance the statistical power for detecting potentially significant differences be - tween the conditions , while also limiting the number of articles that each participant had to review . Given the cognitive burden of reviewing an article while thinking aloud , we decided on 45 in total with the 2 : 1 ratio to fit the practical time limits of interviews . However , this may have led to unan - ticipated effects on ideation outcomes despite having accounted for the difference in sample sizes by measuring the outcomes in ratios . For example , when the results were combined into a single blinded list , the over - representation of analogical results over more purpose - aligned keyword re - sults may have shifted the users\u2019 overall perceived value of the list to be more or less positive . Users\u2019 perception of diverse results may have been further affected by their relative over - representation . For example , increased cognitive load for processing analogical mapping [ 51 , 52 , 102 ] may suggest that results that fully match on the purpose search query may have been perceived even more favorably than analogical results , due to a negative spill over effect from the rest of the articles in the list , which were less likely matched on the purpose . Investigating whether such factors led to compounding effects beyond our ratio - based measures of usefulness remains an open question for future work . 7 . 2 . 3 Controlling the Diversity of Search Results . Our work is also limited by the lack of con - trollability in sampling the search results beyond purpose similarity . As described in Section 2 . 2 . 1 , from pilot tests in our corpus we discovered that even close purpose matches of scientific articles already had high variance in terms of the mechanisms they proposed which allowed us to focus our approach to sampling based solely on purpose similarity . The simplicity of this approach also means fewer hyper parameters in the sampling mechanism compared to other approaches [ 61 , 62 ] . However , all the approaches including this work thus far lacked a mechanism for explicitly con - trolling the diversity in retrieved search results which remains a fruitful avenue for future work . For example , prior research has uncovered the nuanced effects of distance ( e . g . , near vs . far sources of inspiration [ 24 , 97 ] ) , suggesting the benefit of targeting analogs at different distance from the source problem for the right context . Future research may also uncover further complexities in the relationship between novelty and purpose - match . The result of our mediation analysis ( Table 5 ) showed that the novelty of content among the search results in Study 1 was not a significant factor to the same extent that the three levels of purpose match was . However , the relationship between novelty and purpose match may be more complex than the levels of manipulation presented in this work . For example , [ 30 ] suggested a greater importance of novelty than usefulness for predicting creativity scores . Future work may design mechanisms to manipulate the variance in content nov - elty and alignment in the purpose - mechanism schema to uncover dynamics between the two that go beyond the results from mediation analyses presented here ( Section 3 . 3 . 3 ) . Furthermore , chal - lenges with the abstraction of purposes remain open , for example , how core versus peripheral at - tributes of research purposes may be identified , and how they may be selectively matched at a spe - cific level of the conceptual hierarchy . Finally , not all query formulations are created equal in terms of their suitability for analogical search . We observed in the case studies that participants wanted to express different query intent via reformulation ( Section 5 . 2 . 3 ) . While participants could refor - mulate their search queries and examine the returned results from our analogical search engine in real - time , it was unclear whether and how specific query formulations may lead to more or less ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . 57 : 30 H . B . Kang et al . diverse results , and how subsequent queries may be updated after reviewing them . As such , sys - tems that assist users in the potentially tedious process of query reformulation [ 113 ] ( for example , by way of automatic query expansion [ 18 ] ) in the context of analogical search will be important . 7 . 2 . 4 Studying the Effect of Larger Context of Scientific Innovation on Analogical Innovation . Due to our focus on ideation outcomes , our results do not explain how these ideas may be integrated , developed , and shared across the research communities . Studying the lifetime of ideas that goes beyond their inception will deepen our understanding of the factors that currently make analogical innovation such a rare event in sciences ( for example , Hofstra et al . suggested that more seman - tically distant conceptual combinations receive far less uptake [ 58 ] ) . Through interviewing our study participants and other colleagues in academia we found emerging structures related to this challenge . Our interviews informed us that in general the context in which a scientist exists\u2014such as the scientist\u2019s role in a project , the maturity of a project , and the broader academic culture\u2014can ultimately change how they interact with and seek analogical inspirations . For example , a third - year PhD student studying chemical engineering commented \u201cIn the current stage of my project it\u2019s more about parameter - tuning\u2014running multiple experiments at once and comparing which configuration works the best . . . If I were a first year PhD student maybe I would be in a broader field and exploration . \u201d In contrast , a PhD in biology who recently defended noted that \u201canalogical inspirations would perhaps be more useful if you\u2019re looking for a postdoc or a faculty position . \u201d In addition , the underlying career incentive structures in academia may also affect researchers\u2019 perception of and openness to analogical inspirations . One of the study participants commented \u201csince I\u2019m already a third year PhD student and my project is further along and more firmed up , I\u2019m not really looking for really far inspirations . . . first we push the specific way we have in mind with many iterations on the experiments until , say , publication . \u201d In addition to the career - wise incentives there are other types of competitive resourcefulness ( e . g . , social resources such as the advisors\u2019 and colleagues\u2019 expertise that participants can easily tap into ; physical and other forms of resources such as tangible artifacts like previously developed code packages or experimental pro - cesses and setups ) . These factors can influence scientists\u2019 perception of their advantage and lead them to interpret analogical inspirations as more or less useful , feasible , and directly applicable to their research . This observation is further suggested by survey results that asked our participants : \u201c Could this article be useful to you ? , \u201d their ratings were significantly higher for keyword articles than analogy articles despite them having come up with creative adaptation ideas more often with analogy articles . Therefore , future work that studies incentive structures , the quality of ideation outcome , their feasibility , the differences in research context e . g . , frames of research contribution such as discovery - oriented vs . novel system development - oriented , and taking a longitudinal ob - servation of the variation in such factors will add a significant depth to our understanding . 8 CONCLUSION In this article we present our novel human - in - the - loop and fully automated analogical search en - gines for scientific articles . Through a series of evaluations , we found that analogous articles that our systems retrieved were novel and useful for sparking creative adaptation ideas . However , sig - nificant work is needed to continue this trajectory , including additional understanding of the con - text and incentives of scientists as well as advances in the data pipeline and interaction methods beyond those described here for a system to maximize its real - world impact . We imagine a future in which scholars and designers could find inspirations based on deep analogical similarity that can drive innovation across fields . We hope this work will encourage sci - entists , designers , and system builders to collaborate across disciplinary boundaries to accelerate the rate of scientific innovation . ACM Transactions on Computer - Human Interaction , Vol . 29 , No . 6 , Article 57 . Publication date : November 2022 . Augmenting Scientific Creativity with an Analogical Search Engine 57 : 31 APPENDIX A REPRODUCIBILITY Training and validation datasets . The original annotation dataset from [ 21 ] also includes Back - ground and Findings annotations which we exclude due to their relatively high confusion rates among the annotators with the Purpose and Mechanism classes and to balance the number of available training examples per annotation class . Model parameter selection . We experimented with changing the model capacity relative to the signal present in the training dataset by tuning the number of hidden layers and the nodes used in each model architecture . For Model 1 , we found a hidden layer of 100 nodes was sufficient . We opti - mized this model using the cross - entropy loss and the Adam optimizer [ 73 ] with a 0 . 0001 learning rate . For Model 2 , we found three hidden layers with 256 nodes led to an improved accuracy on the validation set . We trained this model with an L2 regularizer ( \u03b1 = 0 . 01 ) , dropouts with the rate of 0 . 3 , and the Adam optimizer with a 0 . 001 learning rate . Span - based model architecture . We adapt SpanRel [ 67 ] as architecture for the span - based Model 2 . SpanRel combines the boundary representation ( BiLSTM ) and the content representa - tion with a self - attention mechanism for finding the core words . More specifically , given a sentence x = [ e 1 , e 2 , . . . , e n ] , of n token embeddings , a span s i = [ \u03c9 s i , \u03c9 s i + 1 , . . . , \u03c9 f i ] is a concatenation of the content representation z i c ( weighted average across all token embeddings in the span ; SelfAttn ) and the boundary representation z i b of the start ( s i ) and end positions ( f i ) of the span : u 1 , u 2 , . . . , u n = BiLSTM ( e 1 , e 2 , . . . , e n ) , z c i = SelfAttn ( e s i , e s i + 1 , . . . , e f i ) , z b i = [ u s i ; u f i ] , z i = [ z c i ; z b i ] . We use the contextualized ELMo 5 . 5B embeddings 15 for token representation , following the near state - of - the - art performance reported on the named entity recognition task on the Wet Lab Proto - col dataset in [ 67 ] . We refer to [ 67 , 79 ] for further details . Other parameters . We use GloVe vectors for input feature representation for Model 1 with 300 dimensions , consistent with the prior work [ 11 , 78 , 88 ] . For Model 2 , we use the contextualized ELMo 5 . 5B embeddings as described above which have pre - determined 1 , 024 dimensions . We use USE [ 20 ] for encoding purposes . A USE embedding vector has pre - determined 512 dimensions . ACKNOWLEDGMENTS We thank our study participants for their valuable insights and feedback . REFERENCES [ 1 ] Random projection in Locality - sensitive hashing . 2008 . Retrieved January 23 , 2022 from https : / / en . wikipedia . org / wiki / Locality - sensitive _ hashing # Random _ projection . [ 2 ] Annoy : How it works . 2018 . 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Publication date : November 2022 .", + "savinainenUsingBridgingRepresentation2005": "Using a Bridging Representation and Social Interactions to Foster Conceptual Change : Designing and Evaluating an Instructional Sequence for Newton\u2019s Third Law ANTTI SAVINAINEN Kuopio Lyseo High School , Puijonkatu 18 , FIN - 70110 Kuopio , Finland PHILIP SCOTT Centre for Studies in Science and Mathematics Education , School of Education , University of Leeds , LEEDS LS2 9JT , UK JOUNI VIIRI Department of Education , University of Joensuu , PL 111 , FIN - 80101 Joensuu , Finland Received 1 September 2003 ; revised 25 April 2004 ; accepted 15 June 2004 DOI 10 . 1002 / sce . 20037 Published online 19 November 2004 in Wiley InterScience ( www . interscience . wiley . com ) . ABSTRACT : This paper offers an account of , and \ufb01ndings from , an approach to designing and evaluating an instructional sequence set in the context of Newton\u2019s third law . The design of the sequence draws upon conceptual change theory and the concept of the \u201cbridging analogy\u201d is extended to introduce the notion of a \u201cbridging representation . \u201d Attention is also given in the instructional design to the proposed social interactions between teacher and students as the teaching and learning activities are played out or \u201cstaged\u201d in the classroom . A range of instruments is used to measure the extent of student learning , and evidence is presented to indicate that the designed sequence leads to enhanced learning gains when compared to those achieved with an equivalent group of students . Although set in the context of teaching and learning about mechanics a number of general points are made in relation to both instructional design and perspectives on conceptual change . C (cid:1) 2004 Wiley Periodicals , Inc . Sci Ed 89 : 175 \u2013 195 , 2005 INTRODUCTION Findings from many studies over the past three decades have shown that students have pre - instructional conceptions or beliefs about the phenomena addressed in science lessons ( see , for example , bibliographies in Duit ( 2004 ) ; McDermott & Redish ( 1999 ) ) . Often Correspondence to : A . Savinainen ; e - mail : antti . savinainen @ kuopio . \ufb01 C (cid:1) 2004 Wiley Periodicals , Inc . 176 SAVINAINEN ET AL . these student ideas differ from accepted scienti\ufb01c views and are resistant to change . This state of affairs poses a great challenge for science teaching : how can conceptual change be fostered ? Theories of conceptual change attempt to describe possible learning pathways from students\u2019 pre - instructional conceptions to the science conceptions to be learned ( Duit & Treagust , 2003 ) . Consequently , the design of instructional sequences can be informed by conceptual change theories and the research on students\u2019 pre - instructional conceptions , or alternative conceptions , in the target domain . However , it is not enough to pay attention only to the design of the instructional sequences : the teacher\u2019s role in staging those activities must also be considered ( Leach & Scott , 2003 ) . In this paper we show how an instructional sequence was designed and staged to improve science teaching , taking Newton\u2019s third law as an example . The design and evaluation is informed by the conceptual change literature , and the staging of the activities is informed by social perspectives on conceptual change . Firstly , we discuss what is involved in learn - ing Newtonian mechanics from a conceptual change perspective . Secondly , we present an instructional approach for teaching Newton\u2019s third law . Here we highlight the underlying principles of the design of the instructional sequence , relate our instructional approach to previous teaching approaches regarding the third law , and introduce the notion of a bridg - ing representation . Thirdly , we present an empirical study of the instructional approach in which its effectiveness in promoting learning is evaluated . We use the notion of contextual coherence to evaluate the extent of each student\u2019s conceptual change in this domain . The contextual coherence is a measure of the extent to which a student can apply a concept or a physical principle in a variety of familiar and novel situations : in such a way that the student\u2019s understanding is not adversely in\ufb02uenced by contextual features that are irrelevant from the physicist\u2019s point of view ( Savinainen & Viiri , 2003 ) . LEARNING THE NEWTONIAN CONCEPT OF FORCE : A CONCEPTUAL CHANGE PERSPECTIVE Many articles have been published on students\u2019 understandings of the force concept ( see , for example , Clement , 1982 ; Hake 1998 ; Halloun & Hestenes , 1985 ; Hestenes , Wells , & Swackhamer , 1992 ; Minstrell , 1982 ) . Several articles focus particularly on students\u2019 understandings of Newton\u2019s third law ( see , for example , Brown , 1989 ; Montanero et al . , 2002 ; Terry & Jones , 1986 ) . The \ufb01ndings commonly indicate that most students have a poor understanding of Newton\u2019s third law and of the force concept in general . In the light of such \ufb01ndings it appears that Newton\u2019s laws are dif\ufb01cult both to teach and to learn . Students tend to have a limited number of fundamental alternative conceptions about forces . They might think of forces as being things in themselves , as events , and as properties of objects ( Terry & Jones , 1986 ) . Perhaps the most common view among students is that of force as an innate or acquired property of objects , which implies that forces are not seen as arising from an interaction between objects . For example , Brown ( 1989 ) used a multiple - choice question in which the student is asked to compare the forces a 16 - pound bowling ball and a 4 - pound stationary pin exert on each other when the ball strikes the pin . Only 5 % of students answered the question correctly ( stating that the forces will be equal ) after a full year of traditional high school physics instruction . Most students seemed to think in terms of a \u201cdominance principle , \u201d where the bowling ball is \u201cmore forceful\u201d because it is moving , is heavier , and is more able to cause damage than the pin . Additionally , many students do not believe that an inanimate and inert object can exert a force . For instance , they may think that a table does not exert a force on a book lying on it , it is just \u201cin the way\u201d ( Minstrell , 1982 ) . The students\u2019 idea of force as an acquired property has close links with the pre - Galilean notion of impetus : it is conceived to be an inanimate \u201cmotive power\u201d or \u201cintrinsic force\u201d BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 177 that keeps things moving ( Hestenes , Wells , & Swackhamer , 1992 ; Sequira & Leite , 1991 ) . In this view , for an object to move it must be supplied with impetus . This is , of course , inconsistent with Newton\u2019s \ufb01rst law as well as with the character of forces arising due to interactions . Hestenes , Wells , and Swackhamer ( 1992 ) argue that the impetus concept of motion and the dominance principle are the most dif\ufb01cult and usually the last of the alternative conceptions to be overcome in the transition to Newtonian thinking . At a more general level , it can be stated that students\u2019 understandings of the force concept are very often context dependent , meaning that a student may show correct understanding in some exercises involving the force concept but fail to apply this in other contexts ( Bao et al . , 2002 ; Finegold & Gorsky , 1991 ; Palmer , 1997 ; Savinainen & Scott , 2002 ; Steinberg & Sabella , 1997 ) . Steinberg and Sabella ( 1997 ) argue that \u201cdifferent contexts and presentations can trigger different responses from a given student , even if the underlying physics is identical . \u201d Bao et al . ( 2002 ) identify four contextual features ( which they call physical features ) that students use in their reasoning regarding Newton\u2019s third law : velocity , mass , pushing , and acceleration . For instance , \u201cpushing\u201d can imply for some students that the object that \u201cpushes\u201d exerts a larger force . So these students recognize that both objects exert a force on each other but they fail to appreciate the fact that the forces arising from an interaction are always symmetrical . Furthermore , students may use combinations of different contextual features in their reasoning and may consider that the features have different levels of signi\ufb01cance for speci\ufb01c questions . We interpret this as a lack of contextual coherence ( Savinainen & Viiri , 2003 ) . These \ufb01ndings indicate that there is a big gap between everyday views ( or alternative con - ceptions ) and scienti\ufb01c views in the case of the force concept . According to the conceptual change literature , it seems that learning in this area requires a form of strong restructur - ing or reconceptualization ( Carey , 1985 ; Dykstra , Boyle , & Monarch , 1992 ; Tyson et al . , 1997 ) . The change from seeing force as an innate property to seeing forces as interactions between objects requires more than just extending existing ideas , it requires changing points of view . Furthermore , it seems that these changes often involve revisions to basic beliefs about the nature of the world : for instance , that inert and inanimate bodies can exert forces ( Minstrell , 1982 ) . Chi , Slotta , and de Leeuw ( 1994 ) refer to such conceptual changes in terms of ontological shifts . The notion of force as an innate property of an object , for in - stance , falls into the ontological category of \u201cmatter , \u201d whereas the scienti\ufb01c view of force as being due to interactions belongs to the category of \u201cprocesses . \u201d According to Chi and colleagues , conceptual change takes place when a concept is shifted between categories ( in this case , when the learner sees forces in terms of interactions and processes rather than in terms of object properties and matter ) . In summary , we see the learning problem in this area as one which entails a signi\ufb01cant movement from everyday to scienti\ufb01c views . In other words , it entails a big learning demand ( Leach & Scott , 2002 ) . Next we discuss how this demand is to be addressed through instruction . AN INSTRUCTIONAL APPROACH FOR TEACHING NEWTON\u2019S THIRD LAW Underlying Principles According to Leach and Scott ( 2002 ) , instructional aims can be identi\ufb01ed by comparing students\u2019 everyday ways of knowing and the scienti\ufb01c ideas to be taught . The differences between everyday and scienti\ufb01c ways of thinking and talking give rise to a \u201clearning de - mand\u201d that is speci\ufb01c to an individual learning in particular domains of science ( Leach & 178 SAVINAINEN ET AL . Scott , 1995 ) . The learning demand may be due to differences in the conceptual tools used , differences which relate to ontological assumptions , and differences in the epistemological underpinnings of the knowledge being used . Based on this approach , instructional design starts with identi\ufb01cation of the school science knowledge to be taught , and consideration of how this area of science is conceptualized in the everyday language of students . Then follows the learning demand analysis , which involves identifying the nature of any differences between the everyday and scienti\ufb01c views . The aspects of school science to be taught and students\u2019 typical views of the force concept are compared in Table 1 ( this learning demand analysis focuses on the aspects which are relevant to Newton\u2019s third law ) . The next step is to develop an instructional sequence , involving a sequence of activities plus information about the staging of those activities , to address each aspect of the learning demand . The staging involves all of the interactions between teacher and students and these are multimodal in nature ( see Kress et al . , 2001 ) since the ideas are communicated through talk , various images , representations , gestures and so on . By these means the school science point of view is gradually developed ( see Mortimer & Scott , 2003 ) during the sequence of lessons with the aim of rendering it intelligible and plausible to the students . To achieve this , the teacher needs to be aware of the existing understandings of the students and to develop convincing lines of argument to engage with those existing understandings . Social perspectives on conceptual change have identi\ufb01ed peer discussions as playing a potentially important role in helping students to explore meanings ( Jones & Carter , 1998 ) . Moreover , group discussions make it possible for students to express their ideas and beliefs , helping them to increase their metaconceptual awareness ( Mason , 1998 ; Vosniadou et al . , 2001 ) . Mason ( 1998 ) argues that in peer discussions the authority for learning and knowing is shared between teacher and students and that this enhances the intrinsic motivation for learning ( see also , Pintrich , Marx , & Boyle , 1993 ) . Review of Previous Approaches to Teaching Newton\u2019s Third Law Most textbooks introduce Newton\u2019s third law after Newton\u2019s \ufb01rst and second laws ( see , for instance , Giancoli , 1998 ; Halliday , Resnick , & Walker , 2001 ) . This order does not give a central role to forces as interactions , which we have identi\ufb01ed as being the crucial feature TABLE 1 Learning Demand Analysis : The Force Concept Aspects of School Science To Be Addressed Students\u2019 Typical Everyday Views Ontological aspect Force is the property of an interaction between two objects . Force is an innate or acquired property of objects ( impetus ) . Conceptual aspect Interaction between two objects implies that they exert forces on each other : forces always come in pairs . Inert or inanimate objects cannot exert forces . Epistemological aspect The notion of symmetrical interaction between two objects ( i . e . , Newton\u2019s third law ) is generally applicable to all situations . Newton\u2019s third law is used in some situations but not others ( where , for example , the dominance principle may be applied ) depending on the contextual features of the situation at hand . BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 179 of the force concept . Moreover , a careful conceptual analysis reveals that Newton\u2019s third law is more complex than one might assume from the standard statement ( Giancoli , 1998 , p . 83 ) : \u201cWhenever an object exerts a force on a second object , the second object exerts an equal and opposite force on the \ufb01rst . \u201d It should be noted that this statement does not explicitly address the notion of interaction . Asthenotionofinteractioniscentraltounderstandingtheforceconcept , someresearchers recommend that the force concept might be more effectively taught by emphasizing forces as interactions between objects ( Brown , 1989 ; Hellingman , 1989 , 1992 ; Jim\u00b4enez & Perales , 2001 ; Reif , 1995a ; Reif & Heller , 1982 ) . For instance , Reif ( 1995a ) recommends analyzing a physical system by describing both motion and interactions . He argues that identifying interactions before forces helps to avoid the inclusion of nonexistent forces . Just \u201ctelling\u201d students that forces arise due to interactions is not going to be very effec - tive . This became clear in our earlier study that was very successful in addressing many dimensions of the force concept , but not Newton\u2019s third law ( Savinainen & Scott , 2002 ) . In that study , many students had serious dif\ufb01culties , after being taught , in generalizing from Newton\u2019s third law to both the accelerated and uniform velocity cases , with many students believing that Newton\u2019s third law does not hold in a dynamic situation . In other words , they lacked contextual coherence in applying Newton\u2019s third law . It is worth noting that the force concept was introduced as a measure of the strength of interaction at the very beginning of the teaching in that study but this idea was not systematically used and developed in the teaching . We believe that this was a signi\ufb01cant reason why many students failed to understand Newton\u2019s third law . Some research studies have demonstrated that analogies or visualizable models can be helpful in teaching Newton\u2019s third law . For instance , Jim\u00b4enez and Perales ( 2001 ) report that the symbolic representation of interactions ( SRI ) notation signi\ufb01cantly improved students\u2019 comprehension of the third law ( the evidence for this was published in Spanish : Jim\u00b4enez & Perales , 1999 ) . In the modeling method of instruction a similar ( but not identical ) rep - resentational tool is referred to as a \u201csystem schema\u201d ( Hestenes , 1996 ; Turner , 2003 ) . Another example is provided by Clement et al . ( 1987 ) who used the simple compression of a spring as a successful anchoring example in teaching Newton\u2019s third law ( see also , Camp & Clement , 1994 ) . The use of bridging analogies and how they link to our notion of a bridging representation is discussed in the next section . Bridging Analogies and Bridging Representations in Promoting Conceptual Change Analogies and \u201canchoring examples\u201d that draw upon students\u2019 intuitive knowledge ( Brown , 1992 ; Brown & Clement , 1989 ; Treagust , Harrison , & Venville , 1996 ) appear to be powerful tools for learning science , though there are also studies which show that they are not always successful in bringing about conceptual change ( Duit et al . , 2001 ) . Brown and Clement ( 1989 ) provide an example of building on students\u2019 existing ideas through a \u201cbridging strategy\u201d which involves four steps : 1 . A target question is used to make explicit students\u2019 alternative conceptions relating to the topic under consideration ( e . g . , forces acting on a book on a table ) . 2 . An analogous case , an anchoring example , is suggested by the instructor ( e . g . , a hand pushing down on a spring ) . 3 . The instructor asks students to make an explicit comparison between the anchor and target cases in order to establish the analogy relation . 180 SAVINAINEN ET AL . 4 . If students do not accept the analogy , the instructor attempts to \ufb01nd an intermediate bridging analogy between the target and anchor ( e . g . , a book on top of a spring , and then on top of a noticeably \ufb02exible board ) . This speci\ufb01c example of a bridging analogy ( using the springy piece of wood ) utilizes a physical system to prompt an intermediate step in thinking . The bridge can also be in the form of a diagrammatic representation . The symbolic representation of interactions ( SRI ; developed by Jim\u00b4enez and Perales , 2001 ) makes identi\ufb01cation of the mechanical interaction between pairs of objects explicit ( this is elaborated later in this paper ; see Figure 2 ) . This can be contrasted with a standard diagrammatic representation in physics instruction\u2014 the free - body diagram\u2014which concentrates on the forces acting on one target object , and consequently does not make the concept of interaction explicit to students . In our view the SRI diagram provides a bridge linking concrete physical situations , more abstract free - body diagrams involving vector notation and the equations of Newton\u2019s laws . In this sense we see the pedagogic function of the SRI diagram as providing a bridge , which we refer to as a \u201cbridging representation\u201d ( Figure 1 ) . The notion of bridging representation is , we believe , very useful and has close links with the concept of a bridging analogy . It plays a central role in our instructional approach to the force concept in general and Newton\u2019s third law in particular . Next we turn to explaining how the bridging representation permits us to emphasize forces as interactions in an instructional sequence on the force concept for upper secondary school students . The Structure and Staging of the Instructional Sequence The instructional sequence includes three general features \ufb01rst developed in an earlier study ( see Savinainen & Scott , 2002 ) . Firstly , there is a strong conceptual focus : before problem solving a thorough qualitative treatment was implemented , as recommended by Van Heuvelen ( 1991 ) . Secondly , the students were provided with plenty of opportunities to explore meanings in carefully framed peer discussions monitored by the teacher . This is in line with research \ufb01ndings which show that teachers have a crucial role to play in promoting productive discussions ( Meloth & Deering , 1999 ; Mercer & Wegerif , 1999 ) . Thirdly , the use of multiple representations and linking between them was explicitly developed throughout the teaching . There is a growing recognition in science education that understanding and conceptually integrating multiple representations play a crucial role in learning scienti\ufb01c concepts ( e . g . , Goldman , 2003 ; van Someren et al . , 1998 ) . Furthermore , Hestenes ( 1996 ) argues that students\u2019 ability to understand physics depends on the representational tools at their disposal . We have used the notion of representational coherence in characteriz - ing students\u2019 ability to use multiple representations correctly and to move between them ( Savinainen & Viiri , 2003 ) . At the start of the instructional sequence , the concept of force was introduced by the teacher ( author AS ) in the context of contact interaction . The students were asked to press down on a table with their thumbs and to observe what happened . This touching was Figure 1 . The SRI diagram as a bridging representation . BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 181 characterized as an interaction between thumb and table . The students were also asked to press their books and notebooks to \ufb01nd out if they too were deformed . This made it easier to believe that the table does deform in an interaction with another object . Minstrell ( 1982 ) has shown that this is far from evident to students . Next the students were asked to press the table gently and then hard , and to observe if there were any changes in the deformation of their thumb . This simple activity gave them a sense that the strength of an interaction can vary and at this point \u201cforce\u201d was de\ufb01ned as a measure of the strength of the interaction . An ordinary scale and a spring balance were then introduced as tools to measure the strengths of interaction , i . e . forces . These activities can be interpreted as building on students\u2019 existing ideas and extending them to new domains in the spirit of a bridging analogy . Next the SRI diagram was introduced as a tool to represent interactions . In the diagram double - sided arrows are used to show an interaction between two objects . It was emphasized that both objects participate in the interaction and that the interaction is symmetrical . In other words , as the force is a measure of the interaction , the same amount of force is necessarily exerted on both objects . As an example of the use of the SRI diagram , Figure 2 illustrates the situation of a hand pushing a block on the horizontal surface of a table . Figure 3 presents a possible corresponding free - body diagram . In the SRI diagram , the block is located at the center because it is the target object in this example . It should be noted that this use of SRI diagrams is not identical to that in the original paper ( Jim\u00b4enez & Perales , 2001 ) . In our version , the perpendicular and parallel components of contact interactions are identi\ufb01ed , to facilitate a better correspondence with free - body diagrams . While the division between contact and distance forces ( or \ufb01eld forces ) is not justi\ufb01ed in the context of modern physics , we believe that it is a useful distinction in an introductory course on mechanics ( Physics Textbook Review Committee , 1998 ) . In Figure 2 the double - headed arrows emphasize that interactions are always symmetrical . This was reinforced in the teaching by explicating all of the interactions in the diagram . For instance , \u201cthe interaction between the hand and the block is symmetrical , hence the Figure 2 . A SRI diagram of a block sliding on a horizontal table . Interaction type is indicated by C or D ( contact or distant interaction ) . 182 SAVINAINEN ET AL . Figure 3 . A free - body diagram representing a block sliding on a horizontal table with constant acceleration . Friction , normal force , and gravitational forces are denoted by the symbols used in Giancoli ( 1998 ) . Directions of velocity and acceleration are also shown . force exerted by the hand on the block has the same magnitude as the force exerted by the block on the hand , but the opposite direction . \u201d The same SRI diagram represents the situation in which the block remains at rest ( friction would be static ) . Furthermore , it is not possible to tell whether the block is accelerating or not . This point was stressed by asking the students to draw SRI diagrams representing the block moving at constant velocity and with decreasing / increasing velocity . This was an illuminating moment for some students , who realized that there was no difference whatsoever in terms of the SRI diagrams . It illustrates the point that it is crucial to consider the same concept from a number of different\u2014but related\u2014perspectives in order to develop a coherent understanding of forces as interactions ( Maloney , 1990 ) . Magnitudes of forces cannot be deduced from the SRI diagram , whilst free - body dia - grams , which were introduced after the SRI diagrams , show the magnitudes of interactions via the length of force vectors ( Figure 3 ) . A free - body diagram allows determination of the direction of possible acceleration from the sum of the force vectors , i . e . the net force . The students were required to draw both SRI and free - body diagrams for multiple situations and were asked to compare the diagrams in peer discussions . This approach provided students with plenty of opportunities to explore meanings together . After the pairs had reached a con - sensus on the diagrams and their verbal explanations , the teacher provided his explanations and checked how many of the pairs had a correct solution and correct explanation . Using the SRI diagram makes it possible to address all the aspects of the learning demand ( see Table 1 ) . The SRI diagram provides a visualizable tool for identifying and representing interactions between objects , which helps students to perceive forces as the property of an interaction instead of a property of an object ( ontological aspect ) . It also shows by means of the double - headed arrows that an interaction between two objects is symmetrical ( conceptual aspect ) . Furthermore , applying the SRI diagram in a variety of situations helps students to realize that Newton\u2019s third law is indeed valid in all situations regardless of contextual features ( epistemological aspect ) . The use of SRI diagrams was discontinued around the middle point of teaching the force concept , since it was felt by the teacher ( author AS ) that the students had gained enough experience of thinking of forces as interactions , and the bridging representation was no longer needed . However , interactions were addressed throughout the course when analyzing free - body diagrams : the students were asked to identify all objects touching the target object , since touching implies interaction . BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 183 DESIGN OF THE STUDY Teaching Context and Research Questions The study group consisted of Finnish students ( aged 16 , n = 23 ) following a preparatory International Baccalaureate program during school year 2002 \u2013 2003 . A very similar pilot group ( n = 22 ) was involved in an earlier study by Savinainen and Scott ( 2002 ) during 2000 \u2013 2001 . The students in the pilot group and those in the study group had encountered mechanics in their lower secondary school studies . Both groups had available to them an American physics textbook based on algebra and trigonometry ( Giancoli , 1998 ) , and they were taught by the same teacher ( author AS ) . All teaching took place through the medium of English . The study group utilized the SRI diagram whereas the pilot group did not ; this was the essential difference in the teaching of the groups . Partially due to the introduction of a new tool ( the SRI diagram ) , slightly more time was devoted to teaching the force concept to the study group than to the pilot group . Our research questions are as follows : 1 . What is the effect of the instructional sequence , which utilizes the SRI Bridging Representation , on the development of the study group students\u2019 ability to apply Newton\u2019s third law in a range of contexts ( i . e . , on their contextual coherence in applying Newton\u2019s third law ) ? 2 . How do the results of the study group compare with those of the pilot group ? Data - Gathering Instruments Since we believe that a thorough evaluation of conceptual understanding should be broadly based , several instruments were used in this study to probe students\u2019 contextual coherence regarding Newton\u2019s third law , i . e . their ability to apply the third law in differ - ent contexts . These instruments included multiple - choice questions , an extended written response question , and interview questions . Multiple - Choice Questions . The \u201cforce concept inventory\u201d ( FCI ) has four multiple - choice questions addressing Newton\u2019s third law ( Halloun et al . , 1995 ) . Two of the questions have the same context of a car pushing a truck ( questions 15 and 16 ) . Question 4 addresses a collision between a large truck and a small car . Question 28 involves two students sitting on identical of\ufb01ce chairs and the heavier student pushes the other with his feet . This question involves two contextual features\u2014mass and pushing\u2014at the same time . Bao et al . ( 2002 ) criticize this question , arguing that if a student answers that the forces are not equal , it is impossible to know ( without asking the students ) whether an incorrect response is generated through consideration of mass , of pushing , or of both . To study the effects of the above - mentioned contextual features , Bao et al . ( 2002 ) de - veloped their \u201cSurvey on Newton\u2019s Third Law . \u201d Each question in it measures students\u2019 reasoning related to a single contextual feature of the third law . The effects of velocity , mass , pushing , and acceleration are evaluated with three questions for each feature . In ad - dition there are four questions involving other contextual features , making 16 questions altogether . Bao et al . validated the instrument by interviewing students . Figure 4 provides a sample question from the survey that involves only the contextual feature of velocity . The correct answer is shown in bold print . The Survey contains some questions that are similar to , but not identical with , those in another research - based multiple - choice test on the force concept , the \u201cforce and motion conceptual evaluation\u201d ( FMCE ) ( Thornton & Sokoloff , 1998 ) . The FMCE has ten questions 184 SAVINAINEN ET AL . Figure 4 . A sample question from the survey on Newton\u2019s third law ( Bao et al . , 2002 ) . This question involves only the contextual feature of velocity . The correct answer is shown in bold print . on the third law , which introduce variations of contextual features of mass , velocity , and acceleration within three main contexts . All of these multiple - choice questions ( FCI , Survey on Newton\u2019s Third Law , and FMCE ) were used in this study ( see Table 2 ) . Extended Written Response Question . In addition to the multiple - choice questions , an extended written response question was used to gain insights into the students\u2019 reasoning ( Figure 5 ) . The main contextual feature of the written question is mass , though the car TABLE 2 Research Instruments Administered to the Study Group Research Instrument Pilot Group Study Group Comments Pre - FCI ( in Finnish ) Yes Yes At the beginning of teaching FMCE Yes Yes Two months after the pre - FCI Post - FCI ( in English ) Yes Yes After completing the teaching of the concept of force ( a month after the FMCE ) Interview questions No Yes Five students were interviewed a week after the post - FCI Extended Response written question Yes Yes A month after the post - FCI Survey on Newton\u2019s third law No Yes After completion of teaching the law of conservation of momentum , which was derived using Newton\u2019s third law ( three months after the post - FCI ) BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 185 Figure 5 . The extended response written question used in the study ( Reif , 1995b , p . 86 ) . and the bug have different magnitudes of velocities as well . The written question involves identifying the correct answer and justifying it . Essentially the same question was answered by the pilot group in the earlier study ( Savinainen & Scott , 2002 ) . Interview Questions . Five students from the study group were chosen for interview on the basis of their success in the early part of the course : they represented the top , middle , and lower levels of the group . A semistructured interview was used and the students were asked to \u201cthink aloud\u201d whilst answering the interview questions , being free to change their answers during the interview if they so wished . The interviews were conducted by author AS . The interview questions relating to Newton\u2019s third law are presented in Figure 6 . In these questions , parts ( a ) and ( b ) have the same general context but the state of the system is changed . Part ( a ) addresses constant velocity , whereas part ( b ) addresses varying velocity ( acceleration ) . From the physics point of view , these questions resemble the situations in the FCI questions 15 and 16 , where the car is pushing a truck with increasing and constant velocity , respectively . Administration of the Research Instruments to the Study Group A Finnish translation of the 1995 version of the Force Concept Inventory ( Halloun , et al . , 1995 ; Koponen , Jauhiainen , & Lavonen , 2000 ) was administered prior to instruction because the students\u2019 competence in English was not suf\ufb01ciently good at the beginning of the program . The students were allowed to see the correct answers after the instruments had been administered but all question and answer materials were subsequently collected up . \u2217 After the pretest , all instruments and questions were in English . An outline schedule for the administration of the research instruments , over a period of six months , is presented in Table 2 . The pilot group students in the earlier study answered the pre - FCI , FMCE , and post - FCI , and essentially the same written question . Measure of the Students\u2019 Contextual Coherence It is possible for a group of students to achieve a relatively high average score for the multiple - choice questions used , even if only a few students correctly answer all of the questions addressing Newton\u2019s third law . Hence , the following measure ( based on four \u2217 Of course , they were not allowed to see the correct answers of the Pre - FCI . 186 SAVINAINEN ET AL . Figure 6 . The interview questions ( derived from McDermott et al . , 1998 , pp . 28 \u2013 29 ) . criteria ) of each student\u2019s contextual coherence was used . A student exhibits contextual coherence if 1 . all four FCI questions addressing the third law are correctly answered ; 2 . at least 9 out of 10 FMCE questions on the third law are correctly answered ( one lapse was allowed since it could be due to carelessness ) ; 3 . the written question has the correct answer with correct reasoning ; 4 . at least 15 out of 16 Survey questions are correctly answered . The above criteria were applied to the different instruments separately . This measure of contextual coherence is taken as an indicator of conceptual change . Because FCI question 15 concerning a dynamic situation proved to be hard for many pilot group students , a written explanation of the reasoning accompanied this question in the post - FCI of the study group . A short text was added to the Survey as well : \u201cExplain brie\ufb02y how you arrived at your answers and what physical principle ( s ) you applied . \u201d RESULTS Multiple - Choice Tests The averages of the FCI and FMCE results are presented in Table 3 . The statistical signi\ufb01cance of possible differences between the groups was tested with the nonparamet - ric Mann - Whitney U test of two independent samples . ( A nonparametric test was used rather than the t - test because the data did not follow the normal distribution . ) There is no BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 187 TABLE 3 Newton\u2019s Third Law Results for the Pilot Group ( n = 22 ) and Study Group ( n = 23 ) N3 Law Tests Pilot Group Study Group ( No . of Questions ) Average ( SD ) Average ( SD ) p - value Pre - FCI ( 4 ) 23 . 9 ( 30 . 4 ) 30 . 4 ( 23 . 8 ) 0 . 194 FMCE ( 10 ) 62 . 2 ( 28 . 1 ) 78 . 7 ( 23 . 8 ) 0 . 038 Post - FCI ( 4 ) 75 . 0 ( 28 . 9 ) 93 . 5 ( 13 . 5 ) 0 . 012 Survey ( 16 ) \u2013 98 . 6 ( 4 . 6 ) \u2013 p - value is calculated using Mann - Whitney U - test for two independent samples . statistically signi\ufb01cant difference between the pre - FCI test results , whereas the FMCE and post - FCI results of the study group are better ( p - values are below 0 . 05 ) . While the averages provide a way of comparing differences between the groups , they do not directly reveal information about the students\u2019 contextual coherence . Figure 7 presents the percentages of students who demonstrated contextual coherence , from different in - struments , according to the criteria given in the preceding section . The pilot group did not answer the questions in the Survey on Newton\u2019s third law . The study group students\u2019 written explanations in the Survey all supported the correct answers . In the pilot group , two students answered all the FCI questions on Newton\u2019s third law correctly at the beginning of the teaching , whereas in the study group none did . There is a clear line of progression in the development of contextual coherence in the study group and most of the students reached contextual coherence at the level of identi\ufb01cation , i . e . they were able to identify the correct answers from non - Newtonian alternatives . Only two study group students failed to answer all the Survey questions correctly . These two had only a few wrong answers but they revealed that these students , just like many in the pilot group , were still struggling to differentiate between Newton\u2019s second and third laws . This conclusion was supported by their written justi\ufb01cations accompanying the Survey . For instance , one student who incorrectly answered two questions addressing a dynamical situation wrote ( the student\u2019s language has not been edited ) : Ifthereisnoacceleration , there\u2019snonetforce , massdoesn\u2019tmatter . Whenyoupushsomeone with some amount of force , other one pushes you with the same amount force . Figure 7 . Percentage of students exhibiting contextual coherence in the questions addressing Newton\u2019s third law for the pilot and study groups . The tests are in chronological order . 188 SAVINAINEN ET AL . It seems that this student thinks that Newton\u2019s third law is valid only if there is no acceleration . Extended Response Written Question Students\u2019 responses to the extended response written question support the \ufb01ndings from the multiple - choice tests since 82 % of students in the study group answered it correctly with correct reasoning . To give some idea of the students\u2019 answers to the written question , we present one correct and one incorrect response from the study group ( the students\u2019 language has not been edited ) . Correct response ( underlining done by the student ) : According to Newton\u2019s third law , forces always come as pairs and objects interacting always exert equal amount of force on each other , no matter what . So the car and the bug exert equal magnitude of force on each other . This response shows a clear understanding of forces as interactions and recognizes that contextual features of an interaction are irrelevant from the point of view of Newton\u2019s third law . Incorrect response : As it says in N III Law , when two object interacts they exert equal amount of force in each other only magnitude varies . In this case magnitude of force exerted on the bug by the car is larger than the magnitude of the force exerted by the bug . The student recalls Newton\u2019s third law correctly but she interprets the term \u201csame amount\u201d incorrectly , enabling her to hold both Newton\u2019s third law and the dominance principle at the same time . Interview Questions Given that all \ufb01ve students interviewed from the study group had correctly answered the four questions on Newton\u2019s third law in the post - FCI a week earlier , the interview results were surprising . Three students out of \ufb01ve answered both questions correctly . One student who ended up with the correct answer was unsure about the Newton\u2019s third law force pair in the case of gravitational force : Butagain , there\u2019sthe . . . Thatmean , meangravitationalforce . Something . . . Ohwell . . . The amount of force the gravitational force exerts on the rocket , it exerts back on the , on the gravitational force . But that doesn\u2019t make any sense . When , when I put it like that . But , could the proper explanation be that it exerts the same amount of force on the Earth ? The fourth student answered both questions incorrectly in the same way . She stated that the normal force and gravitational force exerted on the crate inside the rocket constitute a Newton\u2019s third law force pair regardless of the state of motion . She recalled a slogan used during the lessons \u201cyou can\u2019t touch without being touched , \u201d but this did not help her in answering the questions . The \ufb01fth student did not seem to understand the question at all . When prompted she recalled relevant aspects from the third law but she was unable to apply these aspects in the interview questions : BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 189 Was it that , umm , they come in pairs and they\u2019re symmetrical and . . . [ and a bit later ] I think it was , umm , that forces come in pairs and they\u2019re symmetrical . And , umm , that . . . Was it that you can\u2019t touch without being touched ? It is worth noting that in contrast to the multiple - choice questions , the interview questions went beyond identifying magnitudes of a given interaction force pair . The questions were posedinacontextthatalsorequiredtheuseofNewton\u2019s\ufb01rstandsecondlaws . Inaddition , the interview questions addressed Newton\u2019s third law in the case of a noncontact gravitational force , which was not addressed in any other instruments used in this study . DISCUSSION Evaluation of the Results The \ufb01rst research question addresses the development of the study group students\u2019 con - textual coherence concerning Newton\u2019s third law . Both the study and pilot groups followed the same teaching approach but in the case of the study group the notion of forces as in - teractions was emphasized , through the use of the SRI bridging representation . Learning outcomes were measured in terms of contextual coherence : the student was required to answer correctly almost all the questions , which involved different contexts . The results demonstrate that the instructional approach proved to be very successful in promoting con - textual coherence of the third law , at least at the level of identi\ufb01cation : the students could identify the correct answers from non - Newtonian alternatives and justify their reasoning . The second research question addresses possible differences between the study and pilot groups : the study group students\u2019 results were better than those of the pilot group , the differ - ence being statistically signi\ufb01cant . Similar results have been achieved by author AS with par - allel groups of students following the Finnish upper secondary school syllabus ( Savinainen , 2004 ) . Our \ufb01ndings therefore provide strong support for Jim\u00b4enez and Perales ( 2001 ) , who reported that the SRI notation signi\ufb01cantly improved their students\u2019 comprehension of the third law . Further support can be found in the Finnish syllabus \u201cbaseline\u201d study of post - FCI results . Almost 400 Finnish upper secondary students from different parts of Finland were sampled , and it was found that only 28 % correctly answered the questions on Newton\u2019s third law in all the various contexts addressed by the FCI ( Jauhiainen , Koponen , & Lavonen , 2001 ) . In the light of these results , even the instructional approach for the pilot group , which did not utilize the SRI diagrams , can be considered quite successful , since 45 % of those students exhibited contextual coherence in Newton\u2019s third law at the end of the course . Minstrell ( 1982 ) showed that students may have dif\ufb01culties with the idea of inanimate objects , such as a table , exerting forces . The students in this study had no trouble in accept - ing the idea once they started thinking of forces as interactions . The dominance principle was prevalent in the students\u2019 responses regarding the third law at the beginning of the course . At the end of the course , almost all the students recognized that an interaction is symmetrical regardless of contextual features in the given tasks ( i . e . , they exhibited con - textual coherence in this respect ) . They had , however , much more dif\ufb01culty with Newton\u2019s second law ( Savinainen , 2004 ) . Hence , our \ufb01ndings suggest that the dominance principle is not necessarily \u201cone of the last misconceptions to be overcome in transition to Newtonian thinking\u201d ( Hestenes , Wells , & Swackhamer , 1992 ) . It appears that the prevalence of the dominance principle depends on the instructional sequence . Dykstra , Boyle , and Monarch ( 1992 ) present a successful instructional sequence in which Newton\u2019s \ufb01rst and second laws are dealt with before the third law . Given their instructional sequence , it is likely that students\u2019 would sort out the third law last . 190 SAVINAINEN ET AL . Evaluation of Research Design and Methodology In this study , there are questions that need to be addressed regarding the possible causes of the learning gains observed . Leach and Scott ( 2002 ) conclude that it is not legitimate to claim that \u201cone instructional sequence is better than another in promoting student learning , without taking into account the part played by the teacher . \u201d In this study most of the factors affecting the learning outcomes were kept as constant as was possible : the same experienced teacher ( author AS ) taught approximately the same course to similarly selected preparatory year International Baccalaureate students . Also , almost all the research instruments used for the evaluation of student learning were the same . The major difference between the two instructional approaches was the use of SRI diagrams directly demonstrating the simulta - neous and symmetric nature of interactions . Another difference was linguistic : the terms \u201caction\u201d and \u201creaction\u201d were not employed in the revised approach , as recommended by Hellingman ( 1989 ) , whereas these terms were mentioned but not systematically used with the pilot group . There are also concerns relating to the use of multiple - choice questions , which require only identi\ufb01cation of correct answers . It could be argued that the study group students just learned to do this without real understanding . Given that the students completed many similar types of multiple - choice tests , this is a reasonable worry . However , there are several reasons why the concern is not warranted : 1 . During the course , the pilot group students answered almost as many questions on the third law as the study group students , and saw the correct answers to the FMCE afterwards , but this was not enough to change the incorrect views of over half of the group . 2 . The extended response written question and the written parts of the multiple - choice tests were used to gain insight into students\u2019 reasoning and overall student\u2019s written justi\ufb01cations supported their choices in the multiple - choice tests . 3 . Data were collected over a relatively long time span ( six months ) . An additional point of concern is that the multiple - choice questions used do not address all aspects of Newton\u2019s third law . For instance , they do not test whether a student understands that forces arising from an interaction between two objects are always exactly opposite in direction . Furthermore , the questions involve only verbal representation . There is some evidence that students may not master the third law in vector diagram representation equally well as in verbal representation ( Meltzer , 2002 ) . The interview results suggest that the multiple - choice questions used have limitations : in general , these questions seem to do a good job at the level of identifying the correct answer , but they do not necessarily predict students\u2019 understanding in more complex situations . Other interviews conducted by author AS with different groups of students lend support to this suggestion ( Savinainen , 2004 ) . However , one could also question the extent to which the interviews provide reliable knowledge on students\u2019 thinking . For instance , Schoultz , S\u00a8alj\u00a8o , and Wyndham ( 2001 ) provide evidence suggesting that students\u2019 responses in in - terview studies should be regarded as situated and dependent on the tools available as resources for reasoning . Furthermore , Welzel and Roth ( 1998 ) conclude that interviews can only provide clues on ongoing , dynamic cognitive processes . In this study , the interview questions deliberately avoided referring to the concept of interaction or to the SRI diagram : hence , the interview situation did not make the above - mentioned tools readily available for the students . From this perspective , it could be speculated that the outcome of the inter - views could have been different had these tools been cued in the interviews . Indeed , it is BRIDGING REPRESENTATION IN FOSTERING CONCEPTUAL CHANGE 191 regrettable that only \ufb01ve students were interviewed , and only once , in the case of Newton\u2019s third law ( the same students were interviewed several times on Newton\u2019s \ufb01rst and second laws ) . Nevertheless , our study lends support to the claim made by Dufresne , Leonard , and Gerace ( 2002 ) that multiple sources of information about students\u2019 knowledge and under - standing are needed if we are to obtain a reliable assessment of students\u2019 knowledge and understanding . Whilst acknowledging all of these questions relating to methodological issues , we would nevertheless support the proposition that the instructional approach developed here , based on the SRI representation , offers an effective way of teaching Newton\u2019s third law . Why Was the Bridging Representation Helpful ? It is interesting to re\ufb02ect on the role of the bridging representation in fostering con - ceptual change . There are several possible reasons why the SRI diagram\u2014the bridging representation\u2014was effective in supporting conceptual change in the case of Newton\u2019s third law . Firstly , it helps students to perceive forces as symmetric interactions and provides the means to identify other objects with which the target object is interacting . Secondly , the potential value of the bridging representation can be seen in terms of diSessa\u2019s ( 1993 ) phenomenological primitives ( p - prims ) . The p - prims or core intuitions are not misconcep - tions as such but they can bring about a misconception when applied in physical situations ( Brown , 1993 ) . The most relevant p - prim here is \u201cmore agency means more effect\u201d : the dominance principle can be interpreted as a manifestation of this particular core intuition or p - prim when applied in , say , collisions . As pointed out earlier , almost all the students in this study gave up the dominance principle after utilizing the bridging representation . This suggests that the bridging representation helps to override the core intuition related to the dominance principle . On the other hand , this core intuition ( \u201cmore agency means more effect\u201d ) has a meaningful function in analyzing , for instance , a collision between a heavy truck and a light car : the forces on both vehicles have the same magnitude but the resulting accelerations and damages are not the same , since the masses and the internal structures of the vehicles are different . Thirdly , the bridging representation provides a complementary approach to free - body diagrams , with the potential to support the construction of deeper understandings since information is integrated by the students from more than one representation ( Ainsworth , 1999 ) . Treagust , Chittleborough , and Mamiala ( 2003 ) , for instance , found that effective learning in chemical explanations requires simultaneous use of multiple representations . In the instructional approach developed in this study , students were encouraged to combine diagrammatic representations ( the SRI and free - body diagrams ) with verbal representations , thereby helping to promote representational coherence . It would also be reasonable to assume that the bridging representation fosters students\u2019 ability to identify the forces that must be included in the free - body diagram . However , this was not investigated in this study . Fourthly , the use of the bridging representation in this study \ufb01ts well with Rosenshine , Meister , and Chapman\u2019s ( 1996 ) characterization of scaffolding , drawn from sociocultural perspectives on learning . They interpret scaffolding as \u201ctemporary frameworks or supports usedtoassiststudentsduringinitiallearningofacomplexskillorcognitivestrategy . \u201dItmight thereforebearguedthatthebridgingrepresentationhelpstoscaffoldstudents\u2019understandingofNewton\u2019sthirdlawenablingthemtomovefromconcretephysicalsituationstowardsmoreabstractdiagrammaticandmathematicalrepresentationsasdepictedinFigure1 . The bridging representation acted as a temporary scaffold since the teacher ( author AS ) discontinued its use around the middle point of teaching . It is interesting to note that even after this point the students did not completely abandon it . In retrospective comments , the 192 SAVINAINEN ET AL . interviewed students indicated that they did not draw the diagram anymore but that they used it as an aid to their reasoning when identifying forces . They also commented that the linguistic aspect was important ( i . e . , stressing that forces are due to interactions ) but that the diagram had a crucial role in their understanding and applying Newton\u2019s third law . Finally , the bridging representation was used in a highly interactive manner based on peer discussions . The students had time and opportunity to talk through their developing understandings , with the support of the teacher , as recommended by Scott ( 1998 ) . It is worth noting that the same interactive teaching approach but without an effective tool for representation of interactions was not so successful in promoting learning of the third law in our earlier study ( Savinainen & Scott , 2002 ) . It is , moreover , doubtful that the emphasis on forces as interactions would be very effective in a passive lecture format ( Hake , 1998 ) . Reflections on the Nature of Conceptual Change The results from this study can be interpreted in terms of a conceptual change that involves the students reassigning the force concept to the ontological category of \u201cprocesses\u201d and starting to think of forces in terms of interactions between objects . Furthermore this would qualify as an example of strong restructuring or a \u201cbig\u201d change ( Tyson et al . , 1997 ) . Our data lend support ( see Figure 7 ) to the view that big conceptual changes are often evolutionary rather than revolutionary in nature , involving a gradual process ( Vosniadou & Ioannides , 1998 ) . This suggests that students\u2019 learning progresses from initial , scienti\ufb01cally more or less incorrect views via some intermediate or \u201ctransitional states\u201d towards the scienti\ufb01c perspective ( Thornton , 1995 ) . Our results also support Harrison , Grayson , and Treagust\u2019s ( 1999 ) conclusion that students\u2019 conceptual change requires time and explicit attention for developing the concepts : this was addressed in our approach by applying the \u201cconcepts \ufb01rst\u201d principle ( Van Heuvelen , 1991 ) . Pintrich , Marx , and Boyle ( 1993 ) stress that motivational factors play a signi\ufb01cant role in conceptual change . Students need to be cognitively engaged in order for conceptual change to occur in the \ufb01rst place . The instructional approach taken in our study relied heavily on peer discussions as ways of exploring meanings and it was important that the students recognized and accepted the worth of peer discussions ( Crouch & Mazur , 2001 ; Gunstone , McKittrick , & Mullhall , 1999 ) . Care was taken at the beginning of instruction to motivate the students to participate in peer discussion . The teacher explained that knowing physics means being able to \u201ctalk physics\u201d as well as being able to use other representations in analyzing physical situations . It was also made clear ( as recommended by Crouch and Mazur , 2001 ) that conceptual understanding is required in the examinations . It was gratifying to note that the students were very willing to participate in peer discussions almost from the very beginning . Final Reflections This article provides an account of a study in which various theoretical perspectives have been drawn upon in designing an instructional sequence that was then implemented in the classroom and demonstrated to give rise to enhanced learning outcomes when com - pared with those from a parallel class of students . 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International Journal of Science Education , 20 , 25 \u2013 44 .", + "bernsteinNetworkCentralizationCollective2023": "This article was downloaded by : [ 129 . 2 . 89 . 115 ] On : 03 May 2024 , At : 15 : 27 Publisher : Institute for Operations Research and the Management Sciences ( INFORMS ) INFORMS is located in Maryland , USA Organization Science Publication details , including instructions for authors and subscription information : http : / / pubsonline . informs . org Network Centralization and Collective Adaptability to a Shifting Environment Ethan S . Bernstein , Jesse C . Shore , Alice J . Jang To cite this article : Ethan S . Bernstein , Jesse C . Shore , Alice J . Jang ( 2023 ) Network Centralization and Collective Adaptability to a Shifting Environment . 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Copyright \u00a9 2022 The Author ( s ) Please scroll down for article\u2014it is on subsequent pages With 12 , 500 members from nearly 90 countries , INFORMS is the largest international association of operations research ( O . R . ) and analytics professionals and students . INFORMS provides unique networking and learning opportunities for individual professionals , and organizations of all types and sizes , to better understand and use O . R . and analytics tools and methods to transform strategic visions and achieve better outcomes . For more information on INFORMS , its publications , membership , or meetings visit http : / / www . informs . org Network Centralization and Collective Adaptability to a Shifting Environment Ethan S . Bernstein , a , * Jesse C . Shore , b , * Alice J . Jang c * Contributed equally a Harvard Business School , Organizational Behavior Unit , Boston , Massachusetts 02163 ; b Questrom School of Business , Boston University , Boston , Massachusetts 02215 ; c Pamplin College of Business , Virginia Polytechnic Institute and State University , Blacksburg , Virginia 24061 Contact : e @ hbs . edu , https : / / orcid . org / 0000 - 0001 - 9819 - 0639 ( ESB ) ; jesse . c . shore @ gmail . com , https : / / orcid . org / 0000 - 0003 - 3812 - 0262 ( JCS ) ; ajjang @ vt . edu , https : / / orcid . org / 0000 - 0003 - 1140 - 0598 ( AJJ ) Received : September 15 , 2019 Revised : June 7 , 2020 ; April 15 , 2021 ; August 20 , 2021 ; January 19 , 2022 Accepted : February 4 , 2022 Published Online in Articles in Advance : May 31 , 2022 https : / / doi . org / 10 . 1287 / orsc . 2022 . 1584 Copyright : \u00a9 2022 The Author ( s ) Abstract . We study the connection between communication network structure and an organization \u2019 s collective adaptability to a shifting environment . Research has shown that network centralization \u2014 the degree to which communication \ufb02 ows dispropor - tionately through one or more members of the organization rather than being more equally distributed \u2014 interferes with collective problem - solving by obstructing the integration of existing ideas , information , and solutions in the network . We hypothesize that the mecha - nisms responsible for that poor integration of ideas , information , and solutions would nevertheless prove bene \ufb01 cial for problems requiring adaptation to a shifting environment . We conducted a 1 , 620 - subject randomized online laboratory experiment , testing the effect of seven network structures on problem - solving success . To simulate a shifting environ - ment , we designed a murder mystery task and manipulated when each piece of information could be found : early information encouraged an inferior consensus , requiring a collective shift of solution after more information emerged . We \ufb01 nd that when the communication network within an organization is more centralized , it achieves the bene \ufb01 ts of connectivity ( spread of novel better solutions ) without the costs ( getting stuck on an existing inferior sol - ution ) . We also \ufb01 nd , however , that these bene \ufb01 ts of centralization only materialize in net - works with two - way \ufb02 ow of information and not when information only \ufb02 ows from the center of the network outward ( as can occur in hierarchical structures or digitally mediated communication ) . We draw on these \ufb01 ndings to reconceptualize theory on the impact of cen - tralization \u2014 and how it affects conformity pressure ( lock - in ) and awareness of diverse ideas ( learning ) \u2014 on collectiveproblem - solving thatdemands adaptation . History : This paper has been accepted for the Organization Science Special Issue on Experiments in Organizational Theory . Open Access Statement : This work is licensed under a Creative Commons Attribution 4 . 0 International License . You are free to copy , distribute , transmit and adapt this work , but you must attribute this work as \u201c Organization Science . Copyright \u00a9 2022 The Author ( s ) . https : / / doi . org / 10 . 1287 / orsc . 2022 . 1584 , used under a Creative Commons Attribution License : https : / / creativecommons . org / licenses / by / 4 . 0 / . \u201d Funding : The authors acknowledge \ufb01 nancial support from the Division of Research at the Harvard Busi - ness School for making this research possible . Keywords : network centralization \u2022 adaptation \u2022 organizational structure \u2022 communication networks \u2022 social networks \u2022 problem - solving \u2022 dynamic environments \u2022 collective intelligence How does the centralization of an organization \u2019 s communication network affect its members \u2019 capacity for collective problem - solving ? Because problem - solving is key to organizational success ( Grant 1996 , Nickerson and Zenger 2004 ) , because organizational problem - solving is increasingly collective ( Hong and Page 2004 , Wuchty et al . 2007 , Hackman 2011 ) , and because communication network structure is a key factor in making collective problem - solving effective ( Mason and Watts 2012 , Becker et al . 2017 , Argote et al . 2018 ) , organizational theorists and managers are interested in the link between characteristics of a communication network ( like centralization ) and suc - cessful collective problem - solving . \u201c Network centralization , \u201d following Freeman ( 1978 ) , has been de \ufb01 ned as the degree to which communication \ufb02 ows disproportionately through one or more members of the organization rather than equally through all ( e . g . , Huang and Cummings 2011 ) . Although it has been measured in a variety of ways , from degree ( Shaw 1954 ) to eigenvector centrality ( Bonacich 1987 ) , a highly cen - tralized network is always characterized by ( a ) direct ties between a \u201c core \u201d individual or group and most ( or all ) individuals and ( b ) a lack of direct ties between those not 2064 ORGANIZATION SCIENCE Vol . 34 , No . 6 , November \u2013 December 2023 , pp . 2064 \u2013 2096 ISSN 1047 - 7039 ( print ) , ISSN 1526 - 5455 ( online ) https : / / pubsonline . informs . org / journal / orsc D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . in the core ( i . e . , the \u201c periphery \u201d ) . Such a network all but guarantees , even without status - based hierarchy , 1 that the core will have access to all critical information and can \u201c put the pieces together \u201d ( Bavelas 1950 , Tushman 1979 , Butts et al . 2007 ) , whereas the periphery will have access only to whatever the core has the bandwidth and chooses to share . The hub - and - spoke structure \u2014 with a core leader ( the \u201c hub \u201d ) who brokers all interactions amongst peripheral members ( the \u201c spokes \u201d ) \u2014 is the most extreme of centralized networks , but others ( e . g . , a leadership team with multiple core members ) operate similarly , with the core responsible for integrating the work of the periphery . Prior research \ufb01 nds that such network centraliza - tion of communication harms collective problem - solving because people in the periphery cannot directly share ideas , information , and / or solutions with each other , whereas the core can become a bottleneck to \ufb01 nd - ing a good solution or can lead the whole network astray should it adopt a bad one ( Tushman 1979 , Huang and Cummings 2011 , Becker et al . 2017 , Argote et al . 2018 ) . Some organizations have , therefore , decided that collective problem - solving calls for decentralized communication structures , especially to draw on fast information \ufb02 ows and diverse expertise from across the organization for collective problem - solving requiring adaptation ( Gulati 2007 , Bernstein et al . 2016 , Lee and Edmondson 2017 , Reitzig 2022 , Bernstein 2022 ) . However , prior studies have not considered adapta - tion problems , which have different features than the previously studied collaboration problems . Instead , prior studies have focused on tasks that , although rep - resentative of daily collaborative work , do not re \ufb02 ect the rapidly shifting environments , sudden develop - ments , and need to draw on diverse ideas ( Lee and Edmondson 2017 ) that characterize adaptation tasks . The purpose of this paper is to investigate whether communication network centralization might make organizations not less but rather more capable of col - lective problem - solving requiring adaptation . We build on a tradition of research about how network structure relates to social in \ufb02 uence and the diversity of ideas ( Lazer and Friedman 2007 , Mason et al . 2008 , Mason and Watts 2012 , Shore et al . 2015 , Bernstein et al . 2018 ) to investigate whether the characteristics of centralized networks that impede traditional collabo - rative problem - solving may nevertheless improve col - lective adaptation . Speci \ufb01 cally , we consider whether the inability of peripheral individuals to communicate with each other directly \u2014 although posing an integra - tion risk to collaborative problem - solving \u2014 could actually be bene \ufb01 cial for solving adaptation problems because it could reduce conformity pressure and promote independent thinking in the periphery . Simi - larly , we consider whether the fact that the core is connected to everybody in the periphery \u2014 although posing a bottleneck risk to collaborative problem - solving \u2014 could be bene \ufb01 cial in adaptation problems because the core could learn from differing solutions in the periphery and spread the one they think best . To test these ideas , we ran a 1 , 620 - person experiment on a new experimental platform that we developed for randomized experiments with communication network structure as the independent variable ( i . e . , investigating how network structure causally affects collective per - formance ) . To collectively solve problems , participants could seek and share information \u2014 much as they would do at work with enterprise social network tech - nologies ( e . g . , Slack , Teams , Workspace , Yammer , etc . ) \u2014 and they could enter solutions and access their neighbors \u2019 solutions . We designed the experimental platform to permit the creation of a shifting environ - ment ( analogous to dynamic environments faced by product managers and organization leaders ) by con - trolling when certain information can be discovered . We used this feature to create a collaborative murder mystery task with a pivotal phase in which partici - pants had to collectively shift away from a premature ( although seemingly reasonable ) consensus on an infe - rior solution , adapt to new information , and adopt a new solution . To study the main effect of communi - cation network centralization on problem - solving and to describe the mechanisms through which it works , we tested seven realistic network structures ( four varia - tions on centralized network structures and three decentralized controls ) ( see Figure 5 ) . We randomly assigned participants to nodes , so that our results would isolate the effects of overall network structure from individual attributes rather than our \ufb01 ndings about centralization and problem - solving being con - founded with individual attributes or competency . We \ufb01 nd that centralized communication networks solved adaptation problems better than decentralized networks did . They were less likely to get stuck on a premature consensus and more likely to disseminate the superior solution . They exhibited the bene \ufb01 cial aspects of social in \ufb02 uence in problem - solving ( expo - sure to differing interpretations ) without the harmful aspects ( lock - in on an existing solution because of con - formity pressure ) . By testing both directed network ties ( one way from core to periphery ) and undirected network ties ( two way between core and periphery ) , we also provide a major caveat with respect to that main \ufb01 nding . The bene \ufb01 ts to centralization rely on central nodes learn - ing from the more - independent peripheral nodes . In real - world organizations , however , central nodes often in \ufb02 uence without being in \ufb02 uenced by the periphery , as in traditional bureaucratic / administra - tive pyramidal structures ( Puranam 2018 ) . Such struc - tures tend to prevent the aforementioned bene \ufb01 ts of centralization . Our results , therefore , reinforce the Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2065 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . importance of nurturing behaviors ( Detert and Burris 2007 , Morrison 2011 , Detert et al . 2013 ) to ensure that communication \ufb02 ows both ways . This paper seeks to make three contributions by addressing an open question in organizational theory and organization design about the impact of central - ization on collective problem - solving in tasks requir - ing adaptation and , therefore , on collective intelligence ( Woolley et al . 2010 , Malone 2018 ) . First , we demon - strate that , contrary to popular opinion , centralization is bene \ufb01 cial to tasks requiring adaptation because it promotes social learning but avoids lock - in . Second , by cleanly decoupling the effects of centralization and of hierarchy , we observe the pure effect of structurally - imposed centralization on problem - solving , which is useful for advancing scholarship on both centraliza - tion and hierarchy . Finally , because managers increas - ingly use enterprise social networking technologies ( Majchrzak et al . 2009 , Leonardi 2014 ) to bypass tradi - tional centralized structures ( Gulati and Puranam 2009 ) , our \ufb01 ndings have practical implications for using com - munication technologies to promote \u2014 and not under - mine \u2014 organizational adaptability . Hypothesis Development : The Effect of Centralization on Collective Problem - Solving in Adaptation Tasks The Harmful Effects of Centralization in Previously - Studied Tasks Generally , prior studies have found that structural centralization impedes using knowledge and ideas already in the network . More speci \ufb01 cally , studies of collaboration tasks \ufb01 nd that centralization impedes the collective ability to integrate the information and ideas held in the periphery , whereas studies of wisdom - of - the - crowd tasks \ufb01 nd that collective judg - ments suffer from the excessive in \ufb02 uence of central individuals ( see Table 1 ) . What Is Different in Adaptation Tasks ? Given the long - standing belief that the effect of struc - tural characteristics ( like centralization ) on outcomes will be contingent upon the type of problem to be solved ( Tushman 1979 ) , it may be the case that collective problem - solving tasks requiring adaptation \u2014 which differ in several key ways from tasks requiring collabo - ration or coordination \u2014 respond differently to central - ization , even as the mechanisms identi \ufb01 ed in prior research remain the same ( see Table 2 ) . We , therefore , reinterpret the key harmful mechanisms identi \ufb01 ed in prior research in light of literature on social in \ufb02 uence , social network structure , and adaptability in social net - works . In doing so , we theorize that those same mecha - nisms may improve performance on tasks requiring collective adaptation \u2014 that is , collectively shifting from one solution to a dissimilar , but superior , one . Scholars have found that various adaptation prob - lems share a common trait that distinguishes them from tasks requiring collaboration or coordination ; the existing understanding of a problem can make it very hard to \ufb01 nd other ways to solve it \u2014 that is , to adapt \u2014 because high - quality new solutions are likely to be very different from the current solution ( as is the case , for example , with successful responses to disruptive inno - vations ( Christensen 2013 ) ) . The landscape of solutions to adaptation problems would , therefore , \ufb01 t what scholars call \u201c rugged \u201d ( Levinthal 1997 ) \u2014 meaning that the performance \u201c peaks \u201d ( i . e . , best solutions ) may be very distant from each other in terms of their compo - nent parts . More generally , this happens when a solu - tion has many interdependent components , such that someone trying to solve that problem is likely to go over multiple hills and valleys ( a bumpy , iterative proc - ess of trying diverse combinations of solution compo - nents ) to get from one good solution to the next . There are many examples of such interdependent components \u2014 and therefore \u201c rugged \u201d solution land - scapes \u2014 challenging organizations that attempt to adapt to a shifting environment ( Lavie et al . 2010 ) . In steel production ( Levinthal 1997 , Prennushi et al . 1997 ) , for example , as modern factory work practices ( incentive pay , team - based work , \ufb02 exible job assign - ments , employee training ) began to replace more traditional practices ( hourly wages , narrow job de \ufb01 ni - tions , close supervision ) , factories failed to experience improved performance unless coherent , interdepend - ent sets of modern work practices were adopted together . Experimenting with only one deviation from traditional working structures ( e . g . , shifting from nar - row job de \ufb01 nitions to \ufb02 exible assignments ) might have produced no performance gain ; it might even have seen a decline . To modernize effectively , many interdependent work practices needed to be adjusted in tandem ( Bloom et al . 2012 ) , making the future solu - tion very dissimilar from the past one . Such changes are often described \u2014 and experienced \u2014 as transfor - mational rather than incremental ( Immelt 2017 , Reeves et al . 2018 ) . Similarly , components of a corpo - rate strategy are often highly interdependent . Thanks to tracking customer preferences through personaliza - tion technologies and web search history , Google \u2019 s simultaneous investments in web search , ad auctions , and personalization technologies jointly contribute to its successful overall business model precisely because they are interdependent ; Google \u2019 s auctions maximize the probability of clicking , which they are able to do by tracking customer preferences and interests and personalizing their ads . If one of these strategic ele - ments was no longer viable ( perhaps through regula - tion ) , the whole business model would have to adapt . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2066 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . Conceptually , although not all adaptation problems necessarily require such transformational rather than incremental change , this paper follows prior research in focusing on those that do . Such challenges of solving adaptation problems have been viewed in terms of the trade - off in individuals \u2019 choices between exploration ( searching for new solu - tions ) and exploitation ( re \ufb01 ning a preexisting consen - sus ) ( March 1991 , Levine et al . 2018 ) . However , even holding individual exploratory behavior constant , network structure \u2014 such as centralization \u2014 can make it more or less likely for an organization facing adapta - tion problems to shift from a good solution to a supe - rior ( but dissimilar ) one by ( a ) reducing the probability of sticking to an inferior consensus and ( b ) spreading a superior solution to everyone once it is found . In the following sections , we theorize the role centralization might play in each of those two . Table 1 . Negative Effects of Centralization on Integration and Wisdom of the Crowd Tasks Type of task Task examples Findings \u2014 mechanism by which centralization harms performance Collaboration tasks : Tasks that bene \ufb01 t from integration of the collaborators \u2019 diverse contributions of ideas and information Tushman ( 1979 ) : Research & Development ( R & D ) projects of different types and complexity Work group hierarchical structure and work group task were correlated , implying a contingency theory of centralization . High - performing research groups were less centralized than high - performing technical service groups performing routine work Cummings and Cross ( 2003 ) : Project - based work , requiring coordination and integration of ideas Centralization was negatively associated with performance Huang and Cummings ( 2011 ) : Project - based work , requiring coordination and integration of ideas Centralization prevents peripheral individuals from integrating each other \u2019 s information . Core acts as a bottleneck Argote et al . ( 2018 ) : Laboratory experiment on the generation of new features in a collaborative programming task Centralized networks generated fewer new features Wisdom of the crowd tasks : Tasks based on averaging a group of individual estimates Becker et al . ( 2017 ) : Estimation of various quantities ( coins in a jar , calories in a meal , etc . ) Centralized networks were more likely to decrease in performance than decentralized networks because of the excessive in \ufb02 uence of central individuals Golub and Jackson ( 2010 ) : Mathematical modeling Crowd wisdom occurs only when no single individual is too in \ufb02 uential Yan et al . ( 2021 ) : Crowdsourced clothing design More centralized crowds that did not have shared task experience were worse at predicting market demand for their designs Table 2 . Distinguishing Collective Problem - Solving Tasks Requiring Adaptation from Previously Studied Collective Problem - Solving Tasks Requiring Collaboration Collaboration tasks Adaptation tasks Task context Stable environment Shifting environment Task objective Integration / coordination to reach consensus Departure from prior consensus to a new , superior solution Nature of the information landscape Information available for problem - solving remains relatively static Information available for problem - solving is revealed over time Nature of the solution landscape Relatively smooth with independent components ; improving from one solution to the next - better one generally involves following an incremental path Relatively rugged with interdependent components ; improving from one solution to the next - better one generally involves following a transformational path Success enabled by Free \ufb02 ow of communication , allowing nodes to share and build off of information and interpretations already existing amongst them Preservation of diverse , novel options and multiple interpretations , unconstrained by preexisting knowledge and the consensus interpretation ( s ) Indicators of success Fully elaborated solution Shift from one solution to a new solution Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2067 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . The Effect of Centralization on the Probability of Sticking to an Inferior Consensus in Adaptation Tasks How could network structure affect the probability of sticking to an inferior consensus ? Conformity pres - sure from peers is a determinant of whether organiza - tion members are collectively adaptable or likely to get jointly stuck on a current consensus . Being aware of a network neighbor \u2019 s solution can lead an individ - ual to adopt a matching solution , and this \u201c social in \ufb02 u - ence \u201d can be a positive or negative force . It is often individually rational to copy peers \u2019 solutions when one is not con \ufb01 dent in one \u2019 s own , but this can have the effect of entrenching the current inferior consensus \u2014 because it does not allow more diverse solutions to be considered \u2014 as easily as it could lead to bene \ufb01 cial learning ( Banerjee 1992 , Bikhchandani et al . 1992 ) . For our purposes , social in \ufb02 uence can be divided roughly into two components : conformity pressure to adopt peers \u2019 solutions ( contributing to entrenched consensus ) and transfer of substantive information between nodes ( contributing to bene \ufb01 cial learning ) . In general , holding transfer of information ( and therefore learning ) constant , the stronger the social conformity pressure , the harder it will be to collectively shift from one consensus to a new superior one . We , therefore , begin by focusing \ufb01 rst on conformity pressure ( that increases the probability of sticking to an inferior con - sensus ) and return to the transfer of information in the following section on the probability of spreading superior solutions . Conformity Pressure from Neighbors : Number Effects Prior research shows that network structure can increase conformity pressure when more of a person \u2019 s network neighbors adopt a given solution . Results from Centola ( 2010 ) and Ugander et al . ( 2012 ) suggest that two neighbors with the same solution are approxi - mately twice as in \ufb02 uential as one , with each additional neighbor having a decreasing marginal in \ufb02 uence . See the four curves in Figure 1 for a visualization , with higher curves representing more neighbors . Conformity Pressure from Neighbors : Share Effects When the solutions to a problem are mutually exclu - sive ( e . g . , in a jury , the options are to convict or acquit but not both ) , the share of neighbors who have adopted each solution is important ; there is a strong majority effect , resulting in an S curve of probability of adoption ( see , e . g . , equations 15 and 16 in Stasser and Davis 1981 ) for each individual behavior ( or judgment ) . Conformity Pressure from Neighbors : Combining Number and Share Effects Together , these two effects result in a response curve ( the probability of adopting an alternative solution due to social in \ufb02 uence ) that depends on both number ( different curves in Figure 1 ) and share ( horizontal axis in Figure 1 ) of neighbors . Figure 1 depicts the case in which two competing alternatives are equal in Figure 1 . Relative Amounts of Conformity Pressure to Shift to a Competing and Mutually Exclusive Solution to a Problem Vary by Number and Share of Adopting Peers 1 neighbor 2 neighbors 3 neighbors 8 neighbors 0 . 0 0 . 3 0 . 6 0 . 9 share of neighbors adopting c on f o r m it y p r e ss u r e ( a r b it r a r y un it s ) Notes . Separate curves , as inferred from prior literature ( Stasser and Davis 1981 , Centola 2010 , Ugander et al . 2012 ) , are drawn for groups with one , two , three , and eight ( other ) peers , with points plotted at each whole number of peers . Note that exact parameters vary by setting and that this visualization is intended only to convey qualitatively how number and share matter in in \ufb02 uence . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2068 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . intrinsic in \ufb02 uence such that differences are a conse - quence only of number and of share of peers who adopt each alternative . These results suggest that for network structure to aid in adaptability , it must put people in network positions such that either ( a ) they have relatively few neighbors ( and are thus on a lower curve in Figure 1 ) or ( b ) their neighbors are unlikely to all adopt the same solution ( and thus are located to the left side of their curve ) . How Centralization Might Reduce Conformity Pressure in Adaptation Tasks If we logically apply the results on number and share effects reviewed to centralized networks , they suggest centralization might reduce conformity pressure in adaptation tasks because of the way that number and share effects work in tandem with one another in cen - tralized networks . With respect to number effects , central nodes are by de \ufb01 nition relatively few in number , meaning that peripheral nodes would experience relatively little con - formity pressure by virtue of sitting on a relatively low curve in Figure 1 . In this context , by \u201c relatively low , \u201d we mean low relative to other possible network positions and structures with the same total number of nodes \u2014 in other words , other ways of organizing the same number of people . In short , although central nodes in \ufb02 uence many people , that in \ufb02 uence may not be very strong because there are so few of them ( number effects ) . On the other hand , number effects would predict that central nodes in a centralized network could be highly in \ufb02 uenced by the peripheral nodes \u2014 central nodes in a centralized network have relatively many neighbors and thus sit on a relatively high conformity pressure curve in Figure 1 . However , share effects might limit that con - formity pressure , as peripheral nodes are likely to adopt a variety of different solutions because of ( a ) the low conformity pressure peripheral nodes feel from central nodes and ( b ) the fact that these peripheral nodes are not able to exert conformity pressure on each other ( because they are not directly connected ) . This means that despite sitting on a high curve ( because of number effects ) , central nodes are likely to \ufb01 nd themselves on the left side of their curve ( Figure 1 ) , where a relatively small share of their neighbors has adopted any given solution . Moreover , because conformity is a self - reinforcing phenomenon , low conformity pressure on the core further reduces conformity pressure on the periphery ( see Figure 2 , block A ) . The logic we have built above , based on number and share effects , to theorize how centralization could reduce conformity pressure in adaptation tasks \ufb01 nds some support , albeit by analogy , in prior theoretical and empirical research on network clustering ; that is , when one \u2019 s network neighbors are also neighbors to each other ( Hansen 1999 , Burt 2004 , Centola 2010 ) . Because both number and share of neighbors matter , the formation and maintenance of a single consensus solution are also more likely with network clust - ering . Network clustering makes it more likely for an individual to have more reinforcing exposures to the same solution ( s ) , and thus it both hastens the spread of \u201c complex contagions \u201d ( Centola 2010 ) and impedes deviation from an established consensus ( Stasser and Davis 1981 ) . Clustering ( and its global analog , density \u2014 having many network connections within a set of nodes ) , thus , has been found to inhibit a net - work \u2019 s collective ability to consider different solutions to a complex problem , even as it aids in collecting more information ( Lazer and Friedman 2007 , Shore et al . 2015 ) . Although a high level of centralization does not necessarily imply general lack of clustering , it does imply a lack of clustering and density of con - nections among peripheral nodes , which are connected to central nodes but not to each other . To make our logic more concrete , we built a sim - ulation model of how the number and share effects found in prior literature would work in the context of various network structures . We simulated the probabil - ity of shifting from one consensus solution to another , assuming the conformity pressure rules depicted in Figure 1 and that in each time period , people have a small random chance of changing their solutions ( see Appendix A for details and Figure 5 for network struc - tures ) . Our simulation found centralized networks less likely to get stuck on an initial consensus than other network structures . Speci \ufb01 cally , agents in a complete clique retained the initial consensus 92 % of the time , agents in a locally clustered network retained the ini - tial consensus 80 % of the time , peripheral nodes in a hub - and - spoke network retained the initial consensus 57 % of the time , and peripheral nodes in a core - periphery network retained the initial consensus 72 % of the time . Thus , prior research on network structure and our own simulations based on the microfoundations of social in \ufb02 uence support our logic that members of centralized networks feel less conformity pressure than those in other empirically common network structures and are , therefore , more likely to choose different solutions than their peers when solving com - plex problems . That is consistent with prior work \ufb01 nding that the performance of centralized networks on nonadaptation tasks suffers because peripheral nodes are not tightly integrated ( Huang and Cum - mings 2011 ) , but it is precisely that lack of mutual in \ufb02 uence and resultant conformity pressure that could be a blessing for adaptation tasks because it allows more diverse solutions to be considered collectively . We therefore hypothesize : Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2069 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . Hypothesis 1 . In problems requiring adaptation , central - ization reduces the probability of sticking to an inferior consensus . The Effect of Centralization on the Probability of Spreading Superior Solutions in Adaptation Tasks For collective adaptation , it is not enough to avoid get - ting stuck on an inferior solution . Good solutions \u2014 which can be hard to come by \u2014 must spread . In gen - erating Hypothesis 1 , we argued that centralization might reduce conformity pressure , but we must also argue that it does not impair the positive side of social in \ufb02 uence : the bene \ufb01 cial transfer of information and knowledge ( i . e . , bene \ufb01 cial learning ) ( e . g . , Argote and Ingram 2000 ) . There has been signi \ufb01 cant study of how to promote the spread of established , successful solu - tions in an organization ( e . g . , Reagans and McEvily 2003 ) . It can be particularly valuable to learn from peers when one lacks relevant experience ( Aranda et al . 2017 ) . Given that bene \ufb01 cial transfer of information and counterproductive conformity pressure both result from the spread of solutions through social in \ufb02 uence , is it possible to predictably get the bene \ufb01 ts of social in \ufb02 uence without the costs ? Bernstein et al . ( 2018 ) show that it is indeed possible , in that case by having intermittent breaks in interaction ( i . e . , alternating indi - vidual work with connectivity ) . In this way , people experience independence , which generates diversity of solutions without conformity pressure , while also preserving opportunities to interact , which allows them to learn from that diversity . Theoretically , centralization may create structural differentiation somewhat as intermittent interaction does , creating the conditions for both diversity of ideas ( because of relatively little conformity pressure ) and the spread of good ideas ( through the central nodes ) . Although we have argued that central nodes exert relatively little conformity pressure , they still expose many peripheral nodes to their solutions , creating the opportunity for their ideas to spread . Burt \u2019 s research program on \u201c brokerage \u201d and \u201c struc - tural holes \u201d has long argued that those who bridge otherwise disconnected people are structurally privi - leged , being more exposed to different ways of think - ing ( e . g . , Burt 2004 ) . Again , in Figure 1 , central nodes would sit to the left side , where a small share of their neighbors has adopted any given solution ( Figure 2 , B ) . Knowledge of others \u2019 solutions without conform - ity pressure to adopt a speci \ufb01 c solution compels cen - tral nodes to exercise judgment , albeit with the bene \ufb01 t of being able to ask others to explain their various positions . Thus , in a setting requiring adaptation , it would seem that central nodes are especially well positioned to learn from the periphery ( Figure 2 , C ) and to spread what they learn more widely . In essence , centralized networks may , like intermittent interaction , be able to achieve the in \ufb02 uence necessary for spreading good solutions without creating conformity pressure that would keep those good solutions from being generated by peripheral members in the \ufb01 rst place . We therefore hypothesize : Hypothesis 2 . In problems requiring adaptation , central - ization promotes the spread of superior solutions . Taken together , centralization \u2019 s hypothesized lower probability of sticking to an inferior consensus and its hypothesized increased probability of spreading supe - rior solutions should result in a higher average per - formance . We therefore hypothesize : Hypothesis 3 . In problems requiring adaptation , central - ized networks cause more people to be better on average . We add a \ufb01 nal hypothesis to capture a particularly important boundary condition . The logic behind the bene - \ufb01 ts of centralization for collective adaptation and , there - fore , behind our \ufb01 rst three hypotheses , relies on central nodes being in \ufb02 uenced by peripheral nodes ( Figure 2 , B ) . In practice , communication in some centralized net - works is unidirectional ( for example , in traditional , bureaucratic , hierarchical organizations and self - organ - ized online communities ) ; the core in \ufb02 uences but is not in \ufb02 uenced by the periphery . Thus , it is important to dis - tinguish between centralization with two - way communi - cation between core and periphery and centralization with only one - way communication from the core to the periphery . For the latter , we do not expect the bene \ufb01 ts stipulated in Hypotheses 1 \u2013 3 . Hypothesis 4 . In problems requiring adaptation , central - ized networks with one - way communication from the core to the periphery do not reduce the probability of sticking to an inferior consensus , do not promote the spread of superior solutions , and do not cause more people to be better on average . Experimental Design Using a new online laboratory platform , 2 we con - ducted a randomized experiment of the effect of net - work centralization on problem - solving in a setting that requires adapting from one answer to another because of the emergence of new information ( see Figure 5 for network treatments ) . The Task In developing a suitable experimental task , we sought a task design focused on the collective ability of the organization that was solving the problem \u2014 in the context of different communication structures \u2014 to shift from one idea to a dissimilar one , with the main Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2070 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . dependent variable de \ufb01 ned as the number of partici - pants per trial who were correct in the end . We also sought a protocol that allowed experimental control of important variables and yet was not overly stylized , such that the task was similar to real problem - solving work in shifting environments and that the means for accomplishing the task within the platform had paral - lels in the real world . Following the long tradition of murder mystery protocols in group research ( Stasser and Stewart 1992 , Stasser and Titus 2003 ) and more recently in network research ( Shore et al . 2015 ) , we adopted a traditional \ufb01 ctional murder mystery task ( akin to the popular game Clue or Cluedo ) , in which the task required piecing together facts ( \u201c clues \u201d ) to identify the culprit . However , we added a twist : red herring clues that implicate the wrong suspect and the timed release of critical clues . Information leading to the correct answer would only become available after partici - pants had already come to a reasonable but incorrect conclusion or consensus . Because we were interested in how organizational structure affects collective adaptation to a shifting environment , we measured performance in terms of the number of individuals who \ufb01 nished each trial with the correct answer \u2014 that is , who successfully adapted . Our results are , thus , at the network level , but they aggregate individual - level data , capturing how network structure affects the distribution of the number of participants who \ufb01 nd the best answer . Thus , we build on the paradigm of social organization and collective intelligence research on settings in which individuals work on separate instances of the same task but nonetheless in \ufb02 uence each other such that collective , network - level outcomes differ from those of sets of disconnected individuals ( also known as \u201c nominal groups \u201d ) . As with past research in this paradigm ( e . g . , Lazer and Friedman 2007 , Mason and Watts 2012 , Levine et al . 2014 , Shore et al . 2015 , Becker et al . 2017 ) , our approach allows disaggregated meas - urement of differing solutions and focuses on how social organization affects the emergence of consen - sus \u2014 that is , on the ability to learn from and build on each other \u2019 s solutions . Other work has adopted an alternative paradigm \u2014 with individuals working on a shared task \u2014 and has focused on determinants of Figure 2 . ( Color online ) Diagram of Mechanisms Underlying Performance in Adaptation Tasks Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2071 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . group or team performance , with attention to process gains and losses ( e . g . , Woolley et al . 2010 , Huang and Cummings 2011 , Argote et al . 2018 ) . We return to this distinction in paradigms as a limitation in our discus - sion section . Our experimental interface ( Figure 3 ) allows partici - pants to ( a ) search for clues about the mystery by entering keywords into a search bar , ( b ) share any clues they \ufb01 nd with network neighbors ( with or with - out free - text messages ) , ( c ) send messages \u2014 without clues \u2014 to neighbors , ( d ) reply to or forward messages received from neighbors , ( e ) register an answer ( along with a measure of one \u2019 s con \ufb01 dence in it ) , and ( f ) see the answers ( and con \ufb01 dence levels ) registered by neighbors . Other than the use of one \u2019 s time and the fact that payment depends on registering answers , there are no explicit costs or incentives for any of these actions ( see Appendix B for details on payment ) . Structure of Task At the beginning of the trial , 3 every participant received an initial clue in the form of a \u201c message from headquarters \u201d announcing that the victim is missing and presumed dead . Subsequent clues could be found by a search using keywords from the initial clue , which in turn produced clues about other people with whom the victim had interacted ( i . e . , possible sus - pects ) . Using their names as search terms produced more information about them , including possible motives or connections . Each trial lasted 15 minutes and had three phases in which new information became accessible . The \ufb01 rst set of clues established a red herring by bringing up a speci \ufb01 c suspect and establishing a motive . Five minutes into the \ufb01 rst phase , the second phase began , with a new message from headquarters \u2014 broadcast to all participants \u2014 revealing new information ( and therefore , new search terms ) that led to information not previously accessible . With suf \ufb01 cient search and deduction , it was now possible \u2014 although not straight - forward \u2014 to rule out the original red herring and dis - cover the real culprit . At the 10 - minute mark , the third phase began . A new \u201c message from headquarters \u201d was broadcast with a critical clue that ruled out the most prominent red herring and drew attention to the real culprit . Figure 4 shows the percentage of participants who found the correct answer , the red herring answer , or one of four other answers over time and illustrates the predominant switch toward the correct answer in the last \ufb01 ve minutes . During phase 2 , a majority had reg - istered the red herring , and fewer than 10 % had iden - ti \ufb01 ed the correct answer even though it was logically possible to do so . For 95 % of the participants for whom more than half of their visible peers had entered an answer prior to the third phase , the majority of those visible answers were matching and incorrect ; that is , those participants saw a consensus but on a wrong answer . For 69 % of the participants for whom more than half of their visible peers had entered an answer prior to the third phase , all visible answers were match - ing and incorrect . During the third phase , when it was logically straightforward to deduce the correct answer , a sizeable minority retained incorrect answers . Network Treatments We examine six network treatments with nine nodes ( participants ) each . The two centralized networks that are the focus of our study are the hub - and - spoke net - work and the core - periphery network . The hub - and - Figure 3 . ( Color online ) Screenshot of the Experimental Interface Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2072 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . spoke is the maximally centralized network , with a single central node to which all others are connected , like a group with a single coordinator who brokers interactions between the others . As the most central - ized structure possible , the hub - and - spoke network ( or close variants ) has been used in prior experimental studies of networks ( e . g . , Bavelas 1950 , Becker et al . 2017 , Argote et al . 2018 ) . It is also a representation of the communication pattern commonly associated with hierarchical organization and has been \u2014 and in some cases , remains \u2014 a default in organizations ( Figure 5 ) . The core - periphery network is a less centralized but common form of centralization found in self - organized social networks ( see , e . g . , Colizza et al . 2006 , Kane and Alavi 2007 , Cattani and Ferriani 2008 , Connelly et al . 2011 , Dahlander and Frederiksen 2012 ) and in organizations ( e . g . , a leadership team that brokers interactions between the other group mem - bers ) . It consists of a central group ( the core ) \u2014 rather than a single central individual \u2014 and peripheral indi - viduals connected to the core but not to each other ( Borgatti and Everett 2000 ) . The existence of a central group is of practical importance because of the greater conformity pressure felt in network positions with a clustered pattern of ties ( Centola 2010 ) . We tested two versions of the core - periphery net - work with directed ( one - way ) ties in order to study the mechanisms underlying the performance effects of centralization that we postulated in deriving our hypotheses . One , the \u201c out core - periphery \u201d network , differs from the undirected version of the core - periphery network in that the core has few incoming ties from the periphery . This structure occurs in self - organized networks in which communication and in \ufb02 uence do not \ufb02 ow from the periphery to the core ; for example , digital social media in which one user can unilaterally follow another ( Frahm and Shepe - lyansky 2012 ) or hierarchical organizations in which the leader communicates to the members but the members do not speak upward to the leader ( or alter - natively , the leader fails to listen ) . The second directed core - periphery network \u2014 the \u201c in core - periphery \u201d net - work \u2014 reverses the pattern : the core has many incom - ing ties from the periphery but few outgoing ties ( e . g . , a leadership team that gathers information from but does not communicate back to other organization members , as in organized crime ( Aven 2010 ) and the U . S . Central Intelligence Agency ( Hackman 2011 ) ) . In our analyses , we compare these directed core - periphery networks with both the undirected central - ized networks and the control networks . Finally , we tested three decentralized network struc - tures as controls . The locally clustered network consists , in our case , of three interconnected cliques of three indi - viduals , akin to interconnected teams ( a team of teams ) or groups of friends . The complete clique is a group of nine people , all of whom are connected to each other , like a large work group ( e . g . , a so - called \u201c all hands on deck \u201d collaboration ) or people using a shared digital communication channel . Finally , we constructed a syn - thetic treatment of \u201c isolates , \u201d which are groups of nine individuals who were resampled with replacement from the set of all participants in the core - periphery network and who had no incoming ties ( positions 5 , 7 , and 9 ) . Isolates represent the disconnected nature of \u201c crowds \u201d ( Afuah and Tucci 2012 ) or participants in a competition ( Boudreau et al . 2011 ) . These three decentralized controls represent what we believe are empirically plausible alternatives to centralization , with different levels of social in \ufb02 uence . Although random network structures ( e . g . , Becker et al . 2017 ) or extremal network structures ( e . g . , Mason and Watts 2012 ) have sometimes been used as controls for network experiments , we wanted controls that could also function as plausible null hypotheses \u2014 plausible alternative modes of organiz - ing for problem - solving that exist in the real world . See Appendix E for more detail on the network treatments . We collected 30 independent trials for each experi - mental condition and 90 bootstrap - sampled synthetic trials of isolates . Network treatments were collected in randomly permuted order to avoid systematic bias because of time effects . Fewer than 2 % of participants dropped out midway , but this resulted in approxi - mately 16 % of trials being affected by dropout at Figure 4 . ( Color online ) Percentage of Participants with Each Answer over Time 0 . 0 0 . 2 0 . 4 0 300 600 seconds pe r c en t age answer type red herring correct other Note . \u201c Other \u201d includes four incorrect suspects . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2073 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . some point . In estimating the causal effects of treat - ments , we analyzed all trials , regardless of dropout , to avoid inadvertently conditioning on posttreatment latent variables ( Montgomery et al . 2018 ) . 4 Participants were given no information about their network structure or their position within it other than the fact that communication ties were not neces - sarily symmetrical ; the people they followed ( received information from ) might not follow them back . Participant Pipeline We recruited participants through Amazon Mechani - cal Turk ( MTurk ; https : / / www . mturk . com ) , located in the United States . Building on work by Mason and Suri ( 2012 ) , we constructed a pipeline using a task sequence consisting of three stages : ( Stage 1 ) a 3 - minute individual task in which participants learn to search for clues and enter their answers as well as learn the basic format of a murder mystery task , ( Stage 2 ) a 5 - minute task in which three participants learn to use the social features of the interface , and ( Stage 3 ) the main 15 - minute trial for nine people . Requiring two tasks to be complete before entering the main trial played the dual roles of ensuring that participants were familiar with the platform before the main experiment and \ufb01 ltering out participants who were inattentive , disinterested , unable to com - plete the task , or otherwise likely to drop out of a syn - chronous trial . Low attention among MTurk workers is a rational response to the preponderance of low - paying and poorly designed tasks ( Bigham 2014 ) . However , dropouts and inattentive participants affect the performance of everybody else in the trial , so min - imizing them was a priority . In addition , random dropout ( i . e . , dropout not caused by something spe - ci \ufb01 c to network treatment ) is not a problem from a Figure 5 . Experimental Treatments Notes . ( Left panel ) Visualization of network structures . An arrow from node i to node j indicates that node j can receive clues from node i and see its answers but not the other way around . ( Right panel ) Adjacency matrix visualizations of the same treatments . A \ufb01 lled - in square in position ( i , j ) indicates that node j can receive clues from node i and see its answers . All ties in the hub - and - spoke , core - periphery , locally clustered , and complete clique are bidirectional , with the sole exception of the ties between 1 , 4 , and 7 in the locally clustered network , which are unidirectional for the purpose of preserving degree . Most ties in the out core - periphery and in the in core - periphery are one way between the core and periph - eral nodes , as indicated by the arrows , with the exception of the ties between nodes 1 and 4 , nodes 2 and 6 , and nodes 3 and 8 for the purposes of preserving degree and external validity . See Appendix E for more detail . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2074 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . statistical inference perspective but does introduce prob - lems of validity of interpretation ; if the central node in the hub - and - spoke network drops out , this creates a large de facto change in network structure . We thus took pains to create an effective \ufb01 lter \u2014 prior to the main task \u2014 that was still fair to those whom we \ufb01 ltered out . Participants were paid according to the length of time that they had the correct answers entered into the user interface ( including both the trivial attention check question and the main question of \u201c whodunnit \u201d ) . Median wages were $ 8 . 47 per hour . See Appendix B for full details on the subject pipeline , including task speci - \ufb01 cations , \ufb01 ltering and representativeness of subjects , and payment . The Nature of Communication and Collaboration Among Participants Participants could only receive information from each other or see each other \u2019 s answers if they shared a net - work tie . A network tie from person i to person j meant that j could always see i \u2019 s answers to the prob - lem . Sharing clues or free - text messages was also only possible with a network tie but was not required or explicitly incentivized . However , the instructional text that we provided articulated the collaborative nature of the experiment . Speci \ufb01 cally , the instructions for stage 1 state that \u201c [ t ] he real HIT is a collaborative game \u201d ( \u201c HIT \u201d\u2014 human intelligence task \u2014 is the standard term for a paid task on MTurk ) , and we provided the following information on interacting with other subjects for the stage 3 ( main ) trial : \u201c Like last time , you will be solving a \ufb01 ctional mur - der mystery . Some clues will be given to you , and you must use a search tool to \ufb01 nd other clues . 1 . This time , you will be working with other Turkers in real time . Be sure to watch for mes - sages from other players . Everybody will be given a fake name ; your real identity will be hidden from the other players . 2 . Note that you can only send messages to people listed in the messaging window . Some people you \u201c follow \u201d don \u2019 t \u201c follow \u201d you back . [ \u2026 ] The purpose of the study is to learn about how people solve challenging problems together . You will be collaborating with other participants to solve a \ufb01 ctional murder mystery game . You will receive some clues , and you will have to search for other clues to be used to solve the game . You will be paid based on the time you have entered the correct answers . \u201d In practice , subjects did use the communication and collaboration features frequently . The mean number of messages passed per trial was 75 . 7 ( 25th percentile ( Q1 ) (cid:2) 59 , 75th percentile ( Q3 ) (cid:2) 90 ) ; most messages shared clues that someone had found without any additional text comments . Note that search actions themselves were not visible to peers , but the clues found through search could be voluntarily shared . Participants did use free - text communication as well , with a mean of 14 . 5 messages per trial ( Q1 (cid:2) 7 , Q3 (cid:2) 20 ) . Most free - text messages were collaborative in the sense that participants discussed clues and their implications . Occasionally , process - oriented and con - versational messages were also included . A represen - tative selection of messages includes the following : \u2022 \u201c So the major project launch was in Honolulu , yes ? Where Ms . Moore and Ms . Jones were \u201d \u2022 \u201c Seems a lot of people had dealings with Anderson \u201d \u2022 \u201c Mr . Brown is the only one who got hurt twice by hall \u201d \u2022 \u201c Looks like you have info on Hall and I have info on the suspects \u201d \u2022 \u201c Have you found anything regarding a murder weapon ? \u201d \u2022 \u201c Davis has a gun , but was gone . So that leaves Brown and Lewis \u201d The spread of these free - text messages could be tracked as easily as the spread of the of \ufb01 cial clues , giv - ing us a comprehensive view of how information \ufb02 owed through our network treatments . We provided a channel of nontextual communica - tion for registering answers ; participants were also prompted for a con \ufb01 dence level ( which defaulted to 50 % ) . Our intention was not to measure an outcome variable but rather to enable more effective collective problem - solving . Previous research ( Bahrami et al . 2010 , Engel et al . 2014 , Madirolas and De Polavieja 2014 , Becker et al . 2017 ) shows the importance of com - municating con \ufb01 dence levels to collective intelligence outcomes . We believe that this feature also increases the external validity of the experiment , as most collab - orative settings allow some means of expressing con \ufb01 - dence in a solution . Results Our dependent variable is the number of participants correct at the end of the trial ; thus , it is measured at the network level but is a count of individual - level outcomes within each trial . 5 We study the effects of social in \ufb02 uence within networks and thus expect both the mean and the shape of the distribution of our dependent variable to vary with network treatment . For example , in a trial of isolates , we expect the distri - bution of the number of participants correct per trial to be unimodal and binomially distributed around some mean ( because participants solve the problem independently ) , but in a trial of a complete - clique net - work , we expect the distribution to be bimodal Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2075 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . ( because participants solve the problem with social in \ufb02 uence from every other participant , thus prompting an expectation of high correlation among their ans - wers that will , therefore , be either mostly correct or mostly incorrect ) . Our hypothesis tests rely not only on the means of these distributions but also on their quantiles in order to capture differences in collective behavior , as described . Main Result In this experiment , which required adapting to a shift - ing environment by changing from one temporarily justi \ufb01 able answer to a superior one , centralized networks performed best . They minimized the proba - bility of getting stuck on an incorrect consensus ( Hy - pothesis 1 ) while maximizing the spread of the correct answer ( Hypothesis 2 ) , thus getting the bene \ufb01 ts of collaboration without the costs ( Hypothesis 3 ) but only with two - way versus one - way communication ( Hypothesis 4 ) . We found no signi \ufb01 cant performance differences caused by network position ; the key causal variable was the whole - network structure . Basic descriptive statistics are shown in Table 3 . Figure 6 provides an overview of the distribution of how many participants were correct at the end of the trial \u2014 by experimental condition \u2014 in the form of empirical cumulative distribution functions ( eCDFs ) , which plot the percentage of trials that ended with a certain number of people correct or fewer . The farther the eCDF curves are to the right side of the panel , the better the performance in terms of the percentage of participants correct per trial . Panel ( a ) shows the eCDFs for the nominal groups of isolates ( minimal social in \ufb02 uence ) and complete clique ( maximal social in \ufb02 uence ) conditions . Compared with isolates , the com - plete clique tended to have more trials with very few participants correct ( marked \u201c costs \u201d ) and more trials with many participants correct ( marked \u201c bene \ufb01 ts \u201d ) . Subsequent panels overlay the eCDF from another net - work treatment on those of the isolates and complete clique for easier visual interpretation . Panel ( b ) shows results for the locally clustered network , which has fewer costs and fewer bene \ufb01 ts than the complete clique . Panels ( c ) and ( d ) in Figure 6 show results for the centralized network treatments ( see Figure 11 for results for the directed core - periphery treatments that serve as tests of mechanisms ) . Panel ( c ) shows that the hub - and - spoke network had few trials with few participants correct ( it did not experience the costs of social in \ufb02 uence ) but many trials with many par - ticipants correct ( it did bene \ufb01 t from social in \ufb02 uence ) . Panel ( d ) shows that the core - periphery did suffer some of the costs \u2014 although less than the complete clique treatment did \u2014 while enjoying similar bene \ufb01 ts . Tests of proportions show no signi \ufb01 cant differences in the costs of social in \ufb02 uence ( as measured by the proportion of trials with zero or one participant cor - rect at the end ) between the isolates and both central - ized network treatments . We thus reject the null for Hypothesis 1 . We also found no signi \ufb01 cant differences in the bene \ufb01 ts of social in \ufb02 uence ( as measured by the proportion of trials with greater than six participants correct at the end ) between the complete clique and both centralized network treatments . We thus reject the null for Hypothesis 2 . However , the out core - periphery had a greater proportion of trials with zero or one person correct than did the isolates ( p (cid:2) 0 . 03 , one - tailed test ) and a smaller proportion of trials with greater than six people correct than did the complete clique ( p (cid:2) 0 . 06 , one - tailed test ) , consistent with Hypothesis 4 . Models of the number of participants correct at the end of a trial put these comparisons in a regression framework , reported in Table 4 . Quantile regressions ( which model conditional quantiles instead of the more typical conditional mean ) provide a perspective on the costs and bene \ufb01 ts of social in \ufb02 uence that is complementary to that of the tests of proportions reported above . We expect outcomes to vary , even with the same treatment . If people become stuck on the wrong answer , there will be more trials toward the bottom of the performance distribution with very few participants correct at the end of the trial . There - fore , we use the 25th percentile of the distribution ( models 1 and 5 ) to provide another test of Hypothesis 1 . Similarly , if the correct answer spreads widely , it will increase the number of trials toward the top of the performance distribution with very many participants correct at the end of the trial . Therefore , we use the 75th percentile ( models 4 and 8 ) to provide another test Table 3 . Descriptive Statistics by Network Treatment Hub - and - spoke Core - periphery Isolates Locallyclustered Completeclique Out core - periphery In core - periphery N 30 30 90 30 30 30 30 Mean ( no . correct / network ) 4 . 7 4 . 367 3 . 189 3 . 2 3 . 533 3 . 133 4 Std . dev . ( no . correct / network ) 2 . 706 3 . 079 1 . 381 2 . 497 3 . 391 2 . 417 1 . 965 Median ( no . correct / network ) 4 . 5 4 3 3 1 2 . 5 4 Notes . N refers to the number of independent networks . Other statistics are in terms of the number of individuals per network . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2076 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . of Hypothesis 2 . For completeness , we model the median of the distribution of the dependent variable ( models 2 and 6 ) . Quantile regressions for count varia - bles ( all of the models ) are \ufb01 t through the method of Machado and Silva ( 2005 ) in the R package lqmm ( Ger - aci and Bottai 2014 ) . Regression models for the mean number of participants correct ( models 3 and 7 ) test Hypothesis 3 and were \ufb01 tted as quasi - Poisson generalized linear models to account for overdispersion from social in \ufb02 uence . Appendix D discusses model choice at greater length . Models 1 \u2013 4 use the hub - and - spoke as the refer - ence category ; models 5 \u2013 8 duplicate models 1 \u2013 4 but use the core - periphery network as the reference category . In each model , the only parameters included are the constant ( intercept ) and dummy variables corresponding to the network treatment . Hypothesis 4 is tested by means of the estimated parameters for the out core - periphery and in core - periphery networks . More on the mechanisms underlying performance in these directed networks can be found in the \u201c Mechanism Check \u201d section . Figure 6 . ( Color online ) Empirical Cumulative Distribution Functions of the Number of Individuals Correct per Trial costs benefits 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct ( of 9 ) c u m u l a t i v e pe r c en t age o f t r i a l s Isolates Complete clique ( a ) 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct ( of 9 ) c u m u l a t i v e pe r c en t age o f t r i a l s Isolates Complete clique Locally - clustered ( b ) 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct ( of 9 ) c u m u l a t i v e pe r c en t age o f t r i a l s Isolates Complete clique Hub\u2212and\u2212spoke ( c ) 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct ( of 9 ) c u m u l a t i v e pe r c en t age o f t r i a l s Isolates Complete clique Core\u2212periphery ( d ) Notes . Panel ( a ) shows only the isolates ( no social in \ufb02 uence ) and complete clique ( maximal social in \ufb02 uence ) conditions . The complete clique condition has more trials with few participants correct ( the \u201c costs \u201d of social in \ufb02 uence because of conformity pressure ) and more trials with many participants correct ( the \u201c bene \ufb01 ts \u201d of social in \ufb02 uence because of learning the right answer from peers ) . Panel ( b ) adds the locally clustered network , which is the third control condition , which has less cost and less bene \ufb01 t than the complete clique but maintains the tradeoff between them . Panel ( c ) shows that the hub - and - spoke network produced the bene \ufb01 ts of social in \ufb02 uence without the costs . Panel ( d ) shows that the core - periphery network produced the bene \ufb01 ts with reduced costs . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2077 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . The hub - and - spoke network dominated the three controls : it had fewer very poor trials ( with the major - ity wrong ) than the complete clique and locally clus - tered networks ( model 1 ) and more very good trials ( with the majority correct ) than the isolates ( model 4 ) . The average number of participants correct ( Table 2 and model 3 of Table 3 ) was higher for the hub - and - spoke ( 4 . 7 of 9 ) than for the isolates ( 3 . 19 ) , the complete clique ( 3 . 53 ) , and the locally clustered network ( 3 . 2 ) . Quantile regression results for the core - periphery network were more equivocal . Panel ( d ) of Figure 6 shows an effect similar to that of the hub - and - spoke but with several more trials with few people correct because of the presence of a central cluster . The core - periphery network thus outperformed the isolates and the locally clustered network in disseminating the correct answer ( 75th percentile , model 8 ) and the mean ( model 7 ) , but it was only directionally consis - tent with the hub - and - spoke network and not statisti - cally signi \ufb01 cantly better with respect to getting stuck on the wrong consensus ( model 5 ) . Given that the core - periphery network is less centralized than the hub - and - spoke , a smaller effect is expected and still consistent with our hypotheses . All that said , the core - periphery has the same proportion of trials with zero or one person correct as the isolates condition , consis - tent with a \ufb01 nding of minimal costs because of social in \ufb02 uence ( low probability of sticking to an inferior consensus ) . Figure 7 documents the size of the causal effects of the treatments , relative to the controls , in terms of \u201c common language effect size , \u201d which expresses the probability that the treatment would perform better than the controls if one trial in the treatment condition and one in a control condition were run ( or picked at random from the data ) . 6 It thus accounts for variance in outcomes for control and treatment networks . Compared with the isolates , locally clustered net - works , and complete cliques , the hub - and - spoke would perform better 60 % , 62 % , and 59 % of the time , respectively , and the control network would perform better 29 % , 30 % , and 33 % of the time , respectively . Compared with the isolates , locally clustered net - works , and complete cliques , the core - periphery would perform better 56 % , 56 % , and 52 % of the time , respectively , and the control network would perform better 35 % , 35 % , and 36 % of the time , respectively . Additionally , the distribution of performance of the hub - and - spoke network is \ufb01 rst - order stochastically dominant over that of the locally clustered network , meaning that the dominant distribution should be chosen by all decision makers ( who want high per - formance ) because for any level of performance , x , the probability that the performance of the hub - and - spoke Table 4 . Number of Participants Correct at End of Trial Dependent variable 25th percentile Median Mean 75th percentile 25th percentile median mean 75th percentile ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) ( 7 ) ( 8 ) Hub - and - spoke 0 . 099 0 . 083 0 . 074 0 . 068 ( 0 . 566 ) ( 0 . 354 ) ( 0 . 149 ) ( 0 . 247 ) Core - periphery \u2212 0 . 341 \u2212 0 . 046 \u2212 0 . 074 0 . 058 ( 0 . 536 ) ( 0 . 268 ) ( 0 . 149 ) ( 0 . 196 ) Isolates 0 . 036 \u2212 0 . 327 \u2212 0 . 388 * * * \u2212 0 . 502 * * * 0 . 133 \u2212 0 . 261 \u2212 0 . 314 * * \u2212 0 . 466 * * ( 0 . 165 ) ( 0 . 222 ) ( 0 . 126 ) ( 0 . 187 ) ( 0 . 555 ) ( 0 . 163 ) ( 0 . 129 ) ( 0 . 190 ) Locally clustered \u2212 0 . 602 * * \u2212 0 . 399 \u2212 0 . 384 * * \u2212 0 . 323 * \u2212 0 . 513 \u2212 0 . 329 * \u2212 0 . 311 * \u2212 0 . 291 ( 0 . 295 ) ( 0 . 248 ) ( 0 . 162 ) ( 0 . 193 ) ( 0 . 607 ) ( 0 . 197 ) ( 0 . 165 ) ( 0 . 197 ) Complete clique \u2212 0 . 947 * * * \u2212 0 . 863 \u2212 0 . 285 * 0 . 024 \u2212 0 . 854 \u2212 0 . 898 * \u2212 0 . 212 0 . 070 ( 0 . 266 ) ( 0 . 658 ) ( 0 . 157 ) ( 0 . 186 ) ( 0 . 593 ) ( 0 . 520 ) ( 0 . 160 ) ( 0 . 189 ) Out core - periphery \u2212 0 . 524 * \u2212 0 . 468 \u2212 0 . 405 * * \u2212 0 . 467 * * \u2212 0 . 414 \u2212 0 . 388 \u2212 0 . 332 * * \u2212 0 . 429 * * ( 0 . 277 ) ( 0 . 519 ) ( 0 . 163 ) ( 0 . 194 ) ( 0 . 598 ) ( 0 . 500 ) ( 0 . 166 ) ( 0 . 197 ) In core - periphery 0 . 110 \u2212 0 . 095 \u2212 0 . 161 \u2212 0 . 257 0 . 177 \u2212 0 . 020 \u2212 0 . 088 \u2212 0 . 214 ( 0 . 231 ) ( 0 . 242 ) ( 0 . 152 ) ( 0 . 192 ) ( 0 . 581 ) ( 0 . 189 ) ( 0 . 155 ) ( 0 . 195 ) Constant 0 . 891 * * * 1 . 468 * * * 1 . 548 * * * 1 . 867 * * * 0 . 795 1 . 403 * * * 1 . 474 * * * 1 . 831 * * * ( 0 . 140 ) ( 0 . 218 ) ( 0 . 103 ) ( 0 . 178 ) ( 0 . 548 ) ( 0 . 157 ) ( 0 . 107 ) ( 0 . 182 ) Observations 270 270 270 270 270 270 270 270 Note . All coef \ufb01 cients are on the log scale . * p < 0 . 1 ; * * p < 0 . 05 ; * * * p < 0 . 01 . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2078 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . was at least x was greater than or equal to the proba - bility that the performance of the locally clustered net - work was at least x and , for some x , the performance of the hub - and - spoke network was strictly better . The core - periphery , although not differing as greatly in mean performance from the controls , is still \ufb01 rst - order stochastically dominant over the locally clustered and complete clique controls . With respect to the hub - and - spoke network , our experimental data are suf \ufb01 cient to reject the null for Hypothesis 1 ( reduced probability of getting collectively stuck on an inferior answer as measured by a test of proportions and 25th percentile regression , model 1 ) , Hypothesis 2 ( maintained spread of a superior answer as measured by a test of proportions and the 75th per - centile regression , model 4 ) , and Hypothesis 3 ( overall positive average effect , model 3 ) . For the less - centralized core - periphery network , we reject the null for Hypothe - sis 1 ( test of proportions ) , Hypothesis 2 ( test of propor - tions and model 8 ) , and Hypothesis 3 ( model 7 ) . Overall , given that quantile regression effect sizes are smaller but directionally consistent for the less - centralized net - work \u2014 and given our results from tests of propor - tions \u2014 we interpret the data overall as supporting Hypotheses 1 \u2013 3 . Tests of proportions show that the out core - periphery was signi \ufb01 cantly worse than the isolates in terms of the costs of social in \ufb02 uence and worse than the complete clique in terms of the bene \ufb01 ts . It also had a substantially and statistically signi \ufb01 cantly lower mean and 75th percentile than either of the two undirected centralized treatments ; we , therefore , reject the null for Hypothesis 4 ( see results from tests of proportions and parameter estimate for out core - periphery in models 3 , 4 , 7 , and 8 ) . Mechanisms We have shown that centralized networks can adapt better to new information and spread the correct solu - tion . We also posited certain mechanisms : speci \ufb01 cally , that the inability of peripheral nodes to communicate directly with each other could reduce conformity pressure and allow those individuals to adapt to new information and that the privileged position of the central nodes could allow them to spread good ideas that arose in the periphery . We now present step - by - step evidence of those mechanisms . Boxes A \u2013 C of Figure 2 identify the mechanisms implied by our hypotheses : that is , a more detailed account of what we should expect to observe in our data if the hypothesized outcomes do , in fact , occur . Note that although these mechanisms were part of the logic motivating our hypotheses at the whole - network level , they were never articulated as separate hypotheses prior to running the experiment , although we did intentionally collect detailed data for the purpose of exploring them . Note also that because results at the posi - tional or individual levels are endogenously correlated , an experiment like ours cannot show them as causal , and they should be treated as descriptive correlations . Mechanism A : Peripheral Nodes Are Relatively Independent We begin by providing evidence that peripheral nodes feel less conformity pressure to stay with the consensus solution than those in other network posi - tions . Figure 8 plots the probability of having the same answer as one \u2019 s neighbors for the correct answer ( upper panels ) and for the red herring ( lower panels ) . Peripheral positions covary less with their neighbors than other positions do . Speci \ufb01 cally , the left side of Figure 7 . ( Color online ) Visualization of \u201c Common Language \u201d Effect Size : Choosing One Random Trial of a Centralized Network ( Left Panel : Hub - and - Spoke , Right Panel : Core - Periphery ) and One Random Trial of a Control Network Structure , What Are the Chances That the Treatment Will Be Better Than , Equal to , or Worse Than the Control in Terms of the Number of Participants Correct at the End of the Trial ? 0 . 33 0 . 08 0 . 59 0 . 3 0 . 08 0 . 62 0 . 29 0 . 11 0 . 6 complete clique locally\u2212clustered isolates 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 probability of result c on t r o l hub\u2212and\u2212spoke better equal control better p ( hub\u2212and\u2212spoke network better than controls ) 0 . 36 0 . 12 0 . 52 0 . 35 0 . 09 0 . 56 0 . 35 0 . 09 0 . 56 complete clique locally\u2212clustered isolates 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 probability of result c on t r o l core\u2212periphery better equal control better p ( core\u2212periphery network better than controls ) Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2079 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . Figure 8 . Probability of Having the Same Answer as Neighbors by Position During Phase 3 of the Experiment Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2080 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . each panel shows that peripheral nodes are more likely than other positions to adopt a given answer ( cor - rect or not ) , even if no neighbor has adopted it . Simi - larly , they are less likely than other positions to adopt an answer that all of their neighbors have adopted . Figure 8 shows that both the number and share of neighbors with a given answer matter in deriving the probability of adopting a matching answer , as pre - dicted by others ( Stasser and Davis 1981 , Centola and Macy 2007 ) and shown in Figure 1 . For example , a member of the periphery of the hub - and - spoke net - work has only a single neighbor ( the central node ) ; this is 100 % of what that person can see . Thus , that single answer has a higher weight than one of several answers visible to a position with multiple neighbors . Conversely , when a given share of neighbors adopts an answer , the conformity pressure increases with the actual number of neighbors ; for example , when all of one \u2019 s eight neighbors agree , the probability of match - ing them is 85 % \u2013 100 % , whereas the probability of matching one \u2019 s only neighbor is approximately 55 % . Mechanism B : The Core Sees More Correct Answers , Even When Wrong Itself If peripheral nodes are less subject to conformity pres - sure , then the core should see more diverse solutions . The upper panels in Figure 9 visualize the moving average of the number of unique answers visible to individuals in each network position over time . Although the general trend is similar across positions , the core nodes in the hub - and - spoke and core - periphery conditions saw more unique answers on average ( 3 . 12 and 2 . 65 , respectively ) than did individuals in the complete clique , locally clustered , and isolates con - ditions ( 2 . 44 , 1 . 58 , and 0 , respectively ) . Isolates are not shown in the \ufb01 gure because , by construction , they saw no peer answers . The \u201c Mechanism Check \u201d section dis - cusses results for the directed core - periphery networks . Beyond seeing more diverse answers , core individuals saw more correct answers , even when they were wrong themselves . Of participants with the wrong answer reg - istered in the \ufb01 nal phase , those in the core position of the hub - and - spoke saw 0 . 267 more correct answers among neighbors than those in the complete clique , whereas those in the core of the core - periphery network saw 0 . 203 more ( p (cid:2) 0 : 008 and 0 . 011 , respectively ) . Com - paring core nodes of centralized networks with nodes in the locally clustered network , the core node in the hub - and - spoke saw 1 . 074 more correct alters , and core nodes in the core - periphery network saw 1 . 009 more . Mechanism C : The Core Learns from the Periphery Figure 8 demonstrates that those who saw more correct answers were more likely to be correct themselves . Given the nature of our data , we cannot conclusively show who learned from whom , but to connect mecha - nism C to the main result , we can show that the effects occurred in the sequence described : core nodes see the correct answer in the periphery and adopt it , and then it spreads farther to other peripheral nodes . Put another way , ( a ) individuals in the core adopted the correct answer after some in the periphery did and before others in the periphery did , and ( b ) more participants were correct in those trials in which the core adopted the correct answer . To assess ( a ) , we conducted a random permutation test of the order in which individuals in a given trial adopted a correct answer that they stuck with until the end . We randomly permuted the sequence of adopting the correct answer \u2014 coding anybody who did not end the trial with the correct answer as zero and everyone else with the whole number indicating when in the sequence they adopted the correct answer \u2014 in groups of 30 trials . We repeated this process 10 , 000 times to create a null distribution to which to compare the real data . The central node in the hub - and - spoke was statistically signi \ufb01 cantly ( p (cid:2) 2 \u00d7 10 \u2212 4 ) more likely to be the third person in the trial to adopt the correct answer , and members of the core in the core - periphery network were signi \ufb01 cantly ( p (cid:2) 0 . 01 ) more likely to be the fourth . As for point ( b ) , in the hub - and - spoke network , when the central node adopted the correct answer and maintained it to the end , 64 % of peripheral nodes that had not yet adopted the correct answer when the central node did went on to do so as compared with 29 % for cases in which the central node had not yet adopted the correct answer ( the difference is statisti - cally signi \ufb01 cant at p (cid:2) 10 \u2212 6 ) . In core - periphery net - works , the probability that a peripheral node is correct at the end goes up sharply if two or three core nodes have already adopted the correct answer . The proba - bility of completing the trial with the correct answer is 13 % if no core nodes have already adopted it but 36 % if two core nodes have and 57 % if three core nodes have ( p (cid:2) 0 . 022 and 2 . 92e - 04 , respectively ) . Figure 10 illustrates both of these \ufb01 ndings for the hub - and - spoke network . Mechanism Check The individual - level data discussed in reporting mec - hanisms A \u2013 C in Figure 2 are endogenously correlated within each trial . Thus , although we have detailed de - scriptive data , they do not support causal inferences . However , data from the directed core - periphery network treatments can be studied to increase the plausibility of those causal mechanisms , because those treatments exo - genously manipulate key variables in the mechanism diagram . Because the out core - periphery structure does not allow the core to see the periphery ( and therefore Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2081 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . Figure 9 . ( Color online ) Core Nodes See Diverse Answers , Including Correct Answers , Even When They Are Wrong Themselves U n i que s o l u t i on s C o rr e c t a l t e r s Notes . ( Upper panels ) Moving average of the number of unique answers visible to each position over the course of the experiment . ( Lower panels ) Box plot of the number of correct answers visible to individuals who did not have the correct answer registered during the last minute of the experiment . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2082 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . communication is only one way ) , all statements logi - cally downstream from \u201c core sees periphery \u201d ( includ - ing all of mechanisms A , B , and C ) should be false for that treatment condition ( per Hypothesis 4 ) . For mecha - nism A , as we argued , the core \u2019 s exposure to the pos - sibly differing answers in the periphery lessens the conformity pressure on the periphery ( because of the self - reinforcing nature of conformity ) , so when the core cannot see the periphery , we should not expect the periphery to be as independent . Figure 8 con \ufb01 rms that . As for mechanism B , the core , unable to see the periphery , cannot learn from it . Thus , the core should see a less diverse set of answers \u2014 including fewer cor - rect answers \u2014 than it would in the hub - and - spoke net - work or the ( undirected ) core - periphery network . Figure 9 con \ufb01 rms that . For mechanism C , individuals in the core of the out core - periphery should , therefore , be less likely to be correct than individuals in the core positions of undirected centralized networks . Indeed , we \ufb01 nd that they were correct 32 % of the time compared with 57 % for the core of the hub - and - spoke network and 51 % for the core of the undirected core - periphery network ( p - val - ues from \u03c7 2 tests are 0 . 029 and 0 . 014 respectively ( esti - mated on those who completed the trial ) ) . With fewer trials in which members of the core choose the correct answer , there are fewer in which the core spreads the correct answer to the periphery ( see Figure 11 and results from Table 4 ) , indicating little social learning . The in core - periphery network had a different pat - tern of results . The core could see the periphery but not vice versa . The mechanism diagram , therefore , predicts that the core would see many correct answers among the independent peripheral nodes . Indeed , because peripheral individuals in the in core - periphery had fewer network neighbors than peripheral individu - als in other networks \u2014 or none at all \u2014 they were even more independent ( see Figure 9 , lower panels ) . Individ - uals in the core , like those in the undirected centralized networks , should , therefore , be correct more frequently ; in fact , they were 49 % of the time . Despite these struc - tural bene \ufb01 ts , the core \u2019 s correct answers could not spread as widely , because three peripheral individuals had no incoming information from the core and three only had one link from a single member of the core . Figure 6 and Table 4 show that although results were not signi \ufb01 cantly different from those for decentralized networks , there were fewer trials with many correct individuals , and the 75th percentile of the number of participants correct per trial was only 5 compared with 6 . 47 for the hub - and - spoke and 6 . 24 for the undirected core - periphery network . Discussion and Conclusion Prior research has found centralization harmful for collective problem - solving and collective intelligence because it impedes the integration of ideas and infor - mation held by peripheral individuals . We argue \u2014 and our experimental results show \u2014 that these same effects of centralization nevertheless have bene \ufb01 ts for tasks requiring collective adaptation : that is , for solving problems that require a collective shift from one solution to another in response to a shifting environment . These bene \ufb01 ts \ufb02 ow from the way social in \ufb02 uence is shaped by two de \ufb01 ning features of highly centralized networks , namely that peripheral nodes ( a ) are not Figure 10 . ( Color online ) Illustration of the Time Course of Final Correct Answers for Hub - and - Spoke Network Trials core incorrect core correct 300 600 900 123456789 123456789 time of final correct answer c u m u l a ti v e nu m b e r c o rr ec t Position : periphery core Notes . If the central node is correct , it has typically adopted the correct answer after it had already been found in the periphery and then spreads that answer widely . ( Upper panel ) Trials in which the central node adopted the correct answer . ( Lower panel ) Trials in which the central node did not \ufb01 nish with the correct answer . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2083 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . ( densely ) interconnected with each other and ( b ) are connected with central nodes ( the core ) . Peripheral nodes are , therefore , relatively independent because they do not in \ufb02 uence each other , whereas the core nodes that do in \ufb02 uence them are , in turn , exposed to many \u2014 possibly differing \u2014 solutions . This lack of con - formity pressure makes the periphery more adaptable to new information and less likely to retain a consen - sus ( but inferior ) solution . Core nodes are , therefore , exposed to the diverse solutions of many of these inde - pendent peripheral nodes and thus can learn from the nodes with superior solutions . Once the core nodes adopt a superior solution , it spreads quickly to the periphery . Thus , by limiting conformity pressure but retaining ef \ufb01 cient connectivity , centralization struc - tures use social in \ufb02 uence to promote learning good solutions without undue pressure to stay on a current inferior consensus . However , for the bene \ufb01 ts of cen - tralization to occur , there must be two - way communi - cation between core and periphery . Boundary Conditions and Limitations Our experimental design , although enabling causal inference , does come with important limitations too . Figure 11 . ( Color online ) Empirical Cumulative Distribution Functions of the Number of Individuals Correct per Trial 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct c u m u l a t i v e pe r c en t o f t r i a l s Isolates Complete clique Out core\u2212periphery ( a ) 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct c u m u l a t i v e pe r c en t o f t r i a l s Isolates Complete clique In core\u2212periphery ( b ) 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct c u m u l a t i v e pe r c en t o f t r i a l s Core\u2212periphery Out core\u2212periphery ( c ) 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 number correct c u m u l a t i v e pe r c en t o f t r i a l s Core\u2212periphery In core\u2212periphery ( d ) Notes . Panels ( a ) and ( b ) show results for the out core - periphery and in core - periphery along with the isolates ( no social in \ufb02 uence ) and complete clique ( maximal social in \ufb02 uence ) conditions , similar to Figure 6 . Panels ( c ) and ( d ) show the directed core - periphery networks along with the undirected version . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2084 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . First , it was an intentional feature of our design \u2014 a randomized experiment \u2014 that the core was not made up of \u201c special \u201d individuals but instead was randomly drawn . Thus , our design separates the phenomenon of interest \u2014 network centralization \u2014 from confounds , such as hierarchy and power . However , this depar - ture from realism for the sake of valid causal inference attaches a caveat to our \ufb01 ndings . In practice , central individuals are not typically randomly assigned to such positions but rather gain them through experi - ence , in \ufb02 uence , or other qualities . Although our study is focused on advancing theory in a \ufb01 eld that has evolved through theoretical elaborations ( for another example , see Schilke et al . 2018 ) , it is not clear whether , in real life , central nodes would play the observed role of identifying and spreading good ideas generated by peripheral nodes or if instead would privilege their own ideas ( or indeed if peripheral nodes would decline to volunteer those very ideas out of deference ( Bernstein 2012 ) ) . Put another way , our results point to the necessity of humility among cen - tral individuals ; collective adaptation and perform - ance require them not only to identify and spread the periphery \u2019 s good ideas but also to encourage the periphery \u2019 s voice and speaking - up behaviors ( Detert and Burris 2007 , Morrison 2011 , Detert et al . 2013 ) . Second , our experiment tested centralization at the interindividual level , but in real organizations , cen - tralization can also be interteam or interunit . It is not clear from this experiment how centralization would operate at those other levels of analysis , especially if peripheral units were not separate individuals but rather , say , teams ( i . e . , centralization in a team of teams ) . Ugander et al . ( 2012 ) suggest that core individ - uals may treat teams analogously to individuals in terms of how they feel social in \ufb02 uence , but it may be that clustering within peripheral teams is suf \ufb01 cient to reduce peripheral nodes \u2019 freedom from conformity pressure ( our mechanism A ) . We leave this as an open question that would be very challenging to study with a randomized , controlled experiment . Third , our task allowed each participant to enter his or her own answer to the problem . This allows us to measure in \ufb02 uence among participants , which was a focus of our study and is a realistic representation of many problem - solving settings . However , certain types and scales of problem - solving involve all partic - ipants working on a single shared solution to a single instance of the problem ; for example , multiple pro - grammers contributing code to a single piece of soft - ware . ( Conversely , if the multiple contributions were enabled by code forks and pull requests , as on Github . com , that would again be the paradigm we study . ) It is unclear a priori how this difference in problem structure would change our results , but it is possible that working on a shared solution would increase conformity pressure ( or more generally , lock - in on a current solution ) , which could reduce or elimi - nate the bene \ufb01 ts we see . Implications for Research on Network Structure We contribute to the literature on network structure , in which a major topic of inquiry is how to leverage social in \ufb02 uence for improved collective problem - solving and collective intelligence . Prior work has pre - sented two distinct views of the effects of connectivity , and thus social in \ufb02 uence , in problem - solving ; it can harm performance ( the \u201c herding \u201d perspective ) or help it ( the \u201c learning \u201d perspective ) . In the former , connec - tivity is conceptualized as a threat , because like sheep in a herd , individuals follow the group rather than exercising individual judgment , leading often to suboptimal decisions . In the latter , connectivity is con - ceptualized as a bene \ufb01 t because good solutions to problems are hard for individuals to generate and most often need to be learned from peers . We show that \u2014 in the context of adaptation \u2014 centralization bypasses the trade - offs between these negative and positive aspects of connectivity and that it is possible to structure social in \ufb02 uence to produce collective learning ( from exposure to others \u2019 diverse ideas ) while minimizing herding ( from conformity pressure to stick to a current consensus ) . In essence , for prob - lems requiring adaptation ( speci \ufb01 cally , collective search of a rugged solution space ) , our \ufb01 ndings on centralization contradict the preexisting view that network structure implies a \u201c trade - off between main - taining the diversity necessary for obtaining high per - formance in a system and the rapid dissemination of high - performing strategies \u201d ( Lazer and Friedman 2007 ; see also Uzzi 1997 , Hansen 1999 ) . This conclu - sion joins , and strengthens , nascent related work \ufb01 nd - ing that intermittent interaction ( the on - off cycling of connectivity in a network over time ) can produce superior solutions in collective problem - solving ( Bern - stein et al . 2018 ) . It also connects with established work in innovation - related tasks showing that net - works that enable both diversity and learning ( includ - ing small world networks and other structures that combine local clustering with ties to different knowl - edge pools ) are most conducive to success for individ - ual nodes within the network ( Schilling and Phelps 2007 , Tortoriello 2015 , Ter Wal et al . 2016 ) ( without investigating , as we do , the collective success of the whole network ) . Our contributions to the literature on network struc - ture also include a novel perspective on centralization . Our experimental \ufb01 ndings run exactly opposite to the academic ( Lazer and Friedman 2007 , Becker et al . 2017 ) and popular ( Bernstein et al . 2016 ) narrative of centralization suppressing solution diversity . It is pos - sible that the everyday experience of minimal diversity Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2085 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . of thought in centralized structures is misattributed to centralization and actually is a result of other forces . Another possibility is that individual experience of diversity is lessened in centralized networks precisely because they isolate peripheral nodes from each other , thus maintaining the bene \ufb01 cial aggregate diversity required for adaptability . Also , of course , it is possible that this popular negative view of centralization is simply wrong . Implications for Research on Organizational Adaptation We contribute to scholarship on network - level adapta - tion \u2014 and therefore to the literature on exploration and exploitation in searching rugged solution spaces ( Lazer and Friedman 2007 , Mason and Watts 2012 ) \u2014 by clarifying how networks shape outcomes in \u201c complex \u201d problem - solving by creating the conditions for knowledge transfer without conformity pressure . The literature on network structure has investigated the distinction between \u201c simple \u201d and \u201c complex \u201d problem - solving ( Tushman 1979 , Mason and Watts 2012 ) but has achieved only limited consensus as to what makes a problem complex , with a consequent lack of coherent theory on how group or network structure helps solve complex problems ( Almaatouq et al . 2020 ) . Prior research has generally focused either on how networks shape the diversity of available knowledge or on conformity pressure , complex conta - gions , and norms . Bringing them together as we do in this work highlights the type of complexity that is amenable to network interventions \u2014 when good solu - tions are dissimilar to each other and thus require col - lective adaptation ( rather than only balancing the exploration and exploitation behavior of individuals ) , such as the adaptation requirements experienced by many organizations during the COVID - 19 pandemic . Implications for Research on Hierarchy vs . Centralization Third , we contribute to the literature on hierarchy and organization design . Empirically , centralization is con - founded by hierarchy ( Lin 1999 ) , which is the differ - entiation of individuals by power , rank , or status . Hierarchy and centralization are both common in organizations ( Tiedens and Fragale 2003 , Gruenfeld and Tiedens 2010 , Valentine 2018 ) and have been his - torically linked with the same generally negative effects . Both are generally seen to be good for co - ordination ( Galinsky et al . 2012 ) but bad for colla - borative performance ( Van der Vegt et al . 2010 , Greer et al . 2018 ) . Because of their generally confounded nature , hierarchy and social network centralization are unlikely to be distinguishable via observational data , perpetuating the view that they have similar implications for problem - solving . We , however , mani - pulate centralization without hierarchical differences among participants to focus on the in \ufb02 uence of com - munication network structure per se , rather than of power or status hierarchy . We then \ufb01 nd that central - ization improves performance on tasks requiring adaptation \u2014 our primary contribution to the literature on hierarchy and organization design . However , our results may also imply that hierarchy \u2014 because of the centralized network structures it typically induces \u2014 is likely to be effective at adapting to a changing envi - ronment , so long as the leaders can bring themselves to listen to the periphery , encourage speaking - up behaviors , and then seek and spread good ideas to the rest of the organization ( Detert and Burris 2007 , Mor - rison 2011 , Detert et al . 2013 ) . Alternatively , it may be possible to achieve those bene \ufb01 ts for adaptability by keeping centralization while limiting hierarchy \u2014 as with the random individuals in our centralized networks . Implications for Research on Communication Technologies Fourth , we contribute to the literature on using tech - nology to structure communication . Social communi - cation technologies often create decentralized infor - mation \ufb02 ows ( Majchrzak et al . 2009 , Leonardi 2014 ) . Our results imply that such technology may , there - fore , have distinct advantages for integration of knowledge for problems that do not require adapta - tion but may be counterproductive for the problems that do require adaptation they are supposed to address . Connecting otherwise disconnected people through technology may result in \u201c recombinant innovation \u201d in the short run ( Leonardi 2014 ) , as peo - ple are able to bring together ideas already in the net - work . However , as our results suggest , it also causes people \u2019 s solutions to become more similar to each other , which limits adaptability ( including innovative - ness ) after the initial set of fruitful recombinations has been exhausted . Especially when much work is done virtually ( Neeley 2021 ) , organizational leaders may want to ensure that decentralized information \ufb02 ows do not undermine the ability to solve problems requir - ing adaptation . Conclusion : Centralization Improves Performance on Tasks Requiring Collective Adaptation Centralization has previously been found to impede the integration of knowledge and ideas held by indi - viduals in the network periphery . By designing an experimental protocol that manipulates when each piece of information can and cannot be found , we test Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2086 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . the effects of centralization in a different class of prob - lems . We show experimentally that centralization enhances collective adaptability in shifting environ - ments , so long as the central people in the network can both learn from and in \ufb02 uence the periphery , iden - tifying and spreading the good ideas discovered there . Acknowledgments The authors extend gratitude to the four editors and \ufb01 ve anonymous reviewers for investing considerable time to provide developmental , insightful , and enthusiastic com - ments throughout the review process , even during a pan - demic . The authors thank Akshaya Varghese and Cara Mazzucco for their research assistance , the participants at the Organization Science Special Issue Conference for helpful input at a critical stage in this paper \u2019 s develop - ment , and the Workshop on Information Systems and Economics program committee for nominating an earlier version of this manuscript for best paper . The novel Net - LabExperiments . org platform would never have been built without the dedication of the Harvard Business School Research Computing Services ( RCS ) team , particularly Troy Adair , Bob Freeman , and Gabe Mansur . Appendix A . Nodes in Centralized Structures Are Less Subject to Conformity Pressure Based on the assumptions documented in Figure 1 about the relative amount of in \ufb02 uence that people feel as a con - sequence of the number and share of their neighbors hav - ing an alternative opinion as well the assumption that , in each time step , people have a small random chance of changing their opinions , we simulated a random process of social in \ufb02 uence on the network structures we studied here . Our simulation proceeded as follows : Algorithm A . 1 ( Simple Simulation of Social Influence on Networks ) each node starts with opinion A at t (cid:2) 1 ; possible opin - ions are { A , B , C } ; s \u2190 0 : 04 1 + e \u2212 1 : 5 ( numNeighbors \u2212 0 : 9 ) # vector of node susceptibilities by number of neighbors ; r \u2190 0 : 001 # probability of switching to random opinion independent of in \ufb02 uence at each time step , t ; for t \u2190 2 to 500 do for O \u2208 { A , B , C } do in \ufb02 uence of opinion O \u2190 1 1 + e \u2212 10 ( shareOfNeighborsWithOpinion \u2212 : 5 ) ; chance of switching to O because of in \ufb02 uence \u2190 s \u2217 influence of opinion O sum ( influence of all opinions ) (cid:1) (cid:3) ; end determine opinion for time step t through random draw , according to probabilities calculated above ; end With 1 , 000 simulated trials of 500 time steps each , we found that centralized networks retained an initial consen - sus less than other networks . Speci \ufb01 cally , members of a complete clique had opinion A ( the initial consensus ) 92 . 3 % of the time , and members of the locally clustered network had opinion A 79 . 7 % of the time , whereas peripheral nodes in the hub - and - spoke network and core - periphery network had opinion A only 56 . 6 % and 71 . 6 % of the time , respectively . Each peripheral node in a centralized network is exposed to the same in \ufb02 uences from the center of the net - work . However , the in \ufb02 uence is not very strong because ( a ) central nodes are few in number and ( b ) central nodes are likely to be exposed to differing opinions in the periphery and themselves have a higher probability of changing opinions . Appendix B . Additional Details of Subject Pipeline Participants were eligible to advance to stage 2 if they could complete stage 1 by ( a ) following instructions to operate the search and answer functions and ( b ) deducing the correct answer to the stage 1 problem from the six clues that could be found ( details on the logical task are in this appendix ) and entering it into the answer \ufb01 eld . Participants were eligible to advance to stage 3 ( the main trial ) if they could complete stage 2 by ( a ) deducing the correct answer to the stage 2 problem , a slightly longer task completed synchronously with two other partici - pants , and ( b ) answering an attention check question cor - rectly . Participants who failed to pass a stage 1 or stage 2 trial were allowed to try again up to four times before becoming disquali \ufb01 ed from further attempts . The participant pipeline exhibited a steep drop - off at each stage . Approximately 43 % of the stage 1 ( instruc - tional ) trials were passed ; of these , 74 % opted in to a stage 2 trial ( thus the number of people opting in to the stage 2 trial was 43 % \u00d7 74 % \u2248 32 % of those who had opted in to stage 1 trials ) . Approximately 80 % of second - stage trials were passed ; of these , 78 % opted in to the main stage 3 trial . Overall , only 20 % of participants progressed all the way through a stage 3 trial , which we attribute to effective \ufb01 ltering of attentive , interested , and capable participants . We did not collect any data that would allow us to draw conclusions about the representativeness of our experimental sample . Research has shown that MTurk workers are generally representative of the U . S . popula - tion , although somewhat more politically liberal ( Levay et al . 2016 ) and with somewhat higher negative affect and lower social engagement ( McCredie and Morey 2019 ) . That said , they are more representative of the general population than university subject pools ( Berinsky et al . 2012 ) , and results of experiments run on MTurk match results from of \ufb02 ine experiments ( Horton et al . 2011 ) . It is dif \ufb01 cult to say how well our sample represents the broader population of members of organizations or , for that matter , to de \ufb01 nitively identify the attributes of such a population . We would argue , however , that our multi - stage pipeline , which involved solving complex and uncertain problems with others and appearing at a pre - scheduled time ( for the synchronous start of the experi - ment ) , selected participants for the main trial who are likely to be more similar to typical members of organiza - tions than the broader population of MTurk workers . Online labor market workers review the \u201c requesters \u201d posting tasks to help identify reputable employers ( Gray Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2087 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . et al . 2016 ) ; our experiment retained a strongly positive reputation . On Turkerview . com , our experiments had an average reported overall wage of $ 11 . 11 per hour , which includes pay for time in the waiting room and in the three stages of our experiment . Our experiments received the overall rating of \u201c Workers feel this requester pays fairly \u201d as well as the \u201c good communication , \u201d \u201c approves quickly , \u201d \u201c no rejections , \u201d and \u201c no blocks \u201d tags . We used MTurk \u2019 s quali \ufb01 cations functionality ( assigning a worker a custom quali \ufb01 cation upon successfully completing a task to allow him or her to see further tasks in a series ) to qualify and disqualify workers for speci \ufb01 c tasks . We never blocked workers or rejected their work , as these actions can seri - ously harm their ability to qualify for other work on MTurk . In addition to building on the experience of other re - searchers , many details of our subject pipeline were re - \ufb01 ned through a piloting process conducted in tandem with debugging and testing our new experimental plat - form , such that we could improve participants \u2019 under - standing of the task and platform and calibrate the degree to which our task was engaging and paid well enough . The improvements we made during the pilot phase , using feedback from pilot participants , to make the task clear , engaging , and worthwhile helped us ( a ) attract enough participants and ( b ) keep their attention focused on the experiment . Appendix B . 1 . Details of the Stage 1 Trial Stage 1 was a single - person trial and served to teach partici - pants how to use the basic functions in the experimental interface ( speci \ufb01 cally , searching for clues and entering their answers ) and also to ensure they could both perform basic logical deduction and were paying suf \ufb01 cient attention to do so . We provide details on the logical task here . Participants received the following clue as part of the instructions : \u201c The murderer is either Mr . Smith , Mr . Lee , or Ms . White . The murder weapon was poison . \u201d The instructions continue : Other clues must be found by using the search bar to search for keywords . DO IT NOW : Use the search bar to find more information about each suspect . Tip # 1 : searches should be single words , so type \u201c Smith \u201d instead of \u201c Mr . Smith \u201d . Tip # 2 : you can search the same keyword more than once to find more clues . Tip # 3 : some clues contain useful information and some don \u2019 t . The clues that could be found with the search tool were as follows : \u2022 Mr . Smith was out of town at the time of the murder . \u2022 Mr . Smith was very sad to learn about the murder . \u2022 Ms . White was with Mr . Smith at the time of the murder . \u2022 Ms . White grew up in California . \u2022 Mr . Lee \u2019 s \ufb01 ngerprints were found at the scene of the crime . \u2022 Mr . Lee had poison at his house that matched the poison used in the murder . To pass the stage 1 trial , participants had to correctly identify \u201c Mr . Lee \u201d as the culprit . Stage 1 trials were offered to all MTurk workers . Approximately 40 % of these trials were passed to Stage 2 . Appendix B . 2 . Details of the Stage 2 Trial Stage 2 was a synchronous three - person trial , and it served to teach participants how to use the social features of the experimental interface , including sharing clues with their peers with or without free - text annotations , as well as using free - text messages without sharing clues . The logical puzzle was similar in dif \ufb01 culty to that in stage 1 . At the beginning of the trial , each participant received the following message : \u2022 Message from headquarters : Mr . Walker was found strangled in his bed . Additionally , at the beginning of the session , each par - ticipant was given one of the following clues that was not given to their peers ( i . e . , each of the three partici - pants received a different one of the following clues ) : \u2022 Mr . Walker recently had romantic affairs with Mr . Brown \u2019 s wife and Mr . Taylor \u2019 s wife . \u2022 Mr . Walker had recently insulted Mr . Martin and Mr . Taylor in public . \u2022 Mr . Walker had recently \ufb01 red Mr . Clark and Mr . Taylor from his company . The individualized clues each mention Mr . Taylor ( the real culprit ) as well as an additional suspect not men - tioned in their peers \u2019 clues . Thus , they each had informa - tion and the starting point for additional searches that could be shared with their peers , but this still put all experimental participants on even footing with respect to completing the task . Clues that could be found by search were as follows : \u2022 Mr . Brown was out of town on the day of Mr . Walker \u2019 s death because of a last - minute business trip . \u2022 Mr . Martin was in a car accident and was unconscious at the hospital at the time of Mr . Walker \u2019 s death . \u2022 Security camera footage shows that Mr . Clark spent the night in a hotel and did not leave until after Mr . Walker \u2019 s murder . \u2022 Mr . Taylor could not provide an alibi for the time of Mr . Walker \u2019 s death . \u2022 Mr . Taylor \u2019 s friends say that he hasn \u2019 t been himself recently . \u2022 Witnesses saw Mr . Taylor leaving Mr . Walker \u2019 s house after dark on the day of his death . Clues found by search exonerated all suspects except for Mr . Taylor . In addition to introducing the social features , stage 2 tri - als also acted to \ufb01 lter out participants in several ways . First , stage 2 trials required a synchronous start , which we achieved by emailing individuals who had passed the stage 1 trial and announcing start times ( we provided sev - eral start times through the day , announced times a day in advance , and kept the schedule consistent from day to day ) . Only individuals who would appear at previously scheduled start times could move on to the main trial ( which required nine people in the waiting room to begin ) . Second , to pass this stage and be quali \ufb01 ed to take part in the main trial , participants had to not only identify the correct suspect but also correctly answer an attention Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2088 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . check question ( identify the name of the victim , which is given in the \ufb01 rst shared clue , as well as all of the individ - ualized clues at the start of each session ) . To reduce the probability of participants copying and pasting answers posted elsewhere on the internet , we used multiple ver - sions of the clue set , each with different suspect and vic - tim names . Thus , this stage acted as an additional \ufb01 lter on atten - tion , ability to use the platform , ability to solve simple logical puzzles , and ability to join a trial with a synchro - nous start . Appendix B . 3 . Clues and Design of the Main Trial Problem This section lists the clues that were used in the main trial . Note that like the stage 2 trials , we used multiple versions of this problem set to reduce the possibility that participants could learn from any answers posted else - where on the internet . We randomized names and gen - ders of suspects , murder weapons , city names , location names , event names , and times of day ( while maintaining the internal logic of time - based alibis ) . The design of the problem draws heavily on the one used in Shore et al . ( 2015 ) , which also used keyword - based search and logically interrelated clues requiring interpretation and deduction . We designed the three - phase structure of the information set for the present experiment with the intention of modeling a shifting information environment . The main trial was 15 minutes long and consisted of three effective phases , each 5 minutes long . At the beginning of the trial , all participants received the following message : \u2022 Mr . Hall is missing and presumed dead . Your task is to identify the murderer . Mr . Hall was scheduled to be at a political fundraiser , a major project launch , and a confer - ence in the days around his disappearance . The name of the victim ( Mr . Hall ) as well as the names of the three events listed ( political fundraiser , major project launch , and conference ) provide initial keywords for search - ing . In this \ufb01 rst phase , one suspect \u2019 s name ( Mr . Brown ) is associated with two motives and appears to have multiple connections with the events around the victim \u2019 s death . Clues that could be found by search starting at the beginning of the trial were the following : \u2022 The political fundraiser was scheduled in Chicago . \u2022 The major project launch was scheduled in Honolulu . \u2022 The conference was scheduled in London . \u2022 The conference apparently went well . \u2022 Two witnesses reported that Mr . Hall extorted large sums of money from Ms . Moore , Ms . Jones , and Mr . Brown . \u2022 Ms . Taylor told investigators that Mr . Hall cut Mr . Brown , Mr . Davis , and Ms . Taylor out of a lucrative busi - ness deal . \u2022 Ms . Miller reports that Ms . Miller and Mr . Brown were involved in business with The Panoid Group . \u2022 Ms . Miller reports that The Panoid Group was involved in secret negotiations with Mr . Hall . \u2022 Mr . Davis and Ms . Taylor were con \ufb01 rmed to be in Lon - don since before the victim went missing . \u2022 Mr . Davis and Ms . Taylor were traveling together . \u2022 Mr . Brown , Mr . Davis , and Ms . Taylor have been having frequent private meetings recently . \u2022 Anonymous sources say Ms . Taylor was interested in undermining the conference . \u2022 Ms . Miller claims to have been in Chicago since before Mr . Hall went missing . \u2022 Ms . Moore and Ms . Jones were con \ufb01 rmed to be in Hono - lulu around the time of Mr . Hall \u2019 s disappearance . \u2022 Mr . Brown and Mr . Lewis were con \ufb01 rmed to be in Hon - olulu around the time of Mr . Hall \u2019 s disappearance . \u2022 Ms . Miller and Mr . Brown were friends . \u2022 Search of Ms . Miller \u2019 s home revealed that Ms . Miller owned cyanide and a bolo knife . \u2022 Search of Ms . Moore \u2019 s home revealed that Ms . Moore owned a switchblade . \u2022 Search of Ms . Jones \u2019 s home revealed that Ms . Jones owned a bolo knife . \u2022 Search of Mr . Brown \u2019 s home revealed that Mr . Brown owned a bolo knife . \u2022 Search of Mr . Davis \u2019 s home revealed that Mr . Davis owned a bolo knife . \u2022 Search of Mr . Davis \u2019 s home revealed that Mr . Davis owned a 9 - mm handgun . \u2022 Search of Mr . Lewis \u2019 s home revealed that Mr . Lewis owned a 9 - mm handgun . \u2022 Search of Mr . Brown \u2019 s home revealed that Mr . Brown owned a 9 - mm handgun . \u2022 There is no evidence that Ms . Taylor owned any poten - tial murder weapons . \u2022 The victim , Mr . Hall , had many enemies . From the participants \u2019 points of view , other than receiv - ing messages , there was no obvious division of the trial period into phases . At the \ufb01 ve - minute mark , all partici - pants received the following messages : \u2022 Mr . Hall \u2019 s body was discovered at the Harbor Hotel in Honolulu , after the major project launch . \u2022 Security camera footage shows that Mr . Hall went directly to the Harbor Hotel after the major project launch and never left . \u2022 Initial examination indicates that the time of death was 8 pm . After the \ufb01 ve - minute mark , it is possible to \ufb01 nd clues that provide alibis to all suspects ( including Mr . Brown , the leading suspect from phase 1 ) other than the true cul - prit . Three suspects can be ruled out immediately from the shared information that the murder occurred in Hono - lulu ( Davis , Miller , and Taylor were all in other locations ) , and three additional suspects have alibis for the time of the murder ( Moore , Jones , and Brown were at the busi - ness meeting at the time of death ) . Thus , by process of elimination , it is possible to \ufb01 nd the correct answer dur - ing this phase . Clues that could be found by search after the \ufb01 ve - minute mark were the following : \u2022 Ms . Jones , Mr . Brown , and Mr . Lewis were present at the major project launch , according to a witness . \u2022 Ms . Miller was present at the political fundraiser . \u2022 Ms . Taylor did not attend the conference , according to an alibi witness . \u2022 Mr . Davis attended the conference . \u2022 Ms . Moore did not attend the major project launch , according to an alibi witness . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2089 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . \u2022 The Panoid Group \u2019 s business meeting occurred at the Grand Hotel , which is a 35 - minute drive from the Harbor Hotel . \u2022 The Panoid Group \u2019 s business meeting began at 6 and ended around 9 pm . \u2022 Ms . Moore , Ms . Jones , and Mr . Brown attended The Panoid Group \u2019 s business meeting after the major project launch . \u2022 Mr . Brown and Mr . Lewis attended The Supil Company \u2019 s cocktail hour after the major project launch . \u2022 The Supil Company \u2019 s cocktail hour began at 10 and ended around 12 pm . \u2022 The Supil Company \u2019 s cocktail hour occurred at the Gar - den Hotel , which is a 45 - minute drive from the Harbor Hotel . \u2022 Mr . Brown traveled directly between The Panoid Group \u2019 s business meeting and The Supil Company \u2019 s cocktail hour . \u2022 Mr . Brown was seen speaking to a friend at 7 : 50 pm at The Panoid Group \u2019 s business meeting . \u2022 The Panoid Group often hosted VIP parties . \u2022 Mr . Lewis was hoping to land a big contract with The Supil Company . Finally , at the 10 - minute mark , all participants received the following clue : \u2022 Autopsy results show that the murder weapon was a 9 - mm handgun . This \ufb01 nal clue focuses attention on three suspects who were in possession of a matching murder weapon . Two of those three can be logically ruledout , leavingonlythetrue culprit . Appendix B . 4 . Payment In designing our payment scheme , we worked to bal - ance the need to incentivize attentive participation to gather valid data against the desire to pay participants a fair wage for their time . In addition to using the pipeline to \ufb01 lter out inattentive participants , our solution was to have a three - part pay - ment structure . We provided a guaranteed but modest ( $ 0 . 40 ) base payment ; we paid $ 0 . 14 per minute that they had the correct answer to a simple attention check ques - tion registered . Finally , we paid $ 0 . 14 per minute that they had the correct answer to the murder mystery entered . The attention check ( which simply asked the name of the murder victim ) could be answered based on the \ufb01 rst \u201c message from headquarters \u201d clue provided to everyone at the start of the trial , and thus it accounted for the majority of subject payment , regardless of whether they eventually solved the mystery . If participants took 30 seconds to enter the answer to the attention check and did not solve the mystery , they would earn approximately $ 8 . 58 per hour , assuming that they spent 15 minutes on the trial itself plus 2 minutes reading the consent form and waiting for others to be ready to join the trial . In practice , our median hourly wage for participants who completed a trial and provided an answer to the attention check problem was $ 8 . 47 , assuming a total of 17 minutes spent on the task . That said , participants varied , and the 10th and 90th percentile wages were $ 6 . 52 and $ 10 . 41 , respectively . Prior to the trial starting , they received an email stating the base payment and bonus payment rates , and in the trial instructions and consent page , participants were given as accurate of an assessment of payment as we could generate given pilot testing data . The longer you have correct answers entered in the computer , the higher the payment . The base payment is 40 cents , but the majority of the compensation will be bonus payments for correct answers . Participants who are paying attention earn on average approxi - mately $ 8 per hour , and most participants earn between $ 5 and $ 12 . 25 per hour . Following Mason and Suri ( 2012 ) , we used a waiting room to facilitate synchronous start to multiperson trials . Prior to each trial beginning , we assigned more partici - pants than necessary to a trial , with the understanding that some workers either do not consent to a trial or have other reasons for not continuing , such as disinterest or inattention . If more than the required number of partici - pants consented to a trial and clicked the button indicating they are ready to begin , we randomly assigned the required number to begin the actual trial . All participants were paid $ 0 . 40 regardless of whether they consented and indicated they were ready to join ( an estimated $ 8 \u2013 $ 12 per hour equivalent ) . We paid an additional bonus of $ 0 . 40 to those who indicated they were ready to join but were not assigned to an active trial ( $ 16 \u2013 $ 24 per hour equivalent ) to encourage them to reenter the queue and eventually join a full trial . Appendix C . Results on Full Trials Only In the main results , we presented results \ufb01 tted on the full data without regard for whether participants dropped out of any of the trials . Here , we present regression results \ufb01 t - ted to only those data in which no participants dropped out of trials . Table C . 1 shows that results on this subset closely match the full data . Appendix D . Sample , Power , and Models Randomized , controlled experiments on grouped data present additional challenges with respect to sample size , power , and model selection . We document our decision making on these issues here . Appendix D . 1 . Sample Size Because we analyze outcomes at the collective ( nine - per - son network ) level , we had to obtain nine experimental participants for each independent observation . Accord - ingly , our biggest concern was having a suf \ufb01 cient sample size to draw reliable conclusions , and we decided to col - lect the largest balanced sample that we could given the limited amount of Mechanical Turk workers on the plat - form at any one time and the need to conduct synchro - nous experiments with nine workers at a time . Practically speaking , this meant that as we approached our \ufb01 nal sam - ple size , we were able to run fewer trials per day and had to tolerate an increasing rate of trials not starting because of the lack of nine quali \ufb01 ed participants at the trial \u2019 s start time . Our \ufb01 nal sample size was 30 trials per condition ; we collected a 31st trial for the hub - and - spoke and locally Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2090 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . clustered networks ; however , we made the determination that collecting further samples was no longer feasible , and we stopped data collection . Appendix D . 2 . Data Exclusions Results reported in the main text are based on an analy - sis of the \ufb01 rst 30 trials for each condition ( omitting the 31st observation for the hub - and - spoke and locally clus - tered networks ) to obtain a balanced sample . Among these \ufb01 rst 30 observations per condition , all data were analyzed with no exclusions . Appendix C provides a com - plementary analysis of only those trials that did not expe - rience any dropout . Prior to collecting the data analyzed in this paper , we did conduct earlier experimental trials that were too easy to serve as a test of adaptability to a shifting environment ; the vast majority of all participants immediately switched to the correct answer because the key information pre - sented at the beginning of phase 3 essentially gave the answer away without the need for any further thought . Thus , this \ufb01 rst round of trials failed to adequately model the experimental scenario we were intending to create ( the need to move from one solution to the other in the context of an uncertain and shifting information environment ) , and we have excluded data from these trials without analysis . Appendix D . 3 . Power and Model Selection As noted , our approach was to collect as many inde - pendent observations as we could given the availability of Mechanical Turk workers . Thus , our sample size was not determined by an a priori analysis of power . Using G * Power , we obtain post hoc \u201c achieved powers \u201d of 0 . 84 , 0 . 83 , and 0 . 60 for the differences between the hub - and - spoke network and the isolates , locally clustered network , and complete clique , respectively ( assuming Poisson regression with binomially distributed predictors and effective sample size of 60 ) . Similarly , we obtain achieved powers of 0 . 65 , 0 . 64 , and 0 . 37 for the differences Table C . 1 . Number of People Correct at End of Trial : Trials with No Dropout Only Dependent variable 25th percentile Median Mean 75th percentile 25th percentile Median Mean 75th percentile ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) ( 7 ) ( 8 ) Hub - and - spoke \u2212 0 . 019 0 . 038 \u2212 0 . 000 \u2212 0 . 032 ( 0 . 487 ) ( 0 . 340 ) ( 0 . 167 ) ( 0 . 181 ) Core - periphery \u2212 0 . 173 0 . 105 0 . 000 0 . 116 ( 0 . 558 ) ( 0 . 344 ) ( 0 . 167 ) ( 0 . 161 ) Isolates 0 . 077 \u2212 0 . 163 \u2212 0 . 299 * * \u2212 0 . 403 * * * 0 . 096 \u2212 0 . 201 \u2212 0 . 299 * * \u2212 0 . 472 * * * ( 0 . 187 ) ( 0 . 318 ) ( 0 . 146 ) ( 0 . 142 ) ( 0 . 471 ) ( 0 . 156 ) ( 0 . 146 ) ( 0 . 125 ) Locally clustered \u2212 0 . 602 * * \u2212 0 . 295 \u2212 0 . 333 * \u2212 0 . 271 * \u2212 0 . 589 \u2212 0 . 333 \u2212 0 . 333 * \u2212 0 . 340 * * ( 0 . 285 ) ( 0 . 353 ) ( 0 . 181 ) ( 0 . 150 ) ( 0 . 518 ) ( 0 . 217 ) ( 0 . 181 ) ( 0 . 136 ) Complete clique \u2212 1 . 040 * * * \u2212 1 . 120 * * * \u2212 0 . 379 * * \u2212 0 . 018 \u2212 1 . 040 * \u2212 1 . 150 * * * \u2212 0 . 379 * * \u2212 0 . 040 ( 0 . 330 ) ( 0 . 367 ) ( 0 . 185 ) ( 0 . 311 ) ( 0 . 542 ) ( 0 . 240 ) ( 0 . 185 ) ( 0 . 197 ) Out core - periphery \u2212 0 . 495 \u2212 0 . 425 \u2212 0 . 395 * * \u2212 0 . 448 * * * \u2212 0 . 485 \u2212 0 . 433 \u2212 0 . 395 * * \u2212 0 . 520 * * * ( 0 . 318 ) ( 0 . 506 ) ( 0 . 180 ) ( 0 . 145 ) ( 0 . 537 ) ( 0 . 459 ) ( 0 . 180 ) ( 0 . 130 ) In core - periphery 0 . 311 0 . 088 \u2212 0 . 041 \u2212 0 . 204 0 . 323 0 . 056 \u2212 0 . 041 \u2212 0 . 268 * ( 0 . 192 ) ( 0 . 330 ) ( 0 . 171 ) ( 0 . 145 ) ( 0 . 473 ) ( 0 . 179 ) ( 0 . 171 ) ( 0 . 144 ) Constant 0 . 879 * * * 1 . 350 * * * 1 . 520 * * * 1 . 830 * * * 0 . 860 * 1 . 390 * * * 1 . 520 * * * 1 . 900 * * * ( 0 . 158 ) ( 0 . 312 ) ( 0 . 118 ) ( 0 . 126 ) ( 0 . 460 ) ( 0 . 144 ) ( 0 . 118 ) ( 0 . 108 ) Observations 216 216 216 216 216 216 216 216 * p < 0 . 1 ; * * p < 0 . 05 ; * * * p < 0 . 01 . Table D . 1 . Estimated Number of People Correct Under Different Levels of Social In \ufb02 uence Model No social influence Medium social influence Max social influence Ground truth 2 . 986 3 . 029 3 . 005 Binomial 2 . 986 3 . 029 3 . 005 Logit , clustered standard errors 2 . 986 3 . 029 3 . 005 Logit , random effects 2 . 986 2 . 191 0 . 333 Quasibinomial 2 . 986 3 . 029 3 . 005 Quasi - Poisson 2 . 986 3 . 029 3 . 005 Beta binomial 3 . 012 2 . 753 2 . 381 Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2091 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . between the core - periphery and the isolates , locally clus - tered network , and complete clique , respectively ( again assuming Poisson regression with binomially distributed predictors and effective sample size of 60 ) . However , these power estimates are hard to interpret in terms of whether this research will be reproducible because they are simultaneously overly optimistic and overly pessimistic in different ways . They are overly opti - mistic because standard power calculations do not account for the interference within trials and the resulting changes to the distribution of the dependent variable that are caused by different experimental treatments . They are overly pessimistic because when it comes to reproducibil - ity , we really care most not about power or sensitivity ( the ability to detect true statistical differences if they exist ) but speci \ufb01 city ( the ability to avoid concluding that a difference is real when it is not ) . In other words , we care less about detecting true positives than avoiding false pos - itives . To address this and the intertwined issue of model selection , we conduct simulation analysis to evaluate power , speci \ufb01 city , and model bias , tailored to our setting of network experimentation . Our experimental data on the number of people correct at the end of the trial do not conform neatly to the assumptions of standard statistical models . Individual suc - cesses are dichotomous , but they are nested within trials , within which social in \ufb02 uence occurs . Thus , at a trial level , the data could be considered the results of a binomial process ( measured as the number of successes and failures of nine possible people per trial ) but with overdispersion such that the success or failure of different individuals in the same trial is correlated . Alternatively , the trial - level data could be considered a Poisson - like process but with the caveat that the data are bounded above with a maxi - mum number of possible successes equal to nine . A fur - ther complication is that the amount and pattern of social in \ufb02 uence vary systematically ( but in a way unknown to the experimenters a priori ) by treatment condition . Thus , there is no single statistical model for our data that is obviously best , and we turn to a simulation - based approach to evaluate the merits of different approaches . We simulate 30 trials of nine people for each of three syn - thetic conditions intended to mimic different types of social in \ufb02 uence in networks , such as we observe in our experimental data . Each condition has the same global mean number of individuals correct , but the number of correct individuals per trial is drawn from different distri - butions . The \ufb01 rst is akin to an \u201c isolates \u201d condition , in which each individual is independently and identically a Bernoulli random variable ( each trial is a binomial draw ) . The second is akin to the \u201c complete clique \u201d condition , in which all nine people in a single trial have the same answer , such that the trial as a whole is determined by a single draw of a Bernoulli random variable . The third con - dition is akin to a more moderate level of social in \ufb02 uence , and the number of successes per trial is drawn from the beta - binomial distribution ( i . e . , an overdispersed bino - mial ) . For each condition , the grand mean was set such that , on average , three of nine people were correct ( chosen to approximate the average for the isolates condition in the experimental data ) . We create 1 , 000 simulated data sets and \ufb01 t several stat - istical models in order to assess bias , the false - positive rate ( speci \ufb01 city ) , and the false - negative rate ( power / sensi - tivity ) . We considered variations on standard models for dichotomous or count data . Speci \ufb01 cally , we \ufb01 t 1 . a standard binomial regression ( unit of observation is the trial , data are number of successes and failures per trial ) , 2 . a quasibinomial regression , 3 . a beta - binomial regression , 4 . a logistic regression with standard errors clustered by trial ( unit of observation is the individual ) , 5 . a random effects logistic regression with separate ran - dom variances for the two social in \ufb02 uence conditions , and 6 . a quasi - Poisson regression ( unit of observation is the trial ) . Fixed effects models could not be \ufb01 t on these data because of the perfect separation of the dependent varia - ble in the individual trials in the maximum social in \ufb02 u - ence condition . Our primary concerns in choosing a regression frame - work for analyzing these data were obtaining unbiased estimates of the mean number of people correct at the end of the trial and avoiding false - positive \ufb01 ndings . 7 There - fore , we assessed coef \ufb01 cient bias and the false - positive Table D . 2 . False - Positive Rates for Networks with Same True Mean Model \u03b1 (cid:2) 0 : 10 \u03b1 (cid:2) 0 : 05 \u03b1 (cid:2) 0 : 01 Binomial 0 . 255 0 . 198 0 . 118 Logit , clustered standard errors 0 . 068 0 . 036 0 . 009 Logit , random effects 0 . 424 0 . 391 0 . 358 Quasibinomial 0 . 044 0 . 021 0 . 004 Quasi - Poisson 0 . 044 0 . 022 0 . 004 Beta binomial 0 . 112 0 . 076 0 . 026 Table D . 3 . Estimated Number of People Correct Under Different Levels of Social In \ufb02 uence Model No social influence Medium social influence Max social influence Ground truth 3 . 007 4 . 742 3 . 015 Binomial 3 . 007 4 . 742 3 . 015 Logit , clustered standard errors 3 . 007 4 . 742 3 . 015 Logit , random effects 3 . 007 4 . 895 0 . 311 Quasibinomial 3 . 007 4 . 742 3 . 015 Quasi - Poisson 3 . 007 4 . 742 3 . 015 Beta binomial 3 . 031 4 . 499 2 . 372 Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2092 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . rate on data for which we know there is no true differ - ence in means by construction . Table D . 1 gives the coef \ufb01 cients estimated by each model . Most models give an unbiased estimate of the ground truth quantity of number of people correct , but the estimates from random effects logistic regression and the beta - binomial regression were extremely biased . Although these two models are intended to model endog - enous correlation , they performed poorly , and we rejected them for use in analyzing this experiment . The proportion of data sets for which a false - positive \ufb01 nding of statistical signi \ufb01 cance occurred ( for different decision thresholds , \u03b1 ) is in Table D . 2 . The standard bino - mial model does not account for overdispersion from social in \ufb02 uence within trials , and unsurprisingly , it has an unacceptably high false - positive rate ( almost 20 % at \u03b1 (cid:2) 0 : 05 ) . The remaining models \u2014 logistic regression with clustered standard errors , quasibinomial regression , and quasi - Poisson regression \u2014 had more reasonable false - positive rates . Among these three , logistic regression with clustered standard errors produced more false positives ( approximately 60 % more at \u03b1 (cid:2) 0 : 05 ) , so we reject it in favor of one of the remaining two . To assess power , or sensitivity , we also created simu - lated data in which the moderate social in \ufb02 uence condi - tion had a higher true mean , similar to the mean of the hub - and - spoke network in the real results . Tables D . 3 and D . 4 give mean coef \ufb01 cients and false - negative rates when there is a true difference in means . Results of this second simulated data set are similar to the \ufb01 rst one . Of particu - lar note is that the false - negative rate for the quasibino - mial and quasi - Poisson regressions is substantial : around 81 % for \u03b1 (cid:2) 0 : 05 . Thus , we have reason to believe that in the context of network interference , our power is much lower than standard power calculations ( such as can be obtained from software , such as G * Power ) would suggest . Nevertheless , as we explain , this does not mean that our results are not reproducible . With low false - positive rates and high false - negative rates , these regression methods are conservative tests of our hypotheses . We cannot say for certain what the prob - ability is that there is a true result conditional on a \ufb01 nding of statistical signi \ufb01 cance without making assumptions about the underlying distribution of effects in the world . Nevertheless , for the sake of illustration , assume that this present experiment is drawn from an abstract \u201c population \u201d of potential experiments that the same authors might con - duct . This population has a true rate of positive \ufb01 ndings ( we will call it \u03c4 for \u201c truth \u201d ) , which we do not know but can make arbitrary assumptions about . Given that this experiment \ufb01 nds a difference between treatment and con - trols , we would like to know what the probability that our \ufb01 nding is a true positive is , given \u03c4 , as well as our simu - lated rates for false positives and false negatives ( which are themselves calculated from the experimentally obtained effect sizes ) . Plugging in to Bayes \u2019 rule , we see P ( True | StatSig ) (cid:2) ( 1 \u2212 FalseNegativeRate ) ( \u03c4 ) ( ( 1 \u2212 FalseNegativeRate ) ( \u03c4 ) ) + ( ( FalsePositveRate ) ( 1 \u2212 \u03c4 ) ) : Thus , assuming that half of the experiments we run have a true difference between treatment and control \u2014 that is , that \u03c4 (cid:2) 0 : 5 \u2014 a signi \ufb01 cant \ufb01 nding corresponds to a true \ufb01 nding 89 . 4 % of the time . If \u03c4 (cid:2) 1 = 3 , a signi \ufb01 cant \ufb01 nding corresponds to a true \ufb01 nding 80 . 9 % of the time . If we only pick experiments with true effects one - quarter of the time , making \u03c4 (cid:2) 0 : 25 , a signi \ufb01 cant \ufb01 nding still corre - sponds to a true \ufb01 nding 73 . 8 % of the time because of the low false - positive rate and high false - negative rate . Appendix D . 4 . Conclusions : Model Choice and Reproducibility Both quasibinomial and quasi - Poisson regression pro - vided unbiased estimates and conservative hypothesis tests . In the tables of results , we opted for quasi - Poisson regres - sion so that the coef \ufb01 cients would be on the same scale as the models of conditional quantiles for count data . The true value of \u03c4 is unknown and cannot be known , but our hypotheses build directly on the results of prior experiments and thus are consistent with a relatively high \u03c4 . Therefore , given the low false - positive rate and high false - negative rate , we believe that it is likely that our stat - istical \ufb01 nding of difference corresponds to a true difference between centralized and noncentralized networks . Appendix E . Additional Details on the Design of Network Treatments In the abstract , core - periphery networks are de \ufb01 ned as \u201c blockmodels \u201d ( White et al . 1976 ) . In the idealized block - model of core - periphery networks , nodes in the adjacency matrix representation of the network are permuted such that high - degree ( core ) nodes have low node indices and low - degree ( peripheral ) nodes have high node indices ( where an index is simply the order that a node appears in the permutation ) . In a simple blockmodel , all of the entries in a given quadrant of the adjacency matrix ( see Figure E . 1 ) take the same value \u2014 either ones or zeros . In an idealized version of an undirected core - periphery network , all of the entries in quadrants I , II , and III are ones , and all of the entries in quadrant IV are zeroes . Core nodes are connected to each other , and peripheral nodes are connected to core nodes ; however , peripheral nodes are not connected to each other . In the idealized blockmodels of the directed ver - sions of the core - periphery network , either quadrant II or quadrant III is zeroes ( but not both ) ; in the out core - periphery network , quadrant III is all zeroes , and in the in core - periphery network , quadrant II is all zeroes . Table D . 4 . False - Negative Rates for Trials with Different True Means Model \u03b1 (cid:2) 0 : 10 \u03b1 (cid:2) 0 : 05 \u03b1 (cid:2) 0 : 01 Binomial 0 . 521 0 . 558 0 . 619 Logit , clustered standard errors 0 . 675 0 . 717 0 . 796 Logit , random effects 0 . 454 0 . 492 0 . 564 Quasibinomial 0 . 728 0 . 808 0 . 933 Quasi - Poisson 0 . 731 0 . 814 0 . 945 Beta binomial 0 . 735 0 . 784 0 . 874 Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) 2093 D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d . In the experimental treatments we used , the networks deviated from the idealized blockmodels by including three links ( ones in the adjacency matrix ) in a quadrant that is otherwise all zeroes . In the out core - periphery network , there are three ones in quadrant III , and in the in core - periphery network , there are three ones in quadrant II . We made this choice for two reasons . The \ufb01 rst reason is that adding those extra ties maintains a degree ( number of ties incident to each node ) of three for all nodes in the network . Speci \ufb01 cally , in the out core - periphery network , each node has three incoming ties , which hold availability of information constant within the network ; in the in core - periphery network , each node has three outgoing ties because this network is the exact transpose of the out core - periphery network . The second reason is that we believe it is more realistic to have some information com - ing from the periphery in the out core - periphery network rather than no information at all . Thus , for external valid - ity reasons , we also prefer including a few incoming ties . In addition to the directed core - periphery network , one additional network treatment has directed ties : the locally clustered network . The locally clustered network was cre - ated by creating three clusters ( complete cliques ) of three participants each and then adding ties between the cliques to create a connected network . We started by adding an undirected ( that is , two - way ) tie between each pair of clusters ; speci \ufb01 cally , we added the ties between node pairs ( 2 , 6 ) , ( 3 , 8 ) , and ( 5 , 9 ) . This left three nodes ( 1 , 4 , and 7 ) with only two incoming and two outgoing ties . Con - necting a single pair of these with an undirected tie would leave one additional node with a lesser degree . Therefore , to complete the network , we added directed ties from node 1 to node 7 , from node 7 to node 4 , and from node 4 to node 1 . Thus , our directed ties allow us to create a network in which the number of incoming ties , the number of outgoing ties , and the eigenvector centrality ( Bonacich 1987 ) are all equal ( on these meas - ures , the network \u2014 like the other controls \u2014 is completely decentralized ) . Endnotes 1 Communication network centralization , which has been measured as a structural characteristic of a communication network , can be the cause or consequence of status - or power - based differentiation and hierarchy ( Lin 1999 ) . Being central in a communication network can confer control and influence ; on the other hand , it can be an individual \u2019 s power that makes him or her central in the communi - cation structure . The term centralization has , therefore , been used to mean centralization of communication patterns , centralization of power , or both . In this experimental study , we focus specifically on centralization of communication patterns , disentangling its effects from the influence of power that derives from rank , status , or deci - sion rights . 2 Code is freely available at https : / / github . com / jessecshore / oceler . 3 This protocol was approved by the University Committee on the Use of Human Subjects under IRB17 - 1054 . 4 If dropout is affected by experimental treatment ( e . g . , if one treat - ment was more difficult and caused more dropout ) , then omitting trials risks biasing estimates of the causal effects of the treatment ( e . g . , if we omitted observations in which participants dropped out because of difficulty , our estimates would be positively biased because we ignored observations in which the treatment caused participants to have trouble ) . 5 Replication data are available at https : / / osf . io / 9d3tv / ? view _ only = ecc8f28edba749a0aa6d8ebc0b96de27 or https : / / doi . org / 10 . 7910 / DVN / X2HQXD . 6 Given the complexities of our analytical framework ( see Appendix D for more discussion ) and the natural interpretation for a decision maker who must decide how to structure a communication net - work , common language effect size is more straightforward than alternatives , such as Cohen \u2019 s d . 7 Note that our intention is to provide a conservative test to build theory . 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Bernstein is the Edward W . Conard Associate Professor of Business Administration in the Organizational Behavior Unit at Harvard Business School . He received his doctorate from Harvard University . His research investigates the systems and structures that are being introduced by organizations \u2014 in particular , systems to increase workplace transparency and novel collaboration structures \u2014 to enhance employee effectiveness in an era when the nature of work is changing dramatically . Jesse C . Shore is a research scientist at Meta Platforms , Inc . ; this work was done while he was a professor at Boston University and is unrelated to his work at Meta . He received his doctorate from the Massachusetts Institute of Technology . His primary research focus is collective decision making and how it is shaped by social networks and digital technologies . Alice J . Jang is an assistant professor in the Department of Business Information Technology at Virginia Tech . She received her doctorate from Boston University . Her research focuses on understanding the dynamics of interaction in social networks and the effect of social dynamics on the performance and sustainability of organizations and online communities . Bernstein , Shore , and Jang : Network Centralization and Collective Adaptability 2096 Organization Science , 2023 , vol . 34 , no . 6 , pp . 2064 \u2013 2096 , \u00a9 2022 The Author ( s ) D o w n l o a d e d fr o m i n f o r m s . o r g by [ 129 . 2 . 89 . 115 ] on 03 M a y 2024 , a t 15 : 27 . F o r p e r s on a l u s e on l y , a ll r i gh t s r e s e r v e d .", + "fuExpertRepresentationDesign2013": "Expert representation of design repository space : A comparison to and validation of algorithmic output Katherine Fu , Department of Mechanical Engineering , Carnegie Mellon University , 5000 Forbes Ave . , Pittsburgh , PA 15217 , USA Joel Chan and Christian Schunn , Department of Psychology , University of Pittsburgh , Pittsburgh , PA , USA Jonathan Cagan , Department of Mechanical Engineering , Carnegie Mellon University , Pittsburgh , PA , USA Kenneth Kotovsky , Department of Psychology , Carnegie Mellon University , Pittsburgh , PA , USA Development of design - by - analogy tools is a promising design innovation research avenue . Previously , a method for computationally structuring patent databases as a basis for an automated design - by - analogy tool was introduced . To demonstrate its strengths and weaknesses , a computationally - generated structure is compared to four expert designers\u2019 mental models of the domain . Results indicate that , compared to experts , the computationally - generated structure is sensible in clustering of patents and organization of clusters . The computationally - generated structure represents a space in which experts can \ufb01nd common ground / consensus e making it promising to be intuitive / accessible to broad cohorts of designers . The computational method o\ufb00ers a resource - e\ufb03cient way of usefully conceptualizing the space that is sensible to expert designers , while maintaining an element of unexpected representation of the space . (cid:1) 2013 Elsevier Ltd . All rights reserved . Keywords : computer supported design , design by analogy , design methods , en - gineering design D esign by analogy , in which designers draw inspiration from cross - domain design solutions , is a promising methodology for design prac - tice ( e . g . , Bhatta & Goel , 1996 ; Davies , Goel , & Nersessian , 2009 ; Goel , 1997 ; Herstatt & Kalogerakis , 2004 ; Vattam , Helms , & Goel , 2010 ) . Not all analogies are equally useful ( Casakin & Goldschmidt , 1999 ; Chan et al . , 2011 ; Dunbar , 1997 ; Fu et al . , in press - b ; Gick & Holyoak , 1980 ; Weis - berg , 2009 ) , raising the question of how to organize the very large space of possible analogies to any given problem . A number of methods exist for orga - nizing spaces of possible design analogies ( e . g . , Gentner et al . , 1997 ; Gentner & Markman , 1997 ; Linsey , Markman , & Wood , 2008 ; Mcadams & Wood , 2000 ; Corresponding author : Katherine Fu Katherine . Fu @ gmail . com www . elsevier . com / locate / destud 0142 - 694X $ - see front matter Design Studies - - ( 2013 ) - - e - - http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 1 (cid:1) 2013 Elsevier Ltd . All rights reserved . Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 Murphy , 2011 ; Verhaegen , D\u2019hondt , Vandevenne , Dewulf , & Du\ufb02ou , 2011 ) that make use of varying computational and representational techniques . For example , the computational work presented in this paper considers a diverse set of possible structural types ( e . g . , trees , rings , chains ) and uses Bayesian methods for choosing the best type and the best instance within each type . Because design - by - analogy inherently has a human in the loop , it is useful to understand how meaningful expert designers \ufb01nd the algorithm - generated or - ganizations of possible inspirational examples . We hypothesize that a tech - nique previously found to capture psychologically meaningful structures in other contexts is especially likely to provide structures that align with the mental models that humans develop through the design process . Such align - ment could aid in both the e\ufb00ectiveness of the tool at stimulating designer\u2019s thinking and in easing the transition for its adoption . The study presented here attempts to validate the proposed computational methodology and its output through judgment of its accuracy and adequacy by four design experts and also a direct comparison of its output to the experts\u2019 structuring of the same patent space , which presumably re\ufb02ects their mental model of the space . 1 Background Analogy and external stimuli in engineering design have been studied in a num - ber of ways . Studies have been performed to understand how the introduction of analogies a\ufb00ects the ideation process and outcomes ( e . g . , ( Christensen & Schunn , 2005 ; Dahl & Moreau , 2002 ; Goldschmidt & Smolkov , 2006 ; Linsey et al . , 2008 ) , with some studies speci\ufb01cally examining how the introduction of analogies with di\ufb00erent levels of applicability to the design problem a\ufb00ects indi - vidual designers ( Christensen & Schunn , 2007 ; Tseng , Moss , Cagan , & Kotovsky , 2008 ) . For brevity , we do not dive deeply into the design by analogy literature , here . Most relevant to the current context , the literature \ufb01nds that the distance of analogies to the current design problem can have a large in\ufb02uence on the helpfulness of the analogy , with some distance being useful ( Chan et al . , 2011 ; Dahl & Moreau , 2002 ; Dyer , Gregersen , & Christensen , 2011 ; Fu et al . , in press - b ; Gentner & Markman , 1997 ; Wilson , Rosen , Nelson , & Yen , 2010 ) , but analogies too far away show no bene\ufb01t ( Fu et al . , in press - b ) . Thus , \ufb01nding a tool that organizes the space of possible analogs by distance to the current problem may be especially useful . 1 . 1 Computational design tools The development of computational tools to assist designers during the design process is an area of considerable research in engineering design . To facilitate functional modeling , Stone and Wood created a functional basis that can serve as a universal language in the ideation process ( Stone & Wood , 2000 ) . This work has been extended and adapted a great deal , including , for example , the creation of a biological functional basis ( Cheong , Shu , Stone , & Wood , 2008 ) . 2 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 The U . S . patent database conveniently catalogs existing technology and engi - neering design and , thus , has been used in the development of numerous compu - tational design aids . For example , TRIZ helps engineers overcome di\ufb03culties in functional reasoning by searching through patents based on heuristic rules ( such as use of opposites ) ( Altshuller & Shapiro , 1956 ) . One axiomatic conceptual design model relies on the combination of TRIZ and functional basis ( Zhang , Cha , & Lu , 2007 ) . Patent citation data has been used to identify the interrelated - ness of technologies , and to demonstrate the bene\ufb01ts of tapping into the technol - ogy knowledge base created by competitors within a design \ufb01eld ( Chakrabarti , Dror , & Nopphdol , 1993 ) . The syntactic similarity between patent claims has been investigated as an aid in patent infringement research ( Indukuri , Ambekar , & Sureka , 2007 ) . Businesses haveusedpatentrepositorytools andpat - ent mining to predict potential market trends , identify proli\ufb01c inventors , and for other purposes . Among the characteristics explored in patent mining have been the number of citations , number of claims , average number of words per claim , and number of classes that the patent spans ( Kasravi & Risov , 2007 ) . The poten - tial for using the Design Repository at Oregon State University as a design aid was demonstrated by Bohm et al . by performing a function - based search using Chi Matrix and Morphological Matrix techniques to \ufb01nd components that were present in concepts generated by hand ( Bohm , Vucovich , & Stone , 2005 ) . Morebroadly , designrepositoriesofCADmodelsofcomponentsandassemblies have been explored as resources for designers to streamline product design of complex engineering systems by reusing , revising or gaining insight into previous designs and models ( Szykman , Sriram , Bochenek , Racz , & Senfaute , 2000 ) . The computational work examined in the current study focuses on structuring design repositories , using more open - ended analogical transfer , and using the textual content of the patents , which may generate richer outcomes . In order to assist in avoiding patent infringement , Mukherjea et al . created a BioMedical Patent Semantic Web that discovered semantic associations be - tween important biological terms within biomedical patents , returning a ranked list of patent resources and a Semantic Web showing the relationships between the terms and between resources . Unlike our work , the Semantic Webs generated by Mukherjea et al . are fully connected graphs with no imposed structure , and the data includes only the abstract of the patents being examined . Further , a Bayesian inference approach was not use to generate the webs ( Mukherjea , Bhuvan , & Kankar , 2005 ) . Bayesian inference was used by Chakrabarti et al . , however , to train a model using a small data set of docu - ments , which was then used to categorize the remaining documents into \u201ctopics\u201d and create a hierarchical structure ( Chakrabarti , Dom , Agrawal , & Raghavan , 1998 ) . Structures other than hierarchies were not explored and the structures created were not used as input for analogical design work . PatViz , a tool developed by Koch et al . allows visual exploration of iterative and complex patent searches and queries using a variety of patent data , Expert representation of design repositoxy space 3 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 including full text . Users may create one graph view with PatViz through a guided process , rather than an algorithm . The tool includes three visualiza - tions of interest : Patent Graph , which creates a fully - connected web of patents ; 3D IPD Treemap , which creates a 3D tree structure of the patents based on a prede\ufb01ned classi\ufb01cation scheme ; and Aggregation Tree , which is another tree view that uses prede\ufb01ned adjustable hierarchies ( Koch , Bosch , Giereth , & Ertl , 2009 ) . Unlike the work of Koch et al . , our work does not rely on prede\ufb01ned or user - de\ufb01ned classi\ufb01cation schemes ; rather , it uses an exploration methodology to discover the best - \ufb01tting structures among multiple types of structures to describe the set of patents . The \ufb01t to the data being examined determines the form of the structure . While the U . S . patent database represents a potentially productive repository of analogical or cross - domain design solutions , its size and complexity make it di\ufb03cult for designers to use e\ufb00ectively . There have been many attempts to automate , aid , or streamline the search of the US patent database , including simple key word searches on the United States Patent and Trademark O\ufb03ce ( USPTO ) website or Google Patents , as well as theories like TRIZ and their resulting tools ( Altshuller & Shapiro , 1956 ; Duran - Novoa , Leon - Rovira , Aguayo - Tellez , & Said , 2011 ; Hernandez , Schmidt , & Okudan , 2012a , 2012b ; Houssin & Coulibaly , 2011 ; Krasnoslobodtsev & Langevin , 2005 ; Liang , Tan , & Ma , 2008 ; Nakagawa , 2012 ; Nix , Sherret , & Stone , 2011 ; Zhang et al . , 2007 ) . It is still di\ufb03cult , however , to recognize and appreciate the char - acteristics that might be relevant to a particular design problem within the \u2018space\u2019 of patents . A number of computational \u201cinnovation support tools\u201d have been developed ( CREAX ; Gold\ufb01re ) , as well as a number of research - driven design support tools , proposals for design support tools , and method - ologies ( Bhatta & Goel , 1996 ; Chiu & Shu , 2005 ; Goel , Bhatta , & Stroulia , 1997 ; Verhaegen et al . , 2011 ; Vincent , Bogatyreva , Bogatyreva , Bowyer , & Pahl , 2006 ; Linsey , Wood & Markman , 2008 ) . Many of these tools and methods , however , rely heavily on the users to generate the terms or analogies and comb through search results . Psychology literature suggests that the prob - ability of retrieving similarities is low when surface dissimilarities exist , because retrieval of far - \ufb01eld analogies is cognitively di\ufb03cult ( Gick & Holyoak , 1980 ) and the range of remindings tend to be limited by surface sim - ilarity ( Forbus , Gentner , & Law , 1994 ) . If designers have a means to extract the interrelatedness and interconnectedness of patents in the space , especially with respect to a speci\ufb01c design problem , they may be able to more strategi - cally choose which cross - domain designs to review or to traverse the space in a more intentional and meaningful exploratory way . 1 . 2 Computational basis The computational methodology for discovering structural forms in patent spaces is based on the work of Kemp and Tenenbaum ( 2008 ) . Kemp and Ten - enbaum devised an algorithm that can discover the underlying structure that is 4 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 inherent to a particular data set , enabling the extraction of psychologically meaningful relationships or information . The algorithm discovers 1 ) the best - \ufb01tting form for a given set of data from a space of 8 possible forms , which include a partition , order , chain , ring , tree , hierarchy , grid , and cylinder ; and 2 ) the best - \ufb01tting instantiation of that selected form , which is called a struc - ture . For example , given human similarity judgments for a set of sixteen colors , the structural form discovered by the algorithm that best described the data was a ring , matching the color wheel with which we are all familiar from our elementary school days . Given data regarding votes of Supreme Court justices , the best structural form discovered was a chain , organizing the justices from liberal to conservative ; again , a meaningful structuring . Given data regarding features that a set of animals have , a tree structure was discovered to be the best , similar in form type to the biological classi\ufb01ca - tion scheme proposed by Linnaeus . The form types explored are based on the psychology literature ( Jaynes , 2003 ) , and have been used in formal models in many contexts ( Anderson , 1991 ; Bradley & Terry , 1952 ; Carroll , 1976 ; Collins & Quillian , 1969 ; Fiske , 1992 ; Guttman , 1944 , 1954 ; Huelsenbeck & Ronquist , 2001 ; Inhelder & Piaget , 1969 ; Kohonen , 1997 ; Shepard , 1980 ; Sneath & Sokal , 1973 ; Wiggins , 1996 ) . In addition , the algorithm is based on Bayesian inference , a statistical basis that has a long history of successfully describing and modeling human cognition ( Gri\ufb03ths , Kemp , & Tenenbaum , 2008 ) . Jaynes describes human plausible reasoning as a calculation of the degree of plausibility of a particular hypothesis being true based on previous experience and common sense , and given the facts at hand , corresponding directly to the components that must be considered when calculating the posterior probabil - ity of a hypothesis being true given a set of data using Bayes Rule ( Jaynes , 2003 ) . This basis in human cognition is an important motivation for the choice to use the Kemp and Tenenbaum algorithm in the computation methodology being validated in this study , as it has the potential to be intuitive in the human mind and thus form the basis for an automated design inspiration tool . 2 The current study The goal of the current study was to gain a better understanding of the output structures of the algorithm through comparison to human judgment . Struc - turing algorithms such as Kemp and Tenenbaum\u2019s ( 2008 ) have customarily em - ployed comparisons to pre - existing \u201ccorrect\u201d structures as a method of validation . In a similar vein , we employed comparisons of the algorithm\u2019s output structures to similar structures generated by expert designers , and also solicited their qualitative reactions to particular aspects of the output structure . This method was judged as appropriate for validation for two reasons . First , to our knowledge , there exists no one \u201cpsychologically meaningful\u201d , \u201ccorrect\u201d , or \u201canalytical\u201d solution to the question of how to best structure design repository data or patents . Second , the \ufb01nal application of this structuring work is in - tended to be the basis for an automated design by analogy stimulation tool , making it crucial that the output is intuitive or sensible to human minds . Expert representation of design repositoxy space 5 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 Speci\ufb01cally , the study was performed to address two primary research questions : 1 . In what ways is the algorithm\u2019s patent structure similar to or di\ufb00erent from how an expert designer might structure a space of patents in prep - aration for inspiration through design by analogy with regard to a speci\ufb01c design problem ? 2 . If the structure is di\ufb00erent , can experts nevertheless make sense of it and perhaps see new connections between patents ? The reasoning chain in Figure 1 represents possible outcomes that feed into three main potential inferences from the study . First , suppose that the algo - rithm structures patents in the exact same way that experts do . In that case , we might infer then that ( 1 ) the algorithm does what an expert would be able to do , but more e\ufb03ciently . This would not be an insigni\ufb01cant outcome , as lim - itations of attention and working memory capacity ( Cowan , 2000 ) , and time and e\ufb00ort e\ufb00ectively preclude the possibility of structuring large databases of potential analogical stimuli by hand . Furthermore , supporting new ways of representing the patent space are not the only way to help inspire innova - tion . Experts\u2019 mental representations are often based on deep structural simi - larity between knowledge structures and domains ( Chi , Feltovich , & Glaser , 1981 ) , and experts are often able to draw deep structural analogies from mem - ory ( Ahmed & Christensen , 2009 ; Ball & Christensen , 2009 ; Christensen & Schunn , 2007 ; Holyoak & Thagard , 1996 ; Novick , 1988 ) . Accurate external - izations of experts\u2019 mental representations could potentially inspire innovation by supporting search for non - obvious analogous solutions to design problems that are hidden by surface dissimilarities ( e . g . , application domain , engineering discipline ) . Further , engineering design is inherently and increasingly interdis - ciplinary ( Reich & Shai , 2012 ) ; even experts with one disciplinary focus may be inspired by representations that include insights from other relevant Figure 1 Reasoning chain of possible outcomes of study and main possible inferences . 6 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 engineering disciplines . The possibility of being able to aggregate expert repre - sentations across domains could be useful in catering to the increasingly inter - disciplinary nature of engineering design . These aggregate representations could give access to solutions that might be hidden by surface dissimilarities , given that when one approaches a domain in which she is not expert , process - ing is easily dominated by surface features ( Chi et al . , 1981 ; Hmelo - Silver & Pfe\ufb00er , 2004 ; Novick , 1988 ) . Secondly , suppose that a ) the algorithm produces a structure that is completely di\ufb00erent from that of the experts , but the experts are nevertheless able to make sense of it , or b ) the algorithm produces a structure that bears both similarities and di\ufb00erences to that of the experts , and the di\ufb00erences are not so large as to render the structure incomprehensible . From both these outcomes , we could infer that ( 2 ) the algorithm may structure the analogical stimuli in a way that potentially allows for novel connections to be made between patents . The algo - rithm\u2019s grouping of patents into nodes based on functional similarity could highlight features and functional principles that would otherwise potentially be overlooked ( e . g . , via analogical comparison ; ( Gentner et al . , 1997 ; Kurtz , Miao , & Gentner , 2001 ) ) yielding fresh insights for creative ideation that could inspire innovation . In the cognitive psychology literature , it has been shown that enabling changes in representation of objects and / or ideas , for example by leading problem solvers to attend to previously ignored features , is an e\ufb00ec - tive way of dealing with \u201cfunctional \ufb01xedness\u201d , where problem solvers have di\ufb03culty seeing a potential creative use of an object with which they are familiar ( Kaplan & Simon , 1990 ; McCa\ufb00rey , 2012 ) . Finally , suppose that a ) the algorithm produces a structure that is completely di\ufb00erent from that of the experts , and it is incomprehensible to them , or b ) the algorithm produces a structure that bears both similarities and di\ufb00erences to that of the experts , and the di\ufb00erences are so large as to render the structure incomprehensible . From both these outcomes , we could infer that ( 3 ) the algo - rithm may need revision in order to be useful for supporting design - by - analogy . These outcomes and inferences comprise the set of potential contributions of the study to the research and development of our automated design - by - analogy support tool . 3 Methods To address our research questions , we asked a set of expert designers to 1 ) structure a set of patents , and 2 ) provide focused feedback on our algorithm\u2019s output structure . Structuring of the patents was conducted in the context of preparing to solve a particular design problem , given our interest in comparing the experts\u2019 judgments of patents\u2019 relevance to the design problem to the algo - rithm\u2019s ordering of patents\u2019 relevance as represented in the layout of the struc - ture . The similarity of our algorithm\u2019s output structure to the experts\u2019 structures was evaluated both quantitatively and qualitatively . In the Expert representation of design repositoxy space 7 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 following sections , we describe the details of our methods of data collection , and also the algorithm we used to produce the structure . 3 . 1 Participants This study was conducted with four design experts in Pittsburgh , PA , USA . All participants were males with at least 10 years of experience in the \ufb01eld of product design , and an educational background in engineering or industrial design . Three participants were employed at local product development \ufb01rms , and one was employed at a local biomedical apparatus design and manufac - ture \ufb01rm . The participants were highly experienced and comfortable in review - ing and researching patents . Each participant was compensated with $ 100 for the 2 h of the study . Though there were only a small number of participants , the information is considered valuable because it is based on expert data , and includes deep analysis of interaction between 45 di\ufb00erent pieces of information ( patents ) for each of the four experts . Further , signi\ufb01cant insights in the study of knowledge representations have often been obtained with only a few partic - ipants whose behaviors are examined in depth ( Chase & Simon , 1973 ; Chi et al . , 1981 ; Chi & Koeske , 1983 ; Gobet & Simon , 2000 ; Huth , Nishimoto , Vu , & Gallant , 2012 ; Morais , Olsson , & Schooler , 2013 ) , and a review of research on design revealed that published studies have often been conducted with qualitative analyses of the performance of a small number of experts ( Mehalik & Schunn , 2007 ) . 3 . 2 Materials The experts were supplied with the following materials : (cid:1) Consent form (cid:1) Design problem description ( see Section 3 . 3 ) , on a 8 . 5 in by 5 . 5 in piece of paper , laminated , with mounting putty on the back (cid:1) Title , abstract text and one key \ufb01gure from thirty - seven randomly selected mechanical patents and eight patents selected from previous studies pre - sented in Chan et al . ( 2011 ) and Fu et al . ( in press - b ) ( see Appendix A ) , each on a 8 . 5 in by 5 . 5 in piece of paper , laminated , with mounting putty on the back (cid:1) A 56 in by 107 in sheet of white craft paper , \ufb01xed to a wall (cid:1) Multiple colored magic markers , one black marker , and one pen (cid:1) Pad of sticky notes (cid:1) An 8 . 5 in by 11 in depiction of the algorithm\u2019s output structure for the same 45 patents (cid:1) Title , abstract text and one key \ufb01gure from patents in 3 di\ufb00erent clusters of the algorithm\u2019s output structure , each on a 8 . 5 in by 5 . 5 in piece of paper . 3 . 3 Design problem The design problem supplied to the experts is one that has been used throughout the development and analysis of this work on the structuring of 8 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 patents for use in design by analogy , as presented in Chan et al . ( 2011 ) and Fu et al . ( in press - b ) . The textual design problem description as supplied to the experts of this study is as follows : Design a device to collect energy from human motion for use in developing and impoverished rural communities in places like India and many African countries . Our goal is to build a low - cost , easy to manufacture device tar - geted at individuals and small households to provide energy to be stored in a rechargeable battery with approximately 80 % e\ufb03ciency . The energy is in - tended to be used by small , low power draw electrical devices , such as a radio or lighting device , hopefully leading to an increase in the quality of life of the communities by increasing productivity , connection to the outside world , etc . The target energy production is 1 kW - hour per day , roughly enough to power eight 25 Watt compact \ufb02orescent light bulbs for 5 hours each per day , or enough to power a CB radio for the entire day . For reference , an average adult human can output about 200 watts with full body physical activity for short periods of time , with a signi\ufb01cant reduction for sustained power output . 3 . 4 Patent selection An initial random subset of 10 , 000 patents was selected from the patent data - base . From this initial set , patents were chosen for the \ufb01nal set if they were classi\ufb01ed within the U . S . Patent classi\ufb01cation system as \u201cBody Treatment And Care , Heating And Cooling , Material Handling And Treatment , Me - chanical Manufacturing , Mechanical Power , Static , and Related Arts\u201d . In addition , for continuity with previous studies and an understanding of human judgment of these patents , eight additional patents were included from two previous studies performed by the authors ( Chan et al . , 2011 ; Fu et al . , in press - b ) . Four of these patents were the \u201cfar\u201d analogical stimuli from the study performed to understand how analogical distance , commonness , and modality a\ufb00ect ideation outcomes presented in Chan et al . ( 2011 ) , and the other four were the \u201cfar\u201d analogical stimuli from a study performed to test the use of structures of patents created using Kemp and Tenenbaum\u2019s discov - ery of structural form algorithm to choose \u201cnear\u201d and \u201cfar\u201d patents and mea - sure their e\ufb00ect on ideation outcomes , presented in Fu et al . ( in press - b ) . Both previous studies used the same design problem used here , described in Section 3 . 3 . In total , 45 patents were provided to the experts as described in Section 3 . 2 . 3 . 5 Algorithmic structure generation The method used to produce the structure of patents shown in Figure 2 has been presented in depth in Fu ( 2012 ) and Fu et al . ( in press - a ) . The computa - tional methodology for generating structures of patents has three main parts . Expert representation of design repositoxy space 9 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 First , the set of 45 full - text patents are preprocessed using Latent Semantic Analysis ( LSA ) , a computational text analysis tool that allows for the extrac - tion of contextual similarity and synonymy between words and documents ( Deerwester , Dumais , Furnas , & Landauer , 1990 ; Foltz , Kintsch , & Landauer , 1998 ; Landauer , Foltz , & Laham , 1998 ) . The output from this \ufb01rst step is a similarity matrix , assigning a numeric value between 0 and 1 , connoting semantic similarity , to all pairwise relationships between patents . This similarity matrix is the input to the second portion of the methodology , which is an algorithm for the discovery of structural form in data , devised by Kemp and Tenenbaum ( 2008 ) . The algorithm is a hierarchical Bayesian algo - rithm that discovers the structure and form type , chosen from a prede\ufb01ned set of form types , including the partition , order , chain , ring , hierarchy , tree , grid , and cylinder , that achieves the highest posterior probability value for the description of the data by a particular form type and structure . Within the al - gorithm , the form of the structure itself changes as the data being examined changes and has the potential of revealing deep structural relationships be - tween patents . Finally , LSA is used again to generate labels for the output structure , which describe the clusters of patents in the best structure , allowing a designer viewing the structure to understand the meaning of the clustering of the pat - ents , and characterize the space for navigation and exploration . Labels were not included in the structures used in this study , as the researchers were inter - ested in the alignment of expert - generated labels and computationally - generated labels . A further post - processing step is done to extract \u201cregions\u201d of similarity in the structure , which further facilitate e\ufb03cient understanding and exploration of the space of patents . These regions were also excluded 12 8 1 40 11 7 19 4 39 33 6 30 23 28 2621 24 34 22 36 41 43 14 9 31 10 44 35 42 13 37 32 27 16 18 5 17 25 38 29 45 15 3 20 2 Figure 2 Algorithm output structure of 45 patents . 10 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 from the computational output used in this study . The main goal of this work is a comparison of the judgment of experts to , and a validation of , the struc - ture ( s ) generated by this methodology . The full text of the 45 patents used in the methodology described above , enumerated in Appendix A , were used to create the grid structure in Figure 2 . 3 . 5 . 1 Cluster label generation The structure shown to the experts ( see Figure 2 ) did not include labels to indi - cate the similarity or meaning of the associations of patents within the clusters . Having the experts suggest labels , both for the clusters within the structure generated by the algorithm , as well as for the clusters within their own patent space or structure , was a way to collect data that could then be compared to labels that were generated computationally . The labels were generated by em - ploying LSA , as described in Fu et al . ( in press - a ) . Two di\ufb00erent methods for label generation were employed , the results of which are presented next . The \ufb01rst method , the Highest Average Rank Labeling method , averaged the ranks of each word in the LSA space for each patent within a cluster , and used the top \ufb01ve highest average ranked words for that cluster . The second method , the Highest Cosine Similarity Labeling method , gathered the top twenty words with the highest cosine similarity to each patent within a cluster , pooled and sorted the words in descending cosine similarity value order , and used the top \ufb01ve words with the highest cosine similarity values in the pool of words for the patents within that cluster . The results yielded by Highest Average Rank Labeling method are shown in Figure 3 . The results yielded by the High - est Cosine Similarity Labeling method are shown in Figure 4 . The results of both methods are discussed for the three nodes in particular in Table 6 as compared to expert generated labels . Because four of the patents included in the space from the previous work presented in Chan et al . ( 2011 ) were not 12 8 1 40 11 7 19 4 39 33 6 30 23 28 2621 24 34 22 36 41 43 14 9 31 10 44 35 42 13 37 32 27 16 18 5 17 25 38 29 45 15 3 20 2 mean , show , operate , present , cause ratchet , pawl , guide , travel , house staple , stick , arm , push , magazine pot , support , plant , rod , port part , rotate , shaft , drive , mechanism blood , pump , needle , rotor , hypodermic valve , open , provide , pressure , connect position , provide , engage , support , extend provide , embodiment , form , describe , position insert , chip , hold , port , cut electrode , prosthesis , shaft , core , key provide , port , form , prefer , position Figure 3 Algorithm output structure of 45 patents with cluster labels , highest average rank method . Expert representation of design repositoxy space 11 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 part - of - speech tagged , many unimportant words were included in the text , and thus the LSA space . These words were manually \ufb01ltered out to increase the quality of the labels . This problem would not arise if all patents in the space have been pre - processed with part - of - speech tagging . 3 . 6 Experimental procedure Each participant was allotted 2 h to complete the study , and the \ufb01ve sessions were run in three di\ufb00erent locations . All locations were similar , in that they were large conference rooms , in which experts had ample light , table space on which to work , seating , and a large wall space for building the structure . 3 . 6 . 1 Experimenter\u2019s instructions and overview The study began with one of the experimenters explaining the motivation of the study , the schedule of tasks , and the speci\ufb01c instructions for completing those tasks . The experts were told that the goal of the study was to understand how an expert , or professional product designer , might organize a collection of patents to facilitate searching for potentially relevant inspiration for design - by - analogy , and how this is similar or di\ufb00erent from the output of an 12 8 1 40 11 7 19 4 39 33 6 30 23 28 2621 24 34 22 36 41 43 14 9 31 10 44 35 42 13 37 32 27 16 18 5 17 25 38 29 45 15 3 20 2 coil , anchor , pallet , escape , arbor sheave , rope , ratchet , lever , latch staple , stick , arm , push , magazine pot , support , plant , rod , port product , toy , sort , screw , cam blood , pump , needle , rotor , hypodermic fuel , valve , liquid , diaphragm , combustion bale , steer , envelope , print , member umbrella , rib , towel , tent , fasten insert , chip , hold , port , cut electrode , prosthesis , shaft , core , key valve , ablate , bottle , probe , damp Figure 4 Algorithm output structure of 45 patents with cluster labels , highest cosine similarity method . Table 1 Correlations between participant and algorithm - generated structures of patents E1 E2 E3 E4 Algorithm E1 0 . 22 a 0 . 21 a 0 . 02 0 . 24 a E2 0 . 22 a 0 . 10 a 0 . 01 0 . 11 a E3 0 . 21 a 0 . 10 a 0 . 00 0 . 15 a E4 0 . 02 0 . 01 0 . 00 0 . 17 a Algorithm 0 . 24 a 0 . 11 a 0 . 15 a 0 . 17 a Note : Values are Phi contingency coe\ufb03cients . a denotes statistical signi\ufb01cance at the . 01 level ( 2 - tailed ) 12 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 algorithm we have that tries to do this automatically for large collections of patents . The experts were then tasked with reading , processing , and placing the design problem and the 45 mechanical patents onto a large sheet of paper on the wall , such that the physical proximity between the patents connoted functional similarity , and physical proximity of patents to the design problem connoted relevance to the solving of that design problem . In addition , it was requested that the experts attempt to build the patent space in a way that fa - cilitates searching through the collection of patents for potential inspiration for a design problem . It was emphasized that the space of patents should cap - ture functional similarity , and not necessarily problem or technological domain similarity . To illustrate this point , experts were given the example of placing a patent for a car suspension system near a patent for a running shoe based on the fact that shock absorbing is a function that is key in both patents . This phase of the procedure took approximately 5 min . 3 . 6 . 2 Participant patent space creation The experts were given the design problem description along with the \ufb01rst 5 patents ( in numerical order , see Appendix A ) . They were told to \ufb01rst read the design problem and think about it for a few min . They were explicitly asked to try not to solve the design problem per se , but to think about the design problem as contextualizing information with which to approach the building of the patent space . Table 2 Logistic regression of algorithm pairwise patent links on number of experts judging pairs as linked B S . E . Wald df p Exp ( B ) 95 % C . I . for Exp ( B ) Lower Upper Constant (cid:3) 2 . 59 . 14 339 . 48 1 0 . 00 . 08 Expert links 0 . 93 . 11 68 . 37 1 0 . 00 2 . 53 2 . 03 3 . 15 Table 3 Algorithm structure nodes evaluated by experts during interview Node # of nodes away from design problem Patent # Patent name 1 0 27 Induction loop vehicle detector 16 Escapement mechanism for pendulum clocks 18 Earthquake isolation \ufb02oor 32 Accelerometer 2 1 5 Interactive toy 17 Single drive , multi - screw sorter with pusher means 25 Hanger tilt mechanism for hanging transportation apparatus 3 3 15 Fuel injection system for linear engines 29 Smoke generating apparatus 2 Self - cleaning pressure relief and bypass valve , dispensing apparatus and method 45 Animal waterer Expert representation of design repositoxy space 13 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 After this , experts were told to read each patent , look over the associated key patent \ufb01gure , and place a sticky note on it , writing down key functions or sub - functions of the patent as described . This functionally focused note was meant to serve as a quick reference for them as they built and iterated on the patent space , as well as to remind them to focus on the functionality of the patents as they are placing them in the space . After these six items were read and pro - cessed , the experts were videotaped placing the patents on the large piece of paper on the wall . They were given markers and told that they could annotate the structure as they progressed , and should feel free to modify the placement of the patents or the content of the sticky notes at any point in the patent space building process . After placing the design problem and \ufb01rst \ufb01ve patents in the space , the experts were given the remaining 40 patents in sets of 10 ( in study index number order as shown in Appendix A ) , with a total of four sets of 10 . Each set of 10 patents Table 4 Mean expert reactions to each of the algorithm nodes Participant Node 1 Node 2 Node 3 E1 1 . 00 1 . 00 1 . 00 E2 1 . 00 0 . 25 1 . 00 E3 1 . 00 0 . 00 1 . 00 E4 1 . 00 1 . 00 0 . 75 Mean \u201cSensibility\u201d 1 . 00 0 . 56 0 . 94 % Outright Yes 100 % 50 % 75 % 1 \u00bc Outright Yes , 0 . 75 \u00bc Yes , but there\u2019s an odd one out , 0 . 25 \u00bc Ok , if you insist , but I really wouldn\u2019t put them together unless I had no choice , and 0 \u00bc Outright No Table 5 Summary of expert reactions to algorithm\u2019s distance orderings Participant Summary of response Representative quotes E1 Makes sense The ordering of it makes sense to me , the order of how far away they are . E2 Overall relative ordering makes sense , although he would like to split node 1 into 2 and make part of it ( 16 and 18 ) a little further out , but still closer than node 2 . But I would actually almost do it like , these two , 32 and 27 , then these two 16 , 18 . And then those [ group 2 ] , your other group , and then those [ group 3 ] E3 Ordering of 1 and 3 makes sense ( doesn\u2019t like 2 , so doesn\u2019t want to talk about it ) Um , so yeah that makes sense , although this one [ group 2 ] , I , like I said . So like if this one [ group 1 ] were , if this were in the upper left hand corner , that\u2019s sort of your origin or your center . You know , I\u2019d probably put this [ group 3 ] , you know , closer to that than I would put this , \u2018 cause I just don\u2019t know what the heck this , this group [ group 2 ] means . E4 Makes sense Ok , I understand why that is 14 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 was read , processed , and placed on the wall before proceeding to the next set of 10 . The placement of the patents on the wall was videotaped for all sets . Once \ufb01nished , the experts were told to take a few min to ensure they were satis\ufb01ed with their placement of patents in the space , and to move them around if neces - sary until the placement was found to be satisfactory . The patents were pro - cessed in the same order across experts to eliminate any e\ufb00ect order of exposure or processing of the patents might have on their creation of the pat - ent space . Overall , this phase of the procedure took approximately 1 h and 15 min . 3 . 6 . 3 Participant explanation of structure On video , the experts were then asked to walk the experimenters through the structure , explaining any groups that emerged , the logic behind the organization of the space and regions of patents within the space , and the signi\ufb01cance of distance of patents from each other or from the design problem . They were asked to write down key words or labels on the pa - per itself describing their regions , or clusters of patents , as they talked through them . In addition , the experimenters asked the experts to draw lines indicating borders , clusters , and \ufb02ows , etc . where appropriate to clarify their patent space and the meaning behind it . This phase of the procedure varied between experts , but took approximately 10 min , on average . 3 . 6 . 4 Participant interview regarding algorithm output structure The experts were then asked to examine the structure that constituted the output of the algorithm ( see Figure 2 ) ; the method used to generate this struc - ture is described in Section 3 . 5 . This structure examination was performed sys - tematically for three di\ufb00erent nodes , and audio and video recordings of these interviews were made . The design problem is in the upper left corner , and indi - cated by the shaded circle . The nodes examined were the upper left corner , Table 6 Summary of expert proposed labels and LSA generated cluster labels Participant Node 1 Node 2 Node 3 E1 Motion dampening related to magnetic \ufb01elds Applying small forces to create larger movements Fluid movement and control E2 Energy transfer , energy capture Complex mechanical systems Hydraulic output control E3 Motion conversion N / A Fluid transfer E4 Oscillatory motion as input Rotation as input Dispensing \ufb02uid ( in a discrete amount ) HighestAverageRank Mean , show , operate , present , cause Part , rotate , shaft , drive , mechanism Valve , open , provide , pressure , connect HighestCosine Coil , anchor , pallet , escape , arbor Product , toy , sort , screw , cam Fuel , valve , liquid , diaphragm , combustion Expert representation of design repositoxy space 15 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 containing patents 32 , 18 , 16 , and 27 ; the left middle , containing patents 5 , 17 , and 25 ; and the bottom middle left , containing patents 29 , 2 , 45 , and 15 . Ex - perts were provided with half sheet descriptions of the patents in each of the three nodes , including the title , abstract , and one key \ufb01gure , and were asked the following questions for each of the three nodes , examining one node at a time : Node Grouping Evaluation Questions ( asked regarding three separate nodes ) The algorithm puts patents in the same node that are closely functionally related . Does it make sense to you that these patents are in the same node ? What key functional relationships do you think make them go together ? Are any of these relationships ones you hadn\u2019t thought of before ? Does the grouping of these patents yield any fresh insights or ideas that you think might be useful for the design problem ? Questions Regarding Reactions to Distance of Clusters from Design Problem The algorithm places nodes with respect to the design problem based on potential relevance . So , the patents in Node 1 ( distance 0 ) are supposed to be pretty similar / relevant to the design problem , followed by the patents in Node 2 ( distance 1 ) and then Node 3 ( distance 3 ) . Does this node place - ment make sense ? Why or why not ? General Reactions Question Do you have any overall impressions about this structure with respect to its usefulness for supporting creative design ideation ? If you were to use a structure like this for design by analogy ideation , would you \ufb01nd it useful to have labels describing the functional similarity of the patents within the clusters ? This phase of the procedure also varied between experts , but on average , the interview phase lasted 15 e 20 min . 4 Results The data collected in this study was very rich , allowing for a number of ways to examine the outcomes . The main analyses presented here include a quantita - tive comparison of the structures generated by all experts to one another and to the algorithm\u2019s output structure , a comparison of expert - suggested cluster labels to LSA - generated cluster labels , and a summary of key patterns in the audio and video data . The patent spaces generated by each expert are included in Appendix B . 16 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 4 . 1 Pairwise associations of patents The patent spaces or \u201cstructures\u201d that were created by the experts were diverse in arrangement , meaning , and format . The clear commonality between the participant structures and the algorithm\u2019s output structure is the clustering or grouping of patents . Thus , to examine the degree to which experts ( and the algorithm ) agreed on patent clustering , we examined correlations between structures at the pairwise - link level . Speci\ufb01cally , for all 990 possible pairwise links between the 45 patents , we noted whether each expert ( and the algorithm ) posited a link or not . Table 1 shows that experts 1 e 3 had statistically signi\ufb01 - cant similarities to each other at the pairwise - linking level ; however , correla - tions are small , in part re\ufb02ecting the correlation analysis\u2019s correction for chance agreement ( which is high in binary - to - binary correlations , and espe - cially high when the ratio of non - links to links is very large ) , and in part demonstrating substantive variation among experts\u2019 representations of the space . Some clarity on the reason for these variations is proposed in Section 4 . 5 . 2 , in which the analyses of video and audio data are presented . Notably , the algorithm correlated signi\ufb01cantly with all experts , including E4 . This is important to highlight , as the lack of correlation between E4 and the other 3 experts suggests that this expert was thinking di\ufb00erently about the inter - relationships between the patents and the design problem , and yet the algo - rithm was still able to incorporate some of that perspective into its structure . From the perspective of developing a tool that maps a space of patents and enables automated search for potentially relevant and inspiring patents , correctly not linking two patents that experts generally agree should not be linked is equally as important as correctly linking two patents that experts agree should be linked . A high rate of spurious links would render the tool practically useless , as any query for potentially inspiring patents within the structure would return a deluge of results that included a large number of irrel - evant patents . Furthermore , as there seems to exist nontrivial variation in terms of expert representations of patent spaces ( as indicated by the statisti - cally signi\ufb01cant but small inter - correlations ) , a useful tool should probabilis - tically track the degree of overlap between expert representations , such that patent links that at least one expert proposed should be proposed by the tool with a low but nonzero probability , and patent pairs that more experts agree are linked should be linked by the tool with a higher probability . To formally test whether our algorithm not only correctly proposed expert - agreed - upon links , but also correctly rejected expert - agreed - upon non - links , and whether the algorithm\u2019s link assignments probabilistically tracked the de - gree of agreement among experts for links , we \ufb01tted a logistic regression model predicting the probability of the algorithm linking any two patents , with expert links as the predictor , where expert links indexed the degree of agreement among experts on a given link , ranging from a minimum of 0 if no expert Expert representation of design repositoxy space 17 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 judged the pair as linked , and a maximum of 4 if every expert judged the pair as linked . The results of this analysis are shown in Table 2 . The overall model was statistically signi\ufb01cant , c 2 ( 1 ) \u00bc 70 . 75 , p < . 000 , and a Hosmer and Leme - show test of deviations from the logistic model turned out nonsigni\ufb01cant , c 2 ( 1 ) \u00bc 0 . 31 , p \u00bc . 58 , indicating a model with adequate \ufb01t . As shown in Figure 5 , for patent pairs for which no experts proposed a link , the algorithm proposed links for less than 10 % of those pairs , the proportion of algorithm - proposed links increased monotonically as a function of the number of expert - links , and the algorithm found 100 % of the links that all four experts agreed upon . This function is quanti\ufb01ed in the results of the logistic model , which estimated that , with each additional expert judging a patent pair as linked , the probability of the algorithm linking the pair more than doubles ( exp ( B ) \u00bc 2 . 53 ; see Table 2 ) . Taken together , the results of the pairwise patent link analysis point to the idea that the algorithm clustering of patents can be judged as \u201ccommon ground\u201d or consensus among experts within this study , both in terms of correctly grouping patents together that experts agree should be grouped , and correctly not grouping patents together that experts would agree should not be grouped . Importantly , the algorithm also appears to be able to incorporate an \u201coutlier\u201d perspective in its grouping of the patents , as evidenced by the algorithm\u2019s link correlations with E4 , despite a lack of signi\ufb01cant correlations between that expert and the other experts . 4 . 2 Expert reactions to algorithm output structure The interview of the experts\u2019 reactions to the algorithm output structure was comprised of two parts : 1 ) evaluating the sensibility of the clustering for each node , and 2 ) evaluating the sensibility of the ordering of node distances Figure 5 Proportion of patent pairs linked in algorithm output structure by number of experts judging pairs as linked . 18 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 from the design problem . The condensed \ufb01ndings for these two interview phases are presented next . 4 . 2 . 1 Expert reactions to sensibility of clustering The expert reactions to the algorithm\u2019s structure were obtained as they exam - ined three separate nodes in detail , at varying distances from the design prob - lem . Table 3 shows the contents of each node , along with their distances from the design problem . Table 4 shows the summary of expert reactions to the clus - tering of patents in the three nodes that were examined . The expert judgments were quanti\ufb01ed by the \ufb01rst two authors on a scale from 0 to 1 , where 1 \u00bc \u201cOutright Yes\u201d , 0 . 75 \u00bc \u201cYes , but there\u2019s an odd one out\u201d , 0 . 25 \u00bc \u201cOk , if you insist , but I really wouldn\u2019t put them together unless I had no choice\u201d , and 0 \u00bc \u201cOutright No\u201d . In addition , the proportion of experts that reported that the clustering \u201cmade sense\u201d or not was calculated , also presented in Table 4 . These results indicate that , overall , the experts were generally satis\ufb01ed with the clustering created by the algorithm output structure of patents . Node 2 was the only cluster of which the participants were not in majority agreement with the algorithm clustering . This may be due to di\ufb00erent conceptualiza - tions of the design problem space , or attending to di\ufb00erent features in the patents themselves when considering their clustering . Further discussion of these possible explanations is presented in Section 4 . 5 . Overall , however , the clustering of patents within the nodes examined was a\ufb03rmed by the ex - perts , and we emphasize that only one expert outright rejected the clustering in Node 2 as invalid . 4 . 2 . 2 Expert reactions to sensibility of node distances The second portion of gathering expert reactions to the algorithm output structure was gauging their feelings about the placement of the three clusters being examined in the structure with respect to one another and to the design problem location , indicated by the red circle in Figure 2 . As summarized in Table 5 , the experts generally found the node distances to make sense . Partic - ipant 3 , as seen in the previous section , did not agree with the clustering of Node 2 , and thus did not want to discuss its location in the structure . Partic - ipant 2 would have liked to see Node 1 further subdivided into two smaller clusters , grouping patents 32 and 27 in one , and 16 and 18 in the other . Overall , the locations of the nodes examined within the structure , both relative to one another and to the design problem were a\ufb03rmed as well . 4 . 3 Exploratory quantitative comparison of distance orderings To further explore the degree to which the algorithm\u2019s distance orderings were valid , we undertook an exploratory quantitative comparison of the distance orderings between the algorithm and the experts\u2019 structures . Unfortunately , 3 of the 4 experts did not appear to have an explicit overall similarity space Expert representation of design repositoxy space 19 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 that organized their structures above the cluster level , and also did not explic - itly represent distance of clusters to the design problem . E1 said he was not sure how any of the speci\ufb01c patents were related to the design problem , and did not talk about the relative relevance of each of his clusters to the design problem ; E3 did include a \u201cnot interesting\u201d category of patents ( i . e . , patents unlikely to be relevant to the design problem ) , but the overall spatial ordering of his structure was governed more in terms of a functional \ufb02ow structure than by relevance to the design problem ; E4 explicitly opposed any notion of ordering of relevance , contending that all clusters were potentially relevant . Only E2\u2019s structure bore evidence of spatial ordering of relevance to the design problem , both in the structure itself , and in the verbal content of his explana - tion of the structure . It is unclear to us why only E2 was willing to spatially order the patents by relevance e perhaps further studies with more experts might uncover whether this is due to timing ( e . g . , a general reluctance of expert designers to prune out stimuli as irrelevant in early stages of ideation ) , scale ( e . g . , greater willingness to order by relevance with more stimuli ) , or some other reason . Nevertheless , we thought it would be informative to examine the correlation between the distances of patents from the design problem in E2\u2019s structure as compared to the algorithm\u2019s structure , while bearing in mind that inferences made from this exploratory analyses should be made with care . To measure distances , the following procedure was used . First , an image of E2\u2019s structures was printed out on letter sized ( 8 . 5 (cid:4) 11 in ) paper . Next , dot markers were drawn by one of the authors at the centroid of each patent clus - ter . Finally , a standard metal ruler was used to measure the distance of each dot from the middle of the design problem . Measurements were in cm , in in - crements of 0 . 1 cm . Each patent in the cluster received the distance value of the centroid dot from the design problem . For example , if patents 1 and 4 were in cluster 1 , and the distance of the centroid of cluster 1 from the design problem was 6 cm , then patents 1 and 4 were regarded as being 6 cm from the design problem . In the algorithm\u2019s structure , each patent\u2019s distance from the design problem was the number of nodes ( shortest path ) from the patent\u2019s node location to the design problem . There is a pragmatic and a theoretical motivation for assigning distances at the cluster level rather than the patent level . Pragmatically , we had reason to believe that the experts did not pay much attention to distances at the patent level , due to constraints ( either perceived or real ) of the structuring procedure . For example , our instructions asked the experts to ( if possible ) avoid covering the titles / numbers of patents , which forces a minimum distance on the patents inside a cluster . Theoretically , this cluster - level way of measuring distance is closer to how the algorithm does so ( distance from design problem de\ufb01ned at the node level ) . 20 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 In this exploratory analysis , a statistically signi\ufb01cant and fairly sizable corre - lation was found between the distances of E2 and the algorithm ( Pearson r \u00bc 0 . 55 , p < 0 . 01 ) ( Figure 6 ) . This \ufb01nding provides a useful complement to the interview data , given the rela - tively large e\ufb00ect size and explicit examination of the agreement in distance or - derings for all 45 patents ( as opposed to the 11 in the qualitative reactions in the previous section ) , and lends strength to our conclusion that the algorithm achieves some success in organizing patents in the space in a way that is mean - ingful and sensible to experts . 4 . 4 Cluster label analysis The experts were also asked to propose functional labels for each node . Table 6 shows the labels they proposed , in addition to the labels generated by the two cluster label generation methods presented in Fu et al . ( in press - a ) , Highest Average Rank and Highest Cosine Similarity . In addition , the full cluster label set for both labeling methods are presented on the algorithm output structure in Figures 2 and 3 . On the surface , there seems to be a fair bit of overlap of the text within the labels suggested by the experts for Node 3 , to a slightly lesser extent with Node 1 , and much less with Node 2 . The experts tend to agree that Node 3 is a cluster of patents related to controlling or moving \ufb02uids . The Highest Average Rank method for cluster label generation yielded labels that indicate that \ufb02uid is the topic area common to the patents within Node 3 , but also the functionalities or components that are employed in the patents , like \u201cconnect\u201d and \u201cvalve . \u201d The Highest Cosine Similarity method of gener - ating cluster labels yielded words that are on topic with \ufb02uid applications , but much more speci\ufb01c in terms of actual processes or components , such as \u201ccombustion\u201d and \u201cdiaphragm . \u201d Experts suggested labels for Node 1 that mostly are related to motion , with one expert suggesting more energy - related labels . Similarly , the Highest Figure 6 Scatterplot of patent distances in E2\u2019s vs . algo - rithm\u2019s structures . Expert representation of design repositoxy space 21 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 Average Rank labels for Node 1 seem to be general functional terms , not spe - ci\ufb01c to motion or energy capture ; and the Highest Cosine Similarity labels , like those for Node 3 , are topical to the speci\ufb01c content of the patents within the node e though they reference mechanisms that would be useful when trying to capture energy from motion , like \u201cescape\u201d ( as in escapement ) and \u201ccoil\u201d ( as in induction ) . The expert - suggested labels for Node 2 are diverse in the speci\ufb01c words cho - sen , though they all relate to mechanical systems ( note , node 2 labels from participant 2 were erroneously not collected ) . Correspondingly , the Highest Average Rank labels make clear this common thread , with function and component labels that tie the expert labels together , like \u201cdrive\u201d and \u201cmecha - nism\u201d and \u201cshaft . \u201d The Highest Cosine Similarity labels again contain words that point more toward the speci\ufb01c patents in the cluster , like \u201ctoy . \u201d In general , as exhibited by the detailed node analysis and as can be observed in Figures 2 and 3 , the Highest Average Rank method tends to yield labels that aremoregeneral , indicatingthecommonfunctionalityorcomponentsexplaining the association of the patents within the cluster . In many cases , however , and particularly with nodes that have only one patent in them , the two methods of cluster label generation yield the same results . The Highest Cosine Similarity method , on the other hand , tends to yield labels that are very speci\ufb01c to the con - tent of the individual patents within the cluster , although sometimes containing words that may only pertain to one or two patents in the cluster . It should be noted that the patent text was not part - of - speech tagged for patents 16 , 18 , 27 , and 32 , as these were hand picked patents from the previous design by analogy study presented in Chan et al . ( 2011 ) , which may be a reason for the less descrip - tive label output from the Highest Average Rank labeling method for Node 1 . 4 . 5 Explaining inter - expert and expert - algorithm di\ufb00erences 4 . 5 . 1 Similarities in patent function labels Why did expert structures not completely agree with one another ? Did they agree what the categories of clusters should be but made somewhat di\ufb00erent decisions about which patents below inside each cluster ? An analysis of the overlap among experts between function labels given to each patent can pro - vide some insights . Because of synonyms and near synonym relationships among words , expert e expert similarity between labels should be determined along a semantic overlap dimension rather than exact match dimension . The cosines between words in the LSA vector word representation used by the computational algorithm can serve as an approximation of semantic overlap between words ( Landaurer & Dumais , 1997 ) . Figure 7 plots the mean cosines across patents for three comparisons : ( 1 ) be - tween experts\u2019 function labels for each patent ( 1st column ) , ( 2 ) between 22 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 experts\u2019 function labels and abstracts for each patent ( 2nd column ) , and ( 3 ) be - tween experts\u2019 labels and full - text for each patent . The mean cosine between abstract and full - text across patents is used as a baseline measure against which to compare the mean cosines . This choice of baseline re\ufb02ects our assumption that , in situations where a patent is transformed into a di\ufb00erent , potentially sparser representation ( e . g . , moving between di\ufb00erent experts\u2019 rep - resentations of a patent , summarizing patent in an abstract ) , the transforma - tion from patent full - text to abstract would be a sensible \u201cgold standard\u201d with respect to degree of similarity between transformations , to the extent that pat - ent writers attempt to create a reasonably comprehensive and accurate sum - mary of the patent in the abstract . The \ufb01rst comparison column ( vs . other ) gives the mean cosine of each expert against the other experts , averaged across experts for each patent , and then averaged across patents . If the experts in general were attending to similar as - pects of each patent , one would expect their mean cosine with each other for each patent to at least approximate the mean cosine between the abstract and full - text for that patent . The \ufb01gure clearly shows that this is manifestly not the case : on the contrary , the cosine values clearly indicate that the average simi - larity between experts is dramatically lower than the average abstract e full - text similarity . The second comparison column ( vs . abstract ) gives the mean cosine of each expert\u2019s functional representation of each patent with the patent\u2019s abstract , averaged across patents . The \ufb01gure clearly shows a range of similarities with the abstract , suggesting divergent approaches to representing each patent , perhaps with some experts attending to functional aspects of each patent in a more holistic , high - level manner ( E1 and E2 ) , and others attending to Figure 7 Mean semantic cosine similarity of expert functional patent labels compared to other experts , patent abstracts , and patent full text . Expert representation of design repositoxy space 23 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 much more focused , detailed aspects of each patent ( E3 and E4 ) . The third comparison column yields a similar insight . Overall , the authors interpret these data to suggest that the divergence between experts in the overall structures stems at least in part from their attending to di\ufb00erent aspects of each patent . In support of this interpretation , the inter - expert cosine for each patent was signi\ufb01cantly positively correlated with both average expert vs . abstract ( r \u00bc 0 . 49 , p < 0 . 01 ) and expert vs . full - text co - sines ( r \u00bc 0 . 35 , p < 0 . 05 ) , suggesting that the degree to which , on average , the experts were attending to the patents in a holistic manner ( i . e . , in accord with the full - text or abstract - level summary of the patent , rather than focusing on speci\ufb01c aspects of the patent apart from its core functionality ) was a key deter - minant of the average similarity between experts . Considering the average complexity of patents , this behavior of the experts is not unexpected . 4 . 5 . 2 Protocol video and audio interview analysis It is clear not only from qualitative analysis of the structures , but also from the correlations shown in Table 1 , that the structures created by the experts are diverse . The video and audio explanation of the structures both during the cre - ation and during the \u201cwalk through\u201d shed light on the reasons for this vari - ability . Some representative quotes from the interviews with the experts at di\ufb00erent stages in the experiment are listed as follows . The common thread among these expert quotes is that they all o\ufb00er some indication that the framing of the space prior to working with the patents , or contextualizing the space with the design problem itself , leads to the experts building the space , categorizing the patents , and attending to the content within the patents in di\ufb00erent and highly in\ufb02uenced ways . The quotes have been edited to remove speech dis\ufb02uencies . \u201cWell I guess in general I thought about people , people moving around , and people moving things around and those are things that , those are activities that , that happen , particularly , in the third world there\u2019s a lot of mechanic - , use of the body for labor . So , so when I thought about that , I thought about well how do you attach things to the body , and I thought about that . Let\u2019s see . And then also how do you attach things , how do you attach a cart , or connect a cart , and so you might be carrying water or something . And you might as well carry it on a cart , and it has wheels on it , and maybe those wheels spin , and there\u2019s a small amount of generation of electricity from that , that rotation of the wheels . And then , you\u2019re pushing a cart or you\u2019re pulling a cart . So these are all related to the collecting power from a cart\u201d ( E3 , while explaining structure ) \u201cIt depends what path you were going down . If you said I\u2019m doing something mechanical and you\u2019re describing the escapement mechanism , then that\u2019s \ufb01ne . But otherwise I\u2019m not sure . Again , I would go more with bicycle and 24 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 gears or bicycle and rotary motion . \u201d ( E2 , while evaluating node distances in algorithm output structure ) In addition , there was evidence that the experts experienced signi\ufb01cant cogni - tive load when performing the task , indicating that the automated generation of structures would be bene\ufb01cial in terms of removing the expense of energy and time from the designer during patent exploration and organization . Some representative quotes are below . \u201cSome of it , I think , some of it is wherever your starting \ufb01lter is , how you\u2019re going to sort these things , and what my groupings are . You\u2019ve already , once you start to choose those , you\u2019ve headed down a path , so if you forced a , had some kind of forced starting grouping methodology or some kind of grouping \ufb01lter\u201d ( E1 , while evaluating clustering in algorithm output structure ) \u201cIf I could see all the groups , maybe I\u2019d put it in a di\ufb00erent , di\ufb00erent kind of sort . \u201d ( E4 , while evaluating node distances in algorithm output structure ) 5 General discussion The research questions underlying this work were the following : 1 . How is the algorithm\u2019s patent structure similar to or di\ufb00erent from how an expert designer might structure a space of patents in preparation to be explored for inspiration through design by analogy with regard to a spe - ci\ufb01c design problem ? 2 . If the structure is di\ufb00erent , can experts nevertheless make sense of it and perhaps see new connections between patents ? Our central \ufb01nding was that the algorithm output structure of patents was a good representation of the consensus of experts ( to the extent that such consensus exists ) regarding the associations and clustering of patents in the space . Speci\ufb01cally , the pairwise links generated by the algorithm correlate signi\ufb01cantly with all experts\u2019 links , even with E4 , whose links did not correlate with the other experts , thereby capturing a diversity of expertise , which is important in the increasingly diverse and interdisciplinary nature of engineer - ing design ( Reich & Shai , 2012 ) . Additionally , our logistic regression analysis demonstrated that the algorithm was able to probabilistically track the degree of expert consensus on patent pairwise links , pairing patents experts agreed should be paired , not pairing patents experts agreed should not be paired , and assigning links with nonzero probabilities as a function of degree of expert agreement in areas of potential disagreement . From the verbal accounts given by the experts , both during their working with the patents to create their own patent space , and during the interview Expert representation of design repositoxy space 25 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 regarding the algorithm - generated structure , some additional key \ufb01ndings emerged . First , the general consensus with regard to the algorithm output structure clustering is that it makes sense , and in some cases , was both sensible and surprising e indicating that the output from the algorithm is intuitive to expert designers , and can potentially even help them conceptualize the space in new and useful ways , indicated by some interview data . Second , the node dis - tances with respect to the design problem and the interrelationships between the clusters examined were found to make sense to the experts . The explor - atory quantitative comparison of distance orderings in Section 4 . 3 converges with this \ufb01nding , showing a signi\ufb01cant correlation between the node distances created by E2 and the node distances generated by the algorithm . This strong overlap is of particular interest , as it indicates that the organization of the overall space itself can be meaningful , not just the clustering of patents e moti - vating the use of structures for exploring patents as opposed to simple catego - rization or grouping methods . Thus , the answer to the research questions posed is that the algorithm did pro - duce similar and di\ufb00erent patent spaces when compared to expert thinking , but in bene\ufb01cial ways for both outcomes . The algorithm - generated patent space was similar to expert thinking in that it was strongly correlated with their com - mon ground , and found by the experts to be logical and sensible in clustering , node distances , and node labels . The algorithm - generated patent space was di\ufb00erent from expert thinking in that the clustering of patents by functional similarity was not highly correlated with any individual expert , and found to be surprising at times ( based on interview data ) , while still being sensible to the experts . Most importantly , though , the experts did \ufb01nd the results meaningful . Returning to the potential outcomes outlined in Figure 1 , we interpret the re - sults of this work to be a combination of outcome 1 , the algorithm does what an expert would be able to do ( in this particular case , while further studies using larger expert pools are needed ) , but more e\ufb03ciently , and outcome 2 , the algo - rithm may structure the analogical stimuli in a way that potentially allows for novel connections to be made between patents . The algorithm - generated patent space was similar to the consensus of the experts\u2019 understanding and represen - tation of the patent space , making it much more e\ufb03cient than experts in creating a meaningful patent space . However , the algorithm - generated patent space was also meaningfully di\ufb00erent from each individual expert\u2019s represen - tation of the patent space , and found at times to be surprising to individual experts while still being sensible . This suggests that designers who traverse the algorithm\u2019s structures may be able to discover novel connections between patents . From a practical standpoint , the algorithm output structure o\ufb00ers a more objective conceptualization of the space , while saving the designer time 26 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 and energy . The experts reported that the task of organizing the patents into the space was one in which it was di\ufb03cult not to get locked into a represen - tation early on , and keeping track of all of the information and aspects was challenging . This indicates that the automated patent structuring achieved by the methodology presented in Fu et al . ( in press - a ) could provide signif - icant advantages to designers by presenting them with useful , sensible , and even sometimes surprising representations of patent spaces while saving them signi\ufb01cant mental e\ufb00ort and time . It took the algorithm less than 45 min to optimally generate 8 di\ufb00erent structures , running on a Windows 64 - bit dual core desktop computer , while it took the experts approximately 1 . 25 h to generate one representation of the patent space . As the number of patents grows , the time for the human would increase likely in a non - linear way due to fundamental limitations with human memory ( Cowan , 2000 ) . Perhaps more importantly , it is unlikely that a designer would take the time to structure large numbers of patents by hand , even though this work has shown it potentially valuable . The algorithmic structuring of pat - ents could be viewed as operating like another expert , providing a view that overlaps with that of other experts , while being somewhat di\ufb00erent as well . 6 Conclusion From an inspirational standpoint , the algorithm output structure o\ufb00ers a conceptualization of the space that is sensible to four expert designers , while still maintaining an element of surprise and unexpected representation of the space , making it a promising basis for a computational design by analogy inspiration tool . By allowing for more e\ufb03cient and insightful access to external analogical stimuli , designers have the potential to create more inno - vative design solutions . With a way to extract the interrelatedness and inter - connectedness of patents in the space , designers might be able to strategically choose which cross - domain designs to expose themselves to , or even traverse the space in a more intentional and meaningful exploratory way . Addition - ally , by making novel connections between patents , designers might be able to discover novel solutions via altered representations of the problem or design space . Explicit tests of such e\ufb00ects should be explored in future work . Acknowledgments The authors would like to thank Dr . Kristin Wood for his comments and in - sights on this work . This work was supported by the National Science Foun - dation , under grant CMMI0855326 . Expert representation of design repositoxy space 27 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 Appendix A 45 P atents used in algorithm generated structure Study index number US patent number Patent title 1 6 , 082 , 923 Converging Sphere Joint Assembly b 2 5 , 984 , 148 Self - Cleaning Pressure Relief and Bypass Valve , Dispensing Apparatus and Method 3 5 , 375 , 948 Cutting Insert for Cutting and Grooving Tools 4 6 , 367 , 521 Gravity Feed Fluid Dispensing Valve 5 6 , 497 , 607 Interactive Toy 6 4 , 853 , 977 Patient Garment 7 5 , 993 , 410 Adjustable Probe 8 4 , 223 , 996 Apparatus for Mixing Solid and Liquid Constituents of Mortar or The Like 9 4 , 589 , 668 Wheeled Cart with Removable Skis 10 3 , 962 , 735 Movable Bulkhead with Guiding and Overcanting Prevention Means 11 4 , 124 , 051 Shock Absorbing Wheel Hub 12 4 , 984 , 583 Air Bubbling Mats for Therapeutically Agitating Bath Water 13 7 , 175 , 212 Latch Having Releasable Cinching Mechanism 14 4 , 259 , 034 Bale Handling Apparatus 15 6 , 634 , 325 Fuel Injection System for Linear Engines 16 4 , 139 , 981 Escapement mechanism for pendulum clocks a 17 5 , 909 , 815 Single Drive , Multi - Screw Sorter with Pusher Means 18 4 , 402 , 483 Earthquake isolation \ufb02oor a 19 4 , 103 , 708 Ventilated Poppet Damper 20 3 , 964 , 473 Bone Prosthesis 21 4 , 705 , 064 Safety Seal for an Operating Lever 22 6 , 142 , 689 Envelope Leveler for Printer Feeder 23 5 , 273 , 173 Screw Top 24 5 , 438 , 724 Method for Using Plastic Fasteners for Shoe - Lasting Applications 25 5 , 234 , 096 Hanger Tilt Mechanism For Hanging Transportation Apparatus b 26 4 , 867 , 134 Fluid - Heating Solar Collector 27 4 , 568 , 937 Induction loop vehicle detector a 28 6 , 109 , 282 Self - Erecting Loop Structure 29 4 , 303 , 397 Smoke Generating Apparatus 30 5 , 899 , 571 Beach Towel , Tote Bag and Beach Umbrella System 31 6 , 234 , 452 Hand Operable Motorcycle Stand 32 4 , 335 , 611 Accelerometer a 33 5 , 085 , 240 Shelter Structure b 34 6 , 634 , 044 Compact Stretcher 35 4 , 270 , 310 Support Device for an Upstanding Plant Support Rod in a Plant Pot 36 5 , 423 , 097 Emergency Drop Fowler and Gatch 37 5 , 277 , 276 Compensating rope sheave tie down b 38 3 , 938 , 909 Single Needle Alternating Flow Blood Pump System 39 5 , 647 , 066 Safety Helmet Visor 40 6 , 119 , 041 Apparatus and Method for Linear Lesion Ablation 41 4 , 484 , 762 Ski Binding and Boot 42 4 , 762 , 262 Side - Fed Stapler 43 6 , 164 , 698 Steering Device for Automobiles 44 6 , 062 , 856 Dental Implant Hole Guide Extension 45 4 , 739 , 727 Animal Waterer a Far Analogical Stimuli from Chan et al . ( 2011 ) . b Far Analogical Stimuli from Fu et al . ( 2012 ) . 28 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 Appendix B Expert representation of design repositoxy space 29 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002 References Ahmed , S . , & Christensen , B . T . ( 2009 ) . An in situ study of analogical reasoning in novice and experienced design engineers . Journal of Mechanical Design , 131 ( 11 ) , 111004 . Altshuller , G . S . , & Shapiro , R . B . ( 1956 ) . 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A conceptual design model using axiomatic design , functional basis and TRIZ . Paper presented at the Proceedings of the 2007 IEEE IEEM . 34 Design Studies Vol - - No . - - Month 2013 Please cite this article in press as : Fu , K . , et al . , Expert representation of design repository space : A comparison to and validation of algorithmic output , Design Studies ( 2013 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2013 . 06 . 002", + "roudot2023utrack3d": "Article u - track3D : Measuring , navigating , and validating dense particle trajectories in three dimensions Graphical abstract Highlights d u - track3D provides an improved algorithm and work\ufb02ow for particle tracking d The algorithm quanti\ufb01es molecular traf\ufb01cking , polymerization , and transcription d Dynamic regions of interest reveal collective events nested in the cellular volume d Trackability score enables the automatic localization of tracking errors Authors Philippe Roudot , Wesley R . Legant , Qiongjing Zou , . . . , Reto Fiolka , Eric Betzig , Gaudenz Danuser Correspondence philippe . roudot @ univ - amu . fr ( P . R . ) , gaudenz . danuser @ utsouthwestern . edu ( G . D . ) In brief 3D microscopy of entire cells presents an image complexity that challenges the computation and observation of measurements such as particle trajectories . Roudot et al . introduce u - track3D , a package that combines robust particle tracking with image navigation and track validation algorithms for the discovery and unbiased analysis of dynamic processes . Roudot et al . , 2023 , Cell Reports Methods 3 , 100655 December 18 , 2023 \u00aa 2023 The Authors . https : / / doi . org / 10 . 1016 / j . crmeth . 2023 . 100655 ll Article u - track3D : Measuring , navigating , and validating dense particle trajectories in three dimensions Philippe Roudot , 1 , 4 , 7 , * Wesley R . Legant , 2 , 3 Qiongjing Zou , 1 Kevin M . Dean , 1 Tadamoto Isogai , 1 Erik S . Welf , 1 Ana F . David , 5 Daniel W . Gerlich , 5 Reto Fiolka , 1 Eric Betzig , 6 and Gaudenz Danuser 1 , * 1 Lyda Hill Department of Bioinformatics , UT Southwestern Medical Center , Dallas , TX , USA 2 JointDepartmentofBiomedicalEngineering , UniversityofNorthCarolinaatChapelHill , NorthCarolinaStateUniversity , ChapelHill , NC , USA 3 Department of Pharmacology , University of North Carolina , Chapel Hill , NC , USA 4 Aix Marseille University , CNRS , Centrale Marseille , I2M , Turing Centre for Living Systems , Marseille , France 5 Institute of Molecular Biotechnology of the Austrian Academy of Sciences , Vienna BioCenter , Vienna , Austria 6 Department of Molecular & Cell Biology , University of California , Berkeley , Berkeley , CA , USA 7 Lead contact * Correspondence : philippe . roudot @ univ - amu . fr ( P . R . ) , gaudenz . danuser @ utsouthwestern . edu ( G . D . ) https : / / doi . org / 10 . 1016 / j . crmeth . 2023 . 100655 SUMMARY We describe u - track3D , a software package that extends the versatile u - track framework established in 2D to address the speci\ufb01c challenges of 3D particle tracking . First , we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the stan - dard 3D test dataset of the particle tracking challenge . These analyses indicate that u - track3D presents a tracking solution that is competitive to both conventional and deep - learning - based approaches . We then present the concept of dynamic region of interest ( dynROI ) , which allows an experimenter to interact with dy - namic 3D processes in 2D views amenable to visual inspection . Third , we present an estimator of trackability that automatically de\ufb01nes a score for every trajectory , thereby overcoming the challenges of trajectory vali - dation by visual inspection . With these combined strategies , u - track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences . INTRODUCTION Light - sheet \ufb02uorescence microscopy ( LSFM ) achieves three - dimensional ( 3D ) imaging with minimal phototoxicity , fast sam - pling , and near - isotropic resolution , 1 , 2 allowing the study of dy - namic intracellular processes in the entire cellular volume . 1 \u2013 4 While computer vision techniques are well established for inter - rogating cell biological processes in 2D , 5 these tools do not translate to both the visualization of measurement results and their validation in 3D . A key challenge for image analysis in 3D is the user interaction with the data . The manipulation of time - lapse 3D image volumes is often cumbersome , and any of the projection mechanisms necessary to map the 3D volume into a 2D representation on a screen is prone to artifacts that may cause erroneous conclusions . 6 Thus , computational tools for 3D image analysis must be able to reveal the complexity of 3D cellular and sub - cellular processes while being as automated as possible to avoid selection and perception biases . The most elementary way to measure the behavior of intracel - lular processes is particle tracking . Particles can comprise sub - diffraction - sized objects that appear in the image volume as bona \ufb01de spots , objects of an extended size that appear as a rigid structure , and larger deformable objects . The more complex the object\u2019sshapeis , themoresophisticated the methods neededfor particledetection . Theproblemofparticletrackingisthende\ufb01ned as the reconstruction of plausible trajectories from the coordi - nates [ x ( t ) , y ( t ) , z ( t ) ] oftheidenti\ufb01edparticles . Because the number of hypothetical trajectories grows super - exponentially over time , MOTIVATION Particle tracking is a ubiquitous task in the study of dynamic molecular and cellular pro - cesses through microscopy . Light - sheet microscopy has opened a path to acquiring complete cell volumes for investigation in three dimensions . However , hypothesis formulation and quantitative analysis have re - mained dif\ufb01cult due to fundamental challenges in the visualization and the veri\ufb01cation of large and dense sets of three - dimensional ( 3D ) particle trajectories . New software tools are required that allow microsco - pists to automatically track diverse particle movements in 3D , inspect the resulting trajectories in an infor - mative manner , and receive unbiased assessments of the quality of trajectories . Cell Reports Methods 3 , 100655 , December 18 , 2023 \u00aa 2023 The Authors . 1 This is an open access article under the CC BY - NC - ND license ( http : / / creativecommons . org / licenses / by - nc - nd / 4 . 0 / ) . ll OPEN ACCESS many approaches have been proposed to approximate the best solution . 7 \u2013 9 They typically combine a modeling of intracellular dy - namics and transient disappearances ( or mis - detection ) , statisti - cal approaches , 10 \u2013 16 or recursive neural networks 17 \u2013 19 to esti - mate the likelihood of trajectory - to - measurement associations and discrete optimization 10 , 20 \u2013 22 to select the best set of associ - ations at each time point . Only a few of these methods have been implemented in 3D 7 , 11 , 23 , 24 and even fewer tackle the visualiza - tion and validation challenges in dense sets of trajectories . Several open - source 25 \u2013 29 and commercial 30 \u2013 32 software pack - ages have been released to make volumetric rendering technol - ogy amenable to time - resolved 3D bioimaging data . Together they have proposed advances in accelerating sequence rendering , 26 , 28 modularity and extensibility , 25 , 27 annotation tools , 33 , 34 or scalable raytracing for high - quality rendering . 29 , 35 Those approaches provide an outside view of the 3D data and a range of tools to investigate the volume manually ( e . g . , in focused regions of interest [ ROIs ] or slices ) . They also reveal many chal - lenges associated with visual occlusions , 3D perception on a \ufb02at screen , interactions , and time - consuming volume exploration . BigDataViewer 36 has tackled the occlusion and perception chal - lenges indirectly by focusing on the caching and rendering of 2D slices at arbitrary orientations . The primary effect is to accel - erate rendering at the expense of 3D context , but it also avoids distortion of pixel - level information . As such , the Mamut software has demonstrated that combining BigDataViewer and 3D rendering of vectorial information enables annotation in large and complex 3D trajectories . 37 The immersive quality of virtual re - ality ( VR ) headsets has also been exploited to better perceive and interact with complex 3D datasets . 38 \u2013 42 Finally , a complementary approach to full volume visualization is the presentation of soft - ware - de\ufb01ned ROIs . The embryoMiner package 43 implements this idea by building static ROIs from groups of trajectories Figure 1 . u - track3D is a complete pipeline for the measurement , visualization , and evalua - tion of large sets of 3D trajectories The pipeline is here illustrated on lattice light - sheet imagingofHeLacellsundergoingmitosislabeledwith eGFP - labeled EB3 ( marking microtubule plus - ends , rendered in gray ) and mCherry - labeled centromere protein A ( marking kinetochores , rendered in red ) . thatare thenusedto systematicallyvisualize the data in a series of 3D tiles . The ap - proaches implemented in u - track3D also belong to this category ; however , the ROIs adapt over time to the changing geometry of collective processes . Hence , u - track3D allows the inspection of speci\ufb01c particle dynamics in a crowded and dynamic surrounding . Considerably fewer works have been dedicated to trajectory validation . Previous work can be categorized into ground - truth - based approaches and error - inference - based approaches . Within the former category , a time - consuming but widely accessible approach consists in using manual annotations to build a ground truth to be compared to the measured data - set . 24 , 44 , 45 Already in 2D , these techniques are subject to selec - tion bias , as it is easier to annotate bright and well - separated ob - jects . In 3D , these challenges are compounded by the complexity of visualization and selection of particles . Another ground - truth - based approach relies on the simulation of image sequences that mimic the acquisition . 7 , 46 \u2013 48 In contrast to the annotation - based approach , simulation can automate the pre - diction of tracking performance for simpli\ufb01ed scenarios in which image formation and particle dynamics are known perfectly a priori . The second category of methods attempts to infer the like - lihood of tracking errors directly on the data . Early efforts proposed heuristics that combine motions and density measure - ments to identify error - prone areas . 2 , 49 Arguably , a more elegant approach would be a direct analysis of the optimality of the re - constructed trajectories . This idea has been proposed by Cardi - nale and colleagues 50 and applied to estimating the scale , inten - sity , position , and associated con\ufb01dence intervals of the spindle pole body . While this con\ufb01dence interval re\ufb02ects the uncertainty about positions and velocities , the correctness of trajectory - to - measurement matches is not evaluated . Our goal was to design an algorithm for tracking error inference that does not require annotation or speci\ufb01c simulations , does not rely on heuristics other than those considered by the tracking algorithm , can handle heterogeneous scenarios , scales with the computational complexity of the tracker itself , and provides an interpretable output for mis - matched , spurious , and missing trajectories com - parable to conventional benchmarks . 7 Building upon our previous particle tracking work , 2 , 10 , 13 we thus designed the software package u - track3D to enable the measurement , observation , and validation of dynamic pro - cesses in 3D ( Figure 1 ) . u - track3D can detect and track 2 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS morphologically and dynamically diverse cellular structures , including single molecules , adhesion complexes , and larger macromolecular structures such as growing microtubules . The software design is open , allowing users to import the coordinate \ufb01les from other detection routines and then apply the u - track3D framework only for trajectory reconstruction . We introduce a li - brary for visualization and mapping of dynamic ROIs ( dynROIs ) that move with the biological structure under evaluation and enable an intuitive visualization of particle behaviors . Finally , we present a scalable approach for automatic assessment of trackability of each particle throughout the image volume by evaluating the stability of trajectory - to - assignment associations . RESULTS Measuring , visualizing , and validating 3D trajectories with the u - track3D pipeline Multiple particle tracking To generate a 3D particle tracking package , we adopted and modi\ufb01ed features that were critical for accurate particle tracking in 2D . 10 This includes the breakdown of trajectory reconstruction into a frame - by - frame association of corresponding particles fol - lowed by an association of the resulting track segments into full - length trajectories . Both steps rely on the same solution for optimal one - to - one assignments of particle detections and track segments in a bipartite graph . 10 , 51 The two - step approach per - mits the closing of temporal gaps in the detection of a particle as well as the handling of particle merging and splitting events . U - track3D incorporates a Kalman \ufb01ltering approach to model on the \ufb02y the characteristics of a particle\u2019s Brownian , directed , and heterogeneous motion , which supports both the procedure for frame - by - frame particle association and the one of track segment association . To support the concurrent tracking of ob - jects of variable sizes , we implemented a multiscale particle de - tector equipped with a generalized adaptive thresholding approach ( see section \u2018\u2018Multiscale particle detector\u2019\u2019 in STAR Methods ) . DynROIs Moving from 2D to 3D images complicates the interaction of a human observer with both raw and derived data . Widely used global image projections , including maximum intensity projec - tion ( MIP ) , and other volume rendering techniques are limited by the overlap of many dynamic structures along the viewing axis . 6 However , detailed visualization of 3D images and trajec - tories in their local context is essential for a user to adjust soft - ware control parameters and to interpret the underlying biology . Projection approaches have to be tailored to emphasize a subset of selected voxel or aspects of highest interest . Such projections should not only bring the particle or group of particles of interest into focus but also continuously adapt as the particles move . To meet this requirement , u - track3D incorporates a framework for rendering particle - centric dynROIs , thereby allowing the user to follow the particle behavior throughout its lifetime in a visually comprehensible format . DynROIs are implemented in a hierar - chical object structure across molecular , macromolecular , and cellular scales ( see section \u2018\u2018dynamic region of interest estima - tion\u2019\u2019 in STAR Methods ) . First , u - track3D provides a variety of shapes ( rectangle cuboids , spheres , cones , tubes , and rounded tubes ) to de\ufb01ne an ROI encompassing one , two , or three trajec - tories . Second , to manage larger sets of tracks , dynROIs are built by estimating an af\ufb01ne transform between the associated point cloud in consecutive time points . Finally , the top - level dyn - ROI is de\ufb01ned for the cell . For example , cells embedded in a 3D environment are often randomly oriented , and their orientation changes over time . While image - based registration can be used to correct changes in cell orientation , it is computationally expensive , especially as the size of the volume and length of the sequence grow . To reduce the computational burden , we segment and transform the cell mask into a randomly down - sampled point cloud , which is then used to estimate an af\ufb01ne transform . Trackability score Validation of tracking results is crucial for proper parameter adjustment during image acquisition and analysis as well as the biological interpretation of integrated measurements . How - ever , it remains an extremely challenging task in 3D datasets , particularly when the particle density is high . Contrary to a sce - nario in 2D where a single \ufb01eld of view presents a wide range of trajectories for visual inspection , dynROIs in 3D tend to capture only a few trajectories and cannot represent the hetero - geneity of local image quality , particle density , and dynamic properties , which all affect the tracking accuracy . To solve this problem , we complemented u - track3D with an option to compute a local trackability score . We use Monte Carlo simula - tion to determine for every trajectory and every time point the con\ufb01dence by which the algorithm was able to assign the chosen particle to the trajectory ( see section \u2018\u2018stochastic programming for the evaluation of trackability\u2019\u2019 in STAR Methods ) . Speci\ufb01cally , we exploit the particle history , the detection accuracy , and the associated motion model ( s ) to derive a trackability metric that represents the likelihood of each of the chosen associations vis - a ` - vis the set of alternative associations with neighboring par - ticles . We demonstrate the performance of the resulting score and how it can be used to compare trackability across space , time , and the molecules under study . Measurement of the kinetics of endocytosis in 3D To assess the performance of u - track3D , we investigated the dy - namics of various cellular structures imaged by light - sheet mi - croscopy ( Figure 2 ) . As reported with u - track , 10 gap closing is a crucial step in 2D particle tracking because of frequent , tran - sient disappearances : particles might not be detected , particles move in and out of the microscope\u2019s in - focus volume , or parti - cles can temporarily overlap in space . While the latter two sour - ces of disappearance are largely eliminated by proper 3D imag - ing , the challenges of false or missing detections remain . To test the performance of u - track3D in closing gaps , we examined the lifetimes of clathrin - coated structures forming at the cell plasma membrane ( Figures 2A \u2013 2C ) . These structures represent mostly sub - diffraction objects ; i . e . , they appear in an imaging volume as 3D point - spread functions . We used high - resolution diago - nally swept light - sheet microscopy 2 to sample every second a full volume of puncta generated by the GFP - labeled AP2 subunit of the endocytic coat . U - track3D recovered the canonical life - time distributions of abortive and maturing clathrin - coated pits , 52 , 53 that is , an exponential decay for abortive pits and Cell Reports Methods 3 , 100655 , December 18 , 2023 3 Article ll OPEN ACCESS Figure 2 . u - track3D supports a variety of imaging and biological scenarios ( A ) Maximum intensity projections ( MIPs ) of a rat kidney cell layer imaged with diagonally scanned light - sheet microscopy ( diaSLM ) . Cells are expressing eGFP - labeledalphasubunitoftheAP - 2complex . Greenboxis160 3 40 3 12 m m . Insetshowstrajectoriesofclathrinaggregatesclassi\ufb01edasclathrin - coatedstructures or maturing pits . ( B ) Normalizedmaximumintensityofeachtrajectoryasafunctionoflifetimeplottedforsixcellularlayerscomposedofmultiplecellseach . Thegreenlinedenotes the median of the cumulated distribution ( value T ) . ( legend continued on next page ) 4 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS Rayleigh - like distribution with maximal probability around 20 s for maturing pits ( Figure 2C ; Data S1 ; see section \u2018\u2018clathrin - medi - ated endocytosis study on a glass coverslip\u2019\u2019 in STAR Methods ) . While in 2D the identi\ufb01cation of those two populations relied on extensive trajectory analysis to discount incomplete trajec - tories , 53 our u - track3D software achieves accurate trajectory classi\ufb01cation directly by thresholding the maximum intensity of trajectories in 3D ( Figures 2B and 2C ) . Importantly , the distinc - tion of two lifetime distributions can only be obtained with the support of gap closing ( Figure 2C ) , suggesting that gaps remain a hurdle for accurate tracking in 3D . Measurement of instability in microtubule dynamics across dense 3D mitotic spindles With limited sampling frequency in volumetric imaging , particle tracking can be improved by dynamic motion models through Kalman \ufb01ltering . To assess the performance of a 3D implemen - tation of previously published motion models for 2D tracking of microtubule polymerization dynamics , 54 , 55 we imaged and tracked microtubule dynamics in HeLa cells by following GFP fusions of the microtubule plus - end tracking protein EB1 sampled at 1 Hz by lattice light - sheet imaging . 1 We quanti\ufb01ed metrics such as growth rate , growth lifetime , and pause fre - quency ( see section \u2018\u2018microtubule instability measurement\u2019\u2019 in STAR Methods ) . The latter is a measure for the probability that a stalled or shrinking microtubule , which is accompanied by disappearance of the EB1 particle in the video , is rescued to renewed growth ( see Figure S1 and Data S1 ) . Consistent with our previous observations in 2D , 54 u - track3D faithfully de - tected a dose - dependent decrease in all three metrics upon treatment of cells with the microtubule - destabilizing drug noco - dazole ( Figures 2D \u2013 2E ) . We also investigated the destabilizing effect of nocodazole on the number and duration of pauses or shrinkages ( Figure 2E ) . We then extended our analyses to mitotic cells , where the density of EB1 particles is much higher in central regions of the mitotic spindle ( see Data S1 ) . Both sce - narios show a strong response in nocodazole concentration , indicating that u - track3D properly captures the drug - induced variation of growth rate and lifetime ( Figures 2F and 2G ) , despite strong variations in particle density . Measurement of interaction between transcription factors and chromatin We then sought to investigate the impact of the depth information onthemeasurementofbiologicalquantitieswhencomparedto2D particle tracking . We employed a lattice light - sheet microscope to image the interactions between transcription factors ( TFs ) and chromatin in embryonic stem cells . In a study using the same bio - logical system but performed with 2D imaging , Chen et al . 56 had shown that TFs alternate between short - lived binding events at non - speci\ufb01cchromatinsites ( residencetime (cid:1) 0 . 75s ) , 3Ddiffusion ( average duration (cid:1) 3 s ) , and longer lived transcription events where the TF is bound at speci\ufb01c chromatin sites ( residence time (cid:1) 12 s ) . We performed the same analysis , now applying 3D tracking , and contrasted the results to the tracking of 2D projec - tions of the same 3D volumes ( Figures 2H \u2013 2K and section \u2018\u2018single molecule dynamics study with lattice light - sheet microscopy\u2019\u2019 in STARMethods ) . Ananalysis on2D projections reproduced the re - sultsoftheoriginalstudy . However , with3Danalysis , theresidence timeofspeci\ufb01cbindingeventswasreducedbyone - third ( (cid:1) 7 . 8sin 3D vs . 11 . 9 s in 2D ) . Interestingly , the shorter binding time ex - tracted from 3D trajectories is consistent with measurements per - formedinnuclearreceptorsstudies . 57 , 58 Together , thesedatasug - gest that the overlap caused by axial projections for 2D tracking may bias kinetic measurements . U - track3D leads the \ufb01eld of algorithms evaluated in the particle tracking challenge We evaluated u - track3D\u2019s competitiveness using the standard 3D test dataset of the particle tracking challenge . 7 We compared u - track3D against the top \ufb01ve approaches competing in the orig - inal benchmark as well as two tracking approaches that make use of recent advances in recursive neural networks ( RNNs ) 17 , 18 ( see Figures3andS2 ) . Thedatasetisdesignedtomimicviraldynamics alternating between con\ufb01ned and directed displacements that are large enough to create signi\ufb01cant ambiguities in the densest sce - narios ( Figure 3A ) . The dataset includes 12 sequences represent - ing four signal - to - noise ratios and three density levels ( see Fig - ure S2 ) . The challenge prescribes four metrics to compare the precision and accuracy of each approach . The \ufb01rst precision metric , alpha , relates to the Euclidean distance between the real trajectories and measured trajectories , while beta weighs this by the rate of spurious tracks . The accuracy metrics ignore distance and instead count the points within 5 pixels of a real detection . The Jaccard similarity coef\ufb01cient ( JSC ) or Jaccard index ( JI ) is computed as ( TP / N + FP ) where N is the number of real particles , TP is the number of true - positive matches , and FP is the number of false - positive matches . The JSCt describes the same metric using the count of trajectories that match at least 50 % of the seg - ments ; the rest is considered FP . Ourresultscanbesummarizedasfollows : \ufb01rst , u - track3Dranks \ufb01rst on all metrics and densities for signal - to - noise ratio ( SNR ) 4 ( C ) Probability density of lifetime for the set of trajectories above and below the threshold value T , with and without gap closing ( n = 6 cellular layers , pooled trajectories lifetimes ) . ( D ) MIP of HeLa cells in interphase imaged with lattice light - sheet microscopy ( LLSM ) expressing eGFP - labeled EB1 ( orange area is 30 3 32 3 7 m m ) . Overlay highlights EB1 trajectories . ( E ) Average microtubule lifetimes , microtubule growth rate , as well as average number and duration of pause and shrinkage events per trajectory for increasing concentrations of nocodazole ( n = 5 per conditions ; center line , median ; box limits , 25 and 75 percentiles ; whiskers , extremum ) . ( F ) MIP of HeLa cells in metaphase imaged with LLSM along with 45 (cid:3) rotation around the vertical axis . Overlay highlights EB1 trajectories . ( G ) Same as ( E ) measured for cells in metaphase ( n = 5 per conditions ) . ( H ) MIP of mouse embryonic stem ( ES ) cell nucleus imaged with LLSM expressing GFP - labeled TFs . Green box is 13 3 13 3 3 m m . Overlay highlights SOX2 trajectories . ( I ) MIP of ES cell nucleus imaged with LLSM expressing GFP - labeled TFs . Overlay highlights SOX2 trajectories tracked after MIP transformation . ( J ) Probability density of SOX2 binding time measured in LLSM overlaid with a two - component decay \ufb01t ( n = 1 cell ) . ( K ) Probability density of SOX2 binding time measured in projected LLSM data overlaid with a two - component decay \ufb01t ( n = 1 cell ) . Cell Reports Methods 3 , 100655 , December 18 , 2023 5 Article ll OPEN ACCESS and SNR 7 except for one data point where it ranks second ( Figures 3B and S2 ) . Also , u - track3D ranks \ufb01rst or among the top three approaches for lower SNR data . Strikingly , deep - learning approaches are outperformed by conventional methods , except at the level SNR = 1 . Of note , this scenario represents a breakpoint dataset that is not representative of the typical image quality targeted for a meaningful analysis of trajectory counts and lifetimes . At this low SNR , RNN - based approaches provide the best performance ; however , they are nonetheless insuf\ufb01 - ciently robust to generate accurate trajectory estimates . While a precise ablation study is out of the scope of this paper , ananalysisofthedesignofcompetingapproachescanhelpindis - cussing these performances . First , the most competitive ap - proaches in the original challenge 20 , 23 , 59 present differences in their design but they all rely on similar strategies : object detection is based on some type of adaptive thresholding , they use motion modeling for displacement prediction , they use a discrete optimization approach to assign predictions to measurements , and they employ various techniques to detect and correct for transient object disappearances . The u - track framework pursues the same strategies but incorporates piecewise - stationary motion modeling and gap closing to enhance tracking robustness during transitionsbetweendynamicregimes . Thecompetingapproaches assume stationarity in motion type ( mostly Brownian 20 ) or smooth transitions . 23 , 59 Second , the main difference between u - track3D and RNN - based tracking lies in the method that evaluates the cost of each trajectory - to - measurement association and the detection of transient disappearances . Both approaches employ a conventional detector and temporally greedy assignments . The techniques based on RNNs also use a locally adaptive detector and a discrete combinatorial optimization approach to assign pre - dictiontomeasurement . 51 Assuch , thedifferenceinperformances must lie in the method for motion and gap prediction . In a nutshell , the \ufb01rst RNN technique uses a long / short - term memory ( thus , in Figure 3 it is referred to as model - free LSTM ) network to \ufb01lter and predict particleposition . The second , morerecent , RNN tech - nique by the same authors uses gated recurrent units ( referred to as model - based GRU ) and reduces the parameter space needed for prediction by learning the moment and the covariance of a normal distribution associated with the state of each trajectory . Both approaches are trained on a simulated dataset presenting a mixture of Brownian , directed , and heterogeneous motions . In the original paper , 18 the model - based approach performs better thanboththeconventionalandthemodel - freeRNNonthe\u2018\u2018vesicle scenario , \u2019\u2019 which simulates free diffusion . However , the difference is not as clear in the \u2018\u2018virus scenario , \u2019\u2019 where the model - free approach often outperforms the latter . This suggests that the per - formanceofRNNscan improve uponthe state ofthe artwhentak - ing a priori knowledge into account to constrain the parameter space . However , asdemonstratedinourpreviouswork , 13 thesud - den transition between con\ufb01ned and directed displacement are poorly approximated by a normal distribution and better modeled byapiecewise - stationaryconstraint . Thisqualitativeanalysisdoes not imply that deep - learning approaches cannot be competitive but that further improvement requires the consideration of a priori knowledge on molecular dynamics in the design of the network architecture . Our performance analysis shows that , with the motion models implemented in the package , u - track3D Figure 3 . u - track3D performance in comparison to existing methods evaluated on a standard 3D test dataset with high particle density ( A ) MIP of the simulated virus dynamics overlayed with trajectories reconstructed by u - track3D . Trajectories are colored following a random colormap . ( B ) Performance metrics for precision ( alpha and beta ) and accuracy ( JSC and JSCt ) to compare different tracking pipelines . 6 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS remains a state - of - the - art tracker , despite the advances deep learning and other pipelines deploy . Visualization of adhesion formation in 3D We illustrate the application of a whole - cell dynROI with the study of spatial interactions between cell - matrix adhesions and \ufb02uores - cently labeled 3D collagen \ufb01bers in osteosarcoma cells imaged by axially swept light - sheet microscopy 3 ( Figure 4A ; Data S2 ) . ThedynROI allowed us tovisualize the relationshipbetweenadhe - sion shapes and its proximity to collagen \ufb01brils , showing two pop - ulationsofglobularand elongatedadhesions ( Figures4B \u2013 4D ) . The most elongated adhesions are located predominantly at the tip of pseudopodial extensions and align with the protrusive direction , while globular adhesions concentrate in the quiescent part of the membrane . Our measurements show that this elongation distribu - tion can be decomposed further ( Figure 4E ) . We found a unimodal distribution of mostly globular adhesions in close contact with collagen \ufb01bers ( see section \u2018\u2018adhesions and collagen interaction imaging and analysis\u2019\u2019 in STAR Methods ) . In contrast , adhesions withalesserdegreeofcollagencontactdisplayabimodaldistribu - tion of globular and elongated adhesions . These data suggest\u2014 quite unexpectedly from what is known in 2D\u2014that the most me - chanically engaged adhesions may be the least elongated . 60 We conjecture that adhesion elongation in 3D may be less driven by a zippering of an integrin - mediated plaque along a collagen \ufb01ber but rather dictated by the organization of cell - cortical actin \ufb01bers Figure 4 . DynROIs reveal the behavior of mo - lecular adhesions in 3D environments ( A ) Dual - colored orthogonal MIP of osteocarci - noma cells expressing eGFP - labeled paxillin and embedded in collagen labeled with Alexa Fluor 568 . Overlay highlights dynROI . ( B ) View of the dynROI . ( C and D ) Detection of adhesions colored as a function of the degree of collagen contact and elongation . ( E ) Probability density of elongation for adhesions with high and low degree of contact with collagen \ufb01bers ( n = 1 cell ) . or the local collagen architecture . Indeed , this behavior becomes apparent by replay of time - lapse sequences of the proximity and elongation parameters in the spatially stabilized dynROI ( Data S2 ) . DynROIs are thus a powerful way to assess the spatial distribution and heterogeneity of molecular interactions in highly dynamic cells . DynROI applied to mitotic spindle dynamics Many cellular processes involve a large - scale reorganization of macromolecular structures , which challenges 3D analysis . A goodexampleisthe vertebrate mitoticspin - dle . 61 Thousands of microtubules form a dense bipolar array while the two spindle poles move apart and rotate back and forth . Concurrently , spindle microtubules establish contacts with chro - mosomes at kinetochores and subsequently move chromosomes toward poles or the spindle center . This process is virtually impos - sible to understand by mere visual inspection of volume render - ings . We therefore assessed how u - track3D and dynROIs may facilitate the analysis . The image dataset comprises dual - channel time - lapse sequences of GFP - labeled microtubule plus ends and mCherry - labeled centromeres protein A marking kinetochores in mitotic HeLa cells acquired at 0 . 1 Hz by lattice light - sheet micro - scopy 62 from prometaphase to metaphase . Microtubule plus ends , kinetochores , andspindlepoleswerelocalizedbymultiscale particle detection . Pole trajectories can then be used to de\ufb01ne a dynROIthatfollowsthespindlemotion ( Figure5Aandsection \u2018\u2018dy - namic region of interest estimation\u2019\u2019 in STAR Methods ) . An embedded second dynROI follows the point cloud formed by the kinetochore trajectories ( Figure 5B and section \u2018\u2018dynamic region ofinterestestimation\u2019\u2019inSTARMethods ) . Basedonthepairofdyn - ROIs , we further constructa planardynROI withanorientation that is de\ufb01ned by the interpolar axis and a vector following the kineto - chore - associated dynROI motion ( Figures 5C and 5D ; Data S3 ; section\u2018\u2018dynamicregion ofinterestestimation\u2019\u2019inSTARMethods ) . Our framework for dynROI estimation thus enables the visualiza - tion of mesoscale structures composed of different molecular assemblies . In previous work , 62 using volume acquired at 1 Hz , we showed withspindle - widestatisticsandindirectmodelingthatkinetochore Cell Reports Methods 3 , 100655 , December 18 , 2023 7 Article ll OPEN ACCESS \ufb01berformation isacceleratedbyanaugmin - dependentnucleation and directional growth along the \ufb01ber toward kinetochores . We now use dynROIs todirectly visualize the dynamic space between spindle poles and kinetochores ( Figures 5E \u2013 5G ; Video S1 ) . We de\ufb01ne a kinetochore \ufb01ber assembly dynROI by a cone whose medialaxisconnectsspindlepoleandtargetkinetochore ( seesec - tion \u2018\u2018dynamic region of interest estimation\u2019\u2019 in STAR Methods ) . Using such dynROIs , we noted a directional bias in microtubule polymerization toward kinetochores , consistent with previous ob - servations . 62 However , wealsoobservedmicrotubulepolymeriza - tion branching off a kinetochore \ufb01ber and polymerizing toward another kinetochore ( circled in red in Figure 5G , time 53 \u2013 72 s ) . The branching was followed by rapid poleward movement of the targetedkinetochoreandanincreaseofplus - endcountinthedyn - ROI ( Figure 5G and 5H , time 93 \u2013 102 s ) suggesting that the target kinetochore was captured , generating a new avenue for microtu - bule ampli\ufb01cation . The example underscores how the dynROI li - braryimplementedinu - track3Denablesthevisualdiscoveryofdy - namic processes that are obscured in 3D image volumes . Trackability score to detect tracking ambiguities We developed a pipeline to assign a trackability score to every trajectory , based on the ambiguity of trajectory - to - measure - ment associations ( Figure 6 and section \u2018\u2018stochastic program - ming for the evaluation of trackability\u2019\u2019 in STAR Methods ) . Fig - ure 6A shows an ambiguous association between time t (cid:4) 1 and t where two hypotheses for the assignment of new detec - tions to track heads have a similar association cost . The bipar - tite graph matching identi\ufb01es a single optimal solution ( see Fig - ure 6A . i ) . To determine the level of ambiguity in the solution , we resample all track head predictions N times and test the stabil - ity of the original assignment ( one resampling example is shown in Figure 6A . ii ) . The approach is illustrated in Figures 6B \u2013 6D based on the tracking of a TF in previously published multifocus microscopy data . 63 Each dot indicates a resampled prediction of the particle location at t , and blue versus red de\ufb01nes whether the newly computed local assignment matches or differs from the original solution . The trackability score is de\ufb01ned as the fraction of matching samples . Hence , the score accounts for the local competition among detections for track head associ - ations and the uncertainty of motion prediction for each track head . We evaluated the capacity of our score to predict tracking quality in several scenarios . We simulated trajectory sets of increasing stochasticity along with a noisy detector and applied u - track3D to trace the particle movements ( parameters are described in Tables S1 and S2 ) . Using the ground truth , we then classi\ufb01ed each link of the extracted traces as a TP or FP . Figure 5 . DynROIs drive the visualization of chromosome capture by microtubules and reveal possible interactions between neighboring kinetochore \ufb01bers ( A \u2013 D ) Dual - colored orthogonal MIP of HeLa cells undergoing mitosis labeled with eGFP - labeled EB3 ( marking microtubule plus - ends rendered in gray ) and mCherry - labeledcentromereproteinA ( markingkinetochoresrenderedinred ) . Overlayshighlight ( A ) adynROIbuiltaroundcentrosometrajectories , ( B ) adynROI built around kinetochores trajectories , and ( C ) a plane built to visualize the dynamics of chromosomes relative to the spindle location . ( D ) View of the dynROI following description in ( H ) . ( E ) De\ufb01nition of a conical dynROI between a centrosome and a kinetochore . ( F ) Dual - colored orthogonal MIP of HeLa cells during prometaphase . Overlay highlights the motion of the dynROI . ( G ) Cumulative overlays of the detected microtubule plus - end position for three periods of 10 s between 53 and 102 s post nucleus envelope breakage . ( H ) Plus - ends count function of time and distance from the pole ( n = 1 dynROI ) . 8 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS This classi\ufb01cation allows us to compute for each simulated set the true JI JI = TP TP + FP + FN = TPFP + L , with L denoting the number of simulated links . Figure 6F presents simulated trajectories with increasing diffusion coef\ufb01cients ; the display is truncated to \ufb01ve consecutive time points to improve visibility . With a detection density \ufb01xed to 0 . 1 m m (cid:4) 3 and increasing speed of diffusion , the tracking performance rapidly deteriorates to completely inaccurate links and trajectory lifetime distributions ( see Figures 6G and 6H ) . The trackability score follows the decrease in the JI until it plateaus at 0 . 5 for very challenging conditions ( Figure 6I ) . The initially close match between track - ability score and JI is expected as larger diffusion speeds in - crease ambiguities in parallel to FPs and false negatives . How - ever , beyond a diffusion of 0 . 6 m m 2 / s , the prediction of the particle location in the next frame is less likely centered on the correct detection . As such , during the resampling of the ex - pected particle location , the rate of samples in agreement versus disagreement with the original link is de\ufb01ned by chance ; hence , the score plateaus at 0 . 5 . We also simulated a scenario in which the particle density increases at a diffusion \ufb01xed to 0 . 3 m m 2 / s . Analogous to the increase in diffusion , the trackabil - ity score follows the JI up to a density of 0 . 25 m m (cid:4) 3 where the two performance measurements start to diverge ( see Fig - ure S3 ) . In the case of directed displacements and a given density of 0 . 1 m m (cid:4) 3 , our trackability score also follows the true JI up to a critical velocity of 1 . 8 m m / s , which is more Figure 6 . The trackability score relies on the stochastic footprintofeachtrajectorytoinfer tracking accuracy ( A ) Example of a tracking ambiguity due to three trajectories in close proximity ( orange , blue , and red ) . Dashed lines represent the true motion be - tween track heads at time t (cid:4) 1 and detections at time t , represented by gray dots . Colored gradients represent the likelihood of each expected particle location at time t , estimated using the history of positions up to time t (cid:4) 1 and considering multiple motion model hypotheses . The optimal assignment between the expected and detected particle posi - tions at time t in this case yields an erroneous assignment fromtheorangetrack headtodetection 2 and from theblue track head to detection 3 ( graph A . i ) . Resampling of the expected locations results in a new assignment ( graph A . ii ) , this time without error . ( B ) Orthogonal MIP of ES cells expressing eGFP - labeled Sox2 molecules imaged by multifocus mi - croscopy . Overlaid boxes highlight the ROI enlarged in ( C ) \u2013 ( E ) . ( C ) Orthogonal MIP of ROI . Overlay shows a tra - jectory where two close detections create assign - ment ambiguity . ( D ) Overlay illustrates the stochastic resampling of the predicted particle positions at this time point ; blue circles , assignments in agreement with the original solution ; red circles , assignments that differ from the original solution . ( E ) Overlay shows trajectory segments colored ac - cording to estimated trackability scores . ( G ) Examples ofsimulated trajectories withdiffusion coef\ufb01cients ranging from 0 . 1 to 1 m m 2 / s with a \ufb01xed particle density of 0 . 1 m m (cid:4) 3 . Visualization is limited to \ufb01ve consecutive frames to reduce clutter . ( F ) Lifetime of simulated trajectories ( the change in distribution is due to trajectories leaving the \ufb01eld of view as the diffusion coef\ufb01cient increases ) . ( H ) Lifetime distribution measured through tracking shows a loss of the original distributions when the diffusion coef\ufb01cient exceeds 0 . 2 m m 2 / s . ( I ) Accuracy measured through the Jaccard index ( JI , blue ) ; the trackability score ( orange , dashed ) , which is derived without external ground truth , closely follows the JI up to a diffusion coef\ufb01cient 0 . 6 m m 2 / s beyond which tracking is random . Cell Reports Methods 3 , 100655 , December 18 , 2023 9 Article ll OPEN ACCESS than twice the average distance between a particle and its closest neighbor ( see Figure S4 ) . Finally , we sought to test our approach in a scenario in which trajectories undergo sud - den transitions between diffusive and directed motion ( see Fig - ure S5 ) . Of note , the densities , diffusion coef\ufb01cients , and veloc - ities are \ufb01xed in this scenario and the only parameter that varies is the transition rate , ranging from 0 ( no transition ) to 0 . 5 ( on average one transition every two frames ) . Our results show that the trackability score correctly predicts the reduction in tracking accuracy as increasing transition rates render tracking more ambiguous . A quasi - plateau is reached due to the high frequency of dynamic transitions . In conclusion , the proposed trackability score is able to detect changes in tracking quality in a variety of scenarios . Trackability score to compare tracking quality across time , space , and \ufb02uorescent channels To test the trackability score in real - world tracking , we \ufb01rst analyzed the spatiotemporal variation in the tracking quality of en - docytic pits ( see section \u2018\u2018endosome trackability on cell cultured on top of collagen\u2019\u2019 in STAR Methods ) associated with quiescent and protrusive parts of a cell membrane ( Figure 7A ) . We manually selected dynROIs to capture a quiescent area , a slow \ufb02uctuating protrusion - retraction cycle , and an abrupt and rapid protrusion . These dynROIs were selected within a larger dynROI compen - sating whole - cell movement ( see Video S2 ) . Trackability scores were consistently high in the quiescent dynROI , cyclically decreased in the \ufb02uctuating protrusion , and showed a sharp decrease where and when the fastest protrusion was located Figure 7 . Demonstration of trackability score on experimental data ( A ) Orthogonal MIP of breast cancer cells imaged with diaSLM expressing eGFP - labeled alpha subunit of the AP - 2 complex . Boxes show ROIs with quiescent ( blue ROI ) and slow / fast protrusion - retraction activity ( orange and yellow ROIs ) . Dot overlays show local level of ambiguity . ( B ) Number of track segments over time for the three ROIs ( n = 1 cell ) . ( C ) Trackability score over time for the three ROIs ( n = 1 cell ) . ( D ) Cumulative distribution of the average trackability score of trajectories for both EB3 and kinetochore channels sampling the dynamics of the mitotic spindle shown in Figure 5 . ( E ) Four ROIs ( two for each channel ) showing trajectories colored according to their mean trackability score . Trajectories were selected near the 10th and 90th percentiles of the cumulative distribution . Yellow dots show surrounding detections . 10 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS ( Figures 7B and 7C ) . The score thus accurately re\ufb02ects variations in particle trackability across space and time and detects time points of high ambiguity due to rapid movement . In a second experiment , we analyzed the spindle assembly dataset shown in Figures 5F \u2013 5H . The cumulative distributions of trajectory - averaged trackability scores showed that kineto - chore trajectories overall are more reliably reconstructed compared to microtubule plus - end trajectories ( Figure 7D ) . The score also enables trackability analysis on a per - trajectory basis . Trajectories with a score near the 90th percentile of the cumula - tive distribution appear to be error free for both plus end and kinetochore channels ( Figure 7E ) . In contrast , a plus - end trajec - tory with a score near the 10 th percentile shows a likely erro - neous path in an area of dense , crisscrossing microtubules . Because of the overall much higher trackability of the kineto - chore channel , a trajectory near the 10 th percentile shows only one likely wrong link caused by FP particle detection ( see arrow in Figure 7E detail of ROI 2 ) . Hence , the trackability score is a faithful reporter of the overall accuracy of tracking results , and it assists the selection of correctly tracked objects in dense tra - jectory populations . DISCUSSION We describe a new version of the popular tracking framework u - track , which now enables the study of particle dynamics in 3D live microscopy and tackles key challenges in the exploration and analysis of those complex datasets . The u - track3D software is implemented in MATLAB and distributed with a user - friendly graphical user interface ( GUI ) and tutorial scripts . The GUI is designed for testing the soft - ware and for the interactive visualization of the particle detec - tions , trajectories , and dynROI locations overlaid onto the raw data . In particular , both raw voxels and measurements can be observed using either slice - by - slice visualization or MIPs in the frame of reference of the laboratory or in a frame of refer - ence of a dynROI . The scripts are primarily used for batch pro - cessing and analysis at scale , and they enable the systematic visualization of tracking results across a full dataset . Our rendering engine is designed for automated and parallelized visualization of raw data and overlaid measurements , taking advantage of the asynchronous nature of processing jobs . Montages of raw and overlaid images can be easily speci\ufb01ed and saved in a variety of formats ( png , avi , and gifs ) . The script interface also provides a \ufb01ner control of the shape of dynROIs than the GUI ( cone , tube , rounded tubes , etc . ) . Finally , both detection and tracking can be limited to a dynROI , enabling the rapid adjustment of algorithm parameters before process - ing a full dataset . Two datasets are provided to test the soft - ware , one extracted from the endocytosis imaging introduced in Figure 7A and the other extracted from the mitosis imaging experiment introduced in Figure 5 . Computation time and memory usage for the complete pipeline , including detection , tracking with trackability inference , and dynROI de\ufb01nition , has been benchmarked using the endocytosis dataset on a stan - dard computing workstation and of\ufb01ce laptop ( see \u2018\u2018Bench - marking computational time and memory usage\u2019\u2019 in STAR Methods ) . Limitations of the study While the robustness and applicability of the software have been tested in several studies , 62 , 64 challenges remain toward a generic approach for automated exploration of 3D sequences . A chief bottleneck comes with the multiple sources of motions occurring across scales . While a given frame rate may be suf\ufb01 - cient to sample and track the motion of particles on a static sub - strate , the object may not be trackable when the particle - embedding volume moves rapidly . u - track3D addresses this problem with the estimation of dynROIs , which allow the pre - alignment of particle groups associated with an entire cell or sub - cellular structure . However , the automated estimation of the scale , type , clusters , and magnitude of those displacements remains an open problem for heterogeneous groups of objects . New developments in stochastic \ufb01ltering approaches for multi - scale displacements are thus necessary . Another key challenge in the analysis of dynamic 3D data is the quanti\ufb01cation of the motion of diffuse signaling molecules or macromolecular structures that do not present a well - de\ufb01ned particle in the imaged volume . These motions can be estimated coarsely using 3D optic \ufb02ow approaches , for which a few prom - ising methods tailored to \ufb02uorescence imaging have been proposed . 65 \u2013 67 Finally , the visualization and interaction with large multidi - mensional data remain dif\ufb01cult . While we believe the proposed dynROIs add a powerful tool for exploration of 3D sequences , the underlying rendering engine is limited to MIPs or slide - by - slide visualization . Community efforts are currently underway to provide a generic and versatile graphic library along with GUI interface such as Napari 28 and Sciview . 27 They could complete the capabilities of our renderer with more advanced volumetric rendering ( alpha , ray casting ) as well as surface rendering . We thus introduce u - track3D as feature - complete software for the quanti\ufb01cation and analysis of particle - like trajectories in 3Ds but also as a stepping - stone toward automated and unbiased exploration of any type of dynamic datasets . As we deliver the software to the community , we are continuously improving the software by \ufb01xing bugs and evaluating suggestions for improve - ments made by the community . STAR + METHODS Detailed methods are provided in the online version of this paper and include the following : d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DE - TAILS B Clathrin - mediated endocytosis in an epithelial cell layer B Mitosis in HeLa cells B Transcription factors activity in embryonic stem cells B Adhesions in collagen - embedded osteosarcoma cells Cell Reports Methods 3 , 100655 , December 18 , 2023 11 Article ll OPEN ACCESS B Trackability of endocytic pits in collagen - embedded breast cancer cells d METHOD DETAILS B Multiscale particle detector B Dynamic region of interest estimation B Stochastic programming for the evaluation of track - ability B Clathrin - mediated endocytosis study on a glass cover - slip B Microtubule instability measurement B Single molecule dynamics study with lattice light - sheet microscopy B Adhesions and collagen interaction imaging and anal - ysis B Endosome trackability on cell cultured on top of collagen B Benchmarking computational time and memory usage d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental information can be found online at https : / / doi . org / 10 . 1016 / j . crmeth . 2023 . 100655 . ACKNOWLEDGMENTS The authors are grateful to Yuko Mimori - Kyosue at the RIKEN Institute for the gift of the HeLa cells expressing eGFP - EB1 and fruitful conversations . We are also grateful to Zhe Liu at Janelia Research Campus for giving the ES cells . P . R . was funded by fellowship LT000954 / 2015 from the Human Frontiers in Science Program and the Investissements d\u2019Avenir French government pro - gram managed by the French National Research Agency ( ANR - 16 - CONV - 0001 ) and from Excellence Initiative of Aix - Marseille University - A * MIDEX . Work in the Danuser lab was funded by grant R35GM136428 . Maintenance and dissemination of the software was supported by grant RM1GM145399 . W . R . L . acknowledges support from the Searle Scholars Program , the Beck - man Young Investigator Program , an NIH New Innovator Award ( DP2GM136653 ) , and the Packard Fellows Program . Research in the labora - tory of D . W . G . was supported by the Vienna Science and Technology Fund ( WWTF ; project number LS14 - 009 ) and by the Austrian Science Fund ( FWF special research program SFB Chromosome Dynamics ; project number SFB F34 - 06 ) . R . F . was supported by the Cancer Prevention Research Institute of Texas ( RR160057 ) and the NIH ( R33CA235254 and R35GM133522 ) . Lattice light - sheet imaging of the mitotic spindle data was produced in collaboration with the Advanced Imaging Center , a facility jointly supported by the Gordon and Betty Moore Foundation and Howard Hughes Medical Institute at the Ja - nelia Research Campus . AUTHOR CONTRIBUTIONS P . R . and G . D . designed the research . P . R . wrote the tracking and rendering software and performed data analysis . Q . Z . developed the software\u2019s graph - ical user interface . W . R . L . , K . M . D . , A . D . , T . I . , and E . S . W . performed the biochemical and imaging experiments . K . M . D . , R . F . , and E . B . provided imag - ing resources . P . R . and G . D . wrote the manuscript . All authors read and pro - vided feedback on the \ufb01nal manuscript . DECLARATION OF INTERESTS The authors declare no competing interests . INCLUSION AND DIVERSITY We support inclusive , diverse , and equitable conduct of research . Received : March 12 , 2023 Revised : August 10 , 2023 Accepted : November 9 , 2023 Published : December 1 , 2023 REFERENCES 1 . Chen , B . - C . , Legant , W . R . , Wang , K . , Shao , L . , Milkie , D . E . , Davidson , M . W . , Janetopoulos , C . , Wu , X . S . , Hammer , J . A . , Liu , Z . , et al . ( 2014 ) . Lat - ticelight - sheetmicroscopy : Imagingmoleculestoembryosathighspatio - temporal resolution . Science 346 , 1257998 . 2 . Dean , K . M . , Roudot , P . , Reis , C . R . , Welf , E . S . , Mettlen , M . , and Fiolka , R . ( 2016 ) . Diagonally Scanned Light - Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells . Biophys . 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II - . 14 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS STAR + METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals , peptides , and recombinant proteins DMEM Thermo Fisher Scienti\ufb01c 12491015 DMEM without phenol red Invitrogen N / A Antibiotic / antimycotic for IMCD cells Thermo Fisher Scienti\ufb01c 15240062 DMEM HeLa Cells Institute of Molecular Biotechnology of the Austrian Academy of Sciences N / A penicillin \u2013 streptomycin Sigma - Aldrich P0781 GlutaMAX Thermo Fisher Scienti\ufb01c 35050061 rat tail - derived Collagen Type I Corning 354236 truncated CMV promoter Addgene 110718 mNeonGreen - Paxillin Allele Biotechnology N / A Alexa Fluor 568 Thermo Fisher Scienti\ufb01c A20003 Deposited data Raw test dataset This paper https : / / zenodo . org / record / 6881276 Experimental models : Cell lines Inner medulla collecting duct ( IMCD ) mouse epithelial cells ATCC ATCC : CRL - 2123 , RRID : CVCL _ 0429 U2OS female cells ATCC ATCC : HTB - 96 RRID : CVCL _ 0042 Sum159O breast cancer cells Whitehurst 68 , 69 N / A HeLa Kyoto line S . Narumiya RRID : CVCL _ 1922 Stable Embryonic Stem Cells Liu 56 N / A Software and algorithms u - track3D This paper https : / / doi . org / 10 . 5281 / zenodo . 10055024 MATLAB 2015a - 2023a Mathworks RRID : SCR _ 001622 Gmic D . Tschumperle\u00b4 / GREYC / CNRS https : / / gmic . eu / AMIRA v6 Thermo Fisher Scienti\ufb01c RRID : SCR _ 007353 LabView Emerson RRID : SCR _ 014325 Other FCS Thermo Fisher Scienti\ufb01c A5256701 FBS U2OS Sigma F0926 - 500ML 5 mm diameter coverslips Thomas Scienti\ufb01c 64 \u2013 0700 NA 0 . 71 water dipping illumination objective Special Optics 54 - 10 - 7 25X / NA 1 . 1 water dipping detection objective Nikon Instruments CFI75 Apo LWD 25XY Camera ORCA - Flash 4 . 0 Hamamatsu Photonics C11440 - 22C Bidirectional scan unit Cambridge Technology 6215 Remote focusing system Nikon Instruments CFI S Plan Fluor ELWD Achromatic doublet Edmund Optics 49 \u2013 396 Filters Chroma Technology Corporation ZET405 / 488 / 561 / 640 , ZT568rdc , ET525 / 50m , ET600 / 50m Piezzo Actuator Physik Instrumente P - 603 . 1S2 Piezzo controller Physik Instrumente E (cid:4) 709 . SRG Cell Reports Methods 3 , 100655 , December 18 , 2023 e1 Article ll OPEN ACCESS RESOURCE AVAILABILITY Lead contact Further information and requests for resources should be directed to and will be ful\ufb01lled by the lead contact , Philippe Roudot ( philippe . roudot @ univ - amu . fr ) . Materials availability This study did not generate new unique reagents . Data and code availability d Raw TIFF light - sheet imaging data used in Figures 5 and 7 have been deposited in https : / / zenodo . org / record / 6881276 and are publicly available as of the date of publication . DOIs are listed in the key resources table . They are automatically downloaded by the tutorial scripts . The full imaging datasets used in this paper represent tens of Terabyte of data and are too large to be made available on a server maintained for public access . However , this data can be made available through other means ( such as mail , or large \ufb01le transfer sev ) upon request to the lead contact author . d All original code has been deposited at https : / / zenodo . org / records / 10055024 and is publicly available as of the date of pub - lication . DOIs are listed in the key resources table . A user\u2019s guide for both GUI and scripts and test datasets are available within the same repository . The repository used for update and bug\ufb01xes is at https : / / github . com / DanuserLab / u - track3D . d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request . EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Clathrin - mediated endocytosis in an epithelial cell layer Inner medulla collecting duct ( IMCD ) mouse epithelial cells stably expressing alpha - adaptin GFP 53 were cultured in in Dulbecco\u2019s modi\ufb01ed Eagle\u2019s medium ( DMEM ) supplemented with 10 % fetal calf serum ( FCS ) and 1 % antibiotic / antimycotic at 37 (cid:3) C . Mitosis in HeLa cells HeLa cell lines stably expressing \ufb02uorescent reporter proteins were derived from a HeLa Kyoto line obtained from S . Narumiya ( Kyoto University , Kyoto , Japan ) and cultured in DMEM ( produced in - house at Institute of Molecular Biotechnology of the Austrian Academy of Sciences ) supplemented with 10 % FCS ( Thermo Fisher Scienti\ufb01c ) , 1 % ( v / v ) penicillin \u2013 streptomycin ( Sigma - Aldrich ) , and GlutaMAX ( Thermo Fisher Scienti\ufb01c ) at 37 (cid:3) C with 5 % CO2 in a humidi\ufb01ed incubator . Transcription factors activity in embryonic stem cells Stable Embryonic Stem ( ES ) cell lines were generated and cultured as described in . 56 Brie\ufb02y , ES cell - imaging experiments were per - formed in Dulbecco\u2019s modi\ufb01ed Eagle\u2019s medium ( DMEM ) without phenol red ( Invitrogen ) , 15 % FBS , 1 mM GlutaMAX at 37 (cid:3) C . Adhesions in collagen - embedded osteosarcoma cells Mycoplasma - free U2OS female cells were cultured in DMEM with 10 % FBS ( Sigma ; F0926 - 500ML ) at 5 % CO2 and 37 (cid:3) C . Trackability of endocytic pits in collagen - embedded breast cancer cells Sum159O breast cancer cells stably expressing alpha - adaptin GFP are a derivative of Sum159 cells obtained from Angelique Whitehurst and prepared as in . 68 , 69 Cells were plated on a (cid:1) 2 mm thick bed of rat tail - derived Collagen Type I ( 354236 , Corning ) at 37 (cid:3) C . METHOD DETAILS Multiscale particle detector Three - dimensional microscopy imposes speci\ufb01c constraints on the design of a particle detector . First , the diversity of shapes and sizes of intracellular structures may not be visible to the naked eye in a volumetric rendering , we must thus design a detector that is responsive to those variations . Second , light scattering and variation in signal intensity create large changes in signal - to - noise ratio ( SNR ) across space that are also dif\ufb01cult to assess visually . Our detector must then be adapted to those changes from low to high SNR . Finally , the large dimension of 3D data sets requires the design of computationally ef\ufb01cient approaches . Following , we describe a multiscale detector equipped with an adaptive thresholding approach that tests multiple possible scales at each location through the implementation of multiple iterations of \ufb01ltering . This tool is similar in spirit to other multiscale detectors that combine the local - ization task with the scale selection task using either Gaussian kernels 70 or the wavelet transforms . 71 The main difference is the com - bination of multiple thresholding masks obtained through the evaluation of a statistical test for each voxel and for each of the eval - uated scales . e2 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS We \ufb01rst developed a multiscale adaptive thresholding approach inspired by our previous work focused on the sensitive detection limited to the case of diffraction - limited \ufb02uorescent structures . 53 Let us consider the following image model : M \u00f0 x ; A ; s ; m ; C \u00de = A \u00f0 x \u00de G s ; m \u00f0 x \u00de + C \u00f0 x \u00de + e \u00f0 x \u00de where A denotes the spot amplitude , x the 3D position , G s ; m \u00f0 x \u00de is a Gaussian function with standard deviation s and mean m , C is the background signal and e \u00f0 x \u00de is the additive noise following a Poisson - Gaussian stochastic footprint . The least - square formulation of our optimization problem as argmin A \u00f0 x \u00de ; C \u00f0 x \u00de X x \u02db W (cid:2) A \u00f0 x \u00de G s ; m \u00f0 x \u00de + C \u00f0 x \u00de (cid:4) I \u00f0 x \u00de (cid:3) 2 ; where I \u00f0 : \u00de denotes the image volume and W is a 3D box of size 8 s , can be simpli\ufb01ed to the resolution of a linear system that can be decomposed in multiple \ufb01ltering passes : A \u00f0 x 0 \u00de = \u00f0 I (cid:5) G s ; 0 \u00de\u00f0 x 0 \u00de (cid:4) \u00f0 G s ; 0 (cid:5) 1 w \u00de\u00f0 x 0 \u00de nG s ; 0 2 + nG s ; 0 2 and , C \u00f0 x 0 \u00de = \u00f0 I (cid:5) 1 w \u00de\u00f0 x 0 \u00de (cid:4) nG s ; 0 A \u00f0 x 0 \u00de n where x 0 is the \ufb01xed voxel position 1 w is a unitary convolution kernel along W , n is the number of voxels encompassed in W . The statistical analysis of the local residuals resulting from the \ufb01t r \u00f0 x \u00de = (cid:2) A \u00f0 x 0 \u00de G s ; m \u00f0 x \u00de + C \u00f0 x 0 \u00de (cid:4) I \u00f0 x \u00de (cid:3) ; with x \u02db W , provides a p - value - based threshold for testing for the hypothesis that A \u00f0 x 0 \u00de [ C \u00f0 x 0 \u00de as described in . 53 This approach yields a sensitive binary map H 0 ; s \u00f0 : \u00de for the detection of the voxel describing a \ufb02uorescence object at scale s . This approach avoids the \ufb01tting of an object template in order to reduce computation time . Next , we carry out this adaptive thresholding step at multiple scale to obtain a vote map . V \u00f0 x \u00de = X s \u02db U H 0 ; s \u00f0 x \u00de where U is the scale range , typically ranging between 0 . 120 and 1 m m . The resulting object mask V \u00f0 : \u00de thus summarizes the presence of particles at any scale at a given voxel ( see Figure S6 ) using only \ufb01ltering operations that can process each voxel in a parallelized fashion . In order to re\ufb01ne the localization of objects present in contiguous object masks , we implemented a multiscale Laplacian of a Gaussian \ufb01ltering framework 72 to estimate a map of scale response S \u00f0 : \u00de for each voxel de\ufb01ned as : S \u00f0 x \u00de = argmax s \u02db U s 2 V 2 \u00f0 I \u00f0 x \u00de (cid:5) G s ; 0 \u00f0 x \u00de\u00de where V 2 \u00f0 : \u00de denotes the Laplacian operator . The watershed algorithm is then applied to further segment this scale response map to detecttouchingobjects . Thecenterofobjectisdeterminedthroughtheweightedcentroidofthevoxelsbelongingtoasameobjectmask . Dynamic region of interest estimation In order to visualize and map the molecular processes nested in volumetric time lapse sequences , we propose a framework for the de\ufb01nition of dynamic regions of interest ( dynROI ) from point cloud sequences . Those dynROIs are described by dynamic bounding boxes ( or rectangular cuboids ) that are sized to \ufb01t the data optimally and oriented according to a moving frame of reference . In this note , we describe the general principles underpinning the estimation of those dynROIs from dynamic point clouds and their imple - mentation across scales : from cellular down to molecular dynROIs . Generic point cloud tracking principle We \ufb01rst de\ufb01ne an optimal frame of reference in the \ufb01rst time point of the sequence with an origin O 0 described by the average point cloud position and with unit vectors \u00f0 u 0 ; v 0 ; w 0 \u00de described by the eigenvectors of the covariance matrix of the point positions ( a . k . a . principal component analysis ) . The orientation of the dynROI box in the \ufb01rst frame is described by this frame of reference and its size is de\ufb01ned by the boundaries of the point cloud augmented by a tunable margin ( default is set to 5 voxel ) . The frame of reference at time t is then estimated through a rigid transform as : d \u00f0 O t ; R t \u00de = argmin O t ; R t X x t \u02db U t x 0 \u02db U 0 k x t (cid:4) \u00f0 R t x 0 + O t \u00dek using the Iterative closest point algorithm , 73 where U t denotes the set of points coordinate at time t . The unit vector \u00f0 u t ; v t ; w t \u00de are then estimated by applying the rigid transform to \u00f0 u 0 ; v 0 ; w 0 \u00de . At each time point the size of the box is adjusted to \ufb01t the extension of the Cell Reports Methods 3 , 100655 , December 18 , 2023 e3 Article ll OPEN ACCESS current point cloud in the current orientation with the additional margin . Multiple dynROI shapes have then been implemented to adjust to the local process ( box , sphere , tube , rounded tube , plane and cone ) . Dynamic region of interest estimation for the cell The cell is \ufb01rst segmented using the Otsu algorithm and the point cloud representing the cell mask is downsampled randomly to reduce its density by 90 % and speed up computations . The generic point cloud tracking principle described above is applied to the downsampled sequence with a margin set to 30 voxels and a box - shaped dynROI ( see Figure S7 ) . Dynamic region of interest for the spindle Spindle poles were detected using the multiscale detector with the default p value ( set to 0 . 005 ) and scales ranging from 0 . 4 to 0 . 8 ums . The motion of poles was modeled with a piecewise stationary Brownian and Directed motion model with a maximum instantaneous displacement set to 3 times the process noise estimated from a Kalman \ufb01ltering of the trajectory , a lower bound set at 0 . 5 m m and upper boundsetat0 . 8 m m . Failuretodetectthe verydynamicaggregate onnucleatingmicrotubulesishandledwithgapclosing , themaximum gap is set to 2 s ( or 2 frames ) with a minimum length of 2 s for the track segment . The resulting dynROI was built has a rounded tube center with a fringe of 9 microns around the segment formed by the two brightest objects present during the complete sequence . Dynamic region of interest for the chromosomes The kinetochores marking the center of chromosomes were detected using the multiscale detector with the default p value ( set to 0 . 005 ) and scales ranging from 0 . 15 to 0 . 25 voxels . Motion was modeled with a Brownian motion model with a maximum instanta - neous displacement set to 5 times the process noise estimated by Kalman \ufb01ltering of the trajectory , a lower bound set at 0 . 4 m m and upper bound set at 0 . 6 m m . Variation in SNR were managed with a maximum gap set to 4 s ( or frames ) with a minimum length of 2 s for the track segment . The dynROI was estimated using the generic point cloud tracking principles described above using all the trajec - tories detected inside the spindle dynROI with a box - shaped dynROI and a margin of 0 . 1 m m . Dynamic region of interest estimation for the interpolar region Let \u00f0 O st ; u st ; v st ; w st \u00de and \u00f0 O kt ; u kt ; v kt ; w kt \u00de denote the frames of reference estimated for the spindle and the chromosome respectively . We want to build a frame of reference \u00f0 O it ; u it ; v it ; w it \u00de that follows an interpolar plane showing how microtubule nucleation events inside the spindle are orchestrated to capture chromosomes ef\ufb01ciently . We \ufb01rst set the origin to O it = O st and w it = w st so that one axis is following the spindle at all time . For the plane to describe the motion of the chromosome population , the second unit vector follows a slice of the kinetochore - associated dynROI v kt 0 = cos \u00f0 q \u00de u kt + sin \u00f0 q \u00de w kt projected to ensure orthogonality as v it = v kt 0 : \u00f0 1 (cid:4) w it T w it \u00de . Finally the last unit vector is set as u it = v it 3 w it . The dynROI type is a plane with a lateral fringe of 50 voxels , a height of 4 voxels and an angle q set to p 2 . Dynamic region of interest estimation for the kinetochore \ufb01bers Assuming K - \ufb01bers to span the region between poles and kinetochores as a straight polymer , its associated microtubule dynamics was observed using a conical dynROIs with an angle of p 12 . Stochastic programming for the evaluation of trackability The association of particle detections with trajectory heads is performed in a temporally greedy fashion , i . e . , particles detected at time t are linked to the heads of track segments de\ufb01ned up to time t (cid:4) 1 without consideration of the track segments beyond t and only indirect consideration of track segment before t - 1 . Therefore , our de\ufb01nition of trackability relates to the level of ambiguity in assigning particles detected in time point t to track segment heads in t \u2013 1 . The optimal association is obtained by linear assignment of heads to particles in a bipartite graph : argmin f a ij g X i \u02db U ; j \u02db D t c ij a ij s : t : X i \u02db U a ij = 1 and X j \u02db D t a ij = 1 ; where U is the set of track segment heads , D t is the set of detections measured at time t , a ij \u02db f 0 ; 1 g denotes the assignement of the i th track segment to the j th particle and c ij \u02db R is the cost associated to making that association . The association cost c ij typically re\ufb02ects the distance between the predicted location of the i th track segment at t and the j th detection at this same time point . This assign - ment problem is convex , hence with a guaranteed unique solution , and can be solved using a variety of linear programming algo - rithms . 51 , 74 , 75 However , a key challenge in our framework is the deterministic aspect of this solution . There is no measure of uncer - tainty attributed to the \ufb01nal graph of associations ( see Figure 5A ) . While several algorithms have been proposed to estimate the uncertainty related to the total optimal cost of a linear programming problem , a . k . a . stochastic programming , 76 they do not focus on the detection of local changes in association made in the bipartite graph . In this Section , we will \ufb01rst detail how we consider the randomness present in the history of each track to estimate the probability distribution associated to all assignment costs c ij . We will then describe how these uncertainties can then be exploited to detect local ambiguities in the assignment problem , which subsequently de\ufb01ne a score of trackability . Stochastic \ufb01ltering approaches are routinely used to estimate the parameter describing the dynamic properties of tracked particles from their position history . They enable the prediction of particle location from one frame to the next to re\ufb01ne the cost used for linear assignment . Those temporally recursive algorithms also provide inferences of track segment prediction uncertainty from t - 1 to t . Brie\ufb02y , let x t be a variable describing the state of the track segment . For a particle moving in a directed fashion , it is de\ufb01ned as : x t = \u00f0 x ; y ; z ; dx ; dy ; dz \u00de : e4 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS The associated probability p \u00f0 x t j z 1 : t \u00de can be estimated recursively thanks to the Bayes rule : p \u00f0 x t j z 1 : t \u00de f p \u00f0 z t j x t \u00de Z p \u00f0 x t j x t (cid:4) 1 \u00de p \u00f0 x t (cid:4) 1 j z 1 : t (cid:4) 1 \u00de d x t (cid:4) 1 ; where z 1 : t represents the past measured positions assigned to a particular track . Kalman \ufb01ltering is a scalable and \ufb02exible way to model the motions of thousands of particles in parallel , and as such is used in the majority of tracking approaches , 7 including u - track . In this framework , the relationships between random variables are assumed to be linear and described as follows : x t = Fx t (cid:4) 1 + w t z t = Hx t + v t where F is the state transition matrix between consecutive time points , H is the observation matrix , and w t and v t are the model and measurement noise respectively , bothassumed to be Gaussian with covariance matrices Q t and R t . The Gaussian andlinear assump - tion provides an analytical solution with a computationally ef\ufb01cient implementation to estimate p \u00f0 x t j z 1 : t \u00de(cid:1) N \u00f0 b x t ; b P t \u00de ( see our previous work 13 for a detailed review ) . Before optimal assignment between a track segment at t (cid:4) 1 and the object detected on frame t , the probability distribution of the predicted particle positon at time t is then described by p \u00f0 x t j z 1 : t (cid:4) 1 \u00de(cid:1) N \u00f0 F b x t (cid:4) 1 ; F b P t (cid:4) 1 F T + Q t \u00de . As such the variation of the cost to associate the i th track segment to the j th measurement can be expressed , without loss of generality as : c ij (cid:1) k Hx (cid:4) z jt k s : t : x (cid:1) p (cid:2) x it (cid:4)(cid:4) z i 1 : t (cid:4) 1 (cid:3) : This expression provides us with a direct way to explore the space of possible combination of cost values through Monte Carlo simulations . u - track 3D implements several types of stochastic \ufb01ltering approaches such as unimodal and multimodal Kalman \ufb01ltering as well as piecewise stationary motion \ufb01ltering or smoothing approaches , where the same principles can be straightforwardly applied . The principle underlying the use of our predicted probability distribution to evaluate assignment stability is described graphically in Figure 6 . Our local trackability score is de\ufb01ned as : T it = 1 N X N n = 1 (cid:5) a ij (cid:5) = a ij n (cid:6) where a ij (cid:5) is the initial assignment found for the i th trajectory , a ij n is a newly computed assignment resulting from the n th out of a total of N simultaneous resampling rounds of all costs c ij and [ . ] denotes the Iverson bracket . Each new assignment result , or vote , is considered different if the track segment is assigned to another detection , or determined to be a track termination . As such , a lower score T it re\ufb02ect a larger instability in the optimal assignment , hence a higher ambiguity and lower trackability . In our experiments , the number of resampling rounds is set to N = 20 . Clathrin - mediated endocytosis study on a glass coverslip Cell preparation and imaging Inner medulla collecting duct ( IMCD ) mouse epithelial cells were plated on 5 mm diameter coverslips ( 64 \u2013 0700 , Thomas Scienti\ufb01c ) and mounted to a custom machined holder for imaging with a high - NA version of diagonally scanned light - sheet microscopy . 2 This microscope is equipped with an NA 0 . 71 water dipping illumination objective ( 54 - 10 - 7 , Special Optics ) , and a 25X / NA 1 . 1 water dip - ping detection objective ( CFI75 Apo LWD 25XY , Nikon Instruments ) , and a Hamamatsu Flash 4 . 0 sCMOS camera . Brie\ufb02y , 500 time points were acquired with 30 m W of 488 nm illumination ( measured at the back pupil of the illumination objective ) and a 15 ms camera exposure . Each image stack was 106 . 5 3 39 . 9 3 23 . 1 m m , with a lateral and axial voxel size of 104 and 350 nm , respectively , resulting in a 1 . 008 Hz volumetric image acquisition rate . Clathrin structure trajectory estimation and post - processing Clathrin structure aggregates , labelled by alpha - adaptin GFP were detected using a multiscale particle detector with a p value set to 0 . 05 and scales ranging from 0 . 15 to 0 . 5 m m . For tracking , the motion of particles was modeled with a Brownian motion model with a maximum instantaneous displacement set as three times the process noise estimated by Kalman \ufb01ltering of the trajectory , a lower bound set at 0 . 1 m m and upper bound set at 0 . 3 m m . When detection gaps are enabled , the maximum gap length is set to 3 s ( or frames ) with a minimum length of 3 s for any track segment allowable to be connected by the gap closing algorithm . 10 The median of the maximum intensity reached per track was then used to discriminate between abortive and maturing CCPs . To account for the variation of \ufb02uorescence signal across acquisitions , the maximum intensities were scaled such that the empirical cu - mulative distribution function ( cdf ) of maximum intensities computed for each acquisition matched the median cdf of all acquisitions , as previously described in ref . 53 . Cell Reports Methods 3 , 100655 , December 18 , 2023 e5 Article ll OPEN ACCESS Microtubule instability measurement Cell preparation and imaging HeLa cells stably expressing EB3 - EGFP alone or along with mCherry - CENPA were plated on coverslips mounted on the microscope in CO2 - independent L15 medium containing 10 % FBS , without phenol red , and maintained at 37 (cid:3) C for the duration of the experi - ment . All images were recorded with an Orca Flash 4 . 0 v2 sCMOS camera ( C11440 - 22C ; Hamamatsu ) . Images were acquired in sample - scan imaging mode with a lateral translation of 0 . 4 m m and subsequently deskewed in postprocessing . Final voxel dimen - sions for all lattice light - sheet image datasets were 104 nm 3 104 nm 3 210 \u2013 217 nm . The microscope was controlled by custom - made software . Acquisition frequency was adapted for each experiment and is speci\ufb01ed in the main text . Plus - ends trajectory estimation Plus - ends , labelled through GFP tagging of EB1 , were detected using a multiscale detector with the default p value ( set to 0 . 005 ) and scales ranging from 0 . 15 m m to 0 . 25 m m . The polymerization of microtubule was modeled with a directed displacement estimated through a Kalman \ufb01ltering of the trajectory , similar to , 55 but now in 3D . The random component of this displacement was estimated as 3 times the process noise of the Kalman \ufb01lter with a lower bound of 0 . 3 m m and an upper bound of 0 . 6 m m . The shrinkages and pauses detection framework proposed in 55 has also been translated to 3D plus - ends trajectories . The detec - tion of both shrinkages and pauses is carried out by closing gaps between track segments , which implicitly delineate phases of microtubule growth . In our experiment , the minimum growth duration to consider gap closing was set to 4 s ( or frames ) and the maximum gap duration was set to 8 s . In order to detect pauses , the maximum angle between a speed vector estimated immediately prior and posterior to the pause event was set to 30 (cid:3) , the maximum positional \ufb02uctuation in the plus - ends location during a pause is set to 0 . 5 m m . To detect a shrinkage event between two segments , we \ufb01rst measure the distance D between the termination point of the earlier segment ( which is equivalent to the potential locus of a catastrophe event ) and the initiation point of the later segment ( which is equivalent to the potential locus of a rescue point ) along the path of the earlier segment . The two segments are connected by the gap closer if the distance between the initiation point to the closest point along the trajectory of the \ufb01rst segment does not exceeds Dsin \u00f0 q \u00de with q set to 20 (cid:3) in our experiment . Single molecule dynamics study with lattice light - sheet microscopy Cell preparation and imaging Fluorescently labelled Sox2 transcription factors were imaged over 100 time points with Lattice light - sheet microscopy imaging . Nine planes spaced 500nm apart were acquired at 50 ms of camera exposure , resulting in a 2 Hz volumetric image acquisition rate . Each image stack was 50 3 50 3 5 m m , then cropped around the nucleus , with a lateral and axial voxel size of 100 and 500 nm , respectively . Estimation of transcription factor binding times Transcription factor single molecules were detected using a multiscale detector using a p value of 0 . 01 and a scale ranging from 0 . 15 to 0 . 3 m m . Transcription factor motion was modeled using a Brownian motion model with a maximum instantaneous displacement estimated as 6 times the process noise estimated by Kalman \ufb01ltering of trajectory to account for speed variations during long periods of con\ufb01ned diffusion , a lower bound set at 0 . 3 m m and upper bound set at 0 . 5 m m . The maximum gap is set to 4 s ( or frames ) with a minimum length of 2 s for any track segment allowable to be connected by the gap closing algorithm . We assume that if the single molecule is detectable it immobilized at the DNA . Accordingly , characteristic binding times t are estimated by a double exponential \ufb01t to the lifetime distribution . Adhesions and collagen interaction imaging and analysis Cell preparation and imaging U2OS Cells were lentivirally transduced with a truncated CMV promoter ( Addgene # 110718 ) driving the expression of mNeonGreen - Paxillin ( Allele Biotechnology ) . Cells were seeded into a pH - neutralized collagen solution ( (cid:1) 2 mg / mL ) that , when polymerized , fully embedded cells in a three - dimensional extracellular matrix environment . For visualization of the extracellular matrix , a small concen - tration of the collagen was \ufb02uorescently conjugated with Alexa Fluor 568 NHS Ester ( A20003 , ThermoFisher ) . Samples were imaged with a high - NA variant of Axially Swept Light - Sheet Microscopy using 488 nm and 561 nm lasers for illumination ( OBIS LX , Coherent , Inc . ) . The details of this microscope will be published elsewhere . Brie\ufb02y , lasers are combined , spatially \ufb01ltered , expanded , and shaped into a light - sheet with a cylindrical lens . This light - sheet was relayed to a bidirectional scan unit ( 6215 , Cambridge Technol - ogy ) , a remote focusing system ( CFI S Plan Fluor ELWD , Nikon Instruments ) , and eventually to the illumination objective ( 54 - 10 - 7 , Special Optics ) . Fluorescence was detected in a wide\ufb01eld format with a water - dipping objective ( CFI75 Apo LWD 25SW , Nikon In - struments ) and imaged onto two sCMOS cameras ( ORCA - Flash4 . 0 , Hamamatsu Photonics ) with a 500 mm achromatic doublet ( 49 \u2013 396 , Edmund Optics ) , laser line \ufb01lter , a dichroic , and bandpass \ufb01lters ( ZET405 / 488 / 561 / 640 , ZT568rdc , ET525 / 50m , and ET600 / 50m , Chroma Technology Corporation ) . The laser laterally dithered for shadow reduction and scanned synchronously with the detection objective ( P - 603 . 1S2 and E (cid:4) 709 . SRG , Physik Instrumente ) to acquire a three - dimensional stack of images . All equipment was controlled with custom LabVIEW software , which is available from UTSW upon completion of a material transfer agreement . Adhesion detection and elongation analysis Paxillin aggregates as a surrogate for adhesions were detected using the multiscale detector described in Section \u2018\u2018Multiscale par - ticle detector\u2019\u2019 based on a p value of 0 . 001 and a scale ranging from 0 . 3 to 0 . 5 m m . The elongation of each detected adhesion is computed through a tubularity metric evaluated for each voxel and averaged across all the voxels associated to a single adhesion . e6 Cell Reports Methods 3 , 100655 , December 18 , 2023 Article ll OPEN ACCESS Similar to the classic vesselness estimator by Frangi and colleagues , 77 our tubularity metric is based on the eigen values of the Hes - sian matrix to describe local curvature , . Let \u00f0 l 1 < l 2 < l 3 \u00de be the three eigenvalues of the Hessian matrix computed at each voxel , the tubularity metric T = 1 (cid:4)j l 1 = l 2 j yields a value between 0 and 1 increasing with the elongation of the adhesions . As such , a noteworthy difference between the classic score described in Frangi\u2019s approach is the use of the two lowest eigen - values ( associated with the two axis of lowest curvature direction ) to discriminate between \ufb02at and elongated adhesions . Collagen detection and distance analysis Collagen was detected using the 3D implementation of steerable \ufb01ltering as described in . 78 An adhesion was considered in close contact with collagen if the center of at least one voxel belonging to its mask was less than 100 nm away from any voxels of the collagen mask . Endosome trackability on cell cultured on top of collagen Cell preparation and imaging Sum159O breast cancer cells 69 stably expressing alpha - adaptin GFP were imaged similarly to the one platted on glass coverslip , with the exception that they were plated on a (cid:1) 2 mm thick bed of rat tail - derived Collagen Type I ( 354236 , Corning ) . Clathrin structure trajectory estimation Clathrin structure aggregates , labelled by alpha - adaptin GFP were detected using a multiscale particle detector with a p value set to 0 . 01 and scales ranging from 0 . 125 to 0 . 5 m m . For tracking , the motion of particles was modeled with a Brownian motion model . In order to follow the erratic displacements caused by large protrusive motions , the maximum instantaneous displacement was set to 5 times the process noise estimated by Kalman \ufb01ltering of the trajectory , a lower bound set at 0 . 3 m m and upper bound set at 0 . 6 m m . The maximum gap length is set to 3 s ( or frames ) with a minimum length of 3 s for any track segment allowable to be connected by the gap closing algorithm . 10 Benchmarking computational time and memory usage Computational time and memory consumption have been tested on a computing workstation and of\ufb01ce laptop ( see speci\ufb01cation below ) . The test includes detection , tracking with trackability and dynROI de\ufb01nition on the endocytosis test dataset ( 400 Mb ) pro - vided along with this manuscript . Runtime takes an average of 17 . 3 s ( s . d . 0 . 2 s ) seconds on the workstation and 2 min 29 s ( s . d . 6 s ) on the laptop . The maximum memory used by the pipeline was 12Gb on the workstation and 0 . 5 Gb on the of\ufb01ce laptop . Of\ufb01ce laptop . d Model : X1 Carbon 6 th gen d Year : 2018 d CPU : Intel i7 - 8550U @ 1 . 80GHz , 4 Cores d RAM : 16 Gb d GPU : integrated d Disk : 1 Tb d OS : Windows 11 d MATLAB version : 2023a Computing Workstation . d Model : Colfax SX6300 d Year : 2020 d CPU : Intel Xeon 6242R @ 3 . 10GHz , 80 cores d GPU : Nvidia A6000 d Disk : 16 Tb d OS : Ubuntu 20 . 04 . 3 LTS d MATLAB version : 2021a QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analysis was performed by considering either intracellular processes or cells as independent observations as speci\ufb01ed in the manuscript . Each statistic considering intracellular processes ( either experimental or simulated data ) includes more than a thou - sand events . Statistical details of all experiments at the cellular level can be found in the \ufb01gure legends including exact value of n , clear descriptions of what n represents and box - plot representations . Cell Reports Methods 3 , 100655 , December 18 , 2023 e7 Article ll OPEN ACCESS", + "gilonAnalogyMiningSpecific2018": "Analogy Mining for Speci\ufb01c Design Needs Karni Gilon The Hebrew University of Jerusalem Jerusalem , Israel karni . gilon @ mail . huji . ac . il Joel Chan Carnegie Mellon University Pittsburgh , PA , United States joelchuc @ cs . cmu . edu Felicia Y Ng Carnegie Mellon University Pittsburgh , PA , United States fng @ cs . cmu . edu Hila Lifshitz - Assaf New York University New York , NY , USA h @ nyu . edu Aniket Kittur Carnegie Mellon University Pittsburgh , PA , United States nkittur @ cs . cmu . edu Dafna Shahaf The Hebrew University of Jerusalem Jerusalem , Israel dshahaf @ cs . huji . ac . il ABSTRACT Finding analogical inspirations in distant domains is a pow - erful way of solving problems . However , as the number of inspirations that could be matched and the dimensions on which that matching could occur grow , it becomes challenging for designers to \ufb01nd inspirations relevant to their needs . Fur - thermore , designers are often interested in exploring speci\ufb01c aspects of a product \u2013 for example , one designer might be inter - ested in improving the brewing capability of an outdoor coffee maker , while another might wish to optimize for portability . In this paper we introduce a novel system for targeting analogical search for speci\ufb01c needs . Speci\ufb01cally , we contribute an ana - logical search engine for expressing and abstracting speci\ufb01c design needs that returns more distant yet relevant inspirations than alternate approaches . Author Keywords Computational analogy , innovation , inspiration , creativity , product dimensions , abstraction , focus , text embedding ACM Classi\ufb01cation Keywords H . 5 . 3 Group and Organization Interfaces INTRODUCTION Analogy is a powerful strategy for designing new innovations . Thomas Edison invented the kinetoscope ( the precursor to motion picture projectors that are used in theaters today ) by working out how to do \u201cfor the eye what the phonograph does for the ear\u201d [ 22 ] . The Wright brothers solved a crucial aspect of how to keep their invented aircraft stable during \ufb02ight by analogy to maintaining balance while riding a bicycle [ 1 ] . More recently , a car mechanic created an innovative new way Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for pro\ufb01t or commercial advantage and that copies bear this notice and the full citation on the \ufb01rst page . Copyrights for components of this work owned by others than ACM mustbehonored . Abstractingwithcreditispermitted . Tocopyotherwise , orrepublish , to post on servers or to redistribute to lists , requires prior speci\ufb01c permission and / or a fee . Request permissions from permissions @ acm . org . CHI 2018 , April 21 \u2013 26 , 2018 , Montreal , QC , Canada \u00a9 2018 ACM . ISBN 978 - 1 - 4503 - 5620 - 6 / 18 / 04 . . . $ 15 . 00 DOI : https : / / doi . org / 10 . 1145 / 3173574 . 3173695 to assist with dif\ufb01cult childbirth , drawing on an analogy to a party trick for removing a cork stuck in a wine bottle [ 26 ] . Search engines that could support automatic retrieval of rele - vant analogies for design problems could signi\ufb01cantly increase the rate of innovation and problem solving today . The rise of knowledge databases and repositories on the Internet ( e . g . , the US Patent Database , Google Scholar , Amazon products , etc . ) provides a virtual treasure trove of ideas that could inspire so - lutions across a variety of domains . Research on creativity and innovation suggests that building on analogous inspirations that are not from the same domain as the source problem is a powerful strategy for generating creative ideas [ 6 , 12 , 28 ] . However , \ufb01nding useful distant analogies in large databases of textual documents remains challenging for existing machine learning models of document similarity [ 7 , 4 , 20 , 23 ] , which are largely dependent on surface features like word overlap . An additional challenge is that in real - world contexts with complex problems , designers are often interested in exploring and abstracting speci\ufb01c aspects of a problem rather than con - sidering the problem as a whole . To illustrate , consider the example of the Wright brothers inventing an airplane . Instead of trying to \ufb01nd an analogy for the entire plane , they regularly found analogies for partial problems they needed to solve , such as steering the wings , or controlling balance during \ufb02ight [ 19 ] . For each identi\ufb01ed problem , they then needed to abstract key properties of the problem in order to \ufb01nd useful analogs in other domains . In the example of steering the wings , they needed to look beyond some aspects of the wings \u2013 such as the color or particular material \u2013 while keeping in mind other aspects \u2013 such as the semi - rigid frame and need for the wing material to remain taut on the frame . Doing so may have led them to avoid overly general abstractions of \u201csteering\u201d that ended up less useful ( such as the wings of a bird or the rudder of a ship ) and towards more targeted analogical inspirations including the twisting of a cardboard box which drove their \ufb01nal design of warping the wings to steer [ 1 ] . There are two critical parts of the above example : focusing and targeted abstraction . By focusing we mean identifying CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 1 a particular framing or relation for which we would like to \ufb01nd analogies ; here , steering the wings or stabilizing the plane during \ufb02ight . Importantly , analogies speci\ufb01c to one focus may not be relevant to another ; for example , the problem of keeping the aircraft stable in turbulent air led to a different analogy of riding a bike , suggesting that small unconscious adjustments of the driver could address shifts in air turbulence [ 14 ] . By targeted abstraction we mean choosing the key properties of objects that are important to the core problem ( e . g . , semi - rigid , thin and \ufb02at ) while dropping out other less important properties ( e . g . , color , size ) . For example , in the steering wings problem , the desired abstraction might be something like \u201csteer < something that is semi - rigid , thin , and \ufb02at > \u201d . Targeting the abstraction is necessary in order to avoid \ufb01nding too many irrelevant matches ; for example , previous work has shown that abstracting all domain - speci\ufb01c features of the core relational structure of a problem yields less relevant analogies than retaining some [ 29 ] . Many real world solutions similarly require multiple problem framings that would bene\ufb01t from focusing and targeted ab - straction ; for example , a coffee maker taken camping may bene\ufb01t from distant inspirations that make it more lightweight , resistant to weather , a better grinder , or allow the camper to know when to pull it off the \ufb01re . Together , focus and targeted abstraction make it possible to \ufb01nd inspirations that are ana - logically relevant to very speci\ufb01c design needs , without being restricted to inspirations from the same / similar domains . To address the challenge of targeting analogical search for speci\ufb01c design needs , we present a system in which a de - signer can specify a focus for a given product description , and then abstract that focus beyond its surface features in a tar - geted manner by specifying the key properties of the relations and entities involved that are crucial for understanding the core relational structure . To facilitate expressing this query in a machine - readable way , we leverage a large database of commonsense knowledge ( Cyc ) to provide a set of controlled terms that humans can use to express key properties . Our sys - tem then uses this focus - abstracted query to computationally search a corpus of potential inspirations for analogically rele - vant matches tuned to the designer\u2019s speci\ufb01c design need . We compare this process to previous state - of - the - art approaches for \ufb01nding analogies among product descriptions , and \ufb01nd that using our Focus - Abstracted queries returns inspirations that are high on both relevance ( the results meet the needs of the query ) and domain distance ( the results are from different domains ) ; in contrast , state of the art approaches that operate on the whole document or only on speci\ufb01c keywords , either sacri\ufb01ce relevance or distance . These results have promising implications for creativity - support tools that aim to support designers in solving complex problems through analogy . RELATED WORK Computational Analogy The problem of computational analogy has a long history in arti\ufb01cial intelligence research . Early work focused on devising algorithms for reasoning about analogies between manually created knowledge representations that were rich in relational structure ( e . g . , predicate calculus representations ) [ 8 , 10 ] . While these algorithms achieved impressive human - like accu - racy for analogical reasoning , their reliance on well - crafted representations critically limited their applicability to mining analogies amongst databases of free - text documents . At the same time , much work in machine learning and infor - mation retrieval has devised methods for \ufb01nding documents that are relevant to some query from a user . These methods do not focus on analogy in particular ( and certainly not on far analogies ) : while they differ in the speci\ufb01cs of their methods ( e . g . , using singular value decomposition , or , more recently , neural networks ) , in general , they attempt to learn semantic representations of words based on the way that words are sta - tistically distributed across word contexts in a large corpus of documents ; notable examples include vector - space models like Latent Semantic Indexing [ 7 ] , probabilistic topic model - ing approaches like Latent Dirichlet Allocation [ 4 ] , and word embedding models like Word2Vec [ 20 ] and GloVe [ 23 ] . The semantic representations produced by these methods are quite useful for \ufb01nding very speci\ufb01cally relevant documents / results for a query , but are limited in their ability to \ufb01nd matches that are analogically related to a query ( especially if they do not share domain - speci\ufb01c keywords ) . Recent work by Hope et al [ 15 ] proposes to \ufb01nd analogies among free - text consumer product descriptions by learning to predict an overall representation of a product\u2019s purpose ( what it is good for ) and mechanism ( how it works ) . It uses annotators to mark words related to the purpose / mechanism of the product , and weighs the Glove [ 23 ] values of those words to assign an overall purpose / mechanism representation for each document . It then uses an arti\ufb01cial neural network model ( speci\ufb01cally a bidirectional recurrent neural network , or RNN [ 2 ] ) to learn the mapping between the product de - scription\u2019s word sequence in GloVe representation and the overall - purpose / mechanism representation captures by the pur - pose / mechanism annotations . Hope et al showed that they could use these representations to \ufb01nd analogies at a signi\ufb01 - cantly higher rate than comparison state - of - the - art approaches like TF - IDF - weighted GloVe vectors of the documents . While promising , as noted above , this approach is designed to \ufb01nd analogies for the overall purpose of a given product , and may therefore miss analogies for speci\ufb01c aspects of the product ( the speci\ufb01c focus of our paper ) . Abstraction during Analogy - Finding The essence of analogy is matching a seed document with other documents that share its core relational structure [ 10 ] ; when the analogous documents also have many other details that are very different from the seed document , they are known as far or domain - distant analogies . To \ufb01nd far analogies , it is essential to abstract away these irrelevant details from one\u2019s representation of the seed document ( and possibly also other documents in the corpus ) . Some research has explored how to enable people to construct problem representations that abstract away the surface details of the problem . For example , the WordTree method [ 18 ] has people use the WordNet [ 21 ] lexical ontology to systematically CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 2 \u201cwalk up\u201d levels of abstraction for describing the core desired functionality , leading to the possibility of discovering anal - ogous functions in other domains . For example , a designer who wanted to invent a device to fold laundry for students with very limited \ufb01ne motor skills might abstract the core function of \u201cfolding\u201d to \u201cchange surface\u201d , which could lead to analogous inspirations like \u201cree\ufb01ng\u201d ( rolling up a portion of a sail in order to reduce its area ) . Yu et al [ 27 ] explored how to systematically train crowd workers to convert problem descriptions into an abstracted form that ignored irrelevant surface details . Importantly , these abstraction methods do not blindly abstract all aspects of a problem description . In many cases , humans exert their judgment to select appropriate levels of abstraction , and also do extensive screening of possible matches based on whether they overlap with key properties / constraints in the original problem context . This is important because analogies that are \u201ctoo far\u201d away can actually lead to less creative ideas [ 5 , 9 , 13 ] . Yu et al [ 29 ] recently showed that describing the problem context in abstract terms , but retaining a domain - speci\ufb01c description of its key constraints , enabled crowd work - ers to \ufb01nd more useful far inspirations than a representation that is abstract on both the problem context and its constraints : for example , the description \u201cmake an object ( abstracted prob - lem context ) that does not tip over easily ( concrete constraint ) \u201d yields more useful inspirations for the problem of making a safe chair for kids , compared to \u201cmake an object ( abstracted problem context ) that is safe ( abstracted constraint ) \u201d ( which yields inspirations for safety that cannot be applied to chairs ) . The insight behind this recent innovation is that abstraction should be targeted : rather than completely abstracting away from all the properties of the objects involved in the core rela - tional structure ( e . g . , the wings in the steering problem for the Wright brothers ) , it is critical to retain the key properties of the objects that are important for the core relational structure . For example , in order to \ufb01nd inspirations that can suggest useful mechanisms for angling wings to steer a plane ( e . g . , twisting of a cardboard box ) , designers need to express to a search en - gine that they don\u2019t care about the color and size of wings , but they do care the fact that the wings are \ufb02at , physical objects , or even that they are composed of materials that respond to shear forces in a similar way to cardboard . This insight is consistent with classic cognitive models of analogy ( cited above , e . g . , [ 8 ] ) , which retain key properties of objects during analogical mapping that are essential to the core relational structure : for example , in the atom / solar - system analogy , the absolute size of the sun / planets vs . nucleus / electron doesn\u2019t matter , but the fact that they have mass does . We build on these insights to explore how we might create a focus - abstracted representation that enables a computational semantic model to \ufb01nd more relevant and distant inspirations . DATA SET We use a corpus of product descriptions from [ 15 ] . The prod - ucts in the corpus are from Quirky . com , an online crowd - sourced product innovation website . Quirky is useful for our study because it is large ( the corpus includes 8500 products ) and covers multiple domains , making cross - domain analogies Figure 1 . An example product description from our dataset : Soapy slider possible . Quirky users submit their ideas in free , unstruc - tured ( and often colloquial ) natural language . The ideas are written by non - experts and contain non - technical terms for which abstractions are easy to \ufb01nd . Figure 1 shows an example submission ( \u201cSoapy slider\u201d ) , demonstrating typical language . METHOD When approaching a product different designers may wish to focus on different parts of it . For example , consider the \u201cSoapy slider\u201d product in Figure 1 . One designer ( designer A ) may wish to explore ways of adjusting to different soap bar sizes while another ( designer B ) may be interested in removing soapy water from soap bars . To satisfy their needs , a straightforward approach is for the designers to search the corpus for keyword matches , for example \u201cchange soap size\u201d for one designer , and \u201cremove soap water\u201d for another . We propose a process that enables the designer to focus on a key need , and then abstract the description to include only the properties of that need that are actually important . For example , designer A ( originally interested in changing the size of soap bars ) can indicate that the \u201csoap\u201d domain is not important and can be replaced by the more general property of being a \u201cpersonal product\u201d . The system then \ufb01nds matches in the corpus using a method based on the state - of - the - art purpose representation engine from [ 15 ] . The matches will be analogous products that adjust to different sizes of some personal product , which could hopefully inspire the designer . From a system standpoint , the process can be divided into two phases : 1 ) expressing a focus - abstracted query , and 2 ) using the query to \ufb01nd analogies in a corpus . Below we describe our process in more detail for each phase . Expressing the focus - abstracted query Figure 2 shows a worked example of the overall process . We describe each step in turn . Step 1 : Focus on important sentences We assume the designer begins with an existing complete prod - uct description . Figure 2 shows one such example from the Quirky corpus ( the \u201cSoapy slider\u201d product ) . The designer se - lects the sentences most relevant to their need , thus identifying which aspect in the product they wish to further explore . In the \u201cSoapy slider\u201d example ( Figure 1 ) , designer A ( focusing on product size adjustments ) will choose the sentence \u201cextend - able for different sizes of soap bars\u201d ( see Step 1 in Figure 2 ) . CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 3 Figure 2 . Illustration of the process of expressing focus - abstracted queries in our system . Designers express a focus - abstracted query by ( 1 ) selecting important sentences in a product description that relate to an intended focus , ( 2 ) ignoring irrelevant terms , and ( 3 ) replacing im - portant terms ( where desired ) with appropriate abstracted properties , yielding a sequence of important terms and abstracted properties . Designer B ( interested in removing liquid from things ) will choose the sentence \u201cit removes soapy water away from the bar of soap keeping it dryer to last longer\u201d . Step 2 : Focus on important terms Important sentences from Step 1 may still contain terms or domain details that are irrelevant to the intended purpose of the designer . Ignoring irrelevant terms increases the focus of the query on the intended purpose of the designer . It also achieves a form of abstraction ( e . g . , ignoring irrelevant domain details ) . To achieve this function , the interface allows the designer to take any noun , verb , or adjective from the important sentences and mark them with an \u201cIGNORE\u201d \ufb02ag if they are irrelevant to her speci\ufb01c purpose . For example , designer A ( who is not interested in bars speci\ufb01cally ) , might choose to IGNORE the term \u201cbars\u201d ( see Step 2 in Figure 2 ) . Designer B ( who is interested speci\ufb01cally in how to remove water from a bar of soap ) may IGNORE the term \u201clast\u201d , which describes the ultimate purpose of keeping the bar of soap dry , but may not be shared by other products that also separate water from objects . Step 3 : Abstract important terms After Step 2 , the designer\u2019s needs are still expressed in the original domain ( e . g . , soap ) . In order to \ufb01nd domain - distant analogies it is necessary to replace key terms with their appro - priate abstractions . The designer can abstract a term by clicking on it and selecting the appropriate abstractions from a list . The list is grouped into semantic sets from which the designer chooses the most relevant one . The most useful set is often obvious ( e . g . , Soap - Personal is more relevant than SoapOpera ) , making it easier to narrow the list down . For example , designer A might not be interested in the fact that soap is a ToiletrySubstance , but rather that it is more generally a PersonalProduct , and select that property to abstract the term \u201csoap\u201d ( see Step 3 in Figure 2 ) . In designing this component of our system , we faced and dealt with several design challenges : \u2022 Choosing an appropriate knowledge base . To \ufb01nd abstrac - tions to show the designers , we explored several knowledge bases . WordNet [ 21 ] is a large English lexical database , including relations like synonym , hypernym and hyponym . WordNet is lexical and not focused on facts about the world , rendering it less useful for our purpose . In addition , the number of relations it supports is very small . Another alter - native we considered is ConceptNet [ 25 ] . ConceptNet in - cludes knowledge from crowdsourcing and other resources , rendering it very noisy . We ended up choosing Cyc [ 16 , 17 ] as our main Knowl - edge Base . Cyc is a very large , logic - based knowledge base representing commonsense knowledge . Cyc contains over \ufb01ve hundred thousand terms , seventeen thousand types of relations , and over seven million assertions , i . e . , sim - ple facts about the world . Examples of assertions : \u201c # $ isa # $ DonaldTrump # $ UnitedStatesPresident\u201d ( Donald Trump is the US president ) and \u201c # $ genls # $ Water # $ LiquidTangi - bleThing\u201d ( liquid is a generalization of water ) . If a term is missing from Cyc , we resort to WordNet . Crucially for our purposes , Cyc contains supersets of terms , which are useful for abstraction . For example , \u201cDomes - ticatedAnimal\u201d and \u201cCanisGenus\u201d are supersets of \u201cdog\u201d . \u201csoap\u201d may abstract to its supersets \u201cToiletrySubstance\u201d , \u201cWaterSolubleStuff\u201d , \u201cPersonalProduct\u201d , and many others . Another way of looking at it is that soap has the properties of being a water soluble toiletry substance and a personal product . Thus we use the terms Abstractions and Properties interchangeably . The level of abstraction controls the distance from the do - main , thus allowing the designers to choose far or near analogies . Importantly , the abstractions also give designers control over the direction of the abstraction ( e . g . , ignore all things that are about cleaning , but make sure they share the property of being personal products , or water soluble ) . \u2022 Dealing with natural language descriptions . Quirky prod - uct descriptions are written in unstructured natural language . To obtain and display appropriate abstractions for the de - signers to select from , we \ufb01rst preprocess the corpus and perform part of speech ( POS ) tagging . We then apply NLTK WordNet Morphy [ 3 ] to get the canonical form of each term ( according to its POS ) , and use this form to query Cyc for its associated properties . For example , we change \u201csizes\u201d to \u201csize\u201d before KB lookup . \u2022 Presenting a manageable set of abstractions to choose from . A \ufb01nal design consideration here is the number of abstractions to present to the designer for consideration , CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 4 Figure 3 . Illustration of the process of abstracting the corpus based on a given focus - abstracted query . All terms in each document that match the designer - selected abstracted properties ( shown in monospace font ) are replaced by the matching properties . This brings documents that might be different in domain ( e . g . , about \u201c\u2019knives\u201d ) but are nevertheless similar at the desired level of abstraction ( e . g . , PersonalProduct ) closer to the focus - abstracted query . since words in Cyc are often associated with many potential abstractions ; for example , the term \u201cdog\u201d has over 100 dif - ferent abstractions . To limit the number , we experimented with \ufb01ltering by level of abstraction ; we found that three abstraction levels appeared to be an optimal cutoff , above which the terms become to general and uninteresting . For example , \u201csoap\u201d may be abstracted to \u201cthing\u201d , which is common to all objects and therefore provides little infor - mation and can be replaced by the IGNORE option . The abstractions are sorted from speci\ufb01c to general . We considered sorting within the abstraction levels by mea - suring property prevalence . If a vast number of items share a certain property then it is probably too general ( e . g . , \u201cPhys - ical Entity\u201d ) , and will not be useful . If there are too few items , then maybe the property is too speci\ufb01c and less in - teresting ( e . g . \u201cGoldColor\u201d ) . However , since the relevant abstractions typically appear among the \ufb01rst \ufb01ve to ten abstractions we decided not to implement further ordering . Once the designer selects appropriate abstractions , the expres - sion phase is complete : the end result is a focus - abstracted query derived from the designer\u2019s operations on the origi - nal product description : unchecked sentences are omitted , words in the IGNORE list are omitted , and the words that were abstracted are replaced by their abstractions . For exam - ple , designer A\u2019s selections would yield the following focus - abstracted query : [ extendable , different , SpatialQuantity , Per - sonalProduct ] ( see Figure 2 ) . Finding analogies for the focus - abstracted query Now that the designer expressed their desired focus and ab - straction , we use our analogy engine to \ufb01nd analogies from a corpus of potential matches that are tuned for that particular focus ( while preserving abstraction ) . The most important step is to re - represent the corpus with the designer\u2019s chosen abstractions . Concretely , for each document , we \ufb01nd all terms in it that share the same abstracted properties ( in Cyc ) as those contained in the focus - abstracted query . For example , if the designer abstracted \u201csoap\u201d to \u201cPersonalProduct\u201d ( indicating that it is not the soap they care about , but rather being a personal product ) , the engine looks for other terms in the corpus which share the property of being \u201cPersonalProduct\u201d ( e . g . \u201cknife\u201d ) and abstracts them to \u201cPersonalProduct\u201d as well ( see Figure 3 ) . The goal of this abstraction step is to ensure that products from different domains that nevertheless share key relations or properties with the focus - abstracted query at the right level of abstraction can be seen as close to the query . Next , our engine \ufb01nds matching products in the abstracted corpus . We considered several options for the matching . We build on the work of [ 15 ] , which was shown to \ufb01nd good analo - gies on the same dataset . In short , [ 15 ] takes natural language product descriptions and uses deep learning ( speci\ufb01cally , a bidirectional RNN [ 2 ] ) to learn vector representations for pur - pose ( what is this product good for ? ) and mechanism ( how does it work ? ) . Given the vector representations , the algo - rithm of [ 15 ] \ufb01nds products with similar purpose but different mechanisms , that might serve as analogies . We use a similar algorithm for searching for products ; how - ever , in our case , since we focus on relevance for a speci\ufb01c abstracted need , we change the algorithm to focus only on \ufb01nding similar products with respect to purpose . We do this by computing a similarity score between the purpose represen - tation vector for the focus - abstracted query , and the purpose representations for all documents in the abstracted corpus , and selecting the 10 documents with the highest similarity score . In the case of designer A , who wanted to adjust to different soap bar sizes , the system suggested a knife rolodex , allowing storing knives of different sizes in the same holder ( Figure 3 ) . EVALUATION Our core hypothesis is that our system is able to \ufb01nd analogies for focused queries , while still retaining the ability to \ufb01nd analogies from distant domains . We evaluated this hypothesis across a range of focused query scenarios from seeds sampled from the larger corpus of prod - ucts . The general scenario is that of a designer looking for novel ways to redesign some speci\ufb01c aspect of an existing product . This is a common design task that requires creativity , CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 5 and may especially bene\ufb01t from distant analogies ( in order to maximize novelty ) , since the existence of many domain features may make it dif\ufb01cult for the designer to think of alternative mechanisms or domains . Preliminary Study We \ufb01rst conducted a preliminary usability study to assess the expressiveness of the interface . Four product designers and one non - designer were asked to examine three product descrip - tions randomly selected from the corpus and identify aspects in the product to redesign or generalize ( e . g . , generalize \u201cwater\u201d to \u201cliquid\u201d ) , and then to subsequently express those aspects using our interface . Prior to starting the task the users were given several examples ( using seeds not from the user - study set ) and a short Interface training session . For each aspect the users initiated a new interface session , checked the rele - vant sentences , set to IGNORE the unimportant terms ( e . g . , \u201cblack\u201d or \u201cin the mall\u201d ) , and abstracted terms according to their needs . Users reported they were mostly able to express their needs and in general the users thought the tool was bene\ufb01cial and easy to use . One of the product designers remarked : \u201cFor my own innovation I did not perform such an analogy search . I wish search engines had this Interface\u201d . One interesting \ufb01nding was that the interface helped users identify additional potential aspects and abstractions they had not thought of , and \ufb01nd products in distant domains . For example , one user used \u201cSeparationEvent\u201d ( of liquid ) as an abstraction of \u201cRemoving\u201d , which he had not previously considered ; this abstraction led to a product using hydrophobic coating . Users also cited challenges they encountered in using the interface which included words missing from the description ( e . g . , the option to add \u201clightweight\u201d was not available ) , and bigrams ( e . g . , \u201ccoffee maker\u201d ) that were not abstracted as one unit . One designer suggested marking some words as \u201cnice to have\u201d . These limitations could be addressed by simple extensions of our system ( i . e . , incorporating user - supplied keywords and looking up n - grams in the KB ) . For further discussion see Future Work . Search Scenarios After our usability study , we turned to evaluate the results of our engine . We randomly selected 5 seed products from the corpus , using the following screening criteria : \u2022 Is understandable ( e . g . , grammatical errors and typos do not overly obscure meaning , can actually visualize what it is ) \u2022 Has at least two well - speci\ufb01ed functions / aspects ( so we can de\ufb01ne distinct focus query scenarios ) \u2022 Judged to have corresponding inspirations in the corpus Two members of our research team then identi\ufb01ed two re - design scenarios for each seed . For example , in the \u201cSoapy slider\u201d product ( which was one of the selected seeds ) , the two scenarios were \u201cmake the dish compatible with different sizes of soap bars\u201d , and \u201ckeep excess soapy water away from the Seed product Scenario 1 Scenario 2 Soapy slider . Unique 2 piece horizontal soap dish with a slide that keeps excess soapy water away from the bar of soap . Make the dish com - patible with differ - ent sizes of soap bars Keep excess water away from the bar of soap Camp brew coffee maker . Light weight all in one cof - fee grinder and maker for camping and hiking . Tell when some - thing is done cooking Make food and drink outdoors Laundry folding table . Table that folds down out of the laundry room wall and provides a surface for folding laundry Make compact a pile of \ufb02exible , fold - able garments Make compact a piece of furniture On / off velcro pocket shoe . Attached / detached pocket for any shoe . Attach a small pocket to shoe / ankle comfortably & durably Make the attached pocket inconspicu - ous The restsack . Backpack that doubles as an outdoor chair stool . Carry items on the go Provide a portable seat Table 1 . Seed product descriptions and associated redesign scenarios used for our evaluation experiment . Descriptions shortened . bar of soap\u201d . We therefore have a total of 10 search scenarios ( see Table 1 ) . Constructing the queries The research team members who made the scenarios then used our system to create focus - abstracted queries for each of the scenarios . Figure 2 includes one example scenario and focus - abstracted query . Another example ( for the \u201cSoapy slider\u201d example ) is RemovingSomething , LiquidTangibleThing , SolidTangibleThing for the scenario need \u201ckeep excess soapy water away from the bar of soap\u201d . Measures Our goal is to \ufb01nd relevant analogies for a speci\ufb01c aspect of a seed product without being constrained to the same domain as the seed product ( i . e . , retaining the ability to \ufb01nd domain distant yet relevant analogies for a focused need ) . Therefore , we evaluate the relevance and domain distance of each match for its target query . Both measures were obtained by human judgments of the matches . All ratings were performed blind to the method that produced the match : that is , for each scenario , shared matches were combined across the methods , and the method that produced the match was not shown . Relevance We operationalized relevance as the degree to which the match meets the needs expressed in the query . Judgment of relevance took into account three factors : 1 ) the degree to which it shared the key purpose ( s ) expressed in the query ( e . g . , make compact , adjust ) , 2 ) the degree the objects related to the purpose shared the key properties of the key objects in the query ( e . g . , physical size of soap bars ) , and 3 ) the degree to which the purpose ( and associated mechanism ) was explicitly stated ( since some products state a function as a desirable property of the product , rather than a function it aims to achieve ) . This last factor is included because it is easier for a people to notice and CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 6 use analogies if the mapping to their problem is explicitly described / highlighted [ 24 ] . The judgment scale was a 5 - point Likert - like scale , with the following anchors ( developed after multiple rounds of training and piloting with a separate set of data ) : 1 = Matches none of the key functions and object properties in the query 2 = Implicitly matches a few of the key purpose ( s ) , but none of the key object properties in the query 3 = Implicitly or explicitly matches a few of the key pur - pose ( s ) AND a few of the key object properties in the query 4 = Implicitly or explicitly matches most of the key pur - pose ( s ) AND a most of the key object properties in the query 5 = Explicitly matches most / all of the key purpose ( s ) and key object properties in the query Two members of the research team ( who did not directly de - velop the system ) were trained to use the judgments on a separate dataset until they reached good inter - rater agreement , Cronbach\u2019s alpha = . 82 . They then each evaluated half of the matches independently . Examples of low and high relevance - scored matches are shown in Figure 4 . Domain distance We operationalized relevance as the degree to which the match shared domain features with the query\u2019s seed product . Note that this measure ignores the scenario and instead compares each match with the whole seed product . The judgment scale was also a 5 - point Likert - like scale , rang - ing from 1 ( very similar ) to 5 ( very different ) . One issue with this judgment is that many products could be thought of as being in a number of different domains : for example , the \u201ccamp brew coffee maker\u201d is about food / drink / coffee and camping / outdoors . Different raters might weight each feature differently as core / peripheral to the \u201cdomain\u201d of the prod - uct . For this reason , rather than utilizing single ratings from independent raters , each match received a rating from 2 in - dependent judges ( members of the research team ) , and the average of their ratings yielded the distance score for each match . The inter - rater reliability for this measure was good , Cronbach\u2019s alpha = . 79 ) . Examples of low and high distance - scored matches are shown in Figure 4 . Experiment Design and Hypotheses We compare our method against three other approaches : 1 . OverallPurpMech . This is the purpose - mechanism method from [ 15 ] . It estimates and matches on overall purpose and mechanism vectors . More speci\ufb01cally , we repli - cate the method from their evaluation experiment , which \ufb01nds matches for a given seed based on similarity of pur - pose , and aims to diversify by mechanism . We use this method to \ufb01nd a set of purpose - similar , mechanism - diverse matches for each scenario . The purpose of comparing our system to this method is to determine to what extent we have advanced the state - of - the - art in analogy - \ufb01nding . 2 . OverallGloVe baseline . [ 23 ] This method approximates the status quo for information - retrieval systems , which tend to operate on the whole document . Each document in the corpus is represented by the average of GloVe vectors for all words ( excluding stopwords ) . We use Glove pre - trained on the Common Crawl dataset ( 840B tokens , 300d vectors ) 1 . We then normalize each document vector , and calculate cosine similarity ( which is the same as Euclidean distance in this case ) between the resulting vectors for each seed and all other documents , and choose the 10 nearest as matches . 3 . FocusOnly baseline . This baseline helps tease apart the impact of focusing only versus focusing and abstracting ( with the knowledge base ) . For each scenario , we form a fo - cus query as a bag - of - words containing only the words that were abstracted during the process of making the focused - diverse query with our method , i . e . , the words themselves instead of their abstractions ( stopping at Step 2 in Figure 2 ) . We then calculate the average of the GloVe word vectors for all terms in the query and compare it with the averaged GloVe vectors of all other products . We again use cosine similarity \ufb01nd the 10 nearest products . We therefore have 4 different methods ( FocusAbstracted , Over - allPurpMech , OverallGloVe , and FocusOnly ) of obtaining 10 matches each for 10 different search scenarios . Figure 4 shows illustrative matches from each of these methods . We hypothesize that OverallPurpMech will do relatively poorly on relevance ( since it is tuned to capture \u201cthe\u201d overall purpose / mechanism of each document , which might miss the intended focus of a redesign scenario ) , but well on distance ( as it did in [ 15 ] ) . We hypothesize that OverallGlove will do poorly on relevance and distance ( since it is neither tuned for focus nor abstraction ) , and FocusOnly will do well on rele - vance , but poorly for distance ( since it is tuned for focus , but not abstraction ) . Finally , we hypothesize that our FocusAb - stracted method will do well on both relevance and distance ( since it is tuned for both focus and abstraction ) . Results As a \ufb01rst - pass analysis , we note that the methods return almost completely non - overlapping sets of matches for each scenario , giving us 394 unique matches out of 400 total possible unique matches . This initial result suggests that the methods behave quite differently . Indeed , as Figure 4 illustrates , OverallPurp - Mech appears to return domain - distant inspirations that are analogous on some purpose of the seed product ( though not necessarily the speci\ufb01ed purpose ) , and OverallGlove and Fo - cusOnly appear to return highly relevant inspirations from the same / similar domains , while FocusAbstracted matches appear to be both highly relevant and from distant domains . FocusAbstracted matches more relevant than OverallPurp - Mech , and as relevant as OverallGloVe and FocusOnly We now turn to formal quantitative tests of our hypothe - ses . Figure 5 ( left panel ) shows relevance scores by method , collapsed across scenarios . Using a one - way Analysis of 1 Available here : https : / / nlp . stanford . edu / projects / glove / CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 7 Figure 4 . Illustrative matches from each method for the scenario : \u201cmake the dish compatible with different sizes of soap bars\u201d . The abstraction of \u201csoap\u201d seems to allow the FocusAbstracted method to ignore the domain difference of knives vs . soap . OverallPurpMech \ufb01nds a match from a different domain that is analogous in terms of its overall purpose of keeping something clean / dry , but misses the core purpose of adapting to different sizes . In contrast , both OverallGloVe and FocusOnly \ufb01nd a highly relevant match from the same domain . Figure 5 . FocusAbstracted matches achieve comparable relevance to FocusOnly and GloVe baselines , and more relevance than OverallPurpMech ( left panel ) while being more domain distant than FocusOnly and GloVe baseline matches , and equivalently domain distant as OverallPurpMech matches ( right panel Variance ( ANOVA ) model with method as the sole between - observations factor , and matches as the unit of observation , we \ufb01nd signi\ufb01cant differences on the mean relevance score across the methods , F ( 3 , 396 ) = 14 . 1 , p < . 01 . A follow - up Tukey Honestly Signi\ufb01cant Difference ( HSD ) post - hoc test ( to correct for increased chance of false positives due to multiple comparisons ) shows that only OverallPurpMech has signi\ufb01 - cantly lower relevance compared to the other methods , p < . 01 vs . OverallGloVe , FocusOnly , and FocusAbstracted . FocusAbstracted matches more domain distant than FocusOnly and OverallGloVe , and as distant as OverallPurpMech Figure 5 ( right panel ) shows distance scores by method , col - lapsed across scenarios . Again using a one - way ANOVA model with method as the between - observations factor , we \ufb01nd signi\ufb01cant differences across the methods on mean dis - tance , F ( 3 , 396 ) = 14 . 1 , p < . 01 . A follow - up Tukey HSD post - hoc test shows that only FocusAbstracted has signi\ufb01 - cantly higher domain distance compared to OverallGloVe ( p < . 05 ) and FocusOnly ( p < . 05 ) . Despite being numerically more domain distant than OverallGloVe and FocusOnly , the OverallPurpMech method\u2019s matches are not signi\ufb01cantly more domain distant after the Tukey corrections for multiple com - parisons . Distance of FocusAbstracted matches uncorrelated with rele - vance , in contrast to OverallPurpMech and FocusOnly Finally , we explore the relationship between relevance and domain distance might vary across the methods . Since Over - allGloVe tends to match primarily based on surface features , we expect a strong negative correlation between relevance and distance , such that relevant matches tend to be less domain distant . We expect a similar relationship for FocusOnly ( since it operates in a very similar way to OverallGloVe ) , albeit pos - sibly weaker since it ignores other domain details that were in ignored sentences / terms , but no such relationship for Focus - Abstracted and OverallPurpMech ( since they are designed to abstract away from domain details . Indeed , across all matches from all methods for all scenarios , there is a signi\ufb01cant negative correlation between relevance and distance : on average , the more relevant a match is , the closer it is to the domain of the seed product , r = \u2013 0 . 19 , 95 % CI = [ \u2013 0 . 28 , \u2013 0 . 09 ] , p < . 01 . However , the relationship between CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 8 relevance and distance varies by method . As expected , the relationship is strongest for OverallGloVe matches , r = \u2013 0 . 36 [ \u2013 0 . 52 , \u2013 0 . 18 ] , followed by FocusOnly , r = \u2013 0 . 22 [ \u2013 0 . 40 , \u2013 0 . 03 ] , p < . 05 . In contrast , there is no signi\ufb01cant correlation between relevance and distance for either FocusAbstracted , r = \u2013 0 . 09 [ \u2013 0 . 28 , 0 . 11 ] , p = 0 . 38 , or OverallPurpMech , r = \u2013 0 . 02 [ \u2013 0 . 22 , 0 . 18 ] , p = 0 . 38 . Case Study To give an intuition for what might be driving these quantita - tive difference , we return to examine 4 illustrative matches for the scenario \u201cmake the dish compatible with different sizes of soap bars\u201d ( shown also in Figure 4 ) . OverallPurpMech returns cross - domain matches like a \u201cyoga mat wash stack machine\u201d , which includes drying and cleaning functions for the yoga mats , which match the overall main purpose of the \u201cSoapy slider\u201d product ( i . e . , keeping the bar of soap dry ; in fact , this yoga mat inspiration is relevant for the other \u201cSoapy slider\u201d scenario that focuses on this purpose ) . This illustrates how OverallPurpMech can return interestingly distant but ul - timately irrelevant matches if the designer wants to focus on an aspect of a seed product that is different from its main purpose . On the other extreme , OverallGlove and FocusOnly both return many relevant but near matches , like a \u201csoap saver\u201d device that fuses small used bars of soap together so they don\u2019t slip through the cracks , or a \u201ctouchless soap dispensing unit\u201d with a winding inner tube that expands to reach inside any size bottle . In contrast to both of these extremes , our FocusAbstracted method is able to return matches that are both relevant to the focus need and domain distant , like a \u201cknife rolodex\u201d product that includes multiple slots for different sized knives , or a \u201cmaximizing phone tablet\u201d ( not shown in Figure 4 ) , which uses a telescopic frame to adjust to different - sized phones . In both of these cases , our FocusAbstracted method is able to zero in on the idea of adjusting to different \u201cspatial quantities\u201d , while ignoring differences in the kind of \u201cpersonal product\u201d ( e . g . , knives , phones ) being adjusted to , due to the replacing of the domain - speci\ufb01c terms like knife and phone with abstracted properties that match those of the soap bar . DISCUSSION Summary and Implications of Contributions In this paper , we sought to design a system that can tune computational analogical search to \ufb01nd relevant and distant inspirations for speci\ufb01c design needs . We presented a system that allows designers to focus on a speci\ufb01c aspect of a product description by selecting key terms to form a query , and create a targeted abstraction of those terms by selecting properties from a knowledge base that are important for understanding the core relational structure of the design need . We demon - strated that this focus - abstracted approach led to the retrieval of inspirations that were both relevant and distant , in contrast to alternative state - of - the - art approaches that either sacri\ufb01ced relevance for distance , or vice versa . Thus , we contribute a promising new method \ufb01nding distant analogical inspirations for speci\ufb01c design needs . One speci\ufb01c \ufb01nding that deserves further discussion is the high performance of the OverallGlove condition in terms of relevance . Our initial prediction was that this condition would perform poorly on relevance , since , like the OverallPurpMech method from [ 15 ] , it operates on the whole product description as opposed to a speci\ufb01c focus query . Cognitive theories of analogy suggest one possible explanation . In particular , some researchers point to the \u201ckind world hypothesis\u201d to explain how humans learn abstract concepts : salient surface features ( which tend to be shared by things in the same domain ) tend to be strongly correlated with structural features . As Gentner [ 11 ] notes , \u201cif something looks like a tiger , it is probably a tiger\u201d . One implication of this is that things that are in the same domain likely share many relational features , including purposes . Thus , since OverallGlove is tuned to match based on surface features , it is possible that it found many relevant matches for the speci\ufb01c need simply by \ufb01nding things in the same domain . Limitations and Future Work Supporting more expressive queries We have shown how helpful a focus - abstraction interface can be for a designer wishing to re - design an aspect of a product . However , in our pilot tests of the interface , we noticed that some information needs are still hard to express . An interesting direction is to explore more expressive queries ( by adding more mechanisms to the interface , for example allowing designers to manually add important terms or prop - erties ) . This would also allow designers to explicitly express trade - offs . Improving expressiveness might be especially im - portant for domains with highly technical concepts / terms with very speci\ufb01c meanings ( e . g . , regularization in machine learn - ing ) that have poor coverage in existing knowledge bases like Cyc . Automatically identifying different purposes in a document Our analogy engine calculates a representation of the overall purpose of the focus - abstracted query and all the documents in the corpus . For the abstracted focus description this is a good \ufb01t , as the overall purpose is identical to the speci\ufb01c need . For the rest of the corpus the overall purpose comprises several purposes . We expect that automatically dividing each docu - ment to sub - purposes prior to searching for purpose matches would signi\ufb01cantly improve them . The usage patterns of our tool can serve as annotated set for learning how to segment the documents . Automatically suggesting queries Another interesting future direction might be to use informa - tion obtained from usage of the tool to learn common focus and abstraction patterns , and suggest focus - abstractions au - tomatically to designers . For example , we might learn that soap dishes , phone cases , and cake - cutters often have a focus - abstracted problem of < expanding to \ufb01t objects of different physical sizes > , and suggest these focus - abstractions to other designers creating queries for these ( and similar ) products . Extending to other datasets and domains While we have tested our method on Quirky innovations , it would be useful to explore its utility on other corpora . In CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 9 particular , it would be interesting to test our ideas on a corpus of products from manufacturing companies , which are con - stantly looking for innovations for improving their products , or even to corpora of research papers and patents . The tar - geted abstraction approach could be particularly powerful for \ufb01nding analogies for \ufb01elds of study where the properties of the objects are critical for determining what makes for useful analogies : for example , as we noticed from our experts in mechanical engineering and materials science , someone work - ing on ways to deform stretchable polymers would not likely bene\ufb01t from analogies to deformation techniques for concrete , since polymers ( by virtue of their material properties ) react very differently to physical stresses . Note , further research is required to understand how well the method generalizes to such corpora . As noted above , Cyc does not contain many technical terms , and we may need a different source for their abstractions . CONCLUSION In this paper , we contribute a novel system for tuning analogi - cal search for speci\ufb01c design needs , consisting of an interface for designers to express their speci\ufb01c needs in abstract terms , and an analogy search engine that uses this focus - abstracted query to \ufb01nd inspirations from a corpus that are both relevant and domain - distant . This work contributes a novel path for - ward to computational support for mining large databases of potential inspirations on the Web to improve design work . ACKNOWLEDGEMENTS The authors thank the anonymous reviewers for their helpful feedback , and Amir Shapira for his helpful insights into the design process . Dafna Shahaf is a Harry & Abe Sherman assistant professor . 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DOI : http : / / dx . doi . org / 10 . 1145 / 2818048 . 2835201 CHI 2018 Paper CHI 2018 , April 21 \u2013 26 , 2018 , Montr\u00e9al , QC , Canada Paper 121 Page 11", + "laneEngineeringSerendipityWhen": "R E S E A R C H A R T I C L E Engineering serendipity : When does knowledge sharing lead to knowledge production ? Jacqueline N . Lane 1 | Ina Ganguli 2 | Patrick Gaule 3 | Eva Guinan 4 | Karim R . Lakhani 1 1 Laboratory for Innovation Science at Harvard , Harvard Business School , Boston , Massachusetts 2 Department of Economics , University of Massachusetts Amherst , Amherst , Massachusetts 3 Department of Economics , University of Bath , Bath , UK 4 Dana - Farber Cancer Institute , Boston , Massachusetts Correspondence Jacqueline N . Lane , Laboratory for Innovation Science at Harvard , Harvard Business School , 175 N . Harvard Street , Boston , MA 02134 . Email : jnlane @ hbs . edu Funding information Harvard Catalyst ; Laboratory for Innovation Science at Harvard University ; NIH Clinical Center , Grant / Award Numbers : UL1RR025758 - 02S4 , UL1TR000170 , UL1TR001102 , UL1TR002541 ; Harvard Business School Division of Research and Faculty Development Abstract Research Summary : We investigate how knowledge similarity between two individuals is systematically related to the likelihood that a serendipitous encoun - ter results in knowledge production . We conduct a field experiment at a medical research symposium , where we exogenously varied opportunities for face - to - face encounters among 15 , 817 scientist - pairs . Our data include direct observations of interaction pat - terns collected using sociometric badges , and detailed , longitudinal data of the scientists ' pos - tsymposium publication records over 6 years . We find that interacting scientists acquire more knowl - edge and coauthor 1 . 2 more papers when they share some overlapping interests , but cite each other ' s work between three and seven times less when they are from the same field . Our findings reveal both col - laborative and competitive effects of knowledge simi - larity on knowledge production outcomes . Managerial Summary : Managers often try to stimulate innovation by encouraging serendipitous interactions between employees , for example by using office space redesigns , conferences and similar events . Are such inter - ventions effective ? This article proposes that an effective encounter depends on the degree of common knowledge shared by the individuals . We find that scientists who Received : 4 February 2020 Revised : 27 October 2020 Accepted : 10 November 2020 DOI : 10 . 1002 / smj . 3256 This is an open access article under the terms of the Creative Commons Attribution License , which permits use , distribution and reproduction in any medium , provided the original work is properly cited . \u00a9 2020 The Authors . Strategic Management Journal published by John Wiley & Sons Ltd . Strat Mgmt J . 2020 ; 1 \u2013 30 . wileyonlinelibrary . com / journal / smj 1 attend the same conference are more likely to learn from each other and collaborate effectively when they have some common interests , but may view each other com - petitively when they work in the same field . Hence , when designing opportunities for face - to - face interactions , man - agers should consider knowledge similarity as a criteria for fostering more productive exchanges . K E Y W O R D S innovation , knowledge production , knowledge sharing , knowledge similarity , natural field experiment 1 | INTRODUCTION In 2013 , cell biologist William Earnshaw of the University of Edinburgh happened to attend the same academic conference as systems biologist Job Dekker of the University of Massachusetts Medical School and computational biologist Leonid Mirny from the Massachusetts Institute of Technology . By chance , Earnshaw attended a presentation by Dekker and Mirny about their joint work on mitotic chromosomes , during which he became convinced that his lab could pro - vide bench methods to improve Dekker and Mirny ' s computational models . Earnshaw approached Dekker and Mirny after the talk ; their conversation evolved into a three - lab collab - oration and a 2018 publication in Science ( Pain , 2018 ) . This anecdotal example suggests that serendipitous encounters can play a role in inno - vation , perhaps by exposing individuals to people that they would not otherwise have a chance to meet , and to unfamiliar sources of information that can be combined with their own knowledge stock and lead to new discoveries ( Fleming , Mingo , & Chen , 2007 ; Uzzi , Mukherjee , Stringer , & Jones , 2013 ) . However , the likelihood that serendipitous encounters lead to successful knowledge production remains poorly understood often because the pro - cess is uncertain , complex and relatively understudied . In particular , even if an encounter eventually produces new knowledge , this process may occur slowly and only emerge after a number of years ( Catalini , 2018 ; Fleming , 2001 ) . A significant degree of knowledge within organizations is tacit and is not easily transferred ( Hansen , 1999 ; Nonaka , 1994 ) . Knowl - edge creation via new collaborations can be costly ( Boudreau et al . , 2017 ; Dahlander & McFarland , 2013 ) , requiring coordinated effort , alignment of incentives , establishment of trust , generation of creative synergy , and \u201c matching \u201d criteria , such as personality and scheduling compatibility ( Azoulay , Ding , & Stuart , 2007 ; Catalini , 2018 ) . Even if knowledge transfer or creation is successful , knowledge tends to diffuse slowly through interpersonal networks ( Fleming et al . , 2007 ; Singh , 2005 ) . The question thus arises of when serendipi - tous encounters are more likely to facilitate useful idea exchange and knowledge produc - tion . Previous literature tends to focus on the factors that may increase the amount or intensity of short - term communication and interaction between individuals , rather than the implications of serendipitous encounters on knowledge production ( Allen , 1977 ; Kleinbaum , Stuart , & Tushman , 2013 ) . Given that geographic proximity increases the 2 LANE ET AL . likelihood of serendipitous encounters ( Catalini , 2018 ) , recent research has begun to exam - ine the effects of organizational redesign on knowledge production ( Catalini , 2018 ; Fang , Lee , & Schilling , 2010 ; Lee , 2019 ) . Although it is possible that organizational redesign and greater geographic proximity between individuals can increase knowledge production , most studies do not directly address how knowledge sharing between two individuals affects the path - dependent nature of the knowledge production process . Since prior work tends to focus on a single knowledge production outcome ( Phelps , Heidl , & Wadhwa , 2012 ) , often on the factors that impede knowledge transfer ( Carlile , 2004 ; Hansen , 1999 ; Szulanski , 1996 ; Uzzi , 1997 ) , it is unclear how knowledge sharing may also affect knowledge creation and diffusion . Hence , we know relatively little about whether serendipitous encounters actually lead to meaningful idea exchange that can be applied to one ' s own tasks , or whether these interactions can spark new collaborations and broader diffusion of ideas over time . The goal of our work is to address this gap in literature by examining to what extent a sys - tematic relationship exists between serendipitous encounters and knowledge production outcomes . We posit that an effective encounter hinges not just on the serendipity of the interac - tion but also on the existence of common knowledge that individuals use to make sense of each other ' s specialized knowledge and connect it to what they already know . Common knowledge enhances the ability for new knowledge to be assimilated into the concepts , objects and patterns that are already present in people ' s cognitive structures ( Bower & Hilgard , 1981 ; Cohen & Levinthal , 1990 ) . When new information is related to prior knowl - edge constructs , it is more readily absorbed , integrated , and applied in new settings ( Carlile , 2004 ; Kogut & Zander , 1992 ) without sufficient prior knowledge , individuals may have difficulty integrating new knowledge ( Rosenkopf & Almeida , 2003 ) . In this article , we argue that there are two essential characteristics to people ' s common knowledge : field similarity in their educational backgrounds and training , and intellectual similarity in their interests , passions and pursuits . Serendipitous encounters are more likely to be effec - tive when people share some knowledge similarity in both their field and intellectual interests . To systematically investigate the relationships between serendipitous encounters and knowledge production requires a rich longitudinal dataset that directly observes exogenous face - to - face knowledge sharing between two individuals and subsequent knowledge production outcomes . To draw causal inferences , we designed and executed a field experiment at a research symposium on advanced imaging in early 2012 at a leading academic medical center . We chose this setting due to the centrality of knowledge production to their organizational and individual performance goals and the prevalence of knowledge sharing norms ( Dahlander & McFarland , 2013 ) . We created exogenous variation in opportunities for information - rich , face - to - face encounters between cross - disciplinary scientists interested in applying for an internal grant program promoting the development of advanced imaging solutions to address an unmet clinical need . Using electronic sociometric badges ( Kim , McFee , Olguin , Waber , & Pentland , 2012 ) , we collected a unique dataset of fine - grained , live interactions between pairs of scientists who were randomly assigned to be in the same symposium room for knowledge shar - ing . We then combined this with a rich longitudinal dataset on long - run knowledge production outcomes from the scientists ' written publications over a six - year period , where we linked scien - tists ' pairwise keywords , collaborations and forward citations to examine the extent that the encounters led to knowledge transfer , creation and diffusion , respectively . This design enabled us to causally and systematically identify the relationships between knowledge sharing and LANE ET AL . 3 knowledge production to explain why some serendipitous encounters resulted in knowledge production while others did not . We find both cooperative and competitive effects of serendipitous knowledge sharing encounters on knowledge production . On the one hand , knowledge sharing led to greater knowledge transfer and knowledge creation when people had some overlapping intellec - tual and field interests , respectively . Although knowledge sharing had limited impact on knowledge transfer among pairs with either few or many overlapping interests , we observe a twofold increase in knowledge transfer ( of Medical Subject Headings or MeSH key - words ) for moderately similar pairs , suggesting an inverted U - shaped relationship between knowledge overlap and knowledge transfer . Similarly , we find that knowledge sharing between pairs with some common interests led to 1 . 2 more coauthored peer - reviewed publications , compared to a 0 . 98 decrease in copublications among pairs in the same field , which suggests that the knowledge sharing intervention reduced the search costs associated with forming synergistic collaborations . By comparing the knowledge drivers of near - term collaborations and long - term collaborations , we find that common knowledge is more critical for the former . On the other hand , knowledge sharing reduces knowledge diffusion between people from the same field , with interacting pairs citing each other between 2 . 8 and 6 . 7 times less than noninteracting pairs from more distant fields . We aim to make several contributions to the literature . First , we contribute to the lit - erature on search costs and information frictions associated with the knowledge produc - tion process ( Agrawal & Goldfarb , 2008 ; Boudreau et al . , 2017 ; Catalini , 2018 ; Hansen , 1999 ; Hansen & Haas , 2001 ) , by focusing on the extent that \u201c engineered seren - dipity \u201d can mitigate some of these costs . We emphasize the role of knowledge similarity in shaping different components of knowledge production , and over different time hori - zons . Most prior research has predominantly focused on one component or a single time frame ( Adams , Black , Clemmons , & Stephan , 2005 ; Boudreau et al . , 2017 ; Dahlander & McFarland , 2013 ; Szulanski , 1996 ) . This work is unique because it extends the findings from Boudreau et al . ( 2017 ) on search costs and short - term collaborations using the same field experiment , to show how engineered serendipity may alter the role of knowl - edge similarity in knowledge production over time . Our findings suggest that the magni - tude of these frictions is likely to be more difficult to overcome in the short - run compared to the long - run . Second , we make contributions to the literature on temporary colocation , and the value of conferences , symposia and similar events on the direction of inventive activity on the knowl - edge frontier and the emergent patterns of collaborative activity ( Biancani , McFarland , & Dahlander , 2014 ; Boudreau et al . , 2017 ; Campos , Leon , & McQuillin , 2018 ; Catalini , Fons - Rosen , & Gaul\u00e9 , 2020 ; Chai & Freeman , 2019 ) . Prior research has been limited in its ability to observe actual acts of knowledge sharing between pairs , and its implications on knowledge production . Our study indicates that even brief , information - rich encounters at conferences can benefit knowledge production , with the potential to alter people ' s research directions and collaboration networks . Third , we make methodological contributions by highlighting the benefits of long - term studies that amalgamate multiple forms and uses of data . Prospective experiments can support multiple lines of investigation involving both near - term and long - term outcomes that may not be possible in retrospective , archival studies and suggests the use of multiple sources of data for unpacking the dynamics of knowledge production . 4 LANE ET AL . 2 | THEORY AND HYPOTHESES 2 . 1 | Knowledge production and knowledge sharing According to the knowledge - based view of the firm , knowledge production is a multicomponent process that involves the transfer , creation and diffusion of knowledge ( Grant , 1996 ; Kogut & Zander , 1992 ; Nonaka , 1994 ) . Knowledge transfer involves the movement of facts , relationships , and insights from one setting to another , and becomes evident when the experience acquired by an individual , group or organization in one setting is applied in another setting ( Argote , 2012 ; Hansen , 1999 ; Szulanski , 1996 ) . Knowledge creation refers to the generation of facts , relationships and insights to solve new problems ( Nonaka , 1994 ) . The emphasis on new knowledge is what distinguishes the process of knowledge transfer from creation . Because new knowledge is not held by anyone prior to its creation , it cannot be transferred and applied directly ( McFadyen & Cannella Jr , 2004 ) . Conversely , knowledge diffusion refers to the process through which transferred or newly created knowledge is disseminated and used by other indi - viduals , groups and organizations ( Fleming et al . , 2007 ; Singh , 2005 ) . Knowledge diffusion is a critical component of the knowledge production process because it reduces duplication of effort and promotes efficiency by demarcating what is known from what is yet to be explored on the knowledge frontier ( Boudreau & Lakhani , 2015 ) . Critical to the efficiency of knowledge production is the process of knowledge sharing between individuals possessing different types of knowledge ( Grant , 1996 ; Kogut & Zander , 1992 ; Nonaka , 1994 ) , and their ability to learn from these interactions ( Levinthal & March , 1993 ; Simon , 1991 ) . Common knowledge between partners is important because it enables individuals to absorb the aspects of their knowledge sets that they do not hold in com - mon , or that are unique to each individual ( Carlile , 2004 ; Cohen & Levinthal , 1990 ; Maurer & Ebers , 2006 ) . Building on prior work , we propose that there are at least two separate pathways through which people develop their knowledge bases : their field and intellectual specialties . Because of the path - dependent nature of knowledge ( Hargadon & Sutton , 1997 ) , the overlap in two individuals ' field and intellectual specialties may have different implications on knowledge production . Table 1 provides a summary of our knowledge constructs . 2 . 2 | Two knowledge bases of similarity : Field specialty and intellectual specialty Field specialties are developed through an individual ' s educational background , skills and train - ing ( Bechky , 2011 ; Bourdieu , 1984 ; Haas & Park , 2010 ) . In biomedical research , which is our focal context of study , there are multiple medical field specialties , such as neurology , radiology or oncology that focus on investigations of the biological process and the causes of specific dis - eases . Organizations often structure their departmental and divisional memberships around field specialties ( Biancani et al . , 2014 ) . In universities and academia , this means that field spe - cialties are often the primary channels through which resources , such as salaries , funding , ten - ure , offices and laboratory space , are apportioned to faculty ( Biancani , Dahlander , McFarland , & Smith , 2018 ) . For these reasons , field specialties provide professional reference groups ( Haas & Park , 2010 ) for members to identify appropriate behavior , rigor of scholarship , career goals and aspirations ( Abbott , 2001 ) . In contrast , intellectual specialties are closely aligned to personal interests and passions that evolve over time . Individuals can have LANE ET AL . 5 memberships in multiple intellectual specialties depending on their current pursuits and priori - ties ( Caza et al . , 2018 ) . For example , in biomedical research , intellectual specialties include research topics such as aging , addictions , Alzheimer ' s disease , brain injury , sleep , and stem cells ; each of these intellectual specialties is associated with memberships in professional refer - ence groups ( Haas & Park , 2010 ) for people motivated by common problems , methods , trends , and processes . These intellectual interests can be specific to a field ' s research topics , such as Alzheimer ' s disease , which is typically studied by neurologists , or span different fields , such as stem cell research . In short , field and intellectual specialties shape people ' s identities , language , tastes and affil - iations through professional memberships ( Haas & Park , 2010 ) . Individuals will likely adopt multiple professional identities depending on their field and intellectual memberships : this means that people from different field specialties can share overlapping intellectual interests , while individuals from the same field specialty can have divergent intellectual interests , because field and intellectual interests are two distinct dimensions of individuals ' knowledge constructs . In distinguishing how field and intellectual overlap shape knowledge production , knowl - edge similarity describes the distance between the field specialties and intellectual specialties of two knowledge sharing partners . Individuals are more likely to recognize and absorb new ideas when they already have some existing expertise and find it more difficult when the ideas are outside their realm of expertise ( Carlile , 2004 ; Cohen & Levinthal , 1990 ) . Put differently , even though distant knowledge tends to be novel and valuable ( Jeppesen & Lakhani , 2010 ; Leahey TABLE 1 Summary of knowledge constructs Knowledgeconstruct Definition Citations Knowledgeproduction A multicomponent process that involves knowledge transfer , creation , and diffusion . Grant , 1996 ; Kogut & Zander , 1992 ; Nonaka , 1994 ; Spender , 1996 Knowledge transfer The movement of facts , relationships , and insights from one setting to another Argote , 2012 ; Hansen , 1999 ; Szulanski , 1996 Knowledgecreation The generation of facts , relationships , and insights to solve problems that are new to the knowledge frontier Arrow 1962 ; Boudreau & Lakhani , 2015 ; Nonaka , 1994 Knowledgediffusion The dissemination of facts , relationships , and insights that are subsequently used by others Fleming & Singh , 2010 ; Singh , 2005 Knowledgesimilarity The degree of common knowledge between two individuals ' field of study and intellectual interests Cohen & Levinthal , 1990 ; Carlile , 2004 ; Dahlander & McFarland , 2013 ; Leahey , Beckman , & Stanko , 2017 Fieldsimilarity The dimension of knowledge similarity that emerges from the overlap between two individual ' s educational background , skills , and training Bechky , 2011 ; Carlile , 2004 ; Bourdieu , 1984 ; Haas & Park , 2010 Intellectualsimilarity The dimension of knowledge similarity that emerges from the overlap between two individual ' s personal interests and passions Caza , Moss , & Vough , 2018 ; Dahlander & McFarland , 2013 6 LANE ET AL . et al . , 2017 ) , people are more likely to experience challenges communicating with one another , and may not recognize the value of external information ( Cohen & Levinthal , 1990 ; Grant , 1996 ) . For example , two neurologists are less likely to experience challenges communicating with each other than a neurologist and oncologist pair because they share more similar educational backgrounds , training and clinical expertise . However , there may be greater opportunities for knowledge produc - tion between the neurologist and oncologist pair because their field specialties are more distant . Similarly , although two sleep researchers may communicate and comprehend each other with rela - tive ease because they read and conduct similar research ( Dahlander & McFarland , 2013 ) , there is greater potential for knowledge production between an aging and a sleep researcher , who may offer greater utility and value to one another to solve problems related to both sleep and aging processes \u2014 provided that they have sufficient common knowledge ( Bechky , 2011 ; Grant , 1996 ) . This suggests that there is an inherent trade - off between the greater ease of conversing and absorb - ing ideas locally from individuals with many overlapping interests and the greater potential oppor - tunities for novel ideas and knowledge transfer , creation , and diffusion from sharing and assessing the knowledge of those with more distant interests . 2 . 3 | Knowledge transfer and knowledge similarity Knowledge transfer is a two - sided process because it depends on the efforts of a source to share knowledge with a recipient and the recipient ' s efforts and capacity to acquire , absorb , and learn it ( Argote , 2012 ) . Because of greater common knowledge , local knowledge found within groups of similar individuals tends to be more easily transferred than distant knowledge spanning group boundaries ( Carlile , 2004 ; Kogut & Zander , 1992 ; Rosenkopf & Almeida , 2003 ) . That said , more distant knowledge may present nonredundant ideas that benefit learning and acquiring new concepts . Consequently , during serendipitous idea exchanges , we would expect that part - ners from dissimilar field and intellectual specialties offer each other more novel ideas and opportunities to learn ( Leahey et al . , 2017 ; Lee , 2019 ) . Divergent interests create a wider pool of knowledge to draw upon , which allows for multiple perspectives and problem - solving approaches that increase the likelihood of new discoveries ( Boudreau , Guinan , Lakhani , & Riedl , 2016 ) \u2014 provided that the partners share sufficient common knowledge to make sense of each other ' s knowledge dissimilarities . Based on these reasons , we expect that pairs with less field or intellectual overlap have a greater potential pool of ideas for knowledge transfer , up until a threshold of dissimilarity , after which they will lack sufficient common interests to bene - fit from knowledge transfer . Hypothesis ( H1a ) Field similarity has an inverted U - shaped effect on the relationship between knowledge sharing and knowledge transfer . Hypothesis ( H1b ) Intellectual similarity has an inverted U - shaped effect on the relationship between knowledge sharing and knowledge transfer . 2 . 4 | Knowledge creation and knowledge similarity There is a growing view among innovation scholars that specialization has created a \u201c knowl - edge burden \u201d hypothesis that makes collaborative work combining the increasingly narrow LANE ET AL . 7 niches of specialization imperative to moving the knowledge frontier forward ( Jones , 2009 ; Uzzi et al . , 2013 ) . Scientific discovery is a process that combines individually focused tasks \u2014 such as reading , experimentation , and writing \u2014 with social interactions through joint sense - making with others that can spark new discoveries ( Boudreau et al . , 2016 ; Latour & Woolgar , 2013 ) . That said , collaborations tend to be constrained by social processes , such as preferences for others with shared attributes , as well as search costs that stifle attempts to identify suitable col - laborators . First , principles of homophily suggest that people are attracted to others who hold similar values because their interactions are more rewarding and less uncertain ( McPherson & Smith - Lovin , 1987 ) , as a result of the benefits of security and mutual attraction ( Dahlander & McFarland , 2013 ) . Moreover , joint knowledge creation is a long - term investment in a multiplex relationship ( Uzzi , 1997 ) , where partners engage in joint problem - solving and spend time together discussing , reflecting and interacting to achieve mutual benefit ( McFadyen & Cannella Jr , 2004 ) . Due to these reasons , collaborations are more likely to form among individuals who share overlapping field and intellectual interests ( Biancani et al . , 2014 ; Dahlander & McFarland , 2013 ) . Second , there is a growing view that search costs and resulting information frictions tend to constrain people ' s collaboration patterns ( Boudreau et al . , 2017 ; Catalini , 2018 ) . Engineered , serendipitous interactions can mitigate some of these frictions by providing people the opportu - nity to exchange information and develop intellectual links with others they would not other - wise interact with . Such interactions may enable diverse partners to discover the potential complementarities they offer each other , leading to new collaborations . However , to the extent that serendipitous knowledge sharing leads to joint knowledge creation will also likely require that potential partners share some field or intellectual overlap ; otherwise their interests may be too disparate to establish synergies in incentives ( e . g . , publication or tenure requirements ) or research interests . Accordingly , we expect that increasing field and intellectual similarity should benefit knowledge creation up to a threshold , over which there are decreasing marginal returns on greater levels of knowledge similarity . Hypothesis ( H2a ) Field similarity has an inverted U - shaped effect on the relationship between knowledge sharing and knowledge creation . Hypothesis ( H2b ) Intellectual similarity has an inverted U - shaped effect on the relationship between knowledge sharing and knowledge creation . Another pathway that may lead to joint knowledge creation relies on prior collaborations and tie persistence ( Hasan & Koning , 2019 ; Ingram & Morris , 2007 ; Zhang & Guler , 2020 ) . We examine a specific type of tie persistence , namely the likelihood that an elemental collaboration persists into a complete knowledge product . In science , grant coapplications and copublications represent two essential but opposing ends of knowledge creation , from idea - generation to final output ( McFadyen & Cannella Jr , 2004 ) . Both are also essential to scientists ' research productiv - ity and often used as evaluative criteria for important decisions , such as promotion , tenure , awards and recognition ( Dahlander & McFarland , 2013 ; Stephan , 1996 ) . The knowledge simi - larity requirements that improve the likelihood that elemental collaborations persist into copublications may differ from the processes that shape long - term collaborations on peer - reviewed research publications . In addition to being at the open end of research inquiry , grant applications tend to have finite submission deadlines and these can impose resource , attention , coordination and other constraints on potential partners ( Dahlander , O ' Mahony , & 8 LANE ET AL . Gann , 2016 ) . Although serendipitous knowledge exchanges can reduce information search costs and expose people to promising new contacts , multiplex relationships require repeated interac - tions ( Ingram & Morris , 2007 ) . In the short - term , potential partners may turn to other individ - uals with whom they share significant knowledge overlap , due to fewer frictions and relational uncertainties ( e . g . , priorities , personalities , scheduling constraints ) associated with similar part - ners . Due to these reasons , common knowledge may be even more critical to shaping elemental collaborations . We thereby expect that elemental collaborations are more likely to persist into a final product of knowledge creation when knowledge sharing partners have significant knowl - edge overlap in terms of their field and intellectual interests . Hypothesis ( H2c ) An elemental collaboration is more likely to result in knowledge creation as the field similarity between two individuals increases . Hypothesis ( H2d ) An elemental collaboration is more likely to result in knowledge creation as the intellectual similarity between two individuals increases . 2 . 5 | Knowledge diffusion and knowledge similarity Among research scientists , forward citations to others ' publications are a primary means for dif - fusing knowledge . Beyond knowledge diffusion , forward citations constitute a critical means of social recognition for acknowledging the contributions of predecessors ( Merton , 1973 ) and trac - ing the path of scientific discovery and diffusion ( Stephan , 1996 ) . Some scholars argue that the number of citations a publication has received is perhaps the most common way to measure the importance of an individual ' s contribution to science ( Stephan , 1996 ) . Consistent with this view , citations are a critical currency of scientific credit ( Latour & Woolgar , 2013 ) , both driving research impact and constituting the basis of reward systems in science , including promotion , status , funding , peer esteem , honors , and awards ( Boudreau & Lakhani , 2015 ) . There are generally two views of how knowledge is diffused in science : openness and secrecy . According to the Mertonian norms of communalism or \u201c openness , \u201d publication enables scientists to establish priority of discovery and allows them to be the first to communi - cate an advance in knowledge and allow others to freely use it ( Merton , 1973 ) . Thus , publica - tion promotes the open diffusion of scientific knowledge , as long as scientists ' own internal agents ( i . e . , other scientists ) appropriately recognize and diffuse their work ( Boudreau & Lakhani , 2015 ) . On the other hand , social recognition is a discretionary act among scientists , and strong evidence points to the existence of counter - norms promoting secrecy , competition , and information withholding ( Haas & Park , 2010 ; Haeussler , Jiang , Thursby , & Thursby , 2014 ) . Scientists often compete for similar resources , funding , and recognition . Given limited resources , scientists need to be assured that they will be appropriately remunerated for openly diffusing others ' knowledge ( Hagstrom , 1974 ; Murray & O ' Mahony , 2007 ; Reschke , Azoulay , & Stuart , 2018 ) . On balance , the evidence suggests that scientists do not unequivocally diffuse each other ' s publications . Among scientists with greater knowledge overlap , serendipitous idea sharing may heighten the competition effect , particularly as information exchange can introduce people to novel work they cannot discover on their own via publications or other publicly available sources . These new projects may be similar to their own undertakings , risking duplication of efforts . Because scientists compete for priority ( Merton , 1973 ) , recognition ( Hagstrom , 1974 ; LANE ET AL . 9 Reskin , 1977 ) , and funding ( Stephan , 1996 ) , partners with highly overlapping interests may dis - cover a need to differentiate themselves from one another . Social recognition of highly similar peers would not only highlight redundancies but may also detract attention and resources away from one ' s own work ( Campanario & Acedo , 2007 ) . We expect that partners sharing high field or intellectual similarity may refrain from citing each other ' s research to outperform their peers . Hypothesis ( H3a ) Knowledge sharing is less likely to lead to knowledge diffusion as the field similarity between individuals increases . Hypothesis ( H3b ) Knowledge sharing is less likely to lead to knowledge diffusion as the intellec - tual similarity between individuals increases . 3 | EXPERIMENTAL METHODS In an ideal setting , all of the prior interactions and efforts that go into knowledge production would be fully observable to scholars to theorize and validate through empirical observations . The reality , however , is that the vast majority of prior scholarly work concerned with how knowledge is produced has primarily focused on observed outcomes \u2014 e . g . , papers , funding , pat - ents , citations , team structure , and so forth \u2014 to draw inferences about the mechanisms underly - ing the knowledge production process ( Dahlander & McFarland , 2013 ; Fleming et al . , 2007 ; Staw & Epstein , 2000 ) . For example , research on scientists will include the papers they publish , the collaborators they have worked with , and the knowledge that they have developed and have diffused through citations . However , a concern with relying on published trace data is that it masks all of the work and activities that occurred prior to knowledge production , such as the entire risk set of alters an individual may have interacted with prior to settling on a particular team . The empirical shortcomings of not being able to directly observe knowledge sharing and the drivers of knowledge production may insert biases in our inferences , such as self - selection and survivor bias . Some work aims to address these concerns by extending the risk set to unsuc - cessful collaborations ( e . g . , non - awarded grant applications ) to examine quality - adjusted research output of published ( i . e . , visible ) trace data ( Arora & Gambardella , 2005 ; Ayoubi , Pezzoni , & Visentin , 2019 ; Ganguli , 2017 ) . That said , unobserved heterogeneity between the two groups remains a persistent challenge . A feasible alternative is to design a field experiment that enables the capture of data around interactions between scientists and overcomes concerns around endogeneity of affiliation , team formation , and knowledge exposure by randomizing the encounters that the scientists have with each other . The benefit of this approach is that it can provide causal explanations about the factors that impact knowledge production , weighed against the challenges of drawing infer - ences with smaller sample sizes in an experiment as opposed to relying on all observed data of many more scientists . 3 . 1 | Setting and research design We carried out our study in the context of a medical symposium for research on advanced imag - ing , which is used to detect diseases and other health conditions early , to allow health care 10 LANE ET AL . practitioners to direct patients to the health care services that they need . We collaborated with the administrators of a large U . S . medical school to layer the medical symposium onto the University ' s Clinical and Translational Science Center pilot grant program , which provides seed funding in the form of pilot grants to support nascent research efforts that are awarded compet - itively to faculty within the University . 1 The purpose of the grant opportunity was to solicit pro - posals to improve methods for using advanced imaging technologies to address unmet clinical needs , and offered $ 50 , 000 per award for up to 15 pilot grants . A major challenge in the field of advanced imaging is that furthering the knowledge frontier requires expertise in the latest imaging tools and technologies and a deep understanding of the health problems to which they could be applied . These different types of knowledge are typically held by people from distinct disciplinary backgrounds , which makes advanced imaging an ideal setting for our research . In November 2011 , we invited all life sciences faculty and researchers at the university to a medical research symposium for the unique grant funding opportunity . Our field experiment involved faculty and researchers at the University and its affiliated hospitals and institutions , which are independently owned and managed , with each appearing as a separate entity in hospital rankings and lists of National Institutes of Health ( NIH ) grant recipients . We communicated to applicants that eligibility to submit a grant application was conditional on attending the symposium . In the first stage , investigators interested in applying for the grants submitted a statement of interest in which they briefly described a specific medical problem that advanced imaging techniques could potentially address . We collected basic bio - graphical information ( e . g . , degree , institution , department appointment ) at this stage . 3 . 2 | Participants and randomization of knowledge - sharing partners The symposia were held on January 31 , February 1 , and February 2 , 2012 , at one of the univer - sity ' s innovation labs . Figure 1 summarizes the key details of the randomization : 402 unique participants were randomly assigned to one of three nights of the symposium , one of four breakout rooms , one of two groups , as well as a poster location to stand next to around the perimeter of the room . 2 Participants were provided an electronic device , called a \u201c sociometric badge \u201d to automatically record their face - to - face interactions during the symposium ( Kim et al . , 2012 ) . Each of the three nights featured a 30 - min welcome address , followed by two 45 - min poster sessions in breakout rooms , with a 15 - min social break in between the poster sessions . Within each breakout room , scientists were randomly assigned to a poster location and either the first or second poster group . The scientists presented and exchanged ideas with one another during the poster sessions . The grant administrators prepared the posters to be a standard size and in a standard format , based on each participant ' s state - ments of interest . Thus , treatment pairs assigned to the same breakout rooms had more opportunities for knowledge sharing than control pairs . Shortly after the symposia , all participants received an e - mail invitation to submit applications for the pilot grants or concept awards by the deadline of March 8 , 2012 . At this time , they also 1 The same field experiment was used in Boudreau et al . ( 2017 ) to investigate the effect of search costs on collaborations in the immediate aftermath of the experiment . It did not include long - run knowledge production outcomes or leverage the sociometric badge data , described in Section 3 . 3 . 2 Compared to the entire Harvard Medical School ( HMS ) population ( N = 22 , 625 ) , participants were more likely to be PhDs , had more prior publications on average and were more likely to be instructors , assistant or associate professors relative to the overall distribution at HMS . These differences are consistent with the academic research setting of the symposium and the pilot grant funding opportunity . See Table A1 for summary statistics between participants and the HMS population . LANE ET AL . 11 received PDF booklets with the contact information and posters of all participants so that all researchers had identical information apart from the knowledge acquired in the breakout rooms . 3 . 3 | Data collection and variables We tested our hypotheses using data from a variety of resources from the advanced imaging symposium and the 6 years of publication records from 2013 to 2018 on the attendees . In the analyses , we did not include the year 2012 to remove potential research topics or ideas that were in progress prior to the sym - posium . We used data from the scientists ' registration form for the symposium , which contained infor - mation about their institution , department , academic position , self - identity as an imager or clinician , and statements of interest . The sociometric badges automatically recorded their face - to - face encounters when two badges were facing each other with a direct line of sight within a 30 (cid:1) cone of 1 m . We verified that all recorded interactions were within 10 m using Bluetooth proximity data ( Kim et al . , 2012 ) and required that two badges be in contact with each other over a span of at least 1 min ( Ingram & Morris , 2007 ) . We collected face - to - face interaction data for 306 ( 74 % ) scientists who attended the sym - posium , and the subsequent analyses are based on these scientists . 3 After the symposium , we collected information on the coapplicants and the awardees of the advanced imaging grants and used the Scopus database to collect scientists ' publication records . FIGURE 1 Randomization of participants by night , room , group , and poster location 3 Badges were randomly assigned to all symposium attendees . 26 % of badges malfunctioned and did not record face - to - face interaction data ; participants were not aware of whether they were assigned a working or faulty badge . For each symposium night , the sociometric badges recorded interactions for 64 . 4 , 68 . 2 , and 90 . 4 % of the participants ( Table A2 ) . We conducted balance checks on all available scientist covariates between the complete and observed sample and found no differences between the samples ( Table A3 ) . As robustness , we also perform the main OLS regression analyses for each knowledge production outcome on the full sample of participants to verify that the results are directionally consistent across the full sample and the observed badge sample . 12 LANE ET AL . 3 . 3 . 1 | Dependent variables Knowledge transfer We base our knowledge transfer measure on the MeSH lexicon . In the life sciences , the U . S . National Library of Medicine ( NLM ) uses a controlled MeSH taxonomy of keywords to index biomedical and health - related information for articles appearing in MEDLINE / PubMed , the NLM Catalog , and other NLM databases . Each article is associated with a set of MeSH key - words that describe the content of the citation . MeSH keywords are assigned by professional sci - ence librarians and computer algorithms to ensure global and consistent assignment of keywords across the life sciences ( Coletti & Bleich , 2001 ) . We extracted the unique MeSH key - words associated with each scientist ' s publications to create two vectors of MeSH keywords : the first with all MeSH keywords prior to the advanced imaging symposium ( i . e . , pre - 2012 ) , and the second with all MeSH keywords after the symposium ( i . e . , 2013 \u2013 2018 ) . For each scientist - pair { i , j } , we then counted the number of MeSH terms that j transferred to i and the number of MeSH terms that i transferred to j by taking the intersection of MeSH keywords between i ' s pre - 2012 vector and j ' s post - 2012 vector and between j ' s pre - 2012 vector and i ' s post - 2012 vector , excluding any MeSH terms that were common to both i and j in their pre - 2012 MeSH vectors . Our resulting knowledge transfer measure , MeSH keyword transfer is the count of \u201c transferred \u201d MeSH keywords between pair { i , j } , normalized by the total number of MeSH keywords in scien - tists i and j ' s post - 2012 MeSH vectors , expressed as a percentage . The resulting measure ranges from 0 to 100 % to and is interpreted as the percentage of postsymposium MeSH keywords that were transferred between scientist - pair { i , j } , with 0 % representing no transferred MeSH key - words and 100 % representing complete transfer . Knowledge creation We measure knowledge creation as the postsymposium ( 2013 \u2013 2018 ) count of Copublications between scientist - pair { i , j } in peer - reviewed journals , conference proceedings , and book chapters . Knowledge diffusion We measure knowledge diffusion as the postsymposium ( 2013 \u2013 2018 ) count of noncoauthored Forward citations between scientist - pair { i , j } in peer - reviewed journals , conference proceedings , and book chapters . 3 . 3 . 2 | Independent variables Knowledge sharing We use two alternative variables to capture knowledge sharing . First , we use the dummy vari - able , Same room , to measure whether scientist - pair { i , j } was randomly assigned to the same room ( treatment pairs ) or different breakout rooms ( control pairs ) . Second , we use the dummy variable , F2F ( face - to - face ) communication to measure whether scientist - pair { i , j } engaged in least 1 min of interaction , recorded using sociometric badges . Knowledge similarity Knowledge similarity is comprised of field and intellectual similarity . We measure Field similar - ity using clinical areas ( third - party coded from the scientists ' statements of interest ) , which LANE ET AL . 13 pertain to the primary area of responsibility for \u201c bedside \u201d patient care . There were a total of 24 unique clinical areas ( e . g . , oncology , neurology , immunology ) , and some statements of inter - ests ( 4 . 24 % ) spanned two clinical areas , such as neurology / endocrinology . We then use a cate - gorical variable to indicate whether scientist - pair { i , j } shared Low , Moderate or High field similarity depending on whether their clinical areas shared no ( 85 . 5 % ) , partial ( 1 . 9 % ) , or com - plete ( 12 . 6 % ) overlap , respectively . We measure Intellectual similarity using the count of common MeSH keywords shared by scientist - pair { i , j } prior to 2012 and dichotomize the distribution into three equal - sized groups , to indicate whether scientist - pair { i , j } had low , moderate , or a high number of common MeSH keywords prior to the 2012 symposium . Low intellectual similarity corresponds to 0 \u2013 2 common keywords , Moderate intellectual similarity corresponds to 3 \u2013 11 common keywords , and High intellectual similarity corresponds to more than 11 common keywords . Elemental collaboration We use Grant coapplicant to measure an elemental collaboration , which captured whether scientist - pair { i , j } coapplied on the grant following the symposium . 3 . 3 . 3 | Other variables The analysis strategy relies most critically on the research design ' s randomization . We use dummy variables for each symposium night and room ( i . e . , fixed effects ) to control for unobserved night and room characteristics . To test Hypotheses ( H2c ) and ( H2d ) , we use the dummy variable , Grant awardee to capture whether scientist - pair { i , j } included a grant recipi - ent . Among the 306 scientists , 13 pilot grant proposals were awarded funding , comprising 6 . 54 % of pairs with grant awardees . 3 . 4 | Estimation approach We wish to estimate the effects of field and intellectual similarity between knowledge sharing partners on knowledge production outcomes . The unit of analysis is the scientist - pair { i , j } , and pairs are considered to be \u201c at risk \u201d if they attended the same night of the symposium \u2014 a total of 15 , 817 pairs . First , we analyze the effect of being in the same ( treatment ) versus different ( control ) breakout rooms on knowledge production outcomes using ordinary least squares ( OLS ) regression . We then interact Same room with Field similarity , and Same room with Intel - lectual similarity to examine knowledge similarity effects . Second , we analyze the effect of face - to - face communication on knowledge production outcomes using instrumental variable ( IV ) regression . We use an IV approach to account for the common endogeneity issue in pairwise interaction data . We exploit exogenous variation in the likelihood of interaction between scientists who are assigned to the same versus different breakout rooms by using Same room as an instrument for F2F communication in the first stage , and the estimates for F2F com - munication in the second stage . We then interact F2F communication with Field similarity , and F2F communication with Intellectual similarity to examine knowledge similarity effects . We address the nonindependence , common - person problem of dyadic regressions by esti - mating robust SE s that are simultaneously clustered on both members of the dyad , using 14 LANE ET AL . multiway clustering , developed theoretically by Cameron , Gelbach , and Miller ( 2011 ) and implemented for Stata in clus _ nway . ado ( Kleinbaum et al . , 2013 ) . 4 | RESULTS In this section , we begin by presenting descriptives of the main variables . Table 2 presents the means , standard deviations , and correlations of the main variables . We also note that the sum - mary statistics of the covariates for same room versus different room pairs indicate that the ran - domization achieved balance across covariates ( see Table A4 , Supporting Information ) . Figure 2 shows the yearly trend in copublication ( left ) and forward citation ( right ) rates between different room ( N = 11 , 611 ) and same room ( N = 4 , 206 ) pairs by year , in the 6 years to and since the symposium , with 95 % CIs . The plots show that there were no differences between the same room and different room pairs before the knowledge sharing intervention . After the intervention , we observe an increase in copublication and citation rates for the same room pairs . Next , we present our main regression results in subsections , beginning with knowledge transfer , then creation , and finally , diffusion . We note that the F - statistics for the IV regressions are all above the threshold of 10 for strong instruments ( Table A5 , Supporting Information ) , and that our results are robust to the inclusion of all scientist - pair covariates ( Table A6 , Supporting Information ) and the reduced form ( OLS ) models for the full sample of participants ( Tables A7 \u2013 A9 , Supporting Information ) . 4 . 1 | Knowledge transfer results We present the OLS and IV regression knowledge transfer results in Table 3 . The dependent variable is the percentage of scientist - pair { i , j } ' s MeSH postsymposium keywords that were transferred between i and j , with 0 % being no transfer , and 100 % being complete transfer . TABLE 2 Correlation between main variables ( N = 15 , 817 ) Variable Mean SD 1 2 3 4 5 6 7 1 MeSH keyword transfer 3 . 693 3 . 709 2 Copublications 0 . 031 1 . 004 0 . 010 3 Forward citations 0 . 265 2 . 497 0 . 019 0 . 479 4 Grant coapplicant 0 . 002 0 . 044 0 . 004 0 . 097 0 . 027 5 Same room 0 . 266 0 . 442 0 . 024 \u2212 0 . 004 \u2212 0 . 001 0 . 006 6 F2F communication 0 . 078 0 . 268 0 . 003 0 . 047 0 . 039 0 . 067 0 . 198 7 Intellectual similarity 1 . 076 0 . 810 \u2212 0 . 005 0 . 037 0 . 043 0 . 037 \u2212 0 . 002 0 . 047 8 Field similarity 0 . 272 0 . 673 0 . 437 0 . 029 0 . 074 0 . 017 \u2212 0 . 008 0 . 021 0 . 026 Note : Intellectual and field similarity are categorical variables with the following distributions : intellectual ( low : 32 . 4 % , moderate : 32 . 7 % , high : 35 . 0 % ) and field ( low : 85 . 5 , 1 . 9 , and 12 . 6 % ) . LANE ET AL . 15 Models 1 and 2 present the baseline models with main effects only ; Models 3 and 4 add the field similarity interaction terms ; Models 5 and 6 add the intellectual similarity interaction terms ; and Models 7 and 8 show the full results with both interaction terms . Hypotheses ( H1a ) and ( H1b ) suggest that field and intellectual similarity have an inverted U - shaped effect on the relationship between knowledge sharing and knowledge transfer , respectively . Models 3 and 4 indicate that there is no meaningful association between moderate field similarity and knowledge transfer among same room pairs ( 0 . 712 , p = . 132 ) and communi - cating pairs ( 4 . 088 , p = . 197 ) . Models 5 and 6 show that compared to pairs with high intellectual overlap , scientists with moderate intellectual similarity transferred a higher percentage of MeSH keywords for both same room pairs ( 0 . 362 , p = . 042 ) and communicating pairs ( 3 . 348 , p = . 032 ) , respectively . These results are consistent in Models 7 and 8 , which include both inter - action terms . Figure 3a shows the change in knowledge transfer among communicating versus non - communicating pairs and intellectual similarity from Model 4 , with 95 % CIs , and illustrates the inverted U - shaped relationship . While communication did not meaningfully impact knowledge transfer among pairs with low field overlap , it led to an increase of 3 . 581 % among pairs with moderate intellectual overlap from 4 . 085 % [ 3 . 795 % , 4 . 376 % ] to 7 . 666 % [ 4 . 995 % , 10 . 338 % ] per - cent , a nearly twofold increase , compared to a 0 . 233 % [ \u2212 1 . 239 % , 1 . 705 % ] increase for high intel - lectual overlap pairs . The percentage increase among moderately overlapping pairs corresponds to about six new MeSH keywords per scientist , roughly equivalent to the average number of MeSH keywords in one publication . 4 This result supports Hypothesis ( H1b ) by showing cooper - ative effects of knowledge transfer for pairs with moderate intellectual similarity . 4 . 2 | Knowledge creation We present the OLS and IV knowledge creation regression results in Table 4 . The dependent variable is the number of copublications between scientist - pair { i , j } . Models 1 and 2 present the baseline models with main effects only ; Models 3 and 4 add the field similarity interaction FIGURE 2 Yearly trend in copublication ( left ) and forward citation ( right ) rates between same room ( treatment ) and different room ( control ) pairs with 95 % CIs 4 In the postsymposium period , each scientist has M = 172 . 57 ( SD = 142 . 61 ) MeSH keywords and M = 6 . 85 ( SD = 4 . 28 ) MeSH keywords per publication . 16 LANE ET AL . T A B L E 3 R e g r e ss i o n m o d e l s o f k n o w l e d g e t r a n s f e r \u2014 % o f M e S H k e y w o r d s t r a n s f e rr e d b e t w ee n s c i e n t i s t - p a i r { i , j } ; N = 15 , 817 V a r i a b l e s M o d e l 1 O L S M o d e l 2 I V M o d e l 3 O L S M o d e l 4 I V M o d e l 5 O L S M o d e l 6 I V M o d e l 7 O L S M o d e l 8 I V S a m e r oo m 0 . 107 ( 0 . 0979 ) 0 . 0344 ( 0 . 144 ) 0 . 0336 ( 0 . 138 ) \u2212 0 . 0379 ( 0 . 172 ) F 2 F c o mm u n i c a t i o n 0 . 838 ( 0 . 763 ) 0 . 214 ( 1 . 108 ) 0 . 233 ( 1 . 025 ) \u2212 0 . 512 ( 1 . 352 ) S a m e r oo m \u00d7 l o w f i e l d s i m il a r i t y 0 . 0707 ( 0 . 150 ) 0 . 0686 ( 0 . 150 ) S a m e r oo m \u00d7 m o d e r a t e f i e l d s i m il a r i t y 0 . 712 ( 0 . 471 ) 0 . 681 ( 0 . 468 ) S a m e r oo m \u00d7 l o w i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 175 ( 0 . 218 ) \u2212 0 . 171 ( 0 . 218 ) S a m e r oo m \u00d7 m o d e r a t e i n t e ll e c t u a l s i m il a r i t y 0 . 362 ( 0 . 177 ) 0 . 361 ( 0 . 177 ) F 2 F \u00d7 l o w f i e l d s i m il a r i t y 0 . 621 ( 1 . 204 ) 0 . 804 ( 1 . 239 ) F 2 F \u00d7 m o d e r a t e f i e l d s i m il a r i t y 4 . 088 ( 3 . 169 ) 4 . 059 ( 3 . 379 ) F 2 F \u00d7 l o w i n t e ll e c t u a l s i m il a r i t y \u2212 1 . 459 ( 1 . 753 ) \u2212 1 . 531 ( 1 . 869 ) F 2 F \u00d7 m o d e r a t e i n t e ll e c t u a l s i m il a r i t y 3 . 348 ( 1 . 564 ) 3 . 305 ( 1 . 644 ) L o w f i e l d s i m il a r i t y 0 . 140 ( 0 . 135 ) 0 . 171 ( 0 . 135 ) 0 . 121 ( 0 . 143 ) 0 . 104 ( 0 . 183 ) 0 . 141 ( 0 . 135 ) 0 . 176 ( 0 . 137 ) 0 . 123 ( 0 . 143 ) 0 . 0900 ( 0 . 189 ) M o d e r a t e f i e l d s i m il a r i t y \u2212 0 . 130 ( 0 . 274 ) \u2212 0 . 136 ( 0 . 264 ) \u2212 0 . 316 ( 0 . 269 ) \u2212 0 . 606 ( 0 . 381 ) \u2212 0 . 136 ( 0 . 272 ) \u2212 0 . 174 ( 0 . 274 ) \u2212 0 . 315 ( 0 . 268 ) \u2212 0 . 638 ( 0 . 398 ) L o w i n t e ll e c t u a l s i m il a r i t y \u2212 4 . 130 ( 0 . 238 ) \u2212 4 . 120 ( 0 . 237 ) \u2212 4 . 130 ( 0 . 238 ) \u2212 4 . 123 ( 0 . 238 ) \u2212 4 . 081 ( 0 . 246 ) \u2212 4 . 015 ( 0 . 282 ) \u2212 4 . 082 ( 0 . 246 ) \u2212 4 . 013 ( 0 . 289 ) LANE ET AL . 17 T A B L E 3 ( C o n t i n u e d ) V a r i a b l e s M o d e l 1 O L S M o d e l 2 I V M o d e l 3 O L S M o d e l 4 I V M o d e l 5 O L S M o d e l 6 I V M o d e l 7 O L S M o d e l 8 I V M o d e r a t e i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 880 ( 0 . 153 ) \u2212 0 . 862 ( 0 . 152 ) \u2212 0 . 880 ( 0 . 153 ) \u2212 0 . 866 ( 0 . 152 ) \u2212 0 . 974 ( 0 . 163 ) \u2212 1 . 095 ( 0 . 196 ) \u2212 0 . 974 ( 0 . 163 ) \u2212 1 . 096 ( 0 . 204 ) N i g h t F E Y Y Y Y Y Y Y Y R oo m F E Y Y Y Y Y Y Y Y R - s q u a r e d . 224 . 219 . 224 . 218 . 224 . 201 . 224 . 216 N o t e : M u l t i w ay , r o b u s t S E s i n p a r e n t h e s e s . S i g n i f i c a n c e s t a r s ( * ) a r e o m i tt e d . 18 LANE ET AL . terms ; Models 5 and 6 add the intellectual similarity interaction terms ; Models 7 and 8 show the full results with both interaction terms ; Model 9 \u2013 11 adds Grant coapplicant , followed by the field and intellectual similarity interaction terms , as well as both interaction terms , and controls for Grant awardee . Hypotheses ( H2a ) and ( H2b ) predict that field and intellectual similarity have an inverted U - shaped effect on the relationship between knowledge sharing and knowledge creation , respectively , while Hypotheses ( H2c ) and ( H2d ) predict that knowledge sharing partners who start an elemental collaboration are more likely to copublish when they share greater field and intellectual overlap , respectively . Models 3 and 4 show that compared to same field pairs , mod - erate field similarity has a strong positive relationship with knowledge creation among same room pairs ( 0 . 329 , p = . 047 ) and communicating pairs ( 2 . 159 , p = . 036 ) . There is also a smaller , positive association between low field similarity and knowledge creation among same room pairs ( 0 . 133 , p = . 092 ) and communicating pairs ( 1 . 040 , p = . 101 ) . Turning to intellectual simi - larity , Models 5 and 6 show there is no meaningful association between moderate intellectual similarity and knowledge creation among same room pairs ( 0 . 0409 , p = . 257 ) and communicat - ing pairs ( 0 . 333 , p = . 234 ) . The results are consistent in Models 7 and 8 , which includes both interaction terms . Figure 3b plots the change in copublications between communicating and noncommunicating pairs and field similarity , with 95 % CIs from Model 3 , and illustrates the estimated inverted U - shaped relationship . We observe that while communication did not meaningfully benefit pairs with low field overlap , communication led to an increase of 1 . 174 [ \u2212 0 . 207 , 2 . 556 ] copublications among pairs with moderate field overlap , and \u2212 0 . 984 [ \u2212 1 . 731 , \u2212 0 . 237 ] fewer copublications among pairs with high field overlap . The patterns suggest that communication reduced the search costs of identi - fying synergistic collaborations with scientists sharing some field interests , resulting in a reallocation of resources away from pairs with high field overlap . We also examined the extent that the knowledge sharing intervention impacted the scien - tists ' overall research portfolios . Turning to the scientists ' postsymposium publications , each sci - entist had an average of M = 32 . 394 ( SD = 35 . 412 ) peer - reviewed research articles ; the increase of 1 . 174 publications among pairs with moderate field overlap corresponds to about 7 % of a sci - entist ' s publications , which is of considerable magnitude from a 90 min intervention . Turning FIGURE 3 Change in ( a ) knowledge transfer , ( b ) knowledge creation , and ( c ) knowledge diffusion and knowledge similarity for communicating and noncommunicating pairs LANE ET AL . 19 T A B L E 4 R e g r e ss i o n m o d e l s o f k n o w l e d g e c r e a t i o n \u2014 # o f c o p u b li c a t i o n s b e t w ee n s c i e n t i s t - p a i r { i , j } ; N = 15 , 817 V a r i a b l e s M o d e l 1 O L S M o d e l 2 I V M o d e l 3 O L S M o d e l 4 I V M o d e l 5 O L S M o d e l 6 I V M o d e l 7 O L S M o d e l 8 I V M o d e l 9 O L S M o d e l 10 O L S M o d e l 11 O L S S a m e r oo m \u2212 0 . 00683 ( 0 . 0144 ) \u2212 0 . 127 ( 0 . 076 9 ) \u2212 0 . 0294 ( 0 . 0360 ) \u2212 0 . 148 ( 0 . 0931 ) \u2212 0 . 00784 ( 0 . 0148 ) \u2212 0 . 00822 ( 0 . 0148 ) \u2212 0 . 00799 ( 0 . 0147 ) F 2 F c o mm u n i c a t i o n \u2212 0 . 0535 ( 0 . 111 ) \u2212 0 . 984 ( 0 . 616 ) \u2212 0 . 221 ( 0 . 268 ) \u2212 1 . 147 ( 0 . 746 ) S a m e r oo m \u00d7 l o w f i e l d s i m . 0 . 133 ( 0 . 0788 ) 0 . 132 ( 0 . 0783 ) S a m e r oo m \u00d7 m o d . f i e l d s i m . 0 . 329 ( 0 . 165 ) 0 . 330 ( 0 . 164 ) S a m e r oo m \u00d7 l o w i n t e ll e c t u a l s i m . 0 . 0306 ( 0 . 0378 ) 0 . 0307 ( 0 . 0372 ) S a m e r oo m \u00d7 m o d . i n t e ll e c t u a l s i m . 0 . 0409 ( 0 . 0361 ) 0 . 0397 ( 0 . 0355 ) F 2 F \u00d7 l o w f i e l d s i m . 1 . 040 ( 0 . 634 ) 1 . 048 ( 0 . 637 ) F 2 F \u00d7 m o d . f i e l d s i m . 2 . 159 ( 1 . 029 ) 2 . 110 ( 1 . 022 ) F 2 F \u00d7 l o w i n t e l l e c t u a l s i m . 0 . 240 ( 0 . 297 ) 0 . 205 ( 0 . 306 ) F 2 F \u00d7 m o d . i n t e l l e c t u a l s i m . 0 . 333 ( 0 . 280 ) 0 . 332 ( 0 . 290 ) G r a n t c o a pp l i c a n t 3 . 708 ( 1 . 583 ) 2 . 482 ( 1 . 211 ) 3 . 810 ( 1 . 614 ) C o a pp l i c a n t \u00d7 l o w f i e l d s i m . \u2212 2 . 427 ( 1 . 845 ) \u2212 2 . 299 ( 2 . 109 ) C o a pp l i c a n t \u00d7 m o d . f i e l d S i m . \u2212 3 . 303 ( 1 . 621 ) \u2212 3 . 405 ( 1 . 659 ) C o a pp l i c a n t \u00d7 l o w i n t e l l e c t u a l s i m . \u2212 0 . 639 ( 2 . 085 ) \u2212 0 . 324 ( 2 . 236 ) 20 LANE ET AL . T A B L E 4 ( C o n t i n u e d ) V a r i a b l e s M o d e l 1 O L S M o d e l 2 I V M o d e l 3 O L S M o d e l 4 I V M o d e l 5 O L S M o d e l 6 I V M o d e l 7 O L S M o d e l 8 I V M o d e l 9 O L S M o d e l 10 O L S M o d e l 11 O L S C o a pp l i c a n t \u00d7 m o d . i n t e l l e c t u a l s i m . \u2212 1 . 083 ( 1 . 482 ) \u2212 0 . 571 ( 1 . 632 ) A w a r d ee 0 . 000295 ( 0 . 0140 ) \u2212 0 . 00167 ( 0 . 0137 ) \u2212 2 . 47 e - 05 ( 0 . 0140 ) L o w f i e l d s i m . \u2212 0 . 109 ( 0 . 0540 ) \u2212 0 . 111 ( 0 . 0566 ) \u2212 0 . 14 ( 0 . 073 5 ) \u2212 0 . 220 ( 0 . 120 ) \u2212 0 . 109 ( 0 . 0539 ) \u2212 0 . 112 ( 0 . 0579 ) \u2212 0 . 144 ( 0 . 0733 ) \u2212 0 . 222 ( 0 . 121 ) 0 . 0552 ( 0 . 0422 ) 0 . 0405 ( 0 . 0441 ) 0 . 0552 ( 0 . 0422 ) M o d e r a t e f i e l d s i m . \u2212 0 . 0529 ( 0 . 0659 ) \u2212 0 . 0525 ( 0 . 0655 ) \u2212 0 . 139 ( 0 . 070 1 ) \u2212 0 . 297 ( 0 . 132 ) \u2212 0 . 0534 ( 0 . 0660 ) \u2212 0 . 0567 ( 0 . 0675 ) \u2212 0 . 140 ( 0 . 0706 ) \u2212 0 . 296 ( 0 . 130 ) 0 . 0887 ( 0 . 0536 ) 0 . 0981 ( 0 . 0539 ) 0 . 0887 ( 0 . 0536 ) L o w i n t e l l e c t u a l s i m . \u2212 0 . 0696 ( 0 . 0222 ) \u2212 0 . 0702 ( 0 . 0226 ) \u2212 0 . 0695 ( 0 . 022 1 ) \u2212 0 . 0738 ( 0 . 0240 ) \u2212 0 . 0777 ( 0 . 0297 ) \u2212 0 . 0908 ( 0 . 0437 ) \u2212 0 . 0777 ( 0 . 0294 ) \u2212 0 . 0916 ( 0 . 0455 ) \u2212 0 . 0651 ( 0 . 0222 ) \u2212 0 . 0643 ( 0 . 0224 ) \u2212 0 . 0645 ( 0 . 0224 ) M o d e r a t e i n t e ll e c t u a l s i m . \u2212 0 . 0657 ( 0 . 0209 ) \u2212 0 . 0669 ( 0 . 0219 ) \u2212 0 . 0656 ( 0 . 0207 ) \u2212 0 . 0699 ( 0 . 0231 ) \u2212 0 . 0765 ( 0 . 0272 ) \u2212 0 . 0928 ( 0 . 0399 ) \u2212 0 . 0760 ( 0 . 0268 ) \u2212 0 . 0954 ( 0 . 0418 ) \u2212 0 . 0602 ( 0 . 0209 ) \u2212 0 . 0591 ( 0 . 0209 ) \u2212 0 . 0593 ( 0 . 0210 ) N i g h t F E Y Y Y Y Y Y Y Y Y Y Y R o o m F E Y Y Y Y Y Y Y Y Y Y Y R - s q u a r e d . 003 . 002 . 004 \u2014 . 003 \u2014 . 004 . 012 . 015 . 012 . 015 N o t e : M u l t i w ay r o b u s t S E s i n p a r e n t h e s e s . S i g n i f i c a n c e s t a r s ( * ) a r e o m i tt e d . LANE ET AL . 21 to changes in research direction , we also note that same room pairs were more likely to publish in advanced imaging journals ( e . g . , Magnetic Resonance in Medicine , NMR in Biomedicine ) : 21 % for same room versus 6 % for different room pairs ( t = 2 . 302 , p = . 0249 ) . 5 This suggests that the knowledge sharing treatment not only reduced search costs of finding collaborators but also potentially reshaped the pairs ' research trajectories . Turning to Hypothesis ( H2c ) , Model 9 shows that controlling for Grant awardee , grant coapplicants are more likely to copublish when they are from the same field ( low : \u2212 2 . 427 , p = . 189 ; moderate : \u2212 3 . 303 , p = . 042 ) . Finally , turning to H2d , Model 10 indicates that intel - lectual similarity is not a meaningful predictor of elemental tie persistence ( low : \u2212 0 . 639 , p = . 759 ; moderate : \u2212 1 . 083 , p = . 465 ) . The results remain consistent in Model 11 , which includes both interaction terms . This suggests that although strong field overlap is a catalyz - ing force for initiating early - stage collaborations , these requirements may dissipate over the long - run as these ties persist into copublications . Thus , our results show support for Hypotheses ( H2a ) and ( H2c ) . 4 . 3 | Knowledge diffusion We present the OLS and IV knowledge diffusion regression results in Table 5 . The dependent variable is the number of forward citations between scientist - pair { i , j } . Models 1 and 2 present the baseline models with main effects only ; Models 3 and 4 add the field similarity interaction terms ; Models 5 and 6 add the intellectual similarity interaction terms ; Models 7 and 8 show the full results with both interaction terms . Hypotheses ( H3a ) and ( H3b ) predict that knowledge sharing partners are less likely to dif - fuse knowledge as their field and intellectual similarity increase , respectively . Models 3 and 4 show that increasing field similarity reduced knowledge diffusion among same room pairs ( low : 0 . 366 , p = . 024 ; moderate : 1 . 003 , p = . 098 ) and communicating pairs ( low : 2 . 823 , p = . 033 ; moderate : 6 . 603 , p = . 050 ) . Models 5 and 6 show that there is no evidence that high intellectual similarity lowered knowledge diffusion among same room ( low : \u2212 0 . 0487 , p = . 739 ; moderate : \u2212 0 . 003 , p = . 986 ) or communicating pairs ( low : \u2212 0 . 426 , p = . 712 ; moderate : \u2212 0 . 091 , p = . 936 ) . The results are consistent in Models 7 and 8 , which include both knowledge similarity interac - tion terms . Figure 3c plots the change in knowledge diffusion ( forward citations ) between communicat - ing and noncommunicating pairs from Model 4 , with 95 % CIs . We observe that while commu - nication did alter forward citation trends for pairs with low field overlap , and led to a small increase of 3 . 521 [ \u2212 1 . 465 , 8 . 508 ] citations for pairs with moderate field overlap , we observe a large decrease from 0 . 874 [ 0 . 347 , 1 . 402 ] to \u2212 2 . 207 [ \u2212 4 . 326 , \u2212 0 . 088 ] for same field pairs , corresponding to a reduction of about 3 . 082 citations per pair . To generate greater insight into the types of publications being cited among the low and moderate field overlap pairs , we compared the publication titles of cited papers for pairs assigned to the same versus different rooms . We observe that the same room pairs were more likely to cite papers using advanced imaging technologies ( e . g . , sample paper titles included : \u201c Massively parallel MRI detector arrays \u201d ; \u201c An fMRI study of facial emotion processing patients 5 A journal was coded as an advanced imaging if the journal title included any of the three technologies featured at the symposium ( physiological magnetic resonance , positron emission tomography , and optical imaging ) . There were 48 unique journals for same room versus 169 unique journals for the different room pairs . 22 LANE ET AL . T A B L E 5 R e g r e ss i o n m o d e l s o f k n o w l e d g e d i ff u s i o n \u2014 # o f f o r w a r d c i t a t i o n s b e t w ee n s c i e n t i s t - p a i r { i , j } ; N = 15 , 817 V a r i a b l e s M o d e l 1 O L S M o d e l 2 I V M o d e l 3 O L S M o d e l 4 I V M o d e l 5 O L S M o d e l 6 I V M o d e l 7 O L S M o d e l 8 I V S a m e r oo m \u2212 0 . 0687 ( 0 . 0710 ) \u2212 0 . 399 ( 0 . 165 ) \u2212 0 . 0540 ( 0 . 150 ) \u2212 0 . 384 ( 0 . 211 ) F 2 F c o mm u n i c a t i o n \u2212 0 . 537 ( 0 . 551 ) \u2212 3 . 082 ( 1 . 331 ) \u2212 0 . 391 ( 1 . 115 ) \u2212 2 . 910 ( 1 . 656 ) S a m e r oo m \u00d7 l o w f i e l d s i m il a r i t y 0 . 366 ( 0 . 162 ) 0 . 367 ( 0 . 162 ) S a m e r oo m \u00d7 m o d e r a t e f i e l d s i m il a r i t y 1 . 003 ( 0 . 605 ) 0 . 999 ( 0 . 605 ) S a m e r oo m \u00d7 l o w i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 0487 ( 0 . 146 ) \u2212 0 . 0476 ( 0 . 146 ) S a m e r oo m \u00d7 l o w i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 00251 ( 0 . 145 ) \u2212 0 . 00592 ( 0 . 145 ) F 2 F \u00d7 l o w f i e l d s i m il a r i t y 2 . 823 ( 1 . 324 ) 2 . 835 ( 1 . 332 ) F 2 F \u00d7 m o d e r a t e f i e l d s i m il a r i t y 6 . 603 ( 3 . 364 ) 6 . 679 ( 3 . 334 ) F 2 F \u00d7 l o w i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 426 ( 1 . 154 ) \u2212 0 . 537 ( 1 . 162 ) F 2 F \u00d7 m o d e r a t e i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 0908 ( 1 . 138 ) \u2212 0 . 112 ( 1 . 135 ) L o w f i e l d s i m il a r i t y \u2212 0 . 316 ( 0 . 129 ) \u2212 0 . 336 ( 0 . 137 ) \u2212 0 . 413 ( 0 . 165 ) \u2212 0 . 633 ( 0 . 256 ) \u2212 0 . 316 ( 0 . 129 ) \u2212 0 . 335 ( 0 . 140 ) \u2212 0 . 413 ( 0 . 165 ) \u2212 0 . 632 ( 0 . 258 ) M o d e r a t e f i e l d s i m il a r i t y \u2212 0 . 209 ( 0 . 208 ) \u2212 0 . 205 ( 0 . 214 ) \u2212 0 . 471 ( 0 . 185 ) \u2212 0 . 955 ( 0 . 360 ) \u2212 0 . 209 ( 0 . 208 ) \u2212 0 . 204 ( 0 . 215 ) \u2212 0 . 471 ( 0 . 186 ) \u2212 0 . 962 ( 0 . 358 ) L o w i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 446 ( 0 . 0920 ) \u2212 0 . 452 ( 0 . 0926 ) \u2212 0 . 446 ( 0 . 0918 ) \u2212 0 . 462 ( 0 . 0950 ) \u2212 0 . 433 ( 0 . 102 ) \u2212 0 . 417 ( 0 . 141 ) \u2212 0 . 433 ( 0 . 102 ) \u2212 0 . 418 ( 0 . 143 ) M o d e r a t e i n t e ll e c t u a l s i m il a r i t y \u2212 0 . 353 ( 0 . 0762 ) \u2212 0 . 364 ( 0 . 0791 ) \u2212 0 . 352 ( 0 . 0760 ) \u2212 0 . 373 ( 0 . 0810 ) \u2212 0 . 352 ( 0 . 0866 ) \u2212 0 . 354 ( 0 . 128 ) \u2212 0 . 351 ( 0 . 0862 ) \u2212 0 . 361 ( 0 . 130 ) LANE ET AL . 23 T A B L E 5 ( C o n t i n u e d ) V a r i a b l e s M o d e l 1 O L S M o d e l 2 I V M o d e l 3 O L S M o d e l 4 I V M o d e l 5 O L S M o d e l 6 I V M o d e l 7 O L S M o d e l 8 I V N i g h t F E Y Y Y Y Y Y Y Y R oo m F E Y Y Y Y Y Y Y Y R - s q u a r e d . 011 . 004 . 011 \u2014 . 011 . 004 . 011 \u2014 N o t e : M u l t i w ay , r o b u s t S E s i n p a r e n t h e s e s . S i g n i f i c a n c e s t a r s ( * ) a r e o m i tt e d . 24 LANE ET AL . with schizophrenia \u201d ) . Overall , there was a greater use of advanced imaging technologies in the publication titles of same room pairs : 42 . 6 % for same room pairs versus 31 . 3 % for different room pairs ( t = 2 . 406 , p = . 017 ) . 6 This suggests that the knowledge sharing intervention enabled pairs from different fields to learn about \u201c outside \u201d work , which they subsequently integrated into their own future research . In contrast , the knowledge sharing intervention may have led to a crowding out effect among highly similar pairs . In summary , we find evidence for competitive effects of field similarity on knowledge diffusion , which confirms Hypothesis ( H3a ) . 5 | DISCUSSION The ability to produce and manage knowledge is a critical source of competitive advantage for organizations of all types ( Argote , 2012 ; Hargadon & Sutton , 1997 ) . Yet the knowledge produc - tion process is both time - intensive and uncertain ( Maggitti , Smith , & Katila , 2013 ) . To address this issue , the premise of this article is based on the notion that \u201c engineering serendipity \u201d can promote greater knowledge sharing and more efficient knowledge production . We systemati - cally examine how the degree of field and intellectual similarity between knowledge sharing partners affects the likelihood that engineered encounters affects three knowledge production outcomes : knowledge transfer , creation , and diffusion . We find both cooperative and competitive effects of serendipitous knowledge sharing on knowledge production . On the one hand , knowledge sharing leads to greater knowledge trans - fer and creation when people share some overlapping research interests : knowledge sharing partners are more likely to transfer and acquire MeSH keywords from one another when they already have some intellectual overlap and more likely to copublish when they share field over - lap , with field similarity being more critical to collaborations in the near - term than over longer horizons . On the other hand , knowledge sharing appears to have little benefit for pairs with low knowledge similarity and reduces knowledge diffusion between people from the same field : we observe that pairs from the same field are less likely to cite each other after interacting . These findings make a number of contributions to the literature . First , we contribute to the literature on search costs and information frictions associated with knowledge production ( Agrawal & Goldfarb , 2008 ; Boudreau et al . , 2017 ; Catalini , 2018 ; Hansen , 1999 ; Szulanski , 1996 ) , by focusing on the extent that \u201c engineered serendipity \u201d can mitigate some of these barriers . Engineered , serendipitous encounters differ from traditional knowledge produc - tion mechanisms because they give people a common time and space to meet , thereby removing some of the search costs associated with finding suitable knowledge sharing partners . Our work suggests that engineered serendipity creates opportunities for synergistic collaborations over the long - run that have the potential to broaden collaboration networks and reshape research trajec - tories . Consistent with the information frictions explanation , knowledge overlap is a greater constraint on near - term than long - term knowledge creation . Knowledge similarity can be a cat - alyzing force for near - term research activities because there are fewer start - up costs and uncer - tainties associated with initiating collaborations with similar others , such as people working in the same field . In Boudreau et al . ( 2017 ) , we found that the knowledge sharing intervention ' s effect on grant coapplications was most beneficial for pairs from the same field . This suggests 6 There were a total of 149 ( same room ) and 415 ( different room ) forward citations for pairs with low / moderate field overlap , respectively . A publication title was coded as advanced imaging if it included any of the three technologies featured at the symposium ( physiological magnetic resonance , positron emission tomography , and optical imaging ) . LANE ET AL . 25 that in the short - term , opportunity and discretion remain important factors driving knowledge production ( Dahlander & McFarland , 2013 ) . We expand on these findings to show that while field overlap remains a difficult hurdle to overcome in the near - term , over time , scientists place smaller premiums on knowledge similarity , instead emphasizing synergistic collaborations where partners have both common knowledge and divergent expertise . This shift may be due to the time and repeated interactions needed to develop an initial encounter into a multiplex rela - tionship ( Ingram & Morris , 2007 ; Uzzi , 1997 ) , or the evolving expectations from collaborators as projects move from exploratory research to joint execution of research ideas . They also suggest that homophilous collaborations ( Dahlander & McFarland , 2013 ) can be attributed in part to search costs and information costs . Second , this study advances understanding of how opportunities for temporary colocation , offered by conferences , symposia and similar events potentially impact the direction and quality of scientific knowledge production . Although prior research suggests that temporary colocation is critical to knowledge production ( Campos et al . , 2018 ; Catalini , 2018 ; Catalini et al . , 2020 ; Chai & Freeman , 2019 ) , it has typically inferred face - to - face interaction from observational data , rather than actual acts of knowledge sharing . We suggest that one attractive aspect of con - ferences over informal serendipitous encounters ( e . g . , in hallways , watercoolers ) is that they have a focusing effect on the topic of knowledge sharing and idea exchange . This may be why even brief , highly structured , information - rich interventions can have long - term consequences on knowledge production , most notably by promoting research activities on the conference ' s topics of inquiry . It is important to note that the effects of these events go beyond monetary incentives , which may be an early catalyst to attract participants but the effects are more wide - spread and longer lasting . Third , we make empirical and methodological contributions by designing a prospective study that amalgamates multiple sources and uses of data , namely a natural field experiment , direct observations of face - to - face communications and archival publication data to study long - run knowledge production outcomes . This careful combination of data sources enables us to investigate the causal relationships between knowledge sharing and knowledge production over different time horizons , which are not possible with archival data ( Fleming et al . , 2007 ) . Given the rise in field experiments in innovation research ( Chatterji , Delecourt , Hasan , & Koning , 2019 ) , research designs that at the outset plan for short - and long - term time horizons may yield deeper insights that can help to offset the costs associated with multiple forms of data collection . In addition , our research design can be used as a template for future experiments with multiterm horizons , in terms of learning from its features and its potential shortcomings . In this research , we have made a thorough effort to analyze how knowledge sharing leads to knowledge production in science . However , our study has some empirical limitations . First , our study focused on an interdisciplinary setting in a large , highly selective university . Therefore , it may be interpreted as a best - case scenario in fostering knowledge production , as we must draw a boundary around the network and incentives under consideration . The symposium was highly structured to facilitate knowledge sharing , while knowledge production is a core activity for academics . Second , the geographic proximity of scientists may have facilitated scheduled or serendipitous encounters with greater ease ( Catalini , 2018 ) . Third , our focus on the life sciences also draws upon a specific population , where intragroup competition is normative ( Haas & Park , 2010 ) , in part due to the \u201c winner - takes - all \u201d model of rewards and recognition ( Stephan , 1996 ) . We recognize that other settings may feature greater geographic dispersion or promote more openness and cooperation ; a promising future direction would be to extend this research to different settings . 26 LANE ET AL . We also focused most heavily on archival data ( e . g . , public trace data from publications ) to examine long - term knowledge production outcomes , which provided greater insights into the scientists ' behaviors rather than the reasons behind the observed patterns . Although we made efforts to qualify these findings by examining the content of the knowledge transfer , publica - tions , and citations , future research can seek to supplement archival data with alternative methods ( e . g . , surveys , interviews ) that continue to track the scientists ' interaction patterns , as well as the invisible track of \u201c failed \u201d knowledge production . Such complementary data sources will ultimately provide deeper insights on how engineered serendipitous encounters between people can be cultivated into productive relationships over time . This future work would facili - tate a deeper performative assessment of how knowledge sharing can be \u201c serendipitously engineered \u201d to shape the quality of knowledge outcomes . Overall , this study takes a critical step towards identifying the processes that explain when serendipitous encounters shape knowledge production outcomes among innovating individuals . We show that brief , information - rich interactions between people with some overlapping knowledge interests can have a productive effect on knowledge transfer , creation and diffusion . ACKNOWLEDGEMENTS This work was conducted with support from Harvard Catalyst / The Harvard Clinical and Trans - lational Science Center ( National Center for Advancing Translational Sciences , National Insti - tutes of Health Awards UL1TR001102 , UL1TR000170 , UL1RR025758 - 02S4 , and UL1TR002541 ) , Laboratory for Innovation Science at Harvard University , Harvard Business School Division of Research and Faculty Development , and financial contributions from Harvard University and its affiliated academic health care centers . The authors also acknowledge Harvard Catalyst infrastructure for support and cooperation in implementing the experiment and providing new data for this analysis . The authors are thankful to Charles Ayoubi , Ethan Bernstein , Linus Dahlander , Giada Di Stefano , Adam Kleinbaum , Rory McDonald , Siobhan O ' Mahony , Ryan Raffaelli , Misha Teplitskiy , and the review team at SMJ for comments on earlier drafts . The authors would also like to thank Ruihan Wang for help with data collection . ORCID Jacqueline N . Lane https : / / orcid . org / 0000 - 0002 - 3744 - 9149 Ina Ganguli https : / / orcid . org / 0000 - 0002 - 0691 - 5709 Patrick Gaule https : / / orcid . org / 0000 - 0002 - 8712 - 0405 Eva Guinan https : / / orcid . org / 0000 - 0003 - 3874 - 0007 Karim R . Lakhani https : / / orcid . org / 0000 - 0002 - 5535 - 8304 REFERENCES Abbott , A . ( 2001 ) . Time matters : On theory and method , Chicago , IL : University of Chicago Press . Adams , J . D . , Black , G . C . , Clemmons , J . R . , & Stephan , P . E . ( 2005 ) . Scientific teams and institutional collabora - tions : Evidence from US universities , 1981 \u2013 1999 . 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Engineering serendipity : When does knowledge sharing lead to knowledge production ? Strat Mgmt J . 2020 ; 1 \u2013 30 . https : / / doi . org / 10 . 1002 / smj . 3256 30 LANE ET AL .", + "mwangangi2021structure": "Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 1 of 10 BIOCHEMISTRY The structure of the actin filament uncapping complex mediated by twinfilin Dennis M . Mwangangi 1 , 2 , Edward Manser 1 , 2 , Robert C . Robinson 1 , 3 , 4 * Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from cap - ping protein \u2013 associated filaments ; however , the mechanisms of uncapping leading to rapid disassembly are un - known . Here , we elucidated the x - ray crystal structure of the actin / twinfilin / capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments . The twinfilin / capping protein complex binds to two G - actin subunits in an orientation that resembles the actin filament barbed end . This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G - actin conformation in the two terminal actin subunits . Furthermore , twinfilin disorders critical actin - capping protein interactions , which will assist in the dissociation of capping protein , and may promote filament uncapping through a second mechanism involving V - 1 competition for an actin - binding surface on capping protein . The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments . INTRODUCTION Numerous cellular processes , such as morphogenesis , migration , cytokinesis , endocytosis , and memory , rely on rapid reorganization of actin cytoskeletal networks ( 1 \u2013 3 ) . Coordinated local assembly and disassembly of actin filaments generate force and structure , which is harnessed to drive these specific functions . A number of actin - regulating proteins control the assembly , disassembly , and organization of the filament networks ( 4 , 5 ) . Among the key evolu - tionarily conserved actin regulators are capping protein ( CP ) and the actin depolymerization factor homology ( ADF - H ) domain family of proteins , which include ADF / cofilins and twinfilin ( 6 \u2013 10 ) . While primitive , functional ADF - H domain proteins are found in Asgard archaea ( 7 , 9 ) , CP and twinfilin have only been found , and are ubiq - uitous , in eukaryotes ( 9 , 10 ) . Thus , CP and twinfilin likely arose during eukaryogenesis and were present in the last eukaryotic common ancestor ( LECA ) . The architecture of twinfilin is unique , composed of two ADF - H domains connected by a short linker and followed by a conserved C - terminal tail ( 11 ) . ADF - H proteins generally regulate cytoskeletal reorganization by accelerating the disassembly of actin filaments ( 6 , 12 , 13 ) . Twinfilin - 1 is ubiquitously expressed in almost all tissue types in mammals , where it regulates actin dynamics through mechanisms involving interactions with actin monomers , actin filaments , and CP ( 11 , 14 ) . The biological outputs from twin - filin regulation of actin dynamics include cell motility and synaptic endocytosis ( 15 ) . The reported in vitro roles of twinfilin in actin dynamics are nu - merous , diverse , and somewhat contradictory . Twinfilin binds and sequesters adenosine diphosphate ( ADP ) \u2013 actin monomers with high affinity , inhibiting nucleotide exchange and preventing assem - bly into filaments ( 11 , 14 , 16 ) . Twinfilin also interacts directly with actin filament barbed ends , blocking filament elongation , suggestive of a capping activity ( 17 \u2013 19 ) . Recent studies have also demonstrated that twinfilin accelerates depolymerization of actin filament barbed ends containing ADP - actin subunits ( 20 , 21 ) , and at low pH , twin - filin can sever filaments ( 22 ) . In addition , twinfilin interacts strongly with heterodimeric CP through interactions that include those in - volving the conserved twinfilin C - terminal tail ( 23 ) . CP is a hetero - dimer that binds to the barbed ends of actin filaments to prevent actin subunit exchange ( 24 \u2013 26 ) . Although twinfilin binds to CP with high affinity , its exact biological role in promoting CP capping or uncapping is debated ( 27 , 28 ) . X - ray structural studies of twinfi - lin have been limited to single ADF - H domains , which show high structural conservation , and both domains bind actin monomers ( 19 ) . CP\u2019s interactions with the actin filaments or dynactin fila - ments are resolved to 23 - and 3 . 4 - \u00c5 resolution , respectively , via cryo \u2013 electron microscopy ( cryo - EM ) ( 29 , 30 ) . However , the molec - ular mechanism by which twinfilin interacts with CP at the actin filament barbed ends is unknown . Here , we address the role of twinfilin in uncapping of actin filaments by elucidating the x - ray structure of the twinfilin / CP / actin complex . RESULTS The crystal structure of the twinfilin / CP / actin complex Previous biochemical data have shown that twinfilin\u2019s interaction with CP - capped actin filaments protects CP from displacement by CARMIL , suggesting a stable interaction between twinfilin , CP , and barbed - end actin subunits ( 27 ) . We therefore used purified twinfilin - 1 ( human ) , heterodimeric CP ( mouse CapZ \uf061 1 / \uf062 2 , henceforth CP ) , and skeletal muscle actin ( rabbit ) to reconstitute the complex be - tween twinfilin , CP , and actin monomers . The twinfilin , CP , and actin complex was highly stable in gel filtration chromatography , and this complex was used to prepare protein crystals suitable for structure determination by x - ray crystallography at 3 . 2 - \u00c5 resolu - tion ( fig . S1 ) . The complex consists of two ADP - bound actin subunits , one subunit each of the heterodimeric CP ( CP \uf061 1 and CP \uf062 2 ) and one full - length twinfilin - 1 ( Fig . 1 , A and B ) . In the structure , CP adopts its canonical mushroom - shaped architecture consisting of a cap and stalk and interacts with the barbed - end faces of two G - actin 1 Institute of Molecular and Cell Biology , A * STAR ( Agency for Science , Technology and Research ) , Biopolis , Singapore 138673 , Singapore . 2 Department of Pharmacol - ogy , Yong Loo Lin School of Medicine , National University of Singapore , Singapore 117597 , Singapore . 3 School of Biomolecular Science and Engineering ( BSE ) , Vidya - sirimedhi Institute of Science and Technology ( VISTEC ) , Rayong 21210 , Thailand . 4 Research Institute for Interdisciplinary Science ( RIIS ) , Okayama University , Okayama 700 - 8530 , Japan . * Corresponding author . Email : br . okayama . u @ gmail . com Copyright \u00a9 2021 The Authors , some rights reserved ; exclusive licensee American Association for the Advancement of Science . No claim to original U . S . Government Works . Distributed under a Creative Commons Attribution NonCommercial License 4 . 0 ( CC BY - NC ) . CORRECTED 1 APRIL 2021 ; SEE FULL TEXT D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 2 of 10 subunits via the top surface of the mushroom cap ( Fig . 1 ) . The two actin subunits are in structurally similar conformations and adopt a typical G - actin fold consisting of four subdomains . The actin sub - units do not show the subunit flattening and twisting associated with the G - to - F actin transition ( 31 ) . The deoxyribonuclease I bind - ing loops in the two actin subunits are disordered ( fig . S2 , A and B ) . The relative orientation of the two actin subunits resembles that of barbed - end actin subunits from the F - actin cryo - EM structure ( fig . S2 , C to E ) ( 32 ) . The actin subunits are arranged by the short pitch helical filament relationship , similar to two actin subunits across a filament . They do not adopt the relative positioning of two longitu - dinally related subunits in a single strand . Twinfilin adopts an elon - gated architecture in which the two ADF - H domains each bind one actin subunit , with ADF - H domain 2 ( D2 ) binding to the terminal actin subunit , relative to a filament barbed end . The linker connect - ing the ADF - H domains , which includes an \uf061 - helix , extends across the upper surface ( cap ) of the mushroom - shaped CP and also contacts both actin subunits ( Fig . 1 , A and B ) . The twinfilin C - terminal tail extends from D2 and wraps around the stalk of the CP \uf062 - subunit , which is located opposite to the actin - binding interface , below the CP cap ( Fig . 1 , C and D ) . Binary interactions in the twinfilin / CP / actin complex Each of the two twinfilin ADF - H domains binds to an actin subunit at analogous interfaces , providing basis for monomer sequestration ( Fig . 2A ) . The individual ADF - H domains adopt similar architec - tures except for a difference in the conformation of the \uf062 - sheets . The \uf062 - 3 and \uf062 - 4 strands in D2 form a protrusive extension relative to that in ADF - H domain 1 ( D1 ) ( fig . S3 , A to C ) . The key structural elements of the two ADF - H domains are highly conserved in mouse twinfilin and human twinfilin - 2 ( 33 ) . Despite twinfilin D2 having 10 times higher affinity for G - actin ( 16 ) , its actin - binding interface is markedly similar to that of D1 . Thus , the precise selection of res - idues in the two binding interfaces is likely to explain the differences in actin - binding affinity . Further , the \uf061 - helix in the linker between D1 and D2 loosely associates with the D2 - bound actin subunit , and this likely strengthens D2 interaction with G - actin ( Fig . 2A and fig . S4 , A and C ) . Fig . 1 . The twinfilin / CP / actin complex . ( A ) Front view of the pentameric complex in cartoon representation . CP consists of two subunits , \uf061 - subunit ( CP \uf061 ) and \uf062 - subunit ( CP \uf062 ) . Twinfilin comprises two ADF - H domains ( D1 and D2 ) , a linker between D1 and D2 that includes a helix ( residues 151 to 165 ) , and a C - terminal tail ( Tail ; residues 316 to 342 , the last eight amino acids are disordered ) . Actin subunit 1 is bound to twinfilin D1 , and subunit 2 to twinfilin D2 . The twinfilin secondary structure elements are colored differentially , helices in red , strands in cyan , and loops and extended regions in orange , with the Tail in lime green . ( B ) Front view [ same as in ( A ) ] , in which the actin subunits and twinfilin are represented as surfaces . Twinfilin is shown entirely in orange . ( C and D ) Back and side views , respectively . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 3 of 10 The CP actin - binding interface is located at the top surface of the mushroom cap ( Fig . 2B ) . In the absence of twinfilin , CP interacts with the barbed - end protomers of a filament via C - terminal exten - sions to the \uf061 - and \uf062 - subunits , the \uf061 - and \uf062 - tentacles ( 24 , 26 , 29 ) . In the twinfilin - bound structure , the CP \uf062 - tentacle is mostly disor - dered in the structure and , hence , has no direct contact with either of the actin subunits ( fig . S4 , D and E ) . The \uf061 - tentacle is partially ordered and forms an interaction with actin 1 ( fig . S4F ) . Twinfilin binds CP through its C - terminal tail . This tail wraps around the stalk of CP \uf062 - subunit , a common binding site for filament uncap - ping proteins with CP interaction ( CPI ) motifs ( Fig . 2C ) ( 34 ) . The ordered portion of the tail of twinfilin ( residues 315 to 342 ) includes a basic stretch that interacts with the negatively charged groove be - tween the CP \uf062 - subunit stalk and the underside of the mushroom - shaped cap ( fig . S5 , A to C ) . Furthermore , the complex reveals additional interactions between twinfilin and CP , beyond the C - terminal tail . First , the \uf061 - helix in the linker between D1 and D2 forms an interac - tion with CP that also involves the start of the \uf062 - tentacle ( Fig . 2C and fig . S5 , D to F ) . Second , twinfilin D1 forms a direct interface with the CP \uf062 - subunit ( Fig . 2C and fig . S5G ) . Comparison of the conformations of twinfilin tail \u2013 bound and CARMIL CPI \u2013 bound CP The twinfilin tail - binding site on CP is distant from the actin - binding site , which is centered on the \uf061 - tentacle ( Fig . 3 , A and B ) . Comparison of the tail - binding site with the uncapping CPI motif from CARMIL shows an overlapping interaction on the underside of the CP \uf062 - subunit ( 34 ) ; however , the N termini of the two pep - tides take divergent paths around the CP stalk ( Fig . 3A ) . The CARMIL CPI motif half encircles the CP stalk , with its N terminus making contact with the CP \uf061 - subunit stalk . By contrast , the N ter - minus of the twinfilin tail ( residues 316 to 322 ) follows a straight path and does not form contacts with the CP \uf061 - subunit . This region ( residues 316 to 322 ) is elongated yet ordered with clear electron density ( Fig . 2C and fig . S5K ) , despite not being stabilized by inter - actions , suggesting that it may be under tension to extend the poly - peptide chain into an ordered conformation . The overlapping interface provides a structural basis for the competition between the twinfilin tail and CARMIL CPI for CP binding , and twinfilin\u2019s attenuation of CARMIL - mediated dissociation of CP from filament barbed ends ( 27 ) . Fig . 2 . Binary interactions in the twinfilin / CP / actin complex . ( A ) Front and back views of actin bound to twinfilin D1 or twinfilin D2 . ( B ) Two views of the CP interaction with actin , in which actin 1 and actin 2 are shown in similar orientations . ( C ) Three orientations of the CP / twinfilin interaction . Examples of the electron density of key features are shown in fig . S5 ( H to K ) . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 4 of 10 CP can adopt two known conformations , which are likely to be in dynamic equilibrium in solution . The uncapping CPI motifs of CARMIL , CD2AP , and CKIP stabilize one conformation , actin - free conformation of CP ( 34 ) . Superimposition of the complex with CARMIL - bound CP and unbound CP shows key structural changes that CP subunits undergo in adopting the twinfilin / CP / actin com - plex conformation ( Fig . 3 , C and D , and fig . S6 ) . First , the CP \uf062 - subunit mushroom cap in this complex moves upward relative to the stalk , while the \uf061 - tentacle repositions to adopt the actin - bound con - formation . Second , the \uf061 - subunit adjusts upward flattening the mushroom cap and adopts a similar conformation to that stabilized by V - 1 or Arp1 ( from the dynactin complex ) ( Fig . 3E and fig . S6 ) ( 30 , 35 ) . The binding site of V - 1 , a steric CP inhibitor , overlaps with that of actin in this complex ( fig . S6J ) ( 35 ) . Therefore , the CP struc - ture in the twinfilin / CP / actin complex represents the mushroom cap \u2013 bound conformational state , to either actin , Arp1 , or V - 1 , while the CPI - bound structure of CP represents a stabilized mush - room cap \u2013 unbound conformation . Binding of the CARMIL CPI motif around the stem of CP locks CP in the mushroom cap \u2013 unbound conformation , which is less compatible with actin barbed - end in - teraction , leading to uncapping of the filament ( 34 , 36 ) . The in - ability of the twinfilin tail to induce a change in the CP conformation from mushroom cap \u2013 bound to unbound state suggests that , in the complex , actin binding dominates the CP conformation or the twinfilin tail does not stabilize the mushroom cap \u2013 unbound con - formation . To distinguish between these possibilities , we tested for uncapping activity within the twinfilin tail in isolation . We per - formed pyrene - actin polymerization assays in which an increase in pyrene fluorescence reports on the efficiency of actin assembly from CP precapped actin filament seeds . The CARMIL CPI peptide , a positive control , displayed potent uncapping activity leading to polymer - ization from the CP precapped filament seeds , indicated by the increase in fluorescence ( fig . S7 ) . By contrast , the maltose - binding protein ( MBP ) \u2013 tagged twinfilin tail ( residues Gln 321 - Asp 350 ) had no detect - able uncapping effect , showing a similar polymerization profile to the CP precapped filament seeds alone ( fig . S7 ) . Thus , in this assay , the twinfilin tail alone does not uncap CP - capped filaments , suggesting that it does not strongly stabilize the CP mushroom cap \u2013 unbound conformation . Twinfilin influences CP interaction with actin barbed ends The structure reveals additional interactions between twinfilin and CP that might influence the CP - binding mode to the actin filament barbed ends ( fig . S5 , D to G ) . The dynactin filament ( consisting of Fig . 3 . Conformations of CP . ( A ) Comparison of the twinfilin tail ( yellow ) binding site with CARMIL ( cyan ) on CP . Both CP - binding peptides run in the same direction , and N terminus of the twinfilin tail is labeled N . ( B ) The actin - binding site on the CP \uf061 - tentacle is distant from the twinfilin tail - binding site . ( C and D ) Structural superimposition of the \uf062 - subunits of CP reveals that the conformation of CP in the twinfilin / CP / actin complex is different to the CARMIL - bound and unbound conformations of CP . ( E ) Superimposition reveals that the conformation of CP in the twinfilin / CP / actin complex is similar to the V - 1 \u2013 bound conformation of CP . \uf061 - and \uf062 - subunits of CP are colored light green and dark green , respectively , for the CARMIL , unbound , and V - 1 complexes . Black arrows indicate conformational changes in CP in adopting the twinfilin / CP / actin complex structure . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 5 of 10 Arp1 instead of actin ) is structurally similar to the actin filament and is capped at its barbed end by CP ( 30 ) . Comparison of the CP - binding modes to actin in the twinfilin / CP / actin complex and to Arp1 subunits reveals similar overall geometries ( Fig . 4 and fig . S8 ) . However , there are considerable differences in the binding of the CP \uf061 - and \uf062 - tentacles to their respective actin / Arp1 subunits . These differences arise from the presence of twinfilin , because the binding modes of the \uf061 - and \uf062 - tentacles are similar in the dynactin complex and in the 23 - \u00c5 cryo - EM structure of the CP / actin fila - ment ( 29 , 30 ) . The \uf062 - tentacle , which is bound to the terminal Arp1 subunit in the dynactin complex , is disordered in the twinfilin / CP / actin complex ( Fig . 4A and fig . S8A ) . Structural superimposition indicates that the twinfilin linker obscures the \uf062 - tentacle binding site on actin in the actin / twinfilin / CP complex ( Fig . 4A ) . In the dy - nactin complex , the \uf061 - tentacle is fully ordered and bound to Arp1 . By contrast , the \uf061 - tentacle in the twinfilin / CP / actin complex only forms a partial interaction with actin ( Fig . 4B and fig . S8 , B and C ) . Superimposition reveals that the \uf061 - tentacle \u2013 binding site on actin is partially obstructed by twinfilin D1 ( Fig . 4B ) . The binding of the tentacles to the actin protomers will also be influenced by the actin protomer conformation . A twinfilin - induced shift from an F - actin to G - actin conformation may aid dissociation of the tentacles . In the absence of twinfilin , the CP \uf061 - tentacle is a critical interac - tion with barbed end of a filament , while the \uf062 - tentacle offers a sec - ond important actin - binding interface to stabilize capping ( 36 ) . The obstruction of the CP tentacles by twinfilin in the actin / twinfilin / CP complex will destabilize the CP interaction , leading to weakened CP affinity for actin filament barbed ends . Thus , the CP - binding mode in the dynactin complex represents fully bound CP , conferring strong and stable capping activity , while the CP - binding mode in the actin / twinfilin / CP complex represents a weak and unstable capping activity . This unstable interaction state may be targeted by other regulatory factors , such as the CP - sequestering protein V - 1 . We used the pyrene - actin polymerization assay to test whether V - 1 can enhance CP uncapping in the presence of twinfilin ( 28 ) . Addition of twinfilin into CP precapped actin filament seeds mixed with pyrene - labeled monomers and profilin did not accelerate polymerization , indicat - ing either a lack of uncapping or uncapping followed by recapping ( Fig . 5 ) . Addition of V - 1 alone displayed partial polymerization attributable to CP sequestration ( Fig . 5 ) . However , the presence of both twinfilin and V - 1 accelerated polymerization to near the level of uncapped actin filament seeds , indicating that the cooperative activities of twinfilin and V - 1 can induce dissociation of CP from barbed ends and prevent recapping by CP ( Fig . 5 ) . This indicates that twinfilin is able to remove CP from filaments ; however , the CP - sequestering protein V - 1 is required to prevent recapping of fil - aments . We propose that the high concentration of profilin ( 2 . 8 \uf06d M ) , relative to twinfilin ( 1 \uf06d M ) , used in this assay was sufficient to com - petitively remove actin from the CP / twinfilin complex and allow recapping in the absence of V - 1 . The requirement of both the un - capping agent twinfilin and CP - sequestering protein V - 1 to observe robust uncapping in this in vitro assay partially explains some of the disparities in the reported activities of twinfilin . Uncapping has been difficult to observe in many assays in which recapping has not been excluded . DISCUSSION This analysis of the actin / twinfilin / CP complex reveals the structural basis by which twinfilin interacts with actin and with CP , and pro - vides insight into molecular mechanisms for the regulation of CP in actin filament barbed - end dynamics . The unanticipated geometry Fig . 4 . Comparison of the CP - binding mode to Arp1 in the dynactin complex ( Arp1 / CP ) with the binding mode to actin in actin / twinfilin / CP complex . ( A and B ) Focus on the \uf062 - and \uf061 - tentacles , respectively , which are highlighted by black circles . The \uf062 - tentacle is disordered and unbound in the actin / twinfilin / CP complex , and the \uf061 - tentacle is partially bound and ordered , relative to the dynactin complex . Enlargements of the tentacle regions show the superimpositions of the CP ( \uf061 - and \uf062 - subunits colored red and blue , respectively ) from the dynactin complex on to actin ( green and teal ) and twinfilin ( yellow ) from the actin / twinfilin / CP complex . ( A ) Enlargement , the twinfilin linker ( yellow ) binds to the \uf062 - tentacle \u2013 binding site on actin 2 ( green ) . ( B ) Enlargement , the N terminus of twinfilin D1 ( yellow ) occupies half of the \uf061 - tentacle \u2013 binding site on actin 1 ( teal ) . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 6 of 10 in which twinfilin binds to two actin subunits in a pseudo - filament barbed - end orientation has major implications for its possible mech - anisms of action . Previously proposed mechanisms for twinfilin\u2019s activities assumed that twinfilin contacts two longitudinally related actin subunits at the barbed end of an actin filament ( 17 , 18 , 21 , 27 , 28 ) . However , the structure presented here unambiguously identifies that the two actin subunits are laterally related . Twinfilin binds across the two strands of an actin filament , rather than down the side of a single strand in the filament . We discuss the ramifications of this binding orientation for actin filament regulation . The primary biological role of twinfilin has not been settled . Mammalian twinfilin - 1 was shown to preferentially localize to re - gions of the cell enriched for F - actin ( 14 ) . Loss of twinfilin - 1 in B16 cells leads to their inability to generate lamellipodia ( 27 ) . In vitro and cell - based experiments have led to multiple proposed activities . Twinfilin\u2019s actin - related functions have been variously described as follows : an actin monomer - sequestering protein that forms a 1 : 1 complex with actin that prevents actin polymerization and inhibits nucleotide exchange in actin ( 11 , 14 ) ; a CP - interacting and phos - phatidylinositol 4 , 5 - bisphosphate ( PIP 2 ) \u2013 interacting protein ( 37 ) ; an actin monomer - shuttling protein between the pointed and barbed ends of filaments ( 38 ) ; a protein that does not affect CP - capping activity , nor does CP affect twinfilin\u2019s monomer binding function ( rather , twinfilin localizes actin monomers to sites of assembly through interaction with CP ) ( 23 ) ; a barbed - end actin filament CP ( 17 ) ; an actin filament - severing protein ( 22 ) ; a protein that speeds up actin depolymerization at both ends of the actin filament in con - junction with cyclase - associated protein ( CAP ) ( 20 ) ; a protein that enhances CP actin filament capping , which competes with CARMIL for CP binding ( 27 ) ; and an actin filament CP - uncapping protein ( 28 ) . The actin / twinfilin / CP complex provides the structural basis to reassess these activities . In the complex , twinfilin forms extensive interactions with CP and with two actin protomers , which adopt a pseudo - actin filament barbed - end orientation relative to each other . These multiple inter - actions indicate that twinfilin\u2019s primary and evolutionary conserved role , throughout eukaryotes , is to regulate CP capping at the barbed end of actin filaments . In yeast , the CP : twinfilin ratio is estimated to be 2 . 5 : 1 ( 37 , 39 ) , which indicates that the actin - related cellular activities of twinfilin will be dominated by the CP : twinfilin com - plex , rather than twinfilin acting alone on filaments . Similarly , the molar abundance of mammalian CP : twinfilin - 1 ratio was deter - mined as 2 : 1 ( 27 ) . Twinfilin has been shown to strongly interact with actin filament barbed ends and with CP , dissociation constants ( K d ) of 13 and 50 nM , respectively ( 17 , 27 ) . However , these affinities are at least one order of magnitude weaker than the CP affinity for actin filament barbed ends [ K d = 0 . 1 to 1 . 0 nM ( 40 ) ] . The actin / twinfilin / CP complex structure demonstrates that twinfilin disrupts CP - actin interfaces , sterically competing with the CP \uf061 - and \uf062 - tentacles in binding to actin , indicating that twinfilin\u2019s principal role is to destabilize CP capping ( Fig . 6A ) . We discuss the possible mechanisms that lead to uncapping . The ADP - bound actin protomer conformations in the actin / twinfilin / CP complex are very similar to that in the cofilin - decorated actin filament ( fig . S2B ) ( 41 ) . Cofilin , which consists of a single ADF - H domain , severs actin filaments toward the pointed end of a section of filament decorated with cofilin , by inducing a G - actin \u2013 like conformation in the actin protomers , which are not sta - ble as a filament ( 13 , 41 ) . We hypothesize that the orientation of the two ADF - H twinfilin domains , in binding across the filament , allows twinfilin to induce a G - actin \u2013 like conformation in the final two barbed - end actin protomers . This conformation will destabilize these two terminal actin subunits , leading to them being \u201csevered\u201d from the end of the filament , by an analogous mechanism to cofilin severing ( 13 , 41 ) . Thus , we propose that the two twinfilin ADF domains induce severing at the boundary between ADF - H \u2013 bound and ADF - H \u2013 free portions of F - actin , dissociating CP , twinfilin , and the two terminal actin subunits as a complex ( Figs . 1A and 6 , A and B ) . Immediate reassociation of the actin / twinfilin / CP complex with the filament would be unfavorable because the actin subunits in the complex are held in the ADP - bound G - actin state , and nucleotide exchange is inhibited by the twinfilin ADF - H domains ( 11 ) . We propose that the actin / twinfilin / CP complex will then un - dergo a process of recycling . Twinfilin strongly binds to ADP - bound actin monomers ( K d ~ 40 to 50 nM ) , and this affinity is unaffected by the presence of CP ( 16 , 23 ) . For comparison , CAP , thymosin - \uf062 4 , profilin , and cofilin are characterized in their affinities for ADP - bound actin monomers by K d of 20 nM , 80 to 100 nM , 0 . 17 \uf06d M , and 0 . 4 \uf06d M , respectively [ reviewed in ( 42 ) ] . The two ADP - bound actin subunits , from the actin / twinfilin / CP complex , may then be con - verted to adenosine triphosphate ( ATP ) \u2013 bound actin by the actions of CAP , due to the high affinity of CAP for ADP - bound actin ( 20 , 21 , 33 , 43 ) . Subsequently , the ATP - bound actin monomers will be released to profilin , and possibly to thymosin - \uf062 4 , due to their superior affinities for ATP - bound actin relative to twinfilin , CAP , and cofilin , 0 . 1 , 0 . 1 to 4 . 0 , 0 . 5 , 1 . 9 , and 6 \uf06d M ( 16 , 23 , 42 ) , respectively . This will replenish the polymerization - competent pool of actin monomers ( 42 ) . Because CAP also binds to the pointed ends of actin filaments ( 33 ) , association of the actin / twinfilin / CP complex with the pointed end bound CAP may provide a mechanism to spa - tially separate the twinfilin / CP complex away from elongating actin filament barbed ends close to the membrane . Fig . 5 . V - 1 uncaps filament barbed ends in the presence of twinfilin . Pyrene - actin polymerization assay showing uncapping of F - actin seeds by V - 1 in the presence of twinfilin . Unlabeled F - actin seeds ( 0 . 5 \uf06d M ) were capped with 100 nM CP and subse - quently polymerized in either 2 \uf06d M actin ( 10 % pyrene ) / 2 . 8 \uf06d M profilin ( red ) or 2 \uf06d M actin ( 10 % pyrene ) / 2 . 8 \uf06d M profilin supplemented with either 1 \uf06d M twinfilin ( yellow ) , 5 \uf06d M V - 1 ( purple ) , or a mixture of 1 \uf06d M twinfilin and 5 \uf06d M V - 1 ( blue ) . As a control , F - actin seeds were polymerized in the absence of CP ( black ) . As shown in the pro - file , the presence of both twinfilin and V - 1 induces accelerated polymerization of pre - capped F - actin seeds ( blue ) to almost the same level as the positive control ( black ) . By contrast , twinfilin alone ( yellow ) induces very low level of polymerization , similar to CP alone ( red ) , while V - 1 alone ( purple ) only induces minimal polymerization . AU , arbitrary units . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 7 of 10 The fate of the twinfilin / CP complex may then be several - fold . First , the twinfilin / CP complex may encounter a free barbed end of a filament and undergo a further round of transient binding followed by dissociation of the terminal actin protomers . Thus , the twinfilin / CP complex may depolymerize the filament in two subunit cycles , maintaining transient capping while depolymerizing the filament . Second , twinfilin may competitively dissociate from CP in favor of CP - capped filaments due to the superior affinity for filament ends relative to CP alone , K d of 13 and 50 nM , respectively ( 17 , 27 ) . This mechanism assumes that twinfilin affinity for the CP - capped fila - ment is also high ; however , the value is unknown and possibly diffi - cult to measure due to the uncapping mechanism . Last , the twinfilin / CP complex may be dissociated at the membrane by PIP 2 or by competition with CPI proteins , such as CARMIL ( 27 , 37 ) . The structure of the actin / twinfilin / CP complex suggests that a second possible mechanism of filament uncapping may operate in the presence of V - 1 . In this mechanism , the twinfilin / CP complex \u201cwobbles\u201d ( 44 ) at the barbed end of a filament , remaining attached through the high - affinity actin - binding site on twinfilin D2 , while twinfilin D1 dissociates from actin ( Fig . 6 , A and C ) ( 16 ) . The ex - tended portion of the twinfilin tail ( residues 316 to 322 , between the end of D2 and the first tail residue with significant contact with CP ) , which we hypothesize is under tension , may aid the wobble process . There is homology between twinfilin and CARMIL in this region ( 317 - HAHKQSFAKP - 326 in twinfilin - 1 and 981 - KLEHFTKLRP - 990 in CARMIL1 ) ( 27 ) , which may allow the twinfilin tail to associate more intimately with CP in a manner similar to the CARMIL CPI interaction with CP ( Fig . 3A ) ( 34 ) . Association of this region of twinfilin with CP would hold CP away from the actin subunit , to which it was previously bound via the \uf061 - tentacle , thus freeing the actin - binding site on the \uf061 - tentacle to bind to V - 1 , and preventing the \uf061 - tentacle from reassociating with actin . The partially bound complex would then dissociate as twinfilin / CP / V - 1 bound to a sin - gle actin , with the high - affinity twinfilin D2 inducing a G - actin \u2013 like conformation in the actin subunit to destabilize its association with the filament ( 16 ) . Flow - cell total internal reflection fluorescence microscopy observations of CP uncapping by twinfilin detected en - hanced uncapping in the presence of V - 1 ( 28 ) . This suggests that V - 1 may play an active role in twinfilin - aided uncapping as hypoth - esized in this mechanism ( Fig . 6 , A and C ) . In summary , the actin / twinfilin / CP complex structure clarifies the possible mechanisms by which twinfilin operates as a diffusing uncapping agent . The majority of the in vitro observations outlined above can be rationalized in terms of twinfilin\u2019s proposed role in transforming CP from a strong capping agent into a transiently cap - ping depolymerization complex , which aids in the recycling of actin monomers and is sensitive to the presence of V - 1 . In the bulk cyto - plasm , the outcome of twinfilin\u2019s uncapping activities is likely to be actin depolymerization while maintaining transient capping . This role contrasts starkly to the CPI - containing proteins , which are target - bound uncapping agents that are hypothesized to engender actin polymerization for membrane remodeling ( 45 , 46 ) . The location of uncapping , in either actin assembly - or disassembly - rich regions of a cell , will determine whether filament polymerization or depolym - erization will be the product of the uncapping . Any twinfilin - induced uncapping at a membrane may therefore result in the opposite ac - tivity , actin polymerization . Because twinfilin / CP - bound filaments , relative to CP - bound filaments , have enhanced barbed - end fila - ment dynamics , we propose that cells are able to differentially regu - late these actin filament barbed - end binding states to dictate the Fig . 6 . Models of the filament uncapping . ( A ) Cartoon comparison of CP and CP / twinfilin association at the barbed end of a filament . ( B ) Twinfilin - aided uncapping is a result of twinfilin ADF - H domains inducing G - actin \u2013 like conformations in the terminal two actin protomers , weakening actin : actin interactions in the filament , leading to the dissociation of the complex . ( C ) Twinfilin / V - 1 \u2013 aided uncapping requires space for V - 1 to reach its binding site on CP via a wobble state of the twinfilin - bound complex . Once V - 1 is bound , CP is unable to reassociate with actin . In vitro effects of twinfilin alone on actin filaments and comparisons of uncapping models in the absence of twinfilin are shown in fig . S9 . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 8 of 10 lifetimes of individual actin filaments . The proposed mechanism of depolymerization while maintaining transient filament capping adds to the evidence that actin filament ends are highly controlled and are rarely free in mammalian cells . During depolymerization , filament barbed ends are controlled by twinfilin / CP and filament pointed ends by CAP ( 33 ) . During polymerization modes , the fila - ment barbed ends can be regulated by formins ( 47 ) , or alternatively by the VASP family proteins ( 48 ) , following either ARP2 / 3 nucle - ation or filament uncapping at the membrane ( 34 , 40 , 49 , 50 ) . In each case , the filament end is protected . MATERIALS AND METHODS Protein expression and purification The gene sequences encoding full - length human twinfilin - 1 and profilin - 1 were codon - optimized for Escherichia coli , synthesized ( GenScript ) , and cloned into a pSY5 vector that includes an N - terminal eight - histidine tag followed by a human rhinovirus 3C protease cleavage site ( 51 ) . The mouse full - length CP ( \uf061 1 / \uf062 2 ) con - struct was provided by P . Lappalainen ( University of Helsinki , Finland ) . The construct was in the pRSFDuet - 1 vector designed for coexpres - sion of two interacting target proteins and contains an N - terminal six - histidine tag on the \uf061 - subunit ( 52 ) . All constructs encoding proteins of interest were transformed and recombinantly expressed in phage - resistant E . coli strain BL21 Star ( DE3 ) ( New England Biolabs ) . Fresh LB medium supple - mented with either ampicillin ( pSY5 ) or kanamycin ( pRSFDuet - 1 ) was inoculated with respective overnight cultures and shaken at 37\u00b0C until the cell density reached OD 600 ( optical density at 600 nm ) ~ 0 . 6 . Cells were induced for protein expression with 0 . 25 mM isopropyl - \uf062 - d - thiogalactopyranoside at 16\u00b0C overnight . Cells were harvested via centrifugation at 4000 g at 4\u00b0C for 1 hour , and the pellets were resuspended in 50 ml of His - binding buffer [ 50 mM tris - HCl ( pH 7 . 5 ) , 500 mM NaCl , 20 mM imidazole , and one protease inhibitor tablet ] . The cells were lysed by sonication with a Vibra - Cell ultrasonic processor and clarified by centrifuga - tion at 19 , 000 rpm in SS - 34 rotor using an RC 5C Plus centrifuge ( Sorvall ) at 4\u00b0C for 1 hour followed by filtration through a 0 . 45 - \uf06d m Minisart syringe filter ( Sartorius ) . The proteins were purified on an \u00c4KTAxpress system ( GE Healthcare ) by affinity chromatography using 5 ml of HisTrap FF column with or without ( CP ) on - column cleavage of the His - tag with human rhinovirus 3C protease . Proteins purified with the His - tag were eluted in buffer containing 50 mM tris - HCl ( pH 7 . 5 ) , 500 mM NaCl , and 500 mM imidazole . The eluted proteins were concentrated to 5 ml and subjected to size exclusion chromatography ( SEC ) in a Superdex 75 pg column ( GE Healthcare ) preequilibrated with buffer containing 50 mM tris - HCl ( pH 7 . 5 ) and 150 mM NaCl . All purified proteins were verified by SDS \u2013 polyacrylamide gel electrophoresis ( PAGE ) before being snap - frozen and stored at \u221280\u00b0C . MBP - tagged twinfilin tail constituting residues Gln 321 - Asp 350 was polymerase chain reaction ( PCR ) \u2013 amplified from full - length human twinfilin - 1 expression plasmid and inserted into pSY7 vector , which incorporates a cleavable N - terminal histidine and MBP tag . MBP control was prepared by expressing pSY7 vector alone in BL21 ( DE3 ) E . coli . Both MBP - tagged twinfilin tail and MBP were purified by affinity chromatography using 1 ml of HisTrap FF column with on - column digestion with human rhinovirus 3C protease to remove the histidine tag . The proteins were further purified by gel filtration chromatography with a Superdex 75 pg column ( GE Healthcare ) equilibrated with 50 mM tris - HCl ( pH 9 ) and 150 mM NaCl . Human V - 1 ( pGEX - 6P - 3 vector ) was expressed as a glutathione S - transferase ( GST ) fusion E . coli BL21 Star ( DE3 ) and affinity - purified on an \u00c4KTAxpress system ( GE Healthcare ) by loading cleared lysate onto 1 ml of GSTrap FF Column ( GE Healthcare ) equilibrated with 50 mM tris - HCl ( pH 7 . 4 ) , 150 mM NaCl , and 1 mM dith - iothreitol ( DTT ) . GST tag was removed by on - column digestion with human rhinovirus 3C protease overnight followed by further purification by gel filtration chromatography . CARMIL CP \u2013 binding region ( CBR ) was expressed and purified as previously described ( 34 ) . Preparation of the actin , twinfilin , and CP complex Rabbit skeletal muscle actin was purified from skeletal muscle ace - tone powder ( Pel - Freez ) ( 53 , 54 ) . The protein was subjected to a final SEC using HiLoad Superdex 200 on an \u00c4KTA Prime system . The purity of G - actin was assessed by SDS - PAGE , and concentra - tion was determined by measuring OD at 290 nm . The protein complex was prepared by mixing human twinfilin - 1 , mouse CP \uf061 1 \uf062 2 , and rabbit actin in buffer A [ 2 mM tris - HCl ( pH 7 . 5 ) , 0 . 2 mM ATP , 0 . 5 mM DTT , 1 mM NaN 3 , and 0 . 1 mM CaCl 2 ] at a molar ratio of 1 : 1 : 2 . 5 . The mixture was incubated on ice for 10 min to allow com - plex formation and then purified by SEC ( HiLoad 16 / 60 Superdex 200 preequilibrated with buffer A ) on an \u00c4KTAxpress system ( GE Healthcare ) . Fractions corresponding to ultraviolet absorption peak were analyzed by SDS - PAGE gel to verify complex formation . Crystallization and crystal optimization The fractions corresponding to the peak from SEC were pooled to - gether , concentrated with Vivaspin 20 MWCO 10 , 000 concentrator ( Sartorius ) to approximately 20 mg / ml , and subjected to commer - cial crystallization screens . Crystallization screens were set up as sitting drops in three - drop Intelli - Plate 96 ( Hampton Research ) in 2 : 1 , 1 : 1 , and 1 : 2 ratios consisting of protein solution and precipitant and stored in room temperature ( 25\u00b0C ) . Crystal hits were observed in a number of conditions in the JBScreen Classic HTS I screen after 3 days . One condition [ 12 % polyethylene glycol ( PEG ) 8000 , 10 % glycerol , and 500 mM potassium chloride ] produced the best shaped crystals composed of thin rods . A seed bead kit ( Hampton Research ) was used to generate seed stocks of protein crystals for further opti - mization . Crystals were set up using the hanging - drop vapor diffu - sion method in 2 - \uf06d l drops of 1 : 1 ratio consisting of protein solution and seed stock in the crystallization condition ( 12 % PEG 8000 , 10 % glycerol , and 500 mM potassium chloride ) . Crystal data processing and structure determination Harvested crystals were soaked in 25 % glycerol before being care - fully fished with cryoloops and flash - frozen in liquid nitrogen for subsequent data collection at the National Synchrotron Radiation Research Center , Taiwan . Indexing , scaling , and merging of data - sets were carried out in HKL2000 ( 55 ) . The crystal structure twinfilin / CP / actin complex was solved at a resolution of 3 . 2 \u00c5 by molecular replacement using Phaser ( 56 ) , sequentially searching for one copy of mouse twinfilin - 1 D2 ( 3DAW ) ( 19 ) and one copy of chicken CP hetero - dimer ( 3AA7 ) ( 35 ) followed by a second copy of mouse twinfilin - 1 D2 ( 3DAW ) ( 19 ) . The resultant model was rebuilt by hand , and the structure was subjected to several rounds of refinement using Phenix ( 56 ) and further manual rebuilding in COOT ( 57 ) . The crystal data collection and structure refinement statistics are presented in table S1 . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024 Mwangangi et al . , Sci . Adv . 2021 ; 7 : eabd5271 27 January 2021 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 9 of 10 Pyrene - actin polymerization assays Pyrene - actin polymerization assays to monitor filament uncapping were performed in 100 - \uf06d l reactions containing 2 \uf06d M rabbit skeletal muscle G - actin ( 10 % pyrene \u2013 labeled ) , 0 . 5 \uf06d M F - actin seeds , 2 . 8 \uf06d M profilin ( to prevent pointed - end polymerization ) , 100 nM CP , and variable concentrations of the test proteins . In all pyrene - actin po - lymerization assays , the final concentrations of twinfilin , CP , CARMIL , V - 1 , MBP - tagged tail , and MBP were set at 1 \uf06d M , 100 nM , 250 nM , 5 \uf06d M , 10 \uf06d M , and 10 \uf06d M , respectively . The components were mixed in buffer A [ 2 mM tris - HCl ( pH 7 . 5 ) , 0 . 2 mM ATP , 0 . 5 mM DTT , 1 mM NaN 3 , and 0 . 1 mM CaCl 2 ] to a final volume of 70 \uf06d l in a black flat - bottom 96 - well plate ( Corning ) . To prepare actin filament seed stocks , 5 \uf06d M G - actin was polym - erized for 1 hour at room temperature , after which the filaments were mechanically sheared by repeatedly passing the F - actin solu - tion through a 0 . 7 - mm - diameter needle for 1 min . Thereafter , 10 \uf06d l of the F - actin seed stock was mixed with 10 \uf06d l of 1 \uf06d M CP and left for 5 min to allow barbed - end capping of the F - actin seeds . As a control , the F - actin seed stock was mixed with buffer A , without CP . Then , 20 \uf06d l of this precapped F - seed stock , or control , was add - ed to pyrene - actin polymerization mixture and actin polymeriza - tion initiated by addition of 10 \uf06d l of 10\u00d7 KMEI buffer ( 500 mM KCl , 10 mM MgCl 2 , 10 mM EGTA , and 100 mM imidazole , pH 7 . 4 ) in a total reaction volume of 100 \uf06d l . The pyrene fluorescence intensities were monitored immediately on a Safire 2 fluorimeter plate reader ( Tecan ) with excitation and emission wavelengths set at 365 and 407 nm , respectively . SUPPLEMENTARY MATERIALS Supplementary material for this article is available at http : / / advances . sciencemag . org / cgi / content / full / 7 / 5 / eabd5271 / DC1 REFERENCES AND NOTES 1 . O . L . Mooren , B . J . Galletta , J . A . Cooper , Roles for actin assembly in endocytosis . Annu . Rev . Biochem . 81 , 661 \u2013 686 ( 2012 ) . 2 . T . D . Pollard , J . A . Cooper , Actin , a central player in cell shape and movement . 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Cooper ( Washington University School of Medicine , USA ) for constructs , and the experimental facility and the technical services provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology , Ministry of Science and Technology and the National Synchrotron Radiation Research Center , a national user facility supported by the Ministry of Science and Technology , Taiwan . Funding : This work was supported by Biomedical Research Council of A * STAR under the Singapore International Graduate Award ( SINGA ) scholarship , by NMRC Grant OFIRG / 027 / 2016 , and by Human Frontiers Science Program award RGP0028 / 2018 . Author contributions : D . M . M . and R . C . R . conceived experiments , analyzed data , and wrote the paper . D . M . M . performed experiments . R . C . R . and E . M . supervised the work . Competing interests : The authors declare that they have no competing interests . Data and materials availability : The atomic coordinates and structure factors have been deposited in the Protein Data Bank ( PDB ) under the accession code 7CCC . All other data are available from the corresponding author upon reasonable request . Submitted 26 June 2020 Accepted 8 December 2020 Published 27 January 2021 10 . 1126 / sciadv . abd5271 Citation : D . M . Mwangangi , E . Manser , R . C . Robinson , The structure of the actin filament uncapping complex mediated by twinfilin . Sci . Adv . 7 , eabd5271 ( 2021 ) . D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g on A p r il 09 , 2024", + "chanComparingDifferentSensemaking2016": "Compar ing Different Sensemaking Approaches for Large - Scale Ideation Joel Chan 1 Steven Dang 1 Steven P . Dow 2 1 Human - Computer Interaction Institute Carnegie Mellon University , Pittsburgh , PA USA { joelchuc , stevenda } @ cs . cmu . edu 2 Cognitive Science Department UC San Diego , La Jolla , CA , USA spdow @ ucsd . edu ABSTRACT Large - scale idea generation platforms often expose ideators to previous ideas . However , research suggests people generate better ideas if they see abstracted solution paths ( e . g . , descriptions of solution approaches generated through human sensemaking ) rather than being inundated with all prior ideas . Automated and semi - automated methods can also offer interpretations of earlier ideas . To benefit from sensemaking in practice with limited resources , ideation platform developers need to weigh the cost - quality tradeoffs of different methods for surfacing solution paths . To explore this , we conducted an online study where 245 participants generated ideas for two problems in one of five conditions : 1 ) no stimuli , 2 ) exposure to all prior ideas , or solution paths extracted from prior ideas using 3 ) a fully automated workflow , 4 ) a hybrid human - machine approach , and 5 ) a fully manual approach . Contrary to expectations , human - generated paths did not improve ideation ( as meas - ured by fluency and breadth of ideation ) over simply showing all ideas . Machine - generated paths sometimes significantly improved fluency and breadth of ideation over no ideas ( although at some cost to idea quality ) . These findings suggest that automated sensemaking can improve idea generation , but we need more research to understand the value of human sensemaking for crowd ideation . Author Keywords Creativity ; crowdsourcing ; brainstorming ; sensemaking ACM Classification Keywords Human - centered computing ~ Collaborative and social computing INTRODUCTION Large - scale ideation platforms show potential for solving difficult problems by leveraging the scale and diversity of online crowds [ 4 , 7 , 37 , 59 ] . Current approaches excel at collecting many solutions , especially in a competitive paradigm where crowd innovators work in isolation from each other . However , a significant concern with this ap - proach is that it often results in wasted effort as newcomers retrace obvious but suboptimal solution paths [ 37 ] . For example , in their recent 10 to the 100 th crowdsourced innovation project , Google had to recruit 3 , 000 of their employees to prune the 150 , 000 ideas received from the crowd , pushing the project nine months behind schedule . Cognitive research on creative ideation suggests that people can produce better ideas if they are able to learn from the efforts of others , recombining ideas into new ideas and iterating on new ideas to improve them [ 19 , 21 , 32 , 55 ] . However , at crowd scale , simply exposing all ideas to everyone may not be the most effective strategy . Ideators may not have sufficient time or cognitive resources to sift through potentially hundreds to thousands of other ideas . Consequently , they may only superficially process and build on ideas rather than leveraging them to generate new insights [ 35 , 36 ] . The presence of superficial details in raw ideas might also lead to cognitive fixation [ 34 , 44 , 56 ] . For these reasons , abstracted solution paths \u2014 which distill essential solution approaches from a number of different ideas while avoiding superficial details \u2014 may provide a better way for ideators to interact with prior ideas . Available sensemaking strategies for abstracting solution paths can be placed on a hypothesized cost - quality tradeoff continuum , where sensemaking quality ( especially its benefit for ideation ) is a function of the strategy\u2019s cost . At the high end of the continuum ( high cost , hypothesized high quality ) , there is a mature set of design synthesis strategies ( e . g . , affinity diagramming ) employed effectively by design teams to make sense of a solution space [ 5 , 29 , 38 ] ; these strategies require significant manual human effort , and therefore may be hard to scale to crowd ideation . However , these sensemaking outputs should hypothetically have high value for ideation . At the low end ( low cost , hypothesized low quality ) sit a number of automated approaches ( e . g . , unsupervised machine learning methods like Latent Seman - tic Indexing ( LSI ) [ 18 ] and K - means clustering ) that can extract semantic themes in text - based idea sets very quickly and efficiently . However , the intrinsic quality of these themes ( e . g . , as measured by correspondence with gold Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than the author ( s ) must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from Permissions @ acm . org . CHI ' 16 , May 07 - 12 , 2016 , San Jose , CA , USA Copyright is held by the owner / author ( s ) . Publication rights licensed to ACM . ACM 978 - 1 - 4503 - 3362 - 7 / 16 / 05 \u2026 $ 15 . 00 DOI : http : / / dx . doi . org / 10 . 1145 / 2858036 . 2858178 Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2717 standard human clusters / categories ) is often relatively low ( certainly lower than human - produced themes ) . Thus , these sensemaking outputs should hypothetically have relatively low value for ideation . In between sit hybrid approaches ( medium cost , medium quality ) that combine human and machine effort [ 39 , 58 , 62 , 63 , 65 ] . Some of these approaches may require optimization of complex machine learning parameters to be successful , limiting their accessibility to large scale ideation platforms . But , relatively simple and accessible workflows are also possible , where humans label initial machine clusterings from algorithms like K - means . Ideation platform developers would benefit greatly from knowing the cost - quality tradeoffs of sensemaking ap - proaches and how to use them at scale with limited re - sources , but prior work has rarely compared these ap - proaches directly . To address this important gap , we ex - plore two main research questions : 1 ) Do abstracted solution paths inspire more and better ideas compared to seeing all raw ideas or no ideas at crowd scale ? 2 ) If so , how does the sensemaking strategy for abstract solution paths\u2014from fully automated to hybrid human - machine to fully manual\u2014affect the quantity and quali - ty of generated ideas ? In an online study , 245 Amazon Mechanical Turk ( mTurk ) participants generated ideas for two innovation problems ( generating ideas for a novel fabric technology and for improving mTurk\u2019s mobile experience ) in one of five conditions : 1 ) no stimulation ( control ) , 2 ) viewing all prior ideas for the problem ( all ideas ) , 3 ) with solution paths extracted from human - labeled paths generated for human - generated clusters of ideas ( human - human ) , 4 ) human - labeled paths generated for machine - generated clusters ( machine - human ) , or 5 ) machine - labeled paths generated for machine - generated clusters ( machine - machine ) . Our main finding is that an off - the - shelf machine approach ( as exemplified by our machine - machine condition ) can yield comparable benefits to a costly fully manual approach ( as exemplified by our human - human condition ) . Contrary to expectations , human - generated solution paths do not improve ideation over either simply showing all ideas or no ideas at all , as measured by fluency ( total number of ideas ) and breadth of ideation ( mean pairwise distance in LSI representation of the solution space ) . However , machine - generated solution paths sometimes improve fluency and breadth of ideation over no ideas ( although at a slight cost to idea quality ) . These findings suggest that large - scale ideation could benefit from automated sensemaking , but more research is needed to better understand the value of human - generated sensemaking in a crowd setting . This paper contributes : 1 ) Empirical findings on the relative value of different sensemaking approaches for ideation 2 ) Evidence that a simple , easy to implement approach to automated sensemaking ( using LSI and k - means clus - tering ) can improve fluency and breadth of ideation . RELATED WORK Effective Collaborative Ideation There is consensus in the literature on creative cognition that seeing what others have thought and / or are currently thinking about the problem can increase people\u2019s ability to generate creative ideas ( i . e . , ideas that are both novel and of high quality ) [ 19 , 21 , 32 , 55 ] . For example , people can generate more creative ideas when they have access to example designs [ 41 , 43 , 53 \u2013 55 ] , draw analogies to past experiences [ 11 , 17 , 33 , 64 ] , or see some ideas from others who are working on the same problem [ 8 , 19 ] . Some studies have also shown that building on others\u2019 ideas not only improves individual creativity , but also maximizes the community\u2019s ability to reach an optimal solution [ 8 , 61 ] . However , these benefits of collaborative ideation can be challenging to realize at crowd scale ( typically hundreds to thousands of ideas ) . At this scale , interesting but statistical - ly rare ideas may be less likely to be noticed and built upon , leading the crowd to focus on common ideas . From a cognitive perspective , effective collaborative ideation depends on being able to attend to and deeply process other ideas [ 35 , 55 ] . But people are limited in their capacity to process information [ 16 , 47 ] , and the number of ideas produced at crowd scale certainly exceeds this capacity . When given too many ideas as potential inspiration , people may stop attending to them , or only build on them in superficial ways [ 35 , 55 ] . Ideally , we would like crowd ideators to focus on the primary task of generating ideas , rather than expending most of their effort making sense of a large volume of ideas in order to extract useful inspiration . Another issue is that the presentation of the ideas in their \u201craw form\u201d , which includes many superficial details , can constrain ideators\u2019 search to ideas closely related to the raw idea [ 34 , 44 , 56 ] . Therefore , we hypothesize that simply exposing all ideas to all contributors , while simple to implement , is not likely to be an effective solution . Higher - level abstracted solution paths that distill essential solution approaches shared by a number of different raw ideas may be a good alternative to raw ideas . A classic example of a solution path in the cognitive science litera - ture is the convergence solution schema ( successfully attack a single target by converging from multiple points if a single , focused attack is not feasible ) , abstracted from a variety of situations ( e . g . , generals attacking a fortress via mined bridges radiating from the fortress , a doctor destroy - ing a tumor with radiation rays without also destroying healthy tissue ) [ 25 ] . Abstractions such as categories can be a powerful way to compress large volumes of information into more manageable chunks [ 12 , 46 ] , greatly reducing the number of \u201cbits\u201d that ideators need to process . This should increase the probability that ideators will actually be able to attend to and benefit from them . Also , by representing Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2718 primarily the \u201cessential\u201d information that helps to organize ideas , such as in schemas [ 25 , 64 ] , abstractions could help reduce fixation on superficial details . Some studies have shown that providing one or a few manually generated and selected abstractions can lead to better creative performance compared to showing ideas in their raw form [ 45 , 60 , 64 ] . However , this manual selection may not always be feasible at crowd scale . In this paper , we extend this prior work by examining the value of viewing many ( ~ 15 - 20 ) abstractions rather than just one or two . Automated Sensemaking Strategies Numerous automated sensemaking methods exist , ranging from relatively mature ( e . g . , well - studied , shown to be robust across a range of settings ) and simple ( i . e . , produces reasonable results without requiring significant tuning of many complex model parameters ) vector - space models like Term - Frequency Inverse - Document Frequency ( TF - IDF ) and LSI [ 18 ] models and clustering algorithms like K - means and agglomerative hierarchical clustering , to more sophisticated methods like probabilistic topic models [ 6 ] . Research on these methods has largely focused on improv - ing correspondence with \u201cgold standard\u201d human models of the items being structured ( e . g . , gold standard human - generated categories [ 9 ] ) , or matching human performance on various benchmark tasks ( e . g . , simple A : B , C : D word analogies [ 48 ] ) . Some work has examined the value of automated sensemaking outputs for complex tasks like ideation [ 20 , 24 ] , intelligence analysis [ 57 ] , and conducting scientific literature reviews [ 13 ] . However , relatively little work has compared the value of automated sensemaking outputs to human - produced outputs for complex tasks in general . One notable exception is a study by Andr\u00e9 et al [ 3 ] , who found that simple TF - IDF sensemaking over academic papers could provide suggestions for papers to attendees at an academic conference that were rated at comparable levels of relevance as suggestions provided from a human - generated sensemaking model ( via partial clustering ) . We are not aware of any related studies in the context of idea - tion . This paper explores the cost - quality tradeoffs involved in selecting automated vs . manual sensemaking approaches . SOLUTION PATH EXTRACTION Our goal is to explore the cost - quality curve for current \u201coff - the - shelf\u201d methods that would be readily available to crowd innovation platforms . Here we describe in more detail each of the three points on the cost - quality curve that we select for comparison : 1 ) manual human - human ( highest cost ) , 2 ) machine - human ( medium cost ) , and 3 ) machine - machine ( lowest cost ) . For comparability , all approaches follow the same general process : raw ideas form initial inputs into sensemaking , which includes a cluster phase \u2014 where similar ideas are grouped together \u2014 followed by a label phase to produce \" solution paths \" \u2013 descriptions of key features shared by multiple ideas in a cluster ( often with fewer concrete details than raw ideas ) . Each of the three approaches is defined by whether humans or machines complete each phase , with the intuition that least human involvement ( machines complete both phases ) would be least costly , while maximal human involvement ( in both phases ) would be most costly . Datasets We extracted solution paths for ideas for two problems : 1 ) new product ideas for a novel \u201cfabric display\u201d ( fabric display problem ) , and 2 ) ideas for improving workers\u2019 experience performing HITs on mTurk on mobile devices ( improve mTurk problem ) . Both idea datasets were assem - bled from idea datasets collected in prior studies ( [ 54 ] for the Fabric Display problem , and [ 40 ] for the Improve Turk problem ) . The ideas in those datasets were collected from mTurk workers , and the authors shared the data with us . We randomly sampled 120 ideas for each problem . We chose 120 ideas because it is small enough to apply best practice manual approaches ( e . g . , affinity diagramming ) that would be difficult for 1000s of ideas , yet large enough to motivate the need for sensemaking and to yield intuitions for scala - bility comparisons . Examples of solution paths extracted by each method are shown in Table 1 . Sensemaking Methods Machine - Machine Solution Path Extraction For this method of extracting solution paths , machines complete both the cluster and label phases of solution extraction . Our goal is to design an automated workflow that closely approximates realistic crowd ideation scenarios . In realistic scenarios , one very rarely has \u201cgold standard\u201d data available to assist with evaluation and tuning of complex model parameters . Therefore , we would like to choose relatively mature models with well - known proper - Method Fabric Display Improve Turk Machine - Machine advertisements billboards create shirts / pants cuffs companies time timer site assigned loading notification Machine - Human use for new locations for advertising send timer alerts Human - Human advanced display of advertisements provide notifications for time - sensitive activity Table 1 . Example solution paths extract by each method for both problems . For comparability , we select examples that are close in semantic meaning . Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2719 ties that can work relatively well \u201cout - of - the - box\u201d without significant parametric tuning . To meet these constraints , we chose to do unsupervised feature identification using a combination of TF - IDF ( widely used in information re - trieval ; no parameter settings required ) and LSI ( widely used in information retrieval ; only choose number of dimensions ) , and clustering using the standard K - means algorithm ( widely applicable ; only choose number of clusters ) . In our workflow , we first identify semantic features in the set of ideas using TF - IDF and LSI , and then use these features to achieve unsupervised clustering of the ideas with K - means . We then automatically obtain labels for the clusters by finding the most informative keywords within each cluster . In the cluster phase , ideas are first tokenized , removing uninformative ( stop ) words , and then weighted using TF - IDF . Each idea then is re - represented as a vector of w TF - IDF weights ( where w is the number words in the corpus of ideas ) . We then use LSI to reduce the dimensionality of the vectors . Based on prior experience clustering brainstorming ideas , our intuition is that somewhere between 15 to 25 patterns is often sufficient to adequately describe emerging solution patterns . Therefore , we set our LSI model to retain 15 , 20 and 25 dimensions . For each of these parameter settings , we then identify solution paths by finding the same number of clusters of ideas ( i . e . , 15 , 20 or 25 ) with the K - means clustering algorithm . We use the LSI dimension weights as features for the ideas . Rather than report the running time ( which would vary by machine , dataset , and implementation of the algorithms ) , we leave it to the reader to examine the well - studied computational complexity properties of LSI and K - means and extrapolate accordingly to their desired settings . As a ballpark estimate , all our clustering runs for the 120 ideas took less than 30 seconds . For the label phase , we again select a method that would not require significant parameter tuning . To do this , we treat each cluster as a document , and compute TF - IDF weights for all words . We then choose the top n words with the highest TF - IDF weights within each cluster . N is deter - mined by the average length of human labels ( minus stopwords ) . The intuition behind this method is to identify words that distinguish between the clusters . Machine - Human Solution Path Extraction For this method , the machines complete the cluster phase , but humans are recruited for the label phase . The cluster phase is the same as the machine - machine method ( i . e . , clusters generated by combination of LSI and K - means ) . In the label phase , the labelers received each of the M clusters of ideas identified in the machine - clustering phase . The labeling task was completed in a Google Document , which contained 1 ) instructions for labeling , 2 ) the problem description , and 2 ) each of the M clusters as unlabeled lists with blank headers . Labelers described clusters by typing a description in the blank headers ( see Fig . 1 , left panel ) . Labelers received the following instructions : \u201cHere is a set of 120 ideas for the problem , divided into clusters of ideas . Each cluster of ideas is meant to represent a solution pattern for the problem . Recall that we would like to provide these patterns as inspiration for future brainstorm - ers who will also work on this problem . Your goal is to write a meaningful description for each cluster . Good descriptions capture the essential shared theme of a group of ideas in an appropriately specific fashion ( i . e . , contain enough detail to be useful to future brainstormers , but are not simple restatements of each idea ) . Ideally , they capture a theme shared by all ideas in their cluster ; in some ( rare ) cases , you may find it difficult to discern the common theme : in this case , please do your best to describe a pattern shared by at least 2 - 3 ideas in the cluster . As a good test , good descriptions can fit well into the template \u2018How can we _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ to solve the problem ? \u2019 . \u201d A set of three research assistants \u2014advanced HCI design students \u2014 served as labelers . Labelers on average took approximately 28 minutes to complete labeling for each problem . This approach represents a midpoint on the cost - quality tradeoff curve , and also allows us to potentially tease apart the value provided by clustering ( which influences the semantics of the labels produced ) and labeling ( which vary in such features as coherence , specificity , and phrasing ) provided by machines and humans . Human - Human Solution Path Extraction For this method , humans performed both cluster and label phases in a single task . The clusterer - labelers received all 120 ideas in a single stack . Each idea was printed out on a slip of paper . Clusterer - labelers then sorted ideas into clusters on a 45\u201d by 45\u201d table , and labeled clusters by writing descriptions on Post - It notes . Figure 1 ( right panel ) shows the physical setup of the task . Clusterer - labelers received a description of the problem as well as the following instructions : \u201cHere is a set of 120 ideas for the problem . Please identify solution patterns in the set of ideas . We would like to provide these patterns as inspiration for future brainstormers who will also work on Figure 1 . Screenshot of human labeling task ( left ) physical setup of human cluster - label task ( right ) Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2720 this problem . Do this by grouping ideas on the whiteboard into clusters that define patterns and writing descriptions of those patterns . Good descriptions capture the essential shared theme of a group of ideas in an appropriately specific fashion ( i . e . , contain enough detail to be useful to future brainstormers , but are not simple restatements of each idea ) . As a good test , good descriptions can fit well into the template \u2018How can we _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ to solve the problem ? \u2019 You may identify clusters as large or as small as you like ( even singleton clusters ) . Please do not create miscellaneous clusters or sort ideas by quality . \u201d A different set of three research assistants \u2014 advanced HCI design students \u2014 performed this task . On average , cluster - ing and labeling a single problem took approximately 52 minutes , significantly longer than just labeling . An average of 22 solution paths were identified for the Fabric Display problem , and 16 for the Improve Turk problem . Note that this task could be performed using digital tools ; however , we chose to implement these steps on paper to have as close a match as possible to existing \u201coff - the - shelf\u201d human sensemaking approaches ( e . g . , affinity diagramming ) . IDEATION EXPERIMENT Overview With extracted solution paths , we then conducted an online ideation experiment to compare the relative ideation value of the different sensemaking approaches , compared to simple exposure to all prior ideas , or no stimulation . Method Participants We recruited 139 mTurk workers ( 42 % female , mean age = 32 . 6 years , SD = 9 . 6 ) for this study . To ensure quality data , all participants had to have approval rates of at least 95 % with at least 100 completed HITs . Participants were paid $ 2 . 00 for about 20 minutes of participation ( on average ) , yielding an average hourly rate of $ 6 / hour . Study Design Participants were randomly assigned to one of 5 conditions : 1 ) unconstrained ( control , N = 21 ) , 2 ) viewing all 120 prior ideas for the problem ( all - ideas , N = 24 ) , viewing 3 ) ma - chine - labeled solution paths generated for machine - generated clusters ( machine - machine : least costly , N = 32 ) , 4 ) human - labeled paths generated for machine - generated clusters ( machine - human : somewhat costly , N = 34 ) , or 5 ) with human - labeled paths generated for human - generated clusterings of ideas ( human - human : most costly , N = 28 ) . Within the path conditions , participants were randomly assigned one of the 3 path sets generated using that solution path extraction method . Brainstorming Task Participants generated ideas for both the Fabric Display and Improve Turk problems . Brainstorming Interface Participants generated ideas using a simple ideation inter - face . Inspirations ( whether ideas or solution paths ) were provided to participants in an \u201cinspiration feed\u201d in the right panel of their interface ( see Figure 2 ) . Participants could \u201cbookmark\u201d particular inspirations that they found helpful . No sorting and filtering options were provided . The limited sorting / filtering available in the all - ideas condition might be considered primitive , but it is actually similar to many existing platforms that might provide rudimentary filtering by very broad , pre - defined categories ( that may be reused across problem ) . Participants in the control condition generated ideas with a simpler ideation interface that removed the inspiration feed . Procedure After providing informed consent , participants experienced a brief tutorial to familiarize themselves with the interface . Embedded within the tutorial was an alternative uses task ( where participants were asked to think of as many alterna - tive uses as possible for a bowling pin ) . Participants then Figure 2 . Screenshot of ideation interface . Actual inspirations from the machine - machine condition are shown . Participants enter ideas on the left , and view inspirations on the right . Participants can star inspirations they find useful . Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2721 generated ideas for both the Fabric Display and Improve Turk problems . Participants were given 8 minutes to work on each problem , and the order of the problems was ran - domized across participants . Participants in the inspiration conditions received the following instructions regarding the inspirations : \u201cBelow are some inspirations to boost your creativity . Feel free to create variations on them , elaborate on them , recombine them into new ideas , or simply use them to stimulate your thinking . If you find an inspiration to be helpful for your thinking , please let us know by clicking on the star button ! This will help us provide better inspira - tions to future brainstormers . \u201d After completing both problems , participants then completed a brief survey with questions about demographics and the participants\u2019 experi - ences during the task . Measures Conceptually , we distinguish process ( fluency and breadth of search ) from outcome ( novelty , quality ) measures for exploring the impact of sensemaking on ideation . This distinction follows previous design ideation research [ 52 ] . Theoretically , our process measures also map to Guilford\u2019s [ 28 ] notion of divergent thinking as a process basis for creativity , while our outcome measures map to standard theoretical notions of creative products as a combination of novelty and usefulness / practicality [ 1 , 31 , 50 , 51 ] . Fluency : Number of Ideas Fluency was operationalized as the number of ideas gener - ated by a participant for a given problem . Breadth of Search in Solution Space We used LSI to characterize the nature of participants\u2019 search through the solution space . While LSI can be more accurate with longer texts ( our raw ideas are 8 - 10 words , on average ) , researchers have successfully used LSI to model creativity with similar - length texts [ 23 , 30 ] . To maximize the accuracy of the model , for each problem we enriched the model with the full set of ideas previously collected from mTurk workers for each of the two problems ( 1 , 354 for Fabric Display , and 2 , 287 for Improve Turk ) . Thus , the total sizes of the training corpora for the LSI models were 2 , 354 ideas for Fabric Display ( prior ideas plus 1 , 000 ideas from our experiment ) and 3 , 403 for Improve Turk ( prior ideas plus 1 , 116 ideas from our experiment ) . We used the same procedure to build the LSI space as with the first step of machine clustering ( i . e . , weight words with TF - IDF before estimating LSI ) , except that we retained 200 dimensions ( in keeping with prior rules of thumb for larger datasets [ 42 ] ) . Breadth was operationalized as the mean pairwise distance between a given participant\u2019s ideas . Higher mean pairwise distance indicates that participants\u2019 ideas are sampled from very diverse regions of the solution space . Distances were calculated by subtracting pairwise cosines from 1 , yielding distance scores between 0 ( semantically identical ) and 1 ( semantically very different ) . Novelty and Quality of Ideas To explore impact on novelty and quality , we obtained ratings of novelty and quality for ideas for the Improve Turk problem only , due to concerns about mTurk workers\u2019 ability to provide valid ratings for the Fabric Display problem . MTurk workers are suitable judges for the Im - prove Turk problem , since they have first - hand expertise in the domain of worker experience on mTurk , and are also potential users of mTurk mobile products . 414 workers from mTurk rated the ideas for novelty and quality . Novelty was operationalized as the degree to which an idea was novel , ranging from a scale of 1 ( Extremely Obvious ) to 7 ( Extremely Novel ) . Quality was operational - ized as the degree to which an idea would be useful for solving the problem , assuming it was implemented ( i . e . , separating out concerns over feasibility ) , ranging from a scale of 1 ( Not Useful at All ) to 7 ( Extremely Useful ) . Each worker rated a random sample of approximately 20 ideas . While the raw inter - rater agreement was relatively low ( Krippendorff\u2019s alpha = . 23 and . 25 , respectively ) , the overall aggregate measure had acceptable correspondence with each judges\u2019 intuitions . Computing correlations between each judges\u2019 ratings and the overall aggregate score yielded average correlations of . 52 for novelty and . 58 for quality . To deal with potential differences in usage of High Quality Low Quality High novelty ( 1 . 04 , 1 . 51 ) Allow demographic info to be filled out automatically so it doesn ' t have to be done each time ( 1 . 52 , 1 . 01 ) Virtual keyboard that allows you to have a row of buttons for only the keys that are used in the task , instead of finding them on the normal keyboard ( 1 . 22 , - 1 . 09 ) Fingerprint Captchas are possible . ( 1 . 10 , - 1 . 74 ) music to focus worker Low novelty ( - 0 . 87 , 1 . 04 ) Grey out HITS that have been done before and cannot be repeated ( - 1 . 00 , 1 . 16 ) make it easy to find good hits ( - 1 . 11 , - 1 . 38 ) Be able to complete more hits ( - 1 . 58 , - 1 . 83 ) Have direct contact information such as a phone number clearly listed for MTurk workers . Table 2 . Example ideas at each combination of low and high novelty and quality for the Improve Turk problem . Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2722 the rating scale across raters ( e . g . , some might only use the upper end of the scale ) , we normalized scores within raters ( i . e . , difference between rating and mean rating provide by rater , divided by the standard deviation of the raters\u2019 ratings ) . Table 2 shows examples of low and high novelty and quality ideas in the data . Inspiration Use : Number of Bookmarked Inspirations We also measured the degree to which participants found inspirations to be useful by counting the number of inspira - tions that were bookmarked by each participant . Control Measures Our primary control measure is participants\u2019 performance for the baseline fluency task ( i . e . , number of bowling pin alternative uses generated ) . This measure captures aspects of participants\u2019 base level of creative fluency ( as a proxy for individual creativity [ 27 ] ) , as well as aspects of motiva - tion and conscientiousness ; all of these factors are expected to influence creative performance , and are therefore ac - counted for in our analyses by including baseline fluency as a covariate predictor in our statistical models . Results Participants generated a total of 2 , 116 ideas across the two problems , across conditions ( 1 , 000 ideas for Fabric Display and 1 , 116 for Improve Turk ) . Machine - Generated Paths Stimulate More Ideas Table 3 shows the means and standard errors for each of the conditions . We first estimated separate ANCOVAs with condition as a main effect and baseline fluency as a control covariate for each of the two problems . For Fabric Display , there was no statistically significant overall effect of condi - tion , F ( 4 , 133 ) = 1 . 11 , p = 0 . 35 . However , planned contrasts suggest that both the all - ideas ( model beta = 1 . 9 , p = 0 . 08 , Cohen\u2019s d = 0 . 49 ) and machine - machine conditions ( be - ta = 1 . 8 , p = 0 . 08 , d = 0 . 42 ) trend towards more ideas than control ( see Table 3 , left column ) . That is , adjusting for baseline fluency , participants who saw all prior ideas or machine - generated solution paths generated about 2 more ideas than participants who received no stimulation . In terms of standardized effect sizes , the Cohen\u2019s d of about 0 . 5 corresponds to a medium - sized effect according to Cohen\u2019s [ 15 ] classification of effect sizes in the behavioral sciences . For Improve Turk , there was no significant overall effect of condition , F ( 4 , 133 ) = 1 . 40 , p = 0 . 23 . However , the machine - machine condition did display a significant trend towards more ideas than control , beta = 2 . 5 , p = 0 . 04 , d = 0 . 41 ( see Table 3 , right column ) . Similar to the Fabric Display problem , adjusting for baseline fluency , participants who saw machine - generated paths generated about 2 more ideas than participants who received no stimulation . Machine - Generated Paths Increase Breadth of Search Table 4 shows the means and standard errors for each of the conditions . Baseline fluency was not significantly correlat - ed with breadth of search in either problem ( r = . 06 , p = . 47 for Fabric Display , and r = . 03 , p = . 76 for Improve Turk ) . Therefore , we estimated separate ANOVAs with condition as the only factor for each of the two problems . For the fabric display problem , there was a marginally significant overall effect of condition , F ( 4 , 133 ) = 2 . 12 , p = 0 . 08 . Planned contrasts suggest that both the machine - machine ( be - ta = 0 . 06 , p = . 01 , d = 0 . 65 ) and machine - human paths ( be - ta = 0 . 02 , p = . 02 , d = 0 . 57 ) lead to significantly more breadth of search than control , while human - human paths are marginally significantly better than control ( beta = 0 . 05 , p = . 05 , d = 0 . 52 ) . All effect sizes are medium - sized , by Cohen\u2019s classification . For the Improve Turk problem , the overall main effect of condition is not statistically signifi - cant , F ( 4 , 139 ) = 1 . 53 , p = 0 . 20 . However , planned contrasts suggest that both all - ideas ( beta = 0 . 07 , p = . 04 , d = 0 . 72 ) and machine - machine conditions ( beta = 0 . 06 , p = . 04 , d = 0 . 57 ) are better than control . The all - ideas effect is close to \u201clarge\u201d , by Cohen\u2019s classification ( the suggested cut - off is 0 . 8 ) , while the machine - machine effect is medium - sized . Breadth of Search Fabric display Improve Turk Condition M ( SE ) M ( SE ) Control 0 . 89 ( 0 . 02 ) 0 . 81 ( 0 . 03 ) All - Ideas 0 . 93 ( 0 . 02 ) 0 . 89 ( 0 . 03 ) * Machine - Machine 0 . 95 ( 0 . 02 ) * * 0 . 89 ( 0 . 02 ) * Machine - Human 0 . 95 ( 0 . 02 ) * 0 . 86 ( 0 . 02 ) Human - Human 0 . 94 ( 0 . 02 ) m 0 . 86 ( 0 . 03 ) * p < . 05 vs . control , m p < . 10 vs control Table 4 . External stimulation increases breadth of search by relative to control condition , for both problems . Number of ideas Fabric display Improve Turk Condition M ( SE ) M ( SE ) Control 6 . 46 ( 0 . 79 ) 7 . 13 ( 0 . 93 ) All - Ideas 8 . 40 ( 0 . 77 ) m 8 . 32 ( 0 . 91 ) Machine - Machine 8 . 29 ( 0 . 67 ) m 9 . 59 ( 0 . 78 ) * Machine - Human 7 . 56 ( 0 . 63 ) 7 . 43 ( 0 . 75 ) Human - Human 7 . 30 ( 0 . 68 ) 8 . 13 ( 0 . 81 ) * p < . 05 vs . control , m p < . 10 vs control Table 3 . Machine - generated paths increase number of ideas relative to control condition . Means are adjusted for baseline fluency . Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2723 Solution Paths Do not Impact Novelty of Ideas Table 5 shows the means and standard errors for each of the conditions . Baseline fluency was not significantly correlat - ed with mean novelty . Therefore , we estimated an ANOVA with condition as main effect for mean novelty of ideas . There was no main effect of condition on mean novelty of ideas , F ( 4 , 131 ) = 0 . 79 , p = 0 . 53 . Machine - Generated Paths Reduce Quality of Ideas Table 5 shows the means and standard errors for each of the conditions . Baseline fluency was not significantly correlat - ed with mean quality . Therefore , we estimated an ANOVA with condition as main effect for mean quality of ideas . There was a marginally significant main effect of condition on mean quality of ideas , F ( 4 , 129 ) = 2 . 15 , p = . 08 . Planned contrasts suggested that the mean quality of ideas in the machine - machine ( M = \u2013 0 . 08 , SE = 0 . 06 ) and machine - human conditions ( M = \u2013 0 . 02 , SE = 0 . 05 ) were of signifi - cantly lower quality than in the control condition ( M = 0 . 17 , SE = . 07 ) , beta = - . 25 , p = . 01 , d = 0 . 69 , and beta = \u2013 . 18 , p = . 04 , d = 0 . 54 . Ideas in the human - human condition were of marginally lower quality ( M = 0 . 01 , SE = 0 . 06 ) than in the control condition ( beta = - . 16 , p = . 09 , d = 0 . 47 ) . All effects are approximately medium - sized , by Cohen\u2019s classification . Equal Number of Good Ideas Across Conditions Given the reduction in quality associated with machine labels , we wondered about the impact of those paths on aggregate creativity ( a combination of both novelty and quality ) . Ideation platforms might accept a slight drop in mean quality as long as overall creativity does not suffer . We follow Reinig et al [ 49 ] to measure creative output as the number of good ideas , where a good idea is defined as an idea with novelty and quality scores that are both above the mean ( see Table 2 , upper right quadrant , for examples ) . An ANCOVA controlling for baseline fluency showed no main effect of condition , F ( 4 , 128 ) = 0 . 98 , p = . 42 ; further , the trends approximately track the statistical patterns for total number of ideas , suggesting that participants across condi - tions are generating good ideas at a fairly constant rate . Indeed , when examining the proportion of good ideas as a dependent measure , an ANCOVA again shows no signifi - cant differences across conditions , F ( 4 , 128 ) = . 61 , p = . 66 . DISCUSSION In this research , we examine the relative value of different approaches to inspiring crowd ideators with prior ideas . We empirically test whether abstracted solution paths ( generat - ed by a range of sensemaking methods ) can enable ideators to better benefit from prior ideas ( as measured by impact on fluency , breadth , and novelty and quality of ideas ) . We also empirically explore how the ideation value of solution paths varies with the cost of the sensemaking strategy that pro - duced them . Our study yields two main sets of findings , which we discuss in turn . No Consistent Benefit of Solution Paths The first main set of findings is that solution paths do not consistently improve ideation more than simply showing all raw ideas to ideators . With respect to fluency , for the Improve Turk problem , machine - machine paths ( but not machine - human , human - human , or all - ideas ) improved fluency over control ; however , for the Fabric Display problem , all - ideas and machine - machine paths both improved fluency . With respect to breadth of search , solution paths ( regardless of source ) improved breadth of search over control for the Fabric Display problem ; howev - er , for the Improve Turk problem , only all - ideas and machine - machine paths improved breadth over control . None of the solution paths conditions improved novelty over control , and some ( human - machine and machine - machine ) even reduced quality relative to control , while all - ideas did not . There is a range of possible explanations for why solution paths did not improve ideation in our experiment . First , perhaps the solution paths reduced quality because they were potentially derived from both bad and good ideas ( recall that we randomly sampled the initial set of 120 ideas from the prior datasets ) . However , it seems unlikely to have been the main driving factor . While one participant who saw all ideas did complain in the open - ended portion of our post - task surveys that there were many bad ideas , nobody voiced this complaint in the paths conditions . Further , one Mean Novelty Mean Quality Condition M ( SE ) M ( SE ) Control 0 . 04 ( 0 . 07 ) 0 . 17 ( 0 . 07 ) All - Ideas 0 . 02 ( 0 . 07 ) 0 . 07 ( 0 . 07 ) Machine - Machine 0 . 06 ( 0 . 06 ) - 0 . 09 ( 0 . 06 ) * * Machine - Human 0 . 03 ( 0 . 06 ) - 0 . 02 ( 0 . 06 ) * Human - Human - 0 . 09 ( 0 . 07 ) 0 . 01 ( 0 . 07 ) m * p < . 05 vs . control , m p < . 10 vs control Table 5 . External stimulation does not impact novelty of ideas , but machine - generated paths reduces quality relative to control condition . Figure 3 . Participants generate similar numbers of good ideas across conditions , adjusted for baseline fluency . Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2724 participant ( in the all - ideas condition ) specifically noted that , \u201ca lot of people had some good ideas about different features for a Mturk app . \u201d Indeed , ideas that were more similar to the initial 120 raw ideas were considered to be of slightly higher quality : we found a small but significantly positive correlation between LSI similarity of ideas vs . initial 120 raw ideas and quality ratings , r = . 07 , p = . 01 . Nevertheless , it is possible that the presence of even one or two bad ideas might be sufficient to contribute to fixation . Second , it could be that that there were too many inspira - tions to sift through . However , 15 - 25 inspirations seems like a manageable enough number of items to scan through relatively quickly and find interesting ideas to build on . We also specifically designed the system to be able to fit all of the inspirations on one screen in the paths conditions ( so that there would not need to be much scrolling ) . Indeed , a number of participants in the paths conditions appreciated that they could just glance over to see inspirations , while many participants in the all - ideas condition complained that there were too many inspirations to consider at once . A more interesting alternative explanation might be that , rather than providing bad solutions , the stimuli were steer - ing participants away from good solutions , due to a desire to not repeat ideas ( despite our instructions saying it was ok to build on or recombine inspirations ) . Indeed , some partic - ipants said in the survey that they were influenced by the inspirations in this way . For example , one participant ( in the machine - human condition ) said , \u201cI could just read them and see if an idea I had had already been provided and if it was I would move on to the next idea . \u201d One partic - ipant ( in the same condition ) even said , \u201cI would come up with an idea and then notice it was already listed on the inspiration list , so I felt discouraged about that . \u201d Another participant ( in the machine - machine condition ) said that it was challenging \u201ctrying not to repeat the inspirations . The first inspiration was about a movie theater , which coinci - dentally , was also similar to my first idea . \u201d If this happened often enough , participants might have searched more broadly , but away from any good ideas that happened to be represented in the solution paths . In fact , perhaps the relative ease of gaining a broad sense of the solution space with solution paths\u2014rather than with the 120 raw ideas\u2014 could explain why quality was reduced only in the paths conditions . Relatedly , participants might have felt over - whelmed by the prospect of adding something new to the existing solution space . One participant in the machine - human condition said , \u201cThere were so many inspirations for the second brainstorming task that it was hard to come up with anything new to add . \u201d Overall , these findings suggest that it may not be straight - forward to translate the benefits of abstracted solution paths from individual and group settings into a crowd setting . For example , if providing tens of abstracted solution paths at once as potential inspirations could be overwhelming , alternative delivery mechanisms , such as on - demand delivery of individual inspirations [ 10 , 54 ] , or focusing ideators on a single theme [ 22 , 45 ] . Automated Sensemaking Can Improve Ideation The second main set of findings concerns the value of machine - generated paths . In our data , the only form of stimulation that provided consistent benefits was machine - generated solution paths , which improved fluency and breadth across both problems . However , this increase in fluency and breadth came at the cost of reduced quality of ideas . We did also find , however , that the reduction in quality did not hamper the production of good ideas ( i . e . , ideas that are both novel and of high quality ) . Thus , our findings suggest that machine - generated paths can improve divergent thinking ( generating many possible ideas ) , but not necessarily creativity ( generating high - quality , novel ideas ) . One explanation for why these solution paths ( though hypothesized to be of low intrinsic quality ) , might have benefited ideation is that participants were essentially leveraging them as keyword clouds . For example , one participant in the Machine - Machine condition said , \u201cThe inspirations seemed nonsensical when read as a whole , but one or two words would catch my attention and spark an idea . For example , I think one of the ones for the fabric said \" bathroom \" or \" towel \" or something like that . That gave me an image of a shower curtain and having video play on the shower curtain . The inspirations were jumping off points . \u201d Another participant said , \u201cI would list as many ideas as possible and then browse quickly through key words and run with whatever came to mind . Often I would combine words and that would help me think of an original idea . \u201d In theory , the keywords generated using our ma - chine - machine workflow should identify words that can best discriminate between major patterns of solutions in the set of ideas . It would be valuable to test whether these paths benefit ideators more than simpler ways of generating keyword clouds ( e . g . , frequency - based ) . This set of findings suggests that the relationship between cost and quality ( in terms of value for ideation ) might not be simply linear , monotonic , or even positive . From a practical standpoint , large - scale ideation platforms could gain value from employing very simple automated sense - making methods ( such as the workflow we used in this study ) . These methods might provide the most value in the early stages of the innovation process , where the focus is on quickly exploring as much of the space as possible before focusing in on more promising solution approaches , and some reduction in quality might be acceptable in exchange . Our results suggest that , accounting for our fluency and breadth effects , platforms that use machine paths may need fewer people to achieve comparable coverage of the solu - tion space ( vs . not providing any exposure to prior ideas ) . FUTURE WORK Our finding that solution paths did not consistently benefit ideators ( over providing all ideas or no ideas ) runs counter to theory , and suggests caution when using solution paths in Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2725 a crowd setting . It will be important for future research to test the potential explanations we explored for the lack of ( and even slightly negative ) effect of solution paths , partic - ularly since many of these factors are likely to be present in collective innovation settings ( e . g . , large number of poten - tial inspirations , presence of some bad ideas , pressure to come up with original ideas rather than build on ideas ) . It would also likely be useful to investigate task designs and workflows that could overcome some of those potential challenges . For example , could rephrasing inspirations as questions ( e . g . , \u201cHow can we\u2026 ? \u201d ) mitigate participants\u2019 tendency to move away from solution paths ? Could we also intelligently divide up solution paths to different portions of the crowd such that they can explore a smaller set of solution path in depth and would this help mitigate potential cognitive load issues from seeing many inspirations ? Would it be useful to integrate evaluation into the sense - making process to more quickly weed out bad ideas ? Future efforts to enable more collaborative large - scale ideation could benefit from examination of these questions . Also , in this study we used a fully manual approach to human sensemaking . But , methods exist for sensemaking using distributed human computation [ 2 , 3 , 14 , 26 ] . Future work might fruitfully explore how the value of human sensemaking for crowd ideation might not only depend on task design and workflow factors , but whether such distrib - uted workflows could provide similar benefits . Finally , future work can explore the generality of these findings . Our findings were similar across the two problems we examined ; thus , our results seem robust to domain expertise effects . Also , because our participants were paid mTurkers , our results might map on to other reward - motivated situations ( e . g . , contests ) . However , we are uncertain if they generalize to volunteer communities . Participant motivation may be an important moderating effect in these settings , particularly since the different sensemaking approaches impose varying degrees of cogni - tive load on ideators ( e . g . , less cognitive load for machine - generated paths compared to human - generated paths ) . CONCLUSION In this paper , we examined the ideation value of different approaches to sensemaking over prior ideas , using an online ideation experiment that compares ideation under no stimulation , exposure to all ideas , or abstracted solution paths from fully automated , machine - human hybrid , or fully manual sensemaking approaches . Our results suggest that simple automated sensemaking methods can provide some value ( e . g . , increased fluency , breadth of search ) to large - scale ideation platforms . Our results also motivate further research on how to best enable crowd ideators to benefit from ( human ) sensemaking outputs . ACKNOWLEDGEMENTS We thank all participants , especially Amazon Mechanical Turk workers who provided feedback on our experimental protocol , our volunteer clusterers and labelers , and Michelle Tai for help with data analysis . This research was supported by NSF grants # 1208382 and # 1122206 and a Google Faculty Award . REFERENCES 1 . Teresa M . 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Crowdsourcing and Creation : Large - scale Ideas and Content Production # chi4good , CHI 2016 , San Jose , CA , USA 2728", + "kamrahHowDiverseInitial2023": "Eesh Kamrah Department of Mechanical Engineering , University of Maryland , College Park , MD 20742 e - mail : kamrah @ umd . edu Seyede Fatemeh Ghoreishi Department of Civil and Environmental Engineering & Khoury College of Computer Sciences , Northeastern University , Boston , MA 02115 e - mail : f . ghoreishi @ northeastern . edu Zijian \u201c Jason \u201d Ding College of Information Studies , University of Maryland , College Park , MD 20742 e - mail : ding @ umd . edu Joel Chan College of Information Studies , University of Maryland , College Park , MD 20742 e - mail : joelchan @ umd . edu Mark Fuge 1 Department of Mechanical Engineering , University of Maryland , College Park , MD 20742 e - mail : fuge @ umd . edu How Diverse Initial Samples Help and Hurt Bayesian Optimizers Design researchers have struggled to produce quantitative predictions for exactly why and when diversity might help or hinder design search efforts . This paper addresses that problem by studying one ubiquitously used search strategy \u2014 Bayesian optimization ( BO ) \u2014 on a 2D test problem with modi \ufb01 able convexity and dif \ufb01 culty . Speci \ufb01 cally , we test how providing diverse versus non - diverse initial samples to BO affects its performance during search and introduce a fast ranked - determinantal point process method for comput - ing diverse sets , which we need to detect sets of highly diverse or non - diverse initial samples . We initially found , to our surprise , that diversity did not appear to affect BO , neither helping nor hurting the optimizer \u2019 s convergence . However , follow - on experiments illuminated a key trade - off . Non - diverse initial samples hastened posterior convergence for the underlying model hyper - parameters \u2014 a model building advantage . In contrast , diverse initial samples accelerated exploring the function itself \u2014 a space exploration advan - tage . Both advantages help BO , but in different ways , and the initial sample diversity directly modulates how BO trades those advantages . Indeed , we show that \ufb01 xing the BO hyper - parameters removes the model building advantage , causing diverse initial samples to always outperform models trained with non - diverse samples . These \ufb01 ndings shed light on why , at least for BO - type optimizers , the use of diversity has mixed effects and cautions against the ubiquitous use of space - \ufb01 lling initializations in BO . To the extent that humans use explore - exploit search strategies similar to BO , our results provide a testable conjecture for why and when diversity may affect human - subject or design team experiments . [ DOI : 10 . 1115 / 1 . 4063006 ] Keywords : design of experiments , design optimization , machine learning , metamodeling 1 Introduction and Related Work One open question within design research is when or under what conditions providing diverse stimuli or starting solutions to either humans or algorithms can improve their designs \u2019 \ufb01 nal performance . Researchers have struggled to produce quantitative predictions or explanations for exactly why and when diversity might help or hinder design search efforts . In studies of human designers or teams , there have been numerous empirical results on the effect of diverse stimuli or sets of stimuli on designers , typically referred to under the topic of design \ufb01 xation ( for recent reviews , see Refs . [ 1 , 2 ] ) . In general , available empirical results are mixed and it is dif - \ufb01 cult to quantitatively predict , for a new problem or person , whether or not diversity in problem stimuli will or will not help . For instance , there are a number of empirical demonstrations of positive effects of example diversity on novelty and diversity of ideas [ 3 \u2013 5 ] , but substantially more mixed results on the effects of diversity on solution quality , with some observations of positive effects [ 6 \u2013 8 ] , some null or contingent effects [ 4 , 9 \u2013 14 ] , and even some negative effects on solution quality [ 15 , 16 ] . Likewise , in research focused purely on optimization , common academic and industrial practice initializes search algorithms with different strategies like Latin hypercube sampling ( LHS ) [ 17 ] and others in an attempt to \u201c \ufb01 ll \u201d or \u201c cover \u201d a space as uniformly as pos - sible [ 18 ] or via quasi - random methods [ 19 \u2013 21 ] . Some methods build diversity - encouraging loss functions directly into their core search algorithms , such as in common meta - heuristic optimizers [ 22 ] such as particle swarm optimization , simulated annealing , and genetic algorithms , with one of the most well - known diversity - inducing ones being the non - dominated sorting Genetic Algorithm - II ( NSGA - II ) [ 23 ] . For Bayesian optimization ( BO ) spe - ci \ufb01 cally , a common strategy is to build diversity directly into the acquisition function used in sampling new points from the Gaussian process ( GP ) posterior [ 24 ] . As with human - subjects experiments , the precise effect of diversity on optimization performance is often problem dependent [ 22 ] and dif \ufb01 cult to predict a priori . Nev - ertheless , optimization practitioners take these steps to improve initial sample diversity with the hope that the optimizer will con - verge faster or \ufb01 nd better global optima . But is encouraging initial diversity in this way always a good idea ? If so , when and why is it good ? Are there any times or con - ditions when diversity might hurt rather than help our search for good designs ? 1 Corresponding author . Contributed by the Design Automation Committee of ASME for publication in the J OURNAL OF M ECHANICAL D ESIGN . Manuscript received August 31 , 2022 ; \ufb01 nal manuscript received July 13 , 2023 ; published online August 29 , 2023 . Assoc . Editor : Douglas Allaire . Journal of Mechanical Design NOVEMBER 2023 , Vol . 145 / 111703 - 1 Copyright \u00a9 2023 by ASME D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 ( Spoiler Alert : Yes , it can \u2014 see Sec . 4 for how and Sec . 6 for why . ) To address the above questions , this paper studies one type of commonly used search strategy \u2014 BO \u2014 and how the diversity of its initialization points affects its performance on a search task . We uncover a fascinating dance that occurs between two competing advantages that initial samples endow upon BO \u2014 a model building versus space exploration advantage that we de \ufb01 ne later \u2014 and how the initial samples \u2019 diversity directs the choreography . While the fundamental reason for this interplay will later appear straightfor - ward ( and perhaps even discernible through thought experiments rather than numerical experiments ) , it nevertheless \ufb02 ies in the face of how most practitioners initialize their BO routines or conduct optimal experimental design studies . It also posits a testable prediction about how to induce greater effects of diversity on novice human designers or the conditions under which there may be mixed or even negative effects ( see Sec . 6 ) . Before describing our particular experiment and results , we will \ufb01 rst review why BO is a meaningful and generalizable class of search algorithm to use , as well as past work that has tried to under - stand how diversity affects search processes such as optimization . Why model design search as Bayesian optimization ? While this paper addresses only BO , this is an important algorithm in that it plays an out - sized role within the design research and optimization community . For example , BO underlies a vast number of industri - ally relevant gradient - free surrogate modeling approaches imple - mented in major design or analysis packages , where it is referred to under a variety of names , including Kriging methods or meta - modeling [ 25 , 26 ] . Its use in applications of computationally expen - sive multidisciplinary optimization problems is , while not unilateral [ 27 ] , quite widespread . Likewise , researchers studying human designers often use BO as a proxy model [ 28 ] to understand human search , due to the interplay between exploration and exploi - tation that lies at the heart of most BO acquisition functions like expected improvement ( EI ) . More generally , there is a robust history of fruitful research in cognitive science modeling human cognition as Bayesian processing [ 29 ] , such as concept learning in cognitive development [ 30 ] , causal learning [ 31 ] , and analogical reasoning [ 32 ] . While the bulk of BO - related papers focus on new algorithms or acquisition functions , few papers focus on how BO is initialized , preferring instead the general use of space - \ufb01 lling initializations that have a long history in the \ufb01 eld of optimal experiment design [ 27 ] . In contrast , this paper shows that in certain situations that faith in space - \ufb01 lling designs might be misplaced , particularly when the BO kernel hyper - parameters are adjusted or \ufb01 t during search . What does it even mean for samples to be diverse ? As a practical matter , if we wish to study how diverse samples impact BO , we face a subtle but surprisingly non - trivial problem : how exactly do you quantify whether one set of samples is more or less diverse than another ? This is a set - based ( i . e . , combinatorially large ) problem with its own rich history too large to cover extensively here , however our past work on diversity measurement [ 33 \u2013 35 ] , compu - tation [ 36 ] , and optimization [ 37 ] provides further pointers for inter - ested readers , and in particular the thesis of Ahmed provides a good starting point for the broader literature and background in this area [ 38 ] . For the purposes of understanding how this paper relates to exist - ing approaches , it suf \ufb01 ces to know the following regarding common approaches to quantifying diversity : ( 1 ) most diversity measure - ment approaches focus on some variant of a hyper - volume objective spanned by the set of selected points ; ( 2 ) since this measure depends on a set rather than individual points , it becomes combinatorially expensive , necessitating fast polynomial - time approximation , one common tool for which is a determinantal point process ( DPP ) [ 39 ] ; however , ( 3 ) while sampling the most diverse set via DPPs is easy , sampling percentile sets from the DPP distribution to get the top 5 % , median , or lowest 5 % of diverse sets becomes exceed - ingly slow for a large sample pool . In contrast , for this paper , we created a faster DPP - type sampling method to extract different percentiles of the distribution without actually needing to observe the entire DPP distribution and whose sampling error we can bound using concentration inequalities . Section 2 provides further mathematical background , including information on DPP hyper - parameters and how to select them intel - ligently , and the Supplementary Material on the ASME Digital Collection provides further algorithmic details . With an understand - ing of diversity distribution measures in hand , we can now address diversity \u2019 s speci \ufb01 c effects on optimization more generally . How does diversity in initial inputs affect optimizers ? While there are a number of papers that propose either different initialization strategies or benchmarking of existing strategies for optimization , there is limited prior work addressing the direct effect of initial sample diversity . For general reviews and benchmarking on how to initialize opti - mizers and the effects of different strategies , papers such as Refs . [ 20 , 22 ] compare initialization strategies for particular optimizers and quantify performance differences . An overall observation across these contributions is the inability of a single initialization method to improve performance across functions of varying com - plexity . These studies also do not directly measure or address the role of sample diversity directly , only noting such behavior as it cor - relates indirectly with the sampling strategy . A second body of work tries to customize initialization strategies on a per - problem basis , often achieving faster convergence on domain - speci \ufb01 c problems [ 18 , 19 , 40 \u2013 42 ] . While useful in their designed domain , these studies do not directly address the role of diversity either . In contrast , this paper addresses diversity directly using properties of BO that are suf \ufb01 ciently general to apply across multiple domains and applications . Lastly , how to initialize optimizers has garnered new interest from the machine learning community , for example , in the initial settings of weights and biases in a neural network and the down - stream effects on network performance [ 43 , 44 ] . There is also general interest in how to collect diverse samples during learning , either in an active learning [ 45 ] or reinforcement learning context [ 46 , 47 ] ; however , those lines of work address only diversity throughout data collection , rather than the impact of initial samples considered in this paper . What does this paper contribute beyond past work ? This paper \u2019 s speci \ufb01 c contributions are : ( 1 ) To compute diversity : we describe a fast DPP - based diversity scoring method for selecting diverse initial examples with a \ufb01 xed size k . Any set of size k with these initial examples can be then used to approximate the percentile of diversity that the set belongs to . This method requires selecting a hyper - parameter relating to the DPP measure . We describe a principled method for selecting this parameter in Sec . 2 . 1 and provide numerical evidence of the improved sampling performance in the Supplementary Material . Compared to prior work , this makes percentile sampling of DPP distribu - tions computationally tractable . ( 2 ) To study effects on BO : we empirically evaluate how diverse initial samples affect the convergence rate of a Bayesian opti - mizer . Section 4 \ufb01 nds that low diversity samples provide a model building advantage to BO while diverse samples provide a space exploration advantage that helps BO con - verge faster . Section 5 shows that removing the model build - ing advantage makes having diverse initial samples uniformly better than non - diverse samples . 2 We will next describe our overall experimental approach and common procedures used across all three of our main experiments . 2 For grammatical simplicity and narrative \ufb02 ow , we will use the phrase \u201c non - diverse \u201d throughout the paper to refer to cases where samples are taken from the 5th percentile of diverse sets \u2014 these aretechnically \u201c low - diversity \u201d rather thanbeing abso - lutely \u201c non - diverse \u201d which would occur when all points in the set are identical , but we trust that readers can keep this minor semantic distinction in mind . 111703 - 2 / Vol . 145 , NOVEMBER 2023 Transactions of the ASME D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 We will introduce individual experiment - speci \ufb01 c methods only when relevant in each separate experiment section . To fully repro - duce the below results , the link in the footnote provides the code associated with this paper . 3 2 Overall Experimental Approach This section will \ufb01 rst describe how we compute diverse initial samples , including how we set a key hyper - parameter that controls the DPP kernel needed to measure sample set diversity . It then brie \ufb02 y describes the controllable 2D test problem that we use in our experiments . It ends with a description of how we setup the BO search process and the hyper - parameters that we study more deeply in each individual experiment . 2 . 1 Measuring and Sampling From Diverse Sets Using Determinantal Point Processes . As mentioned above , we measure diversity of a set of points using DPP , which get their name from the fact that they compute the determinant of a matrix referred to as an L - ensemble ( as seen in Eq . ( 1 ) ) that correlates with the volume spanned by a collection or set of samples ( Y ) taken from all possible sets ( Y ) given a diversity / similarity ( feature ) metric . P ( L Y ) \u221d det ( K ( L Y ) ) ( 1 ) Here , L is the ensemble de \ufb01 ned by any positive semi - de \ufb01 nite matrix [ 39 ] , and K is the kernel matrix . For sampling diverse examples , this positive semi - de \ufb01 nite matrix is typically chosen as a kernel matrix ( K ) that de \ufb01 nes the similarity measure between pairs of data points . For this paper , we use a standard and commonly used similarity measure de \ufb01 ned using a radial basis function ( RBF ) kernel matrix [ 48 ] . Speci \ufb01 cally , each entry in L Y for two data points with index i and j is [ L Y ] i , j = exp \u2212 \u03b3 \u00b7 (cid:2) x i \u2212 x j (cid:2) 2 (cid:1) (cid:3) ( 2 ) The hyper - parameter \u03b3 in the DPP kernel can be set in the interval ( 0 , \u221e ) and will turn out to be quite important in how well we can measure diversity . The next section explores this choice in more depth , but to provide some initial intuition : set \u03b3 too high and any selection of points looks equally diverse compared to any other set , essentially destroying the discriminative power of the DPP , while setting \u03b3 too low causes the determinant of L to collapse to zero for any set of cardinality greater than the feature - length of x . With L in hand , we can now turn Eq . ( 1 ) into an equality by using the fact that (cid:4) Y \u2282 Y det ( L Y ) = det ( L + I ) , where I is an identity matrix of the same shape as the ensemble matrix L . Then , using Theorem 2 . 2 from Ref . [ 39 ] , we can write the P ( Y \u2208 Y ) as follows : P ( Y ) = det ( L Y ) det ( L + I ) ( 3 ) This is the probability that a given set of points ( Y ) is highly diverse compared to other possible sets ( Y ) \u2014 that is , the higher P ( Y ) the more diverse the set . The popularity of DPP - type measures is due to their ability to ef \ufb01 ciently sample diverse samples of \ufb01 xed size k . Sampling a set of k samples from a DPP is done using a con - ditional DPP called k - DPP [ 49 ] . k - DPP are able to compute mar - ginal and conditional probabilities with polynomial complexity , in turn allowing sampling from the DPP in polynomial complexity . k - DPPs are also well researched and there exists several different methods to speed up the sampling process using a k - DPP [ 50 , 51 ] . Our approach allows sampling in constant complexity ; however , there is a trade - off in complexity in generating the DPP distribution . The complexity for generating traditional DPP distributions is inde - pendent of \u201c k , \u201d while our approach has linear dependence on \u201c k . \u201d Since existing k - DPP approaches lack the ability to ef \ufb01 ciently sample from different percentiles of diversity and thus make it com - putationally expensive to regenerate the distribution to alternatively sample from different percentiles . To tackle this problem , our approach is designed to sample ef \ufb01 - ciently from different percentiles of diversity . This is made possible by creating an absolute diversity score . This score is generated by taking a log determinant of the kernel matrix de \ufb01 ned over the set Y . Randomly sampling the k - DPP space allows us to bound errors in generating this absolute score through the use of concentration inequalities . The details about how to sample from this distribution and calculate the score are mentioned in the Supplementary Material , so as not to disrupt the paper \u2019 s main narrative . Addition - ally , the Supplementary Material provides empirical results to support our earlier claims regarding ef \ufb01 cient sampling from our approach versus the traditional k - DPP approach , as well as the trade - off in complexity when generating the DPP distribution . Figure 2 shows example sets of \ufb01 ve points and their corresponding DPP score , where the diversity score is monotonic and a positive score corresponds to a more diverse subset . 2 . 1 . 1 Selecting the Hyper - parameter for the Determinantal Point Process Kernel . As mentioned above , the choice of \u03b3 impacts the accuracy of the DPP score , and when we initially \ufb01 xed \u03b3 to | Y i | / 10 , where Y i is the set of data points over which the RBF kernel is calculating the DPP score as suggested by Ref . [ 52 ] , the DPP seemed to be producing largely random scores . To select an appropriate \u03b3 , we designed a kernel - independent diagnostic method for assessing the DPP kernel with four steps . First , we randomly generate M samples of size k sets ( think of these as random k - sized samples from Y ) . Second , we compute their DPP scores for different possible \u03b3 values and then sort those M sets by that score . Third , we compute the rank correlation among these sets for different pairs of \u03b3 \u2014 intuitively , if the rank cor - relation is high ( toward 1 ) , then either choice of \u03b3 would produce the same rank orders of which points were considered diverse , meaning the ( relative ) DPP scores are insensitive to \u03b3 . In contrast , if the rank correlation is 0 , then the two \u03b3 values produce essentially random orderings . This rank correlation between two different \u03b3 settings is the color / value shown in each cell of the matrix in Fig . 1 . Large ranges of \u03b3 with high - rank correlation mean that the rankings of DPP scores are stable or robust to small perturbations in \u03b3 . Lastly , Fig . 1 Correlation matrix showing the relative correlation between two gammas by comparing the way our DPP approach ranks 10 , 000 sampled sets of cardinality , k = 10 . The gamma values in both axes here are logarithmic values with base 10 . 3 https : / / github . com / IDEALLab / JMD - Diversity - in - Bayesian - Optimization Journal of Mechanical Design NOVEMBER 2023 , Vol . 145 / 111703 - 3 D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 we use this \u201c robust \u03b3 \u201d region by choosing the largest range of \u03b3 values that have a relative correlation index of 0 . 95 or higher . We compute the mean of this range and use that as our selected \u03b3 in our later experiments . We should note that the functional range of \u03b3 is dependent on sample size ( k ) , and so this \u201c robust \u03b3 \u201d needs to be recomputed for different initialization sizes . The detailed settings for the results as seen in Fig . 1 are as follows : M = 10 , 000 ; k = 10 ; and \u03b3 \u2208 [ e \u2212 7 , e \u2212 2 ] . The correlation matrix shows a range of \u03b3 with strongly correlating relative ordering of the test sets . All \u03b3 within this range provide a consistent ranking . 2 . 2 A Test Function With Tunable Complexity . A problem that is common across the study of initialization methods is their inconsistency across problems of varying dif \ufb01 culty , motivating the need to test BO \u2019 s search behavior on a problem class with var - iable complexity . Synthetic objective functions are often used to test the ef \ufb01 ciency of different optimizers and there are several libraries online to choose these functions from Ref . [ 53 ] , though these func - tions are largely static , in the sense that there is only a single test function de \ufb01 nition . There has been research into developing objec - tive function generators ; for example , in Ref . [ 54 ] , the author uses a mixture of four features to generate synthetic objective functions . These have been well categorized and the relative performance of different optimizers is documented on each landscape . Similar to this , Ref . [ 55 ] looks at using a mixture of different sinusoidal func - tions to create a noisy 1D function . Both the generators discussed are capable of generating complicated landscapes , but the complex - ity arises from mixing different randomly generated sinusoids and thus are unable to control or quantify a measure of complexity of the generated landscapes . To address this controllable complexity problem directly , we created a simple 2D test function generator with tunable complexity parameters that allow us to instantiate multiple random surfaces of similar optimization dif \ufb01 culty . We modi \ufb01 ed this function from the one used in Ref . [ 56 ] where it was referred to as \u201c Wildcat Wells , \u201d though the landscape is functionally just a normal distribu - tion with additive noise of different frequency spectra . We used four factors to control the synthetic objective functions : ( 1 ) the number of peaks , ( 2 ) noise amplitude , ( 3 ) smoothness , and ( 4 ) distance between peaks and a seed . The number of peaks control the number of layers of multivariate normal with single peaks . The noise amplitude in the range of [ 0 , 1 ] controls the relative height of the noise compared to the height of the peaks . Setting this to 1 would essentially make the noise in the function as tall as the peaks and give the function in \ufb01 nite peaks . Smoothness in the range of [ 0 , 1 ] controls the weighted contribution of the smooth Gaussian function compared to the rugged noise to the wildcat wells landscape . Setting this to 1 would remove the noise from the function because then the normal distribution completely con - trols and dominates the function . The last parameter , the distance between peaks , can be tuned in the range of [ 0 , 1 ] . This parameter prevents overlap of peaks when the function is generated with more than one peak . Some of these parameters overlap in their effects . For example , N controls the number of peaks , and ruggedness amplitude controls the height of the noise in the function , so increasing the noise auto - matically increases the peaks in the function thus we will only look at varying the ruggedness amplitude . Apart from this , ruggedness frequency ( the distance between peaks ) plays the same role as smoothness ( radius of in \ufb02 uence of each individual on its neighbor ) . Thus , for the numerical experiments presented in Secs . 3 \u2013 5 , only the ruggedness amplitude and smoothness will be varied between [ 0 . 2 , 0 . 8 ] with increments of 0 . 2 . To provide some visual examples of the effect of these parameters on the generated functions , Fig . 3 visualizes an example random surface generated with different smoothness and ruggedness amplitude parameters . 2 . 3 Bayesian Optimization . BO has emerged as a popular sample - ef \ufb01 cient approach for optimization of these expensive black - box functions . BO models the black - box function using a sur - rogate model , typically a GP . The next design to evaluate is then selected according to an acquisition function . The acquisition func - tion uses the GP posterior and makes the next recommendation for function evaluation by balancing between exploration and exploita - tion . It allows exploration of regions with high uncertainty in the objective function and exploitation of regions where the mean of the objective function is optimum . At each iteration , the GP gets updated according to the selected sample , and this process contin - ues iteratively according to the available budget . Each data point in the context of Bayesian optimization is extremely expensive ; thus , there is a need for selection of an infor - mative set of initial samples for the optimization process . Toward this , this paper investigates the effect of the level of initial diverse Fig . 2 Scatter plots showing randomly chosen sets with k = 5 high - diversity and low - diversity samples with their diversity score on top of each of the chosen set 111703 - 4 / Vol . 145 , NOVEMBER 2023 Transactions of the ASME D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 coverage of the input space on convergence of Bayesian optimiza - tion policies . For the purpose of numerical experiments , the optimizer used is from the BOTorch Library [ 57 ] . The optimizer uses a single task GP model with expected improvement ; the kernel used is a Mat\u00e9rn kernel . A GP is speci \ufb01 ed by its mean and covariance functions as f ( x ) \u223c GP \u03bc ( x ) , k ( x , x ) (cid:1) (cid:3) ( 4 ) where \u03bc ( . ) and k ( . , . ) are the mean function and a real - valued kernel function encoding the prior belief on the correlation among the samples in the design space . In Gaussian process regression , the kernel function dictates the structure of the surrogate model we can \ufb01 t . An important kernel for Bayesian optimization is the Mat\u00e9rn kernel , which incorporates a smoothness parameter \u03bd to permit greater \ufb02 exibility in modeling functions : k M at \u00b4 ern ( x 1 , x 2 ) = 2 1 \u2212 \u03bd \u0393 ( \u03bd ) (cid:5)(cid:5)(cid:5) 2 \u03bd \u221a (cid:2) x 1 \u2212 x 2 (cid:2) \u03b8 (cid:6) (cid:7) \u03bd H \u03bd (cid:5)(cid:5)(cid:5) 2 \u03bd \u221a (cid:2) x 1 \u2212 x 2 (cid:2) \u03b8 (cid:6) (cid:7) ( 5 ) where \u0393 ( \u00b7 ) and H \u03bd ( \u00b7 ) are the Gamma function and the Bessel func - tion of order \u03bd , and \u03b8 is the length scale hyper - parameter which denotes the correlation between the points within each dimension and speci \ufb01 es the distance that the points in the design space in \ufb02 u - ence one another . Here , we use a constant mean for the mean func - tion . The model building advantage that we refer to in this paper corresponds to learning these hyper - parameters . The hyper - parameters of the Gaussian process , namely , the parameters of the kernel function and the mean function are : Lengthscale of the Mat\u00e9rn Kernel : In Eq . ( 5 ) , \u03b8 is the lengthscale parameter of the kernel . This parameter controls the ruggedness expected by the Bayesian optimizer in the black box function being studied . The effects of the parameter are similar to \u03bd , but \u03bd is not learned during the optimization process while lengthscale is . So , \u03bd is not studied as a parameter that in \ufb02 uences the modeling behavior but rather studied as an additional parameter for sensitivity . Output Scale of Scale Kernel : Output scale is used to control how the Mat\u00e9rn kernel is scaled for each batch . Since our Bayesian opti - mizer uses a single task GP , we do not use batch optimization . Thus , this parameter is unique for us and the way it is implemented using BoTorch can be seen in Eq . ( 6 ) . K scaled = \u03b8 scale K orig ( 6 ) Noise for Likelihood Calculations : The noise parameter is used to model measurement error or noise in the data . So , as the Gaussian process gets more data , the noise term decreases . So , ideally , this term should converge to 0 when the Bayesian optimizer has found an optimal value since our test functions did not have any added noise . Constant for Mean Module : This constant is used as the mean for the normal distribution that forms the prior of the Gaussian process as shown in Eq . ( 4 ) . Further studies and results regarding the effects of the hyper - parameters are available in the Supplementary Material . We now describe the \ufb01 rst experiment where we explore the effects of diversity of initial samples on the convergence of Bayesian optimizers . 3 Experiment 1 : Does Diversity Affect Optimization Convergence ? 3 . 1 Methods . To test the effects of diversity of initial samples on optimizer convergence , we \ufb01 rst generated a set of initial training samples of size ( k ) 10 either from low ( 5th percentile of diversity ) or high - diversity ( 95th percentile of diversity ) using our procedure in Sec . 2 . 1 . Next , we created 100 different instances of the wildcat wells function with different randomly generated seeds for each cell in a 4 \u00d7 4 factor grid of four values each of the smoothness and ruggedness amplitude parameters of the wildcat wells function ( ranging from 0 . 2 to 0 . 8 , in steps of 0 . 2 ) . For simplicity here , we Fig . 3 A grid plot showing how the landscape of wildcat wells changes with smoothness and ruggedness amplitude Journal of Mechanical Design NOVEMBER 2023 , Vol . 145 / 111703 - 5 D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 refer to these combinations as families of the wildcat wells function . This resulted in 1600 function instances . Our experiment consisted of 200 runs of the Bayesian optimizer within each of the smoothness \u2013 ruggedness function families , where each run consisted of 100 iterations , and half of the runs were ini - tialized with a low - diversity training sample , and half were initial - ized with a high - diversity training sample . We then compared the cumulative optimality gap ( COG ) across the iterations for the runs with low - diverse initializations and high - diverse initializations within each smoothness \u2013 ruggedness combination family . We did this by computing bootstrapped mean and con \ufb01 dence intervals within each low - diverse and high - diverse sets of runs within each family . Given the full convergence data , we compute a COG which is just the area under the optimality gap curve for both the 5th and 95th diversity curves . Intuitively , a larger COG corresponds to a worse overall performance by the opti - mizer . Using these COG values , we can numerically calculate the improvement of the optimizer in the 95th percentile . The net improvement of COG value while comparing the 5th and 95th per - centile is also presented as text in each subplot in Fig . 4 . 3 . 2 Results . As Fig . 4 shows , the cumulative optimality gap does not seem to have a consistent effect across the grid . Diversity produces a positive convergence effect for some cells , but is negative in others . Moreover , there are wide empirical con \ufb01 dence bounds on the mean effect overall , indicating that should an effect exist at all , it likely does not have a large effect size . Changing the function rug - gedness or smoothness did not signi \ufb01 cantly modulate the overall effect . As expected , given suf \ufb01 cient samples ( far right on the x - axis ) , both diverse and non - diverse initializations have the same optimality gap , since at that point the initial samples have been crowded out by the new samples gathered by BO during its search . 3 . 3 Discussion . Overall , the results from Fig . 4 seem to indi - cate that diversity helps in some cases and hurts in others , and regardless has a limited impact one way or the other . This seems counter to the widespread practice of diversely sampling the initial input space using techniques like LHS . Figure 4 shows that it has little effect . Why would this be ? Given decades of research into initialization schemes for BO and optimal experiment design , we expected diver - sity to have at least some ( perhaps small but at least consistent ) pos - itive effect on convergence rates , and not the mixed bag that we see in Fig . 4 . How were the non - diverse samples gaining such an upper hand when the diverse samples had a head start on exploring the space \u2014 what we call a space exploration advantage ? The next section details an experiment we conducted to test a hypothesis regarding a potential implicit advantage that non - diverse samples might endow to BO that would impact the convergence of BO \u2019 s hyper - parameter posteriors . As we will see next , this acceler - ated hyper - parameter posterior convergence caused by non - diverse initialization is the Achilles \u2019 heel of diversely initialized BO that allows the non - diverse samples to keep pace and even exceed diverse BO . 4 Experiment 2 : Do Lower Diversity Samples Improve Hyper - parameter Posterior Convergence ? After reviewing the results from Fig . 4 , we tried to determine why the space exploration advantage of diversity was not helping BO as we thought it should . We considered as a thought experiment the one instance where a poorly initialized BO model with the same acquisition function might outperform another : if one model \u2019 s kernel hyper - parameter settings were so grossly incorrect that the Fig . 4 Experiment 1 : optimality gap grid plot showing the difference in current optimality gap between optimizers initialized with 5th versus 95th percentile diverse sample ( y - axis ) as a function of optimization iteration ( x - axis ) . The different factors in the factor grid plot the effects of diversity as the noise amplitude and smoothness are varied in the range [ 0 . 2 , 0 . 8 ] . Each plot also has text indicating the net cumulative optimality gap ( NCOG ) , a positive value corresponds to a better performance by high - diversity samples compared to the low - diversity samples . The plot shows that BO bene \ufb01 ts from diversity in some cases but not others . There is no obvious trends in how the NCOG values change in the grid . The results are further discussed in Sec . 3 . 111703 - 6 / Vol . 145 , NOVEMBER 2023 Transactions of the ASME D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 model would waste many samples exploring areas that it did not need to if it had the correct hyper - parameters . Could this misstep be happening in the diversely sampled BO but not in the non - diverse case ? If so , this might explain how non - diverse BO was able to keep pace : while diverse samples might give BO a head start , it might be unintentionally blindfolding BO to the true function posteriors , making it run ragged in proverbial directions that it need not . If this hypothesis was true , then we would see this re \ufb02 ected in the comparative accuracy of the kernel hyper - parameters learned by the diverse versus non - diverse BO samples . This experiment set out to test that hypothesis . 4 . 1 Methods . The key difference from experiment 1 is that rather than comparing the overall optimization convergence , we instead focus on how the initial samples \u2019 diversity affects BO \u2019 s hyper - parameter posterior convergence , and compare how far each is from the \u201c ground truth \u201d optimal hyper - parameters . As with experiment 1 , we used the same smoothness and rugged - ness amplitude families of the wildcat wells function . To then gen - erate the data for each instance in one of these families , we sampled 20 sets of initial samples . Half of the sampled 20 sets were low ( 5th percentile of diversity ) and the other half from high - diversity ( 95th percentile of diversity ) percentiles . For each initial sample , we then maximized the GP \u2019 s kernel mar - ginal log likelihood ( via BOTorch \u2019 s GP \ufb01 t method ) . We then recorded the hyper - parameters obtained for all 20 initial samples . The mean of the ten samples from low diversity was then used as one point in the box plot \u2019 s low - diversity distribution as seen in Fig . 5 . We then repeated this process for the high - diversity initial samples . Each point in the box plot can then be understood as the mean hyper - parameter learned by BOTorch given just the initial sample of size ( k ) 10 points . To get the full box plot distribution for each family , the above process is repeated over 100 seeds and Fig . 5 provides the resulting box plot for both diverse and non - diverse initial samples for all the 16 families of wildcat wells func - tion as described in experiment 1 . To provide a ground truth for the true hyper - parameter settings , we ran a binary search to \ufb01 nd the size of the sample ( k optimal ) for which BO \u2019 s kernel hyper - parameters converged for all families . The hyper - parameter found by providing k optimal amount of points for each instance in the family was then plotted as a horizontal line in each box plot . An interesting observation is that some fam - ilies have non - overlapping horizontal lines . This is because for Fig . 5 Experiment 2 : box plot showing distribution of \u201c lengthscale \u201d hyper - parameter learned by BO when initiated with diverse ( right - side ) and non - diverse samples ( left - side ) for 16 different families of wildcat wells functions of the same parameters but 100 different seeds . The optimal hyper - parameter for each of the 100 wildcat wells instances from each family is also plotted as hor - izontal lines \u2014 in many but not all cases these overlap . Each cell in the plot also has the 95th percentile con \ufb01 dence bound on MAE for both diverse and non - diverse samples . The results show that MAE con \ufb01 dence bounds for non - diverse samples are smaller compared to diverse samples for all the families of wildcat wells function . Thus , indicating a presence of model building advan - tage for non - diverse initial samples . The results of this \ufb01 gure are further discussed in Sec . 4 . Journal of Mechanical Design NOVEMBER 2023 , Vol . 145 / 111703 - 7 D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 some families there are more than one modes of \u201c optimal hyper - parameters . \u201d The mode chosen as the \u201c optimal hyper - parameter \u201d is the more observed mode . The process for \ufb01 nding the \u201c optimal hyper - parameter \u201d and which mode is chosen as the optimal hyper - parameter have been described in the Supplementary Material . If an initial sample provides a good initial estimate of the kernel hyper - parameter posterior , then the box plot should align well or close to the horizontal lines of the true posterior . Figure 5 only shows the results for the Mat\u00e9rn kernel \u2019 s lengthscale parameter , given its out - sized importance in controlling the GP function poste - riors compared to the other hyper - parameters ( e . g . , output scale , noise , etc . ) , which we do not plot here for space reasons . We provide further details and plots for all hyper - parameters in the Supplementary Material for interested readers . To quantify the average distance between the learned and true hyper - parameters , we also plot in Fig . 5 the mean absolute error ( MAE ) for both highly diverse ( 95th ) and less diverse ( 5th ) points . The MAE is the sum of the absolute distance of each pre - dicted hyper - parameter from the optimal hyper - parameter for the particular surface of each wildcat wells function . The range as seen in each cell in Fig . 5 corresponds to a 95th percentile con \ufb01 - dence bound on the mean absolute error across all the 100 runs . 4 . 2 Results and Discussion . The results in Fig . 5 show that the MAE values for low diversity samples are always lower compared to the MAE for high - diversity samples . This general behavior is also qualitatively visible in the box plot . This means that after only the initial samples , the non - diverse samples provided much more accurate estimates of the kernel hyper - parameters compared to diverse samples . Moreover , BO systematically underestimates the correct lengthscale with diverse samples \u2014 this corresponds to the diverse BO modeling function posteriors that have higher fre - quency components than the true function actually does ( as shown via the pedagogical examples in the Supplementary Material ) . This provides evidence for the model building advantage of non - diverse samples that we de \ufb01 ned in Sec . 2 . 3 . It also con \ufb01 rms our pre - vious conjecture from the thought experiment that diverse samples might be impacting BO by causing slower or less accurate conver - gence to the right BO hyper - parameters . The space exploration advantage of the diverse samples helps it compensate somewhat for its poor hyper - parameters , but BO trained with non - diverse samples can leverage the better hyper - parameters to make more judicious choices about what points to select next . We did not see major differences in the other three kernel hyper - parameters such as output scale , noise , or the mean function ( see Supplementary Material ) ; however , this is not surprising , since BO is not highly sensitive to any of these parameters and the lengthscale parameter dominates large changes in BO behavior . Comparing the different smoothness and ruggedness settings , when the function is more complex ( the top right of the grid at low smoothness and high ruggedness amplitude values ) , the function \u2019 s lengthscale is lower and closer to the value learned by the diverse samples . Looking at the low diversity MAE values ( \u201c MAE 5 \u201d ) , we can see they are much closer to those of the high - diversity samples ( \u201c MAE 95 \u201d ) , in contrast to when the function is less complex ( bottom left side of the grid ) . Under such conditions , low - diversity samples lose some of the relative model building advantage they have over high - diversity samples . This conjecture aligns with exper - iment 1 ( Fig . 4 ) where the COG values on the top right part are positive while those on the bottom left are negative . Figure 5 demonstrated our hypothesized model building advan - tage that non - diverse initial samples confer to BO . But how do we know that this is the actual causal factor that accelerates BO conver - gence , and not just correlated with some other effect ? If correct , our conjecture posits a natural testable hypothesis : if we \ufb01 x the values of the hyper - parameter posteriors to identical values between the non - diverse and diverse samples and do not allow the BOto update or optimize them , then this should effectively eliminate the model building advantage , and diverse samples should always outperform non - diverse samples . Metaphorically , if we were to take away the arrow that Paris used against Achilles , would the Battle of Troy have ended differently ? Our next experiment \ufb01 nds this out . 5 Experiment 3 : Does Diversity Affect Optimization Convergence If Hyper - parameters Are Fixed to Optimal Values ? 5 . 1 Methods . This experiment is identical to experiment 1 , with two key differences : ( 1 ) we now \ufb01 x the kernel hyper - parameters to the \u201c optimal hyper - parameter \u201d values we found in experiment 2 for all the instances in each family of the wildcat wells function ( 2 ) and we do not allow either BO model to further optimize the kernel hyper - parameters . This should remove the hypothesized model building advantage of non - diverse samples without altering any other aspects of experiment 1 and the results in Fig . 4 . 5 . 2 Results and Discussion . Figure 6 shows that once the kernel hyper - parameters are \ufb01 xed \u2014 removing the model building advantage of non - diverse samples \u2014 diverse samples consistently and robustly outperform non - diverse initial samples . This holds for both the initial optimality gap at the beginning of the search as well as the cumulative optimality gap and is not qualitatively affected by the function smoothness or roughness amplitude . Unlike in experiment 1 where diversity could either help or hurt the optimizer , once we remove the model building advantage , diver - sity only helps . 6 General Discussion and Conclusions 6 . 1 Summary and Interpretation of Findings . This paper \u2019 s original goal was to investigate how and when diverse initial samples help or hurt Bayesian optimizers . Overall , we found that the initial diversity of the provided samples created two competing effects . First , experiment 2 showed that non - diverse samples improved BO \u2019 s abilities to quickly converge to optimal hyper - parameters \u2014 we called this a model building advantage . Second , experiment 3 showed that conditioned on the same \ufb01 xed hyper - parameters diverse samples improved BO \u2019 s convergence to the optima through faster exploration of the space \u2014 we called this a space exploration advantage . In experiment 1 , diversity had mixed - to - negligible effects since both of these advantages were in play and competed with one another . This interaction provides insight for academic or industrial BO users since common practice recommends initializing BO with space - \ufb01 lling samples ( to take advantage of the space exploration advantage ) , and ignores the model building advantage of non - diverse samples . Beyond our main empirical result , our improvements to existing diverse sampling approaches ( Sec . 2 . 1 ) provide new methods for studying how different percentile diversity sets affect phenomena . Researchers may \ufb01 nd this contribution of separate technical and scienti \ufb01 c interest for related studies that investigate the impact of diversity . 6 . 2 Implications and Future Work . Beyond the individual results we observed and summarized in each experiment , there are some overall implications and limitations that may guide future work or interpretation of our results more broadly , which we address below . Where does this model building advantage induced by non - diverse samples come from ? As we conjectured in experiment 2 ( Sec . 4 ) and con \ufb01 rmed in experiment 3 ( Sec . 5 ) , the key advantage of using non - diverse initial samples lies in their ability to induce faster and more accurate posterior convergence when inferring the optimal kernel hyper - parameters , such as length scale and others . This allowed the BO to make more judicious and aggressive 111703 - 8 / Vol . 145 , NOVEMBER 2023 Transactions of the ASME D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 choices about what points to sample next , so while the diversely ini - tialized models might get a head start on exploring the space , non - diversely initialized models needed to explore less of the space overall , owing to tighter posteriors of possible functions under the Gaussian process . While we do not have space to include it in the main paper , the Supplementary Material document \u2019 s Sec . 5 shows how this model building advantage occurs as we provide BO with a greater number of initial samples . Brie \ufb02 y , there are three \u201c regimes \u201d : ( 1 ) sample - de \ufb01 cient , where there are too few samples to induce a mod - eling advantage regardless of how diversely we sample the initial points ; ( 2 ) the \u201c modeling advantage \u201d region , where low - diversity samples induce better hyper - parameter convergence than high - diversity samples ; and ( 3 ) sample - saturated , where there are enough initial samples to induce accurate hyper - parameter posteri - ors regardless of how diversely we sample initial points . We direct interested readers to Sec . 5 of the Supplementary Material for a deeper discussion on this . What this behavior implies more broadly is that non - diverse samples , whether given to an algorithm or a person , have a unique and perhaps underrated value in cases where we have high entropy priors over the Gaussian process hyper - parameters or kernel . In such cases , sacri \ufb01 cing a few initial non - diverse points to better infer key length scales in the GP model may well be a worthwhile trade . We also saw that in cases where the BO hyper - parameters were not further optimized ( as in experiment 3 where hyper - parameters were \ufb01 xed to optimal values ) , using diverse points only helped BO . Researchers or practitioners using BO would bene \ufb01 t from care - fully reviewing what kernel optimization strategy their library or implementation of choice actually does since that will affect whether or not the model building advantage of non - diverse samples is actually in play . What if hyper - parameters are \ufb01 xed to non - optimal values ? We showed in experiment 3 that \ufb01 xing BO hyper - parameters to their optimal values ahead of time using an oracle allowed diverse initial samples to unilaterally outperform non - diverse samples . An interesting avenue of future work that we did not explore here for scope reasons would be to see if this holds when hyper - parameters are \ufb01 xed to non - optimal values . In practical problems , we will not often know the optimal hyper - parameters ahead of time as we did in experiment 3 which caused diversity \u2019 s unilateral advantage , so we do not have evidence to generalize beyond this . However , our explanation of the model building advantage would predict that , so long as the hyper - parameters remain \ufb01 xed ( to any value ) , BO would not have a practical mechanism to bene \ufb01 t much from non - diverse samples , on average . What are theimplications for how we currently initialize BO ? One of our result \u2019 s most striking implications is how it might in \ufb02 uence BO initialization procedures that are often considered standard prac - tice . For example , it is common to initialize a BO procedure with a small number of initial space - \ufb01 lling designs , using techniques like LHS before allowing BO to optimize its acquisition function for future samples . In cases where the BO hyper - parameters will remain \ufb01 xed , experiment 3 implies that this standard practice is excellent advice and far better than non - diverse samples . However , in cases where you plan to optimize the BO kernel during search , using something like LHS becomes more suspect . In principle , from experiment 1 , we see that diverse samples may help or hurt BO , depending on how much leverage the model build - ing advantage of the non - diverse samples can provide . For example , in the upper right of Fig . 4 , the function is effectively random noise , and so there is not a strong model building advantage to be gained . In contrast , in the lower left , the smooth and well - behaved functions allowed non - diverse initialization to gain an upper hand . Our results propose a perhaps now obvious initialization strategy : if you plan on optimizing the BO hyper - parameters , use some non - diverse samples to strategically provide an early model building advantage , while leveraging the rest of the samples to diversely cover the space . Fig . 6 Experiment 3 : optimality gap plot showing effects of diversity when the optimizer is not allowed to \ufb01 t the hyper - parameters for the Gaussian process and the hyper - parameters are instead \ufb01 xed to the values found in experiment 2 . The results from this plot show positive NCOG values for all families of wildcat wells function , showing that once the model building advantage is taken away , the diverse samples outperform non - diverse samples . Further dis - cussion on this plot can be read in Sec . 5 . Journal of Mechanical Design NOVEMBER 2023 , Vol . 145 / 111703 - 9 D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024 How might other acquisition functions modulate diversity \u2019 s effect ? While we have been referring to BO as though it is a single method throughout this paper , individual BO implementations can vary , both in terms of their kernel structure and their choice of acqui - sition function \u2014 or how BO uses information about the underlying \ufb01 tted Gaussian process to select subsequent points . In this paper \u2019 s experiments , we used EI since it is one of the most widespread choices and behaves qualitatively like other common improvement - based measures like probability of improvement , posterior mean , and upper con \ufb01 dence bound functions . Indeed , we hypothesize that part of the reason non - diverse initial samples are able to gain a model building advantage over diverse samples due to a faster col - lapse in the posterior distribution of possible GP functions which serves as strong input to EI methods and related variants . Yet EI and its cousins are only one class of acquisition function ; would our results hold if we were to pick an acquisition function that directly attacked the GP \u2019 s posterior variance ? For example , either entropy - based or active learning - based acquisition functions ? This paper did not test this and it would be a logical and valuable future study . Our experimental results and proposed explanation would predict the following : the model building advantage seen by non - diverse samples should reduce or disappear in cases where the acquisition function explicitly samples new points to minimize the posterior over GP function classes since in such cases BO itself would try to select samples that reduced overall GP variance , reducing its dependence on what the initial samples provide . To what extent should we expect these results to generalize to other types of problems ? We selected a simple 2D function with controllable complexity in this paper to aid in experimental simpli - city , speed , replicability , and ease of visualization ; however , this does raise the question of whether or not these results would truly transfer to more complex problems of engineering interest . While future work would have to address more complex problems , we performed two additional experiments studying how the above phenomena change as we ( 1 ) increased the wildcat wells function from two to three dimensions and ( 2 ) how this behavior changes for other types of common optimization test functions \u2014 speci \ufb01 - cally , we chose the N - dimensional sphere , Rastrigin , and Rosen - brock functions from two to \ufb01 ve dimensions . While the existing paper length limits did not allow us to include all of these addi - tional results in the paper \u2019 s main body , we direct interested readers to Secs . 6 and 7 of the Supplementary Material document . Brie \ufb02 y , our results align overall with what we described above for the 2D wildcat wells function , and we do not believe that the phe - nomena we observed are restricted to only our chosen test function or dimension , although clearly future research would need to conduct further tests on other problems to say this with any cer - tainty . Beyond these supplemental results , we can also look at a few critical problem - speci \ufb01 c factors and ask what our proposed explanatory model would predict . For higher dimensional problems , standard GP kernel choices like RBF or Mat\u00e9rn begin to face exponential cost increases due to how hyper - volumes expand . In such cases , having strong con - straints ( via hyper - parameter priors or posteriors ) over possible GP functions becomes increasingly important for fast BO conver - gence . Our results would posit that any model building advantages from non - diverse sampling would become increasingly important or impactful in cases where it helped BO rapidly collapse the hyper - parameter posteriors . For discontinuous functions ( or GP kernels that assumed as much ) , the model building advantage of non - diverse samples would decrease since large sudden jumps in the GP posterior mean and variance would make it harder for BO to exploit a model building advantage . However , in discontinuous cases where there were still common global smoothness parameters that governed the continuous portions , the model building advantage would still accelerate advantages for BO convergence . How might theresultsguide human - subject experimentsor under - standing of human designers ? One possible implication of our results for human designers is that the effects of example diversity on design outcomes may vary as a function of the designer \u2019 s prior knowledge of the design problem . More speci \ufb01 cally , the model building advantage observed in experiment 2 ( and subsequent removal in experiment 3 ) suggests that when designers have prior knowledge of how quickly the function changes in a local area of thedesignspace , theycanmorereliablybene \ufb01 tfromthespaceexplo - ration advantage of diverse examples . This leads to a potentially counter - intuitive prediction that domain experts may bene \ufb01 t more from diverse examples compared to domain novices since domain experts would tend to have prior knowledge of the nature of the design problem ( a model building advantage ) . Additionally , perhaps under conditions of uncertainty about the nature of the design problem , it would be useful to combine the strengths of diverse and non - diverse examples ; this could be accomplished with a cluster - sampling approach , where we sample diverse points of the design space , but include local non - diverse clusters of exam - ples that are nearby , to facilitate learning of the shape of the design function . While these implications might be counter - intuitive in that common guidance suggests that the most informative method is to only diversely sample initial points , the crux of our paper \u2019 s argu - ment is that non - diverse points can , surprisingly , be informative to Bayesian optimization due to their ability to quickly concentrate the posterior distribution of the kernel hyper - parameters , and thus accelerate later optimization . Given this tension , a natural question is \u201c how many non - diverse samples do I really need to take advan - tage of the modeling advantage without giving up the space explo - ration advantage ? \u201d If I have , say , a budget of ten experiments , should I spend only one low - diversity sample ? Or do I need two ? Half of my budget ? We did not explore these practical questions in this work , due to space constraints , but we think this would be an excellent avenue for continued study . Acknowledgment This research was supported by funding from the National Science Foundation through award # 1826083 . Con \ufb02 ict of Interest There are no con \ufb02 icts of interest . Data Availability Statement The data and information that support the \ufb01 ndings of this article are freely available . 4 References [ 1 ] Sio , U . N . , Kotovsky , K . , and Cagan , J . , 2015 , \u201c Fixation or Inspiration ? 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Journal of Mechanical Design NOVEMBER 2023 , Vol . 145 / 111703 - 11 D o w n l oaded f r o m h tt p : / / a s m ed i g i t a l c o ll e c t i on . a s m e . o r g / m e c han i c a l de s i gn / a r t i c l e - pd f / 145 / 11 / 111703 / 7037097 / m d _ 145 _ 11 _ 111703 . pd f b y U n i v e r s i t y o f M a r y l and L i b r a r i e s u s e r on 03 M a y 2024", + "chanFormulatingFixatingEffects2024": "Formulating or Fixating : Effects of Examples on Problem Solving Vary as a Function of Example Presentation Interface Design JOEL CHAN , College of Information Studies , University of Maryland , USA ZIJIAN DING , College of Information Studies , University of Maryland , USA EESH KAMRAH , Department of Mechanical Engineering , University of Maryland , USA MARK FUGE , Department of Mechanical Engineering , University of Maryland , USA Interactive systems that facilitate exposure to examples can augment problem solving performance . However designers of such systems are often faced with many practical design decisions about how users will interact with examples , with little clear theoretical guidance . To understand how example interaction design choices affect whether / how people benefit from examples , we conducted an experiment where 182 participants worked on a controlled analog to an exploratory creativity task , with access to examples of varying diversity and presentation interfaces . Task performance was worse when examples were presented in a list , compared to contextualized in the exploration space or shown in a dropdown list . Example lists were associated with more fixation , whereas contextualized examples were associated with using examples to formulate a model of the problem space to guide exploration . We discuss implications of these results for a theoretical framework that maps design choices to fundamental psychological mechanisms of creative inspiration from examples . Additional Key Words and Phrases : Creativity , Examples , Interface , Problem Solving ACM Reference Format : Joel Chan , Zijian Ding , Eesh Kamrah , and Mark Fuge . 2023 . Formulating or Fixating : Effects of Examples on Problem Solving Vary as a Function of Example Presentation Interface Design . 1 , 1 ( January 2023 ) , 25 pages . https : / / doi . org / 10 . 1145 / 1122445 . 1122456 1 INTRODUCTION Examples \u2014 descriptions or representations of possible solutions ( or parts thereof ) for the same or related problems [ 8 , 18 , 35 , 83 , 84 ] \u2014 are an integral part of the creative problem solving process . Examples can take many forms , such as previous physical prototypes brought to a brainstorm [ 33 ] , search results for patents for related problems [ 25 ] , spoken ideas from collaborators [ 66 ] , UI designs in an accessible gallery [ 50 ] ) , or references from memory to earth animals or science fiction creatures when inventing new fictional alien creatures [ 89 ] ) . Importantly , examples can substantially shape what ideas come to mind [ 84 , 89 ] . This \" structuring of imagination \" [ 89 ] is sometimes helpful \u201c inspiration \" that leads to more creative ideas [ 19 , 23 , 33 , 80 ] . But examples can also have harmful \u201c fixation \u201d effects that constrain novelty and innovation [ 40 , 54 , 84 , 89 ] ( for recent reviews , see [ 83 ] and [ 18 ] ) . Importantly , examples can influence problem solving without conscious effort or recognition [ 54 , 60 , 61 ] , and persist in spite of creators\u2019 explicit intentions not to be influenced by them [ 40 , 84 , 85 ] . Perhaps in recognition of these facts , effective creators take an active role Authors\u2019 addresses : Joel Chan , College of Information Studies , University of Maryland , USA ; Zijian Ding , , College of Information Studies , University of Maryland , USA ; Eesh Kamrah , Department of Mechanical Engineering , University of Maryland , USA ; Mark Fuge , Department of Mechanical Engineering , University of Maryland , USA . Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from permissions @ acm . org . \u00a9 2023 Association for Computing Machinery . Manuscript submitted to ACM Manuscript submitted to ACM 1 a r X i v : 2401 . 11022v2 [ c s . H C ] 23 J a n 2024 2 Chan , et al . in finding , structuring and interacting with examples [ 23 , 35 , 79 ] using a variety of analog and digitally mediated systems and practices , such as search engines [ 35 , 68 , 69 , 97 ] , design workbooks and commonplace notebooks [ 26 ] , online whiteboards [ 58 , 86 ] , mood boards [ 55 ] , and wider interactions with their community of practice , such as trade publications and conventions [ 23 , 33 ] . An important area of HCI research on creativity support tools therefore studies the design of interactive systems that can assist creators in discovering examples [ 9 , 39 , 47 , 48 , 74 , 80 , 82 , 87 , 88 ] , structuring , analyzing , and exploring collections of examples [ 15 , 16 , 53 , 57 , 81 , 93 ] , and adapting and using examples [ 39 , 48 , 50 ] . Designers of such systems need to grapple with an array of very practical interaction design decisions . For example , how should we support interaction with examples over different screen sizes ? Should examples be delivered via recommendation ( in small sets ) , a feed , or some other interaction paradigm ? What information should be presented alongside an example ? We would like to have a consistent theory to draw from to make these decisions . Beyond considerations of usability , we conjecture that such a theory would need to map design decisions ( or classes thereof ) to creativity - relevant behaviors and outcomes , ideally with a nuanced specification of the precise benefits and costs of each design decision for these behaviors and outcomes . A theory of human - example interaction like this could help us design better systems with sensible defaults , prioritize and negotiate design requirements , and guide evaluation . As a step towards developing such a theory , we conducted an experiment with 182 participants solving a controlled analog to an exploratory creativity task [ 7 ] . We varied both the diversity of examples and the types of presentations : overlaid on the search environment ( the \u201c In - Context \" condition ) , presented in a list ( the \u201c List \" condition ) , or in a dropdown selectable menu ( the \u201c Dropdown \" condition ) . The \u201c In - Context \" design was inspired by an emerging pattern of contextualizing examples in the creator\u2019s workspace or problem in HCI systems for example - based creativity [ 39 , 47 , 78 , 91 , 93 ] on the one hand , and theoretical descriptions of the use of examples to ( re ) formulate problems [ 34 , 35 , 43 , 59 , 67 , 79 ] ; the latter \u201c List \" and \u201c Dropdown \" conditions were designed to be representative of common interfaces for interacting with examples ( in search results lists and pages of recommendations ) . Our primary results were threefold : 1 ) \u201c List \" presentation harmed solution quality compared with \u201c In - Context \" or \u201c Dropdown \" presentation ; 2 ) each interface condition was associated with distinct self - reported example usage strategies ( notably , more usage of examples to \u201cmodel \" the problem space to guide exploration in the In - Context vs . List or Dropdown conditions , and more usage of examples to \u201cstimulate \" a specific direction of exploration in the List condition ) ; and 3 ) the List condition\u2019s propensity for stimulation - based strategies was corroborated by an increased usage of \u201chill - climbing \" strategies early on , as evidenced by analyses of Dropdown distance between participants\u2019 moves . We discuss how these results , in conversation with the literature on example - based creativity support systems as well as psychological mechanisms of creativity with examples , could contribute to a theoretical framework for designing interactive systems for creative problem solving with examples . 2 RELATED WORK 2 . 1 Sources of empirical variability in effects of examples Prior work has examined how the consequences of examples for creative problem solving outcomes are related to characteristics of the examples , such as their novelty [ 1 , 6 , 11 , 80 ] ( generally positive effects ) , conceptual distance from the problem domain [ 10 , 19 , 25 , 31 , 90 ] ( mixed or curvilinear effects ) , and example diversity [ 12 , 21 , 29 , 38 , 80 , 94 , 96 ] ( generally positive or contingently positive effects ) . Our work contributes additional empirical results on the relative contributions of ( and potential interactions , in the statistical sense , between ) example characteristics and example Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 3 interface characteristics . In particular , we explore how the example characteristic of diversity might interact with example interface characteristics , such as whether the examples are presented as a list vs . in context of a representation of the design space . To do this , we need to also consider the cognitive mechanisms of inspiration or fixation from examples ( or varying characteristics ) , which might be more or less afforded by example interfaces . We discuss this body of literature in the next section . 2 . 2 Theoretical insights into human - example interaction A number of detailed in - situ studies of creators have documented a range of strategies for working with examples , ranging from simpler , more source - driven strategies like direct source adaptation [ 24 ] , to more complex and reflective strategies associated with more radical transformation of examples , such as source analysis and schema - driven source selection [ 24 ] , analogical reasoning [ 3 , 27 , 28 , 37 ] , and generating novel emergent features that can connect disparate attributes across examples [ 92 ] . These \u201cprocessing strategies \" can be described by a variety of theoretical frames from the psychological literature on creativity . We believe this theoretical level of description could facilitate our goal of synthesizing mappings between interface characteristics and effects of examples on creative problem solving outcomes . Some notable examples include basic memory mechanisms such as priming [ 63 ] and spreading activation [ 17 , 73 ] , and higher level cognitive processes such as conceptual abstraction and analogical transfer [ 20 , 28 ] , conceptual combination [ 92 ] , and problem reformulation based on examples [ 22 , 34 , 57 ] . Of particular interest in our study is a contrast between priming and spreading activation mechanisms on the one hand , which is associated with lower - level conceptual influences , and problem ( re ) formulation , which is associated with more complex , higher - order processing of examples . In this study , we extend this literature by exploring how two specific mechanisms of processing examples ( stimulation , and ( re ) formulation ) might be helped or hindered by different example interaction interfaces . To set the context for our results , we briefly review the literature on each mechanism here . 2 . 2 . 1 Using examples to stimulate ideation . Spreading activation has been invoked to explain the impact of external stimuli on ideation . For instance , the \u201csearch for ideas in associative memory \" ( SIAM ) model [ 66 ] proposes that when ideas come to mind , whether from memory , or through discussion with others or exposure to examples , they also raise the activation level of other associated concepts and features in memory , which can stimulate or inhibit ideation by making certain sets of ideas more or less likely to be generated based on the current network of associations in memory . For example , an example idea \u201cuse as paperweight \" ( for a design prompt to generate alternative use for a brick ) may activate related concepts such as \u201coffice \" , or \u201cis heavy \" , along with their associated concepts ; subsequent ideas such as \u201cconstruct a table \" , or \u201cprop up a bookshelf \" may then be more likely to come to mind , compared to ideas like \u201cuse as a weapon \" or \u201cmakeshift goalposts for soccer \" . In this way , exposure to examples can shape the trajectory of ideation , and the corresponding floor or ceiling of creativity [ 6 , 13 ] . In this paper , we discuss this set of mechanisms under the label \u201c stimulation \" , to capture the intuition of examples stimulating ideation along a particular direction . 2 . 2 . 2 Using examples to ( re ) formulate problems . Past research has documented how people can use examples to construct , refine , and even reformulate their understanding of the creative problem they are trying to solve [ 34 , 35 , 43 , 59 , 67 , 79 ] , through processes such as intentional free - form curation of examples [ 56 ] or on mood boards [ 55 ] . For instance , Okada et al . documented how two artists used individual artworks to shape not just their ideas , but used a process of \u201canalogical modification \" to search for and modify higher - order concepts , and their creative vision over the course of years [ 67 ] . This process of example - influenced problem formulation is related to computational and neurobiological models of cognitive search [ 36 ] which study the range of strategies that intelligent agents can use to Manuscript submitted to ACM 4 Chan , et al . structure their search processes : here , there is an important distinction between \u201cmodel - free \" search , where external feedback from the world on the agents\u2019 actions guide search in a simpler , more local , stimulus - response manner , and \u201cmodel - based \" search , where the agent constructs a model or representation of the task and environment ( in the case of creative problem - solving , this would be the problem and / or design space [ 22 , 30 , 65 ] ) partly on the basis of reflection its own actions and possibly observation of others\u2019 actions , and uses that model to decide where and when to explore in the task environment vs . continue to sample locally . Additionally , insight problem solving research has documented how people not only construct models , but also substantially revise them in radical ways , to solve difficult creative problems [ 43 , 45 ] ; this process is a key source of difficulty for creative problems , where one\u2019s initial problem formulation ( e . g . , key constraints or requirements ) , may be unhelpful [ 45 ] . In this paper , we discuss this set of mechanisms under the label \u201c ( re ) formulation \" , to capture the intuition of creators leveraging examples to ( re ) formulate their understanding of the design space . 2 . 3 Interactive systems for interacting with examples In this study , we are interested in understanding how the impact of examples on problem solving varies as a function of interaction design decisions for how creators will interact with examples . To set the context , we briefly review here some emerging interaction design patterns in HCI systems research into how ( vs . when , as in recommendation systems ) participants interact with sets of examples ( vs . understand or modify a single example ) . One higher - level design pattern involves explorable overviews of examples . For example , MetaMap [ 42 ] supports exploration of examples through keywords and colors and offering a playground to curate examples ; RecipeScape [ 16 ] uses a map UI to present recipe examples ; Sifter [ 70 ] presents large collections of image manipulation tutorials in a faceted view based on their command - level structure ; the Adaptive Ideas Web tool [ 53 ] enables designers to explore and structure collections of web design examples ; the Freed system [ 64 ] empowers design students to spatially organize their digital collection of examples , define relations and reflect on their interrelationships ; and Cabinet [ 44 ] supports collecting and organizing of visual examples for inspiration and reference . Another emerging design pattern can be described as contextualizing examples in the creator\u2019s workspace or problem , enabling designers to curate and reflect on the examples to build an understanding of their design space . For example , ReflectionSpace [ 78 ] interactively contextualizes design artifacts in project timelines ( and associated comments and reflections ) to promote reflection and learning ; MoodCubes [ 39 ] enables designers to curate , compare , and explore suggested 3D design elements in the context of an overall 3D \u201ccube \" room layout ; IdeateRelate [ 93 ] locates design examples in coordinates of similarities corresponding to the users\u2019 original ideas ; the IdeaMache system provides an environment for free - form visual curation and sensemaking of creative materials in the context of a project canvas [ 91 ] ; and ImageSense embeds the process of searching for , exploring , and integrating examples into both individual and shared work spaces [ 47 ] . We build on this work by directly testing how the emerging pattern of contextualizing examples might impact the effects of examples on problem solving . To facilitate downstream theoretical development , we go beyond formulations of problem solving effects and outcomes that are task - specific \u2014 such as writing code [ 9 ] , designing websites [ 50 ] , or designing room layouts [ 39 ] \u2014 and / or removed from creativity - specific mechanisms , such as browsing and searching and exploring , to more theoretically grounded descriptions of psychological mechanisms such as fixation and problem reformulation . Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 5 3 METHODS 3 . 1 The WildCat Wells Task as a Controlled Analog to Exploratory Creative Problem Solving We experimentally investigated our research questions using a controlled analog to exploratory creativity , a term introduced by Margaret Boden\u2019s influential model of creativity [ 7 ] to describe a subset of creative problem solving processes that involve exploration within a conceptual space that is often large and complex . This conception of exploratory creative problem solving as search in a space has deep roots in research on search landscapes and innovation in organization and management science [ 5 ] , as well as psychological models of problem solving [ 65 ] and creativity [ 71 ] ( as reviewed in our discussion of model - based mechanisms for using examples in 2 . 2 . 2 ) . A key insight from this literature is that local search and hill - climbing are insufficient in more rugged and complex search landscapes , because they can trap searchers in local optima ; to overcome this , searchers need to find ways to explore or \u201cjump \" to new regions of the landscape [ 5 ] , such as guiding search through ( re ) modeling of the search space [ 36 ] . This contrast between local and distant exploration is often described in terms of the shift between exploitation and exploration [ 5 ] , where the latter search dynamics are more associated with innovation and creativity [ 71 ] . Note that the notion of exploratory creativity is distinct from another important class of creative processes that involve what Boden [ 7 ] calls transformational creativity : in this form of creativity , creators search for or construct alternative problem spaces ( as discussed in the related work ) [ 22 , 43 ] , rather than search within an existing problem space as given . Our controlled analog is the WildCat Wells task . The name of the task takes inspiration from the real - world task of wildcat drilling 1 , a form of exploratory drilling for oil and gas in an unfamiliar environment where the distribution of resource - rich locations is unknown . Accordingly , in this task , participants can \u201cdrill\u201d for \u201cresources \" in a 2D grid by clicking on locations in the grid ; clicking on a grid location then uncovers a score amount , analogous to the amount of oil / gas uncovered at a drilling site . Like its real - world counterpart , the distribution of resources in this task is unknown ; in our particular instantation , participants\u2019 goal is to uncover the most resource - rich drilling location ( i . e . , the grid location with the highest score ) . Following our conceptualization of examples as descriptions / representations of possible solutions to the same / similar problem , in this task , we operationalized examples as possible grid locations and their associated scores . We chose this task for several reason . First , we had a high degree of parameter control over the properties of the task and examples , which allowed us to precisely control the ruggedness of the task structure and also employ a within - subjects design while mitigating learning effects by constructing and sampling from a set of Wildcat Wells\u2019 tasks with isomorphic ruggedness / complexity properties ( see Section 3 . 1 . 1 ) . The task structure also gave us granular and precise measures of process and outcome dynamics . Finally , the simplicity of the task allowed us to minimize the impact of prior knowledge because the task does not require specialized domain expertise . While the specific task structure in terms of distribution of rewards over the search space is unknown to participants , the generic task structure of searching a space for rewards is probably not unfamiliar to most people . The Wildcat Wells task and its operationalization of examples is also conceptually similar to other instances of exploratory creativity that may draw on example solutions from a very similar problem : for instance , when searching for effective parameter settings for wing airfoil designs , other airfoil designs \u2014 which , like our grid location , are also combinations of parameters \u2014 may serve as relevant examples ; when designing effective ads for a vaccine persuasion campaign , other vaccine persuasion ads \u2014 which are also combinations of design features \u2014 may serve as relevant examples ; and when designing effective UI elements , other websites and their UI elements \u2014 which are also compositions of UI features \u2014 may serve as relevant examples . We also 1 https : / / en . wikipedia . org / wiki / Wildcatter Manuscript submitted to ACM 6 Chan , et al . Fig . 1 . Example Wildcat Wells search environment with color coding of points to indicate their scores ( 0 - 50 : dark blue to light blue , 50 - 100 : light red to dark red ) ( A ) , and example sets of high and low diversity sets of points in this search environment , which are given as examples ( B ) . adapted the Wildcat Wells task specifically from a prior study [ 62 ] of the dynamics of exploration and exploitation in collaborative problem solving . However , because the WildCat wells task is only analogous to exploratory creativity , our results here can only speak to effects of examples on exploratory , but not transformational , creative problem solving . 3 . 1 . 1 Search Environments . Our WildCat Wells search environments consisted of a 100x100 grid of points ( with corresponding scores controlled by a synthetic objective function that determines the distribution of scores ; see Algorithm 1 in Appendix A and our source code for generating search environments ) . Figure 1 ( A ) shows a representative search environment we used in our experiment . Our goal was to more closely match the difficult search spaces that the creativity theorist David Perkins calls \u201cKlondike spaces \" [ 71 ] , which are environments where simple \u201chill - climbing \" exploration strategies are insufficient , and likely outperformed by other creative exploration strategies such as a mix of exploration and exploitation [ 5 , 71 ] . We describe the specific parameter settings and algorithm we used to generate these task environments in Appendix A ( and share the code generating the environments in the Supplementary Material ) ; here , we note that we set the parameters to yield a search environment that was fairly rugged ( adding more false \u201cpeaks \" to incorrectly intuit as the location of the maximum score ) and locally noisy ( reducing the local correlation between scores in the grid , such that searchers would often be surprised by the score of nearby regions in the grid ) . The parameters to generate search environments were determined by a series of rubrics ( e . g . more than one area with scores higher than 80 ) , and pilots for a qualitative sense of difficulty based on the topology of the solution space . To reduce the likelihood that our results were tied to a specific formulation of the search environment , we generated ten search environments with the same synthetic objective function parameters but different random seeds . The resulting search environments were qualitatively similar to Mason & Duncan\u2019s work [ 62 ] . 3 . 1 . 2 Examples . To prepare sets of initial examples with High Diversity ( HD ) or Low Diversity ( LD ) , we randomly generated 10 , 000 sets of 10 examples each ( recall that each example is a \" drilling location \" point in the 100x100 Wildcat Wells search environment , with a corresponding score ) for each of our 10 search environments , and ranked the diversity of each example set with a close variant of the Determinantal Point Process ( DPP ) approach [ 49 ] described in Algorithm 2 in Appendix B proposed by Eesh et al . [ 41 ] ; intuitively , this approach measured the \u201chyper - volume \" spanned by a Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 7 selected set of points , such that larger volumes corresponded to higher levels of diversity , since these points spanned a larger set of the space of possible moves [ 49 ] . We then randomly picked three HD examples sets that were greater than the 99th percentile of the distribution of diversity across the example sets , and three LD examples sets with diversity lower than 1st percentile of the diversity distribution . To ensure that examples would not directly reveal the location of the peak , or provide a high enough score that participants might simply stop after seeing the example instead of searching , we discarded example sets that had any point with a score over 80 ( scores in the search environment ranged from 0 to 100 ) , and resampled example sets as necessary \u2014 subject to the same low / high diversity sampling criteria ) to construct our final example sets for each search environment . Figure 1 ( B ) shows an LD and HD example set used in our experiment . 3 . 2 Experiment Design We conducted a mixed design experiment . Example interface was a between - subjects factor , with three conditions : 1 ) \u201cparallel examples with context \" interface : all 10 examples were shown in the 100x100 space with color coding to denote the score associated with each point , referred to as the \u201c In - Context \" interface 2 ) \u201cparallel examples without context \" interface ( shown in Figure 2 ) : all 10 examples were shown in a list , also with color coding to indicate example score , referred to as the \u201c List \" interface 3 ) \u201cserial examples without context \" interface : only one example was shown at a time and the participant needed to use a dropdown button to see other examples , referred to as the \u201c Dropdown \" interface . Figure 2 shows the experimental interface of the List condition as an example . The In - Context interface was inspired by design patterns of example interfaces that contextualized examples in the creator\u2019s workspace or problem ( e . g . , [ 39 , 47 , 78 , 91 ] The List interface was inspired by the familiar design pattern of a \u201clist \" of examples , often in the context of a search interface ( as search results ) , or list of recommendations in a recommender interface . The Dropdown interface was designed to approximate more constrained interfaces for interacting with examples , such as through chat - based or recommendation systems ( e . g . , popping up one or two examples at a time ) . The three example interfaces were shown in the context of the WildCat Wells task in Figure 3 . We conjectured that interfaces that allowed for comparison between examples ( whether in the context of a task environment , as in the In - Context interface , or just with attributes shown for comparison , as in the List interface ) might facilitate more model - based usage of examples ( what we called a \u201c ( re ) modeling \" mechanism in 2 . 2 ) . Since we designed our Wildcat Wells task to be unsuitable for simpler hill - climbing ( e . g . , \u201cstimulation - based \" mechanism as described in 2 . 2 ) , we also expected that these interfaces might also lead to better performance on the task , through , for example , model - based exploration strategies . Example diversity was a within - subjects factor : each participant attempted the WildCat Wells task twice , once with a set of HD examples , and once with LD examples . Recall that we generated 10 variant search environments , each with their own set of HD and LD example sets . To approximate counterbalancing of our within - subjects factor , we created 2 \u201crun \" variants for each search environment , with each variant having an HD or LD example set as the first trial . Participants were randomly assigned first to an example interface condition , and then randomly assigned to one of the 20 potential \u201cruns \" in each interface condition ( but constraining assignment such that participants would not see the same search environment twice ) . Based on prior research on example diversity , we expected that participants would perform better when given high vs . low diversity example sets . Manuscript submitted to ACM 8 Chan , et al . Fig . 2 . Screenshot of experimental interface , shown for the List condition : the 100x100 grid , which constituted the search environment for the task , was shown on the left panel : participants explored the space by clicking anywhere on the 100x100 grid . The 10 initial examples , moves remaining , the score of current move , the current max score and score legend were shown on the right panel . In the Dropdown condition , the dropdown menu as seen in Figure 3 was shown in the same position as the list of examples in the List condition . In the In - Context condition , examples were instead overlaid as points , with corresponding values , on the search grid , as shown in Figure 3 . 3 . 3 Participants We recruited participants from the Amazon MTurk platform , limiting participants to U . S . residents with more than 500 HITs with at least 99 % approval rate . Each participant was paid US $ 1 . 3 for their participation , which was an effective rate of $ 10 per hour , given the average task completion time of 8 minutes . We aimed for a total sample size of 195 ( 65 per each of the three conditions ) , to achieve target statistical power of over 0 . 80 to detect medium - sized statistical effects in a mixed between - within design experiment analysis . After rejecting invalid work of 42 participants for irrelevant responses ( e . g . , \u201cnice \" ) to the closing survey question about how they used examples ) , we obtained data from 182 participants ( 63 females , 118 males , 1 other ; 65 in context , 56 List , and 61 Dropdown ) in total , yielding an effective statistical power of 0 . 86 for medium - sized effects . 3 . 4 Experimental Procedures Participants experienced the WildCat Wells task as a 100x100 space ( see Figure 2 ) . Their task was to find the square with the highest score . Participants explored squares by clicking on them to reveal their underlying score , shown in color coding , similar to the examples . To simulate the constrained nature of real creative tasks ( which often have some time / budget pressure ) and reduce the likelihood of ceiling effects , participants had a total budget of 60 moves for exploring squares . This budget was estimated from our pilot studies , where on average , most participants found the highest scoring square within 50 moves . We also provided incentives to encourage participants\u2019 exploration : there was a $ 0 . 25 bonus for achieving a highest score greater than 95 , and a $ 0 . 50 bonus for achieving the maximum score of 100 . The information panel on the right side of the experimental interface ( see Figure 2 showed moves remaining , the score of the current exploration and the maximum score the participant had achieved in the current round . Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 9 Fig . 3 . Three conditions of presenting examples : \u201c In - Context \" ( directly on the search environment grid ) , \u201c List \" ( in a list ) and \u201c Dropdown \" ( in a clickable dropdown selector ) . Since the WildCat Wells task does not interact strongly with prior knowledge in any particular domain , we addressed potential pre - existing differences in ability by measuring participants\u2019 baseline divergent thinking ability , a correlate of creative ability [ 75 ] . Before the study , we asked participants to generate as many alternative uses of coffee cup as they could in 2 minutes ( an instance of the commonly used Alternative Uses task [ 32 ] for measuring divergent thinking [ 75 ] ) . Participants were then given one trial round through the WildCat Wells task ( without examples ) to familiarize them with the interface and task . After that , participants completed two formal rounds of the WildCat Wells task , which constituted the main experimental trials in our study . Finally , participants completed a post - study questionnaire , with three free - response questions : 1 ) What strategy did you use for hunting ? 2 ) How did you use initial examples ( the values of ten points given to you ) ? 3 ) What differences did you notice between initial examples given in those two rounds ? Which did you find helpful ? We obtained institutional IRB approval for the whole project prior to the study . 4 RESULTS : PLANNED ANALYSES 4 . 1 No significant differences in baseline divergent thinking ability across interface conditions We first report the results of our check for random assignment with respect to divergent thinking ability and baseline performance on our task . We observed no statistically significant difference in the number of generated alternative uses across three conditions ( \" In - Context \" participants : \ud835\udc40 = 6 . 52 , \ud835\udc46\ud835\udc37 = 3 . 02 ; \u201c List \" participants : \ud835\udc40 = 5 . 75 , \ud835\udc46\ud835\udc37 = 3 . 58 ; \u201c Dropdown \" participants : \ud835\udc40 = 6 . 46 , \ud835\udc46\ud835\udc37 = 3 . 87 , Kruskal - Wallis \ud835\udc3b = 2 . 40 , \ud835\udc5d = 0 . 30 ) . Similarly , we observed no statistically significant difference in participants\u2019 best score on the trial run of the Wildcat Wells task across the conditions ( \" In - Context \" participants : \ud835\udc40 = 90 . 97 , \ud835\udc46\ud835\udc37 = 7 . 52 ; \u201c List \" participants : \ud835\udc40 = 90 . 91 , \ud835\udc46\ud835\udc37 = 7 . 13 ; \u201c Dropdown \" participants : \ud835\udc40 = 91 . 18 , \ud835\udc46\ud835\udc37 = 7 . 37 , Kruskal - Wallis \ud835\udc3b = 0 . 19 , \ud835\udc5d = 0 . 91 ) . This suggests that participants across the interface conditions were comparable in terms of baseline divergent thinking ability as well as baseline task performance . 4 . 2 List presentation of examples and low diversity example sets associated with lower best scores The List condition had slightly lower scores on average compared to the other conditions ( regardless of example diversity ; Fig . 4 , top right ) . There was also an overall slight advantage of HD examples over LD examples ( Fig . 4 , bottom right ) . A linear mixed effects model with best score as the dependent variable , interface condition and example diversity as factors , and random intercepts for participants ( estimated in the \u2018lme4\u2018 package in \u2018R\u2018 ) , showed a significant main Manuscript submitted to ACM 10 Chan , et al . Fig . 4 . Distribution of best scores by interface and example diversity conditions . Participants in the List interface condition had lower best scores than participants in the other interface conditions regardless of example diversity ( top right ) . Best scores were also lower when participants were given low vs . high diversity examples ( bottom right ) . effect of interface condition , F ( 2 , 179 ) = 5 . 92 , \ud835\udc5d < . 01 , \ud835\udc52\ud835\udc61\ud835\udc4e 2 = 0 . 06 . Pairwise post - hoc comparisons with Bonferroni corrections showed that participants in the List interface condition had significantly lower best scores ( est . marginal mean = 90 . 4 , SE = 0 . 65 ) compared to both the In - Context ( est . marginal mean = 92 . 7 , SE = 0 . 61 , contrast t ratio = 2 . 54 , \ud835\udc5d < . 05 ) and Dropdown interface conditions ( est . marginal mean = 93 . 4 , SE = 0 . 63 , contrast t ratio = 3 . 31 , \ud835\udc5d < . 01 ) . There was also a significant main effect of example diversity , F ( 1 , 181 ) = 4 . 59 , \ud835\udc5d < . 05 , \ud835\udc52\ud835\udc61\ud835\udc4e 2 = 0 . 02 ; post - hoc comparisons with Bonferroni corrections showed that participants had higher best scores when they received HD ( est . marginal mean = 92 . 7 , SE = 0 . 46 ) vs . LD examples ( est . marginal mean = 91 . 5 , SE = 0 . 46 , contrast t ratio = 2 . 14 , \ud835\udc5d < . 05 ) . 5 RESULTS : EXPLORATORY ANALYSES We conducted a set of exploratory analyses to better understand the results of our main planned analyses , focusing on understanding process effects of interface conditions that might plausibly explain performance differences . 5 . 1 In - Context presentation of examples associated with early performance advantages , and List presentation of examples with early and persistent performance disadvantages First , for a more granular view of performance , we examined how the participants\u2019 best score changed as a function of their move sequence . This analysis confirmed a cumulative disadvantage for participants in the List condition , but also showed an early advantage for the In - Context interface , particularly with LD examples ( see Figure 5 ) . Using a Kruskal - Wallis H - test on the current max score from the 1st to the 30th move , we observed statistically significant differences from the 1st move to the 6th move and the 8th move except the 7th move ( see Table 1 ) . 5 . 2 Variations in example presentation interfaces associated with different self - reported example usage strategies Next , to understand how participants used the initial examples in their exploration , two researchers coded participants\u2019 responses to the question \u201cHow did you use initial examples ( the values of ten points given to you ) ? \" with three codes : Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 11 Fig . 5 . Maximum score at n - th move for each participant : left ) HD ; right ) LD . We observe ( 1 ) cumulative disadvantages for the List condition , as well as ( 2 ) early advantages for the In - Context interface , especially with LD examples . n - th move In - Context ( \ud835\udc40 ) List ( \ud835\udc40 ) Dropdown ( \ud835\udc40 ) Statistic p - value 1 60 . 06 52 . 30 50 . 64 9 . 13 0 . 01 2 67 . 65 60 . 45 61 . 39 6 . 93 0 . 03 3 74 . 00 66 . 95 67 . 89 9 . 58 0 . 008 4 76 . 65 70 . 54 70 . 84 9 . 06 0 . 01 5 78 . 20 71 . 93 73 . 66 10 . 13 0 . 006 6 79 . 15 73 . 07 74 . 59 9 . 59 0 . 008 7 79 . 98 75 . 07 76 . 87 5 . 93 0 . 06 8 81 . 46 76 . 86 78 . 21 6 . 91 0 . 03 Table 1 . Means ( \ud835\udc40 ) and results of Kruskal - Wallis H - test on the current best score for three interface conditions with LD examples . There were statistically significant differences between the conditions from the 1st to 8th moves ( p < 0 . 05 except the 7th move ) . not using , stimulation - based and model - based . This classification was guided and refined by our initial theoretical interest in the contrast between stimulation - based and ( re ) modeling - based use of examples , as discussed in 2 . 2 . Examples of responses coded as \u201cnot using \" include \" I did not give much thought to it \" , and \" Not much to be honest \" ; examples of \u201cstimulation - based \" responses included \" Start at the reddest one and explore its surroundings \" , and \" I looked around the higher values for boxes that were darker \" ; examples of \u201cmodel - based \" responses include \" To get an overview on which squares would be best \" , and \" They gave a vague idea of whether or not there might be \u201chot \" or \u201ccool \" zones around those points \" . When we could not infer how the participants used the initial examples , the answers were coded as \u201cunclear \" . The researchers were blinded to condition during coding . Inter - rater reliability was substantial , at Cohen\u2019s \ud835\udf05 = 0 . 725 [ 51 ] ; all disagreements were resolved by discussion . The In - Context condition had the largest portion ( 30 . 8 % ) of participants who self - reported using the initial examples to model the space , compared to the List condition ( 10 . 7 % ) and the Dropdown condition ( 11 . 5 % ) ( see Figure 6 ) . In contrast , for self - reported use of initial examples to stimulate their exploration , the List condition had the highest percentage ( 42 . 9 % ) followed by the Dropdown condition ( 29 . 5 % ) and the in context condition ( 29 . 2 % ) . Finally , 17 / 61 ( 27 . 9 % ) Dropdown participants self - reported that they were not using examples , which was higher than participants in the other two conditions . Our log data were consistent with this observation : 37 / 61 ( 60 . 7 % ) participants in the Dropdown Manuscript submitted to ACM 12 Chan , et al . Fig . 6 . Raw proportion of participants expressed \u201cnot using ( examples ) \" , \u201cstimulation - based \" or \u201cmodel - based \" in their answer to \u201cHow did you use initial examples ( the values of ten points given to you ) ? \" . Error bars are standard error of proportion . More participants self - reported using a model - based strategy in the In - Context condition compared to other conditions . condition never clicked the dropdown button to see other examples in both HD and LD conditions . Of the remaining participants who did click on the dropdown button , we infer \u2014 assuming that each subsequent click corresponds to an example view \u2014 that the mean number of examples viewed was M = 7 . 37 ( SD = 3 . 40 ) for HD , and M = 7 . 05 ( SD = 3 . 61 ) for LD . 5 . 2 . 1 In - Context participants more likely than other interface conditions to self - report model - based example usage , and Dropdown participants more likely than other conditions to self - report not - using examples . We first statistically tested these patterns with a series of logistic regressions , one for each example strategy ( not - using , stimulation - based , and model - based ) ( see Table 2 ) . We ran separate logistic regressions rather than a single multinomial regression given our interest at this step in the relative likelihood across interface conditions of self - reporting a particular example strategy , rather than relative differences across strategies within each condition ( best answered by a multinomial logistic regression ) . We first observe that participants in the List and Dropdown conditions were less likely to self - report using a model - based strategy compared to the In - Context condition ( \ud835\udc35 = - 1 . 31 , 95 % CI = [ - 2 . 31 , - 0 . 31 ] , z = - 2 . 57 , \ud835\udc5d < . 05 for List vs . In - Context , and \ud835\udc35 = - 1 . 23 , 95 % CI = [ - 2 . 18 , - 0 . 29 ] , z = - 2 . 55 , \ud835\udc5d < . 01 for Dropdown vs . In - Context ) . In more intuitive terms , In - Context participants were approximately 3x more likely to self - report using a model - based example usage strategy compared to List or Dropdown participants ( Odds Ratio = 3 . 7 and 3 . 4 for In - Context vs . List , and In - Context vs . Dropdown ) . The overall model fit was better than a null model ( \ud835\udc3f\ud835\udc3f \ud835\udc5a\ud835\udc5c\ud835\udc51\ud835\udc52\ud835\udc59 = - 80 . 93 vs . \ud835\udc3f\ud835\udc3f \ud835\udc5b\ud835\udc62\ud835\udc59\ud835\udc59 = - 86 . 16 ) , Likelihood Ratio \ud835\udf12 2 ( 2 ) = 5 . 23 , \ud835\udc5d < . 01 . Next , we observe that participants in the In - Context condition were less likely to self - report not using examples compared to the Dropdown condition ( \ud835\udc35 = - 1 . 01 , 95 % CI = [ - 1 . 94 , - 0 . 09 ] , z = 2 . 14 , \ud835\udc5d < . 05 ) ; in more intuitive odds ratio terms , Dropdown participants were 2 . 7x more likely than In - Context participants to self - report a \u201cnot using \" strategy ( Odds Ratio = 2 . 75 ) . The overall model fit , though better than a null model ( \ud835\udc3f\ud835\udc3f \ud835\udc5a\ud835\udc5c\ud835\udc51\ud835\udc52\ud835\udc59 = - 86 . 62 vs . \ud835\udc3f\ud835\udc3f \ud835\udc5b\ud835\udc62\ud835\udc59\ud835\udc59 = - 89 . 10 ) , was marginally significant , Likelihood Ratio \ud835\udf12 2 ( 2 ) = 5 . 14 , \ud835\udc5d = . 08 . Finally , we observe that there were no significant differences across conditions in the likelihood of self - reporting a stimulation - based strategy . Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 13 Not using Stimulation - based Model - based Intercept - 0 . 951 [ - 1 . 511 , - 0 . 391 ] - 0 . 288 [ - 0 . 817 , 0 . 242 ] - 0 . 811 [ - 1 . 338 , - 0 . 284 ] In - Context - 1 . 013 [ - 1 . 940 , - 0 . 085 ] * - 0 . 597 [ - 1 . 349 , 0 . 156 ] List - 0 . 575 [ - 1 . 459 , 0 . 309 ] - 1 . 309 [ - 2 . 307 , - 0 . 312 ] * * Dropdown - 0 . 583 [ - 1 . 347 , 0 . 180 ] - 1 . 232 [ - 2 . 179 , - 0 . 285 ] * Table 2 . Coefficient estimates from multinomial logistic regressions of probability of self - reported example usage strategy ( 1 each for not - using , stimulation - based , model - based ) on interface condition . Statistics reported as \" coefficient , 95 % CI ( [ lower , upper ] ) \" . When the cell for a given interface condition is blank , that condition was used as the reference class in the regression . * p < . 05 ; * * p < . 01 ; * * * p < . 001 . Indeed , the overall model fit , though nominally better than the null model ( \ud835\udc3f\ud835\udc3f \ud835\udc5a\ud835\udc5c\ud835\udc51\ud835\udc52\ud835\udc59 = \u2212 114 . 52 vs . \ud835\udc3f\ud835\udc3f \ud835\udc5b\ud835\udc62\ud835\udc59\ud835\udc59 = \u2212 116 . 08 was not statistically significant , Likelihood Ratio \ud835\udf12 2 ( 2 ) = 1 . 56 , \ud835\udc5d = . 21 . 5 . 2 . 2 List participants more likely to self - report a stimulation - based example usage strategy compared to not - using or model - based example usage . Next , we focus on statistically evaluating the apparent predominance of a stimulation - based self - reported example usage strategy for List participants . We fitted a multinomial logistic regression , with model - based usage as the reference outcome class . Participants in the List condition were significantly more likely to self - report using a stimulation - based strategy compared to a model - based one , \ud835\udc35 = \u2212 1 . 44 , 95 % CI = [ - 0 . 34 , - 2 . 53 ] , z = - 2 . 58 , \ud835\udc5d < . 01 . In odds ratio terms , participants in the List interface condition were 4x more likely to self - report a stimulation - based vs . model - based strategy ( Odds Ratio = 4 . 21 ) . The overall model fit was statistically significantly better than a null model , Likelihood Ratio \ud835\udf12 2 ( 4 ) = 14 . 39 , \ud835\udc5d < . 01 . 5 . 3 List presentation of examples associated with more local initial exploration of the solution space Finally , we explored how log data might be consistent ( or not ) with participants\u2019 self - reported example usage strategies . We wanted to study how initial examples would affect participants\u2019 exploration behaviors , especially at the beginning of exploration when the examples provided were a major source of information . To explore this , we first constructed an exploration graph for the first 30 moves of each participant trial by computing Euclidean distances between each successive move ; the intuition was that long sequences of low distances between moves would suggest \u201chill - climbing \" , and large distances would suggest \u201cjumps \" . We conjectured that a \u201chill - climbing \" - like exploration graph would be consistent with a stimulation - based strategy , rather than a model - based strategy . Two coders independently coded all 364 exploration graphs ( each of the 182 participants had an HD plot and a LD plot ) , coding whether the exploration behaviors were hillclimbing ( h ) or not ( n ) for sequences of 10 moves ( the 0th - 10th , the 10th - 20th , the 20 - 30th ) . Two examples of coding are shown in Figure 7 . We coded 1092 10 - move instances ( 3 10 - move instances per round x 2 rounds per participant x 182 participants ) with substantial inter - rater reliability , Cohen\u2019s \ud835\udf05 = 0 . 78 . For all trials , a higher proportion of participants in the List interface condition used an exclusively hill - climbing strat - egy for the first 30 moves ( proportion = 0 . 161 , lower bound = 0 . 112 , upper bound = 0 . 210 ) compared to the In - Context ( pro - portion = 0 . 046 , lower bound = 0 . 020 , upper bound = 0 . 072 ) and Dropdown condition ( proportion = 0 . 082 , lower bound = 0 . 047 , upper bound = 0 . 117 ; see Figure 8aa ) . Similarly , for LD trials , the proportion of participants in using an exclusively hill - climbing strategy for the first 30 moves was higher for the List condition ( 34 % ) compared to the In - Context ( 18 % ) and Dropdown condition ( 18 % ) ( Fig . 8ab , left ) . In contrast , for HD trials , the proportion of participants using an Manuscript submitted to ACM 14 Chan , et al . Fig . 7 . Example exploration graphs used for coding hill - climbing strategies : not \u201call hill - climbing \" ( left ) and \u201call hill - climbing \" ( right ) , where each transition between participant moves is plotted on the x - axis , and the Euclidean distance between each move and its immediately preceding move is plotted on the y - axis . In the not \u201call hill - climbing \" example , the first 10 moves ( the 0th - 10th moves ) , where there are substantial variations in Dropdown move distances across the sequences , would be coded as non - hillclimbing ( N ) , while the 10th - 20th moves and the 20th - 30th moves , where Dropdown move distances are consistently low , would be coded as hillclimbing ( H ) . In the \u201call hill - climbing \" example , all first 30 moves would be coded as hillclimbing ( H ) . exclusively hill - climbing strategy for the first 30 moves was similar for the In - Context ( 18 % ) , List ( 25 % ) , and Dropdown conditions ( 23 % ) ( Fig . 8ab , left ) . To statistically test these observations , we fitted a series of logistic regressions , estimated with maximum likelihood , predicting \ud835\udc5d ( \ud835\udc4e\ud835\udc59\ud835\udc59 _ \u210e\ud835\udc56\ud835\udc59\ud835\udc59 ) , the probability of being all hill - climbing in the first 30 moves as a function of \ud835\udc56\ud835\udc5b\ud835\udc61\ud835\udc52\ud835\udc5f\ud835\udc53 \ud835\udc4e\ud835\udc50\ud835\udc52 . Prior work suggests that choice of exploration vs . exploitation is influenced by the \u201cgoodness \" of the current region of the search space ( better scores makes hill - climbing more likely ) [ 5 , 36 ] . Our data confirmed this pattern : the average score of the first move in each of the first three 10 - move blocks was positively correlated with the likelihood of being all hill - climbing in the first 30 moves , Kendall\u2019s \ud835\udf0f = . 17 , \ud835\udc5d < . 01 . Thus , we conditioned our logistic regression models on the average score at the beginning of each 10 - move block . We first analyzed \ud835\udc5d ( \ud835\udc4e\ud835\udc59\ud835\udc59 _ \u210e\ud835\udc56\ud835\udc59\ud835\udc59 ) aggregated across both HD and LD trials ( value would be 1 if both LD and HD trials were 1 ) , and then HD and LD trials separately . Table 3 shows the coefficient estimates for each of these models . For all trials , the coefficient for the contrast between the List and In - Context conditions was \ud835\udc35 = - 1 . 79 , 95 % CI = [ - 3 . 40 , - 0 . 45 ] , z = - 2 . 45 , \ud835\udc5d < . 05 ; in odds ratio terms , participants in the List condition were 6x more likely to use an exclusively hill - climbing strategy for the first 30 moves , compared to participants in the In - Context condition ( Odds Ratio = 5 . 99 ) . Note that this effect was independent of the significant positive coefficient for the average first score in the block . The overall model fit was statistically significantly better than a null model ( \ud835\udc3f\ud835\udc3f \ud835\udc5a\ud835\udc5c\ud835\udc51\ud835\udc52\ud835\udc59 = - 48 . 42 vs . \ud835\udc3f\ud835\udc3f \ud835\udc5b\ud835\udc62\ud835\udc59\ud835\udc59 = - 56 . 48 ) , Likelihood Ratio test \ud835\udf12 2 ( 3 ) = 16 . 12 , \ud835\udc5d < . 01 . Similarly , for LD trials , there was a statistically significant coefficient in the logistic regression model for the contrast between the In - Context and List conditions , \ud835\udc35 = - 0 . 95 , 95 % CI = [ - 1 . 83 , - 0 . 10 ] , z = - 2 . 16 , \ud835\udc5d < . 05 . In odds ratio terms , participants in the List condition were 2 . 5x more likely to use an exclusively hill - climbing strategy for the first 30 moves , compared to participants in the In - Context condition ( Odds Ratio = 2 . 58 ) . There was a similar contrast between the Dropdown and List conditions , \ud835\udc35 = - 0 . 95 , 95 % CI = [ - 1 . 85 , - 0 . 10 ] , z = - 2 . 12 , \ud835\udc5d < . 05 . As with the all trials model , this effect was independent of the significant positive coefficient for the average first score in the block . The overall model fit was statistically significantly better than a null model ( \ud835\udc3f\ud835\udc3f \ud835\udc5a\ud835\udc5c\ud835\udc51\ud835\udc52\ud835\udc59 = 92 . 98 vs . \ud835\udc3f\ud835\udc3f \ud835\udc5b\ud835\udc62\ud835\udc59\ud835\udc59 = \u2212 98 . 32 ) , Likelihood Ratio test \ud835\udf12 2 ( 3 ) = 10 . 67 , \ud835\udc5d < . 05 . In contrast , there were no statistically significant contrasts Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 15 All trials HD trials LD trials Intercept - 7 . 322 [ - 11 . 73 , - 3 . 739 ] - 1 . 734 [ - 3 . 522 , - 0 . 052 ] - 2 . 551 [ - 4 . 409 , - 0 . 870 ] In - Context vs . List - 1 . 789 [ - 3 . 400 , - 0 . 449 ] * - 0 . 431 [ - 1 . 325 , 0 . 448 ] - 0 . 950 [ - 1 . 833 , - 0 . 103 ] * Dropdown vs . List - 0 . 926 [ - 2 . 220 , 0 . 260 ] - 0 . 113 [ - 1 . 325 , 0 . 448 ] - 0 . 946 [ - 1 . 850 , - 0 . 0858 ] * * Avg . first score in block 0 . 089 [ 0 . 035 , 0 . 152 ] * * 0 . 010 [ - 0 . 016 , 0 . 037 ] 0 . 031 [ 0 . 005 , 0 . 059 ] * Table 3 . Coefficient estimates from logistic regressions of probability of predominantly hill - climbing strategy in the first 30 moves on interface condition and average first score in block , across all , HD , and LD trials . Statistics reported as \" coefficient , 95 % CI ( [ lower , upper ] ) \" . * p < . 05 ; * * p < . 01 ; * * * p < . 001 . ( a ) ( b ) Fig . 8 . Raw proportion of participants with a predominantly hillclimbing strategy in the first 30 moves , across interface conditions ( a ) with both HD and LD examples ( b ) . More \u201c List \" participants did hillclimbing in the first 30 moves with both high and low diverse example sets than \u201c In - Context \" participants . More \u201c List \u201d participants did hillclimbing for the first 30 moves with LD examples than other combinations of the presentation and example sets . between conditions in the HD trials , though the numerical pattern of results were similar to the other models ( generally negative coefficients for In - Context and Dropdown vs . List conditions ) . The overall model fit , though nominally better than a null model ( \ud835\udc3f\ud835\udc3f \ud835\udc5a\ud835\udc5c\ud835\udc51\ud835\udc52\ud835\udc59 = \u2212 95 . 14 vs . \ud835\udc3f\ud835\udc3f \ud835\udc5b\ud835\udc62\ud835\udc59\ud835\udc59 = \u2212 95 . 85 , was not statistically significant , Likelihood Ratio \ud835\udf12 2 ( 3 ) = 1 . 42 , \ud835\udc5d = . 70 . Because we were concerned this pattern of differences might be driven by pre - existing individual differences in propensity to hill - climbing , rather than a shift due to the interface condition , we repeated our coding procedure for exploration graphs generated from the initial trial round of exploration , which did not include examples ( described in 3 . 4 . The proportion of participants who displayed a predominant hill - climbing strategy , as described above , was distributed across conditions as follows : In - Context = 0 . 05 ( SE = . 03 ) , List = . 07 ( SE = . 03 ) , and Dropdown = 0 . 15 ( SE = . 05 ) . A logistic regression predicting the probability of a predominant hill - climbing strategy ( yes or no ) as a function of interface did not improve fit over a null model with no predictors , \ud835\udf12 2 ( 2 ) = 4 . 17 , \ud835\udc5d = . 12 ; note , however that the overall frequency of hill - climbing strategies was lower than in the main trials , and the List condition was not the condition with highest frequency ( in contrast to the main trials ) . There was also no significant correlation between the likelihood of hill - climbing predominance and either the number of alternative uses task responses , r = . 04 , \ud835\udc5d = . 61 , or the likelihood of hill - climbing predominance in the main trials , r = . 03 , \ud835\udc5d = . 65 . Altogether , these results are inconsistent with the alternative explanation that List participants were simply more likely ( due to individual differences ) to choose a predominantly hill - climbing strategy overall ; instead , taken together with the survey results , we believe this set of Manuscript submitted to ACM 16 Chan , et al . results suggest a shift in strategy towards hill - climbing ( or , as we describe in this paper , a stimulation - based strategy for using examples ) . 6 DISCUSSION AND CONCLUSION 6 . 1 Summary and Interpretation of Results In this paper , we aimed to contribute to a theory of human - example interaction to guide the design of example - based creativity support tools . Towards this goal , we conducted an experiment with a controlled analog to an exploratory creativity [ 7 ] task to investigate how example presentation interface variations influence whether / how people benefit from examples . We found evidence that List presentation of examples might harm the quality of final solutions . For example we found variations across interface conditions in mean best score obtained at the end of trials across example interface and diversity conditions ( List participants had worse best scores compared to In - Context and Dropdown participants ; Section 4 . 2 ) and cumulative performance differences ( with an early and persistent disadvantage of the List condition compared to the other conditions , with an especially pronounced early disadvantage relative to the In - Context condition ; Section 5 . 1 ) . This result is conceptually significant because the \u201c List \u201d participants received more information ( seeing all 10 examples at the same time ) than the \u201c Dropdown \u201d participants ( only seeing 1 example at a time . and over 60 % of them never checked other examples ) , and approximately equivalent information but different presentation compared with the \u201c In - Context \u201d condition . This suggests that seemingly unimportant , low - level interaction design decisions with respect to presentation of examples can have measurable consequences for creative problem solving performance . Separately , we also observed beneficial effects of diversity for final solution quality , in line with some previous work [ 2 , 4 , 29 , 38 , 80 , 96 ] ; importantly , this effect was similar in magnitude to the example presentation effects , suggesting that example presentation considerations may be just as important to consider as example characteristics when designing example - based creativity support systems . Second , our exploratory analyses suggest that \" In - Context \" and \u201c List \" presentation of examples may lead to distinct patterns of example usage . \u201c In - Context \u201d presentation of initial examples was associated with a greater likelihood of a \u201cmodel - based \" strategy for using examples , where participants self - reported using the examples to gain an overall understanding of the distribution of score in the search environment to guide their exploration , compared to List or Dropdown presentations . Conversely , the \u201c List \u201d presentation of initial examples seemed to encourage a predominant \u201cstimulation - based \" example usage strategy , where participants selected promising examples as starting points for their exploration . Importantly this self - report data was consistent with patterns in our log data : we observed that List participants were more likely to use a predominantly \u201chill - climbing \" strategy ( with low Dropdown distance between their moves ) early in their exploration , relative to the In - Context and Dropdown participants ; this association was independent of the relationship between hill - climbing behavior and the \u201cgoodness \" of initial moves ( hill - climbing in a given block of moves was more likely when the initial move was higher - scoring , consistent with prior empirical work on exploration / exploitation decisions [ 5 , 36 ] ) . Considering these results alongside the performance results suggests that List participants were being fixated [ 40 ] by the examples . A fruitful direction for further research would be to investigate the mechanisms that drive fixation in the List condition . One reason might be the upper limit in scores ( no more than 80 / 100 ) on the examples presented to the participants ; if taken as starting points to begin hill - climbing , those relatively low quality examples could be misleading , and block access to high - quality solutions . Another reason might be the increased effort needed to connect examples of Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 17 the List condition to the search space , which would be consistent with past research on the cognitive load benefits of integrating diagrams and text ( similar to integrating examples and the search space ) in instructional design [ 14 ] . We view the difficulty of transferring from the text modality of lists of examples to the visuospatial modality of the In - Context solution space as a potential mechanism by which example presentation variations might shape their impact on ideation : future work could investigate in more detail how different example presentation designs might shift the cost structure of different processing strategies , in a similar way that variations in environment or interface structure have been shown to shape sensemaking by changing the cost structure of various crucial actions , such as skimming / previewing , moving documents , applying schemas to documents , or adjusting schemas [ 72 , 76 , 77 ] . Separately , we observed that the \u201c Dropdown \u201d presentation was associated with limited usage of the examples : many Dropdown participants self - reported not using examples ( moreso than In - Context participants , for example ) , and this was also corroborated in their log data ( via a lack of interaction with the example interface ) . We do not think that this lack of example usage is indicative of a lack of engagement : recall , for instance , that performance in this condition was on par with the In - Context condition ( i . e . , higher than in the List condition ) . Post - survey comments indicating enjoyment and engagement ( e . g . , \" Fun game . Thank you ! \" ) were also seen across conditions at similar rates , and there were no statistically significant mean differences across the conditions in the trial run of the task . For these reasons , we believe that \u2014 possibly due to the interaction affordances \u2014 the Dropdown condition appeared to act similarly to a \" no - examples \" control condition , where participants used a wider mix of strategies vs . a particular set of example - based strategies tied to an experimental intervention . In light of this , the overall strong performance of the Dropdown condition is akin to past observations of strong performance by control \u201cno - intervention \" conditions in ideation experiments ( see , e . g . , [ 11 , 80 ] ; we thus add to a growing body of evidence that it may be easier to harm rather than help creative ideation by intervening ( as in the List condition ) . Overall , our results suggest that interaction design considerations for human - example interaction go beyond usability : there is indeed a space of mappings to explore between design affordances and fundamental psychological mechanisms of creative inspiration from examples . From a practical standpoint , our empirical results suggest the limitations of only showing examples without the problem space as context , especially if the problem space is large ( a common feature of real world problems ) and there exist some potential solutions far away from the initial examples . This implication is significant since the \u201c List \u201d view of examples - examples presented in a list - is commonly used in current creativity support tools , such as search engines and recommendation systems , yet was associated with substantial negative effects on the usage of examples and task performance relative to In - Context presentation of examples . 6 . 2 Limitations The WildCat Wells task we used in our experiment is simpler than most real - world exploratory creativity tasks\u2014 such as airfoil design , ad design , or UI design \u2014 of which it is an analog . For instance , the task did not require any specialized domain knowledge , and the generic task structure of searching a space for rewards is probably familiar to most people : indeed , one participant in our study noted that in the post - survey that the task was \" very fun and somewhat similar to minesweeper . \" Additionally , although we carefully constructed our Wildcat Wells task surfaces to be rugged , with multiple peaks of good solutions , our task technically has a single best solution ; in contrast , many real - world creative problems \u2014 such as policy design \u2014 lack a single best solution , due to task factors such as intrinsic tradeoffs between different problem requirements ; in these cases , creators often search for and construct \u201cgood enough \" solutions under high uncertainty ( though this might sometimes be a function of feasibility constraints rather than intrinsic properties of the task ) . It is unsurprising , then , that participants performed relatively well as a whole , and \u201c Dropdown \u201d participants Manuscript submitted to ACM 18 Chan , et al . also had competitive performance even though they did not interact or use the examples . We note , however , that performance was not quite at ceiling : only 16 % of participants reached the global max in either trial ( and 42 % reached the threshold score of 95 for the first bonus . Still , caution is warranted when generalizing to other more complex instances of exploratory creativity ; for instance , it may be that the effects of examples , and the corresponding effects of variations in their presentation interactions , will become more pronounced in more sophisticated tasks . Relatedly , the Wildcat Wells task captures aspects of search dynamics ( exploration and exploitation ) in exploratory creative problem solving quite well , but does not enable observation of more sophisticated psychological mechanisms for working with examples . For instance , it is unclear what it might mean to \u201ccombine \" different problem solving moves for this task . Additionally , while participants engaged in modeling of the problem space , they were not able to make larger changes to the problem space , such as questioning assumptions or relaxing constraints [ 45 ] , or even changing the goal / problem altogether [ 43 ] , mechanisms that are common in real - world creative problem solving tasks , such as design [ 22 ] . Thus , we reiterate that our results cannot speak to how example presentation design decisions might influence example usage for transformational creativity [ 7 ] tasks . Thus , more work is needed to extend our exploration of patterns in example interaction design choices to more complex settings : for example , what might it mean to design a \u201ccontextualized \" presentation of examples for UI elements , more complex airfoil designs , ad persuasion campaigns , research papers , or policy ideas ? We are keen to build on existing design patterns similar to this in previous systems such as ReflectionSpace [ 78 ] , MoodCubes [ 39 ] , and ImageSense [ 47 ] , as discussed in Section 2 . 3 . Our implementation of the Dropdown condition may also be quite different from other Dropdown presentations , such as forward / backward interfaces ( e . g . , image suggestions [ 46 ] ) . Future studies can explore the consequences of these differences . For now , we note that our main results on the contrast between In - Context and List conditions are independent of this limitation , and recommend caution in generalizing the results from the Dropdown condition around non - use of examples . Finally , we did not measure demographic information that may have been correlated with task performance or example usage and / or exploration patterns \u2013 for example , personality traits such as disagreeableness or extraversion may be correlated with real - world creative achievement [ 95 ] ; and gender might interact with potential differences in visuospatial reasoning demands between example interfaces , given some existing research on gender differences in spatial ability [ 52 ] . 6 . 3 Towards an interaction - oriented theory of creative inspiration from examples Returning to our higher - level goal of constructing an interaction - oriented theory of human - example interaction , we now reflect on how the empirical results from our study , in conversation with theoretical mechanisms and design patterns from prior work , could contribute to an overall theory that bridges design patterns to psychological mechanisms . We conjecture that a useful theory of human - example interaction could be conceptualized as paths through multiple coordinated spaces of example interaction patterns , example - ideation psychological mechanisms , ideation characteristics and creative outcomes . Paths through this overall set of coordinated spaces could then represent a set of principled design hypotheses about how to best support creative work with examples . For instance , bringing our empirical results in conversation with the literature we reviewed in sections 2 . 2 and 2 . 3 , we could hypothesize that , given a particular exploratory creativity task environment like our instantiation of the WildCat wells task , where the key creative outcome of solution quality is determined at least in part by the ideation characteristic of diversity of search , which is in turn positively influenced by the psychological mechanism of ( re ) modeling , and negatively influenced by the mechanism of stimulation , it may be advantageous to choose example interaction patterns like Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 19 contextualizing examples in the problem space ( which is positively mapped to ( re ) modeling mechanisms ) , over patterns like List viewing of examples ( which is positively mapped to stimulation mechanisms ) . Multiple other hypothesized paths could be generated and refined to map other example interaction patterns from prior work , such as faceted search systems , or example dissection / analysis , to other psychological mechanisms , such as conceptual combination , or analogical abstraction ; each of these mechanisms might then in turn be contextually important for certain kinds of creative problems , such as policymaking or room layout design . We believe that fleshing out these paths through these coordinated spaces towards a theory of human - example interaction can both make contributions to fundamental HCI theory \u2014 by enhancing synthesis of design knowledge about how to best support creative inspiration from examples \u2014 and practice \u2014 by providing a principled framework that is sufficiently granular and directly connected to design decisions , to guide effective design decisions when building example - based creativity support systems , and to practicing creators who wish to more effectively leverage examples in their creative process . We invite the rest of the creativity support systems community to join us in these efforts . REFERENCES [ 1 ] Marine Agogu\u00e9 , Akin Kazak\u00e7i , Armand Hatchuel , Pascal Le Masson , Benoit Weil , Nicolas Poirel , and Mathieu Cassotti . 2014 . The Impact of Type of Examples on Originality : Explaining Fixation and Stimulation Effects . 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APPENDIX A PROCEDURE FOR GENERATING RUGGED WILDCAT WELLS SEARCH ENVIRONMENTS We used four factors to control the synthetic objective functions : ( 1 ) The ruggedness amplitude , in the range of [ 0 , 1 ] , controlled the relative \" height \" of noise added to the search environment , compared to the height of the peaks . Increasing this parameter made the task more difficult by adding more places to incorrectly intuit as the location of the maximum reward . Setting this parameter to 1 would essentially make the search environment have infinite peaks . ( 2 ) The smoothness , in the range of [ 0 , 1 ] , controlled the degree of local correlation of scores in the grid . Intuitively , if smoothness was high ( closer to 1 ) , then a hill - climbing or gradient - based strategy could be viable , as a searcher that saw successive points in increasing score could ( correctly ) intuit that further points down that search path were likely to be of higher score ; at lower values of smoothness , the search environment became very \" bumpy \" , such that searchers would often be surprised by the score of nearby regions in the grid . ( 3 ) The number of peaks controlled the number of \" maxima \" in the search environment ; intuitively , this specified the number of regions in the space where a searcher might ( correctly or otherwise ) intuit as the location of the treasure . Mathematically , this parameter controlled the number of layers of multivariate normal with single peaks . ( 4 ) The distance between peaks , in the range of [ 0 , 1 ] , which prevented overlap of peaks when the function was generated with more than 1 peak . All search environments in this study were generated with ruggedness amplitude set to 0 . 7 ( fairly noisy ) , smoothness level to 0 . 2 ( fairly rugged ) , and the number of peaks to 1 with a maximum of 100 ; distance between peaks was not relevant because we only used a single peak , using the following algorithm : Algorithm 1 Constructing the Wildcat Wells search environment with given ruggedness ( noise ) amplitude ( \ud835\udc45\ud835\udc62\ud835\udc54 \ud835\udc4e\ud835\udc5a\ud835\udc5d ) , smoothness ( \ud835\udc46\ud835\udc5a\ud835\udc61 ) , number of peaks ( \ud835\udc41 ) and distance between peaks ( \ud835\udc45\ud835\udc62\ud835\udc54 \ud835\udc53\ud835\udc5f\ud835\udc52\ud835\udc5e ) . 1 : for \ud835\udc45\ud835\udc62\ud835\udc54 \ud835\udc4e\ud835\udc5a\ud835\udc5d , \ud835\udc46\ud835\udc5a\ud835\udc61 , \ud835\udc41 , \ud835\udc45\ud835\udc62\ud835\udc54 \ud835\udc53\ud835\udc5f\ud835\udc52\ud835\udc5e do 2 : Get \ud835\udc4b \ud835\udc50\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc52\ud835\udc5f\ud835\udc60 = \ud835\udc53 ( \ud835\udc41 , \ud835\udc45\ud835\udc62\ud835\udc54 \ud835\udc53\ud835\udc5f\ud835\udc52\ud835\udc5e ) 3 : Sample (cid:205) \ud835\udc41\ud835\udc56 \ud835\udc60\ud835\udc62\ud835\udc5f\ud835\udc53 \u223c N ( \ud835\udc4b \ud835\udc56 ) 4 : Sample \ud835\udc41\ud835\udc5c\ud835\udc56\ud835\udc60\ud835\udc52 \u223c OpenSimplex ( \ud835\udc46\ud835\udc5a\ud835\udc61 ) 5 : end for 6 : Return \ud835\udc60\ud835\udc62\ud835\udc5f\ud835\udc53 + \ud835\udc5b\ud835\udc5c\ud835\udc56\ud835\udc60\ud835\udc52 \u00d7 \ud835\udc54 ( \ud835\udc45\ud835\udc62\ud835\udc54 \ud835\udc4e\ud835\udc5a\ud835\udc5d ) . Manuscript submitted to ACM Interface Presentation Effects on Example - Based Problem Solving 25 B ALGORITHM FOR SAMPLING DIVERSE AND NON - DIVERSE EXAMPLE POINTS Algorithm 2 Generating a ranked distribution of example sets with Determinantal Point Process ( DPP ) approach [ 49 ] , \ud835\udc40 is batch size ( 10000 ) . \ud835\udc46 \ud835\udc58 is a combinatorial set defined on a finite set \ud835\udc4b \u2208 R 2 , where each element \ud835\udc46 \ud835\udc58\ud835\udc4c \ud835\udc56 \u2208 \ud835\udc46 \ud835\udc58 is k elements long . 1 : for \ud835\udc56 \u2208 \ud835\udc5f\ud835\udc4e\ud835\udc5b\ud835\udc54\ud835\udc52 ( \ud835\udc40 ) do 2 : Sample \ud835\udc46 \ud835\udc58\ud835\udc4c \ud835\udc56 \u223c IID ( \ud835\udc46 \ud835\udc58 ) [ identically sampling unordered sets without replacement ] 3 : Calculate \ud835\udc54 ( \ud835\udc46 \ud835\udc58\ud835\udc4c \ud835\udc56 ) = \ud835\udc54 \ud835\udc66 \ud835\udc56 and append this to \ud835\udc46\ud835\udc50\ud835\udc5c\ud835\udc5f\ud835\udc52\ud835\udc60 \ud835\udc46 \ud835\udc58 4 : end for 5 : Return DPP Score of sets of examples = \ud835\udc46\ud835\udc50\ud835\udc5c\ud835\udc5f\ud835\udc52 \ud835\udc46\ud835\udc58 \u2212 \ud835\udc5a\ud835\udc52\ud835\udc4e\ud835\udc5b ( \ud835\udc46\ud835\udc50\ud835\udc5c\ud835\udc5f\ud835\udc52 \ud835\udc46\ud835\udc58 ) \ud835\udc60 . \ud835\udc51 . ( \ud835\udc60\ud835\udc50\ud835\udc5c\ud835\udc5f\ud835\udc52 \ud835\udc46\ud835\udc58 ) . Manuscript submitted to ACM", + "purcellDesignOtherTypes1996": "Design and Other Types of Fixation or Is Fixation Always Incompatible with Innovation ? A . T . Purcell and J . S . Gero Key Centre of Design Computing Department of Architectural and Design Science Sydney University , 2006 Australia { terry , john } @ arch . su . edu . au Abstract Design educators often comment on the difficulties that result from a premature commitment by students to a solution to a design problem . Similarly practitioners can find it difficult to move away from an idea they have developed or precedents in a field . In the psychology of problem solving this effect is called functional fixedness or fixation . This refers to the observation that people find it very difficult to see , for example , that objects with well known uses or functions can be employed in new or unusual ways to develop an innovative solution to a problem or to solve a problem that requires innovation . Given that fixation refers to situations where innovation is blocked , it is not surprising that these effects should occur in design problem solving . Design problems are essentially ill - defined problems . As such they inherently contain the opportunity for innovation . However , while these types of issues have been discussed in the context of design , there has been little systematic evidence available about whether or not and under what conditions design fixation does occur . The paper reviews the results of a series of recent experiments which begin to addresses these issues . The results of the experiments will then be examined in terms of what insights they provide into the design process and what implications they have for design education and their relevance for the role that computers may play in the design process . 1 . Introduction Through an unusual inversion of everyday ways of thinking , the Gestalt psychologists ( Maier , 1931 ; Wertheimer , 1982 ) sought to understand innovation and creativity in problem solving by studying problems that most people find it difficult to solve . The basic logic attached to this approach appears to have been as follows . If problems are chosen which require innovative solutions , then studying the conditions under which people fail can give insights into why people find it difficult to produce innovative solutions . One of the central concepts to emerge from this approach was the idea of fixation or functional fixedness . For example , people appear to be unable to see new ways of using objects which could lead to an innovative solution to a problem , because they are blocked or fixated on well learnt uses or properties of the object . A typical problem where fixation is exhibited requires that two spatially separated pieces of string hanging from the ceiling of a room be joined . The pieces of string are not long enough for one to be simply picked up and carried to the other . Present in the situation are a variety of everyday objects . Many of these objects are capable of being used as a weight which can be combined with one of the pieces of string to form a pendulum . If a pendulum created in this way is set in motion , it 2 is possible to pick up the end of the other piece of string , catch the end of the pendulum and tie the two together . However this solution is rarely spontaneously produced . The reason for this result , it is argued , lies in the way that the well established , everyday functions of objects prevent the problem solver from seeing this unusual and innovative use . This basic idea is appealing because it taps experiences common to many people and to problem solving in many domains . The area of design is no exception . Here a common and often commented on form of fixation is the premature commitment to a particular problem solution , observed in students and practitioners alike . As in other domains , the designer appears trapped by the characteristics of a possible solution that has been developed or an existing precedent solution . However , in the design domain , the majority of the discussion of this phenomena is essentially anecdotal and not based on either principled argument or the results of empirical research . Design would also appear to offer a particularly suitable domain for this approach to studying these aspects of problem solving . It is well recognised that design problems are inherently ill - defined ( Simon , 1981 ) . Consequently every design problem has the potential for innovative and creative solutions removing the necessity for specifically developing problems that have these characteristics . While all discussion of fixation in design has been largely anecdotal , recently experimental methods have been developed for examining both whether or not the phenomena exists and , perhaps of more interest , the basis for the effect . The aim of this paper is to review the available research results and then to draw out the implications of these results in three areas . First what do the results tell us about the design process ? If there are insights into the design process what are the implications for design education ? Finally there is the question of the implications of the design fixation effect , ( if it exists ) , for the role of computers in the design process . Broadly and briefly computers would appear to potentially have two types of roles in design . In one the aim is to have the computer produce the design ; in the other the computer is used to provide intelligent design support system for a human designer . The first role for computers appears to have been downgraded in the past few years , largely because of the consequences of the ill - defined nature of design problems , while the second has received increasing attention . A central facet of the second computational approach to design would appear to be the reuse in some way of existing designs as in case - based systems . In this approach , a large library of designs of different types and their associated attributes is established . In the context of a particular design problem this library is searched for designs which match , using some measure of similarity , to the requirements of a particular design problem with the human designer specifying what these requirements are . Fixation effects could occur in this situation in a number of ways . However , if the design fixation effect exists , it could be particularly relevant to the development and use of case - based systems . The available evidence indicates that design fixation may be associated with seeing pictorial representations of possible design solutions . It would also appear to be highly likely that the design cases would contain a number of forms of pictorial representation from pictures of the completed design to various types of diagrammatic representations . The form of representation used in such systems would as a result appear to establish the conditions for fixation to occur . However , before exploring this issue further , whether or not fixation occurs and the basis of the effect needs to be established . 2 . Design Fixation Jansson and Smith ( 1991 ) were the first to develop an experimental approach to the problem of fixation in design . They argued that showing designers a picture of a potential design solution to a problem prior to a design session should result in fixation . In effect 3 the picture would act as a precedent , blocking access to other ways of solving the problem . They also extended the argument about the basis of fixation . They suggested that the process of design involves operating on effectively two types of mental representation of the problem . One representation they refer to as the conceptual space which consists of abstract knowledge about principles , concepts and rules which can be used to solve the problem . The other representation takes the form of particular physical objects and elements which could form the physical realisation of a solution to the problem . This representation is referred to as the object space . Jansson and Smith argue that the location of the fixation induced by a pictorial representation is the object space and that innovation is prevented because the designer cannot move to the conceptual space which is where they consider that innovative changes can occur . In order to test this hypothesis Jansson and Smith used three different types of design problem and had advanced undergraduate and practicing mechanical engineers engage in solving the problem . The experimental design was quite straight forward . For each of the design problems two groups of designers were used . One group ( either a student or an expert group ) were simply given a statement of the problem and acted as a control group . Separate groups corresponding in level of expertise to the control groups were given the statement of the problem together with a picture of a possible solution . The example solutions were specifically designed so that aspects were included which were incorrect given the problem statement . A set of features which characterised each pictorial example was developed and the designs produced were scored for the presence or absence of the features . The design sessions lasted for one hour and the designers were allowed to produce as many sketch designs as they liked . It would be expected that , if fixation resulted from exposure to a pictorial representation of a possible design , more features of the example should occur with the groups shown the example . This was found to be the case . For each of the design problems and for student and expert designers , more features associated with the example , including incorrect features , were found with those who had been shown the pictorial representation . 3 . Exploring the Design Fixation Effect The potential significance of this effect is apparent because of the role of precedents , generally presented in the form of pictorial and other forms of visual representations , in the teaching and practice of design . A number of significant issues relating to the effect can be identified . Does the effect occur when pictorial representations of different possible designs are presented prior to a design session ? Is the effect associated specifically with pictorial representations or would similar effects occur if verbal descriptions of possible design solutions were made available ? Does the effect occur with other disciplines and levels of expertise ? In order to begin to explore the basis for the design fixation effect we ( Purcell and Gero , 1991 ) carried out an experiment using one of the Jansson and Smith problems - the design of a bicycle rack for a car . Five different design solutions were identified and these are shown in Figure 1 . Pictures of three of these designs were using as fixating examples ( a , b and c in the Figure ) and for two of these solutions a verbal description was developed ( the verbal description of one of the designs is presented in Appendix 1 ) . Novice students in architecture and industrial design participated in the experiment using the same experimental design as Jannson and Smith . Separate groups of students were shown the example design and were told that it was to illustrate what was meant by a sketch design with corresponding control groups being given only the statement of the problem . Lists of features of each design were developed and the designs produced by the experimental and control groups were scored for the presence or absence of the features . In addition the number of designs developed by each participant was recorded and the participants answered a number of questions at the completion of the session . 4 Differences in the frequency of design features were statistically analysed and part of the results are presented in Table 1 . Effectively no evidence of fixation was found with the exception of one of the pictorial examples for one feature . The fixation effect , on the basis of these results , does not appear to be associated with simply the presentation of a pictorial example nor does it occur with a verbal description of a possible design . Where evidence for fixation was found with one of the examples , an analysis of the results of one of the questions asked at the end of the design session indicated a somewhat different possible basis for the results obtained . Participants were shown the drawings of all of the examples shown in Figure 1 and asked how familiar they were with each . Tabulation of the responses to this question revealed that the most familiar example was also the example where some evidence of fixation was obtained ( see Table 1 ) . What appears to be a fixation effect could as a result simply reflect familiarity with the example . Subsequent enquires with bicycle retailers and a group representing bicyclists demonstrated that the most familiar example was also the type of bicycle rack that predominated in sales . Interestingly this type of design was also different to the rack design that had produced fixation in Jansson and Smith\u2019s work . There is however significant differences between our work and the original Jannson and Smith experiments . They had used advanced student and practicing mechanical engineering designers and we had used novice designers from two other disciples . A possible interpretation of the effect obtained could therefore be that novice designers from these other disciplines , lacking any domain specific knowledge , simply relied on their general , everyday knowledge which was triggered when shown the drawing of the example . This possible explanation is reinforced by the absence of any effects with the drawings of the other types of designs . This result also indicates that the effects obtained with the most familiar example do not simply involve copying as it would have been equally easy to copy each of the other types of design . 4 . The Role of Design Discipline and Expertise in the Design Fixation Effect Our next experiment attempted to examine some of these issues . We again used one of the Jansson and Smith problems - the design of a device to be used by the blind in measuring quantities for cooking . We chose this design problem because it would be unlikely that our designers would have either designed or seen an example of a design for such a device in contrast to the bicycle rack problem . In this way we were able to minimise the effects of familiarity with existing design solutions . Advanced student designers in mechanical engineering and industrial design in their final year participated in the experiment . This choice was made both because we wanted to use the designers from the same discipline as in Jansson and Smith\u2019s work and because we wanted to examine whether or not the effect occurred with different disciplines while using designers with a similar level of expertise to those in the original Jansson and Smith experiment . Industrial designers were chosen as a second discipline because the two disciplines can deal with similar types of problems . This removes a possible effect resulting from using a design discipline , such as architecture , which deals with qualitatively different types of design problems to the problem we had chosen . We also pursued the question of whether or not fixation would be found with other examples to that used by Jansson and Smith . In Australia there is a organisation which represents the blind and we approached them to determine if there were examples of such a device available . They market a device for this propose and the pictorial representations of the two examples are shown in Figure 2 . 5 Again the same experimental design was used as in previous design fixation work . We did however introduce a change in the way the designs were analysed . In previous work features characteristic of each design were identified and each design produced was scored in terms of these features . However , in reflecting on the results of these experiments , it appeared to us that features could be of two types . One type would represent the actual details of the features present in the design example . However it is possible that a design feature could represent the same concept as was present in the design example without having the same detailed characteristics . Consequently for each design two lists of features were developed to reflect this distinction ( for details of the features of each of the designs and the results of the analysis see Purcell , Williams , Gero and Colbron , 1993 ) . In brief the analysis demonstrated that fixation did not occur with the industrial design groups for either of the example designs . However fixation was apparent with the mechanical engineers but only with the example design used in the original Jannson and Smith experiments . This appears to indicate that fixation may occur only where the combination of the design discipline and the example design used are the same as in the original Jansson and Smith experiments suggesting that the effect may only have limited applicability . The presence of fixation in the mechanical engineers and not in the industrial designers could for example reflect differences in approaches to education in the two disciplines . While this would be an interesting result , it would diminish the general significance of the effect . However an examination of the two example designs in Figure 2 suggests another possible basis for this finding . The Royal Blind Society example is a particularly simple device that essentially uses an everyday cup with three dimensional markers identifying discrete quantities . By contrast the Jannson and Smith example is a more complex device which , significantly , appears to involve principles that could be thought of as typical of the mechanical engineering discipline . This suggests the possibility that the fixation effect is not simply discipline specific but involves the use of an example that embodies principles that are specific to a particular domain of knowledge . 5 . Domain Specific Knowledge and Design Fixation In order to test this hypothesis we sought the assistance of experts in mechanical engineering and industrial design . Our aim was to develop a new design problem which would again be the type of problem that could form a part of the practice of each discipline but where it would be unlikely that groups of advanced undergraduate students would have been involved in designing or have seen examples of solutions . We also wanted a problem where there were a number of existing solutions which both involved principles which would be typical of mechanical engineering and also examples which the experts considered unusual and innovative . The problem we identified was the design of a device for assisting the elderly into and out of a bath in a domestic setting . There are a number of existing solutions to this problem four of which are shown in Figure 3 . To initially examine our hypothesis about the role of domain specific knowledge being embodied in the pictorial representation of a design , we chose the example , the Autolift device , labelled as ( c ) in the Figure . We had asked the experts in mechanical engineering to assess whether or not the examples involved typical mechanical engineering principles . In their judgement this example involved the use of such principles and should therefore produce fixation . Our experiment followed the same design as previously . Advanced undergraduate students in their final year in mechanical engineering and industrial design participated in the experiment with two groups from each discipline . Both groups in each discipline received the verbal description of the problem with one group in each discipline also being shown the drawing of the example design . Lists of features related to the principles involved in the example design and the details of the design were again developed . However we 6 considered that measuring the frequency of occurrence of design features was not the only way of assessing design fixation . For example , it could be argued that design fixation should lead to fewer designs being produced because other ways of solving the problem would not be available to the designer . Similarly fixation would be expected to produce a restriction in the range of solution types that were produced . In order to assess these other aspects of fixation , the number of designs produced by each participant was again recorded together with the type of each design produced by each participant . Design type was assessed in the following way . Brief descriptions of approximately 20 % of all the designs were developed independently by two of the researchers working on the project . Similar descriptions were then grouped together to form categories which were then given type labels . This initial list of types was tested on a further sub - set of the designs before the full set of designs was coded . Designs which did not fit into any of the types were classified as \u201cother\u201d . At the completion of the coding the \u201cother\u201d category was reviewed and new categories developed with the \u201cother\u201d category finally referring to unique designs ; that is only one example of the type in the complete data set . The results of the analyses of these various measures are present in Tables 2 , 3 and 4 . Table 2 ( with proportions rather than raw frequencies being reported ) demonstrates quite clearly that fixation had occurred with the mechanical engineering group for both features that involved principles and features involving detail . Of the seven principle features , significant differences were found between the experimental and control groups for the mechanical engineers for five of these features but only for one of these features in the industrial design groups . For the twelve detail features , the numbers of significant differences were eight and three respectively for the two disciplines . Exposing a group of mechanical engineering designers to a pictorial representation of an example design which embodies typical principles of the discipline does , as a result , appear to induce a fixation effect . However this result is contradicted by the analyses of the other two measures of fixation . A comparison between the average number of designs produced showed no differences between the experimental and control conditions for each design discipline ( see Table 3 ) . If fixation was operating to constrain the types of solutions available to the designer such differences would have been expected for the mechanical engineering group . Similarly fewer types of design solutions should have been found where fixation occurred however this was not the case as is apparent from Table 3 . The results of the analysis of the various types of data in this experiment are therefore somewhat paradoxical . In terms of the usual , feature based measures there is quite clear evidence of fixation with the predicted experimental conditions for the mechanical engineers . However with the two other measures , which would appear to be equally as reasonable as the features approach in terms of demonstrating fixation , no evidence for fixation occurred . 6 . Design Fixation and Innovative Design Examples There are a number of other aspects of the results which however are also noteworthy even though they , at least superficially , do not appear to be specifically related to design fixation . Over all of the experiments using advanced student designers there has been no evidence of fixation with the industrial design groups . In this experiment it is apparent that there are large differences between the mechanical engineering groups and the industrial designers . From Table 3 it is clear that the industrial designers produce approximately twice as many designs and twice as many types of design solutions as the mechanical engineers . It is also apparent from Table 4 that , for the industrial designers , by far the most frequently 7 occurring type of design is \u201cother\u201d . That is this group frequently produced , whether under the control or experimental conditions , unique , one off designs and these types of designs were very infrequently found with the mechanical engineers . These differences between the two groups , because they are effectively independent of the experimental manipulations carried out , could be interpreted , for example , as resulting from differences in educational processes in the two disciplines . It could be that industrial design emphasises creativity and difference and associates this with the idea of trying out many ideas . The emphasis on innovation and trying many different ideas would clearly produce many more designs , many more types of design and many more unique designs . However , while these aspects of the results appear to be unrelated to design fixation , they suggest another perspective on what was occurring with the design fixation results . While industrial design may place different emphases on what is important to the outcome of a design process , it is also apparent that the areas of knowledge that make up industrial design are more diverse than those studied in mechanical engineering . It would also appear that many of these areas are associated with less well articulated bodies of knowledge than those that make up the knowledge base of mechanical engineering . For example , aesthetics plays a prominent role in industrial design education , often appearing as a separate subject , while it plays little formal role in mechanical engineering . Aesthetics also has an obviously less articulated and formal knowledge base than for example mechanics in mechanical engineering . It is possible therefore that the absence of highly articulated , formal knowledge bases in industrial design results in the absence of fixation that has been demonstrated in our experiments . While this is a plausible , but after the event , explanation for these results , there is an alternative . It could be that it is not the absence of a highly articulated knowledge base which results in no fixation being found , but that the example used does not emphasise aspects which are considered important in industrial design . If innovation is considered important then the example used in the previous experiments , which was judged by experts from both disciplines as not being innovative , would not emphasise issues which were considered to be important in industrial design . This argument leads to the hypothesis that it may be possible to produce fixation in industrial designers by using an innovative example . One of the designs that our panel of experts considered to be innovative is example b , the Hydrocushion , shown in Figure 3 . This essentially consists of a cushion which can be filled with water to the level of the bath edge to allow access , with the water then being emptied into the bath to lower the person with the process being repeated to exit the bath . If innovation is an important issue in the knowledge base of industrial designers , then this example should produce fixation . For mechanical engineers , the principles involved are not as typical , in the context of lifting weights , as are the principles involved in the Autolift example used in the preceding experiments ( the second example shown in Figure 3 ) . If fixation did not occur with the mechanical engineers it would indicate that the principles involved in an example need to be typical of the task to be performed , whereas if fixation does occur , it would indicate that activation of relevant , although not typical ( in the context of lifting weights ) mechanical engineering principles can produce fixation . We therefore repeated our basic experiment using this example . Table 5 presents the features which were scored for each design that was produced . These are again divided into principle and detail features with an additional group of features relating to aspects of the context that is depicted in the example drawing . In this design example we identified six principle features . However an examination of the list demonstrates a number of the features were not unique to this particular type of design with these features resulting from general constraints on the design eg a removable device or a device which can raise or lower the person in the bath . In effect what characterises this design and makes it innovative is the use a single basic principle - the use of water pressure which is already 8 present in the design context - to resolve many of the design issues . This contrasts with the lists of features associated with the Autolift example . Here the principle features actually identify a series of principles which solve various parts of the problem . The detail features are again those that directly reflect the specific ways in which the principled features are realised in the fixation example . The contextual features are those which are present in the fixation example but either represent expected parts of the design context eg tiles on the adjacent wall or are ancillary drawings which illustrate other views of the device and ways in which the device can be used . Table 5 also presents the proportions representing the occurrence of each of these features in the experimental and control groups for the two design disciplines . Because of the small pool of final year industrial design students that are available in Sydney , only thirteen individuals have participated in this part of the experiment to date with however twenty mechanical engineering students participating . As a result of the unequal numbers in the groups , statistical analyses have not been carried out yet . In order to allow a comparison between the results of the two experiments , the results are expressed in terms of proportions rather than raw frequencies with the potentially significant differences ( based on previous analyses ) being identified by asterisks . A comparison between Tables 3 and 5 identifies a number of interesting differences and similarities between the two experiments . With the Autolift device and the mechanical engineering group , there was clear evidence of what appears to be fixation in the significant differences between the experimental and control groups on the majority of both the principle and detail features . This pattern does not appear to occur with the innovative design example with this group . One principle feature - operates on water pressure - is clearly different between the experimental and control groups with no designs in the control group exhibiting this feature . The features relating to raising and lowering the individual and connecting the device to the existing water tap may demonstrate an effect . With the detail features only one - the presence of a rectangular platform as part of the design - may be significant . It is also apparent that many of these detail features , which show no evidence of differences , are very specifically related to this type of design and are not likely to be present in other types of designs . For example the last six of the detail features are quite specifically associated with a device of the type pictured . However these features are effectively missing from the designs that were produced . The results using the innovative example with this group therefore indicate that fixation is associated with the principles involved in the example design and that the designs produced do not reflect the details of the example . The pattern of results with the industrial design student group is however quite different . There may be a difference between two of the principle features - the device is used to raise and lower the individual from the edge of the bath and the device is removable and two of the detail features - the device has a platform seat with rounded corners . However it is apparent that these features are not specifically associated with the example device that uses water pressure and a water filled container . It therefore appears that , for this group , there is essentially no indication that any of the devices produced in either the experimental or control groups used this innovative approach to the design of the device because neither the principled or detailed features that are specifically associated with this type of design are present in the designs produced . The absence of any fixation effect with this group is therefore similar to the results in the previous experiment however the implications of the result in this case may be quite different . The results with the contextual features are also of interest . For the mechanical engineers differences appear to be associated with two of the types of features which would be consistent with the use of a water filled device - placing the device at the opposite end of the bath to the taps and showing a single bath tap . It is also apparent that many more potentially significant differences are associated with the contextual features than with 9 either the principle or detailed features . Further many of these differences are associated with what might be referred to as conventions associated with the development of a sketch design . For example , the set of features which involve using additional insets which illustrate how the device can be used are quite typical techniques that are used during design development . The reasons why more of these types of features appear in the results of those groups shown the design example across both design disciplines is not immediately apparent . 7 . Conclusions Design Fixation , Design Education and the Design Process The results of this final experiment have potentially important implications . The initial conception of design fixation was that it represented an impediment to innovative design . Designers shown an example demonstrated a lack of flexibility in their design process because they reproduced the characteristics of the example shown . Often this lack of flexibility also extended to reproducing faults that were present in the example design . While this effect resulted from being shown an example design , it could be extended to the more typical design situation . Designers often look at precedent designs and could become fixated in this way . Even more typically , designers , once they have produced a drawing of a potential solution , could become fixated on that solution - an effect often commented on by designers and by those who teach design . However the results of this series of experiments point to a different perspective on the fixation effect . Fixation appeared in our initial experiments to be associated , if it occurred at all , with the absence of domain specific knowledge and a reliance on everyday knowledge activated by exposure to a picture of a familiar example . When the familiarity effect is removed and the type of problem is matched to the discipline of the designer , the results indicated that fixation only occurred with one discipline - mechanical engineering - and only with an example design that embodied principles that formed a part of the knowledge base of that discipline . With the other discipline - industrial design - there was very little evidence of fixation . The role of principles specific to a discipline in producing fixation was demonstrated by using a problem that would not involve the familiarity effect combined with an example solution which was judged by experts to involve knowledge that would be typical of the mechanical engineering discipline . However , while this effect was found when features associated with the example were analysed , two other measures of fixation - the number of designs produced and the number of solution types - showed no fixation effects . Again no fixation effects were found with the industrial designers but there were notable differences between the two disciplines . Industrial designers produced many more designs , many more types of design and many more one off designs . These effects were independent of whether or not an example design was shown . While these differences between the two disciplines could reflect differences in educational processes , it was argued that the absence of the effect with the industrial designers could result from the use of example designs that did not embody aspects relevant to that discipline . The fact that the participants from this discipline produced many more designs , many more types of designs and many more one off designs suggests that there is a focus on innovation in this discipline . If this is correct an innovative example design should produce fixation for this discipline . Once again however no evidence was found for fixation with this discipline but evidence for fixation was again found with the mechanical engineers . 10 However consideration of the attributes of the innovative design and the results with both disciplines suggests that fixation as it has traditionally been conceived may not be the appropriate way to look at the results using this example . The example design used is innovative both because it uses a principle which is not typical of the general approach to lifting weights and because it uses a single principle to resolve a number of the design issues . The results with mechanical engineers demonstrated that what we have normally referred to as fixation was really associated with a concentration predominantly on the core innovative principle involved in the example design - the use of water pressure to raise and lower the individual - with many of the specific aspects of the example design not being found in the designs produced . While the industrial designers showed no evidence of fixation , they also failed to produce any designs which used this innovative approach in either the experimental or control groups . This way of looking at the results raises a number of issues . Is it that the use of typical examples produces the traditional fixation effect while the use of innovative examples focuses the designer on the principles involved ? Does this then result in designs that explore and develop the application of this innovative principle ? We are currently investigating aspects of these designs to determine if they exhibit these characteristics . If this is the case it suggests that , for example , at least part of the use of precedents in design education should involve the deliberate use of and focus on innovative example designs . While innovative designs are discussed and analysed in design education , it may be that this is not effective and that the use of such designs should take the form of showing them prior to a design session with the analysis coming after the session has been completed . The absence of either type of fixation effect with the industrial designers also raises a number of particularly interesting issues . The production of large numbers of designs of many different types would appear to indicate that these designers were attempting to be innovative . The absence of any fixation in the experiments where the \u201ctraditional\u201d fixation effect is found with the mechanical engineers would appear to indicate that this approach perhaps prevents the occurrence of fixation effects of this type . However it is possible that what occurs in fact is a search for difference rather than innovation . The absence of any examples of the innovative design approach represented by the example in the final experiment with this group could reflect just such a process of searching for difference . If this is the case and it represents the results of the educational processes used in this discipline , it indicates that the potential benefits available from innovative design will not be available to the discipline . The results of our research to date may be summarised in the following way . Fixation in the traditional sense may well be found where designers are forced to rely on everyday knowledge . Mechanical engineers become fixated in the traditional sense when the example they are shown embodies typical principles which are characteristic of the knowledge base of the discipline . When shown an innovative example , where the principle involved is unusual in the context and / or resolves many of the design issues using a single principle , mechanical engineers become \u201cfixated\u201d on the principle involved and appear to then explore ways of solving the problem using the principle . Industrial designers appear to show no evidence of fixation under any of the experimental conditions we have employed . However , while showing no evidence of \u201ctraditional\u201d fixation , the industrial designers showed no evidence of producing innovative designs using the principle involved in the innovative example . In a sense these groups may have become \u201cfixated\u201d on being different . \u201cFixation\u201d therefore appears to possibly exist in a number of forms and we as researchers need to be wary of becoming fixated on our conception of what fixation is . Design Fixation and Computational Approaches to Design 11 Given these results from our research it is possible to return to the issue of the relationship between fixation effects and computational approaches to design discussed at the end of the Introduction to this paper . A major finding of this research was that there are differences between the two design disciplines studied in our project . With the mechanical engineers , fixation in the traditional sense of reproducing the characteristics of a design , including incorrect features , occurred where the example shown embodied principles that were typical of the knowledge base of the discipline . If the cases included in a case - based mechanical engineering design support system both embodied principles typical of the discipline and either contained mistakes or features that were inappropriate in relation to the particular design problem being addressed , then fixation effects that are negative in terms of the design outcome for the particular problem are likely to occur . However , if the design examples that are presented are innovative , it appears likely that designers may identify the principle involved and then explore how this could be used in the particular design situation . This latter outcome points towards ways in which such systems might be used to both encourage innovative design and prevent design fixation effects . This could be done in at least two ways . The cases that were included in the system could be deliberately selected so as to include a range of examples that went from the typical to the atypical within any particular type of artefact . In addition the similarity metric which is used to identify relevant cases could be designed so that cases of other types that have relevant features could also be retrieved providing further possibilities for exploration of the implications of these ideas and increasing the possibility of innovation in design . These approaches however would not necessarily prevent the first type of fixation occurring . For example if the cases that are retrieved first all embody typical principles in the field then fixation of the first type could occur with the designer not proceeding to explore the further possibilities of the system . This suggests that it would also be necessary to carefully consider the issue of the order in which such a system retrieves cases and that the process of learning to use such systems should specifically be designed to make users aware of these issues . The results with the industrial designers have equally important but more general implications for the design of such systems . The evidence from our work is that presenting pictorial examples has no effect on the designs produced by the participants from this discipline . Taken at face value this would indicate that there would be little point in developing a case - based design support system for this discipline as it would be unlikely to have any effect . In fact our results would lead to the prediction that such a system would be unlikely to be used as a result of designers from this discipline being \u201cfixated\u201d on difference . This in turn raises some very basic issues about the education of industrial designers which cannot be addressed here but which point to the need for further research in the area of industrial design to establish the generality of the effects we have observed . References Jansson , D . G . and Smith , S . M . ( 1991 ) Design fixation . Design Studies , 12 , 3 - 11 . Maier , N . R . F . ( 1931 ) Reasoning in humans : II . The solution of a problem and its appearance in consciousness . Journal of Comparative Psychology , 12 , 181 - 194 . Purcell , A . T . and Gero , J . S . ( 1991 ) The effects of examples on the results of a design activity . In Artificial Intelligence in Design \u201891 Ed . J . S . Gero , Butterworth - Heinemann : Oxford , pp 525 - 542 . 12 Purcell , A . T . , Williams , P . , Gero , J . S . and Colbron , B . ( 1993 ) Fixation effects : Do they exist in design ? Environment and Planning B : Planning and Design , 20 , 333 - 345 . Purcell , A . T . , Gero , J . S . , Edwards , H . M . and Matka , E . ( 1994 ) Design Fixation and Intelligent Design Aids . In J . S . Gero and F . Sudweeks ( Eds ) Artificial Intelligence in Design \u201894 , 483 - 495 , Kluwer Academic Publishers , The Netherlands . Simon , H . A . ( 1981 ) The Sciences of the Artificial ( 2nd . edition ) M . I . T . Press : Cambridge , Massachusetts . Wertheimer , M . ( 1982 ) Productive Thinking . University of Chicago Press : Chicago . Acknowledgments The research reported in this paper was supported by Grant Number A89231572 from the Australian Research Council to A . T . Purcell and J . S . Gero . Part of the material presented in Section 3 of the paper was reported in Purcell and Gero ( 1991 ) ; part of the material in Section 4 was reported in Purcell , Williams , Gero and Colbron ( 1993 ) and part of the material in Section 5 was reported in Purcell , Gero , Edwards and Matka ( 1994 ) . 13 Figure 1 Illustrations of five bicycle rack designs . 14 Figure 2 Illustrations of the two example designs of devices for use by the blind in measuring quantities for cooking . 15 Figure 3 Illustrations of four devices designed to assist the elderly in entering and leaving a bath . Table 1 Probability of the difference in frequency of occurrence of four bicycle design features between experimental and control groups . 16 Group Location of rack Car attachment Bicycle support Bicycle attachment Control versus single - post picture 0 . 36 0 . 38 0 . 53 0 . 17 Control versus single post description 0 . 83 0 . 88 0 . 72 0 . 06 Control versus A - frame picture 0 . 79 0 . 64 0 . 21 0 . 02 Control versus A - frame description 0 . 3 0 . 13 0 . 61 0 . 12 Control versus boot picture 0 . 89 0 . 99 0 . 86 0 . 26 Table 2 Proportions of two types of features found for the experimental and control groups in two design disciplines for the bath access problem with the Autolift fixating example ( statistically significant differences ( . 05 ) are identified with an asterisk ) . Mechanical Engineers Industrial Designers ExperimentalControl Experimental Control Principles Fixed * . 72 . 42 . 52 . 34 Fixed to floor * . 25 . 06 * . 16 . 04 Column * . 36 . 06 . 14 . 05 Lifting mechanism within * . 23 . 03 . 06 . 04 Handle on column . 03 . 00 . 03 . 00 Boom * . 31 . 03 . 11 . 06 Seat . 67 . 44 . 63 . 55 Details Fixed with base plate * . 22 . 00 * . 09 . 01 Fixed with bolted base plate * . 14 . 00 . 05 . 00 Column - tripartite . 00 . 00 . 03 . 00 Bolts on column . 00 . 00 . 02 . 00 Winder with knob . 03 . 00 . 02 . 00 Rigid boom * . 28 . 00 . 08 . 05 Moulded seat with back * . 44 . 14 * . 38 . 10 Perforated seat * . 17 . 03 . 09 . 03 Arms on seat * . 31 . 08 . 09 . 05 Incorrect orientation to taps . 08 . 03 . 03 . 00 Orientation of bath as in example * . 44 . 11 * . 48 . 13 Tiles on bath as in example * . 25 . 00 . 05 . 00 Table 3 Number of designs and number of solution types produced by the experimental and control groups in two design disciplines for the bath access problem , Autolift fixating example . Industrial Design Mechanical Engineering Control Autolift Inflatable Cushion Control Autolift Inflatable cushion Average number of designs 3 . 8 3 . 4 3 . 1 1 . 8 1 . 7 2 . 1 Average number of solution types 3 . 2 2 . 9 2 . 8 1 . 7 1 . 5 2 . 0 17 Table 4 Frequencies of solution types for the bath access problem produced in the experimental and control groups in two design disciplines , Autolift fixating example . Solution Type Mechanical Engineers Industrial Designers ExperimentalControl Experimental Control Ledge 2 0 2 4 Static rim seat 0 0 1 3 Swivel seat 5 5 9 6 Slide 0 0 1 2 Inflatable cushion 0 2 1 9 Scissor lift 0 4 2 7 Overhead pulley 9 4 2 5 Boom seat with column 9 0 4 2 Door in side of bath 1 1 2 1 Other bath alteration 0 2 3 2 Step and rail 2 5 5 6 Tracks and racks 3 4 7 2 Grip rails 1 4 6 6 Other 4 5 22 28 Total 36 36 64 80 Table 5 Proportions of three types of features for the experimental and control groups in two design disciplines with the Hydrocushion fixating example ( possible significant differences are identified with an asterisk ) . Mechanical Engineers Industrial Designers ExperimentalControl ExperimentalControl Principles Operates on water pressure * . 29 . 00 . 00 . 03 Used to raise and lower person from edge of bath * . 44 . 28 * . 50 . 34 Device is removable . 51 . 50 * . 67 . 55 Device connects to existing faucet * . 15 . 00 . 00 . 01 Valve to direct water flow . 07 . 00 . 00 . 00 Provision made for overflow when device is raised . 02 . 00 . 00 . 00 Details Water filled container . 12 . 00 . 00 . 03 Inflatable cushion . 15 . 06 . 06 . 09 Platform seat , no sides or back . 27 . 19 * . 42 . 30 Rectangular platform * . 27 . 11 . 19 . 15 Rounded corners . 17 . 11 * . 22 . 07 Connection to faucet via an inflatable pressure collar . 02 . 00 . 00 . 00 Collar attached by a hand squeeze pump . 02 . 00 . 00 . 00 Cushion connects to faucet via number of pipes . 02 . 00 . 00 . 00 Fixed piping incorporating a rightangle . 02 . 00 . 00 . 00 18 Insert plug to fill cushion , release plug to drain water . 02 . 00 . 00 . 00 J shaped vertical pipe connected to faucet . 05 . 00 . 00 . 00 Context Unit in intended environment . 90 . 97 * . 92 . 75 Device or user at opposite end to taps * . 27 . 08 * . 19 . 01 Bath positioned in return . 12 . 19 . 14 . 05 Handrail located above bath fixed to tiled wall surface . 02 . 06 . 03 . 00 Tiles shown . 07 . 03 * . 17 . 00 Single bath tap shown * . 12 . 00 . 00 . 00 Insets or series of illustrations used . 73 . 64 * . 86 . 59 i ) User operating the unit * . 39 . 19 * . 44 . 24 ii ) Rear perspective of user seated on unit in bath . 02 . 00 . 03 . 00 iii ) User lowering device * . 22 . 00 * . 14 . 04 iv ) User raising the device . 10 . 00 . 06 . 01 v ) User on raised device ready to leave bath . 07 . 03 . 03 . 00 vi ) Side elevation shown * . 51 . 28 * . 36 . 24 vii ) Schematic representation plan or section view . 56 . 64 * . 61 . 51 19 Appendix 1 Instructions for verbal description fixation group The aim of the design exercise is to come up with a sketch design ( s ) for a bicycle rack for three bicycles for a car . The bicycles have to be held securely , and without damage to either the bicycle or the car . The bicycles must not extend beyond the overall width dimension of the car to avoid potential damage to people or vehicles in passing . There are a number of key issues to be considered in the design of a bicycle rack . The first is the way in which the rack is attached to the car . Then , there has to be a structural system that will support the bicycles . Third , there has to be a way of attaching the bicycles to the support system . Fourth , both the structural system and the way of attaching the bicycles have to have the correct relationship to fulfil the other more general conditions of safety etc . To illustrate , the bicycle rack can be attached to the car at a number of locations : for example at the rear of the car , to a tow bar fitting , or directly to the chassis of the car . The bicycles can be sup [ ported structurally in a number of ways : for example , two steel posts in the shape of an A joined across the bottom of the A can be attached to the fitting fixed to the car . This has to be of sufficient height to provide clearance above the ground . Similarly the bicycles can be attached to the rack in a number of ways : for example , they can be held by the top horizontal section of the bicycle frame in short half sections of pipe of a diameter that allows the bicycle frame section to fit into the pipe with the other half section closing over the bike frame . The bicycles then have to have the appropriate relationship to the car : for example , to the top of the A - frame a small steel section can be attached at rightangles in line with the length of the car . The fitting with the brackets attaching the bicycles can be placed at rightangles to the length of the car on this section of the post , with the bicycles being placed parallel to and clear of , the boot of the car . Detailed and accurate drawings are not required : simple , rough , outline sketches are all that is needed . In addition to the sketches , you can write comments on the drawings to illustrate what you mean . You will be allowed 45 minutes in which to complete the sketch design . If you wish you may complete more than one design . This paper is a copy of : Purcell , T . A . and Gero , J . S . ( 1996 ) Design and other types of fixation , Design Studies 17 ( 4 ) : 363 - 383", + "ulrichs2023multicomponent": "Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Multicomponent regulation of actin barbed end assembly by twin \ufb01 lin , formin and capping protein Heidi Ulrichs 1 , 2 , 3 , Ignas Gaska 1 , 2 , 3 & Shashank Shekhar 1 , 2 Cells control actin assembly by regulating reactions at actin \ufb01 lament barbed ends . Formins accelerate elongation , capping protein ( CP ) arrests growth and twin \ufb01 lin promotes depolymerization at barbed ends . How these distinct activities get integrated within a shared cytoplasm is unclear . Using micro \ufb02 uidics - assisted TIRF microscopy , we \ufb01 nd that formin , CP and twin \ufb01 lin can simultaneously bind \ufb01 lament barbed ends . Three \u2011 color , single - molecule experiments reveal that twin \ufb01 lin cannot bind barbed ends occupied by formin unless CP is present . This trimeric complex is short - lived ( ~ 1 s ) , and results in dissociation of CP by twin \ufb01 lin , promoting formin - based elongation . Thus , the depolymerase twin \ufb01 lin acts as a pro - formin pro - polymerization factor when both CP and formin are present . While one twin \ufb01 lin binding event is suf \ufb01 cient to displace CP from the barbed - end trimeric complex , ~ 31 twin \ufb01 lin binding events are required to remove CP from a CP - capped barbed end . Our \ufb01 ndings establish a paradigm where polymerases , depolymerases and cappers toge - ther tune actin assembly . Cellular actin dynamics are essential in several key processes , such as cell migration , wound healing , cell division , and endocytosis 1 , 2 . Depending upon the requirements of speci \ufb01 c processes , cells dyna - mically tune the rate of assembly , size , and architecture of their \ufb01 la - mentous actin networks . For example , while formins assemble fast - elongatinglinearactinnetworkscomprisingoflongbundled \ufb01 laments , e . g . , in \ufb01 lopodia , stereocilia , and stress \ufb01 bers 3 \u2013 7 , the Arp2 / 3 complex assembles dendritic actin arrays made of short , relatively slowly growing branched actin \ufb01 laments , e . g . , in lamellipodia of motile cells and at sites of endocytosis 1 , 8 , 9 . How cells assemble actin networks with such diverse morphologies and dynamics in a shared cytoplasm remains an open question . Cellular actin assembly is primarily governed by reactions occur - ring at thebarbedend of actin \ufb01 laments ( sometimesalsoreferred toas the plus end ) 2 , 10 . Even though actin \ufb01 laments can elongate by sponta - neous addition of actin monomers , barbed end dynamics can be fur - ther tuned by three distinct classes of proteins that directly bind barbedends . Theseincludepolymerases , cappers , anddepolymerases . Polymerases , like formins and Ena / VASP , nucleate actin \ufb01 laments , and support \ufb01 lament elongation by remaining processively bound to \ufb01 la - ment barbed ends 11 , 12 . Formins can additionally accelerate the rate of barbed - end assembly by up to \ufb01 vefold in the presence of pro \ufb01 lin - bound actin monomers ( referred to as pro \ufb01 lin - actin monomers or PA henceforth ) 13 , 14 . Cappers , like capping protein ( CP ) and gelsolin , cause the complete arrest of \ufb01 lament growth by preventing monomer addition at free barbed ends 15 , 16 . Depolymerases , like twin \ufb01 lin , com - prise the third class of barbed - end binding proteins 17 , 18 . Initially dis - covered as actin monomer - sequestering protein 19 , twin \ufb01 lin induces barbed - end depolymerization in a nucleotide - speci \ufb01 c fashion 17 , 18 . While twin \ufb01 lin accelerates depolymerization of newly assembled ( ADP - P i ) \ufb01 laments , itslowsdowndepolymerizationofaged ( ADP ) actin \ufb01 laments 18 , 20 . Notably , twin \ufb01 lin \u2019 s depolymerization activities persist even in cytosol - mimicking conditions , i . e . , the presence of high con - centrations of actin monomers 17 , 18 , 20 . Althoughtheindividualeffectsofformin , CPandtwin \ufb01 linonactin assembly are relatively well - characterized , how they simultaneously act in multiprotein teams at barbed ends to in \ufb02 uence actin assembly remains unclear . This question is especially important as these factors Received : 22 December 2022 Accepted : 22 June 2023 Check for updates 1 Department of Physics , Emory University , Atlanta , GA 30322 , USA . 2 Department of Cell Biology , Emory University , Atlanta , GA 30322 , USA . 3 These authors contributed equally : Heidi Ulrichs , Ignas Gaska . e - mail : shekhar @ emory . edu Nature Communications | ( 2023 ) 14 : 3981 1 1 2 3 4 5 6 7 8 9 0 ( ) : , ; 1 2 3 4 5 6 7 8 9 0 ( ) : , ; are often found in the same cellular compartments , such as \ufb01 lopodia , lamellipodia and stereocilia 6 , 18 , 21 \u2013 24 . In addition to twin \ufb01 lin \u2019 s monomer sequestration and barbed end depolymerization functions , it can also directly bind CP via its C - terminal tail . The CP - twin \ufb01 lin interaction is requiredforproper intracellularlocalizationofCP but isdispensable for twin \ufb01 lin \u2019 s uncapping activity in vitro 20 , 25 . Moreover , in absence of direct visualization of CP \u2019 s uncapping by twin \ufb01 lin , the underlying mechanism remains unclear . While a number of recent studies have looked attwin \ufb01 lin - CP interaction , twin \ufb01 lin \u2019 s effects on formin have not yetbeenextensivelystudied . Wepreviouslyreportedthatformin \u2019 srate of barbed - end elongation is not affected by a high concentration of twin \ufb01 lin 18 . Nevertheless , twin \ufb01 lin \u2019 s effects on formin \u2019 s long - lived resi - dence time at the barbed end has yet to be investigated . CP and formin were initially thought to bind barbed ends in a mutually exclusive fashion 26 \u2013 28 . Contrary to this assumption , it was discovered that formin and CP simultaneously bind the same barbed end to form a so - called barbed - end decision complex ( henceforth interchangeably referred to as BFC , where B is the barbed end , F is formin , and C is CP ) 29 , 30 . Their concurrent presence at the \ufb01 lament end accelerates barbed - end dissociation of formin and CP byabout 50 - fold and 10 - fold , respectively 29 . While the mechanisms discussed above reducebarbed - endresidencetimesofforminandCPfrom120minand 30min to just a few minutes 29 , these time scales are still too slow to explain the rapid rates at which intracellular actin structures get assembled , arrested , and turned over in a few seconds 1 , 31 . This prompted us to take a fresh look at the multicomponent dynamics between CP , formin , and twin \ufb01 lin at \ufb01 lament barbed ends . Here we show that while twin \ufb01 lin alone has no direct effects on formin \u2019 s processivity , it greatly enhances formin \u2019 s processivity in presence of CP ( Fig . 1 ) . Using micro \ufb02 uidics - assisted total internal re \ufb02 ection \ufb02 uorescence ( mf - TIRF ) microscopy 32 , 33 , we \ufb01 nd that despite its depolymerization of free barbed ends , twin \ufb01 lin effectively promotes actin assembly when both formin and CP are present together ( Fig . 2 ) . We \ufb01 nd that formin , CP and twin \ufb01 lin can all simultaneously bind a \ufb01 lament barbed end in a trimeric com - plex ( Fig . 3 ) . The dynamics of this formin - CP - twin \ufb01 lin complex at the barbed end are visualized by multicolor single - molecule imaging . We discover that twin \ufb01 lin reduces the lifetime of the CP - formin decision complex at the barbed end by about 17 - fold . The trimeric complex formation leads to accelerated transitions between these proteins at the barbed end . We also visualize twin \ufb01 lin \u2019 s uncapping of CP - bound barbed ends and \ufb01 nd that twin \ufb01 lin displaces CP from the formin - CP complex much more ef \ufb01 ciently than from barbed ends bound only to CP ( Fig . 4 ) . While only a single twin \ufb01 lin binding event is suf \ufb01 cient to remove CP from formin - CP complexes , on average it takes about 31 twin \ufb01 lin binding events to displace CP from barbed ends bound only to CP . Using separation - of - function mutants , we \ufb01 nd that twin \ufb01 lin \u2019 s direct interaction with the actin \ufb01 lament is necessary for its ability to rescue formin \u2019 s processivity ( Fig . 5 ) . This provides direct evidence of a depolymerase , polymerase , and capper simulta - neously binding a growing \ufb01 lament end , and demonstrates the multicomponent mechanism by which they can regulate each other \u2019 s barbed end activities . Results Capping protein and twin \ufb01 lin differentially in \ufb02 uence formin \u2019 s processivity We studied how CP and twin \ufb01 lin in \ufb02 uence formin \u2019 s processivity at actin \ufb01 lament barbed ends . Fluorescent actin \ufb01 laments were initiated in a mf - TIRF chamber by exposing coverslip - anchored formins ( mDia1 FH1 - FH2 - C ) to a solution containing \ufb02 uorescent actin monomers and pro \ufb01 lin - actin ( PA ) ( Fig . 1a ) . Then , to con \ufb01 rm these \ufb01 laments were indeed elongating from formins , a solution containing pro \ufb01 lin and unlabeled actin monomers was \ufb02 owed . The use of unlabeled actin prevents any artifacts that might arise from the use of labeled actin . Since elongation occurs by insertion of unlabeled monomers at the formin - bound barbed end , the pre - existing \ufb02 uorescent segment of the \ufb01 lament appears to move in the direction of the \ufb02 ow ( Fig . 1a , b and Supplementary Movie 1 ) , away from the location of formin anchoring . Dissociation of \ufb01 laments from surface - anchored formins caused the immediate disappearance of \ufb01 laments from the \ufb01 eld of view ( BF \u2192 B + F , where B denotes the barbed end , F denotes the formin , and BF is the formin - bound barbed end ) . We recorded the disappearance of actin \ufb01 laments from the \ufb01 eld of view over time . Changes in the time - dependent survival fraction offormin - bound \ufb01 laments were then used to determine the dissociation rate of formin from the barbed end . Using unlabeled actin subunits eliminates the effects of labeled actin on formin \u2019 s processivity . To con \ufb01 rm that \ufb01 lament disappearance was due to detachment of \ufb01 laments from formin rather than due to the detachment of the entire formin - \ufb01 lament complex from the glass coverslip , we re - exposed the surface to a \ufb02 ow containing 1 \u00b5 M Alexa - 488 G - actin and 0 . 5 \u00b5 M pro \ufb01 lin . Consistent with previous studies , over 80 % of formins were able to renucleate new \ufb01 laments following detachment of initially nucleated \ufb01 laments 29 , 34 . First , we asked how CP in \ufb02 uences the processivity of formins at \ufb01 lament barbed ends . Formin - elongated \ufb02 uorescent \ufb01 laments were exposed to a solution containing either pro \ufb01 lin - actin alone or with a range of concentrations of CP . In control reactions , the \ufb02 uorescent segment of actin \ufb01 laments continued to move away from the attached formin , at a constant speed , in the direction of the \ufb02 ow , indicating processive elongation by formin ( Fig . 1b and Supplementary Movie 1 ) . Onlya smallfraction ( ~ 20 % ) of \ufb01 lamentsdissociatedfromforminsover the duration of the experiment ( 500 s ) . The average barbed - end dwell timeof formin mDia1 was ~ 35 min in control experiments ( Fig . 1f , dwell time = 1 / ( dissociation rate ) ) . Formin \u2019 s long barbed - end residence time measured here agrees with previous studies 13 , 14 , 34 . Interestingly , when cappingproteinwasintroduced ( inthepresenceofPA ) , actin \ufb01 laments rapidly dissociated from formins and disappeared from the \ufb01 eld of view ( Fig . 1c , f and Supplementary Movie 2 ) . The rate of dissociation of formins increased with CP concentration ( 5nM to 1 \u00b5 M ) . Compared to control , 1 \u00b5 M CP increased formin \u2019 s rate of dissociation from the bar - bed end by about 30 - fold ( Fig . 1g ) . While the cytoplasmic concentration of CP is around 1 \u00b5 M 35 , majority of it is thought to be sequestrated by V1 / myotrophin 36 . As a result , only about 10 \u2013 20nM free CP is expected to be available for binding barbed ends in cells 36 . We found thateven a low concentration of CP in this range ( ~ 50nM ) was suf \ufb01 cient to accelerate formin dis - sociation by about tenfold compared to the control ( Fig . 1g ) . We then asked if twin \ufb01 lin also in \ufb02 uenced formin \u2019 s dissociation from barbed ends . In contrast to CP , we found that the presence of up to 1 \u00b5 M twin \ufb01 lin ( mouse mTwf1 , referred to as \u201c twin \ufb01 lin \u201d hereafter ) did not signi \ufb01 cantly change formin \u2019 s barbed - end dwell time ( Fig . 1d , h , i ) . Consistent with our earlier study , twin \ufb01 lin also had no observable effect on formin \u2019 s rate of elongation 18 ( Supplementary Fig . 1 ) . Our results imply that unlike capping protein , formins fully protect barbed ends from twin \ufb01 lin , indicating that twin \ufb01 lin cannot associate with a formin - bound barbed end . We then asked how simultaneous presence of twin \ufb01 lin and CP might in \ufb02 uence formin \u2019 s processivity . When formin - nucleated \ufb01 la - mentsareexposedtoCPandtwin \ufb01 linatthesametime ( inthepresence of PA ) , formin dissociated at a rate intermediate between that of control and capping protein ( Fig . 1h , i and Supplementary Movie 3 ) . While there was a tenfold reduction in formin \u2019 s processivity in the presenceof 50 nMCP , thereductionwasjustthreefoldwhen 50nMCP was supplemented with 1 \u00b5 M twin \ufb01 lin . Our data suggest that twin \ufb01 lin \u2019 s presence led to a reduction in the adverse effects of CP on formin \u2019 s processivity . Consistently , actin \ufb01 laments also grew substantially longer prior to their detachment in the presence of twin \ufb01 lin and CP together ( Fig . 1e ) as compared to CP alone ( Fig . 1c ) . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 2 Twin \ufb01 lin accelerates dissociation of the formin \u2013 CP complex at barbed ends X - ray diffraction and EM studies suggest that both CP and twin \ufb01 lin interact directly with \ufb01 lament barbed ends 37 \u2013 40 . Moreover , twin \ufb01 lin destabilizes CP \u2019 s barbed - end localization and causes a sixfold reduc - tion in CP \u2019 s barbed - end lifetime 20 . We , therefore , wondered if twin \ufb01 lin \u2019 s rescue of formin \u2019 s processivity from CP might be due to its destabilizing effects on CP in the formin - CP - barbed end decision complex . To investigate this , we formed decision complexes by exposing formin - nucleated \ufb02 uorescent actin \ufb01 laments to a high con - centration of CP ( 1 \u00b5 M ) . This caused an almost immediate arrest of actin \ufb01 lament elongation and formation of formin - CP - barbed end 0 100 200 300 400 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 ControlmTwf1CPmTwf1 + CP n o i t c a r f l a v i v r u s n i m r o F Time ( s ) Control5 nM 50 nM 250 nM 500 nM 1 \u00b5 M 0 100 200 300 400 500 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 no i t c a r f l a v i v r u s n i m r o F Time ( s ) h b i a f 0 200 400 600 800 1000 0 . 000 0 . 005 0 . 010 0 . 015 s ( e t a r no i t a i c o ss i d n i m r o F - 1 ) [ Capping protein ] ( nM ) Control mTwf1 CP CP + mTwf1 0 . 000 0 . 002 0 . 004 0 . 006 0 . 008 s ( e t a r no i t a i c o ss i d n i m r o F - 1 ) Capping protein Formin Actin ( labeled ) Actin Profilin Flow and / or Twinfilin Coverslip c d e g PA 60s 10 \u03bc m PA * PA 10 \u03bc m 60s PA * PA PA + CP 10 \u03bc m 60s PA * PA PA + CP + mTwf1 10 \u03bc m 60s PA * PA PA + mTwf1 Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 3 decision complexes 29 , 30 . To study the effects of twin \ufb01 lin on BFC com - plexes , BFC complexes were then exposed to a \ufb02 ow containing either pro \ufb01 lin - actin only ( control ) or supplemented with a range of con - centrations of twin \ufb01 lin ( Fig . 2a ) . The BFC complexes can dissociate by two different routes \u2014 ( 1 ) via dissociation of CP ( BFC \u2192 BF + C , with CP departing from the BFC complex with a rate k \u2019 - C ) , which leads to resumption of elongation of formin - bound \ufb01 laments ( Fig . 2b , top ) or ( 2 ) via dissociation of formin ( BFC \u2192 BC + F , withformindetachingfromtheBFCcomplexwitharate k \u2019 - F ) , which leads to detachment and disappearance of the \ufb01 lament ( Fig . 2b , bottom ) . We found that twin \ufb01 lin dramatically accelerated disassembly of BFC complexes in a concentration - dependent manner ( Fig . 2c , d ) . Compared to the control , 1 \u00b5 M twin \ufb01 lin increased the rate of BFC dissociation ( k - BFC ) by about 18 - fold . While 1 \u03bc M twin \ufb01 lin increased CP \u2019 s dissociation rate from the BFC complex by ~ 11 - fold , formin \u2019 s rate of dissociation did not change ( Fig . 2e , f ) . The dissociation kinetics of BFC complexes into BC and BF wereexponentialwithobservedrateconstant k - BFC = k \u2019 - F + k \u2019 - C where k \u2019 - F is the observed rate constant of dissociation of formin from the BFC complex and k \u2019 - C is the observed rate constant of dissociation of CP from the BFC complex ( Fig . 2e and Supplementary Fig . 2 ) . The fraction of \ufb01 laments transitioning to BF and BC states upon BFC dissociation is givenbythe k \u2019 - C / ( k \u2019 - C + k \u2019 - F ) and k \u2019 - F / ( k \u2019 - C + k \u2019 - F ) ( seeMethods ) . Wederived the values k \u2019 - C and k \u2019 - F asa function of twin \ufb01 linconcentration ( Fig . 2e , f , and Supplementary Fig . 2 ) . We also analyzed the route by which the BFC complexes dissociated ( BFC \u2192 BF + C or BFC \u2192 BC + F ) . In absence oftwin \ufb01 lin , slightlymorethanhalfofallBFCcomplexestransitionedto BC ( ~ 54 % ) and the rest transitioned to BF ( ~ 46 % ) . In presence of twin - \ufb01 lin however , this ratio was skewed heavily towards BF ( ~ 90 % at 1 \u03bc M twin \ufb01 lin ) , i . e . , CP dissociated and left formin solely bound to the bar - bed end in majority of BFC complexes ( Fig . 2g ) . These \ufb01 laments immediately returned to rapid elongation , characteristic of formin \u2019 s presence at the barbed end . Taken together , our results indicate that although twin \ufb01 lin is a depolymerase of free barbed ends , it promotes \ufb01 lament assembly by formin when both CP and formin are present . Just as twin \ufb01 lin can uncap CP from free barbed ends 20 , our results indicate twin \ufb01 lin might also be able to destabilize CP from BFC complexes by forming a ternary complex , BFCT , with formin and CP at the barbed end ( T in BFCT denotes twin \ufb01 lin ) . However , due to our inability to directly visualize twin \ufb01 lin , it was not possible to ascertain whether twin \ufb01 lin \u2019 s effects were due to its barbed - end binding or its interactions with \ufb01 lament sides . Single - molecule visualization of twin \ufb01 lin \u2019 s effects on BFC dynamics To directly visualize the effects of twin \ufb01 lin on the dynamics of the BFC complex , we used three - color single - molecule TIRF imaging . SNAP - tagged constructs of CP ( SNAP - CP ) and formin ( SNAP - mDia1 ) were expressed and labeled with benzylguanine functionalized green - excitable ( 549 - CP ) and red - excitable ( 649 - mDia1 ) \ufb02 uorescent dyes . Photobleaching data con \ufb01 rmed that majority of 549 - CP molecules were labeled with only one dye molecule , consistent with a single SNAP - tag per heterodimeric CP molecule 30 ( Supplementary Fig . 3 ) . Photobleaching tests of 649 - mDia1 showed that majority of these molecules exhibited a single - or double - step bleaching pro \ufb01 le , con - sistent with the dimeric nature of formin mDia1 molecules 29 , 41 ( Sup - plementary Fig . 4 ) . Alexa - 488 labeled G - actin actin monomers were incubated with 649 - mDia1 in a non - micro \ufb02 uidic , conventional \ufb02 ow cell . The majority of newly nucleated \ufb01 laments displayed \ufb02 uorescent formins at their barbed ends ( Fig . 3a ) . All \ufb01 lament barbed ends with detectable 649 - mDia1 underwent rapid , continuous elongation with no noticeable pauses . Consistent with long barbed - end dwell times and low photobleaching , the majority of 649 - mDia1 molecules remained bound to elongating actin \ufb01 laments for the duration of the experiment . 649 - mDia1 bound barbed ends were then exposed to a pro \ufb01 lin - actin solution containing either 549 - CP alone or together with unla - beled twin \ufb01 lin . In the absence of twin \ufb01 lin , we routinely observed the following ( Fig . 3a , b ) : \ufb01 rst , the 649 - mDia1 was joined at the barbed end bya549 - CPmoleculetoformtheBFCcomplex ; second , uponarrivalof 549 - CP , the \ufb01 lament immediately stopped elongating ; third , following abriefintervaloftimeduringwhichbothmoleculeswerejointlybound to the barbed end , the 649 - mDia1 : 549 - CP complex dissociated , and one of these two molecules departed from the barbed end , leaving the other behind ( Supplementary Movie 4 ) . In approximately 58 % of the complexes , 649 - mDia1 dissociated ( N = 42 out of 72 complexes ) , leaving 549 - CP at the barbed end , and no further elongation was observed ( Fig . 3a , c ) . In the remaining complexes , 549 - CP departed ( N = 30 out of 72 complexes ) and the \ufb01 lament immediately switched from the paused state to the rapidly elongating state , characteristic of formin - bound barbed ends . The 649 - mDia1 : 549 - CP barbed end com - plex had an average lifetime of 149 \u00b112 . 4s ( mean\u00b1sem ) ( Fig . 3e , f ) . In contrast , when 649 - mDia1 bound \ufb01 laments were exposed to 549 - CP but in presenceof 20nM twin \ufb01 lin , a much shorter pause wasobserved duringwhichboth549 - CPand649 - mDia1wereboundandthe \ufb01 lament elongation was arrested ( Fig . 3d ) . The average complex lifetime reduced to 35 . 4\u00b15s ( mean\u00b1sem ) , a 75 % reduction over control ( Fig . 3e , f ) . In agreement with our mf - TIRF experiments , twin \ufb01 lin also altered the outcome of BFC complex resolution . In presence of 20 nM twin \ufb01 lin , about 73 % of complexes ( 36 out of 49 ) transitioned to the 649 - mDia1 BF state as compared to 42 % of complexes ( 30 out of 72 ) in control experiments . Our mf - TIRF and single - molecule experiments together con - clusively establish that twin \ufb01 lin in \ufb02 uences both the lifetime and eventual outcome ( BF or BC ) of the BFC complex . We then asked if these effects are caused by binding of twin \ufb01 lin along the \ufb01 la - ment length or by its interactions at the barbed end . To do this , we expressed mouse twin \ufb01 lin - 1 as a SNAP - tagged fusion protein and directly visualized its interactions with the BFC complex . Fig . 1 | Effect of twin \ufb01 lin and capping protein ( CP ) on processivity of formin . a Schematic of the experimental strategy . Actin \ufb01 laments were nucleated from coverslip - anchored formins by a \ufb02 ow containing 1 \u00b5 M G - actin ( 15 % Alexa - 488 labeled ) and0 . 5 \u00b5 Mpro \ufb01 lin . Filamentswerethenelongated inthepresenceof1 \u00b5 M unlabeled G - actin and 4 \u00b5 M pro \ufb01 lin to ensure insertional elongation between \ufb02 uorescentfragmentsandsurface - anchoredformins . Filamentswerethenexposed toa \ufb02 owcontaining0 . 2 \u00b5 MunlabeledG - actinand0 . 7 \u00b5 Mpro \ufb01 lin ( PA ) ( control ) with or without a range of concentrations of CP and / or mTwf1 ( alone or together ) . The survival fraction of formin - bound \ufb01 laments attached was monitored as a function of time . b Representative kymographs of a formin - anchored \ufb01 lament elongating from 0 . 2 \u00b5 M unlabeled G - actin and 0 . 7 \u00b5 M pro \ufb01 lin ( PA ) ( see Supplementary Movie 1 ) . c Same conditions as ( b ) but supplemented with 50nM CP ( see Supple - mentary Movie 2 ) . d Same conditions as ( b ) but supplemented with 1 \u00b5 M mTwf1 . e Same conditions as ( b ) but supplemented with 50nM CP and 1 \u00b5 M mTwf1 ( see Supplementary Movie 3 ) . f Survival fraction of formin - bound \ufb01 laments ( BF ) as a function of time in the presence of PA supplemented with a range of CP con - centrations . Experimental data ( symbols ) are \ufb01 tted to a single - exponential decay function ( lines ) todeterminetheformindissociationrate k - F ( BF \u2192 B + F ) . Numberof \ufb01 lamentsanalyzedforeachcondition ( 0to1 \u00b5 MCP ) : 70 , 36 , 75 , 56 , 26 , 26 . g Formin dissociationrate k - F asafunctionofCPconcentration , determinedfromdatain ( f ) . h Survival fraction of formin - bound \ufb01 laments ( BF ) as a function of time in the presence of PA alone ( black symbols , n = 51 \ufb01 laments ) or supplemented with 1 \u00b5 M mTwf1 ( magenta symbols , n = 50 \ufb01 laments ) , 50nM CP ( yellow symbols , n = 51 \ufb01 la - ments ) or 1 \u00b5 M mTwf1 , and 50nM CP together ( orange symbols , n = 50 \ufb01 laments ) . Experimental data ( symbols ) are \ufb01 tted to a single - exponential decay function ( lines ) . i Formin dissociation rate k - F as determined from data in ( h ) . Error bars in g , i indicate 65 % con \ufb01 dence intervals based on \ufb01 ts ( see Methods ) . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 4 SNAP - tagging did not alter mTwf1 \u2019 s ability to uncap CP from CP - bound barbed ends ( Supplementary Fig . 5 ) . Photobleaching records of surface adsorbed 549 - SNAP - mTwf1 showed that almost all molecules exhibited single - step photobleaching , con \ufb01 rming that labeled twin \ufb01 lin molecules were monomeric ( Supplemen - tary Fig . 6 ) . Using labeled twin \ufb01 lin , we further examined the mechanism by which twin \ufb01 lin accelerated dissociation of BFC complexes . We exposed 649 - mDia1 bound Alexa - 488 actin \ufb01 laments to a solution containing \ufb02 uorescently labeled 549 - mTwf1 and unlabeled CP . Expectedly , \ufb01 laments rapidly paused due to BFC complex formation between unlabeled CP and 649 - mDia1 , leading to an abrupt stop in the a c d f 46 % 73 % 89 % 54 % 27 % 11 % 0 nM 50 nM 1 \u03bcM 0 20 40 60 80 100 no i t a i c o ss i d nopu s e x e l p m o c C F B f o e t a F [ mTwf1 ] BC BF 0 50 1000 0 . 00 0 . 01 0 . 02 0 . 03 0 . 04 0 . 05 0 . 06 ( P C f o e t a r no i t a i c o ss i d d e v r e s b O k ' - C r o ) ( n i m r o F k ' - F s ( C F B m o r f ) - 1 ) [ mTwf1 ] ( nM ) k ' - C k ' - F 0 200 400 600 800 1000 0 . 00 0 . 03 0 . 06 0 . 09 0 . 12 0 . 15 s ( e t a r no i t a i c o ss i d C F B - 1 ) [ mTwf1 ] ( nM ) 0 100 200 300 400 500 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 Control15 nM mTwf1 50 nM mTwf1 100 nM mTwf1 250 nM mTwf1 1 \u00b5 M mTwf1 de l b m e ss a s i d s e x e l p m o c C F B f o no i t c a r F Time ( s ) 0 100 200 300 400 500 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 s t ne m a li f f o no i t c a r F Time ( s ) BFC BC BF 0 nM mTwf1 0 100 200 300 400 500 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 s t ne m a li f f o no i t c a r F Time ( s ) BFC BF BC 50 nM mTwf1 0 100 200 300 400 500 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 s t ne m a li f f o no i t c a r F Time ( s ) BFC BF BC 1 \u00b5 M mTwf1 Flow Capping protein Formin Actin ( labeled ) Actin Profilin Twinfilin Coverslip 30s 10 \u03bc m PA * CP PA ( + / - mTwf1 ) 30s 10 \u03bc m PA * CP PA ( + / - mTwf1 ) b e g Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 5 translocation of 649 - mDia1 molecules bound to actin \ufb01 laments . Toour surprise , we observed 549 - mTwf1 molecules were now able to tran - siently join 649 - mDia1 ( and unlabeled CP ) at the barbed end ( Fig . 3g , h ) . Association of 549 - mTwf1 molecules with 649 - mDia1 : unlabeled CP - bound barbed ends were very brief , with an average dwell time of 1 . 4 \u00b10 . 2 s ( mean\u00b1 sd , n = 8 events ) , translating to a dissociation rate of 0 . 71s \u2212 1 . The majority of 549 - mTwf1 : 649 - mDia1 : unlabeled CP BFCT complexes remained intact just for a single frame implying the 0 . 71 s \u2212 1 rate of dissociation of 549 - mTwf1 molecules fromBFCT complex is the lower bound for this rate . Importantly , the departure of 549 - mTwf1 from the BFCT complex led to an immediate transition of the barbed end from the arrested state to the fast formin - based elongation state with visible translocation of the 649 - mDia1 molecule . Notably , in absence of CP , we never observed colocalization of 649 - mDia1 and 549 - mTwf1 at barbed ends . This explains our mf - TIRF results that twin \ufb01 lin does not in \ufb02 uence the rate of elongation or processivity of formins ( Fig . 1h and Supplementary Fig . 1 ) . Together , these observa - tions indicate that the unlabeled CP molecule and twin \ufb01 lin departed the barbed end simultaneously , leaving formin behind . Notably , we never observed twin \ufb01 lin and CP simultaneously arrive at formin - occupied barbed ends . Single - molecule analysis uncovers twin \ufb01 lin \u2019 s uncapping mechanism Although twin \ufb01 lin accelerates CP \u2019 s dissociation 42 , in the absence of simultaneous visualization of these two proteins at barbed ends , the underlying mechanism has remained obscure . We therefore tran - siently exposed actin \ufb01 laments elongating from Alexa - 488 labeled monomers to 649 - CP in a non - micro \ufb02 uidic , conventional \ufb02 ow cell ( Fig . 4a ) . Expectedly , all \ufb01 laments with a visible 649 - CP signal at their barbed end immediately stopped growing following 649 - CP \u2019 s arrival . Upon exposure to 549 - mTwf1 , short - lived associations of twin \ufb01 lin at the 649 - CP - bound barbed ends were observed . To our surprise , unlike inthecaseofBFCcomplexeswhereasingletwin \ufb01 linbindingeventwas suf \ufb01 cient to cause CP \u2019 s departure , 649 - CP remained bound to the barbed end despite repeated arrivals and departures of 549 - mTwf1 molecules at the barbed end ( Fig . 4b ) . Each 549 - mTwf1 : 649 - CP colo - calization event lasted about a second . However , owing to the slow rateofuncappingbytwin \ufb01 lin ( itonlyacceleratestheuncappingrateby sixfold ) , capturing simultaneous departure of 649 - CP and 549 - mTwf1 required frequent , long - term imaging , which in turn caused excessive photobleaching of 649 - CP . As a result , we observed that the dis - appearance of 649 - CP from barbed ends was not always followed by \ufb01 lament depolymerization . These observations suggested that the disappearance of the 649 - CP signal from barbed ends was due to photobleaching and not because of CP \u2019 s dissociation . Owing to photobleaching of 649 - CP , we instead used unlabeled CP . In these experiments , we used the onset of depolymerization of barbed ends to detect the moment when 549 - mTwf1 dissociated unlabeled CP from barbed ends ( Fig . 4c ) . Successful CP dissociation required on average 30 . 9\u00b16 . 5 ( mean \u00b1sem ) successive 549 - mTwf1 binding events , each lasting for 1 . 9\u00b10 . 1 s ( mean\u00b1 sem ) ( Fig . 4d , e ) . Notably , 549 - mTwf1 intensity appeared and disappeared from the barbed end in a single step , suggesting only a single 549 - mTwf1 molecule colocalized with CP at a time at the \ufb01 lament barbed end ( Fig . 4d ) . Twin \ufb01 lin \u2019 s interaction with actin is essential for its effects on BFC dynamics As mentioned earlier , twin \ufb01 lin can directly bind CP as well as interact with terminal F - actin subunits of the barbed end 37 , 43 . Which of these interactions is responsible for twin \ufb01 lin \u2019 s effects on BFC complex dynamics ? To answer this question , we puri \ufb01 ed two twin \ufb01 lin mutants : ( 1 ) the \u201c ADF - domainmutant \u201d , which inhibits twin \ufb01 lin \u2019 s interaction with actin and ( 2 ) the \u201c tail mutant \u201d , which interferes with twin \ufb01 lin \u2019 s direct binding to CP 44 ( Fig . 5a ) . Pre - formed BFC complexes ( similar to the strategy used in Fig . 2a ) were exposed to a solution containing either pro \ufb01 lin - actin only or supplemented with wild - type or mutant twin \ufb01 lin ( Fig . 5b ) . Mutations in the twin \ufb01 lin tail led to BFC dissociation activity partway between the control and the wildtype ( Fig . 5c , d ) . However , the ADF - domain mutant didn \u2019 t cause any visible acceleration of BFC complex dissociation , implying that direct contact between twin \ufb01 lin and actin is essential for these activities . Consistently , an earlier study showedthattheADF - domainmutantisalsoincapableofuncappingCP from free barbed ends 20 . Taken together , our data suggests that while twin \ufb01 lin \u2019 s interaction with CP via its tail domain is bene \ufb01 cial , twin \ufb01 lin \u2019 s binding to actin via its ADF domains is necessary for it to be able to form the BFCT ternary complex at the barbed end and for its ability to rapidly dissociate BFC complexes . Discussion Living cells can rapidly tune actin assembly in response to external stimuli 1 . The actin \ufb01 lament barbed end is considered to be the primary site of regulation of actin assembly . Over the last few decades , several barbed - end interacting proteins , including polymerases ( e . g . , formin ) , cappers ( e . g . , CP ) , and depolymerases ( e . g . , twin \ufb01 lin ) have been dis - covered . Howthesedistinctprotein activities get integrated within the cytoplasm remains an open question . On its own , CP caps free barbed ends and promotes dissociation of formin from formin - bound barbed ends 29 , 30 . Twin \ufb01 lin promotes barbed - end depolymerization and accel - erates the dissociation of CP from barbed ends 17 , 18 , 20 . Here , we have uncovered a mechanism by which twin \ufb01 lin , formin , and CP simulta - neouslybinda \ufb01 lamentbarbedendtoformamulticomponenttrimeric protein complex . We also \ufb01 nd that twin \ufb01 lin does not bind actin \ufb01 la - ment ends occupied by formin mDia1 unless CP is present . Our single - molecule experiments bring valuable insights into twin \ufb01 lin \u2019 s uncapping mechanism ( Fig . 4 ) . We found that unlike co \ufb01 lin , which uncaps by decorating actin \ufb01 lament sides 45 , twin \ufb01 lin directly associates with CP - bound barbed ends to uncap actin \ufb01 laments . Twin \ufb01 lin on its own is , at best , a weak uncapper and only increases the Fig . 2 | Effect of twin \ufb01 lin on capping protein ( CP ) \u2014 formin decision complex . a Experimental strategy schematic . Actin \ufb01 laments were nucleated from coverslip - anchored formins by \ufb02 owing 1 \u00b5 M G - actin ( 15 % Alexa - 488 labeled ) and 0 . 5 \u00b5 M pro \ufb01 lin . Filaments were then exposed to \ufb02 ow containing 1 \u00b5 M unlabeled G - actin , 4 \u00b5 M pro \ufb01 lin , and 1 \u00b5 M CP for ~ 10s to convert formin - bound barbed ends ( BF ) to formin - CP - bound barbed ends ( BF + C \u2192 BFC ) . BFC complexes were then exposed to \ufb02 ow containing PA only or with a range of mTwf1 concentrations . b Representative \ufb01 lament kymographs are transitioning to the BFC state upon exposure to 1 \u00b5 M CP . Upon CP removal from solution and exposure to PA ( with or without mTwf1 ) , \ufb01 laments resume elongation following CP dissociation from BFC ( top , BFC \u2192 BF + C ) or detach from formin ( bottom , BFC \u2192 BC + F ) . White arrow - heads denote BFC complex dissociation . c Dissociation fraction of formin - CP - bound \ufb01 laments ( BFC ) as a function of time in the presence of PA with / without a range of mTwf1 concentrations . Experimental data ( symbols ) are \ufb01 tted to a single - exponential function ( lines ) to determine the rate of BFC disassembly . Number of \ufb01 lamentsanalyzedpercondition ( 0to1 \u00b5 MmTwf1 ) : 31 , 51 , 30 , 44 , 42 , and50 . d BFC dissociation rate into BC or BF as a function of mTwf1 concentration , determined from ( c ) . e Fraction of BFC ( black symbols ) \ufb01 laments transitioning to BF ( magenta symbols ) or BC ( yellow symbols ) . Experimental data ( symbols ) are \ufb01 tted to expo - nential \ufb01 ts ( lines ) , suchthat k - BFC = k \u2019 - F + k \u2019 - C , where k \u2019 - F istheformindissociationrate from BFC ( BFC \u2192 BC + F ) and k \u2019 - C is CP dissociation rate from BFC ( BFC \u2192 BF + C ) . Conditions \u2013 left ( 0nM mTwf1 , 50 \ufb01 laments ) , center ( 50nM mTwf1 , 37 \ufb01 laments ) , right ( 1 \u00b5 M mTwf1 , 30 \ufb01 laments ) . See Supplementary Fig . 2 for the range of mTwf1 concentrations . f Dissociation rate ofCP ( k \u2019 - C ) or Formin ( k \u2019 - F ) from BFC complexes g PercentagesofBFCcomplexesin ( e ) transitioningtoBC ( yellow ) orBF ( magenta ) at different mTwf1 concentrations . Error bars in ( d ) indicate 65 % con \ufb01 dence intervalsbasedon \ufb01 ts ( seemethods ) . SourcedataareprovidedasaSourceData \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 6 0 10 20 0 25 50 75 100 125 150 1a i D m - 94 6 : P C - 945 f o e m i t e f il nae M ) s ( s e x e l p m o c no i s i c ed [ mTwf1 ] ( nM ) 0 s 368 s 184 s mDia1 CP Merge 0 s 92 s 184 s 276 s 368 s 2 \u00b5 m 0 250 500 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 no i t c a r F F r a c t i on F r a c t i on 0 nM mTwf1 5 nM mTwf1 20 nM mTwf1 0 250 500 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 Residence time of 549 - CP & 649 - mDia1 complexes at barbed ends ( s ) 0 250 500 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 a b c d e f 0 1 649 - mDia1 549 - CP 488 - Actin 0 1 e c ne cs e r ou l F i n t en s i t y ( a r b . un i t s ) 0 100 200 300 400 0 4 8 12 t ne m a li F l eng t h ( \u00b5 m ) Time ( s ) 0 1 649 - mDia1 549 - CP 488 - Actin 0 1 e c ne cs e r ou l F y t i s ne t n i ) s t i nu . b r a ( 0 100 200 0 4 8 12 16 t ne m a li F l eng t h ( \u00b5 m ) Time ( s ) BF BFC BC BF BFC BF g 0 1 649 - mDia1 549 - mTwf1 488 - Actin 0 1 e c ne cs e r ou l F i n t en s i t y ) s t i nu . b r a ( 0 50 100 150 0 1 2 3 F il a m en t l eng t h ( \u00b5 m ) Time ( s ) BF BFC BF BFCT 138 s 128 s 133 s mDia1 mTwf1 Merge h Fig . 3 | Direct visualization of formin , CP , and mTwf1 at barbed ends . a Representative time - lapse images of a multicolor single - molecule TIRF experi - ment ( see Supplementary Movie 4 ) . Actin \ufb01 laments were initiated byincubationof 0 . 5 \u00b5 MG - actin ( 15 % Alexa - 488labeled , 0 . 5 % biotin - labeled ) , 1 \u00b5 Mpro \ufb01 lin , and50pM 649 - mDia1 ( magenta ) . 649 - mDia1 bound \ufb01 laments were then exposed to PA and 10nM 549 - CP ( yellow ) withorwithout mTwf1 . The white boxaround each \ufb01 lament end indicates the location of the barbed end . Insets show individual and merged channels localized at the barbed end . Scale bar , 2 \u00b5 m . b Fluorescence images of a 13 \u00d7 13 - pixel box around the barbed end of the \ufb01 lament from ( a ) show the for - mation and dissociation of a mDia1 \u2013 CP complex at the barbed end . The Formin channel is magenta and the CP channel is yellow . c Fluorescence intensity and length records of the \ufb01 lament in ( a ) with formation and resolution of the decision complex in the absence of mTwf1 . d Same as ( c ) but in the presence of 20nM mTwf1 . Gray shaded boxes indicate the period when both 549 - CP and 649 - mDia1 were simultaneously present at the barbed end , i . e . , the BFC complex . e Distributionoflifetimesof649 - mDia1 : 549 - CPdecisioncomplexesatbarbedends in presence of 0nM mTwf1 ( left , n = 110 BFC complexes ) , 5nM mTwf1 ( center , n = 60 BFC complexes ) , and 20nM mTwf1 ( right , n = 57 BFC complexes ) . f Mean lifetimes ( \u00b1sem ) of 649 - mDia1 : 549 - CP decision complexes at barbed ends as a function of mTwf1 concentration , determined from data in ( e ) . g Fluorescence intensity and length records of a \ufb01 lament with formation and resolution of the decision complex formed in the presence of 100pM 649 - mDia1 , 10nM unlabeled CP , and 40nM 549 - mTwf1 . The gray shaded box indicates the time duration when both 549 - mTwf1 , 649 - mDia1 , and unlabeledCPwere simultaneously present atthe barbed end ( BFCT ) . h Cropped \ufb02 uorescence images of a 13 \u00d7 13 - pixel box around thebarbedendofthe \ufb01 lamentin ( g ) showtheformationanddissociationofa649 - mDia1 \u2013 unlabeledCPcomplexinthepresenceof549 - mTwf1 . Theforminchannelis magenta and twin \ufb01 lin channel is yellow . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 7 rate of uncapping by about sixfold . A successful uncapping event , on average , takes about 31 separate twin \ufb01 lin association and disassocia - tion events at the barbed end . In contrast , CARMIL accelerates CP \u2019 s departure by about 180 - fold , bringing down CP \u2019 s barbed - end dwell time from 30 min to just about 10s 46 . As a result , although both CARMIL and twin \ufb01 linbind CP similarlyviatheir CPI motifs 47 , they exert vastly different control on CP . In addition to uncapping , twin \ufb01 lin is also an actin depolymerase 17 , 18 . How does it carry out these two distinct activities ? Our experiments with twin \ufb01 lin variants containing mutations in the actin - binding ADF domains suggest that twin \ufb01 lin \u2019 s interactions with the actin \ufb01 lament are essential for it to rescue formin \u2019 s processivity from CP . Mutations in twin \ufb01 lin \u2019 s tail domain interfere with direct twin \ufb01 lin - CP 20 binding . Although less strongly than the wildtype , we 0 2 4 6 8 10 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 no i t c a r F 549 - mTwf1 dwell times on CP - bound ends ( s ) 1 . 9 \u00b1 0 . 1 s b 15s 1 \u00b5 m 647 - CapZ 488 - Actin Merge 549 - mTwf1 e d c a 125 . 5 s 127 . 1 s mTwf1 0 1 0 50 100 150 0 1 e c ne cs e r ou l F i n t en s i t y ( a r b . un i t s ) Time ( s ) m T w f 1 E v en t s Twinfilin ( labeled ) CP ( unlabeled ) Actin ( labeled ) Twinfilin ( labeled ) CP ( labeled ) Actin ( labeled ) Fig . 4 | Visualization and characterization of twin \ufb01 lin \u2019 s interactions with CP - bound barbedends . a Schematicofthree - colorsingle - molecule experiments with labeledCPandlabeledmTwf1 . Actin \ufb01 lamentswereassembledfromof1 \u00b5 MG - actin ( 15 % Alexa - 488 labeled , 1 . 4 % biotin - labeled ) and then capped by 10nM 649 - CP . These \ufb01 lamentswerethen exposed to 20nM 549 - mTwf1 . The greenarrow denotes polymerization . b Binding of labeled twin \ufb01 lin on CP - bound \ufb01 lament barbed ends recorded at 1s time resolution . Kymographs show Alexa - 488 actin ( top ) , 649 - CP ( secondfromtop ) , 549 - mTwf1 ( thirdfromtop ) , andmerge ( bottom ) . Magentabars denote episodes in whicha 549 - mTwf1 moleculewas present at the 649 - CP - bound barbed end of the \ufb01 lament . c Schematic of the two - color single - molecule experi - ments with unlabeled CP and labeled mTwf1 . Actin \ufb01 laments were polymerized from 1 \u00b5 M G - actin ( 15 % Alexa - 488labeled , 1 . 4 % biotin - labeled ) and0 . 5 \u00b5 M pro \ufb01 ling , then capped by unlabeled CP . The capped \ufb01 laments were then exposed to 15nM 549 - mTwf1 . Arrival and departureof 549 - mTwf1 molecules at the barbed end were recorded until the \ufb01 lament started depolymerizing , i . e . , CP dissociated . The green arrow denotes polymerization , and the red arrow denotes depolymerization . d Distribution of residence times of 549 - mTwf1 on unlabeled CP - bound \ufb01 lament barbed ends ( n = 510 binding events across 16 \ufb01 laments ) . Mean dwell time = 1 . 9\u00b1 0 . 1s ( \u00b1sem ) . The histogram represents 99 % of all binding events ( remaining 1 % outliers are not shown ) . 100 % of binding events were included in calculations ofmeandwelltime . e Top : timerecordsof549 - mTwf1 \ufb02 uorescenceintensityatthe unlabeled CP - bound barbed end of an actin \ufb01 lament . Intensity is integrated over a 5 \u00d7 5 - pixel square centered around the barbed end of the \ufb01 lament . Bottom : pre - sence ( 1 ) or absence ( 0 ) of 549 - mTwf1 molecules at the CP - 649 - bound \ufb01 lament barbed end shown above . Inset : cropped \ufb02 uorescence images of a 10 \u00d7 10 - pixel box around the barbed end of the \ufb01 lament showing the arrival , presence , and departureofasingle549 - mTwf1moleculeattheCP - boundbarbedend . Sourcedata are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 8 \ufb01 nd that the tail mutant can still dissociate CP from the formin - CP barbed end complex and modestly rescue formin \u2019 s processivity from CP . Thiscanbeinterpretedintwoways . Firstly , fortwin \ufb01 lin \u2019 seffectson the BFC complex , its direct binding to CP is bene \ufb01 cial but not neces - sary . Interestingly , a previous study found that the tail mutant uncaps CP from free barbed ends much faster than the wildtype 20 . Alter - natively , thiscouldalsomeanthat thereareadditionalsitesintwin \ufb01 lin , apart from its tail , which participate in its direct binding to CP . Indeed , the linker region between the two ADF domains in twin \ufb01 lin has been proposed as an extra site of contact between CP and twin \ufb01 lin when both are bound to actin 37 . Importantly , mutations in ADF domains , which interfere with twin \ufb01 lin \u2019 s actin binding and extinguish twin \ufb01 lin \u2019 s uncapping activities at free barbed ends 20 , also extinguish twin \ufb01 lin \u2019 s effects on BFC complex dissociation . Together , these results indicate that in the ternary BFCT complex comprising of formin , CP , and twin \ufb01 lin at barbed ends , each factor is directly interacting with the actin \ufb01 lament and not via each other . How would this multicomponent mechanism be relevant in vivo ? Twin \ufb01 lin can depolymerize free barbed ends even in the presence of polymerizable actin monomers 17 , 18 , 20 . In such cytosol - mimicking con - ditions , the simultaneous presence of twin \ufb01 lin and CP would strongly favor \ufb01 lament capping and actin disassembly over actin assembly . How , then , would intracellular actin assembly occur at all ? Combining our observations with previous studies , we present a working model for how polymerases , depolymerases , and cappers can simultaneously regulate actin assembly ( Fig . 6 ) . Eventual barbed - end outcomes would depend upon whether the barbed end was bound to a formin or not . Free barbed ends would \ufb01 rst rapidly get capped by CP ( Fig . 6a ) . Fol - lowing uncapping by twin \ufb01 lin , barbed ends would undergo twin \ufb01 lin - mediated depolymerization 20 . Formin - bound barbed ends , on the other hand , would \ufb01 rst get paused by CP , leading to the formation of BFCdecisioncomplexes ( Fig . 6b ) 29 , 30 . Twin \ufb01 lin \u2019 ssubsequentbindingto BFC complexes to form BFCT complexes would cause CP \u2019 s dissocia - tion and resumption of formin - based \ufb01 lament elongation . As the \ufb01 la - ment ages , other actin - binding proteins with a preference for ADP - F - actin , e . g . , co \ufb01 lin and cyclase - associated protein ( CAP ) , would initiate \ufb01 lament severing and pointed - end depolymerization 45 , 48 \u2013 50 . As a result , \ufb01 lamentswithfreebarbedendswouldrapidlydepolymerizefromboth ends into monomers and formin - bound \ufb01 laments would continue polymerizing at their barbed ends while depolymerizing from their pointed ends , akin to treadmilling . Therefore , the depolymerase twin \ufb01 lin can promote polymerization in the presence of both CP and formin . Although surprising , twin \ufb01 lin \u2019 s ability to promote assembly is not unique . Twin \ufb01 lin is part of the ADF homology ( ADF - H ) family of proteins and contains two ADF domains 51 . Like twin \ufb01 lin , co \ufb01 lin also promotes uncapping and accelerates \ufb01 lament depolymerization ( on top of its severing activities ) 45 , 50 , which in turn promotes assembly by replenishing the pool of monomeric actin . Where in a cell would the mechanisms uncovered here be rele - vant ? Twin \ufb01 lin , CP , and formin operate simultaneously in a number of Control WT mTwf1 Tail mutant ADF mutant 0 . 00 0 . 02 0 . 04 0 . 06 0 . 08 0 . 10 0 . 12 s ( e t a r no i t a i c o ss i d C F B - 1 ) a ADF - H ADF - H ADF - H ADF - H ADF - H ADF - H 1 139 175 313 350 WT mTwf1 Tail mutant ADFdomainmutant b c 0 100 200 300 400 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 ControlmTwf1 WT mTwf1 tail mutant mTwf1 ADF mutant no i t c a r f l a v i v r u s ) C F B ( x e l p m o c no i s i c e D Time ( s ) Flow Formin Actin ( labeled ) Actin Profilin Twinfilin ( WT / mutants ) Coverslip d Capping protein Fig . 5 | Twin \ufb01 lin \u2019 s direct interaction with actin \ufb01 lament is essential for its effects on formin - CP decision complexes . a Domain diagram of wild - type and mutant mTwf1 constructs used here 20 . \u201c x \u201d denotes the location of mutations . b Schematic representation of the experimental strategy . Actin \ufb01 laments were nucleated from coverslip - anchored formins by introducing a \ufb02 ow containing 1 \u00b5 M G - actin ( 15 % Alexa - 488 labeled ) and 0 . 5 \u00b5 M pro \ufb01 lin . The \ufb01 laments were then exposed to a \ufb02 ow containing 1 \u00b5 M unlabeled G - actin , 4 \u00b5 M pro \ufb01 lin , and 500nM CP for about 10s to convert formin - bound barbed ends ( BF ) to formin - CP - bound barbed ends or decision complexes ( BF + C \u2192 BFC ) . These BFC complexes were then exposed to a \ufb02 ow containing PA only or supplemented with wildtype or mutant mTwf1 . c Survival fraction of formin - CP - bound \ufb01 laments ( BFC complexes ) as a function of time in the presence of PA only ( black symbols , 92 \ufb01 laments ) , or supplemented with 1 \u00b5 M wild - typemTwf1 ( black , 36 \ufb01 laments ) , mTwf1ADFmutant ( green , 77 \ufb01 laments ) , or mTwf1 tail mutant ( blue symbols , 22 \ufb01 laments ) . Experi - mental data ( symbols ) were \ufb01 tted to a single - exponential function ( lines ) to determine BFC dissociation rate k - BFC . Error bars indicate 65 % con \ufb01 dence intervals based on \ufb01 ts ( see methods ) . d BFC dissociation rate for wild - type and mutant mTwf1 , determined from data in ( c ) . Error bars in ( d ) indicate 65 % con \ufb01 dence intervalsbasedon \ufb01 ts ( seeMethods ) . SourcedataareprovidedasaSourceData \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 9 cellular compartments , including \ufb01 lopodia , lamellipodia , and stereocilia 6 , 18 , 21 \u2013 24 where size and elongation rates of actin \ufb01 laments are tightly controlled . Given the high barbed - end af \ufb01 nity and intracellular concentration of barbed - end binding proteins like formin , CP , twin \ufb01 - lin , and Ena / VASP , we expect that newly nucleated free barbed ends initiated by Arp2 / 3complex wouldbe rapidly captured by one of these factors . Althoughforminsaremainlythoughtofinthecontextoflinear actin structures , formins like FMNL2 have been shown to play a key role in branched actin networks at the leading edge 24 . In light of our results , we speculate that majority of growing barbed ends in cells might be protected by formins or Ena / VASP . In the future , it will be interesting to explore if other biochemical or mechanical factors which in \ufb02 uence activities of CP and / or formin , e . g . , CARMIL 16 , 36 , V1 / myotrophin 36 , Spire 52 , Bud14 53 , IQGAP 54 , 55 , or Hof1 56 , polyphosphoinositides 42 , 57 orforce 34 mightalsoin \ufb02 uenceBFCcomplex dynamics totune actin assembly . Itwillalsobe important toexamineif the interactions between twin \ufb01 lin , CP , and formin reported here might also play a role in human diseases and disorders , including cancer invasionandprogression 58 , 59 , hearingloss 60 aswellasneuropathiesand cardiac conditions 61 in which these proteins have been implicated . Methods Puri \ufb01 cation and labeling of actin Rabbit skeletal muscle actin was puri \ufb01 ed from acetone powder gen - erated from frozen ground hind leg muscle tissue of young rabbits ( PelFreez , USA ) . Lyophilized acetone powder stored at \u2212 80\u00b0C was mechanically sheared in a coffee grinder , resuspended in G - buffer ( 5 mM Tris - HCl pH 7 . 5 , 0 . 5mM Dithiothreitol ( DTT ) , 0 . 2mM ATP , and 0 . 1 mM CaCl 2 ) , and cleared by centrifugation for 20 min at 50 , 000 \u00d7 g . The supernatant was collected and further \ufb01 ltered with the Whatman paper . Actinwasthenpolymerizedovernightat4 \u00b0C , slowlystirring , by the addition of 2 mM MgCl 2 and 50mM NaCl to the \ufb01 ltrate . The next morning , NaClpowderwasadded toa \ufb01 nal concentration of 0 . 6M and stirring was continued for another 30min at 4 \u00b0C . Then , F - actin was pelleted by centrifugation for 150min at 280 , 000 \u00d7 g , the pellet was solubilized by Dounce homogenization and dialyzed against G - buffer for 48 h at 4\u00b0C . Monomeric actin was then precleared at 435 , 000 \u00d7 g and loaded onto a Sephacryl S - 200 16 / 60 gel - \ufb01 ltration column ( Cytiva , USA ) equilibrated in G - Buffer . Fractions containing actin were stored at 4 \u00b0C . To biotinylate actin , puri \ufb01 ed G - actin was \ufb01 rst dialyzed overnight at 4 \u00b0C against G - buffer lacking DTT . The monomeric actin was then polymerized by the addition of an equal volume of 2X labeling buffer ( 50mM imidazole pH 7 . 5 , 200mM KCl , 0 . 3 mM ATP , 4 mM MgCl 2 ) . After 5 min , the actin was mixed with a \ufb01 vefold molar excess of NHS - XX - Biotin ( Merck KGaA , Germany ) and incubated inthe darkfor 15 h at 4 \u00b0C . The F - actin was pelleted as above , and the pellet was rinsed with G - buffer , then homogenized with a Dounce and dialyzed against G - buffer for 48 h at 4 \u00b0C . Biotinylated monomeric actin was puri \ufb01 ed further on a Sephacryl S - 200 16 / 60 gel - \ufb01 ltration column as above . Aliquots of biotin - actin were snap frozen in liquid N 2 and stored at \u2212 80\u00b0C . To \ufb02 uorescently label actin , G - actin was polymerized by dialyzing overnight against a modi \ufb01 ed F - buffer ( 20mM PIPES pH 6 . 9 , 0 . 2mM CaCl 2 , 0 . 2mMATP , and 100mMKCl ) 32 . F - actin wasincubated for 2h at room temperature with a \ufb01 vefold molarexcessof Alexa - 488 NHS ester dye ( Thermo Fisher Scienti \ufb01 c , USA ) . F - actin was then pelleted by centrifugationat450 , 000\u00d7 g for 40minatroomtemperature , andthe pellet was resuspended in G - buffer , homogenized with a Dounce , and incubatedonicefor2 htodepolymerizethe \ufb01 laments . Themonomeric actin was then re - polymerized on ice for 1 h by the addition of 100 mM KCl and 1 mM MgCl 2 . F - actin was once again pelleted by centrifugation for 40min at 450 , 000 \u00d7 g at 4 \u00b0C . The pellet was homogenized with a Dounce and dialyzed overnight at 4 \u00b0C against 1 L of G - buffer . The solution was precleared by centrifugation at 450 , 000 \u00d7 g for 40 min at 4 \u00b0C . The supernatant was collected , and the concentration and labeling ef \ufb01 ciency of actin was determined . Puri \ufb01 cation and labeling of mTwf1 polypeptides Wildtype and mutant mouse mTwf1 plasmids were a gift from Pekka Lappalainen 20 . All of these proteins were expressed in E . coli BL21 ( pRare ) . Cells were grown in Terri \ufb01 c Broth to log phase at 37\u00b0C . Formin ( F ) ADP - Actin ATP - Actin Capping protein ( C ) ADP - P i - Actin Twinfilin ( T ) Cofilin Cyclase associated protein ( CAP ) b a BC BCT BT B BF BFC BFCT BF Formin - bound barbed ends Free barbed ends Fig . 6 | Working model for regulation of actin dynamics by twin \ufb01 lin , formin , andCP . Barbed - endoutcomeswoulddependuponwhetherbarbedendswerefree or formin - bound . a Free barbed ends ( B ) would rapidly get capped by CP ( C ) , followedbyCP \u2019 sdissociationbytwin \ufb01 lin ( T ) . Thiswouldleavetwin \ufb01 linaloneatthe \ufb01 lamentend , causingitsdepolymerization . Atthesametime , co \ufb01 linwouldbindthe sides of the aging \ufb01 lament and synergize with cyclase - associated protein ( CAP ) to initiate the \ufb01 lament \u2019 s pointed - end depolymerization . The simultaneous depoly - merizationatthetwoendswouldresultinthecompletedisassemblyofthe \ufb01 lament into monomers . b Formin ( F ) bound barbed ends would get paused by CP to form BFC complexes . Twin \ufb01 lin \u2019 s binding to BFC complexes would cause CP \u2019 s dissocia - tion and renewal of formin - based \ufb01 lament elongation . As a result , the \ufb01 lament wouldappeartoatreadmill , i . e . , continueelongatingatthebarbedendwhileatthe same time being disassembled at the pointed end by CAP - co \ufb01 lin synergy . \u201c B \u201d denotesbarbedend , \u201c F \u201d denotesformin , \u201c C \u201d denotesCP , and \u201c T \u201d denotestwin \ufb01 lin . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 10 Expression was induced overnight at 18 \u00b0C by the addition of 1 mM IPTG . Cells were harvested by centrifugation at 11 , 200 \u00d7 g for 15min and the cell pellets were stored at \u2212 80\u00b0C . For puri \ufb01 cation , frozen pellets were thawed and resuspended in 35mL lysis buffer ( 50 mM sodium phosphate buffer pH 8 , 20 mM imidazole , 300mMNaCl , 1 mM DTT , 1 mM PMSF , and protease inhibitors ( pepstatin A , antipain , leu - peptin , aprotinin , and chymostatin , 0 . 5 \u03bc M each ) ) . Cells were lysed using a tip sonicator while kept on ice . The cell lysate was then cen - trifuged at 120 , 000 \u00d7 g for 45min at 4\u00b0C . The supernatant was then incubated with 1 mL of Ni - NTA beads ( Qiagen , USA ) while rotating for 2 h at 4 \u00b0C . The beads were then washed three times with the wash buffer ( 50mM sodium phosphate buffer pH 8 , 300mM NaCl , 20 mM imidazole , and 1 mM DTT ) . The beads were then transferred to a dis - posable column ( Bio - Rad , USA ) . Protein was eluted using the elution buffer ( 50mM phosphate buffer pH 8 , 300 mM NaCl , 250mM imida - zole , and 1mM DTT ) . Fractions containing the protein were con - centrated and loaded onto a size exclusion Superdex 75 Increase 10 / 300 column ( Cytiva , USA ) pre - equilibrated with 20mM HEPES pH 7 . 5 , 1 mM EDTA , 50 mM KCl , and 1 mM DTT . Peak fractions were collected , concentrated , aliquoted , and \ufb02 ash - frozen in liquid N 2 and stored at \u2212 80\u00b0C . Mouse His - SNAP - mTwf1 plasmid was ordered from Twist Bios - ciences . SNAP - mTwf1 was puri \ufb01 ed using the same protocol as above . Puri \ufb01 ed SNAP - mTwf1 was incubated with 5x excess of SNAP - surface - 549dye ( NewEnglandBiolabs , Ipswich , MA ) overnightat4\u00b0C . Freedye was removed using a PD - 10 desalting column ( Cytiva , USA ) . Labeled protein was collected , concentrated , aliquoted , and \ufb02 ash - frozen in liquid N 2 and stored at \u2212 80\u00b0C . Puri \ufb01 cation , labeling , and biotinylation of formin mDia1 Mouse his - tagged mDia1 ( FH1 - FH2 - C ) formin was expressed in E . coli ; BL21 ( DE3 ) pLysScells . CellsweregrowninTerri \ufb01 cBrothtologphaseat 37\u00b0C . Expression was induced overnight at 18\u00b0C by the addition of 1 mM IPTG . Cells were harvested by centrifugation at 11 , 200 \u00d7 g for 15min and the cell pellets were stored at \u2212 80 \u00b0C . For puri \ufb01 cation , frozen pellets were thawed and resuspended in 35 mL lysis buffer ( 50mM sodium phosphate buffer pH 8 , 20mM imidazole , 300 mM NaCl , 1 mM DTT , 1 mM PMSF , and protease inhibitors ( 0 . 5 \u03bc M each of pepstatin A , antipain , leupeptin , aprotinin , and chymostatin ) ) . Cells were lysed using a tip sonicator while being kept on ice . The cell lysate wasthencentrifugedat120 , 000\u00d7 g for45minat4 \u00b0C . Thesupernatant was then incubated with 1 mL of Ni - NTA beads ( Qiagen , USA ) while rotating for 2 h at 4 \u00b0C . The beads were then washed three times with the wash buffer ( 50mMsodium phosphate buffer pH 8 , 300mMNaCl , 20mM imidazole , and 1 mM DTT ) and were then transferred to a dis - posable column ( Bio - Rad , USA ) . Protein was eluted using the elution buffer ( 50mM phosphate buffer pH 8 , 300 mM NaCl , 250mM imida - zole , and 1mM DTT ) . Fractions containing the protein were con - centrated and loaded onto a size exclusion Superdex 200 increase 10 / 300 GL column ( Cytiva , USA ) pre - equilibrated with 20 mM HEPES pH 7 . 5 , 150 mM KCl , 10 % glycerol , and 0 . 5mM DTT . Peak fractions were collected , concentrated , aliquoted , and \ufb02 ash - frozen in liquid N 2 and stored at \u2212 80\u00b0C . SNAP - mDia1 30 was expressed and puri \ufb01 ed using the protocol above . Puri \ufb01 ed SNAP - mDia1 was incubated with 5x excess of SNAP - surface - 649 dye ( New England Biolabs , USA ) overnight at 4 \u00b0C . Free dye was removed using a Superdex 200 increase 10 / 300 GL column ( Cytiva , USA ) . Labeled protein was collected , concentrated , aliquoted , and \ufb02 ash - frozen in liquid N 2 and stored at \u2212 80\u00b0C . Biotin - SNAP - mDia1 was prepared by incubating puri \ufb01 ed SNAP - mDia1 with Benzylguanine - Biotin ( New England Biolabs , USA ) accord - ing to the manufacturer \u2019 s instructions . Free biotin was removed using size - exclusion chromatography by loading the labeled protein on a Superose6gel - \ufb01 ltrationcolumn ( GEHealthcare , Pittsburgh , PA ) eluted with 20mM HEPES pH 7 . 5 , 150 mM KCl , and 0 . 5mM DTT . Puri \ufb01 cation and labeling of capping protein Mouse his - tagged capping protein was expressed in E . coli ; BL21 ( DE3 ) pLysS cells . Capping protein subunits \u03b1 1 and \u03b2 2 were expressed from the same plasmid with a single His - tag on the alpha subunit 29 . Cells were grown in Terri \ufb01 c Broth to log phase at 37\u00b0C . Expression was induced overnight at 18 \u00b0C by the addition of 1 mM IPTG . Cells were harvested by centrifugation at 11 , 200 \u00d7 g for 15min and the cell pellets were stored at \u2212 80\u00b0C . For puri \ufb01 cation , frozenpellets werethawed and resuspended in 35mL lysis buffer ( 50 mM sodium phosphate buffer pH 8 , 20 mM imidazole , 300mM NaCl , 1 mM DTT , 1mM PMSF , and protease inhibitors ( 0 . 5 \u03bc M each of pepstatin A , antipain , leupeptin , aprotinin , and chymostatin ) ) . Cells were lysed using a tip sonicator while being kept on ice . The cell lysate was then centrifuged at 120 , 000 \u00d7 g for 45 min at 4 \u00b0C . The supernatant was incubated with 1 mL of Ni - NTA beads ( Qiagen , USA ) while rotating for 2 h at 4 \u00b0C . The beads were then washed three times with the wash buffer ( 50mM sodium phosphate buffer pH 8 , 300mM NaCl , 20mM imidazole , and 1 mM DTT ) and transferred to a disposable column ( Bio - Rad , USA ) . Proteinwaselutedusingelutionbuffer ( 50mMphosphatebufferpH8 , 300mM NaCl , 250 mM Imidazole , and 1 mM DTT ) . Fractions contain - ing the protein were concentrated and loaded onto a size exclusion Superdex 75 Increase 10 / 300 column ( Cytiva , USA ) pre - equilibrated with 20 mM Tris - HCl , 50 mM KCl , and 1 mM DTT . Peak fractions were collected , concentrated , aliquoted , and \ufb02 ash - frozen in liquid N 2 and stored at \u2212 80\u00b0C . SNAP - CP wasexpressed from a singleplasmidcontaining His - and SNAP - tagged \u03b2 1 subunit and untagged \u03b1 1 subunit 30 . It was puri \ufb01 ed using the protocol above . Puri \ufb01 ed SNAP - CP was incubated with 5x excess of SNAP - surface - 549 dye or SNAP - surface - 649 dye ( New Eng - landBiolabs , USA ) overnightat4 \u00b0C . FreedyeswereremovedusingPD - 10 desalting columns ( Cytiva , USA ) . Labeled protein was collected , concentrated , aliquoted , and \ufb02 ash - frozen in liquid N 2 and stored at \u2212 80\u00b0C . Puri \ufb01 cation of pro \ufb01 lin Human pro \ufb01 lin - 1 was expressed in E . coli strain BL21 ( pRare ) to log phase in LB broth at 37 \u00b0C and induced with 1 mM IPTG for 3 h at 37\u00b0C . Cells were then harvested by centrifugation at 15 , 000 \u00d7 g at 4 \u00b0C and stored at \u2212 80\u00b0C . For puri \ufb01 cation , pellets were thawed and resus - pended in 30mL lysis buffer ( 50mM Tris - HCl pH 8 , 1mM DTT , 1 mM PMSF protease inhibitors ( 0 . 5 \u03bc M each of pepstatin A , antipain , leu - peptin , aprotinin , and chymostatin ) ) was added , and the solution was sonicated on ice by a tip sonicator . The lysate was centrifuged for 45min at 120 , 000 \u00d7 g at 4 \u00b0C . The supernatant was then passed over 20ml of Poly - L - proline conjugated beads in a disposable column ( Bio - Rad , USA ) . The beads were \ufb01 rst washed at room temperature in wash buffer ( 10 mMTrispH8 , 150 mMNaCl , 1 mMEDTA , and1 mMDTT ) and then washed again with two column volumes of 10mM Tris pH 8 , 150 mM NaCl , 1 mM EDTA , 1 mM DTT , and 3 M urea . Protein was then eluted with \ufb01 ve column volumes of 10mM Tris pH 8 , 150 mM NaCl , 1 mM EDTA , 1 mM DTT , and 8 M urea . Pooled and concentrated frac - tions were then dialyzed in 4 L of 2 mM Tris pH 8 , 0 . 2 mM EGTA , 1 mM DTT , and 0 . 01 % NaN 3 ( dialysis buffer ) for 4 h at 4\u00b0C . The dialysis buffer was replaced with fresh 4 L buffer and the dialysis was continued overnight at 4 \u00b0C . The protein was centrifuged for 45min at 450 , 000 \u00d7 g at 4 \u00b0C , concentrated , aliquoted , \ufb02 ash - frozen in liquid N 2 , and stored at \u2212 80 \u00b0C . Conventional TIRF microscopy for single - molecule imaging Glass coverslips ( 60 \u00d7 24 mm ; Thermo Fisher Scienti \ufb01 c , USA ) were \ufb01 rst cleaned by sonication in detergent for 20min , followed by suc - cessive sonications in 1 M KOH , 1M HCl , and ethanol for 20min each . Coverslips were then washed extensively with H 2 O and dried in an N 2 stream . The cleaned coverslips were coated with 2 mg / mL methoxy - polyethylene glycol ( mPEG ) - silane MW 2000 and 2 \u00b5 g / mL biotin - PEG - Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 11 silane MW 3400 ( Laysan Bio , USA ) in 80 % ethanol ( pH 2 . 0 ) and incu - bated overnight at 70 \u00b0C . Flow cells were assembled by rinsing PEG - coated coverslips with water , drying with N 2 , and adhering to \u03bc - Slide VI0 . 1 ( 0 . 1mm \u00d7 17 mm \u00d7 1 mm ) \ufb02 ow chambers ( Ibidi , Germany ) with double - sided tape ( 2 . 5cm \u00d7 2 mm \u00d7 120 \u03bc m ) and epoxy resin for 5min ( Devcon , USA ) . Before each reaction , the \ufb02 ow cell was sequen - tially incubated for 1 min each with 4 \u03bc g / ml streptavidin and 1 % BSA in 20mM HEPES pH 7 . 5 , and 50 mM KCl . The \ufb02 ow cell was then equili - brated with TIRF buffer ( 10mM imidazole , pH 7 . 4 , 50mM KCl , 1mM MgCl 2 , 1 mM EGTA , 0 . 2 mM ATP , 10mM DTT , 2 mM DABCO , and 0 . 5 % methylcellulose [ 4000cP ] ) . For 649 - mDia1 , 549 - CP , and 488 - actin experiments ( Fig . 3a \u2013 f ) , 0 . 5 \u00b5 M G - actin ( 15 % Alexa - 488 labeled , 0 . 5 % biotin - labeled ) , 1 \u00b5 M pro - \ufb01 lin , along with 50pM 649 - mDia1 in TIRF buffer were introduced into the \ufb02 ow cell and \ufb01 laments were allowed to grow for 2 to 3 min . The \ufb02 ow cell was then rinsed with TIRF buffer to remove free formins , and thesolution was replacedwithpro \ufb01 lin - actinand 10nM549 - CP ( with or without mTwf1 ) . In experiments with \ufb02 uorescently labeled twin \ufb01 lin ( Fig . 3g , h ) , 100pM mDia1 was introduced to the \ufb02 ow cell , followed by \ufb02 ows containing 10nM unlabeled CP along with 40nM 549 - mTw1 . Time - lapse images were acquired every 4 s . For three - color uncapping experiments with 549 - mTwf1 , 649 - CapZ , and 488 - actin ( Fig . 4a , b ) , actin \ufb01 laments were elongated from 1 \u00b5 MG - actin ( 15 % Alexa - 488labeledand1 . 4 % biotinylatedG - actin ) . Free actin monomers were removed by rinsing the \ufb02 ow cell with an excess of TIRF buffer and the \ufb01 laments wereexposed to a solution containing 10nM 649 - CP . Following capping , the chamber was once again rinsed with TIRF buffer to remove free 649 - CP and then 20 nM 549 - mTwf1 was introduced . Time - lapse images were acquired every 1s . For two - color experiments involving 549 - mTwf1 , unlabeled CapZ , and 488 - actin ( Fig . 4c \u2013 e ) , actin \ufb01 laments were elongated from 1 \u00b5 M G - actin ( 15 % Alexa - 488 labeled and 1 . 4 % biotinylated G - actin ) and 0 . 5 \u00b5 M pro \ufb01 lin . Free pro \ufb01 lin and actin monomers were removed by rinsing the \ufb02 ow cell with an excess of TIRF buffer and the \ufb01 laments were exposed to a solution containing 10 nM unlabeled CP and 1 \u00b5 M G - actin ( 15 % Alexa - 488 labeled and 1 . 4 % biotinylated G - actin ) with 0 . 5 \u00b5 M pro \ufb01 lin . Following capping , the chamber was once again rinsed withTIRF buffertoremovefreeunlabeledCP , actin , pro \ufb01 ling , andthen 15nM 549 - mTwf1 was introduced . Time - lapse images were acquired either every 200 ms or every 300ms . Micro \ufb02 uidics - assisted TIRF ( mf - TIRF ) imaging and analysis Actin \ufb01 laments were \ufb01 rstassembled inmicro \ufb02 uidics - assisted TIRF ( mf - TIRF ) \ufb02 ow cells 32 , 33 . Coverslips were \ufb01 rst cleaned by sonication in Micro90 detergent for 20min , followed by successive 20min sonica - tions in 1 M KOH , 1M HCl , and 200 - proof ethanol for 20min each . Washed coverslips were then stored in fresh 200 - proof ethanol . Cov - erslips were then washed extensively with H 2 O and dried in an N 2 stream . These dried coverslips were coated with 2 mg / mL methoxy - poly ( ethyleneglycol ) ( mPEG ) - silaneMW2000and2 \u00b5 g / mLbiotin - PEG - silane MW 3400 ( Laysan Bio , USA ) in 80 % ethanol ( pH 2 . 0 ) and incu - bated overnight at 70\u00b0C . A 40 \u00b5 m high PDMS mold with three inlets and one outlet was mechanically clamped onto a PEG - Silane - coated coverslip . The chamber was then connected to a Maes \ufb02 o micro \ufb02 uidic \ufb02 ow - control system ( Fluigent , France ) , rinsed with mf - TIRF buffer ( 10 mM imidazole , pH 7 . 4 , 50 mM KCl , 1 mM MgCl 2 , 1 mM EGTA , 0 . 2 mM ATP , 10mM DTT , and 1 mM DABCO ) and incubated with 1 % BSA and 10 \u00b5 g / mLstreptavidin in 20 mM HEPES pH 7 . 5 , and 50 mM KCl for 5min . About 100pM Biotin - SNAP - mDia1 molecules in TIRF buffer were then \ufb02 owed in and allowed to anchor on the glasscoverslip . Actin \ufb01 laments with barbed ends anchored to the formins were grown by \ufb02 owing in a solution containing 1 \u00b5 M G - actin ( 15 % Alexa - 488 labeled ) and 0 . 5 \u00b5 M pro \ufb01 lin in mf - TIRF buffer . All experiments were carried out atroomtemperatureinaTIRFbuffer . Eachexperimentwasrepeatedat least three times . Data from a single replicate is presented in the \ufb01 gures . Image acquisition and analysis Single - wavelength time - lapseTIRF imaging was performed on a Nikon - Ti2000 inverted microscope equipped with a 40 mW Argon laser , a 60X TIRF - objective with a numerical aperture of 1 . 49 ( Nikon Instru - ments Inc . , USA ) , and an IXON LIFE 888 EMCCD camera ( Andor Ixon , UK ) . One pixel was equivalent to 144 \u00d7 144 nm . Focus was maintained by the Perfect Focus system ( Nikon Instruments Inc . , Japan ) . Time - lapsed images were acquired every 2 or 5s using Nikon Elements imaging software ( Nikon Instruments Inc . , Japan ) . For three - color images , the sample was sequentially excited by 488 , 561 , and 640nm lasers . Imageswereacquiredeithercontinuouslyorwitha1or4sdelay between consecutive images . Images were analyzed in Fiji 62 . Background subtraction was con - ducted using the rolling ball background subtraction algorithm ( ball radius \ufb01 ve pixels ) . Time - lapse images of between 50 and 100 \ufb01 laments were acquired in a single \ufb01 eld of view for each condition and all of these \ufb01 laments were included to determine the cumulative distribu - tion functions ( CDFs ) showing the time - dependent survival fraction of various complexes . For mf - TIRF assays , the kymograph plugin was used to draw kymographs of individual \ufb01 laments . The kymographs were used toidentifythetimepoint of thedetachment of \ufb01 lamentsasa function of time . For three - color images , a 5 \u00d7 5 - pixel box was drawn at the location of the barbed end of the \ufb01 lament and the time - dependent integrated intensity values were recorded for the single - molecule channels . The integrated intensity values were background corrected by subtracting the integrated intensity of a 5 \u00d7 5 - pixel box drawn away from the \ufb01 lament . Data analysisand curve \ufb01 tting werecarried out in MicrocalOrigin . Determination of rates of CP or formin dissociation from BFC complexes ( k \u2019 - F and k \u2019 - C ) The time - dependent fraction of BFC complexes dissociating by either transitioning to BF ( re - elongation ) or BC ( detachment ) upon addition of twin \ufb01 lin to the \ufb02 ow were plotted versus time ( black symbols in Fig . 2e ) . The kinetics of dissociation of BFC complex and simultaneous appearance of BF ( magenta symbols ) and BC ( yellow symbols ) were analyzed using the approach described in our earlier study 29 . Brie \ufb02 y , BFC dissociation can occur via one of the following two routes : BFC (cid:1) ! k 0(cid:1) C BF + C \u00f0 1 \u00de BFC (cid:1) ! k 0 (cid:1) F BC + F \u00f0 2 \u00de These reactions can be described by the following differential equations : d \u00f0 BFC \u00de = dt = (cid:1) BFC \u00f0 k 0(cid:1) F + k 0(cid:1) C \u00de \u00f0 3 \u00de d \u00f0 BF \u00de = dt = k 0(cid:1) C \u00f0 BFC \u00de \u00f0 4 \u00de d \u00f0 BC \u00de = dt = k 0(cid:1) F \u00f0 BFC \u00de \u00f0 5 \u00de The number of \ufb01 laments transitioning out of BFC , and into BF and BC allvaryexponentiallywithrateconstant ( k \u2019 - F + k \u2019 - C ) . Thevalueof k \u2019 - F and k \u2019 - C was derived from the relative fraction of \ufb01 laments in BF and BC states as follows . BFC \u00f0 t \u00de = BFC 0 = e (cid:1)\u00f0 k 0(cid:1) C + k 0(cid:1) F \u00de t \u00f0 6 \u00de Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 12 BF \u00f0 t \u00de = BFC 0 = k 0(cid:1) C k 0(cid:1) C + k 0(cid:1) F (cid:1) (cid:3) * \u00f0 1 (cid:1) e (cid:1)\u00f0 k 0(cid:1) C + k 0(cid:1) F \u00de t \u00de \u00f0 7 \u00de BC \u00f0 t \u00de = BFC 0 = k 0(cid:1) F k 0(cid:1) C + k 0(cid:1) F (cid:1) (cid:3) * \u00f0 1 (cid:1) e (cid:1)\u00f0 k 0(cid:1) C + k 0(cid:1) F \u00de t \u00de \u00f0 8 \u00de BFC 0 is the total number of \ufb01 laments in the BFC state just prior to \ufb02 owing in twin \ufb01 lin . The ratio of the number of \ufb01 laments taking either of the two routes ( BF or BC ) is given by N BF = N BC = k \u2019 - C / k \u2019 - F . Note that k \u2019 - F + k \u2019 - C are observed rate constants as they depend upon the con - centration of twin \ufb01 lin as well as elongation rates of \ufb01 laments asso - ciated with a formin . Statistical analysis and error bars for dissociation rates The uncertainty in dissociation rates of formin bound to barbed ends ( BF \u2192 B + F ) or BFC complexes ( BFC \u2192 BF + C or BFC \u2192 BC + F ) were determined by bootstrapping strategy 34 . The dissociation rate was determined by \ufb01 tting the survival fraction ( or CDF ) data to a single - exponential function ( y = e - k t or y = 1 \u2013 e - k t ) . A custom - written MATLAB code was then used to simulate BF ( or BFC ) complex lifetimes for N \ufb01 laments ( where N is the number of \ufb01 laments in the particular experiment ) based on the rate k determined from the experimental data . The simulation was repeated 1000 times to generate 1000 indi - vidual survival fractions of N \ufb01 laments . Each dataset was then \ufb01 t to an exponential function and an observed rate constant k obs was deter - mined foreachof the 1000 simulated datasets . Thestandard deviation oftheseestimatedratesallowedustodeterminetheuncertaintyinour measured rates . Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article . Data availability Data supporting the \ufb01 ndings of this manuscript are available from the corresponding author upon request . Source data are provided with this paper Source data are provided with this paper . Code availability Code used in this manuscript is available from the corresponding author upon request . References 1 . Lappalainen , P . , Kotila , T . , Jegou , A . & Romet - Lemonne , G . Bio - chemical and mechanical regulation of actin dynamics . Nat . Rev . Mol . Cell Biol . 23 , 836 \u2013 852 ( 2022 ) . 2 . Carlier , M . F . & Shekhar , S . 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S . thanks Marie - France Carlier for introducing him to actin and Jeff Gelles for teaching him single - molecule imaging . S . S . also thanks his graduate advi - sors Vinod Subramaniam and Hans Kanger for their mentorship . We thank Jim bear for critical reading of the manuscript and feedback . We thank Pekka Lappalainen for generously sharing twin \ufb01 lin plas - mids and Jan Faix for sharing VASP chimera plasmids . We thank Ankita and Sandeep Choubey for their help with statistical analysis . We thank Sandeep Choubey , Ekram Towsif , and Surbhi Garg for their comments on the manuscript . We thank Matt Winfree , Thomas Pitta and Lauryn Luderman for their tireless support towards our microscopy efforts . This work was supported by NIH NIGMS grant R35GM143050 to S . S . and startup funds from Emory University to S . S . Author contributions H . U . and I . G . conducted experiments and analyzed data . H . U . and S . S . prepared \ufb01 gures . S . S . designedexperimentsandsupervisedtheproject . S . S . and H . U . wrote the \ufb01 rst draft of the manuscript and all authors contributed to the editing . S . S . acquired funding . Competing interests The authors declare no competing interests . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 14 Additional information Supplementary information The online version contains supplementary material available at https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 . Correspondence and requests for materials should be addressed to Shashank Shekhar . Peer review information Nature Communications thanks Thomas Pol - lard , and the other , anonymous , reviewer ( s ) for their contribution to the peer review of this work . A peer review \ufb01 le is available . Reprints and permissions information is available at http : / / www . nature . com / reprints Publisher \u2019 s note Springer Nature remains neutral with regard to jur - isdictional claims in published maps and institutional af \ufb01 liations . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Creative Commons license , and indicate if changes were made . The images or other third party material in this article are included in the article \u2019 s Creative Commons license , unless indicated otherwise in a credit line to the material . If material is not included in the article \u2019 s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2023 Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 39655 - 3 Nature Communications | ( 2023 ) 14 : 3981 15", + "yuEncouragingOutsideBox2016": "Encouraging \u201cOutside-\u00adthe-\u00adbox\u201d Thinking in Crowd Innovation Through Identifying Domains of Expertise Lixiu Yu , Aniket Kittur , Robert E . Kraut Carnegie Mellon University 5000 Forbes Avenue , Pittsburgh , PA 15213 { lixiuyu , nkittur , robert . kraut } @ cs . cmu . edu ABSTRACT People are more creative at solving difficult design problems when they use relevant examples from outside of the problem\u2019s domain as inspirations . However , finding such \u201coutside - the - box\u201d inspirations is difficult , particularly in large idea repositories such as the web , because without guidance people select domains to search based on surface similarity to the problem\u2019s domain . In this paper , we demonstrate an approach in which non - experts identify domains that have the potential to yield useful and non - obvious inspirations for solutions . We report an empirical study demonstrating how crowds can generate domains of expertise and that showing people an abstract representation rather than the original problem helps them identify more distant domains . Crowd workers drawing inspirations from the distant domains produced more creative solutions to the original problem than did those who sought inspiration on their own , or drew inspiration from domains closer to or not sharing structural correspondence with the original problem . Author Keywords Crowdsourcing ; problem solving ; design ; idea generation ACM Classification Keywords H . 5 . 3 Group and Organization Interfaces INTRODUCTION Important innovations and discoveries often come from drawing upon knowledge from domains outside of a target problem as an inspiration to solve the problem [ 8 , 10 ] . For example , using origami - folding techniques , NASA ' s space engineers designed a space array , which can be folded compactly and then deployed while in outer space . The new design solved the 50 - year - old space problem of transporting large objects in narrow rockets [ 2 ] . The emergence of large online idea repositories has the potential to radically accelerate innovation by increasing designers\u2019 access to analogical ideas . There now exists an enormous selection of ideas that could spark creative ideas through analogy . For example , InnoCentive ( innocentive . com ) contains more than 40 , 000 business , social , policy , scientific , and technical problems and solutions in various domains , hundreds of new product ideas are submitted to Quirky ( quirky . com ) every day by a pool of over a million inventors , and OpenIDEO has collected hundreds of solutions for a variety of social problems since 2010 ( openideo . com ) . More generally , information available online including scientific literature , patents , webpages , and images and videos represent a treasure trove of potential analogies in different domains . For example , a car mechanic adapted a method for extracting a lost cork from a wine bottle seen in a YouTube video to save a baby stuck in the birth canal , described as the most important innovation in birthing since forceps [ 17 , 22 ] . However , our ability to process this deluge of information to find and use analogies is severely bottlenecked by individual cognitive limits , as the speed and capacity with which individuals can learn and explore new domains have not kept up with the rapid growth in online information from which analogies can be discovered . Even when they have the appropriate knowledge , people often become fixated on surface - level details that prevent them from retrieving useful analogs from memory or external repositories or applying them for problem solving [ 9 ] . Previous work has shown that using crowds to mine online idea repositories for such analogous inspirations can enable search at a scale beyond the individual and has identified ways of reducing fixation by having different workers see different representations of the problem [ 24 , 25 ] . However , this work has looked at relatively small repositories of ideas , such as Quirky\u2019s , where searchers were only looking through hundreds of ideas . In contrast , the web holds orders of magnitude more potentially useful analogous ideas , not only in explicit idea repositories , such as the US patent Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from Permissions @ acm . org . CSCW ' 16 , February 27 - March 02 , 2016 , San Francisco , CA , USA \u00a9 2016 ACM . ISBN 978 - 1 - 4503 - 3592 - 8 / 16 / 02 \u2026 $ 15 . 00 DOI : http : / / dx . doi . org / 10 . 1145 / 2818048 . 2820025 1214 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA database , but also in expert - generated content in nearly any domain . In this paper , we describe and evaluate a two - stage process that enables crowds to search the web for useful and novel do design ideas than alternative approaches . In the first stage , crowds identify domains of expertise remote from the initial problem but relevant enough to provide ideas to inspire useful and non - obvious solutions . They are best able to identify relevant but remote domains when the original concrete problem is represented as an abstract problem schema . In the second stage , crowds search in these domains to find inspirational examples they can adapt to solve the original problem . The key insight here is that a rich set of expert - generated ideas , solutions , and skills have already been documented on the web , and non - experts can find and appropriate these resources in the design process even if they did not possess the expertise to generate this knowledge in the first place . Identifying \u201cOutside-\u00adthe-\u00adbox\u201d Domains Although distant analogies can inspire innovation , systematically identifying them in distant domains is challenging . First , people must identify the domains where relevant inspirational sources might be found . The most useful inspirations often come from distant domains that have little surface similarity with the target problem [ 11 , 18 , 21 ] ; for example , in the NASA example , the problem comes from the aerospace field while paper folding is from origami art . Identifying such domains can be challenging for human , because they often become fixated on the surface details of the problem domain . Outside - the - box domains are also challenging for automated search algorithms to identify , since the algorithms also generally search on surface features , leading to same - domain recommendations [ 3 , 16 , 20 , 26 ] . Yu et al . [ 24 ] demonstrated a way for crowds to find distant domains more effectively by re - representing the problem . A critical step was abstracting surface details of the original problem to reduce people\u2019s fixation on surface features , such as domain - specific vocabulary and objects [ 7 , 15 ] . For example , in the previous NASA story , space and rockets are surface features while the abstract representation of the problem can be \u201chow to contain more content within a limited space\u201d . Using this abstract representation rather than the literal description of the problem frees people to identify remote areas of knowledge containing useful inspirations for the problem . Yu et al . then showed that crowd workers given the abstract representation of a problem ( i . e . , the schema ) rather than the concrete representation were able to find analogies in more distant domains that were more useful for solving the problem creatively . However , Yu et al . treated the search process as a black box , with no instructions on how to identify potentially fruitful domains to search within . This approach may have been successful on the tens to hundreds of ideas easily available on Quirky . com , but may not scale well to the billions of web pages available on the Internet , a subset of which could have a potentially relevant solution to the target problem . The difficulty of winnowing distant domains for useful analogs has posed a crucial challenge to many design - by - analogy approaches , causing \u201cthis influential technique to be limited to little more than interesting examples with accompanying direction to simply \u2018try to find analogies . \u2019\u201d [ 14 ] . Exploring \u201cOutside-\u00adthe-\u00adbox\u201d Domains A second problem is finding useful ideas within the identified domain . An expensive but powerful way to do so would be to have a panel of relevant experts on call from that domain . Indeed major design firms like IDEO are successful in part because they hire employees who are diverse in demographics , education and personal interests and can apply ideas from their areas of expertise to the problems clients bring them [ 10 ] . Although retaining experts may be necessary in research - investment intensive industries , like pharmaceuticals , for many more routine problem challenges in engineering and design , experts may have already documents relevant solutions in language accessible to laymen . Crowdsourcing approaches have been increasingly used to expand the pool that innovation companies can draw upon to solve problems , ranging from product ideation ( e . g . , Quirky . com ) to research and development ( e . g . , Innocentive . com ) to societal challenges ( OpenIDEO . com ) Companies describe their problems and then use what is called \u201cbroadcast search\u201d to invite anyone with a good idea to contribute solutions [ 6 , 13 ] . Here instead of contributing original ideas , we aim to use non - expert crowd workers to mine the web for ideas in various domains that might help solve the target problem . Knowledge , methods , principles , skills and tools that experts use in their domains have often been amply documented on the Internet . For example , the origami folding techniques used in the previously mentioned NASA example can be found through a search for \u201corigami patterns\u201d . Thus , problem solvers who might not possess the desired expertise themselves might profitably search for outside - the - box knowledge online . EXPERIMENT 1 : IDENTIFYING OUTSIDE-\u00adTHE-\u00adBOX DOMAINS Our overall goal in this research is to elicit distant domains and useful inspirations from a large , open - ended idea repository such as the web . In particular , we aim to go beyond previous design - by - analogy work in providing a structured process for identifying fruitful domains for exploration , rather than simply asking individuals to find useful examples directly . We hypothesize that doing so requires generating and exploring domains that are distant from the original problem domain , yet contain structurally similar and relevant inspirations . In Experiment 1 we 1215 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA explore a process for generating distant structurally similar domains , and in Experiment 2 we test whether the found domains yield relevant and useful inspirations . In a pilot study we asked workers directly to identify domains that might be useful for solving the schematic representation of the problem . However , the resulting domains were generally too vague to be useful ( e . g . , \u201cengineering\u201d ) . To address this problem we turned to a mechanism that people use in real life when they are looking for knowledge in a different domain : they look for referrals . The intuition is that even if people might not have the desired knowledge a problem needs and are not accustomed to thinking about \u201cdomains\u201d of knowledge , they may be able to identify a type of expert in a different domain who might deal with relevant problems . We thus reframed our elicitation from crowd workers , asking them to identify professions that could have useful perspectives . For example , if the goal were to design a new power strip , one might ask about professions that deal with problems of packing things into a limited space . Such professions could be as diverse as landscapers packing plants into a small yard ; warehouse loaders packing products into crates ; user interface designers packing information into interfaces ; or even contortionists packing their bodies into small containers . Some of these domains might yield interesting solutions , such as a terraced landscaping solution inspiring the design of a power strip with different height levels to avoid plugs obstructing each other . The above example also highlights the importance of abstraction in the domain elicitation process . Providing an abstract representation of a problem can increase the diversity of the resulting domains found [ 25 ] . For example , the abstract representation of the power strip\u2019s problem for \u201cpacking things into a limited space\u201d might elicit more diverse professions than a concrete representation such as \u201cfitting different sized plugs into a power strip\u201d . In Experiment 1 we manipulated the level of abstraction of the problem representation ( i . e . , original , concrete problem or abstracted problem ) and asked people to identify professions that might have interesting perspectives on the problem . Our goal was to find distant yet structurally relevant domains that might yield useful inspirations . Participants One hundred and twenty - two Amazon Mechanical Turk workers [ 12 ] participated in this experiment . Forty percent were women , and 93 % were native English speakers . Their average age was 33 and ranged from 19 to 68 . Design and Procedure We selected two design challenges from a crowd innovation website ( Quirky . com ) to use in the experiment ( see Table 1 ) . For each challenge we generated an abstract schematic representation by first abstracting the goal and the sub - goals , and replacing concrete objects with generic objects sharing essential attributes . For example , the original power strip problem listed on the website talks about plugs and outlets , while the schematic version talks about objects fitting into a container or blocking each other . While in this research the experimenters created the schematic problem representation for convenience and standardizing the input to the experiment , previous research shows schematization of a concrete problem can be done by lay crowd workers following instructions [ 25 ] . After accepting the task , participants were randomly assigned to either the original or schematic representation of the power strip or cup problem and asked to recommend types of experts for a design problem . Specifically , they were asked , \u201c Please read the design problem below and suggest three types of experts who might provide useful or interesting perspectives in solving it and explain why \u201d . Rating the Recommended Experts We hypothesized that the schematic representation would return a more diverse set of experts . Table 2 shows examples of the recommended experts for the two problems in the two conditions . Some professions appeared in both problems ( e . g . , carpenter , construction worker ) but for different reasons . For example , a rationale for a carpenter in the cup problem was that they could \u201c design a rack that separates the cups just enough so they can dry properly , but not take up too much space \u201d while for the power strip problem they \u201c Could design hidden compartments to run cords to other outlets that would also keep the cords hidden . \u201d Original description Schematic representation ( Power strip problem ) Have a look at the power strip under your desk . How many of its outlets are being used ? How many of them would you like to use , but you can ' t , because a giant power brick ( transformer ) in the adjacent outlet is blocking it ? How could you fit all the different plugs in all the outlets ? 1 . How can you fit objects of different sizes into a container ? ( goal ) 2 . Prevent objects blocking each other ( sub - goal ) 3 . Fully make use of a container\u2019s capacity ( sub - goal ) ( Cup problem ) When we finish washing cups and glasses , we have to either spread them out individually , but then they take up all the counter space . Alternatively , we can stack them , but then the cups never dry completely and it is hard to separate them from each other later . How can you dry many cups quickly so that they don\u2019t take up too much space and moisture doesn\u2019t get trapped between them ? 1 . How can you dry multiple stackable objects ? ( goal ) 2 . Prevent multiple similar objects from taking up much space ( sub - goal ) 3 . Separate multiple stackable objects easily ( sub - goal ) Table 1 . Original and schematic representations for the two design problems ( power strip , cup ) used in the experiments . 1216 SESSION : CROWD INNOVATION AND CROWDFUNDING In the original concrete formulation of the cup problem , most recommendations appeared to invoke people who would have direct knowledge about kitchens and cups \u2013 e . g . , housewives , chefs , bartenders , cup designers , and interior designers . Similarly , many participants who saw the original description of the power strip problem recommended electricians and electronic engineers . In contrast , the schematic representations of the cup problem appeared to return a wider variety of experts , including bakers , forklift operators , and sculptors . Participants who saw the power strip problem\u2019s schematic representation recommended a variety of experts who work with problems related to sizes , shapes , and blocking . For example , in the schematic representation of the power strip problem , a participant recommended a topology expert and explained , \u201c This person would know about the mathematical study of shapes and shapes in space which would help with deciding about objects of different sizes . \u201d To analyze whether the conditions differed in generating domains two judges rated each recommendation on two dimensions : whether the expert was unique among all recommendations for the same problem and how distant the expert\u2019s domain was from the original problem domain . To count unique experts , we combined similar recommendations into single one . For example , \u201cmover\u201d and \u201ca moving expert - moving peoples\u2019 possessions from one home to another\u201d were both classified as \u201cmover\u201d . To judge distance , two judges blind to experimental condition rated each recommended expert on a 7 - point Likert scale : \u201c How different is the above power strip problem from the problems that the recommended expert works with in his or her domain ? \u201d By this metric , for example , a \u201cmail sorter\u201d was considered further from the cup problem than was a dishwasher . The judges achieved a good inter - rater reliability of 0 . 78 , calculated using the Intraclass Correlation Coefficient ( ICC ) [ 5 ] . The final distance score was calculated by averaging the scores of the two judges . Analysis and Results Table 3 shows the means and standard errors of the percentages of unique experts and the distance scores . A proportion test showed participants identified a larger proportion of unique experts when shown schematic descriptions of the problem than when shown the more concrete , original problem descriptions ( z = 3 . 21 , p < 0 . 001 ) . Participants shown the schematic problem descriptions also recommended domains more distant from problem domain than did those shown the original problem description ( z = - 5 . 89 , p < 0 . 001 , by a Mann - Whitney U test used because of the skewed distribution ) . EXPERIMENT 2 : GENERATING IDEAS Although Experiment 1 suggests that that converting the original problem description into a schematic representation and eliciting domains in the form of professions led people to identify more distant structurally related domains . However , it fails to answer the key question of whether these distant domains yield more useful inspirations that lead to more creative solutions that the ones found in domains closer to the original problem domain . While more distant domains have the potential to yield inspirations that break fixation with the original problem and suggest interesting approaches from other domains , it is also possible that these domains might not be sufficiently relevant to the target problem to yield useful inspirations . For example , in Table 2 the schematic representation resulted in professions that at first glance might not seem relevant , such as \u201cmagician\u201d for the power strip problem or \u201cparking lot attendant\u201d for the cups problem . However , on closer inspection workers provided rationales that make these potentially plausible sources , such as \u201c Can make objects appear to do many things you might think illogical \u201d for magician , perhaps suggesting that some trick mechanics might help fit things in places you wouldn\u2019t expect ; or Cup problem Power strip problem Original Schematic Original Schematic Chef , Housewife , Bartender , Carpenter , Counter - designer , Cup designer , Dishwasher , Gardener , Glass expert , Home - builder , Maid , HVAC - technician , Interior - designer , Professional - organizer , Rack - specialist , Waiter Baker , Bodybuilder , Carpenter , Cashier , Construction \u2013 worker , Designer , Fireman , Forklift - operator , Landscaper , Mail sorter , Mathematician specializing in geometry , Meteorologist , Parking - lot - attendant , Pianist , Potter , Sculptor Electrician , Building - contractor , Computer - technician , Housewife , Network - engineer , Interior - designer , Artificial - intelligence - scientist , Building - maintainer , Cable installer , Carpenter , Construction - worker , Electrical - engineer Contortionist , Geometry - expert , Graphic - designer , Landscaper , Magician , Physicist , Sculptor , User - interface - designer , Architect , Warehouse - dock - loader , Artist , Expert - of - arithmetic , Expert - on - Japanese - aesthetics , Expert - on - topology Table 2 . Examples of the recommended expert domains from Experiment 1 in the Original and Schematic problem representation conditions . Condition Freq . Percent of unique experts Distance Mean S . E . Mean S . E Original 204 0 . 41 0 . 03 3 . 87 0 . 23 Schematic 162 0 . 60 0 . 04 5 . 74 0 . 16 Note on frequency : There were 68 participants in Original representation and 54 participants in Schematic representation . Each participant provided three recommendations . Table 3 . Experiment 1 : Means and standard errors . 1217 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA \u201c \u2026needs to be wise about how they position cars so that they can get in and out whenever needed\u2026would know the best way to position objects \u201d for the parking lot attendant . In this experiment we explore whether distant domains would yield useful inspirations by having participants generate new ideas using the inspirations found in the previous experiment . We are interested in whether participants can find more useful and interesting inspirational sources from an outside - the - box domain and solve the problem better than when searching on their own or when searching in a domain that shared surface similarities to the problem domain . Our hypothesis is that the benefits of far domains occur only when they are likely to contain ideas that have deep structural correspondences to the original problem . Thus , one purpose of this experiment is to insure that distance from the target domain by itself is not what leads to better problem solutions . The experiment involved four conditions . In the Problem - driven condition , participants searched for inspiration in a domain recommended from the original problem description . In the Schema - driven condition , they searched for inspiration in a domain recommended from the schematic problem representation . The Irrelevant schema - driven control condition tested whether far domains that are not structurally similar to the original problem have the same benefits as far domains that were structurally similar . Specifically , participants searched for inspirations for the cup problem in a domain recommended from the schematic problem representation of the power strip problem and vice versa . Finally , we also included a control condition where participants were asked to search for inspiration on their own , called the Self - selected condition . In all these conditions , participants first searched for inspirations and then tried to solve the original problem using the inspirations they found . Participants Overall 130 Amazon Mechanical Turk workers participated in Experiment 2 . Forty - six percent were women , and 96 % were native English speakers . Their average age was 34 and ranged from 18 to 66 . Participants from Experiment 1 were excluded from participating in Experiment 2 . Design and Procedure After participants accepted the task , they were randomly assigned to one of the four experimental conditions . They were asked to search for two inspirational examples for either the Cup or Power Strip problem and then generate a new product idea that solved the problem . They were either given no hint about where to search for inspiration ( Self - selected ) or were told to look for their inspirations from domains recommended in Experiment 1 from participants who saw the concrete version of an original problem ( Problem - driven ) , the schematized version of the original problem ( Schema - driven ) or a schematized version of an irrelevant problem ( Irrelevant schema - driven ) . Instructions for the conditions are described in more detail below : 1 . Self - selected . Participants saw either the Cup or Power strip problem as described on the left column in Table 1 and then asked to search for useful ideas on the web that would help them design a solution to the problem . Specifically , they were told , \u201c Please go to the Internet and find two useful ideas that could inspire good solutions for the above problem . These ideas could be knowledge , skills or methods other people use to solve a similar problem in their own domain . Please don ' t search for ideas related to this cup [ power strip ] problem . The useful ideas have to be about similar problems in a non - cup related domain . \u201d After they found two useful ideas , they were asked to solve the problem . Specifically they were told , \u201c Please generate a new product idea that solves the above design problem using the ideas you found . \u201d 2 . Problem - driven . After seeing one of the two design problems , participants were asked to search for useful ideas that an expert in the problem domain might have . For example , if they were assigned the housewife domain , their prompt would be , \u201c Please go to the Internet and find two useful ideas a housewife might have that could inspire good Inspiration Expert domains and solutions An architect : Cup with studs on sides , so that they can be stacked while allowing airflow to dry easily . A carpenter : Maybe instead of a linear power strip it could be built as an arc or circle that allows more plugs to fit in basically the same amount of space . By turning a line into a circle it opens up more room around each plug or AC adaptor . An expert on Japanese aesthetics : If you made an outlet that looks like a Miyabi style building . You could go up levels and it would leave more room for the plugs to go around the outlet . Table 5 . Examples of inspirations and resulting solutions from participants . Independent variable % creative Odds Ratio se Z Self - selected 0 . 26 5 . 44 * * 3 . 10 2 . 98 Problem - driven 0 . 12 13 . 53 * * * 8 . 91 3 . 95 Schema - driven 0 . 65 NA NA NA Irrelevant schema 0 . 25 5 . 45 * * 3 . 08 3 . 01 p < . 01 = * * p < . 001 = * * * Table 4 . The effects of inspiration condition on design quality . 1218 SESSION : CROWD INNOVATION AND CROWDFUNDING solutions for the above problem . These ideas could be knowledge , skills , or methods a housewife uses to solve a similar problem in his or her own domain . Please don ' t search for ideas related to this cup problem . The useful ideas have to be about problems in the domain that a housewife deals with . \u201d An expert was randomly selected an expert without replacement from the domains recommended in the Original representation condition in Experiment 1 , with a probability proportional to the number of times the expert was recommended in Experiment 1 . After finding two useful ideas , participants were asked to solve the problem using those ideas as inspiration . 3 . Schema - driven . This condition is identical to the Problem - driven condition except that participants were asked to find inspirations from domains identified in the Schematic representation condition in Experiment 1 . After finding two inspirations , they then solved the problem . 4 . Irrelevant schema - driven . This condition was identical to the Schema - driven condition except that participants were asked to find inspirations from the domains identified in the Schematic representation condition in Experiment 1 for an irrelevant problem . That is , if participants were to solve the cups problem , they were shown domains recommended for the power strip problem and vice versa . After finding two inspirations , they then solved the problem . There were roughly 90 recommendations identified in each condition in Experiment 1 . When selecting domains to give to participants , we coalesced domains to give related ones a common name and excluded generic recommendations such as \u201cscientist\u201d and \u201cengineer\u201d . Quality of the Design Solutions Judging the creative quality of an idea can be difficult problem involving significant subjectivity . To judge the ideas we draw on previous research establishing methods for robustly rating creative idea quality , which considers an idea as being creative if it is both novel and practical [ 4 ] . Novelty was defined as an idea that was not obvious and differed from existing products on the market . Practicality was defined as how realistically an idea achieved its goal and could be designed and manufactured . Two judges blind to experimental condition iterated on a rubric for rating product ideas , then used this rubric to independently rate each idea on two 7 - point Likert scales measuring novelty and practicality . The judges achieved good ICC inter - rater reliabilities of 0 . 79 and 0 . 61 for novelty and practicality respectively . The final creativity score of each idea was computed as the mean of its novelty and practicality scores . Some limitations of this approach are discussed in the discussion section . Below is an example of a solution to the power strip problem rated highly in terms of both novelty ( 6 . 0 ) and practicality ( 6 . 0 ) . Make a power strip that has some outlets raised about the others . That way a large plug can fit into the raised outlet and still leave room for another plug in the outlet that isn ' t raised . In contrast , the design idea below was rated poorly on both dimensions of novelty ( 1 . 0 ) and practicality ( 2 . 5 ) . I would propose a specially made power strip that is longer , and wider than any power strip on the market . Would that has enough space between each socket to fit any plug , or power brick comfortably . Of course it would be bigger , but that is a fair trade off . Following recommendations from [ 4 ] , we then classified each design as creative if it was above a media - split threshold of 3 . 0 for both novelty and practicality or non - creative otherwise . Analysis and Results To determine whether the source of the domains participants used as inspiration influenced the quality of their designs , we conducted a logistic regression model predicting with whether an idea was creative ( 1 ) or not ( 0 ) from the experimental conditions , along with level of education and whether the participants\u2019 language was English as controls . Table 4 summarizes the results , with Schema - driven domain as the reference group . Results indicate that searching for inspiration in domains far from the original problem significantly increased the probability of generating a creative solution compared to searching in Problem - driven domains , in Irrelevant schema - driven domains or when given no direction about which domains to search . The odds of producing a creative idea when given recommendations to search in a Schema - driven domain were 13 . 53 times the odds when given a Problem - driven domain , 5 . 45 times the odds when given an Irrelevant schema - driven domain and 5 . 44 times of the odds when participants searched for inspiration with guidance about domain ( Self - selected ) . To further examine whether the domain of search is responsible for the difference in the quality of ideas , we added the domain distance score ( judged in Experiment 1 ) as a mediator and re - run the logistic regression analysis . The results show that domain distance is significantly correlated with the odds of producing a creative idea : for every one - unit increase in the distance score , the odds of producing a creative idea increases by 2 . 07 times ( p < 0 . 01 ) . After adding domain distance as a mediator , the quality difference between the problem - driven condition and the schema - driven condition became non - signi \ufb01 cant ( b = - 0 . 96 , p = . 23 ) . However , the difference of idea quality still exists between the schema - driven condition and the irrelevant schema - driven condition : even holding domain distance constant , the odds of producing a creative idea when given recommendations to search in a schema - driven 1219 CSCW ' 16 , FEBRUARY 27 \u2013 MARCH2 , 2016 , SAN FRANCISCO , CA , USA domain were 3 . 22 times of the odds when given an irrelevant schema - driven domain ( p < 0 . 05 ) . The mediation analysis suggests that the distance of inspiration from the target domain created by the abstraction of the problem schemas completely explains the higher idea quality in the schema - driven condition compared to the problem - driven condition . That is , compared to the problem - driven condition , people in the scheme - driven condition used examples from distant domains as inspiration , which in terms led them to generate creative ideas . However , the distance of inspiration from the target domain only partially explains why solutions inspired by schema - driven examples were more creative than ones inspired by irrelevant schema - driven ones . This failure of mediation shows that the benefits of far domains occur only when they are likely to contain ideas that have deep structural similarities to the original problem . Search Mechanisms In Experiment 2 , we found that people were able to find relevant inspirational examples for a problem in remote domains and were more likely to generate creative ideas by using the inspirations that was found from remote domains . These results , however , beg the question of how non - expert crowd workers were able to find useful ideas in unfamiliar domains . People found and used inspiration in the schema - driven domains using a variety of mechanisms . To illustrate , Table 5 shows the inspirations people found by searching in the domain of an architect , a carpenter , and an expert on Japanese aesthetics . Searching in an architect\u2019s domain , a participant found and adopted the energy - saving ventilation design of a building for the cup problem ; searching in a carpenter\u2019s domain , a participant was inspired by the circle - shape of a drawer ; and searching in the domain of Japanese aesthetics , a participant borrowed a design of the Japanese Miyabi . Other participants also utilized the mechanisms and principles of other domains . For example , a participant designed a power strip that has some outlets raised about the others by utilizing how warehouse dock loading systems work . Another participant designed a bendable power strip by searching in a contortionist\u2019s domain . These forms of inspiration are much different from those found through domains recommended based on the original problem description or when participants searched for inspirations on their own . In these conditions , people found ideas related to organizing clutter , drying dishware , or rack design . When searching based on irrelevant domains , people often found information that appeared random and their solutions were not well connected to the inspirations they found . The findings support the assumption that knowledge encoded on the web in various expert domains is useful in sparking creative solutions for a related problem in a different domain . However , the outside - the - box knowledge had to be structurally alignable and relevant to the problem as well . The distance and relevance were created through the schematic representation in Experiment 1 : the abstraction reduces the domain fixation leading to a distance between the original problem and the recommended domains , while goals and sub - goals keep the connection between them . The expert domains in Experiment 2 work as a mediation step to bridge the schematic representation and the useful ideas in the domains . DISCUSSION Previous research has shown that creative solutions to problems often emerge when experts from outside a problem domain apply their \u201cforeign\u201d skills , techniques and tools to the problem . This insight inspired our research in developing a systematic process to leverage crowd workers to harvest potentially applicable ideas from fields outside of a problem\u2019s domain . To do so , we had to get the workers to \u201cthink outside of the box\u201d and overcome the challenge of functional fixedness [ 1 ] , the cognitive bias that limits people to using objects only for their traditional purposes . We addressed this challenge by transforming the original , concrete problem statement to a more abstract representation . When combined with an instruction set framed in terms of \u201creferrals\u201d , this schematic representation of the problem allowed people to identify a more diverse set of domains to explore for potential solutions than did the original , concrete problem description . Moreover , when non - expert crowd workers searched for inspirations for solutions in domains primed by the abstract representations , they returned a rich set of relevant examples . Their examples were often very different from the original problem in terms of surface features , but shared structural features . For example , at the surface level a Miyabi style building has little in common with an electrical power strip , but its three dimensional structure and arrangement of rooms might suggest ways to fit different plugs in a power strip . Crowd workers who collected inspirations from these remote domains produced more creative problem solutions than did those who searched for inspiration without guidance about where to look or who looked for inspiration in domains primed by the original concrete problem statement or by an abstract representation of a problem that did not share structural correspondence with the target problem . We asked Amazon Mechanical Turk workers , who were not selected for engineering or design expertise , to solve relatively simple but real design challenges . The participants provided brief text descriptions of the approach they would take to a problem , such as building a power strip in two dimension ( a curve ) or three dimension ( a tower ) ; they were not required to provide details about implementation . Despite these limitations , the Cup and Power Strip problems were authentic design challenges taken from Quirky . com , the crowd innovation company 1220 SESSION : CROWD INNOVATION AND CROWDFUNDING specializing in consumer products . In the past , members of the Quirky community designed detailed solutions for these challenges , and Quirky itself manufactured and sold consumer products to solve these problems . Interestingly , some of the solutions provided by crowd workers , such as a bendable power strip inspired by the contortionist domain , paralleled those from the Quirky community itself , such as the bendable PivotPower ( one of Quirky\u2019s most popular products ) . Similarity , the architecture - inspired method for attaching studs to cups so that air could circulate between stacked cups to dry them is highly reminiscent of Totem Air Dry ( another Quirky product ) . Workers did not indicate being influenced by Quirky products and none returned a Quirky product as an inspiration . When they submitted design ideas that paralleled Quirky products , their designs had clear links to examples retrieved from their assigned source domains , although we cannot guarantee that participants had never seen the relevant Quirky products . However , assuming no direct influence , parallels between crowd workers\u2019 solutions and those manufactured by Quirky suggest that the design ideas participants came up with in this controlled experiment could be commercially valuable in the world . We consider our research , which challenged relatively unsophisticated workers to solve relatively simple design problems , only a first step . One area for profitable future research is investigating whether this process will be effective in crowd innovation sites like InnoCentive , TopCoder , or MathOverflow , which typically challenge sophisticated experts to solve complicated R & D problems . We believe the process of searching for inspirations outside of a problem\u2019s domain should be useful to overcome functional fixedness for sophisticated problems as well as simple ones and may actually be more useful for problems that require deep expertise . Abstracting away some of the non - essential features of the problem should help in identifying new domains in which to search for solutions . However , when the problem is complex or when the solution must be fully elaborated or reduced to practice , one may need to recruit true experts from the distant knowledge domains and not just novices reading what experts had previously written . Another limitation in the current research is that one of the two judges in Experiment 2 was the first author . While both were blind to experimental condition and used rubrics to reduce bias and to standardize their ratings , future experiments could benefit from separate judges to reduce any biases resulting from trying to infer which experimental condition each idea came . Future research could also examine the robustness of using non - designers as judges in this context , though prior work shows high agreement between designers and non - designers in judging consumer products [ e . g . , 23 ] . The approach of using schemas as described here could also benefit from further research into the boundary conditions for where schemas are useful . For example , it is not yet clear whether schemas would be as useful if the design process continued past ideation into the prototyping stage . Prototyping may force people to think more deeply about how to adapt specific mechanisms of inspirational examples to a solution , which may result in poor fits . For example , people may run into configurational or material use problem with designing a Japanese Miyabi - style power strip , which could limit the practicality of manufacturing it . Representing Problems in Crowd Innovation A core contribution of this work is that we use a problem\u2019s abstract representation as a cue to identify domains where a solution might be found , instead of directly searching for analogical ideas [ 25 ] . This opens up the search space from well - defined idea repositories ( as in [ 25 ] ) to the entire web . Starting a search for solutions in experts\u2019 domains can provide more concrete guidance about the type of knowledge needed to solve a problem than more abstract schematic representations of the problem , which can be ambiguous and difficult to interpret . Presenting different domains to different problem solvers opens up the search space and helps solves find more varied inspirations even from the same schematic representation . We argue that problem representation is an important research topic with the rise of crowd innovation . This new problem - solving model opens a rich research area on how to formulate problems so that a distributed group of people can solve them creatively . The schematic representation proposed in this paper is one approach constructing an alternative representation for a problem in order to encourage outside - the - box search for solutions . However , how to identify the most productive level of abstraction for a particular design challenge is still an open question . An interesting area for future study is whether more complex problems on sites such as InnoCentive could be re - represented and classified based on abstract goals and sub - problems , and thereby attract more actual outside - the - box experts to solve them . ACKOWLEDGEMENTS This work was supported by NSF grants IIS - 1526665 , IIS - 1149797 , IIS - 1217559 , OCI - 0943148 , IIS - 0968484 , IIS - 1111124 , Bosch , Google , and Microsoft , Heinz College Center for the Future of Work , and Carnegie Mellon\u2019s Center for the Future of Work . We thank reviewers for useful feedback . REFERENCES 1 . Robert E . Adamson . 1952 . Functional fixedness as related to problem solving : A repetition of three experiments . Journal of experimental psychology 44 , 4 : 288 . 2 . Meg Monk . 2013 . 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Proceedings of the 2005 international ACM SIGGROUP conference on supporting group work , 71 - 80 1222 SESSION : CROWD INNOVATION AND CROWDFUNDING", + "gickAnalogicalProblemSolving1980": "COGNITIVE PSYCHOLOGY 12 , 306 - 355 ( 1980 ) Analogical Problem Solving MARY L . GICK AND KEITH J . HOLYOAK University of Michigan The use of an analogy from a semantically distant domain to guide the problem - solving process was investigated . The representation of analogy in memory and processes involved in the use of analogies were discussed theoretically and explored in five experiments . In Experiment I oral protocols were used to exam - ine the processes involved in solving a problem by analogy . In all experiments subjects who first read a story about a military problem and its solution tended to generate analogous solutions to a medical problem ( Duncker\u2019s \u201cradiation prob - lem\u201d ) , provided they were given a hint to use the story to help solve the problem . Transfer frequency was reduced when the problem presented in the military story was substantially disanalogous to the radiation problem , even though the solution illustrated in the story corresponded to an effective radiation solution ( Experiment II ) . Subjects in Experiment III tended to generate analogous solutions to the radiation problem after providing their own solutions to the military problem . Subjects were able to retrieve the story from memory and use it to generate an analogous solution , even when the critical story had been memorized in the con - text of two distractor stories ( Experiment IV ) . However , when no hint to consider the story was given , frequency of analogous solutions decreased markedly . This decrease in transfer occurred when the story analogy was presented in a recall task along with distractor stories ( Experiment IV ) , when it was presented alone , and when it was presented in between two attempts to solve the problem ( Experi - ment V ) . Component processes and strategic variations in analogical problem solving were discussed . Issues related to noticing analogies and accessing them in memory were also examined , as was the relationship of analogical reasoning to other cognitive tasks . INTRODUCTION Where do new ideas come from ? What psychological mechanisms underlie creative insight ? This fundamental issue in the study of thought has received a great deal of informal discussion , but little empirical psychological investigation . The anecdotal reports of creative scientists and mathematicians suggest that the development of a new theory fre - quently depends on noticing and applying an analogy drawn from a differ - ent domain of knowledge ( Hadamard , 1954 ) . The hydraulic model of the blood circulation system , the planetary model of atomic structure , and the This research was supported by NSF Grant BNS77 - 01211 and NIMH Grant l - ROl - MH33278 - 01 to K . Holyoak . We thank Ellen Junn and Roger Kohlman for their assistance in testing subjects and scoring data , and David McArthur for helpful discussions . Robert Sternberg and an anonymous referee provided careful reviews of an earlier version of this paper . Experiments I and II were reported at the meeting of the Midwestern Psychological Association , May 1979 . Reprint requests should be sent to K . Holyoak at the University of Michigan , Human Performance Center , 330 Packard Road , Ann Arbor , MI 48104 . 306 OOlO - 0285 / 80 / 030306 - 50 $ 05 . 00 / O Copyright @ 1980 by Academic Press , Inc . AU rights of reproduction in any form reserved . ANALOGICAL PROBLEM SOLVING 307 \u201cbilliard ball\u201d model of gases all represent major scientific theories founded on analogies ( Boden , 1977 , Chap . 11 ) . As these examples suggest , fruitful analogies may be based on a map - ping of relations between two very disparate domains . In the brainstorm - ing technique of \u201cSynectics , \u201d problem - solving groups are trained to ac - tively search for analogies in areas other than that of the target problem ( Gordon , 1961 ) . It seems clear , however , that the \u201csemantic distance\u201d between analogous domains can vary a great deal . For example , Polya ( 1957 ) suggests that a useful strategy for solving geometry problems is to search for analogous problems within the domain of geometry . Analogies drawn within a domain may also play a role in categorization tasks . Clas - sification models based on comparisons of instances , which have been developed both in psychology ( Brooks , 1978 ; Medin & Schaffer , 1978 ) and in artificial intelligence ( Winston , 1975 ) , involve comparisons be - tween objects within a single domain ( e . g . , geometric patterns ) . Collins and his associates ( Collins , Warnock , Aiello , & Miller , 1975 ) have used protocol analyses to investigate the role of analogical reasoning by stu - dents who have incomplete knowledge of a problem domain such as geog - raphy . For example , a student might evaluate whether the region around Santiago , Chile is likely to produce wine by comparing it to a known wine - producing area , such as Northern California , on the relevant geo - graphic dimensions ( e . g . , latitude , proximity to ocean , type of terrain ) . While the process of solving analogy test items of the form A : B : : C : D has been studied quite extensively ( Sternberg , 1977a , 1977b ) , there has been little experimental investigation of analogical thinking in more com - plex problem - solving tasks . Some studies have examined transfer be - tween homomorphic or isomorphic versions of puzzle problems , such as the \u201cmissionaries and cannibals\u201d ( Reed , Ernst , & Banerji , 1974 ) and Tower of Hanoi ( Hayes & Simon , 1977 ) puzzles . These are relatively \u201cwell - defined\u201d problems ( Reitman , 1964 ; Simon , 1973 ) , in which the ini - tial conditions , legal operations , and goal state are explicitly specified . In contrast , anecdotal reports of the use of analogies typically involve prob - lems that are much less well defined . The present study was designed to investigate the use of analogies between disparate domains as a guide to finding solutions for an ill - defined problem . THE RADIATION PROBLEM AND ITS ANALOGIES Our basic experimental procedure was to provide subjects with a story analogy , describing a problem and its solution , and then to observe how subjects used the analogy in solving a subsequent target problem . The target problem was Duncker\u2019s ( 1945 ) \u201cradiation problem , \u201d which in our experiments was stated as follows . Suppose you are a doctor faced with a patient who has a malignant 308 GICK AND HOLYOAK tumor in his stomach . It is impossible to operate on the patient , but unless the tumor is destroyed the patient will die . There is a kind of ray that can be used to destroy the tumor . If the rays reach the tumor all at once at a sufficiently high intensity , the tumor will be destroyed . Unfortunately , at this intensity the healthy tissue that the rays pass through on the way to the tumor will also be destroyed . At lower intensities the rays are harm - less to healthy tissue , but they will not affect the tumor either . What type of procedure might be used to destroy the tumor with the rays , and at the same time avoid destroying the healthy tissue ? There are several reasons why the radiation problem seemed especially suitable for use in a study of analogical problem solving . First , it has all the hallmarks of the kind of \u201cill - defined\u201d problem for which an analogy from a remote domain might trigger a creative insight . The desired goal state is specified only at a relatively abstract level , and the permissible operations that might be used to achieve the goal are left very open ended . As a consequence , the possible solution proposals vary considerably . This made it possible to test for the use of analogies by attempting to influence the specific solutions that subjects would generate . In addition , we were able to benefit from Duncker\u2019s analyses of the performance of subjects who worked on the problem without receiving an analogy . Duncker identified three broad categories of proposed solutions to the radiation problem : ( 1 ) reducing the intensity of the rays as they pass through the healthy tissue ; ( 2 ) avoiding contact between the rays and healthy tissue ; and ( 3 ) altering the relative sensitivity to rays of the healthy tissue and the tumor ( e . g . , by immunizing the healthy tissue or sensitizing the tumor ) . Our analogies were designed to guide subjects toward specific versions of the first two classes of proposals . Our general aim in the present study , then , was to explore the process by which subjects use analogies between remote domains to generate problem solutions . Consequently , we wrote a series of stories far re - moved from the medical domain , each involving a military problem and its solution , which were analogous to the radiation problem . We will intro - duce the various stories as we proceed ; all are presented in the Appen - dixes . A FRAMEWORK FOR ANALOGICAL PROBLEM SOLVING It is important to develop a general conceptual framework within which specific issues concerning the role of analogies in problem solving can be formulated . What is meant by analogy , and how can an analogy be used to generate a problem solution ? In order to make our discussion more concrete we will consider the major story analogy used in the present experiments and the correspond - ANALOGICAL PROBLEM SOLVING 309 ing solution to the radiation problem . In the Attack - Dispersion story\u2019 a general wishes to capture a fortress located in the center of a country . There are many roads radiating outward from the fortress . All have been mined so that while small groups of men can pass over the roads safely , any large force will detonate the mines . A full - scale direct attack is there - fore impossible . The general\u2019s solution is to divide his army into small groups , send each group to the head of a different road , and have the groups converge simultaneously on the fortress . The analogous solution to the radiation problem is to simultaneously direct multiple low - intensity rays toward the tumor from different directions . In this way the healthy tissue will be left unharmed , but the effects of the multiple low - intensity rays will summate and destroy the tumor . At an intuitive level the parallels between the Attack - Dispersion story and the radiation problem are clear . Both situations involve an object that must be overcome , surrounded by objects that must be preserved . The target object in each case occupies a topographically central position in its environment . In each situation the protagonist has available a weapon with an effect proportional to the intensity or amount of the weapon that is used , and so on . How might people represent these analogical relationships and use them to generate a solution to the target problem ? This is not an easy question to answer . First of all , both the story and the problem must be read and understood . In attempting to describe this type of analogical problem solving we thus inherit all the problems associated with text comprehension . In particular , perception of analogy hinges on semantic knowledge and inference procedures . Since no general theory of language understanding is available , we must of necessity gloss over many impor - tant issues related to the understanding process . However , recent work on story comprehension ( Black & Bower , in press ; Kintsch , 1974 ; Kintsch & Van Dijk , 1978 ; Rumelhart , 1975 ; Schank & Abelson , 1977 ; Thorndyke , 1977 ) may offer some insights into how our story analogies might be represented in memory . Indeed , there appear to be close ties between the concept of analogy and the concept of \u201cschema , \u201d which has been widely applied in discussions of prose comprehension . In essence , both an analogy and a schema consist of an organized system of relations . Consequently , the framework for analogical problem solving presented here will draw its conceptual vocabulary from various schema - based models , as well as from Sternberg\u2019s ( 1977a , 1977b ) model of component processes involved in analogical reasoning . We will first consider how \u2019 In fact , three versions of this story were used in different experiments ( see Appendixes I - III ) . The versions differed only in minor points of wording of no consequence to the present discussion . 310 GICK AND HOLYOAK analogy might be represented , and then how this representation could be used to generate a solution to a problem . The Representation of Analogy A system of representation for analogy must be able to describe a fundamental property of such relational systems , namely , that analogy may be defined at multiple levels of abstraction . For example , at a rela - tively low level of abstraction the Attack - Dispersion story and the radia - tion problem have a variety of corresponding details ( e . g . , small groups of soldiers correspond to low - intensity rays ) . At a more abstract level , the story and the problem both involve the goal of overpowering an object located in a region that must be preserved . The multileveled nature of analogy can perhaps be understood in the context of Kintsch and Van Dijk\u2019s ( 1978 ) theory of prose representation . They argue that the understanding process may involve the iterative ap - plication of a set of inference rules that generate increasingly abstract \u201cmacrostructure\u201d representations of a prose passage . These macro - structures essentially correspond to summaries of the passage at various levels of generality . In the case of a problem - oriented story such as the Attack - Dispersion story , an abstract level of macrostructure might state a general solution principle ( e . g . , to destroy a target when direct application of a large force is harmful to the surrounding area , disperse the attacking forces , and have them converge at the target ) . The process of extracting a solution principle might thus be viewed as a special case of the process of deriving macrostructures for a body of information . While much remains to be learned about how this process operates , the three specific inference rules proposed by Kintsch and Van Dijk ( which they term \u201cdeletion , \u201d \u201cgeneralization , \u201d and \u201cconstruction\u201d ) would seem readily applicable to the type of story analogies we are considering here . Kintsch and Van Dijk emphasize that control processes are required to select a level of macrostructure analysis consistent with the person\u2019s processing goals . Similarly , we assume there is an optimal level of abstraction at which analogical relations may be represented in order to effectively guide the solution process . Indeed , an important empirical issue is to determine what factors influence this optimal level of abstrac - tion . We will now consider in more detail how an analogy between two relational systems might be represented , assuming an appropriate level of macrostructure has been derived . To pursue our example , Table 1 pre - sents our own summary of the Attack - Dispersion story , as well as a sum - mary of the radiation problem and its dispersion solution . These sum - maries are intended to reflect the major causal connections within both the story and the problem , and to illustrate the major analogical relations ANALOGICAL PROBLEM SOLVING 311 between them . The sentences in Table 1 are numbered to correspond to an approximate propositional analysis presented in Fig . 1 . Propositions from the story and from the radiation problem are matched to indicate analogical relations , and the propositions corresponding to the dispersion solution to the radiation problem ( which a person would be required to generate ) are italicized in both the table and the figure . Note that some of the propositions included in the story summary ( e . g . , proposition 11 ) are inferences , which are not directly stated in the original story ( see Appen - dix I ) . The notation in Fig . 1 consists of propositional functions , in which predicates are followed by one or more arguments ( enclosed in paren - theses ) . The arguments fill various semantic roles , such as agent , ob - TABLE 1 A Summary of Attack - Dispersion Story and of Corresponding Solution to Radiation Problem ( See Fig . 1 ) Proposition number Attack - Dispersion story l - 2 A fortress was located in the center of the country . 2a Many roads radiated out from the fortress . 3 - 4 A general wanted to capture the fortress with his army . 5 - 7 The general wanted to prevent mines on the roads from destroying his army and neighboring villages . 8 As a result the entire army could not attack the fortress along one road . 9 - 10 However , the entire army was needed to capture the fortress . 11 So an attack by one small group would not succeed . 12 The general therefore divided his army into several small groups . 13 He positioned the small groups at the heads of different roads . 14 - 15 The small groups simultaneously converged on the fortress . 16 In this way the army captured the fortress . l\u2019 - 2\u2019 3\u2019 - 4\u2019 5\u2019 - 7\u2019 8\u2019 Radiation problem and dispersion solution\u201d A tumor was located in the interior of a patient\u2019s body . A doctor wanted to destroy the tumor with rays . The doctor wanted to prevent the rays from destroying healthy tissue . As a result the high - intensity rays could not be applied to the tumor along one path . 9\u2019 - 10\u2019 11\u2019 12\u2019 13\u2019 14\u2019 - 15\u2019 16\u2019 However , high - intensity rays were needed to destroy the tumor . So applying one low - intensity ray would not succeed . The doctor therefore divided the rays into several low - intensity rays . He positioned the low - intensity rays at multiple locations around the patient\u2019s body . The low - intensity rays simultaneously converged on the tumor . In this way the rays destroyed the tumor . n Italicized propositions summarize the target dispersion solution . 312 GICK AND HOLYOAK ject , and location . Propositions may themselves serve as arguments , so in several cases one proposition is embedded in another . We make no claims about the logical adequacy or completeness of the representation in Fig . 1 ; indeed , many of the indicated arguments ( e . g . , \u201clow - intensity rays\u201d ) clearly could be further decomposed . However , separation of relations ( predicates ) and arguments serves to highlight the critical properties of analogy between remote domains : similarity of corresponding relations despite dissimilarity of corresponding arguments . The notation in Fig . 1 has been augmented by labeled arcs that repre - sent the major causal connections within the Attack - Dispersion story and the radiation problem . The labels are inspired by but not identical to the analysis of causal types offered by & hank and Abelson ( 1977 ) ( see also Black & Bower , in press ) . Roughly , a goal can be a \u201creason\u201d for an action ; a state or action can \u201cenable\u201d a subsequent action ; an action can \u201cresult\u201d in a subsequent state ; and a state or action can \u201cprevent\u201d a subsequent action . Again , the adequacy of this analysis is not really crit - ical for our present purpose , which is simply to make salient the corre - spondences in causal structure between the story and the problem , par - ticularly with respect to the target solution . Note that the labeled arcs are equivalent to higher - order predicates that embed numbered propositions as arguments . The representation in Fig . 1 can be used to highlight a variety of prop - erties of analogy , as well as some of the issues we have glossed over in constructing this representation . Fundamentally , an analogy consists of a yre @ nerd . ytue brmm fortress ) ) desm & ml . preat & shuy ( mines , army / v ~ llcqsd ) desm & ml . preat & shuy ( mines , army / v ~ llcqsd ) 6 5 6 5 allack ( enhre ormy , fortmrr . ona read ) atlack ( enhre ormy , fortmrr . ona read ) requre kapture hirers ) , sntwe army ) requre kapture hirers ) , sntwe army ) attack bnc small qwp , fwtm . ss ) attack bnc small qwp , fwtm . ss ) tgeenad , ormy , small grwpr ) locate iiatrerr , can * kou ~ trYl1 y2 I radiate ( roods , fortress ) $ s . e kkctw , B\u201d\u201d mys , tumw11 ( dacta , p $ vent $ strny ( roys , healthy t ! ssuaN ( one bw urtawty ray , tumw ) W groups , different roads ) konvenp bmatl groqs . fatressl ) FIG . 1 . Analogical correspondences between the Attack - Dispersion story and the radia - tion problem . ANALOGICAL PROBLEM SOLVING 313 mapping between two sets of propositions . Propositions are matched on the basis of similarity between the corresponding relations . Note that similarity need not imply identity . There need only be a consistent trans - formation that maps one set of relations onto the other . Boden ( 1977 ) gives the example of the Black Mass , which is based on systematic semantic reversals of the Catholic ritual . In the case of our story and problem , we wrote the summaries to maximize the similarity of the rela - tions . Yet dividing an army ( proposition 12 ) , for example , is clearly not quite the same as dividing high - intensity rays ( proposition 12\u2019 ) . To gener - ate this aspect of the parallel solution the person would need to take account of the relevant differences between an army and rays , perhaps explicitly introducing multiple machines that each emit low - intensity rays . In order to map proposition 1 onto 1\u2019 a person must have semantic knowl - edge of the relation between the meanings of \u201ccenter\u201d and \u201cinterior . \u201d Incidentally , note that we assume proposition 1\u2019 is included in the mac - restructure for the problem as an inference based on knowledge of where the stomach is located . In the case of propositions 16 and 16\u2019 , we assume the person will use semantic knowledge to transform the relation of \u201ccapturing\u201d into the relation of \u201cdestroying , \u201d operating on the common semantic core ( i . e . , \u201covercoming\u201d ) that links the two relations . As Fig . 1 indicates , there is clearly a high degree of correspondence between the propositions of the story and of the problem . At the same time , the systems are not perfectly isomorphic . It is probably the case that analogies used to guide problem solving are generally incomplete in some respects ( Gentner , Note 1 ) . For example , proposition 2a in the Attack - Dispersion story , which states that many roads radiate outward from the fortress , has no parallel in the statement of the radiation problem . Note that in the story this proposition serves as an important enabling condition for the solution . The absence of any explicit mention in the radiation problem of multiple paths to the target object would presumably hinder generation of the dispersion solution . For the above example it is plausible to argue that people must infer the fact that there are multiple potential \u201croutes\u201d to the tumor in the course of generating the dispersion solution , even though no such inference is represented in Fig . 1 . But in addition , Fig . 1 reveals at least one clear disanalogy between the story and the problem . In a complete analogy , there is a consistent mapping between pairs of arguments . That is , wher - ever argument A occurs in one relational system , argument A\u2019 occurs in the other . For example , in Fig . 1 the role the fortress plays in the story consistently maps onto the role the tumor plays in the problem . Note that the role of the army usually corresponds to that of the rays . However , this is not the case in propositions 5 and 5\u2019 . In the Attack - Dispersion story , sending the entire army down one road will result in destruction of the 314 GICK AND HOLYOAK army ( as well as neighboring villages ) by mines ; whereas in the radiation problem applying high - intensity rays to the tumor will result in destruc - tion of the healthy tissue by the rays . In other words , the army and the rays do not till corresponding semantic roles in propositions 5 and 5\u2019 ; rather , the army is the object of the process of destruction in 5 , while the rays are the instrument of the destruction in 5\u2019 . This example illustrates that degree of analogy in part depends on the level of abstraction at which the analogy is defined . In macrostructures slightly more abstract than those depicted in Fig . 1 , the fact that an attack by the entire army is impossible would map onto the fact that direct application of high - intensity rays is impossible . At this level the roles of the army and of the rays correspond appropriately . However , the story and the problem are disanalogous at the more specific level depicted in Fig . 1 ( i . e . , the level of the reas0n . s why the two respective courses of action are blocked ) . This observation suggests that for use in solving a problem the optimal level of abstraction for representing an analogy may be that which maximizes the degree of correspondence between the two relational systems . In many cases a very detailed representation will in - clude disanalogous relations , while a very abstract representation will omit information about important correspondences . The Process of Analogical Problem Solving So far we have been discussing how analogical relations may be repre - sented ; we must now consider how this information might be used to generate a solution to a problem . For our story analogy and target prob - lem the solution process appears to require three major steps . ( 1 ) A representation of the story analogy and of the target problem ( i . e . , its initial state and goal state ) must be constructed , as described above . ( 2 ) The representation of the story must be mapped onto that of the problem . If the story and the problem are drawn from remote domains , as in our example , the correspondences between arguments will not be im - mediately obvious . We would therefore expect the mapping process to be initiated by detection of similar relations in the two systems . For example , the person might notice that propositions 2 and 2\u2019 both involve location . Accordingly , a mapping between the two propositions will be established . This will automatically establish a mapping between the corresponding arguments ( i . e . , the fortress and the tumor , the center of the country and the interior of the body ) . Once a few such correspondences have been detected , the mapping process may proceed in a more \u201ctop - down\u201d man - ner , guided by expectations that previously mapped arguments will con - tinue to play parallel roles . For example , having mapped propositions 2 and 2\u2019 , the person might assume that 8 maps onto 8\u2019 because the role of the fortress should correspond to that of the tumor . ANALOGICAL PROBLEM SOLVING 315 ( 3 ) Finally , the person must use the mapping to generate the parallel solution to the target problem . This can be done by constructing a set of solution propositions for the target problem that correspond to the solu - tion propositions of the story . For example , consider how proposition 12\u2019 might be generated on the basis of proposition 12 . The mapping process will have identified the general with the doctor and the army with the rays . Accordingly , \u201cdoctor\u201d and \u201crays\u201d will be used to fill the argument slots corresponding to \u201cgeneral\u201d and \u201carmy . \u201d In addition , the relation be - tween the general and the army in 12 will be used to construct a parallel relation between the doctor and the rays in 12\u2019 . Thus the idea of the general dividing the army into several small groups will be transformed into the idea of the doctor dividing the rays into a number of low - intensity rays , initiating the dispersion solution to the radiation problem . ISSUES AND EXPERIMENTS A number of important questions arise within the framework of the process model we have outlined . A major issue , which we touched upon earlier , concerns the level of macrostructure at which the mapping pro - cess takes place . At one extreme the solution process might amount to abstracting a solution principle from the story and then applying it to the target problem . At the other extreme subjects might map the corre - spondences between the story and the problem at the most detailed possi - ble level . It is possible , of course , that the mapping process may actually proceed partially in parallel on several different levels . Even at a single level of macrostructure , there may be strategic varia - tions in the degree of mapping that takes place during the solution pro - cess . For example , subjects need not derive the entire set of corre - spondences outlined in Fig . 1 in order to generate the dispersion solution . One possibility , which we will term the \u201csolution - focusing\u201d strategy , is that subjects attempting to apply the story analogy will immediately iden - tify the solution propositions of the story . By doing the minimal amount of mapping required to match the arguments in these propositions with ar - guments in the radiation problem , the parallel solution could be gener - ated . Subjects using the solution - focusing strategy might thus solve the target problem without entirely grasping the correspondences between the problem statements in the story and in the radiation problem . Given the lack of empirical research on analogical problem solving , even more basic issues arise . We have sketched a model of how in princi - ple a problem might be solved on the basis of an analogy . However , we do not know whether subjects could actually execute this kind of process for our story analogies and target problem . There seem to be at least three distinct ways in which subjects who have a relevant analogy available might nonetheless fail to derive the parallel solution to a target problem . 316 GICK AND HOLYOAK The first and most basic is that subjects might be unable to successfully apply the story analogy even if they tried . Second , even if a story analogy is potentially useful , subjects might be unable to locate it in memory , especially if it had been encoded in the context of irrelevant distractor stories . Third , subjects might be able to retrieve a potentially useful anal - ogy and yet fail to spontaneously notice its relevance to the target problem . The experiments reported below were designed to explore these and related issues . In Experiments I - III subjects were presented with story analogies and given a hint to use them to solve the radiation problem . In addition to investigating whether subjects can in fact use analogies to generate problem solutions , Experiments I and II were intended to pro - vide some information about the processes involved in analogical problem solving . In Experiment I the oral protocols of subjects solving the target problem were analyzed , and in Experiment II the degree of analogical correspondence between the story and the target problem was varied . In Experiment III subjects were asked to first solve the problem presented in the story themselves ( rather than having its solution presented to them ) , and then to attempt to use their solutions as aids in solving the target problem . Experiments IV and V investigated the ability of subjects to solve the target problem on the basis of story analogies stored in memory . In addition , the latter two experiments varied whether or not subjects were provided with a hint to use a story analogy to help solve the target problem . These experiments thus examined the propensity of subjects to spontaneously notice and apply potential analogies between remote problem domains . EXPERIMENT I Experiment I was designed to demonstrate that subjects can use an analogy from a remote domain as a hint for solving a problem . Subjects first read a story analogy , and then attempted to propose as many solu - tions as possible to the radiation problem . By varying the nature of the solution suggested by the story , we hoped to influence the likelihood that subjects would generate specific solutions to the target problem . Subjects\u2019 \u201cthinking aloud\u201d protocols were tape recorded and later analyzed as a source of evidence regarding the process of analogical problem solving . Subjects in three experimental conditions read one of three stories about a military problem and its solution ( see Appendix I ) . Table 2 infor - mally illustrates the correspondences among the three stories and the radiation problem . The statement of the radiation problem ( see Introduc - tion ) was worded so as to minimize obvious lexical or syntactic corre - spondences with the story analogies . The Attack - Dispersion , Open Sup - ply Route , and Tunnel stories all have identical first paragraphs describing ANALOGICAL PROBLEM SOLVING 317 TABLE 2 Schematic Outline of Duncker\u2019s Radiation Problem Showing Correspondences with Analogous Stories Problem Statement Radiation problem Story analogies - Experiment I Problem setting Doctor has rays . General has army . Patient has tumor . Country has dictator . Tumor in stomach , surrounded Dictator in fortress in center of by healthy tissue . country , surrounded by villages . Roads radiate from fortress like spokes on a wheel . Desired goal Destroy tumor with rays . Capture fortress with army . Problem High - intensity rays destroy tumor Entire army can capture fortress , constraints but also destroy healthy tissue . but large group detonates mines on roads , destroying army and villages . Low - intensity rays destroy Small group of men can pass safely neither tumor nor healthy over roads but can not capture tissue . fortress . Impossible to operate . Solutions Type 1 Reduce intensity of rays on way Reduce size of group traveling to to tumor . fortress on one road . Dispersion ( 1 ) Many low intensity rays ( 1 ) Many small groups of men ( Attack - ( 2 ) From different directions ( 2 ) From different directions Dispersion ( 3 ) Simultaneously ( 3 ) Simultaneously story ) Type 11 Avoid contact between rays and Avoid contact between army and healthy tissue . mines . ( 1 ) Open pas - Send high - intensity rays through General discovers road that is not sage an open route , ( e . g . , esopha - mined , and sends entire army ( Open Supply pus ) . down this road . Route story ) ( 2 ) Operation Make an incision in stomach wall , Dig tunnel under mines , and send ( Tunnel story ) removing healthy tissue from entire army through . path of rays , and apply high intensity rays to tumor . \u201d Resulting goal state Radiation of high - intensity reaches tumor . Tumor destroyed . Healthy tissue intact . Entire army reaches fortress . Fortress captured . Army and villages preserved . n Incision violates constraint . the problem setting , desired goal , and the constraints on a solution . These aspects of the stories are analogous to the radiation problem , as discussed earlier ( see Fig . 1 ) . However , the stories differ in their second paragraphs , which state the 318 GICK AND HOLYOAK general\u2019s solution to his problem . In the Attack - Dispersion story ( de - scribed in the Introduction ) the general divides his army into small groups and sends them simultaneously down different roads to the fortress . The analogous solution to the radiation problem is the \u201cdispersion\u201d solution : have multiple low - intensity rays converge at the tumor . This is a very effective solution , 2 but one which subjects seldom generate spontane - ously . Duncker ( 1945 ) reported that only 2 of 42 subjects arrived at this dispersion solution , and both were prompted by the experimenter . A basic difftculty that appears to block generation of this solution is that people do not spontaneously think of rays as having the property of \u201cdi - visibility . \u201d In fact , Duncker found that the frequency of the dispersion solution increased when the term \u201cparticles\u201d was substituted for \u201crays\u201d ( presumably because particles are more obviously divisible ) . In the Open Supply Route story the general discovers an unblocked road leading to the fortress , and sends the entire army down this open road . An analogous radiation solution is to direct high - intensity rays down the esophagus ( or some other open passage , such as the intestines ) to the stomach . This solution was generated relatively frequently by the subjects tested by Duncker ( 29 % gave the open passage solution as opposed to only 5 % who gave the dispersion solution ) . In the Tunnel story the general digs an underground tunnel and sends his army through it to the fortress . Analogous radiation solutions might be to operate to expose the tumor to the rays , or to insert a tube through the stomach wall and send rays through it to the tumor . Many of Duncker\u2019s subjects ( 40 % ) spontaneously suggested such solutions . However , such procedures to create an open route to the tumor involve operating , and hence conflict with one of the constraints imposed on the radiation problem ( that it is impossible to operate ) . The Tunnel story is therefore a kind of \u201cfalse analogy\u201d to the radiation problem . That is , although the problem statements are analo - gous , the solution suggested by the story is inappropriate . If the analogy is nevertheless applied , subjects given the Tunnel story might be especially likely to momentarily disregard the problem constraints and propose an operation solution to the radiation problem . Although the above analysis of the analogous relationships between various solutions to the military problem and to the radiation problem was 2 This solution is functionally very similar to the standard medical procedure for radiation therapy , which is to rotate the radiation source around the patient ( or vice versa ) in such a way that the tumor is always the focal point of the radiation . The malignancy thus receives a cumulative dose of radiation while other areas receive a lesser amount . One difference is that our dispersion solution involves simultaneous application of the rays , whereas the medical procedure takes advantage of the fact that the effects of radiation summate over time . Even knowledge of the medical procedure would therefore be unlikely to lead to the exact solution corresponding to the Attack - Dispersion story . ANALOGICAL PROBLEM SOLVING 319 initially based on the experimenters\u2019 intuitions , we will see below that subjects\u2019 ratings essentially confirm the validity of this analysis . The primary prediction in Experiment I was that each story analogy would tend to increase the frequency of the analogous solution to the radiation problem , relative to the solution frequencies obtained for con - trol subjects given no prior story . However , there are additional ways in which the story analogies might influence the solutions given to the target problem . First , note that the problem statements for all three stories contain all the enabling conditions ( see Fig . 1 ) for generating the disper - sion solution ( e . g . , the central location of the fortress , the roads radiating outward , the fact that small groups can travel on the roads ) . Accordingly , subjects might spontaneously think of the dispersion solution to the gen - eral\u2019s problem , and then use it to generate the parallel solution to the radiation problem . If so , subjects given the Open Supply Route and Tun - nel stories might also produce the dispersion solution more often than would control subjects . It is also possible that giving subjects a story analogy may actually hinder the generation of nonanalogous solutions . That is , attempting to generate a parallel solution to the target problem may create a kind of \u201cset\u201d effect , so that other possible solutions ( e . g . , immunizing the healthy tissue to protect it from the rays ) will not be discovered . If such a set effect is obtained , control subjects should produce more total solu - tions than experimental subjects , and in addition there should be qualita - tive differences between the solutions produced by control subjects ver - sus subjects given story analogies . Method Subjects were divided into four conditions , each receiving either the Attack - Dispersion story , the Open Supply Route story , the Tunnel story , or no story ( the control group ) , prior to solving the radiation problem . The control condition used no story at all , rather than an irrelevant story , because it seemed possible that an irrelevant story would actually interfere with the solution process . ( A control condition using an irrelevant story was included in Experiment V below . ) Subjects were tested individually by a trained experimenter and sessions were tape recorded . All subjects first solved Duncker\u2019s ( 1945 ) \u201ccandle problem\u201d to familiarize them with the process of thinking aloud while problem solving . Subjects were told that the problems required some creativity for their solutions , and that they should not feel inhibited about making any suggestions that came to mind . They were also encouraged to give an ongoing account of what they were thinking about . Subjects were asked to begin by reading the problem out loud in order to get them used to speaking in front of the experimenter . Following the practice problem subjects in the experimental conditions were told that they would receive two further problems , the first of which would also have a solution given for it . They were told to read the first \u201cstory problem\u201d aloud and then to orally summarize the gist or point of the story . This part of the procedure was omitted for subjects in the control condition . All subjects then read the radiation problem and began to solve it . They were reminded to talk out loud , and encouraged to interrupt their reading of the problem at any 320 GICK AND HOLYOAK time if a solution occurred to them . Experimental subjects were told to try to use the first story problem as a hint in solving the second ( radiation ) problem . However , they were also told that it was not necessary to use the prior story in order to solve the problem . Subjects were allowed to reread the story analogy at any time . As subjects worked on the radiation problem the experimenter was prepared to intervene with an explicit hierarchy of prompts . If subjects were not explicit about the nature of a proposed solution , the experimenter asked them to clarify it , sometimes by drawing a diagram . Subjects in the experimental conditions who at first failed to generate the analo - gous solution were eventually prompted to reread the instructions . If they still did not produce the analogous solution , they were then reminded to use the prior story as a hint . At the end of the session , subjects were asked to rate each of their proposed solutions on two 7 - point scales , as to how \u201ccreative\u201d and how \u201cpractical\u201d the solutions are . A rating of 1 indicated maximum creativity or practicality . Forty undergraduates enrolled in introductory psychology at the University of Michigan served as subjects as part of a course requirement . Ten subjects were assigned to each of the four conditions . Results and Discussion Frequencies of analogous solutions . Subjects\u2019 protocols for the radia - tion problem were transcribed and scored for the presence of various types of proposed solutions , by two independent scorers . For this pur - pose any suggestion , even if it was eventually rejected by the subject , was counted as a proposed solution . The results of major interest concern the three types of proposals that are analogous to the solutions embodied in the story analogies - the dispersion solution , the open passage solution , and operation solutions . Table 3 presents the percentage of subjects in each condition who produced these various types of proposed solutions . The frequency of each solution was highest for subjects who received the relevant story analogy , i . e . , the dispersion solution was most frequent for the Attack - Dispersion condition , the open passage solution was most fre - quent for the Open Supply Route condition , and operation solutions were most frequent for the Tunnel condition . These differences in solution frequencies were most dramatic for the dispersion solution . All 10 subjects who were given the Attack - Dispersion story produced this solution , whereas not a single control subject did so . For this solution the frequency differences among the four conditions were highly significant , G2 ( 3 ) = 33 . 9 , p < . OOl , as was a comparison between the Attack - Dispersion condition versus all others , G2 ( 1 ) = 30 . 9 , p < . 001 . 3 The frequencies of the open passage solution and of operation solutions were also influenced by the story analogies . Seventy percent of subjects in the Open Supply Route condition produced the open passage solution , as opposed to 20 % of subjects in all other conditions , G2 ( 1 ) = 8 . 21 , p < 3 All contingency table analyses reported in the present paper use the GZ statistic ( maximum likelihood x2 ) ( Bishop , Fienberg , & Holland , 1975 ) . ANALOGICAL PROBLEM SOLVING 321 TABLE 3 Percentage of Subjects in Each Condition of Experiment I Who Proposed Various Solutions to the Radiation Problem Proposed solution Condition Dispersion Open Passage Operation\u201d Attack - Dispersion story 100 10 30 Open Supply Route story 10 70b 50 Tunnel story 20 30 80 Control 0 20 50 U This solution type includes proposals to operate to clear a path for the rays or to insert a tube through the stomach wall and send rays through to the tumor . b This value includes one subject who proposed the abstract idea of finding an open pas - sage through which the rays could be directed at the tumor , but who failed to suggest the esophagus or any other concrete possibility as a route . . Ol . Eighty percent of subjects in the Tunnel conditions produced opera - tion solutions , as opposed to 43 % of subjects in all other conditions , G2 ( 1 ) = 4 . 29 , p < . 05 . Ratings of stories as solution prompts . As we noted earlier , we initially used our own intuitions about the semantic parallels between the military and the medical solutions to predict the radiation solutions that would be triggered by the various story analogies . In order to assess whether sub - jects shared our intuitions we administered a rating task to 51 under - graduates , none of whom previously knew of any solutions to the radia - tion problem . For each of the three stories used in Experiment I , 17 subjects first read the story , then the radiation problem , and finally the six possible solution proposals listed in Table 4 . Subjects rated each of the proposals ( which were listed in a random order ) on a 9 - point scale as to how likely they thought it was that the story they had just read would make them think of the proposed solution . A rating of 9 indicated maximum likelihood that the story would suggest the solution . Subjects were instructed not to consider the practicality of the various solutions in making their judgments . The mean ratings are presented in Table 4 . Note that proposal 1 is the dispersion solution , 2 is the open passage solution , and 3 and 4 are opera - tion solutions . Proposals 5 and 6 are two other fairly common suggestions that we expected would be relatively unrelated to all of the three stories . ( Proposal 5 is a type of \u201caccumulation\u201d solution , which will be discussed in connection with Experiment III ) . The proposal ratings differed greatly depending on which story subjects had read , F ( 10 , 240 ) = 8 . 84 , p < . OOl . For each story analogy , Newman - Keuls tests were performed on the differences among the ratings for the six proposals . In the case of the 322 GICK AND HOLYOAK TABLE 4 Mean Ratings of Likelihood that Stories Would Help to Think of Various Radiation Solutions\u201d Proposed solutions 1 . Apply low - intensity rays from several different directions so they simultaneously converge at the tumor . 2 . Send high - intensity rays down the esophagus so they strike the tumor . 3 . Insert a tube through the healthy tissue to the tumor , and then send high - intensity rays through the tube to the tumor . 4 . Make an incision into the stomach to expose the tumor , and then apply high - intensity rays directly to the tumor . 5 . Treat the person with low - intensity rays , repeating the treatment a number of times . 6 . Treat the person with medium - intensity rays . Solution number story 1 2 3 4 5 6 Atack - Dispersion 8 . 41 2 . 53 3 . 71 2 . 94 4 . 88 3 . 47 Open Supply Route 3 . 59 6 . 65 5 . 82 4 . 58 2 . 94 2 . 29 Tunnel 5 . 12 4 . 47 6 . 47 5 . 35 2 . 71 1 . 82 ( 1 A rating of 9 indicated maximum likelihood that the story would suggest the solution . Attack - Dispersion story , subjects gave higher ratings to the dispersion solution than to any other ( p < . Ol ) . For the Open Supply Route story , the open passage solution received the highest rating , although it did not differ significantly from the ratings given the two operation solutions . However , these three solutions were all rated significantly higher than any of the others ( p < . 05 ) . For the Tunnel story , the \u201cinsert a tube\u201d solution ( proposal 3 ) received the highest rating , followed by the other operation solution ( proposal 4 ) , the dispersion solution , and the open passage solution . These four proposals did not differ significantly from each other . However , the two operation solutions received significantly higher ratings than proposals 5 and 6 ( p < . 05 ) . The fact that subjects gave the highest rating of all to the Attack - Dispersion story as a prompt for the dispersion solution is consistent with the fact that this story appeared to be the most effective analogy in Ex - periment I . The overlap in the ratings for the solutions analogous to the Open Supply Route and Tunnel stories ( proposals 2 , 3 , and 4 ) also reflects trends in the observed solution frequencies ( see Table 3 ) . Among the three story conditions , the Open Supply Route condition produced the second highest frequency of operation solutions , while the Tunnel condi - tion produced the second highest frequency of open passage solutions . Notice that both types of solutions involve avoiding contact between the rays and the healthy tissue , by directing the rays through an unobstructed ANALOGICAL PROBLEM SOLVING 323 route . The basic difference between the two solution types is simply that the open passage solution makes use of a preexisting route ( the esophagus ) , while the operation solutions require that the route be con - structed ( by some type of operation ) . At first glance the relatively high rating given to the Tunnel story as a prompt for the dispersion solution is more puzzling , since digging a tunnel seems very disanalogous to dispersion of rays . In fact , however , as Table 3 indicates , two subjects in the Tunnel condition actually did produce the relatively rare dispersion solution . Furthermore , as we will see when we discuss the protocols in more detail below , both of these subjects also spontaneously suggested that the general might have sent his army down multiple roads . Recall that the identical first paragraphs of all three stories contain the enabling conditions for the dispersion solution . However , in the Open Supply Route story the given solution involves use of only one road , which may create a set effect that blocks consideration of how multiple roads might be used . In contrast , the solution given in the Tunnel story avoids use of the roads altogether . Since some kind of attack by road is nonetheless an obvious possibility , the stated enabling conditions may lead subjects who read the Tunnel story to think of the alternative solution of using multiple roads , which in turn could prompt the disper - sion solution to the radiation problem . While some aspects of the above analysis are certainly speculative , it seems clear that subjects in the rating task were highly sensitive to factors that actually influenced the effective - ness of the story analogies in Experiment I . Frequencies of other solutions . The possibility of a set effect , men - tioned above , raises the general issue of whether story analogies actually block generation of qualitatively different solution proposals . To investi - gate this question an analysis of variance was performed on the total number of proposed solutions , other than the primary analogous solution , that were given by subjects in the various conditions . That is , dispersion solutions were excluded for the Attack - Dispersion condition , open pas - sage solutions were excluded for the Open Supply Route condition , oper - ation solutions were excluded for the Tunnel condition , while none of the above solutions were excluded for the control condition . In addition , a small number of proposals ( a mean of 0 . 4 per subject ) that did not involve use of the rays at all ( e . g . , Laetrile treatment ) were excluded for all conditions . The average number of nonanalogous solutions produced was 1 . 10 for the Attack - Dispersion condition , 1 . 60 for the Open Supply Route condi - tion , 2 . 70 for the Tunnel condition , and 2 . 00 for the control condition , F ( 3 , 36 ) = 4 . 17 , p < . 025 . Newman - Keuls tests revealed that only the difference between the Tunnel and Attack - Dispersion conditions was sig - nificant ( p < . Ol ) . As we noted earlier , subjects in the Tunnel condition 324 GICK AND HOLYOAK tended to produce a relatively high number of open passage and disper - sion solutions . The rating results ( Table 4 ) also indicated that the Tunnel story is a moderately effective prompt for several different solutions . The trends toward fewer alternative proposals in the Attack - Dispersion and Open Supply Route conditions , compared to the control condition , at least suggest the possibility of some kind of set effect . In order to obtain further evidence regarding a possible set effect , the frequencies of specific radiation solutions , other than those analogous to the various stories , were tabulated for each condition . The solutions ex - amined were proposals to treat the healthy tissue directly , rather than altering the route that the rays take . Specifically , these solutions suggested decreasing the sensitivity of the healthy tissue to the rays ( e . g . , by a chemical injection , or building up a tolerance to the rays ) , or covering the healthy tissue with a barrier to protect it from the rays ( e . g . , by inserting a lead shield to protect the healthy tissue ) . Such solutions were produced by 30 % of the subjects in the control condition , 10 % of the subjects in the Tunnel condition , and none of the subjects in the Attack - Dispersion and Open Supply conditions . While the numbers involved were too small to be statistically reliable , these results suggest that an analogy may tend to block generation of alternative types of solutions . Practicality and creativity ratings . The practicality and creativity rat - ings that subjects gave for their own solutions were examined . Within each story condition , the ratings given to the analogous solution were compared to the means of the ratings given to other solutions by the same subjects . In the Attack - Dispersion condition seven subjects produced and rated the dispersion solution and at least one other proposal . The disper - sion solution was rated as much more practical than other solutions ( 1 . 43 versus 4 . 64 ) , F ( 1 , 6 ) = 27 . 2 , p < . Ol . However , the creativity ratings did not differ significantly between the dispersion solution and other propos - als ( 3 . 43 versus 3 . 64 ) , F < 1 . It seems likely that subjects did not perceive their dispersion solutions as especially creative because they were aware of using the prior story to generate an analogous solution . Parallel analyses for the Open Supply Route and Tunnel conditions revealed no significant differences . Collapsing over subjects in all condi - tions , the dispersion solution tended to be rated as most practical ( a mean of 2 . 0 ) , followed by the open passage ( 3 . 9 ) and operation solutions ( 5 . 4 ) . We also asked an independent group of 20 undergraduates ( none of whom previously knew of the radiation problem ) to choose which solution was more practical : the dispersion solution ( \u201capply low - intensity rays from several different directions so they simultaneously converge at the tumor\u201d ) , or the open passage solution ( \u201csend high - intensity rays down the esophagus so they strike the tumor\u201d ) . Fifteen of the twenty subjects selected the dispersion solution as more practical @ < . 05 by a sign test ) . ANALOGICAL PROBLEM SOLVING 325 When asked to justify their decision , 11 of these 15 mentioned the prob - lem of preventing the rays from destroying the tissue lining the esophagus . It may be that some subjects in Experiment I implicitly generated the open passage solution ( or an operation solution ) on the basis of the rele - vant story analogy , but then failed to mention it ( despite the instruction to mention any possibility ) because they recognized its inadequacy . We will consider other sources of difficulty in using the Open Supply Route and Tunnel stories below , when subjects\u2019 protocols are discussed in more detail . Problem - solving protocols . The results discussed so far demonstrate that story analogies can play a major role in directing the problem - solving process . However , they reveal little about the process by which subjects arrive at an analogous solution . We therefore supplemented the quantita - tive analysis of solution types , reported above , with a more qualitative analysis of subjects\u2019 problem - solving protocols . Several aspects of the protocols were examined . Occasions when the experimenter prompted the subjects to use the story were noted , as were correspondences be - tween the story and the target problem that subjects mentioned in the course of generating solutions . This analysis was , of course , constrained by the overall quality and quantity of the protocols . For example , some subjects insisted that talking aloud hindered their thinking , and con - sequently did not say very much . Rather than presenting an exhaustive analysis of all the protocols , we will therefore concentrate on particularly suggestive excerpts . While this type of protocol analysis has obvious limitations , it may at least provide some hints about the process of analogical problem solving , and in fact served in part to motivate sub - sequent experiments . A major issue , raised earlier , concerns the degree of mapping subjects perform in the process of generating an analogous solution . Do subjects make use of detailed correspondences between the story and the target problem , or do they focus directly on the solution embedded in the story and attempt to apply it to the target problem ? Of the 10 subjects in the Attack - Dispersion condition , 7 produced the dispersion solution without any prompt , and 3 produced it after being prompted to refer back to the story . In some respects the protocols for prompted subjects are poten - tially more informative , since what such subjects say is more likely to reflect an ongoing solution process , rather than the result of a process already completed . The protocols of 2 of the 3 prompted subjects suggested use of a solution - focusing strategy . Table 5 presents an excerpt from the protocol of one of these subjects , S15 . After the prompt to use the story , this subject clearly focuses on the solution of dividing up the army into groups and immediately generates the parallel solution to the radiation problem . There is no apparent map - 326 GICK AND HOLYOAK TABLE 5 Portion of Protocol for Sl5 ( Attack - Dispersion Condition ) Subject reads radiation problem . S : Alright I , what I most , what I\u2019d probably do is send in the ray at sufficiently high intensity and then taking the risk that the tissues , the healthy tissues that would be destroyed , could be repaired later on . Trying to relate this to the other problem , I could say that you could give multiple treatments of low - intensity ray . But from this problem it seems that they won\u2019t have effect on the tumor so . . . so I don\u2019t think that would work . Later . . . E : Okay . And as a last question can you give me a , tell me ways in which your solution would satisfy the constraints of the experiment ? S : What are the constraints of the experiment ? E : Okay , i . e . , that the healthy tissue will not be destroyed , and the tumor will be ? S : Alright , in that way my first suggestion would probably not be the way to go at it . Because that way you\u2019re getting low intensity so it won\u2019t destroy the tissue and hopefully over a period of time the additive effect of low - intensity rays would kill the tumor . But from reading the article , I don\u2019t know if that would work or not , because it says that a low - intensity ray doesn\u2019t have any effect on the tumor at all . So I don\u2019t know . I don\u2019t know any other possible ways of doing it . E : Would it help to possibly go back to the story and see whether you can apply that ? S : Well , that\u2019s what I was trying to do here . It says here he divides his army into different small groups . Okay , may . . . possibly . What they could do , but this is a whole new solution now , possibly what they could do is attack the tumor from a multiple of directions with lower intensity rays and then , since you\u2019re coming in from all different directions , the healthy , with small - intensity rays you\u2019re not going to be destroying the healthy tissue but you\u2019re , and they\u2019ll all converge at the point of the tumor which will hopefully destroy the tumor . ping between the initial problem stated in the story and the target problem . Notice also that the solution S 15 proposes prior to the prompt involves the idea of applying many low - intensity rays . After the prompt , the sub - ject produces the dispersion solution by augmenting this aspect of the earlier solution with the idea of sending rays from many angles . This pattern of gradual solution development was also evident in the protocol of another prompted subject in the Attack - Dispersion condition . In such cases it appears that the subjects were working in the appropriate direc - tion ( in the sense of Maier , 1930 ) prior to producing the complete disper - sion solution . That is , at first they seemed to have the abstract idea that the solution should involve reducing the intensity of the rays on the way ANALOGICAL PROBLEM SOLVING 327 to the tumor , but only later were able to develop a concrete solution satisfying the problem constraints . S15 appeared to use the story analogy to develop the early partial solution ; however , other subjects apparently generated an abstract partial solution independently of the analogy . The details of the problem - solving process are less evident in the pro - tocols of the seven unprompted subjects , since they expressed the solu - tion all at once . Three of these subjects simply stated the solution and alluded to the usefulness of the prior story ( saying , for example , \u201cconsid - ering the problem before\u201d ) . These subjects did not mention any specific correspondences between the story and the target problem , and hence their protocols were quite unrevealing with respect to the solution pro - cess . However , two other unprompted subjects did spontaneously mention correspondences between the problems . Immediately after reading the radiation problem , S23 stated : Like in the first problem , the impenetrable fortress , the guy had put bombs all around , and the bombs could be compared to the destruction of healthy tissue . And so they had to , they couldn\u2019t go in in mass through one road , they had to split up so as not to destroy the healthy tissue . Because if there\u2019s only a little bit of ray it doesn\u2019t damage the tissue , but it\u2019s all focused on the same spot . Table 6 presents a portion of the protocol for S28 , another unprompted subject . This subject\u2019s protocol is particularly interesting because he claimed that he generated the dispersion solution on the basis of an anal - ogy between the radiation problem and an actual problem he had read about the previous night ( using lasers to fuse the filament in a lightbulb without breaking the glass ) . This may be an unanticipated instance of the use of a \u201creal world\u201d analogy to solve a problem . However , when later questioned by the experimenter , S28 clearly was also aware of corre - spondences between the radiation problem and the prior story\u2019 . It is therefore uncertain which analogy initially triggered generation of the dispersion solution . Also note that at the beginning of this excerpt S28 remarks that the dispersion solution made it hard to think of alternative solutions , suggesting that he was aware of a set effect . No other subject explicitly mentioned such an inhibitory effect of the prior story . It is clear in the above two cases that the subjects noticed some corre - spondences involving the initial conditions and constraints of the story and target problem . However , it is difficult to tell whether these aspects of the mapping process were instrumental in generating the analogous solu - tion , or whether subjects simply mentioned the correspondences to justify the adequacy of the solution , after it had already been generated . In general it was not clear what particular correspondences were central to the solution process . However , several subjects alluded to the importance of the phrase \u201clike spokes on a wheel . \u201d Recall that the existence of 328 GICK AND HOLYOAK TABLE 6 Portion of Protocol for S28 ( Attack - Dispersion Condition ) Subject reads radiation problem and states dispersion solution . Experimenter asks for other solutions and subject suggests operating to expose tumor . S : I like my first solution so much that it\u2019s hard to come up with any others . E : Can you tell me how you arrived at your first solution ? S : To tell you the truth I was thinking about that problem last night . ( Experiment asks for clarification ) . S : I remembered reading an ad at one time on one . Some company has really expensive lightbulbs and when the filament breaks inside it\u2019s really expensive to replace the lightbulb so what they do is take lasers from all different angles and , like shooting through they don\u2019t disturb the glass but when they concentrate they fuse the filament . E : Oh , I see , very interesting . S : A . . . other than that I would , I really couldn\u2019t , don\u2019t think I could come up with other solutions . E : Okay , can you tell me whether you applied any of the hints from the previous story at all ? S : Yeah , I would say , yeah , the fortress is similar to the tumor and the army is the same as X rays or rays that destroy the tumor , and they cannot all pass through the organism , i . e . , countryside , at the same time or they blow up . multiple routes is a critical enabling condition for the solution embodied in the Attack - Dispersion story , and it has no explicit parallel in the state - ment of the radiation problem . This aspect of the story analogy may therefore serve to generate the critical insight that it is possible to send rays from multiple directions . One illustrative example is the following excerpt from the protocol of S7 in the Attack - Dispersion condition , which begins immediately after the subject had read the radiation problem : Well , I already know the answer . I knew it when I read it . I might as well stop and say that . What you do is you have a bunch of rays that are weaker , and you point them so that they concentrate on one point . So you just have many different angles . It could not only be two dimensional , the analogy of the spokes on the fortress . But you could even have it three dimensional , so you could have a whole ball of spokes going in . And you would have a high intensity right at the tumor . In addition , the protocols of all three subjects in the Open Supply Route and Tunnel conditions who produced the dispersion solution suggested that it was triggered by the idea of multiple converging routes . For exam - ple , immediately after S2 in the Open Supply Route condition read the problem , she expressed the idea of using a \u201ccircular approach\u201d ( which in her earlier story summary she explicitly related to the phrase \u201cspokes on a wheel\u201d ) . This idea then led to the multidirectional aspect of the disper - sion solution to the radiation problem . ANALOGICAL PROBLEM SOLVING 329 Two subjects in the Tunnel condition who produced the dispersion solution did so after first spontaneously remarking that the general might have sent his men down multiple roads . One other subject , in the Open Supply Route condition , also suggested the dispersion solution to the military problem , but failed to apply it to the radiation problem . The use of self - generated solutions to help solve an analogous problem will be investigated more systematically in Experiment III . Subjects\u2019 protocols also provided information about some of the dif - ficulties they encountered in attempting to apply analogies based on the Open Supply Route and Tunnel stories . As we pointed out earlier , the open passage solution is not especially practical and some subjects may have thought of this solution without mentioning it . For example , S32 in the Tunnel condition gave the open passage solution as an afterthought at the very end of the interview , and also outlined the problems with it : that the esophagus is not straight , and that there would be \u201crefraction off the esophagal walls , or absorption of the rays , \u201d which would destroy tissue . Three subjects attempted to overcome this difficulty by suggesting that a ray - proof tube through which the rays could be directed should be in - serted down the throat . In addition , the nature of the analogy suggested by the Open Supply Route story is somewhat different from that suggested by the other two stories . The solutions embodied in both of the latter stories suggest pro - cedures that can be used to generate parallel solutions to the radiation problem ( dividing the rays in the case of the Attack - Dispersion story , operating in the case of the Tunnel story ) . In contrast , the Open Supply Route story only suggests that an existing open passage might be used . The subject must then search memory to find a concrete example of such an open passage to the stomach ( e . g . , the esophagus ) . Applying the anal - ogy thus involves two steps : mapping the abstract idea of an open passage from the story to the target problem , and then thinking of a concrete example of such a passage . The difficulty of applying the analogy may account for the fact that four of the seven subjects in the Open Supply Route condition who gave the open passage solution had to be prompted to use the story . Table 7 presents a portion of the protocol for S19 in the Open Supply Route condition . This subject works through a rather detailed mapping of the correspondences between the story and the radiation problem . But while she clearly develops the abstract idea of finding an open passage , she fails in the attempt to think of a concrete example . The partial solution produced by S19 can be contrasted with the complete lack of success apparent in the protocol of S37 in the Open Supply Route condition : The only thing that is apparent to me is that the general had other information that he knew that one of the thoroughfares would be left open , and so he was able to 330 GICK AND HOLYOAK TABLE 7 Portion of Protocol for S19 ( Open Supply Route Condition ) E : It might help if you reread the instructions here . This part . ( S rereads radiation problem . ) S : Okay , so what was the first problem ? The spokes of the wheel - right ? E : Right . S : So the center fortress deal would be the idea of the tumor . That\u2019s . . . E : Okay . S : And then the spokes that blow up a little would be like the healthy tissue that blows up a little bit . And so with that one the guy had one route that was gonna do it . I guess in this one that\u2019s what you have to do is find the one route that would work . E : Okay . S : And , I think , and not use the other ways . E : Okay . What would that be ? S : That would mean we have to find one approach that was going to get to the tumor without getting the healthy tissue . And I don\u2019t see how you could do that . Cause it\u2019s not - it doesn\u2019t seem like it\u2019s the same thing . E : What doesn\u2019t seem like the same thing ? S : Well the idea that road , with a road its possible to have one road unguarded but without , in your body there\u2019s always going to be , unless the tumor was right on the outside , there would always be some tissue you would have to go through to get to it . use that . But , unless the doctor had some new information or some other treat - ment , I don\u2019t see any other applications from the first problem to the second problem . Notice that S37 appears to have mapped the story and the target prob - lem at a very abstract level of macrostructure , so that the perceived analogy ( the general had new information , so perhaps the doctor might also ) was too vague to yield a specific solution proposal for the radiation problem . In the case of the Tunnel condition four of the eight subjects who generated operation solutions received a prompt to use the story before they did so . Some subjects may have been reluctant to suggest an opera - tion solution because they were aware that it violated a constraint given in the problem statement . 4 An excerpt from the protocol of S24 , presented in Table 8 , illustrates the kind of difficulty encountered in this condition . The protocol suggests that the subject was quite carefully mapping com - ponents of the story onto components of the radiation problem . However , 4 Actually , some subjects pointed out that the problem statement is somewhat vague on this point . The statement that \u201cit is impossible to operate on the patient\u201d might be inter - preted as meaning only that it is impossible to operate and remove the tumor , rather than that an operation of any kind is impossible . However , protocols and practicality ratings indicated that most subjects who suggested operating to expose the tumor considered the proposal dubious at best . ANALOGICAL PROBLEM SOLVING 331 TABLE 8 Portion of Protocol for S24 ( Tunnel Condition ) E : If you read your instructions , it says that this story might give you some hints . . . What are you thinking ? S : Well , I remember that the main way they solved this problem was they dug under the fortress and went around it . So , possibly in this situation , you could go around the healthy tissue . I don\u2019t know how you\u2019d do that I see an analogy , it\u2019s not real clear . E : Why isn\u2019t it clear ? S : Because when I picture healthy tissue in my mind , healthy tissue all over and , you know , just like a tumor in between all this healthy tissue . But here , the mines they\u2019re on top near the surface of the ground , so they can , you can dig under those and you won\u2019t really have any problem . But here no matter where you go , like a circle around the healthy tissue . . . maybe an operation . E : Except that one of the constraints of the experiment says that you can\u2019t operate . S : Okay , that\u2019s not possible . . . maybe . . . I was thinking that maybe you could give intervals of high intensity , but I don\u2019t know , that still would probably destroy the healthy tissue . E : Can you think of anything else ? . . . Is this problem , the previous story , is that distracting ? S : ( mumbles ) Again , I\u2019m looking for an analogy between the two . And kind of set up the same problem and solve it the same way , but I don\u2019t know if I can or not . E : So , can you think of any other possibilities ? S : ( long pause ) No . the subject was unable to generate a satisfying parallel solution to the target problem . The overall impression created by the problem - solving protocols is that the generation of analogous solutions involves a conscious process of mapping correspondences between the story and the target problem . The degree of mapping required seems to vary a great deal . Sometimes mapping was done in considerable detail , particularly if the subject was having difficulty producing a parallel solution . In other cases noticing one or two major points of correspondence seemed sufficient to generate the solution . In some instances , particularly for dispersion and open passage solutions , aspects of the solution were clearly generated in sequential steps . The protocols of control subjects differed in several ways from those of subjects in the story conditions . Control subjects more often were prompted to talk . Sometimes they seemed confused and asked if there really was a solution to the problem . In addition , control subjects received fewer prompts to clarify their solutions . The latter finding raises the ques - tion of the extent to which the use of story analogies may depend on verbal or nonverbal feedback from the experimenter . Since a primary concern in Experiment I was to obtain interpretabie oral protocols , a 332 GICK AND HOLYOAK considerable amount of probing by the experimenter was essential . How - ever , it would clearly be desirable to demonstrate that people can use analogies to generate problem solutions without interacting with the ex - perimenter . Experiment II was designed to serve this purpose , and also to provide additional information about the degree of mapping required to produce a solution on the basis of a story analogy . EXPERIMENT II In order to avoid any possibility that subjects could be led to a particu - lar solution by the experimenter , Experiment II used a noninteractive procedure in which story analogies and the radiation problem were ad - ministered in booklet form . Experiment II was also designed to assess the degree of mapping required to generate a solution on the basis of an analogy . For this purpose different subjects were given one of two matched stories . While both stories embodied the same solution ( the dis - persion solution ) , they differed in the degree of correspondence that existed between their problem statements and the radiation problem . If correspondences between the problem statements play an important role in generating a parallel solution to the target problem , lessening the degree of analogy between the story problem and the radiation problem should reduce the probability that subjects will be able to use the story to pro - duce the analogous solution . Method Materials . The stories used in Experiment II were the Attack - Dispersion story ( ver - sion 2 ) and the Parade - Dispersion story ( see Appendix II ) . Table 9 presents a schematic outline of the Parade - Dispersion story , which can be compared to the outlines of the Attack - Dispersion story and radiation problem ( Table 2 ) . The Attack - Dispersion story used in Experiment II is essentially identical to that used in Experiment I . The story was rewritten slightly to match it as closely as possible with the Parade story in length and wording . Note that the setting information is nearly the same in the Attack and Parade stories . In particular , the critical enabling conditions for the dispersion solution ( centrally located fortress , multi - ple roads radiating outward ) are present in both stories . In addition , the second paragraphs of the two stories , which describe the general\u2019s dispersion solution , are identical ( except for the tinal sentences , which state the goal that has been achieved ) . However , the problem statement for the Parade story is substantially different from that for the Attack story , and it is disanalogous to the radiation problem . In the Parade story the genera1 is not trying to attack the dictator in the fortress , but rather to stage a parade that meets the dictator\u2019s specifications . The constraint of the mined roads has been removed ; and use of the entire army is required not to produce a sufftciently strong assault , but rather to produce a sufficiently impressive display . The problem statements of the Parade story and of the radiation problem are thus disanalogous in several respects . The Parade problem lacks the basic element of \u201cdesire to overcome a target object . \u201d The basis of the constraint against sending the entire army down one road ( the genera1 would lose his rank ) does not parallel the basis of the constraint against using high - intensity rays ( healthy tissue would be destroyed ) . Only the setting information and the constraint against sending the full army down one route remain analogous to features of the radiation problem . Accordingly , the mapping process ANALOGICAL PROBLEM SOLVING 333 TABLE 9 Schematic Outline of Parade - Dispersion Story Problem statement Problem setting General has army . Country has dictator . Dictator in fortress in center of country , surrounded by villages . Desired goal Problem constraints Roads radiate from fortress like spokes on a wheel . Produce impressive parade that can be seen and heard throughout entire country . Sending entire army down one road fails to produce impres - sive parade . If parade fails to impress dictator , general will lose his rank . Solution Dispersion Divide up parade so that each part of country sees part of parade . Use Resulting goal state ( 1 ) Many small groups of men ( 2 ) From different directions ( 3 ) Simultaneously Parade seen and heard simultaneously throughout country . General preserves his rank . subjects can perform in order to use the Parade story to generate the radiation dispersion solution will be limited by the lack of correspondence between the problem statements for the story and the target problem . As in the Attack story , the general in the Parade story solves his problem by dividing his troops and sending them down multiple roads to the fortress . But in the Parade story the procession of soldiers to the fortress directly constitutes achievement of the goal state , whereas in the Attack story the movement of troops is simply the means by which the final goal ( capture of the fortress ) is achieved . Furthermore , in the Parade story the fact that the troops converge on the fortress is a more or less incidental aspect of the solution procedure , whereas in the Attack story this aspect of the solution is critical . Thus even though the surface description of the solution is the same in both stories , the solution contexts differ . Such contextual differences may influence the precise interpretation subjects give to the solution . In order to assess whether the Parade and Attack stories in fact suggest similar solutions to the radiation problem , two independent groups of 17 undergraduates rated the stories as prompts for various radiation solutions . ( The Parade story was simply included as an addi - tional condition in the story rating task discussed under Results of Experiment I . ) The six solutions that were rated are listed in Table 4 . While there was a slight trend for the Attack story to be rated higher than the Parade story as a prompt for the dispersion solution , the two stories did not produce significantly different ratings , either as a main effect , F < 1 , or as an interaction , F ( . 5 , 160 ) = 1 . 32 , p > . 25 . These rating results thus confirm that the two stories , despite the differences in their problem statements , suggest essentially the same solution to the radiation problem . Experiment II was designed to determine whether subjects actually solving the radiation problem would be hindered by a disanalogous problem statement . Procedure and subjects . Subjects were divided into three conditions , receiving either the Attack story , the Parade story , or no story ( control condition ) prior to working on 334 GICK AND HOLYOAK the radiation problem . The test booklet consisted of instructions , story analogy ( for experi - mental conditions ) , radiation problem , a solution sheet , and a final questionnaire , each on a separate page . Experimental subjects first read the story and then wrote a brief summary of it , referring back to the story if they wished . All subjects then attempted to solve the radiation problem . Subjects in the experimental conditions were told that the first story might give them some hints for solving the test problem , although it was not necessary to use the prior story to solve the problem . They were allowed to refer back to the story at any time . All subjects were instructed to write as many solutions as possible in the order they came to mind . They were asked to write down every idea they considered , even those later rejected . Subjects were told to make their proposals as explicit as possible , but not to be concerned with technical medical considerations . The final questionnaire asked subjects to indicate how helpful the story problem was in solving the radiation problem ( \u201cnot helpful , \u201d \u201csomewhat helpful , \u201d or \u201cvery helpful\u201d ) , and in what way it was helpful . Subjects were also asked if they had known the solution to the radiation problem prior to the experiment . Subjects were 143 undergraduates tested in five introductory psychology classes . Forty - seven subjects served in the Attack - Dispersion condition , 46 in the Parade - Dispersion con - dition , and 50 in the control condition . Results and Discussion The data for one subject in the Parade - Dispersion condition who was familiar with the radiation problem and dispersion solution were dis - carded . The remaining subjects\u2019 solution proposals were analyzed for the presence of various solution types , particularly the dispersion solution . This analysis was done by two independent scorers , each blind to the conditions in which subjects served . Disagreements were resolved by discussion . Unlike subjects in Experiment I , subjects in Experiment II were not prompted to fully explicate their solutions . As a result , a number of subjects produced incomplete versions of the dispersion solution . In order to be scored as a complete dispersion solution , three features had to be present in the proposal : ( 1 ) the rays are applied to the tumor from different directions , ( 2 ) at low intensity , and ( 3 ) simultaneously . A partial solution had to contain at least the first feature , the critical element of dispersion . However , partial solutions might omit features 2 and / or 3 . In addition , a partial solution might include reference to some degree of damage to healthy tissue . Presence of the latter feature was taken as an indication that the subject did not entirely understand the implications of the dispersion solution , since the problem statement specified that low - intensity rays are harmless . The percentage of subjects producing complete or partial dispersion solutions differed substantially across the three conditions , as shown in Table 10 . Dispersion solutions were produced by 76 % of the subjects in the Attack - Dispersion condition , 49 % of the subjects in the Parade - Dispersion condition , and only 8 % of the subjects in the control condition , G2 ( 2 ) = 53 . 1 , p < . OOl . The Attack story produced significantly more dispersion solutions than did the Parade story , G2 ( 2 ) = 7 . 70 , p < . Ol , while the two story conditions together produced significantly more dis - ANALOGICAL PROBLEM SOLVING 335 TABLE 10 Percentage of Subjects in Each Condition of Experiment II Who Proposed the Dispersion Solution to the Radiation Problem Dispersion solution Condition Complete Partial Total N Attack - Dispersion story Parade - Dispersion story Control 57 19 76 47 31 18 49 45 8 0 8 50 persion solutions than did the control condition , GZ ( l ) = 45 . 5 , p < . OOl . The Attack story also prompted more complete dispersion solutions than did the Parade story . The four subjects in the control condition who spontaneously generated dispersion solutions gave complete solutions . Collapsing over all conditions , dispersion solutions tended to be produced relatively early in the sequence of proposals given by a subject ( i . e . , prior to the proposal of median rank , p < . 025 by a sign test ) . Table 11 presents the percentage of subjects in the two story conditions who rated the story as \u201cnot helpful , \u201d \u201csomewhat helpful , \u201d or \u201cvery helpful , \u201d as a function of whether the subject produced a complete dis - persion solution , partial solution , or no dispersion solution . The results obtained with such post - hoc questionnaires must be interpreted with cau - tion , since they may reflect hindsight rather than accurate memories of the solution process . Nevertheless , these rating results are at least a source of converging evidence . Most subjects who produced a dispersion solution rated the story as \u201cvery helpful . \u201d In contrast , the majority of those subjects in both conditions who failed to produce a dispersion solu - tion rated the story as \u201cnot helpful at all . \u201d There were no reliable differ - ences between the rating patterns produced by subjects in the Attack versus Parade conditions . The total number of proposed solutions ( other than the dispersion solu - tion ) was tabulated for subjects in each of the three conditions . The mean number of proposals was 1 . 17 for the Attack condition , 1 . 40 for the Parade condition , and 2 . 12 for the control condition , F ( 2 , 139 ) = 12 . 7 , p < . OOl . A Newman - Keuls test indicated that only the difference between the control condition and the two story conditions was significant , p < . Ol . This decline in the number of alternative proposals given by subjects prompted to generate the dispersion solution replicated the comparable trend obtained in Experiment I . As in Experiment I , the frequency of a specific class of alternative solutions ( decreasing the sensitivity of the healthy tissue to the rays , or T AB L E 11 P e r c en t age o f S ub j e c t s G i v i ng E a c h H e l p f u l ne ss B a t i ng a s a F un c t i on o f S t o r y C ond i t i on and T y pe o f D i p s e r s i on S o l u t i on G i v en 0 T y pe o f d i s pe r s i on s o l u t i on g i v en 2 C o m p l e t e d i s pe r s i on P a r t i a l d i s pe r s i on N o d i s p e r s i on > s o l u t i on s o l u t i on s o l u t i on 2 N o t S o m e w ha t V e r y N o t S o m e w ha t V e r y N o t S o m e w ha t V e r y ! 3 C ond i t i on he l p f u l he l p f u l he l p f u l he l p f u l he l p f u l he l p f u l he l p f u l he l p f u l he l p f u l 2 A tt a ck - D i s pe r s i on 0 22 78 11 22 67 64 27 0 9 s t o r y k P a r ade - D i s pe r s i on 0 29 71 0 38 63 57 39 4 s t o r y ANALOGICAL PROBLEM SOLVING 337 somehow protecting the tissue ) was tabulated for each condition . Whereas 28 % of the subjects in the control condition produced such solu - tions , only 2 % of those in the Attack condition and 16 % of those in the Parade condition did so , G2 ( 2 ) = 14 . 6 , p < . Ol . Subjects in the Parade condition produced more solutions of this type than did subjects in the Attack condition , G2 ( 1 ) = 5 . 78 , p < . 02 . This result is a significant repli - cation of a trend obtained in Experiment I : the more effective the story analogy is in prompting the analogous solution , the more it inhibits pro - duction of . alternative , disanalogous proposals . The basic results of Experiment II are thus extremely clear . First , subjects can readily use story analogies to guide their attempts to solve the radiation problem , even without feedback from the experimenter . Second , the effectiveness of analogies in prompting a specific solution is a matter of degree . In particular , a story with a problem statement analo - gous to that of the radiation problem ( the Attack story ) was more likely to trigger the dispersion solution than was a story with a problem statement less related to that of the radiation problem ( the Parade story ) . This was true even though both stories embodied similar setting information and solution statements . EXPERIMENT III Experiments I and II demonstrated that subjects can use a story anal - ogy , describing a problem and its solution , to guide generation of an analogous solution to the radiation problem . A natural question is whether subjects could also use their own solutions to the initial story problem to help them solve the target problem . You may recall that Experiment I provided some evidence for this possibility . Three subjects who received stories other than the Attack - Dispersion story spontaneously suggested that the general might have divided his troops and sent them down multi - ple roads ; two of these subjects then went on to produce the dispersion solution to the radiation problem . Accordingly , in Experiment III subjects were first given just the problem statement from the Attack - Dispersion story , and asked to suggest what the general might do to capture the fortress . Subjects were then asked to solve the radiation problem , using their solutions to the initial problem as hints . Method The problems were administered in booklet form as in Experiment II . The instructions stated that the subject would have to solve \u201ctwo verbal problems requiring some creativity for their solutions . \u201d The first problem consisted of the first paragraph of the Attack - Dispersion story ( version 2 , Appendix II ) , followed by the question \u201cWhat could the general do in order to capture the fortress . 7\u201d Subjects were to provide as many possible solutions as they could think of . Following this , subjects proceeded to give solutions to the radiation problem . The instructions stated , \u201c , . . you may find that the first problem that you solved 338 GICK AND HOLYOAK gives you some hints for solving the second problem , so you should try and use it if you can . . . However , it is not necessary to use the first problem in order to solve the second . \u201d Subjects were told that they could look back to the first problem and their solutions to it at any time . On the final page of the booklet subjects were asked to indicate to what extent the first story problem helped in solving the ray problem ( \u201cnot at all , \u201d \u201csomewhat , \u201d \u201cvery much\u201d ) , and in what way it was helpful . They were also asked to indicate if they had known the solution to the radiation problem prior to the experiment . The experiment was administered to 46 students in two introductory psychology classes . Results and Discussion Data from one subject who indicated prior knowledge of the dispersion solution to the radiation problem were discarded . The frequencies of vari - ous solutions to the two problems were tabulated for the remaining 45 subjects by two independent scorers ; disagreements were resolved by discussion . As might be expected given the extremely loose constraints imposed by the problem statement , the proposed solutions to the military problem were quite varied . Many of these ( e . g . , airlifting troops into the fortress by balloon ) had no apparent correspondence to any potential solution to the radiation problem . However , 22 of the 45 subjects ( 49 % ) produced the gist of the dispersion solution : the general should divide the troops into small groups and send them down different roads . The major question of interest was whether those subjects who pro - duced the dispersion solution to the military problem would be especially likely to then produce the dispersion solution to the radiation problem . Of the 22 subjects who produced the dispersion solution to the military problem , 9 ( 41 % ) subsequently produced either complete or partial dis - persion solutions to the radiation problem . Five of these subjects pro - duced a complete solution . In contrast , only 3 of the remaining 23 subjects ( 13 % ) produced this solution G2 ( 1 ) = 4 . 61 , p < . 05 . Two of these subjects produced a complete solution . The value of 41 % is significantly higher than the 8 % of subjects who produced the dispersion solution to the radiation problem without any prior story problem ( Control condition of Experiment II ) , G2 ( 1 ) = 10 . 4 , p < . Ol . The above results are correlational in nature , since subjects who ar - rived at the dispersion solution to the military problem did so of their own accord - no experimental manipulation determined the subjects that would produce the critical solution . It could therefore be argued that subjects who produced dispersion solutions to both problems did so be - cause of some general factor related to problem - solving skills or strategies . However , additional evidence suggests that self - generated so - lutions to the military problem had a causal influence on generation of the radiation dispersion solution . First , dispersion solutions were produced significantly more frequently in Experiment III ( regardless of whether a parallel solution was produced for the military problem ) than in the con - ANALOGICAL PROBLEM SOLVING 339 trol condition of Experiment II . Second , the questionnaire results also reflected the influence of the solutions produced for the military problem . Of the nine subjects who generated the dispersion solution to both prob - lems , all but one indicated that the military story was \u201csomewhat\u201d or \u201cvery\u201d helpful in solving the radiation problem ( one other subject did not respond to this question ) . In contrast , a majority ( 58 % ) of the remaining subjects indicated that the story was not helpful at all . Interestingly , two of the three subjects who produced the radiation dispersion solution with - out first producing the military dispersion solution indicated that the story was somewhat or very helpful ( the third subject failed to answer the question ) . All three of these subjects had suggested that the general could send small groups in succession to the fortress . This solution may have triggered the idea of dividing forces that is critical to the radiation disper - sion solution . While self - generated solutions to the simpler military problem thus facilitated discovery of the radiation dispersion solution , the degree of transfer was less than perfect . The value of 41 % radiation dispersion solutions , based on a self - generated military dispersion solution , is sig - nificantly less than the 76 % radiation dispersion solutions produced by subjects given a military dispersion solution written by the experimenter ( Attack - Dispersion condition of Experiment II ) , G2 ( 1 ) = 8 . 25 , p < . Ol . There are a number of possible explanations for this difference . Subjects in Experiment III almost always proposed more than one solution for the military problem ( a mean of 3 . 04 ) . Consequently , their own dispersion solution , if they produced it , was usually embedded in other \u201cdistractor\u201d solutions . Subjects who did not systematically consider each of their pro - posed military solutions with respect to the radiation problem may there - fore have sometimes missed the critical analogy . Indeed , some subjects may have simply failed to apply their prior solutions at all when working on the radiation problem . In addition , it is possible that the self - generated dispersion solutions did not always entirely capture the analogy to the corresponding radiation solution . All of the self - generated dispersion solutions expressed the basic idea of sending small groups down multiple roads . However , in some versions the element of simultaneity of attack was absent or not clearly expressed . In other cases subjects suggested that the small groups would meet and regroup near the fortress prior to initiating an attack . This added feature of delay is disanalogous to the radiation dispersion solution ( since low - intensity rays cannot \u201cwait and meet up\u201d near the tumor prior to striking it ) . Of the 22 self - generated dispersion solutions , 10 either lacked a clear expression of simultaneity or added the element of delay . How - ever , since 4 of these 10 subjects succeeded in generating the radiation dispersion solution , we were unable to find any effect of quality of the 340 GICK AND HOLYOAK military dispersion solution on subsequent transfer to the radiation prob - lem . Nonetheless , it remains possible that in various ways the subjects\u2019 dispersion solutions ( which were often written in a cryptic fashion ) were incomplete in comparison with our own version of the solution . At any rate , it is clear that further research will be required to assess how problem - solving activity per se affects subsequent transfer of a solution to an analogous problem . In addition to the dispersion solution , another frequently proposed so - lution to the military problem was to have the general send successive small groups of men down a single road . This \u201csuccessive groups\u201d solu - tion appeared to trigger an analogous solution to the radiation problem . We defined proposed solutions to the latter problem that involved spreading out ray applications over time ( regardless of the intensity of the rays or whether treatment was intermittent or continuous ) as \u201caccumula - tion\u201d solutions . Of 24 subjects who gave the successive groups solution to the military problem , 10 ( 42 % ) suggested an accumulation solution to the radiation problem . In contrast , only 3 of the remaining 21 subjects ( 14 % ) suggested an accumulation solution to the radiation problem , G2 ( l ) = 4 . 28 , p < . 05 . Furthermore , 2 of the latter 3 subjects had produced the dispersion solution to the military problem . Their accumulation solutions may therefore have been triggered by a relatively abstract analogy be - tween \u201cdispersion over space\u201d and \u201cdispersion over time . \u201d This possi - bility is supported by the fact that an accumulation solution was rated the second most likely ( after the dispersion solution ) to be prompted by the Attack - Dispersion story ( see Table 4 ) . This tendency for subjects to generate analogous accumulation solu - tions is particularly intersting because such proposals are clearly ineffec - tive solutions . Simply distributing ray treatments over time will not over - come the basic problem that the rays will have equal effects on both the tumor and the healthy tissue . Since the instructions to subjects stated that they should write down any proposal they could think of , even if they later rejected it , we do not know whether subjects who gave an accumu - lation solution actually believed it would work . However , this result at least raises the possibility that a \u201cfalse analogy\u201d between problems ( sim - ilar to the Tunnel condition in Experiment I ) may foster errors in evaluat - ing a parallel solution to a transfer problem . EXPERIMENT IV Our central concern in the experiments reported so far was to deter - mine if people can use an analogy to generate a solution to a target prob - lem , and to investigate how analogical problem solving proceeds . Con - sequently , we simplified the subjects\u2019 task in several important ways . First , subjects were always allowed to reread the story analogy at any ANALOGICAL PROBLEM SOLVING 341 time , so that their performance would not be limited by memory factors . Second , the story was always presented alone , so that subjects would have no problem identifying the relevant analogy . Third , subjects were always explicitly told to try to use the story as an aid in solving the target problem . This hint was quite nonspecific ; at no time were subjects told the nature of the analogous relationship between the story and problem . Nevertheless , the hint eliminated the need for subjects to spontaneously notice the possible analogy . In many cases of everyday problem solving in which an analogy could help , the person would have to spontaneously notice the correspondence between the target problem and some analogous problem , either of which might be stored in memory . The two experiments reported below begin to investigate the effect of such additional processing requirements on analogical problem solving . Method In Experiment IV subjects first memorized the Attack - Dispersion story ( version 3 , Ap - pendix HIS ) in the guise of a story recall experiment , and then went on to work on the radiation problem . In addition , subjects also first memorized two additional \u201cdistractor\u201d stories written by the experimenters ( \u201cThe Wine Merchants\u201d and \u201cThe Identical Twins\u201d ; see Appendix IV ) . These additional stories were matched closely in length with the Attack - Dispersion story , and also describe problems and their solutions . However , the two dis - tractor stories were intended to be as disanalogous to the radiation problem as possible . The experiment was administered in booklet form to small groups of subjects . The initial instructions to all subjects stated that the experiment would have two parts , first a story recall task and then a problem - solving task . For the story recall task , subjects first received one story , and were given 3 min to study it . The stories were then collected , and subjects had up to 15 min to recall the story . They were asked to recall the story in as close to its original form as possible , but to give the gist of it even if they couldn\u2019t remember the exact wording . All subjects completed their recall attempts within 15 min . When all subjects in a group were finished , the next story was distributed and the study - recall procedure was repeated . The stories were always administered in the order \u201cThe Wine Merchants , \u201d the Attack Dispersion story ( entitled \u201cThe General\u201d for subjects ) , and \u201cThe Identical Twins . \u201d The critical story analogy was placed in the middle serial position in order to maximize the difficulty of later using the memorized story to solve the radiation problem . Following the story recall tasks subjects were given a short ( 3 to 5 min ) break , and then asked to write solutions to the radiation problem . However , subjects were divided into two conditions that received slightly different instructions . For subjects in the \u201cHint\u201d condition , the instructions on solving the radiation problem included the following sentence : \u201cIn solv - ing this problem you may find that one of the stories you read before will give you a hint for a solution of this problem . \u201d For subjects in the \u201cNo Hint\u201d condition , this sentence was deleted from the instructions . The instructions used in the Hint condition were thus compa - rable to those used in the earlier experiments , whereas the instructions used in the No Hint condition for the first time did not call attention to the potential relevance of the stories to the target problem . 5 The wording of the story was modified slightly in this version to match it with other story analogies we intended to use in a larger experimental design . For various reasons these additional conditions were never actually tested . 342 GICK AND HOLYOAK After completing their solution attempts subjects were given a final questionnaire . Sub - jects in the No Hint condition were asked whether it occurred to them \u201cto try and use any of the stories from the first experiment to help solve the ray problem\u201d ; and if so , how helpful each of the three stories was ( \u201cnot at all , \u201d \u201csomewhat , \u201d \u201cvery\u201d ) , and in what way . Sub - jects in the Hint condition answered only the latter part of the above question . All subjects were also asked if they had known the solution to the ray problem prior to the experiment . Twenty - seven undergraduates from the Human Performance Center paid subject pool served as paid subjects . Twelve subjects were tested in the Hint condition and fifteen in the No Hint condition . Results and Discussion Two independent scorers were entirely in agreement in identifying dis - persion solutions to the radiation problem . For the Hint condition , 11 out of 12 subjects ( 92 % ) produced the complete dispersion solution . This percentage is , of course , far higher than would be expected for subjects who did not receive the Attack - Dispersion story ( under 10 % in both Ex - periments I and II ) . In addition , 10 of the 12 subjects indicated that the Attack - Dispersion story was \u201cvery helpful , \u201d one indicated that it was \u201csomewhat helpful , \u201d and one did not answer the question . In contrast , 50 % of the subjects indicated that \u201cThe Wine Merchants\u201d was \u201cnot helpful at all , \u201d and 91 % ( 10 of 11 who answered the question ) indicated that \u201cThe Identical Twins\u201d was \u201cnot helpful at all . \u201d Subjects clearly had no serious difficulty in identifying the critical story analogy in memory and applying it to generate the dispersion solution to the radiation problem . However , this picture changes dramatically when the results for the No Hint condition are examined . Whereas 92 % of the subjects in the Hint condition produced the dispersion solution , only 20 % ( 3 out of 15 ) of those in the No Hint condition did so , G2 ( 1 ) = 15 . 5 , ~ < . OOl . Furthermore , 2 of these 3 subjects gave only partial solutions ( as defined in Experiment II ) , and indicated that they did not consider using the stories . It is therefore possible , and in fact rather likely , that only 1 of the 15 subjects spontane - ously noticed the critical analogy and successfully applied it to produce the dispersion solution . In response to the questionnaire , 12 of the 15 No Hint subjects indi - cated that it had not occurred to them to use the stories . The 3 who said they did use the stories all indicated that the Attack - Dispersion story was \u201cvery helpful\u201d ; however , only one of these subjects actually produced the dispersion solution . The written comments of the other two subjects who said they used the Attack - Dispersion story suggested they were at most sensitive to some very vague , abstract analogy with the radiation problem . For example , one wrote that the story \u201cshowed a unique way to attack a problem using methods that were not very obvious or usual . \u201d ( Similar comments were given by subjects who rated the Wine Merchants ANALOGICAL PROBLEM SOLVING 343 story as somewhat or very helpful . ) It therefore seems that no more than 3 subjects , and perhaps only 1 , actually noticed the critical analogy without a hint from the experimenter . Since subjects were randomly assigned to the two conditions , degree of memory for the critical story should have been equalized across the two conditions . To confirm this , the protocols for the Attack - Dispersion story were scored for gist recall . For this purpose the story was divided into 43 propositions ( see Appendix III ) . This propositional division was made using the procedure outlined by Thorndyke ( 1977 ) , with the addition that adjectives and prepositional phrases that seemed intuitively important to the story were counted as separate propositions . As anticipated , the mean number of propositions recalled did not differ significantly between the Hint and No Hint groups ( 32 . 08 versus 33 . 53 ) , f < 1 . While we did not analyze the recall in detail , a few aspects are worth noting . First , performance appeared quite good in an absolute sense , which is consistent with the fact that subjects were almost invariably successful in using the story to generate a solution to the radiation prob - lem ( as long as they were told to try ) . Second , as many investigators of story recall have reported , some propositions were much more memora - ble than others . Three propositions were recalled by all 27 subjects . These were proposition 1 ( \u201cA small country was ruled by a king\u201d ) and proposi - tions 5 and 6 ( \u201cMany roads radiated outward from the fortress like spokes on a wheel\u201d ) . As we argued earlier , the latter pair of propositions estab - lish a critical enabling condition for the general\u2019s solution to his problem . This high level of recall obtained for setting information causally related to the solution is consistent with the problem - based model of story recall proposed by Black and Bower ( in press ) . EXPERIMENT V The results of Experiment IV demonstrated that subjects can identify a relevant story analogy encoded into memory in the context of distractor stories , and can use the analogy to generate a solution to a subsequent target problem . However , when the experimental instructions did not provide a hint that the stories might help to solve the target problem , subjects seldom noticed or used the analogy . This suggests that the pro - cess of analogical problem solving is neither automatic nor invariably applied by college students as a conscious strategy . The knowledge ac - quired in the context of the story recall phase of the experiment seemed to be encapsulated in such a way that its pertinence to the problem - solving task was not recognized . An important question is whether this type of encapsulation of experi - ence is more or less absolute , or whether there are factors that would make a relevant analogy more likely to be noticed even though it was 344 GICK AND HOLYOAK initially encoded in a recall context . Experiment V modified the design of Experiment IV in order to examine two such possible factors . First , the total memory load was reduced by eliminating the two distractor stories from the recall phase ; and second , in one condition the story analogy was presented after subjects had read and begun to work on the radiation problem . The latter condition can be viewed as an experimental analog of a situation in which a person \u201cstumbles upon\u201d relevant information in the course of working on a problem , as is often reported in anecdotes de - scribing the \u201cEureka\u201d experiences of creative thinkers . Method The experiment was administered in booklet form as in Experiment IV . Subjects were assigned to one of three conditions . The initial procedure for the Story First condition was identical to that for the No Hint condition of Experiment IV , except that the recall task involved only the critical Attack - Dispersion story . After working on the radiation problem subjects were asked to indicate whether it had occurred to them to use the story from the first experiment to help solve the problem . If they responded \u201cno , \u201d they were then asked to try and use the story to generate additional solutions to the radiation problem . The proce - dure thus involved three steps : ( 1 ) reading and recall of the story ; ( 2 ) an attempt to solve the radiation problem without a hint to use the story ; and ( 3 ) a final attempt to solve it with a hint . The Story Second condition was presented in the guise of an \u201cincubation\u201d experiment . Subjects were told they would be given a problem to work on , then would be interrupted to perform a different task ( story recall ) , and then would again work on the problem . The actual procedure involved four steps : ( 1 ) an initial IO - min attempt to solve the radiation problem ; ( 2 ) reading and recall of the Attack - Dispersion story ; ( 3 ) a second attempt at the radiation problem , without any hint to use the story ; and ( 4 ) a final attempt to solve the radiation problem after the hint was given . This procedure thus differed from that for the Story First condition only by the addition of step 1 , the initial attempt to solve the problem . If subjects in the Story Second condition were to produce more dispersion solutions than subjects in the Story First condition immediately after recalling the story , this would suggest that initial exposure to the problem makes it more likely that the person will notice a relevant analogy . However , one might argue that such a result could be interpreted as a real \u201cincu - bation effect . \u201d That is , regardless of the nature of the story presented during the intervening recall task , perhaps simply taking a break from the problem would be sufficient to increase the probability of generating the dispersion solution . To control for this possibility , subjects in the Incubation Control condition received exactly the same procedure as did those in the Story Second condition , except that their recall task used one of the distractor stories from Experiment IV ( \u201cThe Identical Twins\u201d ) . Accordingly , any tendency for the Incubation Control condition to produce more dispersion solutions after presentation of the story would presumably be due to a beneficial effect of incubation per se , rather than to use an analogy . Forty - seven undergraduates from the Human Performance Center subject pool served as paid subjects . Seventeen subjects were assigned to the Story First condition , 20 to the Story Second condition , and 10 to the Incubation Control condition . Results and Discussion Table 12 presents the percentage of subjects in each condition who produced the dispersion solution during the various steps of the proce - ANALOGICAL PROBLEM SOLVING 345 TABLE 12 Percentage of Subjects in Experiment V Who Produced Dispersion Solution at Each Step of the Procedure Condition Before story After story ( no hint ) After story ( with hint ) Never N Story First - 41 35 24 17 Story Second 10 35 30 25 20 Incubation Control 10 0 0 90 10 dure . There was no evidence that the manipulation of presenting the problem prior to the story analogy ( Story Second condition ) increased the probability that subjects would notice or use the analogy . In the Story First condition , 41 % of the subjects gave the dispersion solution on their first attempt following recall of the story ; while in the Story Second con - dition , 35 % of the subjects produced this solution immediately after reading the story . One subject in the Story First condition gave a partial solution ; all other solutions were complete . For the Story First condition we cannot clearly separate subjects who used the story to produce the solution from those who may have produced it spontaneously ( as did those subjects in the Story Second condition who gave the dispersion solution prior to seeing the story ) . However , of the seven subjects in the Story First group who gave the dispersion solution immediately after story recall , six reported that they used the story to help solve the prob - lem . If we accept these reports at face value , it appears that the per - centages of subjects in the two conditions who spontaneously noticed and used the analogy were identical ( 35 % in both conditions ) . The percentages of all subjects who reported that it occurred to them to use the story were also similar across the two conditions ( 47 % in the Story First condition , 40 % in the Story Second condition ) . It is clear that in both conditions not all of the subjects who eventually proved able to use the story to generate the dispersion solution did so spontaneously . Collapsing over the Story First and Story Second condi - tions , 43 % of the subjects generated the dispersion solution prior to re - ceiving the hint , while a total of 76 % eventually succeeded in producing the critical solution . Note that there was a trend toward a higher percent - age of subjects generating the solution without a hint in Experiment V ( 43 % ) , where no distractor stories were used , than in Experiment IV ( 20 % ) , where two distractor stories were included in the recall task . How - ever , this trend was not significant , G2 ( 1 ) = 2 . 64 , p = . lO . It should be noted that the above comparison confounds number of distractor stories with serial position of the critical story ( which was always presented in the middle position in Experiment IV ) . It is possible that distractor stories 346 GICK AND HOLYOAK would have a less detrimental effect on the likelihood of subjects noticing the analogy if the critical story were presented last , just prior to the problem - solving task . As in all previous experiments , the story analogy clearly played a crit - ical role in generating the dispersion solution . Whereas 76 % of the sub - jects in the two experimental conditions eventually produced the disper - sion solution , only 10 % ( one subject ) produced it in the Incubation Con - trol condition , G2 ( 1 ) = 15 . 0 , p < . OOl . Since the one successful subject in the latter group produced the target solution prior to receiving the story , there was not the slightest suggestion that simply taking a break from the problem was sufficient to stimulate discovery of the dispersion solution . GENERAL DISCUSSION The present study provides an experimental demonstration that a solu - tion to a problem can be developed by using an analogous problem from a very different domain . Our results substantiate anecdotal descriptions of the role that analogical thinking may play in creative problem solving , and at the same time provide some information about the mental processes involved in analogical problem solving . The results of Experiments I and II indicated that there is considerable variation in the degree of mapping required to generate an analogous solution . In particular , the intermediate frequency of dispersion solutions produced in Experiment II by the Parade story , which was only partially analogous to the radiation prob - lem , supports two important conclusions about the mapping process in - volved in analogical problem solving . First , subjects in the Parade condi - tion were much more likely to generate dispersion solutions than were control subjects . Thus it seems that subjects can often generate an analo - gous solution even though a complete mapping between aspects of the prior story and the target problem is impossible . In such cases it seems that a solution - focusing strategy may be sufficient to produce the parallel solution . Second , the Parade story was not as effective as the more com - pletely analogous Attack story in prompting the dispersion solution . This suggests that subjects can also perform a more detailed mapping between the problem statements of the story and of the target problem , and that these additional correspondences are sometimes critical in determining whether the subject arrives at the analogous solution . However , the types of correspondences between the two problem statements that are most critical in developing a solution are not entirely clear . Numerous subjects in our experiments commented on the impor - tance of the reference in the story to roads radiating outward \u201clike spokes on a wheel . \u201d Intuitively , this phrase seems to elicit a spatial image that represents those essential aspects of the dispersion solution that can be applied to both the military and the medical problems . Even though the ANALOGICAL PROBLEM SOLVING 347 stories and the target problem were always presented verbally in our experiments , the problems essentially describe spatial relationships . Our use of a propositional representation to describe the correspondences between the stories and the radiation problem does not preclude the pos - sibility that some form of analog representation plays an important role in the mapping process . For example , the mapping process may in part depend on interpretive procedures that are applied to a mediating spatial image . Further research is needed to explore the role of spatial repre - sentation in analogical problem solving . It is clear that our understanding of the use of analogies in problem solving remains severely limited in many important respects . We certainly need to be cautious in generalizing on the basis of the present study , which used only one target problem and a very limited set of story analogies . While it seems reasonable to expect that comparable results would be obtained with other ill - defined \u201cinsight\u201d problems , for which a solution hinges on a small number of critical inferences , this remains to be demonstrated . It is still less clear whether analogies can be used in a similar fashion to help solve more \u201ccomputational\u201d problems , for which the solution con - sists of a series of discrete steps . Reed et al . ( 1974 ) were unable to demon - strate positive transfer between two homomorphic \u201criver crossing\u201d problems , except when the correspondences between the arguments of the two problems were described to subjects . In addition , most subjects in the Reed et al . study reported making little or no use of the first problem when solving the second . It is possible that the mapping process required in such multimove problems places excessive demands on memory ca - pacity . However , various procedural differences make it difficult to di - rectly compare the Reed et al . results to those obtained in the present study . For example , subjects in the Reed et al . study solved two succes - sive problems , while in our experiments the solution to the first problem was described in the context of a story ( except in Experiment III , in which the probability of transfering the analogous solution was somewhat re - duced when subjects solved the first problem themselves ) . In addition , it is possible that people are able to use analogies more easily in solving some computational problems than in solving others . For example , Hayes and Simon ( 1977 ) have demonstrated positive transfer between isomor - phic versions of the Tower of Hanoi puzzle , another computational prob - lem . Clearly much remains to be learned about the influence of problem characteristics on problem solving by analogy . In addition to investigating the effects of problem type , we need to learn more about the ways in which the use of analogies may interact with other strategies ( e . g . , means - ends analysis ) used in problem solving . 348 GICK AND HOLYOAK Noticing and Accessing Potential Analogies A number of important questions for future research involve the closely related issues of the spontaneous noticing of analogies , and the accessing of potential analogies stored in memory . The results of Experiments IV and V suggest that one of the major blocks to successful use of an analogy may be failure to spontaneously notice its pertinence to the target prob - lem . When subjects were not told to try to use the prior stories to help solve the radiation problem , only a minority succeeded in generating the analogous solution . This decline in transfer performance cannot be attrib - uted to faulty encoding of the story analogy , since most subjects were able to produce the analogous solution once they were given a hint to apply the story . Also , the problem of spontaneous noticing was not limited to stories previously encoded into memory . In the Story Second condition of Experiment V , many subjects failed to notice the relevance of the story even though they had to read , memorize , and recall it after beginning to work on the target problem . However , even when subjects are not given an explicit hint to use a story analogy to solve a problem , the analogy itself can be viewed as a hint about a possible solution to the target problem . Previous work on the general topic of hints in problem solving , most notably that of Maier ( 1930 , 193 l ) , has used hints that involve objects that are incorporated into the solution directly , rather than analogically . It is therefore difftcult to compare the present results with earlier research on hints . For example , in one investigation of the use of hints in the \u201ctwo - string\u201d problem , the experimenter \u201caccidentally\u201d brushed against one string and set it in mo - tion ( Maier , 1931 ) . This hint was very effective in eliciting the \u201cpen - dulum\u201d solution ( attaching objects as weights and setting the strings in motion ) ; yet subjects rarely reported being aware of using the hint , unless they gave the solution in separate stages , rather than all at once . This experiment is most comparable to the conditions in Experiments IV and V in which an analogy was made available to subjects in the guise of a recall task , but subjects were not told that the story analogy was a hint to help solve the radiation problem . In apparent contrast to Maier\u2019s results , most subjects in these conditions who generated the dispersion solution re - ported using the story to do so . Thus it seems that our subjects were usu - ally aware of the usefulness of the prior story , although the precise time at which they noticed its relevance is unclear from the data ( since these were post - hoc reports ) . Why should subjects so often fail to notice the relevance of a story analogy to a target problem when a hint to use the story is not provided ? One might argue that this result is not particularly surprising , since the ANALOGICAL PROBLEM SOLVING 349 story was presented in a different experimental context ( a story recall experiment ) . The difficulty of the recall context may be related to the problem of identifying the optimal level of abstraction for representing an analogy , as we discussed in the Introduction . A recall task , with its em - phasis on memory for specific wording , may lead the person to represent the story at a level of macrostructure too detailed to maximize its analogi - cal correspondence with the target problem . A hint to use the story may lead the person to derive a more abstract level of macrostructure , better suited for the problem - solving task . But in any case , the issue of how analogies are noticed is a very general one . A potential analogy may often be encoded in a very different context from that in which the target problem appears . Indeed , the basic problem in using an analogy between remote domains is to connect two bodies of information from disparate semantic contexts . More generally , successful transfer of learning generally involves overcoming contextual barriers . This may not be easy ; for example , it is all too common for a student to fail to notice the relevance of knowledge acquired in one class to a problem encountered in another . The problem of how analogies are noticed is closely related to the issue of how analogies are accessed in memory . Noticing that information in memory is relevant to a target problem is part of the process of retrieving an analogy . These problems were side - stepped in Experiments I - III , since subjects received a hint to use the story analogies and were allowed to reread them at any time . The problem of memory access was greatest in Experiment IV , in which the relevant story analogy was memorized in the context of two irrelevant distractor stories . Subjects in this experiment seemed to have little difficulty in identifying the appropriate story in memory , and applying it to the target problem , as long as they were instructed to do so . However , subjects may have performed this task by simply testing each of the three stories to see if it suggested a solution to the target problem . Such a strategy would presumably be impractical in most everyday problem - solving situations , where virtually any piece of information in memory might potentially afford a useful analogy . How might potential analogies be accessed in memory ? Is the memory search process directed , and if so , how ? At one extreme the problem solver may not actually search memory at all ; rather , he or she may simply \u201cstumble upon\u201d an analogy . That is , after a piece of knowledge has for some reason become the focus of attention , the person may spon - taneously notice its analogous relationship to a problem yet to be solved . It also seems plausible , however , that people may sometimes locate use - ful analogies in memory on the basis of a conscious search process . It may be possible to use a representation of the current problem as a retrieval cue for accessing analogous problems . Perhaps in some cases the person 350 GICK AND HOLYOAK first begins working on a problem and arrives at an abstract characteriza - tion of a potential solution , as we discussed in Experiment I . This solution representation might then be used to retrieve an analogous problem with that type of solution , which could then be used to help generate a more concrete solution to the target problem . The latter possibility is related to the solution - focusing strategy discussed in connection with Experiment I . A better understanding of how analogies are retrieved and noticed is clearly essential in order to effectively teach the use of analogies as a heuristic strategy for problem solving ( Polya , 1957 ) . The Generality of the Mapping Process The mapping process involved in the use of analogies may play a role in a variety of cognitive skills . Using an analogy involves mapping the repre - sentations of two ( or perhaps more ) instances onto one another . Similar processes may also be involved in abstracting the relational structure common to a set of particular instances . In the domain of problem solving , for example , a person who encounters several analogies to the radiation problem might eventually derive a schema for \u201cdispersion - type\u201d prob - lems . This schema would presumably be structured much like a concrete instance of a dispersion problem ( cf . Figure l ) , except that the predicates and arguments would be more abstract . A person equipped with such a general schema could then solve new dispersion - type problems by map - ping them directly onto it . These observations suggest that similar map - ping processes may be involved in three distinct but interrelated ac - tivities : ( 1 ) comparing one instance to another ; ( 2 ) deriving a schema for a class of instances ; and ( 3 ) comparing an instance to a general schema . Note that the above description of the role of mapping potentially applies not just to problem solving , but to a wide range of cognitive skills requiring concept learning and classification of instances . Such skills are involved in tasks that vary a great deal in terms of both complexity and cognitive domain . For example , the mapping of correspondences between relational structures is involved in the use of schemata for story under - standing ( Rumelhart , 1975 ) , frames for scene perception ( Minsky , 1975 ) , and scripts for understanding of social behavior ( Abelson , 1975 ) . Such structures all serve to describe our ability to deal with novel instances of familiar situations . Theories in each domain must explain how abstract structures can be derived from a set of instances , and how instances can be related to each other and to abstract structures . If similar mapping processes are involved in analogical problem solving and other cognitive skills , then the study of the use of analogies to solve problems has implications that extend to other domains . We mentioned at the beginning of this paper that an analogy may often serve as a model to guide the development of a new theory . In a similar fashion a theory of ANALOGICAL PROBLEM SOLVING 351 analogical problem solving might serve as a useful model in developing theories in other areas of cognition . APPENDIX I Story Analogies Used in Experiment I First Paragraph ( All Stories ) A small country fell under the iron rule of a dictator . The dictator ruled the country from a strong fortress . The fortress was situated in the middle of the country , surrounded by farms and villages . Many roads radiated outward from the fortress like spokes on a wheel . A great general arose who raised a large army at the border and vowed to capture the fortress and free the country of the dictator . The general knew that if his entire army could attack the fortress at once it could be captured . His troops were poised at the head of one of the roads leading to the fortress , ready to attack . However , a spy brought the general a disturbing report . The ruthless dictator had planted mines on each of the roads . The mines were set so that small bodies of men could pass over them safely , since the dictator needed to be able to move troops and workers to and from the fortress . However , any large force would detonate the mines . Not only would this blow up the road and render it impassable , but the dictator would then destroy many villages in retaliation . A full - scale direct attack on the fortress therefore appeared impossible . Second Paragraph , Attack - Dispersion Story ( Version 1 ) The general , however , was undaunted . He divided his army up into small groups and dispatched each group to the head of a different road . When all was ready he gave the signal , and each group charged down a different road . All of the small groups passed safely over the mines , and the army then attacked the fortress in full strength . In this way , the general was able to capture the fortress and overthrow the dictator . Second Paragraph , Open Supply Route Story The general , however , was undaunted . He knew that one major thoroughfare leading to the fortress was always kept open as a supply route . He led his army to the head of the supply route . When all was ready he gave the signal , and the entire army charged down the open route . The army avoided the mines and attacked the fortress in full strength . In this way , the general was able to capture the fortress and overthrow the dic - tator . Second Paragraph , Tunnel Story The general , however , was undaunted . He and his men dug an under - ground tunnel beneath the mines following the route of the road to the 352 GICK AND HOLYOAK fortress . When the tunnel was dug , the men crawled through it until they arrived safely at the foot of the fortress . Here they all gathered together and attacked the fortress in full strength . In this way , the general was able to capture the fortress and overthrow the dictator . APPENDIX II Story Analogies Used in Experiment II Attack - Dispersion Story ( Version 2 ) A small country was controlled by a dictator . The dictator ruled the country from a strong fortress . The fortress was situated in the middle of the country , surrounded by farms and villages . Many roads radiated out - ward from the fortress like spokes on a wheel . A general arose who raised a large army and vowed to capture the fortress and free the country of the dictator . The general knew that if his entire army could attack the fortress at once it could be captured . The general\u2019s troops were gathered at the head of one of the roads leading to the fortress , ready to attack . However , a spy brought the general a disturbing report . The ruthless dictator had planted mines on each of the roads . The mines were set so that small bodies of men could pass over them safely , since the dictator needed to be able to move troops and workers to and from the fortress . However , any large force would detonate the mines . Not only would this blow up the road and render it impassable , but the dictator would then destroy many villages in retaliation . It therefore seemed impossible to mount a full - scale direct attack on the fortress . The general , however , knew just what to do . He divided his army up into small groups and dispatched each group to the head of a different road . When all was ready he gave the signal , and each group marched down a different road . Each group continued down its road to the fortress , so that the entire army finally arrived together at the fortress at the same time . In this way , the general was able to capture the fortress , and thus overthrow the dictator . Parade - Dispersion Story A small country was controlled by a dictator . The dictator ruled the country from a strong fortress . The fortress was situated in the middle of the country , surrounded by farms and villages . Many roads radiated out - ward from the fortress like spokes on a wheel . To celebrate the anniver - sary of his rise to power , the dictator ordered his general to conduct a full - scale military parade . On the morning of the anniversary , the gen - eral\u2019s troops were gathered at the head of one of the roads leading to the fortress , ready to march . However , a lieutenant brought the general a disturbing report . The dictator was demanding that this parade had to be more impressive than any previous parade . He wanted his army to be ANALOGICAL PROBLEM SOLVING 353 seen and heard at the same time in every region of the country . Further - more , the dictator was threatening that if the parade was not sufficiently impressive he was going to strip the general of his medals and reduce him to the rank of private . But it seemed impossible to have a parade that could be seen throughout the whole country . The general , however , knew just what to do . He divided his army up into small groups and dispatched each group to the head of a different road . When all was ready he gave the signal , and each group marched down a different road . Each group continued down its road to the fortress , so that the entire army finally arrived together at the fortress at the same time . In this way , the general was able to have the parade seen and heard through the entire country at once , and thus please the dictator . APPENDIX III Story Analogy Used in Experiments IV and V Attack - Dispersion Story ( Version 3 ) ( 1 ) A small country was ruled ( 2 ) [ from a strong fortress ] by a king . ( 3 ) The fortress was situated in the middle of the country ( 4 ) surrounded by farms and villages . ( 5 ) Many roads radiated outward from the fortress ( 6 ) like spokes on a wheel . ( 7 ) A rebel general vowed ( 8 ) to capture the fortress . ( 9 ) The general knew that ( 10 ) an attack ( 11 ) [ by his entire army ] would capture the fortress . ( 12 ) He gathered his army ( 13 ) at the head of one of the roads . ( 14 ) However , the general learned that ( 15 ) the king had planted mines ( 16 ) on each of the roads . ( 17 ) The mines were set ( 18 ) so that ( 19 ) [ small ] bodies of men could pass over them safely , ( 20 ) since the king needed ( 21 ) to move his troops and workers to and from the fortress . ( 22 ) However , any ( 23 ) [ large ] force would detonate the mines . ( 24 ) Not only would this blow up the road ( 25 ) and render it impassable , ( 26 ) but it would also destroy many neighboring villages . ( 27 ) It therefore seemed impossible ( 28 ) to mount a ( 29 ) [ full - scale direct ] attack on the fortress . ( 30 ) The general , however , knew just what to do . ( 31 ) He divided his army up into small groups ( 32 ) and dispatched each group ( 33 ) to the head of a different road . ( 34 ) When all was ready ( 35 ) he gave the signal ( 36 ) and each group marched down a different road . ( 37 ) Each group continued down its road to the fortress ( 38 ) so that the ( 39 ) [ entire ] army finally arrived ( 40 ) [ together ] at the fortress at the same time . ( 41 ) The fortress fell ( 42 ) and the king was forced ( 43 ) to flee into exile . APPENDIX IV Distractor Stories Used in Experiment IV The Wine Merchants One day a rich man found that his wine cellar was empty . So he sent out messengers to announce a generous offer . The first person to bring the 354 GICK AND HOLYOAK rich man a barrel of wine would be given a brick of solid gold . However , the offer would expire at sundown . Two wine merchants heard the news . Each had a horse - drawn cart loaded with large barrels of wine . They both set out for the duke\u2019s palace at once . An hour before sundown they came to a place where the bridge had been washed out by a raging river . The first merchant drove his horses and cart into the flood in a desperate attempt to reach the other side . But the horses were already exhausted and could not fight the cur - rent . The cart overturned , and the horses , wine , and driver were washed away . The second merchant tried a different tactic . He poured the wine out of all but one of his barrels , and lashed them together to form a raft ; then he loaded the one full barrel , a horse , and himself on top . He set the raft adrift and floated downstream . In a few minutes the raft came to rest on the shore in front of the town where the rich man lived . The merchant disembarked , loaded the wine barrel on the horse , and led it to the rich man\u2019s house . He arrived just as the sun was setting , and collected the gold brick as a reward for his efforts . The Identical Twins Once there were identical twins who were continually playing pranks on their family , friends , and teachers . The annual school picnic was al - ways a big event for the twins . There were races and other athletic events in which the twins won lots of prizes . One year a new student arrived who was a star runner . The twins wanted to win the main event : the 2 - mile race through the woods behind the school . So they secretly devised a plan which would enable them to outdo the newcomer . The day of the race arrived . Each runner was to pick his own path through the woods to a clearing , where a teacher stood posted to deter - mine the winner . One twin entered the race , while the other excused himself on the grounds that he had hurt his leg in an earlier broadjumping event . The race began and the students rushed into the woods . 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Psychological Review , 1977 , 84 , 353 - 378 . ( b ) Thorndyke , P . W . Cognitive structures in comprehension and memory of narrative dis - course . Cognitive Psychology , 1977 , 9 , 77 - 110 . Winston , P . H . Learning structural descriptions from examples . In P . H . Winston ( Ed . ) , The psychology of computer vision . New York : McGraw - Hill , 1975 . REFERENCE NOTE 1 . Gentner , D . The role of analogical models in learning scientific topics . Technical re - port , Bolt , Beranek and Newman , Inc . , 1979 . ( Accepted October 2 , 1979 )", + "boudreauFieldExperimentSearch2017": "The Review of Economics and Statistics V OL . XCIX O CTOBER 2017 N UMBER 4 A FIELD EXPERIMENT ON SEARCH COSTS AND THE FORMATION OF SCIENTIFIC COLLABORATIONS Kevin J . Boudreau , Tom Brady , Ina Ganguli , Patrick Gaule , Eva Guinan , Anthony Hollenberg , and Karim R . Lakhani * Abstract\u2014 We present the results of a \ufb01eld experiment conducted at Har - vard Medical School to understand the extent to which search costs affect matching among scienti\ufb01c collaborators . We generated exogenous varia - tion in search costs for pairs of potential collaborators by randomly assigning individuals to 90 - minute structured information - sharing ses - sions as part of a grant funding opportunity . We estimate that the treat - ment increases the probability of grant co - application of a given pair of researchers by 75 % . The \ufb01ndings suggest that matching between scien - tists is subject to considerable friction , even in the case of geographically proximate scientists working in the same institutional context . I . Introduction T HE primary unit of scienti\ufb01c knowledge production has become the team or collaboration rather than the lone scientist ( Jones , 2009 ) . Indeed , teams are not only growing in frequency , but also in size and impact relative to single authors ( Wuchty , Jones , & Uzzi , 2007 ) . Unlike set - tings inside \ufb01rms , where executives and managers play a central role in organizing and forming teams ( Lazear & Shaw , 2007 ) , academic scientists have greater freedom and autonomy in selecting their collaborators and their topics of inquiry ( Stephan 2012 ) . Although there is a growing body of research on the productivity and outcomes of scienti\ufb01c teams once formed ( e . g . , Adams et al . , 2005 ; Wuchty , Jones , & Uzzi , 2007 ; Agrawal , Goldfarb , & Teodoridis , 2016 ) , we know relatively little about the largely decentra - lized process by which scienti\ufb01c teams come into existence ( Stephan , 2012 ) . In this paper , we investigate the role of one particular mechanism , search costs and frictions , on these matching outcomes . The role of search costs and resulting frictions in the for - mation of scienti\ufb01c collaborations is not well understood . On the one hand , the growing prominence of teams and fall - ing communications and collaboration costs in science ( Agrawal & Goldfarb , 2008 ; Ding et al . , 2010 ) might sug - gest forces favorable to novel team formation . On the other hand , geography and distance are regularly documented to play a role in shaping collaborations , even today ( e . g . , Rosenthal & Strange , 2001 ; Glaeser , 2010 , Catalini , 2016 ) , and rather than continually forming novel collaborations , scientists most often work with partners in the same institu - tion , in a similar knowledge domain , and within preexisting social networks ( Baccara & Yariv , 2013 ; Freeman , Ganguli , & Murciano - Goroff , 2015 ; Freeman & Huang , 2014 ; Fafchamps , Goyal , & Van der Leij , 2010 ; Azoulay , Liu , & Stuart , 2009 ) . Moreover , past collaborations remain an important predictor of future ones . Although these patterns might be explained by any number of factors , they raise the question of whether search costs play a \ufb01rst - order role in shaping the organization of scientists into teams . The high information requirements for forming matches suggest that search frictions may be an important considera - tion . A large number of factors can play a role in decisions to collaborate , and these factors may be nuanced or dif\ufb01cult to observe . This includes factors such as the complementar - ity of skills of prospective partners , current research interests and priorities , access to broader sets of relevant resources ( funding , equipment , research personnel ) , timing and sche - duling constraints , and personal chemistry and disposition Received for publication March 11 , 2016 . Revision accepted for publi - cation December 2 , 2016 . Editor : Brigitte C . Madrian . * Boudreau : Northeastern University ; Brady : Massachusetts General Hospital and Harvard Medical School ; Ganguli : University of Massachu - setts Amherst ; Gaule : CERGE - EI , a joint workplace of Charles University and the Economics Institute of the Academy of Sciences of the Czech Republic ; Guinan : Dana - Farber Cancer Institute and Harvard Medical School ; Hollenberg : Beth Israel Deaconess Medical Center and Harvard Medical School ; Lakhani : Harvard Business School and NBER . K . J . B . , I . G . , P . G . , E . G . , and K . R . L . designed , developed , and executed the experiment and contributed to the manuscript . I . G . and P . G . con - ducted data analysis . T . B . and A . H . contributed to the experimental devel - opment and execution . We appreciate helpful comments from Pierre Azoulay , Marcel Fafchamps , Lee Fleming , Ben Golub , Shane Greenstein , Ben Jones , Nicola Lacetera , Josh Lerner , Paula Stephan , and Scott Stern , and seminar participants at the NBER Summer Institute Innovation Meet - ings , the NBER Productivity Lunch , the Georgia Tech REER Conference , MIT Sloan School of Management , Northwestern University , University of California , Berkeley , University of Rochester , SITE Stockholm School of Economics , CIRCLE Lund University , Graduate Institute Geneva , and Universidad Carlos III de Madrid . This work was conducted with support from Harvard Catalyst / The Harvard Clinical and Translational Science Center ( National Center for Advancing Translational Sciences , National Institutes of Health Awards UL1TR001102 , UL1TR000170 , and UL1RR025758 - 02S4 ) , NASA Tournament Lab at Harvard University , Harvard Business School Division of Research and Faculty Development , and \ufb01nancial contributions from Harvard University and its af\ufb01liated aca - demic health care centers . We also thank Harvard Catalyst for support and cooperation in implementing the experiment , particularly Lee Nadler , William Chin , Laura Weisel , and David Frank . Amy Webber and Wei Zhong provided valuable assistance throughout the implementation pro - cess . Michael Menietti and Christoph Riedl assisted with execution of the experiment . The Harvard Innovation Laboratory generously provided us with meeting space for the experiment . A supplemental appendix is available online at http : / / www . mitpress journals . org / doi / suppl / 10 . 1162 / REST _ a _ 00676 . The Review of Economics and Statistics , October 2017 , 99 ( 4 ) : 565 \u2013 576 (cid:2) 2017 by the President and Fellows of Harvard College and the Massachusetts Institute of Technology doi : 10 . 1162 / REST _ a _ 00676 ( Stephan , 2012 ) . If acquiring and evaluating this information is costly , signi\ufb01cant search frictions will appear , as has been found in other matching markets ( Mortensen & Pissarides , 1999 ) . Observed patterns of collaboration might then be interpreted as re\ufb02ecting limited information in decision making\u2014and therefore may constitute a suboptimal alloca - tion of human resources . To understand whether and to what extent search costs can affect the formation of collaborations among research collaborators , we carried out a \ufb01eld experiment with the goal of introducing exogenous variation in the informa - tion available to research scientists concerning potential collaborators . Our research team worked closely with Harvard Medical School\u2019s ( HMS ) clinical and administra - tive executives to modify and redesign existing internal grant processes so that causal inferences could be drawn in the context of an $ 800 , 000 grant opportunity for re - searchers at Harvard University and the HMS system of hospitals and research centers to encourage the develop - ment of clinical applications of advanced medical imaging technologies . The \ufb01eld experiment involved designing a research sym - posium ( repeated on three consecutive nights ) that was part of the grant process , where investigators were to get details about the grant rules and administration , learn about advanced technologies underlying the grant , and meet other researchers through structured information - sharing ses - sions . Participation in one of the symposia ( and only one ) was mandatory for submitting a grant application , which was due four weeks after the symposia . Each symposium consisted of a 30 - minute general introduction followed by 90 minutes of information sharing in independent and phy - sically separated breakout rooms . Breakout rooms facili - tated face - to - face interactions by having half of the researchers circulate about the room while the other half \u2018\u2018broadcast\u2019\u2019 their research ideas in a standardized poster format . We reduced the cost of initial face - to - face interac - tions for random subsets of scientists by randomly assigning the roughly 400 researchers who took part to independent breakout rooms . Therefore , we can evaluate the effect of the treatment by simply comparing the likelihood of colla - boration for pairs of researchers assigned to the same room ( treatment ) with the likelihood of pairs assigned to different rooms ( control ) . It is important to note that estimates of search costs in this context might be interpreted as occurring under best - case conditions . We study prospective collaborators operat - ing within a shared institutional context , with potential funding availability , within the same geographic area , and in a context in which information systems and tools facili - tate the search for prospective collaborators . Yet our results suggest that matching between scientists is subject to considerable frictions even in this best - case context . We estimate that assignment to the same breakout room increased the probability of forming a collaboration by 75 % , increasing the probability from 0 . 16 % in the control group to 0 . 28 % in the treatment . We estimate the effect to be signi\ufb01cant at the 5 % or 10 % level , depending upon model speci\ufb01cation . ( The 95 % con\ufb01dence interval around the point estimate ranges from (cid:2) 4 % to (cid:2) 112 % . ) To put this into perspective , the point estimate of the treatment effect is about one - third of the effect of working in the same hospital or of performing research in the same clinical area . This is a substantial effect for what is arguably a relatively small ( 90 - minute information sharing ) treatment . This main \ufb01nding is consistent with the view that large search costs and frictions play a \ufb01rst - order role in shaping the process of searching for collaborators and suggests the important function of information - rich face - to - face encoun - ters in catalyzing collaborations . Consistent with the inter - pretation of a signi\ufb01cant effect of search costs , the treatment effect was especially strong for pairs of researchers working in the same clinical area , where presumably search costs might be construed as lower given similar backgrounds and training . The \ufb01ndings therefore suggest the possibility that current observed patterns of collaborations in academic science are perhaps highly constrained by the availability of information and search costs . This is plausibly an important source of inef\ufb01ciency . However , we cannot observe impli - cations of this inef\ufb01ciency within this analysis . The \ufb01nding of the \ufb01rst - order role played by search costs also offers one plausible explanation for the prevalence of homophily ( McPherson , Smith - Lovin , & Cook 2001 ; Currarini , Jackson , & Pin 2009 ) in forming collaborations , where like scientists tend to coauthor , and repeatedly , as both tendencies may economize on search costs . The \ufb01nd - ings also imply potentially important differences between the formation of collaborations versus the execution of dis - tributed collaborations . The formation and execution of col - laborations may be considered as representing altogether different kinds of coordination problems\u2014one of matching and the other of joint production . Whereas evidence sug - gests research collaborations may be able to be carried out at a distance through decreased communication and travel costs and increasingly sophisticated collaboration platforms ( see Agrawal & Goldfarb 2008 ; Jones , Wuchty , & Uzzi 2008 ; Adams et al . , 2005 ; Catalini , Fons - Rosen , & Gaule 2016 ) , the process of forming collaborations may still be especially highly in\ufb02uenced and informed by information - rich , interpersonal interactions . The paper proceeds as follows . We \ufb01rst describe our experimental design , including details of the grant program and research symposia in section II . In section III , we describe the data . The empirical strategy and results follow in sections IV and V , respectively . Section VI concludes . II . The Field Experiment A . Harvard Medical School and Its Af\ufb01liated Hospitals Our \ufb01eld experiment involved faculty and researchers from Harvard University and its af\ufb01liated hospitals and 566 THE REVIEW OF ECONOMICS AND STATISTICS institutions . Harvard Medical School and its seventeen af\ufb01liated hospitals and research institutes ( including Massa - chusetts General Hospital , Children\u2019s Hospital Boston , Brigham and Women\u2019s Hospital , Beth Israel Deaconess Medical Center , and the Dana - Farber Cancer Institute ) are a major force in biomedical research . Collectively , they employ more than 11 , 000 faculty and receive in excess of $ 1 . 5 billion in annual funding from the U . S . National Insti - tutes of Health ( NIH ) . Harvard researchers account for around 5 % of scienti\ufb01c articles published in the top four medical journals , a larger share than Germany or Canada as a whole . 1 Fifteen researchers have shared in nine Nobel prizes awarded for work done while at Harvard Medical School . While our experiment is set entirely within the Harvard University system , in fact its researchers work in distinct organizations and research centers . The Harvard - af\ufb01liated hospitals are separately owned and managed and appear as separate entities in hospital rankings and lists of NIH grant recipients . Four of the \ufb01ve largest hospitals are located in the Longwood Medical Area campus in Boston , while Mas - sachusetts General Hospital has its own campus about 3 miles away ( and approximately 20 minutes by institutional shuttle bus ) . B . Harvard Catalyst and Advanced Imaging Closing the gap between research \ufb01ndings and clinical applications ( \u2018\u2018bench to bedside\u2019\u2019 ) is a major priority for the NIH . This has resulted in the establishment of a new insti - tute , National Center for Advancing Translational Sciences , that provides signi\ufb01cant research funding to universities and hospitals that undertake collaborative translational activities to accelerate treatment development . As part of Harvard\u2019s efforts to promote clinical and translational research , the Harvard Clinical and Translational Center , Harvard Catalyst , provides seed funding in the form of pilot grants to support nascent research efforts . These pilot grants are awarded competitively to faculty within Harvard Uni - versity . They emphasize early - stage research with the potential to improve human health . Pilot grant funding enables researchers to generate the preliminary data that are essential for larger grant applications to the NIH . Our \ufb01eld experiment was layered onto a Harvard Catalyst pilot grant program . This particular grant opportunity , which offered $ 50 , 000 per award , was centered on proposals to devise or improve methods for using advanced medical ima - ging technologies\u2014speci\ufb01cally , physiological magnetic resonance ( MR ) , positron emission tomography ( PET ) , and optical imaging\u2014to address unmet clinical needs . A major challenge in the \ufb01eld of advanced imaging is that progress requires both expertise in the latest imaging tools and tech - nologies and a deep understanding of the health problems to which they could be applied , with these different types of knowledge typically being held by people with different dis - ciplinary backgrounds . Thus , advanced imaging is an arche - typical example of a problem often found in modern science where advancing the knowledge frontier requires combining knowledge embodied in different individuals ( Jones , 2009 ) . We worked in close collaboration with HMS administra - tors and executives to redesign their pilot grant process so that we could obtain causal inferences about the role of search costs in \ufb01nding collaborators . While the grant process was primarily focused on identifying and funding promising early - stage translational research in the \ufb01eld of advanced imaging , Harvard Catalyst leaders also perceived a need for familiarizing clinicians with recent developments in advanced imaging and for Harvard - wide community build - ing among researchers . This provided us with the opportu - nity to create a new interactive research symposium where we could exogenously shift search costs for certain pairs of individuals by building in randomized face - to - face interac - tions . Hence we modi\ufb01ed the Harvard Catalyst grant process by requiring potential applicants to attend an interactive research symposium that would be a forum to learn about new technologies , understand the grant process , and exchange ideas among fellow researchers across Harvard . This was the \ufb01rst time such an interactive Harvard - wide symposium on a new research grant opportunity was offered . In November 2011 , all Harvard University life sciences faculty and researchers were invited to participate in a unique funding opportunity centered on advanced imaging technologies using a directed e - mail campaign , outreach to departmental clinical and research directors , and marketing messages on various internal websites and through posters across facilities . Up to $ 800 , 000 was available to support \ufb01fteen pilot grants . There was the additional potential for researchers to apply for several concept development prizes of $ 2 , 000 each . The concept prizes were meant to stimulate innovative thinking and future investigation in areas in which imaging had not been previously considered as an intervention and did not require any implementation plan . In the \ufb01rst stage , investigators who were interested in applying for the grants were asked to submit a statement of interest in which they brie\ufb02y described a speci\ufb01c medical problem that advanced imaging techniques could poten - tially address . Basic biographical information ( e . g . , degree , institution , department appointment ) was collected at this stage . Information distributed about the funding opportunity speci\ufb01ed that eligibility to submit a \ufb01nal application was conditional on attending an advanced imaging symposium on one of three preannounced dates . Applicants could indi - cate at this stage if there were any dates during which they could not attend a symposium . It was also communicated to applicants that the symposia would be studied by Harvard Catalyst to develop better insights about scienti\ufb01c team for - mation and that data on interaction patterns among indivi - duals would be collected . 1 Journals included are the New England Journal of Medicine , Journal of the American Medical Association ( JAMA ) , Nature Medicine , and Lan - cet . Authors\u2019 calculations based on research articles published during from 2000 to 2009 . Fractional counting was used when coauthors belonged to different institutions . 567 SEARCH COSTS AND THE FORMATION OF SCIENTIFIC COLLABORATIONS C . Randomization and the Advanced Imaging Symposium The initial call generated 471 statement of interest appli - cations , of which 435 applicants were invited to attend an advanced imaging symposium and thus proceed in the grant application process . 2 Forty - one applicants ( 9 . 4 % ) failed to RSVP or otherwise show up at the event . 3 Invitations were also extended to several individuals with world - class exper - tise in advanced imaging , bringing the total number of par - ticipants to 402 . The symposium was structured so that participants would come to the event prepared to discuss their idea with other participants in small breakout rooms of thirty to forty peo - ple . The treatment was intended to introduce exogenous var - iation in search costs to some pairs of participants at the symposium by having them be present in the same breakout rooms at the event . Each participant was randomly allocated to a breakout room in advance so that a random subset of all possible pairs among all participants would receive the treat - ment . Three symposia were held on sequential nights and were identically structured , with four breakout rooms per night . 4 We also randomized the participants across nights ; however , we respected the blackout dates for which appli - cants had previously indicated they would not be available . 5 The events were held January 31 , February 1 , and Febru - ary 2 , 2012 , at the Harvard Innovation Lab , located on Har - vard\u2019s Allston campus . The program began with a 30 - minute address by the program leadership describing the pilot grant opportunity and the agenda for the evening , including an introduction to advanced imaging tools and technologies . The breakout sessions then began in separate rooms . The number of participants in each room varied from 28 to 43 . The breakout room sessions were split into two periods of 45 minutes each , with a 15 - minute break in the middle dur - ing which all participants could mingle in a common space where refreshments were provided . The rooms provided a venue for presentation of the participants\u2019 ideas in the form of posters . Each poster followed a standard format describ - ing each participant\u2019s submitted idea from the statement of interest ( based on information they had provided prior to the event ) and was placed in the breakout room in advance . 6 The posters were intended to foster information sharing among participants and included the following details related to the statement of interest idea : ( a ) What is your question ? ( b ) Why does it matter ? and ( c ) What is needed for your research to succeed ? A 300 - character limit was imposed for each question . Posters were prepared in a stan - dard size and format by Harvard Catalyst , and each was placed on a separate whiteboard that allowed for the possibi - lity of visual explanations and note taking . Participants within each breakout room were randomly split into two groups . Participants from group 1 were asked to stand by their poster during the \ufb01rst period , while group 2 participants circulated . The two groups then switched roles during period two : group 1 participants circulated around the room , while group 2 participants stood near their own posters . The placement of each individual\u2019s poster in the room was also randomly determined in advance . D . Grant Applications Shortly after the symposia , all participants received via e - mail an invitation to submit applications for the pilot grants or concept awards by the deadline of March 8 , 2012 . At this time , they also received PDF booklets with the names , contact information , and posters of all researchers who participated over the three nights . 7 Note that as of 2008 , Harvard Catalyst had already deployed the Harvard Catalyst Pro\ufb01les website , an online , publicly accessible interactive database that includes contact information , cur - rent appointments , individual publication records , and other information for faculty and researchers across Harvard Medical School , and can be searched by name or keyword . Thus , much of the information contained in the PDF book - let could already easily be acquired online . However , the booklet also included information on current research inter - ests , related to the grant , that was less easily available ; the booklet may also have increased the salience of the publicly available information for the included individuals . Our intention was to provide identical information to all partici - pants apart from information acquired speci\ufb01cally in the breakout rooms at the symposia . Consistent with previous Harvard Catalyst pilot grant processes , applications had to include a principal investiga - tor and at least one co - investigator . Concept award applica - tions similarly had to include at least two individuals . Researchers with faculty appointments could apply as prin - cipal investigator on only one pilot grant but could apply as 2 Thirty - six statements of interests were outside the parameters of the request for applications in terms of area of inquiry ( e . g . , proposing ultra - sound or X - ray computed tomography [ CT ] techniques ) and the submit - ters were not invited to attend the symposium . 3 We do not include these individuals in the analysis . 4 The randomization was carried out by generating a unique random number for each participant , ranking the numbers , and then assigning par - ticipants to breakout rooms on nights based on their rank . We assigned 32 participants to the \ufb01rst three rooms each night , and the remainder ( 41 \u2013 48 ) to the last room , which was slightly larger . 5 Participants with blackout dates were a minority , but to guard against the potential endogeneity of selection into nights for this group , the analy - sis focuses on comparisons within nights . 6 Participants were provided with the following information in the e - mailed invitations to attend a symposium : \u2018\u2018You do not need to bring any particular items with you . We have a poster prepared with your submitted answers to the three questions based on your statement of interest . Posters will be displayed at the symposium to facilitate talking about your idea with other attendees . There will be no formal presentations of any kind . \u2019\u2019 7 The following information was included in e - mail communication with participants immediately following the event : \u2018\u2018Attached to this email is a PDF booklet with the names and contact information of all researchers who participated over the three nights and their posters . We hope this is of use in contacting individuals that you met during the eve - ning and in identifying additional potential collaborations and col - laborators . . . . As described at the symposium , your proposal or your col - laborators can be the same as suggested in your Statement of Interest or can be somewhat or entirely different . You can participate in multiple applications . \u2019\u2019 568 THE REVIEW OF ECONOMICS AND STATISTICS co - investigator on an unlimited number of additional appli - cations . Researchers without a faculty appointment could not be principal investigators on a pilot grant application , but they could be co - investigators on an unlimited number of applications . All attendees were eligible to apply for a concept award grant and could appear on an unlimited num - ber of applications . Finally , at least one applicant on any grant application had to have attended the symposium . The grant application did not need to be based on the initial statement of interest . Extra care was taken to ensure that the symposium pro - cess did not somehow prime participants to seek collabora - tions only in their breakout rooms . Participants were informed that the composition of their teams would not be communicated to reviewers and would not be considered as a criterion for awarding the grant . They were also told to remove any personal identifying information about the submission teams from their proposals ( including self - references and indications of special access to technologies ) . 8 This differed from the typically single - blinded process used in NIH and Catalyst grants , in case the identi\ufb01cation of sub - mission applicants might have an impact on collaboration choices . In the end , the majority of participants chose not to apply with other symposium participants : 66 % of the applications included only one symposium participant as a coapplicant . III . Data A . Sources and Construction Registration data . Faculty and researchers interested in taking part in the funding opportunity were asked to submit a short statement of interest describing in 250 words or less a speci\ufb01c medical problem that advanced imaging techni - ques could potentially address . Registration data also included basic biographical information ( rank , education history , hospital af\ufb01liation , department ) . Participants were also asked to identify themselves as primarily an imager or primarily a clinician . Clinical area and imaging modality were coded from the statement of interest documents . Publications . We matched participants to Harvard Cat - alyst Pro\ufb01les , an online , publicly accessible database that includes individual publication records and other informa - tion for faculty and researchers across Harvard Medical School . Grant applications . Our main outcome variable comes from the pilot grant and concept award applications . We received 224 applications for pilot grants or concept awards . 9 Of those , 148 included one symposium participant in the applicant list , 49 included two symposium participants , and 27 included more than two symposium participants . Constructing the pair - level data . Our analysis is con - ducted at the dyadic level . We constructed dyads by creat - ing every possible pairwise combination of researchers attending a symposium on the same night ( 26 , 604 dyads ) . We constructed our main outcome at the dyadic level , colla - boration , from grant applications . A collaboration was de\ufb01ned as any pairs of symposium participants appearing on the same application . In some parts of the analysis , we restricted our sample to the 52 pairs of scientists who attended on the same night and coapplied . We also con - structed a number of dyadic - level control variables from the registration and publication data , which are described in more detail in section IV . B . Summary Statistics and Randomization Check Table 1 provides individual - level summary statistics for symposium participants . 10 Of the 402 attendees , 29 % were women , 42 % identi\ufb01ed themselves as imagers , and 73 % held Harvard faculty appointments ( the others were post - doctoral fellows or clinical fellows ) . Over 80 % of attendees came from the four largest Harvard - af\ufb01liated hospitals : Massachusetts General Hospital ( MGH ) , Brigham and T ABLE 1 . \u2014S UMMARY S TATISTICS , A TTENDEES Sample Mean Female 0 . 29 Faculty member 0 . 73 Imager 0 . 42 Longwood 0 . 51 HospitalMassachusetts General Hospital 0 . 37 Brigham and Women\u2019s Hospital 0 . 19 Beth Israel Deaconess Medical Center 0 . 14 Children\u2019s Hospital Boston 0 . 13 Other 0 . 17 Clinical area ( SOI ) Neurology 0 . 25 Oncology 0 . 25 Neuropsychiatric 0 . 10 Cardiovascular 0 . 06 Gastroenterology 0 . 04 Transplantation 0 . 04 Ophthalmology 0 . 03 Other 0 . 23 Attended January 31 0 . 35 Attended February 1 0 . 32 Attended February 2 0 . 33 Observations 402 SeesectionIII inthe textfor adetaileddescriptionofthevariables . 8 The following directions to applicants were highlighted in the grant request for applications : \u2018\u2018As the initial review will be blinded in regard to the applicant ( s ) , do not refer to yourself , other participants or institu - tions by name ( e . g . , substitute \u2018our optical imaging experts , \u2019 \u2018our cardiol - ogy collaborators , \u2019 \u2018our laboratory\u2019 or \u2018the genomics core\u2019 for speci\ufb01c individuals or facilities ) . \u2019\u2019 9 Seventy - eight percent of applications were for pilot grants , and 22 % were for concept awards . 10 Across the three nights , 394 individuals were in attendance . How - ever , 5 people with special expertise in advanced imaging attended the event on more than one night ; we count them as different participants on each night , bringing the total number of participants to 402 . 569 SEARCH COSTS AND THE FORMATION OF SCIENTIFIC COLLABORATIONS Women\u2019s Hospital , Children\u2019s Hospital Boston , and the Beth Israel Deaconess Medical Center . The most prevalent clinical expertise areas were neurology ( 25 % ) , oncology ( 25 % ) , and neuropsychiatry ( 10 % ) . We can also compare the participants to the general population of researchers at Harvard Medical School . Appendix table A1 provides summary statistics on partici - pants and nonparticipants based on information in the Har - vard Catalyst Pro\ufb01les database . In terms of degree types , there was no signi\ufb01cant difference in the share of MDs among attendees and the overall HMS population , but there was a larger share of PhDs among attendees ( 49 % PhDs among attendees versus 38 % at HMS ) . We would expect a greater representation of PhDs at the event since it was part of a research grant opportunity , and academic PhDs are very often focused on research , while academic MDs have a larger array of potential roles . Attendees also had more prior publications on average ( approximately four publica - tions more than the typical HMS researcher ) . We also see some signi\ufb01cant differences in the distribution across ranks , with attendees more likely to be instructors and assistant or associate professors relative to the overall dis - tribution at HMS and less likely to be full professors and postdocs . Attendees were also more likely to come from MGH . One reason for this is that MGH houses a large advanced imaging center , the Martinos Center for Biome - dical Imaging , and the focus of the grant opportunity was advanced imaging . For the same reason , individuals from radiology departments were overrepresented among atten - dees . To verify that the randomization generated balance across covariates , we present summary statistics in table 2 for the pairs in our sample assigned to the same breakout room and those assigned to different breakout rooms . The unit of observation here is a dyad of researchers , and the sample is every possible pairwise combination of research - ers attending on the same night ( 26 , 604 dyads ) . Treated pairs and control pairs look very similar , with the exception of pairs of previous coauthors , pairs with both members from the same hospital , and pairs including one woman , which are statistically different across treated and control pairs . 11 In our regression analysis , we control for these cov - ariates . The last row of table 2 includes collaboration , our out - come variable . The incidence of collaboration is signi\ufb01 - cantly larger in the treated group , which we investigate in a regression framework in the next section . It is notable that the incidence of collaboration is less than 0 . 2 % in our sam - ple . While this may seem low , the likelihood that any two HMS faculty members will copublish in a given year is 0 . 06 % and , thus , of the same order of magnitude . 12 Viewed through the lens of all pair - wise combinations of scientists who could collaborate , collaboration is indeed a relatively rare event . Table 3 shows the characteristics of the subset of colla - borating dyads . Here we restrict the sample of every possi - ble pairwise combination of researchers attending on the same night ( 26 , 604 dyads ) to those who coapplied ( 52 dyads ) . Among attendees who attended on the same night but were not in the same breakout room , 33 pairs co - applied . Among pairs in the same room at the event , 19 pairs coapplied . 13 T - tests show that among the same - room collaborations , there was a higher incidence of pairs with one postdoc and of pairs researching the same clinical area . It is important to note that some of the within - room colla - borations would have occurred in the absence of any treat - ment effect . Extrapolating the across - room incidence rate ( 0 . 16 % ) to the number of within - room pairs ( 7 , 149 ) , we would expect eleven collaborations to have occurred within rooms in the absence of any treatment effect . T ABLE 2 . \u2014D YADS BY T REATMENT S TATUS Treatment : Control : Sample Means Same Room Different Room Difference One postdoc 0 . 404 0 . 396 (cid:2) 0 . 007 Both postdocs 0 . 072 0 . 075 0 . 003 One female 0 . 403 0 . 418 0 . 015 * Both female 0 . 085 0 . 082 (cid:2) 0 . 004 Same hospital 0 . 198 0 . 208 0 . 010 * Both Longwood 0 . 266 0 . 258 (cid:2) 0 . 010 * One imager \u00fe one clinician 0 . 492 0 . 489 (cid:2) 0 . 003 Both imagers 0 . 175 0 . 176 0 . 001 Same clinical area ( SOI ) 0 . 123 0 . 119 (cid:2) 0 . 004 Previous coauthor 0 . 001 0 . 002 0 . 001 * Observations 6 , 702 19 , 962 The unit of observation is a dyad of researchers . We construct dyads by creating every possible pairwise combination of researchers attending on the same night ( 26 , 604 dyads ) . The category Treatment : Same Room refers to participants in the same room at the event ; it was randomized across pairs of participants attending on the same night . Collaboration indicates whether the pair appeared on any common pilot grant or conceptawardapplications . See sectionIII foradetaileddescriptionofthevariables . Asterisksindicatethe results oftestsforequalityofmeans . * p < 0 . 10 , * * p < 0 . 05 , * * * p < 0 . 01 . 11 The relatively large difference between the percentage of pairs of previous coauthors across treatment and control groups can be explained by the very small number of pairs of previous coauthors in our sample ( 40 out of more than 20 , 000 ) . Thus , randomization could easily result in a dif - ferent incidence of pairs with coauthors across treatment and control groups , as it did in our case . 12 Authors\u2019 calculation based on publication data from Harvard Catalyst Pro\ufb01les . 13 The nineteen pairs who coapplied from the same room correspond to eighteen separate grant applications . 570 THE REVIEW OF ECONOMICS AND STATISTICS IV . Estimation Strategy A . Speci\ufb01cation We use the simplest possible estimation strategy to describe differences between treatment and control groups\u2014 and the effect of exogenous variation in search costs in our context . The approach of our statistical analysis is to study the incidence of collaborations among all possible pairs of participants attending on the same night within our experi - mental group of 402 individuals . This reduced - form approach suits our interest in studying the extent to which observed behaviors deviate from fully informed equilibrium out - comes . 14 This approach also allows us to deal with relatively small numbers of actual within - room collaborations in a most straightforward and conservative manner . Thus , the unit of analysis is the scientist pair , and the data set includes every possible pair of scientists across all nights . We use a linear probability model to describe how the incidence or probability of collaborations differs across treatment and control groups ( i . e . , those in the same versus different breakout rooms ) . Random assignment of pairs within the research design allows us to interpret differ - ences as causally related to exogenous variation in search costs . We are also able to regress the incidence of colla - borations on other covariates of researcher pairs to further describe associations with the incidence of collaborations . To measure whether treatment effects varied across sub - groups , we interacted Same Room indicator with pair - level variables . Thus , to estimate the impact of being in the same room at the event on the likelihood of collaboration between pairs , we ran linear regressions with the following speci\ufb01cation : Collaboration ij \u00bc a \u00fe b Same room ij \u00fe h Same room ij (cid:3) Distance ij \u00fe p Distance ij \u00fe d X ij \u00fe e ij ; ( 1 ) where the key explanatory variable associated with the treat - ment effect , Same Room ij , is an indicator variable that equals 1 if both researcher i and j were randomly assigned to the same breakout room at the symposium . 15 Collaboration ij is an indi - cator variable for whether i and j appeared on any common pilot grant or concept award applications . X ij is a vector of observable pair - level characteristics that can affect the likeli - hood of collaboration and includes measures of gender and professional rank . The vector Distance ij includes measures of differences in professional rank , as well as geographic , scien - ti\ufb01c , and past coauthoring , described below . The model also includes \ufb01xed effects for each night of the symposium . The estimation of dyadic regressions raises an inference problem : dyadic observations may not be independent of each other as the same individual appears in many dyads . To address this inference problem , Fafchamps and Gubert ( 2007 ) have developed a network inference method adapted from spatial econometrics that corrects dyadic correlation of errors and also possible heteroskedasticity . We estimate and report equation ( 1 ) using an OLS regression with grouped dyadic standard errors according to Fafchamps - Gubert . 16 T ABLE 3 . \u2014C OLLABORATING D YADS BY T REATMENT S TATUS Sample Means Collaborations within the Same Room CollaborationsacrossRooms Difference One postdoc 0 . 421 0 . 212 (cid:2) 0 . 209 Both postdocs 0 . 000 0 . 030 0 . 030 One female 0 . 474 0 . 303 (cid:2) 0 . 140 Both female 0 . 158 0 . 061 (cid:2) 0 . 097 Same hospital 0 . 579 0 . 636 0 . 057 Both Longwood 0 . 158 0 . 303 0 . 145 One imager \u00fe one clinician 0 . 474 0 . 485 0 . 011 Both imagers 0 . 316 0 . 394 0 . 078 Same clinical area ( SOI ) 0 . 579 0 . 273 (cid:2) 0 . 306 * * Previous coauthor 0 . 105 0 . 121 0 . 016 Observations 19 33 The unit of observation is a dyad of researchers . We construct dyads by creating every possible pairwise combination of researchers attending on the same night ( 26 , 604 dyads ) , but here we focus on researchers who attend on the same night and appeared on a common pilot grant or concept award application ( 52 dyads ) . See section III in the text for a detailed description of the variables . Asterisks indicate the r esults of t - testsforequality ofmeans . * p < 0 . 10 , * * p < 0 . 05 , * * * p < 0 . 01 . 14 Structural matching models that contemplate competitive equilibria in matching are an alternative approach to modeling the equilibrium for - mation of collaborations . However , pursuing such an approach requires we make structural assumptions regarding equilibrium search process and outcomes\u2014which goes against our interests in this study , given our inter - est in investigating frictions . Therefore , it is more appropriate in this instance to proceed with a reduced - form description of patterns to better describe any implications of search costs . Although this creates the possi - bility of downwardly biased estimates on the treatment effect , any such effect is likely to be vanishingly small : competition in matching is likely to have played only a small role , if much at all , as the absolute incidence of collaborations in these data is rather low and individuals were not lim - ited in the number of collaborations they could form . 15 There are several other ways to study and model search costs in this setting . We could , for example , study the effect of attending the sympo - sium on the same night . Furthermore , since participants\u2019 posters were also randomized within the breakout rooms , we could study if immediate neighbors in the breakout room at the event had an impact on collabora - tion . However , neither of these approaches had a signi\ufb01cant impact on our outcome of interest , grant coapplications . 16 We implement this using the ngreg ado \ufb01le available on Marcel Faf - champs\u2019s website : http : / / web . stanford . edu / (cid:4) fafchamp / resources . html . Note that we obtain very similar standard errors and con\ufb01dence intervals when using Eicker - White heteroskedasticity - robust standard errors instead . We do not cluster standard errors by night of attendance , since assignment to nights is itself random ( conditional on blackout dates for a minority of participants ) ( Cameron & Miller , 2015 ) . 571 SEARCH COSTS AND THE FORMATION OF SCIENTIFIC COLLABORATIONS B . Covariates Several additional covariates describing pairs are also included in the model . Inclusion of these covariates should not affect the point estimate of the treatment effect but should increase its precision and offer further opportunity for interpretation . Our vector of pair - level covariates , X ij , includes variables for gender and professional rank . Gender is captured by indicator variables Both female , One female , and Both male . Research indicates that women have a greater propensity to work with other women ( Boschini & Sjogren , 2007 ) and more generally have more limited aca - demic networks ( see Ding et al . , 2010 ) . For professional rank , we include indicators for One postoc in the pair and Both postdocs . Postdocs were eligible to apply for either the concept or pilot grants ; however , two postdocs could colla - borate on a pilot grant application only if a third team mem - ber with a faculty appointment assumed the role of princi - pal investigator . The vector Distance ij includes measures of differences in professional rank and geographic , scienti\ufb01c , and past coau - thoring . Given the potential relevance of these various forms of distance to search costs , coef\ufb01cients estimated on these variables provide at least some broad and rough means of judging the importance of any estimated treatment effect by direct comparison with coef\ufb01cients on these variables . Distances in professional range are measured with indi - cator variables corresponding to possible combinations of differences . In relation to geographic distance , we create an indicator for Same hospital , which indicates whether pair members\u2019 primary appointments are in the same Harvard - af\ufb01liated hospital or institute . We also create an indicator for Both Longwood , indicating that both members of the pair work on the same campus , 17 as the largest concentra - tion of researchers are located in hospitals and institutes either on the Longwood Medical Area ( LMA ) campus or at the MGH campus . The campuses are located approximately 3 miles apart ( with a travel time of about 20 minutes during normal traf\ufb01c ) . We also create a direct measure of geo - graphic distance by geocoding exact locations of of\ufb01ces and calculating pairwise distances in miles . In relation to scienti\ufb01c or intellectual distance , we create indicator variables for Both imagers , One imager \u00fe one clinician , and Both clinicians . We construct this variable using the information attendees themselves reported during the initial stage of the application process . We also con - structed indicator variables for Same clinical area and Same imaging modality ( physiological MR , PET , or optical ima - ging ) . These were coded from the statement of Interest documents submitted in the \ufb01rst stage of the application process . We also create measures of scienti\ufb01c distance using overlap in the Medical Subject Heading ( MeSH ) terms from each individual\u2019s publications and overlaps in the keywords of each individual\u2019s statement of interest . 18 A \ufb01nal measure of distances is whether the pair had pre - viously collaborated , indicator variable Previous coauthors . We also distinguish cases of one single past copublication with more than one past copublication with indicator variables . V . Analysis and Results A . Does Reducing Search Costs Increase the Propensity to Collaborate ? We \ufb01rst analyze whether our 90 - minute breakout treat - ment had an effect on the incidence of collaborations and the magnitude of any such effects . OLS estimates with robust standard errors are presented in table 4 . ( The same results are presented using probit estimation in table A2 . ) Column 1 shows the basic result , regressing the incidence of collabora - tions on our treatment effect indicator and a constant . The baseline probability of collaborations is captured by the con - stant coef\ufb01cient of 0 . 0016 or 0 . 16 % . The point estimate shows that the treatment increases the likelihood of collabor - ating on an application by approximately 75 % ( increasing T ABLE 4 . \u2014M AIN E FFECT OF T REATMENT ON C OLLABORATION DV \u00bc Collaboration ( 1 ) ( 2 ) ( 3 ) Same Room 0 . 0012 * 0 . 0012 * 0 . 0014 * ( 0 . 0007 ) ( 0 . 0007 ) ( 0 . 0007 ) One postdoc (cid:2) 0 . 0008 ( 0 . 0005 ) Both postdocs (cid:2) 0 . 0014 * * ( 0 . 0007 ) One is female 0 . 0002 ( 0 . 0005 ) Both are female 0 . 0010 ( 0 . 0011 ) Same hospital 0 . 0042 * * * ( 0 . 0010 ) Both Longwood (cid:2) 0 . 0001 ( 0 . 0007 ) One imager \u00fe one clinician 0 . 0008 * ( 0 . 0005 ) Both imagers 0 . 0025 * * ( 0 . 0010 ) Same clinical area ( SOI ) 0 . 0042 * * * ( 0 . 0014 ) Previous coauthor 0 . 1176 * * ( 0 . 0468 ) Constant 0 . 0016 * * * 0 . 0012 * * * (cid:2) 0 . 0010 ( 0 . 0003 ) ( 0 . 0004 ) ( 0 . 0007 ) Night \ufb01xed effects No Yes Yes R 2 0 . 000 0 . 000 0 . 017 Number of observations 26 , 664 26 , 664 26 , 664 The unit of analysis is a dyad of researchers . We construct dyads by creating every possible pairwise combination of researchers attending on the same night ( 26 , 604dyads ) . Thedependent variable is Colla - boration , an indicator variable for whether the pair appeared on any common pilot grant or concept award applications . The main variable of interest is Same room , which was randomized across pairs attending on the same night . All estimation is by OLS . Grouped dyadic standard errors in parentheses . * p < 0 . 10 , * * p < 0 . 05 , * * * p < 0 . 01 . 17 The LMA includes eight hospitals or institutes in our sample , and the MGH campus includes two hospitals or institutes . The other hospitals or institutes in the sample are considered to be individual campuses . 18 We include these other measures of scienti\ufb01c distance in our regres - sion analysis , but since these measures rely on prior publications ( and some individuals in the sample have no or few publications ) , our pre - ferred measure is self - reported clinical area . 572 THE REVIEW OF ECONOMICS AND STATISTICS the likelihood of a pair collaborating from 0 . 16 % to 0 . 28 % ) . 19 The estimate is signi\ufb01cant at the 10 % level . The advanced imaging symposia were held on three differ - ent nights . We thus include \ufb01xed effects for the night of the event ( January 31 , February 1 , or February 2 ) in column 2 to account for any differences across nights . The night \ufb01xed effects are not signi\ufb01cant , and their inclusion has very little impact on the same room coef\ufb01cient ( or its standard error ) . In column 3 we introduce pair - level variables to account for gender composition , differences in rank , as well as geo - graphic , scienti\ufb01c , and past coauthoring distance . The ran - dom assignment ensures that being in the same room is orthogonal asymptotically to any observable or unobserva - ble pair characteristic . 20 Correspondingly , introducing co - variates does not statistically change the estimated treatment effect . The standard error is not palpably changed , but the signi\ufb01cance marginally increases on account of a small increase in the point estimate . The point estimate for the effect of being in the same room increases slightly from 0 . 0012 to 0 . 0014 . ( Note that we also include additional con - trols in other speci\ufb01cations , including dummies for whether a pair was in the same group within a breakout room\u2014 group 1 or 2\u2014and their proximity to one another in the room\u2014whether the pair had posters next to each other\u2014 but the results do not change . ) We conclude this section by brie\ufb02y discussing the sign of the point estimates of the control variables in table 4 , column 3 . Working in the same clinical area , being af\ufb01liated with the same hospital , and being a coauthor in the past are positively and signi\ufb01cantly correlated with collaboration . Consistent with the related literature , these results suggest that geo - graphic , scienti\ufb01c , and past coauthoring are all positively related to collaboration . Pairs of one imager and one clinician were signi\ufb01cantly more likely to collaborate than pairs of clinicians only , but collaborations were even more likely to form when both members of the pair were imagers . Colla - boration was signi\ufb01cantly less likely to occur between pairs consisting of two postdocs , which is possibly explained by the fact that two postdocs could collaborate on a pilot grant appli - cation only if a third team member with a faculty appointment assumed the role of principal investigator . Overall , the single largest correlate is whether scientists had previously coau - thored a publication . This association is at least an order of magnitude larger than for each of the other correlates . Therefore , our estimated treatment effect of being in the same breakout room on collaboration is over 30 % of the effect of being from the same hospital ( 0 . 0044 ) and of researching the same clinical area ( 0 . 0040 ) . Relative to the single most important correlate , past coauthorship , it is only about 1 % of the magnitude ( 0 . 1126 ) . ( The probit estimates in table A2 of the appendix show similar results . ) B . For Which Pairs Does Reducing Search Costs Have the Greatest Effect ? Next , we investigate whether the treatment had an effect for different types of pairs . Unlike earlier estimates of cor - relations with covariates , interaction terms can be inter - preted causally . Probit estimates are reported in table A3 of the appendix . We introduce the interactions between co - variates with the treatment effect individually in columns 1 to 7 and then simultaneously in column 8 of table 5 . In introducing each of the interaction terms , we also of course reintroduce the direct effect of the covariate in the regres - sions ; however , our focus here is on interactions terms . In the results of columns 1 through 7 , the only interaction term found to be signi\ufb01cant is that in column 6 , which reports a positive and signi\ufb01cant coef\ufb01cient on the interac - tion between the treatment effect and the indicator for researchers being in the same clinical area . The coef\ufb01cient on the direct treatment effect term Same Room becomes sta - tistically indistinguishable from 0 when introducing this interaction . Results in column 8 corroborate this result , as introducing all covariates and all interaction terms at once in the model produces an almost identical estimate on this interaction term . In column 8 , which includes all interac - tions , the treatment increases the likelihood of collaborating for pairs researching the same clinical area from 0 . 35 % to 0 . 94 % relative to pairs researching different clinical areas . 21 There are several possible explanations for the effect , but it suggests that researchers had limited information about these potential collaborations\u2014about either the potential interest of other researchers in certain types of projects or the potential bene\ufb01ts of collaborating with these indivi - duals . If they did , the information provided at the event should not have any independent effect for these pairs . It may also be the case that discussions were more bene\ufb01cial for clinically proximate pairs because they shared common ground , allowing them to convert their discussions into col - laborations . Another possible explanation is that it is quite costly to switch clinical areas ( specialization and training in medicine occurs on the basis of clinical areas\u2014e . g . , dermatology , neurology , oncology ) , and therefore , even if researchers talked to people with interesting ideas in other clinical areas at the event , the bene\ufb01ts to collaboration were highest for those in the same clinical area . We fail to detect evidence of the signi\ufb01cance of other inter - actions . Our results on the interaction between being in the same room at the event and other pair characteristics are not 19 However , our point estimates regarding the magnitude of the effect are imprecise . The con\ufb01dence interval ranges from 4 % to 112 % . 20 Being in the same room is orthogonal to pair characteristics ex ante . However , ex post , being in the same room at the event could be correlated with pair characteristics by chance . While this is much less of a concern than in observational data ( Leamer , 2010 ) , it is nonetheless useful to con - trol for relevant , observable pair characteristics to address the possibility that the effect of being in the same room is affected by differences in observable pair characteristics . Introducing controls has the added bene\ufb01t of improving the precision of the Same room estimate by reducing the unexplained variance . 21 The sample average incidence of collaboration for pairs working in the same clinical area but not in the same room is 0 . 35 . 573 SEARCH COSTS AND THE FORMATION OF SCIENTIFIC COLLABORATIONS conclusive . While the point estimates for some interactions are positive , they are not signi\ufb01cant up to the 10 % level . 22 We also investigated various alternative speci\ufb01cations such as including more \ufb01nely grained measures of geo - graphic distance , scienti\ufb01c distance , or past coauthoring , 23 as well as controlling more \ufb02exibly for ranks and rank dif - ferences between pair members . We included dummies for whether a pair was in the same group ( 1 or 2 ) , proximity in the room ( whether the pair had posters next to each other ) , and the number of total individuals in the room ( to test whether density mattered ) , but the results were not signi\ufb01 - cant , and the same clinical area result is consistent and stable across these speci\ufb01cations . VI . Summary and Conclusions Teams are a primary unit of knowledge production , and scientists in large part self - organize into research teams . Yet we know little regarding the matching of scientists into teams . In this paper , we present the results of a \ufb01eld experi - ment to investigate the role of search costs in the formation of scienti\ufb01c teams by comparing the incidence of collabora - T ABLE 5 . \u2014T REATMENT AND I NTERACTIONS WITH M EASURES OF D ISTANCE DV \u00bc Collaboration ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) ( 7 ) ( 8 ) Same Room 0 . 0008 0 . 0000 0 . 0008 0 . 0018 * 0 . 0012 (cid:2) 0 . 0000 0 . 0011 (cid:2) 0 . 0019 ( 0 . 0009 ) ( 0 . 0009 ) ( 0 . 0006 ) ( 0 . 0009 ) ( 0 . 0009 ) ( 0 . 0006 ) ( 0 . 0007 ) ( 0 . 0014 ) One postdoc (cid:2) 0 . 0016 * * * (cid:2) 0 . 0012 * * ( 0 . 0006 ) * ( 0 . 0005 ) Same room (cid:3) One postdoc 0 . 0015 0 . 0019 ( 0 . 0013 ) ( 0 . 0013 ) Both postdocs (cid:2) 0 . 0016 * (cid:2) 0 . 0012 ( 0 . 0008 ) ( 0 . 0008 ) Same room (cid:3) Both postdocs (cid:2) 0 . 0014 (cid:2) 0 . 0011 ( 0 . 0011 ) ( 0 . 0011 ) One is female (cid:2) 0 . 0008 (cid:2) 0 . 0004 ( 0 . 0006 ) ( 0 . 0006 ) Same room (cid:3) One female 0 . 0021 0 . 0024 ( 0 . 0016 ) ( 0 . 0015 ) Both are female (cid:2) 0 . 0007 (cid:2) 0 . 0000 ( 0 . 0013 ) ( 0 . 0013 ) Same room (cid:3) Both female 0 . 0040 0 . 0035 ( 0 . 0025 ) ( 0 . 0025 ) Same hospital 0 . 0044 * * * 0 . 0037 * * * ( 0 . 0014 ) ( 0 . 0013 ) Same room (cid:3) Same hospital 0 . 0024 0 . 0023 ( 0 . 0026 ) ( 0 . 0025 ) Both Longwood 0 . 0005 0 . 0004 ( 0 . 0007 ) ( 0 . 0007 ) Same room (cid:3) Both Longwood (cid:2) 0 . 0020 (cid:2) 0 . 0018 ( 0 . 0014 ) ( 0 . 0015 ) One imager \u00fe one clinician 0 . 0009 \u00fe 0 . 0008 * ( 0 . 0005 ) ( 0 . 0004 ) Same room (cid:3) One imager (cid:2) 0 . 0000 (cid:2) 0 . 0000 ( 0 . 0014 ) ( 0 . 0012 ) Both imagers 0 . 0031 * * * 0 . 0025 * ( 0 . 0011 ) ( 0 . 0011 ) Same room (cid:3) Both imager 0 . 0002 (cid:2) 0 . 0002 ( 0 . 0026 ) ( 0 . 0025 ) Same clinical area ( SOI ) 0 . 0025 * 0 . 0018 ( 0 . 0011 ) ( 0 . 0012 ) Same room (cid:3) Same clin area 0 . 0095 * * 0 . 0094 * * ( 0 . 0045 ) ( 0 . 0042 ) Previous coauthor 0 . 0917 * 0 . 0889 * * ( 0 . 0450 ) ( 0 . 0442 ) Same room (cid:3) Prev coauthor 0 . 2389 0 . 2363 ( 0 . 1981 ) ( 0 . 1967 ) Night \ufb01xed effects Yes Yes Yes Yes Yes Yes Yes Yes R 2 0 . 001 0 . 000 0 . 003 0 . 000 0 . 001 0 . 002 0 . 019 0 . 024 Number of observations 26 , 664 26 , 664 26 , 664 26 , 664 26 , 664 26 , 664 26 , 664 26 , 664 The unit of analysis is a dyad of researchers . We construct dyads by creating every possible pairwise combination of researchers attending on the same night ( 26 , 604 dyads ) . The dependent variable is Collabora - tion , an indicator variable for whether the pair appeared on any common pilot grant or concept award applications . The main variable of interest is Same room , which was randomized across pairs attending on the samenight . Allestimationis byOLS . Groupeddyadicstandarderrorsin parentheses . * p < 0 . 10 , * * p < 0 . 05 , * * * p < 0 . 01 . 22 The interaction between being in the same room and pairs with one woman are marginally signi\ufb01cant with p - values of 0 . 093 in the probit speci\ufb01cation and 0 . 133 in the OLS speci\ufb01cation . A differential effect for pairs with a woman would be consistent with the \ufb01ndings of Ding et al . ( 2010 ) , who show that the introduction of information technology bene - \ufb01ted collaborations more for female scientists than for male scientists , since women tend to have less diverse networks , lower job mobility , and more constraints to attending conferences and seminars . These factors would similarly lead women to bene\ufb01t more from mixing with other researchers at the event in terms of \ufb01nding coauthors . 23 We considered , for instance , whether pair members investigated the same imaging modality , the extent of the overlap of scienti\ufb01c keywords in previous publications , and whether pair members shared a common coauthor . 574 THE REVIEW OF ECONOMICS AND STATISTICS tions among researchers who participated in the same breakout rooms within an interactive research symposium as part of a grant proposal process versus those who were assigned to different breakout rooms . We thus randomly varied search costs for a set of prospective collaborators , observing both the collaborations that did form along with those that did not . We \ufb01nd that the small , focused treatment signi\ufb01cantly increased the incidence of collaboration on subsequent grant proposals by 75 % in relation to the baseline probabil - ity of collaboration between pairs of researchers ( increasing from 0 . 16 % in the control group to 0 . 28 % in the treated group ) . The magnitude of this effect is equivalent to roughly a third of the boost in the probability of collabora - tion associated with working in the same hospital or , alter - natively , the probability of working in the same clinical area . In this regard , the point estimate can be viewed as rather large , despite the relatively small and focused nature of the treatment : a 90 - minute breakout session . It is in fact notable that we \ufb01nd any effect at all , let alone such a large effect in the context of scientists who are already geograph - ically proximate and working within a common institutional context , where online resources and information systems already exist to facilitate collaboration . We interpret these large effects as showing that even when working in relatively favorable conditions , search costs and frictions continue to powerfully shape ( and limit ) the formation of collaborations between scientists . Whereas a great deal of collaborative work might potentially be per - formed at a distance , the formation of collaborations appears to be highly sensitive to information - rich face - to - face interactions . In this sense , the question of the \u2018\u2018death of distance\u2019\u2019 and the role of collocation and information tech - nology , for example , might be reconsidered at least in rela - tion to questions of forming collaborations . The \ufb01nding is consistent with the complex and manifold set of variables on which collaboration decisions might be based and the effectiveness of face - to - face interactions in rapidly conveying information through high - frequency rapid feedback and visual and nonverbal cues ( Gaspar & Glaeser , 1998 ; Storper & Venables , 2004 ) . For example , given our existing communications technologies , it may remain dif\ufb01cult to wholly codify current research interests , complementarity in knowledge and skills , access to resources , and timing and scheduling constraints , let alone questions of personal chemistry and disposition or subtler questions of one\u2019s intellectual outlook . The result is also consistent with face - to - face interactions potentially trigger - ing or credibly signaling commitments , establishing trust and personal chemistry ( Azoulay et al . , 2009 ) . Further consistent with the role of search costs in our results , the treatment effect was most pronounced on sub - sets of scientist pairs who are less distant , working within the same clinical area , and therefore perhaps needing to overcome lower information and search cost hurdles . We also found positive associations between the likelihood of forming collaborations as prospective collaborators coming from the same hospital , and the single most important pre - dictor of collaborations , in terms of coef\ufb01cient magnitude , was whether individuals had previously collaborated . In documenting an important role played by search costs in in\ufb02uencing the formation of collaborations , we leave open a range of related questions . For example , in this paper , we did not study or observe longer - run outcomes of scienti\ufb01c productivity such as subsequent publications . ( Initial analysis of reviewers\u2019 assessments of the grant applications indicates no statistical difference between scores of applications submitted by pairs in the same room versus pairs not in the same room at the event ; see appendix table A4 . ) Also , we demonstrate what are arguably large effects of the particular treatment we implemented here . However , the treatment exploited here is not necessarily optimal and could be subject to further improvement . Such insights could be relevant in devising improved means of designing supporting information systems and matching facilities . An additional and potentially rather important series of questions falling outside the scope of this study concerns how individuals develop their own stock of matching - relevant information and heuristics in the \ufb01rst place ( apart from effects of situational or episodic shocks in information , as were explored here ) . The patterns documented here also raise questions regarding the extent to which homophily ( Lazarsfeld & Merton , 1954 ; Baccara & Yariv , 2013 ) exempli\ufb01ed by increased likelihood for scientists to form ties with other scientists possessing similar personal characteristics , might , at least in large part , be the result of search costs\u2014rather than re\ufb02ecting collaboration preferences ( Boudreau & Lakhani , 2012 ) or lower coordination costs when collabor - ating with similar partners ( Reagans & Zuckerman , 2001 ; Reagans , Zuckerman , & McEvily , 2004 ) . Despite these limitations , we see this study as a step toward opening the black box of how scienti\ufb01c collabora - tions form . In recent years , there has been considerable interest in the policy arena in fostering collaborations , and especially interdisciplinary collaborations , in particular by the U . S . government agencies funding fundamental research and development ( a combined budget of $ 36 bil - lion in 2011 ) , the NIH and the National Science Founda - tion . Yet there is scant evidence indicating how to do this in practice . On a methodological level , we are\u2014to the best of our knowledge\u2014the \ufb01rst to bring \ufb01eld experimental meth - ods to a workplace setting where the participants are engaged in scienti\ufb01c knowledge production . Evidence from randomized experiments on the scienti\ufb01c community such as ours will presumably be increasingly valuable to policy - makers as they consider reforms to scienti\ufb01c institutions ( Azoulay , 2012 ) , and more generally our study provides a template for the design of randomized control trials in inno - vation research ( Boudreau & Lakhani , 2016 ) . 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SCHUNN 3 1 Center for Advanced Study of Language , University of Maryland , College Park , College Park , USA 2 Human \u2013 Computer Interaction Institute , Carnegie Mellon University , Pittsburgh , USA 3 Learning Research and Development Center , University of Pittsburgh , Pittsburgh , USA Summary : This study explored the association between different types of brief disagreements and subsequent levels of expressed psychological uncertainty , a fundamental cognitive aspect of complex problem solving . We examined 11hours ( 11861 utterances ) of conversations in expert science teams , sampled across the \ufb01 rst 90days of the Mars Exploration Rover mission . Utterances were independently coded for micro - con \ufb02 icts and expressed psychological uncertainty . Using time - lagged hierarchical linear modeling applied to blocks of 25 utterances , we found that micro - con \ufb02 icts regarding rover planning were followed by greater uncertainty . Brief disagreements about science issues were followed by an increase in expressed uncertainty early in the mission . Examining the potential reverse temporal association , uncertainty actually predicted fewer subsequent disagreements , ruling out indirect , third variable associations of con \ufb02 ict and uncertainty . Overall , these \ufb01 ndings suggest that some forms of disagreement may serve to uncover important areas of uncertainty in complex teamwork , perhaps via revealing differences in mental models . Copyright \u00a9 2016 John Wiley & Sons , Ltd . INTRODUCTION The most important scienti \ufb01 c and technological advances are increasingly being produced by teams ( Jones , 2009 ; Singh & Fleming , 2009 ; Wuchty , Jones , & Uzzi , 2007 ) . Organiza - tions are also looking to interdisciplinary teams to address especially dif \ufb01 cult global problems ( Beck , 2013 ; Derry , Schunn , & Gernsbacher , 2005 ) . However , interdisciplinary teams have challenges : they must communicate across un - shared norms and mental models , which may result in dis - agreement and gaps in understanding ( Paletz & Schunn , 2010 ) . Furthermore , real - world problems are notable for their high levels of uncertainty . Problem solving with com - plex , ill - de \ufb01 ned problems often depends on signi \ufb01 cant effort devoted to the detection and resolution of uncertainty ( Schunn & Trafton , 2012 ) : Detection strategies are necessary to identify what is unknown ( either because information is missing or because presented information is misleading ) , and resolution of the uncertainty is important for ultimately solving the complex problem . This project studies how real - world science teams deal with uncertainty in problem solving . By taking a micro - process approach , this study examines how and whether brief intrateam disagreements can help teams detect uncer - tainty and then compares the association between disagree - ments and subsequent uncertainty in conversations at two different time phases within a team \u2019 s life . The past few de - cades have seen an increased interest in examining how teams \u2019 problem - solving processes unfold over time ( Cronin , Weingart , & Todorova , 2011 ; Kurtzberg & Mueller , 2005 ; McGrath , 1991 ; McGrath & Tschan , 2004 ) . At the most micro - temporal level ( at the timescale of seconds ) , brief speech acts , such as disagreement , may cumulate to have large effects on subsequent communication and cognition ( e . g . , Chiu , 2008a , 2008b ; Paletz , Schunn , & Kim , 2013 ) . Uncertainty Research in cognitive psychology has revealed that uncer - tainty is a central aspect of complex problem solving ( for a review , see Schunn & Trafton , 2012 ) . Psychological uncer - tainty is the recognition or feeling of missing , vague , or incomplete information ( Schunn , 2010 ) . Uncertainty is ubiq - uitous within and central to real - world problem solving , including science and engineering , management , medicine , and education ( Kahneman & Tversky , 1982 ; Schunn , 2010 ) . The research \ufb01 eld of naturalistic decision making unpacks in - dividual and team judgment in uncertain situations such as avi - ation ( e . g . , Bearman , Paletz , Orasanu , & Thomas , 2010 ; Klein , 1989 ) . Indeed , one of the main differences between real - world and experimental tasks is the level of uncertainty , as well as uncertainty about uncertainty , in real - world tasks ( Chinn & Malhotra , 2002 ; Kirschenbaum , Trafton , Schunn , & Trickett , 2014 ; Schunn & Trafton , 2012 ) . Across real - world domains , problem - solving success de - pends on the effective detection , understanding , and resolu - tion of uncertainty ( Chan , Paletz , & Schunn , 2012 ; Downey & Slocum , 1975 ; Kahneman & Tversky , 1982 ; Schunn , 2010 ; Schunn & Trafton , 2012 ) . For example , product devel - opment leaders have to deal with design uncertainties , while doctors have to deal with uncertainties regarding the true causes or diseases associated with a group of symptoms . People productively deal with task - related uncertainty in many ways ( Jordan & Babrow , 2013 ; Lipshitz & Strauss , 1997 ; Schunn & Trafton , 2012 ) , especially by taking it into account in decisions ( e . g . , avoiding irreversible action and explicitly noting con \ufb01 dence levels ) , communicating directly about uncertainty , or reducing uncertainty through additional problem solving ( e . g . , collecting additional information and making analogies ; Chan et al . , 2012 ) . Although the importance of the resolution of uncertainty is obvious to problem solving , uncertainty detection is also important . For instance , initial diagnostic certainty can be correct or incorrect . If incorrect , raising uncertainty about the initial idea will enable the re - examination of data and en - courage information search . Indeed , past literature suggests * Correspondence to : Susannah B . F . Paletz , Center for Advanced Study of Language , University of Maryland , College Park , College Park , MD , USA . E - mail : sbfpaletz @ gmail . com Copyright \u00a9 2016 John Wiley & Sons , Ltd . Applied Cognitive Psychology , Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) Published online 17 February 2016 in Wiley Online Library ( wileyonlinelibrary . com ) DOI : 10 . 1002 / acp . 3213 that individuals are often bad at articulating their assumptions and identifying genuine uncertainties , especially in complex situations ( Dunbar , 1997 ; Evans , 1989 ; Gorman , 1986 ) . Uncertainty is also important in the earliest stages of problem solving . Problem \ufb01 nding , or how a problem is initially discovered and structured , is a vital precursor to problem solving ( Mumford , Reiter - Palmon , & Redmond , 1994 ; Paletz & Peng , 2009 ) , especially in ill - de \ufb01 ned domains where un - certainty abounds ( Runco , 1994 ; Runco & Nemiro , 1994 ) . Put simply , teams and individuals need to know what they do not know in order to move forward and innovate . Con \ufb02 ict Researchers from different \ufb01 elds have examined con \ufb02 ict in many different forms , from violent acts perpetrated on behalf of nations to con \ufb02 icts within oneself . We examine con \ufb02 ict as disagreements between individuals within teams , drawing from a tradition within social and organizational psychology ( Barki & Hartwick , 2004 ) . Teams are collections of individ - uals that perform interdependent tasks that affect others , are embedded in a larger social system , and perceive themselves , and are perceived by others , as a social entity ( Guzzo & Dickson , 1996 ) . Intrateam con \ufb02 ict can vary in its focus , length , and intensity , and these variations can change the effects of con \ufb02 ict on team processes ( Paletz , Schunn , & Kim , 2011 ; Todorova , Bear , & Weingart , 2014 ; Weingart , Behfar , Bendersky , Todorova , & Jehn , 2014 ) . Under certain conditions , con \ufb02 ict has the potential to increase creativity ( e . g . , Chiu , 2008a ; Nemeth , 1986 ; Nemeth , Personnaz , Personnaz , & Goncalo , 2004 ; Paletz , Miron - Spektor , & Lin , 2014 ) . However , it can also hinder creativity , including when the con \ufb02 ict is only observed by another ( Miron - Spektor , Efrat - Treister , Rafaeli , & Schwartz - Cohen , 2011 ) . This study focuses on micro - con \ufb02 icts , or brief , expressed disagreements , in order to unpack and illuminate team pro - cesses in detail . Prior research on micro - con \ufb02 icts has exam - ined complex interaction patterns and con \ufb02 ict management ( e . g . , Kuhn & Poole , 2000 ; Poole & Dobosh , 2010 ) , affec - tive con \ufb02 ict interactions within families or married couples ( e . g . , Gottman & Notarius , 2000 ; Vuchinich , 1987 ) , and the immediate effects of brief disagreements on creativity , problem solving , and analogy ( e . g . , Chiu , 2008a , 2008b ; Paletz , Schunn , et al . , 2013 ) . Intragroup con \ufb02 ict researchers distinguish between task con \ufb02 ict , or disagreements about the work being performed ; process con \ufb02 ict , or how to go about doing the task ( e . g . , pri - oritization and scheduling ) ; and relationship con \ufb02 ict , which is about personal issues ( e . g . , Barki & Hartwick , 2004 ; De Dreu & Weingart , 2003 ; Jehn , 1995 , 1997 ) . Relationship con \ufb02 ict is generally thought to be harmful ( e . g . , Amason , 1996 ; De Dreu & Weingart , 2003 ; He , Ding , & Yang , 2014 ; Jehn , 1997 ; De Wit , Greer , & Jehn , 2012 ; De Wit , Jehn , & Scheepers , 2013 ) . Process con \ufb02 ict is also generally thought to be problematic ( e . g . , De Dreu & Weingart , 2003 ) , but how destructive it is may depend on when it comes during the team \u2019 s process ( e . g . , Goncalo , Polman , & Maslach , 2010 ) . Task con \ufb02 ict is now thought to have a cur - vilinear effect on performance on intellectual tasks ( creativ - ity , Fahr , Lee , & Farh , 2010 ; decision quality , Parayitam & Dooley , 2011 ) . These studies suggest that moderate levels of task con \ufb02 ict are best for team outcomes . In addition , the dynamics and implications of con \ufb02 ict may vary across a team \u2019 s life cycle . Con \ufb02 ict occurs throughout the life of a team , but it may be more common during early phases of a team \u2019 s life cycle ( Paletz et al . , 2011 ; Poole & Garner , 2006 ) . Con \ufb02 ict earlier in the team \u2019 s life may be func - tional , helping set up processes and tasks : early process con - \ufb02 ict may help forestall premature collective ef \ufb01 cacy , leading to better team performance later ( Goncalo et al . , 2010 ) . Most research on work teams examines con \ufb02 ict using retro - spective self - report surveys ( e . g . , De Dreu & Weingart , 2003 ; Fahr et al . , 2010 ; Greer , Jehn , & Mannix , 2008 ; Parayitam & Dooley , 2011 ; Shaw et al . , 2011 ) . However , self - report sur - veys or interviews fail to capture phenomena that require exact recall and are too fast or complex to be noticed by participants ( Goh , Goodman , & Weingart , 2013 ; Gottman & Notarius , 2000 ; Weingart , 1997 ) . Retrospection is often inaccurate , and con \ufb01 dence about memories may be unrelated to accuracy ( e . g . , Chua , Hannula , & Ranganath , 2012 ; Wong , Cramer , & Gallo , 2012 ) . Further , surveys lose the \ufb01 ne - grained temporal structure of communication processes that occur during con - versations . For example , micro - process research has found that polite disagreements are positively associated with micro - creativity ( Chiu , 2008a ) and subsequent correct contributions ( Chiu , 2008b ) , whereas rude disagreements after a wrong idea can increase teammate creativity ( Chiu , 2008a ) . This current study follows this growing interest in examin - ing the immediate consequences of disagreement , or micro - con \ufb02 icts , on team processes ( e . g . , Chiu , 2008a , 2008b ; Kauffeld & Lehmann - Willenbrock , 2012 ; Paletz , Schunn , et al . , 2013 ) . We examine individual utterances for the pres - ence of disagreement and then aggregate them into micro - con \ufb02 ict events . Con \ufb02 ict and subsequent uncertainty We embed our research questions within shared mental models theory to propose that expressed micro - con \ufb02 icts , or brief disagreements ( Paletz et al . , 2011 ) , may in \ufb02 uence sub - sequent uncertainty , depending on the type of con \ufb02 ict and phase of the team \u2019 s life cycle . One aspect of teams as they develop is the creation of shared mental models ( e . g . , Burke , Stagl , Salas , Peirce , & Kendall , 2006 ; Johnson - Laird , 1980 ; Mathieu , Heffner , Goodwin , Salas , & Cannon - Bowers , 2000 ) . Shared mental models are the similarity in cognitive schemas regarding teamwork , norms , and tasks . They are important for effective plan execution and timely problem solving , among other team functions ( e . g . , Burke et al . , 2006 ; Jones , Stevens , & Fischer , 2000 ) . Interdisciplinary work is often fraught with con \ufb02 ict , as in - dividuals with different backgrounds , skills , and perspec - tives are brought together ( Mannix & Neale , 2005 ; Paletz & Schunn , 2010 ) . Con \ufb02 ict may be caused by underlying differences in shared mental models ( Bearman et al . , 2010 ; Cronin & Weingart , 2007 ) . Disagreement is generally expressed because an individual thinks or feels something con - tradictory to what was just said or implied . Indeed , disagree - ment can enable team members to hear previously unshared information from team members \u2019 different mental models . 388 S . B . F . Paletz et al . Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e On the one hand , additional information gained via con - \ufb02 ict can decrease team uncertainty . Gathering additional information is , after all , an uncertainty - reduction technique ( Lipshitz & Strauss , 1997 ; Schunn & Trafton , 2012 ) . Members of multidisciplinary teams may have information that resolves others \u2019 uncertainty . New information unco - vered via con \ufb02 ict may enable team members to update their mental models and create new certainties . On the other hand , the non - overlapping mental models that arise from interdisciplinary teams also may increase un - certainty . Individuals may not see ambiguity or errors in their own thinking that are then noticed by other scientists ( Dama & Dunbar , 1996 ) . Particularly in multidisciplinary contexts , a disagreement may raise and question underlying assumptions held by other members of the team , leaving pre - viously ( supposedly ) known information to be suddenly up for debate . A period of uncertainty may then occur when mental models incorrectly thought to be shared are reassessed and re - formed . Team context may affect whether disagreement decreases subsequent uncertainty or not . In this study , we focus on temporal context ( early versus later time periods in a team \u2019 s planned life cycle ) , a contextual factor that seems especially likely to interact with team con \ufb02 ict and uncertainty . Teams often go through iterative cycles of sorting out their pro - cesses , self - evaluating , and focusing on the work ( Gersick , 1988 ; Morgan , Salas , & Glickman , 1994 ; Tuckman , 1965 ; Tuckman & Jensen , 1977 ) . Early in a team \u2019 s life cycle , there is more uncertainty and con \ufb02 ict , as processes are established and iterated ( e . g . , Paletz et al . , 2011 ; Poole & Garner , 2006 ; Tuckman & Jensen , 1977 ) . In other words , early on , the members are formulating their shared mental models of their tasks and processes . During this phase , disagreement may lead to more uncertainty . Later in a team \u2019 s life cycle , dis - agreements are less likely to reveal unknown , major underly - ing differences in assumptions and mental models . Thus , there may be different relations between con \ufb02 ict and uncer - tainty early on , when everything is in \ufb02 ux and being established , versus later in the team \u2019 s lifecycle . Finally , different types of con \ufb02 ict may have different effects on uncertainty , just as they have different impacts on team success ( De Wit et al . , 2012 ) . Task con \ufb02 ict is most relevant to exposing gaps in mental models about the task , and thus most likely to be related to subsequent uncertainty . But process con \ufb02 ict may also reveal gaps , if perhaps more indirectly . For example , because mental models about the task support speci \ufb01 c processes , arguments about processes may en - courage others to share task information about these particular processes . It is unclear whether relationship con \ufb02 icts should have an effect on uncertainty , and these will not be a focus of this study for practical reasons ( as discussed later ) . Research questions This research is one of a small number of studies to examine \ufb01 ne - grained temporal processes of con \ufb02 ict ( e . g . , Chiu , 2008a , 2008b ; Paletz , Schunn , et al . , 2013 ; Poole & Dobosh , 2010 ) and the \ufb01 rst to examine the association between con - \ufb02 ict and subsequent uncertainty . The prior literature reviewed earlier suggests an association is plausible , but the exact form is dif \ufb01 cult to predict , and it is likely depen - dent on both the time phase in the group \u2019 s life cycle and the type of con \ufb02 ict . Our research questions are as follows : Research Question 1 : What is the association between the different types of expressed micro - con \ufb02 icts and immedi - ate , subsequent levels of uncertainty in speech ? Research Question 2 : How might these relations differ during an early phase of a team \u2019 s life cycle versus a later phase ? In addition , to further probe the nature of observed associ - ations , we also examine whether the relations between micro - con \ufb02 icts and subsequent uncertainty are bidirectional or potentially caused by a third factor . We accomplish this by testing for an association between uncertainty and subse - quent micro - con \ufb02 icts . METHODS Research context and data collection Research context This study examined informal , task - relevant conversations occurring between scientists working on the multidisciplin - ary Mars Exploration Rover ( MER ) mission ( Squyres , 2005 ) . The MER mission aimed to , and succeeded in , dis - covering evidence for a history of liquid water on Mars via sending two rovers to separate sides of the planet . The mis - sion was initially scheduled to last 90 Martian days , and so the scientists spent those days working at the Jet Propulsion Laboratory with workstations and screens enabling collabo - ration . Each rover had its own , roughly independent science team ( MER A and B ) , which was further broken up into dif - ferent science sub - teams . The discovery of the history of liq - uid water involved geological and atmospheric science , complex task planning , and general human work processes . Data collection Data were collected on 20 data collection trips over the 90 initial mission days . Researchers placed video cameras on top of large shared screens near different subgroups \u2019 work - station clusters . Each trip involved 3 days of about 8 hours of data collection each day , resulting in roughly 400 h of video . The scientists became habituated to the cameras , at times discussing personal matters . Data sampling and transcription We sampled 11 hours and 25 minutes of informal , task - relevant conversation clips from days early and later in the nominal mission ( \ufb01 rst 90 days ) . Re \ufb02 ecting the common real - ities of complex work unfolding over long timescales and distributed over multiple physical workspaces , much of the 400 hours of video ended up recording individuals working in silence , empty chairs , and / or such poor audio \u2013 video that it could not be transcribed . We excluded structured meetings from the sample because they were conducted in a formal round - robin presentation style , with most participants simply listening to people talking offscreen . During the normal workday , scientists sitting at their workstations would strike Uncovering uncertainty through disagreement 389 Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e up task - relevant conversations , and these would end ( as clips ) when the conversation stopped or the speakers moved away together ( e . g . , to get food ) . Task - relevant talk was de \ufb01 ned as anything relating to the mission at all , including process , re - lational , and organizational issues ( e . g . , organizational strug - gles between subgroups ) , whereas off - task talk was de \ufb01 ned as exclusively personal matters such as family vacations . While the larger MER project consisted of over 100 scientists , \u2018 sub - teams \u2019 of 2 \u2013 10 MER scientists assembled organically through - out the project . These sub - teams were most often task focused , engaged in interdependent tasks , and had a shared social iden - tity ( as MER scientists ) . Because the sub - teams tended to be task focused and \ufb02 uid in number , rather than centrally planned , con \ufb01 gurations of individuals often overlapped imperfectly be - tween sub - team conversations ( Paletz & Schunn , 2011 ) . This study examined conversations from these sub - teams , which occurred in video clips , representing natural within - team , interpersonal con \ufb02 icts and uncertainty as they arose . The over - whelming majority of the conversations sampled ( 93 % ) were inherently multidisciplinary , as they came from video of the Long Term Planning area , the location of a cross - disciplinary group that was the hub for different groups \u2019 conversations re - garding future science activities . The video clips were transcribed into 12 336 utterances , which were then 100 % double coded as to whether they were related to the MER mission at all ( on - task ) or not ( Cohen \u2019 s kappa = 0 . 96 ) . On - task talk was de \ufb01 ned in the same way as in our clip sampling strategy . Trained transcribers broke ( unit - ized ) the conversations into utterances during the course of transcription . Utterances were operationalized as thought state - ments or clauses and are thus often briefer than turns of talk ( Chi , 1997 ) . Our analyses are conducted on the resulting 11861 on - task utterances ( roughly 11hours ) , coming from 114 clips that ranged from 8 to 760 utterances long ( M = 104 . 0 , median = 66 . 5 , SD = 121 . 8 ) . Twenty - six per cent of the video clips had a mix of male and female scientists , whereas 74 % of video clips were all male . The number of speakers in the clip ranged from 2 to 10 . These data have been previously analyzed to examine the nature of micro - con \ufb02 icts ( Paletz et al . , 2011 ) , the temporal relations between micro - con \ufb02 icts and analogy ( Paletz , Schunn , et al . , 2013 ) , and the association between analogy and uncertainty ( Chan et al . , 2012 ) . Measures The primary variables for this study were micro - con \ufb02 icts and their types , psychological uncertainty , and temporal context . To reduce noise from the dif \ufb01 cult coding task , both uncer - tainty and micro - con \ufb02 icts were double coded ( 100 % overlap in assessment by two independent coders ) with mismatches resolved through discussion ( Smith , 2000 ) . To remove the in \ufb02 uence of cross - category coding cues , each variable was coded by a different pair of independent coders . All coders were blind to the results of the other coding and also to the goals and questions of this study . Temporal context : early versus later in the \ufb01 rst 90 days of the mission The scientists were relative novices to real - time Mars opera - tions initially but gained expertise dramatically over the \ufb01 rst 90 days ( Paletz , Kim , Schunn , Tollinger , & Vera , 2013 ) . The mission was initially planned to be 90 Martian days long . We chose 50 days as our cutoff point to divide clips into ear - lier versus later in the mission because the initial few days of each mission involved tests regarding rover health . Also , the scientists experienced a dramatic speedup of their scheduled science planning over the course of these 90 days : 51 days was when one of the rover teams decreased their scheduled shift ( and planning time ) from 6 to 4 . 5 hours ( Paletz , Kim , et al . , 2013 ; Tollinger , Schunn , & Vera , 2006 ) . Early in the mission , the scientists were more likely to grapple with the mission processes . Indeed , process micro - con \ufb02 icts were less likely during the second half of the mission ( Paletz et al . , 2011 ) . Further , by the second half of the mission , not only were the scientists more practiced at running a mission on Mars , but they had also discovered strong evidence of liquid water and had granted a news conference on the topic . Sixty - \ufb01 ve per cent ( 74 ) of the conversation clips were from before Day 50 ( early phase ) , and 35 % ( 40 ) were drawn from after or on Day 50 ( Days 50 \u2013 90 , later phase ) . Finer - grained temporal analyses were not possible : the sampling was uneven owing to mission events intermittently disrupting data collection . Micro - con \ufb02 icts We coded for micro - con \ufb02 icts , or brief disagreements , using a coding scheme detailed elsewhere for this sample ( Paletz et al . , 2011 ) . This scheme involved assessing each utterance for the presence ( 1 ) or absence ( 0 ) of con \ufb02 ict , as contrasted with other schemes that code 30 - second to 1 - minute seg - ments in their entirety ( e . g . , Poole , 1989 , used in Poole & Dobosh , 2010 ) and those that involve coding by sentence into one of several exclusive categories , collectively captur - ing all types of team interactions ( e . g . , act4teams , used in Lehmann - Willenbrock , Meyers , Kauffeld , Neininger , & Henschel , 2011 ) . Each utterance was \ufb02 agged as containing a micro - con \ufb02 ict whenever a speaker explicitly disagreed with something said earlier in the conversational video clip . Simply stating something controversial was not suf \ufb01 cient , but supporting arguments were counted as con \ufb02 ict . The coders read the transcript and referred to the audio \u2013 video re - cording to resolve differences and to clarify what occurred , paying close attention to participants \u2019 body language , tone , and , when possible , facial expressions ( con \ufb02 ict event kappa = 0 . 62 ) . For example , a group of scientists were discussing the probable geological nature of a small area on Mars . One noted that they would be able to \ufb01 nd out more by looking at an outcrop closely . A second scientist disagreed , saying , \u2018 but I think you gonna see , I think if it \u2019 s there , you \u2019 re gonna see it remotely anyway \u2019 ( via viewing by satellites ) . The original scientist tried to explain his posi - tion , \u2018 I think the , uh , I mean the scale of detail \u2019 , and the sec - ond scientist continued his argument with \u2018 no , I think , because up close \u2014 up close you \u2019 re gonna see the broken , you know , jumble \u2019 . The \ufb01 rst disagreed , \u2018 nah , I think it \u2019 s worse than that \u2019 , and the second continued his thought , \u2018 if you can get it \u2019 . At that point , a third scientist laughed and said , \u2018 you \u2019 re , you \u2019 re , I think you \u2019 re both wrong . The prob - lem is from looking at it from just sort of across , I don \u2019 t think you can see close enough to see the lamination \u2019 . This exam - ple is re \ufb02 ective of the natural micro - con \ufb02 icts from this 390 S . B . F . Paletz et al . Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e sample : overlapping ideas , low affect , and spontaneous . All disagreements between coders were resolved by discussion , with a consensus code assigned to each utterance . The coding scheme was developed iteratively on this dataset , with nuances noted in the coding scheme and past clips recoded as necessary ( Paletz et al . , 2011 ) . The micro - con \ufb02 icts were also coded by utterance for type of con \ufb02 ict , using categories described widely in the social and organizational con \ufb02 ict literature ( task , process , and rela - tionship con \ufb02 ict , e . g . , Jehn , 1997 ) . Because the MER con - text clearly distinguished between the two major work task types of science ( e . g . , data interpretation ) and rover planning ( e . g . , which instrument readings to do ) , the coders assessed each con \ufb02 ict utterance as to whether the con \ufb02 ict was about science ( task ) , rover planning ( task ) , work process , or rela - tionship matters ( see Table 1 for examples ) . To reduce noise from reliability issues , the data were exhaustively double coded , and discrepancies were resolved through discussion ( Smith , 2000 ) . These utterances were clustered into con \ufb02 ict events ( and blocks made up of continuous con \ufb02 ict events , as discussed later ) based on speci \ufb01 c topic : for instance , a speci \ufb01 c process micro - con \ufb02 ict could then lead to another process con \ufb02 ict , another type of con \ufb02 ict , or no con \ufb02 ict at all . Expressed relationship con \ufb02 ict was too rare in this sam - ple to be included in the analyses ( only three of the 688 total blocks had relationship con \ufb02 ict ) ; leaving out relationship con \ufb02 ict , the con \ufb02 ict - type coding had a reliability kappa of 0 . 67 at the utterance level ( up from 0 . 48 with relationship con \ufb02 ict included , likely because the rarity of relationship con \ufb02 ict decreased the overall kappa ; Bakeman , Quera , McArthur , & Robinson , 1997 ) . Uncertainty Two coders used a preexisting coding scheme to assess each utterance for the presence of uncertainty , using \u2018 hedge words \u2019 ( e . g . , \u2018 I guess \u2019 , \u2018 I think \u2019 , \u2018 possibly \u2019 , \u2018 maybe \u2019 , \u2018 I believe \u2019 , de - tailed in Trickett , Trafton , Saner , & Schunn , 2007 ) as cues to potential uncertainty . All coder disagreements were resolved Table 1 . Examples of micro - con \ufb02 icts between Mars Exploration Rover scientists ( key words in bold ) , excerpts from Paletz et al . ( 2011 ) Coded as Utterance Example 1 : science con \ufb02 ict No con \ufb02 ict S2 : \u2026 I \u2019 m afraid that this very low angle interior wall re \ufb02 ects that No con \ufb02 ict there is no bedrock there . Science con \ufb02 ict S3 : Well , Bereonies got a low slope also . Science con \ufb02 ict It could be that the bedrock is just not very conical . Example 2 : rover planning con \ufb02 ict No con \ufb02 ict S3 : Then the issue becomes we all , we may not have enough time to do any mineralogy or this spectral stuff No con \ufb02 ict if we \u2019 re going to have a long look at the salt . No con \ufb02 ict S2 : You think of this No con \ufb02 ict as we \u2019 ve only been there for two weeks . Rover planning con \ufb02 ict There \u2019 s a time for everything now . Rover planning con \ufb02 ict How do we spend it such that Rover planning con \ufb02 ict maybe your stuff , half the peoples \u2019 stuff doesn \u2019 t get done on one sol , Rover planning con \ufb02 ict but the other half can really get something that \u2019 s good . Rover planning con \ufb02 ict S3 : Right , but I need to do the stuff I want to do , which is \u2026 Example 3 : process con \ufb02 ict ( with \u2018 no \u2019 ) No con \ufb02 ict S2 : You showed me that already . Process con \ufb02 ict S3 : No , I showed you a single frame . Example 4 : relationship con \ufb02 ict Rover planning con \ufb02 ict S1 : Well , what I \u2019 m saying is common things occur commonly Rover planning con \ufb02 ict and rare things occur rarely Rover planning con \ufb02 ict and to say that we landed on the rare thing rather than the common thing Relationship con \ufb02 ict is a totally subjective decision that we make that goes against logic . Relationship con \ufb02 ict That \u2019 s all I \u2019 m saying . Rover planning con \ufb02 ict S2 : No , I agree with that , but \u2014 [ is cut off ] Example 5 : con \ufb02 ict with no con \ufb02 ict embedded Science con \ufb02 ict S6 : But to me they look like each one of these things No con \ufb02 ict Even though they \u2019 re really wide , Science con \ufb02 ict They do sort of look like single event types of features Science con \ufb02 ict Because the sides of each of them are sort of parallel to each other Science con \ufb02 ict which you wouldn \u2019 t get if there was a swarm coming in from the same place . Example 6 : use of \u2018 no \u2019 without con \ufb02 ict No con \ufb02 ict S3 : Is there any change in the plan here ? No con \ufb02 ict S1 : No . Example 7 : use of \u2018 but \u2019 without con \ufb02 ict ( additions rather than disagreement ) No con \ufb02 ict S3 : Let me ask . No con \ufb02 ict S2 : Well , we need some , No con \ufb02 ict but I think we need to pick a number , you know , No con \ufb02 ict and I think we need to pick a number of rocks No con \ufb02 ict and there \u2019 s certainly at least two types that I can think of . No con \ufb02 ict But we need to have \u2026 S2 , S1 , and so on are speaker numbers . The \ufb01 rst speaker in the clip is S1 and so on . S2 in one clip may not be S2 in another clip . Uncovering uncertainty through disagreement 391 Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e by discussion , with a consensus code assigned . The uncer - tainty coding in this dataset is described in more detail else - where , in a paper that found that problem - related analogies may serve an uncertainty - reduction function ( Cohen \u2019 s kappa = 0 . 75 ; Chan et al . , 2012 ; see Table 2 for examples ) . Schunn ( 2010 ) established the validity of this measure - ment approach using convergent and discriminant validity methods . For example , use of these hedge words corresponded also to the use of uncertainty gestures in team conversations at the utterance level , well above base rates in the local temporal context ( Schunn , 2010 ) . Further , uncertainty and imprecision ( purposeful approximations like \u2018 thirty something \u2019 ) could be reliably separated in speech and gestures , and they had differ - ent relations to other cognitive processes ( Schunn , 2010 ; Schunn , Saner , Kirschenbaum , Trafton , & Littleton , 2007 ) . 1 Both con \ufb02 ict and uncertainty were analyzed at the block level ( i . e . , aggregated across utterances ) . We describe our block creation approach in the subsequent section . Analyses Blocks and video clips To unpack temporal patterns , we broke the video clips ( con - versations ) into segmented blocks made up of continuous , multiple utterances . Intuitively , segmenting video clips into blocks of utterances allows us to track the ebb and \ufb02 ow of uncertainty through conversations by examining how prop - erties of one block relate to uncertainty in subsequent blocks . We created blocks by \ufb01 rst identifying contiguous con \ufb02 ict ut - terances and events . These events would become the initial blocks . For example , the con \ufb02 ict described previously would become a single block made up of multiple utterances . We then identi \ufb01 ed the 25 utterances before and after each con \ufb02 ict event as two additional blocks ( Figure 1 ) . The rest of the clips were broken up into successive blocks of 25 utterances each , each ending at the 25th utterance , the beginning / end of a video clip , or with the next con \ufb02 ict event . Analyses of temporal patterns in uncertainty in this and other complex problem - solving data ( e . g . , Chan et al . , 2012 ; Christensen & Schunn , 2009 ) suggest that uncertainty tends to show signi \ufb01 cant rises and declines in windows of 15 \u2013 30 utterances . This approach to creating utterance blocks also enables the transcript and analyses to be relatively homoge - nous on the independent variable ( con \ufb02 ict ) and standardized in length on the dependent variable blocks ( as with Paletz , Schunn , et al . , 2013 ) . This segmentation method resulted in 688 blocks nested within 114 video clips . Block - level measures As noted , we used aggregated measures of con \ufb02 ict subtype and uncertainty at the block level in our analyses . We \ufb02 agged each con \ufb02 ict event block as to whether it contained at least one of each speci \ufb01 c type of con \ufb02 ict ( at the utterance level ) to create three separate , dichotomous con \ufb02 ict - type vari - ables ( science , planning , and process , 1 = present , 0 = absent ) . Uncertainty was de \ufb01 ned as the number of utterances in a given block coded as uncertain . While the con \ufb02 ict subtype codes at the block level were theoretically not mutually exclusive ( the same block could be marked as a 1 , present , for multiple of the three con \ufb02 ict - type variables ) , blocks were often relatively homogeneous with respect to con \ufb02 ict subtype . Using chi - squared tests , lagged process con \ufb02 icts were signi \ufb01 cantly negatively associ - ated with both lagged planning con \ufb02 icts , \u03c7 2 ( 1 , 115 ) = 32 . 84 , p < . 001 , phi = (cid:1) 0 . 53 , and lagged science con \ufb02 icts , \u03c7 2 ( 1 , 115 ) = 21 . 30 , p < . 001 , phi = (cid:1) 0 . 43 , and planning and science lagged micro - con \ufb02 icts were also negatively related to each other , \u03c7 2 ( 1 , 115 ) = 17 . 97 , p < . 001 , phi = (cid:1) 0 . 40 . 1 To rule out the possibility that our uncertainty coding scheme merely re \ufb02 ected politeness norms , we tested differences by gender and status . Men in our dataset expressed uncertainty ( proportion of uncertainty out of all utterances spoken by men , M = 0 . 14 , SE = 0 . 003 ) at a slightly higher rate than women ( proportion of uncertainty out of utterances spoken by women , M = 0 . 11 , SE = 0 . 01 ) , z = 1 . 96 , p = . 05 . Facultyexpresseduncertainty ( M = 0 . 14 , SE = 0 . 003 ) at asimilar ratetograduate students ( M = 0 . 10 , SE = 0 . 02 ) , z = 1 . 44 , p = . 15 , and older and younger faculty ( M = 0 . 14 , SE = 0 . 00 ) used uncertainty at the same rate , z = 0 . 07 , p = . 94 . Table 2 . Examples of uncertainty from Mars Exploration Rover scientists ( key words in bold ) in conversational contexts , excerpts from Chan et al . ( 2012 ) Speaker Turn Excerpt 1 1 I would have thought that the ventifacting would have been very late and it would have destroyed the surface . Actually maybe that \u2019 s why we don \u2019 t see the black surface of the other two rocks , which also were ventifacts , right ? Because maybe they just happened , we happened to hit faces where it had been scarred 2 And of course that , all that , would mean , if that is an injected block , all of that took place after ; it did not take place at < missing words > 1 Yea , well I mean , I don \u2019 t think that we can rule out that this isn \u2019 t some kind of desert varnish , although I don \u2019 t understand how desert varnish forms Excerpt 2 4 Can you do something with an acid fog ? I know we got sulfate and sulfurs and chlorines ; is there some way we can < missing words > 1 Certainly , yea , maybe that \u2019 s a good explanation of it 4 Maybe it \u2019 s a coating of some sort of sulfate chlorine , you know , crude Excerpt 3 1 Ok , so the idea is go , and then we \u2019 re going to at the end of sol [ Martian day ] 90 . At the end of the afternoon we deploy the M\u00f6ssbauer to capture it . 2 Is this going to be the < missing words > 1 I don \u2019 t know , I think so . And then we let it integrate all night . And then , or maybe , we let it integrate , we let it integrate until some other pass at 3 o \u2019 clock in the morning or something and then 392 S . B . F . Paletz et al . Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e Indeed , 96 % of the 118 con \ufb02 ict blocks had only one subtype . Thus , our data enable us to tease apart the associations between con \ufb02 ict , uncertainty , and temporal context by con \ufb02 ict subtype . Statistical analyses We addressed our main research questions using time - lagged , variable exposure overdispersed Poisson models . These models were tested with two - level hierarchical linear modeling ( Raudenbush & Bryk , 2002 ) via the HLM 7 ( 7 . 01 ) software , which uses penalized quasi - likelihood estimation ( Raudenbush , Bryk , & Congdon , 2013 ) . We report the unit - speci \ufb01 c models with robust standard errors , as is appro - priate for these analyses . Dependent variables that are count data ( i . e . , number of uncertainty utterances in the block ) require Poisson models , and we used the overdispersed setting because the variance of uncertainty ( 5 . 0 ) was greater than the mean ( 2 . 3 ; \u03c3 2 was 1 . 3 instead of 1 ) . Because the blocks varied in their number of utterances because of the endings of clips , beginnings of new con \ufb02 ict events , and so on , we used variable exposure Poisson . As an offset variable , we used ( 1 + ln [ number of utterances ] ) because the offset variable ( i . e . , ln [ 1 ] ) cannot equal zero . We utilized two - level hierarchical linear modeling of blocks ( Level 1 ) within video clips ( Level 2 ) to capture the dependence inherent in the data ( greater similarity in , and non - independence of , conversational dynamics within clips ) and manage unequal cell sizes . Supporting our assertion that uncertainty was in \ufb02 uenced by conversation - level factors , a null model of the uncertainty dependent variable 2 showed that there was signi \ufb01 cant variance at Level 2 ( video clip level ) , tau = 0 . 096 , \u03c7 2 ( 113 ) = 223 . 68 , p < . 001 ( intraclass correlation = 6 . 8 % ) . Because our research questions centered on temporal as - sociations , we used time - lagged analyses to test whether un - certainty in one block was predicted by the presence / absence of con \ufb02 ict , or a particular type of con \ufb02 ict , in the block ( 25 utterances ) before . In creating the models to test our research questions , the time - lagged con \ufb02 ict - to - uncertainty tests were at Level 1 ( blocks ) . Temporal context ( early versus later in the mission ) was at the conversational clip level ( Level 2 ) , and the moderation effect of early / later phase was a cross - level interaction between the clip and block level ( interaction between Level 2 and Level 1 variables ; Figure 1 ) . Before examining our independent variables , we tested for signi \ufb01 cant associations between uncertainty and several co - variates . These covariates were plausibly connected to levels of uncertainty or con \ufb02 ict and therefore represent possible confounds . First , the covariates at Level 1 ( block level ) were tested , then those that were signi \ufb01 cant at Level 2 ( video clip ) , controlling for the signi \ufb01 cant ones at Level 1 . These tested variables had to be signi \ufb01 cant to be retained in the \ufb01 nal model . Three Level 1 variables passed these tests : the average number of words per utterance ( across all the blocks , M = 7 . 0 , median = 6 . 86 , SD = 2 . 16 ; t ( 563 ) = 6 . 75 , B = 0 . 11 , SE = 0 . 02 , event ratio = 1 . 12 [ 1 . 08 , 1 . 15 ] , p < . 001 ) and the two topic vector variables to account for the three topics of discussion ( vector # 1 : t ( 563 ) = (cid:1) 1 . 66 , B = (cid:1) 0 . 14 , SE = 0 . 09 , event ratio = 0 . 87 ( 0 . 73 , 1 . 03 ) , p = . 098 ; vector # 2 , t ( 563 ) = (cid:1) 2 . 21 , B = (cid:1) 0 . 25 , SE = 0 . 11 , event ratio = 0 . 78 [ 0 . 63 , 0 . 97 ] , p = . 027 ) . Although one of the topic vectors was not signi \ufb01 - cant , we kept both vectors because together they represented all three topics of discussion ( science , planning , and work process ; see Paletz et al . , 2013 , for more detail on this covariate ) . The number of words per utterance was a control because the simple length of an utterance could in \ufb02 ate the likelihood of any feature , including uncertainty , in the block . The topic vectors were controlled for because topic alone could have been a third variable that could affect both uncer - tainty and con \ufb02 ict : different topics may elicit or have more con \ufb02 ict or uncertainty regarding them . We tested the following covariates , any of which could have reasonably been related to uncertainty or con \ufb02 ict , and found them not to be signi \ufb01 cant ( p s > . 05 ) : at Level 1 ( block ) , the average number of people present ; and at Level 2 ( video clip ) , the general age / status composition of the scientists present in the clip using two vector variables representing graduate students , young faculty , and older faculty ; the average number of speakers in the clip ; gender composition ; type of science team ( Long Term Planning versus other types of science teams ) ; number of utterances in the clip ; the rover team ( A versus B ) ; and whether the clip was from earlier or later in the initial 90 days . As appropriate to testing interaction effects , nonsigni \ufb01 cant relevant main effects ( i . e . , temporal context ) are included in models to control for main effects when testing interaction effects . 2 A null hierarchical linear model tests the dependent variable without any predictor variables using chi - squared estimation to determine whether there are signi \ufb01 cant higher - level components . Figure 1 . Data structure : blocks nested within clips , and time - lagged analyses of micro - con \ufb02 icts to uncertainty moderated by temporal context Uncovering uncertainty through disagreement 393 Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e RESULTS Frequency of uncertainty and micro - con \ufb02 icts At the block level , uncertainty occurred relatively frequently , with 75 % ( 518 ) of the 688 blocks having at least one uncertain utterance ( M = 2 . 3 , median = 2 . 0 , SD = 2 . 2 , range is 0 \u2013 10 ) . The frequency of the lagged versions of these different task and process con \ufb02 ict types is shown in Table 3 ( see also Paletz et al . , 2011 ; Paletz , Schunn , et al . , 2013 ) . Associations between types of con \ufb02 ict , team factors , and subsequent uncertainty Overall con \ufb02 ict We \ufb01 rst tested whether lagged con \ufb02 ict had a signi \ufb01 cant asso - ciation with subsequent uncertainty and had a signi \ufb01 cant in - teraction with temporal context , as well as whether temporal context had a main effect . The interaction of overall con \ufb02 ict with temporal context was not signi \ufb01 cant , and the model removing it showed that overall lagged con \ufb02 ict was only a marginally signi \ufb01 cant predictor of uncertainty ( Table 4 ) . We expected different forms of con \ufb02 ict to have different effects on uncertainty , so we then unpacked the effects by subtype . Micro - con \ufb02 ict by subtype Next , we conducted moderated tests of temporal context ( early / later phase ) for the three topic subtypes of lagged con - \ufb02 ict : science con \ufb02 ict , planning con \ufb02 ict , and process con \ufb02 ict . Lagged process con \ufb02 icts were unrelated to uncertainty either as a main effect , t ( 462 ) = 1 . 18 , B = 0 . 15 , SE = 0 . 13 , event ra - tio = 1 . 17 [ 0 . 90 , 1 . 51 ] , p = . 239 , or in interaction with tempo - ral context , t ( 462 ) = (cid:1) 1 . 07 , B = (cid:1) 0 . 35 , SE = 0 . 33 , event ratio = 0 . 70 [ 0 . 37 , 1 . 35 ] , p = . 287 ( Figure 2A ) . Planning con \ufb02 ict had a signi \ufb01 cant , positive main effect on subsequent uncertainty , but no signi \ufb01 cant interaction with temporal con - text , t ( 462 ) = (cid:1) 0 . 25 , B = (cid:1) 0 . 05 , SE = 0 . 21 , event ratio = 0 . 95 [ 0 . 63 , 1 . 43 ] , p = . 801 ( see Table 5 for the \ufb01 nal model without the interaction vector ; Figure 2B ) . In contrast , lagged science con \ufb02 ict had a signi \ufb01 cant interaction with temporal context ( early / later phase in the \ufb01 rst 90 days of the mission ) . Early in the mission , lagged science con \ufb02 ict had a positive associ - ation with uncertainty ( similar to planning con \ufb02 ict ) , whereas the association was not signi \ufb01 cant later in the mission ( Table 6 , Figure 2C ) . Two examples of con \ufb02 ict ( planning micro - con \ufb02 icts and early science micro - con \ufb02 icts ) leading to increased uncer - tainty are in Table 7 . In the \ufb01 rst example , two scientists are debating what a sensible plan of instrument readings for Table 3 . Frequency of presence of lagged science , planning , pro - cess , quickly resolved , positive , and negative micro - con \ufb02 icts by block Micro - con \ufb02 ict type Frequency % ( n of 569 lagged blocks ) Any micro - con \ufb02 ict 20 . 2 ( 115 ) Process micro - con \ufb02 ict 8 . 0 ( 46 ) Rover planning ( task ) micro - con \ufb02 ict 8 . 2 ( 47 ) Science ( task ) micro - con \ufb02 ict 4 . 4 ( 25 ) Table 4 . Any lagged micro - con \ufb02 ict and temporal context on uncertainty Variable B SE t Event rate ratio [ 95 % con \ufb01 dence ratio ] df p Intercept , \u03b3 000 (cid:1) 1 . 20 0 . 15 (cid:1) 8 . 21 0 . 30 [ 0 . 23 , 0 . 40 ] 97 < . 001 Average words per utterance 0 . 11 0 . 02 6 . 23 1 . 11 [ 1 . 08 , 1 . 15 ] 463 < . 001 Topic Vector 1 (cid:1) 0 . 09 0 . 09 (cid:1) 1 . 01 0 . 91 [ 0 . 76 , 1 . 09 ] 463 . 315 Topic Vector 2 (cid:1) 0 . 22 0 . 12 (cid:1) 1 . 80 0 . 81 [ 0 . 64 , 1 . 02 ] 463 . 073 Early / later phase 0 . 14 0 . 08 1 . 76 1 . 15 [ 0 . 98 , 1 . 35 ] 97 . 082 Lagged con \ufb02 ict 0 . 13 0 . 07 1 . 67 1 . 13 [ 0 . 98 , 1 . 31 ] 463 . 095 Figure 2 . Predicted number of uncertain utterances controlling for covariates by time - lagged ( A ) process micro - con \ufb02 icts , ( B ) rover planning micro - con \ufb02 icts , and ( C ) science micro - con \ufb02 icts for early and later in the \ufb01 rst 90days of the mission 394 S . B . F . Paletz et al . Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e the rover would be . After the \ufb01 rst speaker strongly explains his or her opinion and the second responds with disagree - ment , the \ufb01 rst starts to express uncertainty about his or her proposed plan . In the second example , a scientist disagrees using uncertainty hedge words , but then after the disagree - ment , that scientist and two others express uncertainty about the nature of the geology they are observing . In both cases , the disagreements precede a signi \ufb01 cant increase in uncer - tainty rates . In summary , micro - con \ufb02 icts about rover planning and science con \ufb02 icts ( the aspects most central to work in the MER context ) were predictive of increases in subsequent psychological uncertainty , but there was no relation between process con \ufb02 icts and subsequent uncertainty . The associa - tion between science con \ufb02 icts and subsequent uncertainty depended on whether the science micro - con \ufb02 ict came earlier or later in the mission , with only early science micro - con \ufb02 icts increasing uncertainty . Association between uncertainty and subsequent con \ufb02 ict Testing the predictiveness of con \ufb02 ict for subsequent uncer - tainty is only half the story in understanding the ebb and \ufb02 ow of con \ufb02 ict and uncertainty . It is theoretically possible that high levels of uncertainty before con \ufb02 icts were related to high levels after the con \ufb02 ict and that the uncertainty was causing the con \ufb02 ict . Therefore , we analyzed our data using the same multilevel analysis approach ( except with logistic regression to account for the binary nature of the dependent variable , the presence of con \ufb02 ict in blocks , and using R \u2019 s multilevel modeling module lme4 ) , and controlling for the same set of covariates ( adding lagged within - domain analo - gies to predict process and science micro - con \ufb02 icts because they were found in prior research to be signi \ufb01 cantly related to these micro - con \ufb02 icts ; Paletz , Schunn , et al . , 2013 ) . We found that uncertainty was , in general , signi \ufb01 cantly nega - tively related to subsequent and concurrent micro - con \ufb02 icts . Indeed , we found that concurrent and previous uncertainty were signi \ufb01 cantly negatively related to rover planning : con - current uncertainty , t ( 462 ) = (cid:1) 2 . 54 , B = (cid:1) 0 . 23 , SE = 0 . 09 , odds ratio = 0 . 79 ( 0 . 66 , 0 . 95 ) , p = . 011 , and previous uncer - tainty , t ( 462 ) = (cid:1) 3 . 02 , B = (cid:1) 0 . 29 , SE = 0 . 10 , odds ratio = 0 . 75 ( 0 . 62 , 0 . 90 ) , p = . 003 . Similarly , process con \ufb02 icts were nega - tively predicted by concurrent uncertainty , t ( 461 ) = (cid:1) 3 . 36 , B = (cid:1) 0 . 40 , SE = 0 . 12 , odds ratio = 0 . 67 ( 0 . 53 , 0 . 84 ) , p < . 001 , and previous uncertainty , t ( 461 ) = (cid:1) 2 . 65 , B = (cid:1) 0 . 27 , SE = 0 . 10 , odds ratio = 0 . 76 ( 0 . 62 , 0 . 93 ) , p = . 008 . Previous uncertainty , but not concurrent uncertainty ( p > . 20 ) , was neg - atively related to subsequent science con \ufb02 ict , t ( 461 ) = (cid:1) 2 . 60 , B = (cid:1) 0 . 34 , SE = 0 . 13 , odds ratio = 0 . 71 ( 0 . 55 , 0 . 92 ) , p = . 010 . These \ufb01 ndings suggest that con \ufb02 ict of any type is less likely to occur at the same time as or immediately following relatively high levels of uncertainty . DISCUSSION Unpacking brief temporal relations between micro - processes is important for gaining a deeper understanding of team problem - solving processes . This study analyzed conversa - tions as a key medium for unpacking team problem solving , focusing on exploring the temporal association between expressed brief disagreements and subsequent uncertainty . Summary and interpretation of \ufb01 ndings We discovered that two different types of preceding task con \ufb02 ict predicted subsequent increases in psychological uncertainty : Planning task con \ufb02 ict was associated with an increase in uncertainty without any interaction with temporal phase , and science con \ufb02 ict was associated with an increase in uncertainty , but only early in the mission . Process con \ufb02 ict was unrelated to subsequent uncertainty . Testing for a poten - tial reverse association revealed that process , rover planning , and science con \ufb02 ict all decreased following uncertainty , suggesting that our \ufb01 ndings for con \ufb02 ict predicting increases in expressed uncertainty could not be due to a general Table 5 . Final model of lagged planning con \ufb02 ict and covariates on uncertainty Variable B SE t Event rate ratio [ 95 % con \ufb01 dence ratio ] df p Intercept , \u03b3 000 (cid:1) 1 . 18 0 . 15 (cid:1) 8 . 00 0 . 31 [ 0 . 23 , 0 . 41 ] 97 < . 001 Average words per utterance 0 . 11 0 . 02 6 . 27 1 . 11 [ 1 . 08 , 1 . 15 ] 463 < . 001 Topic Vector 1 (cid:1) 0 . 12 0 . 09 (cid:1) 1 . 29 0 . 89 [ 0 . 74 , 1 . 07 ] 463 . 198 Topic Vector 2 (cid:1) 0 . 22 0 . 12 (cid:1) 1 . 85 0 . 80 [ 0 . 63 , 1 . 01 ] 463 . 065 Early / later phase 0 . 13 0 . 08 1 . 69 1 . 14 [ 0 . 98 , 1 . 33 ] 97 . 095 Lagged planning con \ufb02 ict 0 . 22 0 . 11 2 . 09 1 . 25 [ 1 . 01 , 1 . 54 ] 463 . 037 Table 6 . Final model of lagged science con \ufb02 ict , temporal context , their interaction , and covariates on uncertainty Variable B SE t Event rate ratio [ 95 % con \ufb01 dence ratio ] df p Intercept , \u03b3 000 (cid:1) 1 . 19 0 . 15 (cid:1) 7 . 97 0 . 31 [ 0 . 23 , 0 . 41 ] 97 < . 001 Average words per utterance 0 . 11 0 . 02 6 . 10 1 . 11 [ 1 . 08 , 1 . 15 ] 462 < . 001 Topic Vector 1 (cid:1) 0 . 08 0 . 09 (cid:1) 0 . 91 0 . 92 [ 0 . 77 , 1 . 10 ] 462 . 366 Topic Vector 2 (cid:1) 0 . 22 0 . 12 (cid:1) 1 . 82 0 . 81 [ 0 . 64 , 1 . 02 ] 462 . 069 Early / later phase 0 . 16 0 . 08 2 . 05 1 . 17 [ 1 . 01 , 1 . 37 ] 97 . 043 Lagged science con \ufb02 ict 0 . 30 0 . 18 1 . 64 1 . 35 [ 0 . 94 , 1 . 93 ] 462 . 102 Lagged Science Con \ufb02 ict\u00d7 Early / Later Phase (cid:1) 0 . 51 0 . 23 (cid:1) 2 . 23 0 . 60 [ 0 . 39 , 0 . 94 ] 462 . 027 Uncovering uncertainty through disagreement 395 Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e positive association between uncertainty and con \ufb02 ict through some alternate factor . Taken together , these \ufb01 ndings suggest that individuals were more likely to disagree when they expressed more con \ufb01 dence and fewer hedge words and that con \ufb02 ict exposed underlying team uncertainties . Uncertainty is central to problem solving ( Kahneman & Tversky , 1982 ; Schunn , 2010 ; Schunn & Trafton , 2012 ) . Resolving uncertainty often entails sharing information previously unknown , whereas detecting uncertainty involves discovering what is unknown ( Schunn & Trafton , 2012 ) . Task con \ufb02 ict is commonly argued to be the least harmful to team success , at best ( e . g . , de Wit et al . , 2012 ; Nemeth , 1986 ) . Uncertainty is generally thought to be problematic , a relatively weak information state that needs to be improved . Does this \ufb01 nding imply that task con \ufb02 ict has yet another potentially unproductive function ? Such a value judgment is unwarranted given the grain size of the current analysis . Micro - con \ufb02 icts and expressed uncertainty are part of complex team dynamics that iterate many times and gradually unfold . In fact , the discovered dynamic may simply be a core characteristic of normal team functioning . To interpret these \ufb01 ndings , we examine the MER context more closely and draw on the shared mental models theory ( e . g . , Burke et al . , 2006 ; Mathieu et al . , 2000 ) , which de - scribes the development of team cognition . We suggest that task con \ufb02 ict in this case may have uncovered disconnects between the multidisciplinary scientists \u2019 mental models , and the rise in uncertainty following task con \ufb02 ict may have re \ufb02 ected the identi \ufb01 cation of newly exposed problems . There was a great deal of uncertainty in conducting science on Mars in general . Although levels of uncertainty were not generally different between early and later in the \ufb01 rst 90 days of the mission , disagreements about science were more likely to involve an increase in subsequent uncertainty Table 7 . Examples of micro - con \ufb02 icts preceding increases in uncertainty Coded con \ufb02 ict Coded uncertainty a Speaker number Utterance Example 1 : planning micro - con \ufb02 ict preceding more uncertainty Planning 0 1 No , no , no , no Planning 0 1 If you want to do the integration on the RAT [ Rock Abrasion Tool ] , Planning 0 1 you \u2019 re going to have to do some additional brushing Planning 0 1 because you don \u2019 t want crap in the brushing in the gravel . Planning 0 1 So you pretty much go through the standard sequence , Planning 0 1 and the advantage of that is that you now have looked at all the different depths . Planning 0 2 But what do you , what do you do with that ? ? Planning 0 2 So after you \u2019 ve done the integration , Planning 0 2 then you do the \ufb01 nal brush with backup . None 0 1 Right , you can \u2019 t do any more , more brushing None 0 1 You don \u2019 t want to contaminate the hole None 1 1 So that could be one way you could get that Minitest [ another instrument ] observation , None 0 1 and not a lot of < missing word > in one sol . None 0 1 The only thing that \u2019 s scary about that is that you drive away not knowing what you got in the Minitest observation , None 0 1 but it \u2019 s just a backup you know of 185 . None 0 1 I \u2019 ll show you what we \u2019 re talking about . None 0 1 So that \u2019 s one option . None 0 1 Then after you start to drive , None 1 1 but I guess you \u2019 re on for what , two more sols , so pretty much . Example 2 : science micro - con \ufb02 ict early in mission ( sol 16 ) preceding more uncertainty None 0 3 So these aren \u2019 t exactly perfect . None 0 3 These are actually old < missing words > weathered out . Science 0 4 Well , but if you think about it , Science 1 4 the history of this thing post - impact , you know , maybe , maybe these have , um , formed over time . None 0 1 Well , do you think it could be impact generated ? None 1 4 No , not impact generated , \u2019 cause they maybe a certain , well I don \u2019 t know . Science 0 1 Well , the problem is they look very speci \ufb01 c , it \u2019 s not even penetrative < missing word > . None 0 4 No , you \u2019 re right , None 1 4 there would be if , there would have to be some fundamental difference in the hardness of those layers . None 0 2 You know , one thing you can do on that is to say None 1 2 that maybe these were , that there is a sulfate cement in there < missing words > , None 1 2 but they don \u2019 t really look like < missing words > . None 0 1 See , I love all this < missing words > None 0 1 If you were on Earth you would just simply call that a nodular fabric None 0 1 and go about your business None 0 1 and then you \u2019 d speculate about whether it was nodular because of early cementation , pearling , or pressure solution None 1 1 and probably here we can rule out the last two . a Zero is not coded as having uncertainty , 1 is coded as having uncertainty . 396 S . B . F . Paletz et al . Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e early in the mission . The scientists during that phase were likely still developing their mental models ( individually and jointly ) of how and whether liquid water had existed on Mars \u2014 the high - level topic of the science con \ufb02 icts . Roughly a third of the way through the original mission , the scientists felt con \ufb01 dent that they had discovered evidence of historical liquid water on Mars . At this stage , mental models regarding the science may have become more shared across the scientists , affecting the interpretation of new \ufb01 nd - ings . This turning point in scienti \ufb01 c discovery may have constrained the types of new information that could arise from disagreement , such that disagreements after that point were no longer positively , signi \ufb01 cantly related to uncertainty . However , issues of what instrument readings and activi - ties to do with the rovers ( i . e . , planning activities ) remained both contentious and uncertain throughout the mission . The different subgroups of scientists ( e . g . , soil science and atmospheres ) each wanted to conduct their own instrument readings , and each rover team had to prioritize and plan what the rovers would do . For instance , the scientists would debate whether they should have a rover ( i . e . , Rover A , Spirit ) stay in one location and conduct additional instrument readings or drive to another location . Thus , these kinds of arguments could be due to differences in explicit goals in addition to differences in perceptions of the utility of doing instrument readings in a particular location . Furthermore , there was still a great deal unknown about what the best course of action would be , either scienti \ufb01 cally or logistically . The scientists did not know at the time that the rovers would last several years past their initial 90 - day life . Thus , the underlying differences in goals , assumptions , and mental models of what the best courses of action would be may have continued throughout the \ufb01 rst 90 days of the mission , unchanged by the discovery of historical liquid water . Disagreements would still lead to greater uncertainty , because they would point out the missing information inherent in trying to plan rover activities on Mars . Uncertainty could , and did , lead to effective problem solv - ing , even within the moment . Indeed , previous research we conducted on this dataset found a signi \ufb01 cant positive associa - tion between uncertainty and subsequent problem - related anal - ogy , with a return to baseline uncertainty after the analogy was mentioned ( Chan et al . , 2012 ) . This study also found that the most common topic of uncertainty was science problem solv - ing , suggesting that much of the uncertainty was central to their primary problem solving . For example , one scientist expressed uncertainty about the cause of observed ventifacting ( a geomorphic feature on rocks ) and uncertainty about a poten - tial mechanism for the ventifacting , desert varnish ( Chan et al . , 2012 , p . 1361 ) . That scientist and two others proceeded to discuss the problem . One of the scientists made an analogy to how desert varnish works on Earth and a later analogy to \ufb01 ndings from a Viking mission . Between the three scientists , they identi \ufb01 ed several possible reasons for ventifacting and the known causes of desert varnish that could be occurring on Mars . This anecdote and the data supporting it illustrate how uncertainty in the MER context could , and did , precede problem solving ( Chan et al . , 2012 ) . Thus , if task con \ufb02 ict is leading to uncertainty in this domain , that uncertainty then has the potential to lead to problem solving . Previous \ufb01 ndings suggest that self - reported assessments of task con \ufb02 ict have a curvilinear ( inverse - U ) association with team performance ( e . g . , Fahr et al . , 2010 ; Shaw et al . , 2011 ) . This study unpacks one possible function of task con \ufb02 ict at the process level : uncovering areas of uncertainty . Relating to the curvilinear pattern with success , it may be that low levels of task con \ufb02 ict leave critical misunderstan - dings or mental model gaps hidden and that very high levels of task con \ufb02 ict unearth more uncertainty than can be resolved . Alternately , these very high levels of con \ufb02 ict inhibit the resolution of the uncertainty via limiting the team \u2019 s ability to build shared mental models . These hypo - theses should be investigated in future research . Strengths and limitations A bene \ufb01 t of this study is that it studied real - world problem solving at the micro - temporal level . Too often , detailed pro - cess studies of actual behaviors are limited to the research lab with undergraduate participants completing arti \ufb01 cial tasks over limited time periods , and self - report methods ( survey or interviews ) are used to study actual work contexts . Even the rare communication study of con \ufb02 ict as it arises from natural conversations does not ask the research ques - tions posed here ( e . g . , Poole & Dobosh , 2010 ) . However , one limitation of the methods used in this study involves the lower reliabilities associated with coding micro - con \ufb02 ict types . There are multiple factors underlying lower measure reliability than that typically found with some other approaches . For one , the coding scheme for this study follows traditional examinations of con \ufb02 ict types as distinct ( i . e . , task , process , and relationship ; Jehn , 1995 ) , but these dimensions were developed on self - report measures , inter - views , and larger grain sizes in observations of teams . It can be dif \ufb01 cult for even trained coders to distinguish between these types at the utterance . To reduce the negative effects of lowered reliability on the statistical power of the analyses , the target behavior was 100 % double coded , with all disagreements resolved by discussion , giving us more con \ufb01 dence in our eventual resolutions ( Smith , 2000 ) . Further , the operationalization of micro - con \ufb02 icts was at an aggregated level , not the utterance level , helping to reduce noise due to lower reliabilities . Another bene \ufb01 t of the methods used in this study is the di - versity of individuals and tasks . For example , it examined in - dividuals across a range of professions ( university scientist , government lab scientist , and industry engineer ) and along a range of skill levels ( e . g . , from famous full professors to early graduate students ) . The scientists also accomplished a large variety of tasks , including dealing with anomalies , planning , scheduling , data analysis , e - mailing , taking photo - graphs , and so on . Thus , even though these conversations were taken from one larger context ( i . e . , the MER mission ) , the sample was quite diverse in many other ways . At the same time , there were some unique features of the sample that may limit its generalizability . The scientists were overall an extremely successful team . They had complex processes and worked during the \ufb01 rst 90 days on Mars time schedules , getting progressively out of sync with Earth day / night cycles . While some of these features may be Uncovering uncertainty through disagreement 397 Copyright \u00a9 2016 John Wiley & Sons , Ltd . Appl . Cognit . Psychol . 30 : 387 \u2013 400 ( 2016 ) 10990720 , 2016 , 3 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / ac p . 3213 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 03 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e generalizable ( complex processes and expert team ) , some are unique ( e . g . , working on Mars time ) , which means the \ufb01 nd - ings may not transfer to other settings . Furthermore , even given transcribing and coding over 11 hours of conversa - tions , we acknowledge that these conversations were only a sample of what the scientists discussed , on and off camera . Future studies could examine data evenly across the life of a team and so uncover with greater precision when the temporal moderation effects occur . Implications and future directions Scienti \ufb01 c problem solving often attempts to resolve uncer - tainty , and uncertainty may drive scienti \ufb01 c discovery ( Schunn , 2010 ; Schunn & Trafton , 2012 ) . Even before prob - lem solving is used to resolve uncertainty , however , what is unknown needs to be identi \ufb01 ed . Problem \ufb01 nding is a neces - sary precursor to problem solving . This paper illuminates a possible , contingent association between brief disagreements and immediate , subsequent psychological uncertainty , join - ing with other studies that examine the immediate conse - quences of brief disagreements ( e . g . , Chiu , 2008a , 2008b ; Kauffeld & Lehmann - Willenbrock , 2012 ; Paletz et al . , 2013 ) . Future research could continue to unpack potential contextual and other moderators of this micro - con \ufb02 ict / uncertainty connection . This study supports theorizing that different types of con \ufb02 ict may play different roles in team processes . For instance , process con \ufb02 ict early in a team \u2019 s life cycle may be positively related to eventual team perfor - mance , perhaps because it prevents premature closure ( Goncalo et al . , 2010 ) . A \ufb01 ne - grained analysis , as conducted in this study , of multiple teams \u2019 process micro - con \ufb02 icts in their early stages can shed light onto how they may in \ufb02 uence uncertainty and closure on speci \ufb01 c decisions . This paper also has implications for the role of uncertainty in problem - solving conversations . Future directions might also include qualitative studies of different types of uncer - tainty ( e . g . , Lipshitz & Strauss , 1997 ) and how the uncer - tainty that arises after a con \ufb02 ict may serve a different role in problem - solving conversations compared with other types of uncertainty . For instance , how is decision making impacted by whether or not a con \ufb02 ict \u2013 uncertainty pair is present ? Additional studies could also examine whether the uncertainty that arises after a micro - con \ufb02 ict is related to problem \ufb01 nding and restructuring , exposing gaps in informa - tion and / or freeing individuals to share their concerns . This paper advances future theorizing about team pro - cesses by examining a new association at a micro - temporal level . Of all the potential functions of disagreement , uncertainty reduction or increase has been barely studied . Examining micro - con \ufb02 icts serves to shift the lens of scienti \ufb01 c examination from mostly static snapshots of broad team perceptions to examining the interplay of different types of quick conversational acts . Practically , such research and theory could help guide the creation of team facilitation techniques and suggestions for how teams can better leverage their disagreements for speci \ufb01 c problem - solving processes . Much of the literature on con \ufb02 ict focuses on reducing or mitigating it . This paper suggests that con \ufb02 ict resolution training could also be expanded to emphasize the useful cognitive functions of small disagreements , such as questioning underlying assumptions and restructuring problems . Once uncertainty is brought into the open , new problems can be explicitly addressed . Problem solving , particularly in multidisciplinary teams , is vital for scien - ti \ufb01 c innovation , economic progress , and \ufb01 nding of solutions to pressing societal problems . By unpacking problem - solving conversations as they occur in real - world successful expert teams , we discover more about their functioning . ACKNOWLEDGEMENTS This research was supported in part by the United States National Science Foundation ( NSF ) Science of Science and Innovation Policy Program grants # SBE - 1064083 to the \ufb01 rst author and NSF grant # SBE - 0830210 to the \ufb01 rst and third authors when the \ufb01 rst author was at the Learning Research and Development Center at the University of Pittsburgh . We wish to thank Kevin Kim and Mathilda du Toit for their statistical advice . The authors are grateful to Alonso Vera , Irene Tollinger , Preston Tollinger , Mike McCurdy , and Tyler Klein for their assistance in data collection ; Lauren Gunn , Julia Cocchia , Carl Fenderson , Justin Krill , Kerry Glassman , Tiemoko Ballo , Rebecca Sax , Michael Ye , and Candace Smalley for coding ; and Carmela Rizzo for research management support . These data were coded while the \ufb01 rst and second authors were at the University of Pittsburgh . REFERENCES Amason , A . C . ( 1996 ) . 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Joel Chan , Learning Research and Development Center , University of Pittsburgh , LRDC Room 823 , 3939 O\u2019Hara St , Pittsburgh , PA 15260 , USA Steven P . Dow , Human e Computer - Interaction Institute , Carnegie Mellon University , Pittsburgh , PA , USA Christian D . Schunn , Learning Research and Development Center , University of Pittsburgh , Pittsburgh , PA , USA Design ideas often come from sources of inspiration ( e . g . , analogous designs , prior experiences ) . In this paper , we test the popular but unevenly supported hypothesis that conceptually distant sources of inspiration provide the best insights for creative production . Through text analysis of hundreds of design concepts across a dozen di\ufb00erent design challenges on a Web - based innovation platform that tracks connections to sources of inspiration , we \ufb01nd that citing sources is associated with greater creativity of ideas , but conceptually closer rather than farther sources appear more bene\ufb01cial . This inverse relationship between conceptual distance and design creativity is robust across di\ufb00erent design problems on the platform . In light of these \ufb01ndings , we revisit theories of design inspiration and creative cognition . (cid:1) 2014 Elsevier Ltd . All rights reserved . Keywords : innovation , design cognition , creative design , conceptual design , sources of inspiration W here do creative design ideas come from ? Cognitive scientists have discovered that people inevitably build new ideas from their prior knowledge and experiences ( Marsh , Ward , & Landau , 1999 ; Ward , 1994 ) . While these prior experiences can serve as sources of inspiration ( Eckert & Stacey , 1998 ) and drive sustained creation of ideas that are both new and have high potential for impact ( Hargadon & Sutton , 1997 ; Helms , Vattam , & Goel , 2009 ) , they can also lead designers astray : for instance , de - signers sometimes incorporate undesirable features from existing solutions ( Jansson & Smith , 1991 ; Linsey et al . , 2010 ) , and prior knowledge can make it di\ufb03cult to think of alternative approaches ( German & Barrett , 2005 ; Wiley , 1998 ) . This raises the question : what features of potential inspi - rational sources can predict their value ( and / or potential harmful e\ufb00ects ) ? In this paper , we examine how the conceptual distance of sources relates to their inspirational value . Corresponding author : Joel Chan joc59 @ pitt . edu joelchuc @ cs . cmu . edu www . elsevier . com / locate / destud 0142 - 694X Design Studies - - ( 2014 ) - - e - - http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 1 (cid:1) 2014 Elsevier Ltd . All rights reserved . Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 1 Background 1 . 1 Research base What do we mean by conceptual distance ? Consider the problem of e - waste accumulation : the world generates 20 e 50 million metric tons of e - waste every year , yielding environmentally hazardous additions to land\ufb01lls . A designer might approach this problem by building on near sources like smaller - scale electronics reuse / recycle e\ufb00orts , or by drawing inspiration from a far source like edible food packaging technology ( e . g . , to design re - usable electronics parts ) . What are the relative bene\ufb01ts of di\ufb00erent levels of source conceptual distance along a continuum from near to far ? Many authors , principally those studying the role of analogy in creative prob - lem solving , have proposed that conceptually far sources d structurally similar ideas with many surface ( or object ) dissimilarities d are the best sour - ces of inspiration for creative breakthroughs ( Gentner & Markman , 1997 ; Holyoak & Thagard , 1996 ; Poze , 1983 ; Ward , 1998 ) . This proposal d here called the Conceptual Leap Hypothesis d is consistent with many anecdotal accounts of creative breakthroughs , from Kekule\u2019s discovery of the structure of benzene by visual analogy to a snake biting its tail ( Findlay , 1965 ) , to George Mestral\u2019s invention of Velcro by analogy to burdock root seeds ( Freeman & Golden , 1997 ) , to more recent case studies ( Enkel & Gassmann , 2010 ; Kalogerakis , Lu , & Herstatt , 2010 ) . However , empirical support for this proposal is mixed . Some studies have shown an advantage of far over near sources for novelty , quality , and \ufb02ex - ibility of ideation ( Chan et al . , 2011 ; Chiu & Shu , 2012 ; Dahl & Moreau , 2002 ; Gonc\u00b8alves , Cardoso , & Badke - Schaub , 2013 ; Hender , Dean , Rodgers , & Jay , 2002 ) ; but , some in vivo studies of creative cognition have not found strong connections between far sources and creative mental leaps ( Chan & Schunn , 2014 ; Dunbar , 1997 ) , and other experiments have demonstrated equivalent bene\ufb01ts of far and near sources ( Enkel & Gassmann , 2010 ; Malaga , 2000 ) . Relatedly , Tseng , Moss , Cagan , and Kotovsky ( 2008 ) showed that far sources were more impactful after ideation had already begun ( vs . before ideation ) , providing more functionally distinct ideas than near or control , but both far and near sources led to similar levels of novelty . Similarly , Wilson , Rosen , Nelson , and Yen ( 2010 ) showed no advantage of far over near sources for novelty of ideas ( although near but not far sources decreased variety of ideas ) . Fu et al . ( 2013 ) even found that far sources led to lower novelty and quality of ideas than near sources . Thus , more empirical work is needed to determine whether the Conceptual Leap Hypothesis is well supported . Further , Fu et al . ( 2013 ) argue there is an inverted U - shape function in which moderate distance is best , suggesting 2 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 the importance of conceptualizing and measuring distance along a continuum . 1 . 2 Impetus for the current work Key methodological shortcomings in prior work further motivate more and better empirical work . Prior studies may be too short ( typically 30 min to 1 h ) to convert far sources into viable concepts . To successfully use far sources , designers must spend considerable cognitive e\ufb00ort to ignore irrelevant surface details , attend to potentially insightful structural similarities , and adapt the source to the target context . Additionally , many far sources may yield shallow or unusable inferences ( e . g . , due to non - alignable di\ufb00erences in structural or surface features ; Perkins , 1997 ) ; thus , designers might have to sift through many samples of far sources to \ufb01nd \u2018hidden gems . \u2019 These higher processing costs for far sources might partially explain why some studies show a negative impact of far sources on the number of ideas generated ( Chan et al . , 2011 ; Hender et al . , 2002 ) . In the context of a short task , these processing costs might take up valuable time and resources that could be used for other important as - pects of ideation ( e . g . , iteration , idea selection ) ; in contrast , in real - world design contexts , designers typically have days , weeks or even months ( not an hour ) to consider and process far sources . A second issue is a lack of statistical power . Most existing experimental studies have N (cid:1) 12 per treatment cell ( Chiu & Shu , 2012 ; Hender et al . , 2002 ; Malaga , 2000 ) ; only four studies had N (cid:3) 18 ( Chan et al . , 2011 ; Fu et al . , 2013 ; Gonc\u00b8alves et al . , 2013 ; Tseng et al . , 2008 ) , and they are evenly split in support / opposition for the bene\ufb01ts of far sources . Among the few correlational studies , only Dahl and Moreau ( 2002 ) had a well powered study design in this regard , with 119 participants and a reasonable range of concep - tual distance . Enkel and Gassmann ( 2010 ) only examined 25 cases , all of which were cases of cross - industry transfer ( thus restricting the range of con - ceptual distance being considered ) . This lack of statistical power may have led to a proliferation of false negatives ( potentially exacerbated by small or potentially zero e\ufb00ects at short time scales ) , but possibly also severely over - estimated e\ufb00ect sizes or false positives ( Button et al . , 2013 ) ; more adequately powered studies are needed for more precise estimates of the e\ufb00ects of con - ceptual distance . A \ufb01nal methodological issue is problem variation . Many experimental studies focused on a single design problem . The inconsistent outcomes in these studies may be partially due to some design problems having unique characteristics , e . g . , coincidentally having good solutions that overlap with concepts in far sources . Indeed , Chiu and Shu ( 2012 ) , who examined multiple design prob - lems , observed inconsistent e\ufb00ects across problems . Other investigations of design stimuli have also observed problem variation for e\ufb00ects ( Goldschmidt & Smolkov , 2006 ; Liikkanen & Perttula , 2008 ) . Inspiration source distance and design ideation 3 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 This paper contributes to theories of design inspiration by 1 ) reporting the re - sults of a study that addresses these methodological issues to yield clearer ev - idence , and 2 ) ( to foreshadow our results ) re - examining theories of design inspiration and conceptual distance in light of accumulating preponderance of evidence against the Conceptual Leap Hypothesis . 2 Methods 2 . 1 Overview of research context The current work is conducted in the context of OpenIDEO ( www . openideo . - com ) , a Web - based crowd - sourced innovation platform that addresses a range of social and environmental problems ( e . g . , managing e - waste , increasing accessibility in elections ) . The OpenIDEO designers , with expertise in design processes , guide contributors to the platform through a structured design pro - cess to produce concepts that are ultimately implemented for real - world impact ( \u2018Impact Stories , \u2019 n . d . ) . For this study , we focus on three crucial early stages in the process : \ufb01rst , in the inspiration phase ( lasting between 1 . 5 and 4 weeks , M \u00bc 3 . 1 ) , contributors post inspirations ( e . g . , descriptions of solutions to analogous problems and case studies of stakeholders ) , which help to de\ufb01ne the problem space and identify promising solution approaches ; then , in the concepting phase ( lasting the next 2 e 6 weeks , m \u00bc 3 . 4 ) , contributors post con - cepts , i . e . , speci\ufb01c solutions to the problem . Figure 1 shows an example concept ; it is representative of the typical length and level of detail in concepts , i . e . , w 150 words on average , more detail than one or two words / sentences / sketches , but less detail than a full - \ufb02edged design report / presentation or patent application . Finally , a subset of these concepts is shortlisted by an expert panel ( composed of the OpenIDEO designers and a set of domain experts / stake - holders ) for further re\ufb01nement , based on their creative potential . In later stages , these concepts are re\ufb01ned and evaluated in more detail , and then a sub - set of them is selected for implementation . We focus on the \ufb01rst three stages given our focus on creative ideation ( the later stages involve many other design processes , such as prototyping ) . The OpenIDEO platform has many desirable properties as a research context for our work , including the existence of multiple design problems , thousands of concepts and inspirations , substantive written descriptions of ideas to enable e\ufb03cient text - based analyses , and records of feedback received for each idea , another critical factor in design success . A central property for our research question is the explicit nature of sources of inspiration in the OpenIDEO work\ufb02ow . The site encourages contributors to build on others\u2019 ideas . Importantly , when posting concepts or inspirations , contributors are prompted to cite any concepts or inspirations that serve as sources of inspira - tion for their idea . Also , when browsing other concepts / inspirations , they are able to also see concepts / inspirations the given concept / inspiration \u2018built upon\u2019 ( i . e . , cited as explicit sources of inspiration ; see Figure 2 ) . This culture 4 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 Figure 1 Example concept illustrating the typical amount of detail per concept Figure 2 Depiction of OpenIDEO citation work\ufb02ow . When posting concepts / inspirations , users are prompted to cite concepts / inspirations they \u2018build upon\u2019 by dragging bookmarked concepts / inspirations ( middle panel ) to the citation area ( left panel ) . Users can also search for related concepts / inspirations at this step ( middle panel ) . These cited sources then show up as metadata for the concept / inspiration ( right panel ) Inspiration source distance and design ideation 5 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 of citing sources is particularly advantageous , given that people generally forget to monitor or cite their sources of inspiration ( Brown & Murphy , 1989 ; Marsh , Landau , & Hicks , 1997 ) , and our goal is to study the e\ufb00ects of source use . While users might still forget to cite sources , these platform fea - tures help ensure higher rates of source monitoring than other naturalistic ideation contexts . We note that this operationalization of sources as self - identi\ufb01ed citations precludes consideration of implicit stimulation ; however , the Conceptual Leap Hypothesis may be more applicable to conscious inspi - ration processes ( e . g . , analogy , for which conscious processing is arguably an important de\ufb01ning feature ; Schunn & Dunbar , 1996 ) . 2 . 2 Sample and initial data collection The full dataset for this study consists of 2341 concepts posted for 12 completed challenges by 1190 unique contributors , citing 4557 unique inspira - tions ; 241 ( 10 % ) of these concepts are shortlisted for further re\ufb01nement . See Table 2 for a description of the 12 challenges ( with some basic metadata on each challenge ) . Figure 3 shows the full - text design brief for two challenges . Figure 3 Full - text of challenge briefs from two OpenIDEO challenges 6 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 With administrator permission , we downloaded all inspirations and concepts ( which exist as individual webpages ) and used an HTML parser to extract the following data and metadata : 1 ) Concept / inspiration author ( who posted the concept / inspiration ) 2 ) Number of comments ( before the re\ufb01nement phase ) 3 ) Shortlist status ( yes / no ) 4 ) List of cited sources of inspiration 5 ) Full - text of concept / inspiration Not all concepts cited inspirations as sources . Of the 2341 concepts , 707 ( posted by 357 authors ) cited at least one inspiration , collectively citing 2245 unique inspirations . 110 of these concepts ( w 16 % ) were shortlisted ( see Table 1 for a breakdown by challenge ) . This set of 707 concepts is the primary sample for this study ; the others serve as a contrast to examine the value of explicit building at all on prior sources , and to aid in interpretation of any negative or positive e\ufb00ects of variations in distance . Because we only collected publicly available data , we do not have complete information on the expertise of all contributors : however , based on their public pro\ufb01les on OpenIDEO , at least 1 / 3 of the authors in this sample are professionals in design - related disci - plines ( e . g . , user experience / interaction design , communication design , archi - tecture , product / industrial design , entrepreneurs and social innovators , etc . ) and / or domain experts or stakeholders ( e . g . , urban development researcher Table 1 Descriptions and number of posts for OpenIDEO challenges in final analysis sample Name / description # of Inspirations # of Concepts ( shortlisted ) How might we increase the number of registered bone marrow donors to help save more lives ? 186 71 ( 7 ) How might we inspire and enable communities to take more initiative in making their local environments better ? 160 44 ( 11 ) How can we manage e - waste & discarded electronics to safeguard human health & protect our environment ? 60 26 ( 8 ) How might we better connect food production and consumption ? 266 147 ( 10 ) How can technology help people working to uphold human rights in the face of unlawful detention ? 248 62 ( 7 ) How might we identify and celebrate businesses that innovate for world bene\ufb01t and inspire other companies to do the same ? 122 24 ( 13 ) How might we use social business to improve health in low - income communities ? 131 46 ( 11 ) How might we increase social impact with OpenIDEO over the next year ? 67 40 ( 12 ) How might we restore vibrancy in cities and regions facing economic decline ? 558 119 ( 13 ) How might we design an accessible election experience for everyone ? 241 47 ( 8 ) How might we support web entrepreneurs in launching and growing sustainable global businesses ? 88 49 ( 7 ) How can we equip young people with the skills , information and opportunities to succeed in the world of work ? 118 32 ( 3 ) Inspiration source distance and design ideation 7 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 contributing to the vibrant - cities challenge , education policy researcher contributing to the youth - employment challenge , medical professional contributing to the bone - marrow challenge ) . Collectively , these authors ac - counted for approximately half of the 707 concepts in this study . We analyze the impact of the distance of inspirations ( and not cited concepts ) given our focus on ideation processes during \u2018original\u2019 or non - routine design , where designers often start with a problem and only \u2018inspirations\u2019 ( e . g . , infor - mation about the problem or potentially related designs ) rather than routine design ( e . g . , con\ufb01guration or parametric design ) , where designers might be modifying or iterating on existing solutions rather than generating novel ones ( Chakrabarti , 2006 ; Dym , 1994 ; Gero , 2000 ; Ullman , 2002 ) . The Concep - tual Leap Hypothesis maps most clearly to non - routine design . 2 . 3 Measures 2 . 3 . 1 Creativity of concepts We operationalize concept creativity as whether a concept gets shortlisted . Shortlisting is done by a panel of expert judges , including the original chal - lenge sponsors , who have spent signi\ufb01cant time searching for and learning about existing approaches , and the OpenIDEO designers , who are experts in the general domain of creative design , and who have spent considerable time upfront with challenge sponsors learning about and de\ufb01ning the problem space for each challenge . An expert panel is widely considered a \u2018gold standard\u2019 for measuring the crea - tivity of ideas ( Amabile , 1982 ; Baer & McKool , 2009 ; Brown , 1989 ; Sawyer , 2012 ) . Further , we know from conversations with the OpenIDEO team that the panel\u2019s judgments combines consideration of both novelty and useful - ness / appropriateness ( here operationalized as potential for impact ; A . Jablow , personal communication , May 1 , 2014 ) , the standard de\ufb01nition of creativity ( Sawyer , 2012 ) . Since OpenIDEO challenges are novel and unsolved , success - ful concepts are di\ufb00erent from ( and , perhaps more importantly , signi\ufb01cantly better than ) existing unsatisfactory solutions . We use shortlist ( rather than win status ) given our focus on the ideation phase in design ( vs . convergence / re\ufb01nement , which happens after concepts are shortlisted , and can strongly in\ufb02uence which shortlisted concepts get selected as \u2018winners\u2019 for implementation ) . 2 . 3 . 2 Conceptual distance 2 . 3 . 2 . 1 Measurement approach . Measuring conceptual distance is a major methodological challenge , especially when studying large samples of ideation processes ( e . g . , many designs across many design problems ) . The complex and multifaceted nature of typical design problems can make it di\ufb03cult to distin - guish \u2018within\u2019 and \u2018between\u2019 domain sources in a consistent and principled 8 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 manner . Further , using only a binary scale risks losing variance information that could be critical for converging on a more precise understanding of the e\ufb00ects of conceptual distance ( e . g . , curvilinear e\ufb00ects across the continuum of distance ) . Continuous distance measures are an attractive alternative , but can be extremely costly to obtain at this scale , especially for naturalistic sour - ces ( e . g . , relatively developed text descriptions vs . simple sketches or one - to - two sentence descriptions ) . Human raters may su\ufb00er from high levels of fa - tigue , resulting in poor reliability or drift of standards . We address this methodological challenge with probabilistic topic modeling ( Blei , 2012 ; Steyvers & Gri\ufb03ths , 2007 ) , a major computational approach for understanding large collections of unstructured text . They are similar to other unsupervised machine learning methods d e . g . , K - means clustering , and Latent Semantic Analysis ( Deerwester , Dumais , Furnas , & Landauer , 1990 ) d but distinct in that they emphasize human understanding of not just the relationship between documents in a collection , but the \u2018reasons\u2019 for the hy - pothesized relationships ( e . g . , the \u2018meaning\u2019 of particular dimensions of vari - ation ) , largely because the algorithms underlying these models tend to produce dimensions in terms of clusters of tightly co - occurring words . Thus , they have been used most prominently in applications where understanding of a corpus , not just information retrieval performance , is a high priority goal , e . g . , knowl - edge discovery and information retrieval in repositories of scienti\ufb01c papers ( Gri\ufb03ths & Steyvers , 2004 ) , describing the structure and evolution of scienti\ufb01c \ufb01elds ( Blei & La\ufb00erty , 2006 , 2007 ) , and discovering topical dynamics in social media use ( Schwartz et al . , 2013 ) . We use Latent Dirichlet Allocation ( LDA ; Blei , Ng , Jordan , & La\ufb00erty , 2003 ) , the simplest topic model . LDA assumes that documents are composed of a mixture of latent \u2018topics\u2019 ( occurring with di\ufb00erent \u2018weights\u2019 in the mixture ) , which in turn generate the words in the documents . LDA de\ufb01nes topics as probability distributions over words : for example , a \u2018genetics\u2019 topic can be thought of as a probability distribution over the words { phenotype , popula - tion , transcription , cameras , quarterbacks } , such that words closely related to the topic { phenotype , population , transcription } have a high probability in that topic , and words not closely related to the topic { cameras , quarter - backs } have a very low probability . Using Bayesian statistical learning algo - rithms , LDA infers the latent topical structure of the corpus from the co - occurrence patterns of words across documents . This topical structure in - cludes 1 ) the topics in the corpus , i . e . , the sets of probability distributions over words , and 2 ) the topic mixtures for each document , i . e . , a vector of weights for each of the corpus topics for that document . We can derive concep - tual similarity between any pair of documents by computing the cosine be - tween their topic - weight vectors . In essence , documents that share dominant topics in similar relative proportions are the most similar . Inspiration source distance and design ideation 9 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 Here , we used the open - source MAchine Learning for LanguagE Toolkit ( MALLET ; McCallum , 2002 ) to train an LDA model with 400 topics for all documents in the full dataset , i . e . , 2341 concepts , 4557 inspirations , and 12 challenge briefs ( 6910 total documents ) . Additional technical details on the model - building procedure are available in Appendix A . Resulting cosines be - tween inspirations and the challenge brief ranged from 0 . 01 to 0 . 91 ( M \u00bc 0 . 21 , SD \u00bc 0 . 18 ) , a fairly typical range for large - scale information retrieval applica - tions ( Jessup & Martin , 2001 ) . 2 . 3 . 2 . 2 Validation . Since we use LDA\u2019s measures of conceptual distance as a substitute for human judgments , we validate the adequacy of our topic model using measures of \ufb01t with human similarity judgments on a subset of the data by trained human raters . Five trained raters used a Likert - type scale to rate 199 inspirations from one OpenIDEO challenge for similarity to their challenge brief , from 1 ( very dissim - ilar ) to 6 ( extremely similar ) . Raters were given the intuition that the rating would approximately track the proportion of \u2018topical overlap\u2019 between each inspiration and the challenge brief , or the extent to which they are \u2018about the same thing . \u2019 The design challenge context was explicitly deemphasized , so as to reduce the in\ufb02uence of individual di\ufb00erences in perceptions of the \u2018relevance\u2019 of sources of inspiration . Thus , the raters were instructed to treat all the docu - ments as \u2018documents\u2019 ( e . g . , an article about some topics , vs . \u2018problem solution\u2019 ) and consciously avoid judging the \u2018value\u2019 of the inspirations , simply focusing on semantic similarity . Raters listed major topics in the challenge brief and eval - uated each inspiration against those major topics . To ensure internal consis - tency , the raters also sorted the inspirations by similarity after every 15 e 20 judgments . They then inspected the rank ordering and composition of inspira - tions at each point in the scale , and made adjustments if necessary ( e . g . , if an inspiration previously rated as \u20181\u2019 now , in light of newly encountered inspira - tions , seemed more like a \u20182\u2019 or \u20183\u2019 ) . Although the task was di\ufb03cult , the mean ratings across raters had an acceptable aggregate consistency intra - class corre - lation coe\ufb03cient ( ICC ( 2 , 5 ) ) of 0 . 74 ( mean inter - coder correlation \u00bc 0 . 36 ) . LDA cosines correlated highly , at r \u00bc 0 . 51 , 95 % CI \u00bc [ 0 . 40 , 0 . 60 ] , with the contin - uous human similarity judgments ( see Figure 4A ) . We note that this correlation is better than the highest correlation between human raters ( r \u00bc 0 . 48 ) , reinforc - ing the value of automatic coding methods for this di\ufb03cult task . For comparability with prior work , we also measure \ufb01t with binary ( within - vs . between - domain ) distance ratings . Two raters also classi\ufb01ed 345 inspirations from a di\ufb00erent challenge as either within - or between - domain . Raters \ufb01rst collaboratively de\ufb01ned the problem domain , focusing on the question , \u2018What is the problem to be solved ? \u2019 before rating inspirations . Within - domain inspi - rations were information about the problem ( e . g . , stakeholders , constraints ) 10 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 and existing prior solutions for very similar problems , while between - domain inspirations were information / solutions for analogous or di\ufb00erent problems . Reliability for this measure was acceptable , with an overall average kappa of 0 . 78 ( 89 % agreement ) . All disagreements were resolved by discussion . Similar to the continuous similarity judgments , the point biserial correlation between the LDA - derived cosine and the binary judgments was also high , at 0 . 50 , 95 % CI \u00bc [ 0 . 42 , 0 . 58 ] . The mean cosine to the challenge brief was also higher for within - domain ( M \u00bc 0 . 49 , SD \u00bc 0 . 25 , N \u00bc 181 ) vs . between - domain inspi - rations ( M \u00bc 0 . 23 , SD \u00bc 0 . 20 , N \u00bc 164 ) , d \u00bc 1 . 16 , 95 % CI \u00bc [ 1 . 13 , 1 . 19 ] ( see Figure 4B ) , further validating the LDA approach to measuring distance . Figure 5 shows examples of a near and far inspiration ( from the e - waste chal - lenge ) , along with the top 3 LDA topics ( represented by the top 5 words for that latent topic ) , computed cosine vs . its challenge brief , and human similarity rat - ing . The top 3 topics for the challenge brief are { waste , e , recycling , electronics , electronic } , { waste , materials , recycling , recycled , material } , and { devices , elec - tronics , electronic , device , products } , distinguishing e - waste , general recycling , and electronics products topics . These examples illustrate how LDA is able to e\ufb00ectively extract the latent topical mixture of the inspirations from their text ( inspirations with media also include textual descriptions of the media , miti - gating concerns about loss of semantic information due to using only text as input to LDA ) and also capture intuitions about variations in conceptual dis - tance among inspirations : a document about di\ufb00erent ways of assigning value to possessions is intuitively conceptually more distant from the domain of e - waste than a document about a prior e\ufb00ort to address e - waste . The near and far examples depicted in Figure 5 also represent the range of con - ceptual distance measured in this dataset , with the near inspiration\u2019s cosine of 0 . 64 representing approximately the 90th percentile of similarity to the chal - lenge domain , and the far inspiration\u2019s cosine of 0 . 01 representing approxi - mately the 10th percentile of similarity to the challenge domain . Thus , the range of conceptual distance of inspirations in this data spans approximately Figure 4 ( A ) Scatterplot of LDA cosines vs . averaged human continuous similarity judgments for inspirations in the e - waste challenge . ( B ) . Mean cosine against the challenge brief for within - vs . between - domain inspirations Inspiration source distance and design ideation 11 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 from sources that are very clearly within the domain ( e . g . , an actual solution for the problem of electronic waste involving recycling of materials ) to sources that are quite distant , but not obviously random ( e . g . , an observation of how people assign emotional value to relationships and artifacts ) . This range most likely excludes the \u2018too far\u2019 example designs studied in Fu et al . ( 2013 ) or the \u2018opposite stimuli\u2019 used in Chiu and Shu ( 2012 ) . 2 . 3 . 2 . 3 Final distance measures . The challenge briefs varied in length and speci\ufb01city across challenges , as did mean raw cosines for inspirations . But , these di\ufb00erences in mean similarity were much larger , d \u00bc 1 . 90 , 95 % CI \u00bc [ 1 . 85 e 1 . 92 ] ( for 80 inspirations from 4 challenges with maximally di\ufb00erent mean cosines ) , than for human similarity judgments ( coded sepa - rately but with the same methodology as before ) , d \u00bc 0 . 18 , 95 % CI \u00bc [ e 0 . 05 to 0 . 43 ] . This suggested that between - challenge di\ufb00erences were more an artifact of variance in challenge brief length / speci\ufb01city . Thus , to ensure meaningful comparability across challenges , we normalized the cosines by computing the z - score for each inspiration\u2019s cosine relative to other inspi - rations from the same challenge before analyzing the results in the full dataset . However , similar results are found using raw cosines , but with more uncer - tainty in the statistical coe\ufb03cient estimates . We then subtracted the cosine z - score from zero such that larger values meant more distant . From these \u2018reversed\u2019 cosine z - scores , two di\ufb00erent distance Figure 5 Topics found by LDA within examples of near and far inspirations for the e - waste challenge 12 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 measures were computed to tease apart possibly distinct e\ufb00ects of source dis - tance : 1 ) max distance ( DIST MAX ) , i . e . , the distance of a concept\u2019s furthest source from the problem domain and 2 ) mean distance ( DIST MEAN ) of the concept\u2019s sources . DIST MAX estimates \u2018upper bounds\u2019 for the bene\ufb01ts of dis - tance : do the best ideas really come from the furthest sources ? DIST MEAN cap - italizes on the fact that many concepts relied on multiple inspirations and estimates the impact of the relative balance of relying on near vs . far sources ( e . g . , more near than far sources , or vice versa ) . 2 . 3 . 3 Control measures Given our correlational approach , it is important to identify and rule out or adjust for other important factors that may in\ufb02uence the creativity of concepts ( particularly in the later stages , where prototyping and feedback are especially important ) and may be correlated with the predictor variables . Feedback . Given the collaborative nature of OpenIDEO , we reasoned that feedback in the form of comments ( labeled here as FEEDBACK ) in\ufb02uences success . Comments can o\ufb00er encouragement , raise issues / questions , or pro - vide speci\ufb01c suggestions for improvement , all potentially signi\ufb01cantly enhancing the quality of the concept . Further , feedback may be an alternate pathway to success via source distance , in that concepts that build on far sour - ces may attract more attention and therefore higher levels of feedback , which then improve the quality of the concept . Quality of cited sources . Concepts that build on existing high - quality concepts ( e . g . , those who end up being shortlisted or chosen as winners ) have a partic - ular advantage of being able to learn from the mistakes and shortcomings , good ideas , and feedback in these high - quality concepts . Thus , as a proxy mea - sure of quality , the number of shortlisted concepts a given concept builds upon ( labeled SOURCESHORT ) could be a large determinant of a concept\u2019s success . 2 . 4 Analytic approach We are interested in predicting the creative outcomes of 707 concepts , posted by 357 authors for 12 di\ufb00erent design challenges . Authors are not cleanly nested within challenges , nor vice versa ; our data are cross - classi\ufb01ed , with concepts cross - classi\ufb01ed within both authors and challenges ( see Figure 6 ) . This cross - classi\ufb01ed structure violates assumptions of uni - form independence between concepts : concepts posted by the same author or within the same challenge may be more similar to each other . Failing to account for this non - independence could lead to overestimates of the sta - tistical signi\ufb01cance of model estimates ( i . e . , make unwarranted claims of sta - tistically signi\ufb01cant e\ufb00ects ) . This issue is exacerbated when testing for small e\ufb00ects . Additionally , modeling between - author e\ufb00ects allows us to separate author - e\ufb00ects ( e . g . , higher / lower creativity ) from the impact of sources on Inspiration source distance and design ideation 13 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 individual concepts Thus , we employ generalized linear mixed models ( also called hierarchical generalized linear models ) to model both \ufb01xed e\ufb00ects ( of our independent and control variables ) and random e\ufb00ects ( potential varia - tion of the outcome variable attributable to author - or challenge - nesting and also potential between - challenge variation in the e\ufb00ect of distance ) on short - list status ( a binary variable , which requires logistic , rather than linear , regression ) . An initial model predicting the outcome with only the intercept and between - challenge and - author variation con\ufb01rms the presence of signi\ufb01cant non - independence , with between - author and between - challenge variation in short - list outcomes estimated at 0 . 44 , and 0 . 50 , respectively . The intra - class correla - tions for author - level and challenge - level variance in the intercept are w 0 . 11 and 0 . 13 , respectively , well above the cuto\ufb00 recommended by Raudenbush and Bryk ( 2002 ) . 1 3 Results 3 . 1 Descriptive statistics On average , 16 % of concepts in the sample get shortlisted ( see Table 2 ) . DIS - T MEAN is centered approximately at 0 , re\ufb02ecting our normalization procedure . Both DIST MAX and DIST MEAN have a fair degree of negative skew . SOUR - CESHORT and FEEDBACK have strong positive skew ( most concepts either have few comments or cite 0 or 1 shortlisted concepts ) . There is a strong positive relationship between DIST MAX and DIST MEAN ( see Table 3 ) . All variables have signi\ufb01cant bivariate correlations with SHORT - LIST except for DIST MAX ; however , since it is a substantive variable of inter - est , we will model it nonetheless . Controlling for other variables might enable us to detect subtle e\ufb00ects . Figure 6 Illustrated cross - classi\ufb01ed structure of the data 14 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 3 . 2 Statistical models We estimated separate models for the e\ufb00ects of DIST MAX and DIST MEAN , each controlling for challenge - and author - nesting , FEEDBACK , and SHORTSOURCE . 3 . 2 . 1 Max distance Our model estimated an inverse relationship between DIST MAX and Pr ( shortlist ) , such that a 1 - unit increase in DIST MAX predicted a 0 . 33 decrease in the log - odds of being shortlisted , after accounting for the e\ufb00ects of FEEDBACK , SHORTSOURCE , and challenge - and author - level nesting , p < . 05 ( see Appendix B for technical details on the statistical models ) . How - ever , this coe\ufb03cient was estimated with considerable uncertainty , as indi - cated by the large con\ufb01dence intervals ( coe\ufb03cient could be as small as (cid:4) 0 . 06 or as large as (cid:4) 0 . 60 ) ; considering also the small bivariate correlation with SHORTLIST , we are fairly certain that the \u2018true\u2019 coe\ufb03cient is not pos - itive ( contra the Conceptual Leap Hypothesis ) , but we are quite uncertain about its magnitude . Figure 7 visually displays the estimated relationship between DIST MAX and Pr ( shortlist ) , evaluated at mean values of feedback and shortlisted sources . To aid interpretation , we also plot the predicted Pr ( shortlist ) for concepts that cite no sources using a horizontal gray bar ( bar width indicates uncertainty in estimate of Pr ( shortlist ) ) : concepts with approximately equivalent amounts of feedback ( i . e . , mean of 8 . 43 ) , have a predicted Pr ( shortlist \u00bc 0 . 09 , 95 % CI \u00bc [ 0 . 07 e 0 . 11 ] ; using a logistic model , the coe\ufb03cient for \u2018any citation\u2019 ( con - trolling for feedback ) is 0 . 31 , 95 % CI \u00bc [ 0 . 01 e 0 . 62 ] ) . This bar serves as an approximate \u2018control\u2019 group , allowing us to interpret the e\ufb00ect not just in terms of the e\ufb00ects of far sources relative to near sources , but also in comparison with using no sources . Comparing the \ufb01tted curve with this bar highlights how the Table 2 Descriptive statistics Variable Valid N Min Max Mean Median SD SHORTLIST 707 0 . 00 1 . 00 0 . 16 0 . 00 0 . 36 DIST MAX 707 (cid:4) 3 . 85 1 . 90 0 . 45 0 . 76 0 . 85 DIST MEAN 707 (cid:4) 3 . 85 1 . 67 (cid:4) 0 . 10 0 . 01 0 . 85 SOURCESHORT 707 0 11 0 . 51 0 0 . 96 FEEDBACK 707 0 67 8 . 43 6 9 . 45 Table 3 Bivariate correlations Variable DIST MAX DIST MEAN SOURCESHORT FEEDBACK SHORTLIST (cid:4) 0 . 05 (cid:4) 0 . 10 * 0 . 11 * * 0 . 33 * * * DIST MAX 0 . 77 * * * 0 . 05 0 . 07 m DIST MEAN (cid:4) 0 . 05 0 . 01 SOURCESHORT 0 . 12 * * m p < . 10 ; * p < . 05 ; * * p < . 01 ; * * * p < . 001 . Inspiration source distance and design ideation 15 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 advantage of citing vs . not citing inspirations seems to be driven mostly by cit - ing relatively near inspirations : Pr ( shortlist ) for concepts that cite far inspira - tions converges on that of no - citation concepts . We emphasize again that , despite the uncertainty in the degree of the negative relationship between DIS - T MAX and Pr ( shortlist ) , the data do not support an inference that the best ideas are coming from the farthest inspirations : rather , relying on nearer rather than farther sources seems to lead to more creative design ideas . Importantly , this pattern of results was robust across challenges on the platform : the model esti - mated essentially zero between - challenge variation in the slope of DIST MAX . c 2 ( 2 ) \u00bc 0 . 05 , p \u00bc . 49 ( see Figure 8 ) . 3 . 2 . 2 Mean distance Similar results were obtained for DIST MEAN . There was a robust inverse rela - tionship between DIST MEAN and Pr ( shortlist ) , such that a 1 - unit increase in DIST MEAN was associated with a decrease of approximately 0 . 40 in the log - odds of being shortlisted , p < . 05 . The estimates of this e\ufb00ect were obtained with similarly low precision regarding the magnitude of the e\ufb00ect , with 95 % CI upper limit of at most B \u00bc (cid:4) 0 . 09 ( but as high as (cid:4) 0 . 71 ) . As shown in Figure 9 , as DIST MEAN increases , Pr ( shortlist ) approaches that of non - citing concepts , again suggesting ( as with DIST MAX ) that the most bene\ufb01cial sources appear to be ones that are relatively close to the challenge domain . Again , as with DIST MAX , this pattern of results did not vary across challenges : our model estimated essentially zero between - challenge variation in the slope of DIST MEAN , c 2 ( 2 ) \u00bc 0 . 07 , p \u00bc . 48 ( see Figure 10 ) . 4 Discussion 4 . 1 Summary and interpretation of \ufb01ndings This study explored how the inspirational value of sources varies with their conceptual distance from the problem domain along the continuum from near to far . The study\u2019s \ufb01ndings provide no support for the notion that the best ideas come from building explicitly on the farthest sources . On the Figure 7 Model - \ufb01tted rela - tionship between DIST MAX and Pr ( shortlist ) , evaluated at mean values of feedback and source shortlist . Grayed lines are \ufb01ts with upper and lower limits for 95 % CI for e\ufb00ect of DIST MAX 16 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 Figure 8 Overall and by - challenge model - \ufb01tted rela - tionship between DIST MAX and Pr ( shortlist ) . Fitted values evaluated at mean values of feedback and source shortlist . Grayed lines are \ufb01ts for each individual challenge Figure 9 Model - \ufb01tted rela - tionship between DIST MEAN and Pr ( shortlist ) , evaluated at mean values of feedback and source shortlist . Grayed lines are \ufb01ts with upper and lower limits for the 95 % CI for the e\ufb00ect of DIST MEAN Figure 10 Overall and by - challenge model - \ufb01tted rela - tionship between DIST MEAN and Pr ( shortlist ) . Fitted values evaluated at mean values of feedback and source shortlist . Grayed lines are \ufb01ts for each individual challenge Inspiration source distance and design ideation 17 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 contrary , the bene\ufb01ts of building explicitly on inspirations seem to accrue mainly for concepts that build more on near than far inspirations . Impor - tantly , these e\ufb00ects were consistently found in all of the challenges , addressing concerns raised about potential problem variation , at least among non - routine social innovation design problems . 4 . 2 Caveats and limitations Some caveats should be discussed before addressing the implications of this study . First , the statistical patterns observed here are conditional : i . e . , we \ufb01nd an inverse relationship between conceptual distance of explicitly cited inspiration sources and Pr ( shortlist ) . Our data are silent on the e\ufb00ects of dis - tance for concepts that did not cite sources ( where lack of citation could indi - cate forgetting of sources or lack of conscious building on sources ) . There is a potential concern over range restriction or attrition due to our reli - ance on self - identi\ufb01ed sources . However , several features of the data help to ameliorate this concern . First , concepts that did not cite sources were overall of lower quality ; thus , it is unlikely that the inverse e\ufb00ects of distance are solely due to attrition ( e . g . , bene\ufb01cial far inspirations not being observed ) . Second , the integration of citations and building on sources into the overall OpenI - DEO work\ufb02ow and philosophy of ideation also helps ameliorate concerns about attrition of far sources . Finally , the dataset included many sources that were quite far away , providing su\ufb03cient data to statistically test the e\ufb00ects of relative reliance on far sources ( even if they are overall under - reported ) . Nevertheless , we should still be cautious about making inferences about the impact of unconscious sources ( since sources in this data are explicitly cited and therefore consciously built upon ) . However , as we note in the methods , Table 4 Model estimates and fit statistics for cross - classified multilevel logistic regressions of Pr ( shortlist ) on DIST MAX , with comparison to baseline model ( controls only ) Baseline model ( controls only ) DIST MAX , \ufb01xed slope DIST MAX , random slope Fixed e\ufb00ects g 00 , intercept (cid:4) 2 . 66 [ (cid:4) 3 . 28 , (cid:4) 2 . 03 ] (cid:4) 2 . 57 [ (cid:4) 3 . 29 , (cid:4) 2 . 05 ] (cid:4) 2 . 57 [ (cid:4) 3 . 29 , (cid:4) 2 . 05 ] g 10 , FEEDBACK 0 . 09 * * * [ 0 . 07 , 0 . 12 ] 0 . 10 * * * [ 0 . 07 , 0 . 12 ] 0 . 10 * * * [ 0 . 07 , 0 . 12 ] g 20 , SOURCESHORT 0 . 14 [ (cid:4) 0 . 08 , 0 . 36 ] 0 . 15 [ (cid:4) 0 . 07 , 0 . 38 ] 0 . 15 [ (cid:4) 0 . 07 , 0 . 38 ] g 30 , DIST MAX (cid:4) 0 . 33 * [ (cid:4) 0 . 60 , (cid:4) 0 . 06 ] (cid:4) 0 . 32 * [ (cid:4) 0 . 59 , (cid:4) 0 . 06 ] Random e\ufb00ects u 0 authorj for intercept 0 . 29 0 . 31 0 . 32 u 0 challengek for intercept 0 . 75 0 . 76 0 . 74 u 3 challengek for DIST MAX 0 . 00 Model \ufb01t statistics Deviance 511 . 39 506 . 04 505 . 99 AIC 521 . 39 518 . 04 521 . 99 m p < . 10 ; * p < . 05 ; * * p < . 01 ; * * * p < . 001 ; 95 % CI ( Wald ) \u00bc [ lower , upper ] . 18 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 the Conceptual Leap Hypothesis maps most cleanly to conscious inspiration processes ( e . g . , analogy ) . Finally , some may be concerned that we have not measured novelty here . Conceivably , the bene\ufb01ts of distance may only be best observed for the novelty of ideas , and not necessarily quality , consistent with some recent work ( Franke , Poetz , & Schreier , 2014 ) . However , novelty per se does not produce creativity ; we contend that to fully understand the e\ufb00ects of distance on design creativity , we must consider its impacts on both novelty and quality together ( as our shortlist measure does ) . 4 . 3 Implications and future directions Overall , our results consistently stand in opposition to the Conceptual Leap Hypothesis . In tandem with prior opposing \ufb01ndings ( reviewed in the introduc - tion ) , our work lends strength to alternative theories of inspiration by theorists like Perkins ( 1983 ) , who argues that conceptual distance does not matter , and Weisberg ( 2009 , 2011 ) , who argues that within - domain expertise is a primary driver of creative cognition . We should be clear that our \ufb01ndings do not imply that no creative ideas come from far sources ( indeed , in our data , some creative ideas did come from far sources ) ; rather , our data suggest that the most crea - tive design ideas are more likely to come from relying on a preponderance of nearer rather than farther sources . However , our data do suggest that highly creative ideas can often come from relying almost not at all on far sources ( as evidenced by the analyses with maximum distance of sources ) . These good ideas may arise from iterative , deep search , a mechanism for creative breakthroughs that may be often overlooked but potentially at least as impor - tant as singular creative leaps ( Chan & Schunn , 2014 ; Dow , Heddleston , & Table 5 Model estimates and fit statistics for cross - classified multilevel logistic regressions of Pr ( shortlist ) on DIST MEAN , with comparison to baseline model ( controls only ) Baseline model ( controls only ) DIST MEAN , \ufb01xed slope DIST MEAN , random slope Fixed e\ufb00ects g 00 , intercept (cid:4) 2 . 66 [ (cid:4) 3 . 28 , (cid:4) 2 . 03 ] (cid:4) 2 . 74 [ (cid:4) 3 . 36 , (cid:4) 2 . 11 ] (cid:4) 2 . 74 [ (cid:4) 3 . 36 , (cid:4) 2 . 11 ] g 10 , FEEDBACK 0 . 09 * * * [ 0 . 07 , 0 . 12 ] 0 . 10 * * * [ 0 . 07 , 0 . 12 ] 0 . 10 * * * [ 0 . 07 , 0 . 12 ] g 20 , SOURCESHORT 0 . 14 [ (cid:4) 0 . 08 , 0 . 36 ] 0 . 13 [ (cid:4) 0 . 09 , 0 . 35 ] 0 . 13 [ (cid:4) 0 . 09 , 0 . 35 ] g 30 , DIST MEAN (cid:4) 0 . 40 * [ (cid:4) 0 . 71 , (cid:4) 0 . 09 ] (cid:4) 0 . 40 * [ (cid:4) 0 . 73 , (cid:4) 0 . 07 ] Random e\ufb00ects u 0 authorj for intercept 0 . 29 0 . 31 0 . 30 u 0 challengek for intercept 0 . 75 0 . 73 0 . 73 u 3 challengek for DIST MEAN 0 . 03 Model \ufb01t statistics Deviance 511 . 39 505 . 13 505 . 06 AIC 521 . 39 517 . 13 521 . 06 m p < . 10 ; * p < . 05 ; * * p < . 01 ; * * * p < . 001 ; 95 % CI ( Wald ) \u00bc [ lower , upper ] . Inspiration source distance and design ideation 19 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 Klemmer , 2009 ; Mecca & Mumford , 2013 ; Rietzschel , Nijstad , & Stroebe , 2007 ; Sawyer , 2012 ; Weisberg , 2011 ) . In light of this and our \ufb01ndings , it may be fruitful to deemphasize the privileged role of far sources and mental leaps in theories of design inspiration and creative cognition . How might this proposed theoretical revision be reconciled with the relatively robust \ufb01nding that problem solvers from outside the problem domain can often produce the most creative ideas ( Franke et al . , 2014 ; Hargadon & Sutton , 1997 ; Jeppesen & Lakhani , 2010 ) ? Returning to our re\ufb02ections on the potential costs of processing far sources , one way to reconcile the two sets of \ufb01ndings might be to hypothesize that expertise in the distant source domain enables the impact of distant ideas by bypassing the cognitive costs of deeply understanding the far domain , and \ufb01lters out shallow inferences that are not likely to lead to deep insights . Hargadon and Sutton\u2019s ( 1997 ) \ufb01nd - ings from their in - depth ethnographic study of the consistently innovative IDEO design \ufb01rm are consistent with an expertise - mediation claim : the \ufb01rm\u2019s cross - domain - inspired innovations appeared to \ufb02ow at the day - to - day process level mainly from deep immersion of its designers in multiple disciplines , and \u2018division of expertise\u2019 within the \ufb01rm , with brainstorms acting as crucial cata - lysts for involving experts from di\ufb00erent domains on projects . However , studies directly testing expertise - mediation are scarce or non - existent . Further , the weight of the present data , combined with prior studies showing no advantage of far sources , suggests that considering alternative mechanisms of outside - domain advantage may be more theoretically fruitful : for instance , perhaps the advantage of outside - domain problem - solvers arises from the di\ufb00erent perspectives they bring to the problem d allowing for more \ufb02exible and alternative problem representations , which may lead to breakthrough in - sights ( Kaplan & Simon , 1990 ; Knoblich , Ohlsson , Haider , & Rhenius , 1999 ; \u20ac Ollinger , Jones , Faber , & Knoblich , 2012 ) . Domain - outsiders may also have a looser attachment to the status quo or prior successful solutions by virtue of being a \u2018newcomer\u2019 to the domain ( Choi & Levine , 2004 ) d leading to higher readiness to consider good ideas that challenge existing assumptions within the domain d rather than knowledge and transfer of di\ufb00erent solutions per se . Finally , it would be interesting to examine potential moderating in\ufb02uences of source processing strategies . In our data , closer sources were more bene\ufb01cial , but good ideas also did come from far sources ; however , as we have argued , it can be more di\ufb03cult to convert far sources into viable concepts . Are there common strategies for e\ufb00ective conversion of far sources , and are they di\ufb00erent from strategies for e\ufb00ectively building on near sources ? For example , one e\ufb00ective strategy for building on sources while avoiding \ufb01xation is to use a schema - based strategy ( i . e . , extract and transfer abstract functional principles rather than concrete solution features ; Ahmed & Christensen , 2009 ; Yu , Kraut , & Kittur , 2014 ) . Are there processing strategies that expert creative 20 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 designers apply uniquely to far sources ( e . g . , to deal with potentially un - alignable di\ufb00erences ) ? Answering this question can shed further light on the variety of ways designers can be inspired by sources to produce creative design ideas . We close by noting the methodological contribution of this work . While we are not the \ufb01rst to use topic modeling to explore semantic meaning in a large collection of documents , we are the \ufb01rst to our knowledge to validate this method in the context of large - scale study of design ideas . We have shown that the topic model approach adequately captures human intuitions about the semantics of the design space , while providing dramatic savings in cost : indeed , such an approach can make more complex research questions ( e . g . , exploring pairwise distances between design idea or , tracing conceptual paths / moves in a design ideation session ) much more feasible without sacri - \ufb01cing too much quality . We believe this approach can be a potentially valuable way for creativity researchers to study the dynamics of idea generation at scale , while avoiding the ( previously inevitable ) tradeo\ufb00 between internal validity ( e . g . , having adequate statistical power ) and external validity ( e . g . , using real , complex design problems and ideas instead of toy problems ) . Appendix A . Topic model technical details A . 1 . Document preprocessing All documents were \ufb01rst tokenized using the TreeBank Tokenizer from the open - source Natural Language Tool Kit Python library ( Bird , Klein , & Loper , 2009 ) . To improve the information content of the document text , we removed a standard list of stopwords , i . e . , highly frequent words that do not carry semantic meaning on their own ( e . g . , \u2018the\u2019 , \u2018this\u2019 ) . We used the open - source MAchine Learning for LanguagE Toolkit\u2019s ( MALLET ; McCallum , 2002 ) stopword list . A . 2 . Model parameter selection We used MALLET to train our LDA model , with asymmetric priors for the topic - document and topic - word distributions , which allows for some words to be more prominent than others and some topics to be more prominent than others , typically improving model \ufb01t and performance ( Wallach , Mimno , & McCallum , 2009 ) . Priors were optimized using MALLET\u2019s in - package optimization option . LDA requires that K ( the number of topics ) be prespeci\ufb01ed by the modeler . Model \ufb01t typically improves with K , with diminishing returns past a certain point . Intuitively , higher K leads to \ufb01ner - grained topical distinctions , but too high K may lead to uninterpretable topics ; on the other hand , too low K would yield too general topics . Further , traditional methods of optimizing K ( computing \u2018perplexity\u2019 , or the likelihood of observing the distribution of Inspiration source distance and design ideation 21 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 words in the corpus given a topic model of the corpus ) do not always correlate with human judgments of model quality ( e . g . , domain expert evaluations of topic quality ; Chang , Gerrish , Wang , Boyd - graber , & Blei , 2009 ) . We explored the following settings of K : [ 12 , 25 , 50 , 100 , 200 , 300 , 400 , 500 , 600 , 700 ] . Because the optimization algorithm for the prior parameters is nondeterministic , models with identical K might produce noticeably di\ufb00erent topic model solutions , e . g . , if the optimization search space is rugged , the al - gorithm might get trapped in di\ufb00erent local maxima . Therefore , we ran 50 models at each K , using identical settings ( i . e . , 1000 iterations of the Gibbs sampler , internally optimizing parameters for the asymmetric priors ) . Figure 11 shows the mean \ufb01t ( with both continuous and binary similarity judg - ments ) at each level of K . Model \ufb01t is generally fairly high at all levels of K , with the continuous judg - ments tending to increase very slightly with K , tapering out past 400 . Fit with binary judgments tended to decrease ( also very slightly ) with K , probably re\ufb02ecting the decreasing utility of increasingly \ufb01ner - grained distinctions for a binary same / di\ufb00erent classi\ufb01cation . Because we wanted to optimize for \ufb01t with human judgments of conceptual distance overall , we selected the level of K at which the divergent lines for \ufb01t with continuous and binary judgments \ufb01rst begin to cross ( i . e . , at K \u00bc 400 ) . Subsequently , we created a combined \u2018\ufb01t\u2019 mea - sure ( sum of the correlation coe\ufb03cients for \ufb01t vs . continuous and binary judg - ments ) , and selected the model with K \u00bc 400 that had the best overall \ufb01t measure . However , as we report in the next section , the results of our analyses are robust to di\ufb00erent settings of K . Figure 11 Mean \ufb01t ( with (cid:5) 1 SE ) vs . human judgments for LDA cosines by level of K Appendix B . Statistical modeling technical details B . 1 . Statistical modeling approach All models were \ufb01tted using the lme4 package ( Bates , Maechler , Bolker , & Walker , 2013 ) in R ( R Core Team , 2013 ) , using full maximum likelihood esti - mation by the Laplace approximation . The following is the general structure of these models ( in mixed model notation ) : 22 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 h i \u00f0 authorjchallengek \u00de \u00bc g 00 \u00fe X q g q 0 X qi \u00fe u 0 authorj \u00fe u 0 challengek where (cid:6) h i \u00f0 authorjchallengek \u00de is the predicted log odds of being shortlisted for the ith concept posted by the j th author in the k th challenge (cid:6) g 00 is the grand mean log odds for all concepts (cid:6) g q 0 is a vector of q predictors ( q \u00bc 0 for our null model ) (cid:6) u 0 authorj and u 0 challengek are the random e\ufb00ects contribution of variation between - authors and between - challenges for mean g 00 ( i . e . , how much a given author or challenge varies from the mean ) A baseline model with only control variables and variance components was \ufb01rst \ufb01tted . Then , for the models for both DIST MAX and DIST MEAN , we \ufb01rst estimated a model with a \ufb01xed e\ufb00ect of distance , and then a random e\ufb00ect ( to test for problem variation ) . These random slopes models include the addi - tional parameter u 1 challengek that models the between - challenge variance component for the slope of distance . B . 2 . Model selection Estimates and test statistics for each step in our model - building procedure are shown in Tables 4 and 5 . We \ufb01rst \ufb01tted a model predicting Pr ( shortlist ) with our control variables to serve as a baseline for evaluating the predictive power of our distance measures . The baseline model estimates a strong positive e\ufb00ect of FEEDBACK , estimated with high precision : each additional comment added 0 . 10 [ 0 . 07 , 0 . 12 ] to the log - odds of being shortlisted , p < . 001 . The model also estimated a positive e\ufb00ect of SHORTSOURCE , B \u00bc 0 . 14 [ e 0 . 08 , 0 . 36 ] but with poor precision , and falling short of conventional statistical signi\ufb01 - cance , p \u00bc . 21 ; nevertheless , we leave it in the model for theoretical reasons . The baseline model is a good \ufb01t to the data , reducing deviance from the null model ( with no control variables ) by a large and statistically signi\ufb01cant amount , c 2 ( 1 ) \u00bc 74 . 35 , p \u00bc . 00 . For the \ufb01xed slope model for DIST MAZ , adding the coe\ufb03cient for results in a signi\ufb01cant reduction in deviance from the baseline model , c 2 ( 2 ) \u00bc 0 . 13 , p \u00bc . 47 . The random slope model did not signi\ufb01cantly reduce deviance in com - parison with the simpler \ufb01xed slope model , c 2 ( 2 ) \u00bc 0 . 05 , p \u00bc . 49 ( p - value is halved , heeding common warnings that a likelihood ratio test discriminating two models that di\ufb00er on only one variance component may be overly conser - vative , e . g . , Pinheiro & Bates , 2000 ) . Also , the Akaike Information Criterion ( AIC ) increases from the \ufb01xed to random slope model . Thus , we select the \ufb01xed slope model ( i . e . , no problem - variation ) as our best estimate of the e\ufb00ects of DIST MAX . This \ufb01nal model has an overall deviance reduction vs . null at c 2 ( 3 ) \u00bc 79 . 71 , p \u00bc . 00 . Inspiration source distance and design ideation 23 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001 We used the same procedure for model selection for the DIST MEAN models . The \ufb01xed slope model results in a small but signi\ufb01cant reduction in deviance from the baseline model , c 2 ( 1 ) \u00bc 6 . 27 , p \u00bc . 01 . Adding the variance compo - nent for the slope of DIST MEAN increases the AIC , and does not signi\ufb01cantly reduce deviance , c 2 ( 2 ) \u00bc 0 . 07 , p \u00bc . 48 ( again , p - value here is halved to correct for overconservativeness ) . Thus , again we select the \ufb01xed slope model as our \ufb01nal model for the e\ufb00ects of DIST MEAN . This \ufb01nal model has an overall reduc - tion in deviance from the null model of about c 2 ( 3 ) \u00bc 80 . 61 , p \u00bc . 00 . B . 3 . Robustness and sensitivity We tested the robustness of our coe\ufb03cient estimates by calculating outlier in - \ufb02uence statistics using the in\ufb02uence . measures method in the stats package in R , applied to logistic regression model variants of both the DIST MEAN and DIST MAX models ( i . e . , without author - and challenge - level variance compo - nents ; coe\ufb03cient estimates are almost identical to the \ufb01xed slope multilevel models ) : DFBETAS and Cook\u2019s Distance measures were below recommended thresholds for all data points ( Fox , 2002 ) . Addressing potential concerns about sensitivity to topic model parameter set - tings , we also \ufb01tted the same \ufb01xed slope multilevel models using recomputed conceptual distance measures for the top 20 ( best - \ufb01tting ) topic models at K \u00bc 200 , 300 , 400 , 500 , and 600 ( total of 100 models ) . All models produced negative estimates for the e\ufb00ect of both DIST MEAN and DIST MAX , with poorer precision for lower K . Thus , our results are robust to di\ufb00erent settings of K for the topic models . We also address potential concerns about interactions with expertise by \ufb01tting a model that allowed the slope of distance to vary by authors . In this model , the overall mean e\ufb00ect of distance remained almost identical ( B \u00bc (cid:4) 0 . 46 ) , and the model\u2019s \ufb01t was not signi\ufb01cantly better than the \ufb01xed slope model , c 2 ( 3 ) \u00bc 3 . 44 , p \u00bc . 16 , indicating a lack of statistically signi\ufb01cant between - author variability for the slope of distance . Finally , we also \ufb01tted models that considered not just immediately cited inspi - rations , but also indirectly cited inspirations ( i . e . , inspirations cited by cited in - spirations ) , and they too yielded almost identical coe\ufb03cient estimates and con\ufb01dence intervals . References Ahmed , S . , & Christensen , B . T . ( 2009 ) . An in situ study of analogical reasoning in novice and experienced designer engineers . Journal of Mechanical Design , 131 ( 11 ) , 111004 . Amabile , T . M . ( 1982 ) . Social psychology of creativity : a consensual assessment technique . 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Endnote Although concept - level variance is not estimated in mixed logistic regressions , we follow Zeger , Liang , and Albert\u2019s ( 1988 ) suggestion of ( 15 / 16 ) p 3 / 3 as a reason - able approximation for residual level - 1 variance ( the concept level in our case ) . 28 Design Studies Vol - - No . - - Month 2014 Please cite this article in press as : Chan , J . , et al . , Do the best design ideas ( really ) come from conceptually distant sources of inspiration ? , Design Studies ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . destud . 2014 . 08 . 001", + "deguchi2023direct": "BIOPHYSICS Direct observation of motor protein stepping in living cells using MINFLUX Takahiro Deguchi 1 , Malina K . Iwanski 2 , Eva - Maria Schentarra 1 , 3 , Christopher Heidebrecht 1 , 3 , Lisa Schmidt 1 , 3 , Jennifer Heck 4 , Tobias Weihs 5 , Sebastian Schnorrenberg 6 , Philipp Hoess 1 , Sheng Liu 1 , 7 , Veronika Chevyreva 1 , 8 , Kyung - Min Noh 4 , Lukas C . Kapitein 2 , Jonas Ries 1 * Dynamic measurements of molecular machines can provide invaluable insights into their mechanism , but these measurements have been challenging in living cells . Here , we developed live - cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super - resolution technique MINFLUX . Using this approach , we resolved the precise stepping motion of the motor protein kinesin - 1 as it walked on microtubules in living cells . Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution . M olecular machines drive many pro - cesses essential for life . For example , members of the myosin , kinesin , and dynein families are adenosine 5 \u2032 - triphosphate ( ATP ) \u2013 driven processive molecular motors that recognize the intrinsic structural polarity of cytoskeletal filaments to drive directed movement important for cellular processes such as intracellular transport and cell division ( 1 \u2013 3 ) . Over the past decades , the mi - crotubule plus - end directed motor kinesin - 1 , hereafter called kinesin , has served as a key model both for the understanding of motor dynamics and for the development of improved single - molecule methods such as optical tweez - ers and single - particle tracking ( 4 \u2013 7 ) . These studies have revealed that kinesinmoves in a hand - over - hand manner , in which each step along the microtubule encompasses a 16 - nm displacement of the N - terminal motor do - main , which leads to an 8 - nm displacement of the C - terminal cargo - binding domain ( Fig . 1A ) . Despite the great success of existing single - molecule techniques in studying purified mo - tors in well - controlled in vitro reconstitution experiments , performing such experiments in thelivingcellhasprovenchallenging . Thelarge labels required for optical tweezers typically bind multiple motors in undefined ways , and single fluorophores do not provide sufficient spatial and temporal resolution to resolve the fast stepping behavior of motors under phys - iologicalATPconcentrations . Asaworkaround , bright nanoparticles taken up through endo - cytosis into transport vesicles have been used as proxies to study motor dynamics in living cells ( 8 , 9 ) . However , in such experiments , the identity and copy numbers of the motors that drive transport are unknown . Therefore , the stepping dynamics of specific motors inside living cells has remained experimen - tally inaccessible . MINFLUX , a super - resolution microscopy technology ( 10 ) , holds great potential to over - come these limitations . By efficiently using the limited photon budget of single fluoro - phores , MINFLUX enables high spatial res - olution for imaging ( 10 \u2013 12 ) and temporal resolutionfor fluorophore tracking ( 13 , 14 ) . In MINFLUX tracking , a donut - shaped excitation beam is scanned around a single fluorophore ( Fig . 1B ) . From the intensities measured at specific positions , the coordinate of the fluo - rophore is calculated and the scanning pattern is recentered on this position before the next iteration . Keeping the fluorophore close to the dark center of the beam results in a high localization precision and minimizes photo - bleaching . In a companion paperinthisissue , MINFLUX was used to dissect the conforma - tional stages of kinesin stepping on in vitro polymerized microtubules ( 15 ) . We now show that this technique can also enable the high - resolution tracking of fast molecular motors in living cells . Optimization of MINFLUX for motor protein tracking To establish MINFLUX tracking of molecular motors in living cells , we first optimized the workflow on single molecules and fluorescent beads in vitro to maximize precision and speed ( fig . S1 , A to C ) and achieved a localization precision of \u2248 2 nm with a submillisecond tem - poral resolution . We then optimized kinesin tracking using motor - PAINT ( 16 ) . In this method , cells are permeabilized and fixed before fluorescently labeled kinesin motors are added that walk along microtubules toward the plus end ( Fig . 1C and movie S1 ) . Unlike high - resolution in vitro assays that use large beads as labels ( 5 , 17 ) , we used small fluorescent tags that reduced the linkage error to \u2248 3 nm [ as predicted by Alphafold2 ( 18 , 19 ) ] , which was comparable to the system resolution . Motor - PAINT is less challenging than live - cell tracking because it allows us to precisely control the concentration of kinesin motors and , importantly , their speed , by ad - justingtheATPconcentration . UsingMINFLUX motor - PAINT , we were able to reconstruct cel - lular microtubules with a precision of \u2248 2 nm ( Fig . 1 , D and E , and table S1 ) . Additionally , the directionality of kinesin reveals the ori - entation of the microtubules . Compared with standard motor - PAINT with a wide - field mi - croscope , the use of MINFLUX improved the localization precision 5 - fold , the tempo - ral resolution 50 - fold , and the number of localizations per track by more than one order of magnitude . In neurons , this allowed us to better resolve individual microtubules inside dendrites compared with our earlier motor - PAINT study ( Fig . 1D ) ( 16 ) . In human osteosarcoma ( U2OS ) cells , we could resolve individual trajectories of the purified motors in the crowded area around the centrosome with near - protofilament resolution ( Fig . 1 , E and F ) . Tracks , which were just 12 nm apart , were easily resolvable , and we regularly ob - served side stepping between different pro - tofilaments ( Fig . 1 , F and G , and movie S2 ) . These side steps often occurred after stalling events , suggesting that motors were circum - venting obstructions such as microtubule - associated proteins ( MAPs ) that became fixed to the microtubules or microtubule defects from the fixation process . A closer inspection of individual tracks showed clusters of localizations that cor - respond to the 8 - nm steps of the labeled C terminus . Indeed , these steps become obvious when plotting the position of the motor along the microtubule over time ( Fig . 1H ) . This al - lowed us to quantify the precise step - size and dwell - timedistributionsundersaturating ( phys - iological ) ATP concentrations ( Fig . 1 , I and J ) . From956stepsin49tracks , wemeasuredastep size of 7 . 8 \u00b1 2 . 7 ( SD ) \u00b1 0 . 09 ( SEM ) nm and an averagedwelltimeof30 . 8ms . Toinvestigatethe stepping behavior in greater detail , we reduced the ATP concentration to slow down the motors ( 20 ) , resulting in a similar step size but a re - duced rate constant for ATP binding ( fig . S2 , A and E , and tables S1 and S2 ) . Under these conditions , we could measure hundreds to thousands of localizations per step ( Fig . 1 , K and L , and movie S3 ) . Averaging over the co - ordinates allowed us to calculate the position of each step with subangstrom precision ( SEM ) . RESEARCH Deguchi et al . , Science 379 , 1010 \u2013 1015 ( 2023 ) 10 March 2023 1 of 6 1 Cell Biology and Biophysics Unit , European Molecular Biology Laboratory , Heidelberg , Germany . 2 Cell Biology , Neurobiology and Biophysics , Department of Biology , Faculty of Science , Utrecht University , Utrecht , Netherlands . 3 Faculty of Biosciences , University of Heidelberg , Heidelberg , Germany . 4 Genome Biology Unit , European Molecular Biology Laboratory , Heidelberg , Germany . 5 Abberior Instruments GmbH , G\u00f6ttingen , Germany . 6 EMBL Imaging Centre , European Molecular Biology Laboratory , Heidelberg , Germany . 7 Department of Physics and Astronomy , University of New Mexico , Albuquerque , NM , USA . 8 The FIRC Institute of Molecular Oncology , Milano , Italy . * Corresponding author . Email : jonas . ries @ embl . de D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023 Deguchi et al . , Science 379 , 1010 \u2013 1015 ( 2023 ) 10 March 2023 2 of 6 1 2 3 4 5 6 7 L permeabilization & fixation kinesin motor addition A B 8 nm 16 nm 8 nm 0 1 2 3 4 5 6 7 8 9 0 50 100 150 po s i t i on ( n m ) time ( s ) 100 ms 68 7 9 7 8 117 888 7 88 10 8 6 7 18 nm 6 nm 29 nm 8 nm 25 nm 12 nm 12 nm 26 nm 12 16 step size ( nm ) 00 4 8 100 c ou n t s dwell time ( ms ) c oun t s 20 40 60 80 20 40 60 80 100 0 40 20 0 60 80 100120 0 200 400 600 time ( ms ) 0 50 100 po s i t i on ( n m ) 9 9 7 8 7 8 117 8 7 8 7 low [ ATP ] saturating [ ATP ] x ( nm ) 00 20 40 60 10 20 30 40 50 c oun t s d = 12 nm \u03c3 = 5 nm E H D K F G C datamoving average ( 20 ms ) steps I J L M 1 2 towards cell body towards periphery 2 3 4 4 3 1 1 2 3 Fig . 1 . MINFLUX tracking of kinesin in fixed cells . ( A ) Kinesin walks on microtubules in a hand - over - hand manner . The apparent step size is 8 nm when the label is attached to the C - terminal tail domain and 16 nm when it is attached to the N - terminal motor domain . ( B ) 2D MINFLUX tracking of a single molecule . A donut beam probes seven positions around a fluorophore to determine its location with nanometer precision . The scan pattern is iteratively centered on the fluorophore during tracking . ( C ) Motor - PAINT approach to track kinesin in fixed cells . Cells are first permeabilized to extract cell contents and then gently fixed to preserve microtubules . Purified fluorescently labeled kinesins [ Dm KHC ( 1 - 421 ) - SNAP - tag - 6xHis ] are added and tracked as they walk toward the plus ends of the microtubules ( movie S1 ) . Panels ( D ) to ( M ) show MINFLUX motor - PAINT in fixed cells . ( D ) Confocal images of a neuron and overlaid kinesin trajectories in four neurites . Most of the neurites show kinesin trajectories in both directions , i . e . , toward ( magenta ) and away ( cyan ) from the soma , as expected for dendrites . ( E ) Confocal microscopy images of green fluorescent protein ( GFP ) \u2013 a - tubulin in U2OS cells showing what appears to be a centrosome and overlaid kinesin trajectories with color - coded walking directions . ( F ) Tracks as indicated in ( E ) show side stepping . ( G ) Tracks as close as 12 nm are clearly resolved and display kinesin switching laterally between neighboring microtubules or protofilaments ( movie S2 ) . ( H ) Representative track and the corresponding time versus position plot at saturating ATP concentrations ( > 1 mM , here 6 mM ) showing 8 - nm walking steps . ( I ) Histogram of step size at saturating ATP concentrations from seven experiments , 49 tracks , and 956 steps with a Gaussian fit ( 7 . 8 nm \u00b1 2 . 7 SD \u00b1 0 . 09 nm SEM ; red line ) . ( J ) Dwell time histogram and fit with a convolution of two exponential functions ( average dwell time of 30 . 8 ms ; red line ) . ( K and L ) Representative track at low ATP concentrations ( 10 m M ) and a corresponding time versus position , raw data ( gray ) , and 20 - ms running mean ( black ) clearly showing 8 - nm walking steps ( see movie S3 ) . ( M ) Representative track showing a zigzag trajectory , indicating an asymmetric arrangement of the label within a kinesin molecule ( see fig . S2 , A and E , and movie S4 ; see fig . S3 for additional examples ) . Scale bars : ( L ) and ( M ) , 10 nm ; ( F ) to ( H ) , 100 nm ; ( D ) and ( E ) , 1 m m . RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023 Deguchi et al . , Science 379 , 1010 \u2013 1015 ( 2023 ) 10 March 2023 3 of 6 GFP - \u03b1 - tubulin 21 16 1714 13 15 16 15 1713 16 161817 915 161620 14 1019 18 16715 17 16 16 14 15 19 17 20 16 18 1518 17 13 18 16 16 15 1916 1912 17 17181616 13 16 19131619 1812 19 19 16 22 1417 18 19 20 1416 15 1816 16 161913 16 13 21 8 15 14 21 17 1517 19 18 12 15 po s i t i on time 16 n m 100 ms a c d b a a c c d d e f g b b a c d e f g b HaloTag ( JF646 ) - K560 kinesin - 1 16 nm GFP - \u03b1 - tubulin HaloTag ( JF646 ) - FL kinesin - 1 B 00 1 po s i t i on ( n m ) 50 100 150 200 13 18 14 15 16 14 15 16 16 17 18 17 0 . 5 1 . 5 time ( s ) A H G F J K L M I D E po s i t i on time 16 n m 100 ms 17191816 1421 16 1915 13 17 18 151713191714 12 19 1616 1713 181416 161217132113 191321 17 11 15 15 161516 17 15 1419161915 161615 a a b b c c B a c b step size ( nm ) c oun t s c oun t s 50 100 150 0 dwell time ( ms ) 40 20 0 60 80 100 120 C 8 4 0 12 16 20 24 28 32 0 50 100 150 200 Fig . 2 . MINFLUX tracking of kinesin in live cells . Panels ( A ) to ( D ) show tracking of full - length kinesin labeled N - terminally with a HaloTag bound to JF646 in live U2OS cells . ( A ) Confocal images of GFP \u2013 a - tubulin in untreated live U2OS cells and overlaid full - length human kinesin trajectories . ( B ) Kinesin RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023 Currently , the accuracy of the measurements is not limited by the detected photons , but rather by the stability of the sample and microscope , as well as the offset of the label from the microtubule - binding site . We observed that 58 % ofthetracksdisplayedzigzag motion , with every other step displaced perpendicular to the track center by , on average , 3 . 6 nm ( Fig . 1M ; fig . S3 , A and C ; and movie S4 ) . This motion is likely caused by an asymmetric positioning of the fluorophore with respect tothe two motor domains imaged in a top view ( fig . S3D ) ( 21 ) , demonstrating that MINFLUX can reveal in - tricate details of the conformational dynamics of individual motor proteins . Deguchi et al . , Science 379 , 1010 \u2013 1015 ( 2023 ) 10 March 2023 4 of 6 track in which the localizations are rendered as a super - resolution image in the region indicated in ( A ) . ( C ) Line plot connecting each localization . ( D ) Time versus position plot of the highlighted portion of the track in ( C ) showing steps of 16 nm . Panels ( E ) to ( J ) show tracking of truncated kinesin ( HaloTag - K560 ) in Taxol - treated live U2OS cells . ( E ) Confocal images of GFP \u2013 a - tubulin and overlaid kinesin tracks . ( F and G ) The tracks indicated in ( E ) rendered as a super - resolution image ( F ) and line plots connecting each localization ( G ) ( see movie S9 ) showing clear walking steps ( localization precision : 2 nm ; temporal resolution : 1 ms ) . ( H ) Time versus position plots of representative kinesin tracks as indicated in ( E ) showing clear 16 nm stepwise movements . ( I ) Step - size histogram ( 161 experiments , 330 tracks , and 2887 steps ) and a Gaussian fit ( 16 . 2 \u00b1 3 . 8 SD \u00b1 0 . 07 SEM nm ) . ( J ) Dwell - time histogram , fit with a convolution of four exponential functions ( average dwell time of 27 . 5 ms ; red line ) . Panels ( K ) to ( M ) show tracking of kinesin ( HaloTag - K560 ) in untreated live primary mouse cortical neurons . ( K ) Confocal images of GFP \u2013 a - tubulin and overlaid kinesin tracks . ( L and M ) Representative tracks corresponding to those indicated in ( K ) as line plots ( L ) and time versus position plots ( M ) showing 16 - nm stepwise movements ( see fig . S2 , C and G , for step size and dwell time histograms ) . Scale bars : ( B ) , ( C ) , ( F ) , ( G ) , and ( L ) , 100 nm ; ( A ) , ( E ) , and ( K ) , 1 m m . xy z x z A C D 90\u00b0 90\u00b0 90\u00b0 90\u00b0 x y z motor - PAINT a c b B E F x - y x - y x - z x - y x - z x - y x - z x - z po s i t i on time 16 n m 100 ms 11 1614 15 12 1617 15 16 15 15 16 13 1710 19 14 13 16 19 17 12 12 16 11 15 13 12 14 20 17 21 18 live - cell a b c Fig . 3 . 3D MINFLUX tracking of kinesin . Panels ( A ) and ( B ) show 3D MINFLUX tracking . ( A and B ) A 3D donut beam ( A ) probes the intensity at seven 3D - distributed positions around a fluorophore ( B ) . Panels ( C ) and ( D ) show 3D tracking with motor - PAINT in fixed U2OS cells . ( C ) 3D rendering of kinesin tracks at crossing microtubules with a volumetric size of 1 . 2 \u00d7 1 . 2 \u00d7 1 m m . ( D ) Selected tracks from ( C ) in top and side views , including ascending and descending trajectories and two trajectories in which motors switch microtubules ( arrows ) . Panels ( E ) and ( F ) show 3D tracking in live cells . ( E ) Representative kinesin tracks in live U2OS cells in top and side views , showing stepwise movements both in the x \u2013 y plane and along the z axis ( see fig . S2 , D and H , for histograms and fig . S9 for confocal overview images ; also see movie S10 ) . ( F ) Position versus time plots of the tracks from ( E ) showing 16 - nm steps . Scale bars : ( E ) , 100 nm ; ( C ) and ( D ) , 200 nm . RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023 Application of MINFLUX in living cells We next attempted MINFLUX tracking of kinesin in living cells . To this end , we ex - pressed HaloTag - kinesin ( full - length ) in U2OS cells ( movie S5 ) and labeled at most a single motor domainper dimer by addition of the dye JF646 at very low concentrations . Individual tracks clearly revealed the 16 - nm steps of the motor domains ( Fig . 2 , A to D , and movie S6 ) . On average , we found a step size of 15 . 7 \u00b1 3 . 8 SD \u00b1 0 . 25 SEM nm and an average dwell time of 46 . 8 ms ( fig . S2 , B and F ) . We also observed tracks with frequent switching between microtubules , back - slipping potentially caused by multiple competing mo - tors ( movie S7 ) , and , unlike in motor - PAINT , tracks without clear steps ( fig . S4 ) . The latter potentially stem from kinesins that are attached to dynamic microtubules or cargoes driven by other motors and are thus passively dragged along . However , the number of tracks that we couldacquirewaslow , likelybecauseofkinesins assuminganautoinhibitedformwithonlyalowfractionintheprocessivestate ( 22 , 23 ) . To increasethethroughput , weusedthetruncated kinesin variant K560 , in which cargo binding and autoinhibition are removed , and treated cells with Taxol to increase the number of stabilized microtubules preferred by kinesin ( movie S8 ) ( 24 ) . With these changes , we could readily observe multiple tracks in a single field of view ( Fig . 2 , E to H , and movie S9 ) . This allowed us to measure precise in vivo step - size anddwell - timedistributionsfrom2887stepsin 330 tracks ( Fig . 2 , I and J ) . These measure - ments revealed that although the average step sizeof16 . 2\u00b13 . 8SD\u00b10 . 07SEMnmwassimilar to that of full - length kinesin , the average dwell time of 27 . 5 ms was much shorter , consistent with the higher speeds that we observed with K560 ( table S2 ) . To test whether our approach could be extended to more complex and sen - sitive cell types , we examined kinesin dynam - ics in the axons of live primary mouse cortical neurons ( Fig . 2 , K to M ) , which critically depend onmotor - driventransport . Here , wecouldclearly quantify the 16 - nm stepping dynamics of kinesin withoutTaxoltreatment ( stepsize = 15 . 7\u00b13 . 7SD\u00b1 0 . 21 SEM nm ; average dwell time 29 . 2 ms ; fig . S2 , C and G ) , demonstrating that MINFLUX re - veals conformational dynamics of individual motor proteins in complex cellular systems . Three - dimensional tracking in live cells Because most biological structures extend into three dimensions , only three - dimensional ( 3D ) tracking can capture the true dynamics and avoid projection artifacts that limit the accu - racy in 2D tracking . Unfortunately , standard single - particle tracking provides , at best , poor z resolution ( 25 ) . MINFLUX has been used to image cellular structures in 3D ( 11 ) , but for tracking , it has so far remained limited to 2D . We therefore adapted MINFLUX for 3D track - ing by scanning a3D donut beam in 3D ( Fig . 3 , A and B ) . We achieved a localization precision of 2 . 5 , 3 . 1 , and 3 . 9 nm in the x , y , and z di - rections , respectively ( fig . S1D ) . When used with motor - PAINT , we could resolve many tracks on crossingmicrotubules ( Fig . 3C ) , including jumps between microtubules ( arrow in Fig . 3D ) . We could also establish 3D tracking in live cells with a similar spatial and temporal resolution ( 3 . 9 nm and 3 . 0 ms , respectively ; table S1 ) , al - lowing us to resolve the 16 - nm steps of kinesin in 3D ( Fig . 3 , E and F ; fig . S2 , D and H ; and movie S10 ) . When we investigated these tra - jectories in the cross - sectional views , we found dynamic movements along the z axis , includ - ing side steps and vertical trajectories . 3D tracking allowed us to extract accurate step sizes from a strongly inclined trajectory ( aver - age 15 . 1 nm ) , which , when analyzed in 2D , showed a bias toward smaller step sizes ( average 9 . 0 nm ) ( fig . S5 ) . Thus , MINFLUX tracking opens the possibility to quantify the precise 3D dynamics of molecular machines in living cells . Discussion Here , we established MINFLUX tracking of kinesin with nanometer spatial and submilli - second temporal resolution and demonstrated that we could directly resolve steps of individ - ual motors in live cells . In contrast to recent in vitro MINFLUX measurements ( 15 ) , we did not observe clear 4 - nm substeps of kinesin in motor - PAINT , likely because of insufficient spatial resolution . However , in live - cell exper - iments with our N - terminally labeled motor , we could occasionally observe 8 - nm substeps ( fig . S6 ) . This encouraged us to also image C - terminally labeled kinesin in living cells . Here , the faster dynamics and higher back - ground fluorescence made the measurements challenging , but we could observe tracks with the expected 8 - nm step size ( fig . S7 ) . We found that these C - terminally labeled motors moved with a slightly higher average velocity ( table S2 ) , an effect that could be the result of motor domain labeling , an important consideration for future experiments . Our study paves the way for investigating how the stepping kinetics of motors in cells are modulated by the presence or absence of different MAPs or cargo adaptors ( 26 ) . Such measurements could help to explain the ob - served discrepancy of kinesin stepping be - haviors such as dwell time , stalling , and side stepping in motor - PAINT ( in vitro \u2013 like ) and live cells ( e . g . , the presence of regulatory MAPs such as MAP7 , higher salt concentrations , etc . ) ( tables S1 and S2 ) . One parameter that was consistent across studies was the average step size despite the fact that in live cells the mo - tion of the microtubules themselves could contribute to the measured step size . As ex - pected , the average step size was \u2248 16 nm , both in U2OS cells [ which have some micro - tubule sliding ( 27 ) ] and in neuronal axons [ where microtubules are largely immobile ( 28 ) ] ( table S1 ) . The impact of microtubule movement is thus likely to be minimal at the time scale of kinesin steps . MINFLUX tracking is not limited to kinesin , but can also be used to study the precise motion of any protein in living cells with high spatiotemporal resolution and minimal perturbance because of its compatibility with single - fluorophore labels ( seefig . S8 for track - ing of Myosin - V ) . In the future , developing MINFLUX to simultaneously track two colors will enable monitoring of the relative 3D po - sitions of labeled protein domains with nano - meter spatial and submillisecond temporal resolution . Such measurements of confor - mational changes of molecular machines in their native environment will provide im - portant insights into their function and regulation . 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Pacheco , U . Matti , and L . Perez for help with sample preparations ; and the EMBL Deguchi et al . , Science 379 , 1010 \u2013 1015 ( 2023 ) 10 March 2023 5 of 6 RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023 Imaging Centre for access to the MINFLUX instrument . JF646 and JFX646 HaloTag ligands were a kind gift of L . Lavis ( HHMI Janelia Research Campus ) . Funding : This work was supported by H2020 Marie Sk \u0142 odowska - Curie Actions ( RobMin grant no . 101031734 to T . D . , EMBL ARISE fellowship no . 945405 to S . L . , and EIPOD4 program grant no . 847543 to J . H . ) ; the European Research Council ( grant no . ERC CoG - 724489 to J . R . and grant no . CoG - 819219 to L . C . K . ) ; and the European Molecular Biology Laboratory ( T . D . , E . - M . S . , C . H . , L . S . , J . H . , S . S . , P . H . , S . L , V . C . , K . - M . N . , and J . R . ) . Authorcontributions : J . R . conceivedtheproject . L . S . , P . H . , and V . C . designed and generated constructs . M . K . I . purified proteins for motor - PAINT . J . H . cultured neurons . T . D . , M . K . I . , E . - M . S . , C . H . , L . S . , and J . H . prepared samples . T . D . , T . W . , and S . S . established MINFLUX trackingprotocols . T . D . andS . L . acquired MINFLUX tracking data . E . - M . S . and C . H . acquired fluorescence images . J . R . wrote the analysis software . T . D . , M . K . I . , E . - M . S . , C . H . , L . C . K . , and J . R . analyzedthe data . K . - M . N . , L . C . K . , and J . R . supervised the research . T . D . , M . K . I , E . - M . S . , C . H . , J . H . , P . H . , L . C . K . , and J . R . wrote the manuscript with input from all authors . Competing interests : The authors declare no competing interests . Data and materials availability : All MINFLUX tracking data are available at BioStudies ( https : / / www . ebi . ac . uk / biostudies / ) under accession no . S - BIAD608 . License information : Copyright \u00a9 2023 the authors , some rights reserved ; exclusive licensee American Association for the Advancement of Science . No claim to original US government works . https : / / www . science . org / about / science - licenses - journal - article - reuse SUPPLEMENTARY MATERIALS science . org / doi / 10 . 1126 / science . ade2676 Materials and Methods Figs . S1 to S9 Tables S1 and S2 References ( 29 \u2013 37 ) MDAR Reproducibility Checklist Movies S1 to S10 View / request a protocol for this paper from Bio - protocol . Submitted 8 August 2022 ; accepted 23 January 2023 10 . 1126 / science . ade2676 Deguchi et al . , Science 379 , 1010 \u2013 1015 ( 2023 ) 10 March 2023 6 of 6 RESEARCH | RESEARCH ARTICLE D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023 Use of this article is subject to the Terms of service Science ( ISSN ) is published by the American Association for the Advancement of Science . 1200 New York Avenue NW , Washington , DC 20005 . The title Science is a registered trademark of AAAS . Copyright \u00a9 2023 The Authors , some rights reserved ; exclusive licensee American Association for the Advancement of Science . No claim to original U . S . Government Works Direct observation of motor protein stepping in living cells using MINFLUX Takahiro Deguchi , Malina K . Iwanski , Eva - Maria Schentarra , Christopher Heidebrecht , Lisa Schmidt , Jennifer Heck , Tobias Weihs , Sebastian Schnorrenberg , Philipp Hoess , Sheng Liu , Veronika Chevyreva , Kyung - Min Noh , Lukas C . Kapitein , and Jonas Ries Science , 379 ( 6636 ) , . DOI : 10 . 1126 / science . ade2676 Zeroing in on motor proteins The super - resolution microscopy technique MINFLUX enables localization of fluorophores using a minimal number of photons . Two studies now expand on the development and implementation of MINFLUX to track motor protein dynamics in vitro and in cells ( see the Perspective by Fei and Zhou ) . Wolff et al . refined the precision of MINFLUX such that single - fluorophore tracking with nanometer precision was possible with only tens of photons . They tracked the movement of kinesin - 1 on microtubules and were able to see individual 4 - nanometer substeps and rotation of the protein during stepping in their analysis . Deguchi et al . applied MINFLUX with a labeling and tracking approach called motor - PAINT to monitor stepping of motor proteins on microtubules in living and fixed cells in both two and three dimensions . \u2014MAF View the article online https : / / www . science . org / doi / 10 . 1126 / science . ade2676 Permissions https : / / www . science . org / help / reprints - and - permissions D o w n l o a d e d fr o m h tt p s : / / www . s c i e n ce . o r g a t U n i v e r s it y o f W a s h i ng t on on A p r il 15 , 2023", + "chanSemanticallyFarInspirations2017": "Semantically Far Inspirations Considered Harmful ? Accounting for Cognitive States in Collaborative Ideation Joel Chan 1 , Pao Siangliulue 2 , Denisa Qori McDonald 3 , Ruixue Liu 3 , Reza Moradinezhad 3 , Safa Aman 3 , Erin T . Solovey 3 , Krzysztof Z . Gajos 2 , Steven P . Dow 4 , 1 Carnegie Mellon University , 2 Harvard University , 3 Drexel University , 4 University of California San Diego , joelchuc @ cs . cmu . edu , { paopow , kgajos } @ seas . harvard . edu , { denisa . qori , rl498 , reza . mora , sra68 , erin . solovey } @ drexel . edu , spdow @ ucsd . edu ABSTRACT Collaborative ideation systems can help people generate more creative ideas by exposing them to ideas different from their own . However , there are competing theoretical views on whether and when such exposure is helpful . Associationist theory suggests that exposing ideators to ideas that are semantically far from their own maximizes novel combinations of ideas . In contrast , SIAM theory cautions that systems should offer far ideas only when ideators reach an impasse ( a cognitive state in which they have exhausted ideas within a particular category ) , and offer near ideas during productive ideation ( a cognitive state in which they are actively exploring ideas within a category ) , which maximizes exploration within categories . Our research compares these theoretical recommendations . In an online experiment , 245 participants generated ideas for a themed wedding ; we detected and validated participants\u2019 cognitive states using a combination of behavioral and neuroimaging data . Receiving far ideas during productive ideation resulted in slower ideation and less within - category exploration , without significant benefits for novelty , compared to receiving no inspirations . Participants were also more likely to hit an impasse when receiving far ideas during productive ideation . These findings suggest that far inspirational ideas can harm creativity if received during productive ideation . Author Keywords Creativity ; creativity support tools ; brainstorming ; examples ; collaborative ideation ACM Classification Keywords Human - centered computing ~ Collaborative and social computing INTRODUCTION Large - scale collaborative ideation platforms , like Climate CoLab and OpenIDEO , draw hundreds to thousands of contributors to collaboratively generate and develop solutions for creative problems . The promise of these platforms is that more breakthrough ideas can be developed by facilitating collaboration and remixing ideas at much higher levels of scale and diversity than before . However , the scale and diversity of crowd ideation also presents unique challenges for collaborative inspiration . In small groups , ideation can be improved by simply exposing all contributors to all ideas [ 14 ] ; however , at crowd scale , it is not uncommon to have hundreds or thousands of contributions . Ideators do not have sufficient time or cognitive resources to sift through that many ideas to select and build on ideas that are most helpful for their thinking [ 26 , 27 ] . Instead , ideators in these settings often resort to superficial processing of a few ideas [ 26 , 27 ] . Consequently , systems that can find and deliver potentially inspiring content to ideators are an important area of technical research and development for large - scale collaborative ideation platforms . From a technical standpoint , significant progress has been made on the problem of how to structure large collections of ideas to enable exploration and navigation of the solution space [ 1 , 18 , 23 , 33 , 43 , 44 ] . Other studies have explored how dedicated community managers [ 6 ] or facilitators [ 8 ] might deliver appropriate inspiration to ideators . In this paper , we consider a central human factors question facing designers of these inspiration delivery systems : how should inspiration delivery systems take into account the semantic distance of other people\u2019s ideas from the target user\u2019s ideas ? Are the benefits of collaborative inspiration maximized by promoting cross - pollination of ideas ( e . g . , exposing ideators to ideas that are very different from their own ) , or by promoting deeper exploration of shared solution approaches ( e . g . , iterating on each other\u2019s ideas Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than the author ( s ) must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from Permissions @ acm . org . C & C ' 17 , June 27 - 30 , 2017 , Singapore , Singapore Copyright is held by the owner / author ( s ) . Publication rights licensed to ACM . ACM 978 - 1 - 4503 - 4403 - 6 / 17 / 06\u2026 $ 15 . 00 DOI : http : / / dx . doi . org / 10 . 1145 / 3059454 . 3059455 Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 93 within a particular semantic region of the solution space ) ? Understanding the relative impact these approaches on the quantity , novelty , and diversity of generated ideas can inform the design of these platforms . To preview our results , we find that semantically far inspirations can negatively impact creative processes ( speed , amount of within - category exploration ) and products ( novelty of ideas ) if they are received during particular cognitive states . Theoretical Foundations Before we describe our experiment and results in detail , it is useful to consider the theoretical foundations of our investigation . Two prominent theories of creativity offer different answers to the question of semantic distance of inspirational stimuli from target user\u2019s own ideas . We selected these theories for relevance to the specific question of inspiration delivery . Our goal is not to arbitrate between competing overall theories of creativity , but rather to advance theoretical foundations for designing effective inspiration delivery systems . Associationist Theory of Creativity On the one hand , the associationist theory of creativity [ 21 , 31 , 34 , 42 ] argues that creativity arises from combining ideas that are very different from one another . From a cognitive standpoint , the associationist view notes a few key mechanisms by which this can be accomplished . First , creators can use serendipity to activate disparate portions of semantic memory at the same time . Mednick [ 34 ] noted an illustrative example of a physicist who \u201creduced serendipity to a method by placing in a fishbowl large numbers of slips of paper , each inscribed with a physical fact . He regularly devotes some time to randomly drawing pairs of these facts from the fishbowl , looking for new and useful combinations\u201d ( p . 221 - 222 ; emphasis ours ) . A related mechanism is mediation , which connects disparate concepts by finding deep structural similarities between them ( e . g . , by analogy [ 19 ] ) . It is worth mentioning that the original associationist theory also cites a mechanism of similarity , which connects disparate concepts by \u201cspreading activation\u201d through similar / related concepts in between . This mechanism is not shared by other associationist theories ( e . g . , conceptual combination , analogy ) , so we focus on the first two mechanisms\u2014serendipity and mediation\u2014that emphasize directly connecting semantically disparate concepts . These theoretical assertions imply that , to improve creativity , inspiration should maximize the probability of making interesting remote associations , either by directly providing candidate combinations , or by activating distant portions of semantic memory through semantic priming [ 12 , 35 ] . This can be accomplished by delivering ideas that are semantically distant from the user\u2019s own ideas . According to the associationist theory , presenting ideators with such semantically far stimuli should increase the diversity and novelty of ideas they subsequently generate . Search for Ideas in Associative Memory ( SIAM ) Model On the other hand , the SIAM ( Search for Ideas in Associative Memory ) model of creative idea generation [ 37 ] argues that the answer depends on the user\u2019s cognitive state . The SIAM model posits that ideation proceeds by alternating between two kinds of cognitive states : productive ideation and impasses . During productive ideation , an ideator fluently accesses idea components from memory , and actively develops new ideas from those idea components . In this state , temporally adjacent ideas are often relatively close in semantic space , with some being close variations or elaborations of prior ideas , and some assembled from neighboring components in associative memory . In this state , ideators can develop more novel and useful ideas through deeper iteration and elaboration [ 11 , 15 , 36 , 41 ] . After exhausting resources within a semantic region , people enter an impasse state , and commence search for new semantic regions of memory from which to sample idea components . Idea generation during this state is slower and more effortful ( as measured by time intervals between ideas , and subjective reports and / or neurophysiological measures of cognitive effort ) , and temporally adjacent ideas tend to be semantically distant from each other . Overall , SIAM posits that , in addition to exploring new semantic regions ( when appropriate ) , creativity can be maximized with fluent exploration within categories , which enables people to move past common , shallow ideas to more interesting , less obvious ideas . The theoretical assertions of the SIAM model imply that semantic distance should be accounted for in different ways for these states . During productive ideation , inspiration should enrich the local semantic region directly , or activate other ( potentially valuable ) idea components in the near neighborhood through semantic priming [ 12 , 35 ] . This can be accomplished by delivering conceptually near stimuli , which can help people more deeply explore the local region ( beneficial for reaching more creative ideas [ 11 , 15 , 36 , 41 ] ) . In this state , exposure to far stimuli might even be harmful : attending to those stimuli could shift attention away from the current memory region ( again , through processes like semantic priming ) , perhaps prematurely terminating productive chains of thought . Further , understanding and adapting far stimuli may require significant cognitive effort , and ideators may not be motivated to expend this cognitive effort if they are productively ideating within a category . During impasses , inspiration should suggest new semantic regions to explore , rather than refocusing ideators on the depleted semantic region . This can be accomplished by delivering semantically far stimuli , which could help accelerate the process of finding new productive regions to explore by activating more diffuse portions of memory . From Theories to Inspiration Delivery System Designs The associationist and SIAM theories therefore predict very different best and worst inspiration delivery approaches ( see Table 1 ) . Associationist theory suggests an ALWAYS - FAR inspiration approach would be best for ideation , where Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 94 the system strives to deliver semantically distant ideas to the user , without accounting for cognitive states . According to the associationist theory , the least helpful approach would be the ALWAYS - NEAR inspiration strategy , which would be predicted to harm ideation by always constraining the user to a semantically adjacent region . In contrast , SIAM suggests a MATCH - STATE inspiration approach would be best for ideation , where the system delivers near stimuli during productive ideation , and far stimuli during impasses . SIAM further predicts that a MISMATCH - STATE inspiration approach will be least helpful , since it presents the user with the opposite of their theoretically predicted inspiration needs during each state ( i . e . , potentially distracting far stimuli during productive ideation , and constraining near stimuli during impasses ) . Interestingly , SIAM also offers a competing prediction for the associationist\u2019s best and worst approaches : the ALWAYS - FAR and ALWAYS - NEAR approaches should yield similar levels of novelty . This is because SIAM predicts that , in the ALWAYS - FAR approach , the increase in novelty from providing pointers to new areas of exploration should be offset by losses in novelty due to hindered within - category exploration . In the ALWAYS - NEAR approach , SIAM predicts that decreased novelty from lack of pointers to new areas of exploration should be offset by increased novelty from fluent within - category exploration [ 5 , 36 ] . To advance research and development of inspiration delivery systems for collaborative ideation platforms , empirical work is needed to tease apart whether the associationist or SIAM theories ( or neither ) are more useful theoretical guides for how to appropriately account for semantic distance of potential inspirational stimuli . Overview and Contributions of The Present Study In this paper , we report the results of an empirical test of these theories by comparing each of their predicted best and worst inspiration delivery approaches against a NO - STIMULI baseline . In an online ideation experiment , 245 participants generated ideas for themed weddings in one of the four inspiration conditions ( ALWAYS - FAR , ALWAYS - NEAR , MATCH - STATE , and MISMATCH - STATE ) , or in the NO - STIMULI baseline condition . We detected changes between productive ideation and impasse states through a simple self - report mechanism . This approach was validated by behavioral ( ideation was significantly slower right before an impasse ) and neuroimaging data ( which showed neuroimaging markers of significantly elevated cognitive effort right before an impasse ) . Consistent with SIAM predictions , MISMATCH - STATE and ALWAYS - FAR participants generated ideas at a slower rate than NO - STIMULI participants , and ALWAYS - FAR participants iterated less within categories ( as measured by mean similarity between subsequent ideas ) compared to NO - STIMULI participants . Further , participants who received near stimuli during productive ideation ( MATCH - STATE , ALWAYS - NEAR ) were less likely to face impasses than participants who received far stimuli during productive ideation ( ALWAYS - FAR , MISMATCH - STATE ) . Contrary to associationist predictions , ALWAYS - FAR ideas were not significantly more diverse or novel than NO - STIMULI ideas ; instead , ALWAYS - FAR ideas were marginally statistically significantly less novel than NO - STIMULI ideas . This paper contributes new insights for how to best promote creative inspiration on collaborative ideation platforms . Specifically , our findings show that far inspirational ideas\u2014though considered to be generally useful for creative inspiration\u2014can harm creativity if received during productive ideation . Our findings also imply that the SIAM model\u2019s state - contingent view of inspiration needs is more useful as a theoretical starting point than the associationist theory of creativity for guiding the design of collaborative inspiration systems . Predicted best approach Predicted worst approach Associationist theory ALWAYS - FAR : deliver far stimuli , regardless of cognitive state ; increases novelty and diversity of ideas by promoting remote associations ( SIAM predicts neutral effect on novelty : gains in novelty from pointers to new ideas offset by hindering exploration within categories ) ALWAYS - NEAR : deliver near stimuli , regardless of cognitive state ; decreases novelty and diversity of ideas by suppressing remote associations ( SIAM predicts neutral effect on novelty : loss of novelty from lack of pointers to new ideas offset by gains from fluent within - category exploration ) SIAM model MATCH - STATE : deliver near stimuli during productive ideation , and far stimuli during impasses ; increases novelty of ideas by promoting fluent exploration within categories , and providing pointers to new areas of exploration at the appropriate time MISMATCH - STATE : deliver far stimuli during productive ideation , and near stimuli during impasses ; decreases novelty of ideas by hindering fluent exploration within categories , and suppressing pointers to new areas of exploration at the appropriate time Table 1 . Best and worst approaches for choosing semantic distance of inspirational stimuli , as predicted by the associationist and SIAM theories of creativity . Associationist theory predicts that an ALWAYS - FAR approach is best , and an ALWAYS - NEAR approach is worst ; SIAM predicts that a MATCH - STATE approach is best , and a MISMATCH - STATE approach is worst . SIAM also makes competing predictions for the associationist theory\u2019s predicted best and worst approaches . Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 95 EXPERIMENT Participants We recruited 245 participants ( mean age = 33 . 5 years , SD = 10 . 4 , 51 % female ) from Amazon Mechanical Turk ( MTurk ) . All participants were located in the U . S . and had 95 % approval on at least 100 MTurk tasks . Participants were paid $ 1 . 25 for their time ( approximately $ 6 / hr wage , given average completion times of 12 \u2013 13 minutes ) . Study Design We used a between - subjects design . Participants were randomly assigned to one of the 5 conditions : 1 ) the NO - STIMULI baseline ( N = 54 ) , 2 ) associationist theory\u2019s predicted best ALWAYS - FAR condition ( N = 47 ) , 3 ) associationist theory\u2019s predicted worst ALWAYS - NEAR condition ( N = 48 ) . 4 ) SIAM theory\u2019s predicted best MATCH - STATE condition ( N = 51 ) , and 5 ) SIAM theory\u2019s predicted worst MISMATCH - STATE condition ( N = 45 ) . Brainstorming Task Participants generated ideas for a themed wedding , where each idea consisted of 1 ) a theme , 2 ) a main prop to be used for guest activities , and 3 ) a freeform description of how the prop would be incorporated into the wedding . We chose this task structure to maximize our ability to accurately tailor conceptual similarity based on participants\u2019 current thinking ( and therefore experimentally isolate our intervention ) in real - time . Achieving real - time semantic tailoring of potential stimuli to unstructured participant ideas of varying length and specificity is challenging to accomplish with a high degree of accuracy . To address this concern , our brainstorming task is semi - structured : participants separately specify theme ( e . g . , \u201cmedieval\u201d ) and prop ( e . g . , \u201csilver spoons\u201d ) components of their themed wedding idea . This allows us to perform fast and accurate tailoring based on those single or compound words where computational similarity measurements tend to do better . For example , models like Pennington et al\u2019s [ 38 ] Global Vectors for Word Representation ( GloVe ) model \u2014 which uses an unsupervised learning algorithm to learn vector representations for words from global word - to - word co - occurrence statistics within a corpus \u2014 are able to achieve between 60 % and 84 % accuracy on word analogy tasks . This brainstorming task also achieves a degree of ecological validity since developing ideas for themed weddings is a common real - world creative task . Sampling Inspirations based on Conceptual Distance We used pre - trained GloVe vectors ( trained on approximately 6 billion tokens ( Wikipedia 2014 and Gigaword 5 corpora , with 300 dimensions ) provided by Pennington et al [ 38 ] to perform similarity matching . While other vector - space models like Latent Semantic Analysis ( LSA ) have a longer history in cognitive science for measuring semantic distance [ 32 ] , we opted to use GloVe , a recent state - of - the - art model that typically agrees well with classic models like LSA [ 17 ] , while being capable of modeling more nuanced semantics , such as simple four - term analogies ( e . g . , man : woman = king : queen ) [ 38 ] . Our database of potential inspirational stimuli consisted of 455 themes and 655 props collected from pilot runs of this study ( with 207 MTurk workers ; none of these workers also participated in the main study ) . We showed inspirations as sets of 3 themes and 3 props , assembled in real - time and tailored to participants\u2019 last generated idea . Near stimuli are sampled to be near in semantic space , but no nearer than cosine similarity of 0 . 5 in the GloVe vector space . We selected this threshold to avoid duplicates and very close matches that are likely already activated , and also to activate the periphery of the current location in the semantic network in order to enrich the semantic region with additional potentially active idea components . Far stimuli were sampled to be as far from current thinking as possible . To ensure our sampling approach focused on conceptual distance , we also controlled for diversity of inspiration sets ( the relative distance between inspirations in a set ) because the diversity of inspirational examples has been shown to impact creative performance [ 4 , 43 ] . Conceptual distance and diversity tend to be positively correlated , but it is hard to generate diverse sets for near stimuli . All things being equal , ideas that are all close to a seed idea will be relatively close to each other , compared to ideas sampled from distant semantic regions . Therefore , we restricted the diversity of sets to be relatively low . We use a simple sampling algorithm to ensure low diversity of sets : For each query , we first sampled a seed inspiration ( whether near or far ) . Then , we found two nearest neighbors of that seed inspiration ( where the cosine similarity of those neighbors to the seed and each other were less than 0 . 5 ) . This completed a set of 3 inspirations for the query . The following are examples of near and far inspirations sampled by our approach for two different themes : For \u201cfootball\u201d , Near : [ season , fun and games , fourth of July ] , and Far : [ toga , hula , prom ] . For \u201csteam punk\u201d , Near : [ album , light of love , rock ] , and Far : [ minions , knight and damsel , ghostbusters ] . Validating Stimuli Sampling Approach To validate our approach , we randomly sampled 100 themes generated by participants in the study , along with the near and far sets of inspirations actually retrieved for those themes during the experiment . We then generated a new set of inspirations that was the opposite distance ( either near or far ) from each theme . A trained research assistant ( blind to which sets were deemed near or far by the algorithm ) then went through each theme and marked which of the two sets ( left or right ) was \u201cnearest\u201d to the theme . A second judge ( one of the authors , also blind to the algorithm\u2019s predictions ) completed judgments for a random subset of 40 of the items , and agreement between the human judges was very high , Cohen\u2019s \ud835\udf05 = 0 . 95 . The Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 96 research assistant judged the remaining items . The model\u2019s selection of near stimuli corresponded well to the judge\u2019s selection , Cohen\u2019s \ud835\udf05 = 0 . 84 , validating our semantic tailoring manipulation . In terms of \u201cabsolute\u201d distance , the near stimuli in our dataset were , on average , 4 nodes away from the participant\u2019s last idea in Wordnet\u2019s association network ( e . g . , WOLF - - > canine - - > carnivore - - > feline - - > CAT ) , compared to 9 nodes away for far stimuli . To validate our diversity control mechanism , we sampled 855 inspiration sets actually provided to participants during our experiment , and measured their pairwise distances using GloVe . In this sample , far sets were not more diverse than near sets ; in fact , there was a tendency for near sets to be more diverse ( M = . 39 , SE = . 00 ) than far sets ( M = . 27 , SE = . 00 ; t = 18 . 67 , p < . 01 ) . This suggests that our diversity control mechanism successfully removed the usual coupling between distance and diversity of inspiration sets . Inferring Ideators\u2019 Cognitive States Our experiment requires that we accurately infer participants\u2019 cognitive state at each moment . Automatic detection is likely to be noisy ; people can be idle for different reasons , and may be productively thinking while not typing . In contrast , prior work shows that people can notice when they are stuck [ 45 ] . However , ideally we do not want to burden participants with constantly monitoring their own cognitive state when they are productive . Therefore , we designed a partially user - driven approach to infer cognitive states . The default state is productive ideation . The participant triggers a state change to impasse by requesting a set of inspirations , with the intuition that participants in this state would naturally seek out new stimulation . The system then infers a state change back to productive ideation once the participant submits a new idea . Validating User - Driven Inferring of Cognitive States Since this user - driven approach is novel , we sought to validate that it succeeds at differentiating between cognitive states . We conducted a small pilot study in which participants brainstormed while wearing functional near - infrared spectroscopy ( fNIRS ) brain sensors . fNIRS is a neuroimaging method that detects changes in the concentration of oxygenated and deoxygenated blood in the brain , relative to a reference point ( e . g . , during resting state , or a fixed time interval prior to an \u201cevent\u201d ) . These changes in blood oxygen concentration can be used to infer changes in brain activity in particular regions of the brain [ 49 ] , similar to the blood oxygen level dependent ( BOLD ) measure used in fMRI . Neuroimaging Validation . The fNIRS device , manufactured by ISS , Inc . contained six measurement channels with 3 - cm source - detector distances . Participants experienced the same set of procedures as our online participants , except they generated ideas for 20 minutes ( to maximize the amount of data points per participant ) , and participated in a post - task semi - structured interview . We preprocessed the fNIRS data for analysis using Homer2 [ 24 ] , a MATLAB - based application for processing fNIRS data . Preprocessing involved converting the raw light intensity data to oxygenated ( HbO ) and deoxygenated hemoglobin ( HbR ) changes . A high - pass filter was also applied at 0 . 5 Hz . Our sample consists of 6 participants , with a total of 23 instances of inferred impasses . We focused our analysis on comparing brain activity 10 seconds immediately before an inspiration request ( which we assume would be an impasse state ) and 10 seconds immediately after the first idea submission after an inspiration request ( which we assume would be a productive ideation state ) . Our hypothesis is that we should see brain activity that indicates higher levels of cognitive effort during the inferred impasse state , compared to the productive ideation state . The expected hemodynamic response would be a negative change in HbR during increase cognitive load [ 25 , 46 ] . Thus , we operationalize cognitive effort as the maximum change in HbR relative to the 2s just prior to the event . We found a significant difference in the maximum HbR signal between the two states , with the pre - stuck period being lower than the post - stuck period across all of the channels we measured ( see Figure 1 ; all p < . 01 ) . This finding suggests participants were exerting high levels of cognitive effort during inferred impasses ( i . e . , right before requesting inspirations ) . Behavioral Validation . These neuroimaging results were further corroborated by comments participants made during the post - task semi - structured interview . For example , one participant said she clicked to request more inspirations \u201cmostly just like after I had like exhausted the ideas in my mind and I was like OK I don\u2019t know what could possibly Figure 1 . Participants in our pilot study showed higher levels of cognitive effort during system - inferred impasses ( green bars ) compared to system - inferred productive ideation ( red bars ) , as indicated by lower levels of \u0394 HbR ( in micromolars , \u03bc M ) in regions of the prefrontal cortex ( measured using functional near - infrared spectroscopy ) . Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 97 be next\u201d . Another participant said he requested inspirations when he \u201cwas just running out of ideas\u201d . Further , participants in our online experiment took significantly more time between subsequent idea submissions just before an inferred impasse ( M = 79 . 6 seconds , SE = 7 . 3 ) , compared to idea transitions not temporally adjacent to an inferred impasse event ( M = 55 . 8 , SE = 5 . 0 ) , t ( 19 . 4 ) = 3 . 3 , p < . 01 ( see Figure 2 ) . Note that these do not include the interval between the last idea submission before an inspiration request and the request , or the interval between the request and the first idea submission after the request . The intervals are therefore indicative of the speed with which ideas are generated , and not the time it takes to perform extra tasks , such as ( deciding to ) request more inspirations . This difference in inter - idea interval is consistent with SIAM\u2019s predictions of faster ideation during productive ideation and slower ideation during impasses . Altogether , these results suggest that our user - driven approach successfully detects cognitive state changes ( i . e . , inspiration requests signal a transition to an impasse state ) . System and Interface Figure 3 shows the ideation interface used in our experiment . Participants enter ideas in a semi - structured format ( separate fields for themes , props , and descriptions ) . In the left pane , the system automatically retrieves a new set of themes and props after each idea entry . The participant is assumed to be in a productive ideation state unless they click the \u201cGive me other inspirations\u201d button . When this button is clicked , the system infers the participant is in an impasse state , and retrieves a new set of inspirations accordingly . Participants can refresh the inspiration feed during the impasse state as many times as they wish . Each button click during this state retrieves a new set of inspirations . Once a participant submits a new idea after requesting inspirations , the system infers the participant has returned to a productive ideation state . The system was built in Meteor . js ( a Node . js - based web application framework ) . The system communicates with a similarity engine via a RESTful API to select inspirations based on semantic relatedness to participants\u2019 current idea focus . Inspiration retrievals took about 1 \u2013 2 seconds . Procedure After providing informed consent , participants were randomly assigned to one of the 5 conditions . Next , they entered a warm - up task screen where they generated alternative uses for a brick for one minute . After this , they completed a short tutorial that highlighted the key features of the interface . Finally , they generated ideas for the main problem for 8 minutes . The system automatically took them to a final survey page after 8 minutes . MEASURES Within - Category Fluency We operationalize two measures of within - category fluency that capture related but distinct aspects of the theoretical construct of within - category fluency : inter - idea interval ( probability of an impasse ) , and transition similarity . Inter - Idea Interval SIAM posits that ideation within a category is more rapid than generating ideas in - between categories ; thus , participants who have higher within - category fluency should , on average , have shorter intervals between idea submissions . Thus , we operationalize inter - idea interval as the median number of seconds between subsequent idea submissions , as logged by our system . We report median inter - idea interval because this measure is insensitive to long inter - idea intervals during impasses and instead , reflects how rapidly a participant was generating ideas while they were in a productive ideation state . We also statistically control in our analysis for the number of ideas generated . This allows us to more cleanly capture the degree of within - category fluency . Transition Similarity Ideas within a category tend to be more semantically similar to each other than ideas between categories : thus , participants who have higher within - category fluency should , on average , have higher similarity between successive ideas . We operationalized transition similarity as Figure 3 . Participants enter ideas in a semi - structured interface . Inspirations in the left pane automatically update during productive ideation ; when participants request new inspirations , this signals an impasse state to the system , and new inspirations are retrieved . The control condition interface is identical except for the absence of an inspiration feed . Figure 2 . Participants generated ideas more slowly just before an impasse compared to other points in their ideation session . Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 98 the median GloVe cosine between themes and props of subsequent ideas . As with inter - idea interval , reporting the median allows us more cleanly capture the degree of within - category fluency . Overall Fluency : Number of Ideas Generated Fluency was operationalized as the number of ideas generated for the problem . Diversity : Mean Pairwise Distance Between Ideas Diversity was operationalized as mean pairwise distance ( the reverse of similarity ) between a participant\u2019s ideas as measured by GloVe . Novelty We recruited 185 workers from MTurk to rate the novelty of the generated ideas on a scale of 1 ( Extremely Obvious ) to 7 ( Extremely Novel ) . Each worker rated a random subset of approximately 30 ideas . Computing correlations between each judges\u2019 ratings and the overall aggregate score yielded an average aggregate - judge correlation of r = . 64 . To deal with potential differences in usage of the rating scale across raters ( e . g . , some might only use the upper end of the scale ) , we compute standardized scores ( a . k . a . \u201cz - scores\u201d ) within raters ( i . e . , a mean score of a rater was subtracted from that rater\u2019s each individual score and the difference was divided by the standard deviation of that rater\u2019s scores ) . An example of a high novelty idea is \u201c [ Chemistry ] [ Lab experiment ] The couple could conduct a common laboratory experiment combining two substances to create a third as part of their ceremony symbolizing and celebrating their union . \u201d ( z - score = 1 . 61 ) . An example of a low novelty idea is \u201c [ formal ] [ gift ] It would be a typical wedding\u201d ( z - score = \u2013 1 . 94 ) Each participant\u2019s novelty score was the highest novelty score across his / her ideas . This conceptualization of novelty is a good fit for the predictions of both associationist and SIAM theories for novelty , which emphasize novelty as an outcome of the ideation process ( i . e . , the most novel idea that was generated , instead of average novelty of all ideas ) . Control Measure : Baseline Fluency In ideation studies , it is important to control for pre - existing differences in participants\u2019 creative capacities , such as baseline fluency of ideation [ 7 , 9 ] . Baseline fluency was operationalized as the number of alternative uses generated for a brick during the warm - up task that participants completed prior to the main ideation task . This measure is intended to capture both aspects of baseline creative fluency [ 20 ] , and aspects of participant motivation and comfort with the interface ( all important for creative productivity ) . RESULTS System - Usage Statistics Participants generated a total of 1 , 574 ideas across conditions . 85 % of participants in the inspiration conditions reported using the inspiration feature in some way ( e . g . , attending to inspirations , requesting inspirations ) . Of those who did , 68 % self - reported interacting with the inspirations ( e . g . , attending to , using as inspiration ) at least \u201csomewhat frequently\u201d ( 3 on a scale of 1 to 5 ) when they weren ' t actively clicking to get more inspirations , M = 3 . 0 ( SE = 0 . 1 ) . Across the inspiration conditions , 49 % of participants requested inspirations at least once . Interestingly , however , likelihood of an inspiration request was not equal across conditions . Participants who received near stimuli during productive ideation ( ALWAYS - NEAR and MATCH - STATE ) were less likely to request an inspiration at least once ( M = . 42 , SE = . 05 ) than participants who received far stimuli during productive ideation ( ALWAYS - FAR and MISMATCH - STATE , M = . 57 , SE = . 05 ) . A logistic regression model , predicting the probability of inspiration request as a function of stimuli distance during productive ideation , showed that the difference between the near and far distance groups was statistically significant , z = 1 . 96 , p = . 05 . Since this analysis was conducted in response to seeing the data ( vs . hypothesized in advance , as with the primary analyses in the subsequent section ) , we wish to clearly mark this finding as exploratory ( rather than confirmatory ) . We revisit this finding in the Discussion . Overall , these numbers suggest that the features of the system relating to inspiration were used reasonably frequently , providing an adequate test of our manipulations . Primary Analyses For each dependent measure , we estimate an ANCOVA model with baseline fluency as a control covariate ( if it is a statistically significant predictor of the dependent measure ) . For median inter - idea interval , we also include number of ideas generated as a theoretically motivated control covariate ( if it is statistically significant ) . All significant main effects of condition are followed up with planned contrasts against the NO - STIMULI condition , using Dunnett\u2019s procedure [ 16 ] to control Type I error inflation from multiple comparisons . Table 2 summarizes model - adjusted means and standard errors for each dependent measure by condition . Slower Ideation with Always - Far and Mismatch - State To investigate ideation pace in each condition , we estimated an ANCOVA predicting median inter - idea interval as a function of condition , controlling for number of ideas ( which was significantly negatively correlated with inter - idea interval , r = \u2013 . 63 , p < . 01 ) . Recall that controlling for number of ideas provides a finer - grained measure of within - category fluency , allowing us to discern qualitative differences ( low vs . high within - category fluency ) between quantitatively similar ( overall number of ideas ) ideation traces . The model showed a significant main effect of condition on median seconds between subsequent ideas , F ( 4 , 233 ) = 3 . 2 , p = . 01 . Planned contrasts showed that ALWAYS - FAR and MISMATCH - STATE participants had significantly longer median inter - idea intervals ( t = 2 . 8 , p = . 02 and t = 3 . 1 , p = . 01 , respectively ) compared to NO - STIMULI participants . Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 99 Lower Transition Similarity in Always - Far Condition Transition similarity was not significantly correlated with baseline fluency ( r = . 02 , p = . 79 ) . Therefore , we estimated an ANOVA with condition as the only factor . The model showed a significant effect of condition , F ( 4 , 218 ) = 4 . 9 , p < . 01 . Planned contrasts showed that ALWAYS - FAR participants had significantly lower median transition similarity ( t = - 3 . 1 , p < . 01 ) than participants in the NO - STIMULI condition . A post - hoc Tukey test also showed that ALWAYS - FAR participants had lower median transition similarity than both MATCH - STATE ( p = . 02 ) and ALWAYS - NEAR participants ( p < . 01 ) . Fluency : Equal Number of Ideas Across Conditions An ANCOVA controlling for baseline fluency showed no significant main effect of condition on the number of ideas , F ( 4 , 239 ) = 0 . 5 , p = 0 . 77 . Always - Far Leads to More Diversity than Always - Near No theoretical covariates were statistically significantly related to diversity . Therefore , we estimated an ANOVA with condition as the only factor . This ANOVA showed a significant main effect of condition on diversity , F ( 4 , 233 ) = 2 . 97 , p < . 01 , but planned contrasts did not show any differences between the inspiration conditions and the control condition . However , a post - hoc Tukey test showed that participants in the ALWAYS - NEAR conditions had significantly lower diversity of ideas compared to the ALWAYS - FAR condition ( p = . 01 ) . No Benefits for Novelty in Always - Far Condition Baseline fluency was not significantly correlated with novelty ( r = . 03 , p = . 68 ) . Thus , we estimated an ANOVA with condition as the only factor . This model showed a significant main effect of condition , F ( 4 , 239 ) = 2 . 5 , p = . 04 . Planned contrasts showed no significant differences between ALWAYS - FAR and NO - STIMULI participants , t = \u2013 2 . 3 , p = . 07 ; however , the mean trends were in the opposite direction predicted by the associationist theory , with the most novel ideas of ALWAYS - FAR participants rated as less novel ( M = 0 . 64 , SE = 0 . 07 ) than NO - STIMULI participants\u2019 most novel ideas ( M = 0 . 88 , SE = 0 . 07 ) . DISCUSSION In this study , we explored how the semantic distance of inspirations from the target user\u2019s own ideas impacts their creative performance . Specifically , we compared two competing theoretical recommendations from creativity theories : 1 ) the associationist view , which predicted that always providing far stimuli would be most beneficial , and 2 ) the SIAM model of creative ideation , which predicted that a state - contingent inspiration delivery ( where near stimuli are delivered during productive ideation , and far stimuli during impasses ) would be most beneficial . Figure 3 summarizes our findings and their implications for the two competing theories . Consistent with the associationist view , ideas generated in the ALWAYS - FAR condition were significantly more diverse than those generated in the ALWAYS - NEAR condition ; however , ALWAYS - FAR ideas were not significantly more diverse than NO - STIMULI ideas . Further , contrary to associationist predictions , ALWAYS - FAR ideas were not significantly more novel than NO - STIMULI ideas ; instead , the mean trends showed that ALWAYS - FAR ideas were possibly less novel than NO - STIMULI ideas . In contrast , consistent with SIAM predictions , MISMATCH - STATE and ALWAYS - FAR participants generated ideas at a slower rate than NO - STIMULI participants , and ALWAYS - FAR participants iterated less within categories compared to NO - STIMULI participants ( indicating that far stimuli hinder within - category exploration ) . Finally , an exploratory analysis showed that participants who received near stimuli during productive ideation ( MATCH - STATE , ALWAYS - NEAR ) were less likely to request inspirations ( which our system used to detect impasses ) than participants who received far stimuli during productive ideation ( ALWAYS - FAR , MISMATCH - STATE ) , suggesting that near stimuli could extend productive ideation chains ( relative to far stimuli ) . However , MATCH - STATE participants did not have greater within - category fluency , overall fluency , or novelty of ideas than NO - STIMULI participants . In summary , we conclude that the SIAM model\u2019s state - contingent view may be more useful as a theoretical starting Inter - idea interval Transition similarity Overall Fluency Diversity Novelty NO - STIMULI 64 . 2 ( 5 . 3 ) 0 . 19 ( 0 . 01 ) 6 . 5 ( 0 . 5 ) 0 . 84 ( 0 . 01 ) 0 . 88 ( 0 . 07 ) ALWAYS - FAR 86 . 2 ( 5 . 7 ) * 0 . 12 ( 0 . 02 ) * * 6 . 1 ( 0 . 5 ) 0 . 86 ( 0 . 01 ) 0 . 64 ( 0 . 07 ) m ALWAYS - NEAR 74 . 3 ( 5 . 6 ) 0 . 20 ( 0 . 02 ) 6 . 4 ( 0 . 5 ) 0 . 81 ( 0 . 01 ) 0 . 67 ( 0 . 07 ) MATCH - STATE 76 . 6 ( 5 . 5 ) 0 . 19 ( 0 . 01 ) 7 . 0 ( 0 . 5 ) 0 . 83 ( 0 . 01 ) 0 . 88 ( 0 . 07 ) MISMATCH - STATE 88 . 7 ( 5 . 8 ) * * 0 . 14 ( 0 . 02 ) 6 . 5 ( 0 . 5 ) 0 . 84 ( 0 . 01 ) 0 . 79 ( 0 . 07 ) Table 2 . Model - adjusted means and standard errors for each dependent measure by condition . m p < . 10 , * p < . 05 , and * * p < . 01 for contrasts with the NO - STIMULI baseline , with Dunnett\u2019s correction for multiple comparisons . Contrasts show that ALWAYS - FAR resulted in significantly longer inter - idea intervals and significantly lower transition similarity and novelty than NO - STIMULI ; MISMATCH - STATE resulted in significantly longer inter - idea intervals than NO - STIMULI . Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 100 point than the associationist theory of creativity for guiding the design of inspiration delivery systems . In practical terms , our findings suggest that following the associationist recommendation to deliver semantically far inspirations throughout ideation ( including during productive ideation , as in the ALWAYS - FAR condition ) is inadvisable , as it provides uncertain benefits for diversity , and relatively certain costs for novelty and within - category exploration . The theoretical assertions of SIAM suggest that the reduction in novelty arises from disruption of the deep - exploration pathway to novel ideas [ 11 , 36 , 41 ] . However , it is still unclear if delivering near stimuli during productive ideation and far stimuli during impasses maximizes benefits for ideation . In the limitations section , we suggest methodological changes that might help future studies explore this issue further . We remind the reader that we make no claims about the relative merits of the associationist or SIAM theories for explaining creativity in general : we restrict our claims to their relative merits for guiding the design of effective inspiration delivery systems . Limitations One limitation of our study is that not all participants entered an impasse state , partially limiting our ability to observe the effects of stimuli during that state . This might be one reason we did not detect an advantage for SIAM\u2019s hypothesized best condition ( MATCH - STATE ) , since\u2014 possibly due to the benefits of receiving near stimuli during productive ideation\u2014many of those participants did not have the opportunity to receive interesting pointers to new areas of exploration ( only available in the impasse state in that condition ) . Perhaps a longer time scale than 8 minutes ( to increase the probability that most participants would run out of ideas ) or induced breaks in the session would provide a better opportunity to study the effects of stimuli during impasses . Relatedly , a longer time scale might provide a clearer test of the potential cumulative benefits of inspiration during productive ideation ( which might come at a slight cognitive cost ) . Finally , our sample consisted of MTurk workers paid $ 6 / hr : while many participants expressed enjoyment in the task , low baseline levels of skill / knowledge are still a possibility , which may have suppressed positive effects of inspirations ( to the extent that skill / motivation is required to adequately benefit from the inspirations ) . These are possible reasons that the four treatment conditions did not significantly outperform the NO - STIMULI participants on any measure . We therefore urge caution generalizing these results to other settings with a longer time scale and with more skilled / knowledgeable / motivated participants . Our system also had a relatively slow response time ( ~ 1 - 2s ) when retrieving inspirations . While this response time is likely to be acceptable from a usability perspective , the inspirations may not always have arrived before participants started writing down their next idea , potentially adding noise to the intervention . A quicker response time might enable a cleaner test of the potential benefits of near stimuli during productive ideation . Broader Implications and Future Work Inspirations should be Delivered at the Right Moments Our study is consistent with previous work that suggests that potentially helpful inspirations ( e . g . , analogous ideas from other domains [ 48 ] , simple hints [ 28 ] , or diverse ideas of others [ 45 ] ) are only helpful when delivered under particular circumstances . These findings underscore the importance of considering not just which inspirations should be delivered to improve creative ideation , but also when they should be delivered . Considering this larger body of findings yields important issues for further research . Figure 3 . Summary of model - adjusted mean contrasts across dependent measures for each inspiration condition against the NO - STIMULI baseline condition ( vertical dashed lines ) . Mean contrasts are reported on the original scale of the dependent measure . Error bars are 95 % confidence intervals . Significant contrasts from the NO - STIMULI baseline ( by Dunnett\u2019s t - test ) are shown in orange . Contrasts that support a theory\u2019s prediction are marked green ; contrasts that contradict ( i . e . , go in the opposite direction of ) a theory\u2019s prediction are marked red . Here , SIAM\u2019s predictions for the ALWAYS - FAR ( for inter - idea interval , transition similarity ) , ALWAYS - NEAR ( for inter - idea interval , transition similarity , fluency ) , and MISMATCH - STATE conditions ( for inter - idea interval ) are supported , while the associationist theory\u2019s predictions for ALWAYS - NEAR is contradicted ( for novelty ) . Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 101 One important issue to consider is whether ideators should receive any inspiration during productive ideation . Does receiving any stimuli during productive ideation amount to mere disruptive interruptions [ 2 , 3 , 45 ] ) ? One state - contingent strategy might be to avoid offering any stimuli during productive ideation , and only offer far stimuli during impasses . Our findings are only partially consistent with this view of inspirations : rather than global deficits associated with any stimulation , we observed meaningful theoretically predicted differences between the conditions , finding that far stimuli was harmful , but near stimuli did not lead to statistically significant deficits . The SIAM model of ideation still provides some theoretical reason to doubt this ( ideation depends on having \u201cidea elements\u201d to recombine ; having more \u201cidea elements\u201d should improve ideation ) , as does prior studies on the benefits of seeing the ideas of others during collaborative brainstorming in small groups [ 14 ] . Future studies that , like our study , vary not just timing but theoretically meaningful variations in the kind of stimuli presented could provide more clarity on this issue . Another important issue is the implications of a state - contingent view for computational creativity support tools that operate at longer time scales ( e . g . , weeks , the lifespan of a project ) than what we examined in this body of work on timing ( i . e . , seconds and minutes ) . At longer time scales , the lines between cognitive states and between real - time inspiration and user - driven search for inspiration might be blurred . For example , how do users get \u201cstuck\u201d , or enter a state of creative flow , within the larger context of a project ? How might we we appropriately tailor the behavior of the inspiration tools to these states at those time scales ( e . g . , patent database search engines [ 30 ] ) ? Finally , if accounting for cognitive states of users is important , how might systems effectively detect when users are in particular cognitive states ? In this study , we used a partially user - driven approach to detecting users\u2019 cognitive states . While this is a reasonable approach for detecting switches to impasse states , future work might explore the use of behavioral markers that predict the onset of an impasse ( e . g . , slowed inter - idea interval , excessively high inter - idea similarity ) and prevent , rather than respond to it . These behavioral markers could be augmented with physiological markers ( e . g . , the fNIRS signals we obtained in our validation study ) , to obtain more nuanced and accurate representations of user states . Advances in the portability and wearability of these physiological sensors open up exciting new avenues for designing creativity support systems that respond to users\u2019 \u201cimplicit input\u201d [ 47 ] . This approach could be especially productive to the extent that changes in cognitive states happen more on a continuum than a binary state shift : systems that can detect early / mild stages of impasse and pre - emptively introduce interventions to prevent impasses might represent a new class of creativity support tools that promote extended states of creative \u201cflow\u201d [ 13 ] . Far Stimuli Should be Used with Caution Our findings also have broader implications for the role of semantic distance in creative inspiration . Far ( rather than near ) stimuli have generally been thought to be more useful for provoking creative mental leaps [ 19 , 22 , 29 , 40 , 50 ] . However , recent investigations are beginning to challenge and refine that claim [ 10 , 39 , 51 ] , pointing out , for example , that overreliance on semantically far stimuli can harm creative performance [ 10 ] . Much of this recent work has focused on the impact of inspirations that are far from one\u2019s problem domain ( e . g . , drawing inspiration from pendulum motions in grandfather clocks when generating ideas for a new approaches to generating electricity in developing countries ) . In this work , we extend the notion that far stimuli should be used with caution to the related but distinct notion of semantic distance of inspirations from one\u2019s current thinking . We do not mean to argue that far stimuli are unimportant or that they should be avoided entirely ; rather , our findings , together with other work on semantic distance , suggest that future research should explore when and how creators can best take advantage of semantically far inspirations . Dual Pathways to Creative Outcomes Finally , our finding that far stimuli during productive ideation not only reduced within - category fluency , but also reduced novelty of ideas ( as predicted by SIAM ) , lends support to the \u201cdual pathway\u201d view of creative ideation [ 11 , 36 , 41 ] , which posits that iteration and deep exploration within categories is an important pathway to creative ( not just better quality ) ideas , perhaps just as important as creative \u201cmental leaps\u201d to remote regions of a solution space [ 50 ] , or combining semantically very different ideas [ 34 ] . Future research on large - scale collaborative ideation could build on this view to explore how to coordinate the crowd to deeply explore within solution approaches , as a complement to promoting cross - pollination of ideas . CONCLUSION In this paper , we empirically examined competing theoretical recommendations for how inspirational delivery systems on collaborative ideation platforms should account for semantic distance of inspirational stimuli . In an online ideation experiment , we find that following the associationist theory\u2019s recommendation to always provide far stimuli yields uncertain benefits for idea diversity and relatively certain costs for within - category fluency and idea novelty . Our research suggests that far inspirations can be harmful for creativity if delivered during productive ideation , and that collaborative inspiration systems could be improved by accounting for ideators\u2019 cognitive states . ACKNOWLEDGEMENTS We thank all of the people ( both in - person and on MTurk ) who participated in the experiment , particularly those who gave valuable feedback on early versions of the experiment . 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Session : Crowd Ideation C & C 2017 , June 27 \u2013 30 , 2017 , Singapore 105", + "chanBenefitsPitfallsAnalogies2011": "Joel Chan University of Pittsburgh , LRDC Room 823 , 3939 O\u2019Hara Street , Pittsburgh , PA 15260 e - mail : joc59 @ pitt . edu Katherine Fu Carnegie Mellon University , 5000 Forbes Avenue , Pittsburgh , PA 15213 e - mail : kfu @ andrew . cmu . edu Christian Schunn University of Pittsburgh , LRDC Room 821 , 3939 O\u2019Hara Street , Pittsburgh , PA 15260 e - mail : schunn @ pitt . edu Jonathan Cagan Carnegie Mellon University , Scaife Hall 419 , 5000 Forbes Avenue , Pittsburgh , PA 15213 e - mail : jcag @ andrew . cmu . edu Kristin Wood University of Texas - Austin , 1 University Station , ETC 4 . 146B , M / C C2200 , Austin , TX 78712 - 1063 e - mail : ood @ mail . utexas . edu Kenneth Kotovsky Carnegie Mellon University , Baker Hall 342F , Pittsburgh , PA 15213 e - mail : kotovsky @ andrew . cmu . edu On the Benefits and Pitfalls of Analogies for Innovative Design : Ideation Performance Based on Analogical Distance , Commonness , and Modality of Examples Drawing inspiration from examples by analogy can be a powerful tool for innovative design during conceptual ideation but also carries the risk of negative design outcomes ( e . g . , design \ufb01xation ) , depending on key properties of examples . Understanding these properties is critical for effectively harnessing the power of analogy . The current research explores how variations in analogical distance , commonness , and representa - tion modality in\ufb02uence the effects of examples on conceptual ideation . Senior - level engi - neering students generated solution concepts for an engineering design problem with or without provided examples drawn from the U . S . Patent database . Examples were crossed by analogical distance ( near - \ufb01eld vs . far - \ufb01eld ) , commonness ( more vs . less - common ) , and modality ( picture vs . text ) . A control group that received no examples was included for comparison . Effects were examined on a mixture of ideation process and product var - iables . Our results show positive effects of far - \ufb01eld and less - common examples on novelty and variability in quality of solution concepts . These effects are not modulated by modal - ity . However , detailed analyses of process variables suggest divergent inspiration path - ways for far - \ufb01eld vs . less - common examples . Additionally , the combination of far - \ufb01eld , less - common examples resulted in more novel concepts than in the control group . These \ufb01ndings suggest guidelines for the effective design and implementation of design - by - anal - ogy methods , particularly a focus on far - \ufb01eld , less - common examples during the ideation process . [ DOI : 10 . 1115 / 1 . 4004396 ] Keywords : design cognition , design methods , conceptual design , innovation , analogy 1 Introduction Innovation , de\ufb01ned as the capacity to generate ideas or products that are both novel and useful , is a critical component of success - ful design in today\u2019s economy [ 1 , 2 ] . A number of investigators have argued that innovation can be best managed in the \u201cfuzzy front end\u201d of the design process [ 3 , 4 ] , notably in the ideation phase , where concepts are created either intuitively or through systematic processes . While many approaches exist to create ideas and concepts as part of ideation , the search for and use of analo - gies have been shown to be quite powerful [ 5 \u2013 8 ] . Analogy is a mapping of knowledge from one domain to another enabled by a supporting system of relations or representations between situa - tions [ 9 ] . This process of comparison between situations fosters new inferences and promotes construing problems in new insight - ful ways . This process likewise is dependent on how the problem is represented , encouraging multiple representations to more fully enable analogical reasoning [ 10 , 11 ] . As an illustrative example , the design concept for the bipolar plate of a fuel cell could be use - fully informed by analogy to a plant leaf due to its similarity in functionality . The most signi\ufb01cant functions affecting the current generation capability of a bipolar plate are \u201cdistribute \ufb02uid , \u201d \u201cguide \ufb02uid , \u201d and \u201cdisperse \ufb02uid . \u201d The plant leaf possesses a sim - ilar function chain , where the veins and lamina perform the func - tions . As a result of this analogy , the bipolar plate \ufb02ow \ufb01eld can be designed to mimic the structure of a leaf [ 10 , 11 ] . Design - by - analogy is clearly a powerful tool in the conceptual design process , and a number of methods have been developed to harness its power , such as Synectics [ 12 ] \u2014group design through analogy types ; French\u2019s work on inspiration from nature [ 13 ] ; Biomimetic concept generation [ 14 ] \u2014a systematic tool to index biological phenomena that links to textbook information ; and analogous design using the Function and Flow Basis [ 15 , 16 ] \u2014 analogous and nonobvious product exploration using the func - tional and \ufb02ow basis . However , fundamental questions surround the proper use of design - by - analogy methods . Most critical , and the problems that are the focus in our work , are what should one analogize over , and what reasoning modalities and associated rep - resentations make innovative design - by - analogy more likely ? While these questions have remained largely unanswered in speci\ufb01c knowledge domains such as engineering design , there is related research literature in the domain of psychological studies of creativity , reasoning , and problem solving . In what follows , we review the relevant literature that motivate our present hypothe - ses , describe the methods and \ufb01ndings of our cognitive study , and then discuss the insights and implications of our work . 2 Background 2 . 1 Analogical Distance of Example Designs . One key vari - able of interest with respect to the question of what one should analogize over is analogical distance . This variable can be Contributed by the Design Education Committee of ASME for publication in the J OURNAL OF M ECHANICAL D ESIGN . Manuscript received December 20 , 2010 ; \ufb01nal manuscript received June 7 , 2011 ; published online August 1 , 2011 . Assoc . Editor : Janis Terpenny . Journal of Mechanical Design AUGUST 2011 , Vol . 133 / 081004 - 1 Copyright V C 2011 by ASME Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm conceptualized as ranging over a continuum from far - \ufb01eld ( from a different problem domain ) to near - \ufb01eld ( from the same or very similar problem domain ) , where analogies closer to the far - \ufb01eld end point share little or no surface features with the target domain , while analogies closer to the near - \ufb01eld end point share a signi\ufb01 - cant number of surface features . The potential for creative insights seems clearest when the two domains being compared are very different on the surface [ 17 ] . Classic accounts of creative discov - eries and inventions often highlight the potential of far - \ufb01eld anal - ogies for creative insights , including George Mestral\u2019s invention of Velcro via analogy to burdock root seeds , and Niels Bohr\u2019s dis - covery of the structure of atoms via analogy to the solar system . Empirical work has also supported a link between far - \ufb01eld analo - gies and innovative outcomes . For instance , it has been shown that the number of far - \ufb01eld analogies used by designers during ideation is positively related to the originality of proposed solu - tions , as rated by a sample of potential customers [ 18 ] . Further , exposure to surface dissimilar design examples increases idea novelty relative to using no examples , and exposure to surface similar examples decreases the variety of ideas generated relative to surface dissimilar examples [ 19 ] . On the other hand , far - \ufb01eld analogies can be dif\ufb01cult to retrieve from memory [ 20 ] or notice as relevant to one\u2019s target problem [ 5 ] . In addition , some investigators have disputed the privileged role of far - \ufb01eld analogies in prominent inventions and discoveries [ 21 , 22 ] . As such , it is an open question whether far - \ufb01eld analogies are always bene\ufb01cial to the design process . One way to tease apart possible ways in which far - \ufb01eld and near - \ufb01eld analogies might help or hinder designers is to use multiple measures of ideation processes , including novelty and variety of ideas , as well as aver - age quality and variance in idea quality . An initial testable hy - pothesis is that providing far - \ufb01eld examples would allow one to generate more novel ideas relative to near - \ufb01eld or no examples . 2 . 2 Commonness of Example Designs . Another potential variable of interest is the commonness of example designs ( i . e . , how common the designs are found in designers\u2019 worlds ) . The commonness of the example design in its respective design space increases the probability that a designer would have had prior ex - posure and / or experience with the design . Psychologically , the commonness of an example design is related to the degree to which it activates relevant prior knowledge of a designer . This knowledge can come from exposure to instances ( since designed objects exist in the world ) , or from deliberately structured experi - ences , such as in engineering coursework or in the course of pro - fessional design [ 23 ] . The psychological literature on creativity and problem solving suggests that prior experience with an artifact might in\ufb02uence one\u2019s ability to \ufb02exibly re - represent and use it and combine it with other concepts in a novel fashion . Take for instance , Duncker\u2019s [ 24 ] classic candle problem , where the task is to \ufb01x a lighted candle on a wall in such a way that the candle wax will not drip onto a table below , and the given materials are a can - dle , a book of matches , and a box of thumb - tacks . A correct solu - tion involves emptying the box of tacks and using it as a platform for the candle ; however , this solution eludes most solvers because it requires recognizing an unconventional use of the box as a plat - form . In fact , when the box is presented to solvers empty , with the tacks beside it , solvers are much more likely to \ufb01nd the unconven - tional solution [ 25 ] . Similarly , in Maier\u2019s [ 26 ] two string problem , where the task is to tie two strings together that are hanging from the ceiling just out of arm\u2019s reach from each other using various objects available ( e . g . , a chair , a pair of pliers , etc . ) , people often fail to recognize the solution of tying the pair of pliers to one string and swinging it like a pendulum and catching it while stand - ing on a chair between the strings . These \ufb01ndings demonstrate the phenomenon of \u201cfunctional \ufb01xedness , \u201d where individuals have dif\ufb01culty seeing unusual alternative uses for an artifact . Another potentially relevant \ufb01nding in the psychological litera - ture is that individuals who acquire experience with classes of in - formation and procedures tend to represent them in relatively large , holistic \u201cchunks\u201d in memory , organized by deep functional and relational principles [ 27 \u2013 29 ] . Many researchers have argued that this ability to \u201cchunk\u201d underlies expertise and skill acquisi - tion [ 27 , 30 , 31 ] . However , if the task at hand requires the individ - ual to perceive or represent information in novel ways , e . g . , to stimulate creative ideation in design , representation of that infor - mation in chunks might become a barrier to success , particularly if processing of component parts of the information chunks helps with re - representation [ 32 \u2013 34 ] . These \ufb01ndings lead to a hypothesis that less - common example designs , which designers are less likely to have been exposed to , might present a unique advantage over more - common example designs in terms of the potential for stimulating creative ideation . Speci\ufb01cally , it could be that less - common examples are more likely to support multiple interpretations , and thus facilitate broader search through the space of possible solutions . Addition - ally , given that the commonness of example designs in the world ( e . g . , in practice , curriculum , etc . ) is related to its representation in designers\u2019 long - term memory , e . g . , ease / probability of recall , one could hypothesize that less - common examples might confer an advantage in terms of the novelty of solution paths they inspire . However , the literature gives no a priori reason to expect effects of commonness on mean quality of solution concepts . 2 . 3 Modality of Example Designs . With respect to the question of optimal reasoning modalities , a potential variable of interest is the contrast between pictorial and text - based represen - tations of examples . One possible reason to investigate this con - trast is that pictorial representations , e . g . , sketches , photographs , and engineering drawings , often contain a higher degree of su - per\ufb01cial features than text - based representations of the same in - formation . This might be detrimental to conceptual design , as the presence of representations with a high degree of super\ufb01cial detail , such as in detailed prototypes , in the physical design envi - ronment tend to restrict the retrieval of far - \ufb01eld analogies from memory [ 7 ] . On the other hand , some investigators argue that pictorial - based representations are better for conceptual design ; for example , it has been shown that novice designers who are presented with sketches of example designs produce more novel and higher quality solution concepts on average relative to being presented with text - based example designs [ 35 ] . At a pragmatic level , too , in creating design - by - analogy tools , one ultimately has to decide on a representation format for potential analogies ; thus , it is important to investigate if it matters whether they are represented in pictorial or text - based formats [ 10 , 11 ] . Addition - ally , it is important to know if the effects of example analogical distance or commonness are modulated by their representation modality . 2 . 4 Summary . In summary , a review of the relevant psycho - logical literature suggests that investigating variations in example analogical distance , commonness , and modality might shed some important light on the questions regarding what to analogize over and whether there are optimal reasoning modalities . Prior work tentatively supports a hypothesis favoring far - \ufb01eld over near - \ufb01eld examples . With respect to commonness , to our knowledge , no studies have directly tested the effects of example commonness on conceptual ideation ; however , the literature does suggest a hy - pothesis favoring less - common over more - common examples . Importantly , the theoretical and empirical literature suggest that there might be different effects of example analogical distance and commonness along different dimensions of the ideation pro - cess , thus motivating a \ufb01ne - grained analytic approach to ensure that the effects of these variables can be clearly understood . Finally , the literature appears to be relatively equivocal about the contrast between pictorial and text - based representations ; thus , our investigation of this variable in the present study is more ex - ploratory than hypothesis - driven . 081004 - 2 / Vol . 133 , AUGUST 2011 Transactions of the ASME Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm 3 Experimental Methods 3 . 1 Design . To investigate the effects of example analogical distance and commonness on conceptual design processes and possible interactions with modality , we conducted a 2 ( distance : far - \ufb01eld vs . near - \ufb01eld ) ! 2 ( commonness : more - common vs . less - common ) ! 2 ( modality : pictures vs . text ) factorial experiment , where participants , i . e . , senior - level engineering students , were given a real - world design problem and were asked to generate so - lution concepts \ufb01rst brie\ufb02y without examples , such that they understood the problem , and then with examples , to evaluate the effects of examples on problem solving . To establish whether examples of different types enabled or hindered problem solving , a control group of students executed a similar procedure but received no examples . 3 . 2 Participants . Participants were 153 students ( predomi - nantly mechanical engineering undergraduates ) enrolled at two research universities in the United States . Participants were recruited from classes and were given either extra credit or com - pensation of $ 15 for their participation . Participants ranged from 20 to 38 years in age ( M \u00bc 22 , SD \u00bc 1 . 89 ) . 70 % were male . 87 % were undergraduate engineering students ( 95 % mechanical engi - neering , 5 % electrical engineering and others ) and 13 % masters students in disciplines related to product design ( e . g . , mechanical engineering , product development , business administration ) . 66 % of the participants had at least 1 \u2013 6 months of engineering intern - ship experience , and all but 2 out of the 153 students had experi - ence with at least one prior design project in their engineering curriculum . Approximately 82 % of the students had taken at least one course where a structured approach to design was taught . Thus , most of the participants had relevant mechanical engineer - ing domain knowledge and design experience . Participants were randomly assigned to one of the nine possible conditions in each class by distributing folders of paper materials prior to students arriving in class . The obtained distribution of par - ticipants across the nine conditions is shown in Table 1\u2014the sam - ple populations , N s , are unequal not because of dropout but rather from stochasticity in where students chose to sit down . With these sample populations , statistical power for detecting three - way interactions ( not our theoretical goal ) is modest , but power for detecting two - way interactions and main effects is good . 3 . 3 Design Problem . The design problem was to design a low cost , easy to manufacture , and portable device to collect energy from human motion for use in developing and impover - ished rural communities , e . g . , India , many African countries . This design problem was selected to be meaningful and challenging to our participants . The problem was meaningful in the sense that real - world engineering \ufb01rms are seeking solutions to this problem and the problem involves social value ; thus , students would be appropriately engaged during the task [ 36 \u2013 38 ] . The problem was challenging in the sense that a dominant or accepted set of solu - tions to the problem has yet to be developed ( so students would not simply retrieve past solutions ) , but it was not so complex as to be a hopeless task requiring a large design team and very detailed task analysis . 3 . 4 Selection of Examples . Examples were patents selected from the U . S . Patent Database . Candidate patents were retrieved using keyword search on the U . S . Patent and Trade Of\ufb01ce web - site . The keywords used were basic physical principles , such as induction , heat transfer , potential energy , as well as larger cate - gorical terms like mechanical energy . The \ufb01nal set of eight patents was selected by two PhD - level mechanical engineering faculty based on two sets of criteria : ( 1 ) balanced crossing of the analogi - cal distance and commonness factors , such that there would be two patents in each of the four possible combinations , and ( 2 ) overall applicability to the design problem , over and above ana - logical distance and commonness . Each participant in the analogy conditions received two examples of a particular type , roughly balanced across conditions for applicability . The patents for each of the conditions are shown in Table 2 . With respect to the \ufb01rst set of criteria , the speci\ufb01c guidelines for selection were as follows : 1 . Distance : Far - \ufb01eld patents were devices judged to be not directly for the purpose of generating electricity , while near - \ufb01eld patents were those judged to be directly for the purpose of generating electricity . 2 . Commonness : More - common patents were devices judged likely to be encountered by our target population in their standard engineering curriculum and / or everyday life , while less - common patents were those judged unlikely to be seen previously by the participants under typical circumstances . With respect to the modality factor , in the picture conditions , participants received a representative \ufb01rst \ufb01gure from the patent , which typically provides a good overview of the device , while in the text conditions , participants received the patent abstract . In some cases , abstracts differed substantially in length ; to equate for quantity of text across conditions , overly brief abstracts were aug - mented with additional text from the body of the patent , which elaborated on the details of the design and technology . To provide some foundational context , all text - and - picture - condition partici - pants also received the patent title . 3 . 5 Experimental Procedure . The experiments were con - ducted during class . Participants generated solution concepts in three phases and subsequently completed a background survey . Participants proceeded through the phases using a sequence of envelopes to carefully control timing of the task and exposure to examples across conditions . In particular , we wanted to ensure that design examples were received only after participants had made some substantial progress in ideation , since prior work has shown that examples and potential analogies are most helpful when received after ideation has already begun [ 39 , 40 ] . The over - all time allowed for this task was suf\ufb01cient to allow for broad ex - ploration of the concept space , but not enough to develop Table 1 Distribution of participants across conditions Near - field Far - field More - common Less - common More - common Less - common Picture 13 17 15 16 Text 17 16 16 17 Control 24 Table 2 Patents for each condition Near - field Far - field More - common - Waterwheel - driven generating assembly ( 6208037 ) - Escapement mechanism for pendulum clocks ( 4139981 ) - Recovery of geothermal energy ( 4030549 ) - Induction loop vehicle detector ( 4568937 ) Less - common - Apparatus for producing electrical energy from ocean waves ( 4266143 ) - Accelerometer ( 4335611 ) - Freeway power generator ( 4247785 ) - Earthquake isolation \ufb02oor ( 4402483 ) Journal of Mechanical Design AUGUST 2011 , Vol . 133 / 081004 - 3 Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm particular ideas in depth , matching our focus on the ideation process . Analogy and control groups executed the same overall sequence , but differed in the particular activities in the second phase of ideation ( see Fig . 1 for a comparison of the procedures ) . In general , the sequence of phases was to : ( 1 ) read design problem and generate solution concepts , ( 2 ) either ( a ) review two patents and write / draw solutions / ideas that come to mind when looking at the patents or ( b ) continue generating concepts , and ( 3 ) generate more solution concepts . Each phase lasted 10 min . With respect to idea generation , participants were instructed to generate and record as many solution concepts to the design prob - lem as they could , including novel and experimental ones , using words and / or sketches to describe their solution concepts . 4 Ideation Metrics The experiment generated 1321 total ideas . To thoroughly explore the range of effects of varying the analogical distance , commonness , and modality of design examples on conceptual design processes , we applied a range of ideation metrics to these ideas : ( 1 ) the extent to which solution features were transferred from examples , ( 2 ) quantity of ideation , ( 3 ) breadth of search through the space of possible solutions , ( 4 ) quality of solution concepts , and ( 5 ) novelty of solution concepts . The \ufb01rst three met - rics provided measures of the ideation process of participants and how they processed the examples : examining solution transfer provides insight into the mechanisms by which participants might be stimulated by the examples , e . g . , did they actually use solution elements ; measuring quantity of ideation gave a sense of how par - ticipants were exploring the design space , i . e . , whether they were generating and re\ufb01ning a small number of ideas , or exploring mul - tiple concepts and variations of concepts , which is associated with higher likelihood of generating high - quality concepts [ 4 ] ; \ufb01nally , breadth of search was taken to be a measure of the ability to gen - erate a wide variety of ideas , which is associated with the ability to restructure problems , an important component of creative abil - ity [ 41 \u2013 43 ] . The \ufb01nal two metrics focused on the ideation prod - ucts of participants . We investigated quality because in design , a baseline requirement is that concepts must meet customer speci\ufb01 - cations ; design concepts that are novel but do not meet customer speci\ufb01cations cannot be considered acceptable designs , let alone creative ones [ 41 ] . We investigated novelty because there is a high degree of consensus in the literature that creative products are at least novel [ 41 , 42 ] . 4 . 1 Data Preprocessing . The raw output of each participant was in the form of sketches and / or verbal descriptions of concepts . Examples of participant - generated solution concepts are shown in Fig . 2 . A number of preprocessing steps were necessary to prepare the data for coding and analysis . First , each participant\u2019s raw output was segmented by a trained coder into solution concepts . A sketch and / or verbal description was segmented as one solution concept if it was judged to describe one distinct solution to the design problem . Variations of solutions ( e . g . , with minor modi\ufb01cations ) were counted as distinct solution concepts . Segmentation was independently checked by a second coder . Inter - rater agreement was high ( 96 % ) , and all dis - agreements were resolved by discussion . Next , sets of two senior mechanical engineering students rated each solution concept as meeting or not meeting the minimum constraints of the design problem , as described above , to remove off - topic inspirations gen - erated by the patent examples , especially in the second phase . Inter - rater agreement was acceptable , with an average Cohen\u2019s kappa of 0 . 72 . All disagreements were resolved through discus - sion . The 1066 solution concepts remaining after preprocessing constituted the \ufb01nal data set for analysis . 4 . 2 Solution Transfer . Solution transfer was de\ufb01ned as the degree to which a given participant\u2019s idea set contained solution features from the examples she / he received . The process of pro - ducing a solution transfer score for each participant was as fol - lows . First , key features were generated by one of the co - authors for each of the eight patent examples , and the list was cross - checked for relevance by the other co - authors . Recall that each participant received two examples ; however , since picture and text examples were essentially the same examples ( only in differ - ent representations ) , the 2 ! 2 ! 2 design reduced to a 2 ! 2 design , leaving a total of eight examples . A total of 39 key fea - tures were identi\ufb01ed . Because some features overlapped across examples ( e . g . , \u201cbuilt into ground , stationary , or permanent\u201d was Fig . 1 Comparison of experimental procedures for analogy vs . control groups Fig . 2 Example participant solution concepts 081004 - 4 / Vol . 133 , AUGUST 2011 Transactions of the ASME Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm associated with four patent examples ) , there was not a simple one - to - one mapping of features to examples . The number of features associated with each of the eight examples ranged from 4 to 7 ( M \u00bc 4 . 9 , SD \u00bc 1 . 0 ) . Second , each participant solution concept was coded for the presence or absence of a set of the features found in the full set of patent examples presented to participants . The \ufb01rst 50 % of solution concepts was double - coded by two sen - ior mechanical engineering students to establish reliability . Later , all coding was completed by one student only . Test - retest meas - ures of reliability were obtained in lieu of inter - rater reliabilities . Cohen\u2019s kappa averaged across features was 0 . 57 . Because some features had low coding reliability or high overlap of features across many of the patents or simply were common elements of most proposed solutions across all conditions , the initial set of 39 features was \ufb01ltered down to 23 features according to three criteria : 1 . Acceptable inter - rater agreement , i . e . , Cohen\u2019s kappa greater than 0 . 4 . 2 . Not shared by more than three examples . 3 . Not too common , i . e . , base rate ( collapsed across conditions ) less than 0 . 5 . After \ufb01ltering , the number of features ranged from 1 to 5 ( M \u00bc 2 . 9 , SD \u00bc 1 . 4 ) per example and from 4 to 8 ( M \u00bc 5 . 8 , SD \u00bc 1 . 7 ) per each of the four conditions in the distance by com - monness 2 ! 2 design . Cohen\u2019s kappa averaged across the \ufb01ltered set of features was 0 . 66 . To produce solution transfer scores for each participant , the fol - lowing procedure was used . First , for each cell in the 2 ! 2 ( distance ! commonness ) , we computed for each participant the proportion of his / her ideas that had at least one solution feature from the examples she / he received . Next , this proportion was con - verted into a standardized z - score by subtracting the mean and dividing by the standard deviation of proportion scores for all par - ticipants who were not in that 2 ! 2 cell . The reason for using this transformation was that solution features from examples could occur in participants\u2019 ideas even if they never saw the relevant examples ; this transformation allows us to separate the probability of participants using solution features from examples they have seen from the probability of using those solution features even if they had never seen the examples . For each participant , the trans - fer score was the z - score of each feature relevant to the examples they actually received . The solution transfer score thus gave a measure of the degree to which a given participant\u2019s idea set differed from \u201cnormal\u201d in terms of the proportion of ideas with at least one feature from the examples she / he received . To illustrate , suppose participant 1001 had a z - score of 1 . 34 for far - \ufb01eld , more - common examples . This number would say that the proportion of 1001\u2019s ideas with at least one solution feature from the examples s / he received was 1 . 34 standard deviations higher than the mean proportion of ideas with at least one solution from those examples under \u201cnormal\u201d circum - stances ( i . e . , without having seen either of the two far - \ufb01eld , more - common examples ) . 4 . 3 Quantity of Ideation . Quantity of ideation was de\ufb01ned as the number of solution concepts generated post analogy , i . e . , from the second phase of ideation onwards , that met the minimum constraints of the design problem , viz . ( 1 ) the device generates electricity , and ( 2 ) it uses human motion as the primary input . As noted in the introduction , quantity is often taken to be a key com - ponent of creativity . Quantity was de\ufb01ned at the level of the par - ticipant , i . e . , each participant received a single quantity score . Because we were primarily interested in the effects of examples on quantity , analyses concentrated on the number of solution con - cepts generated after receiving examples ( i . e . , after the \ufb01rst phase ) adjusting for the number of solution concepts generated in the \ufb01rst phase ( which acted as a covariate to adjust for baseline variation in quantity across participants ) . 4 . 4 Breadth of Search . Breadth of search was conceptual - ized in our study as the proportion of the space of possible solu - tions searched by a given participant . To determine the space of possible solutions , the design problem was \ufb01rst functionally decomposed into potential subfunctions by one of the authors , drawing from the reconciled function and \ufb02ow basis of Hirtz and colleagues [ 16 ] . Due to the open - ended nature of the design problem , a rela - tively large number of subfunctions were initially generated , as follows : 1 . Import / accept human interaction 2 . Transform human energy to mechanical energy 3 . Transform human energy to alternative energy 4 . Import other material 5 . Contain / store other material 6 . Transfer other material 7 . Import alternative energy source 8 . Transform alternative energy source into mechanical energy 9 . Transform alternative energy source to alternative energy 10 . Transform collected energy to mechanical energy 11 . Transmit mechanical energy 12 . Transform mechanical energy 13 . Store mechanical energy 14 . Transform mechanical to alternative energy 15 . Transform alternative energy to electrical energy 16 . Actuate / deactuate energy 17 . Transform mechanical energy to electrical energy 18 . Condition electrical energy 19 . Store electrical energy 20 . Supply electrical energy 21 . Transmit electrical energy 22 . Convert electrical to light or EM Each subfunction solution consisted of a how and what compo - nent , where the former speci\ufb01es the component of the solution concept that implements the subfunction , and the latter speci\ufb01es either the input or the output of the subfunction ( whichever is the less speci\ufb01ed ) . For example , a solution for the subfunction \u201cimport human\u201d might be \u201c foot with pedals . \u201d Two senior mechanical engineering students independently coded the solutions to the subfunctions for each solution concept . The solution types for the how and what components of each sub - function were generated bottom - up by the students as they coded , with each new solution type being added to a running list of solu - tion types ; the running list of solution types for each subfunction constituted the coding scheme . Inter - rater reliability was high , with an average Cohen\u2019s kappa across subfunctions of 0 . 84 . All disagreements were resolved by discussion . While the nature of the design problem was open - ended , a core set of subfunctions emerged from the dataset : only a small subset of the initial set of subfunctions occurred often enough for stable estimates of breadth and novelty ( i . e . , base rate greater than 0 . 1 , collapsed across conditions ) : 1 . Import human 2 . Transform human energy to mechanical energy 3 . Import alternative energy 4 . Transform alternative energy to mechanical energy 5 . Transform mechanical energy to electrical energy 6 . Store electrical energy Upon more detailed analysis , it turned out that there were only two solution types for the subfunction \u201cstore electrical energy , \u201d namely \u201cbattery\u201d or \u201ccapacitor , \u201d and the frequency of occurrence for each solution type was relatively equivalent ; thus , novelty scores for this subfunction would be unlikely to differentiate between participants . Furthermore , since the design problem was focused on the problem of harvesting ( vs . storing ) energy , data for this subfunction were not included in computations of breadth . Journal of Mechanical Design AUGUST 2011 , Vol . 133 / 081004 - 5 Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm We de\ufb01ned the space of possible solutions for each of the what and how components of each subfunction by enumerating the number of distinct solution types generated by participants across all phases of ideation . A breadth score b j for each participant on subfunction j was then computed with b j \u00bc X n k \u00bc 1 w jk ! C jk T jk ( 1 ) where C jk is the total number of solution types generated by the participant for level k of subfunction j , T jk is the total number of solution types produced by all participants for level k of subfunc - tion j , and w k is the weight assigned level k . To give priority to breadth of search in the what space ( types of energy / material manipulated ) , we gave a weight of 0 . 66 to the what level ( which was assigned to k \u00bc 1 ) , and a weight of . 33 to the how level ( which was assigned to k \u00bc 2 ) . An overall breadth score for each partici - pant was given by the average of breadth scores for each of the three subfunctions j . 4 . 5 Quality . Quality of solution concepts was measured using holistic ratings on a set of subdimensions of quality . Two other senior mechanical engineering students independently coded solution concepts on 5 - point scales ranging from 0 to 4 ( 0 is unac - ceptable and 4 is excellent ) for six subdimensions of quality , cor - responding to a set of possible customer speci\ufb01cations : 1 . Cost 2 . Feasibility of materials / cost / manufacturing 3 . Feasibility of energy input / output ratio 4 . Number of people required to operate device at a given moment 5 . Estimated energy output 6 . Portability 7 . Time to set up and build , assuming all parts already avail - able at hand These subdimensions were generated by the second author , who is a Ph . D . candidate in mechanical engineering focusing on design methods and cognition , and checked for validity by two other authors , who are mechanical engineering faculty specializing in en - gineering design . For each subdimension , each point on the 5 - point scale was anchored with a unique descriptor . For example , for the \u201cfeasibility of energy input / output ratio\u201d subdimension , 0 was \u201cunfeasible design or input energy completely dwarfs output , \u201d 1 was \u201cinput less than output\u201d , 2 was \u201cI / O about even , \u201d 3 was \u201csustainable / little surplus output ; human input easy , \u201d and 4 was \u201coutput signi\ufb01 - cantly higher than input . \u201d Inter - rater agreement was computed using a Pearson correlation between the ratings of the two coders for each subdimension . The average of correlations across subdimensions was 0 . 65 , and the range was from 0 . 49 to 0 . 77 . An overall quality score was computed for each solution concept , as given by Q \u00bc P n j \u00bc 1 q j ! r j Q max ( 2 ) where q j is the quality score for quality subdimension j , r j is the reliability of the coding for that subdimension , and Q max is the max - imum possible overall quality score , which would be given by set - ting q j to 4 for each subdimension . The contributions of subdimension scores to the overall quality score were weighted by reliability to minimize the in\ufb02uence of measurement error . Since the overall quality score was a proportion of the maximum possible quality score , the score ranged from 0 to 1 . Agreement between coders at the level of this composite score was acceptable ( r \u00bc 0 . 68 ) . 4 . 6 Novelty . Novelty was de\ufb01ned as the degree to which a particular solution type was unusual within a space of possible solutions . This approach allowed us to avoid the dif\ufb01culties of judging the novelty of thousands of solution concepts via holistic rating methods . Recall that for the breadth metric , the space of possible solutions was de\ufb01ned in terms of a set of \ufb01ve core sub - functions for the design problem ; recall further that each subfunc - tion was decomposed further into what and how components , where the former speci\ufb01es the component of the solution concept that implements the subfunction , and the latter speci\ufb01es either the input or the output of the subfunction ( whichever is the less speci - \ufb01ed ) . Rather than computing novelty scores for solutions to each level of each subfunction ( the what and how levels ) , we chose to compute novelty scores for the conjunction of what and how solu - tion components for each subfunction . For example , rather than computing the relative unusualness of the solution components \u201cfoot\u201d and \u201cpedals\u201d separately for the solution \u201cfoot with pedals\u201d for the subfunction \u201cimport human interaction , \u201d the relative unusualness of the solution \u201cfoot with pedals\u201d relative to other solutions would be computed . The rationale for this choice was that these words in conjunction as a solution have a speci\ufb01c mean - ing that needed to be considered . Novelty scores were computed for each subfunction solution using Eq . ( 3 ) , which is a formula adapted from Ref . [ 39 ] N i \u00bc T i # C i T i ( 3 ) where T i is the total number of solution tokens generated for sub - function i in the \ufb01rst phase of ideation ( collapsed across all partic - ipants ) , and C i is the total number of solution tokens of the current solution type in the \ufb01rst phase of ideation . Because this measure was essentially a measure of proportion , the novelty score for each idea ranged from 0 to 1 , with 0 representing solution types found in every solution ( this extreme was never observed ) and 1 representing solution types that never occurred in the \ufb01rst phase . The initial set of solution concepts ( generated in the \ufb01rst phase of ideation ) was taken to be the original design space of the partici - pants since it corresponded to concepts generated prior to receiv - ing examples . The \ufb01nal novelty score for each solution concept was the average of its subfunction novelty scores . 5 Results 5 . 1 Relationships Between Metrics . Analysis of the inter - relationships between the ideation metrics suggested a preliminary process model that could account for these correlations and help to conceptually organize the results ( see Fig . 3 ) . Of course , corre - lations per se do not guarantee causation and other causal models are possible . The preliminary process model is as follows : \u2022 Increased solution transfer results in decreased quantity , pos - sibly because many participants had trouble thinking of solu - tions beyond the ones presented . \u2022 A high quantity of ideation allows for greater breadth of search , even if only on a statistical sampling basis . Fig . 3 Summary of intermetric correlations . Numbers shown are Pearson\u2019s r . All correlations are signi\ufb01cant at p < 0 . 01 . 081004 - 6 / Vol . 133 , AUGUST 2011 Transactions of the ASME Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm \u2022 Greater breadth of search , perhaps also only on a statistical sampling basis , in turn allows for the generation of higher novelty and higher quality solution concepts . \u2022 Repeatedly searching on the fringes of the design space ( as measured by high average novelty ) further increases the probability of \ufb01nding a highly novel concept . \u2022 Finally , increasing the variability of the quality of solution concepts increases the probability of generating a high - qual - ity solution concept . This last relationship is in accord with the work of Ulrich and colleagues in the \ufb01eld of innovation management , who have argued and showed empirically that one way to increase the likelihood of \ufb01nding high market potential product concepts is to increase the variance of the quality of the concepts that are generated [ 4 , 44 ] . 5 . 2 Effects of Analogy Manipulations on Ideation Metrics . We now present our \ufb01ndings by manipulation ( distance , commonness , and modality ) , using the preliminary process model as an organizational framework . Effects of manipulations on the ideation metrics will be described following the \ufb02ow of the process model , \ufb01rst considering solution transfer , quantity , and breadth , followed by consideration of effects on quality and novelty of ideation . Separate 3 - way ( distance ! commonness ! modality ) analysis of variance ( ANOVA ) models were computed for each process variable in the model . In some cases ( indicated in each case ) , the level of that variable during the pre - analogy phase was used as a covariate in the analysis because the baseline measure was a signi\ufb01cant predictor of postanalogy performance . 5 . 2 . 1 Analogical Distance of Examples . There was a main effect of example distance ( p < 0 . 01 , g 2 \u00bc 0 . 08 ) on solution trans - fer , where participants who received far - \ufb01eld examples were much more likely than participants who received near - \ufb01eld exam - ples to use solution elements from the examples they received ( d \u00bc 0 . 60 ) ; 1 in fact , solution features from near - \ufb01eld examples were no more likely to be present in participant solutions after processing examples relative to the pre - example phase ( see Fig . 4 , bottom left ) . There was also a main effect on quantity ( p < 0 . 01 , g \u00bc 0 . 05 ) , where participants who received far - \ufb01eld examples generated sig - ni\ufb01cantly fewer solution concepts relative to participants who received near - \ufb01eld examples ( p < 0 . 05 , d \u00bc # 0 . 30 ; see Fig . 4 , upper left ) . There were no signi\ufb01cant differences in terms of quantity between receiving no examples ( control ) and receiving either far - or near - \ufb01eld examples . However , the small effect of distance on quan - tity did not translate into an effect on breadth : there were no reliable effects of distance on breadth of search ( p \u00bc 0 . 78 , g 2 \u00bc 0 . 00 ) . With respect to quality of solution concepts , there were no effects of distance on either mean or maximum quality . However , there was a main effect of distance of the variability in quality of participants\u2019 solution concepts ( p < 0 . 05 , g 2 \u00bc 0 . 06 ; see Fig . 4 , lower right ) , where participants who received far - \ufb01eld examples had a larger standard deviation in quality of solution concepts Fig . 4 Summary of effects of example distance . * p < 0 . 05 and * * p < 0 . 01 . Control group data are shown in white bars . Error bars are 6 1 standard error . 1 d statistics estimate the size of the difference in group means in terms of the av - erage standard deviation of the two groups in the contrast ; in this case , d \u00bc 0 . 60 esti - mates that the mean probability of transfer is greater with far - \ufb01eld vs near\ufb01eld examples by 0 . 60 of a standard deviation ( a moderate to large difference ) . Journal of Mechanical Design AUGUST 2011 , Vol . 133 / 081004 - 7 Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm than participants who received either near - \ufb01eld examples ( p < 0 . 05 , d \u00bc 0 . 64 ) or no examples ( p < 0 . 05 , d \u00bc 0 . 78 ) . There were no signi\ufb01cant differences between receiving near - \ufb01eld examples vs . no examples . Finally , there was a main effect of distance on mean novelty ( p < 0 . 05 , g 2 \u00bc 0 . 04 ) , where participants who received far - \ufb01eld examples generated solution concepts that were more novel on av - erage relative to participants who received near - \ufb01eld examples ( p < 0 . 05 , d \u00bc 0 . 56 ; see Fig . 4 , upper right ) . Similar patterns of effects were found with maximum novelty of solution concepts ( p < 0 . 05 , g 2 \u00bc 0 . 04 ) , where the most novel solution concept of participants who received far - \ufb01eld examples was more novel on average relative to the most novel solution concept of participants who received near - \ufb01eld examples ( p < 0 . 05 , d \u00bc 0 . 56 ) . There were no signi\ufb01cant differences between participants who received no examples ( control ) vs . near - or far - \ufb01eld examples on either mean or maximum novelty . In summary ( see Fig . 4 ) , example distance appeared to have signi\ufb01cant effects on multiple aspects of ideation . Speci\ufb01cally , novelty and variability in quality of concepts increased as a func - tion of receiving far - \ufb01eld examples , although only in the latter case was the contrast with control statistically signi\ufb01cant . The so - lution transfer metric suggests that these increases might be asso - ciated with incorporating solution elements from the far - \ufb01eld examples . However , the bene\ufb01ts of far - \ufb01eld examples came with a slight cost , viz . a reduction in quantity : in meaningful terms , the cost of processing far - \ufb01eld examples given a standard time for ideation appeared to be , on average , about one solution concept . 5 . 2 . 2 Commonness of Examples . Turning now to the main effects of commonness in the same ANOVAs , there were no reliable effects on solution transfer ( p = 0 . 30 , g 2 \u00bc 0 . 01 ) . How - ever , there was a main effect on quantity ( p < 0 . 01 , g 2 \u00bc 0 . 12 ) , where participants who received more - common examples gen - erated signi\ufb01cantly fewer solution concepts relative to partici - pants who received either more - common examples ( p < 0 . 01 , d \u00bc # 0 . 67 ) or no examples ( p < 0 . 01 , d \u00bc # 0 . 76 ; Fig . 5 , upper left ) . There were no signi\ufb01cant differences in quantity between participants who received less - common vs . no examples ( con - trol ) . There was also a main effect of on breadth of search ( p < 0 . 01 , g 2 \u00bc 0 . 07 ) , where participants who received more - common examples searched less of the design space than par - ticipants who received either less - common examples ( p < 0 . 05 , d \u00bc # 0 . 61 ; Fig . 5 , lower middle ) or no examples ( p < 0 . 01 , d \u00bc # 1 . 03 ) . There were no signi\ufb01cant differences in breadth of search between participants who received less - common vs . no examples ( control ) . With respect to quality of solution concepts , there were no reli - able effects of commonness on either mean or max quality . How - ever , there was a main effect on variability in quality of participants\u2019 solution concepts ( p < 0 . 05 , g 2 \u00bc 0 . 06 ; see Fig . 5 , lower right ) , where participants who received less - common exam - ples had a larger standard deviation in quality of solution concepts than participants who received either more - common examples ( p < 0 . 05 , d \u00bc 0 . 62 ) or no examples ( p < 0 . 05 , d \u00bc 0 . 68 ) . There were no signi\ufb01cant differences between receiving more - common examples vs . no examples . Fig . 5 Summary of effects of example commonness . * p < 0 . 05and * * p < 0 . 01 . Control group data are shown in white bars . Error bars are 6 1 standard error . 081004 - 8 / Vol . 133 , AUGUST 2011 Transactions of the ASME Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm Finally , there were main effects on mean novelty ( p < 0 . 01 , g 2 \u00bc 0 . 10 ) , where participants who received less - common exam - ples generated solution concepts that were more novel on average relative to participants who received more - common examples ( p < 0 . 01 , d \u00bc 0 . 61 ; see Fig . 5 upper right ) and maximum novelty ( p < 0 . 01 , g 2 \u00bc 0 . 96 ) , where the most novel solution concept of participants who received less - common examples was more novel on average relative to the most novel solution concept of partici - pants who received more - common examples ( p < 0 . 01 , d \u00bc 0 . 61 ) . There were no signi\ufb01cant differences between participants who received no examples ( control ) vs . more - or less - common exam - ples on either mean or maximum novelty . In summary ( see Fig . 5 ) , example commonness also appeared to have signi\ufb01cant effects on ideation . Less - common examples were associated with more positive ideation processes and prod - ucts relative to more - common examples , with bene\ufb01ts for quan - tity and breadth of ideation , variability in solution quality , and novelty of solution concepts , although only in the case of vari - ability in solution quality was the contrast with control statisti - cally signi\ufb01cant . 5 . 2 . 3 Joint Effects of Example Distance and Commonness on Novelty . While far - \ufb01eld and less - common examples separately increased novelty of ideas , neither far - \ufb01eld examples as a whole nor less - common examples as a whole were signi\ufb01cantly different from control , which sat in the middle . To examine whether the combination of far - \ufb01eld and less - common properties increased novelty over control , we used a Dunnett\u2019s multiple comparison post hoc test . Since there were no effects of modality on novelty ( described below ) , we collapsed across the picture and text factors and conducted the post hoc test comparing each of the combina - tions in the 2 ! 2 matrix ( distance x commonness ) with the control condition as a reference group . The post hoc test showed that the combination of far - \ufb01eld , less - common examples did in fact increase novelty vs . control , for both mean ( d \u00bc 1 . 14 ; see Fig . 6 ) and max ( d \u00bc 1 . 29 ) . 5 . 3 Effects of Example Modality . Turning to the effects of modality in the overall ANOVAs , there was a main effect of example modality ( p < 0 . 01 , g 2 \u00bc 0 . 09 ) on solution transfer , where participants who received their examples in text form were more likely to use solution elements from the examples they received , regardless of distance or commonness of the example ( d \u00bc 0 . 60 ; Fig . 7 , lower left ) . There was also a main effect of on quantity ( p < 0 . 01 , g \u00bc 0 . 12 ; Fig . 7 , upper left ) , where participants who received text examples generated signi\ufb01cantly fewer solution concepts relative to partici - pants who received either picture examples ( p < 0 . 01 , d \u00bc # 0 . 67 ) or no examples ( control ; p < 0 . 05 , d \u00bc # 0 . 56 ) . There were no sig - ni\ufb01cant differences between participants who received picture examples vs . no examples ( control ) . Thus , receiving examples in text form increased the likelihood of being able to use solution Fig . 6 Mean novelty of solution concepts by example distance and commonness . * p < 0 . 05 . Error bars are 6 1 standard error . Fig . 7 Summary of effects of example modality . * p < 0 . 05 and * * p < 0 . 01 . Control group data are shown in white bars . Error bars are 6 1 standard error . Journal of Mechanical Design AUGUST 2011 , Vol . 133 / 081004 - 9 Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm elements from those examples relative to picture form , but also decreased quantity by an average of about two concepts relative to receiving either picture or text examples . There were no additional effects of modality on the other de - pendent measures ( breadth , p \u00bc 0 . 11 , g 2 \u00bc 0 . 03 ; mean novelty , p \u00bc 0 . 20 , g 2 \u00bc 0 . 02 ; max novelty , p \u00bc 0 . 49 , g 2 \u00bc 0 . 00 ; quality vari - ability , p \u00bc 0 . 44 , g 2 \u00bc 0 . 01 ) . Thus , modality had little impact on the key end - state outputs of the ideation process , unlike the effects of example commonness or example analogical distance . 6 Discussion 6 . 1 Optimal Example Types . Our \ufb01ndings demonstrate that the analogical distance and commonness of examples signi\ufb01cantly in\ufb02uences their impact on designers\u2019 ideation . With respect to an - alogical distance , augmenting ideation with far - \ufb01eld examples brings signi\ufb01cant bene\ufb01ts vis - a ` - vis the kinds of concepts that can be generated ; speci\ufb01cally , ideation with far - \ufb01eld examples enhan - ces the ability to generate highly novel solution concepts and also allows for more variability in the quality of concepts , which may increase the likelihood of generating high quality concepts . It is interesting to note that , even though the far - \ufb01eld examples we gave participants were not energy - generating devices , they were still able to bene\ufb01t from the concepts and solution elements in the devices . This sort of transfer is greater in distance than typically seen in the analogy literature , where far - \ufb01eld analogies in problem solving are usually from cases in other domains that are surface dissimilar but still solve the same basic problem [ 20 , 45 ] . However , the use of far - \ufb01eld examples was not without some cost . Far - \ufb01eld examples reduced overall quantity of ideation rela - tive to near - \ufb01eld or no examples . This \ufb01nding can be interpreted in terms of processing dif\ufb01culty . When we computed an addi - tional 3 - way ANOVA model on quantity for only the \ufb01nal phase of ideation , removing from consideration quantity of ideation while processing examples , the effects of distance were no longer present ( p \u00bc 0 . 47 ) . This suggests that the reduction in quantity can be attributed to the time taken to map the far - \ufb01eld example to the design problem . Thus , it appears that far - \ufb01eld examples not only carry with them the potential to increase novelty and quality of design concepts generated but also carry an initial processing cost in terms of time taken to map them to the target problem . With respect to commonness of examples , we found that the use of less - common examples positively impacts ideation . Less - common examples resulted in increased quantity of ideation , breadth of search , and higher novelty of ideas relative to more - common examples . In a follow - up analysis analyzing quantity for only the \ufb01nal phase of ideation , the positive effects of less - com - mon examples relative to more - common examples were still pres - ent ( p < 0 . 05 , d \u00bc 0 . 56 ) , suggesting that the effects cannot be explained simply in terms of initial processing costs , as in the case of distance effects on quantity . Thus , it seems that less - common examples might be more bene\ufb01cial for stimulating ideation , par - ticularly in terms of novelty of concepts generated . This \ufb01nding is in accord with some work in the domain of artistic creativity , where it has been shown that copying novel artworks has a posi - tive effect on the ability of art students to \ufb02exibly re - interpret art - work and increases the novelty of the artworks produced [ 46 ] . While distance and commonness had some similar effects on ideation processes and products , our \ufb01ne - grained analytic approach suggests some potentially important distinctions . The critical contrast seems to be with respect to effects on quantity and breadth of ideation . Far - \ufb01eld examples increased novelty of solutions and variability in solution quality , but appeared to do so via solution transfer , and resulted in decreased quantity ; in con - trast , less - common examples also increased novelty and quality variability , but appeared to do so via broadening the search space and increasing quantity . One way to interpret this contrast is that example distance and commonness have different mechanisms of inspiration . Based on the results , one could hypothesize that far - \ufb01eld examples inspire designers by moving them into one or two novel regions of the design space ( high solution transfer , high novelty ) , which they then explore in more depth ( low quantity , no bene\ufb01ts on breadth ) ; in contrast , one could hypothesize that less - common examples inspire designers by moving them into multi - ple different regions of the design space via re - interpretation of design functions and features ( low solution transfer , high breadth , and quantity ) . 6 . 2 Optimal Representation Modality of Examples . With regard to the outcome measures of novelty and quality of solution concepts , we found that the representation modality of examples did not change the effects of the distance and commonness factors on ideation . However , we did \ufb01nd evidence for a negative effect of text representations on overall quantity of ideation relative to picture or no examples . Similar to the effects of distance on quan - tity , this suppression effect of text representations can be inter - preted in terms of initial processing costs : when we analyzed only the last phase of ideation , the effect of modality was weaker ( pic - tures vs . text , d \u00bc 0 . 32 ; pictures vs . control , d \u00bc 0 . 45 ) and no lon - ger statistically signi\ufb01cant ( p \u00bc 0 . 07 ) . As an ancient proverb puts it , one picture may be worth 10 , 000 words with respect to convey - ing design concepts . 6 . 3 Caveats . The current work comes with a number of cav - eats . First , we have examined only one design problem . Although a real design problem of some complexity , examples may have different effects on more complex design problems . Second , we examined the effects of particular examples rather than a range of examples sampled multiple times from a class of examples . This experimental design choice made it more feasible to analyze solu - tion transfer but raises possibilities of effects being caused by odd examples or example descriptions . To reduce this threat , we had two examples per condition , and the factorial design of the study permits for multiple replications of main effects . Third , our partic - ipants were senior - level engineering students , for the most part , rather than expert designers , and there is some research to suggest that novices have more dif\ufb01culty with analogical mappings [ 5 , 47 ] . However , design teams sometimes include less experienced designers . Finally , our study focused only on the earliest ideation phase , and future work will have to examine the effects of exam - ples on downstream , and in particular \ufb01nished , solutions . This restriction was most salient in the analyses of quality in that many of the ideas were not feasible or not \ufb02eshed out suf\ufb01ciently to determine feasibility . However , a number of studies point to early ideation as a key moment for intervention to generate innovative designs [ 3 , 4 ] . 6 . 4 Practical Implications and Future Work . The overall focus of this study was on whether particular kinds of examples are more helpful than others for stimulating ideation . However , with the inclusion of a control group , which received no exam - ples , we were able to answer a separate but related question : all things considered , does analogizing over examples confer bene\ufb01ts over and above ideating without examples ? In other words , is design - by - analogy worth the extra time and effort ? Our \ufb01ndings suggest that if the goal of conceptual ideation is to ultimately gen - erate and develop a concept that is high quality and novel , then the answer is yes . There are also implications for the design of tools and methods to support design - by - analogy . As noted in the introduction , a range of previous design - by - analogy methods have been devel - oped ; of particular interest is the development of computational tools that automate the search for analogies [ 48 ] . It is well known in the psychological literature that retrieving far - \ufb01eld analogies is cognitively dif\ufb01cult ; remindings tend to be signi\ufb01cantly con - strained by surface similarity [ 49 ] , reducing the probability of retrieving potentially relevant surface dissimilar analogies . Thus , computational tools that are able to de\ufb01ne and compute functional 081004 - 10 / Vol . 133 , AUGUST 2011 Transactions of the ASME Downloaded 03 Aug 2011 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm and surface similarity between items in a design space in a prin - cipled manner relative to the current design problem would hold excellent potential as aids for inspiration . These tools might be able to maximize the potential bene\ufb01ts of analogies by retrieving and delivering to the designer in a timely manner surface dissimi - lar analogies and potentially ( as our \ufb01ndings suggest ) even analo - gies that do not necessarily provide direct solutions to the target problem . Additionally , if these systems are able to give priority to analogies that are relatively unusual or infrequently encountered , the potential for inspiration might be even higher . Currently , the state of the art for computational design - by - anal - ogy tools has not reached the point of being able to provide \ufb02exi - ble and real - time support in this manner . The present work provides an impetus for investment into this important research area , as the potential bene\ufb01ts to engineering practice and to soci - ety via increased innovation is high Acknowledgment This work is supported by grants from the National Science Foundation , Grant Nos . CMMI - 0855326 , CMMI - 0855510 , and CMMI - 0855293 , and , in part , by the University of Texas at Austin Cockrell School of Engineering and the Cullen Trust Endowed Professorship in Engineering No . 1 . Any opinions , \ufb01ndings , or recommendations are those of the authors and do not necessarily re\ufb02ect the views of the sponsors . References [ 1 ] Pisano , G . , and Shih , W . , 2009 , \u201cRestoring American Competitiveness , \u201d Harv . Bus . Rev . , 87 , pp . 114 \u2013 125 . [ 2 ] National Academy of Engineering , 2005 , Engineering Research and America\u2019s , Future : Meeting the Challenges of a Global Economy , National Academies Press , Washington , DC . [ 3 ] Vogel , C . 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Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm", + "chanSituativeCreativityLarger2016": "29 docs . lib . purdue . edu / jps 2016 | Volume 9 Journal of Problem Solving Special iSSue Situative Creativity : Larger Physical Spaces Facilitate Thinking of Novel Uses for Everyday Objects Joel Chan 1 and Timothy J . Nokes - Malach 2 1 Human - Computer Interaction Institute , Carnegie Mellon University , 2 Learning Research and Development Center , University of Pittsburgh People often use spatial metaphors ( e . g . , think \u201claterally , \u201d \u201coutside the box\u201d ) to describe explo - ration of the problem space during creative problem solving . In this paper , we probe the potential cognitive underpinnings of these spatial metaphors . Drawing on theories of situ - ative cognition , semantic foraging theory , and environmental psychology , we formulate and test the hypothesis that larger physical spaces can facilitate divergent ( but not convergent ) processes in problem space exploration . Across two experiments , participants worked on a battery of problem solving tasks intended to represent divergent ( alternative uses , shape invention ) and convergent ( remote associates , letter extrapolation ) problem solving processes in either a large or a small room . In Experiment 1 , participants in the larger room produced more novel alternative uses for everyday objects , and created more novel shape inventions , but generated less practical alternative uses , than participants in the smaller room . In Experi - ment 2 , participants in the larger room ( including a variant larger room ) also produced more novel alternative uses for everyday objects , and less practical alternative uses , than partici - pants in a small room , but did not create more novel shape inventions . These results sug - gest that spatial metaphors for problem space exploration may reflect meaningful cognitive phenomena : People may be able to search more broadly in a problem space if they are in an environment where broad physical search is a salient affordance ; however , this effect appears to be relatively small and may depend on having sufficiently motivated participants . Correspondence : Correspondence concerning this article should be addressed to Joel Chan at the Human - Computer Interaction Institute , Carnegie Mellon University , 2504B Newell - Simon Hall , 5000 Forbes Ave , Pittsburgh , PA 15213 , via email to joelchuc @ cs . cmu . edu , or via telephone to ( 479 ) 647 - 0575 . Keywords : situative cognition , creative problem solving , metaphor Acknowledgments : We thank Chris Wiltrout , Emily Schmidt , Courtney Stein , and Jeremy Addison for help with data collection and coding , and Efosa Osazuwa and Tina Liu for help with data coding . We also thank the edi - tor and our two anonymous referees for very helpful comments on earlier drafts of the manuscript . INTRODUCTION A key component of creative problem solving is exploration of the problem space . The problem space is typically described as the mental representation of the problem , including the initial problem description , goal , and operators ( i . e . , strate - gies ) to move from the initial state to the goal state ( Newell & Simon , 1972 ) . Theories of creative problem solving posit that the effective initial exploration of the problem space\u2014some - times called \u201cdivergence\u201d or \u201cdivergent thinking\u201d ( Guilford , 1956 ) \u2014is critical to produce a successful solution ( Amabile , 1983 ; Finke , Ward , & Smith , 1996 ; Sawyer , 2012 ; Simonton , 2011 ; Wallas , 1926 ; Warr & O\u2019Neill , 2005 ) . Effective problem space exploration can be supported by considering many dif - ferent solution approaches ( Ad\u00e1nez , 2005 ; Chan et al . , 2011 ; Parnes & Meadow , 1959 ; Shah , Millsap , Woodward , & Smith , 2012 ; Torrance , 1988 ) , increasing the variance in the quality of solutions considered ( e . g . , being willing to consider \u201cwild\u201d ideas ; Chan et al . , 2011 ; Girotra , Terwiesch , & Ulrich , 2010 ; Terwiesch & Ulrich , 2009 ) , considering solutions and perspec - tives from outside one\u2019s discipline or problem domain ( Chan et al . , 2011 ; Gentner & Markman , 1997 ; Ward , 1998 ) , relaxing inferred constraints about the problem description ( Knoblich , Ohlsson , Haider , & Rhenius , 1999 ; Ohlsson , 1992 ) , and explor - ing alternative conceptualizations of the problem ( Kaplan & Simon , 1990 ; MacGregor & Cunningham , 2009 ) . Divergence can also be facilitated by modulation of attention : For example , a reduction of attentional control or focus has been identified as a key mechanism for achieving divergent thinking and mak - ing remote associations in creative problem solving ( Aiello , Jarosz , Cushen , & Wiley , 2012 ; Haarmann , George , Smaliy , & Dien , 2012 ; Martindale , 1997 ; Wiley & Jarosz , 2012 ) . Successful exploration is often described with spatial lan - guage and imagery . For example , people commonly encour - age one another to think \u201claterally , \u201d not \u201cvertically\u201d ( Bono , 1970 ) , and \u201coutside the box , \u201d or to explore \u201cbroadly\u201d ( Wiley , 1998 ) and make \u201cremote\u201d associations in semantic memory ( Mednick , 1962 ) . In this paper , our goal is to probe the poten - tial cognitive underpinnings of these spatial metaphors . Are these metaphors arbitrary , or merely artifacts of human convention ? Or do they identify real cognitive phenomena ? Could embodying variations in these spatial metaphors ( e . g . , large vs . small physical environments ) influence the nature of people\u2019s search patterns in semantic space ? The present investigation is inspired by a growing body of literature across a diverse range of tasks that suggests that http : / / dx . doi . org / 10 . 7771 / 1932 - 6246 . 1184 docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 30 people\u2019s embodied physical context can have significant impli - cations for information processing . For example , people per - ceive slopes as steeper if they wear a heavy vs . a light backpack ( Bhalla & Proffitt , 1999 ) , can transfer knowledge and skills across contexts when there is high interconnectedness across activities and practices in those contexts ( Brown , Collins , & Duguid , 1989 ) , and make more \u201cholistic\u201d decisions ( i . e . , inte - grating multiple sources of data and abstraction ) in rooms with higher vs . lower ceilings ( Meyers - Levy & Zhu , 2007 ) . The literature provides two potential theoretical motivations for suspecting that the spatial metaphors of creative search have grounding in cognitive phenomena . The first account , which we call \u201cdirect priming , \u201d is exemplified by Hills and col - leagues\u2019 argument that goal - directed search for resources in external spaces and search for resources in internal spaces ( e . g . , semantic memory ) share a common neural substrate ( Hills , 2006 ; Hills , Jones , & Todd , 2012 ; Hills , Todd , & Goldstone , 2008 ) . Specifically , that they share dopaminergic modulation of area - restricted search such that search is narrow in situa - tions where the target resources have been frequently found in the past and search becomes broad in situations where the target resources are encountered less frequently . One intrigu - ing implication of this argument is that expectations about the structure of search environments in external spaces can shape search patterns in internal spaces , or vice versa . To test this implication , Hills et al . ( 2008 ) studied how search patterns on an anagram task ( i . e . , search for as many words as possible for a given letter set ) might be shaped by prior experience with a spatial foraging task ( i . e . , search for high - value pixels in a simple 2 - D computer maze ) . In their experiment , participants completed spatial foraging tasks with either \u201cclumpy\u201d\u2014many pixels concentrated in a few patches\u2014or \u201cdiffuse\u201d\u2014pixels evenly distributed in the envi - ronment\u2014resource distributions , and subsequently tried to find as many anagrams as they could . They found that par - ticipants who had just experienced a \u201cclumpy\u201d distribution of pixels took longer to switch between letter sets when search - ing for anagrams , consistent with expectations for a \u201cclumpy\u201d distribution of anagrams ( i . e . , expecting letter sets to contain more anagrams . They inferred from this that the distribution of resources in the spatial environment primed expectations for the distribution of \u201cresources\u201d in the semantic space . This analysis suggests that the affordances in the external , physical environment ( e . g . , the distribution of resources ) may shape the mind\u2019s internal search in semantic space . Following this line of thought , we reason that , to the extent that large physical environments afford free movement and exploration , they may also better facilitate divergent problem solving ( i . e . , exploration of semantic space ) relative to smaller , constrained spaces . Rather than simply fostering increased performance via increased effort , people might be sensitive to how larger physical spaces afford freer exploration , and consequently adopt a semantic search strategy that better matches this resource distribution , such as by relaxing their focus of attention from more clearly relevant or high - quality responses to more semantically distant and varied ( and likely more novel ) associations ( Aiello et al . , 2012 ; Haarmann et al . , 2012 ; Martindale , 1997 ; Wiley & Jarosz , 2012 ) . This direct priming of attentional focus might occur without conscious awareness , similar to some varieties of top - down modulation of visual attention ( Koch & Tsuchiya , 2007 ) . This direct prim - ing mechanism can also be related to the notion of \u201cframes\u201d in research on situative cognition , for example , expectations ( whether explicit or tacit ) about a given situation that are influ - enced by the affordances and constraints of particular environ - ments , and go to shape cognition and interaction ( Goffman , 1974 ; Greeno , 1994 ; Greeno & Middle - School Mathemat - ics through Applications Project Group , 1998 ; Maclachlan & Reid , 1994 ; Nokes - Malach & Mestre , 2013 ; Scherr & Hammer , 2009 ) . Because both varieties of direct priming mechanisms can occur without conscious awareness , we do not expect facilitation of divergent performance to be associated with more effortful performance . Indeed , to the extent that people relax their focus of attention to search more broadly , we might even expect to see a decreased perception of task difficulty as measured by cognitive load ( e . g . , Antonenko , Paas , Grabner , & Gog , 2010 ; Chandler & Sweller , 1991 ) . The second line of reasoning , which we call the \u201cconcept activation\u201d account , comes from research in environmental psychology that explores how certain configurations of physical environments can prime certain psychological states or ideas , which can then influence later information processing . For example , Hall ( 1966 ) argues that small and contained spaces ( e . g . , chapels ) can evoke notions of confinement or restricted - ness , while larger spaces ( e . g . , cathedrals ) can prime notions of freedom and openness . Similarly , Moore , Lane , Hill , Cohen , and McGinty ( 1994 ) suggest that lower ceilings may invoke more restricted play , while higher ceilings may encourage \u201cfreer\u201d play . In the Meyers - Levy and Zhu ( 2007 ) study men - tioned previously , the effect of the ceiling height manipulation on decision making was mediated by activation of the concept of \u201cfreedom\u201d vs . \u201cconfinement . \u201d This line of reasoning presents an indirect mechanism by which larger spaces prime concepts of \u201cfreedom\u201d and \u201cbroadness , \u201d which in turn induces infor - mation processing that is also \u201cless constrained , \u201d for example , more holistic , as in Meyers - Levy & Zhu ( 2007 ) , thereby facili - tating divergent processing during problem solving . In contrast to the direct priming account , concept activa - tion may also be marked by affective changes ( e . g . , increases in positive affect , decreases in negative affect ) , since concepts related to \u201cfreedom\u201d may have positive valence , while con - cepts related to \u201cconfinement\u201d may have negative valence . For example , a recent affective norming project found that the word \u201cfreedom\u201d had a highly positive valence score of docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 31 7 . 72 on a 1 to 9 valence scale ( 1 is highly negative , 9 is highly positive ) , while the word \u201crestrict\u201d had a much more nega - tive valence of 3 . 48 out of 9 ( Warriner , Kuperman , & Brys - baert , 2013 ) . Therefore , measuring changes in affect may be a way to distinguish between direct priming or concept activation accounts of potential associations between physi - cal surroundings and divergent / convergent problem solving processes : Increased divergent performance in large physi - cal spaces accompanied by increases in positive affect ( and decreases in negative affect ) would be more consistent with a concept activation account of the cognitive basis of spatial imagery for divergent exploration . Synthesizing these ideas , we test the hypothesis that larger spaces will have a facilitation effect on divergent problem solving processes ( i . e . , processes that have similar cognitive characteristics to the exploration stage of the creative pro - cess ) , but not \u201cconvergent\u201d problem solving processes ( i . e . , processes that focus on \u201cconverging\u201d on a single \u201ccorrect\u201d or canonical answer ) . To the extent that increased divergence may be at odds with convergent processes ( Goldenberg , Lar - son , & Wiley , 2013 ) , larger spaces might also hinder con - vergent problem solving . We further hypothesize that this facilitation would be accompanied by decreases in perceived task difficulty . Measures of affect might help distinguish between the direct priming and concept activation expla - nations of observed effects . In summary , in this paper , we examine the following three main hypotheses . H1 : Divergent performance will be higher in larger vs . smaller physical spaces . H2 : Convergent performance will be lower in larger vs . smaller physical spaces . H3 : Perceived task difficulty for divergent tasks will be lower in larger vs . smaller physical spaces . We conducted two experiments to test these hypotheses , first with a sample of paid volunteers , and then with a larger sample of psychology subject pool participants and an expan - sion of the range of physical spaces and problem solving stimuli that are tested . To preview our results , we find partial support for the first two hypotheses across both experiments , and find that these effects are not associated with changes in affect . EXPERIMENT 1 In this study , we provide a first test of the three hypotheses . The basic experimental approach is to have participants work on a battery of problem solving tasks intended to represent both divergent and convergent processing in either large or small rooms . As noted , our hypotheses are that divergent problem solving performance will be facilitated by being in a large ( vs . small ) room , while convergent performance will be hindered by being in the large room . METhODs Participants Forty - seven people ( 20 males , 27 females ; ages 19 \u2013 66 , average age 27 ) from the community at a large research university in the northeastern United States participated in this study . Thirty - five of the participants were undergraduate or graduate students at the university . Most of the other participants were recent gradu - ates or employees of the university or businesses on campus . All participants were recruited through fliers posted around cam - pus and were compensated 10 dollars for their time . Four participants didn\u2019t produce valid data on one of the problem solving tasks ( 3 did not produce any valid inven - tions , and 1 did not produce any valid uses ) , and were there - fore dropped from our analyses : There were two each from the large and small rooms respectively . 1 Therefore , our final dataset consisted of 43 participants . Materials Room size manipulation . To manipulate room size , we had partici - pants complete their problem solving tasks in one of two rooms on campus . The \u201clarge\u201d room was a conference auditorium ( see Fig 1 , left panel ) . The dimensions of the room were approximately 15\u2019 W \u00d7 30\u2019 L \u00d7 15\u2019 H . Participants completed their tasks on a desk in the front of the auditorium facing toward the audience seats so that the size of the room would be salient . Other than the desk and chair the participants used , and the other chairs facing the front of the auditorium , the auditorium was empty . The \u201csmall\u201d room was a former office space that was emp - tied out for the experiment ( Fig 1 : next page , right panel ) . The dimensions of the room were approximately 8\u2019 W \u00d7 10\u2019 L \u00d7 8\u2019 H . Participants completed their tasks on a desk facing one of the walls . It was empty except for the desk and chair the participants used . Other than the size of the room , we made sure that the two rooms were similar in a number of important ways , including amount of stimuli encountered on the walk to the room ( both rooms were in the same building ) , ambient noise ( we chose rooms that were far from other offices in the build - ing ) , and temperature ( both rooms shared the same central air conditioning system ) . The one potentially salient differ - ence was the tone of lighting : the large room used incandes - cent lighting , while the small room used fluorescent lighting . Problem - solving tasks Participants completed a battery of four problem - solving tasks intended to represent both divergent and convergent processing : 1 ) an alternative uses task , 2 ) a shape invention task , 3 ) a version of the Remote Associates Test ( RAT ) , and 4 ) a letter series extrapolation task . In this section we describe each task along with the hypothesized processes involved . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 32 Alternative uses . The alternative uses task is patterned after Guilford\u2019s ( 1967 ) classic alternate uses task in which the problem solver is asked to list as many uses as possible for a common object ( e . g . , think of as many uses as you can for a brick ) . It has been hypothesized to measure divergent thinking processes because the output of the task is a range of responses rather than one correct or incorrect response . Task output is typically measured in terms of the fluency and flexibility ( e . g . , novelty ) of the responses . However , this task may also involve convergent processes . People may initially think of a variety of responses , and subsequently evaluate and select only the uses that are both novel and practical . This corresponds to the selection / evaluation processes / phases in various theories of cognitive and creative production , such as the convergent production component of Guilford\u2019s ( 1956 ) \u201cstructure of intellect\u201d theory , the response validation stage of Amabile\u2019s ( 1983 ) process model of creativity , and the Explore phase of Finke and colleagues\u2019 ( 1996 ) Geneplore model . Therefore , we hypothesize that fluency and novelty are mea - sures of divergent thinking on this task , whereas practical - ity is a measure of convergent thinking . It is useful to note that convergence may not necessarily always follow diver - gence : Convergence can also reflect attentional focus on and rapid selection of the most readily accessible responses , which are often the most successful or appropriate ( Bilali\u0107 , McLeod , & Gobet , 2008 ; Guilford , 1956 ; Luchins , 1942 ) . We used \u201cSHOE\u201d and \u201cNEWSPAPER\u201d as our common object items and gave the following instructions to partici - pants : \u201cIn this part of the experiment , your task is to list as many uses as you can for an object ( named below ) . For exam - ple , if the object is \u2018BRICK , \u2019 you could say \u2018building material , doorstop , anchor , etc . \u2019 The goal is to come up with as many uses of an object as possible . There are 2 of these problems , and you will have 4 minutes for each . \u201d Shape invention . In the shape invention task ( Finke et al . , 1996 ) , the problem solver is given three three - dimensional shapes to combine together to create as many useful objects that belong to one of three given categories ( e . g . , toys and games , transportation ) . Similar to the alternative uses task , we hypothesize that this task includes elements of both diver - gent and convergent processing . Again , we hypothesize that Figure 1 Picture of large room ( left ) and small room ( right ) in Experiment 1 . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 33 the novelty of the items generated would reflect divergent thinking processes , whereas the practicality of the objects would reflect convergent processes . Some versions of this task allow the problem solver to choose the shapes and / or the object categories ; we elected to randomly select both shapes and categories and present them as givens for all participants . Part of the motivation for this was that Finke and colleagues ( 1996 ) found that this con - dition stimulated the most creative responses , and we wanted to give our participants the best chance of displaying creative performance . Finke and colleagues\u2019 original set of shapes includes 15 different shapes , ranging from spheres to cylin - ders , to wires , wheels , and flat squares ; each shape belongs to a subcategory set of 5 shapes , ordered by how difficult it is ( normed from their studies ) to incorporate into an invented object ( easy , medium , and hard ) . We randomly sampled 3 shapes from this list , with \u201ceasy\u201d shapes having a probability = 0 . 10 of being selected , \u201cmedium\u201d shapes having a probability = . 07 of being selected , and \u201chard\u201d shapes having a probabil - ity = 0 . 03 of being selected . Finke and colleagues\u2019 original set of object categories consisted of 8 categories : weapons , toys and games , appliances , transportation , scientific instru - ments , tools and utensils , furniture , and personal items . We randomly selected three categories from this list to give to participants . We ended up with the sphere , tube , and cone objects ( see Figure 2 ) , and the following object categories : tools and utensils , toys and games , and personal items . We gave the following instructions to participants : \u201cIn this part of the experiment , your task is to try to use the follow - ing \u2018parts\u2019 to \u2018construct\u2019 as many useful objects as you can . These objects can be existing things or things you invent . You will have 8 minutes to do this . The rules for using the parts to construct objects are as follows : 1 ) you are allowed to vary the size , position , or orientation of any part , but you may not bend or deform the parts ( except the tube ) , 2 ) the parts can be put inside one another , 3 ) you decide if the parts are hollow or solid , and 4 ) you decide what material the parts are made of\u2014 they can be made of any material , including wood , metal , plas - tic , rubber , or glass , or any combination of these materials . \u201d RAT . To complete the RAT ( Bowers , Regehr , Balthazard , & Parker , 1990 ; Mednick , 1962 ; Mednick & Mednick , 1967 ) , solvers must generate a target word that is related to a list of three cue words . For example , a correct response for the cue words Elephant - Lapse - Vivid would be the target word \u201cmemory . \u201d While the RAT is most commonly used as a mea - sure of creativity ( e . g . , Wiley , 1998 ) , and several prior studies have examined the role of divergent processes in RAT per - formance ( Aiello et al . , 2012 ; Haarmann et al . , 2012 ) , we rea - soned that it also heavily taps convergent processes since the final output is a single answer that is compared to a predeter - mined correct answer . We are not the first to treat the RAT in this way : Other recent studies have also studied the RAT as primarily a convergent task in contrast to the alternate uses task ( treated as primarily measuring divergent processes ) , and found both dissociable effects on these tasks from their manipulations and a lack of correlation between performance on these tasks ( Colzato , Ozturk , & Hommel , 2012 ; Oppezzo & Schwartz , 2014 ; Radel , Davranche , Fournier , & Dietrich , 2015 ) . We gave participants 32 items , of varying difficulty , drawn from Mednick and Mednick ( 1967 ) and Bowers et al . ( 1990 ) , chosen to reflect a range of difficulty levels . The full list of items can be seen in the appendix . Letter series . In the letter series task , the problem solver is given a series of letters and is asked to generate additional letters to \u201cN\u201d places to complete the pattern exemplified in the given series . For example , given the series \u201caaabbbccc , \u201d the correct extrapolation to N = 3 places is \u201cddd . \u201d Similar to the RAT , this task requires the identification and genera - tion of a single answer . Although participants may consider several possible patterns when solving the problem , they must eventually converge or decide on one to extrapolate . We gave participants 18 items drawn from prior studies with the letter series task ( Nokes , 2009 ; Simon & Kotovsky , 1963 ) , which were chosen to reflect a range of difficulty levels . The items had initial series ranging from 9 to 16 letters in length ; all items required participants to extrapolate the series to N = 10 places . The full list of items can be seen in the appendix . Dependent measures In the previous section we described each task we used for the experiment . Some of the tasks ( specifically the uses and invention tasks ) were hypothesized to include both divergent and convergent processing . We now describe how we mea - sured divergent and convergent processing across the tasks . Divergent measures . Both the uses and invention tasks were scored for fluency and novelty to yield our primary diver - gent measures . Fluency was defined as the number of uses or inventions generated . Novelty was rated on a scale from 1 ( not at all novel ) to 4 ( extremely novel ) . Examples of low and high novelty uses are \u201cuse SHOE to protect feet\u201d and \u201cuse SHOE as boat for termites\u201d ; examples of low and high Situative Creativity 13 following object categories : tools and utensils , toys and games , and personal items . We gave the following instructions to participants : \u201cIn this part of the experiment , your task is to try to use the following \u2018parts\u2019 to \u2018construct\u2019 as many useful objects as you can . These objects can be existing things or things you invent . You will have 8 minutes to do this . The rules for using the parts to construct objects are as follows : 1 ) you are allowed to vary the size , position , or orientation of any part , but you may not bend or deform the parts ( except the tube ) , 2 ) the parts can be put inside one another , 3 ) you decide if the parts are hollow or solid , and 4 ) you decide what material the parts are made of\u2014they can be made of any material , including wood , metal , plastic , rubber , or glass , or any combination of these materials . \u201d RAT . To complete the RAT ( Bowers , Regehr , Balthazard , & Parker , 1990 ; Mednick , 1962 ; Mednick & Mednick , 1967 ) , solvers must generate a target word that is related to a list of three cue words . For example , a correct response for the cue words Elephant - Lapse - Vivid would be the target word \u201cmemory . \u201d While the RAT is most commonly used as a measure of creativity ( e . g . , Wiley , 1998 ) , and several prior studies have examined the role of divergent processes in RAT performance ( Aiello et al . , 2012 ; Haarmann et al . , 2012 ) , we reasoned that it also heavily taps convergent processes since the final output is a single answer that is compared to a predetermined correct answer . We are not the first to treat the RAT in this way : Other recent studies have also studied the RAT as primarily a convergent task in contrast to the alternate uses task ( treated as primarily measuring divergent processes ) , and found both dissociable effects on Figure 2 . Shapes for invention task in Experiment 1 . Figure 2 Shapes for invention task in Experiment 1 . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 34 novelty inventions are shown in Figure 3 . Two trained coders evaluated the uses , with high inter - rater reliability , ICC ( 2 , 2 ) = . 89 . Three trained coders evaluated the inventions , with high inter - rater reliability , ICC ( 2 , 3 ) = . 85 . Each use / invention\u2019s novelty score was the arithmetic mean of all judges\u2019 scores for that use / invention . Novelty scores were then aggregated into participant - level measures in the following ways : mean nov - elty ( how novel their uses were , on average ) , and max novelty ( what was the highest novelty score they achieved ) . Convergent measures . Both the uses and invention tasks were also scored for practicality , to reflect convergent processing on those tasks . Practicality scoring for the uses task was initially done with a 4 - point scale ( 1 \u2013 unlikely to work at all , 2 \u2013 will work less well than conventional means , 3 \u2013 will work as well as conventional means , 4 \u2013 will work better than conven - tional means ) , but was collapsed to a 3 - point scale because there were almost no ( agreed - upon ) generated uses that war - ranted a 4 . Two trained coders evaluated the uses , with good inter - rater reliability , ICC ( 2 , 2 ) = . 79 . In contrast , scoring for the invention category had slightly higher variance , allowing us to code inventions on a 5 - point scale ( 1 \u2013 extremely bad example of its invention category , to 5 \u2013 exceeds expectations for a good example of its invention category ) . Seven trained coders evaluated the inventions for practicality , with accept - able inter - rater reliability , ICC ( 2 , 7 ) = . 61 . Examples of low and high practicality uses are \u201cuse SHOE as tent stake\u201d and \u201cuse SHOE as slapping device to bring someone back to their senses\u201d ; examples of low and high practicality inventions are shown in Figure 3 . Participant - level practicality measures were created by taking the arithmetic mean of practicality scores achieved to create a mean practicality measure for both uses and invention tasks , separately . Performance on the RAT and letter series tasks were intended to primarily reflect convergent processing , since both tasks sought the production of a single \u201cbest\u201d response . One trained coder scored the RAT responses as either correct or incorrect , using the answer key from Mednick & Mednick ( 1967 ) and Bowers et al . ( 1990 ) . Percent correct was used for analysis . Letters series task performance was measured by marking responses as either correct or incorrect , using canonical answers from the prior references ( Nokes , 2009 ; Simon & Kotovsky , 1963 ) , and the percent correct was used for analysis . Other measures Perceived task difficulty . To measure perceived task difficulty , we adapted two items from prior research with cognitive load ( Jang & Schunn , 2012 ) . The measure was about the task just completed . The first item asked , \u201cHow easy or difficult was this task ? \u201d and participants were asked to answer using a 1 to 9 scale , where 1 was anchored as \u201cVery , very easy , \u201d and 9 was anchored as \u201cVery , very difficult . \u201d The second item asked \u201cHow much mental effort ( e . g . , searching , remembering , thinking , deciding ) did the task take ? \u201d and participants were asked to answer using a 1 to 9 scale , with 1 anchored as \u201cVery , very low mental effort , \u201d and 9 anchored as \u201cVery , very high mental effort . \u201d Positive and negative affect . To measure affect , we used the Positive and Negative Affect Schedule ( PANAS ; Watson , Clark , & Tel - legen , 1988 ) , in which a subject is given 20 words that describe different feelings and emotions , and\u2014using a scale of 1 ( very slightly or not at all ) to 5 ( extremely ) \u2014rates the extent to which she feels that way \u201cright now\u201d ( i . e . , at the present moment ) . Situative Creativity 15 inventions generated . Novelty was rated on a scale from 1 ( not at all novel ) to 4 ( extremely novel ) . Examples of low and high novelty uses are \u201cuse SHOE to protect feet\u201d and \u201cuse SHOE as boat for termites\u201d ; examples of low and high novelty inventions are shown in Figure 3 . Two trained coders evaluated the uses , with high inter - rater reliability , ICC ( 2 , 2 ) = . 89 . Three trained coders evaluated the inventions , with high inter - rater reliability , ICC ( 2 , 3 ) = . 85 . Each use / invention\u2019s novelty score was the arithmetic mean of all judges\u2019 scores for that use / invention . Novelty scores were then aggregated into participant - level measures in the following ways : mean novelty ( how novel their uses were , on average ) , and max novelty ( what was the highest novelty score they achieved ) . Convergent measures . Both the uses and invention tasks were also scored for practicality , to reflect convergent processing on those tasks . Practicality scoring for the uses task was initially done with a 4 - point scale ( 1 \u2013 unlikely to work at all , 2 \u2013 will work less well than conventional means , 3 \u2013 will work as well as conventional means , 4 \u2013 will work better than conventional Figure 3 . Example of low and high novelty / practicality inventions . The low and high novelty inventions are a \u201cfunnel\u201d and a device that \u201cslows elevators with centripetal force . \u201d The low and high practicality inventions are an \u201cunstable martini glass\u201d and a \u201ctool to catch water to measure the rain . \u201d Note that the shapes used for invention are a cone , sphere , and tube . Figure 3 Example of low and high novelty / practicality inventions . The low and high novelty inventions are a \u201cfunnel\u201d and a device that \u201cslows elevators with centripetal force . \u201d The low and high practicality inventions are an \u201cunstable martini glass\u201d and a \u201ctool to catch water to measure the rain . \u201d Note that the shapes used for invention are a cone , sphere , and tube . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 35 Procedure Participants were greeted and brought to either the large or small room , depending on their assignment . They were then informed that they would complete four problem - solving tasks , grouped into two blocks with two problem sets each . The uses and invention tasks formed one block , while the RAT and letter series task formed the other block . The tasks were counterbalanced by block , and specific problem solving tasks within the block ( RAT and letters ; uses and invention ) . They had eight minutes to complete each problem set , and a one - minute warning was given before the time was up . After each problem set , participants completed the untimed cognitive load survey . Before they began the problem sets , participants were asked to complete the first PANAS to get a baseline measure . They were asked to complete a second and third PANAS after the first and second blocks of problem sets , respectively . Overall , the experiment ran no longer than 45 minutes . The experimenter remained in the room during the length of the experiment , seated behind the participant . Design This study had a between - subjects design . The independent variable was room size , with two levels ( large or small ) . Par - ticipants were randomly assigned to conditions . In the final dataset , there were 21 participants in the large room and 22 participants in the small room . REsUlTs Table 1 shows the descriptive statistics for all problem solv - ing measures . Table 2 shows the intercorrelations between participants\u2019 problem solving task performance measures , collapsed across Experiments 1 and 2 . In general , the correla - tions were in the expected directions ( e . g . , significant positive correlations between divergent measures ; significant positive correlations between letter series and RAT measures ; sig - nificant negative correlations between uses practicality and the divergent measures ) . However , the correlations are gen - erally low , explaining small amounts of common variance , and some correlations are missing ( e . g . , no significant cor - relations between practicality and the letters series and RAT measures , and no significant correlations between invention practicality and any of the other measures ) . This suggests that the measures do not necessarily primarily reflect the two constructs of divergence and convergence ( as we had hypoth - esized ) . Therefore , we analyze each measure separately . Alternative uses There was an effect of room size on mean fluency , with par - ticipants in the large room generating more uses ( M = 25 . 0 , SE = 1 . 8 ) than participants in the small room ( M = 19 . 3 , SE = 1 . 8 ) , d = 0 . 68 , 95 % CI = [ 0 . 03 , 1 . 33 ] , F ( 1 , 41 ) = 5 . 0 , p = . 03 . There was also an effect of room size on mean novelty , with participants in the large room producing higher mean novelty with their uses ( M = 1 . 8 , SE = . 09 ) than those in the small room ( M = 1 . 5 , SE = . 08 ) , d = 0 . 78 [ 0 . 13 , 1 . 44 ] , F ( 1 , 41 ) = 6 . 6 , p = . 01 ( Fig . 4 , left panel ) . The results were similar for max novelty : Participants in the large room achieved marginally higher max novelty scores ( M = 3 . 4 , SE = . 19 ) than those in the small room ( M = 2 . 9 , SE = . 18 ) , d = 0 . 61 [ - 0 . 04 , 1 . 26 ] , F ( 1 , 41 ) = 4 . 0 , p = . 05 ( Fig . 4 , middle panel ) . In contrast , participants in the large room generated alter - native uses that were significantly less practical ( M = 2 . 5 , SE = . 04 ) than those from the small room ( M = 2 . 7 , SE = . 04 ) , d = \u2013 1 . 00 [ - 1 . 68 , - 0 . 33 ] , F ( 1 , 41 ) = 10 . 9 , p = . 00 ( Fig . 4 , right panel ) . Shape invention Participants in the large room generated slightly more inven - tions ( M = 5 . 5 , SE = . 42 ) than participants in the small room ( M = 4 . 6 , SE = . 41 ) , d = 0 . 47 [ - 0 . 17 , 1 . 11 ] , but this difference was not statistically significant , F ( 1 , 41 ) = 2 . 3 , p = . 13 . However , there Table 1 Descriptive statistics for Experiment 1 . Situative Creativity 19 the intercorrelations between participants\u2019 problem solving task performance measures , collapsed across Experiments 1 and 2 . In general , the correlations were in the expected directions ( e . g . , significant positive correlations between divergent measures ; significant positive correlations Table 1 Descriptive statistics for experiment 1 . Mean Median Min Max SE Divergent measures Uses fluency 22 . 07 21 7 42 1 . 32 Uses novelty mean 1 . 67 1 . 59 1 2 . 58 0 . 06 Uses novelty max 3 . 12 3 1 4 0 . 13 Invention fluency 5 . 07 5 2 9 0 . 29 Invention novelty mean 2 . 74 2 . 83 1 . 56 3 . 83 0 . 08 Invention novelty max 3 . 58 3 . 67 2 4 0 . 08 Convergent measures Uses practicality 2 . 62 2 . 64 2 . 03 3 0 . 04 Invention practicality 3 . 15 3 . 21 1 . 79 3 . 86 0 . 06 RAT 0 . 59 0 . 62 0 1 0 . 04 Letters 0 . 61 0 . 71 0 1 0 . 05 Table 2 Intercorrelations between measures , collapsed across Experiments 1 and 2 . Divergent Convergent Uses novel mean Uses novel max Invent fluen . Invent novel mean Invent novel max Uses pract . Invent pract . RAT Letters U fluency 0 . 50 * 0 . 50 * 0 . 37 * 0 . 24 * 0 . 26 * - 0 . 43 * - 0 . 07 - 0 . 04 - 0 . 01 U novel mean 0 . 79 * 0 . 31 * 0 . 27 * 0 . 29 * - 0 . 89 * - 0 . 02 0 . 01 - 0 . 04 U novel max 0 . 17 * 0 . 25 * 0 . 22 * - 0 . 66 * 0 . 01 0 . 03 - 0 . 12 I fluency 0 . 09 0 . 46 * - 0 . 32 * 0 . 00 - 0 . 07 - 0 . 06 I novel mean 0 . 75 * - 0 . 23 * - 0 . 11 - 0 . 02 - 0 . 03 I novel max - 0 . 28 * - 0 . 05 - 0 . 01 - 0 . 02 U practicality 0 . 06 - 0 . 06 0 . 00 I practicality 0 . 14 m 0 . 00 RAT 0 . 21 * m p < . 10 ; * p < . 05 docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 36 was an effect of room size on mean novelty , with participants in the large room , on average , creating more novel inventions ( M = 2 . 9 , SE = . 10 ) than participants in the small room ( M = 2 . 6 , SE = . 10 ) , d = 0 . 76 [ 0 . 10 , 1 . 41 ] , F ( 1 , 41 ) = 6 . 2 , p = . 02 . Similarly , there was an effect of max novelty , with the most novel inventions of participants in the large room being , on average , more novel ( M = 3 . 8 , SE = . 10 ) than the most novel inventions of participants in the small room ( M = 3 . 4 , SE = . 10 ) , d = 0 . 76 [ 0 . 10 , 1 . 41 ] , F ( 1 , 41 ) = 6 . 2 , p = . 02 . For practi - cality , there was no effect of room size , with inventions from the large room condition about as practical ( M = 3 . 2 , SE = . 08 ) as those from the small room ( M = 3 . 1 , SE = . 08 ) , d = 0 . 32 [ - 0 . 31 , 0 . 96 ] , F ( 1 , 41 ) = 1 . 1 , p = . 29 . RAT There was no effect of room size , with participants in the large room having about the same mean proportion cor - rect ( M = . 57 , SE = . 05 ) as participants in the small room ( M = . 60 , SE = . 05 ) , d = - 0 . 12 [ - 0 . 75 , 0 . 51 ] , F ( 1 , 41 ) = 0 . 2 , p = . 69 . Letter series Participants in the large room generated a slightly higher proportion of correct responses ( M = . 65 , SE = . 07 ) than participants in the small room ( M = . 57 , SE = . 07 ) , d = 0 . 22 [ - 0 . 41 , 0 . 85 ] , but this difference was not statistically signifi - cant , F ( 1 , 41 ) = . 05 , p = . 48 . Perceived task difficulty There was a marginal effect of room size on perceived dif - ficulty for the uses task , with participants in the large room self - reporting slightly lower levels of difficulty ( M = 4 . 1 , SE = . 36 ) compared to participants in the small room ( M = 5 . 1 , SE = . 36 ) , d = - 0 . 53 [ - 1 . 15 , 0 . 08 ] , F ( 1 , 45 ) = 3 . 4 , p = . 07 . For the invention task , there was a main effect of room size , with lower self - reported difficulty in the large ( M = 5 . 2 , SE = 0 . 3 ) vs . small room ( M = 6 . 8 , SE = 0 . 3 ) , d = - 1 . 16 [ - 1 . 81 , - 0 . 51 ] , F ( 1 , 45 ) = 15 . 7 , p = 0 . 00 . In contrast , for the RAT , participants in the large room self - reported about the same levels of perceived difficulty ( M = 7 . 1 , SE = . 30 ) as participants in the small room ( M = 7 . 4 , SE = . 29 ) , d = - 0 . 16 [ - 0 . 77 , 0 . 43 ] , F ( 1 , 45 ) = 0 . 34 , p = . 56 . Similarly , for the letters task , participants in the large room self - reported about the same levels of perceived difficulty ( M = 6 . 3 , SE = . 36 ) as participants in the small room ( M = 5 . 9 , SE = . 36 ) , d = 0 . 24 [ - 0 . 37 , 0 . 84 ] , F ( 1 , 45 ) = 0 . 65 , p = . 42 . Positive and negative affect There was no effect of room size on positive affect , with par - ticipants in the large room self - reporting about the same levels of positive affect ( M = 27 . 6 , SE = 1 . 5 ) as participants in the small room ( M = 27 . 4 , SE = 1 . 5 ) , d = 0 . 04 [ - 0 . 59 , 0 . 68 ] , F ( 1 , 41 ) = 0 . 02 , p = . 89 . Similarly , there was no effect of room size on negative affect , with participants in the large room self - reporting about the same levels of negative affect ( M = 12 . 3 , SE = . 47 ) as participants in the small room ( M = 11 . 8 , SE = . 46 ) , d = 0 . 22 [ - 0 . 41 , 0 . 86 ] , F ( 1 , 41 ) = 0 . 53 , p = . 47 . DIsCUssION Experiment 1 yielded evidence consistent with Hypothesis 1 ( see Figure 5 for a summary of the observed effects ) . As pre - dicted , participants\u2019 performance was higher on the diver - gent problem solving measures in the larger room than in the smaller room ( e . g . , uses fluency , uses novelty , invention nov - elty ) . In contrast , we found only partial support for Hypothesis 2 , that is , that participants in the large rooms would perform worse on convergence measures than participants in the small rooms . Consistent with the hypothesis , we found that partic - ipants in the larger room showed lower performance on the Situative Creativity 19 the intercorrelations between participants\u2019 problem solving task performance measures , collapsed across Experiments 1 and 2 . In general , the correlations were in the expected directions ( e . g . , significant positive correlations between divergent measures ; significant positive correlations Table 1 Descriptive statistics for experiment 1 . Mean Median Min Max SE Divergent measures Uses fluency 22 . 07 21 7 42 1 . 32 Uses novelty mean 1 . 67 1 . 59 1 2 . 58 0 . 06 Uses novelty max 3 . 12 3 1 4 0 . 13 Invention fluency 5 . 07 5 2 9 0 . 29 Invention novelty mean 2 . 74 2 . 83 1 . 56 3 . 83 0 . 08 Invention novelty max 3 . 58 3 . 67 2 4 0 . 08 Convergent measures Uses practicality 2 . 62 2 . 64 2 . 03 3 0 . 04 Invention practicality 3 . 15 3 . 21 1 . 79 3 . 86 0 . 06 RAT 0 . 59 0 . 62 0 1 0 . 04 Letters 0 . 61 0 . 71 0 1 0 . 05 Table 2 Intercorrelations between measures , collapsed across Experiments 1 and 2 . Divergent Convergent Uses novel mean Uses novel max Invent fluen . Invent novel mean Invent novel max Uses pract . Invent pract . RAT Letters U fluency 0 . 50 * 0 . 50 * 0 . 37 * 0 . 24 * 0 . 26 * - 0 . 43 * - 0 . 07 - 0 . 04 - 0 . 01 U novel mean 0 . 79 * 0 . 31 * 0 . 27 * 0 . 29 * - 0 . 89 * - 0 . 02 0 . 01 - 0 . 04 U novel max 0 . 17 * 0 . 25 * 0 . 22 * - 0 . 66 * 0 . 01 0 . 03 - 0 . 12 I fluency 0 . 09 0 . 46 * - 0 . 32 * 0 . 00 - 0 . 07 - 0 . 06 I novel mean 0 . 75 * - 0 . 23 * - 0 . 11 - 0 . 02 - 0 . 03 I novel max - 0 . 28 * - 0 . 05 - 0 . 01 - 0 . 02 U practicality 0 . 06 - 0 . 06 0 . 00 I practicality 0 . 14 m 0 . 00 RAT 0 . 21 * m p < . 10 ; * p < . 05 Table 2 Intercorrelations between measures , collapsed across Experiments 1 and 2 . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 37 uses practicality measure . However , no differences were found across the two groups on invention practicality and perfor - mance on the RAT and letter series task . In the general discus - sion we discuss possible reasons for why we did not observe stronger negative effects of a large space on convergence mea - sures . Taken together , these results show that the benefits of the larger room for divergent performance were not simply due to a general facilitation effect of being in the larger room ; rather , there seems to be a specific effect of being in a larger room on the cognitive processes that enable divergent performance . Analysis of the additional measures yielded additional insights . In support of Hypotheses 3 , the perceived task dif - ficulty results suggest that participants in the larger room not only performed better on the uses and invention tasks ( in terms of divergent performance measures ) , but also found the task overall to be less cognitively taxing ( compared to participants in the smaller rooms ) , suggesting that some of the performance benefits might be due to unconscious mechanisms ( e . g . , automatic attunement of semantic search patterns to search affordances in the physical environment ) . Further , analysis of the survey responses for PANAS suggests that the differences are not explained by positive boosts to affect in the larger room ( or increased negative affect in the smaller room ) . This result is consistent with the direct prim - ing hypothesis and not the concept activation account . EXPERIMENT 2 : REPlICATION AND EXTENsION Given the novelty of our hypotheses , we conducted a second study to replicate and extend the results of Experiment 1 . The focus of the extension is to ensure that the effects were not due to idiosyncrasies of the particular configurations of the large room or problem solving stimuli . To this end , we slightly altered the large room manipulation from Experiment 1 ( par - ticipants sat at the top of the auditorium rather than at the bot - tom ) , and added a second new large room that had a lower ceiling height but was still spacious horizontally . To maximize statistical power , we treated them as a single condition in our analyses . 2 We also changed the objects used for the uses task , as well as the categories and shapes used for the invention task . METhODs Participants One hundred and nine undergraduates ( 61 females ; ages 18 \u2013 31 , average age 19 ) enrolled in Introduction to Psy - chology at a large research university in the northeastern Situative Creativity 20 between letter series and RAT measures ; significant negative correlations between uses practicality and the divergent measures ) . However , the correlations are generally low , explaining small amounts of common variance , and some correlations are missing ( e . g . , no significant correlations between practicality and the letters series and RAT measures , and no significant correlations between invention practicality and any of the other measures ) . This suggests that the measures do not necessarily primarily reflect the two constructs of divergence and convergence ( as we had hypothesized ) . Therefore , we analyze each measure separately . Alternative uses There was an effect of room size on mean fluency , with participants in the large room generating more uses ( M = 25 . 0 , SE = 1 . 8 ) than participants in the small room ( M = 19 . 3 , SE = 1 . 8 ) , d = 0 . 68 , 95 % CI = [ 0 . 03 , 1 . 33 ] , F ( 1 , 41 ) = 5 . 0 , p = . 03 . There was also an effect of room size on mean novelty , with participants in the large room producing higher mean novelty with Figure 4 . Novelty and practicality of alternative uses by room size in Experiment 1 . Error bars are \u00b11 SE . Figure 4 Novelty and practicality of alternative uses by room size in Experiment 1 . Error bars are \u00b11 SE . Situative Creativity 24 showed lower performance on the uses practicality measure . However , no differences were found across the two groups on invention practicality and performance on the RAT and letter series task . In the general discussion we discuss possible reasons for why we did not observe stronger negative effects of a large space on convergence measures . Taken together , these results show that the benefits of the larger room for divergent performance were not simply due to a general facilitation effect of being in the larger room ; rather , there seems to be a specific effect of being in a larger room on the cognitive processes that enable divergent performance . Analysis of the additional measures yielded additional insights . In support of Hypotheses 3 , the perceived task difficulty results suggest that participants in the larger room not only performed better on the uses and invention tasks ( in terms of divergent performance measures ) , but also found the task overall to be less cognitively taxing ( compared to participants in the smaller rooms ) , suggesting that some of the performance benefits might be due to unconscious mechanisms ( e . g . , automatic attunement of semantic search patterns to search affordances in the Figure 5 . Summary of effects in Experiment 1 . Error bars are 95 % CIs . Figure 5 Summary of effects in Experiment 1 . Error bars are 95 % CIs . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 38 United States participated in this study . All participants were recruited through the university\u2019s psychology subject pool , and were compensated with course credit . Three participants ( assigned to the large rooms ) did not produce any valid responses to the invention task , and were dropped from all analyses , leaving us with 106 total partici - pants in our final dataset . 3 Materials Room size manipulation . In this study , the \u201clarge\u201d setting of our room size manipulation included two different rooms : 1 ) the same conference auditorium as in Experiment 1 , with the only difference being that participants sat at the top of the room , rather than the bottom of the room , and 2 ) another conference room in the same building ( see Figure 6 ) . The dimensions of the new large room were approximately 15\u2019 W \u00d7 30\u2019 L \u00d7 8\u2019 H . Other than the desk and chair the participants used , along with the other desks and chairs in the room , the room was empty . Note also that the lighting here is fluores - cent , similar to the small rooms in both experiments . The \u201csmall\u201d room was another former office space in the same building that was emptied out for the experiment . The dimensions of the room were the same as in Experiment 1 , approximately 8\u2019 W \u00d7 10\u2019 L \u00d7 8\u2019 H . As in Experiment 1 , participants completed their tasks on a desk facing one of the walls . It was empty except for the desk and chair the partici - pants used . Problem - solving tasks . As in Experiment 1 , participants com - pleted the alternative uses , shape invention , RAT , and let - ter series tasks . The only differences from Experiment 1 are with the stimuli for the alternative uses and shape invention tasks . The objects used for the alternative uses were \u201cCUP\u201d and \u201cTABLE . \u201d A different set of categories and shapes were randomly sampled for the invention task , using the same procedure as in Experiment 1 . The new categories were \u201cTransportation , \u201d \u201cFurniture , \u201d and \u201cWeapons , \u201d and the new shapes were \u201crectangular block , \u201d \u201cring , \u201d and \u201chalf - sphere\u201d ( see Figure 7 ) . Dependent measures As in Experiment 1 , we obtained measures of fluency and novelty for both the uses and invention tasks . Inter - rater reli - ability was high for novelty scoring across both tasks , ICC ( 2 , 2 ) = . 83 for uses novelty , and ICC ( 2 , 3 ) = . 84 for invention nov - elty . We then aggregated scores at the participant level into mean and max novelty . Also as in Experiment 1 , we evaluated uses and inventions for practicality . Inter - rater reliability was high for uses practicality , ICC ( 2 , 2 ) = . 82 , and acceptable for invention practicality , ICC ( 2 , 4 ) = . 67 . We then aggregated scores into participant - level measures of mean uses and invention practicality . Both the RAT and letter series tasks were scored identically to Experiment 1 ( i . e . , percent correct ) . Procedure The procedure was identical to Experiment 1 , except that partic - ipants did not complete the PANAS measurement at any point . Design This experiment had a between - subjects design . The inde - pendent variable was room size , with two levels ( large or small ) . Participants were randomly assigned to conditions . In the final dataset , there were 68 participants in the large rooms and 38 participants in the small room . REsUlTs Descriptive statistics for all measures are shown in Table 3 . Note that performance on all divergent measures ( except for uses fluency ) was significantly lower than that observed in Experiment 1 . In contrast , performance on the practi - cality measures for both the uses and invention tasks were Situative Creativity 26 Three participants ( assigned to the large rooms ) did not produce any valid responses to the invention task , and were dropped from all analyses , leaving us with 106 total participants in our final dataset . 3 Materials Room size manipulation . In this study , the \u201clarge\u201d setting of our room size manipulation included two different rooms : 1 ) the same conference auditorium as in Experiment 1 , with the only difference being that participants sat at the top of the room , rather than the bottom of the room , and 2 ) another conference room in the same building ( see Fig . 6 ) . The dimensions of the new large room were approximately 15\u2032 \u00d7 30\u2032 \u00d7 8\u2032 . Other than the desk and chair the participants used , along with the other desks and chairs in the room , the room was empty . Note also that the lighting here is fluorescent , similar to the small rooms in both experiments . The \u201csmall\u201d room was another former office space in the same building that was emptied 3 Results are the same with partial data from these participants included . Figure 6 . Picture of new large room in Experiment 2 . Figure 6 Picture of new large room in Experiment 2 . Situative Creativity 27 out for the experiment . The dimensions of the room were the same as in Experiment 1 , i . e . , approximately 8 \u2032 W \u00d7 10 \u2032 L \u00d7 8 \u2032 H . As in Experiment 1 , participants completed their tasks on a desk facing one of the walls . It was empty except for the desk and chair the participants used . Problem - solving tasks . As in Experiment 1 , participants completed the alternative uses , shape invention , RAT , and letter series tasks . The only differences from Experiment 1 are with the stimuli for the alternative uses and shape invention tasks . The objects used for the alternative uses were \u201cCUP\u201d and \u201cTABLE . \u201d A different set of categories and shapes were randomly sampled for the invention task , using the same procedure as in Experiment 1 . The new categories were \u201cTransportation , \u201d \u201cFurniture , \u201d and \u201cWeapons , \u201d and the new shapes were \u201crectangular block , \u201d \u201cring , \u201d and \u201chalf - sphere\u201d ( see Fig . 7 ) . Dependent measures As in Experiment 1 , we obtained measures of fluency and novelty for both the uses and invention tasks . Inter - rater reliability was high for novelty scoring across both tasks , ICC ( 2 , 2 ) = . 83 for uses novelty , and ICC ( 2 , 3 ) = . 84 for invention novelty . We then aggregated scores at the participant level into mean and max novelty . Also as in Experiment 1 , we evaluated uses and inventions for practicality . Inter - rater reliability was high for uses practicality , ICC ( 2 , 2 ) = . 82 , and acceptable for invention practicality , ICC ( 2 , 4 ) = . 67 . We then aggregated scores into Figure 7 . Shapes for invention task in Experiment 2 . Figure 7 Shapes for invention task in Experiment 2 . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 39 significantly higher than in Experiment 1 . However , letters performance was also lower than in Experiment 1 . We return to this issue in the discussion when interpreting the relation - ship between the results across the two experiments . Alternative uses In contrast to Experiment 1 , there was no effect of room size on mean fluency , F ( 1 , 1 04 ) = 0 . 4 , p = . 53 , with participants in the large rooms generating about the same number of uses ( M = 20 . 3 , SE = 0 . 9 ) as participants in the small room ( M = 19 . 3 , SE = 1 . 2 ) , d = 0 . 13 [ - 0 . 28 , 0 . 54 ] . In contrast , similar to Experiment 1 , mean trends for novelty of uses were in the hypothesized direction . However , the mean differences did not reach statistical significance . Mean novelty of uses was nonsignificantly higher in the large room ( M = 1 . 4 , SE = 0 . 0 ) compared to the small room ( M = 1 . 3 , SE = 0 . 0 ) , d = 0 . 34 [ - 0 . 07 , 0 . 75 ] , F ( 1 , 104 ) = 2 . 8 , p = . 10 ( Fig . 8 , left panel ) . Max novelty was marginally higher in the large rooms ( M = 2 . 9 , SE = 0 . 1 ) compared to the small room ( M = 2 . 6 , SE = 0 . 1 ) , d = 0 . 37 [ - 0 . 03 , 0 . 78 ] , F ( 1 , 104 ) = 3 . 4 , p = . 07 ( Fig . 8 , middle panel ) . Similar to Experiment 1 , there was an effect of room size on uses practicality , F ( 1 , 104 ) = 4 . 1 , p = . 04 , with participants in the larger rooms generating less practical uses ( M = 2 . 8 , SE = 0 . 01 ) than participants in the small room ( M = 2 . 9 , SE = 0 . 02 ) , d = - 0 . 41 [ - 0 . 82 , 0 . 00 ] ( Fig 8 , right panel ) . Shape invention There was no effect of room size on mean fluency , F ( 1 , 104 ) = 0 . 9 , p = . 36 , with participants in the large room generating about the same number of inventions ( M = 3 . 4 , SE = 0 . 3 ) as participants in the small room ( M = 3 . 8 , SE = 0 . 3 ) , d = - 0 . 19 [ - 0 . 59 , 0 . 22 ] . In contrast to Experiment 1 , there were no reli - able effects of room size on novelty of inventions : Participants in the larger rooms had similar mean novelty scores ( M = 2 . 5 , SE = 0 . 1 ) as participants in the small room ( M = 2 . 5 , SE = 0 . 1 ) , d = 0 . 14 [ - 0 . 26 , 0 . 55 ] , F ( 1 , 104 ) = 0 . 5 , p = . 49 . Similarly , partici - pants in the larger rooms achieved about the same max novelty scores ( M = 3 . 2 , SE = 0 . 1 ) as participants in the small room ( M = 3 . 1 , SE = 0 . 1 ) , d = 0 . 06 [ - 0 . 35 , 0 . 46 ] , F ( 1 , 104 ) = 0 . 1 , p = 0 . 79 . There was no effect of room size on invention practical - ity , with participants in the large room generating inventions that were about as practical ( M = 3 . 4 , SE = 0 . 1 ) as partici - pants in the small room ( M = 3 . 5 , SE = 0 . 1 ) , d = - 0 . 15 [ - 0 . 56 , 0 . 25 ] , F ( 1 , 104 ) = 0 . 6 , p = . 46 . RAT There was no effect of room size , F ( 1 , 104 ) = 0 . 02 , p = . 90 . Participants in the larger rooms had about the same mean proportion correct ( M = . 56 , SE = . 03 ) as participants in the small room ( M = . 55 , SE = . 04 ) , d = 0 . 03 [ - 0 . 37 , 0 . 43 ] . Situative Creativity 29 Alternative uses In contrast to Experiment 1 , there was no effect of room size on mean fluency , F ( 1 , 1 04 ) = 0 . 4 , p = . 53 , with participants in the large rooms generating about the same number of uses ( M = 20 . 3 , SE = 0 . 9 ) as participants in the small room ( M = 19 . 3 , SE = 1 . 2 ) , d = 0 . 13 [ - 0 . 28 , 0 . 54 ] . In contrast , similar to Experiment 1 , mean trends for novelty of uses were in the hypothesized direction . However , the mean differences did not reach statistical significance . Mean novelty of uses was nonsignificantly higher in the large room ( M = 1 . 4 , SE = 0 . 0 ) compared to the small room ( M = 1 . 3 , SE = 0 . 0 ) , d = 0 . 34 [ - 0 . 07 , 0 . 75 ] , F ( 1 , 104 ) = 2 . 8 , p = . 10 ( Fig . 8 , left panel ) . Max novelty was marginally higher in the large rooms ( M = 2 . 9 , SE = 0 . 1 ) compared to the small room ( M = 2 . 6 , SE = 0 . 1 ) , d = 0 . 37 [ - 0 . 03 , 0 . 78 ] , F ( 1 , 104 ) = 3 . 4 , p = . 07 ( Fig . 8 , middle panel ) . Similar to Experiment 1 , there was an effect of room size on uses practicality , F ( 1 , 104 ) = 4 . 1 , p = . 04 , with participants in the larger rooms generating less practical uses ( M = 2 . 8 , SE = Table 3 Descriptive statistics for experiment 2 . Mean Median Min Max SE Uses fluency 19 . 93 19 6 37 0 . 69 Uses novelty mean 1 . 38 V 1 . 35 1 2 . 12 0 . 02 Uses novelty max 2 . 77 V 3 1 4 0 . 08 Invention fluency 3 . 51 V 3 1 13 0 . 21 Invention novelty mean 2 . 51 V 2 . 52 1 . 25 3 . 78 0 . 05 Invention novelty max 3 . 17 V 3 . 33 1 . 67 4 0 . 07 Uses practicality 2 . 83 ^ 2 . 86 2 . 47 3 0 . 01 Invention practicality 3 . 43 ^ 3 . 50 1 . 5 4 . 33 0 . 04 RAT 0 . 56 0 . 55 0 1 0 . 02 Letters 0 . 50 V 0 . 52 0 0 . 78 0 . 01 V p < . 05 lower than Experiment 1 ; ^ p < . 05 higher than Experiment 1 . Table 3 Descriptive statistics for Experiment 2 . Situative Creativity 30 0 . 01 ) than participants in the small room ( M = 2 . 9 , SE = 0 . 02 ) , d = - 0 . 41 [ - 0 . 82 , 0 . 00 ] ( Fig 8 , right panel ) . Shape invention There was no effect of room size on mean fluency , F ( 1 , 104 ) = 0 . 9 , p = . 36 , with participants in the large room generating about the same number of inventions ( M = 3 . 4 , SE = 0 . 3 ) as participants in the small room ( M = 3 . 8 , SE = 0 . 3 ) , d = - 0 . 19 [ - 0 . 59 , 0 . 22 ] . In contrast to Experiment 1 , there were no reliable effects of room size on novelty of inventions : Participants in the larger rooms had similar mean novelty scores ( M = 2 . 5 , SE = 0 . 1 ) as participants in the small room ( M = 2 . 5 , SE = 0 . 1 ) , d = 0 . 14 [ - 0 . 26 , 0 . 55 ] , F ( 1 , 104 ) = 0 . 5 , p = . 49 . Similarly , participants in the larger rooms achieved about the same max novelty scores ( M = 3 . 2 , SE = 0 . 1 ) as participants in the small room ( M = 3 . 1 , SE = 0 . 1 ) , d = 0 . 06 [ - 0 . 35 , 0 . 46 ] , F ( 1 , 104 ) = 0 . 1 , p = 0 . 79 . Figure 8 . Novelty and practicality of alternative uses by room size in Experiment 2 . Error bars are \u00b11 SE . Figure 8 Novelty and practicality of alternative uses by room size in Experiment 2 . Error bars are \u00b11 SE . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 40 Letter series There was no effect of room size , F ( 1 , 104 ) = 0 . 07 , p = . 79 . Participants in the larger rooms had about the same mean proportion correct ( M = . 50 , SE = . 02 ) as participants in the small room ( M = . 50 , SE = . 02 ) , d = - 0 . 05 [ - 0 . 46 , 0 . 35 ] . Perceived task difficulty In contrast to Experiment 1 , there was no effect of room size on perceived difficulty for the uses task , F ( 1 , 104 ) = 0 . 0 , p = . 98 . Participants in the larger rooms self - reported the same levels of cognitive load ( M = 5 . 1 , SE = . 18 ) as participants in the small room ( M = 5 . 1 , SE = . 24 ) , d = - 0 . 00 [ - 0 . 40 , 0 . 41 ] . Similarly , for the invention task , participants self - reported the same level of difficulty in the large ( M = 5 . 9 , SE = 0 . 21 ) and small rooms ( M = 5 . 9 , SE = 0 . 28 ) , d = - 0 . 00 [ \u2013 0 . 41 , 0 . 40 ] , F ( 1 , 104 ) = 0 . 0 , p = 0 . 98 . Results were the same as Experiment 1 for the RAT and letters tasks . For the RAT , perceived difficulty was about the same in the large ( M = 7 . 5 , SE = 0 . 15 ) and small rooms ( M = 7 . 3 , SE = 0 . 20 ) , d = 0 . 13 [ - 0 . 28 , 0 . 53 ] , F ( 1 , 104 ) = 0 . 4 , p = 0 . 53 . Similarly , perceived difficulty of the letters task was the same in the large ( M = 0 . 50 , SE = 0 . 02 ) and small rooms ( M = 0 . 50 , SE = 0 . 02 ) , d = - 0 . 05 [ - 0 . 46 , 0 . 35 ] , F ( 1 , 104 ) = 0 . 1 , p = 0 . 79 . DIsCUssION In Experiment 2 , we sought to replicate and extend the findings from Experiment 1 . See Figure 8 for a summary of the effects . We observed very similar patterns of effects for the alternative uses , RAT , and letter series tasks . Similar to Experiment 1 , novelty ( both mean and max ) of uses was higher in the larger vs . small room , although the effect size was substantially smaller than Experiment 1 ( approximately half the size ) . These trends are in the predicted direction of Hypothesis 1 , and consistent with both the direct priming and concept activation accounts . Overall performance on the divergent measures were worse in both conditions in this experiment compared to Experiment 1 , which may suggest floor effects . In the general discussion we further consider possible reasons for the partial replication . Partial support and replication was found for Hypothesis 2 , with participants in the large room showing worse perfor - mance on the practicality measure of the alternative uses task compared to those in the small room . Also similar to Experi - ment 1 , there was no effect of room size on the RAT or letter series task . However , perceived difficulty patterns did not rep - licate from Experiment 1 . The failure to replicate the difference in perceived difficulty means that findings do not support the direct priming hypothesis more so than the concept activation account . In sum , we observed a partial replication of the results in Experiment 1 ( mainly with novelty and practicality of uses ) . GENERAl DIsCUssION In this paper , we sought to explore the potential cognitive bases of the spatial metaphor that initial exploration of a creative space should be \u201cbroad . \u201d Across two experiments , we tested the hypothesis that larger physical spaces facilitate divergent , but not convergent , processes in problem - solving . Experiment 1 provided support for Hypothesis 1 and 2 , and Experiment 2 partially replicated the findings . Smaller room sizes facilitated the generation of more practical uses of everyday objects across both experiments . Larger room sizes facilitated the generation of more novel uses of everyday objects ( specifically mean and max novelty ) in Experiment 1 ; although the same trends were seen in Experiment 2 , the effects did not reach significance . As noted in our discussion of the descriptive statistics in Experiment 2 , there was a significant drop in performance across many of our measures from Experiment 1 , suggest - ing that there may have been important differences in the two samples . One potential explanation is that Experiment 2 participants were exclusively undergraduate students par - ticipating for course credit , whereas Experiment 1 partici - pants were paid volunteers and included a wider range of demographics ( not just undergraduate students ) . Reduced motivation may have led to floor effects , potentially reducing the sensitivity of our measures . Motivation differences might have been especially important since we did not provide direct instructions to \u201cbe creative\u201d in either experiment . For example , while the mean rated novelty of alternative uses was close to \u201cnot at all novel\u201d across both experiments , the mean and variability was higher in Experiment 1 ( M = 1 . 67 , SD = 0 . 42 ) compared to Experiment 2 ( M = 1 . 38 , SD = 0 . 24 ) . The generally low novelty scores ( and high practicality scores ) with relatively low variance suggest that participants were in general defaulting to more convergent processing . This Situative Creativity 32 7 . 3 , SE = 0 . 20 ) , d = 0 . 13 [ - 0 . 28 , 0 . 53 ] , F ( 1 , 104 ) = 0 . 4 , p = 0 . 53 . Similarly , perceived difficulty of the letters task was the same in the large ( M = 0 . 50 , SE = 0 . 02 ) and small rooms ( M = 0 . 50 , SE = 0 . 02 ) , d = - 0 . 05 [ - 0 . 46 , 0 . 35 ] , F ( 1 , 104 ) = 0 . 1 , p = 0 . 79 . Discussion In Experiment 2 , we sought to replicate and extend the findings from Experiment 1 . See Figure 8 for a summary of the effects . We observed very similar patterns of effects for the alternative uses , RAT , and letter series tasks . Similar to Experiment 1 , novelty ( both mean and max ) of uses was higher in the larger vs . small room , although the effect size was substantially smaller than Experiment 1 ( approximately half the size ) . These trends are in the predicted direction of Hypothesis 1 , and consistent with both the direct priming and concept activation accounts . Overall performance on the divergent measures were worse in both conditions in this experiment compared to Experiment 1 , which may suggest floor effects . In the general Figure 9 . Summary of effects in Experiment 2 . Error bars are 95 % CIs . Figure 9 Summary of effects in Experiment 2 . Error bars are 95 % CIs . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 41 observation is consistent with prior research on the \u201cpath of least resistance\u201d in creative production ( Ward , 1994 ) , and other work that has shown that instructions to \u201cbe creative\u201d can yield substantial improvements to creative output ( Nus - baum , Silvia , & Beaty , 2014 ) : People generally need to expend cognitive effort to overcome initial biases toward less cre - ative responses ( e . g . , using cognitive control to inhibit more accessible but less creative responses ; Beaty , Silvia , Nusbaum , Jauk , & Benedek , 2014 ) . In light of this , it may be useful to think of these results as describing the effects of room size on \u201cdefault\u201d problem solving ( i . e . , when participants are not necessarily actively trying to be creative in their responses ) . We believe that pooling the data from the two experi - ments provides the clearest picture ( e . g . , robust across a wide range of participants and problem solving stimuli ) of whether there is a relationship between room size and diver - gent and convergent problem solving processes . The pooled data indicate reliable evidence that larger physical spaces facilitate novelty and hinder practicality of solutions on the alternative uses task ( see Figure 9 ) . In the pooled data , mean novelty of uses is higher in the larger rooms ( M = 1 . 5 , SE = 0 . 03 ) compared to the small rooms ( M = 1 . 4 , SE = 0 . 04 ) , d = 0 . 34 [ 0 . 01 , 0 . 67 ] , F ( 1 , 147 ) = 4 . 13 , p = . 04 . Similarly , max novelty of uses is higher in the larger rooms ( M = 3 . 0 , SE = 0 . 09 ) compared to the small rooms ( M = 2 . 7 , SE = 0 . 11 ) , d = 0 . 37 [ 0 . 04 , 0 . 71 ] , F ( 1 , 147 ) = 5 . 13 , p = . 03 . In contrast , mean practicality of uses was lower in the larger rooms ( M = 2 . 7 , SE = 0 . 02 ) compared to the small rooms ( M = 2 . 8 , SE = 0 . 02 ) , d = - 0 . 38 [ - 0 . 71 , - 0 . 04 ] , F ( 1 , 147 ) = 5 . 11 , p = . 03 . Our primary goal in this study is to document a psycholog - ical phenomenon : We provide an initial test of whether there is an association between room size and divergent problem Situative Creativity 35 We believe that pooling the data from the two experiments provides the clearest picture ( e . g . , robust across a wide range of participants and problem solving stimuli ) of whether there is a relationship between room size and divergent and convergent problem solving processes . The pooled data indicate reliable evidence that larger physical spaces facilitate novelty and hinder practicality of solutions on the alternative uses task ( see Figure 9 ) . In the pooled data , mean novelty of uses is higher in the larger rooms ( M = 1 . 5 , SE = 0 . 03 ) compared to the small rooms ( M = 1 . 4 , SE = 0 . 04 ) , d = 0 . 34 [ 0 . 01 , 0 . 67 ] , F ( 1 , 147 ) = 4 . 13 , p = . 04 . Similarly , max novelty of uses is higher in the larger rooms ( M = 3 . 0 , SE = 0 . 09 ) compared to the small rooms ( M = 2 . 7 , SE = 0 . 11 ) , d = 0 . 37 [ 0 . 04 , 0 . 71 ] , F ( 1 , 147 ) = 5 . 13 , p = . 03 . In contrast , mean practicality of uses was lower in the larger rooms ( M = 2 . 7 , SE = 0 . 02 ) compared to the small rooms ( M = 2 . 8 , SE = 0 . 02 ) , d = - 0 . 38 [ - 0 . 71 , - 0 . 04 ] , F ( 1 , 147 ) = 5 . 11 , p = . 03 . Our primary goal in this study is to document a psychological phenomenon : We provide an initial test of whether there is an association between room size and divergent problem solving Figure 10 . Novelty and practicality of alternative uses by room size , pooled across experiments . Error bars are \u00b11 SE . solving performance . While this effect appears to be relatively small and may depend on having sufficiently motivated par - ticipants , some aspects of our results provide hints for future theoretical refinement . While our results from the problem solving measures ( and non - replication of the hypothesized perceived task difficulty results from Experiment 1 to Experi - ment 2 ) are consistent with both a direct priming explanation ( Hills , 2006 ; Hills et al . , 2008 , 2012 ) , as well as the concept activation explanation ( Meyers - Levy & Zhu , 2007 ) , the affect results in Experiment 1 help to partially arbitrate between the explanations . The lack of effect on positive / negative affect is more consistent with a direct priming explanation , since concepts of \u201cfreedom\u201d or \u201copenness\u201d are expected to engen - der more positive affect , whereas attunement to resource distribution patterns are not . We therefore suggest that , to the extent that this effect proves reliable , it may be a conse - quence of automatic attunement of semantic search patterns to search affordances in the physical environment . That is , people may be responding to the physical search affordances of the physical environment by defocusing their attention to enable broader search in semantic memory , shifting from a tight focus on a few highly relevant responses to consider - ing more semantically distant and varied responses . However , alternative explanations are possible : For example , it is possi - ble that the small room reminded people of traditional office environments , which may have invoked a \u201cwork schema\u201d that primed more focused attention , impairing divergent think - ing . Further investigations are necessary to tease apart the psychological underpinnings of this effect . Although we intended the letter series task and RAT to be measures of convergent problem solving processes based on the nature of the response required ( many = divergent , single Figure 10 Novelty and practicality of alternative uses by room size , pooled across experiments . Error bars are \u00b11 SE . docs . lib . purdue . edu / jps 2016 | Volume 9 J . Chan & T . J . Nokes - Malach Situative Creativity 42 = convergent ) , in hindsight , it is probably best to consider both the RAT and the letter series task as a more even mix of diver - gent and convergent processes than the fluency and novelty measures for the uses and invention tasks ( which quite cleanly measure divergent processes ) . For example , in the RAT , one might first search broadly for possible meanings ( strong and weak associates ) of the target words and then only later con - verge on the common target that links across all three . Similarly , the letter series task may first require divergent search for sev - eral possible patterns before converging on the single , correct pattern to extrapolate . The mixture of both divergent and con - vergent processes in the letter series task and RAT might explain why we did not find a harmful effect of large rooms on letter series and RAT performance , and why we only found a harmful effect of large rooms on practicality of uses ( which turned out empirically to be our cleanest measures of convergent processes , based on the intercorrelations between measures ) . Our data have broader implications for the psychology of creative problem solving . For example , our observed strong negative correlations between novelty and practicality of uses corroborate prior arguments that originality and prac - ticality in creative thought are cognitively at odds with each other ( Goldenberg et al . , 2013 ) . Our results also have impli - cations for how we should think about the RAT as a measure of creativity . In this study , we departed from a number of prior studies that have examined the divergent aspects of the RAT , for example , studying the relationship between defo - cused attention and RAT performance ( Aiello et al . , 2012 ; Haarmann et al . , 2012 ) . However , the differing patterns of results and lack of correlation between the RAT and diver - gent problem solving measures for the invention and uses task suggest that the RAT may involve more convergence than is typically described . Noting these findings might lead to more fruitful theoretical examinations of the relation - ship between the RAT and problem solving and creativity . For example , Goel , Eimontaite , Goel , and Schindler ( 2015 ) recently argued that insight problems ( such as the RAT ) are a subset of well - structured problems , while divergent prob - lem solving tasks ( such as the alternative uses and invention tasks ) are a subset of ill - structured problems . Our results also echo a number of recent studies that have demonstrated the psychological separability of divergent and convergent prob - lem solving processes ( Chermahini & Hommel , 2010 ; Col - zato et al . , 2012 ; Hommel , Colzato , Fischer , & Christoffels , 2011 ; Oppezzo & Schwartz , 2014 ; Radel et al . , 2015 ) . We join these more nuanced arguments to call for more careful anal - yses of the components of creative performance ( e . g . , sepa - rating divergent vs . convergent processes ) in future research on creativity . 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Sofia , Bulgaria . Warr , A . , & O\u2019Neill , E . ( 2005 ) . Understanding design as a social creative process . In Proceedings of the 5th Confer - ence on Creativity & Cognition ( pp . 118 \u2013 127 ) . New York : ACM . http : / / doi . org / 10 . 1145 / 1056224 . 1056242 Warriner , A . B . , Kuperman , V . , & Brysbaert , M . ( 2013 ) . Norms of valence , arousal , and dominance for 13 , 915 English lemmas . Behavior Research Methods , 45 ( 4 ) , 1191 \u2013 1207 . http : / / doi . org / 10 . 3758 / s13428 - 012 - 0314 - x Watson , D . , Clark , L . A . , & Tellegen , A . ( 1988 ) . Develop - ment and validation of brief measures of positive and negative affect : The PANAS scales . Journal of Personality and Social Psychology , 54 ( 6 ) , 1063 \u2013 1070 . http : / / dx . doi . org / 10 . 1037 / 0022 - 3514 . 54 . 6 . 1063 Wiley , J . ( 1998 ) . Expertise as mental set : The effects of domain knowledge in creative problem solving . 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Nokes - Malach Situative Creativity 45 APPENDIX A : FUll lIsT OF ITEMs FOR RAT TAsK APPENDIX B : FUll lIsT OF ITEMs FOR lETTER sERIEs TAsK Situative Creativity 46 APPENDIX A : FULL LIST OF ITEMS FOR RAT TASK Situative Creativity 47 APPENDIX B : FULL LIST OF ITEMS FOR LETTER SERIES TASK 1 . aaabbbcccdd _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2 . atbataatbat _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 3 . abmcdmefmghm _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 4 . defgefghfghi _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 5 . qxapxbqxa _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 6 . aduacuaeuabuafua _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 7 . mabmbcmcdm _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 8 . urtustuttu _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 9 . abyabxabwab _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 10 . rscdstdetuef _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 11 . npaoqapraqsa _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 12 . wxaxybyzczadab _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 13 . jkqrklrslmst _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 14 . pononmnmlmlk _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 15 . lmzmlymnx _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 16 . efsferfgq _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 17 . cdqdcpdeo _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 18 . ijwjivjku _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _", + "chen2023novo": "Article De novo protein identi\ufb01cation in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking Graphical abstract Highlights d In situ cryo - ET revealed native structures of doublet microtubules in mammalian sperm d Subtomogram averaging led to 6 - A\u02da reconstruction of microtubule doublets d Protein discovery by matching structures with the AlphaFold2 - predicted mouse proteome d Sperm doublets feature a plastic and partially redundant Tektin 5 network Authors Zhen Chen , Momoko Shiozaki , Kelsey M . Haas , . . . , Robyn M . Kaake , Ronald D . Vale , David A . Agard Correspondence zhen . chen @ ucsf . edu ( Z . C . ) , valer @ janelia . hhmi . org ( R . D . V . ) , david @ agard . ucsf . edu ( D . A . A . ) In brief Chen et al . reported the reconstruction of microtubule doublets in mammalian sperm using in situ cryoelectron tomography . Unbiased matching of sperm - speci\ufb01c structures with the AlphaFold2 library allowed de novo protein identi\ufb01cation of microtubule inner proteins . A transgenic mouse model revealed the functional importance and partial redundance of Tektin 5 in sperm . Chen et al . , 2023 , Cell 186 , 5041 \u2013 5053 November 9 , 2023 \u00aa 2023 The Authors . Published by Elsevier Inc . https : / / doi . org / 10 . 1016 / j . cell . 2023 . 09 . 017 ll Article De novo protein identi\ufb01cation in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking Zhen Chen , 1 , 2 , * Momoko Shiozaki , 3 Kelsey M . Haas , 2 , 4 , 5 Will M . Skinner , 6 Shumei Zhao , 3 Caiying Guo , 3 Benjamin J . Polacco , 2 , 5 Zhiheng Yu , 3 Nevan J . Krogan , 2 , 4 , 5 Polina V . Lishko , 6 , 7 Robyn M . Kaake , 2 , 4 , 5 Ronald D . Vale , 2 , 3 , * and David A . Agard 1 , 5 , 8 , * 1 Department of Biochemistry and Biophysics , University of California , San Francisco , San Francisco , CA , USA 2 Department of Cellular and Molecular Pharmacology , University of California , San Francisco , San Francisco , CA , USA 3 Janelia Research Campus , Howard Hughes Medical Institute , Ashburn , VA , USA 4 J . David Gladstone Institutes , San Francisco , CA , USA 5 Quantitative Biosciences Institute ( QBI ) , University of California , San Francisco , San Francisco , CA , USA 6 Department of Molecular and Cell Biology , University of California , Berkeley , Berkeley , CA , USA 7 Present address : Department of Cell Biology and Physiology , Washington University School of Medicine in St . Louis , St . Louis , MO , USA 8 Lead contact * Correspondence : zhen . chen @ ucsf . edu ( Z . C . ) , valer @ janelia . hhmi . org ( R . D . V . ) , david @ agard . ucsf . edu ( D . A . A . ) https : / / doi . org / 10 . 1016 / j . cell . 2023 . 09 . 017 SUMMARY To understand the molecular mechanisms of cellular pathways , contemporary work\ufb02ows typically require multiple techniques to identify proteins , track their localization , and determine their structures in vitro . Here , we combined cellular cryoelectron tomography ( cryo - ET ) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ . Our cellular cryo - ET and subtomogram averaging provided 6 . 0 - A\u02da reconstructions of axonemal microtubule structures . The well - resolved tertiary structures allowed us to unbiasedly match sperm - speci\ufb01c densities with 21 , 615 AlphaFold2 - predicted pro - tein models of the mouse proteome . We identi\ufb01ed Tektin 5 , CCDC105 , and SPACA9 as novel microtubule - associated proteins . These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules , suggesting their roles in modulating the mechanical properties of the \ufb01la - ments . Indeed , Tekt5 (cid:1) / (cid:1) sperm possess more deformed \ufb02agella with 180 (cid:3) bends . Together , our studies pre - sented a cellular visual proteomics work\ufb02ow and shed light on the in vivo functions of Tektin 5 . INTRODUCTION Natural fertilization requires the rhythmic beating motion of sperm \ufb02agella to propel the cell toward the egg . 1 , 2 This coordi - nated and repetitive bending of sperm \ufb02agella relies on macro - molecular machinery to generate periodic force and endure me - chanical stresses . Genetic analyses of infertility have so far offered only an incomplete list of protein candidates in sperm . 1 Additionally , we currently lack high - resolution information of sperm macromolecular complexes to understand their assem - blies and functions at the molecular level . Eukaryotic motile cilia and \ufb02agella share a conserved \ufb01lamen - tous structure known as the axoneme , characterized by an over - all architecture of nine doublet microtubules ( doublets ) sur - rounding two singlet microtubules . 3 \u2013 6 These cytoskeletal \ufb01laments are extensively decorated externally and internally by proteins required for the various beating motions and the struc - tural integrity of \ufb02agella . 3 , 4 Notably , sperm from different species can differ substantially in their morphologies , functions ( e . g . , swimming behaviors ) , and genetics . 7 \u2013 9 In particular , mammalian sperm \ufb02agella are much longer and wider and must withstand larger bending torques compared with other motile cilia . 10 , 11 Despite the crucial roles of sperm axonemes in fertility and speciation , our understanding of their unique adaptations re - mains limited . Isolation of axonemal complexes from non - sperm motile cilia combined with single - particle cryoelectron microscopy ( cryo - EM ) analyses has led to high - resolution reconstructions ( better than4A\u02da ) . 12 \u2013 16 Therichstructural information onthetertiarystruc - tures and side chains has allowed con\ufb01dent assignments of pro - teinidentitiesintheEMreconstructions . However , carefuloptimi - zation of puri\ufb01cation strategies is required to avoid partial loss of components . 14 , 16 , 17 The truly intact structures are not guaran - teed by the end of puri\ufb01cation . On the other hand , direct visuali - zation of macromolecular complexes in mammalian sperm using cryogenic focused ion beam - scanning electron microscopy ( cryo - FIB - SEM ) and in situ cryoelectron tomography ( cryo - ET ) indicated that there are indeed mammalian sperm - speci\ufb01c ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 , November 9 , 2023 \u00aa 2023 The Authors . Published by Elsevier Inc . 5041 This is an open access article under the CC BY - NC - ND license ( http : / / creativecommons . org / licenses / by - nc - nd / 4 . 0 / ) . features . 18 \u2013 20 However , thecurrentreconstructionsfromcryo - ET subtomogram averaging are limited to (cid:4) 10 \u2013 20 A\u02da resolutions , mainly due to alignment inaccuracies . At such resolutions , ter - tiary structures of the proteins are rarely resolved , and multi - pro - tein complexes appear as blobs , making it challenging to deter - mine the identities of individual sperm proteins . Here , our in situ cryo - ET and subtomogram averaging has achieved up to 6 . 0 - A\u02da reconstructions of native microtubule structures in mouse and human sperm . The well - resolved ter - tiary structures in our cryo - EM maps allowed us to survey 21 , 615 AlphaFold2 - predicted protein models of the mouse pro - teome and unbiasedly identify matching ones . Such a visual pro - teomics approach helped us to discover novel microtubule - associated proteins in mammalian sperm , localize them in cells , and determine their native structures and interaction network without cell disruption and biochemical puri\ufb01cation . We also generated CRISPR - knockout mouse lines and showed that the newly identi\ufb01ed Tektin 5 is important for the structural integrity of the \ufb02agella . RESULTS In situ structures of sperm doublets at subnanometer resolutions Freshly extracted mouse sperm were treated with the dynein in - hibitor erythro - 9 - ( 2 - hydroxy - 3 - nonyl ) adenine ( EHNA ; 10 mM ) , which immediately stopped the beating motion of sperm \ufb02agella . 21 Subsequently , the inhibited sperm were vitri\ufb01ed on EM grids . To facilitate cryo - ET imaging , which is limited by sam - ple thickness , lamellae of (cid:4) 300 nm thickness were generated by cryo - FIB - SEM milling . Tilt series were recorded using a 300 kV Krios cryo transmission electron microscope ( cryo - TEM ) and a dose - symmetric scheme with a tilting increment of 4 (cid:3) . The 4 (cid:3) tilt increment , instead of the commonly used 1 (cid:3) \u2013 3 (cid:3) , 18 , 19 , 22 , 23 was used to improve the signal - to - noise ratio of each tilt image while keeping the similar angular ranges and total dose ( Fig - ure S1 ) . Three - dimensional classi\ufb01cation and re\ufb01nement of sub - tomograms corresponding to the 96 nm - repeating units were carried out as reported previously . 19 We then performed local re\ufb01nement on the 48 nm - repeating structures of doublets , focusing on the microtubules , aiming to reach the highest possible resolution ( see the work\ufb02ow shown in Figure S1 ) . The newly developed RELION4 was used to re\ufb01ne the 3D recon - structions and achieved 7 . 7 A\u02da overall resolution ( Fourier shell correlation [ FSC ] = 0 . 143 ) ( Figures 1A , S2A , and S2B ; see STAR Methods ) . 24 The improvement of resolution compared with RELION3 comes from more accurate contrast transfer func - tion ( CTF ) estimation and alignment of tilt series as these param - eters of each tilt image were iteratively re\ufb01ned relative to the 3D reconstructions ( Figure S2A ) . 24 Densities of microtubule inner proteins ( MIPs ) from mouse axonemes that repeat every 16 nm were observed despite of the overall periodicity of 48 nm ( Figures 1C \u2013 1E ) . In addition , we reprocessed a previously collected human sperm dataset and achieved 10 . 3 A \u02da for the 48 nm - repeating structures of the doublets ( FSC = 0 . 143 ) ( Figures 1B , S3A , and S3B ) . 19 Individual a - helices for the tubulins and MIPs are resolved in both maps . These maps were then compared with the published cryo - EM map of isolated doublets from bovine trachea reconstructed by single - particle cryo - EM , 13 which were low - pass \ufb01ltered to comparable resolutions of 7 . 5 and 10 A\u02da , respectively ( Figures S2C and S3C ) . Similar levels of detail in secondary and tertiary structures were resolved , vali - dating the resolution estimates of our 3D reconstructions . To our knowledge , these resolutions are currently the highest achieved by cryo - ET for any in situ axonemal structures ( 12 - A\u02da maps were reported previously for equivalent structures from Tetrahymena cilia 23 , 25 ) . Our 3D reconstructions of mouse and human sperm doublets reveal densities similar to the ones from bovine trachea dou - blets , 13 as well as sperm - speci\ufb01c densities ( colored densities in Figure 1 ) . Inside the A - tubule of mouse sperm axonemes , twelve helical bundles form a \ufb01lamentous core parallel to the lon - gitudinal axis , whereas only eight helical bundles , identi\ufb01ed as Tektins 1 \u2013 4 , are present in bovine trachea cilia ( Figures 1A and S2D ) . 13 Among the four mouse sperm - speci\ufb01c helical bundles , only one is a continuous 3 - helix bundle that runs along the entire length of the doublets ( Figures 1A and 1C ) . This continuous bundle is also observed in human sperm doublets ( Figures 1A \u2013 1C ) . The other three bundles , the two broken bundles and the curved bundles , all have breaks within the 48 - nm periodic struc - ture and appear different in mouse and human sperm ( Figures S2D and S3D \u2013 S3G ) . In particular , the two broken straight 3 - helix bundles have very low occupancy in the human sperm doublet ( Figures 1B , S3D , and S3E ) , while one of the curved helical bundles is connected to the microtubule lumen in human but not mouse sperm ( Figures S2D , S3F , and S3G ) . In the mouse sperm doublet , we also observed unique \u2018\u2018oblique\u2019\u2019 helical densities oriented (cid:4) 45 (cid:3) relative to the \ufb01lament axis and a globular domain next to it every 16 nm ( Figure 1C ) . These com - parisons indicate there are sperm - speci\ufb01c MIPs compared with mammalian trachea cilia and also diversi\ufb01cations among mammalian sperm in the A - tubule of the doublets . Outside the A - tubule , novel densities that are conserved in both mouse and human sperm doublets were resolved . A contin - uous 3 - helix bundle with multiple protrusions is situated at the external interface between A11 and A12 proto\ufb01laments , previ - ously named the \u2018\u2018ribbon\u2019\u2019 region of doublets ( Figures 1A , 1B , and 1D ) . 26 Inside the B - tubule , there are groups of 4 - helix bun - dles lining the inner surface of tubulins from the B4 to B9 proto - \ufb01laments . These 4 - helix bundles are stacked along the helical pitch of the microtubule , consistent with the previously reported striation density at lower resolutions ( Figure 1E ) . 19 Together , these data reveal that the mammalian sperm doublets have the most extensive MIP network of any microtubule structure observed to date . Although the B - tubule appears similar , mouse sperm have more MIPs than human sperm in the A - tubule . De novo protein identi\ufb01cation using AlphaFold2 The clearly resolved secondary and tertiary structures allowed us to interpret maps and build pseudo - atomic models . First , we were able to identify densities corresponding to the 29 MIPs observed in bovine trachea cilia ( Figure S4 ) , 13 suggesting their orthologs or homologs are likely present in sperm axo - nemes . We then sought to identify proteins contributing to the conserved sperm densities in mouse and human doublets ( high - lighted in Figures 1C \u2013 1E ) . Because most of these features repeat ll OPEN ACCESS 5042 Cell 186 , 5041 \u2013 5053 , November 9 , 2023 Article Figure 1 . The 3D reconstructions of mouse and human sperm doublets revealed novel MIPs ( A and B ) Transverse cross - section views of the doublets of mouse ( A ) and human ( B ) sperm . Conserved sperm MIP densities are highlighted ( pink , blue , and green ) , andthecorrespondingviewinganglesof ( C ) \u2013 ( E ) areindicated ( coloredarrowheads ) . The3 - helixdensitiesinA - tubulesharedwithbovinetracheadoublets ( EMD - 24664 ) are colored ( yellow ) . 13 Divergent sperm densities are also indicated ( red dashed shapes ) . Individual proto\ufb01laments of the doublets are labeled as A1 \u2013 A13 and B1 \u2013 B10 . ( C \u2013 E ) Zoom - in views of the conserved sperm MIP densities along the longitudinal axis . In ( C ) , mouse sperm - speci\ufb01c densities are indicated and labeled ( red dashed shapes ; see more in Figures S2 and S3 ) . In ( E ) , although the striations are 8 nm apart from one another , the overall periodicity is 48 nm . See also Figures S1 , S2 , and S3 . ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 , November 9 , 2023 5043 Article every 16 nm along the axoneme axis , we performed focused re\ufb01nement of the 16 nm - repeating structures in the A - and B - tubules of mouse doublets separately , and the larger number of subtomograms further improved the resolutions of recon - structions to 6 . 0 and 6 . 7 A \u02da , respectively ( Figure S5 ) . We then aimed to develop a general strategy to assign or narrow down the protein identities of the densities in the 6 \u2013 7 - A \u02da reconstructions . Unassigned densities from our maps were manually isolated and unbiasedly matched to the predicted models from the AlphaFold2 mouse proteome library ( 21 , 615 proteins ) using the COLORES program from the SITUS package ( Figure 2A ) . 27 , 28 The best poses for 21 , 615 mouse proteins were scored and ranked by the cross - correlation scores calculated by COLORES . 28 We tested this work\ufb02ow using densities corre - sponding to conserved single - domain MIPs ( CFAP20 and PACRG ) and a multi - domain MIP ( NME7 ) as controls . The cor - rect PDBs corresponding to the selected densities all came up as the top hits using this unbiased proteome - wide search ( Data S1 ) , indicating this visual proteomic approach can reliably identify proteins with matching tertiary structures . Figure 2 . De novo protein identi\ufb01cation of sperm MIPs assisted by AlphaFold2 ( A ) Conserved densities in mouse and human sperm were segmented from the averages of 16 - nm repeats of mouse sperm doublets and searched in the AlphaFold2 library of the mouse proteome ( 21 , 615 proteins ) . ( B ) The predicted structure of Tektin 5 based on AlphaFold2 was \ufb01tted into the continuous 3 - helix bundle . ( C ) Modeling of a complex formed by a full - length Tektin 5 and a truncated one ( N - Tekt 5 : aa 1 \u2013 149 ) using ColabFold . 32 ( D ) Fitting andmodelingof Tektin5s into the3 - helix bundle densities intheA - tubule . Thenearby densities accounted forby other proteins arealso shown ( yellow ribbon ) . ( E ) An unbiased search in the AlphaFold2 library identi\ufb01ed CCDC105 as the candidate for the continuous 3 - helix density at the ribbon . The three conserved proline - rich loops among CCDC105 orthologs could account for the protrusion densities but were not modeled ( see Figures S6C and S6D ) . ( F ) Modeling of a complex formed by a full - length CCDC105 and a truncated one ( N - CCDC105 : aa 1 \u2013 135 ) using ColabFold . 32 ( G ) Fitting and modeling of CCDC105 into the 3 - helix bundle density at the ribbon . The nearby densities accounted for by other proteins are also shown ( yellow ribbon ) . ( H ) The AlphaFold2 model for SPACA9 was directly \ufb01tted into the density and viewed from different angles . ( I ) TwoorthogonalviewsofthestriationsofSPAC9intheB - tubule . DifferentSPACA9moleculesarecoloredwithdifferentshadesofgreen . Theleftpanelshowed a particular striation indicated in the right panel ( the dashed rectangle ) . See also Figures S1 \u2013 S6 . ll OPEN ACCESS 5044 Cell 186 , 5041 \u2013 5053 , November 9 , 2023 Article For the continuous 3 - helix densities in the A - tubule , the best hit was Tektin 5 ( Figure S6A ) , previously found only in the mammalian testis and sperm in previous proteomic studies ( Figures 2B , S6A , and S6B ; see STAR Methods for details ) . 29 \u2013 31 Although Tektin 5 has no reported structure , AlphaFold2 pre - dicted that it possesses single - helix , 3 - helix , and 2 - helix seg - ments ( Figure 2B ) , 27 a tertiary structure that is almost identical to the ones reported for Tektin 1 \u2013 4 in bovine trachea cilia . 13 The ColabFold , an AlphaFold2 - based Google notebook , was then used to model how two copies of Tektin 5 molecules interact . 32 The resulting complexes suggest the single - helix N - terminal region of one Tektin 5 could interact with the 2 - helix C termini of the other molecule ( Figure 2C ) , indicating its poten - tial to self - polymerize and form a quasi - continuous 3 - helix bundle . Indeed , multiple Tektin 5 could be \ufb01tted into the contin - uous 3 - helix densities with 16 - nm periodicity , with minor adjustments of the orientations of individual a - helices of the orig - inal AlphaFold2 model ( Figure 2D ) . Upon manual inspection of the hit list , Tektin 1 \u2013 4 were also among the top 10 hits ( Figures S6A and S6B ) , and this \ufb01nding corroborated the robust - ness of our search method in \ufb01nding proteins with matching tertiary structures . We assigned these densities as Tektin 5 because it is uniquely present in mammalian sperm based on previous proteomic studies , 30 , 31 and such densities are absent in bovine trachea cilia that only contain Tektin 1 \u2013 4 . 13 For the continuous 3 - helix densities with protrusions at the ribbon , CCDC105 ( coiled - coil - domain - containing protein 105 ) was identi\ufb01ed in the top 20 hits from the unbiased search in AlphaFold2 mouse proteome library , along with Tektin 1 \u2013 5 ( see Data S2 for the top 30 hits ) . Previous proteomic studies revealed that CCDC105 was found in mammalian sperm and the testis but not other tissues . 29 \u2013 31 CCDC105 adopts a similar overall tertiary structure as Tektins 1 \u2013 5 based on AlphaFold2 prediction ( Fig - ure 2E ) , 27 suggesting it is a yet uncharacterized Tektin homolog . However , there are three proline - rich loops in CCDC105 that are uniquely conserved across CCDC105 orthologs ( Figures 2E , S6C , and S6D ) , 33 and they likely form structured loops like the ones observed in other axonemal complexes . 34 , 35 We also modeled how two copies of CCDC105 would interact using AlphaFold2 / ColabFold . 32 The predicted interface again involves coiled - coil interactions between the single - helix segment of one CCDC105 and the 2 - helix segment of the other ( Figure 2F ) . Furthermore , CCDC105 \ufb01ts well into the continuous 3 - helix densities at the ribbon , with their characteristic proline - rich loops matching the protrusions in our density maps ( Figure 2G ) . Notably , we could not swap the \ufb01tting of Tektin 5 and CCDC105 into these two 3 - helix bundles after extensive trials , mostly due to the different orientations and lengths of the a - helices ( Figure S6F ) . We also extracted the 4 - helix bundles at the B - tubule striations and performed an unbiased search against the AlphaFold2 library of the mouse proteome . Sperm acrosome - associated protein 9 ( SPACA9 ) was found to be the best hit ( Figures 2H and 2I ; see Data S3 for the top 30 hits ) . SPACA9 was previously found in various ciliated organs in humans ( testis , fallopian tubes , and lung ) using proteomics . 29 The tertiary fold of SPACA9 is so unique that no other homologous protein was found in the top 50 ranked structures from the unbiased search . Interestingly , no match was found when the search was done against the CATH library that curates non - redundant domains of published PDBs . 36 Thus , the capability of AlphaFold2 to pre - dict protein structures accurately , especially for the ones without published homologous structures , is critical to carrying out the unbiased proteome - wide survey . We next focused on the mouse sperm - speci\ufb01c densities . There are two 3 - helix bundles that appear to be similar to the ones formed by Tektins 1 \u2013 5 , apart from the discontinuous sec - tions ( Figures 3A and S2D ) . We also applied the unbiased search method to the slanted and curved helical bundles ( Figures 1C and S2D ) . Intriguingly , Tektin 1 \u2013 5 and CCDC105 were found to be among the top 30 hits in both cases , whereas no other PDB among the top 200 \ufb01ts better , albeit only parts of the structures are observed for the densities ( see Data S4 ) . Other hits among the top 200 do not match the secondary structures of the target densities upon visual inspections , suggesting Tektin 1 \u2013 5 and CCDC105 are the only proteins in mouse proteome that adopt such conformations . For the slanted helical densities , there is an additional a - helix connecting to the position where the missing single helix was expected to originate and is folded back by (cid:4) 180 (cid:3) ( Figures 3B and 3C ) . Interestingly , Tektin 5 , but not Tektin 1 \u2013 4 , has multiple conserved Gly residues among its orthologs at this turning region , making it plausible that Tektin 5 could adopt the bent - helix conformation . At a lower threshold , this bent helix is connected to a nearby globular domain that also repeats every 16 nm ( Figure S7A ) . The unbiased proteome - wide search suggests this globular domain matches the tertiary struc - ture of multiple dual speci\ufb01city phosphatase ( DUSP ) proteins ( DUSP 3 , 13 , 14 , 18 , 21 , and 29 ) ( Figures 3C and S7B ) . For the curved helical bundles , there are three 16 - nm groups of densities within every 48 - nm repeat ( Figures 3D and S2D ) . These densities can be explained by three modi\ufb01ed Tektin 5 mol - ecules , in which the two intermolecular interfaces near the two NME7s ( a previously known MIP shared with bovine trachea cilia ) are disrupted ( Figure 3D ) . The \ufb01rst and second Tektin 5s lack densities for the single - helix segment before the conserved glycine residues near Gly137 ( mouse ) , whereas the second and third Tektin 5s possess curved 2 - helix segments ( Figure 3D ) . Both modi\ufb01cations of Tektin 5s are necessary to avoid direct ste - ric clashes with the two NME7s , which adopt similar conforma - tions in bovine trachea and mouse sperm doublets . As curved bundles were not observed in the bovine trachea cilia that only contain Tektins 1 \u2013 4 , 13 we hypothesize that Tektin 5 has evolved to adopt multiple conformations and positions within sperm ax - onemes ( Figure 4E ) . In summary , the various helical conformers of Tektin 5 , together with the more uniform 3 - helix bundles of Tektin 1 \u2013 4 , are arranged with different polarities ( Figures 4A and 4B ) and ori - entations ( Figure 4C ) , forming the most extensive MIP network inside microtubules discovered to date . Mass spectrometry analyses of mouse sperm To further validate our de novo protein assignments , we used mass - spectrometry - based proteomics . Mouse sperm were iso - lated and extracted using salt buffers with increasing denaturing capabilities ( Extraction1 or E1 : 0 . 1 % Triton , E2 : NaCl , E3 : KSCN , E4 : urea , and E5 : 10 % SDS ) , and the extractions were analyzed ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 , November 9 , 2023 5045 Article using SDS - PAGE and western blotting ( Figure S8A ) . a - Tubulins could be detected in KSCN and urea extractions but not in others ( Figure S8B ) , suggesting the microtubule doublets are disas - sembled and the MIP candidates are likely present in these two extractions . After analyzing the E1 \u2013 E5 fractions by mass spectrometry ( MS ) , proteins with signi\ufb01cant changes in abun - dance between fractions were clustered into six distinct groups based on the correlation of intensity pro\ufb01le ( Figures S8C and S8D ; Tables S1 and S2 ) . 37 , 38 Gene ontology ( GO ) analyses sug - gest that cluster 4 is enriched for proteins involved in cilium , cilium assembly , cytoskeleton , and the axoneme and shows increased intensities in fraction E3 , whereas cluster 6 is enriched for cilia assembly and shows increased intensities in fraction E4 ( Figure S8E ; Table S3 ) . 39 The overall change in protein Figure 3 . Conformational plasticities of Tektin 5 ( A ) The two broken 3 - helix bundles could be explained by two complete and a third partial copies of Tektin 5 ( dashed rectangles ) per 48 - nm repeat , instead of three Tektins in the continuous 3 - helix bundle . ( B ) TheAlphaFold2modelof mouseTektin 5was\ufb01tted into theslantedhelicaldensities . Sequence alignmentofTektin5from M . musculus , H . sapiens , B . taurus , and F . catus is shown from Q133 to F151 ( the numbering of amino acids is based on M . musculus Tektin 5 ) . The conserved Gly137 , Gly143 , and Gly150 are near the turning point of the bent a - helix . ( C ) The \ufb01tting of Tektin 5 and DUSP3 protein ( its homologs are also possible candidates ) into the 16 nm - repeating features ; see the same view of the map in Figure 1C . ( D ) Three modi\ufb01ed Tektin 5 were \ufb01tted into the densities of the curved bundle in the mouse sperm doublet ( as indicated in Figure 1A ) . The intact intermolecular interaction interface , N termini of the Tektin 5s , and curved 2 - helix segments are indicated ( arrows ) . Nearby MIPs shared between mouse sperm \ufb02agella and bovine trachea cilia are also colored and labeled ( NME7 , CFAP161 , and SPAG8 ) . ( E ) The cross - section schematic is shown . The highlighted models of ( A ) \u2013 ( D ) are indicated using arrows . See also Figures S1 , S2 , S4 , S6 , S7 , and S8 . ll OPEN ACCESS 5046 Cell 186 , 5041 \u2013 5053 , November 9 , 2023 Article abundance is consistent with the idea that E3 and E4 buffers ex - tracted microtubule - associated proteins in the axonemes . Indeed , 28 of the 29 previously identi\ufb01ed MIPs in bovine trachea doublets were reproducibly identi\ufb01ed in all three biological repli - cates of mouse sperm extractions ( Table S4 ) . The almost com - plete list of MIPs highlighted the coverage of our biochemical and MS analyses . Importantly , SPACA9 was reproducibly iden - ti\ufb01ed in fraction E3 , whereas Tektin 1 \u2013 5 and CCDC105 were reproducibly identi\ufb01ed in fractions E3 and E4 , with high protein intensity and a range of 3 to 34 unique peptide identi\ufb01cations per replicate ( Figure S8F ; Table S4 ) . Only one DUSP protein , DUSP3 , was identi\ufb01ed in two of three replicates in fraction E4 fraction ( Figure S8F ; Table S4 ) . However , additional analyses are required to identify these proteins . Moreover , AlphaFold2 models for the other candidates from fractions E1 \u2013 E5 were in - spected , but no additional candidates could explain the various densities of helical bundles described above . Tektin 5 strengthens the \ufb02agella and is partially redundant In order to analyze the functions of Tektin 5 , we generated knockout mice carrying null alleles of Tekt5 using CRISPR tech - nologies . To our surprise , the F2 homozygous knockout males are still fertile when they are mated with the wild - type ( WT ) fe - males ( litter size : 7 . 3 \u00b1 1 . 4 , n = 6 mating trials ) , suggesting some levels of functional redundancy . However , the sperm ex - tracted from the mutant males have a lower fraction of motile cells compared with WT controls ( 64 % \u00b1 3 % vs . 77 % \u00b1 4 % ) and a higher percentage of defective \ufb02agella with 180 (cid:3) bends ( 30 % \u00b1 3 % vs . 13 % \u00b1 3 % ) ( Figures 5A and 5B ) , suggesting the mechanical integrity of the cellular structures inside \ufb02agella is compromised in the mutant sperm . We then analyzed the doublet structure of the mutant sperm and compared it with the WT counterpart . We \ufb01rst focused on the various densities that were assigned to be Tektin 5 ( as shown in Figure 3 ) . The continuous 3 - helix bundle remains in the mutant Figure 4 . Sperm doublets are composed of microtubules and extensive coiled - coil bun - dles ( A ) The plus and minus ends of Tektin 5 were named based on the N and C termini of the protein . ( B ) The cross - section view of the mouse sperm doublets shows the polarities of 3 - helix bundles pointing toward the readers . ( C ) The orientations for each 3 - helix bundle were represented by a vector starting from the middle point of the2 - helix segment and pointingtoward the single - helix segment of the other Tektin molecule . ( Figure 5C ) , suggesting a Tektin homolog could substitute on the same position in the absence of Tektin 5 . By contrast , the occupancy of densities corresponding to broken and curved helical bundles is much lower compared with the surround - ing proteins , such as the tubulins , in the mutant sperm ( Figures 5D and 5E ) , whereas in WT sperm the occupancies are comparable ( Figures 3C and 3D ) . These comparisons suggest the compensation for the lack of Tektin 5 by other Tektin homologs is low at these sites . For the slanted helical densities that repeat every 16 nm in WT sperm , we observed that two of the slanted helical densities are partially occupied while the last one is almost absent ( Fig - ure 5C ) . Interestingly , the densities corresponding to the DUSP domains next to the slanted helical densities were barely resolved , suggesting the lack of Tektin 5 would decrease the recruitment of the neighboring MIP . We did not observe addi - tional differences in densities corresponding to other MIPs . Together , these results suggest that Tektin homologs could partially re\ufb01ll docking sites of Tektin 5 in the mutant sperm . DISCUSSION Our in situ cryo - ET studies have provided high - resolution recon - structions of native microtubule structures within mouse and hu - man sperm , enabling us to identify Tektin 5 , SPACA9 , and CCDC105 as novel components in sperm doublets . Alignment of gold beads has traditionally been the method of choice to align tomographic tilt series , but the positions of gold beads undergo heterogeneous motions due to the sample defor - mation induced by the electron beam during imaging . 40 The sig - ni\ufb01cant improvement in resolutions made possible by RELION4 ( Figures S1 and S2A ) highlights the bene\ufb01ts of aligning 3D recon - structions of protein complexes with the individual 2D projection views on different tilt images to re\ufb01ne tilt series alignment . 24 Thus , new methods of aligning the molecular features directly from tilt series while considering their local motions have the po - tential to further improve the initial alignment of tilt series and set an even better starting point for subtomogram averaging . 41 The application of AlphaFold2 has facilitated structural modeling based on cryo - EM reconstructions below 10 A \u02da , where secondary structures are resolved . 42 However , most studies have focused on protein complexes with known components ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 , November 9 , 2023 5047 Article identi\ufb01ed through other approaches such as MS analyses of puri\ufb01ed complexes . However , this information may not be readily available in other less - studied cell biology systems . Our studies provided the \ufb01rst demonstration that high - resolution cellular cryo - ET combined with unbiased proteome - wide searches could identify previously unknown components of cellular complexes in their native context . This integrative struc - tural modeling approach offers a powerful alternative to the con - ventional genetic and cell biology approaches to identify protein components and localize them inside cells . The comparison of MIPs in WT and mutant sperm doublets with bovine tracheal doublets suggests that the assembling of MIPs is modular , and novel MIPs discovered in this study ( SPACA9 , CCDC105 , Tektin 5 , and DUSP ) are recruited after the commonly shared MIPs ( common MIPs ) . First , homologs or orthologs of MIPs identi\ufb01ed from bovine tracheal doublets Figure 5 . Characterization of mutant Tekt5 (cid:1) / (cid:1) sperm ( A ) The percentages of motile sperm from wild - type and Tekt5 knockout mice ( > 200 cells were counted for each mouse , three knockout (cid:1) / (cid:1) mice and two wild - type mice were analyzed , and the pool percentage and 95 % con\ufb01dence intervals [ Wilson / Brown method ] were shown ) . ( B ) The percentages of bent sperm from wild - type and Tekt5 knockout mice ( > 200 cells were counted for each mouse , three knockout (cid:1) / (cid:1) mice and two wild - type mice were analyzed , and the pool percentage and 95 % con\ufb01dence intervals by Wilson / Brown method were shown ) . Two examples of bent sperm are shown . ( C ) An overlay of wild - type models with the densities of Tekt5 (cid:1) / (cid:1) sperm around the slanted bundles . The continuous 3 - helix bundle assigned as Tektin 5 ( high occupancies ) and slanted helical bundles ( low occupancies ) are shown . The densities corresponding to the DUSP proteins are barely resolved . Note that there are substantially less densities for these models compared with Figure 3C . ( D ) An overlay of wild - type models with the densities of Tekt5 (cid:1) / (cid:1) sperm around the curved bundles . The occupancies of the curved bundles are lower than the other MIPs and tubulins . Note that there are substantially less densities for these models compared with Figure 3D . ( E ) The twobroken 3 - helix bundles have lower occupancies compared withthesurroundingMIPs and tubulins . Notethatthere aresubstantially less densities for these models compared with Figure 3A . ll OPEN ACCESS 5048 Cell 186 , 5041 \u2013 5053 , November 9 , 2023 Article are all present in WT and mutant mouse sperm , and they adopt indistinguishable conformations ( Figure S4 ) . 13 Second , SPACA9 and CCDC105 crosslink the tubulin dimers only at the exposed lumen sites observed in tracheal doublets , without displacing any common MIPs ( Figures 2G , 2I , and S4 ) . Interest - ingly , the remarkable conformational plasticity of Tektin 5 is partially molded by the doublets and common MIPs , as shown by the bent single helix that would otherwise clash with the microtubule wall ( Figures 3B and 3C ) , as well as the missing single helix and curved 2 - helix segment that would otherwise clash with NME7 , a conserved common MIP ( Figure 3D ) . Lastly , knockout of Tektin 5 decreases the occupancies of the bent Tektin 5 and the nearby DUSP densities ( Figure 5C ) . Such de - pendency or modularity could be further tuned during evolu - tion , as we observed that both the bent Tektin 5 and DUSP densities are absent in human sperm doublets . Bending of the axonemes would stress the nine microtubule \ufb01laments in nine different directions , and mammalian axonemes have to bend to various directions to generate the 3D beating waveforms . 43 The non - uniform arrangement of helical bundles in sperm doublets could be built to reinforce the doublets to with - stand mechanical stress from different directions ( Figures 4 and 6 ) . From a structural perspective , the intramolecular and inter - molecular coiled - coil interaction interfaces of Tektins are parallel to the microtubule axis so that bending would not expand the gap of these interfaces ( Figure 6A ) . Instead , the bending force would distort the straight helical bundles and the ideal bond an - gles / lengths . The transition of releasing such molecular strains could provide a restoring force that allows the curved \ufb01lament to return to the straight conformation . By contrast , the interface between tubulin dimers along the proto\ufb01laments is at a plane perpendicular to the \ufb01lament axis , and bending would open the interface and lower the af\ufb01nity and the potential restoring force . Therefore , the helical bundles are arranged at different an - gles to provide an effective means to bear the bending force , sta - bilizing the axoneme and \ufb02agella ( Figure 6B ) . From a functional perspective , we discovered that mutant mice lacking Tektin 5 have more deformed sperm \ufb02agella , yet they remain fertile . This observation is consistent with previous studies on knockout mouse models lacking Tektin - 3 or Tektin - 4 , which also showed deformed sperm but normal fertility , 44 , 45 underscoring the functional redundancy of Tektins . Our structural analyses of Tekt5 (cid:1) / (cid:1) sperm doublets uncov - ered the compensatory mechanisms of Tektin 5 at the molecu - lar level . Moreover , we discovered that different Tektin 5 con - formers were compensated to varying extents , likely due to the differential dependencies of the distinctive interaction interfaces . The human sperm doublets revealed that the densities formed by Tektin 5 are signi\ufb01cantly less resolved compared with the other MIPs ( Figure 1 ) , adopt different conformations ( Figure S3 F ) , or are completely absent compared with mouse sperm dou - blets ( Figure 1C ) . This is also consistent with the idea that the bundles formed by Tektin 5 are partially redundant and plastic during evolution so that some extent of degeneration is tolerable . Still , such degeneration would be selected against in the wild , particularly in species where sperm from multiple males in the fe - male reproductive tracts competing for fertilization . Our studies combined high - resolution in situ cryo - ET and AlphaFold2 modeling and de\ufb01ned a visual proteomic approach of precisely placing proteins in their native cellular environment without the need for labeling , cellular disruption , or puri\ufb01cation . This work\ufb02ow has allowed us to uncover the cellular locations and interaction networks of several MIPs , providing insights into how they contribute to the mechanics of \ufb02agellar bending . Moreover , this visual proteomic work\ufb02ow could potentially be applied to other cell biology problems , such as membrane re - modeling by viruses and identi\ufb01cation of their in situ interactors . Limitations of the study The majority of the densities in sperm doublets feature well - de\ufb01ned domains that could be isolated and identi\ufb01ed using Figure 6 . Coiled - coil interfaces are suitable to withstand mechanical stress from orthogonal directions ( A ) A model of how 3 - helix bundles would be able to bear mechanical stress differently compared with the microtubules . The bending curvatures and gaps are exaggerated for illustration purposes . ( B ) A schematic of wild - type and mutant sperm doublet structures highlighting the Tektin 5 bundles and the partial redundancy . ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 , November 9 , 2023 5049 Article our visual proteomics approach . However , it is possible that there are unknown MIPs that are composed of coiled coils without substantial intramolecular interactions . These proteins may adopt conformations that are determined by intermolec - ular interactions in the context of native complexes , which are not accounted for by AlphaFold2 predictions . Furthermore , map segregation for these types of proteins could be chal - lenging at (cid:4) 6 \u2013 10 - A\u02da resolutions . Improvement of resolutions for cellular cryo - ET is also desirable to resolve the side - chain densities in the EM reconstructions and distinguish the proteins with similar tertiary structures . While this manuscript was under revision , two single - particle cryo - EM studies of splayed mammalian sperm doublets were reported . 46 , 47 The single - par - ticle cryo - EM reconstructions of microtubule doublets from mouse and bovine sperm are indistinguishable from our cryo - ET reconstruction at our resolutions , suggesting that the sperm doublets are robust enough to withstand gentle biochemical treatments . Importantly , their assignments of protein identity based on side - chain densities are consistent with our identi\ufb01 - cation of CCDC105 , SPACA9 , and the different forms of Tektin 5 , validating our cellular visual proteomics approach for de novo protein discovery . In the future , a more systematic characterization of MIPs using transgenic mice will be needed to elucidate the functions of individual MIPs . Additionally , ge - netic analyses of patients with infertility are likely to identify more essential and redundant components relevant to sperm functions . STAR + METHODS Detailed methods are provided in the online version of this paper and include the following : d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS B Mouse models B Human sample d METHOD DETAILS B Sample preparation B Grid preparation B Cryogenic focused ion beam ( cryoFIB ) milling B Image acquisition and tomogram reconstruction B Subvolume averaging B Model building and unbiased matching of density maps to protein candidates B Sequence alignment and search for homologous proteins B Biochemical extractions of mouse sperm B Mass spectrometry ( MS ) - based global protein abun - dance of mouse sperm B Generation of Tektin - 5 knockout mice and functional / - structural analyses of mutant sperm d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplementalinformationcanbefoundonlineathttps : / / doi . org / 10 . 1016 / j . cell . 2023 . 09 . 017 . ACKNOWLEDGMENTS We are grateful to members of the Agard and Vale laboratories for the discus - sions and critical reading of the manuscript . We thank Xiaowei Zhao , Shixin Yang , and Rui Yan from the CryoEM Facility at the Janelia Research Campus for their assistance with data collection . We thank Zanlin Yu and Hao Wu at UCSF for their suggestions on sample processing and model building . We thank Garrett Greenan , Shawn Zheng , and Sam Li at UCSF for discussions on cryo - ET data processing . We thank Willy Wrigger from Old Dominion Uni - versity for his input and suggestions on the SITUS package . EM data process - ingutilizedcomputingresourcesatboththeworkstationsattheCryoEMFacil - ity at the Janelia Research Campus and the UCSF HPC Wynton cluster . We also thank David Bulkley , Glenn Gilbert , and Matt Harrington from the UCSF cryo - EM facility for their discussion on data collection and processing . We also thank Colin Morrow , Gillian Harris , Crystall Lopez , and Catherine Lindsey from Janelia Vivarium for mouse experiments . Z . C . was supported by the Helen Hay Whitney Foundation Postdoctoral Fellowship . W . M . S . was sup - ported by the National Science Foundation Graduate Research Fellowship ProgramundergrantnumbersDGE1752814andDGE2146752 . Anyopinions , \ufb01ndings , and conclusions or recommendations expressed in this material are those of the author ( s ) and do not necessarily re\ufb02ect the views of the National Science Foundation . P . V . L . received funding from a Pew Biomedical Scholars award and a GCRLE grant from the Global Consortium for Reproductive Longevity and Equality made possible by the Bia - Echo Foundation . D . A . A . received funding from NIH R35GM118099 . R . D . V . received funding from NIH R35GM118106 and the Howard Hughes Medical Institute . The UCSF cryo - EM facility was supported by NIH instrumentation grants 1S10OD026881 , 1S10OD020054 , and 1S10OD021741 . AUTHOR CONTRIBUTIONS Conceptualization , Z . C . , R . D . V . , and D . A . A . ; mouse sample preparation , S . Z . , C . G . , and W . M . S . ; cryo - EM sample preparation , Z . C . and M . S . ; data process - ing , Z . C . ; biochemical extraction , Z . C . ; mass spectroscopy , K . M . H . , R . M . K . , B . J . P . , and N . J . K . ; sperm analyses , W . M . S . , Z . C . , and P . V . L . ; writing \u2013 original draft , Z . C . , K . M . H . , and R . M . K . ; writing \u2013 review & editing , Z . C . , R . D . V . , and D . A . A . DECLARATION OF INTERESTS We declare that one or more authors have a competing interest as de\ufb01ned by Nature Portfolio . The Krogan Laboratory has received research support from Vir Biotechnology , F . Hoffmann - La Roche , and Rezo Therapeutics . N . J . K . has previously held \ufb01nancially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai , New York , and Twist Bioscience Corp . He currently has \ufb01nancially compensated consulting agreements with Maze Therapeutics , Interline Therapeutics , Rezo Therapeutics , and GEn1E Lifesciences , Inc . He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics , Maze Therapeutics , Rezo Therapeu - tics , and Interline Therapeutics . INCLUSION AND DIVERSITY We support inclusive , diverse , and equitable conduct of research . Received : October 19 , 2022 Revised : August 2 , 2023 Accepted : September 16 , 2023 Published : October 20 , 2023 ll OPEN ACCESS 5050 Cell 186 , 5041 \u2013 5053 , November 9 , 2023 Article REFERENCES 1 . Sironen , A . , Shoemark , A . , Patel , M . , Loebinger , M . R . , andMitchison , H . M . ( 2020 ) . Sperm defects in primary ciliary dyskinesia and related causes of male infertility . Cell . 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Symp , 141 \u2013 154 . ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 , November 9 , 2023 5053 Article STAR + METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Mouse ( Mus musculus ) sperm Gene Targeting and Transgenics Center , Janelia Research Campus N / A Human ( Homo sapiens ) sperm Lishko Laboratory , UC Berkeley N / A Chemicals , peptides , and recombinant proteins NaCl Sigma - Aldrich Cat # 71376 KH 2 PO 4 Sigma - Aldrich Cat # P0662 MgSO 4 , 7H 2 O Sigma - Aldrich Cat # M3409 Dextrose Sigma - Aldrich Cat # D9434 CaCl 2 Sigma - Aldrich Cat # 21097 KCl Sigma - Aldrich Cat # 60128 NaHCO 3 Sigma - Aldrich Cat # S5761 DTT Sigma - Aldrich Cat # DTT - RO TCEP Sigma - Aldrich Cat # C4706 Erythro - 9 - ( 2 - hydroxy - 3 - nonyl ) adenine Santa Cruz Biotechnology Cat # sc - 201184 KCSN Sigma - Aldrich Cat # 60178 Urea Sigma - Aldrich Cat # U5128 SDS Sigma - Aldrich Cat # 62862 Triton Sigma - Aldrich Cat # X100PC Deposited data Cryo - EM map of the 16 nm - repeating A - tubule ( mouse ) This paper EMDB : EMD - 41315 Cryo - EM map of the 16 nm - repeating B - tubule ( mouse ) This paper EMDB : EMD - 41316 Cryo - EM map of the 48 nm - repeating doublets ( mouse ) , the composite map This paper EMDB : EMD - 41431 Cryo - EM map of the 48 nm - repeating doublets ( mouse ) , submap 1 of EMDB : EMD - 41431 This paper EMDB : EMD - 41450 Cryo - EM map of the 48 nm - repeating doublets ( mouse ) , submap 2 of EMDB : EMD - 41431 This paper EMDB : EMD - 41451 Cryo - EM map of the 48 nm - repeating doublets ( human ) This paper EMDB : EMD - 41317 Cryo - EM map of the 48 nm - repeating doublets ( Tekt5 - / - ) This paper EMDB : EMD - 41320 Model of the mouse sperm doublets This paper PDB : 8TO0 PRIDE partner repository for MS data This paper PXD036885 R package source materials for MSstats from Krogan Lab This paper https : / / github . com / kroganlab Experimental models : Organisms / strains Mouse : C57BL / 6J The Jackson Laboratory https : / / www . jax . org / strain / 000664 Tekt5 - / - mouse This paper N / A Software and algorithms Prism v8 GraphPad https : / / www . graphpad . com / SerialEM 3 . 8 Mastronarde 48 https : / / bio3d . colorado . edu / SerialEM / Etomo Kremer et al . 49 https : / / bio3d . colorado . edu / imod / doc / UsingEtomo . html TOMOCTF Fernandez et al . 50 https : / / sites . google . com / site / 3demimageprocessing / tomoctf TOMO3D 2 . 0 Agulleiro and Fernandez 51 https : / / sites . google . com / site / 3demimageprocessing / tomo3d ( Continued on next page ) ll OPEN ACCESS e1 Cell 186 , 5041 \u2013 5053 . e1 \u2013 e6 , November 9 , 2023 Article RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to the lead contact , David A . Agard ( david @ agard . ucsf . edu ) . Materials availability Experimental reagents generated in this study are available from the lead contact with a completed material transfer agreement . Data and code availability Cryo - EM maps of 48 nm - repeating structures of doublets from wildtype mouse , Tekt5 - / - mouse and human sperm have been deposited in the Electron Microscopy Data Bank ( EMDB ) with accession codes : EMD - 41431 , EMD - 41320 and EMD - 41317 , respec - tively . The EMD - 41431 is a composite map with its two submaps deposited with accession codes : EMD - 41450 and EMD - 41451 . Maps of focused re\ufb01nement of 16 nm - repeating structures of A - and B - tubules from wildtype mouse have been deposited also : EMD - 41315 and EMD - 41316 . The atomic model of the 48 - nm repeat of the mouse sperm doublets has been deposited in the Protein Data Bank ( PDB ) with accession codes 8TO0 . MS data are shared and available through the ProteomeXchange Consortium via the PRIDE partner repository under the dataset identi\ufb01er : PXD036885 ( username : reviewer _ pxd036885 @ ebi . ac . uk ; password : tMEZ90MC ) . 56 R package source materials for MSstats ( version 3 ) are publicly available through the Krogan Lab GitHub : https : / / github . com / kroganlab . After downloading the AlphaFold2 library of the mouse proteome , this code is used to distribute PDB \ufb01les into subdirectories . i = 0 ; for f in * ; do # # Splitting 50 PDBs in each subdirectory d = dir _ $ ( printf % 03d $ ( ( i / 50 + 1 ) ) ) ; mkdir - p $ d ; mv \" $ f \" $ d ; let i + + ; done This code is used to unbiasedly match all PDBs with the target densities in each subdirectory : for \ufb01le in * do echo $ \ufb01le # # CCDC105 _ \ufb02ipped _ b150 . mrc is the target densities , the options could be found in the situs website colores . . / CCDC105 _ \ufb02ipped _ b150 . mrc $ { \ufb01le } - res 6 . 0 - cutoff 0 . 0048 - deg 15 . 0 mkdir . . / output / $ { \ufb01le } _ out mv col _ * . . / output / $ { \ufb01le } _ out / . mv Continued REAGENT or RESOURCE SOURCE IDENTIFIER Chimera Pettersen et al . 52 https : / / www . cgl . ucsf . edu / chimera / ChimeraX Goddard et al . 53 https : / / www . rbvi . ucsf . edu / chimerax / RELION - 4 . 0 Zivanov et al . 24 https : / / relion . readthedocs . io / en / release - 4 . 0 / AlphaFold2 Jumper et al . 27 https : / / alphafold . ebi . ac . uk / Situs Wriggers et al . 28 https : / / situs . biomachina . org / fguide . html Coot 0 . 9 . 8 . 1 Emsley et al . 54 http : / / www2 . mrc - lmb . cam . ac . uk / personal / pemsley / coot MaxQuant 1 . 6 . 3 . 3 Cox and Mann 37 https : / / www . maxquant . org / R Bioconductor package artMS 1 . 14 . 0 Jimenez - Morales et al . 55 https : / / doi . org / 10 . 18129 / B9 . bioc . artMS Other Quantifoil holey carbon grids ( R2 / 2 , 200 - mesh gold ) Quantifoil MicroTools GmbH https : / / www . quantifoil . com / products EM GP2 Automatic Plunge Freezer Leica Microsystems https : / / www . leica - microsystems . com / products ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 . e1 \u2013 e6 , November 9 , 2023 e2 Article done The cross - correlation scores could then be extracted using the following script : for f in * _ outt do echo $ f grep structure $ f / * . pdb > > TheResultFile grep Unnormalized $ f / * . pdb > > TheResultFile done grep \" correlation \" TheResultFile > JustCCResults The \ufb01nal output could then be sorted based on the cross - correlation scores in Excel . Note each PDB would be matched to the target densities with multiple orientations , resulting in multiple entries with the same PDB but different cross - correlation scores . The duplicate items for each PDB could be deleted in Excel . Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request . EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Mouse models Wild - type and transgenic male C57BL / 6J mice at ages of 10 to 16 weeks were used for this study . All mice were cared for in compli - ance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals . All experiments were approved by the Janelia Research Campus ( JRC ) IACUC . JRC is an AAALAC - accredited institution . Mice were maintained under SPF conditions . Human sample A man aged 25 - 39 years old was recruited and consented to participate in this study . We did not bias ancestry , race or ethnicity throughout the recruitment process . We only checked the samples under the microscope to make sure the sperm were normozoo - spermicwithacellcountofatleast30millionspermcellspermilliliter . Allexperimentalproceduresusinghuman - derivedsampleswere approved by the Committee on Human Research at the University of California , Berkeley , under IRB protocol number 2013 - 06 - 5395 . METHOD DETAILS Sample preparation Mouse sperm were collected from 10 to 16 - week - old C57Bl / 6J mice based on the published protocol . 19 , 57 Brie\ufb02y , the sperm were extracted from vasa deferentia by applying pressure to cauda epididymides in 1x Krebs buffer ( 1 . 2 mM KH 2 PO 4 , 120 mM NaCl , 1 . 2 mM MgSO 4 , 7H 2 O , 14 mM dextrose , 1 . 2 mM CaCl 2 , 5 mM KCl , 25 mM NaHCO 3 ) . The sperm were washed and resuspended in (cid:4) 100 m L Krebs buffer for the following experiments . For human sperm samples , freshly ejaculated semen samples were obtained by masturbation . Grid preparation EM grids ( Quantifoil R 2 / 2 Au 200 mesh ) were glow discharged to be hydrophilic using an easiGlow system ( Pelco ) . The grid was then loaded onto a Leica GP2 plunge freezer ( pre - equilibrated to 95 % relative humidity at 25 (cid:3) C ) . The mouse sperm suspension was then mixed with 10 nm gold beads ( Electron Microscopy Science , cat # 25487 ) to achieve \ufb01nal concentrations at 2 - 6 million cells / mL . EHNA ( erythro - 9 - ( 2 - hydroxy - 3 - nonyl ) adenine ) ( Santa Cruz Biotechnology , CAS 51350 - 19 - 7 ) was added to a \ufb01nal concentration of 10 mM . Next , 3 . 5 m L of the sperm mixture was loaded onto each grid , followed by a 15 - second incubation period . The grids were then blotted for 4 sec and plunge - frozen in liquid ethane . Cryogenic focused ion beam ( cryoFIB ) milling CryoFIB was performed using an Aquilos II cryo - FIB / SEM microscope ( Thermo Fisher Scienti\ufb01c ) . A panorama SEM map of the whole grid was \ufb01rst taken at 377x magni\ufb01cation using an acceleration voltage of 5 kV with a beam current of 13 pA , and a dwell time of 1 m s . Targets with appropriate thickness for milling were selected on the grid . A platinum layer ( (cid:4) 10 nm ) was sputter coated and a gas injection system ( GIS ) was used to deposit the precursor compound trimethyl ( methylcyclopentadienyl ) platinum ( IV ) . The stage was tilted to 15 - 20 (cid:3) , corresponding to a milling angle of 8 - 13 (cid:3) relative to the plane of grids . FIB milling was performed using stepwise decreasing current as the lamellae became thinner ( 1 . 0 nA to 30 pA , \ufb01nal thickness : (cid:4) 300 nm ) . The grids were then stored in liquid nitrogen before data collection . Image acquisition and tomogram reconstruction Tilt series of mouse sperm were collected on a 300 - kV Titan Krios transmission electron microscope ( Thermo Fisher Scienti\ufb01c ) equip - ped with a high brightness \ufb01eld emission gun ( xFEG ) , a spherical aberration corrector , a Bioquantum energy \ufb01lter ( Gatan ) , and a K3 ll OPEN ACCESS e3 Cell 186 , 5041 \u2013 5053 . e1 \u2013 e6 , November 9 , 2023 Article Summit detector ( Gatan ) . The images were recorded at a nominal magni\ufb01cation of 26 , 000x in super - resolution counting mode using SerialEM . 48 After binning over 2 x 2 pixels , the calibrated pixel size was 2 . 612 A\u02da on the specimen level . For each tilt series , images were acquired using a modi\ufb01ed dose - symmetric scheme between - 48 (cid:3) and 48 (cid:3) relative to the lamella with 4 (cid:3) increments and grouping of two images on either side ( 0 (cid:3) , 4 (cid:3) , 8 (cid:3) , - 4 (cid:3) , - 8 (cid:3) , 12 (cid:3) , 16 (cid:3) , - 12 (cid:3) , - 16 (cid:3) , 20 (cid:3) . ) . 58 At each tilt angle , the image was recorded as movies divided into fourteen subframes . The total electron dose applied to a tilt series was 100 e - / A\u02da 2 . The defocus target was set to be - 2 to - 5 m m . All movie frames were corrected with a gain reference collected in the same EM session . Movement between frames was corrected using MotionCor2 without dose weighting . 59 All tilt series were aligned using AreTomo and the tomograms were inspected to identify good tilt series . 41 Tilt series with crystalline ice and big ice blocks , or possessing less than \ufb01ve doublets of the axonemes were dis - carded . Alignment of the good tilt series was then performed in Etomo using the gold beads as \ufb01ducial markers . 49 The AreTomo is less labor - intensive for screening purposes , while the Etomo work\ufb02ow allowed us to achieve high - resolution reconstructions . The aligned tilt series were then CTF - corrected using TOMOCTF 50 and the tomograms were generated using TOMO3D 51 ( bin4 , pixel size : 10 . 448 A\u02da ) . In total , we started with eight milling grids of mouse sperm and obtained 159 lamellae . Ultimately , the \ufb01nal reconstruc - tions of the consensus averages were based on 77 usable tomograms . Subvolume averaging Subvolume extraction , classi\ufb01cation and re\ufb01nement were \ufb01rst performed using RELION3 as reported previously . 60 Brie\ufb02y , subvo - lumes from the doublets were manually picked every 24 nm and extracted at binning of 6 ( pixel size : 15 . 672 A\u02da , box size : 80 pixels , dimension : 125 . 376 nm ) . These subvolumes were aligned to a map of non - treated mouse sperm doublet structure ( EMDB : EMD - 27444 ) lowpass \ufb01ltered to 80 A\u02da and the resulting map was used as the reference for further processing . Supervised 3D classi\ufb01cation on radial spokes gave rise to four class averages of the 96 - nm repeating units at four different registers . All four class averages were recentered at the base of Radial spoke 2 and re - extracted at the same point at the binning of 4 ( pixel size : 10 . 448 A\u02da , box size : 120 pixels , dimension : 125 . 376 nm ) . All subvolumes were combined and aligned to one reference and duplicate subvolumes were removed based on minimum distance ( < 40 nm ) . The remaining subvolumes were aligned to yield the consensus average for all nine doublets . Subvolumes of the 96 - nm repeating units were recentered on MIP features that repeat every 48 nm or 16 nm to obtain the coordinates of these subvolumes . The tomograms and coordinates were imported in RELION4 without binning . 24 Pseudo - sub - tomograms were extracted ( pixel size : 2 . 612 A\u02da , box size : 220 pixels , dimension : 57 . 464 nm ) and the \ufb01rst round of Re\ufb01ne3D jobs yield the initial reference for the following re\ufb01nement of geometric and optical parameters of the tilt series . TomoFrameAlign and CtfRe\ufb01neTomo jobs were executed alternatively for two rounds and new pseudo - subtomograms were extracted ( see FSC curves in Figure S2A ) . The same \u2018\u2018Re\ufb01ne3D - TomoFrameAlign - CtfRe\ufb01neTomo - TomoFrameAlign - CtfRe\ufb01neTomo\u2019\u2019 process was repeated again and new pseudo - subtomograms were extracted . The \ufb01nal Re\ufb01ne3D job yields the reported maps after three iterations . Further re\ufb01nement did not improve the resolutions and quality of maps . In order to generate a map covering the entire 48 nm - repeating struc - ture of the doublets , the run _ data . star \ufb01le from the \ufb01nal Re\ufb01ne3D job was shifted along the longitudinal axis and another round of re\ufb01nement yielded averages with the shifted register . These reconstructions were aligned to the reported 48 nm repeating structure of doublets from bovine trachea cilia and a composite map was generated to match the register of the periodic structure . The resolutions of the maps were estimated based on the FSC of two independently re\ufb01ned half datasets ( FSC = 0 . 143 ) . Local resolution maps for doublets of both human and mouse sperm were calculated by RELION4 and displayed in UCSF Chimera . 52 These local - resolution maps represent relative differences in resolution across the maps but the absolute values may not be precise . IMOD was used to visualize the tomographic slices . 49 UCSF Chimera was used to manually segment the maps for various structural fea - tures and these maps were colored individually to prepare the \ufb01gures using UCSF ChimeraX . 52 , 53 , 61 Model building and unbiased matching of density maps to protein candidates Model building was performed in Coot v0 . 9 . 8 . 1 54 and rigid body \ufb01tting was achieved using UCSF Chimera . The interpretation of the mouse sperm doublet map started with the atomic model of the bovine trachea doublet ( PDB : 7RRO ) . 13 Densities matching tubulins and 29 bovine MIPs in the bovine trachea doublet were found in the mouse sperm doublet map so all of these densities were consid - ered to be formed by M . musculus orthologs ( Figure S4 ) . We cannot exclude the possibilities that they are formed by sperm - speci\ufb01c homologs with similar tertiary structures . These orthologs were identi\ufb01ed using UniProt 62 or the NCBI protein database 63 based on the sequences of bovine proteins . The atomic models of bovine trachea MIPs were mutated to match the sequence of the mouse proteins using the Chainsaw plugin in Coot . The resulting models of individual proteins were then \ufb01t into the mouse sperm doublet map as rigid bodies in Chimera . MIP densities that are unique in sperm doublets were segmented from the corresponding maps using UCSF Chimera manually . At 6 - 8 A \u02da resolutions , a - helix is well - resolved and b - strands appear as curved sheets and we focused on the unassigned densities with well - de\ufb01ned tertiary structures or domains ( as the various colored densities shown in Figure 1 ) . Meanwhile , the PDB library of 21 , 615 mouse proteins based on AlphaFold2 prediction was downloaded . 27 The unbiased matching was carried out using the COLORES program ( Situs package , see the Data and code availability section in the STAR Methods ) . 28 , 64 The matching was scored and ranked by the cross - correlation scores and the top 200 hits were inspected individually with the target densities in UCSF Chimera . We noticed that matching of densities to much larger PDBs could lead to unrealistic cross correlations ( > 1 ) . Setting the box sizes of the maps to be two or three times larger compared to the isolated densities does not solve the issue since COLORES would cut ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 . e1 \u2013 e6 , November 9 , 2023 e4 Article off the zero valued edges by default to reduce computational loads . We thus used \u2018\u2018voledit\u2019\u2019 command from SITUS to edit the voxels at the corner of the cubic map . Speci\ufb01cally , we edited the \ufb01rst and the last values in the sit \ufb01le to be slightly larger than the threshold values to avoid the cropping . To test the unbiased proteome - wide search approach , we used densities corresponding to known MIPs identi\ufb01ed in bovine tracheal doublets , including PACRG , CFAP20 and NME7 as controls for the method . Indeed , PDB models cor - responding to the respective mouse orthologs were identi\ufb01ed as the top hits ( Data S1 ) , suggesting this visual proteomics approach can identify protein with matching tertiary structures . We then applied this method to identify candidates for the mouse sperm - spe - ci\ufb01c densities ( Data S2 \u2013 S4 ) . For the 4 - helix bundle densities , the CATH library , which curated non - redundant PDBs of published structural domains , 36 was also used and no homologous proteins of SPACA9 were found . The AlphaFold2 predicted PDBs were then used as starting models and initial \ufb01tting pose discovered by COLORES were inspected in Chimera . The various Tektin 5 and CCDC105 models were built in Coot to match the corresponding densities . Unresolved loops were deleted . We observed densities corresponding to 24 copies of SPACA9 for the 48 nm - repeating units of the doublet microtu - bules , albeit with varied occupancies . We rigid - body \ufb01tted all 24 SPACA9 in these densities in Coot to re\ufb02ect the stacking oligomer - ization in the deposited model . We also performed rigid - body \ufb01tting of DUSP3 ( although other DUSP proteins could be \ufb01tted too ) into the globular densities next to the slanted Tektin 5s . These models were combined in ChimeraX and all side chains were stripped using the phenix . pdbtools command . Sequence alignment and search for homologous proteins Sequence alignment was performed using Clustal Omega 33 server and displayed in Jalview . 65 M . musculus Tektin 1 sequence was used as input to search for Tektin homologs using the HHpred server . 66 Biochemical extractions of mouse sperm For each of the three biological replicates , sperm from two mice were washed with PBS and pelleted at 2000x g for 5 min . Then , the E1 - E5 buffers were used to extract proteins from the pellets [ 0 . 1 % Triton in PBS ( E1 ) , 0 . 6 M NaCl in PBS ( E2 ) , 0 . 6 M KCSN in PBS ( E3 ) , 8 M urea ( E4 ) and 10 % SDS ( E5 ) ] . For E1 to E4 , 100 m L of the buffer was added to the pellets and the resuspension was mixed by pipetting up and down using a p200 pipette . Then the solution was incubated at room temperature for 10 min and the pellet was spun down at 21 , 000x g for 10 min . For E5 , after 10 % SDS was added and mixed , the resuspension was heated at 95 (cid:3) for 5 min . After the pellets were spun down , the supernatant was taken as the extraction . 20 m L and 2 m L of the extractions were used for SDS - PAGE analyses , either stained with AcquaStain ( Fisher Scienti\ufb01c , NCO170988 ) and blotted with an antibody against a - tubulins ( ThermoFisher Scienti\ufb01c , DM1A , # 62204 ) . The remaining extractions were used for mass spectrometry analysis . Mass spectrometry ( MS ) - based global protein abundance of mouse sperm Proteins in biochemical fractions E1 , E2 , E3 , E4 and E5 from three biological replicates were reduced and alkylated in 4 mM \ufb01nal concentration tris ( 2 - carboxyethyl ) phosphine ( TCEP ) and 10 mM \ufb01nal concentration iodoacetamide by 20 - minute incubation in the dark , after which excess iodoacetamide was quenched with 10 mM \ufb01nal concentration dithiothreitol ( DTT ) . Proteins were then subjected to methanol chloroform precipitation . Brie\ufb02y , 1 part sample was combined and vortexed sequentially with 4 parts meth - anol , 1 part chloroform , and 3 parts water for phase separation , after which samples were spun for 2 minutes at top speed ( 14 , 000 g ) in a bench - top centrifuge ( Centrifuge 5424R , Eppendorf ) . The upper phase was removed and discarded , and 4 parts methanol were combined and vortexed with the interphase and lower phase and subsequently centrifuged for 3 minutes at 14 , 000 g . The superna - tant was removed and discarded , and the pellet was washed three times in 80 % ice - cold acetone followed by centrifugation for 3 minutes at 14 , 000 g . Extracted proteins were air dried , resuspended in 8 M urea buffer ( 8 M urea , 150 mM NaCl , 50 mM NH 4 HCO 3 , cOmplete Mini EDTA - free protease inhibitor ( Roche , 11836170001 ) ) , and quanti\ufb01ed using Bradford reagent ( Sigma , B6916 ) following Coomassie ( Bradford ) Protein Assay Kit\u2019s protocol ( Thermo Fisher , 23200 ) . Following quanti\ufb01cation , protein sam - ples were diluted 4 - fold to 2 M urea concentration with 0 . 1 M NH 4 HCO 3 pH 8 , digested with trypsin ( Promega , V5111 ) at a protea - se : protein ratio of 1 : 100 ( weight / weight ) , and incubated overnight at 37 (cid:3) C in a thermomixer at 750 rpm . After tryptic digest , samples were acidi\ufb01ed to pH < 3 with 1 % \ufb01nal concentration formic acid , and desalted for MS analysis using HPLC - grade reagents and 100 m L OMIX C18 tips ( Agilent Technologies , A57003100 ) according to the manufacturer\u2019s protocol with the following adjustments . Brie\ufb02y , OMIX tips were conditioned by sequential washes of 100 % acetonitrile and 50 % acetonitrile , 0 . 1 % formic acid , and equilibrated with two washes of 0 . 1 % formic acid . Peptides were bound to the C18 polymer by repeated pipetting , subsequently washed three times with 0 . 1 % formic acid , and sequentially eluted in 50 % acetonitrile , 0 . 1 % formic acid followed by 90 % acetonitrile , 0 . 1 % formic acid . Peptides were dried by vacuum centrifugation ( CentriVap Cold Trap , Labconco ) and stored at - 80 (cid:3) C until MS analysis . Digested , desalted peptides were resuspended to 0 . 125 - 2 m g / m L \ufb01nal concentration in 2 % acetonitrile , 0 . 1 % formic acid . 1 - 2 m L were injected in technical singlet onto an Easy - nLC 1200 ( Thermo Fisher Scienti\ufb01c ) interfaced via a nanoelectrospray source ( Nano - spray Flex ) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer ( Thermo Fisher Scienti\ufb01c ) . Peptides were separated on a PepSep reverse - phase C18 column ( 1 . 9 m m particles , 1 . 5 m m x 15 cm , 150 m m ID ) ( Bruker ) with a gradient of 5 - 88 % buffer B ( 0 . 1 % formic acid in acetonitrile ) over buffer A ( 0 . 1 % formic acid in water ) over a 100 - minute data acquisition . Spectra were acquired contin - uously in a data - dependent manner . One full scan in the Orbitrap ( scan range 350 - 1350 m / z at 120 , 000 resolution in pro\ufb01le mode with a custom AGC target and maximum injection time of 50 milliseconds ) was followed by as many MS / MS scans as could be acquired ll OPEN ACCESS e5 Cell 186 , 5041 \u2013 5053 . e1 \u2013 e6 , November 9 , 2023 Article on the most abundant ions in 2 seconds in the dual linear ion trap ( rapid scan type with \ufb01xed HCD collision energy of 32 % , custom AGC target , maximum injection time of 50 milliseconds , and isolation window of 0 . 7 m / z ) . Singly and unassigned charge states were rejected . Dynamic exclusion was enabled with a repeat count of 1 , an exclusion duration of 25 seconds , and an exclusion mass width of \u00b1 10 ppm . Liquid chromatography 67 and MS acquisition parameters are reported in ( Table S1 ) . Raw MS \ufb01les were searched using MaxQuant ( version 1 . 6 . 3 . 3 ) against a database of the mouse proteome ( SwissProt Mus mus - culus reviewed protein sequences , downloaded 07 May 2022 ) with a manual addition to include mouse piercer of microtubule wall 2 protein ( protein sequence from NCBI Reference Sequence NP _ 001185718 . 1 , manually assigned the UniProt identi\ufb01er \u2018\u2018ZCC15orf65\u2019\u2019 in our database after its bovine homolog ) . 37 MaxQuant settings were left at default , with the following exceptions : LFQ was enabled with skip normalization enabled ; and match between runs was enabled with a 1 . 5 - minute matching time window and 20 - minute align - ment window . Trypsin ( KR | P ) was selected and allowed up to two missed cleavages , and variable and \ufb01xed modi\ufb01cations were as - signed for protein acetylation ( N - terminal ) , methionine oxidation and carbamidomethylation . Statistical analysis of protein quantitation was completed with R Bioconductor package artMS ( version 1 . 14 . 0 ) 55 and its function artmsQuanti\ufb01cation , which is a wrapper around the R Bioconductor package Mass Spectrometry Statistics and Quanti\ufb01cation ( MSstats ) ( version 4 . 4 . 0 ) as follows 38 ( Table S2 ) . Peptide intensities from the MaxQuant evidence \ufb01le were summarized to protein intensities using the MSstats function dataProcess with default settings . The differences in log 2 - transformed intensity between biochemical fractions were scored using the MSstats function groupComparison , which \ufb01ts a single linear model for each protein with a single categorical variable for condition , or fraction in our case . From these models , MSstats reports pairwise differences in means between conditions as log 2 fold change ( log2FC ) with a p - value based on a t - test assuming equal variance across all con - ditions , and reports adjusted p - values using the false discovery rate ( FDR ) estimated by the Benjamini - Hochberg procedure . Proteins with signi\ufb01cant changes in abundance between fractions were de\ufb01ned as : ( 1 ) absolute ( log 2 FC ) > 1 ; and ( 2 ) adjusted p - value < 0 . 05 . Proteins with signi\ufb01cant changes in abundance were tested for enrichment of Gene Ontology terms ( Table S3 ) . The over - represen - tation analysis was performed using the enricher function from R package clusterPro\ufb01ler ( version 4 . 4 . 1 ) . 39 Gene Ontology ( GO Bio - logical Process , Molecular Function and Cellular Component ) terms and annotations were obtained from the R annotation package org . Mm . eg . db ( version 3 . 15 . 0 ) . From among all signi\ufb01cantly enriched terms , we selected a set of non - redundant terms following a clustering procedure . We \ufb01rst constructed a term tree based on distances ( 1 - Jaccard Similarity Coef\ufb01cients of shared genes in KEGG or GO ) between the signi\ufb01cant terms . The term tree was cut at a speci\ufb01c level ( h = 0 . 99 ) to identify clusters of non - redundant gene sets ( Table S4 ) . For results with multiple signi\ufb01cant terms belonging to the same cluster , we selected the most signi\ufb01cant ( lowest adjusted p - value ) term . Generation of Tektin - 5 knockout mice and functional / structural analyses of mutant sperm To create an easily detected frameshift mutation in the Tekt5 gene , we used two gRNAs located in exon 1 of the gene . There is a pseudogene located on chromosome 19 with 85 . 8 % homology to the Tektin5 coding region but has a truncated C termini so that the 2 - helix segment is incomplete . Three gRNAs were carefully selected to avoid cutting the pseudogene . They are gRNA1 ( CGCTGGGTCTCCACGCGTTCAGG ) , gRNA2 ( AGTTTCTGTGGCCCCAAGAAAGG ) , and gRNA3 ( CCGAGGAATGCTCAGGC ATCCGG ) . The gRNAs were in vitro transcribed using the MEGA shortscript T7 kit ( Life Tech Corp AM1354 ) . Two combinations of the gRNAs were used : gRNA1 + gRNA2 , and gRNA2 + gRNA3 . The gRNAs and Cas9 protein ( Invitrogen True - cut Cas9 protein V2 , cat # A36498 ) with a concentration of 125 ng each were co - electroporated into 1 - cell C57Bl / 6J embryos using a BEX Genome Editor . A total of 75 pups were weaned and genotyped by PCR . Thirty - nine of them with an obviously shorter PCR band were sequenced , and nine of them with a frameshift deletion were selected for germline testing . Germline transmission was found in 7 out of 9 , and 2 lines were selected for further breeding : line 6 with a 166 bp deletion and line 7 with a 181 bp deletion . Both lines were then mated with wild - type females to generate F1 animals . Mating of heterozygous F1 male and female led to F2 homozygotes , which were further con\ufb01rmed by sequencing . Sperm motility was recorded at 37 (cid:3) C on a Hamilton Thorne IVOS II CASA machine using a Zeiss 10x NH objective , at a frame rate of 60 Hz , in the presence of 1 % polyvinyl alcohol to prevent cell adhesion to glass . The motility and sperm morphologies were inspected and counted manually . The structural analyses of the mutants were performed using the same work\ufb02ow described for wild - type sperm , except omitting the EHNA treatment . Sperm from two different Tektin5 - knockout lines were processed , imaged and analyzed independently . The two independent reconstructions show low occupancies of speci\ufb01c Tektin 5 densities and only the higher - resolution reconstruction is shown . QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses of Tekt5 - / - mouse were prepared using Prism9 ( GraphPad ) . We performed six mating trials by using six Tekt5 - / - males ( 3 from each line of two lines ) and six wildtype females . The average and standard deviation are presented ( 7 . 3 \u00b1 1 . 4 , n = 6 ) . For the sperm analyses , we counted enough videos so the number of sperm from each mouse is > 200 . Sperm from three Tekt5 - / - knockout mutants and two wild - type mice were analyzed functionally . ll OPEN ACCESS Cell 186 , 5041 \u2013 5053 . e1 \u2013 e6 , November 9 , 2023 e6 Article Supplemental \ufb01gures ( legend on next page ) ll OPEN ACCESS Article Figure S1 . Work\ufb02ow of data processing , related to Figures 1 , 2 , and 3 and STAR Methods ( A ) Tilt series composed of 25 2D projections were recorded ( scale bar : 100 nm ) . The image shows the midpiece of sperm \ufb02agella that contains mitochondria around the axoneme . Gold beads on the tilt images are indicated ( green arrowhead ) . The \ufb01ducial gold beads were used to align the tilt images . ( B ) 3D tomograms were reconstructed , and subvolumes were picked along the microtubules ( scale bar : 100 nm ) . ( C ) 3Dclassi\ufb01cationandre\ufb01nementwereperformedtoalignandaveragethesubtomogramforthe96nm - repeatingstructuresofthemousespermdoublets . Four views of the 96 nm - repeating structure of doublets from EHNA - treated sperm are shown for the 3D reconstruction generated using RELION3 as reported previously . 19 Gold - standardFouriershellcorrelation ( FSC ) curvecalculatedbetweenhalfmapsofmousespermdoubletsisshown . Theresolutionwasestimated as 26 A\u02da ( FSC = 0 . 143 ) . ( D ) Two slices of the 96 nm - repeating structure of doublets looking along and perpendicular to the \ufb01lament axis . Note the red line in the top panel indicates the planeofthebottomslice , andperiodicstructuresareobservedinsidethemicrotubules . Thecoordinateswererecenteredonthe48 - nmrepeatsandimportedinto RELION4 . In the top panel , note that the features further away from the microtubules are blurrier , suggesting that there are conformational heterogeneities and that they are resolved at lower resolutions . ( E ) The initial Re\ufb01ne3D job of the 48 nm - repeating structures was performed using RELION4 . 24 ( F ) The 3D reconstructions were matched to the 2D projections of individual particles in the raw tilt images , and this step re\ufb01ned both the geometric and optical parameters of the tilt series ( i \u2013 iii ) . ( G ) Another round of subtomogram averaging was performed based on re\ufb01ned tilt series . No additional improvement was observed after 3 rounds of re\ufb01nement and Re\ufb01ne3D as shown in ( F ) \u2013 ( G ) . ll OPEN ACCESS Article ( legend on next page ) ll OPEN ACCESS Article Figure S2 . Characterization of the 48 nm - repeating structure of doublets from mouse sperm , related to Figures 1 , 2 , and 3 ( A ) Gold - standard Fourier shell correlation ( FSC ) curves were calculated between half maps of mouse sperm doublets . The resolutions were reported as FSC = 0 . 143 . Note the FSC curves resulting from the iterative frame alignment and CTF re\ufb01nement between the second and third Re\ufb01ne3D jobs were not shown for the clarity of the \ufb01gure . Further re\ufb01nement after the third Re\ufb01ne3D did not improve the resolution or the quality of the map . ( B ) The local resolution map of mouse sperm doublets was calculated by RELION4 . The ribbon region has the highest resolutions . Densities in the A - tubule have higher resolutions than the ones from the B - tubule . ( C ) Equivalentlongitudinalcross - sectionviewsofdoubletsfrommousespermandbovinetracheacilia ( EMDB : EMD - 24664 ) areshown . 13 Thelatterwaslow - pass \ufb01ltered to 7 . 5 A\u02da , and comparable details of the secondary and tertiary structures of the MIPs are observed . ( D ) The reconstruction of mouse sperm doublet ( gray ) is overlaid with the bovine trachea doublets ( yellow ) . The mouse sperm - speci\ufb01c densities are highlighted ( dashedovals ) . ThebrokenhelicalbundlesandthecurvedhelicalbundlesinsidetheA - tubuleofmousespermdoubletsalongthemicrotubuleaxisareshown . The discontinuous parts of the broken helical bundles are indicated ( dashed rectangles ) . Note that the curved bundles have one straight and two curved groups of densities in every 48 - nm repeat ( outlined using dashed shapes ) . ll OPEN ACCESS Article ( legend on next page ) ll OPEN ACCESS Article Figure S3 . Characterization of the 48 nm - repeating structure of doublets from human sperm , related to Figure 1 ( A ) The gold - standard Fourier shell correlation ( FSC ) curve was calculated between half maps of mouse sperm doublets . The resolution was estimated as 10 . 3 A\u02da ( FSC = 0 . 143 ) . ( B ) The local resolution map of human sperm doublets was calculated by RELION4 . The ribbon region has the highest resolutions . Densities in the A - tubule have higher resolutions than the ones from the B - tubule . ( C ) Equivalent views of doublets from human sperm and bovine trachea cilia ( EMDB : EMD - 24664 ) are shown . 13 The latter was low - pass \ufb01ltered to 10 A\u02da , and comparable details of the secondary and tertiary structures of the MIPs are observed . ( D ) The reconstruction of human sperm doublet ( blue ) is overlaid with the bovine trachea doublets ( yellow ) at low and high thresholds . ( E ) The two broken bundles inside the A - tubule in human sperm are shown at a low threshold ( see the corresponding mouse densities in Figure S2D ) . ( F ) ThecurvedhelicalbundlescontainonestraightandtwocurvedgroupsofdensitiesinsidetheA - tubuleofhumansperm . Humansperm - speci\ufb01cdensitieswere observed to connect one curved bundle to the lumen of A - tubule . ( G ) The human sperm doublets overlaid with mouse sperm doublets are shown . The inconsistent densities are outlined ( dashed line ) ( also see Figures S2D and S3F ) . ll OPEN ACCESS Article ( legend on next page ) ll OPEN ACCESS Article FigureS4 . Rigid - body\ufb01ttingof29identi\ufb01ed MIPsfrombovinetrachea ciliaintothedensitymapofmousespermdoublet , relatedtoFigures2 and 3 and STAR Methods ( A \u2013 F ) Modelsof29knownMIPsfrombovinetrachea cilia ( PDB : 7RRO ) 13 are\ufb01ttedintothedensitymapofmousespermdoublet . Theviewing anglesforallpanels are shown . For proteins that have multiple a - helices ( CFAP161 , RIBC2 , CFAP53 , MNS1 , CFAP21 , NME7 , CFAP141 , EFHC1 , EFHC2 , ENKUR , CFAP210 , EFCAB6 , CFAP45 , PACRG , andTEKTIN1 \u2013 4 ) , thearrangementofsecondarystructuresmatchesdensitiesinspermdoublets . Theoverallshapesof b - sheetsin b - sheet - richproteins ( CFAP52 and CFAP20 ) matchthedensities , andtheseproteinsarehighly conservedinaxonemes . Fortheproteins thatcontainrandom coils , we did observe matching features in the maps and were able to trace some of the main chains at the current resolution ( CFAP95 , SPAG8 , CFAP107 , FAM166B , Pierce1 , Pierce2 , CFAP126 , CFAP276 , and TEKTIP1 ) . ll OPEN ACCESS Article Figure S5 . Characterization of the 16 nm - repeating structures of doublets from mouse sperm , related to Figure 2 ( A and B ) Gold - standard Fourier shell correlation ( FSC ) curves were calculated using half maps of 16 nm - repeating structures of A - tubule and B - tubule . The resolution was estimated as 6 . 0 and 6 . 7 A\u02da , respectively ( FSC = 0 . 143 ) . The Nyquist limit is 5 . 26 A\u02da . ( C \u2013 E ) The local resolution map was calculated from the two half maps of 16 nm - repeating structures of A - tubule using RELION4 . The viewing angles for ( D ) and ( E ) are shown in ( C ) ( black arrow ) . These viewing angles are similar to Figures 1A , 1D , and 1E , respectively . ( F \u2013 H ) The local resolution map was calculated using the two half maps of 16 nm - repeating structures of B - tubule using RELION4 . The viewing angles for ( G ) and ( H ) are shown in ( F ) ( black arrow ) . The viewing angles of ( F ) and ( G ) are similar to Figures 1A and 1F , respectively . ll OPEN ACCESS Article ( legend on next page ) ll OPEN ACCESS Article Figure S6 . Tektin 5 and CCDC105 likely form sperm - speci\ufb01c 3 - helix bundles associated with the A - tubule , related to Figures 2 and 3 ( A ) After unbiased matching , Tektin 5 was scored as the # 5 hit of the predicted structures out of 21 , 615 proteins from the mouse proteome , ranked by cross - correlation scores ( the top 10 are shown ) . Tektin 1 \u2013 4 were ranked at # 7 \u2013 10 due to their similar tertiary structures . ( B ) Typicalfalsepositives ( # 1 \u2013 4and # 6 ) fromthesamesearch . Usually , theseareproteinswithlongsinglehelicesthatdonotmatchthegapsobservedinthemap . Also , they do not explain the 3 - helix bundles . The \ufb01tting of Tektin 5 into the same densities is shown for comparison . ( C ) ThestructureofCCDC105directlypredictedbyAlphaFold2 ( left ) iscomparedwiththepredictedcomplexformedbytwoCCDC105molecules ( right ) . Thefull - length CCDC105 molecule in the complex is colored based on the per - residue con\ufb01dence scores ( predicted local distance difference test [ pLDDT ] , red : high con\ufb01dence ) from the AlphaFold2 prediction . The three P - loops have medium con\ufb01dence scores ( green ) , suggesting the exact conformations of these loops may not be accurately predicted . However , the presence of these structured loops is conceivably con\ufb01dent based on the conserved proline residues ( see the sequence alignment in D ) and the matching protrusion densities observed in our maps ( Figure 2G ) . Note that the conformations of the three proline - rich loops differ in these two predictions . These differences could be caused by the presence of neighboring molecules during AlphaFold2 analyses . 27 ( D ) The sequence alignment of CCDC105 from \ufb01ve mammals ( H . sapiens , M . musculus , B . taurus , S . scrofa , and F . catus ) , zebra\ufb01sh ( D . rerio ) , and sea urchins ( S . purpuratus ) . The three proline - rich loops are marked above the sequences . ( E ) ThemodelsofCCDC105andTektin5are\ufb01ttedintothedensitiesofthe3 - helixbundleattheribbon , wheretheformermodelexplainstheextraprotrusionsand orientation / lengths of helices of the densities , but the latter does not . ll OPEN ACCESS Article Figure S7 . DUSP proteins in the A - tubule , related to Figure 3 ( A ) At a lower threshold compared with Figure 3C , densities connecting the N - terminal residues of the slanted Tektin 5s ( magenta models ) and the DUSPs ( blue models ) are observed . ( B ) The DUSP3 is \ufb01tted into the globular domain , and three orthogonal views are shown . Other homologous DUSP proteins \ufb01t well into the density because of similar tertiary structures ( DUSP 3 , 13 , 14 , 18 , 21 , and 29 ) . ll OPEN ACCESS Article Figure S8 . Biochemical extractions of proteins from mouse sperm , related to Figure 3 ( A ) SDS - PAGE analyses of protein extractions from mouse sperm using 0 . 1 % Triton in PBS ( E1 ) , 0 . 6 M NaCl in PBS ( E2 ) , 0 . 6 M KCSN in PBS ( E3 ) , 8 M urea ( E4 ) , and 10 % SDS ( E5 ) . ( B ) Westernblotanalysesofproteinextractionsfrommousespermusinganantibodyagainst a - tubulins . NotethatstrongbandsweredetectedonlyinE3andE4 , suggesting the microtubule structures were stable in Triton and high NaCl buffers and dissembled completely in KCSN / urea solutions . ( C ) Bar chart of the number of proteins identi\ufb01ed by MS ( protein count ) in each fraction ( E1 \u2013 E5 ) and biological replicate . We identi\ufb01ed a total of 1 , 677 mouse proteins , with a range of 772 to 1 , 326 proteins identi\ufb01ed in each individual fraction and replicate . ( D ) Heatmap of proteins with signi\ufb01cant changes between any two fractions ( absolute log 2 fold change [ log 2 FC ] > 1 , adjusted p value < 0 . 05 ) , listed by fractions ( E1 \u2013 E5 ) and biological replicate , and clustered by correlation of intensity pro\ufb01le . Proteins are colored by the log 2 FC in protein intensity normalized to the row median ( red , increased intensity ; blue , decreased intensity ; gray , not detected ) . Cluster identi\ufb01cation numbers ( cluster ID ) are labeled ( left ) . ( E ) Heatmapofgeneontology ( GO ) enrichmentsamongthesigni\ufb01cantlychangingproteinsidenti\ufb01edineachclusterfrom ( D ) ( lefttoright : clusterID1 \u2013 6 , aslabeled in D ) . GO terms were curated from the top 4 enrichment terms per cluster , and non - redundant terms were selected by an automated clustering procedure ( see STAR Methods ) . Increased shading re\ufb02ects increased signi\ufb01cance of the enrichment term . The number of proteins per enrichment term is shown in white if signi\ufb01cant ( adjusted p value < 0 . 05 ) and gray if not signi\ufb01cant ( adjusted p value > 0 . 05 ) . A bar chart plotting the number of total genes in each cluster ID is included . 68 ( F ) Log 2 protein intensities ( y axis ) for eight mouse proteins as quanti\ufb01ed by MS in each fraction ( E1 \u2013 E5 ) and biological replicate ( colored dots ; maximum n = 3 ) . ll OPEN ACCESS Article", + "mikolovEfficientEstimationWord2013": "Ef\ufb01cient Estimation of Word Representations in Vector Space Tomas Mikolov Google Inc . , Mountain View , CA tmikolov @ google . com Kai Chen Google Inc . , Mountain View , CA kaichen @ google . com Greg Corrado Google Inc . , Mountain View , CA gcorrado @ google . com Jeffrey Dean Google Inc . , Mountain View , CA jeff @ google . com Abstract We propose two novel model architectures for computing continuous vector repre - sentations of words from very large data sets . The quality of these representations is measured in a word similarity task , and the results are compared to the previ - ously best performing techniques based on different types of neural networks . We observe large improvements in accuracy at much lower computational cost , i . e . it takes less than a day to learn high quality word vectors from a 1 . 6 billion words data set . Furthermore , we show that these vectors provide state - of - the - art perfor - mance on our test set for measuring syntactic and semantic word similarities . 1 Introduction Many current NLP systems and techniques treat words as atomic units - there is no notion of similar - ity between words , as these are represented as indices in a vocabulary . This choice has several good reasons - simplicity , robustness and the observation that simple models trained on huge amounts of data outperform complex systems trained on less data . An example is the popular N - gram model used for statistical language modeling - today , it is possible to train N - grams on virtually all available data ( trillions of words [ 3 ] ) . However , the simple techniques are at their limits in many tasks . For example , the amount of relevant in - domain data for automatic speech recognition is limited - the performance is usually dominated by the size of high quality transcribed speech data ( often just millions of words ) . In machine translation , the existing corpora for many languages contain only a few billions of words or less . Thus , there are situations where simple scaling up of the basic techniques will not result in any signi\ufb01cant progress , and we have to focus on more advanced techniques . With progress of machine learning techniques in recent years , it has become possible to train more complex models on much larger data set , and they typically outperform the simple models . Probably the most successful concept is to use distributed representations of words [ 10 ] . For example , neural network based language models signi\ufb01cantly outperform N - gram models [ 1 , 27 , 17 ] . 1 . 1 Goals of the Paper The main goal of this paper is to introduce techniques that can be used for learning high - quality word vectors from huge data sets with billions of words , and with millions of words in the vocabulary . As far as we know , none of the previously proposed architectures has been successfully trained on more 1 a r X i v : 1301 . 3781v3 [ c s . C L ] 7 S e p 2013 than a few hundred of millions of words , with a modest dimensionality of the word vectors between 50 - 100 . We use recently proposed techniques for measuring the quality of the resulting vector representa - tions , with the expectation that not only will similar words tend to be close to each other , but that words can have multiple degrees of similarity [ 20 ] . This has been observed earlier in the context of in\ufb02ectional languages - for example , nouns can have multiple word endings , and if we search for similar words in a subspace of the original vector space , it is possible to \ufb01nd words that have similar endings [ 13 , 14 ] . Somewhat surprisingly , it was found that similarity of word representations goes beyond simple syntactic regularities . Using a word offset technique where simple algebraic operations are per - formed on the word vectors , it was shown for example that vector ( \u201dKing\u201d ) - vector ( \u201dMan\u201d ) + vec - tor ( \u201dWoman\u201d ) results in a vector that is closest to the vector representation of the word Queen [ 20 ] . In this paper , we try to maximize accuracy of these vector operations by developing new model architectures that preserve the linear regularities among words . We design a new comprehensive test set for measuring both syntactic and semantic regularities 1 , and show that many such regularities can be learned with high accuracy . Moreover , we discuss how training time and accuracy depends on the dimensionality of the word vectors and on the amount of the training data . 1 . 2 Previous Work Representation of words as continuous vectors has a long history [ 10 , 26 , 8 ] . A very popular model architecture for estimating neural network language model ( NNLM ) was proposed in [ 1 ] , where a feedforward neural network with a linear projection layer and a non - linear hidden layer was used to learn jointly the word vector representation and a statistical language model . This work has been followed by many others . Another interesting architecture of NNLM was presented in [ 13 , 14 ] , where the word vectors are \ufb01rst learned using neural network with a single hidden layer . The word vectors are then used to train the NNLM . Thus , the word vectors are learned even without constructing the full NNLM . In this work , we directly extend this architecture , and focus just on the \ufb01rst step where the word vectors are learned using a simple model . It was later shown that the word vectors can be used to signi\ufb01cantly improve and simplify many NLP applications [ 4 , 5 , 29 ] . Estimation of the word vectors itself was performed using different model architectures and trained on various corpora [ 4 , 29 , 23 , 19 , 9 ] , and some of the resulting word vectors were made available for future research and comparison 2 . However , as far as we know , these architectures were signi\ufb01cantly more computationally expensive for training than the one proposed in [ 13 ] , with the exception of certain version of log - bilinear model where diagonal weight matrices are used [ 23 ] . 2 Model Architectures Many different types of models were proposed for estimating continuous representations of words , including the well - known Latent Semantic Analysis ( LSA ) and Latent Dirichlet Allocation ( LDA ) . In this paper , we focus on distributed representations of words learned by neural networks , as it was previously shown that they perform signi\ufb01cantly better than LSA for preserving linear regularities among words [ 20 , 31 ] ; LDA moreover becomes computationally very expensive on large data sets . Similar to [ 18 ] , to compare different model architectures we de\ufb01ne \ufb01rst the computational complex - ity of a model as the number of parameters that need to be accessed to fully train the model . Next , we will try to maximize the accuracy , while minimizing the computational complexity . 1 The test set is available at www . fit . vutbr . cz / \u02dcimikolov / rnnlm / word - test . v1 . txt 2 http : / / ronan . collobert . com / senna / http : / / metaoptimize . com / projects / wordreprs / http : / / www . fit . vutbr . cz / \u02dcimikolov / rnnlm / http : / / ai . stanford . edu / \u02dc ehhuang / 2 For all the following models , the training complexity is proportional to O = E \u00d7 T \u00d7 Q , ( 1 ) where E is number of the training epochs , T is the number of the words in the training set and Q is de\ufb01ned further for each model architecture . Common choice is E = 3 \u2212 50 and T up to one billion . All models are trained using stochastic gradient descent and backpropagation [ 26 ] . 2 . 1 Feedforward Neural Net Language Model ( NNLM ) The probabilistic feedforward neural network language model has been proposed in [ 1 ] . It consists of input , projection , hidden and output layers . At the input layer , N previous words are encoded using 1 - of - V coding , where V is size of the vocabulary . The input layer is then projected to a projection layer P that has dimensionality N \u00d7 D , using a shared projection matrix . As only N inputs are active at any given time , composition of the projection layer is a relatively cheap operation . The NNLM architecture becomes complex for computation between the projection and the hidden layer , as values in the projection layer are dense . For a common choice of N = 10 , the size of the projection layer ( P ) might be 500 to 2000 , while the hidden layer size H is typically 500 to 1000 units . Moreover , the hidden layer is used to compute probability distribution over all the words in the vocabulary , resulting in an output layer with dimensionality V . Thus , the computational complexity per each training example is Q = N \u00d7 D + N \u00d7 D \u00d7 H + H \u00d7 V , ( 2 ) where the dominating term is H \u00d7 V . However , several practical solutions were proposed for avoiding it ; either using hierarchical versions of the softmax [ 25 , 23 , 18 ] , or avoiding normalized models completely by using models that are not normalized during training [ 4 , 9 ] . With binary tree representations of the vocabulary , the number of output units that need to be evaluated can go down to around log 2 ( V ) . Thus , most of the complexity is caused by the term N \u00d7 D \u00d7 H . In our models , we use hierarchical softmax where the vocabulary is represented as a Huffman binary tree . This follows previous observations that the frequency of words works well for obtaining classes in neural net language models [ 16 ] . Huffman trees assign short binary codes to frequent words , and this further reduces the number of output units that need to be evaluated : while balanced binary tree would require log 2 ( V ) outputs to be evaluated , the Huffman tree based hierarchical softmax requires only about log 2 ( Unigram perplexity ( V ) ) . For example when the vocabulary size is one million words , this results in about two times speedup in evaluation . While this is not crucial speedup for neural network LMs as the computational bottleneck is in the N \u00d7 D \u00d7 H term , we will later propose architectures that do not have hidden layers and thus depend heavily on the ef\ufb01ciency of the softmax normalization . 2 . 2 Recurrent Neural Net Language Model ( RNNLM ) Recurrent neural network based language model has been proposed to overcome certain limitations of the feedforward NNLM , such as the need to specify the context length ( the order of the model N ) , and because theoretically RNNs can ef\ufb01ciently represent more complex patterns than the shallow neural networks [ 15 , 2 ] . The RNN model does not have a projection layer ; only input , hidden and output layer . What is special for this type of model is the recurrent matrix that connects hidden layer to itself , using time - delayed connections . This allows the recurrent model to form some kind of short term memory , as information from the past can be represented by the hidden layer state that gets updated based on the current input and the state of the hidden layer in the previous time step . The complexity per training example of the RNN model is Q = H \u00d7 H + H \u00d7 V , ( 3 ) where the word representations D have the same dimensionality as the hidden layer H . Again , the term H \u00d7 V can be ef\ufb01ciently reduced to H \u00d7 log 2 ( V ) by using hierarchical softmax . Most of the complexity then comes from H \u00d7 H . 3 2 . 3 Parallel Training of Neural Networks To train models on huge data sets , we have implemented several models on top of a large - scale distributed framework called DistBelief [ 6 ] , including the feedforward NNLM and the new models proposed in this paper . The framework allows us to run multiple replicas of the same model in parallel , and each replica synchronizes its gradient updates through a centralized server that keeps all the parameters . For this parallel training , we use mini - batch asynchronous gradient descent with an adaptive learning rate procedure called Adagrad [ 7 ] . Under this framework , it is common to use one hundred or more model replicas , each using many CPU cores at different machines in a data center . 3 New Log - linear Models In this section , we propose two new model architectures for learning distributed representations of words that try to minimize computational complexity . The main observation from the previous section was that most of the complexity is caused by the non - linear hidden layer in the model . While this is what makes neural networks so attractive , we decided to explore simpler models that might not be able to represent the data as precisely as neural networks , but can possibly be trained on much more data ef\ufb01ciently . The new architectures directly follow those proposed in our earlier work [ 13 , 14 ] , where it was found that neural network language model can be successfully trained in two steps : \ufb01rst , continuous word vectors are learned using simple model , and then the N - gram NNLM is trained on top of these distributed representations of words . While there has been later substantial amount of work that focuses on learning word vectors , we consider the approach proposed in [ 13 ] to be the simplest one . Note that related models have been proposed also much earlier [ 26 , 8 ] . 3 . 1 Continuous Bag - of - Words Model The \ufb01rst proposed architecture is similar to the feedforward NNLM , where the non - linear hidden layer is removed and the projection layer is shared for all words ( not just the projection matrix ) ; thus , all words get projected into the same position ( their vectors are averaged ) . We call this archi - tecture a bag - of - words model as the order of words in the history does not in\ufb02uence the projection . Furthermore , we also use words from the future ; we have obtained the best performance on the task introduced in the next section by building a log - linear classi\ufb01er with four future and four history words at the input , where the training criterion is to correctly classify the current ( middle ) word . Training complexity is then Q = N \u00d7 D + D \u00d7 log 2 ( V ) . ( 4 ) We denote this model further as CBOW , as unlike standard bag - of - words model , it uses continuous distributed representation of the context . The model architecture is shown at Figure 1 . Note that the weight matrix between the input and the projection layer is shared for all word positions in the same way as in the NNLM . 3 . 2 Continuous Skip - gram Model The second architecture is similar to CBOW , but instead of predicting the current word based on the context , it tries to maximize classi\ufb01cation of a word based on another word in the same sentence . More precisely , we use each current word as an input to a log - linear classi\ufb01er with continuous projection layer , and predict words within a certain range before and after the current word . We found that increasing the range improves quality of the resulting word vectors , but it also increases the computational complexity . Since the more distant words are usually less related to the current word than those close to it , we give less weight to the distant words by sampling less from those words in our training examples . The training complexity of this architecture is proportional to Q = C \u00d7 ( D + D \u00d7 log 2 ( V ) ) , ( 5 ) where C is the maximum distance of the words . Thus , if we choose C = 5 , for each training word we will select randomly a number R in range < 1 ; C > , and then use R words from history and 4 w ( t - 2 ) w ( t + 1 ) w ( t - 1 ) w ( t + 2 ) w ( t ) SUM INPUT PROJECTION OUTPUT w ( t ) INPUT PROJECTION OUTPUT w ( t - 2 ) w ( t - 1 ) w ( t + 1 ) w ( t + 2 ) CBOW Skip - gram Figure 1 : New model architectures . The CBOW architecture predicts the current word based on the context , and the Skip - gram predicts surrounding words given the current word . R words from the future of the current word as correct labels . This will require us to do R \u00d7 2 word classi\ufb01cations , with the current word as input , and each of the R + R words as output . In the following experiments , we use C = 10 . 4 Results To compare the quality of different versions of word vectors , previous papers typically use a table showing example words and their most similar words , and understand them intuitively . Although it is easy to show that word France is similar to Italy and perhaps some other countries , it is much more challenging when subjecting those vectors in a more complex similarity task , as follows . We follow previous observation that there can be many different types of similarities between words , for example , word big is similar to bigger in the same sense that small is similar to smaller . Example of another type of relationship can be word pairs big - biggest and small - smallest [ 20 ] . We further denote two pairs of words with the same relationship as a question , as we can ask : \u201dWhat is the word that is similar to small in the same sense as biggest is similar to big ? \u201d Somewhat surprisingly , these questions can be answered by performing simple algebraic operations with the vector representation of words . To \ufb01nd a word that is similar to small in the same sense as biggest is similar to big , we can simply compute vector X = vector ( \u201d biggest \u201d ) \u2212 vector ( \u201d big \u201d ) + vector ( \u201d small \u201d ) . Then , we search in the vector space for the word closest to X measured by cosine distance , and use it as the answer to the question ( we discard the input question words during this search ) . When the word vectors are well trained , it is possible to \ufb01nd the correct answer ( word smallest ) using this method . Finally , we found that when we train high dimensional word vectors on a large amount of data , the resulting vectors can be used to answer very subtle semantic relationships between words , such as a city and the country it belongs to , e . g . France is to Paris as Germany is to Berlin . Word vectors with such semantic relationships could be used to improve many existing NLP applications , such as machine translation , information retrieval and question answering systems , and may enable other future applications yet to be invented . 5 Table 1 : Examples of \ufb01ve types of semantic and nine types of syntactic questions in the Semantic - Syntactic Word Relationship test set . Type of relationship Word Pair 1 Word Pair 2 Common capital city Athens Greece Oslo Norway All capital cities Astana Kazakhstan Harare Zimbabwe Currency Angola kwanza Iran rial City - in - state Chicago Illinois Stockton California Man - Woman brother sister grandson granddaughter Adjective to adverb apparent apparently rapid rapidly Opposite possibly impossibly ethical unethical Comparative great greater tough tougher Superlative easy easiest lucky luckiest Present Participle think thinking read reading Nationality adjective Switzerland Swiss Cambodia Cambodian Past tense walking walked swimming swam Plural nouns mouse mice dollar dollars Plural verbs work works speak speaks 4 . 1 Task Description To measure quality of the word vectors , we de\ufb01ne a comprehensive test set that contains \ufb01ve types of semantic questions , and nine types of syntactic questions . Two examples from each category are shown in Table 1 . Overall , there are 8869 semantic and 10675 syntactic questions . The questions in each category were created in two steps : \ufb01rst , a list of similar word pairs was created manually . Then , a large list of questions is formed by connecting two word pairs . For example , we made a list of 68 large American cities and the states they belong to , and formed about 2 . 5K questions by picking two word pairs at random . We have included in our test set only single token words , thus multi - word entities are not present ( such as New York ) . We evaluate the overall accuracy for all question types , and for each question type separately ( se - mantic , syntactic ) . Question is assumed to be correctly answered only if the closest word to the vector computed using the above method is exactly the same as the correct word in the question ; synonyms are thus counted as mistakes . This also means that reaching 100 % accuracy is likely to be impossible , as the current models do not have any input information about word morphology . However , we believe that usefulness of the word vectors for certain applications should be positively correlated with this accuracy metric . Further progress can be achieved by incorporating information about structure of words , especially for the syntactic questions . 4 . 2 Maximization of Accuracy We have used a Google News corpus for training the word vectors . This corpus contains about 6B tokens . We have restricted the vocabulary size to 1 million most frequent words . Clearly , we are facing time constrained optimization problem , as it can be expected that both using more data and higher dimensional word vectors will improve the accuracy . To estimate the best choice of model architecture for obtaining as good as possible results quickly , we have \ufb01rst evaluated models trained on subsets of the training data , with vocabulary restricted to the most frequent 30k words . The results using the CBOW architecture with different choice of word vector dimensionality and increasing amount of the training data are shown in Table 2 . It can be seen that after some point , adding more dimensions or adding more training data provides diminishing improvements . So , we have to increase both vector dimensionality and the amount of the training data together . While this observation might seem trivial , it must be noted that it is currently popular to train word vectors on relatively large amounts of data , but with insuf\ufb01cient size 6 Table 2 : Accuracy on subset of the Semantic - Syntactic Word Relationship test set , using word vectors from the CBOW architecture with limited vocabulary . Only questions containing words from the most frequent 30k words are used . Dimensionality / Training words 24M 49M 98M 196M 391M 783M 50 13 . 4 15 . 7 18 . 6 19 . 1 22 . 5 23 . 2 100 19 . 4 23 . 1 27 . 8 28 . 7 33 . 4 32 . 2 300 23 . 2 29 . 2 35 . 3 38 . 6 43 . 7 45 . 9 600 24 . 0 30 . 1 36 . 5 40 . 8 46 . 6 50 . 4 Table 3 : Comparison of architectures using models trained on the same data , with 640 - dimensional word vectors . The accuracies are reported on our Semantic - Syntactic Word Relationship test set , and on the syntactic relationship test set of [ 20 ] Model Semantic - Syntactic Word Relationship test set MSR Word Relatedness Architecture Semantic Accuracy [ % ] Syntactic Accuracy [ % ] Test Set [ 20 ] RNNLM 9 36 35 NNLM 23 53 47 CBOW 24 64 61 Skip - gram 55 59 56 ( such as 50 - 100 ) . Given Equation 4 , increasing amount of training data twice results in about the same increase of computational complexity as increasing vector size twice . For the experiments reported in Tables 2 and 4 , we used three training epochs with stochastic gradi - ent descent and backpropagation . We chose starting learning rate 0 . 025 and decreased it linearly , so that it approaches zero at the end of the last training epoch . 4 . 3 Comparison of Model Architectures First we compare different model architectures for deriving the word vectors using the same training data and using the same dimensionality of 640 of the word vectors . In the further experiments , we use full set of questions in the new Semantic - Syntactic Word Relationship test set , i . e . unrestricted to the 30k vocabulary . We also include results on a test set introduced in [ 20 ] that focuses on syntactic similarity between words 3 . The training data consists of several LDC corpora and is described in detail in [ 18 ] ( 320M words , 82K vocabulary ) . We used these data to provide a comparison to a previously trained recurrent neural network language model that took about 8 weeks to train on a single CPU . We trained a feed - forward NNLM with the same number of 640 hidden units using the DistBelief parallel training [ 6 ] , using a history of 8 previous words ( thus , the NNLM has more parameters than the RNNLM , as the projection layer has size 640 \u00d7 8 ) . In Table 3 , it can be seen that the word vectors from the RNN ( as used in [ 20 ] ) perform well mostly on the syntactic questions . The NNLM vectors perform signi\ufb01cantly better than the RNN - this is not surprising , as the word vectors in the RNNLM are directly connected to a non - linear hidden layer . The CBOW architecture works better than the NNLM on the syntactic tasks , and about the same on the semantic one . Finally , the Skip - gram architecture works slightly worse on the syntactic task than the CBOW model ( but still better than the NNLM ) , and much better on the semantic part of the test than all the other models . Next , we evaluated our models trained using one CPU only and compared the results against publicly available word vectors . The comparison is given in Table 4 . The CBOW model was trained on subset 3 We thank Geoff Zweig for providing us the test set . 7 Table 4 : Comparison of publicly available word vectors on the Semantic - Syntactic Word Relation - ship test set , and word vectors from our models . Full vocabularies are used . Model Vector Training Accuracy [ % ] Dimensionality words Semantic Syntactic Total Collobert - Weston NNLM 50 660M 9 . 3 12 . 3 11 . 0 Turian NNLM 50 37M 1 . 4 2 . 6 2 . 1 Turian NNLM 200 37M 1 . 4 2 . 2 1 . 8 Mnih NNLM 50 37M 1 . 8 9 . 1 5 . 8 Mnih NNLM 100 37M 3 . 3 13 . 2 8 . 8 Mikolov RNNLM 80 320M 4 . 9 18 . 4 12 . 7 Mikolov RNNLM 640 320M 8 . 6 36 . 5 24 . 6 Huang NNLM 50 990M 13 . 3 11 . 6 12 . 3 Our NNLM 20 6B 12 . 9 26 . 4 20 . 3 Our NNLM 50 6B 27 . 9 55 . 8 43 . 2 Our NNLM 100 6B 34 . 2 64 . 5 50 . 8 CBOW 300 783M 15 . 5 53 . 1 36 . 1 Skip - gram 300 783M 50 . 0 55 . 9 53 . 3 Table 5 : Comparison of models trained for three epochs on the same data and models trained for one epoch . Accuracy is reported on the full Semantic - Syntactic data set . Model Vector Training Accuracy [ % ] Training time Dimensionality words [ days ] Semantic Syntactic Total 3 epoch CBOW 300 783M 15 . 5 53 . 1 36 . 1 1 3 epoch Skip - gram 300 783M 50 . 0 55 . 9 53 . 3 3 1 epoch CBOW 300 783M 13 . 8 49 . 9 33 . 6 0 . 3 1 epoch CBOW 300 1 . 6B 16 . 1 52 . 6 36 . 1 0 . 6 1 epoch CBOW 600 783M 15 . 4 53 . 3 36 . 2 0 . 7 1 epoch Skip - gram 300 783M 45 . 6 52 . 2 49 . 2 1 1 epoch Skip - gram 300 1 . 6B 52 . 2 55 . 1 53 . 8 2 1 epoch Skip - gram 600 783M 56 . 7 54 . 5 55 . 5 2 . 5 of the Google News data in about a day , while training time for the Skip - gram model was about three days . For experiments reported further , we used just one training epoch ( again , we decrease the learning rate linearly so that it approaches zero at the end of training ) . Training a model on twice as much data using one epoch gives comparable or better results than iterating over the same data for three epochs , as is shown in Table 5 , and provides additional small speedup . 4 . 4 Large Scale Parallel Training of Models As mentioned earlier , we have implemented various models in a distributed framework called Dis - tBelief . Below we report the results of several models trained on the Google News 6B data set , with mini - batch asynchronous gradient descent and the adaptive learning rate procedure called Ada - grad [ 7 ] . We used 50 to 100 model replicas during the training . The number of CPU cores is an 8 Table 6 : Comparison of models trained using the DistBelief distributed framework . Note that training of NNLM with 1000 - dimensional vectors would take too long to complete . Model Vector Training Accuracy [ % ] Training time Dimensionality words [ days x CPU cores ] Semantic Syntactic Total NNLM 100 6B 34 . 2 64 . 5 50 . 8 14 x 180 CBOW 1000 6B 57 . 3 68 . 9 63 . 7 2 x 140 Skip - gram 1000 6B 66 . 1 65 . 1 65 . 6 2 . 5 x 125 Table 7 : Comparison and combination of models on the Microsoft Sentence Completion Challenge . Architecture Accuracy [ % ] 4 - gram [ 32 ] 39 Average LSA similarity [ 32 ] 49 Log - bilinear model [ 24 ] 54 . 8 RNNLMs [ 19 ] 55 . 4 Skip - gram 48 . 0 Skip - gram + RNNLMs 58 . 9 estimate since the data center machines are shared with other production tasks , and the usage can \ufb02uctuate quite a bit . Note that due to the overhead of the distributed framework , the CPU usage of the CBOW model and the Skip - gram model are much closer to each other than their single - machine implementations . The result are reported in Table 6 . 4 . 5 Microsoft Research Sentence Completion Challenge The Microsoft Sentence Completion Challenge has been recently introduced as a task for advancing language modeling and other NLP techniques [ 32 ] . This task consists of 1040 sentences , where one word is missing in each sentence and the goal is to select word that is the most coherent with the rest of the sentence , given a list of \ufb01ve reasonable choices . Performance of several techniques has been already reported on this set , including N - gram models , LSA - based model [ 32 ] , log - bilinear model [ 24 ] and a combination of recurrent neural networks that currently holds the state of the art performance of 55 . 4 % accuracy on this benchmark [ 19 ] . We have explored the performance of Skip - gram architecture on this task . First , we train the 640 - dimensional model on 50M words provided in [ 32 ] . Then , we compute score of each sentence in the test set by using the unknown word at the input , and predict all surrounding words in a sentence . The \ufb01nal sentence score is then the sum of these individual predictions . Using the sentence scores , we choose the most likely sentence . A short summary of some previous results together with the new results is presented in Table 7 . While the Skip - gram model itself does not perform on this task better than LSA similarity , the scores from this model are complementary to scores obtained with RNNLMs , and a weighted combination leads to a new state of the art result 58 . 9 % accuracy ( 59 . 2 % on the development part of the set and 58 . 7 % on the test part of the set ) . 5 Examples of the Learned Relationships Table 8 shows words that follow various relationships . We follow the approach described above : the relationship is de\ufb01ned by subtracting two word vectors , and the result is added to another word . Thus for example , Paris - France + Italy = Rome . As it can be seen , accuracy is quite good , although there is clearly a lot of room for further improvements ( note that using our accuracy metric that 9 Table 8 : Examples of the word pair relationships , using the best word vectors from Table 4 ( Skip - gram model trained on 783M words with 300 dimensionality ) . Relationship Example 1 Example 2 Example 3 France - Paris Italy : Rome Japan : Tokyo Florida : Tallahassee big - bigger small : larger cold : colder quick : quicker Miami - Florida Baltimore : Maryland Dallas : Texas Kona : Hawaii Einstein - scientist Messi : mid\ufb01elder Mozart : violinist Picasso : painter Sarkozy - France Berlusconi : Italy Merkel : Germany Koizumi : Japan copper - Cu zinc : Zn gold : Au uranium : plutonium Berlusconi - Silvio Sarkozy : Nicolas Putin : Medvedev Obama : Barack Microsoft - Windows Google : Android IBM : Linux Apple : iPhone Microsoft - Ballmer Google : Yahoo IBM : McNealy Apple : Jobs Japan - sushi Germany : bratwurst France : tapas USA : pizza assumes exact match , the results in Table 8 would score only about 60 % ) . We believe that word vectors trained on even larger data sets with larger dimensionality will perform signi\ufb01cantly better , and will enable the development of new innovative applications . Another way to improve accuracy is to provide more than one example of the relationship . By using ten examples instead of one to form the relationship vector ( we average the individual vectors together ) , we have observed improvement of accuracy of our best models by about 10 % absolutely on the semantic - syntactic test . It is also possible to apply the vector operations to solve different tasks . For example , we have observed good accuracy for selecting out - of - the - list words , by computing average vector for a list of words , and \ufb01nding the most distant word vector . This is a popular type of problems in certain human intelligence tests . Clearly , there is still a lot of discoveries to be made using these techniques . 6 Conclusion In this paper we studied the quality of vector representations of words derived by various models on a collection of syntactic and semantic language tasks . We observed that it is possible to train high quality word vectors using very simple model architectures , compared to the popular neural network models ( both feedforward and recurrent ) . Because of the much lower computational complexity , it is possible to compute very accurate high dimensional word vectors from a much larger data set . Using the DistBelief distributed framework , it should be possible to train the CBOW and Skip - gram models even on corpora with one trillion words , for basically unlimited size of the vocabulary . That is several orders of magnitude larger than the best previously published results for similar models . An interesting task where the word vectors have recently been shown to signi\ufb01cantly outperform the previous state of the art is the SemEval - 2012 Task 2 [ 11 ] . The publicly available RNN vectors were used together with other techniques to achieve over 50 % increase in Spearman\u2019s rank correlation over the previous best result [ 31 ] . The neural network based word vectors were previously applied to many other NLP tasks , for example sentiment analysis [ 12 ] and paraphrase detection [ 28 ] . It can be expected that these applications can bene\ufb01t from the model architectures described in this paper . Our ongoing work shows that the word vectors can be successfully applied to automatic extension of facts in Knowledge Bases , and also for veri\ufb01cation of correctness of existing facts . Results from machine translation experiments also look very promising . In the future , it would be also interesting to compare our techniques to Latent Relational Analysis [ 30 ] and others . We believe that our comprehensive test set will help the research community to improve the existing techniques for estimating the word vectors . We also expect that high quality word vectors will become an important building block for future NLP applications . 10 7 Follow - Up Work After the initial version of this paper was written , we published single - machine multi - threaded C + + code for computing the word vectors , using both the continuous bag - of - words and skip - gram archi - tectures 4 . The training speed is signi\ufb01cantly higher than reported earlier in this paper , i . e . it is in the order of billions of words per hour for typical hyperparameter choices . We also published more than 1 . 4 million vectors that represent named entities , trained on more than 100 billion words . Some of our follow - up work will be published in an upcoming NIPS 2013 paper [ 21 ] . References [ 1 ] Y . Bengio , R . Ducharme , P . Vincent . A neural probabilistic language model . Journal of Ma - chine Learning Research , 3 : 1137 - 1155 , 2003 . [ 2 ] Y . Bengio , Y . LeCun . Scaling learning algorithms towards AI . 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Empirical Evaluation and Com - bination of Advanced Language Modeling Techniques , In : Proceedings of Interspeech , 2011 . 4 The code is available at https : / / code . google . com / p / word2vec / 11 [ 18 ] T . Mikolov , A . Deoras , D . Povey , L . Burget , J . \u02c7Cernock\u00b4y . Strategies for Training Large Scale Neural Network Language Models , In : Proc . Automatic Speech Recognition and Understand - ing , 2011 . [ 19 ] T . Mikolov . Statistical Language Models based on Neural Networks . PhD thesis , Brno Univer - sity of Technology , 2012 . [ 20 ] T . Mikolov , W . T . Yih , G . Zweig . Linguistic Regularities in Continuous Space Word Represen - tations . NAACL HLT 2013 . [ 21 ] T . Mikolov , I . Sutskever , K . Chen , G . Corrado , and J . Dean . Distributed Representations of Words and Phrases and their Compositionality . Accepted to NIPS 2013 . [ 22 ] A . Mnih , G . Hinton . Three new graphical models for statistical language modelling . ICML , 2007 . [ 23 ] A . Mnih , G . Hinton . A Scalable Hierarchical Distributed Language Model . Advances in Neural Information Processing Systems 21 , MIT Press , 2009 . [ 24 ] A . Mnih , Y . W . Teh . A fast and simple algorithm for training neural probabilistic language models . ICML , 2012 . [ 25 ] F . Morin , Y . Bengio . Hierarchical Probabilistic Neural Network Language Model . AISTATS , 2005 . [ 26 ] D . E . Rumelhart , G . E . Hinton , R . J . Williams . Learning internal representations by back - propagating errors . Nature , 323 : 533 . 536 , 1986 . [ 27 ] H . Schwenk . Continuous space language models . Computer Speech and Language , vol . 21 , 2007 . [ 28 ] R . Socher , E . H . Huang , J . Pennington , A . Y . Ng , and C . D . Manning . Dynamic Pooling and Unfolding Recursive Autoencoders for Paraphrase Detection . In NIPS , 2011 . [ 29 ] J . Turian , L . Ratinov , Y . Bengio . Word Representations : A Simple and General Method for Semi - Supervised Learning . In : Proc . Association for Computational Linguistics , 2010 . [ 30 ] P . D . Turney . Measuring Semantic Similarity by Latent Relational Analysis . In : Proc . Interna - tional Joint Conference on Arti\ufb01cial Intelligence , 2005 . [ 31 ] A . Zhila , W . T . Yih , C . Meek , G . Zweig , T . Mikolov . Combining Heterogeneous Models for Measuring Relational Similarity . NAACL HLT 2013 . [ 32 ] G . Zweig , C . J . C . Burges . The Microsoft Research Sentence Completion Challenge , Microsoft Research Technical Report MSR - TR - 2011 - 129 , 2011 . 12", + "mund2023clathrin": "ARTICLE Clathrin coats partially preassemble and subsequently bend during endocytosis Markus Mund 1 , 2 * \ue840 , Aline Tschanz 1 , 3 * \ue840 , Yu - Le Wu 1 , 3 \ue840 , Felix Frey 4 \ue840 , Johanna L . Mehl 1 \ue840 , Marko Kaksonen 2 , 5 \ue840 , Ori Avinoam 1 , 6 \ue840 , Ulrich S . Schwarz 7 , 8 \ue840 , and Jonas Ries 1 \ue840 Eukaryotic cells use clathrin - mediated endocytosis to take up a large range of extracellular cargo . During endocytosis , a clathrin coat forms on the plasma membrane , but it remains controversial when and how it is remodeled into a spherical vesicle . Here , we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites . Through pseudo - temporal sorting , we determine the average trajectory of clathrin remodeling during endocytosis . We find that clathrin coats assemble first on flat membranes to 50 % of the coat area before they become rapidly and continuously bent , and this mechanism is confirmed in three cell lines . We introduce the cooperative curvature model , which is based on positive feedback for curvature generation . It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane . Introduction Endocytosis is an essential function of eukaryotic cells to in - ternalize molecules from their surface . The major endocytic pathway is clathrin - mediated endocytosis ( CME ) , which sup - ports the uptake of many diverse cargos including nutrients , signaling molecules , membrane proteins , and pathogens . During CME , a dense coat of proteins self - assembles on the inner leaflet of the plasma membrane . The membrane itself is bent into an \u03a9 - shaped invagination that is eventually pinched off to form a coated vesicle , completing endocytosis ( Kaksonen and Roux , 2018 ) . The major component of the coat is clathrin triskelia , which comprises three heavy and three light chains ( Fotin et al . , 2004 ) . When triskelia are bound to the plasma membrane by adaptor proteins , they form ordered clathrin lattices . The structural flexibility of triskelia allows these lattices to adapt variable ra - tios of predominantly pentagonal and hexagonal faces , forming both flat and curved geometries . Both geometries have been observed in vivo and in vitro , and their structure has been well characterized in vitro from a structural biology perspective ( Cheng et al . , 2007 ; Dannhauser and Ungewickell , 2012 ; Fotin et al . , 2004 ; Heuser and Kirchhausen , 1985 ; Morris et al . , 2019 ; Pearse , 1976 ; Smith et al . , 1998 ; Takei et al . , 1998 ; Ungewickell and Branton , 1981 ) . However , it remains elusive how clathrin coat formation and membrane bending are temporally and causally related during endocytosis in cells . In EM micrographs , it was observed early on that clathrin lattices can assume many different curvatures in cells ( Heuser , 1980 ) . Since then , two main models of clathrin coat formation during endocytosis have been put forward . In the constant area model ( CAM ) , clathrin grows to its final surface area as a flat coat , which then becomes continuously more curved until ves - icle formation is complete . This model assumes that all the dif - ferently curved clathrin structures are endocytic intermediates . Early observations suggested the presence of pentagonal and hexagonal faces in isolated coated vesicles ( Kanaseki and Kadota , 1969 ) . Combined with the observation of flat lattices being en - riched in hexagons , it was suggested that the integration of at least 12 pentagonal faces is a prerequisite for the formation of a spherical structure ( Heuser , 1980 ) . However , this would require extensive lattice remodeling , which was deemed thermody - namically and structurally unfavorable and thus unlikely to occur ( Kirchhausen , 1993 ) . The constant curvature model ( CCM ) was therefore formulated , which assumes that flat clathrin structures are not endocytic precursors . Instead , it was proposed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Cell Biology and Biophysics , European Molecular Biology Laboratory , Heidelberg , Germany ; 2 Department of Biochemistry , University of Geneva , Geneva , Switzerland ; 3 Candidate for Joint PhD Programme of EMBL and University of Heidelberg , Heidelberg , Germany ; 4 Kavli Institute of Nanoscience , Department of Bionanoscience , Delft University of Technology , Delft , Netherlands ; 5 NCCR Chemical Biology , University of Geneva , Geneva , Switzerland ; 6 Department of Biomolecular Sciences , Weizmann Institute of Science , Rehovot , Israel ; 7 Institute for Theoretical Physics and Bioquant , Heidelberg University , Heidelberg , Germany ; 8 Bioquant , Heidelberg University , Heidelberg , Germany . * M . Mund and A . Tschanz contributed equally to this paper . Correspondence to Jonas Ries : Jonas . ries @ embl . de J . L . Mehl \u2019 s current affiliation is ETH Zurich , Switzerland . F . Frey \u2019 s current affiliation is Institute of Science and Technology Austria , Klosterneuburg , Austria . \u00a9 2023 Mund , Tschanz , et al . This article is available under a Creative Commons License ( Attribution 4 . 0 International , as described at https : / / creativecommons . org / licenses / by / 4 . 0 / ) . Rockefeller University Press https : / / doi . org / 10 . 1083 / jcb . 202206038 1 of 14 J . Cell Biol . 2023 Vol . 222 No . 3 e202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 that the endocytic clathrin coat assumes its final curvature , i . e . , the curvature of the vesicle , directly from the start , while continuously growing in surface area over time . The constant curvature model had been prevalent in the field but was recently challenged by reports that flat clathrin coats can indeed change curvature during endocytosis ( Avinoam et al . , 2015 ; Bucher et al . , 2018 ; Scott et al . , 2018 ) . This has once again boosted interest in clathrin remodeling ( Chen and Schmid , 2020 ; Kaksonen and Roux , 2018 ; Sochacki and Taraska , 2018 ) . Recent years have seen numerous studies based on diverse methods that did not converge on a common result . Reports supported either the constant area model ( Avinoam et al . , 2015 ; Sochacki et al . , 2021 ) , the constant curvature model ( Willy et al . , 2021 ) , a combination of both models ( Bucher et al . , 2018 ; Tagiltsev et al . , 2021 ; Yoshida et al . , 2018 ) , or the simultaneous existence of both models within cells ( Scott et al . , 2018 ) . To conclusively understand this complex process , it would be de - sirable to directly visualize the 3D nanoscale structure of the endocytic clathrin coat in living cells . Unfortunately , however , to date , no method offers the necessary spatial and temporal resolution to do that . Here , we aim to circumvent this limitation by densely sampling the entire endocytic process in fixed cells , subsequently reconstructing the dynamic information . We developed a superresolution microscopy approach to quantitatively study the 3D clathrin coat architecture at endo - cytic sites . We used a novel model fitting framework to extract geometric parameters that allowed us to sort static images of clathrin lattices according to their progression along the endo - cytic timeline . The inferred endocytic dynamics allowed us to reconstruct the stereotypic remodeling of clathrin during en - docytosis at the nanoscale . In summary , we found that a clathrin coat first partially as - sembles on a flat membrane and then simultaneously grows in surface area and coat curvature . While initial bending occurs rapidly , it later slows down and endocytic sites are eventually paused in a state of high curvature before vesicle scission . This trend is conserved across cell lines , suggesting it is a common attribute that is not affected by cell type . Based on this data , we developed a new kinetic growth model , the cooperative curva - ture model ( CoopCM ) . It describes coat area growth as the ad - dition of clathrin to the lattice edge with a constant rate and assumes that the curvature of the coat increases toward a pre - ferred curvature , driven by the cooperative interplay of clathrin triskelia within the lattice . The CoopCM predicts a fast initial curvature increase that is slowed down progressively as the coat becomes spherical and shows excellent agreement with experimental data . Results Quantitative 3D superresolution imaging of clathrin structures Here , we used 3D single - molecule localization microscopy ( SMLM ) to systematically determine the precise geometry of individual clathrin - coated structures at the plasma membrane . For this , we optimized the sample preparation to label clathrin at endocytic sites as densely as possible using indirect immuno - fluorescence with polyclonal antibodies against clathrin light and heavy chains , which was crucial to achieve high - quality SMLM ( Mund and Ries , 2020 ) . We then localized sparsely ac - tivated single fluorophores by fitting an experimentally derived model of the astigmatic point - spread function using a method we developed previously ( Li et al . , 2018 ) . This improved the resolution to about 10 nm in x / y and 30 nm in z ( based on modal values of the localization precision at 3 . 9 nm in x / y and 12 . 5 nm in z ; see Materials and methods ) and reduced typically observed image distortions . The achieved image quality allowed us to clearly visualize and quantify the 3D clathrin coat shape at dif - ferent stages of endocytic site maturation ( Fig . 1 , A \u2013 C ) . The large majority of sites were single structures that were well - isolated from each other and exhibited great structural diversity . In addition , we noted several clusters of closely jux - taposed endocytic sites ( Fig . S1 A ) . In the isolated sites , we ob - serve a variety of shapes including flat , curved , dome - shaped , and spherical structures of different sizes ( Fig . 1 C ) , indicating that endocytosis has been arrested by fixation at different time points during endocytic progression . To quantify the size and shape of individual clathrin coats , we used LocMoFit , a computational pipeline based on a maximum - likelihood model fitting framework that we developed recently ( Wu et al . , 2023 ) . This framework directly fits 3D geometric models to the 3D point cloud of localizations ( Fig . 1 , D and E ) instead of rendered images and thereby harnesses the full in - formation contained in the SMLM data , including , for instance , the localization precision , rather than just spatial coordinates . We describe the clathrin coat as a spherical cap that is defined by a radius r and a closing angle \u03b8 ( Fig . 1 E ) . Our model also accounts for coat thickness , antibody size , and blurring due to the localization precision ( Materials and methods ) . Hence , it describes both shallow and highly curved structures equally well . Moreover , because \u03b8 increases during endocytosis from 0\u00b0 at flat sites to 180\u00b0 at complete vesicles , we could use this pa - rameter to sort the individual images according to endocytic progress ( Fig . 1 F ) . The clathrin coat grows in area and becomes more curved during endocytosis We first imaged immunostained clathrin in chemically fixed SK - MEL - 2 cells , where we focused on the bottom plasma membrane that was densely covered by endocytic sites . These cells have been extensively studied and are established model cells for clathrin - mediated endocytosis with well - characterized endo - cytic dynamics ( Doyon et al . , 2011 ; Aguet et al . , 2013 ; Avinoam et al . , 2015 ; Kaplan et al . , 2022 ) . Using the 3D model fitting pipeline , we determined radius R , closing angle \u03b8 , position , and rotation of 1 , 798 endocytic sites from 13 cells ( Fig . 2 A ) with high accuracy ( Fig . S2 ) . We found that two structural intermediates were enriched , while others were rare and only a small fraction of sites were completely flat ( Fig . 2 B ) . Slightly curved sites with \u03b8 \u2248 70 \u00b0 and strongly curved sites with \u03b8 \u2248 130 \u00b0 were enriched , indicating that those structural states are more frequently found in cells . We only rarely obtained sites with \u03b8 \u2248 180 \u00b0 , which would be expected for complete spherical coats , even though fully formed vesicles are found in our data ( Fig . S1 B ) . This indicates that at the time point of scission , the clathrin coat of nascent Mund et al . Journal of Cell Biology 2 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 vesicles is still incomplete at the neck or that the kinetics of scission are too transient for detection . Deeply invaginated clathrin - coated pits could further be sheared off during sample preparation , thus evading our detection . Curvatures H = 1 / R ranged from 0 to 0 . 022 nm \u2212 1 with a median of 0 . 011 nm \u2212 1 , corresponding to a median radius of 87 nm , and the surface areas A ranged from 9 , 000 to 140 , 000 nm 2 with a median of 54 , 000 nm 2 . These values agree well with previous measure - ments of the vesicular coat using EM ( Avinoam et al . , 2015 ) , where a median curvature of 0 . 015 nm \u2212 1 and median surface area of 54 , 500 nm 2 was measured . We then wanted to understand how the clathrin coat geom - etry changes during endocytosis . To this end , we used the closing angle parameter \u03b8 to sort endocytic sites relative to each other in time . Irrespective of whether endocytosis follows a constant curvature or constant area model , \u03b8 monotonically increases from early to late endocytic time points ( Fig . 2 C ) , and can thus be used as an unbiased proxy for endocytic progression ( Avinoam et al . , 2015 ) . The curvature H was strongly correlated with \u03b8 , indicating that coat curvature increases continuously during endocytosis ( Fig . 2 D ) . Similarly , the surface area A increased from 32 , 000 nm 2 ( median of 5 % of sites with smallest \u03b8 ) to 50 , 000 nm 2 ( median of 5 % of sites with highest \u03b8 ) . The projected area A p decreased from 31 , 000 to 13 , 000 nm 2 ( median of 5 % of sites with lowest and highest \u03b8 respectively ) , which is in close agreement with previous EM measurements ( Bucher et al . , 2018 ) . It is readily obvious that our data are incompatible with the constant curvature model , as the curvature is not constant , but increases monotonically with \u03b8 . Just as clearly , our data do not support the constant area model because the coat surface also increases during endocytosis . Almost all data points are part of one continuous point cloud ( Fig . 2 D ) , indicating a continuous transition between structural states during endocytosis . We noticed an additional small , dis - connected set of data points representing 8 . 5 % of all sites that correspond to endocytic sites with curvatures above 0 . 016 nm \u2212 1 and \u03b8 of 80\u00b0 \u2013 180\u00b0 ( Fig . 2 D , and example structures in Fig . S3 , A and B ) . In a control experiment to check whether these are endocytic structures , we selectively analyzed clathrin structures that colocalized with AP - 2 , a bona fide marker for CME . We did not observe AP - 2 in any of the disconnected sites , from which we conclude that they indeed do not belong to CME . These small structures could represent coated vesicles from the trans Golgi ( Fig . S3 ) . The cooperative curvature model of clathrin coat remodeling Our data clearly showed that clathrin coats grow and become more curved as the plasma membrane gets bent to produce a vesicle . Since the data quantitatively describes all 3D geometries that the endocytic clathrin coat assumes , it allowed us to move toward a mathematical model of clathrin coat formation during endocytosis . Here , we introduce the Cooperative Curvature Model ( CoopCM ) , which describes coat growth based on known struc - tural and dynamical properties of clathrin coats ( Fig . 3 A ) . First , Figure 1 . 3D single - molecule localization microscopy ( SMLM ) of clathrin - coated structures . ( A ) 3D SMLM image of clathrin immunolabeled with AF647 at the bottom membrane of a SK - MEL - 2 cell . ( B ) Enlarged view of the region indicated in A . ( C ) All structures found in B are shown in top view ( xy ) and as 50 nm - thick z - slices ( xz ) , and orientation of slice is indicated by a dotted line . Scale bars are 10 \u00b5m ( A ) ; 1 \u00b5m ( B ) ; and 100 nm ( C ) . ( D ) Geometric analysis pipeline . All clathrin coats are segmented , and their localizations are directly fitted with a spherical cap model using the fitting framework LocMoFit ( Wu et al . , 2023 ) . ( E ) InLocMoFit , individualclathrincoats are parametrizedbytheirsize ( radius R ) , closing angle ( \u03b8 ) , position ( x 0 , y 0 , z 0 ) , and 3D orientation . ( F ) Using \u03b8 asa proxy for endocytic progression , the relative endocytic time point for each structure can be determined . Mund et al . Journal of Cell Biology 3 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 we assume that the clathrin coat starts growing on a flat mem - brane . While triskelia have been shown to exchange dynamically during endocytosis ( Avinoam et al . , 2015 ) , net growth of the area A occurs via the addition of triskelia at the lattice edge \u0190 with a constant growth rate k on ( Eq . 1 ) . d dtA (cid:2) \u02d9 A (cid:2) k on \u0190 . ( 1 ) Moreover , we assume that the intrinsic pucker angle of in - dividual triskelia and their interactions in a lattice together translate into an overall preferred curvature H 0 of the clathrin Figure 2 . Quantitative analysis of clathrin - coated structures in SK - MEL - 2 cells . ( A ) Clathrin coat geometry is quantified using LocMoFit . The fitted spherical cap model is drawn as a circle with its outer radius ( top row , xy ) , as cross - sectional profile ( 50 nm xz slices , middle row ) , and as surface with the corresponding closing angle \u03b8 ( bottom row ) . Scale bar , 100 nm . ( B ) Distributions of closing angle \u03b8 ( median = 108\u00b0 ) , curvature H ( median = 0 . 011 nm \u2212 1 ) , and surface area A ( median = 54 , 000 nm 2 ) of endocytic sites in SK - MEL - 2 cells ( n = 1 , 798 sites , N = 13 cells ) as determined from the model fit . ( C ) Two previously proposed mechanistic models for clathrin coat assembly during endocytosis ( for details , see text ) . In both scenarios , \u03b8 increases monotonically and is thus a proxy for endocytic progression . ( D ) Development of curvature , surface area , and projected area of the clathrin coat during endocytosis . Color indicates point density . Mund et al . Journal of Cell Biology 4 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 coat as a whole . However , the initially flat assembly suggests that this preferred curvature cannot be realized in an immature lattice . We hypothesize that cooperative processes in the coat are required to increase curvature . Coat curvature H then increases asymptotically toward H 0 at an initial rate \u03b3 , slowing down to - ward zero when the preferred curvature is reached ( Eq . 2 ) . d d \u03b8 H (cid:2) \u03b3 1 \u2212 H 2 H 20 (cid:1) (cid:3) . ( 2 ) Choosing a quadratic dependence of the rate of curvature change on curvature is a simple way to represent cooperativity in the lattice , which recently has been demonstrated in experi - ments ( Sochacki et al . , 2021 ; Zeno et al . , 2021 ) and fits our data more accurately than the linear relationship , which would cor - respond to a less cooperative process ( Appendix ) . Eq . 2 can be solved analytically to yield an expression of the curvature H depending on \u03b8 ( Eq . 3 ) , which can then be fitted to our data . H \u03b8 ( ) (cid:2) H 0 tanh \u03b3 \u03b8 H 0 (cid:1) (cid:3) . ( 3 ) Analogous expressions for other geometric parameters like surface area can be derived straightforwardly . The fit of this CoopCM to the data of curvature H in relation to the closing angle \u03b8 shows excellent agreement ( Fig . 3 B ) . In comparison , the CAM fitted the curvature data slightly worse than the CoopCM , and the CCM agreed poorly with the data . The improved fit of the CoopCM compared with the CAM or CCM is not a result of the additional fitting parameter , as we showed using the Bayesian Information Criterion ( see Appendix ) . From the curvature fits , we calculated the corresponding curves for surface area ( Fig . 3 C ) and the edge length of the clathrin coat ( Fig . 3 D ) . The edge length decreases monotonically and approaches zero at \u03b8 = 180 \u00b0 , thereby stopping growth ac - cording to Eq . 1 . Both graphs again highlight the very close agreement of the model prediction with the experimental data . From the fit , we determined that invagination occurs when about half of the total coat area has grown ( A 0 = 0 . 51 ) , and that the preferred curvature of a clathrin coat is H 0 = 0 . 014 nm \u2212 1 , corresponding to a radius of R 0 = 72 nm . The model yields nearly identical parameters when the surface area or edge length is fitted instead of curvature ( Table 1 ) , highlighting its robustness . We then decided to test if the observed mode of clathrin re - modeling is specific for SK - MEL - 2 cells . For this , we analyzed the endocytic clathrin ultrastructure also in U2OS cells and 3T3 mouse fibroblasts ( Fig . S4 ; and Tables 2 and 3 ) . In these cell lines , just like in SK - MEL - 2 cells , the curvature as well as the surface area continuously increase throughout endocytosis . We ob - served that the preferred curvature of the clathrin coat is smaller in U2OS ( H 0 = 0 . 012 nm \u2212 1 , R 0 = 85 nm ) and 3T3 cells ( H 0 = 0 . 011 nm \u2212 1 , R 0 = 89 . 7 nm ) compared with SK - MEL - 2 ( H 0 = 0 . 014 nm \u2212 1 , R 0 = 72 nm ) . This suggests that the average size of vesicles formed in CME is cell - line specific . The fraction of the surface area acquired on the flat membrane is very similar for all three cell lines , with U2OS - derived sites initiating curvature at A 0 = 0 . 52 and 3T3 sites at A 0 = 0 . 45 ( Tables 2 and 3 ) . Taken together , we have shown in several cell lines that clathrin coats neither grow via constant curvature or constant area pathways , but rather first grow flat and then acquire curvature and more surface area simultaneously , with a nonlinear mode of curvature generation . Temporal reconstruction of structural dynamics in endocytosis We systematically segmented all endocytic sites in the super - resolution images and thereby obtained a large dataset where all endocytic time points were sampled homogeneously . The dis - tribution of structural states within the dataset is thus repre - sentative of their lifetime , with long - lived , stable curvature states being overrepresented in the data compared to transient states . This opens up the possibility to reconstruct the temporal progression of clathrin remodeling during endocytosis and to ask whether clathrin coats acquire their curvatures at a constant rate or if there are certain curvature transitions that occur more rapidly than others . For this , we sorted all endocytic sites by \u03b8 . The rank of an endocytic site thus corresponds to its pseudotime , which de - scribes its relative time point between 0 and 1 along the endo - cytic trajectory ( Fig . 4 A ) . As our model ( Eq . 1 ) describes the dynamic growth of the clathrin coat , we can solve it to derive an expression for \u03b8 over time t ( Eq . 4 ) , where the coat starts out flat and then starts to generate curvature , increasing \u03b8 over time . Figure 3 . Model for clathrin coat growth . ( A ) Schematic of the cooperative curvature model , where clathrin lattices grow by the addition of triskelia to the edge at a constant growth rate k on . Curvature generation is driven toward a preferred curvature , ultimately creating a spherical vesicle . ( B ) Distinct clathrin growth models and rolling median ( window width = 82 sites ) fitted to curvature over \u03b8 . ( C and D ) The resulting fitting parameters are then used to map the same models also over ( C ) surface area and ( D ) edge length . ( n = 1 , 645 sites , N = 13 cells ) . Mund et al . Journal of Cell Biology 5 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 \u03b8 t ( ) (cid:2) \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 24 \u03b3 k on 8 \u03b3 2 H \u2212 20 \u2212 1 t s . ( 4 ) The square root dependence of \u03b8 on time t reflects the slowing down of curvature generation as the clathrin coat ap - proaches its preferred curvature . This expression fits the pseudotime - resolved data remarkably well ( Fig . 4 B ) . Consistent with our previous reasoning , a linear model did not agree well with the data ( Appendix ) , since it gives a linear invagination speed for small t , emphasizing the validity of our cooperative curvature model , which leads to the characteristic square root dependence . The data slightly deviates from the model in the early phase and more notably in the very end for pseudotimes close to 1 . This potentially indicates that clathrin geometry is influenced by other factors besides the coat itself close to vesicle scission . Since this pseudotime - resolved dataset was generated from a large number of endocytic sites in many cells , we effectively generated the average trajectories of how curvature , surface area , projected area , and lattice edge change during endocytosis in SK - MEL - 2 cells ( Fig . 4 , C \u2013 F ) . We observe comparatively few flat clathrin coats . This shows that clathrin lattices are only transiently flat at the beginning of endocytosis and might rep - resent an energetically unfavorable conformation ( Fig . 4 B ) . A fast transition from a flat to a curved structure could further be mediated independently of clathrin coat ( Zhao et al . , 2017 ) , e . g . , by the action of BAR domain proteins ( Henne et al . , 2010 ) or at the base of filopodial projections . We further find comparatively many structures with a curvature of \u03b8 \u2248 70 \u00b0 and \u03b8 \u2248 130 \u00b0 , indi - cating more long - lived endocytic stages . As the surface area is constantly increasing over pseudotime , we assume that the en - richment at \u03b8 \u2248 130 \u00b0 represents a stalling point , where vesicles are formed by the addition of the final clathrin triskelia and potentially other factors that enable the recruitment and me - chanic function of dynamin during vesicle scission . Similarly , the enrichment at \u03b8 \u2248 70 \u00b0 could be indicative of further re - cruitment of regulatory components , potentially supporting a previously suggested endocytic checkpoint ( Loerke et al . , 2009 ) . Table 1 . Summary of clathrin coat growth model fits in SK - MEL - 2 . Curvature H ( \u03b8 ) Surface area A ( \u03b8 ) Edge length \u0190 ( \u03b8 ) Projected area A p ( \u03b8 ) CAM A 58 , 300 \u00b1 400 nm 2 56 , 600 \u00b1 400 nm 2 49 , 400 \u00b1 500 nm 2 48 , 800 \u00b1 500 nm 2 CCM R 94 . 5 \u00b1 0 . 7 nm 84 . 8 \u00b1 0 . 4 nm 97 . 3 \u00b1 0 . 8 nm 92 . 8 \u00b1 0 . 7 nm CoopCM \u03b3 ( 9 . 4 \u00b1 0 . 1 ) 10 - 3 nm \u2212 1 ( 9 . 0 \u00b1 0 . 1 ) 10 - 3 nm \u2212 1 ( 9 . 4 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 9 . 1 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 H 0 ( 13 . 9 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 13 . 8 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 13 . 4 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 13 . 6 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 R 0 72 . 0 \u00b1 0 . 6 nm 72 . 2 \u00b1 0 . 6 nm 74 \u00b1 0 . 8 nm 73 . 4 \u00b1 0 . 9 nm A 0 0 . 51 0 . 55 0 . 49 0 . 52 k on 78 . 1 nm \u02dc s \u2212 1 72 . 2 nm \u02dc s \u2212 1 83 . 4 nm \u02dc s \u2212 1 78 nm \u02dc s \u2212 1 Fitted parameter values for the constant area model ( CAM ) , the constant curvature model ( CCM ) and the cooperative curvature model ( CoopCM ) when fitting curvature H ( \u03b8 ) , surface area A ( \u03b8 ) , edge length \u0190 ( \u03b8 ) , and projected area A p ( \u03b8 ) . A : Surface area fitted with CAM ; R : Radius fitted with CCM ; \u03b3 : Constant rate of curvature increase fitted with CoopCM ; H 0 : Preferred curvature of the clathrin coat fitted with CoopCM ; R 0 : preferred radius of the clathrin coat fitted with CoopCM ; A 0 : Fraction of surface area growing as a flat lattice before curvature initiation , defined as A 0 = A ( \u03b8 = 0 . 01 ) / A ( \u03b8 = 180\u00b0 ) ; k on : local growth rate obtained from \u03b8 ( t ) fitted with CoopCM and measured in nm per pseudotime units \u02dc s - 1 . N = 13 cells ; n = 1 , 645 sites . Table 2 . Summary of clathrin coat growth model fits in U2OS . Curvature H ( \u03b8 ) Surface area A ( \u03b8 ) Edge length \u0190 ( \u03b8 ) Projected area A p ( \u03b8 ) CAM A 80 , 800 \u00b1 1 , 600 nm 2 76 , 900 \u00b1 1 , 700 nm 2 65 , 900 \u00b1 1 , 600 nm 2 65 , 400 \u00b1 1 , 600 nm 2 CCM R 119 . 1 \u00b1 2 . 5 nm 101 . 6 \u00b1 1 . 5 nm 122 . 8 \u00b1 2 . 7 nm 115 \u00b1 2 . 4 nm CoopCM \u03b3 ( 7 . 9 \u00b1 0 . 2 ) 10 \u2212 3 nm - 1 ( 7 . 7 \u00b1 0 . 2 ) 10 \u2212 3 nm \u2212 1 ( 8 . 1 \u00b1 0 . 2 ) 10 \u2212 3 nm \u2212 1 ( 7 . 9 \u00b1 0 . 2 ) 10 \u2212 3 nm \u2212 1 H 0 ( 11 . 8 \u00b1 0 . 2 ) 10 \u2212 3 nm \u2212 1 ( 11 . 5 \u00b1 0 . 2 ) 10 \u2212 3 nm - 1 ( 11 . 0 \u00b1 0 . 3 ) 10 \u2212 3 nm - 1 ( 11 . 1 \u00b1 0 . 3 ) 10 \u2212 3 nm - 1 R 0 85 . 0 \u00b1 1 . 8 nm 87 . 1 \u00b1 1 . 7 nm 90 . 5 \u00b1 2 . 7 nm 90 . 2 \u00b1 2 . 6 nm A 0 0 . 52 0 . 52 0 . 45 0 . 47 k on 89 . 3 nm \u02dc s \u2212 1 91 . 5 nm \u02dc s \u2212 1 110 . 3 nm \u02dc s \u2212 1 105 . 5 nm \u02dc s \u2212 1 Fittedparameter valuesfor the constantareamodel ( CAM ) , the constantcurvature model ( CCM ) , and the cooperative curvaturemodel ( CoopCM ) whenfitting curvature H ( \u03b8 ) , surface area A ( \u03b8 ) , edge length \u0190 ( \u03b8 ) , and projected area A p ( \u03b8 ) . A : Surface area fitted with CAM ; R : Radius fitted with CCM ; \u03b3 : Constant rate of curvature increase fitted with CoopCM ; H 0 : Preferred curvature of the clathrin coat fitted with CoopCM ; R 0 : preferred radius of the clathrin coat fitted with CoopCM ; A 0 : Fraction of surface area growing as a flat lattice before curvature initiation , defined as A 0 = A ( \u03b8 = 0 . 01 ) / A ( \u03b8 = 180\u00b0 ) ; k on : local growth rate obtained from \u03b8 ( t ) fitted with CoopCM and measured in nm per pseudotime units \u02dc s \u2212 1 . N = 3 cells ; n = 241 sites . Mund et al . Journal of Cell Biology 6 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 This functional interpretation is supported by the observation of similar peaks in U2OS ( at \u03b8 \u2248 60 \u00b0 and \u2248 130 \u00b0 ) as well as 3T3 cells ( at \u03b8 \u2248 50 \u00b0 and \u2248 130 \u00b0 ) . Within the first 10 % of pseudotemporal progression , the clathrin coat rapidly acquires shallow curvature of H = 0 . 007 nm \u2212 1 ( R = 134 nm ) . It then becomes gradually more bent up to Table 3 . Summary of clathrin coat growth model fits in 3T3 mouse fibroblasts . Curvature H ( \u03b8 ) Surface area A ( \u03b8 ) Edge length \u0190 ( \u03b8 ) Projected area A p ( \u03b8 ) CAM A 90 , 100 \u00b1 1 , 000 nm 2 83 , 700 \u00b1 1 , 000 nm 2 65 , 800 \u00b1 1 , 000 nm 2 66 , 200 \u00b1 1 , 000 nm 2 CCM R 115 . 8 \u00b1 1 . 5 nm 98 . 3 \u00b1 0 . 7 nm 121 . 1 \u00b1 2 nm 108 . 5 \u00b1 1 . 5 nm CoopCM \u03b3 ( 8 . 2 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 7 . 8 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 7 . 9 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 7 . 8 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 H 0 ( 11 . 3 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 11 . 2 \u00b1 0 . 1 ) 10 \u2212 3 nm \u2212 1 ( 11 . 2 \u00b1 0 . 2 ) 10 \u2212 3 nm \u2212 1 ( 11 . 2 \u00b1 0 . 2 ) 10 \u2212 3 nm \u2212 1 R 0 88 . 8 \u00b1 0 . 7 nm 89 . 3 \u00b1 0 . 7 nm 89 . 1 \u00b1 1 . 4 nm 89 . 5 \u00b1 1 . 3 nm A 0 0 . 45 0 . 49 0 . 47 0 . 49 k on 128 . 4 nm \u02dc s \u2212 1 120 . 8 nm \u02dc s \u2212 1 123 . 9 nm \u02dc s \u2212 1 121 nm \u02dc s \u2212 1 Fittedparameter valuesfor the constantareamodel ( CAM ) , the constantcurvature model ( CCM ) , and the cooperative curvaturemodel ( CoopCM ) whenfitting curvature H ( \u03b8 ) , surface area A ( \u03b8 ) , edge length \u0190 ( \u03b8 ) , and projected area A p ( \u03b8 ) . A : Surface area fitted with CAM ; R : Radius fitted with CCM ; \u03b3 : Constant rate of curvature increase fitted with CoopCM ; H 0 : Preferred curvature of the clathrin coat fitted with CoopCM ; R 0 : preferred radius of the clathrin coat fitted with CoopCM ; A 0 : Fraction of surface area growing as a flat lattice before curvature initiation , defined as A 0 = A ( \u03b8 = 0 . 01 ) / A ( \u03b8 = 180\u00b0 ) ; k on : local growth rate obtained from \u03b8 ( t ) fitted with CoopCM and measured in nm per pseudotime units \u02dc s \u2212 1 . N = 7 cells ; n = 688 sites . Figure 4 . Temporal reconstruction of clathrin coat remodeling . ( A ) Endocytic sites are sorted by \u03b8 to reconstruct pseudotime . Enriched \u03b8 states , for example the peak at 135\u00b0 , represent long - lived states that remain for extended periods in pseudotime . Color code represents the same number of sites in both histograms . ( B ) The square - root dependencebetween \u03b8 and pseudotimeapproximatedby the cooperative curvature model ( redline ) . ( C ) Curvaturegeneration over pseudotime is approximated by the cooperative curvature model . ( D \u2013 F ) Fit results in C were used to describe ( D ) edge length , ( E ) surface area , and ( F ) projected area change over pseudotime . A rolling median ( window of 82 sites ) is plotted alongside ( black line ) . ( G ) Superresolution averages for distinct endocytic stages , resulting from all collected snapshots . Each bin contains the same number of snapshots of clathrin - coated structures sorted along their pseudotime ( n = 163 per bin ) , so that all bins represent equally long pseudotime intervals . Individual sites were rescaled to the median bin radius and aligned by their center coordinates as well as rotation angles . Scale bar is 100 nm . ( n = 1 , 645 sites , N = 13 cells ) . Mund et al . Journal of Cell Biology 7 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 \u2248 60 % of the endocytic timeline , when the sites reach an average curvature of 0 . 012 nm \u2212 1 ( R = 83 nm ) . During the last \u2248 40 % of progression , the curvature increase almost stops , until ves - icle scission occurs . Interestingly , the earliest sites in our dataset already contain \u2248 50 % of the surface area of the final vesicles , which is also reflected in the fitting results ( A 0 = 0 . 51 ; Fig . 4 E ) . This indicates that the initial assembly of the clathrin coat occurs very rapidly or is in part below our detection limit . Finally , we made a 3D nanoscale movie , which vividly illus - trates the assembly and remodeling of the clathrin coat during endocytosis from pseudotime - resolved averages of our data ( Fig . 4 G ) . This yielded a nanoscale pseudo - temporal movie of endocytosis in SK - MEL - 2 cells ( Video 1 ) . We performed identical analyses for U2OS - and 3T3 - derived clathrin sites and obtained highly similar results as for SK - MEL - 2 cells , indicating that this trajectory represents a general pathway for clathrin coat remodeling during endocytosis ( Fig . S4 ; and Tables 2 and 3 ) . Also , these data can be represented as high - resolution averages ( Fig . 5 ) . Discussion Quantitative description of clathrin coat ultrastructure The nature of clathrin assembly during endocytosis has become a classical question in cell biology that remains unresolved ( re - viewed in Chen and Schmid , 2020 ; Sochacki and Taraska , 2018 ) . There have been two main competing mechanistic models of how triskelia form a coat : the constant curvature model predicts that the clathrin coat assumes its final curvature directly from the start , while continuously increasing in surface area over time . In contrast , the constant area model predicts that clathrin grows to its final surface area as a flat coat , which then becomes continuously more curved until vesicle formation is complete . Each model is supported by a number of studies , which mostly relied on electron microscopy techniques . Among them , 3D correlative electron tomography yields exquisite 3D endo - cytic membrane shapes ( Avinoam et al . , 2015 ) but can be tedious to extend toward large numbers of cells . Platinum replica elec - tron microscopy offers large fields of view and thus superb throughput , but gives limited 3D resolution ( Bucher et al . , 2018 ; Sochacki et al . , 2017 ) . We reason that the trajectory of clathrin coat assembly dur - ing endocytosis could best be determined by systematically imaging large numbers of endocytic sites with high 3D resolu - tion . Therefore , we used SMLM , which combines high 3D res - olution with high throughput . Although lower than in electron microscopy , the resolution we achieved allowed us to resolve the precise 3D coat ultrastructure of clathrin - coated pits . Impor - tantly , due to the molecular specificity for clathrin , we were able to segment all endocytic sites at the bottom surface of the cells in our images in an unbiased way , thus ensuring homogenous sampling of the structural variety of clathrin coats . We applied a novel maximum - likelihood - based fitting framework that we developed recently ( Wu et al . , 2023 ) , which allows fitting complex geometric models to localization point clouds . Since the fit is applied directly to the localizations , it considers all quantitative parameters of all localizations , most notably localization uncertainty . This results in the precise and reliable quantification of the underlying structures ( Wu et al . , 2020 ) , even when taking linkage error through indirect im - munolabeling into account ( Fig . S5 , A \u2013 L ) . We described the clathrin coat as a spherical cap , which matched the large majority of sites very well . Additionally , in our data , we observed that some sites are asymmetric , ellipsoi - dal , or deformed more irregularly ( Fig . S1 C ) . We do not currently evaluate this asymmetry . Rather , we introduced rotational symmetry during the averaging ( Fig . 4 G ) , where we aligned Figure 5 . Temporal reconstruction of clathrin coat remodeling across three different cell lines . ( A and B ) Superresolution averages for distinct en - docytic stages , resulting from all collected snapshots for ( A ) U2OS ( n = 241 sites , N = 3 cells ) and ( B ) 3T3 ( n = 688 sites , N = 7 cells ) . Each bin contains the same number of snapshots of clathrin - coated structures sorted along their pseudotime ( n U2OS = 24 per bin , n 3T3 = 68 ) and represent equally long pseudotime intervals . Mund et al . Journal of Cell Biology 8 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 each site based on their model parameters and thus averaged out many irregularities . A deviation of the cross - sectional profile from a circle is nevertheless preserved in the averag - ing ( Fig . S2 , C \u2013 G ) , and in future studies , more complex geo - metric models will enable analyzing the structure and asymmetries of the coat in more detail . Flat clathrin - coated structures In the first step of our analysis pipeline , we segmented all clathrin - coated structures in the superresolution images . We thereby aimed to achieve homogenous sampling to capture all endocytic intermediates with the same probability irrespective of time point , size , and orientation . Interestingly , the surfaces of the earliest detectable endocytic sites already contained half the surface area of sites in the latest CME stages . How do these earliest sites assemble ? We cannot sort the very flat sites , which are comparatively rare in our dataset , in time using \u03b8 because their \u03b8 is close to zero . However , their relative fraction among all sites is still informative . As short - lived states are underrepre - sented , the relative absence of small flat sites in our data in - dicates that the initial assembly occurs rapidly . In addition to the short lifetimes of small flat sites , two technical reasons could potentially contribute to their rare oc - currence in our data . Due to their small size and potentially sub - stoichiometric labeling , which is also noticeable as holes in larger structures ( Fig . S1 D ) , they might not have sufficient immunostaining signal and thus they might be hard to be differentiated from small clusters of primary and secondary antibodies that are routinely observed in SMLM images . Ad - ditionally , the very first small clathrin assemblies might be meta - stable with mostly loosely bound triskelia , and thus might not be stabilized well by chemical fixation . However , it is im - portant to note that very small sites are also rarely found in previously published electron microscopy datasets ( Avinoam et al . , 2015 ; Bucher et al . , 2018 ; Sochacki et al . , 2021 ) . Since EM should not suffer from the same technical limitations , it seems likely that these states are indeed short - lived . Generally , completely flat clathrin coats are rare in our data . Instead , we observed that clathrin coats quickly acquire curva - ture already early in endocytosis . This agrees with the early observation made using EM ( Heuser , 1980 ) that even flat structures already had some small degree of curvature . Inter - estingly , an enrichment of pentagons was observed toward the edges of flat lattices , leading to the hypothesis that even without additional formation of pentagons these flat lattices have the built - in capacity to become curved and are not very stable . This notion is in agreement with recent demonstrations that flat clathrin coats store energy , which can drive coat bending without additional factors ( Sochacki et al . , 2021 ; Tagiltsev et al . , 2021 ) . The importance of curvature generation for clathrin coat maturation is supported by previous studies that suggest failure to initiate significant curvature as a hallmark of abortive events ( Loerke et al . , 2009 ; Wang et al . , 2020 ) . Observed in live - cell studies , these dim and short - lived events fail to significantly increase in intensity and do not undergo scission ( Loerke et al . , 2009 ; Mettlen et al . , 2009 ) . As structures in our data set mostly contain at least shallow curvatures at minimally half the final coat area , we are likely not capturing these abortive events , and they have negligible impact on our analysis . The cooperative curvature model Here , we visualized the shapes of clathrin coats with high 3D resolution . This was crucial to precisely measure the clathrin curvature throughout the endocytic timeline ranging from flat to highly curved structures . Especially shallow curvatures are hard to accurately assess with 2D imaging . Thus , our data en - abled us to robustly test different growth models for clathrin during endocytosis . The constant curvature model predicts a continuous increase in surface area , constant coat curvature , as well as a edge length that increases until a half - spherical geometry is reached and then decreases again . Our experimental data , most notably , the increase in curvature and monotonical decrease in edge length are incompatible with these predictions , thus ruling out the constant curvature model ( Fig . 3 , green lines ) . The constant area model , in a strict interpretation , on the other hand , predicts the coat to assemble completely on a flat membrane , after which its area stays constant and only curva - ture increases to form a spherical vesicle . These predictions agree reasonably well with the data for curvature propagation during CME but fail to explain the monotonic increase in coat surface over time . Thus , the continuous increase of surface area that we observed here rules out the constant area model as well ( Fig . 3 , blue lines ) . Interestingly , earlier work compatible with the constant area model already suggested the presence of shallow curvatures in even the flattest clathrin - coated struc - tures ( Avinoam et al . , 2015 ; Bucher et al . , 2018 ; Heuser , 1980 ) . We found that around half of the clathrin coat area has preassembled when plasma membrane invagination begins ( Fig . 2 D and Fig . 4 C ) , agreeing well with previous reports ( Bucher et al . , 2018 ; Scott et al . , 2018 ) , after which the coat keeps growing gradually . Based on this observation , we developed a mathematical model that for the first time considers that cur - vature is generated in a positive ( nonlinear ) feedback loop . Our cooperative curvature model assumes that net area growth of the clathrin coat occurs via triskelia addition to the many available binding sites at the coat edge . We proposed that this process depends on the number of free binding sites , which scale with the edge length and can be described by a single kinetic constant k on . Triskelia addition within the coat is still likely to occur at a lower rate , as clathrin lattices can be unsaturated and have defects ( Frey et al . , 2020 ; Sochacki et al . , 2021 ) , and tris - kelia can exchange ( Avinoam et al . , 2015 ) , which is energetically favorable for curvature changes within these lattices ( Frey and Schwarz , 2020 ; Sochacki et al . , 2021 ; Tagiltsev et al . , 2021 ) . While growth at the edge accounts for an increase in surface area , the curvature is most likely generated in the bulk of the coat . Here , we assumed a nonlinear relation between the rate of curvature increase and curvature , which reflects cooperativity in the lattice , e . g . , due to rearrangements of neighboring tris - kelia or larger regions thereof . This assumption of cooperativity is supported by recent experiments , which suggest that clathrin exhibits curvature - sensing properties , preferentially assembling Mund et al . Journal of Cell Biology 9 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 at prebent membranes ( Sochacki et al . , 2021 ; Zeno et al . , 2021 ) . The model described above shows an excellent fit between theory and experiment and further predicts a square root de - pendence of \u03b8 over time . This describes curvature generation during clathrin coat maturation as a nonlinear mechanism driven by a positive feedback of multiple triskelia , slowing down once the coat approaches a preferred degree of curvature . Even though our model agrees very well with the data , it still exhibits certain limitations worth mentioning : First , it only considers the coat itself and ignores the plethora of other proteins within the endocytic machinery . These include numerous BAR domain proteins that are dynamically recruited and disassembled during endocytosis and thus are bound to influence membrane curvature at endocytic sites , as well as a variety of clathrin adaptor proteins , whose presence or absence could explain the cell - type specific differences in average vesicle sizes that we observed ( Fig . S4 ) . Taken together , these factors could explain the imperfection of our model in the very begin - ning and the final part of the timeline ( Fig . 4 A ) , where vesicle scission is driven by the fast - acting mechanoenzyme dynamin . Additionally , we only modeled clathrin recruitment and ignored clathrin disassembly , which could be mediated by adaptor un - binding ( Taylor et al . , 2011 ) and uncoating factors , including auxilin ( He et al . , 2020 ) , that are recruited before the end of vesicle scission . We also assumed that the clathrin coat has constant properties , most notably that the intrinsic coat - driven curvature generation toward its preferred curvature occurs unidirectionally and remains the same throughout endocytosis ( Eq . 3 ) . It is however likely that the properties of the clathrin coat change during endocytosis , e . g . , by coat stiffening or in - creasingly tight interactions between triskelia ( Frey and Schwarz , 2020 ; Frey et al . , 2020 ; Sochacki et al . , 2021 ) . Second , we reconstructed an average trajectory of clathrin coat remodeling generated from many individual static snapshots , thereby averaging conceivable different pathways of CME . How - ever , different pathways with substantially changed relationships between the parameters like curvature and \u03b8 would be visible in the corresponding plots as separate point clouds . This is not the case , rather weobserved acontinuous correlationbetweencurvatureand \u03b8 following a single trajectory , indicating that CME follows a single , stereotypic pathway . We did identify a small , disconnected popu - lation of sites in our data set that most likely originate from a dis - tinct cellular mechanism ( Fig . S3 ) . While this indicates that our approach should capture potentially different pathways of clathrin - coated vesicle formation , we cannot exclude minor mechanistic variations that are included in the average trajectory . Clathrin recruitment has been quantified extensively using live - cell microscopy , resolving the sequential recruitment and dynamic characteristics of many important endocytic compo - nents ( Aguet et al . , 2013 ; Cocucci et al . , 2012 ; Doyon et al . , 2011 ; He et al . , 2017 ; Jin et al . , 2021 Preprint ; Loerke et al . , 2009 ; Mettlen et al . , 2009 ; Saffarian et al . , 2009 ; Sch\u00a8oneberg et al . , 2018 ; Taylor et al . , 2011 ) . We wondered if it is possible to cor - relate our pseudotime reconstruction with previously reported real - time dynamics of clathrin , which shows an initial fluores - cence intensity increase , a plateau , and finally a sharp intensity decrease after vesicle scission . While we observed a correlation between the number of clathrin localizations and surface area ( Fig . S5 , M \u2013 O ) , we note that indirect immunolabeling is not al - ways quantitative , which complicates a direct comparison of previously reported live - cell fluorescence intensity over time and our pseudotime trajectory data . Nevertheless , we speculate that the initial fast intensity increase in live - cell studies likely corresponds to the initial growth of the flat clathrin coat that escapes our detection due to the fast dynamics and small size . The slower fluorescence increase and subsequent plateauing then coincide with curvature generation and the final addition of triskelia at the coat edge , as resolved in detail in our pseu - dotime data . To better understand the correlation between changes in the nanoscale architecture of clathrin coats and dy - namic consequences , we would ultimately require a method that combines high structural with temporal resolution . In a recent publication , this was attempted using a live - cell 2D super - resolution microscopy approach ( Willy et al . , 2021 ) . This study reported an increasing projected area of clathrin over time , suggesting that curvature is present in the earliest detectable clathrin structures , and concluded that CME follows the CCM . Although we also find that completely flat structures are rare and curvature is initiated before the final surface is acquired , our data is entirely incompatible with the CCM . This is espe - cially true in the first half of endocytosis with shallow but progressively increasing curvatures ( Fig . 3 ) , which is challeng - ing to measure using 2D imaging . This shows that it remains highly desirable to ultimately image the 3D nanoscale architec - ture of the clathrin coat in living cells in real - time . In summary , we characterized the dynamics and geometries of the clathrin coat in endocytosis by combining 3D super - resolution microscopy , quantitative analysis , and mathematical modeling . We found that clathrin bending and assembly occur simultaneously along a precisely defined trajectory . We anticipate that this work will be foundational to further study the structural mechanism of endocytosis , both under physiological conditions , and in diseases , where changes in CME likely occur very fre - quently and have just recently been shown to have profound functional consequences ( Moulay et al . , 2020 ; Xiao et al . , 2018 ) . Materials and methods Cell culture SK - MEL - 2 cells ( gift from David Drubin , UC Berkeley , described in Doyon et al . [ 2011 ] ) were cultured adherently in DMEM ( Gibco , no . 10565 - 018 ) , supplemented with 10 % ( v / v ) FBS ( Gibco , no . 10270 - 106 ) , and ZellShield ( Biochrom AG ) at 37\u00b0C , under an atmosphere with 5 % CO 2 and 100 % humidity . U2OS cells ( # 300174 ; Cell Line Services ) were cultured ad - herently as described previously ( Thevathasan et al . , 2019 ) in DMEM ( Gibco , no . 11880 - 028 ) supplemented with 10 % FBS ( Gibco , no . 10270 - 106 ) , 1x GlutaMAX ( Gibco , no . 35050 - 038 ) , nonessential amino acids ( Gibco , no . 11140 - 035 ) , and ZellShield ( Biochrom AG ) at 37\u00b0C under an atmosphere with 5 % CO 2 and 100 % humidity . 3T3 mouse fibroblasts ( gift from Alba Diz - Mu\u00f1oz , EMBL Heidelberg ) were cultured adherently in DMEM ( 4 . 5 g / l D - Glucose ) supplemented with 1\u00d7 MEM NEAA ( Gibco , no . 11140 - 035 ) , 1\u00d7 GlutaMAX ( Gibco , no . 35050 - 038 ) , and 10 % Mund et al . Journal of Cell Biology 10 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 ( v / v ) FBS ( Gibco , no . 10270 - 106 ) at 37\u00b0C under an atmos - phere with 5 % CO 2 , 100 % humidity . Sample preparation for superresolution imaging of clathrin - coated pits Cells were seeded onto high - precision 24 - mm round glass cov - erslips ( No . 1 . 5H , catalog no . 117640 ; Marienfeld ) . Coverslips were previously cleaned by incubating them overnight in a methanol / hydrochlorid acid ( 50 : 50 ) solution while stirring . The coverslips were then repeatedly rinsed with water until a neu - tral pH was reached . They were then placed into a laminar flow cell culture hood overnight to dry . In a final cleaning step , the coverslips are irradiated with ultraviolet light for 30min . Cells were fixed as described previously ( Li et al . , 2018 ) using 3 % ( w / v ) formaldehyde , 10 mM MES pH 6 . 1 , 150 mM NaCl , 5 mM EGTA , 5 mM glucose , and 5 mM MgCl 2 for 20 min . Fix - ation was quenched in 0 . 1 % ( w / v ) NaBH 4 for 7 min . The sample was washed three times with PBS and permeabilized for 15 min with 0 . 01 % ( w / v ) digitonin ( Sigma - Aldrich ) in PBS . The sample was then washed twice with PBS and blocked for 1 h with 2 % ( w / v ) BSA / PBS , washed with PBS , and stained for 3 \u2013 12 h with anti - clathrin light chain ( sc - 28276 ; Santa Cruz Biotechnology ) and anti - clathrin heavy chain rabbit polyclonal antibodies ( ab21679 ; Abcam ) in 1 % ( w / v ) BSA / PBS . After three washes with PBS , the sample was incubated for 3 \u2013 4 h with a secondary donkey anti - rabbit antibody ( 711 - 005 - 152 ; Jackson ImmunoResearch ) that was conjugated to Alexa Fluor 647 \u2013 NHS at an average degree of labeling of 1 . 5 . The sample was then washed three times and mounted for imaging in blinking buffer ( 50 mM Tris / HCl pH 8 , 10 mM NaCl , 10 % ( w / v ) D - glucose , 500 \u00b5g ml \u2212 1 glucose oxidase , 40 \u00b5g ml \u2212 1 glucose catalase , and 35 mM MEA in H 2 O ) . For the analysis of the disconnected population of sites , 3T3 cells were transfected with a plasmid encoding the sigma 2 sub - unit , fused to GFP ( gift from Steeve Boulant , University of Florida , # 53610 ; Addgene ) , to obtain cells transiently expressing AP2 - GFP . The transfection was performed using a Lipofectamine 2 , 000 re - agent ( Life Technologies ) according to the manufacturer \u2019 s rec - ommendations : 1 \u00b5g DNA was mixed with 50 \u00b5l OptiMEM I ( Thermo Fisher Scientific ) . The same was done for 3 \u00b5l Lipo - fectamin in 50 \u00b5l OptiMEM I . Both solutions were incubated for 5 min and then mixed together and incubated for an additional 10 min at room temperature . The media of previously seeded cells was exchanged to prewarmed OptiMEM I , to which the DNA - Lipofectamin solution ( 100 \u00b5l ) was added dropwise . After \u223c 24 h of incubation ( at 5 % CO 2 , 37\u00b0C ) , the medium was exchanged with fresh growth medium . After additional incubation for \u223c 16 h , cells were fixed according to the same protocol described above . Superresolution microscopy SMLM images were acquired at room temperature ( 24\u00b0C ) on a custom - built microscope ( Mund et al . , 2018 ) with a 160\u00d7 NA 1 . 43 oil - immersion objective ( Leica ) . The sample was illumi - nated using a LightHub laser combiner ( Omicron - Laserage La - serprodukte ) with Luxx 405 , 488 , and 638 and Cobolt 561 lasers , which were triggered using a Mojo FPGA ( Embedded Micro ) for microsecond pulsing control of lasers . The lasers were guided through a speckle reducer ( LSR - 3005 - 17S - VIS ; Optotune , Dietikon , Switzerland ) and coupled into a multimode fiber ( M105L02S - A ; Thorlabs ) . The output of the fiber was magni - fied and imaged in the sample . Fiber - generated fluorescence was removed using a laser clean - up filter ( 390 / 482 / 563 / 640 HC Quad ; AHF ) . The focus was stabilized using a closed - loop system based on reflecting the signal of a near - infrared laser on the coverslip and detecting the resulting signal on a quadrant photodiode , re - sulting in focus stabilities of \u00b1 10 nm over several hours . Fluo - rescence emission was filtered using a 676 / 37 or a 700 / 100 bandpass filter ( AHF ) and recorded by an EMCCD camera ( Evolve512D ; Photometrics ) . Typically , 100 , 000 \u2013 300 , 000 frames were acquired using 15 - or 30 - ms exposure times and laser power densities of \u223c 15 kW / cm 2 . 405 - nm laser intensity was adjusted automatically by changing pulse duration to keep the number of localizations per frame constant during the acquisition . For the analysis of the disconnected population of sites , one diffraction - limited image was additionally acquired before SMLM imaging . For this , a 488 laser at 1 . 4 kW / cm 2 was used to take a single frame at 30 ms exposure time . Emission was fil - tered using a 525 / 50 bandpass filter ( AHF ) . The microscope hardware and data acquisition was handled via Micro - Manager 1 . 4 . 22 using custom - written software ( Edelstein et al . , 2010 , 2014 ; Deschamps and Ries , 2020 ) . Data analysis All data analysis was conducted in SMAP ( [ Ries , 2020 ] based on MATLAB and available as open source under https : / / github . com / jries / SMAP ) . Superresolution image reconstruction For fitting the localizations , peaks were detected as maxima in raw camera images after applying a difference - of - Gaussian fil - ter . At those positions , cropped images of 13 \u00d7 13 pixels were fitted with an experimentally derived PSF model ( free fitting parameters : x , y , z , photons per localization , background per pixel ) using an MLE ( Maximum likelihood estimation ) fitter ( Li et al . , 2018 ) . The x , y , and z positions were corrected for residual drift by a custom algorithm based on redundant cross - correlation . In short , the data were distributed into 10 - time bins of equal length . For each bin , a superresolution image was reconstructed . We then calculated the image cross - correlations among all superresolution images and extracted the relative displacements in x and y from the position of the maximum in the cross - correlation images . We then calculated the drift tra - jectory that best describes the relative displacements . In a second step , the z - drift was measured in an analogous way using in - tensity profiles in z instead of images . Localizations persistent over consecutive frames ( detected within 35 nm from one an - other and with a maximum gap of one dark frame ) were merged into one localization by calculating the weighted average of x , y , and z positions and the sums of photons per localization and background . Localizations were filtered by the localization pre - cision in x , y ( 0 \u2013 20 nm ) and z ( 0 \u2013 30 nm ) to exclude dim local - izations . The modal value for the localization precision \u03c3 was 3 . 9 nm in x / y and 12 . 5 nm in z , leading to a resolution estimate ( calculated using the Full Width Half Maximum ( FWHM ) using FWHM = 2 \u221a ( 2 ln 2 ) \u03c3 ) of 9 . 2 nm in x / y and 29 . 4 nm in z ( typical Mund et al . Journal of Cell Biology 11 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 values based on the representative image ) . Superresolution images were constructed with every localization rendered as a two - dimensional spherical Gaussian with a sigma of 3 nm . The red hot color map used represents the density of localizations and is scaled in a way that 0 . 03 % of the pixel values are saturated . Quantitative geometric analysis of clathrin - coated structures Clathrin - coated structures were segmented semiautomatically . First , we manually defined a region of interest excluding the edges of the cell . Then , the image was blurred using a Gaussian filter with a sigma of 100 nm and peaks were detected using a manually set threshold . This typically yielded several hundreds of sites in a region of 30 \u00d7 30 \u00b5m . These candidate sites were curated manually , and only single , well - isolated clathrin - coated structures were retained in the dataset . Next , these structures were analyzed using LocMoFit , an MLE - based model fitting framework that we developed recently ( Wu et al . , 2023 ) . LocMoFit directly fits localization coordinates with the probability density function ( PDF ) of a parametrized geometric model . In this study , we modeled clathrin - coated structures with a hollow spherical cap parameterized by the surface area A and the closing angle \u03b8 . \u03b8 is defined as the angle between the two vectors that point to the pole and the edge , respectively , from the center of the sphere . The position of the model is defined as the center of mass of the cap . In practice , we discretized the cap by placing spiral points ( Saff and Kuijlaars , 1997 ) or the spherical Fibonacci lattice on the surface of the cap to approximate an even distribution . In LocMoFit , these points were treated as discrete fluorophore coordinates when con - structing the PDF of the model . During fitting , additional pa - rameters including the center position and the orientation of the model were determined with respect to the fluorophore coor - dinates , and an extra uncertainty and the background weight were applied to the PDF . After fitting , the sphere radius is de - rived as r (cid:2) \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 A / 2 \u03c0 ( 1 \u2212 cos \u03b8 ) p , projected area as A p = \u03c0 sin 2 \u03b8 , and edge length as \u03b5 = 2 \u03c0 sin \u03b8 . For some flat sites where the fit pro - duced slightly negative curvature values , curvature H and \u03b8 were set to 0 nm \u2212 1 and 0 , \u00b0 respectively , to approximate them as completely flat . After model fitting , a second curation step was performed . With this , we ensured that only well - fitted sites are included in the final data set . Sites were excluded if they were upside down ( clearly originating from an upper membrane ) , double - sites with two sites clearly connected to each other or an adjacent flat structure , and large plaques or structures nondistinguishable from an antibody cluster . Pseudo - temporal reconstruction of clathrin remodeling during endocytosis To sort endocytic sites in pseudotime , they were sorted by the closing angle \u03b8 , assigning each site a rank index . Flat sites with a manually assigned H = 0 nm \u2212 1 and \u03b8 = 0 were all assigned an index of 0 . As pseudo - temporal sorting assumes that all sites are part of the same endocytic trajectory , endocytic sites with cur - vatures above a cell line \u2014 specific threshold ( H > 0 . 016 nm \u2212 1 for SK - MEL - 2 ; H > 0 . 013 nm \u2212 1 for U2OS ; H > 0 . 014 nm \u2212 1 for 3T3 ) that form a visibly disconnected point cloud ( Figs . S3 and S4 ) were excluded for this analysis ( Fig . S5 ) . To compute pseudo - temporal averages , the sites were spatially and rotationally aligned and rescaled by the average radius of all sites within the respective bin . As the geometric model is rotationally symmet - ric , we then performed rotational averaging by generating 72 duplicates of each structure , rotating them by 5\u00b0 with respect to each other , and averaging them . Video 1 was computed using a sliding window approach , where each frame shows an average of 30 sites , and the frame - to - frame increment is 20 sites . The median pseudotime of those 30 sites is indicated . Further data analysis All data that resulted from the quantitative geometric descrip - tion of clathrin - coated structures were further analyzed in R ( R Core Team , 2020 ) . Fitting of the growth models ( Tables 1 , 2 , and 3 ) was performed according to the equations described in the Appendix using a nonlinear least square fit . All analyses were then performed on filtered data sets , excluding disconnected sites above a cell line \u2014 specific threshold ( H > 0 . 016 nm \u2212 1 for SK - MEL - 2 ; H > 0 . 013 nm \u2212 1 for U2OS ; H > 0 . 014 nm \u2212 1 for 3T3 ) , and sites of negative curvature . During fitting , for sites with \u03b8 = 0\u00b0 , we set \u03b8 = 0 . 0001\u00b0 to avoid division by 0 . For depicting the growth models in Figs . 3 and 4 , as well as Fig . S5 , parameters resulting from the H ( \u03b8 ) fit were used and mapped to the A , \u0190 , and A p data . Simulations Simulations were performed using the simulation engine for SMLM data implemented in SMAP and LocMoFit as described in Thevathasan et al . ( 2019 ) . The realistic simulations were based on a two - state ( bright and dark ) fluorophore model plus bleaching ( Sage et al . , 2019 ) , and parameters ( number of pho - tons , background photons , and fluorophore on - time t l ) were extracted from our experiment . ( 1 ) First , we defined an equally distributed closing angle \u03b8 from 0 to 180\u00b0 and calculated the surface area A ( \u03b8 ) = 2 \u03c0 ( 1 \u2212 cos \u03b8 ) / H ( \u03b8 ) 2 ( see Appendix for details ) . Here , H ( \u03b8 ) is defined as in Eq . 3 , with fitting parameters de - termined in SK - MEL - 2 ( see Table 1 ) . ( 2 ) With the defined model parameters , we generated protein positions for each simulated structure by taking randomly drawn N } A samples from the PDF of the hollow spherical cap with no uncertainty . ( 3 ) With a probability p label = 0 . 6 , a fluorescent label was created at a pro - tein position . ( 4 ) Linkage displacements in x , y , and z were added to a label and were determined as normally distributed random variables with a variance corresponding to the linkage error of 5 nm . The fluorophore is assumed to be freely rotating three - dimensionally between different blinks . ( 5 ) Each fluo - rophore appeared at a random time and lived for a time t l , de - termined as a random variable from an exponential distribution with a mean of 1 . 6 frames . ( 6 ) A label had a probability p react = 0 . 5 to be reactivated and then appeared at a random later time point , otherwise it was bleached . ( 7 ) When it was on , a fluorophore had a constant brightness . Thus , the brightness in each frame was proportional to the fraction of the time the fluorophore was on in each frame . ( 8 ) The emitted photons in each frame were determined as a random Poisson variable with a mean value corresponding to the average brightness in the frame . ( 9 ) For Mund et al . Journal of Cell Biology 12 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 each frame , we calculated the CRLB ( Cram\u00b4er - Rao lower bound ) in x , y , and z from the number of photons ( with a mean of 11 , 000 ) and the background photons ( 130 per pixel ) based on the theoretical Gaussian PSF ( Mortensen et al . , 2010 ) or a 3D cspline PSF model derived from beads calibrations ( Li et al . , 2018 ) . This error was added to the true x , y , and z positions of the fluo - rophores as normally distributed random values with a variance corresponding to the respective calculated CRLB . Online supplemental material Fig . S1 provides examples of diverse clathrin coat structures . Fig . S2 provides simulations . Fig . S3 shows non - endocytic clathrin structures . Fig . S4 shows clathrin coat remodeling in three dif - ferent cell lines . Fig . S5 provides linkage error investigation and number of localizations per clathrin coat . Video 1 shows a pseudo - temporal movie of clathrin coat remodeling during en - docytosis in SK - MEL - 2 cells . Appendix shows a detailed de - scription of the cooperative curvature model . All presented data are available in the BioStudies database ( https : / / www . ebi . ac . uk / biostudies / ) under accession number S - BIAD566 . Acknowledgments We thank the entire Ries and Kaksonen labs for fruitful dis - cussions and support . This work was supported by the European Research Council ( ERC CoG - 724489 to J . Ries ) , the National Institutes of Health Common Fund 4D Nucleome Program ( Grant U01 to J . Ries ) , the Human Frontier Science Program ( RGY0065 / 2017 to J . Ries ) , the EMBL Interdisciplinary Postdoc Programme ( EIPOD ) under Marie Curie Actions COFUND ( Grant 229597 to O . Avinoam ) , the European Molecular Biology Laboratory ( M . Mund , A . Tschanz , Y . - L . Wu and J . Ries ) , and the Swiss National Sci - ence Foundation ( grant 310030B _ 182825 and NCCR Chemical Biology to M . Kaksonen ) . O . Avinoam is an incumbent of the Miriam Berman Presidential Development Chair . Author contributions : M . Mund , O . Avinoam , M . Kaksonen and J . Ries conceived the study , M . Mund , A . Tschanz , J . L . Mehl and O . Avinoam performed experiments , M . Mund , A . Tschanz , Y . - L . Wu , J . L . Mehl , O . Avinoam and J . Ries analyzed superresolution data ; F . Frey and U . S . Schwarz developed the cooperative curvature model ; J . Ries supervised the study . M . Mund , A . Tschanz , F . Frey , J . Ries wrote the manuscript with input from all authors . Disclosure : The authors declare no competing financial interests . 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Cui , and B . Cui . 2017 . Nanoscale manipulation of membrane curvature for probing endocytosis in live cells . Nat . Nanotechnol . 12 : 750 \u2013 756 . https : / / doi . org / 10 . 1038 / nnano . 2017 . 98 Mund et al . Journal of Cell Biology 14 of 14 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 Supplemental material Mund et al . Journal of Cell Biology S1 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 Figure S1 . Examples of diverse clathrin coat structures . ( A ) Large clusters of clathrin molecules excluded from further analysis . Shown in top view ( xy ) with the dotted line indicating a 50 nm - thick z - slice ( xz ) shown below . ( B ) Vesicular structures are sometimes fitted with a lower \u03b8 than expected . ( C ) While a spherical model describes the structure of most endocytic clathrin coats faithfully , there are few cases , as exemplified here , where the elliptical and irregular shape of an assembling coat is difficult to approximate with a simple geometric model . Two orthogonal 50 - nm - thick z - slices are shown here in xz and yz , and the respective spherical model fit is plotted as a dotted line . ( D ) Non - continuous labeling of clathrin manifests itself as holes in the coat , indicated with a blue arrow . All scale bars are 100 nm . Mund et al . Journal of Cell Biology S2 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 FigureS2 . Simulations . ( Aand B ) Estimation error of the closingangle \u03b8 ( N = 1 , 800 sites ) . ( A ) The comparison offitted \u03b8 of simulated clathrin coat structures to corresponding ground - truth \u03b8 shows no systematic bias and a narrow spread of the error across the ground truth although the spread increases when \u03b8 < 20 \u00b0 or \u03b8 > 160 \u00b0 . We reasoned that in the earlier range , where the structures are flat , the slightly increased error corresponds to the insensitivity of \u03b8 to flat structures . In the later range , where the vesicles are almost closed , the error was caused by indistinguishable tiny holes corresponding to the real vesicle openings and unlabeled clathrin in the coat . The fitted structures were simulated to have similar quality as the experimental data and to distribute evenly across \u03b8 . ( B ) The distribution of \u03b8 shows no significant error compared to the expectation corresponding to the evenly distributed \u03b8 , except for the small potential underestimation of entirely closed coats . ( C \u2013 G ) Averaging preserves U - shapes in simulated endocytic sites . ( C ) A ground - truth structure was used for simulating U - shaped clathrin coats . The model for simulation was built by combining a hemisphere with a radius of R = 97 nm and a cylinder with a height D = 0 . 5 R . We choose the radius value according to the median radius of bin 6 in Fig . 4 G . This bin has a closing angle slightly larger than 90\u00b0 . 20 - nm - thick cross - sections of the averages ( N = 100 sites ) registered based on ( D ) the ground truth and ( E ) the spherical fit are shown with the ground - truth U - shaped model ( dotted line ) . Histograms of normalized estimation errors are shown for the parameters ( F ) surface area and ( G ) radius , with mean values of \u2212 2 . 6 and 2 . 5 % respectively . The scale bar is 100 nm . Mund et al . Journal of Cell Biology S3 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 FigureS3 . Non - endocytic clathrinstructures . Results ofLocMoFitanalysis of clathrinstructures inSK - MEL - 2cells ( N = 13cells , n = 1 , 798 sites ) . ( A ) A strong correlation between curvature and \u03b8 can be observed for most structures ( n = 1 , 645 sites , black ) . A disconnected point cloud ( n = 153 sites , 8 . 5 % , red ) indicates the presence of endocytosis - unrelated clathrin structures . ( B \u2013 D ) The same distinct population of data points can be observed for the ( B ) surface area , ( C ) edge length , and ( D ) projected area . ( B ) Example structures from the disconnected population of sites in top view ( xy ) and 50 nm - thick z - slices ( xz ) and their respective fitted \u03b8 values . Scale bar , 100 nm . ( C \u2013 F ) Analysis of clathrin coats not following general trajectory of curvature generation . ( C ) 3T3 cell transiently overexpressing AP2 - GFP . Left : Single - molecule localization microscopy image of immunolabeled clathrin . Middle : Diffraction - limited image of the AP2 - GFP signal . Right : Overlay of the two targets ( scale bar , 10 \u00b5m ) . ( D ) Enlarged image of the section indicated in C ( scale bar , 1 \u00b5m ) . ( E ) Example sites indicated in B ( scale bar , 100 nm ) . 1 : Example for a structure annotated as \u201c GFP positive . \u201d 2 : Example for an \u201c inconclusive \u201d GFP signal . 3 and 4 : Example of \u201c GFP negative \u201d structures . \u201d ( F ) Analysis results when estimating \u03b8 and curvature from clathrin structures and annotating them depending on their AP2 signal ( N = 3 cells and n = 277 sites ) . No AP2 - GFP positive structures are found in the disconnected population of sites , suggesting that they are most likely not generated via clathrin - mediated endocytosis . Mund et al . Journal of Cell Biology S4 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 Figure S4 . Clathrin coat remodeling in three different cell lines . ( A \u2013 C ) Results of LocMoFit analysis for clathrin structures . Different growth models are fitted to curvature H over \u03b8 . The resulting fitting parameters are then used to map the same models also over the surface area , edge length , and projected area ( left to right ) . Purple : Completely flat sites with H = 0 nm \u2212 1 and \u03b8 = 0 . Red : Disconnected sites that were excluded from the fitting . Black line : Rolling median ( window width = 5 % of total number of sites ; A ) U2OS ( N = 3 cell , n grey = 241 sites , n red = 53 disconnected sites , n purple = 1 completely flat sites ) , ( B ) 3T3 mouse fibroblasts ( N = 7 cells , n grey = 688 sites , n red = 51 disconnected sites , n purple = 8 completely flat sites ) , and ( C ) SK - MEL - 2 cells ( N = 13 cells , n grey = 1 , 631 sites , n red = 153 disconnected sites , n purple = 14 completely flat sites ) . ( D \u2013 F ) Temporal reconstruction of clathrin coat remodeling . ( i ) Distribution of \u03b8 slightly differs between cell lines , especially in the earlier states . Median \u03b8 shown as dotted lines correspond to 99 . 6\u00b0 for U2OS ; 121 . 4\u00b0 for 3T3 ; and 108 . 5\u00b0 for SK - MEL - 2 cells . ( ii ) The cooperative curvature model ( red line ) highlights the square - root dependence between \u03b8 and pseudotime . ( iii ) The cooperative curvature model is used to describe the curvature H propagation over pseudotime . Resulting fitting parameters are then used to map the same model to surface area A , edge length \u0190 , and projected area A p . A rolling median is plotted in black ( window width = 5 % of total number of sites ) . Mund et al . Journal of Cell Biology S5 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 Figure S5 . Linkage error investigation and number of localizations per clathrin coat . ( A \u2013 L ) Impact of linkage error on geometric model fit . Indirect immunolabeling displaces the label from the target molecule by two antibodies . This generates a so - called linkage error of on average \u00b1 10 nm , and resulting localizations might not accurately represent the underlying structure of interest ( Fr\u00fch et al . , 2021 ) . ( A ) An ideal case of no linkage error , where the model ( dotted black line ) with radius R M accurately represents the underlying clathrin coat ( red ) with a radius R C . ( B ) Histogram of quantified curvature with a median of 0 . 011 nm \u2212 1 . ( C ) Models fitted to curvature propagation over \u03b8 . ( D ) Uniform displacement of localizations ( light red , \u00b1 10 nm ) due to unbiased labeling by the antibodies . The radius R M still accurately represents the true radius R C . ( E ) Histogram of the curvature , assuming no needed correction of R M . Median : 0 . 011 nm \u2212 1 . ( F ) Models fitted to curvature propagation over \u03b8 remain the same as for C . ( G ) Biased labeling of the antibodies ( light red , + 10 nm ) could result in an overestimation of R M by 10 nm . ( H ) Histogram of the curvature corrected by subtracting 10 nm from quantified R M . Median : 0 . 013 nm \u2212 1 . ( I ) Models fitted to corrected curvature propagation over \u03b8 . ( J ) Biased labeling could result in an underestimation of R M by 10 nm . ( K ) Histogram of the curvature corrected for the overestimation in radius by adding 10 nm from quantified R M . Median : 0 . 010 nm \u2212 1 . ( L ) Models fitted to corrected curvature propagation over \u03b8 . ( C , F , I , and L ) Fitting parameters are A 0 : Fraction of surface area growing as a flat lattice before curvature initiation , defined as A 0 = A ( \u03b8 = 0 . 01 ) / A ( \u03b8 = \u03c0 ) ; R 0 : Preferred radius of the clathrin coat fitted with CoopCM ; H 0 : Preferred curvature of the clathrin coat fitted with CoopCM . Analysis of N = 13 SK - MEL - 2 cells , n = 1 , 645 sites . While the fitting parameters scale with the error in radius estimation , the relationships among the parameters and thus our mechanistic interpretation by the cooperative curvature model still holds true . ( M \u2013 O ) Number of localizations versus surface area . For N = 6 SK - MEL - 2 cells , n = 700 sites the number of localizations found in one clathrin - coated structure was extracted . This is plotted against the quantified surface area determined for each coat . Mund et al . Journal of Cell Biology S6 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023 Video 1 . Pseudo - temporal movie ofclathrin coatremodelingduring endocytosis inSK - MEL - 2 cells . Eachframe correspondsto a slidingwindowaverage of 30 sites , with a frame - to - frame increment of 20 sites . The median pseudotime of those 30 sites is indicated . Provided online is an Appendix , containing a detailed description of the cooperative curvature model . Further , all data shown here are also available in the BioStudies database ( https : / / www . ebi . ac . uk / biostudies / ) under accession number S - BIAD566 . Mund et al . Journal of Cell Biology S7 Remodeling of the clathrin coat during endocytosis https : / / doi . org / 10 . 1083 / jcb . 202206038 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 3 / e202206038 / 1447430 / j c b _ 202206038 . pd f b y gue s t on 09 A ugu s t 2023", + "linEffectivenessTeachingScience1996": "Research in Science Education , 1996 , 26 ( 4 ) , 495 - 511 The Effectiveness of Teaching Science with Pictorial Analogies Huann - shyang Lin and Bih - ruh Shiau National Kaohsiung Normal University Frances Lawrenz University of Minnesota Abstract This study used a conceptual problem solving test to investigate the effect of a series of pictorial analogies on the concepts of density , pressure , and atmospheric pressure in Year 8 classrooms . The analogies were taught following Glynn ' s teaching with analogies model . It was found that the students taught with the pictorial analogies scored significantly higher than their counterparts ( p < . 01 ) . In addition , the low achievers benefited more from this teaching strategy than did the high achievers . Further , qualitative analysis revealed that most of the students ' alternative conceptions were from preexisting naive intuitions rather than arising from analog instruction . In recent years , teaching science with analogies has attracted attention from science educators . Part of the reason for the interest in analogies is that most theoretical scientific concepts are creative inventions or from scientists ' imaginations . The invention may or may not actually exist in the real world . When students need examples in order to acquire understanding , it is believed that analogies can play a role in helping them get an initial sense of the concepts ( Lawson , 1993 ) . Moreover , analogies can be used to help students develop further understanding through applications in different situations . Analogies can be classified in many different ways . Dagher ( 1995 ) grouped them according to whether the analogies were included in text ( text - based ) , or presented by teachers ( teacher - based ) . Gabel and Sherwood ( 1980 ) divided analogies according to whether they were physical objects and manipulated by students ( physical ) , or descriptions of phenomena to which one related mentally ( verbal ) . This study investigated the effect of using teacher - based verbal analogies ( most of them were pictures ) on the physical science achievement of Year 8 students . The effects of teacher - based verbal analogies have been reported by many researchers . However , the results are not consistent . Most researchers found that analogies are capable of enhancing students ' conceptual understanding ( Brown , 1994 ; Clement , 1993 ; Dupin & Johsua , 1989 ; Raven & Cole , 1978 ; Stavy , 1991 ) . But some studies concluded that there were no significant effects at all ( Dowell , 1968 ; Drugge & Kass , 1978 ; Friedel , Gabel , & Samuel , 1990 ) . The reason why students have not profited from analog teaching has been investigated by researchers . It is concluded that both types of analogy ( Drugge & Kass , 1978 ) and students ' levels of understanding of analogies ( Gabel & Sherwood , 1980 ) are two of the key factors affecting students ' learning performance . Additional research was conducted by Thiele and Treagust ( 1994 ) to understand how teachers use analogies in natural classrooms . The two researchers indicated that there was little evidence that teachers preplanned their analogies . In addition , teachers tended to draw upon their experiences or their own professional readings as a source of analogs , which may account for students ' difficulty in understanding the relationship between analog and target concept . Furthermore , when students encounter the work of mapping from an analog to a target concept , if the mapping support from teachers is not sufficient , the potential fbr misrepresentation is created ( Duit , 1991 ; Zook , 1991 ; Zook & Maier , 1994 ) . 496 LIN , SHIAU AND LAWRENZ In contrast to the above studies where analogies were presented by teachers , Wong ( 1993 ) asked students to create , apply , and modify their own analogies for a given scientific phenomenon . In the process of conducting self - generated analogies , students were asked to specify similarities and differences between the analogies they created and the scientific phenomenon . Although factors related to the effectiveness of using teacher - based verbal analogies are many and varied , researchers have reached consensus that the students ' thorough understanding of the analogy used is one of the critical factors . Friedel , Gabel and Samuel ( 1990 ) indicated that teachers had always assumed that their students understood the analogies being taught , saw the relationship between the analog and the corresponding scientific concept , and were able to transfer their problem solving skills from one problem to another . Actually , it is not necessarily true that students understand the meaning of the analogies teachers use . For instance , in the study by Gabel and Sherwood ( 1980 ) , the two researchers were surprised that there was a large number ( 48 % ) of students who did not understand 90 % of the analogies they used in an experimental treatment . A further analysis revealed that students who understood the analogies scored significantly higher than those who did not . In order to increase students ' understanding of the analogies used in teaching , Glynn ( 1989 ) developed the teaching - with - analogies ( TWA ) model which included the following six operations : 1 . Introduce target 2 . Cue retrieval of analog 3 . Identify relevant features of target and analog 4 . Map similarities 5 . Draw conclusions about target 6 . Indicate where analogy breaks down Harrison and Treagust ( 1993 ) followed the TWA model and used it successfully in a Year 10 optics class on refraction of tight . Glynn , Harrison , and Treagust all stressed the importance of helping students understand the meaning of analogies . With a purpose similar to the TWA model , Brown ( 1994 ) and Clement ( 1993 ) developed bridging analogies to facilitate students ' conceptual change ~ They saw these bridging analogies as conceptually intermediate between situations drawing out valid anchoring intuitions and target problems drawing out naive conceptions . For example , in the teaching of a unit on force , a sequence of analogies was used . The anchor analogy is a hand pressing down on a spring , which is used to let students believe that there is an upward tbrce . The intermediate analogy is putting a book on a flexible board between two sawhorses , in which students still can see the bending of the board . The target problem is putting the same book on a table and asking whether the table pushes up on the book . This teaching strategy of bridging analogies has been used with apparent success ( Clement & Brown , 1984 ; Brown , 1992 ) . A review of the inconsistent results of the research studies described above indicates that more research is needed to determine the effect of using teacher - based verbal analogies . A closer look reveals that most of the studies focused on students ' \" algorithmic problem solving ability . \" Students ' \" conceptual problem solving ability ' , was rarely measured . Science educators have assumed that success in solving mathematical problems indicates mastery of a scientific concept . Consequently , more algorithmic manipulation problems than conceptual problems were included in school tests and research studies . Recently , however , researchers have become more aware of the situation and found that students ' conceptual problem solving ability lagged tar behind their algorithmic problem solving ability ( Nakhleh , ~ \" 1993 ; Nakhleh & Mitchell , 1993 ) . Nakhleh and Mitchell concluded that science teaching must take on a much more concept - based approach . Teacher - based verbal analog teaching is much more closely related to concept - based teaching than algorithm - based teaching . Therefore , the measurement of students ' achievement in these settings should be focused on their conceptual problem solving ability . TEACHING SCIENCE WITH PICTORIAL ANALOGIES 497 Since Brown ' s ( 1994 ) and Clement ' s ( I 993 ) bridging analogy and Glynn ' s ( 1989 ) TWA model have already shown positive results , they were used in this study . Pictorial analogies on the topic of density , pressure , water pressure and atmospheric pressure were developed and used with junior high school students who were struggling to grasp the meaning of these theoretical physical science concepts . The primary purpose of this study was to determine the effect of using teacher - based verbal analogies on students ' conceptual problem solving ability . A secondary purpose of the study was to identify whether high or low achievers profited more from the treatment . The final purpose of the study was to find out what prevents students from correctly solving conceptual problems . Methodology Instrument The test used to monitor students ' conceptual problem solving ability consisted of tour open - ended , essay - type items ( see Appendix ) . For each item , students applied appropriate concepts of density , pressure , water pressure , and atmospheric pressure to explain or predict a phenomenon . Content validity of the test was assured by asking four experts ( two faculty chemists and two science educators ) and two high school science teachers to rate the degree of the representativeness of the content covered , the readability , and the clarity of the items . After rating the students ' responses , the Cronbach alpha reliability of the test was found to be document . 76 . Item I was based on the principle that the boiling point of water depends on the magnitude of the atmospheric pressure on the system . The lowered air pressure caused by the cooling of the ice on the tube , is the main reason that the water boils again . Item 2 asked students in which direction a drop of mercury inside a glass tube connected to a flask would move when the flask was submitted to varying temperatures . The students with only rote learning about the nature of gases could get the first and second parts of the item correct . However , on the third part , they needed a deeper understanding . When the surrounding pressure is kept constant , the volume of the air inside the flask sealed by the mercury ( i . e . , the volume of the system ) will expand or shrink depending on the temperature of the air in the flask . Item 3 dealt with the balance of atmospheric pressure and water pressure . While the bottle is closed by the hand , the atmospheric pressure overcomes the water pressure and prevents the water flowing out the small hole . When the bottle is open , the top of the bottle and the small hole have equal air pressure . Consequently , the water pressure itself will push the water out of the bottle from the small hole . Item 4 examined students ' ability to apply the concept of density in a practical situation . Since the density of gold is given and the two pieces have the same size and shape , the piece with a density of 19 . 3 gcm \" 3 would be the real gold . Scoring Scheme of the Instrument The students ' answers were graded by three science educators with the following scheme for each item : answers with irrelevant statements or misconceptions were given 0 points ; answers with partial misconceptions but that indicated some degree of relevance toward the target concept were assigned 1 point ; answers with sound arguments but minor mistakes of using apparatus or concepts were assigned 2 points ; answers with correct statements and use of target concepts were assigned as 3 points . 498 LIN , SHIAU AND LAWRENZ ( a ) ( b ) ( c ) 1 C ~ ~ Figure 1 . Pictorial analogy of density . TEACHING SCIENCE WITH PICTORIAL ANALOGIES 499 Treatment The pictorial analogies were developed by the authors of this paper . For the concept of density , the analog used to enhance students ' understanding is shown in Figure 1 . In Figure 1 , there are three parts . Part A : Suppose a volley ball has the same mass ( 100g ) as a baseball . Now we have two weightless containers with the same volume . One can be filled with 5 volley balls and the other one can be filled with 30 baseballs . The first one weighs 500g and the other one 3000g . The two containers are then compared to wood and iron . Part B : If we can evenly cut 1 cm 3 from each of the two containers , then the weight of the wood piece ( 0 . 6g ) would be much less than the iron piece ( 7 . 9g ) . At this time , the definition of density was introduced . Part C : When iron is manufactured in different shapes , a 1 cm 3 cut from each of them would have the same mass and the same density . This situation is analogous to that when a small cup of sugar water is taken from a pitcher , it still has the same sweetness . I w I / / / / / I / I ( A ) ( B ) w / I / I / I W / I I I I I I I I Figure 2 . Pictorial analogy of pressure . 500 LIN , SHIAU AND LAWRENZ In order to make sense of the difference between force and pressure , the picture in Figure 2 was shown to students . They were asked : Which way would be easier for lifting the wood ? How much more weight should the people in ( B ) push up than the people in ( A ) ? Subsequently , the bridging case of the spring replacing the person was introduced . Students were asked for the similarities and differences between it and the people case . Finally , students discussed how putting the same piece of wood on a piece of foam in different ways would cause it to sink to different depths in the foam . Toward the end of the lesson , students were reminded , in the above three cases , the pi ' essure that the people , the spring , and the foam experienced are related to the area and the force pushing on that area . In the three cases , the force which resulted from gravity was equal to the weight of the wood . After in ~ oducing the equation P = F / A , students discussed some applications in small groups : for example , why does it hurt more when stepped on by a high heel shoe than by a flat sports shoe ? Students were asked to figure out and explain the relationship of force , pressure and area in this question . latin ( a ) 1033 wa 76cm U ~ ( b ) ( c ) 0 i 0 0 0 t , O Figure 3 . Torricetli ' s experiment , pictorial analogy , and demonstration of atmospheric pressure . TEACHING SCIENCE WITH PICTORIAL ANALOGIES 501 It is common in textbooks to introduce the concept of atmospheric pressure by presenting Torricelli ' s 1643 experiment ( as shown in Figure 3 part a ) . However , there is no guarantee that this will help students understand the existence of atmospheric pressure . In this study , after showing the Torricelli experiment , in order to stress the existence of air pressure , an analog like Figure 3 part b was introduced . The teacher showed and explained that the weight of air pushes down on the surface of the mercury ( like the compressed spring ) in the container , but not on the top of the mercury in the tube ( like the uncompressed spring ) . Toward the end of the lesson , students were asked whether the mercury or the water would stay higher in the tube of Torricelli ' s experiment . They predicted the height of different liquids with various densities . At the end of the discussion , a demonstration was used to let students see the \" push action \" of atmospheric pressure . Shortly after burning a piece of paper inside a I000 mL flask as in part C of Figure 3 , a cooked and shell - peeled egg was put on the mouth of the flask . Students were able to see the egg was quickly pushed by the atmospheric pressure into the flask , because the pressure inside the flask was lower than the outside atmospheric pressure . They were asked why it happened and how the egg could be taken out of the flask . For the concept of water pressure , the pictorial analogy in Figure 4 was shown to students . In part a , the water is referred to as a well - sequenced pile of people . \" The deeper the water , the higher the pressure \" is analogised so that the people at the lowest level of the pile feel the most pressure . Their pressure is also related to the average weight of people above them . After students had an initial understanding , the meaning of the formula P ( pressure ) = H ( height ) x D ( density of the liquid ) was explained . The demonstration of part b was followed by an explanation to stress the relationship between height of the water and its pressure . ( a ) ( b ) Figure 4 . Pictorial analogy and demonstration of water pressure . 502 LIN , SHIAU AND LAWRENZ Procedure Three physical science teachers and six classes of Year 8 students ( N = 225 ) participated in this study . All of them were from a typical school located in central Kaohsiung City of Taiwan . In this school , students were from families representing a broad range of socioeconomic backgrounds . The three teachers were recommended by the school principal and were interested in trying out new teaching strategies . Two of them had 12 years and one had eight years of teaching experience . Originally , it was intended to select two teachers only . However , when the two teachers were asked if they would teach a class without using the pictorial analogies , they refused because they felt morally obligated to use the materials once they had learned about them . They simply could not keep these materials away from their students . Therefore , the authors were forced to use another teacher ' s two classes as the control group . Two classes were selected from each of the two teachers ' sets of classes as the experimental group . In the selection of the classes , an attempt was made to minimise the initial differences between them . Classes were matched on students ' learning achievement ( grade point average ) for each class , and the students ' grades on the first midterm examination of physical science . Each student was classified either as a high achiever or a low achiever based on her / his performance on the first term examination . The cut off point for forming these groups within the experimental and control groups was the median score for each group . After the selection and assignment of the groups was completed , the two teachers of the experimental group participated in a half day workshop . The TWA model was discussed and the pictorial analogies were reviewed until the teachers were comfortable and confident in their use . The teachers were asked strongly to remind their students about the unshared attributes between an analog and its target concept . For example , in Figure 1 of the pictorial analogy for density , students were to be reminded that neither the shape nor the structure of the particles ( molecules ) of wood or iron were like volleyballs or baseballs . All of the pictorial analogies were produced as transparencies for the teachers ' convenience . The teacher in the control group taught the same way as before , which did not include pictorial analogies . It should be noted that in this one and one - half month long quasi - experimental study , all of the six classes devoted the same amount of time to the learning of these concepts . In addition , classroom transactions of each teacher were all video - taped and analysed to assure that the experimental group students were taught with the pictorial analogies . The control group teacher rarely used analog teaching . Instead , her students engaged in extra problem solving . Although both of the control and the experimental group students are all in the same school , because the recess time for each period is as short as 10 minutes , it is not likely that there was cross - talk between students who received the verbal - pictorial analogies and those who did not . At the end of the experiment , one class was randomly selected from each of the three teachers ' classes to take the conceptual problem solving test . In order to assure that every student understood the meaning of each test item , the authors set up the equipment and demonstrated the test , item by item for each class . Students were encouraged to state freely their answers on the answer sheet . It took one hour for each class test . Data Analysis Three science educators were asked to grade the conceptual problem solving test . The first author explained the scoring scheme and gave examples for each criterion . Then they pilot - graded ten students ' answersfor the first item of the test and together checked their gradings . If a student ' s score differed by more than one point , a discussion tbllowed and continued until a consensus was needed about how to assign an appropriate score tbr that situation . Finally , they separately graded the rest of the students ' item one answers . The second , third , and the fourth item of the test were graded using TEACHING SCIENCE WITH PICTORIAL ANALOGIES 503 the same procedure as for the first item . The correlational analysis on the three science educators ' grading revealed a high consistency . The inter - rater correlational coefficients ranged from . 93 to . 95 . Each student ' s total score was counted as the average of the three graders ' scores . Using the students ' scores on the school ' s first term examination ( well - developed and conducted by the school authority ) as the covariate , and the students ' conceptual problem solving scores as the dependent variable , a two - way analysis of covariance ( ANCOVA ) was conducted to investigate whether the experimental group was significantly different from the control group and if the teaching strategy benefited certain ability level students . In addition , qualitative analysis of students ' answers was used to understand students ' alternative conceptions and the barriers that prevented them from correctly solving a problem . The first term examination score was used as the covariate because it was the most highly correlated with conceptual understanding and there was a homogeneity of the slopes of regression on the covariate and the dependent variable . This meant that the slopes of the control group and the experimental group were parallel and their initial difference on the covariate could be adjusted for the comparison of the dependent variable ( Howell , 1982 ) . Quantitative Results and Discussion The two - way analysis of covariance revealed that both of the two independent variables had statistically significant effects . First , there was a significant effect associated with the instructional approach of the teacher - based pictorial analogies ( F = 9 . 11 ; df = 1 ; p < . 01 ) ; and second , there was a significant difference between various learning ability levels ( F = 6 . 22 ; df = 1 ; p < . 05 ) . There was , however , no interaction between the two factors ( F = 2 . 08 ; dr = I ; p > . 05 ) . The result also revealed that five percent of the variance was accounted for by the instructional approach , three percent by learning ability level , and 29 % by the covariate ( i . e . , the first term examination ) . Table 1 shows the means and standard deviations of the control and experimental group for students ' achievement on the conceptual problem solving test and the covariate ( Science 1 ) . It can be seen that the adjusted mean of the experimental group ( 3 . 94 ) is significantly higher than the adjusted mean of the control group ( 2 . 55 ) . In order to examine the effect on low and high achievers , mean analysis and t - tests were conducted . Table 2 shows the high and low achievers ' means and standard deviations on the covariate and the conceptual problem solving test . For the high achievers the adjusted mean score of the experimental group is higher than the adjusted mean of its counterpart ( 5 . 04 vs . 4 . 08 , p = . 20 ) , but the difference is not statistically significant . Table I Means and Standard Deviations of the Conceptual Problem Solving Test for Experimental and Control Groups Mean scores ( SD ) Group N Science 1 Adjusted Experiment 69 49 . 07 3 . 94 \" * ( 22 . 43 ) ( 0 . 25 ) Control 36 59 . 80 2 . 55 ( 18 . 56 ) ( 0 . 36 ) * * t - test for difference in adjusted means , p < . 01 504 LIN , SHIAU AND LAWRENZ Table 2 The High and Low Achievers ' Means and Standard Deviations the Conceptual Problem Solving Test Mean scores ( SD ) Ability Group N Science I Adjusted High Experiment 35 67 . 31 5 . 04 ( 13 . 81 ) ( 0 . 34 ) High Control 18 74 . 83 4 . 08 ( 9 . 77 ) ( 0 . 48 ) Low Experiment 34 30 . 29 2 . 70 \" * ( 11 . 13 ) ( 0 . 37 ) Low Control 18 44 . 78 1 . 16 ( 11 . 03 ) ( 0 . 54 ) * * t - test for difference in adjusted means , p < . 01 In contrast , the comparison of low achievers on the control and experimental groups showed a statistically significant difference . The low achievers in the experimental group outperformed their counterparts ( 2 . 70 vs . 1 . 16 , p < . 01 ) . This indicates that the main effect is due to the low achievers . As mentioned earlier , the design of the study assigned different teachers for the control and experimental groups . A major concern arises from this design ; that is , how can we be sure that the difference between the experimental and control groups is really from the treatment instead of from the different teacher ' s teaching ' ? . There is no easy way to answer this question . However , in order to be more confident , we observed the control and the experimental group students ' learning progress pattern to check if there were any difference in the patterns . The results of term examinations conducted by the school authority were used for comparison . Since the correlational analysis revealed that the scores on the term examinations are highly related to the study ' s conceptual problem solving scores ( r ranged from . 68 to . 70 , p < . 0001 ) , it is reasonable to analyse the term examination scores for reference . As can be seen from Figure 5 , among the first , second , and third term examinations , the comparison of the control and the experimental groups ' achievement reveals that the second one has the narrowest margin ( mean difference = 6 ) , while the first and the third term examinations both have an 11 point margin . This result indicates that the treatment ( which occurred between the first and the second term examinations ) resulted in an effect on students ' performance on the school examinations . Apparently , without the treatment , the students ' performance returned to the original level ( mean difference = 11 ) . Two further comparisons between the control and the experimental groups on the second and the third term examinations provide more evidence on the effect of the treatment . The first comparison uses the first term examination score as a covariate , and the second term examination score as the dependent variable . It is apparent that there was a significant treatment effect ( F = 5 . 87 ; df = 1 ; p < . 05 ) . Looking at the adjusted means of the two groups in Table 3 reveals that the experimental group outperformed its c ' 6unterpart ( 46 . 78 vs . 42 . 79 , p < . 05 ) . The same procedure was used for the second comparison except that the third term examination score was used as the dependent variable . TEACHING SCIENCE WITH PICTORIAL ANALOGIES 505 Mean 80orr 60 , o 55 ' 50 ' x 45 ' o : control group x : experimental group Ist 2nd 3rd Term examination Figure 5 . The learning progress pattern of the control and the experimental group . The ANCOVA result indicates that there was no statistically significant difference on the third term examination score between the two groups . Again , Table 3 provides the adjusted means of the two groups . The adjusted mean score of the experimental group ( 52 . 10 ) is very close to the adjusted mean score of the control group ( 51 . 87 ) . The above two comparisons and the pattern of Figure 5 provides more confidence in the treatment effect for the following reasons . If the experimental group ' s performance on the second term exam resulted from teacher effects , then why didn ' t it happen on the third term examination which dealt with different content without using analogies ? In addition , how would one account for the consistent differences between the two groups on the first and the third term examinations , while the difference on the second term examination is unique ? Table 3 Means and Standard Deviations of the Second and Third Term Examinations for Experimental and Control Groups Mean scores ( SD ) Group N Science 1 Adjusted - second Adjusted - third examination examination Experiment 69 49 . 07 46 . 78 * 52 . I0 ( 22 . 43 ) ( 1 . 37 ) ( 1 . 61 ) Control 36 59 . 80 42 . 79 51 . 87 ( 18 . 56 ) ( 1 . 89 ) ( 2 . 26 ) * t - test for difference in adjusted means , p < . 05 Results and Discussion Table 4 shows the main alternative conceptions about boiling and atmospheric pressure , pressure , and density as well as the problematic aspects of understanding held by the students . As can be seen , most of the alternative conceptions are from students ' naive intuitions instead of instruction . For example , in Item 1 , even if students were taught that the boiling point of water will change when the water is heated at different altitudes , some students still answered that the boiling of water at a 506 LIN , SHIAU AND LAWRENZ temperature under 100 \" C was not really boiling . Despite this alternative conception , some students correctly performed the lbllowing algorithmic problem solving item that appeared in the school term examination : What is the atmospheric pressure at 2000 metres up on a mountain ? Would the boiling point of the water heated on the mountain be higher or lower than 100 \" C ? The students successfully used the mathematical formula and calculated the atmospheric pressure at that height : P = 1033 . 6 gcm ~ - - 10 . 9gcm z x H / t 00 , where the H is the height of the mountain . They could solve the problem algorithmically but had alternative conceptions . This result seems to support the ' conclusion of Anderson and Smith ( 1987 ) , Gussarsky and Gorodetsky ( 1990 ) and Helm and Novak ( 1983 ) . These researchers concluded that students bring to instruction a variety of naive alternative conceptions that are often at odds with scientific ideas , In a problem solving situation , the students are likely to make their own associations based on concepts used in their daily living to justify an answer . For Item 2 , it is also apparent that students ' predictions were based on their daily experiences rather than scientific reasoning : \" Heating the empty flask wilt create steam inside the flask to push the mercury outward , \" and \" The moving motion of the mercury will be like the moving of the mercury in a working body thermometer . \" It can be seen that students ' familiarity with cooking food and reading thermometers influenced their explanations . In addition , students had difficulty in identifying the variables involved in the problem . Most students answered that \" The mercury won ' t move , since both the left side and the right side of the mercury have the same temperature . \" They regarded the situation of part three of the item as different from part one or part two , in which , the flask has been submitted to a different temperature . In fact , the \" system \" is the air inside the flask sealed by the mercury . Since the system ' s volume is not a constant ( i . e . , the mercury is moveable ) , when the temperature around the \" system \" is decreased at the condition of n ( number of moles of air ) and p ( pressure of the surrounding ) being fixed ( p = I atm ) , the volume of the \" system \" will be decreased . In other words , it is cooling the \" system \" at constant pressure . Table 4 Summary of Students ' Alternative Conceptions and Difficulties About Density , Atmospheric Pressure , and Pressure 1 . Water can only be boiled at 100 \" C . Any building happened below 100 \" C is a fake phenomenon . 2 . A turbulence caused by a confrontation between a cold front and a hot front will result in building of the liquid . 3 . When heat is trapped inside a test tube , the water inside the test tube will boil , even without providing any additional heat . 4 . When water flows out from the bottle , the space originally occupied by the water must be replaced by air , otherwise the water can ' t flow . 5 . Heating the empty flask in item 2 will create steam inside the flask to push the mercury outward . 6 . The moving motion of the mercury in item 2 will like the moving of the mercury in a working body thermometer . 7 . General problematic aspects of problem solving ( a ) verbalisation and language difficulties ( b ) improper handling of the density definition ( c ) lack of , mastery of the pressure concept ( d ) difficulty of identifying variables ( factors ) involved in problem solving situation TEACHING SCIENCE WITH PICTORIAL ANALOGIES 507 In Item 3 , the students ' alternative conception number 4 in Table 4 deserves a special note . Again , this is a student ' s spontaneous naive intuition and coincidentatly , this conception is much like the seventeenth century doctrine of \" horror vacui . \" At that time the Aristotelian science tbllowers believed that nature abhors a vacuum . Because today students are experienced in the use of straws to drink and the use of a pipette to transfer fluids , it is easy for them to believe that the space originally occupied by the water in a bottle must be replaced by air . They simply believe that when the water flows out from the bottle , it is like the use of a straw . The air sucked from the straw is soon replaced by the drink . The finding of this alternative conception provides support for the argument of Matthews ( 1994 ) who pointed out the similarity between the students ' naive intuition and ancient scientists ' beliefs about air pressure . Theretbre , the addition of history of science in science teaching may help more students construct scientific concepts from naive concepts . For Item 4 , the majority of the students have difficulty in making hypotheses and designing experimental procedures to identify the real gold . These two scientific processing skills are rarely practised in most classrooms and laboratories . Most experiments were carried out by students as recipes like pertbrming cooking with the required procedures and directions provided . If students do not have the chance to explore inquiry procedures , they may remember the knowledge successfully from their teacher ' s instruction . However , it would be difficult for them to apply this knowledge in a different situation , because they are not equipped with the two critical scientific processing skills . Although quite a few students still held naive conceptions and did not construct the scientific concepts successfially even after the treatment with pictorial analogies , the significant progress of the experimerltal group should not be ignored . Especially , it is encouraging that there is almost no indication of negative results such as those described by Zook and Di Vesta ( 1991 ) . They pointed out that some students ' misconceptions were the products of teaching because of the analogies used by both teachers and textbooks . The results of this study show that if the analogies are appropriately presented , such as the systematic use of analogies in the TWA model and the bridging analogies , the negative effect can be avoided . While looking at the positive effect of the study resulting from the treatment of analogies , readers are reminded that the novelty effect ( Gay , 1981 ) should be considered . As mentioned in the methodology part , the experimental group teachers refused to teach a class for comparison without using the pictorial analogies . This ethical issue indicates that these teachers might quickly recognise the power of the pictorial analogies to enhance understanding . This raises an important question in the study : How much did this enthusiasm affect their teaching ? Although the teaching of analogies lasted for one and one - half months and enthusiasm may have diminished , this study did not investigate whether the gains were due to \" cold \" rationalism or \" hot \" motivational factors . This can be investigated in future studies by changing the experimental design or extending the treatment to a longer period of time ( Gay , 1981 ) . Conclusion and Implications for Science Education The results of the study appear to confirm the research report of Dreistadt ( 1969 ) , Dupin and Johsua ( 1989 ) and Raven and Cole ( I 978 ) . However , the above researchers concluded that the effect of analogies was mostly on students ' algorithmic problem solving ability . This study added the improvement of students ' conceptual problem solving ability as one more of the benefits of using teacher - based pictorial analogies in science teaching . A further analysis revealed that the low achievers benefited more from the teaching strategy than did the high achievers . The reason could be as indicated by Gabel and Sherwood ( 1980 ) : that is , since the capable students are able to think at the formal level , it is not necessary to make the theoretical concepts concrete for them . 508 LIN , SHIAU AND LAWRENZ The quantitative and qualitative results together indicate the fruitful efficacy of using teacher - based verbal analogies . In the quantitative analysis , the statistically significant difference between the experimental and the control group students ' achievement on the conceptual problem solving test revealed the effectiveness of this teaching strategy . The additional analysis on the students ' progress pattern on the three term examinations confirmed that the difference of achievement between the two groups was mainly attributable to the treatment and not to a teacher effect . Finally , the qualitative results indicated that no negative effect was found from the analog teaching61 It is apparent that if analogies are presented as research studies recommend , students ' learning outcomes can be enhanced . Unfortunately , analogies are not used by science teachers as often as expected ( Treagust , Duit , Joslin , & Lindauer , 1992 ) . In addition , Treagust ( 1993 ) points out that the majority of science teachers have no formal training in the use of analogies . In order to provide empirical evidence , this study followed the recommendations of former analog studies in developing pictorial analogies and providing a halt - day inservice training for the teachers involved with the experimental group . The fruitfulness of the study can be used to encourage the implementation of teacher - based pictorial verbal analogies in science classrooms . Despite the positive et - ti ~ ct of teacher - based verbal analogies , the students still performed poorly on the conceptual problem solving test . The tact that students ' conceptual problem solving ability lags tar behind algorithmic problem solving ability has been also noted by Nakhleh ( 1993 ) . In this study , the students scored only a mean of four points out of a possible total of 12 points on the conceptual problem solving test . Their difficulties were varied in many ways , ranging from various alternative conceptions to improper handling of a concept ' s definition . It may not be easy for students to describe , predict , and explain phenomena in the natural world , since they were rarely asked to do so in previous examinations . Most examinations , including standardised tests , centre on computational skills and recalling definitions . Questions that require students to synthesise information and apply concepts are not very common in such examinations . The students ' low achievement on the conceptual problem solving test may serve to remind science educators that more efforts are needed to focus on conceptual - teaching pedagogies . Goswami ( 1991 ) indicated that even young children can reason analogically in both classical and problem analogy tasks as long as they have knowledge of the relations used in the analogies . However , even a simple analog leaves great scope for students to arrive at their own conclusion about the related science content . Most analogies in textbooks are provided with limited extent of mapping between analog and target ( Thiele , Venville , & Treagust , 1995 ) . In addition , not only do teachers tend to use analogies spontaneously rather than in a pre - planned way , but they always draw on their own experiences ( not students ' prior knowledge ) as a source of those analogies ( Thiele & Treagust , 1994 ) . This appears to result in difficulties tbr some students . If positive effects are expected from the use of analogies in science teaching , based on the results of this study , then pre - planned and systematically presented bridging analogies are strongly recommended . Acknowledgement The authors gratefully thank the two anonymous reviewers of this article for their constructive contributions and helpful comments . In addition , this research was made possible by the financial aid from the National Science Council ( NSC 84 - 2511 - S - 017 - 005 ) , Taiwan , Republic of China . Correspondence : Huann - shyang Lin , Department of Chemistry , National Kaohsiung Normal University , Ho - ping 1st Road , Kaohsiung , Taiwan . Internet email : t 1666 @ nknucc . nknu . edu . tw TEACHING SCIENCE WITH PICTORIAL ANALOGIES 509 References Anderson , C . W . , & Smith , E . L . ( 1987 ) . Teaching science ( Report Series No . 169 ) . East Lansing , MI : Michigan State University Institute for Research on Teaching . 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Journal of Educational Psychology , 86 ( 4 ) , 589 - 600 . Appendix The Conceptual Problem Solving Test Give reasons supporting each answer or describe your answer to the following questions . 1 . As shown in the following figure , a test tube is filled with ~ / 5 volume of water and heated to boiling point ( 100 \" C ) . The test tube is then removed from the burner and a rubber stopper ( equipped with a thermometer ) is inserted in the end of the test tube . Why does the water boil again if a sack of ice is put around the upper end of the test tube ( the reading of the thermometer is below 100 ~ 4 f TEACHING SCIENCE WITH PICTORIAL ANALOGIES 511 . As shown in the following figure , an empty flask is sealed with a rubber stopper which includes a glass tube ( at the end of the glass tube , there is a drop of mercury ) . ( a ) Can you predict and explain the movement of the mercury if the flask is immersed in the beaker filled with water of 3 ~ ( b ) Can you predict and explain the movement of the mercury if the flask is immersed in the beaker filled with water of 80 \" C ? ( c ) Can you predict and explain the movement of the mercury if the flask ( not including the beaker ) is put inside the refrigerator of 5 ~ . As shown in the Ibllowing figure , a plastic bottle with a small hole on the side is filled with water . The water flows out from the small hole . As soon as you put your hand on the top of the bottle , the water stops flowing . Why does the water stop flowing ? 4 . In a jewelry store , there are two pieces of golden metal with the same size , shape , and appearance . Knowing that the density of pure gold is 19 . 3gcm 3 , how can you use the following equipment to identify which piece is the real gold ? e e", + "rhyscoxDirectedDiversityLeveraging2021a": "Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation Samuel Rhys Cox \u2217 National University of Singapore , Singapore samuel . cox @ u . nus . edu Yunlong Wang \u2217 National University of Singapore , Singapore yunlong . wang @ nus . edu . sg Ashraf Abdul National University of Singapore , Singapore ashrafabdul @ u . nus . edu Christian von der Weth National University of Singapore , Singapore vonderweth @ nus . edu . sg Brian Y . Lim \u2020 National University of Singapore , Singapore brianlim @ comp . nus . edu . sg ABSTRACT Crowdsourcing can collect many diverse ideas by prompting ideators individually , but this can generate redundant ideas . Prior methods reduce redundancy by presenting peers\u2019 ideas or peer - proposed prompts , but these require much human coordination . We introduce Directed Diversity , an automatic prompt selection ap - proach that leverages language model embedding distances to maxi - mize diversity . Ideators can be directed towards diverse prompts and away from prior ideas , thus improving their collective creativity . Since there are diverse metrics of diversity , we present a Diver - sity Prompting Evaluation Framework consolidating metrics from several research disciplines to analyze along the ideation chain \u2014 prompt selection , prompt creativity , prompt - ideation mediation , and ideation creativity . Using this framework , we evaluated Di - rected Diversity in a series of a simulation study and four user studies for the use case of crowdsourcing motivational messages to encourage physical activity . We show that automated diverse prompting can variously improve collective creativity across many nuanced metrics of diversity . CCS CONCEPTS \u2022 Human - centered computing ; \u2022 Collaborative and social computing ; \u2022 Collaborative and social computing theory , concepts and paradigms ; \u2022 Computer supported cooperative work ; KEYWORDS Diversity , Collective Creativity , Crowdsourcing , Ideation , Motiva - tional messaging , Collective Intelligence , Creativity Support Tool \u2217 Co - first authors , ordered alphabetically \u2020 Corresponding author This work is licensed under a Creative Commons Attribution International 4 . 0 License . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan \u00a9 2021 Copyright held by the owner / author ( s ) . ACM ISBN 978 - 1 - 4503 - 8096 - 6 / 21 / 05 . https : / / doi . org / 10 . 1145 / 3411764 . 3445782 ACM Reference Format : Samuel Rhys Cox , Yunlong Wang , Ashraf Abdul , Christian von der Weth , and Brian Y . Lim . 2021 . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation . In CHI Conference on Human Factors in Computing Systems ( CHI \u201921 ) , May 08 \u2013 13 , 2021 , Yokohama , Japan . ACM , New York , NY , USA , 34 pages . https : / / doi . org / 10 . 1145 / 3411764 . 3445782 1 INTRODUCTION Crowdsourcing has been used to harness the power of human creativity at scale to perform creative work such as text editing [ 7 , 21 , 78 ] , iterating designs [ 27 ] , information synthesis [ 54 ] , and motivational messaging [ 4 , 50 , 95 ] . In such tasks , empowering crowd workers to ideate effectively and creatively is key to achiev - ing high - quality results . Different prompting techniques have been proposed to stimulate creativity and improve the diversity of ideas [ 2 , 27 , 50 , 95 ] , but they suffer from ideation redundancy , where multiple users express identical or similar ideas [ 10 , 48 , 76 , 80 ] . Current efforts to avoid redundancy include iterative or adaptive task workflows [ 99 ] , constructing a taxonomy of the idea space [ 40 ] , and visualizing a concept map of peer ideas [ 80 ] , but these re - quire much manual effort and are not scalable . Instead , we propose an automatic prompt selection mechanism \u2014 Directed Diversity \u2014 to scale crowd ideation diversity . Directed Diversity composes prompts with one or more phrases to stimulate ideation . It helps to direct workers towards new ideas and away from existing ideas with the workflow : 1 ) extract phrases from text corpuses in a target domain , 2 ) embed phrases into a vector embedding , and 3 ) automat - ically select phrases for maximum diversity . These phrases are then shown as prompts to ideators to stimulate ideation . The phrase em - bedding uses the Universal Sentence Encoder ( USE ) [ 14 ] to position phrases within an embedding vector space . Using the embedding vectors , we calculated distances between phrases to optimally select phrases that are farthest apart from one another ; this maximizes the diversity of the selected phrases . Hence , Directed Diversity guides ideators towards under - utilized phrases or away from existing or undesirable phrases . The embedding space provides a basis to calculate quantitative , distance - based metrics to estimate diversity in selected phrases and prompts , and subsequently ideated messages . These met - rics can complement empirical measurements from user studies evaluate prompts and ideations . We curate multiple measures CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . and evaluation techniques and propose a Diversity Prompting Evaluation Framework to evaluate perceived and subjective cre - ativity and objective , computed creativity , and diversity of crowd ideations . We demonstrate the framework with experiments on Di - rected Diversity to 1 ) evaluate its efficacy to select diverse prompts in a simulation study , 2 ) measure the perceived diversity of selected prompts and effort to generate ideas in an ideation study , and 3 ) evaluate the creativity and diversity of generated ideas in valida - tion studies using quantitative and qualitative analyses . The exper - iments were conducted with the application use case of writing motivational messages to encourage physical activity [ 2 , 3 , 50 , 95 ] , though we discuss how Directed Diversity can apply to other crowd ideation tasks . In summary , our contributions are : 1 . We present Directed Diversity , a corpus - driven , automatic approach that leverages embedding distances based on a language model to select diverse phrases by maximizing a diversity metric . Using these constrained prompts , crowd - workers are directed to generate more diverse ideas . This results in improved collective creativity and reduced redun - dancy . 2 . A Diversity Prompting Evaluation Framework to evaluate the efficacy of diversity prompting along an ideation chain . This draws constructs from creativity and diversity literature , metrics computed from a language model embedding , and is validated with statistical and qualitative analyses . 3 . We applied the evaluation framework in a series of four experiments to evaluate Directed Diversity for prompt selec - tion , and found that it can improve ideation diversity without compromising ideation quality , but at a cost of higher user effort . 2 BACKGROUND AND RELATED WORK We discuss related research on supporting crowd ideation with the cognitive basis for creative ideation , how creativity support tools help crowd ideation , and how artificial intelligence can help collective intelligence . 2 . 1 Cognitive Psychology of Creative Ideation Different cognitive models of creativity have been proposed to explain how ideation works . Memory - based explanation models describe how people retrieve information relevant to a cue ( prompt ) from long - term memory and process it generate ideas [ 1 , 26 , 53 , 67 , 68 ] . Since retrieval is dependent on prompts , they need to be sufficiently diverse to stimulate diverse ideation [ 68 ] , otherwise people may fixate on a few narrow ideas [ 42 ] . Ideation - based models [ 64 ] explain how individuals can generate many ideas through complex thinking processes , including analogical reasoning [ 36 , 46 , 61 ] , problem constraining [ 84 ] , and vertical or lateral thinking [ 35 ] . We focus on prompting to promote memory - based retrieval than these other reasoning processes . Besides cue - based retrieval and thinking strategies , other factors influence ideation creativity , such as personal traits , motivation to perform the task , and domain - relevant skills that can affect individual creativity [ 90 ] . We provide technological support to improve the creative mental process , rather than to select creative personalities , recruit domain experts , or improve task motivation . Next , we discuss how different cognitive factors have been leveraged at scale to support creative ideation with the crowd . 2 . 2 Creativity Support Tools for Crowd Ideation Creativity Support Tools have been widely studied in HCI to en - able crowdworkers to ideate more effectively and at scale [ 31 , 32 ] . Showing workers ideas from their peers has been very popular [ 18 , 33 , 79 , 81 ] , but can have limited benefit to creativity if peer ideas are too distant from the ideators\u2019 own ideas [ 18 ] . Other ap - proaches include employing contextual framing to prompt ideators to imagine playing a role for the task [ 69 ] or using avatars for vir - tual interactions while brainstorming [ 57 ] . While these methods focus on augmenting individual creativity , they do not coordinate the crowd , so multiple new ideations may be redundant . More re - cent approaches apply provide more explicit guidance to workers . IdeaHound [ 80 ] visualizes an idea map to encourage workers to focus on gaps between peer ideas , but does not inform what ideas or topics will fill the gaps . BlueSky [ 40 ] and de Vries et al . [ 95 ] use crowd or expert annotators to construct taxonomies to constrain the sub - topics for ideation , but these taxonomies require significant manual effort to construct and are difficult to scale . Chan et al . [ 16 ] employed latent Dirichlet allocation ( LDA ) to automatically identify topics , but this still requires much manual curation which does not scale to many topics . With Directed Diversity , we automatically extract a phrase corpus and embed the phrases as vectors , and se - lect diverse phrases for focused prompting . We employ pre - trained language model to provide crowd ideation support , thus we next dis - cuss how artificial intelligence can support collective intelligence . 2 . 3 Supporting Collective Intelligence with Artificial Intelligence Collective Intelligence is defined as groups of individuals ( the collec - tive ) working together exhibiting characteristics such as learning , judgement and problem solving ( intelligence ) [ 56 ] . Crowdsourcing is a form of collective intelligence exhibited when crowdworkers work towards a task mediated by the crowdsourcing platform . How - ever , managing crowdwork to ensure data quality and maximize efficiency is difficult because of the nature and volume of the tasks , and varying abilities and skills of workers [ 97 ] . HCI research has contributed much towards this with interfaces to improve crowd - worker efficiency , designing incentives for workers , and workflows to validate work quality [ 9 , 38 , 62 , 89 , 97 ] . Furthermore , recent de - velopments in artificial intelligence ( AI ) provides opportunities to complement human intelligence to improve the quality and effi - ciency of crowd work [ 47 , 97 ] , optimize task allocation [ 22 , 28 ] , adhere to budget constraints [ 45 ] , and dynamically control quality [ 11 ] . With Directed Diversity , we used AI to optimize ideation di - versity by shepherding the crowd towards more desired and diverse ideation with diverse prompt selection . 3 TECHNICAL APPROACH We aim to improve the collective diversity of crowdsourced ideas by presenting crowdworker ideators , with carefully selected prompts that direct them towards newer ideas and away from existing ones . The prompts presented to the ideators consist of one or more phrases Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 1 : Pipeline of the overall technical approach to extract , embed , and select phrases to generate diverse prompts . a ) Phrase extraction by collecting phrases from online articles and discussion forums ( shown as pages ) , filtering phrases to select a clean subset ( shown as the black dash for each phrase ) ; b ) Phrase embedding using the Universal Sentence Encoder [ 14 ] to compute the embedding vector of each phrase ( shown as scatter plot ) ; c ) Phrase Selection by constructing the minimal spanning tree to select optimally spaced phrases ( see Figure 2 for more details ) . that represent ideas that are distinct and different from prior ideas . Prompts can have one or more phrases . As a running example throughout the technical discussion and experiments , we apply our approach to the application of motivational messages for healthy physical activity , where it is important to collect diverse motiva - tional messages [ 50 , 95 ] . Figure 1 shows the 3 - step overall approach to extract , embed , and select phrases . We next describe each of these steps in detail . 3 . 1 Phrase Extraction We extracted phrases from selected sources of documents with the following semi - automatic data - driven process : 1 ) collect a corpus of documents , 2 ) tokenize documents into sentences , 3 ) extract phrases as constituent structures , 4 ) filter for length , slang , emoti - cons . We collected documents about exercising , weight loss , and healthy living from two types of sources : i ) credible , authoritative health news articles 1 to obtain texts relevant to the domain ( health and fitness ) , and ii ) discussion posts from popular subreddits 2 of online health communities related to fitness and physical activity to obtain texts relevant to the task ( motivational messaging [ 50 , 95 ] ) . Together , the combined corpus contained 3 , 235 articles and 32 , 721 user posts . To extract phrases , we tokenized each document into sentences and performed Part - of - Speech ( POS ) tagging using Python Spacy to select phrases that form syntactic constituents [ 13 ] . From each sentence ( e . g . , \" Regular exercising helps to improve people\u2019s health at any age . \" , we extract verb phrases ( e . g . , \" helps to improve \" ) , noun phrases ( e . g . , \" regular exercising , \" \" people\u2019s health , \u201d \u201cage\u201d ) and prepo - sitional phrases ( e . g . , \u201cat any age\u201d ) . To provide more context to each phrase , we combined adjoining verb and noun phrases to generate noun - verb phrases ( e . g . \u201cregular exercising helps to improve\u201d ) and verb - noun phrases ( e . g . , \u201chelps to improve people\u2019s health\u201d ) . After extracting the phrases , we filtered phrases for length , quality , and relevance . We kept phrases that were 3 to 5 words long , since short phrases may not sufficiently stimulate creativity and long prompts may restrict creativity . Since user posts often contain typographical errors , slang , or other stylistic devices ( e . g . , emoticons ) , we kept phrases that only contain words from a dictionary 3 of American 1 Source of three authoritative websites on health : www . health . harvard . edu , www . medicinenet . com , www . webmd . com . 2 Source of 20 subreddits from www . reddit . com : 90daysgoal , advancedfitness , advance - drunning , bodyweightfitness , c25k , crossfit , fitness , gainit , getmotivated , ketogains , kettlebells , leangains , loseit , motivation , powerlifting , running , selfimprovement , swim - ming , weightroom , xxfitness . 3 Debian Wordlist pagkage . packages . debian . org / es / sid / wordlist and British words . To reduce repetition of phrases , we removed shorter phrases that overlapped with longer phrases ( e . g . , excluded \u201cfederal exercise recommendations\u201d , kept \u201cfederal exercise recom - mendations and guidelines\u201d ) . The final corpus contained clean 3 , 666 phrases . We next describe the construction of the multi - dimensional idea space to characterize how the phrases are separated or similar . 3 . 2 Phrase Embedding The corpus of extracted phrases provides a large set of poten - tial phrases for prompting , but we seek to select phrases that are least similar to one another . For each phrase , we obtain a multi - dimensional vector representation , called an embedding , so that the phrase is a data point in an idea space . Similar work by Siangliu - lue et al . [ 79 ] obtained embeddings of N = 52 ideas by training a Crowd Kernel model [ 91 ] from 2 , 818 triplet annotations is not scalable to our corpus of N = 3 , 666 phrases , since that would need N ( N \u2212 1 ) ( N \u2212 2 ) / 3 = 16 . 4 million triplets . Instead , similar to Chan et al . \u2019s [ 18 ] use of GloVE [ 71 ] , we use pre - trained language models based on deep learning to encode each word or sentence as a vector representation . Specifically , we use the more recent Universal Sen - tence Encoder ( USE ) [ 14 ] to obtain embeddings for phrases in our corpus , compute their pairwise distances , and selected a maximally diverse subset of phrases . Our approach is generalizable to other language embedding techniques [ 98 ] . To obtain the phrase embedding presentation , we use a pre - trained USE model 4 to obtain embedding vectors for each phrase . With USE , all embeddings are 512 - dimensional vectors are located on the unit hypersphere , i . e . , all vectors are unit length , and only their angles are different . Hence , the dissimilarity between two phrase embeddings x i and x j is calculated as the angular distance arccos ( x i , x j ) , which is between 0 and \u03c0 . For our phrase corpus , the pairwise distance between phrases ranged from Min = 0 . 06 to Max = 0 . 58 , Median = 0 . 4 , inter - quartile range 0 . 39 to 0 . 46 , SD = 0 . 043 ; see Appendix Figure 10 . We use the same USE model to compute embeddings and distances for ideated messages . For a dataset of 500 motivational messages ideated in a pilot study with no prompting , the pairwise distance between ideations ranged from Min = 0 . 169 to Max = 0 . 549 , Median = 0 . 405 , inter - quartile range 0 . 376 to 0 . 432 , SD = 0 . 043 ; see Appendix Figure 11 . Table 1 shows example phrases 4 Pre - trained Universal Sentence Encoder model ( https : / / tfhub . dev / google / universal - sentence - encoder / 4 ) , which was trained using both unsupervised learning on Wikipedia , web news , web question - answer pages , and discussion forums , and super - vised learning on Stanford Natural Language Inference ( SNLI ) corpus . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Table 1 : Demonstration of pairwise embedding angular distances between an example text items ( first data row ) and neighbor - ing text items . Text items with semantically similar words have smaller distances . For interpretability , we highlighted words to indicate darker color with higher cosine similarity to the first phrase . a ) Example extracted Phrases Distance Phrase tofirstPhrase app with yoga poses 0 ( self ) yoga really taking off 0 . 284 popular form of yoga today 0 . 304 yoga pants or sweats 0 . 351 of handstand push - ups 0 . 406 on the road to diabetes 0 . 475 b ) Example Ideations from Ideation User Study Distance Ideated Message tofirstIdeation Exercise will release endorphins and you will feel good for a while after doing it . 0 ( self ) Exercise releases endorphins and makes you feel better ! 0 . 171 Exercise relieves stress in both the mind and the body . It\u2019s the best way to get your mental health in check . 0 . 301 We are the leading country in obesity . Do you want to be part of ? 0 . 509 and messages and their corresponding pairwise dissimilarity dis - tances . With the embedding vectors and pairwise distances for all phrases , the next step selects diverse phrases with which to prompt ideators . 3 . 3 Phrase Selection Given the embeddings of the curated phrases , we want to select the subset of phrases with maximum diversity . Mathematically , this is the dispersion problem or diversity maximization problem of \u201carranging a set of points as far away from one another as pos - sible\u201d . Among several diversity formulations [ 20 ] , we choose the Remote - MST diversity formulation [ 37 ] ( also called Remote - tree [ 20 ] or functional diversity [ 72 ] ) that defines diversity as the sum of edge weights of a minimum spanning tree ( MST ) over a set of vertices . It is robust against nonuniformly distributed data points ( e . g . , with multiple clusters , see Table 4 ) . We construct the minimum spanning tree by performing agglomerative hierarchical clustering on the data points with single linkage [ 82 ] . Next , we describe how we select phrases as prompts to direct ideators towards diverse phrases , or away from prior ideas . Figure 2 illustrates the technical approach . 3 . 3 . 1 Directing towards Diverse Phrases . For phrase selection , we aim to select a fixed number of points n from the corpus with maximum diversity . This is equivalent to finding a maximal edge - weighted clique in a fully connected weighted graph , which is known to be NP - hard [ 39 ] . Hence , we propose a scalable greedy approach that uses the dendrogram representation of the MST re - sulting from the hierarchical clustering . Starting from the root , we set the number of clusters to the desired number of phrases n . For each cluster C r , we select the phrase that is most distant from other points , with largest minimum pairwise distance from all points from outside the cluster , i . e . , x r = argmax i \u2208 C r (cid:18) min j (cid:60) C r d (cid:0) x i , x j (cid:1)(cid:19) where x r is the diverse phrase selected in cluster C r , x i is a point in cluster C r and x j is a point in the corpus not in C r , and d is the pairwise distance between x i and x j . This method has O ( n 2 ) time complexity and runs in less than one second on a desktop PC for 3 . 6k phrases ; it is generalizable and can be substituted for other approximate algorithms to select most diverse points [ 20 , 41 ] . Figure 2 ( top row ) illustrates the phrase selection method to direct towards areas without ideations : a ) Start with all phrases in a corpus represented as USE embed - ding points . b ) Construct a dendrogram ( MST ) from all points , using single - linkage hierarchical clustering . c ) Set # clusters equal to desired number of diverse phrases . For each cluster , find the most distant phrase . d ) Selected phrases are the approximately most diverse from the corpus , for the desired number of phrases . 3 . 3 . 2 Directing Away from Prior Ideas . Other than directing ideators towards new ideas with diverse prompts , it is important to help them to avoid prior ideas written by peers . We further propose a method to remove corpus phrases that are close to prior ideas so that ideators do not get prompted to write ideas similar to prior ones . The method , illustrated in Figure 2 ( bottom row ) , is similar as before , but with some changes : a ) Add the embedding points of prior ideas to the corpus . b ) Calculate phrase - ideation distance d ( x Pi , x Ij ) for each phrase x Pi and ideation x Ii and exclude phrases too close to the ideas , i . e . , d < \u03b4 , where \u03b4 is an application - dependent threshold , \u03b4 = 0 . 29 in our case . c ) Same as step ( c ) , but different clusters , since fewer points are clustered . d ) Same as step ( d ) , but different prompts would be selected , even if the number of phrases is the same . 3 . 3 . 3 Directing with Prompts of Grouped Phrases . Instead of prompting with only one phrase , prompting with multiple related terms can help ideators to better understand the concept being prompted and generate higher quality ideas [ 17 , 67 , 83 ] . We extend the phrase selection method to group multiple phrases in a sin - gle prompt using the following greedy algorithm . After step ( a ) , we i ) sorted phrases by descending order of minimum pairwise distance for each phrase to produce a list of seed candidates , ii ) for each seed phrase , perform a nearest neighbors search to re - trieve a specified prompt size ( number of phrases \u0434 in a prompt ) Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 2 : Procedure to direct ideation towards diverse phrases ( top ) and away from prior or redundant ideas ( bottom ) . To attract ideation with diverse prompts : a ) start with embeddings of corpus - extracted phrases ; b ) construct minimum spanning tree ( MST ) ; c ) traverse tree to select distant prompts from clusters ( most distant points as green dots , in clustered phrases as green ellipses ) ; d ) selected prompts are the most diverse . To repel ideation from prior ideas , e ) compute embeddings of prior ideas ( red hollow dots ) ; f ) compute prompt - ideation pairwise distances of all prompts from each prior ideation , exclude phrases ( dotted black circles ) with pairwise distance less than a user - defined threshold ( red bubble ) , and construct the MST with remaining phrases ; g ) traverse MST to select a user - defined number of prompts ; h ) selected prompts are diverse , yet avoids prior ideas . and remove the selected neighbors from the seed list , iii ) repeat seed neighbor selection until n seed phrases have been processed . We grouped the phrases into a prompt and calculate its embed - ding point x Pri as the angular average of all phrases x Pk in the prompt , i . e . , x Pri = \u0434 (cid:205) k = 1 x Pk / Z , where Z = \u0434 (cid:205) k = 1 x Pk 2 is the magni - tude of the vector sum and x Pri is also a unit vector . We then perform steps ( b ) to ( d ) with the prompts x Pri instead of individual phrases . Note that the corpus of prompts will be smaller than the corpus of phrases . This approach has disjoint prompts that do not share phrases , but there can be alternative approaches to group phrases 5 . 4 DIVERSITY PROMPTING EVALUATION FRAMEWORK To evaluate the effectiveness of the Directed Diversity prompt se - lection technique to improve the collective creativity of generated ideas , we define an ideation chain as a four step process ( 3 top ) : 1 ) setting the prompt selection technique will influence 2 ) the creativ - ity of selected prompts ( prompt creativity ) , 3 ) the ideation process 5 Analternativeapproachis , afterstep ( c ) , tosimplygroupnearestneighbors . However , this will cause the prompt embeddings to be shifted after the diversity is maximized , so it may reduce the diversity of the selected prompts . of the ideators ( prompt - ideation mediation ) , and 4 ) the creativ - ity of their ideation ( ideation creativity ) . We propose a Diversity Prompting Evaluation Framework , shown in 3 , to measure and track how creative and diverse information propagates along this ideation chain to evaluate how and whether a creativity prompting technique improves various measures of creativity and diversity in outcome ideas . Note that our proposed framework is descriptive to curate many useful metrics , but not prescriptive to recommend best metrics . 4 . 1 Research Questions and Experiments Prompt stimuli act along the ideation chain to increase ideation diversity , but it is unclear how well they work and at which point along the chain they may fail . We raise three research questions between each step in the ideation chain , which we answer in four experiments ( Section 2 ) with various measures and factors . RQ1 . How do the prompt techniques influence the perceived di - versity of prompts ? ( RQ1 . 1 ) How do they affect diversity in prompts ? ( RQ1 . 2 ) How well can users perceive differences in creativity and diver - sity in these prompts ? These questions relate to the prompt selection technique effectiveness and serve as a manipulation check . We an - swer them in a Characterization Simulation Study ( Section 2 . 1 ) with objective diversity measures , and an Ideation User Study ( 2 . 2 ) with subjective measures perceived prompt diversity measures . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 3 : Diversity prompting evaluation framework to evaluate prompting to support diverse ideation along the ideation chain . We pose research questions ( RQ1 - 3 ) between each step to validate the ideation diversification process . For each step , we manipulate or measure various experiment constructs to track how well ideators are prompted to generate creative ideas . Except for prompt selection , each construct refers to a statistical factor determined from factor analyses of multiple dependent variables . Constructs are grouped in colored blocks indicating different data collection method ( \u25a1 Computed embedding - based metric , \u25a1 ratings from ideators , \u25a1 ratings from validators , \u25a1 thematic coding of ideations ) . RQ2 . How does diversity in prompts affect the ideation process for ideators ? ( RQ2 . 1 ) Do differences in diversity affect ideation ef - fort ? ( RQ2 . 2 ) How well do ideators adopt and apply the content of the prompts ? ( RQ2 . 3 ) How does prompt creativity affect diversity in ideations ? We answer these questions as a mediation analysis in the Ideation User Study ( Section 5 . 2 ) with objective measures of task time and similarity between ideations and stimulus prompts , the - matically coded creativity metrics , and perceived ease of ideation . RQ3 . How do prompt selection techniques affect diversity in ideations ? Having validated the manipulation checks , we evalu - ate the effectiveness of prompt selection techniques in questions in the Ideation User Study ( Section 5 . 2 ) with subjective measures self - assessed creativity and thematically coded creativity metrics , and two Validation User Studies ( Section 2 . 3 ) with subjective mea - sures of perceived creativity . 4 . 1 . 1 Independent variables of Prompt Specifications . We manip - ulated prompt selection technique , prompt count , and prompt size as independent variables ; these are detailed in Appendix Table 6 . We chose Random prompt selection as a key baseline where selec - tion is non - trivial and data - driven based on our corpus , but not intelligently selected for diversity . 4 . 2 Diversity and Creativity Measures of Prompting and Ideation We measured diversity and creativity for selected prompts and generated ideas with embedding - based and human rated metrics . We color code variable names based on data collection method as in 3 4 . 2 . 1 Embedding - based Diversity Metrics for Prompts and Ideations . Although crowd creativity research has focused on the mean pair - wise distance as a metric for idea diversity , our literature review has revealed many definitions and metrics . Here , we describe com - putational metrics calculated from the embedding - based distances . Inspired by Stirling\u2019s general framework diversity framework [ 87 ] , we collect definitions from crowd ideation [ 15 , 27 , 40 , 79 , 80 ] , ecol - ogy [ 24 , 73 , 94 ] , recommender systems [ 29 , 44 , 60 , 93 ] , and theoreti - cal computer science [ 20 , 37 ] . These cover many aspects of diversity to characterize the mean distance and minimum Chamfer distance between points , MST - based dispersion , sparseness of points around the median , span from the centroid , and entropy to indicate the even - ness of points in the embedding vector space . Table 2 and Table 3 describe distance metrics for individual and collective text items , respectively . These metrics describe nuances of diversity , which we illustrate with example distributions in Table 4 . Other measures of Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Table 2 : Metrics of distances between two points in a multi - dimensional vector space . Each metric can be calculated for an individual text item . These metrics can apply to the embedding of phrases or ideations . Metric Definition Interpretation MeanPairwise Distance 1 N \u2212 1 N (cid:205) j = 1 d ( x i , x j ) Average distance of all other points to the current point . MinimumPairwiseDistance min j (cid:60) i d ( x i , x j ) Distance of closest neighbor to current point . This focuses on redundancy and ignores points that are very far from the current point . Table 3 : Metrics of diversity of phrases or ideation embeddings in a vector space . These capture more characteristics of diversity than average distances in Table 2 . Each metric can only be calculated collectively for multiple items . Metric Definition Interpretation Remote - Clique 1 N 2 (cid:205) i , j d ( x i , x j ) Average of mean pairwise distances . While commonly used in crowd ideation studies [ 27 , 44 , 80 ] , it is insensitive to highly clustered points . ChamferDistance 1 N N (cid:205) i = 1 min j (cid:60) i d ( x i , x j ) Average of minimum pairwise distances . Chamfer distance [ 43 ] ( or Remote - pseudoforest [ 20 ] ) measures the distance to the nearest neighbor . However , it is biased when points are clustered . MSTDispersion Mean of MST edge distances 1 | E MST | (cid:205) ( x i , x j ) \u2208 E MST d ( x i , x j ) Popular in ecology research as functional diversity [ 72 ] , and called Remote - tree or Remote - MST [ 20 , 37 ] , this learns a minimum spanning tree ( MST ) of the points , and calculates the sum of edge weights . Span percentile P % d ( x i , \u00af x M ) P th percentile distance to centroid ( \u00af x M = N (cid:205) i = 1 x Mi / N ) ; i . e . , \u201cradius\u201d of distribution [ 12 , 65 ] . We calculate 90 th percentile to centroid ( vs . medoid ) to be robust against outliers and skewed distributions , respectively . Sparseness Mean distance to medoid 1 N N (cid:205) i = 1 d ( x Mi , \u02dc x M ) Sparsity of points positioned around the medoid ( \u02dc x M = argmin x i { N (cid:205) j = 1 d ( x i , x j ) } ) [ 51 , 52 , 77 ] . If points cluster around the medoid , then this metric will be small ( i . e . , not sparse ) . Entropy Shannon - Wiener index for points in a grid partition (cid:205) b f b log ( f b ) This index [ 75 , 86 ] indicates how evenly points are distributed ; more even is more diverse . We calculated entropy for a 2D projection of the USE feature space to avoid high time complexity 6 and divided the space into a 5 \u00d7 5 grid 7 , and counted the frequency f b of points in each bin b . diversity and divergence [ 20 ] can be included in the framework , which we defer to future work . Next , we describe human - subjects ratings to validate these embedding - based metrics with measures that do not depend on the embeddings to avoid circular dependency . 4 . 2 . 2 Creativity Measures for Ideations . Along with the computed diversity metrics , we evaluate with qualitative characteristics of creativity . From creativity literature , we draw from Torrance\u2019s [ 92 ] description of several measures for creativity , including quality , flexibility and originality . Quality measures whether an ideation is \u201cusable , practical , or appropriate\u201d [ 66 ] . We asked ideators to self - assess on a 5 - point Likert scale their message\u2019s effectiveness ( towards motivation ) and creativity . We ask validator crowdworkers to rate each individual ideation on a 7 - point Likert scale whether it is effective ( motivating [ 95 ] ) , helpful 8 [ 50 , 88 ] , and informative [ 50 ] 8 Note that a message could be helpful but written with negative impressions and thus not motivating . towards encouraging physical activity ; rank collections of ideations on effectiveness , informativeness and unrepetitiveness . ; and rate the pairwise difference between ideation pairs from each collection . Note that Directed Diversity was not designed to improve quality , since these metrics were not explicitly modeled . Flexibility [ 85 ] measures how many unique ideas were generated , and originality [ 100 ] measures how infrequently each idea occurs . These require expert annotation to identify distinct categories . We conducted a thematic analysis on the messages using open coding [ 34 ] to derive categories and affinity diagramming [ 8 ] to consolidate categories to themes ( see details in Appendix Table 19 ) . We calculate the flexibility and originality measures based on the coded categories ( fine - grained ) and themes ( coarser ) described in Appendix Table 7 4 . 2 . 3 Creativity Measures for Prompts . As a manipulation check , it is important to verify that prompts that are computed as more diverse , are perceived by ideators as more creative . Since perceived CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Table 4 : Comparison of diversity metrics for canonical examples of distributed points in a 2D space . Points farther apart mean higher diversity . Here , we calculate Euclidean instead of angular distance , but intuitions are similar . creativity encompasses more qualitative effects , computed diver - sity may not be correlated with creativity . Thus , we measure the creativity and usefulness of prompts by asking about prompt under - standability , relevance to domain topic ( physical activity ) , relevance to task 9 ( motivation ) , helpfulness to inspire ideation , and unexpect - edness [ 66 ] along 7 - point Likert scales . 4 . 2 . 4 Mediating Variables for Prompt - Ideation Process . Even if more diverse prompts can facilitate more creative ideation , it is important to understand whether this requires more effort and time , how the consistency of phrases within prompts affect ideation , and how well ideators adopt words and concepts from the phrases into their ideations . We measure effort as ease of ideation with a 7 - point Likert scale survey question . For individual creativity , fluency [ 30 ] is defined as the number of ideas an individual writes within a fixed time . Chan et al . had also measured fluency for an 8 - minute crowd ideation task [ 18 ] . In contrast , we asked ideators to only write one idea per prompt without time constraint , so we measure the in - verse relation of ideation task time to generate one ideation [ 5 ] . Specifically , since task time is skewed , we use \u2013 Lo\u0434 ( ideation time ) to represent fluency . For prompts with more than one phrase , the similarity between phrases can affect their perceived consistency . Therefore , we measure the intra - prompt mean phrase and prompt average phrase Chamfer distances ( Appendix Table 8 ) to indicate the similarity between intra - prompt phrases . We measure the adoption of the prompt ideas by calculating the proportion of words from phrases in the ideations as prompt recall and prompt precision , and computing the prompt - ideation distance between the embeddings of the prompt and ideation ( Appendix Table 9 ) . 4 . 3 Factor analyses to draw constructs from experiment variables With the numerous variables from our experiments , we observed some may be correlated since they measure similar notions or participants may confound questions to have similar meanings . We employed an iterative design - analytical process to organize and consolidate variables into factors with the following steps . 9 Note that a prompt could be relevant to the domain , but not motivating . \u2022 Identify metrics of creativity and diversity from a literature review from various research domains , such as ecology , cre - ativity , crowdsourcing , theoretical computer science , recom - mender systems ( Section 1 . 8 . 1 ) . Ideate additional measures and questions to capture user behavior and opinions when generating and validating ideas . We refine and reduce mea - sures based on survey pilots and usability testing . \u2022 Collect measurements of each metric with different meth - ods : a ) Compute embedding - based metrics from prompts shown and messages written . This was computed individ - ually for each text item ( e . g . , mean pairwise distance ) and collectively for all text items in each prompt technique ( e . g . , Remote - MST diversity ) . b ) Measure perception ratings and behavioral measures regarding reading prompts and ideating messages and rating messages . We asked text ra - tionale to help with interpretations . c ) Measure subjective thematic measures to qualitatively assess the collective creativity with thematic analysis and idea counting . \u2022 Perform factor analysis on quantitative data to organize correlated variables into fewer factors . Variables are first grouped by data collection method 10 and analyzed together . To determine the number of factors , we examined scree plots and verified grouped variables as consistent with constructs from literature . The final number of factors are statistically significant by the Bartlett Test of Sphericity ( all p < . 0001 ) . See Appendix Tables 10 - 17 for the results of the factor analysis , including factor loadings and statistical significance . Table 5 summarizes the learned factors from 42 variables that we developed . \u2022 Perform statistical hypothesis testing using these learned factors to answer our research questions . 5 EVALUATION : APPLYING FRAMEWORK TO STUDY DIRECTED DIVERSITY We have described a general descriptive framework for evaluat - ing diversity prompting . We applied it to evaluate our proposed 10 E . g . , individual text item metrics , collective text items metrics , ratings of text item from ideators , ratings of text item from validators , ratings of collection of text items from validators . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Table 5 : Constructs from factor analyses of variables along ideation chain . Factor loadings in Appendix Tables 10 - 17 . Chain Factor Construct Interpretation P r o m p t C r e a t i v i t y Prompt Distance How distant and isolated the prompt is from other prompts . Prompt Consistency How similar ( consistent ) the phrases in a prompt are . Prompt Dispersion How spread out the selected prompts are from one another . Prompt Evenness How evenly spaced the selected prompts are among themselves . Prompt Unexpectedness Ideator rating of how unexpected a prompt was on a 5 - pt Likert scale . Prompt Understandability Ideator rating of how understandable a prompt was on 5 - pt Likert scale Prompt Relevance Ideator rating of prompt relevant to the domain ( i . e . exercise ) on 5 - pt Likert scale Prompt Quality Ideator rating of the overall quality of prompt on 5 - pt Likert scale . P r o m p t - I d e a t i o n M e d i a t i o n Ideation Fluency Ideator speed to ideate ( reverse of time taken ) . Ideation Ease Ideator ease of ideating based on multiple 5 - point Likert scale ratings . Phrase Adoption Measures the extent of phrase usage from the prompts in the ideation . I d e a t i o n C r e a t i v i t y Ideation Distance How distant and isolated the ideation is from other ideations . Ideation Dispersion How spread out the ideations are from one another . Ideation Evenness How evenly spaced the ideations are among themselves . Ideation Flexibility Count of unique categories / themes across all ideations . Ideation Originality How rare each category / theme is across all ideations . Ideation Self - Quality Ideator self - rating of the overall quality of the ideation on 5 - pt Likert scale . Ideation Quality Validator rating of overall quality of individual ideation on 7 - pt Likert scale . IdeationInformative - Helpfulness Validator rating of informativeness and helpfulness of individual ideation on 7 - pt Likert scale . Ideations Unrepetitive Validator cumulative rating of non - redundancy in collection of ideations . Ideations Informative Validator cumulative rating of informativeness in collection of ideations . Ideations Motivating Validator cumulative rating of overall quality of collection of ideations . Ideations Pairwise Difference Validator rating of difference between a pair of ideations in collection . Directed Diversity prompt selection technique against baseline ap - proaches ( no prompting , random prompt selection ) in a series of experiments ( characterization , ideation , individual validation , col - lective validation ) , for the use case of crowd ideating motivational messages for physical activity . Here , we describe the procedures for each experiment and their results . 5 . 1 Characterization Simulation Study The first study uses computational methods to rapidly and scalably evaluate prompt selection techniques . This helps us to fine tune prompt parameters to maximize their potential impact in later human experiments . 5 . 1 . 1 Experiment Treatments and Method . We varied three inde - pendent variables ( prompt selection , prompt count , prompt size ) to measure the impact on 7 dependent variables of distance and diver - sity metrics . We varied Prompt Selection technique ( None , Random , or Directed ) to investigate how much Directed Diversity improves prompt diversity with respect to baseline techniques . For None prompt selection , we simulated ideation with 500 ideas collected from a pilot study where crowd ideators wrote messages without prompts . We simulated Random selection by randomly selecting phrases from the phrase corpus ( Section 1 . 4 ) and Directed selec - tion with our technical approach ( Sections 1 . 4 to 1 . 6 ) . If we assume that prompt embeddings are an unbiased estimator for ideation embeddings , then this gives an approximation of ideation diver - sity due to prompting . We conducted experiments for directing towards diverse prompts and for directing away from the 500 pilot prior ideations . We varied the number of prompts ( Prompt Count , n = 50 , 150 , . . . , 950 ) to simulate how diversity increases with the number of ideation tasks performed . This investigates how diversity increases as the budget for crowd tasks increases . To investigate how well Directed selection avoids prior ideations , we varied the number of repeller prior ideations ( Repeller Prior Ideations Count , n R = 50 , 100 , 150 , 200 ) . We varied the number of phrases in prompts ( Prompt Size , \u0434 = 1 to 5 ) to simulate ideating on one or more phrases in each prompt . We computed the prompt embedding as the average of all phrases in the prompt . For Random selection , we randomly chose phrases to group together for each prompt . This random neighbor selection will lead to variation in prompt consistency , but does not bias the prompt embedding on average . For Directed selection , phrases in each prompt were chosen as described in Sec - tion 1 . 6 . 3 . 5 . 1 . 2 Results on Manipulation Efficacy Analysis ( RQ1 . 1 ) . We visu - alized ( Figure 4 ) the phrase embeddings to help to interpret how the selected prompts are distributed , whether they are well spread out , clustered , etc . We used Uniform Manifold Approximation and Projection ( UMAP ) [ 59 ] to reduce the 512 dimensions of USE to a 2D projection . Hyperparameters were selected such that the 2D CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 4 : 2D UMAP projection showing how diversely selected prompts and resulting ideation messages are distributed based on Directed or Random prompt selection technique and prompt size ( number in brackets ) . Each point represents the embed - ding of a text item . Light grey points represent all phrases in the extracted corpus , dark grey points represent selected phrases from the simulation study ( Section 5 . 1 ) and blue dots represent the ideated messages written by crowdworkers in the ideator user study ( Section 5 . 2 ) . Gradient lines connect ideation messages to their stimulus prompts . points in UMAP had pairwise distances correlated with that of the 512 - dimension USE embeddings . We can see that Directed prompt selection led to prompts that were more spread out , and less redundant from prior ideation . This is more pronounced for higher prompt size ( \u0434 = 3 ) . Random ( 3 ) had lower diversity than None with tighter clustering of prompts ( grey points in middle - bottom graph ) than of messages ( blue points in left graph ) . This was because Random ( 3 ) prompts averaged their embeddings from multiple phrases , such that this variance of means of points is smaller than the variance of points 11 . We further conducted a characterization study with 50 simulations for each prompt configuration to confirm that Directed Diversity improves diversity and reduces redundancy from prior ideations for various embedding - based metrics ( see Appendix E and Figure 12 ) . 5 . 2 Ideation User Study The Ideation User Study serves as a manipulation check that higher prompt diversity can be perceived by ideators , and as an initial evaluation of ideation diversity based on computed and thematically coded metrics . 5 . 2 . 1 Experiment Treatment and Procedure . We conducted a between - subjects experiment with two independent variables prompt selection technique ( None , Random , Directed ) and prompt size ( \u0434 = 1 and 3 ) , and kept constant prompt count n = 250 . The None condition ( no prompt ) allows us to measure if the quality of ideations become worse due to the undue influence of phrases in prompts . The Random condition provides a strong baseline since it also leverages the extracted phrases in the first step of Directed Diversity . A prompt size of \u0434 > 1 can provide more contexts to help 11 This is analogous to standard error is to standard deviation ideators understand the ideas in the phrases , but may also lead to more confusion if the phrases are not consistent ( too dissimilar ) . Figure 5 shows example prompts that ideator participants see in different conditions . The experiment apparatus and survey ques - tions were implemented in Qualtrics ( see Appendix Figures 13 - 19 for instructions and question interface ) . 5 . 2 . 2 Experiment Task and Procedure . Participants were tasked to write motivational messages and answer questions with the following procedure : Read the introduction to describe the experiment objective and consent to the study . 1 . Complete a 4 - item word associativity test [ 19 ] to screen for English language skills . 2 . Write 5 messages to motivate for physical activity for a fitness mobile app . For each message , one at a time , a ) On the first page , depending on condition , see no prompt or a prompt with one or three phrases selected randomly or by Directed Diversity ( see Figure 5 ) , then write a motivational message in one to three sentences . This page is timed to measure ideation task time . b ) Rate on a 5 - point Likert scale the experience of ideating the current message : ease of ideation ( described in Section 4 . 2 . 4 ) , self - assessed success in writing motivationally , and success in writing creatively ( Section 4 . 2 . 2 ) ; perception of the prompt on : understandability , relevance to domain topic ( physical activity ) , relevance to task ( motivation ) , helpfulness for inspiration , and unexpectedness ( Section 4 . 2 . 3 ) . c ) Reflect and describe in free text on their rationale , thought process , phrase word usage , and ideation effort . We analyze Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 5 : Example prompts shown to participants in different conditions : None ( left , \u0434 = 0 ) , Directed ( 1 ) ( center , \u0434 = 1 ) , and Directed ( 3 ) ( right , \u0434 = 3 ) . Phrase texts would be different for Random ( 1 ) and Random ( 3 ) selection techniques . these quotes to verify our understanding of the collected quantitative data . \u2022 Answer demographics questions , and end the survey by receiving a completion code . 5 . 2 . 3 Experiment Data Collection and Statistical Analyses . We re - cruited participants from Amazon Mechanical Turk with high qual - ification ( \u2265 5000 completed HITs with > 97 % approval rate ) . Of 282 workers who attempted the survey , 250 passed the test to complete the survey ( 88 . 7 % pass rate ) . They were 45 . 2 % female , between 21 and 70 years old ( M = 38 . 6 ) ; 76 . 4 % of participants have used fitness apps . Participants were compensated after screening and were ran - domly assigned to one prompt selection technique . Participants in the None condition were compensated with US $ 1 . 80 , while others with US $ 2 . 50 due to more time needed to answer the additional sur - vey questions about prompts . Participants completed the survey in median time 15 . 4 minutes and were compensated > US $ 8 / hour . We collected 5 messages per participant , 50 participants per condition , 250 ideations per condition , and 1 , 250 total ideations . For all response variables , we fit linear mixed effects models de - scribed in Appendix Tables 20 - 23 . To allow a 2 - factor analysis , we divided responses in the None ( 0 ) condition ( no prompt , 0 phrases ) randomly and evenly to None ( 1 ) and None ( 3 ) . Results are shown in Figure 6 . We performed post - hoc contrast tests for specific dif - ferences identified . Due to the large number of comparisons in our analysis , we consider differences with p < . 001 as significant and p < . 005 as marginally significant . Most significant results reported are p < . 0001 . This is stricter than a Bonferroni correction for 50 comparisons ( significance level = . 05 / 50 ) . We next describe the sta - tistically significant results for prompt mediation check ( RQ1 . 2 ) , mediation analysis ( RQ2 . 1 , 2 . 2 ) , and ideation evaluation ( RQ3 . 1 , 3 . 2 ) . We include participant quotes from their rationale text response where available and relevant . 5 . 2 . 4 Results of Manipulation Check on Creativity and Mediation on Ideation Effort ( RQ1 . 2 , 2 . 1 ) . We discuss findings on how ideators perceived creativity factors in prompts and how prompt config - urations affected their ideation effort . Figure 6 ( Top ) shows that compared to Random , Directed Diversity selected prompts that were more unexpected ( good for diversity ) ; but were slightly more difficult to understand ( by half unit on 5 - point Likert scale ) , very slightly less relevant ( 1 / 4 unit ) , and of slightly lower quality ( 1 / 2 unit ) . However , the relevance of the selected diverse prompts was not explicitly controlled . P173 in Directed ( 1 ) felt that the phrase \u201cfirst set of challenges is\u201d was \u201cstraightforward and gave me the idea of what to write . It was very easy\u201d ; whereas P157 in felt that the phrase \u201creview findings should be\u201d \u201cdidn\u2019t really have anything I could think to tie towards a motivational message . I tried to think of it as looking back to see progress in terms of reviewing your journey . \u201d Random prompts with more phrases were harder to understand , perhaps , because they were randomly grouped and are less seman - tically similar . P128 in Random ( 3 ) found that \u201cthese [ phrases ] were hard to combine since they deal with different aspects of exercise . Also the weight lifting seems to be not the best thing for addressing obesity , so that was hard to work in . \u201d We found that ideation effort was mediated by prompt factors . Figure 6 ( Bottom ) shows that Directed prompts were least easy to use for ideation , and less adopted than Random selected prompts . This is consistent with Directed prompts being less understandable than Random . Ideating with 1 - phrase prompts increased ideation time from 44 . 1s by 21 . 6s ( 48 . 9 % ) compared to None , and viewing 3 phrase increased time further by 11 . 9s . In summary , Directed Diver - sity may improve diversity by selecting unexpected prompts , but at some cost of ideator effort and confusion . This cost compromises prompt adoption and suggests that directing diversity may not work . Yet , as we will show later , Directed Diversity does improve ideation creativity . We analyzed the confound of understandability further in Appendix Section K . Next , we investigate if prompts characteristics mediate more ideation creativity . 5 . 2 . 5 Results of Mediation Analysis of Diversity Propagation from Prompt to Ideation ( RQ2 . 3 ) . We found that prompt configuration and perceived prompt creativity mediated the individual diver - sity of ideated messages ( RQ2 . 2 ) . Appendix Table 21a ( in ) shows that Ideation Mean ( or Min ) Pairwise Distance increased with Prompt Mean ( or Min ) Pairwise Distance by + 0 . 176 ( or + 0 . 146 ) , and marginally with Intra - Prompt Phrase Mean Distance by + 0 . 021 ( or + 0 . 020 ) . This means that farther Prompts stimulated farther Ideations , and higher variety of Phrases within each prompt drove slightly farther Ideations too . Hence , prompt diversity ( mean pair - wise distance ) influenced ideation diversity , and prompt redundancy ( minimum pairwise distance ) influenced ideation redundancy . Ap - pendix Table 21b shows that as Prompt Relevance decreased by one Likert unit ( on 5 - point scale ) , ideation mean pairwise distance decreased by 0 . 0034 ( 7 . 9 % of ideation pairwise distance SD of 0 . 043 ) CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 6 : Results of ideators\u2019 perceived prompt creativity ( Top ) and ideation effort ( Bottom ) for different Prompt Selection technique and Prompt Size . All factors values on a 5 - point Likert scale ( \u2013 2 = \u201cStrongly Disagree\u201d to 2 = \u201cStrongly Agree\u201d ) . Dotted lines indicate extremely significant p < . 0001 comparisons , otherwise very significant with p - value stated ; solid lines indicate no significance at p > . 01 . Error bars indicate 90 % confidence interval . and ideation minimum pairwise distance decreases by 0 . 0056 ( 13 % of SD ) . This suggests that prompting with irrelevant phrases slightly reduced diversity , since users had to have to conceive their own inspiration ; e . g . , P165 in Directed ( 1 ) \u201ccouldn\u2019t make sense of the given messages , so I tried my best to make something somewhat mo - tivational and correct from them . \u201d . Prompt understandability and quality did not influence ideation individual diversity ( p = n . s . ) . In summary , selecting and presenting computationally diverse and less redundant prompts increased the likelihood of crowdworkers ideating messages that are more computationally diverse and less redundant . 5 . 2 . 6 Results on Evaluating Individual , Collective Objective , The - matic Ideation Diversity ( RQ3 ) . Having shown the mediating effects of diverse prompts , we now evaluate how prompt selection tech - niques affect self - assessed creativity ratings , objective diversity metrics of ideations , and thematically coded diversity metrics of ideations . To carefully distinguish between the commonly used mean pairwise distance with the less used minimum pairwise dis - tance , we performed our analyses on them separately . We calculated one measurement of each collective diversity metric in Table 3 for all messages in each prompt selection condition , and computed uncertainty estimations from synthesized 50 bootstrap samples 12 to generate 50 readings of each diversity metric . We performed factor analyses on the metrics as described in Section 1 . 9 , and per - formed statistical analyses on these factors as described in Appendix Table 22 . Analyses on both individual diversity and collective diver - sity measures had congruent results ( Figure 7 ) , though results for collective diversity had more significant differences ( p < . 001 ) . For collective diversity , our factor analysis found that Ideation Disper - sion was most correlated with mean pairwise distance , and Ideation Evenness with entropy and mean of Chamfer distance . Directed ( 3 ) 12 For each dataset , randomly sample with replacement from the original dataset until the same dataset size is reached . improved Ideation Dispersion from None , while Random reduced Dispersion ( even more for 3 vs . 1 phrases ) . Directed prompts im - proved Ideation Evenness more than Random with respect to None . There was no significant difference for self - assessed Ideation Qual - ity ( p = n . s . , Table 23a in Appendix ) . The previous ideation diversity metrics were all computational . We next assess diversity with human judgement based on the - matic analysis . To conserve manpower to evaluate ideations , we limited thematic coding and crowdworker validation to ideations from three conditions of prompts with 1 phrase , i . e . , None , Ran - dom ( 1 ) , and Directed ( 1 ) . From the results of computational met - rics , we expect bigger differences between Directed ( 3 ) and Ran - dom ( 3 ) for this analysis too . From our thematic analysis , we coded 239 categories 13 which we consolidated to 53 themes ( see Table 19 in Appendix ) . Figure 8 shows results from our statistical anal - ysis . We found that ideations generated with Directed prompts had higher Flexibility and Originality in categories and themes than with Random or None . Ideations from Random prompts mostly had higher Flexibility and Originality compared to None , but the theme Originality was significantly lower . This could be because Random prompts primed ideators to fixate on fewer broad ideas ( themes ) , instead of the higher number of fine - grained idea categories . In summary , despite lower ideation ease and understandabil - ity with Directed prompts ( Section 2 . 2 . 4 ) , we found objective and thematic evidence that Directed Diversity improved ideation di - versity compared to Random and None . Next , we describe how crowdworkers would rate these ideations . 13 Example categories ( in themes ) : Pull - ups ( Exercise Suggestion ) , Strong immune system ( Health Benefits ) , Set daily exercise goal ( Goals ) . See Appendix 8 . 7 for full list of categories and themes . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 7 : Results of computed individual and collective diversity from ideations for different prompt configurations . See Figure 6 caption for how to interpret charts . Figure 8 : Results of diversity in categories and themes derived from thematic analysis of ideations . 5 . 3 Validation User Studies The third and fourth studies employed third - party crowdworkers to assess the creativity of ideated messages from the Ideation User Study , to answer ( RQ3 ) How do prompt selection techniques affect diversity in ideations ? This provides a less biased validation than asking ideators to self - assess . We conducted three experiments with different questioning format to strengthen the experiment design . Appendix Figures 20 - 24 details the questionnaires . 5 . 3 . 1 Individual Validation : Experiment Treatment and Procedure . For the individual validation study , we conducted a within - subjects experiment with prompt selection technique ( None , Random , Di - rected ) as independent variable , and controlled prompt size ( \u0434 = 1 ) . Each participant assessed 25 ideation messages chosen randomly from the three conditions . Participants went through the same pro - cedure as in the Ideation user study , but with a different task in step 3 : 3 . Assess 25 messages regarding how well they motivate for physical activity . For each message , a ) Read a randomly chosen message . b ) Rate on a 7 - point Likert scale , whether the message is moti - vating ( effective ) , informative , and helpful ( as described in Section 4 . 2 . 4 ) . c ) Reflect and write the rationale in free text on why they rated the message as effective or ineffective . This was only asked randomly two out of 25 times , to avoid fatigue . As we discuss later , we found that participants confounded the three ratings questions and answered them very similarly ( responses were highly correlated ) , thus , we designed collective validation user studies to pose different questions and distinguish between the measures . 5 . 3 . 2 Collective Ranking Validation : Experiment Treatment and Pro - cedure . The collective validation study had the same experiment design as before , but different procedure step 3 : 1 . Complete 5 trials to rate collections of ideation messages , where for each trial , a ) Study three groups of 5 messages each ( 3 \u00d7 5 messages ) to b ) Rank message groups as most , middle or least motivating , informative , and unrepetitive ( Section 4 . 2 . 4 ) . Instead of rating messages individually , participants viewed grouped messages from each condition side - by - side and answered ranking questions . Messages in each group were selected from those ideated with the same prompt selection technique . By asking participants to assess collections rather than individual messages , we explicitly measured perceived diversity , since the user perceived the differences between all ideations in the collection ; this is more direct than asking them about the \u201cinformativeness\u201d of an ideation , since this could be confounded with \u201chelpfulness\u201d , \u201cteaching some - thing new\u201d , \u201ctelling something different from other messages\u201d , etc . This approach differs from the triplet similarity comparison [ 55 , 91 ] employed by Siangliulue et al . [ 79 ] , and benefits from requiring fewer assessments . We asked participants to rank groups rather than rate them relatively to obtain a forced choice [ 25 ] . Another method to assess diversity involves longitudinal exposure ( e . g . , [ 50 ] ) , but this is expensive and difficult to scale . 5 . 3 . 3 Collective Pairwise Rating Validation : Experiment Treatment and Procedure . The collective pairwise rating validation study fur - ther validates our results with an existing , commonly used measure to rate the difference between pairs of messages , both from the same prompt selection technique [ 27 , 79 ] . We randomly selected 200 message - pairs from None , Random ( 1 ) and Directed ( 1 ) , yielding CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 9 : Results of perceived individual and collective creativity from the three validation user studies . a pool of 600 message - pairs . All steps in the procedure are identical as before except for Step 3 : 3 . Rate 30 message - pairs randomly selected from the message - pair pool , where for each message - pair , a ) Read the two messages b ) Rate their difference on a 7 - point Likert scale : 1 \u201cNot at all different ( identical ) \u201d to 7 \u201cVery different\u201d This complements the previous study by having participants focus on two messages to compare , which is more manageable than assessing 5 messages , but is limited to a less holistic impression on multiple messages . 5 . 3 . 4 Experiments Data Collection and Statistical Analysis . For all validation studies , we recruited participants from Amazon Mechan - ical Turk with the same high qualification as the ideation study . Of 348 workers who attempted the surveys , 290 passed the screening tests to complete the surveys ( 83 . 3 % pass rate ) . They were 50 . 2 % female , between 22 and 71 years old ( M = 38 . 1 ) ; 67 . 5 % of participants have use fitness apps . For the individual validation study , Partici - pants completed the survey in median time 14 . 7 minutes and were compensated US $ 1 . 50 ; for the collective ranking validation study , participants completed the survey in median time 12 . 7 minutes and were compensated US $ 1 . 80 ; for the collective pairwise rating validation study , participants completed the survey in median time 8 . 4 minutes and were compensated US $ 1 . 00 . In total , 740 messages were individually rated 3 , 375 times ( M = 4 . 56x per message ) , 450 message groups were ranked 1 , 350 times ( M = 3 . 00x per message group ) , and 600 message pairs were rated 2 , 430 times ( M = 4 . 05x per message pair ) . To assess inter - rater agreement , we calculated the average aggregate - judge correlations [ 18 ] as r = . 59 , . 62 , . 63 for mo - tivation , informativeness and helpfulness for individual validation ratings , respectively ; these were comparable to Chan et al . \u2019s r = . 64 for idea novelty [ 18 ] . We performed the same statistical analyses as in the Ideation User Study ( see Section 2 . 2 . 3 ) , report the linear mixed effects models in Appendix Table 23 , and include participant quotes from their rationale text response where relevant . For the collective ranking validation study , we counted how often each Prompt Selection technique was ranked first or last across the 5 trials , performed factor analyses on the counts for best and worst ranks for the three metrics ( motivating , informative , unrepetitive ) to derive three orthogonal factors ( Ideations Unrepetitive , Ideations Informative , Ideations Motivating ) , and performed the statistical analysis on the factors ( see Table 23b in Appendix ) . 5 . 3 . 5 Results on Evaluating Individual and Collective Ideation Cre - ativity ( RQ3 ) . We investigated whether Directed prompts stimulate the highest ideation diversity and whether 3 rd - party validations agree with our computed and thematic results . For illustration , Ap - pendix Table 25 shows examples of message - groups with high and low factor values . Figure 9 shows results of our statistical analysis . We found that ideations from Directed prompts were most different and least repet - itive , ideations from Random were no different and as repetitive as None . Ideations generated with prompts were more informative and helpful than without prompts , but there was no difference whether the prompts were Directed or Random . For example , P4 reviewed the message \u201cExercise and live longer , and prosper more ! \u201d ideated with None , and felt that \u201cit\u2019s basically telling you what you already know . It\u2019s a rather generic message . \u201d ; P63 reviewed the message \u201cWaking up early and working out will help you get into shape , and is a great way to have more energy and better sleep . \u201d from the Directed ( 1 ) prompt \u201cinto a habit of sleep\u201d and felt \u201cit\u2019s effec - tive because it gives me a goal and tells me why this is a good goal\u201d . There were no significant differences in ideation quality or moti - vation , though there was a marginal effect that Random prompts could hurt quality compared to None . Therefore , Directed Diversity helped to reduce ideation redundancy compared to Randomly se - lected prompts , improved informativeness , and did not compromise quality . 5 . 4 Summary of Answers to Research Questions We summarize our findings to answer our research questions with results from multiple experiments . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan RQ1 . How did prompt selection techniques affect diversity in prompts ? Compared to Random , Directed Diversity : a ) selected more diverse prompts , b ) with less redundancy from prior ideation , c ) that ideators perceived as more unexpectedness , but d ) of poorer quality and understandability . RQ2 . How did diversity in prompts affect the ideation process for ideators ? Compared to Random , prompts selected with Directed Diversity were : a ) harder to ideate with , b ) less applied for ideation , c ) but their higher prompt diversity somewhat drove higher ideation diversity . RQ3 . How did prompt selection techniques affect diversity in ideations ? Compared to None and Random , Directed Diversity : a ) improved ideation diversity and reduced redundancy , b ) increased the flexibility and originality of ideated categories , c ) without com - promising ideation quality . 6 DISCUSSION We discuss the generalization of our technical approach , evaluation framework , and experiment findings . 6 . 1 Need for Sensitive and Mechanistic Measures of Creativity We have developed an extensive evaluation framework for two key reasons : 1 ) to precisely detect effects on diversity , and 2 ) to track the mechanism of diversity prompting . We have sought to be very diverse in our evaluation of prompt technique to carefully identify any benefits or issues . We have found that some popu - lar metrics ( e . g . , mean pairwise distance ) were less sensitive than others ( e . g . , MST Dispersion / Remote - tree ) . Therefore , a null re - sult in one metric ( e . g . , [ 79 ] ) may not mean that diversity was not changed ( if measured by another metric ) . Instead of only depend - ing on the \u201cblack box\u201d experimentation of prompt treatment on ideation ( e . g . , [ 18 , 40 , 79 , 80 ] ) , investigating along the ideation chain is interpretable and helpful for us to identify potential issues or breakdowns in the diversity prompting mechanism . Had our evalu - ation results on ideation diversity been non - significant , this would be helpful to debug the lack of effectiveness . Conversely , we may find that an ideation diversity effect may be due to contradictory or confounding effects . Indeed , we found that Directed Diversity improved diversity , despite poorer prompt understandability and adoption . Ideators could not directly use the selected prompts , but still managed to conceive ideas that were more diverse than not having seen prompts or seeing random ones . This suggests that they generated ideas sufficiently near the prompts . The findings also suggested that the increased effort helped to im - prove diverse ideation [ 5 , 6 , 96 ] , but the ideator user experience should be improved . Future work is needed to improve Directed Diversity to reduce ideator effort and improve the relevance of selected prompts , such as by limiting the distance of new prompts from prior ideations , or using idea - based embeddings [ 79 , 80 ] in - stead of language models , as discussed next . 6 . 2 Generalization of Directed Diversity to other Domains The full process of Directed Diversity ( Figure 1 ) allows us to gen - eralize its usage to other domains , such as text creativity tasks beyond motivational messages ( e . g . , birthday greetings [ 79 ] ) by changing the document sources in the phrase extraction step . In the phrase embedding step , we used the Universal Sentence En - coder [ 14 ] , but other text embedding models ( e . g . , word2vec [ 63 ] , GloVe [ 71 ] , ELMo [ 74 ] , BERT [ 23 ] ) could be used that model lan - guages slightly differently . In the third step , we selected phrases based on the Remote - tree diversity formulation using an efficient greedy algorithm that approximates the diversity maximization . Other diversity criteria and maximization algorithms could be used ( see review [ 20 ] ) . Note that since USE and similar language mod - els are domain - independent , which do not model the semantics of specific domains and semantic quality , Directed Diversity cannot guarantee improving quality . A domain - specific model trained with human - annotated labels of quality could be used to improve both diversity and quality . Furthermore , instead of representing text with language models , the idea space could be explicitly modelled to obtain embeddings from annotated semantic similarity [ 55 , 79 ] . Finally , since Directed Diversity operates on a vector representation of prompting and ideations , it can also be used for ideation tasks beyond text as long as they can be represented in a feature vector by feature engineering or with deep learning approaches , such as furniture [ 58 ] , mood boards [ 49 ] , and emojis [ 101 ] . 6 . 3 Generalization of Evaluation Framework Our Evaluation Framework is a first step towards the goal of stan - dardizing the evaluation of crowd ideation . This requires further validation and demonstration on existing methods of supporting crowd ideation . Due to the costs of engineering effort , set - up prepa - ration , and recruitment , we defer it to future work . Just as the Directed Diversity pipeline is generalizable , we discuss how the Diversity Prompt Evaluation Framework is generalizable . We had identified many diversity metrics , but only measured some of them ; see [ 20 ] for a review of other mathematical metrics . If applying the framework to non - text domains , the vector - based distance metrics should still be usable if the concepts can be embedded with a do - main model . While we analyzed diversity in terms of mathematical metrics [ 20 ] and several measures for creativity [ 92 ] , other criteria may be important to optimize , such as serendipity for recommender systems to avoid boredom [ 44 ] . To measure creativity , just as in prior research [ 50 ] , we had used several Likert scale ratings ( e . g . , helpfulness and informativeness ) and found evidence that participants confound them . Furthermore , it may be excessive to apply all our measures , therefore the re - searcher is advised to use them judiciously . For example , we found that individually rating ideations tends to lead to poor statistical significance , so this data collection method should be avoided . The thematic analysis coding is also very labor intensive for the re - search team , but provides rich insights into the ideas generated . We had proposed using ranking and pairwise rating validations of collections of ideations as a scalable way to measure collective diversity . While our evaluations based on generating motivational mes - saging for physical activity helped to provide a realistic context , it was limited to measuring preliminary impressions of validators . The social desirability effect may have limited how accurately par - ticipants rated the effectiveness of the messages . While our focus CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . was on evaluating diversity , future work that also seeks to improve and evaluate motivation towards behavior change should conduct longitudinal trials with stronger ecological validity [ 50 ] . 7 CONCLUSION In this paper , we presented Directed Diversity to direct ideators to generate more collectively creative ideas . This is a generalizable pipeline to extract prompts , embed prompts using a language model , and select maximally diverse prompts . We further proposed a gener - alizable Diversity Prompting Evaluation Framework to sensitively evaluate how Directed Diversity improves ideation diversity along the ideation chain \u2014 prompt selection , prompt creativity , prompt - ideation mediation , and ideation creativity . We found that Directed Diversity improved collective ideation diversity and reduce redun - dancy . 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Variable Definition Interpretation PromptSelection None : no prompt , other than task instructions Random : randomly selected phrase from corpus Directed : prioritized phrase from corpus Selection algorithm for selecting phrases to include in prompts . PromptCount Number of prompts { 50 , 100 , 150 , 200 , 250 , . . . , n prompts } Indicates how many prompts shown to generate new messages . A prompt may contain \u2265 1 phrases . This was only tested in the simulation study . PromptSize Number of phrases per prompt { 1 , 2 , 3 , 4 , 5 } Prompts selected depends on Prompt Selection . B ADDITIONAL DEFINITIONS OF DIVERSITY METRICS B . 1 Thematic Analysis Method for Flexibility and Originality Metrics Flexibility [ 85 ] measures how many unique ideas ( conceptual cat - egories ) was generated , and originality [ 100 ] measures how infre - quently each conceptual category occurs . These require expert annotation to identify distinct categories . We conducted a thematic analysis of ideated messages using open coding of grounded theory [ 34 ] to derive categories . These categories were added , reduced , merged , and refined by iteratively assessing the messages . We then consolidated the categories into themes using affinity diagram - ming [ 8 ] . This was done separately for different prompt techniques . The thematic analysis was primarily performed by one co - author researcher with regular discussion with co - authors who are ex - perienced HCI researchers with experience in Amazon Mechan - ical Turk experiments and research on health behavior change . We calculated inter - rater reliability on a random 10 % subset of messages was coded independently by another co - author to ob - tain a Krippendorff\u2019s alpha with MASI distance [ 70 ] of \u03b1 = 0 . 82 , which indicated good agreement . Note that while thematic anal - yses and affinity diagramming are popular methods to interpret qualitative data , we use them here for data pre - processing . Finally , we calculate the flexibility and originality measures based on the coded categories ( fine - grained ) and themes ( coarser ) described in Table 7 . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Table 7 : Metrics of creativity of ideation based on categories and themes derived from a thematic analysis of generated ideas . Metrics are shown for categories , but are the same for themes . Metric Definition Interpretation MessagesFlexibility Number of categories coded (cid:205) c [ f c > 0 ] This counts how many unique categories / themes were observed in messages for each Prompt Technique . A higher count indicates qualitatively more diversity . MessagesOriginality Category originality o c = ( 1 \u2212 f c / N p ) How original o c each theme is , where f c is the frequency for the category c , N p is the number of messages with the Prompt Technique p . B . 2 Intra - Prompt Diversity Metrics based on Embedding Distances Table 8 : Metrics of prompt diversity for all phrases in a single prompt . Metric Definition Interpretation Intra - Prompt MeanPhrase Distance Intra - prompt mean phrase - phrase distance 1 \u0434 (cid:205) i , j \u2208 Prompt d ( x Pi , x Pj ) Indicates how similar ( consistent ) all phrases are to one another in the same prompt . Prompts with better consistency would be easier to understand and use . Prompt Phrase Chamfer distance 1 \u0434 (cid:205) i \u2208 Prompt min j (cid:60) i d ( x Pi , x Pj ) Average distinctiveness of phrases in prompt . C DEFINITIONS OF PROMPT ADOPTION METRICS Table 9 : Metrics indicating how much of prompt text and concepts are adopted into the ideations . Metric Definition Interpretation Prompt Recall 1 \u0434 (cid:205) Phrase \u2208 Prompt n word \u2208 Ideation \u2227 word \u2208 Phrase n word \u2208 Phrase The proportion of words from phrases that were used in the ideated message . Prompt Precision (cid:205) phrase \u2208 prompt n word \u2208 Ideation \u2227 word \u2208 Phrase n word \u2208 Ideation The proportion of ideated message words that were from phrases in the shown prompt . Prompt - Ideation Distance Prompt - Ideation distance d ( x Pri , x Ij ) Indicates how similar the written ideation message is to the prompt , as a measure of how the phrase ( s ) ideas were adopted . D PAIRWISE EMBEDDING DISTANCES OF PHRASES AND MESSAGES These figures show the distribution of pairwise distances based on the embeddings of phrases and messages . Figure 10 : Distribution of pairwise distances between the extracted phrases ( N = 3 , 666 ) . The pairwise distances ranged from Min = 0 . 057 to Max = 0 . 586 , Median = 0 . 430 , inter - quartile range 0 . 394 to 0 . 460 , SD = 0 . 047 . Figure 11 : Distribution of pairwise distances between the messages ( N = 250 ) ideated in the pilot study with no prompt - ing ( None ) . The pairwise distances ranged from Min = 0 . 169 to Max = 0 . 549 , Median = 0 . 405 , inter - quartile range 0 . 376 to 0 . 432 , SD = 0 . 043 . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . E RESULTS OF CHARACTERIZATION SIMULATION STUDY We created 50 simulations for each prompts configuration to get a statistical estimate of the performance of each prompt selection technique . Figure 12 shows the results from the simulation study . Error bars are extremely small , and not shown for simplicity . Span and Sparseness results not shown , but are similar to Mean Dis - tance . Note that we computed the mean of MST edge distances instead of sum , which is independent of number of prompts . In general , Directed Diversity selects prompts to be more diverse for fewer prompts ( smaller prompt count ) , but after a threshold , Ran - dom selection can provide for better diversity . This demonstrates directing is useful for small crowd budgets . Note that the actual threshold depends on corpus and application domain . We found an interaction effect where single - phrase prompts benefit most with Directed Diversity , since for low prompt count , and Directed ( 1 - phrase ) has highest diversity , followed by Directed ( 3 ) , Random ( 3 ) , and Random ( 1 ) with lowest diversity . Figure 12 : Influence of prompt selection technique , prompt size , and prompt count on various distance and diversity metrics . Higher values for all metrics indicate higher diversity . Span and Sparseness results are not shown , but are similar to Mean Distance . Note that we computed the mean of MST edge distances instead of sum , which is independent of number of prompts . Error bars are extremely small , and not shown for simplicity . F FACTOR LOADINGS FROM FACTOR ANALYSIS IN USER STUDIES Table 10 : The rotated factor loading of factor analysis on metrics of prompt distance and consistency . Factors explained 73 . 6 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 5810 , p < . 0001 ) . PromptDistance Prompt Consistency Phrase Minimum Pairwise Distance 0 . 95 0 . 08 Prompt Minimum Pairwise Distance 0 . 95 0 . 05 Intra - Prompt Mean Phrase Distance - 0 . 05 - 0 . 69 Table 11 : The rotated factor loading of factor analysis on metrics of perceived helpfulness of prompts . Factors explained 68 . 9 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 2575 , p < . 0001 ) . PromptQuality Prompt Unexpectedness PromptRelevance Prompt Understandability Phrase Helpfulness rating 0 . 85 - 0 . 13 0 . 2 0 . 15 Phrase Relevance to Task ( Motivation ) rating 0 . 89 - 0 . 2 0 . 23 0 . 14 Phrase Understanding rating 0 . 62 - 0 . 21 0 . 27 0 . 47 Phrase Relevance to Domain ( Exercise ) rating 0 . 59 - 0 . 14 0 . 54 0 . 18 Phrase Unexpectedness rating - 0 . 11 0 . 63 - 0 . 06 - 0 . 05 Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Table 12 : The rotated factor loading of factor analysis on metrics of prompt adoption . Factors explained 65 . 1 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 1315 , p < . 0001 ) . PhraseAdoption Prompt Precision 0 . 82 Prompt Recall 0 . 67 Prompt - Ideation Distance - 0 . 92 Table 13 : The rotated factor loading of factor analysis on diversity metrics of generated messages . Factors explained 75 . 2 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 2676 , p < . 0001 ) . IdeationDispersion IdeationEvenness Message Remote - clique 0 . 99 0 . 16 Message Sparseness 0 . 99 0 . 16 Message Span 0 . 77 - 0 . 05 Message MST Dispersion 0 . 29 0 . 96 Message Chamfer Distance - 0 . 02 0 . 91 Message Entropy 0 . 01 0 . 3 Table 14 : The rotated factor loading of factor analysis on metrics of perceived quality of the generated messages . Factors ex - plained 80 . 9 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 5810 , p < . 0001 ) . Ideation Informative - Helpfulness IdeationQuality Informativeness rating 0 . 8 0 . 39 Helpfulness rating 0 . 66 0 . 65 Motivation rating 0 . 39 0 . 79 Table 15 : The rotated factor loading of factor analysis on metrics of group ranking of the generated messages . Factors explained 93 . 9 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 366 , p < . 0001 ) . For usability , \u201cunrepetitive\u201d was measured with the word \u201crepetitive\u201d in the survey . IdeationsUnrepetitive Ideations Informative IdeationsMotivating Sum ( Most Unrepetitive ( Rank = 1 ) ) 1 . 32 0 . 40 0 . 10 Sum ( Most Informative ( Rank = 1 ) ) 0 . 48 0 . 72 0 . 05 Sum ( Least Unrepetitive ( Rank = 3 ) ) - 0 . 73 - 0 . 50 0 . 02 Sum ( Least Informative ( Rank = 3 ) ) - 0 . 26 - 0 . 89 - 0 . 03 Sum ( Most Motivating ( Rank = 1 ) ) 0 . 17 - 0 . 04 1 . 00 Sum ( Least Motivating ( Rank = 3 ) ) 0 . 05 - 0 . 06 - 0 . 54 Table 16 : The rotated factor loading of factor analysis on metrics of message distinctness . Factors explained 74 . 8 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 1022 , p < . 0001 ) . IdeationDistance Ideation Min Pairwise Distance 0 . 86 Ideation Mean Pairwise Distance 0 . 86 CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Table 17 : The rotated factor loading of factor analysis on metrics of ideation effort . Factors explained 59 . 0 % of the total variance . Bartlett\u2019s Test for Sphericity to indicate common factors was significant ( \u03c7 2 = 1008 , p < . 0001 ) . Ideation Self - Quality IdeationEase Message Creativity Self - Rating 0 . 76 0 . 19 Message Motivation Self - Rating 0 . 63 0 . 55 Message Writing Ease 0 . 76 0 . 19 G SURVEY SCREENSHOTS IN USER STUDIES G . 1 Ideation User Study Figure 13 : The instructions in the Ideation User Study for the None condition . Figure 14 : For the None , users are asked to write a message that is at least one to three sentences long . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 15 : The instructions of Ideation User Study for the Random ( 1 ) and Directed ( 1 ) conditions . Figure 16 : Random ( 1 ) and Directed ( 1 ) prompts consisted of one phrase per prompt . Note that selected phrase for each trial will be different . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 17 : The instructions of Ideation User Study for the Random ( 3 ) and Directed ( 3 ) conditions . Figure 18 : Random ( 3 ) and Directed ( 3 ) prompts consist of three phrases per prompt . Note that selected phrases for each trial will be different . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 19 : Ideators are asked to evaluate the message they wrote by providing Likert scale ratings for many different fac - tors along with a short reflection about the message writing process . The screenshot above shows the evaluation screen for Directed ( 3 ) . G . 2 Validation User Studies Figure 20 : The instruction for individual message rating tasks . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 21 : Validators rated a randomly selected message on a Likert scale and gave a justification . Figure 22 : The instruction for group message ranking tasks . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Figure 23 : Validators were asked to rank groups of messages for motivation , informativeness and repetitiveness . Note that while we used the word \u201crepetitive\u201d for usability in the survey , we analyzed this dependent variable as \u201cunrepetitive\u201d to be consistent with other diversity metrics . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Figure 24 : Validators were asked to rate the difference of two messages in a message - pair . H EXAMPLES OF PROMPTS AND MESSAGES WRITTEN BY IDEATORS Table 18 : Messages generated in our study and the phrase prompt ( s ) that were shown to ideators . PromptSelection Phrase ( s ) Shown Message Written Random ( 1 ) daily club swim workout Do you want a way to train your whole body ? Try a swim workout ! You can even join a club to help challenge you to reach your goals ! Random ( 1 ) like a barrier of insecurity Get out and try a new exercise today . Don\u2019t let not doing it be a barrier or insecurity . Even pro athletes have to try new exercises for the first time . Directed ( 1 ) snooze button repeatedly isn\u2019t exercise Reminder that hitting the snooze button repeatedly is NOT considered an exercise ! Make sure to wake up first thing , and get your legs moving ! Directed ( 1 ) next set of stats Not happy with what you see on the scale or the number of calories you burned ? Don\u2019t let one day\u2019s data ruin your mood . Give it time and you\u2019ll see better results if you keep at it ! Random ( 3 ) ( 1 ) hard workout may feel ( 2 ) multiple exercise interventions in terms ( 3 ) exercise program for clients plagued Hard workouts may feel uncomfortable . However , those carry the most enjoyment and success for you ! Random ( 3 ) ( 1 ) religious institution offers exercise classes ( 2 ) workout program because people ( 3 ) other forms of water aerobics Your religious institution offers exercise classes and your local pool offers water aerobics . Exercise with people for motivation ! Directed ( 3 ) ( 1 ) in the risk of diabetes ( 2 ) for the development of diabetes ( 3 ) from complications of diabetes Exercising will help you stay in shape . It will prevent health issues in the future and it can stop the risk of developing diabetes . Directed ( 3 ) ( 1 ) book and workout videos ( 2 ) mechanics and workout plans ( 3 ) exercise tapes or videos Watching tapes and videos are good ways to try out new exercises . Follow along and impress your loved ones with your new moves ! Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan I THEMATIC ANALYSIS OF MESSAGES Table 19 : Themes and categories identified with the qualitative coding of ideated messages . Theme Categories Ambiguous Benefits Ambiguous benefits Anecdote Anecdote Appeal to \" Obvious \" Knowledge Appeal to \" obvious \" knowledge Appeal by Cohorts Appeal to children | Appeal to older ages | Appeal to overweight Appeal to Fear Appeal to fear Appeal to Guilt Appeal to guilt Appeal to Shame Appeal to shame Appeal to Social Approval Appeal to social approval Barrier to Ability Barrier to ability Barrier to Boredom Encourage exercise variety | Prompt to try something new | Tips to make exercise less boring / more fun Barrier to Comfort Barrier to comfort Barrier to Cost Cheap exercises | Lower healthcare / insurance costs Barrier to Effort It will get easier | Recommending less effortful routines or exercises | Take a short break then carry on Barrier to Energy Barrier to energy Barrier to Enjoyment Prompt to research fun exercise | Recommending enjoyable activity Barrier to Motivation Barrier to motivation Barrier to Resources At home exercises | No equipment needed | No gym available Barrier to Self - Efficacy Don\u2019t feel bad if confused | Don\u2019t need certificate / qualifications | Improving self - confidence | Recommending exercises within ability Barrier to Time Barrier to time Call to Action Call to action Call to Authority Citing health experts | Unspecified authority Collective Societal Benefits Collective societal benefits Equipment Bench press | Exercise machine | Exercise machines ( unspecified | Rubber exercise tubing / band | Swimming gear | Treadmill | Vertical or horizontal press | Work - standing desk Exercise Suggestion Aerobic exercises | Aerobics | Anaerobic exercise | Biking | Body weight exercises | Cardiovascular exercise | Climbing | Competitive cycling | Dance | Diving | Double clean | Exercise through chores | Handstand | High intensity exercise | Hot yoga | Internal rotation workouts | Iron Yoga | Jump on bed | Jumping jacks | Lift weights | Lifting luggage | Meditation | Pull - ups | Pushing kids on swings | Push - ups | Resistance exercises | Ring Pull - ups | Running | Seated leg - raises | Sit - ups | Snatches | Sports | Squats | Strength training | Strenuous / moderate / vigorous exercise | Stretching | Swimming | Tennis | Using stairs | Vertical and horizontal presses | Volleyball | Walk your dog | Walking | Water exercises | Work / desk exercise | Yoga Fear of Injury Don\u2019t overexert yourself | Recommending exercises to avoid injury | Research good techniques to avoid injury | Take breaks | Tips to avoid injury for outdoor activities Food and Drink Avoid steroids / pills / drugs | Avoid unhealthy food | Exercise supplements | Exercise to avoid medication / drugs | Food recommendation | Staying hydrated | Stress eating advice Future Life Improve quality of life | Live longer Goals Journaling to track your goal | Set Actionable goals | Set daily exercise goal | Set goals based on health recommendations | Set unspecified goal | Set weight goal | Tips to reach goals | Visualizing meeting goals Health Advice Advice for diabetics | See a doctor if you are worried Health Benefits Better mobility | Bone health | Cardio Health | Fluid regulation | Help with foot problems | Lowers blood pressure | More stamina | Pain / strain relief | Slows aging process | Strong immune system | Unspecified health benefit CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Health Risks Arthritis | Breathing difficulty | Cancer | Depression | Diabetes | Heart disease | Hernias | Obesity | Unspecified health risk Improving Appearance \" Look better \" | Beach ready | Chest and back | Improved posture | Look more appealing to potential partners | Nice butt | Six - pack abs Inspirational Phrase Inspirational phrase Lack of Knowledge Research exercise routines | Research nutrition | Study exercise form Lack of Social Support Exercise with an expert | Exercise with friends | Family want you to be healthy | Find places to support you | Impress your doctor | Interacting with others | Join a health club | Join an exercise class | Meeting new people | Playing / exercising with children | Social competition Mental Health Benefits \" Feeling \" better | Happier | Improved sleep | Improves cognitive abilities | Lower depression | Lowers anxiety | Relaxing | Release endorphins | Self - esteem | Stress reduction | Unspecified mental health benefits Muscle Building Biceps | Core strength | Leg Muscles | Physically stronger | Shoulder muscles | Triceps | Unspecified Muscle building | Upper body strength Overcoming Beliefs Changing mindset Overcoming Family Obligations Overcoming family obligations OvercomingSelf - Consciousness Overcoming self - consciousness Push to Do More Prompt to increase | Prompt to steadily increase Push to Start Prompt to start exercising | Prompt to stop sedentary lifestyle Rewards Prizes from exercise competitions | Reward with food | Reward with new clothes | Reward with new exercise equipment | Unspecified reward Self - Empowerment Self - empowerment Self - Forgiving Self - forgiving Self - Reflection Self - reflection Social Comparison Avoid social comparisons | Downwards Social comparison | Upwards Social Comparison Specific Locations Around the neighbourhood | At desk exercises | At home | At school | Beach | Church / community centre | Front yard | Gym | Outdoors | Park | Travelling / airport | Walk to train station Specificity Appropriate Exercises ( e . g . \" Try what\u2019s best for you \" | Developing habits | Even small amounts of exercise | Exercise daily | Exercise regularly | Follow exercise plan / routine | Specific amount / distance to exercise | Specific days a week to exercise | Specific minutes to exercise Time to See Results Dedicate time | Fast results | Promise of results | Tips to progress faster Time to Exercise Anytime | End of the day | Morning exercise | Spring | Summer / hot weather Use of Technology Exergame | Experts review your exercises from an app | Follow videos | Listen to music / podcast | Reflect on progress | Use apps for exercise tips | Use apps for workout schedules | Use apps to track progress | Watch TV while exercising Weight Loss Aid digestion | Boost metabolism | Burning calories | Burning cellulite | Maintaining weight | Slimming down Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan J LINEAR MIXED MODELS AND STATISTICAL ANALYSIS RESULTS OF PROMPT CREATIVITY , PROMPT - IDEATION MEDIATION , AND IDEATION DIVERSITY Table 20 : Statistical analysis of responses due to effects ( one per row ) , as linear mixed effects models , all with Participant as random effect , Prompt Selection and Prompt Size as fixed effects , their interaction effect . a ) model for manipulation check analysis of how prompt configurations affect perceived prompt creativity ( RQ1 . 2 ) ; b ) model for mediation analysis of how prompt configurations affect ideation effort ( RQ2 . 2 ) . clr23n . s . means not significant at p > . 01 . p > F is the significance level of the fixed effect ANOVA . R 2 is the model\u2019s coefficient of determination to indicate goodness of fit . a ) Prompt Creativity Manipulation Check ( RQ1 . 2 ) Response Linear Effects Model ( Participant random effect ) p > F R 2 PromptUnexpected - ness Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size < . 0001 n . s n . s . 523 PromptUnderstand - ability Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size . 0008 < . 0001 . 0316 . 500 PromptRelevance Prompt Selection + Prompt Size + Selection \u00d7 Size < . 0001 < . 0001 n . s . . 450 Prompt Quality Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size < . 0001 < . 0001 n . s . . 572 b ) Prompt - Ideation Effort Mediation Analysis ( RQ2 . 2 ) Response Linear Effects Model ( Participant as random effect ) p > F R 2 IdeationFluency Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size < . 0001 < . 0001 . 0042 . 542 Ideation Ease Prompt Selection + Prompt Size + Selection \u00d7 Size < . 0001 n . s n . s . 546 PromptAdoption Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size < . 0001 n . s n . s . 575 Table 21 : Statistical analysis and results of mediation effects ( RQ2 . 3 ) of how prompt configurations ( a ) and perceived prompt creativity ( b ) affect ideation diversity . See Table 20 caption to interpret tables . Positive and negative numbers in second column represent estimated model coefficients indicating how much each fixed effect influences the response . a ) Prompt Distance to Ideation Mediation Response Linear Mixed Effects Model ( Participant as random effect ) p > F R 2 IdeationMeanPairwiseDistance + 0 . 18 + 0 . 06 + 0 . 01 + 0 . 02 Prompt Mean Distance + Prompt Min Distance + Pr . P . Chamfer Dist . + Intra - Pr . P . Mean Dist . < . 0001 . 0205 n . s . < . 0001 . 399 IdeationMinimumPairwiseDistance + 0 . 10 + 0 . 15 + 0 . 06 + 0 . 02 Prompt Mean Distance + Prompt Min Distance + Pr . P . Chamfer Dist . + Intra - Pr . P . Mean Dist . . 0241 < . 0001 . 0115 . 0041 . 315 b ) Prompt Creativity to Ideation Mediation Response Linear Mixed Effects Model ( Participant as random effect ) p > F R 2 IdeationMeanPairwiseDistance \u2013 0 . 0014 + 0 . 0001 \u2013 0 . 0034 + 0 . 0018 Pr . Unexpectedness + Pr . Understandability + Prompt Relevance + Prompt Quality . 0315 n . s . 0020 n . s . 367 IdeationMinimumPairwiseDistance + 0 . 0024 \u2013 0 . 0026 \u2013 0 . 0056 + 0 . 0052 Pr . Unexpectedness + Pr . Understandability + Prompt Relevance + Prompt Quality . 0087 n . s . 0003 . 0431 . 272 CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . Table 22 : Statistical analysis of how prompt selection influences ideation diversity defined by different metrics ( RQ3 ) : a ) indi - vidual diversity , b ) collective diversity , and c ) thematic diversity . See Table 20 caption for how to interpret tables . a ) Ideation Individual Diversity Response Linear Effects Model ( Participant as random effect ) p > F R 2 IdeationMeanPairwisedistance Prompt Selection + Prompt Size + Selection \u00d7 Size < . 0001 n . s . 0005 . 361 IdeationMinPairwisedistance Prompt Selection + Prompt Size + Selection \u00d7 Size < . 0001 n . s n . s . 296 IdeationSelf - Quality Prompt Selection + Prompt Size + Selection \u00d7 Size n . s . 0292 . 0152 . 570 b ) Ideation Collective Diversity Response Linear Effects Model ( Sample as random effect ) p > F R 2 IdeationDispersion Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size < . 0001 n . s < . 0001 . 873 IdeationEvenness Prompt Selection + Prompt Size + Prompt Selection \u00d7 Size < . 0001 . 0030 . 0061 . 984 c ) Ideation Collective Diversity ( Thematic Coding ) Response Linear Effects Model ( Sample as random effect ) p > F R 2 Category Flexibility Prompt Selection < . 0001 . 979 Category Originality Prompt Selection < . 0001 . 933 Theme Flexibility Prompt Selection < . 0001 . 911 Theme Originality Prompt Selection < . 0001 . 396 Table 23 : Statistical analysis of how prompt selection influences ideation creativity as validated by different methods ( RQ3 . 1 ) : a ) individual rating , b ) collective ranking , and c ) collective pairwise rating . See Table 20 caption for how to interpret tables . a ) Individual Rating Validation Response Linear Effects Model ( Participant + Ideation as random effects ) p > F R 2 Ideation Informative Helpfulness Prompt Selection < . 0001 . 559 Ideation Quality Prompt Selection n . s . . 467 b ) Collective Ranking Validation Response Linear Effects Model p > F R 2 Ideations Unrepetitive Prompt Selection < . 0001 . 284 Ideations Informative Prompt Selection < . 0001 . 340 Ideations Motivating Prompt Selection . 0426 . 028 c ) Collective Pairwise Rating Validation Response Linear Effects Model p > F R 2 DifferenceRating Prompt Selection < . 0001 . 279 Figure 25 : Results of computed individual diversity from ideations for different prompt configurations for ( left ) prompts that users understood ( > 0 ) or did not and ( right ) ideations that were fast or slow . Directed Diversity : Leveraging Language Embedding Distances for Collective Creativity in Crowd Ideation CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan K INVESTIGATING CONFOUND OF PROMPT UNDERSTANDABILITY ON IDEATION DIVERSITY Having found that prompt understanding difficulty is correlated with ideation diversity , we investigated the alternative hypothesis that the difficulty of interpreting the prompts was a key reason for improved ideation because of increased ideation determination , rather than the content diversity in phrases due to the prompt selec - tion technique . We argue that the increase in ideation diversity due to Directed Diversity is evidenced by increased perceived diversity ratings from validators and the higher number of idea categories from the thematic analysis . This shows that Directed Diversity did stimulate more diverse ideas due to some knowledge transfer from prompt to ideations , albeit with difficulty . We identify three more sources of evidence next . First , we qualitatively analyzed ideation rationales and found that while prompts could be rated hard to understand or irrelevant , participants still adopted some ideas . Ideators cherry - picked parts that were usable or conceived tangential ideas : e . g . , P1 read \u201cor - thopaedic surgeons and exercise specialists\u201d and decided to \u201ccut out the bit about surgeons . . . I focused on the idea of specialists . . . \u201d ; P2 read \u201cballistic stretch uses vigorous momentum\u201d , commented that \u201cthis isn\u2019t a phrase that I\u2019m familiar with\u201d , yet could write about stretching : \u201cStretch , breathe , and feel mindful . \u201d Second , we quantitatively analyzed the Ideation Mean Pairwise Distance for prompts that participants understood ( Phrase Under - standing factor > 0 ) . Table 24a describes the statistical analysis of the linear mixed effects model . We found that although dis - tance was slightly higher ( i . e . , less diverse ) when ideators under - stood phrases less , regardless of understanding , ideations from Directed ( 1 ) prompts had higher distances than ideations from Ran - dom ( 1 ) prompts ( Figure 25 , left ) . The effect due to Prompt Type was larger than due to Phrase Understanding . Furthermore , we an - alyzed whether the difficulty to understand may manifest as slower ideation speed due to more thinking time to ideate to lead to better diversity , but did not find a correlation between phrase understand - ing and ideation speed ( \u03c1 = . 046 , p = n . s . ) , and found the opposite effect that slower ideations led to lower distances ( Table 24a and Figure 25 right ) . These suggest that prompt selection is a primary factor . Third , we investigated if Directed Diversity helped to stimulate ideas closer to prompts than would be done naturally without prompts ( None ) or accidentally with Random prompts . We analyzed this by calculating the prompt - ideation distance between Directed prompts and their corresponding ideated message , and their closest None and Random messages . Table 24b describes the statistical analysis of the linear mixed effects model . Figure 26 shows that the directed ideations were closest to the prompts , indicating the efficacy of Directed Diversity to transfer knowledge for ideation diversity . Table 24 : Statistical analysis of a ) how ideators\u2019 understanding of phrases influences ideation diversity and b ) how similar Directed ideations are to their prompts compared to other None and Random Messages . a ) Ideation Individual Diversity Response Linear Effects Model ( Participant as random effect ) p > F R 2 Ideation Mean Pairwisedistance Prompt Selection + Prompt Size + Selection \u00d7 Size + Phrase Understanding > 0 + Selection \u00d7 ( Understanding > 0 ) + Log ( Ideation Speed ) > Median + Selection \u00d7 ( Speed > Median ) < . 0001 n . s . 0001 . 0074 n . s . . 0043 . 0213 . 289 b ) Prompt - Ideation Closeness Response Linear Effects Model ( Participant random effect ) p > F R 2 Prompt - MessageDistance Message Type < . 0001 . 047 Figure 26 : Results of prompt - message distance ( how dissimilar a prompt is from a message ) comparing different messages with respect to Directed ( 3 ) prompts . CHI \u201921 , May 08 \u2013 13 , 2021 , Yokohama , Japan Samuel Cox et al . L EXAMPLES OF MESSAGE - GROUP RANKING The factors of message - group ranking were derived from the sum of rankings ( for each of the three condition ) per validator for his five ranking trials ( see the factor loadings in Table 15 The rotated factor loading of factor analysis on metrics of group ranking of the generated messages . ) . Therefore , these factors reflect the probabil - ity of how a validator ranked the message - groups of each condition . The following table shows examples of the factors and the corre - sponding message - group samples . Table 25 : Examples of the factors with low ( < Median ) and high ( \u2265 Median ) scores for \u201cIdeations Unrepetitive\u201d and \u201cIdeations Informative\u201d . I d e a t i o n s U n r e p e t i t i v e I d e a t i o n s I n f o r m a t i v e Example Message - Group of 5 Ideations High High \u2022 Exercise can help you have really good sleep . \u2022 Why don\u2019t you try something new ? Shake it up a little ? Maybe lift a few small weights , or add in some squats - variety keeps things interesting . \u2022 You have 24 hours in a day \u2013 think about how much time you spend on social media or doing something that\u2019s not going to benefit you in the long run and use that time to workout by prioritizing your health ! \u2022 Go for the goal , do not stop , do not think you cannot do it . YOU CAN ! \u2022 Summer is coming up and you want to look good when you are outside . Exercising at a health club is a good way to meet other people . Have a friend to work out with you and have each other motivate each other . High Low \u2022 Just get moving . It\u2019s that simple . \u2022 Your dog is bored . Take him for a walk ! It\u2019s good for both of you and he\u2019ll be thrilled ! \u2022 Work out more . You will feel and look better . You will get more toned . \u2022 Exercising can improve your cardio health , thus helping you to live a more fulfilling life . \u2022 Start exercising more ! You\u2019ll improve your mood and boost your self confidence . You\u2019ll feel great ! Low High \u2022 Switch off an air conditioner while working out . Let the sweat out , and burn some calories . \u2022 Not happy with what you see on the scale or the number of calories you burned ? Don\u2019t let one day\u2019s data ruin your mood . Give it time and you\u2019ll see better results if you keep at it ! \u2022 Sleep is when the body recovers and is very important . Rest early and run tomorrow ! \u2022 Overcome your anger and your fear by going to the gym and working out ! \u2022 Always stretch so that you perform at your best . You can do it ! Low Low \u2022 Exercise helps build strong muscles as well as well as making your body more flexible . You will reduce your risk of disease and injury by keeping up with your program . \u2022 The first page of every book is the hardest to grasp , the first drink tastes the most sour and the first minute of every exercise is the hardest . All things get easier as you press on . \u2022 Walking to the train station is better as it gets you more active . Avoid lifts to the train station . \u2022 Keep exercising to keep your mind off difficult personal issues , like college admissions . \u2022 By using the proper squat position , you can train muscles that take pressure off of your knee and back to help with pain in both areas .", + "kaplan2022load": "Molecular Biology of the Cell \u2022 33 : ar50 , 1 \u2013 16 , May 15 , 2022 33 : ar50 , 1 MBoC | ARTICLE Load adaptation by endocytic actin networks ABSTRACT Clathrin - mediated endocytosis ( CME ) robustness under elevated membrane ten - sion is maintained by actin assembly \u2013 mediated force generation . However , whether more actin assembles at endocytic sites in response to increased load has not previously been investigat - ed . Here actin network ultrastructure at CME sites was examined under low and high mem - brane tension . Actin and N - WASP spatial organization indicate that actin polymerization initi - ates at the base of clathrin - coated pits and that the network then grows away from the plasma membrane . Actin network height at individual CME sites was not coupled to coat shape , rais - ing the possibility that local differences in mechanical load feed back on assembly . By manipu - lating membrane tension and Arp2 / 3 complex activity , we tested the hypothesis that actin assembly at CME sites increases in response to elevated load . Indeed , in response to elevated membrane tension , actin grew higher , resulting in greater coverage of the clathrin coat , and CME slowed . When membrane tension was elevated and the Arp2 / 3 complex was inhibited , shallow clathrin - coated pits accumulated , indicating that this adaptive mechanism is especially crucial for coat curvature generation . We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions . INTRODUCTION Actin networks produce force for a wide variety of cellular processes through a Brownian ratchet mechanism ( Mogilner and Oster , 1996 , 2003 ; Pollard , 2016 ) . Live - cell studies of lamellipodia ( Mueller et al . , 2017 ) , biochemical reconstitutions ( Bieling et al . , 2016 ; Funk et al . , 2021 ; Li et al . , 2021 ) , and modeling studies ( Akamatsu et al . , 2020 ) have shown that actin networks nucleated by the Arp2 / 3 complex respond to increased load by becoming more dense , which en - hances force production . This phenomenon has been demonstrated in the context of actin networks producing force on the lamelli - podum , whose membrane is essentially flat on the length scale of an individual actin filament branch . However , actin networks can also produce force on membranes that change shape over time , for ex - ample during vesicle formation . Whether actin networks in the latter context show load adaptation has not been investigated . From yeast to humans , transient actin assembly is associated with the formation of clathrin - coated endocytic vesicles . In yeast cells , actin assembly is required to generate forces to invaginate the plasma membrane against a high intrinsic turgor pressure for clath - rin - mediated endocytosis ( CME ) ( Kaksonen et al . , 2005 ; Aghamo - hammadzadeh and Ayscough , 2009 ; Idrissi et al . , 2012 ; Kukulski et al . , 2012 ) . When actin assembly is perturbed in mammalian cells , CME typically slows in a manner that depends on cell type ( Fujimoto et al . , 2000 ; Merrifield et al . , 2005 ; Yarar et al . , 2005 , 2007 ; Grassart et al . , 2014 ; Dambournet et al . , 2018 ; Sch\u00f6neberg et al . , 2018 ) . A potential cause of this reported variation between cell types might Monitoring Editor Alex Mogilner New York University Received : Nov 29 , 2021 Revised : Mar 28 , 2022 Accepted : Mar 29 , 2022 This article was published online ahead of print in MBoC in Press ( http : / / www . molbiolcell . org / cgi / doi / 10 . 1091 / mbc . E21 - 11 - 0589 ) on April 7 , 2022 . Author contributions : C . K . and D . G . D . conceived the study and experiments . C . K . performed live cell data acquisition , SRM data analysis and live cell data analysis . S . J . K . , X . C . , and K . X . performed SRM and super - resolution data reconstruction and supervised SRM imaging . J . S . supported the SRM data analysis . E . S . and A . D . - M . performed membrane tether pulling experiments by atomic force microscopy and data analysis and supervised AFM tether pulling experiments . C . K . , and M . A . prepared the plot layouts and figures . C . K . , M . A . , and D . G . D . wrote the manuscript with feedback from the other authors . \u2020 Present address : Department of Biology , University of Washington , Seattle , WA 98195 . * Address correspondence to : David G . Drubin ( drubin @ berkeley . edu ) , Matthew Akamatsu ( akamatsm @ uw . edu ) , or Ke Xu ( xuk @ berkeley . edu ) . \u00a9 2022 Kaplan et al . This article is distributed by The American Society for Cell Biol - ogy under license from the author ( s ) . Two months after publication it is available to the public under an Attribution \u2013 Noncommercial - Share Alike 4 . 0 International Cre - ative Commons License ( http : / / creativecommons . org / licenses / by - nc - sa / 4 . 0 ) . \u201cASCB\u00ae , \u201d \u201cThe American Society for Cell Biology\u00ae , \u201d and \u201cMolecular Biology of the Cell\u00ae\u201d are registered trademarks of The American Society for Cell Biology . Abbreviations used : CCP , clathrin - coated pit ; CCS , clathrin - coated structure ; CME , clathrin - mediated endocytosis ; SI , shape index . Charlotte Kaplan a , Sam J . Kenny b , Xuyan Chen b , Johannes Sch\u00f6neberg a , c , Ewa Sitarska d , e , Alba Diz - Mu\u00f1oz d , Matthew Akamatsu a , \u2020 , * , Ke Xu b , f , * , and David G . Drubin a , * a Department of Molecular and Cell Biology and b Department of Chemistry , University of California , Berkeley , Berkeley , CA 94720 - 3220 ; c Department of pharmacology and Department of chemistry and biochemistry , University of California , San Diego , La Jolla , CA 92093 ; d Cell Biology and Biophysics Unit , European Molecular Biology Laboratory Heidelberg , 69117 Heidelberg , Germany ; e Collaboration for joint PhD degree between EMBL and Heidelberg University , Faculty of Biosciences ; f Chan Zuckerberg Biohub , San Francisco , CA 94158 2 | C . Kaplan et al . Molecular Biology of the Cell be differences in plasma membrane tension ( Pontes et al . , 2017 ; Djakbarova et al . , 2021 ) . Indeed , in mammalian cells , actin assembly becomes increasingly critical as plasma membrane tension increases ( Yarar et al . , 2005 ; Liu et al . , 2009 ; Batchelder and Yarar , 2010 ; Boulant et al . , 2011 ) . Actin perturbation results in the accumulation of \u201cU - shaped\u201d membrane invaginations , reflecting difficulty in pro - gressing to the subsequent \u201c \u03a9 - shaped\u201d membrane stage ( Fujimoto et al . , 2000 ; Yarar et al . , 2005 ; Boulant et al . , 2011 ; Almeida - Souza et al . , 2018 ) . In total , these findings suggest that actin assembly improves the efficiency of CME in mammalian cells , potentially com - pensating for changes in plasma membrane tension . Despite the fact that actin assembly appears to be associated with CME in all eukaryotes , and over a large range of membrane tension values , whether it adapts to changes in membrane tension is not known . Knowing whether the actin cytoskeleton at CME sites adapts to changes in membrane tension will facilitate elucidation of the fundamental mechanisms by which cytoskeletal complexes pro - duce force . Platinum replica electron microscopy of cultured cells led to the proposal that actin networks assemble in a collar - like arrangement around the vesicle neck ( Collins et al . , 2011 ) . This actin organization would imply that a constricting force is generated toward the neck of the pit , supporting fission . However , actin filaments interact not only with the vesicle neck but also with the bud surface , given that the clathrin coat is impregnated with actin - binding linker proteins like Hip1R and Epsin ( Engqvist - Goldstein et al . , 2001 ; Messa et al . , 2014 ; Sochacki et al . , 2017 ; Clarke and Royle , 2018 ) . Such an ar - rangement and evidence from photobleaching studies imply that actin filaments also apply a force that pulls the clathrin - coated pit ( CCP ) into the cell interior ( Kaksonen et al . , 2003 ; Akamatsu et al . , 2020 ) . In yeast , such a pulling mechanism is likely . Actin filaments are nucleated in a ring surrounding the pit and grow from the plasma membrane toward the cell interior . The resulting actin filaments are coupled to the clathrin coat surface , generating an inward force orthogonal to the plane of the plasma membrane ( Kaksonen et al . , 2003 ; Carroll et al . , 2012 ; Skruzny et al . , 2012 , 2015 ; Picco et al . , 2015 ; Mund et al . , 2018 ) . Because the endocytic machinery is highly conserved from yeast to mammals , a similar mechanism for actin force generation during CME seems likely ( Akamatsu et al . , 2020 ) . However , ultrastructural evidence for actin organization through dif - ferent stages of mammalian CME , and for how this organization might respond to changing membrane tension , is lacking . Several competing models for actin organization at CME sites in mammalian cells have been proposed , so it is important to distin - guish between these models ( Engqvist - Goldstein et al . , 2001 ; Boulant et al . , 2011 ; Messa et al . , 2014 ; Sochacki et al . , 2017 ; Clarke and Royle , 2018 ) . Recent advances in superresolution microscopy ( SRM ) permit examination of cellular ultrastructure with large sample sizes , low invasiveness , and high molecular specificity to reveal the ultrastructure of membrane cytoskeletal systems in mammalian cells ( Xu et al . , 2012 , 2013 ; Hauser et al . , 2018 ) . Here , we combined two - color , three - dimensional stochastic opti - cal reconstruction microscopy ( 2c - 3D STORM ) with live - cell fluores - cence imaging to determine how filamentous actin is organized at CME sites with different coat geometries under various values of plasma membrane tension . These measurements led to the conclu - sion that the actin network grows from the base of the pit inward , supporting a pulling mechanism for mammalian endocytic actin fila - ments . The size of the actin network is not tightly coupled to the geometry of the endocytic coat , implying that assembly is modu - lated by factors uncoupled from coat shape . Importantly , under el - evated membrane tension , the actin network grows higher relative to the pit at all endocytic coat geometries . These observations sup - port a mechanism in which the actin network adapts to load by in - creasing in size to enhance force production and ensure robust CME across a wide range of membrane tension values . RESULTS Actin organization at CME sites suggests that force generation can be either parallel or orthogonal to the axis of CCP formation , or both , at different sites in the same cell We used 2c - 3D STORM ( Rust et al . , 2006 ; Huang et al . , 2008 ) to determine the ultrastructural organization of the actin cytoskeleton at sites of CME . Henceforth , we refer to clathrin - coated structures ( CCSs ) as relatively flat clathrin structures and CCPs as curved , in - vaginating clathrin structures . Our 2c - 3D STORM method preserves cellular ultrastructure by chemical fixation of intact cells , provides high molecular specificity due to immunofluorescence labeling , and allows large numbers of sites to be imaged ( Xu et al . , 2012 ) . We conducted our experiments on a skin - melanoma cell line ( SK - MEL - 2 ) wherein \u223c 87 % of dynamin2 - eGFP EN ( DNM2 - eGFP EN ) spots coaccu - mulate with actin ( Grassart et al . , 2014 ) . We used this SK - MEL - 2 cell line endogenously expressing DNM2 - eGFP EN and clathrin light chain A - tagRFP - T ( CLTA - TagRFP - T EN ) for both live - cell and super - resolution experiments ( Doyon et al . , 2011 ) . In these cells , we resolved CF680 - labeled CCSs in the X - Y plane as discrete , round or elliptical shapes on the ventral surface ( Figure 1A ) . Most of the CCSs appeared connected to filamentous actin vi - sualized by Alexa Fluor 647 \u2013 tagged phalloidin . These superresolu - tion reconstructions resolve the association between clathrin coats and actin networks for hundreds of pits with high resolution in all three dimensions . The SDs of positions of single fluorophores were 10 nm in plane for XY and 19 nm in depth for the Z dimension ( Supplemental Figure S1 ; Materials and Methods ) . Knowing how actin networks are organized spatially in three di - mensions at CME sites provides insights into its force generation mechanisms . It was important to show that we could distinguish ac - tin specifically associated with CCSs from actin in the cell cortex . In Supplemental Figure S2 , we show STORM images to compare actin at CCSs with actin at randomly selected regions of the cell cortex . We found examples of actin that specifically accumulates at the CCP ( Supplemental Figure S2 , D and I ) . Here , actin builds up higher into the cell interior compared with the actin extending horizontally away from the CCP and at randomly selected sites on the cell cortex ( Sup - plemental Figure S2 , E and J ) . This observation is consistent with recent cryoelectron tomograms in this cell type distinguishing branched actin networks around CCPs from the largely unbranched cortical actin network ( Serwas et al . , 2021 ) . To then investigate actin organization at multiple CCSs , we ren - dered the CCSs in three dimensions by cropping a tight area around each clathrin and actin structure in x - y STORM image pro - jections and generated an x - z STORM image projections from the selected regions of interest ( Figure 1 , B and C ) . To our surprise , we observed strikingly different actin filament spatial organizations in the clathrin coats that we examined , even when they were near each other in the same cell and even when they were at the same morphological stage of CME ( see below ) ( Figure 1C ) . In the first example shown , a thin layer of actin filaments resided along the base of the clathrin coat ( Figure 1C , inset 1 ) , reminiscent of struc - tures observed in electron micrograph by Collins et al . ( 2011 ) . In contrast , in the second example , actin filaments covered the CCP completely ( Figure 1C , inset 2 , and Supplemental Figure S2D ) . This organization resembles actin interacting with the entire clathrin Volume 33 May 15 , 2022 Actin load adaptation in endocytosis | 3 coat as in yeast ( Mulholland , 1994 ; Idrissi et al . , 2008 ; Ferguson et al . , 2009 ; Kukulski et al . , 2012 ; Buser and Drubin , 2013 ; Mund et al . , 2018 ) and in recent cryoelectron tomograms ( Akamatsu et al . , 2020 ) . These micrographs indicate that distinct CME - associ - ated actin structures can coexist in the same cell , consistent with models for force generation both parallel and orthogonal to the invagination axis . CME site \u2013 associated actin networks grow from the CCP base to the tip of the coat , and their organization is not coupled to clathrin coat geometry We used quantitative analysis of these STORM reconstructions to determine how actin is organized at CME sites and how actin orga - nization relates to coat geometry . We first selected 989 high - resolu - tion clathrin coats by visual inspection based on quality control cri - teria explained in Materials and Methods and determined which ones had associated actin filaments . When cropping CCSs for analy - sis , we could clearly distinguish single sites from double CCSs . In Supplemental Figure S3 we present views of 20 sites identified as singles and 20 sites identified as doubles in this manner . We ex - cluded 86 % of the visually inspected images ( in total 6858 ) that ac - counted for double CCSs and CCSs of noncircular shape , as well as very large and very small clathrin antibody \u2013 positive immunostained structures in the XY plane ( Supplemental Figure S4A ) . We detected actin associated with 74 % of the clathrin coats selected for further analysis , which is comparable to previous measurements for the same cell type that we made from endocytic traces for events in live cells in which dynamin2 - GFP associated with actin - RFP , considering that our superresolved images are snapshots of time - lapse events ( Grassart et al . , 2014 ) . We classified the coats by stage based on their shape in the XZ dimension , similar to earlier analyses of electron micrographs ( Avinoam et al . , 2015 ) . For presentation purposes , clathrin coat shapes were binned into three geometrical categories based on their aspect ratio ( which we call the shape index [ SI ] ) : shallow ( flat - ter coat ) , U - shaped ( intermediate aspect ratio ) , or \u03a9 - shaped ( coat with height similar to its width ) ( Figure 1D ; Supplemental Figure S4 , B and C ) . This measurement of SI was consistent whether we carried out the measurement on XZ or YZ projections of the coat ( r 2 = 0 . 92 ) ( Supplemental Figure S5 ) . We found a surprisingly wide variety of actin organizations in each of the three geometries ( Figure 1E and Supplemental Figure S4D ) . To quantify the relation - ship between actin organization and endocytic coat geometry , the SI of the coat was plotted as a function of the extent of actin cover - ing clathrin . When the actin network is larger in size than the clath - rin coat , we define this state as 100 % coverage ( see Materials and Methods ; Supplemental Figure S4B ) . There was no significant cor - relation between actin / coat coverage and coat shape for any of the three geometries ( Figure 1F ) . We also observed a lateral mean displacement between the peak signals of clathrin and actin of 74 \u00b1 42 nm , indicating an asymmetry in actin localization around the pits ( Yarar et al . , 2005 ; Collins et al . , 2011 ; Jin et al . , 2021 ; Serwas et al . , 2021 ) . This asymmetry value did not significantly change between CME geometries ( Supplemental Figure S4 , E and F ) . We conclude that irrespective of coat geometry , some pits have a thin actin network at the base of the pit , others have an interme - diate level of coverage around the clathrin coat , and others have actin completely covering the pit . Thus , actin assembly at CCSs is not coupled to a particular stage of coat curvature development but might be recruited to promote curvature development . In our reconstructions , we observed that whenever actin only partially covers the clathrin coat , the network is always located at the base of the pit ( Figure 1E ) . This observation suggests that actin polymerization is nucleated at the base of the pit and that the net - work then grows inward around and over the tip of the clathrin coat . To test the generality of this observation , we calculated the differ - ence in average axial ( Z ) position between the clathrin and actin signals for each site . We define this difference , D z , such that nega - tive D z corresponds to actin residing nearer the base of the pit ( Figure 1G ) . To determine whether actin grows from the base or tip of the pit , we plotted D z as a function of the extent of coverage between actin and the clathrin coat . Increasing values of D z would indicate that the network grows from the base of the pit toward the cell interior ( Figure 1G ) . Indeed , as a function of actin / coat cover - age , D z increased from negative values to near zero ( Figure 1H ) . We conclude that actin polymerization is initiated at the base of clathrin coats . Given our finding that actin growth originates from the base of the pit , we next investigated the spatial distribution of the actin nu - cleation factor N - WASP at clathrin coats by 2c - 3D STORM . Consis - tent with our conclusions about where actin assembly occurs at CCPs , N - WASP localized to the base of both shallow and highly curved clathrin coats ( Supplemental Figure S6A ) . More unexpect - edly , at some CME sites , N - WASP covered the entire clathrin coat irrespective of coat geometry ( Supplemental Figure S6B ) . In summary , we conclude that actin polymerization is nucleated at the base of CCPs and grows toward the coat\u2019s tip ( Figure 1I ) . Unexpectedly , actin nucleation is not coupled to coat geometry . A possible explanation for the variety of actin organizations that we observed associated with clathrin coats is that actin network organi - zation responds to differences in load at CME sites . Dynamics of CME slow down under elevated membrane tension We next combined osmotic shock with live - cell fluorescence micros - copy and 2c - 3D STORM to determine how actin - mediated force generation contributes to CCP formation under elevated mem - brane tension ( Figure 2A ) . Previous EM studies identified a require - ment for actin filaments at the \u201cU\u201d to \u201c \u03a9 \u201d transition ( Boulant et al . , 2011 ) . However , for a mechanistic understanding , the quantitative relationship between membrane tension and endocytic dynamics must be elucidated ( Akamatsu et al . , 2020 ) . Our quantitative light microscopy \u2013 based analysis of a large number of sites at different CME stages provided the necessary sensitivity to detect effects throughout the process . We first needed to establish conditions un - der which CME dynamics are affected by elevated membrane ten - sion in these cells . To determine how membrane tension is affected by changes in media osmolarity , we performed membrane tether pulling experi - ments by atomic force microscopy ( AFM ) on SK - MEL - 2 cells cultured under isotonic and hypotonic conditions ( 75 mOsm ) ( Figure 2B ) . In isotonic media , the force required to maintain a pulled membrane tether was 33 . 0 \u00b1 7 . 4 pN . Under hypotonic conditions the tether force increased to 48 . 0 \u00b1 17 . 1 pN ( Figure 2B ) . These measurements allowed us to quantitatively relate a given hypotonic environment in these cells to changes in membrane tension . To decipher the relationship between CME dynamics and membrane tension , we used total internal reflection fluorescence ( TIRF ) microscopy to image SK - MEL - 2 cells under isotonic and hy - potonic conditions ( Figure 2C ) . CLTA - TagRFP - T EN and DNM2 - eGFP EN fluorescence lifetimes were determined by single - particle tracking ( Hong et al . , 2015 ) . In isotonic media most clathrin tracks were relatively short ( 47 \u00b1 32 s ) with a burst of DNM2 - eGFP EN signal peaking at the end ( DNM2 lifetime 39 \u00b1 32 s ) ( Figure 2C , 4 | C . Kaplan et al . Molecular Biology of the Cell Volume 33 May 15 , 2022 Actin load adaptation in endocytosis | 5 Supplemental Movie 1 , and Supplemental Table S1 ) . Neither iso - tonic media nor exchange to slightly hypotonic 225 mOsm media noticeably affected DNM2 - eGFP EN and CLTA - TagRFP - T EN life - times or CME initiation and completion rates ( Supplemental Figure S7 , A \u2013 D ) . Only 1 \u2013 2 % of these CLTA - TagRFP - T EN and DNM2 - eGFP EN fluorescence tracks persisted over the entire 4 . 8 min movie under these conditions ( Supplemental Figure S7 , B and D ) . Upon treatment with moderately hypotonic ( 150 mOsm ) media for 10 min , CLTA - TagRFP - T EN lifetime in cells increased by 20 % ( 62 \u00b1 51 vs . 49 \u00b1 31 s ) ( Supplemental Figure S7E ) . This moderate treatment also had mild effects on the CME initiation rate ( 15 \u00b1 5 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 vs . 25 \u00b1 6 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 ) , completion rate ( 11 \u00b1 4 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 vs . 19 \u00b1 6 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 ) , and stalling rate ( events that persist the entire length of our 4 . 8 min movies ) ( 3 \u00b1 1 % vs . 0 . 8 \u00b1 0 . 9 % ( Supplemental Figure S7F ) . Treatment of cells with more strongly hypotonic ( 75 mOsm ) me - dia dramatically perturbed CME dynamics . After 2 min in 75 mOsm hypotonic media , tracks elongated and very often lasted the entire duration of the 4 . 8 min movie ( Figure 2C , Supplemental Movie 2 , and Supplemental Table S1 ) . The mean lifetimes of CLTA - TagRFP - T EN and DNM2 - eGFP EN tracks were 128 \u00b1 112 s ( Figure 2D ) and 125 \u00b1 112 s ( Figure 2E ) , respectfully . We also observed a substantial decrease in the CME initiation rate ( 10 . 3 \u00b1 1 . 5 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 vs . 22 . 2 \u00b1 3 . 5 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 ) and completion rate ( 5 . 3 \u00b1 1 . 7 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 vs . 17 . 8 \u00b1 3 . 4 \u00d7 10 \u2013 2 \u03bcm \u2013 2 min \u2013 1 ) and an increase in track stalling ( 19 \u00b1 9 % vs . 0 . 7 \u00b1 0 . 3 % ) ( Figure 2 , F \u2013 H ; Supplemental Figure S7H ) . After 10 min of culturing , the CLTA - TagRFP - T EN and DNM2 - eGFP EN lifetimes began to recover , most likely reflecting cellular adaptation to the hypotonic treatment ( Figure 2 , C \u2013 H , and Supplemental Movie 3 , 4 , and 5 ) . We did not detect effects of hypo - tonic media treatment on lifetimes of tracks containing only CLTA - TagRFP - T EN , characteristic of clathrin structures not associated with CME ( Supplemental Figure S7 , A , C , E , and G ) ( Hong et al . , 2015 ) . DNM2 - eGFP EN - only events , presumably representing non \u2013 clathrin mediated endocytosis events , showed a moderate response to el - evated membrane tension with 75 mOsm hypotonic media ( Supple - mental Figure S7G ) . We conclude that elevating plasma membrane tension with hypotonic shock perturbs CME dynamics in a dose - dependent manner . Actin force generation assists clathrin coat remodeling under elevated membrane tension Next , we determined the role of branched actin filament assembly on endocytic outcome under elevated membrane tension . Branched actin networks are generated by activated Arp2 / 3 complex ( Mullins et al . , 1998 ) . We inhibited Arp2 / 3 complex \u2013 mediated actin polym - erization using the small molecule CK666 ( Nolen et al . , 2009 ; Hetrick et al . , 2013 ) . We reasoned that inhibition of the Arp2 / 3 complex might have less effect on membrane tension than global actin in - hibitors such as latrunculin and jasplakinolide often used in previous CME studies . Nevertheless , because Arp2 / 3 complex inhibition can affect membrane tension ( Diz - Mu\u00f1oz et al . , 2016 ) , we first carefully established experimental conditions in which effects of high mem - brane tension or CK666 would not mask one another . Treatment of cells with 100 \u00b5M CK666 did not affect the membrane tether force in these cells ( p > 0 . 5 ) during the time the experiment was per - formed ( Supplemental Figure S8A ) . Under these optimized condi - tions , we performed live - cell fluorescence microscopy and STORM to learn more about the clathrin coat remodeling steps at which branched actin network assembly is required . We first titrated CK666 and monitored the effect on CME dy - namics after 2 min of treatment . We aimed to identify a minimal CK666 concentration that induced a rapid effect on CME dynamics . CLTA - TagRFP - T EN lifetimes to 79 \u00b1 66 s after 2 min of 100 \u00b5M CK666 treatment compared with 56 \u00b1 40 s for the dimethyl sulfoxide ( DMSO ) control ( Supplemental Figure S8B and Supplemental Table S2 ) . Similarly , DNM2 - EGFP EN lifetimes increased to 65 \u00b1 64 s upon CK666 treatment , compared with 49 \u00b1 38 s for the DMSO control ( Supplemental Figure S8B ; Supplemental Table S2 ) . CK666 at 100 \u00b5M did not affect CME completion frequency or the percentage of persistent CLTA - TagRFP - T EN tracks ( Supplemental Figure S8 , C and D ) , though we observed a small decrease in the CME initiation rate ( Supplemental Figure S8E ) . The elongation of DNM2 - eGFP EN and CLTA - TagRFP - T EN lifetimes upon 100 \u00b5M CK666 treatment was exacerbated upon simultaneous incubation in moderately hypotonic media ( 150 mOsm ) . Compared with controls , the combination of 100 \u00b5M CK666 and 150 mOsm hyopotonic media markedly lengthened the clathrin lifetimes to 96 \u00b1 86 s ( compared with DMSO control , 59 \u00b1 52 s ) and dynamin2 to FIGURE 1 : 2c - 3D STORM resolves clathrin structures highly connected to actin networks at different stages of endocytosis . ( A ) STORM image of the ventral surface of an SK - MEL - 2 cell immunolabeled with the CF - 680 antibody ( clathrin coats in red ) and phalloidin - AF647 ( actin in cyan ) . Orange squares are areas shown in panel B . Color bar shows the z position of actin . Scale bar : 5 \u00b5m . ( B ) Magnification of highlighted areas 1 and 2 in panel A . Magenta squares are shown in panel C . Scale bars : 250 nm . ( C ) X - Z projections of the regions highlighted in panel B . Scale bars : 100 nm . ( D ) Illustration of binning clathrin coats ( red ) into three geometric stages based on their aspect ratio ( shape index SI ) . Shallow : SI < 0 . 7 ; U - shape : 0 . 7 < SI > 0 . 9 and \u03a9 : SI > 0 . 9 . ( E ) X - Z projections of representative STORM images showing clathrin coats ( red ) with different actin ( cyan ) coverages around clathrin . Calculated SI of shallow CCSs from left to right image : 0 . 56 , 0 . 53 , 0 . 51 , 0 . 55 ; for U - shaped CCPs from left image to right image : 0 . 87 , 0 . 89 , 0 . 86 , 0 . 82 ; for \u03a9 - shaped CCPs from left image to right image : 1 . 31 , 1 . 06 , 1 . 31 , 1 . 52 . Scale bars : 100 nm . ( F ) Graph of endocytic coat SI as a function of actin coverage for shallow ( black dots ) , U - shaped ( blue dots ) , and \u03a9 - shaped ( gray dots ) pits . Categories of shape indices are chosen similar to E . Pits with actin coverage > 5 % are shown . R = \u2013 0 . 04 , n = 719 . Events accumulated from six cells . ( G ) Cartoon depicting the clathrin coat with actin either at the tip of the coat ( top ) , covering the clathrin coat completely ( middle ) , or at the base of the clathrin coat ( bottom ) . Dashed black lines indicate the average Z position of actin and clathrin . D z is the difference between average actin and clathrin Z positions . D z < 0 is defined as the average actin Z position nearer the base of the pit . Schematic is a hypothetical plot of D z vs . actin coverage for scenarios in which actin grows from the tip of the coat ( red line ) or the base of the pit ( black line ) . ( H ) D z as a function of actin coverage ( for actin coverage > 5 % , R = 0 . 66 , n = 719 , N = 6 cells ) . ( I ) Cartoon of actin ( blue ) growing from the base of the pit ( black lines ) to cover clathrin coat ( red ) from a shallow membrane invagination to a fully formed membrane vesicle . X - Z projection ( side profile ) is shown . Dashed arrows indicate that growth of the actin network is not tightly coupled to the endocytic coat geometry and is variable in extent . 6 | C . Kaplan et al . Molecular Biology of the Cell 84 \u00b1 85 s ( compared with DMSO control , 47 \u00b1 49 s ) ( Figure 3 , A \u2013 F , and Supplemental Table S3 ) . We conclude that in these cells , Arp2 / 3 complex \u2013 mediated actin assembly is required to maintain normal CME dynamics under elevated membrane tension . Next , we used STORM to determine which endocytic coat ge - ometries are enriched upon a combination of Arp2 / 3 complex inhi - bition and osmolarity treatment . Cells were cultured under the CK666 and osmolarity conditions described above , chemically fixed , and then immunolabeled for clathrin . Clathrin coat height served as a metric for the membrane invagination stage . As in the above 2c - 3D STORM experiments , the full progression from a flat clathrin coat to a rounded vesicle could be clearly resolved in 3D ( Figure 3G , bottom image panel ) . Control cells treated with DMSO showed an average clathrin coat height of 98 \u00b1 21 nm ( Figure 3H and Supplemental Figure S8F ) . The average height increased to 106 \u00b1 27 nm when cells were treated with 100 \u00b5M CK666 ( Figure 3H and Supplemental Figure S8F ) . Thus , when Arp2 / 3 complex activity was inhibited in these cells , clathrin pits accumulated at a greater height . This observation suggests that more clathrin pits may stall at a later stage of progression , consistent with observations of accu - mulated coat geometries from actin inhibitor studies ( Yarar et al . , 2005 ; Boulant et al . , 2011 ; Yoshida et al . , 2018 ) . Interestingly , when Arp2 / 3 - mediated actin polymerization was inhibited in cells with elevated membrane tension , the average clathrin coat height decreased to 96 \u00b1 24 nm ( Figure 3 , G and H , and Supplemental Figure S8F ) . This height decrease was also reflected in FIGURE 2 : Quantitative analysis of CME mechanosensitivity under elevated membrane tension . ( A ) Schematic of cells in isotonic media ( top ) or hypotonic media ( bottom ) , which causes water influx and stretches the cell membrane . In this figure , hypotonic treatment is 75 mOsm . ( B ) Mean membrane tether force values measured by AFM of cells in isotonic media ( n = 18 ) or in hypotonic media ( n = 17 ) . Mean values were obtained by pulling at least three tethers ( three independent experiments ) . In hypotonic treatment , circles are mean tether values from 2 to 10 min after hypotonic media exchange and triangles are mean tether values obtained between 10 and 16 min after hypotonic media exchange . Bars are mean \u00b1 SD . p = 0 . 002 by two - tailed Mann \u2013 Whitney test . ( C ) Kymographs of TIRF micrographs of live SK - MEL - 2 cells endogenously expressing CLTA - TagRFP - T EN ( magenta ) and DNM2 - eGFP EN ( green ) . Time is on the X axis . Kymographs are 4 . 8 min long . Cells were imaged in isotonic media ( top ) or hypotonic media for 2 min ( middle ) or 10 min ( bottom ) . ( D ) Cumulative distribution plot of clathrin lifetimes marked by CLTA - TagRFP - T EN in isotonic media ( red ) , hypotonic media for 2 min ( violet ) , and hypotonic media for 10 min ( orange ) . These tracks were associated with DNM2 - eGFP EN . ( E ) Cumulative distribution plot of dynamin2 lifetime marked by DNM2 - eGFP EN in isotonic media ( light green ) , hypotonic media for 2 min ( blue ) , and hypotonic media for 10 min ( dark green ) . These tracks were associated with CLTA - TagRFP - T EN . n = 5831 tracks in 17 cells across four experiments for D \u2013 H . ( D , E ) Detailed statistics in Supplemental Table S1 . ( F ) Plot of endocytic initiation rate for the three conditions . p < 0 . 05 by two - tailed Mann \u2013 Whitney test for both comparisons . ( G ) Endocytic completion rate in the three conditions . p < 0 . 05 by two - tailed Mann \u2013 Whitney for both comparisons . ( H ) Percentage of persistent tracks ( defined as tracks lasting the entirety of the image acquisition ) for the three conditions . p < 0 . 05 by two - tailed Mann \u2013 Whitney for both comparisons . ( F \u2013 H ) Barplots show mean \u00b1 SD . Statistics to plots in Supplemental Figure S7H . Volume 33 May 15 , 2022 Actin load adaptation in endocytosis | 7 FIGURE 3 : Importance of Arp2 / 3 complex \u2013 mediated actin polymerization during CME increases under elevated membrane tension . In this figure , CK666 ( Arp2 / 3 complex inhibitor ) treatment is 100 \u00b5M and hypotonic shock is 150 mOsm . ( A ) Kymographs of cells expressing CLTA - TagRFP - T EN ( magenta ) and DNM2 - eGFP EN ( green ) imaged by TIRF . Cells were imaged in isotonic media . The media was then exchanged to include CK666 ( bottom panel ) and imaged after 4 min . ( B ) Cumulative distribution plots of clathrin lifetimes in control , DMSO - treated conditions ( orange , n = 4219 ) , and CK666 - treated ( red , n = 3124 ) conditions . ( C ) Cumulative distribution plots for control , DMSO - treated ( dark green , n = 4219 ) , and CK666 - treated ( light green , n = 3124 ) dynamin2 lifetimes associated with clathrin lifetimes in B . ( BC ) Control , N = 10 cells , and CK666 treatment , N = 10 cells , measured in three independent experiments . Complete statistics in Supplemental Table S3 . ( D ) Kymographs of cells in hypotonic media . In the top panel , cells were placed in hypotonic media and imaged after 4 min . In the bottom panel , cells were treated with CK666 in hypotonic media and imaged after 4 min . ( E ) Cumulative distribution plots of clathrin lifetimes for control , DMSO - treated conditions ( magenta , n = 1405 ) , and CK666 - treated conditions ( blue , n = 2783 ) in hypotonic media . ( F ) Cumulative distribution plots of DMSO - treated ( black , n = 1405 ) and CK666 - treated ( olive , n = 2783 ) dynamin2 lifetimes in hypotonic media associated with clathrin lifetimes in E . ( E , F ) Control , N = 9 cells , and CK666 treatment , N = 10 cells , measured in three independent experiments . Complete statistics in Supplemental Table S3 . ( G ) Representative STORM images of immunolabeled clathrin - coated structures in control cells arranged by coat height . Top panel shows the x - y projections and bottom panel the corresponding x - z projections . The white square in the x - y projections shows the area that was cropped to generate the x - z projections . The heights of clathrin coats in the x - y projection from left to right image are 61 , 96 , 124 , 158 , and 177 nm . Scale bars : 100 nm . ( H ) Clathrin coat heights when cells were treated with DMSO ( n = 154 ) or CK666 ( n = 158 ) in isotonic media or CK666 in hypotonic media ( n = 159 ) . Clathrin coat images for quantitative analysis were collected from at least three different cells for each condition from a single experiment . Statistics are given in Supplemental Figure S8F . p < 0 . 05 in both comparisons by Mann \u2013 Whitney test . 8 | C . Kaplan et al . Molecular Biology of the Cell an accumulation of smaller shape indices and a very mild effect on clathrin coat width ( Supplemental Figure S8H ) . This result , along with our observation of slowed CME dynamics , suggests that upon membrane tension elevation in response to hypotonic conditions in SK - MEL - 2 cells , inhibition of Arp2 / 3 complex activity may cause an enrichment of shallow endocytic coat geometries . Actin organization adapts to elevated membrane tension by increasing clathrin coat coverage Finally , we used STORM to determine the relationship between el - evated membrane tension and actin cytoskeleton organization at CME sites ( Figure 4 ) . We treated cells with strong ( 75 mOsm ) hypo - tonic shock for 5 min and then chemically fixed them for 2c - 3D STORM . When we superresolved clathrin and actin by 2c - 3D STORM in these cells , the actin cytoskeleton remained intact ( Pan et al . , 2019 ) and associated with CCSs ( Supplemental Figure S9 , A \u2013 D ) . Strikingly , in response to hypotonic media treatment , the aver - age actin height increased for all endocytic geometries ( Figure 4 , B , C , and E ; Supplemental Figure S9 , E \u2013 G ) . When we quantified actin cell cortex height in randomly selected regions of the cell cortex , we did not observe any increase in cortical actin height , reinforcing the conclusion that any observed effects are specific for CME sites ( Sup - plemental Figure S10 , A \u2013 J and P \u2013 R ) . Overall , the average actin height at CME sites increased from 130 \u00b1 30 nm under isotonic me - dia conditions to 160 \u00b1 40 nm ( Figure 4C ) . This increase corre - sponded to an increase of actin growth over the clathrin coat from covering 66 \u00b1 23 % of the coat to covering 73 \u00b1 21 % under 75 mOsm hypotonic media ( Figure 4D ) . The increase in actin height was ob - served for all coat shapes ( e . g . , shallow or curved pits ) ( Figure 4E ) . Similarly , for different extents of clathrin coverage by actin , the aver - age actin height was greater in hypotonic conditions across different levels of coat coverage ( Figure 4F ) . However , the extent of asym - metry between actin and clathrin signals did not significantly change compared with that in the isotonic condition ( Supplemental Figure S9H ) . These observations of greater average actin height and cover - age over the clathrin coat suggest that the force contribution of ac - tin at CME sites is increased when membrane tension is elevated such that a greater load is carried by the actin network . Overall , these observations led us to conclude that under ele - vated plasma membrane tension , actin grows higher in the z - dimen - sion around clathrin coats ( Figure 4G ) . Such an adaptive mechanism for actin organization presumably generates the required forces to ensure the efficient progression of mammalian CME under various levels of membrane tension . DISCUSSION By combining 2c - 3D STORM imaging and quantitative live - cell TIRF microscopy for thousands of CME sites , with AFM tether pulling membrane tension measurements , and manipulations of membrane tension and Arp2 / 3 - mediated actin assembly , we showed that actin assembly adapts to changes in load at individual CME sites . This mechanism likely ensures that sufficient forces are generated for ro - bust endocytic progression over a range of membrane tension re - gimes . While STORM of individual CCSs cannot attain the resolution of EM , our approach had several advantages that allowed us to gain new mechanistic insights : 1 ) it allowed us to sample much larger numbers of CME sites with a preserved actin network than is possi - ble by EM , thus permitting rigorous quantitative analysis , 2 ) we im - aged CME sites and associated actin in intact cells that had not been subjected to unroofing or extraction protocols , and 3 ) we were able to use antibodies and fluorescent phalloidin to unambiguously identify specific proteins at CME sites . Actin assembly and organization adapt to elevated membrane tension Elevating membrane tension can have a dramatic impact on CME progression in mammalian cells ( Raucher and Sheetz , 1999 ; Boulant et al . , 2011 ; Ferguson et al . , 2016 , 2017 ; Willy et al . , 2017 ; Bucher et al . , 2018 ) . However , how cells adapt to compensate for increased load at CME sites has not been elucidated . Our results provide criti - cal mechanistic insights into how the CME machinery adapts to el - evated membrane tension to maintain robust CME progression . We showed quantitatively that actin assembly and organization adapt to changes in membrane tension , which we measured by AFM mem - brane tether pulling ( Figure 2 ) . Changes in membrane tension can in principle occur globally ( entire cells ) or locally ( in different regions of one cell , or even within different regions of a single endocytic site ) ( Houk et al . , 2012 ; Rangamani et al . , 2014 ; Shi et al . , 2018 ) . Because we detect different actin organizations at individual CME sites with similar clathrin coat geometries within a single cell , such differences might reflect subcellular , local load variation resulting from variance in such factors as membrane tension and cell adhesion . The obser - vation in other studies that actin assembles late in CME progression ( Merrifield et al . , 2002 ; Grassart et al . , 2014 ; Akamatsu et al . , 2020 ; Jin et al . , 2021 ) , coupled with the observation here that actin can associate with clathrin coats of different geometries , is consistent with the possibility that clathrin lattices of low curvature can develop high curvature under the \u201cconstant area\u201d model ( Avinoam et al . , 2015 ; Sochacki et al . , 2021 ) . Moreover , our data provide evidence that the low to high coat curvature transition becomes more depen - dent on actin assembly forces as load increases . Measurement of membrane tension changes using AFM . Using AFM , we measured the membrane tether force for SK - MEL - 2 cells in isotonic and hypotonic conditions ( Figure 2 ) . We measured tension on the dorsal cell surface while imaging endocytic events on the ventral surface . Local differences in membrane tension have been observed in some cell types ( Shi et al . , 2018 ) but not others ( Houk et al . , 2012 ) . Nevertheless , AFM remains the gold standard for mea - suring apparent membrane tension in cells ( Sitarska and Diz - Mu\u00f1oz , 2020 ) . In isotonic conditions , the tether force was 33 \u00b1 7 pN ( Figure 2B ) . This tether force is within an intermediate range measured for other cell types such as NIH3T3 cells , HeLa cells , and macrophages ( Sitarska and Diz - Mu\u00f1oz , 2020 ; Roffay et al . , 2021 ) . Assuming a 100 pN * nm bending rigidity of the plasma membrane , this value corresponds to a membrane tension of 0 . 14 \u00b1 0 . 04 pN / nm ( Diz - Mu\u00f1oz et al . , 2013 ) . In hypotonic conditions , the membrane tether force increased to 48 \u00b1 17 pN , which corresponds to a dou - bling of membrane tension to 0 . 29 \u00b1 0 . 04 pN / nm . Higher mem - brane tether values have been reported for other cell types ( Sitarska and Diz - Mu\u00f1oz , 2020 ) . Below , we describe implications of earlier results and our new results reported here on actin\u2019s role in CME un - der three different plasma membrane tension regimes ( Figure 4G ) . Low - membrane - tension regime . When membrane tension is low , clathrin coat assembly provides sufficient energy to bend the under - lying plasma membrane into a full spherical shape , similar to the constant - curvature model ( Figure 4G ) ( Saleem et al . , 2015 ; Willy et al . , 2021 ) . We indeed found in our STORM data that 26 % of clath - rin coats lack actin in shallow , U - shape and \u03a9 - shape CME geome - tries . This observation is consistent with mathematical modeling , which indicates that the coat can provide sufficient energy to bend the plasma membrane when membrane tension is low ( 0 . 002 pN / nm ) ( Hassinger et al . , 2017 ) . Here , we consider low membrane tension to be of a value lower than those we measured for SK - MEL - 2 cells in Volume 33 May 15 , 2022 Actin load adaptation in endocytosis | 9 FIGURE 4 : The actin network at CME sites increases in size in response to elevated membrane tension . In this figure , hypotonic refers to 75 mOsm media . ( A ) Schematic of cells in hypotonic media , which increases plasma membrane tension . The response of the actin network ( blue ) to elevated plasma membrane tension ( purple ) was previously unknown . ( B ) Representative STORM images of clathrin ( red ) and actin ( cyan ) in x - z projections for cells fixed after treatment in the hypotonic media for 5 min ( bottom ) . Coated pits are classified as shallow , U - shaped , or \u03a9 - shaped based on the aspect ratio of the coat . Scale bars : 100 nm . ( C ) Plots of actin Z height at CCPs from cells in the isotonic ( n = 736 ) and hypotonic ( n = 527 ) media measured from STORM x - z projections . Lines are median \u00b1 interquartile range . p < 0 . 0001 determined by Mann \u2013 Whitney test . ( D ) Plots of actin coverage over the clathrin coat in pits found in STORM x - z projection images in isotonic ( n = 719 ) and hypotonic ( n = 509 ) conditions . Pits with actin coverage > 1 % are plotted . Lines are median \u00b1 interquartile range . p < 0 . 0001 determined by Mann \u2013 Whitney test . ( E ) Actin Z height as a function of coat shape in isotonic ( gray , n = 736 ) and hypotonic ( purple , n = 527 ) conditions . ( F ) Actin Z height as a function of actin coverage over the clathrin coat in isotonic ( gray , n = 719 ) and hypotonic ( purple , n = 509 ) conditions . The data for isotonic conditions were also used to generate the plots in Figure 1 . Three independent STORM experiments with N _ = 6 cells in isotonic and N _ = 7 cells in hypotonic media . ( G ) Cartoon depicting an adaptive actin force \u2013 generating mechanism that counteracts elevated membrane tension to ensure robust CME progression . This schematic describes three scenarios in which membrane tension is low , intermediate , or high and how CME is accomplished efficiently by adaptive actin network organization . Under low tension ( bottom ) , the clathrin coat provides sufficient force to advance CME . At intermediate tension ( middle ) , actin polymerization is required for the transition from U to \u03a9 shape . At high tension ( top ) , endocytic progression slows . More pits stall at the shallow conformation . In response to increased resistance , the actin network grows to envelop the coat and provide additional force against high membrane tension . 10 | C . Kaplan et al . Molecular Biology of the Cell standard media ( Figure 2 ) . Given that actin polymerization appears to be dispensable for CME in some cell types , even though it still might make CME more efficient , we hypothesize that the basal membrane tension may be lower in those cell types . However , we urge caution with the interpretation of experiments with harsher actin drug treat - ments , as prolonged actin inhibitor treatment of cells can perturb the actin cortex and reduce the tether force measurements ( membrane tension ) by a factor of \u223c 50 % ( Pontes et al . , 2017 ) . In future studies , strategies should be developed to relate local membrane tension to specific endocytic site geometry and actin organization . Intermediate tension regime . In the intermediate tension regime , defined as the resting tension of SK - MEL - 2 cells in isotonic condi - tions ( Figure 2 ) , clathrin coat assembly and membrane curvature \u2013 in - ducing proteins still appear to provide sufficient energy to drive clathrin coats to adopt the U shape ( Figure 4G , intermediate mem - brane tension ) . When we inhibited Arp2 / 3 - mediated actin polymer - ization using CK666 , clathrin coat progression stalled at an aspect ratio most likely reflecting the U - shaped stage , consistent with the effects of actin assembly inhibition reported for other cell types ( Yarar et al . , 2005 ; Boulant et al . , 2011 ; Almeida - Souza et al . , 2018 ) . Thus , at intermediate membrane tension it appears that actin force generation is primarily required for the U - to \u03a9 - shaped clathrin coat transition . The actin network observed near the base of the pit may reflect a role driving plasma membrane neck constriction and scis - sion by generating forces orthogonal to the direction of membrane invagination as proposed previously ( Bucher et al . , 2018 ; Scott et al . , 2018 ; Mund et al . , 2021 ) . High - membrane - tension regime . Our live - cell and STORM obser - vations indicate that as membrane tension is elevated further , coat deformation and membrane invagination become increasingly de - pendent on actin force generation ( Figure 4 ) . When membrane ten - sion was elevated to an intermediate level ( e . g . , 150 mOsm media ) , CME lifetimes slowed modestly ( Supplemental Figure S7 ) . When we inhibited Arp2 / 3 - mediated actin polymerization using CK666 in cells treated with 150 mOsm media , the average clathrin coat height was lower than in CK666 - treated cells cultured under isotonic condi - tions , likely reflecting an enrichment of shallow pits ( Figure 3H ) . Un - der elevated membrane tension , we observed no change in actin cortex thickness , while others reported that the actually actin cortex tends to become thinner and less dense ( Supplemental Figure S10 ) ( Houk et al . , 2012 ; Chugh et al . , 2017 ; Roffay et al . , 2021 ) . However , our study shows that the actin network associated with CCPs instead increases in height under elevated membrane tension ( Figure 4 ) . Actin assembly from the base of the CCP continues until the net - work covers the clathrin coat completely , allowing it to interact with proteins linking the actin network to the clathrin coat ( Engqvist - Goldstein et al . , 2001 ; Messa et al . , 2014 ; Sochacki et al . , 2017 ) . Actin - binding linker proteins such as Hip1R and Epsin1 cover the clathrin coat completely and are thus positioned to provide high internalization efficiency by harnessing actin assembly forces per - pendicular to the plasma membrane ( Akamatsu et al . , 2020 ; Serwas et al . , 2021 ) . It may be that the \u201cconstant area\u201d model ( Avinoam et al . , 2015 ; Sochacki et al . , 2021 ) , in which a flat or shallow coat grows to full size and then bends , applies under the high load re - gime ( Bucher et al . , 2018 ; Scott et al . , 2018 ; Mund et al . , 2021 ) , and it is here that actin assembly forces are most important for remodel - ing the coat into a curved vesicle . An important question for future studies concerns the nature of the adaptive mechanism that increases actin assembly in response to elevated membrane tension . Possible mechanisms include the following : 1 ) stalling works passively to allow actin to assemble lon - ger ; or 2 ) high load works actively through mechanisms documented for other contexts , for example by increasing contact of filaments associated with the coated pit with membrane - associated N - WASP - Arp2 / 3 complex , leading to more assembly , alongside load - depen - dent decreases in the rate of filament capping ( Bieling et al . , 2016 ; Akamatsu et al . , 2020 ; Funk et al . , 2021 ; Li et al . , 2021 ) . As endocy - tosis and other actin - mediated trafficking events operate within a membrane geometry and local protein and lipid environment differ - ent from that in lamellipodia , a high priority is now to determine whether similar adaptive mechanisms operate during such local membrane bending processes . When the actin network fully covers the clathrin coat , it resem - bles the radial organization described by mathematical modeling for mammalian CME and the actin network organization described for budding yeast ( Ferguson et al . , 2009 ; Hassinger et al . , 2017 ; Mund et al . , 2018 ; Akamatsu et al . , 2020 ) . In yeast this actin organi - zation drives endocytic membrane invagination against the high resistance that results from turgor pressure . Mathematical modeling showed that this organization produces high forces perpendicular to the plasma membrane ( Hassinger et al . , 2017 ) . Actin - generated forces parallel and orthogonal to the membrane invagination at high tension may coexist to drive membrane invagination followed by neck constriction and scission . CME dynamics dramatically slow down when cells are in this high - membrane - tension regime , resulting in only \u223c 40 % of endocytic lifetimes shorter than 50 s compared with 80 % at low membrane tension ( Figure 2D ) . We also detected \u223c 19 % of lifetimes being lon - ger than the 4 . 8 min movies that we captured , a 20 - fold increase in stalled ( persistent ) tracks ( Figure 2H ) . We suggest that this tension regime pushes this adaptive mechanism to the limit . N - WASP spatial organization suggests an actin force generation control mechanism N - WASP spatial organization at CCSs and CCPs provides valuable mechanistic insight into how actin network assembly contributes to force generation during CME . We found that N - WASP localizes at the base of early and late CCPs , where it likely interacts with SH3 domain \u2013 containing proteins present at the endocytic membrane neck ( Sch\u00f6neberg et al . , 2017 ; Sochacki et al . , 2017 ; Almeida - Souza et al . , 2018 ) . This organization is similar to that of the ho - mologous nucleation - promoting factor Las17 in budding yeast ( Mund et al . , 2018 ) . Filaments nucleated at the base of CCPs would be able to interact with coat proteins such as Hip1R and Epsin1 / 2 / 3 to generate forces to invaginate the plasma membrane ( Hassinger et al . , 2017 ; Mund et al . , 2018 ; Akamatsu et al . , 2020 ; Joseph et al . , 2020 ) . Intriguingly , we also sometimes observed a strikingly different N - WASP spatial organization in which it was distributed over the full clathrin coat . The type II nucleation factors Abp1 and cortactin bind to actin filaments and to the Arp2 / 3 complex and could serve as binding partners for N - WASP around the CCP ( Le Clainche et al . , 2007 ; Pinyol et al . , 2007 ; Helgeson and Nolen , 2013 ; Guo et al . , 2018 ) . WASP located on the clathrin coat might reflect a distinct path for filament nucleation , originating from the coat rather than the base , that is potentially important to generate higher forces when actin already surrounds the CCP . A late burst of actin assembly was shown previously to often accompany CME ( Merrifield et al . , 2002 ) and to facilitate CME progression , especially when membrane tension is high ( Fujimoto et al . , 2000 ; Yarar et al . , 2005 ; Batchelder and Yarar , 2010 ; Boulant et al . , 2011 ; Grassart et al . , 2014 ; Li et al . , 2015 ; Ferguson et al . , 2017 ; Volume 33 May 15 , 2022 Actin load adaptation in endocytosis | 11 Yoshida et al . , 2018 ; Akamatsu et al . , 2020 ; Jin et al . , 2021 ) . In this study we observed that load - adapted , CME - associated actin assem - bly buffers changes in plasma membrane tension , thereby ensuring clathrin - coated vesicle ( CCV ) formation over a range of membrane tension regimes . We expect that the ability of actin assembly to re - spond to load is a common feature critical to many membrane re - modeling processes . MATERIALS AND METHODS Request a protocol through Bio - protocol . Cell culture SK - MEL - 2 cells from clone Ti13 ( hCLTA EN \u2013 1 / hDNM2 EN \u2013 1 ) were cul - tured in DMEM / F12 with GlutaMax supplement ( 10565 - 018 ; Thermo Fisher Scientific ) containing 10 % fetal bovine serum ( FBS ) and a 1000 U / ml penicillin \u2013 streptomycin mix ( 15140122 ; Thermo Fisher Scientific ) and kept in a 37\u00b0C humidified incubator with 5 % CO 2 ( cell source information in Doyon et al . , 2011 ) . After each cell vial was thawed , cells were checked after two passages for myco - plasma contamination . Cell line authentication was performed by short tandem repeat validation . Antibodies and reagents The primary antibodies used were mouse anti - clathrin light chain ( AB CON . 1 , MA5 - 11860 ; Thermo Fisher Scientific ) , mouse anti - clathrin heavy chain ( AB X - 22 , MA1 - 065 ; Thermo Fisher Scientific ) , and rabbit anti \u2013 N - WASP ( ab126626 ; Abcam ) . The secondary anti - bodies used were Alexa Fluorophore 647 chicken anti - rabbit ( A21443 ; Thermo Fischer Scientific ) , goat anti - mouse ( 115 - 005 - 205 ; Jackson ImmunoResearch ) conjugated to CF680 - NHS ester ( Bio - tium ; 92139 ) . Reagents and small molecule inhibitors used were DMSO ( D2650 ; Sigma Aldrich ) , CK666 ( SML0006 , batch # 0000012761 ; Sigma Aldrich ) , and phalloidin - AF647 ( A22287 ; Fisher Scientific ) . Preparation of CF680 - labeled secondary goat anti - mouse antibody CF680 NHS ester was dissolved at a concentration of 3 mM in anhy - drous DMSO . One microliter of dye solution , 80 \u00b5l of a 1 . 25 mg / ml suspension of unlabeled goat anti - mouse immunoglobulin G1 sec - ondary antibody ( 115 - 005 - 205 ; Jackson ImmunoResearch Labora - tories ) , and 10 \u00b5l of 1 M sodium bicarbonate solution were mixed and allowed to react for 15 min at room temperature . The reaction mixture was added to an equilibrated NAP - 5 column ( Sigma ; GE17 - 0853 - 01 ) and flushed with phosphate - buffered saline ( PBS ) . The dye - conjugated antibody was collected from the first colored eluent fraction , and a concentration of 0 . 12 mg / ml was determined with a NanoDrop spectrophotometer . Sample preparation for two - color clathrin and actin imaging Round coverslips ( 18 mm ) were cleaned 20 min in 70 % ethanol ( Electron Microscopy Science ; Cat # 72222 - 01 ) . Cells were detached with 500 \u00b5l 0 . 05 % trypsin ( 25300 - 054 ; Life Technologies ) , washed once in DMEM / F12 , and collected by centrifugation . Cells were counted using a hemocytometer , and 20 , 000 cells / ml were seeded onto 18 mm round coverslips in 12 - well plates . Cells were incubated for 16 \u2013 24 h in culture media before preparation for imaging . Cells were fixed first for 1 \u2013 2 min in 0 . 3 % ( vol / vol ) glutaraldehyde ( GA ) solution containing 0 . 25 % ( vol / vol ) Triton in cytoskeleton buffer ( CB : 10 mM MES , 150 mM NaCl , 5 mM ethylene glycol - bis ( 2 - aminoethylether ) - N , N , N \u2032 , N \u2032 - tetraacetic acid ( EGTA ) , 5 mM glucose , 5 mM MgCl 2 , 0 . 005 % NaN 3 , pH 6 . 1 ) and then immediately fixed for 10 min in 2 % ( vol / vol ) GA solution in CB . Both solutions were pre - pared fresh from a 70 % GA stock ( Electron Microscopy Science ; cat # 16365 ) ( protocol follows Xu et al . , 2012 ) . After fixation , samples were washed once in CB and then incubated for 7 min in freshly prepared CB containing 0 . 1 % ( wt / vol ) NaBH 4 . Subsequently , sam - ples were washed three times for 10 min in CB with gentle agitation on a shaker . Samples were then blocked for 30 min in 5 % ( wt / vol ) bovine serum albumin ( BSA ) in CB ( Sigma Aldrich ; A3733 ) . For dense clathrin labeling , light ( diluted 1 : 200 ) and heavy ( diluted 1 : 200 ) chain antibodies were used together in a 1 % ( wt / vol ) BSA CB solution . Primary antibody immunostaining was performed over - night at 4\u00b0C . The next day , samples were washed twice in 1 % ( wt / vol ) BSA CB for 5 min . The mouse secondary antibody CF680 was used at a final concentration of 0 . 40 \u2013 0 . 60 \u00b5g / ml in a 1 % BSA \u2013 1 \u00d7 CB solution . Samples were stained for 30 min at room temperature in the dark and washed twice for 5 min in 1 % ( wt / vol ) BSA CB solution and then for 10 min in CB solution . Samples were then placed into a solution of CB containing 0 . 5 \u00b5M phalloidin - AF647 and kept at room temperature in the dark for a minimum of 2 h . Samples were washed once with PBS before STORM imaging . Sample preparation for single - color clathrin and dual - color N - WASP imaging Cells were prepared as for the two - color sample preparation on cov - erslips and then fixed for 20 min in 3 % ( vol / vol ) paraformaldehyde ( PFA ; 15710 ; Electron Microscopy Sciences ) in CB ( protocol follows Li et al . , 2018 ) . Samples were washed quickly in CB and subse - quently were incubated for 7 min in freshly prepared 0 . 1 % ( wt / vol ) NaBH 4 in CB solution . Subsequently , samples were washed three times for 10 min in CB with gentle agitation on a shaker and per - meabilized afterward in a 0 . 1 % Triton - PBS solution for 1 \u2013 2 min . For single - antibody clathrin staining , subsequent washing , blocking , and antibody incubation steps were similar to those of the two - color clathrin and actin sample preparation protocol . Dual - color immunolabeling was performed with primary anti - body against N - WASP ( diluted 1 : 200 ) and clathrin heavy and clath - rin light chain ( diluted 1 : 600 \u2013 1 : 1000 ) in 1 % ( wt / vol ) BSA in PBS over - night at 4\u00b0C . Samples were washed the next day twice for 5 min in 1 % ( wt / vol ) BSA in PBS . Secondary antibody staining was first per - formed with Alexa Fluorophore 647 anti - rabbit antibody ( diluted 1 : 200 ) in 1 % BSA ( wt / vol ) in PBS for 30 min at room temperature and kept in the dark . After two 10 - min - long washes in PBS containing 1 % ( wt / vol ) BSA , secondary antibody staining was performed with CF680 anti - mouse antibody ( diluted 1 : 600 ) . The samples were given three final washes in PBS for 10 min each . SRM imaging Dye - labeled cell samples were mounted on glass slides in a standard STORM imaging buffer consisting of 5 % ( wt / vol ) glucose , 100 mM cysteamine , 0 . 8 mg / ml glucose oxidase , and 40 \u00b5g / ml catalase in 1 M Tris - HCl ( pH 7 . 5 ) ( Rust et al . , 2006 ; Huang et al . , 2008 ) . Cover - slips were sealed using Cytoseal 60 . STORM imaging was per - formed on a homebuilt setup ( Wojcik et al . , 2015 ) based on a modi - fied Nikon Eclipse Ti - U inverted fluorescence microscope using a Nikon CFI Plan Apo \u03bb 100 \u00d7 oil immersion objective ( NA 1 . 45 ) . Dye molecules were photoswitched to the dark state and imaged using a 647 - nm laser ( MPB Communications ) ; this laser was passed through an acousto - optic tunable filter and introduced through an optical fiber into the back focal plane of the microscope and onto the sample at an intensity of \u223c 2 kW cm 2 . A translation stage was used to shift the laser beam toward the edge of the objective so the light reached the sample at incident angles slightly smaller than the 12 | C . Kaplan et al . Molecular Biology of the Cell critical angle of the glass \u2013 water interface . A 405 - nm laser was used concurrently with the 647 - nm laser to reactivate fluorophores into the emitting state . The power of the 405 - nm laser ( typical range 0 \u2013 1 W cm \u2013 2 ) was adjusted during image acquisition so that at any given instant , only a small , optically resolvable fraction of the fluorophores in the sample was in the emitting state . For 3D STORM imaging , a cylindrical lens was inserted into the imaging path so that images of single molecules were elongated in opposite directions for mole - cules on the proximal and distal sides of the focal plane ( Huang et al . , 2008 ) . The raw STORM data were analyzed according to pre - viously described methods ( Rust et al . , 2006 ; Huang et al . , 2008 ) . Data were collected at a frame rate of 110 Hz for a total of \u223c 80 , 000 frames per image . Single - and two - color imaging was performed on cells labeled with Alexa Fluor 647 only or Alexa Fluor 647 and CF680 with 647 - nm excitation based on a ratiometric detection scheme ( Bossi et al . , 2008 ; Testa et al . , 2010 ; Gorur et al . , 2017 ) . In the two - color imaging scheme , light emitted from the AF647 and CF680 fluorophores was collected concurrently and split into two light paths using a long - pass dichroic mirror ( T685lpxr ; Chroma ) . Each light path was projected onto half of an Andor iXon Ultra 897 EM - CCD camera . Dye assignment was performed by localizing and recording the intensity of each single molecule in each channel . Conventional imaging of 560 - and 488 - nm dyes was performed im - mediately before STORM imaging using the appropriate laser and filter set . Emission data were collected through the short - wave - length reflected path of the aforementioned optical setup and over - laid directly onto the final STORM image . Details of selection and analysis of SRM images are found in the Supplemental Information . Our 3D - STORM setup was based on the same design as in Huang et al . ( 2008 ) and so we achieved comparable resolutions . The experimental STORM resolution was measured by repeatedly measuring the position of a single fluorophore and determining the SD of the localization distribution ( Huang et al . , 2008 ; Xu et al . , 2015 ) . We accordingly examined our STORM data in this work and overlaid the localization distributions of 24 representative single molecules from three different samples , as shown ( Supplemental Figure S1 ) . Gaussian fits ( red curves ) gave SDs of 10 nm in plane for the XY directions and 19 nm in depth for the Z direction . These re - sults are similar to those reported in Figure 1C of Huang et al . ( 2008 ) , where SDs are 9 nm in X , 11 nm in Y , and 22 nm in Z . TIRF microscopy TIRF imaging was carried out on a Nikon Eclipse Ti2 inverted micro - scope with a CFI60 60 \u00d7 Apo TIRF objective and a Hamamatsu Orca - Flash 4 . 0 V2 sCMOS camera . eGFP and Tag . RFP - T fluorescence was excited using 488 and 561 nm lasers and detected using a Chroma HC TIRF Quad Dichroic ( C - FL TIRF Ultra Hi S / N 405 / 488 / 561 / 638 ) and Chroma HC Quad emission filters BP 525 / 50 and BP600 / 50 , respectively ( Bellows Falls , VT ) . Unless mentioned specifically , chan - nels were acquired sequentially at a 1 . 2 s interval and 400 ms expo - sure time over 4 . 8 to 6 min . Real - time acquisition was achieved by a National Instruments ( PXI 1033 ; Austin , TX ) controller . The system was controlled with NIS - Elements software and maintained at 37 \u00b0 C by an OkoLab environmental chamber ( Burlingame , CA ) . Hypo - osmotic media treatment SK - MEL - 2 cells were plated on glass coverslips 1 d before osmotic treatment and imaging : 20 , 000 cells / m were seeded 16 \u2013 24 h before the experiment on 25 mm round # 1 . 5 glass coverslips that had been cleaned with 70 % ethanol ( Warner Instruments ; 64 - 0715 ) . Isotonic imaging media contained DMEM and Ham\u2019s F - 12 medium ( DMEM / F12 ) without phenol red ( 11039 ; Thermo Fisher Scientific ) with 5 % vol / vol FBS . The media was diluted with an inorganic salt solution containing 10 mM CaCl 2 0 . 3 mM MgCl 2 and 0 . 1 mM MgSO 4 ( CMM ) to maintain concentrations of critical ions , while obtaining hypo - os - motic conditions by diluting the media containing components such as d - glucose . The 225 mOsm hypotonic imaging media contained 1 : 4 vol / vol CMM solution in DMEM / F12 , the 150 mOsm hypotonic imaging media contained 1 : 1 vol / vol CMM solution in DMEM / F12 , and the 75 mOsm hypotonic imaging media contained 4 : 1 vol / vol CMM solution in DMEM / F12 . Five percent vol / vol FBS was present in all hypotonic solutions . For live - cell fluorescence microscopy CLTA - TagRFP - T EN and DNM2 - eGFP EN fluorescence in SK - MEL - 2 cells was acquired first in isotonic media over a course of 4 . 8 min . Subsequently , media was exchanged on the stage to hypotonic media ( either 225 , 150 , or 75 mOsm ) and movies were acquired for 4 . 8 min , starting 2 and 10 min after media exchange . Media exchange on the stage did not affect CME initiation rates or fluores - cence lifetimes beyond the existing experimental intrinsic variability ( Supplemental Figure S7 , A and B ) . For STORM imaging , 75 mOsm hypotonic buffer treatment was performed in the cell culture dish for 5 min . Cells were immediately chemically fixed after the 5 min treatment and further treated with the STORM sample preparation protocol as described above . Methods to analyze TIRF data can be found in the Image analysis section . CK666 concentration titration SK - MEL - 2 cells ( 20 , 000 / ml ) were seeded in eight - well chambers 16 h\u201424 h before the experiment ( 80826 ; ibidi , Fitchburg , WC ) . A CK666 ( SML0006 , batch # 0000012761 ; Sigma Aldrich ) stock solu - tion was prepared at 50 mM in DMSO and kept at \u2013 20\u00b0C . CK666 ( 25 , 50 , and 100 \u00b5M ) and equivalent 0 . 5 % vol / vol DMSO , 1 % vol / vol DMSO , and 2 % DMSO vol / vol solutions for controls were prepared fresh in DMEM / F12 containing 5 % FBS and kept at 37\u00b0C until used . Cells were first imaged in DMEM / F12 containing 5 % FBS solution as a baseline control for 4 . 8 min . Subsequently , imaging solution was exchanged on the microscopy stage to CK666 - or DMSO - contain - ing imaging solution and another 4 . 8 - min movie was acquired after 2 min of treatment . Each treatment was repeated twice , and an area of 1024 pixels \u00d7 1024 pixels was used to record 3 \u2013 6 cells per experiment . CK666 in combination with hypo - osmotic media Cells were prepared as for the CK666 concentration titration experi - ment described above . Solutions of 2 % vol / vol DMSO in DMEM / F12 , 100 \u00b5M CK666 in DMEM / F12 , 2 % vol / vol DMSO in 1 : 1 vol / vol CMM solution in DMEM / F12 ( 150 mOsm hypotonic media ) , and 100 \u00b5M CK666 1 : 1 vol / vol CMM solution in DMEM / F12 ( 150 mOsm hypotonic media ) were prepared fresh and kept at 37\u00b0C until used . All solutions contained 5 % FBS . Cells were first imaged in DMEM / F12 - 5 % FBS solution as a baseline control for 6 min . Subsequently , the imaging solution was exchanged on the microscopy stage to the desired experimental solutions and a 6 - min movie was recorded af - ter 4 min of incubation . Tether pulling experiments using AFM Custom - cut 35 - mm glass - bottom dishes ( Greiner Bio - One ; # 627860 ) were coated with fibronectin ( 50 \u00b5g / ml ; Corning # 356008 ) for 30 min and washed with Dulbecco\u2019s phosphate buffered saline ( DPBS ) shortly before use . SK - MEL - 2 cells were seeded at a density of 0 . 15 \u2013 0 . 20 \u00d7 10 5 cells / ml in DMEM / F12 GlutaMax supplement Volume 33 May 15 , 2022 Actin load adaptation in endocytosis | 13 with 1 % FBS and a penicillin \u2013 streptomycin mix ( Gibco ; # 15140 - 122 ) in a 37\u00b0C humid incubator with 5 % CO 2 for 2 \u2013 4 h and used directly for membrane tether pulling experiments . OBL - 10 cantilevers ( Bruker ) were mounted on a CellHesion 200 AFM ( Bruker ) integrated into an Eclipse Ti inverted light microscope ( Nikon ) , calibrated using the thermal noise method and coated with 2 . 5 mg / ml concanavalin A ( C5275 ; Sigma ) for 1 h at 30\u00b0C . After the cantilever was rinsed with DPBS , it was positioned at any location over the cell for tether pull - ing using bright - field imaging . The approach velocity was set to 1 \u00b5m / s , contact force to 100 \u2013 300 pN , contact time to 300 ms \u2013 10 s , and retraction speed to 10 \u00b5m / s . After a 10 \u00b5m tether was pulled , the cantilever position was held constant until the moment of tether breakage and at least 2 s afterward . The sampling rate was set to 2000 Hz . After measurements of tether forces in control conditions , an inorganic salt solution containing 10 mM CaCl 2 , 0 . 3 mM MgCl 2 , and 0 . 1 mM MgSO 4 was added to the medium ( 4 : 1 vol / vol ) to achieve 75 mOsm hypotonic treatment . Tether forces were mea - sured after media dilution for 2 \u2013 16 min . Tether forces per cell are the average of at least three tethers . Cells were not used longer than 1 h for data acquisition . Force - time curve analysis was performed us - ing JPKSPM Data Processing Software . Data analysis , statistical analysis , and data plotting For statistical analysis , data plotting , and image analysis , Prism ver - sion 7 . 0e and python module matplotlib , numpy , pandas modules , and scikit library in Jupyter notebook ( Python 3 . 8 . 3 ) were used . For some parts of the image analysis we used FIJI / ImageJ ( version 2 . 1 . 0 / 1 . 53c ) . Image analysis Selection of clathrin - coated superresolved structures for image analysis . Clathrin - containing structures were extracted from pro - cessed STORM images using a custom MATLAB routine . Briefly , a kernel convolution with a disk of \u223c 80 nm radius was performed on the superresolved clathrin image channel to accentuate possible CCPs . The resulting image was median filtered , and peaks were de - tected by sequentially identifying and cropping out regions corre - sponding to the local image maxima . Regions 310 nm \u00d7 310 nm wide square centered at these peaks were cropped from all color channels and aligned . Subsequently , we selected superresolved clathrin coats by visual inspection for further quantitative image analysis based on the following criteria : We excluded clathrin - la - beled structures for image analysis that appeared deformed , that covered nearly the entire 310 nm \u00d7 310 nm wide square , or that contained small punctuated structures that were not distinguishable from background noise or small clathrin seeds ( Supplemental Figure S4A ) . Clathrin - coated structures selected for analysis were first in - spected to determine whether they appeared round - shaped , ellipti - cal - shaped , or triangle - shaped in the x - y projected superresolved image . These images were 3D rendered to determine whether the x - z projection resulted in the typical wide - crescent shape , U - shape , or round - shape appearance for the clathrin coat . If we could identify the stereotypical clathrin - coat shapes in both projections , we in - cluded the clathrin coat in the pool for further image analysis . We define stereotypical shapes of individual clathrin coats based on platinum replica EM data of CCPs in the same cell type ( Supplemen - tal Figure S3 ) ( courtesy of Justin Taraska ) . We then assigned the clathrin coats as being actin negative when the number of localiza - tions in the actin channel in the 310 nm \u00d7 310 nm region of interest ( ROI ) was below 50 , which is close to the background level . Clathrin - coated structures were classified as actin positive when the number of localizations in the actin channel in the 310 nm \u00d7 310 nm ROI was above 50 and actin signal overlapped with the clathrin signal ( Supplemental Figure S9 , E \u2013 G ) . STORM image data display in figures . Reconstructed superreso - lution images were visualized using the \u201cinsight\u201d software package and saved in a . png file format ( Huang et al . , 2008 ) . These images were loaded into ImageJ , converted from an RGB image into an 8 bit image , pseudocolored for clathrin = red hot lookup table , actin = cyan lookup table , and then converted back into an RGB image . Image analysis of reconstructed clathrin and actin side views . Se - lected reconstructed superresolved images of clathrin and actin were treated as digital images for further analysis . A custom - written Jupyter notebook script was used to project reconstructed side views of clathrin and actin onto their x - and z - axes to obtain histo - grams of the clathrin and actin pixel intensity signals normalized to their respective maximum intensity value ( Supplemental Figure S4 , B and C ) . From these normalized z - axis and x - axis pixel intensity histograms , we read out the height and width of clathrin and actin at the 30th percentile . This process resulted in more robust results than the readout at the full width at half maximum ( Supplemental Figure S4 , B and C ) . Actin and clathrin coat height : The z - axis intensity histograms were used to report the actin and clathrin coat heights in the x - z projections . Before extracting the values , a mean filter set to 50 was run over the histograms to reduce signal fluctuations that interfere with the size measurement ( Supplemental Figure S4 , B and C ) . Actin - clathrin overlap calculation : We calculated the total over - lap between clathrin and actin using the indices obtained at the upper position of clathrin and lower position of actin at the 30th percentile of the respective z axis \u2013 projected intensity histograms ( Supplemental Figure S4B ) . We then reported the overlap relative to the clathrin height in units of percentage . Clathrin - coat width : We used the 30th percentile of x - axis inten - sity histogram to report the clathrin coat width in the x - z projection . Before extracting the values , a median filter set to100 was run over the histogram to smooth out signal fluctuations that interfere with the correct size determination ( Supplemental Figure S4 , B and C ) . Shape index calculation : Shape indices of individual clathrin structures are defined as the ratio between clathrin coat height and clathrin coat width . This value allowed us to sort flat ( low shape in - dex ) and rounded ( high shape index ) clathrin coats from each other . Asymmetry of actin signal around clathrin coat : To evaluate the asymmetry of the spatial actin organization around the clathrin coat , we determined the difference in the positions of the peak actin and clathrin signals on both x - z and y - z projections of our images ( Sup - plemental Figure S4E ) . We obtained the center of the clathrin coat in nanometers by rendering the clathrin superresolved image into a diffraction - limited image , as explained in the section Selection of clathrin - coated superresolved structures for image analysis . We first identified the position of the actin maximum intensity in the x - z pro - jection profile and y - z projection profile in nanometers . Then we measured the distance of the obtained position to the middle posi - tion of the clathrin intensity profile . This distance measurement is proportional to the asymmetry of the actin position with respect to clathrin ; namely , a low distance corresponds to high symmetry and a high distance corresponds to high asymmetry in the position of the actin signal . Image analysis of TIRF live - cell microscopy data . Fluorescent dif - fraction - limited spots of DNM2 - eGFPEN and CLTA - TagRFP - TEN in SK - MEL - 2 cells were tracked in each channel over the course of a 14 | C . Kaplan et al . Molecular Biology of the Cell movie using the detection and tracking features of the cmeAnalysis software package in Matlab ( Aguet et al . , 2013 ) . Subsequently , we cleaned up the background signal and , on the basis of the x and y positions of associated CLTA - TagRFP - TEN and DNM2 - eGFPEN flu - orescent tracks , separated them from CLTA - TagRFP - TEN and DNM2 - eGFPEN that were not associated with each other , using a custom - written Matlab script ( 10 , 36 , 42 ) . We measured fluores - cence lifetimes for DNM2 - eGFPEN and CLTA - TagRFP - TEN tracks that were associated and not associated with each other and that appeared and disappeared within the duration of a movie . We clas - sified the tracks as \u201cpersistent\u201d when they were already present in the first movie frame and lasted longer than the movie . CME initiation rate and completion rate measurement . We de - fined a track as initiated if it appeared within the course of the acqui - sition , excluding the first frame . We classified a track as complete when it appeared and disappeared within the course of the acquisi - tion . To calculate rates of initiation and completion , we used these values along with measurement of the area of the tracked region , which was obtained from the binary cell mask image generated dur - ing the detection step of the cmeAnalysis software that highlights the cell area in which particle tracking was performed . ImageJ was then used to calculate the area of the cell from the binary mask image . The final metric is defined as the number of initiations ( or completions ) per area per time . . Actin cortex height quantification . To measure the height of the actin cortex in our 2c - 3D STORM images , we cropped an approxi - mately 6 \u00b5m \u00d7 6 \u00b5m ROI of the superresolved clathrin image in a cell in which both actin and clathrin were imaged . Using this mask we removed all coordinates from the actin X , Y , and Z localization fluo - rophore list that overlapped with clathrin in that ROI . Then , we re - ported the SD of those Z coordinates not associated with clathrin and their median as a metric for actin cortex height . The clathrin mask was generated as follows : An approximately 4 \u00b5m \u2013 6 \u00b5m \u00d7 4 \u00b5m \u2013 6 \u00b5m ROI of the superresolved clathrin image of a cell was convolved with Gaussian distribution ( sigma = 5 ) ( Supple - mental Figure S2K ) . We ran a local maxima finder on the Gaussian - rendered image ( Scikit Image , Python ) . We generated a 2D histo - gram array of the local maxima and saved these as an image file . To this image we applied an outline finding algorithm ( FIJI ) , followed by a maximum filter ( px = 3 , FIJI ) , and finally with a hole - filling algo - rithm ( FIJI ) . The hole - filled image was inverted such that all clathrin areas had pixel value zero and all nonclathrin areas had pixel value 1 ( Supplemental Figure S2L ) . This mask was subsequently applied to a 2D histogram \u2013 generated array of actin X , Y , and Z superre - solved coordinates from the same ROI ( Supplemental Figure S2 , M and O ) . Both the clathrin mask and the 2D histogram actin array were adjusted to the same pixel size and rescaled by a factor of four . We then calculated the SDs of binned Z coordinates . Bins , respec - tively pixels , with fewer than four localizations were rejected for the analysis . The SDs of binned Z coordinates were converted from pix - els into nanometers for data visualization and plotted in histograms in Supplemental Figure S2 , P and Q . In Supplemental Figure S2R we report the median SD of the actin Z coordinates as a metric for actin cortex height . ACKNOWLEDGMENTS C . K . was funded by the German Research Foundation ( DFG KA4305 / 1 - 1 ) . D . G . D . was funded by National Institutes of Health ( NIH ) grant R35GM118149 . K . X . was funded by the National Sci - ence Foundation under CHE - 1554717 and the Pew Biomedical Scholars Award . A . D . M . was funded by the European Molecular Bi - ology Laboratory ( EMBL ) , Human Frontiers Science Program ( HFSP ) grant number RGY0073 / 2018 , and Deutsche Forschungsgemein - schaft ( DFG ) grant numbers DI 2205 / 2 - 1 and DI 2205 / 3 - 1 . M . A . was funded by NIH grant 1 K99 GM132551 - 01 . E . S . was funded by the EMBL and the Joachim Herz Stiftung Add - on Fellowship for Inter - disciplinary Science . 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In this article , we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome - like adhesions and src - induced podosomes . We further demonstrate that these sites comprise microdomains of highly or - dered lipid enriched in GRAF1 endocytic cargo . GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells . Depletion of GRAF1 , which inhibits CLIC generation , resulted in profound defects in cell spreading and migration . We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers , facilitating membrane turnover during cell morphological changes . INTRODUCTION Cells interact with their immediate environments through the liga - tion of plasma membrane - anchored or transmembrane receptors for soluble molecules , such as growth factors , extracellular matrix components , and proteins presented on the surface of neighboring cells . Cell - matrix adhesions are local , dynamic attachments of cell - surface proteins , including integrins and glycophosphatidylinositol - anchored proteins ( GPI - APs ) , to extracellular matrix components that allow indirect bridging of this matrix to the internal cytoskele - ton . These dynamic anchor points determine the position of the cell in space and allow cells to undergo shape changes , including those required during cell division , spreading , and migration ( Doherty and McMahon , 2008 ) . Initial sites of such adhesion can produce focal complexes , which can 1 ) be disassembled ( if conditions so dictate ) ; 2 ) become stabilized ; or 3 ) grow into larger , more mature ( and less dynamic ) adhesion sites , known as focal adhesions , that allow strong connection of the matrix to actin stress fibers . The diversity and dy - namics of these integrin - based adhesions , including podosomes and invadopodia , are coordinated by the activity of rho - family small G proteins that transduce internal and external cues into signals for the development , maintenance , growth , and disassembly of these anchor points . The turnover of adhesion - associated lipid domains and proteins has the potential to regulate adhesion sites , and several studies have suggested an important role for endocytosis in their dynamics ( reviewed in Caswell et al . [ 2009 ] ) . Adhesive sites strongly affect lipid order and promote the formation of microdomains necessary for certain endocytic events . Interestingly , loss of adhesion correlates with rapid endocytosis of molecules enriched in microdomains , such as cholera toxin B subunit ( CTxB ; del Pozo et al . , 2004 ; Schlunck et al . , 2004 ; Gaus et al . , 2006 ) . It has been shown that focal adhesion Monitoring Editor Yu - Li Wang Carnegie Mellon University Received : Dec 2 , 2010 Revised : Aug 16 , 2011 Accepted : Sep 20 , 2011 * These authors contributed equally to this work . The authors declare no competing interests . Address correspondence to : Richard Lundmark ( Richard . lundmark @ medchem . umu . se ) . \u201cASCB \u00ae , \u201c \u201cThe American Society for Cell Biology \u00ae , \u201d and \u201cMolecular Biology of the Cell \u00ae \u201d are registered trademarks of The American Society of Cell Biology . \u00a9 2011 Doherty et al . This article is distributed by The American Society for Cell Biology under license from the author ( s ) . Two months after publication it is avail - able to the public under an Attribution \u2013 Noncommercial \u2013 Share Alike 3 . 0 Unport - ed Creative Commons License ( http : / / creativecommons . org / licenses / by - nc - sa / 3 . 0 ) . This article was published online ahead of print in MBoC in Press ( http : / / www . molbiolcell . org / cgi / doi / 10 . 1091 / mbc . E10 - 12 - 0936 ) on September 30 , 2011 . Abbreviations used : BAR , Bin / amphiphysin / Rvs ; CLIC , clathrin - independent car - rier ; CTxB , cholera toxin B subunit ; DA , dominant - active ; DTT , dithiothreitol ; FAK , focal adhesion kinase ; GFP , green fluorescent protein ; GIT1 , G protein \u2013 coupled receptor kinase \u2013 interacting ArfGAP 1 ; GPI - AP , glycophosphatidylinositol - an - chored protein ; GRAF1 , GTPase regulator associated with focal adhesion ki - nase - 1 ; GST , glutathione S - transferase ; MEF , mouse embryonic fibroblast ; PBS , phosphate - buffered saline ; pFAK , phosphorylated FAK ; PH , pleckstrin homology ; PIP2 , phosphatidyl inositol 4 , 5 bisphosphate ; PLA , podosome - like adhesion ; RFP , red fluorescent protein ; rhoGAP , rho GTPase - activating protein ; ROCK , rho ki - nase ; siRNA , small interfering RNA ; Tfn , transferrin . Gary J . Doherty a , * , Monika K . \u00c5hlund b , * , Mark T . Howes c , Bj\u00f6rn Mor\u00e9n b , Robert G . Parton c , Harvey T . McMahon a , and Richard Lundmark b a MRC Laboratory of Molecular Biology , Cambridge CB2 0QH , United Kingdom ; b Medical Biochemistry and Biophys - ics , Ume\u00e5 University , 901 87 Ume\u00e5 , Sweden ; c Institute for Molecular Bioscience and Centre for Microscopy and Microanalysis , University of Queensland , Brisbane QLD 4072 , Australia Volume 22 November 15 , 2011 GRAF1 trafficking directs cell spreading | 4381 turnover can be induced by regrowth of microtubules after their de - polymerization in a process that requires the endocytic protein dy - namin and focal adhesion kinase ( FAK ; Ezratty et al . , 2005 , 2009 ) . Dynamin is also found at podosomes . These are rather small and highly active adhesion sites comprising adhesion proteins with an actin core . Here , dynamin is thought to function in the interplay be - tween membrane and actin dynamics ( Ochoa et al . , 2000 ) . The best understood endocytic process is mediated by the coat protein clathrin , but cells use a variety of alternative , clathrin - inde - pendent endocytic routes ( Doherty and McMahon , 2009 ) . The most prevalent of these in fibroblasts is mediated by clathrin - inde - pendent carriers ( CLICs ; reviewed in Mayor and Pagano [ 2007 ] ; Hansen and Nichols [ 2009 ] ; Howes et al . [ 2010b ] ) . We have recently shown that the membrane remodeling protein GTPase regulator associated with focal adhesion kinase - 1 ( GRAF1 ) regulates GPI - APs and CTxB uptake into CLICs , as well as a large proportion of fluid - phase uptake ( Lundmark et al . , 2008 ) . These pleiomorphic , tubulo - vesicular carriers lack an obvious protein coat and form in the ab - sence of clathrin and caveolin ( Kirkham et al . , 2005 ) . Internalization of GPI - APs and other microdomain - enriched molecules via these carriers depends upon the activity of cdc42 , GRAF1 , Arf1 , ordered lipid microdomains , and actin polymerization ( Sabharanjak et al . , 2002 ; Chadda et al . , 2007 ; Kumari and Mayor , 2008 ; Lundmark et al . , 2008 ) . GRAF1 has a rho GTPase - activating protein ( rhoGAP ) domain that stimulates the GTPase activity of cdc42 in vitro ( Hildebrand et al . , 1996 ) , as well as Bin / amphiphysin / Rvs ( BAR ) and pleckstrin homology ( PH ) domains required for membrane binding and CLIC formation . We describe a link between the en - docytic activity of GRAF1 and adhesion modulation . GRAF1 was found enriched at podosome - like adhesions induced in HeLa cells . Our investigations of the activity of these structures support a central role for GRAF1 and CLIC production in cell spreading and migration through modulation of the dynamics of these adhe - sion sites . RESULTS The GRAF1 interactome links endocytosis and cell adhesion Monomers of the dimeric protein GRAF1 are composed of BAR , PH , rhoGAP , and SH3 domains ( Figure 1A ) . Our previous studies have shown that GRAF1 is necessary for the generation of clathrin - inde - pendent endocytic carriers that endocytose a large amount of extra - cellular fluid and cargoes that include CTxB and GPI - APs ( Lundmark et al . , 2008 ) . GRAF1 has also been implicated in regulation of focal adhesions and cytoskeletal rearrangements ( Hildebrand et al . , 1996 ) . To address how these seemingly distinct roles might be linked , we first sought to identify additional proteins that function together with GRAF1 . We immunoprecipitated GRAF1 from rat brain cytosol and identified dynamin , FAK , and G protein \u2013 coupled receptor kinase \u2013 interacting ArfGAP 1 ( GIT1 ) as interacting partners using mass spectrometry and immunoblotting ( Figure 1B ) . We also tested whether GRAF1 could be isolated by immunoprecipitating dynamin , FAK , or GIT1 . Under these conditions , GRAF1 coimmuno - precipitated with dynamin , but not with either FAK or GIT1 , sug - gesting that these interactions were weaker or that the antibodies perturbed the binding ( Figure 1C , top panel ) . GRAF1 could , how - ever , be coimmunoprecipitated with the active , phosphorylated form of FAK , pFAK ( Figure 1C , bottom panel ) . Pulldown studies us - ing the GRAF1 SH3 domain as bait in rat brain cytosol verified that the interaction to dynamin and FAK was dependent on this domain ( Figure 1D ) . We then showed that GRAF1 , GIT1 , and dynamin are found together at a subset of basal plasma membrane structures in HeLa cells , suggesting that GRAF1 also interacts with these proteins in vivo ( Figure 1E ) . GIT1 is an Arf GTPase - activating protein that lo - calizes to cell - matrix adhesions ; it has been proposed that GIT1 pro - motes adhesion down - regulation and cell spreading ( Zhao et al . , 2000 ; Manabe et al . , 2002 ) and influences endocytosis and recycling of G protein \u2013 coupled receptors ( Premont et al . , 1998 ; Claing et al . , 2000 ; Lahuna et al . , 2005 ) . Interestingly , the endocytic membrane fission protein dynamin is also implicated in cell - matrix adhesion site turnover and is present at podosomes and focal complexes ( Ochoa et al . , 2000 ; Ezratty et al . , 2005 ) . The known interactions of GRAF1 are illustrated in an interactome in Figure 1F . Taken together with previous results , our observations suggest that the N - terminal BAR and PH domains endow GRAF1 with the ability to generate / stabilize highly curved endocytic membranes from ordered , phosphatidyl inositol 4 , 5 - bisphosphate ( PIP2 ) - enriched plasma membrane re - gions , while the C - terminal rhoGAP and SH3 domains modulate rho - family small G - protein activity and interact with dynamin and FAK . GRAF1 is not a general component of focal adhesions but localizes to podosome - like adhesions Given the nature of the GRAF1 interactome , we examined whether GRAF1 localization / function was linked to adhesion sites . When GRAF1 was expressed at very high levels , cells underwent a drastic morphological rearrangement , developing long protrusions and collapsing around the nucleus ( Taylor et al . , 1999 ; Supplemental Figure S1 , A and B ) . In these cells , the typical focal adhesions and actin stress fibers seen in low - level , overexpressing cells or control cells were absent , and only a few , small punctate adhesions contain - ing vinculin and actin could be detected . When cells with endoge - nous or low levels of overexpressed GRAF1 were costained with markers for adhesions such as vinculin , we found that GRAF1 was not detected at large , elongated adhesions known as mature focal adhesions ( see Figure S1 , C and D ) . Instead , both endogenous and overexpressed GRAF1 were found enriched at small , round , vincu - lin - positive structures in a subset of cells ( see Figure S1 , C and D ) . Our data suggested that GRAF1 might be recruited to a specific intermediate state of adhesive structures . To determine whether its interacting partner GIT1 , which is normally found at cell - matrix ad - hesion sites , could promote the localization of GRAF1 to adhesion sites , we co - overexpressed these proteins . This resulted in fewer mature focal adhesions and a large increase in the number of vincu - lin - and GRAF1 - positive structures ( Figure 2 , A and B ) . This is consis - tent with previous observations that GIT1 promotes turnover of focal adhesions ( Zhao et al . , 2000 ) and promotes podosome forma - tion ( Wang et al . , 2009 ) . GRAF1 - positive adhesive structures closely resembled podo - some - like adhesions ( PLAs ) previously described to be induced by expression of dominant - active cdc42 in HeLa cells ( Dutartre et al . , 1996 ) . Therefore , to further verify that GRAF1 localized to cdc42 - induced adhesions , we coexpressed dominant active cdc42 ( cdc42 Q61L ; herein named cdc42 - DA ) with GRAF1 and costained these cells for vinculin . Indeed , we found that co - overexpressing cells ex - hibited GRAF1 in cdc42 - and vinculin - positive PLAs ( Figure 2C ) . Interestingly , these structures were found to contain punctate actin and dynamin , consistent with their similarity to podosomes and the fact that dynamin has a role at adhesions ( see Figure S1 , E and F ) . To verify that it is prolonged local cdc42 activity that promotes ad - hesion localization of GRAF1 , we expressed the GRAF1 arginine finger mutant , GRAF1 R412D , in cells . This mutant , which is inca - pable of stimulating GTP hydrolysis by cdc42 , significantly in - creased the proportion of cells with abundant PLAs containing both vinculin and GRAF1 ( Figures 2B and S1G ) . The R412D mutation 4382 | G . J . Doherty et al . Molecular Biology of the Cell FIGURE 1 : GRAF1 interacts with proteins involved in membrane remodeling and cell adhesion . ( A ) Domain model of GRAF1 . ( B ) Immunoprecipitation of GRAF1 from rat brain cytosol showing that GRAF1 interacts with dynamin , GIT1 , and FAK as identified by mass spectrometry and confirmed by Western blotting with the indicated antibodies . ( C ) Immunoprecipitates of GIT1 , FAK , dynamin , and pFAK from rat brain cytosol were analyzed by SDS \u2013 PAGE and immunoblotting with antibodies against the indicated proteins . ( D ) Coomassie - stained gel of pulldown experiments against brain lysates with beads bound to GST or GST - tagged GRAF1 SH3 domain ( GST - SH3 ) . Indicated proteins were identified by mass spectrometry . ( E ) Confocal micrograph of a HeLa cell expressing GFP - GIT1 and myc - GRAF1 , costained for dynamin . Merged image shows section views ( as indicated by yellow lines ) . Note that structures with all three proteins colocalizing are found at the basal surface , while dynamin is also found at the top surface . Scale bar : 5 \u03bcm . ( F ) Schematic representation of the GRAF1 interactome , showing the interactions that link cell adhesion , small G - protein regulation , and GRAF1 - mediated membrane trafficking ( Hildebrand et al . , 1996 ; Zamir and Geiger , 2001a , 2001b ; Hoefen and Berk , 2006 ; Lundmark et al . , 2008 ) . Dotted lines show interactions known to be directly activating ( arrowheads ) or inhibiting ( no arrowheads ) the active state of depicted small G protein . Volume 22 November 15 , 2011 GRAF1 trafficking directs cell spreading | 4383 also significantly abrogated GRAF1\u2019s ability to cause profound cellular morphological changes ( Figure S1B ) . GRAF1 localizes to src - induced podosomes Podosome formation is highly stimulat ed by src kinase through activation of cdc42 , and active src has been used to promote podosome formation in a number of cell lines ( Moreau et al . , 2006 ) . To assay whether src - induced podosomes contained GRAF1 , we expressed the dominant active version of src ( src Y527F ) in HeLa cells . This clearly induced both small and rosette - like basal adhesions that , similarly to podosomes , were positive for vinculin , cortactin , and pFAK ( Figures 2D and S2 ) . When costained for GRAF1 , the majority ( 76 % ) of cells with such podosomes were found to have GRAF1 highly enriched in these structures ( Figure 2D ) . These results verified the similarity be - tween podosomes and GRAF1 - positive adhesive structures and indicated that GRAF1 might have a role at bona fide po - dosomes . On the basis of size and appear - ance ; the presence of dynamin , punctate actin , and cortactin ; and the dependence on cdc42 and src activity , we propose that GRAF1 is present at a subset of adhesions most accurately described to date as PLAs . Acute adhesion site modulation by small molecules results in clustering of CTxB in GRAF1 - positive PLAs Cell - matrix adhesions are dependent upon the contractive force mediated by myosin and actin filaments . This process is controlled by cdc42 , rhoA , and rac1 , and induction of PLAs is thought to rely upon cdc42 activity ( Nobes and Hall , 1995 ; Moreau et al . , 2006 ) . To favor cdc42 activ - ity over rhoA activity acutely , we inhibited the rhoA effector protein rho kinase ( ROCK ) , a key regulator of adhesions ( Totsukawa et al . , 2000 ) . Inhibition of ROCK using the small molecule Y - 27632 led to a dramatic loss of actin stress fibers and focal adhesions in favor of PLAs ( Figure S3A ) . In GRAF1 - expressing cells , we observed a profound increase in the proportion of cells exhibiting PLA - local - ized GRAF1 on their basal surfaces ( Figures 2 , B and E , and S3 , B \u2013 D ) . Similarly , the drug blebbistatin , which induces loss of stress fibers , resulted in a significantly in - creased number of PLAs positive for GRAF1 ( Figures 2B and S3E ) . The membrane at cell - matrix adhesions is composed of highly ordered lipids that are also necessary for endocytosis via CLICs FIGURE 2 : GRAF1 is not a general component of focal adhesions but localizes to PLAs . ( A ) Fluorescence micrograph of HeLa cells coexpressing myc - tagged GRAF1 and GFP - tagged GIT1 and costained for myc and vinculin . Insets show magnifications of the areas indicated by yellow squares . ( B ) Bar graph showing the percentage of cells in which myc - GRAF1 or myc - GRAF1 R412D was found localized with vinculin in PLAs following the indicated treatments or overexpression of GFP - GIT1 . Cells expressing myc - GRAF1 were treated with Y - 27632 , an inhibitor of rhoA kinase ( 5 min ) , or blebbistatin , an inhibitor of nonmuscle myosin II ( 10 min ) , and the number of cells where GRAF1 colocalized with vinculin was counted . Bars and error bars correspond to mean and SEM calculated from three independent experiments ( n > 250 ; \u03b1 = 0 . 05 ; two - tailed Fisher\u2019s exact test , * * * , p < 0 . 001 ) . ( C ) Fluorescence micrograph of cells coexpressing myc - tagged GRAF1 and GFP - cdc42 - DA and costained for myc and vinculin . ( D ) Merged confocal micrograph of a cell expressing src Y527F and GFP - GRAF1 and costained for vinculin . Inset below shows three - dimensional view of the section indicated by the dotted line . Bar graph depicting the percentage of cells with untagged or GFP - tagged GRAF1 localized to vinculin - defined , src - induced podosomes . Bars and error bars correspond to mean and SEM calculated from six independent experiments , each including 30 cells . ( E ) Fluorescence micrographs of cells treated with the ROCK inhibitor Y - 27632 for 15 min or untreated before fixation and staining for vinculin and overexpressed myc - GRAF1 . Insets in the top panel show magnification and three - dimensional rotation of the area indicated in the vinculin panel . Arrows indicate basal structures in which GRAF1 and vinculin colocalize . Scale bars : 10 \u03bcm . 4384 | G . J . Doherty et al . Molecular Biology of the Cell Membrane remodeling by GRAF1 and uptake of CTxB is stimulated at the leading edge of migrating cells As previously shown ( Lundmark et al . , 2008 ) , CTxB was internalized by tubular GRAF1 - positive structures ( Figure 4A ) . We ob - served that these structures communicated with adhesion sites , supporting a role for GRAF1 in promoting endocytosis of lipids enriched in sites of high membrane order ( Figure 4A ) . Similarly , the long , \u201ctrapped\u201d membrane tubules generated by the over - expression of GRAF1 BARPH truncation mutant ( Lundmark et al . , 2008 ) spanned long stretches along the basal surface of cells , linking mature focal adhesions ( Figure S4C ) . When tracked by live - cell imaging , the formation of GRAF1 R412D - and GRAF1 BARPH - positive tubules ( which are more stable then GRAF1 - positive tubules ) was detected . These tubules were found to both originate from , and specifically communi - cate with , sites of adhesions ( Figure S4 and Supplemental Movies S1 \u2013 S4 ) . This sug - gested that GRAF1 - mediated membrane trafficking is coupled to adhesion modula - tion . To study how GRAF1 - mediated and clathrin - dependent endocytic events are in - fluenced by the movement of cells , we ex - amined the uptake of the CLIC cargo CTxB and the clathrin - mediated endocytosis cargo transferrin ( Tfn ) by migrating fibro - blasts ( which are more migratory than HeLa cells ; Figure 4B ) . We found that the uptake of CTxB was concentrated at the leading edge of the cell . By contrast , Tfn , a clathrin - dependent endocytic cargo , appeared to be endocytosed homogeneously from the plasma membrane , consistent with previous studies ( Howes et al . , 2010a ) . Furthermore , CTxB - enriched endocytic structures were found to colocalize with paxillin , a marker of integrin - based adhesions , to a significantly higher extent than with Tfn ( Figure 4C ) . Given these data , one might therefore expect that GRAF1 - medi - ated endocytic activity would be affected by adhesion and spread - ing of cells . Rac1 promotes cell spreading and lamellopodia / ruffles at the leading edges of cells ( Nobes and Hall , 1995 ) . We therefore examined whether rac1 - induced cell spreading could also induce GRAF1 - mediated endocytosis . Cells expressing dominant - active rac1 ( rac1 Q61L ; herein named rac1 - DA ) had a round , spread - out morphological phenotype with prevalent ruffled structures contain - ing rac1 . Strikingly , when GRAF1 was co - overexpressed in these cells , we found it on large numbers of membrane tubules at the ruffled regions ( Figure 4D and Movie S5 ) . These tubular structures contained CTxB ( Figure 4D , right panel ) . In contrast with both cdc42 and rac1 , rhoA activity is known to promote cell adhesion and contraction . Expression of DA rhoA ( rhoA Q63L ; herein named rhoA - DA ) results in the rounding up of cells and promotion of ma - ture focal adhesions . Co - overexpression of rhoA - DA and GRAF1 likewise led to contracted cells ( owing to actin stress fiber formation and maintenance ; Figure 4E ) . GRAF1 - positive tubular structures ( Sims and Dustin , 2002 ; Gaus et al . , 2006 ) . We therefore exam - ined whether induced PLAs and podosomes contained the GM1 receptor , a glycosphingolipid that is enriched in microdomains . When CTxB , which binds GM1 specifically , was added to Y - 27632 - treated cells , we found that it clustered in vinculin - posi - tive structures on the membrane , overlapping closely with GRAF1 localization ( Figure 3A ) . Treatment with blebbistatin like - wise resulted in clustering of CTxB at PLAs containing GRAF1 ( Supplemental Figure S3E ) . We measured the amount of colocal - ization and found that approximately one - half the area of acutely induced PLAs contained CTxB ( Figure 3B ) . Our data show that acute induction of adhesion turnover affects membrane order and GRAF1 activity , which supports the view that plasma mem - brane dynamics are closely coupled to cell - matrix adhesion turn - over . The presence of GRAF1 at src - induced podosomes indi - cates that these structures might have a similar membrane composition . Indeed , we found that CTxB was clustered in src - induced podosomes together with GRAF1 ( Figure 3C ) . FIGURE 3 : Adhesion reorganization results in clustering of CTxB at the cell surface in GRAF1 - positive structures . ( A ) Fluorescence micrographs of myc - GRAF1 \u2013 expressing HeLa cells incubated with Y - 27632 together with CTxB - Alexa555 for 5 min as indicated before washing , fixation , and costaining for vinculin . Insets are magnifications of the area marked by a rectangle in panel 1 . ( B ) Vinculin - and myc - GRAF1 - positive PLA areas were selected from four cells from two different experiments , and the percent colocalization of CTxB was measured as described in the text . The mean is indicated by a red line and the error bars represent standard deviation ( SD ) above and below the mean . ( C ) Confocal micrograph of cell expressing src Y527F and GFP - GRAF1 and incubated together with CTxB - Alexa555 for 5 min before washing , fixation , and costaining for vinculin . The merged image is rotated to highlight the basal localization of GRAF1 - positive structures . Scale bars ; 10 \u03bcm . Volume 22 November 15 , 2011 GRAF1 trafficking directs cell spreading | 4385 overexpression of the dominant - negative GRAF1 variant GRAF1 BARPH ( Lundmark et al . , 2008 ) , suggesting that the associated membrane trafficking is similarly abrogated . GRAF1 is necessary for efficient spreading and migration of HeLa cells To determine whether GRAF1 activity was necessary for spreading and migration of cells , we depleted cells of GRAF1 using small interfering RNAs ( siRNAs ) previously shown to profoundly abrogate CLIC / endo - cytic membrane manufacture ( Lundmark et al . , 2008 ) . Immunoprecipitation of GRAF1 from cell lysates and detection by immunob - lotting showed that GRAF1 was efficiently depleted using either of two different siRNAs ( Figure 5A ) . When cells were seeded sparsely and allowed to spread after deattachment , we noted that GRAF1 - depleted cells were elongated and unable to spread fully , in con - trast with validated controls ( Figure 5B ) . We measured the area of experimental cells and found that GRAF1 - depleted cells had significantly smaller surface - connected areas compared with control cells . We also found that cells lacking GRAF1 had approximately double the average length : width ratios of controls ( Figure 5C ) . Interestingly , cells lack - ing rac1 and rac2 displayed a similar elon - gated phenotype ( Wheeler et al . , 2006 ) . Two large - scale siRNA screens have identified the gene encoding GRAF1 as being involved in migration and adhesion ( Simpson et al . , 2008 ; Winograd - Katz et al . , 2009 ) . We ob - served that GRAF1 - depleted cells were defi - cient in migrating into an induced wound in a cell monolayer ( Figure 5D ) , and the distri - bution of mature focal adhesions was fre - quently disturbed ( Figure 5E ) . To obtain real - time quantitative data on migration , we assayed the ability of cells to \u201cheal\u201d an elec - trically induced wound . While control cells closed the wound within 5 \u2013 6 h , GRAF1 - de - pleted cells were profoundly deficient in their abilities to spread and migrate into this wound ( Figure 5F ) . Taken together , our data demonstrate a role for GRAF1 in cell migra - tion , due to regulation of cell - matrix adhe - sion sites through endocytic turnover of membranes . DISCUSSION The mechanisms by which plasma mem - brane morphological changes and mem - brane trafficking are coupled in processes such as cell migration are greatly debated . Many studies have high - lighted the importance of specific rho - family , small G proteins in both cytoskeletal dynamics and endocytic events ( Mayor and Pagano , 2007 ; Doherty and McMahon , 2008 ) . Our studies reported here on membrane remodeling and rhoGAP protein GRAF1 have revealed that a prevalent clathrin - independent endocytic pathway is were found only in a small subset of rhoA - DA \u2013 expressing cells ( 14 . 3 \u00b1 3 . 7 % ) . These tubules were much longer than GRAF1 - positive tubules in rac1 - expressing cells and communicated with focal adhe - sions ( Figure 4E ) . However , GRAF1 was not enriched at the abun - dant , mature focal adhesions in rhoA - DA \u2013 expressing cells . The tubular phenotype was strikingly reminiscent of that observed upon FIGURE 4 : Membrane remodeling by GRAF1 and uptake of CTxB are stimulated at the leading edge of migrating cells . ( A ) Fluorescence micrographs of myc - GRAF1 \u2013 expressing HeLa cells incubated with CTxB - Alexa555 for 5 min before washing , fixation , and costaining for vinculin . ( B ) Confluent wild - type MEF monolayers were wounded by scratching , and cells were allowed to migrate into the wound for 4 \u2013 6 h . CTxB - 555 and Tfn - 647 were then added to migrating cells for 2 min of uptake at 37\u00b0C . Cells were acid - stripped and fixed and were then labeled for endogenous paxillin . Arrows indicate colocalization between paxillin and CTxB but not Tfn . ( C ) Twenty - four cells across three independent experiments were treated as in ( B ) , and the percentage of paxillin ( green ) pixels that colocalized with either CTxB ( red ) or Tfn ( blue ) pixels was calculated using Volocity version 3 . 0 . Bars and error bars correspond to mean and SEM ( n = 12 \u2013 15 ; \u03b1 = 0 . 05 ; Student\u2019s t test , * * , p < 0 . 01 ) . ( D ) Fluorescence micrographs of HeLa cells expressing myc - GRAF1 and DA rac1 ( GFP - Rac - DA ; left panels ) and incubated with CTxB - Alexa555 for 5 min ( right panels ) . The length of GRAF1 tubules was measured in fluorescence micrographs of seven different cells ( n = 193 ) as described . The mean length is indicated by a red line and the error bars represent standard deviation ( SD ) above and below the mean . ( E ) Fluorescence micrographs of cells expressing myc - GRAF1 and DA rhoA ( GFP - rhoA - DA ) and costained for vinculin . GRAF1 - positive tubules were found in 14 . 3 \u00b1 3 . 7 % , as determined from three independent experiments ( n = 124 ; error values represent SEM ) . Length of GRAF1 tubules in \u03bcm was measured from nine different cells ( n = 108 ) and depicted as in Figure 4D . Scale bars : 10 \u03bcm . 4386 | G . J . Doherty et al . Molecular Biology of the Cell intimately coupled to adhesion site regula - tion . GRAF1 - dependent CLICs are induced by cell spreading , enriched at the leading edge of cells , and necessary for cell spread - ing and migration . Interestingly , proteomic analysis has recently revealed enrichment for cargo molecules linked to adhesions ( Howes et al . , 2010a ) . Although the exact role of GRAF1 in receptor trafficking is not known , our data illuminate an important cell physiological function for GRAF1 - depen - dent CLICs , and provide further insight into how cells coordinate membrane and protein redistribution with changes in their morphologies . While it has previously been suggested ( Hildebrand et al . , 1996 ) that GRAF1 func - tions at focal adhesions , owing to its interac - tion with FAK , we find no evidence that GRAF1 is a structural component of such ad - hesions . However , we found GRAF1 at what , at present , can be best described as PLAs . These structures could be induced by pro - longed src and cdc42 activity ( through ex - pression of active src , cdc42 - DA , or GRAF1 R412D , or by inhibition of ROCK using small molecules ) and are enriched in cortactin , ac - tin , dynamin , actin , vinculin , FAK , and GIT1 . Although the exact role of GRAF1 - positive adhesions is not clear , the localization to src - induced podosomes indicates a relevant function of GRAF1 at such sites . Podosomes are classically found in cells of the monocytic lineage but are becoming increasingly rec - ognized as being present in a number of cell FIGURE 5 : GRAF1 is necessary for cell spreading and migration . ( A ) Immunoprecipitation and immunodetection of GRAF1 from HeLa cells treated with either a control siRNA or siRNAs against GRAF1 . Tubulin was detected in the cell lysates as an immunoprecipitation control . GRAF1 expression following siRNA treatment ( GRAF1 expr . ( % ) ) was quantified as described from three independent experiments . Values correspond to normalized means \u00b1 SD . ( B ) Micrographs of cells treated with either a control siRNA or siRNAb against GRAF1 for 72 h before fixation and imaging . Inset 1 ( left panel ) shows magnification of the marked square . Inset 1 ( right panel ) exemplifies cell area indicated in blue , and length and width indicated in red and green , respectively . ( C ) Bar graphs showing quantification of the cell area and length : width ratios , as described in the text , of cells treated with control siRNA or siRNAs against GRAF1 , as in ( B ) . In the left panel , bars and error bars correspond to mean and SEM calculated from five or six independent experiments ( n = 50 \u2013 60 ; \u03b1 = 0 . 05 ; Student\u2019s t test , * * * , p < 0 . 001 ) . In the right panel , bars and error bars correspond to mean and SEM calculated from two independent experiments ( n > 100 ; a \u03b1 = 0 . 05 ; Student\u2019s t test , * * * , p < 0 . 001 ) . Length : width ratio was scored as the ratio between length ( longest straight line within a cell ( red line in ( B ) ) and width ( the broadest region perpendicular to the measured length ( green line in ( B ) ) . ( D ) Micrograph showing the regrowth of control siRNA - treated and GRAF1 siRNA - treated cells into an induced wound in the cell monolayer . ( E ) Fluorescence micrograph of control cells and GRAF1 - depleted cells stained for vinculin . ( F ) Principle of electrically induced and monitored wound - healing assay performed as described ( left panel ) . Graph showing the recovery from electrical wound healing of a confluent HeLa cell layer . Cells were previously transfected with a control siRNA or GRAF1 - siRNA , as indicated . An increase in impedance reflects the migration of surrounding healthy cells onto an electrode through which ( at time zero ) a high current has passed to irreversibly injure the cells on the electrode . Note the delay in wound healing observed in GRAF1 - depleted cells and their slowed and incomplete recovery ( even after 9 h ) , compared with control cells . Impedance values were normalized to postwounding nadirs , and the shaded areas surrounding each curve represent one SD above and below the mean values for each condition . Scale bars : 10 \u03bcm . Volume 22 November 15 , 2011 GRAF1 trafficking directs cell spreading | 4387 of cells . Indeed , it may be that it is the permissive state of lipids that allows for the spatiotemporal coupling of adhesion site turnover and membrane trafficking . GRAF1 might be recruited to such domains preferentially , owing to both their distinct membrane composition and local , small G - protein activity . The extent of GRAF1 - mediated endocytosis linked to adhesion sites versus endocytosis at nonadhe - sive microdomains appears high , and it appears that this ratio is co - ordinated with the migratory behavior of the cell . Taken together , our data support a role for GRAF1 at adhesive microdomains , where its membrane remodeling activity promotes the manufacture of CLICs . This activity is promoted at the leading edge of cells , where it is required for membrane spreading and cell migration . MATERIALS AND METHODS Reagents , antibodies , and cDNA contructs Polyclonal antisera against GRAF1 ( RA - 83 and RaZ1 ) were generated as previously described ( Lundmark et al . , 2008 ) . Purchased antibod - ies were : mouse anti - myc clone 9E10 and rabbit anti - myc ( Cell Sig - naling Technology , Danvers , MA ) , mouse anti - dynamin ( Hudy ) , mouse anti - GIT1 ( BD Biosciences ) , and mouse anti - paxillin , mouse anti - vin - culin , rabbit anti - FAK , rabbit anti - pFAK , and rabbit anti - cortactin ( Abcam , Cambridge , MA ) . All secondary antibodies were conjugated to Alexa Fluor 488 , 546 , or 647 ( Invitrogen , Carlsbad , CA ) . cDNA constructs encoding human GRAF1 and derivatives were as de - scribed previously ( Lundmark et al . , 2008 ) , except for the amino acid substitution R412D , which was created using PCR - directed mutagen - esis ( Stratagene , Agilent , Santa Clara , CA ) . Red fluorescent protein ( RFP ) - tagged GRAF1 and untagged GRAF1 was cloned using the Gateway system ( Invitrogen ) . Green fluorescent protein ( GFP ) - tagged cdc42 Q61L ( 12600 ; Nalbant et al . , 2004 ) , rac1 Q61L ( 12981 ) , rhoA Q63L ( 12968 ; Subauste et al . , 2000 ) , and untagged src Y527F ( 13660 ) were purchased from Addgene ( Cambridge , MA ) . GFP - GIT1 and RFP - paxillin were kind gifts from A . F . Horwitz and E . E . Marcantonio , respectively . Y - 27632 and blebbistatin was purchased from Sigma - Aldrich ( St . Louis , MO ) and used at 10 and 20 \u03bcM , respectively . Immunoprecipitation and pulldown For immunoprecipitation experiments , rat brain cytosol was gener - ated by homogenization of rat brains in buffer ( 25 mM HEPES , 150 mM NaCl , 1 mM dithiothreitol [ DTT ] , 0 . 1 % Triton X - 100 , and protease inhibitors ) , before centrifugation at 50 , 000 rpm for 30 min at 4\u00b0C . The supernatant was removed and added to protein A Sep - harose 4B beads ( GE Healthcare ) , to which antibodies had been pre - viously bound , and incubated at 4\u00b0C for 3 h . Beads were washed three times in buffer ( 25 mM HEPES , 150 mM NaCl ) supplemented with 1 % NP - 40 , and once in buffer without NP - 40 , before analysis by SDS \u2013 PAGE combined with immunoblotting or Coomassie Blue stain - ing . Immunoprecipitation of GRAF1 to determine siRNA efficiency was performed as described above , except that HeLa cells were ly - sed in buffer ( 25 mM HEPES , 150 mM NaCl , 1 % NP - 40 , and protease inhibitors ) , before centrifugation at 14 , 000 rpm for 30 min at 4\u00b0C . Recombinant proteins were expressed in a BL21 ( DE3 ) pLysS Escher - ichia coli strain as glutathione S - transferase ( GST ) - fusion proteins and purified using glutathione - Sepharose 4B beads ( Amersham Bio - sciences ) and gel filtration on a sephacryl S - 200 column ( GE Health - care ) . Pulldown experiments against rat brain cytosol using purified proteins and identification by mass spectrometry were performed as previously described ( Lundmark et al . , 2008 ) . Cell culture and transfections HeLa cells and Balb3T3 cells were grown in DMEM media ( Gibco , Invitrogen ) supplemented with l - glutamine , 10 % fetal bovine types . These enigmatic structures are much more dynamic than focal adhesions and resemble the intermediates formed during focal adhesion formation and turnover ( Block et al . , 2008 ) . Ordinarily , GRAF1 - mediated endocytic activity is very dynamic and most preva - lent at the leading edge of cells , suggesting that GRAF1 - positive PLAs enriched with GM1 might represent a stabilized intermediate that occurs during adhesion turnover ( favored by prolonged cdc42 activity or alterations in the integrity of membrane microdomains ) . Truncated GRAF1 , lacking the regulatory GAP and SH3 domains ( GRAF1 BARPH ) , generates long , tubular membrane structures in cells ( Lundmark et al . , 2008 ) . Interestingly , we found that these tu - bules frequently spanned long stretches between mature focal adhe - sions , suggesting that truncated GRAF1 is unable to appropriately regulate the dynamics of membrane carriers at adhesive sites . The real time formation of GRAF1 - positive carriers showed that such structures both originate at and are targeted to adhesion sites , suggesting that CLICs might control bidirectional trafficking to regu - late membrane turnover . Our biochemical analyses identified interacting partners of GRAF1 that include FAK , GIT1 , and dynamin , supporting a role for GRAF1 at the interface between membrane dynamics and adhesion . Both GIT1 and FAK are known to regulate adhesion sites and influ - ence endocytosis ( Premont et al . , 1998 ; Claing et al . , 2000 ) . In addi - tion to the established role of dynamin during fission of endocytic carriers , dynamin also localizes to podosomes and invadopodia ( Ochoa et al . , 2000 ; Lundmark et al . , 2008 ) . Interestingly , a GTPase - deficient mutant of dynamin was found to perturb actin dynamics at such sites ( Ochoa et al . , 2000 ; Baldassarre et al . , 2003 ) and a mutant lacking its proline - rich domain ( and therefore unable to interact with SH3 domains ) was found to disrupt podosome formation ( Lee and De Camilli , 2002 ) . Dynamin and GRAF1 interact avidly , with both re - sulting in plasma membrane invagination . While fission of CLICs from the plasma membrane has been shown to be capable of func - tioning without dynamin , this does not necessarily mean that all clathrin - independent events are dynamin independent . We show that GRAF1 and dynamin colocalize at PLAs , suggesting that these proteins might organize molecular networks and membrane dynam - ics at such sites . The membrane architecture at podosomes / PLAs is also presently unclear , but it has been suggested that formation of both podosomes and invadopodia involves membrane invagina - tions , and our results support this . Uptake of gold - labeled gelatin via narrow membrane tubules has been described as strongly associ - ated with podosome localization , and it has been suggested that dynamin coats membrane tubules in the central region of podo - somes ( Gawden - Bone et al . , 2010 ; Ochoa et al . , 2000 ) . Further ultra - structural studies will assess the precise proteolipid architecture of PLAs , podosomes , and GRAF1 - positive tubules and determine whether GRAF1 - mediated endocytosis is directly coupled with the internalization of any particular adhesion molecule ( s ) . Previous work has shown that integrin - based adhesions influence membrane order and control the recruitment of active rac1 to cho - lesterol - dependent microdomains at the leading edge , where it is found together with the CTxB receptor GM1 ( del Pozo et al . , 2004 ; Gaus et al . , 2006 ) . Furthermore , detachment of cells from their sub - strata triggers the internalization of highly ordered regions of the plasma membrane ( del Pozo et al . , 2004 ) . We show that GRAF1 - positive PLAs include highly ordered membranes . CLIC manufacture is likewise dependent upon the presence of highly ordered mem - branes , and the removal of these lipids from the plasma membrane could promote adhesion turnover through dissipation of adhesion proteins that preferentially accumulate in microdomains . GRAF1 - mediated CLIC generation is promoted by rac1 at the leading edge 4388 | G . J . Doherty et al . Molecular Biology of the Cell serum , and nonessential amino acids ( for MEM ) , and transfected using Lipofectamine 2000 ( Invitrogen ) or Neon transfection system for electroporation ( Invitrogen ) for transient protein expression . Mouse embryonic fibroblasts ( MEFs ) were generated and grown as previously described ( Kirkham et al . , 2005 ) . For GRAF1 depletion , HeLa cells were transfected with stealth siRNA specific against hu - man GRAF1 ( Invitrogen ) , using Lipofectamine 2000 or Neon trans - fection system for electroporation according to the manufacturer\u2019s instructions . Cells were cultured for 72 h for efficient silencing of the GRAF1 expression . Stealth negative control medium GC Block - it siRNA ( Invitrogen ) was used as a control . GRAF1 siRNAa : GUA AU - CUGUGCUGAAUGGGAGAUAA ; GRAF1 siRNAb : CCACUCAU - GAUGUACCAGUUUCAAA . Fixed - sample and real - time imaging For immunofluorescence analysis , HeLa cells were fixed in 3 % para - formaldehyde in phosphate - buffered saline ( PBS ) for 15 min at 37\u00b0C , then washed and blocked in 5 % goat serum with 0 . 05 % saponin in PBS before staining with the appropriate antibodies in 1 % goat se - rum with 0 . 05 % saponin in PBS using standard protocols . Confocal images were taken sequentially using either a TCS SP5 system con - focal laser - scanning microscope ( Leica Microsystems ) or a fully mo - torized A1 R Laser Scanning Confocal Microscope system ( Nikon Instruments , USA ) using a 60 \u00d7 lens ( Plan Apochromat VC Oil DIC N2 , Nikon ) at appropriate excitation and emission wavelengths un - der control of the NIS - Elements Microscope Imaging Software . Epi - fluorescence and phase - contrast images were taken using a Zeiss Axioimager Z1 system with AxioVision software . Images were pro - cessed using Adobe Photoshop CS2 ( San Jose , CA ) . For immuno - fluorescence trafficking assays in HeLa cells , Alexa Fluor 546 / 555 - con - jugated CTxB ( Invitrogen ) was diluted in prewarmed media , added to cells , and incubated for the time periods and temperatures de - scribed in the figure legends . After being washed , cells were fixed and subjected to immunofluorescence analysis as described above . For polarized uptake experiments , confluent monolayers of MEFs , grown on 12 - mm , round , glass coverslips ( Lomb Scientific , Australia ) were wounded by scratching with a 200 \u03bcl pipette tip . Cells were allowed to migrate into the wound for 4 h before addition of 10 \u03bcg / ml CTxB - 555 ( Invitrogen ) and 20 \u03bcg / ml Tfn - 647 ( Invitrogen ) for 2 min at 37\u00b0C . Cells were fixed in 4 % paraformaldehyde and la - beled with anti - paxillin antibodies ( BD Transduction Laboratories ) . An axiovert 200 m SP LSM 510 META confocal laser - scanning micro - scope ( Zeiss ) was used to capture images , which were processed using Volocity , version 3 . 7 . Quantification and image analysis For quantification of the proportion of cells with protrusions , PLA - lo - calized GRAF1 , GRAF1 localization to src - induced podosomes , and the length of GRAF1 - positive structures , HeLa cells were transfected with constructs for overexpression as indicated in the figure legends and processed for immunofluorescence analysis as described in the preceding section . Protrusive structures ( > 20 \u03bcm long and < 10 - \u03bcm wide protrusions ) were measured using Axiovision software ( Zeiss ) , and the proportion of cells with such protrusions was calculated for each condition ( n > 200 for each condition ) . The percentage of cells with PLA - localized GRAF1 was counted in three independent ex - periments for each condition using the Axiovision software ( n > 250 for each condition ) . The percentage of cells with src - induced podo - somes in which GRAF1 was also localized was calculated from six independent experiments ( 30 cells per experiment ) using the NIS Elements software ( Nikon ) . Length of GRAF1 structures was mea - sured using the ImageJ segmented lines tool . To calculate the length : width ratio of cells , phase - contrast images were captured and the length ( longest straight line within a cell ) and width ( the broadest region perpendicular to the measured length ) was measured using ImageJ . For cell area determination , cell borders from 50 \u2013 60 cells from at least five independent experiments were manually defined in captured phase - contrast images , and the area was calculated using the \u201cOutline\u201d function of the AxioVision software . Creation of bar graphs and statistical analysis was performed using Graphpad Prism ( La Jolla , CA ) . For colocalization experiments in migrating cells , 12 \u2013 15 cells in three independent experiments were processed using Volocity , version 3 . 7 , and colocalization was determined using the colocalization function with automatic threshold . Automatic thresh - olds were applied to individual cells , and percentage of overlapping pixels was calculated for CTxB and Tfn channels against paxillin . For quantification of the colocalization between CTxB and PLAs , 3 \u2013 10 separate images of each condition were thresholded for the bright - est areas ; overlapping areas were then transferred into a new chan - nel . GRAF1 - and vinculin - positive PLA areas were manually defined , and the amount of CTxB colocalization in these areas was calculated using Adobe Photoshop . For determining the amount of GRAF1 fol - lowing siRNA treatment , intensity of GRAF1 and tubulin bands iden - tified by immunoblotting from three independent experiments was quantified using ImageJ . GRAF1 intensity was related to tubulin in - tensity for each sample , and the amount of GRAF1 in control cells was set to 100 % . Biophysical cell recordings HeLa cells ( 10 5 ) were transfected with siRNA against GRAF1 or con - trol siRNA for 24 h before being plated into chambers of 8W1E elec - trode arrays ( Applied Biophysics , Troy , NY ) and incubated at 37\u00b0C with 5 % CO 2 . Impedance values between the electrode and coun - terelectrode were recorded continuously from each array at a 15 - kHz oscillator frequency using an ECIS 1600 system with elevated field module ( Electric Cell - substrate Impedance Sensing , Applied Bio - physics ) . Cell attachment , spreading , and layer confluence were verified electrically and microscopically before electrical wounding at 45 kHz , 4 V for 10 s with subsequent recording from electrodes using the same parameters as prewounding . Data were normalized to initial electrode impedance value for each wounding experiment . ACKNOWLEDGMENTS This work was supported by the Swedish Cancer Society ; the Swed - ish Medical Research Council ; Swedish foundation for strategic re - search ; the Medical Faculty , Ume\u00e5 University ; the Royal Swedish Academy of Sciences ; the Magn Bergvall Foundation ; the Harald Jeanssons Foundation ; the \u00c5ke Wibergs Foundation ; and the Na - tional Health and Medical Research Council of Australia ( R . G . P . ) . Gary Doherty was supported by a Trinity College , Cambridge , Internal Graduate Studentship and Research Scholarship and an MRC post - doctoral award . Many thanks to Sven Carlsson and all members of the McMahon lab for help and support . 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Mol Cell Biol 20 , 6354 \u2013 6363 .", + "cureton2010length": "The Length of Vesicular Stomatitis Virus Particles Dictates a Need for Actin Assembly during Clathrin - Dependent Endocytosis David K . Cureton 1 , Ramiro H . Massol 2 , Sean P . J . Whelan 3 * , Tomas Kirchhausen 1 * 1 Department of Cell Biology , Harvard Medical School , and Immune Disease Institute at Children\u2019s Hospital , Boston , Massachusetts , United States of America , 2 The Division of Gastroenterology and Nutrition , Children\u2019s Hospital , Boston , Massachusetts , United States of America , 3 Department of Microbiology and Molecular Genetics , Harvard Medical School , Boston , Massachusetts , United States of America Abstract Microbial pathogens exploit the clathrin endocytic machinery to enter host cells . Vesicular stomatitis virus ( VSV ) , an enveloped virus with bullet - shaped virions that measure 70 6 200 nm , enters cells by clathrin - dependent endocytosis . We showed previously that VSV particles exceed the capacity of typical clathrin - coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization . To understand why the actin system is required for VSV uptake , we compared the internalization mechanisms of VSV and its shorter ( 75 nm long ) defective interfering particle , DI - T . By imaging the uptake of individual particles into live cells , we found that , as with parental virions , DI - T enters via the clathrin endocytic pathway . Unlike VSV , DI - T internalization occurs through complete clathrin - coated vesicles and does not require actin polymerization . Since VSV and DI - T particles display similar surface densities of the same attachment glycoprotein , we conclude that the physical properties of the particle dictate whether a virus - containing clathrin pit engages the actin system . We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process . Since some enveloped viruses have pleomorphic particle shapes and sizes , our work suggests that they may use altered modes of endocytic uptake . More generally , our findings show the importance of cargo geometry for specifying cellular entry modes , even when the receptor recognition properties of a ligand are maintained . Citation : Cureton DK , Massol RH , Whelan SPJ , Kirchhausen T ( 2010 ) The Length of Vesicular Stomatitis Virus Particles Dictates a Need for Actin Assembly during Clathrin - Dependent Endocytosis . PLoS Pathog 6 ( 9 ) : e1001127 . doi : 10 . 1371 / journal . ppat . 1001127 Editor : John A . T . Young , The Salk Institute for Biological Studies , United States of America Received June 3 , 2010 ; Accepted September 1 , 2010 ; Published September 30 , 2010 Copyright : (cid:2) 2010 Cureton et al . This is an open - access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use , distribution , and reproduction in any medium , provided the original author and source are credited . Funding : This work was supported by NIH ( http : / / www . nih . gov / ) grants U54 AI057159 ( New England Regional Center of Excellence in Biodefense and Emerging Infectious Disease ( NERCE BEID ) ) to TK and AI081842 to SPJW . The funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript . Competing Interests : The authors have declared that no competing interests exist . * E - mail : kirchhausen @ crystal . harvard . edu ( TK ) ; swhelan @ hms . harvard . edu ( SPJW ) Introduction Eukaryotic cells internalize constituents of the plasma mem - brane and extracellular cargos by entrapping them in membrane - bound carriers . The most prominent and well - characterized endocytic carriers are clathrin - coated vesicles ( reviewed in [ 1 \u2013 3 ] ) . Coated vesicles transport lipids , proteins , and other essential macromolecules from the cell surface to endosomal organelles . Extensive biochemical and cell biological research supports the following model for conventional coated vesicle formation in higher eukaryotes . The AP - 2 clathrin adaptor complex recruits clathrin to the cytosolic leaflet of the plasma membrane and sequesters cargos at the endocytic site [ 4 , 5 ] . The continued assembly of clathrin into a lattice - like configuration helps deform the underlying membrane and ultimately creates an invagination , or \u2018pit\u2019 [ 2 ] . Recruitment of the GTPase , dynamin , then facilitates scission of the coated pit from the plasma membrane [ 6 ] , and clathrin is rapidly removed from the cargo - loaded vesicle by the combined action of the heat shock cognate protein 70 ( Hsc70 ) and its co - chaperone auxilin [ 7 , 8 ] . The entire process is typically complete within 30 \u2013 60 s [ 9 , 10 ] . Coated pits incorporate and internalize soluble cargos of various sizes , such as transferrin ( 5 nm ) [ 9 , 11 ] and low density lipoproteins ( 25 nm ) [ 9 , 12 ] . Many viruses and intracellular bacteria are also internalized by the clathrin machinery [ 9 , 13 \u2013 16 ] . We previously evaluated how cells internalize the 70 6 200 nm bullet - shaped vesicular stomatitis virus ( VSV ) . We found that VSV internalization occurs through elongated , partially clathrin - coated structures that have longer lifetimes ( , 2 min . ) than typical endocytic clathrin - coated vesicles and require local actin polymerization for uptake [ 15 ] . During VSV internalization , the clathrin coat first assembles as a partially closed dome at one end of the virion [ 15 , 17 ] , and growth of the coat stalls when it encounters the long particle axis . Actin assembly then drives one or more late stage ( s ) of the internalization process , as recruitment of the actin machinery peaks during completion of clathrin assembly , and pharmacological inhibition of actin polymerization blocks VSV internalization without interfering with clathrin coat assembly [ 15 ] . Relatively small , spherical viruses like dengue virus ( 50 nm ) [ 13 ] and some influenza A viruses ( X - 31 strain , , 120 nm ) [ 14 , 18 ] also enter using a clathrin - dependent route , but it is unclear whether actin function is required for their uptake . Our observations with VSV led us to PLoS Pathogens | www . plospathogens . org 1 September 2010 | Volume 6 | Issue 9 | e1001127 hypothesize that the physical dimensions of the virion block the ongoing polymerization of clathrin during its uptake , and that the stalled structure recruits regulators of actin assembly whose activity is required to complete the internalization process . Defective interfering ( DI ) particles arise spontaneously during virus replication . Such particles depend upon coinfecting helper virus to support their replication but contain all the essential cis - acting regulatory elements for genome replication and assembly . One such well - characterized DI particle of VSV is termed DI - T , which lacks 82 % of the viral genome [ 19 , 20 ] . Since the length of a VSV particle is dictated by the genome size [ 21 ] , DI - T particles are 75 nm long and appear as truncated bullets by electron microscopy [ 22 ] . DI - T particles contain normal proportions of the viral structural proteins [ 23 ] , including the viral surface glycopro - tein ( G ) , which mediates VSV attachment and entry into host cells . Here we took advantage of significant differences in the physical dimensions of VSV and DI - T to investigate how the geometry of a viral cargo influences the actin - dependency of clathrin internal - ization . Using live cell fluorescence microscopic imaging , we compared the uptake mechanisms of VSV and DI - T at the single particle level . We report that in contrast to the clathrin - and actin - dependent uptake of VSV , the shorter DI - T particles enter cells through fully coated clathrin carriers that do not require actin dynamics for vesicle budding . These observations highlight the plasticity of the clathrin endocytic system , where clathrin coats serve as a scaffold to direct actin assembly when the clathrin machinery alone is not sufficient to mediate internalization . Results Biological properties of DI - T particles To generate a clonal population of VSV DI - T particles , we recovered DI - T from cDNA ( Figure 1A ) [ 24 ] and amplified the particles by co - infection of cells with VSV [ 25 ] . We separated DI - T particles from VSV by rate zonal centrifugation in a sucrose density gradient . Electron microscopic analysis ( Figure 1B , C ) confirmed that VSV virions measure 70 + / 2 8 nm by 204 + / 2 14 nm ( n = 114 ) [ 21 ] , while the shorter DI - T particles have a length of 76 + / 2 8 nm ( n = 81 ) [ 22 ] . DI - T particles , like VSV , are covered with spike - like projections that correspond to homotrimers of G protein ( Figure 1B ) , and SDS - PAGE analysis of purified particles confirmed that VSV and DI - T particles contain similar ratios of G protein to core virion components ( Figure 1D ) [ 23 ] . The purified stocks of DI - T lacked full - length virions ( Figure 1B ) , with only a single VSV virion observed amongst more than 3 , 000 DI - T particles . Limited dilutions of the purified DI - T stock contained , 1x10 6 plaque forming units of virus per microgram of total viral protein , or 10 - 5 times fewer infectious particles than for an equivalent protein quantity of VSV particles ( not shown ) . Thus , we have successfully purified relatively homogeneous populations of VSV and DI - T particles , which differ only in their physical dimensions . DI - T particles enter cells by clathrin - dependent endocytosis To visualize VSV and DI - T by fluorescence microscopy , we covalently labeled the G proteins with spectrally separable fluorescent dye molecules ( Alexa Fluor 568 and 647 , respectively ) using conditions that do not reduce viral infectivity [ 15 ] . Spinning disk confocal images of labeled particles adsorbed onto glass coverslips showed diffraction - limited objects with single - peaked distributions of fluorescence intensity values ( Figure 1E ) , indicating that the DI - T and VSV populations primarily consist of individual particles [ 15 ] . We tracked the entry of DI - T particles into BSC1 cells stably expressing an eGFP - tagged s 2 subunit of the AP - 2 adaptor complex ( s 2 - eGFP ) , which incorporates into all clathrin - coated structures that form on the plasma membrane [ 9 , 15 , 26 ] . Single DI - T particles readily attached to cells and progressed through the following set of defined events ( see Figures 2A , B ; Video S1 for examples ) : ( 1 ) membrane - bound DI - T particles diffused slowly ( D = 5 6 10 2 11 \u2013 5 6 10 2 12 cm 2 s 2 1 ) and with the random directionality characteristic of Brownian motion ; ( 2 ) shortly after DI - T attachment , a dim spot of AP - 2 signal arose and remained colocalized with the particle , signifying incorporation of DI - T into an assembling clathrin - coated pit ; ( 3 ) the AP - 2 signal steadily increased over time until it peaked as coat assembly completed , and the DI - T particle then underwent an abrupt movement into the cell , after which the AP - 2 signal disappeared due to clathrin uncoating . This sequence of events is identical to what we previously observed for VSV entering cells by clathrin - dependent endocytosis [ 15 ] . Moreover , the efficiency of DI - T uptake via the clathrin pathway is similar to that of the full - length VSV particles , as 89 % ( 55 / 62 ) of DI - T particles that attached to 3 individual cells during imaging entered by clathrin - dependent endocytosis . These data show that DI - T efficiently enters cells through the clathrin pathway and validate the use of DI - T and VSV as comparative endocytic cargos . Clathrin structures capture VSV and DI - T particles with similar kinetics To directly compare how DI - T and VSV particles engage the clathrin machinery , we simultaneously inoculated BSC1 cells with the two spectrally distinct particle forms and then analyzed their mode of incorporation into AP - 2 containing clathrin structures on a single cell basis ( Figure 2C ) . For each complete virus uptake event , we quantified the kinetics of particle capture by measuring the elapsed time between virion attachment and the appearance of an AP - 2 signal that colocalized with the bound particle . The capture time for DI - T particles was 110 + / 2 80 s ( n = 121 ) , which is statistically indistinguishable ( Student\u2019s t - test , p = 0 . 2 ) to that measured for VSV ( 130 + / 2 125 s ( n = 87 ) ) and agrees well with our prior measurements ( Figure 2D ) [ 15 ] . The AP - 2 structures that captured DI - T or VSV initiated within a , 250 nm zone ( the resolution limit of the optical system ) of the attached particle . We therefore conclude that DI - T and VSV particles engage the clathrin system in an indistinguishable manner , which likely reflects a shared mechanism triggered by the same viral glycoprotein - receptor interactions . Author Summary We present a detailed comparison between the clathrin - dependent entry mechanisms of a parental virus ( VSV ) and its smaller defective interfering particle ( DI - T ) . We used the difference in virion length to probe why actin assembly is required for the uptake of full - length VSV particles by nonpolarized mammalian cells . By imaging the entry of single particles in an unbiased manner , we resolved differences in the maturation kinetics , clathrin content , and actin dependency of clathrin endocytic structures internalizing VSV or DI - T virions . Our principal finding is that , unlike VSV uptake , DI - T internalization does not induce or require robust actin polymerization . We have also established , for the first time , that the geometry of an endocytic cargo can alter the mechanism of clathrin uptake . We propose that VSV - containing clathrin struc - tures display characteristics of \u2018frustrated\u2019 endocytic intermediates that cells resolve by using the force of actin assembly to deform the plasma membrane into a complete endocytic vesicle . Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 2 September 2010 | Volume 6 | Issue 9 | e1001127 Cells internalize DI - T particles using conventional clathrin - coated vesicles To investigate the characteristics of the clathrin coat responsible for DI - T internalization , we imaged the uptake of both particle forms by the same BSC1 cell . We found that DI - T particles are internalized through AP - 2 containing structures significantly faster than full - length virions ( Figure 3A , B ; Video S2 ) . Quantitative analysis of data compiled from 4 cells showed that pits incorporating DI - T ( n = 36 ) form in 43 + / 2 14 s , which is similar to the assembly kinetics of pits that lack virus particles ( n = 212 , 35 + / 2 10 s ) ( Figure 3C , Table 1 ) . As expected , AP - 2 structures that capture VSV ( n = 29 ) require longer ( 75 + / 2 22 s ) to complete ( Figure 3C , Table 1 ) . A similar analysis conducted in cells transiently expressing eGFP - tagged clathrin light chain A1 ( eGFP - LCa ) yielded analogous results ( Figure 3D , E , Table 1 , Videos S3 , S4 ) . The interaction of full - length VSV with a cell had no impact on the uptake kinetics of DI - T into the same cell ( Table 1 ) . The different kinetics of DI - T versus VSV internalization suggests that DI - T enters cells through conventional fully coated clathrin structures and not the partially coated vesicles responsible for VSV uptake . To further investigate this possibility , we measured the maximum fluorescent signal of eGFP - tagged AP - 2 or clathrin molecules , as this peak signal is known to be proportional to the overall size of a clathrin coat [ 9 , 15 , 26 ] . We compared this value for structures that contained DI - T with those that contained VSV or lacked either particle . Similar quantities of coat components were present in structures associated with DI - T to those lacking viral particles ( Figure 3C , E , Table 1 ) . As expected , VSV - containing structures accumulate more AP - 2 and LCa molecules than structures lacking virus ( Figure 3C , E , Table 1 ) . Taken together , the above experiments suggest that DI - T enters cells through pits that acquire a full clathrin coat . Consistent with this , electron micrographs of DI - T particles captured during cell entry show particles present in circular pits entirely surrounded by a clathrin coat ( Figure 4A ) . This is in marked contrast to the partial clathrin coat found at one end of the endocytic carriers that internalize VSV ( Figure 4B ) [ 15 ] . Clathrin structures containing VSV recruit more cortactin that pits that internalize DI - T During the final phase of coat assembly , endocytic clathrin structures associated with VSV show a strong recruitment of cortactin , an F - actin and dynamin binding protein that activates Arp2 / 3 - mediated assembly of branched actin filaments [ 15 , 27 , 28 ] . To determine whether DI - T uptake is associated with an acute recruitment of cortactin , we monitored internalization Figure 1 . Biological properties of DI - T particles . ( A ) Structure of VSV and DI - T genomic RNAs . The single - stranded negative - sense RNA genomes are shown in a 3 9 - 5 9 orientation . The five viral genes are colored as follows : red nucleocapsid ( N ) protein gene ; orange , phosphoprotein ( P ) gene ; yellow , matrix ( M ) protein gene ; green , glycoprotein ( G ) gene ; blue , large ( L ) polymerase protein gene . The noncoding genomic terminal leader ( Le ) and trailer ( Tr ) regions , which serve as promoters for RNA synthesis and genomic RNA encapsidation , are shown in gray . The DI - T genome comprises 2 , 208 nts . The 5 9 terminal 2 , 163 nts derive from the 5 9 terminus of the parental VSV genome ( 11 , 161 nts ) , and the 3 9 terminus contains a 45 nt inverted complement of the wild - type Tr ( TrC ) . ( B ) Electron micrographs of purified VSV and DI - T particles negatively stained with PTA . Middle panel , inset shows an expanded view of virions from the boxed region to facilitate visualization of the viral glycoprotein spikes . Inset scale bar , 100 nm . ( C ) Virion geometry . The dimensions of individual DI - T ( blue ) and VSV ( red ) particles measured from electron micrographs of negatively stained particles . Each open circle represents the measurement for a single particle . Horizontal red lines denote the mean value of each population , and the numerical means ( + / 2 SD ) are provided above each plot . ( D ) Protein composition of purified VSV and DI - T particles . Viral proteins ( L , G , P , N , and M ) were separated by SDS - PAGE and visualized with Coomassie blue staining . The ratio of N protein to G protein was quantified using ImageJ and is displayed below the gel as a comparative measure of average virion surface glycoprotein density in each particle population . ( E ) Fluorescence intensity of virus particles labeled with Alexa Fluor dye molecules . Purified DI - T or VSV particles were labeled with Alexa Fluor 647 or 568 and imaged on separate glass coverslips using a spinning disk confocal microscope . The fluorescence intensity of individual spots in a single field of view was quantified , and the distribution of intensity values ( in arbitrary units , a . u . ) for DI - T ( blue ) and VSV ( red ) particles is shown . doi : 10 . 1371 / journal . ppat . 1001127 . g001 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 3 September 2010 | Volume 6 | Issue 9 | e1001127 into BSC1 cells transiently co - expressing monomeric Cherry - LCa ( mCherry - LCa ) and low levels of cortactin - eGFP . As previously shown [ 15 , 26 , 29 ] , conventional clathrin - coated pits exhibit minimal cortactin recruitment that typically peaks just before completion of clathrin assembly ( Figure 5A , Video S5 ) . Cortactin recruitment is similarly sparse during the uptake of DI - T particles ( Figure 5B , Video S6 ) . In marked contrast , and as expected [ 15 ] , large bursts of cortactin accompany the internalization of VSV ( Figure 5C , Video S7 ) . Quantitative analysis revealed that the peak fluorescence intensity of cortactin detected in the late phase of VSV uptake averaged 3 - fold higher than the signal associated with pits containing DI - T or pits that did not capture a virus particle ( Figure 5D , Table 1 ) . These data suggest that while formation of short - branched actin filaments is required during the late stages of clathrin - mediated VSV entry , this need is obviated during clathrin - dependent DI - T entry . Actin polymerization is not required for DI - T internalization To directly test whether actin assembly is required for DI - T entry , we treated BSC1 cells with latrunculin B ( latB ) , a chemical inhibitor of actin filament assembly [ 30 ] , and tracked the endocytic fate of DI - T and VSV in the same cells . Treatment of cells with 6 . 3 m M latB did not change the efficiency of DI - T entry ( Figure 6A \u2013 C , Video S8 ) , but it reduced the internalization of VSV by . 75 % ( Figure 6A \u2013 C ) . As expected [ 15 ] , latB treatment did not affect the capture efficiency of either particle type by clathrin ( Figure 6C ) . The lifetimes and AP - 2 content of pits lacking particles or containing DI - T was similarly unaffected by latB ( Figure 6D ) . We conclude that the shorter DI - T particles bypass the actin requirement displayed by the larger VSV for efficient clathrin - based uptake . Figure 2 . Clathrin structures capture VSV and DI - T particles with similar kinetics . ( A ) Schematic of clathrin - dependent virus internalization . 1 . A particle ( blue ) attaches to receptor moieties ( orange ) on the cell surface ( black horizontal line ) , and the virus - receptor complex diffuses in the plane of the membrane . 2 . The virus particle is captured by the clathrin endocytic machinery ( AP - 2 , green ; clathrin , red ) after diffusion into an existing clathrin structure ( e . g . Dengue virus ) or entrapment within a clathrin structure that initiates in close proximity to the virion ( e . g . VSV and influenza A virus ) . 3 . Clathrin assembly completes , and the virus - containing pit is severed from the cell surface in a dynamin - dependent process . Internalization of VSV also requires local actin assembly . Clathrin is rapidly removed from the nascent vesicle , and the vesicle is actively transported further into cell . ( B ) Example of a complete DI - T internalization event . A single DI - T particle ( red ) attaches to a BSC1 cell expressing s 2 - eGFP ( green ) and diffuses on the cell surface . A dim spot of AP - 2 appears beneath the virion , signifying capture of the particle . The AP - 2 fluorescence intensity increases as the clathrin coat assembles , and the virus disappears into the cell shortly after the AP - 2 signal reaches a maximum ( Video S1 ) . Numbered stages correspond to the events described in A . The path of particle motion is depicted as a linear , color - coded trace that progresses with time from blue to red . ( C ) VSV and DI - T particle capture by clathrin structures in the same cell . BSC1 cells stably expressing s 2 - eGFP ( green ) were inoculated with Alexa Fluor 647 - labeled DI - T ( blue , blue arrowheads ) and Alexa Fluor 568 - labeled VSV ( red , red arrowheads ) . Time - lapse images were acquired at 4 s intervals using a spinning disk confocal microscope . Left , snapshot of a cell depicting coated pits lacking ( white arrowheads ) or containing ( blue / red arrowheads ) virus particles . Right , expanded split - channel views of the region within the dashed box at left . ( D ) Kinetics of virus capture . BSC1 cells stably expressing s 2 - eGFP ( 7 cells ) or eGFP - LCa ( 12 cells ) were inoculated with VSV and DI - T particles . Images were acquired at 3 - 4 s intervals as in C , and the time interval between virus attachment and detection of AP - 2 or LCa beneath a virion was quantified for productive internaliza - tion events . The distribution of capture times is shown for DI - T ( blue ) and VSV ( red ) particles , and the mean time to capture ( + / 2 SD ) for each particle population is provided at right . The kinetics of VSV and DI - T capture are not significantly different ( Student\u2019s t - test p value = 0 . 2 ) . doi : 10 . 1371 / journal . ppat . 1001127 . g002 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 4 September 2010 | Volume 6 | Issue 9 | e1001127 Discussion The major conclusion of our study is that the physical properties of a virus particle dictate the need for engagement of the actin system during its clathrin - dependent uptake into cells . We formulate this conclusion based on tracking the clathrin - depen - dent internalization of VSV and its shorter DI - T particle into live cells . Internalization of VSV is accompanied by the recruitment of Figure 3 . Cells internalize DI - T particles using conventional clathrin - coated vesicles . ( A ) Internalization of VSV and DI - T by the same cell . BSC1 cells stably expressing s 2 - eGFP ( green ) were inoculated with VSV ( red ) and DI - T ( blue ) particles , and confocal images were captured at 4 s intervals . The images show the sequential internalization of single VSV ( red , red arrowheads ) and DI - T ( blue , blue arrowheads ) particles , followed by the formation of a canonical coated vesicle ( white arrowhead ) within a 3 . 5 6 3 . 5 m m 2 area of the plasma membrane ( Video S2 ) . The first acquired frame of the time - lapse series is designated + 0 s , and the capture time of the subsequent images is shown . ( B ) AP - 2 recruitment during the uptake events shown in A . Left , image quadrants depicting AP - 2 accumulation over time ( left panels ) and at the time of maximum AP - 2 signal during each event ( right panels ) . Upper panels in each quadrant show the AP - 2 channel alone , and the lower panels show overlays of the virus and AP - 2 channels . The highlighted pit from A . is indicated with a white arrowhead , and virus particles are colored as in A . Right , fluorescence intensity ( in arbitrary units , a . u . ) of AP - 2 over time for the events shown at left and in A . The time of AP - 2 detection above the local background was set to t = 0 s for each event . ( C ) Kinetics and AP - 2 content of endocytic structures . The plots show the relative lifetime ( left ) and maximum fluorescence intensity ( right ) of AP - 2 during the uptake of coated pits lacking virus ( pits , black ) or structures that internalized DI - T ( blue ) or VSV ( red ) particles ( from 4 cells ) . Values are expressed as percentages to facilitate comparison of viral and nonviral uptake events across multiple cells . Approximately 50 pits lacking virus were analyzed in each cell , and the mean of the measured values was calculated for each parameter . The values for each nonviral and viral uptake event were divided by the mean for pits lacking virus in the same cell , and the resulting values were multiplied by 100 . Each open circle represents a single uptake event , and horizontal red lines demark the mean of the compiled population . The number of events is provided above each plot . Numerical values and statistical analyses are provided in Table 1 . ( D ) Clathrin recruitment during virus entry . Left , kymograph views of internalization events from a single BSC1 cell transiently expressing eGFP - LCa ( Videos S3 , S4 ) . Images were captured as in A . and displayed as described in B . Right , fluorescence intensity of eGFP - LCa over time for the events shown at left . ( E ) Kinetics and clathrin content of endocytic structures . The plots show the relative lifetime ( left ) and maximum fluorescence intensity ( right ) of clathrin during the uptake of coated pits lacking virus ( pits , black ) or structures that internalized DI - T ( blue ) or VSV ( red ) particles ( from 3 cells ) . Data were calculated and plotted as described in C . Numerical values and statistical analyses are provided in Table 1 . doi : 10 . 1371 / journal . ppat . 1001127 . g003 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 5 September 2010 | Volume 6 | Issue 9 | e1001127 cortactin at a late step of the endocytic process , and chemical inhibition of actin polymerization blocks virus uptake ( this study and [ 15 ] ) . By contrast , internalization of the shorter DI - T particles is insensitive to chemical inhibition of actin polymerization and is not accompanied by the same spike of cortactin recruitment observed for the full - length VSV particles . VSV and DI - T particles differ in their length and not the density of the glycoprotein spike that dictates their entry . We suggest that the shape of the full - length VSV particles presents a physical barrier that frustrates completion of the clathrin - coated pit . The stalled clathrin structure then recruits the actin machinery required to finalize internalization . The shorter DI - T particles no longer provide such a physical barrier during engulfment , which results in their actin - independent internalization through coated vesicles that acquire a full complement of clathrin . The importance of particle length for the clathrin - dependent uptake of VSV Receptor - dependent signaling events lead to actin filament assembly during the clathrin - independent uptake of other viruses , including poliovirus and coxsackie B virus [ 31 , 32 ] . Here we show that the initial interactions of DI - T with the cell are indistinguish - able to those of VSV , as both particle types diffuse slowly on the cell surface and are captured by coated pits with similar kinetics ( Figure 2 ) . Such similar behavior suggests that DI - T and VSV engage an as yet unknown viral receptor in an analogous manner . Since initial receptor interactions appear indistinguishable , it seems remote that VSV G - receptor interactions induce a signaling cascade that leads to actin polymerization at sites of VSV but not DI - T uptake . We therefore conclude that the glycoprotein is not the primary trigger of actin assembly during clathrin - dependent VSV internalization . We previously showed that endocytic structures containing VSV do not acquire a full clathrin coat [ 15 ] , which agreed with earlier electron micrographs depicting virus particles in tubular invagi - nations with clathrin at the cytosolic tip [ 17 ] . The morphology of those structures suggests that cells initially capture one tip of the virus particle , and clathrin assembly stalls when the constricting coat encounters the long axis of the virion ( Figure 7 ) . Here we found that DI - T particles do not alter the process of clathrin - coated vesicle formation ( Figures 3 , 4 ) . This finding implies that clathrin can fully enclose cargos displaying VSV G provided that the particle shape does not physically prohibit clathrin assembly or closure of the plasma membrane ( Figure 7 ) . Consequently , the physical properties of the VSV particle captured by a clathrin - coated pit are what dictate the altered mode of actin - dependent uptake . We therefore propose the model ( Figure 7 ) that it is the incomplete clathrin structure formed during VSV uptake that elicits the actin - based response to rescue the endocytic process and leads to the successful engulfment of the trapped virus particle . Actin - dependent clathrin - mediated endocytosis The importance of actin function during clathrin - mediated endocytosis varies . In yeast cells , actin polymerization drives invagination and endocytosis of long - lived ( . 2 min . ) clathrin assemblies through tubular intermediates with clathrin at the cytosolic tip [ 33 \u2013 35 ] . Mammalian cells also form large ( 0 . 2 \u2013 1 m m ) , relatively flat arrays of clathrin , or \u2018plaques , \u2019 on the adherent cell surface [ 26 , 36 ] . These structures also exhibit long lifetimes ( 2 - 15 + min . ) and require local actin assembly for internalization [ 26 ] . Coated pit internalization from the apical ( but not the basolateral ) surface of polarized mammalian cells [ 37 , 38 ] and lamprey neuronal synapses [ 39 , 40 ] is also actin - dependent . By contrast , actin polymerization is only required for the uptake of clathrin Table 1 . Summary of kinetic and fluorescence intensity data . Particle ( s ) Fluorescentprotein ( s ) Treatment # Expts / # Cells Eventsanalyzed # Events Lifetime % Lifetime % Max fluorescence DI - T s 2 1 / 3 DI - T 62 47 + / 2 13 s ND ND DI - T + VSV s 2 1 / 4 pits 212 35 + / 2 10 s 100 + / 2 24 100 + / 2 23 DI - T 36 43 + / 2 14 s 128 + / 2 37 * * 119 + / 2 38 * * VSV 29 75 + / 2 22 s 224 + / 2 68 * * 148 + / 2 48 * * LCa 1 / 3 pits 209 44 + / 2 19 s 100 + / 2 34 100 + / 2 26 DI - T 48 44 + / 2 14 s 100 + / 2 30 99 + / 2 26 VSV 34 89 + / 2 36 s 222 + / 2 104 * * 130 + / 2 38 * * DI - T + VSV s 2 + latB 2 / 4 pits 193 47 + / 2 14 s 100 + / 2 27 100 + / 2 27 + latB DI - T 46 54 + / 2 21 s 118 + / 2 46 * 133 + / 2 42 * * + latB VSV entry 20 141 + / 2 104 s 298 + / 2 198 * * 207 + / 2 59 * * + latB trapped VSV 31 615 + / 2 216 s 1283 + / 2 430 * * 192 + / 2 73 * * DI - T LCa + cortactin 1 / 3 pits 155 51 + / 2 19 s 100 + / 2 34 100 + / 2 50 DI - T 30 65 + / 2 28 s 129 + / 2 63 * 130 + / 2 63 * 1 / 3 pits 220 52 + / 2 24 s 100 + / 2 36 100 + / 2 66 VSV LCa + cortactin VSV 21 166 + / 2 63 s 335 + / 2 105 * * 300 + / 2 164 * * Kinetic and fluorescence values are provided as the mean + / 2 SD for all events in a given context . The number of experiments and cells analyzed is indicated . The absolute lifetimes are expressed in seconds ( s ) . The % lifetime and % maximum fluorescence intensity values were calculated as described in the legend of Figure 3C . The % maximum fluorescence intensity of cortactin is provided for events analyzed in cells co - expressing mCherry - LCa and cortactin - eGFP . A two - tailed Student\u2019s t - test was used to determine whether data from 2 categories differ in a statistically significantly manner . ( * ) denotes a statistical difference with a p - value , 0 . 005 and . 0 . 00005 when comparing data in a given category to data for pits lacking virus in the same category . ( * * ) similarly denotes a p - value , 0 . 00005 . All values for VSV are significantly different ( p , 0 . 005 for % max AP - 2 fluorescence ; p , 0 . 00005 for all other categories ) from those measured for DI - T particles in the same context . ND , not determined . doi : 10 . 1371 / journal . ppat . 1001127 . t001 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 6 September 2010 | Volume 6 | Issue 9 | e1001127 plaques and not conventional coated pits in several types of nonpolarized mammalian cells [ 26 , 41 ] . Thus , while actin dynamics play an evolutionarily conserved role in clathrin - dependent endocytosis , mammalian cells regulate the interplay between the clathrin and actin systems . Our analyses of VSV and coated plaque internalization reveal two properties that correlate with their actin - dependent uptake mechanisms . First , plaques and VSV - containing structures remain on the plasma membrane for . 2 - fold longer than standard coated pits [ 15 , 26 ] ( Figure 3C , E ) . The prolonged presence of a clathrin lattice might promote interactions between clathrin - associated proteins and regulators of actin polymerization . Second , clathrin plaques and VSV - containing structures physically differ from standard coated pits . Plaques fail to constrict their outer boundaries during the final phase of clathrin assembly [ 26 ] , and pits containing VSV lack a complete clathrin coat [ 15 , 17 ] . Such unusual structural features might attract proteins that associate with exposed lipids , such as dynamin . Indeed , significantly more dynamin molecules accumulate during the final stages of VSV uptake [ 15 ] . This localized increase in dynamin may enhance the recruitment of dynamin - interacting proteins with the capacity to bind lipids and activate the Arp2 / 3 complex through N - WASP , including endophilin , syndapin , and SNX9 [ 42 \u2013 46 ] . Although these proteins may link the clathrin and actin systems and facilitate localized membrane remodeling during endocytosis ( reviewed in [ 47 ] ) , further studies are needed to determine whether they play a role in clathrin - dependent VSV endocytosis . Comparative studies of VSV and DI - T uptake may provide a useful tool to dissect the mechanisms that regulate actin assembly during clathrin - mediated endocytosis . Implications for the internalization mechanisms of other viruses The dimensions of the actin - dependent VSV particle and the actin - independent DI - T particle fall within the range of shapes present in many pleomorphic viruses . Our work suggests that this may lead to important distinctions in their mode of uptake . For example , some influenza A virus strains , such as X - 31 , produce spherical particles that measure 80 \u2013 120 nm in diameter [ 18 ] . By contrast , the Udorn strain forms filamentous particles that measure 80 \u2013 100 nm wide and up to 30 microns in length [ 48 , 49 ] . Although influenza A virus can enter cells by clathrin - dependent and - independent mechanisms [ 14 ] , the impact of particle shape on the entry pathway remains unknown . It seems likely that remodeling of the cortical actin cytoskeleton will be important for uptake of filamentous influenza particles , and clathrin may facilitate local membrane deformation during the endocytic process . The Arenaviridae generate roughly spherical particles that range in diameter from 40 \u2013 300 nm [ 50 , 51 ] , and it is known that clathrin function is important for efficient infection of cells by some New World arenaviruses [ 52 ] . It will now be of interest to determine whether spherical particles of different diameter employ altered modes of clathrin - based endocytosis . Pseudotyping is often used to study the entry pathway of highly pathogenic viruses , including the long filamentous filoviruses , Ebola and Marburg , as well as several arenaviruses . Such pseudotypes are frequently based on VSV or retroviral virions in which the endogenous entry proteins have been replaced with the surface glycoproteins of the pathogenic virus [ 53 \u2013 56 ] . Although viral pseudotypes are useful for studying the entry process , VSV and the spherical virions of retroviruses ( , 100 nm in diameter ) do not accurately recapitulate the sizes or shapes of the pleomorphic viruses . Our studies of VSV and DI - T clearly show that virion geometry can fundamentally alter aspects of the viral internaliza - tion process . Therefore , it is critically important to study viral endocytosis using pseudotyped or virus - like particles that closely approximate the physical properties of a virus in question . Materials and Methods Cells and viruses African green monkey kidney BS - C - 1 cells ( herein BSC1 , American Type Culture Collection ( ATCC ) CCL - 26 ; Manassas , VA ) and Vero cells ( ATCC ) were maintained at 37 u C and 5 % CO 2 in Dulbecco\u2019s Modified Eagle Medium ( DMEM , Invitrogen Corporation ; Carlsbad , CA ) supplemented with 10 % fetal bovine serum ( Tissue Culture Biologicals ; Tulare , CA ) . BSC1 cells stably expressing rat s 2 adaptin - eGFP ( s 2 - eGFP ) [ 9 ] were maintained as above in the presence of 0 . 4 mg mL 2 1 geneticin ( G418 , Invitrogen ) . Figure 4 . Electron microscopic images of DI - T and VSV particles in clathrin - coated pits . ( A ) Electron micrographs depicting DI - T particles at early ( left ) or late ( right ) stages of clathrin - dependent endocytosis . BSC1 cells were incubated with , 1000 particles per cell for 10 min . at 37 u C . Cells were then fixed , and samples were processed for ultra thin sectioning and viewed as described in the materials and methods . ( B ) Electron micrographs of VSV particles at sequential ( left to right , top to bottom ) stages of clathrin - dependent endocytosis . Vero cells were inoculated with VSV at an MOI of 5 , and samples were prepared for analysis at 6 h post - infection . doi : 10 . 1371 / journal . ppat . 1001127 . g004 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 7 September 2010 | Volume 6 | Issue 9 | e1001127 Recombinant VSV ( rVSV ) [ 25 ] was amplified and purified as before [ 15 ] . Defective interfering T ( DI - T ) particles of VSV were recovered from a cDNA clone of the DI - T genome [ 24 ] . The DI - T particles were amplified by co - infecting baby hamster kidney cells ( BHK - 21 , ATCC C - 13 ) with rVSV ( multiplicity of infection ( MOI ) 50 ) . A subsequent passage was performed by inoculating cells with filtered , undiluted supernatant from the primary amplification and rVSV ( MOI of 50 ) . Viruses were concentrated by centrifugation at 44 , 000 6 g , and the virus pellet was resuspended in NTE ( 10 mM Tris pH 7 . 4 , 100 mM NaCl , 1 mM EDTA ) . The two particle forms were separated on a 15 \u2013 45 % sucrose gradient prepared in NTE by centrifugation at 77 , 000 6 g for 5 h . The DI - T particles were extracted from the upper virus band , concentrated as before , and resuspended to 1 mg mL 2 1 of total protein in PBS . Dye conjugation to virus particles Purified DI - T and VSV particles were labeled with Alexa Fluor dye molecules ( Molecular Probes , Invitrogen ) as previously described [ 15 ] except that the final dye concentration in the labeling reaction was reduced to 25 m g ml 2 1 . Plaque assays of virus preps before and after labeling showed that dye conjugation did not affect the infectivity of VSV particles or the capacity of DI - T virions to inhibit plaque formation by VSV . The surface density of G protein on VSV or DI - T particles was estimated by measuring the ratio of G protein to N protein in each particle population . To separate and visualize the viral proteins , purified virions were subjected to SDS - PAGE using 10 % polyacrylamide and 0 . 13 % bis - acrylamide and stained with Coomassie blue . The relative amounts of N or G protein were established using ImageJ ( U . S . National Institutes of Health , Bethesda , Maryland ; http : / / rsb . info . nih . gov / ij / ) . Nucleic acid transfection BSC1 cells were seeded into 6 - well plates at , 60 , 000 cells per well 16 \u2013 20 h prior to transfection . Plasmid DNA was introduced into the cells using FuGENE 6 ( Roche Diagnostics ; Indianapolis , Figure 5 . Clathrin structures containing VSV recruit more cortactin that pits that internalize DI - T . ( A ) Cortactin recruitment during coated pit formation . Left , snapshot showing the surface of a BSC1 cell transiently expressing cortactin - eGFP ( green ) and mCherry - LCa ( red ) at 18 h post - transfection . Time - lapse images were acquired at 3 s intervals , and frame 83 is shown . Middle , split channel kymographs of coated pit formation in the cell at left . White arrowheads highlight pits in which cortactin recruitment is clearly visible above the local background . Right , example plot of cortactin and clathrin fluorescence intensity over time during the formation of a single clathrin - coated pit in the cell shown at left ( Video S5 ) . ( B and C ) Examples of cortactin recruitment during DI - T ( B ) and VSV ( C ) uptake . BSC1 cells transiently expressing mCherry - LCa ( red ) and cortactin - eGFP ( green ) were inoculated with Alexa Fluor 647 - labeled DI - T or VSV , and images were acquired as in A . Left , split - channel kymograph views of protein and virion ( blue ) fluorescence intensity over time ( Videos S6 , S7 ) . Images in the right - hand panels show a snapshot of the maximal cortactin or clathrin fluorescence , and white arrowheads highlight the peak cortactin signal . Right , plots of the cortactin and clathrin fluorescence intensity over time for each internalization event . ( D ) Relative peak fluorescence intensity of cortactin in cells co - expressing mCherry - LCa and cortactin - eGFP . At 18 h post - transfection , samples were separately inoculated with DI - T or VSV particles , and images were acquired as in A . For each cell that was imaged , the maximum cortactin fluorescence associated with , 50 pits lacking virus particles ( pits , black ) and all pits that internalized a DI - T ( left , blue , 3 cells ) or VSV ( right , red , 5 cells ) particle was measured . The data are plotted as described in the legend of Figure 3C , and the number of events is shown above each plot . Numerical values and statistical analyses are provided in Table 1 . doi : 10 . 1371 / journal . ppat . 1001127 . g005 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 8 September 2010 | Volume 6 | Issue 9 | e1001127 IN ) according to the manufacturer\u2019s instructions . Prior to trans - fection , media on the cells was replaced with 1 ml OPTIMEM ( Invitrogen ) . After addition of the transfection mixture , cells were incubated at 37 u C for 5 h , and the existing media was supplemented with 2 ml DMEM containing 10 % FBS . To ensure optimal replacement of endogenous clathrin light chain molecules with rat eGFP - clathrin light chain A1 ( eGFP - LCa ) [ 9 ] , cells were transfected with 0 . 75 m g of plasmid DNA encoding eGFP - LCa . The cells were cultured for , 36 h and seeded onto glass coverslips , 14 h prior to image acquisition . Co - expression of mCherry - LCa ( constructed as described for tomato - LCa [ 8 ] ) and mouse cortactin - eGFP [ 57 , 58 ] was achieved by transfection of cells on glass coverslips with 1 m g of each plasmid , and the cells were imaged , 18 h later . Figure 6 . Actin polymerization is not required for DI - T internalization . ( A ) The endocytic fate of virus particles after inhibition of actin polymerization . BSC1 cells stably expressing s 2 - eGFP ( green ) were treated with 6 . 3 m M latB for 12 min . and inoculated with DI - T and VSV particles in the continued presence of latB . Time - lapse images of a single cell were acquired at 4 s intervals for 692 s , and an 8 . 8 6 5 . 0 m m 2 area of the cell surface is shown . The upper panels show the complete internalization of a DI - T particle ( blue , blue arrowheads ) , where + 0 s indicates the first frame of the time - lapse series . The lower panels show the subsequent capture but failed internalization of 2 VSV ( red , red arrowheads ) particles on the same area of cell membrane ( time scale continued from above ) ( Video S8 ) . ( B ) AP - 2 fluorescence intensity for the events shown in A . Note that the adaptor fluorescence associated with the DI - T particle ( blue ) and a conventional coated pit ( black ) that formed within the same membrane area peak and disappear normally , while the adaptor signal associated with the upper - most VSV particle ( red ) does not , signifying failed internalization . ( C ) Effect of latB on the efficiency of virus capture and internalization . BSC1 cells stably expressing s 2 - eGFP were treated and imaged as described in A . Left , the percentage of virus particles that were captured by a clathrin structure after attachment . Right , the percentage of captured virus particles that were successfully internalized within 300 s of capture ( see D . for details ) . Cumulative data are from 5 cells . ( D ) Effect of latB on the lifetime and peak fluorescence intensity of AP - 2 in clathrin structures . Data were acquired as described in A . and displayed as in the legend of Figure 3C . Left , relative lifetime of AP - 2 in structures that lack ( pits , black ) or capture a virus particle . Inset shows a rescaled distribution of the pit and DI - T internalization events . Right , maximum fluorescence intensity of AP - 2 in the events at left . Data are from 4 of the 5 cells analyzed in C , as thermal drift during imaging prevented accurate fluorescence intensity measurements in one cell . The number of events in each category is shown above the corresponding plots at right . DI - T ( blue ) data consists only of productive internalization events . VSV events are categorized as productive internalizations ( VSV entry , red ) or non - productive captures ( trapped VSV , red ) . A non - productive capture is defined as a stable colocalization between a spot of AP - 2 and a VSV particle that began at least 300 s before the last captured image and did not result in virus uptake before cessation of imaging . The 300 s cutoff was chosen because a majority ( 22 / 24 ) of productive internalizations occurred within 300 s of capture . Captures in which the final image frame was acquired before 300 s elapsed were excluded from the analysis , as the eventual endocytic fate of the particle cannot be predicted . doi : 10 . 1371 / journal . ppat . 1001127 . g006 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 9 September 2010 | Volume 6 | Issue 9 | e1001127 Live cell imaging Cells on 25 mm coverslips ( No . 1 . 5 , Electron Microscopy Sciences ; Hatfield , PA ) were placed into a perfusion chamber and overlaid with a - MEM lacking phenol red ( Invitrogen ) and supplemented with 20 mM HEPES pH 7 . 4 and 2 % FBS . The chamber was placed in a heated sample holder ( 20 / 20 Technology Inc . ; Wilmington , NC ) mounted on the stage of a Mariana imaging system ( Intelligent Imaging Innovations , Denver , CO ) based on an Axiovert 200M inverted microscope ( Carl Zeiss , Inc . ; Thornwood , NY ) . The microscope stage and objective lenses were maintained at 37 u C within an environmental chamber , and the air above the cells was supplied with 5 % CO 2 . The position of the sample holder with respect to the objective lens was manipulated using a PZ - 2000 automated stage ( Applied Scientific Instrumen - tation ; Eugene , OR ) . Samples were illuminated using 40 \u2013 50 mW solid state lasers ( l = 488 , Coherent , Inc . ; Santa Clara , CA , l = 561 , Cobolt AB ; Solna , Sweden , l = 640 , 40 mW ; Coherent ) directed through an acousto - optic tunable filter ( AOTF ; Gooch and Housego L . L . C . ; Melbourne , FL ) . Laser illumination was imparted on the sample through a CSU - X1 spinning disk confocal unit ( Yokogawa Electric Corporation ; Tokyo , Japan ) and a 63X objective lens ( Plan - Apochromat , NA 1 . 4 , Carl Zeiss ) . Emission spectra were selected using single band width emission filters ( LF405 / 488 / 561 / 635 - A , Semrock ; Rochester , NY ) , and after transmission through a spherical aberration correction device ( Infinity Photo - Optical ; Boulder , CO ) , the emission photons were collected using a Cascade II : 512B back - illuminated EMCCD camera ( Roper Scientific , Photometrics ; Tuscon , AZ ) . Under this configuration , a single pixel corresponds to 0 . 07 6 0 . 07 m m 2 . Slidebook 4 . 2 . 13 ( Intelligent Imaging Innovations , Inc . ( III ) ; Denver , CO ) was used to command the hardware devices and visualize the acquired data . To image virus internalization , Alexa - labeled virus was centrifuged briefly to remove aggregates , and cells were inoculated with virus to result in attachment of , 50 particles per cell within 20 min . ( , 1 6 10 7 p . f . u . of VSV , , MOI 100 , 0 . 005 \u2013 0 . 05 particles per m m 2 of cell surface area ) . Time - lapse acquisitions were typically carried out for 8 \u2013 10 min . per cell , and images were captured at 3 \u2013 4 s intervals after sequentially illuminating the sample with the appropriate laser for 50 \u2013 100 ms per wavelength . To assess the effect of latrunculin B ( latB ) ( Sigma - Aldrich , Inc . ; St . Louis , MO ) on coated vesicle formation and virus entry , cells were treated with 6 . 3 m M of latB for , 10 min . at 37 u C prior to the addition of virus particles . Image analysis Image analysis was performed as previously described [ 15 ] with the following modifications . Slidebook 4 . 2 . 13 ( III ) was used to view , scale , and export images for publication . Movies were generated by exporting a series of continuous TIFF files from a Slidebook time - lapse acquisition and compiling the images into a single AVI file using ImageJ ( NIH ) . SigmaPlot 8 . 0 ( SYSTAT ; Point Richmond , CA ) was used to plot data . Microsoft Excel was used to determine whether data from 2 categories differ in a statistically significant manner using two - tailed Student\u2019s t - tests . An automated image analysis application ( IMAB ) [ 8 ] developed within MATLAB ( Mathworks ; Natick , MA ) was used to track the formation of clathrin - coated structures and the internalization of single virus particles . Images were processed for analysis as previously described , and established criteria were used to exclude incomplete endocytic events and events in which pixels assigned to one clathrin structure merged with or split from an adjacent structure [ 8 ] . For each cell of interest , the first , 50 pits lacking virus particles were analyzed in detail to measure the coat lifetime and protein composition ( see below ) . All complete virus uptake events that occurred in these cells were also analyzed in a similar manner . With the exception of internalization events blocked by latB , incomplete internalization events or events truncated by the start / end of image acquisition were not analyzed . Aggregates of virus were identified as objects with fluorescence intensities greater than that of single particles and were excluded from all analyses . IMAB was used to measure the fluorescence intensity of coat components or virus particles in the following manner . A roughly spherical mask encompassing 69 pixels was centered on the peak fluorescence intensity of the object in all frames that the object was detectable above the local background fluorescence . The contri - bution of the local background fluorescence was estimated by measuring the average intensity of pixels within a ring of single pixel width that extended from the outer boundary of the object mask . The intensity of the pixels within the object mask was then summed , and the average intensity value of pixels in the local background was subtracted from each pixel within the object mask to estimate the fluorescence contributed by proteins ( or dye molecules ) within the object of interest . Electron microscopy Purified virus particles were deposited onto carbon - coated copper grids and stained with 2 % phosphotungstic acid ( PTA ) in H 2 O ( pH 7 . 5 ) . To visualize DI - T particles in clathrin - coated pits , BSC1 cells were inoculated with unlabeled DI - T particles using a dose that yielded , 1000 attached particles per cell after 10 min . Samples were then processed for ultra - thin sectioning as previously described [ 15 , 59 ] . Electron micrographs of VSV particles in clathrin endocytic structures were obtained from cells infected with VSV for 6 h , where the entry of newly released particles could readily be visualized . Virus particles and ultra - thin sections of cells were viewed using a Tecnai G 2 Spirit BioTWIN transmission electron microscope ( FEI , Hillsboro , OR ) . Figure 7 . Model of DI - T and VSV entry . DI - T ( above ) and VSV ( below ) particles engage host cells through interactions between the viral surface glycoproteins and unknown cellular receptor moieties . Following attachment , both particle types undergo slow diffusion ( diffusion coefficient , 5 6 10 2 11 cm 2 s 2 1 ) on the cell surface for an average of , 2 min . before being captured by a clathrin - coated pit . For DI - T , continued clathrin assembly drives complete particle envelopment by the plasma membrane and leads to virus endocytosis . In contrast , the presence of a VSV particle in a coated pit physically prevents complete membrane constriction by the clathrin machinery and causes clathrin assembly to halt prematurely . The force provided by actin polymerization then further remodels the plasma membrane and thereby encloses the virus particle in a partially clathrin - coated vesicle . doi : 10 . 1371 / journal . ppat . 1001127 . g007 Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 10 September 2010 | Volume 6 | Issue 9 | e1001127 Supporting Information Video S1 Clathrin - dependent DI - T internalization . BSC1 cells stably expressing s 2 - eGFP ( green ) were inoculated with DI - T particles , and time - lapse images were acquired at 4 s intervals . A 4 . 2 6 4 . 2 m m 2 area of the cell surface is shown as in Figure 2B . The video depicts a single DI - T particle ( red ) that attaches to the plasma membrane and diffuses slowly . A clathrin - coated pit captures the virus particle when a dim adaptor spot colocalizes with the virus signal . The adaptor fluorescence intensity increases as coated pit assembly proceeds , and particle internalization occurs shortly after the adaptor signal peaks . Disappearance of the adaptor signal signifies clathrin uncoating , and the virus - containing vesicle is then transported toward the cell interior . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s001 ( 0 . 31 MB AVI ) Video S2 Sequential internalization of single VSV and DI - T particles by the same cell . BSC1 cells stably expressing s 2 - eGFP ( green ) were simultaneously inoculated with wild - type VSV and DI - T particles , and time - lapse images were acquired at 4 s intervals . A 3 . 5 6 3 . 5 m m 2 area of the cell surface is shown as in Figure 3A . At the outset of imaging ( t = 0 ) , one VSV ( red ) particle and one DI - T particle ( blue ) are visible on the cell surface . The VSV particle enters first , followed by the DI - T particle . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s002 ( 0 . 39 MB AVI ) Video S3 Uptake of a single DI - T particle by a cell expressing eGFP - LCa . BSC1 cells transiently expressing eGFP - LCa were inoculated with DI - T and full - length VSV particles , and images were captured as for Video S2 . At the onset of imaging , a DI - T particle is present within a 3 . 5 6 3 . 5 m m 2 area of the cell surface . Shortly thereafter , the particle briefly colocalizes with a spot of eGFP - LCa but does not enter . As visualized in Figure 3B , a clathrin - coated pit subsequently initiates near the virion , and the particle disappears into the cell after the clathrin signal peaks . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s003 ( 0 . 77 MB AVI ) Video S4 Uptake of a single VSV particle by a cell expressing eGFP - LCa . The movie depicts a separate area ( of equal size ) of the plasma membrane from the same cell that internalized the particle shown in Video S3 . A VSV particle ( red ) attaches to the cell surface , and a clathrin endocytic structure subsequently internal - izes the particle ( Figure 3D ) . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s004 ( 0 . 77 MB AVI ) Video S5 Cortactin recruitment during conventional clathrin - coated pit formation . Time - lapse images were acquired at 3 s intervals from a cell transiently co - expressing mCherry - LCa ( red ) and cortactin - eGFP ( green ) ( Figure 5A ) . A 7 . 0 6 7 . 0 m m 2 area of the cell surface is shown . Note that the cortactin signal is nearly indistinguishable from the local background during most clathrin endocytic events . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s005 ( 3 . 01 MB AVI ) Video S6 Cortactin recruitment during the internalization of a single DI - T particle . BSC1 cells transiently co - expressing mCherry - LCa ( red ) and cortactin - eGFP ( green ) for 18 h were inoculated with DI - T particles ( blue ) , and time - lapse images were acquired at 3 s intervals . The video shows the internalization of a single DI - T particle by a clathrin - coated vesicle . The internaliza - tion event is centered within a 3 . 5 6 3 . 5 m m 2 area of the cellular plasma membrane ( Figure 5B ) . The left panel shows an overlay of the 3 channels , and the right panel shows only the cortactin channel displayed in monochrome . Note that the cortactin fluorescence intensity during DI - T entry is nearly indistinguishable from the local background . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s006 ( 1 . 42 MB AVI ) Video S7 Cortactin recruitment during the internalization of a wild - type VSV particle . BSC1 cells transiently co - expressing mCherry - LCa ( red ) and cortactin - eGFP ( green ) for 18 h were inoculated with VSV particles ( blue ) , and time - lapse images were acquired at 3 s intervals . The video is displayed as described for Video S3 and shows the clathrin - dependent uptake of a single VSV particle in a 3 . 5 6 3 . 5 m m 2 area of the plasma membrane ( Figure 5C ) . Note the visible burst of cortactin that occurs in the final stages of VSV internalization . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s007 ( 1 . 51 MB AVI ) Video S8 BSC1 cells stably expressing s 2 - eGFP were treated with 6 . 3 m M latB for 12 min . Cells were inoculated with DI - T ( blue ) and VSV ( red ) particles , and images were acquired at 4 s intervals . The movie shows an 8 . 8 6 5 . 0 m m 2 area of the plasma membrane . The 2 DI - T particles ( the lower particle is already in a coated pit at the onset of imaging ) enter in the presence of latB , while the 2 VSV particles are subsequently captured by coated pits but fail to enter the cell ( Figure 6A , B ) . Found at : doi : 10 . 1371 / journal . ppat . 1001127 . s008 ( 4 . 65 MB AVI ) Acknowledgments We express gratitude to Eric Marino for maintaining the imaging resource used in this study . We also gratefully acknowledge Maria Ericsson and Irene Kim for preparation of samples for electron microscopic analysis and members of the Kirchhausen and Whelan labs for thought - provoking discussions . Author Contributions Conceived and designed the experiments : DKC RHM SPJW TK . Performed the experiments : DKC . Analyzed the data : DKC SPJW TK . Contributed reagents / materials / analysis tools : DKC RHM SPJW TK . Wrote the paper : DKC SPJW TK . References 1 . Conner SD , Schmid SL ( 2003 ) Regulated portals of entry into the cell . 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Maupin P , Pollard TD ( 1983 ) Improved preservation and staining of HeLa cell actin filaments , clathrin - coated membranes , and other cytoplasmic structures by tannic acid - glutaraldehyde - saponin fixation . J Cell Biol 96 : 51 \u2013 62 . Virus Length Alters Clathrin - Dependent Endocytosis PLoS Pathogens | www . plospathogens . org 12 September 2010 | Volume 6 | Issue 9 | e1001127", + "vasconcelosCopyNotCopy2017": "Design Computing and Cognition DCC\u201916 . J . S . Gero ( ed ) , pp . xx - yy . \u00a9 Springer 2016 1 To copy or not to copy : the influence of instructions in design fixation experiments Luis Vasconcelos , Chih - Chun Chen , Eloise Taysom and Nathan Crilly University of Cambridge , UK Design fixation experiments often require participants to solve a design problem whilst being exposed to an example solution and instructions for how to treat that example . However , little is known about the influence of such instructions , leading to difficulties in interpreting results and under - standing how the introduction of examples affects idea generation . In our experiment , participants were all provided with the same design problem and example solution , but were presented with different instructions , rang - ing from strongly encouraging copying the example to strongly discourag - ing copying . Analyses of participants\u2019 work indicated that only the instruc - tions encouraging copying had an effect . When encouraged to copy , participants tended to only copy the structural features of the example ra - ther than the underlying concept . By contrast , the number of features cop - ied was not reduced when participants were discouraged from copying . These findings suggest that there are subtle interactions between instruc - tions and stimuli that influence design fixation . Introduction Inspiration is vital to creative design . This has driven design researchers to conduct many studies into inspiration , for instance to find out what materi - als inspire designers [ 1 ] , how designers achieve inspiration [ 2 ] , and how inspiration can improve designers\u2019 performance [ 3 ] . Many of these studies have observed the use of external stimuli during idea generation , and have reported that whilst external stimuli can assist idea generation , they can al - so hinder it . This detrimental effect of inspiration is described in the design literature as \u2018design fixation\u2019 [ 4 ] . Design fixation was originally studied in L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 2 situations where the stimuli represented possible design solutions to the problem that was being addressed . It is often measured in the solutions that the designers propose according to the repetition of features from the stim - uli that the designers see [ 4 - 10 ] . Although reproducing the features of good designs might be beneficial or efficient , the researchers have found that repetition is still present when the stimuli contain flawed inspiration sources , which they had thought designers would identify and avoid [ 4 , 7 , 10 ] . In general , fixation research suggests that blindly copying features from stimuli is harmful to idea generation , and therefore to the design process it - self . As such , some studies have incorporated constraining textual instruc - tions into the stimuli given to participants , to prevent them from copying features . However , the efficacy of the instructions seemed to vary across different accounts : they were effective in some cases [ 11 , 12 ] and ineffec - tive in others [ 4 , 13 ] . Thus , it is still uncertain how textual instructions can influence the copying behaviour , and this uncertainty might be attributed to different factors . For instance , people may tend to overlook instructions presented along with example material [ 14 ] , they can interpret the instruc - tions in different ways if instructions are not strict , or wonder why they are being exposed to the stimuli and change their idea generation process ac - cordingly . Whatever the reason might be , it is important to understand the relevance of the instructions provided to designers as part of the inspiration material . Methodologically , this would help in determining how fixation studies should be conducted and the results interpreted . More practically , it would also help to better understand how stimuli should be presented in software tools that aim to help idea generation by providing external stimuli to designers , and in other contexts in which designers are stimulat - ed with examples ( for such tools , see [ 15 - 17 ] ) . In order to better understand the influence of instructions on idea gen - eration , we conducted an experiment in which participants in different treatment conditions were provided with the same design problem and the same stimuli , but the instructions they were given with respect to that solu - tion differed between the groups . In reporting on that experiment we offer insights that are useful for interpreting the existing inspiration and fixation studies , and for designing new ones . These insights are also relevant for considering how inspirational material might best be presented to design - ers outside of experimental contexts , for example where computer tools are used to index and retrieve inspirational material . To copy or not to copy 3 Inspiration , fixation and the introduction of inspiration sources Design researchers have been studying many different aspects of the inspi - ration process , including properties of the inspiration sources ( e . g . quantity of sources ) and aspects of the design process ( e . g . total time available ) [ 18 ] . One of the stimuli characteristics that has been studied is whether good and bad examples would be copied indiscriminately . Researchers wanted to know whether designers would fixate on the examples they see and would copy them to some extent , irrespective of their quality . Indeed , a series of studies has found that participants still replicated features from previous examples even though they were flawed [ 4 , 7 , 10 , 11 , 19 , 20 ] . To counteract this indiscriminate replication , some studies have tried warning participants about the flaws in the examples [ 4 , 11 , 20 ] , while others have tried instructing participants not to copy the examples [ 13 , 21 , 22 ] . Con - sidering these two approaches , Chrysikou & Weisberg [ 11 ] found that warning participants of flaws in the examples was not enough ; they had to be told to avoid repeating those flaws . Yilmaz et al . [ 22 ] also reported tell - ing participants not to reproduce the examples and found that feature repe - tition was reduced . Conversely , Jansson & Smith [ 4 ] and Perttula & Sipil\u00e4 [ 13 ] found fixation effects even when participants were instructed to avoid using features from the examples provided . As such , surveying the pub - lished literature reveals conflicting results relating to the use of instruc - tions when providing stimuli to designers . Whilst many variables have been manipulated in fixation experiments , and some studies have already tested the effectiveness of using textual in - structions to some extent , the way the stimuli are introduced in such exper - iments has not yet been studied systematically . Such stimuli introductions can typically be divided into two components : a descriptive statement on what the stimulus is ( e . g . \u201chere is an example solution\u201d ) and a prescriptive instruction for how the stimulus should be used ( e . g . \u201cdon\u2019t copy its fea - tures\u201d ) . Currently , the stimuli introductions ( i . e . descriptions and instruc - tions ) given to participants vary from study to study . For instance , partici - pants have been told that the example should be considered a solution for the given problem [ 7 ] , that it was provided to help them get started [ 6 ] , that it was there to raise thoughts [ 23 ] , and that it should be used to awak - en thoughts but should not be reproduced [ 13 ] ( also , sometimes the studies do not report how the stimuli were introduced ) . Such variation in the way the stimuli are provided can be attributed to a lack of agreement across studies about which \u2018real world\u2019 situation is being simulated ( e . g . contexts in which examples are accidentally seen , intentionally searched for , or al - ready known ) . Regardless of the reason , the variation in the way copying L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 4 is encouraged or discouraged makes it difficult to know to what extent the design work is being influenced by such instructions . It could even be pos - sible that designers will incorporate features from the examples provided irrespective of how constraining the instructions provided to them are . To investigate the influence of stimuli instructions on idea generation , Smith et al . [ 21 ] did an experiment in which participants were asked to ei - ther conform to or diverge from the example solutions provided in a crea - tive task . Some participants ( group 1 ) were told that the examples were great ideas previously created for that task , and that participants should create ideas like those whilst not copying them exactly . Other participants ( group 2 ) were told that the examples restricted people\u2019s creativity , and that participants should create ideas that were different to the examples . Finally , other participants ( control group ) were given the same examples but without any instructions . It was found that when compared to the ideas generated by participants in the control group , those in group 1 and in group 2 generated more ideas containing features from the examples and that groups 1 and 2 were not different in this respect . Based on these re - sults , the researchers proposed that the participants did not conform to ex - amples because they had assumed that they should ; they conformed be - cause they could not forget the examples that they had seen . However , the instructions used in the experiment were not strict ( i . e . they suggested how ideas should be created , but did not forbid or require the use of features from the examples , allowing participants to interpret the information in different ways ) and both the description of the stimuli and the instruction for their use varied between groups , making it difficult to infer the influ - ence of each piece of information in isolation . We believe that a different experimental setup could provide research with more data , complementing the results previously found and helping the field to clarify the influence of the textual instructions used in experimental research and professional de - sign practice . To investigate this , we designed and conducted an experi - ment to find out the effects of instructions in design inspiration and fixa - tion , be the instructions neutral or either \u2013 slightly or very \u2013 encouraging or discouraging Methodology Objective and hypothesis This experiment investigates how textual instructions accompanying ex - ternal inspiration sources can shape the design work of the participants . In To copy or not to copy 5 particular , we hypothesise that instructions provided along with external sources will not influence the ideas that participants generate . Experimental method Participants were randomly allocated to different experimental conditions . They were verbally asked to be creative and to design , individually , as many ideas as possible to a given problem . They were also asked to sketch and describe in writing their ideas on sheets of paper . Except for a control group that designed without any stimuli , all other experimental groups re - ceived a sketch of one example solution and a description of what the sketch represented . These stimulated groups received instructions for the use of features from the example , and these instructions varied with re - spect to how constraining or encouraging they were ( see the materials sec - tion for the complete instructions ) . Finally , the authors assessed the partic - ipants\u2019 ideas to evaluate the influence of the instructions on the level of fixation demonstrated . Participants One hundred and sixty - eight first - year students in Engineering from the University of Cambridge , UK , were assigned to six experimental groups ( n = 28 ) . Participation in the experiment was part of the students\u2019 education , and was aimed at collecting data that could later be used to introduce them to the concept of design fixation . No demographic data was collected from the participants , but as first year undergraduate students they were broadly similar in age and design experience , drawn from a cohort with a male - female ratio of 3 : 1 . Task and problem The participants were told to solve the following problem . \u201c Bicycles are a popular mode of transportation and recreation for many people . While growing up , a person might go through a series of ever - larger bikes , some - times having several models , one after the other . However , having several bikes can be a problem for many reasons . Your task is to generate as many ideas as possible to eliminate the need to have multiple bikes as people grow up . \u201d This problem was selected because it was expected to satisfy the follow - ing three criteria . First , it was unlikely that the participants had designed solutions to it before , although they were likely to have experienced the situation described in the problem previously ( i . e . while growing up , they probably had multiple bikes ) , therefore helping their understanding of it . L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 6 Second , the problem could be solved in many different ways , with many different underlying principles being applied , thus leaving enough room for creativity . Finally , both the design brief and the potential ideas held a low level of complexity , thus being suitable for a quick experiment fitting with the constraints of the course . Procedure ( overview ) The experiment took place in a large lecture theatre with all the partici - pants present . During the first five minutes , the participants listened to an oral explanation about the activities to follow and received all the material they needed . Participants in the five stimulated groups ( SGs ) received the design brief , the inspiration source , and blank sheets of paper , whilst par - ticipants in the control group ( CG ) received the brief without any inspira - tion source . Then , the participants were asked to think of ideas for three minutes without actually committing any designs to paper ( because differ - ent participants had different materials and content , this ensured they all had enough time to read all the materials and start developing some ideas ) . Finally , for the remaining ten minutes , all participants individually gener - ated as many ideas as possible in silence , ideally including both a sketch and a written description for each idea . Materials All participants received the same design problem written on an A4 sheet , as well as blank A4 sheets to sketch and annotate their own ideas . Except for the participants in the control group , all participants received one addi - tional sheet with an example solution , i . e . an annotated sketch of a bike ( Figure 1 ) . To copy or not to copy 7 Fig1 . Example solution provided to participants along with the following descrip - tion . \u201c A modular bike with parts of various sizes that can be connected and swapped to fit people with very different heights . Apart from the socketing parts and expansible / contractible wheels , the angles between tubes can also be modified in specific joints \u201d . The sketch used is a modification of the ECO 07 Compactable Urban Bicycle [ 24 ] . The example solution was preceded with the description : \u201cBelow is an ex - ample of how you should present your ideas ( i . e . annotated sketches ) \u201d . This description was either immediately followed by an instruction regard - ing the use of features from the examples ( constraining or encouraging ) or by no instruction whatsoever . The instructions for the different experi - mental groups are listed below against a code for each experimental group . \u2022 SG2\u22122 ( strictly forbidding ) : \u201c make sure you do not use features from this example in your own work \u201d . \u2022 SG\u22121 ( constraining ) : \u201c avoid using features from this example in your own work \u201d . \u2022 SG0 ( neutral ) : no instruction was given . \u2022 SG + 1 ( encouraging ) : \u201c consider using features from this example in your own work \u201d . \u2022 SG + 2 ( strictly requiring ) : \u201c make sure you use features from this exam - ple in your own work \u201d . L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 8 Analysis The assessment of the participant\u2019s ideas was conducted by the first three authors of this study , with backgrounds in design , computer science , and mechanical engineering respectively . Initially , the three evaluators agreed on the metrics to be included in the analysis . After that , the three evalua - tors analysed the design work of a random experimental group together in order to agree on the assessment method , ultimately reaching a consensus with respect to how to interpret and assess the ideas . Finally , each of the evaluators individually judged a subset of the remaining ideas . If any eval - uator had trouble judging an idea , this idea was then discussed collectively . This interactive assessment method was intended to offer a reliable analy - sis of the ideas ; but because there was no redundancy in the assessment , no inter - rater agreement could be calculated . We considered \u2018one idea\u2019 either to be a sketch or a written description ( usually both ) that presented an un - derstandable way to solve the problem . Participants often generated more than one idea , but in some cases all ideas could be considered as one , par - ticularly when the idea was a bike . For instance , if the ideas could all be incorporated onto the same bike without interference , then they were con - sidered as a single idea . Conversely , if there were two or more ideas for the same bike component ( e . g . frame , wheel , handle bar ) , then they were considered to be distinct ideas . The metrics used in the assessment were \u2018idea fluency\u2019 and idea \u2018repeti - tion\u2019 . Idea fluency is the total number of ideas generated , also called \u2018quantity\u2019 elsewhere [ 25 ] . Idea repetition might happen in different levels , for instance , the repetition of idea types , conceptual features , or structural features . With respect to the idea type , we divided the ideas into two broad categories : bike and non - bike ideas , thus by designing a bike the partici - pant would be repeating the idea type . With respect to their conceptual fea - tures , we also divided the ideas into two categories : modular or non - modular ideas , thus by designing a modular idea the participant would be repeating the conceptual feature . Finally , we examined the incorporation of structural features in the participants\u2019 ideas . These features were intention - ally included into the example design in order to permit a measure of fixa - tion . There were five structural features : swappable components to change bike size ; frame joints ( lugs ) that act as sockets for the tubes ; wheels with bendable spokes ; an hourglass - shaped frame ; and a saddle that cannot be adjusted in height directly . Eight participants ( 4 . 8 % ) either did not generate any idea or generated ideas that could not be interpreted by the authors ; the results from such participants were not included in our analysis . The adjusted number of par - ticipants per experimental group is indicated in the following section . To copy or not to copy 9 Results and discussion Idea fluency Instructions had no effect on the number of ideas generated . The data did not satisfy the assumptions for a standard ANOVA , therefore a non - parametic Kruskal \u2013 Wallis test equivalent to a one - way ANOVA was im - plemented instead . The results show that the number of ideas generated did not vary significantly across the five stimulated groups ( SGs ) ( H ( 4 ) = 2 . 63 , p = . 62 ) . However , there is a significant difference in the idea fluency be - tween participants in these groups and those in the control group ( CG ) ( H ( 5 ) = 18 . 27 , p < . 01 ) , with participants in the control group generating a greater number of ideas . Additionally , although the majority of participants in the stimulated groups had an idea fluency of 1 , participants in the non - constraining groups ( SG , SG + 1 , and SG + 2 ) had a higher frequency of flu - encies greater than 1 ( i . e . more participants in those groups generated more than one idea ) . However , this difference was not shown to be significant ( X 2 ( 4 ) = 19 . 85 , p = . 47 ) . A significant difference in the frequency of idea flu - encies was found between the stimulated groups and the control group ( X 2 ( 5 ) = 55 . 38 , p < . 001 ) , with participants in the control group having a higher frequency of fluencies greater than 1 . Table 1 shows summary sta - tistics for these results . Table 1 Summary of ideas generated per participant and ideas frequencies across groups Generated ideas SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 CG Mean ( and SD ) for the number of ideas per participant 1 . 50 ( 1 . 27 ) 1 . 30 ( 0 . 61 ) 1 . 48 ( 0 . 49 ) 1 . 41 ( 0 . 56 ) 1 . 54 ( 1 . 10 ) 2 . 39 ( 1 . 31 ) Range of ideas per participant 1 - 6 1 - 3 1 - 4 1 - 4 1 - 6 1 - 5 Total number of ideas 39 35 40 38 43 67 Participants with 1 idea ( and % ) 21 ( 80 . 8 % ) 21 ( 77 . 8 % ) 16 ( 59 . 3 % ) 19 ( 70 . 4 % ) 19 ( 67 . 9 % ) 11 ( 39 . 3 % ) Participants with 2 ideas ( and % ) 2 ( 7 . 7 % ) 4 ( 14 . 8 % ) 10 ( 37 % ) 6 ( 22 . 2 % ) 7 ( 25 % ) 3 ( 10 . 7 % ) Participants with 3 ideas ( and % ) 1 ( 3 . 9 % ) 2 ( 7 . 4 % ) 0 ( 0 . 0 % ) 1 ( 3 . 7 % ) 0 ( 0 . 0 % ) 7 ( 25 % ) Participants with 4 ideas ( and % ) 0 ( 0 . 0 % ) 0 ( 0 . 0 % ) 1 ( 3 . 7 % ) 1 ( 3 . 7 % ) 1 ( 3 . 6 % ) 6 ( 21 . 4 % ) L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 10 Participants with 5 ideas ( and % ) 1 ( 3 . 9 % ) 0 ( 0 . 0 % ) 0 ( 0 . 0 % ) 0 ( 0 . 0 % ) 0 ( 0 . 0 % ) 1 ( 3 . 6 % ) Participants with 6 ideas ( and % ) 1 ( 3 . 9 % ) 0 ( 0 . 0 % ) 0 ( 0 . 0 % ) 0 ( 0 . 0 % ) 1 ( 3 . 6 % ) 0 ( 0 . 0 % ) Total number of participants 26 27 27 28 28 28 These results reveal that the idea generation rate was not influenced by how encouraging or constraining the instructions were . However , the pres - ence of an example design affected idea generation : designing without ex - posure to stimuli resulted in more ideas being generated , which we inter - pret as a benefit of isolation from examples . This effect is consistent with other studies in which seeing an example caused reduction in the idea flu - ency [ 7 ] , although studies have also reported an increase in the idea fluen - cy as a result from external stimulation [ 26 ] or even no effect at all [ 4 ] . Fi - nally , we should mention that although all groups were asked to present their ideas with both sketches and textual descriptions , the control group produced many ideas that were presented only in text . Thus , it is possible that the control group created more ideas because they did not spend their time sketching every idea . Repetition of the idea type Instructions had no effect on the number of bike ideas . The data did not satisfy the assumptions for a standard ANOVA , therefore a Kruskal \u2013 Wallis test was is implemented instead . The results show that the number of bike ideas generated did not vary significantly across the five stimulated groups ( SGs ) ( H ( 4 ) = 2 . 98 , p = . 56 ) , nor between these groups and the con - trol group ( CG ) ( H ( 5 ) = 3 . 55 , p = . 62 ) . Consistent with these results , there is also no significant difference in the proportion of bike ideas generated ( compared to non - bike ideas ) across the five stimulated groups ( X 2 ( 4 ) = 2 . 53 , p = . 64 ) . However , there is a significant difference in the pro - portions between these groups and the control group ( X 2 ( 5 ) = 32 . 73 , p = . 001 ) , with participants in the control group having a greater proportion of non - bike ideas , such as other transportation means or policies to dis - courage the use and acquisition of bikes . Table 2 shows summary statistics for these results . Table 2 Summary of bike and non - bike ideas generated across groups Bike Ideas SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 CG Mean ( and SD ) for the number of bike ideas per 1 . 27 ( 0 . 92 ) 1 . 15 ( 0 . 36 ) 1 . 33 ( 0 . 83 ) 1 . 33 ( 0 . 55 ) 1 . 29 ( 0 . 53 ) 1 . 43 ( 0 . 84 ) To copy or not to copy 11 participant Range of bike ideas per participant 0 - 5 1 - 2 0 - 4 1 - 3 0 - 2 0 - 3 Number of bike ideas ( and % ) 33 ( 85 % ) 31 ( 89 % ) 36 ( 90 % ) 36 ( 95 % ) 36 ( 84 % ) 40 ( 60 % ) Number of non - bike ide - as ( and % ) 6 ( 15 % ) 4 ( 11 % ) 4 ( 10 % ) 2 ( 5 % ) 7 ( 16 % ) 27 ( 40 % ) If we adopt a bike and non - bike categorisation for the ideas generated , these results reveal that the type of idea generated was not influenced by the instructions . Additionally , the presence of the bike example did not in - crease the number of bike ideas either . However , whilst the number of bike ideas was roughly the same for all groups , there was a large difference in the proportion of bike ideas between the control and stimulated groups . Only 60 % of all ideas generated by the control group were bikes , whereas the stimulated groups had a much greater proportion of bike ideas ( 89 % on average ) . The results indicate that bike ideas were equally likely to be gen - erated irrespective of the experimental condition , but not seeing the exam - ple allowed participants from the control group to explore different areas of the solution space , again confirming the beneficial isolation effect . This effect is broadly consistent with other studies in which seeing an example caused participants to conform to certain types of solutions , thus reducing the diversity of ideas [ 4 , 5 , 7 ] . Repetition of the conceptual feature Instructions had no effect on the number of modular ideas . The data did not satisfy the assumptions for a standard ANOVA , therefore a Kruskal \u2013 Wallis test was is implemented instead . The results show that the number of modular ideas generated did not vary significantly across the five stimu - lated groups ( SGs ) ( H ( 4 ) = 4 . 21 , p = . 38 ) . However , there appears to be a significant difference in the repetition of modularity between the stimulat - ed groups and the control group ( CG ) ( H ( 5 ) = 11 . 40 , p < . 05 ) , with partici - pants in the control group creating a greater number of modular ideas . When looking at the frequencies , there is no significant difference in the proportion of modular ideas generated ( compared to non - modular ideas ) across the five stimulated groups ( X 2 ( 4 ) = 5 . 12 , p = . 27 ) , nor even when the control group is included in the comparison ( X 2 ( 5 ) = 5 . 68 , p = . 34 ) . Table 3 shows summary statistics for these results . Table 3 Summary of modular and non - modular ideas generated across groups Modular ideas SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 CG L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 12 Mean ( and SD ) for the number of modular ideas per participant 0 . 08 ( 0 . 27 ) 0 . 3 ( 0 . 47 ) 0 . 19 ( 0 . 40 ) 0 . 19 ( 0 . 40 ) 0 . 18 ( 0 . 39 ) 0 . 43 ( 0 . 50 ) Range of modular ideas per participant 0 - 1 0 - 1 0 - 1 0 - 1 0 - 1 0 - 1 Number of modular ideas ( and % ) 2 ( 5 % ) 8 ( 23 % ) 5 ( 13 % ) 5 ( 13 % ) 5 ( 12 % ) 12 ( 18 % ) Number of non - modular ideas ( and % ) 37 ( 95 % ) 27 ( 77 % ) 35 ( 87 % ) 33 ( 87 % ) 38 ( 88 % ) 55 ( 82 % ) If we adopt a modular and non - modular categorisation for the ideas gener - ated , these results reveal that the type of idea generated was not influenced by the instructions . In fact , modular ideas were extremely rare across all groups . However , the results suggest that the presence of an example de - sign affected idea generation and the control group created on average more modular ideas than the stimulated groups . This apparently surprising result can be attributed to a higher overall number of ideas generated by the control group , which is supported by there being no difference in the proportion of modular ideas generated among all groups . Still , the fixation literature would suggest that the stimulated groups would generate more modular ideas , an effect similar to the repetition of the idea type ( i . e . bikes ) . In particular , participants in SG + 1 and SG + 2 were encouraged to use features from the example in their own work ( and modularity was a visible feature on the example provided ) but the results from those groups do not indicate that they acted accordingly . Similarly , participants in SG\u22122 and SG\u22121 were discouraged from using features from the example but generated as many modular ideas as the other groups . One possible expla - nation for this is that instructions have no influence on idea generation , as we had previously hypothesised . However , as there was no difference in the proportion of modular ideas between control and stimulated groups , we believe that the general principle of modularity ( included in the example as a conceptual feature ) was less obvious than the structural features and thus did not induce fixation effects . Repetition of structural features Instructions had a significant effect on the number of ideas that contained the structural features of the example provided . The data did not satisfy the assumptions for a standard ANOVA , therefore a Kruskal \u2013 Wallis test was implemented instead . The results show that the number of structural fea - tures incorporated into the participants\u2019 ideas varied significantly across the five stimulated groups ( SG ) ( H ( 4 ) = 41 . 62 , p < . 001 ) and between these To copy or not to copy 13 groups and the control group ( CG ) ( H ( 5 ) = 54 . 63 ; p < . 001 ) , with participants in the encouraged groups ( SG + 1 and SG + 2 ) incorporating a greater num - ber of structural features . Consistent with these results , there is also a sig - nificant difference in the relative number of ideas with structural features across the stimulated groups ( X 2 ( 4 ) = 37 . 32 , p < . 001 ) , with a higher number of features being associated with positive instructions to copy . There is al - so a significant difference between these groups and the control group ( X 2 ( 5 ) = 49 . 74 , p < . 001 ) , with the control group\u2019s repetition rate being close to the neutrally stimulated group ( SG0 ) . Table 4 shows summary statistics for these results . Table 4 Summary of features incorporated into the participants\u2019 ideas and fre - quencies of ideas with features across groups Feature SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 CG Mean ( and SD ) for the number features in - corporated per idea 0 . 08 ( 0 . 27 ) 0 . 17 ( 0 . 38 ) 0 . 10 ( 0 . 30 ) 0 . 71 ( 0 . 98 ) 0 . 93 ( 0 . 99 ) 0 . 13 ( 0 . 42 ) Range of features in - corporated 0 - 1 0 - 1 0 - 1 0 - 3 0 - 3 0 - 2 Ideas with 0 features ( and % ) 36 ( 92 % ) 29 ( 83 % ) 36 ( 90 % ) 22 ( 58 % ) 19 ( 44 % ) 60 ( 90 % ) Ideas with 1 features ( and % ) 3 ( 8 % ) 6 ( 17 % ) 4 ( 10 % ) 8 ( 21 % ) 11 ( 26 % ) 5 ( 7 % ) Ideas with 2 features ( and % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 5 ( 13 % ) 10 ( 23 % ) 2 ( 3 % ) Ideas with 3 features ( and % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 3 ( 8 % ) 3 ( 7 % ) 0 ( 0 % ) Ideas with 4 features ( and % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) Ideas with 5 features ( and % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) Ideas with features in - corporated ( and % ) 3 ( 8 % ) 6 ( 17 % ) 4 ( 10 % ) 16 ( 42 % ) 23 ( 56 % ) 7 ( 10 % ) These results reveal that encouraging instructions influenced the repetition or incorporation of conceptual features , an outcome that does not support our hypothesis . On average , participants in SG + 1 and SG + 2 incorporated more features per idea and generated more ideas that incorporated any fea - ture . Additionally , participants in the strictly forbidden group , SG\u22122 , did not incorporate any structural feature from the example . However , partici - pants in SG\u22121 did not follow this trend and produced results similar to the neutral and control groups . This result is partially consistent with research L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 14 from Smith et al . [ 21 ] but inconsistent with Chrysikou & Weisberg [ 11 ] , as encouraging instructions increased fixation , whereas constraining instruc - tions did not decrease it . However , contrary to the results from Smith et al . [ 21 ] , our constrained groups did not replicate more features than a control group , contradicting the idea that participants could not forget the example they had seen . It seems that a few structural features were naturally likely to be incorporated into the ideas generated ( supported by the similar results from SG\u22121 , SG0 and CG ) , with SG + 1 and SG + 2 incorporating more fea - tures because instructed to do so . When comparing the results from the repetition of structural features to the repetition of the conceptual feature used in this study , we infer that concrete structural features can be more easily copied or can fixate more than abstract conceptual features from ex - amples . This is similar to what has been suggested in previous studies [ 27 , 28 , 29 ] . Again , we should highlight that the control group produced pro - portionally less sketches than the other groups . This puts the other groups in an unfavourable position with respect to the count of structural features incorporated , since these groups had to represent a shape of the bike in which repetition could be more easily recognised \u2013 it is difficult to identify structural repetition when the idea is represented only by text \u2013 thus possi - bly biasing the results . Study limitations The main limitations of this study involve the duration of the generation session , the pool of participants chosen , the design problem used , and the assumptions for inter - rater agreement . These limitations are discussed next . The idea generation session in this study was ten minutes long , which can be considered short when compared to other fixation studies in which generation sessions lasted for 30 or 60 minutes [ 30 ] , and shorter still com - pared to professional practice [ 31 ] . Also , past research suggests [ 32 ] , novel ideas tend to occur later in the idea generation session . As a result , the short session adopted for this study might have contributed to inflated fixa - tion scores . The participants in this study were undergraduate students and the gen - eration session was part of an ongoing engineering course . This might have resulted in a more diligent participant behaviour when compared to other studies in which participants and experimenters did not have a stu - dent - lecturer relationship . As a result , the setup adopted for this study might have contributed to an increased participant adherence to the in - structions . The design problem used in this study was chosen because it was unlikely that the participants had designed solutions to it before . As To copy or not to copy 15 such , it is possible that using familiar problems will produce different re - sults , although research has demonstrated that fixation effects can be ob - served with both familiar and unfamiliar problems [ 4 ] . Finally , in this study we did not measure the inter - rater agreement for the assessment , a test which is often performed for similar studies . Such measurement is important to demonstrate that the reliability of the evalua - tors\u2019 assessment when working individually . Whereas the assessment per - formed in this study involved many interactions between evaluators ( thus we expected a high agreement between evaluators ) , we cannot quantify how good this agreement might have been , or even if there were varying levels of agreement for the different ideation metrics used in this study . Conclusion and future work In this study we have tested the influence of instructions on idea genera - tion . In particular , we analysed how instructions may affect the number of ideas generated and the repetition or incorporation of the example or its parts into the participants\u2019 ideas . The instructions used were provided along with an external stimulus and its description . It is important to dif - ferentiate the descriptions from instructions because in this study we have controlled the former but manipulated the latter . We found that instructions had some influence on the idea generation of our participants . When asked to use features from the example , participants copied structural features but failed to copy a more abstract conceptual feature . When asked not to use features from the example , however , most participants did not reduce the number of features copied . This result allows us to infer that more concrete features are easier to recognise \u2013 and thus reproduce \u2013 than more abstract features , such as modularity . Also , it might indicate that positive instruc - tions are more effective than negative ones , which can tell us how to frame future instructions , whether that is with respect to experimental stimuli in research or inspirational stimuli in design practice . Irrespective of how constraining the instructions were , participants ex - hibited fixation effects due to their exposure to the example design ( in comparison to the control group , all stimulated groups created more ideas of the same type as the example provided ) . This result is in line with many other design fixation experiments in which participants become stuck on a particular idea type . However , it is important to emphasise that the descrip - tion of the stimulus itself could also be causing the fixation effect as the stimulus was presented to the participants as \u201c an example of how they should present their ideas \u201d . Thus , perhaps there is an implicit suggestion L . A . Vasconcelos , C - C . Chen , E . Taysom and N . Crilly 16 for the participants to produce ideas similar to the example , i . e . a bike . Fu - ture studies could investigate such possibility and complement our under - standing about stimuli introduction with the influence of the descriptions provided to designers . In this study we observed that some results might have been influenced by participants communicating their ideas in different ways ( some of them drawing , some writing , and some doing both ) , an issue that cannot be con - firmed until the analysis of the results . In inspiration and fixation studies , participants are asked to provide their ideas according to a content tem - plate , but that is rarely compulsory . It is fundamental that all ideas contain the same elements ( e . g . text and sketch ) in order to be analysed and com - pared , so studies should make sure every idea has a similar content . Addi - tionally , future studies could investigate the difference between providing written , pictorial or spoken instructions to designers , but also how they as - similate instructions . For instance , it is possible that people need instruc - tions about the instructions ( i . e . \u201cread the instructions carefully\u201d ) , as one way to make sure that they will read and fully understand what is required from them . Finally , when considering design practice , the results reported here are relevant to how inspirational stimuli should be framed when presented to designers . This is particularly important for the development and imple - mentation of computer - aided design tools that provide designers with ex - ternal stimuli . Much has been researched on how such software tools might be structured and interacted with , and what form the inspirational stimuli should take [ 33 , 34 ] . However , it is also important to understand how those stimuli should be introduced , whether by description , instruc - tion , or both . Should designers be steered towards or away from the repeti - tion of structural features , directed to identify conceptual features , or simp - ly left alone to interpret and respond as they see fit ? By developing a better understanding of how stimuli instructions influence idea generation , we will move closer to answering such questions and thereby be more capable of supporting design activities . Acknowledgement The authors would like to thank Carlos Coimbra Cardoso for commenting on earlier drafts of this paper , to all the students for taking part in the ex - periment , and to the three DCC reviewers for improving this work with their feedback . This work was supported by the CAPES Foundation , Min - istry of Education of Brazil , under grant / process : BEX 11468 / 13 - 0 and by the UK\u2019s Engineering and Physical Sciences Research Council To copy or not to copy 17 ( EP / K008196 / 1 ) . Research data supporting this publication is available from the DSpace @ Cambridge repository at the web address given below . It contains data from the participants and from our analysis . The data from the participants consists of all the annotated sketches that participants gen - erated in the experiment . The data from the analysis consists of the evalua - tion for all ideas generated by the participants in the experiment . https : / / www . repository . cam . ac . uk / handle / 1810 / 254702 References 1 . Gon\u00e7alves M , Cardoso C , Badke - Schaub P ( 2014 ) What inspires designers ? Preferences on inspirational approaches during idea generation . Des Stud 35 : 29 \u2013 53 . 2 . Zhao M ( 2013 ) Seek it or let it come : how designers achieve inspirations . In : CHI13 Ext . Abstr . Hum . Factors Comput . Syst . ACM , pp 2779 \u2013 2784 3 . Eckert CM ( 1997 ) Design inspiration and design performance . In : Proc . 78th World Conf . Text . Inst . 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Kudrowitz B , Dippo C ( 2013 ) Getting to the novel ideas : exploring the alter - native uses test of divergent thinking . In : ASME 2013 International Design Engineering Technical Conferences and Computers and Information in Engi - neering Conference . American Society of Mechanical Engineers , pp V005T06A013 \u2013 V005T06A013 . To copy or not to copy 19 33 . T\u00f6re Yargin G , Crilly N ( 2015 ) Information and interaction requirements for software tools supporting analogical design . Artificial Intelligence for Engi - neering Design , Analysis and Manufacturing 29 : 203 \u2013 214 . 34 . Shneiderman B ( 2000 ) Creating Creativity : User Interfaces for Supporting In - novation . ACM Trans Comput - Hum Interact 7 : 114 \u2013 138 .", + "kordeAlternatingIndividualGroup2016": "Alternating individual and group idea generation : Finding the elusive synergy Runa Korde , Paul B . Paulus \u204e The University of Texas at Arlington , United States H I G H L I G H T S \u2022 Alternating individual and group ideation was better than group ideation . \u2022 Alternating individual and group ideation was better than solitary ideation . \u2022 The bene \ufb01 ts of alternation are only evident in the alone sessions . \u2022 The results provide evidence for the synergistic bene \ufb01 ts of group ideation . \u2022 The results support an association model of group ideation . a b s t r a c t a r t i c l e i n f o Article history : Received 3 May 2016 Revised 9 November 2016 Accepted 9 November 2016 Available online 23 November 2016 Three experiments were designed to test theef \ufb01 cacy ofideation procedures that involvedalternation ofindivid - ual and group idea generation sessions ( hybrid brainstorming ) as compared to traditional individual and group ideation . Thehybridconditionledtothebestperformanceintermsofnumberofideasgenerated . Thiseffectwas strongest in comparison to the group condition . A meta - analytic comparison involving all three experiments in - dicated that the hybrid condition outperformed both the alone and the group conditions . Since after each group ideaexchangesession there wasanenhancementinthenumberofideas generatedinthealonesession , thepat - tern of performance in the hybrid condition supported the cognitive perspective of group creativity ( Nijstad & Stroebe , 2006 ; Paulus & Brown , 2007 ) . Social cues in the form of the co - presence of other participants in the alonecondition , theadditionofpracticesessionstoallconditions , andanadditionalphasedidnotchangethepat - tern ofresults . Theresults oftheexperiments supportthe original suggestion by Osborn ( 1953 ) thatthemostef - fective brainstorming process is one that involves a variation in individual and group ideation . \u00a9 2016 Elsevier Inc . All rights reserved . Keywords : Group creativity Brainstorming Innovation Brainwriting Creativity Synergy 1 . Introduction Collaborative idea generation is a frequently used approach in many settings . Itis typicallyassumed thatsharingideasingroupswillincrease thenumberandnoveltyofideas ( Paulus , Dzindolet , Poletes , & Camacho , 1993 ; Sutton & Hargadon , 1996 ) . However , studies that have compared collaborative ideation or brainstorming with the performance of similar numbers of individual performers ( nominal groups ) in various formats have typically found that interactive groups tend not to be better than nominal groups ( cf . , Paulus & Coskun , 2012 ) . It has been suggested that alternating group and individual ideation may be a more optimal procedure than either alone or group ideation . However , research with this paradigm has been very limited thus far , and the results have been mixed . In a series of three experiments we examine more precisely than past research the variation in idea generation over the course of the alone and group alternation process in order to demon - strate the synergistic bene \ufb01 t of group ideation . A review of traditional ideation methods shows that the bene \ufb01 ts of group ideation depend on the type of paradigm used . In face - to - face ( FTF ) settingswhereinideas aresharedverbally , groupstypicallygener - ate fewer ideas than comparable size nominal groups , with the differ - ence increasing with group size ( Bouchard & Hare , 1970 ; Diehl & Stroebe , 1987 ) . However , this performance de \ufb01 cit for groups can disap - pear when ideas are exchanged either electronically ( electronic brain - storming - EBS ) or by writing ( brainwriting - BWr ) . These techniques help overcome the production blocking and evaluation apprehension that are some of the factors responsible for the low performance of FTF groups ( Dennis & Williams , 2003 ; Heslin , 2009 ) . Although the EBS and BWr techniques appear to be useful for group ideation , their bene \ufb01 ts are limited . EBS groups can typically outperform Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 \u204e Corresponding author at : Department of Psychology , The University of Texas at Arlington , Arlington , TX 76019 - 0528 , United States . E - mail address : Paulus @ uta . edu ( P . B . Paulus ) . http : / / dx . doi . org / 10 . 1016 / j . jesp . 2016 . 11 . 002 0022 - 1031 / \u00a9 2016 Elsevier Inc . All rights reserved . Contents lists available at ScienceDirect Journal of Experimental Social Psychology journal homepage : www . elsevier . com / locate / jesp nominals only if groups are size 8 or larger ( Derosa , Smith , & Hantula , 2007 ) . However , these effects are not large ( Paulus , Kohn , & Arditti , 2011 ) . Research has found that BWr groups can generate as many or more ideas than comparable nominal groups ( Goldenberg , Larson , & Wiley , 2013 ; Paulus , Korde , Dickson , Carmeli , & Cohen - Meitar , 2015 : Paulus & Yang , 2000 ) . The positive effects of BWr appear to depend on the exact procedure used ( Goldenberg et al . , 2013 ) . Even though EBS and BWr can eliminate the production losses ob - served in FTFbrainstorming , it is apparentthattheevidence for thesyn - ergistic effect of group collaboration using these paradigms is quite limited . A demonstration of synergy would require that the perfor - mance of the groups exceed those of the nominals ( Larson , 2010 ) . There are a number of potential reasons for this state of affairs . Sharing ideas in a group should be cognitively stimulating ( Nijstad & Stroebe , 2006 ; Paulus & Brown , 2007 ) . There is in fact evidence that exposure to the ideas of others can stimulate additional ideas because of associa - tive processes ( Dugosh , Paulus , Roland , & Yang , 2000 ; Nijstad , Stroebe , & Lodewijkx , 2002 ) and the exposure to different categories of ideas ( cf . , Deuja , Kohn , Paulus , & Korde , 2014 ) . However , the group exchange process can also slow down the rate of exchanging ideas . The ef \ufb01 ciency of the group exchange process is limited by the task or coordination de - mands of this process ( Steiner , 1972 ) . This is most evident in the FTF case since group members have to take turns to present their ideas . Al - though turn taking is not required in EBS and BWr , the time spent read - ing ideas from others takes away from generating one ' s own ideas . Furthermore , although ideas from others can be stimulating , they can also distract from one ' s own train of thought or may lead to free riding ( Kerr & Bruun , 1983 ) in that one may feel less responsibility for gener - ating ideas if others are generating many ideas . One solution to this dilemma is to have individuals participate in both aloneand groupsessions . Infact , Osborn ( 1953 ) noted \u201c an alterna - tion between groupideation and individualideation is desirable , since a combination of these two methods has produced maximum results in almost every case . \u201d ( p . 209 ) . Although Osborn provided no empirical data to support this claim , it seems reasonable to expect that combining individual and group brainstorming into a single paradigm or \u201c hybrid brainstorming \u201d may be an optimal procedure . This would allow both for unconstrained ideation in individual brainstorming and stimulation of additional ideas by exposure to ideas of others . This might be most evident with the EBS and BWr paradigms since these signi \ufb01 cantly re - duce production blocking . There has been very limited work on brainstorming procedures that combine the individual and group paradigms . Some studies have varied theorder of aloneand groupbrainstorming to determinewhichorder is most bene \ufb01 cial . One might expect that group brainstorming would be most bene \ufb01 cial after a period of individual brainstorming . This allows participants to generate their ideas in an unconstrained fashion \ufb01 rst , and then when thinking of new ideas becomes more dif \ufb01 cult , group in - teraction may provide cues to additional ideas or categories of ideas . One study has in fact found that this order is most bene \ufb01 cial ( cf . , Baruah & Paulus , 2008 ) . However , one can also argue that having group interaction before a solitary ideation session would be bene \ufb01 cial since this provides participants a chance to re \ufb02 ect on the shared ideas and build on them while the shared ideas are still salient in the associa - tive network ( Brown & Paulus , 2002 ) . Evidence for the superiority of this order has been obtained in two studies using BWr ( Paulus & Yang , 2000 ; Paulus et al . , 2015 ) and also in several studies with verbal brain - storming ( Dunnette , Campbell , & Jaastad , 1963 ; Leggett , Putman , Roland , & Paulus , 1996 ) . One of the more famous examples of this type of effect is Einstein ' s writing of the theory of relativity . After a longdiscussion session with his friends on this topic , heisolatedhimself in a room to write out the basics of his theory ( Brian , 1996 ) . Given the different \ufb01 ndings on the effect of the order of alone and group brainstorming and the fact that some studies \ufb01 nd no effect of order ( Paulus , Larey , & Ortega , 1995 ; Rotter & Portugal , 1969 ; Taylor , Berry , & Block , 1958 ) , it would seem that the most important consideration might be simply the importance of alternating alone and group brainstorming rather than focusing on the bene \ufb01 ts of one particularorder . However , only asmall number of studies have assessed this possibility and each of these have methodological limitations . A number of studies used different procedures for generating ideas in dif - ferent conditions . Rotter and Portugal ( 1969 ) compared nominal and interactive groups with those that worked both alone and as a group ( in two different orders ) . All of the participants wrote their ideas on a sheet of paper . The participants in the alone condition performed best , with the alternating condition leading to better performance than the groupcondition . However , thealonesessionsweredoneonlywithwrit - ing whereas the group session also involved discussion of the written ideas that took time away from idea generation . Several EBS studies have used a form of asynchronous brainstorming group in which mem - bers submit their ideas at different points in time . That is , ideas are sub - mittedindividuallybuttoacentrallocationorforumthatisaccessibleto all members of the group . This paradigm should allow group members to follow their train of thought without being disrupted , while also being able to gain stimulation from other members ' ideas . Dornburg , Stevens , Hendrickson , and Davidson ( 2009 ) examined asynchronous brainstorming over the course of four days using a large EBS group that was compared to individual brainstorming . There was no differ - ence in the number of ideas generated between the individuals and the group . Individuals outperformed the group in originality , feasibility and effectiveness . But the duration of brainstorming was not controlled , and the study only involved one interactive group of 30 . Ocker , Fjermestad , Hiltz , and Johnson ( 1998 ) compared FTF brainstorming with two differenttypes of EBS procedures ( synchronous andasynchro - nous ) and a combination group . However , the synchronous groups worked atthesame timein the same room , while members of the asyn - chronous groups worked from different locations at different times . In the combination group , participants \ufb01 rst worked FTF and later worked asynchronously . All groups received two weeks to work on the given task . The amount of time allowed for communication among group members in the asynchronous conditions was not controlled . The com - bined groups generated signi \ufb01 cantly more creative solutions and a bet - ter quality solution than the asynchronous , the synchronous , and the FTF groups . Some studies did not have alone or nominal control conditions , Girotra , Terwiesch , and Ulrich ( 2010 ) compared a \u201c hybrid \u201d condition to a group condition . Participants in the hybrid condition were asked to write their ideas individually for the \ufb01 rst 10 min and then share and discuss the ideas as a group ( FTF ) for the remaining time . Partici - pants using the hybrid process generated three times more ideas than those in the real groups . Since the hybrid condition used both BWr and FTF techniques , while the real group only used FTF , it is possible that the bene \ufb01 t of the hybrid condition may simply be due to a differ - ence in techniques across conditions . De Vreede and Reiter - Palmon ( 2010 ) examined the asynchronous brainstorming process using multiple groups . Large EBS groups were comprised of subgroups that either completed the entire brainstorming process on their own ( parallel mode ) or built on the work provided by the previoussubgroups ( serial mode ) . They found that serial processing wasbettersuitedfortasksthatrequirein - depthprocessingandelabora - tion , butparallelprocessingwasappropriatefortasksthatdemandmul - tiple new ideas . However , there was no individual control group to determine the relative effectiveness of group interaction . Although there is evidence that using a hybrid procedure may have some bene \ufb01 t over traditional group brainstorming , the results are not clear because of lack of control groups and lack of comparability of pro - cedures . In thepresentseries of experiments thesame technique ( BWr ) was used in all conditions . Alone and group BWr conditions were com - pared to hybrid conditions that alternated alone and group brainstorm - ing sessions . This design allowed us to determine to what extent the alternating process enhanced the number of ideas relative to the alone and group conditions . Eight - minute sessions were employed since 178 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 prior unpublished research has suggested that bene \ufb01 ts of hybrid brain - storming can be observed with this type of timing ( Korde , 2012 ) . BWr was used since we felt that this procedure was more likely than the FTF and EBS ones to demonstrate a bene \ufb01 t of the hybrid procedure be - cause of the more ef \ufb01 cient group exchange process . There are several demonstrations of such small BWr groups exceeding the performance of nominal groups ( Paulus et al . , 2015 ; Paulus & Yang , 2000 ) . However , small EBS groups of size b 8 tend to show production losses relative to same size nominal groups . These differences in outcome for EBS and BWr groups may be due to that fact that a collaborative stimulation ef - fect can occur only if participants pay attention to the shared ideas ( Dugosh et al . , 2000 ; Paulus & Yang , 2000 ) . In the typical EBS programs there is no way to be sure that participants are reading all the ideas pre - sented to them . In the BWr procedure used in the present experiments , reading shared ideas is part of the process . Recent research has shown that BWrcan producemore ideasthanEBSwhenparticipants are forced to make an effort to read the ideas ( Michinov , 2012 ) . However , there has to be reasonable balance between time spent reading others ideas and generating one ' s own ideas ( Brown & Paulus , 2002 ) . It is expected that hybrid brainstorming will be more effective than both group and alone brainstorming , but this could occur for a variety of reasons . There are least two theoretical perspectives , which suggest that individual ideation will be elevated after group ideation . The cogni - tive models suggest that the cognitive stimulation from group ideation sessions can yield associations that can be the basis for enhanced idea generation in a subsequent solitary ideation session . In fact , Brown and Paulus ( 2002 ) reported simulations based on their cognitive matrix model ( cf . , Brown , Tumeo , Larey , & Paulus , 1998 ) that demonstrated spikes in performance in an alone phase that followed a group phase . A production blocking / constraint perspective suggests that perfor - mance in a group session will be inhibited because the time taken to at - tend to others ' ideas will take away from one ' s own idea generation time . But , in the alone session performance is no longer constrained bythis demand , andthis mayprovidesomeextra motivationtotakead - vantage of this new freedom to generate ideas at will ( Amabile , 1996 ) . We are not aware of any demonstrations of such effects , but there is a literaturesuggestingthatvariouscognitiveandtaskconstraintscanmo - tivate individual creative behavior ( Medeiros , Partlow , & Mumford , 2014 ; Stokes , 2005 ) . However , this research has not addressed the pos - sible positive effects of eliminating the constraint on subsequent creativity . In contrast to the above discussion , a social comparison perspective suggeststhattheperformanceinthealonephasesmaybecriticalforen - hancing performance in the subsequent group phases ( Paulus & Dzindolet , 1993 ) . The typically higher rate of performance in the alone session may become a reference point for participants in a subsequent group session and result in an elevated level of performance relative to a condition in which participants share ideas only as a group . PaulusandDzindolet ( 1993 ) foundthatperformanceinsubsequentses - sions is positively correlated with the performance rate in earlier ses - sions ( Studies 3 & 4 ) . Thus those who started with a task that yielded a higher rate of idea generation performed better on a subsequent dif - ferent idea generation task than participants who started off with a lower rate task . This type of pace entrainment effect was stronger for group ideation than individual ideation , consistent with the entrain - mentperspectiveofKelly ( 1988 ) aswellasasocialcomparisonperspec - tive . Thus according to a social comparison perspective , a preliminary alone session may enhance the performance of a subsequent group ses - sion and this pattern may be entrained during the sessions that follow . In a preliminary experiment we examined the potential bene \ufb01 ts of hybrid ideation and the impact of starting a session with alone or group ideation ( see Korde , 2014 and supplementary materials for de - tails ) . The experiment employed a brainwriting paradigm for 4 eight - minute sessions , either all alone or in an alone - group - alone - group ( AGAG ) or a group - alone - group - alone ( GAGA ) sequence . It was found that the AGAG sequence led to more ideas than the other two conditions . The performance in the alone phases was elevated relative to the other phases , but only the second alone phase in the AGAG con - dition was signi \ufb01 cantly different from same phase in the alone condi - tion . Thus this study provides some support for the perspectives which suggest that the bene \ufb01 t of hybrid ideation will be primarily due to enhancement of performance in the alone phases . However , a possi - ble sampling bias in the AGAG condition and the fact that hybrid condi - tion participants received additional training may have in \ufb02 uenced the results . Thus we conducted two similar experiments to more de \ufb01 nitive - ly assess the effects of hybrid ideation . For these experiments all condi - tions , measures , and exclusions are reported . 2 . Experiment 1 Experiment 1 included the three conditions of the preliminary study and a group condition , with four phases in each condition . Participants either switched back and forth from group brainstorming to working alone or generated ideas alone or as a group for the entire session . There were two hybrid conditions depending on which phase was \ufb01 rst \u2013 alone or group . The four conditions were 1 ) alone - group - alone - group ( AGAG ) 2 ) group - alone - group - alone ( GAGA ) , 3 ) alone - alone - alone - alone ( Alone ) and 4 ) group - group - group - group . Each phase ( alone or group ) within the conditions lasted for 8 min and the total brainstormingsessionforeachconditionwas32minlong . Inallthecon - ditions , participants performed in groups of three . However , in the alone condition they performed the task individually but in the pres - ence of other members of their group . Based on previously discussed research and theory , the hybrid con - ditions should lead to more ideas than the nominal and group conditions . H1 . Participants in the hybrid conditions will generate more ideas than those in the alone and group conditions . Prior researchmight lead one to expect differences between the two different hybrid conditions . Given the \ufb01 ndings that either sequence can be bene \ufb01 cial , there is not a strong empirical basis for making a differen - tial prediction . However , both the cognitive and blocking / constraint perspectives suggest that a condition that begins with a group session may have an advantage . However , the social comparison and entrain - ment perspectives suggest that the condition that begins with an alone session will lead to the better performance . Furthermore , the cog - nitive and blocking / constraint perspectives both suggest that the alone sessions that follow the group sessions will demonstrate elevations in performancein comparison tothe alonecondition . In contrast the social comparison and entrainment perspectives suggest that a group session that follows an alone session will be elevated relative to the group condition . H2a . In accord with the cognitive and blocking / constraint perspectives , the hybrid condition that begins with a group session will lead to a bet - ter performance than one that begins with an alone session since group sessions should be followed by elevations in the performance during alone sessions . H2b . In line with the social comparison and entrainment perspectives the hybrid condition that begins with an alone session should lead to the best performance since this will lead to elevations in the perfor - mance in the subsequent phases and elevations in performance the group sessions that follow the alone sessions . H3a . In accord with the cognitive and constraint perspectives the alone sessions that follow group sessions in both of the hybrid conditions should show enhanced performance relative to the comparable phases in the alone condition . H3b . In accord with the social comparison and entrainment perspec - tives the group sessions that follow an alone session in both of the 179 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 hybrid conditions should show enhanced performance relative to the comparable phases in the group condition . A blocking / constraint perspective would suggest that the more constrained the performance is in the group phase in terms of number of ideas , the greater will be the subsequent enhancement in the alone phase . This could be examined by examining the extent to which the performance in the \ufb01 rst group phase of the hybrid condition is lower than the prior alone phase . The greater the discrepancy , the greater should be the experience of constraint and the subsequent increased motivation to perform in the alone session that follows the group session . H4 . If blocking / constraint plays a role in the elevation of the perfor - mance of the alone phases after the group phases in the hybrid condi - tion , the performance in the second alone session will be positively related to the degree of constraint experienced in the group phase in comparison to the prior alone phase . Studies that have used BWr techniques have obtained mixed results asto the bene \ufb01 t of sharingideas in a group . Paulus and Yang ( 2000 ) and Coskun ( 2005 ) both found that real groups generated more ideas than nominal groups when the BWr technique was used . However , thenom - inal groups generated ideas on a single sheet of paper , and the interacting groups generated ideas on slips of paper . A study by Goldenberg et al . ( 2013 ) found that when participants in the nominal condition are also asked to write their ideas on separate slips of paper , the difference in number of ideas generated by the interacting groups and nominal groups disappears . Since the BWr technique involved sep - arate slips in all conditions , it was anticipated that there would be no difference between the alone and group condition . However , Paulus et al . ( 2015 ) found that group BWr led to more ideas than alone writing foremployeesof atechnologycompanywhenall participants usedindi - vidual slips . This result was only marginally signi \ufb01 cant with the rela - tively small sample . Thus given the mixed \ufb01 ndings in past studies , we did not make a directional prediction for alone and group brainwriting . 2 . 1 . Method 2 . 1 . 1 . Participants Participants were undergraduate students from a large university in theSouthwest U . S . whoparticipated in thestudy in order to ful \ufb01 llintro - ductorypsychologyclassrequirements . 189students participatedinthe study , and data from nine participants were not used as they failed to follow instructions correctly . Six of these participants belonged to the Alone condition and three to the Group condition . Participants were randomly assigned to one of the four experimental conditions , each consisting of 15 groups of three . Data from 45 individual participants were pooled together to create 15 nominal groups . In our previously published studies on brainwriting , we have found effects related to alone and group sequence with 10 or 12 groups per condition ( Paulus et al . , 2015 ; Paulus & Yang , 2000 ) . All data were collected prior to our analyses . The average age of the participants was 20 . 16 years ( SD = 3 . 45 ) . There were 110 females and 70 males . 2 . 1 . 2 . Design and procedure The experiment used a 4 ( Condition ) \u00d7 4 ( Phases ) mixed design . In - formed consent was obtained from the participants before providing them with the instruction packet . Participants were provided instruc - tions about the task , the rules of brainstorming ( Osborn , 1957 ) , and the procedure to be used . All the participants wrote their ideas on col - ored slips of paper and were instructed to write only one idea per slip . Each participant received a different set of colored slips ( pink , white , or yellow ) . In the group phases , participants were seated at a table , fac - ing each other and were asked to pass the ideas to the person on their right and were told to read the ideas that they would receive from their left . Once the participants received their own ideas back , they were asked to place them at the center of the table . During the alone phases , participants were asked to write their ideas and simply place the slip of paper next to them without passing it on . At the end of each phase the slips were collected and placed into separate envelopes and labeled by group and phase . The participants were given different colors of pen for each phase . The colors of pens used were constant across condition and were always used in the same order ( blue , red , purple , black ) regardless of phase type ( alone or group ) . Participants in all of the conditions were given a group practice session with the \u201c al - ternate uses of a paper clip \u201d topic , even though those in the Alone con - dition would be performing alone , to ensure that all conditions had similar training . For the brainstorming session , participants were asked to generate ideas to the \u201c thumbs problem \u201d\u2014 the implications of having an extra thumb on one ' s hand . At the end of the session , partic - ipants completed a questionnaire about the task and their performance . The number of non - redundant ideas for each phase was used as the dependent variable . This was the only performance measure obtained in this experiment . We also obtained responses to a post - experimental questionnaire . This included items that asked participants to rate their performance in the different phases . However , since only the hybrid condition involved different phases , the items were not comparable across the conditionsand thus werenot analyzed . In thesubsequent ex - periment , all of the other measures obtained and all of the conditions employed are reported as well as any exclusion of participants . 2 . 2 . Results The data were analyzed at the group level using a 4 ( Condition ) \u00d7 4 ( Phases ) mixed ANOVA ( refer to Table 1 for descriptive statistics ) . The results showed that there was a signi \ufb01 cant main effect of condition , F ( 3 , 58 ) = 2 . 854 , p = 0 . 045 , \u03b7 p2 = 0 . 129 . Participants in the AGAG con - ditiongeneratedmore ideasthanthoseinthegroupcondition , although this difference was marginally signi \ufb01 cant ( p = 0 . 056 ) . The other condi - tions were not signi \ufb01 cantly different from one another . Thus , there was only partial support for H1 and no support for H2a or H2b . There was also a signi \ufb01 cant main effect of phases , F ( 3 , 174 ) = 99 . 365 , p b 0 . 001 , \u03b7 p2 = 0 . 631 . Signi \ufb01 cantly more ideas were generated in Phase 1 across all conditions as compared to phases 2 , 3 and 4 ( p b 0 . 001 ) . A signi \ufb01 cant interaction effect wasalso observed , F ( 9 , 174 ) = 13 . 008 , p b 0 . 001 , \u03b7 p 2 = 0 . 402 . This is primarily due to the elevation of the performance in the alone sessions of the hybrid conditions that followed the group sessions in support of H3a . ( see Fig . 1 ) . Pairwise comparisons using the Bonferroni adjustment indicated that quantity was signi \ufb01 cantly higher in Phase 1 of the AGAG condition as compared to the GAGA ( mean dif - ference = 10 . 113 , SE = 3 . 247 , p = 0 . 017 ) and Group conditions ( mean difference = 11 . 008 , SE = 3 . 247 , p = 0 . 008 ) . For phase 2 , the perfor - mance in the GAGA condition was signi \ufb01 cantly higher than the AGAG ( meandifference = 8 . 804 , SE = 3 . 145 , p = 0 . 042 ) andGroup ( meandif - ference = 12 . 312 , SE = 3 . 094 , p = 0 . 001 ) conditions . In phase 3 , signif - icantly more ideas were generated in the AGAG condition as compared to the GAGA ( mean difference = 11 . 479 , SE = 3 . 417 , p = 0 . 008 ) and Group ( mean difference = 12 . 729 , SE = 3 . 417 , p = 0 . 003 ) conditions . There were signi \ufb01 cantly more ideas in the phase 4 of the GAGA condi - tion as compared to the same phase of the Group condition ( mean dif - ference = 10 . 938 , SE = 2 . 979 , p = 0 . 003 ) . More ideas were generated in phase 3 of the AGAG condition as compared to the same phase of the Alone condition , but this difference was only marginally signi \ufb01 cant , t ( 58 ) = 2 . 592 , p = 0 . 072 . A speci \ufb01 c planned comparison of the com - bined quantity in the alone phases ( Phases 2 and 4 ) of the GAGA condi - tion was signi \ufb01 cantly higher than the combined quantity of the corresponding phases in the Alone condition F ( 2 , 58 ) = 4 . 860 , p = 0 . 011 , \u03b7 p2 = 0 . 144 , insupportofH3a . Contrarytotheblocking / constraint perspective ( H4 ) , the extent to which performance was lower in the \ufb01 rst group phase of the AGAG condition relative performance in the prior alone phase was negatively related to performance in the 180 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 subsequentalone phase , b = \u2212 0 . 383 ( CI \u2212 0 . 748 , \u2212 0 . 017 ) , SE = 0 . 169 , t ( 13 ) = \u2212 2 . 261 , p = 0 . 042 . 2 . 3 . Discussion As in the preliminary study , there was an elevation in performance during the alone phases that followed the group phases of the hybrid conditions . This pattern of results supports the prediction made on the basis the cognitive / associational models of collaborative ideation ( Brown & Paulus , 2002 ; Nijstad & Stroebe , 2006 ) . However , it could also re \ufb02 ect the enhancement of idea generation after constraint due to production blocking . As a result of the elevated performance during these phases the hybrid conditions performed better than the other two conditions , but these differences were not statistically signi \ufb01 cant . The results did not support the social comparison perspective since there was not an elevation of performance in the group phases after the alone phases in the hybrid conditions in comparison to the compa - rable phases in the group condition . The group condition did not generate signi \ufb01 cantly more ideas than the alone condition . This is consistent with the \ufb01 ndings of Goldenberg et al . ( 2013 ) who also found no differences in quantity when slips of paper were used to write the ideas instead of a single sheet of paper . This result may seem to indicate that generating ideas in a group has no bene \ufb01 t as compared to generating ideas individually . When partici - pants are working in a group , their attention and time is divided be - tween generating their own ideas and reading others ' ideas . This may lead to distraction and some production loss ( Santanen , Briggs , & de Vreede , 2004 ; Valacich , Dennis , & Connolly , 1994 ) . In fact , in the \ufb01 rst alone phase of the AGAG condition was signi \ufb01 cantly higher than the \ufb01 rst group phases in the Group and GAGA condition . However , this re - sult does not necessarily indicate a lack of stimulation in the group phases . The effect of stimulation from group ideation can be seen more clearly when comparing within the conditions rather than across the conditions . For the hybrid conditions , the data clearly show an in - crease in ideas in the alone phases after the group phases , whereas the number of ideas in the nominal condition does not increase at any point during the session . Thus , this may re \ufb02 ect the bene \ufb01 t of group stimulation ( Dugosh et al . , 2000 ; Paulus & Brown , 2007 ; Paulus & Yang , 2000 ) and this bene \ufb01 t is only visible after the group phase . The key is to allow the participants to work individually after a group phase so that they may utilize the group derived stimulation more effectively . Another interesting \ufb01 nding from Experiment 1 is that the number of ideas generated by participants in the alone condition drops consider - ably from phase 1 to phase 2 . But after phase 2 , the rate of idea genera - tion stays almost constant until the end . Prior research with verbal brainstorming has found a more consistent decline in the performance of the alone participants over time so that the difference in quantity of ideas between the nominal and interacting groups almost disappears ( Paulus & Dzindolet , 1993 , Experiment5 ) . More of adeclineinthenum - ber of ideas was expected from the nominals in Experiment 1 because they received no ideas from other participants in the 32 - minute long session . It is possible that participants continued to generate ideas due to the motivating effects of the social cues present . When participants generated ideas and kept the slips to their side , the stacks of slips ( but not the ideas ) were visible to the other participants . Previous research has shown that this social comparison of performance can lead to an in - crease in the number of ideas generated ( Dugosh & Paulus , 2005 ; Paulus , Larey , Putman , Leggett , & Roland , 1996 ) . If these participants were generating ideas without the social cues ( i . e . , without being able to see each other ) , their rate of ideation may have shown a steeper drop . If that is true , the decline should be evident in all of the alone phases regardless of condition \u2013 alone or hybrid . Isolating the group members for this phase in the hybrid condition may help determine if the previously seen increase in quantity of the alonephases that follow - ed the group phase was purely due to stimulation they received from the group phase or was in \ufb02 uenced by the effect of social cues from the alone phase . If participants in the hybrid condition are able to utilize the stimulation from the group phase , quantity in the alone phases of the AGAG condition should not drop as drastically as it might in the alone condition . The hybrid condition was designed to mimic the way in which idea - tion often occurs in the real world . Group meetings can occur a few times a week with time in between for individuals to generate more ideas and discuss them at the next meeting and so on . However , these individual brainstorming phases occur when the individual is away from other group members . The individual phases in Experiment 1 oc - curred in full view of the other group members . Therefore , it may be in - structive to examine the effect of the hybrid condition when the group members are separated or unable to view each other ' s performance during the alone phases . In Experiment 2 social cues were eliminated in the alone condition and alone phases of the hybrid condition , and we added another eight - minute phase to lengthen the session to enhance the chances of seeing a decline in the alone condition . This could result in the group condition matching or exceeding the performance of the alone condi - tion since alone participants could begin to run out of ideas without ex - ternal stimulation ( Dennis et al . , 2005 ; Nijstad , Stroebe , & Lodewijkx , 1999 ) . In Experiment 1 we only measured the number of ideas generat - ed since that has been the main measure of choice in this type of re - search . However , in the second experiment we added additional performance measures to obtain a richer set of data on the effects of hy - brid brainwriting and to allow for discrimination between the cognitive and blocking / constraint perspectives . Table 1 Experiment 1 : means and standard deviations of quantity by conditions and phases . Phase Alone AGAG GAGA Group Overall Phase 1 41 . 267 ( 11 . 931 ) 45 . 133 ( 9 . 257 ) 35 . 000 ( 7 . 874 ) 34 . 125 ( 6 . 386 ) 38 . 742 ( 9 . 924 ) Phase 2 27 . 267 ( 9 . 924 ) 26 . 133 ( 11 . 370 ) 34 . 938 ( 8 . 029 ) 22 . 625 ( 4 . 365 ) 27 . 774 ( 9 . 693 ) Phase 3 26 . 667 ( 10 . 661 ) 35 . 667 ( 12 . 715 ) 24 . 188 ( 8 . 727 ) 22 . 938 ( 4 . 057 ) 27 . 242 ( 10 . 527 ) Phase 4 24 . 133 ( 10 . 302 ) 22 . 333 ( 8 . 558 ) 30 . 438 ( 8 . 981 ) 19 . 500 ( 5 . 151 ) 24 . 129 ( 9 . 186 ) Overall 119 . 333 ( 39 . 776 ) 129 . 267 ( 35 . 692 ) 124 . 563 ( 29 . 003 ) 99 . 188 ( 15 . 285 ) \u2013 Note . Standard deviations ( SD ) are listed inparentheses . There were 15 groups in each condition . 15 20 25 30 35 40 45 50 Phase 1 Phase 2 Phase 3 Phase 4 U n i qu e I d e a s AGAG GAGA ALONE GROUP Fig . 1 . Number of unique of ideas generated in the different conditions across phases in Experiment 1 . 181 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 3 . Experiment 2 Experiment 2 compared a hybrid and a group condition to an alone condition , and group members were isolated in the alone condition as well as all alone phases . The overall procedure remained similar to the prior two experiments . The session time was increased from 32 min to 40 min by adding a \ufb01 fth phase . To prevent the participants from seeing each other , partitions were placed between participants during all the individual phases , across all the conditions . So only the group phases occurred in full view off all the group members without the partition . Since the participants would be isolated during the individual phases to eliminate both cogni - tive and social cues , it was expected that fewer ideas would be generat - ed in the alone condition as compared to the hybrid condition . The hybrid condition would receive some stimulation from the group phases , whereas the alone condition would not . The \ufb01 rst two hypotheses are similar to those of the prior experiments . H1 . More ideas will be generated in the hybrid condition as compared to the alone and group conditions . H2 . The alone phases that come after the group exchange in the hybrid condition will demonstrate an elevated level of idea generation com - pared to the same phases in the alone condition . For thisexperimentwe also developed hypotheses for theadditional measures of the novelty of ideas , their variety , and the depth of catego - ries explored . The number of categories ( variety ) is often but not neces - sarily correlated with number of ideas ( Deuja et al . , 2014 ) , average novelty is typically fairly independent of the number of ideas ( Paulus et al . , 2011 ) , but depth of categories ( number of ideas per category ) can be related both to enhanced novelty and quantity ( cf . , Nijstad , De Dreu , Rietzschel , & Baas , 2010 ; Rietzschel , Nijstad , & Stroebe , 2007 ) . For example , a number of studies have found that a focusingon onecat - egoryatatimeasanindividualoragroupenhances thenumber ofideas generated ( Baruah & Paulus , 2011 ; Coskun , Paulus , Brown , & Sherwood , 2000 ; Deuja et al . , 2014 ) . The dual pathway model of Nijstad et al . ( 2010 ) suggests that idea - tional creativity ( novelty ) involves both persistence ( category depth ) and \ufb02 exibility ( category variety ) pathways . 1 Both persistence and \ufb02 ex - ibility are important when generating ideas in a group . Since the hybrid condition has both individual and group phases , these factors become important in understanding cognitive processes involved in the hybrid condition . Persistence , thatis , focusingonacategoryanddiggingdeeper may be more feasiblewhen workingalone . The stimulation fromshared ideaswhenworkinginagroup should increasethe \ufb02 exibility ornumber of categories explored in the subsequent alone phases . Prior , research has typically found that as the number of ideas generated increases so do thenumberof categories ( e . g . , Kohn & Smith , 2010 ) . Based on there - sultsfromExperiment1 , theDeDreu , Nijstad , Baas , Wolsink , andRoskes ( 2012 ) \ufb01 ndings , and the Nijstad et al . ( 2010 ) dual pathway model , the following hypotheses are proposed : H3 . The hybrid condition should demonstrate enhanced depth and va - riety because . of the bene \ufb01 t of both group stimulation and private re \ufb02 ection time . H4 . The hybrid condition will have the highest novelty since partici - pants should beable toutilizeboth \ufb02 exibility and persistence pathways . The exposure to the ideas of others in the group phase of the hybrid condition should increase the awareness of a broader range of catego - ries and increase the extent to which participants generate ideas in a broader range of categories in the subsequent alone phase . H5 . The alone phases that follow the group sessions in the hybrid con - dition will show more category variety ( \ufb02 exibility ) than the corre - sponding phases of the alone condition . The generation of ideas is also related to the extent to which to which categories are tapped more deeply ( Baruah & Paulus , 2011 ; Deuja et al . , 2014 ) . H6a . We expect that the ideas in the alone phases that follow group sessions in the hybrid conditions will demonstrate greater depth than the same phases in the alone condition . Persistence or focusing on a category and digging deeper may be more feasible when working alone since the train of thought is not disrupted by exposure to the ideas of others . H6b . The alone phases will show more category depth than the group phases , regardless of condition . We will also analyze the extent to which the performance in one phase predicts performance in a subsequent phase . For the alone condi - tion intersession correlations should primarily re \ufb02 ect individual differ - ences in ability or motivation . These factors also should play a role in the other conditions . However , in the group condition the tendency for social comparison and normative in \ufb02 uence may lead to even stron - ger correlations among the sessions as in the Paulus and Dzindolet ( 1993 ) study . In the hybrid conditions , the correlations may be some - what lower since the contiguous sessions involve a change in the idea - tion process . Of primary interest will be the patterns in the hybrid condition and their theoretical implications . We would expect the quantity , variety , depth and novelty of the group phase to in \ufb02 uence thesamemeasures inthealonephasebecause of related cognitive stim - ulation and the carry - over of social in \ufb02 uence and entrainment effects from one phase to the other . If the enhanced performance in the alone phases that follow group phases were due to various cognitive process - es , one would expect a number of other relationships . The greater the variety of categories ( \ufb02 exibility ) experienced in the group phase , the better should be the performance in the alone phases because the cate - gories can prime new semantic domains ( e . g . , Coskun et al . , 2000 ) . The greater category depth in the group phase may increase the novelty of the ideas in the subsequent alone phase as well the number of ideas . Category depth has been associated increased novelty in alone and dy - adic conditions ( Nijstad et al . , 2010 ; Rietzschel et al . , 2007 ) . However , category depth has also been associated with an increase in the number of ideas generated since focusing on one idea at a time is an ef \ufb01 cient cognitive strategy ( Deuja et al . , 2014 ) . The novelty of ideas in the group phase may not enhance the number of ideas generated in the subsequent alone session since more common ideas may yield more as - sociations ( Dugosh & Paulus , 2005 ) . H7 . The performance in the alone phases after the group phases will be predicted by the quantity , variety and depth of ideas in the prior group phases . The blocking / constraint perspective is not well suited for making predictions about the different measures of performance since it in - volves a non - speci \ufb01 c motivational process . However , as suggested for Experiment 1 , the degree of constraint experienced in the \ufb01 rst group phase of AGAG condition could be related to enhanced performance in the following group phase . This did not occur in Experiment 1 , but we again assessed this possibility in Experiment 2 . 1 DeDreuetal . ( 2012 ) assessedtheserelationshipsusingindividualbrainstormingand the role of working memory . In experiment 3 , the effects of working memory capacity were tested on the alone , group , and hybrid conditions . Working memory was assessed using three different measures \u2013 OSPAN , RSPAN and WCST . Several mediation models and moderated regression analyses were conducted to examine the effects based on the dualpathwaymodel . TheresultsshowedthatonlylowerscoresonOSPAN ( lowerworking memory capacity ) signi \ufb01 cantly predicted higher novelty in the hybrid condition via the \ufb02 exibilitypathway . Scoresonpersonalityvariableslikeopennesstoexperience , extraver - sion , andperspectivetakingalsodidnothavesigni \ufb01 canteffectsonquantity , varietydepth , or novelty ( see Korde , 2014 ) . 182 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 H8 . If blocking / constraint plays a role , the performance in the second alone session will be positively related to the degree of constraint expe - rienced in the group phase in comparison to the prior alone phase . 3 . 1 . Method 3 . 1 . 1 . Participants ParticipantswererecruitedfromalargeuniversityintheSouthwest - ern U . S . and all participants received research credits to help complete requirements for their courses . If a complete group of three students ar - rived for the session , they were randomly assigned to either the group or hybrid brainstorming conditions . If fewer than three people arrived for the session , they were automatically assigned to the individual brainstorming condition . Data from three individual participants were pooled together to form a nominal group . A total of 270 students partic - ipated in the idea generation and data from all the participants were used for the analyses . The number of groups per condition was doubled in number compared to Experiment 1 since we anticipated needing ad - ditional power for planned path analyses . All of the data were gathered before the analyses of the data were begun . The mean age was 20 . 03 ( SD = 3 . 664 ) ; the youngest participant was 17 and the oldest was 62 years of age . 54 . 1 % of the sample consisted of females . 30 % of the sample was Hispanic , 28 . 5 % were Caucasian , 21 . 1 % Asian , 15 . 2 % African American , and the remaining 1 % comprised of Native Americans , Paci \ufb01 c Islanders and those who reported more than one race . Only one partic - ipant did not disclose their race . 3 . 1 . 2 . Design and procedure The experiment used a 3 ( Condition ) \u00d7 5 ( Phases ) mixed design . Participants were recruited to come tothe labingroups of threetocom - plete the brainstorming task . Similar to the prior experiments , brainwriting was the procedure of choice and slips of paper were used to write ideas . Each participant received a different set of colored of slips ( pink , white , or yellow ) to easily identify as their own . The ideas were written using a different colored pen for each phase ( blue , red , purple , black , and orange ) . Participants in all the conditions received a three - minute group practice session and topic was \u201c alternate uses of a paper clip \u201d . The thumbs problem was used again for the brainstorming task . Participants sat at a table , facing each other . A partition was placed betweenthoseparticipants thatwere starting the \ufb01 rst phaseindividual - ly . Those in the alone condition continued the remaining phases with the partitions in place , stopping every 8 min for a change of pens . Each phase lasted for 8 min just as the previous experiments . The parti - tionwasremovedfortheparticipantsinthehybridconditionjustbefore every group phase . All the phases for the group condition took place without the partition . At the end of the session the participants were asked to complete a post - experiment questionnaire and were debriefed . 3 . 1 . 3 . Dependent variables The dependent variables obtained for this experiment were quanti - ty , novelty , category depth , and category variety of the ideas . The scores on all of these variables were calculated for each phase within each group . The operational de \ufb01 nitions of these variables are as follows : \u2022 Quantity : Total number of non - redundant ideas . Ideas that were re - peated across phases and within a phase were considered as redun - dant and were not counted . Ideas were counted and checked for redundancy by two trained raters . Both raters counted all the ideas for all the sessions and removed redundant ideas by consensus . \u2022 Novelty : Novelty was determined based on the infrequency of the ideas . Ideas that were produced frequently by many participants were considered less novel . Whereas ideas that were rarely produced by participants were rated as novel . Two trained raters rated novelty of the ideas independently . The ideas were rated on a scale of 1 to 5 , where one meant \u201c very common \u201d and \ufb01 ve stood for \u201c very uncom - mon \u201d . \u2022 Categoryvariety ( Flexibility ) : Ideaswerealsosortedintodifferentcat - egories . Using previous data , 24 categories were developed for the thumbs problem ( Korde , 2014 ) . Some of those categories were so - cial / discrimination , sports , gestures , etc . The total number of catego - ries explored by each participant and by the group was recorded . \u2022 Category depth ( Persistence ) : For each category that was explored by aparticipant , thetotalnumberof ideasgeneratedwithinthatcategory andtheaveragenumberof ideaspercategorywascalculatedbydivid - ing quantity for the phase by the corresponding category variety score . An overall category depth score was also calculated for each group . As inthecase ofExperiment1 , thequestionnaire responses were not analyzed since the items were not designed to tap the processes rele - vant to the hybrid condition . All of the other measures obtained in this experiment and all conditions employed are reported as well as any ex - clusion of participants . 3 . 1 . 4 . Coding of ideas Data from the brainstorming session were screened to eliminate re - dundant ideas by consensus . The ideas were coded for novelty and cat - egories by two independent raters . Both raters coded the ideas blind to the conditions and phases . Each rater coded 25 % of the ideas for novelty on a scale of oneto \ufb01 ve as described earlier . Scoresthat were within one point of each other were considered as indicators of inter - rater agree - ment and the ratings were adjusted for analysis ( Diehl & Stroebe , 1991 ; Kohn , Paulus , & Choi , 2011 ) . Inter - rater reliability for the novelty ratings on 25 % of the data was calculated using ICCs resulting in a value of 0 . 834 . One rater then coded the remainder of the data for novelty . Bothratersalsocoded 25 % of thedata for categories . Theratersinde - pendently assigned one of 24 categories to each idea . Cohen ' s Kappa was used to test inter - rater reliability and the resulting value was 0 . 808 . The second rater coded the remaining 75 % of data for categories . To calculate category variety , the number of non - redundant categories explored within each phase was counted . Additionally , the number of non - redundant categories explored throughout the entire session was counted . Therefore , each group had six separate category variety scores \u2013 one for each phase and one overall for the entire session . Category depth was computed by dividing the number of non - redundant ideas by category variety for the corresponding phase and / or session . 3 . 2 . Results The data were analyzed at the group level . Examination of the corre - lations among the variables for the three conditions combined showed that all four measures were signi \ufb01 cantly correlated with one another . Quantity was signi \ufb01 cantly correlated with the remaining three mea - sures ( variety : 0 . 509 , p b 0 . 001 ; depth : 0 . 957 , p b 0 . 001 ; novelty : 0 . 494 , p b 0 . 001 ) . Variety was signi \ufb01 cantly correlated with depth ( 0 . 251 , p = 0 . 017 ) and novelty ( 0 . 407 , p b 0 . 001 ) , and depth was signif - icantly correlated with novelty ( 0 . 427 , p b 0 . 001 ) . Even though quantity and depth were highly correlated , given their distinct theoretical impli - cations , we included both of these measures in our various analyses . 3 . 2 . 1 . Quantity Four separate 3 ( Condition ) \u00d7 5 ( Phases ) mixed ANOVAs were used to test the differences among conditions and phases on each of the four dependent variables \u2013 quantity , novelty , category variety , and category depth ( refer to Table 2 for descriptive statistics ) . The ANOVA for quanti - ty yielded a signi \ufb01 cant main effect of condition , F ( 2 , 87 ) = 3 . 430 , p = 0 . 037 , \u03b7 p 2 = 0 . 073 . Pairwise comparisons using the Bonferroni adjust - ment indicated that the hybrid condition had the highest mean number of ideas ( M = 37 . 407 , SE = 1 . 962 ) , followed by the alone condition 183 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 ( M = 34 . 980 , SE = 1 . 962 ) , and then the group condition ( M = 30 . 260 , SE = 1 . 962 ) . However , the hybrid condition was only signi \ufb01 cantly dif - ferent from the group condition , p = 0 . 035 . The other differences be - tween conditions were not signi \ufb01 cant . H1 was only partially supported . There was also a main effect of phases , F ( 4 , 348 ) = 86 . 179 , p b 0 . 001 , \u03b7 p 2 = 0 . 498 . As expected , the most ideas were generated in the \ufb01 rst phase and quantity declined as the phases progressed . Phase 1wassigni \ufb01 cantlydifferentthanalltheotherphases ( p b 0 . 001 ) . Thein - teraction between condition and phasewasalso signi \ufb01 cant , F ( 8 , 348 ) = 8 . 829 , p b 0 . 001 , \u03b7 p 2 = 0 . 169 ( see Fig . 2 ) . Signi \ufb01 cantlymore ideaswere in generated intheAGAG conditionascompared tothe Group conditionin phase 1 ( mean difference = 9 . 433 , SE = 3 . 000 , p = 0 . 007 ) , phase 3 ( mean difference = 11 . 500 , SE = 2 . 964 , p = 0 . 001 ) and phase 5 ( mean difference = 12 . 067 , SE = 3 . 423 , p = 0 . 002 ) . The within condi - tion comparisons showed enhanced performance in the alone phases after the group phases in the hybrid condition ( see Fig . 2 ) . The com - binedquantityofphase3andphase5wassigni \ufb01 cantlyhigherinthehy - brid condition than the alone condition , F ( 1 , 87 ) = 4 . 590 , p = 0 . 035 , \u03b7 p2 = 0 . 050 . These results support H2 . Quantity in the Group condition was highest in phase 1 ( phase 2 : mean difference = 6 . 0 33 , SE = 1 . 297 , p b 0 . 001 ; phase 3 : mean difference = 9 . 033 , SE = 1 . 360 , p b 0 . 001 ; phase 4 : mean difference = 8 . 800 , SE = 1 . 547 , p b 0 . 001 ; phase 5 : mean difference = 12 . 167 , SE = 1 . 749 , p b 0 . 001 ) . Quantity in phase 2 was also signi \ufb01 cantly higher than the quantity in phase 5 ( mean difference = 6 . 133 , SE = 1 . 609 , p = 0 . 003 ) . 3 . 2 . 2 . Categories Testing for differences in the number of categories explored or cate - gory variety yielded a main effect of condition , F ( 2 , 87 ) = 7 . 849 , p = 0 . 001 , \u03b7 p2 = 0 . 153 . The group condition explored fewer categories than the alone ( mean difference = \u2212 1 . 413 , SE = 0 . 466 , p = 0 . 010 ) and hy - brid conditions ( mean difference = \u2212 1 . 733 , SE = 0 . 466 , p = 0 . 001 ) . However , since the alone and hybrid conditions did not differ signi \ufb01 - cantly from each other , there was only partial support for H3 . There was a main effect of phases , F ( 4 , 348 ) = 19 . 641 , p b 0 . 001 , \u03b7 p2 = 0 . 184 ; category variety decreased from phase 1 to phase 5 . Variety in phase 1 was signi \ufb01 cantly higher than variety in the remaining phases ( Phase 2 : p = 0 . 003 ; Phase 3 , 4 , and 5 : p b 0 . 001 ) . The interaction effect of conditions and phases was signi \ufb01 cant , F ( 8 , 348 ) = 2 . 846 , p = 0 . 004 , \u03b7 p2 = 0 . 061 in support of H5 ( see Fig . 3 ) . Even though category variety Table 2 Experiment 2 : means and standard deviations of quantity , novelty , category variety and category depth by conditions and phases . Condition Quantity ( SD ) Novelty ( SD ) Variety ( SD ) Depth ( SD ) Alone Phase 1 44 . 433 ( 10 . 190 ) 2 . 356 ( 0 . 265 ) 14 . 967 ( 2 . 008 ) 2 . 965 ( 0 . 532 ) Phase 2 35 . 500 ( 10 . 582 ) 2 . 727 ( 0 . 315 ) 13 . 900 ( 2 . 369 ) 2 . 550 ( 0 . 633 ) Phase 3 34 . 633 ( 11 . 263 ) 2 . 777 ( 0 . 258 ) 13 . 067 ( 2 . 728 ) 2 . 646 ( 0 . 687 ) Phase 4 30 . 800 ( 11 . 223 ) 2 . 920 ( 0 . 292 ) 12 . 067 ( 2 . 392 ) 2 . 551 ( 0 . 702 ) Phase 5 29 . 533 ( 12 . 586 ) 2 . 911 ( 0 . 298 ) 11 . 900 ( 2 . 695 ) 2 . 470 ( 0 . 894 ) Overall 174 . 900 ( 49 . 700 ) 2 . 738 ( 0 . 209 ) 21 . 100 ( 1 . 845 ) 8 . 235 ( 2 . 072 ) AGAGA Phase 1 46 . 900 ( 13 . 422 ) 2 . 250 ( 0 . 216 ) 14 . 667 ( 2 . 249 ) 3 . 179 ( 0 . 636 ) Phase 2 33 . 567 ( 9 . 964 ) 2 . 556 ( 0 . 311 ) 13 . 533 ( 2 . 161 ) 2 . 467 ( 0 . 580 ) Phase 3 39 . 933 ( 11 . 756 ) 2 . 893 ( 0 . 343 ) 13 . 967 ( 2 . 646 ) 2 . 857 ( 0 . 654 ) Phase 4 29 . 267 ( 11 . 298 ) 3 . 009 ( 0 . 441 ) 11 . 900 ( 2 . 551 ) 2 . 440 ( 0 . 750 ) Phase 5 37 . 367 ( 14 . 829 ) 3 . 229 ( 0 . 970 ) 13 . 433 ( 2 . 208 ) 2 . 764 ( 0 . 920 ) Overall 187 . 033 ( 55 . 333 ) 2 . 787 ( 0 . 367 ) 21 . 233 ( 1 . 832 ) 8 . 770 ( 2 . 263 ) Group Phase 1 37 . 467 ( 11 . 004 ) 2 . 193 ( 0 . 240 ) 12 . 700 ( 2 . 037 ) 2 . 928 ( 0 . 653 ) Phase 2 31 . 433 ( 11 . 464 ) 2 . 762 ( 0 . 376 ) 12 . 000 ( 3 . 118 ) 2 . 645 ( 0 . 826 ) Phase 3 28 . 433 ( 11 . 416 ) 2 . 929 ( 0 . 387 ) 11 . 333 ( 2 . 412 ) 2 . 465 ( 0 . 752 ) Phase 4 28 . 667 ( 12 . 408 ) 3 . 079 ( 0 . 358 ) 11 . 500 ( 2 . 446 ) 2 . 459 ( 0 . 838 ) Phase 5 25 . 300 ( 12 . 200 ) 3 . 152 ( 0 . 464 ) 11 . 300 ( 2 . 842 ) 2 . 211 ( 0 . 978 ) Overall 151 . 300 ( 56 . 079 ) 2 . 823 ( 0 . 306 ) 20 . 067 ( 2 . 132 ) 7 . 504 ( 2 . 520 ) Phase 1s combined 43 . 202 ( 11 . 968 ) 2 . 269 ( 0 . 248 ) 14 . 157 ( 2 . 281 ) 3 . 036 ( 0 . 604 ) Phase 2s combined 33 . 685 ( 10 . 613 ) 2 . 687 ( 0 . 117 ) 13 . 157 ( 2 . 696 ) 2 . 567 ( 0 . 677 ) Phase 3s combined 34 . 539 ( 12 . 208 ) 2 . 871 ( 0 . 335 ) 12 . 843 ( 2 . 763 ) 2 . 663 ( 0 . 710 ) Phase 4s combined 29 . 753 ( 11 . 504 ) 3 . 004 ( 0 . 372 ) 11 . 865 ( 2 . 427 ) 2 . 491 ( 0 . 758 ) Phase 5s combined 30 . 899 ( 14 . 028 ) 3 . 100 ( 0 . 655 ) 12 . 225 ( 2 . 733 ) 2 . 493 ( 0 . 948 ) Alone phases 37 . 387 ( 6 . 128 ) 2 . 758 ( 0 . 319 ) 13 . 496 ( 1 . 115 ) 2 . 748 ( 0 . 242 ) Group phases 30 . 591 ( 3 . 978 ) 2 . 811 ( 0 . 339 ) 12 . 038 ( 0 . 819 ) 2 . 516 ( 0 . 221 ) Note . Standard deviations ( SD ) are listed inparentheses . There were 30 groups in each condition . 15 20 25 30 35 40 45 50 Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 U n i q u e I d e a s Alone AGAGA Group Fig . 2 . Number of unique of ideas generated in the different conditions across phases in Experiment 2 . 8 10 12 14 16 18 20 Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 C a t e g o r y V a r i e t y Alone Hybrid Group Fig . 3 . Number of categories explored in the different conditions across phases in Experiment 2 . 184 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 declined over time , the pattern was different in the hybrid condition as compared to the alone and group conditions ( see Fig . 3 ) . There was a sharp increase in category variety for the hybrid condition in the third and \ufb01 fth phase . These were the individual brainstorming sessions that occurred right after group brainstorming suggesting that they were ex - posedtonewerormorecategoriesduringthegroupsession . Thedepen - dent t - test yielded a signi \ufb01 cant difference among these phases , t ( 29 ) = 4 . 050 , p b 0 . 001 . Combined variety from phase3 and phase5 wassignif - icantly higher in the hybrid condition than the alone condition , F ( 1 , 87 ) = 4 . 697 , p = 0 . 033 , \u03b7 p2 = 0 . 051 , in support of H5 . 3 . 3 . Category depth The analysis for category depth revealed no main effect of condition , F ( 2 , 87 ) = 0 . 790 , p = 0 . 457 , \u03b7 p2 = 0 . 018 , contrary to H3 . However , there was a main effect of phases , F ( 4 , 348 ) = 20 . 668 , p b 0 . 001 , \u03b7 p2 = 0 . 192 . The category depth decreased from one phase to the next as the session progressed . Category depthin phase1 wassigni \ufb01 cantly higher than cat - egory depth in all the other phases ( p b 0 . 001 ) . The other differences were not signi \ufb01 cant . The interaction between phase and condition on category depth was signi \ufb01 cant , F ( 8 , 348 ) = 3 . 201 , p = 0 . 002 , \u03b7 p2 = 0 . 069 ( see Fig . 4 ) . Similar to the pattern of interaction seen for category variety and consistent with H6a , category depth increased in Phase 3 and Phase 5 of the hybrid condition , after the group brainstorming phase . However , a planned contrast of the combined depth in Phase 3 and Phase 5 showed that those phases in the hybrid condition were not signi \ufb01 cantly different from those in the alone condition , F ( 1 , 87 ) = 1 . 709 , p = 0 . 195 , \u03b7 p2 = 0 . 019 . It was also suggested that category depth could be greater in the alone phases as compared to the group phases , regardless of condition ( H6b ) . Two t - tests were used to test this hypothesis \u2013 one independent t - test and one dependent t - test . The independent t - test compared the alonecondition to the groupcondition , whilethedependent t - test com - paredthealonephasestogroupphaseswithinthehybridcondition . The independent t - test showed that there was no signi \ufb01 cant difference in category depth between the alone and group conditions , t ( 58 ) = 1 . 228 , p = 0 . 224 . The dependent t - test for the hybrid condition showed that the alone phases had signi \ufb01 cantly higher category depth than the group phases , t ( 29 ) = 5 . 776 , p b 0 . 001 . Therefore , H6b was partially supported . Categorydepthwashigherinthealonephase , butonlywith - inthehybridcondition . H3was alsopartiallysupportedsincethehybrid conditiondemonstrated signi \ufb01 cantlyhighervariety butnot asigni \ufb01 cant increase in category depth as compared to the alone condition . 3 . 4 . Novelty AnANOVA tested fordifferences innovelty of theideasacrosscondi - tions and phases . There was no signi \ufb01 cant effect of condition , F ( 2 , 87 ) = 0 . 604 , p = 0 . 549 , \u03b7 p2 = 0 . 014 . Thus H4 was not supported . How - ever , there was a main effect of phases , F ( 4 , 348 ) = 102 . 329 , p b 0 . 001 , \u03b7 p2 = 0 . 540 . Novelty of the ideas increased as the session progressed , leading to highest novelty in the fourth and \ufb01 fth phases . Novelty in Phase 5 was signi \ufb01 cantly higher than novelty in Phase 1 and Phase 2 ( p b 0 . 001 ) , and Phase 3 ( p = 0 . 001 ) . There was an interaction effect of condition and phase on novelty of the ideas , F ( 8 , 348 ) = 3 . 972 , p b 0 . 001 , \u03b7 p2 = 0 . 084 ( see Table 2 ) . In the \ufb01 rst phase , participants in the alone condition generated ideas with signi \ufb01 cantly higher average novelty than participants in the group condition ( mean difference = 0 . 163 , SE = 0 . 062 , p = 0 . 032 ) . By the \ufb01 fth phase , the hybrid and group conditions showed higher novelty than the alone condition , con - sistent with \ufb01 ndings of Baruah and Paulus ( 2016 ) . However , the com - parison was not signi \ufb01 cant ( hybrid \u2013 p = 0 . 176 ; group \u2013 p = 0 . 450 ) . None of the other comparisons were signi \ufb01 cant . 3 . 5 . Mediation model It was expected that the hybrid condition would have the highest novelty since participants would be able to use both \ufb02 exibility and per - sistence ( H4 ) . Based on the previously discussed results of the ANOVA , there was no signi \ufb01 cant main effect of condition on novelty . Even so , the mediation model was examined to test the second part of the hy - pothesis . Since the independent variable ( condition ) was a categorical variable with three levels , two dummy codes ( k - 1 ) were created and the alone condition was used as a reference group ( Hayes & Preacher , 2014 ) . The \ufb01 rst dummy code ( d 1 ) indicated the hybrid condition and the second ( d 2 ) indicated the group condition . Category depth and vari - ety were centered on the mean before they were added to the model . The \u201c create a new estimand \u201d command in AMOS was used to produce each of the speci \ufb01 c indirect effects and the total indirect effect for each dummy coded condition . The chi - square for the base model ( Fig . 5 ) was signi \ufb01 cant , \u03a7 2 ( 3 ) = 12 . 846 , p = 0 . 005 . The GFI ( 0 . 947 ) and AGFI ( 0 . 735 ) were below the cut off , as was the CFI ( 0 . 856 ) . The RMSEA was 0 . 192 ( CI : 0 . 093 , 0 . 305 ) and signi \ufb01 cantly different than zero ( p = 0 . 013 ) . Based on thesevalues the base model was not consid - ered a good \ufb01 t to the data . The modi \ufb01 cation indices suggested that a path was needed from d 2 to novelty , i . e . the direct effect of the group condition on novelty . Theoretically , condition can predict novelty ; but there is no reason to expect that only the group condition should have an effect on novelty . A second model was tested after adding direct paths from both indicator variables for the condition to novelty , and the chi - square was still signi \ufb01 cant , \u03a7 2 ( 1 ) = 3 . 927 , p = 0 . 048 ( Fig . 6 ) . The GFI ( 0 . 983 ) was above the cut off , but the AGFI was not ( 0 . 745 ) . The CFI ( 0 . 957 ) was above the cut off but the RMSEA ( 0 . 181 , CI : 0 . 016 , 0 . 384 ) was marginally signi \ufb01 cant ( p = 0 . 073 ) . The results showed that both models were not a very good \ufb01 t , but the second model had slight better values based on the \ufb01 t indices . Additionally , the AIC of the second model with the direct paths was lower ( 31 . 927 ) than the AIC of the base model ( 36 . 846 ) . Therefore , parameter estimates were examined from the second model . Category depth ( b = 0 . 050 , CI : 0 . 026 , 0 . 068 , SE = 0 . 010 , p = 0 . 001 ) and category variety ( b = 0 . 057 , CI : 0 . 037 , 0 . 081 , SE = 0 . 011 , p b 0 . 001 ) both predicted novelty signi \ufb01 - cantly . The a paths , total paths , direct paths , and indirect paths are de - scribed separately for each indicator variable . 3 . 5 . 1 . Hybrid condition The hybrid condition did not signi \ufb01 cantly predict category depth ( b = 0 . 535 , CI : \u2212 0 . 594 , 1 . 609 , SE = 0 . 561 , p = 0 . 348 ) or variety ( b = 0 . 133 , CI : \u2212 0 . 724 , 1 . 110 , SE = 0 . 470 , p = 0 . 774 ) . The relative total effect of the hybrid condition on novelty was not signi \ufb01 cant ( b = 0 . 049 , CI : \u2212 0 . 090 , 0 . 207 , SE = 0 . 076 , p = 0 . 482 ) , but it was in the expected direction . A one - unit increase ( in this case from the alone condition to the hybrid condition ) was related to an increase in novelty but the relationship was not signi \ufb01 cant . The relative direct ef - fect ( b = 0 . 015 , CI : \u2212 0 . 103 , 0 . 170 , SE = 0 . 069 , p = 0 . 784 ) was also 1 1 . 5 2 2 . 5 3 3 . 5 4 Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 C a t e g o r y D e p t h Alone Hybrid Group Fig . 4 . Number of ideas exploredper category inthe differentconditionsacrossphases in Experiment 2 . 185 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 not signi \ufb01 cant . The relative speci \ufb01 c indirect effects of the hybrid condi - tion ( viadepth : b = 0 . 027 , CI : \u2212 0 . 027 , 0 . 088 , SE = 0 . 028 , p = 0 . 297 ; via category : b = 0 . 008 , CI : \u2212 0 . 047 , 0 . 061 , SE = 0 . 027 , p = 0 . 761 ) and the relative total indirect effect were also not signi \ufb01 cant ( b = 0 . 034 , CI : \u2212 0 . 053 , 0 . 122 , SE = 0 . 044 , p = 0 . 432 ) . The results did not support the 4th hypothesis \u2013 the hybrid condition did not signi \ufb01 cantly predict novelty via the persistence and \ufb02 exibility pathways . 3 . 5 . 2 . Group condition The group did not signi \ufb01 cantly predict category depth ( b = \u2212 0 . 731 , CI : \u2212 1 . 947 , 0 . 435 , SE = 0 . 602 , p = 0 . 220 ) , butitdidpredictcategoryva - riety ( b = \u2212 1 . 033 , CI : - 2 . 014 , \u2212 0 . 012 , SE = 0 . 515 , p = 0 . 048 ) . The rel - ative total effect was not signi \ufb01 cant ( b = 0 . 085 , CI : \u2212 0 . 048 , 0 . 221 , SE = 0 . 068 , p = 0 . 203 ) but the relative direct effect was signi \ufb01 cant ( b = 0 . 181 , CI : 0 . 079 , 0 . 291 , SE = 0 . 053 , p b 0 . 001 ) . A one - unit change ( from the alone condition to the group condition ) signi \ufb01 cantly predict - ed higher novelty when controlling for category depth and category va - riety . The relative speci \ufb01 c indirect effect via depth was not signi \ufb01 cant ( b = \u2212 0 . 036 , CI : \u2212 0 . 112 , 0 . 017 , SE = 0 . 032 , p = 0 . 184 ) , but it was sig - ni \ufb01 cant via variety ( b = \u2212 0 . 059 , CI : \u2212 0 . 128 , \u2212 0 . 003 , SE = 0 . 031 , p = 0 . 039 ) . The relative total indirect effect was also signi \ufb01 cant ( b = \u2212 0 . 096 , CI : \u2212 0 . 192 , \u2212 0 . 004 , SE = 0 . 048 , p = 0 . 041 ) . As com - pared to the control condition , the group condition predicted novelty as a result of the negative effect of the group condition on category variety . 3 . 6 . Additional analyses We also examined the within session correlations to determine whether there were unique patterns in the hybrid condition . Of partic - ular interest is how various measures of performance in the second and fourth group phases predictthe performance in thefollowingaloneses - sions . These results are presented in Tables 3 and 4 . In addition we per - formed regression analyses to determine the relation of the performance in the second and fourth sessions to the \ufb01 nal session ( Table 5 ) . In general the correlations were the strongest for the group condition . For the hybrid condition signi \ufb01 cant correlations and regres - sion effects occurred primarily for the quantity and depth predictors of the four variables , providing partial support for H7 . To examine the potential impact of constraint , we regressed the dif - ference between the \ufb01 rst alone and group session in the hybrid condi - tion on the performance in the third alone phase . The bigger the gap between performance in the alone and subsequent group phase ( lower performance ) , the greater the number of ideas generated in the next phase , b = 0 . 528 , SE = 0 . 232 , t = 2 . 273 , p = 0 . 031 . This same comparison for the other two conditions was not signi \ufb01 cant ( alone condition , b = \u2212 0 . 505 , SE = 0 . 312 , t = \u2212 1 . 616 , p = 0 . 117 ; Fig . 5 . Effect of condition on novelty using the dual - pathway model . The model shows the effect of the hybrid and group conditions as compared to the alone condition . Fig . 6 . Effectofconditiononnoveltyusingthedual - pathwaymodelwithdirectpaths . Themodelshowstheeffectofthehybridandgroupconditionsascomparedtothealonecondition . 186 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 group condition , b = \u2212 0 . 217 , SE = 0 . 408 , t = \u2212 0 . 530 , p = 0 . 600 ) . These results provide some evidence that the performance constraint experienced in the group phase of the hybrid condition motivated in - creased performance in the subsequent alone phase ( H8 ) . 4 . Discussion The results of Experiment 2 are consistent with those of Experiment 1 . The hybridcondition wasthebest in termsof number of ideasbut not signi \ufb01 cantlybetter thanthealonecondition . The mainbene \ufb01 t in thehy - brid condition appears in the alone phases after exposure to ideas of others . This pattern occurred for number of ideas , number of categories and depth of categories but was only signi \ufb01 cant for the within phase analyses in the case of the latter . In terms of number of ideasgenerated , thehybrid condition wassig - ni \ufb01 cantly better than thegroupcondition in the experiments in whicha group condition was employed . However , to demonstrate a synergistic bene \ufb01 t of hybrid ideation the hybrid condition needs to perform better than the alone condition . The trends in the three experiments ( includ - ing the preliminary study ) were in support of such an outcome . A meta - analysis was conducted to examine the difference in the hybrid andaloneconditions acrossthethreeexperiments . Themeansandstan - dard deviations were used to conduct the meta - analysis ( Borenstein , Hedges , Higgins , & Rothstein , 2005 ) . Across the three experiments the total samplesizefor thetwoconditionswas114 . The \ufb01 xedeffects statis - tics revealed a signi \ufb01 cant mean effect of 0 . 402 ( SE = 0 . 187 , 95 % CI = 0 . 036 / 0 . 768 ) , z = 2 . 154 , p = 0 . 013 . The degree of heterogeneity was not signi \ufb01 cant , Q ( 2 ) = 3 . 257 , p = 0 . 196 , indicating that the data from the three experiments were similar to each other . Since the bene \ufb01 cial impact was most evident in the alone phases after group interaction , we also did a meta - analytic comparison across the three experiments for the performance in the third alone phase of the AGAG conditions versus thealoneconditions . The \ufb01 xed effect statisticsshowed a main ef - fect of 0 . 6994 ( SE = 0 . 191 , 95 % CI = 0 . 326 / 1 . 072 ) , z = 3 . 672 , p = 0 . 0002 , providing strong support for the performance enhancement after group idea exchange . Thus a consistent pattern of results has been obtained across the three experiments in support of the hybrid ideation and its synergistic bene \ufb01 t relative to the alone condition . The bene \ufb01 t of hybrid ideation and the elevated performance in the alone phases of the hybrid conditions are consistent with the predic - tions based on the association models of group ideation ( Brown & Paulus , 2002 ; Nijstad & Stroebe , 2006 ) . This perspective suggests that group ideation generates associations that can be the basis for the gen - eration of additional ideas . Only some of these can be expressed in the group session , so the alone session provides an opportunity for the \u201c spreading activation \u201d of the associations to in \ufb02 uence the idea genera - tion in the alone session . The analyses of the relationships between the sessions within the three conditions indicated that the performance was most consistent over time for the group condition , with all four measures of perfor - mance in one phase predicting the performance on all four measures in the subsequent phase in most of the comparisons . This is consistent with the entrainment ( Kelly , 1988 ; Kelly & Karau , 1993 ) and social in - \ufb02 uence perspective ( Paulus & Dzindolet , 1993 ) in that performance Table 3 Experiment 2 . Phase 2 group performance correlations with Phase 3 alone performance . Conditions Alone Hybrid Group Phase 2 quantity Quantity 0 . 828 \u204e\u204e\u204e 0 . 731 \u204e\u204e\u204e 0 . 912 \u204e\u204e\u204e Novelty 0 . 568 \u204e\u204e\u204e 0 . 260 0 . 510 \u204e\u204e Flexibility 0 . 539 \u204e\u204e 0 . 431 \u204e 0 . 634 \u204e\u204e\u204e Depth 0 . 656 \u204e\u204e 0 . 551 \u204e\u204e 0 . 814 \u204e\u204e\u204e Phase 2 novelty Quantity 0 . 305 0 . 050 0 . 717 \u204e\u204e\u204e Novelty 0 . 457 \u204e 0 . 524 \u204e\u204e 0 . 679 \u204e\u204e\u204e Flexibility 0 . 116 0 . 332 0 . 606 \u204e\u204e\u204e Depth 0 . 269 \u2212 0 . 162 0 . 531 \u204e\u204e\u204e Phase 2 variety Quantity 0 . 366 \u204e 0 . 429 \u204e 0 . 559 \u204e\u204e Novelty 0 . 057 0 . 382 0 . 424 \u204e Flexibility 0 . 599 \u204e\u204e\u204e 0 . 269 0 . 481 \u204e\u204e Depth 0 . 054 0 . 314 0 . 453 \u204e Phase 2 depth Quantity 0 . 764 \u204e\u204e\u204e 0 . 618 \u204e\u204e\u204e 0 . 598 \u204e\u204e\u204e Novelty 0 . 642 \u204e\u204e\u204e 0 . 083 0 . 277 Flexibility 0 . 277 0 . 324 0 . 348 Depth 0 . 761 \u204e\u204e\u204e 0 . 494 \u204e\u204e 0 . 595 \u204e\u204e\u204e \u204e\u204e\u204e p b 0 . 001 . \u204e\u204e p b 0 . 01 . \u204e p b 0 . 05 . Table 4 Experiment 2 : Phase 4 group performance correlations with Phase 5 alone performance . Conditions Alone Hybrid Group Phase 4 quantity Quantity 0 . 921 \u204e\u204e\u204e 0 . 700 \u204e\u204e\u204e 0 . 953 \u204e\u204e\u204e Novelty 0 . 466 \u204e\u204e 0 . 099 0 . 625 \u204e\u204e\u204e Flexibility 0 . 348 0 . 290 0 . 661 \u204e\u204e\u204e Depth 0 . 858 \u204e\u204e\u204e 0 . 682 \u204e\u204e\u204e 0 . 732 \u204e\u204e\u204e Phase 4 novelty Quantity 0 . 558 \u204e\u204e 0 . 518 \u204e\u204e 0 . 595 \u204e\u204e\u204e Novelty 0 . 504 \u204e\u204e 0 . 515 \u204e\u204e 0 . 612 \u204e\u204e\u204e Flexibility 0 . 066 0 . 285 0 . 405 \u204e Depth 0 . 699 \u204e\u204e\u204e 0 . 456 \u204e 0 . 426 Phase 4 variety Quantity 0 . 447 \u204e 0 . 491 \u204e\u204e 0 . 543 \u204e\u204e Novelty 0 . 229 \u2212 0 . 085 0 . 292 Flexibility 0 . 167 0 . 198 0 . 568 \u204e\u204e\u204e Depth 0 . 394 \u204e 0 . 463 \u204e\u204e 0 . 326 Phase 4 depth Quantity 0 . 809 \u204e\u204e\u204e 0 . 545 \u204e\u204e 0 . 850 \u204e\u204e\u204e Novelty 0 . 430 \u204e 0 . 071 0 . 573 \u204e\u204e Flexibility 0 . 319 0 . 221 0 . 517 \u204e\u204e Depth 0 . 765 \u204e\u204e\u204e 0 . 549 \u204e\u204e 0 . 709 \u204e\u204e\u204e \u204e\u204e\u204e p b 0 . 001 . \u204e\u204e p b 0 . 01 . \u204e p b 0 . 05 . Table 5 Experiment 2 : Regression results for the relationship of performance in the two alone phases on the \ufb01 nal group phase for the hybrid condition . Conditions Alone Hybrid Group Ph2 & Ph4 quantity Ph5 quantity 78 . 139 \u204e\u204e\u204e 14 . 649 \u204e\u204e\u204e 143 . 522 \u204e\u204e\u204e Ph5 variety 2 . 157 1 . 58 10 . 613 \u204e\u204e\u204e Ph5 novelty 4 . 509 \u204e\u204e 0 . 581 10 . 366 \u204e\u204e\u204e Ph5 depth 41 . 760 \u204e\u204e\u204e 15 . 483 \u204e\u204e\u204e 17 . 520 \u204e\u204e\u204e Ph2 & Ph4 variety Ph5 quantity 4 . 157 \u204e 7 . 243 \u204e 8 . 727 \u204e Ph5 variety 0 . 643 0 . 563 10 . 790 \u204e\u204e\u204e Ph5 novelty 0 . 754 0 . 785 1 . 615 Ph5 depth 3 . 093 8 . 630 \u204e 2 . 001 Ph2 & Ph4 novelty Ph5 quantity 9 . 442 \u204e\u204e 5 . 729 \u204e 14 . 022 \u204e\u204e\u204e Ph5 variety 1 . 202 1 . 224 4 . 031 \u204e Ph5 novelty 4 . 883 \u204e 8 . 340 \u204e 15 . 254 \u204e\u204e\u204e Ph5 depth 11 . 912 \u204e\u204e\u204e 4 . 144 \u204e 5 . 078 \u204e Ph2 & Ph4 depth Ph5 quantity 35 . 905 \u204e\u204e\u204e 8 . 296 \u204e 35 . 672 \u204e\u204e\u204e Ph5 variety 1 . 594 0 . 805 6 . 307 Ph5 novelty 3 . 058 0 . 077 7 . 413 \u204e Ph5 depth 32 . 301 \u204e\u204e\u204e 7 . 956 \u204e 14 . 343 \u204e\u204e\u204e \u204e\u204e\u204e p b 0 . 001 . \u204e\u204e p b 0 . 01 . \u204e p b 0 . 05 . 187 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 becomes more consistent or normative over time in the social context than in solitary sessions . In the hybrid condition the quantity and depthmeasuresinthegroupphasesweretheprimarypredictorsofsub - sequent performance in the subsequent alone phases on all four mea - sures . The number of ideas being shared and the extent to which people dig deeply into speci \ufb01 c categories has been associated with in - creased creativity in groups in a number of studies ( e . g . , Dugosh & Paulus , 2005 ; Rietzschel et al . , 2007 ) . It now appears that these factors alsoaffecttheability tocontinuegeneratingideasinsubsequentsolitary sessions , in support of the cognitive models of group creativity ( Nijstad & Stroebe , 2006 ; Paulus & Brown , 2007 ) . Our research was framed in part in relation to the dual process the - ory of creativity ( Nijstad et al . , 2010 ) , and some of our \ufb01 ndings were consistent that that theory , such as the correlations among \ufb02 exibility , depth and novelty . However , the path models for the hybrid condition did not \ufb01 t with this particularperspective for predictingnovelty . The el - evation in performance in the hybrid condition re \ufb02 ected both enhance - ment of \ufb02 exibility and depth , but this effect does not require an explicit dual process of \ufb02 exibility and persistence . The semantic network model of Brown and Paulus ( 2002 ) has predicted this type of outcome based on a category transition matrix model ( Brown , et al . , 1998 ) . Thus indi - viduals in a group are exposed to ideas and categories of ideas , which will in turn in \ufb02 uence the subsequent ideation process . In generating ideas there is a tendency to stick in the same category until it has been somewhat depleted before moving on to other categories . So this model predicts that group exposure can increase both divergent ( \ufb02 exi - bility ) and convergentthinking ( depth ) . This model has been supported in a number of simulations of existing data ( Brown et al . , 1998 ; Coskun et al . , 2000 ) , and several simulations found evidence of a strong spike in performance of an alone session that followed a group session , includ - ing an increase in the number of categories in comparison to an alone only condition ( Brown & Paulus , 2002 ) . There was some support for the blocking constraint perspective in the hybrid condition as well . In contrast to the \ufb01 ndings of Experiment 1 , the more that the performance in the group phase was lower than that in the prior alone phase , the better was the performance in the alone phase that followed the group phase . These cases should have ex - perienced the most sense of constraint , which may have increased the motivation level when they were again \u201c free \u201d in the third session . How - ever , since the overall high performance of the second and fourth group phases was related to increased performance in the subsequent alone phases , constraint cannot be the sole factor . This pattern \ufb01 ts most clear - ly with the cognitive perspectives . However , our study was not de - signed a priori to clearly differentiate between the cognitive and constraint perspective . Additional research is required to evaluate the potential motivational effects of constraint in these types of paradigms . For example , the constraint effect mightbe observed even if theconsec - utive phases involve different problems . The cognitive perspective would not predict any elevations in performance in this case . Althoughwehavefoundenhancedperformanceinthehybridcondi - tion , the boundary conditionsforthis effectremain to be discovered . For example , how will it be affected by problems varying in dif \ufb01 culty ? The \u201c thumbs problem \u201d and other problems like generating ideas to improve a university are almost unlimited in the ideas that can be generated . They also have many different categories or types of ideas . Thus , ideas from others can be quite bene \ufb01 cial in suggesting new avenues for idea - tion . However , in more constrained problems with limited realistic op - tions ( such as generating ideas for reduced energy consumption ) , the potential stimulatingeffectof sharing ideaswithothers may be lessdra - matic . However , since one may quickly run of ideas on such a task , hav - ing input from others may be even more important . The length of thesession may also in \ufb02 uence the outcomes . As we in - dicated earlier , idea generation tends to slow down rather dramatically in a short period of time for most problems . Brief breaks appear to re - duce the pace of this decline ( Paulus , Nakui , Putman , & Brown , 2006 ) . The alone sessions served not only as breaks for the constraints of the demanding group exchange process but also provided an opportunity for unrestrained expression of ideas . Although there is a theoretical basis for predictingthepattern we observed , there are node \ufb01 nitive the - oretical bases for predicting the optimal length of the sessions . Shorter sessions may be too disruptive , but longer sessions may be too tiring . Having longer group sessions and shorter alone sessions may reduce the bene \ufb01 ts of the group - to - alone carry - over effects . However , if the alone sessions are too long relative to the group sessions , there may not be enough stimulation to have a strong impact on the alone sessions . The hybrid effect has implications for collaborative creativity in real world settings . It suggests that some balance of alone and group idea - tion will be ideal . Although we cannot provide precise suggestions as to the ideal length of the sessions , it is likely that having the alone ses - sion follow the group sessions immediately is best so as to not lose the associational potentialof thepriorgroupsession whichshould dissipate or decay over time ( Brown & Paulus , 2002 ) . EventhoughFTFsessions tend to reduce the numberof ideas generat - edingroupsrelativetootherwaysofsharingideas , thisisstillaverycom - monandpopularapproach . Thehybridapproachshouldalsobebene \ufb01 cial for enhancing the bene \ufb01 ts of FTF brainstorming . This could involve inter - spersing the verbal exchange sessions with ones in which participants generate ideas privately ( either in written form or electronically ) . Ideas generated in the alone sessions can be shared subsequently with the group and provide cues for additional ideas . This type of paradigm should increase the number of ideas generated relative to using only FTF brain - storming . However , for this type of paradigm to yield more ideas than nominal control condition may require longer sessions ( cf . , Nijstad et al . , 1999 ) in which those in the control conditions begin running out of ideas before those in the hybrid condition . Hybrid FTF sessions may also havesomeadvantagesoversolitarysessionsinotherareassuchasthede - velopment of group based skills ( Sutton & Hargadon , 1996 ) , facilitating subsequent decisions about which ideas to select for implementation , and producing higher levels of task enjoyment ( cf . , Paulus et al . , 1993 ) . The modi \ufb01 cations in Experiment 2 of reducing social cues in the alone condition and adding another phase did not appear to change the pattern of results among the conditions . Whether the participants in the alone condition work in full view of each other or in isolation doesnotseemtochangetheperformanceofthealoneconditionrelative to the other conditions ( in comparison to Experiment 1 ) . It was antici - patedthatprovidingextratimemightbene \ufb01 tthegroupandhybridcon - ditions relative to the alone condition . Nijstad et al . ( 1999 ) found that given enough time , group performance can achieve the level of individ - ual performance . That is , individuals and groups should start running out of ideas over time , but the cues provided in the group setting ( both the group and the hybrid conditions ) should enable them to con - tinue coming up with new ideas for a longer period of time . We did not \ufb01 nd results consistent with that expectation since the pattern of results in Experiment 2 wassimilarto that of Experiment1 . A substantially lon - gersessionmightberequireddemonstrateanenhancedperformanceof the group condition relative to the alone one and a stronger effect for the hybrid condition relative to the alone condition . There were no differences in novelty across the three conditions for Experiment 2 , but there was an effect of time . Average novelty of the ideas increased as the session progressed across all three conditions . These results are consistent with previous studies suggesting that the more common ideas are generated earlier in the session ( Kohn & Smith , 2010 ; Paulus , Kohn , Arditti , & Korde , 2013 ) . The interaction be - tween condition and time was also signi \ufb01 cant . The alone condition had the highest novelty at the beginning of the session and lowest nov - elty at the end of the session . But the patterns were different for the groupandhybrid conditions . Thus there is someevidencethatgroup in - teraction can enhance the novelty of idea generation , especially in the later phases of brainstorming sessions ( see also Baruah & Paulus , 2016 ) . The results from the mediation models echo the correlation and re - gressions patterns seen in Tables 3 - 5 . The correlations are stronger for 188 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190 the group condition , and predictions based on the dual pathway model apply better to group condition when usingthe alone condition as com - parison . Although it was expected that participants in the hybrid condi - tion would be able to utilizeboth pathways to attain novelty , the results indicate that the dual pathway model may not apply as well to the hy - brid condition since this condition involves alternatingbetween solitary and group ideation and results in generally lower correlations in perfor - mance from one phase to the other . The \ufb01 ndings , however , highlight that engaging in hybrid brainstorming entails processes that are differ - entfromaloneorgroupbrainstorminganddonot \ufb01 twiththedualpath - way model but allow for higher levels of performance . The fact that the \ufb02 exibility and persistence pathways were not related to novelty in the hybrid condition is consistent with that conclusion . In sum , the experiments have demonstrated the bene \ufb01 ts of hybrid brainwriting compared to alone and group brainwriting . This bene \ufb01 t was re \ufb02 ected in number of ideas and the number of categories sur - veyed . These effects were mostly due to the elevated performance dur - ing the alone sessions that followed the group sessions in the hybrid conditions . Apparently , the sharing of ideas in the group phases en - hanced both the \ufb02 exibility and but not the persistence and novelty for individuals in the subsequent alone phases . This series of studies is the \ufb01 rst to demonstrate the synergistic bene \ufb01 t of alternating alone and group ideation . Our research suggests in both short - term sessions and longer - term team interactions , the effective integration of group and solitary ideation is important for effectively tappinggroup ' s creative po - tential . Although hybrid ideation did not increase the novelty of the ideas , past research has found that an increase in the number of ideas is related to an increase in the number of ideas that are both novel and feasible ( e . g . , Paulus et al . , 2011 ) . Thus this pool of good ideas can then be the basis for developing the best ideas for potential implemen - tation ( Kohn et al . , 2011 ) . Acknowledgements The research reported in this paper was supported by collaborative grants to the second author from the National Science Foundation ( CreativeIT 0855825and INSPIREBCS 1247971 ) . Any opinions , \ufb01 ndings , and conclusions or recommendations expressed in this material are those of the authors and do not necessarily re \ufb02 ect the views of the Na - tional Science Foundation . This paper is based in part on a dissertation by the \ufb01 rst author under the direction of the second author . We would like to thank Tyler Hunt and Jaclyn West for their assistance in this research . Appendix A . Supplementary data Supplementary data to this article can be found online at http : / / dx . doi . org / 10 . 1016 / j . jesp . 2016 . 11 . 002 . References Amabile , T . M . ( 1996 ) . Creativity in context . Boulder , CO : West - view Press . Baruah , J . , & Paulus , P . B . ( 2008 ) . Effects of training on idea generation . Small Group Research , 39 ( 5 ) , 523 \u2013 541 . http : / / dx . doi . org / 10 . 1016 / j . jesp . 2011 . 04 . 007 . Baruah , J . , & Paulus , P . B . ( 2011 ) . Category assignment and relatedness in the group idea - tion process . 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Group processes and productivity . New York : Academic Press . Stokes , P . D . ( 2005 ) . Creativityfromconstraints : Thepsychologyofbreakthrough . NewYork , NY : Springer . Sutton , R . I . , & Hargadon , A . ( 1996 ) . Brainstorming groups in context : Effectiveness in a product design \ufb01 rm . Administrative Science Quarterly , 41 ( 4 ) , 685 \u2013 718 . http : / / dx . doi . org / 10 . 2307 / 2393655 . Taylor , D . W . , Berry , P . C . , & Block , C . H . ( 1958 ) . Does group participation when using brainstorming facilitate or inhibit creative thinking ? Administrative Science Quarterly , 3 , 23 \u2013 47 . http : / / dx . doi . org / 10 . 2307 / 2390603 . Valacich , J . S . , Dennis , A . R . , & Connolly , T . ( 1994 ) . Idea generation in computer - based groups : A new ending to an old story . Organizational Behavior and Human Decision Processes , 57 ( 3 ) , 448 \u2013 467 http : / / dx . doi . org . ezproxy . uta . edu / 10 . 1006 / obhd . 1994 . 1024 . 190 R . Korde , P . B . Paulus / Journal of Experimental Social Psychology 70 ( 2017 ) 177 \u2013 190", + "lundmark2008gtpaseactivating_supplement": "Current Biology Volume 18 1 Supplemental Data The GTPase - Activating Protein GRAF1 Regulates the CLIC / GEEC Endocytic Pathway Richard Lundmark , Gary J . Doherty , Mark T . Howes , Katia Cortese , Yvonne Vallis , Robert G . Parton , and Harvey T . McMahon Supplemental Experimental Procedures cDNA construct preparation cDNA constructs encoding human GRAF1 ( amino acids 1 - 759 ) , GRAF1 - BAR + PH ( amino acids 1 - 383 ) , GRAF1 PH + GAP ( amino acids 267 - 576 ) , and GRAF1 - SH3 ( 694 - 759 ) were amplified from IMAGE clone 30343863 using PCR and cloned into the pGEX - 4T - 2 vector for bacterial expression ( Amersham Biosciences ) . GRAF2 SH3 domain was amplified similarly from IMAGE clone 6188298 and cloned into the pGEX - 4T - 2 vector . Fragments were also cloned into the pCMVmyc vector with added Not1 site ( a kind gift from JGW Anderson ) or EGFP - C3 ( Clontech ) for mammalian expression . Amino acid substitutions K131E , K132E and R412D were created using PCR directed mutagenesis ( Stratagene ) . The GFP - tagged Cdc42 L61 consrtruct was a kind gift from M . F\u00e4llman . pEGFP - GPI was a generous gift from S . Mayor . caveolin1 - GFP and flotillin1 - GFP were kind gifts from Ben Nichols . Protein expression , protein purification and antibodies Recombinant proteins were expressed in a BL21 ( DE3 ) pLysS E - coli strain as Glutathione S - transferase ( GST ) - fusion proteins and purified using glutathione - Sepharose 4B beads ( Amersham Biosciences ) and gel filtration on a sephacryl S - 200 column ( Amersham ) as previously described [ 1 ] . Polyclonal antisera against GRAF1 were generated by immunising Rabbits ( Ra83 / Ab1 directed against the SH3 domain ; Ra84 / Ab2 directed against the PH + GAP domains ; and RaZ1 / Ab3 directed against the full length protein ; all of these were used for Western blotting analyses , and Ab1 / 3 also used for immunofluorescence analyses ) and a chicken ( CH - 9798 / Ab4 , directed against the full length protein , used for immunofluorescence analyses ) with recombinantly expressed human GRAF1 proteins . Purchased antibodies were : mouse anti - myc clone 9E10 , mouse anti - tubulin ( Sigma - Aldrich ) , Rabbit anti - myc ( Cell Signalling Technology ) , mouse anti - Dynamin , ( BD Transduction Laboratories ) , Rabbit anti - synaptojanin Ra59 [ 1 ] , ( Affinity Bioreagents ) . All secondary antibodies and streptavidins were conjugated to Alexa Fluor 488 , 546 or 647 ( Invitrogen ) . Expression , immunoprecipitation and pull down experiments For analysis of endogenous protein expression , cell lines were grown according to instructions from American Tissue Culture Collection , harvested and lysed in 1 % NP - 40 in PBS supplemented with protease inhibitors . After a 20 , 000g centrifugation the supernatant was analysed by SDS - PAGE and immunoblotting . For immunoprecipitation experiments , rat brain cytosol was generated by homogenization of rat brains in buffer ( 25mM HEPES , 150mM NaCl , 1mM DTT , 0 . 1 % Triton X - 100 and protease inhibitors ) , before centrifugation at 50 , 000rpm for 30 minutes at 4\u00b0C . The supernatant was removed and added to protein A Sephorose 4B beads ( Amersham Biosciensis ) to which antibodies had been previously bound and incubated at 4\u00b0C for 3 hours . Beads were washed three times in buffer ( 25mM HEPES , 150mM NaCl ) supplemented Current Biology Volume 18 2 with 1 % NP - 40 , and once in buffer without NP - 40 before analysis by SDS - PAGE combined with immunoblotting or Coomassie staining . Pull - down experiments against rat brain cytosol using purified proteins and identification by mass - spectrometry were performed as previously described [ 1 ] . Protein and lipid interaction assays Liposomes from total brain lipids ( FOLCH fraction I ) ( Sigma Aldrich ) or synthetic lipids ( Avanti Polar Lipids ) , and liposomes of a specified diameter or phosphoinositide enrichment were generated as previously described [ 2 ] . Liposome binding assays for lipid specificity and curvature sensitivity was performed as previously described [ 2 ] . Briefly , proteins were incubated together with liposomes followed by centrifugation and analysis of the pellet and supernatant by SDS - PAGE and Coomassie staining . In vitro liposome tubulation assays were performed and analysed as previously described [ 2 ] . Isothermal titration calorimetry The binding of synthetic peptides from Dynamin1 to purified GRAF1 SH3 domain was measured by isothermal titration calorimetry ( ITC ) using a VP - ITC ( MicroCal Inc . , USA ) . All experiments were performed in 100mM HEPES / NaOH pH 7 . 4 , 50mM NaCl and 2mM DTT at 10\u00b0C . Protein concentrations were determined by absorbance at 280nm . 1 . 36ml of 51\u00b5M GRAF1 SH3 domain was loaded into the cell . The peptides ( at 1mM which were custom - designed and manufactured at the Institute of Biomolecular Sciences , University of Southampton , UK ) were injected from a syringe in 5\u00b5l steps every 3 . 5 minutes . The heat of the dilution of the ligand was subtracted from the data prior to fitting . Titration curves were fitted to the data using the ORIGIN program ( MicroCal Inc . ) which yielded the stoichiometry ( ~ 1 ) , the binary association constant K a ( = K d - 1 ) and the enthalpy of binding . The entropy of binding ( \u2206 S\u00b0 ) was calculated from the relationship \u2206 G\u00b0 = - RT \u2022 ln ( K a ) and the Gibbs - Helmholtz equation . Cell culture and transfections HeLa cells were grown in RPMI 1640 or MEM media ( GIBCO ) supplemented with LGlutamine , 10 % foetal bovine serum , non - essential amino acids ( for MEM ) , and transfected using Genejuice ( Novagen ) for transient protein expression . For primary cultures , rat hippocampal neurons / astrocytes were prepared by trypsin digestion and mechanical trituration from E18 or P1 Sprague - Dawley rats and plated onto poly L - lysine coated coverslips . Cells were cultured in B27 - supplemented Neurobasal media . For GRAF1 depletion , HeLa cells were transfected with Stealth siRNAs specific against human GRAF1 using Lipofectamine 2000 ( Invitrogen ) according to manufacturers instructions . The Invitrogen siRNA duplex sequences used were siRNAa ( UUA UCU CCC AUU CAG CAC AGA UAU C / GAU AUC UGU GCU GAA UGG GAG AUA A ) , and siRNAb ( UUU GAA ACU GGU ACA UCA UGA GUG G / CCA CUC AUG AUG UAC CAG UUU CAA A ) . Cells were cultured for an additional 48 hours for efficient silencing of the GRAF1 expression . Stealth Block - it siRNA ( Invitrogen ) was used as a control . AP2 siRNA was used as previously described [ 3 ] . Caveolin - 1 knock - out mouse embryonic fibroblasts ( KO MEFs ) were generated and grown as previously described [ 4 ] . NIH 3T3 cells were cultured as per ECACC guidelines . Trafficking assays For immunofluorescence trafficking assays , biotinylated holo - transferrin , ( Sigma Aldrich ) , Alexa Fluor 647 - conjugated transferrin ( Invitrogen ) , Alexa Fluor 546 / 555 - conjugated CTxB ( Invitrogen ) , DiI ( Invitrogen ) , FITC - dextran ( 10kDa MW , used for fluorimetric uptake assay , Invitrogen ) , and biotinylated dextran ( 10kDa MW , used for immunofluorescent uptake assays , Invitrogen ) , were diluted in pre - warmed media , added to cells and incubated for time periods and temperatures as described in figure legends . After washing , cells were fixed and subjected to Current Biology Volume 18 3 immunofluorescence analysis as described below . For quantitative analysis of dextran endocytosis , HeLa cells in 35mm dishes were transfected with siRNAs / control siRNAs 48 hours prior to the experiment . Fluorescein isothiocyanate ( FITC ) - dextran ( Sigma - Aldrich ) was diluted in media to a concentration of 1mg / ml and added to cells before incubation for 15 minutes at the indicated temperature . Cells were washed twice in media and once in PBS before lysis in 1 % NP - 40 in PBS supplemented with protease inhibitors . The lysate was centrifuged at 20 , 000g for 20 minutes at 4\u00b0C and the protein concentration in the supernatant was measured using the BCA Protein Assay Kit ( Pierce ) for normalization . The amount of FITC - dextran in the supernatant was measured as the emission at 515nm after exciting at 488nm using a FP - 6500 spectrofluorometer with Spectra Manager software ( JASCO ) . The MHC Class I uptake assay was performed according to reference [ 5 ] . Briefly , HeLa cells transiently transfected with GFP - tagged GRAF1 or GRAF1 BAR + PH for 16 hours were incubated with W6 / 32 anti - MHC Class I antibody ( American Type Culture Collection ) diluted in culture media for 5 or 15 minutes at 37\u00b0C to allow endocytosis . Cells were washed in PBS and surface - bound antibody was removed by a 30 second acid wash followed by a wash in culture media to re - adjust the pH . Cells were fixed and MHC Class I was visualised using Alexa568 - conjugated antibodies . Fixed sample and real time imaging For immunofluorescence analysis , HeLa cells were fixed in 3 % paraformaldehyde in phosphate - buffered saline ( PBS ) for 15 minutes at 37\u00b0C ( to preserve intracellular tubules which are disrupted by fixation at lower temperatures ) , or 4\u00b0C ( to demonstrate this temperature dependence ) , then washed and blocked in 5 % goat serum , with 0 . 1 % saponin , in PBS before staining with the appropriate antibodies in 1 % goat serum , 0 . 1 % saponin in PBS using standard protocols . Confocal images were taken sequentially using a BioRad Radiance system and LaserSharp software ( BioRad ) . Epifluorescence images were taken using a Zeiss Axioimager Z1 system with AxioVision software . For real time microscopy of the dynamics of GRAF1 - and GRAF1 BAR + PH - positive tubules , transfected cells on glass - bottom Petri dishes ( WillCo Wells BV , Amsterdam ) were washed with buffer ( 125mM NaCl , 5mM KCl , 10mM D - glucose , 1mM MgCl2 , 2mM CaCl2 and 25mM HEPES ) and images were taken using a 5 - live scanning microscope ( Zeiss ) or spinning disc confocal system ( Improvision ) with subsequent analysis in LSM Image Browser ( Zeiss ) , ImageJ ( freeware ) or Volocity ( Improvision ) . Caveolin1 - KO MEFs and control MEFs grown on 12mm coverslips were cotransfected with GFP - GPI and myc - tagged GRAF1 or GRAF1 BAR + PH . Anti - GFP was bound to cells on ice for 30 minutes in unsupplemented CO2 - independent medium ( Gibco ) . Cells were washed in CO2 - independent medium to remove unbound antibody prior to internalization in pre - warmed growth media ( 10 % foetal bovine serum ( Cambrix ) , 2mM L - glutamine in DMEM ( Gibco ) ) at 37\u00b0C , 5 % CO2 for 2 , 10 or 40 minutes . Post internalization , cells were washed in ice - cold CO2 - independent medium and acid stripped using 500mM glycine pH2 . 2 in ice cold PBS for 1 minute . To remove intensive cytosolic labeling , acid - stripped live cells were permeablized in ice - cold PBS containing 0 . 05 % saponin ( Sigma ) for 5 minutes . Cells were washed 2 x 1 minute in ice cold PBS before fixation in 2 % paraformaldehyde . Internalized anti - GFP was labeled with Alexa Fluor - 660 - conjugated goat anti - Rabbit secondary antibody . Myc - tagged GRAF1 FL or BAR + PH was labeled with anti - myc ( 9B11 ) primary and Alexa - Fluor - 555 goat - anti mouse secondary antibodies . Fluorescence microscopy was carried out using an Axiovert 200m SP LSM 510 META confocal laser - scanning microscope ( Zeiss ) . Images were captured under oil with a 63x plan - APOCHROMAT objective , at appropriate excitation and emission wavelengths . Images were processed using Adobe Current Biology Volume 18 4 Photoshop CS2 . Quantification of anti - GFP and GRAF1 / GRAF1 BAR + PH colocalization was carried out using Volocity 3 . 7 . 0 . In brief , target cells were cropped and blue and red channels overlaid to generate a colocalization coefficient based on a percentage of blue voxels that identify with red voxels . Background was subtracted using the automatic threshold feature . Five to seven images across three independent experiments were used to calculate average colocalization and standard error of the mean ( SEM ) . Relative pixel numbers of anti - GFP in each image was calculated in Adobe Photoshop CS2 based on the histogram of each image . For real - time endocytosis experiments , NIH 3T3 cells grown in 35mmM glass bottom dishes were transfected with GFP - tagged GRAF1 or GRAF1 BAR + PH constructs . Cells were washed in ice - cold CO2 - independent medium before binding Alexa Fluor - 555 - conjugated CTxB and / or Alexa Fluor - 647 - conjugated transferrin for 30 minutes on ice . Appropriate cells were identified using an Axiovert 200m SP LSM 510 META confocal laser - scanning microscope in ice - cold CO2 - independent medium . Medium was exchanged for prewarmed CO2 - independent medium plus 10 % heat - inactivated Serum Supreme and images captured using Lasersharp 2000 4 . 0 . Frames were captured every 10 seconds for 50 - 100 frames . Images were processed using Image J v1 . 37 and converted to Quicktime files . Supplemental References 1 . Praefcke , G . J . , and McMahon , H . T . ( 2004 ) . The Dynamin superfamily : universal membrane tubulation and fission molecules ? Nat . Rev . Mol . Cell Biol . 5 , 133 - 147 . 2 . Peter , B . J . , Kent , H . M . , Mills , I . G . , Vallis , Y . , Butler , P . J . , Evans , P . R . , and McMahon , H . T . ( 2004 ) . BAR domains as sensors of membrane curvature : the amphiphysin BAR structure . Science 303 , 495 - 499 . 3 . Motley , A . , Bright , N . A . , Seaman , M . N . , and Robinson , M . S . ( 2003 ) . Clathrin - mediated endocytosis in AP - 2 - depleted cells . J . Cell Biol . 162 , 909 - 918 . 4 . Kirkham , M . , Fujita , A . , Chadda , R . , Nixon , S . J . , Kurzchalia , T . V . , Sharma , D . K . , Pagano , R . E . , Hancock , J . F . , Mayor , S . , and Parton , R . G . ( 2005 ) . Ultrastructural identification of uncoated caveolin - independent early endocytic vehicles . J . Cell Biol . 168 , 465 - 476 . 5 . Caplan , S . , Naslavsky , N . , Hartnell , L . M . , Lodge , R . , Polishchuk , R . S . , Donaldson , J . G . , and Bonifacino , J . S . ( 2002 ) . A tubular EHD1 - containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane . EMBO J . 21 , 2557 - 2567 . Current Biology Volume 18 5 Figure S1 | Localisation of GRAF1 to tubular membrane structures is temperature sensitive and dependent on the BAR and PH domains . A , Western blots showing the different forms of GRAF1 detected in adult rat brain and their differential presence / absence in cultured SH - SY5Y ( human neuroblastoma ) , HeLa ( human fibroblast ) , K562 ( human Chronic Myeloid Leukaemia ) , and MEF ( mouse embryonic fibroblast ) , cells . Western blots of purified GRAF1 and myc - tagged GRAF1 ( from lysates of HeLa cells overexpressing this protein ) are shown for comparison . B , Confocal micrograph of an NIH 3T3 cell stained for endogenous GRAF1 distribution . C , Confocal micrographs of HeLa cells fixed either at 4\u00b0C or 37\u00b0C for 10 minutes in 4 % paraformaldehyde and then stained for endogenous GRAF1 . Note the absence of GRAF1 - positive tubules in the 4\u00b0C fixation image . D , Confocal micrographs showing the tubular localization of overexpressed myc - tagged GRAF1 BAR + PH protein in HeLa cells and the cytoplasmic localization of a similarly overexpressed protein with a BAR domain mutation ( KK131 / 132EE ) . E , Confocal micrograph showing the cytoplasmic and punctate localization of overexpressed myc - tagged GRAF1 BAR protein in HeLa cells . Scale bars = 10 \u00b5 m . Current Biology Volume 18 6 Figure S2 A - D | Supplementary biochemical data on the binding between GRAF1 and Dynamin . A , Coomassie - stained gel and confirmatory Western blots of co - immunoprecipitation experiments in rat brain cytosol performed with either control pre - immunization serum ( pre - serum ) or the Ab3 antibody . Bands in the Coomassie - stained gel were identified by mass spectrometry as described . B and C , Coomassie - stained gel and Western blots of pull - down experiments from mouse brain lysate ( B ) or HeLa cell cytosol ( C ) with beads bound to GST ( control ) or GST - tagged GRAF1 BAR + PH , or SH3 proteins . The bands in the Coomassie - stained gel were identified by mass spectrometry as described . Note the major band of Dynamin present in the SH3 lanes , which is not present in the control or BAR + PH condition . \u2018cyt\u2019 marks the HeLa cell lysate ( positive control ) lane . D , The upper panel shows a raw trace from isothermal titration calorimetry performed as described . The lower panel shows the fitting of this data to a one - site binding model from which the affinity ( shown ) can be calculated . GRAF1 SH3 domain and peptide concentrations , as well as injection volumes and times are shown . Current Biology Volume 18 7 Figure S2 E | Dynasore inhibits the uptake of dextran and affects the localization of GRAF1 . E , Confocal micrographs ( maximum projections ) of HeLa cells treated with either DMSO ( vehicle ) or 100\u00b5M dynasore for 1 hour before addition of dextran for 15 minutes , fixation , and immunostaining for dextran and the focal adhesion marker paxillin . Scale bars = 10 \u00b5 m . Current Biology Volume 18 8 Figure S3 | GRAF1 - positive endocytic structures are Clathrin - independent and exclude transferrin A - C , Confocal fluorescent micrographs of HeLa cells stained for endogenous GRAF1 and Clathrin ( A ) , transferrin ( B ) or transferrin receptor ( B ) . The depicted images are used to show some of the different morphologies of GRAF1 - positive structures that are observed in these cells . Scale bars = 10 \u00b5 m . Current Biology Volume 18 9 Figure S4 | GRAF1 BAR + PH overexpression affects CTxB uptake but not transferrin uptake . A and B , Confocal micrographs of HeLa cells transfected with myc - tagged GRAF1 BAR + PH and incubated with CTxB or transferrin for 15 minutes before fixation and staining . C , The graph shows the quantification of images such as depicted in ( A ) . Cells were scored for expression of GRAF1 BAR + PH ( over a threshold corresponding to maximum autofluorescence ) and CTxB internalization ( over an arbitrarily - set threshold above background ) . Note the reduction of the number of transfected cells internalising CTxB compared with controls . D , Live cell microscopy of NIH 3T3 cells expressing GFP - tagged GRAF1 and incubated with CTxB and transferrin ( Tfn ) at 4\u00b0C before chasing their internalization from the time of warming to 37\u00b0C ( time = 00 : 00 ) . Note the internalising GRAF1 - positive tubule containing CTxB . Note also the lack of colocalization of GRAF1 - positive tubules with internalized transferrin . Time is given as minutes : seconds . This sequence is taken from Movie S3 . Scale bars = 10 \u00b5 m . Current Biology Volume 18 10 Figure S5 | Nature of GRAF1 - positive endocytic structures . A and B , Confocal micrographs of HeLa cells overexpressing GFP - tagged flotillin1 ( A ) or GFP - tagged caveolin1 ( B ) and co - stained for endogenous GRAF1 . Note the lack of colocalization . C and D , Confocal micrographs of HeLa cells overexpressing myc - tagged GRAF1 BAR + PH and flotillin1 ( E ) or caveolin1 ( F ) incubated with CTxB for 5 minutes . Note the colocalization of GRAF1 BAR + PH and flotillin1 in CTxB - positive tubular structures . Scale bars = 10 \u00b5 m . Current Biology Volume 18 11 Figure S6 | GRAF1 BAR + PH overexpression does not affect the uptake of MHC Class I which enter GRAF1 - negative compartments . A and B , Epifluorescence micrographs of HeLa cells , transiently transfected with GFP - tagged GRAF1 FL ( A ) or GRAF BAR + PH ( B ) were pulsed with anti - MHC Class I antibody for 5 ( A ) or 15 minutes ( B ) at 37\u00b0C followed by a brief acid wash to remove surface - bound antibody . MHC Class I was visualized using Alexa568 - conjugated secondary antibodies . C , Quantitation of surface GFP - GPI levels in cells overexpressing this protein with myc - tagged GRAF1 FL or GRAF1 BAR + PH . D , Confocal micrographs of HeLa cells treated with siRNA against GRAF1 or a control siRNA and stained for endocgenous GRAF1 . Scale bars = 10 \u00b5 m .", + "mcfarland2008rna": "RNA Interference - Mediated Knockdown of Dynamin 2 Reduces Endocannabinoid Uptake into Neuronal dCAD Cells Matthew J . McFarland , Tamera K . Bardell , Marla L . Yates , Ekaterina A . Placzek , and Eric L . Barker Department of Medicinal Chemistry and Molecular Pharmacology , Purdue University , West Lafayette , Indiana Received January 3 , 2008 ; accepted April 23 , 2008 ABSTRACT The precise mechanism by which the cellular uptake of the endocannabinoid anandamide ( AEA ) occurs has been the source of much debate . In the current study , we show that neuronal differentiated CAD ( dCAD ) cells accumulate anand - amide by a process that is inhibited in a dose - dependent manner by N - ( 4 - hydroxyphenyl ) arachidonylamide ( AM404 ) . We also show that dCAD cells express functional fatty acid amide hydrolase , the enzyme primarily responsible for anandamide metabolism . Previous data from our laboratory indicated that anandamide uptake occurs by a caveolae - related endocytic mechanism in RBL - 2H3 cells . In the current study , we show that anandamide uptake by dCAD cells may also occur by an endocytic process that is associated with detergent - resistant membrane microdomains or lipid rafts . Nystatin and progester - one pretreatment of dCAD cells significantly inhibited anand - amide accumulation . Furthermore , RNA interference ( RNAi ) - mediated knockdown of dynamin 2 , a protein involved in endocytosis , blocked the internalization of the fluorescently labeled anandamide analog SKM 4 - 45 - 1 ( [ 3 (cid:1) , 6 (cid:1) - bis ( acetyloxy ) - 3 - oxospiro [ isobenzofuran - 1 ( 3 H ) , 9 (cid:1) - [ 9 H ] xanthen - 5 - yl ] - 2 - [ [ 1 - oxo - 5 Z , 8 Z , 11 Z , 14 Z - eicosatetraenyl ] amino ] ethyl ester carbamic acid ) . RNAi - mediated knockdown of the (cid:1) 2 subunit of the clathrin - associated activator protein 2 complex had no effect on SKM 4 - 45 - 1 internalization . We were surprised to find that dynamin 2 knockdown in dCAD cells did not affect [ 3 H ] AEA uptake . However , dynamin 2 knockdown caused a significant increase in the overall levels of intact [ 3 H ] AEA associated with the cells , suggesting that trafficking of [ 3 H ] AEA to FAAH had been disrupted . This finding may be the result of an accumu - lation of the anandamide carrier protein in detergent - resistant membranes after dynamin 2 knockdown . Our studies provide evidence that the cellular uptake of anandamide may occur by a dynamin 2 - dependent , caveolae - related endocytic process in dCAD cells . The endocannabinoid anandamide ( AEA ) is an agonist of the cannabinoid 1 and 2 receptors ( Matsuda et al . , 1990 ; Munro et al . , 1993 ) and some vanilloid type ion channels ( Di Marzo et al . , 2001 ; Voets and Nilius , 2003 ) . AEA is internal - ized by most cell types and is thought to be metabolized primarily by the intracellular enzyme fatty acid amide hy - drolase ( FAAH ) ( Deutsch and Chin , 1993 ; Di Marzo et al . , 1994 ; Cravatt et al . , 1996 ) . Previous studies from our labo - ratory have indicated that AEA uptake potentially occurs by a caveolae - related , or clathrin - independent , endocytic pro - cess ( McFarland et al . , 2004 ) . Interestingly , neurons are not thought to express caveolin - 1 but do exhibit lipid raft - related endocytic processes ( Cameron et al . , 1997 ) . Thus , our studies seek to validate a neuronal - like cell line as a useful model in the study of AEA uptake and to show that AEA uptake can be disrupted by using molecular inhibitors that are specifically targeted to endocytic processes . Cath . a cells display neuronal properties and express pan - neuronal markers but lack classic neuronal morphology ( Suri et al . , 1993 ; Lazaroff , 1996 ) . Cath . a differentiated ( CAD ) cells are derived from the Cath . a cell line and are characterized by the loss of the immortalizing oncogene present in Cath . a cells ( Qi et al . , 1997 ) . CAD cells also undergo a reversible morpho - logical differentiation that is initiated when serum is re - moved from the cell culture media ( Qi et al . , 1997 ) . Differen - tiated CAD ( dCAD ) cells stop proliferating and extend long neuronal processes characteristic of primary neuronal cell cultures ( Qi et al . , 1997 ) . Accompanying the morphological This works supported by National Institutes of Health grants R21 - DA13268 and R21 - DA018844 ( to E . L . B . ) . Article , publication date , and citation information can be found at http : / / molpharm . aspetjournals . org . doi : 10 . 1124 / mol . 108 . 044834 . ABBREVIATIONS : AEA , anandamide ; FAAH , fatty acid amide hydrolase ; CAD , Cath . a differentiated ; cCAD , cycling CAD ; siRNA , short interfering RNA ; AP , activator protein ; Dyn 2 , dynamin 2 ; KRH , Krebs - Ringer - HEPES ; AM404 , N - ( 4 - hydroxyphenyl ) arachidonylamide ; PBS , phosphate - buffered saline ; MAFP , methyl arachidonyl fluorophosphonate ; RNAi , RNA interference ; 488 - CT , cholera toxin B subunit - conjugated Alexa Fluor 488 ; 488 - Tf , transferrin - conjugated Alexa Fluor 488 ; TLC , thin layer chromatography ; CB , cannabinoid ; NP , nystatin / progesterone ; SKM 4 - 45 - 1 , ( [ 3 (cid:1) , 6 (cid:1) - bis ( acetyloxy ) - 3 - oxospiro [ isobenzofuran - 1 ( 3 H ) , 9 (cid:1) - [ 9 H ] xanthen - 5 - yl ] - 2 - [ [ 1 - oxo - 5 Z , 8 Z , 11 Z , 14 Z - eicosatetraenyl ] amino ] ethyl ester carbamic acid ) . 0026 - 895X / 08 / 7401 - 101 \u2013 108 $ 20 . 00 M OLECULAR P HARMACOLOGY Vol . 74 , No . 1 Copyright \u00a9 2008 The American Society for Pharmacology and Experimental Therapeutics 44834 / 3357609 Mol Pharmacol 74 : 101 \u2013 108 , 2008 Printed in U . S . A . 101 a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m differentiation of CAD cells is an observed change in the expression and function of several neuronal proteins . For example , dCAD cells express higher levels of tyrosine hydrox - ylase ( Lazaroff et al . , 1998 ) and somatostatin receptor sub - type 2a ( Hashemi et al . , 2003 ) , exhibit a decrease in sodium current accompanied by an increase in potassium current ( Wang and Oxford , 2000 ) , and display alterations in cyclic AMP - mediated signaling ( Hashemi et al . , 2003 ) . Despite re - ported differences in their cellular signaling and protein ex - pression profiles , both cycling CAD ( cCAD ) and dCAD cells express neuron - specific proteins and display the biochemical characteristics of neuronal cells ( Qi et al . , 1997 ) . In RBL - 2H3 cells , AEA transport occurs via a clathrin - independent , or caveolae - related endocytic process , substan - tiated by studies using pharmacological inhibitors of endocy - tosis ( McFarland et al . , 2004 ) . We tested the hypothesis that the disruption of lipid rafts / detergent - resistant membranes in dCAD cells would also decrease the cellular accumulation of AEA to confirm that endocytosis may play a role in AEA uptake in neuronal cells . To avoid the potential problems associated with biochemical disruption of lipid rafts , we examined AEA uptake in dCAD cells after treatment with short interfering RNA ( siRNA ) to knockdown expression of the (cid:1) 2 subunit of the AP2 complex , a protein necessary for clathrin - mediated endocytosis ( Huang et al . , 2004 ) , as well as dynamin 2 ( Dyn 2 ) , a protein involved in both clathrin - mediated and clathrin - independent endocytic processes ( Altschuler et al . , 1998 ; Maxfield and McGraw , 2004 ) . The results of our studies are consistent with a role for endocy - tosis in the neuronal uptake of endocannabinoids . Materials and Methods Cell Culture . cCAD cells were maintained in a 1 : 1 ratio of Ham\u2019s F - 12 / Dulbecco\u2019s modified Eagle\u2019s medium with 5 % bovine calf serum and 5 % fetal clone 1 supplemented with 1 % penicillin / streptomycin , and 2 mM L - glutamine . Cells were grown in a humidified environ - ment containing 5 % CO 2 and held at the constant temperature of 37\u00b0C . cCAD cells were maintained in serum - free 1 : 1 Ham\u2019s F - 12 / Dulbecco\u2019s modified Eagle\u2019s medium and were allowed to differenti - ate for 36 h before experiments to generate dCAD cells . [ 3 H ] AEA Uptake and SKM 4 - 45 - 1 Internalization Assays . CAD cells were plated at approximately 50 , 000 cells per well in 24 - well culture dishes and then allowed to differentiate in serum - free media for 36 h . The resulting dCAD cells were then transfected with siRNA oligonucleotides as described below . After the transfec - tion period , media were removed and 1 nM [ 3 H ] AEA ( labeled on the arachidonate portion of the molecule ; PerkinElmer Life and Analyt - ical Sciences , Waltham , MA ) in Krebs - Ringer - HEPES ( KRH ) buffer ( 120 mM NaCl , 4 . 7 mM KCl , 2 . 2 mM CaCl 2 , 10 mM HEPES , 1 . 2 mM KH 2 PO 4 , and 1 . 2 mM MgSO 4 , pH 7 . 4 ) was added to all samples and allowed to incubate for 5 min at 37\u00b0C . To determine the effects of cholesterol depletion on AEA accumulation in dCAD cells , uptake assays were performed after a 30 - min incubation in KRH buffer containing nystatin ( 25 (cid:2) g / ml ) and progesterone ( 10 (cid:2) g / ml ) . Control cells were incubated for 30 min in 1 (cid:2) KRH buffer . AM404 ( 100 (cid:2) M ) was used to define nonspecific uptake and was added 10 min before the addition of [ 3 H ] AEA at 37\u00b0C . Cells were then washed once with 1 (cid:2) KRH buffer containing 1 % bovine serum albumin , and MicroScint - 20 ( PerkinElmer Life and Analytical Sciences ) was added to each well . The amount of tritium present was determined using a TopCount microplate scintillation and luminescence counter ( PerkinElmer Life and Analytical Sciences ) . To verify morphological changes , cells were seeded in clear 24 - well tissue culture plates for uptake assays in untransfected dCAD cells . To quantify tritium from the wells of clear plates , cells were solubilized in 1 % SDS and transferred to vials containing 10 ml of EcoLite scintillation fluid ( MP Biomedicals , Irvine , CA ) for subsequent scintillation counting . For experiments examining SKM 4 - 45 - 1 internalization , CAD cells were treated with SKM 4 - 45 - 1 ( 25 (cid:2) M ) and increasing concentra - tions of AM404 for 10 min in prewarmed 37\u00b0C buffer . Fluorescence was quantified using a FUSION microplate reader ( PerkinElmer Life and Analytical Sciences ) . Determination of Plasma Membrane Cholesterol Content . Membrane cholesterol content was determined according to a method described previously ( Millard et al . , 2005 ) . CAD cells were plated in triplicate at 5 (cid:2) 10 4 cells per 35 - mm well in complete culture medium . Twenty - four hours later , cells were washed three times with PBS and then incubated in complete medium that had been supplemented with 10 % lipoprotein - deficient fetal calf serum and 1 (cid:2) Ci / ml [ 3 H ] cholesterol ( 40 Ci / mmol ; GE Healthcare , Chalfont St . Giles , UK ) . Cells were labeled to equilibrium for 48 h . Cells were then washed three times with PBS and then treated with nystatin / progesterone as described above . Membrane cholesterol was ex - tracted by a 10 min incubation with medium containing 4 % methyl - (cid:1) - cyclodextrin . Media was collected , cells were washed three times with PBS , and proteins were extracted . Protein concentrations were determined using the bicinchoninic acid assay . [ 3 H ] Cholesterol ex - tracted into the medium was quantified by scintillation counting and normalized to total protein values . FAAH Activity Assays . FAAH activity was determined by a modification of a previously published method ( Day et al . , 2001 ) . In brief , CAD cells were homogenized in buffer containing 20 mM Tris - HCl , 1 mM EDTA , 0 . 7 (cid:2) g / ml pepstatin A , and 0 . 5 (cid:2) g / ml leupep - tin . Cell homogenates were then incubated with 5 nM [ 3 H ] AEA [ ethanolamine 1 - 3 H ] ( ARC , St . Louis , MO ) in the presence or absence of 500 nM methyl arachidonyl fluorophosphonate ( MAFP ) with a reaction volume of 250 (cid:2) l . After the incubation , the reaction was terminated by adding 500 (cid:2) l of 1 : 1 chloroform / methanol ( 2 (cid:2) assay volume ) to each tube . Samples were vortexed for 30 s and centrifuged at 3900 g for 1 min to separate the aqueous and organic phases . Then , 100 - (cid:2) l aliquots were collected from both the aqueous and organic phases , and radioactivity was quantified using a TopCount Micro - plate scintillation and luminescence counter ( PerkinElmer Life and Analytical Sciences ) . The [ 3 H ] AEA used in these experiments was labeled on the ethanolamine portion of the molecule and not the arachidonate backbone ; therefore , as FAAH activity increased so did the level of radioactivity in the aqueous phase . Western Blot Analysis . Western blot analysis was performed as described previously ( McFarland et al . , 2004 ) . The presence of FAAH , dynamin 2 , and the (cid:1) 2 subunit of the AP2 complex was detected using rabbit polyclonal anti - FAAH , rabbit polyclonal anti - dynamin 2 ( Abcam Inc . , Cambridge , MA ) , and mouse monoclonal anti - (cid:1) 2 subunit ( Novus Biologicals , Inc . , Littleton , CO ) primary an - tibodies , respectively , followed by an incubation with horseradish peroxidase - labeled goat anti - rabbit or goat anti - mouse secondary antibodies ( Bio - Rad Laboratories , Hercules , CA ) , and enhanced chemiluminescence detection reagents . Membranes were then ex - posed to X - ray film . RNAi Transfections . CAD cells were transfected with siRNA oligonucleotides ( Stealth RNAi ; Invitrogen , Carlsbad , CA ) directed against either Dyn2 or the (cid:1) 2 subunit of the AP2 complex mRNA using the Lipofectamine 2000 ( Invitrogen ) RNAi transfection proto - col for mammalian cells provided by Invitrogen ( 16 pmol of each siRNA oligo was added for every 1 (cid:2) l of Lipofectamine 2000 used ) . For later experiments , the siLentFect Lipid Reagent was used fol - lowing the manufacturer\u2019s protocol ( Bio - Rad Laboratories ) . Protein knockdown was achieved by cotransfection of two different siRNA oligonucleotides targeted at each individual protein . The siRNA se - quences were as follows : Dyn 2 : NM _ 007871 _ stealth _ 1410 , GUG - GACCUGGUUAUCCAGGAGCUAA , and NM _ 007871 _ stealth _ 2009 , GGCAGAGAAUGAGGAUGGAGCACAA , (cid:1) 2 / AP2 : NM _ 027915 _ 102 McFarland et al . a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m stealth _ 1591 , GCUGGUCCAACAGGUCUUGAGCUUA , and NM _ 027915 _ stealth _ 2400 , CCAUACAUACUCCACUGAUGCCAAA . Stealth RNAi negative control duplex was purchased from Invitro - gen . Sequences of the negative ( mock ) control contained medium GC content and matched no known mRNA sequence in the vertebrate genome . Confocal Microscopy . dCAD cells plated in a four - well Lab - Tek Chambered Coverglass slide ( Nalge Nunc International , Rochester , NY ) were transfected with the appropriate siRNA oligonucleotides . Then , 72 h after transfection , the cells were treated with the B subunit of cholera toxin conjugated to Alexa Fluor 488 ( 488 - CT ) ( Invitrogen ) , transferrin conjugated to Alexa Fluor 488 ( 488 - Tf ) ( In - vitrogen ) , or SKM 4 - 45 - 1 ( Muthian et al . , 2000 ) for 10 min and imaged using oil immersion confocal microscopy at 60 (cid:2) magnifica - tion with a krypton ( 488 nm ) / argon ( 568 nm ) laser , band pass filter at 522 to 535 nm , and a long pass filter at 588 nm . Thin Layer Chromatography Analysis for Intact [ 3 H ] AEA . dCAD cells transfected with either mock or dynamin 2 - targeted siRNA were treated with [ 3 H ] AEA ( 1 nM ) for 5 min and washed three times with KRH containing 1 % bovine serum albumin . After the removal of the wash buffer , transfected dCAD cells were exposed to 1 ml of lysis buffer ( 0 . 5 % Triton X - 100 , 50 mM Tris - HCl , 150 mM NaCl , and 5 mM EDTA ) on ice for 20 min . The cells were then scraped and added to a 15 - ml centrifuge tube kept on ice that con - tained 1 . 5 ml of 2 : 1 CHCl 3 / MeOH ( v / v ) and 8 (cid:2) l of 1 M HCl . The aqueous and organic phases were mixed by vortexing and then separated by centrifugation ( 3000 g for 15 min at 4\u00b0C ) . The organic layer was removed from each sample , placed in a clean glass tube , and 1 ml of ice - cold CHCl 3 was added to the remaining aqueous layer . The phases were again mixed by vortexing and separated by centrifugation ( 3000 g for 15 min at 4\u00b0C ) . The resulting organic layer was removed and combined with the previously extracted organic layer . The solvent from the sample was evaporated under a stream of argon . The tubes were capped and kept overnight at (cid:3) 20\u00b0C . The concentrated samples were resuspended in 52 (cid:2) l of ice - cold CHCl 3 and then divided into two spots of 20 (cid:2) l each onto 20 - (cid:2) 20 - cm glass - backed silica gel thin layer chromatography ( TLC ) plates . The mobile phase in which the TLC plates were developed consisted of 11 : 5 ethyl acetate / isooctane saturated with H 2 O and acetic acid . After the plates were dried , the lipids were visualized with iodine vapor . The location of the [ 3 H ] AEA spot was identified by comparison with the unlabeled AEA standard . The silica corresponding to the AEA spots was then scraped off of the TLC plate into scintillation vials and allowed to equilibrate for 72 h in scintillation fluid . Radio - activity was then quantified by liquid scintillation counting . Results Endocannabinoid Uptake Properties of dCAD and cCAD Cells . Differentiated and undifferentiated CAD ( dCAD and cCAD , respectively ) cells displayed robust uptake of radio - labeled anandamide . The accumulation of AEA by CAD cells was inhibited by the AEA uptake inhibitor AM404 , with a K i value in the low micromolar to high nanomolar range ( Fig . 1A ) . SKM 4 - 45 - 1 is a fluorescent analog of AEA ( Muthian et al . , 2000 ) . Previous results using AM404 indicate that SKM 4 - 45 - 1 is internalized by the same mechanism as AEA in C6 glioma cells ( Muthian et al . , 2000 ) . SKM 4 - 45 - 1 internalization in CAD cells was also inhibited by AM404 , with K i values similar to those previously reported for inhibition of [ 3 H ] AEA uptake ( Fig . 1B ) ( Muthian et al . , 2000 ) . Furthermore , Western blot analysis of whole cell lysates from dCAD and cCAD cells revealed that both cell types express the AEA - metabolizing enzyme FAAH ( Fig . 2A ) . FAAH activity assays performed on CAD cell homog - enates revealed FAAH activity that was inhibited in the pres - ence of the FAAH inhibitor MAFP ( Fig . 2B ) . FAAH has been implicated in playing an important role in AEA uptake ( Day et al . , 2001 ; Glaser et al . , 2003 ) . We were surprised to find that although FAAH was present in CAD cells , inhibition of FAAH by phenylmethylsulfonyl fluoride did not reduce AEA uptake in CAD cells ( data not shown ) . This finding suggests that FAAH may not contribute to AEA uptake in these neuronal cells . Effect of Nystatin / Progesterone Pretreatment on AEA Uptake . As described above , both cCAD and dCAD cells display endocannabinoid properties . However , dCAD cells are morphologically similar to primary neuronal cell cultures . Thus , dCAD cells were used for our additional stud - ies to assess the role that endocytosis might play in the AEA uptake by a neuronal cell line . dCAD cells were treated with nystatin and progesterone to deplete membrane cholesterol levels and thereby disrupt detergent - resistant lipid raft do - mains . Based on the results from previous studies with RBL - 2H3 cells ( McFarland et al . , 2004 ) , we expected that this treatment would decrease the specific uptake of AEA by dCAD cells . Indeed , the disruption of detergent - resistant membrane microdomains in dCAD cells resulted in an (cid:4) 50 % decrease in the specific uptake of [ 3 H ] AEA ( Fig . 3 ) . Control experiments confirmed that nystatin and progesterone treat - Fig . 1 . Dose - dependent inhibition of AEA uptake and SKM 4 - 45 - 1 inter - nalization by AM404 . Both dCAD ( f ) and cCAD ( circf ) cells were treated with increasing concentrations of AM404 for 10 min at 37\u00b0C . A , after the incubation with AM404 , [ 3 H ] AEA was added at a final concentration of 1 nM as described under Materials and Methods . Data shown are means (cid:5) S . E . M . and are representative of four separate experiments performed in triplicate . K i values ( means (cid:5) S . E . M . ; n (cid:6) 3 ) were 850 (cid:5) 600 nM ( cCAD ) and 930 (cid:5) 150 nM ( dCAD ) . The cpm values for cCAD cells were total (cid:6) 6440 (cid:5) 914 and nonspecific (cid:6) 850 (cid:5) 86 . The cpm values for dCAD cells were total (cid:6) 5124 (cid:5) 1456 and nonspecific (cid:6) 794 (cid:5) 35 . B , cells were treated with SKM 4 - 45 - 1 ( 25 (cid:2) M ) and increasing concentrations of AM404 for 10 min , and fluorescence was determined on a FUSION microplate reader ( PerkinElmer Life and Analytical Sciences ) . IC 50 val - ues were converted to K i values using the Cheng - Prusoff equation ( K m value for SKM 4 - 45 - 1 (cid:6) 16 (cid:2) M ) . K i values ( means (cid:5) S . E . M . ; n (cid:6) 3 ) were 24 (cid:5) 12 (cid:2) M ( cCAD ) and 16 (cid:5) 10 (cid:2) M ( dCAD ) . Data shown are represen - tative of three separate experiments performed in duplicate . Arbitrary fluorescence unit values for cCAD cells were total (cid:6) 13 , 858 (cid:5) 1856 , nonspecific (cid:6) 7521 (cid:5) 573 , and background (cid:6) 4313 (cid:5) 625 . Arbitrary fluorescence unit values for dCAD cells were total (cid:6) 23 , 137 (cid:5) 6590 , nonspecific (cid:6) 11 , 309 (cid:5) 4024 , and background (cid:6) 2006 (cid:5) 74 . AEA Uptake via Endocytosis in dCAD Cells 103 a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m ment reduced membrane cholesterol by approximately 50 % , consistent with the effect on [ 3 H ] AEA uptake ( Fig . 3 , inset ) . RNAi - Mediated Knockdown of Endocytic Machin - ery . The studies described above with cholesterol depletion , along with our previous work ( McFarland et al . , 2004 ) , suggested that AEA uptake occurs via a caveolae - related endocytic process . The nonspecific nature of the cholesterol depletion treatments limits interpretation of our results . Therefore , we assessed the role of clathrin - independent ver - sus clathrin - dependent endocytosis of AEA in dCAD cells using molecular inhibitors . Specifically , we used an RNAi approach to knockdown expression of either the (cid:1) 2 subunit of the AP2 complex or dynamin 2 . Knockdown of the (cid:1) 2 subunit of the AP2 complex should only inhibit clathrin - mediated endocytosis and have no effect on clathrin - independent , or caveolae - related , endocytic processes ( Johannes and Lamaze , 2002 ; Huang et al . , 2004 ) . Dynamin 2 , however , is a GTPase that has been shown to play a role in both clathrin - mediated and clathrin - independent endocytosis ( Altschuler et al . , 1998 ; Maxfield and McGraw , 2004 ) . After transfection with siRNA oligonucleotides using Lipofectamine 2000 ( Invitro - gen ) , the cells were incubated for 72 h to allow for the deg - radation of resident protein . Western blot analysis confirmed that we had successfully reduced the expression of both pro - teins by 70 to 90 % ( Fig . 4 ) . Control transfection experiments using green fluorescent protein ( pEGFP - N1 ; Clontech , Moun - tain View , CA ) confirmed transfection efficiency in CAD cells to be greater than 80 % ( data not shown ) , consistent with our observed knockdown effects . To verify the effects of both (cid:1) 2 subunit and dynamin 2 knock - down , we used the Alexa Fluor 488 - conjugated probes trans - ferrin ( 488 - Tf ) and the B subunit of cholera toxin ( 488 - CT ) , which are endocytosed by clathrin - dependent and lipid raft / caveolae - related endocytic processes , respectively . As was ex - pected , after a 10 - min treatment with either probe at 37\u00b0C , RNAi knockdown of the (cid:1) 2 subunit of the AP2 complex signif - icantly reduced the internalization of 488 - Tf ( Fig . 5 ) but had no effect on 488 - CT endocytosis in dCAD cells compared with con - trol ( Fig . 6 ) . In the case of dynamin 2 , RNAi knockdown inhib - ited internalization of both 488 - Tf ( Fig . 5 ) and 488 - CT ( Fig . 6 ) in dCAD cells . These control experiments confirm that by knock - ing down expression of the (cid:1) 2 subunit of the AP2 complex we are only inhibiting clathrin - mediated endocytosis ( i . e . , 488 - Tf internalization ) , whereas knockdown of dynamin 2 inhibits both clathrin - dependent endocytosis as well as a lipid raft - related endocytic process that is clathrin - independent . AEA Uptake after RNAi Transfection . Our results above showing AM404 inhibition of both [ 3 H ] AEA and SKM 4 - 45 - 1 internalization ( Fig . 1 ) suggest that both compounds are trans - ported by a common process in the CAD cells . Because the fluorescence associated with SKM 4 - 45 - 1 is only observed after internalization of the compound , it is a true marker of actual uptake into the cell ( Muthian et al . , 2000 ) . To assess the effects that RNAi knockdown of endocytic machinery might have on Fig . 2 . Expression of FAAH in CAD cells . A , cCAD and dCAD cells express FAAH . Western blots were performed as described under Mate - rials and Methods . Data are representative of three separate experi - ments . B , cCAD and dCAD cells were homogenized and then incubated at 37\u00b0C for 10 min . Cell homogenates were then incubated with 5 nM [ 3 H ] AEA in the presence or absence of 500 nM MAFP . After the incuba - tion , the reaction was terminated , and the aqueous and organic phases were separated . Bars represent AEA - derived [ 3 H ] ethanolamine present in the aqueous phase ( i . e . , metabolized AEA ) after the termination of the reaction as a direct measure of FAAH activity . Data shown are means (cid:5) S . D . of three separate experiments performed in duplicate . Fig . 3 . Effect of lipid raft disruption on AEA uptake in dCAD cells . dCAD cells were pretreated for 30 min with 25 (cid:2) g / ml nystatin and 10 (cid:2) g / ml progesterone ( NP treatment ) . After NP treatment , AEA uptake assays were performed in 1 (cid:2) KRH at 37\u00b0C in the presence or absence of 100 (cid:2) M AM404 to define nonspecific transport . Transport assays were performed for 5 min with 1 nM [ 3 H ] AEA as described under Materials and Methods . Data are means (cid:5) S . E . M . combined from three separate experiments performed in triplicate . The cpm values for control cells were total (cid:6) 5755 (cid:5) 118 and nonspecific (cid:6) 502 (cid:5) 41 . The cpm values for NP - treated cells were total (cid:6) 3117 (cid:5) 40 and nonspecific (cid:6) 729 (cid:5) 61 . Inset , effect of NP treatment on membrane cholesterol content . Cholesterol content in CAD cell membranes was determined as described under Materials and Methods . Data are means (cid:5) S . E . M . from two separate experiments per - formed in triplicate . (cid:1) , p (cid:7) 0 . 05 . Fig . 4 . RNAi - mediated knockdown of the (cid:1) 2 subunit of the AP2 complex and dynamin 2 . dCAD cells were transfected with Stealth RNAi ( Invitro - gen ) using Lipofectamine 2000 ( Invitrogen ) and then incubated at 37\u00b0C in serum - free media for 72 h to allow for protein degradation . The expres - sion of both the (cid:1) 2 subunit of the AP2 complex and the protein dynamin 2 was decreased by (cid:8) 90 % compared with mock siRNA ( nonsense se - quence ) transfection . siRNA oligonucleotides had no effect on the level of control protein (cid:1) - actin ( data not shown ) . To control for protein loading , equal amounts of total protein ( 20 (cid:2) g ) from the cell lysates were added to each well for Western blot analysis as described under Materials and Methods . Both proteins resolved at the expected molecular weights . Data are representative of three separate experiments . 104 McFarland et al . a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m AEA uptake , cells were treated with 25 (cid:2) M SKM 4 - 45 - 1 at 37\u00b0C for 10 min after knockdown of the expression of either the (cid:1) 2 subunit of the AP2 complex or dynamin 2 . Compared with control , RNAi knockdown of the (cid:1) 2 subunit of the AP2 complex caused no observable change in the internalization of SKM 4 - 45 - 1 ( Fig . 7 ) . However , knockdown of dynamin 2 expression abolished the SKM 4 - 45 - 1 fluorescence accumulation in dCAD cells ( Fig . 7 ) . In addition to examining SKM 4 - 45 - 1 accumulation , we investigated the effects of RNAi - mediated knockdown of both the (cid:1) 2 subunit of the AP2 complex and dynamin 2 on the specific uptake of [ 3 H ] AEA . We were surprised to find that neither the (cid:1) 2 subunit of the AP2 complex nor dynamin 2 knockdown displayed any significant effect on the specific uptake of [ 3 H ] AEA ( Fig . 8 ) . To explain the potentially con - flicting result between [ 3 H ] AEA and SKM 4 - 45 - 1 internaliza - tion , we hypothesized that dynamin 2 siRNA - transfected dCAD cells might be accumulating more [ 3 H ] AEA at the plasma membrane instead of internalizing the AEA . If this were true , then FAAH would be unable to metabolize the [ 3 H ] AEA that has been retained at the plasma membrane . Such an effect would be revealed by an increase in intact [ 3 H ] AEA in the dynamin 2 siRNA - transfected cells after a 5 - min uptake assay . dCAD cells transfected with either mock or dynamin 2 siRNA were treated with [ 3 H ] AEA for 5 min in the presence or absence of AM404 . After [ 3 H ] AEA treatment , each treat - ment group of cells was lysed , and the lipids were extracted using chloroform / methanol . TLC was performed on the lipid Fig . 6 . Effect of RNAi knockdown of dynamin 2 and the (cid:1) 2 subunit of the AP2 complex on the internalization of 488 - CT in dCAD cells . Seventy - two hours after transfection with either mock , (cid:1) 2 subunit , or dynamin 2 siRNA , dCAD cells were incubated with 488 - CT for 10 min and imaged by confocal microscopy . Dynamin 2 siRNA - transfected cells showed a reduc - tion of 488 - CT internalization ( indicated by arrows ) compared with mock - transfected dCAD cells . (cid:1) 2 subunit siRNA - transfected cells did not display any difference in 488 - CT internalization compared with mock - transfected dCAD cells , confirming the specificity of the treatments . Images were obtained using confocal microscopy as described under Ma - terials and Methods . The internal fluorescence intensity of the intracel - lular space of all cells in the field of view was quantified using Meta - Morph software ( bottom right ) . Fifteen to 20 cells per experiment were analyzed . Data are representative of three separate experiments . Aster - isks denote a significant difference in intracellular mean fluorescence intensity compared with mock - transfected cells ( analysis of variance and Dunnett\u2019s multiple comparison test ; p (cid:7) 0 . 05 ) . Fig . 5 . Effect of RNAi knockdown of dynamin 2 and the (cid:1) 2 subunit of the AP2 complex on the internalization of 488 - Tf in dCAD cells . Seventy - two hours after transfection with either mock , (cid:1) 2 subunit , or dynamin 2 siRNA , dCAD cells were incubated with 488 - Tf for 10 min and imaged by confocal microscopy . Both (cid:1) 2 subunit - and dynamin 2 siRNA - transfected cells showed a reduction of 488 - Tf internalization ( indicated by arrows ) compared with mock - transfected dCAD cells . Images were obtained using confocal microscopy as described under Materials and Methods . The internal fluorescence intensity of the intracellular space of all cells in the field of view was quantified using MetaMorph software ( Molecular De - vices , Sunnyvale , CA ) ( bottom right ) . Fifteen to 20 cells per experiment were analyzed . Data are representative of three separate experiments . Asterisks denote a significant difference in intracellular mean internal fluorescence intensity compared with mock - transfected cells ( analysis of variance and Dunnett\u2019s multiple comparison test ; p (cid:7) 0 . 01 ) . Fig . 7 . SKM 4 - 45 - 1 internalization by dCAD cells after siRNA transfec - tion . dCAD cells were treated with SKM 4 - 45 - 1 ( 25 (cid:2) M ) for 10 min at 72 h after transfection with mock , (cid:1) 2 subunit , or dynamin 2 siRNA . Knock - down of the (cid:1) 2 subunit of the AP2 complex had no effect on the SKM 4 - 45 - 1 - derived fluorescence signal compared with mock . Knockdown of dynamin 2 in dCAD cells significantly decreased the accumulation of SKM 4 - 45 - 1 - derived fluorescence . Images were obtained using confocal microscopy as described under Materials and Methods . Phase contrast images are shown below each corresponding fluorescence image . Data are representative of three separate experiments . AEA Uptake via Endocytosis in dCAD Cells 105 a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m samples of each treatment group , and the levels of intact [ 3 H ] AEA were quantified by scintillation counting . TLC anal - ysis revealed that the specific levels of intact [ 3 H ] AEA were approximately 2 - fold higher in dCAD cells in which dynamin 2 had been knocked down compared with mock - transfected dCAD cells ( Fig . 9A ) . One explanation for the above - mentioned results could be that the knockdown of dynamin 2 altered FAAH activ - ity . Examination of FAAH protein by Western blot analysis ( Fig . 9B ) and FAAH activity ( Fig . 9C ) after dynamin 2 knockdown indicated that the dynamin 2 knockdown had no detectable effect on FAAH . The lack of effect on FAAH and actin levels also confirms the specificity of our dynamin 2 siRNA ( Fig . 9B ) . Discussion dCAD cells represent a biochemically and morphologically neuronal cell line that rapidly accumulates the endocannabi - noid AEA and displays robust FAAH activity . Thus , dCAD cells may present a novel system useful in the study of endocannabinoid biosynthesis and inactivation . Pharmacological inhibition of endocytosis in RBL - 2H3 cells suggests that an endocytic process is involved in AEA uptake and that this process is clathrin - independent , potentially involving detergent - resistant membrane microdomains such as lipid rafts ( McFarland et al . , 2004 ) . Our present results confirm these findings in the neuronal dCAD cell line as well . Pretreatment with nystatin / progesterone to disrupt detergent - resistant membrane microdomains significantly reduced the specific uptake of [ 3 H ] AEA by dCAD cells by (cid:4) 50 % . An alter - native explanation for our results with nystatin / progesterone could be that the treatment is generally toxic to the cells . Be - cause a major factor determining nonspecific uptake relates to cell number , it is unlikely that the brief treatments caused a decrease in cell viability because nonspecific uptake of AEA was unaltered by the treatment ( data not shown ) . We recognize that there is a lack of specificity associated with the chemical dis - ruption of lipid rafts . Thus , a means of specifically disrupting endocytic processes without causing the potential nonspecific effects associated with the chemical disruption of lipid rafts was needed to assess the role that endocytosis may play in the cellular uptake of AEA . The (cid:1) 2 subunit of the AP2 complex allows for the associa - tion of this complex with clathrin and is necessary for clath - rin - mediated endocytosis to occur ( Huang et al . , 2004 ) . Transferrin is internalized by a clathrin - mediated endocytic process ( Johannes and Lamaze , 2002 ) . As expected , RNAi knockdown of the (cid:1) 2 subunit of the AP2 complex inhibited transferrin internalization in dCAD cells but did not disrupt Fig . 8 . [ 3 H ] AEA uptake by dCAD cells after siRNA transfection . Seventy - two hours after transfection with mock , dynamin 2 , or (cid:1) 2 subunit siRNA , [ 3 H ] AEA uptake assays were performed in 1 (cid:2) KRH at 37\u00b0C as described under Materials and Methods . AM404 ( 100 (cid:2) M ) was used to define nonspecific transport . Data represent mean (cid:5) S . E . M . for three separate experiments performed in triplicate . The cpm values for mock cells were total (cid:6) 2887 (cid:5) 136 and nonspecific (cid:6) 1125 (cid:5) 128 . The cpm values for dynamin 2 knockdown cells were total (cid:6) 2973 (cid:5) 89 and nonspecific (cid:6) 905 (cid:5) 83 . The cpm values for (cid:1) 2 subunit knockdown cells were total (cid:6) 2638 (cid:5) 104 and nonspecific (cid:6) 777 (cid:5) 27 . Fig . 9 . Metabolism of [ 3 H ] AEA in dynamin 2 siRNA - transfected dCAD cells . A , 72 h after transfection with either mock or dynamin 2 siRNA , dCAD cells were treated with [ 3 H ] AEA for 5 min and then lysed . Cellular lipids were extracted from the cell lysates using chloroform / methanol and analyzed by TLC for intact [ 3 H ] AEA . Nonspecific uptake was determined using 100 (cid:2) M AM404 . The graph represents data from three separate experiments . Statistical analysis was performed using a one - sample t test comparing with the value of 100 ( mock control ) . (cid:1) , p (cid:7) 0 . 05 . The cpm values from the TLC analysis for mock cells were total (cid:6) 2986 (cid:5) 795 and nonspecific (cid:6) 2243 (cid:5) 407 . The cpm values from the TLC analysis for dynamin 2 knockdown cells were total (cid:6) 3257 (cid:5) 545 and nonspecific (cid:6) 1793 (cid:5) 198 . B , FAAH expression after RNAi - mediated knockdown of dynamin 2 . Knockdown of dynamin 2 in cCAD cells was performed as described under Materials and Methods except siLentFect Lipid Reagent ( Bio - Rad ) was used as the transfection reagent for 48 h with 10 nM siRNA oligo . Western blots were performed using the polyclonal dynamin 2 antibody , monoclonal FAAH antibody ( 1 : 1000 ; Abnova Corporation , Tai - pei City , Taiwan ) , monoclonal actin antibody ( 1 : 2000 ; Sigma - Aldrich , St . Louis , MO ) . C , FAAH activity after RNAi - mediated knockdown of dy - namin 2 . FAAH activity assays were performed on whole cell lysates as described under Materials and Methods . Data shown represent means (cid:5) S . D . from two experiments performed in quadruplicate . 106 McFarland et al . a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m the cellular accumulation of the cholera toxin B subunit or SKM 4 - 45 - 1 , the fluorescently tagged analog of AEA . The cholera toxin B subunit is known to be internalized via a clathrin - independent ( lipid raft - related ) endocytic process ( Johannes and Lamaze , 2002 ) . Whereas its role in neurons is not completely clear , the small GTPase dynamin 2 is probably involved in both clath - rin - dependent and clathrin - independent endocytic recycling ( Altschuler et al . , 1998 ; Maxfield and McGraw , 2004 ) . Thus , both transferrin and cholera toxin should require dynamin 2 expression for internalization to occur . RNAi knockdown of dynamin 2 significantly reduced both transferrin and cholera toxin accumulation by dCAD cells . Furthermore , dynamin 2 knockdown in dCAD cells inhibited the cellular internaliza - tion of SKM 4 - 45 - 1 . Because the fluorescence associated with SKM 4 - 45 - 1 is only observed after internalization of the compound , it can be used as a marker for actual uptake ( Muthian et al . , 2000 ) . One caveat of using only [ 3 H ] AEA uptake is that conventional uptake assays do not differenti - ate between the tritium bound to the cells or membranes and the AEA that is actually transported into the cells . Taken together , our data suggest that actual internalization of AEA by dCAD cells occurs via a clathrin - independent endocytic process . Furthermore , dynamin 2 seems to play a role in the cellular uptake of AEA by the neuronal dCAD cells . Internal - ization of the B subunit of cholera toxin occurs by a lipid raft - mediated endocytic process ( Johannes and Lamaze , 2002 ) . Dynamin 2 knockdown significantly reduced 488 - CT internalization in dCAD cells consistent with this dynamin 2 - dependent , clathrin - independent endocytic process involv - ing detergent - resistant membrane microdomains . An alter - native approach to the knockdown of dynamin 2 or the (cid:1) subunit of the AP2 complex could be to use siRNA targeted to clathrin itself . We were concerned about potential membrane effects reported with clathrin knockdowns and as such , we opted to focus on proteins downstream of clathrin in the endocytic pathways ( Hinrichsen et al . , 2003 , 2006 ) . Dynamin 2 knockdown displayed a profound inhibitory effect on SKM 4 - 45 - 1 internalization in dCAD cells , and yet it did not have any observable effect on the specific uptake of [ 3 H ] AEA . This discrepancy may be reconciled with the obser - vation that [ 3 H ] AEA uptake assays cannot discriminate be - tween [ 3 H ] AEA that has actually been internalized and [ 3 H ] AEA that is binding to a target on the plasma membrane . Interestingly , a study conducted by Schmid and colleagues showed that expression of dominant - negative mutants of dy - namin proteins inhibited endocytic processes but had no ef - fect on endosomal recycling of membrane components to the cell surface ( Altschuler et al . , 1998 ) . dCAD cells that have lost functioning dynamin 2 protein during the 72 - h incuba - tion after transfection with siRNA may be accumulating the AEA carrier in the detergent - resistant membrane microdo - mains . Precedence for such an effect comes from studies showing that expression of dominant - negative dynamin 2 mutant causes an accumulation of transferrin receptor at the cell surface ( Altschuler et al . , 1998 ) . A similar phenomenon could be occurring with the putative AEA carrier after dy - namin 2 knockdown . The accumulation of detergent - resis - tant membrane components at the cell surface with dynamin 2 knockdown could create a scenario in which dCAD cells , although they are not internalizing [ 3 H ] AEA , are accumulat - ing [ 3 H ] AEA at the membrane . This effect would be due to more [ 3 H ] AEA being able to associate with the plasma mem - brane that is now enriched with the putative carrier protein . This experimental limitation is not an issue in the case of SKM 4 - 45 - 1 internalization assays . Fluorescence from SKM 4 - 45 - 1 is not observed until after the compound is trans - ported into the cell , and the fluorescent component of the molecule is liberated by esterases in the cell ( Muthian et al . , 2000 ) . Thus , excess SKM 4 - 45 - 1 bound only at the membrane is not detectable . We propose that knockdown of dynamin 2 in dCAD cells causes an accumulation of the AEA carrier in detergent - resistant membrane domains at the cell surface . There is no observable difference between the [ 3 H ] AEA uptake in mock siRNA - transfected and dynamin 2 siRNA - transfected dCAD cells because more [ 3 H ] AEA is able to associate with the plasma membrane carrier in the dynamin 2 siRNA - transfected cells . If this is true , then the majority of the radioactivity obtained from the dynamin 2 siRNA - transfected cells should be represented by intact [ 3 H ] AEA . This is because AEA that is only associating with the cell surface and is not internalized will not be available to FAAH for metabolism . Indeed , TLC analysis of lipid extracts from dynamin 2 siRNA - transfected dCAD cells that were treated with [ 3 H ] AEA for 5 min revealed levels of intact [ 3 H ] AEA that were 2 - fold greater than the levels of [ 3 H ] AEA extracted from mock siRNA - transfected dCAD cells treated in the same manner . Furthermore , our results indicate that dy - namin 2 knockdown does not alter FAAH protein or activity . These data support the hypothesis that dynamin 2 siRNA - transfected dCAD cells are able to accumulate more [ 3 H ] AEA on their cell surface than mock - transfected dCAD cells due to an enrichment of the putative AEA carrier in the microdo - mains . If this is the case , then [ 3 H ] AEA uptake assays may not be an ideal method for characterizing AEA internalization after the knockdown of proteins , such as dynamin 2 , that are impor - tant in recycling events that maintain the composition of the plasma membrane . One alternative explanation for our results involves a possible role for the CB1 cannabinoid receptor in the uptake process . CB1 receptors have been shown to signal through lipid raft domains ( Bari et al . , 2005 ) ; thus , binding to CB1 receptors and subsequent internalization would be one possible mechanism for AEA uptake . Unlike RBL - 2H3 cells , we do not expect that dCAD cells contain caveolin - 1 , but we have preliminary evidence that dCAD cells express caveolin - 3 ( data not shown ) . Thus , caveo - lae - related endocytic processes may still be present in neu - rons and are dependent on intact detergent - resistant mem - brane microdomains . In summary , our data , combined with previous experiments in RBL - 2H3 cells ( McFarland et al . , 2004 ) , offer evidence that endocannabinoids are internalized by a lipid raft - or caveolae - related endocytic process . We speculate that the yet unidentified anandamide transporter will in fact be enriched in lipid rafts and functionally partic - ipate in endocytosis . Acknowledgments We thank Jason Parish for helpful discussion and technical assis - tance regarding TLC analysis of intact [ 3 H ] AEA and the Purdue Cytomics Laboratory for technical assistance with microscopic imaging . AEA Uptake via Endocytosis in dCAD Cells 107 a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m References Altschuler Y , Barbas SM , Terlecky LJ , Tang K , Hardy S , Mostov KE , and Schmid SL ( 1998 ) Redundant and distinct functions for dynamin - 1 and dynamin - 2 isoforms . J Cell Biol 143 : 1871 \u2013 1881 . Bari M , Battista N , Fezza F , Finazzi - Agro A , and Maccarrone M ( 2005 ) Lipid rafts control signaling of type - 1 cannabinoid receptors in neuronal cells . J Biol Chem 280 : 12212 \u2013 12220 . Cameron PL , Ruffin JW , Bollag R , Rasmussen H , and Cameron RS ( 1997 ) Identifi - cation of caveolin and caveolin - related proteins in the brain . J Neurosci 17 : 9520 \u2013 9535 . Cravatt BF , Giang DK , Mayfield SP , Boger DL , Lerner RA , and Gilula NB ( 1996 ) Molecular characterization of an enzyme that degrades neuromodulatory fatty - acid amides . Nature 384 : 83 \u2013 87 . 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Lazaroff M ( 1996 ) A CNS catecholaminergic cell line expresses voltage - gated cur - rents . J Membr Biol 151 : 279 \u2013 291 . Lazaroff M , Qi Y , and Chikaraishi DM ( 1998 ) Differentiation of a catecholaminergic CNS cell line modifies tyrosine hydroxylase transcriptional regulation . J Neuro - chem 71 : 51 \u2013 59 . Matsuda LA , Lolait SJ , Brownstein MJ , Young AC , and Bonner TI ( 1990 ) Structure of a cannabinoid receptor and functional expression of the cloned cDNA . Nature 346 : 561 \u2013 564 . Maxfield FR and McGraw TE ( 2004 ) Endocytic recycling . Nat Rev Mol Cell Biol 5 : 121 \u2013 132 . McFarland MJ , Porter AC , Rakhshan F , Rawat DS , Gibbs RA , and Barker EL ( 2004 ) A role for caveolae / lipid rafts in the uptake and recycling of the endogenous cannabinoid anandamide . J Biol Chem 279 : 41991 \u2013 41997 . 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Suri C , Fung BP , Tischler AS , and Chikaraishi DM ( 1993 ) Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase - SV40 T antigen transgenic mice . J Neurosci 13 : 1280 \u2013 1291 . Voets T and Nilius B ( 2003 ) TRPs make sense . J Membr Biol 192 : 1 \u2013 8 . Wang H and Oxford GS ( 2000 ) Voltage - dependent ion channels in CAD cells : a catecholaminergic neuronal line that exhibits inducible differentiation . J Neuro - physiol 84 : 2888 \u2013 2895 . Address correspondence to : Dr . Eric L . Barker , Department of Medicinal Chemistry and Molecular Pharmacology , Purdue University , 575 Stadium Mall Dr . , West Lafayette , IN 47907 - 2091 . E - mail : ericb @ pharmacy . purdue . edu 108 McFarland et al . a t A SP ET J ou r n a l s on M a r c h 17 , 2015 m o l ph a r m . a s p e tj ou r n a l s . o r g D o w n l o a d e d fr o m", + "pandit2020force": "Force and phosphate release from Arp2 / 3 complex promote dissociation of actin filament branches Nandan G . Pandit a , b \ue840 , Wenxiang Cao a , Jeffrey Bibeau a , Eric M . Johnson - Chavarria a , Edwin W . Taylor a , Thomas D . Pollard a , b , c , d \ue840 , and Enrique M . De La Cruz a , b , 1 a Department of Molecular Biophysics and Biochemistry , Yale University , New Haven , CT 06520 ; b Program in Physical and Engineering Biology , Yale University , New Haven , CT 06520 ; c Department of Molecular , Cellular , and Developmental Biology , Yale University , New Haven , CT 06520 ; and d Department of Cell Biology , Yale University , New Haven , CT 06520 Edited by Harry Higgs , Geisel School of Medicine at Dartmouth , Hanover , NH , and accepted by Editorial Board Member Yale E . Goldman April 16 , 2020 ( received for review June 28 , 2019 ) Networks of branched actin filaments formed by Arp2 / 3 complex generate and experience mechanical forces during essential cellular functions , including cell motility and endocytosis . External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells . Considerably less is known about how mechanical forces influence the disassembly of actin filament networks , specifically , the dissociation of branches . We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2 / 3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy . Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude , from hours to < 1 min . Nei - ther force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching . Arp2 / 3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force , which we name \u201c young / strong \u201d and \u201c old / weak . \u201d The \u201c young / strong \u201d state 1 has adenosine 5 \u2032 - diphosphate ( ADP ) \u2212 P i bound to Arp2 / 3 complex . Phosphate re - lease converts Arp2 / 3 complex into the \u201c old / weak \u201d state 2 with bound ADP , which is 20 times more sensitive to force than state 1 . Branches with ADP \u2212 Arp2 / 3 complex are more sensitive to debranching by fission yeast GMF ( glia maturation factor ) than branches with ADP \u2212 P i \u2212 Arp2 / 3 complex . These findings suggest that aging of branch junctions by phosphate release from Arp2 / 3 complex and mechanical forces contribute to disassembling \u201c old \u201d actin filament branches in cells . actin | Arp2 / 3 complex | branched filament | debranching | force A rp2 / 3 complex forms networks of branched actin filaments that generate and sustain mechanical forces that power cell motility , endocytosis , and vesicle trafficking ( 1 , 2 ) . Membrane - bound proteins , called nucleation - promoting factors , such as WASP activate Arp2 / 3 complex , which then nucleates a branch when it binds to the side of a preexisting \u201c mother \u201d filament ( 3 ) . The new \u201c daughter \u201d filament elongates and pushes against the membrane until it is capped . All of the filaments , including branches formed by Arp2 / 3 complex , must disassemble for recycling to form new filaments and branches . Similar to actin , Arp2 / 3 complex is an ATPase ( 4 \u2013 6 ) . Hydrolysis of bound ATP and subsequent phosphate release have been implicated in controlling branched network dy - namics ( 5 , 7 \u2013 10 ) , but mechanistic details are lacking . The assembly and architecture of branched actin networks are sensitive to force in vitro and in cells ( 11 , 12 ) . Under load , branched actin networks assembled from purified proteins grow more slowly and with a higher branch density ( 13 , 14 ) , but how these mechanical forces directly affect the biochemical interac - tions of branched actin network protein components has not been firmly established . Similar to measurements with purified protein components , branched actin networks in cells respond to external load by increasing density of branched filaments while also reorganizing relative to the membrane ( 15 ) . Networks of branched actin filaments turn over much more rapidly in cells than in vitro when assembled from purified proteins ( 16 \u2013 19 ) . The regulatory proteins cofilin and glia matu - ration factor ( GMF ) accelerate debranching and have been im - plicated in accelerating branched network remodeling and turnover ( 20 \u2013 24 ) . Although not investigated previously , mechanical forces may also affect network disassembly through debranching . We report that mechanical forces promote dissociation of branches formed by Arp2 / 3 complex and that phosphate bound to Arp2 / 3 complex regulates the sensitivity to force . Phosphate release from Arp 2 / 3 complex at branch junctions also regulates debranching by GMF . Thus , phosphate release from the Arp2 / 3 complex could target \u201c older \u201d adenosine 5 \u2032 - diphosphate ( ADP ) \u2212 Arp2 / 3 branches for dissociation while sparing \u201c younger \u201d branches with ADP \u2212 P i \u2212 Arp2 / 3 complex . Results Microfluidics Assay to Measure Dissociation of Branches Formed by Arp2 / 3 Complex under Force . We used fluid flowing through a microfluidics apparatus to apply force to actin filament branches formed by purified fission yeast Arp2 / 3 complex and muscle actin monomers ( Fig . 1 A ) as we observed the dissociation of the branches by fluorescence microscopy . Starting with short fila - ment seeds tethered to the surface of the slide , we assembled branched filaments from purified ATP - actin monomers and Significance Arp2 / 3 complex is an ATPase that binds to the side of a pre - existing actin filament and nucleates an actin filament branch . Growing branched networks experience variable resistance and respond by adapting growth speed , power , and architec - ture . How force influences the dissociation of actin filament branches was not known . We used microfluidics to show that mechanical force promotes the dissociation of actin filament branches and that Arp2 / 3 complex adopts two distinct me - chanical states with different responses to force . Phosphate release from Arp2 / 3 complex increases the sensitivity to both force and the debranching protein GMF . Thus , phosphate re - lease from Arp2 / 3 complex may regulate debranching by force and debranching proteins . Author contributions : N . G . P . and E . M . D . L . C . designed research ; N . G . P . performed re - search ; N . G . P . , W . C . , J . B . , E . M . J . - C . , E . W . T . , T . D . P . , and E . M . D . L . C . contributed new re - agents / analytic tools ; N . G . P . , W . C . , J . B . , E . W . T . , T . D . P . , and E . M . D . L . C . analyzed data ; and N . G . P . , W . C . , J . B . , E . W . T . , T . D . P . , and E . M . D . L . C . wrote the paper . The authors declare no competing interest . This article is a PNAS Direct Submission . H . H . is a guest editor invited by the Editorial Board . Published under the PNAS license . 1 To whom correspondence may be addressed . Email : enrique . delacruz @ yale . edu . Thisarticlecontainssupportinginformationonlineathttps : / / www . pnas . org / lookup / suppl / doi : 10 . 1073 / pnas . 1911183117 / - / DCSupplemental . First published May 27 , 2020 . www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1911183117 PNAS | June 16 , 2020 | vol . 117 | no . 24 | 13519 \u2013 13528 B I O P H Y S I C S A ND C O M P U T A T I O N A L B I O L O G Y Arp2 / 3 complex for 2 min to 4 min . After washing out the soluble proteins , the filaments were allowed to \u201c age \u201d for an additional , variable time with very slow fluid flow . Thereafter , we started the debranching process under constant force by flowing buffer over the surface at a rate of 2 \u03bc L \u00b7 min \u2212 1 to 500 \u03bc L \u00b7 min \u2212 1 until the end of the experiment , while we recorded a series of images . The seeds were immobilized on the slide , but both the mother fila - ments and branches were free to fluctuate ( Fig . 1 A ) . Rapid flow rates flattened branches against the mother fila - ments until they dissociated ( Fig . 1 B and Movies S1 \u2013 S3 ) . Neither the angle of the applied force ( relative to mother filament ori - entation ) , the tension in the mother filament , nor fluctuations in mother filament shape had a strong influence on debranching ( SI Appendix , Fig . S1 and Movies S6 and S7 ) . We added a snap tag to the Arpc5 subunit of Arp2 / 3 complex for labeling with Alexa 488 and simultaneous viewing with Alexa 647 - labeled actin . Force dissociated Arp2 / 3 complex and the daughter filament concurrently , within the 0 . 1 - s time resolution of our imaging ( Fig . 1 D and Movies S4 and S5 ) . Buffer flowed across the sample at 500 \u03bc m \u00b7 s \u2212 1 dissociated both the daughter filament and Arp2 / 3 complex from the field of view by the next frame , making it impossible to determine whether the labeled Arp2 / 3 complex remained bound to the dissociated daughter filament . Piconewton Forces Decreased the Time for Branch Dissociation from Hours to < 1 Min . Branches formed by ATP \u2212 Arp2 / 3 com - plex were stable for many minutes without buffer flow ( Fig . 2 A ) but had a higher probability of dissociating when subjected to the forces produced by the range of buffer flow rates in our exper - iments ( Fig . 2 A ) . The following sections explain how the time course of debranching depends on the applied force and how long the newly formed branches were aged after assembly . At a given flow rate , the force on a branch scales with its length . In some experiments , we show the force on individual branches ( SI Appendix , Fig . S2 ) , but , in most experiments , we report flow rates from which we calculate the average force on branches ( e . g . , Fig . 2 A ) . In samples of branches formed by ATP \u2212 Arp2 / 3 complex and aged for 30 min , the time course of debranching followed a single exponential that depended on the applied force ( Fig . 2 A ) . The observed lifetimes of these branches decreased with force ( Fig . 2 D ) , suggesting a slip bond behavior , so we used the Bell \u2019 s equation ( 25 ) ( Eq . 1 ) to estimate the force sensitivity , \u03c4 obs = \u03c4 0 e \u2212 FdkBT . [ 1 ] Here k B is the Boltzmann constant , T is the absolute tempera - ture , F is force , \u03c4 0 is the branch lifetime in the absence of force , and d is the characteristic distance to the transition state ( 26 ) . The value of d is typically considered a force sensitivity param - eter for bond rupture . An alternative estimate of force depen - dence is the force that reduces the branch lifetime by half ( half - force , F 1 / 2 = 0 . 693 k B T / d ) , which we estimate to be 0 . 054 ( \u00b1 0 . 008 ) pN . Without force , the branch lifetime ( \u03c4 0 ) , estimated from ex - trapolation of the fit of the force dependence ( Fig . 2 D ) , was 106 ( \u00b1 8 ) min [ Table 1 ; k 2 \u2248 1 / \u03c4 0 = 0 . 01 ( \u00b1 0 . 0007 ) min \u2212 1 ] , indicating - 2 0 2 4 time ( s ) - 20 0 20 40 60 80 100 y t i s ne t n I e c ne cs e r ou l F ARP2 / 3 BackgroundDebranching - 2 0 2 4 time ( s ) 0 100 200 300 400 500 y t i s ne t n I e c ne cs e r ou l F Actin Branch Debranching A w o l F 2 \u00b5 L m i n - 1 ( S l o w ) w o l F 500 \u00b5 L m i n - 1 ( F a s t ) 0 s 30 s 300 s B C No Flow Flow D Debranching Glass coated with sparse biotin - BSA ( blue ) to anchor the mother filament via neutravidin ( orange ) Biotinylated Alexa - 568 actin filament seed bound to surface Freely fluctuating Alexa - 647 actin filaments grown from seed Branch formed by Arp2 / 3 complex Pointed End Barbed End Arp2 / 3 complex ointed End Arp2 / 3complexx Fig . 1 . Microfluidics assay to measure Arp2 / 3 complex debranching under force . ( A ) Diagram showing a short segment of an actin filament containing 10 % biotinylated and 15 % Alexa 568 - labeled ( red ) actin subunits immobilized on the neutravidin - coated surface . The surface is passivated with 0 . 2 % tween ( illustrated with gray vertical lines ) . This seed was elongated at its barbed end with 1 . 5 \u03bc M 15 % Alexa 647 - labeled Mg - ATP - actin ( green ) , and Arp2 / 3 complex formed a branch with Alexa 647 - labeled Mg - ATP - actin . The green filaments fluctuate freely and are subject to viscous drag forces applied by fluid flow . ( B ) TIRF microscopy images of representative branched filaments under slow flow ( 2 \u03bc L \u00b7 min \u2212 1 , \u223c 0 . 004 pN of force for a 1 . 5 - \u03bc m branch ; Top ) and fast flow ( 500 \u03bc L \u00b7 min \u2212 1 , \u223c 1 . 02 pN of force for a 1 . 5 - \u03bc m branch ; Bottom ) . Branches are aligned in the direction of flow . ( Scale bar , 1 \u03bc m . ) ( C ) The Arpc5 subunit of the Arp2 / 3 complex was labeled with Alexa 488 via snap tag and tracked during debranching . Time - lapse images with the actin filaments represented in red and the Alexa 488 \u2212 Arp2 / 3 complex located at the junction of the daughter branch and mother filament represented in green . ( Scale bar , 1 \u03bc m . ) ( D ) Top shows the spatially integrated fluorescence intensity of actin at a branch junction as a function of time , used to determine the ob - served debranching event time ( t = 0 ) . Middle shows a kymograph measured across a branched actin filament . Bottom shows the time course of spatially integrated fluorescence intensity of Arpc5 subunit at a branch junction with time aligned to its corresponding actin frame . The fluorescence intensity from Arp2 / 3 complex reproducibly decreased in a single step for all 12 debranching events observed . 13520 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1911183117 Pandit et al . that sustained pN forces reduce \u03c4 obs more than two orders of magnitude to < 30 s . The force dependence of individual branch lifetimes ( SI Appendix , Fig . S2 ) yielded a half - force ( F 1 / 2 ) of 0 . 054 ( \u00b1 0 . 008 ) pN and \u03c4 0 = 96 ( \u00b1 6 ) min , comparable to the values estimated from exponential fits of debranching time courses ( Fig . 2 A and D ) . Branches Assembled from ATP \u2212 Arp2 / 3 Complex Dissociate Faster under Force as They Age . As they age , branches assembled from ATP \u2212 Arp2 / 3 complex dissociated progressively faster under force produced by a buffer flow rate of 500 \u03bc L \u00b7 min \u2212 1 ( Fig . 2 B ) . Immediately after the 2 . 6 - min assembly reaction , when the dom - inant nucleotide bound to both Arp2 / 3 complex and the actin filaments is expected to be ADP \u2212 P i , the time course of branch dissociation followed a single exponential with a slow observed lifetime ( \u03c4 s , F , where the subscript F indicates under flow force ) of 3 . 08 ( \u00b1 0 . 05 ) min corresponding to a first - order rate constant k s , F of 0 . 32 ( \u00b1 0 . 005 ) min \u2212 1 . After aging for 30 min , when both Arp2 / 3 complex and the subunits in the actin filaments are expected to have bound ADP , the time course of debranching also followed a single exponential with a 20 - fold shorter lifetime ( \u03c4 f , F ) of 0 . 15 ( \u00b1 0 . 01 ) min ( k f , F = 6 . 67 ( \u00b1 0 . 44 ) min \u2212 1 ) . At intermediate aging times , the time courses followed double exponentials ( Fig . 2 B ) with distinguishable fast and slow phases , indicating that ( at least ) two reactions contributed to debranching . To evaluate how force affects slow and fast debranching , we aged samples for a short time ( \u223c 4 min ) , so the sample would include mixtures of branches with Arp2 / 3 complex with bound A B C D Fig . 2 . Effects of mechanical force and nucleotide bound to Arp2 / 3 complex on the time course of dissociation of actin filament branches . Arp2 / 3 complex branches were assembled in the flow chamber before applying flow as described in Materials and Methods . In A and C , the force on each observed branch was calculated from its length and the flow rate , and then binned at the indicated average forces values ( see Materials and Methods ) . ( A ) The effect of force on the time course of dissociation of actin filament branches formed by ATP - actin monomers and ATP \u2212 Arp2 / 3 complex and aged for 30 min , when most branches had ADP bound to Arp2 / 3 complex . The fraction of branches remaining is plotted . Smooth curves are the best fits of single exponentials to the data . Each trace includes at least 14 branches . ( B ) Dependence of the time course of dissociation of branches formed with ATP - actin monomers and ATP \u2212 Arp2 / 3 complex with different aging times ( 2 . 6 , 4 , 12 , and 30 min ) and the presence of BeF x . For all time courses , 500 \u03bc L \u00b7 min \u2212 1 of buffer flow was applied to the branches for debranching , producing a force of \u223c 1 pN for a branch of 1 . 5 \u03bc m . The force on each branch was not calculated for the data shown . Smooth curves are the best global fits of double exponentials to the data for aging branches and yielded two shared rate constants for debranching : slow k s , F = 0 . 32 ( \u00b1 0 . 005 ) min \u2212 1 and fast k f , F = 6 . 67 ( \u00b1 0 . 44 ) min \u2212 1 . Smooth curves are the best single exponential fits to the data for branches aged for 4 and 30 min with BeF x with lifetimes of 13 . 9 ( \u00b1 0 . 2 ) min \u2212 1 for the sample aged for \u223c 4 min and 14 . 8 ( \u00b1 0 . 09 ) min \u2212 1 for the sample aged for 30 min . Fig . 4 D presents the fractional amplitudes obtained from the double exponential fits . ( C ) The effect of force on the time course of the dissociation of actin filament branches formed by ATP - actin monomers and ATP \u2212 Arp2 / 3 complex and aged for \u223c 4 min using the same data collection and analysis methods as in A . The smooth curves are best fits of the data to single ( F \u2264 0 . 6 pN ) or double ( F > 0 . 6 pN ) exponentials . The fractional amplitudes of the slow phase in the time courses that follow the double exponentials are 0 . 71 \u00b1 0 . 05 ( F = 0 . 98 pN ) , 0 . 39 \u00b1 0 . 06 ( F = 1 . 29 pN ) , 0 . 81 \u00b1 0 . 05 ( F = 1 . 60 pN ) , 0 . 38 \u00b1 0 . 02 ( F = 1 . 93 pN ) , and 0 . 17 \u00b1 0 . 02 ( F = 2 . 86 pN ) . Each trace includes at least 19 branches . ( Inset ) Time courses of branch dissociation under a range of forces for branches formed from ATP \u2212 Arp2 / 3 complex in the presence of 2 mM BeF x and aged for \u223c 4 min . Each trace includes at least 13 branches . The smooth curves are single exponential fits to the time courses . Branches dissociate slowly under 0 . 2 pN of force , so the debranching time course cannot be reliably fitted to obtain the branch lifetime . ( D ) Dependence of branch lifetimes on force for four different conditions : ( filled black circles ) branches formed from ATP \u2212 Arp2 / 3 complex and aged for 30 min to form branches with ADP \u2212 Arp2 / 3 complex ( time courses in A ) ; ( filled red squares ) branches formed from ADP \u2212 BeF x \u2212 Arp2 / 3 complex and aged for \u223c 4 min ( time courses in C , Inset ) ; ( filled pink triangles ) slow debranching phase of branches formed from ATP \u2212 Arp2 / 3 complex and aged for \u223c 4 min ( ADP \u2212 P i branch population ; time courses in C ) ; and ( filled blue triangle ) fast debranching phase of branches formed from ATP \u2212 Arp2 / 3 complex and aged for \u223c 4 min ( ADP branch population ; time courses in C ) . The uncertainty bars for all data represent the SDs from the fits to exponentials shown in A and C . The smooth black curve is the best single exponential fit ( Eq . 1 ) to the ADP \u2212 Arp2 / 3 complex debranching data points , yielding a half - force ( F 1 / 2 ) of 0 . 054 ( \u00b1 0 . 008 ) pN and branch lifetime in the absence of force ( \u03c4 0 ) of 106 ( \u00b1 8 ) min ( observed rate constant = \u03c4 0 \u2212 1 = 0 . 01 ( \u00b1 0 . 007 ) min \u2212 1 ) . Inset shows that 1 ) fast phase lifetimes ( blue triangles ; 0 . 3 min to 0 . 8 min at F > 1 pN ) differ from the slow phase lifetimes ( pink triangles ; 2 min to 4 min at F > 1 pN ; t = 6 . 85 , one - tail t critical = 2 . 13 and P = 0 . 001 by Welch \u2019 s unequal variances t test ) ; 2 ) the slow phase lifetimes ( pink triangles ; 2 min to 4 min at F > 1 pN ) differ from the debranching lifetimes with BeF x ( red squares ; 8 min to 9 min at F > 1 pN ; t = 6 . 65 , one - tail t critical = 2 . 02 and P = 0 . 0006 by Welch \u2019 s unequal variances t test ) ; and 3 ) the fast phase lifetimes ( blue triangles ) do not differ significantly from the debranching with ATP aged for 30 min lifetimes ( black circles ) at F > 1 pN ( t = \u2212 0 . 86 , two - tail t critical = 2 . 45 and P = 0 . 42 by Welch \u2019 s unequal variances t test ) . Pandit et al . PNAS | June 16 , 2020 | vol . 117 | no . 24 | 13521 B I O P H Y S I C S A ND C O M P U T A T I O N A L B I O L O G Y ADP \u2212 P i and ADP and thus exhibit both slow and fast phases of dissociation over a range of forces applied with different flow rates . However , the actual aging times prior to making obser - vations varied . This uncertainty influenced the observed ampli - tudes but not the observed lifetimes , so we only analyzed the lifetimes . The debranching time courses at forces of > 1 pN fol - lowed double exponential decays ( Fig . 2 C ) , yielding the force dependence of the slow and fast phase lifetimes ( Fig . 2 D and Inset ; triangles ) . The lifetimes of the slow phase ( 2 - to 4 - min range ) differed from the fast phase ( 0 . 3 min to 0 . 8 min ) in this force range ( > 1 pN ) , but neither depended strongly on the ap - plied force . At low forces of < 0 . 6 pN , dissociation of branches formed from ATP \u2212 Arp2 / 3 complex followed single exponentials , with lifetimes similar to branches with ADP \u2212 Arp2 / 3 complex ( Fig . 2 D ) . The debranching model presented below accounts for these different time courses . The fast phase lifetime behaved simi - lar to ADP \u2212 Arp2 / 3 complex branches ( i . e . , assembled from ATP \u2212 Arp2 / 3 complex and aged for 30 min ) , consistent with fast debranching population corresponding to ADP \u2212 Arp2 / 3 complex branches . We discuss the force dependence of the slow phase below . The Nucleotide Bound to Arp2 / 3 Complex Influences the Sensitivity of Branches to Force . ATP \u2212 Arp2 / 3 complex hydrolyzes the bound nucleotide upon or soon after branch formation ( 5 , 7 ) followed by dissociation of the \u03b3 - phosphate with an unknown rate con - stant . Therefore , branches formed by ATP \u2212 Arp2 / 3 complex rapidly transition to the ADP \u2212 P i state for an unknown duration before the release of P i . Actin filament branches formed by Arp2 / 3 complex with mutations that slow ATP hydrolysis are more stable than those formed by native Arp2 / 3 complex , so Martin et al . ( 10 ) proposed that the hydrolysis of ATP bound to Arp2 / 3 complex destabilizes branches . We performed a series of experiments to determine whether the nucleotide state of Arp2 / 3 complex could explain the slow and fast debranching states ( Fig . 2 B and C ) . Like Acanthamoeba Arp2 / 3 complex ( 5 ) , Schizosaccharomyces pombe ADP \u2212 Arp2 / 3 complex did not form branches from ATP - actin monomers nor did the ADP \u2212 Arp2 / 3 complex form branches in the presence of 20 mM phosphate ( SI Appendix , Fig . S3 ) . A likely interpretation of this behavior is that the transient intermediate ADP \u2212 P i \u2212 Arp2 / 3 complex is competent to form branches , but ADP \u2212 Arp2 / 3 com - plex binds P i very weakly ( K d > 20 mM ) . On the other hand , S . pombe ADP \u2212 Arp2 / 3 complex formed branches with 2 mM be - ryllium fluoride ( BeF x ) in the buffer ( SI Appendix , Fig . S3 ) , as originally shown for Acanthamoeba Arp2 / 3 complex ( 5 ) . ATP \u2212 Arp2 / 3 complex also forms branches with 2 mM BeF x in the buffer . Branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex dissociate with indistinguishable time courses whether assembled from ATP \u2212 Arp2 / 3 complex or ADP \u2212 Arp2 / 3 complex in the presence of 2 mM BeF x and aged for \u223c 4 min ( SI Appendix , Fig . S4 ) . We assume that , in both cases , BeF x binds to the ADP \u2212 Arp2 / 3 complex and stabilizes conformations similar to ADP \u2212 P i , as established for actin ( 27 , 28 ) , so we used ADP \u2212 BeF x \u2212 Arp2 / 3 complex branches made with either method . ADP \u2212 BeF x \u2212 Arp2 / 3 complex branches aged for 4 or 30 min in the presence of 2 mM BeF x dissociate with similar time courses and follow single exponen - tials ( Fig . 2 B ) . Thus , branches did not convert from the slowly to the rapidly dissociating state when aged with BeF x . Force is required to dissociate , within our experimental ob - servation period , ADP \u2212 BeF x \u2212 Arp2 / 3 complex branches formed from ATP \u2212 Arp2 / 3 complex in the presence of 2 mM BeF x ( Fig . 2 C , Inset ) . Under low forces ( i . e . , < < 0 . 6 pN ) , < 20 % of branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex dissociated within 5 h , so branching lifetimes could not be measured reliably . With forces > 0 . 6 pN , branches dissociated with single exponential time courses and observed rate constants ( inverse of lifetimes ) that depended on the applied force ( Fig . 2 C , Inset ) . Therefore , branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex were more stable under force than branches with ADP \u2212 Arp2 / 3 complex ( formed from ATP \u2212 Arp2 / 3 complex and aged for 30 min ; Fig . 2 D ) . Under a given force , the lifetimes of ADP \u2212 BeF x \u2212 Arp2 / 3 complex branches were longer ( \u223c twofold ) than the slow debranch - ing phase of ATP \u2212 Arp2 / 3 complex branches that had been aged for \u223c 4 min ( Fig . 2 D ) . The observed rate constant of the slow phase debranching reflects the sum of the ADP \u2212 P i state debranching rate constant and the rate constant for conversion of the ADP \u2212 P i \u2212 Arp2 / 3 complex to the ADP state ( see Discussion ) . It is therefore expected to be faster than ADP \u2212 BeF x \u2212 Arp2 / 3 complex debranching , which converts more slowly to ADP \u2212 Arp2 / 3 complex ( Fig . 2 B ) . The Nucleotide State of the Actin Filaments Does Not Influence Debranching . We assembled branches from ATP \u2212 Arp2 / 3 com - plex with ADP or ADP \u2212 P i bound to the subunits in the actin filaments and observed that all had similar time courses of debranching after aging for a given time ( SI Appendix , Fig . S5 ) . Samples with ADP - actin filaments were prepared by assembly from ATP - actin monomers and ATP - actin Arp2 / 3 complex fol - lowed by aging for 30 min . Samples with ADP \u2212 P i actin filaments were prepared by assembly from ATP - actin monomers and ATP \u2212 Arp2 / 3 complex in buffer containing 20 mM phosphate followed by aging for 4 or 30 min ( SI Appendix , Fig . S5 ) . This concentration of phosphate is well above the K d for binding ADP - actin subunits ( 29 ) , so subunits in the filaments likely had ADP \u2212 P i in the active site , assuming that Arp2 / 3 complex binding does not dramatically change the affinity of actin for phosphate . Samples assembled from ATP - actin monomers and ATP \u2212 Arp2 / 3 complex followed by \u223c 4 min of aging had a mixture of ADP and ADP \u2212 P i nucleotide states for both actin subunits and Arp2 / 3 complex . Samples with AMPPNP actin ( a nonhydrolyzable an - alog of ATP ) were assembled from AMPPNP actin monomers and ATP - actin Arp2 / 3 complex in buffer containing 2 mM AMPPNP followed by aging for \u223c 4 min . Since AMPPNP \u2212 Arp2 / 3 complex does not form branches ( ref . 5 and SI Appendix , Fig . S3 ) , all of the branches formed from Arp2 / 3 complex with bound ATP , while the actin filaments had bound AMPPNP . A previous study of bovine Arp2 / 3 complex ( 16 ) reported more branches on mother filament segments with ADP \u2212 P i than segments with ADP . This difference was attributed to slower dissociation of branches aged in buffer with 25 mM phosphate allowing time for them to be stabilized by binding to the slide coated with N - ethylmaleimide ( NEM ) - myosin anchors . However , that study did not measure time course of the dissociation of branches , and we did not compare the rate of branch formation on ADP and ADP \u2212 P i mother filaments , so we do not know whether the sources of Arp2 / 3 complex , the presence of NEM - myosin an - chors on the surface , or other factors account for the apparent difference . Table 1 . Rate constants for branch formation and dissociation in the absence of force and related figures Conversion k conv ( min \u2212 1 ) State 1 debranching k 1 * ( min \u2212 1 ) State 2 debranching k 2 \u2020 ( min \u2212 1 ) Branch formation k form * ( \u03bc M \u2212 1 \u00b7 s \u2212 1 ) 0 . 14 \u00b1 0 . 03 0 . 012 0 . 01 \u00b1 0 . 007 0 . 02 Fig . 4 Fig . 4 Fig . 2 D Fig . 4 * The equations used to determine these parameters are approximations , so uncertainties are not reported . \u2020 From the fit of force - dependent debranching data to Eq . 1 ; k 2 = 1 / \u03c4 0 . The error was calculated from the SD \u03c4 0 in the fit . 13522 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1911183117 Pandit et al . Since the debranching kinetics are independent of the nucle - otide ( i . e . , ADP , ADP \u2212 P i , or AMPPPNP ) bound to the mother and daughter filaments , the stabilization of branches by BeF x ( Fig . 2 B \u2013 D ) is likely due to BeF x bound to Arp2 / 3 complex in the branch junction . This suggests that the ADP \u2212 P i \u2212 Arp2 / 3 complex intermediate has different mechanical properties than the ADP \u2212 Arp2 / 3 complex after phosphate dissociation . The lack of an effect of 20 mM phosphate on debranching ( SI Appendix , Fig . S5 ) is consistent with the affinity of S . pombe ADP \u2212 Arp2 / 3 complexes in branch junctions for phosphate being very weak ( K d > 20 mM ) . Force Promotes but BeF x Inhibits Debranching by GMF . GMF ( 20 \u2013 22 ) promotes the dissociation of actin filament branches without applied force , so we measured how the concentration of fission yeast GMF influences the rates of dissociation of actin filament branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex or ADP \u2212 Arp2 / 3 complex at a low buffer flow rate ( 15 \u03bc L \u00b7 min \u2212 1 ) . This flow rate exerts very little force on branches and had little or no effect on debranching ( Fig . 2 ) . Concentrations of GMF up to 1 \u03bc M did not dissociate branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex ( Fig . 3 A ) , but nanomolar con - centrations of GMF promoted dissociation of branches with ADP \u2212 Arp2 / 3 complex ( Fig . 3 ) . Time courses of dissociation of branches with ADP \u2212 Arp2 / 3 complex followed single exponentials ( Fig . 3 A ) with lifetimes ( \u03c4 obs ) that depended hyperbolically on the concentration of GMF ( Fig . 3 B ) . Force increased the rate at which GMF dissociated branches with ADP \u2212 Arp2 / 3 complex ( SI Appendix , Fig . S6 ) , but dissoci - ation was slower than predicted if GMF and force increased the rate of dissociation independently ( i . e . , their energetic contri - butions to debranching were additive ) . This raises the possibility that the reactions catalyzed by force and GMF are coupled and / or that debranching follows different pathways in the presence absence of force . Discussion Quantitative Analysis of the Two - State Model for Dissociation of Actin Filament Branches . Our experiments show that the nucleo - tide bound to Arp2 / 3 complex determines the sensitivity of branches to dissociation by physical force . We use a formal de - scription of a simple two - state model ( Fig . 4 A ) to analyze our data and estimate the rate constants for the three reactions and their sensitivities to force . Description of the model . The model ( Fig . 4 A ) assumes that branches form when ATP \u2212 Arp2 / 3 complex binds to the side of a mother filament and nucleates a daughter filament growing at its barbed end . Hydrolysis of ATP in the active sites of Acantha - moeba Arp2 / 3 complex is closely associated with nucleation of the daughter filament ( 5 , 7 ) , so we assume that state 1 branches have Arp2 / 3 complex with bound ADP \u2212 P i . Dissociation of the \u03b3 - phosphate with a rate constant k conv converts branch state 1 to branch state 2 with ADP bound to Arp2 / 3 complex . Branches in either state can dissociate from the mother filament . The model predicts that , overall , net debranching time courses follow dou - ble exponentials with two rate constants . One observed rate constant is the sum of the rate constants for state 1 debranching and conversion ( k obs , 1 ( F ) = k 1 ( F ) + k conv ( F ) ; ( F ) indicates optional force ) . The second observed rate constant corresponds to debranching of state 2 ( k obs , 2 ( F ) = k 2 ( F ) ; SI Appendix , Eqs . S25 and S29 ) . Force accelerates the dissociation of branches with either ADP \u2212 Arp2 / 3 and ADP \u2212 P i \u2212 Arp2 / 3 complex , but branches with ADP \u2212 P i or ADP \u2212 BeF x \u2212 Arp2 / 3 ( mimicking the ADP \u2212 P i state ) complex are much more resistant to force than branches with ADP \u2212 Arp2 / 3 complex . As branches age , phos - phate release converts slowly dissociating , \u201c young and strong \u201d state 1 branches with ADP \u2212 P i \u2212 Arp2 / 3 complex into rapidly dissociating , \u201c old and weak \u201d state 2 branches with ADP \u2212 Arp2 / 3 complex . Formulation of the model . We analyzed the experimental data us - ing a parallel debranching pathway ( Fig . 4 A and SI Appendix , Part 2 and Scheme S1 ) where \u201c young \u201d ( strong , state 1 ) branches convert to \u201c old \u201d ( weak , state 2 ) branches as they age ( Fig . 4 A ) . We fit the aging time dependence of the fractions of slowly and rapidly dissociating branches observed under force as the amplitudes ( A s , F and A f , F , respectively ; Fig . 4 C ) of double A B Fig . 3 . BeF x inhibits debranching by GMF . ( A ) Dependence of the time course of dissociation of branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex or ADP \u2212 Arp2 / 3 complex on the concentration of GMF . Branches with ADP \u2212 - BeF x \u2212 Arp2 / 3 complex were assembled in buffer containing 0 . 2 mM ATP , 2 mM BeSO 4 , and 10 mM NaF and aged for \u223c 4 min . Branches with ADP \u2212 Arp2 / 3 complex were assembled in buffer with 0 . 2 mM ATP and aged for 30 min to allow for ATP hydrolysis and phosphate dissociation ( Fig . 2 B ) . Debranching was initiated by flowing buffer with GMF at 15 \u03bc L \u00b7 min \u2212 1 and continued throughout the debranching measurements . The smooth curves are the best fits of single exponentials to the data , yielding the ( average ) branch lifetimes ; n = 30 branches for each trace . A concentration of 1 \u03bc M GMF did not dissociate branches with ADP \u2212 BeF x \u2212 Arp2 / 3 complex assembled from ATP - actin and ATP \u2212 Arp2 / 3 complex with BeF x and aged for \u223c 4 min ( open black circles ) . ( B ) Dependence of the lifetimes of branches with ADP \u2212 Arp2 / 3 complex on the concentration of GMF at a buffer flow rate of 15 \u03bc L \u00b7 min \u2212 1 . The line is the best fit of Eq . 3 to the data , yielding a GMF binding affinity ( K d , GMF ) of 40 ( \u00b1 10 ) nM and a maximum debranching rate constant ( k diss , GMF ) of 0 . 31 ( \u00b1 0 . 05 ) min \u2212 1 . At this low flow rate , branches with ADP \u2212 Arp2 / 3 complex ( without GMF ) dissociated with a rate constant ( k diss , 0 = 0 . 014 \u00b1 0 . 0002 min \u2212 1 , indicated by an open circle ) similar to that under zero force ( Fig . 2 B and Table 1 ) . The uncertainty bars are within the data points and represent the SDs of lifetimes in the best single exponential fits of time traces in A . Pandit et al . PNAS | June 16 , 2020 | vol . 117 | no . 24 | 13523 B I O P H Y S I C S A ND C O M P U T A T I O N A L B I O L O G Y exponential fits to the following functions ( SI Appendix , Eqs . S34 and S37 ) : A s , F = be \u2212 ( k conv + k 1 \u2212 k 2 ) ( t age \u2212 t 1 ) 1 + ae \u2212 ( k 1 + k conv \u2212 k 2 ) ( t age \u2212 t 1 ) A f , F = 1 + ( a \u2212 b ) e \u2212 ( k conv + k 1 \u2212 k 2 ) ( t age \u2212 t 1 ) 1 + ae \u2212 ( k 1 + k conv \u2212 k 2 ) ( t age \u2212 t 1 ) = 1 \u2212 A s , F [ 2 ] where t 1 is the 2 . 6 min during which branches formed from ATP \u2212 Arp2 / 3 complex and ATP - actin monomers , t age is aging time , the intrinsic debranching rate constants are k 1 for state 1 and k 2 for state 2 in the absence of force , and k conv is the rate constant for conversion of state 1 to state 2 in the absence of force ( Fig . 4 A and SI Appendix , Part 2 and Scheme S1 ) . The constants a and b are unitless composites of rate constants and initial branch concentrations defined in SI Appendix , Part 2 and Scheme S1 ( SI Appendix , Eqs . S39 and S40 ) . The constant a is force independent and determined only by intrinsic debranching in the absence of an applied force ( SI Appendix , Eq . S39 ) . The constant b is a function of debranching and conversion under force ( SI Appendix , Eq . S40 ) , yielding force - dependent ampli - tudes ( A s , F and A f , F ) when debranching and / or conversion are force dependent . Estimation of the rate constants for branch formation and phosphate dissociation . Global analysis of the data ( Figs . 2 B and 4 C and SI Appendix , Part 2 ) yielded an overall pseudo \u2212 first - order associa - tion rate constant for branch formation of 0 . 12 min \u2212 1 ( SI Ap - pendix , Part 2 ) . Given 100 nM Arp2 / 3 complex used in our experiments , we estimate the second - order association rate constant for Arp2 / 3 complex binding to mother filaments and subsequent activation ( k form ) to be \u223c 0 . 02 \u03bc M \u2212 1 \u00b7 s \u2212 1 ( Table 1 ) , consistent with published reports ( 30 \u2013 32 ) . The best fit of the fractional amplitudes to Eq . 2 ( Fig . 4 C ) yielded a rate constant ( k conv ) of 0 . 14 ( \u00b1 0 . 03 ) min \u2212 1 ( corre - sponding to a lifetime of > 7 min ) for conversion of state 1 branches to state 2 branches . We interpret the conversion re - action as the release of P i from the Arp2 / 3 complex , and thus interpret state 2 branches as ADP \u2212 Arp2 / 3 complex branches . This rate constant inferred for P i release from ADP - P i Arp2 / 3 complex is similar to the rate constant of \u223c 0 . 18 min \u2212 1 for P i release from actin filaments ( 29 ) . We can place a limit on the rate constant for phosphate binding to ADP - Arp2 / 3 complex in a branch junction ( k + P i ) . We interpret conversion to reflect P i release , so k \u2212 P i = k conv = 0 . 14 ( \u00b1 0 . 03 ) min \u2212 1 , and know that P i binds Arp2 / 3 complex in branches with a low affinity ( K d > 20 mM ; SI Appendix , Fig . S5 A ) . Therefore , the second order association rate constant of k + P i is less than 7 M \u2212 1 min \u2212 1 = 0 . 12 M \u2212 1 s \u2212 1 . This value is two orders of magnitude slower than P i binding to ADP - actin subunits in the interior of filaments [ 600 M \u2212 1 min \u2212 1 = 10 M \u2212 1 s \u2212 1 ( 29 ) ] . Estimation of the rate constants for branch dissociation at low force . The rate constant for dissociation of state 2 branches at zero force is k 2 \u2248 0 . 01 min \u2212 1 , based on the intercept of the force - dependence of ADP - Arp2 / 3 complex ( state 2 ) branch lifetimes ( Fig . 2 D ) . The rate constant for debranching from state 1 at zero force ( k 1 ) is \u223c 0 . 012 min \u2212 1 , from the analysis of the aging time - dependence of State 1 Arp2 / 3 - ADP - P i State 2 Arp2 / 3 - ADP Arp2 / 3 - ATP 0 50 100 150 200 250 300 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 state 1 ) de z il a m r on ( s eh c na r b # Aging time ( min ) state 2 net debranching 0 5 10 15 20 25 30 0 . 0 0 . 5 1 . 0 state 1 state 2 f o r m a t i on 0 10 20 30 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 e c r o f r ednu edu t il p m a . c a r F Aging time ( min ) slow / strong fast / weak A B C Fig . 4 . Model and simulations of the pathways of branch formation , aging , and debranching . ( A ) Schematic of our hypothesis . Formation of a branch by ATP \u2212 Arp2 / 3 complex ( red ) is coupled to hydrolysis of ATP bound to the Arps ( 5 , 7 ) with rate constant k form , yielding ADP \u2212 P i \u2212 Arp2 / 3 complex ( orange ) in state 1 . Irreversible phosphate dissociation with rate constant k conv converts state 1 to ADP \u2212 Arp2 / 3 complex ( blue ) in state 2 . Branches dissociate from mother filaments with rate constants k 1 for state 1 and k 2 for state 2 , both sensitive to force . ( B ) Simulated time course of the model showing how the populations of state 1 branches , state 2 branches , and dissociated branches evolve over time in the absence of force . We assumed that Mg - ATP - actin monomers and Mg - ATP \u2212 Arp2 / 3 complex formed branches for 2 . 6 min , when the free proteins were removed , and the reactions continued without ad - ditional branch formation . The red line represents the best single expo - nential fit to the observed debranching ( i . e . , combined from both states ) starting with a normalized value of 1 at the end of branch formation ( 2 . 6 min ) . The experimentally determined or estimated rate constants used in the simulation are k form [ Arp ] = 0 . 12 min \u2212 1 , k conv = 0 . 14 min \u2212 1 , k 1 = 0 . 012 min \u2212 1 , and k 2 = 0 . 01min \u2212 1 . ( C ) Aging time dependence of the fractional the slow and fast phase amplitudes in the debranching time courses under 500 \u03bc L \u00b7 min \u2212 1 of buffer flow , obtained from double exponential fits to the time courses ( Fig . 2 B ) . These data were used for extraction of fundamental rate constants in Table 1 . The best global fits of the two - state model ( Eq . 2 ) to the fractional amplitude data gave rate constants for conversion k conv of 0 . 14 min \u2212 1 , state 1 branch dissociation k 1 of 0 . 012 s \u2212 1 , and branch formation k form , of 0 . 02 \u03bc M \u2212 1 \u00b7 s \u2212 1 without force ( Table 1 ) . The one negative fast phase amplitude at 2 . 6 min results from a net increase in the state 2 branch population during debranching under force after 2 . 6 min aging time . The state 2 branch population is the net sum of depletion from debranching ( negative contri - bution to population , exponential decay ) and gain from conversion of state 1 branches ( positive contribution , exponential rise ) . For short aging times , little or no state 2 branches exist for depletion , and the conversion from state 1 branches , represented by an exponential rise ( negative amplitude ) , dominates the time course ( 54 , 55 ) . The uncertainty bars are SDs of the fractions of branches from the global double exponential fits of debranch - ing time courses with different aging times in Fig . 2 B . 13524 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1911183117 Pandit et al . time course of dissociation of branches ( Figs . 2 B and 4 C , Ta - ble 1 , and SI Appendix , Part 2 and Eq . S42 ) . As a result of these two rate constants being similar ( k 1 \u2248 k 2 ) , debranching without force follows a single exponential with a rate constant of \u223c k 2 ( SI Appendix , Part 2 and Eq . S25 and Fig . 4 B ) , even though three reactions ( conversion and debranching from state 1 and state 2 ) occur simultaneously . Time courses also follow single exponentials at low forces ( < 0 . 6 pN ) , independent of the aging time ( Fig . 2 C and D ) . This behavior arises because dissociation is much slower than con - version ( k 1 < < k conv ) , so ADP - P i branches convert to ADP be - fore dissociating . At forces > 1 pN , the observed dissociation time courses follow double exponentials because k conv < k 1 and debranching occurs from both ADP - P i and ADP branches . Estimation of the force sensitivity of the branch dissociation rate constants . Force increases the dissociation rate constants of both branch states , but has a larger effect on state 2 branches with ADP - Arp2 / 3 complexes than state 1 branches with ADP - P i - Arp2 / 3 complexes ( Fig . 2 D ) . The best global fits of the double expo - nential time courses at intermediate aging times yielded a \u223c 20 - fold difference in the lifetimes of the species that dissociated slowly ( k s , F = 0 . 32 ( \u00b1 0 . 005 ) min \u2212 1 ) and rapidly ( k f , F = 6 . 67 ( \u00b1 0 . 44 ) min \u2212 1 ) at a buffer flow rate of 500 \u03bc L min \u2212 1 ( Fig . 2 B ) . Under this force , the observed fast rate constant is the rate constant for the state 2 branch dissociation ( i . e . , k f , F = k 2 , F ) and the observed slow rate constant is the sum of the rate constants for dissociation for state 1 branches and conversion from state 1 to state 2 ( i . e . , k s , F = k 1 , F + k conv , F ; SI Appendix , Part 2 and Eqs . S29 and S41 ) . Force produced by a flow of 500 \u03bc L min \u2212 1 increased the ob - served dissociation of branches \u223c 670 - fold ( from k 2 = 0 . 01 to k 2 , F = 6 . 67 min \u2212 1 ) for those with ADP - Arp2 / 3 complex but only 32 - fold ( from k 1 = 0 . 012 to k 1 , F = k s , F \u2212 k conv , F < k s , F = 0 . 32 min \u2212 1 ; Fig . 2 B and SI Appendix , Fig . S5 ) for those with ADP - P i - Arp2 / 3 complex . This difference in force sensitivity of the two states explains the dramatic acceleration in overall debranching with aging ( Fig . 2 B ) . Furthermore , we estimate that force produced by 500 \u03bc L min \u2212 1 flow increased k conv only two - fold from k conv = 0 . 14 min \u2212 1 in the absence of force to k conv , F = k s , F \u2212 k 1 , F < k s , F = 0 . 32 min \u2212 1 in the presence of force ( SI Appendix , Fig . S5 ) . Implications of the Force Sensitivity of Branches for Their Turnover in Cells . Implications for debranching by myosin motor proteins . A sustained flow force of \u223c 1 pN dissociates ADP - Arp2 / 3 complex branches in < 30 s ( Fig . 2 D ) , raising the possibility that pN contractile forces generated by myosin motors could rapidly debranch and reorganize Arp2 / 3 complex networks . Myosin accelerates net - work disassembly and reorganization in both biomimetic systems ( 33 , 34 ) and in cells ( 35 , 36 ) . We note , however , that the work ( i . e . , energy term ) from the applied force determines the overall debranching acceleration , not the force per se . Implications of Nucleotide - Dependent Force Sensitivity of Arp2 / 3 Complex Debranching . \u201c Young \u201d branches with ADP \u2212 P i \u2212 Arp2 / 3 complex are more resistant to dissociation by force than \u201c old \u201d branches with ADP \u2212 Arp2 / 3 complex . Nucleation - promoting fac - tors associated with membranes ( plasma membrane , vesicles , in - tracellular bacteria ) activate Arp 2 / 3 complex , so \u201c young \u201d branches are located closer to these surfaces than \u201c old \u201d branches . If force is uniformly distributed across the filament network , old branches farthest from the surface with nucleation - promoting factors would be preferentially debranched . This differential response to force may contribute to the ob - served remodeling of cellular branched actin networks under load , including local changes in branch density ( 13 \u2013 15 ) . We apply a simple a minimal kinetic model ( SI Appendix , Part 3 ) to analyze how selective , force - sensitive debranching might influence the distribution of branches in actin networks at the leading edge of cells . The model assumes Arp 2 / 3 complex is activated uniformly at the membrane , such that the branch density distribution is also uniform , on average , along the plane parallel to the cell mem - brane at the leading edge . The model with the experimental pa - rameters determined here predicts that the density of branches decays exponentially ( see also refs . 37 \u2013 39 ) along the axis per - pendicular to the membrane , toward cell interior in both the absence and presence of force , but preferential debranching of old Arp 2 / 3 complexes shortens the branch density decay length and can , under some conditions , increase the local branch density near the membrane ( SI Appendix , Part 3 and Figs . S9 and S10 ) . Thus , external force on the leading edge of cells fa - vors debranching of old branches that have migrated toward the cell interior over young branches still near the membrane and can thus contribute to spatial and vectorial network turnover . External force may also influence binding of cofilin ( 40 ) and GMF ( Fig . 3 ; discussed below ) , both of which display debranching activity ( 20 , 24 ) . GMF Selectively Dissociates ADP \u2212 Arp2 / 3 Complex Branches . Fission yeast GMF increases the rate constant for dissociation of branches with ADP \u2212 Arp 2 / 3 complex up to \u223c 20 - fold , but does not disso - ciate branches with ADP \u2212 BeF x \u2212 Arp 2 / 3 complex ( Fig . 3 ) . The following parallel reactions describe GMF - catalyzed dissociation of branches with ADP \u2212 Arp2 / 3 complex : Arp + GMF \u21cc Arp - GMF \u2193 k diss , 0 \u2193 k diss , GMF , debranched Scheme 1 where k diss , 0 and k diss , GMF are the dissociation rate constants of branches formed by Arp2 / 3 complex alone ( Arp ) and GMF - bound Arp2 / 3 complex ( Arp - GMF ) , and K d , GMF is the affinity of GMF for Arp 2 / 3 complex in a branch junction . This scheme assumes that GMF binding equilibrates rapidly compared to the rate of debranching ( and k + GMF [ GMF ] , k \u2212 GMF > > k diss , 0 , k diss , GMF ) and that [ GMF ] > > [ Arp2 / 3 complex ] . Debranching from the parallel pathway in Scheme 1 follows a single exponential ( 41 ) . The dependence of the observed branch lifetime ( \u03c4 obs ) on the concentration of GMF can be expressed in two ways , first , \u03c4 obs = \u03c4 0 \u2212 ( \u03c4 0 \u2212 \u03c4 GMF ) [ GMF ] K d , GMF ( \u03c4 GMF \u03c4 0 ) + [ GMF ] . [ 3 ] Alternatively , the relationships can also be expressed in terms of observed rate constants ( 41 , 42 ) , k obs = k 0 + ( k GMF \u2212 k 0 ) [ GMF ] K d , GMF + [ GMF ] . [ 4 ] The best fit of Eq . 3 to the dependence of the observed branch lifetime ( \u03c4 obs ) on the concentration of GMF ( Fig . 3 B ) yielded an apparent affinity of GMF for ADP \u2212 Arp2 / 3 complex ( K d , GMF ) of 40 ( \u00b1 10 ) nM and a lifetime of GMF bound to ADP \u2212 Arp2 / 3 complex in branches ( \u03c4 GMF ) of 3 . 3 ( \u00b1 0 . 5 ) min . This corre - sponds to a debranching rate constant ( k GMF = 1 / \u03c4 GMF ) of 0 . 31 ( \u00b1 0 . 05 ) min \u2212 1 . In Eqs . 3 and 4 , k 0 is the debranching rate con - stant and \u03c4 0 is the branch lifetime without GMF under very low flow rate ( 15 \u03bc L \u00b7 min \u2212 1 ) , that is , very low average force . The best fit yielded k 0 = 1 / \u03c4 0 = 0 . 014 ( \u00b1 0 . 0002 ) min \u2212 1 , corresponding to \u03c4 0 = 71 ( \u00b1 1 ) min . These values are slightly faster than the in - trinsic dissociation rate constant for branches with ADP \u2212 Arp2 / 3 complex in the absence of force ( > 100 min ; Fig . 3 ) due to the 15 \u03bc L \u00b7 min \u2212 1 flow rate employed in this experiment . We note , for general readers , that the GMF concentration at half - maximum Pandit et al . PNAS | June 16 , 2020 | vol . 117 | no . 24 | 13525 B I O P H Y S I C S A ND C O M P U T A T I O N A L B I O L O G Y effect is given by K d , GMF ( \u03c4 GMF / \u03c4 0 ) in Eq . 3 and K d , GMF in Eq . 4 , so its value differs depending on how the data are plotted . Reported affinities of GMF for soluble Arp2 / 3 complex ( K d , GMF ) vary widely : > > 10 \u03bc M for ATP \u2212 Arp 2 / 3 complex ( 43 ) ; 0 . 7 \u03bc M for ADP \u2212 Arp2 / 3 complex ( 43 ) ; and 13 nM ( 20 \u2013 22 ) or \u223c 1 \u03bc M ( 44 ) for Arp2 / 3 complex without a specified nucleotide . We found that the measured affinity of GMF for Arp2 / 3 complex in branch junctions bound to both a mother and daughter filament depended on the bound nucleotide . Arp2 / 3 complex used in these studies came from different organisms : Saccharomyces cerevisiae ( 20 \u2013 22 ) , bovine brain ( 43 ) , and S . pombe ( this study and ref . 44 ) . Thus , GMF preferentially dissociates \u201c old \u201d branches with ADP \u2212 Arp2 / 3 complex rather than \u201c young \u201d branches with ADP \u2212 P i \u2212 Arp2 / 3 complex . Given that GMF has a much lower affinity for ATP \u2212 Arp2 / 3 complex in solution than ADP \u2212 Arp2 / 3 complex ( 43 ) , weak binding of GMF to ADP \u2212 P i \u2212 Arp2 / 3 com - plex in branches is the most likely explanation for the resistance of young branches and branches with ADP \u2212 BeF x \u2212 Arp2 / 3 com - plex to dissociation by GMF . The nucleotide bound to Arp2 / 3 complex might also affect the debranching reaction directly . Saturating GMF destabilizes ADP \u2212 Arp2 / 3 complexes , reducing their lifetime from more than 60 min to \u223c 3 min ( Fig . 3 ) . While GMF strongly promotes debranching , these long lifetimes ( 3 min ) at saturating GMF concentrations place limits on the role of GMF in mediating debranching and network turnover in cells . More - rapid debranching may be achieved by combining GMF with force and / or from contributions from other debranching proteins such as cofilin , which dissociates branches on a second time scale at micromolar concentrations without force ( 24 ) . Arp2 / 3 Complex Likely Dissociates with the Daughter Filament . A daughter filament and its associated Arp2 / 3 complex dissociate simultaneously ( within the 100 ms time resolution of our ex - periments ) during debranching events . We cannot eliminate a pathway in which the daughter filament detaches first , followed by rapid release of Arp2 / 3 complex . However , we favor a mechanism where the interface between Arp2 / 3 complex and the mother filament ruptures and simultaneously releases the daugh - ter filament with Arp2 / 3 complex on its pointed end . This mech - anism is consistent with actin filaments rarely fragmenting under debranching conditions , so the rate constant for rupture of the actin \u2212 actin interface is hundreds of times slower than the rate constant for rupturing a branch junction . Given that the interface between Arp2 / 3 complex and the daughter filament is structurally similar on many levels to an actin \u2212 actin interface ( 45 ) , it is likely to rupture slowly like actin filaments . Materials and Methods Protein Purification . Rabbit skeletal muscle actin was purified from back and legmusclesandlabeledonlysineresidueswithNHSestersofAlexa568 , Alexa 647 , or biotin ( 46 , 47 ) or on Cys - 374 with pyrene iodoacetamide ( 48 ) . Actin monomers with bound ATP were passed twice through a desalting column to exchange into G - Buffer ( 20 mM Tris pH 8 , 2 mM CaCl 2 , 10 mM NaN 3 , 0 . 2 mM ATP , and 0 . 5 mM dithiothreitol [ DTT ] ) containing 0 . 2 mM AMPPNP instead of ATP and incubated for 1 h at 25 \u00b0C . Remaining free nucleotides were removed with a desalting column in G - Buffer containing AMPPNP in - stead of ATP prior to polymerization experiments . Arp2 / 3 complex was stored in ATP buffer solution composed of 10 mM Pipes at pH 6 . 8 , 100 mM KCl , 1 mM MgCl 2 , 0 . 25 mM ethylene glycol bis ( \u03b2 - aminoethyl ether ) - N , N , N \u2032 , N \u2032 - tetraacetic acid [ EGTA ] , 0 . 1 mM ATP , and 1 mM DTT . Recombinant fission yeast glutathione S - transferase ( GST ) - VCA was puri - fied from bacteria ( 49 ) . S . pombe Arp2 / 3 complex was purified from the TP150 strain , a protease - deleted S . pombe strain ( 49 ) . Fluorescent Arp2 / 3 complex was generated by conjugating Alexa 488 to a snap tag on the C terminus of the Arpc5 subunit . Bulk polymerization assays with pyrenyl actin and GST - VCA showed that Arp2 / 3 complex with 82 % labeling of the snap tag nucleated 91 % as many actin filaments as unlabeled Arp2 / 3 complex ( 4 . 2 nM vs . 4 . 6 nM of barbed ends at half - maximal polymerization ; SI Ap - pendix , Fig . S6 ) ( refs . 48 and 49 and SI Appendix , Fig . S8 ) . A complementary DNA for S . pombe GMF was cloned and inserted into the pet28a plasmid that includes GST and Tobacco Etch Virus ( TEV ) protease sites . Recombinant GMF was expressed in Rosetta2 BL21 Escherichia coli cells ( Novagen ) and purified using glutathione affinity chromatography . After cleaving off GST with TEV protease , a second round of glutathione chro - matography removed the protease and GST . After fast protein liquid chro - matography on a MonoQ column ( 20 ) , the GMF was greater than 95 % pure when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis . Microscopy and Microfluidics . A total internal reflection fluorescence ( TIRF ) microscopy system with a Till iMic digital microscope equipped with a \u00d7 100 objective ( Olympus ) and an Andor iXon897 electron multiplying charge - coupled device ( EMCCD ) camera was used in this study . Images were ac - quired at a rate of 5 to 30 frames per second . Coverslips were cleaned with the following solutions , all incubated in a sonicator for 30 min and rinsed in - tensively with Milli - Q water in between steps : 2 % Hellmanex , water , acetone , 1 M HCl , 5 M KOH , and hexane , then silanized with 650 \u03bc L of dichlorodimethyl silane in 500 mL of hexane for 30 min , and subsequently rinsed and sonicated in hexane for 3 \u00d7 1 min . Coverslips were dried using a stream of nitrogen gas and stored in falcon tubes at \u2212 20 \u00b0C for up to 2 mo ( 50 ) . A glass microfluidic chamber was constructed as described in ref . 51 . Briefly , the input and output ports for flow solution were formed in poly - dimethylsiloxane ( PDMS ) using a flat - tip needle . Holes connecting the PDMS to parallel sample chambers were drilled through the glass with a diamond tip bit . Subsequently , a plasma cleaner was employed to bond PDMS to slide glass . Immediately before use , chambers were assembled using parafilm on the glass slide opposite to PDMS ports , and a coverslip was placed over the parafilm and sealed with heat . A fixed rate hydrodynamic flow exerted by an automatic syringe pump applied pullingforcesonactinfilaments . Themagnitudeofpullingforceona branch joint ( F d ; SI Appendix , Fig . S1 A and B ) scales with daughter filament branch length and flow rate . These values span a range up to a few pico - newtons for the daughter filament lengths and flow rates examined here . Pulling force was calculated according to the Batchelor ( 52 ) equation , F d = \u03b7 2 \u03c0 lv ln ( 2 hr ) . [ 5 ] Eq . 5 estimates drag force on a cylindrical filament with correction for the height ( h ) of the filaments from the surface of the flow chamber ( 53 ) . So - lution viscosity ( \u03b7 ) is assumed to equal unity ; l is the ( daughter ) filament length and varies for individual branches . The average actin filament radius is r = 4 nm , and v is the linear fluid velocity in the plane of the filament . We assume an average height ( h ) of 200 nm from the flow chamber surface . The fluid velocity ( v ) is proportional to bulk flow rate and was determined from the movement of 100 nM TetraSpek beads ( Thermo Fisher ) through the sample chamber ( 52 ) . Only beads moving parallel to the surface were employed in our analysis , as the flow velocity profile changes with the height from the chamber surface . Bead flow velocity was measured via ImageJ \u201c Manual Tracking \u201d and used in force calculations ( 52 ) . In the ex - periments presented in Fig . 2 A and C , multiple experiments at different flow rates were performed , and the force on each branch was calculated using the flow rate and length of the branch . Then , debranching data from different flow cells with a range of flow rates were binned according to the force on each branch and plotted as survival time courses , given by the fraction of branches remaining at each time point . For example , the branches in Fig . 2 A were divided into bins of at least 13 branches . The bin size for each data point is approximately the midpoint between the point itself and its neighboring data points ( Fig . 2 D ) . At low forces ( 0 . 002 pN to 0 . 4 pN ) , the bin sizes were small , for example , 0 pN to 0 . 04 pN , 0 . 1 pN to 0 . 2 pN , etc . , and , at higher forces ( > \u223c 1 pN ) , the bin sizes were larger , for example , 0 . 8 pN to 1 . 4 pN , 1 . 4 pN to 2 . 4 pN , etc . Someoftheseparametersareuncertain , sotheestimatedforcevaluesmay be offset systematically for all of our experiments . The least certain pa - rameter is the distance of individual filaments from the surface , which may vary by < 50 % from the average distance of 200 nm assumed here . A 500 \u03bc L \u00b7 min \u2212 1 flow rate produces forces on a 1 . 5 - \u03bc m branch of 0 . 94 pN at a height of 300 nm , 1 . 02 pN at 200 nm , and 1 . 20 pN at 100 nm ( SI appendix in ref . 52 ) . Therefore , the absolute forces and parameters calculated from them may be imprecise , but no more than 20 % , and the relative forces between experi - ments can be compared . Preparation of Branched Actin Filament Networks and Experimental Procedures . KMIE buffer ( 6 , 23 ) ( 10 mM imidazole pH 7 . 0 , 50 mM KCl , 2 mM MgCl 2 , 1 mM EGTA , 0 . 2 mM ATP , 2 mM DTT ) supplemented with 13526 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1911183117 Pandit et al . 15 mM glucose , 0 . 02 mg \u00b7 mL \u2212 1 catalase , and 0 . 1 mg \u00b7 mL \u2212 1 glucose oxidase was used for all microscope experiments , unless noted otherwise . Filaments of Alexa 568 \u2212 labeled actin ( Fig . 1 A , red filament segment ; 15 % labeled with Alexa 568 and containing 10 % biotinylated actin subunits ) were polymer - ized in KMIE buffer , sheared by vigorous pipetting , and subsequently mixed with 1 . 5 \u03bc M Alexa 647 - labeled actin monomers ( Fig . 1 A , green segment and branch ) , 0 . 1 \u03bc M Arp2 / 3 complex , and 0 . 5 \u03bc M GST - VCA in KMIE buffer . This solution was incubated for 1 min to 2 min to initiate branch formation . During the branching reaction , green actin monomers elongated from ei - ther the barbed end of short Alexa 568 actin segments or mother filament bound Arp2 / 3 complex VCA , forming mother or daughter filaments , re - spectively . This mixture was pipetted into flow chambers where the short , red actin segments bind the neutravidin - coated coverslip surface , and the branching reaction was allowed to continue for 1 min to 2 min . The total time of branch formation and aging for a given experiment ( e . g . , 2 . 6 min ) is indicated in the text . Branch formation and filament elongation was then terminated by removing untethered proteins ( labeled actin , Arp2 / 3 complex , and GST - VCA ) from the sample chamber with gentle flow of KMIE buffer . Tethered filaments were further aged for different time periods after the removal of unbound protein . Low ( 2 \u03bc L \u00b7 min \u2212 1 ) flow applied during aging ensured irreversible debranching . Filament segments with Alexa 568 actin subunits ( Fig . 1 A , red ) were tethered to the surface , while Alexa 647 actin ( Fig . 1 A , green ) mother and daughter filaments were allowed to freely fluctuate and align with flow . Unlabeled actin monomer ( 0 . 2 \u03bc M ) was in - cluded in flow buffer solution to prevent filament depolymerization . The \u201c aging time \u201d specified in each experiment is the time between the initial mixing of the proteins and application of debranching flow , including the time for branch formation and further aging . The age times were precisely measured in the experiment in Fig . 2 B but are approximate times in all other experiments . Debranching under force was performed by applying flow at a fixed rate ( specified in text ) throughout the experiment . Since unbound proteins were removed after the branch formation period , actin filament elongation was terminated , and individual branch length ( l ) and conse - quently applied force ( F ) on the branch joint were constant ( Eq . 5 ) . Experiments with BeF x were performed two different ways . 1 ) For ex - periments with ATP \u2212 Arp2 / 3 complex ( Fig . 2 C , Inset ) , branches were formed from ATP - actin monomers and ATP \u2212 Arp2 / 3 complex as described above but with 2 mM BeSO 4 and 10 mM NaF included in the buffer at all times , and aged for \u223c 4 min before applying various rates of flow with KMIE buffer supplemented with 2 mM BeSO 4 and 10 mM NaF . For the experiment with BeF x and 30 - min aging ( Fig . 2 B , gray ) , ATP \u2212 Arp2 / 3 complex branches were formed as described above and aged for 30 min with 2 mM BeSO 4 and 10 mM NaF before applying flow with KMIE buffer . 2 ) For experiments with ADP \u2212 Arp2 / 3 complex and BeF x ( Fig . 2 B , brown ) , mother filaments with ATP - actin ( 10 % biotinylated ) monomers were immobilized on the surface as described above , and the KMIE buffer ( which includes ATP ) was washed out and replaced with KMIE ADP buffer supplemented with 2 mM BeSO 4 and 10 mM NaF . Then ADP - actin monomers , ADP \u2212 Arp2 / 3 complex , and GST - VCA in KMIE buffer with 2 mM BeSO 4 and 10 mM NaF and 2 mM ADP instead of 2 mM ATP were gently flowed into the chamber and allowed to form branches . After a \u223c 4 - min branch formation period , all unbound proteins were washed out with KMIE ADP and 2 mM BeSO 4 and 10 mM NaF and maintained under flow as noted . ADP - actin and ADP \u2212 Arp2 / 3 complex were prepared by exchanging nucleotide from ATP - actin and ATP \u2212 Arp2 / 3 com - plex , respectively , using desalting columns . ADP \u2212 Arp2 / 3 complex never formed branches unless 2 mM BeSO 4 and 10 mM NaF was present as reported previously and shown here ( SI Appendix , Fig . S3 ) ( 5 , 6 ) . For experiments with AMPPNP mother filaments , filaments were poly - merized from AMPPNP actin monomers and immobilized on the surface as described above . Branch formation was initiated with ATP \u2212 Arp2 / 3 complex as described above in KMIE buffer with 0 . 2 mM AMPNP instead of 0 . 2 mM ATP . Since AMPPNP \u2212 Arp2 / 3 complex does not form branches ( 5 ) , verified in our experiments ( SI Appendix , Fig . S3 ) , all branches formed from Arp2 / 3 complex with bound ATP . Since the flow buffer included 0 . 2 mM AMPPNP , all actin monomers remained bound to AMPNP . The solution with Arp2 / 3 complex accounted for less than 1 % of the final solution volume , so it in - troduced a negligible amount of ATP . In debranching experiments with GMF with orwithout BeF x , samples were prepared as above except that debranching was initiated by gentle flow of buffer ( 15 \u03bc L \u00b7 min \u2212 1 , very low force ) containing a range of concentrations of GMF , and the flow was maintained throughout the experiment . Data Analysis . Analysis of branch dissociation was done by manual tracking with ImageJ ( https : / / imagej . nih . gov / ) . Branches that were fluctuating at all times and did not stick to the glass surface were selected randomly to minimize bias . These branches were labeled , cataloged , and observed for their entire lifetimes . Origin ( https : / / www . originlab . com / ) was used to fit all data and make plots . For the Arp2 / 3 complex debranching images in Fig . 1 , a Gaussian blur with a sigma radius of 1 . 25 pixels was applied . 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Funding : See page 34 Received : 02 July 2019 Accepted : 16 January 2020 Published : 17 January 2020 Reviewing editor : Patricia Bassereau , Institut Curie , France Copyright Akamatsu et al . This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited . Principles of self - organization and load adaptation by the actin cytoskeleton during clathrin - mediated endocytosis Matthew Akamatsu 1 , Ritvik Vasan 2 , Daniel Serwas 1 , Michael A Ferrin 1 , Padmini Rangamani 2 * , David G Drubin 1 * 1 Department of Molecular and Cell Biology , University of California , Berkeley , Berkeley , United States ; 2 Department of Mechanical and Aerospace Engineering , University of California , San Diego , La Jolla , United States Abstract Force generation by actin assembly shapes cellular membranes . An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension . Around 200 activated Arp2 / 3 complexes are required for robust internalization . A newly developed molecule - counting method determined that ~ 200 Arp2 / 3 complexes assemble at sites of clathrin - mediated endocytosis in human cells . Simulations predict that actin self - organizes into a radial branched array with growing ends oriented toward the base of the pit . Long actin filaments bend between attachment sites in the coat and the base of the pit . Elastic energy stored in bent filaments , whose presence was confirmed by cryo - electron tomography , contributes to endocytic internalization . Elevated membrane tension directs more growing filaments toward the base of the pit , increasing actin nucleation and bending for increased force production . Thus , spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints . Introduction Cells polymerize actin filaments to produce force and provide mechanical integrity for a variety of cellular processes , from cytokinesis and cell migration , to membrane reshaping and trafficking ( Pol - lard , 2016 ) . For each cellular process , actin filaments organize into a specific geometry that confers structural integrity and force - generation capacity . Most membrane deformation processes use branched actin networks nucleated by the Arp2 / 3 complex , a branched actin filament network nucle - ator ( Carlsson , 2018 ; Rotty et al . , 2013 ) . On a large ( m m ) length scale , branched actin networks drive the plasma membrane forward during cell migration , such that on the scale of individual actin branches , the membrane shape can be thought of as more or less constant ( Keren et al . , 2008 ; Mueller et al . , 2017 ; Schaus et al . , 2007 ) . However , on a smaller ( sub - micron ) length scale , branched actin networks deform many cellular membranes as part of organelle and vesicle biogene - sis and function ( Rottner et al . , 2017 ) . The relationship between cellular membrane curvature and local actin assembly for each of these \u2018local\u2019 membrane - deformation processes remains relatively unexplored ( Daste et al . , 2017 ) . Clathrin - mediated endocytosis ( CME ) is an especially attractive process for studies of actin\u2019s role in membrane shape changes due to the relatively complete parts list and available quantitative infor - mation about the positions , recruitment timing and biochemical function of many of the participating proteins ( Arasada et al . , 2018 ; Idrissi et al . , 2012 ; Kaksonen et al . , 2005 ; Kaksonen et al . , 2003 ; Mund et al . , 2018 ; Picco et al . , 2015 ; Sochacki et al . , 2017 ; Taylor et al . , 2011 ) . CME is a ubiqui - tous and essential cellular process by which cells take macromolecules from the extracellular space and the plasma membrane into the cell interior ( Kaksonen and Roux , 2018 ) . During CME , the Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 1 of 40 RESEARCH ARTICLE plasma membrane is bent , pinched , and pulled inward in a time frame of ~ 60 s thereby transitioning from a flat sheet into a spherical vesicle ~ 100 nm in diameter . Clathrin and its adaptor proteins establish a coat that generates initial membrane curvature ( Chen et al . , 1998 ; Pearse , 1976 ; Stachowiak et al . , 2012 ) , and BAR ( bin - amphiphysin - rvs ) - domain proteins bind curved membranes and support further membrane curvature ( Buser and Drubin , 2013 ; David et al . , 1996 ; Kishimoto et al . , 2011 ) . During yeast endocytosis , branched actin filaments provide the force required for membrane tubule formation ( Engqvist - Goldstein and Drubin , 2003 ; Idrissi et al . , 2012 ; Kukulski et al . , 2012 ; Picco et al . , 2018 ; Sun et al . , 2006 ; Wang and Carlsson , 2017 ) . In metazoan cells , endocytic pits under high tension stall at a \u2018U\u2019 - shaped intermediate in the absence of functional actin ( Boulant et al . , 2011 ) , implying that actin is required to generate plasma mem - brane shape changes late in CME ( Hassinger et al . , 2017 ; Yarar et al . , 2005 ; Yoshida et al . , 2018 ) . The molecular mechanism by which a network of polarized , branched actin filaments assembles at these sites for productive force generation is poorly understood . Actin network assembly is known to play a key role in membrane shape change in some contexts . For example , mathematical modeling ( Berro et al . , 2010 ; Carlsson and Bayly , 2014 ; Dmitrieff and Ne\u00b4de\u00b4lec , 2015 ; Liu et al . , 2009 ; Mund et al . , 2018 ; Wang et al . , 2016 ) and quantitative fluores - cence imaging in yeast ( Wu and Pollard , 2005 ; Sirotkin et al . , 2010 ; Berro and Pollard , 2014 ; Picco et al . , 2015 ) have established the relationship between actin filament assembly and plasma membrane shape particular to fungi , which have unique mechanical requirements due to very high ( ~ 10 atm ) hydrostatic turgor pressure . However , less is known about actin organization and function in the lower force regime characteristic of metazoan cells . A multiscale modeling effort that accounts for the mechanics of single actin filaments and that is constrained by experimental measurements of actin dynamics , spatial organization of the filaments , and tension in the plasma membrane is required to gain insight into actin organization and force generation capacity . We hypothesize that in localized membrane - reshaping processes such as endocytosis , branched actin networks assemble under specific spatial \u2018boundary conditions , \u2019 which serve as geometrical constraints dictated both by the shape of the membrane and the spatial segregation of membrane - associated proteins that inter - act with actin . These unique spatial boundary conditions on a curved surface , combined with the knowledge of numbers of molecules in cells and known reaction rate constants , provide the eLife digest The outer membrane of a cell is a tight but elastic barrier that controls what enters or leaves the cell . Large molecules typically cannot cross this membrane unaided . Instead , to enter the cell , they must be packaged into a pocket of the membrane that is then pulled inside . This process , called endocytosis , shuttles material into a cell hundreds of times a minute . Endocytosis relies on molecular machines that assemble and disassemble at the membrane as required . One component , a protein called actin , self - assembles near the membrane into long filaments with many repeated subunits . These filaments grow against the membrane , pulling it inwards . But it was not clear how actin filaments organize in such a way that allows them to pull on the membrane with enough force \u2013 and without a template to follow . Akamatsu et al . set about identifying how actin operates during endocytosis by using computer simulations that were informed by measurements made in living cells . The simulations included information about the location of actin and other essential molecules , along with the details of how these molecules work individually and together . Akamatsu et al . also developed a method to count the numbers of molecules of a key protein at individual sites of endocytosis . High - resolution imaging was then used to create 3D pictures of actin and endocytosis in action in human cells grown in the laboratory . The analysis showed the way actin filaments arrange themselves depends on the starting positions of a few key molecules that connect to actin . Imaging confirmed that , like a pole - vaulting pole , the flexible actin filaments bend to store energy and then release it to pull the membrane inwards during endocytosis . Finally , the simulations predicted that the collection of filaments adapts its shape and size in response to the resistance of the elastic membrane . This makes the system opportunistic and adaptable to the unpredictable environment within cells . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 2 of 40 Research article Cell Biology Physics of Living Systems necessary information for multiscale modeling and a mechanistic framework to understand the rela - tionship between plasma membrane mechanics and branched actin assembly and mechanics associ - ated with CME . Using this framework , we sought to answer the following questions : How do branched actin net - works assemble , organize , and produce force around an endocytic pit ? How does the spatial segre - gation of Arp2 / 3 complex activators ( Almeida - Souza et al . , 2018 ; Mund et al . , 2018 ) and actin - binding proteins associated with endocytic coats ( Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; Sochacki et al . , 2017 ) influence this organization ? And finally , how do endo - cytic actin networks adapt to changing loads due to the stochastic environment and changes in membrane tension ? To answer these questions , we combined live - cell molecule counting methods in genome - edited diploid human cells and cryo - electron tomography of intact cells with multiscale modeling of plasma membrane mechanics and actin filament dynamics . Our results show that a mini - mal branched actin network is sufficient to create sustained internalization of an endocytic pit against physiological membrane tension . Actin filament self - organization and bending , which arise from the spatial distribution of actin - coat attachments around the curved endocytic pit , allow the actin net - work to adapt to changing loads . We anticipate that the mechanistic insights gained for actin in mammalian endocytosis will also apply to a variety of local membrane - bending processes carried out by branched actin throughout the cell . Results Multiscale modeling shows that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension We combined a continuum - mechanics model of the plasma membrane , an agent - based model of actin filament dynamics , quantitative fluorescence microscopy , and electron tomography in cells to determine the molecular mechanism by which branched actin networks produce force during mam - malian clathrin - mediated endocytosis ( Figure 1 , Scheme 1 ) . First , we used a continuum - mechanics model of the plasma membrane ( Alimohamadi et al . , 2018 ; Hassinger et al . , 2017 ; Rangamani et al . , 2014 ) to determine the force - extension relation - ship for clathrin - coated pits stalled at a U - shaped intermediate under high membrane tension ( Figure 1B ) . Here , the extension refers to the extent of pit internalization , which is a displacement in the - Z direction ( Figure 1A \u2013 B ) . Previously , we showed that membrane curvature generation by the endocytic coat during vesicle formation could snap the membrane into a pinched \u2018omega\u2019 shape as a function of membrane tension and the curvature induced by the coat ( Hassinger et al . , 2017 ) , but we did not focus on force produced by the actin cytoskeleton . Here , we modeled the coated mem - brane based on the Helfrich ( 1973 ) energy and applied a linear force to the clathrin - coated pit in increasing value over successive simulations , corresponding to a simplified actin force . Simulations demonstrated that a clathrin - coated pit experiences a nearly linear force - extension relationship until an internalization of ~ 100 nm , at which point the pit can also adopt a pinched ( or \u2018omega\u2019 ) shape , which requires a lower force ( Figure 1C and Figure 1\u2014video 1 ) . We calculated the resistance to internalization as the slope of the force - extension plot for the linear regime and found that it is directly proportional to plasma membrane tension for a wide range of coat rigidities ( Figure 1D ) . Importantly , this direct scaling between resistance to internalization and membrane tension allowed us to treat this step of endocytic pit internalization as a linear spring , with the spring constant cali - brated using measurements of plasma membrane tension in mammalian cells ( Diz - Mun\u02dcoz et al . , 2016 ; Kaplan et al . , in preparation ) . The simple spring - like relationship uncovered above between force and endocytic pit internaliza - tion ( Figure 1D ) allowed us to simplify our mechanical treatment of the plasma membrane while modeling individual actin filaments and actin - binding proteins with realistic kinetics and mechanics ( Figure 1E \u2013 G , Supplementary file 3 ) . We used Cytosim ( Nedelec and Foethke , 2007 ) to construct a filament - based model of the endocytic actin network . This agent - based model allowed us to simu - late the emergent architecture and mechanical functions of branched actin for realistic endocytic geometries . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 3 of 40 Research article Cell Biology Physics of Living Systems 0 - 20 - 40 - 60 - 80 - 100 Internalization ( nm ) 0 5 10 15 20 25 30 35 40 45 F o r c e ( p N ) growing end capped end Hip1R nucleator Arp2 / 3 Actin 3 s 7 s 9 s 11 s 5 s Membrane tension ( pN / nm ) S p r i ng c on s t an t ( p N / n m ) A B E F Y ( n m ) Z ( n m ) X ( n m ) C Actin Arp2 / 3 complex Clathrin Capping protein Hip1R Plasma membrane vesicle A r p2 / 3 c o m p l e x H i p1 R T en s i on c apped end nu c l ea t o r D Tension Number Arp2 / 3 complex G U - shaped pit Transition - shaped pit ? 0 . 15 0 . 2 0 . 25 0 . 3 0 . 35 0 . 4 0 . 45 1 . 6 1 . 4 1 . 2 1 0 . 8 0 . 6 0 . 4 0 . 2 0 Tension ( pN / nm ) N - WASP N - N - N - WA N - WASP Base Coat \u03a9 0 - 50 - 100 0 5 10 15 Time ( s ) I n t e r na li z a t i on ( n m ) 1x2x5x7 . 5x 10x Coat rigidity 0 . 45 0 . 40 0 . 35 0 . 30 0 . 25 0 . 20 0 . 15 0 . 10 0 . 05 S p r i ng c on s t an t Figure 1 . Multiscale modeling shows that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension . ( A ) Schematic of a section of the cell\u2019s plasma membrane being internalized during mammalian endocytosis depicts plasma membrane deformation against membrane tension ( purple arrows ) countered by the clathrin coat ( yellow ) and the actin cytoskeleton ( red ) . ( B ) Shape of the membrane and pit internalization from continuum mechanics simulations of the endocytic pit experiencing axial ( Z ) forces corresponding to simplified actin forces . To begin with , the plasma membrane ( yellow ) is deformed by a coat with preferred curvature that expands in area until the pit stalls . A net Figure 1 continued on next page Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 4 of 40 Research article Cell Biology Physics of Living Systems We simplified the endocytic pit as a solid , impermeable structure , initially a hemisphere , attached to a flat plasma membrane corresponding to the \u2018U - shaped\u2019 intermediate ( Avinoam et al . , 2015 ; Boulant et al . , 2011 ; Messa et al . , 2014 ; Yarar et al . , 2005 ; Figure 1E ) . The following rules were prescribed for actin filament dynamics . Initially , actin filament - nucleating proteins seed a small num - ber of actin filaments near the endocytic pit . These randomly - oriented \u2018mother filaments\u2019 serve as templates for binding pre - activated Arp2 / 3 complexes , which correspond to the coincidence of Arp2 / 3 complex and its activator N - WASP , arranged in a ring ( Almeida - Souza et al . , 2018 ; Mund et al . , 2018 ) at the base of the endocytic pit ( Idrissi et al . , 2008 ; Kaksonen et al . , 2003 ; Picco et al . , 2015 ; Kaplan et al . , in preparation ) . When an active Arp2 / 3 complex comes in proximity Figure 1 continued force ( red arrows ) is applied downward from the coat and upward into the base of the endocytic pit ( red dotted lines ) . In this simulation , membrane tension was 0 . 2 pN / nm , and the coated area was rigid ( 2400 pN ! nm ) . ( C ) Force versus pit internalization relationships for different values of membrane tension . Internalization is defined as the pit displacement in Z . Shading delineates linear force - internalization regime ( blue ) ; \u2018transition point\u2019 from U to omega shape ( orange ) ; \u2018omega - shaped\u2019 regime where the neck is narrower than the pit diameter and the force required for internalization is lower than at the transition point ( for tensions > 0 . 1 pN / nm ) ( yellow ) . Color matches the three snapshots in B . Parameters are given in Supplementary files 1 and 2 . ( D ) Resistance of pit to internalization versus membrane tension . Resistance ( spring constant ) is defined as absolute value of slope in C for the \u2018U - shaped\u2019 region . Each curve is calculated for a different value of membrane rigidity ( where 1x = 320 pN ! nm , the rigidity of the uncoated plasma membrane ) . ( E ) Computational model of branched actin filament polymerization coupled to endocytic pit internalization . An internalizing endocytic pit is modeled as a sphere with a neck attached to a flat surface by a spring . Active Arp2 / 3 complex ( blue ) is distributed in a ring around the base of the pit . An actin nucleation protein ( pink ) generates an actin filament ( white ) , which polymerizes , stalls under load , and is stochastically capped ( red ) . Arp2 / 3 complexes bind to the sides of actin filaments and nucleate new filaments at a 77 - degree angle , creating new branches . Linker Hip1R ( purple ) is embedded in the pit and binds to actin filaments . Model parameters are given in Supplementary file 3 . ( F ) Graphical output of the simulations from Cytosim ( Nedelec and Foethke , 2007 ) at 2 s intervals . Scale bar : 100 nm . ( G ) Pit internalization over simulated time as a function of the number of available molecules of Arp2 / 3 complex . Average of 16 simulations per condition . Shaded bars are standard deviations . The online version of this article includes the following video and figure supplement ( s ) for figure 1 : Figure supplement 1 . Effect of different actin - and simulation - related parameters on pit internalization dynamics . Figure supplement 2 . Initiation from a pool of diffusing cytoplasmic actin filaments leads to variable timing of internalization . Figure 1\u2014video 1 . Simulations of continuum membrane mechanics model . https : / / elifesciences . org / articles / 49840 # fig1video1 Figure 1\u2014video 2 . Simulation of actin in endocytosis using Cytosim . https : / / elifesciences . org / articles / 49840 # fig1video2 Scheme 1 . Flow chart of multiscale modeling and experimental strategy combining membrane mechanics , actin spatiotemporal dynamics , molecule counting , and cryo - electron tomography . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 5 of 40 Research article Cell Biology Physics of Living Systems with an actin filament , it can bind to the filament and nucleate the growth of a new branched fila - ment at an ~ 77\u02da angle ( Blanchoin et al . , 2000 ) . Growing actin filaments can polymerize , diffuse under thermal fluctuations , and bend under force , and their growing ends are capped stochastically . Filament growth decreases with load according to the Brownian ratchet mechanism ( Mogilner and Oster , 1996 ; Peskin et al . , 1993 ) . Growth of the actin network is coupled to internalization of the endocytic pit by an actin - linking protein ( Hip1 / Hip1R / Epsin , simplified here as Hip1R ) , which is embedded in the coated pit and binds to actin filaments ( Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; Engqvist - Goldstein et al . , 1999 ; Sochacki et al . , 2017 ) . Importantly , most of the parameters in this model have been determined with measurements in vitro or in vivo , includ - ing the dimensions of the endocytic pit , its resistance to internalization ( modeled as a spring , Figure 1D ) , rates of association and dissociation of different proteins , branching angles , capping rates , filament persistence length , and stall force ( Supplementary file 3 and Materials and methods ) . Stochastic simulations of the model showed that this minimal branched actin network internalizes endocytic pits up to ~ 60 nm against physiological membrane tension ( Figure 1F and Figure 1\u2014 video 2 ) . In order to compare different conditions , we used two metrics \u2013 internalization of the pit ( in nm ) over time ( Figure 1G ) and the 95th percentile of internalization ( Figure 1\u2014figure supple - ment 1A ) . Then , we evaluated the robustness of the model to different parameters by conducting a series of parameter sweeps ( Figure 1\u2014figure supplement 1 ) . We found that the extent of internali - zation is robust to a wide range of parameters , including filament stiffness , stall force , and affinity between Hip1R attachments and actin filaments ( Figure 1\u2014figure supplement 1 ) . Initiating the sim - ulations from a cytoplasmic pool of linear actin filaments ( Raz - Ben Aroush et al . , 2017 ) allowed for endocytosis but the timing of the onset of internalization was more variable ( Figure 1\u2014figure sup - plement 2 ) . The extent of internalization was particularly sensitive to the number of available Arp2 / 3 complexes ( Figure 1G ) , indicating a need for precise measurements of this molecule at mammalian endocytic sites . Molecule counting of endogenously GFP - tagged Arp2 / 3 complex in live mammalian cells Motivated by our prediction that internalization rate is sensitive to the number of Arp2 / 3 complexes , we developed a method to count the number of molecules of endogenously GFP - tagged proteins in living mammalian cells ( Figure 2 ) . We adapted the self - assembling GFP - tagged protein nanocages developed by Hsia et al . ( 2016 ) for expression in live cells to create a fluorescence - based calibration curve relating fluorescence intensity of endogenously GFP - tagged proteins to numbers of molecules per endocytic site . Given that the nanocages are derived from bacterial glycolytic enzymes , we made point mutations known to abolish enzymatic activity of the proteins . To slow the diffusion of the intracellular nanocages and facilitate fluorescence measurements , we introduced an inducible dimerization motif to the plasma membrane by fusing the construct to FKBP and coexpressing a pal - mitoylated and myristoylated FRB variant ( Figure 2A ) . The resulting two - component fusion protein transiently associated with the plasma membrane even without rapamycin analog AP21967 , but the extent of association with the plasma membrane increased in a dose - dependent manner with the concentration of AP21967 ( Figure 2\u2014figure supplement 1A \u2013 B and Figure 3\u2014video 1 ) . We mea - sured the fluorescence intensity of four GFP - tagged nanocages of copy number ranging from 12 to 120 copies of GFP per structure using spinning disk confocal microscopy ( Figure 2B ) . After correct - ing for exposure time ( Figure 2\u2014figure supplement 1E \u2013 F ) , uneven illumination intensity , and local background ( Materials and methods ) , the fluorescence intensity per spot was unitary ( Figure 2C ) and directly proportional to the predicted numbers of molecules per structure ( R 2 = 0 . 996 ) ( Figure 2D ) . Using this calibration curve , we measured the numbers of molecules of an E . coli flagel - lar motor protein eGFP - MotB , which resulted in measurements similar to previously published meas - urements ( Figure 2\u2014figure supplement 1G \u2013 I ) . Thus , we established the suitability of this method to relate fluorescence intensity of endogenously GFP - tagged proteins to numbers of molecules inside live mammalian cells . To measure the timing , frequency , and numbers of Arp2 / 3 complexes assembling at sites of cla - thrin - mediated endocytosis , we used CRISPR / Cas9 - mediated genome editing to endogenously tag the C terminus of ArpC3 , a subunit of the Arp2 / 3 complex , with the fluorescent protein tagGFP2 in human induced pluripotent stem cells ( Figure 2E , Figure 2\u2014figure supplement 2A \u2013 B ) . Human Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 6 of 40 Research article Cell Biology Physics of Living Systems 12mer 0 1000 2000 3000 4000 0 20 40 24mer 0 1000 2000 3000 4000 0 5 10 60mer 0 1000 2000 3000 4000 0 20 40 120mer 0 1000 2000 3000 4000 0 20 40 0 50 100 150 Molecules per structure 0 1000 2000 3000 I n t en s i t y pe r s po t ( A . U . ) ! \" # $ % & \u2019 ( ) * ) + , - . / ) * ) 0122 , 220 \u00b1 190 390 \u00b1 130 1000 \u00b1 240 1800 \u00b1 400 12mer 24mer 60mer 120mer O cc u rr en c e s \" 2424me 6060me ! \" # $ % # & % \u2019 % ( ) # * \u2019 FRBFKBPnanocageGFP Intensity ( A . U . ) 120m ArpC3 - GFP ArpC2Arp3Arp2ArpC4 ArpC5 ArpC1 - , 0 60mer - 200 - 175 - 150 - 125 - 100 - 75 - 50 - 25 0 Time ( s ) 0 0 . 2 0 . 4 0 . 6 0 . 8 1 R e l a t i v e I n t e n s i t y ( A . U . ) 3 - 20 0 20 Time ( s ) 0 100 200 300 400 M o l e c u l e s o f A r p C 3 - G F P ! . 4 & 563 # 7 89 . : 9 ! 7 / 6 ; # 7 < 60mer - GFP 120mer - GFP ArpC3 - GFP ArpC3 - GFP AP2 - RFP # = : > . 9 ) / Figure 2 . Molecule counting of endogenously GFP - tagged Arp2 / 3 complex in live human induced pluripotent stem cells . ( A \u2013 D ) Development of a calibration curve relating fluorescence intensity to numbers of molecules in live cells . ( A ) Cartoon of intracellular GFP - tagged 60mer nanocage with inducible plasma membrane tether . Each subunit ( blue ) is tagged with GFP ( green ) and FKBP ( orange ) . FRB ( T2098L ) ( Purple ) is targeted to the plasma membrane by a palmitoylation and myristoylation sequence and dimerizes with FKBP in the presence of rapamycin analog AP21967 . Cartoon showing one of 60 tagged subunits is based on PDB structures 5kp9 , 2y0g , and 4dri . Scale bar 10 nm . ( B ) Inverse contrast fluorescence intensity images of human induced pluripotent stem cells expressing GFP - tagged plasma membrane - bound nanocages . Sum projection of nine 300 nm confocal images . Scale bar : 2 m m . ( C ) Histograms of fluorescence intensity per spot for the four calibration constructs showing mean \u00b1 standard deviation . Images were corrected for uneven illumination and intensity was background - corrected . Data from 305 spots in 15 cells over three experiments . ( D ) Calibration curve relating fluorescence intensity to numbers of molecules in mammalian cells . Line is a linear fit through zero . Error bars are standard deviations . ( E ) Cartoon drawn to scale of Arp2 / 3 complex tagged with GFP at the flexible C - terminus of ArpC3 . Known binding and activation sites are distal to this site . Based on PDB 2p9l . ( F ) Montage of CME event marked by AP2 - tagRFP - T and ArpC3 - tagGFP2 from TIRF imaging . Montage shows 4 s intervals from a movie taken at 2 s intervals . ( G ) Relative fluorescence intensity over time of AP2 - tagRFP - T and ArpC3 - tagGFP2 in endocytic events imaged by TIRF microscopy . Traces were normalized to maximum intensity and averaged . 121 traces from 8 cells in four experiments . Shading is \u00b1 1 s . d . ( H ) Fluorescence micrographs of ( left ) 60mer - tagGFP2 , ( left - center ) 120mer - tagGFP2 , ( right - center ) ArpC3 - tagGFP2 , and ( right ) ArpC3 - tagGFP2 and AP2 - tagRFP - T . White arrows mark spots in which ArpC3 - tagGFP2 and AP2 - tagRFP - T colocalize . Scale bar 2 m m . ( I ) Numbers of molecules of ArpC3 over time . The online version of this article includes the following video and figure supplement ( s ) for figure 2 : Figure 2 continued on next page Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 7 of 40 Research article Cell Biology Physics of Living Systems induced pluripotent stem cells are diploid and thus suitable for molecule - counting measurements when both alleles of the ArpC3 gene are fused to the gene for GFP . C - terminal GFP tags on ArpC3 are more functional than on other subunits of the Arp2 / 3 complex ( Egile et al . , 2005 ; Picco et al . , 2015 ; Sirotkin et al . , 2010 ; Smith et al . , 2013 ) . Cells tagged at both alleles of the ArpC3 gene had twice the fluorescence intensity of cells with a single allele tagged , suggesting direct proportionality between GFP fluorescence intensity and numbers of molecules ( Figure 2\u2014figure supplement 2C \u2013 D ) . These cells also endogenously express a tagRFP - T fusion with the m 2 subunit of the adaptor pro - tein AP2 , allowing us to identify sites of clathrin - mediated endocytosis ( Hong et al . , 2015 ) . We determined the relative timing of AP2 and ArpC3 appearance at endocytic sites using time - lapse TIRF imaging and automated two - color particle tracking ( Dambournet et al . , 2018 ; Hong et al . , 2015 ; Figure 2F ) . The vast majority ( 81 \u00b1 10 % , n = 136 ) of CME events marked by AP2 - RFP culminated in a burst of ArpC3 - GFP fluorescence , prior to internalization of the pit , persisting until the pit internalized ( Figure 2G and Figure 2\u2014video 2 ) . In addition , 24 \u00b1 4 % of ArpC3 - GFP tracks ( n = 145 ) did not colocalize with AP2 . We hypothesize that these are sites of clathrin - indepen - dent endocytosis . Then , using spinning - disk confocal fluorescence microscopy , we compared the fluorescence intensities of ArpC3 - GFP spots and GFP - tagged nanocage proteins in cells to deter - mine the numbers of ArpC3 - GFP molecules at clathrin - mediated endocytosis sites ( Figure 2H ) . Thus , we determined that ~ 200 molecules of Arp2 / 3 complex accumulate at clathrin - mediated endo - cytosis sites over time ( Figure 2I ) . Self - organization of actin filaments into a radial dendritic network drives endocytic internalization Incorporating the Arp2 / 3 molecule number we determined experimentally into our multiscale model , we next conducted simulations of the model to investigate the spatial organization of actin and force generation capacity of the endocytic network ( Figure 3 ) . Strikingly , the actin network self - organized around the endocytic pit . This self - organized network drove the assembly of 150 \u00b1 30 actin filaments ( Figure 3\u2014figure supplement 1A ) containing 5700 \u00b1 1100 monomers ( Figure 3\u2014figure supple - ment 1B ) . Interestingly , only a small number of actin filaments ( < 5 ) grew at any given time because the filaments became capped soon after they were nucleated ( Figure 3\u2014figure supplement 1C ; Berro et al . , 2010 ; Rangamani et al . , 2011 ; Xiong et al . , 2010 ) . Filament lengths were expo - nentially distributed with a final length of 90 \u00b1 80 nm ( Figure 3\u2014figure supplement 1D \u2013 E ) . Actin fil - aments bound to 120 \u00b1 10 Hip1R molecules in the coat ( Figure 3\u2014figure supplement 1F ) . The endocytic pit internalized ~ 60 nm in 10 \u2013 15 s ( Figure 3A and D ) . Based on the initial geometry of the endocytic pit and activated Arp2 / 3 complex , branched actin filaments self - organized into a radial dendritic network : the network attached to the clathrin coat by binding to Hip1R , the pointed ( minus ) ends localized close to the pit and the barbed ( plus ) ends near the base of the pit were ori - ented to grow toward the base of the pit ( Figure 3A \u2013 C and Figure 3\u2014video 1 ) . The axial self - organization of this branched actin network resembles that at the leading edge of cells ( Figure 3\u2014figure supplement 1G \u2013 I ; Maly and Borisy , 2001 ; Mueller et al . , 2017 ; Schaus et al . , 2007 ; Svitkina and Borisy , 1999 ) , with an important difference . Because actin fila - ment attachment sites are located on the coat of the endocytic pit , filaments radiate away from the center of the pit , such that most of the barbed ends orient radially away from the center of the pit rather than toward the coat or neck ( Figure 3E ) . The radial orientation of barbed ends gradually increases from the center of the pit , where there is no preferred orientation , to the periphery , where the barbed end radial orientation is highest ( Figure 3F ) . The extent of radial distribution of the Figure 2 continued Figure supplement 1 . Optimization and validation of fluorescence calibration method . Figure supplement 2 . Generation of genome - edited human induced pluripotent stem cell lines endogenously expressing AP2 - RFP and ArpC3 - GFP . Figure 2\u2014video 1 . Time lapse images of human induced pluripotent stem cells transiently expressing FKBP - 60mer - GFP and treated with 0 . 5 nM AP21967 . https : / / elifesciences . org / articles / 49840 # fig2video1 Figure 2\u2014video 2 . Time - lapse TIRF microscopy image of a human induced pluripotent stem cell endogenously expressing ArpC3 - GFP and AP2 - RFP . https : / / elifesciences . org / articles / 49840 # fig2video2 Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 8 of 40 Research article Cell Biology Physics of Living Systems R Z Axial orientation C D XZ B (cid:16)(cid:28)(cid:19)(cid:219) (cid:14)(cid:28)(cid:19)(cid:219) (cid:19)(cid:219) (cid:14)(cid:28)(cid:19)(cid:221) (cid:14)(cid:23)(cid:24)(cid:221) (cid:19)(cid:221) (cid:16)(cid:23)(cid:24)(cid:221) (cid:16)(cid:28)(cid:19)(cid:221) Radial orientation XY BE R Y X 1 - 1 F E H I I G + 1 + 0 . 5 0 - 0 . 5 - 1 random filaments endocytosis Base Neck p i nch i n g pu lli ng A Z ( n m ) X ( nm ) Barbed Barbed Pointed Pointed Base Neck + + 1 s 3 s 5 s 7 s 9 s 11 s 13 s 15 s Axial orientation Radial orientation 0 - 50 - 100 - 150 6420 - 2 - 4 - 6 - 200 0 200 - 200 0 200 - 200 0 200 - 200 0 200 - 200 0 200 - 200 0 200 - 200 0 200 - 200 0 200 R e l a t i v e nu m be r o f end s I n t e r n a li z a t i on ( n m ) Figure 3 . Self - organization of actin filaments into a radial dendritic network drives endocytic internalization . ( A ) ( Left ) Schematic depicting actin barbed ( plus ) or pointed ( minus ) ends . ( Right ) Heat maps of the positions of barbed ends ( red ) or pointed ends ( blue ) relative to the endocytic pit . Color code represents the relative number of ends . Each graph is averaged across 96 simulations and 1 s of simulation time . ( B ) Simulation output of endocytic actin filaments color - coded for axial ( Z ) orientation . Blue filaments orient toward the base of the pit ( + 90\u02da ) and green filaments orient parallel to the base of the pit ( 0\u02da ) . ( C ) Axial orientation of barbed ends . ( Left ) Schematic of axes . R is radial position of barbed end . ( Right ) Heat map of axial orientation of barbed ends as a function of R and Z position . Average of 96 simulations . ( D ) Pit internalization over time ( n = 96 simulations ) . ( E ) Simulation output of endocytic actin filaments color - coded for radial orientation . ( F ) Radially oriented endocytic actin filaments . ( Left ) Schematic of axes . Radial orientation is defined such that + 1 = barbed end oriented away from the center of the pit , and \" 1 = barbed end oriented toward the center of the pit . ( Right ) Heat map of radial orientation of barbed ends as a function of X and Y position ( n = 96 simulations ) . Barbed ends radiate outward . ( G ) Radial orientation of barbed ends over time for 96 simulations . Gray curve is negative control of randomly oriented filaments ( n = 50 filaments in one simulation ) . ( H ) Concentration of barbed ends near the base of the endocytic pit . ( Left ) Schematic of positions of the neck and base of the pit . ( Right ) Number of barbed ends near base ( green ) or neck ( blue ) of pit , defined as within 7 . 5 nm of each surface . ( I ) The majority of forces are directed orthogonal to the base of the pit based on positions of barbed ends in simulations . Shaded bars are standard deviations . The online version of this article includes the following video and figure supplement ( s ) for figure 3 : Figure supplement 1 . Assembly and self - organization of endocytic actin network . Figure 3\u2014video 1 . Simulation of actin in endocytosis with actin filaments color coded for axial orientation . https : / / elifesciences . org / articles / 49840 # fig3video1 Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 9 of 40 Research article Cell Biology Physics of Living Systems filaments increases rapidly after time 0 ( Figure 3G ) . An important consequence of this self - organiza - tion is that , based on the position of Hip1R and the Arp2 / 3 complex , more barbed filament ends localize near the base ( 10 \u00b1 4 ends ) than near the neck of the endocytic pit ( 1 \u00b1 1 ends ) ( Figure 3H ) . These data result in an important prediction from our model : an actin network self - organized as described here will produce an axial force during pit internalization ( Figure 3I ) . We predict that the radial dendritic self - organization is a powerful mechanism that makes endocytic actin networks resil - ient to biochemical and mechanical perturbations . Spatial distribution of actin / coat attachments and Arp2 / 3 complex , but not Arp2 / 3 complex density , strongly affects actin self - organization and pit internalization Our finding that self - organized endocytic actin networks grow toward the base of the pit prompted us to explore the molecular mechanism by which actin filaments self - organize . Actin dynamics in association with the endocytic machinery can be thought of as a polymerization engine constrained by two spatial boundary conditions \u2013 active Arp2 / 3 complex at the base of the pit ( Almeida - Souza et al . , 2018 ; Idrissi et al . , 2008 ; Kaksonen et al . , 2003 ; Mund et al . , 2018 ; Picco et al . , 2015 ; Kaplan et al . , in preparation ) and Hip1R / actin attachments on the curved pit surface ( Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; Engqvist - Goldstein et al . , 1999 ; Sochacki et al . , 2017 ; Figure 4A ) . Given that such spatial boundary conditions confer unique mechanical properties and adaptation to loads under flat geometries in vitro ( Bieling et al . , 2016 ) , we aimed to understand how the boundary conditions corresponding to the curved endocytic pit affect endocytic actin organization and internalization . We tested two different scenarios : varying the surface density of Arp2 / 3 complex at the base of the pit and varying Hip1R surface coverage around the pit itself . First , we tested whether the surface density of the Arp2 / 3 complex at the base of the pit affects endocytic internalization because recent studies in vitro and in yeast suggest that the local concen - tration of Arp2 / 3 complex activators is critical for the timing of Arp2 / 3 complex activation and endo - cytic progression ( Case et al . , 2019 ; Sun et al . , 2017 ) . In a series of simulations , we distributed 200 molecules of active Arp2 / 3 complex in a ring of increasing outer diameter to vary the surface den - sity . Surprisingly , we found that varying the surface density of Arp2 / 3 complex along the base of the pit by a factor of 20 had little impact on endocytic outcome ( Figure 4\u2014figure supplement 1 ) . We also explored whether localization of a fraction of Arp2 / 3 complexes at the neck of the pit provided an additional advantage for the endocytic outcome . In this scenario , we distributed 50 of the 200 molecules of the active Arp2 / 3 complex near the neck of the pit . We found that localizing some of the active Arp2 / 3 complex near the neck of the pit did not have an impact on the outcome of simu - lations ( p > 0 . 5 ) ( Figure 4\u2014figure supplement 2D \u2013 E ; Figure 4\u2014figure supplement 2 ) . We next conducted a series of simulations in which we varied the surface distribution of a con - stant number of Hip1R molecules to cover between 1 % ( localized to the tip of the pit ) and 80 % ( up to the neck of the pit ) of the pit ( Figure 4B ) and found that the surface distribution of Hip1R around the endocytic pit strongly impacted endocytic outcome ( Figure 4 ) . Simulations in each of these con - ditions revealed that endocytic internalization depends on the surface distribution of actin - coat attachments around the endocytic site ( Figure 4C and Figure 4\u2014video 1 ) . Both the rate and extent of internalization increased with increasing surface area of Hip1R around the pit ( Figure 4D ) . From a functional standpoint , increased Hip1R surface coverage around the pit drove more barbed ends toward the base of the pit ( Figure 4E ) . This increase in Hip1R surface coverage resulted in an increase in Arp2 / 3 complexes bound in the endocytic actin network ( Figure 4F ) , which in turn nucle - ated more actin filaments ( Figure 4G ) . Simulations showed that a threshold of ~ 100 Hip1R molecules on the pit is necessary for endocytic internalization ( Figure 4\u2014figure supplement 3A ) . The high impact of Hip1R surface distribution on actin filament organization implies that Hip1R molecules dis - tributed broadly around the pit allow for multivalent attachments between the pit and actin fila - ments , resulting in filaments being captured in an orientation conducive to force production . Further examination of the simulations revealed that the Hip1R surface distribution supports a self - organized dendritic actin network via a mechanism of stochastic self - assembly and selection for actin filaments growing toward the base of the pit ( Figure 4\u2014figure supplement 3B ) . Mother fila - ments initially bind and unbind the coat in random orientations ( Figure 4\u2014figure supplement 3B \u2013 C ) . Filaments growing toward the interior of the cell do not template the growth of new branched Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 10 of 40 Research article Cell Biology Physics of Living Systems actin filaments . However , filaments growing toward the base of the pit encounter active Arp2 / 3 com - plex , which catalyzes dendritic nucleation of new actin filaments growing in a similar direction ( Fig - ure 4\u2014figure supplement 3B and D ; Carlsson , 2001 ) . As a result , near the base of the pit , filaments increasingly orient toward the base of the pit over time ( Figure 4\u2014figure supplement 3E \u2013 F ) . Our observations therefore establish an important principle for actin organization in endocytosis : the positions of active Arp2 / 3 complexes are critical for organizing the actin network and determin - ing the direction of force production , while the Hip1R linker distribution is critical for recruiting 40 - 40 - 20 0 20 40 A r p2 / 3 c o m p l e x H i p1 R Boundary 1 : Active Arp2 / 3 complex Boundary 2 : Hip1R attachments B C G A D E C F Time ( s ) N u m be r A r p2 / 3 bound Hip1R coverage ( % ) Hip1R coverage ( % ) Z ( n m ) X ( n m ) Y ( n m ) N u m be r f il a m en t s bound barbed end Hip1RActin 80604020101 11020406080 - 40 - 20 0 20 20 - 40 Z ( nm ) 40 40 0 0 - 40 - 40 40 40 0 0 - 40 - 40 40 40 0 0 - 40 - 40 40 40 0 0 - 40 - 40 40 40 0 0 - 40 - 40 40 40 0 0 - 40 - 40 0 5 10 15 100 50 0 95 % i n t e r na li z a t i on ( n m ) Time ( s ) 0 5 10 15 Time ( s ) Time ( s ) 0 5 10 15 0 5 10 15 B a r bed end s nea r ba s e 200 150 100 50 0 200 150 100 50 0 20 10 0 1 % coverage 10 % coverage 20 % coverage 40 % coverage 60 % coverage 80 % coverage 1 % coverage 10 % coverage 20 % coverage 40 % coverage 60 % coverage 80 % coverage Hip1R coverage Figure 4 . Spatial distribution of actin / Hip1R attachments strongly affects actin self - organization and pit internalization . ( A ) Schematic of spatial boundary conditions from endocytic actin - binding proteins . Positions of active Arp2 / 3 complex ( blue ) and actin / pit attachments via linker proteins such as Hip1R ( purple ) . ( B ) Initial positions of Hip1R around increasingly large pit surface area , from 1 % to 80 % of a sphere . The top ~ 20 % of the sphere is occluded by the neck . ( C ) Snapshots of a series of simulations for different values of Hip1R coverage showing actin distribution at t = 13 s . ( D \u2013 G ) Changes in the endocytic actin network over time as a function of Hip1R coverage ( colors ) . n = 96 simulations . ( D ) Internalization ; ( E ) Number of barbed ends near the base of the pit ( within 7 . 5 nm ) ; ( F ) Number of Arp2 / 3 complexes bound in the endocytic network ; ( G ) Number of actin filaments bound in the endocytic network . Scale bar : 50 nm . The online version of this article includes the following video and figure supplement ( s ) for figure 4 : Figure supplement 1 . Relationship between endocytic outcome and active Arp2 / 3 complex surface density or mother filament nucleating protein surface density at the base of the pit . Figure supplement 2 . A collar of active Arp2 / 3 complex near the neck the pit does not affect endocytic outcome . Figure supplement 3 . Internalization as a function of the number of Hip1R molecules and mechanism of self - organization of endocytic actin filaments . Figure 4\u2014video 1 . Simulations in which the coverage of linker Hip1R around the pit was varied from 1 % to 80 % of a sphere . https : / / elifesciences . org / articles / 49840 # fig4video1 Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 11 of 40 Research article Cell Biology Physics of Living Systems mother filaments that activate the Arp2 / 3 complex to direct filament growth to the area surrounding the base of the pit . Bending of endocytic actin filaments contributes to endocytic robustness Given that self - organized actin filaments help to nucleate new actin filaments that grow toward the base of the pit , questions remained about how these filaments contribute to and sustain force pro - duction . Closer examination of the simulations revealed that long actin filaments bend between their attachment sites in the clathrin coat and the base of the pit as a result of assembly confined by the membrane at the base of the pit ( Figure 5A ) . We predict that these bent filaments provide a previ - ously unrecognized means of force production by endocytic actin filaments . To test the prediction that actin filaments bend at sites of mammalian endocytosis , we used cryo - electron tomography on intact vitrified mammalian ( SK - MEL - 2 ) cells . SK - MEL - 2 cells grown on electron - microscopy grids are thin at their periphery ( < 1 m m ) , which makes them suitable for electron tomography studies . Indeed , we found bent actin filaments present at sites of clathrin - mediated endocytosis , between the clathrin coat ( Figure 5\u2014figure supplement 1 ) and the base of the pit ( Figure 5B and Figure 5\u2014video 1 ) , in extended \u2018U\u2019 - shaped clathrin - coated pits similar to the stage modeled in our simulations ( Figure 5C \u2013 D ) . What could be a functional consequence of such bent filaments ? We hypothesized that the bent actin filaments store elastic energy that promotes endocytic internalization . We first quantified the filament bending in simulations and found that many ( 13 \u00b1 3 % ) of the actin filaments bend further than can be accounted for by thermal fluctuations ( Boal and Boal , 2012 ; Mogilner and Oster , 1996 ; Figure 5E and Figure 5\u2014figure supplement 2A ) . Most ( 92 % ) of the bent filaments bent less than the minimum energy expected to sever the filaments ( De La Cruz et al . , 2015b ; Sept and McCammon , 2001 ; Figure 5E ) . Importantly , the bent filaments stored elastic energy by collectively continuing to bend over time , storing up to ~ 750 pN ! nm of elastic energy \u2013 mostly in capped fila - ments ( Figure 5F ) . In the context of pit internalization , the amount of elastic energy stored was larger than the magnitude of work required to internalize endocytic pits ( Figure 5F and Figure 5\u2014 figure supplement 2B ) . The elastic energy stored in bent filaments was ~ 1 % of the total energy required to polymerize the endocytic actin network ( Figure 5\u2014figure supplement 2C \u2013 D ) , with pit internalization constituting ~ 0 . 5 % of the total energy from actin filament assembly ( Figure 5\u2014figure supplement 2E ) . The majority ( 62 \u00b1 20 % ) of filament bending energy came from filaments directly bound to Hip1R , and 78 \u00b1 25 % of the bending energy came from filaments with barbed ends > 5 nm from the coat surface ( Figure 5\u2014figure supplement 2F \u2013 H ) . 17 \u00b1 16 % of bending energy came from filaments with barbed ends near the base of the pit ( Figure 5\u2014figure supplement 2I ) . For fila - ments near the base of the pit , the bending energy was distributed radially such that filaments with barbed ends ~ 130 nm from the center of the pit contribute the most bending energy ( Figure 5\u2014 figure supplement 2J ) . Filament bending serves as an important functional consequence of the self - organization of actin filaments at endocytic sites ( Figure 4 ) . With high Hip1R surface coverage around the pit , filaments directed to grow toward the base of the pit bend , storing elastic energy ( Figure 5\u2014figure supple - ment 2K \u2013 L ) . This elastic energy can be harnessed gradually under thermal fluctuations to drive endocytic internalization through a ratchet mechanism ( Mogilner and Oster , 1996 ) . To test the hypothesis that energy stored in bent actin filaments can promote endocytic internali - zation , we conducted simulations in which the resistance from membrane tension was released at a late time point ( t = 10 s , internalization ~ 50 nm ) along with capping filament growth ( Figure 5G ) . This scenario allowed us to test how the stored energy in the bent filaments ( rather than force gener - ated by growing filaments ) can promote internalization in response to an abrupt decrease in tension . We found that once membrane tension decreases , pit internalization sharply increases ( Figure 5H and Figure 5\u2014video 2 ) and filament bending near the base of the pit gradually decreases by 50 % with wide variance ( Figure 5I ) . Thus , we found that in addition to generating force actively by fila - ment growth ( Figure 3 ) , the endocytic actin network stores potential energy in the form of bent fila - ments that can promote internalization even after filaments have stopped growing . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 12 of 40 Research article Cell Biology Physics of Living Systems ! \" # $ \" % & \u2019 ( # $ ) & \u2019 ( # $ * + , ( - . + / 0 , + 1 , + / - 2 + - ( - . + / 0 , + 1 , + / - 2 + - ( - . + / 0 , + + , ( - . + / 0 , + 3 4 5 6 f il a m en t s 21 - 07 . ( 8 . + 90 + : ; 2 / / 07 . 8 . + 90 + : / . 7 . < 0 + : Z = 0 nm Z = 37 nm = > ? @ A . B8 < 2 + . ( B . 1C2 + 01 / ( / 0BDE2 - 0 , + = 1 - 0 + ( B . 1C2 + 01 / ( / 0BDE2 - 0 , + ? < F , % . E . 1 - < , + ( - , B , : < 2B ? D - ( / ; < 0 + : 4 + - . < + 2E0G . / HD < - C . < I ? 2 ; H0E2B . + - / ? D - ( 12 ; ; . 9 0 + - . < + 2E0G2 - 0 , + : < , J0 + : 1500 1000 500 0 200 100 0 90 75 60 45 30 15 0 0 5 10 15 0 5 10 15 0 - 50 - 100 0 5 10 15 0 100 200 300 400 500 Time ( s ) Time ( s ) Time ( s ) Free filament length ( nm ) B end i ng ene r g y ( p N (cid:278) n m ) B end i ng ene r g y ( p N (cid:278) n m ) I n t e r n a li z a t i on ( n m ) B end i ng ang l e ( deg r ee s ) Figure 5 . Bending of endocytic actin filaments stores elastic energy for pit internalization . ( A ) Snapshot of simulation showing filaments bent between the attachment site in the coat and the base of the pit . Also see Figure 1F . Yellow arrowheads point to a bent actin filament . ( B ) Tomographic slice of cryo - electron tomogram of an SK - MEL - 2 cell . Long actin filaments ( yellow arrowheads ) bend along the clathrin - coated pit between the coat and the base of the pit . ( C ) Snapshot of membrane mechanics simulation under an internalization force with 60 nm internalization . ( D ) Slice of the same tomogram as shown in B at a different Z - level ( + 37 nm ) in which the coated plasma membrane ( white arrowheads ) is visible . Scale bar for A - D : 50 nm . ( E ) Heat map of the bending angle and free filament length of endocytic actin filaments in simulations . Color code is number of filaments ( summed for all time points , average of 24 simulations ) . Lines demarcate the magnitude of energy stored in these filaments , based on the theory of elastic beam rigidity for filaments of persistence length 10 m m ( Materials and methods ) , in units of k B T ( 4 . 1 pN ! nm ) . Purple lines : filament conformations expected from thermal fluctuations ( passive bending ) . White lines : filament bending greater than from thermal fluctuations ( active bending ) . Magenta lines : lower Figure 5 continued on next page Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 13 of 40 Research article Cell Biology Physics of Living Systems Inhibiting Arp2 / 3 complex activity stalls endocytosis We next investigated how inhibiting the activity of Arp2 / 3 complex would affect endocytosis ( Fig - ure 6 ) . Our simulations , conducted by varying the nucleation rate of Arp2 / 3 complex , predicted that inhibiting Arp2 / 3 complex activity stalls endocytosis ( Figure 6A ) . Endocytosis was inhibited when Arp2 / 3 complex nucleation rates fell below the basal value of 1 filament per second ( Beltzner and Pollard , 2008 ) , and was insensitive to increased rates of nucleation ( Figure 6B ) . We validated this relationship with experiments modulating Arp2 / 3 complex activity in cells . The small molecule inhibi - tor CK - 666 prevents the Arp2 / 3 complex from nucleating actin filaments ( Hetrick et al . , 2013 ; Figure 5 continued limit for bending energy expected to sever filaments ( De La Cruz et al . , 2015b ) . ( F ) Total elastic energy stored in bent capped ( red ) or growing ( green ) endocytic actin filaments during simulation over time compared to mean energy necessary for internalization ( black ) ( n = 96 simulations ) . ( G ) Schematic of an in silico experiment to test the mechanical function of bent endocytic actin filaments . At t = 10 s , the membrane tension was reduced to zero , and the filaments were capped . ( H ) Internalization ( green ) after spring cut and filament capping , compared to simulation with no change in tension ( black , same data as Figure 3D ) . n = 48 simulations . ( I ) Bending energy of endocytic actin filaments with barbed ends near base of pit over time . Release of tension and filament capping at t = 10 s ( green ) compared to no change in tension ( black ) . The online version of this article includes the following video and figure supplement ( s ) for figure 5 : Figure supplement 1 . Hexagonal and pentagonal lattices in tomogram of clathrin - coated pit . Figure supplement 2 . Energetics of endocytic actin network . Figure 5\u2014video 1 . Cryo - electron tomogram of SK - MEL - 2 cell grown on holey carbon grid and vitrified , related to Figure 5 . https : / / elifesciences . org / articles / 49840 # fig5video1 Figure 5\u2014video 2 . Simulation of actin in endocytosis in which , at t = 10 s , filaments were all capped and the membrane tension was reduced to 0 pN / nm . https : / / elifesciences . org / articles / 49840 # fig5video2 ! \" # Arp2 / 3 nucleation rate ( filaments per second ) A $ % & \u2019 ( ) * + , - & . / 0 12 + . / 345 , 6 # 7888 100 75 50 25 0 0 0 . 2 0 . 4 0 0 . 2 0 . 4 0 0 . 2 0 . 4 0 50 100 150 200 250 0 0 . 2 0 . 4 Lifetime ( s ) Time ( s ) F r equen cy 9 * : ; * # 7888 < * : ; * # 7888 \u2019 < * : ; * # 7888 < 9 * : ; * # 7888 # . 34 = % 56 > ? @ A * ! B63 - 56\u2019 > C @ A 95 % i n t e r na li z a t i on ( n m ) 0 5 10 ref - track lifetimes : 10 . 00 - 87 . 34 ( n = 182 ) , 87 . 34 - 164 . 67 ( n = 14 ) , 164 . 67 - 242 . 01 ( n = 18 ) , - 200 - 150 - 100 - 50 0 0 0 . 2 0 . 4 0 . 6 0 . 8 A v e r age I n t en s i t y ref - track lifetimes : 14 . 00 - 90 . 00 ( n = 19 ) , 90 . 00 - 166 . 01 ( n = 3 ) , 166 . 01 - 242 . 01 ( n = 3 ) , - 200 - 150 - 100 - 50 0 0 0 . 2 0 . 4 0 . 6 0 . 8 A v e r age I n t en s i t y ref - track lifetimes : 10 . 00 - 87 . 34 ( n = 71 ) , 87 . 34 - 164 . 67 ( n = 10 ) , 164 . 67 - 242 . 01 ( n = 13 ) , - 200 - 150 - 100 - 50 0 0 0 . 2 0 . 4 0 . 6 0 . 8 A v e r age I n t en s i t y cor - track lifetimes : 10 . 00 - 87 . 34 ( n = 21 ) , 87 . 34 - 164 . 67 ( n = 8 ) , 164 . 67 - 242 . 01 ( n = 6 ) , - 200 - 150 - 100 - 50 0 0 0 . 2 0 . 4 0 . 6 0 . 8 A v e r age I n t en s i t y # . 34 = % 56 > ? @ A * ! B63 - 56\u2019 > C @ A N o r m a li z ed i n t en s i t y ( A . U . ) 9 * : ; * # 7888 < * : ; * # 7888 \u2019 < * : ; * # 7888 < 9 * : ; * # 7888 Figure 6 . Inhibiting Arp2 / 3 complex nucleation activity stalls endocytosis . ( A ) Schematic of the model parameter corresponding to Arp2 / 3 nucleation activity , and the step inhibited by the small molecule CK - 666 . ( B ) Internalization as a function of Arp2 / 3 complex nucleation rate . Orange region highlights parameter sensitivity , and green region highlights parameter insensitivity . n = 96 simulations . Reducing Arp2 / 3 nucleation rate reduces internalization as seen in the orange region . ( C ) Histograms of endocytic lifetime in SK - MEL - 2 cells endogenously expressing clathrin light chain CLTA - tagRFP - T and dynamin2 - eGFP and treated with CK - 666 . n = 368 tracks from 10 cells . ( D ) Fluorescence intensity over time for endocytic tracks marked by clathrin - RFP and dynamin2 - GFP in SK - MEL - 2 cells treated with 0 . 1 % DMSO ( 0 m M ) or the indicated concentration of CK - 666 for 45 min . Fluorescence events were tracked automatically ( Materials and methods ) . Tracks in which GFP and RFP colocalized are shown . Each track was normalized to its maximum intensity and then all tracks were averaged and aligned to the time of the disappearance of the clathrin - RFP signal . The lifetimes of these events are plotted in D . Shaded bars are standard deviations . The online version of this article includes the following figure supplement ( s ) for figure 6 : Figure supplement 1 . Effect of Arp2 / 3 complex inhibitor CK - 666 on lifetimes of endogenously tagged markers of endocytosis . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 14 of 40 Research article Cell Biology Physics of Living Systems Nolen et al . , 2009 ) . Treatment of SK - MEL - 2 cells with CK - 666 inhibited endocytic progression , as marked by the lifetimes of endogenously tagged AP2 - RFP or dynamin2 - GFP at endocytic sites , in a dose - dependent and time - dependent manner ( Figure 6C \u2013 D and Figure 6\u2014figure supplement 1A ) . Adaptation of the endocytic actin network to changes in membrane tension Because we and others previously modeled that membrane tension plays an important role in mem - brane bending during the formation of an endocytic pit ( Hassinger et al . , 2017 ; Rangamani et al . , 2014 ; Walani et al . , 2014 ) we next varied the value of membrane tension in simulations to under - stand the relationship between tension , actin filament bending , and actin assembly ( Figure 7A ) . In our simulations , endocytic progression attenuated in a tension - dependent manner ( Figure 7A \u2013 E ) , consistent with previous modeling ( Hassinger et al . , 2017 ) and experimental observations ( Boulant et al . , 2011 ; Ferguson et al . , 2017 ; Ferguson et al . , 2016 ; Wu et al . , 2017 ) . However , at higher membrane tensions , endocytosis persisted better than expected for a non - adapting network , suggesting the existence of an adaptive mechanism ( Figure 7E ) . Therefore , we sought to under - stand how endocytic actin networks adapt to increases in load . We found that under low tension ( 0 . 015 pN / nm ) , endocytic pits internalize strongly ( Figure 7B and E ) and few barbed ends encounter the base of the pit ( Figure 7F ) , with fewer Arp2 / 3 complexes recruited to the network ( Figure 7\u2014figure supplement 1A ) and a correspondingly low filament bending energy ( Figure 7H ) . Under > 50 x higher membrane tension ( 1 pN / nm ) , endocytic ! \" # $ % & \u2019 ( ) * + , - & + . * / - 01 ( ) * + , - & + 2 - 34 ( ) * + , - & + 5 6 7 2 8 * + , - & + ! 9 ) - + : / : ; ) : ) - & + < 6 - = : 1 * + ) > * + / - + 3 B a r bed end s nea r ba s e A c t i n f il a m en t s 95 % i n t e r na li z a t i on ( n m ) Tension ( pN / nm ) Tension ( pN / nm ) Tension ( pN / nm ) Tension ( pN / nm ) 100 50 0 0 . 01 0 . 1 1 10 0 . 01 0 . 1 1 10 0 . 01 0 . 1 1 10 0 . 01 0 . 1 1 10 B end i ng ene r g y ( p N (cid:278) n m ) Figure 7 . Adaptation of endocytic actin network to changes in membrane tension . ( A ) Schematic depicting possible adaptation of the actin network to membrane tension via self - organization and bending . ( B \u2013 D ) Snapshots of simulations from the same time point ( 14 s ) for ( B ) low membrane tension ( 0 . 015 pN / nm ) ; ( C ) medium membrane tension ( 0 . 15 pN / nm ) ; ( D ) high membrane tension ( 1 pN / nm ) . Scale bar is 50 nm . ( E \u2013 H ) Changes in the endocytic actin network as a function of membrane tension . n = 144 simulations . ( E ) Internalization ; ( F ) Number of barbed ends near base of pit ; ( G ) Number of actin filaments in Hip1R - bound network ; ( H ) Bending energy for filaments with barbed ends near base of pit . Mean \u00b1 standard deviation of time points in the last 5 s of simulations . Dashed line in ( E ) is expected internalization based on constant energy usage with 0 . 01 pN / nm condition as reference ( see Methods ) . The online version of this article includes the following video and figure supplement ( s ) for figure 7 : Figure supplement 1 . Membrane tension - dependent adaptation by the actin network . Figure 7\u2014video 1 . Simulations of actin in endocytosis with different values of membrane tension ( Low , 0 . 01 pN / nm ; Medium , 0 . 15 pN / nm ; High , 1 pN / nm ) . https : / / elifesciences . org / articles / 49840 # fig7video1 Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 15 of 40 Research article Cell Biology Physics of Living Systems internalization slowed but was not abolished ( Figure 7E ) . For these pits , more barbed ends encoun - tered the base of the pit ( Figure 7F ) , binding more Arp2 / 3 complexes ( Figure 7\u2014figure supple - ment 1A ) to nucleate more actin filaments ( Figure 7G ) and increasing the total actin filament bending energy near the base of the pit ( Figure 7H ) . As a result , upon increasing membrane ten - sion , the overall endocytic energy efficiency increased ( Figure 7\u2014figure supplement 1B ) . Thus , the self - organization of the endocytic actin network allows it to adapt to elevated membrane tension by nucleating more filaments at the base of the pit . Arp2 / 3 complex activity and Hip1R / actin attachments are critical for allowing actin filaments to drive endocytic pit internalization and adapt to changing tension Having established that endocytic internalization depends on two spatially confined boundary condi - tions \u2013 Hip1R / actin attachments at the curved pit ( Figure 4 ) and active Arp2 / 3 complex activity at the base of the pit ( Figure 6 ) \u2013 we next investigated how these boundary conditions alter the endo - cytic response to membrane tension ( Figure 8A ) . We systematically varied membrane tension and Arp2 / 3 complex activity in our model to gener - ate a phase diagram of endocytic internalization as a function of membrane tension and Arp2 / 3 com - plex activity ( Figure 8B ) . This phase diagram shows that cells with high membrane tension are especially sensitive to changes in Arp2 / 3 complex nucleation rate ( Kaplan et al . , in preparation ) , whereas cells with low membrane tension carry out endocytosis even with low Arp2 / 3 complex activ - ity , consistent with experimental observations ( Boulant et al . , 2011 ) . We hypothesized that actin network self - organization arising from the broad Hip1R distribution around the pit ( Figure 4 ) and filament bending ( Figure 5 ) might allow for the endocytic actin net - work to change its organization and force - producing capacity under elevated loads ( Figure 7 ) . To test this hypothesis , we conducted simulations in which Hip1R coverage was varied for different val - ues of plasma membrane tension ( Figure 8A and Figure 7\u2014video 1 ) . We found that the endocytic actin network\u2019s ability to adapt to load ( Figure 7 ) depends on Hip1R coverage around the pit ( Figure 8D \u2013 F ) . As the coverage of Hip1R around the pit increased , actin\u2019s ability to adapt to changes in membrane tension also increased , as measured by the number of barbed ends near the base of the pit ( Figure 8D ) , the binding of active Arp2 / 3 complex at the base of the pit ( Figure 8E ) , subsequent nucleation of additional actin filaments ( Figure 8F ) , and bending of actin filaments near the base of the pit ( Figure 8G ) . We conclude that sufficient Hip1R coverage around the pit ( Clarke and Royle , 2018 ; Sochacki et al . , 2017 ) allows endocytic actin filaments to orient in such a way that they can encounter more Arp2 / 3 complexes at the base of the pit to nucleate more actin fil - aments . This spatial organization allows the actin network to adapt to sustain force production under a range of opposing loads ( Figure 8H ) . Discussion Understanding the relationship between actin filament assembly , actin network organization , and force generation on the plasma membrane requires iterative feedback between experimental meas - urements and computational modeling . An ultimate goal of this study was to relate single actin fila - ment mechanics to force generation by the collective actin filament network in CME ( Lacayo et al . , 2007 ) . We integrated modeling and quantitative cellular measurements to show that a minimal actin network composed of actin , the Arp2 / 3 complex and capping protein , with linker attachments in the clathrin coat and rates constrained by cellular and biochemical measurements , is able to generate sufficient force to internalize endocytic pits against mammalian plasma membrane tension . Approxi - mately 200 Arp2 / 3 complexes constitutively assemble at sites of endocytosis in human induced plu - ripotent stem cells . Endocytic actin filaments self - organize into a radial dendritic array , in which filaments grow toward the base of the pit . These filaments bend and store elastic energy , which sup - ports internalization . The endocytic actin network adapts to changes in membrane tension by driving more filaments to the base of the pit and increasing filament bending , which supports a higher load and nucleates more actin filaments . Four lines of experimental evidence support our model ( Figure 8\u2014figure supplement 1 ) . Two pieces of evidence serve as model validation based on published data and two more are based on experiments conducted in this study . Previous experiments from our lab showed that knocking down Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 16 of 40 Research article Cell Biology Physics of Living Systems Hip1R in HeLa cells inhibited endocytosis ( Engqvist - Goldstein et al . , 2004 ) . Consistent with these observations , our simulations showed that a threshold number of actin linkers such as Hip1R is nec - essary for endocytic internalization ( Figure 4\u2014figure supplement 3A and Figure 8\u2014figure supple - ment 1A \u2013 B ) . This threshold appears necessary to allow efficient filament capture by the coat and force transmission from the actin network to the coat . Second , experiments showed that capping actin filament elongation with the small molecule compound Cytochalasin inhibits CME , reflected by an increase in stalled endocytic sites marked by clathrin - RFP ( Brady et al . , 2010 ) or slower A r p2 / 3 c o m p l e x D Low Medium High E F Hip1R coverage ( % ) Hip1Rcoverage Tension Tension 0 20 40 60 80 100 50 0 95 % i n t e r na li z a t i on ( n m ) 3 . Nucleates new filaments Actin Arp 2 / 3 complex Clathrin N - WASP Hip1R 1 . Broad Hip1R coverage in clathrin coat 2 . Drives barbed ends to base 4 . Filaments bend to store elastic energy T en s i on ( p N / n m ) 0 . 1 0 . 01 0 . 001 0 . 0001 Arp2 / 3 nucleation rate ( filaments per second ) 0 1 10 H A B C 95 % internalization ( nm ) Hip1R coverage ( % ) Hip1R coverage ( % ) Hip1R coverage ( % ) Hip1R coverage ( % ) G B a r bed e n d s nea r ba s e A c t i n f il a m en t s B ound A r p2 / 3 c o m p l e x B end i ng e n e r g y ( p N (cid:278) n m ) Figure 8 . Arp2 / 3 complex activity and Hip1R / actin attachments are critical for allowing actin filaments to drive endocytic pit internalization and adapt to changing tension . ( A ) Schematic of Arp2 / 3 complex activity and Hip1R coverage along with membrane tension . ( B ) Phase diagram of endocytic internalization as a function of membrane tension and Arp2 / 3 complex nucleation rate shown on a log - log plot . Dotted lines are values taken from the literature ( Beltzner and Pollard , 2008 ; Diz - Mun\u02dcoz et al . , 2016 ) . ( C \u2013 G ) Changes in the endocytic actin network as a function of Hip1R coverage for different values of membrane tension . Low tension = 0 . 015 pN / nm ; medium tension = 0 . 15 pN / nm ; high tension = 1 pN / nm . n = 288 simulations . ( C ) Internalization ; ( D ) Number of barbed ends near base of pit ; ( E ) Number of Arp2 / 3 complexes bound in network ; ( F ) Number of actin filaments bound in network ; ( G ) Bending energy of filaments with barbed ends near the base of the pit . Mean \u00b1 standard deviation of time points in the last 5 s of simulations . ( H ) Summary of load - dependent adaptation of self - organizing endocytic actin network due to spatial segregation of active Arp2 / 3 complex at the base and Hip1R in a broad distribution within the clathrin coat . The online version of this article includes the following figure supplement ( s ) for figure 8 : Figure supplement 1 . Summary of predictions of the model supported by experimental data in the current manuscript and in the literature . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 17 of 40 Research article Cell Biology Physics of Living Systems accumulation of dynamin2 - GFP at endocytic sites ( Grassart et al . , 2014 ) . We also showed in our model that capping rate is an important parameter for progression of CME ; our simulations show that increasing the capping rate of actin filaments inhibits CME , presumably because increasing cap - ping decreases the total amount of actin ( Figure 1\u2014figure supplement 1C and Figure 8\u2014figure supplement 1C \u2013 D ) . In this study , our simulations predicted that actin filaments bend around endo - cytic pits . These bent filaments store elastic energy for subsequent force production much as a pole vaulter\u2019s pole bends and stores energy for delayed force production . Using cryo - electron tomogra - phy of intact cells , we observed bent actin filaments at sites of endocytosis in mammalian cells ( Fig - ure 5 and Figure 8\u2014figure supplement 1E \u2013 F ) . Finally , we also predicted that inhibiting Arp2 / 3 complex activity below its basal nucleation rate of 1 filament per second inhibits endocytosis in silico and this prediction was validated in cells using pharmacological agents ( Figure 6C , D and Figure 8\u2014 figure supplement 1G \u2013 H ) . Without sufficient Arp2 / 3 complex , CME fails due to insufficient force production . Three main conclusions resulted from our study . First , we found that the spatial segregation of Arp2 / 3 complex activation and Hip1R linker proteins on the clathrin coat are important factors for effective force generation . Unlike actin organization at the leading edge of a migrating cell wherein only one boundary condition at the plasma membrane is sufficient to enable force - generation capac - ity to be inferred ( Abercrombie , 1980 ; Bieling et al . , 2016 ; Mogilner and Edelstein - Keshet , 2002 ) , in CME two boundary conditions are required \u2013 one at the base of the pit for actin polymeri - zation against the plasma membrane and the second on the coat of the pit for attachment of the growing actin filaments so forces are transmitted to the pit to drive internalization . In our model , we used Hip1R as a surrogate for Hip1 , Hip1R , and Epsin1 / 2 / 3 , which cooperatively link actin filaments to the coat ( Brett et al . , 2006 ; Chen et al . , 1998 ; Messa et al . , 2014 ; Senetar et al . , 2004 ; Skruzny et al . , 2012 ) . We conclude that based on the relative positions of Arp2 / 3 complex activa - tors and actin filament linkers , the resultant self - organized actin network orients to produce force orthogonal to the base of the pit rather than producing a pinching force on the neck ( Collins et al . , 2011 ; Hassinger et al . , 2017 ) . Pinching forces are achieved by the spontaneous curvature of the rigid coat ( Alimohamadi et al . , 2018 ; Foret , 2014 ; Hassinger et al . , 2017 ) . Any constriction forces generated by actin polymerization at the neck would likely occur at a later stage of endocytosis than is the focus of our model , and the filaments would need to be nucleated by a spatially distinct set of Arp2 / 3 activating proteins around the neck , or by an interaction between other actin filaments and dynamin , but the mechanism for arranging and anchoring such a network has not been elucidated ( Ma and Berro , 2018 ) . Second , the effective anchoring of actin filaments to the surface of the pit depends on the distri - bution of linker proteins on the pit surface . Since these linker proteins are embedded within the cla - thrin coat ( Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; Sochacki et al . , 2017 ) , their surface coverage is directly proportional to the coat coverage on the endocytic pit . This observation suggests that one possible function of a large coat is for the actin - linking proteins Hip1 , Hip1R and Epsin to cover enough surface area to provide leverage for internalization . The role of linker protein coverage in force generation also has implications for the flat - to - curved transition earlier in endocy - tosis , when the membrane either begins to bend from a flat pre - assembled coat or continually deforms while the coat assembles ( Avinoam et al . , 2015 ; Bucher et al . , 2018 ; Scott et al . , 2018 ; Sochacki and Taraska , 2019 ) . In cases when the clathrin coat gradually increases in area during membrane deformation , our findings imply that actin polymerization may be ineffective until the coat reaches a threshold size ( Avinoam et al . , 2015 ; Sun et al . , 2017 ) , with membrane tension con - trolling a switch for the flat - to - curved transition ( Bucher et al . , 2018 ; Scott et al . , 2018 ) . Future work will investigate the relationship between coat topology and actin forces during the initiation of endocytosis . Third , we showed a significant fraction of endocytic actin filaments bend under force . We predict that the bent filaments , whose existence we confirmed by cryo - electron tomography of intact cells , contribute to successful endocytic internalization in at least two ways . First , they might contribute to the resilience of endocytosis by preventing backsliding of the pit . Second , we expect that they con - tribute to internalization by releasing stored elastic energy when they straighten out under thermal fluctuations , consistent with the elastic Brownian ratchet mechanism for actin - mediated force pro - duction ( De La Cruz and Gardel , 2015a ; Mogilner and Oster , 1996 ) . Here , filament bending occurs to a greater extent and for a longer time than previously described for coherent flat surfaces like the Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 18 of 40 Research article Cell Biology Physics of Living Systems leading edge , possibly due to the curved geometry of endocytic pits . Fixing the filament orientation at one end increases bending energy ( De La Cruz et al . , 2015b ; Ferna\u00b4ndez et al . , 2006 ) , which is accomplished here by multivalent attachments from Hip1R . Previous studies overlooked the role of actin filament bending at endocytic sites because of the predicted short length of filaments based on population averages ( Berro et al . , 2010 ) and the possible loss of less densely branched filaments during the preparation process for platinum - replica electron microscopy ( detergent extraction or sonication - based unroofing ) ( Collins et al . , 2011 ) . The load response of branched actin networks in vitro can be reversible due to filament bending ( Chaudhuri et al . , 2007 ) , or permanent from a change in filament architecture ( Bieling et al . , 2016 ; Parekh et al . , 2005 ) . In our simulations , some of the elastic energy from bent filaments is released as internalization increases , suggesting a revers - ible compression of the network to store elastic energy ( Figure 5 ) . However , a significant fraction of filament bending is retained after the spring is released , which suggests that load also changes the intrinsic structure of the network ( Bieling et al . , 2016 ) . Importantly , the results presented here demonstrate a mechanism of active adaptation by the endocytic actin network to changes in load ( Figure 7 ) . Different cell types , different locations in the same cell , and different stages of endocytosis at the same location can have different membrane tension values at different times ( Shi et al . , 2018 ) . Under flat geometries , branched actin networks adapt to load ( Bieling et al . , 2016 ; Mueller et al . , 2017 ) . Here , the distribution of Hip1R linkers around the pit directs more filaments to grow toward the base of the pit ( Figure 4 ) , which nucleates more filaments autocatalytically and increases filament bending ( Figure 5 ) , thereby supporting greater internalization ( Figure 7 ) . It is now important to determine whether the principles of actin filament self - organization and load adaptation identified here also apply to endocytic actin in the higher force regime characteristic of fungi . An agent - based model of endocytic actin networks in yeast predicted that barbed filament ends radiate away from the center of pit in the XY plane ( Mund et al . , 2018 ) . However , the > 200 x larger force requirements in this organism lead to a different axial organization of the filaments , less filament bending , and a distinct mechanism of force production sufficient to counteract high turgor pressure . Understanding the mechanical function and load adaptation in the \u2018soft\u2019 regime studied here is likely to apply to a variety of cellular membrane bending processes employing branched actin networks , including the assembly and maturation of endosomes , lysosomes , and autophagosomes ( Rottner et al . , 2017 ) . Finally , we acknowledge that our model represents a minimal core actin machinery necessary for endocytic internalization in mammalian cells . This feature of our study was necessary so the number of free parameters could be limited . Future models will add complexity to test roles for filament crosslinking , filament severing , and myosin - I motor activity , among other factors . It is anticipated that these additional features will increase our understanding of the force - generation capability and overall efficiency of the endocytic actin network . Materials and methods Key resources table Reagent type ( species ) or resource Designation Source or reference Identifiers Additionalinformation Gene ( H . sapiens ) ArpC3 HGNC : 706 ARPC3 Cell line ( human ) WTC - 10 hiPSC AP2 - tagRFP - t ArpC3 - tagGFP2 This study Cell line maintained in D . Drubin lab Transfected construct ( H . sapiens ) 12mer - tagGFP2 - FKBP This study Plasmid to transiently express calibration construct Transfected construct ( H . sapiens ) 24mer - tagGFP2 - FKBP This study Plasmid to transiently express calibration construct Continued on next page Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 19 of 40 Research article Cell Biology Physics of Living Systems Continued Reagent type ( species ) or resource Designation Source or reference Identifiers Additionalinformation Transfected construct ( H . sapiens ) 60mer - tagGFP2 - FKBP This study Plasmid to transiently expresscalibration construct Transfected construct ( H . sapiens ) 120mer - tagGFP2 - FKBP This study Plasmid to transiently expresscalibration construct Antibody mouse monoclonal anti - GAPDH ProteinTech 10494 \u2013 1 - AP ( RRID : AB _ 2263076 ) ( 1 : 5000 dilution ) Antibody tag ( C , G , Y ) FP Evrogen 12101231265 ( 1 : 2500 dilution ) Sequence - based reagent ArpC3 crRNA This paper crRNA CCGGGCUCCCUUCACUGUCC Sequence - based reagent ArpC3 _ sequencing primer This paper PCR primers ACTTATTCTTATTAAGCGCCAGC Sequence - based reagent ArpC3 _ sequencing primer This paper PCR primers CAGGGCTCTGGAGACGGT Commercialassayorkit Lipofectamine Stem Thermo Fisher STEM00003 Chemical compound , drug AP21967 Clontech 635056 Chemical compound , drug CK - 666 Sigma SML0006 Software , algorithm Cytosim Nedelec and Foethke , 2007 https : / / github . com / DrubinBarnes / Akamatsu _ CME _ manuscript Software , algorithm MATLAB Mathworks , Inc R2017b Software , algorithm Python Python . org 3 . 7 Software , algorithm m - Track Jaqaman et al . , 2008 https : / / github . com / DrubinBarnes / Akamatsu _ CME _ manuscript Mathematical modeling We combined a continuum membrane mechanics model with filament - based simulations of actin polymerization coupled to endocytic pit internalization to develop a multiscale model of actin in mammalian endocytosis . In the continuum model , the bending of the membrane was explicitly mod - eled and the contributions of the actin cytoskeleton were simplified to an applied localized force , while the agent - based model simplified the membrane and explicitly modeled actin and associated binding proteins ( Scheme 1 ) . We iteratively ran simulations in each module . The results from each module improved the assumptions of the other , over successive rounds of simulation and comparison to experimental measurements . In particular , experiments focused our attention on the internalization of the U - shaped pit and its transition to omega shape for both the membrane and actin modules . Experi - ments and the actin module informed the location of actin forces for the membrane mechanics model . The membrane mechanics simulations in turn informed the initial pit shape and force / exten - sion relationship for the actin module . Thus , these two modules synergistically provided collective information about how actin organization and dynamics couple to the bending and internalization of the clathrin - coated pit . Membrane mechanics module Continuum mechanics modeling of the plasma membrane allows a quantitative understanding of the relationship between applied forces and the shape of the membrane ( Dere\u00b4nyi et al . , 2002 ; Rangamani et al . , 2013 ) . Bending the membrane requires energy , such that pulling a tether from a flat membrane requires increasing force until the membrane adopts a tubule shape ( Dere \u00b4 nyi et al . , 2002 ; Alimohamadi et al . , 2018 ) . Adding a region with spontaneous curvature ( corresponding to the endocytic coat ) can help lower this energy barrier against moderate membrane tension ( Rangamani et al . , 2013 ; Hassinger et al . , 2017 ) . Forces due to actin polymerization can also help overcome the energy barrier ( Hassinger et al . , 2017 ) , but the relationship between applied actin Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 20 of 40 Research article Cell Biology Physics of Living Systems forces and coated membrane shape has not been explored quantitatively . The following assump - tions guide our model of the coated plasma membrane : . Membrane curvature generated due to forces or protein - induced spontaneous curvature is much larger than the thickness of the bilayer . Based on this assumption , we model the lipid bilayer as a thin elastic shell with a bending energy given by the Helfrich - Canham energy , which is valid for radii of curvatures much larger than the thickness of the bilayer ( Helfrich , 1973 ) . . We neglect the surrounding fluid flow or inertial dynamics and assume that the membrane is at mechanical equilibrium at all times ( Naghdi , 1957 ; Steigmann et al . , 2003 ) . This assump - tion is commonly used in the modeling of membrane curvature to keep the mathematics trac - table ( Alimohamadi et al . , 2018 ; Hassinger et al . , 2017 ; Rangamani et al . , 2014 ; Steigmann et al . , 2003 ; Vasan et al . , 2018 ) . . The membrane is incompressible because the energetic cost of stretching the membrane is high ( Steigmann et al . , 2003 ; Steigmann , 1999 ) . This constraint is implemented using a Lagrange multiplier ( Alimohamadi et al . , 2018 ; Rangamani et al . , 2014 ; Rangamani et al . , 2013 ) . . Finally , for simplicity in the numerical simulations , we assume that the membrane in the region of interest is rotationally symmetric . The following derivation can also be found in Hassinger et al . ( 2017 ) . We use a modified form of the Helfrich energy defined as W \u00f0 H ; K ; ! a \u00de \u00bc k \u00f0 H \" C \u00f0 ! a \u00de\u00de 2 \u00fe ! k K A general force balance on the membrane can be written as ! ! s \u00fe p n \u00bc f ; where is ! ! surface divergence , s is the stress vector , p is the pressure difference between the inside and outside of the volume bounded by the membrane , and f is any externally applied force per unit area on the membrane . The stress vector can be split into normal and tangential components as s a \u00bc T a \u00fe S a n ; where T a \u00bc T ab a b ; T ab \u00bc s ab \u00fe b b \" M \" a ; S a \u00bc \" M ab ; b : s ab and M ab can also be written as s ab \u00bc # q F \u00f0 # ; H ; K ; x a \u00de q a ab \u00fe q F \u00f0 # ; H ; K ; x a \u00de q a ba ! \" ; M ab \u00bc # 2 q F \u00f0 # ; H ; K ; x a \u00de q b ab \u00fe q F \u00f0 # ; H ; K ; x a \u00de q b ba ! \" ; Here \u00f0 a ab \u00de \u00bc \u00f0 a ab \u00de is the dual metric or first fundamental form , b ab is the second fundamental form and # is the surface mass density . Using the first and second fundamental form , we can define H ( mean curvature ) and K ( Gaussian curvature ) as H \u00bc 1 2 a ab b ab ; K \u00bc 1 2 \" ab \" l \" b al b b \" : where \" ab is the permutation tensor defined by \" 12 \u00bc \" \" 21 \u00bc 1 \ufb03\ufb03 a p ; \" 11 \u00bc \" 22 \u00bc 0 . We then define an area incompressibility constraint by rewriting the free energy density as F \u00f0 # ; H ; K ; x a \u00de \u00bc ~ F \u00f0 H ; K ; x a \u00de \" g \u00f0 x a ; t \u00de # : where g \u00f0 x a ; t \u00de is a Lagrange multiplier field required to impose invariance of # on the whole of the surface . This free energy density relates to the Helfrich energy density as Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 21 of 40 Research article Cell Biology Physics of Living Systems W \u00bc # ~ F Combining these equations , we can get the stress equations s ab \u00bc \u00f0 l \u00fe W \u00de a ab \" \u00f0 2 HW H \u00fe 2 k W K \u00de a ab \u00fe W H ~ b ab ; and M ab \u00bc 1 2 W H a ab \u00fe W k ~ b ab ; where l \u00bc \" \u00f0 g \u00fe W \u00de Simplifying this further , we can get the shape equation ( normal balance ) p \u00fe f ! n \u00bc D 1 2 W H \u00fe\u00f0 W K \u00de ; ab ~ b ab \u00fe W H \u00f0 2 H 2 \" K \u00de\u00fe 2 H \u00f0 KW K \" W \u00de \" 2 l H ; and the tangential balance q W q x a j exp \u00fe l ; a ! a ba \u00bc f : a s : where \u00f0\u00de j exp denotes the explicit derivative respect to coordinate ! a . To further simplify the equa - tions , we define the coordinate system as axisymmetric using r \u00f0 s ; ! \u00de \u00bc r \u00f0 s \u00de e r \u00f0 ! \u00de\u00fe z \u00f0 s \u00de k : We define a such that r 0 \u00f0 s \u00de \u00bc cos \u00f0 \u00de , z 0 \u00f0 s \u00de \u00bc sin \u00f0 \u00de and n \u00bc \" sin e r \u00f0 ! \u00de\u00fe cos k Using this , we can write the mean curvature ( H ) and Gaussian curvature ( K ) as H \u00bc 1 2 \u00f0 k v \u00fe k t \u00de \u00bc 1 2 \u00f0 0 \u00fe r \" 1 sin \u00de K \u00bc k t k v \u00bc 0 sin r : We also introduce L \u00bc 1 2 k r \u00f0 W H \u00de 0 allowing us to formulate a system of ordinary differential equations ( ODE\u2019s ) as function of arc length s r 0 \u00bc cos ; z 0 \u00bc sin ; r 0 \u00bc 2 rH \" r 0 \u00bc \" sin ; rH 0 \u00bc L \u00fe rC 0 ; L 0 r \u00bc pk \u00fe f ! n k \u00fe 2 H \u00f0 H \" C \u00de 2 \u00fe lk \" 2 \u00f0 H \" C \u00de h i H 2 \u00fe\u00f0 H \" r \" 1 sin \u00de 2 h i l 0 \u00bc 2 k \u00f0 H \" C \u00de C 0 \" f ! a s : This can also be written as a function of membrane area using a \u00f0 s \u00de \u00bc 2 p Z s 0 r \u00f0 $ \u00de d $ ! da ds \u00bc 2 p r : Here , we choose to non - dimensionalize the system using a \u00bc a 2 p R 2 0 ; x \u00bc r R 0 ; y \u00bc y R 0 ; h \u00bc HR 0 ; c \u00bc CR 0 ; l \u00bc LR 0 l \u2019 \u00bc l R 20 k 0 ; p \u2019 \u00bc pR 30 k 0 f \u2019 \u00bc fR 30 k 0 ; k \u2019 \u00bc k k 0 giving us the system of equations Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 22 of 40 Research article Cell Biology Physics of Living Systems x _ x \u00bc cos ; x _ y \u00bc sin ; x 2 _ \u00bc 2 xh \" sin ; x 2 _ h \u00bc l \u00fe x 2 _ c ; _ l \u00bc p \u2019 k \u2019 \u00fe f \u2019 ! n k \u2019 \u00fe 2 h \u00f0 h \" c \u00de 2 \u00fe l \u2019 k \u2019 h i \" 2 \u00f0 h \" c \u00de h 2 \u00fe\u00f0 h \" x \" 1 sin \u00de 2 h i ; _ l \u2019 \u00bc 2 k \u2019 \u00f0 h \" c \u00de _ c \" f \u2019 ! a s x : We define a spatially varying spontaneous curvature as c \u00bc c 0 \u2019 0 : 5 \u00f0 1 \" tanh \u00f0 g \u2019\u00f0 a \" a 0 \u00de\u00de\u00de where a is the non - dimensional membrane area , a 0 is the non - dimensional membrane area of the protein coat , g is a constant and c 0 is the coat spontaneous curvature . The parameters used for the spontaneous curvature simulations are specified in Supplementary file 1 : To perform the coat pulling simulations , we applied an axial force acting downward along the protein coat and upward along the base of the pit such that the net force integrates to 0 ( we do this by scaling the applied force by the area over which it is applied ) . This force function was defined as f \u00bc f 0 \u2019\u00f0 0 : 5 \u00f0\u00f0 1 \" tanh \u00f0 g \u2019\u00f0 a \" a 0 \u00de\u00de\u00de = a 0 \" \u00f0 tanh \u00f0 g \u2019\u00f0 a \" a in \u00de\u00de \" tanh \u00f0 g \u2019\u00f0 a \" a out \u00de\u00de\u00de = \u00f0 a out \" a in \u00de\u00de\u00de where a 0 is the non - dimensional coat area , a is the non \" dimensional membrane area , a out \" a in is the area of force applied at the base of the pit . a in corresponds to an inner radius r in and a out corre - sponds to an outer radius r out within which the upward force is applied . These parameters are specified in Supplementary file 2 . To simulate the pinched ( \u2018omega - shaped\u2019\u201d ) curves at high membrane tension , we provided an ini - tial guess of an \u2018omega - shaped\u2019 membrane from a lower membrane tension . We did this because the simulations stalled at U shapes at an internalization of about 100 nm . Providing this initial guess led to solutions for membrane shape and force beyond 100 nm , as seen in Figure 1C . Further , to fully explore the space of solutions , we ran the simulations backward by starting from an \u2018omega shaped\u2019 pit at a large internalization and then decreasing the internalization . In Figure 1C , we plot - ted the curves for negative internalization > = the farthest internalization for the U shaped pit ( generally ~ 100 nm ) . Values of membrane tension in Figure 1C are [ 0 : 051 : 0 . 05 : 0 . 451 ] pN / nm , and rounded to two significant digits in the figure for clarity . Actin module We used Cytosim ( Nedelec and Foethke , 2007 ) to model the polymerization of a branched actin network coupled to the internalization of a clathrin - coated pit . This approach simplified the pit as a bead attached to a flat boundary ( the plasma membrane ) by a spring . This assumption of a linear force - extension relationship was validated in Figure 1 . Actin filaments and actin - binding proteins ( Arp2 / 3 complex , Hip1R ) were explicitly simulated as individual objects ( agents ) . Cytosim calculates the forces on each segment of actin from rules such as diffusion , confinement , growth , and binding based on Brownian dynamics . Assumptions in Cytosim Cytosim simulates the movement of actin model points within a boundary according to constrained Langevin dynamics ( Nedelec and Foethke , 2007 ) , which accounts for the diffusion , bending , and forces of actin filaments , as well as the diffusion and binding of actin - binding proteins detailed below . 1 . Force balance equations ( Nedelec and Foethke , 2007 ) section 7 . 1 : a . All points in the simulation follow constrained Langevin dynamics : b . dx \u00bc \" F \u00f0 x ; t \u00de dt \u00fe dB \u00f0 t \u00de i . where m is defined as an effective mobility , which takes on a different value for each type of object . 2 . Mobilities of diffusing objects : a . The simulated Brownian motion dB \u00f0 t \u00de of objects of radius r is governed by a uniformly dis - tributed random \u00bd 0 ; 1 ) \u2019 \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 k B T = 3 ph p number at each time point , where h is the viscosity of the cytoplasm . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 23 of 40 Research article Cell Biology Physics of Living Systems b . Their movement is governed by a mobility m i . For model points of an actin filament , \" \u00bc log \u00f0 L = d \u00de $ \u00f0 3 ph L \u00de for a rod of diameter d , length L and cytoplasmic viscosity h . This mobility term ignores the bending of the filaments . c . The endocytic pit is modeled as a solid , with bulk fluid viscosity associated with pit trans - lational movement and a viscoelastic confinement to the cell surface : dx \u00bc \" F \u00f0 x ; t \u00de dt \u00fe dB \u00f0 t \u00de , where \" \u00bc 6 ph r . 3 . Confinement of objects : a . Objects are confined within a boundary ( cell surface ) according to a harmonic spring potential F \u00bc kx . i . The endocytic pit a distance z from the cell surface experiences a force F \u00bc kz . ii . Actin filaments are confined inside the cell wherein each model point at distance z outside the cell experiences a force F \u00bc kz . 4 . Bending elasticity of filament model points : a . Filament model points are connected via linear elasticity according to a flexural rigidity k , which is the persistence length L p multiplied by k B T . b . The bending elasticity is treated as linear ( see Limitations ) such that for three connected actin model points m 0 ; m 1 ; m 2 the force for those points is F \u00bc k \u00f0 p = L \u00de 3 \u00f0 m 0 \" 2 m 1 \u00fe m 2 \u00de , where k is the flexural rigidity , p is the number of model points , and L is the length of the filament . 5 . Actin - binding proteins a . Hip1R binds actin filaments according to a binding rate and binding radius ( probability of binding when a filament is within the binding radius ) . This general actin - binding protein is a simplification of the multiple interacting proteins that link actin to the coat , including Hip1 and Epsin1 / 2 / 3 ( Brett et al . , 2006 ; Chen et al . , 1998 ; Messa et al . , 2014 ; Senetar et al . , 2004 ; Skruzny et al . , 2012 ) . b . Arp2 / 3 complex was developed as a special - case \u2018fork\u2019 class with two coupled ends . One end binds actin filaments , and the other nucleates a new actin filament , provided the first end bound an actin filament ( this is defined as trans - activation in Cytosim ) . In the \u2018fork\u2019 class , the two ends are constrained geometrically at a resting angle with a given resis - tance to torque ( angular stiffness ) similar to 4b above ( Mund et al . , 2018 ) . Assumptions for modeling mammalian clathrin - mediated endocytosis in cytosim Geometry Endocytic pit : We used our membrane mechanics simulations ( Figure 1 ; Hassinger et al . , 2017 ) to estimate the dimensions of the endocytic pit for physiological values of membrane tension and rigid - ity of the membrane and clathrin coat . Under these conditions the clathrin coat bends the plasma membrane into a U - shaped hemisphere ( Figure 1 ; Boulant et al . , 2011 ; Messa et al . , 2014 ; Yarar et al . , 2005 ) . We initialized the pit as a hemisphere 90 nm in diameter ( Avinoam et al . , 2015 ; Collins et al . , 2011 ) . As the pit internalizes , a smaller neck is exposed ( Figure 1 ) , which is modeled as a sphere with a cylindrical neck of diameter 60 nm . Internalization is defined as a displacement in the - Z direction ( Figure 1A ) . Active Arp2 / 3 complex : We collapsed the activation steps of Arp2 / 3 complex into a single spe - cies , active Arp2 / 3 complex , that resides on the plasma membrane from the beginning of the simula - tion . This models the cellular process , in which soluble Arp2 / 3 complex is inactive until it encounters its activator N - WASP at the plasma membrane . N - WASP binds the plasma membrane via a PI ( 4 , 5 ) P2 - binding site ( which relieves its own autoinhi - bition ) ( Rohatgi et al . , 2000 ) . Additional proteins can bind different regions of N - WASP to increase its level of activation , including the GTPase Cdc42 , actin nucleator cortactin , and BAR protein SNX9 . Because the activation rate and concentrations of these proteins are not yet known , we considered fully active N - WASP ( similar to the VCA region alone ) rather than modeling the individual activation steps . Furthermore , rather than explicitly modeling N - WASP , we used pre - activated Arp2 / 3 com - plex , which models the coincidence of active N - WASP with soluble Arp2 / 3 complex on the plasma membrane . This active Arp2 / 3 complex can template new branched actin filaments when in proxim - ity of an existing \u2018mother\u2019 actin filament . Thus , this model aims to functionally capture Arp2 / 3 Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 24 of 40 Research article Cell Biology Physics of Living Systems complex activation and the geometry of branched actin filament nucleation , rather than explicitly modeling each molecule involved in the process of Arp2 / 3 complex activation . N - WASP ( or its homologues Las17 / WASP in yeast ) accumulates earlier in endocytosis ( Taylor et al . , 2011 ) until a \u2018threshold\u2019 concentration triggers actin assembly ( Sun et al . , 2017 ) ; here we initialize all the active Arp2 / 3 complex on the plasma membrane at the beginning of the simula - tion , and it is used over the course of the simulation . Therefore we model the phase in which a threshold value of Arp2 / 3 complex activators has accumulated at the endocytic site and is ready to trigger actin polymerization . We assumed that activated Arp2 / 3 complex resides in a ring around the base of the endocytic pit . This feature has been shown for the budding yeast homologue of N - WASP , Las17 ( Picco et al . , 2015 ) . Endocytic actin polymerizes from the base of the pit in budding yeast ( Idrissi et al . , 2008 ; Kaksonen et al . , 2003 ; Mund et al . , 2018 ; Picco et al . , 2015 ) and in mammalian cells ( Kaplan et al . , in preparation ; Almeida - Souza et al . , 2018 ) , consistent with active Arp2 / 3 complex residing in a ring at the base of the pit . In our fluorescence micrographs , Arp2 / 3 complex is diffraction - limited , so the outer diameter of this ring is * 250 nm . The inner diameter of the ring of Arp2 / 3 complex corre - sponds to the width of the neck of the pit , 60 nm . In budding yeast the Las17 ring outer diameter is ~ 140 nm ( Mund et al . , 2018 ) , which corresponds to a surface density of ~ 3000 molecules / m m 2 . We conservatively set the outer radius of the ring to be 240 nm , which also corresponds to a surface density of ~ 3000 molecules / m m 2 . Estimates of the surface density of in vitro and in vivo patterned activators of Arp2 / 3 complex ( also called nucleation - promoting factors ) range from ~ 3000 \u2013 19000 molecules / m m 2 ( Bieling et al . , 2018 ; Case et al . , 2019 ; Ditlev et al . , 2012 ) . Filament attachments to endocytic pit In mammalian cells , Hip1R and Epsin connect actin filaments to the endocytic pit ( Brett et al . , 2006 ; Chen et al . , 1998 ; Engqvist - Goldstein et al . , 2001 ; Messa et al . , 2014 ; Senetar et al . , 2004 ; Skruzny et al . , 2012 ) . Both are present throughout the clathrin coat ( Clarke and Royle , 2018 ; Sochacki et al . , 2017 ) . We wrote a script in Matlab to uniformly distribute Hip1R molecules around a desired coverage of a sphere , from 1 % to 80 % of a sphere ( Figure 4 ) . In most simulations Hip1R was distributed in 60 % of a sphere . Modeling actin filament dynamics Stall force : Filament polymerization slows under applied load according to the Brownian Ratchet the - ory ( Peskin et al . , 1993 ) . This is treated in Cytosim as growth velocity that slows exponentially under applied load , which is reasonable within the time scales of endocytosis . Modeling filament capping Previous filament - based models of actin in endocytosis modeled actin filaments with uniform lengths or that grow until a maximum length ( Mund et al . , 2018 ) , while others took into account stochastic capping without diffusion or bending ( Wang et al . , 2016 ) . We adapted an existing property in Cyto - sim to model stochastic filament capping , such that the capping events were exponentially distrib - uted . We modeled actin filaments using Cytosim\u2019s \u2018classic\u2019 fiber class , which treats the filament as growing from its plus end , with a stochastic probability of depolymerizing ( corresponding to catas - trophe for microtubules ) . We set the depolymerization rate of shrinking filaments to be 0 with no recovery rate . Thus these filaments become irreversibly capped after an exponential wait time char - acterized by the parameter catastrophe _ rate ( which we define as the capping rate ) . Because the probability that capping protein binds to the barbed end of the filament is exponentially distributed , filament lengths are exponentially distributed ( Figure 3\u2014figure supplement 1F ) . We set the rate of capping to achieve a desired mean filament length based on the expected distributions of actin fila - ment lengths for a given capping rate . In Cytosim , the catastrophe rate can be set to depend on a combination of applied load and growth velocity , which we did not include in our model of actin fila - ment capping . Source of actin mother filaments Active Arp2 / 3 complex requires a mother filament from which to nucleate a new actin filament at a defined angle ( Amann and Pollard , 2001 ; Mullins et al . , 1997 ) . Therefore , the polarity of the Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 25 of 40 Research article Cell Biology Physics of Living Systems mother filament defines the polarity of the resultant branched actin network . Our study uses diffus - ing linear actin filament nucleating proteins ( Balzer et al . , 2018 ; Basu and Chang , 2011 ; Wagner et al . , 2013 ) to seed a defined number of randomly oriented mother filaments near the endocytic site . Alternatively , simulations using a pool of cytoplasmic linear actin filaments ( Raz - Ben Aroush et al . , 2017 ) allowed for similar internalization , but with less reliable timing of initiation . More detailed studies of the mechanism of actin nucleation and mother filament generation are necessary . Limitations Endocytic pit internalization is simplified as an elastic spring , with a linear relationship between force and extension . We show in Figure 1 that this linear relationship is characteristic of coated plasma membranes under force up until a threshold internalization of ~ 100 nm . Future studies will treat the coated membrane as a 3D , force - dependent curving surface ; such an approach is outside the scope of the present work . We focus our model on the minimal actin machinery required to produce force . We have not included crosslinkers ( Ma and Berro , 2018 ) or myosin I , both of which are expected to increase the network\u2019s ability to produce force . The effects of these two proteins on mammalian endocytosis will be treated in a future study . The treatment of filament bending elasticity as linear is valid for small deflections of individual actin model points . Importantly , the outcomes of our simulations did not depend on the frequency of segmentation of actin model points ( which change the magnitude of deflection between individ - ual actin model points ) . Filament twist or twist - bend coupling , which increases the total energy stored in bent actin filaments ( De La Cruz et al . , 2015b ) , is not considered in Cytosim , and requires a more detailed modeling approach considering each subunit . Cytosim does not implement hydro - dynamics for curved filaments , so the diffusion of these filaments is approximated as the motion for a linear filament . Arp2 / 3 complex preferentially binds to the curved sides of actin filaments ( Risca et al . , 2012 ) . We do not include this assumption in our model . We expect that the self - organization and robustness exhibited by our minimal actin network would be enhanced by this assumption , given that Arp2 / 3 complex at the base of the pit encounters many curved sides of actin filaments . Parameter values We derived most parameters from experimental data in the literature , and made measurements for some measurements not available ( Supplementary file 3 ) . We varied the remaining parameters to show their effect on the outcome of the simulations . Discussion of each parameter follows below . Membrane tension We used the relationship between internalization resistance and membrane tension ( Figure 1 ) to cal - ibrate the spring stiffness in our agent - based simulations . We relied on values of membrane tension measured in human skin melanoma SK - MEL - 2 cells , based on atomic force microscope membrane tether rupture forces and the assumption that the rigidity of the plasma membrane is 320 pN ! nm ( Dimova , 2014 ; Diz - Mun\u02dcoz et al . , 2016 ; Kaplan et al . , in preparation ) . Association rate constants The biochemical association rate constant , k on , is given in units of m M \" 1 s \" 1 . In Cytosim , we input the association probability between actin and a binding protein as a binding _ rate , in units of s \" 1 . These two rates can be related by the following relationship : k on \u00bc binding rate \u2019 capture volume ; which is defined as p \u2019 capture radius 2 \u2019 filament length ( Francois Nedelec , personal communication ) . This gives an order - of - magnitude scaling relationship to convert between k on and binding rate , considering that cytosim does not treat explicit binding sites on the filament ( Francois Nedelec , personal communication ) . Arp2 / 3 complex Branching angle : Based on Blanchoin et al . ( 2000 ) we set the branching angle of Arp2 / 3 complex to be 77 \u00b1 13\u02da , as measured for bovine Arp2 / 3 complex . Acanthomeba Arp2 / 3 complex adopts closer Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 26 of 40 Research article Cell Biology Physics of Living Systems to a 70\u02da branching angle . In NIH3T3 cells preserved by cryo - fixation , branch angles in the lamellipo - dium are 77 \u00b1 8\u02da ( Vinzenz et al . , 2012 ) . Importantly , Blanchoin et al . ( 2000 ) was the only in vitro study we are aware of that measured the variance of branching angles , which was converted to an angular stiffness ( 0 . 076 pN / rad 2 ) . Therefore we set the resting angle of Arp2 / 3 branches as 1 . 344 rad with an angular stiffness of 0 . 076 pN / rad 2 . Unbinding rate We used the k off measured by Beltzner and Pollard ( 2008 ) : 0 . 003s - 1 . Nucleation rate Based on the association rate constant between activated Arp2 / 3 complex and actin filaments of 0 . 00015 m M \" 1 s \" 1 ( Beltzner and Pollard , 2008 ) , we set the binding _ rate of Arp2 / 3 to actin to be 7 s \" 1 , given a capture radius of 10 nm and filament length of 100 nm ( see relationship between these parameters above ) . We note that this calculation of binding rate depends inversely with the filament length ( and number of filaments ) which are not directly comparable in our simulations given that the filaments and Arp2 / 3 are generally not freely diffusing . Still , it was remarkable that our best estimate for binding rate gave reasonable nucleation kinetics , and served as a threshold for timely internaliza - tion of the endocytic pit , whereas previous deterministic models needed to increase the association rate constant by 300 - 600x for efficient nucleation ( Beltzner and Pollard , 2008 ; Berro et al . , 2010 ) . These ODE models did not have spatial considerations , so this suggests that the spatial and tempo - ral confinement of actin filaments and the high local concentration of active Arp2 / 3 complex in our simulations accounted for most of this difference . Thus the local geometry has a significant ( > 2 orders of magnitude ) effect on the effective nucleation rate . Actin Growth rate : In cells the cytoplasmic concentration of actin is 60 \u2013 100 m M ( Haugwitz et al . , 1994 ; Wu and Pollard , 2005 ) . In mammalian cells , a subset of this actin is available as polymerizable actin , both due to monomer - sequestering proteins ( thymosin B4 ) and due to only a subset of monomers being ATP - bound . We conservatively set the concentration of available polymerizable actin to be 20 m M , which given the association rate constant of ATP - actin of 11 . 6 subunits / m M / s ( Pollard , 1986 ) corresponds to a polymerization rate of 500 nm ( 182 subunits ) per second . Capping rate : The mean length of filaments in mammalian endocytosis has not been measured . We relied on the estimates from Berro et al . ( 2010 ) ; Sirotkin et al . ( 2010 ) which showed that for fis - sion yeast filaments were an average of 150 nm in length . We set the capping rate to be 6 . 3 / s , which set the mean filament length at 150 nm . We varied the rate of capping in our simulations . Less actin capping resulted in greater internalization , due to more actin ( Figure 1\u2014figure supplement 1 ) . However , the resultant amount of actin is larger than the amount of actin measured in CME in other organisms ( Picco et al . , 2015 ; Sirotkin et al . , 2010 ) . Stall force : The stall force scales with the load applied and the concentration of actin monomers available ( Peskin et al . , 1993 ) . At 4 m M actin the filaments\u2019 stall force was measured to be 1 \u2013 2 pN ( Footer et al . , 2007 ) . With 40 m M actin the filaments could theoretically stall at up to 9 pN force per filament ( Dmitrieff and Ne\u00b4de\u00b4lec , 2016 ) . For the ~ 20 m M actin that we assumed was available for polymerization , the stall force was ~ 5 pN . Surprisingly , the extent of internalization varied weakly with stall force ( Figure 1\u2014figure supplement 1 ) , suggesting that actin used another mode of force generation than elongation directly against the membrane ( Figure 5 ) . Persistence length : We set the persistence length of actin filaments to be 10 m m , which corre - sponds to a flexural rigidity of 0 . 041 pN ! m m 2 ( McCullough et al . , 2008 ) . Previous modeling studies used a value of 20 m m , based on measurements of actin filaments labeled with phalloidin ( Gittes et al . , 1993 ) , which stiffens actin filaments ( Isambert et al . , 1995 ; Pfaendtner et al . , 2010 ) . Changing the persistence length of actin between 1 and 20 m m had a minor effect on pit internaliza - tion ( Figure 1\u2014figure supplement 1 ) . Hip1R In mammalian endocytosis , several proteins link actin filaments to the clathrin coat via phosphoinosi - tide - and clathrin - binding domains and actin - binding domains , including Hip1 , Hip1R , and Epsin Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 27 of 40 Research article Cell Biology Physics of Living Systems ( Brett et al . , 2006 ; Chen et al . , 1998 ; Messa et al . , 2014 ; Senetar et al . , 2004 ; Skruzny et al . , 2012 ) . Our general linker protein , named in the text as Hip1R , is a surrogate for all proteins that link actin to the coat . Endocytic actin - binding proteins Hip1 and Hip1R use a conserved domain to bind actin with sur - prisingly weak affinity . This domain , which is alternately named the THATCH ( Talin - Hip1 / R / Sla2p Actin - Tethering C - terminal Homology ) , Talin - family , or I / LWEQ domain , has been isolated and stud - ied by several groups . We fit the binding results of previous Hip1R binding experiments in our lab ( Engqvist - Goldstein et al . , 1999 ) to estimate a binding affinity between Hip1R and actin as ~ 400 nM , and the affinity between clathrin cages and Hip1R to be ~ 1 nM . Both sets of data were fit well by the quadratic binding curve ( Pollard , 2010 ) , \u00bd LR ) = \u00bd L ) \u00bc \u00f0\u00f0\u00bd R ) \u00fe\u00bd L ) \u00fe K d \u00de \" \u00f0\u00f0\u00bd R ) \u00fe\u00bd L ) \u00fe K d \u00de 2 \" 4 \u2019\u00bd R ) \u2019\u00bd L ) \u00de 1 = 2 = \u00f0 2 \u2019\u00bd L ) \u00de where \u00bd L ) is the concentration of actin or clathrin and \u00bd R ) is the concentration of Hip1R , with r 2 = 0 . 94 and 0 . 99 , respectively ( data not shown ) . Other studies measured a weaker affinity between Hip1R and actin : K d = 3 . 4 m M , or 2 . 5 m M for Hip1 ( Senetar et al . , 2004 ) . In the presence of the first helix of the five - helix bundle comprising the THATCH domain , actin affinity further decreases ( Senetar et al . , 2004 ) . Epsin has two actin - binding domains with very weak ( K d > 10 m M ) or unknown affinity to actin ( Messa et al . , 2014 ; Skruzny et al . , 2012 ) . For our linker protein we used a combina - tion of rate constants such that k off / k on ~ K d of 400 nM . Compared with dilute reactions , in an endo - cytic geometry actin filaments grow near the coat , so the actin filaments bind Hip1R more frequently . As a result we found that a relatively low binding rate was sufficient for binding between actin and Hip1R in our simulations . We varied the off rate of Hip1R and found that that , surprisingly , the internalization was robust to Hip1R off rate between 0 . 001 and 10 s \" 1 ( Figure 1\u2014figure supple - ment 1 ) . With such weak affinity , + 100 linking molecules are required for robust endocytosis ( Figure 4 ) . The following considerations support the likelihood that a sufficient number of actin - linking proteins reside in the clathrin - coated pit . In yeast , endocytic sites accumulate ~ 100 \u2013 150 molecules of the Hip1R homologue End4p and 230 molecules of the actin - binding protein Pan1 ( with K d to actin = 2 . 9 m M ) . Estimation from platinum replica images of clathrin - coated pits in SK - MEL - 2 cells ( Sochacki et al . , 2017 ) suggest that clathrin cages have approximately 55 \u00b1 12 \u2018faces\u2019 ( pentagons and hexagons ) , or up to 90 faces in HeLa cells . If the cage accumulates one Hip1R dimer per face , this would lead to 110 \u2013 180 molecules of Hip1R , plus molecules of Hip1 , Epsin1 , Epsin2 , and Epsin3 . From a similar analysis , SK - MEL - 2 cells have ~ 66 \u00b1 12 vertices ( triskelia ) , which corresponds to ~ 200 \u00b1 40 clathrin heavy chains and ~ 200 \u00b1 40 light chains . Since Hip1R binds clathrin light chain , a 1 : 1 ratio of these proteins would again suggest ~ 200 Hip1R molecules in the clathrin coat . Addi - tionally , actin binding by these linker proteins is likely highly multivalent . Mammalian Epsin proteins hexamerize in vitro via their membrane - binding ENTH domains , and Hip1 and Hip1R can dimerize with each Epsin through its ANTH domain by sharing a Pi ( 4 , 5 ) P2 molecule ( Garcia - Alai et al . , 2018 ; Skruzny et al . , 2015 ) . Adding an additional layer of multivalency , Hip1R and Hip1 hetero - and homodimerize via coiled - coil domains and a C - terminal dimerization motif ( Brett et al . , 2006 ; Chen and Brodsky , 2005 ; Engqvist - Goldstein et al . , 2001 ; Niu and Ybe , 2008 ; Senetar et al . , 2004 ) . Therefore , it is quite likely that a sufficient number of actin - linking proteins cover the coat . Simulation environment parameters The internalization of endocytic pits was not sensitive to other simulation parameters including the segmentation of actin ( 1 and 100 nm per model point ; 1 m m model points introduced additional vari - ability ) , confinement force for actin within the cell , and time step of the simulation ( Figure 1\u2014figure supplement 1 ) . The viscosity of the cytoplasm and the endocytic pit weakly affected extent of inter - nalization ( Figure 1\u2014figure supplement 1 ) . Modifications to source code We added a method \u2018confine _ first _ surface\u2019 in which only the first segment of the multi - point bead is under confinement , and that the bead does not undergo angular displacements . We added data reporting methods , including ( 1 ) reporting the Arp2 / 3 complex branch angle ( Francois Nedelec ) ; ( 2 ) reporting the Hip1R - attached filament ID numbers ; ( 3 ) visualizing the axial Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 28 of 40 Research article Cell Biology Physics of Living Systems orientation of actin segments with respect to the plasma membrane ( implemented as 75 % of the hsv colormap ) . Comparison to theory Our calculation of the elastic energy stored in bent actin filaments is derived from the theory of deforming elastic beams ( Boal ) . Specifically , the bending energy E is determined by \u00f0 k B TL p ! 2 \u00de = \u00f0 2 l \u00de , where k B is the Boltzmann constant , T is temperature , L p is the filament persistence length ( 10 m m for actin ) , ! is the bending angle , and l the free filament length ( contour length of the filament between the attachment site and barbed end of the filament ) ( Boal , equations 3 . 15 and 3 . 21 ) . In Figure 5 we reference ( Sept and McCammon , 2001 ) and ( De La Cruz et al . , 2015b ) to esti - mate the bending energy associated with fragmentation of bare actin filaments . Based on the energy associated with removal of two longitudinal contacts and one lateral contact between mono - mers in an actin filament ( Sept and McCammon , 2001 ) , the elastic energy associated with fragment - ing an actin filament is 26 \u2013 28 k B T ( De La Cruz et al . , 2015b ) . The rate constants in De La Cruz and Gardel ( 2015a ) associated with the probability of severing are not considered in this study . Running simulations We wrote custom scripts in bash to run parallel simulations on a high - performance computing server . Analysis of simulations We wrote custom code in Python ( 3 . 7 ) with Jupyter Notebook ( Project Jupyter ) to read , analyze , and plot the simulations obtained from Cytosim . X , Y = 0 is defined as the center of the pit . Energy associated with polymerization was defined as 5 pN * 2 . 75 nm = 13 . 5 pN ! nm per binding event . This code is available at the following website : https : / / github . com / DrubinBarnes / Akamatsu _ CME _ manuscript ( Akamatsu , 2019 ; copy archived at https : / / github . com / elifesciences - publications / Aka - matsu _ CME _ manuscript ) . Internalization energy We used the relationship E \u00bc 1 = 2 kx 2 to estimate the expected internalization for a non - adapting machine . From the internalization for k = 0 . 01 pN / nm ( low tension ) , we calculated the expected internalization using the same energy for different values of k . Specifically , for a single value of load , we calculated the work output based on energy ( work output ) E \u00bc 1 = 2 kx 2 , where k is the load ( in pN / nm ) and x is the internalization ( in nm ) . For an adapting network , the work output changes with load . For a non - adapting network , it does not , so as load k increases , internalization x decreases . This relationship of x as a function of k for constant E is plotted as the dotted line in Figure 7E . Energy efficiency is defined as the energy ( of internalization or stored in bent filaments ) divided by the total polymerization energy . Radial orientation metric : We defined the radial orientation as the sum of the dot products of the direction of the barbed end with the unit vector in X and Y , such that \" 1 = barbed ends oriented toward the origin ( the center of the pit ) , 0 = oriented tangent to the pit , and + 1 = oriented away from the center of the pit . 95 % internalization is defined as the 95th percentile of internalization . We use the first 12 s of sim - ulations to allow for comparison with simulations that terminated after t = 12 s . For Figure 4\u2014figure supplement 2 , we compared 95 % internalization in two conditions using the Welch\u2019s t - test in Python ( 3 . 7 ) with scipy . stats . ttest _ ind ( equal _ var = False ) . Barbed ends near base / neck : We chose a distance of 7 . 5 nm as a metric for proximity to the membrane ( base or neck ) as this was the width of 1 \u2013 2 actin monomers and less than the radial dis - tance between the neck surface and the pit diameter . The absolute value of this metric did not change the results appreciably . Experimental method details Cell line maintenance WTC - 10 human induced pluripotent stem cells ( hiPSCs ) were obtained from the lab of Bruce Conklin and genome edited using TALENs to endogenously express AP2 - tagRFP - T at one allele at an Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 29 of 40 Research article Cell Biology Physics of Living Systems internal loop of the m 2 subunit ( Hong et al . , 2015 ) . We grew these cells on matrigel ( hESC - Qualified Matrix , Corning ) ( 80 m g / mL , 1 mL / well ) in StemFlex ( Thermo Fisher ) with Penicillin / Streptomycin ( Thermo Fisher ) , and passaged with Gentle Cell Dissociation reagent ( EDTA - based clump passaging ; StemCell Technologies ) . Parental and genome - edited cells were tested for mycoplasma and authen - ticated by STR profiling . For single - cell applications ( genome editing , flow cytometry , transfections ) we trypsinized the cells with the recombinant trypsin TrypLE Select ( Thermo Fisher ) and grew the cells in StemFlex supplemented with the specific rho kinase inhibitor RevitaCell ( Thermo Fisher ) . SK - MEL - 2 cell culture SK - MEL - 2 cells endogenously expressing clathrin light chain CLTA - RFP and dynamin2 - eGFP ( Doyon et al . , 2011 ) were cultured in DMEM / F12 ( Thermo Fisher ) supplemented with 10 % FBS ( HyClone ) and Penicillin / Streptomycin ( Thermo Fisher ) . A day before imaging , the cells were seeded on 25 mm diameter glass coverslips ( Fisher Scientific ) . Cell line construction We followed ( Dambournet et al . , 2014 ) to create lines human induced pluripotent ( hiPS ) cells with genetically encoded fluorescent fusion proteins . To the AP2 - RFP cells described above , we used the following Cas9 ribonuclear protein electroporation method for genome editing . Gibson assembly of DNA G - blocks ( IDT ) was used to construct a donor plasmid containing the tagGFP2 gene , codon optimized for mammalian cell expression , between 500 base pair homology arms flanking the 3\u2019 ter - minus of the ArpC3 gene . S . pyogenes NLS - Cas9 was purified in the University of California Berkeley QB3 MacroLab and frozen at \" 80 \u02daC until use . TracrRNA and crRNAs were purchased from IDT . Cells were trypsinized in TrypLE select ( Thermo Fisher ) , mixed with donor plasmid ( final concentra - tion 3 . 8 m M ) and 240 pmol Cas9 ( final concentration 1 . 75 m M ) complexed with 100 m M crRNA and tracrRNA ( final concentration 3 . 7 m M ) , and electroporated with an Amaxa nucleofector in stem cell nucleofector reagent ( Lonza ) . The sequence of the crRNA to target the C terminus of the ArpC3 gene was CCGGGCUCCCUUCACUGUCC . Cells were seeded on matrigel - coated 6 - well plates in StemFlex supplemented with the rho - kinase inhibitor RevitaCell and media was changed 24 hr later . Three days after electroporation the cells were bulk sorted for GFP fluorescence with a BD Biosci - ence Influx sorter ( BD Bioscience ) . Fluorescence intensities of cell populations were analyzed using the flow cytometry software FlowJo ( FlowJo , LLC ) . Around one week later the cells were re - sorted into matrigel - coated 96 - well plates at densities of 5 , 10 , or 20 cells per well . Positive clones were confirmed by PCR and sequencing of the genomic DNA locus . From genomic DNA and fluorescent cell sorting analysis we determined that both alleles of ArpC3 were tagged with tagGFP2 . We sequenced genomic DNA extracts from the cell lines at the insertion sites to confirm that there were no insertions or deletions at the site of GFP insertion . We isolated genomic DNA using the DNEasy blood and tissue DNA isolation kit ( Qiagen ) . The primers used for PCR amplification of genomic DNA were TCAGGGTGGCTTTCTCTCCT and CCAGAGCTGCAACCAGTACA . The primers used for sequencing the ArpC3 allele were ACTTATTCTTATTAAGCGCCAGC and CAGGGCTCTGGA - GACGGT . Western blotting We pelleted 1 \u2013 2 wells of cells from a 6 - well plate ( ~ 10 6 cells ) at 4 \u02daC and lysed the cells in 50 mM Hepes pH 7 . 4 , 150 mM NaCl , 1 mM MgCl 2 , 0 . 1 % Triton X - 100 , and cOmplete Mini EDTA - free prote - ase inhibitor ( Sigma Aldrich ) . Extracts were separated electrophoretically in sample buffer with 80 mM DTT on 8 % polyacrylamide gels and transferred to nitrocellulose membranes . Membranes were blocked with 5 % nonfat milk in PBS , probed with mouse monoclonal anti - GAPDH ( ProteinTech 10494 \u2013 1 - AP ) at 1 : 5000 dilution and rabbit polyclonal antibody against tag ( C , G , Y ) FP ( Evrogen AB121 Lot 12101231265 ) at 1 : 2500 dilution in PBS with 0 . 1 % TWEEN - 20 and 1 : 100 PBS / milk , followed by incubation in donkey anti - mouse CF680 and donkey anti - rabbit CF800 ( Li - Cor Bioscences ) . Washed membranes were imaged on a Li - Cor Odyssey Clx infrared fluorescence imager ( Li - Cor Biosciences ) . Choice of subunit to tag : We chose the location of the GFP tag based on available structural , biochemical , cell biological , and genetic data on the functionality of fluorescent fusion proteins of subunits of the Arp2 / 3 complex . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 30 of 40 Research article Cell Biology Physics of Living Systems ArpC3 ( p21 ; Arc18 in budding yeast ) tags are more functional than tags on Arp2 , Arp3 , or ArpC5 ( Arc15 in budding yeast ) ( Egile et al . , 2005 ; Sirotkin et al . , 2010 ; Smith et al . , 2013 ; Picco et al . , 2015 ) . Single - particle electron microscopy reconstructions and the crystal structure ( PDB : 2p9l ) of Arp2 / 3 complex show that the C terminus of ArpC3 is flexible and does not sterically interfere with Arp2 / 3 complex\u2019s binding site to actin filaments or its activators ( VCA ) . Constructs for intracellular fluorescence - based standard curve We adapted self - assembling protein nanocages of defined copy number ( Hsia et al . , 2016 ) to con - struct a fluorescence calibration standard in live mammalian cells . These trimeric proteins were engi - neered by Hsia et al . ( 2016 ) at protein - protein interfaces to self - assemble into a 60mer ( KPDG aldolase ) that can be tagged at the N or and C termini with GFP to yield an average of 60 or 120 copies of GFP . Alternatively a two - component 24mer ( alamin adenosyl transferase and 5 - carboxy - methyl - 2 - hydroxymuconate isomerase [ King et al . , 2014 ] ) can be tagged one or both components with GFP to yield an average 12 or 24 copies of GFP per structure ( Hsia et al . , 2016 ) . DNA con - structs codon - optimized for mammalian expression were synthesized ( IDT ) with alanine mutations at K129 ( KPDG aldolase ) and R72 ( 5 - carboxymethyl - 2 - hydroxymuconate isomerase ) to abolish enzy - matic activity . An E126L mutation in the transferase ( King et al . , 2014 ) is predicted to abolish its enzymatic activity . The synthetic construct included GS repeat linkers and tagGFP2 codon optimized for mammalian cell expression . We inducibly tethered the nanocages to the plasma membrane using an N - terminal myristolation and palmitoylation motif and the FKBP / FRB * dimerization system , where FRB * is a T2098L variant of FRB that binds a rapamcyin analog , AP21967 ( Clontech ) , and does not bind endogenous mTOR . These constructs bound weakly to the plasma membrane even in the absence of AP21967 , presumably due to multivalent weak affinity binding . We transiently expressed these plasmid constructs into hiPS ( or SK - MEL - 2 ) cells with Lipofectamine Stem ( Thermo Fisher ) . After 2 days we imaged the cells along with the genome edited cells using similar imaging settings . TIRF microscopy Cells were imaged on either an Olympus IX - 81 or Nikon Ti - 2 inverted microscope fitted with TIRF optics . The IX - 81 microscope used a 60 , 1 . 49 NA objective ( Olympus ) and an Orca Flash 4 . 0 sCMOS camera ( Hamamatsu ) . Cells were illuminated with solid - state lasers ( Melles Griot ) with simul - taneous acquisition by a DV - 2 image splitter ( MAG Biosystems ) . The microscope was maintained at 37 \u02daC with a WeatherStation chamber and temperature controller ( Precision Control ) and images were acquired using Metamorph software . The Nikon Ti2 microscope was equipped with a motor - ized stage ( Nikon ) , automated Z focus control , LU - N4 integrated four - wavelength solid state laser setup , TIRF illuminator ( Nikon ) , quad wavelength filter cube , NI - DAQ triggering acquisition ( National Instruments ) , an Orca Flash 4 . 0 sCMOS camera ( Hamamatsu ) , and triggerable filter wheel ( Finger Lakes Intstrumentation ) with 525 / 50 and 600 / 50 wavelength emission filters . Cells were seeded on autoclaved 25 mm # 1 . 5 round coverslips coated with 1 mL matrigel ( 80 m g / mL ) or recombinant Vitronectin - N diluted in PBS ( Thermo Fisher ) . Cells were maintained at 37 \u02daC with a stage top incuba - tor ( OKO Lab ) and images were acquired with Nikon Elements . CK - 666 experiments CK - 666 ( Sigma ) was reconstituted in DMSO and diluted in imaging media prior to treatment . Cells were treated prior to or during imaging . Cells were treated for 45 min prior to imaging unless other - wise indicated in the text . \u20180 m M\u2019 treatment corresponds to 0 . 1 % DMSO treatment . Confocal microscopy We imaged cells on a Nikon Eclipse Ti inverted microscope ( Nikon Instruments ) fitted with a CSU - X spinning disk confocal head ( Yokogawa ) , four solid - state lasers ( Nikon ) , IXon X3 EMCCD camera ( Andor ) , and emission filter wheel ( Sutter Instruments ) . The imaging area was kept at 37 \u02daC with 5 % CO2 ( In Vivo Scientific , LCI ) . We used a 100 , 1 . 45 NA Plan Apo oil immersion objective ( Nikon ) . Images were acquired using Nikon Elements . We generally imaged 3 \u2013 7 z slices with 300 nm z spac - ing at 3 s time intervals . Cells were seeded on sterile 4 - chambered or 8 - chambered # 1 . 5H ( 0 . 170 \u00b1 0 . 005 mm ) cover glasses ( CellVis ) . We imaged the cells in media supplemented with HEPES and the antioxidant oxyrase ( OxyFluor ) with substrate lactate . For quantitative fluorescence Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 31 of 40 Research article Cell Biology Physics of Living Systems experiments we limited the cells\u2019 exposure to ambient light , brightfield light and 488 nm laser light prior to acquisition , and used the same laser power during acquisition to compare experiments ( gen - erally 10 % acousto - optic tunable filter ( AOTF ) power ) . For most experiments , we used DMEM / F12 without phenol red ( Thermo Fisher ) supplemented with the protein supplement used in StemFlex media ( Thermo Fisher ) , which gave similar fluorescence intensity results as cells imaged in StemFlex ( which has phenol red ) . Time - lapse imaging of nanocages We treated cells with a range of concentrations of AP21967 for > 45 min and then imaged them on a spinning disk confocal microscope at 0 . 3 s intervals for 1 min . Image correction We took images of dilute GFP or autofluorescent Luria Broth ( LB ) to correct for uneven illumination in the imaging plane . The exposure time and laser power both scaled linearly on our instrument ( Fig - ure 2\u2014figure supplement 1 ) which allowed us to adjust for dimmer or brighter signals by changing the exposure time . Cryo - electron tomography sample preparation Holey carbon grids ( Quantifoil R2 / 1 , 200 mesh , gold ) were glow discharged using a Pelco SC - 6 sput - ter coater and sterilized in 70 % ethanol in H 2 O . SK - MEL - 2 cells were plated in DMEM / F12 ( Gibco ) supplemented with 10 % fetal bovine serum ( premium grade , VWR Seradigm Life Science ) and 1 % Penicillin / Streptomycin ( Gibco ) onto grids and incubated overnight at 37\u02daC and 5 % CO 2 in a cell cul - ture incubator . Samples were blotted and plunge frozen using a Vitrobot Mark IV ( FEI ) after addition of 10 nm BSA Gold Tracer ( Electron Microscopy Sciences ) . Cryo - electron tomography data recording and processing Tilt - series were recorded on a Titan Krios operated at 300 kV ( FEI ) equipped with a Quantum energy filter ( Gatan ) and a K2 direct electron detecting device ( Gatan ) at 2 . 97 A\u02da pixel size and a target defo - cus of \" 2 m m . SerialEM ( Mastronarde , 2005 ) in low - dose mode was used for automated tilt series acquisition using a bidirectional tilt scheme covering a whole tilt range from + 60\u02da to \" 60\u02da with a base increment of 2\u02da and a total electron dose of 100e - / A\u02da 2 . Tomograms were generated in IMOD ( Kremer et al . , 1996 ) using the gold beads as fiducials for tilt - series alignment . For Figure 5 , tomo - grams were reconstructed using the simultaneous iterative reconstruction technique ( SIRT ) recon - struction algorithm , then filtered using the Nonlinear Anisotropic Diffusion ( NAD ) filter in IMOD and binned by a factor of 2 . For Figure 5\u2014video 1 , we used the backprojection algorithm for tomogram reconstruction followed by a smoothing filter ( Clip smooth function in IMOD ) . The U - shaped pit shown is one of six sites of CME we have identified in our tomograms , all of which have bent actin filaments around the pit to varying degrees . The bent filaments are especially prominent in the tomogram shown because of the orientation of the pit with respect to the missing wedge effect . A detailed treatment of these tomograms will be the subject of a subsequent study . Data analysis Calibration curve : We used a combination of custom - written and publicly available image analysis software in Fiji ( 1 . 52i ) and Matlab ( r2017b ) to analyze the traces of fluorescence intensity per spot , for multiple Z slices and time points . To measure fluorescence intensity per spot of GFP - tagged nanocages , we wrote a toolset in Fiji to select and circular regions of interest eight pixels ( 1 . 1 m m ) in diameter , and used cross - correlation to center the regions around the intensity - based center of mass . We selected as background regions of concentric circles one pixel larger than the original region of interest . The toolset measured the fluorescence intensity per spot , which we subtracted by the area - corrected background intensity to yield fluorescence intensity per spot for the four con - structs . Some analysis functions were adapted from published software ( Akamatsu et al . , 2017 ; Epstein et al . , 2018 ; McCormick et al . , 2013 ) . We measured only spots that were contained within the slices imaged and were single stationary spots . For comparison of fluorescence to eGFP - MotB , we used smaller ( 6 - pixel ) ROIs . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 32 of 40 Research article Cell Biology Physics of Living Systems We plotted background - subtracted fluorescence intensity per spot as a function of predicted copy number per structure to obtain a calibration curve relating fluorescence intensity per spot to numbers of molecules per structure . For the curve in Figure 3D , we combined data from three experiments with different imaging conditions by defining the average 60mer - GFP intensity per experiment as 1000 arbitrary units . Lines are linear fits through zero with r 2 calculated by linear least - squares fitting . Time - lapse fluorescence quantification : We made modifications to automated MATLAB - based tracking software ( Aguet et al . , 2013 ; Hong et al . , 2015 ) to track and analyze fluorescence - intensity time lapse data of genome - edited cells . The core tracking program ( based on the software package m - track ) automatically identifies fluorescent spots and connects them as tracks by minimizing the lin - ear assignment problem ( Jaqaman et al . , 2008 ) . We used stringent tracking parameters with gap size 0 and search radius 0 \u2013 2 . 3 pixels ( 248 nm ) . GFP and RFP tracks with high variability in the inten - sity / time profile were automatically rejected ( Ferguson et al . , 2017 ) as well as tracks * 3 s in dura - tion ( Dambournet et al . , 2018 ) and the remaining tracks were associated spatiotemporally according to a cost matrix ( Hong et al . , 2015 ) . We used two track rejection schemes . In the first , users were presented with fluorescence montages and XY coordinates of the tracks to assess the fidelity of tracking for each event ( Hong et al . , 2015 ) . In the second , tracks were automatically rejected based signal - to - noise ratio ( > 1 . 05 ) and proximity to neighboring tracks ( > 525 nm ) ( Hong et al . , 2015 ) . We checked that the manual and automatic track rejection schemes yielded similar results ( lifetime distributions and intensity versus time plots ) as well as to manual , kymograph - based quantification of lifetimes ( below ) . From the above workflow ( Dambournet et al . , 2018 ) we increased throughput by connecting all steps into an automated tracking pipeline requiring minimal user input . For SK - MEL - 2 cells expressing CLTA - RFP and DNM2 - GFP , we tracked regions that were not near the nucleus ( which has a concentration of Golgi - derived clathrin budding ) and that did not have large , bright , persistent structures containing invariant RFP and GFP signals ( \u2018plaques , \u2019 which are likely sites of adhesion ) . Alignment method : For Figure 2G , we aligned tracks based on the time point after the peak intensity in which 50 % of the fluorescence remained in the GFP channel . We normalized the fluores - cence intensity to compare movies from different imaging conditions . For Figure 2I , we aligned the tracks based on the maximum intensity . For Figure 6D , we aligned the tracks based on the disap - pearance of the RFP signal . This code is available at the following website : https : / / github . com / Dru - binBarnes / Akamatsu _ CME _ manuscript . Manual track analysis : We wrote a Fiji toolset that generated two - color kymographs from user - defined regions of interest , and then quantified the lifetime based on the lengths of the kymographs ( from user - defined regions on the kymographs ) . This manual analysis was used in Figure 6\u2014figure supplement 1A and for verification of the automated tracking scheme . This code is available at the following website : https : / / github . com / DrubinBarnes / Akamatsu _ CME _ manuscript . Nanocage particle tracking : To track the membrane - tethered nanocages in 2D in cells we used TrackMate , a plugin available in Fiji that optimizes the Linear Assignment Problem ( LAP ) ( Jaqaman et al . , 2008 ) . We detected spots using an estimate of 0 . 5 m m and threshold of 15 , using a median filter and sub - pixel localization . We used the simple LAP tracker with a maximum linking and gap closing distance of 1 m m and two frames and a minimum track length of 4 frames . Calculating numbers of molecules per endocytic site : We calculated the fluorescence intensity of background - subtracted ArpC3 - GFP spots colocalized with AP2 - RFP spots from single time - point images using the same background correction approach described for the calibration curve . We used the slope of the calibration curve to convert fluorescence intensity to numbers of molecules of ArpC3 - GFP . Because the standard is inside cells , this standard controls for fluorescence environment and fluorescent protein folding and maturation . We used the resultant histogram of numbers of ArpC3 - GFP per spot and the time - lapse fluorescence intensity data in Figure 2G to create the graph in Figure 2I of numbers of molecules of ArpC3 - GFP over time . Acknowledgements We would like to thank Dan Fletcher , Johannes Schoeneberg , and Jasmine Nirody for insightful com - ments on the manuscript ; Julian Hassinger for advice on the continuum mechanics model and gener - ating movies of the membrane simulations ; Francois Nedelec for training and advice in Cytosim , Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 33 of 40 Research article Cell Biology Physics of Living Systems discussion of the relationship between association rate constants and binding rates , and sharing the \u2018Fork . cc\u2019 class for modeling Arp2 / 3 complex ; Johannes Schoeneberg and Logan Akamatsu for help generating parallel simulation shell scripts ; Sun Hong and Meiyan Jin for generating and validating the AP2 - tagRFP - T human induced pluripotent cell line ; and Elizabeth Li for contributions to automat - ing the particle tracking pipeline . In addition , we would like to thank the UC Berkeley High Perfor - mance Computing cluster for training and server space for parallel simulations ; the UC Berkeley QB3 MacroLab for purified S . pyogenes NLS - Cas9 ; the UC Berkeley Cancer Research Laboratory Molecu - lar Imaging Center with support from the Gordon and Betty Moore Foundation ; Karen M Davies and Jonathan Remis , Lawrence Berkeley National Labs Donner Building Cryo EM facility ; Daniel B Toso and Paul Tobias , UC Berkeley Berkeley Bay Area Cryo - EM facility ; NIH MIRA R35GM118149 to DGD ; a postdoctoral fellowship from the Arnold and Mabel Beckman Foundation to MA ; postdoc - toral fellowship LT000234 / 2018L from the Human Frontier Science Program to DS ; ARO W911NF1610411 and Office of naval research N00014 - 17 - 1 - 2628 to PR . Additional information Funding Funder Grant reference number Author National Institutes of Health R35GM118149 David G Drubin Arnold and Mabel Beckman Foundation Matthew Akamatsu Human Frontier Science Pro - gram LT000234 / 2018 - L Daniel Serwas Army Research Office W911NF1610411 Padmini Rangamani Office of Naval Research N00014 - 17 - 1 - 2628 Padmini Rangamani The funders had no role in study design , data collection and interpretation , or the decision to submit the work for publication . Author contributions Matthew Akamatsu , Conceptualization , Software , Formal analysis , Funding acquisition , Investigation , Visualization , Methodology , Project administration ; Ritvik Vasan , Michael A Ferrin , Software , Formal analysis , Investigation , Methodology ; Daniel Serwas , Investigation , Methodology ; Padmini Ranga - mani , Conceptualization , Supervision , Funding acquisition , Visualization , Project administration ; David G Drubin , Conceptualization , Resources , Supervision , Funding acquisition , Visualization , Proj - ect administration Author ORCIDs Matthew Akamatsu https : / / orcid . org / 0000 - 0002 - 0286 - 5310 Daniel Serwas http : / / orcid . org / 0000 - 0001 - 9010 - 7298 Michael A Ferrin https : / / orcid . org / 0000 - 0002 - 9899 - 1169 Padmini Rangamani https : / / orcid . org / 0000 - 0001 - 5953 - 4347 David G Drubin https : / / orcid . org / 0000 - 0003 - 3002 - 6271 Decision letter and Author response Decision letter https : / / doi . org / 10 . 7554 / eLife . 49840 . sa1 Author response https : / / doi . org / 10 . 7554 / eLife . 49840 . sa2 Additional files Supplementary files . Supplementary file 1 . Model parameters for continuum membrane mechanics model . . Supplementary file 2 . Parameters for coat pulling continuum mechanics simulations . Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 34 of 40 Research article Cell Biology Physics of Living Systems . Supplementary file 3 . Parameters in the model . . Transparent reporting form Data availability All code associated with simulation and analysis is available at https : / / github . com / DrubinBarnes / Akamatsu _ CME _ manuscript ( copy archived at https : / / github . com / elifesciences - publications / Aka - matsu _ CME _ manuscript ) . References Abercrombie M . 1980 . The crawling movement of metazoan cells . Proceedings of the Royal Society of London Series B Biological Sciences 207 : 129 \u2013 147 . DOI : https : / / doi . org / 10 . 1098 / rspb . 1980 . 0017 Aguet F , Antonescu CN , Mettlen M , Schmid SL , Danuser G . 2013 . 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DOI : https : / / doi . org / 10 . 1371 / journal . pbio . 2004786 , PMID : 29723197 Akamatsu et al . eLife 2020 ; 9 : e49840 . DOI : https : / / doi . org / 10 . 7554 / eLife . 49840 40 of 40 Research article Cell Biology Physics of Living Systems", + "okrut2015allosteric": "Allosteric N - WASP activation by an inter - SH3 domain linker in Nck Julia Okrut a , b , Sumit Prakash a , Qiong Wu c , Mark J . S . Kelly d , and Jack Taunton a , b , 1 a Department of Cellular and Molecular Pharmacology , University of California , San Francisco , CA 94158 ; b The Howard Hughes Medical Institute Summer Institute , Marine Biological Laboratory , Woods Hole , MA 02543 ; c Department of Biophysics , University of Texas Southwestern Medical Center , Dallas , TX 75390 ; and d Department of Pharmaceutical Chemistry , University of California , San Francisco , CA 94158 Edited by Brenda A . Schulman , St . Jude Children \u2019 s Research Hospital , Memphis , TN , and approved October 20 , 2015 ( received for review June 3 , 2015 ) Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott \u2013 Aldrich - syndrome pro - tein ( N - WASP ) and the Arp2 / 3 complex . An inactive conformation of N - WASP is stabilized by intramolecular contacts between the GTPase binding domain ( GBD ) and the C helix of the verprolin - homology , connector - helix , acidic motif ( VCA ) segment . Multiple SH3 domain - containing adapter proteins can bind and possibly activate N - WASP , but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2 / 3 complex . Here , we have used purified components to reconstitute a signaling cascade driven by membrane - localized Src homology 3 ( SH3 ) adapters and N - WASP , resulting in the assembly of dynamic actin networks . Among six SH3 adapters tested , Nck was the most potent activator of N - WASP \u2013 driven actin assembly . We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains . This linker sequence , reminiscent of bacterial virulence fac - tors , directly engages the N - WASP GBD and competes with VCA bind - ing . Our results suggest that animals , like pathogenic bacteria , have evolved peptide motifs that allosterically activate N - WASP , leading to localized actin nucleation on cellular membranes . signal transduction | actin cytoskeleton | SH3 adapter | Nck | N - WASP A ctin polymerization provides the force that drives membrane protrusion during cell motility as well as the propulsion of endocytic vesicles and intracellular pathogens . Branched actin networks are assembled on the surface of cellular membranes , where actin monomers are incorporated into the membrane - apposed ends of growing actin filaments ( 1 \u2013 3 ) . The formation of new branches is initiated by the Arp2 / 3 complex , which requires allosteric activation by membrane - associated nucleation - promoting factors . Neural Wiskott \u2013 Aldrich - syndrome protein ( N - WASP ) is an essential nucleation - promoting factor that integrates and trans - duces membrane - localized signals to the Arp2 / 3 complex . N - WASP constitutes a regulatory hub whose localization and activation state govern the spatiotemporal dynamics of actin network formation . Under resting conditions , N - WASP exists in an autoinhibited conformation in the cytoplasm . Signaling from tyrosine kinases , GTPases , and acidic phospholipids cooperatively activates N - WASP on the membrane ( 4 \u2013 7 ) . Two principal themes have emerged to describe N - WASP regulatory mechanisms : allo - steric activation and oligomerization ( 8 ) . Allosteric activation dis - rupts intramolecular autoinhibitory contacts between the C helix and the GTPase binding domain ( GBD ) . These interactions maintain N - WASP in a closed conformation that sterically occludes its carboxyl - terminal verprolin - homology , connector - helix , acidic motif ( VCA ) segment ( 9 , 10 ) ( Fig . 3 A ) . The small GTPase Cdc42 is the archetypal allosteric N - WASP activator . Cdc42 binds directly to the GBD and releases the VCA segment ( 11 ) , which subsequently binds the Arp2 / 3 complex and promotes actin filament nucleation from the side of a preexisting actin filament . N - WASP oligomeri - zation or clustering , mediated by signaling adapter proteins and acidic phospholipids , facilitates simultaneous interaction of two N - WASP molecules with one Arp2 / 3 complex . Simulta - neous engagement of the constitutively inactive Arp2 / 3 complex by two VCA - type ligands is required for Arp2 / 3 - mediated actin nucleation ( 12 , 13 ) . Src homology 3 ( SH3 ) domain - containing adapter proteins have also been shown to activate N - WASP . Such signaling adapters often harbor multiple SH3 domains , each capable of binding a canonical polyproline motif ( 14 ) . Genetic , cell biological , and biochemical evidence supports a role for the SH2 / SH3 adapter protein Nck in the activation of N - WASP ( 15 \u2013 22 ) . Phosphorylated tyrosine residues on membrane receptors , such as the podocyte adhesion receptor nephrin and the vaccinia virus membrane protein A36R , localize Nck to the plasma membrane through its SH2 domain . Nck then directly binds N - WASP and the N - WASP \u2013 associated protein WIP , leading to localized actin polymerization ( 15 , 20 , 21 , 23 ) . Similar to Nck , Grb2 is an SH2 / SH3 adapter that binds and activates N - WASP ( 24 , 25 ) , acting in concert with Nck to promote actin - dependent vaccinia virus motility ( 26 ) . Other SH3 adapter proteins implicated in N - WASP activation include Crk - II ( 27 ) , cortactin ( 28 ) , Toca / CIP4 ( 29 , 30 ) , and Tks4 / 5 ( 31 ) ( Fig . 1 A ) . The mechanism by which SH3 adapters activate N - WASP is only partially understood . Although SH3 - mediated oligomerization has been shown to increase N - WASP activity , these experiments were performed with a constitutively active N - WASP mutant lacking the GBD \u2013 VCA autoinhibitory in - teraction ( 32 ) . Key unresolved questions are whether and how SH3 adapters counteract the autoinhibitory interactions that restrain access to N - WASP \u2019 s VCA segment . Here , we systematically compare the ability of distinct SH3 adapters to assemble actin and Arp2 / 3 networks while localized to a membrane surface . To this end , we have biochemically Significance Actin is a monomeric protein that can polymerize into branched networks . Actin polymerization acts like an engine to drive cell movement and is regulated by multiple interacting proteins on the cell membrane . To understand the molecular details of how cells transmit signals from the membrane to the actin polymer - ization engine , we reconstituted this process in a test tube using seven purified proteins and membrane - coated glass beads . Us - ing this \u201c biomimetic \u201d system , we discovered a sequence motif in the human protein Nck that activates a core component of the actin polymerization engine . This motif shares similarity with certain bacterial virulence factors that stimulate actin polymeri - zation in infected human cells , suggesting that similar activation mechanisms have evolved in humans and bacterial pathogens . Author contributions : J . O . and J . T . designed research ; J . O . , Q . W . , and M . J . S . K . performed research ; J . O . and S . P . contributed new reagents / analytic tools ; J . O . , S . P . , Q . W . , and M . J . S . K . analyzed data ; and J . O . and J . T . wrote the paper . The authors declare no conflict of interest . This article is a PNAS Direct Submission . Datadeposition : TheNMRchemicalshiftshavebeendepositedin theBiologicalMagnetic Resonance Data Bank ( accession no . 26643 ) . 1 To whom correspondence should be addressed . Email : jack . taunton @ ucsf . edu . Thisarticlecontainssupportinginformationonlineatwww . pnas . org / lookup / suppl / doi : 10 . 1073 / pnas . 1510876112 / - / DCSupplemental . E6436 \u2013 E6445 | PNAS | Published online November 9 , 2015 www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1510876112 D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . reconstituted the N - WASP / actin signaling cascade from pure components using a membrane bilayer supported on silica micro - spheres . We find that membrane - localized actin assembly varies dramatically depending on the SH3 adapter , with only Nck showing robust actin assembly . Structure \u2013 function analysis revealed a pre - viously unidentified N - WASP activation motif embedded within a 45 - aa linker that connects the first two SH3 domains of Nck . This conserved inter - SH3 domain linker binds directly to the N - WASP GBD and in concert with the SH3 domains , potently stimulates actin network assembly on membranes . Nck thus uses both allo - steric and oligomerization - based mechanisms to activate N - WASP . Results Polarized Actin Network Assembly Promoted by SH3 Adapters and N - WASP . We initially compared six full - length SH3 adapter pro - teins ( Fig . 1 A ) using a well - established reconstituted motility system that contains purified N - WASP ( amino acids 151 \u2013 501 ; lacking the N - terminal EVH1 domain ) , Arp2 / 3 complex , actin , and the actin Nck1 1 % NTAD 2 . 5 % NTAD 5 % NTAD Grb2 Crk - II cortactin Phase Alexa - actin Phase Alexa - actin Phase Alexa - actin T ks4 CIP4 CIP4 BAR SH3 SH3A SH3B SH3D SH3C PH cortactin Arp2 / 3 Actin Binding Repeats SH3 Tks4 SH3A SH3B SH2 Grb2 SH3A SH3B SH3C SH2 Nck1 SH3A SH3B SH2 Crk - II N ck 1 G r b2 C r k - II c o r t a c t i n C I P 4 T ks 4 0 . 0 5 . 0 10 . 0 15 . 0 20 . 0 25 . 0 T a il L e ng t h [ \u00b5 m ] * * NTAD 1 % 2 . 5 % 5 % A C B D E F NTAD + actin , Arp2 / 3 , cofilin , profilin , capping protein lipid - coated glass bead S H 3 a d a p t e r N - W A SP S H 3 a d a p t e r s N ck 1 G r b2 C r k - II c o r t a c t i n C I P 4 T ks 4 0 10000 20000 30000 40000 50000 60000 70000 A l exa568 - S H 3 a d a p t e r [ AU ] NTAD 1 % 2 . 5 % 5 % NTAD : 1 % 2 . 5 % 5 % N ck 1 G r b2 C r k - II c o r t a c t i n C I P 4 T ks 4 0 500 1000 1500 2000 2500 A l exa488 - ac t i n [ AU ] * * * Fig . 1 . Membrane - associated actin networks assembled by SH3 adapters and N - WASP . ( A ) Domain organization of SH3 adapter proteins used in this study . ( B ) Experimental strategy for localization of His - tagged SH3 adapters using NTAD - doped membranes supported on silica microspheres . ( C ) Phase contrast and fluorescence images of lipid - coated beads ( NTAD density : 1 % , 2 . 5 % , or 5 % ) incubated with the indicated SH3 adapter ( 250 nM ) , N - WASP ( 50 nM ) , Alexa488 actin , and actin regulatory components . After 15 min , reactions were fixed with glutaraldehyde . ( Scale bar : 5 \u03bc m . ) ( D ) Integrated fluorescence intensity of Alexa488 actin tails . ( E ) Actin tail length . ( F ) Integrated fluorescence intensity of bead - localized Alexa568 SH3 adapters ( 250 nM ) . ( Mean \u00b1 SD ; n > 20 . ) Asterisks indicate a significant difference between Nck1 and Grb2 at 1 % and 5 % NTAD : * * P < 0 . 01 ; * * * P < 0 . 001 , respectively . Okrut et al . PNAS | Published online November 9 , 2015 | E6437 B I O C H E M I S T RY P N A S P L U S D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . regulatory proteins profilin , cofilin , and capping protein ( 33 , 34 ) . As described in the Introduction , all six SH3 adapters have been implicated in cellular N - WASP activation and have been shown to bind the N - WASP proline - rich region . Because N - WASP \u2013 medi - ated actin assembly occurs on the cytosolic surface of cellular membranes , we used lipid bilayers supported on silica microspheres ( 7 , 35 , 36 ) . Supported lipid bilayers were supplemented with a nickel - chelating lipid [ 1 , 2 - dioleoyl - sn - glycero - 3 - [ ( N - ( 5 - amino - 1 - carboxypentyl ) iminodiacetic acid ) succinyl ] ( NTAD ) ] to localize His 6 - tagged versions of the SH3 adapter proteins ( Fig . 1 B ) . To test a range of SH3 adapter densities , we varied the molar fraction of NTAD in the lipid bilayers ( 1 % , 2 . 5 % , and 5 % NTAD , mol % ) . Lipid - coated beads were incubated in a solution containing the purified full - length SH3 adapter ( 250 nM ) , Alexa488 - labeled actin , N - WASP , and the actin regulatory components used at concentrations that were previously shown to support bead motility ( 7 , 33 ) . After 15 min , reactions were fixed with glutar - aldehyde , and the bead - associated actin structures were analyzed by wide - field epifluorescence imaging . Two measurements were used to quantify bead - associated actin : integrated fluorescence intensity of Alexa488 actin and actin tail length . Nck was the only SH3 adapter that induced polarized actin tails on > 95 % of the beads at the lowest NTAD density tested ( Fig . 1 C and Fig . S1 ) . Image analysis revealed that , in reactions driven by Nck , bead - associated actin intensity and tail length were already maximal at the lowest NTAD density ( Fig . 1 D and E ) . Moreover , Nck - coated beads displayed robust actin - dependent motility when imaged live in a flow chamber containing N - WASP , Arp2 / 3 complex , and actin regulatory components ( Movie S1 ) . By contrast , Grb2 showed substantially weaker ac - tivity . Grb2 - induced tails were not detected at 1 % NTAD , and only diffuse actin \u201c clouds \u201d or short tails were observed at 2 . 5 % and 5 % NTAD , respectively ( Fig . 1 C , row 2 ) . The Grb2 - induced tails at 5 % NTAD density contained one - half as much actin as the Nck - induced tails obtained at 1 % NTAD and were signifi - cantly shorter ( Fig . 1 D and E ) . A thin shell of bead - associated actin was visible in reactions containing cortactin ( Fig . 1 C and D ) , consistent with its ability to directly activate Arp2 / 3 and bind actin filaments ( 37 ) . However , cortactin did not promote the assembly of polarized actin tails . Reactions containing Crk - II , CIP4 , or Tks4 were devoid of visible actin structures . To ensure that the observed effects in the actin tail assembly assay were not caused simply by differential recruitment of the SH3 adapters , we used live imaging to quantify the fluorescence intensity of Alexa568 - labeled adapters on the bead surface . Despite the common recruitment mechanism through hexahistidine tag and NTAD lipids , the adapters localized to different extents . At an NTAD density of 1 % , the fluorescence intensities measured for CIP4 and Tks4 were significantly higher than for Nck , Grb2 , Crk - II , and cortactin , all of which were used at a concentration of 250 nM ( Fig . 1 F , blue bars ) . We speculate that these differences arise from intrinsic lipid binding and / or a propensity to oligomerize on membrane surfaces . Most importantly , the Nck density at 1 % NTAD was significantly lower than the density of the other five adapters at 5 % NTAD . Despite achieving higher membrane den - sities than Nck , the other SH3 adapters were significantly less active or inactive . These results show that , of six SH3 adapters tested , Nck has by far the strongest ability to assemble polarized actin networks on the surface of supported membranes . A potential explanation for Nck \u2019 s superior activity is that it re - cruits more N - WASP to the membrane compared with the other SH3 adapters . To test this , we measured the density of Alexa568 - labeled N - WASP recruited to lipid beads in the presence of the His - tagged adapters . In initial experiments , Nck and Grb2 recruited similar levels of N - WASP , but the other SH3 adapters recruited substantially lower amounts . To compensate for these differences in apparent affinity , we increased the N - WASP concentration to 200 nM . Additionally , we increased the NTAD density to 5 % for Crk - II , cortactin , CIP4 , and Tks4 , while maintaining 1 % NTAD for Nck and Grb2 . Under these conditions , N - WASP levels recruited by all of the SH3 adapters were within approximately twofold of each other ( Fig . 2 A ) . Despite the similar N - WASP density observed with all six SH3 adapters , Nck showed the strongest actin assembly activity ( Fig . 2 B ) . Tails assembled by Grb2 were approximately threefold shorter and incorporated approximately twofold less actin compared with Nck ( Fig . 2 C ) . Similar to our previous results ( Fig . 1 C and D ) , Crk - II , cortactin , CIP4 , and Tks4 did not assemble any detectable actin structures . These data indicate that the increased ability of Nck to assemble membrane - associated actin networks cannot be explained solely by enhanced N - WASP recruitment . We therefore considered the possibility that , among the SH3 adapters tested , Nck might deploy a unique N - WASP activation mechanism . Discovery of an Inter - SH3 Domain N - WASP Activation Motif . Nck is recruited to cellular membranes through its SH2 domain , which binds phosphorylated tyrosine residues on integral membrane receptors or membrane - associated adapter proteins . Nephrin is an Nck receptor on the plasma membrane of podocytes . Three phosphorylated tyrosine residues on nephrin bind the SH2 domain of Nck , and this interaction is essential for regulating podocyte actin dynamics in an N - WASP \u2013 dependent manner ( 20 , 21 ) . To explore the mechanism of Nck - mediated N - WASP activation on membranes , we used a His - tagged , triphosphorylated nephrin peptide ( His 8 - pY 3 - nephrin ) , which recruits untagged Nck to the surface of NTAD - containing membranes ( 38 ) . Lipid - coated beads with 1 % NTAD were preincubated with His 8 - pY 3 - nephrin and subsequently added to a solution containing untagged full - length Nck , N - WASP , and the actin regulatory components described above ( Fig . 3 A ) . Similar to His - tagged Nck recruited through NTAD lipids ( Fig . 1 C ) , untagged Nck recruited through pY 3 - nephrin promoted the assembly of actin tails on > 95 % of the lipid - coated beads ( Fig . 3 C and Fig . S2 ) . Actin assembly promoted by untagged Nck was strictly dependent on the addition of His 8 - pY 3 - nephrin , and Nck mutants lacking the SH2 domain were inactive in this system . Using the pY 3 - nephrin system , we performed a deletion analysis of Nck ( Fig . 3 B ) . Whereas deletion of the amino - terminal SH3A domain had no effect , additional deletion of the inter - SH3AB linker led to a drastic reduction in actin tails ( Fig . 3 C ) . The few residual actin structures formed by this deletion construct con - tained threefold lower actin fluorescence , and the average tail length was reduced by 85 % ( Fig . 3 D and E ) . To exclude the possibility that decreased actin assembly was a trivial result of re - duced membrane recruitment , we measured surface densities of the three Nck constructs in a separate experiment . Both deletion mutants were recruited similarly to one another and to a slightly higher extent than full - length Nck ( Fig . 3 F ) . Hence , the Nck mutant lacking the inter - SH3AB linker is recruited to pY 3 - nephrin but is specifically defective in promoting actin assembly ( Fig . 3 F ) . These experiments identify for the first time , to our knowledge , a 45 - aa linker motif between the first two SH3 domains of Nck that strongly potentiates N - WASP \u2013 dependent actin assembly on membranes . Direct Binding of the Inter - SH3 Linker to the N - WASP GBD . We hy - pothesized that the inter - SH3AB linker ( Nck1 amino acids 61 \u2013 105 ) might interact directly with N - WASP . Within this linker is a 9 - aa motif ( amino acids 67 \u2013 75 ) that is highly conserved across species ( Fig . 4 A and B ) . Moreover , the spacing of the hydrophobic resi - dues suggested the possibility of forming an amphipathic helix ( Fig . 4 B and Fig . S3 ) . Several amphipathic helical peptides have been shown to bind N - WASP through its GBD . In the autoinhibited state , the N - WASP GBD binds intramolecularly to the amphipathic C helix in the carboxyl - terminal VCA region ( 10 ) ( Fig . 3 A ) . Am - phipathic helices can also activate N - WASP : helical motifs within the Escherichia coli virulence factors , EspF U and EspF , bind the GBD in competition with the inhibitory C helix ( 39 \u2013 41 ) . To our E6438 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1510876112 Okrut et al . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . knowledge , such an activation mechanism has only been docu - mented with bacterial pathogens and has not been found with endogenous eukaryotic activators . To test whether the inter - SH3AB linker can directly interact with the N - WASP GBD , we first performed GST pull - down ex - periments with purified proteins . Full - length Nck bound to gluta - thione beads loaded with N - WASP GST - GBD but not to beads loaded with a structurally and functionally related GST - GBD de - rived from the PAK1 kinase ( Fig . 4 C ) . These results indicate a specific , direct , and previously undocumented interaction between Nck and the N - WASP GBD . Deletion of the SH2 and the SH3A domains from Nck did not affect the interaction ( Fig . 4 D ) . How - ever , additional deletion of only 18 amino acids from the inter - SH3AB linker , including the putative amphipathic helix , com - pletely abrogated binding to the GBD along with deletion of the entire linker ( Fig . 4 D ) . To further assess the importance of the inter - SH3AB linker , we used a construct lacking the SH2 domain ( Nck amino acids 1 \u2013 270 ) . Replacing either the entire linker ( amino acids 67 \u2013 105 ) or only the hydrophobic motif ( amino acids 67 \u2013 77 ) with a Gly - Ser linker of equal length abolished the interaction ( Fig . 4 E ) . Finally , GBD binding was completely prevented by four ala - nine mutations at hydrophobic positions within the putative am - phipathic helix [ Ile67 / Val68 / Leu71 / Leu75 ( 4A ) ] . These results strongly suggest that the inter - SH3AB linker , implicated above in promoting N - WASP \u2013 dependent actin assembly ( Fig . 3 C \u2013 E ) , mediates direct interactions between Nck and the N - WASP GBD . We next evaluated actin comet tail assembly by full - length Nck containing the four Ala mutations in the inter - SH3AB linker . In the presence of lipid - coated beads with a high NTAD density ( 1 % ) , 4A Nck formed tails similar to the WT ( Fig . S4 ) . However , with a lower density of 0 . 25 % NTAD , 4A Nck was defective , producing two - to threefold less actin than WT Nck ( P < 0 . 001 ) ( Fig . S4 A \u2013 C ) . We hypothesized that , at higher NTAD densities , polyvalent SH3 - mediated interactions could potentially compen - sate for the defective linker in 4A Nck . To test this , we introduced the four Ala mutations in a construct lacking the SH3A domain B CIP4 cortactin Tks4 Nck1 Crk - II Phase Alexa - actin A C Grb2 0 . 0 10 . 0 20 . 0 30 . 0 N ck 1 , 1 % N T A D G r b2 , 1 % N T A D C r k - II , 5 % N T A D c o r t a c t i n , 5 % N T A D C I P 4 , 5 % N T A D T ks 4 , 5 % N T A D T a il L e ng t h [ \u03bc m ] * * * 0 400 800 1200 1600 2000 2400 A l exa488 - ac t i n [ AU ] * * * NTAD 1 % 5 % N ck 1 , 1 % N T A D G r b2 , 1 % N T A D C r k - II , 5 % N T A D c o r t a c t i n , 5 % N T A D C I P 4 , 5 % N T A D T ks 4 , 5 % N T A D 0 1000 2000 3000 4000 5000 6000 7000 A l exa568 - N - W A SP [ AU ] Fig . 2 . Nck is the most effective SH3 adapter in promoting N - WASP \u2013 dependent actin assembly . ( A ) Integrated fluorescence intensity and representative images of bead - localized Alexa568 N - WASP ( 200 nM ) incubated with NTAD - doped lipid - coated beads and the indicated His - SH3 adapters ( 250 nM ) . To compensate for differences in N - WASP recruitment efficiency , the NTAD density was increased to 5 % for Crk - II , cortactin , CIP4 , and Tks4 , whereas 1 % NTAD was used for Nck1 and Grb2 . ( B ) Representative phase contrast and fluorescence images of actin assembly reactions under conditions reported in A . ( Scale bars : 5 \u03bc m . ) ( C ) Integrated fluorescence intensity of Alexa488 actin tails and tail lengths ( mean \u00b1 SD ; n > 20 ) . Asterisks indicate significant differences between Nck and Grb2 : * * * P < 0 . 001 . Okrut et al . PNAS | Published online November 9 , 2015 | E6439 B I O C H E M I S T RY P N A S P L U S D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . ( Nck amino acids 61 \u2013 377 ) . Here , the 4A mutant was strongly compromised in its ability to assemble actin tails , displaying a threefold reduction in polymerized actin and a fivefold decrease in tail length , even at 1 % NTAD ( P < 0 . 001 ) ( Fig . 4 F and Fig . S5 A and B ) . Collectively , these data establish a role for the amphi - pathic motif in actin comet tail formation , which becomes essen - tial under conditions of low pY 3 - nephrin density or when the first SH3 domain is removed . Structural studies of the WASP GBD ( 68 % sequence identity to N - WASP GBD ) have revealed a high degree of conforma - tional plasticity . In the absence of a binding partner , the WASP GBD is partially folded or unstructured . Binding of the auto - inhibitory C helix ( 10 ) or an activating EspF U - derived peptide ( 40 ) causes the GBD to adopt a mostly helical globular fold . To test whether the Nck linker behaves similarly , we recorded heter - onuclear single - quantum coherence ( HSQC ) spectra of 15 N - labeled N - WASP GBD in the presence and absence of the unlabeled Nck linker peptide ( amino acids 61 \u2013 106 ) . The GBD spectrum in the presence of the Nck linker peptide revealed greater peak disper - sion and more uniform peak intensities and line widths compared with the GBD alone ( Fig . 5 A ) , consistent with a more compact , folded domain . Based on chemical shift differences from NMR spectra recorded with increasing concentrations of the Nck pep - tide , we determined the equilibrium K d to be 33 \u00b1 8 \u03bc M ( Fig . 5 B and Fig . S6 ) . We also recorded the N - WASP GBD spectrum with the VCA peptide and observed similar chemical shifts to those recorded in the presence of the Nck linker peptide : 80 % of the peaks show a weighted chemical shift difference of less than 0 . 1 ppm ( Fig . S7 ) . To characterize the GBD \u2013 Nck linker complex in greater detail , we used standard triple - resonance experiments to assign the back - bone ( 86 % completion ) of a construct comprising the N - WASP GBD covalently linked to the inter - SH3AB segment from Nck ( Fig . S8 ) ( Biological Magnetic Resonance Data Bank accession no . 26643 ) . Analysis of the HN , N , CO , C \u03b1 , and C \u03b2 chemical shifts using Talos + ( 42 ) indicates secondary structure similar to the WASP GBD in complex with the autoinhibitory C helix ( Fig . 5 C and Fig . S9 A ) ( 10 ) . Importantly , the Talos + analysis predicts a helical conformation for the Nck hydrophobic motif ( amino acids 66 \u2013 75 ) . Moreover , the difference between the secondary C \u03b1 and C \u03b2 chemical shifts for residues within the hydrophobic motif is positive ( \u0394\u03b4 [ C \u03b1 \u2212 C \u03b2 ] > 0 ) ( Fig . 5 D and Fig . S9 B ) , consistent with a helical conformation ( 43 ) . Collectively , these data suggest that the structure of the GBD \u2013 Nck linker complex is similar to that of the GBD \u2013 VCA complex . A prediction of this model is that binding of the C helix and the Nck linker to the GBD are mutually exclusive . Consistent with competitive binding , addition of in - creasing concentrations of VCA abrogated binding of full - length Nck to the GBD ( Fig . 5 E and F ) . Based on these results , we conclude that Nck binds N - WASP through not only its SH3 do - mains but also , its inter - SH3AB linker . These interactions may act in a cooperative manner to destabilize autoinhibitory contacts between the GBD and VCA regions , resulting in allosteric acti - vation of N - WASP ( Fig . 6 ) . Phase actin SH3A SH3B SH3C SH2 SH3B SH3C SH2 SH3B SH3C SH2 Nck1 full - length Nck1 aa 61 - 377 Nck1 aa 106 - 377 A C B Alexa488 D Nck fl . Nck aa 61 - 377 Nck aa 106 - 377 G B D V C A P RR N - W A SP + actin , Arp2 / 3 , cofilin , profilin , capping protein lipid - coated glass bead p Y 3 - n e ph r i n pY N ck NTAD F E SH2 SH3 SH3 SH3 0 2000 4000 6000 8000 N ck f l . N ck aa 61 - 377 N ck aa 106 - 377 A l e x a568 - N ck [ A U ] 0 . 0 4 . 0 8 . 0 12 . 0 16 . 0 20 . 0 24 . 0 28 . 0 N ck f l . N c k aa 61 - 377 N ck aa 106 - 377 T a il Leng t h [ m ] * * * 0 400 800 1200 1600 2000 2400 N ck f l . N c k aa 61 - 377 N c k aa 106 - 377 A l e x a488 - a c t i n [ A U ] * * * Fig . 3 . An inter - SH3 linker in Nck promotes actin network assembly by N - WASP . ( A ) Experimental strategy for localization of Nck to membranes con - taining pY 3 - nephrin . ( B ) Nck constructs used in the deletion analysis . ( C ) Representative images of actin tails formed by Nck deletion constructs . Motility reactions contained 200 nM N - WASP and 100 nM Nck . ( Scale bar : 5 \u03bc m . ) ( D ) Integrated fluorescence intensity of Alexa488 actin tails . ( E ) Actin tail length . ( F ) Integrated fluorescence intensity of bead - localized Alexa568 Nck constructs . ( Mean \u00b1 SD ; n > 20 ; * * * P < 0 . 001 . ) fl . , Full length ; PRR , proline - rich region . E6440 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1510876112 Okrut et al . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . Discussion In response to membrane - localized signals , N - WASP binds and activates the Arp2 / 3 complex , promoting the assembly of branched actin networks and the physical movement and deformation of cellular membranes . In addition to the canonical activators , Cdc42 and PIP2 , membrane - localized SH3 adapter proteins have been implicated in N - WASP activation . It has remained unclear how SH3 adapters disrupt the intramolecular interactions that main - tain N - WASP in an autoinhibited state . Using purified components , we reconstituted N - WASP \u2013 dependent actin assembly on a lipid bilayer supported on silica microspheres , allowing side by side comparison of multiple SH3 adapters . We find that the SH2 / SH3 adapter Nck activates N - WASP more po - tently than all of the other SH3 adapters , including Grb2 , Crk - II , cortactin , CIP4 , and Tks4 . Although previous studies have focused on SH3 / polyproline interactions to explain N - WASP activation by Nck , we identify here an activation motif in the linker between the first two SH3 domains ( amino acids 67 \u2013 75 ) . This motif , which had no assigned function before this work , binds directly to the N - WASP GBD and competes with the autoinhibitory C helix in the VCA region , leading to allosteric activation of N - WASP . The inter - SH3AB activation motif is conserved among Nck orthologs , suggesting that the N - WASP GBD interaction and activation mechanism is also evolutionarily conserved . N - WASP activation by the inter - SH3 linker is reminiscent of the mechanism used by the bacterial virulence factors , EspF U and EspF , to nu - cleate actin assembly in host cells . The activation motif in EspF U and EspF has the same pattern of hydrophobic residues as the Nck motif ( Fig . 4 B ) ( \u03a6 - \u03a6 - x - x - \u03a6 - x - x - x - \u03a6 ; \u03a6 is any hydrophobic residue and x is any residue ) ( 39 \u2013 41 ) . Structural studies by mul - tidimensional NMR have shown that the EspF U peptide adopts a helical conformation when bound to the WASP GBD ( 40 ) . Likewise , our analysis of secondary chemical shifts strongly sug - gests that the Nck hydrophobic motif also adopts a helical con - formation with the side chains of Ile67 , Val68 , Leu71 , and Leu75 lying on one face of the helix ( Fig . S3 ) . Similar to linker segments in many other proteins ( 44 ) , the Nck inter - SH3AB linker is multifunctional . In addition to acti - vating N - WASP through GBD binding , the inter - SH3AB linker has been shown to bind intramolecularly to the SH3B domain ( 45 ) and mediate Nck homo - oligomerization and phase separa - tion ( 46 ) . The N - WASP C - helix motif is similarly multifunc - tional , binding in a mutually exclusive manner to G actin and the Arp2 / 3 complex ( 47 ) in addition to mediating autoinhibition by intramolecular GBD interactions . Our results , combined with previous studies , suggest that Nck can activate N - WASP by two complementary mechanisms . First , the Nck inter - SH3 linker can bind directly to the N - WASP GBD , NCK1 , HUMAN RKASIVKNLKDTLGIGK NCK1 , FISH RKASIVKNLKDTLGIGK NCK2 , HUMAN RKGSLVKNLKDTLGLGK NCK , CHICKEN KKGSLVKNLKDTLGLGK NCK , FLY - KPSLFDSIKKKVKKGS NCK , WORM - KESIVDKAKGTIKGLA N - WASP , C - helix - TSGIVGALMEVMQ - - - EspFu , helix - LPDVAQRLMQMLA - - - EspF , helix - LPPIAQALKDMLA - - - consensus \u03a6\u03a6xx\u03a6xxx\u03a6 SH3A SH3B SH3C SH2 A aa 2 - 60 115 - 165 190 - 252 282 - 377 61 71 81 91 101 SARKASIVKN LKDTLGIGKV KRKPSVPDSA SPADDSFVDP GERLY N - WASP activation motif Nck1 PAK1 GST - GBD N - WASP GST - GBD B S B S B 10075 50 37 25 Nck GST - GBD GST - GBD w t G S aa 67 - 77 4 A G S aa 67 - 105 Nck aa 1 - 270 D aa 61 - 270 aa 79 - 270 aa 106 - 270 C B S B S B S N - WASP GST - GBD 37 25 E B S B S B S N - WASP GST - GBD B S Nckaa 61 - 377 wt Nckaa 61 - 377 4A Phase Alexa - actin F Fig . 4 . The inter - SH3AB linker contains a conserved motif and binds directly to the N - WASP GBD . ( A ) Nck domain structure and sequence of the inter - SH3AB linker . ( B ) Sequence alignment comparing the Nck inter - SH3 linker from various organisms and previously known N - WASP GBD ligands . ( C ) Coomassie - stained gel of GST pull - down samples . Immobilized GST - GBD from N - WASP or PAK1 was used to pull down full - length Nck . ( D ) N - WASP GST - GBD was used to pull down the indicated Nck deletion constructs . ( E ) N - WASP GST - GBD was used to pull down Nck amino acids 1 \u2013 270 WT and mutants . Samples comprising 1 . 25 % of the unbound supernatant ( S ) and 12 . 5 % of the bound fraction ( B ) were separated by SDS / PAGE and stained with Coomassie . GS aa 67 \u2013 105 , substitution of the entire SH3AB linker with a Gly - Ser linker ; GS aa 67 \u2013 77 , substitution of the hydrophobic motif with a Gly - Ser linker . ( F ) Representative images of actin tails formed by Nck amino acids 61 \u2013 377 WT and 4A mutant . Motility reactions contained 200 nM N - WASP and 100 nM Nck . ( Scale bar : 5 \u03bc m . ) Okrut et al . PNAS | Published online November 9 , 2015 | E6441 B I O C H E M I S T RY P N A S P L U S D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . thereby releasing the critical VCA segment . We note that the affinity of this interaction ( K d = 33 \u00b1 8 \u03bc M ) is probably insufficient to activate autoinhibited N - WASP on its own ; cooperative binding of the linker , along with one or more SH3 domains , is likely re - quired to disrupt N - WASP \u2019 s autoinhibitory interactions . Second , Nck can promote N - WASP oligomerization . When N - WASP binds to a poly - SH3 adapter that is itself oligomerized by a third multivalent adapter , phase separation can occur , enforcing a high density of N - WASP molecules ( 38 , 48 ) . On recruitment to the membrane by pY 3 - nephrin / Nck complexes , N - WASP molecules can presumably equilibrate between auto - inhibited and activated states . At high local densities of pY 3 - nephrin and Nck , the density of activated N - WASP molecules bound to the Arp2 / 3 complex may be sufficient to nucleate actin assembly without GBD engagement by the Nck hydrophobic motif . This model may explain our observation that , at high NTAD densities , the Nck 4A mutant ( with a defective hydrophobic motif ) assembles actin comet tails similar to the WT ( Fig . S4 ) . By contrast , a functional hydro - phobic motif is essential at low NTAD densities or when the SH3A domain is deleted ( Fig . 4 F and Figs . S4 and S5 ) . In the cellular context , a similar dependency on the Nck hydrophobic motif may occur under conditions where the local density of Nck is below the critical concentration required for phase separation ( for example , when the balance of kinase and phosphatase activity is shifted to produce low phosphotyrosine levels on membrane - associated receptors that recruit Nck ) . Consistent with previous observations ( 48 ) , Nck and N - WASP phase separate in our hands when mixed together at micromolar concentrations . However , under the conditions of our bead motility assays ( 100 \u2013 250 nM Nck and 50 \u2013 200 nM N - WASP ) , we do not observe phase separation in solution . We also have not detected macroscopic phase separation on the lipid - coated beads in contrast to a recent study , in which macroscopic , phase - sep - arated domains containing pY 3 - nephrin , Nck , and N - WASP were observed on planar lipid bilayers ( 38 ) . Nevertheless , it is likely that membrane - associated Nck and N - WASP can form higher - order oligomers in our system , especially at high densities of NTAD ( and pY 3 - nephrin ) ; such oligomeric assemblies may be important for promoting localized actin nucleation ( Fig . 6 ) . The quantitative relationships between Nck / N - WASP signal - ing inputs on the one hand \u2014 tyrosine kinase / phosphatase activ - ities , phosphotyrosine valency , and phase separation \u2014 and actin 0 0 . 05 0 . 1 0 . 15 0 . 2 0 100 200 300 400 500 600 \u2206 \u03b4 ( HN , N ) Nck linker [ \u03bc M ] E Nck fl VCA 0 2 . 5 5 10 25 50 GST - GBD 50 37 25 20 VCA [ \u03bc M ] F C D - 2 - 1 0 1 2 3 4 5 6 7 8 A R K A S I V K N L K D T L G I G K V \u2206 \u03b4 ( C \u03b1 - C \u03b2 ) N ck aa 62 - 80 I V L L WASP - C - helix SGFKHVSHVGWDPQNGFDVNNLDPDLRSLFSRAGI N - WASP - Nck SNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGI Talos bb hhhhhhhhhh WASP - C - helix SEAQLTDAETSKLIYDFIEDQGGLEAVRQEMRRQ N - WASP - Nck SEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQ Talos hhhh hhhhhhhhhhhhh hhhhhhhhhh WASP - C - helix GGSGGSQSSEGLVGALMHVMQKRSRAIH N - WASP - Nck GGSGGSARKASIVKNLKDTLGIGKVKRK Talos hh hhhhhhhhhh 0 0 . 2 0 . 4 0 . 6 0 . 8 1 1 . 2 0 10 20 30 40 50 60 R e l a t i ve B a nd I n t e n s i t y VCA concentration [ \u03bc M ] Nck VCA 7 . 9 8 . 0 8 . 1 114 . . 0 114 . . 5 Nck linkerequiv 00 . 2 0 . 4 0 . 7 11 . 5 23458 \u03b4 1 H [ ppm ] \u03b4 15 N [ pp m ] \u03b4 15 N [ pp m ] GBD alone GBD + Nck aa 61 - 106 \u03b4 1 H [ ppm ] 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 9 . 0 9 . 5 110 115 120 125 130 A B Fig . 5 . NMR analysis reveals direct binding of the Nck inter - SH3 linker and the N - WASP GBD . ( A ) [ 15 N , 1 H ] - heteronuclear single - quantum coherence ( HSQC ) NMR spectra of 15 N - labeled N - WASP GBD acquired in the presence and absence of unlabeled Nck linker peptide ( amino acids 61 \u2013 106 ) . ( B ) NMR titration of 15 N - labeled GBD ( 65 \u03bc M ) with the Nck linker peptide . Chemical shift changes , along with the corresponding curve fit , are shown for a representative peak from the [ 15 N , 1 H ] - HSQC spectrum . A binding K d of 33 \u00b1 8 \u03bc M was determined by fitting titration curves to a total of 17 peaks . ( C ) Sequence alignment of a WASP - GBD C - helix construct ( 10 ) and the N - WASP GBD Nck linker fusion protein . The secondary structure of the WASP \u2013 C helix complex ( 1EJ5 . pdb ) is in - dicated . ( D ) Secondary structure prediction of the N - WASP Nck fusion protein by Talos + is shown . ( E ) Coomassie - stained gel from a competition pull - down experiment ( bound fraction ) . N - WASP GST - GBD was used to pull down Nck in the presence of increasing N - WASP VCA . ( F ) Integrated band intensities from the Coomassie - stained gel showing Nck and VCA bound to GST - GBD . E6442 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1510876112 Okrut et al . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . network assembly and force production on the other hand re - main to be elucidated . We anticipate that reconstituted signaling and actin assembly systems of even higher complexity will be essential for obtaining a complete mechanistic description of emergent phenomena at the intersection of signaling and cyto - skeletal dynamics . Materials and Methods ProteinExpressionandPurification . Expression plasmids , constructedfrompET vectors , were transformed into E . coli BL21 ( DE3 ) CodonPlus RIL ( construct details are in Table S1 ) . Cultures ( 2 \u2013 4 L ) were grown in LB at 18 \u00b0C overnight after induction with 0 . 5 mM isopropyl \u03b2 - D - 1 - thiogalactopyranoside . For isotope labeling , M9 minimal medium was supplemented with 13 C - glucose and / or 15 NH 4 Cl , and bacteria were induced for 6 h at 30 \u00b0C . Bacterial cell pellets were lysed using a microfluidizer ( Microfluidics ) in 20 mM Hepes , pH 7 . 5 , 500 mM NaCl , 5 \u2013 10 % glycerol , and 4 mM \u03b2 - mercapto - ethanol with protease inhibitor ( 4 mM Benzamidine or 1 mM di - isopropyl - fluorophosphate ) . For Nck and Grb2 , 30 mM arginine was added to the lysis buffer . Lysates were cleared ( 24 , 000 \u00d7 g for 40 min at 4 \u00b0C ) and incubated in batch with Ni - nitriloacetic acid - agarose ( Qiagen ) . The beads were washed with ATP buffer ( lysis buffer supplemented with 10 mM MgCl 2 , 2 mM ATP , 30 mM KCl ) and a high - salt buffer ( lysis buffer with 1 M NaCl ) , and the protein was eluted with 400 mM imidazole . Poly - His tags were cleaved from Nck , N - WASP ( amino acids 151 \u2013 501 ) , cofilin , and profilin by incubation with tobacco etch virus ( TEV ) protease overnight . Tks4 and N - WASP were further purified with a heparin column ( HiTrap ; GE Healthcare ) , eluting with 50 \u2013 1500 mM NaCl in storage buffer . N - WASP was stored in 20 mM Hepes , pH 7 . 5 , 300 mM NaCl , and 1 mM tris ( 2 - carboxyethyl ) phosphine ( TCEP ) . Tks4 was stored in 20 mM MES , pH 6 . 5 , 250 mMNaCl , and 5 % glycerol . Allother proteinswere furtherpurified by size exclusion chromatography on S75 or S200 columns ( GE Healthcare ) equilibrated with storage buffer . The following storage buffers were used : Grb2 and Nck constructs ( 20 mM Hepes , pH 7 . 5 , 500 mM NaCl , 0 . 5 mM TCEP , 10 % glycerol ) , CIP4 ( 20 mM Hepes , pH 7 . 5 , 150 mM NaCl ) , cortactin ( 50 mM Tris , pH 7 . 4 , 100 mM NaCl , 10 % glycerol ) , cofilin ( 20 mM Tris , pH 8 , 50 mM NaCl , 1 mM DTT ) , and profilin ( 20 mM Tris , pH 8 , 1 mM DTT , 1 mM EDTA ) . GST - tagged N - WASP GBD and covalently linked GBD \u2013 Nck linker complex were purified on glutathione - Sepharose 4B ( GE Healthcare ) . GST fusion protein was eluted with 30 mM glutathione ( GSH ) followed by dialysis in 20 mM Hepes , pH 7 . 5 , 100 mM NaCl , and 1 mM DTT . For NMR experiments , the GST tag was cleaved by incubation with TEV protease followed by size exclusion chromatography . To purify N - WASP VCA and Nck inter - SH3AB linker , cell pellets were lysed by incubation with 3 M guanidinium HCl overnight at room temperature ( RT ) . The cleared lysate was applied to a Ni - chelating column ( GE Healthcare ) and washed with 1 M NaCl . Proteins were eluted with 0 \u2013 400 mM imidazole followed by dialysis in storage buffer ( Nck linker : 20 mM Hepes , pH 7 . 5 , 200 mM NaCl ; VCA : 20 mM Hepes , pH 7 . 5 , 200 mM NaCl , 1 mM TCEP ) . The following proteins were purified as previously described : actin ( 49 ) , capping protein ( 50 ) , and Arp2 / 3 complex ( 51 ) , except that Arp2 / 3 complex was purified from frozen bovine thymus ( Pel - Freez Biologicals ) . Fluorescent Labeling . Unless stated otherwise , proteins ( 5 \u2013 50 \u03bc M ) were la - beled with 1 M eq Alexa568 maleimide ( Invitrogen ) on ice for 1 h . Crk - II was labeled overnight at 4 \u00b0C with 10 eq Alexa568 NHS ( Invitrogen ) . Unreacted dye was removed by gel filtration . F - actin was labeled for 6 h on ice with 2 eq Alexa488 - TFP ( Invitrogen ) ; unreacted dye was removed by pelleting the actin filaments followed by actin depolymerization . The extent of labeling ( percentage of dye - labeled protein ) was calculated by dividing the concen - tration of the protein - bound dye ( determined by measuring the fluores - cence emission intensity relative to a standard dilution series of the free dye ) by the protein concentration . Protein concentrations were calculated from the absorbance at 280 nm after subtracting the dye contribution . Preparation of Lipid - Coated Beads . Chloroform solutions of 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine , NTAD ( nickel salt ; Avanti Polar Lipids ) , and Atto390 - 1 , 2 - dioleoyl - sn - glycero - 3 - phosphoethanolamine ( Atto390 - DOPE ; ATTO - TEC ) were mixed in molar ratios of 94 : 5 : 1 , 96 . 5 : 2 . 5 : 1 , and 98 : 1 : 1 of 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine : NTAD : Atto390 - DOPE , evaporated in a stream of argon , and dried under high vacuum for at least 2 h . Lipids were resus - pended in vesiclebuffer ( 20 mMHepes , pH7 . 5 , 100 mMNaCl , 330 mMsucrose ) to a final concentration of 5 mM , sonicated for 20 s , subjected to five freeze \u2013 thaw cycles , and stored at \u2212 80 \u00b0C . A suspension of glass beads ( 3 . 5 \u03bc L ; 2 . 3 - \u03bc m diameter ; Total 2 . 8 \u00d7 10 7 ; Bangs Laboratories ) was mixed with 5 \u03bc L lipid suspension and diluted to a final volume of 45 \u03bc L in vesicle buffer ( 6 . 2 \u00d7 10 8 beads mL \u2212 1 ) . The suspension was sonicated ( 3 \u00d7 10 s ) in a bath sonicator ( Branson 2510 ) and incubated while rotating for 30 min at RT . The beads were pelleted by pulse centrifugation , washed two times with 200 \u03bc L vesicle buffer , resuspended in 45 \u03bc L vesicle buffer , and used within 8 h . Motility Assay with Different SH3 Adapters . Lipid - coated beads ( 6 . 1 \u00d7 10 6 beads mL \u2212 1 ) containing 1 % , 2 . 5 % , and 5 % NTAD were incubated with 0 . 25 \u03bc M His 6 - tagged SH3 adapter ( 20 % Alexa568 labeled ) , 0 . 05 \u03bc M N - WASP , and motility components [ 9 \u03bc M actin ( 5 % Alexa488 labeled ) , 0 . 075 \u03bc M Arp2 / 3 , 0 . 05 \u03bc M capping protein , 2 . 6 \u03bc M profilin , 3 . 5 \u03bc M cofilin ] in motility buffer ( 10 mM Hepes , pH 7 . 5 , 2 mM MgCl 2 , 50 mM KCl , 50 mM NaCl , 1 mg / mL BSA , 2 . 5 mM ATP , 5 mM TCEP , 1 . 3 mM Dabco , 20 mM ascorbic acid , 0 . 2 % methylcellulose ) . Samples were incubated for 15 min at RT while rotating . For quantitative image analysis , the reaction was diluted 1 : 1 in 3 % glutaraldehyde ( Ricca Chemical Company ) , spotted on a microscopy slide , covered with a coverslip , and imaged within 1 h . Motility Reactions Using pY 3 - nephrin and Nck . For each assay , lipid - coated beads ( 7 . 6 \u00d7 10 7 mL \u2212 1 ) were preincubated ( 15 min at RT ) with 0 . 5 \u03bc M His 8 - pY 3 - nephrin ( 38 ) in buffer containing 10 mM Hepes , pH 7 . 5 , 1 mg / mL BSA , and 50 mM KCl . Phosphonephrin - loaded beads were diluted eightfold in motility buffer containing 0 . 1 \u03bc M Nck ( 20 % Alexa568 labeled ) , 0 . 2 \u03bc M N - WASP , and actin motility components as described above . N - WASP and SH3 Adapter Density Measurements . To compare the surface densities of SH3 adapters or N - WASP , we quantified the Alexa568 signal on the lipid - coated beads . Indicated amounts of Alexa568 - labeled SH3 adapters or unlabeled SH3 adapter and Alexa568 N - WASP were incubated with lipid - coated beads in motility buffer for 15 min while rotating . The suspension ( \u223c 6 \u03bc L ) was transferred to a flow chamber that had been passivated with \u2013 poly - ( L - lysine ) - PEG ( 3 mg / mL ) ( 52 ) and imaged live as described below . Microscopy and Image Analysis . Epifluorescence and phase contrast images of fixed motility reactions were acquired with a 60 \u00d7 objective ( N . A . 1 . 4 ) on an Olympus IX70 Inverted Microscope equipped with a Coolsnap HQ CCD Camera ( Photometrics ) . Flat field - corrected images were analyzed using a CellProfiler Pipeline ( 53 ) to automatically locate beads , threshold images ( Background Global Method ) , and determine the integrated fluorescence intensity of Alexa488 actin associated with each bead . Live confocal images were acquired to quantify the density of N - WASP or SH3 adapters on lipid - coated beads . Images were obtained using a 100 \u00d7 objective ( N . A . 1 . 49 ) on a Nikon ECLIPSE Ti Inverted Microscope equipped with a Yokoyama CSU - X1 Spinning Disk , a Hamamatsu EM CCD 9100 \u2013 13 Camera , and 50 - mW lasers [ solid state , 405 nm ; diode - pumped solid - state ( DPSS ) , 561 nm ] . For density measurements , flat field - corrected images were analyzed for bead fluorescence using a Mathematica script . The script inactive G BD V P RR C A active membrane G BD V C A SH3A SH2 SH3B SH3C PxxP PxxP Nck pY N - WASP Fig . 6 . Proposed mechanism of N - WASP activation by Nck . In the auto - inhibited conformation , the N - WASP C helix binds intramolecularly to the GBD . Nck binds to tyrosine - phosphorylated receptors on the membrane and activates N - WASP by competitively displacing the C helix from the GBD with its inter - SH3AB linker . The released VCA segment is available to activate the Arp2 / 3 complex . Polyvalent interactions between the SH3 domains of Nck and the proline - rich region ( PRR ) of N - WASP can lead to higher - order olig - omer formation , increasing the local density of activated N - WASP molecules . Okrut et al . PNAS | Published online November 9 , 2015 | E6443 B I O C H E M I S T RY P N A S P L U S D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S A RC S - S E R I A L S on S e p t e m b e r 20 , 2023 fr o m I P a dd r e ss 205 . 175 . 106 . 245 . identifies bead positions based on the Atto390 - DOPE lipid fluorescence , quantifies bead fluorescence in the Alexa568 channel ( SH3 adapter or N - WASP ) , and subtracts the median background value of a circular region outside of the bead . P values were calculated using an unpaired , two - tailed Student \u2019 s t test . Pull - Down Assays . GST - tagged N - WASP GBD ( 1 . 5 nmol ; amino acids 196 \u2013 274 ) or PAK1 - GBD ( 1 . 5 nmol ; amino acids 67 \u2013 150 ; Cytoskeleton Inc . ) was immo - bilized on GSH Sepharose 4B ( 20 \u03bc L per sample ; GE Healthcare ) for 15 min at 4 \u00b0C . The beads were washed two times with Nck storage buffer . Nck con - structs ( 1 . 5 nmol total ) were added to the beads and incubated for 2 h at a final concentration of 5 \u03bc M . The beads were washed two times , and the supernatant as well as the bead samples were subjected to SDS / PAGE . The Coomassie - stained gels were analyzed using a Licor Gel Imaging System . For competitive pull - down assays , N - WASP VCA ( 0 \u2013 50 \u03bc M ) was added to the binding reaction . NMR Spectroscopy . All NMR chemical shift assignment experiments were performed at 298 K onan Agilent DD2 600 - MHz spectrometer equipped with a cold probe using NMRPipe for data processing ( 54 ) and NMRView for analysis ( 55 ) . Backbone chemical shift assignments were obtained using standard 3D 15 N - edited triple - resonance experiments including HNCACB , CBCA ( CO ) NH , and HNCO spectra ( 56 ) . 13 C - , 15 N - labeled N - WASP Nck fusion protein in 20 mM Hepes , pH 7 . 5 , 100 mM NaCl , 1 mM DTT , 1 mM EDTA , 0 . 01 % NaN 3 , and 5 % D 2 O was used at a concentration of 550 \u03bc M . NMR titration data were acquired at 300 K on a Bruker DRX500 spectrometer ( Topspin , version 1 . 3 ) equipped with a cryogenic probe . 15 N - labeled N - WASP GBD ( 65 \u03bc M ; in 20 mM Hepes , pH 7 . 5 , 200 mM NaCl , 1 mM DTT , 0 . 01 % NaN 3 , 5 % D 2 O ) was titrated with the Nck linker peptide ( 0 \u2013 520 \u03bc M ) . Titration curves from 17 peaks were fit using CCPN Analysis ( 57 ) . Weighted chemical shift differences were calculated using the following equation : \u0394\u03b4 = ( ( \u0394\u03b4 N \u00d7 0 . 2 ) 2 + \u0394\u03b4 H2 ) 1 / 2 . ACKNOWLEDGMENTS . 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( a - b ) Endocytic 7 route of SecGFP - GPI in AGS cells . Single confocal plane shows a 3 minute pulse of \u03b1 - GFP 8 Fab [ a - b : magenta in Merge ] at 37\u00b0C ( upper panel ) and 30\u00b0C ( lower panel ) along with TMR - 9 dextran ( a : green in Merge ) or A647 - Tf ( b : green in Merge ) in AGS cells transiently 10 transfected with SecGFP - GPI . Insets show a magnified view of the marked areas . ( c ) Plot 11 ( top ) showing quantification of the fraction of GFP - GPI endocytic vesicles containing fluid 12 or Tf . The number of cells is shown below the graph . ( d ) Schematic of pH pulsing analysis , 13 steps ( 1 - 3 ) used for identifying and quantifying the fluorescence spots associated with newly 14 formed endocytic vesicles in the sequential frames of the pH 5 montage . Step 1 - New spot 15 identification : Each spot ( green ) in i th frame is compared with the previous frame and is 16 considered new if no nearest neighbour is found by euclidean distance search within 5 pixels 17 ( 1 pixel = 84nm ) . Random ( red ) are generated randomly within the cell mask . Step 2 - Spot 18 mask ( green filled ) and background mask ( green dashed ) considered around the centroid the 19 new spot identified in the step - 1 [ also see pH 5 frame in the middle panel of ( Figure 1a ) ] . 20 The process is repeated for Random spot mask ( red filled ) and background mask ( red 21 dashed ) . Step 3 - schematic of the temporal profile of a new spot ( black ) and a random spot . 22 Multiple traces pooled from different spots and cells over different days was averaged and 23 shown in rest of the figures . The x - axis represents time and y - axis represents fold change in 24 intensity in the spot over the background . See S . I . for a detailed description . ( e ) AGS cells 25 were pre - treated with either pH 7 . 3 or pH 5 . 5 buffer for 5 minutes followed by 5 - minute fluid 26 uptake in pH 7 . 3 buffer . Data was pooled from 2 independent experiments and the number of 27 cells indicated in the graph . ( f ) Untreated AGS ( Control , ( i ) ) or LG186 - treated AGS ( ii ) were 28 incubated for 2 minutes at 37\u00b0C with 10mg / ml HRP as a fluid phase marker before 29 processing for electron microscopy . Endocytic structures close to the plasma membrane ( PM ) 30 are filled with the electron dense peroxidase precipitate . Control cells show a range of 31 endocytic structures including vesicular structures ( CCP / Cav & EE ) ( a pair of small 32 arrowheads ) and tubular / ring - shaped putative CLIC / GEECs ( large arrowhead ) but the drug - 33 treated cells show predominant labelling of vesicular profiles . Histogram shows mean 34 endocytic structures quantified per cell ( n = 5 ) . CCP / Cav represent vesicles derived from 35 clathrin mediate or caveolar endocytosis and EE represent early endosomes ( See S . I . ) . ( g ) 36 The histogram shows quantification of 5 - minute fluid - uptake in AGS cells when treated with 37 indicated concentrations of LG186 or DMSO . Data was pooled from 2 independent 38 experiments and the number of cells indicated in the graph . Scale bar , 5\u00b5m ( a - b ) , 4\u00b5m ( a - b , 39 inset ) , 1 \u00b5m ( f ) & 20\u00b5m ( e and g ) respectively . Error bars ( c ) represent s . e . m . and ( e - g ) s . d . 40 respectively . p - value < 0 . 001 ( * * ) 2 - sample student\u2019s T - test ( f ) and Mann - Whitney U test 41 ( e ) . 42 43 Supplementary Figure 2 : Characterization of the pH pulsing assay for visualizing 44 SecGFP - GPI endocytic vesicle formation . ( a - b ) Graphs show the average normalized 45 fluorescence intensity versus time traces for the recruitment of TagRFPt - CDC42 ( CDC42 46 All ) to all forming SecGFP - GPI endocytic sites ( a ) or endocytic sites that do not co - detect 47 TagRFPt - CDC42 ( CDC42 NoColoc ; 44 % ) and its corresponding random intensity trace ( n , 48 Table 1 ) . ( c - g ) Graphs show the average normalized fluorescence intensity versus time trace 49 for the recruitment of mCherry - dynamin to forming SecTfR endocytic sites [ c ; ( n = 21 50 SecTfR and 1448 random spots from 6 cells , 2 experiments ) , mCherry - clathrin to forming 51 SecGFP - GPI endocytic sites [ d ; n , Table 1 ] , mCherry - dynamin [ e ; n , Table 1 ] to forming 52 SecGFP - GPI endocytic sites , mCherry - ARF1 to forming SecGFP - GPI endocytic sites [ f ; ( n = 53 228 , as in Figure 1e ) ; Note the extended time axis ] , mCherryGBF1 to forming SecGFP - GPI 54 endocytic sites [ g ; ( n = 75 , as in Figure 1e ) ; Note the extended time axis ] , and their 55 corresponding random intensity trace . A representative montage depicted below ( c - e ) . 56 Arrowheads indicate the spot . The random traces were derived from randomly assigned spots 57 of the same radius as the endocytic regions , as detailed in S . I . Endocytic distribution at each 58 time point was compared to the random distribution by Mann - Whitney U test and the log 10 59 ( p ) [ log 10 ( 0 . 05 ) is - 1 . 3 and log 10 ( 0 . 001 ) is - 2 . 5 ] is plotted below each trace ( a , & c - g ) . Error 60 bars represent s . e . m . for ( a , & c - g ) . Scale bar , 1 . 5\u00b5m ( c - e ) . 61 62 Supplementary Figure 3 : RNAi screen reveals BAR domain proteins involved in CG 63 endocytosis . Representative images for data shown in Figure 2b . Scale bar is 20\u00b5m . 64 Supplementary Figure 4 : IRSp53 is involved in CG endocytosis . ( a ) Histogram ( top ) 65 showing quantification of mean 5 - minute normalized TfR uptake in IRSp53 WT cells when 66 treated with 10\u00b5M LG186 for 45 minutes along with the representative images ( bottom ) . 67 Data was pooled from 2 independent experiments and the number of cells is indicated in the 68 figure . ( b ) Histogram ( top ) shows mean number of endocytic structures per cell from the 69 electron microscope images ( below ) . Data pooled from 3 independent blocks with 5 cells 70 each . Untreated WT MEFs ( WT , i ) , IRSp53 null MEFs ( IRSp53 - / - , ii ) or LG186 - treated WT 71 MEFs ( LG186 , iii ) were incubated for 2 minutes at 37\u00b0C with 10mg / ml HRP as a fluid phase 72 marker before processing for electron microscopy . Endocytic structures close to the plasma 73 membrane ( PM ) are filled with the electron dense peroxidase precipitate . WT cells ( left ) 74 show a range of endocytic structures including vesicular structures ( double arrowheads ) and 75 tubular / ring - shaped putative CLIC / GEECs ( large arrowheads ) but the KO cells ( middle ) and 76 LG186 - treated ( right ) cells show predominant labelling of vesicular profiles . ( c ) Electron 77 micrographs of AGS cells co - transfected GFP - IRSp53 and GBP - Apex . ( i - ii ) The reaction 78 product is highly patched ( arrowheads ) on the plasma membrane ( PM ) along with zoomed 79 regions on the right . ( iii ) Double arrowheads indicate specific labelling within defined 80 microdomains of filopodia . ( d ) Plot ( top ) showing quantification of co - localizing IRSp53 81 with p21 ( ARP2 / 3 complex subunit ) using ImageJ plugin ( Van Steensel\u2019s CCF , See 82 Methods . ) when compared with its random . Representative ( bottom ) images of AGS cells co - 83 expressing mEmerald - p21 subunit with mCherry - IRSp53 , which were fixed and imaged with 84 TIRFM , with zoomed inset at top left corner . Data was pooled from 2 independent 85 experiments and the cell number is indicated below the graph . ( e ) ( 1a - c ) Wide - field images of 86 cells at 1x ( 1a ) , 4x ( 1b ) along with a representative confocal slice of a cell ( 4x , 1c ) . ( 2a - c ) 87 Inset from the 4x cell ( Supplementary Movie 5 ) depicting enrichment of IRSp53 ( magenta , 88 2b ) within filopodia ( CD44 , green , 2a ) along with the merge ( 2c ) . ( 3 - 6 ) Insets of CD44 89 ( green ) labelled invaginations showing recruitment of IRSp53 ( magenta ) at various stages . 90 Each example depicts from left to right CD44 , IRSp53 and Merge respectively . Scale bar , 91 20\u00b5m ( a ) , 1\u00b5m ( b - c ) , 5\u00b5m ( d ) , 20\u00b5m ( e , 1a - b ) , 5\u00b5m ( e , 1c ) , 1\u00b5m ( e , 2a - c & 3 - 6 ) 92 respectively . p - value < 0 . 01 ( * ) , and 0 . 001 ( * * ) by Mann - Whitney U Test ( d ) and 2 - sample 93 student\u2019s T - test ( b ) . 94 95 Supplementary Figure 5 : ARP2 / 3 is negatively regulated by PICK1 for CG endocytosis 96 independent of N - WASP . ( a ) Histogram ( left ) showing quantification of 5 - minute pulse 97 fluid - phase in AGS cells treated with either DMSO or 10\u00b5M SMIFH2 along with 98 representative images ( right ) . Data was pooled from 2 independent experiments and the cell 99 number is indicated in the figure . ( b ) Plot ( left ) showing quantification of co - localizing 100 PICK1 with ARP3 or ARF1 using ImageJ plugin ( Van Steensel\u2019s CCF , See Methods ) when 101 compared with its random . Data was pooled from 2 independent experiments and the number 102 the cell number is indicated below the graph . Representative ( right ) images of AGS cells co - 103 expressing GFP - ARF1 with TagRFP - PICK1 or GFP - PICK1 and mCherry - ARP3 which were 104 fixed and imaged with TIRFM , with zoomed inset at bottom right corner . ( c ) Histogram ( left ) 105 showing quantification of mean 5 - minute pulse fluid - phase in AGS cells overexpressing 106 either pIRES - PICK1 WT or PICK1 KK - EE mutant . Data was pooled from 2 independent 107 experiments and the number of cells is indicated in the figure . Error bar represents s . d . ( a , & 108 c ) Scale bar , 20\u00b5m ( a & c ) , 5\u00b5m ( b ) . p - value < 0 . 01 ( * ) , and 0 . 001 ( * * ) by Mann - Whitney U 109 Test ( a - c ) . 110 Supplementary Figure 6 : Fold change over time profiles of SecGFP - GPI spots that did 111 not show a co - detected X - FP spot . ( a - h ) Graphs show the average normalized fluorescence 112 intensity versus time traces for the recruitment of mCherry - ARF1 NoColoc ( a ) , mCherry - 113 GBF1 NoColoc ( b ) , mCherry - IRSp53 NoColoc ( c ) , mCherry - ARP3 NoColoc ( d ) , pRuby - 114 Lifeact NoColoc ( e ) , TagRFPt - PICK1 NoColoc ( f ) , mCherry - NWASP NoColoc ( g ) 115 compared to their respective random trace . The random traces were derived from randomly 116 assigned spots of the same radius as the endocytic regions , as detailed in S . I . They represent 117 the fraction of SecGFP - GPI that failed to show a co - detected the X - FP spots . For n see Table 118 1 and S . I . for further information . 119 Supplementary Methods and Materials 120 Chemicals and reagents : All reagents were purchased from Sigma unless otherwise 121 mentioned . TMR - Dextran ( 10kDa ) and Alexa TM dyes were purchased from Molecular Probes 122 ( Eugene , OR ) . Transfection was carried out using FuGENE6 reagent ( Promega , USA ) as per 123 the manufacturer\u2019s instructions . LG186 was synthesized in the laboratory of Dr . Ram A . 124 Vishwakarma ( Indian Institute of Integrative Medicine ( CSIR ) , Canal Road , Jammu 180001 , 125 India ) . Heat inactivated fetal bovine serum ( 16000044 ) . Hygromycin B ( 10687 - 010 ) was 126 obtained from Invitrogen . CK - 666 ( SML0006 - 5MG ) , SMIFH2 ( S4826 - 5MG ) , FSC231 127 ( 529531 - 10MG , Millipore ) , Puromycin ( P2233 ) . PI - PLC ( purified in - house , used at 128 50\u00b5g / ml ) . hTf was Iron loaded and purified in gel filtration 1 and was fluorescently labelled 129 with ( A 10235 ) , Alexa - 568 ( A 10238 ) or Alexa - 647 ( A 20173 ) as per manufacturer\u2019s 130 instructions . Acryloyl X \u2013 SE ( Life technologies : A20770 ) . Sodium acrylate ( 408220 , 131 Sigma ) . 132 Cell culture : FR - AGS ( adenogastric carcinoma cells stably transfected with FR - GPI ) 2 were 133 maintained in 10 % FBS HF12 ( HiMedia , India ) . S2R + TfR cells 2 , 3 were grown in 10 % FBS 134 Schneider\u2019s Drosophila Media ( 21720 - 024 , Gibco TM ) . IRSp53 - / - ( pBABE - puro empty ) and 135 IRSp53 - / - pBABE - puro - IRSp53WT mouse embryonic fibroblasts 4 were grown in 20 % FBS 136 DMEM ( 31053 - 028 , Gibco TM ) supplemented by with 1 \u00b5g / ml Puromycin . All media were 137 supplemented with L - Glutamine Penicillin Streptomycin Solution ( Sigma , G1146 - 100ml ) . 138 CK - 666 treatment was done for 90 minutes . SMIFH2 treatment was done for 60 - 90 minutes 139 until lamellipodia formation was observed . FSC231 treatment was done for 60 minutes . 140 LG186 treatment was done in serum free media for 45 minutes ( MEFs ) and 30 minutes ( FR - 141 AGS ) . 142 Antibodies : \u03b1 - hTfR monoclonal antibody was purified from mouse hybridoma , OKT9 5 143 ( National Centre for Cell Science , India , used at 1 : 100 ) , \u03b1 - PICK1 antibody ( PA1 - 073 , 144 Thermo Pierce , used at 1 : 50 ) . \u03b1 - mTfR antibody ( 553264 , Becton Dickinson , used at 1 : 50 ) 145 and Cy3 - Mov18 ( 5\u00b5g / mL ) 5 , 6 . Fab fragments of \u03b1 - GFP monoclonal antibodies generated 146 using papain digestion subsequently fluorescently labelled with Alexa - 647 ( used at 1 : 100 ) . \u03b1 - 147 GFP antibody ( ab290 , abcam , 1 : 200 ) . \u03b1 - CD44 antibody ( IM7 clone , 14 - 0441 - 82 , 148 eBiosciences , 1 : 150 ) . \u03b1 - IRSp53 ( HPA023310 , Sigma , 1 : 50 ) . 149 Plasmids : SecGFP - GPI was made by site - directed mutagenesis , F64L and S65T in ecliptic - 150 GPI 7 was a gift from Gero Miesenb\u04e7ck ( University of Oxford ) . pIRES - EGFP - PICK KK - EE 151 and pIRES - EGFP - N - WASP - CA domain were a gift from J . Hanley ( University of Bristol , 152 UK ) 8 . GFP - N - WASP - VCA and GFP - N - WASP\u0394VCA were a gift from Mike Way ( Francis 153 Crick Institute ) . ARF1 - mCherry was a gift from Paul Melan\u00e7on 9 ( University of Alberta ) . 154 GBF1 - mCherry was a gift from Catherine Jackson ( Institut Jacques Monod ) 10 . pRuby - 155 Lifeact was a gift from Roland Wedlich - Soeldner ( Max Planck Institute of Biochemistry ) 11 . 156 IRSp53 - mCherry , SecGFP - TfR 12 , psPAX2 and pMD2 . G were gifts from Dr Marcus J . 157 Taylor ( UCSF / NCBS ) . GFP - PICK1 and TagRFPt - PICK1 was a gift from Harvey McMahon 158 ( Medical Research Council , UK ) . Tag - RFPt - CDC42 was made in the laboratory by sub - 159 cloning GFP - CDC42 into a Tag - RFPt vector . Tag - RFPt vector was purchased from Evrogen 160 ( Catalogue # FP141 ) . GFP - IRSp53 and mutants were generated in the laboratory of Giorgio 161 Scita ( IFOM ) . Dyn2 - pmCherryN1 ( Addgene plasmid # 27689 ) , CLC - pmCherryC1 ( Addgene 162 plasmid # 27680 ) and Arp3 - pmCherryC1 ( Addgene plasmid # 27682 ) , mCherry - WASP - N - 18 163 ( Addgene plasmid # p55164 ) . GFP - binding peptide conjugated to APEX2 ( Addgene plasmid 164 # 67651 ) . shRNA against hPICK1 ( V3LHS _ 347038 , RHS4531 - EG9463 , 165 5\u2019TTCTTCAACACAATGTCCA3\u2019 ) and GIPZ Non - silencing Lentiviral shRNA Control 166 ( RHS4346 ) were purchased from Open Biosystems . 167 Generation of lentiviral stable lines : To generate GFP - IRSp53 and its different mutant 168 addback stable lines in IRSp53 - / - mouse embryonic fibroblasts and shRNA stable line for 169 FR - AGS , second generation lentiviral packaging systems was used following the protocol 170 from Trono lab 13 . HEK293T cells was used for virus generation and PEI ( polyethylenimine ) 171 was used for transfection . The ratio of DNA to PEI used for transfection is gene of interest : 172 packaging ( psPAX2 ) : envelope ( pMD2 . G ) in 1200ng : 1200ng : 1200ng for 3 wells of a 6 - well 173 plate . This DNA mix is added to serum free DMEM ( 327 \u00b5L / well ) and PEI ( 18 . 8 \u00b5L / well ) 174 and the transfection mix is incubated at room temperature for 20 minutes and added to the 175 cells . PEI is used at 1mg / mL and is heated to 70\u2103 for 10 minutes before transfection . 176 Viruses were collected after 2 - 3 days and syringe filtered using 200 \u00b5m filter and added to 177 60 % confluent culture . Typically after 3 - 4 days of infection gene expression was observed . 178 A similar procedure was followed to generate stable shRNA lines by in FR - AGS . 179 Immunostaining : Antibodies were tested for their specificity by western blotting if it was 180 suitable . Additionally expression was verified with published reports and was compared with 181 the expression of the fluorescently tagged version of the gene . Cells were fixed using 4 % 182 paraformaldehyde for 20 minutes at room temperature . Cells were permeablised with 0 . 1 % 183 TritonX at for 10 minutes followed by blocking for 1 hour using 2mg / mL BSA in PBS . \u03b1 - 184 PICK1 was diluted in blocking buffer ( 1 : 50 ) and added to the cells overnight at 4\u00b0C , 185 followed by A647 - Goat - \u03b1 - Rabbit for 1 hour at room temperature . 186 Image Analysis 187 pH pulsing assay : A semi - automated analysis was developed on MATLAB ( M . S . ) to identify 188 newly formed endosomes in the pH pulsing assay and trace their intensity over time in pH 7 , 189 pH 5 and RFP channels . The following steps ( a - g ) describe the procedure used : 190 a ) Identification of new endocytic structures : Two consecutive pH 5 frames were segmented 191 using MATLAB based tracking software which segments using sub - pixel localisation of 192 particles using radial symmetry centre 14 . The x - y coordinate of each particle in the i th 193 frame was compared with the rest of the particles in the i - 1 frame and the nearest 194 neighbour was located . If the nearest neighbour for a particle in the i th frame was found in 195 the i - 1 frame within 5 - pixel radius of its current co - ordinate , then that particle was 196 rejected . If the nearest neighbour was not found within a 5 - pixel radius of its current co - 197 ordinate and it stayed on for the next frame , then that particle was considered \u2018new\u2019 . 198 b ) Intensity calculation and bleach correction : A mask of radius 3 pixels ( 252 nm ) grown 199 from the centroid of the new spot was used for all the calculations . For the local 200 background , an annulus of radius 6 - 8 pixels ( 8 - 10 pixels for PICK1 ) was taken from the 201 centroid of the new spot . The average intensity of a spot is normalised to the average 202 intensity of the local annulus . ( mean intensity of spot - 20th percentile of local 203 background ) / ( mean intensity of local background - 20 percentile of local background ) 204 ( \u2211\ud835\udc3c \ud835\udc60 \ud835\udc5b \ud835\udc60 \u2212 20\ud835\udc61\u210e \ud835\udc5d\ud835\udc52\ud835\udc5f\ud835\udc50\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc56\ud835\udc59\ud835\udc52 \ud835\udc5c\ud835\udc53 \ud835\udc3c \ud835\udc4f ) ( \u2211\ud835\udc3c \ud835\udc4f \ud835\udc5b \ud835\udc4f \u2212 20\ud835\udc61\u210e \ud835\udc5d\ud835\udc52\ud835\udc5f\ud835\udc50\ud835\udc52\ud835\udc5b\ud835\udc61\ud835\udc56\ud835\udc59\ud835\udc52 \ud835\udc5c\ud835\udc53 \ud835\udc3c \ud835\udc4f ) . \u2044 Each spot was 205 considered if its average intensity was at least 20 % higher than the local background to 206 throw out low signal to noise spots . Additionally , we demanded that in the subsequent 207 frame the average intensity of the spot should be within 10 % so rule out a spot coming 208 into the TIRF plane , instead of it being recently pinched endocytic vesicle . A spot\u2019s 209 intensity will increase as it approaches the plasma membrane , while a recently pinched 210 endocytic vesicle will not show a rise in the intensity . These newly identified structures in 211 the TIRF plane were used as fiducial markers of the event . 212 c ) Random spots generation : Arbitrary regions using the masks defined in step ( b ) within 213 the cell boundary was chosen . The intensity of such spots is henceforth called \u201cRandom\u201d 214 was calculated as discussed in Step ( b ) and was used to compare the behaviour of X - FP at 215 the new SecGFP - GPI spots vs . random locations in the cell . 216 d ) False positive removal : In addition to the criteria mentioned in Step ( b ) , separately , 217 montages of each event depicting frames from - 18s to + 18s were generated and used to 218 manually verify the appearance of a new event . In addition to it , a spot can be mistakenly 219 identified as new due to the sudden lateral movement into the frame or growth of a sub - 220 threshold spot , appearing in the TIRF plane from the inside of the cell . These events are 221 screened out by manually verifying the absence of such occurrences in the frames prior to 222 0s in the montages . 223 e ) Manual classification of the X - FP spots : In addition to removing the false positives , the 224 montages generated at the Step ( d ) underwent manual check to classify the new SecGFP - 225 GPI spots into two groups based on whether X - FP co - detection was observed or not . One 226 population exhibited co - localisation of X - FP with SecGFP - GPI during the time window 227 of - 18 to + 18s for at least 1 frame , while the second failed to register any co - localization . 228 We called them X - FP Coloc ( Co - detection of X - FP and SecGFP - GPI ) and X - FP NoColc 229 ( the remainder ) . The X - FP NoColoc profile was comparable to Random ( Supplementary 230 Fig . 2b and 6 ) indicative of lack of recruitment in that fraction . This was supported by X - 231 FP All and X - FP Coloc having similar profiles for most CG pathway molecules 232 ( Compare Supplementary Fig . 2a and Fig . 1d ) . Although the fraction of X - FP NoColc was 233 variable , we observed that for molecules that were involved in CG endocytosis , it was 234 typically between 20 - 40 % . On the other hand , for the non - CG pathway molecules like 235 Clathrin , Dynamin and N - WASP it was around 60 - 70 % . Thus , the endocytic sites 236 detected by our assay consisted of two populations wherein one fraction exhibited an 237 accumulation of X - FP while the second fraction failed to show a discernable 238 accumulation . As the removal of the events that did not coincide with the presence of X - 239 FP did not alter their recruitment profile , they were discarded from further analysis . 240 Despite removing , the false positive via both automatic and manual methods there exists a 241 fraction of SecGFP - GPI endocytic sites that fails to associate with X - FP . The reasons for 242 not detecting X - FP at every endocytic event is both a function of both the signal and noise 243 in the data , and it reflects a genuine lack of recruitment at some endocytic events . 244 The number of SecGFP - GPI spots obtained for every molecule , the fraction of X - FP that 245 were associated with the SecGFP - GPI spots , the number of random spots along with the 246 number of cells and experiments is detailed in Table 1 . 247 f ) Plotting : Intensity , corrected for bleaching and normalised , was plotted against time for 248 each event and then averaged across all spots . The average data was compared to average 249 intensity obtained from multiple randomly picked regions from inside the mask of the 250 cell . The random data points ranged from 1800 to 5000 . Y - axis represents fold change 251 over the local background . All the pH pulsing trace used circular mask of radius 3 pixels 252 ( 250 nm ) whose mean intensity was normalized to the local background mask donut of 253 size 6 - 8 pixels ( 470 - 672 nm ) except for IRSp53 ( additional masks of circular radius 2 254 pixels , ( 168 nm ) , donut 3 - 5 pixels ( 250 - 420 nm normalized to background donut 6 - 8 255 pixels ( 470 - 672 nm ) ) and PICK1 which was normalized to background donut 8 - 10 pixels 256 ( 672 - 840 nm ) . 257 g ) Correlation analysis : The average intensity trace over time between two molecules was 258 compared by using MATLAB function called \u2018corrcoef\u2019 that calculates Pearson 259 correlation coefficient wherein the p - value is calculated by t - statistic . 260 Microscope : For population - based endocytic assays quantification of uptake of endocytic 261 tracers , images were obtained using either low magnification objective . For pH pulsing assay 262 the imaging was performed in a TIRF setup with custom designed chamber to maintain 263 constant temperature . The imaging was done at 30\u00b0C . For residence time imaging the 264 imaging was done on a TIRF setup at 37\u00b0C . Confocal imaging was done with spinning disk 265 setup . 266 A detailed list of microscopes used for this manuscript is below . 267 1 . Nikon TE300 - Objectives used in this setup were 20x 0 . 75 NA , 40x 0 . 65 NA . The 268 images collected using an EMCCD camera Ixon ( Andor Technologies ) using \u00b5Manager 269 15 . This microscope was utilized for population level endocytic assay imaging . 270 2 . Nikon TE 2000 equipped with TIRF - Objective used in this setup were 20x 0 . 75 NA , 40x 271 0 . 65 NA and 100x 1 . 49 NA oil . The camera in this setup is CCD cascade camera 272 ( Photometrics Inc . , USA ) . Images were acquired using Metamorph TM and \u00b5Manager . This 273 microscope was utilized for pH pulsing assays , residence time , TIRF based co - 274 localization and population level endocytic assay imaging . 275 3 . Nikon Eclipse Ti equipped with TIRF setup fed by Agilent laser combiner MCL400 276 ( Agilent technologies ) . Laser lines used were 488 and 561 along with epi - fluorescence 277 lamp . Objectives used in this setup were 20x 0 . 75 NA , 40x NA and 100x 1 . 49 NA 278 objective with EMCCD camera ( Photometrics Inc . , USA ) . Image acquisition was done 279 with \u00b5Manager . This microscope was utilized for residence time , TIRF based co - 280 localization and population level endocytic assay imaging . 281 4 . Nikon Eclipse Ti equipped with confocal spinning disk unit ( Yokogawa CSU - 22 scan 282 head ) . Images were collected with 100x 1 . 4 NA oil Nikon objective with an EMCCD 283 camera ( Andor ixon + 897 ) . Images were acquired using Andor iQ2 with Python scripting . 284 This microscope was utilized for confocal imaging . 285 5 . Perkin Elmer equipped with confocal spinning disk unit ( Yokogawa CSU - X1 scan head ) . 286 Images were collected with 100x 1 . 4 NA oil Olympus objective with Hamamatsu 287 IMAGE EM X2 EMCCD camera . Images were acquired using Volocity software . This 288 microscope was utilized for confocal imaging . 289 Supplementary References 290 1 . Mayor , S . , Presley , J . F . & Maxfield , F . R . Sorting of Membrane - Components From 291 Endosomes and Subsequent Recycling To the Cell - Surface Occurs By a Bulk Flow 292 Process . J . Cell Biol . 121 , 1257 \u2013 1269 ( 1993 ) . 293 2 . Gupta , G . D . et al . Population distribution analyses reveal a hierarchy of molecular 294 players underlying parallel endocytic pathways . PLoS One 9 , ( 2014 ) . 295 3 . Gupta , G . D . et al . Analysis of endocytic pathways in Drosophila cells reveals a 296 conserved role for GBF1 in internalization via GEECs . PLoS One 4 , ( 2009 ) . 297 4 . Disanza , A . et al . CDC42 switches IRSp53 from inhibition of actin growth to 298 elongation by clustering of VASP . 2735 \u2013 2750 ( 2013 ) . doi : 10 . 1038 / emboj . 2013 . 208 299 5 . Kumari , S . & Mayor , S . ARF1 is directly involved in dynamin - independent 300 endocytosis . Nat . Cell Biol . 10 , 30 \u2013 41 ( 2008 ) . 301 6 . Coney , L . R . et al . Cloning of a tumor - associated antigen : MOv18 and MOv19 302 antibodies recognize a folate - binding protein . Cancer Res . 51 , 6125 \u2013 6132 ( 1991 ) . 303 7 . Miesenb\u00f6ck , G . , De Angelis , D . A . & Rothman , J . E . Visualizing secretion and 304 synaptic transmission with pH - sensitive green fluorescent proteins . Nature 394 , 192 \u2013 5 305 ( 1998 ) . 306 8 . Rocca , D . L . , Martin , S . , Jenkins , E . L . & Hanley , J . G . Inhibition of Arp2 / 3 - mediated 307 actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor 308 endocytosis . Nat . Cell Biol . 10 , 259 \u2013 71 ( 2008 ) . 309 9 . Chun , J . , Shapovalova , Z . , Dejgaard , S . Y . , Presley , J . F . & Melan\u00e7on , P . 310 Characterization of class I and II ADP - ribosylation factors ( Arfs ) in live cells : GDP - 311 bound class II Arfs associate with the ER - Golgi intermediate compartment 312 independently of GBF1 . Mol . Biol . Cell 19 , 3488 \u2013 500 ( 2008 ) . 313 10 . Bouvet , S . , Golinelli - Cohen , M . - P . , Contremoulins , V . & Jackson , C . L . Targeting of 314 the Arf - GEF GBF1 to lipid droplets and Golgi membranes . J . Cell Sci . 126 , 4794 \u2013 805 315 ( 2013 ) . 316 11 . Riedl , J . et al . Lifeact : a versatile marker to visualize F - actin . Nat . Methods 5 , 605 \u2013 7 317 ( 2008 ) . 318 12 . Taylor , M . J . , Perrais , D . & Merrifield , C . J . A high precision survey of the molecular 319 dynamics of mammalian clathrin - mediated endocytosis . PLoS Biol . 9 , e1000604 320 ( 2011 ) . 321 13 . Barde , I . , Salmon , P . & Trono , D . Production and titration of lentiviral vectors . Curr . 322 Protoc . Neurosci . 1 \u2013 23 ( 2010 ) . doi : 10 . 1002 / 0471142301 . ns0100s37 323 14 . Parthasarathy , R . Rapid , accurate particle tracking by calculation of radial symmetry 324 centers . Nat . Methods 9 , 724 \u2013 726 ( 2012 ) . 325 15 . Stuurman , N . , Edelstein , A . D . , Amodaj , N . , Hoover , K . H . & Ronald , D . Computer 326 Control of Microscopes using \u03bc Manager . 1 \u2013 22 ( 2011 ) . 327 doi : 10 . 1002 / 0471142727 . mb1420s92 . Computer 328", + "messa2014epsin": "elifesciences . org Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 1 of 25 Epsin deficiency impairs endocytosis by stalling the actin - dependent invagination of endocytic clathrin - coated pits Mirko Messa 1 , Rub\u00e9n Fern\u00e1ndez - Busnadiego 1\u2020 , Elizabeth Wen Sun 1 , Hong Chen 1\u2021 , Heather Czapla 1 , Kristie Wrasman 2 , Yumei Wu 1 , Genevieve Ko 1 , Theodora Ross 3 , Beverly Wendland 2 , Pietro De Camilli 1 * 1 Program in Cellular Neuroscience , Neurodegeneration and Repair , Department of Cell Biology , Howard Hughes Medical Institute , Yale University School of Medicine , New Haven , United States ; 2 Department of Biology , Johns Hopkins University , Baltimore , United States ; 3 Department of Internal Medicine , UT Southwestern Medical Center , Dallas , United States Abstract Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function ( s ) in clathrin coat dynamics remain ( s ) elusive . To elucidate such function ( s ) , we generated embryonic fibroblasts from conditional epsin triple KO mice . Triple KO cells displayed a dramatic cell division defect . Additionally , a robust impairment in clathrin - mediated endocytosis was observed , with an accumulation of early and U - shaped pits . This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton , which we confirmed in a cell - free assay of endocytosis . Our results indicate that a key evolutionary conserved function of epsin , in addition to other roles that include , as we show here , a low affinity interaction with SNAREs , is to help generate the force that leads to invagination and then fission of clathrin - coated pits . DOI : 10 . 7554 / eLife . 03311 . 001 Introduction Clathrin - mediated endocytosis involves a complex set of factors besides clathrin itself and the classical clathrin adaptors . These factors help coordinate nucleation of the clathrin coat with cargo selection , membrane invagination and fission ( Schmid , 1997 ; Slepnev and De Camilli , 2000 ; Kaksonen et al . , 2003 ; Merrifield et al . , 2005 ; Ferguson and De Camilli , 2012 ) . An important player in these pro - cesses is epsin , the collective name for a family of evolutionarily conserved clathrin - associated pro - teins ( Chen et al . , 1998 ; Wendland , 1999 , 2002 ; Ford et al . , 2002 ; Chen and De Camilli , 2005 ) , which in mammals is represented by three isoforms : epsin 1 , 2 , and 3 ( encoded by the Epn1 , Epn2 , and Epn3 genes , respectively [ Ko et al . , 2010 ] ) . Epsin was identified as a major interactor of Eps15 ( Chen et al . , 1998 ) , another clathrin coat associated protein . It comprises a membrane binding N - terminal ENTH ( Epsin N - Terminal Homology ) domain , which is followed by ubiquitin - interacting motifs ( UIMs [ Polo et al . , 2002 ] ) and a long sequence ( tail ) predicted to be primarily unfolded and flexible ( Wendland , 2002 ) . The core of the ENTH domain is preceded by a short sequence that is unfolded in solution but folds into an amphipathic \u03b1 - helix upon binding to PI ( 4 , 5 ) P 2 . The hydrophobic portion of the helix partially penetrates the bilayer , thus conferring membrane curvature generation and sensing properties to the protein ( Itoh et al . , 2001 ; Ford et al . , 2002 ) . Epsin ' s disordered tail binds compo - nents of the clathrin coat via multiple short amino acid motifs : \u2018clathrin boxes\u2019 bind clathrin , DPW / F motifs bind the appendage domain of AP - 2 , and NPF motifs bind the EH domains of Eps15 and intersectin ( Chen et al . , 1998 ; Rosenthal et al . , 1999 ; Drake , 2000 ; Shih et al . , 2002 ; Overstreet et al . , 2003 ) . * For correspondence : pietro . decamilli @ yale . edu Present address : \u2020 Max - Planck - Institut f\u00fcr Biochemie , Martinsried , Germany ; \u2021 Cardiovascular Biology Research Program , Oklahoma Medical Research Foundation , Oklahoma City , United States Competing interests : The authors declare that no competing interests exist . Funding : See page 21 Received : 07 May 2014 Accepted : 12 August 2014 Published : 13 August 2014 Reviewing editor : Suzanne R Pfeffer , Stanford University , United States Copyright Messa et al . This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited . RESEARCH ARTICLE Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 2 of 25 Research article As epsin binds ubiquitin and genetically interacts with enzymes of ubiquitin metabolism ( Cadavid et al . , 2000 ; Chen et al . , 2002 ; Polo et al . , 2002 ; Shih et al . , 2002 ; Chen et al . , 2003 ; Sigismund et al . , 2005 ) , it was proposed to function as a clathrin adaptor for ubiquitinated cargo . Strong evidence for such a role came from the demonstration of Notch signaling defects in epsin ( liquid facets ) mutant flies , as Notch signaling is critically dependent upon ubiquitin - dependent endocytosis of Notch ligands ( Overstreet et al . , 2003 ; Xie et al . , 2012 ) . However , other findings pointed to a general house - keeping role of epsin in clathrin - mediated endocytosis . Absence of the two epsins ( Ent1 and Ent2 ) in yeast is lethal , while hypomorphic Ent1 or Ent2 mutations result in defects in endocytosis and actin dynamics ( Wendland , 1999 ; Aguilar et al . , 2003 ; Skruzny et al . , 2012 ) . Impairments in clathrin and actin function were also observed in epsin null Dictyostelium mutants ( Brady et al . , 2008 ; 2010 ) . In both these unicellular organisms , epsin functions in close cooperation with Sla2 / Hip1R , another evolu - tionarily conserved clathrin accessory factor ( Brady et al . , 2008 ; 2010 ; Skruzny et al . , 2012 ) . However , a link between epsin and Hip1R in metazoan cells has not been reported . Hip1 family members ( Hip1 and Hip1R in mammals ) comprise an N - terminal ANTH domain fol - lowed by unfolded regions that bracket a coiled - coil region and a C - terminal THATCH ( talin - HIP1 / R / Sla2p actin - tethering C - terminal homology ) domain ( Engqvist - Goldstein et al . , 1999 ; Wilbur et al . , 2008 ; Skruzny et al . , 2012 ) . The coiled - coil region can homo - heterodimerize and also binds clathrin light chain ( Engqvist - Goldstein et al . , 2001 ; Metzler et al . , 2001 ; Legendre - Guillemin et al . , 2002 ; Gottfried et al . , 2010 ) . The THATCH domain is an actin - binding module ( Yang et al . , 1999 ; Engqvist - Goldstein et al . , 2001 ; Brett et al . , 2006 ; Wilbur et al . , 2008 ) . Accordingly , Sla2 / Hip1R binds actin and is thought to function as a major link between the clathrin coat and actin . Studies in yeast have additionally shown that the ENTH domain of epsin and the ANTH domain of Sla2 interact with each other , and the two proteins function together in providing a link between the endocytic coat and the actin cytoskeleton ( Skruzny et al . , 2012 ) . eLife digest Clathrin - dependent endocytosis is one of the mechanisms used by cells to internalize specific proteins ( cargo ) from their surface . First , the cargo interacts with adaptor proteins that help cluster them in the cell ' s outer membrane , called the plasma membrane . This causes the protein clathrin to assemble into a lattice at the cytosolic side of the plasma membrane and deform the membrane into a pit . The pit grows deeper over time as more clathrin molecules assemble , eventually resulting in a deeply invaginated clathrin - coated pit that encloses the cargo to be taken up by the cell . The clathrin - coated pit then pinches off inside the cell in a process called fission to form a bubble - like structure called a vesicle , which transports the molecule to its destination . The deep invagination of clathrin - coated pits that leads to fission is assisted by actin , a protein that assembles into filaments that are suggested to generate the forces needed for this process . Many other factors are also involved . One of them is epsin , the collective name for a family of three very similar proteins in mammalian cells . Epsin binds to several other proteins implicated in clathrin - dependent endocytosis , including clathrin itself , and to plasma membrane proteins specifically \u2018tagged\u2019 for internalization . In addition , a portion of the epsin molecule can insert into the plasma membrane and help it to curve , which is important for forming the invaginated pit . However , due to the number of possible functions epsin could perform , its main role has remained elusive . Messa et al . created mouse cells that lack all three epsin proteins . Although these cells can form clathrin - coated pits , they struggle to develop into vesicles . The normal linking of the actin filaments to the clathrin coat does not occur , and another protein called Hip1R that also participates in clathrin - mediated endocytosis and links clathrin to actin , no longer accumulates at the clathrin - coated pits . Messa et al . also find that epsins can bind directly to actin . Overall , these results suggest that a main role of epsin is to help actin interact with the clathrin - coated pits and generate the force required for a pit to develop into a vesicle . However , epsin also performs many other roles , including recruiting a membrane protein ( a so - called SNARE ) that directs the fate of the vesicle to the clathrin - coated pit . Additionally , Messa et al . find that cells lacking all three epsins have problems dividing correctly . More research is required to establish whether this effect is also due to epsin ' s interaction with the cell ' s actin cytoskeleton . DOI : 10 . 7554 / eLife . 03311 . 002 Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 3 of 25 Research article In addition to roles of epsin mediated by protein \u2013 protein interactions , membrane remodeling prop - erties resulting from the amphipathic helix at the N - terminus of its ENTH domain have been implicated in the clathrin - dependent endocytic reaction . In vitro studies showed that this helix confers , upon the ENTH domain , the property to induce bilayer curvature and even to fragment bilayer tubules into vesicles , thus pointing to a potential role of the epsin in fission ( Itoh et al . , 2001 ; Ford et al . , 2002 ; Boucrot et al . , 2012 ) . Surprisingly , in view of this evidence for an important housekeeping role of epsin in endocytosis , the germline knockout ( KO ) of the mouse Epn1 and Epn2 genes that encode the two major ubiqui - tously expressed mammalian epsins , epsin 1 and 2 , did not block the early embryonic development ( Chen et al . , 2009 ) . Arrest of embryonic development occurred only at E9 . 5 \u2013 E10 , with a pattern sug - gestive of impaired Notch signaling , while no obvious defects in clathrin - mediated endocytosis were observed in fibroblasts derived from these embryos ( Chen et al . , 2009 ) . Moreover , studies of epsin 1 and 2 conditional double KO endothelial cells revealed a selective defect in the internalization of ubiq - uitinated VEGF receptor ( Pasula et al . , 2012 ) . However , a recent study based on RNAi - mediated knock - down ( KD ) in fibroblastic cells reported that the KD of all the three epsins produces a global impairment of clathrin - mediated endocytosis , which was attributed to a defect of the fission reaction ( Boucrot et al . , 2012 ) . The goal of the present study was to provide conclusive evidence about the function ( s ) and sites of action of epsin in endocytosis using a gene KO strategy to completely eliminate all epsins . Our results , which capitalize on triple KO ( TKO ) cells generated from conditional epsin TKO mice , show that epsin provides a link between the clathrin coat and actin and is needed for the transition of pits from a shallow to a deeply invaginated state . As in unicellular organisms , epsin acts in concert with Hip1R but with differences from yeast to mammals . An additional function of epsin is a low affinity interaction of its ENTH domain with synaptobrevin 2 / VAMP2 ( Syb2 ) that may help ensure the presence of a vesic - ular SNARE in the budding vesicle . Results Generation of conditional epsin triple knockout cells As the germline deletion of even only two Epn genes results in embryonic lethality , a conditional approach was used to generate Epn1 , Epn2 , and Epn3 triple KO cells . Towards this goal , Epn1 loxP / loxP ( Figure 1\u2014figure supplement 1A , see also [ Pasula et al . , 2012 ] ) mice were crossed with Epn2 ( Chen et al . , 2009 ) and Epn3 KO ( Ko et al . , 2010 ) mice to generate Epn1 loxP / loxP ; Epn2 \u2212 / \u2212 ; Epn3 \u2212 / \u2212 animals , which were viable and fertile with no obvious pathological phenotypes . These mice were subsequently interbred with mice transgenic for 4 - hydroxy - tamoxifen ( OHT ) - inducible Cre recombinase [ Cre - ER , Badea et al . , 2003 ] to obtain Epn1 loxP / loxP ; Epn2 \u2212 / \u2212 ; Epn3 \u2212 / \u2212 ; Cre - ER + / 0 animals . These mice did not exhibit obvious defects either . Conditional epsin TKO mouse embryonic fibroblasts ( MEFs ) were derived from these animals via treatment with OHT , whose action was confirmed by the translocation of Cre into the nucleus ( Figure 1\u2014figure supplement 1B ) , and typically examined after 7 days of OHT treatment ( after 9 days of treatment cell death started to occur ) . Treated MEFs derived from the same litters but harboring a wild - type ( WT ) epsin 1 allele did not exhibit any obvious differences from OHT - treated WT fibroblasts in terms of proliferation , endocytosis and actin organization . In the experiments described below OHT - treated WT MEFs were used as controls . Immunoblotting with epsin isoform - specific antibodies demonstrated the near complete disap - pearance of epsin 1 from conditional TKO cell extracts in response to OHT ( Figure 1A ) and confirmed the absence of epsin 2 and 3 ( Figure 1B ) . The extremely small amount of residual epsin 1 was likely explained by delayed gene recombination in a few cells , where residual epsin 1 immunoreactivity , much lower than in controls , was observed by immunofluorescence . A C - terminal epsin 2 fragment ( MW about 40 kDa ) was detected in cells containing the epsin 2 KO allele , possibly reflecting an alter - native start site ( Figure 1\u2014figure supplement 1C ) . However , the KD of this fragment by RNAi in TKO cells did not produce phenotypic changes in addition to the ones described below , indicating that it does not play a relevant role . OHT - dependent loss of epsin 1 expression was also validated by the anti - epsin 1 immunofluorescence , as the typical punctate epsin 1 signal ( reflecting clathrin - coated pits [ Chen et al . , 1998 ] ) was completely absent ( Figure 1C ) . We also mated mice with mutations in all the three Epn genes with nestin - Cre transgenic mice ( Tronche et al . , 1999 ) to obtain brain - specific TKO mice ( genotype : Epn1 loxP / loxP ; Epn2 \u2212 / \u2212 ; Epn3 \u2212 / \u2212 ; Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 4 of 25 Research article Figure 1 . Mitotic defects in epsin TKO fibroblasts . ( A ) Anti - epsin 1 immunoblot shows the disappearance of epsin 1 from Epn 1 loxP / loxP ; Epn 2 \u2212 / \u2212 ; Epn 3 \u2212 / \u2212 ; Cre - ER + / 0 cells after 7 - day treatment with 4 - hydroxy - tamoxifen ( OHT ) . Tubulin was used as a loading control ( top ) . Densitometric analysis of the epsin 1 band during OHT - treatment ( bottom ) . ( B ) Anti - epsin immunoblots with isoform - specific antibodies showing OHT - treatment for 7 days results in the loss of epsin 1 in epsin 2 / 3 double knock - out ( DKO ) thus generating triple KO ( TKO ) cells . Wild type ( WT ) cell lysate was Figure 1 . Continued on next page Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 5 of 25 Research article Nestin - Cre + / 0 ) and their controls with a WT epsin 1 allele ( Epn1 loxP / + ; Epn2 \u2212 / \u2212 ; Epn3 \u2212 / \u2212 ; Nestin - CRE + / 0 ) . Nestin - Cre epsin TKO animals were born , but at a lower than expected Mendelian ratio . A few days after birth they began to develop locomotor dysfunction , failed to gain weight ( Figure 1\u2014figure supplement 1D ) , and rapidly deteriorated , typically dying before the end of the fourth week . Western blot analysis of extracts of their brains at the beginning of the fourth week , confirmed the absence of all the three epsin genes ( Figure 1\u2014figure supplement 1E ) . In this study , we focused on the impact of the lack of epsin on basic cellular properties using MEFs and cell - free assays . Brain specific epsin TKO mice will be characterized in a future study , but we made use of their brains as the source of epsin TKO brain cytosol in the cell - free assay described below . Epsin TKO cells have defects in cytokinesis Microscopy of the TKO MEFs revealed them to be abnormally large compared to WT cells ( contour in Figure 1C ) . Furthermore , these cells contained multiple clustered nuclei or very large and abnormally shaped nuclei , as shown by both DAPI staining and Cre immunofluorescence ( Figure 1D , Figure 1\u2014 figure supplement 1B , F ) , in contrast to the single nuclei present in the smaller WT cells . This finding could be explained by the reported role of epsin 1 and 2 in mitotic spindle organization ( Liu and Zheng , 2009 ) , a defect that can result in abnormal chromosome segregation and impaired cytokinesis . Accordingly , analysis of WT and TKO cells during 48 - hours of OHT treatment revealed that the increase in either cell size or size / number of nuclei of TKO cells was accompanied by a strong cell number reduction ( Figure 1E ) . Additionally , immunostaining of cells synchronized by double - thymidine treat - ment ( Banfalvi , 2011 ) for anti - Aurora B kinase , a marker of midbodies of telophase ( [ Banerjee et al . , 2014 ] inset of Figure 1D ) , showed a striking decrease of cytokinesis profiles in TKO ( Figure 1F ) . As the focus of our study is the role of epsin in endocytosis , we did not explore mechanistic aspects of this cytokinesis defect further . We note , however , that other endocytic proteins , including clathrin itself , have been implicated in the organization of mitotic scaffolds ( Royle , 2013 ; Kaur et al . , 2014 ) . Endocytic delay at early / mid - stage clathrin - coated pits We next analyzed the impact of the complete absence of the three epsins on clathrin - mediated endo - cytosis . Immunostaining of TKO cells for \u03b1 - adaptin ( a subunit of the clathrin adaptor complex AP - 2 ) and for clathrin - light chain ( CLC ) showed an increase in the density of clathrin - coated pits relative to controls ( Figure 2A \u2013 D ) . Additionally , most pits occurred in small clusters rather than as single - puncta ( insets in Figure 2A \u2013 D ) . Total levels of clathrin and \u03b1 - adaptin , however , were not changed ( Figure 2\u2014 figure supplement 1A ) , suggesting that the increase in pits reflects an increase in the pool of assem - bled clathrin coats ( Figure 2E ) . Clathrin - coated pit dynamic was also altered , as live imaging with spinning disk confocal microscopy of cells transfected with \u03bc 2 - adaptin - GFP ( another subunit of the AP - 2 complex ) revealed that the turnover rate of pits in TKO cells was much lower than in WT cells ( Figure 2F , G ) . In agreement with these morphological changes , an impairment of the internalization of fluorescent transferrin , a cargo of endocytic clathrin - coated pits , was also observed in TKO cells . Following preincu - bation with Alexa594 - transferrin on ice and subsequent incubation at 37\u00b0C for 15 min , the bulk of transferrin remained at the cell surface in TKO cells , while remaining transferrin was intracellular in used as a control . ( C ) Anti - epsin 1 immunofluorescence shows that the typical punctate epsin 1 signal of WT cells ( top ) was completely absent in TKO cells ( bottom ) . The perimeter of the TKO cell is indicated by a dotted white line and demonstrates a very large size relative to WT cells . The insets show higher magnification of the boxed regions . ( D ) DAPI staining showing single nuclei in WT and multiple nuclei in a TKO cell . The inset of the WT field shows the accumulation of AuroraB kinase immunoreactivity at the mid - body during cytokinesis . ( E ) The increase in cell number during a 3 - day incubation is lower in TKO cells . Cells were counted at day 7 and 9 after addition of OHT ( * * p < 0 . 01 , Student ' s t test , n = 3 experiments ) . ( F ) As shown by a morphometric analysis , cytokinesis events are only rarely observed in TKO cells . Scale bar represents 10 \u03bc m . Data are represented as mean \u00b1 SEM . See also Figure 1\u2014figure supplement 1 . DOI : 10 . 7554 / eLife . 03311 . 003 The following figure supplement is available for figure 1 : Figure supplement 1 . The generation of triple KO cells , nestin Cre brain specific triple KO mice , and the observation of nuclear defect in TKO cells . DOI : 10 . 7554 / eLife . 03311 . 004 Figure 1 . Continued Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 6 of 25 Research article Figure 2 . Absence of epsin stalls endocytic clathrin - coated pit maturation at an early stage . Confocal microscopy . ( A \u2013 D ) Immunofluorescence staining for \u03b1 - adaptin ( A and B ) and clathrin light chain ( C and D ) indicates an increase of clathrin - coated pits ( CCPs ) number in cells that lack all three epsins . In Figure 2 . Continued on next page Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 7 of 25 Research article control cells ( Figure 2H , I ) . This change correlated with a major shift of overexpressed and endogenous transferrin receptor from a punctate localization in the cytoplasm ( WT cells ) to a plasma membrane localization ( TKO cells ) , as demonstrated by both immunofluorescence ( Figure 2J , K ) and a cell surface biotinylation assay ( Figure 2L ) . Transferrin internalization impairment could be rescued by electropo - ration of epsin1 - GFP cDNA ( Figure 2\u2014figure supplement 1B \u2013 D ) . Likewise , a major shift of the local - ization of synaptobrevin 2 / VAMP2 ( Syb2 ) , a vesicular SNARE that is internalized by clathrin - mediated endocytosis , was observed in TKO MEFs stably expressing Syb2 - HA , ( this construct harbors the HA epitope at the very short C - terminal non - cytosolic portion of Syb2 ) . Total Syb2 immunoreactivity had a predominant punctate intracellular distribution in control MEFs , and an additional strong plasma membrane localization in TKO ( Figure 2M , O ) . The presence of an abundant surface exposed pool of Syb2 selectively in TKO MEFs was confirmed by the surface immunofluorescence for the HA epitope ( Figure 2N , P ) and by a cell surface biotinylation assay ( Figure 2Q , R ) . When cells were examined by electron microscopy to assess the stage at which clathrin - mediated endocytosis is impaired in TKO cells , the major increase was observed for shallow and U - shaped pits ( Figure 2S , T ) . This observation may seem in contrast with the reported accumulation of the so - called multiheaded coated - structures at the plasma membrane of cells subjected to RNAi - dependent KD of all the three epsins ( Boucrot et al . , 2012 ) . Such a phenotype was interpreted as a defect in the fission reaction . However , multiheaded - coated structures , which were not clearly detected in our cells ( Figure 2S , T ) , could instead reflect clustering of shallow / intermediate stage pits at the pre - fission stage . In agreement with an arrest at early stage , dynamin 2 , endophillin 2 , and myosin 1E ( a myosin implicated in clathrin - mediated endocytosis [ Cheng et al . , 2012 ] ) , three proteins whose localization at pits peaks at the time of fission ( Taylor et al . , 2011 ) were present at the subset of very late pits in WT cells , while in TKO cells they had a diffuse cytosolic distribution ( Figure 2U ) . Enhanced actin accumulation at epsin - deficient clathrin - coated pits A stalling of endocytic clathrin - coated pits prior to the deep invagination stage has been observed upon manipulations affecting actin nucleation and dynamics ( Shupliakov et al . , 2002 ; Ferguson et al . , 2009 ) , leading us to explore actin localization in TKO cells . Phalloidin staining for F - actin revealed only few of the actin stress fibers typically observed in control cells ( Figure 3A , B ) and a strong accumulation of F - actin foci in TKO cells ( Figure 3B , D ) , sometimes in the form of elongated curved structure at the cell cortex ( inset in Figure 3B ) . Similar results were observed upon immu - nostaining for Arp 2 / 3 ( an actin nucleating complex that functions with N - WASP [ Figure 3E ] ) . A differ - ence in actin organization relative to WT cells was also observed by live total internal reflection TKO cells , clathrin - coated pits generally occur in small clusters . Insets show the boxed regions at high magnification . Note the large size of TKO cells relative to control . ( E ) CCP number in WT and TKO cells as assessed by \u03b1 - adaptin immunofluorescence ( * * * p < 0 . 001 , Student ' s t test , n = 10 cells / genotype ) . ( F ) Kymographs from a time series of WT and TKO cell expressing \u03bc 2 - adaptin - GFP . Each line represents a single \u03bc 2 - GFP spot . Note the short length of the lines for WT , reflecting the turnover of the pits and the continuous lines in TKO cells , reflecting an arrest of the pit maturation . ( G ) Clathrin - coated pit turnover ( appearance and disappearance events ) as analyzed by spinning - disk confocal imaging of \u03bc 2 - adaptin - GFP fluorescence in WT and TKO cells ( n = 5 cells / genotype ) . ( H and I ) Impaired uptake of pre - bound Alexa594 - transferrin ( Tf ) in TKO cells during a 15 - min incubation . In WT , the bulk of Tf was internalized , while in TKO Tf remained at the cell surface . ( J and K ) Transferrin receptor ( TfR ) - GFP predominantly localizes in intracellular vesicles in WT but at the cell surface in TKO cells . ( L ) A surface biotinylation assay reveals elevated amounts of endogenously expressed TfR at the plasma membrane of TKO cells relative to WT , as assessed by anti - TfR immunoblotting of streptavidin affinity - purified material . ( M \u2013 P ) Increased surface localization of stably expressed Syb2 - HA in TKO fibroblasts as shown by total ( M \u2013 O ) and surface - only ( N \u2013 P ) immunofluorescence . ( Q and R ) A surface biotinylation performed as in ( L ) demonstrating an increased fraction of cell surface exposed Syb2 - HA in TKO cells relative to WT ( * * p < 0 . 01 , Student ' s t test , n = 4 experiments , Surf : surface , Int : internal ) . ( S and T ) Representative electron microscopy images of different stage endocytic clathrin - coated intermediates in TKO cells ( S ) and quantification of the corresponding stages ( T , * * p < 0 . 01 , * * * p < 0 . 001 , n = 33 cells / genotype , one - way ANOVA ) . ( U ) Comparative analysis of the localization of clathrin immunoreactivity ( CLC ) with the localization of dynamin 2 , endophillin 2 , and myosin 1E immunoreactivities . In WT cells , these three proteins co - localize with a subset of clathrin - coated pits ( examples are indicated by small white arrows ) , which represent late - stage pits . In TKO cells , where more numerous clathrin - coated pits are observed , the punctate localization of dynamin 2 , endophillin 2 , and myosin 1E is completely lost . Scale bars : 10 \u03bc m for ( A \u2013 D , H \u2013 K ) , 20 \u03bc m for ( M \u2013 P ) , 5 \u03bc m for ( U ) , and 200 nm for ( S ) . In E , G , R , and T black bars indicate WT and red bars epsin TKO . See also Figure 2\u2014figure supplement 1 . DOI : 10 . 7554 / eLife . 03311 . 005 The following figure supplement is available for figure 2 : Figure supplement 1 . Endocytic defect in epsin TKO cells is not due to alteration in endocytic protein levels and it is rescued by epsin1 \u2013 GFP . DOI : 10 . 7554 / eLife . 03311 . 006 Figure 2 . Continued Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 8 of 25 Research article ( TIRF ) microscopy of TKO cells , co - expressing the calponin homology domain of utrophin fused to mCherry ( CH Utr \u2013 mCh ) and CLC fused to GFP ( CLC \u2013 GFP ) , as there was a general increase in the pool of actin associated with clathrin spots ( Figure 3F ) . The expression of full - length epsin1 \u2013 GFP ( Figure 2\u2014figure supplement 1B \u2013 D ) rescued actin cytoskeleton changes ( Figure 3C , D ) . Epsin is required for the recruitment of Hip1R at endocytic pits Interestingly , while no significant alterations of the levels of several major components of the clathrin - dependent endocytic machinery were observed in TKO cells ( Figure 2\u2014figure supplement 1A ) , the level of Hip1R was increased ( Figure 4A ) . This observation strengthened the idea that epsin and Hip1R are functionally interconnected , as suggested by studies in yeast and Dictyostelium ( Baggett et al . , 2003 ; Brady et al . , 2010 ; Skruzny et al . , 2012 ) , and prompted us to explore the interplay of epsin and Hip1R . Pull - down experiments from rat brain homogenate , using ENTH domain of epsin 1 as bait revealed an enrichment of Hip1R in the affinity - purified material in the sample also containing PI ( 4 , 5 ) P 2 ( Figure 4B ) . Additionally , the typical clathrin - coated pit - like punctate localization ( Engqvist - Goldstein et al . , 1999 ) of endogenous ( immunofluorescence , Figure 4C ) and exogenous ( TIRF microscopy of Hip1R - GFP , Figure 4F ) Hip1R was replaced in TKO cells by a diffuse signal ( Figure 4D , F ) . Expression of epsin1 - GFP in TKO cells ( Figure 2\u2014figure supplement 1B \u2013 D ) rescued Hip1R localization ( Figure 4E ) . We conclude that the localization of HipR at endocytic clathrin - coated pits is heavily dependent on epsin . Figure 3 . Abnormal actin distribution in epsin deficient cells . ( A \u2013 C ) Confocal microscopy images . Phalloidin staining of WT and epsin TKO cells shows a major loss of stress fibers with a corresponding accumulation of elongated F - actin foci ( see inset ) in TKO cells . These changes were rescued by the expression of epsin1 \u2013 GFP ( C ) . ( D ) Quantification of the actin foci shown in ( A \u2013 C , * * * p < 0 . 001 , n = 8 cells / conditions , one - way ANOVA ) . ( E ) Phalloidin staining and Arp 2 / 3 immunoreactivity in a WT and a TKO cell showing the co - localization of Arp 2 / 3 with the actin foci , as visualized by confocal microscopy . ( F ) TIRF microscopy of WT and TKO cells expressing clathrin light chain \u2013 GFP ( CLC \u2013 GFP ) and the F - actin binding protein utrophin \u2013 mCherry ( CH Utr \u2013 mCh ) . Note the increase in F - actin ( CH Utr \u2013 mCh signal ) typically surrounding the clathrin - coated pits , in the cortical region ( TIRF plane ) of the TKO cell . Virtually all pits are positive for CH Utr \u2013 mCh . Scale bars : 10 \u03bc m for ( A \u2013 C ) , 5 \u03bc m for ( E and F ) . Data are represented as mean \u00b1 SEM . DOI : 10 . 7554 / eLife . 03311 . 007 Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 9 of 25 Research article Figure 4 . Epsin is required for the recruitment of Hip1R at the endocytic clathrin - coated pits . ( A ) Immunoblot analysis of Hip1R ( left ) shows an increase ( as quantified in the right ) of its levels in epsin TKO cells . ( B ) Pull down experiments from rat brain homogenate , using ENTH \u2013 GST or GST alone as bait , shows the affinity - purification of Hip1R in the presence of diC8 - PI ( 4 , 5 ) P 2 [ PI ( 4 , 5 ) P 2 , top ] . Coomassie blue stained gel of the baits ( bottom ) . ( C \u2013 E ) Anti - Hip1R immunofluorescence of representative WT and TKO cells . The clathrin - coated pit pattern of Hip1R in WT ( see also high magnification insets ) was replaced by a diffuse localization in the TKO cell , but was rescued by expression of epsin1 \u2013 GFP . ( F ) WT and TKO cells were transfected with Hip1R \u2013 GFP and imaged by live TIRF microscopy ( fluorescence is shown in black ) . The punctate accumulation ( clathrin - coated pits ) of Hip1R at the cortex of WT was lost in the TKO cell . ( G and H ) siRNA - mediated knockdown of Hip1R ( G , Ctrl : non - transfected control ; NC : scramble control ; O1 : Hip1R specific double - stranded siRNA ) does not affect epsin localization in HeLa cells as shown by epsin immunofluorescence ( H ) . Scale bars represent 10 \u03bc m . Data are represented as mean \u00b1 SEM . DOI : 10 . 7554 / eLife . 03311 . 008 Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 10 of 25 Research article In contrast , the localization of epsin at pits was independent of Hip1R , as Hip1R KD in HeLa cells by RNAi ( Figure 4G ) did not affect epsin localization ( Figure 4H ) . Absence of epsin impairs the coupling of clathrin - dependent budding to actin growth in a cell - free assay To address the role of epsin at the interface of endocytic clathrin coats and actin dynamics , we turned to a cell - free system developed in our laboratory ( Wu et al . , 2010 ; Wu and De Camilli , 2012 ) . The approach consists of incubating glass - attached plasma membrane sheets , produced by cell sonication , with purified brain cytosol in the presence of ATP and the non - hydrolyzable GTP analogue GTP \u03b3 S . Under these conditions , where endocytic invagination is not compensated by internal pressure , the incubation triggers the formation of endocytic tubules that are capped by clathrin - coated pits and are surrounded by F - BAR proteins of the FBP17 family . Consistent with the role of these proteins in actin nucleation , elongation of the tubules is actin - dependent ( Wu et al . , 2010 ; Wu and De Camilli , 2012 ) . Plasma membranes obtained by sonication of PTK2 cells expressing PM - anchored GFP exhibit a homogeneous fluorescence when kept in cytosolic buffer ( Wu and De Camilli , 2012 ) . Upon incuba - tion with WT mouse brain cytosol and nucleotides , growth of the tubular invaginations was revealed in en face views by the resulting GFP puncta ( Figure 5\u2014figure supplement 1A ) , and \u2018in side\u2019 views by the growth of short columns of GFP fluorescence perpendicular to the plane of the coverslip ( Figure 5A , left PM - GFP panel ) . The presence of clathrin at the tips of the tubules was confirmed by immunofluo - rescence ( Figure 5A , left clathrin panel ) . Clathrin signal had an elongated appearance \u2018in side\u2019 views , given the low resolution of confocal microscopy in the Z dimension . Epsin immunoreactivity precisely overlapped with clathrin ( Figure 5A , left epsin panel ) indicating epsin localization throughout the clathrin coat . Clathrin - capped invaginations also formed on plasma membrane sheets incubated with epsin TKO brain cytosol ( Figure 5A , right panels and Figure 5\u2014figure supplement 1B ) . However , important dif - ferences relative to incubations with control cytosol were observed . In membrane sheets incubated with control brain cytosol , F - actin polymerized in alignment with the invaginations thus generating a well - organized scaffold that contributes to maintain their parallel orientation perpendicular to the plasma membrane ( Figure 5B , left panels ) . On the contrary , in membranes incubated with TKO cytosol , the F - actin network around the invaginations was exaggerated and disorganized ( Figure 5B , right panels ) , leading to clumping of the tips of the invaginations in a meshwork of actin . As a result of this clumping , invaginations often had an oblique orientation ( Figure 5A , B , right panels and Figure 5\u2014figure supplement 1B ) . Such disorganization was confirmed by electron microscopic observations of sections cut parallel to the substrate . Regularly spaced cross - sectioned tubules interspersed within an actin meshwork were observed with WT cytosol , but numerous obliquely cut tubules embedded in a dense actin matrix were visible with TKO cytosol ( Figure 5\u2014figure supplement 1C , D ) . Another striking modification related to an abnormal actin cytoskeleton was the mislocalization of myosin 1E . As shown by immunofluorescence , this protein was localized at the bottom of the tubular invaginations in sheets incubated with WT cytosol , but at the tips in sheets incubated with epsin TKO brain cytosol ( Figure 5C and higher magnifications ) . Epsin is required for the recruitment of Hip1R to endocytic invaginations in the cell - free assay The differences in the cell - free assay using WT vs TKO cytosol were consistent with abnormal actin nucleation observed at clathrin - coated pits of intact TKO cells . Furthermore , in agreement with these observations , Hip1R immunoreactivity was present at the tips of the invaginations in sheet prepara - tions incubated with WT cytosol , but was largely absent from the tubules incubated with TKO cytosol ( Figure 5D ) . This was in spite of the higher levels of Hip1R in the TKO brain cytosol ( Figure 5\u2014figure supplement 1E ) in agreement with the higher levels of Hip1R in TKO fibroblasts ( Figure 4A ) . Thus , also in this system , Hip1R recruitment requires epsin . We also prepared cytosol from brains of mice lacking Hip1R and its close homologue Hip1 ( Hip1 / Hip1R double KO ( DKO ) mice [ Bradley et al . , 2007 ] ) . These mice are typically dwarfed , afflicted with severe spinal defects , and die in early adulthood ( Bradley et al . , 2007 ) . When membrane sheets were incubated with Hip1 / Hip1R DKO brain cytosol , differences from WT were observed that were qualita - tively similar to those obtained with the epsin TKO cytosol , but quantitatively milder ( Figure 5E , F ) . Tubular invaginations were longer and some exaggerated and disorganized actin was also observed Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 11 of 25 Research article Figure 5 . Coupling of clathrin - dependent budding to actin dynamics in a cell - free assay is perturbed by the absence of epsin . Plasma membrane sheets of PTK2 cells expressing PM - anchored GFP ( PM \u2013 GFP ) were incubated in the presence of WT , epsin TKO or Hip1 / Hip1R double KO ( DKO ) brain cytosol , and nucleotides as described in Wu et al . ( 2010 ) , fixed , immunostained , and observed by confocal microscopy . Views orthogonal to the substrate are shown . ( A ) Immunofluorescence staining for clathrin light chain and epsin 1 . The GFP - positive columns represent narrow tubular invaginations of the plasma membrane capped by clathrin - coated pits , as revealed by the presence of clathrin and epsin immunoreactivity ( Wu et al . , 2010 ) . In the control preparation ( left ) tubules are straight and perpendicular to the substrate , while in the preparation incubated with TKO cytosol ( right , note the absence of epsin immunoreactivity ) they have a more disordered orientation . ( B ) Phalloidin staining of the membrane sheets revealing a well - organized actin scaffold around the tubules after incubation with WT cytosol ( left ) and an exaggerated and disorganized F - actin network in sheets incubated with TKO cytosol ( right ) . ( C ) Myosin 1E immunoreactivity localizes at the bottom of tubular invaginations in sheets incubated with WT cytosol ( left ) , and at the tip of the invaginations after incubation with TKO cytosol ( right ) . Details of the sheets are shown at high magnification below the main panels . ( D ) Hip1R immunoreactivity is present at the tip of tubular invaginations in the control samples ( left ) , but is absent in sheets incubated with epsin TKO cytosol ( right ) . ( E ) Quantification of F - actin polymerization upon incubation with the cytosol of the three tested genotypes ( WT , epsin TKO , and Hip1 / Hip1R DKO ) . The average phalloidin fluorescence per unit area of membrane sheets was calculated ( n = 10 sheets / conditions , * * * * p < 0 . 0001 , one - way ANOVA ) . ( F ) Phalloidin staining of sheets incubated with Hip1 / Hip1R double KO ( right ) cytosol reveals an exaggerated and disorganized F - actin network relative to WT ( left ) , but not as prominent as that observed in preparations incubated with epsin TKO cytosol . ( G ) Immunofluorescence staining for epsin 1 and Hip1R shows that the presence of epsin 1 at the tips of the invagination is not strongly modified by the absence of Hip1 and Hip1R . ( H ) The disordered C - terminal tail of epsin binds F - actin . GST fused epsin 1 fragments ( ENTH domain , DPW , NPF , and DPW - NPF containing regions ) were incubated ( 5 \u03bc M final concentration ) with previously polymerized F - actin ( 15 \u03bc M ) and then subjected to ultracentrifugation followed by SDS - PAGE and anti - GST immunoblotting of the supernatant ( S ) and pellet ( P ) materials . Scale bars : 5 \u03bc m . Data are represented as mean \u00b1 SEM . See also Figure 5\u2014figure supplement 1 . DOI : 10 . 7554 / eLife . 03311 . 009 The following figure supplement is available for figure 5 : Figure supplement 1 . Nestin Cre epsin TKO brain cytosol has increased Hip1R level and disrupted membrane tubulation in a cell - free assay . DOI : 10 . 7554 / eLife . 03311 . 010 Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 12 of 25 Research article ( Figure 5E , F ) . Importantly , localization of epsin 1 at the tips of the invaginations was not affected by the absence of Hip1 / Hip1R in the cytosol ( Figure 5G ) , once again as in the case of intact cells . Direct binding of epsin to actin Altogether , the results discussed above support a role for epsin as a critical factor required for the temporal and spatial coordination between clathrin - mediated endocytosis and actin dynamics . We thus explored whether epsin interacts directly with actin by incubating recombinant epsin 1 fragments ( Figure 5\u2014figure supplement 1F ) with purified F - actin followed by co - sedimentation . This analysis revealed that both the DPW and NPF motif containing regions of the protein ( Chen et al . , 1998 ) , but not the ENTH domain , bind actin ( Figure 5H ) . Thus , there appear to be at least two actin - binding sites in epsin . The site in the NPF motif containing region likely corresponds to the actin cytoskeleton - binding ( ACB ) site , previously identified in yeast epsin ( Ent1 , Skruzny et al . , 2012 ) . A low affinity binding between epsin and synaptobrevin 2 / VAMP2 Collectively , previous findings from the literature and the findings described above are consistent with roles for epsin from early to late stage clathrin - coated pits , and more generally for an essential role of this protein in clathrin - mediated endocytosis in mammalian fibroblasts . It was therefore of interest to determine whether epsin , like several clathrin adaptors with ENTH / ANTH domains ( Hirst et al . , 2004 ; Miller et al . , 2007 , 2011 ; Koo et al . , 2011 ) , binds SNAREs . SNARE binding by ENTH / ANTH family proteins helps coordinate bud formation with the incorporation of an appropriate SNARE in the nas - cent vesicle to direct its fate after fission . Such binding in the case of epsin would likely be of very low affinity , as it was not detected in previous investigations . However , even a low affinity binding could be of physiological significance in the context of other synergistic interaction between epsin , the mem - brane bilayer and other coat proteins . Pull - downs from mouse brain lysate using GST \u2013 ENTH domain of epsin 1 as bait , followed by analysis of the affinity purified material by mass spectroscopy , identified Syb2 as one of the top hits ( Figure 6\u2014 figure supplement 1A ) . A direct interaction was next explored by assessing binding to the ENTH domain of epsin 1 ( with an His tag at the C - terminus ) to increasing concentrations of the cytosolic portion of Syb2 fused to GST ( Figure 6A ) . The very low affinity of this interaction ( K D 470 \u00b1 30 \u03bc M ) was enhanced by the presence in the incubation medium of IP 6 ( K D 80 \u00b1 7 . 9 \u03bc M , Figure 6B , D and Figure 6\u2014figure supplement 1B ) , which induces the folding of helix zero of the ENTH domain ( Itoh et al . , 2001 ; Ford et al . , 2002 ) . In contrast , the deletion of helix zero from the ENTH construct abolished Syb2 binding in either the presence or the absence of IP 6 ( Figure 6C ) . Further analysis of the interaction of Syb2 with the ENTH domain using Syb2 fragments ( Figure 6E ) identified the SNARE motif of Syb2 ( Figure 6F , G ) , and more specifically the N - terminal portion of the motif ( residues 29 \u2013 60 , Figure 6H , I ) , as the region respon - sible for binding . Interestingly , previously reported interactions of vesicular SNAREs with ENTH / ANTH proteins also involve their SNARE motifs ( Koo et al . , 2011 ; Miller et al . , 2011 ) , pointing to an interaction mutually exclusive with SNARE complex formation , as expected for an \u2018endocytic\u2019 interaction . The interaction of epsin with Syb2 also occurs in intact cells , as shown by anti - HA co - immunoprecipitation experiments from HeLa cells expressing FLAG - tagged epsin 1 and either HA - tagged full - length Syb2 ( Figure 6\u2014figure supplement 1C ) , or deletion constructs of Syb2 ( Figure 6\u2014figure supplement 1D ) . Only constructs including the N - terminal portion of the SNARE motif of Syb2 co - precipitate epsin 1 ( Figure 6\u2014figure supplement 1E ) . The low affinity interaction of epsin with an exocytic SNARE is evolutionarily conserved as yeast Snc1 ( a Syb2 homologue ) bound the ENTH domain of Ent1 in a recombinant pull - down assay ( Figure 6J ) . Discussion Previous reports have addressed the role of epsin in endocytosis . However , studies in different organ - isms and in cell - free systems had emphasized the different aspects of its function , so that a complete picture of its physiological role ( s ) has remained elusive . Elucidation of epsin function in mammalian cells had been complicated by the existence of the three Epn genes with overlapping functions . To overcome this problem , we have generated cells that lack all the three epsins by a conditional KO approach involving the OHT - dependent disruption of the Epn1 gene in epsin 2 and 3 double KO mice . A main conclusion of our study is the occurrence of a close coupling between the function of epsin and the dynamics of the actin cytoskeleton . While such coupling had been suggested by studies in unicellular organisms , studies of epsin in cells of metazoan had emphasized its role as a bilayer Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 13 of 25 Research article Figure 6 . Epsin directly binds synaptobrevin 2 / VAMP2 . ( A ) Interaction of purified epsin ENTH \u2013 His6 ( 800 ng ) with increasing amounts of GST - cytosolic portion of synaptobrevin2 ( Syb2 ) as revealed by anti - His immunoblotting of bound material in a GST pull - down ( top ) . Coomassie blue stained gel of the baits ( bottom ) . ( B ) The interaction of epsin ENTH \u2013 His6 with equal amounts of GST \u2013 Syb2 ( Syb2 ) is enhanced by the presence of soluble diC8 - PI ( 4 , 5 ) P 2 ( PI ( 4 , 5 ) P 2 ) , IP 3 or IP 6 ( final concentration 50 \u03bc M ) . Top : anti - His immunoblotting . Bottom : Coomassie blue stained Figure 6 . Continued on next page Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 14 of 25 Research article deforming protein ( Ford et al . , 2002 ; Boucrot et al . , 2012 ) and as a cargo - specific adaptor ( Polo et al . , 2002 ; Overstreet et al . , 2003 ; Chen and De Camilli , 2005 ; Meloty - Kapella et al . , 2012 ) . A link of epsin to actin function at endocytic clathrin - coated pits , and in particular a role as a factor that ensures a \u2018controlled\u2019 actin growth at sites of endocytosis , is supported by several observations . First , Hip1R , which had so far been considered the major link of endocytic clathrin coats to actin in mamma - lian cells ( Engqvist - Goldstein et al . , 2001 ; Le Clainche et al . , 2007 ) , is not recruited to endocytic clathrin - coated pits in the absence of epsin . This is consistent with the interaction and functional part - nership of epsin with Sla2 / Hip1R , previously demonstrated in yeast and Dictyostelium , although in yeast it is Sla2 that recruits epsin to clathrin - coated pits ( Skruzny et al . , 2012 ) . Second , the lack of epsin results in a disruption of the normal actin cytoskeleton with a major loss of stress fibers and an accumulation of actin foci at the cell surface , typically in proximity of arrested endocytic clathrin - coated pits . Third , epsin binds actin directly via its disordered tail region . Fourth , a cell - free assay of endocytosis involving plasma membrane sheets and brain cytosol revealed that relative to WT , epsin - deficient cytosol results in an exaggerated and abnormal growth of the actin cytoskeleton . These find - ings suggest a role for epsin in orchestrating a coupling between actin and endocytic clathrin coats that ( 1 ) limits excessive actin growth and ( 2 ) helps mediate an effect of actin polymerization on bud invagination . A functional partnership between epsin and Hip1R explains why the KD of Hip1R results in a similar enhanced and abnormal actin nucleation at endocytic sites , with the formation of large actin bundles projecting away from these sites ( Engqvist - Goldstein et al . , 2004 ) . The excess of actin nucleation in the absence of epsin is of special interest as in yeast the ENTH domains of Ent1 and 2 bind two Cdc42 \u2013 GAPs ( GTPase activating proteins ) , Rga1 and Rga2 , and this interaction is essential for function ( Aguilar et al . , 2006 ) . Interestingly RLIP76 / RalBP1 , a protein with Cdc42 GAP activity , interacts with the ENTH domain of epsin ( Ross\u00e9 et al . , 2003 ) , although there is no clear sequence homology between RLIP76 / RalBP1 and Rga proteins . In view of the modulatory role of Cdc42 in the nucleation of N - WASP - Arp 2 / 3 - dependent actin nucleation at endocytic clathrin - coated pits , the recruitment of a Cdc42 \u2013 GAP by epsin could help explain why actin nucleation is enhanced in epsin null cells . A very strong link between epsin and actin is consistent with evidence for a general and evolution - arily conserved role of actin in clathrin - mediated endocytosis , which is more prominent under condi - tions of high plasma membrane tensions ( Merrifield et al . , 2005 ; Kaksonen et al . , 2006 ; Ferguson et al . , 2009 ; Boulant et al . , 2011 ; Taylor et al . , 2012 ) . The force of actin polymerization may help drive the deep invagination of the pits and also assist in dynamin - dependent fission ( Itoh et al . , 2005 ; Roux et al . , 2006 ) by developing a force that drives the deeply invaginated bud away from the plasma membrane . The occurrence of this force is well demonstrated by the dramatic actin - dependent elon - gation of the clathrin - coated pit necks in living cells and in cell - free systems ( Ferguson et al . , 2009 ; Wu et al . , 2010 ) , when the fission reaction is impaired by the lack of dynamin ( living cells ) or block of its GTPase activity ( cell - free system ) , respectively . Clearly , however , epsin does not simply function as gel of the baits . EB : empty beads . ( C ) The interaction of epsin ENTH \u2013 His6 with equal amounts of GST \u2013 Syb2 is lost in a construct missing the N - terminal helix zero ( \u0394 Helix0 ) irrespective of the presence of IP 6 ( final concentration 50 \u03bc M ) . EB : empty beads . ( D ) Quantitative analysis of the binding of ENTH \u2013 His6 to increasing amounts of GST \u2013 Syb2 in the presence or absence of IP 6 , as revealed by densitometry of anti - His immunoreactivity in western blots of bound material . K D s are also indicated . ( E ) Schematic representation of constructs used for ( F \u2013 I ) . SNARE motif and transmembrane region ( TM ) are denoted by dotted lines . ( F and G ) Pull - down of ENTH \u2013 His6 by GST fusions of different cytosolic fragments of Syb2 . Anti - His immunoblotting ( F ) shows binding only to SNARE motif containing fragments ( top ) . Coomassie blue stained gel of the baits ( Bottom ) . Quantitative analysis of the results is shown in ( G ) . ( H and I ) ENTH \u2013 His6 domain binding to SNARE motif fragments ( H ) and corresponding quantification ( I ) . Anti - His immunoblotting detects ENTH interaction with the N - terminal portion of the SNARE motif . ( J ) Purified yeast His - Snc1 binds a GST fusion of the ENTH domain of Ent1 ( yeast epsin 1 ) in a pull down assay ( top ) . Coomassie blue stained gel of the baits ( Bottom ) . Corresponding quantification is shown . Data are represented as mean \u00b1 SEM . See also Figure 6\u2014figure supplement 1 . DOI : 10 . 7554 / eLife . 03311 . 011 The following figure supplement is available for figure 6 : Figure supplement 1 . Epsin binding to Syb2 : mass spectroscopy analysis and interaction in intact cells . DOI : 10 . 7554 / eLife . 03311 . 012 Figure 6 . Continued Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 15 of 25 Research article a negative regulator of actin polymerization , but it is required for proper spatial organization of the actin - based cytoskeleton , as exemplified by the abnormal localization of myosin 1E at sites of endo - cytic invaginations on plasma membrane sheets incubated with epsin TKO cytosol . We note that partial deficiency of epsin function has a major impact on intercellular signaling medi - ated by Notch ( Overstreet et al . , 2003 ; Chen et al . , 2009 ) . It was proposed that Notch activation requires a pulling force applied to Notch by the endocytosis of its ligand in the neighboring cell and that epsin may be essential for the generation of such force ( Meloty - Kapella et al . , 2012 ; Musse et al . , 2012 ; Shergill et al . , 2012 ) . Based on our findings , such a requirement for epsin may be linked to its role in the control of actin dynamics at clathrin - coated pits . Reported roles of epsin in clathrin - independent endocytosis may also be linked to its function in actin regulation ( Sigismund et al . , 2005 ) . Another important finding of our study is a weak interaction of epsin ' s ENTH domain with the SNARE synaptobrevin 2 / VAMP2 . As intracellular vesicles need SNAREs to fuse with their target mem - branes , a coupling must exist between vesicle budding and incorporation of the proper SNARE ( s ) in the bud . Accordingly , other coat adaptors with ANTH or ENTH modules , such as AP180 / CALM and epsinR were shown to bind SNAREs ( Hirst et al . , 2004 ; Miller et al . , 2007 , 2011 ; Koo et al . , 2011 ) . We hypothesize that the very low affinity of the binding of synaptobrevin 2 to epsin , which explains why such an interaction had not been described so far , may nevertheless be physiologically relevant when compounded by other interaction of epsin with the bilayer , other coat components , and ubiqui - tinated cargo proteins . Recently , it was proposed that a main role of epsin in endocytic clathrin - coated pit dynamics is to help mediate membrane fission via the membrane remodeling properties of the amphipathic helix zero of its ENTH domain ( Boucrot et al . , 2012 ) . While this action seems plausible , our study strongly suggests that epsin becomes critically important at the earlier stages of clathrin coat maturation , as we have observed an accumulation of shallow and U - shaped endocytic clathrin - coated pits in epsin TKO cells . The diffuse , rather than punctate distribution of proteins that assemble at the necks of deeply invaginated endocytic clathrin - coated pits , such as dynamin 2 , endophilin 2 , and myosin 1E further supports a stalling of pits at an early stage in epsin TKO cells . In contrast to the observations made in epsin triple KD cells , we have not observed clearly multiheaded pits , although the dome of some pits was irregular . Multiheaded pits observed in epsin triple KD cells had been interpreted as reflecting a defect in fission . However , they do not have the constricted neck of multiheaded pits observed in dynamin mutant cells ( Ferguson et al . , 2007 , 2009 ) and at least some of them could represent a clus - tering of shallow or U - shaped pits . We note that the multiple endocytic functions of epsin are con - sistent with its localization throughout the coat ( Hawryluk et al . , 2006 ; Sochacki et al . , 2014 ) , rather than a selective localization at the bud neck to mediate fission . Finally , our work also demonstrates a dramatic impact of the lack of epsin on cell division , extend - ing previous observations made in epsin 1 and 2 double KD cells , where abnormal spindles were detected ( Liu and Zheng , 2009 ) . Surprisingly , we did not find a major effect on cell division in cells derived from epsin 1 and 2 double KO embryos possibly because of long - term adaptation to decreased levels of epsin . For example , we have detected an upregulation of epsin 3 in epsin 1 and 2 double KO cells ( our unpublished observations ) . Interestingly , epsin undergoes phosphorylation and mono - or oligo - ubiquitination ( Stukenberg et al . , 1997 ; Chen et al . , 1999 , 2003 ; Polo et al . , 2002 ) in mito - sis and these covalent modifications impair its binding to clathrin and AP - 2 , suggesting a switch of its function in the mitotic cytosol ( Chen et al . , 1999 ) . A role of epsin in the dynamics of the cytoskeleton may underlie its role in mitosis , for example by affecting its anchoring to the cell cortex . Several other endocytic proteins , including clathrin , are also implicated in mitosis , and , in particular , in cytokinesis ( Royle , 2013 ) . In conclusion , our results reveal new aspects of the collective function of the three epsin genes and point to epsin as an important coordinator of the dynamics of endocytic clathrin coats from early to late stages . Our findings , along with previous studies suggest the models depicted in Figure 7 . Epsin is an early component of endocytic clathrin - coated pits , which interacts directly with PI ( 4 , 5 ) P 2 , AP - 2 , clathrin , and other early clathrin - coated pit components such as intersectin and Eps15 . The partial bilayer penetration of its ENTH domain makes it optimally suited to function at a site where membrane buckling occurs ( as a generator , sensor or stabilizer of curvature ) , while its low affinity binding to syn - aptobrevin 2 / VAMP2 may collaborate with AP180 / CALM to ensure the coupling of bud nucleation to a membrane patch containing SNAREs . It may additionally participate in the recruitment to the pit of ubiquitinated cargo proteins via its UIMs , a function that does not apply to Dictyostelium epsin , Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 16 of 25 Research article which expresses a single epsin family protein lacking UIMs . As the bud expands , epsin along with Hip1R provide a link between actin nucleation and coat maturation , which is required for deep invag - ination . Finally , the membrane insertion properties of its helix zero may assist dynamin in the fission Figure 7 . Functions of epsin in clathrin - mediated endocytosis . ( A ) Schematic representation of the role of epsin in the coupling of the endocytic clathrin coat to actin in cooperation with Hip1R ( left ) . This coupling helps invaginate the pit . Only epsin localized at the equator of the bud is shown to emphasize its actin - related function in pit invagination , but epsin is not restricted to the equator . Higher magnification representation of the interactions of epsin ( right ) : the N - terminal ENTH domain binds ( 1 ) the PI ( 4 , 5 ) P 2 - rich membrane bilayer of the plasma membrane ( and partially penetrates it ) , ( 2 ) the ANTH domain of Hip1R , and ( 3 ) synaptobrevin2 / VAMP2 ; its tail binds ( 1 ) ubiquitinated cargo proteins ( via UIMs ) , ( 2 ) AP - 2 and clathrin heavy chain ( via DPF / DPW motifs and \u2018clathrin boxes\u2019 , respectively ) , and ( 3 ) the EH domain region of Eps15 ( via NPF motifs ) . Epsin ' s tail also binds F - actin and co - operates with the THATCH domain of Hip1R in the coupling of the actin cytoskeleton to the clathrin - coated pit . In both fields Hip1R is depicted as a monomer but functions as a dimer , or heterodimer with Hip1 . ( B ) Schematic represen - tation of the multiple functions of epsin in clathrin - mediated endocytosis . Epsin participates in the early stages of the reaction as a coat nucleator , curvature inducer / sensor and also helps coupling bud formation to SNARE incorporation . As the coat matures , ubiquitinated cargo is recruited . Additionally , the link between epsin / Hip1R and actin is required for the deep invagination of the pit , and this is the process at which the action of epsin becomes essential . Subsequently , the force produced by actin and the bilayer destabilizing properties of epsin ' s ENTH domain may co - operate with dynamin in fission . DOI : 10 . 7554 / eLife . 03311 . 013 Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 17 of 25 Research article reaction . As we show here , however , its most critical function is at the transition between shallow / U - shaped and deep invaginated pits . Materials and methods Antibodies and reagents Antibodies were obtained from the following commercial sources : mouse anti - tubulin , rabbit anti - FLAG , and mouse anti - Actin ( Sigma - Aldrich , St . Louis , MO , USA ) ; mouse anti - transferrin receptor and rabbit anti - GFP ( Life Technologies , Carlsbad , CA , USA ) ; goat anti - epsin 1 and mouse anti - GST ( Santa Cruz Biotechnology , Santa Cruz , CA , USA ) ; rabbit anti - clathrin light chain , mouse anti - Arp 2 / 3 , rabbit anti - Hip1R , and mouse anti - Hip1 ( EMD - Millipore , Billerica , MA , USA ) ; rat HRP - conjugated anti - HA ( Roche , Mannheim , Germany ) ; rabbit anti - His tag ( GenScript , Piscataway , NJ , USA ) ; mouse anti - Aurora B , rabbit anti - caveolin - 1 , and mouse anti - adaptin \u03bc 2 subunit ( BD Transduction Laboratories , San Jose , CA , USA ) . The following antibodies were kind gifts : mouse anti - epsin 1 and mouse anti - epsin 3 ( Pier - Paolo Di Fiore , IFOM , Milan , Italy ) ; rabbit anti - Hip1R ( David Drubin , University of California , Berkeley , CA , USA ) ; rabbit anti - Myosin 1E ( Mark Mooseker , Yale University , New Haven , CT , USA ) and rabbit anti - synaptophysin ( Paul Greengard , The Rockefeller University , New York , NY , USA ) . Rabbit anti - epsin 2 , mouse anti - clathrin heavy chain , mouse anti - adaptin \u03b1 subunit , rabbit anti - dynamin 2 and rabbit anti - endophillin 2 were generated in our lab . Alexa594 - phalloidin , Alexa594 - mouse anti - HA and Alexa488 , Alexa594 , and Alexa647 conjugated secondary antibodies were from Life Technologies ; Alexa405 - phalloidin was from AAT Bioquest ( Sunnyvale , CA , USA ) while DAPI was from Sigma - Aldrich . Plasmids C - terminal GFP - tagged epsin 1 was obtained by PCR amplification of the epsin 1 coding sequence from brain cDNA library ( Clontech Laboratory Inc . , Mountain View , CA , USA ) followed by ligation into the pEGFP \u2013 N3 plasmid ( Clontech Laboratory Inc . ) . For the generation of the GST tagged ENTH , the rat epsin 1 ENTH coding sequence was PCR amplified and ligated in the pGEX4T - 1 vector ( GE Healthcare , Pittsburgh , PA , USA ) . The GST - tagged epsin fragments ( ENTH , DPW , NPF ) were generated by the ligation of the corre - sponding PCR products to pGEX4T - 1 ( GE Healthcare ) , while for the GST - tagged DPW \u2013 NPF tandem fragment the ligation was with pGEX6 ( GE Healthcare ) . The 6xHis - tagged ENTH of rat epsin 1 was produced by cloning ENTH coding sequence in pET21a ( + ) ( EMD - Millipore ) , and the resulting coding plasmids was mutagenized by site directed mutagenesis ( QuikChange II XL , Agilent Technology , Santa Clara , CA , USA ) to remove the helix zero coding sequence in order to obtain \u0394 Helix0 - ENTH His6 . GST - tagged Syb2 was created by PCR amplification of the cytosolic portion of human Syb2 , the corresponding SNARE motif or the N - terminal extra SNARE region . PCR products were ligated in the pGEX4T - 1 vector ( GE Healthcare ) . FLAG - tagged full - length epsin 1 was generated by cutting epsin 1 coding sequence from the above reported epsin1 - GFP followed by ligation in the pFLAG - CMV4 ( gift from Angus Nairn , Yale University , New Haven , CT , USA ) . For the generation of a cell line stably expressing Syb2 - HA , human Syb2 coding sequence in fusion with HA was cut from pCI neo Syb2 - HA and cloned into pBABE puro . Syb2 - HA fragments ( \u0394 1 \u2013 31 , \u0394 1 \u2013 50 , \u0394 1 \u2013 70 ) were generated by site directed mutagenesis ( QuikChange II XL , Agilent Technology ) of pCI neo Syb2 - HA . For study of yeast Ent1 ENTH domain \u2013 Snc1 interaction , the following plasmids were used : GST control ( pBW1546 pGEX - 5X - 1 ) , WT ENTH1 ( pBW1800 pGEX - 5X - 1 : ENTH1 ) , and Snc1 cytosolic tail ( pBW1916 pET28a : Scn1 [ aa1 - 93 ] ) . pGST 29 / 60 and pGST 61 / 93 Syb2 SNARE were a gift from Karin Reinisch ( Yale University , New Haven , CT , USA ) ; Hip1R \u2013 GFP was a gift from David Drubin ( University of California , Berkeley , CA , USA ) ; and clathrin light chain \u2013 GFP was a gift from James Keen ( Thomas Jefferson University , Philadelphia , PA , USA ) . Adaptin \u03bc 2 subunit - GFP , mCherry - tagged utrophin , and GFP - tagged human transferrin receptor were previously described ( Merrifield et al . , 2005 ; Zoncu et al . , 2007 ; Ferguson et al . , 2009 ) . Generation of epsin conditional triple knock out mice Conditional Epn1 KO mouse ( Epn1 fl / fl ) was generated by homologous recombination at the Epn1 locus with a targeting construct containing loxP sequence and a Frt - flanked neomycin cassette for selection Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 18 of 25 Research article ( removed by breeding with Flp mice , see also [ Pasula et al . , 2012 ] ) . Epn1 fl / fl mice were interbred with Epn2 \u2212 / \u2212 ( Chen et al . , 2009 ) and Epn3 \u2212 / \u2212 ( Ko et al . , 2010 ) mice and with either 4 - hydroxy - tamoxifen ( OHT ) - inducible Cre strain mice ( Badea et al . , 2003 ) , expressing Cre recombinase under the control of the estrogen receptor promoter , or nestin - Cre mice ( Tronche et al . , 1999 ) to generate OHT - inducible and brain - specific epsin triple KO mice , respectively . All mice were of a C57BL / 6J congenic background . Animal care and use was carried out in accordance with our institutional guidelines . Fibroblast cultures Primary fibroblasts were isolated form conditional KO and WT mice from E18 embryos or P1 pups and cultured by standard methods . To achieve gene recombination and obtain epsin triple KO cells and their controls , cells were cultured for 7 days with addition of OHT ( Sigma - Aldrich ) to the culture medium at day 1 ( 3 \u03bc M ) and day 4 ( 1 \u03bc M ) , a two - step 7 - day long process . For cell counting , cells were plated at 50 , 000 cells per 35 - mm dish . Two counts were performed for each of the three dishes of WT and epsin TKO cells using a hemocytometer , both at 1 and 3 days post - plating . The cell proliferation index represents the ratio between cell numbers at these two time points . Transfection Plasmids were transfected by electroporation ( Nucleofector , Amaxa , Cologne , Germany ) for imaging experiments , or using Lipofectamine 2000 ( Life Technologies ) for immunoprecipitation experiments . siRNA oligos were transfected by using Lipofectamine RNAi MAX ( Life Technologies ) , and cells were cultured for 48 \u2013 72 hours before analysis . Double - stranded siRNAs were derived from the following references : Hip1R ( mouse Hip1R , MMC . RNAI . N145070 . 12 . 1 , and 12 . 2 from IDT , Coralville , IA , USA ) ; control ( NC1 negative control duplex from IDT ) . Immunofluorescence Cells were plated and grown on 5 \u03bc g / ml human fibronectin ( EMD - Millipore ) - coated glass coverslips and fixed with 4 % paraformaldehyde \u2013 4 % sucrose in 0 . 1 M sodium phosphate buffer ( pH 7 . 2 ) at room temperature . Coverslips were washed with 50 mM NH 4 Cl pH 7 . 2 , then blocked and permeabilized in 0 . 1 % Triton X - 100 and 3 % bovine serum albumin in PBS . Primary and secondary antibody incubations were performed using the same buffer . Coverslips were finally rinsed in PBS and mounted in Prolong Gold + DAPI ( Life Technologies ) . For surface staining , cells were incubated with an Alexa594 - conjugated rat anti - HA antibody ( Life Technologies ) for 60 min on ice at 4\u00b0C . After extensive washing with cold PBS , cells were fixed as described above and counterstained with DAPI ( Sigma - Aldrich ) before coverslip mounting . Immunofluorescence data acquisition was performed using either a Zeiss Axioplan2 micro - scope ( for the epifluorescence images ) or a spinning disk confocal microscope ( see below ) . Live imaging For live imaging , cells were incubated at 37\u00b0C in the following buffer : 136 mM NaCl , 2 . 5 mM KCl , 2 mM CaCl 2 , 1 . 3 mM MgCl 2 , 3 mM D - glucose , and 10 mM Hepes - Na pH 7 . 4 . Spinning - disk con - focal microscopy was performed using the Improvision UltraVIEW VoX system ( Perkin - Elmer , Walthman , MA , USA ) built on a Nikon Ti - E inverted microscope , equipped with PlanApo objec - tives ( 60 \u00d7 1 . 45 - NA ) and controlled by Volocity ( Improvision , Coventry , UK ) software . Total internal reflection fluorescent ( TIRF ) microscopy was carried out on a Nikon TiE microscope equipped with 60 \u00d7 1 . 49 - NA and 100 \u00d7 1 . 49 - NA objectives . Excitation lights were provided by 488 nm ( for GFP ) and 561 nm ( mCherry ) diode - pumped solid - state lasers . An optical fiber coupled the lasers to the TIRF illuminator and an acousto - optic tunable filter controlled the output from the lasers . Fluorescent signals were detected with an EM - CCD camera ( DU - 887 ; Andor , Belfast , NIR ) and acquisition was controlled by Andor iQ software . Images typically were sampled at 0 . 2 Hz with exposure times in the 4 to 6 s range . Transferrin uptake and transferrin receptor localization For transferrin uptake , cells were electroporated with a plasmid encoding GFP - tagged human trans - ferrin receptor ( Merrifield et al . , 2005 ) . Cells were then starved for 2 hours with serum - free DMEM , incubated at 4\u00b0C for 30 min with 10 \u03bc g / ml Alexa594 - conjugated human transferrin ( Life Technologies ) , and then warmed up to 37\u00b0C for 15 min to allow internalization . Uptake was ended by a brief rinse with ice - cold PBS followed by fixation with 4 % parafolmaldehyde \u2013 4 % sucrose in 0 . 1 M sodium phosphate buffer pH 7 . 2 at room temperature . Cells were counterstained with DAPI to visualize nuclei . Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 19 of 25 Research article Cell surface protein biotinylation Cells were rinsed with PBS and labeled on ice for 60 min with 1 mg / ml EZ - link Sulfo - NHS - SS - Biotin ( Thermo Fisher , Walthman , MA , USA ) , rinsed on ice with 50 mM glycine in PBS pH 8 . 0 , and then in PBS only . Cells were then lysed in modified RIPA buffer ( 1 % Triton X - 100 , 0 . 1 % SDS in 20 mM TRIS pH 7 . 5 , 50 mM NaCl , EDTA 0 . 5 mM with protease and phosphatase inhibitors cocktails ) and lysates were cen - trifuged at 16 . 200\u00d7 g , at 4\u00b0C for 5 min . Biotinylated proteins were recovered on NeutrAvidin beads ( Thermo Fisher ) . After washing with modified RIPA buffer , proteins were eluted with Laemmli SDS - PAGE sample buffer and boiling . Evaluation of protein levels in starting material , biotinylated ( cell surface ) and non - biotinylated ( intracellular ) fractions was assessed by Western blotting . Electron microscopy Control and epsin TKO cells ( \u223c 80 % confluent ) in 60 - mm dishes were fixed in 2 % glutaraldehyde \u2013 0 . 1 M sodium cacodylate . They were post - fixed with 1 % OsO 4 in 1 . 5 % K 4 Fe ( CN ) 6 and 0 . 1 M sodium caco - dylate , en bloc stained with 0 . 5 % uranyl magnesium acetate , dehydrated and embedded in Embed 812 . For morphometric analysis , cells were selected at a low magnification allowing confirmation that the entire outer perimeter was intact , but not visualizing clathrin - coated pits to make the selection process blind with respect to the phenotype of interest . Once selected , higher magnification images were taken around the periphery of the cell , and clathrin - coated structures within the categories defined in Figure 2T were counted all the way around the perimeter of each cell . Electron microscopy reagents were purchased from Electron Microscopy Sciences ( Hatfield , PA , USA ) . Immunoprecipitation HeLa cells co - expressing FLAG - tagged epsin1 , or FLAG alone and HA - tagged Syb2 or HA alone , were washed in ice - cold PBS and lysed in the lysis buffer ( 150 mM NaCl , 0 . 1 % SDS , 0 . 5 % Triton X - 100 , 10 mM EDTA , 20 mM Hepes pH 7 . 4 , and protease inhibitor cocktail [ Roche ] ) . Cell lysates were then centrifuged at 16 , 000\u00d7 g for 20 min at 4\u00b0C and supernatants were incubated , under rotation , with agarose - conjugated anti - HA beads ( Roche ) for 1 hour at 4\u00b0C . Beads were then extensively washed in cold lysis buffer and bound proteins were eluted in Laemmli sample buffer and boiled for 5 min . Mouse brain cytosol preparation 4 weeks - old WT , epsin TKO or Hip1 / Hip1R double KO mouse brains were homogenized in homogeni - zation buffer ( 25 mM Tris pH 8 . 0 , 500 mM KCl , 250 mM sucrose , 2 mM EGTA , 1 mM dithiothreitol ) in the presence of a protease inhibitor cocktail ( Roche ) . The lysate was centrifuged at 160 , 000\u00d7 g for 2 hours in a TL100 . 2 rotor ( Beckman Coulter , Indianapolis , IN , USA ) . The resulting supernatant was buffer - exchanged on PD - 10 columns ( GE Healthcare ) at room temperature into cytosolic buffer ( 25 mM Hepes pH 7 . 4 , 120 mM potassium glutamate , 20 mM potassium chloride , 2 . 5 mM magnesium acetate , 5 mM EGTA ) . Protease inhibitors were added and aliquots of cytosol were immediately frozen in liquid nitrogen and stored at \u221280\u00b0C for up to 2 months . Plasma membrane sheets preparation PTK2 cells stably expressing PM \u2013 GFP ( Chen and De Camilli , 2005 ) were cultured at 37\u00b0C in 10 % CO 2 in minimum essential Eagle medium ( Life Technologies ) supplemented with 10 % ( vol / vol ) fetal bovine serum , 100 \u03bc g / ml penicillin / streptomycin , and 0 . 5 mg / ml G418 . Cells were grown for 24 \u2013 48 hours until confluent in poly - d - lysine coated MatTek dishes ( MatTek , Ashland , MA , USA ) . These dishes were prepared by exposing them to 20 \u03bc g / ml poly - d - lysine ( Sigma - Aldrich ) for 30 min and washed overnight . To prepare plasma membrane sheets , cells were rinsed with PBS and then sheared in ice - cold cytosolic buffer by a brief pulse of sonication ( about 0 . 5 s ) using a cell disruptor ( VirTis Ultrasonics ) set at 20 % of output power with a 1 / 8 - inch microprobe positioned at about 15 mm above the dish . Membrane sheets were rinsed in cytosolic buffer and used for the in vitro assay within 20 min . Generation of endocytic budding intermediates from plasma membrane sheets was achieved by incubating them at 37\u00b0C with cytosol in the presence of nucleotides in the following concentrations : 2 mg / ml cytosol , 1 . 5 mM ATP and 150 \u03bc M GTP \u03b3 S . Samples were supplemented with an ATP - regenerating system consisting of 16 . 7 mM creatine phosphate and 16 . 7 U / ml creatine phosphokinase . The reaction was stopped by washing with cytosolic buffer and fixing samples with 4 % paraformaldehyde \u2013 4 % sucrose in 0 . 1 M sodium phosphate buffer pH 7 . 2 for 15 min at room temperature . Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 20 of 25 Research article Actin co - sedimentation assay 250 \u03bc g of rabbit muscle G - actin ( Sigma - Aldrich ) were polymerized for 30 min at 25\u00b0C in polymerization buffer ( 50 mM KCl , 2 mM MgCl 2 , 1 mM ATP ) . Recombinant GST - epsin fragments ( final concentration 5 \u03bc M ) were pre - cleared by centrifugation at 150 , 000\u00d7 g for 1 hour at 4\u00b0C and incubated with F - actin ( final concentration : 15 \u03bc M ) . After 30 min of incubation at room temperature , samples were centri - fuged at 150 , 000\u00d7 g for 1 . 5 hours at 4\u00b0C . Supernatants and pellets were brought to equal volumes of SDS / PAGE Laemmli buffer , and samples were analyzed by SDS / PAGE using 4 \u2013 12 % Bis - Tris NuPAGE gel in MES buffer ( Life Technologies ) followed by anti - GST immunoblotting . Recombinant protein purification For GST - fusion protein production , BL21 - DE3 E . coli cells were transformed by heat - shock at 42\u00b0C with pGEX - 4T alone or pGEX - 4T constructs . Large - scale cultures were grown to log phase ( about 3 hours ) in Luria - Bertani broth containing ampicillin ( 100 mg / ml ) at 37\u00b0C , under shaking at 200 \u2013 250 rpm . Cultures were then induced with 500 mM isopropyl - \u03b2 - D - 1 - thiogalactopyranoside ( IPTG ) at 37\u00b0C for 3 hours and pelleted by centrifugation at 3000\u00d7 g for 15 min at 4\u00b0C . Bacteria were lysed in 150 mM NaCl , 4 mM DTT , 10 mM Hepes pH 7 . 4 , and protease inhibitor cocktail ( Roche ) in the presence of 1 % Triton X - 100 . Lysates were cleared by centrifugation at 22 , 000\u00d7 g for 20 min at 4\u00b0C , and the resulting supernatants were incubated with Glutathione - Sepharose 4 Fast Flow beads ( GE Healthcare ) for 1 hour at 4\u00b0C . After thorough washings with ice - cold buffer ( without Triton X - 100 ) , proteins were eluted by incubating the beads with 30 mM glutathione , 150 mM NaCl , 4 mM DTT , and 10 mM HEPES pH 8 . For His - tagged protein generation , BL21 - DE3 pLys - S E coli were transformed as described above with a pET21a ( + ) encoding a 6xHis - tagged fusion protein . After reaching the log phase at 37\u00b0C , bac - teria were induced overnight at 18\u00b0C with 100 mM IPTG . Bacterial pellet was lysed in buffer ( 150 mM NaCl , 1 mM \u03b2 - mercaptoethanol , 20 mM HEPES pH 7 . 4 , 1 % Triton X - 100 ) containing 15 mM imidazole , and the cleared supernatants were incubated with Ni 2 + \u2013 NTA agarose beads for 1 hour at 4\u00b0C . Samples were washed in the same buffer , but containing 30 mM imidazole , and finally recombinant proteins were eluted in the same buffer containing 300 mM imidazole . Both GST - and His - tagged proteins were subsequently purified by gel filtration using a Superdex S200 or a Superdex S75 column ( GE Healthcare ) on the Akta Pure FPLC system ( GE Healthcare ) . Finally , proteins were buffer exchanged in 150 mM NaCl , 4 mM DTT , 10 mM Hepes pH 7 . 4 by High - Prep 26 / 10 Desalting Column ( GE Healthcare ) . Protein purity was assessed by Coomassie Blue stain - ing of samples run on 4 \u2013 20 % gradient Tris / Glycine or 16 . 5 % Tris - Tricine MiniProtean gels ( Biorad , Hercules , CA , USA ) . GST pull - down assays to assess protein \u2013 protein interactions GST - tagged bait proteins bound to glutathione beads were incubated overnight at 4\u00b0C with constant agitation with 6xHis - tagged prey proteins in 150 mM NaCl , 4 mM DTT , 4 mM \u03b2 - mercaptoethanol , 0 . 1 % NP - 40 , 0 . 05 % BSA , 20 mM HEPES pH 7 . 4 . Reaction mixtures were subsequently cleared by centrifugation at 3000\u00d7 g for 5 min at 4\u00b0C ; supernatants were removed and beads were washed three times with ice - cold buffer . Bound proteins were eluted in Laemmli buffer and analyzed by separation on 16 . 5 % Tris - Tricine gradient Mini - Protean gels ( Biorad ) followed by Western blotting with anti - His antibody . In experiments where the effect of phosphoinositol groups was tested , the incubation mixture was supplemented with 50 \u03bc M final of diC8PI ( 4 , 5 ) P 2 , IP 3 ( 1 , 4 , 5 ) ( Avanti Polar Lipids , Alabaster , AL , USA ) or IP 6 ( 1 , 4 , 5 ) ( phytic acid dipotassium salt , Sigma - Aldrich ) . Generation of Syb2 \u2013 HA stable cell line Mouse fibroblasts stably expressing Syb2 \u2013 HA were generated by transducing WT and conditional epsin double KO mouse fibroblasts with a Syb2 \u2013 HA encoding retrovirus . The virus was produced in the Phoenix helper - free retrovirus producer line ( 293T cell line transformed with adenovirus E1a and carrying a temperature sensitive T antigen co - selected with neomycin ) . Briefly 10 \u03bc g of pBABE puro - Syb2 \u2013 HA were transfected by Lipofectamine 2000 ( Life Technologies ) into Phoenix cells . In parallel WT and conditional epsin double KO fibroblasts were plated . 24 hours after transfection , the medium from the Phoenix cell dishes was removed , supplemented with 8 \u03bc g / ml polybrene ( cationic polymer used to increase the retroviral infection efficiency in cell culture ) and filtered to remove cell debris . The filtrate was added to the mouse fibroblasts and removed after 4 \u2013 6 hours incubation at 37\u00b0C . 48 hours after the initial viral incubation fibroblasts were splitted in Dulbecco ' s Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 21 of 25 Research article Modified Eagle Medium ( Life Technologies ) . pBABE puro vector carries a puromycin resistance gene that was used to select the Syb2 \u2013 HA expressing cells by supplementing the culture medium with 4 \u03bc g / ml puromicyn ( [ Ferguson et al . , 2009 ] , Sigma - Aldrich ) . Quantification and statistical analysis Fluorescent signals were quantified with Fiji ImageJ ( http : / / fiji . sc / wiki / index . php / Fiji ) or the Volocity 3D Image Analysis software ( Improvision ) . Immunoblots were analyzed by Fiji ImageJ or Image Studio ( Licor Bioscience , Lincoln , NE , USA ) . Graphical presentations were made using Graph Pad Prism ( Graph Pad Software , La Jolla , CA , USA ) . Statistical analyses were performed by Graph Pad Prism using Student ' s t test for independent samples or one - way Anova . Miscellaneous procedures Bicinchoninic acid protein quantification ( Thermo Fisher ) , SDS - PAGE electrophoresis , immunoblotting , and GST - pulldowns from brain homogenates were performed by standard procedures ( Ferguson et al . , 2009 ) . Acknowledgements We thank Shawn Ferguson ( Yale University ) and Yixian Zheng ( Carnegie Institution for Science , Baltimore , MD ) for discussion and suggestions ; Frank Wilson , Lijuan Liu , and Louise Lucast for superb technical assistance and Alanna Coughran and Abigail Soyombo for help with the Hip1 / Hip1R DKO mice . Generous gifts of key reagents are acknowledged in the Materials and methods section . This work was supported in part by grants R37NS036251 , DK082700 , DK45735 and DA018343 to PDC , R01GM60979 to BW , R01CA098730 to TR and a Grant from the American Heart Association ( # 0835544N ) to HC . RF - B was the recipient of a Feodor Lynen postdoctoral fellowship from the Alexander von Humboldt Foundation , EWS was supported by NIH 5T32GM007223 - 38 / 39 and KW by NIGMST32007231 . Additional information Funding Funder Grant reference number Author Howard Hughes Medical Institute Mirko Messa , Rub\u00e9n Fern\u00e1ndez - Busnadiego , Elizabeth Wen Sun , Hong Chen , Heather Czapla , Yumei Wu , Genevieve Ko , Pietro De Camilli National Institute of Neurological Disorders and Stroke R37NS036251 Pietro De Camilli National Institute on Drug Abuse DA018343 Pietro De Camilli National Institute of Diabetes and Digestive and Kidney Diseases DK082700 , DK45735 Pietro De Camilli National Institute of General Medical Sciences R01GM60979 Beverly Wendland National Cancer Institute R01CA098730 Theodora Ross American Heart Association 0835544N Hong Chen Alexander von Humboldt - Stiftung Rub\u00e9n Fern\u00e1ndez - Busnadiego National Institute of General Medical Sciences NIGMST32007231 Kristie Wrasman National Institutes of Health 5T32GM007223 - 38 / 39 Elizabeth Wen Sun The funders had no role in study design , data collection and interpretation , or the decision to submit the work for publication . Cell biology Messa et al . eLife 2014 ; 3 : e03311 . DOI : 10 . 7554 / eLife . 03311 22 of 25 Research article Author contributions MM , RF - B , PDC , Conception and design , Acquisition of data , Analysis and interpretation of data , Drafting or revising the article , Contributed unpublished essential data or reagents ; EWS , Acquisition of data , Analysis and interpretation of data , Drafting or revising the article ; HC , TR , Drafting or revising the article , Contributed unpublished essential data or reagents ; HC , YW , Acquisition of data , Analysis and interpretation of data ; KW , Conception and design , Acquisition of data , Analysis and interpreta - tion of data ; GK , Acquisition of data , Contributed unpublished essential data or reagents ; BW , Conception and design , Analysis and interpretation of data , Drafting or revising the article , Contributed unpublished essential data or reagents Ethics Animal experimentation : The institutional animal care and use committee ( IACUC ) of the Yale University and the approved animal protocol is 2012 - 07422 . 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Zoncu R , Perera RM , Sebastian R , Nakatsu F , Chen H , Balla T , Ayala G , Toomre D , De Camilli PV . 2007 . Loss of endocytic clathrin - coated pits upon acute depletion of phosphatidylinositol 4 , 5 - bisphosphate . Proceedings of the National Academy of Sciences of USA 104 : 3793 \u2013 3798 . doi : 10 . 1073 / pnas . 0611733104 .", + "arasada2018highspeed": "Volume 29 February 1 , 2018 295 MBoC | ARTICLE High - speed superresolution imaging of the proteins in fission yeast clathrin - mediated endocytic actin patches ABSTRACT To internalize nutrients and cell surface receptors via clathrin - mediated endocy - tosis , cells assemble at least 50 proteins , including clathrin , clathrin - interacting proteins , actin filaments , and actin binding proteins , in a highly ordered and regulated manner . The mole - cular mechanism by which actin filament polymerization deforms the cell membrane is un - known , largely due to lack of knowledge about the organization of the regulatory proteins and actin filaments . We used high - speed superresolution localization microscopy of live fis - sion yeast cells to improve the spatial resolution to \u223c 35 nm with 1 - s temporal resolution . The nucleation promoting factors Wsp1p ( WASp ) and Myo1p ( myosin - I ) define two independent pathways that recruit Arp2 / 3 complex , which assembles two zones of actin filaments . Myo1p concentrates at the site of endocytosis and initiates a zone of actin filaments assembled by Arp2 / 3 complex . Wsp1p appears simultaneously at this site but subsequently moves away from the cell surface as it stimulates Arp2 / 3 complex to assemble a second zone of actin fila - ments . Cells lacking either nucleation - promoting factor assemble only one , stationary , zone of actin filaments . These observations support our two - zone hypothesis to explain endocytic tubule elongation and vesicle scission in fission yeast . INTRODUCTION Clathrin - mediated endocytosis recycles membrane receptors and takes up nutrients . Studies of budding yeast , fission yeast , and ani - mal cells identified many proteins that assemble and disassemble at endocytic sites . Recruitment of membrane proteins that recognize the endocytic cargo initiates the process at nascent endocytic sites . These sites mature with the assembly of a clathrin coat and recruit - ment of nucleation promoting factors and Arp2 / 3 complex that stim - ulate actin polymerization . Yeast cells use mechanical force provided by actin polymerization to overcome the internal turgor pressure and deform the membrane ( Aghamohammadzadeh and Ayscough , 2009 ; Minc et al . , 2009 ; Schaber et al . , 2010 ; Basu et al . , 2014 ) . However , it is still unclear how actin polymerization generates force to reshape the membrane , because conventional fluorescence microscopy has not resolved the organization of actin filaments or established the sites of actin polymerization . One model based on the localization of the nucleation promoting factors , myosin - I and WASp ( Las17 ) in budding yeast , proposes that the actin filaments polymerize against the plasma membrane at the neck of endocytic invaginations . Consequently , the barbed ends of the elongating ac - tin filaments are oriented toward the plasma membrane at the base of the membrane invagination with their pointed ends anchored to the clathrin coat via the adapter proteins at the invagination tip ( Kaksonen et al . , 2005 ; Skruzny et al . , 2012 ; Picco et al . , 2015 ) . Actin polymerization primarily at the plasma membrane together with some myosin motor activity is proposed to generate force to elon - gate the membrane tubule . On the basis of the localization of the nucleation promoting fac - tors in fission yeast , we proposed another model to explain how actin polymerization drives endocytosis ( Arasada and Pollard , 2011 ) . In our model , actin polymerization occurs in two distinct zones : one Monitoring Editor David G . Drubin University of California , Berkeley Received : Jun 21 , 2017 Revised : Nov 28 , 2017 Accepted : Nov 28 , 2017 This article was published online ahead of print in MBoC in Press ( http : / / www . molbiolcell . org / cgi / doi / 10 . 1091 / mbc . E17 - 06 - 0415 ) on December 6 , 2017 . * Address correspondence to : Thomas D . Pollard ( thomas . pollard @ yale . edu ) . \u00a9 2018 Arasada et al . This article is distributed by The American Society for Cell Biology under license from the author ( s ) . Two months after publication it is avail - able to the public under an Attribution \u2013 Noncommercial \u2013 Share Alike 3 . 0 Unported Creative Commons License ( http : / / creativecommons . org / licenses / by - nc - sa / 3 . 0 ) . \u201cASCB \u00ae , \u201d \u201cThe American Society for Cell Biology \u00ae , \u201d and \u201cMolecular Biology of the Cell \u00ae \u201d are registered trademarks of The American Society for Cell Biology . Abbreviations used : ARPC5 , Arp2 / 3 complex subunit 5 ; CHD , calponin homology domain ; DIC , differential interference contrast ; EMM5S , Edinburgh minimal me - dium 5 supplements ; Fim , fimbrin ; FPALM , fluorescence photoactivation localiza - tion microscopy ; mEGFP , monomeric enhanced green fluorescent protein ; Myo1p , myosin - I ; PALM , photoactivated localization microscopy ; sCMOS , scien - tific complementary metal - oxide - semiconductor ; STORM , stochastic optical re - construction microscopy ; Wsp1p , Wiskott - Aldrich syndrome protein . Rajesh Arasada a , Wasim A . Sayyad a , Julien Berro b , c , d , and Thomas D . Pollard a , b , c , * a Departments of Molecular Cellular and Developmental Biology , b Departments of Molecular Biophysics and Biochemistry , c Department of Cell Biology , and d Nanobiology Institute , Yale University , New Haven , CT 06520 - 8103 296 | R . Arasada et al . Molecular Biology of the Cell Over time , all of the fluorescent proteins within the \u223c 400 - nm - thick imaging plane were localized with an average radial precision of 21 \u00b1 4 nm , and the centroids of each molecule were plotted as two - dimensional histograms ( Figure 1B , middle panel ) . Software rejected molecules outside the imaging plane during image processing . To aid visualization the two - dimensional histograms of centroids of at the base of the membrane invagination and the other traveling with the tip of the invagination . This model postulates that the ex - panding actin networks in the two zones repel each other as they grow , driving invagination of the plasma membrane tubule . This two - zone model is based on quantitative measurements by confocal fluorescence microscopy of fission yeast actin patch pro - teins fused to mEGFP or mYFP ( Sirotkin et al . , 2010 ; Arasada and Pollard , 2011 ) . Two activators of Arp2 / 3 complex , type - I myosin Myo1p and WASp homologue Wsp1p , localize and initiate actin po - lymerization together but then separate with Myo1p at the base of plasma membrane invagination and Wsp1p at the tip of the invagi - nation ( Figure 1A ) . These nucleation promoting factors recruit very high densities ( \u223c 20 , 000 molecules per \u00b5m 2 ) of two F - BAR ( Fes / CIP4 homology Bin - Amphiphysin - Rvs ) proteins , Cdc15p and Bzz1p , onto the invaginating membrane tubule . Cdc15p interacts with Myo1p at the base of the membrane tubule and Bzz1p interacts with Wsp1p at the invagination tip . The F - BAR proteins interact with the nucle - ation promoting factors to stimulate Arp2 / 3 complex in two zones along the invaginating tubule . However , in yeast cells expressing GFP - actin or GFP - tagged proteins that bind actin filaments , the dense network of \u223c 5000 polymerized actin molecules appears as a single mass of homogeneous fluorescence \u223c 500 nm in diameter at sites of endocytosis rather than as two distinct polymerization zones ( Arasada and Pollard , 2011 ) . Better spatial resolution was required to determine whether actin polymerizes in two zones . Since the two yeast cells diverged from a common ancestor \u223c 400 million years ago and have adapted differently during their subse - quent evolution , they may control actin assembly during endocyto - sis in different ways . On the other hand , it is worth considering whether endocytosis in the two yeasts has more in common than suggested by these two models . We used high - speed fluorescence photoactivation localization microscopy ( FPALM ) of live Schizosaccharomyces pombe cells ex - pressing photoconvertible fluorescent proteins ( Huang et al . , 2013 ; Laplante et al . , 2016a , b ) to study the organization of proteins in ac - tin patches . We obtained 1 - s temporal resolution and \u223c 35 - nm spa - tial resolution and observed that actin filaments assemble in two zones : one at the base of the membrane invagination and the other at the tip of the invagination . Polymerization of actin in both zones depends on Arp2 / 3 complex , which is activated by type - I myosin Myo1p at the base of the invagination and WASp homologue Wsp1p along the path of the invagination from the cell surface to the tip of the membrane tubule . These observations support the two - zone hypothesis for the assembly of actin in association with endocytosis in fission yeast . RESULTS Observations of protein turnover in actin patches of live fission yeast cells by FPALM We examined actin filaments and four components of the actin as - sembly machinery in live fission yeast using a single - molecule , su - perresolution method , FPALM ( also called STORM or PALM ) ( Huang et al . , 2013 ; Laplante et al . , 2016a ) . We tagged proteins with the photoconvertible fluorescent proteins mMaple3 or mEOS3 . 2 at their gene loci unless indicated otherwise . The cell in Figure 1 , B and C expressed the actin patch component capping protein Acp2p tagged with mEOS3 . 2 . We used epifluorescence illumination to ex - pose live S . pombe cells expressing photoconvertible fluorescent proteins continuously with both a near UV laser ( 405 nm ) to photo - convert the fluorescent proteins randomly to the state that emits red light and a yellow laser ( 564 nm ) to image the red light emitted by individual , spatially separated , photoconverted fluorescent proteins . FIGURE 1 : FPALM improves the spatial resolution of actin patches over conventional fluorescence microscope . ( A ) Two - zone model based on quantitative confocal fluorescence microscopy . The cartoon represents an actin patch at \u2013 2 s when the membrane tubule elongates and at 0 s when the vesicle pinches away from the plasma membrane . The plasma membrane is a black line , the clathrin coat is gray , nucleation - promoting factors Wsp1p and Myo1p are yellow , and zones with actin filaments are blue . Growth of the branched filaments from two distinct zones of NPFs is postulated to push the tip of the invaginating tubule away from the cell surface contributing to the elongation of the tubule and scission of the coated vesicle . ( B , C ) Fluorescence micrographs of an S . pombe cell expressing capping protein Acp2p - mEOS3 . 2 with focusing in the middle plane of the cell . We used continuous epifluorescence illumination to photoconvert mEOS3 . 2 with 405 - and 564 - nm lasers to excite the photoconverted mEOS3 . 2 through the entire cell . Top panels , wide - field epifluorescence images reconstructed from the total fluorescence emission . Middle panels , raw FPALM images constructed from the localizations of single molecules . Bottom panels , each localized emitter in the raw data set was convolved with a Gaussian kernel ( \u03c3 = 1 . 5 pixel ) and color coded for density in a heat map . ( B ) Whole cell during a 1 - s interval . Scale bar is 1 \u00b5m . ( C ) Time series of images of one actin patch at 1 - s intervals each reconstructed from 200 sequential frames . Top panel , inverted contrast wide - field epifluorescence images . Scale bar is 250 nm . Volume 29 February 1 , 2018 Organization of S . pombe a ctin patches | 297 ( Figure 2B , center micrograph ) . The clusters of mMaple3 - Wsp1p maintained a constant size with a width of 100 \u00b1 38 nm and length of 200 nm ( Figure 2 , C and D , center graphs ) as they moved from the membrane proximal zone to the membrane distal zone ( a box \u223c 250 nm wide located in the cytoplasm between 200 and 550 nm from the cell edge ) ( Figure 2E , middle panel , and Supplemental Figure S1B ) . mMaple3 - Wsp1p appeared in the membrane proximal zone and peaked there at \u223c 50 localizations per second . The peak of \u223c 50 mMaple3 - Wsp1p localizations per second in the membrane distal zone was \u223c 6 s later . The localizations of Arc5p - mMaple3 ( the marker for Arp2 / 3 com - plex ) appeared first at the plasma membrane and then deeper in the cytoplasm at later times ( Figure 2A , bottom panel ) , as illustrated by composite images with temporal color coding ( Figure 2B , right micrograph ) . Throughout this shift in the localizations over time , clusters of Arc5p - mMaple3 were 122 \u00b1 62 nm wide and 300 nm long ( Figure 2 , C and D , right panels ) . Like mMaple3 - Wsp1p , the localiza - tions of Arc5p - mMaple3 molecules first peaked at \u223c 150 per second in the membrane proximal zone followed \u223c 5 s later by a peak in membrane distal zone ( Figure 2E , right panel , and Supplemental Figure S1C ) . Actin assembles in two distinct zones in an actin patch We localized actin filaments indirectly with two different probes : 1 ) an actin filament binding protein fimbrin ( Fim1p ) tagged to mEOS3 . 2 at its C - terminus and expressed from the endoge - nous locus ; and 2 ) calponin homology domain ( CHD ) from the S . pombe IQGAP protein Rng2p fused to mEOS3 . 2 at its N termi - nus and expressed ectopically from a repressible promoter at the leu1 + locus ( Figure 3 ) . Fim1p - mEOS3 . 2 expressed from the native promoter has minimal impact on the organization of actin filaments at endocytic sites ( Berro and Pollard , 2014b , a ) . CHD tagged with a fluorescent protein binds actin filaments , but it also appears to bundle actin filaments and may influence cell mor - phology when overexpressed . The time course of the appear - ance and disappearance of Fim1p - mEOS3 . 2 in patches ( Figure 3H ) was similar to a trace of Fim1p - mGFP or Fim1p - mCherry ob - served by confocal microscopy ( Sirotkin et al . , 2010 ; Berro and Pollard , 2014a ) . Both actin filament probes appeared in two distinct zones at sites of endocytosis ( Figure 3 , A and B ) , first close to the plasma mem - brane and then in a second zone further from the plasma mem - brane . Throughout their existence , the clusters of single molecule localizations of mEOS3 . 2 - CHD in patches were 110 \u00b1 55 nm wide ( along the cell long axis ) and 350 nm long , while those of Fim1p - mEOS3 . 2 were 101 \u00b1 36 nm wide and 300 nm long , both with two distinct peaks ( Figure 3 , C \u2013 F ) . Reconstructions of series of images ( Figure 3A ) with temporal color - coding emphasize that actin assem - bly begins at the plasma membrane and shifts to a site deeper in the cytoplasm over time ( Figure 3 , G and H ) . Like Arp2 / 3 complex , the mEOS3 . 2 - CHD and Fim1p - mEOS3 . 2 first peaked at \u223c 150 localiza - tions per second in the membrane proximal zone followed \u223c 4 s later by a peak in the membrane distal zone ( Figure 3 , I and J , and Sup - plemental Figure S1 , D and E ) . Starting at time 0 s , the localizations of each actin patch protein declined and appeared further from the plasma membrane . At the onset of patch assembly and up to time zero , the localizations of the patch proteins were tightly packed in a column normal to the plasma membrane . After time zero , the positions of these localizations be - came more variable and more spread out . The localizations colored in red at the end of the disassembly phase were more spread out and noisy due to fewer localizations . localized molecules were convolved with a two - dimensional Gauss - ian kernel ( \u03c3 = 7 . 5 nm ) and color coded for localization density ( Figure 1B , bottom panel ) . It is important to note that photoactivation localization mi - croscopy depends on irreversibly photobleaching each fluores - cent protein after it is imaged and localized , so a time series of FPALM images reveals the position of each molecule when it is photoconverted . Photobleaching occurs in less than 2 s under our conditions ( Laplante et al . , 2016b ) . However , the method does not reveal the subsequent history of the photobleached protein , such as its persistence at the site of localization or any motion . Actin patches concentrate at the poles of the cells but are spread around the surface , so they are viewed from many differ - ent angles . Therefore , we used differential interference contrast ( DIC ) microscopy to identify the midplane of the cells , where ac - tin patches moving in the XY - plane of the microscope were con - tained in the optical section throughout their lifetimes . To obtain enough single - molecule localizations to form an image , we com - bined groups of 200 sequential 5 - ms frames . These 1 - s compos - ite images were linked into movies or montages to show actin patch dynamics . These FPALM images highlight structural details of actin patches that appear as blurred , pixelated spots of fluo - rescence by wide - field fluorescence micro scopy ( Figure 1C , top panel ) . Localization of Arp2 / 3 complex activators and Arp2 / 3 complex We tagged three proteins that control the assembly of actin fila - ments at sites of endocytosis and established their distributions over time in actin patches . We inserted the open reading frame for mMaple3 ( Wang et al . , 2014 ) into the genome so the fluorescent protein was fused to the N - termini of Arp2 / 3 complex activators myosin - I ( Myo1p ) and Wiskott - Aldrich syndrome protein ( Wsp1p ) and on the C - terminus of the Arc5p ( ARPC5 ) subunit of Arp2 / 3 complex , so all of the tagged proteins were expressed from their endogenous promoters . Schizosaccharomyces pombe cells ex - pressing these fusion proteins were viable and had wild - type morphologies at 25\u00b0 and 36\u00b0C . In wild - type cells , localizations of mMaple3 - Myo1p appeared in a small , stationary region 65 \u00b1 18 nm ( mean \u00b1 SD ) wide and extending 85 \u00b1 18 nm from the plasma membrane ( Figure 2 , A , C , and D , left graphs ) as actin patches assembled and disassembled over time ( Figure 2A , top panel ) . A composite image with tempo - ral color coding according to the time of localization shows the time course of the whole process ( Figure 2B , left micrograph ) . Counts of mMaple3 - Myo1p localizations in actin patches over time established that 82 % of Myo1p localizations appeared and disappeared within membrane proximal zone ( defined as box \u223c 250 nm wide , extending 200 nm into the cytoplasm from the cell edge ) ( left panels in Figure 2B and and Supplemental Figure S1A ) . These observations confirmed at much higher resolution the lack of mobility of Myo1p tagged with mEGFP ( monomeric enhanced green fluorescent protein ) and imaged by confocal microscopy ( Sirotkin et al . , 2005 , 2010 ) . Over \u223c 15 s , mMaple3 - Wsp1p appeared at the plasma mem - brane and subsequently was localized along a trajectory normal to the membrane ( middle column of Figure 2 , B \u2013 E ) , similarly to lower - resolution confocal micrographs of cells expressing mEGFP - Wsp1p ( Sirotkin et al . , 2010 ) . Reconstructions of time series with temporal color - coding illustrate that mMaple3 - Wsp1p molecules were local - ized up to 300 nm from the plasma membrane at late time points 298 | R . Arasada et al . Molecular Biology of the Cell Assembly of two zones of actin is required for endocytic tubule elongation and vesicle scission from the plasma membrane Both Myo1p and Wsp1p contribute to ac - tivating Arp2 / 3 complex and the assembly actin filaments at sites of endocytosis in fis - sion yeast . Both \u2206 wsp1 and \u2206 myo1 mutant cells are viable with defects in endocytosis , but double mutant \u2206 wsp1 \u2206 myo1 cells are not viable ( Lee et al . , 2000 ; Sirotkin et al . , 2005 ) . We investigated the relationships among Myo1p , Wsp1p , Arp2 / 3 complex , and actin filaments by analyzing \u2206 wsp1 yeast cells lacking Wsp1p or \u2206 myo1 cells lacking Myo1p ( Figure 4 ) . We compared the distribution of Arc5p - mMaple3 and mEOS3 . 2 - CHD in the mutant strains with wild - type cells . Both single deletion strains differed from wild - type cells in two regards : 1 ) they accumulated far less Arc5p - mMaple3 than FIGURE 2 : Localization of nucleation promoting factors and Arp2 / 3 complex in actin patches by FPALM in wild - type cells . ( A ) Time series of FPALM images color coded for the density of localizations : top , mMaple3 - Myo1p ; middle , mMaple3 - Wsp1p ; and bottom , Arc5p - mMaple3 a subunit of Arp2 / 3 complex . The micrographs were aligned to time 0 s when the vesicle pinches away from the plasma membrane . For mMaple3 - Myo1p the peak localizations were aligned to the peak localizations of mMaple3 - Wsp1p . Yellow dashed lines show the position of the plasma membrane . Scale bar is 100 nm . ( B ) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches : left , 18 s from a cell expressing mMaple3 - Myo1p ; center , 16 s from a cell expressing mMaple3 - Wsp1p ; and right , 21 s from a cell expressing Arc5p - mMaple3 a subunit of Arp2 / 3 complex . The blue and red lines indicate the membrane - proximal and membrane - distal regions . Scale bar is 200 nm . ( C , D ) Spatial distributions of single molecule localizations in FPALM images of actin patches . FWHM is full - width of the distributions at half - maximums . ( C ) Width distributions and ( D ) length distributions of localizations of left , mMaple3 - Myo1p in 20 actin patches ; center , mMaple3 - Wsp1p in 12 actin patches ; and right , Arc5p - mMaple3 in 18 actin patches . The data from separate patches were aligned to their peaks and averaged . Full - widths at half - maximum were calculated from the distributions of the localizations along the x - axis . The lengths of the patches are given as the distances that include 90 % of the localizations from the cell edge . The gray area in the graphs represents the localizations detected outside the cell . ( E ) Time courses of the average number of localizations detected in ( blue line ) the membrane proximal zone ( 200 \u00d7 250 - nm region next to the cell edge ) and ( red line ) the membrane distal zone ( 350 \u00d7 250 - nm region 200 nm away from the cell edge ) : left , mMaple3 - Myo1p ; middle , mMaple3 - Wsp1p ; and right , Arc5p - mMaple3 . Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations . Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches . Time 0 s indicates vesicle scission . Error bars are SDs . Every third error bar is shown . Volume 29 February 1 , 2018 Organization of S . pombe a ctin patches | 299 wild - type cells ( Figure 4A ) ; and 2 ) the localizations of Arc5p - mMaple3 appeared and remained next to the plasma membrane ( Figure 4 , A and B ) as actin patches assembled and disassembled over time . Thus , few Arc5p - mMaple3 molecules were localized in the membrane distal zone at any time ( Figure 4E and Supplemen - tal Figure S2A ) . In cells lacking either Myo1p or Wsp1p , the actin filament marker mEOS3 . 2 - CHD remained close to the plasma membrane and per - sisted there longer than in wild - type cells ( Figure 4F and Suppleme - mental Figure S2B ) . The peak numbers of mEOS3 . 2 - CHD localiza - tions in membrane proximal zones of actin patches were similar in wild - type cells and cells without Myo1p or Wsp1p , but few of the localizations appeared in the membrane distal zone . DISCUSSION High - speed FPALM increases the spatial resolution nearly 10 - fold compared with confocal microscopy and is fast enough to follow the assembly and disassembly of actin patches . We used the estab - lished timing of GFP - tagged proteins ( Sirotkin et al . , 2010 ; Arasada and Pollard , 2011 ) to align the timing of the superresolution micro - scopy data , with 0 s as the time when some of the patch compo - nents start to move away from the cell surface after vesicle scission ( Figure 5 ) . High - resolution images aligned in this way at 1 - s intervals helped us distinguish elongation of the membrane tubule from ves - icle scission and support the two - zone hypothesis . Note that after random photoconversion FPALM irreversibly bleaches each fluorescent protein in the imaging beam in \u223c 2 s ( Laplante et al . , 2016b ) , so the method does not reveal how long each molecule persists at its detected location . Thus , the absence of localizations does not necessarily mean that the tagged protein has turned over . For example , actin may persist longer in the membrane proximal zone than the detection of newly localized proteins . Status of the two - zone hypothesis The key feature of the two - zone hypothesis is that the two major nucleation promoting factors , Myo1p and Wsp1p , are distributed differently on the membrane tubules , so they can drive the reactions FIGURE 3 : Localization of actin filaments in two zones in endocytic actin patches in wild - type S . pombe cells . ( A , B ) Time series of fluorescence micrographs at 1 - s intervals of representative actin patches in cells expressing proteins that bind actin filaments . The images are regions of interest of \u223c 500 \u00d7 500 nm around one actin patch for each protein . Scale bars are 250 nm . ( A ) GFP - CHD and mEOS3 . 2 - CHD and ( B ) Fim1p - GFP and Fim1p - mEOS3 . 2 . Top panels , maximum intensity projections of five confocal z - slices in the XY - plane of individual actin patches from cells expressing GFP - CHD or Fim1p - GFP . Bottom panels , FPLAM images of cells expressing mEOS3 . 2 - CHD or Fim1p - mEOS3 . 2 . Each localized emitter was convolved with a Gaussian kernel ( \u03c3 = 1 . 5 pixel ) and color coded for density in a heat map . Yellow dashed lines show the location of the plasma membrane ( PM ) . ( C \u2013 F ) Spatial distributions of single molecule localizations of mEOS3 . 2 - CHD and Fim1p - mEOS3 . 2 in FPALM images of actin patches . FWHM is full - width of the distributions at half - maximums . ( C ) Width distribution and ( E ) length distribution of mEOS3 . 2 - CHD in 20 actin patches . ( D ) Width distribution and ( F ) length distribution of Fim1p - mEOS3 . 2 in 14 actin patches prepared as in Figure 2 . ( G , H ) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches . The micrographs are aligned to time 0 s when the vesicle pinches off from the plasma membrane . Scale bars are 200 nm . The blue and red lines indicate the membrane - proximal and membrane - distal regions . ( G ) Twenty - one seconds from a cell expressing mEos3 . 2 - CHD . ( H ) Seventeen seconds from a cell expressing Fim1p - mEos3 . 2 . ( I , J ) Time courses of the average number of localizations of ( I ) CHD - mEOS3 . 2 and ( J ) Fim1p - mEOS . 2 detected in ( blue line ) the membrane proximal zone ( 200 \u00d7 250 - nm region next to the cell edge ) and ( red line ) the membrane distal zone ( 350 \u00d7 250 - nm region 200 nm away from the cell edge ) . Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations . Time zero seconds indicates vesicle scission . Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches . Error bars are SDs . Every third error bar is shown . 300 | R . Arasada et al . Molecular Biology of the Cell that produce two zones of actin polymeriza - tion . Confocal microscopy showed that Myo1p and Wsp1p have lifetimes of \u223c 15 s in actin patches . They appear at the same time , peak together at time \u2013 3 s with \u223c 165 molecules of Myo1p and \u223c 140 molecules of Wsp1p , and disappear together around time + 6 s ( Sirotkin et al . , 2010 ; Arasada and Pollard , 2011 ) . Superresolution images show that Myo1p is stationary at the base of the membrane invagination , while Wsp1p first appears at the same place as Myo1p followed by new localizations further from the plasma membrane . This relocation of FIGURE 4 : Actin patch assembly in cells lacking nucleation promoting factors Myo1p or Wsp1p . ( A , B ) Time series of FPALM images color coded for localization density showing the ( A ) Arc5p - mMaple3 a subunit of Arp2 / 3 complex or ( B ) mEOS3 . 2 - CHD to visualize actin filaments . Top panels , wild - type cells ; middle panels , \u2206 myo1 cells ; and bottom panels , \u2206 wsp1 cells . Yellow dotted lines indicate the cell membrane . The micrographs are aligned to time 0 s when the vesicle pinches off from the plasma membrane . Scale bars are 250 nm . ( C , D ) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches . The blue and red lines indicate the membrane - proximal and membrane distal regions . ( C ) Cells expressing Arc5p - mMaple3 : left , 16 s of an actin patch in a wild - type cell ; center , 18 s of an actin patch in a \u2206 myo1 cell ; and right , 18 s of an actin patch in a \u2206 wsp1 cell . ( D ) Cells expressing mEOS3 . 2 - CHD : left , 15 s of an actin patch in a wild - type cell ; center , 27 s of an actin patch in a \u2206 myo1 cell ; and right , 28 s of an actin patch in a \u2206 wsp1 cells . ( E , F ) Time courses of the average number of localizations of ( E ) Arc5p - mMaple3 and ( F ) mEOS . 2 - CHD detected in actin patches in ( blue line ) the membrane proximal zone ( 200 \u00d7 250 - nm region next to the cell edge ) and ( red line ) the membrane distal zone ( 350 \u00d7 250 - nm region 200 nm away from the cell edge ) : left , wild - type cells ; middle , \u2206 myo1 cells ; and right , \u2206 wsp1 cells . Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations . Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches . Time 0 s indicates the vesicle scission . Error bars are SDs . Every third error bar is shown . Volume 29 February 1 , 2018 Organization of S . pombe a ctin patches | 301 the ability of Myo1p to stimulate actin polymerization by Arp2 / 3 complex in fission yeast ( Arasada and Pollard , 2011 ) and phosphory - lation of the Myo1p TEDS site ( Serine 361 ) by Pak1p appears to be the upstream signal that localizes Myo1p at clathrin - coated pits ( Attanapola et al . , 2009 ) . In both yeasts , WASp initially concentrates near the plasma membrane and recruits the F - BAR protein Bzz1p , which increases the ability of Wsp1p to activate Arp2 / 3 complex to assemble actin filaments ( Padrick et al . , 2008 ; Arasada and Pollard , 2011 ) . The main difference is the subsequent behavior of WASp . The current work confirms that fission yeast Wsp1p moves away from the cell surface along with Arp2 / 3 complex and actin filaments ( Arasada and Pollard , 2011 ) , while multiple papers ( Kaksonen et al . , 2005 ; Sun et al . , 2006 , 2017 ; Rajmohan et al . , 2009 ; Picco et al . , 2015 ) show that budding yeast Las17 - GFP remains close to the cell sur - face during endocytosis . Consistent with a single zone of nucleation promoting factors in budding yeast , elegant photobleaching ex - periments showed that actin filaments appear at the base of the membrane invagination ( Picco et al . , 2015 ) . The accumulated work suggests that the behavior of WASp dif - fers in the two yeasts , supporting a single zone of actin assembly in budding yeast and two zones in fission yeast . Exploring this differ - ence is worthwhile for two reasons . First , tagging Las17 or Wsp1p can influence their behavior . Growth and the time course of patch assembly are normal in fission yeast cells dependent on mGFP - Wsp1p ( Sirotkin et al . , 2005 ) , rather than growing slowly like mis - shapen , temperature - sensitive \u2206 wsp1 cells . However , the GFP - Wsp1p strain has defects in mating and sporulation , as observed in \u2206 wsp1 cells . Tagging either end of Las17 with GFP may compro - mise patch mobility ( Galletta et al . , 2008 ) . Second , electron micros - copy of sections of chemically fixed budding yeast cells treated with gold - labeled antibodies showed that Las17 with a small C - terminal HA peptide tag distributed significantly further than myo - sin - I along endocytic membrane invaginations as they elongated ( p < 0 . 001 ) . However , chemical fixation may have compromised the preservation . High - pressure freezing followed by freeze substitu - tion improves the preservation and is compatible with antibody staining for electron microscopy ( Buser and Drubin , 2013 ) or cor - relative fluorescence and electron tomography ( Kukulski et al . , 2012 ) , but neither Las17 nor myosin - I have been localized by these methods . Wsp1p begins at time \u2013 3 s coincident with the peak of Wsp1p local - izations and the accumulation of localizations in the membrane dis - tal zone of Arp2 / 3 complex ( Arc5p ) and actin filaments ( CHD and Fim1p ) . Localizations of both Arp2 / 3 complex and actin filaments peak at time 0 s . Both Arp2 / 3 complex ( Figure 3B ) and actin ( Figure 2 , H and I ) form two spatially and temporally separated networks of about the same size . Judging from their sudden increase in mobility , patches pinch off from the membrane with most of the actin network at time 0 s ( Berro and Pollard , 2014b , a ) . After time 0 , patches diffuse in the cytoplasm at increasing rates as they disassemble their actin filament coat over 6 s ( Berro and Pollard , 2014b , a ) . Localizations of Arc5p , CHD , and Fim1p decline more rapidly in the membrane proximal zone than the membrane distal region . These proteins may dissociate from the patch or persist after they are localized . The sudden collapse of the membrane tubule after vesicle scission might be a factor in dispers - ing the actin . How much does endocytosis in fission and budding yeast have in common ? In spite of differences in a few observations , most evidence sup - ports the conclusion that the mechanisms of endocytosis in bud - ding yeast and fission yeast are fundamentally the same . The two yeasts have a common ancestor and use most of the same pro - teins to form a membrane invagination \u223c 150 nm long surrounded by actin filaments ( Kaksonen et al . , 2005 ; Sirotkin et al . , 2010 ; Arasada and Pollard , 2011 ; Goode et al . , 2015 ; Lu et al . , 2016 ) . Actin polymerization in both yeasts depends on Arp2 / 3 complex and two types of nucleation promoting factors , myosin - I ( MYO3 and MYO5 in budding yeast and Myo1p in fission yeast ) and WASp homologues ( LAS17 in budding yeast and Wsp1p in fission yeast ) . In both the yeasts , myosin - I appears near the plasma membrane at the base of the tubular invagination ( Kaksonen et al . , 2005 ; Sun et al . , 2006 ; Galletta et al . , 2008 ; Idrissi et al . , 2008 ; Sirotkin et al . , 2010 ) and recruits Arp2 / 3 complex to nucleate branched actin fila - ments that give rise to the membrane - proximal network of fila - ments . In both yeasts , interactions of myosin - Is with adapter and coat proteins ( Sun et al . , 2006 ; Barker et al . , 2007 ; Arasada and Pollard , 2011 ) are important for efficient endocytosis . For example , recruitment of F - BAR protein Cdc15p to the endocytic site enhances FIGURE 5 : Hypothesis for the contributions of the nucleation promoting factors Myo1p and Wsp1p to endocytosis . Different stages of endocytosis with time zero defined as vesicle scission from the plasma membrane . The plasma membrane is a black line , clathrin is gray , nucleation promoting factors Wsp1p and Myo1p are blue , and actin filaments are red , green , and yellow . Clathrin is recruited 2 min prior to invagination . Nucleation promoting factors are recruited beginning at \u2013 6 s and peak prior to patch movement at \u2013 3 s . Myo1p and Wsp1p both stimulate actin polymerization at the base of the invagination near the cell surface . As the actin zones around Myo1p and Wsp1p grow , they repel each other , driving membrane invagination and redistribution of Wsp1p away from the base along the membrane invagination creating new sites of actin polymerization at the invagination tip . Myo1p remains associated with the plasma membrane at the base of the invagination . The expansion of the two zones triggers vesicle scission between the two zones of actin at time 0 s . 302 | R . Arasada et al . Molecular Biology of the Cell Models of actin assembly Understanding the forces produced by actin assembly during endo - cytosis depends on knowing the sites of assembly and the orienta - tions of the filaments . Electron microscopy of chemically fixed and freeze - substituted yeast cells ( Idrissi et al . , 2008 ) , platinum replicas of fixed and extracted animal cells ( Collins et al . , 2011 ) , and quanti - tative fluorescence microscopy of live cells ( Kaksonen et al . , 2005 ; Sirotkin et al . , 2005 , 2010 ; Berro et al . , 2010 ; Berro and Pollard , 2014b , a ; Arasada and Pollard , 2011 ; Picco et al . , 2015 ) have sug - gested three possible orientations of filaments in actin patches as follows : 1 ) actin filaments are assembled from the plasma mem - brane with barbed ends oriented toward the plasma membrane ( Kaksonen et al . , 2005 ; Picco et al . , 2015 ) ; 2 ) actin filaments are oriented with the barbed ends pointed toward the tip of the mem - brane invagination ( Collins et al . , 2011 ) ; and 3 ) very short actin filaments ( Young et al . , 2004 ; Kaksonen et al . , 2005 ; Sirotkin et al . , 2005 , 2010 ; Berro et al . , 2010 ; Berro and Pollard , 2014b , a ; Arasada and Pollard , 2011 ; Picco et al . , 2015 ) are oriented randomly in one zone ( Young et al . , 2004 ; Kaksonen et al . , 2005 ; Sirotkin et al . , 2005 , 2010 ; Berro et al . , 2010 ; Berro and Pollard , 2014b , a ; Arasada and Pollard , 2011 ; Picco et al . , 2015 ) or two zones ( Arasada and Pollard , 2011 ) . Model 3 with two zones is simpler and more appealing to us , because it does not require mechanisms to orient the filaments . Dis - tinguishing these hypotheses definitively will require methods to document the arrangement of individual filaments . MATERIALS AND METHODS Strain construction , growth conditions , and yeast methods Supplemental Table S1 lists the S . pombe strains used in this study . Fluorescent protein ( FP ) tag sequences were integrated into the genome by PCR - based gene tagging ( Bahler et al . , 1998 ) . All ge - nomic integrations were confirmed by PCR and fluorescence mi - croscopy . We used plasmid pFA6a - mEos3 . 2 - kanMX6 to tag pro - teins with mEOS3 . 2 at their endogenous genomic loci ( Laplante et al . , 2016b ) . We constructed plasmid pFA6a - mMaple3 - KanMX6 to tag proteins at their endogenous loci with mMaple3 . mMaple3 cDNA was PCR amplified and replaced mEOS3 . 2 in pFA6a - mEos2 - kanMX6 . We constructed pFA6a - kanMX6 - Pmyo1 - mMaple3 and pFA6a - kanMX6 - Pwsp1 - mMaple3 plasmids for N - terminal tagging of Myo1p and Wsp1p . We PCR amplified the coding sequence of mMaple3 ( Wang et al . , 2014 ) and replaced the mEGFP coding sequence of pFA6a - kanMX6 - Pmyo1 - mEGFP and pFA6a - kanMX6 - Pmyo1 - mEGFP using the restriction sites Pac I and Asc I ( Sirotkin et al . , 2005 ) . Actin filaments were visualized indirectly with either with Fim1p - mEOS3 . 2 or with Rng2p CHD . To express mEOS3 . 2 - CHD or GFP - CHD , cells expressing mEOS3 . 2 - CHD or GFP - CHD under the control of a 41x nmt1 promoter were grown in Edinburgh minimal media ( EMM5S ) for 18 h before imaging . Preparation of cells for imaging All S . pombe cells expressing a fluorescently tagged protein were grown in YE5S at 25\u00b0C to OD 595 0 . 2 \u2013 0 . 5 , washed in EMM5S filtered through a 0 . 45 - \u00b5m filter , and mounted on 25 % gelatin pads in fil - tered EMM5S . DIC images were collected prior to superresolution imaging . Superresolution data acquisition , data analysis , and display Superresolution imaging was performed with a custom - built two - dimensional FPALM system ( Huang et al . , 2013 ; Laplante et al . , 2016a , b ) equipped with a scientific complementary metal - oxide - semiconductor ( sCMOS ) camera ( ORCA - Flash4 . 0 ; Hamamatsu ) . Wide - field epifluorescence optics were used to illuminate cells simultaneously with a near - UV laser ( 405 nm ) to photoconvert small numbers of random fluorescent proteins and 564 - nm laser to excite the fluorophores through the entire cell . The average intensity of the 564 - nm laser used to excite photoconverted mEOS3 . 2 and mMaple3 for imaging was \u223c 1 . 2 kW / cm 2 . To main - tain an optimal density of photoconverted mEOS3 . 2 or mMaple3 for single molecule localization , the power of the 405 - nm photo - conversion laser was increased every 5 s during data acquisition with a computer - controlled ramp to compensate for irreversible photobleaching of molecules that were photoconverted and im - aged . Single - molecule images were acquired at a frame rate of 200 frames / s for 40 \u2013 60 s from the medial focal plane of a cell with an sCMOS camera ( ORCA - Flash4 . 0 ; Hamamatsu ) using HCimage software ( Hamamatsu ) . Acquired frames were analyzed using a custom sCMOS - specific localization algorithm based on a maximum likelihood estimator ( Huang et al . , 2013 ; Laplante et al . , 2016a , b ) . Localizations were plotted to construct a two - dimensional histogram FPALM image . To aid visualization of the two - dimensional histogram images , the im - ages were convolved with a two - dimensional Gaussian kernel ( \u03c3 = 7 . 5 nm ) . Time - lapse images showing dynamics were built by com - bining sequential 200 frame reconstructions ( 1 - s intervals ) and color coded for localization density . To display temporal information in a single composite image , the localizations in each 1 - s time point was assigned a Matlab Jet Color over the lifetime of the patch . We ex - tracted from the Matlab images of individual patches the numbers of localized emitters detected each second overall and from two spatial zones . Dimensions of an immobile mMaple3 - Myo1p patch that is confined the plasma membrane is used as a reference for the membrane proximal zone . Most of the mMaple3 - Myo1p localiza - tions are confined to a region 250 nm wide and 200 nm deep from the cell edge . Localizations that were detected within a 250 - nm - wide and 200 - nm - deep box from the cell edge were classified as membrane proximal zone , and any localizations detected outside the region are defined to be in the membrane distal region . The membrane distal region is limited to 250 nm wide and 350 nm deep starting 200 nm away from the cell edge . Images acquired in each data set ( typically 38 \u2013 40 s at 200 frames / s ) were sum projected , and the boundary of the localizations was identified as the cell edge . The raw time courses were aligned to the time course of a reference patch using the temporal superresolution realignment method ( Berro and Pollard , 2014a ) and averaged to obtain the average time course of localized emitters in the membrane proximal and mem - brane distal regions . The time courses were aligned to the vesicle scission event at time 0 s . To align the data , we first obtained the time courses of the total number of localizations detected in the actin patch ( sum localizations in the membrane proximal region and in the membrane distal regions ) . We then aligned the peak number of localizations detected to the peak number of GFP / YFP molecules assembled in an actin patch reported previously ( Sirotkin et al . , 2010 ; Arasada and Pollard , 2011 ) . To measure the dimensions of the actin patch , a 100 \u00d7 100 pixel region with an actin patch was selected , and the coordinates of the localizations for the patch were calculated from the 100 \u00d7 100 two - dimensional matrix using Matlab . The patches in all of the figures were oriented with the cell surface at the top with the cell interior below . DIC images acquired prior to FPALM imaging were used to align patches in this coordinate system . To calculate the width of the patch , localization densities in each column of the two - dimensional matrix were summed and projected onto an x - axis . The width of the patch represents the width of the localization density distribution at half the maximum . 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Dev Cell 11 , 33 \u2013 46 . Wang S , Moffitt JR , Dempsey GT , Xie XS , Zhuang X ( 2014 ) . Characterization and development of photoactivatable fluorescent proteins for single - molecule - based superresolution imaging . Proc Natl Acad Sci USA 111 , 8452 \u2013 8457 . Young ME , Cooper JA , Bridgman PC ( 2004 ) . Yeast actin patches are net - works of branched actin filaments . J Cell Biol 166 , 629 \u2013 635 . ACKNOWLEDGMENTS Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R01GM026132 and R01GM026338 to T . D . P . and grant R01GM115636 to J . B . The content is solely the re - sponsibility of the authors and does not necessarily represent the official views of the National Institutes of Health . We thank Steven Wang for providing a plasmid with mMaple3 and especially Jeorg Bewersdorf of Yale University for the use of his superresolution microscope . 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The length distributions of the aligned patches were averaged .", + "francis2015endocytic": "RESEARCH ARTICLE Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1 Monika K . Francis 1 , 2 , Mikkel R . Holst 1 , Maite Vidal - Quadras 1 , Sara Henriksson 2 , 3 , Rachel Santarella - Mellwig 4 , Linda Sandblad 3 and Richard Lundmark 1 , 2 , * ABSTRACT Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics , processes that are regulated by Rho GTPases . Here , we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase - activating protein ( GAP ) GRAF1 ( also known as ARHGAP26 ) , facilitates rapid cell surface turnover at the leading edge . Both Cdc42 and GRAF1 were required for fluid - phase uptake and regulated the generation of transient GRAF1 - coated endocytic carriers , which were distinct from clathrin - coated vesicles . GRAF1 was found to transiently assemble at discrete Cdc42 - enriched punctae at the plasma membrane , resulting in a corresponding decrease in the microdomain association of Cdc42 . However , Cdc42 captured in its active state was , through a GAP - domain - mediated interaction , localised together with GRAF1 on accumulated internal structures derived from the cell surface . Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing . We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism . KEY WORDS : Clathrin - independent endocytosis , GRAF1 , Cdc42 , Cell surface , Actin INTRODUCTION Cell surface dynamics are fundamental to a variety of basic biological processes , such as migration , polarisation and division , as well as protein and lipid trafficking . Intimate coupling to an underlying meshwork of cortical actin keeps the plasma membrane under high tension , providing the cell surface with its vital strength to withstand mechanical stress ( Gauthier et al . , 2012 ) . Consequently , structural alterations of the cell surface can only be achieved through the intricate coordination of membrane remodelling events and cytoskeletal rearrangements ( de Curtis and Meldolesi , 2012 ) . Small G - proteins of the Rho GTPase family are known as master regulators of the cytoskeleton and have been shown to greatly influence membrane tension and plasma membrane turnover by regulating endocytic and exocytic events ( Jaffe and Hall , 2005 ; Ridley , 2001 ) . Rho GTPases are peripherally attached to the membrane through lipid modifications and are active in their GTP - loaded state , where they interact with various effector molecules and function as molecular switches through rounds of GTP hydrolysis . The activity , which is stimulated by external cues , is strictly regulated by GTPase - activating proteins ( GAPs ) and guanine - nucleotide - exchange factors ( GEFs ) , which promote GTP hydrolysis and facilitate loading of GTP , respectively ( Jaffe and Hall , 2005 ) . However , the intricate mechanisms that allow for a cyclical nucleotide exchange in small G - proteins to synchronise membrane and cytoskeletal dynamics are still not understood . The clathrin - independent carrier ( CLIC ) pathway , a major pinocytic endocytic route in fibroblasts , facilitates polarised and rapid uptake of fluid , bacterial toxins , glycosylphosphatidylinositol ( GPI ) - linked receptors and receptors involved in cell adhesion ( Howes et al . , 2010a ) . CLICs were originally defined by their dependence on the Rho GTPase Cdc42 and tubular morphology , separating them from clathrin - coated vesicles and caveolae ( Hansen and Nichols , 2009 ; Howes et al . , 2010b ; Mayor and Pagano , 2007 ) . Carriers derived from this pathway have been shown to fuse into GPI - enriched endosomal compartments ( GEECs ) , which subsequently merge with early endosomes or recycling endosomes ( Kalia et al . , 2006 ) . The formation of CLICs depends on a poorly characterised molecular system , involving the small G - proteins Cdc42 and Arf1 , and their regulatory proteins ( Chadda et al . , 2007 ; Kumari and Mayor , 2008 ; Lundmark et al . , 2008 ; Sabharanjak et al . , 2002 ) . Although CLICs account for a major portion of the endocytic turnover of the plasma membrane , very little mechanistic detail is available regarding their formation . It has previously been shown that GRAF1 ( also known as ARHGAP26 ) , a GAP active against Cdc42 ( Hildebrand et al . , 1996 ; Jelen et al . , 2009 ; Longenecker et al . , 2000 ) , is essential for CLIC uptake ( Lundmark et al . , 2008 ) . GRAF1 is a dimeric multidomain protein composed of Bin , amphiphysin , RVS161 / 167 ( BAR ) , pleckstrin homology ( PH ) , GAP and Src homology 3 ( SH3 ) domains . The BAR and PH domains can generate and / or stabilise highly curved endocytic membranes from phosphatidylinositol 4 , 5 - bisphosphate ( PIP 2 ) - enriched regions , whereas the C - terminal GAP and SH3 domains modulates Cdc42 activity and interacts with regulatory proteins , respectively ( Doherty et al . , 2011a ; Hildebrand et al . , 1996 ; Lundmark et al . , 2008 ; Okada et al . , 2011 ) . Loss of GRAF1 downregulates endocytosis and affects cellular processes such as cortical actin remodelling , integrin trafficking , adhesion , spreading , migration and fusion , suggesting that this protein is an important regulator of cell surface dynamics ( Doherty et al . , 2011a , b ; Lundmark et al . , 2008 ; Nonnenmacher and Weber , 2011 ; Simpson et al . , 2008 ; Stergiou et al . , 2013 ) . In this work , we studied the interplay between GRAF1 and Cdc42 to decipher its importance for coordination of membrane and Received 13 May 2015 ; Accepted 28 September 2015 1 Integrative Medical Biology , Ume\u00e5 University , Ume\u00e5 901 87 , Sweden . 2 Medical Biochemistryand Biophysics , Ume\u00e5 University , Ume\u00e5 901 87 , Sweden . 3 Molecular Biology , Ume\u00e5 University , Ume\u00e5 901 87 , Sweden . 4 European Molecular Biology Laboratory , Meyerhofstr . 1 , Heidelberg 69117 , Germany . * Author for correspondence ( richard . lundmark @ umu . se ) This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http : / / creativecommons . org / licenses / by / 3 . 0 ) , which permits unrestricted use , distributionandreproductioninanymediumprovidedthattheoriginalworkisproperlyattributed . 4183 \u00a9 2015 . Published by The Company of Biologists Ltd | Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e actin dynamics during cell surface turnover at the leading edge . We characterise GRAF1 as a molecular marker of CLICs and demonstrate how this protein , through a direct interaction , regulates Cdc42 activity during endocytosis . This study also defines a temporal and spatial restriction of active Cdc42 as a prerequisite for the internalisation and further trafficking of cell surface components . RESULTS Correlative light and electron microscopy reveals highly curved membrane carriers decorated by GRAF1 and GTPase - deficient Cdc42 Consistent with the proposed involvement of the GTPase Cdc42 and the GAP GRAF1 in the generation of CLICs , GRAF1 localised with a dominant - active form of Cdc42 ( Cdc42 - Q61L , which is deficient in GTP hydrolysis ) to discrete assemblies in fixed cells ( Fig . 1A ) . As a step towards determining their identity , correlative light and electron tomography microscopy ( CLEM ) was used to resolve the ultrastructure of these assemblies . High - pressure frozen cells transfected with GFP \u2013 GRAF1 and mCherry \u2013 Cdc42 - Q61L were further prepared for CLEM as described in the Materials and Methods ( Kukulski et al . , 2011 ; Nixon et al . , 2009 ) . By overlaying the captured epifluorescence micrograph with high - resolution electron tomograms acquired from the same sample and region of interest ( ROI ) , it was apparent that all the fluorescent spots containing both proteins overlapped with small membranous structures ( Fig . 1B ) . Cdc42 - Q61L fluorescence alone , commonly seen lining the cell edge , was instead associated with the plasma membrane . Three - dimensional ( 3D ) models of the membranous structures , reconstructed from the tomograms , revealed that the GRAF1 - and Cdc42 - Q61L - positive structures detected by epifluorescence microscopy actually corresponded to tightly packed clusters of membrane carriers ( Fig . 1B ) . Most of the carriers were separated from the plasma membrane , but examples of tubular structures connected to the cell surface were also found ( Fig . 1B ) . Although they were quite morphologically diverse , the modelled carriers could roughly be divided into being either disc - like or tube - like on the basis of their shape ( Fig . 1C ; compare to the GRAF1 - and Cdc42 - Q61L - negative clathrin - coated vesicle ) . The measurement of the minor axis of each structure further indicated a surprisingly conserved diameter , spanning 20 \u2013 60 nm in 97 % of the carriers ( Fig . 1D ) . The close proximity of the GRAF1 - and Cdc42 - Q61L - positive carriers to the basal cell surface , evident using confocal microscopy ( Fig . 1A ) , was further confirmed by the reconstructed 3D models . The carrier clusters were detected within 500 nm of the basal membrane , and their distance to the cell edge , as measured in the x - y plane , ranged between 360 and 2200 nm . Taken together , the information collected from light and electron microscopy shows that GRAF1 and GTPase - deficient Cdc42 localise to carriers formed near the cell surface that have a morphology consistent with the membrane compartments previously defined as CLICs . GRAF1 is a marker of CLICs that are rapidly forming at the leading edge of cells To enable the dissection of a potential molecular interplay between Cdc42 and GRAF1 during CLIC formation , a cell model system was established utilising GRAF1 as a pathway marker . In a Flp - In T - REx HeLa cell line , untagged or GFP - tagged GRAF1 was placed under the control of a tet promoter modulated by doxycycline , resulting in an inducible system for the expression of a controlled amount of protein ( Fig . 2A ) . The protein expression level adapted for all subsequent experiments was chosen as to enable the detection of fluorescently tagged GRAF1 by microscopy , inevitably resulting in protein levels above the comparatively low endogenous level in this specific HeLa cell line . However , in contrast to using transient overexpression of GRAF1 , the developed Flp - In T - REx cell lines importantly allowed more reproducible and homogenous protein levels within the cultures ( Fig . S1A ) . In fixed cells , confocal microscopy showed that GRAF1 , independent of the presence of a fluorescent tag , assembled into punctate and , much less frequently , tubular structures at the cell surface ( Fig . S1B ) . In agreement with previous results detecting endogenous and transiently expressed GRAF1 in HeLa cells ( Lundmark et al . , 2008 ) , these assemblies colocalised with a fraction of the promiscuous cargo cholera toxin B - subunit ( CTxB ) , as assayed after 2 min internalisation at 37\u00b0C ( Fig . S2A ) . Moreover , these assemblies were only on very rare occasions found to overlap with clathrin - associated markers like transferrin ( Tfn ) and AP - 2 ( the adaptin for clathrin - mediated endocytosis ) , or the early endosomal marker EEA1 ( Fig . S2A ) . Analysis by live - cell spinning disc confocal microscopy revealed the formation of abundant short - lived GRAF1 \u2013 GFP - positive punctae at the leading edge of cells ( Fig . 2B ; Movie 1 ) . Software was used to create tracks corresponding to each detected structure , and from these the duration time of each track was derived . The average lifetime ( track duration ) of the dynamic GRAF1 assemblies was thereby determined to be \u223c 10 s ( Fig . 2C ) , showing that the protein was transiently assembled at the leading edge . This is in agreement with GRAF1 being associated with CLICs , which are dynamically formed at the cell periphery during cell spreading and migration ( Doherty et al . , 2011a ; Howes et al . , 2010a ) . To determine whether GRAF1 assembled at the plasma membrane , cells were followed by live - cell total internal reflection fluorescence ( TIRF ) microscopy . Structure tracks were created from the acquisitions and the duration of each track was calculated . The lifetimes of GRAF1 \u2013 GFP and GFP \u2013 GRAF1 assemblies were comparable to that detected by spinning disc confocal microscopy , suggesting that the burst in GRAF1 fluorescence corresponded to activity very near or at the cell surface ( Fig . 2D ; Fig . S1C ) . In agreement with this , detecting the surface binding and uptake of fluorescently tagged CTxB by performing live - cell TIRF microscopy revealed the recruitment of GFP \u2013 GRAF1 to CTxB clusters and the subsequent disappearance of both markers from the surface , indicative of endocytic carrier formation ( Fig . S2B ) . Importantly , in GRAF1 \u2013 GFP cells co - expressing mCherry - tagged clathrin light chain , live - cell TIRF microscopy followed by track analysis showed no overlap between the two proteins ( Fig . 2E ; Movie 2 ) , implying that GRAF1 does not aid the process of clathrin - mediated endocytosis . It should be noted that GRAF1 assemblies were not exclusively observed at the leading edge of cells and that an increased fraction of the assemblies in the centre and rear of cells displayed duration times longer than 30 s ( Fig . S1D , E ) . LossofCdc42andactinpolymerisation impairGRAF1 carrier processing and the fluid - phase endocytic capacity Cdc42 was identified as a key regulator of CLIC - mediated endocytosis on the basis of the effects that overexpressed Cdc42 activity mutants exerted on the uptake through this pathway ( Sabharanjak et al . , 2002 ) . To test the importance of Cdc42 and GRAF1 for clathrin - independent endocytosis in our established Flp - In T - REx HeLa cell lines , fluid - phase internalisation was quantified in cells depleted of either of these two proteins , or of AP - 2 by small interfering RNA ( siRNA ) . To control for effects on clathrin - mediated endocytosis , internalisation of transferrin was 4184 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e also assayed . The fluorescence of transferrin - S - S - CW800 or dextran - S - S - CW800 was measured after MesNa - reduction to remove surface - exposed fluorophores , and the internalisation was quantified in relation to that of cells transfected with control siRNA ( Fig . 3A , B ) . Although dextran uptake was impaired in all three samples , transferrin uptake was only significantly reduced in AP - 2 - depleted cells ( Fig . 3A ) , verifying the importance of Cdc42 and GRAF1 in clathrin - independent endocytosis . The \u223c 50 % reduction of dextran uptake seen in all samples further indicated that clathrin - independent and - dependent pathways , under these conditions , contribute to roughly equal amounts of internalised fluid volume . To investigate the importance of Cdc42 function on GRAF1 - mediated carrier formation , the localisation and behaviour of GRAF1 \u2013 GFP was followed by live - cell spinning disc confocal microscopy in cells depleted of Cdc42 . Although GRAF1 in these cells could still be found in dynamic assemblies at the leading edge , there was a marked increase of GRAF1 - decorated tubular structures associated with the cell surface ( Fig . 3C ; Fig . S3A , B ) . This implies that GRAF1 in cells with a decreased available pool of Cdc42 can exert its sculpting and / or stabilising function on the plasma membrane , but in the absence of the GTPase that this activity is not properly regulated . Interestingly , active Cdc42 has been shown to be important for recruitment of the Wiskott \u2013 Aldrich syndrome protein ( WASP ) - dependent actin machinery to aid CLIC formation ( Chadda et al . , 2007 ) . Similar to the effect seen after Cdc42 depletion , cells treated with the WASP - inhibitor wiskostatin showed a time - dependent increase of GFP \u2013 GRAF1 - decorated tubes associated with the cell surface ( Fig . 3D , E ) . The same phenotype was also observed after treatment with the actin - depolymerising drugs latrunculin A and cytochalasin D ( Fig . S3C ) , previously shown to inhibit uptake through CLICs ( Chadda et al . , 2007 ) . The altered processing of GRAF1 - mediated carriers in Cdc42 - depleted cells is likely to be at least a contributing factor to their impaired endocytic capacity , and a result of dysregulated Cdc42 - dependent actin dynamics . Fig . 1 . GRAF1 and GTPase - deficient Cdc42 localise to surface - detached pleomorphic membrane compartments . ( A ) Confocal stack of a Myc \u2013 GRAF1 - and GFP \u2013 Cdc42 - Q61L - expressing HeLa cell , presented as a maximum intensity projection and a 90\u00b0 tilted 3D volume . Scale bar : 10 \u03bc m . ( B ) Correlative microscopy analysis of a GFP \u2013 GRAF1 and mCherry \u2013 Cdc42 - Q61L - expressing HeLa cell . The left panel depicts the epifluorescence and electron micrographs . White arrowheads mark the analysed structures positive for both proteins . Colour - coded 3D models derived from the reconstructed electron tomograms and magnifications of two of the analysed clusters are visualised in the right panel . Thewhite arrow points at atube connected to the cell surface . Scale bars : 100 nm . ( C ) Representative examples of the two types of membrane compartments recognised within the analysed clusters in B ( disc - like and tube - like ) and a clathrin - coated vesicle ( CCV ) . Scale bars : 50 nm . ( D ) Widest diameterofminoraxis measurementsfromthe analysed membrane compartments inB . 136 structuresfrom 11 clusters in one cell were included in the analysis . The mean\u00b1s . d . is indicated . 4185 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e GRAF1 assembly at Cdc42 - enriched microdomains antagonises the local plasma membrane association of the GTPase To study the dynamics of the interplay between Cdc42 and GRAF1 during carrier formation , mCherry - tagged wild - type Cdc42 was co - expressed in GRAF1 \u2013 GFP Flp - In T - REx HeLa cells , and the two proteins followed over time with live - cell spinning disc microscopy ( Fig . 4A ; Movie 3 ) . Remarkably , 94 % of the GRAF1 assemblies that transiently appeared at the leading edge overlapped with membrane - associated Cdc42 . Moreover , 88 % of these assemblies were recruited to existing microdomains enriched with the GTPase . Further dissection of the indicated interdependency between the two proteins was accomplished by detailed analysis of fluorescence intensity plots created for each detected structure and channel over time ( Fig . 4B ) . The lack of major lateral movements in the detected GRAF1 punctae allowed the intensity value calculations to be restricted to circular ROIs ( Fig . 4B ) . The peak range and maximum was determined for each structure and channel , as described in the Materials and Methods . An average GRAF1 assembly lifetime of 18 s was approximated on the basis of the determined peak ranges , which was slightly longer , but in the same range , as the \u223c 10 s calculated from the track durations ( Fig . 4C , compare to Fig . 2C ) . Quantification of the lifetime overlap between GRAF1 and Cdc42 assemblies averaged 99 % , consistent with the visual assessment above ( Fig . 4D ) . Interestingly , there was a clear trend of Cdc42 reaching the maximum intensity value before GRAF1 ( on average 5 s before the GRAF1 intensity maximum , and 3 s after the start of the GRAF1 peak ) ( Fig . 4E ) . Consistent with this , for 49 % of the analysed structures , it was evident that the Cdc42 intensity decreased as the GRAF1 intensity increased ( compared to 82 % when analysing the GRAF1 intensity decrease ) ( Fig . 4F ) . This suggests that GRAF1 somehow antagonises the existing cell surface enrichment of Cdc42 as it assembles on the membrane , likely by mediating its internalisation and / or transiently interacting with the GTPase to switch it off and thereby destabilising its association with the microdomain . An interaction between the GAP domain and transition state Cdc42 enforces membrane assembly of GRAF1 To investigate the impact of the Cdc42 activity state on GRAF1 membrane assembly , GFP \u2013 GRAF1 Flp - In T - REx HeLa cells co - expressing CoralHue - tagged dominant - negative ( nucleotide - binding deficient T17N ) , wild - type and dominant - active Cdc42 were analysed by confocal microscopy . In comparison to the other two samples , Cdc42 - Q61L - transfected cells showed a striking accumulation of GRAF1 in bright punctate and tubular Fig . 2 . Dynamic assembly of GRAF1 at the leading edge . ( A ) Immunoblots detecting endogenous and recombinant GRAF1 in Flp - In T - REx HeLa GFP \u2013 GRAF1 , GRAF1 and GRAF1 \u2013 GFP cell lysates after induction with the stated doxycycline ( Doxy ) concentrations for 24 h . A doxycycline concentration of 1 ng / ml was chosen for all subsequent experiments . ( B ) Fluorescence micrograph detecting GRAF1 \u2013 GFP , corresponding to the last frame of a live - cell spinning disc confocal microscopy acquisition . At the leading edge , tracks of structure movement and duration over time are illustrated as colour - coded lines . Representative frames from the same acquisition are presented as a time series . Scale bar : 10 \u03bc m . ( C ) Duration time of GRAF1 \u2013 GFP structure tracks derived from acquisitions exemplified in B . 19 cells from three independent experiments were analysed . The bar graph depicts the distribution of the mean\u00b1s . d . values derived from each evaluated cell . ( D ) Duration time of GRAF1 \u2013 GFP and GFP \u2013 GRAF1 structure tracks derived from live - cell TIRF microscopy acquisitions . Bar graphs depict the distribution of the mean\u00b1s . d . values derived from each cell included in the respective analysis . Three independent experiments were analysed ( Mann \u2013 Whitney U test , n = 11 \u2013 12 cells , \u03b1 = 0 . 05 , ns , not significant ) . ( E ) Fluorescent micrograph showing GRAF1 \u2013 GFP and mCherry - tagged clathrin light chain ( CLC ) at a protrusion , corresponding to one frame from a live - cell TIRF microscopy acquisition . Tracksvisualise thedetectedstructuremovementin the respective channel . The edge of the cell is outlined in the upper panels . Arrowheads highlight GRAF1 assemblies . Scale bar : 2 \u03bc m . 4186 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e structures ( Fig . 5A , B ) , reminiscent of the clustered carriers formed in HeLa cells transiently co - expressing the two proteins ( compare with Fig . 1A ) . Moreover , this phenotype was dependent on the membrane localisation of the GTPase because it was abolished after introducing a point mutation of the cysteine residue ( C188A ) responsible for anchoring Cdc42 to the membrane ( Fig . 5A , B ) . Notably , no accumulation of GRAF1 structures was seen in cells co - expressing dominant - active RhoA - Q63L or Rac1 - Q61L ( Fig . 5A ) , two other GTPases within the same Rho family of actin regulators , confirming the specificity of the induced phenotype . In vitro pulldown experiments were used to determine whether the Cdc42 - Q61L mutation somehow influenced the otherwise inherently transient interaction between GRAF1 and Cdc42 , to thereby so profoundly affect GRAF1 membrane assembly . Using purified GST - tagged GRAF1 GAP domains as bait and Cdc42 or Cdc42 - Q61L loaded with GTP \u03b3 S ( a more stable GTP analogue ) as prey , revealed a preference in binding to the mutant ( Fig . 5C ) . However , repeating the assay with GDP - loaded GTPase in the presence of AlF x , to mimic the transition state conformation ( Mittal et al . , 1996 ) , resulted , in addition , to detectable binding to wild - type Cdc42 ( Fig . 5C ) . The Q61L mutation in Cdc42 increased the affinity to GRAF1 , likely by imposing an active site conformation , suggesting that the accumulation of GRAF1 assemblies in cells overexpressing this mutant is the effect of an enforced prolonged direct interaction . To test this hypothesis in vivo , HeLa cells transiently co - expressing CoralHue - tagged Cdc42 - Q61L and a GFP - tagged GAP domain containing or lacking truncates of GRAF1 , were analysed by confocal microscopy . Like full - length GRAF1 , the BAR - PH - GAP truncate showed an extensive accumulation in punctate and tubular structures together with the mutated GTPase ( Fig . 5D , E ) . Importantly , this effect was not seen with GFP \u2013 BAR - PH , confirming that it was dependent on the presence of the GAP domain . To avoid potential averse consequences of overexpressing truncated proteins , a Flp - In T - REx HeLa cell line was established with an inducible expression of GFP \u2013 GRAF1 - R412D , a point mutant disrupted in the so - called arginine finger known to be vital for the GAP activity ( Jelen et al . , 2009 ) ( Fig . S3D , E ) . Co - expression of Cdc42 - Q61L in this cell line did not result in any accumulation of GRAF1 - R412D structures ( Fig . 5A ; Fig . S3G ) , verifying that the enforced GRAF1 membrane accumulation induced by GTPase - deficient Cdc42 is a consequence of a direct interaction between the two proteins . GTP hydrolysis deficiency traps Cdc42 together with GRAF1 on endocytic vesicles The confirmed interaction between GRAF1 and Cdc42 in vivo , begged the question as to whether GRAF1 - mediated inactivation of Cdc42 was important during endocytic carrier formation . To resolve how Cdc42 activity influenced the formation of endocytic carriers , the dynamics of GFP \u2013 GRAF1 assemblies in Flp - In Fig . 3 . Inhibition of Cdc42 function and actin polymerisation affects GRAF1 carrier processing . ( A , B ) Relative internalisation of aminodextran - S - S - CW800 and transferrin - S - S - CW800 after 30 min uptake in Flp - In T - REx HeLa GFP \u2013 GRAF1 cells depleted of AP - 2 , Cdc42 or GRAF1 by siRNA transfection ( A ) , as confirmed by the immunodetection of the respective protein in comparison to clathrin heavy chain ( CHC ) in the cell lysates ( B ) . The cargo uptake was normalised to the corresponding control siRNA - transfected sample and the significance in relation to this sample was determined by analyses of at least three independent experiments ( mean\u00b1s . d . ; Mann \u2013 Whitney U tests , n = 3 \u2013 4 samples , \u03b1 = 0 . 05 ; ns , not significant ; * P < 0 . 05 ) . ( C ) Fluorescent micrographs depicting GRAF1 \u2013 GFP in control cells and cells depleted of Cdc42 . The ratio of cells with GRAF1 in at least one tubular structure ( i . e . a structure with a length > 2 \u03bc m ) was quantified on the basis of three independent experiments ( mean\u00b1s . d . ; Chi - square test , n > 450 cells , \u03b1 = 0 . 05 ; * * * P < 0 . 0001 ) . ( D ) Ratio of cells with GFP \u2013 GRAF1 in tubular structures after treatment with DMSO ( Vehicle ) or wiskostatin ( Wisk ) , for the indicated time intervals . Cells from three independent experiments were included in the analysis ( mean\u00b1s . d . ; Chi - square tests , n > 300 cells , \u03b1 = 0 . 05 , * * * P < 0 . 0001 ) . ( E ) Confocal stack of a GFP \u2013 GRAF1 - expressing cell after 20 min wiskostatin treatment , with presented top and slice views positioned as indicated ( white arrows ) . Scale bar : 10 \u03bc m . 4187 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e T - REx HeLa cells co - expressing CoralHue - tagged wild - type Cdc42 , Cdc42 - T17N or Cdc42 - Q61L were analysed by live - cell TIRF microscopy . Individual GRAF1 structures detected over the whole basal membrane were tracked over time in the acquisitions ( Fig . 6A ) and used to calculate the number of assemblies , their lifetime ( track duration ) , the distance between their appearance and disappearance ( track displacement ) , and the maximum speed of the structures ( Fig . 6B \u2013 E ) . Consistent with the results from fixed cells , co - expression of Cdc42 - Q61L resulted in four times more GRAF1 assemblies than detected in the other samples ( Fig . 6B ) . Notably , no clear difference in lifetime ( track duration ) of GRAF1 assemblies was found between Cdc42 - and Cdc42 - Q61L - expressing cells ( Fig . 6C ) , showing that the GRAF1 membrane accumulation enforced by Cdc42 - Q61L was not trapping the protein at the cell surface . However , the GRAF1 structures in cells co - expressing the GTPase - deficient mutant exhibited both an increased lateral mobility ( track displacement ) and a higher maximum speed ( Fig . 6D , E ) . Although the directional movement revealed by the track pattern of GRAF1 structures in Cdc42 - Q61L co - expressing cells ( Fig . 6A ) was unlikely to represent diffusion within the membrane , it could be explained by enforced GRAF1 assembly on intracellular vesicles trafficked along cytoskeletal tracks near the cell surface that are in close enough proximity to the plasma membrane to be detected within the TIRF field . To further explore the mobility of the GRAF1 assemblies enforced by co - expression of GTPase - hydrolysis - deficient Cdc42 , live - cell spinning disc microscopy was employed to increase the depth of the fluorescence probe detection in the focal plane of interest ( i . e . a plane close to , but just above , the basal membrane ) . In GFP \u2013 GRAF1 cells co - expressing mCherry \u2013 Cdc42 - Q61L , the proteins localised together both on budding carriers and intracellular laterally mobile vesicles ( Fig . 6F ; Movie 4 ) . The appearance of the latter type of structures in the TIRF and captured confocal fields indicates that carriers formed from the cell surface , positive for both proteins , later stay or reappear in a plane near the plasma membrane . From live - cell spinning disc microscopy acquisitions of cells co - expressing either wild - type Cdc42 or Cdc42 - Q61L , the detected structures of GRAF1 and respective GTPase protein were tracked and the time range of colocalisation was calculated . The colocalisation of GRAF1 and Cdc42 was significantly more transient than that of GRAF1 and Cdc42 - Q61L ( Fig . 6G ) , suggesting that the prolonged interaction between GRAF1 and GTP - hydrolysis - deficient Cdc42 results in a longer co - existence of the proteins on internalised vesicles ( i . e . an entrapment of both proteins on budded vesicles ) . Fig . 4 . GRAF1 recruitment to Cdc42 microdomains coincides with a local decreasing plasma membrane association of the GTPase . ( A ) Time series from a live - cell spinning disc confocal microscopy acquisition , visualising a protrusion from a Flp - In T - REx HeLa GRAF1 \u2013 GFP cell co - expressing mCherry \u2013 Cdc42 . White arrows mark detected GRAF1 assemblies . ( B ) Intensity profiles of the green and red channels , calculated over time as described in the Materials and Methods , for the first - appearing GRAF1 assembly in A . The grey area highlights the peak duration for the green channel . The insert visualises the ROI used to derive the plotted fluorescence intensity curves . ( C \u2013 F ) Parameters calculated for single GRAF1 assemblies on the basis of the corresponding intensity peak recorded within the intensity profiles derived from live - cell acquisitions , as exemplified in A and B . The time points fortheintensitypeakstart , maximumand end were defined for each structure and channel as described in the Materials and Methods . 34 \u2013 36 GRAF1 structures from three cells were included in the analysis and results are mean\u00b1s . d . 4188 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e Inactivation of Cdc42 by GRAF1 is not required for fluid - phase uptake but for the intracellular maturation of CLICs The potential effects on endocytosis inferred by overexpression of Cdc42 - Q61L were furtherassayed byanalysing the uptake of dextran . Dextran \u2013 Alexa - Fluor - 555 was added to GFP \u2013 GRAF1 Flp - In T - REx HeLa cells co - expressing Myc \u2013 Cdc42 - Q61L as a 5 - min pulse , then a quick wash was used to remove the excess extracellular marker and probes were followed by live - cell confocal microscopy . Immediately after the pulse , dextran was enriched in approximately one third of the accumulated GRAF1 structures ( Fig . 7A ) , verifying that these correspond to plasma - membrane - derived and budded endocytic carriers . The same protocol was used to prepare fixed samples for epifluorecence microscopy . Dextran could also be visualised in GRAF1 - positive carriers under fixed conditions ( Fig . 7B ) , but it should be noted that permeabilisation of fixed samples was avoided , given that the detection of this marker deteriorates with detergent treatment . Using software to define dextran compartments from the captured images , it was determined that the total uptake of fluid - phase was not significantly altered in cells affected by the Cdc42 - Q61L transfection ( Fig . 7C ) . Also in line with this , GFP \u2013 GRAF1 - R412D cells showed a higher prevalence of membrane assemblies in comparison to GFP \u2013 GRAF1 cells ( Fig . S3H ) , without any detectable effect on dextran endocytosis ( Fig . 7E ; Fig . S3F ) , further verifying that the GRAF1 GAP activity against Cdc42 was not required for endocytic carrier formation . The entrapment of GRAF1 and Cdc42 - Q61L on erratically behaving internalised vesicles , pointed towards a post - fission trafficking defect . Strikingly , in GRAF1 and Cdc42 - Q61L co - Fig . 5 . Interaction with transition state Cdc42 enforces membrane assembly of GRAF1 . ( A ) Ratio of Flp - In T - REx HeLa cells showing a phenotype of abundant ( \u2265 15 ) GFP \u2013 GRAF1 or GFP \u2013 GRAF1 - R412D structures in the absence ( \u2212 ) or presence of co - expressed CoralHue - tagged small GTPases and mutants thereof . For statistical analysis , cells from at least three independent experiments were included . The results were compared to the GRAF1 samples expressingwild - typeCdc42 , orinthecaseofGRAF1 - R412Dcells , wererelatedtothecorrespondingGRAF1samples ( mean\u00b1s . d . ; Chi - squaretests , n \u2265 300cells , \u03b1 = 0 . 05 ; ns , not significant ; * * * P < 0 . 0001 ) . ( B ) Maximum intensity projections of confocal stacks detecting GFP \u2013 GRAF1 in cells co - expressing the indicated CoralHue - tagged Cdc42 proteins . ( C ) Coomassie - stained SDS - PAGE gel from a pulldown experiment using purified GST ( GST ) or a GST - tagged GRAF1 GAP domain ( GAP ) as bait , and purified Cdc42 or Cdc42 - Q61L loaded with GTP \u03b3 S or GDP in the presence of AlF x ( GDP / AlF x ) as prey . The bound ( B ) and unbound GTPases ( U ) weredetected . ( D ) Maximumintensity projectionsofconfocalstacksdetectingGFP \u2013 GRAF1orindicatedtruncates ( theBAR - PH - GAPandBAR - PH domains ) and CoralHue \u2013 Cdc42 - Q61L in transfected HeLa cells . ( E ) Percentage volume of GFP \u2013 GRAF1 proteins colocalised with CoralHue \u2013 Cdc42 - Q61L , as visualised in D . Three independent experiments were analysed ( mean\u00b1s . d . ; Kruskal - Wallis test , Dunn \u2019 s post test , n = 15 cells , \u03b1 = 0 . 05 ; ns , not significant ; * * * P > 0 . 0001 ) . Scale bars : 10 \u03bc m . 4189 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e expressing cells with accumulated GRAF1 assemblies , significantly more , but smaller , dextran - positive compartments were detected ( Fig . 7D ) . This indicated that these internal carriers were stalled in their endosomal maturation or that early endosomes were fragmented under these conditions . However , there was no significant effect on the number of early endosomes or the total intensity of the early Fig . 6 . Cdc42 GTPase deficiency affects GRAF1 carrier dynamics after their formation . ( A ) Fluorescence micrographs depicting the start frames from live - cell TIRF microscopy acquisitions of GFP \u2013 GRAF1 structures in Flp - In T - REx HeLa cells transfected with the indicated CoralHue - tagged Cdc42 constructs ( upper panels ) . The corresponding structure tracks over time , derived from the acquisitions , are represented as colour - coded lines ( lower panels ) . Structures detected over the entire basal cell surface were included in the track analysis . ( B \u2013 E ) Quantification of the number and dynamic parameters of GFP \u2013 GRAF1 structure tracks from acquisitions exemplified in A . Three independent experiments were analysed [ Student \u2019 s t - tests on log 10 - transformed data , n \u2265 136 structure tracks ( fromfourcells ) , \u03b1 = 0 . 05 ; ns , notsignificant ; * * * P > 0 . 0001 ] . BargraphsinC \u2013 Edepictthedistributionofthemean\u00b1s . d . valuesderivedforeachcellincludedin the respective analysis . ( F ) Fluorescence micrograph showing the first frame from a live - cell spinning disc confocal microscopy acquisition of GFP \u2013 GRAF1 structures in cells transfected with the indicated mCherry - tagged Cdc42 - Q61L . Speeds measured over the detected GRAF1 tracks are represented as colour - codedlines . Magnificationsofareas1 and2arevisualised asatime seriestohighlight themobile membranestructurespositiveforbothproteins . Thewhitearrow marks a structure at the cell surface , the yellow arrow follows the lateral movement of an internal structure and the red arrow points out the fission of a tubular structure from the cell surface . ( G ) Quantification of duration of colocalisation between GFP \u2013 GRAF1 membrane assemblies and respective indicated mCherry - tagged Cdc42 protein , based upon track analyses performed on live - cell spinning disc confocal microscopy acquisitions like that exemplified in F . Cells from two independent experiments were analysed and the mean\u00b1s . d . is indicated [ Student \u2019 s t - test on log 10 - transformed data , n \u2265 29 structure tracks ( from three cells ) , \u03b1 = 0 . 05 ; * * * P > 0 . 0001 ) . Scale bars : 10 \u03bc m . 4190 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e endosomal marker EEA1 ( Fig . 7F ) . Furthermore , carriers decorated by GRAF1 and Cdc42 - Q61L were never positive for EEA1 ( Fig . 7G ) , suggesting that these more likely represented a trapped intermediate membrane compartment . To explore this in more detail , dextran trafficking was captured in GFP \u2013 GRAF1 cells co - expressing BFP \u2013 Rab5 alone , or together with Myc \u2013 Cdc42 - Q61L , by live - cell spinningdiscmicroscopy . Cells were pulsedwith the cargofor 5 min , quickly washed and the dynamics of the probes were monitored over 5 min . At the start of the acquisition , there was a significantly lower ratio of dextran - filled Rab5 vesicles in cells affected by the co - expression of GTPase - deficient Cdc42 ( Fig . 7H ; Fig . S4A ) , showing that trafficking of internalised dextran to this early endosomal compartment was negatively affected . It should also be noted that therewasa highnumberofdextran - containing vesiclesthat , at the end of the acquisition , were still devoid of Rab5 , even in cells not co - expressing Cdc42 - Q61L , likely corresponding to alternative trafficking routes . Although fusion events could be distinguished between dextran - filled Rab5 compartments ( Fig . S4B ) , and between Fig . 7 . See next page for legend . 4191 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e separate GRAF1 compartments in cells co - expressing Cdc42 - Q61L ( Fig . 7I ) , no example of fusion between GRAF1 and Rab5 compartments was found despite frequent transient engagements observed between trapped GRAF1 compartments and labelled early endosomes ( Fig . 7I ) . As reflected by the Cdc42 - Q61L - enforced clustering of GRAF1 carriers resolved by CLEM , taken together , our study shows that GRAF1 - mediated inactivation of Cdc42 is not vital for the formation and budding of endocytic carriers , but for their further intracellular maturation . DISCUSSION Cell spreading and migration involve dynamic alterations in cell surface turnover supporting local membrane rigidity and flexibility through directed trafficking of lipids and surface receptors . This is reflected in the polarised activities of endocytic pathways including caveolae concentrated at the lagging edge , and fast endophilin - mediated endocytosis ( FEME ) and CLICs enriched at the leading edge ( Boucrot et al . , 2015 ; Chaudhary et al . , 2014 ) . Here , we show that direct interplay between GRAF1 and Cdc42 coordinates maturation of CLICs at the cell front and hence constitutes an efficient machinery for aiding in the turnover of membrane constituents . We found that GRAF1 dynamically assembled at the plasma membrane in discrete areas that did not overlap with the formation of clathrin - coated pits . The assemblies of GRAF1 were short lived and preceded by local membrane enrichment of Cdc42 , suggesting that GRAF1 is transiently recruited to membrane microdomains imposed by upstream Cdc42 activity . The concentration of GRAF1 will increase the local curvature of the membrane through the BAR domain , promoting membrane invagination . At the same time , this results in high spatially confined GAP activity to stimulate GTP hydrolysis by Cdc42 . The inactivation of Cdc42 has been shown to reduce the membrane domain association and thereby results in a more dynamic pool of Cdc42 ( Bendez\u00fa et al . , 2015 ; Chadda et al . , 2007 ) . Indeed , the progressive assembly of GRAF1 correlated with a decrease in local Cdc42 enrichment . Given that we could not detect Cdc42 on internal carriers together with GRAF1 , we suggest that the transient interaction between the two proteins results in inactivation and a subsequent decrease in the membrane association of Cdc42 . This spatial restriction of Cdc42 activity could promote the identity transition from plasma membrane to an internal membrane compartment and facilitate further maturation . Based on this , we propose that the key role of GRAF1 in regulating turnover at the leading edge explains the previously described defect in the fusion of myoblasts and impaired migratory behaviour of cells lacking GRAF1 ( Doherty et al . , 2011a , b ; Lenhart et al . , 2014 ) . The initiation of the budding process and activation of Cdc42 might be facilitated by GEFs in response to extracellular signals , cellular polarisation or alterations in membrane tension . There are likely to be additional factors that are involved in the initiation and progression of CLIC formation . Previously , Arf1 has been shown to be essential for CLIC uptake , and the activation of Arf1 at the cell surface has been proposed to recruit ARHGAP10 to modulate Cdc42 activity ( Kumari and Mayor , 2008 ) . It is not yet known whether GRAF1 is directly coupled to these components or whether they regulate similar or distinct subtypes of CLICs . Actin polymerisation appears to play a central role in CLIC formation . Local actin - driven organisation of the cell surface has been suggested to promote endocytosis through clustering of lipids and proteins ( Gowrishankar et al . , 2012 ; Romer et al . , 2010 ) . In addition , actin polymerisation has been proposed to facilitate the release of carriers from the surface in a process controlled by Cdc42 ( Chadda et al . , 2007 ) . Interestingly , we found that Cdc42 and the downstream WASP - mediated actin polymerisation were vital for processing of GRAF1 - positive carriers . Depletion of Cdc42 or the acute inhibition of actin polymerisation resulted in extended GRAF1 - decorated tubules associated with the cell surface . This shows that Cdc42 is not essential for GRAF1 membrane assembly , but suggests that Cdc42 - driven actin polymerisation plays a central role for processing of membrane invaginations generated by this mechanism . The effect on carriers could be due to impaired scission from the cell surface or reduced support from local cortical actin that relieves membrane tension and thereby promotes tubulation of the cell surface . Interestingly , it has recently been shown that perturbation of the cortical actin by inhibition or depletion of Cdc42 results in reduced membrane tension ( Bretou et al . , 2014 ) . We show that the depletion of either Cdc42 or GRAF1 results in a striking reduction in fluid uptake , consistent with that the cycling activities of Cdc42 substantially regulate the dynamics and turnover of the plasma membrane ( Kumari et al . , 2010 ) . However , the precise influence and effects of GTP - binding and hydrolysis has so far not been understood . Here , we show that the inactivation of Cdc42 was not required for the generation or the internalisation of GRAF1 - Fig . 7 . Cdc42 GTPase deficiency disrupts the maturation of internalised GRAF1carriers . ( A ) Fluorescencemicrographrepresentingthefirstframeofa live - cell confocal acquisition detecting dextran \u2013 Alexa - Fluor - 555 and GFP \u2013 GRAF1 in Flp - In T - REx HeLa cells co - expressing Myc \u2013 Cdc42 - Q61L ( upper panels ) . The acquisition was started after a 5 - min incubation with the cargo followed by medium exchange . Analysis on the basis of four captured cells revealed dextran enrichment in 31\u00b116 % ( mean\u00b1s . e . m . ) of the identified GRAF1 structures . Areas 1 and 2 from the acquisition are presented as time series ( lower panels ) . Red stars indicate cells affected by Myc \u2013 Cdc42 - Q61L . Scale bar : 10 \u03bc m . ( B ) Epifluorescence micrograph of GFP \u2013 GRAF1 and dextran structures after a 5 - min uptake in two cells , one defined as phenotypically unaffected ( control ) and the other defined as affected by Myc \u2013 Cdc42 - Q61L ( red star ) . Scale bar : 10 \u03bc m . ( C , D ) Quantification of the total dextran uptake and descriptive parameters for dextran - positive compartments for comparison of GFP \u2013 GRAF1 cells unaffected and affected by the Cdc42 - Q61L transfection , as defined in B , to circumvent inter - sample variation of cargo fluorescence . Error bars represent the s . e . m . from three independent experiments ( Mann \u2013 Whitney U tests , n = 12 \u2013 14 cells , \u03b1 = 0 . 05 ; ns , not significant ; * * P < 0 . 01 ) . ( E ) Relative internalisation of aminodextran - S - S - CW800 and transferrin - S - S - CW800 after a 30 - min uptake in Flp - In T - REx HeLa GFP \u2013 GRAF1 - R412D cells . The cargo uptake of the mutant - expressing cells collected from at least three independent experiments was normalised to the corresponding wild - type sample and the significance in relation to this samplewas assessed ( mean\u00b1s . d . ; Mann \u2013 WhitneyU tests , n = 3 \u2013 4 , \u03b1 = 0 . 05 ; ns , not significant ) . ( F ) EEA1 intensity per endosome and number of EEA1 - positive endosomes per GFP \u2013 GRAF1 cell transfected with mCherry - tagged Cdc42 or Cdc42 - Q61L . Three independent experiments wereanalysed [ mean \u00b1s . d . ; left diagram , Student \u2019 s t - test , n > 13 , 500 EEA1 - stained endosomes ( from 50 cells ) , \u03b1 = 0 . 05 ; ns , not significant ; right diagram , Mann \u2013 Whitney U test , n = 3 cells , \u03b1 = 0 . 05 ; ns , not significant ) . Bars in the left graph depict thedistribution of the mean values derived from each experiment . ( G ) Confocal fluorescence micrographs of GFP \u2013 GRAF1 cells transfected with mCherry \u2013 Cdc42 - Q61L and immunostained with anti - EEA1 antibody . Scale bar : 5 \u03bc m . ( H ) Ratio of dextran - filled Rab5 compartments in BFP \u2013 Rab5 - transfected GFP - GRAF1 cells without or with co - expressed Myc \u2013 Cdc42 - Q61L after a 5 - min cargo uptake . Cells from three independent experiments were included in the analysis [ mean\u00b1s . d . ; Mann \u2013 Whitney U test , n \u2265 5 captured frames ( at least 5 cells ) , \u03b1 = 0 . 05 ; * P < 0 . 05 ] . ( I ) Representative time series from a live - cell spinning disc confocal microscopy acquisition visualising the dynamics of dextran - filled internal compartments in GFP \u2013 GRAF1 cells co - expressing BFP \u2013 Rab5 and Myc \u2013 Cdc42 - Q61L . The yellow arrow marks a Rab5 vesicle devoid of dextran , adjacent to a cargo - filled GRAF1 compartment pointed out by the magenta arrow . The turquoise arrow marks a second GRAF1 - labelled structure devoid of dextran , which over the depicted time range fuses with the first GRAF1 compartment ( specifically note the redistribution of cargo between these two compartments ) . Scale bar : 2 \u03bc m . 4192 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e positive CLICs given that neither the duration time of GRAF1 assemblies at the surface nor the total amount of internalised fluid was influenced by expression of Cdc42 - Q61L or GRAF1 mutated in the GAP domain . However , we show that inability of Cdc42 to hydrolyse GTP results in the accumulation of rapidly moving internal carriers coated by both GRAF1 and Cdc42 . Analysis of these carriers by CLEM showed that they were composed of multiple small vesicular and tubular structures with a mean diameter of 50 nm . This is in agreement with the previously described morphology of CLICs , and the proposed membrane curvature promoted by the BAR domain of GRAF1 ( Howes et al . , 2010a ; Lundmark et al . , 2008 ) . We show that the Q61L mutation increases the affinity of Cdc42 for the GAP domain of GRAF1 , which could explain the prolonged time of residence of GRAF1 and Cdc42 on the carrier membrane . Our results suggest that this affects the maturation of carriers , possibly due to altered recruitment of endosomal proteins and lipid turnover . We found that the stalled carriers were able to fuse with each other but not with Rab5 - positive endosomes , suggesting that they were trapped in their membrane identity . CLIC and GEEC carriers have been shown to undergo homotypic and endosomal fusion ( Kalia et al . , 2006 ) . We did not detect GRAF1 in EEA1 - positive endosomes , suggesting that this protein likely comes off the nascent vesicle to allow fusion with endosomal compartments . Taken together , the transient interaction between GRAF1 and Cdc42 will terminate Cdc42 activity and promote transition of the local PIP 2 - enriched membrane environment to enable release of GRAF1 and promote endosomal maturation . This might be facilitated by the phosphoinositide 3 - kinase - driven conversion of the lipid environment . Indeed GRAF1 , but not Cdc42 , has been detected on CLICs stalled by wortmannin treatment , which inhibits phosphoinositide 3 - kinase ( Howes et al . , 2010a ) . We have previously shown that GRAF1 is detected in pleomorphic structures ( Lundmark et al . , 2008 ) . Because there is no definite cargo known for CLICs , it is difficult to know whether these structures represent distinct compartments or different steps of maturation of carriers . Characterisation of GRAF1 - positive carriers using the Flp - In T - Rex HeLa cell system revealed that the majority of GRAF1 structures detected at the cell periphery were small and transient . We also observed longer tubes , likely corresponding to the previously described endogenous GRAF1 - containing tubes ( Lundmark et al . , 2008 ) , suggesting that GRAF1 is involved in further membrane maturation events under some conditions . The stabilisation of CLIC and GEEC intermediates upon the expression of Cdc42 - Q61L phenotypically resembles the previously described trapping of an endosomal compartment induced by constant Arf6 activity ( Naslavsky et al . , 2004 ) . Interestingly , the analogous Arf GAPs of the ASAP family are involved in regulating cytoskeletal and membrane reorganisation ( Randazzo et al . , 2007 ) , and might influence Arf activity and membrane curvature through a similar co - operative regulatory mechanism as proposed here . We believe that these types of systems should not be considered canonical means for receptor sorting and vesicle generation . Instead , they are optimised to regulate cell surface dynamics and the recycling of membrane reservoirs in response to external cues such as changes in adhesion or membrane tension . MATERIALS AND METHODS Antibodies , probes and plasmids The polyclonal anti - GRAF1 rabbit antibody RA - 83 was produced as previously described ( Lundmark et al . , 2008 ) . Commercially acquired antibodies included rabbit anti - Myc ( 2272S , Cell Signaling Technology ) , mouse anti - clathrin heavy chain ( clone 23 , 610499 , BD Transduction Laboratories ) , mouse anti - AP50 ( clone 31 , 611351 , BD Transduction Laboratories ; an AP - 2 subunit ) , rabbit anti - Cdc42 ( ab109553 , Abcam ) , mouse anti - EEA1 ( 610456 , Clone 14 , BD Transduction Laboratories ) , mouse anti - GFP ( JL - 8 , 632381 , Living Colours ) and goat anti - aldolase ( AB1809 , Chemicon International , Inc . ) antibodies . Secondary antibodies conjugated to Alexa Fluor molecules ( Molecular Probes ) , horseradish peroxidase ( HRP ; Sigma - Aldrich and Agrisera ) and IRDye 800CW or 680RD ( LI - COR Biosciences ) were used for immunofluorescence and western blot detections . The fluorescent DNA probe DRAQ5 was obtained from BioStatus Limited , and the 10 kDa dextran \u2013 Alexa - Fluor - 555 and CTxB \u2013 Alexa - Fluor - 647 were purchased from Molecular Probes . Cy3 \u2013 transferrin , 800CW - S - S - transferrin and 800CW - S - S - aminodextran were produced by covalently coupling respective cargo ( Sigma - Aldrich ) to amine - reactive Cy3 ( Amersham ) and 800CW - S - S ( LI - COR Biosciences ) according to the manufacturer \u2019 s recommendations . The plasmids used in this study are summarised in Table S1 . Constructs were created by PCR amplification of inserts with flanking restriction sites for ligation into corresponding vectors . Amino acid mutations were generated through site - directed PCR mutagenesis with PCR primers specified in Table S2 . Protein purification and pulldown experiments GST fusions of wild - type GRAF1 , its arginine finger mutated GAP truncate and Cdc42 proteins were purified as previously described ( Doherty et al . , 2011a ) . Thrombin ( GE Healthcare Life Sciences ) was used for tag removal . GST \u2013 GAP and GAP - R412D fusions were purified in 25 mM HEPES pH 7 . 4 and 150 mM NaCl , and Cdc42 proteins in the same buffer supplemented with 5 mM MgCl 2 . The nucleotide loading of GTPases was performed at 30\u00b0C for 20 min in the presence of EDTA ( twofold molar excess to MgCl 2 ) and nucleotide ( 18 - fold molar excess to protein ) ( GTP \u03b3 S and GDP from Sigma - Aldrich ) . MgCl 2 ( twofold molar excess to EDTA ) was added to stop the reaction and the experimental buffer conditions were reached by buffer exchange in Micro Bio - Spin P - 6 Gel columns ( Bio - Rad ) . For the in vitro protein interaction experiments , glutathione \u2013 Sepharose - 4Bbead - bound GST - tagged protein baits were incubated with prey proteins for 1 \u2013 3 h at 4\u00b0C under rotation . The unbound protein was collected by centrifugation ( 400 g for 2 min ) , the bound protein was washed , and both fractions were boiled in sample buffer for analysis by SDS - PAGE . All assays including GDP - loaded GTPase were performed in the presence of AlF x ( 1 mM AlCl 3 , and 25 mM NaF ) . Cell culture and transfection HeLa cells ( ATCC - CRM - CCL - 2 ) were cultured in Dulbecco \u2019 s modified Eagle \u2019 s medium ( DMEM ; low glucose , L - glutamine , sodium pyruvate , HEPES and Phenol Red ) , supplemented with 10 % fetal bovine serum ( FBS ; Gibco ) . Flp - In T - REx HeLacell lines with tetracycline - inducible expression of GRAF1 , GRAF1 \u2013 GFP , GFP \u2013 GRAF1 and GFP \u2013 GRAF1 - R412D were generated as previously described ( Mohan et al . , 2015 ) . Established cultures were grown in the mentioned DMEM , exceptions being during 800CW - S - S - cargo internalisation assays and live - cell acquisitions when basic medium containing 4 . 5 g / l glucose was used ( Gibco ) . The culturing media were further supplemented with 100 \u03bc g / ml hygromycin B and 5 \u03bc g / ml blasticidin S HCl ( Gibco ) for plasmid selection , and recombinant protein expression was induced by incubation in doxycycline hyclate ( Sigma - Aldrich ) for 20\u00b14 h . Targeted protein silencing was accomplished with siRNA against GRAF1 ( \u2018 siRNAb \u2019 Stealth RNAi , Invitrogen ) ( Lundmark et al . , 2008 ) , Cdc42 # 1 ( J - 005057 - 05 - 0020 ON - TARGET plus siRNA , Dharmacon ) , Cdc42 # 2 ( VHS40393 , Stealth siRNA , Life Technologies ) or AP - 2 ( AP2M1HSS101955 Stealth RNAi , Invitrogen ) , and a Medium GC Duplex RNA ( Stealth RNAi , Invitrogen ) served as the negative control . Transfections for transient expression of plasmids ( 16\u00b14 h ) and siRNA ( 60 \u2013 96 h ) were performed using Lipofectamine 2000 ( Invitrogen ) according to the manufacturer \u2019 s recommendations . Protein expression levels were analysed in cleared cell lysates by western blotting and HRP or IRDye antigen detection using a Medical X - ray Processor ( Kodak ) or an Odyssey Sa reader ( LI - COR Biosciences ) , respectively . Cellular uptake and drug treatments To capture cargo uptake after a given time interval , cells were incubated at 37\u00b0C with fluorescently labelled CTxB ( 3 \u03bc g / ml ) , transferrin ( 2 \u03bc g / ml ) or 4193 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e dextran ( 2 mg / ml ) and washed in preheated medium before fixation . For the quantification of internalised IRDye - S - S - linked cargo , cells seeded in 24 - well plates were incubated for 30 min at 37\u00b0C with 8 \u03bc g / ml 800CW - S - S - transferrin or 0 . 18 mg / ml 800CW - S - S - aminodextran and washed in preheated medium before fixation . At room temperature , the samples were washed in stripping buffer ( 50 mM Tris - HCl pH 8 . 7 , 100 mM NaCl , and 2 . 5 mM CaCl 2 ) , reduced for 1 . 5 h in the same buffer supplemented with 60 mM MesNa and further washed in phosphate - buffered saline ( PBS ) . Cells were finally stained with 1 \u03bc M DRAQ5 for 30 min and washed in PBS before recording probe fluorescence using the Odyssey Sa reader . The uptake was defined as the ratio between the cell number ( the measured DRAQ5 fluorescence ) and the amount of internalised cargo ( the measured 800CW fluorescence ) . Cargo trafficking was also monitored by live - cell fluorescent microscopy directly after the addition of 3 \u03bc g / ml CTxB \u2013 Alexa - Fluor - 647 or after 5 min of incubation with 0 . 5 \u2013 2 mg / ml dextran \u2013 Alexa - Fluor - 555 followed by a wash with preheated medium . The effects of actin polymerisation drugs on GRAF1 structures were analysed by fluorescence microscopy of fixed cells after treatment with 2 \u03bc M cytochalasin D , 2 \u03bc M latrunculin A ( Sigma - Aldrich ) , 5 \u03bc M wiskostatin ( Calbiochem ) or DMSO . Fluorescence microscopy Cellswerefixedandpreparedforimmunofluorescence analysisaspreviously described ( Lundmark et al . , 2008 ) . Images of fixed cell samples were captured using an epifluorescence Axioimager Z1 system ( AxioCam MRm camera ) ( Zeiss ) with the ZEN Software and a 63\u00d7 lens ( Plan - Apochromat 1 . 40 Oil DIC 0 . 17 ) or an A1 R Laser Scanning Confocal Microscope system ( ANDOR iXon EMCCD camera ) ( Nikon Instruments ) under control of the NIS - Elements Microscope Imaging Software and a 60\u00d7 lens ( Apochromat 1 . 40 Oil \u03bb S 0 . 17 WD 0 . 14 , Nikon ) , at the appropriate excitation and emission wavelengths . Live - cell confocal movies were recorded using the 63\u00d7 lens in the Nikon system or the 63\u00d7 lens ( Plan - Apochromat 1 . 40 Oil DIC M27 ) in a Cell Observer Spinning Disc Confocal Microscope system ( ANDOR iXon Ultra ) ( Zeiss ) controlled by the ZEN Software . Real - time TIRF acquisitions were captured byemploying the 100\u00d7 lens in respective system [ Apochromat 1 . 49 Oil 0 . 13 - 0 . 20 DIC N2 ( Nikon ) or Plan - Apochromat 1 . 46 Oil DIC M27 ( Zeiss ) ] . Micrographs and the acquired movies were prepared ( cropped , rotated , linearly adjusted for intensity and converted ) using Adobe Photoshop or ImageJ . Correlative microscopy For correlative fluorescence and electron microscopy , cells were high - pressure frozen on sapphire discs ( 0 . 7 % low - melt agarose and DMEM ; HPM100 ) and processed by freeze substitution . 250 - \u00b5m sections were placed on 200 mesh copper grids with a carbon support film ( TAAB ) and immediately imaged by epifluorescence microscopy , with all above steps performed according to Kukulski et al . ( 2011 , 2012 ) . Grids were further dried , coated with 15 - nm gold particles conjugated to protein A ( BioCell ) and post - stained with 2 % uranyl acetate and Reynolds lead citrate . Grids were placed in a high - tilt dual - axis holder ( Fischione Instruments ) and electron tomograms corresponding to the fluorescent structures of interest ( determined from recorded grid positions ) were acquired using a Tecnai F30 electron microscope ( FEI ) at 300 kV , equipped with an Eagel 4 k CCD camera ( FEI ) using serial electron microscopy ( Mastronarde , 2005 ) . The 9400\u00d7 magnification tomograms were acquired as single - axis and the 15 , 500\u00d7 magnification tomograms as dual - axis tilt series over \u2212 60\u00b0 to 60\u00b0 ( 1 . 5\u00b0 increment ) . All tomograms were reconstructed , modelled and quantified with the IMOD package version 4 . 7 . 10 ( Kremer et al . , 1996 ) . The 15 , 500\u00d7 magnification tomograms used for the detailed models have a voxel size of 7 . 67 \u00c5 . Fluorescent micrographs , models and tomograms were overlaid using Adobe Photoshop . Image analysis and quantifications Protein bands on western blots detected with HRP - conjugated antibodies were determined from scanned images in ImageJ to derive the relative sample protein content . Estimations of the fraction of fixed cells with GFP \u2013 GRAF1 proteins in abundant or tubular structures were performed by epifluorescent visual assessment . The Imaris Software V7 . 5 ( Bitplane ) was utilised to analyse micrographs captured from fixed and live - cell samples as specified in Table S3 . The recruitment of GRAF1 \u2013 GFP to mCherry \u2013 Cdc42 membrane domains was analysed by comparing the fluorescence intensity profiles of each channel over time , from the live - cell confocal spinning disc acquisitions . The mean intensity value within a five - unit - diameter circular ROI , spanning each structure of interest , was obtained for each channel and frame using the \u2018 Plot Z - axis profile \u2019 tool in ImageJ . The start and end of each intensity peak were defined as the time points where the calculated mean intensity values rose above and declined below , respectively , the background . Parameters of GFP \u2013 GRAF1 and mCherry \u2013 Cdc42 - Q61L carriers calculated from the correlative microscopy were derived from measurements on the constructed 3D image using the IMOD package . Statistics Statistical tests were performed using Prism 5 ( GraphPad Software ) with the indicated sample size and number of independent experiments . All quantifications are visualised as the mean\u00b1s . d . unless otherwise stated . Acknowledgements The authors would like to thank the Biochemical Imaging Centre Ume\u00e5 , Ume\u00e5 Core facility for Electron Microscopy and the electron microscopy facility at EMBL - Heidelberg . Competing interests The authors declare no competing or financial interests . Author contributions M . K . F . , M . R . H . , M . V . Q . , S . H . , R . S . M . , L . S . and R . L . designed and performed experiments . M . K . F . and R . L . wrote the manuscript . All authors commented on the final manuscript . Funding This work was supported by the Swedish Cancer Society [ grant number 2012 / 751 ] ; the Swedish Research Council [ grant number 821 - 2013 - 2241 ] ; the Swedish Foundation for Strategic Research [ grant number FFL09 - 0181 ] ; the Kempe foundations ; the Baltic Group foundations ; Molecular Infection Medicine Sweden ( MIMS ) ; and Ume\u00e5 Centre for Microbial Research ( UCMR ) . Deposited in PMC for immediate release . Supplementary information Supplementary information available online at http : / / jcs . biologists . org / lookup / suppl / doi : 10 . 1242 / jcs . 174417 / - / DC1 References Bendezu \u0301 , F . O . , Vincenzetti , V . , Vavylonis , D . , Wyss , R . , Vogel , H . and Martin , S . G . ( 2015 ) . Spontaneous Cdc42 polarization independent of GDI - mediated extraction and actin - based trafficking . 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Integrin - mediated signaling induced by simian virus 40 leads to transient uncoupling of cortical actin and the plasma membrane . PLoS ONE 8 , e55799 . 4195 RESEARCH ARTICLE Journal of Cell Science ( 2015 ) 128 , 4183 - 4195 doi : 10 . 1242 / jcs . 174417 J o u r n a l o f C e ll S c i e n c e", + "rohatgi1999interaction": "Cell , Vol . 97 , 221 \u2013 231 , April 16 , 1999 , Copyright \u00aa 1999 by Cell Press The Interaction between N - WASP and the Arp2 / 3 Complex Links Cdc42 - Dependent Signals to Actin Assembly 1999 ) . Because of their central role in signal transduc - tion , a great deal of interest has focused on the identifi - cation of downstream pathways that link these G pro - teins to the actin cytoskeleton ( Hall , 1998 ) . Progress has also been made in identifying compo - Rajat Rohatgi , * (cid:107) Le Ma , * (cid:107) Hiroaki Miki , \u2021 Marco Lopez , * \u2020 Tomas Kirchhausen , * \u2020 Tadaomi Takenawa , \u2021 and Marc W . Kirschner * \u00a7 * Department of Cell Biology \u2020 The Center for Blood Research Harvard Medical School nents closer to the process of actin polymerization at the plasma membrane . Actin polymerization can be reg - Boston , Massachusetts 02115 \u2021 Department of Biochemistry ulated by uncapping filament barbed ends ( Hartwig et al . , 1995 ) , by severing filaments ( Arber et al . , 1998 ; Yang Institute of Medical Science University of Tokyo et al . , 1998 ) , or by de novo nucleation ( Zigmond , 1998 ) . The Arp2 / 3 complex is the best candidate to generate Tokyo 108 Japan new barbed ends by stimulating nucleation ( Bi and Zig - mond , 1999 ; Machesky and Gould , 1999 ) . The complex is composed of seven polypeptides and has been puri - fied from Acanthamoeba castellanii ( Machesky et al . , Summary 1994 ) , Saccharomyces cerevisiae ( Winter et al . , 1997 ) , humans ( Welch et al . , 1997b ) , and Xenopus laevis ( Ma Although small GTP - binding proteins of the Rho family et al . , 1998b ) . The Arp2 / 3 complex is required for actin havebeenimplicatedinsignaling totheactincytoskel - assembly stimulated by the motile pathogen Listeria eton , the exact nature of the linkage has remained monocytogenes ( Welch et al . , 1997b ) , implicated in the obscure . We describe a novel mechanism that links polarized organization and motility of actin patches of one Rho family member , Cdc42 , to actin polymeriza - yeast ( Winter et al . , 1997 ) and required for Cdc42 - induced tion . N - WASP , a ubiquitously expressed Cdc42 - inter - actin assembly in Xenopus egg extracts ( Ma et al . , acting protein , is required for Cdc42 - stimulated actin 1998b ) . The complexnucleatesfilamentsin vitro ( Mullins polymerization in Xenopus egg extracts . The C terminus et al . , 1998 ) and is localized to actin - rich structures ( Kel - of N - WASP binds to the Arp2 / 3 complex and dramati - leher et al . , 1995 ; Welch et al . , 1997a ; Winter et al . , 1997 ) . cally stimulatesits abilityto nucleateactin polymeriza - Although the actin nucleation activity of the Arp2 / 3 tion . Although full - length N - WASP is less effective , its complex is weak ( Mullins et al . , 1998 ) , it can be stimu - activity can be greatly enhanced by Cdc42 and phos - lated by the Listeria protein ActA ( Welch et al . , 1998 ) . phatidylinositol ( 4 , 5 ) bisphosphate . Therefore , N - WASP This result points to the presence of analogous cellular and the Arp2 / 3 complex comprise a core mechanism mechanisms , presumably tied to signal transduction that directly connects signal transduction pathways pathways , for regulating the complex ( Beckerle , 1998 ) . to the stimulation of actin polymerization . Our demonstration that the Arp2 / 3 complex is required for Cdc42 - induced actin assembly in Xenopus extracts Introduction suggests that the Cdc42 signaling pathway might be such a cellular mechanism ( Ma et al . , 1998b ) . However , The actin cytoskeleton is a dynamic filament network intermediate components between Cdc42 and Arp2 / 3 that is essential for cell movement , polarization , mor - are clearly required because the complex does not di - phogenesis , and cell division ( Drubin and Nelson , 1996 ; rectly bind to Cdc42 , and it is not sufficient to mediate Mitchison and Cramer , 1996 ; Field et al . , 1999 ) . To en - Cdc42 - induced actin polymerization ( Ma et al . , 1998b ) . gage in these complex behaviors , cells must direct actin Among all the Cdc42 - interacting proteins identified to assembly with a high degree of spatial and temporal date , the WASP family proteins , especially WASP and resolution in response to extracellular signals . Over the N - WASP , are the best candidates for mediating the last 10 years , extensive research using genetic , cell bio - effects of Cdc42 on the actin cytoskeleton . WASP logical , and biochemical approaches has focused on ( W iskott - A ldrich s yndrome p rotein ) , which is only ex - the signal transduction pathways that link cell surface pressed in hematopoietic cells , was originally identified receptors to actin assembly at the plasma membrane . as a protein mutated in patients with Wiskott - Aldrich In the early 1990s , members of the Rho family of small syndrome ( WAS ) . N - WASP , which is ubiquitously ex - GTP - binding proteins ( G proteins ) , including Rac , Rho , pressed , shares (cid:122) 50 % homology with WASP ( Fukuoka and Cdc42 , were found to cause distinct morphological et al . , 1997 ) . Both proteins possess several domains : a changes in the actin cytoskeleton upon injection into pleckstrin homology ( PH ) domain that binds phosphati - cultured cells ( Ridley and Hall , 1992 ; Ridley et al . , 1992 ; dylinositol ( 4 , 5 ) bisphosphate ( PI ( 4 , 5 ) P 2 ) , a Cdc42 - bind - Nobes et al . , 1995 ) . Since then , Rho family G proteins ing ( GBD ) domain , a proline - rich region , a G - actin - bind - have been shown to regulate actin - dependent pro - ing verprolin homology ( V ) domain , a domain ( C ) with cesses in many different systems ( Hall , 1998 ; Johnson , homology to the actin - depolymerizing protein cofilin , and finally a C - terminal acidic segment ( A ) ( Figure 1A ) ( Miki et al . , 1996 ) . Both WASP and N - WASP induce actin \u00a7 To whom correspondence should be addressed ( e - mail : marc @ polymerization when overexpressed in fibroblasts ( Miki hms . harvard . edu ) . (cid:107) These authors contributed equally to this work . et al . , 1996 ; Symons et al . , 1996 ) . Cell222 Figure 1 . N - WASP Is Required for Cdc42 - Induced Actin Assembly in High - Speed Su - pernatants of Xenopus Egg Extracts ( A ) Schematic diagram of N - WASP showing its domain structure . Abbreviations for the domains used throughout the text are indi - cated inside the boxes . Numbers refer to amino acid residues . The H208D and (cid:68) cof mutantsareshownbelowalongwiththesitesofthemutations . ( B ) Immunodepletion of N - WASP from Xeno - pus HSS . Affinity - purified (cid:97) - N - WASP anti - body or antibody buffer ( mock ) was used to immunodeplete N - WASP , and the material left in the supernatant or captured on the beadswasanalyzedbyimmunoblottingusingtheaffinity - purified (cid:97) - N - WASP antibody . 0 . 6 % of the input material ( HSS ) , 0 . 7 % of the supernatant material , and 2 % of the material captured on beads was loaded on the gel . ( C ) Comparison of actin assembly stimulated by250nMGTP (cid:103) S - chargedGST - Cdc42inun - treated HSS , mock - depleted HSS , (cid:97) - N - WASP depleted HSS , and in depleted HSS reconsti - tutedwith50nMrecombinantN - WASP . Poly - merization kinetics were monitored in the HSS using the pyrene actin assay in which fluorescence increase indicates filament for - mation . ( D ) Comparison of actin assembly stimulated by250nMGTP (cid:103) S - chargedGST - Cdc42in (cid:97) - N - WASP depleted HSS to which the indicated concentrations of recombinant N - WASP were added . The initial rate of actin assembly and the maximum level of F - actin attained were quantitated from curves of the type shown in ( C ) . ( E ) Relative ability of wild - type N - WASP , the H208D mutant , and the (cid:68) cof mutant to re - storeactinassemblyactivitytoHSSdepletedofendogenousN - WASP . In all cases , actin assembly was initiated by the addition of 250 nM GTP (cid:103) S - charged GST - Cdc42 . N - WASP is a particularly good candidate for mediat - Results ing the effects of Cdc42 on the cytoskeleton because it can interact with a Cdc42 mutant ( Y40C ) that still N - WASP Is Required for Cdc42 - Induced Actin Polymerization in Xenopus Egg Extracts induces filopodia but which cannot bind to a variety of other targets , including WASP ( Miki et al . , 1998a ) . In We have previously described the reconstitution of Cdc42 - stimulated actin assembly in high - speed super - addition , dominant - negative mutations in N - WASP block Cdc42 - induced filopodium formation , and wild - type natants from Xenopus egg extracts ( \u201c Xenopus HSS\u201d or \u201c Xenopus extract\u201d in the rest of the text ) ( Ma et al . , N - WASP potentiates Cdc42 - induced microspike forma - tion ( Miki et al . , 1998a ) . Finally , in search for other com - 1998a ) . Thekineticsofactinpolymerizationcanbemoni - tored by supplementing the extracts with pyrene actin , a ponents ( Ma et al . , 1998b ) required for Cdc42 - induced actin assembly in vitro , we found that N - WASP partially fluorescent derivative of actin that exhibits much higher fluorescence intensity when actin is assembled into fila - copurified with MCAP2 ( m ediator of C dc42 - induced a ctin p olymerization ; unpublished observation ) . ments . Using this cell - free system , we have demon - strated a requirement for the Arp2 / 3 complex in Cdc42 - We show here that N - WASP can activate the Arp2 / 3 complex to stimulate actin polymerization in vitro using induced actin polymerization ( Ma et al . , 1998b ) . In order to test the requirement for N - WASP in this purified components . This N - WASP \u2013 Arp2 / 3 interaction is also required for Cdc42 - stimulated actin assembly in system , we raised a polyclonal rabbit antiserum against a full - length , recombinant rat N - WASP protein that is Xenopus egg extracts . Although full - length N - WASP is only weakly active , a C - terminal fragment confers a dra - (cid:46) 90 % identical to the bovine and human forms . After affinity purification , the antibody detected a single (cid:122) 65 matic 30 - fold acceleration of actin assembly kinetics . The activity of full - length N - WASP can be increased to kDa band in Xenopus extracts on immunoblots and also immunoprecipitated it ( Figure 1B ) . Using this affinity - levels similar to the C - terminal fragment in the presence of two putative regulators of N - WASP , Cdc42 and purified antibody , we immunodepleted (cid:46) 95 % of N - WASP from Xenopus HSS ( Figure 1B ) . The depletion of N - WASP PI ( 4 , 5 ) P 2 . Thus , the N - WASP \u2013 Arp2 / 3 interaction seems to be a critical link in Cdc42 - dependant signaling path - eliminates the ability of the extract to support Cdc42 - stimulated actin polymerization ( Figure 1C ) . The activity ways that control actin assembly . N - WASP Interaction with the Arp2 / 3 Complex 223 Figure 2 . Full - Length N - WASP Cooperates with the Arp2 / 3 Complex to Accelerate Actin Polymerization In Vitro ( A ) The composition of the Arp2 / 3 complex purified from bovine brain is shown on a 12 % polyacrylamidegelstainedwithGelcodeBlue ( Coomassie G - 250 ) . The thin , unlabeled lines on the right indicate the seven subunits of the complex . Listed in descending order of molecular weight , the subunits are Arp3 , Arp2 , p41 - ARC , p34 - ARC , p21 - ARC , p20 - ARC , and p16 - ARC . ( B ) Thepyrene actin assaywas usedto moni - tor the polymerization of 2 . 5 (cid:109) M G - actin ( 1 . 5 (cid:109) M unlabeled actin (cid:49) 1 (cid:109) M pyrene actin ) in the presence of 250 nM Arp2 / 3 complex alone , 400 nM N - WASP alone , or both com - ponents added together . In all the curves shown here and in the remainder of the fig - ures , polymerizationwasinitiatedattime (cid:53) 0 . ( C ) Comparison of the abilities of wild - type N - WASP ( 400 nM ) and the (cid:68) cof mutant ( 400 nM or 800 nM ) to stimulate actin polymeriza - tionin thepresence of60 nMArp2 / 3 complex underconditionsdescribedin ( B ) . Thecontrol curve depicts actin polymerization in the presence of Arp2 / 3 alone . can be rescued by adding back purified recombinant Full - Length N - WASP Cooperates with the Arp2 / 3 Complex to Produce a Modest Acceleration N - WASP protein at approximately physiological con - of Actin Polymerization with Purified centrations ( (cid:122) 20 \u2013 50 nM ) ( Suzuki et al . , 1998 ) . Adding Components In Vitro back increasing amounts of N - WASP leads to a dose - Since we had identified requirements for both N - WASP dependant increase both in the initial rate and in the and the Arp2 / 3 complex in Cdc42 - induced actin assem - final level of actin polymerization induced by a fixed bly , we wanted to determine whether N - WASP could amount of Cdc42 ( Figure 1D ) . synergize with the Arp2 / 3 complex to accelerate actin Thespecificity oftheadd - backexperiment wastested polymerization as observed for the ActA protein of Liste - using two previously characterized mutants of N - WASP , ria ( Welch et al . , 1998 ) . For these assays , we used the H208D and (cid:68) cof ( Figure 1A ) . H208D cannot bind to Arp2 / 3 complex purified from bovine brain extracts ( Fig - Cdc42 due to a point mutation in its Cdc42 - binding ure 2A ) . When we used pyrene actin fluorescence to domain and does not potentiate Cdc42 - induced filo - monitor actin assembly in the presence of either pure podia formation when introduced into cells ( Miki et al . , N - WASP or pure Arp2 / 3 complex , the kinetics of assem - 1998a ) . (cid:68) cof , containing a four \u2013 amino acid deletion in bly were not significantly different from those of actin the C domain of N - WASP , does not induce actin reorga - alone ( Figure 2B ) . Actin assembly kinetics demonstrate nization when overexpressed in Cos - 7 cells and acts in an initial lag phase , reflecting the kinetic barrier to nucle - a dominant - negative fashion to block Cdc42 - induced ation , followed by a rapid , linear phase during which filopodium formation ( Miki et al . , 1998a ) . In Xenopus filaments elongate by monomer addition . In the pres - HSS depleted of N - WASP , these mutants cannot rescue ence of both Arp2 / 3 and N - WASP , the slope of the elon - Cdc42 - induced actin assembly even when added back gation phase is increased by (cid:122) 4 - fold and the lag phase at concentrations 20 - fold above the wild - type protein is shortened , although not completely eliminated ( Figure ( Figure 1E ) . These results show that regions of N - WASP 2B ) . Addition of increasing amounts of N - WASP to a implicated in interactions with both upstream regulators fixed concentration of Arp2 / 3 caused a dose - dependant ( Cdc42 ) and downstream targets are necessary for its acceleration of actin polymerization ( Figure 5C ) . ability to support Cdc42 - induced actin assembly in The (cid:68) cof mutantdescribed abovedoes notaccelerate actin assembly in the presence of the Arp2 / 3 complex Xenopus HSS . Cell224 Figure 3 . N - WASP Binds to and Activates the Arp2 / 3 Complex via Its C Terminus ( A ) Schematic showing the domain structure of full - length N - WASP and the various C - terminal fragments of N - WASP constructed as GST fusion proteins . All fusion proteins are named according to the C - terminal segments ( V , C , or A ) of N - WASP that they contain . ( B ) The GST fusion proteins shown in ( A ) were immobilized on glutathione - Sepharose beads and tested for their abilities to pull down either theArp2 / 3complexorG - actinfromsolution . ThepresenceoftheArp2 / 3complexorG - actinboundtothebeadswasassayedbyimmunoblotting with (cid:97) - Arp2 or (cid:97) - actin , respectively . ( C ) Actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) was followed in the presence of GST - VCA ( 50 nM ) , GST - V ( 200 nM ) , GST - VC ( 200 nM ) , GST - CA ( 200 nM ) , or in the absence of any additions ( control ) . ( D ) The initial phases of actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) , including the lag phase , are shown in the presence of indicated concentrations of GST - VCA . ( E ) Summary of the G - actin binding , Arp2 / 3 binding , and Arp2 / 3 activation properties of full - length N - WASP and the fragments shown in ( A ) . Binding of full - length N - WASP to Arp2 / 3 was determined by coimmunoprecipitation rather than GST pull down . Binding of GST - V and GST - VCA was determined by both methods . ( Figure 2C ) , suggesting a biochemical mechanism for its the p21ARC subunit of the Arp2 / 3 complex ( Machesky and Insall , 1998 ) . phenotypes in tissue culture cells as well as in Xenopus extracts ( Figure 1E ) . Unexpectedly , the H208D mutant , We expressed and purified GST fusion proteins con - taining various portions of this VCA region ( diagrammed which has only one point mutation in the GBD domain , does not accelerate actin assembly ( data not shown ) , in Figure 3A ) and tested their abilities to interact with the Arp2 / 3 complex in a GST pull - down assay . The complex suggesting that this mutation leads to defects in func - tions other than just Cdc42 binding . bound to GST - CA and GST - VCA but not to GST , GST - V , and GST - VC ( Figure 3B ) . As a control , the fragments were tested for binding to G - actin and , as expected , the The C Terminus of N - WASP Strongly Accelerates Arp2 / 3 - Mediated Actin Assembly presence of the V domain perfectly correlated with this property ( Figure 3B ) . We conclude that the C - terminal To define the region of N - WASP required for activity , we concentrated on a C - terminal region of N - WASP 55 amino acids of N - WASP ( 450 \u2013 505 ) are necessary and sufficient to bind to the Arp2 / 3 complex and that the ( amino acids 392 \u2013 505 ) , which contains a G - actin - bind - ing V domain , a cofilin homology sequence ( C ) , and an ability to bind G - actin through the V domain is not re - quired . We were unable to detect any interaction be - acidic segment ( A ) ( Figures 1A and 3A ) . The VCA region is thought to mediate interactions of N - WASP with tween full - length N - WASP and Arp2 / 3 by coimmuno - precipitation under conditions where the binding of downstream effectors because mutations in this region abrogate its ability to induce actin rearrangements upon GST - VCA to Arp2 / 3 is readily detectable ( data not shown ) . injection into cells andoften function as dominant - nega - tive mutations ( Miki et al . , 1998a ) . In addition , a homolo - Since the C - terminal region of N - WASP could bind to the Arp2 / 3 complex and actin , we wanted to determine gous region from other WASP family proteins ( Scar1 / WAVE and WASP ) was recently found to interact with whether it could confer the activity of the full - length N - WASP Interaction with the Arp2 / 3 Complex 225 Figure 4 . The GST - CA Fragment Can Inhibit the Interaction between N - WASP and the Arp2 / 3 Complex ( A ) Stimulation of actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) by N - WASP ( 200 nM ) is inhibited by GST - CA but not GST - V . ( B ) Stimulation of actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) by GST - VCA ( 1 nM ) is inhibited by GST - CA in a dose - dependent manner . ( C ) Cdc42 - stimulated actin polymerization in Xenopus HSS is inhibited by GST - CA , but not GST - V or GST - VC ( 200 nM each ) . ( D ) Dose - dependent inhibition of Cdc42 - stimulated actin polymerization in Xenopus HSS by GST - CA as compared using the initial rate and maximal level of F - actin assembly . The control depicts assembly in the absence of any GST - CA . protein to accelerate actin polymerization . When the N - WASP \u2013 Arp2 / 3 interaction ( Figure 3E ) . When this model was tested using purified components , GST - CA purified fragments were mixed with pure Arp2 / 3 com - plex , only the GST - VCA fusion protein accelerated as - ( but not GST - V , GST - VC , or GST ) inhibits the ability of both full - length N - WASP and the GST - VCA fragment to semblykinetics , shorteningthelagphaseandincreasing the elongation rate by 30 - fold ( Figure 3C ) . Figure 3D stimulate actin polymerization in the presence of the Arp2 / 3 complex ( Figures 4A and 4B ) . clearly shows that the GST - VCA fragment shortens the lag phase to polymerization in a dose - dependent man - The efficacy of the GST - CA fragment as a dominant negative was tested in Xenopus HSS for its effects on ner , suggesting that it can lower the kinetic barrier to nucleation . These effects are dependent on the pres - Cdc42 - induced actin assembly . When used in extracts at concentrations roughly equivalent to the estimated ence of the Arp2 / 3 complex and can be inhibited by cytochalasin D ( data not shown ) . The GST - V , GST - VC , endogenousArp2 / 3concentration ( (cid:122) 100 \u2013 300nM ) , GST - CA specifically inhibited Cdc42 - induced actin assembly and GST - CA fragments have no effect , even when used at 4 - fold higher concentrations ( Figure 3C ) . The GST - ( Figures 4C and 4D ) . This result strongly suggests that the kind of interaction between N - WASP and the Arp2 / 3 VCA fragment is both much more potent and effective in stimulating actin polymerization when compared to complex that we have observed using purified compo - nents in vitro is also required for Cdc42 - induced actin the full - length N - WASP , a point we will return to later . Thus , the requirements for binding to the Arp2 / 3 com - polymerization in a more complete and physiological system . plex are different from the requirements for activation , as summarized in Figure 3E . The C - Terminal Domain of N - WASP Is at Least 100 - Fold More Potent Than A C - Terminal Fragment of N - WASP Can Act as a Dominant Inhibitor and Block the Function the Full - Length Protein Using purified components , N - WASP ( 200 nM ) can stim - of N - WASP and the Arp2 / 3 Complex The observation that the GST - CA fragment can bind to ulate actin polymerization in the presence of only the Arp2 / 3 complex and G - actin ( Figure 2 ) . However , when the Arp2 / 3 complex but cannot activate it suggests that it can be used as a competitive inhibitor to block the added to Xenopus HSS at concentrations ( 400 nM ) as Cell226 Figure 5 . Full - Length N - WASP and the GST - VCA Fragment Activate Actin Polymerization with Different Potencies ( A ) Comparion of spontaneous ( Cdc42 - independent ) actin polymerization in Xenopus HSS treated with the indicated concentrations of full - length N - WASP , GST - CA , or GST - VCA . ( B ) Actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) stimulated by full - length N - WASP ( 200 nM ) or GST - VCA ( 10 nM ) . ( C ) Dose \u2013 response curves showing the variation in the maximum rate of filament elongation as a function of increasing concentrations of either GST - VCA or full - length N - WASP in the presence of 60 nM Arp2 / 3 complex . The elongation rate was calculated from the linear phase of polymerization curves of the type shown in ( B ) . The main graph depicts the concentrations ( abscissa ) on a log scale , whereas the inset depicts them on a linear scale . At higher concentrations of GST - VCA , the elongation rate saturates and is no longer a good indicator of activity . Instead , the shortening of the lag phase becomes more dramatic ( Figure 3D ) . high as 8 - fold above the estimated endogenous N - WASP difference between the full - length protein and the GST - VCA fragment in their ability to stimulate actin polymer - concentration ( 50 nM ) , full - length N - WASP did not stim - ulate spontaneous actin assembly in the absence of ization in the presence of the Arp2 / 3 complex . When full - length N - WASP was directly compared to the GST - activated Cdc42 ( Figure 5A ) . To test whether this differ - ence in the behavior of N - WASP in the purified system VCA fragment , a large difference in activity was observed : 10 nM GST - VCA stimulates polymerization in vitro far compared to the extract is due to inhibitory factors that interact with its N terminus , we added the GST - VCA more effectively than 200 nM N - WASP ( Figure 5B ) . To obtain a more quantitative measure of the activity fragment to Xenopus HSS . We reasoned that GST - VCA would bypass the Cdc42 requirement and act in a domi - difference between full - length N - WASP and the GST - VCA fragment , we generated dose \u2013 response curves for nant active fashion in the extract . Indeed , when GST - VCA was added to the HSS ( in the absence of any acti - both proteins , using elongation rates as an indicator of activation ( Figure 5C ) . The concentration required for vated Cdc42 ) even at very low concentrations , actin polymerization was dramatically stimulated : the effect half maximal activation is (cid:122) 250 nM for the full - length protein and (cid:122) 2 nM for GST - VCA ( Figure 5C ) . Based on of 1 nM GST - VCA was comparable to that of 800 nM N - WASP ( Figure 5A ) . This observation suggests that elongation rates , the C - terminal VCA domain is at least 100 times more potent than full - length N - WASP . This N - WASP is regulated in extracts , a property that is con - ferred by the N terminus of the molecule . potency difference is also demonstrated by the (cid:122) 20 - fold higher molar ratio of GST - CA needed to inhibit GST - How might the activity of N - WASP be regulated ? It hasbeen proposedthatN - WASPis intrinsicallyinhibited VCA compared to the full - length protein ( Figures 4A and 4B ) . The efficacy of GST - VCA at inducing actin polymer - and is activated when the VCA region is unmasked , perhaps by the binding of upstream regulators to the N ization is also about 5 - fold higher than that of N - WASP ( Figure 5C ) . The difference in activity between GST - VCA terminus ( Miki et al . , 1998a ) . This model predicts a large N - WASP Interaction with the Arp2 / 3 Complex 227 Figure 6 . Cdc42 and PI ( 4 , 5 ) P 2 Coordinately Activate Full - Length N - WASP In Vitro ( A ) The effect of GTP (cid:103) S - Cdc42 ( 500 nM ) , PI ( 4 , 5 ) P 2 - containing vesicles ( 100 (cid:109) M , PC : PI : PI ( 4 , 5 ) P 2 , 48 : 48 : 4 ) , or both on actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) stimulated by N - WASP ( 200 nM ) . The solid line shows stimulation of actin polymerization under the same conditions by GST - VCA ( 200 nM ) alone . ( B ) Dose \u2013 response curves showing the maximum elongation rate of actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) as a function of the following molecules added : GST - VCA , full - length N - WASP , and full - length N - WASP stimulated with GTP (cid:103) S - Cdc42 ( 50 nM ) and PI ( 4 , 5 ) P 2 - containing vesicles ( 100 (cid:109) M , PC : PI : PI ( 4 , 5 ) P 2 , 48 : 48 : 4 ) . Note that the curve of N - WASP activated by PI ( 4 , 5 ) P 2 and GTP (cid:103) S - Cdc42 starts to taper off between 8 and 80 nM because the Cdc42 is at a relatively low concentration in the experiment ( 50 nM ) ; addition of higher concentrations of Cdc42 will drive activation to higher levels approaching those of GST - VCA ( Figure 6A ) . ( C ) Effects of Cdc42 bound to different nucleotides on N - WASP . Actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) was stimulated by N - WASP at the indicated concentrations in the presence of Cdc42 ( 50 nM , GTP (cid:103) S or GDP (cid:98) S charged ) and PI ( 4 , 5 ) P 2 - containing vesicles ( 10 (cid:109) M , PC : PI : PI ( 4 , 5 ) P 2 , 48 : 48 : 4 ) . ( D ) Actin polymerization ( 2 . 5 (cid:109) M actin and 60 nM Arp2 / 3 complex ) was stimulated by N - WASP ( 5 nM ) in the presence of Cdc42 ( 50 nM , GTP (cid:103) S charged ) and the indicated concentrations of lipid vesicles containing PC / PI ( 50 : 50 ) or PC / PI / PI ( 4 , 5 ) P 2 ( 48 : 48 : 4 ) . and full - length N - WASP is not due to the presence of and PH domains of N - WASP , respectively ( Figure 1A ) . Both domains are necessary forN - WASP to induce actin the GST domain fused to VCA . In the presence of 60 nM Arp2 / 3 and 2 . 5 (cid:109) M actin , 50 nM GST - VCA and 50 reorganization in fibroblasts ( Miki et al . , 1996 , 1998a ) . Addition of GTP (cid:103) S - charged Cdc42 had only a small nM VCA ( when cleaved from the GST moiety ) stimulated the elongation rate by 35 - fold and 25 - fold , respectively , effect on the activity of full - length N - WASP , increasing the elongation rate by (cid:122) 2 - fold and having no effect on compared to the 2 . 5 - fold stimulation obtained with a 4 - fold higher concentration ( 200 nM ) of full - length the duration of the lag phase ( Figure 6A ) . Likewise , lipid vesicles containing phosphatidylcholine ( PC ) , phospha - N - WASP . tidylinositol ( PI ) , and PI ( 4 , 5 ) P 2 in a 48 : 48 : 4 ratio failed to activate N - WASP ( Figure 6A ) . However , the addition of Cdc42 and Phosphatidylinositol ( 4 , 5 ) Bisphosphate Can Synergistically Activate N - WASP In Vitro both PI ( 4 , 5 ) P 2 and GTP (cid:103) S - Cdc42 dramatically stimu - lated actin assembly ( Figure 6A ) , an effect that de - Theabovedatastronglysupport thehypothesisthatfull - length N - WASP is inhibited both in vitro and in Xenopus pended on N - WASP and the Arp2 / 3 complex ( data not shown ) . In fact , the ability of 200 nM N - WASP to activate extracts ( Miki et al . , 1998a ) . To identify potential up - stream regulators that may activate N - WASP in vitro , actin assembly in the presence of PI ( 4 , 5 ) P 2 and GTP (cid:103) S - Cdc42 was equal to that achieved by 200 nM GST - VCA we focused on Cdc42 and PI ( 4 , 5 ) P 2 , which have been implicated in actin assembly in Xenopus extracts ( Ma ( Figure 6A ) . To more precisely determine the activation potency of PI ( 4 , 5 ) P 2 and GTP (cid:103) S - Cdc42 , we compared et al . , 1998a ) . Cdc42 and PI ( 4 , 5 ) P 2 interact with the GBD Cell228 Figure 7 . Signal - DependentActinNucleation A speculative model for the mechanism by which signals , such as Cdc42 and lipids , may regulate actin assembly at membrane - proxi - mal sites by recruitment and activation of the Arp2 / 3 complex via N - WASP - like proteins . the elongation rate as a function of the concentration N - WASP and Actin Nucleation Although N - WASP has been implicated in many actin - of N - WASP , GST - VCA , or N - WASP activated by GTP (cid:103) S - Cdc42 and PI ( 4 , 5 ) P 2 ( Figure 6B ) . In the presence of dependent processes , such as filopodium formation ( Miki et al . , 1998a ) and the actin - based motility of Shi - Cdc42 and PI ( 4 , 5 ) P 2 , the potency of N - WASP was in - creased to a level approaching that of GST - VCA , sug - gella ( Suzuki et al . , 1998 ) , the mechanism by which it mediates actin polymerization has remained elusive . In gesting that N - WASP was fully activated by the two molecules . this report , we provide evidence that N - WASP stimu - lates actin nucleation , a kinetically unfavored step of The coordinate stimulation by Cdc42 and PI ( 4 , 5 ) P 2 favors GTP (cid:103) S - over GDP (cid:98) S - charged Cdc42 , but it is actin polymerization , by interacting with the Arp2 / 3 complex . not completely specific for the GTP form ( Figure 6C ) . Selectivity is most apparent under conditions where the An N - WASP fragment consisting of the C - terminal 55 amino acids ( CA region ) directly binds to the Arp2 / 3 concentration of N - WASP is below that of Cdc42 ( Figure 6C ) . At these low concentrations of N - WASP , the po - complex in vitro ( Figure 3 ) . This association is consistent with the observation that C - terminal segments of Scar1 / tency differenceof GTP (cid:103) S - overGDP (cid:98) S - charged Cdc42 is (cid:122) 5 - fold . Since the PH domain of N - WASP has been WAVE and WASP , both of which share homology with the corresponding region in N - WASP , can bind to the reported to bind specifically to PI ( 4 , 5 ) P 2 , we tested Arp2 / 3 complex ( Machesky and Insall , 1998 ) . However , whether the stimulation seen above is specific for a longer segment of N - WASP , including both the Arp2 / PI ( 4 , 5 ) P 2 - containing vesicles ( Miki et al . , 1996 ) . While 3 - binding CA region and the actin - binding V region , is control lipid vesicles containing only PC and PI ( 50 : 50 ) required for the activation of the Arp2 / 3 complex . The gave only an (cid:122) 2 - fold activation above the no lipid con - homologous VCA region of Scar1 / WAVE has recently trol , similarvesicles containing 4 % PI ( 4 , 5 ) P 2 can activate been shown to disrupt Arp2 / 3 localization and actin as - by (cid:46) 10 - fold ( Figure 6D ) . Finally , GTP (cid:103) S - charged Rho is sembly at the cell periphery when overexpressed in ineffective at mediating stimulation ( data not shown ) . Swiss 3T3cells ( Macheskyand Insall , 1998 ) . The authors Thus , the coordinate activation of N - WASP is Cdc42 and also observed an increase in diffuse perinuclear phalloi - PI ( 4 , 5 ) P 2 specific and selective for the GTP form of Cdc42 . din staining , consistent with our observation that the VCA region should both bind to and stimulate the Arp2 / 3 Discussion complex ( Machesky and Insall , 1998 ) . Disruption of pe - ripheral actin assembly is likely to be a consequence of The study of complex signal transduction networks competition for the Arp2 / 3 complex or actin or both . comprising many direct and indirect pathways requires The Listeria protein ActA can dramatically stimulate an integrated use of biochemical , genetic , and cell bio - the actin nucleation activity of the Arp2 / 3 complex , as logical approaches . Biochemical reconstitution experi - evidenced by a shortening of the lag phase and an in - ments can isolate the minimal set of components essen - crease in the elongation rate ( Welch et al . , 1998 ) . The tialfor biologicalfunctionandhelp elucidatethedetailed VCA region of N - WASP has a very similar effect on actin mechanism by which these components interact . Be - polymerization kinetics in the presence of the Arp2 / 3 cause small G proteins and phosphoinositides have complex , suggesting that N - WASP - like proteins might many targets in the cell ( Toker and Cantley , 1997 ; Van be the cellular counterparts of ActA ( Figures 2 and 3 ) . and D\u2019Souza , 1997 ) , the pathways that connect them to Other than activation of nucleation , these kinetic changes actin polymerization have been difficult to delineate . In in actin assembly could be produced by filament sev - this paper , we have demonstrated one such pathway in ering or an increased rate of elongation . We can essen - vitro , using both a cell - free system and purified compo - tially rule out both these alternative explanations be - nents . WehaveshownthattheArp2 / 3complex , required causewefailedtodetectanysignificantseveringactivity for actin nucleation in a variety of systems , is regulated or any change in the elongation rate of actin filaments by N - WASP . This interaction mediates Cdc42 - induced in the presence of N - WASP , the Arp2 / 3 complex , or both actin polymerization in extracts and can be modulated ( data not shown ) . Although ActA and N - WASP share no significant sequence homology , they do share a short by both PI ( 4 , 5 ) P 2 and Cdc42 . N - WASP Interaction with the Arp2 / 3 Complex 229 segment of similarity in the cofilin homology domain , N - WASP ( Miki et al . , 1996 ) . We have previously demon - strated thatboth Cdc42and PI ( 4 , 5 ) P 2 canstimulate actin which is also present in WASP , Scar1 / WAVE , and Bee1 ( Bi and Zigmond , 1999 ) . The (cid:68) cof mutant , which is de - polymerization in Xenopus egg extracts ( Ma et al . , 1998a ) . Our in vitro data suggests that PI ( 4 , 5 ) P 2 may fective in Arp2 / 3 activation ( Figure 2C ) , contains a four \u2013 amino acid deletion ( (cid:68) 473 KRSK 476 ) within this segment . play an important role in N - WASP activation . HowisN - WASPregulatedby both Cdc42andPI ( 4 , 5 ) P 2 ? How do N - WASPand the Arp2 / 3 complexact together to stimulate actin nucleation ? Since the entire VCA do - Miki et al . have suggested that N - WASP is regulated by an allosteric mechanism ( 1998a ) . Full - length N - WASP main of N - WASP is required for its ability to stimulate actin polymerization , we favor a model ( Machesky and may exist in two conformations that are in equilibrium : an inactive conformation , in which the VCA domain is Insall , 1998 ) in which actin monomers and the Arp2 / 3 complex could be brought into proximity as a result of masked , and an active one in which the VCA domain is exposed and able to interact with the Arp2 / 3 complex . their interactions with the V and CA regions , respectively ( Figure 7 ) . In fact , the V domain is required for the ability At rest , N - WASP largely populates the inactive confor - mation . This is supported by two observations : ( 1 ) full - of N - WASP to remodel the actin cytoskeleton when overexpressed in cells ( Miki and Takenawa , 1998 ) . lengthN - WASPonlymodestlyactivatestheArp2 / 3com - plex ( in comparison to the isolated VCA fragment ) ; and ( 2 ) the full - length protein does not bind to the Arp2 / 3 Cdc42 Signaling and the N - WASP \u2013 Arp2 / 3 complex under conditions where the association be - Complex Interaction tween its VCA fragment and Arp2 / 3 is readily detectable . Our results also shed light on the detailed mechanism However , since full - length N - WASP is completely inac - by which Cdc42 regulates actin polymerization . We pre - tive when added to extracts , we believe that other trans - viously demonstrated a requirement for the Arp2 / 3 com - acting factors may further suppress even the modest plex in Cdc42 - induced actin assembly ( Ma et al . , 1998b ) . activity we have observed with the purified protein in Here , we extend this work to show that N - WASP relays vitro . Indeed , the majority of N - WASP from bovine brain Cdc42 signals and directly activates the Arp2 / 3 complex extracts is present in high molecular weight complexes in Xenopus extracts and in vitro with purified compo - ( unpublished observation ) . In any case , binding of up - nents . Most importantly , using purified N - WASP and stream regulators , such as Cdc42 and PI ( 4 , 5 ) P 2 , to the purified Arp2 / 3 complex , we find that actin polymeriza - N terminus would activate N - WASP by stabilizing the tion can be coordinately stimulated by Cdc42 and active conformation of the molecule ( Figure 7 ) . In addi - PI ( 4 , 5 ) P 2 ( Figure 6 ) . tion to Cdc42 and PI ( 4 , 5 ) P 2 , the N - WASP protein binds This novel finding strongly suggests that the Cdc42 to calmodulin and to SH3 domain \u2013 containing proteins signaling pathway represents one cellular mechanism and may be regulated in other ways ( Miki et al . , 1996 ) . that stimulates actin polymerization by regulating the Unmasked in this active conformation , the VCA domain nucleation activity of the Arp2 / 3 complex ( Ma et al . , would promote nucleation by recruiting Arp2 / 3 via its 1998b ) . The intracellular bacteria Shigella and Listeria CA region and G - actin via its V region . These speculative appear to hijack the cellular machinery for actin assem - models of N - WASP activation and filament nucleation bly by intersecting this pathway at different points . The ( summarized in Figure 7 ) await rigorous testing using ActA protein of Listeria bypasses N - WASP and interacts kinetic analysis as well as by higher resolution mutagen - with the Arp2 / 3 complex directly ( Welch et al . , 1998 ) , esis . However , the results presented here establish whereas the VirG protein of Shigella functions by re - N - WASP and the Arp2 / 3 complex as a core for signal - cruiting N - WASP ( Suzuki et al . , 1998 ) . However , the regulated actin assembly . Cdc42 \u2013 N - WASP pathway probably represents only one of several cellular mechanisms that control actin assem - bly via the Arp2 / 3 complex . N - WASP belongs to a family Experimental Procedures of proteins , including WASP , Scar1 / WAVE ( Bear et al . , Preparation of Xenopus Egg Extracts 1998 ; Miki et al . , 1998b ) , and Bee1 ( Li , 1997 ) , which and Phospholipid Vesicles share sequence homology at their C termini . Since the Xenopus HSSwaspreparedasdescribedpreviously ( Maetal . , 1998b ) . N termini of these proteins are quite different ( Scar1 / Phospholipid vesicles containing PC : PI ( 50 : 50 ) or PC : PI : PI ( 4 , 5 ) P 2 WAVE and Bee1 do not have the GBD or PH domains ) , ( 48 : 48 : 4 ) were prepared as described previously ( Ma et al . , 1998a ) they may transduce diverse upstream signals to a com - with the following modifications . Chloroform - dissolved phospholip - ids ( PC , PI from Avanti Polar Lipids [ Alabaster , AL ] ; PI [ 4 , 5 ] P 2 from moneffector , theArp2 / 3 complex , throughtheirhomolo - Calbiochem ) were mixed in the appropriate ratios and dried under gous C termini . More generally , our results firmly estab - nitrogen . The dried lipid mixture was resuspended in lipid buffer ( 10 lish that de novo actin nucleation is a step at which mMHEPES [ pH7 . 7 ] , 100mMNaCl , 5mMEGTA , and50mMsucrose ) signaling pathways can regulate the actin cytoskeleton . to a final concentration of 10 mM and then extruded through a 100 nm pore polycarbonate filter ( Avanti ) using the Mini - Extruder . Signal - Dependent Actin Polymerization In Vitro The difference in potency between the full - length Preparation of Recombinant Proteins The GST - Cdc42 and GST - Rho fusion proteins were prepared and N - WASP and its C - terminal VCA fragment both in vitro loaded with different nucleotides ( GTP (cid:103) S or GDP (cid:98) S ) while still and in extracts suggests that N - WASP\u2019s activity is sup - bound to glutathione - Sepharose beads according to established pressed by 2 \u2013 3 orders of magnitude . However , using procedures ( Ma et al . , 1998a ) . All excess unbound nucleotide was purified components , we can maximally activate full - washed away , and the G proteins were eluted in 20 mM HEPES ( pH length N - WASP in the presence of two signaling mole - 7 . 6 ) , 5 mM MgCl 2 , 100 mM NaCl , 0 . 1 % sodium cholate , and 10 mM cules , Cdc42 and PI ( 4 , 5 ) P 2 , that have previously been glutathione . Recombinant forms of full - length rat N - WASP , the H208D mutant , shown to interact with the GBD and PH domains in Cell230 and the (cid:68) cof mutant ( deletion of amino acids 473 \u2013 476 ) were pre - were eluted with SDS sample buffer . G - actin pull downs were per - formed in the same manner except that G - actin was used at a pared according to a published protocol , with modifications ( Miki et al . , 1998a ) . Proteins were expressed in insect ( Sf9 ) cells and concentration of 0 . 05 (cid:109) M , below the barbed end critical concen - tration . purified sequentially over heparin , Q - Sepharose , and Superdex 200 columns . GST fusion proteins containing various C - terminal regions of bovine N - WASP ( shown in Figure 3 ) were prepared as described Actin Polymerization Assays previously ( Miki et al . , 1996 ) . The VCA fragment was cleaved from Pyrene actin was used to follow actin polymerization in Xenopus GST - VCA on glutathione - Sepharose beads using Factor Xa ( Phar - extracts as described previously ( Ma et al . , 1998b ) . Polymerization macia ) , and the Factor Xa was separated from VCA using Benzami - wasinitiatedbyaddingGTP (cid:103) S - charged GST - Cdc42ataconcentra - dine Sepharose ( Pharmacia ) . tion of 250 nM ( unless otherwise noted ) . All proteins were quantitated by the Bradford assay ( Bio - Rad , To follow actin polymerization using purified components , pyrene Hercules , CA ) or by scanning densitometry of Gelcode Blue ( Coo - G - actin or unlabeled G - actin was isolated by incubating freshly massie G - 250 ) stained gels , using BSA as a standard in both cases . thawed proteins in G buffer ( 5 mM Tris - HCl [ pH 8 . 0 ] , 0 . 2 mM CaCl 2 , Proteins were stored at concentrations (cid:36) 1 mg / ml at (cid:50) 80 (cid:56) C and 0 . 2 mM ATP , 0 . 2 mM DTT ) for (cid:122) 10 hr at 4 (cid:56) C and then removing thawed and diluted immediately before use . residualF - actinbycentrifugationat400 , 000 (cid:51) gfor1hr . Polymeriza - tion reactions contained 1 . 5 (cid:109) M unlabeled actin , 1 (cid:109) M pyrene actin Preparation and Purification of Antibodies ( 50 % labeled ) , 0 . 2 mM ATP , and various proteins in 80 (cid:109) l of XB . All Affinity - purified peptide antibodies against Arp3 and p34ARC were reaction components except the actin were first mixed together in kindly provided by Matt Welch ( University of California , San Fran - XB and preincubated for 5 min . The reaction was started by adding cisco , CA ) . The anti - actin monoclonal antibody was purchased a mixture of actin and pyrene actin to the preincubated protein mix , from Amersham ( Arlington Heights , IL ) and the anti - Glutathione and fluorescence changes were measured in a fluorometer . In all S - Transferase polyclonal antibody from Santa Cruz Biotech ( Santa the curves shown in the figures , actin polymerization was initiated Cruz , CA ) . at time (cid:53) 0 ; the curves are shifted along the abscissa to account Full - length , Sf9 - expressed rat N - WASP purified according to the for the delay time between the addition of actin and the first fluores - proceduresgivenabovewasusedtoraiseantiserainrabbits ( Zymed cence reading . Labs , SouthSanFrancisco , CA ) . Theantibodieswereaffinitypurified The curves shown in Figure 2B indicate an increase in the steady - essentially as described ( Harlow and Lane , 1988 ) . state pyrene fluorescence in the presence of both Arp2 / 3 and Anti - Arp2 serum was generated by injecting rabbits ( Zymed ) with N - WASP . This is not due to a change in filaments mass at steady a Maltose - binding protein ( MBP ) fusion of human Arp2 , which was state because the addition of phalloidin after the steady state is expressed in E . coli andpurified onan amylose affinitycolumn ( New reached did not change the signal ( data not shown ) . In cases where England Biolabs , Beverly , MA ) . The specificity of the antiserum was a clear steady state was attained , steady - state signals have been confirmedby itsability torecognize ( whenused forimmunoblotting ) normalized to avoid underestimating the slopes of the control the Arp2 subunit from the purified bovine Arp2 / 3 described below . curves . Purification of the Arp2 / 3 Complex from Bovine Brain Extracts Data Analysis The Arp2 / 3 complex was purified from bovine brain extracts by All kinetic analyses were performed using either the software pro - modifying a previously described protocol for the purification of vided with the fluorescence spectrometer ( SLM - Aminco ) or using Arp2 / 3 from Xenopus egg extracts ( Ma et al . , 1998b ) . High - speed Origin ( Microcal Software , Northampton , MA ) . Maximum elongation extracts from calf brain were sequentially fractionated on Butyl rates from the polymerization curves using the purified system have Sepharose , DEAE Sepharose , S Sepharose , Phenyl Sepharose , and been calculated by performing a linear regression on the linear , Superdex 200 columns , and the Arp2 / 3 complex was followed by elongation phase of the actin assembly curve . For pyrene assays immunoblottingwithanti - Arp3oranti - p34ARC . Thebovinecomplex in extracts , the initial rate and the maximum F - actin were calculated runs as a symmetric peak at the expected ( (cid:122) 200 kDa ) molecular as described previously ( Ma et al . , 1998a ) . In all the curves shown weight on a gel filtration column and contains all seven polypeptide in the figures , solid lines are drawn through all the data points subunits ( Figure 2A ) . The identities of four of these polypeptides collected ; a symbol is only placed on every fifth or tenth point for ( Arp2 , Arp3 , p41 - ARC , and p34 - ARC ) were confirmed by immu - clarity . All data shown in the figures were taken from experiments noblotting . The bovine complex is also functional because it can performed at least twice . When normalized to controls , data varied support Cdc42 - induced actin assembly when substituted for a Xen - by (cid:35) 20 % between independent experiments . opus Arp2 / 3 - containing fraction ( Ma et al . , 1998b ) . Immunodepletion Acknowledgments For immunodepletion of N - WASP , high - speed supernatants of Xen - opus egg extracts were mixed with affinity - purified anti - N - WASP We thank Jeff Peterson for his help in the purification of the Arp2 / 3 antibodies at a concentration of 75 (cid:109) g / ml and incubated on ice for complex from bovine brain extracts and for his preparation of lipid 2 hr . The immune complexes were captured by adding one - tenth vesicles . We thank Teresita Bernal , Louise Evans , and Ann Georgi volume of packed protein - A Affiprep beads ( Bio - Rad ) and gently for their help in expressing recombinant proteins in Sf9 cells . We rockingat 4 (cid:56) Cfor 2hr . Mockdepletions wereperformed inthe same thank Tim Mitchison and Phil Allen for many helpful discussions way except that antibody buffer ( PBS (cid:49) 40 % glycerol ) was used in - and Matt Welch for providing antibodies against Arp3 and p34ARC . stead of the anti - N - WASP . The efficacy of the depletion was con - We also thank Jeff Peterson , Susannah Rankin , and Todd Stuken - firmed to be (cid:36) 95 % by immunoblotting with the anti - N - WASP an - berg for comments on the manuscript . R . R . is a member of the tibody . Medical Scientist Training Program at Harvard Medical School . This work was supported in part by grants from the National Institutes of Health to M . W . K . ( GM26875 ) and T . K . ( 5PO1 HL 59561 - 02 ) . Binding Assays GST pull - down assays were performed by immobilizing equal amounts of the GST fusion proteins on glutathione - Sepharose Received February 25 , 1999 ; revised March 24 , 1999 . beads ( Pharmacia ) at a concentration of approximately 5 mg / ml packed beads . For the Arp2 / 3 complex pull downs , (cid:122) 5 (cid:109) l of beads were incubated with 200 (cid:109) l of bovine Arp2 / 3 complex ( 0 . 8 (cid:109) M ) in References XB ( 10 mM HEPES [ pH 7 . 6 ] , 100 mM KCl , 1 mM MgCl 2 , 0 . 1 mM EDTA , 1 mM DTT ) plus 0 . 1 % ( v / v ) Tween - 20 , 0 . 2 mM ATP , and 0 . 5 Arber , S . , Barbayannis , F . A . , Hanser , H . , Schneider , C . , Stanyon , C . A . , Bernard , O . , and Caroni , P . ( 1998 ) . Regulation of actin dynam - mg / ml chicken albumin ( binding buffer ) . 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The small GTP - binding protein rho", + "serwas2022mechanistic": "Article Mechanistic insights into actin force generation during vesicle formation from cryo - electron tomography Graphical abstract Highlights d Native - state description of force - producing actin networks during endocytosis d Branched actin \ufb01lament assembly is triggered from multiple mother \ufb01laments d Actin force production is robust despite considerable network variability d Filament anchorage points are key to pulling force generation and ef\ufb01ciency Authors Daniel Serwas , Matthew Akamatsu , Amir Moayed , . . . , Karen M . Davies , Padmini Rangamani , David G . Drubin Correspondence daniel . serwas @ berkeley . edu ( D . S . ) , drubin @ berkeley . edu ( D . G . D . ) In brief Actin \ufb01lament polymerization generates forces essential for numerous cellular processes including vesicle formation , cell motility , and cytokinesis . Serwas et al . combined cryo - electron tomography of intact mammalian cells with mathematical modeling to gain mechanistic insights into how actin assembly forces pull on the plasma membrane to support endocytic vesicle formation . Serwas et al . , 2022 , Developmental Cell 57 , 1132 \u2013 1145 May 9 , 2022 \u00aa 2022 Elsevier Inc . https : / / doi . org / 10 . 1016 / j . devcel . 2022 . 04 . 012 ll Article Mechanistic insights into actin force generation during vesicle formation from cryo - electron tomography Daniel Serwas , 1 , * Matthew Akamatsu , 1 , 4 Amir Moayed , 1 Karthik Vegesna , 1 Ritvik Vasan , 2 , 5 Jennifer M . Hill , 1 Johannes Scho\u00a8neberg , 1 , 6 Karen M . Davies , 1 , 3 , 7 Padmini Rangamani , 2 and David G . Drubin 1 , 8 , * 1 Department of Molecular and Cell Biology , University of California , Berkeley , Berkeley , CA , USA 2 Department of Mechanical and Aerospace Engineering , University of California , San Diego , La Jolla , CA , USA 3 Molecular Biophysics and Integrative Bioimaging Division , Lawrence Berkeley National Laboratory , Berkeley , CA , USA 4 Present address : Department of Biology , University of Washington , Seattle , WA , USA 5 Present address : Allen Institute of Cell Science , Seattle , WA , USA 6 Present address : Department of Pharmacology and Department of Chemistry & Biochemistry , University of California , San Diego , La Jolla , CA , USA 7 Present address : Electron Bio - Imaging Centre , Diamond Light Source , Harwell Science and Innovation Campus , Didcot , UK 8 Lead contact * Correspondence : daniel . serwas @ berkeley . edu ( D . S . ) , drubin @ berkeley . edu ( D . G . D . ) https : / / doi . org / 10 . 1016 / j . devcel . 2022 . 04 . 012 SUMMARY Actin assembly provides force for a multitude of cellular processes . Compared to actin - assembly - based force production during cell migration , relatively little is understood about how actin assembly generates pulling forces for vesicle formation . Here , cryo - electron tomography identi\ufb01ed actin \ufb01lament number , orga - nization , and orientation during clathrin - mediated endocytosis in human SK - MEL - 2 cells , showing that force generation is robust despite variance in network organization . Actin dynamics simulations incorporating a measured branch angle indicate that suf\ufb01cient force to drive membrane internalization is generated through polymerization and that assembly is triggered from (cid:1) 4 founding \u2018\u2018mother\u2019\u2019 \ufb01laments , consistent with tomography data . Hip1R actin \ufb01lament anchoring points are present along the entire endocytic invagi - nation , where simulations show that it is key to pulling force generation , and along the neck , where it targets \ufb01lament growth and makes internalization more robust . Actin organization described here allowed direct translation of structure to mechanism with broad implications for other actin - driven processes . INTRODUCTION Actin \ufb01laments are structurally polarized linear polymers that preferentially grow at one end , called the plus or barbed end ( Pollard , 2016 ) . Polymerization of individual actin \ufb01laments can produce forces in the range of 1 \u2013 9 pN ( Dmitrieff and Nedelec , 2016 ) . These \ufb01laments can organize into higher order assemblies that facilitate a multitude of essential cellular functions including clathrin - mediated endocytosis ( CME ) ( Rottner et al . , 2017 ) . During CME , the plasma membrane is deformed to produce cargo - containing clathrin - coated vesicles ( CCVs ) . This mem - brane remodeling is promoted by assembly of a clathrin - contain - ing protein coat and forces provided by the actin cytoskeleton ( Kaksonen and Roux , 2018 ) . Although how a growing actin network can push on a cellular membrane , for example , during cell migration , is well understood , how assembly can aid in mem - brane pulling during endocytosis and intracellular traf\ufb01cking is much less well understood . CME is well suited to studies of membrane pulling through actin assembly as nearly complete lists of the components involved and detailed information on their dynamics exist ( Kaksonen and Roux , 2018 ) . However , mo - lecular - scale positional information about these components in their native state , which is essential for attaining a mechanistic understanding of their activities , is lacking . During CME , clathrin coat assembly initiation is followed by recruitment of the actin \ufb01lament nucleating Arp2 / 3 complex ( Tay - lor et al . , 2011 ) . This complex can bind to the sides of existing \u2018\u2018mother\u2019\u2019 \ufb01laments to induce assembly of new \u2018\u2018daughter\u2019\u2019 \ufb01la - ments , leading to formation of branched actin networks ( Rottner et al . , 2017 ) . The clathrin and plasma membrane - binding coat protein Hip1R can tether actin \ufb01laments to CME sites to harness \ufb01lament polymerization forces for plasma membrane deforma - tion ( Akamatsu et al . , 2020 ; Engqvist - Goldstein et al . , 1999 , 2001 ) . Using agent - based models , we previously found that actin self - assembles into a branched network during CME , with \ufb01la - ments oriented orthogonal to the plasma membrane , surround - ing , and attached to , the clathrin coat , and their growing plus ends oriented toward the plasma membrane . This geometry was dependent on the experimentally constrained spatial distri - bution of activated Arp2 / 3 complexes and actin \ufb01lament - binding 1132 Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 \u00aa 2022 Elsevier Inc . ll Hip1R linkers embedded in the clathrin coat ( Akamatsu et al . , 2020 ; Mund et al . , 2018 ; Sochacki et al . , 2017 ) . The resulting network geometry was consistent with previously proposed models and predicted the range for the numbers of \ufb01laments that could be involved in force generation in CME ( Akamatsu et al . , 2020 ; Kaksonen et al . , 2006 ) . In contrast , platinum replica electron microscopy ( EM ) of unroofed mammalian cells showed branched actin \ufb01laments surrounding only the neck region of CME sites in a collar - like fashion with \ufb01laments aligned parallel to the plasma membrane ( Collins et al . , 2011 ) . However , this method , likeotherclassicEMmethods , mightnotpreservenative actin cytoskeleton organization as it might result in partial removal of some of the actin network during unroo\ufb01ng and might not allow entire actin \ufb01laments in dense networks to be traced ( Collins et al . , 2011 ; Maupin - Szamier and Pollard , 1978 ; Resch et al . , 2002 ; Small , 1981 ) . In addition , in silico experiments sug - gest that both parallel and orthogonal \ufb01lament arrangements can facilitate CME progression ( Hassinger et al . , 2017 ) . These predictions emphasize the need to determine the orientation and numbers of actin \ufb01laments at CME sites before a quantitative understandingof theprecisemechanism ofactin - mediated force generationduringCMEcanbeachieved . TheplatinumreplicaEM study further suggested that the branched actin networks origi - natefromasinglefoundingmother\ufb01lament , butitsoriginremains unclear ( Collins et al . , 2011 ) . In addition to the orientation of branched actin \ufb01laments and the origin of mother \ufb01laments , the precise localization of the critical actin - CME linker Hip1R is ambiguous since partially contradicting data have been pub - lished ( Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; Sochacki et al . , 2017 ) . Obtaining the structural information described above requires a method that allows visualization of native three - dimensional ( 3D ) actin networks at the single - \ufb01la - ment level , determination of each \ufb01lament\u2019s orientation , and the precise localization of the aforementioned linker protein Hip1R . In recent years , in situ cryo - electron tomography ( cryo - ET ) has been shown to be an extremely powerful approach to visualize the 3D organization of cellular features , including actin networks and protein - coated vesicles , at near - native - state conditions with unprecedented detail ( Bykov et al . , 2017 ; F \u20ac a\u00dfler et al . , 2020 ; Jas - nin and Crevenna , 2016 ; Mahamid et al . , 2016 ; Vinzenz et al . , 2012 ) . Here , to elucidate the mechanism of pulling force gener - ation through actin network assembly , we integrated cryo - ET on organization of actin networks involved in mammalian cell CME with mathematical modeling . RESULTS Clathrin coat identi\ufb01cation in cryo - electron tomograms of intact cells Toinvestigatethestructural organization ofactinduring CME , we used cryo - ET of vitri\ufb01ed intact human - derived SK - MEL - 2 cells growing on EM grids ( Serwas and Davies , 2021 ) . We identi\ufb01ed honeycomb - like arrangements in our tomography data , which were reminiscent of clathrin coats seen in previous EM studies ( Figure 1A ) ( Avinoam et al . , 2015 ; Cheng et al . , 2007 ; Heuser , 1980 ) . To test whether these arrangements are indeed clathrin coats , we applied correlative cryo - \ufb02uorescence lightmicroscopy ( cryo - FLM ) andcryo - ETusingSK - MEL - 2cellsthatendogenously express GFP - tagged clathrin light chain A ( CLTA - GFP ) and were incubated with \ufb02uorescent transferrin ( TF ) cargo to precisely pinpoint CME events ( Figures S1A and S1B ) . Tomograms ob - tained by this method showed the same structural features as in the randomly collected datasets ( Figure S1B ) . The randomly collected tomograms were of higher quality than the correlative data , likely due to fewer manual handling steps , and were there - fore used for the further analysis . Subtomogram averaging of clathrin coat vertices from a total of 8 CME sites and CCVs iden - ti\ufb01ed additional details of the native hub structure ( Figure 1B ; Video S1 ) . The resolution of our density map was 2 . 7 nm based on the 0 . 5 Fourier shell correlation criterion ( Figure 1C ) . Compu - tational \ufb01ttingoftherecently published structural model obtained by single particle cryo - EM ( PDB : 6SCT ; Morris et al . , 2019 ) into our in situ map resulted in a cross - correlation score of 0 . 94 ( Fig - ure 1D ) . Our structural analysis together with our correlative mi - croscopy results thus veri\ufb01es the identity of the protein coats in our tomograms as clathrin coats . Actin organization at different CME stages DuringCME , therelatively\ufb02atplasmamembraneinvaginatesand thenaconstrictedneckformsatthebaseoftheformedpitpriorto CCV scission ( Avinoam et al . , 2015 ; Roth and Porter , 1964 ) . We classi\ufb01ed the clathrin structures in our tomograms according to the shape of their underlying membrane as early \ufb02at , early , and late invaginated clathrin - coated pits ( CCPs ) and as CCVs ( Figures 2A , 2B , S2A , and S2B ; Videos S2 , S3 , S4 , and S5 ) . CCVs had a mean membrane and a clathrin coat diameter of 46 . 7 \u00b1 7 . 5 and 93 . 4 \u00b1 4 . 2 nm , respectively , whereas the CCP mean membrane and coat diameter were 90 . 2 \u00b1 10 . 4 and 136 . 1 \u00b1 17 . 7 nm ( Figure S1C ) . We then generated segmentation models to visualize the spatial relationship between actin \ufb01la - ments , the membrane , and the clathrin coat in 3D ( Figures 2B and S2B ) . Our analysis showed that individual clathrin triskelia tended to be somewhat disconnected in early clathrin coats compared with late CME sites and CCVs , indicating \ufb02exibility of earlyclathrincoats , whichisconsistentwitharecentstudyonun - roofed , chemically \ufb01xed cells ( Sochacki et al . , 2021 ) . Using live cell microscopy , it was previously shown that actin assembly oc - curs at (cid:1) 87 % of CME events in SK - MEL - 2 cells ( Grassart et al . , 2014 ) . Of 13 clathrin - coated structures in our tomograms ( Table S1 ) , only two did not show actin \ufb01lament association , and these two were released CCVs . It is important to note that duetotechnicallimitationsincryo - ET , ouranalysiswasrestricted tothinperipheralcellregions . Perhaps , inthesecellregions , CME sites are more likely to assemble actin . It will be important to determine whether CME events in thicker cell regions are more likely to be free of actin \ufb01laments or if they have amounts of actin \ufb01laments that are below the detection limit of light microscopy . Actin branch junctions could clearly be identi\ufb01ed based on the presence of an arc - like density at the junctions , most likely repre - senting the Arp2 / 3 complex branch nucleator ( Figures 3A and S3A ) ( Rouiller et al . , 2008 ; Vinzenz et al . , 2012 ) . To our surprise , CME - associatedactin\ufb01lamentsdidnotconsistofbranchedactin \ufb01lamentsexclusivelyaspreviouslyproposed ( Collinsetal . , 2011 ) . Instead , we observed a mixture of branched and unbranched \ufb01l - aments at all stages of CME ( Figures 2B and S2B ) . While branched \ufb01laments accumulated directly adjacent to CME sites , unbranched \ufb01laments were distributed across the entire tomographic volume . Unbranched \ufb01laments were organized in ll Article Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 1133 bundles close to CME sites , in dense meshworks of cortical actin \ufb01laments covering and surrounding CME sites , or as separated individual \ufb01laments . Branched actin \ufb01laments appeared asym - metrically distributedaround CMEsites , as suggested previously ( Collins et al . , 2011 ; Yarar et al . , 2005 ) . Themechanicalproperties ofindividual actin \ufb01lamentsdepend ontheirlength , withshorter\ufb01lamentsbeingmorerigidthanlonger ones ( De La Cruz and Gardel , 2015 ) . Our previous simulations predicted that branched actin \ufb01laments grow to a median length of 64 nm during CME and that branched \ufb01lament length was exponentially distributed , which can be explained by stochastic capping ( Figure S2C ; Tables S2 and S3 ; Akamatsu et al . , 2020 ) . Here , we measured a median branched \ufb01lament length in tomo - grams of 59 nm , indicating that these \ufb01laments could have assembled over the course of a CME event ( Figure 2C ; Table S3 ) . This possibility is further supported by the relatively low number of branched \ufb01laments in the early - stage CME tomo - gram shown in Figures 2A and 2B . As predicted by our previous simulations , branched \ufb01lament length in our tomograms was exponentially distributed ( Table S2 ) . Unbranched \ufb01laments were longer and had a median length of 108 nm ( Figure 2C ; Table S3 ) . Some of the unbranched \ufb01laments likely represent \ufb01l - aments that existed before the onset of the CME event , for example , as part of the actin cortex . However , a subset of these unbranched \ufb01laments could have been newly polymerized and alsocontributedtoforcegenerationtosupportCMEprogression . CME is robust to variations in the actin \ufb01lament branch angle As described above , branch junctions were identi\ufb01ed based on the presence of an additional density , likely the Arp2 / 3 complex , at the connection between the mother and daughter \ufb01lament . Figure 1 . Native clathrin coat architecture and structure ( A ) TomographicslicesofaCCVatZ - positionsrelativetocentralslice ( Z = 0nm ) . Topandbottomsliceshowcharacteristiccage - likeclathrincoatarchitecturewith threeleg - like extensions emanating fromeachvertex ( yellowarrowheads ) . Yellowand whitecirclesincentralslice indicate vesicleandcoatedarea , respectively . ( B ) Density map of the clathrin coat determined within intact cells . ( C ) Resolution determination of the clathrin density map based on Fourier shell correlation ( FSC ) . ( D ) Structuralmodeloftheclathrinhub ( PDB : 6SCT , inrainbowcolors , fromMorrisetal . [ 2019 ] ) \ufb01ttedintosubtomogramaverageisosurfacemodelofcytoplasmic clathrin vertex . Scale bars , 50 nm in ( A ) and 10 nm in ( B ) and ( D ) . See also Figure S1 . ll Article 1134 Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 Figure 2 . CME - actin networks consist of branched and unbranched \ufb01laments ( A ) Tomographic slices at Z - positions relative to central slice ( Z = 0 nm ) of CME events at indicated stages . Clathrin coat and individual actin \ufb01laments are high - lighted with yellow and red arrowheads , respectively . ( legend continued on next page ) ll Article Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 1135 We used subtomogram averaging to further test whether the identi\ufb01ed junctions were indeed Arp2 / 3 complex - based branch junctions . A low - resolution density map was obtained , which was suf\ufb01cient to computationally \ufb01t the recently published in situ branch junction structure , indicating that the junctions were Arp2 / 3 complex based ( Figures S3B \u2013 S3D ; F \u20ac a\u00dfler et al . , 2020 ) . Branch angles in a range of about 60 (cid:3) \u2013 80 (cid:3) , obtained from various sample types and methods , have been reported previously for Arp2 / 3 complex - nucleated actin \ufb01lament net - works ( Blanchoin et al . , 2000 ; F \u20ac a\u00dfler et al . , 2020 ; Jasnin et al . , 2019 ; Mueller et al . , 2014 ; Mullins et al . , 1998 ; Rouiller et al . , 2008 ; Svitkina and Borisy , 1999 ; Vinzenz et al . , 2012 ) . The branch angle of the structure that we used to \ufb01t into our subtomogram average is 71 (cid:3) . However , this average value does not capture the range of branch angle values at CME sites . Therefore , we measured the angles of individual branch junctions . The average branch angle from these measurements was 68 (cid:3) \u00b1 9 (cid:3) , which is in close agreement with recently published in situ structures ( Fig - ure 3B ) ( F \u20ac a\u00dfler et al . , 2020 ; Jasnin et al . , 2019 ) . Physical limita - tions ( e . g . , limited tilt range due to sample holder geometry and sample thickness ) restrict electron tomography data recording , resulting in a wedge - shaped lack of information in 3D Fourier space ( Lu (cid:2) ci (cid:3) c et al . , 2013 ; Wan and Briggs , 2016 ) . The so - called \u2018\u2018missing wedge\u2019\u2019 results in anisotropic resolution such that the tomography data are elongated in the electron beam direction ( z direction ) ( Lu (cid:2) ci (cid:3) c et al . , 2013 ; Wan and Briggs , 2016 ) . To test whether the missing wedge strongly affected our branch angle measurements , we plotted the measured branch angles against the orientation of the branch junctions in our to - mography data ( Figure S3E ; see STAR Methods for details ) . Branch angle values were independent of branch orientation . We therefore concluded that within the precision of our measure - ments , our branch angle measurements were not affected by the missing wedge . The native branch angles were on average smaller and the junctions slightly stiffer ( indicated by the stan - dard deviation ) compared with the average branch angle of 78 (cid:3) \u00b1 16 (cid:3) used in our previous mathematical model ( Akamatsu et al . , 2020 ) . To test whether the smaller branch angles affect Figure 3 . CME is robust against branch angle variation ( A ) Tomographic slicesof an individual branch junction atZ - positions , left toright , relative tocentral slice ( Z = 0nm ) . Positions of mother , daughter \ufb01laments , and plus and minus ends are indicated . Arrow points to the arc - like density of the Arp2 / 3 complex . ( B ) Branch angle distribution measured in tomograms . Mean value and standard deviation are displayed . ( C ) Resultofsimulations showing theeffect ofbranchangle variationonthenumberofplusendsatthebase ofCME invaginations ( greenand orangecurves ) and the internalization rate ( cyan and purple curves ) . Mean and SD are shown . Scale bar , 5 nm in ( A ) . See also Figure S3 . ( B ) Segmentation models of the tomograms in ( A ) , bottom row shows branched actin \ufb01laments only . Color - coded legend describes elements shown in the models . ( C ) Filament length distribution of unbranched and branched \ufb01laments across all CME events shown in this publication . Median \ufb01lament length for unbranched and branched actin \ufb01laments are highlighted . Scale bars , 50 nm in ( A and B ) . See also Figure S2 . ll Article 1136 Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 the force production capability , we modi\ufb01ed our computational model of branched actin \ufb01lament assembly at CME sites to re\ufb02ect the newly measured branch angle ( Figure S3F ) . In this model , an endocytic pit is modeled as a solid hemispherical ob - ject that resists internalization elastically ( Hassinger et al . , 2017 ) . Activated Arp2 / 3 complex resides in a ring around the base of the pit ( Almeida - Souza et al . , 2018 ; Mund et al . , 2018 ) , whereas Hip1R linkers are embedded in the surface of the curved pit ( So - chacki et al . , 2017 ) . Linear \ufb01laments diffusing near the pit serve as mother \ufb01laments that , when in proximity to the active Arp2 / 3 complex , generate new actin \ufb01laments branching from the mother \ufb01lament ( Blanchoin et al . , 2000 ) . 3D stochastic simu - lations of the model showed that actin \ufb01laments self - assembles into a polarized branched network that internalizes the pit against physiological values of plasma membrane tension ( Aka - matsu et al . , 2020 ; Kaplan et al . , 2021 ; Nedelec and Foethke , 2007 ) . We found that for both ranges of branch angles , the num - ber of plus ends polymerizing against the base of the CME site remained the same ( 10 \u00b1 4 ) , leading to similar internalization rates ( Figure 3C ) . In light of this result , we next tested the effect of a wider range of branch angles ( 10 (cid:3) \u2013 170 (cid:3) ) on internalization ef\ufb01ciency . We found that endocytosis remained robust ( Figures S3G and S3H ) . Although the branch angle might affect some architectural features , we conclude that overall polymeri - zation - based force production for CME is robust against branch angle variation , which contrasts with results reported for simula - tions of lamellipodia formation where a branch angle of (cid:1) 70 (cid:3) \u2013 80 (cid:3) was predicted to be optimal ( Garner and Theriot , 2020 ) . Branched actin assembly is nucleated from multiple mother \ufb01laments Next , we tested the previously postulated notion that branched actin network assembly at CME sites is initiated from a single Figure 4 . Branched actin \ufb01laments are orga - nized into clusters ( A ) Simulation snapshot highlighting clustered or - ganization of branched actin \ufb01laments in mathe - matical model . Individual clusters are color coded . ( B ) Simulation of mean cluster number during inter - nalization . ( C ) Segmentation model of late CME site from Fig - ure 2A highlighting clustered organization of branched actin \ufb01laments . Individual clusters are color coded . ( D ) Number of clusters found at individual CME events . Mean and SD are shown in ( B ) and ( D ) . Scale bar , 50 nm in ( C ) . See also Figure S4 . \u2018\u2018founding\u2019\u2019 mother \ufb01lament ( Collins et al . , 2011 ) . Simulations using the conditions we de\ufb01ned for our model predicted that branched actin network assembly can be initiated from several \u2018\u2018founding\u2019\u2019 mother \ufb01l - aments , each giving rise to a distinct branchedactin\ufb01lamentcluster . Thenumber of clusters and number of \ufb01laments per cluster increased during CME progression to an average of 4 \u00b1 2 clusters with 49 \u00b1 21 \ufb01laments in each cluster ( Figures 4A , 4B , and S4A ) . The predicted clustered branched actin \ufb01lament orga - nizationwasconsistentwiththearrangementofbranchedactin\ufb01l - aments in the tomograms , where theaverage cluster numberwas 8 \u00b1 6 ( Figures 3C and 3D ) . However , individual clusters only con - sisted of an average of 2 . 2 \u00b1 0 . 5 \ufb01laments in the tomography data ( FigureS4B ) . Thediscrepancyinthe\ufb01lamentnumberperclusterin the model versus experiment can be understood as follows : although the model shows 49 \u00b1 21 \ufb01laments per cluster at the end of a CME event , the number of \ufb01laments at the base is only 10 \u00b1 4 , suggesting that not all \ufb01laments in the model contribute directly to force generation ( Figure 3C ) . Since the model only cap - tures select actin interactions , it is also possible that the excess \ufb01lament number per cluster in the model re\ufb02ects certain limita - tions in the model . For example , it has been shown previously that in lamellipodia individual branch junctions are separated by multiples of the actin helix repeat of 36 nm along individual \ufb01la - ments ( Vinzenz et al . , 2012 ) . In the current version of the model , the spacing between individual branches along a \ufb01lament cannot be controlled . As a result , branching occurred at a higher fre - quency , with a median inter - branch distance of 6 . 5 nm , than is possible in cells due to geometrical and crowding - related spatial restrictions ( Figure S4C ) . It will be important for future models to controlthemaximumspacingbetweenbranchesinordertobetter understand the relationship between local branch and cluster ge - ometryandoverallendocyticactinnetworkarchitecture . Theearly CME site in the tomogram in Figure 2 showed the lowest number of branched actin \ufb01lament clusters , suggesting that more branched actin clusters are initiated and assembled during the plasma membrane internalization phase . Actin \ufb01lament orientation at CME sites Next , we set out to analyze the polarity and orientation of the actin \ufb01laments in the tomograms to assess the direction of force ll Article Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 1137 production through polymerization . The polarity of branched \ufb01l - aments can be determined based on branch junction geometry ( Figure 3A ) ( Narita et al . , 2012 ) . To analyze the polarity of un - branched \ufb01laments , we adapted a previously published method , which is based on cross - correlation analysis of the \ufb01laments in tomograms against simulated reference \ufb01laments with known polarity ( Figures S5A \u2013 S5E ) ( Narita et al . , 2012 ) . We developed an analysis software package that allowed us to calculate and plot the orientation of the \ufb01laments relative to the normal vector of a simulated reference plane representing the position of the plasma membrane or individual CCVs . Orientation of the vector was de\ufb01ned such that 0 (cid:3) orientation indicates actin \ufb01lament plus end pointing toward the reference plane , and 180 (cid:3) orientation in - dicates actin \ufb01lament plus end pointing away from the plane . The average \ufb01lament orientation across all tomograms was 86 (cid:3) \u00b1 31 (cid:3) for unbranched and 81 (cid:3) \u00b1 36 (cid:3) for branched \ufb01laments , indicating that a large proportion of \ufb01laments were oriented parallel to the plasma membrane ( Figures S5F and S5G ) . Further examination of individual CME sites identi\ufb01ed two CME archetypes for branched actin \ufb01lament organization , one in which \ufb01laments were oriented orthogonal to the plasma membrane , similar to the predictions from our simulations , and a second in which \ufb01la - ments were oriented largely parallel to the plasma membrane , similar to what was observed in previous work using platinum replica EM ( Figures 5A \u2013 5D , S5H , and S5I ) ( Akamatsu et al . , 2020 ; Collins et al . , 2011 ) . On average , 11 \u00b1 8 branched \ufb01lament plus ends and 36 \u00b1 7 unbranched \ufb01lament plus ends were ori - ented toward the base of individual CME sites ( relative \ufb01lament orientation angle < 90 (cid:3) ) during the membrane internalization phase ( Figure 5E ) . In contrast to our simulations , we also observed up to two branched \ufb01lament plus ends pointing toward the neck of CME invaginations for both types of actin arrange - ments , where they could potentially produce a squeezing force to support neck constriction and scission ( Figures 5B , 5C , and 5E ) . We also found branched actin \ufb01laments oriented with their plus ends toward CCVs , suggesting a role for \ufb01lament polymer - ization in CCV transportation inside cells ( Figures S5J and S5K ) . Hip1R localizes to the CME neck , where it is predicted to increase internalization ef\ufb01ciency Our previous model identi\ufb01ed the localization and distribution of the actin - binding protein Hip1R as a main determinant for actin organization and function ( Akamatsu et al . , 2020 ) . There , we had assumed that Hip1R exclusively localizes to the tip of CME invaginations , based on Sochacki et al . ( 2017 ) , in which averaging of FLM data were used ( Akamatsu et al . , 2020 ; So - chacki et al . , 2017 ) . However , individual images from the work of Sochacki et al . ( 2017 ) and other published work show variable Hip1R localization , including at the neck of CME invaginations ( Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; So - chacki et al . , 2017 ) . Importantly , only the methodology in the study of Clarke and Royle ( 2018 ) allowed visualization of the entire CME invagination , and it is likely that the other localization data gave an incomplete picture . Therefore , we asked if we could identify Hip1R in our tomograms and whether its localization might play a role in targeting actin \ufb01laments to the neck of CME sites . Hip1R forms dimers that by quick - freeze deep - etch EM appear as (cid:1) 60 nm rod - shaped densities with two globular actin - binding domains at one end and two globular mem - brane - binding domains at the other ( Engqvist - Goldstein et al . , 2001 ) . We identi\ufb01ed densities resembling Hip1R in size and structure over the invagination surface including the neck of CME invaginations as well as at the plasma membrane adjacent to the pit , which was consistent with the work from Clarke and Royle ( 2018 ) ( Figures 6A and S6A ) . To further test whether these densities could be Hip1R dimers , we subjected the putative cytoplasmic actin - binding domains to subtomogram averaging , yielding a low - resolution density map ( Figures 6B , S6B , and S6C ) . The size and shape of the density was suf\ufb01cient to house two actin - binding domains plus the dimerization domains of the homologous protein talin and to \ufb01t onto an actin \ufb01lament ( Fig - ure 6B ) ( Gingras et al . , 2008 ) . This analysis provides further sup - port for the likelihood that these densities are Hip1R dimers . We then tested in our model the consequence of Hip1R localization at the neck by conducting simulations , wherein Hip1R molecules were positioned along the neck surface in addition to the invag - ination tip . The number of \ufb01lament plus ends at the base ( 10 \u00b1 4 ) remained the same with or without additional Hip1R dimers at the neck . However , an additional 4 . 5 \u00b1 2 plus ends were now found in the neck region , which is similar to the branched \ufb01la - ment plus end number in our tomograms ( Figures 5E , 6C , S6D , and S6E ) . Strikingly , in our simulations , Hip1R neck localization not only directed \ufb01laments to the neck region but also strongly improved internalization ef\ufb01ciency , indicating that Hip1R at the neck can help with actin - mediated force generation ( Figure 6D ) . DISCUSSION Forces produced by actin \ufb01lament assembly are harnessed for many membrane remodeling processes , but much remains to be learned about the underlying assembly regulation and force - producing mechanisms . CME is a particularly attractive process for elucidating these mechanisms because it consists of a predictable series of morphological changes , each of which is coupled to recruitment of speci\ufb01c proteins . This work set out to de\ufb01ne the 3D organization of individual actin \ufb01laments at different CME stages because this information directly informs the force - producing mechanism . What follows is an attempt to synthesize our cryo - ET and simulation results into a harmonious model for how actin assembly forces are generated and har - nessed during CME . Our \ufb01nding that branched actin networks are organized in branched actin \ufb01lament clusters at individual CME sites supports the conclusion that these clusters originate from multiple found - ing mother \ufb01laments ( 4 \u00b1 2 in the simulations and 8 \u00b1 6 in the to - mograms ) , which is a different conclusion from the single mother \ufb01lament model that was suggested previously ( Collins et al . , 2011 ) . This clustered organization may provide \ufb02exibility for branched actin \ufb01laments to assemble in a crowded environment like the cell cortex , which also suggests that excluded volume ef - fects may affect branched actin network assembly ( Schreiber et al . , 2010 ) . Individual branch clusters consisted of an average of 2 . 2 \u00b1 0 . 5 \ufb01laments . Accordingly , most clusters in our tomograms were ar - rangements of a mother and one daughter \ufb01lament . This number of branch junctions seems low at \ufb01rst glance . As described above , cryo - ET data are affected by the missing wedge problem . We therefore cannot completely exclude the possibility that ll Article 1138 Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 Figure 5 . Actin \ufb01lament orientation at CME sites ( A ) Color code indicating \ufb01lament orientation relative to the normal ( 0 (cid:3) ) of a reference plane representing the plasma membrane . At 0 (cid:3) , \ufb01lament plus ends point toward the membrane . ( B and C ) Color - coded \ufb01lament orientation at two late - stage CME invaginations ( green ) , ( C ) shows a site toward the end of vesicle scission . Note the presence of \ufb01laments with their plus ends oriented toward the neck region ( red arrowheads ) . ( D ) Relative \ufb01lament to reference plane orientation of late CME sites 1 and 2 shown in ( B ) and ( C ) , respectively . ( E ) Number of \ufb01lament plus ends pointing toward base and neck region of CME sites . Mean and SD are shown in ( D ) and ( E ) . See also Figure S5 . ll Article Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 1139 some \ufb01laments were not detected in our tomograms ( i . e . , when they are parallel to the electron beam direction and are posi - tioned completely in the missing wedge ) . However , given that the branch number per individual mother \ufb01lament across multi - ple tomograms is very consistent , we are convinced that our data reliably represent the branched actin \ufb01lament organization at CME sites in cells . A possible biological explanation for the sparse number of branch junctions concerns the spacing of Arp2 / 3 branch junctions . Previous work showed that the inter - branch spacing along individual mother \ufb01laments in lamellipodia corresponds to multiples of the actin helix repeat of 36 nm ( Vin - zenz et al . , 2012 ) . The reason for this inter - branch spacing is not clear but might be because more branch junctions cannot be initiated due to structural or steric restrictions or it might be due to the interplay between \ufb01lament growth and Arp2 / 3 binding dynamics ( i . e . , the mother \ufb01lament grows faster than new Arp2 / 3 complexes can bind ) . The median branched actin \ufb01lament length in our data is 59 nm . Based on the work by Vinzenz et al . ( 2012 ) , we would not expect that more than one branch junction would form along such short \ufb01laments . The number of branched actin \ufb01laments and the number of branched actin \ufb01lament clusters were greater during the Figure 6 . Hip1R - like linkers observed at the neck of CME invaginations increase internalization ef\ufb01ciency ( A ) Tomographic slices of the regions indicated in the segmentation model of the tomogram shown in Figures 2A and 2B . Slices highlight putative Hip1R dimers ( cyanboxes ) thatareassociatedwiththecoatedmembraneoftheCMEinvagination ( greenarrowheads ) andtheirpositionsarehighlightedbycyanspheresinthe segmentation model correspond . Red arrowheads in slice 1 point at an actin \ufb01lament associated with a putative Hip1R dimer . ( B ) Superimposition of the surface model of the density map in ( Figure S6B ) with previously published atomic models of two actin - binding domains ( ABD ) ( PDB : 2JSW , Gingras et al . [ 2008 ] ) and two dimerization domains ( PDB : 2QDQ , Gingras et al . [ 2008 ] ) of the homologous protein talin . Model of actin \ufb01lament ( PDB : 6DJN , Chouand Pollard [ 2019 ] ) was added asa reference . Mathematical model readoutshowing\ufb01lament distribution between CME invagination base and neck in the presence or absence of Hip1R molecules at the neck . ( C ) Result of simulations showing the effect of Hip1R neck localization on the number of plus ends at the base ( orange and green curves ) and neck regions ( cyan and purple curves ) of CME invaginations . Mean and SD are shown . ( D ) Simulated internalization ef\ufb01ciency with and without Hip1R molecules at the neck . Mean and SD are shown . Scale bars , 50 nm in ( A ) and 5 nm in ( B ) . See also Figure S6 . ll Article 1140 Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 internalization phase compared with early stage , consistent with ongoing assembly ( Figures 2B and S2B ) . In addition , the branched actin \ufb01lament length distribution indicates that these \ufb01laments could have polymerized over the course of a CME event and are thus capable of providing assembly force ( Figures 2B , 2C , S2B , and S2C ; Akamatsu et al . , 2020 ) . Variation in branched actin \ufb01lament organization and in the number of clus - ters and \ufb01laments between individual CME sites likely re\ufb02ects the stochastic nature of \ufb01lament assembly as well as local adap - tive responses to variable conditions . High variability in actin network organization and density from one CME site to the next might result from such factors as variance in mother \ufb01la - ment number and orientation , Hip1R localization , active Arp2 / 3 complex , N - WASP position , and differences in plasma mem - brane tension ( this study ; Akamatsu et al . , 2020 ; Clarke and Royle , 2018 ; Engqvist - Goldstein et al . , 2001 ; Kaplan et al . , 2021 ; Sochacki et al . , 2017 ) . Work in the CME \ufb01eld has mainly focused on assembly of branched actin networks and did not take the presence of un - branched \ufb01laments at CME sites into account when investigating actin function . Also , unbranched actin \ufb01laments were not identi - \ufb01ed by the previous EM work , likely due to the inability to trace every individual \ufb01lament along its entire length by platinum replica EM ( Collins et al . , 2011 ) . Given the high number of un - branched actin \ufb01laments observed in close proximity to CME sites in our tomograms , two important questions arise : where do they come from and what are their functions ? The presence of unbranched \ufb01laments at early stages of CME suggests that they might represent pre - existing cortical actin \ufb01laments ( early - stage site in Figure 2 , CME invaginations in Figure S2 ) or \ufb01la - ments originating from other nearby actin structures like the bundle in the late - stage tomogram in Figure 2 . These dense cortical actin arrangements might represent a physical barrier that needs to be cleared by \ufb01lament severing , disassembly , or repositioning for CME progression . Shorter \ufb01laments might be ones that are diffusing through the cytoplasm ( Chen and Pollard , 2013 ; Aroush et al . , 2017 ) . Importantly , some of the unbranched \ufb01laments might be actively producing assembly forces at CME sites as well as acting as mother \ufb01laments for new branch forma - tion . This possibility is supported by the presence of the Arp2 / 3 nucleation promoting factor SPIN90 at CME sites . SPIN90 pro - motes nucleation of unbranched actin \ufb01laments and plays a role in epidermal growth factor receptor endocytosis ( Kim et al . , 2006 ; Luan et al . , 2018 ; Oh et al . , 2013 ) . In addition , un - branched actin \ufb01laments might dissociate from pre - existing actin structures that were involved in other processes and have undergone dynamic remodeling , representing an additional potential source of mother \ufb01laments required for Arp2 / 3 - medi - ated , SPIN90 - independent , branched actin \ufb01lament nucleation ( Pollard , 2007 ) . The abundance of potential sources of mother \ufb01l - aments and pathways for actin \ufb01lament assembly may allow for a safety net that allows actin assembly and adaptation to ensure robust CME under variable conditions within a cell . We acknowl - edge that not all of these unbranched \ufb01laments necessarily have a CME - speci\ufb01c function . Because our cryo - ET approach allowed individual actin \ufb01la - ments to be fully traced and oriented in the volume surrounding CME sites , we were able to use simulations to assess the force - producing capabilities of these networks . We found in our cryo - ET data that on average , during the invagination formation and neck constriction stages , 11 \u00b1 8 branched and 36 \u00b1 7 un - branched \ufb01lament plus ends were pointing toward the base of the CME sites ( relative angle < 90 (cid:3) ) , and up to two additional branched actin \ufb01lament plus ends were pointing toward the neck region ( Figure 5 ) . In the simulations , we found that 10 \u00b1 4 \ufb01lament plus ends assembled at the base of CME invaginations , which was suf\ufb01cient for successful internalization ( Figures 3C , 6C , and 6D ; Akamatsu et al . , 2020 ) . In previous simulations , the number of plus ends varied between 2 at low and 22 at high membrane tension conditions ( Akamatsu et al . , 2020 ) . The force requirement to support the transition from a U - shaped to an omega - shaped invagination with a constricted neck through actin polymerization was predicted to be < 1 pN when the force is applied in parallel to the plasma membrane and 15 pN when applied orthogonally ( Hassinger et al . , 2017 ) . Polymerization of individual actin \ufb01laments can provide between 1 and 9 pN of force ( Dmitrieff and Nedelec , 2016 ) . Despite the variability in actin network organization observed in the tomograms , our sim - ulations predicted that the internalization ef\ufb01ciency was robust under different conditions . This conclusion suggests that actin \ufb01lament assembly can harness the local variability and stochas - ticity at the \ufb01lament level to generate ef\ufb01cient , robust force - generating machineries at the network level for CME . The actin - binding linker Hip1R might provide a spatial constraint for actin assembly to ensure robust internalization . Here , we \ufb01nd that Hip1R is not only at the tip surface of the CME invagination but also at the neck region . In simulations , Hip1R neck localiza - tion directs \ufb01laments toward the neck constriction and improves internalization ef\ufb01ciency . Actin \ufb01lament growth toward the neck also supports a possible role for actin polymerization during vesicle scission . Our modeling approach currently does not allow us to test whether an effective neck constriction force can be produced by the observed actin arrangements . Previous work showed that the total force requirement for constriction can be less than 1 pN when the force is applied in a collar - like fashion . In principle , this force could be provided by a single polymerizing actin \ufb01lament ( Dmitrieff and Nedelec , 2016 ) . How - ever , additional experiments and modeling developments will be required to test under which cellular conditions an effective constriction force can be generated . The principles identi\ufb01ed here are expected to apply to other actin - driven processes where cellular membranes are being pushed , pulled , or squeezed ( Figure 7 ) . The \ufb01nding that branched actin \ufb01lament networks at individual CME sites are organized in multiple discrete clusters is similar to force - produc - ing branched actin assemblies in lamellipodia , which push the plasma membrane outward during cell migration ( Vinzenz et al . , 2012 ) . However , the presence of actin \ufb01lament anchoring points at CME sites allows conversion of pushing force into pull - ing and squeezing forces . Internalization ef\ufb01ciency and \ufb01lament orientation strongly depend on the distribution of these anchor points ( this study ; Akamatsu et al . , 2020 ) . The same mechanism might facilitate budding and \ufb01ssion at intracellular membranes , for example , during vesicle budding from the trans - Golgi , where Hip1R anchoring points are important , or endosomes where the actin - and lipid - binding protein moesin could mediate anchorage ( Carreno et al . , 2004 ; Fehon et al . , 2010 ; Muriel et al . , 2016 ) . Actin \ufb01laments in \ufb01lopodia are also anchored to the plasma ll Article Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 1141 membrane , which might be important for \ufb01lopodia formation and maintenance ( Medalia et al . , 2007 ) . However , instead of being pulled inward , the plasma membrane is pushed outward . Be - sides of the position of anchoring points , we identi\ufb01ed the posi - tion of actin \ufb01lament assembly factors as a second constraint that de\ufb01nes the geometry of CME - actin networks , which is also likely to be the case for \ufb01lopodia extension ( Akamatsu et al . , 2020 ) . The nucleation promoting factor N - WASP arranges in a ring around CME sites ( Almeida - Souza et al . , 2018 ; Mund et al . , 2018 ) . In contrast , actin assembly factors accumulate at the tip of \ufb01lopodia ( Rottner et al . , 2017 ) . Interestingly , previous structural work also suggests that , similar to our results , about 10 \ufb01laments are involved in \ufb01lopodia extension ( Medalia et al . , 2007 ) . Moreover , analysis of baculovirus - induced actin comet tails by electron tomography of membrane extracted cells suggests that the intracellular pathogen is pushed through the cytoplasm by the simultaneous assembly of 2 \u2013 6 branched actin \ufb01laments ( Mueller et al . , 2014 ) . We found that endocytic vesicles are pushed through the cytoplasm by a comparable number of branched actin \ufb01laments ( Figures S5J and S5K ) . In conclusion , we provided a comprehensive description of the complex native actin architecture during mammalian CME in unprecedented detail . We have precisely quanti\ufb01ed the \ufb01la - ment number and their orientation at CME events allowing for un - ambiguous predictions on their force production capabilities , which has not been achieved for other actin - driven processes in unperturbed cells . Our experimental and modeling data both highlight the remarkable \ufb02exibility and robustness of productive actin network organization . We conclude that assembly of actin networks in human cells produces suf\ufb01cient force to account for robust endocytic membrane internalization and neck constric - tion . By combining structural analysis and mathematical modeling , we gained previously inaccessible mechanistic in - sights into CME - actin regulation and function , which has impli - cations for many other actin - driven processes . Limitations of the study Manual segmentation of actin \ufb01laments and identi\ufb01cation of actin \ufb01lament branch junctions , as we did in this study , are well - established methods for negative staining electron tomo - graphy and for cryo - ET data ( Mueller et al . , 2014 , 2017 ; Vinzenz et al . , 2012 ) . In addition to manual approaches , automatic actin \ufb01lament segmentation and branch junction detection ap - proaches have also been developed ( F \u20ac a\u00dfler et al . , 2020 ; Jasnin and Crevenna , 2016 ; Jasnin et al . , 2019 ; Rigort et al . , 2012 ) . Automatic approaches can speed up the data analysis pro - cedure but are not necessarily more precise compared to manual approaches executed by expert users ( Rigort et al . , 2012 ) . Additionally , these approaches still require some level of user interference and , likemanual approaches , are not completely bias free . Although we are convinced that we did not miss a signi\ufb01cant number of authentic branch junctions , we cannot completely exclude the existence of additional junc - tions that might have been picked up using automatic ap - proaches . Special care needs to be taken , either manually or computationally , to ensure that identi\ufb01ed branches are indeed bona \ufb01de branch junctions . For a cell - biology - based project like ours , false - positive branch junctions would result in misleading conclusions . Two promising , freely available actin convolutional neural - network - based segmentation tools were published after we \ufb01nalized our analysis ( Dimchev et al . , 2021 ; Martins et al . , Figure 7 . Harnessing actin polymerization force for the pushing , pulling , and squeezing of cellular membranes Actin polymerization can generate a pushing force to extend the plasma membrane during the formation of \ufb01lopodia or lamellipodia and to push vesicles and intracellular pathogens through the cytoplasm . Proteins with both actin - and membrane - binding abilities ( e . g . , Hip1R ) can anchor actin \ufb01laments to the mem - brane , converting pushing force into pulling and squeezing forces based on their spatial localization . In addition to the distribution of anchoring points , actin network morphology and polymerization force direction are de\ufb01ned by a second geometrical constraint , the position of actin assembly factors . Note that the precise localization of assembly factors and actin - membrane linkers is not clear for most of the displayed processes . ll Article 1142 Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 2021 ) . One of these tools also allows actin \ufb01lament polarity to be determined , possibly with greater precision than was possible using our approach ( Martins et al . , 2021 ) . Thus , this software tool might have allowed \ufb01lament polarity determination for some of the unbranched \ufb01laments in our study , for which a clear polarity assignment was not possible ( see STAR Methods ) , though a head - to - head comparison of the two approaches would be required to de\ufb01nitively test this possibility . Given that we already detected an excess of \ufb01lament plus ends at CME sites over what is required for CME progression , this tool in all likelihood would not have changed our overall conclusions , though it might have yielded a more precise quanti\ufb01cation . Given the prospects for new convolutional neural - network - based tech - nologies , we are eager to implement such approaches in our future studies . STAR + METHODS Detailed methods are provided in the online version of this paper and include the following : d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell lines B Cell culture B Cell line authentication d METHOD DETAILS B Cell culture B Cryo - sample preparation B Cryo - \ufb02uorescence light microscopy B Cryo - electron tomography data acquisition B Tomogram reconstruction , segmentation model gen - eration and subtomogram averaging B Size measurement of clathrin - coated features B Filament length analysis B Branch angle measurements B Filament polarity and orientation analysis B Mathematical modeling using Cytosim d QUANTIFICATION AND STATISTICAL ANALYSIS B Image analysis and quanti\ufb01cation B Distribution analysis SUPPLEMENTAL INFORMATION Supplemental information can be found online at https : / / doi . org / 10 . 1016 / j . devcel . 2022 . 04 . 012 . ACKNOWLEDGMENTS We would like to thank Elizabeth Montabana , Daniel Toso , and Jonathan Re - mis for technical assistance with cryo - ET method development and data collection ; Paul Tobias for computation support ; and Sun Hae Hong for CLTA - GFP cell line generation and validation . Cryo - ET sample preparation was optimized at the LBNL cryo - EM resource , and image data were collected at the Bay Area Cryo - EM facility at the University of California Berkeley . Daniel Fletcherprovidedvaluableremarks onthemanuscript . Funding : thisworkwas supported through National Institutes of Health MIRA grant R35GM118149 to D . G . D . , Human Frontier Science Program long term fellowship LT000234 / 2018 - L to D . S . , Arnold and Mabel Beckman Foundation fellowship and Na - tional Institutes of Health grant 1 K99 GM132551 - 01 to M . A . , and National In - stitutes of Health grant R01 - GM132106 to P . R . . Method development was partially supported through Departments of Energy Early Career Award DE - AC02 - O5CH11231 to K . M . D . The funders had no role in study design , data collectionandinterpretation , orthedecisiontosubmittheworkforpublication . AUTHOR CONTRIBUTIONS Conceptualization , D . S . , M . A . , P . R . , and D . G . D . ; methodology , D . S . and M . A . ; investigation , D . S . , M . A . , A . M . , K . V . , and J . M . H . ; visualization , D . S . ; funding acquisition , D . S . , M . A . , K . M . D . , P . R . , and D . G . D . ; project administration , D . S . and D . G . D . ; software , D . S . , R . V . , M . A . , and J . S . ; supervision , D . G . D . and P . R . ; writing\u2014original draft , D . S . ; writing\u2014review & editing , D . S . , M . A . , P . R . , and D . G . D . DECLARATION OF INTERESTS The authors declare no competing interests . 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Actin branching in the initiation and maintenance of lamellipodia . J . Cell Sci . 125 , 2775 \u2013 2785 . Wan , W . , and Briggs , J . A . G . ( 2016 ) . Chapter thirteen\u2014cryo - electron tomogra - phy and subtomogram averaging . In Methods Enzymol , R . A . Crowther , ed . ( Academic Press ) , pp . 329 \u2013 367 . Yarar , D . , Waterman - Storer , C . M . , and Schmid , S . L . ( 2005 ) . A dynamic actin cytoskeleton functions at multiple stages of clathrin - mediated endocytosis . Mol . Biol . Cell 16 , 964 \u2013 975 . ll Article Developmental Cell 57 , 1132 \u2013 1145 , May 9 , 2022 1145 STAR + METHODS KEY RESOURCES TABLE RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be ful\ufb01lled by the lead contact , David G Drubin . REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals , peptides , and recombinant proteins Alexa Fluor 647 ChromPure human transferrin Jackson Immuno Research Inc . 009 - 600 - 050 ; RRID : AB _ 2337114 Deposited data Tomogram This paper EMDB - 26483 EM density map clathrin hub This paper EMDB - 26484 Experimental models : Cell lines SK - MEL - 2 ( sex : male ) ( endogenously expressing Dynamin2 tagged with eGFP clathrin light chain A ( CLTA ) tagged with TagRFP ) Drubin lab N / A SK - MEL - 2 ( sex : male ) ( endogenously expressing clathrin light chain A ( CLTA ) tagged with eGFP ) Drubin lab N / A Software and algorithms SerialEM https : / / bio3d . colorado . edu / SerialEM / download . html ( Mastronarde , 2005 ) N / A IMOD 4 . 9 . 12 https : / / bio3d . colorado . edu / imod / ( Kremer et al . , 1996 ) N / A PEET 1 . 13 . 0 https : / / bio3d . colorado . edu / PEET / , ( Heumann et al . , 2011 ; Nicastro et al . , 2006 ) N / A Leica Application Suite 3 . 7 Leica N / A actinPolarity This paper ( https : / / github . com / JohSchoeneberg / actinPolarity ) https : / / doi . org / 10 . 5281 / zenodo . 6410857 Actincme This paper ( https : / / github . com / kvegesna / polarity - analysis / tree / paper _ submission ) https : / / doi . org / 10 . 5281 / zenodo . 6413181 Other Leica EM Cryo CLEM ( Leica DM6 FS ) Leica N / A 50x Leica EM Cryo CLEM ceramic tipped objective Leica N / A GFP ET \ufb01lter ( Excitation : 470 / 40 , Dichroic : 495 , Emission 525 / 50 ) Leica 11504164 Y5 ( Excitation : 620 / 60 , Dichroic : 660 , Emission 700 / 75 ) Leica 11504171 Leica DFC9000 GT Leica 11547006 Titan Krios G2 FEI ( now Thermo Fisher ) N / A K2 direct electron detector Gatan N / A K3 direct electron detector Gatan N / A BioQuantum energy \ufb01lter Gatan N / A Vitrobot Mark IV ( FEI , now Thermo Fisher ) N / A Holey carbon grids ( R2 / 1 , 200 mesh , gold ) Quantifoil Q35430 ll Article e1 Developmental Cell 57 , 1132 \u2013 1145 . e1 \u2013 e5 , May 9 , 2022 Materials availability Mammalian cell lines that were used in this study are available from the lead contact upon request . Data and code availability d One representative tomogram and the EM density map of the clathrin hub have been deposited in the EMDB and are publicly available as of the date of publication . Accession numbers are listed in the key resources table . d Additional data reported in this paper will be shared by the lead contact upon reasonable request . d All original code has been deposited at GitHub and is publicly available as of the date of publication . URLs are listed in the key resources table . EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell lines All cell lines used in this study are derivates of commercially available SK - MEL - 2 cells ( https : / / www . atcc . org / products / htb - 68 ) . These are human melanoma cells . Constructions of recombinant cell lines and culture conditions are described in detail below . Cell culture Double tagged SK - MEL - 2 cells endogenously expressing clathrin light chain CLTA - TagRFP and dynamin2 - eGFP , and single tagged SK - MEL - 2 cells endogenously expressing clathrin light chain CLTA - eGFP , were cultured in DMEM / F12 ( Gibco (cid:2) ) supplemented with 10 % fetal bovine serum ( premium grade , VWR Life Science Seradigm ) and 1 % Penicillin / Streptomycin ( Gibco (cid:2) ) . Cell line authentication Genome - edited cells were authenticated by short tandem repeat pro\ufb01ling . METHOD DETAILS Cell culture The double - tagged CLTA - TagRFP and dynamin2 - eGFP cell line was generated using zinc - \ufb01nger nuclease - mediated genome editing and was published previously ( Doyon et al . , 2011 ; Grassart et al . , 2014 ) . The same reagents were used for the single - tagged cell line , except that the TagRFP in the donor plasmid used to construct the double tagged cell line was replaced with eGFP ( Doyon et al . , 2011 ; Grassart et al . , 2014 ) . SK - MEL - 2 cell lines were used because of their robust endocytic dynamics , good adherence and spreading behavior on EM grids . Cryo - sample preparation Holey carbon grids ( Quantifoil R2 / 1 , 200 mesh , gold ) were washed in acetone while agitating on a rocker for 15 to 30 min to remove potential residual plastic backing , washed in H 2 O and dried . Grids were then glow discharged using a Pelco SC - 6 sputter coater , sterilized in 70 % ethanol in H 2 O . Grids were placed in a 6 - well plate containing cell culture medium ( one grid per well ) under sterile conditions in a biosafety cabinet . Grids were incubated in cell culture medium at 37 (cid:3) C and 5 % CO 2 in a cell culture incubator over - night . Cell culture medium was replaced the next day . SK - MEL - 2 cells from a 75 \u2013 90 % con\ufb02uent 10 cm plastic cell culture dish were transferred to wells using a 1 : 2 \u2013 1 : 3 dilution . Cells were further incubated overnight at 37 (cid:3) C and 5 % CO 2 in a cell culture incubator . Before vitri\ufb01cation , grids were removed from the 6 - well plate , washed by continuously dripping a total of 10 m l of 10 nm BSA Gold Tracer ( Electron Microscopy Sciences ) solution and removing the drops with \ufb01lter paper . Next , 3 m l 10 nm BSA Gold Tracer were pipetted onto the sample side . Using a Vitrobot Mark IV ( FEI , now Thermo Fisher ) , samples were blotted either from both sides or from the backside of the grid by replacing the \ufb01lter paper on the sample side with a same - sized Te\ufb02on sheet , and vitri\ufb01ed by plunging into liquid ethane . See ( Serwas and Davies , 2021 ) for a step - by - step protocol . For cryo - correlative light and electron microscopy im - aging , SK - MEL - 2 cells expressing CLTA - eGFP were serum starved in medium without fetal bovine serum for 10 \u2013 30 min . Grids were then incubated in a 3 m l drop of medium containing 25 m g / ml Alexa Fluor 647 ChromPure human transferrin ( Jackson Immuno Research Inc . ) for 2 \u2013 6 min prior to initiating the washing steps in BSA Gold Tracer solution . Chamber conditions for the Vitrobot Mark IV were set to 37 (cid:3) C and 90 % humidity . To ensure that suitable grids were produced for each imaging session , a range of plot forces between 0 and 10 , blotting times of 2 . 5 to 4 s , and a 1 s drain time , were used . Grids were \ufb01xed into AutoGrid carrier ( Thermo Fisher ) and stored in liquid nitrogen until they were imaged . Cryo - \ufb02uorescence light microscopy Cryo - samples prepared from single - tagged CLTA - GFP expressing SK - MEL - 2 cells and incubated with Alexa Fluor 647 ChromPure human transferrin ( Jackson Immuno Research Inc . ) were imaged on a Leica EM Cryo CLEM system ( Leica ) . The system consists of a Leica DM6 FS wide\ufb01eld microscope that is equipped with a motorized Leica EM Cryo stage and a short working distance ( < 0 . 28 mm ) 50x Leica EM Cryo CLEM ceramic tipped objective ( numerical aperture = 0 . 90 ) . These speci\ufb01cations allow sample imaging at liquid nitrogen temperatures . A halogen lamp powered by a CTR6 halogen supply unit was used as light source . We used GFP ll Article Developmental Cell 57 , 1132 \u2013 1145 . e1 \u2013 e5 , May 9 , 2022 e2 ET ( Excitation : 470 / 40 , Dichroic : 495 , Emission 525 / 50 ) , Y5 ( Excitation : 620 / 60 , Dichroic : 660 , Emission 700 / 75 ) \ufb01lter cubes for imaging . For cryo - correlative light and electron microscopy , a grid overview map was recorded using transmitted light and GFP channels . The map was used to identify suf\ufb01ciently spread cells in regions with good ice quality . The CLTA - GFP signal appeared diffuse in regions of thick or crystalline ice . Z - stacks ( total Z = (cid:1) 6 m m in 0 . 6 m m steps ) of cells with clear CLTA - GFP foci were recorded using the transmitted light , GFP ( CLTA ) and Y5 ( transferrin ) channels . The same imaging conditions were used for cells on individual grids . Imaging conditions were varied between grids to obtain suf\ufb01cient signal to allow correlation in later steps . Note that some cells or CME sites did not show a transferrin signal most likely either because not all CME sites were loaded with transferrin cargo or due to inconsistent labeling . Images for panel generation in Adobe Illustrator were prepared using FIJI and Adobe Photoshop . No nonlinear gamma correction was applied during image processing . Cryo - electron tomography data acquisition Samples were imaged on a Titan Krios transmission electron microscope ( FEI ) operated equipped with a X - FEG electron source , a BioQuantum energy \ufb01lter ( Gatan ) and a K2 or K3 direct electron detecting device ( Gatan ) ( see Table S1 for details on the used de - tector ) and operated at 300 kV . Samples were visually inspected for ice quality using the FEI \ufb02u cam . Overview grid maps were ac - quired at (cid:1) 0 . 2 m m pixel size . If samples were imaged by cryo - \ufb02uorescence light microscopy before , these grid maps were used to identify cells of interest from the cryo - \ufb02uorescence light microscopy overview maps . For random data collection , cells with thin cell regions were located . For both types of data collection , polygon maps of the regions of interest with an overlap of 20 - 25 % between individual images were recorded . The polygon maps were used to pick tilt series acquisition points either at random or based on \ufb02uorescent signal from cryo - \ufb02uorescence light microscopy . The hole pattern of the carbon \ufb01lm was used as a guide . Acquisition points were chosen with adequate distance between individual points to prevent electron dose exposure damage prior to data collection . Only cellular regions without obvious sample damage ( e . g . , through blotting ) were used for data collection . SerialEM ( Mastronarde , 2005 ) in low - dose mode was used for automatic tilt - series recording using a bidirectional tilt scheme starting at + 20 (cid:3) and with a typical tilt range from + 60 (cid:3) to - 60 (cid:3) and base increment of 2 (cid:3) . Pixel sizes between 2 . 97 and 3 . 72 A\u02da were used . Target defocus was varied between - 2 and - 8 m m and the targeted total electron dose was 100 e - / A\u02da 2 ( see Table S1 for acquisition parameter details of individual tomograms presented in here ) . Data was collected in super resolution mode on the K2 detector or in 0 . 5 binning mode on the K3 detector . Frame time was 0 . 2 \u2013 0 . 25 s . Note that at the beginning of the project , the Leica cryo - CLEM microscope was not available to us and we therefore used the double - tagged \ufb02uorescent SK - MEL - 2 cell line to verify that CME occurs in cells growing on holey carbon grids by \ufb02uorescence microscopy . Tomogram reconstruction , segmentation model generation and subtomogram averaging Tilt series alignment and tomogram reconstruction were done using the freely available IMOD software package ( Kremer et al . , 1996 ) . Tilt series were aligned using 10 nm gold particles as \ufb01ducials and tomograms were reconstructed using the backprojection algorithm combined with the SIRT - like \ufb01lter . Tomograms were then \ufb01ltered using the Nonlinear Anisotropic Diffusion \ufb01lter and binned by a fac - tor of 2 using the binvol function to further increase contrast . Tomograms from a total of 7 independent datasets were analyzed ( see Table S1 for details ) . Subtomogram averaging was performed using PEET . The tomograms used are indicated in Table S1 ( Heumann et al . , 2011 ; Ni - castro et al . , 2006 ) . Subtomograms were picked manually and initial motive lists with starting orientations for alignment were gener - ated using the spikeInit or the slicer2MOTL functions . A single subtomogram was used as the starting reference . Subtomograms were iteratively aligned and averaged and an updated coarse aligned average was used after each iteration . For the subtomogram average of the clathrin hub , 3 - fold rotational symmetry was applied . Fourier shell correlation was performed by using the calcFSC function and plotted using the plotFSC function . ChimeraX ( https : / / www . rbvi . ucsf . edu / chimerax / ) was used for subtomogram average visualization and docking of PDB structures . The clathrin hub isosurface model that was used in the segmentation models was generated in IMOD . Segmentation model generation in IMOD was done by manually tracing actin \ufb01laments and membrane shapes and plotting the clathrin hub isosurface model to the original subtomogram positions that were re\ufb01ned during the subtomogram averaging procedure . The \ufb01ltered and binned tomograms were used for this procedure . Filament segmentation and branch junction identi\ufb01cation were car - ried out by two individuals ( DS and AM ) to reduce bias . Branch junctions were modeled as a scattered point object ( spherical object ) that was placed in the center of the junction density . Branch junctions were identi\ufb01ed manually in a two - step process : First , candidate branch junctions were marked during the initial manual actin \ufb01lament segmentation . Then , we searched our segmentation models for actin \ufb01lament pairs where one \ufb01lament end was close to another \ufb01lament and resembled branch junction geometry . Tomograms were re - inspected in these positions and we re - evaluated whether these \ufb01lament pairs were connected through branch junctions . Tomograms and corresponding segmentation models were cropped to a volume that was centered on the CME site or CCV and had a size of about 500 nm x 500 nm in x and y dimension while the zdimension was kept at its original value . Actin \ufb01laments were only considered for further analysis when they were contained in the cropped volume and thus in close proximity to the clathrin - coated feature . For \ufb01lament length analysis ( see below ) , the original length of the uncropped \ufb01lament was used if only a part of that \ufb01lament was contained in the cropped volume . Images for panel preparation in Adobe Illustrator were generated in IMOD and further processed in Adobe Photoshop . No nonlinear gamma correction was applied during image processing . ll Article e3 Developmental Cell 57 , 1132 \u2013 1145 . e1 \u2013 e5 , May 9 , 2022 Size measurement of clathrin - coated features CCV and CCP membrane and coat diameters were measured in the slicer view mode in IMOD . Rotation angles were adjusted to bring the CCV / CCP into full view as a symmetric object . A total of 3 measurements per CCV / CCP were performed on the central tomo - graphic slice and averaged to compensate for imprecision . Measurements for CCPs with a constricted neck were performed at the widest point of the invagination . Further analysis and graph generation were done in Prism8 . Filament length analysis Filament length was extracted from segmentation models of the \ufb01ltered and binned tomograms using the imodinfo function in IMOD . Further analysis and graph generation were done in Prism8 . Branch angle measurements Subtomograms of the branch junctions were extracted using the junction model points and the boxstartend function in IMOD . Sub - tomograms were displayed in the slicer view mode in IMOD . Rotational angles were adjusted to bring both \ufb01laments at the junction into view as shown in Figures 3A and S3A . Tiff images of these views were saved and opened in FIJI . Branch angle measurement was performed using the Angle tool . To minimize the effect of \ufb01lament \ufb02exibility , measurements were performed close to the junction and minimal parts of the \ufb01laments were included . To assess the effect of the missing wedge , which is most prominent in the z direction , on the branch angle measurements , we plotted the measured branch angle against the x and y rotations that had to be applied to bring the junction into view ( mother and daughter \ufb01lament visible ) . The equal distribution of branch angles across the rotation angles shows that our branch angle measurements were not strongly affected by the missing wedge at this level of precision . Filament polarity and orientation analysis Branched actin \ufb01lament polarity was analyzed based on branch junction geometry as indicated in Figure 3A . For analysis of un - branched \ufb01lament polarity , we adapted a previously published cross - correlation based method ( Narita et al . , 2012 ) . For the cross - correlation analysis , we \ufb01rst generated an arti\ufb01cial 3D actin \ufb01lament from a previously published structure ( PDB : 6DJM ; ( Chou and Pollard , 2019 ) ) using ChimeraX and IMOD . We then used the relion _ image _ handler function in Relion to rescale and low - pass \ufb01lter the arti\ufb01cial \ufb01lament to the pixel size of our binned tomography data ( 5 . 943 A\u02da ) and a resolution of 30 A\u02da . Next , a tiff image stack of 2D projection images of the arti\ufb01cial actin \ufb01lament was generated using the xyzproj function in IMOD . Each of the projection images was taken after rotating the \ufb01lament by 5 (cid:3) around the \ufb01lament axis ( F ) relative to the previous image . The complete image stack contained a total of 36 images covering a 180 (cid:3) rotation of the arti\ufb01cial actin \ufb01lament . The image stack was then cropped to 69 pixels x 23 pixels , corresponding to the length of a 13 - subunit actin \ufb01lament . This size was picked because of the structural or - ganization of actin \ufb01laments . The symmetry of an actin \ufb01lament can be described as a single strand left - handed helix with about 13 monomers repeating every six turns ( Dominguez and Holmes , 2011 ) . Accordingly , a 13 - subunit reference projection series contains suf\ufb01cient information about the helical organization of actin \ufb01laments to serve as a reference structure . We then generated a second reference image stack with opposing polarity by rotating the original stack by 180 (cid:3) ( Figure S4A ) . Hereafter we refer to the reference with the plus pointing up as ref + angle - i and the one with the minus end pointing up as ref - angle - i . Filaments for polarity analysis were extracted from the tomograms using the corresponding segmentation models and the imodmop function in IMOD . 2D projection im - ages of these \ufb01laments were generated in FIJI . Images were oriented as shown as in Figure S4B and cropped so the resulting image was 23 pixels wide ( = width of the reference images ) . Note was taken on the rotation that was applied to the images , and only straight parts of the \ufb01laments were included . From these test \ufb01laments ( test ) , sub - images of the size of the reference images ( 69 pixels x 23 pixels ) were extracted with a frequency of 5 pixels ( test i ) , corresponding to about one actin subunit in the \ufb01lament ( Figure S4B ) . The cross - correlation coef\ufb01cient ( R ) was calculated for each of the test sub - images test i with each of images in both of the reference stacks ref + angle - i and ref - angle - i ( Figure S4C ) . Then , the correlation curves with ref + angle - i and ref - angle - i were subtracted from each other and a difference curve was generated ( Figure S4D ) . The mean average difference was calculated from these values . If the value was negative , the tested \ufb01lament was determined to have the same polarity as ref - angle - i , if the value was positive , the tested \ufb01lament was determined to have the same polarity as ref + angle - i . The cross - correlation calculation algorithm was prototyped on ImageJ with the Image CorellationJ plugin ( https : / / www . gcsca . net / IJ / ImageCorrelationJ . html ) . To increase throughput and ease of use , the software was automated and reimplemented into a custom python - based command line tool called \" actinPolarity\u2019\u2019 . The accuracy of this method depends on test \ufb01lament length . Therefore , only unbranched \ufb01laments of at least 80 nm length ( > two 13 - subunit repeats ) were included in the analysis . Branched \ufb01laments were included without length consideration because the \ufb01lament junction identi\ufb01es the polarity . To calculate and display the orientation of the \ufb01laments in the tomograms based on the determined polarity ( either by branch junc - tion geometry or cross - correlation analysis ) relative to a user - de\ufb01ned reference plane , we developed an analysis pipeline with Jupyter notebooks ( Python 3 . 7 ) called \u2018\u2018actincme\u2019\u2019 . In general , the reference plane was parallel to the plasma membrane for CME invagina - tions or tangential to the surface for CCVs . The reference plane was manually segmented using IMOD . We calculated a vector normal to the reference plane , such that 0 (cid:3) orientation was de\ufb01ned as the vector pointing from the \ufb01laments toward the reference plane , and 180 (cid:3) was identi\ufb01ed as pointing away from the plane . Filaments with model points crossing within 10 pixels ( 6 nm ) from the reference plane were excluded from the analysis . The directionality of the \ufb01laments ( from minus end to plus end ) was calculated as the arcco - sine of the dot product between the \ufb01lament vector and the vector normal to the reference plane . Filament positions and orientations were plotted using matplotlib ( 3 . 0 . 2 ) . Further analysis and graph generation were done in Prism8 . ll Article Developmental Cell 57 , 1132 \u2013 1145 . e1 \u2013 e5 , May 9 , 2022 e4 Mathematical modeling using Cytosim We used the agent - based model in Akamatsu et al . 2020 to run 3D stochastic simulations of the mammalian endocytic actin network ( Akamatsu et al . , 2020 ) . We used similar parameters and initial conditions with the following modi\ufb01cations : To test effects of the branch angle variability on force production and internalization ef\ufb01ciency , we changed the average branching angle of the Arp2 / 3 complex to be 70 (cid:3) rather than 77 (cid:3) , to closer match the measured branch angle in the tomograms ( Akamatsu et al . , 2020 ) . In both cases the branch \ufb02exibility was set to 0 . 076 pN m m / rad . In general , to average multiple stochastic simulations , we ran 24 simulations per condition . To add Hip1R molecules at the neck , we repurposed Matlab code from Akamatsu et al . ( 2020 ) in order to distribute Hip1R molecules uniformly around a cylinder of radius 30 nm to match the shape and diameter of a typical CME neck . Sixty Hip1R molecules were distributed along 45 nm of the surface of the neck . This conservative value for the number of Hip1R molecules at the neck corresponds to a lower molecular surface density relative to the bud , and results in more tractable simulation runs that proceed to completion . QUANTIFICATION AND STATISTICAL ANALYSIS Image analysis and quanti\ufb01cation A total of 12 CME events from 11 tomograms were analyzed ( Table S1 ) , which is the range of similar studies done previously ( Jasnin et al . , 2019 ; Mueller et al . , 2017 ) . Image analysis and quanti\ufb01cation was done as detailed in the Method details section . Statistical analysis was done in GraphPad Prism8 unless otherwise noted . Statistical details can be found in the Results section , \ufb01gure legends , STAR Methods , Tables S2 and S3 . Distribution analysis The Prism8 curve \ufb01tting function was used for distribution analysis . We tested for Gaussian , lognormal and exponential ( one phase decay ) distributions ( Table S2 ) . R 2 values were used to determine the best \ufb01t . For Gaussian distributed data , we used mean and stan - dard deviation values for description in the main manuscript text , and we used the median for non - Gaussian distributed data . ll Article e5 Developmental Cell 57 , 1132 \u2013 1145 . e1 \u2013 e5 , May 9 , 2022", + "pedersen2023endocytic": "REPORT Endocytic myosin - 1 is a force - insensitive , power - generating motor Ross T . A . Pedersen 1 * \ue840 , Aaron Snoberger 2 * \ue840 , Serapion Pyrpassopoulos 2 \ue840 , Daniel Safer 2 \ue840 , David G . Drubin 1 \ue840 , and E . Michael Ostap 2 \ue840 Myosins are required for clathrin - mediated endocytosis , but their precise molecular roles in this process are not known . This is , in part , because the biophysical properties of the relevant motors have not been investigated . Myosins have diverse mechanochemical activities , ranging from powerful contractility against mechanical loads to force - sensitive anchoring . To better understand the essential molecular contribution of myosin to endocytosis , we studied the in vitro force - dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5 , a motor whose role in clathrin - mediated endocytosis has been meticulously studied in vivo . We report that Myo5 is a low - duty - ratio motor that is activated \u223c 10 - fold by phosphorylation and that its working stroke and actin - detachment kinetics are relatively force - insensitive . Strikingly , the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin - 1s found on endosomal membranes . We , therefore , propose that Myo5 generates power to augment actin assembly - based forces during endocytosis in cells . Introduction During clathrin - mediated endocytosis ( CME ) , the plasma mem - brane invaginates and undergoes scission to become a cytoplas - mic vesicle . Coat proteins like clathrin can deform membranes under low tension ( Dannhauser and Ungewickell , 2012 ; Busch et al . , 2015 ; Cail et al . , 2022 ) , but when bending is resisted by membrane tension ( Hassinger et al . , 2017 ) , the actin cytoskeleton drives membrane invagination ( Boulant et al . , 2011 ; Kaplan et al . , 2022 ) . In yeasts , including Saccharomyces cerevisiae and Schizo - saccharomyces pombe , turgor pressure opposes plasma mem - brane invagination , so actin is required at every CME site ( Aghamohammadzadeh and Ayscough , 2009 ; Basu et al . , 2014 ) . The actin cytoskeleton can produce pushing and pulling force , both of which are required for CME in S . cerevisiae ( Sun et al . , 2006 ) . When actin filament ends grow against a surface , they push the surface forward ( Mogilner and Oster , 1996 , 2003 ) . During CME , actin filaments , bound by coat proteins , grow against the plasma membrane to drive invagination ( Picco et al . , 2015 ; Kaksonen et al . , 2005 , 2003 ; Skruzny et al . , 2012 , Fig . 1 ) . Modeling of the homologous CME machinery in mammalian cells demonstrated that such actin networks generate sufficient power for CME ( Akamatsu et al . , 2020 ) , but whether actin as - sembly alone can overcome turgor pressure in yeast cells is debated ( Nickaeen et al . , 2019 ; Carlsson , 2018 ) . Additional power may be provided by myosins , which gen - erate tension on actin filaments . The myosins critical for CME \u2014 Myo3 and Myo5 in budding yeast and Myo1e in vertebrates \u2014 are type I myosins ( Geli and Riezman , 1996 ; Cheng et al . , 2012 ; Krendel et al . , 2007 ) . Some type I myosins are suited to generate power \u2014 i . e . , they carry out mechanical work over time by consuming ATP to execute a power stroke . Other type I myosins are suited to serve as force - sensitive anchors in that mechanical load locks them in a low energy \u2013 requiring , tension - maintaining state ( Greenberg and Ostap , 2013 ) . The possible roles of type I myosins in CME depend on whether endocytic myosins are power generators or force - sensitive anchors . If endocytic type I myosins are acutely force sensitive , they might organize the actin filaments of the endocytic actin net - work , while if they are less force sensitive , they could power plasma membrane invagination ( Evangelista et al . , 2000 ; Fig . 1 ) . Myosin - 1 motors form a ring at the base of CME sites , where the invaginated membrane meets the plasma membrane ( Mund et al . , 2018 ; Fig . 1 ) . Yeast type I myosins serve at least one or - ganizational function as a membrane anchor for the actin as - sembly machinery , a function associated with the non - motor tail of the molecules ( Lewellyn et al . , 2015 ) , but motor activity is required in addition to membrane anchorage ( Pedersen and . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Department of Molecular and Cell Biology , University of California , Berkeley , Berkeley , CA , USA ; 2 Pennsylvania Muscle Institute , Perelman School of Medicine , University of Pennsylvania , Philadelphia , PA , USA . Correspondence to David G . Drubin : drubin @ berkeley . edu ; E . Michael Ostap : ostap @ mail . med . upenn . edu * R . T . A . Pedersen and A . Snoberger contribution equally to this paper . R . T . A . Pedersen \u2019 s current affiliation is Department of Embryology , Carnegie Institution for Science , Baltimore , MD , USA . \u00a9 2023 Pedersen et al . This article is available under a Creative Commons License ( Attribution4 . 0 International , as describedat https : / / creativecommons . org / licenses / by / 4 . 0 / ) . Rockefeller University Press https : / / doi . org / 10 . 1083 / jcb . 202303095 1 of 12 J . Cell Biol . 2023 Vol . 222 No . 10 e202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 Drubin , 2019 ) . If endocytic myosin - 1s are force - sensitive an - chors , they may serve a further organizational role by holding growing filaments in an optimal orientation for force generation ( Fig . 1 , left ) . If the myosins are power - generating motors , they may pull actin filament ends away from the plasma membrane , deepening the plasma membrane invagination and creating space for monomer addition and filament elongation ( Fig . 1 , right ) , a model supported by the observation that the actin as - sembly rate at CME sites depends on type I myosin motors in a dose - dependent manner ( Manenschijn et al . , 2019 ) . To distinguish between these possibilities , we measured the force sensitivity of the endocytic myosin Myo5 ( not to be confused with the vertebrate type V myosin ) . Myo5 is insensitive to resis - tive force compared to related myosins . We , therefore , propose that Myo5 actively powers CME . Because actin and myosin col - laborate in a variety of membrane remodeling processes , we ex - pect that these results will be instructive beyond CME . Results and discussion Heavy chain phosphorylation activates Myo5 ATPase activity To determine Myo5 force sensitivity , we first needed to measure its unloaded kinetics . We purified a Myo5 construct containing the motor and lever domains from S . cerevisiae ( Fig . 2 A ) . Because phosphorylation of Myo5 at the TEDS site is required for most CME events and is thought to regulate Myo5 \u2019 s motor activity ( Grosshans et al . , 2006 ; Sun et al . , 2006 ; Bement and Mooseker , 1995 ) , we purified a phosphory - lated version and an unphosphorylated version of the protein ( see Materials and methods ) . The p21 - activated kinase was used to phosphorylate the myosin at the TEDS site ( S357 ) , as determined by control experiments with an S357A mutant ( Fig . S1 ) . The phosphorylation state of preparations was judged to be uniform when ATP - induced actoMyo5 dissociation transients were well fit by single exponential functions ( see below ) . The yeast light chain for Myo5 , calmodulin ( Cmd1 , Geli et al . , 1998 ) , was purified from E . coli and included in excess in all experiments ( Fig . 2 A ) . We measured the steady - state actin - activated ATPase activ - ities of phosphorylated and unphosphorylated Myo5 using the NADH - coupled assay ( De La Cruz and Ostap , 2009 ) in the presence of 0 \u2013 80 \u00b5M phalloidin - stabilized actin filaments . Un - phosphorylated Myo5 ATPase activity was largely insensitive to actin filaments : the ATPase rate at 0 \u00b5M actin was 0 . 14 s \u2013 1 , while the maximum ATPase rate measured was 0 . 39 s \u2013 1 at 40 \u00b5M actin . ( Fig . 2 B ) . Phosphorylation activated Myo5 ATPase activity by \u223c 10 - fold ( Fig . 2 B ) . The actin concentration dependence of the phosphorylated Myo5 ATPase rate ( k obs ) was well fit by : k obs (cid:1) v o V max [ Actin ] K ATPase + [ Actin ] . ( 1 ) From the fit , the actin concentration at half - maximum of the ATPase rate ( K ATPase ) was determined to be 5 . 1 \u00b1 0 . 88 \u00b5M , and the maximum ATPase rate ( V max ) was found to be 3 . 3 \u00b1 0 . 15 s \u2013 1 ( Fig . 2 B ; Table 1 ) . ATP binding and ADP release are non - rate limiting for Myo5 ATPase activity Resistive force impacts the rate of myosin detachment from actin and two biochemical transitions , ADP release and subsequent ATP binding , determine the detachment rate . Therefore , we used stopped - flow kinetics to measure ADP release from ( Fig . 2 C , k + 5 9 ) and ATP binding to ( Fig . 2 C , K 1 9 and k + 2 9 ) actoMyo5 . We found that yeast Myo5 does not quench the fluorescence of actin labeled at cys - 374 with pyrene iodoacetamide , which is the probe most used to measure myosin binding ( De La Cruz and Ostap , 2009 ) . Thus , we measured actoMyo5 detachment by monitoring light scattering , which decreases as myosin unbinds actin filaments . To determine the rate constant for ATP binding , we mixed nucleotide - free actoMyo5 ( 100 nM ) with varying concentrations of ATP and monitored 90\u00b0 light scattering . Time courses fol - lowed single exponential functions ( Fig . 2 D ) . For phosphorylated Myo5 , the observed rates determined from the fits increased linearly with ATP concentration ( Fig . 2 E ) . At concentrations of > 1 mM ATP , the actomyosin complex dissociated within the re - sponse time of the instrument , precluding measurement . For unphosphorylated Myo5 , the observed rates fit a rectangular hyperbola with increasing ATP concentration ( Fig . 2 E ) . Scheme 1 The mechanism was modeled as in Scheme 1 ( De La Cruz and Ostap , 2009 ) , where K 1 9 is a rapid equilibrium binding step , k 2 9 is Figure 1 . Models for the functions of actin assembly and myosin ac - tivity during membrane deformation for clathrin - mediated endocytosis . Cartoon diagram illustrating the organization of actin filaments and Myo5 molecules at endocytic sites . Actin filaments are bound by coat proteins at the tip of the growing membrane invagination and oriented with their growing ends toward the plasma membrane , powering membrane invagi - nation . The type I myosin Myo5 could either anchor the actin network in a favorable orientation ( left ) or provide an assisting force ( right ) . Pedersen et al . Journal of Cell Biology 2 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 Figure 2 . In - solution , population biochemical characterization of Myo5 . ( A ) Coomassie - stained SDS - polyacrylamide gels showing example preparations of the purified Myo5 motor / lever construct and calmodulin ( Cmd1 , light chain ) used in all experiments . ( B ) The actin concentration dependence of the steady - state ATPase activity of 100 nM unphosphorylated ( gray circles ) and phosphorylated Myo5 ( black circles ) . Each data point represents the average of 6 \u2013 7 time courses , which were 100 s each . The orange line is the best fit of the phosphorylated Myo5 data to a rectangular hyperbola . ( C ) Schematic pathway for the Myo5 ATPase cycle . Blue motors are in tightly bound conformations and green motors are weakly bound / unbound . ( D ) Example of light scattering transients reportingon ATP - induced dissociation of phosphorylated ( left , k obs = 17 s \u2212 1 ) and unphosphorylated ( right , k obs = 64 . 1 s \u2212 1 ) actoMyo5 , obtained by mixing 100 nM actoMyo5 ( AM ) with94and 72 \u00b5M ATP , respectively , as shown intheinset schematic . Theblacklineisthefit of a singleexponential function to thedata . ( E ) ATP Pedersen et al . Journal of Cell Biology 3 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 a rate - limiting isomerization to the AM . ATP state , and k diss is the rapid actin dissociation step . The apparent second - order rate constant for ATP binding to phosphorylated actoMyo5 was de - termined by a linear fit to the data ( K 1 9 k 2 9 = 0 . 39 \u00b1 0 . 017 \u00b5m \u2013 1 s \u2013 1 ) . The unphosphorylated actoMyo5 data were fit by k obs (cid:1) K 1 \u2019 ATP [ ] 1 + K 1 \u2019 ATP [ ] (cid:1) (cid:3) k 2 \u2019 ( 2 ) and the maximum rate of isomerization ( k 2 9 = 290 \u00b1 24 s \u2013 1 ) and ATP affinity ( K 1 9 = 0 . 006 \u00b1 0 . 0016 \u00b5M \u2013 1 ) were determined . The apparent second - order rate constant for ATP binding ( K 1 9 k 2 9 ) was determined from a linear fit of the observed rates below 100 \u00b5M ATP to be 1 . 1 \u00b1 0 . 28 \u00b5M \u2013 1 s \u2013 1 ( Table 1 ) . The rate constant for ADP dissociation ( k + 5 9 ) was measured by preincubating 100 \u00b5M ADP with 200 nM actoMyo5 and then rapidly mixing with 2 . 5 mM ATP , as shown in Scheme 2 . Scheme 2 When myosin active sites are saturated with ADP , the rate of ATP - induced dissociation of actomyosin is limited by ADP \u2019 s slow dissociation . Light scattering transients were fitted by single exponential functions , yielding rates for ADP release for phosphorylated actoMyo5 ( k + 5 9 = 74 \u00b1 2 . 0 s \u2013 1 ) and for un - phosphorylated actoMyo5 ( k + 5 9 = 107 \u00b1 5 . 9 s \u2013 1 ; Fig . 2 F and Table 1 ) . The signal - to - noise ratio of the fast light scattering transients is low , resulting in large uncertainties on these fits . However , these rates are substantially faster than the steady - state ATPase values but slower than the maximum rate of ATP - induced actomyosin dissociation . ADP release for actoMyo5 ADP is much faster than ADP release for vertebrate Myo1b and Myo1c ( Greenberg et al . , 2012 ; Lewis et al . , 2006 ) . It is more similar to the vertebrate en - docytic myosin - 1 , Myo1e ( El Mezgueldi et al . , 2002 ) . Because ADP release is rate limiting for detachment of Myo5 and Myo1e from actin , fast ADP release by these molecules means that the unloaded actin - attachment lifetimes for endocytic type I myosins are < 15 ms . This property may make these motors particularly well - suited to function in dynamic actin networks like those at CME sites , where actin filaments elongate and \u201c treadmill \u201d into the cytoplasm ( Kaksonen et al . , 2003 , 2005 ) . Actin gliding is dependent on Myo5 phosphorylation state Our results suggest that both phosphorylated and unphosphorylated Myo5 have low duty ratios ( i . e . , the motor spends a small fraction of its ATPase cycle bound to actin ) . Since ADP release limits the rate of phosphorylated Myo5 detachment from actin at saturating ATP ( k + 5 9 = 74 \u00b1 2 . 0 s \u2013 1 ) and since we have measured the overall ATPase rate ( V max = 3 . 3 \u00b1 0 . 15 s \u2013 1 ) , we can estimate the duty ratio as follows : DutyRatio (cid:1) 1 k + 5 \u2019 (cid:4) (cid:5) 1 V max (cid:4) (cid:5) ( 3 ) The calculated duty ratio of phosphorylated Myo5 is 0 . 045 . Unphosphorylated Myo5 has a lower duty ratio ( < 0 . 004 ) . To assess the effect of phosphorylation on Myo5 motility , we performed in vitro motility assays at 1 mM ATP . Motors were attached site - specifically to coverslips coated with anti - His 6 antibody . Coverslips were incubated with a range of concentrations of phosphorylated and unphosphorylated Myo5 , creating a titration series of surface densities . At low Myo5 surface densities ( incubation with \u2264 30 nM phosphory - lated Myo5 , \u2264 150 nM unphosphorylated Myo5 ) , actin fila - ments failed to bind the coverslip ( Fig . 2 G ; and Videos 1 and 2 ) . At higher concentrations , phosphorylated Myo5 moved actin filaments at velocities ranging from 720 \u00b1 40 nm / s ( 100 nM phosphorylated Myo5 ) to 880 \u00b1 90 nm / s ( 40 nM ; Fig . 2 G and Video 1 ) . These gliding velocities are considerably higher than those reported by Sun et al . , ( 2006 ) , possibly reflecting differences in the phosphorylation state of the purified Myo5 protein ( see below ) or differences in other motility assay conditions , such as light chain availability . Higher ( greater concentration dependence of dissociation of 100 nM unphosphorylated ( gray circles ) and phosphorylated actoMyo5 ( black circles ) . Each data point represents 3 \u2013 6 time courses averaged and fit to a single exponential decay function . The orange line is a linear best fit of the phosphorylated Myo5 data . The purple line is the best fit of the unphosphorylated Myo5 data to a rectangular hyperbola . ( F ) Example light scattering transients reporting ATP - induced dissociation of ADP - saturated phosphorylated ( left ) and unphosphorylated ( right ) actoMyo5 , obtained by preincubating 200 nM actoMyo5 ( AM ) with 100 \u00b5M ADP , then mixing rapidly with 2 . 5 mM ATP , as shown in the inset schematic . The black line is the fit of a single exponential function to the data . ( G ) Velocity of actin filament gliding , measured at varying surface densities of Phospho - Myo5 ( black circles , orange line ) and unphosphorylated Myo5 ( gray circles , purple line ) in in vitro motility assays . Myosin concentrations indicate the quantity of protein incubated in the flow chamber before washing . Each data point represents the average velocity of 30 \u2013 60 filaments , and the error bars are standard deviations . Source data are available for this figure : SourceData F2 . Table 1 . Rate and equilibrium constants of the Myo5 ATPase cycle Phosphorylated Myo5 Unphosphorylated Myo5 Steady - state actin - activated ATPase V max ( s \u2212 1 ) 3 . 3 ( \u00b10 . 15 ) ND K ATPase ( \u00b5M ) 5 . 1 ( \u00b10 . 88 ) ND ATP binding K 1 9 ( \u00b5M \u2212 1 ) ND 0 . 006 ( \u00b10 . 0016 ) k 2 9 ( s \u2212 1 ) \u2265 335 290 ( \u00b124 ) K 1 9 k 2 9 ( \u00b5M \u2212 1 s \u2212 1 ) a 0 . 39 ( \u00b10 . 017 ) b 1 . 1 ( \u00b10 . 28 ) c ADP release k + 5 9 ( s \u2212 1 ) 74 \u00b1 2 . 0 107 ( \u00b15 . 9 ) Summary ofrateand equilibriumconstants measuredforMyo5inthisstudy . Errors are standard errors of the fits . a Determined from a linear fit of the unbinding rates . b Linear fit of all data for phosphorylated Myo5 in Fig . 2 E . c Linear fit of observed rates below 100 \u00b5M ATP for unphosphorylated Myo5 in Fig . 2 E . ND : Not determined . Pedersen et al . Journal of Cell Biology 4 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 than fivefold ) surface densities of unphosphorylated Myo5 were required to achieve smooth motility , but this motility occurred at a substantially slower speed , \u223c 120 nm / s ( Fig . 2 G and Video 2 ) . While it is possible that residual phosphorylated Myo5 in the unphosphorylated preparation contributed to this motility , Sun et al . , 2006 similarly reported that Myo5 harboring TEDS site mutations moved actin filaments much more slowly . The slower actin gliding speed for unphosphorylated myosin was unexpected given the similar rates of ADP release between phosphorylated and unphosphorylated Myo5 ( Table 1 ) . It is possible that our kinetics experiments have not determined the rate - limiting step for detachment , but it is more likely that the motility of the unphosphorylated myosin is limited by the slow attachment rate of the motor ( Stewart et al . , 2021 ) , as suggested by the slow actin - activated ATPase rate . The activation of Myo5 motility by phosphorylation could explain why fast , cargo - induced endocytosis , which involves rapid and dynamic actin turnover , requires phosphorylated Myo5 , while slower constitutive endo - cytosis does not ( Grosshans et al . , 2006 ) . Myo5 \u2019 s working stroke comprises two substeps that are consistent with unloaded kinetic measurements The kinetics of actin attachment and mechanics of single myosin molecules were measured by optical trapping ( Woody et al . , 2018 ; Snoberger et al . , 2021 ) . We used the three - bead optical trapping geometry in which a biotinylated actin filament is held between two laser - trapped polystyrene beads coated with neu - travidin , creating a bead - actin - bead dumbbell ( Fig . 3 A ) . Dumb - bells were lowered onto pedestal beads that were sparsely coated with phosphorylated Myo5 - His 9 bound to a surface - adsorbed anti - His 6 tag antibody . The positions of trapped beads were detected , and single actomyosin binding events were identified by a de - crease in the covariance of the bead positions ( Fig . 3 , B \u2013 D ) . Traces acquired at 1 , 10 , and 1 , 000 \u00b5M ATP reveal clear displacements and drops in covariance during actomyosin binding events . Event durations decreased with increasing ATP concentrations ( Fig . 3 , B \u2013 D , blue lines ) . The myosin - 1 working stroke occurs in two discrete substeps , with the first substep occurring with phosphate release and the second with ADP release ( Jontes et al . , 1995 ; Veigel et al . , 1999 , Fig . 3 E ) . The substeps can be characterized in optical trapping assays by ensemble averaging single interactions ( Veigel et al . , 1999 ; Chen et al . , 2012 ; Laakso et al . , 2008 ) , where the detected events are aligned at their beginnings and forward - averaged in time ( Fig . 3 , F \u2013 H , left ) , or aligned at their ends and reverse - averaged in time ( Fig . 3 , F \u2013 H , right ) . Ensemble averages of Myo5 interactions showed a two - step working stroke at the three ATP concentrations but the step size was most accurately resolved at 10 \u00b5M ATP ( see Materials and methods ) . In this condition , an initial substep of 4 . 8 nm was followed by a second substep of 0 . 2 nm ( Fig . 3 G ) . We deter - mined the lifetimes of the substeps by fitting the ensemble averages with single exponential functions . At 1 \u00b5M ATP ( Fig . 3 F , left trace ) , the measured rate ( > 30 s \u2212 1 ) of the time - forward average was limited by the covariance smoothing window , but at 10 and 1 , 000 \u00b5M ATP ( Fig . 3 , G and H , left traces ) , the rates were 49 \u00b1 1 . 6 and 50 \u00b1 0 . 2 s \u2212 1 , respectively ( Fig . 3 K ) , which are similar to the measured ADP release rate ( k + 5 9 , 74 \u00b1 2 . 0 s \u2212 1 , Table 1 ) supporting the model that the tran - sition from State 1 to State 2 accompanies ADP release . The kinetics of time - reversed averages reveal the lifetime of State 2 ( Fig . 3 , F \u2013 H , right traces ) . Fitting single exponential functions to these traces reveals rates of 0 . 59 \u00b1 0 . 003 and 7 . 34 \u00b1 0 . 1 s \u2212 1 at 1 and 10 \u00b5M ATP , respectively ( Fig . 3 K ) . At 1 , 000 \u00b5M ATP , the observed rate ( > 187 s \u2212 1 ) was limited by the covariance smoothing window ( 5 . 25 ms ; Fig . 3 K ) . The observed rates at 1 and 10 \u00b5M ATP are consistent with the second - order rate constant for ATP binding of 0 . 39 \u00b1 0 . 017 \u00b5M \u2212 1 s \u2212 1 measured by stopped - flow kinetics ( K 1 9 k 2 9 , Table 1 ) . We determined the detachment rates of actomyosin events by plotting the cumulative frequency of individual attachment durations and fitting a single exponential function to the data by maximum likelihood estimation ( MLE ; Fig . 3 I ) . Data from 1 and 10 \u03bc M ATP were well fit by single exponentials with rates of 0 . 88 and 6 . 87 s \u2212 1 , respectively ( Fig . 3 , I \u2013 K ) . These rates match well with the observed rate of ATP binding ( Table 1 ) , as well as the fits for the reverse ensemble averages , indicating that at sub - saturating ATP ( 1 and 10 \u00b5M ) , detachment is limited by ATP binding ( Fig . 3 , I \u2013 K ) . Data from 1 , 000 \u03bc M ATP were best described as the sum of two exponentials , with the major rate of 67 . 8 s \u2212 1 comprising 92 . 1 % of the total , and a minor rate of 11 . 6 s \u2212 1 comprising 7 . 9 % of the total ( Fig . 3 , I and K ) . The major rate is consistent with both the observed ADP release rate and the measured forward ensemble average rates , indicating that at saturating ATP , ADP release limits detachment of actomyosin interactions ( Fig . 3 , J \u2013 K ) . Myo5 is a relatively force - insensitive motor We measured how the actin detachment rate of Myo5 was af - fected by mechanical force opposing the power stroke using an isometric feedback system ( Takagi et al . , 2006 ) . The initial force applied to Myo5 in this system depends on where along the actin filament Myo5 stochastically binds , allowing measurement of attachment durations at a range of resistive forces ( Fig . 4 A ) . Plotting attachment durations as a function of force revealed a trend of longer attachment durations at higher resisting forces . At each force , attachment durations are exponentially distributed and , as expected , the data appear noisy when plotted ( Fig . 4 A ) . Con - verting these data to detachment rates by binning by force , aver - aging , and taking the inverse clearly reveals the trend ( Fig . 4 B ) . The force dependence of the Myo5 detachment rate was fit by the Bell Equation : k ( F ) (cid:1) k 0 e \u2212 Fd kB \u2219 T , ( 4 ) where k ( F ) is the detachment rate at force F , k 0 is the detachment rate in the absence of load , d is the distance parameter ( the distance to the force - dependent transition state and a measure of force sensitivity ) , k B is Boltzmann \u2019 s constant , and T is the temperature . Best fit parameters for k 0 and d were determined by MLE of the unaveraged data from Fig . 4 A , incorporating the instrument response time ( Woody et al . , 2016 ) . The esti - mated detachment rate in the absence of force is 67 . 6 s \u2212 1 , in agreement with the measured detachment rate under low load Pedersen et al . Journal of Cell Biology 5 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 Figure 3 . Single molecule , optical trap analysis of Myo5 step size and kinetics . ( A ) Cartoon schematic of the three - bead optical trapping setup . A bi - otinylated actin filament is tethered between two neutravidin - coated beads that are trapped in a dual - beam optical trap . This bead - actin - bead \u201c dumbbell \u201d is lowered onto pedestal beads that have been sparsely coated with His 6 antibody to attach Myo5 - motor / lever - Avi - Tev - His 9 . ( B \u2013 D ) Single Myo5 displacements of a single bead position and covariance traces , calculated using both beads , showing single molecule interactions acquired in the presence of 1 \u00b5M ( B ) 10 \u00b5M ( C ) , and 1 , 000 \u00b5M ATP ( D ) . Blue bars indicate attachment events as identified by covariance ( gray ) decreases . The threshold of event detection by the co - variance traces are indicated by dashed gray lines . ( E ) Schematic of displacement traces depicting the two - step nature of actomyosin displacements in the optical trap . ( F \u2013 H ) Binding events were synchronized at their beginnings ( left ) or ends ( right ) and averaged forward or backward in time , respectively . The Pedersen et al . Journal of Cell Biology 6 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 conditions at 1 , 000 \u00b5M ATP ( 67 . 8 s \u2212 1 , Fig . 3 K ) , and the value for d was 1 . 14 nm . To put Myo5 \u2019 s force sensitivity in context , we replotted the function describing the force - dependent actin detachment rate of Myo5 alongside the same curves for vertebrate Myo1b , Myo1c , and \u03b2 - cardiac myosin , which were determined by the same experimental approach ( Fig . 4 C , Laakso et al . , 2010 ; Greenberg et al . , 2012 ; Woody et al . , 2018 ) . The mechanochemistry of Myo5 ( d = 1 . 14 nm ) is most like that of \u03b2 - cardiac ( muscle ) myosin ( d = 1 . 3 nm ) , suggesting that it is well - suited for generating power . The difference between Myo5 and acutely force - sensitive Myo1b , a tension - sensitive anchor myosin ( d = 15 nm ) , is dramatic . From 0 to 2 pN of resistance , Myo1b at - tachment lifetimes slow from \u223c 600 ms to \u223c 45 s , resulting in negligible power generation ( Fig . 4 D ) . Over the same in - terval , Myo5 attachment lifetimes slow modestly from \u223c 15 to \u223c 25 ms , allowing it to generate considerable power ( Fig . 4 D ) . Thus , Myo5 is unlikely to act as a force - sensitive anchor in measured total displacement of Myo5 was 5 . 0 nm at 10 \u00b5M ATP , with the first substep contributing a 4 . 8 nm displacement ( arrow 1 in G ) and the second substep contributing a 0 . 2 - nm displacement ( arrow 2 in G ) . ( F \u2013 H ) Left : Forward - averaged ensembles synchronized at the beginnings of events . Right : Reverse - averaged ensembles synchronized at the ends of events . Black and gray lines are single exponential fits in the forward and reverse ensembles , respectively . ( I ) Cumulative distributions of attachment durations for Myo5 at 1 , 10 , and 1 , 000 \u00b5M ATP . Blue lines show the cumulative frequency of attachment durations at the indicated ATP concentrations , and the red , yellow , and green lines indicate fitted exponential distributions at 1 , 10 , and 1 , 000 \u00b5M ATP , respectively . 1 and 10 \u00b5M ATP were fit well to single exponentials , and the 1 , 000 \u00b5M ATP data were best described by the sum of two exponentials . ( J ) Summary of rates at 1 , 10 , and 1 , 000 \u00b5M ATP calculated from F \u2013 H . Blue boxes are the fitted exponential distributions from I , black diamonds are forward ensemble fits from F \u2013 H ( left ) , and gray diamonds are reverse ensemble fits from F \u2013 H ( right ) . At lower concentrations of ATP ( 1 and 10 \u00b5M ) , the rate of detachment is limited by ATP association , corresponding to the reverse ensemble fits , while at saturating ATP concentration ( 1 , 000 \u00b5M ) , the detachment rate is limited by the rate of ADP dissociation , corresponding to the forward ensemble fits . ( K ) Summary of rates determined via single - molecule optical trapping . Errors for detachment rates are 95 % confidence intervals . Errors for forward and reverse ensemble fits are standard errors of the fits . * Detachment rates at 1 , 000 \u00b5M ATP were best fit to the sum of two exponents . The major component of the fit ( 67 . 8 s \u2013 1 ) comprises 92 . 1 % of the total with the remaining 7 . 9 % having a rate of 11 . 6 s \u2013 1 . Figure 4 . Myo5 attachment lifetimes are substantially less force - dependent than other known type I myosins . An isometric optical force clamp was utilized to determine the force sensitivity of the detachment of Myo5 from actin . ( A ) Durations of individual actomyosin attachments as a function of force , plotted on a semi - log scale . ( B ) The solid black line shows the force dependence of the detachment rates determined by MLE fitting of unaveraged points in A . For illustration purposes , attachment durations from A were binned by force at every 10 points , averaged , and converted to rates . Best - fit parameters were determined by MLE fitting and 95 % confidence intervals were calculated via bootstrapping . The solid black line is calculated from best - fit parameters ( k = 67 . 6 s \u2013 1 , d = 1 . 14 nm ) , while the gray shaded region is the 95 % confidence interval ( k = 62 . 4 \u2013 72 . 9 s \u2013 1 , d = 1 . 03 \u2013 1 . 26 nm ) . All MLE fitting was performed on unaveraged data and was corrected for instrument deadtime . ( C ) The force dependent detachment rate of Myo5 ( from B ) plotted alongside the force de - pendent detachment rates for Myo1b , Myo1c , and \u03b2 - cardiac muscle myosin , Myh7 . ( D ) Power output for the same four myosins is calculated over a range of forces by multiplying the functions from C by the applied force F , and the step size and duty ratios of each myosin . Pedersen et al . Journal of Cell Biology 7 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 cells and is more likely to power movements against a resisting load . Proposed function of type I myosin in clathrin - mediated endocytosis Myo5 is one of the best - studied myosin - 1 proteins in vivo . Ex - tensive imaging has revealed that it is recruited to CME sites at the initiation of actin assembly , where it concentrates at the base of the site during membrane invagination ( Jonsdottir and Li , 2004 ; Idrissi et al . , 2008 ) . Although it was known that the mechanochemical activity of type - 1 myosins is required for CME ( Geli and Riezman , 1996 ; Goodson et al . , 1996 ; Sun et al . , 2006 ) , the mechanistic contribution of motor activity was unknown . When it was discovered that some type I myosins are acutely force - sensitive ( Laakso et al . , 2008 ) , it became apparent that these motors could have mechanochemical activities that range from force - dependent anchoring to power generation . It is clear that mutant Myo5 molecules with disrupted mechanochemistry block CME , but these results do not reveal the molecular role of Myo5 ( Lewellyn et al . , 2015 ; Idrissi et al . , 2012 ) . Perhaps , the most informative finding in cells has been the observation that varying the number of type I myosins at CME sites results in altered actin assembly rates ( Manenschijn et al . , 2019 ) . How - ever , because load influences growing branched actin networks in complex ways ( Bieling et al . , 2016 ) , even this finding did not clarify the molecular roles of endocytic myosin - 1s . Here , we have shown that Myo5 \u2019 s motor generates power rather than forming force - sensitive catch bonds . The overall ATPase rate of Myo5 is slow relative to other power - generating myosins , but its power stroke and detachment from actin are fast , and they slow only modestly under load ( Fig . 4 C ) . Myo5 \u2019 s relative force insensitivity means it generates power against resistance ( Fig . 4 D ) . Because Myo3 and Myo5 can each support CME in the absence of the other , we suspect that Myo3 is sim - ilarly force - insensitive . Given the homology between Myo5 and vertebrate Myo1e , together with the close agreement of their unloaded kinetics ( El Mezgueldi et al . , 2002 ) , we also predict that Myo1e generates biologically relevant power . Our finding that Myo5 \u2019 s kinetics are relatively force insen - sitive led us to interpret the previously described dose depen - dence of actin assembly on the number of myosin - 1s at endocytic sites to mean that this motor moves actin filaments at CME sites to power plasma membrane invagination and create space for new monomers to assemble ( Manenschijn et al . , 2019 ; Fig . 1 , right ) . On the order of 300 myosin molecules ( Myo3 and Myo5 combined ) are present at CME sites , mostly where the invagi - nating membrane meets the plasma membrane ( Mund et al . , 2018 ; Idrissi et al . , 2008 ; Sun et al . , 2019 ; Picco et al . , 2015 ) . Myo5 \u2019 s diffusion is likely to be impeded by the many proteins at the base of CME sites and it may move actin filaments at an angle to the membrane to which it is bound , both conditions that allow a related myosin to generate and sustain sub - piconewton forces ( Pyrpassopoulos et al . , 2016 ) . Actin networks grow at the plasma membrane at \u223c 50 \u2013 100 nm / s ( Kaksonen et al . , 2003 , 2005 ) , so Myo5 \u2019 s motility rate of 700 \u2013 900 nm / s ( Fig . 2 G ) , which we would expect resistance to slow only modestly , is fast enough to do work on the actin network as it assembles . We therefore expect that the myosins power membrane invagination and relieve the load to accelerate actin assembly during CME . Type I myosins are involved in a variety of membrane - reshaping events in cells , where they often interact with growing branched actin networks ( Sokac et al . , 2006 ; Almeida et al . , 2011 ; Joensuu et al . , 2014 ; Krendel et al . , 2007 ; Cheng et al . , 2012 ) , but the relative contributions of myosin motor activity and actin assembly have rarely been resolved . Here , we dem - onstrated that a type I myosin critical for CME , a process well - known to be driven by actin assembly , generates power . The implication of endocytic type I myosin as a force - insensitive motor suggests that actin assembly and myosin power genera - tion can be coordinated to do coherent work in membrane re - modeling processes . Materials and methods Reagents , proteins , and buffers ATP concentrations were determined spectrophotometrically after each experiment by absorbance at 259 nm , 2 259 = 15 , 400 M \u2212 1 cm \u2212 1 . For all ATP solutions , 1 M equivalent of MgCl 2 was included to make MgATP . Rabbit skeletal muscle actin was prepared and gel - filtered ( Spudich and Watt , 1971 ) . Actin con - centrations were determined spectrophotometrically by absor - bance at 290 nm , 2 290 = 26 , 600 M \u2212 1 cm \u2212 1 . All actin was stabilized with 1 M equivalent of phalloidin ( Sigma - Aldrich ) . Steady - state , transient , and single molecule experiments were performed at 20\u00b0C in KMg25 buffer ( 60 mM MOPS pH 7 , 25 mM KCl , 1 mM EGTA , 1 mM MgCl 2 , 1 mM DTT ) . Apyrase VII was obtained from Sigma - Aldrich . The purity and concentration of purified pro - teins were determined by comparing in - gel Coomassie blue staining to staining of known amounts of bovine serum albumin ( Pierce ) . Expression and purification of Cmd1 The S . cerevisiae calmodulin gene CMD1 was cloned from genomic DNA into a bacterial expression plasmid with a sequence en - coding His 6 - TEV situated at the 5 9 end to generate pDD2743 . pDD2743 was transformed into Rosetta E . coli , optimized for expression ( Novagen ) . A saturated overnight culture in LB ( 10 g / L Bacto tryptone , 5 g / L Bacto yeast extract , 10 g / L NaCl ) was used to inoculate a 1 - L culture in LB to OD 600 = 0 . 1 . Cells were grown to OD 600 = 0 . 6 \u2013 1 , induced with 0 . 5 mM IPTG for 5 h at 37\u00b0C , pelleted at 4 , 225 \u00d7 g for 20 min at 4\u00b0C in a Sorvall SLA - 3000 ( fixed angle ) rotor , washed with cold 20 mM HEPES pH 7 . 5 , and re - pelleted at 2 , 250 \u00d7 g for 10 min at 4\u00b0C in a Jouan CR3i ( swinging bucket ) centrifuge . Cell pellets were flash - frozen in 45 ml lysis buffer ( 20 mM HEPES pH 7 . 5 , 1 M KCl , 20 mM Im - idazole ) . Upon thawing , cells were lysed by sonication , 2 mg DNase I ( Roche ) and Triton X - 100 to 1 % were added , and the resulting lysate was incubated on ice for 30 min , then spun at 92 , 000\u00d7 g for 25 min in a Beckman Type 70 Ti rotor . The su - pernatant was loaded onto a 1 - ml HisTrap HP column ( GE healthcare ) pre - equilibrated with binding buffer ( 20 mM HEPES pH 7 . 5 , 500 mM KCl , 20 mM imidazole ) . The column was washed with 20 ml binding buffer , and Cmd1 was eluted using a 30 ml linear gradient from 0 \u2013 100 % elution buffer ( 20 mM Pedersen et al . Journal of Cell Biology 8 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 HEPES pH 7 . 5 , 500 mM KCl , 500 mM imidazole ) . Fractions containing Cmd1 were pooled , Cmd1 was cleaved from His 6 with TEV protease , and dialyzed overnight at 4\u00b0C into a low salt buffer ( 10 mM Tris pH 7 , 25 mM NaCl , 2 mM MgCl 2 , 5 mM DTT ) . Fol - lowing dialysis , purified , cleaved Cmd1 was bound to a MonoQ column and eluted using a 10 ml linear gradient from 0 \u2013 70 % high salt buffer ( 10 mM Tris pH 7 , 1 M NaCl , 2 mM MgCl 2 , 5 mM DTT ) . Fractions containing Cmd1 were pooled , dialyzed into KMg50 buffer ( 60 mM MOPS pH 7 , 50 mM KCl , 1 mM MgCl 2 , 1 mM EGTA , 1 mM DTT , 5 % glycerol ) , and stored at \u2212 80\u00b0C . Expression and purification of Myo5 Myo5 was coexpressed with the myosin chaperone She4 in S . cerevisiae . The MYO5 open reading frame ( ORF ) from S . cerevisiae was cloned from genomic DNA and truncated at Gly 763 , gener - ating a construct containing the motor domain and both Cmd1 - binding IQ motifs of the lever arm . The SHE4 ORF was cloned in its entirety from S . cerevisiae genomic DNA . Both ORFs were li - gated into a 2 - \u00b5m expression plasmid with a partially defective LEU2 gene ( leu2d ) to ensure a high copy number , creating plasmid pDD2744 ( parent vector described in Roy et al . , 2011 ) . The MYO5 ORF was situated with a sequence encoding AviTag - TEV - His 9 at the 3 9 end . Expression of the MYO5 and SHE4 ORFs was driven by a bidirectional Gal 1 / 10 promotor . pDD2744 was transformed into D1074 yeast ( Roy et al . , 2011 ) . Saturated overnight cultures in synthetic minimal medium ( 1 . 5 g / L Difacto yeast nitrogen base , 5 g / L ammonium sulfate , supplemented with 2 % glucose 20 \u00b5g / ml adenine , L - histadine , L - methionine , and 30 \u00b5g / ml L - lysine ) were used to inoculate 1 . 5 liters of cultures in the same media with raffinose substituted for glucose to OD 600 = 0 . 1 . After 18 h of growth at 30\u00b0C , cultures were induced with 2 % galactose , Bacto yeast extract was added to 10 g / L , and Bacto peptone to 20 g / L . After 8 h of expression , the cells were harvested at 4 , 225 \u00d7 g for 20 min at 4\u00b0C in a Sorvall SLA - 3000 rotor , washed with 25 ml cold Milli - Q water , re - pelleted at 2 , 250 \u00d7 g for 10 min at 4\u00b0C in a Jouan CR3i centri - fuge , resuspended in 0 . 2 vol of cold Milli - Q water , and drop frozen into liquid nitrogen . Lysis was achieved through cry - omilling ( 10 cycles of 3 min grinding with one min cooldown ) in the large vials of a 6870 freezer / mill ( SPEX Sample Prep ) . Cell powders were thawed in binding buffer ( 10 mM Tris pH 7 , 500 mM NaCl , 4 mM MgCl 2 , 2 mM ATP , 20 mM imidazole , 5 mM DTT ) supplemented with 1 mM PMSF , 1 \u00d7 cOmplete protease inhibitor cocktail without EDTA ( Roche ) , and 1 \u00b5M Cmd1 . For purification of phosphorylated Myo5 , 1 \u03bc g Pak1 ( Sigma - Aldrich , Brzeska et al . , 1997 ; Fig . S1 ) was included in the lysis buffer and 10 mM \u03b2 - glycerophosphate , 5 mM sodium py - rophosphate , and 50 mM sodium fluoride were included in all purification buffers . For the purification of unphosphorylated Myo5 , 4 , 000 units of lambda phosphatase ( NEB ) and 1 mM MnCl 2 were included in the lysis buffer . The lysate was then spun at 345 , 000 \u00d7 g for 10 min at 4\u00b0C in a Beckman TLA100 . 3 rotor , filtered through a 0 . 22 - \u00b5m filter , and loaded onto a 1 ml HisTrap HP column . The column was washed with wash buffer ( binding buffer with only 200 mM NaCl ) , and Myo5 was eluted using a 20 ml linear gradient from 0 - 100 % elution buffer ( wash buffer with 1 M imidazole ) . Fractions containing Myo5 were pooled and supplemented with Cmd1 to 1 \u00b5M . For unphosphorylated Myo5 purification , a further 20 , 000 units of lambda phosphatase were added along with MnCl 2 to 1 mM , and the fractions were incubated at 30\u00b0C for 30 min . Purified protein was dialyzed through a 3 . 5 KD MWCO membrane into 1 L storage buffer ( KMg50 with 50 % glycerol ) overnight at 4\u00b0C and again into 500 ml of the same buffer for 2 h at 4\u00b0C and then stored at \u2212 20\u00b0C . Kinetic measurements Steady - state actin - activated ATPase activity was measured us - ing the NADH enzyme - linked assay in an Applied Photophysics SX . 18 MV stopped - flow apparatus ( De La Cruz and Ostap , 2009 ) . In one reaction , the syringe contained the ATP mix ( 200 \u00b5M NADH , 20 U / ml lactic dehydrogenase , 100 U / ml pyruvate ki - nase , 500 \u00b5M phopho ( enol ) pyruvate , 2 mM MgCl 2 , 2 mM ATP in KMg25 ) and the other syringe contained the mixture of actin ( 0 \u2013 80 \u00b5M ) and myosin ( 100 nM ) in KMg25 . The concentrations above are after mixing . After mixing , the concentration of NADH loss due to ATP hydrolysis was monitored by absorbance at 340 nm ( 2 340 = 6 , 220 M \u2212 1 cm \u2212 1 ) , and the linear regions of the curve were fitted to a straight line to determine ATPase activity . ATP - induced dissociation of actoMyo5 was measured and analyzed as described ( De La Cruz and Ostap , 2009 ) . Briefly , one reaction syringe contained ATP ( 0 \u2013 2 . 7 mM ) in KMg25 and the other syringe contained 100 nM Myo5 and 100 nM phalloidin - stabilized actin . Reactants were rapidly mixed by the instru - ment , and light scattering at 90\u00b0 was acquired using a 450 - nm excitation light and a 400 - nm emission filter . Experimental transients were fit by single exponentials using the software provided with the stopped - flow apparatus . One to seven traces were averaged together to generate each data point . 0 . 04 U / ml apyrase was added to solutions of actoMyo5 before mixing to remove contaminating ADP and ATP . Unphosphorylated acto - Myo5 required prolonged treatment with apyrase to achieve sufficient signal , presumably because a larger fraction of the population was bound to ATP left over from purification and because the actin - activated ATPase rate of unphosphorylated Myo5 is slow . ADP release transients were acquired and ana - lyzed as above by preincubating 100 \u00b5M ADP with 200 nM actoMyo5 and then rapidly mixing with 2 . 5 mM ATP . The concentrations reported are after mixing . Motility assays Motility assays were carried out essentially as in Lin et al . ( 2005 ) . Double - sided Scotch tape and vacuum grease were used to create flow chambers from a clean glass coverslip ( 22 mm \u00d7 40 mm , # 1 . 5 ; Thermo Fisher Scientific ) and a glass coverslip coated with 20 \u03bc l nitrocellulose ( catalog number 11180 ; Ernest F . Fullam , Inc . ) . A mouse monoclonal antibody against His 6 ( Sigma - Aldrich ) , made with 0 . 2 mg / ml in motility buffer ( 10 mM MOPS pH 7 , 25 mM KCl , 1 mM EGTA , 1 mM MgCl 2 , 1 mM DTT ) , was first added to the flow chamber and incubated there for 5 min to coat the nitrocellulose - coated coverslip with the antibody . The flow chamber was then blocked for 2 min with 2 mg / ml casein . Blocking coverslips with bovine serum albumin ( BSA ) led to in - ferior gliding . Phosphorylated or unphosphorylated Myo5 in Pedersen et al . Journal of Cell Biology 9 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 motility buffer with 2 mg / ml casein , diluted to a range of con - centrations as indicated in Fig . 2 G , was added to the flow chamber and incubated for 2 min , then the chamber was washed once with motility buffer containing 1 mM ATP and 5 \u00b5M Cmd1 and three more times with the same buffer without ATP . Motility was initiated by loading the chambers with 5 nM rhodamine - phalloidin - labeled actin filaments in motility buffer with 1 \u00b5M ATP , 5 \u00b5M Cmd1 , 2 mg / ml casein , 0 . 4 mg / ml glucose oxidase , 0 . 08 mg / ml catalase , and 5 mg / ml glucose . Movies of actin mo - tility in the flow chambers were recorded at room temperature ( \u223c 20\u00b0C ) on a Leica DMI3000 B microscope outfitted with a 100\u00d7 , 1 . 4 NA plan apo objective and a Retiga R6 CCD camera ( Teledyne ) , controlled by Metamorph software . The rate of actin filament gliding was determined using the manual tracking plugin in Fiji . Optical trapping Flow chambers for optical trapping were constructed with double - sided tape and vacuum grease as previously described ( Snoberger et al . , 2021 ; Greenberg et al . , 2017 ) . Briefly , the coverslip was coated with a 0 . 1 % mixture of nitrocellulose and 2 . 47 \u03bc m diameter silica beads . Coverslips were dried for at least 30 min and were used within 24 h of preparation . To define the walls of the flow cell , two strips of double - sided tape were placed \u223c 5 mm apart , and a 1 - mm thick glass slide was placed on top and carefully sealed with vacuum grease after the addition of the final buffer . Trapping buffer ( KMg25 with 1 mM DTT freshly added ) was used for all trapping assays . A 100\u00d7 stock of glucose oxidase + catalase ( GOC ) was freshly prepared by centrifuging catalase ( > 30 , 000 U \u00b7 ml \u2212 1 ; Sigma - Aldrich ) at 15 , 000 \u00d7 g for 1 min and adding 2 \u03bc l of catalase supernatant to 20 \u03bc l of 19 . 1 U / \u03bc l glucose oxidase ( Sigma - Aldrich ) . 0 . 01 mg / ml anti - His 6 antibody ( Sigma - Aldrich ) was flowed in the chamber and incubated between 30 s and 3 min and then immediately blocked with 2 , 3 - min incubations of 1 \u2013 2 mg / ml BSA . Stocks of phosphorylated His 9 - tagged Myo5 were diluted to 1 nM in trapping buffer with 300 mM added KCl and incubated in the flow cell for 2 min . The number of myosins bound to the surface was limited by the surface concentration of anti - His 6 antibody and the incubation time of anti - His 6 antibody was adjusted daily between 30 s and 3 min such that one of the three to five pedestals tested showed clear myosin interactions with the actin dumbbell . Following incubation with Myo5 , a second blocking step with two 3 - min incubations of 1 \u2013 2 mg / ml BSA was performed . The final buffer added to the flow cell contained trapping buffer with the indicated amount of ATP , 1 \u03bc l of GOC added immediately prior to addition to the chamber , and 0 . 1 \u2013 0 . 25 nM rabbit skeletal muscle actin polymerized with 15 % biotinylated actin ( Cyto - skeleton ) stabilized by rhodamine - phalloidin ( Sigma - Aldrich ) at a 1 . 1 \u2013 1 . 2 molar ratio with G - actin concentration . Neutravidin - coated beads were prepared by incubating 0 . 4 ng of 0 . 8 \u03bc m di - ameter polystyrene beads ( Polysciences ) and coated with 5 mg / ml neutravidin ( Thermo Fisher Scientific ) . 3 \u03bc l of neutravidin - coated beads were added to one side of the chamber prior to sealing . All trapping data were acquired within 90 min of the addition of the final buffer to the chamber . Optical trapping experiments were performed at room tem - perature ( 20 \u00b1 1\u00b0C ) using a dual beam 1 , 064 - nm trapping laser as previously described ( Woody et al . , 2017 , 2018 ) . A single laser beam was split into two beams using polarizing beam splitters and steered into a 60\u00d7 water immersion objective ( Nikon ) . Laser light was projected through an oil immersion condenser and into quadrant photodiodes ( JQ - 50P ; Electro Optical Components , Inc . ) , each of which were conjugate to the back focal plane of the objective . Direct force detection from the quadrant photodiodes was achieved using a custom - built high - voltage reverse bias and an amplifier . Data acquisition , beam position control output , and isometric feedback calculations were controlled with custom - built virtual instruments ( Labview ; Matlab ) . Individual 0 . 8 - \u03bc m - diameter neutravidin - coated beads were caught in the two traps and held \u223c 5 \u03bc m apart . Trap stiffnesses were adjusted to 0 . 05 \u2013 0 . 1 pN / nm for each trap . A biotinylated actin filament visualized by rhodamine - phalloidin was bound to the two trapped beads , creating a bead - actin - bead dumbbell . The dumbbell was pretensioned ( 3 \u2013 5 pN ) by steering one beam using a piezo - controlled mirror conjugate to the back focal plane of the objective , and the surface of the pedestal beads was probed for myosins . Putative myosin interactions were detected via drops in the variance of the two beads , and the three - dimensional position of the dumbbell relative to the myosin was refined further by maximizing the rate and size of the observed power stroke deflections . Every 30 \u2013 60 s , the dumbbell was moved axially along the actin filament in \u223c 6 - nm steps between trace acquisition to ensure even accessibility of actin - attachment target zones . Stage drift was corrected via a feedback system using a nano - positioning stage and imaging the position of the pedestal bead with nm precision ( Woody et al . , 2017 ) . In ex - periments using 1 \u03bc M ATP , due to the longer actomyosin in - teractions , stage drift was still observed even with the stage feedback engaged , leading to a presumed underestimation of the displacement size . All data were acquired at a sampling rate of 250 kHz . Isometric optical clamping experiments were performed as previously described ( Woody et al . , 2018 ; Takagi et al . , 2006 ) using a digital feedback loop and a 1 - D electro - optical deflector ( EOD , LTA4 - Crystal ; Conoptics ) to steer the beam position using input from a high - voltage source ( Model 420 Amplifier ; Con - optics ) . Briefly , the position of one bead ( the \u201c transducer \u201d bead ) was maintained at a constant position by adjusting the position of the other bead ( referred to as the \u201c motor \u201d bead ) during ac - tomyosin interactions . The response time of the feedback loop during actomyosin interactions was \u223c 15 \u2013 30 ms . Optical trap data analysis Actomyosin interactions for non - isometric optical clamping experiments were detected using the single - molecule compu - tational tool SPASM ( Software for Precise Analysis of Single Molecules , Blackwell et al . , 2021 ) , which uses a calculation of the dumbbell bead covariances and a change - point algorithm . Data collected at 1 , 000 \u03bc M ATP were analyzed at 250 kHz , while data collected at 1 and 10 \u03bc M ATP were downsampled to 2 kHz by averaging every 125 points to enhance analysis speed . Events were detected by calculating the covariance of the two beads Pedersen et al . Journal of Cell Biology 10 of 12 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 using a smoothing window of 33 . 3 , 15 , and 5 . 25 ms and an av - eraging window 60 , 36 , and 12 ms at 1 , 10 , and 1 , 000 \u03bc M ATP , respectively . The instrument deadtime was calculated to be two times the covariance averaging window . For each 15 - s trace , the detected covariance was plotted and fit to double Gaussian dis - tributions , with the smaller mean Gaussian corresponding to the actomyosin \u201c bound \u201d portion and the larger mean Gaussian corresponding to the \u201c unbound \u201d portion of events . A putative event was defined as an event where the covariance starts above the unbound peak mean , drops below the bound peak mean , and remains below the unbound peak mean for at least the length of the instrument deadtime prior to returning back above the unbound peak mean . Event starts and ends were further refined using a changepoint algorithm as described ( Blackwell et al . , 2021 ) . Attachment durations and ensemble averages of single events were determined using built - in features in the SPASM software . Exponential fits for forward and reverse ensemble averages were performed in Origin 2019 graphing and analysis software ( OriginLab ) . Events detected in isometric optical clamping experiments were detected as described in ( Takagi et al . , 2006 ) using a zero crossing analysis via custom MATLAB scripts . Briefly , when a myosin is actively engaged with the dumbbell , force is applied to the transducer bead , a feedback loop is engaged , and opposing force is applied to the motor bead until the position of the trans - ducer bead is restored . Beginnings of events are defined at the point at which the feedback signal increases from the baseline in the motor bead and ends of events are defined when the feedback signal decreases back below the baseline in the motor bead . Online supplemental material Fig . S1 shows the results of a kinase assay demonstrating that Pak1 , used in purifications of phosphorylated Myo5 , specifically phosphorylates Myo5 serine - 357 . Video 1 shows motility assays with phosphorylated Myo5 . Video 2 shows motility assays with unphosphorylated Myo5 . Acknowledgments We thank M . Greenberg , E . Lewellyn , and A . Kunibe , each of whom played important roles at the inception of this study . We thank Y . E . Goldman for optical trap instrumentation . We thank M . Ferrin for his helpful comments on the manuscript . This work was funded by the National Institute of General Medical Sciences ( NIGMS ) grant R35 GM118149 to D . G . Drubin and grant 5R37GM057247 to E . M . Ostap . R . T . A . Pedersen is currently funded by NIGMS F32 GM142145 . Author contributions : R . T . Pedersen , A . Snoberger , S . Pyr - passopoulos , D . G . Drubin , and E . M . Ostap conceived of the ex - periments . R . T . Pedersen , A . Snoberger , S . Pyrpassopoulos , and D . Safer generated the reagents . R . T . Pedersen , A . Snoberger , S . Pyrpassopoulos , D . Safer , and E . M . Ostap performed the ex - periments and analyzed the data . R . T . Pedersen , A . Snoberger , D . G . Drubin , and E . M . Ostap wrote the manuscript . D . G . Drubin and E . M . Ostap secured funding . Disclosures : The authors declare no competing interests exist . Submitted : 23 March 2023 Revised : 17 May 2023 Accepted : 24 July 2023 References Aghamohammadzadeh , S . , and K . R . 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P21 - activated Kinase 1 ( Pak1 ) phosphorylates Myo5 on S357 . Crude preparations of wild type and S357A Myo5 motor / lever constructs were mixed with 250 \u00b5M ATP including 20 \u00b5Ci of ATP \u03b3 P32 in kinase assay buffer ( 5 mM MOPS pH 7 , 2 . 5 mM \u03b2 - glycerophosphate , 5 mM MgCl 2 , 400 \u00b5M EDTA , 1 mM EGTA , and 50\u00b5M DTT ) in eitherthe presenceor absence of Pak1 . Reactions were incubated at 25\u00b0C for 60min , thenquenchedby adding an equal volumeof 2\u00d7 Tris urea sample buffer ( 125 mM Tris pH 6 . 8 , 6 M urea , 2 % SDS , 0 . 1 % bromophenol blue , 10 % \u03b2 - mercaptoethanol ) and resolved on a 10 % polyacrylamide gel . The gel was stained with Coomassie , then dried onto Whatman paper and exposed to a storage phosphor screen ( Amersham ) . The Coomassie - stained gel was imaged on a standard photo scanner and the phosphor screen on a Typhoon gel imager ( Amersham ) . Note that there are differences in baseline labeling in the absence of added kinase between the two different protein preps , but the addition of Pak1 clearly results in radiolabeling of wild type but not mutant Myo5 . Pedersen et al . Journal of Cell Biology S1 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023 Video 1 . Motility assays with phosphorylated Myo5 . Rhodamine - phalloidin - labeled actin filaments gliding over coverslips coated with a concentration series of phosphorylated Myo5 protein in motility buffer with 1 mM ATP . Videos were collected at one frame per second and are played back at 16 frames per second . Video 2 . Motility assays with unphosphorylated Myo5 . Rhodamine - phalloidin - labeled actin filaments gliding over coverslips coated with a concentration series of unphosphorylated Myo5 protein in motility buffer with 1 mM ATP . Short movies of motility at 100 and 150 nMunphosphorylated Myo5 were collected because no motility was observed . Movies at all other concentrations were collected at one frame every 4 s and are played back at 16 frames per second . The playback rate of Video 2 is four times faster than the playback rate of Video 1 . Pedersen et al . Journal of Cell Biology S2 An endocytic myosin - 1 generates power https : / / doi . org / 10 . 1083 / jcb . 202303095 D o w n l oaded f r o m h t t p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 222 / 10 / e202303095 / 1916174 / j c b _ 202303095 . pd f b y gue s t on 18 A ugu s t 2023", + "kosmalska2015physical_supplement": "Supplementary figures Supplementary Figure 1 . Stretch system . a , Schematic of stretch system . A ring clamping a PDMS membrane ( shown in red ) is placed on a support containing a central loading circular post and an external ring , with an opening in between . Imaging objectives can then be placed either below ( inverted microscope ) or above ( upright microscope ) . Cells are then cultured on the membrane after coating with fibronectin . b , Once vacuum is applied through the opening , it deforms and stretches the membrane . In some cases , cells were seeded on polyacrylamide and soft silicone gels previously attached to the membrane ( see methods ) . c , Level of strain in the X and Y axis obtained after applying different vacuum suction pressures . Stretch was biaxial . Supplementary Figure 2 . Response of cells to 2 % strain . Response of a peYFP - mem transfected cell to the application of 2 % strain for three minutes . No visible effects were observed either upon stretch application or upon stretch release . Insets display magnifications of the areas in red , showing that membrane ruffles were not flattened upon stretch application , and reservoirs were not formed upon stretch release . Scale bar is 20 \u03bcm . Supplementary Figure 3 . Dynamic formation and resorption of membrane structures . a , Quantification of VLD fluorescence after re - application of iso - osmotic medium ( 1 : maximum fluorescence , 0 : background ) . n = 20 VLDs from 2 cells . Imaging was carried out using an inverted microscope ( 60x objective ) which allowed to visualize the initial VLD formation period . b , Quantification of reservoir fluorescence after stretch release ( 1 : initial fluorescence , 0 : background ) . n = 100 / 80 / 30 reservoirs from 10 / 3 / 3 cells . Imaging was carried out using an upright microscope ( 60x objective ) . Both decreasing temperature ( pink symbols ) and increasing stretch magnitude ( black symbols ) slowed the process of reservoir formation . Images to the right show corresponding examples of membrane structures at the times indicated in the graph . c , Quantification of VLD fluorescence after re - application of iso - osmotic medium ( 1 : initial fluorescence , 0 : background ) . n = 100 / 50 / 60 VLDs from 10 / 5 / 4 cells . Imaging was carried out using an upright microscope ( 60x objective ) . Both decreasing temperature ( pink symbols ) and increasing the magnitude of the hypo - osmotic shock ( black symbols ) slowed the process of VLD formation . Images to the right show corresponding examples of membrane structures at the times indicated in the graph . All insets have a size of 10x10 \u03bcm . Supplementary Figure 4 . Co - localization of actin and membrane structures before , during and after stretch and hypo - osmotic shocks . a , cells transfected with pEYFP - mem and Lifeact - Ruby before , during , and after application of 6 % stretch for 3 minutes . b , equivalent images obtained before , during , and after application of a 50 % hypo - osmotic shock for 3 minutes . Insets ( 10x10 \u03bcm ) show zoomed views of membrane and actin structures . Scale bars are 20\u03bcm . Supplementary Figure 5 . Poroelasticity of polyacrylamide gels . a , graph showing the height of polyacrylamide gels during and after application of 6 % stretch for three minutes . Values are shown normalized to the height before stretch application . As gels are stretched , they first decrease their height to maintain volume , but they progressively incorporate water and swell . Once stretch is released , the progressive reduction in thickness is indicative of water expulsion flow . b , As a control , gels submitted to a 50 % decrease in media osmolarity did not modify their height . ( n = 5 gels ) Supplementary Figure 6 . Reservoirs and VLDs are unrelated to caveolae . a , Time sequence of pEYFP - mem and Cav1 - mcherry transfected cells before , during , and after application of 6 % stretch for three minutes . Image to the right shows the lack of co - localization between pEYFP - mem ( green ) and Cav1 - mcherry ( red ) . b , pEYFP - mem transfected cell ( green in merged image ) fixed right after restoring iso - osmotic media and stained for caveolin 1 ( red in merged image ) . No co - localization was observed between caveolin and VLDs . c , Time sequence of pEYFP - mem and Cav1 - mcherry transfected cells before , during , and after application of 50 % hypo - osmotic media for three minutes . Image to the right shows the lack of co - localization between peYFP - mem ( green ) and Cav1 - mcherry ( red ) . d , pEYFP - mem transfected cell ( green in merged image ) fixed right after releasing 6 % stretch and stained for caveolin 1 ( red in merged image ) . No co - localization was observed between caveolin and reservoirs . e , Time sequence of pEYFP - mem transfected cells before , during , and after application of 50 % hypo - osmotic media during three minutes . Zoomed insets show the formation and evolution of VLDs . Caveolin 1 knock - out cells were reconstituted either with caveolin 1 - GFP or an empty control vector ( IRES - GFP ) . f , Quantification of VLD fluorescence after re - application of iso - osmotic media ( 1 : initial fluorescence , 0 : background ) . n = 50 / 25 VLDs from 5 / 3 cells . g - h , Corresponding quantification of mean VLD diameter ( g ) ( n = 100 / 60 VLDs from 5 / 3 cells ) and density ( h ) ( n = 30 / 20 regions from 5 / 3 cells ) . No significant differences were observed . i , Time sequence of peYFP - mem transfected cells before , during , and after application of constant 6 % stretch during three minutes . Zoomed insets show the formation and evolution of membrane reservoirs . Caveolin 1 knock - out cells were reconstituted either with caveolin 1 - GFP or an empty control vector ( IRES - GFP ) . j , Quantification of reservoir fluorescence after stretch release ( 1 : initial fluorescence , 0 : background ) ( n = 30 / 60 reservoirs from 3 / 4 cells ) . k , Corresponding quantification of mean reservoir diameter ( n = 250 / 100 reservoirs from 3 / 3 cells ) . No significant differences were observed . l , Corresponding quantification of mean reservoir density ( n = 40 / 20 regions from 3 / 3 cells ) . No significant differences were observed . Scale bars are 20\u03bcm . Supplementary Figure 7 . Actin and temperature dependence of reservoirs and VLDs . a , Time sequence of peYFP - mem transfected cells before , during , and after application of constant 6 % stretch during three minutes for control cells and cells treated with 0 . 5 \u03bcM cytochalasin D . Zoomed insets show the formation and evolution of membrane reservoirs . b , Quantification of reservoir fluorescence after stretch release ( 1 : initial fluorescence , 0 : background ) ( n = 100 / 100 reservoirs from 10 / 4 cells ) . c , Corresponding quantification of mean reservoir diameter ( n = 250 / 100 reservoirs from 8 / 4 cells ) . No significant differences were observed . d , Corresponding quantification of mean reservoir density ( n = 30 / 30 regions from 5 cells ) . e , Time sequence of peYFP - mem transfected cells before , during , and after application of 50 % hypo - osmotic media during three minutes for control cells and cells treated with 0 . 5 \u03bcM cytochalasin D . Zoomed insets show the formation and evolution of membrane VLDs . f , Quantification of VLD fluorescence after re - application of iso - osmotic media ( 1 : initial fluorescence , 0 : background ) . n = 100 / 100 VLDs from 10 / 5 cells . g , Corresponding quantification of mean VLD diameter ( n = 100 / 60 VLDs from 10 / 3 cells ) . h , Corresponding quantification of mean VLD density ( n = 50 / 30 regions from 8 / 3 cells ) . i , Time sequence of peYFP - mem transfected cells before , during , and after application of constant 6 % stretch during three minutes for control cells at 37\u00ba and cells at 26\u00ba . Zoomed insets show the formation and evolution of membrane reservoirs . j , Quantification of reservoir fluorescence after stretch release ( 1 : initial fluorescence , 0 : background ) n = 100 / 60 reservoirs from 10 / 3 cells . k , Corresponding quantification of mean reservoir diameter ( n = 250 / 140 reservoirs from 8 / 3 cells ) . No significant differences were observed . l , Corresponding quantification of mean reservoir density ( n = 30 / 30 zones from 5 / 3cells ) . No significant differences were observed . m , Time sequence of peYFP - mem transfected cells before , during , and after application of 50 % hypo - osmotic media during three minutes for control cells at 37\u00ba and cells at 26\u00ba . Zoomed insets show the formation and evolution of membrane VLDs . n , Quantification of VLD fluorescence after re - application of iso - osmotic media ( 1 : initial fluorescence , 0 : background ) . n = 100 / 24 VLDs from 10 / 3 cells . o , Corresponding quantification of mean VLD diameter ( n = 100 / 45 VLDs from 10 / 3 cells ) . No significant differences were observed . p , Corresponding quantification of mean VLD density ( n = 50 / 25 zones from 8 / 3 cells ) . No significant differences were observed . Scale bars indicate 20 \u03bcm . N . s . , non - significant , * , p < 0 . 05 , * * * , p < 0 . 001 . Scale bars are 20 \u03bcm . Supplementary Figure 8 . Membrane reservoirs and VLDs are observed across cell types and species . Images of Chinese Hamster Ovary ( CHO ) cells , A431 human squamous carcinoma cells , and human keratinocytes ( HaCaT ) transfected with pEYFP - mem before and after being submitted to biaxial stretch ( a ) or hypo - osmotic shock ( b ) . All cell types consistently showed the formation of reservoirs or VLDs , respectively . Zoomed insets ( 10x10 \u03bcm ) show a magnification of both membrane structures . Scale bar indicates 20 \u03bcm . Supplementary Figure 9 . Adhesion dependence of reservoirs and VLDs . a , Time sequence of pEYFP - mem transfected cells before , during , and after application of constant 6 % stretch during three minutes for control cells and cells treated with 10 \u00b5g mL - 1 \u03b15\u03b21 antibody . b , Quantification of reservoir fluorescence after stretch release ( 1 : initial fluorescence , 0 : background ) ( n = 100 / 150 reservoirs from 10 / 6 cells ) . c , Corresponding quantification of mean reservoir diameter ( n = 250 / 200 reservoirs from 8 / 6 cells ) . d , Corresponding quantification of mean reservoir density ( n = 30 / 60 regions from 5 / 5 cells ) . e , Time sequence of pEYFP - mem transfected cells before , during , and after application of 50 % hypo - osmotic media during three minutes for control cells and cells treated with 10 \u00b5g mL - 1 \u03b15\u03b21 antibody . f , Quantification of VLD fluorescence after re - application of iso - osmotic media ( 1 : initial fluorescence , 0 : background ) . n = 100 / 40 VLDs from 10 / 5 cells . g , Corresponding quantification of mean VLD diameter ( n = 100 / 150 VLDs from 10 / 7 cells ) . h , Corresponding quantification of mean VLD density ( n = 50 / 70 regions from 8 / 7 cells ) . Scale bars indicate 20 \u03bcm . N . s . , non - significant , * , p < 0 . 05 , * * * , p < 0 . 001 . Scale bars are 20 \u03bcm . In all cases , Zoomed insets ( 10x10 \u03bcm ) show the formation and evolution of membrane structures . Supplementary Figure 10 . Model predictions . a , Model prediction for reservoir length upon stretch release as a function of applied stretch . b , model prediction for VLD diameters formed after restoring iso - osmotic medium from osmotic shocks of different magnitude . In a and b , red line corresponds to the bending modulus used throughout the work , dashed black line shows the prediction for a 5 - fold increase in bending modulus . c , VLD shape after restoring osmolarity from hypo - osmotic shocks of different magnitude and d , VLD shape after restoring osmolarity from a 50 % hypo - osmotic shock in a pre - stretched membrane ( top ) and then de - stretching the membrane by 6 % ( top ) . Supplementary Notes Supplementary Note 1 \u2013 Dynamics of reservoir and VLD formation . Throughout the figures , images of stretch - induced reservoirs and osmolarity - induced VLDs generally show fully formed structures at the first frame after their appearance . In the case of reservoirs , this is because their formation was faster than the few seconds required to re - position and re - focus cells after being moved by the stretch device . In the case of VLDs , a few seconds to re - focus after changing the medium were also required . This is because we used an upright microscope with a water - immersion objective that imaged through the medium , which lost focus after medium changes . Whereas the time lag required after stretch operations was unavoidable , we could eliminate the lag required after osmolarity changes by imaging with an inverted microscope . In this case , we observed a VLD formation time of approximately 20 s ( Supplementary Figure 3a ) . Interestingly , the formation period of both reservoirs and VLDs became observable even with the upright microscope after either increasing the magnitude of stretch / hypo - osmotic shock or after decreasing cell activity by reducing temperature ( Supplementary Figure 3b - c ) . This shows that the dynamics of VLD / reservoir formation are governed both by the magnitude of the applied stimulus and the active ability of the cell to recover its original state . Supplementary Note 2 \u2013 Driving forces behind VLD formation and maintenance . It is important to distinguish that VLDs form due to the need to store water volume at the cell - substrate interface , not due to changes in the volume of cells themselves . In earlier work , VLDs had been hypothesized to initiate as membrane invaginations driven by cellular shrinking , then enlarge due to hydrostatic pressure transients , and finally become stabilized by a spectrin cytoskeletal lining 1 , 2 , 3 . However , we believe that hydrostatic pressure of confined water at the cell - substrate interface accounts for all stages of the VLD cycle for different reasons . First , exposure to 50 % osmotic shocks for 3 minutes only generates small membrane compressions of approximately 2 % , which do not form any visible membrane invaginations ( Supplementary Figure 2 ) . Second , elimination of water confinement by using polyacrylamide gels abrogates VLD formation altogether . Finally , decreasing osmolarity after VLD formation collapses VLDs ( Fig . 7e ) , showing that water flows are essential to their maintenance . Similarly , VLDs in cells with ATP depletion ( Fig . 5e - f ) , cytochalasin D treatment ( Supplementary Figure 7e - f ) , or reduced temperature ( Supplementary Figure 7m - n ) gradually collapse , but do not disappear entirely . This suggests that progressive flow from VLDs to the exterior bath reduces pressure and collapses the structure , while active resorption of membrane accumulation is impaired by the different treatments . Thus , hydrostatic pressure driven by confined water generates and maintains VLDs . A detailed analysis of the process would nevertheless require the consideration of the specific and non - specific adhesion between cells and their substrate , and the complex dynamics of confined flows through this interface . Another important aspect to clarify is that the formation of VLDs , while driven by water volume storage , also requires the recruitment of membrane area . This is exemplified by the collapsed VLDs observed after either decreasing osmolarity ( Fig . 7e ) , depleting ATP ( Fig . 5e - f ) , treating with cytochalasin D treatment ( Supplementary Figure 7e - f ) , or reducing temperature ( Supplementary Figure 7m - n ) . In fact , collapsed VLDs are membrane accumulations akin to reservoirs , which like reservoirs can be eliminated through application of stretch ( Fig . 7e ) . Supplementary Note 3 \u2013 Model assumptions , parameters , and predictions . We compare the experimental observations with the predictions of the model described in detail in 4 . In this model , a circular membrane patch with one protrusion at its center is considered , and the optimal shape is obtained by minimizing the energy of the system , consisting of elastic ( stretching and bending ) and adhesive contributions . Depending on the excess membrane area , given by the applied strain , and the interstitial volume , or alternatively the osmotic gradient across the membrane , various optimal shapes emerge , ranging from shallow caps when very little membrane area is available to very thin tubules when very little interstitial volume is available . The nominal separation between membrane structures is modeled through the size of this circular patch , and can be obtained from experiments . For instance , consistent with a density of between 1 and 2 VLDs per 100 \u03bcm 2 , we choose a domain size of 5 \u03bcm . The higher density of reservoirs is modeled with a domain size of 1 . 25 \u03bcm . For the adhesion energy , we use a cell - measured value of 4 mJ / m 2 , reflecting the adhesion strength of a cell membrane to an RGD - coated substrate 5 . We use the same parameters as in 4 for the lipid bilayer stretching ( K s = 0 . 12 J / m 2 ) and bending moduli ( k = 10 - 19 J ) . We note that unlike the model bilayer membranes in 4 , cell membranes are lined with an actin cortex . This cortex has a stretching modulus of the order of 10 - 4 J / m 2 , as obtained from a Young\u2019s modulus and thickness of the order of 10 3 Pa and 100 nm , respectively 6 , 7 . This is three orders of magnitude below the value of the membrane itself , indicating that the contribution of the cortex to the overall stretching modulus is negligible . In contrast , the cortex would be expected to significantly increase membrane bending modulus . Indeed , it has been shown that cells with impaired membrane - actin links have a membrane bending stiffness comparable to that of lipid bilayers , whereas this bending stiffness increases 5 - fold in cells with a normal membrane - cortex connection 8 . However , a 5 - fold increase in bending stiffness did not significantly alter model predictions of the experimental observables , such as tube length or VLD diameter ( Supplementary Figure 10a - b ) . Given the very high membrane stretching modulus and the strong osmolarity differences across the bilayer , the main factors determining the shape of tubes or VLDs were simply the excess area ( compressive strain ) and the volume enclosed in the interstitial space . Consistently with a negligible effect of bending stiffness , depolymerizing the actin cytoskeleton with cytochalasin did not impair reservoir or VLD formation ( Supplementary Figure 7e and 1 ) , and barely affected reservoir diameter ( Supplementary Figure 7c ) . The more marked effect of cytochalasin on VLDs ( reduction in diameter of 30 % , and a 2 - fold increase in density , Supplementary Figure 7g ) is probably due not to the cortex but to stress fibers and focal adhesions , through which VLDs have to make their way ( Supplementary Figure 4 ) . Cytochalasin treatment would reduce cytoskeletal resistance from focal adhesions and stress fibers , leading to the generation of many small VLDs throughout the cell . In contrast , increased cytoskeletal resistance in control cells would allow VLD formation only in specific sites with low resistance , which would then have to accommodate a larger water volume . The only parameter that we adjust to fit our experimental results is the equilibrium separation of the adhesion potential , which in 4 was set to 3 nm . Here , best results are obtained with a value of 30 nm , reflecting the additional separation likely provided by integrins ( extending approximately 20 nm from the membrane 9 ) and the fibronectin coat . With these parameters , the model closely replicates the observations . For instance , the model predicts tubule length to increase linearly with applied destretch , see Fig . 6b . The VLD diameter predicted by the model for a 2 % excess area and various degrees of osmolarity reduction from baseline osmolarity ( M 0 = 300 mOsm ) agrees well with the experimental quantification in Fig . 6c ( see Supplementary Figure 10a ) . Mimicking Fig . 7g , j , increasing the membrane area by de - stretch after VLD formation in pre - stretched cells results in bud - like protrusions with a small neck and a slightly smaller apparent diameter , which store the same volume as the hemispherical VLDs but significantly more membrane area , see Supplementary Figure 10b . In agreement with Fig . 5p , we find that a three - fold increase in the density of VLDs ( modelled through a corresponding decrease in the size of the membrane patch ) results in a decrease of VLD diameter from 2 . 2 \u03bcm to 1 . 6 \u03bcm . Supplementary references 1 . Herring TL , Cohan CS , Welnhofer EA , Mills LR , Morris CE . F - actin at newly invaginated membrane in neurons : implications for surface area regulation . J Membr Biol 1999 , 171 ( 2 ) : 151 - 169 . 2 . Herring TL , Juranka P , McNally J , Lesiuk H , Morris CE . The spectrin skeleton of newly - invaginated plasma membrane . J Muscle Res Cell Motil 2000 , 21 ( 1 ) : 67 - 77 . 3 . Morris CE , Homann U . Cell surface area regulation and membrane tension . J Membr Biol 2001 , 179 ( 2 ) : 79 - 102 . 4 . Staykova M , Arroyo M , Rahimi M , Stone HA . Confined Bilayers Passively Regulate Shape and Stress . Physical Review Letters 2013 , 110 ( 2 ) : 028101 . 5 . Rico F , Roca - Cusachs P , Sunyer R , Farre R , Navajas D . Cell dynamic adhesion and elastic properties probed with cylindrical atomic force microscopy cantilever tips . Journal of Molecular Recognition 2007 , 20 ( 6 ) : 459 - 466 . 6 . Clark AG , Dierkes K , Paluch EK . Monitoring actin cortex thickness in live cells . Biophys J 2013 , 105 ( 3 ) : 570 - 580 . 7 . Salbreux G , Charras G , Paluch E . Actin cortex mechanics and cellular morphogenesis . Trends Cell Biol 2012 , 22 ( 10 ) : 536 - 545 . 8 . Simson R , Wallraff E , Faix J , Niewohner J , Gerisch G , Sackmann E . Membrane bending modulus and adhesion energy of wild - type and mutant cells of Dictyostelium lacking talin or cortexillins . Biophys J 1998 , 74 ( 1 ) : 514 - 522 . 9 . Eng ET , Smagghe BJ , Walz T , Springer TA . Intact \u03b1IIb\u03b23 Integrin Is Extended after Activation as Measured by Solution X - ray Scattering and Electron Microscopy . J Biol Chem 2011 , 286 ( 40 ) : 35218 - 35226 .", + "vojnovic2024combining": "royalsocietypublishing . org / journal / rsob Research Cite this article : Vojnovic I , Caspari OD , Ho \u015f kan MA , Endesfelder U . 2024 Combining single - molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level . Open Biol . 14 : 230414 . https : / / doi . org / 10 . 1098 / rsob . 230414 Received : 8 November 2023 Accepted : 4 December 2023 Subject Area : cellular biology / microbiology / structural biology / molecular biology / biophysics Keywords : single - molecule localization microscopy , photoactivated localization microscopy , expansion microscopy , Schizosaccharomyces pombe , protein retention yield , correlative expansion microscopy Author for correspondence : Ulrike Endesfelder e - mail : endesfelder @ uni - bonn . de \u2020 Present address : Institut f\u00fcr Mikrobiologie und Biotechnologie , Rheinische Friedrich - Wilhelms - Universit\u00e4t , Bonn , Germany . \u2021 These authors contributed equally to this study . \u00b6 Present address : Department of Medical Microbiology and Immunology , University of Wisconsin - Madison , Madison , WI , USA . One contribution to a Special Feature \u2018 Bioimaging \u2019 . Electronic supplementary material is available online at https : / / doi . org / 10 . 6084 / m9 . figshare . c . 7007803 . Combining single - molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level Ilijana Vojnovic 1 , 2 , \u2020 , \u2021 , Oliver D . Caspari 1 , 3 , \u2020 , \u2021 , Mehmet Ali Ho \u015f kan 1 , \u00b6 and Ulrike Endesfelder 1 , 2 , \u2020 1 Department of Systems and Synthetic Microbiology , Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology ( SYNMIKRO ) , Marburg , Germany 2 Department of Physics , Carnegie Mellon University , Pittsburgh , PA , USA 3 Department of Microbiology , Institute Pasteur , Paris , France ODC , 0000 - 0001 - 8235 - 0503 ; UE , 0000 - 0002 - 7801 - 6278 In this work , we have developed an expansion microscopy ( ExM ) protocol that combines ExM with photoactivated localization microscopy ( ExPALM ) for yeast cell imaging , and report a robust protocol for single - molecule and expansion microscopy of fission yeast , abbreviated as SExY . Our optimized SExY protocol retains about 50 % of the fluorescent protein signal , doubling the amount obtained compared to the original protein retention ExM ( proExM ) protocol . It allows for a fivefold , highly isotropic expansion of fis - sion yeast cells , which we carefully controlled while optimizing protein yield . We demonstrate the SExY method on several exemplary molecular tar - gets and explicitly introduce low - abundant protein targets ( e . g . nuclear proteins such as cbp1 and mis16 , and the centromere - specific histone protein cnp1 ) . The SExY protocol optimizations increasing protein yield could be ben - eficial for many studies , when targeting low abundance proteins , or for studies that rely on genetic labelling for various reasons ( e . g . for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality ) . 1 . Introduction The resolution of optical microscopy is constrained by the diffraction limit of light at about 200 nm [ 1 ] . However , most of molecular organization occurs in dimensions below this limit and therefore cannot be resolved by conventional light microscopic techniques . Super - resolution fluorescence microscopy ( SRM ) can be used to circumvent the diffraction limit by modulating the fluorescence of fluorescently labelled molecules at a sub - diffraction level , thereby discrimi - nating between them [ 2 ] . This is achieved either by precisely defined illumination patterns ( e . g . in STED [ 3 ] and SIM [ 4 ] imaging ) or by single - mol - ecule localization microscopy ( SMLM ) methods , in which the fluorescence of individual , on - and off - blinking or on - and off - binding fluorophores is separ - ated in time ( e . g . in PALM [ 5 ] , ( d ) STORM [ 6 , 7 ] or PAINT [ 8 ] techniques ) . More recently , expansion microscopy ( ExM ) techniques have been developed that physically expand the biological sample , increasing resolution by practi - cally a factor of 3 \u2013 20 - fold as a result of inflating the structures [ 9 \u2013 13 ] . In contrast to the \u2018 classical \u2019 SRM methods , where preparing a good sample mostly relies on choosing the right fluorophores ( e . g . bright ( all ) and either photostable ( SIM , STED ) or well - controlled in photoswitching ( SMLM ) ) and optimizing for efficient and specific labelling ( e . g . small labels for dense label - ling , non - sticking labels for high specificity or genetic , covalent or high affinity labels for high efficiency ) [ 2 ] , ExM poses additional demands to the sample preparation due to the desired physical expansion : in ExM , the sample is \u00a9 2024 The Authors . Published by the Royal Society under the terms of the Creative Commons Attribution License http : / / creativecommons . org / licenses / by / 4 . 0 / , which permits unrestricted use , provided the original author and source are credited . D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 anchored to a gel mesh and expanded upon incubation in aqueous media [ 9 ] . To achieve an isotropic expansion of the sample and thus a preservation of the underlying biological ultrastructure , all physical connections within the structure must be efficiently removed by , e . g . cell wall removal [ 14 \u2013 17 ] and protein digestion steps [ 10 , 18 \u2013 21 ] , and all target molecules must be properly linked to the gel mesh before expansion by , for example , anchoring [ 22 \u2013 24 ] or linkage by fixation [ 22 ] . In this context , implementing ExM in organisms with rigid cell walls [ 14 \u2013 17 , 25 \u2013 27 ] or maintaining isotropic expansion within samples of heterogeneous \u2018 rigidity \u2019 [ 20 , 28 \u2013 31 ] is a particular challenge . Recent work has shown that samples can show different macro - and micro - expansion factors ( e . g . factors that vary within a gel or tissue or differ between different organelles or domains within organelles [ 20 , 28 \u2013 31 ] ) . Finally , protocols are often not directly transfer - able between different samples . For example , an ExM method that preserved isolated centrioles from Chlamydomo - nas reinhardtii failed to isotopically expand chromatin in root tips of barley [ 28 , 32 ] . A second challenge in ExM is to retain the molecular information of the sample while breaking physical connec - tions for expansion . The protein retention yield is typically measured by how many target molecules can still be detected via fluorescent markers in an expanded sample [ 23 ] . It is mainly affected by the protocol steps of protein digestion and gelation . Protein digestion involves homogenization of the sample by cleavage of the proteins \u2019 peptide chains with proteinase K or collagenase type II , heat denaturation or homogenizing agents such as SDS [ 9 , 10 , 18 , 19 , 21 ] . Gelation involves the generation of radicals during the polymerization chain reaction of monomers to form a polyacrylamide gel ( PAA ) . Only recently , a radical - free gelation method using novel custom - synthesized monomers was developed [ 33 ] . Both , protein digestion and gelation , thus have potential to reduce the protein retention yield by degrading target sites for staining or by degrading genetically encoded markers . Protocols combining ExM with SRM techniques to date have achieved an overall resolution of 1 \u2013 30 nm [ 34 \u2013 37 ] . While a combination of ExM and SRM yields superior resol - ution , fluorophore choice and labelling strategy must suit both techniques , i . e . withstand and perform under the com - bined sample preparation and imaging protocols . To date , ExM was successfully combined with SIM [ 34 ] , STED [ 35 ] , dSTORM [ 36 , 37 ] and fluctuation - based techniques [ 38 , 39 ] . These ExM \u2013 SRM protocols rely on extrinsic labelling tech - niques of the targets using labels with organic dyes , mainly immunofluorescence [ 34 \u2013 39 ] . Furthermore , dSTORM uses ionic switching buffers needed for the on - and off - blinking of dyes . These buffers however shrink expanded gels from deionized water . Thus , ExSTORM relies on expansion in ionic buffers , which achieves only lower expansion of about threefold [ 36 ] . An alternative is Ex - SMLM [ 37 ] . Here , gels expanded in deionized water are embedded in an uncharged secondary gel . These double - layer gels achieve an about three - fold increase and tolerate incubation in ionic switching buffers . While current ExM \u2013 SRM protocols work well for abun - dant targets and larger ( polymeric ) structures in various samples , staining background signal from non - specific adher - ent labels severely compromises imaging of sparse targets . Additionally , factors that hinder efficient extrinsic labelling , such as a cell wall or a highly crowded and charged cytosol like in microbial organisms , can complicate labelling [ 40 ] and can introduce artefacts [ 41 ] . To date , there are only a handful of publications on combined ExM and SRM strat - egies for microbiology , namely two studies using ExSIM of fungi [ 16 ] and bacteria [ 42 ] , and one has combined ExM with SRRF imaging for viral SARS - Cov - 2 particles [ 38 ] . In this work , we set out to establish a protocol for com - bined ExM and SMLM imaging in yeast . Here , our SMLM method of choice was PALM microscopy . In contrast to dSTORM , PALM microscopy makes use of genetic labelling using fluorescent proteins ( FP ) [ 2 ] . Thus , PALM samples intrinsically possess high labelling efficiency and specificity and do not rely on switching buffers which makes an implementation of ExPALM attractive for microbiological studies ( at the price that current FPs offer lower photostabil - ity and photon yield compared to dyes ) . Nevertheless , as fluorescent proteins are affected and at least partially degraded by the ExM sample preparation protocols , so far , no protocols that combine ExM with PALM ( ExPALM ) exist . Here , we combine PALM imaging with a proExM protocol [ 23 ] optimized for isotropyand protein retention for the fission yeast Schizosaccharomyces pombe . Using our optimized sample preparation protocols , we have established an ExPALM proto - col that preserves about 50 % of the FP signal , doubling the retained amount compared to applying the protein retention ExM ( proExM ) protocol to the tested S . pombe cell samples . Our optimized protocol achieves a fivefold , highly isotropic expansion of fission yeast cells which we tested for several molecular targets . Taken together , this protocol is the first demonstration of combined single - molecule and expansion microscopy of yeast ( SExY microscopy ) . 2 . Material and methods 2 . 1 . Strain construction C - terminal tagging of cbp1 with mEos2 [ 43 ] and sad1 with mScarlet - I [ 44 ] was adapted from [ 45 ] . First , two intermediate plasmids were created . For creating cbp1 - mEos2 , a pBlue - script II SK + plasmid containing the Saccharomyces cerevisiae ADH1 terminator , a kanamycin resistance gene and the mEos2 gene was constructed using primers with 20 bp over - lap sequences ( primers 9 \u2013 14 ; electronic supplementary material , table S1 ) [ 45 ] . Similarly , for creating sad1 - mScarlet - I , a pFA6 - mScarlet - I - ADH1 - hphMX6 plasmid was con - structed amplifying mScarlet - I from pFA6a - mScarlet - I - hphNT1 [ 46 ] ( primers 24 \u2013 25 ) and pFA6 - ADH1 - hphMX6 ( primers 15 \u2013 16 ) from pFA6 - hphMX6 [ 47 ] . DNA fragments were combined by Gibson Assembly ( New England Biolabs , cat . no . E5510S ) , transformed into competent DH5 \u03b1 cells , streaked onto LB Amp R plates and incubated overnight at 37 \u00b0C . Single colonies were picked , grown in LB Amp R cul - tures for 2 h at 37 \u00b0C and plasmids were extracted ( ZymoPure II Plasmid Midiprep kit , Zymo Research , cat . no . D4200S ) and checked by sequencing ( Eurofins Genomics Germany GmbH ) . In a second step , 200 \u2013 600 bp DNA frag - ments up - and downstream of the respective insertion site were amplified from isolated genomic DNA from h \u2212 S . pombe cells with 20 bp overlaps to the flanking genes using primers 1 \u2013 4 for cbp1 and 17 , 19 & 22 \u2013 23 for sad1 . The DNA fragments were combined with the corresponding FP - ADH1 - antibiotic resistance fragment using overlap - extension PCR [ 48 ] for sad1 - mScarlet - I or SLiCE [ 49 ] for cbp1 - mEos2 . r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 2 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 10 \u00b5l of the PCR mix were transformed into either competent h + ade6 - 210 leu1 - 32 ura4 - D18 mEos2 : cnp1 or h - WT strain using the Frozen - EZ Yeast Transformation II Kit ( Zymo Research , cat . no . T2001 ) . Cells were streaked onto YES agar plates , incubated overnight at 32 \u00b0C , replica plated onto either YES Kan R or YES Hyg R agar plates and grown at 32 \u00b0C for another two days . Successful genomic integration was checked by colony - PCR and DNA sequencing ( Eurofins Genomics Germany GmbH ) . For N - terminal tagging of mis16 , DNA fragments up - and downstream of the insertion site where generated using primers 5 \u2013 8 . The DNA fragments were combined with mEos2 - mis16 using SLiCE [ 49 ] . 10 \u00b5l of the PCR mix were transformed into either competent h + ade6 - M210 leu1 - 32 ura4 - D18 ura4nm41 : mis16 cells using the Frozen - EZ Yeast Transformation II Kit ( Zymo Research , cat . no . T2001 ) . Cells were streaked out onto YES agar plates containing 1 g l \u2212 1 5 - Fluoroorotic Acid and 50 mg ml \u2212 1 uracil and grown at 32 \u00b0C for 2 days . Successful genomic integration was checked by colony - PCR and DNA sequencing ( Eurofins Genomics Germany GmbH ) . 2 . 2 . Protease enzyme activity assay The colorimetric assay to measure protease activities was adapted from [ 50 , 51 ] . In short , casein ( Sigma - Aldrich , cat . no . C7078 - 500G ) was digested to release tyrosine . After stop - ping the digestion with tricholoroacetic acid ( Sigma - Aldrich , cat . no . T0699 - 100ML ) , released tyrosine was measured by absorption at 660 nm using Folin & Ciocalteu \u2019 s Phenol reagent ( Sigma - Aldrich , cat . no . F9252 - 100ML ) . Enzymes were tested at their respective optimal working temperature and digestion buffer for 10 min . Concentration ranges were 0 . 2 \u2013 1 . 0 U ml \u2212 1 for proteinase K , 0 . 2 \u2013 1 . 0 , 10 U ml \u2212 1 and 50 U ml \u2212 1 for Zymo - lyase and \u03b2 - glucoronidase and 0 . 02 \u2013 0 . 1 U ml \u2212 1 for lysing enzyme . Controls with 20 mM of the protease inhibitor phe - nylmethylsulfonylfluorid ( PMSF , ThermoFisher Scientific , cat . no . 36978 ) were conducted for proteinase K and lysing enzyme and a standard curve of free L - tyrosine ( Sigma - Aldrich , cat . no . T3754 - 50G ) was measured . 2 . 3 . Schizosaccharomyces pombe cell culture S . pombe strains were grown on YES or EMM - leu agar plates at 32 \u00b0C for two days . Single colonies were picked and inocu - lated overnight in YES or EMM - leu medium at 25 \u00b0C . Overnight cultures were diluted to OD 0 . 05 for YES and OD 0 . 1 for EMM - leu cultures and grown at 25 \u00b0C to OD 0 . 5 \u2013 0 . 8 . For imaging non - expanded cells expressing cytosolic mEos2 , the EMM - leu overnight cultures were diluted to OD 0 . 2 in EMM containing 5 \u00b5M thiamine to repress mEos2 expression and were grown for 3 h at 25 \u00b0C . 2 . 4 . Sample preparation using the SExY protocol The SExY protocol was adapted from the proExM method [ 23 ] and optimized for PALM and expansion microscopy in fission yeast . The ideal cell density for sample preparation was deter - mined to be 6 OD ml \u2212 1 for all fluorescently - labelled protein of interest ( POI ) strains ( electronic supplementary material , table S2 ) and 12 OD ml \u2212 1 for SP16 cells which express cytosolic mEos2 and were added to all samples for drift correction . The appropriate volumes were transferred to fresh Erlenmeyer flasks and fixed with 3 . 2 % paraformaldehyde ( Sigma - Aldrich , cat . no . F8775 - 4X25ML ) and 0 . 1 % glutaraldehyde ( Sigma - Aldrich , cat . no . G5882 - 50ML ) at 25 \u00b0C for 10 min . The cells were harvested by centrifugation ( 358 g , 2 min , room tempera - ture ) , resuspended in 1 ml 1x PBS and washed twice in 1 ml 1x PBS for 5 min . The cells were washed twice with 200 \u00b5l 1x S - c / pH 5 . 8 ( 1 . 2 M D - Sorbitol ( Sigma - Aldrich , cat . no . S1876 - 500G ) in citrate / phosphate buffer ( Sigma - Aldrich , cat . no . C0759 - 1KG and cat . no . S7907 - 500G ) , pH 5 . 8 ) for 10 min each . The pellet after centrifugation was resuspended in 1 ml per 6 OD / ml Lallzyme MMX mix ( 100 mg ml \u2212 1 Lallzyme MMX ( Lallemand , cat . no . ElO11 - 2240 - 15 ) , 1 . 2 M D - Sorbitol in citrate / phosphate buffer , pH 5 . 8 ) and incubated at 30\u00b0C for 2 h for cell wall digestion . Subsequently , the cells were washed twice in 1 ml 1x S - PBS ( 1 . 2 M D - Sorbitol in 1x PBS , pH 7 . 4 ) . To anchor the amine groups of the proteins to the PAA gel mesh , the cell pellets were resuspended in 200 \u00b5l per 6 OD ml \u2212 1 AcX solution ( 0 . 1 mg ml \u2212 1 Acryloyl X ( Invitro - gen , cat . no . A20770 ) , 1 % ( v / v ) DMSO ( Carl Roth , cat . no . A994 . 1 ) in 1x S - PBS ) . Each POI strain was mixed with SP16 cells in a 1 : 2 ratio and incubated at 25 \u00b0C overnight . The next day , the sample was washed twice with 500 \u00b5l 1x S - PBS for 15 min . Round coverslips were cleaned in 1 M KOH for 30 min and rinsed with MilliQ water . Air - dried coverslips were incubated with 100 \u00b5l poly - L - lysine ( Sigma - Aldrich , cat . no . P8920 ) for 20 min at room temperature ( RT ) and then assembled into custom made imaging gel cassettes made of polyoxymethylene ( POM ) . Since a previous study found that an initial incubation of the monomer solution with the sample without the catalysator yielded higher expansion factors [ 31 ] , the cell pellets were resuspended in half of the 1 . 06x Monomer solution ( 8 . 625 % ( w / w ) Sodium Acrylate ( Sigma - Aldrich , cat . no . 408220 - 25G ) , 2 . 5 % ( w / w ) 37 : 1 Acrylamide : Bisacrylamide ( Sigma - Aldrich , cat . no . A6050 - 100ML ) , 0 . 15 % ( w / w ) N , N 0 - methylenebisacrylamide ( Sigma - Aldrich , cat . no . M1533 - 25ML ) , 2 M NaCl ( Sigma - Aldrich , cat . no . S3014 - 500G ) in 1x PBS ) together with 0 . 2 % TEMED ( Research Products Int , cat . no . T18000 - 0 . 05 ) and 0 . 05 % of freshly prepared L - glutathione ( Carl Roth , cat . no . 6832 . 3 ) . This pre - gelation mix was pipetted onto the coverslip in the imaging gel cassette and incubated for 10 min at 37 \u00b0C in the dark . Gelation was initiated by addition of 0 . 2 % APS ( Carl Roth , cat . no . 9178 . 3 ) and the rest of the 1 . 06x Monomer solution to the pre - gelation mix and thoroughly mixed . The gel cassettes were placed into a humid environment using Petri dishes with wet paper towels wrapped in aluminium foil and incubated at 37 \u00b0C for 1 h . The gels were then incu - bated in 2 ml digestion buffer ( 50 mM Tris ( Carl Roth , cat . no . AE15 . 2 ) pH 7 . 5 , 1 mM EDTA ( Sigma - Aldrich , cat . no . EDS - 100G ) , 0 . 2 % Triton X - 100 ( Sigma - Aldrich , cat . no . T8787 - 50ML ) , 1 M NaCl ) with 3 U ml \u2212 1 effective proteinase K ( ThermoFisher , cat . no . EO0491 ) at 37 \u00b0C for 30 min in the aluminium foil wrapped mini Petri dishes ( Sarstedt , cat . no . 82 . 1135 . 500 ) . Thereafter , gels were washed with MilliQ water , transferred to Petri dishes , wrapped in aluminium foil and fully expanded in 100 ml MilliQ water for 4 h at RT , exchan - ging MilliQ water every hour . Ibidi 8 - well glass bottom slides ( Ibidi , cat . no . 80807 ) , which were previously cleaned with 1 M KOH ( Sigma - Aldrich , cat . no . 221473 - 2 . 5KG - M ) for 30 min and washed twice with MilliQ water , were incubated with poly - L - lysine for 20 min and air dried . The expanded gel was cut using a scalpel , transferred into an Ibidi well , excess water was removed and the gel was incubated for r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 3 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 10 min . After that , the gel was secured in place by embedding it in 2 % agarose ( Carl Roth , cat . no . 3810 . 3 ) and allowing the agarose to dry for 10 min . The gel was submerged in 300 \u00b5l MilliQ water and then imaged . 2 . 5 . Tests of various sample preparation steps while optimizing the protocol 2 . 5 . 1 . Cell wall digestion To lyse the cell wall , the chemically fixed cells were treated with various enzymes and tested for a homogeneous macro - scale expansion via fluorescence microscopy . The used enzymes and conditions for cell wall digestion can be found in electronic supplementary material , table S3 . All incubations were done agitating the samples in a thermo block . 2 . 5 . 2 . Permeabilization For permeabilizing the cell membrane , the fixed and in 1xPBS washed cells were resuspended in 200 \u00b5l 1xPBS containing 0 . 1 % Triton X - 100 and incubated at 37 \u00b0C for 30 min . After - wards the cell wall was digested and the initial proExM protocol for fission yeast was continued . 2 . 5 . 3 . Protein digestion by heat denaturation The initial proExM protocol adapted for fission yeast was performed until the gelation step , where 0 . 05 % glutathione were added to the gelation as described above . Afterwards , the gel was submerged into 2 ml of the 50 mM Tris . HCl buffer at pH 8 . 0 and incubated at 65\u00b0C for 15 min . Then the gel was incubated in the renaturing buffer ( 35 mM KCl 2 , 1 mM MgCl 2 , 1 mM DTT , 30 % glycerol , 50 mM Tris . HCl , pH 8 . 0 ) at RT for 30 min . 2 . 5 . 4 . Protein digestion by homogenization with SDS and heat denaturation The sample was prepared the same way as for the heat dena - turation until protein digestion . The gel was then submerged in 2 ml freshly prepared SDS solution ( 200 mM SDS , 200 mM NaCl , 50 mM Tris . HCl , pH 8 . 0 ) and incubated at 37 \u00b0C for 30 min with a subsequent incubation at 65 \u00b0C for 15 min . After washing the gel with deionized water , it was fully sub - merged in renaturing buffer and incubated at RT for 30 min . 2 . 5 . 5 . Gelation including glutathione To determine the effect of glutathione , 0 \u2013 0 . 5 % freshly prepared glutathione was added to the pre - gelation mix and incubated with the sample for 10 min at 37 \u00b0C in the dark . After initiation of the gelation , samples were taken for fluorescence intensity measurement , and the rest of the sample was digested and expanded as described above . The gel rigidity of the expanded gels was determined visually and haptically . 2 . 6 . Microscope set - up For all imaging experiments , a custom build set - up based on an automated Nikon Ti Eclipse microscopy body with suitable dichroic and emission filters ( ET dapi / Fitc / cy3 dichroic , ZT405 / 488 / 561rpc rejection filter , ET525 / 50 for GFP and the green form of mEos2 or ET610 / 75 for mScar - let - I and the red photoconverted form of mEos2 , all AHF Analysentechnik , Germany ) and a CFI Apo TIRF 100 \u00d7 oil objective ( NA 1 . 49 , Nikon ) was used . A perfect focus system ( Nikon , Germany ) was used for z - axis control , except for ExPALM imaging , where the sample was imaged at 10 \u2013 80 \u00b5m depth . All lasers ( 405 nm OBIS , 488 nm Sapphire LP and 561 nm OBIS , Coherent Inc . USA ) were modulated by an acousto - optical tunable filter ( AOTF ; Gooch and Housego , USA ) . Fluorescence detection was performed by an emCCD camera ( iXON Ultra 888 , Andor , UK ) at a pixel size of 129 nm . Image acquisition was controlled by a customized version of \u00b5Manager [ 52 ] . 2 . 7 . Fluorescence imaging of GFP - nup132 Expanded and non - expanded GFP - nup132 cells were ident - ified using a 488 nm laser ( 21 W cm \u2212 2 at the sample level ) . A fluorescence video of 50 frames with an exposure time of 100 ms per frame was acquired using 81 W cm \u2212 2 at the sample level . 2 . 8 . Fluorescence imaging and analysis of sad1 - mScarlet - I While optimizing the ExPALM protocol , a sample was taken after every step of the protocol and placed on a cleaned and poly - L - lysine coated Ibidi 8 - well glass bottom slide . After 15 min of settling time , the sample was either washed twice ( 1x PBS for fixation / permeabilization steps or 1x S - PBS for cell wall digestion / anchoring steps ) or prevented from drying / shrinking by adding one to two drops of MilliQ water in gelation / expansion steps . Only cells in mitosis with two sad1 - mScarlet - I spots were imaged . A video of 10 imaging frames of the sad1 - mScarlet - I fluorescence was taken with an exposure time of 100 ms per frame and in epi - illumination using a 561 nm laser . Laser intensities as measured at the sample level were 3 W cm \u2212 2 ( live - anchoring ) and 6 W cm \u2212 2 ( gelation - protein digestion ) . The integrated intensity of the fluorescent spots and a close - by background area were measured in a 14 \u00d7 22 pixel ROI in the first three imaging frames using ImageJ [ 53 ] . The final intensity measure was obtained by calculating the mean of the three frames for both ROIs and subtracting the background . 2 . 9 . PALM imaging and analysis of expanded cells Expanded cbp1 - mEos2 and mis16 - mEos2 cells were ident - ified in the gel by their fluorescence in epi - illumination using the 488 nm laser ( 21 W cm \u2212 2 at the sample level ) and PALM videos of 3000 - 5000 frames were acquired with an exposure time of 60 \u2013 80 ms per frame photoconverting and imaging mEos2 by continuous illumination of 405 nm laser ( 3 \u2013 12 W cm \u2212 2 at the sample level ) and 561 nm laser ( 1 . 2 kW cm \u2212 2 at the sample level ) . For imaging the DNA , the DNA was stained after mEos2 read - out using 100 nM TO - PRO - 3 Iodide ( Invitrogen , cat . no . T3605 ) which was added to the imaging well on the microscope stage and incu - bated for 30 min . A fluorescence video of 50 frames with an exposure time of 100 ms per frame was acquired using the 561 nm laser ( 0 . 5 kW cm \u2212 2 at the sample level ) . Expanded mEos2 - cnp1 / sad1 - mScarlet - I cells where identified by r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 4 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 mScarlet - I fluorescence using the 561 nm laser . A fluor - escence movie of 50 frames with an exposure time of 100 ms per frame was acquired using the 561 laser ( 0 . 5 kW cm \u2212 2 at the sample level ) . Remaining mScarlet - I flu - orescence was photobleached ( 1 . 2 kW cm \u2212 2 at the sample level , 561 nm laser ) and the mEos2 signal and the TO - PRO - 3 Iodide DNA stain were imaged as described above . All samples contained cells expressing cytosolic mEos2 , which served as marker for drift correction . Each field of view contained at least one cell expressing cytosolic mEos2 and was drift - corrected by cross - correlation or by relative frame - to - frame shift ( supplementary code and https : / / github . com / Endesfelder - Lab ) . Data with unreliable drift cor - rection , which typically occurred at the end of PALM videos at low localization densities , were discarded . Images were reconstructed in Rapidstorm 3 . 2 with a pixel size of 10 nm [ 54 ] . Localization precision was determined using the NeNA method [ 55 , 56 ] . For image representation , images were blurred by a Gaussian using their NeNA value as the sigma value . PALM images were overlaid with corre - sponding fluorescence images of sad1 - mScarlet - I and DNA in ImageJ 1 . 52p . 2 . 10 . PALM imaging and analysis of non - expanded cells Cells expressing cytosolic mEos2 were fixed with 3 . 2 % paraf - ormaldehyde ( Sigma - Aldrich , cat . no . F8775 - 4X25ML ) and 0 . 1 % glutaraldehyde ( Sigma - Aldrich , cat . no . G5882 - 50ML ) and embedded in 0 . 5 % agarose gels in an Ibidi 8 - well glass bottom slide together with a 1 : 500 dilution of sonicated , dark - red , 0 . 2 \u00b5m sized FluoSpheres ( Molecular probes , cat . no . F8807 ) . PALM videos of 3000 - 5000 frames were acquired with an exposure time of 60 ms per frame photoconverting and imaging mEos2 by pulsed illumination of 405 nm laser ( 3 \u2013 12 W cm \u2212 2 at the sample level ) and 561 nm laser ( 1 . 2 kW cm \u2212 2 at the sample level ) . Each field of view was corrected by fiducial drift correc - tion using a custom script ( Python 3 . 7 ) . Images were reconstructed in Rapidstorm 3 . 2 with a pixel size of 10 nm [ 54 ] . Localization precision was determined using the NeNA method [ 55 , 56 ] . For image representation , images were tracked in Rapidstorm 3 . 2 with an allowed blinking interval of five frames and a sigma set to the NeNA value . After filtering out all trajectories of three steps and less , a PALM image was reconstructed in Rapidstorm 3 . 2 and blurred by a Gaussian using the NeNA value as sigma value . 2 . 11 . Determination of expansion factor The expansion factor was measured by the cytosol width and the nuclear size of expanded and non - expanded cells . For the cytosol width , mean and standard error of cells in recon - structed PALM images of fixed and expanded cells expressing cytosolic mEos2 were measured . Raw SMLM data were used for this purpose , as cell peripheries appear sparser post - processing ( e . g . due to combining multiple local - izations of the same fluorescence event into one ) . For measuring the nuclear expansion factor , the mean and stan - dard error of the nuclear diameter were determined in fluorescence images of expanded and non - expanded GFP - nup132 cells were taken . Cells with non - spherical nuclei were excluded from the analysis . The expansion factor was calculated by dividing the expanded cytosol width or nuclear size by the non - expanded cytosol width or nuclear size . 2 . 12 . Assessment of microscale isotropy of individual expanded cells by evaluating the distribution of cytosolic mEos2 molecules Nearest - neighbour distances between cytosolic mEos2 local - izations in expanded cells were compared with cytosolic mEos2 localizations in non - expanded cells in R , v3 . 6 . 1 ( https : / / www . r - project . org / ) , using RStudio , 2022 . 07 . 2 + 576 ( https : / / www . rstudio . com / ) and the spatstat package [ 57 ] . To be able to compare the distances , first , point densities ( number of points per cell area ) were evaluated for all cells . Next , for a given expanded cell , areas of the 30 non - expanded cells were enlarged in silico until point densities matched . Then , the average nearest - neighbour distances were calcu - lated . Finally , the mean ( \u03bc ) across non - expanded cells was subtracted from the value of the expanded cell ( x i ) , and nor - malized to the standard deviation ( \u03c3 ) of the distribution of non - expanded cells : ( x i \u2212 \u03bc ) / \u03c3 . A two - sided Student \u2019 s t - test was performed . 2 . 13 . Data reproducibility statement All experiments were performed at least in duplicate for negative results that did not lead anywhere further and at least in triplicate for forward - leading results from different days of sample preparation . 3 . Results and discussion In this work , we established a sample preparation and imaging protocol which combines the super - resolution tech - nique PALM with a proExM protocol optimized for isotropy and protein retention for the fission yeast S . pombe , which we named Single - molecule and Expansion microscopy in fis - sion Yeast ( SExY ) . Our final protocol ( figure 1 a ) consists of six steps : chemical fixation of the cells and subsequent cell wall digestion , protein anchoring and gelation of the PAA , which is then followed by protein digestion and finally expansion . SExY achieves an about fivefold expansion , as demonstrated by cells that cytosolically express the FP mEos2 whose positions were super - resolved by PALM ima - ging ( figure 1 b ) . While cytosolic , free diffusive mEos2 molecules are generally distributed throughout the cell , SExY also clearly resolves their ( expected ) non - random , small - scale substructured distribution ( e . g . small omitted regions of round vesicles and a maximum of FPs near the nucleus ) . Essential aspects of the SExY protocol are , first , an isotro - pic and satisfactory expansion , and second , a high protein retention yield to preserve protein yield ( i . e . the FP signal for PALM read - out ) . Importantly , neither was the case when using the original proExM protocol on S . pombe cells . For verifying the isotropic expansion of the cells , we measured the cytosol width of cells expressing cytosolic mEos2 as seen in PALM images ( figure 1 c ( i ) ) . For verifying the isotropic expansion of organelles , we measured the nuclear diameter of cells in interphase that expressing GFP - tagged nuclear pore protein Nup132 ( figure 1 c ( ii ) ) . Non - expanded cells yielded a cytosol width of 2 . 4 \u00b5m \u00b1 0 . 19 \u00b5m r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 5 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 and a nuclear diameter of 2 . 36 \u00b5m \u00b1 0 . 34 \u00b5m , cells expanded with the SExY protocol showed a cytosol width of 11 . 8 \u00b5m \u00b1 1 . 8 \u00b5m and a nuclear diameter of 11 . 6 \u00b5m \u00b1 1 . 4 \u00b5m . This resulted in a macroscale expansion factor of 4 . 9 ( standard error ( s . e . ) 0 . 2 , standard deviation ( s . d . ) 0 . 8 ) as measured by the cytosol width and a factor of 4 . 9 ( s . e . 0 . 2 , s . d . 0 . 9 ) for 5 . protein digestion 6 . expansion in MQ water 1 . fixation 2 . cell wall digestion 3 . protein anchoring 4 . gelation of PAA gel O macroscale expansion ( d ) microscale expansion 2 \u00b5m isotropic ( e ) FP survival 2 \u00b5m non - expanded expanded 0 2 4 6 8 10 12 14 non - expanded expanded 0 2 4 6 8 10 12 14 post optimization 2 \u00b5m non - isotropic pre optimization 0 500 1000 1500 2000 n = 143 n = 19 n = 68 n = 28 n = 21 n = 41 n = 35 c y t o s o l w i d t h ( \u00b5 m ) nu c l e u s d i a m e t e r ( \u00b5 m ) f l uo r e s ce n ce s po t i n t e n s it y ( a r b . un it s ) fixedcells pre post optimization ( a ) ( b ) ( c ) ( i ) ( ii ) 2 \u00b5m 5 \u00d7 expansion Figure 1 . Principle and performance of the SExY method . ( a ) Principle of SExY sample preparation , a correlative ExPALM protocol for expanding yeast . Shown are cell wall ( black ) , cell membrane ( grey ) , inner and outer nuclear membrane ( magenta ) , target protein ( blue ) , FP marker ( red ) and the PAA gel mesh ( orange ) . The acrylate group of the protein anchoring agent AcX is highlighted in orange . ( b ) Fission yeast cells expand approximately fivefold using the SExY protocol . ( c ) SExY provides a macroscale isotropic expansion as determined by two different samples , measuring ( i ) cytosol widths and ( ii ) nuclear diameter ( Nup132 ) of non - expanded and expanded cells imaged over a total of nine different experiments . The expansion factor was determined to be 4 . 9 ( s . e . 0 . 2 , s . d . 0 . 8 ) ( cytosol width ) and 4 . 9 ( s . e . 0 . 2 , s . d . 0 . 9 ) ( nuclear diameter ) , respectively . Note that nuclear widths were estimated from diffraction limited epifluorescence data and cannot be compared directly to cytosolic widths derived from SMLM data . ( d ) Additionally , the isotropic microscale expansion of the SExY protocol was probed investigating cell borders . Here , cells expanded with the SExY protocol show no mEos2 localizations being pulled outside of the cell area and hence an isotropic expansion on the microscale . ( e ) SExY retains 46 % ( s . e . 3 . 1 % , s . d . 21 . 8 % ) of FP signal retention , whereas the original proExM protocol retained 22 % ( s . e . 2 . 0 % , s . d . 12 . 4 % ) . r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 6 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 the nuclear diameter . Notably , the achieved expansion is higher than in current protocols that combine ExM with dSTORM and achieve an about threefold expansion [ 36 , 37 ] . To assess isotropic expansion on the microscale , we examined the cell boundaries of cells expressing cytosolic mEos2 which were expanded using either the initial proExM protocol ( adding the step of using Lallzyme MMX for cell wall diges - tion ) or the optimized SExY protocol . When applying the original protocol , we found a substantial fraction of mEos2 molecules located outside the main cell area within the poly - acrylamide mesh network , apparently pulled outward due to heterogeneous expansion and resulting in \u2018 fuzzy \u2019 cell bound - aries . By contrast , cell boundaries were clearly defined when the SExY protocol was applied ( figure 1 d ) , indicating a more defined and homogeneous microscale expansion . Further - more , statistical testing for changes in cytosolic mEos2 FP distribution upon expansion did not show any changes ( elec - tronic supplementary material , figure S1 ) . While prior to optimization of the protocol , individual expanded cells show a relatively high variation in cytosolic mEos2 distri - bution relative to non - expanded cells ( up to 1 . 88 s . d . ) , post - optimization all expanded cells lie well within the range expected for isotropic expansion ( up to 0 . 5 s . d . in relation to the mean of non - expanded cells ) . To measure the protein retention yield , we quantified the fluorescent signal retained from the spindle pole protein sad1 tagged with mScarlet - I from cells during anaphase B in mitosis . For our initial proExM protocol , we found that only 22 % ( s . e . 2 . 0 % , s . d . 12 . 4 % ) of the fluorescence signal was retained when compared to fixed cells ( figure 1 e ) . However , using the SExY protocol , about 46 % ( s . e . 3 . 1 % , s . d . 21 . 8 % ) of the sad1 - mScarlet - I signal was preserved , doubling the retained amount . To gauge whether an increase in resolution could be achieved by expansion , localization precisions were estimated using the NeNA method [ 55 ] to be 13 . 9 nm ( s . e . 0 . 8 , s . d . 3 . 5 , n = 22 ) for non - expanded cells and 26 . 5 nm ( s . e . 0 . 7 , s . d . 5 . 4 , n = 55 ) for expanded cells ( electronic supplementary material , figure S2 ) . This implies a 1 . 9x ( s . e . 0 . 1 , s . d . 0 . 6 ) worse localiz - ation precision in SExY compared to conventional PALM . This change can be attributed to the reduced brightness of individual spots due to imaging in a larger depth of several micrometres to which our set - up is not adapted to . However , the 1 . 9x worse localization precision is compensated by the 4 . 9x expansion , which leaves us with an overall increase in optical resolution of 2 . 5x when going from conventional PALM imaging to SExY on our set - up . By using a set - up better suited for imaging at depth , this loss could be compensated further . Altogether , these quality measures indicate that the SExY protocol is a promising approach to map protein structures in fission yeast . To achieve this , we have optimized the cell wall digestion , gelation and protein digestion steps in the develop - ment of the final SExY protocol . We report on these optimizations and discuss their rationale in detail in the following sections . 3 . 1 . Efficient and complete cell wall digestion of fission yeast We tested various cell wall removal enzymes commonly used in fission yeast studies [ 58 \u2013 60 ] and visualized expanded S . pombe cells that cytosolically expressed mEos2 . We found that zymolyase , zymolyase in combination with lysing enzyme , snail enzyme and lyticase caused partial expansion of yeast cells visible by \u2018 hour - glass \u2019 like shapes failing to expand the septum or earlier cytokinesis sites ( figure 2 a ) . Lallzyme MMX , chitinase and \u03b2 - glucoronidase completely expanded S . pombe cells and showed comparable mEos2 signal to the non - expanded cells ( figure 1 b ; electronic supplementary material , table S3 ) . The fact that we were able to fully expand fission yeast cells using a chitinase for cell wall digestion may indicate that chitin is present at the septum . The fission yeast septum consists of a primary septum composed predomi - nantly of \u03b2 - ( 1 , 3 ) - glucan and a flanking secondary septum composed of \u03b1 ( 1 , 3 ) - glucan and branched \u03b2 ( 1 , 6 ) - glucan [ 61 , 62 ] . Chitin synthase genes ( chs1 , chs2 ) are present , but their function is still controversial [ 63 , 64 ] . On the one hand , chs2 has been shown to be localized to the growing septal edges in vegetative S . pombe cells , yet chs2 possesses several mutations at sites critical for chitin synthesis [ 64 \u2013 66 ] . We decided not to further investigate the \u2018 hourglass - like pheno - types \u2019 and the potential localization of chitin at the septum and instead proceeded to the next steps of protein digestion and gelation to be optimized , using Lallzyme MMX as our standard for cell wall digestion , as it was the cheapest and most reliable alternative among the three enzymes that resulted in complete expansion in our hands . In a recent study , S . pombe was successfully expanded using zymolyase for cell wall removal [ 67 ] . We speculate that this may be explained by the generally harsher conditions of this proto - col , e . g . an additional fixation step with ice - cold acetone and replacement of proteinase K digestion with SDS treat - ment , which may be at the expense of protein retention yield , but this was not specified . 3 . 2 . Increasing the protein retention yield To evaluate how much fluorescent signal of the fluorescent proteins is retained after each step of sample preparation , we measured the fluorescent spot intensity of the mScarlet - I - tagged spindle pole protein sad1 , whose structure and protein copy numbers is well defined during anaphase B in mitosis [ 68 ] . For our initial protocol , following the proExM protocol with an added step for cell wall digestion using Lall - zyme MMX , only 22 % ( s . e . 2 . 0 % , s . d . 12 . 4 % ) of the signal was retained compared to chemical fixed samples ( figure 1 e ) . This overall loss of fluorescent protein signal was mainly caused by three processing steps : A decrease of 18 % ( s . e . 2 . 8 % , s . d . 29 . 6 % ) after permeabilization with 0 . 1 % Triton X - 100 , a decrease of another 22 % ( s . e . 2 . 5 % , s . d . 23 . 9 % ) after cell wall digestion using Lallzyme MMX compared to the pre - vious step in the protocol and a decrease of another 46 % ( s . e . 2 . 0 % , s . d . 12 . 4 % ) after protein digestion ( electronic sup - plementary material , figure S3 ) . We therefore targeted these three steps for optimization of the protocol . In a first optimization , we excluded a separate permeabi - lization step using Triton X - 100 as we hypothesized that the cell membrane is sufficiently disintegrated combining the steps of fixation with 3 . 2 % paraformaldehyde and 0 . 1 % glu - taraldehyde and of protein digestion in a buffer containing Triton X - 100 . Cells expanded with a protocol excluding this step indeed expanded normally ( examples in figure 1 ) . Fur - thermore , we tested proteinase K as well as the different enzymes for cell wall removal for their proteinase activity r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 7 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 in a colorimetric assay measuring tyrosine release in casein digestion ( figure 2 b and electronic supplementary material , figure S4 ) . For proteinase K , we found that the proteinase activity for 1 U ml \u2212 1 of our stock was reduced to only 64 % for freshly bought proteinase K and lower for older stocks . A control with protease inhibitor PMSF showed no residual proteinase activity . Therefore , for all further experiments , we determined the effective proteinase K activity using the colorimetric assay and adjusted the used concentrations accordingly . Among the cell wall removal enzymes , only the lysing enzyme and Lallzyme MMX showed proteinase activity , whereas zymolyase and \u03b2 - glucoronidase showed no activity at 1 U ml \u2212 1 and even at higher concentrations of 50 U ml \u2212 1 . Compared to proteinase K at a 10 - fold higher con - centration , lysing enzyme at a concentration of 0 . 1 U ml \u2212 1 showed rather strong activity . For Lallzyme MMX , we detected a ( low ) proteinase activity at 10 mg ml \u2212 1 . Impor - tantly , we found no significant decrease in fluorescence intensity of sad1 - mScarlet - I after the exclusion of a separate permeabilization step with Triton X - 100 ( electronic sup - plementary material , figure S3b ) . We thus speculate that Lallzyme MMX without the detergent step at this early stage of the protocol might be hindered to freely access the cell , in particular the nucleus , and thus does not show visible proteinase activity on sad1 - mScarlet - I fluorescence . When adjusting the proteinase K concentration for protein digestion ( see below ) , we nevertheless could measure an effect when blocking the proteinase activity of Lallzyme MMX by PMSF . In general , different proteinase K activities in different laboratories could be one cause for inhomogeneous expansions [ 20 , 28 \u2013 31 ] . On the basis of excluding a separate permeabilization step and using 10 mg ml \u2212 1 of Lallzyme MMX for cell wall digestion , we then optimized protein digestion and tested different methods that have been reported in the ExM literature : protein digestion using proteinase K [ 9 ] , heat denaturation [ 10 ] and denaturation using SDS [ 19 ] . Hom - ogenization by heat denaturation or treatment with SDS resulted in expanded fission yeast cells , but no or very low fluorescent signal for sad1 - mScarlet - I or cytosolically expressed mEos2 was subsequently detected ( data not shown ) . Using the proteinase K concentration from the proExM protocol , we were able to expand and image fluores - cently labelled fission yeast , albeit with severe signal loss . As sufficient sample homogenization is critical for isotropic expansion , we tested different proteinase K concentrations to determine the minimal needed concentration of use . We found that the lowest effective concentration that allowed iso - tropic expansion on both , the macro - and the microscale of the sample was 3 U ml \u2212 1 ( controlled for efficiency by the snail enzyme lyticase zymolyase + lysing enzyme 5 \u00b5m released tyrosine ( \u00b5mol ) (cid:2) 0 . 5 0 0 . 5 1 . 0 1 U ml \u2013 1 proteinase K 1 U ml \u2013 1 proteinase K + 20 mM PMSF 1 U ml \u2013 1 zymolyase 50 U ml \u2013 1 zymolyase 1 U ml \u2013 1 (cid:3) - glucoronidase 50 U ml \u2013 1 (cid:3) - glucoronidase 0 . 1 U ml \u2013 1 lysing enzyme 0 . 1 U ml \u2013 1 lysing enzyme + 20 mM PMSF 10 mg ml \u2013 1 lallzyme MMX ( a ) cell wall digestion zymolyase ( b ) effective proteinase activity 0 0 . 05 0 . 10 0 . 20 0 . 30 glutathione ( % ) 0 . 40 0 . 50 0 500 1000 1500 2000 f l uo r e s ce n ce s po t i n t e n s it y ( a r b . un it s ) gel rigidity ( c ) gelation n o g e l a ti o n n = 34 n = 143 n = 34 n = 35 n = 35 Figure 2 . Protocol optimizations for SExY ( a ) Examples of incomplete cell wall digestion using zymolyase , snail enzyme , a combination of zymolyase and lysing enzyme and lyticase . All tested conditions lead to a partial and non - isotropic expansion constrained by remaining cell wall . ( b ) The proteinase activity of proteinase K ( red ) in comparison to different cell wall digesting enzymes measured by the release of tyrosine during casein digestion . Zymolyase ( blue ) , \u03b2 - glucoronidase ( green ) , lysing enzyme ( yellow ) and Lallzyme MMX ( magenta ) . Adding proteinase inhibitor PMSF successfully suppressed tyrosine release . ( c ) sad1 - mScarlet - I signal shows an increase of 27 % ( s . e . 12 . 8 % , s . d . 85 % ) in FP retention when adding 0 . 05 % glutathione during gelation . Gel rigidity decreases with increasing glutathione concentration . Thus , no gel formation when adding 0 . 4 % glutathione or higher was achieved . r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 8 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 activity assay ) . Lower concentrations of proteinase K or a cell wall digest of Lallzyme MMX in the presence of the protein - ase inhibitor PMSF followed by 3 U ml \u2212 1 proteinase K during protein digestion resulted in heterogeneous samples with a symptomatically increasing fraction of non - expanded cells cumulating at the bottom of the gel , only semi - expanded ( two - to threefold expansion ) or non - isotropic expanded cells , and only partially expanded gels ( data not shown ) . For the final SExY protocol , we thus use the protease activity of 3 U ml \u2212 1 proteinase K combined with the protease activity of Lallzyme MMX which represents the minimum protein digestion level for homogeneous expansion of fission yeast to maintain a robust ExM protocol while retaining the maximum possible fluorescence signal . In a final optimization , we then focused on the gelation step of the protocol as the free radicals that emerge during the gelation of the PAA gel have been shown to reduce the protein retention yield as well [ 23 , 33 ] . While we were not able to detect this loss in the initial protocol , we could detect the negative effect of the gelation process on protein retention after having optimized the other protocol steps ( figure 2 c ) . To minimize the loss , we tested whether a pre - sumably milder gelation by adding the antioxidant glutathione ( GSH ) , an efficient radical scavenger , would yield a higher retention yield , since glutathione can accept a radical at its sulfur moiety by hydrogen atom transfer ( GS ) , adding a hydrogen at the former radical site of proteins [ 69 , 70 ] . To test this hypothesis , we measured the fluorescence intensity of sad1 - mScarlet - I after PAA gelation for different glutathione concentrations and found a fluorescence increase of about 27 % ( s . e . 12 . 8 % , s . d . 85 % ) when adding 0 . 05 % gluta - thione ( figure 2 c ) . At higher glutathione concentrations , no further increase in fluorescence spot intensity but a decrease in PAA gel rigidity was observed until gelation was comple - tely abolished at concentrations of 0 . 4 % glutathione and higher ( figure 2 c ) . Presumably , the lower PAA gel rigidity stems from overall shorter PAA chains in the gel mesh , e . g . by glutathione reacting with growing PAA radical chains and thus terminating the chains early or by glutathione rad - icals ( GS ) forming glutathione disulfide ( GSSG ) , decreasing the overall radical concentration . In the final SExY protocol , we thus use 0 . 05 % glutathione during gelation . Finally , during this work , we tested several commonly used FPs for their fluorescence retention in ExM sample prep - aration as it is a known problem that chromophores often do not withstand treatments in correlative imaging protocols , e . g . when combining fluorescence microscopy with electron microscopy [ 71 ] . For conventional fluorophores , we tested GFP and mCherry , commonly used FPs of different origin from jellyfish and coral , in a dual - colour strain expressing two nuclear pore protein fusions [ 72 ] . While GFP - Nup132 was clearly visible , we could not detect mCherry - Nup131 in the expanded cells ( figure 3 d ) . Interestingly , we then found that mScarlet - I , a synthetic FP constructed by rational design based on several red FPs but with a high sequence identity of 86 % to mCherry , survives the protocol and we could obtain images of expanded cells with mScarlet - I labelled spindle pole protein sad1 ( figure 3 e , f ) . Owing to mScarlet - I \u2019 s superior brightness and red colour ( allowing to image deeper into the samples than when using GFP ) , we decided to use it for the characterization of protein retention ( figure 2 ) [ 73 ] . For PALM imaging , mainly FPs from Anthozoan corals , either DsRed - type like PAmCherry or Kaede - type like the Eos / Dendra / Maple FP family , are used [ 40 ] . We tested photoactivatable PAmCherry which showed no residual fluorescence already after fixation ( data not shown ) . From previous studies , it is known that PAmCherry photoactivation is substantially reduced for fixation protocols including glutaraldehyde [ 74 ] . Within the ExM protocol , a comparably high glutaraldehyde concentration is used . We thus hypothesize that PAmCherry was fully quenched by glutaraldehyde when fixing the samples . By contrast , the green - to - red photoconvertible FP mEos2 survives the ExM protocol just fine . We therefore tagged different low and high abundant proteins with different cellular localizations with mEos2 to validate our imaging sensitivity and FP survival during SExY imaging . Using mEos2 , we could suc - cessfully super - resolve proteins in expanded cells such as the nuclear DNA binding protein cbp1 ( figure 3 b ) [ 75 ] and the nuclear protein mis16 ( figure 3 c ) [ 76 ] as well as rather low - abundant proteins such as the centromere - specific his - tone protein cnp1 ( figure 3 e , f ) , which is only present in several tens to a few hundred copies per cell [ 41 , 77 ] . Using TO - PRO - 3 Iodide , we co - visualized DNA ( figure 3 c , e ) . On a small technical note , we also explored different drift correction strategies for PALM imaging of expanded gels . Drift correction of PALM data is commonly needed , as the recording of a PALM movie can take up to several minutes in which the sample can physically drift . This drift is often corrected by adding fiducial markers to the sample , e . g . flu - orescent polystyrene beads or gold nanoparticles [ 78 ] . For expanded gels , we found that fiducial markers ( even when covalently linking them to the gel mesh by amine groups ) to a large extend either ended up at the bottom of the expanded gel and in case of polystyrene beads , high amounts of individual dyes leached out into the gel and resulted in cells coated by fuzzy highly fluorescent background signal ( data not shown ) . Thus , we decided to use cross - correlation drift correction [ 79 ] , which was previously also used in ExSTORM and Ex - SMLM [ 36 , 37 ] . While cross - correlation approaches work well for large statistics , thus directly on samples with high abundant proteins such as e . g . microtu - bules ( as imaged in the ExSTORM and Ex - SMLM works ) , the correction of low - abundant protein signal fails due to insufficient statistics . Therefore , we mixed all samples with cells that cytosolically expressed mEos2 at high amounts and used the latter as markers for cross - correlation . Even at this for microbial PALM studies rather high level of several thousands of localizations per cell per movie , available cross - correlation approaches that cross - correlate sub - stacks of data fail to work reliably in many cases due to still too low statistics . Therefore , we implemented an additional drift correction method that determines the relative frame - to - frame shift between frames using tracked data of the cyto - solically expressed mEos2 ( supplementary code and https : / / github . com / Endesfelder - Lab ) . 4 . Conclusion With the combination of ExM with PALM ( ExPALM ) , we close a gap in available super - resolution and ExM correlative protocols . We report the homogeneous expansion of fission yeast S . pombe cells to a fivefold expanded size while retaining about 50 % of FP signal by several optimization steps of the original proExM protocol . The final protocol , which we r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 9 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 termed SExY ( Single - molecule and Expansion Microscopy of Yeast ) is robust and reproducible , and extends the ExM tool - box by a correlative protocol for ExPALM imaging in microbiology . SExY explicitly excludes a separate permeabili - zation step with a detergent , uses Lallzyme MMX for digestion of the cell wall as an alternative to pricier enzymes and / or lysing components that only show heterogeneous or partial expansion ( i . e . most striking \u2018 hour - glass \u2019 shaped phe - notypes ) , and includes a fine - tuned protein digest using proteinase K at a controlled effective potency of 3 U ml \u2212 1 and a gentler PAA gelation by adding 0 . 05 % glutathione . We suppose that the improved microscale isotropy in expan - sion as seen in SExY ( figure 1 d ) stems from the dual effect of optimized homogeneous protein digestion and overall shorter PAA chains due to the milder gelation . SMLM imaging has high demands for labelling specificity and efficiency . SExY as a correlative PALM imaging tech - nique is especially suitable for low - abundant proteins as it does not rely on indirect target labelling via organic dyes using immunofluorescence and covalent bioconjugation methods ( which are prone to unspecific staining due to the charge and molecular crowding in microbes [ 41 ] ) and is opti - mized for a high protein retention yield retaining 50 % of the FP signal . Cells expanded by the SExY protocol show a high fivefold expansion compared to Ex - SMLM and ExSTORM that both report only a threefold expansion , which can be attributed to the needed photoswitching buffers . Thus , for some research , using SExY as an ExPALM method might diminish the need for additional expansion steps of the sample in iterative ExM protocols . Possible future optimizations should be to implement a non - radical , highly defined lattice - like gel into the method , as recently introduced by the Tetragel [ 33 , 80 ] . This might lower loss of FP signal during gelation . Second , the range of suitable FPs should be extended . As the selection of FPs suitable for high - quality PALM imaging is rather small [ 2 , 40 ] , we believe that engineering optimized FP variants for correlative ExM approaches based on current FPs might be an effective strategy . Engineering FPs that are not further affected by ExM protocol steps ( e . g . that are less sensitive ( a ) ( b ) ( c ) ( d ) ( e ) ( f ) ( i ) GFP - Nup132 cytosolic mEos2 mEos2 - mis16 cbp1 - mEos2 1 \u00b5 m sad1 - mScarlet - I mEos2 - cnp1 DNA i ) sad1 - mScarlet - I mEos2 - cnp1 5 \u00b5m 5 \u00b5m 5 \u00b5m 5 \u00b5m 1 \u00b5m 1 \u00b5m DNA Figure 3 . Examples of SExY microscopy SExY imaging of high ( a \u2013 c ) and low ( d \u2013 f ) abundant proteins in fission yeast . Super - resolved targets are marked in italic , targets imaged by conventional epifluorescence in regular script . ( a ) mEos2 expressed in the cytosol , ( b ) nuclear DNA binding protein cbp1 , ( c ) nuclear protein mis16 and DNA ( TO - PRO - 3 Iodide ) , ( d ) nuclea pore protein Nup132 , ( e , f ) centromeric histone protein cnp1 relative to the spindle pole body protein sad1 ; combined with visualizing DNA ( TO - PRO - 3 Iodide ) ( e , inset i ) ) . r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 10 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024 to a specific protein digestion method ) offers a significant advantage [ 81 ] . This strategy has been shown successful for other correlative methods ( e . g . in the development of mEos4 FPs ) . Here , nucleophilic amino acid residues that cross - link with aldehydes were replaced so that mEos4 var - iants tolerate osmium ( OsO4 ) fixation \u2014 a treatment needed for electron microscopy [ 82 ] . Third , microscopes should be set - up for imaging at 50 \u2013 80 \u00b5m depths to limit losses in local - ization precision that run counter to the gain in resolution provided by sample expansion . Overall , the new correlative protocol is valuable for all yeast researchers who wish to combine ExM imaging with super - resolution imaging . In particular , we believe that the optimizations in protein retention yield , encompassing reten - tion yield for fluorescent proteins , will also be of interest to a larger audience in microbial cell biology research and will extend to even more distant research areas if their interest involves studying low abundance proteins or for any reason relies on genetic labelling , e . g . when proteins are not readily accessible by extrinsic staining . Ethics . This work did not require ethical approval from a human subject or animal welfare committee . Data accessibility . Raw image data can be obtained from the authors on request . Custom relative frame - to - frame drift correction code is added as supplementary code and provided in the electronic sup - plementary material [ 83 ] as well as on github https : / / github . com / Endesfelder - Lab / frametoframedrift . Declaration of AI use . We have not used AI - assisted technologies in creating this article . Authors \u2019 contributions . I . V . : data curation , investigation , methodology , supervision , validation , visualization , writing \u2014 original draft , writ - ing \u2014 review and editing ; O . D . C . : conceptualization , data curation , formal analysis , funding acquisition , investigation , methodology , software , supervision , validation , visualization , writing \u2014 original draft , writing \u2014 review and editing ; M . A . H . : data curation , investi - gation , methodology ; U . E . : conceptualization , formal analysis , funding acquisition , investigation , methodology , project adminis - tration , resources , software , supervision , validation , visualization , writing \u2014 original draft , writing \u2014 review and editing . All authors gave final approval for publication and agreed to be held accountable for the work performed therein . Conflict of interest declaration . Authors declare no competing interests . Funding . This work was supported by funds from the Max Planck Society , the SYNMIKRO Post - Doc short - term fellowship to O . C . and start - up funds at Carnegie Mellon University . Acknowledgements . We thank Dr David Lando , Dr David Virant and Prof . Dr Haruhiko Asakawa for sharing their strains for this study . References 1 . Abbe E . 1873 Beitr\u00e4ge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung . Archiv f . mikrosk . Anatomie 9 , 413 \u2013 468 . ( doi : 10 . 1007 / BF02956173 ) 2 . 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( doi : 10 . 1038 / nprot . 2017 . 017 ) 83 . Vojnovic I , Caspari OD , Ho \u015f kan MA , Endesfelder U . 2024 Combining single - molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level . Figshare . ( doi : 10 . 6084 / m9 . figshare . c . 7007803 ) r o y a l s o c i e t y pub l i s h i ng . o r g / j o u r n a l / r s o b O p e n B i o l . 14 : 230414 13 D o w n l o a d e d fr o m h tt p s : / / r oy a l s o c i e t ypub li s h i ng . o r g / o n 28 A p r il 2024", + "wilsonEffectsBiologicalExamples2010": "The e\ufb00ects of biological examples in idea generation Jamal O . Wilson 1 and David Rosen , Woodru\ufb00 School of Mechanical Engineering , Georgia Institute of Technology , Atlanta , GA 30332 - 0405 , USA Brent A . Nelson 2 and Jeannette Yen , Center for Biologically Inspired Design , Georgia Institute of Technology , Atlanta , USA In engineering design , analogies can be used in conceptual design to aid in generating new and novel design ideas . In this paper , a cognitive study was performed to assess the impact of biological examples , which serve as surface dissimilar analogies , in the idea generation process during conceptual design . In this study , participants were exposed to biological examples during the idea generation process . These results were then compared to those of participants receiving no examples and to those receiving human - engineered examples . The results suggest that exposure to biological examples in idea generation can increase the novelty of design ideas generated after exposure without inhibiting the variety of the design ideas generated , unlike human - engineered examples which resulted in decreased variety . (cid:2) 2009 Elsevier Ltd . All rights reserved . Keywords : innovation , design techniques , conceptual design , creative design , evaluation I n the conceptual design phase of the engineering design process ( Pahl & Beitz , 1996 ) , the designer is tasked with searching for novel and innovative solutions to engineering problems . However , humans are imperfect search engines ( Busby & Lloyd , 1999 ) and tend to focus on a narrow range of solution approaches and overlook many valuable solutions ( Perttula , 2006 ) . According to the theory of bounded rationality ( Simon , 1976 , 1983 , 1996 ) , the space searched is bounded by the limited cognitive abilities of the designer ( Schild , Herstatt , & Luthje , 2004 ) . To overcome this limitation , designers often employ various techniques to aid in idea generation , which are listed elsewhere ( Pahl & Beitz , 1996 ) . These techniques aid the designer in expanding and exploring her / his design space more e\ufb03ciently . In engineering design , analogies are often used in idea generation to transfer knowledge through analogical mapping from a source domain containing the analogous phenomena to a target domain containing the problem to be solved by the analogy ( Mak & Shu , 2004 ) . Analogies can be classi\ufb01ed by the similarity , or the conceptual distance , between the source and target Corresponding author : Jamal O . Wilson jamal _ o _ wilson @ whirlpool . com www . elsevier . com / locate / destud 0142 - 694X $ - see front matter Design Studies 31 ( 2010 ) 169 e 186 doi : 10 . 1016 / j . destud . 2009 . 10 . 003 169 (cid:2) 2009 Elsevier Ltd . All rights reserved . domain . Local analogies are analogies where the source domain is similar to that of the target , where surface - level attributes and relations between these attributes can be easily mapped ( Dahl & Moreau , 2002 ) . Surface - level attributes are easily retrievable aspects of representation , such as color and shape ( Gentner & Markman , 1997 ) . In distant analogies , the source and target domains are very di\ufb00erent and are surface dissimilar . In this case , few surface - level attributes can be mapped and structural similarity must be relied upon ( Dahl & Moreau , 2002 ) . For illustration , consider the design of an air condi - tioning system for a hotel . A local , or surface similar , analog would be consid - ered commercial air conditioning systems from other companies , whereas a distant , or structurally similar , analogy would be self - cooling termite mounds . In the distant case , the termite mounds and commercial air condi - tioning systems share no surface - level attributes and are considered surface dissimilar . However , at the structural level , they both are analogous in provid - ing a cooling function . Tseng and co - authors ( Tseng , Moss , Cagan , & Kotovsky , 2008 ) studied the role of timing and similarity in the idea generation process . The authors found that distantly related information presented after participants had begun problem solving increased both the novelty and number of functionally dis - tinct solutions . This agrees with other work in the literature where the number of distant analogies used during design was positively correlated with the originality ( novelty ) of the resulting design ( Dahl & Moreau , 2002 ) and these distant analogies are considered the main drivers of innovative thought ( Ward , 1998 ) . However , the use of distant analogies does have challenges . Because distant analogies are surface dissimilar and involve analogous relationships at the structural level , it is often di\ufb03cult to transfer knowledge or solutions ( Johnson - Laird , 1989 ) . These analogies are more di\ufb03cult to access than local analogies because the nature of the similarity is at a more abstract , relational level , thus causing an increase in cognitive e\ufb00ort ( Dahl & Moreau , 2002 ) . In this work , we extend the work of Tseng and co - authors ( Tseng et al . , 2008 ) and prior literature by ( 1 ) testing the e\ufb00ect of surface dissimilar analogies in the form of biological systems and ( 2 ) more completely evaluating the e\ufb00ect of these analogs on the ideation process . In this research , we believe that biological examples provide a source of structurally similar ( and surface dis - similar ) analogies . Biologically inspired design ( BID ) describes the process of transferring solution approaches embodied in biological adaptations into other domains , such as engineering , materials science , and design . Because millions of years of evolution re\ufb01ned these locally optimized adaptations , such solutions may be useful for engineers in their search for energy - e\ufb03cient answers to similar problems raised by their technologies ( Vincent , 2002 ) . By adapting principles from these natural solutions , engineers have developed many innovative and novel products , including Velcro based on the hooked seeds of the burdock plant ( Velcro , 1955 ) , dry reusable tape based on Gecko 170 Design Studies Vol 31 No . 2 March 2010 feet ( Autumn et al . , 2002 ) and self - cleaning paint based on the leaves of the lotus plant ( Barthlott & Neinhuis , 1997 ) . Several researchers have begun de - veloping systematic approaches for leveraging biological examples in engineer - ing design , including functional keyword searches through biological literature ( Chiu & Shu , 2004 , 2005 ) , data repositories containing biological examples ( Bruck et al . , 2006 ; Chakrabarti , Sarkar , Leelavathamma , & Nataraju , 2005 ; Vincent , 2002 ; Vincent , Bogatyreva , Bogatyrev , Bowyer , & Pohl , 2006 ; Vincent & Mann , 2002 ; Yim , Wilson , & Rosen , 2008 ) , BID methods ( Benyus & Baumeister , 2007 ; Wilson & Rosen , 2007 , 2008 ) , and cognitive analyses of BID ( Helms , Vattam , & Goel , 2009 ) . However , there has been little empirical evidence on the value of bio - inspired techniques and the cognitive mechanisms behind these techniques in idea generation . In this work , we evaluate the e\ufb00ect of surface dissimilar analogies in the form of biological examples on the ideation process . Theories on the e\ufb00ects of expo - sure to examples in idea generation have converged on a dual in\ufb02uence model of both negative ( design \ufb01xation ) and positive ( cognitive stimulation ) e\ufb00ects ( Perttula , 2006 ) . Design \ufb01xation refers to design conformity that results from exposing designers to example solutions . Cognitive stimulation refers to the stimulation of new ideas that occurs as a result of exposure to the ideas of others . Jansson and Smith found that participants generated a larger amount of ideas containing features of examples to which they were exposed than did a control group not exposed to the examples ( Jannson & Smith , 1991 ) . Similar results were found in studies by Purcell and co - authors ( Purcell & Gero , 1996 ; Purcell , Williams , Gero , & Colbron , 1993 ) , but only when the principles from the example problem were considered to involve the same knowledge base as the expertise of the mechanical engineering participants ( Purcell & Gero , 1996 ; Purcell et al . , 1993 ) . In contrast , students in industrial design did not show these conformity e\ufb00ects ( Purcell & Gero , 1996 ; Purcell et al . , 1993 ) . Several other studies have shown similar conformity e\ufb00ects ( Marsh , Bink , & Hicks , 1999 ; Marsh , Landau , & Hicks , 1996 ; Ward , 1993 ) . Exposure to examples can also result in cognitive stimulation , thus having a positive in\ufb02uence on idea generation . In cognitive stimulation , the knowl - edge embodied in the examples stimulate ideas that designers would not other - wise have accessed ( Perttula , 2006 ) . In a study on idea generation in groups , Paulus and Yang found enhanced performance in group brainstorming due to cognitive stimulation ( Paulus & Yang , 2000 ) . Nijstad found that partici - pants exposed to ideas from a wide variety of categories surveyed more cate - gories of ideas than participants not exposed to any ideas or participants exposed to ideas from fewer categories ( Nijstad , Stroebe , & Lodewijkx , 2002 ) . The authors also argued that design problems with larger design spaces were more likely to show these stimulation e\ufb00ects than ones with smaller spaces . Dugosh also found stimulation e\ufb00ects in group brainstorming and The e\ufb00ects of biological examples in idea generation 171 increased amounts in cases where participants were asked to attend to the ideas of others ( Dugosh , Paulus , Roland , & Yang , 2000 ) . The main goal of this research was to examine the e\ufb00ect of surface dissim - ilar analogies in the form of biological examples on the ideation process . Speci\ufb01cally , the impact of analogies on design space exploration and expan - sion was quanti\ufb01ed . De\ufb01ned metrics ( Nelson , Wilson , Rosen , & Yen , 2009 ; Shah , Vargas - Hernandez , & Smith , 2002 ) relating the novelty and variety of ideas generated in ideation to the expansion and exploration of the design space were used . These metrics for novelty and variety were used to assess the value of surface dissimilar analogies in the form of biological examples in ideation . To examine this , an experimental study was conducted in which participants were exposed to biological examples in the idea generation pro - cess . These results were then compared to that of participants receiving no examples and to those receiving surface similar examples . 1 Hypotheses This study tested the following speci\ufb01c hypotheses : ( 1 ) exposure to surface dissimilar analogies in the form of biological design examples in ideation will increase the novelty of design ideas generated and ( 2 ) exposure to surface dissimilar analogies in the form of biological design examples in ideation will increase the variety of design ideas generated . 2 Experimental methods To test the hypotheses presented in Section 2 , a study was performed in which participants generated conceptual designs to solve con\ufb02icting design require - ments . In testing hypotheses 1 and 2 , participants were placed into one of three potential conditions : ( 1 ) a condition in which participants received a surface dissimilar biological example after some time in ideation , ( 2 ) a condition in which participants received a surface similar human - engineered example after some time in ideation , and ( 3 ) an un - aided condition where no example was presented . The un - aided condition established a baseline for un - aided concept generation . The surface similar condition established a baseline for concept generation using easily accessible , human made design examples , and the sur - face dissimilar condition assessed the e\ufb00ect of biological design examples in concept generation . At the onset of the study , participants were given a design problem , several sheets of paper , and a single pen color by the experimenters . Participants were randomly assigned to one of the experimental conditions . The assigned condition was designated by the color paper the participants received , al - though the participants were unaware of this . After instruction and time for questions , the participants were given 20 min of un - aided concept gener - ation time , then given their respective design aids if applicable , and then given another 20 min for further concept generation . At the beginning of 172 Design Studies Vol 31 No . 2 March 2010 the study , the participants were verbally instructed to generate and label distinct design ideas , but no instructions were given regarding quantity or creativity of the design ideas so as not the bias the participants . Participants were also given a new color of pen for the second 20 min of idea generation to enable the researchers to distinguish between designs generated during the initial 20 min and those from the second 20 min . When the participants were given their respective design aids , they were verbally instructed that the design aids may or may not prove useful to them and that they were not re - quired to use the materials . To test our hypotheses , comparisons were made both within groups and be - tween groups . The within - group analysis compared the design ideas generated before and after exposure to the design example , while the between - group analysis compared the ideas generated after exposure to the design example across each of the di\ufb00erent conditions . 3 Leg immobilization versus portability 3 . 1 Participants Twenty - six undergraduate mechanical engineering students from our institu - tion participated in Study 1 . The students were recruited from a senior level capstone design course . To be eligible for the course , students needed at least 3 years of undergraduate engineering coursework and at least one semester of a formal undergraduate design course . All participants were volunteers and did not receive payment or additional course credit for participation in the study . 3 . 2 Materials All participants were asked to individually solve the same design problem . The design problem given in this study , displayed in Figure A - 1 of the Appendix , asked participants to solve a design problem with con\ufb02icting design require - ments of leg immobilization and portability . 3 . 3 Experimental procedure The design study was carried out in a classroom setting in the second meeting of the senior capstone design course . At the onset of the study , participants were given the design problem , several sheets of paper , and a black pen by the experimenters . Participants were assigned to either the biological ( N \u00bc 9 ) , the human - engineered ( N \u00bc 9 ) , or the un - aided ( N \u00bc 8 ) condition in an alternating fashion based on seating location . Speci\ufb01c procedures for each condition were as follows . 3 . 3 . 1 Biological condition Participants in the biological condition were given a biological design ex - ample at the midpoint of the study that described the variable - sti\ufb00ness be - havior of the mutable connective tissue of the sea cucumber . The design The e\ufb00ects of biological examples in idea generation 173 example included a pictorial and textual description of the biological sys - tem and is displayed in Figure A - 2 of the Appendix . 3 . 3 . 2 Human - engineered condition Participants in the human - engineered condition received a design example with a pictorial and textual description of a human - engineered system with similar behavior to that of the biological system . The design example described the variable - sti\ufb00ness behavior of electro - rheological \ufb02uids and is displayed in Figure A - 3 of the Appendix . The human - engineered and biological examples were intentionally chosen to match each other very closely in functional principles . 3 . 3 . 3 Un - aided condition After the initial 20 min of concept generation , participants in the un - aided condition received no design example and were instructed to continue with concept generation after receiving a new pen color . After the allotted 40 min , the students were stopped and given a post survey to evaluate their prior knowledge and experience as well as their understanding of the design examples given to them . Figure 1 displays some examples of design solutions generated in this study . 3 . 4 Data analysis The impact of surface dissimilar examples on idea generation was measured by evaluating the novelty and variety of the conceptual designs generated by the participants . The novelty indicated the degree to which exposure to examples aided designers in expanding their design space , while the va - riety indicated how examples a\ufb00ected the degree to which designers ex - plored their design space ( Shah et al . , 2002 ) . Metrics for novelty and variety were assessed using the drawings and descriptions generated by the participants . 3 . 4 . 1 Novelty Novelty was assessed as de\ufb01ned in the literature ( Nelson et al . , 2009 ; Shah et al . , 2002 ) . First , the design problem was decomposed into its key functions . Next , each design idea generated by the participants was categorized on the basis of the principles used to address the key functions and characteristics of the design problem . Finally , the used principles of the designs were given a score corresponding to the degree to which they were unusual . For our study , the score was determined by the frequency of occurrence of the given design principle among the designs generated by all the participants in the \ufb01rst 20 min of the study , before exposure to design examples . This initial group of ideas was considered to be the original design space of the participants since it corresponded to ideas generated before design aids were given , and most stu - dents had stopped generating new ideas by the end of the initial 20 min . 174 Design Studies Vol 31 No . 2 March 2010 Such a measure of novelty by frequency of occurrence was shown to be similar to subjective novelty scores assigned by external judges ( Shah et al . , 2002 ) . The novelty score , N , for an idea with m functions or characteristics was calculated for each design idea using the equation . N \u00bc X m j \u00bc 1 f j T j (cid:2) C j T j \u00f0 1 \u00de where T j is the total number of participants in the study , C j is the total num - ber of participants that utilized the particular design principle in their initial designs , and f j is an optional weighting factor for function j and was set to unity in this study . The average novelty score was then calculated for each in - dividual by averaging the novelty scores for their designs . For example , con - sider the sample set of design concepts displayed in Figure 1 . In the initial idea generation session ( before exposure to examples ) , if many participants gener - ated ideas with the working principle \u2018in\ufb02atable\u2019 , the novelty score would be low ( C j (cid:3) T j ) . Alternatively , if no one generated a design idea containing a working principle of a variable sti\ufb00ness , composite \ufb02uid ( electro - rheological \ufb02uid ) , then the idea would be considered fully novel ( C j (cid:3) 0 ) and get a novelty score of 1 . 3 . 4 . 2 Variety Variety indicates the total design space explored , and was calculated using a genealogy tree of the design ideas ( Shah et al . , 2002 ) . The genealogy tree Figure 1 Sample of design ideas generated by students . ( a ) In\ufb02atable ( b ) multiple - part snap ( c ) electro - rheolog - ical \ufb02uid chambers with power source ( d ) chemically - rigidizable The e\ufb00ects of biological examples in idea generation 175 was constructed based on di\ufb00erentiation between designs at four levels of cat - egorization : physical principles , working principles , embodiment , and detail . While most of the concept of the variety metric was generated by Shah et al . ( Shah et al . , 2002 ) a re\ufb01nement of that metric was used in this study ( Nelson et al . , 2009 ) . Variety , V , was calculated using the equation . V \u00bc X m j (cid:2) 1 f j S 1 \u00f0 b 1 (cid:2) 1 \u00de \u00fe X 4 k \u00bc 2 S k X b k (cid:2) 1 l \u00bc 1 d l ! \u00f0 2 \u00de where f j is a weighting factor for function j , S k is the score given for di\ufb00er - entiation at level k , b k is the number of branches in the tree at level k , and m is the total number of functions . Di\ufb00erentiation between designs at higher levels of the genealogy tree receive a higher weighting , S k , than di\ufb00erentia - tion at lower levels of hierarchy . Weightings of 10 , 5 , 2 , and 1 were used at the physical principle , working principle , embodiment , and detail hierarchy levels , respectively , to ensure that di\ufb00erentiation at higher levels of hierar - chy were valued at least twice that of the level beneath it ( Nelson et al . , 2009 ) . For example , consider again the design solutions from Figure 1 as a single set of ideas . Figure 2 displays the genealogy tree that would represent this set . De - sign ideas ( a ) , ( c ) , and ( d ) have the same physical principle of using \u2018variable sti\ufb00ness\u2019 to satisfy the immobilization vs . portability con\ufb02ict . However , they have di\ufb00erent working principles to achieve variable sti\ufb00ness : ( a ) in\ufb02ation , ( c ) controllable , composite \ufb02uid , and ( d ) chemical hardening . Idea ( b ) uses a separate physical principle , \u2018assembly of multiple parts\u2019 , to solve the con\ufb02ict . The variety score would be calculated as 10 (cid:4) ( 1 node separating the two phys - ical principles ) \u00fe 5 (cid:4) ( 2 nodes separating the 3 working principles having a \u2018variable - sti\ufb00ness\u2019 physical principle ) \u00bc 10 (cid:4) ( 1 ) \u00fe 5 (cid:4) ( 2 ) \u00bc 20 . Note that unlike novelty , the variety score is not normalized by the total number of de - signs , which yields a truer measure of total design space exploration ( Nelson et al . , 2009 ) since variety is a metric that inherently only applies to a set of multiple ideas . ( b ) Assemblage ( a , c , d ) Variable Stiffness ( d ) Chemical hardening ( c ) controllable , composite fluid ( a ) inflation Physical Principle Working Principle Figure 2 Genealogy tree for sample design solutions in Figure 1 176 Design Studies Vol 31 No . 2 March 2010 For this study , two judges , who were also authors on this study , independently coded genealogy trees for the four level categorization scheme based on the idea set generated by the participants . These trees were then compared and showed mostly similar groupings , with one of the trees di\ufb00ering primarily in that it had fewer branches but most of the same descriptions . Unaligned points of the independent trees were discussed and reconciled by discussion and con - sensus between the judges . This helped to assure accuracy in the \ufb01nal master genealogy tree , which was used to calculate the variety metric . The partici - pants\u2019 ideas were then allocated within the genealogy tree and the variety score was calculated . The \ufb01nal genealogy tree used to calculate variety in the study is displayed in Figure 3 . 4 Results The novelty and variety of the design ideas generated by the participants were calculated using the metrics de\ufb01ned in Section 3 . 3 . The results were analyzed using nonparametric Wilcoxon signed - ranked tests and Mann e Whitney U tests at an alpha level of 0 . 05 ( a \u00bc 0 . 05 ) . The more conservative nonparametric tests were chosen because the small sample sizes resulted in non - normally distrib - uted results . 4 . 1 Novelty The \ufb01rst hypothesis stated that exposure to surface dissimilar analogies in the form of biological design examples would lead to design ideas of greater nov - elty . This hypothesis was tested by comparing the novelty scores of the partic - ipants in the biological condition before and after the design example was introduced . These results were then compared to that of the human - engineered and un - aided conditions . The mean and the standard deviation ( SD ) of the novelty scores for the three conditions are displayed in Table 1 . Immobolize Leg / Storage Elastic helix brace wrap Collapsible Rigid Structure tent - pole Sliding Telescoping Other sliding Folding / Ratcheting / Hinging Wooden plates and adjustable rings Shape memory Detachable multiple / segmented crutch rigid / soft elements Sleeve : soft w / rigid inserts Splint : rods / straps Thin / rollable Aluminum tape rolled splint ( Slap / tape meas . ) Variable Stiffness Inflatable Inflatable splint inflatable sleeve Chemical stiff 2 - part fiberglass / bondo epoxy thermal - set foaming cement crystal structure Composite fluid ER stiff Fibrils Dual purpose materials tie legs together gear backpack frame hiking stick scavenge environmental wood splint makeshift stretcher makeshift crutch Medical paralyze leg anesthetic Figure 3 Genealogy tree for the entire set of designs produced in the study The e\ufb00ects of biological examples in idea generation 177 Analysis of the results from Table 1 yields the following : (cid:5) Participants in both the biological and human - engineered condition showed statistically signi\ufb01cant increases in novelty ( W \u00bc 2 , p < 0 . 01 and W \u00bc 0 , p < 0 . 01 , respectively ) after being exposed to the example ; participants in the un - aided condition showed no signi\ufb01cant change in the novelty of their design ideas . (cid:5) Participants in both the biological and human - engineered conditions gener - ated design ideas with higher novelty ( U \u00bc 68 , p < 0 . 01 and U \u00bc 68 , p < 0 . 01 , respectively ) than participants in the un - aided condition . The dif - ference between the novelty scores of participants in the biological and human - engineered condition approached signi\ufb01cance ( U \u00bc 55 , p < 0 . 07 ) . These results supported the hypothesis that exposure to surface dissimilar analogies in the form of biological examples leads to design ideas of greater novelty . The same e\ufb00ect also occurred with participants exposed to the surface similar ( human - engineered ) example . 4 . 2 Variety The second hypothesis examined was that exposure to surface dissimilar anal - ogies in the form of biological examples would lead to a greater variety of de - sign ideas . This hypothesis was tested by comparing the variety of the design ideas produced by the participants before and after the design examples were introduced . Table 2 displays the variety scores of participants in the three conditions . The results from Table 2 are summarized as follows : (cid:5) Participants in the biological and un - aided conditions showed no statisti - cally signi\ufb01cant change in the variety of design ideas during the two phases of the study , while participants in the human - engineered condition showed a signi\ufb01cant decrease ( W \u00bc 0 , p < 0 . 01 ) in the variety of design ideas gener - ated after receiving the example . (cid:5) The variety of design ideas of participants in the biological and un - aided conditions did not di\ufb00er signi\ufb01cantly . The hypothesis that exposure to surface dissimilar analogies in the form of bi - ological examples would lead to a greater variety of design ideas was Table 1 Novelty scores Unaided ( SD ) Bio - inspired ( SD ) Human - engineered ( SD ) Before 0 . 32 ( 0 . 26 ) 0 . 36 ( 0 . 08 ) 0 . 28 ( 0 . 19 ) After 0 . 41 ( 0 . 33 ) 0 . 76 ( 0 . 24 ) 0 . 94 ( 0 . 11 ) 178 Design Studies Vol 31 No . 2 March 2010 unsupported with these results . However , while the biological example did not increase the variety of design ideas relative to the un - aided condition , it did not signi\ufb01cantly decrease the variety of the design ideas , as had resulted in the hu - man - engineered ( surface similar ) condition . 5 Discussion The results from the studies presented in this paper supported the hypoth - eses that exposure to surface dissimilar analogies in the form of biological examples in the idea generation process increases the novelty of design ideas . The results from the study did not support the hypothesis that ex - posure to surface dissimilar analogies in the form of biological examples in the idea generation process would increase the variety of design ideas generated . However , the surface similar examples negatively a\ufb00ected the observed variety , and this did not occur for the surface dissimilar condition . In this work , novelty was de\ufb01ned as the uniqueness of an idea with respect to the un - aided set of ideas generated by the group of participants . Hypothesis 1 stated that exposure to surface dissimilar analogies in the idea generation process would increase the novelty of ideas generated , and the results indi - cated that exposure to biological examples did indeed increase the novelty of ideas generated . This increase in novelty signi\ufb01es that participants were able to generate ideas that otherwise would not have been accessed without exposure to the biological example . A signi\ufb01cant increase in the novelty of participants receiving the surface similar example was also found . The in - crease in novelty cannot be attributed solely to increased ideation time , since participants not receiving any examples showed no increase in the novelty of their design ideas during the second ideation phase . The increase in novelty can be attributed to the use of a non - obvious solution approach in the stimulation examples given at the midpoint of the study . Most students had limited familiarity with variable - sti\ufb00ness materials , and those with \ufb01rst aid training would have been taught about rigid splints already , so the ma - jority of their pre - example design concepts did not utilize the idea of variable sti\ufb00ness . Because the baseline for the novelty calculation was determined from only those concepts generated before the example was given , the later ideas utilizing variable - sti\ufb00ness materials yielded a high novelty score . This implies that merely the presence of external stimulation in the ideation process increases the novelty of the design ideas generated . Shah et al . Table 2 Variety scores Unaided ( SD ) Bio - inspired ( SD ) Human - engineered ( SD ) Before 5 . 8 ( 5 . 6 ) 12 . 1 ( 7 . 8 ) 13 . 3 ( 9 . 3 ) After 5 . 6 ( 6 . 8 ) 9 . 4 ( 7 . 6 ) 2 . 4 ( 3 . 7 ) The e\ufb00ects of biological examples in idea generation 179 ( Shah et al . , 2002 ) correlated this increase in novelty to a broadening of the design space of the designer . The type of analogy ( surface similar vs . surface dissimilar ) was also found to have an e\ufb00ect on novelty . Participants receiving surface similar examples produced more novel ( near signi\ufb01cance ) designs than those receiving surface dissimilar examples . One possible explanation lies in the manner in which participants used the design examples . Because surface dissimilar analogies have a larger conceptual distance to the design problem , participants are less likely to view them as relevant and thus , less likely to utilize them . In this case , participants receiving the surface similar example transferred some attribute of the human - engineered example to 100 % of their design ideas . In contrast , participants receiving the surface dissimilar example only transferred some attribute of the biological example 52 % of the time . Since the use of external stimuli helps increase the novelty of design ideas , this only holds when these external stimuli are utilized . In this case , the larger conceptual distance of the surface dissimilar examples leads to a lower utilization rate versus that of the surface similar examples where attributes can be easily transferred . Hypothesis 2 stated that exposure to surface dissimilar analogies would in - crease the variety of the design ideas generated . This hypothesis was un - supported by the results . The variety before and after exposure to the biological example showed no statistically signi\ufb01cant di\ufb00erence in variety . However , even though exposure to the biological examples did not in - crease the variety of ideas generated , such exposure also did not signi\ufb01 - cantly decrease the variety of the generated ideas . Exposure to examples in idea generation has been shown to cause \ufb01xation ( Jannson & Smith , 1991 ; Purcell & Gero , 1996 ; Purcell et al . , 1993 ) , which decreases the va - riety of the generated ideas . When comparing the variety scores between the bio - inspired and human - engineered condition in Table 2 , participants exposed to surface dissimilar analogies had a signi\ufb01cantly higher variety ( U \u00bc 17 . 5 , p < . 05 ) than participants exposed to surface similar examples . This suggests that the participants exposed to the surface similar example \ufb01xated more so than those exposed to the surface dissimilar example . A possible explanation for this phenomenon can be found by examining the \ufb01xation e\ufb00ect in \ufb01ner detail . As mentioned previously , participants exposed to the surface dissimilar example transferred less attributes from the example problem to their de - sign ideas than participants exposed to the surface similar example . This lower utilization rate leaves way for ideas that are on di\ufb00erent ideation pathways , thus keeping the design space open and fostering variation in the ideas . An additional explanation for the larger variety in participants exposed to surface dissimilar examples can also be found by looking at the 180 Design Studies Vol 31 No . 2 March 2010 level at which attributes were transferred . Knowledge can be transferred from design examples at varying levels of abstraction . Higher levels of ab - straction when transferring from an analogical source would correspond to utilizing the principles and mechanisms from the source ( i . e . structural relationships ) , whereas lower levels of abstraction would correspond to utilizing speci\ufb01c materials and devices from the source ( i . e . surface - level attributes ) . To assess the level of transfer of ideas , the design examples given to the students were \ufb01rst decomposed into their key characteristics ( i . e . physical principles , working principles , etc . ) by the evaluators . Next , the ideas generated by the participants were compared to the char - acteristics of the design example . The number of design ideas that utilized the physical principle and working principle of the example was then de - termined for each participant . The results are displayed in Table 3 . The values in Table 3 represent the average percentage of the participants\u2019 ideas generated after exposure to the example that utilized characteristics of the example at either the physical principle or working principle level of abstraction . As seen in Table 3 , and as mentioned above , participants in the surface sim - ilar condition transferred more characteristics at both the physical princi - ple , and the more basic working principle level of abstraction . The signi\ufb01cance of the results in Table 3 lies in that participants exposed to the surface similar examples transferred signi\ufb01cantly more characteristics ( U \u00bc 61 . 5 , p < 0 . 01 ) at the working principle level of abstraction than those exposed to biological examples . For example , participants in the surface similar condition primarily suggested designs speci\ufb01cally utilizing electro - rheological \ufb02uids whereas participants in the biological condition suggested designs that utilized the general principle of a variable - sti\ufb00ness material . This means that those exposed to the surface similar example transferred more surface - level ( working principle ) characteristics . In this case , \ufb01xation on surface - level characteristics could have constrained the variety of ideas in idea generation . In the surface dissimilar case , transferring principles at a higher level of abstraction ( structural level ) allowed a greater variety of ideas to still be generated . An additional reason that biological analogical sources encourage higher level abstraction is that biological analogies typically use mechanisms and Table 3 Level of transfer scores Level of transfer Bio - inspired Human - engineered Physical principle 52 % 100 % Working principle 19 % 80 % The e\ufb00ects of biological examples in idea generation 181 materials that are not commercially available products . A designer utilizing sea cucumber connective tissue as an analogical source cannot plausibly im - plement sea cucumber connective tissue within a design , and thus the de - signer is forced to understand the design at higher functional levels . In contrast , designers presented with electro - rheological \ufb02uids can implement such \ufb02uids within their designs and are not forced to determine the higher level physical principle e that of a variable - sti\ufb00ness material . The greater do - main distance of the biological example forces analogical transfer at higher levels of abstraction . Participants in both the surface similar and dissimilar cases \ufb01xated on their respective examples , but at di\ufb00erent levels of abstraction . Those receiving surface similar examples \ufb01xated and transferred characteristics from both the surface and structural levels , while those receiving surface dissimilar ex - amples transferred characteristics at the structural level . This \ufb01xation at lower levels constrained the searchable design space and the \ufb01xation e\ufb00ect was greater than that of stimulation . In the case of the surface dissimilar ex - ample , while the participants still \ufb01xated , the higher level of abstraction still allowed stimulation of a broad range of new ideas and outweighing the \ufb01x - ation e\ufb00ect . 6 Conclusions Currently , there is little empirical evidence as to the e\ufb00ects of surface dis - similar analogies from the biological domain in the idea generation process , despite the existence several research e\ufb00orts devoted to the process of ana - logical mapping from the biological domain . The aim of this work was to quantify the value of these techniques in ideation as it relates to design space expansion and exploration . In this study , we describe experimental re - sults for the concept generation demonstrated by senior mechanical engi - neering students exposed to biological examples during the idea generation process . We compared these results to participants receiving no examples as well as to those receiving human - engineered ( surface simi - lar ) examples . The results indicated that , in this case , exposure to biological examples increased the novelty of design ideas generated after exposure without decreasing the variety of design ideas generated . The results also indicated that the type of example used ( surface dissimilar vs . surface sim - ilar ) had a signi\ufb01cant impact on the novelty and variety of design ideas gen - erated . When compared to surface similar examples , the biological examples showed a lower utilization rate , which negatively impacted the novelty of the design idea set . However , this lower utilization rate coupled with the transfer of attributes from the biological example at a high level of abstraction positively impacted the variety of design ideas generated . For this reason , biological examples thus show potential as a resource for iden - tifying surface dissimilar analogies . 182 Design Studies Vol 31 No . 2 March 2010 The results of the work presented in this paper have a number of implications in engineering design . One of the primary goals of engineering design is to dis - cover new and innovative solutions to encountered problems . In the develop - ment of these solutions , idea generation is paramount . Dylla has demonstrated signi\ufb01cant correlation between the amount of design space considered in idea generation and the quality of the \ufb01nal design ( Dylla , 1991 ) . It follows that methods for aiding designers in expanding and exploring their design space should yield better designs . Search strategies that include surface dissimilar analogies in the form of biological examples may help expand the design space of the designer while also limiting some of the restrictive e\ufb00ects of \ufb01xation typ - ically associated with exposure to examples . These results have been found for senior mechanical engineering students , but are anticipated to be extendable beyond the current sample group . The scope of this research was limited to the investigation of biological anal - ogies as a potential source of surface dissimilar analogies . Future research in this area should include an investigation and comparison amongst a variety of sources of surface dissimilar analogies . A larger sample of participants would also be useful in the expanding the validity space of this research . Acknowledgements JOW acknowledges \ufb01nancial support from the O\ufb03ce of Naval Research and the David and Lucille Packard Foundation . 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A systematic method for transferring biological strat - egiesinengineeringdesign : applicationtothedesignofvariable - sti\ufb00nessmaterials , Biological Approaches for Engineering Conference , Southampton , UK . Yim , S . , Wilson , J . , & Rosen , D . ( 2008 ) . Development of an Ontology for Bio - In - spired Design Using Description Logics , International Conference on Product Lifecycle Management , Seoul , Korea . 186 Design Studies Vol 31 No . 2 March 2010", + "sitarska2023sensing": "Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles Ewa Sitarska 1 , 2 , Silvia Dias Almeida 1 , 8 , Marianne Sandvold Beckwith 1 , Julian Stopp 3 , Jakub Czuchnowski 1 , Marc Siggel 4 , 5 , Rita Roessner 4 , 5 , Aline Tschanz 1 , 2 , Christer Ejsing 1 , 6 , Yannick Schwab 1 , Jan Kosinski 4 , 5 , 7 , Michael Sixt 3 , Anna Kreshuk 1 , Anna Erzberger 1 & Alba Diz - Mu\u00f1oz 1 To navigate through diverse tissues , migrating cells must balance persistent self - propelled motion with adaptive behaviors to circumvent obstacles . We identify a curvature - sensing mechanism underlying obstacle evasion in immune - like cells . Speci \ufb01 cally , we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature - sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature . The genetic perturbation of this machinery reduces the cells \u2019 capacity to evade obstructions combined with faster and more persistent cell migration in obstacle - free environments . Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment , allowing them to adaptively decide if they should move ahead or turn away . On the basis of our \ufb01 ndings , we propose that the natural diversity of BAR domain proteins may allow cells to tune their curva - ture sensing machinery to match the shape characteristics in their environment . Cell migration drives many developmental , physiological , and patho - logical processes . While the mechanisms underlying propulsion are largely known , it remains unclear how cells steer their movement to navigate complex and dynamic environments 1 and circumvent obsta - cles within tissues 2 , 3 . These adaptive behaviors are particularly important for fast and dynamic cell types , such as immune cells and disseminating tumor cells . Switching between actin - based protrusions and blebbing has been shown to aid cell steering in development 4 , but cells often display only actin - rich protrusions such as lamellipodia and ruf \ufb02 es . These need to be long - lived enough for cells to explore and then persistently move through their surroundings , while \u2018 unstable \u2019 enough to allow them to adapt and change direction when confronted with an obstacle 5 , 6 . During migration , the outmost boundary of the cell \u2014 the plasma membrane \u2014 is deformed by the changing microenvironment . How - ever , it remains unclear whether membrane curvature encodes infor - mation that cells use to choose their migration path 7 , 8 , or if membrane topography is only a side - effect of the forces acting at the cell surface . Many migrating cells indeed express a variety of curvature - sensing proteins , which can directly interact with both the plasma membrane and the subjacent actin cytoskeleton 9 . In particular , the family of BAR domain proteins could facilitate membrane curvature sensing . These Received : 18 October 2022 Accepted : 22 August 2023 Check for updates 1 Cell Biology and Biophysics Unit , European Molecular Biology Laboratory , 69117 Heidelberg , Germany . 2 Collaboration for joint PhD degree between EMBL andHeidelbergUniversity , FacultyofBiosciences , EMBLandHeidelbergUniversity , Heidelberg , Germany . 3 InstituteofScienceandTechnologyAustria , 3400 Klosterneuburg , Austria . 4 EMBLHamburg , EuropeanMolecularBiologyLaboratory , 22607Hamburg , Germany . 5 CentreforStructuralSystemsBiology , 22607 Hamburg , Germany . 6 Department of Biochemistry and Molecular Biology , Villum Center for Bioanalytical Sciences , University of Southern Denmark , Cam - pusvej 55 , 5230 Odense , Denmark . 7 Structural and Computational Biology Unit , European Molecular Biology Laboratory , 69117 Heidelberg , Germany . 8 Present address : Division of Medical Image Computing , German Cancer Research Center ( DKFZ ) , 69120 Heidelberg , Germany . e - mail : diz @ embl . de Nature Communications | ( 2023 ) 14 : 5644 1 1 2 3 4 5 6 7 8 9 0 ( ) : , ; 1 2 3 4 5 6 7 8 9 0 ( ) : , ; proteins form crescent - shaped membrane - binding dimers that can sense and generate curvature 9 \u2013 11 , and are known to interact with reg - ulators of the actin cytoskeleton such as the Arp2 / 3 complex , formins , or Rho GTPases through several auxiliary domains 9 , 12 . In fact , changes in surface topography induce recruitment of some cytosolic BAR domain proteins to regions of highly positive ( inward ) curvature 13 , and can trigger the formation of actin structures and endocytosis hotspots 14 , 15 . Here , we show that the curvature - sensing BAR domain protein Snx33 inhibits actin polymerization and rearranges the locali - zation of WAVE2 to limit the persistence of the leading edge and steer migration . By this mechanism Snx33 allows cells to adaptively decide if they should move ahead or turn away . Results Snx33 is preferentially excluded from outward - curved mem - brane regions at the leading edge of migrating cells During the differentiation process immune - like HL - 60 cells undergo substantial changes in gene expression , initiate rapid migration , and acquire a complex membrane topography , resembling what occurs in the bone marrow in vivo 16 ( Supplementary Fig . 1a ) . The terminally differentiated , motile cells ( dHL - 60 cells ) display both actin - rich lamellipodia and membrane ruf \ufb02 es at the leading edge ( Fig . 1a , b ) . To analyze membrane topography in more detail , we used scanning electron microscopy ( SEM ) , for ultrastructural detail of natively \ufb01 xed cell membranes , and polarized total internal re \ufb02 ection \ufb02 uorescence microscopy ( p - TIRFM ) , which enables probing membrane curvature dynamics in live cells . We observed curved membrane patterns , as seen by SEM in the upper plasma membrane ( Fig . 1c ) , that are very dynamic , as visualized by p - TIRFM in the basal plasma membrane ( Fig . 1d , Supplementary Fig . 1b \u2013 d , and Supplementary Video 1 ) . These curved membrane patterns likely constitute binding sites for curvature - sensitive proteins , such as those from the BAR domain family . To identify candidates from this large family that could sense indentingobstacles andthusbe relevant foradaptive behaviorsduring cell migration , we measured their expression pro \ufb01 le before and after HL - 60 differentiation to a migratory state , i . e . , in a curvature - poor ( blasts are uniformly round ) versus a curvature - rich state ( Supple - mentary Fig . 1a ) . The expression of the BAR domain protein Snx33 increased 16 - fold during this differentiation process ( Fig . 1e ) . We imaged its subcellular localization by confocal microscopy after \ufb02 uorescent tagging ( eGFP - Snx33 ) and found that Snx33 is enriched throughout membrane ruf \ufb02 es ( Fig . 1f , g ) , the highly curved structures at the leading edge of dHL - 60 cells . Based on the similarity within its protein family , Snx33 is predicted to bind shallow curvatures in com - parison to many other BAR domain proteins from the BAR subgroup 9 . The membrane - binding domain of Snx33 ( PX - BAR ) contains a posi - tivelychargedpatchontheconvexsurface , whichstronglysuggestsan inward curvature - dependent membrane - binding on this surface ( Supplementary Fig . 2a \u2013 d ) . To assess its curvature - sensing properties in more detail , we performed long coarse - grained molecular dynamics ( MD ) simulations of the PX - BAR domain ( Fig . 1h \u2013 j ) on buckled mem - branes with plasma membrane - derived lipid composition ( Table S1 ; see Methods for details ) . This computational assay has been used extensively to characterize curvature - sensing properties of a wide variety of proteins 17 , 18 , including other BAR domains 19 . During the 30 \u00b5 s long simulation , the PX - BAR domain of Snx33 remained tightly bound to the membrane and showed a strong preference for membrane regions with inward local curvature ( Fig . 1i , j , Supplementary Fig . 2e , f ) . Snx33 binds the plasma membrane with a surface of basic residues such as lysine and arginine that mediate electrostatic interactions ( Supplementary Figs . 2a \u2013 d , 3a , b , 4a \u2013 g , see Supplemental Discussion for details ) , which is common for plasma membrane binding 20 . To further test the curvature sensitivity of Snx33 in cells , we imaged its subcellular localization by lattice light sheet microscopy and , sup - porting our MDsimulations , we found that Snx33 isexcluded from the ruf \ufb02 e tips and lamellipodium edge , both structures with highly nega - tive ( outward ) curvature ( Fig . 1k \u2013 m , Supplementary Fig . 5 , and Sup - plementary Video 2 , see Methods for details ) . To corroborate the tip exclusionofSnx33 , wecompareditslocalizationtothatofIRSp53 ( also known as BAIAP2 ) , acanonicaloutward curvature - bindingprotein 21 . As previously reported , in neutrophil - like cells IRSp53 accumulates at the tip of the advancing front and retracting \ufb01 bers 22 , 23 , known outward - curved structures ( Supplementary Fig . 6a \u2013 c and Supplementary Video 3 ) . Altogether , we show that the BAR domain - containing protein Snx33 has a strong preferencefor membrane regions with inward local curvature and is enriched at the leading edge but excluded from outward - curved membrane structures . Snx33 controls leading edge growth and modulates plasma membrane tension TostudytheroleofSnx33inadaptivemigration , wegeneratedaSnx33 knockout ( Snx33 - / - ) cell line using CRISPR / Cas9 genome editing ( SupplementaryFig . 7a , b ) . Propulsioninimmune - likecellsdependson the active polymerization of actin at the leading edge of the cell 24 , 25 . Given that cell shape re \ufb02 ects changes in motion - driving actin - rich protrusions 26 , we performed a quantitative and unbiased comparison of selected cell morphometric parameters , i . e . , cell spreading , cell eccentricity , and leading - edge characteristics . As immune cells radi - cally change their morphology in short periods of time , we averaged time - lapse movies to capture their dynamics . To this end , we trained and used machine - learning - based algorithms in ilastik 27 to analyze movies of cells imaged by total internal re \ufb02 ection \ufb02 uorescence microscopy ( TIRFM ) ( see Methods for details ) . Snx33 - / - cells spread to a larger extent , and showed a more elongated morphology and bigger leading edge ( Fig . 2a \u2013 f , Supplementary Fig . 8a \u2013 e , and Supplementary Videos 4 and 5 ) , independent of adhesion to the substrate ( Supple - mentary Fig . 8f \u2013 i ) . Moreover , the increase in spread area and leading edge area could be rescued by expressing \ufb02 uorescently tagged Snx33 proving the speci \ufb01 city of the observed phenotypes ( Supplementary Fig . 8b , c ) . Altogether , these results indicate that Snx33 - / - cells have a more stable leading edge during migration . Leading edge growth and increased cell spreading during persis - tent migration is known to increase plasma membrane tension 28 , 29 . To test if the more stable leading edge induced by the loss of Snx33 leads to higher membrane tension , we measured it in the leading edge by static tether pulling using atomic force spectroscopy , where a plasma membrane tether is heldwith a constant length until itbreaks ( Fig . 2g ) . We found that the static tether force was signi \ufb01 cantly increased in Snx33 - / - dHL - 60 cells ( from 61 . 58 to 75 . 25 pN , Fig . 2h ) , which corre - sponds to an almost 50 % increase in apparent membrane tension ( from 177 . 87 to 265 . 62 \u03bc N / m ; see Methods for details ) . Moreover , the increase in membrane tension could be rescued by stably expressing \ufb02 uorescently tagged Snx33 , excluding Snx33 - independent functions as the origin of this phenotype ( Fig . 2h ) . Notably , overexpression of Snx33 - GFP on the wild - type background did not decrease membrane tension , suggesting that a gain of function is not suf \ufb01 cient to alter the leading edge or its effects on membrane mechanics ( Supplementary Fig . 9a ) . Last , these phenotypes were not a consequence of defective differentiation , as the neutrophil differentiation marker CD11b was unperturbed in Snx33 - / - cells , nor of potentialoff - target effects caused by cell line generation by CRISPR / Cas9 technology ( Supplementary Fig . 9b , c ) . Altogether , these results con \ufb01 rm that Snx33 - / - cells have a more stable leading edge showing all the characteristics expected for persistent migration . Snx33 regulates actin polymerization Our \ufb01 ndings suggest that the curvature - sensing protein Snx33 nega - tively regulates actin polymerization and thereby limits leading edge size . Thus , we quanti \ufb01 ed the amount of \ufb01 lamentous actin ( F - actin ) by phalloidin staining upon chemoattractant ( fMLP ) addition by \ufb02 ow Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 2 cytometry and observed a signi \ufb01 cant increase in the total amounts of F - actin in Snx33 - / - cells when compared with their wild - type counter - parts ( Fig . 2i ) . Thissuggests thatSnx33 plays an inhibitoryrole onactin polymerization , particularly during the initial burst after stimulation . But how does Snx33 inhibit actin polymerization ? Some BAR domain proteins remodel actin by binding to it directly or by the recruitment of NucleationPromoting Factors ( NPFs ) that activate the Actin Related Protein 2 / 3 ( Arp2 / 3 ) complex 9 , 30 \u2013 34 . To understand how Snx33 inhibits actinpolymerization , wecoimmunoprecipitatedfull - lengthGFP - Snx33 and its bindingpartnersinthe knockout background followed by mass spectrometry . As it is common among BAR domain proteins to have intramolecular autoinhibitory interactions , we also performed coim - munoprecipitation of two truncations of Snx33 containing or missing the PX - BAR domain , but altogether spanning the full - length protein PX - BAR Membrane t = 30\u03bcs t = 0 . 0\u03bcs 10nm Mean curvature H [ \u03bcm - 1 ] Probability 0 . 02 0 . 01 0 . 04 0 . 03 0 . 05 0 . 00 0 50 - 50 0 . 06 8s 16s 0s b r i gh t f i e l d 0s 8s 16s p - po l a r i z a t i on a b c d Pearson correlation ( Snx33 , membrane ) R e l a t i v e po s i t i on z / h 0 . 8 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 2 0 . 4 0 . 6 0 . 0 h upper plane bottom plane S n x 33 M e m b r ane f z - 4 4 0 4 3 2 1 0 - 4 - 3 - 2 - 1 e SNX33 ARHGAP26FNBP1LSRGAP1 SH3GLB2 GAS7 ARFIP1FCHSD1 SNX18PSTPIP2ACAP2PSTPIP1DNMBP ASAP1ARHGAP44 APPL2 SNX9SNX30 ARHGAP4SH3GLB1FESFCHO2 OPHN1FNBP1 SH3BP1 PACSIN2BIN3SNX2FCHO1SNX1SH3GL1SNX6 FERARHGAP42 SNX8FCHSD2SRGAP2BACAP3 SRGAP2C SRGAP2PICK1TRIP10 SRGAP3 ARHGAP17ASAP2 BIN1 APPL1ARFIP2 ARHGAP10 SH3GL2 ACAP1 BAIAP2L2 BAIAP2L1 BAIAP2 SNX4 SNX5 MTSS1L - 8 - 7 - 6 - 5 h i j Membrane Snx33 Membrane Snx33 Snx33 Membrane k m z z lines top membrane postion in z always 0 Snx33 signal 0 . 00 0 . 05 0 . 10 on ruffle off ruffle 0 . 00 0 . 05 0 . 0 0 . 1 0 . 2 on ruffles off ruffles S n x 33 d i s t an c e i n z f r o m c e ll m e m b r ane [ no r m a li z ed ] * top membrane postion 0 . 00 1 . 00 BAR domain proteins Fold change [ log2 ] H > 0 inward H < 0 outward l t = 15\u03bcs g Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 3 ( Fig . 2j ) . Several members of the Arp2 / 3 complex as well as two sub - units of capping proteins coimmunoprecipitated with Snx33 ( limma p - value \u2266 0 . 01 , fold - change \u2265 50 % ) ( Fig . 2j ) . In particular , we identi \ufb01 ed positive fold - changes for ARPC2 , ARPC4 - TTLL3 , ACTR2 , ACTR3 , CAPZA1 and CAPZB1 in the full - length GFP - Snx33 or its truncations , suggesting that Snx33 binds to the Arp2 / 3 complex and to capping proteins . The Arp2 / 3 complex is the actin nucleator responsible for lamellipodia and ruf \ufb02 es formation at the leading edge of neutrophil cells 35 , while capping proteins terminate the growth of actin \ufb01 laments by binding to them , and potentiate their branching by the Arp2 / 3 complex 36 \u2013 39 . Interestingly , we could observe that some Arp2 / 3 com - ponents and capping protein subunits were substantially enriched with either domain compared to the full - length protein ( Supplemen - taryFig . 10a ) , suggestingthatdiversedomainsofSnx33bindthemwith different af \ufb01 nities , possiblybecause ofautoinhibitory interactionsthat mayoccludebindingsitesoraffectmembranebinding , as reportedfor other BAR domain proteins 40 \u2013 42 . In summary , Snx33 negatively reg - ulatesactinpolymerization , likely throughtheregulationof theArp2 / 3 complex and / or capping proteins . Snx33 affects WAVE2 localization and ruf \ufb02 e morphology WAVE2 isthe only knownactin NPF upstreamof the Arp2 / 3 complex in neutrophils 24 , 35 . Thus , to further characterize the leading edge of Snx33 - / - cells we imaged a component of the WAVE2 complex ( Hem - 1 ) during migration by TIRFM ( hereafter referred to as WAVE2 ) ( Fig . 3a and Supplementary Video 6 and 7 ) . WAVE2 earned its name because it localizes in waves on the basal membrane during cell migration 24 . To generate such patterns , WAVE2 waves deposit an inhibitor in their wake that transiently inhibits WAVE2 recruitment 24 . Interestingly , polymerized actin is a key component of this inhibitory feedback but what regulates WAVE2 binding to the membrane and determines its patch morphology remains poorly understood 43 . Thus , we quanti \ufb01 ed WAVE2 pattern characteristics using machine - learning - based seg - mentation . Strikingly , Snx33 - / - cells showed a 40 % increase in the WAVE2 area and the size of its patches at the plasma membrane ( Fig . 3b \u2013 d ) . To determine whether this is due to a difference in the expression levels of WAVE2 components or due to increased mem - brane binding , we performed RNAseq . We observed none or a very minor difference in expression in Snx33 - / - cells when compared with their control counterparts ( Supplementary Fig . 10b ) , suggesting that Snx33 affects the localization of the WAVE2 complex rather than its expression . This is particularly interesting as , to date , only very drastic WAVE2 patch phenotypes have been reported ( complete abrogation or increase in number ) , which lead to a severely disrupted capacity to migrate 44 , 45 . We next sought to test the functional relevance of the change inWAVE2 patch morphology for leading - edge morphology . To this end , we quanti \ufb01 ed the effective ruf \ufb02 e wavelength from SEM ima - ges and observed a signi \ufb01 cantly less tight arrangement of ruf \ufb02 es for Snx33 - / - cells when compared with their wild - type counterparts ( Fig . 3e , f , Supplementary Fig . 8j , Supplementary Fig . 11a ) . Last , to determinewhethertheincreaseineffectiveruf \ufb02 ewavelengthismerely the consequence of increase membrane tension in Snx33 - / - cells , we performed SEM on PLD2 knock - down ( KD ) cells , which also display increased membrane tension 44 . Notably , PLD2 KD cells showed no differences in effective ruf \ufb02 e wavelength compared to their Nonsense counterparts ( SupplementaryFig . 11b , c ) . These \ufb01 ndingsidentifySnx33 as a link between membrane shape and the actin polymerization fac - tors that modulate it . Snx33 enables curvature - dependent object evasion Our data thus far reveal that the membrane curvature - binding protein Snx33 regulates actin polymerization , leading - edge morphology , and stability . Leading edge patterning may facilitate adaptive motility by reducing persistence and promoting random evasive maneuvers . But the particular form of Snx33 - mediated membrane - actin coupling suggests an additional , more direct effect , in which propulsion is reduced speci \ufb01 cally where the presence of an obstacle in the path of thecellis likely , i . e . , whereexternalindentationsgenerateanincreased fraction of regions with inward curvature . Thus , Snx33 could facilitate cell steering , becausemembranedeformationsgenerated by collisions would locally down - regulate propulsion and thereby reorient the leading edge . To test this hypothesis , we \ufb01 rst positioned the cells in micro \ufb02 uidic devices 46 , 47 where they migrated through channels and encountered obstacles in the form of differently sized pores . Snx33 - / - dHL60 cells required almost 20 % more time than their wild - type counterparts to navigate these decision - points and to \ufb01 nd the path of leastresistance ( Fig . 4a , Supplementary Fig . 12a , b , and Supplementary Video 8 ) . As the nucleus has been shown to function as a mechanical guide along the path of least resistance 46 , we measured nuclear stiff - nessby atomic forceindentation and ruled out that nuclear mechanics wasaffected in Snx33 - / - cells ( Supplementary Fig . 12c , d ) , inagreement with their retained ability to read out pore size ( Supplementary Fig . 12b ) . However , in decision - free channels , Snx33 - / - cells migrated 80 % faster than theirwild - type counterparts , including during passage through a single constriction ( Fig . 4b , Supplementary Fig . 12e , and Supplementary Video 9 ) . Next , we quanti \ufb01 ed uncon \ufb01 ned migration on 2D planar substrates with a homogeneous chemoattractant , promot - ing cell motility without requiring the cells to adapt to any obstacle ( Fig . 4c , see Methods for details ) . As in decision - free channels , Snx33 - / - cells migrated faster than wild - type cells ( Fig . 4d ) . Moreover , while the motility of Snx33 - / - cells was more persistent , wild - type cells were more prone to perform large spontaneous turns ( Fig . 4e ) . Thus , the lossofSnx33appearstorendercellslesspronetospontaneousturning and less effective in circumnavigating an object while displaying more persistent migration in decision - free environments . These migratory phenotypes are consistent with the increase in leading edge size and plasma membrane tension we have observed in Snx33 - / - cells ( Fig . 2d \u2013 h ) . Together , these observations suggest that Snx33 - / - cells Fig . 1 | Curvature patterning in the lamellipodium and the curvature - sensitive protein Snx33 . a The leading edge of migrating cells is characterized by intricate curvature patterns . The arrow indicates the direction of cell movement . Lines depict a complex 3D environment . b Time - lapse bright - \ufb01 eld imaging shows migrating immune - like differentiated HL - 60 cells . c Scanning electron microscopy ( SEM ) image of a wild - type cell with zoom - in at the leading edge . n = 175 . d Time - lapse p - polarization of p - TIRFM imaging reveals dynamic membrane waves at the leading edge . e Up - regulated ( orange ) and down - regulated ( blue ) BAR domain genesbetweendifferentiated ( migratory ) andundifferentiated ( non - migratory ) HL - 60cells . f FluorescentlytaggedSnx33 ( eGFP - Snx33 ) andmembranemarker ( CAAX - mcherry ) inupper ( close - to - the - cell - top ) and lower ( near - surface plane ) z - planesin a cell . g Pearson correlation coef \ufb01 cient of \ufb02 uorescently tagged Snx33 and mem - brane marker at different heights acquired by confocal microscopy . n = 10 . Error bars denote the standard error of the mean . h Structure of the Snx33 PX - BAR domain as modeled with AlphaFold - multimer ( green ) compared with the incomplete crystal structure ( PDB ID : 4AKV ; gray ) . i Probability histogram of the mean curvature ( H ) sampled at the center of mass of PX - BAR ( green , mean curvature PX - BAR = 16 \u03bc m \u2212 1 ) and at a random lipid phosphate position ( yellow ) . Ker - nel density estimates to smooth the distributions are shown . The mean values are indicated as dashed vertical lines . j Top and side views from snapshots of the coarse - grained simulation of a buckled membrane with the PX - BAR domain . Pro - tein backbone beads ( green ) , phosphate beads ( yellow spheres ) , and lipid tails ( gray sticks ) are shown . Water and ions are omitted for clarity . A top and side view are shown . Time points are indicated for the respective frames . k , l Cross - sections ( xz ) of migrating cells using lattice - light - sheet microscopy visualizing eGFP - Snx33 and CAAX - mcherry with a zoom - in on a membrane ruf \ufb02 e ( k ) , and on a lamellipo - dium ( l ) . n = 6 . m Quanti \ufb01 cation of the average z - position of Snx33 relative to the plasma membrane on - and off - ruf \ufb02 es ( p TopMembrane = 0 . 01635 , t = \u2212 3 . 5524 , df = 5 , paired , two - sided ) . n = 6cellswhichdata wereobtained from two - color3D movies . Each point represents the average for 1 cell . Scale bars = 10 \u03bc m . p < 0 . 05 ( * ) . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 4 haveimprovedpropulsionbutlacktheabilitytoadaptwhenfacedwith an obstacle . To gain further insights into the object - evasion response of Snx33 - de \ufb01 cient cells , we devised a reductionistic assay to test contact inhibition of locomotion ( CIL ) , a common phenomenon in many cell types , including dHL - 60s , where cells stop moving in a particular direction when they come into contact with another cell or an object 24 , 48 . To test if CIL as a response to cellular obstacles is controlled by Snx33 , we seeded a higher density of dHL - 60 cells and imaged their 2D migration in the presence of cell \u2013 cell interactions by TIRFM . Snx33 - / - cells formed larger cell \u2013 cell contacts compared to wild - type cells ( Fig . 4f , g ) , in agreement with their increased decision time when faced with an inert obstacle ( Fig . 4a ) . Our computational results together with the observed localization of Snx33 ( Fig . 1h \u2013 m ) suggest preferential binding to inward - curved regions . To assess what changes in plasma membrane geometry , Fig . 2 | Snx33 knockout cellsdisplayalteredcelland leading edge morphology due to an increase in actin polymerization . a TIRFM images of a wild - type and a Snx33 - / - cell . b Cellspreadarea ( p = 1 . 505e - 7 , Mann \u2013 Whitney - U - test , two - sided ) and c eccentricity differ between wild - type and Snx33 - / - cells during movement ( p = 0 . 004989 , Mann \u2013 Whitney - U - test , two - sided ) . d Leading edge segmentationof a wild - type and a Snx33 - / - cell . Time frames ( 5s ) are color - coded . e Leading edge area ( p = 1 . 634e - 5 , Mann \u2013 Whitney - U - Test , two - sided ) and f length ( p = 0 . 2111 , Mann \u2013 Whitney - U - test , two - sided ) . n = 82 ( wt ) , n = 78 ( Snx33 - / - ) . g Schematic of statictether pulling experiments . h Meanstatic tetherforce of wt ( n = 24 ) , Snx33 - / - ( n = 26 ) and Snx33 - / - with overexpressed eGFP - Snx33 ( n = 25 ) from 3 independent experiments ( p wt vs . Snx33 - / - = 7 . 883e - 6 , t = \u2212 5 . 0144 , df = 47 . 376 ; p wt vs . Snx33 - / - + eGFP - Snx33 = 0 . 2141 , t = \u2212 1 . 2601 , df = 45 . 515 ; p Snx33 - / - vs . Snx33 - / - + eGFP - Snx33 = 0 . 2141 , t = \u2212 1 . 2601 , df = 45 . 515 , two - sided ) . i Median value of phalloidin \ufb02 uorescence intensity in \ufb01 xed wt and Snx33 - / - dHL - 60 cells quanti \ufb01 ed by \ufb02 ow cytometry . Data from 3 independent experiments ( p t00 = 0 . 184 , Mann \u2013 Whitney - U - test , two - sided ; p t01 = 0 . 0373 , t = \u2212 4 . 26 , df = 2 . 375 , two - sided ; p t30 = 0 . 1298 , t = \u2212 1 . 9728 , df = 3 . 5035 , two - sided ) . j Schematic of full - length Snx33 with its domains and two Snx33 trun - cations ( \u0394 PXBAR and PXBAR ) . Volcano plots from co - immunoprecipitation experiments comparing Snx33 - / - with Snx33 - / - + GFP - Snx33 , Snx33 - / - + GFP - Snx33 \u0394 PXBAR and Snx33 - / - + GFP - Snx33PXBAR . Enriched hits ( limma p - value \u2266 0 . 01 , fold - change \u2267 50 % ) are shown green , and the rest gray . Scale bars = 10 \u03bc m . p < 0 . 001 ( * * * ) , p < 0 . 01 ( * * ) , p < 0 . 05 ( * ) . Box - plots : lower and upper hinges corre - spondtothe25thand75thpercentile . Theupperwhiskerextendsfromthehingeto the largest value , but no further than 1 . 5 * IQR . The lower whisker extends from the hingetothesmallestvalue , butnolowerthan1 . 5 * IQRofthehinge . Databeyondthe whiskers : black dots . Black line : median . Black dot : mean . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 5 occur upon collisions between cells , we quanti \ufb01 ed the curvature differences at the leading edge of freely moving cells and of pairs after collision . We performed serial block - face scanning electron microscopy ( SBEM ) , manually segmented the plasma membrane , and quanti \ufb01 ed the curvatures at free and contacting areas in the front of colliding and single cells in the xz plane ( Fig . 4h \u2013 k ) . Addi - tionally , to rule out potential \ufb01 xation artefacts , we analyzed leading edges in freely migrating and colliding live cells using a membrane marker ( CAAX ) and lattice light sheet ( LLS ) imaging in the xy plane ( Fig . 4l \u2013 n and Supplementary Fig . 13f \u2013 i ) . In both datasets , we observed a change in the curvature distribution at the front between single and collided cells . While single cells displayed a broader distribution biased towards outward curvatures , the con - tact surfaces were characterized by narrower distributions centered at zero , in line with an overall \ufb02 attening at the contact site . Cell \u2013 cell contacts suppressed both positive and negative curvatures ( Sup - plementary Fig . 14a \u2013 d ) . To further dissect the molecular role of Snx33 in CIL , we simulta - neously imaged the neutrophil WAVE2 complex ( using Hem1 ) and Snx33 at the leading edge of live cells . While both proteins largely colocalized in most parts of the cell , they were anticorrelated in the highly negatively curved rim of the leading edge ( Supplementary Fig . 15a , b ) . Here , Snx33 levels decreased while WAVE2 accumulated ( Supplementary Fig . 15c , d ) , consistent with Snx33 being excluded from outward - curved regions and restricting WAVE - binding to such areas . Moreover , when cells collided , Snx33 localized to the cell contact area , whereupon WAVE2 disappeared and relocated to contact - free zones of the plasma membrane ( Fig . 4o , p ) . Subsequently , thecellrepolarizedby forming a new leading edge ( Fig . 4o , p ) . To assess whether the dysfunctional CIL response in Snx33 - / - cells is due to impaired WAVE2 inhibition at the contact area , we followed WAVE2 localization before and after collision in Snx33 - / - cells ( Supplementary Videos 10 ) . In con - trast to wild - type cells , Snx33 - / - cells indeed failed to remove WAVE2 from the contact site ( Fig . 4q and Supplementary Fig . 15e ) . Altogether , weshowakeyroleforthecurvature - sensingproteinSnx33inregulating actin polymerization during CIL . Speci \ufb01 cally , Snx33 displaces WAVE from cell \u2013 cell contact areas and thus leads to cell repolarization towards a contact - free zone upon collision with an obstacle . Discussion Object evasion is key not only for the migration of immune and cancer cells in complex tissue environments , but also fundamental during embryogenesis and collective migration in vivo 49 . Combin - ing genetic perturbations , microscopy , and micro \ufb02 uidics we iden - ti \ufb01 ed a curvature - sensing mechanism underlying adaptive motility in immune - like cells . We show that the BAR domain protein Snx33 reads out changes in the membrane curvature landscape and feeds back to the actin polymerization machinery to regulate complex patterns at the leading edge of migrating cells . Speci \ufb01 cally , Snx33 inhibits actin polymerization and tunes the localization of WAVE2 , the main nucleation - promoting factor in neutrophils . Thereby , Snx33 limits the persistence of the leading edge and reduces migration directionality . This mechanism for promoting sponta - neous reorientation during cell migration complements the one described for abundantly expressed I - BAR domain proteins , where outward curvature - sensing coupled to local activation of actin polymerization enhances exploratory cell behaviors 22 . While few other BAR domain proteins contain domains with inhibitory effects Fig . 3 | WAVE2 pattern width and ruf \ufb02 e wavelength are increased in Snx33 knockout cells . a TIRFM images of a wild - type and Snx33 - / - cell show the dis - tribution of WAVE2 , the only actin nucleator promoting factor upstream of the Arp2 / 3 complex in neutrophils . b The total area occupied by WAVE2 patches in the leading edge increases upon loss of Snx33 ( p = 0 . 000408 , Mann \u2013 Whitney - U - test , two - sided ) . n = 82 ( wt ) , n = 78 ( Snx33 - / - ) . c Segmentation of dynamic WAVE2 patches in a wild - type and Snx33 - / - cell . Time frames ( 5s ) are color - coded . d The size of WAVE2 patches increases upon loss of Snx33 ( p = 0 . 000464 , Mann \u2013 Whitney - U - test , two - sided ) . n = 82 ( wt ) , n = 78 ( Snx33 - / - ) . e SEM images withruf \ufb02 esegmentations ( red ) showtheleadingedgesofawild - typeandSnx33 - / - cells fromthe50 th percentileofthe distribution . f Theeffectiveruf \ufb02 e wavelength increases upon loss of Snx33 ( p = 3 . 171e - 7 , Mann \u2013 Whitney - U - test , two - sided ) . n = 175 ( wt ) , n = 170 ( Snx33 - / - ) . Statistics : t - testornon - parametricMann \u2013 Whitney - U - test . Scale bars = 10 \u03bc m . p < 0 . 001 ( * * * ) , p < 0 . 01 ( * * ) , p < 0 . 05 ( * ) . Box - plots : the lower and upper hinges correspond to the 25th and 75th percentile . The upper whisker extends from the hinge to the largest value , but no further than 1 . 5 * IQR . Thelowerwhiskerextendsfromthehingetothesmallestvalue , butnolowerthan 1 . 5 * IQR of the hinge . Data beyond the whiskers : black dots . Black line : median . Black dot : mean . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 6 on actin polymerization , they are likely not fully redundant . We envision that their mechanism of action will be the result of several properties , including curvature sensitivity , strength of binding , binding partners , and what other domains are present . Thus , BAR domain proteins emerge as versatile tools that locally activate or inhibit actin polymerization and thus endow cells with functional control of their leading edge . The ability to spontaneously reorient and explore the micro - environment with dynamic protrusions in and of itself contributes to more ef \ufb01 cient navigation in crowded contexts 6 , 22 , 44 , 46 , 50 , 51 . A curvature - dependent downregulation of protrusive activity can furthermore aid object evasion more directly , by steering the cell away from the direction in which the presence of an obstacle on the outside is most likely . Indeed , we show that Snx33 is key for cells to circumnavigate both inert and cellular obstacles and thus migrate through complex three - dimensional environments . To date , little is known about the molecular machinery orchestrating CIL . srGAP2 , a BAR domain protein that binds outward membrane curvature , was Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 7 shown to induce protrusions during CIL 52 \u2013 55 . Our study shows a key role for the inward curvature - sensing protein Snx33 in inhibiting actin polymerization during CIL . Speci \ufb01 cally , we show that the multi - domain protein Snx33 localizes to cell \u2013 cell contacts and redistributes WAVE2 to the free surfaces , thereby reorienting the cell . This likely occurs in a curvature - dependent manner as Snx33 preferentially localizes to inward - curved membrane regions while WAVE2 is restricted to outward - curved areas at the leading edge 23 . At cell \u2013 cell contacts we identify a distinct shift in the curvature distribution using two datasets that differ in measured curvature values by an order of magnitude . Speci \ufb01 cally , upon collision , the curvature distributions are narrower compared to that of the free leading edge of migrating single cells , consistent with membrane \ufb02 attening upon collision . The expected curvature sensitivity of an Snx33 PX - BAR dimer corresponds to a higher curvature than that measured at cell \u2013 cell collisions . However , it is important to note that BAR domain proteins have auxiliary domains and can form oligomers as well as linear aggregates that bind membranes with considerably lower curvature 56 \u2013 58 . Furthermore , our observations in cells are restricted to radii of curvature with magnitudes above the respective image resolution limits and limited by detectable cur - vature length scales . Thus , in the future , it will be important to explore how forming assemblies as well as the presence / absence of auxiliary domains may permit BAR domain proteins to span larger radii of curvatures to respond to larger - scale changes in membrane shape within the molecular complexity of cells 19 , 59 . Here , we show that regulation of actin polymerization by Snx33 has important consequences for cell migration . This , in conjunction with the inherent curvature - sensing properties of BAR domain pro - teins and the observed shift in membrane curvature distribution upon cell \u2013 cellcollisionduringCILsuggestamechanismbywhichmembrane curvature changes direct adaptive cellular behaviors . Together , our study supports the notion that cells use their membrane topography to encode information about the external environment they encounter . Given the diversity of BAR domain proteins present in cells , we expect that this regulatory principle is likely to be used in many bio - logical functions that have to react to shape changes , from the sub - cellular level ( organelle homeostasis , membrane traf \ufb01 cking ) , via the single - cell level ( immune surveillance , tumor dissemination ) , to the multicellular level ( gastrulation , tissue folding ) . Methods Cell culture HL - 60 cells were grown in RPMI 1640 media with 10 % heat - inactivated FBS ( # 10500 - 064 , Gibco ) and 1 % Penicillin - Streptomycin ( # 15140 - 122 , Gibco ) in a humidi \ufb01 ed incubator at 37\u00b0C with 5 % CO 2 . Cells were differentiated by adding 1 . 5 % DMSO ( # D2438 , Sigma Aldrich ) and used after 5 days . Each independently differentiated batch was treated as a biological replicate . For starvation , cells were kept for 1h in FBS - free RPMI 1640 media with 0 . 3 % fatty acid - free BSA ( # A7030 - 10G , Sigma Aldrich ) . For imaging or \ufb01 xation , dHL - 60 cells were plated on \ufb01 bronectin - coated ( 0 . 01 mg / ml , # 356008 , Corning ) glass - bottom dishes ( # 627860 , Greiner bio - one ) and allowed to adhere for 10min in growth media . Next , cells were washed and stimulated with 10nM fMLP ( # F3506 - 5MG , Sigma Aldrich ) . For lowering adhesion , the coat - ing was supplemented with 5 % molar BSA ( # A7030 - 10G , Sigma Aldrich ) . To generate stable cell lines with \ufb02 uorescently tagged Snx33 and its truncations ( PXBAR and \u0394 PXBAR ) , as well as Hem1 and CAAX lentiviral transduction was used as described previously 44 . Cells were sorted on a BD FACS Aria \u2122 Fusion at the EMBL \ufb02 ow cytometry core facility . Generation of knockout cell line by CRISPR / Cas9 CRISPR / Cas9 generation in HL - 60 cells was performed as described previously 45 . Brie \ufb02 y , cloning of the target guide sequence to target Snx33 was performed 60 , 61 ( Forward : CACCGctgggacgacGGATGC ACAG ; Reverse : aaacCTGTGCATCCgtcgtcccagC ) . Cells expressing BFP - tagged Cas9 were single - cell sorted in 96 - well plates on a BD FACS Aria TM Fusion at the EMBL \ufb02 ow cytometry core facility . Single - cell clones were veri \ufb01 ed by genomic DNA ampli \ufb01 cation by Touch - down PCR 62 and sequencing , followed by a Western blot of selected clonal lines . RNA sequencing Total RNA samples obtained from 3 biological replicates were pur - i \ufb01 ed using a RNeasy Mini Kit ( # 74104 , Qiagen ) according to the manufacturer \u2019 s instructions with a DNase digestion step ( # 79254 , Qiagen ) . To ensure high quality , samples were analyzed on an Agi - lent 2100 Bioanalyzer ( Agilent Technologies ) . RNA sequencing was performed on an Illumina NextSeq 500 platform as NextSeqHigh - 75 SE at the EMBL genomics core facility . For sequence alignment , the hg19 reference genome was used . Differential expression ana - lysis was performed with a custom - made Galaxy pipeline using a DESeq2 package . The RNAseq data have been deposited to the ArrayExpress collections from BioStudies with the accession num - ber E - MTAB - 12436 . Imaging TIRFM images of live cells were acquired on a Nikon TiEclipse inverted microscopewithaCFIPlanApoLambda100x Oil ( # MRD01905 , Nikon ) silicone objective and a sCMOS camera controlled by NIS - Elements ( Nikon ) . Sample drift was reduced using an autofocus system ( Perfect Focus , Nikon ) for time - lapse imaging . Fig . 4 | Snx33 steers cell movement in single - cell 2D and 3D migration by inhibiting the WAVE2 complex . a Decision channel passage time ( n wt = 159 , mean = 1 . 67min ; n Snx33 - / - = 9 ; mean = 1 . 96min ) in cells ( p = 0 . 02 , Mann \u2013 Whitney - U - t est , two - sided ) . b Constriction passage time ( n wt = 234 , n Snx33 - / - = 158 ) in cells ( p = 2 . 2e - 16 , Mann \u2013 Whitney - U - test , two - sided ) . c Cellbody displacement over time in wt and Snx33 - / - cells . Time frames ( 5s ) are color - coded . d Cell speed in wt and Snx33 - / - cells ( p = 7 . 661e - 5 , Mann \u2013 Whitney - U - test , two - sided ) e Distribution of anglesatwhichcellsturnduringmigration ( p = 0 . 0007583 , Mann \u2013 Whitney - U - Test , two - sided ) . N wt = 82 , n Snx33 - / - = 78 . Data from 3 independent biological replicates . f Segmentation of a contact event between two wt or Snx33 - / - dHL - 60 cells . Shade highlights the contact duration . g Percentage ofcellcircumference in contact with another cell in wt ( n = 18 ) and Snx33 - / - ( n = 20 ) dHL - 60 cells ( p = 0 . 0001704 , Mann \u2013 Whitney - U - test , two - sided ) . h Visualization of curvature magnitudes mea - sured using various techniques . i Cross - section of a single and collided migrating dHL - 60 cell by SBEMimaging . N single = 6 , n collided = 6 . j Visualization of the absolute curvature value in the leading edge of single and collided migrating cell ( from i ) . k Histogram showing the distribution of absolute curvature values in the leading edge of single and collided cells ( n single = 6 , n collided = 6 ) . Data points denote an average value per cell . l xy plane of a membrane marker ( CAAX ) of a single and collided migrating dHL - 60 cells by lattice - light - sheet . m Visualization of the abso - lute curvature value in the leading edge of single and collided migrating cell ( from l ) . n Histogram showing absolute curvature in the leading edge of single and col - lidedcellsfromlatticelightsheetimages ( n single = 5 , n collided = 5 ) . Datapointsdenote an average per cell . o Bright - \ufb01 eld and p TIRFM imaging of cell \u2013 cell contact in wt dHL - 60 cells with \ufb02 uorescently tagged Snx33 and Hem1 . Arrow points towards cell \u2013 cellcontact . n = 3 . q WAVE2fold - changeaftercell \u2013 cellcontactinwt ( n = 9 ) and Snx33 - / - ( n = 10 ) dHL - 60cells ( p = 0 . 01816 , t = \u2212 2 . 5877 , df = 18 . 789 , two - sided ) . Scale bars = 10 \u03bc m . p < 0 . 001 ( * * * ) , p < 0 . 01 ( * * ) , p < 0 . 05 ( * ) . Box - plots : the lower and upper hinges correspond to the 25th and 75th percentile . The upper whisker extends from the hinge to the largest value , but no further than1 . 5 * IQR . The lower whisker extends from the hinge to the smallest value , but no lower than 1 . 5 * IQR of the hinge . Data beyond the whiskers : black dots . Black line : median . Black dot : mean . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 8 Confocal images of \ufb01 xed cells were obtained with a UPLSAPO 60X S ( NA 1 . 3 ; WD 0 . 3 mm ) silicone objective on an Olympus FV3000 inverted microscope at the EMBL advanced light micro - scopy facility . Epi \ufb02 uorescent and bright - \ufb01 eld imaging of \ufb01 xed cells was per - formed using a 40x objective ( # MRD00405 , Nikon ) , a SOLA SE II , and 100W halogen lamps ( Nikon ) using appropriate \ufb01 lter sets . Polarized TIRFM ( pTIRFM ) modality was implemented based on previous work 63 \u2013 68 . For imaging , dHL - 60 cells were stained before plating with carbocyanine dye DiI ( # D3911 , ThermoFisher Scienti \ufb01 c ) . Lattice light sheet imaging of live cells was performed on a Zeiss Lattice Light sheet 7 ( Zeiss , Oberkochen , Germany ) using appropriate \ufb01 lter sets and a 10\u00d7550 \u03bc m base beam . Image analysis For confocal images ( Fig . 1g and Supplementary Fig . 15b ) , only the z - planes that contained the top 80 % intensity of mCherry - CAAX were considered based on line scans covering the entire resliced maximum intensity z projection . A channel of interest ( ChoF1 ) was used for mask generation based on automatic Otsu segmentation . A custom - made ImageJ script allowed us to calculate the Pearson correlation coef \ufb01 - cient ( PCC ) forevery z - planeofChoF1withChoF2basedonthemaskof ChoF1usingthein - builtColoc2ImageJplugin . Z - sliceswereassignedto 10binsandthemeanwithstandarderrorofthemeanforevery binwas calculated . For analysis of migrating cells imaged by TIRFM ( Fig . 2b \u2013 f , Fig . 3b \u2013 d , and Fig . 4c \u2013 e ) a segmentation of the cell mask ( based on mCherry - CAAX signal ) and of the WAVE2 mask ( based on eGFP - Hem1 signal ) were acquired using the machine - learning - based ilastik soft - ware ( Versions 1 . 3 . 1b1 and 1 . 3 . 3post3 ) 27 . Further image analysis was achieved using an in - house built program implemented in Python . The angle at which cells are moving was calculated based on the center of mass for three consecutive frames . The leading edge was de \ufb01 ned as the difference between two consecutive frames where at least one pixel of WAVE2 mask is present per cluster . The leading edge length was de \ufb01 ned as the number of pixels in the outside perimeter of the leading edge . For the analysis of cell \u2013 cell contacts the same segmen - tation strategy was used to segment individual cells . Cell \u2013 cell contact was de \ufb01 ned as the perimeters \u2019 intersection of both cells . For membrane topography analysis of SEM data ( Fig . 3e , f , Sup - plementary Fig . 8j , Supplementary Fig . 11a \u2013 c ) , the leading edge area with ruf \ufb02 es was manually segmented on median \ufb01 ltered images . Next , ridges were detected within the segmented regions using the Meijer - ing \ufb01 lter 69 . Ridges were later segmented using automatic Otsu thresholding and skeletonized using a custom Python script . Inversion of the number of pixels within the skeletonization per leading edge area corresponds to the effective ruf \ufb02 e wavelength . For analysis of membrane curvature of SBEM and lattice light sheet images ( Fig . 4i \u2013 n and Supplementary Fig . 13b \u2013 e , g , i ) , the plasma membrane was manually segmented at the leading edge ( de \ufb01 ned as the area in front of the nucleus ) or segmented using Otsuthreshold on the \ufb02 uorescence channel with membrane signal followed by a manual curation , respectively . Next , the curvature was quanti \ufb01 ed by a circle \ufb01 tting to a window with a user - de \ufb01 ned length ( 2 times the speci \ufb01 ed half - width + 1 pixel ) sliding along the segmented membrane , using a custom Python script . For determining the sign of the curvature , \ufb01 ducialsweremanuallyaddedtotheimagestopointtothecellinterior ( Supplementary Fig . 14a \u2013 d ) . For analysis of protein position in relation to membrane based on lattice light sheet images ( Fig . 1m and Supplementary Fig . 5 ) only cells with visible membrane ruf \ufb02 es in MIP wereincluded in the analysis . The ruf \ufb02 es were segmented in the MIP by Mejiering \ufb01 ltering followed by Otsu thresholding . For all pixels in the user - de \ufb01 ned ROI z - lines of the membraneandSnx33channelswereextractedandnormalizedtotheir peakintensity . To account for changes incell thicknessat different x \u2013 y positions the z - lines were normalized by localizing the peaks in the membrane channel corresponding to the top and bottom cell mem - branes ( all z - lines with more than 2 peaks detected are excluded from further analysis ) . The normalized z - lines were then separated into two halves corresponding to the bottom and top cell membrane , each of the halves was then assessed for Snx33 colocalization by calculating the overlap between the Snx33 and membrane channels . All z - line halves showing low overlap are excluded from further analysis . Next , all z - lines corresponding to one cell were pooled and separated into the in - ruf \ufb02 e and off - ruf \ufb02 e groups as well as the top - and bottom - membrane subgroups . To allow unbiased comparison between the in - ruf \ufb02 e and off - ruf \ufb02 e groups the distributionof z - linesin eachgroup was adjusted to containthe same frequencyof cellthicknesses . In practice , as the off - ruf \ufb02 e group contains more z - lines than the in - ruf \ufb02 e group the off - ruf \ufb02 e group was randomly subsampled to reconstitute the distribution in the in - ruf \ufb02 e group . Finally , as the SNR of single z - lines does not allow for accurate assessment of Snx33 to membrane dis - tance we employ averaging by bootstrap where random 100 z - lines from each group were averaged and renormalized to their peak intensity . The distance was then measured between the positions where the signal \ufb01 rst reaches 20 % in the membrane and Snx33 chan - nels . This process was then repeated multiple times to generate a distribution of distances that captures the heterogeneity of raw data . For each cell , the mean value for each of the 4 subgroups was extracted and they were compared to validate the robustness of the approach . Cell migration assays in PDMS - based devices PDMS - based micro \ufb02 uidic devices were prepared as previously described 46 , 70 , 71 . Thedevicesused forthemigrationofdHL - 60cellshad heights of 2 . 8 and 3 . 13 \u03bc m for channels with decision points and channels with constriction , respectively . The decision channels had constrictions of 2 , 3 , 4 , and 5 \u03bc m in two arrangements . The channels with single constrictions were 2 \u03bc m . To visualize nuclei and cell body , Hoechst 33342 ( # 62249 , Thermo Fisher Scienti \ufb01 c ) and TAMRA ( Invi - trogen ) were added before the introduction of cells into the PDMS device . Cell migration towards chemoattractant ( fMLP ) was imaged on an inverted wide - \ufb01 eld Nikon Eclipse microscope using a 20x / 0 . 5 PH1 airobjective , equipped with aLumencorlight source ( 390nm , 475 nm , 542 / 575 nm ) , an incubation chamber and a heated stage with CO 2 . The acquired data were analyzed using ImageJ and manually curated . Only single cells that moved through the entire channel were con - sideredforanalysis . Allparameterswerequanti \ufb01 edbasedonthenuclei signal . Tether extrusion using atomic force spectroscopy Apparent membrane tension was measured by extruding plasma membrane tethers . For measurements , a Olympus BioLever ( k = 60 pN / nm ) from Bruker was mounted on a CellHesion 200 AFM ( Bruker ) with JPK SPM Software 6 . 1 . 183 , which is integrated into an Eclipse Ti inverted light microscope ( Nikon ) . Cantilevers were calibrated using the thermal noise method and coated with 2 . 5mg / ml Concanavalin A ( # C5275 , Sigma Aldrich ) . Prior to the measurements , cantilevers were rinsed in dPBS . For tether measurement , the canti - lever was positioned over the cell , preferably over the leading edge . Measurements parameters for static tether pulling experiments were as follows : approach velocity was set to 1 \u03bc m / s , contact force to 100 \u2013 300 pN , contact time to 5 \u2013 10s , and retraction speed to 10 \u03bc m / s . After a 10 \u03bc m tether was pulled , the cantilever position was held con - stant until it broke , but no longer than 30s . In every experimental repetition , the conditions \u2019 order was randomized . For every cell at least 3 different tether measurements were taken . The data analysis was performed using the JPK Data Processing Software 6 . 1 . 183 . For assessing the magnitude of membrane tension based on tether force measurements , the following formula was Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 9 used 44 : T = F 20 8 B \u03c0 2 \u00f0 1 \u00de where F 0 is the tether force measured by the AFM and B is the bending rigidity of the plasma membrane , which we assume to be invariable between different experimental conditions ( 2 . 7\u00d7 10 \u2212 19 Nm based on previous measurements 72 , 73 . F - actin staining of non - adherent dHL - 60 cells upon fMLP stimulation Cells were starved and \ufb01 xed by adding an equal volume of 2x \ufb01 xation buffer to 10 6 cells in growth media or stimulated with 10 nM fMLP and \ufb01 xed 0 - , 1 - , and 30 - min post - stimulation . Fixation buffer ( 1x ) contains 3 . 7 % paraformaldehyde ( # 28908 , Thermo Scienti \ufb01 c ) , 1x intracellular buffer ( 140mM KCL , 1mM MgCl 2 , 2 mM EGTA , 20mM HEPES , pH 7 . 5 ) , 320 mM sucrose ( # S0389 - 500G , Sigma Aldrich ) and 0 . 2 % BSA ( # A7030 - 10G , SigmaAldrich ) . Next , cellswere washed carefully with1x intracellular buffer by plate centrifugation and stained with phalloidin coupled with TRITC ( # P1951 , Sigma Aldrich ) for 1 h in intracellular buffer ( 1x ) containing 0 . 2 % of Triton X - 100 ( # T8787 , Sigma Aldrich ) . Cellswerethen again washed with intracellularbuffer ( 1x ) . Finally , they were re - suspended in 1 ml of 0 . 1 % BSA , 2 . 5mM EDTA in dPBS , and analyzed using a Cytek\u00ae Aurora ( Cytek ) at the EMBL Flow Cytometry Core Facility . Data were further analyzed and plotted using the FlowJo software ( Version 10 . 9 . 0 ) . Co - immunoprecipitation For coimmunoprecipittion , GFP - tagged Snx33 and its truncation were added to the Snx33 - / - cell line by viral transduction followed by cell sorting . Harvesting and lysis of dHL - 60 cells was performed as recom - mended for cytoplasmic proteins following the ChromoTek GFP - Trap Magentic Particles M - 270 with protease inhibitor supplementation ( # gtd , Chromotek ) . GFP - nanotrap beads were used to precipitate GFP - tagged proteins from the lysate ( full - length Snx33 and two truncations : PXBAR and \u0394 PXBAR ) afterovernightrotation . Elutionwasperformedin 2xSDS - sample buffer and submitted for mass spectrometry . Protein mass spectrometry Sample preparation . Reduction and alkylation were performed with dithiothreitol ( 56 \u00b0C , 30min , 10 mM in 50 mM HEPES , pH 8 . 5 ) and 2 - chloroacetamide ( room temperature , in the dark , 30min , 20mM in 50 mM HEPES , pH 8 . 5 ) . Samples were prepared according to the SP3 protocol ( https : / / doi . org / 10 . 15252 / msb . 20145625 , https : / / doi . org / 10 . 1038 / s41596 - 018 - 0082 - x ) . In short , sequencing - grade trypsin ( Pro - mega ) was added in an enzyme - to - protein ratio of 1 : 50 for overnight digestion at 37\u00b0C . Peptide recovery was performed in 50 mM HEPES , pH 8 . 5 by collecting the supernatant on the magnet and combining it with a second elution . Peptides were labeled with TMT16plex Isobaric Label Reagent ( ThermoFisher ) according to the manufacturer \u2019 s instructions . Inshort , 0 . 8 mg reagent was dissolved in 42 \u00b5 l acetonitrile ( 100 % ) and 8 \u00b5 l of stock was added and incubated for 1 h at room temperature . The reaction was quenched with 5 % hydroxylamine for 15min at RT . Sam - ples were combined and cleaned up with an OASIS\u00ae HLB \u00b5 Elution Plate ( Waters ) . Mass spectrometry analysis . An UltiMate 3000 RSLC nano - LC system ( Dionex ) \ufb01 tted with a trapping cartridge ( \u00b5 - Precolumn C18 PepMap 100 , 5 \u00b5 m , 300 \u00b5 m i . d . x 5 mm , 100 \u00c5 ) and an analytical column ( nanoEase \u2122 M / Z HSS T3 column 75 \u00b5 m x 250 mm C18 , 1 . 8 \u00b5 m , 100\u00c5 , Waters ) was coupled to an Orbitrap Fusion \u2122 Lumos \u2122 Tribrid \u2122 Mass Spectrometer ( Thermo ) using the Nanospray Flex \u2122 ion source in positive ion mode . Peptides were concentrated with a constant \ufb02 ow rateof30 \u00b5 l / min ( 0 . 05 % tri \ufb02 uoroaceticacidinwater ) ontothetrapping column for 4 min . Subsequently , peptides were eluted via the analy - tical column running using solvent A ( 0 . 1 % formic acid in water , 3 % DMSO ) with a constant \ufb02 ow of 0 . 3 \u00b5 l / min and with an increasing per - centage of solvent B ( 0 . 1 % formic acid in acetonitrile , 3 % DMSO ) from 2 % to 8 % in 4 min , from 8 % to 28 % for a further 104 min , in another 4 min . from 28 % to 40 % , and \ufb01 nally 40 % \u2013 80 % for 4 min followed by re - equilibration back to 2 % B in 4 min . MS instrument parameters were as follows : spray voltage of 2 . 4 kV , capillary temperature 275 \u00b0C , MS1 mass range 375 \u2013 1500 m / z , pro \ufb01 le mode , in the orbitrap with a resolution of 120000 . The max - imum \ufb01 ll time 50 ms , with an AGC target set to standard . Data - dependentacquisition ( DDA ) wasperformed withtheresolutionofthe Orbitrap set to 30000 , with a \ufb01 ll time of 94ms and a limitation of 1\u00d710 5 ions . A normalized collision energy of 34 was applied . MS2 data was acquired in pro \ufb01 le mode . Fixed \ufb01 rst mass at 110 m / z . Mass spectrometry data analysis \u2014 Isobarquant . IsobarQuant 74 and Mascot ( v2 . 2 . 07 ) were utilized to process the acquired data . Data was searched against the Homo sapiens proteome database ( UP000005640 ) containing common contaminants and reversed sequences . The following modi \ufb01 cations were included in the search parameters : Carbamidomethyl ( C ) andTMT10 ( K ) ( \ufb01 xedmodi \ufb01 cation ) , Acetyl ( Protein N - term ) , Oxidation ( M ) and TMT10 ( N - term ) ( variable modi \ufb01 cations ) . Forthefullscan ( MS1 ) amasserrortoleranceof10ppm and for MS / MS ( MS2 ) spectra of 0 . 02 Da was set . Further parameters were set : Trypsin as protease with an allowance of a maximum of two missed cleavages : a minimum peptide length of seven amino acids ; at leasttwo unique peptides were required for protein identi \ufb01 cation . The false discovery rate on peptide and protein levels was set to 0 . 01 . Raw output \ufb01 les of IsobarQuant ( protein . txt \u2013 \ufb01 les ) were analyzed using R programming language ( ISBN 3 - 900051 - 07 - 0 ) . Only proteins with at least two unique peptides were included in the analysis and quanti \ufb01 cation . In total , 561 proteins passed the quality control \ufb01 lters . Raw signal - sums ( signal _ sum columns ) were cleaned for batch effects using limma ( PMID : 25605792 ) and later normalized using vsn ( var - iance stabilization normalization - PMID : 12169536 ) . To test proteins for differential enrichment limma package was employed . The replicate information was appended as a factor in the design matrix given as an argument to the \u2018 lmFit \u2019 function of limma . A hit was de \ufb01 ned as a pro - tein annotated with a false discovery rate ( fdr ) smaller 5 % and a fold - change of at least 100 % and as a candidate with a fdr below 20 % and a fold - change of at least 50 % . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 75 partner repository with the dataset identi \ufb01 er PXD033666 . MD simulations Coarse - grained molecular dynamics simulations . Molecular dynamicssimulationswereperformedusingGROMACS2021 . 4 76 , using the coarse - grained Martini2 . 2 force \ufb01 eld 77 , 78 and applying an estab - lished scaling procedure ( alpha = 0 . 7 ) 79 to all protein beads . As a basis for the structural model , an existing crystal structure ( PDB ID : 4AKV ) of the Snx33 membrane - binding domain ( PX - BAR ) was used . Since someloops were missing inthis structure we predicted the same sequence using AlphaFold - multimer 80 , 81 with default settings but increasing the number of recycles to 6 . The resulting model was in excellent agreement ( RMSD : 2 . 3 \u00c5 ) with the crystal structure and was directly used for simulations . The structuralmodel wascoarse - grained using the \u2018 martinize . py \u2019 script 78 applying secondary structure restraints assigned by DSSP and using an elastic network 82 across both subunits with f c = 500 kJ / mol \u2212 2 and a cut - off c = 1 . 2 nm as previously reported for simulations of other extended BAR proteins 19 . For all disordered coil regions , all elastic bonds were removed . The proteinwas then placed ontoa buckled membranewhich was generated from compression of a \ufb02 at membrane according to the Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 10 following procedure . A \ufb02 at symmetric bilayer with a size of 70x 35x 20nm 3 was prepared using the \u2018 insane . py \u2019 tool 83 . A plasma membrane - derived lipid composition determined by mass spectro - metry was used as input ( Table S1 ) . The membrane was solvated and Na + - ions were added to make the system charge neutral . The mem - brane was then energy minimized using the steepest decent algorithm for 1000 steps . Subsequently , the bilayer was simulated in an NPT ensemble ( semi - isotropic pressure coupling ) for 50 ns with a timestep of 20fs using the Berendsen barostat 84 and velocity rescaling thermostat 85 . Coupling times of 1 ps and 4ps were used , respectively . The tem - perature wasset to 310K in all simulations . The Verlet neighbor search algorithm was used to update the neighbor list , with the length and update frequency being automatically determined . Lennard - Jones and Coulomb forces were cutoff at r c = 1 . 1 nm with the potential shifted to 0 using the Verlet - shift potential modi \ufb01 er 86 . Then , lateral pressure was set to 10bar to generate buckled membrane structures ( P x , y = 10bar , P z = 1 bar ) . Two membranes with different degrees of curvature were extracted from the trajectory of compression with a box size of 58 . 9\u00d7 29 . 5\u00d725 . 9 nm 3 ( excess mem - brane area = 25 . 1 nm 2 ) and 56 . 3 \u00d7 28 . 2 \u00d728 . 3 nm 3 ( excess membrane area = 31 . 4nm 2 ) . In both systems , the protein was placed above the buckled membrane and re - solvated and charge - neutralized . A brief equilibra - tion was performed using the above settings and maintaining position restraints on the protein with a f c = 1000 kJmol \u2212 1 nm \u2212 2 . The equilibra - tion was run for 250 ns . Production simulations were then carried out using anisotropic pressure coupling with \ufb01 xed x , y dimensions and P z = 1 bar . Production simulations were carried out for 30 \u00b5 s . The same simulation parameters were used as above except the pressure was controlled using instead the Parrinello - Rahman barostat with a cou - pling time of 20ps 87 . Images and visualizations were made with VMD ( Version 1 . 9 . 4 ) 88 . Analyses were carried out with MDAnalysis 1 . 1 . 1 89 , 90 and python 3 . 6 . To analyze the curvature preference of the protein on the buckled membrane a previously established protocol by Bhaskara et al . 17 was used ( see : https : / / github . com / bio - phys / MemCurv ) . A 2D Fourier expansion of a height function h ( x , y ) was \ufb01 t to the phosphate beads of the membrane every 1 ns by optimizing a least - squared \ufb01 t . Subse - quently , the meancurvature H wasderived from the shape operatorof the \ufb01 t height function h ( x , y ) . The mean curvature H was calculated for the x , y position of the center of mass of the protein as well as a ran - domly selected phosphate bead position to sample the background membrane at every considered frame . Scanning electron microscopy ( SEM ) After 30min of 10nM fMLP stimulation , cells were \ufb01 xed in 2 . 5 % GA ( # 16220 , EMS ) in 0 , 1M PHEM buffer by adding 37\u00b0C double strength \ufb01 xative ( 5 % GA in 0 , 1 M PHEM ) directly 1 : 1 to the cell medium . After 10min incubation , the \ufb01 xative was replaced by fresh single - strength \ufb01 xative , and cells were further \ufb01 xed at room temperature for 1h . After \ufb01 xation , cells were washed 2 times in 0 , 1 M PHEM and 2 times in 0 , 1 M cacodylate buffer . Next , they were post \ufb01 xed for 2 h on ice in freshly prepared and \ufb01 ltered 1 % OsO 4 ( # 19190 , EMS ) and 0 . 8 % potassium ferrocyanide ( K 4 [ Fe ( CN ) 6 ] * 3H 2 O , # 4984 , Merck ) in 0 . 1 M cacodylate buffer . Afterpost \ufb01 xation , thecellswerewashed4timesinH 2 O , andleft at 4 \u00b0C until further processing . Next , cells were treated with freshly prepared and \ufb01 ltered 1 % tannic acid ( TA , CAS # 1401 - 55 - 4 , EMS ) in water using a Pelco Bio - Wave microwave for seven 1 - min cycles alternating between 150 W and 0 W power . Steady temperature was set to 23 \u00b0C and the vacuum to on for all steps . After TA treatment , cells were washed 2x in H 2 O on the bench and 2x in H 2 O in the microwave for 40 s per step at 250 W power . Cells were then treated with 1 % UA ( # 77870 , Serva ) in H 2 O using the same microwave program as for TA . After washing once in H 2 O and twice in 25 % EtOH , cells were dehydrated in a graded series of ethanol ( 25 % \u2013 50 % \u2013 75 % \u2013 90 % \u2013 100 % \u2013 100 % ) using a microwave program with a step length of 40 s and 250 W power , with a steady temperature at 4 \u00b0C and without vacuum . Finally , the cells were in \ufb01 ltrated with a graded series of Hexamethyldisilizane ( HMDS , CAS # 999 - 97 - 3 , Sigma Aldrich ) in ethanol ( 25 % \u2013 50 % \u2013 75 % \u2013 100 % \u2013 100 % ) using a microwave program with 6 steps of 1 min each , with a power of 150 W for step 1 , 3 , 4 and 6 , and 0 W for steps 2 and 5 . After the \ufb01 nal 100 % HMDS in \ufb01 ltration , all HMDS was removed , and coverslips were left to dry overnight . Silica gel with moisture indicator ( Merck ) was added in 4 empty wells ( corners ) in the 24 - well plate to remove excess humidity . After drying , coverslips were mounted in aluminum stubs ( Agar Scienti \ufb01 c G301F ) using carbon tape , and sputter coated with a layer of gold for 180 s at 30mA current using a Quorum sputter coater model Q150RS . Imaging was performed on a Zeiss Crossbean 540 microscope , using 5kV acceleration voltage and 700pA current for the electron beam , with a working distanceof 5 mm . A secondary electron detector ( SESI ) was used for signal detection , and all images were acquired with a pixel size of 28 , 9 nm / pixel . Serial block - face scanning electron microscopy ( SBEM ) After 30 min of 10nM fMLP stimulation in a MatTek dish , cells were \ufb01 xed in 2 . 5 % GA ( # 16220 , EMS ) in 0 . 1M PHEM buffer by adding 37\u00b0C double strength \ufb01 xative ( 5 % GA in 0 . 1 M PHEM ) directly 1 : 1 to the cell medium . After 10 min incubation , the \ufb01 xative was replaced by fresh single - strength \ufb01 xative , and cells were incubated in \ufb01 xative at 4 \u00b0C overnight . After \ufb01 xation , cells were washed 2 times in 0 . 1 M PHEM and 2 times in 0 . 1 M cacodylate buffer . Next , they were post \ufb01 xed for 1 . 5h on ice in 1 % OsO 4 ( # 19190 , EMS ) and 0 . 8 % potassium ferrocyanide ( freshly prepared and \ufb01 ltered ) ( K 4 [ Fe ( CN ) 6 ] * 3H 2 O , # 4984 , Merck ) in 0 . 1 M cacodylate buffer . After post \ufb01 xation , the cells were washed 4 times in H 2 O , and left in H 2 O at 4 \u00b0C until further processing ( 5 days ) . Next , the cells were stained in three consecutive steps in the fol - lowing order : 1 % Thiocarbihydrazide ( TCH , # 21900 , EMS ) in water , 2 % OsO 4 in water , and 1 % UA ( # 77870 , Serva ) in water . For all three staining steps the cells were processed in a Pelco BioWave microwave with a 7 - step program of 2 min each , with power alternating between 100Wand0W ( startingwith100 ) , steadytemperaturewassetto23 \u00b0C and vacuum on for all steps . Between each staining step , the cells were washed 4 times in H 2 O , two times on the bench , and two times in the microwave ( 40s , 250W power , vacuum off ) . After UA staining , the cells were washed once in H 2 O and twice in 25 % EtOH , then further dehydrated in the microwave in a graded ethanol series ( 50 % \u2013 70 % \u2013 90 % \u2013 100 % \u2013 100 % ) . Microwave settings for the dehydration steps were : 40s time , 250W power , vacuum off , steady temp 4 \u00b0C . After dehydration the cells were in \ufb01 ltrated in a graded series of durcupan resin ( Durcupan ACM from Sigma , # 44611 - # 44614 ( four components ) ) in ethanol ( 25 % \u2013 50 % \u2013 75 % \u2013 100 % \u2013 100 % \u2013 100 % durcu - pan ) , using the microwave for 3 min per step at 150W power , 23 \u00b0C steady temp and vacuum cycle . Finally , the cells were put on a small amount of fresh resin ( cov - ering the center of the MatTek dish ) and left for ~ 1 \u2013 2 h to evaporate residual solvent and air bubbles . Before polymerization , most of the resin was removed ( just enough to cover the center was left ) . A drop of durcupan was added to an 18 \u00d718 mm coverslip ( to avoid trapping air bubbles ) , and the coverslip was put on top of the center of the MatTek dish . The assembly was polymerized in the oven at 60\u00b0C for 2 days . After polymerization , the MatTek dish was removed my sawing close to the coverslip , and the glass on both sides of the round center peace was removed by dipping the assembly in liquid nitrogen and warm water . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 11 A small piece was cut out from the sample using a razor blade and mounted on a SEM pin ( Micro to Nano Gatan3View pins , # 10 - 006003 - 50 ) using two - component silver conductive epoxy ( TedPella , # 16043 ) . Thesamplewasalsocoveredwithsilverepoxyonthesides , and \ufb01 nally , sputter coated with a layer of gold for 180s at 30mA current using a Quorum sputter coater model Q150RS . For curing the silver epoxy , the sample was cured in total for an additional day at 60\u00b0C . Image acquisition was performed on a Zeiss GeminiSEM 450 with 3View from Gatan ( DigitalMicrograph Version 3 . 51 . 3720 . 0 ; SmartSEM version 6 . 06 with Service Pack 4 ) , with the program SBEM Image ( 2021 . 08 . dev ) installed for controlling the acquisition 91 . For image acquisition an acceleration voltage of 1 . 5 kV was used , a beam current of 300 pA , pixel size 10 nm and a dwell time of 1 . 6 \u00b5 s . An on - point BSE detector was used for detection , with the contrast set to 99 . 9 % and brightness to 11 . 8 ( with inverted LUT ) , BSD bias \u2212 5V . Slice thickness was set to 40 nm . Between each cycle an overview image was acquired at 165 . 76nm pixel size and 0 . 8 \u00b5 s dwell time . Statistical analysis Statistical analyses were performed using R ( Version 3 . 2 . 1 ) , while data visualization by both R and Adobe Illustrator\u00ae . The normality of data distribution was tested by Shapiro \u2013 Wilk test . A two - tailed t - test was used for normal distribution . Otherwise , a non - parametric Mann \u2013 Whitney - U - test was used , if not indicated differently . In all box plots , the lower and upper hinges correspond to the \ufb01 rst and third quartiles ( the 25th and 75th percentiles ) . The upper whisker extends from the hinge to the largest value , but no further than 1 . 5 * IQR ( distance between the \ufb01 rst and third quartiles ) . The lower whisker extends from the hinge to the smallest value , but no lower than 1 . 5 * IQR of the hinge . Data beyond the end of the whiskers are plotted as black dots . The black line and dot correspond to the median and mean , respectively . All measurements were taken from distinct samples . Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article . Data availability RCSB PDB database was used in the study with accession number 4AKV . The RNAseq data have been deposited to the ArrayExpress collections from BioStudies with the accession number E - MTAB - 12436 . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner reposi - tory with the dataset identi \ufb01 er PXD033666 . The MD simulation data are available through \ufb01 gshare [ https : / / \ufb01 gshare . com / articles / dataset / Supporting _ data _ for _ Sensing _ their _ plasma _ membrane _ curvature _ allows _ migrating _ cells _ to _ circumvent _ obstacles _ by _ Ewa _ Sitarska _ Silvia _ Dias _ Almeida _ Marianne _ Sandvold _ Beckwith _ Julian _ Stopp _ Jakub _ Czuchnowski _ Marc _ Siggel _ Rita _ Roessner _ Aline _ Tschanz / 22109204 ] . The raw numbers for charts and graphs are available in the Source Data \ufb01 le whenever possible . All other data and unique reagents that support this study are available from the correspond - ing authors upon request . Source data are provided in this paper . Code availability The source code for MD simulations and image analysis are available through \ufb01 gshare [ https : / / \ufb01 gshare . com / articles / dataset / Supporting _ data _ for _ Sensing _ their _ plasma _ membrane _ curvature _ allows _ migrating _ cells _ to _ circumvent _ obstacles _ by _ Ewa _ Sitarska _ Silvia _ Dias _ Almeida _ Marianne _ Sandvold _ Beckwith _ Julian _ Stopp _ Jakub _ Czuchnowski _ Marc _ Siggel _ Rita _ Roessner _ Aline _ Tschanz / 22109204 ] and on github 92 [ https : / / github . com / JakubCzuchnowski / Sensing - their - plasma - membrane - curvature - allows - migrating - cells - to - circumvent - obstacles . git ] . References 1 . Sarris , M . & Sixt , M . Science direct navigating in tissue mazes : chemoattractant interpretation in complex environments . Curr . Opin . Cell Biol . 36 , 93 \u2013 102 ( 2015 ) . 2 . Stoitzner , P . , St\u00f6ssel , H . , Romani , N . & Pfaller , K . 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SBEMimage : versatile acquisition control software for serial block - face electron micro - scopy . Front . Neural Circuits 12 , 54 ( 2018 ) . 92 . Czuchnowski , J . Supporting data for \u201c Sensing their plasma mem - brane curvature allows migrating cells to circumvent obstacles \u201d . GitHub , https : / / doi . org / 10 . 5281 / zenodo . 8169105 ( 2023 ) . Acknowledgements We thank Jan Ellenberg , Leanne Strauss , Anusha Gopalan , and Jia Hui Li for critical feedback on the manuscript and the Life Science Editors for editing assistance . The plasmid with hSnx33 was a kind gift from Duanqing Pei . Cell line with GFP - tagged IRSp53 was a kind gift from Orion Weiner . We thank Brian Graziano for providing protocols , reagents , and key advice to generate CRISPR knockout HL - 60 cells . We thank the EMBL \ufb02 ow cytometry core facility , the EMBL advanced light microscopy facility , the EMBL proteomics facility , and the EMBL geno - mics core facility for support and advice . We thank Anusha Gopalanand Martin Bergert for their support during mechanical measurements by AFM . We thank Estela Sosa Osorio for technical assistance for the co - immunoprecipitation . We thank the EMBL genome biology computa - tional support ( and specially Charles Girardot and Jelle Scholtalbers ) for critical assistance during RNAseq analysis . We thank Hans Kristian Hannibal \u2010 Bach for his technical assistance during the lipidomic analysis of plasma membrane isolates . We thank Steffen Burgold for their sup - port with LLS7 microscope in the ZEISS Microscopy Customer Center Europe . We acknowledge the \ufb01 nancial support of the European Mole - cular Biology Laboratory ( EMBL ) to A . D . - M . , Y . S . , A . K . , and A . E . , the EMBL Interdisciplinary Postdocs ( EIPOD ) program under Marie Sklodowska - Curie COFUND actions MSCA - COFUND - FP to M . S . B . and M . S . ( grant agreement number : 847543 ) , the BEST program funding by FCT ( SFRH / BEST / 150300 / 2019 ) to S . D . A . and the Joachim Herz Stiftung Add - on Fellowship for Interdisciplinary Science to E . S . Author contributions A . D . - M . and E . S . conceived the project and designed the experiments . E . S . , M . S . B . , and J . S . performed experiments with EM advice from Y . S . and micro \ufb02 uidic support from M . S . A . D . - M . and C . S . E . isolated plasma membranes and performed lipidomic analysis . M . S . and R . R . performed MD simulations with support from J . K . E . S . and S . D . A . analyzed the data with support from J . C . , A . E . , and A . K . A . D . - M . , E . S . , and A . E . wrote the manuscript . Allauthorscontributedtotheinterpretationofthedata , and read and approved the \ufb01 nal manuscript . Funding Open Access funding enabled and organized by Projekt DEAL . Competing interests The authors declare no competing interests . Additional information Supplementary information The online version contains supplementary material available at https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 . Correspondence and requests for materials should be addressed to Alba Diz - Mu\u00f1oz . Peer review information Nature Communications thanks Chetan Poojari and the anonymous reviewer ( s ) for their contribution to the peer review of this work . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 14 Reprints and permissions information is available at http : / / www . nature . com / reprints Publisher \u2019 s note Springer Nature remains neutral with regard to jur - isdictional claims in published maps and institutional af \ufb01 liations . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Creative Commons license , and indicate if changes were made . The images or other third party material in this article are included in the article \u2019 s Creative Commons license , unless indicated otherwise in a credit line to the material . If material is not included in the article \u2019 s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2023 Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 41173 - 1 Nature Communications | ( 2023 ) 14 : 5644 15", + "day2015microtubule": "doi : 10 . 1111 / tra . 12269 Microtubule Motors Power Plasma Membrane Tubulation in Clathrin - Independent Endocytosis Charles A . Day 1 , 2 , Nicholas W . Baetz 1 , Courtney A . Copeland 1 , Lewis J . Kraft 3 , Bing Han 1 , Ajit Tiwari 1 , Kimberly R . Drake 1 , Heidi De Luca 4 , Daniel J . - F . Chinnapen 4 , Michael W . Davidson 5 , Randall K . Holmes 6 , Michael G . Jobling 6 , Trina A . Schroer 7 , Wayne I . Lencer 4 , 8 and Anne K . Kenworthy 1 , 3 , 9 , 10 \u2217 1 Department of Molecular Physiology and Biophysics , Vanderbilt University School of Medicine , Nashville , TN , USA 2 Current address : Hormel Institute , University of Minnesota , Austin , MN , USA 3 Chemical and Physical Biology Program , Vanderbilt University , Nashville , TN , USA 4 GI Cell Biology , Department of Pediatrics , Boston Children\u2019s Hospital , Boston , MA , USA 5 National High Magnetic Field Laboratory , The Florida State University , Tallahassee , FL , USA 6 Department of Immunology and Microbiology , University of Colorado School of Medicine , Aurora , CO , USA 7 Department of Biology , The Johns Hopkins University , Baltimore , MD , USA 8 Harvard Medical School and the Harvard Digestive Diseases Center , Boston , MA , USA 9 Department of Cell and Developmental Biology , Vanderbilt University School of Medicine , Nashville , TN , USA 10 Epithelial Biology Program , Vanderbilt University School of Medicine , Nashville , TN , USA \u2217 Corresponding author : Anne K . Kenworthy , Anne . kenworthy @ vanderbilt . edu Abstract How the plasma membrane is bent to accommodate clathrin - independent endocytosis remains uncertain . Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin - independent carriers by binding and cross - linking multiple copies of their glycosphingolipid receptors on the plasma membrane . But it remains unclear if toxin - induced sphin - golipid crosslinking provides suf\ufb01cient mechanical force for deforming the plasma membrane , or if host cell factors also contribute to this process . To test this , we imaged the uptake of cholera toxin B - subunit into surface - derived tubular invaginations . We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules , and that toxin binding was entirely dispensable for membrane tubulations to form . Unexpectedly , the driving force for tubule extension was supplied by the combination of microtubules , dynein and dynactin , thus de\ufb01ning a novel mechanism for generating membrane curvature during clathrin - independent endocytosis . Keywords cholera toxin , clathrin - independent endocytosis , dynactin , dynein , membrane curvature , microtubules Received 30 January 2015 , revised and accepted for publication 6 February 2015 , uncorrected manuscript published online 18 February 2015 , published online 27 April 2015 Endocytosis , the process of internalizing vesicles or tubules and their associated cargo from the cell surface , serves essential functions for all cell types . Two general mechanisms of membrane uptake have been described . One involves the assembly of a rigid clathrin coat on the cytoplasmic surface of the plasma membrane that gener - ates a highly curved membrane invagination , sequesters cargo and facilitates membrane fission and budding from the cell surface for trafficking into the cell ( 1 ) . The other mechanism is clathrin - independent , and consists of multiple pathways , but how clathrin - independent carriers are generated remains poorly understood ( 2 \u2013 9 ) . The AB 5 subunit toxins cholera ( CTx ) and Shiga toxin ( STx ) have become widely used as model cargoes for study - ing clathrin - independent endocytosis , including uptake via tubular endocytic carriers ( 10 \u2013 25 ) . Both toxins bind plasma membrane glycosphingolipids , and contain five or fifteen receptor binding sites for high - avidity association with cells via their respective B - subunits ( 26 , 27 ) . Receptor 572 www . traf\ufb01c . dk \u00a9 2015 The Authors . Traf\ufb01c published by John Wiley & Sons Ltd . This is an open access article under the terms of the Creative Commons Attribution - NonCommercial License , which permits use , distribution and reproduction in any medium , provided the original work is properly cited and is not used for commercial purposes . Microtubules Tubulate the Plasma Membrane cross - linking is dispensable for CTx intoxication of host cells ( 28 , 29 ) , but it is clear that CTx and STx binding to multiple glycosphingolipid receptors affects the membrane dynamics of uptake and intracellular trafficking in ways that enhance their toxicity ( 18 , 30 ) . One way this is thought to occur is via a novel clathrin - independent mechanism in which toxin binding induces negative curvature of the plasma membrane ( 18 , 31 ) . This mechanism has been best described for the B - subunit of Shiga toxin ( STxB ) , but is also utilized by the B - subunit of cholera toxin ( CTxB ) and the glycosphingolipid - binding virus simian virus 40 ( 18 , 30 ) . According to this model , toxin binding deforms the mem - brane beneath the toxin by compacting the glycosphin - golipids and / or by reorienting them to form a curved sur - face ( 18 , 30 ) . In support of this idea , STxB or CTxB binding to giant unilamellar lipid vesicles ( GUVs ) causes inwardly directed tubulations of these artificial lipid membranes . Similarly , in live cells subjected to treatments that block membrane scission , toxins accumulate in tubular invagi - nations originating from the plasma membrane , analo - gous to those seen in GUVs ( 18 , 30 ) . These microns - long structures are devoid of markers of clathrin - dependent endocytosis and can form even under conditions where cellular ATP is depleted ( 18 ) . Toxin - induced changes in membrane organization are thought to be important for their formation , operating from outside the cell to form plasma membrane tubules in the absence of active cel - lular processes . But it remains unclear how toxin bind - ing could supply the mechanical force needed to form the microns - long membrane invaginations observed in cells under these conditions . To what extent host cell fac - tors contribute to this process is also uncertain , as few endogenous cellular regulators of this pathway have been identified . To address these questions , we investigated the mechanisms responsible for plasma membrane deforma - tion during clathrin - independent endocytosis of CTxB in live cells . Results Assay for analysis of tubulation of the plasma membrane To study mechanisms that participate in bending the plasma membrane during clathrin - independent endocytosis , we took advantage of a previously described assay that traps STxB and CTxB in surface attached tubules thought to correspond to stalled endocytic intermediates ( 18 , 30 ) . Several conditions facilitate the growth of surface attached tubular invaginations containing fluorescently labeled toxins , including ATP depletion , actin disruption , cholesterol depletion and inhibition of dynamin . These conditions are thought to be permissive for tubule growth , but prevent tubule scission , leading to the accumulation of toxin within these structures . CTxB accumulated in linear , microns - long tubules in ATP - depleted cells and / or when actin was either disrupted or stabilized ( Figures 1A \u2013 D and S1 ) . Control experiments revealed that STxB and CTxB localized to the same tubules in cells incubated with both toxins , supporting the idea they are formed by a common mechanism ( Figure S1D ) . We also found that the tubules were rapidly labeled by a second application of CTxB after they had formed , confirming that the tubules originated from the plasma membrane and were surface attached for the time course of these studies ( Figure S1E ) . In cells treated with the dynamin inhibitor Dynasore , irregular branched networks of tubules were observed ( Figure 1E ) , resembling those seen before in cells expressing a Dynamin1 K44A mutant ( 17 ) . These findings confirm that CTxB readily associates with membrane tubules under a variety of conditions that inhibit scission , thus allowing us to further study requirements for tubule formation . Binding to a single GM1 is suf\ufb01cient to support the association of cholera toxin with tubular invaginations Current models propose compaction of glycosphingolipids by toxin binding plays an essential role in initiating mem - brane curvature and tubulating the membrane to form plasma membrane invaginations ( 18 ) . A mutant form of STxB lacking the Gb 3 binding site III demonstrated greatly reduced ability to drive tubule formation in cells , suggesting multiple glycosphingolipid binding sites enhance the ability of toxins to deform membranes ( 18 ) . To test if cholera toxin binding causes plasma mem - brane tubulations by compacting or reorienting multiple glycosphingolipids into a curved surface in a similar manner , we first studied a mixture of chimeric cholera toxins ( CTx chimera ) that bind 0 , 1 or 2 glycosphingolipid Traf\ufb01c 2015 ; 16 : 572 \u2013 590 573 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . Figure 1 : Legend on next page . 574 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane receptors ( ganglioside GM 1 ) instead of the usual 5 ( 28 ) . Unexpectedly , the chimeric mutant toxins were readily observed in tubular invaginations in ATP - depleted cells , and the average number of invaginations per cell and length of the invaginations were similar for wild type and chimeric toxins ( Figure 1F \u2013 I ) . We next asked if a toxin with only a single GM 1 binding site ( monovalent CTx ) ( 29 ) can be directed to tubules . Strikingly , this monovalent CTx was localized within tubular invaginations as well ( Figure 1J \u2013 O ) , suggesting that toxin - induced crosslinking of GM 1 is dispensable for tubule formation . Toxin binding is dispensable for tubule formation The finding that extensive crosslinking of GM 1 is not required for tubules to form suggests that the elongated tubules containing CTx may be generated by machin - ery endogenous to the host cell . If so , cellular plasma membrane proteins should also be found in the tubu - lar invaginations in the absence of bound toxin . To test this , we first screened several cell surface markers for co - localization with CTxB in tubular invaginations . Sev - eral cell surface proteins were found to extensively label tubular structures even in the absence of CTxB binding ( Figures 2 and S2 ) . As one example , the cytosolic plasma membrane - associated protein HRas ( GFP - HRas ) strongly localized to the tubular invaginations both in the pres - ence of CTxB and independently of CTxB in response to ATP - depletion , actin disruption or actin stabilization ( Figure 2A \u2013 F ) . In ATP - depleted cells , a similar number of GFP - HRas positive tubules were found in the pres - ence ( 18 \u00b1 15 , n = 26 cells ) or absence ( 18 \u00b1 11 , n = 23 cells ) of CTxB , suggesting they form by the same mecha - nism . Tubule formation was not stimulated by the GTPase activity of Ras , because a minimal membrane targeted form of GFP , GFP - HRas tail , also labeled tubules ( Figure 2G , H ) . Thus , tubulation of the plasma membrane can occur in the absence of toxin - induced cross - linking of glycolipids , indi - cating that the driving force ( s ) for tubule extension can be generated by factors endogenous to the host . An intact microtubule network is required for the formation of extended tubular invaginations It is well known that microtubules and microtubule motors are capable of deforming membranes ( 32 \u2013 34 ) . Such mech - anisms are not currently thought to contribute to the early stages of endocytosis ( 32 ) . However , CTxB has previously been found to localize within microtubule - dependent tubular invaginations of intact BSC1 cells , suggesting a microtubule - dependent process of toxin uptake ( 13 ) . Consistent with these findings , we noticed that the tubular invaginations containing CTxB in ATP - depleted cells were often directed toward the cell center in an orientation typifying the microtubule network ( Figures 1F , J and 2A ) and that the microtubule networks remained intact after ATP depletion ( Figure 3A ) . Tubular invaginations con - taining CTxB were also often found aligned closely with taxol - stabilized microtubules ( Figure 3B , C ) . When imaged over time ( Movies S1 and S2 ) , the tubules sometimes grew smoothly ( Figure 3D \u2013 F ) , but were often observed to pause and undergo bi - directional motions ( Figure 3G \u2013 I ) and branching events ( Figure 3J ) character - istic of microtubule - dependent motions . We thus asked if the microtubule network was required for tubular invagi - nations to form . Remarkably , disruption of microtubules prior to CTxB binding led to a complete loss of tubular Figure 1 : No more than one functional GM 1 binding site is required to target cholera toxin to plasma membrane invaginations . A \u2013 E ) CTxB accumulates in either linear extended tubules ( A \u2013 D ) or branched tubules ( E ) under conditions that block scission . Bar , 10 \u03bc m . ( F \u2013 M ) Cholera toxin binding mutants accumulate in tubular invaginations . F ) Cy3 - CTx chimera labels tubules in ATP depleted COS - 7 cells . G \u2013 I ) Quanti\ufb01cation of invaginations in ATP - depleted cells labeled with Cy3 - CTx chimera or Alexa555 - CTxB . G ) Percentage of cells displaying invaginations ( mean \u00b1 SD from 117 \u2013 119 cells ) . * , p < 0 . 05 , chi - squared test . H ) Average number of invaginations per cell ( mean \u00b1 SD of 42 \u2013 46 cells ) . n . s . , p > 0 . 05 ; Student t - test . I ) Length of invaginations ( mean \u00b1 SD for 219 \u2013 332 invaginations ) . n . s . , p > 0 . 05 ; Student t - test . J \u2013 M ) Both wild type CTxB and monovalent CTx accumulate in tubular invaginations in cells subjected to Jasplakinolide pretreatment prior to ATP depletion . L ) Percentage of cells displaying invaginations . ( mean \u00b1 SD of 59 \u2013 63 cells ) . n . s . , p > 0 . 05 ; chi - squared test . M ) Average number of invaginations per cell . ( mean \u00b1 SD of 59 \u2013 63 cells ) . n . s . , p > 0 . 05 ; Student t - test . N and O ) Similar to wild type CTxB , monovalent CTx accumulates in branched tubules in Dynasore - treated cells . Bars , 10 \u03bc m . Traf\ufb01c 2015 ; 16 : 572 \u2013 590 575 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . Figure 2 : Toxin binding is not necessary for tubular invaginations to form . A , B ) EGFP - HRas ( green ) is found in plasma membrane invaginations in ATP - depleted cells in both the presence ( A ) and absence ( B ) of Alexa555 - CTxB ( red ) . C \u2013 F ) Similar results were obtained for GFP - HRas in cells subjected to actin disruption ( C and D ) or actin stabilization ( E and F ) . G and H ) A construct containing only the C - terminal 10 amino acids of HRas , EGFP - HRas - tail ( green ) , also localized to tubules in both the presence and absence of CTxB . Bars , 10 \u03bc m . invaginations containing the toxin in ATP - depleted cells ( Figure 4A , C ) . Microtubule disruption also inhibited the formation of tubules containing CTxB or monovalent CTx in cells subjected to dynamin inhibition , actin disruption or actin stabilization ( Figure 4E , F ; Figure S3A , B ) . Thus , the extended tubular invaginations are strongly microtubule dependent . Microtubule plus end dynamics are not required for the growth of tubular invaginations To elucidate how microtubules might support tubulation of the plasma membrane , we first considered a mechanism in which interactions between membranes and dynamic microtubules are mediated by plus - end binding proteins to drive endomembrane translocation ( 35 \u2013 39 ) . To test this possibility , we monitored the plus - end binding pro - tein GFP - EB3 ( 40 ) . In ATP - depleted cells , GFP - EB3 still labeled microtubules , but was no longer concentrated at their tips ( Movie S3 ) , indicating enrichment of plus - end binding proteins at microtubule ends cannot be required for tubule formation . Furthermore , pretreating cells with low doses of nocodazole ( 150 nM ) to suppress micro - tubule plus - end dynamics ( 40 \u2013 42 ) had no detectable effect on the number of ATP - depleted cells that contained CTxB - positive invaginations ( Figure 4B , D ) . Dynamic microtubule growth thus cannot explain tubule extension . 576 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane Figure 3 : Tubular invaginations align along microtubules and undergo complex motions including bidirectional motility and branching events . A ) Microtubules persist in RFP - \u03b1 - tubulin expressing HeLa cells following ATP depletion . B ) CTxB positive invaginations ( green ) align with taxol - stabilized microtubules ( red ) in stably expressing RFP - \u03b1 - tubulin HeLa cells under ATP depletion . C ) Percentage of CTxB - positive tubules that align with microtubules ( black ) , partially align with microtubules ( gray ) , or do not colocalize with microtubules ( light gray ) in ATP - depleted HeLa cells expressing RFP - \u03b1 - tubulin . N = 84 cells . D \u2013 J ) CTxB - enriched tubules exhibit complex motions in live cells as illustrated by representative frames from time series and corresponding kymographs . Time stamps are in minutes : seconds . D \u2013 F ) CTxB - positive invaginations often grow in \ufb02uid directed motions . G \u2013 I ) CTxB - positive invaginations also extend , retract and regrow along the same axis . J ) Occasionally , the CTxB positive tubules undergo branching events . Bars , 10 \u03bc m . Low levels of microtubule motor activity are retained in ATP - depleted cells Another way microtubules could support the growth of in - vaginations would be through the activity of microtubule - based motors ( 32 \u2013 34 , 43 ) . Microtubule - based motility involves two classes of motor proteins , dynein and mem - bers of the kinesin family ( 44 , 45 ) . Given that the tubular invaginations underwent preferential growth toward the center of the cell , where microtubule minus ends are located ( Figure 1 , Movies S1 and S2 ) , we hypothesized that the minus - end directed motor dynein might be involved . Dynein is an ATPase ( 44 ) whose activity is expected to be attenuated in ATP - depleted conditions . We thus asked if dynein still functions as a motor in cells depleted of ATP by monitoring the intracellular movement of lysosomes labeled with mCherry - tagged LAMP1 ( 46 ) . Traf\ufb01c 2015 ; 16 : 572 \u2013 590 577 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . Figure 4 : An intact microtubule network is required for the formation of tubular invaginations . A and C ) Microtubule disruption with high dose nocodazole prevents the formation of tubular invaginations in ATP - depleted cells ( mean \u00b1 SD , N = 74 cells . ) * * p < 0 . 01 , chi - squared test . B and D ) Inhibition of microtubule dynamics with low dose nocodazole has no effect on the formation of tubular invaginations in ATP - depleted cells ( mean \u00b1 SD , N = 135 \u2013 169 cells . ) n . s . p > 0 . 05 , chi - squared test . E and F ) Microtubule disruption prior to Dynasore treatment blocks the formation of branched tubules in cells labeled with either wild type CTxB ( E ) or monovalent CTx ( F ) . Bars , 10 \u03bc m . Although strongly reduced compared to control cells , some long - range motions of lysosomes persisted in ATP - depleted cells ( Figure 5A \u2013 G ) ( Movie S4 ) . These data imply that motor proteins remain active at low levels in ATP - depleted cells , consistent with dynein\u2019s known activity under low ATP conditions in vitro ( 47 , 48 ) . We also asked whether dynein is localized appropriately to assist with tubule extension . Due to incompatibility of fix - ation conditions required to preserve dynein staining and tubule morphology , we were unable to determine whether endogenous dynein was present on tubular invagina - tions . We therefore instead used a HeLa cell line stably expressing low levels of multifunctional green fluorescent protein ( mfGFP ) - tagged 74 - kDa dynein intermediate chain ( IC74 ) ( 49 ) to visualize both simultaneously . Some mfGFP - IC74 could be observed at the plasma membrane in ATP - depleted cells , and a few dynein intermediate chain - positive puncta co - localized with the tubular invaginations ( Figure S4 ) . These findings further support the possibility that at least a small number of dyneins are localized correctly to facilitate plasma membrane tubulation under these conditions . The ATPase activity of dynein and an intact dynactin complex are required for tubule extension To test if dynein contributes to the formation of extended tubular plasma membrane invaginations , we used a small molecule inhibitor of dynein , ciliobrevin A ( 50 ) . In the presence of ciliobrevin A , we observed impaired formation of tubular invaginations containing CTxB in ATP - depleted cells ( Figure 6A , B ) . This result implicates dynein in the extension of tubular invaginations . To test this hypothe - sis in another way , we inhibited the function of dynactin , a complex required for dynein function ( 44 , 51 ) . We per - turbed dynactin by overexpressing either a GFP - tagged form of p50 / dynamitin ( 51 ) ( Figure 6C ) or a dsRed - tagged form of the dynein - binding portion of the dynactin subunit 578 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane Figure 5 : Motor - based motions persist in ATP - depleted cells . A \u2013 C ) A subset of lysosomes labeled with mCherry - LAMP1 display long range directed motions in ATP - depleted cells . B ) Time lapse of zoomed in region of the cell in panel A . An example of a lysosome undergoing long - range directed motion is marked with the arrowhead . C ) Tracings of lysosome movement in the cell shown in A . Each track is indicated by a different color . D and E ) Frequent long - range directed motions are observed under control conditions . F and G ) mCherry - LAMP1 positive lysosomes are immobile in PFA \ufb01xed cells . Elapsed time in F , H and J = 470 s . Bars , 10 \u03bc m . p150 Glued , CC1 - dsRed ( 51 ) ( Figure 6D , E ) . Overexpression of either protein strongly inhibited the formation of tubular invaginations in response to ATP depletion ( Figure 6C \u2013 E ) . p50 expression also blocked extended branched tubules from forming in Dynasore - treated cells ( Figure S3E , F ) . Thus , dynactin is required for tubulation of the membrane , further implicating dynein as a host cell factor that under - lies tubule extension . Bulk uptake of CTxB is unaffected by disruption of microtubules or the dynactin complex , suggesting the tubular carriers de\ufb01ne a low capacity endocytic pathway Given our findings that microtubules , dynactin and dynein are required for plasma membrane tubulation , we won - dered if they are also necessary for the uptake of CTxB . CTxB can be internalized by multiple mechanisms ( 12 , 13 ) , including a high capacity pathway that involves morpho - logically distinct clathrin - independent carriers ( 16 , 17 ) and tubular endocytic intermediates that contain CTxB ( 13 ) . Interestingly , we found that uptake of CTxB into ATP replete cells was unaffected by the expression of GFP - p50 ( Figure 7A , B ) or pretreatment of cells with nocodazole ( Figure 7C , D ) . Thus , the microtubule - dependent pathway cannot be highly efficient or high capacity . However , these results do not exclude the possibility that uptake of toxin by plasma membrane tubules is physiologically relevant \u2013 as cholera toxin - induced toxicity is poorly correlated with overall levels of toxin internalization ( 13 ) . Indeed , we found that intact microtubules were required for full tox - icity of CTx in polarized human intestinal epithelial T84 cells , as measured by an electrophysiological assay that monitors toxin - induced Cl \u2212 secretion ( 52 ) ( Figure 7E ) . This is consistent with a role for microtubules in the uptake or trafficking of CT . Discussion Here , we show that microtubules , dynein and dynactin pro - vide an important source of mechanical force that tubulates Traf\ufb01c 2015 ; 16 : 572 \u2013 590 579 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . Figure 6 : The ATPase activity of dynein and an intact dynactin complex are required for the formation of tubular invaginations . A and B ) Inhibition of dynein ATPase activity with ciliobrevin - A ( Cilio - A ) signi\ufb01cantly reduces the percent of cells displaying tubular invaginations ( mean \u00b1 SD from 95 \u2013 122 cells ) . * * p < 0 . 01 , chi - squared test . C ) Expression of GFP - p50 reduced the prevalence of cells with invaginations as compared to untransfected cells ( \u2212 ) or cells expressing EGFP . ( mean \u00b1 SD from 42 \u2013 101 cells ) . n . s . , p > 0 . 05 ; * * p < 0 . 01 , chi - squared test . D and E ) Expression of CC1 - dsRed ( red ) signi\ufb01cantly reduces the percent of cells with CTxB positive invaginations ( green ) as compared to untransfected cells ( \u2212 ) or control cells expressing mCherry ( red ) . ( mean \u00b1 SD from 40 \u2013 64 cells ) . n . s . , p > 0 . 05 ; * * p < 0 . 01 , chi - squared test . Bars , 10 \u03bc m . the plasma membrane during clathrin - independent endocytosis of CTxB . Interestingly , similar tubules were observed in the presence and absence of bound toxin , sug - gesting they demarcate an endogenous , toxin - independent pathway . Very recent evidence indicates microtubules and dynein are also involved in the clathrin - independent uptake of STxB via endophilin A2 - containing tubules ( 53 ) . Formation of these endophilin A2 - positive tubules is strongly induced by toxin binding ( 53 ) . Thus , the mecha - nism of dynein - and microtubule - dependent membrane tubulation that we describe here appears to be a general one utilized by multiple classes of clathrin - independent carriers , inclusive of both constitutive and cargo - induced pathways . On the basis of our findings , we propose a model wherein dynein and dynactin can interact with the plasma mem - brane and adjacent microtubules to allow formation and extension of nascent endocytic tubules ( Figure 8 ) . Dynein likely drives tubule extension by pulling the membrane along existing microtubules , albeit only slowly under conditions where ATP is limiting as in ATP - depleted cells . Other minus end directed motors , such as kinesins - 14 might also contribute to these processes . By providing an internal pulling force that drives membrane curvature , motor - driven bending of the plasma membrane could facilitate recruitment of curvature - sensitive / generating proteins that help stabilize and elongate tubules ( 54 , 55 ) . Pulling forces could also participate in other steps in 580 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane Figure 7 : Legend on next page . Traf\ufb01c 2015 ; 16 : 572 \u2013 590 581 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . clathrin - independent endocytosis . For example , they could help sort cargo into tubules in a curvature - dependent manner ( 56 \u2013 58 ) , as well as contribute to the process of membrane scission ( 53 , 59 ) . The exact mechanism by which dynein and dynactin bind the plasma membrane and how this process is regulated in clathrin - independent endocytosis remains to be deter - mined . It could involve recruitment to sites of local mem - brane bending induced by endogenous cellular factors , similar to that previously described for recruitment of dynein by retromer ( 56 ) , or in the case of the toxins , by membrane bending at the site of toxin binding as discussed further below ( 18 , 30 , 53 ) . Caveolae could potentially serve as a preferential site for dynein / dynactin recruitment to the plasma membrane , as caveolin - 1 has been observed in long , toxin - positive tubules under conditions that inhibit endocytosis ( 24 , 60 ) . The mechanism of attachment of dynein / dynactin to the plasma membrane may alternatively be related to those involved in positioning of the mitotic spindle ( 61 ) . This scenario seems less likely given that stable anchor - ing of dynein at the cell cortex is thought to facilitate microtubule - dependent pulling forces ( 62 ) . Interestingly however , microtubule - dependent plasma membrane invaginations have been reported to form in C . elegans embryos at cortical sites where spindle poles are tethered ( 63 ) . These invaginations are observed at low frequency in unperturbed embryos , but are more readily evident under conditions where the acto - myosin cortex is weakened ( 63 ) . This suggests that cortical actin reorganization may repre - sent a key event that determines whether microtubule - and dynein - dependent tubulation of the plasma membrane can occur . For example , cortical actin may normally act as a physical barrier between microtubules and the cell surface . Reorganization of cortical actin , either through endogenous processes or in response to actin perturb - ing agents ( 64 ) , may permit microtubules access to the plasma membrane , facilitating the formation of the extended tubular carriers into which endocytic cargo such as CTxB enters . Changes in cortical actin organi - zation could also potentially decrease plasma membrane tension , favoring the formation of long invaginations by microtubule - dependent pulling . Actin dynamics may in turn ultimately also contribute to the tubule scission process that releases tubules from the plasma membrane ( 21 , 53 ) . Our findings also have important implications for our understanding of how glycosphingolipid - binding toxins such as cholera toxin and Shiga toxin manipulate cell mem - branes to facilitate their uptake and subsequent cellular intoxification . It is clear that CTx and STx binding to mul - tiple copies of their glycosphingolipid receptors enhances their toxicity ( 18 , 28 , 29 , 30 , 65 ) and that toxins can induce membrane bending in vitro ( 18 , 30 ) . Toxin binding can also initiate the recruitment of curvature - sensing proteins to the plasma membrane ( 53 ) . However , cross - linking of membrane lipids is dispensable for both plasma mem - brane tubulation ( this study ) and CTx intoxication of host cells ( 28 , 29 ) . While CTxB binding is not required for extended tubular invaginations to form , membrane reor - ganizations and local bending induced by the AB5 tox - ins binding to their sphingolipid receptors ( 18 , 30 ) may function cooperatively with the microtubule - based mech - anisms of membrane tubulation we propose here . Toxin binding may for example contribute to the induction or Figure 7 : Bulk endocytosis of CTxB is largely unaffected by disruption of microtubules or the dynactin complex . A and B ) Endocytosis of dextran , transferrin and CTxB in cells expressing GFP - p50 ( gray bars ) compared to cells expressing GFP ( black bars ) . B shows mean \u00b1 SD for 37 \u2013 278 cells . n . s . , p > 0 . 05 , Student\u2019s t - test . C and D ) Effect of microtubule disruption with 5 \u03bc g / mL nocodazole ( gray bars ) on the uptake of dextran , transferrin and CTxB . Control cells were treated with DMSO ( black bars ) . D shows mean \u00b1 SD for 78 \u2013 358 cells . n . s . , p > 0 . 05 , * , p < 0 . 05 , Student\u2019s t - test . Bars , 10 \u03bc m . E ) Representative time course of toxin - induced chloride secretion in T84 cells in response to treatment with 20 nM wt CTx . Cells were either pretreated with NZ ( closed symbols ) or with DMSO ( open symbols ) prior to toxin addition to either the apical ( blue ) or basolateral ( red ) surface at t = 30 min as described in the MaterialsandMethods . Forskolin was added to control monolayers ( green or gray diamonds ) at 90 min in order to demonstrate equivalency of the secretory response and monolayer viability . The error bars indicate the variance calculated as the standard deviation ( n = 3 ) . Data are representative of the results of two independent experiments . 582 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane Figure 8 : Working model : microtubules , dynactin and cytoplasmic dynein facilitate plasma membrane tubu - lation . Dynein and dynactin provide attachment sites or generate tugging forces on the plasma membrane , leading to microtubule - dependent tubulation . Glycolipid - binding toxins may further sense , induce or stabilize membrane curvature , enabling their ef\ufb01cient sorting into tubular structures . stabilization of membrane curvature ( 53 ) or favor sort - ing of the glycosphingolipid - toxin complex into curved structures ( 66 ) . Indeed , we observed a slight preference of wild type CTxB for sorting into tubules compared to mutant toxins containing only 1 or 2 lipid GM 1 bind - ing sites . Scission of toxin - containing tubules may also depend on cooperative actions of curvature generating pro - teins and microtubule - based motors ( 53 ) . Under physio - logical conditions , STxB and CTxB could also potentially regulate cellular machinery that controls membrane cur - vature , either by signaling - based mechanisms ( 67 ) or by directly influencing microtubule dynamics ( 68 ) . Still , the cross - linking of membrane glycosphingolipids by toxin is not absolutely required for sorting into tubules or internal - ization . Our results implicate other mechanisms of mem - brane tubulation in this pathway , one of which we propose is facilitated by microtubules and the microtubule motor dynein . Materials and Methods Cells and reagents COS - 7 and HeLa cells were acquired from ATCC ( Manassas , VA ) . Stable RFP - \u03b1 - tubulinexpressingHeLacellswereagiftfromPaulChang ( M . I . T . ) . HeLacellsstablyexpressingmultifunctionalGFP ( mfGFP ) - tagged74kDa dynein intermediate chain ( IC74 ) were kindly provided by Takashi Murayama ( Department of Pharmacology , Juntendo University School of Medicine , Tokyo , Japan ) . T84 cells were cultured as previously described ( 52 ) . COS - 7 cells were maintained in Dulbecco\u2019s modified Eagle medium ( DMEM ) containing 10 % fetal bovine serum ( Life Technologies ) at 37 \u2218 C and5 % CO 2 . HeLacellsweremaintainedinRoswellParkMemorialInsti - tute medium ( RPMI ) containing 10 % fetal bovine serum at 37 \u2218 C and 5 % CO 2 . Media for RFP - \u03b1 - tubulin HeLa cells additionally contained G418 ( Corning ) , andhygromycin ( 400 \u03bc g / mL ) ( Invitrogen ) wasaddedtomedia for the IC74 cells . Cells were plated on coverslips or into MatTek cham - bers ( MatTek Corporation ) 2 days prior to experiments . Transient trans - fections were performed 24 h prior to imaging using FuGENE 6 as per manufacturer instructions ( Roche Diagnostics ) . Alexa488 - CTxBandAlexa555 - CTxBwereobtainedfromInvitrogen . CTx chimerawasgeneratedaspreviouslydescribed ( 28 ) . MonovalentCTxwas madeessentiallyasdescribed ( 29 ) . Briefly , an E . coli expressionstraincon - taining three plasmids encoding native CTA , CTB - G33D ( non - binding mutant ) and C - terminally GS - H6 - tagged wt - CTB was induced with 0 . 0005 % L - arabinose and 400 \u03bc M IPTG and grown overnight at 30 \u2218 C . Each subunit is secreted to the periplasm where holotoxins with mixed CTB pentamers assemble from a random assortment of G33D mutant and GS - H6 - tagged wt CTB monomers with CTA . This mixture of assem - bled holotoxins and free B pentamers containing from 0 to 5 GS - H6 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 583 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . tagged B subunits ( native binding sites ) was purified from a cell extract by Talon affinity chromatography , and separated into individual species by three rounds of ion - exchange chromatography . Free pentamers were first removed by binding to a cationic resin ( HS20 ) ; the mixture of holo - toxins eluting in the unbound fraction was then bound to an HQ20 anion exchange column , and individual species were eluted with a 0 \u2013 1M NaCl gradient , peak fractions were pooled , concentrated and repuri - fied by anion exchange . Densitometry of protein bands separated by SDS - PAGEshowedtheexpectedratioof1Aand1taggedwtBsubunitto4 native - sized ( G33D ) B subunits . Purified holotoxins were stored at 4 \u2218 C in 50mM Tris \u2013 HCl , pH8 . 0 . Before labeling , several preparations of single binding site holotoxin were pooled , concentrated and buffer - exchanged into PBS , pH7 . 5 by ultrafiltration using an Amicon Ultra - 4 ( Millipore ) 10K cutoff centrifugal filter . Alexa568 - labeled monovalent CTx was prepared by reacting Alexa568 - succinimidyl ester ( Invitrogen ) to 300 \u03bc g toxin chimera ( 29 ) in 100mM sodium bicarbonate buffer pH8 . 3 for 1 h under stirring at room temperature and purified using provided size exclusion chro - matography resin . Alexa568 - labeled wild type CTxB was prepared as above using recombinant B - subunit purified from periplasmic E . coli extracts . Alexa 488 - STxB and Cy3 - STxB along with a plasmid for Gb 3 transferase ( 69 , 70 ) were gifts from Ludger Johannes ( Institut Curie ) . Plasmids encoding EGFP - HRas , EGFP - HRas - tail ( consisting of GFP fused to the C - terminal 10 amino acid residues of HRas ) , and GFP - Fyn were provided by Mark Philips ( NYU School of Medicine ) ( 71 ) ; GFP - EB3 ( 40 ) was a gift from Anna Akhmanova ( Utrecht University ) ; and CC1 - dsRed and GFP - p50 ( 72 ) were gifts from Trina Schroer ( Johns Hopkins ) . LYFPGT46 ( referred to as YFP - GT46 ) was as previously described ( 73 , 74 ) . MyrPalm - mCFP was obtained from Roger Tsien ( 75 ) . A plasmid encoding mCherry - LAMP1 was generated by standard tech - niquesbyfusingthemammalianexpressionplasmidformCherrytoDNA encoding rat lysosomal membrane glycoprotein 1 ( LAMP1 ; NM _ 012857 ; gift from George Patterson , NIH ) . A rabbit anti - caveolin - 1 antibody was obtained from BD Biosciences . A mouse anti - transferrin receptor anti - body was purchased from Life Technologies . A rabbit anti - GFP antibody was obtained from Abcam . An anti - Myc ( 9B11 ) mouse monoclonal anti - bodywas from Cell Signaling . Fluorescently labeled secondaryantibodies were from Jackson ImmunoResearch and Life Technologies . D ( + ) glucose , sodium azide , 2 - deoxyglucose , bovine serum albumin ( BSA ) , trichloroacetic acid , Nocodazole ( NZ ) , Dynasore , Latrunculin A , methyl \u03b2 - cyclodextrin and ciliobrevin A ( HPI - 4 ) were purchased from Sigma Aldrich . Taxol was from Alexis Biochemical . HEPES and TAE buffers were purchased from Mediatech , Inc . . Alexa546 - dextran was from Life Technologies . Jasplakinolide and Alexa647 - transferrin were obtained from Invitrogen . Confocal microscopy Confocal microscopy was carried out on a Zeiss LSM 510 confo - cal microscope ( Carl Zeiss MicroImaging , Inc . ) using a 40X 1 . 4 NA Zeiss Plan - Neofluar oil immersion objective or 100X 1 . 4 NA Zeiss Plan - Apochromat oil immersion objective . Images were collected using 1 Airy unit confocal slices unless otherwise indicated . Cells were maintained in media supplemented with 25mM HEPES and any indicated drugs as described above for live - cell imaging experiments . Cells were maintained at 37 \u2218 C using a stage heater and objective heater during imaging . EGFP and Alexa488 were excited using the 488nm line of a 40mW Argon laser . YFP was excited using the 514nm line of the Argon laser . Alexa546 , Alexa555 , Alexa568 , Cy3 , RFP and mCherry were excited at 543nm using a HeNe laser . Alexa647 was excited at 647nm using a HeNe laser . Fluorescence emission was detected using filter sets provided by the manufacturer . For presentation purposes , images were exported in tiff format and brightness and contrast were adjusted using ImageJ or Fiji ( 76 ) . ATP depletion ATP depletion was performed by pre - incubating cells at 37 \u2218 C and 5 % CO 2 for 15min in ATP depletion medium , composed of glucose - free DMEM containing 50mM 2 - deoxy - D - glucose , 0 . 02 % sodium azide , 25mM HEPES , and 1mg / mL BSA as described in ( 77 ) . Control cells were incubated in ATP control medium ( composed of glucose - free DMEM supplemented with 50mM D - ( + ) - glucose , 25mM HEPES , and 1mg / mL BSA ) . Cells were rinsed twice , incubated for 5 min at room temperature with CTxB ( 100 or 500 nM ) , STxB ( 99 nM ) , CTx chimera ( 500 nM ) or monovalent CTx ( 400 or 800 nM ) , rinsed twice , then imaged live at 37 \u2218 C in either ATP depletion or control media . ATP depletion also induces the formation of abundant actin - rich membrane protrusions ( 78 ) , so ATP - depleted cells were always imaged in 3 - dimensions by confocal microscopy to definitively identify invaginations ( Figure S1 ) . For some experiments , cells were pretreated with Jasplakinolide ( 250 nM ) for 30min prior to ATP depletion , then imaged in the continued presence of both Jasplakinolide and ATP depletion medium . Quanti\ufb01cation of cellular ATP levels ATPdepletionwasverifiedtodecreaseATPlevelsto < 5 % ofcontrolvalues using the commercially available ENLITEN \u00ae ATP assay kit ( Promega ) . For this assay , COS - 7 cells were split into 12 well plates . After 2 days the medium was removed and some cells were incubated directly in 500 \u03bc L ATP - extraction solution [ 1 % TCA in Tris - Acetate - EDTA ( TAE ) buffer ] to collect baseline ATP readings . The remaining cells were rinsed twice with ATP depletion media and incubated at 37 \u2218 C and 5 % CO 2 . At the indicated times after the initiation of ATP depletion , the depletion media was replaced with 500 \u03bc L ATP - extraction solution . Cells were incubated in ATP - extraction solution for 30 min at RT , as described before ( 79 ) . Aliquots of the cell extract were then moved to 96 well plates and diluted tenfoldinTAEbuffer . Reactionreagentcontainingluciferasewasaddedto each well and the chemiluminescence was read on a Synergy H4 Hybrid Multi - Mode Microplate Reader ( BioTek ) . Actin disruption To disrupt actin , cells were first washed with imaging buffer , incubated for 5 min in 1 \u03bc M Alexa546 - CTxB in imaging buffer , and washed again . 584 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane Actin depolymerization was then performed by incubating the cells at 37 \u2218 C for 5min in imaging buffer containing 1 \u03bc M Latrunculin A . Control cells wereincubated inimagingbuffercontaining0 . 1 % DMSO . Cells were maintained in their respective buffer during imaging and all imaging was performed within 30 min of treatment . Where indicated , cells were preincubated with 5 \u03bc g / mL NZ for 15min on ice , followed by a 1 h incubation at 37 \u2218 C prior to Latrunculin A treatment and CTxB labeling , thenimagedliveinthecontinuedpresenceofbothLatrunculinAandNZ . Actin stabilization Cell culture media was replaced with complete imaging buffer containing 250 nM Jasplakinolide or DMSO and incubated at 37 \u2218 C and 5 % CO 2 for 30 min . Cells were then labeled with 100 nM Alexa - labeled CTxB for 5 min at RT in complete imaging buffer containing 250 nM Jasplakinolide or DMSO . Cells were rinsed and imaged in complete imaging buffer containing 250 nM Jasplakinolide or DMSO . Where indicated , cells were ATP depleted following Jasplakinolide treatment as described above . In some experiments , cells were preincubated with 5 \u03bc g / mL NZ for 15min on ice followed by a 1 h incubation at 37 \u2218 C prior to Jasplakinolide treatment and CTxB labeling , then imaged live in the continued presence of both Jasplakinolide and NZ . Dynamin inhibition Dynamin 2 was inhibited using the small molecule inhibitor , Dynasore ( 80 ) . Cell culture media was replaced with DMEM supplemented with 25 \u03bc MHEPESandeither80 \u03bc MDynasoreorDMSO . Cellswereincubated in Dynasore or DMSO at 37 \u2218 C and 5 % CO 2 for 30 min . Cells were then labeled with either 100 nM Alexa568 - CTxB or 400 \u2013 800 nM Alexa568 monovalent CTx for 5 min at RT , rinsed and imaged in DMEM supple - mented with 25 \u03bc M HEPES and either 80 \u03bc M Dynasore or DMSO . Where indicated , cells were preincubated with 5 \u03bc g / mL NZ for 15min on ice fol - lowed by a 1 h incubation at 37 \u2218 C prior to Dynasore treatment and CTxB or monovalent CTx labeling , then imaged live in the continued presence of both Dynasore and NZ . In other experiments , cells were transfected with GFP or GFP - p50 prior to Dynasore treatment and toxin labeling as described above , then imaged live . Analysis of surface accessibility of tubules ATPdepletedCOS - 7werelabeledwith25nMAlexa555 - CTxBandplaced on the stage at 37 \u2218 C . A highly concentrated dose of Alexa488 - CTxB was added to bring the final Alexa488 - CTxB concentration in the imaging buffer to 50 nM while continuously imaging . Immuno\ufb02uorescence labeling For immunostaining of caveolin - 1 and transferrin receptor , cells grown in MatTek dishes were subjected to ATP depletion and labeled with CTxB as described above . After CTxB labeling they were incubated for 30min 37 \u2218 C and 5 % CO 2 in the continued presence of the ATP depletion media . Theywerethenfixedat37 \u2218 Cwith4 % PFA / 0 . 2 % glutaraldehydefor 15min . After rinsing several times they were blocked in PBS containing 10 % FBS and 0 . 1 % saponin for 15min and then labeled with transferrin receptor or caveolin - 1 antibodies for 30min . They were again washed and then labeled with secondary antibody for 30min . After several additional washes they were imaged . In IC74 cells , immunolabeling of the tagged dynein intermediate chain subunit was performed using an anti - myc antibody in order to amplify the fluorescence signal . For these experiments , cells grown on coverslips were either ATP depleted for 15min or left untreated . They were then rinsed twice and incubated for 5 min at room temperature with 100 nM Alexa488 - CTxB or Alexa555 - CTxB . The cells were then rinsed twice and fixedat37 \u2218 Cfor15mininpre - warmed4 % PFA / 0 . 2 % glutaraldehyde . Post fixation , the cells were quenched by three rinses in 100mM glycine in PBS . Permeabilization and blocking for 60min at RT was performed in blocking buffer composed of 0 . 1 % TX - 100 in PBS containing 5 % glycine and 5 % normal goat or donkey serum . Cells were incubated with rabbit anti - myc antibody for 2h at RT . After rinsing in PBS coverslips were incubatedfor1hina1 : 200dilutionoffluorescently - conjugatedsecondary antibodies , andmountedusingProLongGold ( Invitrogen , Carlsbad , CA ) . Microtubule stabilization HeLa cells expressing RFP - \u03b1 - tubulin were incubated with imaging buffer containing 1 \u03bc M taxol for 4 h at 37 \u2218 C . They were then rinsed twice with ATP depletion media containing 1 \u03bc M taxol , incubated at 37 \u2218 C for 15 min , labeled with 100 nM Alexa - labeled CTxB for 5 min at RT , rinsed and imaged in ATP depletion media containing 1 \u03bc M taxol . Suppression of microtubule plus end dynamics Cells were incubated with imaging buffer containing 150 nM NZ ( 40 \u2013 42 ) or DMSO at 37 \u2218 C for 5 min . The media was then replaced with ATP depletion media supplemented with 150 nM NZ or DMSO and incubated at 37 \u2218 C for 15 min . Cells were labeled with 100 nM Alexa - labeled CTxB for 5 min at RT , rinsed and imaged in ATP depletion media containing 150 nM NZ or DMSO . Microtubule disruption Todisruptmicrotubules , cellswereincubatedwithimagingbuffer ( phenol red - free DMEM , 10 % BSA , 25mM HEPES , and 1mg / mL BSA ) contain - ing 5 \u03bc g / mL ( 16 . 7 \u03bc M ) NZ ( 81 , 82 ) or DMSO on ice for 15 min . Cells were then shifted to 37 \u2218 C for 1 h and subjected to further drug treatments and CTx or CTxB labeling as indicated above . Cells were imaged in the continued presence of either NZ or DMSO . The efficacy of microtubule disruptionwasconfirmedbyvisualizingthedistributionoftubulinincon - trol experiments . Analysis of \ufb02uid phase and transferrin uptake in ATP - depleted cells Control experiments were carried out to verify the efficacy of ATP deple - tionbytestingitseffectsonfluidphaseandtransferrinuptake ( FigureS1 ) . For the fluid phase uptake experiments , COS - 7 cells were preincubated in ATP control media or ATP depletion media for 15 min . They were then labeled with 100 nM Alexa488 - CTxB ( to mark the position of cells ) and Traf\ufb01c 2015 ; 16 : 572 \u2013 590 585 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . Alexa546 - Dextran ( 1mg / mL ) for 20 min at 37 \u2218 C , rinsed 10 times with the respective media and imaged live . To measure transferrin uptake in ATP - depleted cells , cells were serum starved for 1h prior to incubation in ATP control media or ATP depletion media for 15min . They were then labeled with 100 nM Alexa488 - CTxB ( to mark the position of cells ) and 25 \u03bc g / mL Alexa647 transferrin for 20 min at 37 \u2218 C , rinsed 5 times with respective media , and imaged live . Inhibition of dynein motor activity In control experiments , we defined conditions in which ciliobrevin A inhibited the dynein - based delivery of CTxB to the perinuclear region in cells with physiological levels of ATP ( Figure S3C , D ) . To generate ciliobrevinAdosedependencecurves , ciliobrevinAwasdilutedinDMSO and stocks were produced by serial dilution in DMEM + 10 % fetal bovine serum . Cells were incubated in medium containing ciliobrevin A or DMSOat37 \u2218 Cfor1h . Theywerethenlabeledwith100nMAlexa - labeled CTxB in ciliobrevin A or DMSO containing media for 5 min at RT , rinsed and either fixed immediately or shifted to 37 \u2218 C and 5 % CO 2 for 30 min priortofixationwith3 . 4 % PFAatRTfor15min . Samplesweremountedin ProLong Gold ( Invitrogen , Carlsbad , CA ) and fields of cells were imaged and image analysis performed as indicated below . For experiments examining the effects of ciliobrevin A on tubule for - mation , cell culture media was replaced with imaging buffer containing 500 \u03bc M ciliobrevin A or DMSO and incubated at 37 \u2218 C and 5 % CO 2 for 1 h . Cells were rinsed twice with ATP depletion or control media con - taining ciliobrevin A or DMSO and incubated at 37 \u2218 C and 5 % CO 2 for 15 min . Cells were labeled with 100 nM Alexa - labeled CTxB for 5 min at RT , rinsed and imaged in ATP depletion or control media containing cil - iobrevin A or DMSO . Care was taken to protect ciliobrevin - A from light throughout experiment . Analysis of \ufb02uid phase uptake in cells expressing GFP - p50 COS - 7 cells transiently expressing EGFP or EGFP - p50 were loaded with Alexa - 546 dextran ( 1mg / mL ) in 100 \u03bc L of serum free imaging buffer and incubated at 37 \u2218 C for 20 min , rinsed with imaging buffer 10 times and imagedlive . Imageswerecollectedusinga1Airyunitconfocalsliceunder identical imaging conditions for cells expressing EGFP and EGFP - p50 . Analysis of transferrin and CTxB uptake in cells expressing GFP - p50 COS - 7cellsplatedoncoverslipsweretransfectedwithEGFPorGFP - p50 . To quantify the cellular uptake of CTxB , cells were rinsed with cold imaging buffer and labeled with 100 nM A555 - CTxB for 5 min on ice . For quantification of transferrin uptake , cells were serum starved using DMEM containing 25mM HEPES for 1 h at 37 \u2218 C and 5 % CO 2 . Serum starved cells were then rinsed with cold DMEM supplemented with 10 % FBS and 25mM HEPES and labeled with 5 \u03bc g / mL Alexa568 - transferrin for 5 min on ice . Cells were rinsed with imaging buffer and shifted to 37 \u2218 C and 5 % CO 2 for 20 min . Acid stripping was performed on ice by incubating cells with 2mL of 100mM glycine , pH2 . 0 , for 5 min and then with HBSS , pH7 . 4 , for 5 min . Acid - stripped samples were subsequently incubated with 37 \u2218 C HBSS , pH7 . 4 , for 10 s to promote release of remaining surface - bound toxin or transferrin ( 83 ) . This process was repeated 3 times . Cells were fixed in 3 . 4 % PFA at room temperature for 15 min and rinsed with 1x PBS . Cells were labeled with an anti - GFP primary antibody and an Alexa488 secondary antibody . Samples were mounted in ProLong Gold . Cells were imaged at 1x zoom and multiple fields were collected using a 1 Airy unit confocal slice for CTxB and a 2 Airy unit slice for transferrin . Experiments were performed at least three times . Analysis of \ufb02uid phase uptake in NZ - treated cells COS - 7 cells were transiently transfected with EGFP ( to mark the position ofcells ) thedaybeforetheexperiment . Thenextday , cellswerepre - chilled on ice in serum free imaging buffer for 5min , then incubated with serum free imaging buffer containing 5 \u03bc g / mL ( 16 . 7 \u03bc M ) NZ or DMSO on ice for 15 min . Cells were then shifted to 37 \u2218 C for 1 h . They were then labeled with Alexa546 dextran ( 1mg / mL ) for 20 min at 37 \u2218 C in the continued presence of NZ or DMSO , rinsed 10 times with the respective media and imagedlive . Imageswerecollectedusinga1Airyunitsliceunderidentical imaging conditions for the NZ and DMSO - treated samples . Analysis of transferrin and CTxB uptake in NZ - treated cells COS - 7 cells were transiently transfected with EGFP ( to mark the position ofcells ) thedaybeforetheexperiment . Thenextday , cellswerepre - chilled on ice in serum free imaging buffer for 5min , then incubated with serum free imaging buffer containing 5 \u03bc g / mL ( 16 . 7 \u03bc M ) NZ or DMSO on ice for 15 min . Cells were then shifted to 37 \u2218 C for 1 h . Cells were then labeled with either CTxB or transferrin exactly as described above except that NZ or DMSO were included in all labeling and washing steps . Acid stripping and further processing of cells was performed exactly as described above . Electrophysiology Measurements of short circuit currents ( Isc ) and resistance ( R ) were performed on confluent monolayers of human intestinal T84 cells grown on 0 . 33cm 2 filters , as previously described ( 52 ) . Monolayers were treated with 5 \u03bc g / mL nocodazole or DMSO for 30min on ice followed by further incubation for 30min at 37 \u2218 C prior to addition of 20 nM cholera toxin . Image analysis To quantify the percentage of a population of ATP - depleted cells con - taining invaginations , full field ( 512 \u00d7 512 ) z - stacks of fields of cells were taken at 1 . 7 \u00d7 zoom on a 40 \u00d7 objective , with line averaging of 4 and opti - mal overlay in z - direction . z - Stacks were collected consecutively for 50 min after labeling with toxin . Cells were then scored by hand as display - ing or not displaying invaginations . For cells expressing a plasmid , that is CC1 - dsRed , the cells were scored in the CTxB channel with the exper - imentalist blind to which cells were expressing the plasmid and which were not . 586 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Microtubules Tubulate the Plasma Membrane To determine the number and length of invaginations z - sections were taken of individual ATP - depleted cells consecutively for 50 min after labeling with toxin . The JFilament plugin ( Dimitrios Vavylonis and XiaoleiHuang ; LehighUniversity ) forImageJwasusedtotraceindividual invaginations in 2D . The automated snake tracking feature was used to align the snakes with the invaginations . Tracings were examined and cor - rected by hand . Kymographs were produced using the MultipleKymo - graph plugin for ImageJ . To quantify the effects of ATP depletion , NZ treatment and GFP - p50 expressionontheuptakeofendocyticcargo , outlinesofcellsweremadein theGFPorCTxBchannelusingImageJ . Theaveragefluorescenceintensity in the endocytic cargo channel was then recorded from the same regions . Backgroundfluorescencewasmeasuredfromregionsdevoidofcells . After subtracting background , the data were normalized to 100 % of control values . For lysosomal tracking experiments , particle detection was per - formed using a custom - written MATLAB algorithm ( available upon request ) and tracking was performed using u - track 2 . 0 software ( http : / / lccb . hms . harvard . edu / software . html ) ( 84 ) . Both programs were run using MATLAB 7 . 11 . 0 ( Mathworks ) . The ciliobrevin A dose dependence curve was generated by collecting full field ( 512 \u00d7 512 ) 16 bit images using a 40x objective with line averaging of 8 . ROIs were then drawn around the perinuclear space , flat membraneregionsandbackgroundinImageJ . Perinuclearandmembrane per pixel fluorescence were corrected for background fluorescence and then divided to produce a perinuclear to membrane ratio per cell . Statistical analysis All experiments were performed at least twice and most were carried out three or more times . Chi - square tests were performed using Excel ( Microsoft ) , and Student t - tests were performed using Excel or OriginPro 8 . 6 ( OriginLab ) . Acknowledgments We thank Dana Hardbower , Carol Landerman and Bradley Clarke for assistance with experiments ; Drs . Ludger Johannes , Ramiro Massol , Irina Kaverina , Julie Donaldson , Mark McNiven , Stephen King and Puck Ohi for stimulating discussions ; Dr . Aurelio Galli for comments on the manuscript ; Jacob Dowler for assistance with figure preparation ; and all those who contributed reagents . Supported by NIH R01 GM106720 ( AKK ) , NIH RO1 GM073846 ( AKK ) , R01 HL111259 ( AKK ) , NIH RO1 AI31940 ( RKH ) , NIH R01 GM44589 ( TAS ) , NIH grants DK48106 , DK084424 and DK090603 and the Harvard Digestive Diseases Cen - ter P30 DK34854 ( WIL ) , the Vanderbilt Molecular Biophysics training grant ( NIH T32 GM08320 ) ( CAD ) , a Vanderbilt Discovery Grant ( Dr . Todd Graham and AKK ) , and NIH grant R44 - EB008589 ( to J . Dami - ano ) . It also utilized the core ( s ) of the Vanderbilt Diabetes Research and Training Center funded by grant DK020593 from the National Insti - tute of Diabetes and Digestive and Kidney Disease . MWD was sup - ported by the National High Magnetic Field Laboratory , which is sup - ported by NSF DMR - 1157490 and the State of Florida . The funding sources had no role in the study design , collection , analysis or interpre - tation of data , writing the report or the decision to submit the paper for publication . Supporting Information Additional Supporting Information may be found in the online version of this article : Figure S1 : Control experiments characterizing the properties of the tubular invaginations and documenting the efficacy of ATP depletion . ATP depletion impairs clathrin - dependent and - independent endocyto - sis , resultingintheaccumulationofSTxBandCTxBinplasmamembrane tubular invaginations . A ) Alexa488 - STxB and Alexa488 - CTxB accumu - late in tubular invaginations in ATP depleted COS - 7 cells . To facilitate STxB labeling , cells were transfected with Gb 3 synthase . B and C ) As reported previously ( 78 ) , fluorescent CTxB labels both plasma mem - brane invaginations ( arrowheads ) and plasma membrane protrusions ( arrows ) in ATP - depleted cells . B and C are single frames from a con - focal z - stack . Dashes mark the position of the xz - sections shown below . D ) Alexa488 - STxB and Alexa555 - CTxB colocalize in invaginations in ATPdepletedCOS - 7cellsexpressingGb 3 synthase . E ) Tubules prelabeled with Alexa555 - CTxB filled at approximately the same rateas newly added Alexa488 - CTxBaccumulatesattheplasmamembrane , indicatingthatthe tubulesareopentothefluidphaseinATP - depletedcells . Timestampsare in minutes : seconds . F \u2013 H ) ATP depletion inhibits uptake of dextran and internalization of transferrin ( Tfn ) ( Mean \u00b1 SD , N = 325 \u2013 417 cells ) . * * * , p < 0 . 001 , Student\u2019s t - test . Bars , 10 \u03bc m . ( Related to Figure 1 . ) Figure S2 : Plasma membrane derived tubules are selective for mem - brane markers . A and B ) CFP - Myr - palm and GFP - Fyn accumulate in tubular invaginations in either the ( A ) presence or ( B ) absence of CTxB in ATP - depleted cells , whereas YFP - GT46 is excluded from tubules . C and D ) Endogenous transferrin receptor ( C ) and caveolin - 1 ( D ) typically did not colocalize with tubules in immunostained ATP - depleted cells , although occasionally caveolin - 1 staining was seen at the end of a tubule ( arrowhead ) . ( Related to Figure 2 ) Figure S3 : Invaginations present following Lat A , Jasplak , or Dyna - sore treatment are dependent on microtubules and dynein . A and B ) Pretreatment of cells with 16 . 6 \u03bc M nocodazole prior to LatA ( A ) or Jasplakinolide treatment ( B ) blocks tubule formation . C and D ) Pretreatment with ciliobrevin A ( Cilio - A ) disrupts delivery of CTxB to perinuclear compartments in ATP replete cells in a dose - dependent manner . n = 36 \u2013 108 cells ; error bars = SD . E and F ) Expression of GFP - p50 ( E ) , but not GFP alone ( F ) blocked the formation of long branched tubules in Dynasore - treated cells . Similar results were obtained for both wild type CTxB or monovalent CTx . Bars , 10 \u03bc m . ( Related to Figures 4 and 6 ) Figure S4 : Some dynein is associated with the tubular invaginations . A ) Distribution of mfGFP - dynein 74kDa intermediate chain in a stably expressing HeLa cell line . Cells were fixed and immunostained using Traf\ufb01c 2015 ; 16 : 572 \u2013 590 587 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e Day et al . a myc antibody to enhance the fluorescence signal . B ) Following ATP depletion , dynein 74kDa intermediate chain staining is apparent at the plasma membrane ( arrowheads ) . C ) mfGFP - IC74 expressing cells were ATP depleted , labeled with CTxB , fixed and immunostained for tagged dynein intermediate chain . D ) Zoom of boxed region of cell shown in C . Some mfGFP - IC74 - positive puncta align along CTxB - containing tubular invaginations . Bars , 5 \u03bc m . ( Related to Figure 5 ) MovieS1 : Dynamics of growth of CTxB - positive tubular invaginations in ATP depleted COS - 7 cells . Correspond to cells shown in Figure 3 . Time stamps are in minutes : seconds . Bar , 10 \u03bc m . ( Related to Figure 3 ) . MovieS2 : Dynamics of growth of CTxB - positive tubular invaginations in ATP depleted COS - 7 cells . Correspond to cells shown in Figure 3 . Time stamps are in minutes : seconds . Bar , 10 \u03bc m . ( Related to Figure 3 ) . Movie S3 : EB3 - GFP is not enriched at microtubule plus ends in ATP - depleted cells . 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Robust single - particle tracking in live - cell time - lapse sequences . Nat Methods 2008 ; 5 : 695 \u2013 702 . 590 Traf\ufb01c 2015 ; 16 : 572 \u2013 590 16000854 , 2015 , 6 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1111 / t r a . 12269 by U n i v e r s it y O f W a s h i ng t o n , W il e y O n li n e L i b r a r y on [ 28 / 03 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti o n s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e g ov e r n e d by t h e a pp li ca b l e C r e a ti v e C o mm on s L i ce n s e", + "debelly2023cell": "Article Cell protrusions and contractions generate long - range membrane tension propagation Graphical abstract Highlights d Forces engaging actin cortex generate rapid long - range membrane tension propagation d Forces applied to cell membrane alone fail to propagate membrane tension d Unifying mechanical model explains the requirements for membrane tension propagation d Membrane tension is consistent with a long - range integrator of cell physiology Authors Henry De Belly , Shannon Yan , Hudson Borja da Rocha , . . . , Herve\u00b4 Turlier , Carlos Bustamante , Orion D . Weiner Correspondence herve . turlier @ college - de - france . fr ( H . T . ) , carlosjbustamante2 @ gmail . com ( C . B . ) , orion . weiner @ ucsf . edu ( O . D . W . ) In brief Experiments and modeling reveal the requirements for rapid and robust membrane tension propagation . De Belly et al . , 2023 , Cell 186 , 3049 \u2013 3061 July 6 , 2023 \u00aa 2023 The Authors . Published by Elsevier Inc . https : / / doi . org / 10 . 1016 / j . cell . 2023 . 05 . 014 ll Article Cell protrusions and contractions generate long - range membrane tension propagation Henry De Belly , 1 , 2 , 10 Shannon Yan , 3 , 4 , 10 Hudson Borja da Rocha , 5 Sacha Ichbiah , 5 Jason P . Town , 1 , 2 Patrick J . Zager , 1 , 2 Dorothy C . Estrada , 1 , 2 Kirstin Meyer , 1 , 2 Herve\u00b4 Turlier , 5 , * Carlos Bustamante , 3 , 4 , 6 , 7 , 8 , 9 , * and Orion D . Weiner 1 , 2 , 11 , * 1 Cardiovascular Research Institute , University of California , San Francisco , San Francisco , CA , USA 2 Department of Biochemistry and Biophysics , University of California , San Francisco , San Francisco , CA , USA 3 Department of Molecular and Cell Biology , University of California , Berkeley , Berkeley , CA 94720 , USA 4 California Institute for Quantitative Biosciences , University of California , Berkeley , Berkeley , CA 94720 , USA 5 Center for Interdisciplinary Research in Biology ( CIRB ) , Colle ` ge de France , CNRS , Inserm , Universite\u00b4 PSL , Paris , France 6 Jason L . Choy Laboratory of Single - Molecule Biophysics , University of California , Berkeley , Berkeley , CA , USA 7 Department of Physics , University of California , Berkeley , Berkeley , CA , USA 8 Howard Hughes Medical Institute , University of California , Berkeley , Berkeley , CA , USA 9 Kavli Energy Nanoscience Institute , University of California , Berkeley , Berkeley , CA , USA 10 These authors contributed equally 11 Lead contact * Correspondence : herve . turlier @ college - de - france . fr ( H . T . ) , carlosjbustamante2 @ gmail . com ( C . B . ) , orion . weiner @ ucsf . edu ( O . D . W . ) https : / / doi . org / 10 . 1016 / j . cell . 2023 . 05 . 014 SUMMARY Membrane tension is thought to be a long - range integrator of cell physiology . Membrane tension has been proposed to enable cell polarity during migration through front - back coordination and long - range protrusion competition . These roles necessitate effective tension transmission across the cell . However , con\ufb02icting observations have left the \ufb01eld divided as to whether cell membranes support or resist tension propagation . This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endoge - nous forces . We overcome this complication by leveraging optogenetics to directly control localized actin - based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual - trap optical tweezers . Surprisingly , actin - driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation , whereas forces applied to cell membranes alone do not . We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid , robust membrane tension propagation through long - range mem - brane \ufb02ows . INTRODUCTION For proper physiology , cells need a way to link short - range biochemical signaling events to long - range integration of cell - wide behaviors . Membrane tension is thought to serve as this global coordinator during cell migration . Membrane tension is the resistance of membrane to deformations . In cells , membrane tension is thought to be a combination of in - plane tension and adhesion between the membrane and underlying actin cytoskel - eton . 1 , 2 It has been proposed that membrane tension guides shape determination in motile cells by relaying actin - based pro - trusive forces at the front to the disassembly and contraction of the rear . 1 , 3 \u2013 14 Conversely , the retraction of the trailing edge ap - pears to modulate actin organization at the cell front through propagated membrane tension changes . 15 Long - range mem - brane tension propagation may similarly enable protrusions to communicate with one another for the winner - take - all competi - tion that establishes the axis of cell movement . 16 \u2013 19 Membrane tension is thought to serve as a central regulator in many other facets of cell and tissue physiologies , including cell spreading and membrane traf\ufb01cking , 11 , 20 \u2013 25 immune response , 26 , 27 cell fate , 28 , 29 cell division , 30 and organ homeostasis . 31 \u2013 33 To operate as a long - range integrator of cell shape and move - ment , membrane tension needs to propagate rapidly and ef\ufb01 - ciently across the cell . However , the actin cortex\u2019s attachment to the plasma membrane appears to inhibit membrane \ufb02ow and tension propagation when external forces are applied to the plasma membrane . 26 , 34 \u2013 36 It remains a source of signi\ufb01cant debate as to whether cell membranes support or resist long - range membrane tension propagation . This is a crucial point to resolve for understanding the role of membrane tension as a global integrator of cell shape and movement . Several factors could potentially underlie the discrepancies among these con\ufb02icting studies of membrane tension propaga - tion . For instance , tension propagation could be cell - type dependent , perhaps more ef\ufb01cient in migrating cells than in ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 , July 6 , 2023 \u00aa 2023 The Authors . Published by Elsevier Inc . 3049 This is an open access article under the CC BY license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . non - motile cells . 35 , 37 , 38 Alternatively , the origin of discrepancies could stem from the limitations of traditional tools for manipu - lating and analyzing membrane tension . For example , exoge - nously applied mechanical perturbations of the plasma membrane may elicit tension responses that are different from those elicited by the endogenously generated mechanical forces that are exerted during cell migration . To overcome these limita - tions , we implemented optogenetics to control localized actin - based cell protrusions or actomyosin contractions while simulta - neously monitoring membrane tension response at multiple locations around the cell using high - precision force measure - ments with dual - trap optical tweezers . We \ufb01nd that optogeneti - cally activated cell protrusions and actomyosin contractions both induce long - range membrane tension propagation within seconds . In contrast , perturbations affecting only the plasma membrane fail to elicit membrane tension propagation\u2014consis - tent with previous results . 34 , 35 , 37 We propose a simple unifying mechanical model in which the cortex resists membrane \ufb02ow when forces are applied to the plasma membrane alone . In contrast , when forces engage the cortex , the membrane and cortex act as an integrated system to ef\ufb01ciently transmit mem - brane tension throughout the cell . Our work demonstrates that membrane tension has the properties expected of a long - range integrator of cell physiology , critical for its role in regulating cell shape and movement . RESULTS Local cell protrusions elicit a rapid long - range increase in membrane tension To investigate membrane tension propagation upon endoge - nous force generation , we used an optogenetic approach ( Opto - PI3K ) 39 , 40 to activate localized actin - driven membrane protrusions in neutrophil - like HL - 60 cells ( Figures 1A , 1B , and S1A \u2013 S1E ; Video S1 ) and increase membrane tension at the pro - truding site . 9 , 16 , 41 The propagation of membrane tension can be A E F G B C D Figure 1 . Local cell protrusions elicit a sharp increase in membrane tension on the opposite side of the cell within seconds ( A ) Optogenetic control for light - induced activation of phosphoinositide 3 - kinase ( PI3K ) via localized recruitment of inter SH2 domain ( iSH2 ) , resulting in Rac GTPase activation that initiates actin - driven cell protrusions ( see STAR Methods ) . ( B ) Time - lapseconfocalimagesofaneutrophil - likeHL - 60cellexpressingopto - construct ( Opto - PI3K ) andmembranemarker ( CAAX - HaloTag ) , showinglocalized membrane protrusion upon light activation . ( C ) After light - activated protrusion on one side of the cell ( top of frame ) , changes in membrane tension on the opposite side ( bottom of frame ) are measured via a membrane tether held by an optical trap . ( Right ) Bright\ufb01eld image of a protruding cell during tether pulling assay . ( D ) After tether pulling measurements , the trapping laser is turned off , and the elastic recoil of the bead toward the cell is observed to con\ufb01rm the absence of cytoskeleton in the tether ( means \u00b1 SD ; n > 15 , N = 5 ) . ( E ) Representative time trace of trap force ( a direct readout of cell membrane tension change ) reveals robust and sharp increase in membrane tension over repeating cycles of light - activated protrusion on the opposite end of the cell ( as in C ) ; light : 90 s on ( shaded area ) . ( F ) Red : averaged time trace of trap force before ( steady state ) , during ( light ) , and after activating cell protrusion ( means \u00b1 SD ; n > 60 , N = 8 ) . Gray : as a control , averaged trace from cells treated with actin polymerization inhibitor ( 10 m M latrunculin B ) shows little membrane tension change upon optogenetic activation . ( G ) Averaged trap force before ( steady state ) and during activation . Box and whiskers : median and min to max ; p values from Wilcoxon paired Student\u2019s t test . Scale bars : 5 m m . See also Figure S1 and Video S1 . ll OPEN ACCESS 3050 Cell 186 , 3049 \u2013 3061 , July 6 , 2023 Article probed via a membrane tether pulled out on the opposite side of cell body using a bead ( coated with lectin to bind carbohydrate groups on the membrane ) and held by an optical trap ( a . k . a . trap - based tether pulling assay ; Figure 1C ; see STAR Methods ) . To verify that our optical trap experiments measure the forces exerted by the plasma membrane as opposed to potential actin polymerization within or along the membrane tether , 42 , 43 we ensured that the trapped beads linked to a membrane tether snap back to the cell within seconds upon the release of the op - tical trap at the end of our experimental measurements ( Fig - ure 1D ; Video S1 ; this was a standard control in our operation protocol for all sets of optical trap experiments ; see STAR Methods ) . In response to light - induced actin - driven protrusions , we observed a rapid long - range increase in membrane tension ( Figures 1E , 1F , and S1F \u2013 S1K ; Video S2 ) . The long - range rise in tension within (cid:1) 5 \u2013 15 s of light activation is in stark contrast to the conclusion arrived at in recent studies 34 that \u2018\u2018cell mem - branes resist \ufb02ow . \u2019\u2019 We also veri\ufb01ed that the observed increase in tension correlates with the local activation of the actin regu - lator , Rac GTPase , which is downstream of phosphoinositide 3 - kinase ( PI3K ) activation and precedes actin - driven protrusion ( Figures S1A \u2013 S1E ) . As an additional control , we treated the cells with the actin inhibitor latrunculin B and observed a lack of mem - brane tension increase after light activation ( Figures 1F , 1G , and S1L ) . These results demonstrate that actin - based protrusions elicit a rapid long - range propagation of membrane tension . Actin - driven protrusions stimulate global , unattenuated membrane tension propagation To examine the dynamics of membrane tension propagation in more detail , we performed a dual - tether pulling assay and simul - taneously monitored membrane tension on the side and back of the cell ( at 90 (cid:3) and 180 (cid:3) from the site of illumination , respectively ) throughout multiple cycles of light - induced protrusion ( Figures 2A \u2013 2C and S2 ; Video S2 ) . Interestingly , the two membrane tethers exhibit a near - simultaneous increase in A E B C D F Figure 2 . Actin - driven protrusions stimulate global , nearly undampened membrane tension propagation ( A ) Adual - tether pullingassaytosimultaneouslymonitormembrane tension onthefarend ( left , trap1at180 (cid:3) ) and onthesideofthecell ( top , trap2at90 (cid:3) ) during light - activated protrusion . ( B ) Representative time traces of dual trap forces over successive cycles of light - activated protrusion show coinciding tension increases on both membrane tethers adjacent to ( trap 2 ) and at the opposite cell surface from ( trap 1 ) protrusion ; light : 90 s on ( shaded area ) , 180 s off . ( C ) Correlation between trap forces at the two tether positions during activation ( blue ) remains robust from \ufb01rst activation cycle to the next ; for comparison , minimal correlation is seen between the two tethers before optogenetic activation ( gray ) . Dashed line : linear regression . ( D ) ( Left ) Time delay measured between tension rise on membrane tethers adjacent to ( trap 2 at 90 (cid:3) , blue ) and opposite from ( trap 1 at 180 (cid:3) , red ) cell protrusion . ( Right ) Inmostcells , thetrapsdetect membrane tension increaseonbothtetherswithin asecondorlessofoneanother , indicating arapidpropagationoftension across the cell . ( E ) Averaged traces of dual trap forces before , during ( light ) , and after activation ( means \u00b1 SD ; n > 25 , N = 4 ) . ( F ) Pearson correlation coef\ufb01cient between dual trap forces measured at steady state , during light activation , and recovery afterward ( 70 s post light ) . Error bar : means \u00b1 SD ; p values from Welch\u2019s unpaired Student\u2019s t test ( n > 10 , N > 4 ) . See also Figure S2 and Video S2 . ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 , July 6 , 2023 3051 Article A D G J K B E H C F I Figure 3 . Membrane tension does not propagate upon direct mechanical pulling on the cell membrane ( A ) A dual - tether assay to detect tension propagation ( static tether , left ) while anearby force is exerted through the use of an optically trapped bead to pull on the membrane (cid:1) 2 - m m away ( moving tether , right ) . ( B ) Anexample timetrace oftrapforcefor dualmembranetension measurements , inwhichonemovingtrap ( T2 , gray ) dynamicallypullson thecellmembrane by continuouslypullingandextendingthemembranetether , whereastheothertrapcontrolsasecondstaticmembranetether ( T1 , black ) tomonitornearbychanges in membrane tension . The increase in the length of the extending tether from the cell body is plotted in gray along the right y axis . ( C ) Correlation plots of normalized trap forces between the moving and static tethers . Five representative measurements from different cells are shown ; dashed lines : linear regression . ( D \u2013 F ) Similarto ( A \u2013 C ) , butprobingtensioninblebs ( membranedetachedfromactincortexgeneratedbyusinglatrunculinBtreatmenttoweakentheactincortex ) ; here , a high correlation is observed between static and moving tethers . ( G \u2013 I ) Similar to ( A \u2013 C ) , but probing tension in cells where the actin cortex has been signi\ufb01cantly disassembled using a combination of latrunculin B treatment and osmotic shock ; a high correlation is observed between static and moving tethers even at a signi\ufb01cant distance from one another ( here , 90 (cid:3) , but in Figures S3H \u2013 S3J , 180 (cid:3) ) . ( J ) Pearson correlation coef\ufb01cient between dual trap forces measured before perturbations ( none ; light gray ) , upon light - activated protrusions ( purple ; Figure 2 ) , during cell membrane pulling ( pink ; A \u2013 C ) , during membrane pulling on a bleb ( light green ; D \u2013 F ) , and during cell membrane pulling in cells with heavily dis - assembled actin cortex ( dark green ; G \u2013 I ) . Error bar : means \u00b1 SD ; p values from Welch\u2019s unpaired Student\u2019s t test ( n > 15 , N > 3 ) . ( legend continued on next page ) ll OPEN ACCESS 3052 Cell 186 , 3049 \u2013 3061 , July 6 , 2023 Article tension , with a delay , on average , of 1 . 2 \u00b1 1 . 2 s between the two ( Figure 2D ) . Readouts on both tethers plateau toward similar ten - sion levels ( Figures 2B , 2E , and S2A \u2013 S2C ) . Furthermore , mem - brane tension measurements of the two tethers remain highly correlated during light - activated protrusion and during recovery ( Figure 2F ) . Our experiments indicate that endogenous actin - based protrusions generate a long - range increase in membrane tension , which is transmitted virtually unattenuated across the cell within seconds . The actin cortex resists membrane tension propagation when external forces are applied to the membrane alone The contradictory observations between this study and some previous studies 34 , 35 , 37 , 38 may originate from how a mechanical perturbation is applied to cell membranes . Here , we optogeneti - cally induce cellular membrane protrusion ( i . e . , endogenous actin driven ) , eliciting rapid global membrane tension propaga - tion . In this approach , the forces of actin polymerization are potentially applied to both the cortex and the plasma membrane . In contrast , previous studies concluding that membrane tension is locally constrained by the actin cytoskeleton 34 used a pair of membrane tethers to pull on the cell membrane ( i . e . , exogenous bead pulling ) , thereby applying forces to the plasma membrane alone . To test whether the membrane - tether - induced forces also fail to propagate in our cells , we repurposed our dual - tether assay to dynamically pull one tether by actively moving the \ufb01rst trap while measuring membrane tension on a nearby membrane tether held in place by the second trap ( i . e . , Figure 2A versus Fig - ure 3A ) . In line with analogous experiments performed in epithe - lial cells , we observe no propagation of membrane tension from the extending tether to the static one ( Figures 3A \u2013 3C , 3J , 3K , S3A , and S3D ; Video S2 ) \u2014even with the two tethers in close proximity ( < 2 m m apart ) . In contrast , when we performed the same dual - tether assay on cellular blebs ( membrane detached from actin cortex , achieved in latrunculin - treated cells ) , tension propagates almost instantly ( < 100 m s , i . e . , below the temporal resolution of the optical tweezers instrument ; Figures 3D \u2013 3F , 3J , 3K , S3B , S3C , S3E , and S3F ; Video S2 ) , in agreement with similar measurements in epithelial cells . 34 These bleb - based ex - periments can only test tension propagation at the size scale of cellular blebs ( < 4 m m ) . To test whether tension can propagate for longer distances , we performed tether experiments in cells treated with inhibitors that ef\ufb01ciently disassemble the actin cyto - skeleton . Because latrunculin does not suf\ufb01ce to depolymerize a population of latrunculin - resistant actin \ufb01laments , we used a combination of latrunculin treatment and osmotic shock , which has previously been shown to adequately depolymerize the actin cortex , 44 as we verify in our cells ( Figure S3G ) . These cortex - free cells exhibited rapid long - range propagation of membrane ten - sion , both for traps at 90 (cid:3) ( Figures 3G \u2013 3I ) and traps at opposite ends of the cell ( Figures S3H \u2013 S3J ; Video S2 ) . Both blebs and cortically depolymerized cells propagate tension when forces are applied to the plasma membrane alone , but cells with an intact cortex do not ( Figures 3J and 3K ) . Our observations that effective membrane tension propagation depends on how me - chanical perturbations are exerted within the context of the same cell type ( Figures 2 , 3 , S2 , and S3 ) suggest that existing disagreements in the \ufb01eld are at least partially methodological in nature . While mechanical perturbations via exogenous tether pulling fail to elicit membrane tension propagation ( consistent with the \u2018\u2018picket fence\u2019\u2019 model of cortex adhesion to the plasma membrane 34 , 38 ) , endogenous actin - based force generation ef\ufb01 - ciently promotes membrane tension propagation across the cell . Long - range tension propagation coincides with directed membrane and actin \ufb02ows toward the protrusion Next , we investigated the mechanism of tension propagation from the site of protrusion to the rest of the cell . We observed the enrichment of our plasma membrane probe in the opto - induced protrusions ( Figure 1B ; Video S3 ) on a similar timescale to that of cellular deformation and tension increase ( 10 \u2013 15 s ) following optogenetic protrusion generation ( Figures S1C \u2013 S1E ) . We hypothesized that membrane and cortical \ufb02ows could underlie the rapid propagation of membrane tension from the site of protrusion to the rest of the cell . To resolve the time - depen - dent \ufb02ow of the plasma membrane and actin cytoskeleton rela - tive to light - activated protrusions , we used \ufb02uorescent markers of the plasma membrane ( CAAX - HaloTag ) and actin cytoskel - eton ( Actin - HaloTag ) ; these markers were respectively examined in separate cells . During protrusion formation , the intensity of the plasma membrane probe is increased at the site of protrusion while decreasing elsewhere ( Figures 4A , 4B , and S4A \u2013 S4G ; Video S3 ) . Because the true width of the plasma membrane is likely to be constant during our experiments , these apparent shifts in intensity presumably represent the bunching and unfold - ing of sub - resolution plasma membrane folds . 45 Neutrophils have more than twice the amount of plasma membrane needed for their apparent cell size , and this excess is held in wrinkled plasma membrane reservoirs . 46 \u2013 48 The actin probe similarly accumulated at the site of protrusion and decreased on the side of the cell opposite from the protrusion ( Video S3 ) . To characterize the \ufb02ows of membrane and actin over time , we developed a novel \ufb02ow inference method based on kymographs to predict a \ufb02ow \ufb01eld that can explain the spatiotemporal redis - tribution of membrane ( or actin ) intensity ( Figure 4C ) . A model to rationalize our experimental observations is that the protrusion resulting from the actin polymerization pulls the actin cortex to - ward the protrusion front , which , in turn , drags the membrane around the cell at each point to which it is connected . In this case , it is reasonable to assume that the \ufb02ows resulting from the actin - driven protrusions are accompanied by dissipation generated by the friction between the membrane and its under - lying cortex . Under these conditions , the observed \ufb02ow reduces to a case of optimal transport 49 , 50 , which minimizes the dissipa - tion . Thus , it is possible to infer membrane and cortical velocity \ufb01elds from the experimental kymographs using the optimal ( K ) Relativeforcechanges ( yaxis ) formembranetensionmonitoredonthestatictetherasafunctionoftheextendingtetherlength ( xaxis ) uponcontinuouspulling . Inthecaseof blebsorcells withheavily disassembled actin cortex ( light and dark green ) , thetension onstatictether increases astheextending tether lengthens ; however , thereareno perceptible tension changes on thestatictethertensionfrom thecellbody ( pink , intactcortex ) even whentheother tetherhasextended by more than 60 m m ( n > 14 , N > 3 ) . Graphical data represent means \u00b1 SDs . See also Figure S3 and Video S2 . ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 , July 6 , 2023 3053 Article transport theory ( see Methods S1 ; Figures S4H and S4I ) . We veri\ufb01ed that our inference method is able to recover velocity \ufb01elds from various simulated kymographs with high accuracy ( Figure S4J ; Methods S1 ) . Our analysis revealed the presence of a cell - wide \ufb02ow of both plasma membrane and actin cortex to - ward the protruding front during light - induced protrusion , and these \ufb02ows reverse direction during recovery ( Figures 4D , 4E , S4F , S4G , and S4K ; Video S3 ) . We used membrane photo - bleaching ( Figures 4F \u2013 4I and S4L \u2013 S4P ) and the tracking of microvilli movement ( Figure S4Q ; Video S3 ) to further validate plasma membrane \ufb02ows toward the protrusion . These directed membrane and cortex \ufb02ows provide a potential mechanism to mediate tension propagation upon cell protrusion . Actomyosin contractions also generate rapid long - range membrane tension propagation and membrane \ufb02ows Because actin - based protrusions elicit membrane and actin \ufb02ows ( Figure 4 ; Video S3 ) that mediate tension propagation ( Fig - ures 1 and 2 ; Video S2 ) , but forces applied to the membrane alone do not ( Figures 3 and S3 ; Video S2 ) , our data suggest that forces applied to the actin cortex could be central to A D E I F G H B C Figure 4 . Long - range tension propagation is accompanied by directed membrane and actin \ufb02ows toward the protrusion ( A ) Confocal images of opto - PI3K cells expressing membrane marker ( CAAX - HaloTag ) : before and during light - activated protrusion . Scale bars : 5 m m . ( B ) Kymographs of membrane \ufb02uorescence along the normalized cell circumference ( y axis ) show that over time ( x axis ) membrane accumulates toward the protruding cell front and is depleted from the back ( n > 50 , N = 6 ; Figure S4 ; see STAR Methods ) . ( C ) Flows of membrane and actin during protrusion are calculated assuming optimal transport ( see STAR Methods ) . ( D ) Membrane\ufb02ow\ufb01eldinferredusingoptimaltransportfromkymographintensitychangesovertime : shortlyafteractivationbegins ( t = 70s , darktealtraces ) , the magnitude of membrane \ufb02ow speed increases ( red dashed arrows ) , with positive speed for clockwise \ufb02ow along the cell upper half and negative speed for counter - clockwise \ufb02ow along the bottom half ( G ) , all moving toward the cell protruding front ( p ) . During recovery ( t = 170 s , light green traces ) , the direction of membrane \ufb02ow reverses ( blue dashed arrows ) . ( E ) Membrane \ufb02ow around the cell before , during , and after ( t = 30 , 70 , and 170 s ) right - side protrusion ; the \ufb02ow magnitude is denoted by the arrow size ( red : forward \ufb02ow , blue : backward ) . Membrane \ufb02ows toward the protrusion in the protruding phase and away from the protrusion during the recovery phase . ( F ) Alternative membrane diffusion assay in which we bleach the membrane marker CellMask across a wide section of the cell ( sparing a small section of the membrane maker ) , opto - activate a portion of the cell angled 90 (cid:3) from the unbleached area ( or use no light as control ) , and monitor the diffusion pattern of the unbleached area over time . ( G ) ( Top ) Example kymograph of unbiased diffusion in a control cell ( no activating light ) . ( Bottom ) Same as top but in a protruding cell , showing biased diffusion and bulk \ufb02ow of the unbleached membrane signal toward the protrusion . Heatmap similar as in ( B ) . ( H ) Sample\ufb01tsofindividualtimepointsofkymographdata ( pointscoloredbyrespectivetimepoints ) withagaussianequation ( thickcurves , coloredbyrespective time points ) . Shifts in the means of the gaussian \ufb01ts , quanti\ufb01ed bulk membrane \ufb02ow , are shown as vertical lines ( colored by respective timepoints ) . ( I ) Quanti\ufb01cation of mean shifts \ufb01t by linear regression to assay membrane \ufb02ow rate in control cells ( gray , no apparent \ufb02ow , u = 3 . 34 nm / s ) and protruding cells ( red , biased \ufb02ow toward side of protrusion , u = 35 . 51 nm / s ) ( N = 3 , n = 3 ) . See also Figure S4 and Video S3 . ll OPEN ACCESS 3054 Cell 186 , 3049 \u2013 3061 , July 6 , 2023 Article membrane tension propagation . As optogenetically activated protrusions exert forces on both the cortex and plasma mem - brane , we next sought to investigate the consequences of applying forces directly to the actin cortex . For this purpose , we leveraged an optogenetic approach to induce local actomy - osin contractility through the local recruitment of the Rho - acti - vating domain of LARG ( leukemia - associated Rho guanine nucleotide exchange factor ) ( Figures 5A \u2013 5C ) . 51 Focal Rho acti - vation elicited the local \ufb02attening of the cell ( Figure 5B ) , as ex - pected from local myosin activation . 52 , 53 Similar to light - acti - vated protrusions ( Figures 1 and 2 ) , light - activated actomyosin contractility also generated a long - range transmission of mem - brane tension ( Figure 5D and 5E ) that rapidly propagated virtually unattenuated across the cell ( Figures 5F \u2013 5H ) . As we observed for actin - based protrusions , the local generation of actomyosin contractility also generated \ufb02ows of both plasma membrane ( Figures 5I \u2013 5L ; Video S4 ) and actin cortex ( Figures S5E \u2013 S5H ; Video S4 ) toward the site of contractions . As an additional con - trol , we also used the speckle tracking of focal enrichments of the actin cortex to demonstrate cortical \ufb02ows toward the site of contractions ( Figure S5I ; Video S4 ) . These data suggest that forces applied to the actin cortex are suf\ufb01cient for ef\ufb01cient mem - brane \ufb02ows and membrane tension propagation in cells Mechanical forces engaging the actin cortex drive robust membrane tension propagation in cells To infer the critical requirements for cellular membrane tension propagation , we constructed a simple composite mechanical model in which an elastic plasma membrane is coupled to a viscous and contractile gel - like actomyosin cortex 52 via adhe - sive linkers ( Figure 6A ; see Methods S1 ) . The tension of a 2D membrane is overall of an entropic origin and corresponds to the unfolding of membrane \ufb02uctuations . In such entropic regime , membrane tension is proportional to the exponential of the area strain , as found experimentally 54 and predicted theoretically . 55 Our model assumes small strains , where this exponential be - haves approximately as an af\ufb01ne function and where the mem - brane can be considered as linearly elastic . For the simplicity of our model , we neglected the contribution of membrane reservoirs , as we do not envision that these domi - nate tension propagation . The presence of multiple membrane reservoirs that can unfold above a given tension threshold would simply limit the ability of tension to increase above this value . Our experimental data are consistent with reservoirs being accessed at the plateau phase ( maximum tension values ) of tension prop - agation . At early , pre - plateau phases of protrusion extension , membrane tension increases rapidly even for relatively small protrusions and then plateaus at a maximum even as the protru - sion continues to expand ( Figure S1F ) . Neutrophils have much larger plasma membrane reservoirs than other cells such as \ufb01 - broblasts ( Figures S6A and S6B 56 ) , making it unlikely that we are exhausting local reservoirs during early , pre - plateau phases of protrusion / contraction or during our tether pulling experi - ments . Intriguingly , both optogenetically induced protrusions and optogenetically induced contractions reach similar maximal membrane tension values , likely re\ufb02ecting the threshold of ac - cessing membrane tension buffers ( Figures 2 and 5 ) . Therefore , membrane tension propagation observed in the pre - plateau phases\u2014the focus of our study here\u2014is unlikely to be affected by the presence of folded membrane reservoirs as tension buffers , which manifest mostly in the plateau phase . In our model , the membrane displacement ( x i ) \u2014upon cortical \ufb02ows ( y i ) \u2014is determined by the overall friction imposed through the interconnecting layer of adhesive linkers ( e . g . , membrane - to - cortex attachment proteins [ MCAs ] , such as Ezrin ) . This friction m exerts a drag force on the cell membrane with a magnitude that is proportional to the relative tangential velocity between the cortex and membrane . Given a moderate membrane - cortex friction , this model adequately captures the known tension re - sponses upon different types of mechanical perturbations ( Figures 6B and 6C ) , including the absence of tension transmis - sion when only the membrane is pulled ( e . g . , exogenous tether pulling ) and rapid propagation of tension upon actin - based cell contraction / protrusion ( e . g . , endogenous force generation ) . Furthermore , the model suggests that perturbations engaging both membrane and cortex not only lead to tension propagation but also exhibit a robust tension transmission over a much wider range of membrane - cortex coupling conditions than perturba - tions engaging either component alone . To test how membrane tension propagation is affected by weakening MCA , we utilized NSC668394 , an inhibitor of Ezrin phosphorylation and Ezrin - actin binding . In accordance with the predictions of our model ( Figure 6C ) , this Ezrin inhibitor elicited only mild defects in protru - sion - mediated tension propagation ( Figures 6D , 6E , and S6C ) and elicited more signi\ufb01cant defects in contraction - mediated tension propagation ( Figures 6F , 6G , and S6C ) . Our modeling suggests that the key determinant of long - range membrane response is not the endogenous or exoge - nous application of force but rather whether the mechanical forces directly engage the actin cortex and whether the cortex is suf\ufb01ciently attached to the membrane ( i . e . , suf\ufb01cient friction / coupling ) to effectively transmit forces to produce membrane displacement upon cortex displacement . To test whether exog - enously applied forces can mediate membrane tension propa - gation , we implemented micropipette aspiration to apply me - chanical pulling on both the actin cortex and plasma membrane and monitored tension propagation using our dual - tether assay ( Figure 6H ; see STAR Methods ) . We detected a rapid , robust , and global increase in membrane ten - sion with little to no attenuation across the cell ( Figures 6I , 6J , and S6D \u2013 S6L ; Video S5 ) . Our unifying model indicates that the plasma membrane and actin cortex act as an integrated sys - tem for robust membrane tension propagation . DISCUSSION By combining optogenetics for local endogenous control of cell protrusion / contraction and optical trapping for direct membrane tension measurements in tether pulling assays , we demonstrate that local mechanical force generation such as through cellular protrusions and contractions elicits rapid long - range propaga - tion of membrane tension throughout the cell . In addition , our \ufb01ndings resolve the long - standing dispute as to whether the actin cortex facilitates or impedes tension propagation . When forces are applied to membranes alone ( e . g . , tether pull - ing ) , the actin cortex opposes membrane \ufb02ow and tension ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 , July 6 , 2023 3055 Article A E I J K L F G H B C D Figure5 . Optogeneticallyinducedactomyosincontractionsgeneraterapidlong - rangemembranetensionpropagationandmembrane\ufb02ows ( A ) Optogenetic approach for light - induced activation of leukemia - associated Rho guanine nucleotide exchange factor ( LARG ) , resulting in Rho GTPase acti - vation to initiate actomyosin - driven cell contraction ( see STAR Methods ) . ( B ) Time - lapse confocal images of a neutrophil - like HL - 60 cell expressing opto - construct ( Opto - LARG ) and membrane marker ( CellMask ) , showing localized membrane contraction and cell \ufb02attening upon light activation . ( C ) Afterlight - activatedcontractionononesideofthecell ( top ) , changesinmembranetensionontheoppositeside ( bottom ) aremeasuredviaamembranetether held by an optical trap . ( D ) Averaged time trace of trap force before ( steady state ) , during ( light ) , and after activating cell contraction ( means \u00b1 SD ; n > 55 , N = 7 ) . ( E ) Averaged trap force before ( steady state ) and during activation . Box and whiskers : median and min to max ; p values from Wilcoxon paired Student\u2019s t test . ( F ) A dual - tetherpullingassayto simultaneouslymonitor membrane tension on thefar end ( left , trap1at180 (cid:3) ) and on theside ofthecell ( top , trap2at 90 (cid:3) ) during light - activated contraction . ( G ) Averagedtracesofdualtrapforcesbefore , during ( light ) , andafteractivationshowingcoincidingtensionincreasesonbothmembranetethersadjacentto ( trap 2 ) and at the opposite cell surface from ( trap 1 ) contraction ( means \u00b1 SD ; n = 25 , N = 4 ) . ( H ) Pearson correlation coef\ufb01cient between dual trap forces measured at steady state and during light activation . Error bar : means \u00b1 SD ; p values from Welch\u2019s unpaired Student\u2019s t test ( n > 20 , N > 4 ) . ( I ) Confocal images of opto - LARG cells stained with membrane marker ( CellMask ) before and during light - activated contraction . ( J ) Kymographs of membrane \ufb02uorescence along the normalized cell circumference ( y axis ) show that over time ( x axis ) membrane accumulates toward the contracting cell front and is depleted from the back ( n = 40 , N = 3 ; see STAR Methods ) . ( K ) Membrane \ufb02ow \ufb01eld inferred using optimal transport from kymograph intensity changes over time : shortly after activation begins ( t = 120 s , teal traces ) , the magnitude of membrane \ufb02ow speed increases ( red dashed arrows ) , with positive speed for clockwise \ufb02ow along the cell upper half and negative speed for counter - clockwise \ufb02ow along the bottom half , all moving toward the site of cell contraction ( p ) . During recovery ( t = 200 s , light green traces ) , the direction of membrane \ufb02ow reverses ( blue dashed arrows ) . ( L ) Membrane \ufb02ow around the cell before , during , and after ( t = 30 , 120 , and 240 s ) right - side contraction ; the \ufb02ow magnitude is denoted by the arrow size ( red : forward\ufb02ow , blue : backward ) . Membrane \ufb02ows towardthecontractioninthecontractingphaseand away fromthecontractionduringtherecoveryphase . Scale bars : 5 m m . See also Figure S5 and Video S4 . ll OPEN ACCESS 3056 Cell 186 , 3049 \u2013 3061 , July 6 , 2023 Article propagation . However , when forces engage the actin cortex un - derneath the membrane\u2014either upon optogenetically induced actin polymerization or actomyosin contraction or upon micropi - pette aspiration\u2014tension rapidly propagates nearly undamp - ened across the cell through the generation of actin - driven mem - brane \ufb02ows ( Figure 6K ) . A D H J K E F G B C I Figure 6 . Mechanical forces acting on the actin cortex drive rapid long - range membrane tension propagation in cells ( A ) A3 - tiercompositemodelformembranetensionpropagationincells : membranedisplacements ( x i ) asareadoutfortensionpropagationuponcortical\ufb02ows ( y i ) depend on the membrane elasticity ( k ) and the membrane - cortex friction m imposed through the adhesive linkers . ( B ) Modelpredictionsofmembranetensionresponseatmoderatemembrane - cortexfriction ( seeMethodsS1 ) : onlyactin - basedpullingleadstotensionincrease and propagation ( red rectangles ) ; external pulling on the membrane alone is inef\ufb01cient ( blue circles ) . ( C ) Predicted membrane tension transmission as a function of membrane - cortex friction ( x axis ) for different targets of force application : plasma membrane only ( blue ) and actin cortex only ( red ) . ( D ) Membrane tension measurements during light - induced protrusions in cells with decreased MCA by using 25 m M of Ezrin inhibitor NSC668394 . ( E ) Red : averaged time trace of trap force before ( steady state ) , during ( light ) and after activating cell protrusion in control cells ( same data as Figure 1F ) . Orange : averaged trace from cells with decreased MCA by using 25 m M of Ezrin inhibitor NSC668394 , showing slight defects in membrane tension propagation during light - activated protrusions ( means \u00b1 SD ; n > 25 , N = 3 ) . ( F and G ) Similar to ( D and E ) but using light - induced actomyosin contractions , in which decreases in MCA lead to severe defects in membrane tension prop - agation across the cell ( red : same data as Figure 5D ; means \u00b1 SD ; n > 25 , N = 4 ) . ( H ) A dual - tether assay to simultaneously monitor membrane tension on the far end ( bottom , trap 1 at 180 (cid:3) ) and on the side of the cell ( right , trap 2 at 90 (cid:3) ) during micropipette aspiration ( top ) , which mechanically pulls on both the membrane and underlying actin cortex ( see STAR Methods ) . ( I ) Representative time traces of dual trap forces over successive cycles of aspiration ( shaded area ) show coinciding tension increases and decreases on both membrane tethers , similar to that in Figure 2B . ( J ) Averaged trap forces measured before ( steady state ) and during aspiration . The robust increase in membrane tension upon aspiration of both membrane and cortex is consistent with our model prediction ( B ) . Box and whiskers : median and min to max ; p values from Wilcoxon paired Student\u2019s t test ( n > 25 , N = 5 ) . ( K ) Schematic of requirements for effective membrane tension propagation : in the presence of membrane - to - cortex attachments , force application to plasma membrane alonedoes not generate tension propagation , in agreement with the picket fence model . However , mechanical stimuli acting on actin cortex , such as contraction , lead to rapid , long - range membrane tension propagation in the presence of signi\ufb01cant membrane - to - cortex attachments . Perturbations affecting bothactin cortex and plasma membrane ( such asprotrusionsor micropipette aspiration ) leadtorobustlong - rangemembranetensionpropagation regardless of membrane - to - cortex attachment levels . See also Figure S6 , Methods S1 , and Video S5 . ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 , July 6 , 2023 3057 Article It is noteworthy that the propagation of membrane tension af - ter cell protrusion / contraction is not only rapid but also unattenu - ated , an optimal behavior for coordinating processes at the scale of the entire cell . Our experiments and modeling suggest that one essential prerequisite for this ef\ufb01cient tension propagation is that the force transmits through the cortex . Accordingly , we propose that the cortical propagation of tension across the cell is supported by a continuous cortical network and that interac - tions between the cortex and cellular substrates other than the low - stiffness , highly compliant plasma membrane must be suf\ufb01 - ciently weak so as to minimize dissipative losses in tension propagation . Any discontinuities in the cortex or physical barriers that disrupt cortical \ufb02ow ( e . g . , the division between the apical versus basolateral portions of epithelia cells ) would be ex - pected to impede tension propagation . Consistent with this idea , we observe more robust membrane \ufb02ows toward the pro - trusion for the portions of the cell away from the substrate sur - face compared with the substrate - adhered ventral region of the cell ( Figures S6M \u2013 S6P ) . Actin - based protrusions and actomyosin contractions both mediate long - range membrane tension propagation and \ufb02ows of both actin and membrane toward the site of protrusion / contraction ( Figures 4 , 5 , S4 , and S5 ) . For actomyosin contrac - tion , the primary force is the myosin contractility that generates the actin \ufb02ows . In this case , the \ufb02ow of the plasma membrane and propagation of membrane tension depend on high MCA ( Figures 6C , 6F , and 6G ) . Compared with actomyosin contrac - tion , we have less of an understanding of why the cortex \ufb02ows toward the protrusion . We speculate that the newly polymerized actin at the leading edge generates a pushing force on the mem - brane while also generating a pulling force on the preexisting actin cortex . Future high - resolution electron microscopy images of protrusive and cortical actin could help reveal the relative or - ganization of these actin networks during motility . 15 , 57 Our modeling suggests that forces that engage both the cortex and plasma membrane could ensure robust membrane tension propagation over a wide range of membrane - to - cortex adhesion strengths ( Figures 6C \u2013 6E ) . During light - activated cell protrusions , forces from actin polymerization are exerted on both the plasma membrane and actin cortex , as can be observed by the \ufb02ow of membrane and cortex toward the site of protrusion ( Figure 4 ) . Membrane \ufb02ows enable membrane ten - sion propagation in regions of low MCA , cortical \ufb02ows permit membrane tension propagation in areas of high MCA , and forces applied to both networks should propagate tension in both set - tings . The ability of protrusions to engage both the plasma mem - brane and cortex may be particularly important for long - range tension propagation in motile cells with discontinuous MCAs . 58 We envision that actin polymerization at the leading edge , where the attachment between the cortex and plasma membrane is weak 58 , 59 , would extend the plasma membrane perpendicular to actin cortex and cause the plasma membrane to \ufb02ow toward the protrusion , whereas at the periphery of the cells ( where MCA goes back up ) , membrane tension would be propagated via pull - ing forces from the actin cortex . Our work indicates that membrane tension has the properties expected of a long - range integrator of cell physiology . Long - range propagation of membrane tension could mediate the competition among multiple protrusion sites for a \u2018\u2018winner - take - all\u2019\u2019 establishment of a dominant front 16 , 18 , 60 and could enable the front - back coordination that maintains cell shape and movement . 5 , 7 , 9 , 14 , 15 In contrast , the coordination of cellular processes that do not apply signi\ufb01cant forces to actin cortex may be more dependent on local membrane reservoirs ; this property could explain why \ufb01lopodia can coexist in adjacent re - gions of the cell without substantially affecting one another . 61 , 62 In future work , it will be critical to examine how cells modulate the dynamic range of membrane tension propagation based on the origin of the forces as well as the continuity and mechanical properties of the cortex . Limitations of the study Inthisstudy , weleveragemultiple modesofforce generation ( op - togenetic protrusion formation , optogenetic cell contractility , op - tical - trap - based tether pulling , and micropipette aspiration ) to probe the requirements for membrane tension propagation in cells . Our model system for this work is neutrophil - like HL - 60 cells . The mechanical model we propose explains our experi - mental results , correctly predicts the effects of MCA perturba - tions , and is consistent with both our experimental observations and those from other groups . Therefore , our general conclusions on membrane tension propagation are likely to translate to other cellular settings . However , it is likely that some of the quantitative featuresweobserveinourcells , inparticular , thenearlyunattenu - atedpropagation oftensionacrossthecellandtheprecisespeed of tension propagation , may differ in other cells where the cortex has different mechanical properties or where there is active me - chanosensory - based regulation of the membrane or cortex . Therefore , it will be important to extend our approach\u2014in partic - ular , the optogenetic engagement of endogenous membrane / cortex forces and direct measurement of membrane tension propagation\u2014to a broader diversity of cell types . STAR + METHODS Detailed methods are provided in the online version of this paper and include the following : d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS d METHOD DETAILS B Transduction of HL - 60 cells B Microscopy hardware B Preparation of Opto - PI3K and Opto - LARG cells for confocal imaging B Preparation , settings , and operation procedures for membrane tethering pulling experiments on C - trap (cid:2) optical tweezers with confocal imaging B Optical trapping \u2013 setting and operations B Micropipette aspiration d QUANTIFICATION AND STATISTICAL ANALYSIS B Image and membrane tension analysis ll OPEN ACCESS 3058 Cell 186 , 3049 \u2013 3061 , July 6 , 2023 Article B Statistical analysis SUPPLEMENTAL INFORMATION Supplementalinformationcanbefoundonlineathttps : / / doi . org / 10 . 1016 / j . cell . 2023 . 05 . 014 . ACKNOWLEDGMENTS TheauthorsthankM . WuforkindlysharingplasmidsandreagentsandProf . S . X . Liuforcarefulreading andcommentsonourmanuscript . Theythankallpre - sent and past members of the Weiner , Turlier , and Bustamante labs for critical discussions . They also thank the support from Lumicks on C - trap application inlivecellstudies , speci\ufb01cally Drs . S . Leachman , N . Hadizadeh , H . Kelkar , and J . Janmaat for technical assistance in instrumentation and Drs . E . Lissek , W . Peutz , M . Johnson , and P . Wheeler for support with operational sustainability . Data for this study were acquired at the Center for Advanced Light Microscopy - Nikon Imaging Center at UCSF on instruments obtained using grantsfromtheUCSF ProgramforBreakthroughBiomedicalResearchfunded in part by the Sandler Foundation , the Strategic Advisory Committee , and the EVCP Of\ufb01ce Research Resource Program Institutional Matching Instrumenta - tion Award . This work was funded by The National Institutes of Health grant GM118167 ( O . D . W . ) ; the National Science Foundation / Biotechnology and Biological Sciences Research Council grant 2019598 ( O . D . W . ) ; the National Science Foundation Center for Cellular Construction grant DBI - 1548297 ( O . D . W . ) ; a Novo Nordisk Foundation grant for the Center for Geometrically Engineered Cellular Systems , NNF17OC0028176 ( O . D . W . ) ; the Labex Memo - Life , France under the program \u2018\u2018Investissements d\u2019Avenir\u2019\u2019 ANR - 10 - LABX - 54 ( H . B . d . R . ) ; the European Research Council ( ERC ) under the European Union\u2019s Horizon 2020 research and innovation programme , grant agreement no . 949267 ( H . B . d . R . and H . T . ) ; a QLife ( ANR - 17 - CONV - 0005 ) / QBio grant ( O . D . W . and H . T . ) ; EMBO ALTF 203 - 2021 ( H . D . B . ) and T32EB009383 ( P . J . Z . ) ; an American Heart Association Postdoctoral Fellowship ( K . M . ) ; the National Institutes of Health grant K99GM137074 ( S . Y . ) ; and the Howard Hughes Medical Institute ( C . B . ) . AUTHOR CONTRIBUTIONS Conceptualization , O . D . W . , H . D . B . , and S . Y . ; Methodology , H . D . B . , S . Y . , O . D . W . , H . T . , J . T . , S . I . , H . B . d . R . , P . J . Z . , D . C . E . , and K . M . ; Investigation , H . D . B . , S . Y . , S . I . , and H . B . d . R . ; Visualization , H . D . B . and S . Y . ; Funding acqui - sition , O . D . W . , H . T . , H . D . B . , H . B . d . R . , S . Y . , and C . B . ; Supervision , O . D . W . , C . B . , and H . T . ; Writing , H . D . B . , S . Y . , O . D . W . , C . B . , and H . T . DECLARATION OF INTERESTS The authors declare no competing interests . INCLUSION AND DIVERSITY We support inclusive , diverse , and equitable conduct of research . Received : October 17 , 2022 Revised : March 10 , 2023 Accepted : May 11 , 2023 Published : June 12 , 2023 REFERENCES 1 . Pontes , B . , Monzo , P . , Gole , L . , Le Roux , A . - L . , Kosmalska , A . 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Methods 17 , 261 \u2013 272 . https : / / doi . org / 10 . 1038 / s41592 - 019 - 0686 - 2 . ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 , July 6 , 2023 3061 Article STAR + METHODS KEY RESOURCES TABLE RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be ful\ufb01lled by the lead contact , Orion Weiner ( orion . weiner @ ucsf . edu ) REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals , peptides , and recombinant proteins RPMI 1640 supplemented with L - glutamine and 25 mM HEPES Corning 10 - 041 - CM Bovine Serum Albumin ( endotoxin - free , fatty acid free ) Sigma - Aldrich A8806 Heat - inactivated fetal bovine serum Gibco 16140071 DMEM Corning 10 - 017 - CV Bovine Calf Serum Sigma - Aldrich 12138C Latrunculin B Sigma - Aldrich 76343 - 94 - 7 NSC668394 Sigma - Aldrich 341216 Carboxyl latex bead Invitrogen C37278 Concanavalin A Sigma - Aldrich C2272 SPY650 - FastAct (cid:3) Cytoskeleton CY - SC505 CellMask (cid:3) Deep Red Thermo\ufb01sher C10046 Janelia Fluor 646 Janelia JF646X Lenti - X Concentrator Clontech 631231 TransIT - 293 Transfection Reagent Mirus Bio MIR2705 96 - well # 1 . 5 glass - bottom plates Azenta Life Sciences 4ti - 0223 u - Flux (cid:3) \ufb02ow cell ( 70 - mm chips ) Lumicks C1 Glass capillary tube King Precision Glass KG - 33 Deposited data Optimal transport and model GitHub Zenodo Github : https : / / github . com / VirtualEmbryo / membrane - cortex - tension ; Zenodo : https : / / doi . org / 10 . 5281 / zenodo . 7894202 Bleaching & gaussian \ufb01tting GitHub Zenodo Github : https : / / github . com / weinerlab / Inverse _ Photobleach _ Flow ; Zenodo : https : / / doi . org / 10 . 5281 / zenodo . 7894212 Experimental models : Cell lines HL - 60s Bourne lab N / A Opto - PI3K HL - 60s Weiner lab N / A Opto - LARG HL - 60s Weiner lab N / A 3T3 - Swiss Albino UCSF cell culture facility CCLZR083 HEK293T UCSF cell culture facility CCLZR076 Software and algorithms Fiji Schindelin et al 63 N / A Prism 9 Graphpad software , Inc N / A Adobe Illustrator Adobe N / A Excel Microsoft N / A Napari Sofroniew et al 64 N / A ll OPEN ACCESS e1 Cell 186 , 3049 \u2013 3061 . e1 \u2013 e5 , July 6 , 2023 Article Materials availability All unique / stable reagents generated in this study are available from the lead contact without restriction . Data and code availability d All data reported in this paper will be shared by the lead contact upon request . d All original code has been deposited on GitHub and Zenodo and is publicly available as of the date of publication . d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request . EXPERIMENTAL MODEL AND SUBJECT DETAILS HL - 60 cells are from the laboratory of Henry Bourne and were recently veri\ufb01ed via STR pro\ufb01ling in . 39 HL - 60s were cultured in R10 growth medium , which is RPMI 1640 supplemented with L - glutamine and 25 mM HEPES ( Corning ; Corning , NY ) and containing 10 % ( v / v ) heat - inactivated fetal bovine serum ( Gibco ; Waltham , MA ) . Cultures were kept at a density of 0 . 2 \u2013 1 . 0 million cells / mL at 37 (cid:3) C / 5 % CO 2 . HEK293T cells ( used to make lentivirus for transduction of HL - 60s ) are from UCSF cell culture facility ( CCLZR076 ) and were grown in DMEM ( Corning ; Corning , NY ) containing 10 % ( v / v ) heat - inactivated fetal bovine serum ( Gibco ; Waltham , MA ) and maintained at 37 (cid:3) C / 5 % CO 2 . All media were 0 . 22 - um \ufb01ltered . Opto - PI3K cells ( iLid - BFP - CAAX , iSH2 - GFP , Pak - PBD - mCherry ) were obtained from . 39 Plasmids used to generate Opto - LARG cells ( iLid - BFP - CAAX , DHPH - ARHGEF1 - GFP , AnillinRBD - mCherry ) , Opto - PI3K expressing CAAX - HaloTag , and Actin - HaloTag were assembled using a Golden - Gate - based modular cloning toolkit . 65 . 3T3 - Swiss Albino were obtained from UCSF cell culture facility ( CCLZR083 ) and were cultured in DMEM ( Corning ; Corning , NY ) supplemented with 10 % Bovine Calf Serum ( Sigma ; St . Louis , MO , 12138C ) and maintained at 37 (cid:3) C / 5 % CO 2 METHOD DETAILS Transduction of HL - 60 cells HEK293T cells were used to generate lentivirus and were seeded into 6 - well plates until approximately 80 % con\ufb02uent . For each transduction , 1 . 5 m g pHR vector ( containing the appropriate transgene ) , 0 . 167 m g vesicular stomatitis virus - G vector , and 1 . 2 m g cytomegalovirus 8 . 91 vector were prepared for transfection using TransIT - 293 Transfection Reagent ( Mirus Bio ; Madison , WI ) . Three days post transduction virus - containing supernatants were harvested and concentrated approximately 40 - fold using Lenti - X Concentrator ( Clontech ; Mountainview , CA ) . Concentrated viruses were frozen and stored at (cid:4) 80 (cid:3) C until needed . For all transduc - tions , thawed virus was mixed with approximately 0 . 3 million cells in growth media supplemented with polybrene ( 8 m g / mL ) and incubated overnight . Cells expressing desired transgenes were isolated using \ufb02uorescence - activated cell sorting ( FACS ) as appro - priate ( FACSAria2 ; BD Biosciences ; Franklin Lakes , NJ ) . Microscopy hardware Imaging depicted in Figures 1B ; 4A , 5B , S4A , S4D , S4F , S5E , S5I , and S6N and Videos S1 , S3 , and S4 were performed at 37 (cid:3) C on a Nikon Eclipse Ti inverted microscope equipped with a Borealis beam conditioning unit ( Andor ) , a CSU - W1 Yokogawa spinning disk ( Andor ; Belfast , Northern Ireland ) , a 100X PlanApo TIRF 1 . 49 numerical aperture ( NA ) objective ( Nikon ; Toyko , Japan ) , an iXon Ultra EMCCD camera ( Andor ) , and a laser merge module ( LMM5 , Spectral Applied Research ; Exton , PA ) equipped with 405 , 440 , 488 , and 561 - nm laser lines . All hardware was controlled using Micro - Manager ( UCSF ) . Optogenetic activation was performed using a LED ( 470 - nm ) via a custom DMD ( Andor Technology ) . Illumination intensities were varied by connecting the LEDs to the analog outputs of a digital - to - analogue converter and setting the LED voltages using serial commands via custom Python code . The microscope is equipped with two stacked dichroic turrets such that samples can be simultaneously illuminated with LEDs and imaged using a 488 - nm long - pass dichroic \ufb01lter ( Chroma Technology Corp . ) Preparation of Opto - PI3K and Opto - LARG cells for confocal imaging For experiments in which we monitored cells by confocal imaging , cells were seeded in a 96 - well # 1 . 5 glass - bottom plates ( Azenta Life Sciences ) in R + B imaging media , which is RPMI 1640 supplemented with L - glutamine and 25 mM HEPES ( Corning ; Corning , NY ) and containing 0 . 2 % Bovine Serum Albumin ( BSA , endotoxin - free , fatty acid free ; A8806 , Sigma ; St . Louis , MO ) . For optoge - netic activation , cells were illuminated using DMD ( see above ) at a chosen location ( using custom Python code ) in a circular pattern of varying size ( (cid:1) 2 microns radius for Opto - PIK , (cid:1) 1 micron radius for Opto - LARG ) for a duration of 90 seconds . For Figures S6M \u2013 S6P , we imaged the cells using a two - step Z - stack of the ventral side ( (cid:1) TIRF plane ) and mid - section of the cell . For plasma membrane and actin imaging using HaloTag ( Figures 1B , 4A , S4F , S4M , S4P , S4Q , and S6N ) , cells were stained with 100nM of JF646X for 10 min before being pelleted at 300 g for 3 min and resuspended in R + B imaging media ( RPMI + 0 . 2 % BSA ) . ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 . e1 \u2013 e5 , July 6 , 2023 e2 Article For plasma membrane imaging using the membrane dye CellMask ( Figures 5B , 5I , and S4D ) , cells were \ufb01rst incubated with (cid:1) 2 - 5 m g / ml of CellMask (cid:3) Deep Red ( C10046 , Thermo\ufb01sher ) for 3 minutes at 37 (cid:3) C / 5 % CO 2 . Cells were then pelleted at 300 g for 3 min and resuspended in R + B imaging media ( RPMI + 0 . 2 % BSA ) . For actin imaging of Opto - LARG ( Figures S5E and S5I ) , cells were incubated with the actin dye SPY650 - FastAct (cid:3) ( CY - SC505 ) for 1h at 37 C / 5 % CO 2 . Cells were then pelleted at 300 g for 3 min and resuspended in R + B imaging media ( RPMI + 0 . 2 % BSA ) . Preparation , settings , and operation procedures for membrane tethering pulling experiments on C - trap (cid:2) optical tweezers with confocal imaging Cell preparation Opto - PI3K & Opto - LARG : 1 - 1 . 5 ml cells ( from culture at density of 0 . 6 \u2013 0 . 8 million cells / mL ) were stained ( with 0 . 5 m l of CellMask (cid:3) Deep Red or 100nM of JF646X ) , then pelleted down and resuspended in either R + B imaging medium ( RPMI + 0 . 2 % BSA ) or R10 medium ( all media 0 . 22 - um \ufb01ltered ) in the absence or presence of actin inhibitor ( 10 m M Latrunculin B ) for samples used in tether pulling assay . To heavily depolymerize the cortex ( Figures 3G \u2013 3K and S3G \u2013 S3J ) Cells were resuspended in a hypotonic media 60 % H2O and 40 % R10 ) containing 10 m M Latrunculin B . To decreases membrane - to - cortex attachment ( Figures 6D \u2013 6G and S6C ) , cells were resuspended in media ( R10 ) containing 25 m M of Ezrin inhibitor NSC668394 ( Sigma - Aldrich , 341216 ) . Bead preparation In a 1 . 7 ml Eppendorf tube , the following solutions were added : 9 m l of ultrapure water ( Corning , 46 - 000 - CM ) , 9 m l of carboxyl latex bead ( 4 % w / v , 2 m m ; Invitrogen , C37278 ) , and 2 m l of Concanavalin A ( 1 mg / ml ; Sigma - Aldrich , C2272 ) ; sample was vortexed at low speed at room temperature for 45 - 60 min ; 1 - 2 m l of this bead mixture stock was added into 1 ml of RPMI 1640 buffer ( 0 . 22 - um \ufb01ltered ) for samples used in tether pulling assay . Micro\ufb02uidics An u - Flux (cid:3) \ufb02ow cell ( 70 - mm chips ; Lumicks , C1 ) , installed on an heat - insulating PVC holder , was passivated with R + B imaging media ( 0 . 22 - um \ufb01ltered ) and pre - warmed at 35 - 37 (cid:3) C for 1 - 2 hours . A custom - made microchamber integrated with micropipettes ( descriptions on the assembly provided at the end ) was used in place of u - Flux (cid:3) \ufb02ow cell to apply aspiration in tether pulling assay performed on C - trap (cid:2) ( instrument operation procedures in the next section ) . During the assay , an air - pressured micro\ufb02ui - dics \ufb02ow system ( u - Flux (cid:3) , Lumicks ) , with pre - cleaned and proper dimensions of tubing connections , was used to deliver cell samples , bead solutions , and blank media / buffers ( for \ufb02ushing ) into the \ufb02ow cell or microchamber . Speci\ufb01cally , a tubing with large ID ( 1 / 32 inch ; Idex , 1520L ) was used to deliver cells at the lowest pressure setting ( 0 . 04 - 0 . 12 mbar , or sometimes just gravity \ufb02ow ) so as to minimize the shear force exerted to the cells during delivery . The delivery of beads and media was made with a narrower tubing ( ID 0 . 004 inch ; Upchurch Scienti\ufb01c , PM - 1148 - F ) . After \ufb02owing (cid:1) 200 - 500 m l ( suf\ufb01cient to displace dead volumes combined within the micro\ufb02uidics system ) of cells into the C - trap (cid:2) system pre - warmed at (cid:1) 36 (cid:3) C , incubated for 15 - 20 min so that the cells settle and stably attach to the bottom surface of the u - Flux (cid:3) \ufb02ow cell . Cell locations were then marked prior to the subsequent tether pulling experiments with optical traps . The cell samples were replenished every 1 . 5 - 2 hours , with abundant \ufb02ushing of R + B imaging medium in between ( which ensures the \ufb02ow cell surface remains properly passivated ) . Optical trapping \u2013 setting and operations A commercial dual - trap optical tweezers with 3 - color confocal imaging , aka C - trap (cid:2) , from Lumicks was used to perform the tether pulling assay with concurrent \ufb02uorescence imaging . The \ufb02ow cell , or microchamber , held paralleled to the table surface was aligned perpendicular between a water objective ( 60x , NA 1 . 2 ; Nikon , MRD07602 ) coming from the bottom and a matching condenser ( 60x , NA 1 . 4 , used with Type A immersion oil ; Leica ) coming from the top . The \ufb02ow cell was positioned in between the two such the IR laser beams ( 1064 nm ) focused down by the objective were formed inside the \ufb02ow cell (cid:1) 10 - 20 m m above the inner bottom surface ( with the \ufb02ow cell nano - stage set at the middle position ) . After the \ufb02ow cell , the condenser can adequately collect photons from the IR trapping beams and project on to position - sensitive detectors ( PSDs ) for accurate trap force measurement ( data acquisition at a rate of 78125 Hz and later down sampled to 10 Hz for analysis ) . The objective also di - rects \ufb02uorescence excitations in the visible wavelength range ( 488 , 532 , and 642 nm respectively for opto - tool , Rac / RhoA biosensor , and CellMask / HaloTag ) into the \ufb02ow cell ( or microchamber ) . The two set of light sources ( IR and visible ) were controlled by separate telescopes and mirror - steering systems upstream from the objective . The same objective collected the emission pho - tons from the imaging / optical trapping sample plane inside the \ufb02ow cell for \ufb02uorescence imaging ( bandpass \ufb01lters : 512 / 25 , 582 / 75 , and 680 / 42 ; camera pixel size : 100 nm ; frame rate depends on confocal scanning area size ) , whereas the condenser provided bright \ufb01eld imaging ( 850 nm LED light source ) recorded at 10 Hz . Both the objective and the condenser were pre - warmed to 35 - 37 (cid:3) C ( temperature control unit , Lumicks ) for at least 2 - 3 hours prior to cell experiments . The IR trapping power was typically set at 100 % trapping laser , 10 % overall power , and 50 - 50 split between trap 1 ( T1 ) and trap 2 ( T2 ) , which is about (cid:1) 175 mW per trap ( measured at the objective front ) and (cid:1) 0 . 2 pN / nm in trap stiffness for a 2 - m m bead ( bead corner frequency (cid:1) 2500 Hz ) . Low settings of excitation laser were suf\ufb01cient for \ufb02uorescence imaging ( typically (cid:1) 2 - 5 % of total power gives (cid:1) 0 . 02 - 0 . 04 m W measured at the objective front ) , minimizing the photo - toxicity to the cell during experiments . ll OPEN ACCESS e3 Cell 186 , 3049 \u2013 3061 . e1 \u2013 e5 , July 6 , 2023 Article At the beginning of each cell recording in the tether pulling assay , 2 - m m Concanavalin - coated beads were \ufb02owed into the micro - chamber ( e . g . , at 0 . 4 mbar via channel 5 in u - Flux (cid:3) C1 \ufb02ow cell ) and single beads were captured in either one or both traps ; we then moved the \ufb02ow cell stage to bring the beads to a cell location marked after incubation ( as described earlier ) . With beads in the vicinity of the cell , i . e . , in z - axis at the same confocal imaging plane for the cell ( (cid:1) 2 - 6 m m from the \ufb02ow cell bottom surface ) and (cid:1) 4 - 6 m m away from the cell body in the x - y plane , the trap stiffness was calibrated , and any residual force readout were zeroed before engaging the bead with the cell body to form membrane tethers . Region of interest ( ROI ) was cropped for bright \ufb01eld imaging ( typically an area of 35x45 m m ) , and continuous recording at 10 Hz was initiated . 1 . Tether pulling assay with light - activated cell protrusions : as seen from the bright \ufb01eld camera , we approached beads to position them in direct contact with the cell body ( even pressing a little , judging from the counter force acted on the bead in the trap ) , then we waited for several seconds before carefully ( slowly ) pulling out membrane tethers ( (cid:1) 4 - 10 m m in length ) at the desired con Figuration ( e . g . , two tethers right angle from each other ) . We monitored steady state tension for at least 1 min ( Figure S1G ) before local 488 - nm excitation ( ROI : 6x10 m m ) continuously for 90 sec on the opposite site of ( or right angle from ) the membrane tether . Upon localized 488 - nm illumination , the local recruitment of opto - controls ( iSH2 labelled with EGFP ) to trigger cell protrusions was also imaged simultaneously ( (cid:1) 1 - 1 . 3 sec / frame scanned ) . Post pro - trusion activation , we monitored cell membrane tension recovery for 180 sec and repeated activation cycles for as long as the tethers last ( see Video S2 ) . At desired time points , i . e . , before , during , or after 488 - nm light activation , the activated Rac was speci\ufb01cally imaged via 532 - nm illumination to visualize the distribution of the Rac biosensor ( Pak - PDB - mCherry ) inside the cell ( see Figures S1B \u2013 S1E and Video S1 ) . Similarly , the changes in cell membrane morphology were imaged over time with 642 - nm illumination ( for CellMask Deep Red or Halo - tag 660 if cells were stained earlier ) . 2 . Other experimental conditions in tether pulling assay , including controls : following the same bead engagement procedure described above , membrane tethers were pulled out from the cell body or from small patches of vesicle - like , outward budding membrane blebs that are detached from actin cortex upon Latrunculin treatment . Speci\ufb01cally , after the \ufb01rst membrane tether was formed , the second tether was pulled from a nearby location (cid:1) 2 m m away . The membrane tension was recorded in the same fashion as detailed earlier but for the following conditions : light activation on wild - type cells or drug - treated opto - PI3K cells ; in the absence of any light illumination , we moved one trap to extend the length of one tether on the cell body ( or bleb ) and monitor the tension response on the other ( see Video S2 ) ; or instead of 488 - nm illumination ( which triggers actin - driven cell protrusion ) , the cell was engaged with micropipette aspiration , which exerts mechanical pulling on both membrane and cortex , and the membrane tension was recorded over cycles of aspiration and relaxation ( see below ; Video S5 ) . For Figures S6A \u2013 S6B , we pulled tethers from either HL - 60s or 3T3 cells at a constant rate until eventual tether breakage ( following method from Raucher and Sheetz 56 ) . We then measured the bead - to - cell distance at the point of tether rupture as detected by our force measurements . At the end of each measurement , unless tethers had already broken on their own or by debris falling into the trap , trapping lasers were turned off to observe the tether and bead elastic recoil toward the cell as a control of the absence of cytoskeleton in the tethers ( Figures 1D , S3C , and S3J and Video S1 ) . Micropipette aspiration A custom - made microchamber was used to implement micropipette aspiration on the C - trap (cid:2) system . Speci\ufb01cally , a micropipette of 2 - 6 um tip diameter was prepared by gravity pulling a thin glass capillary tube ( ID 0 . 040 + / - 0 . 010 mm , OD 0 . 080 + / - 0 . 010 mm , length 150 mm ; King Precision Glass , KG - 33 ) that was threaded through a heated platinum coil ( (cid:1) 2 mm in diam . ; Pt wire is 0 . 3 mm in diam . , Alfa Aesar ) upon application of a desired voltage . The micropipette tip size generally correlates with the heating time required to pull the glass tube apart ; the faster heating , the more rapid the pull , giving micropipette tips in smaller diameters . The micropipette was then sandwiched between a 1 - mm glass cover slide ( 3\u2019\u2019x1\u2019\u2019x 1 mm ; VWR ) and a # 1 . 5 glass cover slip ( 24x60 mm ; VWR ) , held together with two pieces of melted Nesco\ufb01lm ( 100 um in thickness ; Karlan ) as sealant and spacer in the microchamber . 6 holes were drilled prior on the glass slide to provide inlets , which are connected to valves and uFlex (cid:3) pressurized syringe reservoirs ( for cell and bead samples delivery as well as buffer \ufb02ushing ) , and outlets towards the waste collection . The micropipette was con - nected to a separated micro\ufb02uidic pressurized system ( MFCS (cid:3) - EZ from Fluigent ; input : - 600 mbar , output : - 69 to 0 mbar ) powered by a small \ufb02oor pump ( KNF , model : N86KN . 18 , with manual regulator ) to provide aspiration control in the tether pulling assay on C - trap (cid:2) . The aspiration pressure zero point for each micropipette was carefully calibrated and set to have no outward nor inward \ufb02ow detectable to a laser trapped bead that was placed at the tip opening of the micropipette . During the experiments , cells were delivered into the microchamber at the same gentle \ufb02ow rate ( 0 . 04 - 0 . 12 mbar , or sometimes just gravity \ufb02ow ) and captured by the optical trap , which quickly brings the cell to the micropipette tip . A minute amount of suction was applied to keep the cell stably engage with the tip ( so it neither \ufb02oats away from the tip nor falls back into the optical trap ) but without any visible deformation of the cell morphology ( as seen in bright \ufb01eld camera ) . Then following the same bead calibration procedure and membrane tether pulling process as described earlier , consecutive rounds of aspiration and relaxation were performed on the cell for as long as the membrane tethers persist ( see Video S5 ) . ll OPEN ACCESS Cell 186 , 3049 \u2013 3061 . e1 \u2013 e5 , July 6 , 2023 e4 Article QUANTIFICATION AND STATISTICAL ANALYSIS Image and membrane tension analysis Fiji ( NIH ) , 63 Excel ( Microsoft ; Redmond , WA ) , custom Python code , and Prism ( Graphpad software , Inc ) were used for image and membrane tension analysis . Average trap force plots ( Figures 1F , 2E , 5D , 5G , 6E , 6G , S1J , S2A \u2013 S2B , and S5C ) were obtained by aligning trap force traces at time of light induction . Average linked trap force plots were made using Prism Graphpad ( software , Inc ) . In Figures 1G and 5E , average trace trap force was measured for 60 seconds before light induction ( steady state ) and for the duration of the light induction ( 90 seconds , Light ) . For Figure 6J , average trace trap force shown here for 30 seconds before aspiration ( steady state ) , for the duration of aspiration ( 15 - 30 seconds ) , and for intervening recovery periods . Pearson correlation coef\ufb01cients between T1 and T2 were calculated using Prism Graphpad software , Inc ) ( Figures 2F , 3J , 5H , and S6K ) . For Figures 2F and 3J , we used 30 seconds before activation for steady state , 30 seconds of light induction for opto - activated protrusion , and (cid:1) 10 - 30 seconds of active tether pulling on cell membrane ( tether length > 30 m m ) and on blebs . In Figure 2F , for \u2018 + Light\u2019 we used the full duration of light activation ( 90sec ) and for \u2018Recovery\u2019 70sec post light induction . In Figure S6K we used 15 - 30 sec pre - aspiration for steady state value and full duration of aspiration ( (cid:1) 15 - 30sec ) for aspiration . Delay between T2 and T1 during light induced protrusion ( Figures 2D , S5D , and S6J ) was calculated by measuring the time differ - ence between light induction and change in trap force slope for each trap . Of note , measuring time difference from light induction to plateau in force increase yields similar results ( i . e . , delay time between the two traps is still of (cid:1) 1 sec ) . For measurement of relative tether force over distance of moving tether ( Figure 3K ) , we normalized the trap force of static tether by its average when the extending tether was at distance < 1 m m ( namely , before any active pulling ) . For Figures S5E \u2013 S5F , we observed that FastAct ( see above ) intensity linearly increases during imaging . To make sure that these increases did not interfere with our quanti\ufb01cations , we used a set of (cid:1) 40 unstimulated cells , acquired in parallel of every Opto - LARG experiments using FastAct and corrected our measured \ufb02uorescence intensity to compensate for this passive intensity increase . For Figure S1F , in combination to trap forces measurements ( see above ) , cell diameter at the long axis was measured using a custom Fiji plugin and bright\ufb01eld imaging from optical trap setup as a proxy to roughly approximate cell shape changes during light - induced protrusions . For Figure S4Q , microvilli tracking was achieved by manually tracking microvilli over consecutive time frames using HaloTag - CAAX ( see above ) as membrane marker . For Figure S5I , Actin speckle tracking was achieved by using actin dye FastAct ( see above ) and by manually tracking distinct actin features ( e . g . , high intensity points ) over consecutive time frames . For tether length tracking ( Figures 1D , 3B , 3E , 3H , 3K , S3A \u2013 S3C , S3I , and S3J ) , we used a custom - made Fiji macro tracking the position of the bead overtime using bright\ufb01eld recordings during optical trapping experiments . The timestamps for turning the trap off were also recorded . In Figure 4G , kymographs were generated by segmenting the cell body through the HT - CAAX ( JFX - 549 ) channel and \ufb01nding the three - pixel wide boundary pixels that best capture the membrane of the CellMask channel . This segmented cell outline is unraveled and averaged over every three values to provide 1 x N arrays which are stacked to show the evolution of membrane signal over time . Image segmentation code utilized the Python package Scikit - Image . 66 In Figure 4H , f \u00f0 x \u00de = m , e (cid:4) \u00f0 x (cid:4) c \u00de 2 2 r 20 ! + o CellMask signal along the membrane is \ufb01tted with the parametric extension of the gaussian equation de\ufb01ned as : where x is the distance along the cell\u2019s perimeter in m m , m is the peak of the CellMask signal , r0 is the width of the CellMask signal , c position of the peak CellMask signal , and o is the offset of the CellMask signal from zero . The shift in the peak CellMask signal along the membrane was quanti\ufb01ed over time for both control and protruding cells in Figure 4I . Membrane \ufb02ow rates were calculated by taking the slope of the \ufb01tted linear regression lines and averaging the \ufb02ow rates within the control and protruding groups . Code for gaussian and linear regression \ufb01tting utilized the curve _ \ufb01t and linregress functions in the Python package Scipy . 67 Image analysis and gaussian \ufb01tting code in available on Github and Zenodo . Statistical analysis For all statistical analysis , PRISM 9 ( Graphpad software , Inc ) was used . Statistical details can be found in the legend of each \ufb01gure . N represents number of independent biological replicates . Pooled independent experiments are used in dot plots . Unless speci\ufb01ed otherwise , error bars always represent SD . ll OPEN ACCESS e5 Cell 186 , 3049 \u2013 3061 . e1 \u2013 e5 , July 6 , 2023 Article Supplemental \ufb01gures 0 20 40 60 0 10 20 30 40 50 T r ap f o r c e ( p N ) Time ( sec ) Light @ t = 0s A c t i v a t i ng li gh t ( C on f o c a l 488n m ) B r i gh tf i e l d + 2s + 10s + 15s + 30s iSH2 - EGFP 0 1 2 3 4 5 0 10000 20000 30000 40000 Distance ( \u03bc m ) I n t en s i t y ( a . u . ) 0s 2s 10s 30s 15s T 0 30s C D E 0 1 2 3 4 5 Distance ( \u03bcm ) A B Light Anchor Anchor Rac Local protrusion iSH2 iSH2 PI3K Pak - PBD B i nd s a c t i v e R a c PIP3 High Low H a l o T ag - C AAX R a c b i o s en s o r P a k PB D - m C he rr y Before activation After activation Other example post activation High Low Steady state membrane tension measurments Time ( sec ) WT cells + light ISH2 - GFP only cells ( no membrane anchor ) G H K L Light Light Light + 10\u03bcm LatB Light + 10\u03bcm LatB Pre - activation ( steady state ) Activation ( protruding ) Recovery Return to steady state Light Light Light Light Light eeeee Light ) ) Light 50 G F I J \u00d8 Light Risingforce Force plateauing ( legend on next page ) ll OPEN ACCESS Article Figure S1 . Optogenetic control of PI3K leads to local Rac activation , which triggers localized actin - driven cell protrusion and rapid mem - brane tension increase , related to Figure 1 ( A ) Membrane - anchored optogenetic control for light - induced activation of phosphoinositide 3 - kinase ( PI3K ) : upon localized 488 - nm excitation , the membrane anchor protein ( iLid - BFP - CAAX ) undergoes a conformational change , which results in the binding of inter SH2 domain ( iSH2 ) to the illuminated region . iSH2 proceedstorecruitPI3K , whoselipidproduct ( PIP 3 ) inducestheactivationofRacGTPase ( Rac ) . ActiveRacthentriggersactinpolymerizationleadingtolocalized membrane protrusion . By imaging the mCherry - labeled Rac biosensor ( Pak - PBD - mCherry ) , which recognizes and binds the active GTP - bound Rac , we can monitor Rac activation during light - induced protrusions ( see STAR Methods ) . ( B ) Time - lapse confocal images of HL - 60 cells expressing opto - construct ( Opto - PI3K ) , membrane marker ( CAAX - HaloTag , imaged on top ) , and Rac biosensor ( PAK - PBD - mCherry , imagedonbottom ) . Middleand right : localized recruitments ofactiveRaciscon\ufb01rmedatthesiteoflightactivationforcellprotrusion ( boxin black dashed line ) . Scale bar : 5 m m . ( C ) Time - lapse bright\ufb01eld ( top ) and confocal images ( bottom ) of an opto - PI3K cell during light activation . The speci\ufb01c recruitment of PI3K activator , ( iSH2 - EGFP ) to the illuminated area ( box in white dashed line ) is monitored upon 488 - nm excitation . Within 2 s ( between the \ufb01rst two frames ) , iSH2 has redistributed from the cytoplasm to the plasma membrane . Scale bar : 1 m m . ( D ) Fluorescence intensity line scans ( along the white dashed line in ( C ) show the enrichment of opto - construct ( iSH2 - GFP ) at the cell protruding site over time . ( E ) Kymograph of the above line scan ( white dashed line in C ) shows that after iSH2 is recruited to the membrane , the cell contour ( i . e . , its membrane ) rapidly expands outward . ( F ) In red , average time trace of cells before and during light - induced protrusion . In green , apparent cell diameter ( long axis ) over time as proxy of cell shape change and increases in apparent surface area during protrusion . Trap force and shape change are correlated during the initial phase of the protrusion ( rising phase ) but then are decoupled as the cell access its membrane reservoirs limiting further increases in membrane tension ( plateau phase ) even as the protrusion continues to extend ( means \u00b1 SD ; n = 15 , N = 5 ) . ( G ) Representative time trace of trap force measured from the tether pulling assay with a cell at steady state : membrane tension remains stable with low magnitude of stochastic \ufb02uctuations . ( H ) As a control , we light activate the wild - type ( WT ) cells , which lack opto - constructs , and use the same tether pulling assay described above to monitor membranetensionresponsebefore , during , andafter488 - nmillumination ( purpleshadedarea ) . Representativetimetraceoftrapforceforcellmembranetension recorded from WT cells with light activation . The activation light alone does not elicit any changes in cell morphology or membrane tension responses . ( I ) Inanothercontrol , welightactivatecellslackingthemembraneanchorproteinforopto - control ( iLid - BFP - CAAX ) andmonitortheirmembranetensionresponse upon 488 - nm illumination ( purple shaded area ) . No perceptible changes in cell morphology or membrane tension were observed . ( J ) Averaged time trace of trap force ( red ) for cell membrane tension recorded before ( steady state ) , during ( activation ) , and after ( recovery and return to steady state ) light - inducedprotrusionontheoppositesideofthecell ( seeFigure1C ) . Individualdatatraces areshowninlightgray ( samedataasinFigure1F , n > 60 , N = 8 ) . Cells at steady state exhibit stochastic \ufb02uctuations in membrane tension , similar to that shown earlier in ( G ) . Upon light activation ( purple shaded area ) , membrane tension rapidly increases and levels off to a plateau toward the end of activation ( total 90 s ) . The presence of a plateau potentially indicates that membranereservoirsunfoldtoprovideextramembrane , thusbufferingthetensionrise . Shortlyaftertheactivationlightisturnedoff , membranetensiongradually decreases to the steady - state level . ( K ) Two example time traces of trap force for membrane tension before , during , and after light - induced cell protrusion . ( L ) Same as ( K ) but recorded from cells treated with actin inhibitor ( 10 m M latrunculin B ) . We veri\ufb01ed that latrunculin B treatment neither impairs the opto - tool recruitment nor the subsequent Rac activation . This control shows that the increase in membrane tension measured at the opposite side of cell protrusion is dependent on the actin cytoskeleton . ll OPEN ACCESS Article Figure S2 . Membrane tension propagates within seconds across the cell during actin - driven protrusion , related to Figure 2 ( A ) Red and blue : averaged time traces of trap force for dual membrane tensionmeasurements before ( steady state ) , during ( light ) , and after ( recovery ) activating cell protrusion . A nearly coinciding tension increase is observed between the membrane tether adjacent to ( trap 2 , blue ) and opposite from ( trap 1 , red ) cell protrusion . Gray : as a control , averaged trace from cells treated with actin inhibitor ( 10 m M latrunculin B ) shows no membrane tension change upon activation ( means \u00b1 SD ; n > 15 , N > 4 ) . ( B ) Zoom - in on traces in ( A ) : increases in membrane tension emerge on both tethers within the \ufb01rst 5 \u2013 10 s of light activation . ( C ) Three example time traces of trap force for dual membrane tension measurements before , during , and after light - induced cell protrusion . At steady state , tensions from the two tethers show little correlation , but they becomehighly correlated upon light activation ( purple shaded area ) . During the recovery phase , we often observe a lag in time between the two tethers\u2019 tension drop , with the tether opposite from the protrusion site recovering more slowly ( red ) . ( D ) Three example time traces of trap force for dual membrane tension measurements with cells treated with actin inhibitor ( 10 m M latrunculin B ) before , during ( purple shaded area ) , and after light activation of cell protrusion . ll OPEN ACCESS Article ) m \u03bc ( ll e c - d a e b e c n a t s i D Moving trap away from cell Trap OFF elastic recoil of the tether T r a p f o r c e ( p N ) Time ( sec ) D i s t a n c e p u ll i n g t e t h e r ( \u03bc m ) Move trap 1 Move trap 2 T r a p 1 f o r c e ( n o r m ) Trap 1 force ( norm ) A G D E Move trap 1 Move trap 2 T r a p 1 f o r c e ( n o r m ) F S i R - A c t i n Trap 1 force ( norm ) Untreated Latrunculin B Latrunculin B + 60 % H20 * T r ap 2 pu lli ng T r a p f o r c e ( p N ) Time ( sec ) D i s t a n c e p u ll i n g t e t h e r ( \u03bc m ) Step - wise pulling * B T r ap 2 pu lli ng Bleb D i s t a n c e p u ll i n g t e t h e r ( \u03bc m ) T r a p f o r c e ( p N ) Time ( sec ) S t a t i c t r a p M o v i ng t r a p S t a t i c t r a p M o v i ng t r a p H I J R e l a t i v e d i s t an c e o f bead - c e ll Trap ON Trap OFF Elastic recoilof the tether Time ( sec ) C Time ( sec ) Pull on membrane in the absence of actin cortex ) m \u03bc ( ll e c - d a e b e c n a t s i D Moving trap away from cell Time ( sec ) Trap OFF elastic recoil of the tether Trap 1 static Trap 1 static Figure S3 . Mechanical perturbations affecting only the plasma membrane do not result in measurable membrane tension propagation in cells but do in blebs detached from actin cortex , related to Figure 3 ( A ) An example timetrace oftrap force for dual membrane tensionmeasurements , where onemovingtrap ( T2 , blue ) mechanically perturbs on thecellmembrane bycontinuouslypullingandextending themembrane tether , andtheothertrapremains static ( T1 , red ) tomonitorchangesandpropagationintensiontoanearby membranetether . Theincreaseinlengthoftheextendingtetherfromthecellbodyisplottedingrayalongtherightyaxis . \u2018\u2018 * \u2019\u2019annotateswhentheextendingtether broke . Note that a sudden tension release upon breakage of the extending tether ( blue , at t (cid:1) 50 s ) does not lead to changes in tension on the static tether ( red ) , whichisincloseproximitytotheextendingtether ( % 2 m m ) . Thisobservationshowsthatmechanicalperturbationsaffectingonlytheplasmamembraneincellsare locally constrained and inadequate to generate measurable tension propagation between the two tethers . ( B ) Similar operations as ( A ) but monitoring tension propagation between two membrane tethers on cellular blebs ( i . e . , a vesicle - like , small section of membrane detached from actin cortex upon latrunculin B treatment ) . The tension readouts between the extending and the static tethers on blebs appear highly correlated , ( legend continued on next page ) ll OPEN ACCESS Article unlike those on cell body in ( A ) . Speci\ufb01cally , during the \u2018\u2018step - wise pulling\u2019\u2019 to extend tether in trap 2 ( blue ) , the static tether held in trap 1 ( red ) exhibits immediate spikyrisesintension , mirroringthepatternintrap2 . Whenasmoothincreaseisexertedontheextendingtetherbytrap2 ( blue , att (cid:1) 13s ) , thetensionincreaseon statictether ( red ) accordinglybecomesgradual . Furthermore , thesuddendropintensionbacktoinitiallevelonthestatictether ( red , t (cid:1) 26s ) \u2014inresponsetothe sudden tether breakage ( * ) and thus tension release of the extending tether ( blue ) \u2014re\ufb02ects a direct tension transmission and rapid propagation ( see E ) within a membrane bleb detached from the constraining actin cortex . ( C ) Average time trace of relative distance between bead and cell in untreated cells and cells treated with 10 m M of the actin inhibitor latrunculin B . After tether pulling measurements , the trapping laser is turned off and the elastic recoil of the bead toward the cell is observed to con\ufb01rm the absence of cytoskeleton in the tether . Similar tether recoil is observed between untreated and latrunculin - treated cells ( means \u00b1 SD ; n > 13 , N > 3 ) . ( D ) Similar to ( A ) but we alternate which tether is pulling and which tether is static . Trap forces ( readout of membrane tension response ) from static tether is uncorrelated to that of moving tether ( i . e . , little to no change in tension on the static tether during pulling of the moving tether ) . ( E ) Similar to ( C ) but probing tension in blebs ( membrane detached from actin cortex ) ; here , a high correlation is observed between static and moving tethers . ( F ) Example zoom - in traces of dual trap forces ( raw data at 78 kHz ) showing the time difference between a sudden tension release upon breakage ( * ) of the extendingtether ( blue ) andthesubsequentreduction ( \u2726 ) intensiononthestatictether ( red ; tracesslightlyoffsetinyaxisforillustrationclarity ) . Typically , thistime delay observedis % 100 m s ( measured between thein\ufb02ection points , * and \u2726 , on each trace ) , which is right aroundthe temporal resolution of our opticaltrapping instrument ( limitedbythecornerfrequencyofa2 - m mbeadheldbyatrapwithstiffnessof (cid:1) 0 . 2pN / nm ) , indicatingthattheactualtimescaleoftensionpropagation on cellular blebs is likely too fast to be resolved in our experiments . ( G ) Representative confocal images of actin in cells using actin dye SiR - actin , comparing untreated cells as control with cells treated with either 10 m M of la - trunculin B or with a combination of 10 m M of latrunculin B and in hypotonic media ( + 60 % H20 ) . Scale bar : 10 m M . ( H ) Bright\ufb01eld image of dual - tether pulling from opposite sides of a cell treated with a combination of 10 m M of latrunculin B and hypotonic shock . ( I ) Representative force trace of a cell treated with a combination of 10 m M of latrunculin B and a hypotonic shock showing long - range membrane tension propagation in cells with heavily depolymerized cytoskeleton . ( J ) Two example timetraces ofdistancebetweenbead andcellincells treatedwith10 m Moftheactininhibitor latrunculin Bandwithanhypotonicosmoticshock toheavilydepolymerize theactincytoskeleton . Aftertetherpullingmeasurements , thetrappinglaseristurnedoffandtheelastic recoilofthebeadtowardthecell is observed to con\ufb01rm the absence of cytoskeleton in the tether . We observe similar tether recoil as with untreated and latrunculin - treated cells . ll OPEN ACCESS Article Pre - activation Protruding A F r on t B a ck B a ck A ppa r en t m e m b r ane t h i ck ne ss Thick Thin + Light B C E H I Cost Matrix Transport Plan Distance matrix Displacements a b c d e + Light Pre - activation High Low C e ll M a sk H ea t m ap Protruding Apparent membrane thickness Decrease in apparent membrane thickness Membrane unfolding / reservoirs being accessed D J \u22120 . 2 \u22120 . 1 0 . 0 0 . 1 0 . 2 Uniform Distributiont = 0 Flow Flow inference \u22120 . 2 \u22120 . 1 0 . 0 0 . 1 0 . 2 0 . 003 0 . 004 0 . 005 0 . 006 0 . 007 0 . 008 0 . 009 0 . 010 S pa t i a l d i m en s i on Time Time Time S pa t i a l d i m en s i on S pa t i a l d i m en s i on Simulated Velocity field Ground truth Velocity field OptimalTransport reconstruction Kymograph * K Bleaching H a l o T a g - C AA X UnbleachedCellMask Bleaching H a l o T a g - C AA X UnbleachedCellMask L i gh t i ndu c ed p r o t r u s i on Bleaching H a l o T o a g TT - C AA X g Time ( sec ) C e ll M a sk H a l o T ag - C AAX C e ll M a s k hea t m ap C e ll M a sk H a l o T ag - C AAX C e ll M a sk hea t m ap 0s 2 . 5s 5s L M N O Bleaching H a l o T a g - C AA X UnbleachedCellMask L i gh t i ndu c ed p r o t r u s i on Bleaching H a l o T o a g TT - C AA X P C e ll M a sk H a l o T ag - C AAX C e ll M a sk hea t m ap 0s 2 . 5s 5s 7 . 5s A c t i n - H a l o T ag H ea t m ap High Low Steady state ( pre - activation ) Protruding + Light Average membrane intensity ( CellMask ) R e l a t i v e m e m b r ane f l uo r e sc n c e i n t en s i t y High Low F r on t B a ck B a ck Time ( sec ) + Light F G R e l a t i v e a c t i n f l uo r e sc n c e i n t en s i t y High Low F r on t Time ( sec ) B a ck B a ck + Light Front Back Back 0 . 04 0 . 02 0 . 00 - 0 . 02 - 0 . 04 S peed v a l ue ( \u03bc m / s ) 0 Actin \u22120 . 2 \u22120 . 1 0 . 0 0 . 1 - 0 . 02 - 0 . 04 S pee Back0 acBa T = 30sec T = 70sec T = 170sec Protruding Recovery 0s 200s 100s 50s 175s 0s 10s 20s 30s 0s 30s 50s 70s Q ( legend on next page ) ll OPEN ACCESS Article Figure S4 . Long - range tension propagation coincides with directed membrane \ufb02ows toward the protrusion , related to Figure 4 ( A and B ) Apparent membrane thickness is measured based on the width of \ufb02uorescence intensity pro\ufb01le across the cell contour , e . g . , on the side of cell protrusion ( blackline ) . Atsteadystate ( pre - activation ) , thecellmembranecontourappearsrugged ( topimage ) andthickinwidth ( lightgreencurveinB ) , likelydue to the presence of membrane reservoirs . As the cell protrudes , the membrane intensity outside of the protruding region drops ( bottom image ) and becomes thinner in width ( purple curve in B ) . ( C ) Kymograph of averaged apparent membrane thickness along the normalized cell circumference ( y axis ) over time ( x axis ) : before , during , and after localized light - activated protrusion ( box in white dashed line ) . Apart from the protruding site , apparent membrane thickness reduces on average throughout the cell , likely re\ufb02ecting a decrease in membrane reservoirs and a redistribution of extra membranes toward the protrusion site . ( D ) Representative confocal images of an opto - PI3K cell stained with plasma membrane dye ( CellMask ) before light activation or during protrusion . ( E ) Kymograph of membrane \ufb02uorescence intensity ( from cells stained with CellMask ) along the normalized cell circumference ( y axis ) over time ( x axis ) : before , during , and after localized light - activated protrusion ( box in white dashed line ; n > 25 , N = 4 ) . ( F ) Confocal images of opto - PI3K cells expressing actin marker ( actin - HaloTag ) : before and during light - activated protrusion . ( G ) Kymographs of actin \ufb02uorescence along the normalized cell circumference ( y axis ) show that over time ( x axis ) actin accumulates toward the protruding cell front and is depleted from the back ( n > 30 , N = 6 ; see STAR Methods ) . ( H ) Left , evolutionofthetotalmembraneintensityacrossthecellcontour ( means \u00b1 SD ; n > 30 , N = 6 ) . Exceptforasmallintensitydecreaseduetothebleachingof the\ufb02uorophore , the membrane quantityis conserved . Right , evolution ofthe total actin intensity acrossthe cell contour ( means \u00b1 SD ; n > 50 , N = 6 ) . Bleaching of the \ufb02uorophore across time is visible . Actin intensity is conserved across time , with a higher standard deviation than the membrane intensity . ( I ) ( a \u2013 e ) An illustrative example of optimal transport between two discrete 1 - dimensional distributions , at time t ( blue ) and time t + 1 ( orange ) , which represent the amounts of membrane ( or actin ) along the membrane contour at two different time points . ( a ) Cost matrix C , in which C [ i , j ] indicates the value of the cost to displace an element from position i to the position j . Here , the cost function shown is the square of the curvilinear distance . ( b ) Transport Plan to go from the distribution at time t to the distribution at time t + 1 , minimizing the total cost of the displacement , computed from the cost matrix in ( a ) . ( c ) Distance matrix D , in whichD [ i , j ] indicatesthevalueofthedistancebetweenanelementattheposition i andanelementatthepositionj . Thedistancechosenisthecurvilineardistance . ( d ) The transport plan and the distance matrix allow to compute the mean displacement for every position between times t and t + 1 . ( e \u2013 g ) Matrices in the case of periodic boundary conditions , such as the circular contour of the cell . ( e ) Cost matrix with periodic boundary conditions . The cost function chosen is still the square of the curvilinear distance , but as the topology of the curve is periodic , the matrix is changed to re\ufb02ect this new topology . To keep track of the direction of the displacement , the distances can be positive or negative ( and subsequently the positive and negative speed shown in Figures 4D and 4E ) . A displacement in the clockwise direction ( increasing angle coordinate ) is positive , whereas a displacement in the counter - clockwise direction is negative . ( J ) Pipeline and example of \ufb02ow inference validation using computer simulated distributions ( see Methods S1 ) . Using Optimal Transports , \ufb02ows are inferred with minimal errors . ( K ) ( Top ) Actin \ufb02ow \ufb01eld inferred from kymograph intensity change over time using optimal transport . ( Bottom ) Actin \ufb02ow around the cell as inferred by optimal transportbefore , during , andafter ( t = 30 , 70 , and170s ) right - sideprotrusion ; the\ufb02owmagnitudeisdenotedbythearrowsize ( red : forward\ufb02ow , blue : backward ) . ( L ) Alternative membrane diffusion assay inwhichwe sequentially bleach the membrane marker CellMask across awide section of thecell , opto - activate thecell on the side of the unbleached area and monitor the diffusion pattern of the unbleached area over time . We use cells with no activating light as control . ( M ) ExampleconfocalimagesofthemembranemarkersHaloTag - CAAXandCellMaskinacellwithnoactivatinglight ( control , top ) andalight - inducedprotruding cell ( bottom ) . ( N ) Quanti\ufb01cation of shift centroid of signal intensity in control cells ( top , no apparent \ufb02ow ) and protruding cells ( bottom , biased \ufb02ow toward side of protrusion ) . ( O and P ) Similar to ( L and M ) but with an overlap between the unbleached and activation area . ( Q ) Two examples of microvilli tracking during light - induced cell protrusion . Tracked microvilli are circled in red and their trajectory is represented by lines of different colors . Scale bars : 5 m m . ll OPEN ACCESS Article Figure S5 . Optogenetically induced actomyosin contractions generate rapid long - range membrane tension propagation and actin \ufb02ows , related to Figure 5 ( A and B ) Representative time traces of trap force ( a direct readout of cell membrane tension change ) during light - induced actomyosin contraction . Revealing robust increase in membrane tension during light - activated contractions on the opposite end of the cell ; light : 90 s on ( shaded area ) . ( C ) Averaged time trace of trap force before ( steady state ) , during ( Light ) , and after activating cell contraction , measured at the side ( 90 (cid:3) ) of the contraction ( means \u00b1 SD ; n > 30 , N = 7 ) . ( D ) ( Left ) Time delay measured betweentension rise on membrane tethers adjacentto ( trap 2at 90 (cid:3) , blue ) and oppositefrom ( trap 1 at180 (cid:3) , red ) cellcontraction . ( Right ) Inmostcells , thetrapsdetect membrane tensionincreaseonbothtetherswithinasecondorless ofoneanother , indicatingarapid propagation oftension across the cell . Error bar : means \u00b1 SD . ( E ) Confocal images of opto - LARG cells stained with actin marker ( SPY650 - FastAct ) : before and during light - activated contraction . ( F ) Average kymograph of relative actin \ufb02uorescence intensity along the normalized cell circumference ( y axis ) show that over time ( x axis ) . Actin accumulates toward the contracting cell front ( n > 25 , N = 3 ; see STAR Methods ) . ( G ) Actin \ufb02ow \ufb01eld inferred using optimal transport from kymograph intensity changes over time : shortly after activation begins ( t = 120 s , teal traces ) , the magnitude of membrane \ufb02ow speed increases ( red dashed arrows ) , with positive speed for clockwise \ufb02ow along the cell upper half and negative speed for counter - clockwise \ufb02ow along the bottom half , all moving toward the cell contracting front ( p ) . During recovery ( t = 230 s , light yellow traces ) , the direction of membrane \ufb02ow reverses ( blue dashed arrows ) . ( H ) Actin \ufb02ow around the cell before , during , and after ( t = 30 , 80 , and 230 s ) right - side contraction ; the \ufb02ow magnitude is denoted by the arrow size ( red : forward \ufb02ow , blue : backward ) . Membrane \ufb02ows toward the contraction in the contracting phase and away from the protrusion during the recovery phase . ( I ) Twoexamplesofactinspeckletrackingduringlight - inducedcellcontraction . Trackedactinpatchesarecircledinredandtheirtrajectoryisrepresentedbylines of different colors . Scale bars : 5 m m . ll OPEN ACCESS Article ( legend on next page ) ll OPEN ACCESS Article Figure S6 . Mechanical perturbations applied on both membrane and cortex lead to rapid tension propagation across the cell , related to Figure 6 ( A ) Tether pulling assayin which tethers are pulled atconstantspeeduntil they break . Maximum tether length is used as aproxy for local membrane reservoirs 56 . ( B ) Maximum tether length comparison of 3T3s \ufb01broblasts versus HL - 60s cells . In red are cells for which the maximum pulling length was reached on our setup without tether breaking occurring , suggested high local membrane reservoir availability . Error bar : means \u00b1 SD ; n > 15 , N > 3 . ( C ) Average trap force of different opto - cells ( OptoPI3K - based protrusion induction and OptoLARG - based actomyosin contractility ) , before and after light in the absenceorpresenceoftheEzrininhibitorNSC668394 ( 25 m M ) . ThesedatashowthatloweringMCAonlyslightlyaffectsmembranetensionincreaseinprotruding cellsbutseverelyimpedesmembranetensionincreasesincontractingcells . Errorbar : means \u00b1 SD ; pvaluesfromWelch\u2019sunpairedStudent\u2019sttest ( n > 25 , N > 3 ) . ( D ) A dual - tether pulling assay to simultaneously monitor membrane tension on the far end ( bottom , trap 1 at 180 (cid:3) ) and on the side of the cell ( right , trap 2 at 90 (cid:3) ) during micropipette aspiration ( top , (cid:1) 4 \u2013 5 m m in tip diameter ) , which mechanically pulls on both the membrane and actin cortex underneath . ( E ) Representative time traces of dual trap forces over successive cycles of aspiration ( shaded area ) and relaxation ; the magnitude of aspiration progressively increased in the last two cycles ( + and + + ; the \ufb01rst three cycles were also shown in Figure 6I ) . The nearly superimposable tension rise and fall on the two membrane tethers show that membrane tension propagates rapidly across the cell upon mechanical perturbations exerted to both the cortex and membrane . Note that the pro\ufb01les of tension rise upon aspiration and of tension drop during relaxation resemble those observed with light - activated actin - driven protrusions ( Figure 2B ) . ( F ) Zoom - in on the \ufb01rst aspiration event shows that the trap force for membrane tension on the tether closer ( pink ) to the aspiration site started increasing slightly earlier and ended up slightly higher compared with that measured on the tether opposite from the aspiration ( purple ) . ( G ) An example trace of tether tension response monitored on the opposite side of micropipette aspiration ( trap 1 at 180 (cid:3) ) . Here , the recording lasted for six rounds of aspiration and relaxation . ( H ) Another example of dual - tether membrane tension measurement upon micropipette aspiration ; the tether in trap 2 broke ( * ) shortly after the aspiration stopped . ( I ) An example time trace of trap force for cell membrane tension exhibits robust responses over three aspiration cycles using a micropipette of slightly smaller diameter ( (cid:1) 2 m m ) . ( J ) ( Left ) Timedelay measuredbetweentensionriseonmembrane tethers adjacent to ( trap2at90 (cid:3) , pink ) and oppositefrom ( trap1at180 (cid:3) , purple ) cellaspiration using micropipettes . ( Right ) In most cells , the traps detect membrane tension increase on both tethers within a second or less of one another , indicating a rapid propagation of tension across the cell . ( K ) Pearson correlation coef\ufb01cient between dual trap forces measured before any perturbations ( steady state ) and during mechanical pulling upon micropipette aspiration . Error bar : means \u00b1 SD ; p values from Welch\u2019s unpaired Student\u2019s t test ( n > 15 , N > 3 ) . ( L ) Correlation plots of normalized trap forces between the two tethers during micropipette aspiration . Five representative measurements from different cells are shown ; dashed lines : linear regression . ( M ) Comparing membrane \ufb02ows of light - induced protrusions at the mid and ventral plane of the cell . ( N ) Confocal images ofacellmembrane ( visualizedusing CAAX - HaloTag ) before and duringprotrusion attwo different zplanes ( mid - section and ventralplane of the cell ) . Scale bar : 5 m m . ( O ) Average kymograph of relative membrane \ufb02uorescence intensity along the normalized cell circumference ( y axis ) at the ventral and mid - plane of the cell over time ( x axis ) showing a decreased membrane \ufb02ow at the ventral side of the cell , likely due to friction between the cell and the substrate ( n > 30 , N = 3 ) . ( P ) Normalized membrane \ufb02uorescence intensity across the blue dotted line in ( O ) . ll OPEN ACCESS Article", + "day2015microtubule_supplement3": "Supplementary Figure 3 . ( Related to Figures 4 and 6 ) This figure provides additional evidence that intact microtubules and an intact dynactin complex are required for tubule elongation . It also shows the results of control experiments determining the concentration of ciliobrevin A required to inhibit delivery of CTxB to perinuclear compartments . ( A , B ) Pretreatment of cells with 16 . 6 (cid:0) M nocodazole prior to LatA ( A ) or Jasplakinolide treatment ( B ) blocks tubule formation . ( C , D ) Pretreatment with ciliobrevin A ( Cilio - A ) disrupts delivery of CTxB to perinuclear compartments in ATP replete cells in a dose - dependent manner . n = 36 - 108 cells ; error bars = SD . ( E , F ) Expression of GFP - p50 ( E ) , but not GFP alone ( F ) blocked the formation of long branched tubules in Dynasore - treated cells . Similar results were obtained for both wild type CTxB or monovalent CTx . Bars , 10 (cid:0) m .", + "milewska2018entry": "Entry of Human Coronavirus NL63 into the Cell Aleksandra Milewska , a , b Paulina Nowak , a , b Katarzyna Owczarek , a , b Artur Szczepanski , a , b Miroslaw Zarebski , c Agnieszka Hoang , c Krzysztof Berniak , c Jacek Wojarski , d Slawomir Zeglen , d Zbigniew Baster , e Zenon Rajfur , e Krzysztof Pyrc a , b a Microbiology Department , Faculty of Biochemistry , Biophysics and Biotechnology , Jagiellonian University , Krakow , Poland b Laboratory of Virology , Malopolska Centre of Biotechnology , Jagiellonian University , Krakow , Poland c Department of Cell Biophysics , Faculty of Biochemistry , Biophysics and Biotechnology , Jagiellonian University , Krakow , Poland d Department of Cardiac Surgery and Transplantology , Silesian Center for Heart Diseases , Zabrze , Poland e Institute of Physics , Faculty of Physics , Astronomy and Applied Computer Sciences , Jagiellonian University , Krakow , Poland ABSTRACT The \ufb01rst steps of human coronavirus NL63 ( HCoV - NL63 ) infection were previously described . The virus binds to target cells by use of heparan sulfate pro - teoglycans and interacts with the ACE2 protein . Subsequent events , including virus internalization and traf\ufb01cking , remain to be elucidated . In this study , we mapped the process of HCoV - NL63 entry into the LLC - Mk2 cell line and ex vivo three - dimensional ( 3D ) tracheobronchial tissue . Using a variety of techniques , we have shown that HCoV - NL63 virions require endocytosis for successful entry into the LLC - MK2 cells , and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin . Subsequent vesicle scission by dynamin results in virus internalization , and the newly formed vesicle passes the actin cortex , which requires active cytoskeleton rearrangement . Finally , acidi\ufb01cation of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm . For 3D tra - cheobronchial tissue cultures , we also observed that the virus enters the cell by clathrin - mediated endocytosis , but we obtained results suggesting that this pathway may be bypassed . IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells . Here , using the most ad - vanced 3D tissue culture system mimicking the epithelium of conductive airways , we systematically mapped HCoV - NL63 entry into susceptible cells . The data obtained al - low for a better understanding of the infection process and may support develop - ment of novel treatment strategies . KEYWORDS HCoV - NL63 , clathrin , Coronaviridae , coronavirus , endocytosis , entry , infection , internalization H uman coronavirus NL63 ( HCoV - NL63 ) was discovered shortly after the emergence of the severe acute respiratory syndrome coronavirus ( SARS - CoV ) ( 1 ) . Extensive studies on the pathogen\u2019s biology and epidemiology revealed that it is prevalent worldwide , appearing with a seasonal distribution similar to that of other human coronaviruses . The clinical presentation may vary depending on the general health status of the patient . Usually , the virus causes a relatively mild respiratory tract disease , but fatal cases have been reported ( 2 \u2013 5 ) . Furthermore , broad studies on the association between infection and clinical symptoms reveal that HCoV - NL63 is associated with croup in young children ( 6 \u2013 9 ) . Phylogenetically , HCoV - NL63 clusters within the genus Alphacoronavirus , which Received 7 November 2017 Accepted 7 November 2017 Accepted manuscript posted online 15 November 2017 Citation Milewska A , Nowak P , Owczarek K , Szczepanski A , Zarebski M , Hoang A , Berniak K , Wojarski J , Zeglen S , Baster Z , Rajfur Z , Pyrc K . 2018 . Entry of human coronavirus NL63 into the cell . J Virol 92 : e01933 - 17 . https : / / doi . org / 10 . 1128 / JVI . 01933 - 17 . Editor Julie K . Pfeiffer , University of Texas Southwestern Medical Center Copyright \u00a9 2018 American Society for Microbiology . All Rights Reserved . Address correspondence to Krzysztof Pyrc , k . a . pyrc @ uj . edu . pl . A . M . and P . N . contributed equally to this article . VIRUS - CELL INTERACTIONS crossm February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 1 Journal of Virology on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m also includes another human pathogen , HCoV - 229E . Initially , these two viruses were considered distant relatives that diverged at some point due to an unknown reason . More recent research shows , however , that these two species most likely emerged in the human population during two separate zoonotic transmission events ( 10 \u2013 12 ) . From the perspective of genome structure , HCoV - NL63 is similar to other alphac - oronaviruses in that the 5 = - terminal two - thirds of the genome encodes a large poly - protein , which is cleaved to yield several nonstructural proteins . Five genes ( encoding S , ORF3 , E , M , and N ) are located at the 3 = terminus and encode structural proteins . The spike protein ( S ) is a class I fusion protein comprising a rod - like domain anchored to the virion via its C terminus and a globular head responsible for the interaction with cellular entry receptors ( 13 ) . It is generally assumed that alphacoronaviruses interact with and enter host cells using CD13 ( aminopeptidase N ) . However , HCoV - NL63 utilizes the ACE2 protein for this purpose , a characteristic shared only with SARS - CoV ( 14 , 15 ) . Virus tropism not only depends on the presence of a certain entry receptor but also may be modulated by other factors , e . g . , attachment receptors , protease availability , and the activity of pathways responsible for internalization and traf\ufb01cking of the virus particle ( 16 , 17 ) . While binding to their cognate entry receptor provides suf\ufb01cient stimulus for some viruses to initiate fusion between the viral and cellular membranes , most internalize via endocytosis ; acidi\ufb01cation and / or processing by cathepsins is then a prerequisite for fusion ( 13 ) . For a long time , endocytic entry of virions was classi\ufb01ed as clathrin dependent , clathrin independent , or clathrin and caveolin independent . During recent years , a number of other pathways were identi\ufb01ed , and this complex machinery has become better understood . The occurrence , abundance , and mechanistic details of these pathways appear to vary between cell types , tissues , and species . Most often , the selection of a speci\ufb01c endocytic route is linked to cargo - directed traf\ufb01cking and receptor - dependent traf\ufb01cking . Nevertheless , many receptors / cargoes allow \ufb02exibility due to their capacity to enter a cell via multiple pathways . The early stages of HCoV - NL63 infection have been described by us and others ( 18 \u2013 20 ) . Here , we made an effort to delineate events that occur early during HCoV - NL63 infection . First , the virus anchors to ciliated cells via heparan sulfate proteoglycans before interacting with the ACE2 entry receptor . Our results show that the virus - ACE2 interaction triggers recruitment of clathrin , followed by clathrin - mediated , dynamin - dependent endocytosis , which requires actin cortex remodeling . To ensure that our results were reliable , we used ex vivo cultured human airway epithelium ( HAE ) , which mimics the microenvironment at the infection site . RESULTS HCoV - NL63 enters the cell via endocytosis . We \ufb01rst determined whether entry of HCoV - NL63 requires endocytosis and acidi\ufb01cation of endosomes . For this , we studied the effect of ammonium chloride ( NH 4 Cl ) and ba\ufb01lomycin A , lysosomotropic agents that inhibit acidi\ufb01cation of endosomes ( 21 \u2013 23 ) , using two models of HCoV - NL63 infection : permissive LLC - Mk2 cells and HAE cultures . Cells were preincubated with NH 4 Cl ( 50 mM ) , ba\ufb01lomycin A ( 100 nM ) , or control dimethyl sulfoxide ( DMSO ) for 1 h at 37\u00b0C and subsequently incubated with the virus at a 50 % tissue culture infective dose ( TCID 50 ) of 100 / ml for LLC - Mk2 cells or 400 / ml for HAE fo r 2 h at32\u00b0C in the presence of the inhibitor . Subsequently , supernatants were removed , and cells were washed thrice with acidic buffer to inhibit the fusogenic activity of the virions retained on the surface ( 24 ) . Next , LLC - Mk2 cells were washed with 1 (cid:2) phosphate - buffered saline ( PBS ) ( pH 7 . 4 ) , overlaid with culture medium , and incubated at 32\u00b0C for 4 days . Supernatant samples were collected for virus replication analysis . Simultaneously , HAE cultures were washed with 1 (cid:2) PBS ( pH 7 . 4 ) and further maintained at an air - liquid interphase at 32\u00b0C for 5 days . During this time , HAE cultures were washed every 24 h with 1 (cid:2) PBS supplemented with a given inhibitor for 10 min at 32\u00b0C , and apical washes were collected for virus replication analysis . Subsequently , viral RNA was isolated and reverse Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 2 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m transcribed ( RT ) , and the HCoV - NL63 yield was determined using a quantitative real - time PCR ( qPCR ) . Ba\ufb01lomycin A and NH 4 Cl inhibited HCoV - NL63 infection in LLC - Mk2 cells , proving that acidi\ufb01cation is a requirement for the virus infection in vitro . No inhibition was observed in HAE cultures ( Fig . 1A ) . No cytotoxic effect was observed in the presence of these inhibitors ( Fig . 1B ) . Next , we analyzed HCoV - NL63 colocalization with early endosome antigen 1 ( EEA1 ) , a hydrophilic protein localizing exclusively to early endosomes ( 25 ) . LLC - Mk2 cells were \ufb01xed after 10 , 20 , 30 , or 40 min postinoculation ( p . i . ) with gradient - puri\ufb01ed virus , stained with antibodies speci\ufb01c to HCoV - NL63 N protein and EEA1 , and analyzed under a confocal microscope . Measured colocalization , expressed as Manders\u2019 coef\ufb01cient , increases with time and reaches 0 . 68 at 40 min p . i . ( n (cid:3) 6 cells ) ( Fig . 1C ) . We validated the obtained results using the HAE model . Brie\ufb02y , HAE cultures were inoculated with gradient - puri\ufb01ed HCoV - NL63 and incubated at 32\u00b0C for 2 h . For this culture model , a longer incubation was required to observe virus attachment and entry , most likely due to the requirement to cross the mucus layer . Subsequently , cells were \ufb01xed and labeled with speci\ufb01c antibodies against HCoV - NL63 N protein and EEA1 . Colocalization of HCoV - NL63 virus particles with EEA1 protein was analyzed using a confocal microscope . Colocalization of virus and EEA1 was observed in inoculated cells ( Fig . 1D ) . Endocytosis of virus particles is induced by binding to the entry receptor . HCoV - NL63 virus employs the ACE2 protein for cellular entry , while heparan sulfate proteoglycans serve as attachment receptors ( 19 ) . Here , we analyzed the consequence FIG 1 Importance of endosomal entry for HCoV - NL63 infection . ( A ) Inhibition of HCoV - NL63 infection in LLC - Mk2 cells and HAE cultures by the lysosomotropic agents ammonium chloride ( NH 4 Cl ) ( 50 mM ) and ba\ufb01lomycin A ( Baf A ) ( 100 nM ) , as determined by RT - qPCR . Data on the y axis represent LRVs . The assay was performed in triplicate , and average values with standard errors are presented . P values of (cid:4) 0 . 05 were considered signi\ufb01cant and are denoted with an asterisk . ( B ) The cytotoxicity of the tested inhibitors was measured with an XTT assay . Data on the y axis represent viability of the treated cells compared to the untreated reference samples . The assay was performed in triplicate , and average values with standard errors are presented . ( C and D ) Confocal images showing colocalization of HCoV - NL63 virions with the early endosomal marker EEA1 on LLC - Mk2 cells ( C ) and HAE cultures ( D ) . Scale bars (cid:3) 5 (cid:2) m . Green , HCoV - NL63 ; red , EEA1 . Entry of HCoV - NL63 Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 3 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m of interaction between the virus particle and ACE2 . First , we inoculated naturally permissive LLC - Mk2 cells with HCoV - NL63 and incubated them for 40 min at 4\u00b0C to enable virus adhesion to a cell surface . Subsequently cells were \ufb01xed , the virus was labeled with speci\ufb01c antibodies , and its colocalization with ACE2 and clathrin was studied . As shown in Fig . 2A , HCoV - NL63 particles attach ef\ufb01ciently to the cell surface . However , only a proportion of virions colocalize with the ACE2 ( Manders\u2019 coef\ufb01cient (cid:3) 0 . 573 ; n (cid:3) 5 ) , suggesting that binding to the heparan sulfate precedes interaction with FIG 2 HCoV - NL63 binding to ACE2 triggers clathrin - mediated endocytosis . Precooled LLC - Mk2 cells were incubated with gradient - puri\ufb01ed HCoV - NL63 for 40 min at 4\u00b0C following 0 min ( A and B ) or 5 min ( C ) of incubation at 32\u00b0C . Colocalization of the virus ( green ) and ACE2 ( red ) was analyzed using confocal microscopy ( A ) . No colocalization with clathrin was observed after 0 min of incubation ( B ) . Triple colocal - ization of virus with ACE2 and clathrin ( blue ) is visible in panel C . Images on the right side are zoomed - in regions indicated by white rectangles on the left - side slides . A representative image is shown . Scale bars (cid:3) 10 (cid:2) m . Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 4 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m the entry receptor . At that point , there is no colocalization of virus particles and clathrin - coated pits ( Manders\u2019 coef\ufb01cient (cid:3) 0 . 140 ; n (cid:3) 5 ) ( Fig . 2B ) . Next , we tested whether the virus binding to the adhesion or entry receptor triggers recruitment of common cellular proteins responsible for pit formation by incubating cells for 5 min at 32\u00b0C . Immunostaining showed that the virus particles bound to ACE2 start to colocalize with clathrin ( Manders\u2019 coef\ufb01cient (cid:3) 0 . 849 ; n (cid:3) 6 ) ( Fig . 2C ) , while there is no colocal - ization between non - ACE2 - bound virions and clathrin ( Manders\u2019 coef\ufb01cient (cid:3) 0 . 189 ; n (cid:3) 6 ) . HCoV - NL63 colocalizes with clathrin during entry . To determine whether colo - calization with clathrin following the ACE2 binding is relevant and the virus indeed enters the cell by use of clathrin - coated pits , we analyzed colocalization of intracellular virions with clathrin . Brie\ufb02y , LLC - Mk2 cells were incubated at 32\u00b0C for 5 to 20 min with gradient - puri\ufb01ed HCoV - NL63 , \ufb01xed , immunostained , and analyzed by confocal micros - copy . The results showed colocalization of virions entering the cell with clathrin ( Manders\u2019 coef\ufb01cient (cid:3) 0 . 584 ; n (cid:3) 7 ) ( Fig . 3A ) , whereas no colocalization with caveolin 1 was observed ( Manders\u2019 coef\ufb01cient (cid:3) 0 . 053 ; n (cid:3) 5 ) ( Fig . 3B ) . HCoV - NL63 colocaliza - tion with clathrin and caveolin was also studied in the HAE model . For this , cultures were incubated with gradient - puri\ufb01ed HCoV - NL63 at 32\u00b0C for 2 h ; the virus and the cellular proteins were immunostained and analyzed by confocal microscopy . HCoV - NL63 virions also colocalized with clathrin in this model , whereas no colocalization was observed for caveolin 1 ( Fig . 3 ) . Clathrin and dynamin are important for HCoV - NL63 entry . As we already knew that HCoV - NL63 virions migrate to clathrin - coated pits , in the subsequent step we aimed to determine whether the clathrin - mediated endocytosis is indeed important for the virus entry . For this reason , we blocked the pathway using Pitstop 2 { N - [ 5 - ( 4 - bromobenzylidene ) - 4 - oxo - 4 , 5 - dihydro - 1 , 3 - thiazol - 2 - yl ] naphthalene - 1 - sulfonamide } , a selective clathrin inhibitor targeting its amino - terminal domain , and tetradecyltrim - ethylammonium bromide ( MiTMAB ) , a dynamin I and II GTPase inhibitor . Activity of these compounds was veri\ufb01ed with the positive control , \ufb02uorescently labeled transfer - rin ( 26 , 27 ) . LLC - Mk2 cells were treated with Pitstop 2 , MiTMAB , or control DMSO for 30 min at 37\u00b0C following transferrin uptake for 45 min at 37\u00b0C . Confocal images showed that both inhibitors blocked transferrin endocytosis , as the protein was present only on the cell surface ( Fig . 4A to D ) . Subsequently , LLC - Mk2 cells were incubated with one of the inhibitors at 37\u00b0C for 30 min and inoculated with gradient - puri\ufb01ed HCoV - NL63 at 32\u00b0C for 5 min . Following immunostaining of the HCoV - NL63 N protein and actin , virus endocytosis was analyzed using confocal microscopy . The results showed that virus internalization was hampered in cells pretreated with clathrin and dynamin inhibitors compared to the DMSO - treated cells ( Fig . 4D to G ) . Simultaneously , a cytotoxicity test of the entry inhibitors was performed , which showed no toxic effect of the tested compounds on LLC - Mk2 cells FIG 3 HCoV - NL63 colocalizes with clathrin but not caveolin . ( A and B ) LLC - Mk2 cells were incubated with gradient - puri\ufb01ed HCoV - NL63 for 40 min at 4\u00b0C following 5 min ( A ) or 20 min ( B ) of incubation at 32\u00b0C . HAE cultures were incubated with gradient - puri\ufb01ed HCoV - NL63 for 40 min at 4\u00b0C following 120 min of incubation at 32\u00b0C . HCoV - NL63 colocalization with clathrin ( A ) or caveolin ( B ) was analyzed with confocal microscopy ( HCoV - NL63 , green ; clathrin and caveolin , red ; nuclei , blue ) . ( C ) Cells mock incubated and stained with isotypic antibodies were used as a control . Scale bars (cid:3) 5 (cid:2) m . Entry of HCoV - NL63 Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 5 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m ( Fig . 5 ) . In order to ensure that our observations were not biased , statistical analysis of virus entry was performed . For this , an algorithm was prepared for image analysis , a 3D representation of the cell was prepared , and the virus position in the cell was deter - mined ( Fig . 6 ) . FIG 4 Clathrin and dynamin inhibitors hamper internalization of HCoV - NL63 . ( A to C ) In order to verify the effectiveness of inhibitors , LLC - Mk2 cells were incubated with control DMSO ( A ) , 10 (cid:2) M Pitstop 2 ( B ) , or 10 (cid:2) M MiTMAB ( C ) for 30 min at 37\u00b0C and inoculated with Alexa Fluor 488 - labeled transferrin . Following incubation ( 45 min , 37\u00b0C ) , cells were \ufb01xed and stained for actin ( red ) . Transferrin entry was evaluated with confocal microscopy . ( E to G ) LLC - Mk2 cells were incubated with control DMSO ( E ) , 10 (cid:2) M Pitstop 2 ( F ) , or 10 (cid:2) M MiTMAB ( G ) for 30 min at 37\u00b0C . Cells were inoculated with puri\ufb01ed HCoV - NL63 and incubated at 32\u00b0C for 1 h . Subsequently , cells were \ufb01xed and immunostained for HCoV - NL63 particles ( green ) and actin ( red ) . ( D ) Mock - infected cells were used as a control . Scale bars (cid:3) 10 (cid:2) m . FIG 5 Cytotoxicity of Pitstop 2 and MiTMAB on LLC - Mk2 cells . The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay . Cells were incubated with control DMSO , 10 (cid:2) M Pitstop 2 , or 10 (cid:2) M MiTMAB for 2 h at37\u00b0C Data on the y axis represent viability of the treated cells compared to the untreated reference samples . The assay was performed in triplicate , and average values with standard errors are presented . Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 6 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m A similar experiment was conducted using HAE cultures . For this , cultures were incubated for 1 h at 37\u00b0C with the inhibitors described above following incubation with gradient - puri\ufb01ed HCoV - NL63 at 32\u00b0C for 2 h . A strong inhibition of virus internalization in cultures preincubated with clathrin or dynamin inhibitors compared to control cells was observed ( Fig . 7 ) . No cytotoxicity to HAE was observed for the tested inhibitors afte r 3 h ofincubation at 37\u00b0C ( Fig . 8 ) . Clathrin - mediated endocytosis is the main entry route for HCoV - NL63 . Even though certain cargo is usually internalized by a single route , frequently other pathways may be used as alternatives . We therefore aimed to test whether inhibition of clathrin - mediated entry with chemical inhibitors results in inhibition of virus replication . To address this , we incubated LLC - Mk2 cells with a given inhibitor at 37\u00b0C for 1 h and infected them with HCoV - NL63 ( TCID 50 (cid:3) 400 per ml ) for 2 h at32\u00b0C . Subsequently , media were removed and cells were washed thrice with acidic buffer following washing with 1 (cid:2) PBS ( pH 7 . 4 ) . Next , cells were overlaid with culture medium containing a given inhibitor and incubated at 32\u00b0C for 4 days . Cells were \ufb01xed and immunostained for HCoV - NL63 N protein to assess the number of infected cells . To assess the nonspeci\ufb01c effect of entry inhibitors , control cells were also treated with these at 4 h p . i . Clearly , in the presence of clathrin - mediated endocytosis inhibitors ( Pitstop 2 and MiTMAB ) , the number of HCoV - NL63 - infected cells was much lower than in the control . However , MiTMAB also inhibited virus replication at later stages of the infection ( Fig . 9 ) . To ensure FIG 6 Numerical image analysis : clathrin and dynamin inhibitors block HCoV - NL63 entry . ( B to D ) LLC - Mk2 cells were incubated with DMSO ( B ) , 10 (cid:2) M MiTMAB ( C ) , or 10 (cid:2) M Pitstop 2 ( D ) for 30 min at 37\u00b0C and subsequently inoculated with puri\ufb01ed HCoV - NL63 and incubated for 45 min at 32\u00b0C . Confocal images were digitalized , and the localization of each virus particle relative to the cellular membrane was assessed . ( A ) Number of internalized virus particles relative to number of virions on the cell surface ( y axis ) for cells treated with DMSO ( control ) , Pitstop 2 , or MiTMAB . In panels B , C , and D , raw data for cells treated with DMSO , Pitstop 2 , or MiTMAB , respectively , are presented . Histograms show the average number of virus particles ( y axis ) versus the distance from the cell surface ( x axis ) . Values of (cid:4) 0 on the x axis indicate that the virus is inside the cell , while for extracellular virions , the x value is (cid:3) 0 . Entry of HCoV - NL63 Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 7 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m that entry inhibitors affected HCoV - NL63 infection in LLC - Mk2 cells , we analyzed by RT - qPCR virus replication at 120 h p . i . in the presence of the tested compounds . The analysis showed an (cid:5) 2 - log decrease in virus progeny production in the presence of Pitstop 2 and MiTMAB compared to that in DMSO - treated cells and a slight increase of RNA copy levels in the presence of nystatin ( Fig . 10A ) . Importantly , no cytotoxic effect was observed for the tested inhibitors applied to LLC - Mk2 cells for 4 days at 32\u00b0C ( Fig . 10B ) . The in\ufb02uence of the tested inhibitors on HCoV - NL63 infection was also analyzed in HAE cultures . For this , cultures were preincubated with a given inhibitor ( Pitstop 2 , MiTMAB , nystatin , or control DMSO ) for 1 h at37\u00b0C and infected with HCoV - NL63 at a TCID 50 of 400 per ml for 2 h at32\u00b0C . Subsequently , noninternalized virions were inactivated by acid washing , and cultures were washed with 1 (cid:2) PBS and incubated with a given inhibitor for 10 min . After that time , supernatants were discarded and cultures were incubated for 5 days at 32\u00b0C . During this period , cultures were incubated with a given inhibitor for 10 min at 32\u00b0C every 24 h . Viral RNA from these samples was quanti\ufb01ed by RT - qPCR . Virus replication in HAE was not affected by any of the tested inhibitors ( Fig . 10A ) . TMPRSS2 is important during early stages of the infection . It was previously suggested that coronaviruses may bypass the endocytic entry route employing trans - membrane protease serine 2 ( TMPRSS2 ) , which primes the fusion protein and enables fusion of viral and cellular membranes on the cell surface ( 28 , 29 ) . We have tested FIG 7 Clathrin and dynamin inhibitors prevent HCoV - NL63 from entering the cell in the HAE model . HAE cultures were incubated with control DMSO ( A ) , 10 (cid:2) M Pitstop 2 ( B ) , or 10 (cid:2) M MiTMAB ( C ) fo r 1 h at37\u00b0C . Cells were then inoculated with puri\ufb01ed HCoV - NL63 and incubated at 32\u00b0C for 2 h . Subsequently , cells were \ufb01xed and immunostained for HCoV - NL63 particles ( green ) , actin ( red ) , and nuclei ( blue ) . Scale bars (cid:3) 5 (cid:2) m . FIG 8 Cytotoxicity of Pitstop 2 and MiTMAB on HAE cultures . The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay . Cells were incubated with control DMSO , 10 (cid:2) M Pitstop 2 , or 10 (cid:2) M MiTMAB for 2 h at37\u00b0C . Data on the y axis represent the viability of the treated cells compared to the untreated reference samples . The assay was performed in triplicate , and average values with standard errors are presented . Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 8 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m whether inhibition of TMPRSS2 with camostat affects the HCoV - NL63 infection . We observed that inhibition of TMPRSS2 hampers virus infection in HAE cultures , while it has no effect on virus replication in LLC - MK2 cells ( Fig . 11A ) . No inhibition of virus entry was observed in any of the models , as tracked with confocal microscopy visualizing the nucleoprotein ( Fig . 11B ) . As only single entry events per view were observed , several images for camostat - treated and control cells are presented . In total , 500 entry events into HAE cells were tracked , and no difference between the camostat - treated sample and the control sample was noted . HCoV - NL63 entry requires actin remodeling . We studied traf\ufb01cking of HCoV - NL63 inside the cell . As entry by endocytosis would probably require remodeling of the cytoskeleton , we evaluated virus internalization in the presence of cytochalasin D , jasplakinolide , or nocodazole . The \ufb01rst chemical inhibits actin polymerization , whereas the second binds F - actin and stabilizes actin \ufb01laments ( 30 , 31 ) . The last compound interferes with microtubule formation . The analysis showed that actin inhibitors pre - vented virus particles from penetrating the cell , with visible viral particle accumulation on actin cortex or unstructured actin deposits . The microtubule inhibitor did not affect virus entry ( Fig . 12 ) . No cytotoxicity was observed for the tested inhibitors ( Fig . 13 ) . DISCUSSION Previously , we and others described the \ufb01rst steps of the HCoV - NL63 infection process , showing that it begins with the virus binding to the cellular membrane via heparan sulfate proteoglycans , which then enable / facilitate interaction with the entry receptor , ACE2 ( 14 , 18 , 19 ) . Little is known about the subsequent virus internalization FIG 9 Clathrin and dynamin inhibitors limit the number of LLC - Mk2 infected cells . ( A to D ) LLC - Mk2 cells were incubated with control DMSO ( A ) , 5 (cid:2) g / ml nystatin ( B ) , 10 (cid:2) M MiTMAB ( C ) , or 10 (cid:2) M Pitstop 2 ( D ) fo r 1 h at37\u00b0C and inoculated with HCoV - NL63 ( TCID 50 (cid:3) 100 / ml ) . After 2 h ofincubation at 32\u00b0C , virions that were not internalized were inactivated with acidic buffer ( pH 3 ) , and cells were incubated for 4 days at 32\u00b0C in the presence of the tested inhibitors or control DMSO . ( E and F ) The identical procedure was applied to cells , but in these MiTMAB ( E ) and Pitstop 2 ( F ) were applied after the acid wash . Fixed cells were immunostained with anti - NL63 nucleocapsid protein ( green ) and nuclei ( blue ) , and confocal images were collected . Scale bar (cid:3) 200 (cid:2) m . Entry of HCoV - NL63 Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 9 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m and its traf\ufb01cking through the cytoplasm , and some published data are contradic - tory . For example , the role played by cathepsins and acidi\ufb01cation of the microen - vironment during transition of the HCoV - NL63 S protein to its fusogenic form remains unclear . We made an effort to systematically examine every step of the process . First , we tested whether the virus requires endocytosis for successful entry . To do this , we carried out experiments using chemical inhibitors of endosome acidi\ufb01cation ( ammonium chloride and ba\ufb01lomycin A ) . Both blocked virus infection in LLC - MK2 cells , suggesting a requirement for transport of virions to endosomes , which then undergo acidi\ufb01cation . However , such an approach may have several disadvantages . First , we examined the role of endosome acidi\ufb01cation based on virus replication ; thus , we cannot rule out interference with virus infection at later stages ( as shown for MiTMAB ) . Second , the speci\ufb01city and selectivity of chemical inhibitors are questionable . An indirect proof of the pH dependence of HCoV - NL63 entry may be provided by the fact that acidi\ufb01cation of the environment ( acid wash ) results in inactivation of the virus , suggesting a pH - directed structural switch in the S protein . To further con\ufb01rm our observations , we FIG 10 Clathrin and dynamin inhibitors hamper replication of HCoV - NL63 in LLC - MK2 cells . ( A ) HCoV - NL63 replication in LLC - Mk2 cells and HAE cultures in the presence of entry inhibitors or control DMSO was analyzed using RT - qPCR . Cultures were incubated with 10 (cid:2) M Pitstop 2 , 10 (cid:2) M MiTMAB , 5 (cid:2) g / ml nystatin , or DMSO for 1 h at37\u00b0C and inoculated with HCoV - NL63 ( TCID 50 (cid:3) 400 / ml ) . After 2 h of incubation at 32\u00b0C , virions that were not internalized were inactivated with acidic buffer ( pH 3 ) , and cells were incubated for 5 days at 32\u00b0C . The data are presented as log reduction value ( LRV ) compared to the control sample . The assay was performed in triplicate , and average values with standard errors are presented . P values of (cid:4) 0 . 05 were considered signi\ufb01cant and are denoted with an asterisk . ( B ) The cytotoxicity of the inhibitors was tested with an XTT assay . Cells were incubated with 10 (cid:2) M Pitstop 2 , 10 (cid:2) M MiTMAB , 5 (cid:2) g / ml nystatin , or DMSO for 5 days at 32\u00b0C . Data on the y axis represent viability of the treated cells compared to the untreated reference samples . The assay was performed in triplicate , and average values with standard errors are presented . Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 10 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m developed a method of visualizing single virions as they entered the cell . Efforts to stain for virus surface proteins yielded poor results , most likely due to the lack of highly speci\ufb01c antibodies and posttranslational modi\ufb01cation of surface proteins , and the best results were obtained when antibodies speci\ufb01c to the N protein were used . Incubation of cells with puri\ufb01ed virions resulted in virus attachment , which was visualized by confocal microscopy and costaining for markers of the most commonly employed endocytic pathways and allowed us to study the colocaliza - tion . If signi\ufb01cant colocalization was detected , the results were con\ufb01rmed with chemical inhibitors . The results showed that HCoV - NL63 binding to ACE2 initiates recruitment of clathrin and subsequent formation of clathrin - coated pits ; no colocalization of the virus with other markers ( e . g . , caveolin ) was noted . Transferrin was used as a positive control for clathrin - mediated endocytosis ( 32 , 33 ) . Importantly , chemical inhibitors of clathrin completely blocked virus internalization , and the virus remained on the cell surface . Analysis of HAE cultures yielded identical results . The inhibitors of endocytosis also hampered virus infection on LLC - Mk2 cells , highlighting that this pathway is relevant and the lack of an equally effective alternative entry route in this culture model . Clathrin - mediated endocytosis requires a number of other proteins , such as dynamin , the GTPase responsible for scission of clathrin - coated vesicles from the cell surface ( 34 ) . Inhibiting dynamin also hampered virus internalization into LLC - MK2 cells and HAE cultures , con\ufb01rming our previous observations . However , in this case the MiTMAB compound blocked replication of HCoV - NL63 also during subsequent stages of the infection . It is noteworthy that we were not able to block virus infection of HAE cultures using inhibitors of endocytosis . This may be related to the fact that the cultures were exposed to inhibitors for a very short time during apical washes , which is not suf\ufb01cient to permanently block the infection . On the other hand , it is also possible that in HAE , HCoV - NL63 is able to enter the cell by an alternative route . Recent reports on other coronaviruses ( 28 , 29 , 35 ) suggested that these viruses may bypass the endocytic entry route using TMPRRS2 as the priming protease , enabling entry directly from the cell surface . Our experiments showed that inhibition of this protease indeed inhibited virus infection . Interestingly , it did not hamper virus internalization into the cell . Our data are consistent with the data presented by others ( 28 , 29 , 35 ) , yet we believe that there is a different mechanistic explanation for the observed phenomenon . We believe that FIG 11 TMPRSS2 is required for entry into HAE cells but does not enable virus - cell fusion on the cell surface . ( A ) HCoV - NL63 replication in LLC - Mk2 cells and HAE cultures in the presence of camostat or control DMSO was analyzed using RT - qPCR . Cultures were incubated with 100 (cid:2) M camostat or DMSO fo r 1 h at37\u00b0C and inoculated with HCoV - NL63 ( TCID 50 (cid:3) 400 / ml ) . After 2 h ofincubation at 32\u00b0C , virions that were not internalized were inactivated with acidic buffer ( pH 3 ) , and cells were incubated for 5 days at 32\u00b0C . The data are presented as log reduction value ( LRV ) compared to the control sample . The assay was performed in triplicate , and average values with standard errors are presented . P values of (cid:4) 0 . 05 were considered signi\ufb01cant and are denoted with asterisks . ( B ) HAE cultures were incubated with control DMSO or 100 (cid:2) M camostat for 1 h at37\u00b0C . Further , cells were inoculated with puri\ufb01ed HCoV - NL63 and incubated at 32\u00b0C for 2 h . Subsequently , cells were \ufb01xed and immunostained for HCoV - NL63 particles ( green ) , actin ( red ) , and nuclei ( blue ) . Scale bars (cid:3) 5 (cid:2) m . Entry of HCoV - NL63 Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 11 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m TMPRRS2 indeed is required for the virus - cell fusion , acting similarly to cathepsins , but it does not enable fusion on the cell surface , and the acidi\ufb01cation of the microenvi - ronment is required . Our \ufb01nal research question was about virus traf\ufb01cking . The endosome typically translocates through the depolymerizing actin cortex and is subsequently sorted at the endosomal hub and directed to different destinations . This sorting is highly dependent on the cargo . Using two chemical inhibitors ( jasplakinolide and cytochalasin B ) ( 31 , 36 ) , we showed that actin plays a vital role in virus entry . Stabilization of the actin cortex using jasplakinolide resulted in immobilization of the virus at the cell surface , similarly to the case for inhibition of actin polymerization using cytochalasin D . These two experiments suggest a scenario in which virus - carrying endosomes pass along the actin cortex , which actively unwinds and interacts with virions . In summary , we show that HCoV - NL63 enters the cell by clathrin - mediated endo - cytosis , but the pathway may be bypassed to some extent during infection ex vivo . HCoV - NL63 entry into the susceptible cell is summarized in Fig . 14 . FIG 12 Actin is important for HCoV - NL63 entry . LLC - MK2 cells were incubated with DMSO ( A ) , 10 (cid:2) M cytochalasin D ( B and E ) , 1 . 5 (cid:2) M jasplakinolide ( C and F ) , or 400 nM nocodazole ( D and G ) fo r 1 h at37\u00b0C and then inoculated with puri\ufb01ed HCoV - NL63 and incubated at 32\u00b0C for 1 h . Actin and virus localization was veri\ufb01ed with confocal microscopy ; \ufb01xed cells were immunostained for HCoV - NL63 particles ( green ) , actin ( red ) , and nuclei ( blue ) . Scale bars (cid:3) 10 (cid:2) m . Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 12 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m MATERIALS AND METHODS Cell culture . LLC - Mk2 cells ( ATCC CCL - 7 ; Macaca mulatta kidney epithelial ) were maintained in minimal essential medium ( MEM ) ( two parts Hanks\u2019 MEM and one part Earle\u2019s MEM ; Thermo Fisher Scienti\ufb01c , Poland ) supplemented with 3 % heat - inactivated fetal bovine serum ( Thermo Fisher Scienti\ufb01c , Poland ) , penicillin ( 100 U / ml ) , streptomycin ( 100 (cid:2) g / ml ) , and cipro\ufb02oxacin ( 5 (cid:2) g / ml ) . Cells were cultured at 37\u00b0C under 5 % CO 2 . Ethics statement . Human tracheobronchial epithelial cells were obtained from airway specimens resected from adult patients undergoing surgery under Silesian Center for Heart Diseases - approved protocols . This study was approved by the Bioethical Committee of the Medical University of Silesia in Katowice , Poland ( approval no . KNW / 0022 / KB1 / 17 / 10 dated 16 February 2010 ) . Participants provided their written informed consent to participate in the study , as approved by the Bioethical Committee . HAE cultures . Primary human tracheobronchial epithelial cells were expanded on plastic to generate passage 1 cells and plated on permeable Transwell insert ( 6 . 5 - mm - diameter ) supports . Human airway epithelium ( HAE ) cultures were generated by provision of an air - liquid interface for 6 to 8 weeks to form well - differentiated , polarized cultures that resemble in vivo pseudostrati\ufb01ed mucociliary epithelium . Cultures were prepared and maintained as previously described ( 24 ) . Cell viability assay . LLC - Mk2 cells were cultured on 96 - well plates , and HAE cultures were prepared as described above . Cell viability assay was performed by using the 2 , 3 - bis - ( 2 - methoxy - 4 - nitro - 5 - sulfophenyl ) - 2H - tetrazolium - 5 - carboxanilide salt ( XTT ) cell viability assay ( Biological Industries , Israel ) according to the manufacturer\u2019s instructions . Brie\ufb02y , on the day of the assay , 100 (cid:2) l of the culture medium ( for LLC - Mk2 ) or 1 (cid:2) PBS ( for HAE ) with 30 (cid:2) l of the activated XTT solution was added to each well / culture insert . Following 2 h ofincubation at 37\u00b0C , the solution was transferred onto a 96 - well plate FIG 13 Cytotoxicity of the cytoskeleton - modifying compounds . The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay . Cells were incubated with DMSO , 10 (cid:2) M cytochalasin D , 1 . 5 (cid:2) M jasplakinolide , or 400 nM nocodazole fo r 2 h at37\u00b0C . Data on the y axis represent viability of the treated cells compared to the untreated reference samples . The assay was performed in triplicate , and average values with standard errors are presented . FIG 14 Early events during HCoV - NL63 infection . Entry of HCoV - NL63 Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 13 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m and the signal was measured at (cid:4) (cid:3) 490 nm using a colorimeter ( Spectra Max 250 ; Molecular Devices ) . The results obtained were further normalized to the control sample , for which cell viability was set to 100 % . Virus preparation and titration . The HCoV - NL63 stock ( isolate Amsterdam 1 ) was generated by infecting monolayers of LLC - Mk2 cells . The virus - containing liquid was aliquoted and stored at (cid:6) 80\u00b0C . A control LLC - Mk2 cell lysate from mock - infected cells was prepared in the same manner . The virus yield was assessed by titration on fully con\ufb02uent LLC - Mk2 cells in 96 - well plates according to the method described by Reed and Muench ( 37 ) . Puri\ufb01cation of HCoV - NL63 . The virus stock was concentrated 25 - fold using centrifugal protein concentrators ( Amicon Ultra , 10 - kDa cutoff ; Merck , Poland ) and subsequently overlaid on 15 % iodixanol solution in 1 (cid:2) PBS ( OptiPrep medium ; Sigma - Aldrich , Poland ) . Following virus concentration using an iodixanol cushion ( centrifugation at 175 , 000 (cid:2) g for 3 h at4\u00b0C ) , it was overlaid on a 10 to 20 % iodixanol gradient in 1 (cid:2) PBS and centrifuged at 175 , 000 (cid:2) g for 18 h at 4\u00b0C . Fractions ( 1 ml ) collected from the gradient were analyzed by Western blotting using anti - HCoV - NL63 N IgGs ( 0 . 25 (cid:2) g / ml ; Ingenansa , Spain ) and a secondary antibody coupled with horseradish peroxidase ( 65 ng / ml ; Dako , Denmark ) . The virus - containing fractions were aliquoted and stored at (cid:6) 80\u00b0C . The control cell lysate ( mock ) was concentrated and prepared in the same manner as the virus stock . Inhibition of virus entry . LLC - Mk2 cells were seeded on coverslips in six - well plates ( TPP , Switzer - land ) and cultured for 2 days at 37\u00b0C . Subsequently , cells were incubated with a given inhibitor for 30 min at 37\u00b0C and later with 50 (cid:2) l of puri\ufb01ed HCoV - NL63 or mock sample for 1 h at 32\u00b0C . For the ex vivo experiment , HAE cultures were exposed to the tested inhibitor or control PBS for 1 h at37\u00b0C following inoculation with iodixanol - concentrated HCoV - NL63 or mock sample . Followin g 2 h ofincubation at 32\u00b0C , unbound virions were removed by washing with 1 (cid:2) PBS . Cells were then washed with 1 (cid:2) PBS and \ufb01xed with 4 % paraformaldehyde ( PFA ) . Transferrin and albumin were used as positive controls , as they were previously described to serve as a cargo in clathrin - and caveolin - dependent endocytosis , respectively ( 38 , 39 ) . LLC - Mk2 cells were seeded on coverslips in six - well plates ( TPP , Switzerland ) and cultured for 2 days at 37\u00b0C . Subsequently , cells were incubated with a given inhibitor for 30 min at 37\u00b0C following incubation with Alexa Fluor 488 - labeled transferrin ( 100 (cid:2) g / ml ; Molecular Probes ) , \ufb02uorescein isothiocyanate ( FITC ) - labeled albumin ( 500 (cid:2) g / ml ; Sigma - Aldrich , Poland ) , or control PBS for 45 min at 32\u00b0C . Cells were then washed with 1 (cid:2) PBS and \ufb01xed in 4 % PFA . Immunostaining and confocal imaging . Fixed cells were permeabilized with 0 . 1 % Triton X - 100 in 1 (cid:2) PBS and incubated overnight at 4\u00b0C in 1 (cid:2) PBS supplemented with 5 % bovine serum albumin ( BSA ) and 0 . 5 % Tween 20 . To visualize HCoV - NL63 particles , cells were incubated fo r 2 h atroom temperature with mouse anti - HCoV - NL63 N IgGs ( 0 . 25 (cid:2) g / ml ; Ingenansa , Spain ) , followed b y 1 h ofincubation with Alexa Fluor 488 - labeled goat anti - mouse IgG ( 2 . 5 (cid:2) g / ml ; Thermo Fisher Scienti\ufb01c , Poland ) . The following antibodies were used for endosomal markers : polyclonal goat anti - human clathrin HC coupled with tetramethylrhodamine ( 10 (cid:2) g / ml ; Santa Cruz Biotechnology ) , polyclonal rabbit anti - human early endo - some antigen 1 ( 2 (cid:2) g / ml ; Santa Cruz Biotechnology ) , polyclonal rabbit anti - human caveolin 1 ( 2 (cid:2) g / ml ; Sigma - Aldrich , Poland ) , and Alexa Fluor 633 - labeled goat anti - rabbit ( 2 . 5 (cid:2) g / ml ; Thermo Fisher Scienti\ufb01c , Poland ) . Actin \ufb01laments was stained using phalloidin coupled with Alexa Fluor 633 ( 0 . 2 U / ml ; Thermo Fisher Scienti\ufb01c , Poland ) . Nuclear DNA was stained with DAPI ( 4 = , 6 = - diamidino - 2 - phenylindole ) ( 0 . 1 (cid:2) g / ml ; Sigma - Aldrich , Poland ) . Immunostained cultures were mounted on glass slides in ProLong Gold antifade medium ( Thermo Fisher Scienti\ufb01c , Poland ) . Fluorescent images were acquired under a Leica TCS SP5 II confocal microscope ( Leica Microsystems GmbH , Mannheim , Germany ) and a Zeiss LSM 710 confocal microscope ( Carl Zeiss Microscopy GmbH ) . Images were acquired using Leica Application Suite Advanced Fluorescence LAS AF v . 2 . 2 . 1 ( Leica Microsystems CMS GmbH ) or ZEN 2012 SP1 software ( Carl Zeiss Microscopy GmbH ) deconvolved with Huygens Essential package version 4 . 4 ( Scienti\ufb01c Volume Imaging B . V . , The Netherlands ) and processed using ImageJ 1 . 47v ( National Institutes of Health , Be - thesda , MD , USA ) . Flow cytometry . LLC - Mk2 cells were seeded on 6 - well plates ( TPP ) and cultured for 2 days at 37\u00b0C with 5 % CO 2 . Cells in monolayer were incubated with each entry inhibitor fo r 1 h at37\u00b0C following infection with HCoV - NL63 at a TCID 50 of 100 / ml or inoculation of the mock sample . On day 4 p . i . , cells were washed with sterile PBS , \ufb01xed with 3 % PFA , permeabilized with 0 . 1 % Triton X - 100 in 1 (cid:2) PBS , and incubated for 1 h with 3 % BSA in 1 (cid:2) PBS with 0 . 1 % Tween 20 . To quantify HCoV - NL63 infection , \ufb01xed cells were scraped from the plastic and incubated fo r 2 h atroom temperature with mouse anti - HCoV - NL63 N IgG antibodies ( 1 (cid:2) g / ml ; Ingenansa ) , followed b y 1 h ofincubation with Alexa Fluor 488 - labeled goat anti - mouse antibody ( 2 . 5 (cid:2) g / ml ; Molecular Probes ) . Cells were then washed , resuspended in 1 (cid:2) PBS , and analyzed with a FACSCalibur instrument ( Becton Dickinson ) using Cell Quest software . Isolation of nucleic acids and reverse transcription . Viral nucleic acids were isolated from cell culture supernatants ( LLC - Mk2 cells ) or apical washes ( HAE cultures ) using the viral RNA / DNA isolation kit ( A & A Biotechnology , Poland ) according to the manufacturer\u2019s instructions . Reverse transcription was carried out with a high - capacity cDNA reverse transcription kit ( Thermo Fisher Scienti\ufb01c , Poland ) , according to the manufacturer\u2019s instructions . RT - qPCR . The HCoV - NL63 yield was determined by RT - qPCR ( 7500 Fast Real - Time PCR machine ; Life Technologies , Poland ) . Viral cDNA ( 2 . 5 (cid:2) l per sample ) was ampli\ufb01ed in a 10 - (cid:2) l reaction mixture containing 1 (cid:2) master mix ( RT Mix Probe ; A & A Biotechnology , Poland ) , a speci\ufb01c probe labeled with 6 - carboxy\ufb02uorescein ( FAM ) and 6 - carboxytetramethylrhodamine ( TAMRA ) ( 100 nM ) ( 5 = - ATG TTA TTC AGT GCT TTG GTC CTC GTG AT - 3 = ) , and primers ( 450 nM each ) ( sense , 5 = - CTG TGG AAA ACC TTT GGC ATC - 3 = ; antisense , 5 = - CTG TGG AAA ACC TTT GGC ATC - 3 = ) . Rox was used as the reference dye . The reaction Milewska et al . Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 14 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m conditions were as follows : 2 min at 50\u00b0C and 10 min at 92\u00b0C , followed by 40 cycles of 15 s at 92\u00b0C and 1 min at 60\u00b0C . In order to assess the copy number for the N gene , DNA standards were prepared . Brie\ufb02y , the N gene of HCoV - NL63 was ampli\ufb01ed and cloned into pTZ57R / T ( Thermo Fisher Scienti\ufb01c , Poland ) plasmid using the InsTAclone PCR cloning kit ( Thermo Fisher Scienti\ufb01c , Poland ) . Subsequently , DNA vectors were ampli\ufb01ed and linearized with EcoRI restriction enzyme . Linear nucleic acids were further puri\ufb01ed with the GeneJET PCR puri\ufb01cation kit ( Thermo Fisher Scienti\ufb01c , Poland ) according to the manufacturer\u2019s instructions , and its concentration was assessed using a spectrophotometer . The number of DNA copies per milliliter was assessed using Avogadro\u2019s constant and the molecular mass of RNA molecules . Samples were serially diluted and used as an input for real - time PCR . In this article , the data from quantitative PCR are presented as log removal values ( LRVs ) in order to enable comparison of results obtained from different assays . The LRV was calculated according to the formula LRV (cid:3) (cid:6) log ( c i / c 0 ) , where c i is the number of viral RNA copies per milliliter in the sample from the culture treated with a given polymer and c 0 is the number of viral RNA copies per milliliter in the control sample ( untreated cells ) . Image analysis . To evaluate the infection inhibition in the presence of various endocytosis inhibitors , image analysis was performed on 2 - mm by 2 - mm tile scan images . On each image , the number of nuclei ( expressed as a number of cells ) and the mean pixel intensity for the virus were calculated . For that , histograms of all images were adjusted to the minimum / maximum value , excluding signal from the virus derived from images with no infected cells . Results are presented as mean intensity of \ufb02uorescence per cell . Colocalization analyses were performed under ImageJ using the JACoP plugin ( 41 ) , where Manders\u2019 coef\ufb01cient was calculated for 3D images of more than 5 cells . Quantitative analysis of virus internalization in the presence of inhibitors was performed with the algorithm previously described by Berniak et al . with modi\ufb01cations ( 40 ) . The cell surface was estimated on each image slice manually using the polygon selection tool in ImageJ , and based on this information , the 3D cell surface was modeled . Coordinates of virus particles were determined using the 3D Object Counter ImageJ plugin . The relative localization and distance between the virus particle and cell surface were calculated . Results are presented as a ratio between virus particles inside a cell and the particles on the surface ( up to 1 . 5 (cid:2) m above ) . Statistical analysis . All the experiments were performed in triplicate , and the results are presented as mean (cid:7) standard deviation ( SD ) . To determine the signi\ufb01cance of the obtained results , a comparison between groups was conducted using the Student t test . P values of (cid:4) 0 . 05 were considered signi\ufb01cant . ACKNOWLEDGMENTS This work was supported by grants from the National Science Center ( UMO - 2012 / 07 / E / NZ6 / 01712 , UMO - 2012 / 07 / N / NZ6 / 02955 , and 2015 / 19 / N / NZ1 / 00323 to K . P . , A . M . , and K . B . , respectively ) and the Ministry of Science and Higher Education ( 0058 / DIA / 2015 / 44 to P . N . ) . K . P . acknowledges a networking contribution from the COST Action CM1407 \u201cChallenging Organic Syntheses Inspired by Nature\u2014from Natural Products Chemistry to Drug Discovery . \u201d The Faculty of Biochemistry , Biophysics and Biotechnol - ogy of Jagiellonian University is a bene\ufb01ciary of structural funds from the European Union ( grant no . POIG . 02 . 01 . 00 - 12 - 064 / 08 , \u201cMolecular Biotechnology for Health\u201d ) and is a partner of the Leading National Research Center supported by the Ministry of Science and Higher Education of the Republic of Poland . The funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript . REFERENCES 1 . van der Hoek L , Pyrc K , Jebbink MF , Vermeulen - Oost W , Berkhout RJ , Wolthers KC , Wertheim - van Dillen PM , Kaandorp J , Spaargaren J , Berk - hout B . 2004 . Identi\ufb01cation of a new human coronavirus . 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Journal of Virology February 2018 Volume 92 Issue 3 e01933 - 17 jvi . asm . org 16 on J anua r y 23 , 2018 b y ABE - I PS h tt p : / / j v i . a s m . o r g / D o w n l oaded f r o m View publication stats", + "jin2022branched": "ARTICLE Branched actin networks are organized for asymmetric force production during clathrin - mediated endocytosis in mammalian cells Meiyan Jin 1 , 6 , Cyna Shirazinejad 1 , 2 , 6 , Bowen Wang 3 , Amy Yan 1 , Johannes Sch\u00f6neberg 1 , 5 , Srigokul Upadhyayula 1 , 4 , Ke Xu 3 & David G . Drubin 1 \u2709 Actin assembly facilitates vesicle formation in several traf \ufb01 cking pathways , including clathrin - mediated endocytosis ( CME ) . Interestingly , actin does not assemble at all CME sites in mammalian cells . How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed , are not fully understood . Here , branched actin network geometry at CME sites was analyzed using three different advanced imaging approaches . When endocytic dynamics of unperturbed CME sites are compared , sites with actin assembly show a distinct signature , a delay between completion of coat expansion and vesicle scission , indicating that actin assembly occurs preferentially at stalled CME sites . In addition , N - WASP and the Arp2 / 3 complex are recruited to one side of CME sites , where they are positioned to stimulate asymmetric actin assembly and force production . We pro - pose that actin assembles preferentially at stalled CME sites where it pulls vesicles into the cell asymmetrically , much as a bottle opener pulls off a bottle cap . https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 OPEN 1 Department of Molecular and Cell Biology , University of California , Berkeley , CA , USA . 2 Biophysics Graduate Group , University of California Berkeley , Berkeley , CA , USA . 3 Department of Chemistry , University of California , Berkeley , CA , USA . 4 Chan Zuckerberg Biohub , San Francisco , CA , USA . 5 Present address : Department of Pharmacology , and Department of Chemistry and Biochemistry , University of California , San Diego , CA , USA . 6 These authors contributed equally : Meiyan Jin , Cyna Shirazinejad . \u2709 email : drubin @ berkeley . edu NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications 1 1 2 3 4 5 6 7 89 0 ( ) : , ; F ormation of clathrin - coated vesicles requires forces to \ufb01 rst bend the membrane into a sphere or tube , and to then break the thin neck that connects the vesicle to the plasma membrane . These forces are generated through the combined actions of proteins that directly bend the membrane and actin \ufb01 lament assembly 1 \u2013 5 ( Supplementary Fig . 1a ) . Several studies have demonstrated that dependence of CME on actin assembly increases under elevated membrane tension 6 \u2013 10 . Interestingly , actin does not assemble at all CME sites in mammalian cells , suggesting highly localized differences in requirement for actin assembly , the nature of which is obscure 6 , 11 , 12 . A detailed understanding of how actin forces are harnessed to aid vesicle formation and scission depends on understanding which CME sites assemble actin , where \ufb01 lament assembly occurs around the endocytic membrane , and when . In yeast cells , where turgor pressure is particularly high , super - resolution data suggest that actin assembles symmetrically around CME sites and indicate that actin regulators including Las17 , which is yeast WASP , are present in a ring surrounding the base of the clathrin coat symmetrically 13 . On the other hand , studies on \ufb01 xed mammalian cells raised the possibility that actin assembly may at least in some cases be initiated asymmetrically at clathrin coats 14 , 15 . However , methods used for these studies prevented analysis of large numbers of sites , and suffered from possible loss of actin \ufb01 la - ments during unroo \ufb01 ng and extraction of the cells . Which CME sites assemble actin , and how actin networks are organized with respect to CME sites , has not been determined systematically in a large - scale , unbiased manner , particularly in live mammalian cells . This information is essential to understanding how and why actin assembly forces are harnessed for CME . Here , by combining \ufb01 xed and live - cell imaging of triple - gen - ome - edited , human induced pluripotent stem cells ( iPSCs ) , and newly developed machine - learning - based computational analysis tools , we report that N - WASP and the Arp2 / 3 complex localize at one side of the coat and neck of invaginating endocytic sites until vesicle scission . Most importantly , by comparing recruitment dynamics of proteins from three distinct endocytic modules for over one thousand unperturbed endocytic events , we found that branched actin assembly occurs predominantly at sites that have stalled between coat expansion and vesicle scission . We propose that these branched actin networks rescue stalled CME . Results Super - resolution imaging reveals asymmetric actin distribu - tion around endocytic sites . To investigate the physiological roles and spatiotemporal regulation of actin assembly at CME sites in mammalian cells , we applied genome - editing techniques to generate a human iPSC line ( hereafter referred to as ADA cells ) that co - expresses a TagRFP - T fusion of the mu subunit of the AP2 adaptor complex ( AP2M1 ) , a TagGFP2 fusion of dynamin2 ( DNM2 ) , and a HaloTag fusion of the ARPC3 subunit of the Arp2 / 3 complex as representatives of the CME coat , scission and actin modules , respectively 2 , 16 , 17 ( Supplementary Fig . 1a , b ) . Previous studies showed that endogenously tagged AP2M1 , DNM2 and ARPC3 can serve as reliable markers of these CME functional modules that avoid disruption of physiological spa - tiotemporal organization of the process as might be caused by overexpression of \ufb02 uorescently labeled proteins 12 , 18 \u2013 21 . We observed dynamic CME events on the basal plasma membrane of the genome - edited cells using Total Internal Re \ufb02 ection Fluores - cence ( TIRF ) microscopy ( Supplementary Fig . 1c and Supple - mentary Movie 1 , 2 ) . Consistent with previous studies , AP2 is recruited at early CME stages while DNM2 is recruited in two phases 11 , 16 , 20 , 22 . At the early stage of CME , a relatively small amount of DNM2 is recruited to CME sites . Shortly before the end of a CME event , the DNM2 recruitment rate increases rapidly with DNM2 levels reaching a peak concomitant with vesicle scission 16 , 20 , 23 ( Supplementary Fig . 1c ) . This later rapid - recruitment phase represents the assembly of the dynamin helix on the highly curved neck of the budding vesicle after the U to \u03a9 shape transition of the endocytic membrane 20 , 23 \u2013 27 . Super - resolution imaging of \ufb01 xed human skin melanoma SKMEL cells suggested that actin is arranged asymmetrically around CME sites 7 , consistent with indications from other previous studies 14 , 15 . To systematically analyze how actin networks are organized at CME sites in iPSCs , we \ufb01 rst performed two - color 3D Stochastic Optical Reconstruction Microscopy ( STORM ) imaging 28 on \ufb01 xed ADA cells , localizing either AF647 phalloidin - labeled actin \ufb01 laments 29 or HaloTag - fused ARPC3 at CME sites . Due to the dense phalloidin labeling of cortical actin \ufb01 laments under the plasma membrane , it was often challenging to unambiguously identify the CME - speci \ufb01 c actin structures in iPSCs . However , in regions with thinner cortical actin layers , we observed that actin was typically distributed asymmetrically around CME sites ( Fig . 1a \u2013 c ) . Antibody labeling of ARPC3 - Halotag in the ADA cells had the advantage of a less complex staining pattern . Besides being highly concentrated in lamellipodia , ARPC3 was associated with CME sites asymme - trically , like actin ( Fig . 1d \u2013 f ) . These data suggest that the Arp2 / 3 - mediated actin network is arranged asymmetrically around CME sites . Actin networks assembled at CME sites remain asymmetric through scission . We next used ADA cells to investigate actin assembly at CME sites in live cells , which has several advantages over studies in \ufb01 xed cells . During the \ufb01 xation and subsequent sample preparation , actin structures may not be faithfully pre - served . In addition , in live cells it is easier to identify the stage of CME , so the timing , geometry and dynamics of actin assembly can be related to the endocytic stage . More importantly , only by using live cells is it possible to trace a single CME event from start to \ufb01 nish , and to therefore identify those CME events wherein no detectable actin is ever assembled , so key parameters can be compared between events with and without associated actin assembly . By visualizing endogenously tagged AP2M1 to mark the coat and CME initiation , and DNM2 to mark the neck and scission , together with ARPC3 to speci \ufb01 cally label Arp2 / 3 - nucleated , branched actin \ufb01 laments ( Supplementary Fig . 1a ) , we were able to precisely study the spatial and temporal regulation of actin assembly during CME . Three - color labeling and analysis of the displacement between markers for the three modules allowed us to distinguish bona \ufb01 de asymmetric actin assembly from events that might artifactually appear asymmetric because the invagina - tions were elongated and tilted ( Fig . 2a ) . Using TIRF live - cell imaging , we observed ARPC3 - labeled branched actin networks at lamellipodia and at a subpopulation of CME sites ( Fig . 2b , c ) . Dynamic actin assembly and disassembly occurred at CME sites with different spatio - temporal characteristics , including discrete CME sites , clathrin plaques and at clathrin coat splitting sites , as previously reported 14 ( Fig . 2d and Supplementary Fig . 2a , b ) . In the analysis described below , we focus on the discrete CME events and not the more complex ones ( plaques and splitting events ) ( Supplementary Fig . 3a ) . Analysis of the discrete events with 1 s / frame temporal resolution revealed that ARPC3 is most robustly recruited during the late stages of CME shortly before scission 12 ( Fig . 2c , d ) . Interestingly , we observed clear spatial displacement between ARPC3 ( actin module ) and AP2 ( coat module ) as well as between ARPC3 and DNM2 ( neck ) before vesicle scission ( Fig . 2d ) . This observation supports the conclusion that ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 2 NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications asymmetric branched actin networks provide forces at endocytic sites through the time of scission . To investigate the spatiotemporal relationship between actin and dynamin with higher resolution , we used live - cell Airyscan2 confocal microscopy of live cells ( Fig . 3 and Supplementary Movie 3 ) . We observed clear displacement between ARPC3 and DNM2 signals at the time of vesicle scission at most CME sites ( Fig . 3b ) . Notably , ARPC3 remained on one side of DNM2 throughout the last second of CME until scission ( Fig . 3c , d ) . These observations of asymmetric branched actin network assembly at CME sites are consistent with the ones we made with TIRF live - cell imaging ( Fig . 2 ) . a clathrin top view actin c clathrin ARPC3 top view top view side view top view side view top view side view top view side view top view side view A c t i n C l a t h r i n M e r ge p l a s m a m e m b r ane cy t o s o l z top view side view top view side view top view side view top view side view top view side view A R P C 3 C l a t h r i n M e r ge p l a s m a m e m b r ane cy t o s o l z b d 0 20 40 60 80 100120140160180 0 5 10 15 20 Clathrin - actin Distance ( nm ) C oun t s N = 67 71 . 15 \u00b1 36 . 10 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 10 20 30 Clathrin - ARPC3 Distance ( nm ) C oun t s N = 161 107 . 26 \u00b1 49 . 66 e f 0 nm 1500 nm 0 nm 1500 nm NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 ARTICLE NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications 3 Fig . 1 Two - color , 3D stochastic optical reconstruction microscopy ( STORM ) shows that actin structures are off - centered with respect to clathrin coats . a , b Two - color 3D STORM image of bottom membrane of ADA cells immunolabeled with clathrin light chain antibody ( clathrin , CF - 680 , magenta ) and phalloidin ( actin , AF647 , rainbow ) . c A histogram of distances between centroids of clathrin and actin signals . Mean and standard deviation are reported on the graph . Data were analyzed using Prism 9 . Source data are provided in the Source Data \ufb01 le . d , e Two color 3D STORM image of the bottom membrane of ADA cells immunolabeled with clathrin light chain antibody ( clathrin , AF647 , magenta ) and HaloTag antibody ( ARPC3 - HaloTag , CF - 680 , rainbow ) . Dotted lines label lamellipodia . b , e The highlighted CME sites , which are labeled by white arrows in a , d , are rotated and shown in magni \ufb01 ed top and side view projections . Color bar shows the z position of ARPC3 - HaloTag . f A histogram of distances between centroids of clathrin and ARPC3 signals . Mean and standard deviation are reported on the graph . Data were analyzed using Prism 9 . Source data are provided in the Source Data \ufb01 le . Scale bars : a , d : 2 \u00b5 m , b , e : 100 nm . DNM2 - tagGFP2 AP2 - tagRFP - T ARPC3 - HaloTag Merge b DNM2 - tagGFP2 AP2 - tagRFP - T ARPC3 - HaloTag Merge 4 min c d ARPC3 - HaloTag AP2 - tagRFP - T ARPC3 - HaloTag DNM2 - tagGFP2 AP2 - tagRFP - T 1s DNM2 - tagGFP2 ARPC3 - HaloTag * * * * * Center of signal Coat module ( AP2 ) Scission module ( DNM2 ) Actin module ( ARPC3 ) Plasma membrane a Model 1 Model 2 Model 3 Fig . 2 Triple - genome - edited iPSCs reveal dynamic actin organization at CME sites . a Models of branched actin assembly at invaginating CME sites . Model 1 : Asymmetric actin assembly at CME sites results in separated actin - coat and actin - neck signals . Model 2 : Symmetric actin assembly at tilted CME sites results in separated actin - coat signals but overlapped actin - neck signals . Model 3 : Symmetric actin assembly at perpendicularly invaginating CME sites will result overlapped actin , coat and neck signals . b A representative single time frame image of a TIRF movie ( Supplementary Movie 2 ) of AP2M1 - tagRFP - T ( magenta ) , DNM2 - tagGFP2 ( green ) and JF635 ligand 51 - conjugated ARPC3 - HaloTag ( cyan ) in ADA cells . The highlighted region is boxed by a dashed line . Scale bar : 5 \u00b5 m . c A representative kymograph of AP2M1 - tagRFP - T ( magenta ) , DNM2 - tagGFP2 ( green ) and JF635 ligand - conjugated ARPC3 - HaloTag ( cyan ) at CME sites in ADA cells . Scale bar : 5 \u00b5 m . d Montage of a representative ARPC3 positive CME site in ADA cells . Individual channels and pair - wise merges are shown . * : Images from one frame before scission ( maximum DNM2 intensity ) are marked to show the displacement between the CME coat ( AP2 ) - ARPC3 and CME neck ( DNM2 ) - ARPC3 . Size of \ufb01 eld of view : 2 \u00b5 m \u00d7 2 \u00b5 m . Intervals : 1 s . ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 4 NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications To analyze the intrinsic recruitment order and timing for three endocytic proteins at CME sites quantitatively and systematically , we developed an automated , high - throughput method to analyze TIRF live - cell imaging data that avoids bias because it does not involve manual selection of CME sites ( see Materials and Methods ) . Brie \ufb02 y , AP2 tracks were identi \ufb01 ed using standard particle - tracking algorithms 30 . Novel \ufb01 ltering methods then extracted DNM2 - positive events marked by one or more DNM2 burst . The AP2 and DNM2 tracks were decomposed into dynamic features describing each events \u2019 position and brightness . These features were used for clustering via unsupervised machine learning , which enabled grouping of similarly - behaved tracks ( Supplementary Fig . 3a , b ) . DNM2 - positive events were re \ufb01 ned by a detection scheme that determined the number of DNM2 peaks using various characteristics of a single DNM2 - peak : the peak height , width , and minimum peak - to - peak distance ( Supplementary Fig . 3c ) . Events with a single DNM2 peak were analyzed as described below . The method detects low signals from endogenously tagged CME proteins , such as the low - level recruitment of DNM2 at the early CME stages , and accurately reveals the different CME stages ( Supplementary Fig . 3d ) . Next , the timing of actin network assembly at CME sites was determined using ARPC3 as a branched actin \ufb01 lament marker by analyzing over one thousand CME events . Although actin appearance early in CME has been reported 14 , determining the actin assembly timing can be challenging because it is dif \ufb01 cult to distinguish newly assembled branched actin at CME sites from the nearby cortical actin \ufb01 laments or actin \ufb01 laments associated with other vesicles or organelles . Also , whether actin functions during the early stage of CME has not yet been shown conclusively due to the potential side effects such as changes in membrane tension caused by actin inhibitors . Our endogenous ARPC3 tagging and large - scale computational analysis approach sidesteps these problems . We classi \ufb01 ed CME events into three groups : one group without ARPC3 appearance , one group with ARPC3 appearance early in CME , and one group with ARPC3 appearance late in CME ( Supplementary Fig . 4 ) . We observed that in most of the ARPC3 positive events a sharply increasing ARPC3 signal appears with similar timing to the rapid - recruitment phase of DNM2 concomitant with the U to \u03a9 membrane shape transition ( Fig . 4a and Supplementary Fig . 4b ) . This timing is consistent with the previously proposed role for actin in membrane invagination , as studies showed that actin inhibitors block the U to \u03a9 endocytic membrane shape transition 6 , 14 . In some cases we did detect ARPC3 signals at early CME stages ( Supplementary Fig . 4b ) . To test whether random overlap between nearby actin structures and CME sites might be responsible for the apparent early actin recruitment , we generated a randomized data set by pairing ARPC3 images with AP2 and DNM2 images from an unrelated movie ( Supplementary Fig . 4a ) . In this data set , we detected a signi \ufb01 cantly reduced fraction of ARPC3 positive events . However , early \u201c assembly \u201d of actin was observed in a similar fraction of CME events as in the real data set ( Supplementary Fig . 4b ) . Based on these observations we conclude that the presence of actin early in CME is very likely due to nearby actin structures overlapping with CME sites randomly . Our live - cell analysis allowed the timing of branched actin network assembly to be compared to the scission timing , and the spatial offset between the clathrin coat and the associated actin network to be determined . Super - resolution imaging of yeast CME sites suggested that actin and actin nucleators localize * * * DNM2 - tagGFP2 ARPC3 - HaloTag Merge 0 . 2s a c d 010020030040050060070080090010001100120013001400 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0 Distance ( nm ) I n t en s i t y ( a r b . un i t s ) DNM2 ARPC3 b 0 20 40 60 80100120140 160 180200220240260280300320 0 5 10 15 DNM2 - ARPC3 distance ( nm ) C oun t s N = 72 110 . 94 \u00b1 71 . 08 6000 2002500 200 DNM2 - tagGFP2 ARPC3 - HaloTag Merge Fig . 3 Airyscan live - cell imaging reveals asymmetric actin organization at CME sites . a A representative single time frame image of an Airyscan movie ( Supplementary Movie 3 ) of DNM2 - tagGFP2 ( red ) and JF635 ligand - conjugated ARPC3 - HaloTag ( cyan ) on the ventral plasma membrane surface of ADA cells . The highlighted region is boxed by a dashed line . Scale bar : 1 \u00b5 m . b Histogram of distance between the center of mass of DNM2 and ARPC3 at frame corresponding to scission . Mean and standard deviation are shown on the graph . Data were analyzed using Prism 9 . Source data are provided in the Source Data \ufb01 le . c Montage of average intensity projection of representative ARPC3 positive CME sites . Frame corresponding to scission was determined by maximum DNM2 intensity . 33 \u00d7 33 pixels square region centered at DNM2 maximum intensity pixel was cropped for six continuous frames ending in scission . Cropped frame series were rotated by 90 or 180 degrees as needed to align the ARPC3 signal to the left of the center of the image at the frame corresponding to scission . * : Averaged image of the frame corresponding to scission ( maximum DNM2 intensity ) is marked to show the displacement between the CME neck ( DNM2 ) and branched actin ( ARPC3 ) . Scale bar : 1 \u00b5 m . Intervals : 0 . 2 s . N = 72 . d The line scan function in ImageJ software was used to measure the \ufb02 uorescence intensity along a one pixel wide , 33 pixel long horizontal line drawn across the center of the averaged intensity image of the frame corresponding to scission * in c . The signals were normalized to the minimum signal along the line , and intensity was calculated as a ratio to the maximum signal along the line . Source data are provided in the Source Data \ufb01 le . NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 ARTICLE NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications 5 symmetrically in a ring around CME sites , and computational modeling suggested that an asymmetric actin arrangement would not provide suf \ufb01 cient force for the membrane invagination during yeast CME 13 . In contrast , in mammalian cells , which require less actin force production during CME , imaging of \ufb01 xed cells suggested that actin structures associate adjacent to apparent \ufb02 at clathrin coats 14 , 15 . However , these studies proposed that at the later CME stages the actin structures become larger and more symmetric to provide suf \ufb01 cient force for membrane deformation and scission 14 , 15 . Surprisingly , in our live cell studies designed to highlight sites of new actin assembly , we observed off - centered branched actin networks at CME sites throughout even the latest CME stages ( Fig . 2d and Fig . 3b \u2013 d ) . Furthermore , most ARPC3 - positive CME sites accomplish scission within 30 s of the initiation of ARPC3 recruitment ( Fig . 4b ) . The actin networks ( ARPC3 ) we observed were off center from the coat ( AP2 ) and neck ( DNM2 ) signals by approximately 150 nm at the time of vesicle scission ( Fig . 4c ) . Imaging \ufb02 uorescent beads using the same settings indicated that the displacement is not an artifact caused by misalignment between different imaging channels ( Supplementary Movie 4 and Supplementary Fig . 5a ) . By further analyzing \ufb02 uorescent bead images , we concluded that chromatic aberration contributes only a small portion of the AP2 - ARPC3 and AP2 - DNM2 separation we observed by TIRF imaging ( Supplementary Fig . 5b ) . This chromatic aberration might be the reason for slightly increased neck - actin separation we detected by TIRF microscopy ( Fig . 4c ) compared to Airyscan imaging ( Fig . 3b ) . Given the temporal separation between channel acquisition and the movement of AP2 spots , it was important to assess whether the spatial separation between channels can be attributed in part to an imaging artifact caused by puncta movement between subsequent channel acquisitions . When we measured the average movement of AP2 spots leading up to scission , we found that over 95 % of the events had AP2 - ARPC3 separations that exceed the frame - to - frame motility of AP2 ( Supplementary Fig 5c ) . Also , the puncta positional uncertainties indicated by the standard deviations determined when measuring the \ufb01 tted position of AP2 , range up to 40 nm , which is less than the determined displacements . Therefore , we utilized the AP2 - DNM2 separation , which is expected to be small , as the basis for comparison to the AP2 - ARPC3 and DNM2 - ARPC3 separations ( Fig . 4d ) . These results further support our conclusion that branched actin networks assemble asymmetrically at CME sites through the time of scission ( Fig . 2d ) . This observation is consistent with the observation that ring - shaped actin structures at clathrin coats were rarely observed in the high - resolution , live - cell imaging reported in a previous study 31 . In total , these live - cell data suggest that in mammalian cells , asymmetric actin network assembly can provide enough force to assist membrane deformation and scission during the late stages of CME . Asymmetric branched actin networks facilitate CME at stalled sites . To gain additional insights into the function of this asym - metric actin network assembly , we quantitatively compared kinetics of CME events with or without ARPC3 recruitment . We observed that about 30 % of CME events are completed in the absence of detectable actin assembly ( Supplementary Fig . 4b ) , which is consistent with the hypothesis that in mammalian cells a b c D i s t an c e ( \u00b5 m ) F r e quen cy den s i t y ARPC3 lifetime ( s ) 0 . 00 0 . 01 0 . 02 0 . 03 0 . 04 0 . 06 0 . 05 20 0 40 60 100 80 AP2 ARPC3DNM2 DNM2 - ARPC3 AP2 - ARPC3 0 100 200 300 400 500 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 Time ( s ) 0 - 10 - 20 10 20 - 30 AP2 ARPC3DNM2 Lifetime : 7 . 73 % 17 . 33 % 27 . 36 % 47 . 51 % 0 - 10 - 20 10 20 0 - 10 10 0 - 10 - 20 10 20 - 30 30 0 - 10 - 20 10 20 - 30 30 - 40 - 50 - 60 Time ( s ) 0 100 200 300 400 500 I n t en s i t y ( a r b . un i t s ) DNM2 - AP2 < 40s 40 - 60s 60 - 80s > 80s I n t en s i t y ( a r b . un i t s ) Fig . 4 Computational analysis of ARPC3 positive CME sites reveals asymmetric actin network assembly at the late stage of CME . a Averaged intensity vs time plots of cohorts of CME sites with late ARPC3 assembly in ADA cells . Events are grouped into cohorts by the lifetimes of AP2 and aligned to the frames showing the maximum DNM2 intensity ( time = 0 s ) . Percentage of the number of the CME sites in each cohort is shown next to the plot . b Histogram of ARPC3 - mediated actin network assembly duration . The assembly duration is measured from the \ufb01 rst frame of the ARPC3 signal to the presumed scission time ( the peak of DNM2 signal ) . Source data are provided in the Source Data \ufb01 le . c Averaged intensity ( solid lines ) and distance ( dashed lines ) vs time plots of ARPC3 positive CME sites in ADA cells . Events are aligned to the frames showing the maximum DNM2 intensity ( time = 0 s ) . Distance between centers of two signals are shown from - 10 s to 3 s when DNM2 and ARPC3 signals are relatively high . a \u2013 c N = 1 , 385 . a , c Error bar : \u00bc standard deviation . . ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 6 NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications actin assembly is required for CME only under certain conditions , such as relatively high membrane tension or speci \ufb01 c cargo internalization , which can vary regionally within cells 6 , 7 , 9 , 10 , 32 , 33 . Consistent with the possibility that unfavorable conditions such as increased membrane tension , might stall membrane defor - mation during CME 4 , 6 , 8 \u2013 10 , 34 \u2013 36 , CME lifetimes were markedly longer for ARPC3 positive events compared to the ARPC3 negative events ( Fig . 5a ) . In addition , when the AP2M1 intensity vs time pro \ufb01 les were compared between ARPC3 positive and negative CME sites , a plateau , which lasts for approximately 10 s , was observed for the ARPC3 positive events ( Fig . 5b ) . Based on these observations and previous experimental and computational modeling data 4 , 6 , 7 , we propose that this plateau in branched actin - positive CME events represents stalled membrane bending due to an unfavorable local membrane environment . We next tested the hypothesis that the asymmetric actin network might affect the lateral movements of endocytic coats on the plasma membrane . Interestingly , the ARPC3 positive CME sites showed signi \ufb01 cantly slower , but greater directional lateral movement before vesicle scission compared to the ARPC3 negative CME sites ( Fig . 5c , d ) . After scission both ARPC3 positive and negative vesicles showed fast , apparently random movements ( Fig . 5c , d ) . These data suggest that the asymmetric actin can stabilize the forming endocytic coat while pushing it in the plane of the plasma membrane with a lateral directional force . To test the function of actin network assembly on CME , we treated the cells with an Arp2 / 3 inhibitor , CK666 . We observed CME dynamics immediately after the treatment to minimize non - speci \ufb01 c side effects that might be caused by prolonged actin assembly disruption . Even with moderate inhibition of actin network assembly at CME sites , indicated by reduced ARPC3 intensity ( Supplementary Fig . 6a ) , we detected a small but signi \ufb01 cant increase in CME lifetimes ( Supplementary Fig . 6b ) . This result is consistent with results from previous studies in SKMEL cells 7 , 12 and supports the hypothesis that Arp2 / 3 - mediated actin assembly facilitates CME . a b d c F r equen cy den s i t y CME lifetime ( s ) AP2 ARPC3DNM2 ARPC3 - ARPC3 + Time ( s ) F l uo r e sc en c e i n t en s i t y ( a r b . un i t s ) 0 100 200 300 400 500 0 100 200 300 400 500 0 - 10 - 20 10 Average AP2 movement ( \u00b5 m ) C u m u l a t i v e f r equen cy 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 0 . 01 0 . 1 ARPC3 + before ARPC3 + after ARPC3 - after ARPC3 - before ARPC3 + before ARPC3 + after ARPC3 - after ARPC3 - before Straightness - index C u m u l a t i v e f r equen cy 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 ARPC3 - ARPC3 + 0 . 000 0 . 005 0 . 010 0 . 015 0 . 020 0 40 80 120 160 20 60 100 140 Fig . 5 Actin positive CME sites show distinct dynamics . a Histograms of ARPC3 negative ( blue ) and positive ( orange ) CME lifetimes . CME lifetime is measured from the \ufb01 rst frame of the AP2 signal to the presumed scission time ( the peak of DNM2 signal ) . ARPC3 positive CME events have longer lifetimes . p - value from two - sided Welch \u2019 s t - test : 2 . 11e - 76 . b Averaged intensity vs time plots of ARPC3 negative ( top ) and positive ( bottom ) CME sites in ADA cells . Events were aligned to the frames showing the maximum DNM2 intensity . Error bar : \u00bc standard deviation . c Lateral motility of ARPC3 negative ( blue ) and positive ( yellow ) CME sites before ( solid line ) and after ( dashed line ) vesicle scission . ARPC3 positive CME sites move slower than ARPC3 negative ones . p - value from two - sided Kolmogorov - Smirnov test : ARPC3 + before vs ARPC3 - before : 1 . 27e - 14 , ARPC3 + after vs ARPC3 - after : 5 . 18e - 08 . d Straightness - index of ARPC3 negative ( blue ) and positive ( yellow ) CME sites before ( solid line ) and after ( dashed line ) scission . The straightness - index is de \ufb01 ned by the ratio between the sum of frame - to - frame distances to the end - to - end distance of a single event \u2019 s trajectory , where a perfectly straight - lined trajectory would have an index of 1 . APRC3 positive CME sites move with a more straight trajectory . p - value from two - sided Kolmogorov \u2013 Smirnov test : ARPC3 + before vs ARPC3 - before : 1 . 07e - 11 , ARPC3 + after vs ARPC3 - after : 2 . 06e - 6 . a \u2013 d ARPC3 - : N = 840 , ARPC3 + : N = 1 , 385 . a , c , d Source data are provided in the Source Data \ufb01 le . NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 ARTICLE NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications 7 N - WASP is recruited asymmetrically to stalled CME sites . To further explore how asymmetric assembly of actin networks at CME sites is regulated , we endogenously tagged N - WASP , an actin nucleation promoting factor ( NPF ) that plays roles in CME , in AP2M1 - tagRFP - T / DNM2 - tagGFP2 genome - edited iPSCs ( hereafter referred to as ADW cells , Fig . 6a , Supple - mentary Fig . 7a and Supplementary Movie 5 ) . Quantitative imaging of budding yeasts demonstrated that initiation of productive actin assembly at CME sites requires the accumu - lation of yeast WASP and WIP ( WASP Interacting Protein ) to a certain level 37 . In our genome - edited iPSCs , we observed that N - WASP is recruited asymmetrically to CME sites mostly at the late stage of CME ( Fig . 6b , c and Supplementary Fig . 7b , c ) . Longer lifetimes and a plateau in the AP2 intensity vs time plot were observed speci \ufb01 cally for the N - WASP positive CME events ( Fig . 6d , e ) , similar to the ARPC3 positive events ( Fig . 5a , b ) . These data indicate that asymmetric NPF recruit - ment underlies the asymmetric architecture of branched actin networks at CME sites . Discussion Using unbiased analysis of thousands of CME sites in unper - turbed live cells , our study demonstrates that in mammalian cells clathrin coat assembly dynamics predict which sites will assemble actin , and show that at apparently stalled sites , actin assembles asymmetrically to facilitate successful vesicle formation . Based on the data presented here , we propose an updated model for actin assembly at mammalian CME sites in which , beyond global tension - dependent changes in the requirement for actin assembly , highly localized differences give rise to hetero - geneity even within the same patch of plasma membrane in the same cell ( Fig . 7 ) : ( 1 ) Where the local conditions are favorable for membrane deformation by coat proteins ( Fig . 7 upper scenario ) , the membrane can undergo \ufb02 at - U - \u03a9 shape transitions in a relatively short time without actin assembly . When the coat grows large enough to form a \u03a9 - shaped bud , suf \ufb01 cient dynamin can be recruited to perform scission , and there is little delay between coat expansion and scission ; ( 2 ) Where the local conditions are not favorable , presumably under high membrane tension or other AP2 - tagRFP - T HaloTag - N - WASP DNM2 - tagGFP2 Merge a b c M e r ge 4 min N - W ASP DN M 2 AP 2 S c a l ed i n t en s i t y ( a r b . un i t s ) D i s t an c e ( \u00b5 m ) Time ( s ) 0 - 10 - 20 10 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 d F r equen cy den s i t y CME lifetime ( s ) N - WASP - N - WASP + e AP2 N - WASP DNM2 N - WASP - N - WASP + F l uo r e sc en c e i n t en s i t y ( a r b . un i t s ) 0 100 200 300 0 100 200 300 Time ( s ) 0 - 10 - 20 10 AP2 N - WASP DNM2 DNM2 - N - WASP AP2 - N - WASP AP2 - DNM2 0 . 0000 0 . 0025 0 . 0050 0 . 0075 0 . 0100 0 . 0125 0 . 0150 50 0 100 150 200 - 30 20 1 . 25 Fig . 6 Asymmetric N - WASP recruitment to stalled CME sites . a A representative single time frame image of a TIRF movie ( Supplementary Movie 5 ) of AP2M1 - tagRFP - T ( magenta ) , DNM2 - tagGFP2 ( green ) and JF635 ligand - conjugated HaloTag - N - WASP ( cyan ) in ADW cells . The highlighted region is boxed by a dashed line . Scale bar : 5 \u00b5 m . b A representative kymograph of CME sites in ADW cells over a 4 min movie . Scale bar : 5 \u00b5 m . c Averaged intensity ( solid line ) and distance ( dashed line ) vs time plots of N - WASP positive CME sites in ADW cells . Events are aligned to the frames showing the maximum DNM2 intensity . Intensity is scaled to 1 at peaks for each channel . Error bar : \u00bc standard deviation . d N - WASP positive CME events have longer lifetimes . p - value from two - sided Welch \u2019 s t - test : 9 . 06e - 20 . e Intensity vs time plots of averaged N - WASP negative ( top ) and positive ( bottom ) CME sites in ADW cells . Events were aligned to the frames showing the maximum DNM2 intensity . Error bar : \u00bc standard deviation . Source data are provided in the Source Data \ufb01 le . c , d N - WASP negative CME sites : N = 299 , N - WASP positive CME sites : N = 1 , 199 . ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 8 NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications impediments , the coat protein - membrane interaction does not generate suf \ufb01 cient force to curve the membrane ( Fig . 7 lower scenario ) . Here , extra force generation from actin assembly is required 4 , 6 , 10 . Asymmetric N - WASP recruitment activates actin nucleation mostly at one side of the clathrin coat , generating an asymmetric force that pulls the membrane into the cell with a similar action to a bottle cap opener . We speculate that this asymmetrical force contributes to asymmetric membrane defor - mation at endocytic sites observed by high - speed atomic force microscopy 38 and may act with dynamin 39 to twist the clathrin pit to promote scission at the neck . CME events with associated actin assembly have longer lifetimes , likely due to a delay between the end of coat expansion and progress toward vesicle scission , requiring adaptive recruitment of actin regulators followed by actin network assembly and membrane remodeling . This result establishes that site - to - site heterogeneity in actin dependence and involvement can be observed without manipulating actin assembly . Our model provides further insights into the basis for incon - sistent effects of actin drugs on CME 6 , 14 , 18 , 40 \u2013 45 . Actin plays crucial roles in membrane shaping , cell adhesion , and membrane tension . Global disruption of actin dynamics is expected to dra - matically change membrane tension and the available pool of actin and associated proteins and therefore to have both direct and indirect effects on CME . Here , we focused on in - depth analysis of the unperturbed process and detected preference for actin assembly at stalled CME events . We used genome - edited ARPC3 - HaloTag protein expressed at endogenous level as a marker of branched actin networks . This \ufb02 uorescent protein allowed us to study the spatiotemporal dynamics of Arp2 / 3 - nucleated actin structures at CME sites speci \ufb01 cally ( Fig . 1a , d ) . A large population of unbranched corti - cal actin \ufb01 laments at CME sites has been observed by cryo - EM 46 and distinct effects of cortical actin - based structures on CME have been reported 36 . Previous studies on actin \u2019 s spatio - temporal dynamics and function at CME sites applied actin probes such as Lifeact , which label actin \ufb01 laments nucleated by different mechanisms , and which can perturb dynamic actin properties , possibly complicating conclusions reached in those studies 14 , 31 , 47 , 48 . WASP family proteins play a central role in regulation of Arp2 / 3 - mediated actin assembly both spatially and temporally during various cellular processes . We observed that N - WASP is recruited asymmetrically to stalled CME sites ( Fig . 6 ) . We pro - pose this asymmetric recruitment underlies the asymmetric actin architecture at CME sites . Quantitative live - cell imaging studies in yeast suggested concentration of yeast WASP at CME sites through multivalent protein interactions is important for robust , switch - like actin assembly at CME sites 37 . Future modeling together with quantitative in vivo and in vitro studies are needed to determine whether concentrating N - WASP into a small space , which is asymmetrically located relative to CME sites , is a more ef \ufb01 cient way to generate suf \ufb01 cient actin force for productive CME , compared to recruiting more N - WASP into a larger space that encircles the CME sites . Future computational modeling studies on how asymmetric actin network assembly provides forces and generates a torque for vesicle formation and mem - brane remodeling will deepen our understanding of actin \u2019 s functions in a host of actin - mediated processes . While this study provides evidence that actin assembles asymmetrically at mammalian CME sites , some questions remain open . For example , what is the molecular mechanism that couples recruitment of N - WASP and other actin assembly factors to the stalled CME sites ? Many factors , including membrane tension 6 , 9 , 10 , cell cycle 9 , cell adhesion 8 , 49 and cargo size and composition 32 , 33 appear to contribute to the actin requirement for CME . How do these different factors regulate the actin assembly machinery ? Answering these questions in the future will deepen our understanding of dynamic actin assembly regulation in membrane traf \ufb01 cking . Methods Cell culture . The WTC10 hiPSC line was obtained from the Bruce Conklin Lab at UCSF . hiPSCs were cultured on Matrigel ( hESC - Quali \ufb01 ed Matrix , Corning ) in StemFlex medium ( Thermo Fisher ) with Penicillin / Streptomycin in 37\u00b0C , 5 % CO 2 . Cultures were passaged with Gentle Cell Dissociation reagent ( StemCell Technologies , Cat # : 100 \u2013 0485 ) twice every week . Genome - editing . The AP2M1 gene was edited in WTC10 hiPSCs as previously described using TALENs targeting exon 7 of the AP2M1 gene 50 . Both alleles of AP2M1 were tagged with tagRFP - T . The Cas9 - crRNAtracrRNA complex electro - poration method was used sequentially to edit DNM2 and ARPC3 gene in AP2M1 - tagRFP - T genome - edited hiPSCs , as previously described 12 , 18 . The same method was used to edit the WASL gene in AP2M1 - tagRFP - T / DNM2 - tagGFP2 genome edited hiPSCs . S . pyogenes NLS - Cas9 was puri \ufb01 ed in the University of California Berkeley QB3 MacroLab . TracrRNA and crRNA that target CCTGCTCGAC - TAGGCCTCGA ( DNM2 ) , CCTGGACAGTGAAGGGAGCC ( ARPC3 ) and AGCTCATGGTTTCGCCGGCG ( WASL ) , were purchased from IDT . Gibson assembly ( New England Biolabs ) was used to construct donor plasmids containing DNM2 5 \u2032 homology - ggtaccagtggcggaagc - tagGFP2 - DNM2 3 \u2032 homology , ARPC3 5 \u2032 homology - ggatccggtaccagcgatccaccggtcgccacc - HaloTag - ARPC3 3 \u2032 homology , and WASL 5 \u2032 homology - HaloTag - agcgatccaccggtcgccaccggatcc - WASL 3 \u2032 homology sequences , respectively . Three days after electroporation ( Lonza , Cat # : VPH - 5012 ) Actin + CytosolTIRF field CytosolTIRF field Coat module ( AP2 ) Scission module ( DNM2 ) Actin module ( ARPC3 ) Plasma membrane Membrane tension Actin force NPF module ( N - WASP ) Actin - Fig . 7 An updated schematic model of actin - negative and actin - positive clathrin - coated pits in human cells . Actin assembly is induced at stalled CME sites , where asymmetric forces pull , bend and possibly twist the plasma membrane against membrane tension to drive membrane invagination and vesicle scission . NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 ARTICLE NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications 9 of the Cas9 - crRNA - tracrRNA complex and donor plasmid , the tagGFP2 or HaloTag positive cells were single cell sorted using a BD Bioscience In \ufb02 ux sorter ( BD Bioscience ) into Matrigel - coated 96 - well plates . Clones were con \ufb01 rmed by PCR and Sanger sequencing of the genomic DNA locus around the insertion site . Both alleles of DNM2 and ARPC3 were tagged with tagGFP2 and HaloTag , respectively , and one allele of WASL was tagged with HaloTag in the hiPSC lines used in this study . Western blotting . Cells were dissociated from the well using Gentle Cell Dis - sociation reagent ( StemCell Technologies , Cat # : 100 - 0485 ) . Total proteins were extracted by adding 1ml of cold 10 % TCA to the cell pellets , incubated on ice for 30min , and spun down by centrifuging at 4\u00b0C , 13400 \u00d7 g for 10min . Protein pellets were dissolved in loading buffer ( 50 mM HEPES , pH 7 . 4 , 150 mM NaCl , 1mM MgCl2 , 5 % BME , 5mM DTT and protease inhibitor ) and loaded onto an acrylamide gel for SDS - PAGE and transferred to nitrocellulose membranes for immunoblotting . Blots were incubated overnight at 4 \u00b0C with primary antibodies targeting Tag ( CGY ) FP ( 1 : 2000 dilution in 2 % milk , Evrogen , Cat # : AB121 ) , HaloTag ( 1 : 2000 dilution in 2 % milk or 1 : 1000 dilution in 0 . 5 % milk , Promega , Cat # : G9211 ) , GAPDH ( 1 : 100 , 000 dilution in 0 . 5 % milk , Proteintech , Cat # : 10494 - 1 - AP ) , respectively , and subsequently incubated in the dark at room temperature for 1 hr with secondary antibodies . TIRF live - cell imaging . Two days before imaging , hiPSCs were seeded onto Matrigel - coated 4 - well chambered cover glasses ( Cellvis ) . Halotag was labeled by JF635 - HaloTag ligand 51 . Cells were incubated in StemFlex medium with 100 mM JF635 - HaloTag for 45 min and the unbound ligands were washed away by three washes with 5min incubation in prewarmed StemFlex medium . Cells were imaged on a Nikon Ti - 2 inverted microscope \ufb01 tted with TIRF optics and a sCMOS camera ( Hamamatsu ) . Cells were maintained at 37\u00b0C with a stage top incubator ( OKO Lab ) in StemFlex medium with 10 mM HEPES . Images were acquired with Nikon Elements . Channels were acquired sequentially at a 1s interval and 300 ms expo - sure time over 4 min . For the CK666 treatment experiments , a 50 mM CK666 ( SML0006 , Sigma Aldrich ) stock solution was prepared in DMSO and kept at \u2212 80\u00b0C . 200 \u03bc M CK666 solution and 4 % DMSO ( v / v ) solution for a control were prepared fresh in StemFlex medium with 10mM HEPES and incubated at 37\u00b0C prior to use . Live - cell imaging was performed 1min after adding an equal volume of medium containing CK666 or DMSO into the imaging chamber to achieve a \ufb01 nal concentration of 100 \u03bc M CK666 or 2 % DMSO . TIRF image processing and analysis . Events ( i . e . , tracked diffraction - limited spots ) were extracted from cmeAnalysis 30 and processed in Python Jupyter Notebooks . AP2 - tagRFP - T was used as the \ufb01 ducial marker for clathrin - mediated endocytosis in cmeAnalysis tracking experiments . DNM2 was used as a secondary channel to mark vesicle scission and the termination of vesicle formation . ARPC3 - HaloTag was tracked separately from paired AP2 / DNM2 movies and linked to CCPs downstream of cmeAnalysis to allow for determining discrete ARPC3 nucleation and disassembly events . Four generalized processing steps were applied to identify clathrin - coated pits ( AP2 tracks ) with single DNM2 peaks : track feature abstraction , feature dimensionality reduction , event clustering , and DNM2 - peak detection . First , tracks that are de \ufb01 ned by \ufb01 tted positions and intensities for single events were generated using cmeAnalysis . Then , AP2 and DNM2 tracks were decomposed into dynamic features describing the dynamics of the position and brightness for each event . Each tracked event , which was once an arbitrary array of intensities and positions , was now a discrete vector of \ufb01 xed length . The mapping of each track to discrete features was done to generalize the dynamics of heterogeneous tracked events into a set of interpretable coordinates . Following feature abstraction , the output array is a 2 - dimensional matrix with N rows ( N tracked events ) and M columns ( M discrete features per track ) . These features were individually scaled to normal distributions to remove the variability in scale and dampen the effects of outliers . For instance , the \u2018 lifetime \u2019 feature ( AP2 lifetime ) ranged from a few seconds to several minutes on a scale of seconds , whereas the \u2018 DNM2 - peak fraction \u2019 feature ( where the DNM2 peak is located within one AP2 event ) ranges from 0 to 1 . Following feature re - scaling , these events , which each contain over thirty features , were projected to a lower - dimensional space via principal component analysis . The derived clusters were separated using a Gaussian Mixture Model and events were assigned to clusters based on their highest probability of identity to one cluster . DNM2 - positive events represented a distinct cluster of tracks that had detectable DNM2 throughout the event , were long lived , and were below the threshold of motility expected for transient , non - CME - derived clathrin - coated vesicle \u201c visitors \u201d 5 0 to the TIRF \ufb01 eld . To characterize a single - DNM2 peak , DNM2 - positive events were surveyed over a range of values set for the minimum DNM2 peak height , width , and peak - to - peak temporal distance . For each peak - de \ufb01 ning parameter combination , all DNM2 - positive events were categorized as having zero , one , or two or more peaks . After \ufb01 nding single - peaked events in a \ufb01 xed peak - parameter combination , the lifetime distribution of lifetimes for single peak events was \ufb01 t to the expected underlying distribution , a Rayleigh distribution 52 , where the best - \ufb01 tting parameter combination was chosen to identify single - peaked events . Single DNM2 - peaked events were kept as CME sites for the remainder of the analysis . All code associated with this analysis , generating Figs . 4 \u2013 6 , and a detailed step - by - step protocol , are available at https : / / github . com / DrubinBarnes / Jin _ Shirazinejad _ et _ al _ branched _ actin _ manuscript . Two - color 3D STORM imaging and analysis . 12mm round coverslips were sonicated in distilled water and sterilized for 20min in 70 % ethanol , air - dried and coated with Matrigel in 24 - well plates . Cells were seeded onto Matrigel - coated coverslips two days before \ufb01 xation . For clathrin and actin two - color imaging , cells were \ufb01 xed \ufb01 rst for 1min in 0 . 3 % ( v / v ) glutaraldehyde ( GA ) solution containing 0 . 25 % ( v / v ) Triton in cytoskeleton buffer ( CB : 10mM MES , 150 mM NaCl , 5mM EGTA , 5 mM Glucose , 5 mM MgCl 2 , 0 . 005 % NaN 3 , pH 6 . 1 ) and then immediately \ufb01 xed for 20min in 2 % ( v / v ) GA solution in CB . Both solutions were prepared fresh from a 10 % GA stock ( Electron Microscopy Science , Cat # : 16120 ) . After \ufb01 xation , samples were incubated twice for 5min in freshly prepared 0 . 1 % ( w / v ) NaBH4 in PBS . For clathrin and ARPC3 - HaloTag imaging , cells were \ufb01 xed for 20 min in 4 % ( v / v ) PFA ( Electron Microscopy Sciences , Cat # : 15710 ) in CB . Subsequently , both types of samples were washed 3 times for 10min in PBS . Samples were then blocked for 20min in blocking buffer [ 3 % ( w / v ) BSA and 0 . 1 % ( w / v ) Saponin in PBS ] . Clathrin light chain ( Invitrogen , Cat # : MA5 - 11860 , 1 : 200 dilution ) and Halotag ( Promega , Cat # : G9281 , 1 : 200 dilution ) antibodies were used in blocking solution . Primary antibody immunostaining was performed overnight at 4\u00b0C . On the next day , samples were washed three times in washing buffer ( 0 . 1\u00d7 blocking buffer in PBS ) for 10min . Samples were incubated with secondary antibody in blocking buffer for 30min at room temperature in the dark and were washed three times for 10min in washing buffer , and then three times for 10 min in PBS . Mouse secondary antibody ( Jackson ImmunoResearch , Code # : 715 - 005 - 151 ) conjugated with CF680 ( Biothium , Cat # : 92139 ) ( 1 : 50 ) was used to stain clathrin and actin samples . Commercial mouse secondary antibody - AF647 ( ThermoFisher , Cat # : A32787 ; 1 : 400 ) and rabbit secondary antibody ( Jackson ImmunoResearch , Code # : 711 - 005 - 152 ) conjugated with CF680 ( Biothium , Cat # : 92139 ) ( 1 : 50 ) were used to stain the clathrin and ARPC3 - HaloTag . Clathrin and actin samples were then stained with 0 . 5\u00b5M Phalloidin - AF647 ( Fisher Scienti \ufb01 c , Cat # : A22287 ) in PBS and kept at room temperature in the dark for 2 h . Samples were washed three times with PBS before STORM imaging . STORM imaging was performed as previously described on a homebuilt STORM setup 7 , 53 . Samples labeled by AF647 and CF680 were excited by an 647 nm laser . The emission of both AF647 and CF680 was then split into two light paths as two channels using a dichroic mirror ( Chroma , Cat # : T685lpxr ) , and each channel was projected onto one - half of an EMCCD camera ( Andor iXon Ultra 897 ) . Color assignment of each localization was based on its intensity in the two channels . A cylindrical lens was inserted into the transmitted channel to acquire 3D localization 28 . 3D position of each localization was determined from the ellipticity of each point spread function . The raw STORM data was processed according to previously described methods 28 , 54 and single - molecule localization and optical reconstruction were performed using the Insight3 software ( developed by Dr . Bo Huang at University of California , San Francisco and Dr . Xiaowei Zhuang at Harvard University ) . To quantify the distances between centroids of clathrin and actin or ARPC3 signals , we \ufb01 rst manually cropped regions that contain single CME sites associated with actin or ARPC3 structures using Insight3 software . Then we identi \ufb01 ed and saved the xy positions of single - molecule localizations in cropped regions as txt \ufb01 les . We next calculated the centroid of signals by averaging xy positions of each channel and calculated the distance between the centroids of two channels using MATLAB . Airyscan imaging and processing . Live cell sample preparation was performed as described in \u201c TIRF live - cell imaging \u201d . Cells were imaged on a Zeiss LSM 900 inverted microscope using an Airyscan 2 detector and Multiplex 4Y line scanning mode . Cells were maintained at 37\u00b0C , 5 % CO 2 in StemFlex medium . Images were acquired and processed using the ZEN 3 . 1 system . Channels were acquired sequentially for each line with 0 . 2s / frame intervals over 3 min . Images were pro - cessed with 3 . 7 deconvolution strength 2D Airyscan processing . Alignment between channels was corrected using \ufb02 uorescent bead images . Reporting summary . Further information on research design is available in the Nature Research Reporting Summary linked to this article . Data availability The data that support this study are available from the corresponding author upon reasonable request . Source data are provided with this paper . Code availability The Jupyter Notebooks used for live - cell imaging analysis can be found at https : / / doi . org / 10 . 5281 / zenodo . 6575159 . ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 10 NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications Received : 11 September 2021 ; Accepted : 8 June 2022 ; References 1 . Rottner , K . , Faix , J . , Bogdan , S . , Linder , S . & Kerkhoff , E . Actin assembly mechanisms at a glance . J . Cell Sci . 130 , 3427 \u2013 3435 ( 2017 ) . 2 . Lu , R . , Drubin , D . G . & Sun , Y . Clathrin - mediated endocytosis in budding yeast at a glance . J . Cell Sci . 129 , 1531 \u2013 1536 ( 2016 ) . 3 . Lacy , M . M . , Ma , R . , Ravindra , N . 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Graphene - enabled electron microscopy and correlated super - resolution microscopy of wet cells . Nat . Commun . 6 , 7384 ( 2015 ) . 54 . Rust , M . J . , Bates , M . & Zhuang , X . Sub - diffraction - limit imaging by stochastic optical reconstruction microscopy ( STORM ) . Nat . Methods 3 , 793 \u2013 795 ( 2006 ) . Acknowledgements M . J . was funded by American Heart Association Postdoctoral Fellowship ( 18POST34000029 ) . D . G . D . was funded by NIH MIRA grant R35GM118149 . K . X . is a NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 ARTICLE NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications 11 Chan Zuckerberg Biohub investigator and acknowledges support from NIH ( DP2GM132681 ) . S . U . is a Chan Zuckerberg Biohub Investigator and acknowledges support from the Philomathia Foundation , and Chan Zuckerberg Initiative Imaging Scientist program . We would like to thank Dr . Yidi Sun and Dr . Matthew Akamatsu for insightful comments on the manuscript ; the Conklin Lab at UCSF for providing the WTC10 human iPSC line ; the Lavis Lab at Janelia Research Campus for providing JF635 HaloTag ligand ; Dr . Sun Hae Hong for generating the AP2 - tagRFP - T iPSC line ; the UC Berkeley QB3 MacroLab for puri \ufb01 ed S . pyogenes NLS - Cas9 ; the Luo Lab at UC Berkeley for sharing their electroporator and the UC Berkeley Cancer Research Laboratory Flow Cytometry Facility for iPSC sorting . Author contributions M . J . , C . S . and D . G . D . conceived the study and experiments . M . J . and A . Y . generated the genome - edited cell lines . M . J . performed live cell data acquisition and sample preparation for super - resolution microscopy . B . W . and K . X . performed super - resolution microscopy and super - resolution data reconstruction . C . S . developed computational analysis tools and S . U . , J . S . , D . G . D . and M . J . supported the data analysis . M . J . , C . S . , and D . G . D . prepared the \ufb01 gures and wrote the manuscript with feedback from the other authors . Competing interests The authors declare no competing interests . Additional information Supplementary information The online version contains supplementary material available at https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 . Correspondence and requests for materials should be addressed to David G . Drubin . Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work . Peer reviewer reports are available . Reprints and permission information is available at http : / / www . nature . com / reprints Publisher \u2019 s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional af \ufb01 liations . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Creative Commons license , and indicate if changes were made . The images or other third party material in this article are included in the article \u2019 s Creative Commons license , unless indicated otherwise in a credit line to the material . If material is not included in the article \u2019 s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2022 ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 12 NATURE COMMUNICATIONS | ( 2022 ) 13 : 3578 | https : / / doi . org / 10 . 1038 / s41467 - 022 - 31207 - 5 | www . nature . com / naturecommunications", + "day2015microtubule_supplement1": "Supplemental Figure 1 . ( Related to Figure 1 ) This figure shows a series of control experiments that characterize the properties of the tubular invaginations generated in response to ATP depletion . It also documents the efficacy of the ATP depletion treatment . ( A ) Alexa488 - STxB and Alexa488 - CTxB accumulate in tubular invaginations in ATP depleted COS - 7 cells . To facilitate STxB labeling , cells were transfected with Gb3 synthase . ( B , C ) As reported previously ( 78 ) , fluorescent CTxB labels both plasma membrane invaginations ( arrowheads ) and plasma membrane protrusions ( arrows ) in ATP depleted cells . B and C are single frames from a confocal z - stack . Dashes mark the position of the xz - sections shown below . ( D ) Alexa488 - STxB and Alexa555 - CTxB colocalize in invaginations in ATP depleted COS - 7 cells expressing Gb3 synthase . ( E ) Tubules prelabeled with Alexa555 - CTxB filled at approximately the same rate as newly added Alexa488 - CTxB accumulates at the plasma membrane , indicating that the tubules are open to the fluid phase in ATP depleted cells . Time stamps are in minutes : seconds . ( F - H ) ATP depletion inhibits uptake of dextran and internalization of transferrin ( Tfn ) ( Mean \u00b1 SD , N = 325 - 417 cells ) . * * * , p < 0 . 001 , Student\u2019s t - test . Bars , 10 \u00b5 m .", + "sathe2018small": "ARTICLE Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin - independent endocytosis Mugdha Sathe 1 , Gayatri Muthukrishnan 1 , James Rae 2 , 3 , Andrea Disanza 4 , 5 , Mukund Thattai 1 , 6 , Giorgio Scita 4 , 5 , Robert G . Parton 2 , 3 & Satyajit Mayor 1 , 7 Using real - time TIRF microscopy imaging , we identify sites of clathrin and dynamin - independent CLIC / GEEC ( CG ) endocytic vesicle formation . This allows spatio - temporal localisation of known molecules affecting CG endocytosis ; GBF1 ( a GEF for ARF1 ) , ARF1 and CDC42 which appear sequentially over 60 s , preceding scission . In an RNAi screen for BAR domain proteins affecting CG endocytosis , IRSp53 and PICK1 , known interactors of CDC42 and ARF1 , respectively , were selected . Removal of IRSp53 , a negative curvature sensing protein , abolishes CG endocytosis . Furthermore , the identi \ufb01 cation of ARP2 / 3 complex at CG endocytic sites , maintained in an inactive state reveals a function for PICK1 , an ARP2 / 3 inhibitor . The spatio - temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin - independent endocytic process . Coincident with the loss of PICK1 by GBF1 - activated ARF1 , CDC42 recruitment leads to the activation of IRSp53 and the ARP2 / 3 complex , resulting in a burst of F - actin polymerisation potentially powering scission . DOI : 10 . 1038 / s41467 - 018 - 03955 - w OPEN 1 National Centre for Biological Science ( TIFR ) , Bellary Road , Bangalore 560065 , India . 2 Institute for Molecular Bioscience , University of Queensland , Brisbane , QLD 4072 , Australia . 3 Centre for Microscopy and Microanalysis , University of Queensland , Brisbane , QLD 4072 , Australia . 4 IFOM , Fondazione Istituto FIRC di Oncologia Molecolare , Milan 20139 , Italy . 5 Department of Oncology and Hemato - Oncology , University of Milan , Milan 20122 , Italy . 6 Simons Centre for the Study of Living Machines , National Centre for Biological Sciences ( TIFR ) , Bellary Road , Bangalore 560065 , India . 7 Institute for Stem Cell Biology and Regenerative Medicine , Bellary Road , Bangalore 560065 , India . These authors contributed equally : Mugdha Sathe , Gayatri Muthukrishnan . Correspondence and requests for materials should be addressed to S . M . ( email : mayor @ ncbs . res . in ) NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 1 1 2 3 4 5 6 7 89 0 ( ) : , ; There are amendments to this paper M ultiple endocytic pathways function in a eukaryotic cell 1 , 2 ; however , our understanding of the endocytic process is mainly derived from studies on clathrin - mediated endocytosis ( CME ) 3 \u2013 5 . Dynamin is responsible for vesicle scission in CME 6 , 7 and a host of clathrin - independent endocytic ( CIE ) pathways , such as the caveolar and fast endophilin - mediated endocytic pathway 8 \u2013 10 . On the other hand , among CIE pathways , the CLIC / GEEC [ clathrin and dynamin - independent carriers which form GPI - enriched endocytic com - partments ; CG ] pathway functions independent of both clathrin and dynamin in multiple cell types and contexts 11 \u2013 17 , while it is not present in others 18 . The actin polymerisation machinery has been implicated in the functioning of many CIE pathways at different stages 13 , 19 . Our focus , the CG pathway , is regulated by the small GTPases , ARF1 ( ADP - ribosylation factor 1 ) and CDC42 ( cell division control protein 42 ) 11 \u2013 16 . It is responsible for the uptake of many glycosylphosphotidylinositol ( GPI ) - anchored proteins , a major fraction of the \ufb02 uid phase , toxins such as Helicobacter pylori vacuolating toxin A 20 , cholera toxin 21 and viruses like adeno - associated virus 2 22 . The CLICs are formed in a polarised manner at the leading edge of migrating cells 23 and , the resulting GEECs subsequently fuse with the sorting endocytic vesicles via a Rab5 / phosphatidylinositol - 3 - kinase - dependent mechanism 24 . The CLICs / GEECs are high capacity endocytic carriers turning over the entire membrane surface in 12 min in \ufb01 broblasts , highlighting the role of CG pathway in regulating membrane homoeostasis 23 . Recent evidence suggests that this is required for generating a tubular vesicular endocytic network during cytokinesis 25 and serves to deliver ligands to their signalling receptors in a common endocytic compartment 26 . The molecular machinery to form a CG endocytic vesicle involves activating ARF1 at the plasma membrane by GBF1 ( Golgi - speci \ufb01 c brefeldin A resistance factor 1 ) 16 , a speci \ufb01 c ARF - GEF ( guanine nucleotide exchange factor ) . GTP \u2013 ARF1 recruits ARHGAP21 ( a RhoGAP for CDC42 ) , which removes CDC42 from the membrane 14 . Cholesterol removal , in addition , reduces the recruitment of ARF1 and CDC42 , along with accelerated cycling of CDC42 13 , 14 . Lastly , the CG pathway requires dynamic actin since both stabilisation and de - polymerisation of actin \ufb01 laments was found to affect endocytosis 13 . By visualising a forming CG endocytic vesicle , we wanted to understand the molecular mechanism responsible . We adapted a pH pulsing protocol that exploits the pH - sensitive properties of super ecliptic GFP ( SecGFP ) 27 , previously deployed to study CME 4 , 28 , 29 . We tagged the GPI - anchor with SecGFP to make model cargo SecGFP - GPI to assay , in real time , the sites of endocytic vesicle formation . We found that the CG endocytic vesicle formation was initiated by the accumulation of ARF1 / GBF1 followed by CDC42 and F - actin while dynamin and cla - thrin did not associate with forming endosomes . Hence , in the absence of a discernable coat 23 , alternate candidate proteins by generating / stabilising membrane curvature can assist in endocytic vesicle formation such as Bin / Amphiphysin / Rvs ( BAR ) domain - containing proteins ( BDPs ) 30 . Although several BDPs are involved in the CME pathway , only one has been identi \ufb01 ed to be associated with the CG endocytic pathway so far 31 . Using RNAi - screening , we identi \ufb01 ed two BDPs in particular , that affected CG endocytosis downstream of ARF1 and CDC42 . First , a CDC42 interaction partner and I - BAR protein , IRSp53 ( Insulin - responsive protein of mass 53 kDa ) was found to be necessary for CG endocytosis . Importantly , IRSp53 removal resulted in the disappearance of CLICs and loss of a GBF1 - dependent endocytic pathway . Second , an ARF1 interactor , PICK1 ( protein interacting with C kinase 1 ) emerged as a regulator of ARP2 / 3 activity in the early phases of CG vesicle formation . Lastly , ARP2 / 3 , an interaction partner of both IRSp53 and PICK1 , accumulated at the forming CG endocytic site and decreased CG endocytosis when inhibited . Together , the spatio - temporal dynamics of these proteins provided a mechanistic understanding of the forming CG endocytic vesicle . Results pH pulsing assay detects nascent CG endocytic sites . To monitor endocytic vesicle formation in real time , we employed the pH - sensitive \ufb02 uorescence of super ecliptic pHlourin GFP ( SecGFP ) 27 attached to a GPI - anchor ( SecGFP - GPI ) to differ - entiate cell surface - resident molecules from the newly internalised molecules . The \ufb02 uorescence of SecGFP is quenched reversibly when exposed to pH 5 . 5 4 , 27 \u2013 29 . SecGFP - GPI expressed in AGS ( human gastric cell line ) was endocytosed along with the CG cargo , \ufb02 uid phase ( 10 kDa dextran ) , but not with CME cargo , TfR , at both 37 \u00b0C and 30 \u00b0C ( Supplementary Fig . 1a \u2013 c and Supplementary Information ( S . I . ) ) as shown previously 15 , 32 . Endocytic events were identi \ufb01 ed by alternately exposing the cells to buffers equilibrated to pH 7 . 3 ( pH 7 ) or pH 5 . 5 ( pH 5 ) every 3 s at 30 \u00b0C ( Fig . 1a , Schematic and Supplementary Movies 1 \u2013 2 ) . SecGFP - GPI - containing endocytic events occurring during exposure to pH 7 remained \ufb02 uorescent due to their near neutral luminal pH right after formation . However , the buffer exchange from pH 7 to pH 5 quenched the \ufb02 uorescence of cell surface SecGFP - GPI . This enabled visualisation of the newly formed endocytic vesicle . The identi \ufb01 cation of the site of endocytic vesicle formation paved the way for the character - isation of the spatial and temporal dynamics of molecular players by the co - expression of a ( mCherry / TagRFPt / pRuby ) - tagged molecule of interest , \u2018 X - FP \u2019 ( Fig . 1a and Supplementary Movies 1 \u2013 2 ) . The dynamics of X - FP were extracted by looking at the history of the region , where vesicle formation was detected ( Fig . 1a montage ( bottom ) , see Methods and S . I . ) . To rule out the effect of pH 5 on the rate of endocytosis , we pre - treated the cells with either pH 5 or pH 7 buffer , followed by 5 - min pulse in pH 7 buffer and found no difference in CG endocytosis ( Supplemen - tary Fig . 1e ) . The pH pulsing movies were analysed using semi - automated scripts ( see Methods and S . I . ) . Brie \ufb02 y , the centroid of new spots appearing in the pH 5 channel provided a \ufb01 duciary marker for the time and location of the nascent endocytic vesicle ( Supplementary Fig . 1d , step 1 and S . I . ) . The relative enrichment of SecGFP - GPI and X - FP at the endocytic site was determined by normalising the average \ufb02 uorescence of the nascent endocytic spot to its local background annulus ( Supplementary Fig . 1a , d , step 2 \u2013 3 and see S . I . ) . The spots were then put through a series of automated and manual checks . The automated check ensured that the pH 5 intensity of the new spot had ( i ) signi \ufb01 cantly higher intensity than the background , ( ii ) persisted for at least 6 s and ( iii ) did not show an increase in intensity in the subsequent frame . Subsequently , a manual check was performed on the montages , ( i ) to remove any false positives that might have been missed by the automated check and ( ii ) to classify the new SecGFP - GPI spots into two groups based on whether X - FP co - detection was observed or not ( see S . I . ) . The data at the site of the spot were represented as the average fold change over the surrounding background , as a function of time ( Fig . 1b , solid traces ) , and compared to the average fold change of arbitrary regions within the cell ( \u2018 Random \u2019 ) ( Fig . 1b , dashed traces ) . The pro \ufb01 le obtained ( pooled from multiple cells ) represented a spatial and temporal pro \ufb01 le of the X - FP at SecGFP - GPI endocytic sites . Using the pH pulsing assay , we found that the rise in pH 7 SecGFP - GPI intensity occurred only ~ 3 s prior to vesicle ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 2 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications generation as opposed to nearly 40 s for CME , followed by monitoring SecGFP - TfR internalisation ( ref . 4 and Supple - mentary Fig . 2c ) . Furthermore , we observed a poor correlation ( r = 0 . 3 ) between pH 7 vs . pH 5 intensity per endosome , indicating that SecGFP - GPI is endocytosed without a major concentration in the endocytic vesicle ( Fig . 1c ) . In comparison to CME , it should be noted that we faced two main challenges , ( i ) lack of a cytoplasmic marker ( for e . g . , clathrin ) for the endocytic site , ( ii ) lack of strong concentration of the cargo prior to the endocytic vesicle pinching . Regardless , the protocol developed provided a reliable real - time assay for studying the spatio - temporal dynamics of the internalisation of GPI - anchored proteins ( see , S . I . ) . Cytoplasm a pH 7 . 0 pH 5 . 0 pH 7 . 0 pH 7 . 0 pH 5 . 0 secGPI - quenched Cytoplasm Extracellular milieu Cytoplasm Extracellular milieu Cytoplasm Extracellular milieu Extracellular milieu b c e d f \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 \u2013 30 \u2013 25 \u2013 20 \u2013 15 \u2013 10 \u2013 5 0 Time ( s ) ARF1 Random \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 1 . 0 1 . 2 1 . 4 1 . 6 \u2013 9 \u2013 6 \u2013 3 0 Time ( s ) CDC42 Random F o l d c hange ( a . u . ) \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 \u2013 8 \u2013 6 \u2013 4 \u2013 2 0 Time ( s ) GBF1 Random Log10 ( p ) pH 5 pH 5 Random pH 7 Random pH 7 r = 0 . 3 secGPI - fluorescent N o r m a li s ed p H 7 i n t en s i t y ( a . u . ) Normalised pH 5 intensity ( a . u . ) Time ( s ) F o l d c hange ( a . u . ) 2 . 4 2 . 2 2 . 0 1 . 8 1 . 6 1 . 4 1 . 2 1 . 0 0 . 8 \u2013 42 \u2013 36 \u2013 30 \u2013 24 \u2013 18 \u2013 12 \u2013 6 0 6 12 18 50 100 150 200 250 120 100 80 60 40 20 0 1 . 0 1 . 2 1 . 4 1 . 6 1 . 0 1 . 2 1 . 4 1 . 6 pH 5 pH 7 ARF1 GBF1 pH 5 pH 7 pH 7 pH 5 CDC42 Time ( s ) \u2013 18 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 Time ( s ) \u2013 18 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 Time ( s ) \u2013 18 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 pH 5 pH 7 X - FP pH 7 pH 5 X - FP \u2013 12 \u2013 9 \u2013 3 12 15 18 Time ( s ) 6 9 \u2013 18 3 0 \u2013 6 \u2013 15 NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w ARTICLE NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 3 GBF1 , ARF1 and CDC42 are recruited to nascent CG endo - cytic sites . We visualised the temporal dynamics of co - expressed CDC42 at the site of formation of nascent SecGFP - GPI endocytic vesicles . TagRFPt - CDC42 recruitment was quanti \ufb01 ed as average fold accumulation of CDC42 relative to the local background in all the endocytic events recorded over a 40 s time window straddling the endocytic event . A signi \ufb01 cant change in the intensity of CDC42 began at around \u2212 9 s and peaked around 0 to + 3 s ( Supplementary Fig . 2a ) . Based on the presence or absence of a co - detected CDC42 spot ( within \u2212 18 to + 18 s time window ) at the endocytic site , we identi \ufb01 ed two populations via manual classi \ufb01 cation ( see S . I . for a detailed description ) . We called them CDC42 Coloc ( co - detection of CDC42 and SecGFP - GPI ) and CDC42 NoColc ( the remainder ) . We compared the fold accu - mulation of all the CDC42 spots ( CDC42 All ) , CDC42 Coloc and CDC42 NoColoc with the Random . While the CDC42 Coloc pro \ufb01 le was similar to that of CDC42 All , the CDC42 NoColoc pro \ufb01 le was comparable to the Random ( Supplementary Fig . 2b ) . Thus , the endocytic sites detected by our assay consisted of two populations wherein one fraction exhibited an accumulation of CDC42 while the second fraction failed to show a discernable accumulation . CDC42 Coloc corresponded to the 56 % ( of the CDC42 All ) SecGFP - GPI endocytic sites . As the removal of the events which did not coincide with the presence of CDC42 did not alter CDC42 recruitment pro \ufb01 le ( compare , Fig . 1d and Supplementary Fig . 2a ) , they were discarded from further ana - lysis . While the reasons for not detecting CDC42 at all endocytic events is a function of both the signal and noise in the data , it may also re \ufb02 ect a genuine lack of recruitment at some endocytic events ( see S . I . for a detailed explanation ) . Henceforth , for all X - FPs , we report pH pulsing traces that were classi \ufb01 ed as co - detected with the SecGFP - GPI ( see Methods , S . I . and Table 1 , for methods and statistical details and analysis ) . We next examined another CG pathway regulator , ARF1 14 , and found that mCherry - ARF1 was already accumulated at the forming CG endocytic sites at \u2212 36 s and peaked at 0 s ( Fig . 1e , Supplementary Fig . 6a and Table 1 ) . Predictably , mCherry - GBF1 , an ARF1 - GEF 16 , was also recruited to SecGFP - GPI spots ( Fig . 1f , Supplementary Fig . 6b and Table 1 ) . The temporal pro \ufb01 le of GBF1 was correlated to ARF1 ( r = 0 . 6 , Table 2 ) . When we extended the time window of observation for ARF1 and GBF1 further back in time ( nearly 60 s before scission ) , we concluded that the accumulation of ARF1 and GBF1 at CG endocytic sites initiates as early as \u2212 54 s ( Supplementary Fig . 2f \u2013 g ) . In absence of other molecules upstream of GBF1 and ARF1 , this pair currently serves as the earliest initiators of the CG endocytic pathway . Furthermore , when we assessed the ultrastructure of newly formed \ufb02 uid - \ufb01 lled endosomes by electron microscopy ( EM ) 23 in cells treated with the small - molecule inhibitor of GBF1 , LG186 33 , the number of CLICs and \ufb02 uid uptake was drastically reduced , whereas , CME - derived vesicles and uptake appeared relatively unaffected ( Supplementary Fig . 1e \u2013 f ) . In contrast to ARF1 , GBF1 and CDC42 , we did not observe a frequent recruitment of clathrin and dynamin to the CG endocytic sites . The recruitment pro \ufb01 le was comparable to random for > 65 % endocytic events for both mCherry - clathrin and dynamin ( Supplementary Fig . 2d \u2013 e and Table 1 ) . The remainder of the pro \ufb01 les exhibited high levels of clathrin and dynamin at SecGFP - GPI endocytic sites . Never - theless , both fraction showed a temporal trend similar to that observed for random ( Supplementary Fig . 2d \u2013 e , compare grey , blue and random traces ) . Conversely , mCherry - dynamin was recruited to SecGFP - TfR endocytic sites proximal to scission in 80 % of the cases ( Supplementary Fig . 2c ) in agreement with a previous study 4 . Despite being responsible for the timed removal of CDC42 from the plasma membrane 14 , ARF1 ( and GBF1 ) was recruited long before the recruitment of CDC42 . The pH pulsing analysis thus revealed a surprising facet of the recruitment of ARF1 ( and GBF1 ) , which suggested a CDC42 - independent function ( s ) for ARF1 in CG endocytosis . Taken together , these results establish a reliable real - time imaging assay to follow newly formed CG endocytic vesicles containing SecGFP - GPI , correlated with recruitment of its known regulators , ARF1 , GBF1 and CDC42 . Identi \ufb01 cation of BAR domain proteins in CG endocytosis . CLICs , visualised within 15 s of their formation , have a pleomorphic tubular appearance and lack a discernable protein coat when visualised by EM 21 , 23 . This prompted us to investigate Fig . 1 Identi \ufb01 cation of newly formed SecGFP - GPI endocytic vesicles using a pH pulsing assay . a Schematic ( top panel ) of the pH pulsing assay depicting the effect of pH on SecGFP - GPI \ufb02 uorescence during endocytic vesicle budding . Note the \ufb02 uorescence of SecGFP - GPI is retained at high pH ( top and bottom left panel ) or when exposed to low pH if sequestered in a newly formed endocytic vesicle ( bottom right panel ) , and quenched only when exposed to low pH ( top right panel ) . Sequential TIRF images of AGS cell co - expressing SecGFP - GPI and mCherry - ARF1 collected at pH 7 , pH 5 and in the RFP channels ( middle panel ) . Newly formed endocytic vesicles ( inset ) ( identi \ufb01 ed as in Supplementary Figure 1c ) are used to construct a single frame ( yellow rectangle ) of the montage depicted ( bottom panel ) . b Average of the normalised \ufb02 uorescence intensities of pH 5 and pH 7 traces at the site of newly formed SecGFP - GPI endocytic vesicles compared to their respective random traces constructed from 120 endocytic events ( pH 5 and pH 7 ) and 3428 random spots , derived from 17 cells pooled from four independent experiments . c The graph shows the fold enrichment of \ufb02 uorescence intensity over the local background of pH 5 vs . pH 7 at the time of formation of the endocytic vesicles ( data from 1b ) . d \u2013 f Graphs show the average normalised \ufb02 uorescence intensity vs . time traces for the recruitment of TagRFPt - CDC42 ( d ) , mCherry - ARF1 ( e ) and mCherry - GBF1 ( f ) to the forming SecGFP - GPI endocytic sites , and its corresponding random intensity trace ( n , Table 1 ) . The random traces were derived from randomly assigned spots of the same radius as the endocytic regions , as detailed in S . I . Endocytic distribution at each time point was compared to the random distribution by Mann \u2013 Whitney U test , and the log 10 ( p ) [ log 10 ( 0 . 05 ) is \u2212 1 . 3 and log 10 ( 0 . 001 ) is \u2212 2 . 5 ] is plotted below each trace ( d \u2013 f ) . Representative montages from the respective data sets are depicted below the graphs ( d \u2013 f ) . Arrowheads indicate the newly formed endocytic vesicle . Error bars , s . e . m . ( b , d \u2013 f ) . Scale bar , 1 . 5 \u00b5 m ( a , d \u2013 f ) Table 1 pH pulsing assay data set Molecule % Coloc # Spots SecGPI # Spots Random # Cell and Experiment CDC42 56 219 3428 17 and 6 ARF1 62 411 4952 12 and 3 GBF1 62 132 1917 6 and 2 IRSp53 60 309 4439 7 and 3 ARP3 48 170 3277 16 and 6 Lifeact 61 244 4277 7 and 3 PICK1 77 121 1968 22 and 10 N - WASP 27 256 1827 9 and 3 Clathrin 30 437 1801 8 and 4 Dynamin 34 130 1866 10 and 4 See section titled pH pulsing analysis ( Methods and S . I ) for details ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 4 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications the role of BAR domain proteins ( BDPs ) in CG endocytosis due to their capability to sense or stabilise membrane curvature . Additionally , BDPs contain domains that can bind lipids and / or regulate actin machinery , including RhoGTPases 30 . Thus , we performed a dsRNA screen in S2R + cells 15 , 16 to identify the BDPs involved in CG endocytosis using GBF1 ( garz ) and GFP dsRNA as positive and negative control , respectively . The screen yielded 10 \u2018 hits \u2019 which affected \ufb02 uid - phase uptake ( Fig . 2a , b and Supplementary Fig . 3 ) . Predictably , endophilin A required for dynamin - dependent CIE endocytosis of GPCRs and Shiga Toxin 9 , 10 was not selected , whereas , GRAF1 31 previously shown to be involved in CG endocytosis was identi \ufb01 ed as a \u2018 hit \u2019 . The other hits , sorting nexin 6 34 and centaurin \u03b2 1A 35 have been implicated in late stages of endosomal traf \ufb01 cking ; CIP4 36 and NWK 37 being dynamin interactors were not pursued in this study . We focused , instead , on two classes of BDPs , MIM / CG32082 ( I - BAR domain ) and PICK1 ( BAR domain ) , primarily due to their interactions with CDC42 and ARF1 , respectively . IRSp53 is necessary for CG endocytosis . IRSp53 , the mamma - lian orthologue of CG32082 38 , has been implicated in \ufb01 lopodia formation . IRSp53 has a multi - domain architecture consisting of I - BAR , CRIB , SH3 and PDZB domains . Using its SH3 domain , IRSp53 is known to interact with many actin regulatory proteins such as WASP - family verprolin - homologous protein 2 ( WAVE2 ) 39 \u2013 41 , Mena / VASP ( vasodilator - stimulated phosphoprotein ) 42 , 43 , Eps8 42 , 44 \u2013 46 , mDia 40 ( Fig . 3a ) . Furthermore , the recruitment of a b GFP garz PICK1 SNX6 CG32082 CG8176 FER ortholog SNX1 ENDO A AMPH GRAF CENT \u03b2 1A ARFAPTIN RHO92B ICA69 SYNDAPIN CIP4 NWK MIM Serial no . FlyBase ID Gene name ( D . melanogaster ) Mamalian ortholog ( DIOPT ) 1 . 2 Normalised endocytosis 1 . 0 0 . 8 0 . 6 0 . 4 0 . 2 0 . 0 1 FBgn0264560 Gartenzwerg ( garz ) GBF1 2 FBgn0032447 PICK1 ( Protein interacting with C kinase 1 ) PICK1 3 FBgn0032005 Sortin Nexin 6 ( SNX6 ) SNX6 , SNX32 , SNX5 4 FBgn0052082 CG32082 BAIAP2 / IRSp53 , BAIAP2L1 , BAIAP2L2 5 FBgn0037702 CG8176 FCHO2 , FCHO1 , 6 FBgn0000723 FER ortholog FER , FES 7 FBgn0031534 Sortin Nexin 1 ( SNX1 ) SNX2 , SNX1 , SNX33 , SNX18 , SNX30 8 FBgn0038659 Endophilin A ( Endo A ) SH3GL3 , SH3GL2 , SH3GL1 9 FBgn0027356 Amphiphysin ( Amph ) BIN1 , AMPH 10 FBgn0030685 GRAF ortholog ARHGAP26 11 FBgn0039056 Centaurin beta 1A ACAP2 , ACAP3 , ACAP1 12 FBgn0037884 Arfaptin ARFIP2 , ARFIP1 13 FBgn0038747 Rho GTPase activating protein at 92B ARHGAP17 , ARHGAP44 14 FBgn0037050 ICA69 ICA1L , ICA1 15 FBgn0053094 Syndapin PASCIN1 , PASCIN2 16 FBgn0035533 CIP4 ( Cdc42 - interacting protein 4 ) CIP4 17 FBgn0263456 Nervous wreck ( Nwk ) FCHSD1 , FCHSD2 18 FBgn0053558 Missing - in - metastasis ( MIM ) MTSS1L , MTSS1 3327 2791 985 737 779 884 848 736 704 450 539 671 606 844 834 572 794 747 707 Fig . 2 RNAi screen reveals BAR domain proteins involved in CG endocytosis . a List of Drosophila proteins in the PFAM database that contained one of the following BAR domains , PFAM IDs \u2014 PF06456 , PF09325 , PF06456 , PF00611 and PF08397 . The list was \ufb01 ltered to remove duplicates to give 18 genes . b The histogram shows normalised 5 - min \ufb02 uid - phase uptake in S2R + cells treated with 10 \u00b5 g of dsRNA for 4 days as indicated with dsRNA against GBF1 ( garz ) as positive , and GFP as negative controls . In a single experiment , mean uptake of one of GFP dsRNA coverslip was used to normalise the mean for rest of the coverslips . Data were pooled from three independent experiments and the cell numbers are indicated in the graph . The bars in green are signi \ufb01 cantly different from GFP dsRNA using two - sample t - test ( p value < 0 . 05 ) . Version 27 of the PFAM database was used to generate the list Table 2 Cross - correlation calculated for indicated traces Molecule pair Time interval ( s ) ( \u2212 36 s to \u2212 12 s ) Speci \ufb01 c interval GBF1 vs CDC42 0 . 5767 GBF1 vs LIFEACT n . s . GBF1 vs ARP3 n . s . GBF1 vs IRSp53 n . s . ARF1 vs GBF1 0 . 6323 ARF1 vs CDC42 0 . 564 ARF1 vs IRSp53 n . s . ARF1 vs ARP3 0 . 6347 ARF1 vs LIFEACT 0 . 7478 CDC42 vs IRSp53 0 . 65 a , b 0 . 5698 a ( \u2212 33 s to 0 s ) CDC42 vs LIFEACT 0 . 6087 0 . 54 ( \u2212 36 s to + 6 s ) CDC42 vs ARP3 n . s . ARP3 vs IRSp53 n . s . 0 . 7739 a ( \u2212 9 s to + 9 s ) ARP3 vs LIFEACT n . s . 0 . 6292 ( \u2212 33 s to 0 s ) ARF1 vs PICK1 n . s . 0 . 5698 ( \u2212 36 s to \u2212 12 s ) PICK1 vs IRSp53 \u2212 0 . 5353 Rest of the calculations are done with traces with spot radius = 3 pixels and background donut = 6 \u2013 8 pixels 1 pixel = 84nm n . s . not signi \ufb01 cant p value considered here is 0 . 05 calculated via t - statistic a Performed for traces with spot radius = 2 pixels and background donut = 6 \u2013 8 pixels b Performed with one frame shift NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w ARTICLE NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 5 IRSp53 to the plasma membrane was compromised following ARF1 depletion 47 . Hence , IRSp53 was a good candidate to act as a signalling platform , linking CDC42 activation , membrane cur - vature and actin regulation for CG endocytosis . To address the function of IRSp53 we compared endocytosis between mouse embryonic \ufb01 broblasts ( MEFs ) generated from IRSp53 \u2212 / \u2212 mice ( IRSp53 \u2212 / \u2212 MEFs ) and IRSp53 \u2212 / \u2212 IRSp53WT addback MEFs ( IRSp53WT MEFs ) 42 . Loss of IRSp53 caused a signi \ufb01 cant reduction in \ufb02 uid - phase uptake , without affecting TfR internalisation ( Fig . 3b ) . We next addressed the nature of endocytosis in IRSp53 \u2212 / \u2212 MEFs and found that the \ufb02 uid - phase uptake in IRSp53 \u2212 / \u2212 MEFs remained refractory to * * n . s . d IRSp53 \u2013 / \u2013 LG186 PM PM PM 1 \u03bc m 1 \u03bc m 5 - min HRP pulse a b I - BAR CRIB / GBD SH3 WW Dimer inactive Dimer active PDZ - BD c IRSp53 WT IRSp53 \u2013 / \u2013 F L U I D TF TF R 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 1 . 4 * FluidTf / TfR n . s . IRSp53 \u2013 / \u2013 IRSp53 WT N o r m a li s ed endo cy t o s i s ( a . u . ) 1 . 6 n . s . IRSp53 WT LG186 ( 10 \u03bc M ) \u2013 \u2013 + + \u2013 + + \u2013 0 . 0 0 . 8 1 . 0 1 . 2 1 . 4 N o r m a li s ed endo cy t o s i s ( a . u . ) I R S p53 W T I R S p53 \u2013 / \u2013 DMSO LG186 WT PM CLICs EE CCV / Cav 0 2 4 6 8 10 12 14 16 WT MEFs IRSp53 \u2013 / \u2013 WT MEFs LG186 n . s . n . s . n . s . * * * N u m be r pe r f i e l d 319 273 305 235 418 325 403 327 Fig . 3 IRSp53 is involved in CG endocytosis . a Schematic depicting the domain organisation of IRSp53 . IRSp53 exists in an inactive dimer state , which upon binding to GTP - CDC42 is activated , allowing SH3 domain to bind to its effectors . b Histogram ( left ) shows 5 - min uptake of TfR and \ufb02 uid phase in IRSp53 WT cells normalised to IRSp53 \u2212 / \u2212 cells , along with representative images ( right ) . Data were pooled from two independent experiments and the number of cells indicated in the \ufb01 gure . c Histogram ( top ) shows normalised 5 - min \ufb02 uid uptake in IRSp53 \u2212 / \u2212 and IRSp53WT cells when treated with LG186 or vehicle ( DMSO ) along with representative images ( bottom ) . Data were pooled from two independent experiments and the number of cells indicated in the \ufb01 gure . d Histogram ( top ) shows an average number of endocytic structures quanti \ufb01 ed per \ufb01 eld from the electron microscope images ( bottom ) . Data pooled from three independent blocks . Untreated WT MEFs ( WT , top row ) , IRSp53 null MEFs ( IRSp53 \u2212 / \u2212 , bottom left ) or LG186 - treated WT MEFs ( LG186 , bottom right ) were incubated for 5 min at 37 \u00b0C with 10 mg / ml HRP as a \ufb02 uid - phase marker before processing for electron microscopy . Endocytic structures close to the plasma membrane ( PM ) are \ufb01 lled with the electron dense peroxidase precipitate . WT cells show a range of endocytic structures including vesicular structures ( double arrowheads ) and tubular / ring - shaped putative CLIC / GEECs ( large arrowheads ) but the IRSp53 \u2212 / \u2212 cells and LG186 - treated cells only show predominant labelling of vesicular pro \ufb01 les . p value < 0 . 05 ( * ) , 0 . 001 ( * * ) Mann \u2013 Whitney U test ( b \u2013 c ) and two - sample Student \u2019 s t test ( d ) . Error bars , s . d . ( b \u2013 d ) . Scale bar , 20 \u00b5 m ( b \u2013 c ) , 1 \u00b5 m ( d ) , respectively . Schematic ( a ) was adapted with permission from MBInfo ( www . mechanobio . info ) Mechanobiology Institute , National University of Singapore ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 6 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications LG186 - mediated GBF1 inhibition ( Fig . 3c ) . By contrast , GBF1 inhibition in IRSp53WT MEFs decreased \ufb02 uid - phase uptake to the levels observed in IRSp53 \u2212 / \u2212 MEFs ( Fig . 3c ) while endocytosed TfR remained unaffected ( Supplementary Fig . 4a ) . We con \ufb01 rmed the lack of the CG endocytic pathway in IRSp53 \u2212 / \u2212 MEFs by ultrastructural analysis of endocytic structures marked by the \ufb02 uid - phase marker , HRP using EM 14 , 23 . We observed a signi \ufb01 cant reduction of CLICs in IRSp53 \u2212 / \u2212 MEFs when compared with WT MEFs , while the number of clathrin and caveolae - derived structures was relatively unaffected . Similar to Supplementary Fig . 1e , f , the CLICs were reduced signi \ufb01 cantly upon LG186 treatment in WT MEFs as well ( Fig . 3d and Supplementary Fig . 4b ) . This led us to hypothesise that CG cargo would traf \ufb01 c via CME in the absence of IRSp53 . Therefore , we looked at the traf \ufb01 cking of GPI - AP ( GFP - GPI ) , \ufb02 uid phase and TfR ( CME ) in the absence of IRSp53 at high resolution . We \ufb01 rst counted the number of GFP - GPI and \ufb02 uid endosomes , and found them to be signi \ufb01 cantly lower in IRSp53 \u2212 / \u2212 MEFs relative to IRSp53WT MEFs ( Fig . 4a ) . Conversely , TfR endosomal number was unaffected ( Fig . 4a ) . We next , looked at co - localisation of GPI - AP / \ufb02 uid phase with TfR . A relatively higher fraction of GFP - GPI I - BAR CRIB / GBD SH3 + \u2013 / \u2013 + \u2013 / \u2013 + \u2013 / \u2013 + \u2013 / \u2013 + \u2013 / \u2013 a b c d 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 F r a c t i on o f F l d w i t h T f IRSp53 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 IRSp53 Cargo F r a c t i on o f G F P - G P I w i t h F l d / T f 300 600 900 1200 1500 # o f E ndo s o m e s / c e ll IRSp53 Cargo n n n GPI / Tf I R S p53 \u2013 / \u2013 I R S p53 W T GPI / Fluid Fluid Tf Merge I R S p53 W T I R S p53 \u2013 / \u2013 Fluid Transferrin N o r m a li s ed endo cy t o s i s ( a . u . ) 4KE I408P V522G I268N WT Fluid GFP - GPI Transferrin \u03b1 - GFP Tf I R S p53 \u2013 / \u2013 I R S p53 W T Fluid 0 . 0 0 . 2 0 . 8 1 . 0 1 . 2 1 . 4 1 . 6 * * * * * * * * * n . s . * * * WT I268N V522G 4KE I408P PDZ - BD I268N 4KE I408P V522G * * * * * n . s . 0 25 25 58 56 32 31 25 25 32 31 + \u2013 / \u2013 46 36 22 1 21 2 28 4 33 6 29 3 NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w ARTICLE NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 7 ( Fig . 4b ) and \ufb02 uid phase ( Fig . 4c ) co - localised with co - pulsed Tf in IRSp53 \u2212 / \u2212 MEFs than IRSp53WT MEFs . Thus , removal of IRSp53 speci \ufb01 cally affected \ufb02 uid phase and GPI - AP endocytosis , while CME remained unaffected . Moreover , in cells lacking IRSp53 , GPI - AP and the residual \ufb02 uid phase is endocytosed via CME . We next analysed the contribution of different domains of IRSp53 on CG endocytosis by re - introducing into IRSp53 \u2212 / \u2212 MEFs , GFP - tagged IRSp53WT and a number of mutants of IRSp53 speci \ufb01 cally defective in various domains ( Fig . 4d , schematic ) . We found that GFP - IRSp53WT and GFP - IRSp53V522G rescued endocytosis while the rest of the mutants failed to do so ( Fig . 4d ) . In conclusion , these results indicated that IRSp53 is an essential and speci \ufb01 c regulator of CG endocytosis that requires functional I - BAR , CRIB and SH3 domains . IRSp53 is recruited to forming CG endocytic vesicles . The complete absence of CG endocytosis in IRSp53 \u2212 / \u2212 led us to hypothesise that IRSp53 has a direct role to play in CG vesicle formation . Hence , we used pH pulsing assay and examined the recruitment of mCherry - IRSp53 to the forming SecGFP - GPI endocytic sites . A majority of ( > 60 % ) endocytic events exhibited prominent recruitment of IRSp53 ( Fig . 5a , Supplementary Fig . 6c and Table 1 ) . Since the I - BAR domain of IRSp53 has been shown to sense / induce negative curvature in a membrane tension and protein concentration - dependent manner 48 , we looked at changes in its spatial distribution during the formation of endocytic vesicle using two types of masks \u2014 a spot and a ring mask ( Fig . 5a , schematic ) . Unlike the intensity pro \ufb01 les of CDC42 that did not exhibit any differential temporal patterns of recruitment between the two types of masks ( Fig . 5b , black vs . red trace ) , IRSp53 displayed a biphasic recruitment pattern ( Fig . 5a ) . In phase I ( \u2212 36 to \u2212 15 s ) , IRSp53 was \ufb01 rst recruited over a large area indicated by an increase in intensity in both spot and ring traces . Subsequently , in phase II ( \u2212 15 to 0 s ) , IRSp53 was con \ufb01 ned at the centre indicated by a concerted decrease in the ring mask intensity and increase in the spot mask intensity trace ( Fig . 5a , black vs . red trace ) . The increase of IRSp53 was more prominent in phase II toward the centre ( Fig . 5a , black vs . purple trace ) and correlated with the recruitment of CDC42 ( r = 0 . 6 ; Table 2 ) . To visualise the recruitment of GFP - IRSp53 at high spatial resolution , we co - expressed a GBP - APEX reagent ( GFP - binding protein soybean ascorbate peroxidase ) and processed for EM as described previously 49 ( Fig . 5c , Supplementary Fig . 4c and Supplementary Movies 3 \u2013 4 ) . GBP - APEX binds to GFP and converts 3 , 3 \u2032 - diamino - benzamidine into an osmiophilic polymer in the presence of H 2 O 2 49 . Images of 3D rendering from the electron densities revealed that IRSp53 associated with tubular structures characteristic of CLICs as described previously 23 and as expected , with \ufb01 lopodial structures 38 ( Fig . 5c , see Methods ) . In the 2D sections , IRSp53 was observed to accumulate as discrete patches at the plasma membrane ( PM ) ( Supplementary Fig . 4c i \u2013 ii , arrowheads ) and was frequently associated with tubulovesi - cular invaginations or tubular structures close to PM and \ufb01 lopodial tips ( Supplementary Fig . 4c iii , double arrowheads ) . We further validated recruitment of GFP - IRSp53 with an alternate technique , protein - retention expansion microscopy ( ProExM ) 50 , a derivative of expansion microscopy 51 . Expansion microscopy enables imaging of diffraction - limited structures by physically separating them using a swellable polymer cross - linked with the cell . This allows multi - colour super - resolution imaging of a sample with conventional regents and microscope . Thus , structures of around 250 nm will be scaled to 1 \u03bc m in a 4\u00d7 expanded sample ( see Methods ) , providing a potential apparent resolution of around 70 nm 51 with conventional imaging technology . We stained IRSp53 \u2212 / \u2212 GFP - IRSp53 MEFs for CD44 and IRSp53 , processed the cells ( Methods and ref . 50 ) , and imaged the samples using \u00d7100 objective in spinning disk microscope [ Supplementary Fig . 4e ( 1a \u2013 b ) and Supplementary Movie 5 ) ] . In accordance with our EM images , we observed enrichment of IRSp53 at the tips of the \ufb01 lopodia [ Supplementary Fig . 4e ( 2a \u2013 c ) and Supplementary Movie 5 ] . Additionally , we could identify several invaginations of CD44 [ Supplementary Fig . 4e ( 3 \u2013 8 , see arrowheads ) and Supplementary Movie 5 ] and found enrichment of IRSp53 . As the neck constriction is expected to develop [ Supplementary Fig . 4e ( 3 \u2013 4 and 6 , arrowheads ) and Supplementary Movie 5 ] , we could see progressive enrichment of IRSp53 around the region of the constriction , relative to other regions of the invagination . The pH pulsing trace showed a speci \ufb01 c pattern of enrichment of IRSp53 over time . This is consistent with two scenarios : ( 1 ) that IRSp53 enrichment occurs speci \ufb01 cally at the neck which constricts over time and , ( 2 ) that IRSp53 coats the entire tubule with higher enrichment around the necks which in turn constricts over time . Our interpretation of EM and proExM data support the latter scenario . Thus , the complete loss of CG endocytosis in IRSp53 null cells , and localisation of IRSp53 to forming CG endocytic vesicles suggests a role of IRSp53 in the vesicle scission . Branched actin nucleation is required for CG endocytosis . A functional CG endocytic pathway requires dynamic actin since inhibition of actin polymerisation ( Latrunculin A ) , or \ufb01 lament stabilisation ( Jasplakinolide ) , inhibited CG endocytosis 13 . CDC42 and IRSp53 are core components of a signalling axis that indir - ectly controls the location and activity of the ARP2 / 3 actin nucleation complex 39 , 52 , 53 . More pertinent , CK666 54 - mediated inhibition of ARP2 / 3 complex , impaired both \ufb02 uid - phase and TfR uptake in a dose - dependent manner . However , the extent of Fig . 4 CG pathway is abolished in the absence of IRSp53 . a The box plot shows the number of endosomes per cells ( left ) for endocytosed GFP - GPI ( \u03b1 - GFP Fab ) , \ufb02 uid phase and TfR in IRSp53 \u2212 / \u2212 and IRSp53 WT cells when pulsed for 2 min along with representative images ( right ) . Data were pooled from two independent experiments and the number of cells indicated below the graph . b Plot ( left ) shows quanti \ufb01 cation of the fraction of GFP - GPI endocytic vesicles containing \ufb02 uid phase or Tf . Images ( right ) show representative single confocal slices of a 2 - min pulse of \u03b1 - GFP Fab ( green ) / TMR - Dextran ( magenta ) and \u03b1 - GFP Fab ( green ) / A568 - Tf ( magenta ) in IRSp53 \u2212 / \u2212 ( top row ) and IRSp53WT ( bottom row ) cells . The inset depicts magni \ufb01 ed views of the indicated region ; single - channel images are in panel 4a . Data were pooled from two independent experiments and the number of cells is indicated below the graph . c Plot ( left ) showing quanti \ufb01 cation of the fraction of 1 - min \ufb02 uid - phase endocytic vesicles containing Tf . Images show representative single confocal slices of a 1 - min pulse of TMR - Dextran ( green ) and A647 - Tf ( magenta ) in IRSp53 \u2212 / \u2212 ( top row ) and IRSp53WT ( bottom row ) cells . Inset depicts magni \ufb01 ed views of the indicated region . Data were pooled from two independent experiments and the number of cells indicated below the graph . d Histogram ( left ) shows 5 - min uptake of \ufb02 uid phase in IRSp53 \u2212 / \u2212 MEFs transduced with virus expressing GFP - IRSp53 WT , GFP - IRSp53 4KE , GFP - IRSp53 I268N , GFP - IRSp53 I408P and GFP - IRSp53 V522G , normalised to that in IRSp53 \u2212 / \u2212 MEFs , along with representative images ( right ) . Data were pooled from two independent experiments and the number of cells indicated in \ufb01 gure except for IRSp53 \u2212 / \u2212 ( 381 ) . p value < 0 . 01 ( * ) and 0 . 001 ( * * ) by Mann \u2013 Whitney U test ( a \u2013 d ) . Scale bar , 20 \u00b5 m ( d ) , 5 \u00b5 m ( a \u2013 c ) , respectively . Error bars ( d ) represent s . d . ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 8 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications / / \u2013 40 / / / a c b Time ( s ) pH 7 pH 5 CDC42 pH 5 pH 7 IRSp53 Time ( s ) F o l d c hange ( a . u . ) IRSp53 Random Log10 ( p ) RAW THRESHOLDED 1 1a 2 3 Time ( s ) Time ( s ) \u2013 30 \u2013 20 \u2013 10 0 10 20 1 . 0 1 . 1 1 . 2 1 . 3 1 . 4 1 . 5 1 . 6 \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 0 . 95 1 . 00 1 . 05 1 . 10 1 . 15 1 . 20 1 . 25 1 . 30 CDC42Random \u2013 6 \u2013 4 \u2013 2 0 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 \u2013 18 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 \u2013 18 Fig . 5 IRSp53 is recruited to forming CG endosomes . a Graphs show the average normalised \ufb02 uorescence intensity vs . time traces for the recruitment of three different regions [ circles , violet , r = 170 nm and black ( r = 250 nm ) and annulus , orange ( r = 250 \u2013 420 nm ) ] for the recruitment of IRSp53 - mCherry to the forming SecGFP - GPI endocytic sites and its corresponding random intensity trace ( n , Table 1 ) . b Graphs show the average normalised \ufb02 uorescence intensity vs . time traces for the recruitment of TagRFPt - CDC42 to the forming SecGFP - GPI endocytic sites and its corresponding random intensity trace to two different regions [ circle , black , r = 250 nm ; and annulus , orange ( r = 250 \u2013 420 nm ) ] . Error bars , ( a \u2013 b ) represent s . e . m . ( n , Table 1 ) . The random traces were derived from randomly assigned spots of the same radius as the endocytic regions , as detailed in S . I . Endocytic distribution at each time point was compared to the random distribution ( a ) by Mann \u2013 Whitney U test and the log 10 ( p ) is plotted below each trace [ log 10 ( 0 . 05 ) is \u2212 1 . 3 and log 10 ( 0 . 001 ) is \u2212 2 . 5 ] . Representative montages are depicted below the graphs ( a \u2013 b ) . Arrowheads indicate the newly formed endocytic vesicle . c Electron micrographs of AGS cells co - transfected - GFP - IRSp53 and GFP - binding protein coupled to Apex ( GBP - Apex ) . The DAB reaction was performed and the cells were processed for electron tomography . A single section of the original tomogram ( left ) and density - based thresholded of the same plane ( middle ) reveal electron dense structures containing IRSp53 at membrane surfaces . The whole of PM of the tomographic volume was rendered and different examples of enlarged tubular regions of interest show GFP - IRSp53 recruitment patterns ( right ) . Scale bar , 1 . 5 \u00b5 m ( a \u2013 b ) and 0 . 5 \u00b5 m ( c ) , respectively NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w ARTICLE NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 9 inhibition of CG internalisation was markedly more prominent ( Fig . 6a ) . By contrast SMIFH2 55 - mediated inhibition of formins , failed to inhibit CG endocytosis ( Supplementary Fig . 5a ) . We next explored the spatio - temporal dynamics of F - actin and ARP2 / 3 complex using pRuby - lifeact 56 and mCherry - ARP3 , respectively , during the formation of the endocytic vesicle . ARP3 recruitment ( Fig . 6b , Supplementary Fig . 6d and Table 1 ) began earlier than \u2212 35 s and peaked at \u2212 6 s . This was unexpected since CDC42 , a key regulator of ARP2 / 3 57 was not recruited until \u2212 9 s . Instead , the ARP3 pro \ufb01 le was correlated with IRSp53 between \u2212 9 s and + 9 s ( r = 0 . 8 ; Table 2 ) . This indicated that , at least initially , the ARP2 / 3 complex might be recruited in a CDC42 - independent manner . On the other hand , F - actin accumulation began around \u2212 9 s and continued even after the scission event ( Fig . 6c , Supplementary Fig . 6e and Table 1 ) , highly correlated to the CDC42 pro \ufb01 le ( r = 0 . 6 ; Table 2 ) . Thus , F - actin pH 5 pH 7 Arp3 a b N o r m a li s ed endo cy t o s i s ( a . u . ) FluidTf / TfR CK ( \u03bc M ) 1 . 2 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 * * * * F l u i d T f T f R DMSO 50 \u03bc M CK 150 \u03bc M CK 300 \u03bc M CK c Time ( s ) \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 1 . 0 1 . 1 1 . 2 1 . 3 1 . 4 1 . 5 1 . 6 F o l d c hange ( a . u . ) Time ( s ) Arp3Random 1 . 6 1 . 4 1 . 2 1 . 0 0 . 8 0 . 6 0 . 4 0 . 2 0 . 0 N o r m a li s ed f l u i d u p t a k e ( a . u . ) N - WASP \u0394 VCA N - WASPVCA N - WASPCA * * * * n . s . d \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 1 . 0 1 . 1 1 . 2 1 . 3 1 . 4 \u2013 4 \u2013 3 \u2013 2 \u2013 10 F o l d c hange ( a . u . ) Time ( s ) N - WASP Coloc ( 27 % ) RandomN - WASP All Log 1 0 ( p ) Time ( s ) pH 5 pH 7 N - WASP e Time ( s ) pH 5 pH 7 Lifeact F o l d c hange ( a . u . ) \u2013 9 \u2013 6 \u2013 3 0 Log10 ( p ) 385 308 306 n 442 319 379 n 380 \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 1 . 0 1 . 1 1 . 2 1 . 3 \u2013 3 \u2013 2 \u2013 10 Time ( s ) LifeactRandom Log10 ( p ) 300 150 50 0 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 \u2013 18 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 \u2013 18 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 \u2013 18 ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 10 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications was generated coincident with the recruitment of CDC42 , a known regulator of actin polymerisation 57 . These observations suggested that ARP2 / 3 complex might be \ufb01 rst recruited in an inactive state , and then activated following the arrival of CDC42 . ARP2 / 3 is inhibited by PICK1 at nascent CG endocytic sites . To address how ARP2 / 3 complex was perhaps maintained at the forming endocytic pit in an inactive state , we analysed the role of PICK1 , another \u2018 hit \u2019 in the screen ( Fig . 2 ) . PICK1 , a highly con - served protein , possesses PDZ and BAR domain ( Fig . 7a ) that by intra - molecular interaction , maintains PICK1 in an auto - inhibited state . This auto - inhibited state is further stabilised upon GTP \u2013 ARF1 binding to the PDZ domain 58 . Additionally , activated PICK1 negatively regulates ARP2 / 3 - mediated actin polymerisa - tion 58 \u2013 60 . The ability of PICK1 to inhibit ARP2 / 3 complex is suppressed by GTP \u2013 ARF1 58 . To con \ufb01 rm a role of PICK1 in CG endocytosis in mammalian cells , we utilised a speci \ufb01 c small - molecule inhibitor of PICK1 , FSC231 61 . CG endocytosis ( \ufb02 uid phase ) was inhibited in a dose - dependent fashion ( Fig . 7b ) by this inhibitor . Additionally , in stable PICK1 knockdown lines , \ufb02 uid phase and folate receptor ( FR - GPI , another GPI - AP ) endosomal number were lower than that measured in scrambled shRNA stable lines , while TfR endocytosis remained unaffected ( Fig . 7c , d ) . Predictably , GFP - ARF1 and TagRFP - PICK1 co - localised in punctate spots at the TIRF plane in accordance with previous reports 58 ( Supplementary Fig . 5b ) . To test the effect of ARF1 activity on PICK1 recruitment we co - expressed TagRFP - PICK1 with either ARF1 - WT or dominant - negative ( ARF1 - DN ; T31N ) and active mutant ( ARF1 - DA ; Q71L ) and tracked the number and dynamics of PICK1 spots using TIRF microscopy ( see Methods ) . The residence time of PICK1 increased signi \ufb01 cantly in the presence of ARF1 - DN while it was reduced in the presence of ARF1 - DA ( Fig . 7e ) . ARF1 - DN and DA have been shown to decrease and increase endocytosis , respectively 14 . Thus , the local presence of ARF1 - GTP resulted in the removal of PICK1 from the membrane . Thus , we hypothesised that PICK1 was recruited to forming CG endosome at the early stage , rendering ARP2 / 3 inactive . To explore this possibility , we utilised the pH pulsing assay to visualise PICK1 at the CG endocytic site . We found that TagRFP - PICK1 was recruited to the forming CG endocytic sites ( Fig . 7f , Supplementary Fig . 6f and Table 1 ) in a pulsatile fashion . Maximum enrichment occurred at \u2212 12 s , with an eventual loss corresponding to the time of the rapid rise in ARF1 recruitment around \u2212 9 s ( Fig . 1e , r = 0 . 6 , Table 2 ) . Thus , the pH pulsing assay has led to the realisation that in CG endocytosis , the interplay of two BDPs , PICK1 and IRSp53 , regulate ARP2 / 3 complex . These BDPs interact with the ARP2 / 3 complex ( Supplementary Fig . 5b , upper panel and Supplementary Fig . 4d ) regulate its activity at the forming CG endocytic sites , in opposing fashion under the in \ufb02 uence of ARF1 and CDC42 . Discussion CG endocytosis was initially discovered as a route for the entry of toxins 62 , \ufb02 uid phase and GPI - anchored proteins when CME was perturbed 11 , 12 , 63 , raising some concerns regarding its physiological role in unperturbed cells 18 . Using a pH pulsing assay we show , here , that a majority of SecGFP - GPI - containing endocytic vesicles form due to a stereotypical and temporally orchestrated recruit - ment of the key molecular machinery namely , CDC42 , ARF1 and GBF1 , which , in turn , mediate the coordinated assembly of speci \ufb01 c BAR - containing , membrane deforming and actin regulatory pro - teins , IRSp53 and PICK1 . Notably , however , the vast majority of the endocytic vesicles are devoid of clathrin and dynamin . Thus , a dedicated complex protein machinery drives CG internalisation , similarly to that being observed in CME . The ability of the pH pulsing assay to provide a temporal pro \ufb01 le for the recruitment dynamics of the molecular players has considerably extended our understanding of CG endocytic vesicle formation . The GBF1 / ARF1 pair is the earliest module to be assembled and judging by their recruitment pro \ufb01 les , it takes around 1 min to assemble the molecular machinery for CG endocytosis . How this pair is concentrated at a forming CG endocytic site is an open question . The CG machinery includes CDC42 , ARP2 / 3 and F - actin along with BDPs , PICK1 and IRSp53 , and in the model ( Fig . 8 ) we propose a biphasic mechanism correlated with the ARF1 recruitment kinetics . In the \ufb01 rst phase , the accumula - tion is slow , accompanied by the presence of PICK1 and absence of CDC42 . In the second phase , beginning around \u2212 9 s ( before scission ) , the accumulation of ARF1 speeds up concomitant with the arrival of CDC42 and loss of PICK1 . How the kinetics of ARF1 recruitment is regulated is unclear since its GAP ( GTPase - activating protein ) is presently unknown , as is the GEF for CDC42 . Nevertheless , the presence of PICK1 provides an expla - nation behind ARP2 / 3 recruitment in an inactive state to the forming CG endocytic vesicles long before CDC42 . ARP2 / 3 is then induced to promote actin branching only upon the arrival of IRSp53 and CDC42 . The role of ARP2 / 3 in CG endocytosis is reminiscent of the endocytic process occurring in the budding yeast . In this system , the endocytic machinery strictly depends on Las17 , the yeast homologue of N - WASP but not so much on clathrin and dynamin 64 , 65 . There are however important differences . In CG endocytosis , the ARP2 / 3 complex appears to be activated inde - pendent of its canonical NPF , N - WASP , a CDC42 effector 57 . First , not only did N - WASP fail to recruit to forming CG endocytic sites ( Fig . 6d and Supplementary Fig . 6g ) , over - expression its dominant - negative mutants also failed to inhibit CG endocytosis ( Fig . 6e ) . By contrast , in CME , both ARP2 / 3 complex and N - WASP are recruited to budding CME vesicles , and in \ufb02 uence endocytosis in some cell types 28 . The unexpected recruitment pro \ufb01 le of ARP2 / 3 and the iden - ti \ufb01 cation of two BDPs , PICK1 and IRSp53 as upstream regulators Fig . 6 Arp2 / 3 - based actin machinery is required for CG endocytosis . a Histograms ( top ) show quanti \ufb01 cation of \ufb02 uid phase and TfR uptake in AGS cells treated with DMSO alone ( 0 \u00b5 M ) or the indicated concentrations of ARP2 / 3 inhibitor , CK666 , normalised to DMSO - treated controls , along with its representative images ( below ) . Data are pooled from two independent experiments and the number of cells shown indicated the graph . b \u2013 d Graphs show the average normalised \ufb02 uorescence intensity vs . time traces for the recruitment of mCherry - ARP3 ( b ) , pRuby - Lifeact ( c ) and mCherry - NWASP ( d ) to the forming SecGFP - GPI endocytic sites , and its corresponding random intensity trace ( n , Table 1 ) . The random traces were derived from randomly assigned spots of the same radius as the endocytic regions , as detailed in S . I . Endocytic distribution at each time point was compared to the random distribution by Mann \u2013 Whitney U test and the log 10 ( p ) [ log 10 ( 0 . 05 ) is \u2212 1 . 3 and log 10 ( 0 . 001 ) is \u2212 2 . 5 ] is plotted below each trace ( b \u2013 d ) . Representative montages are depicted below the graphs . Arrowheads indicate the newly formed endocytic vesicle . e Histogram ( left ) shows normalised 5 - min mean \ufb02 uid - phase uptake in AGS cells overexpressing pIRES - CA domain , GFP - VCA domain and GFP - N - WASP \u0394 VCA from N - WASP compared to un - transfected cells and representative images ( right ) . The transfected cells are outlined . Data were pooled from two independent experiments and the number of cells shown below the graph . Error bars represent s . e . m . ( b \u2013 d ) and s . d . ( a , e ) . p value < 0 . 01 ( * ) , and 0 . 001 ( * * ) by Mann \u2013 Whitney U test ( a , e ) . Scale bar , 1 . 5 \u00b5 m ( b \u2013 d ) , 20 \u00b5 m ( a , e ) NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w ARTICLE NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 11 of ARP2 / 3 activity also suggests a reason for this biphasic mechanism . PICK1 operates in the early phases , and may func - tion as an inhibitor of ARP2 / 3 , consistent with the modest recruitment of F - actin in the presence of PICK1 observed here and as reported previously 59 , 60 . PICK1 recruitment occurs via its BAR domain since when mutated and overexpressed , it acts as a dominant negative for CG endocytosis ( Supplementary Fig . 5c ) . PICK1 recruitment is not only rapid , but also transient . We \ufb01 nd that the localisation of PICK1 to the membrane was negatively correlated to the activity of ARF1 , similar to that observed in neuronal cells 58 , wherein GTP \u2013 ARF1 interaction with PICK1 rendered PICK1 incapable of inhibiting ARP2 / 3 complex . This is followed by the second phase characterised by the simultaneous recruitment of CDC42 / IRSp53 effector complex enabling the activation of ARP2 / 3 and subsequent polymerisation of actin at the site of endocytosis . However , the NPF linking IRSp53 and ARP2 / 3 activation is not yet characterised and is the subject of investigation . There is no single unifying theme for vesicle scission in CIE and multiple modules may co - exist . Recently , endophilin A has been shown to facilitate tubule scission by a combination of scaffolding , dynamin recruitment and dynein - mediated elonga - tion of membrane tubules leading to an increase in friction 66 . In the CG pathway , IRSp53 emerges as a major player . This protein a b e d Time ( s ) pH 5 pH 7 PICK1 PDZ BAR Acidic Acidic 4 1220 110 152 362 380 \u2013 390 0 200 600 800 * * * Transferrin Fluid Cargo N u m be r o f endo s o m e s / c e ll PICK1 shRNA FR - GPI * * * n . s . P I G P Z P I C K 1 s h RN A Transferrin Fluid FR - GPI DMSO 50 \u03bc M 100 \u03bc M 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 1 . 4 * N o r m a li s ed endo cy t o s i s ( A . U . ) FluidTf / TfR * * * * * * D M S O 100 \u03bc M F S C 231 FLUID TF TFR P I G P Z P I C K 1 s h RN A PICK shRNA 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 N o r m a li s ed P I C K 1 l e v e l s ( a . u . ) * * \u2013 40 \u2013 30 \u2013 20 \u2013 10 0 10 20 1 . 0 1 . 2 1 . 4 1 . 6 F o l d c hange ( a . u . ) Time ( s ) PICK1 Random Log10 ( p ) f GFP ARF1 WT ARF1 DN HA ARF1 DA < R e s i den c e t i m e ( P I C K 1 ) , s > 186 82 68 98 n 63 64 77 32 17 20 13 * * * \u2013 4 \u2013 3 \u2013 2 \u2013 10 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 n 560 361 474 FSC231 n c \u2013 + \u2013 + \u2013 + 18 15 12 9 6 3 0 \u2013 3 \u2013 6 \u2013 9 \u2013 12 \u2013 15 \u2013 18 ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 12 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications may function by multiple mechanisms : it can couple negative curvature with membrane tension , it can scaffold membrane at moderate densities 48 , and it can regulate actin polymerisation as it does in \ufb01 lopodia formation 38 . A minimal model accounting for all these activities suggests that IRSp53 might be enriched at the vesicle neck , where it would regulate the actin machinery necessary to trigger CG vesicle scission . The spatio - temporal dynamics and ultrastructure analysis of IRSp53 recruitment at CG sites are consistent with such a model . However , the data does not permit an unequivocal picture ; better resolution and reagents are necessary to verify this speculation . Lastly , the complete and speci \ufb01 c loss of CG endocytosis ( but not CME ) in the absence of IRSp53 makes the requirement for IRSp53 necessary for CG endocytic process . In summary , we propose that CG endocytic vesicle formation begins with GBF1 / ARF1 concentrating at sites of endocytic pits . Following this , though ARP2 / 3 is recruited , it is held in an inactive state by PICK1 ( Fig . 8a ) . Meanwhile , IRSp53 is recruited ( potentially via its I - BAR domain ) and activated by CDC42 leading to ARP2 / 3 activation via unknown effector ( s ) ( Fig . 8b ) . The loss of IRSp53 and ARP2 / 3 from the membrane as the endocytic vesicle is pinched is consistent with their role in endosomal neck dynamics , providing a new candidate for mole - cular machinery of the pinching process in the absence of dynamin . It is conceivable that the IRSp53 provides a scaffold for a friction - based scission mechanism as recently suggested 66 , with actin polymerisation providing driving force for tubule elonga - tion . Alternatively , this force could arise from the involvement of a microtubule - based machinery as recently advocated in the internalisation of cholera toxin 67 . The assays developed here and the identi \ufb01 cation of a number of molecular players and their temporal recruitment pro \ufb01 le provides a path towards under - standing a molecular mechanism for the formation of a CG endocytic vesicle . Methods Cell culture , reagents , and plasmids . See Supplementary Information . pH pulsing assay . pH pulsing assay was adapted for Nikon TE 2000 TIRF microscope ( for the details of the microscope , see microscope section in S . I . ) from a similar assay as described previously 4 . Brie \ufb02 y , FR - AGS cells were plated on custom - designed coverslip bottom dishes and were transfected with SecGFP - GPI and X - FP constructs 12 \u2013 14 h before the assay . The dishes were then \ufb01 tted with a custom - designed holder to place inlet and outlet tubing . Tubing from a pH 7 . 4 ( HEPES ) and pH 5 . 5 ( MES ) buffers kept in a water bath at 38 \u00b0C went through a peristaltic \ufb02 ow controller ( Bioscience Tools ) into the cell chamber . The tempera - ture of the cells in the dish was maintained at 30\u00b0C by maintaining the buffers , the objective and the microscope ( using a chamber ) at appropriately high tempera - tures . Imaging at 30 \u00b0C slows the endocytic process to match the time resolution achieved by the \ufb02 ow setup ( 3 s ) . Images were captured using a script written in open source imaging software , Micromanager , to control the time of \ufb02 ow and imaging . Typically , buffers are exchanged every 3 s and three images are collected sequentially before the end of 3s in two channels , GFP and RFP , using camera exposure of 100ms . The chamber around the microscope was built with the help of NCBS Mechanical Workshop , AC department and Dr Manoj Matthew ( NCBS - CIFF ) . Endocytic assay . Mammalian endocytic assays : All population - based endocytic assays were performed as described 14 . For endocytic assays in mammalian cells , 2 - day plated cells on poly - D - Lysine - coated dishes were used . The assays were performed in the water bath maintained at 37\u00b0C . The media was removed and replaced with media containing \ufb02 uorescently labelled probes at appropriate dilu - tions for the required time ( TMR - Dextran was used at 1 mg / ml , \u03b1 - GFP was used at 20 \u00b5g / ml , Cy3 - Mov18 was used at 5\u00b5g / ml and Tf was used at 10\u00b5g / ml ) . The cells were then transferred to ice and washed with ice - cold medium 1 buffer ( 140mM NaCl , 20 mM HEPES , 1mM CaCl 2 , 1mM MgCl 2 , 5 mM KCl , pH 7 . 4 ) . The cells were then stripped for surface - bound Tf with ascorbate buffer ( 160mM sodium ascorbate , 40 mM ascorbic acid , 1mM MgCl 2 , 1mM CaCl 2 , pH 4 . 5 ) . In the case of GPI - AP ( GFP - GPI and FR - GPI ) uptake , the surface was removed using PI - PLC which cleaves GPI anchor 14 . Cells were treated with PI - PLC ( 50 \u00b5g / ml ) for 1h on ice . The cells were \ufb01 xed with 2 . 5 % paraformaldehyde and stained for surface TfR . BAR domain screen : RNAi screen for BDPs in Drosophila genome was done on S2R + cells stably expressing TfR 15 . Brie \ufb02 y , the cells were plated in a 12 - well plate ( 0 . 5 million cells / well ) for 1h . The media was then replaced with 600 \u00b5l of serum - free media supplemented with appropriate dsRNA at ( \ufb01 nal amount , 10 \u00b5g ) for 1h , post which 600 \u00b5l of serum containing media was added . After 4 days of depletion , the cells were assayed for endocytosis . On the 4th day , cells were deadhered from the well by manual pipetting and plated on coverslip bottom dishes . Cells were pulsed with TMR - dextran diluted in serum containing media for 5min . The cells were subsequently transferred to ice and washed with ice - cold medium 1 buffer ( supplemented with 1mg / ml BSA and glucose ) . The cells were then \ufb01 xed using 2 % paraformaldehyde ( 5 min on ice and 15 min at room temperature ) . dsRNA was prepared from the Drosophila Open Biosystems library v1 15 . HRP uptake and electron microscopy : WT and IRSp53 \u2212 / \u2212 MEFs were serum starved for 45min in the presence or absence of 10 \u03bc M LG186 compound . Cells were treated for 2 or 5 min at 37 \u00b0C with 10mg / ml HRP in serum - free medium , rapidly washed with complete medium on ice and subsequently \ufb01 xed with 2 . 5 % glutaraldehyde in PBS . Peroxidase development and further processing were performed as described previously 23 . Cells were sectioned parallel to the substratum and viewed unstained on a Jeol 1011 ( Tokyo , Japan ) transmission electron microscope equipped with a Morada Soft Imaging camera ( Olympus ) at two - fold binning . Quantitation was performed as follows : for 5 - min HRP uptake , images were captured at random across the monolayer by moving a de \ufb01 ned distance across the grid to avoid user bias . For 2 - min uptake , whole cells positive for HRP were imaged and montaged to generate a high - resolution image encompassing the entire cell . Five cells were imaged and montaged for each replicate . HRP - labelled elements per image or per cell pro \ufb01 le were classi \ufb01 ed using the following criteria ; vesicular elements including clathrin - coated vesicles and caveolae \u2014 circular pro \ufb01 les < 200nm in diameter ; early endosomes \u2013 circular and ring - shaped pro \ufb01 les , including multivesicular structures > 200nm in diameter ; CLIC / GEEC \u2013 other pro \ufb01 les including tubules and small ring - shaped structures < 200 nm in diameter . Ultrastructural localisation of IRSp53 . Thin sections : Localisation of GFP - tagged IRSp53 was performed as described previously 49 . Cells ( FR - AGS ) were seeded onto 30 mm tissue culture dishes ( TRP ) and transfected 24 h later using Lipofectamine Fig . 7 PICK1 is involved in CG endocytosis and is negatively regulated by ARF1 . a Schematic depicts domain organisation of PICK1 . b Histograms ( top ) show quanti \ufb01 cation of \ufb02 uid - phase and TfR uptake in AGS cells treated with DMSO alone ( 0 \u00b5 M ) or the indicated concentrations of PICK1 inhibitor , FSC231 , normalised to DMSO - treated controls , along with its representative images ( below ) . Data were pooled from two independent experiments with the cell numbers shown below the graph . c Box plot ( top ) shows the number of endosomes per cells for FR - GPI ( Cy3 - Mov18 ) , \ufb02 uid phase and TfR in scrambled ( PIGPZ ) and PICK1 shRNA - infected AGS cells when pulsed for 2 min along with representative images ( bottom ) . Data are pooled from two independent experiments and the number of cells indicated below the graph . d Histogram ( left ) shows normalised PICK1 levels measured by immunostaining in PICK1 shRNA - infected AGS cells along with representative images ( right ) . Data were pooled from two independent experiments with the cell numbers indicated in the \ufb01 gure except for PIGPZ ( 292 ) . e Box plot ( top ) shows the residence time of TagRFPt - PICK1 spots at the TIRF plane ( see Methods ) , averaged in an individual cell expressing either GFP , GFP - ARF1 WT , GFP - ARF1 DN or HA - ARF1 DA . The data are pooled from two independent experiments with cell number indicated below the graph . f The graph shows the average normalised \ufb02 uorescence intensity vs . time trace for the recruitment of TagRFPt - PICK1 to the forming SecGFP - GPI endocytic sites and its corresponding random intensity trace ( n , Table 1 ) . The random traces were derived from randomly assigned spots of the same radius as the endocytic regions , as detailed in S . I . Endocytic distribution at each time point was compared to the random distribution by Mann \u2013 Whitney U test and the log 10 ( p ) [ log 10 ( 0 . 05 ) is \u2212 1 . 3 and log 10 ( 0 . 001 ) is \u2212 2 . 5 ] is plotted below . Representative montage is depicted below . Arrowheads indicate the newly formed endocytic vesicle . Error bars represent s . e . m . ( f ) and s . d . ( b , d ) . p value < 0 . 01 ( * ) , 0 . 001 ( * * ) and 0 . 0001 ( * * * ) by Mann \u2013 Whitney U test ( b \u2013 e ) . Scale bar , 1 . 5 \u00b5 m ( f ) , 20 \u00b5 m ( b , d ) and 5 \u00b5 m ( c ) NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w ARTICLE NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications 13 3000 ( as per the manufacturer \u2019 s instructions ) . Cells were subsequently processed for electron microscopy 24 h after transfection . Brie \ufb02 y , AGS cells were double transfected with GFP - tagged IRSp53 and an expression vector encoding for a GFP - binding peptide conjugated to APEX2 ( Addgene plasmid # 67651 ) . Cells were washed in PBS , \ufb01 xed in 2 . 5 % glutaraldehyde in 0 . 1M sodium cacodylate buffer and subjected to the 3 , 3 \u2032 - diaminobenzidine ( DAB ; Sigma - Aldrich ) reaction in the presence of H 2 O 2 for 30 min . DAB reaction product was contrasted by 1 % osmium tetroxide for 2min . Cells were processed in situ and embedded in LX112 resin before sectioning parallel to the culture dish . Tomogram : Thick plastic sections ( 200nm ) were cut on an ultramicrotome ( UC6 , Leica ) and collected onto formvar - coated copper slot grids and lightly carbon coated . Dual - axis tilt series were acquired on a 120 kV TECNAI 12 ( FEI ) transmission electron microscope \ufb01 tted with a LC - 1100 4k x 4k lens coupled CCD camera ( Direct Electron ) and a tilt rotate holder ( Fischione ) utilising a tilt range of \u2212 60 to + 60\u00b0 . Microscope control and image acquisition was accomplished using the software SerialEM 68 . Tilt series were later reconstructed using weighted back projection and \ufb01 ducial markers ( 10 nm ) into a single volume with IMOD 69 . To examine areas with the greatest electron density in an unbiased manner , density - based thresholding was employed with the Isosurface render programme in IMOD as previously described 49 for APEX / DAB reaction product . The whole PM of the tomographic volume was rendered and tubular regions of interest were highlighted at greater magni \ufb01 cation . Protein - retention expansion microscopy . The ProExM protocol was adapted from previous reports 50 , 51 . IRSp53 \u2212 / \u2212 GFP - IRSp53 addback cells were grown on coverslips for 2 days . The cells were \ufb01 xed using 4 % PFA for 15min at room temperature ( RT ) . The surface CD44 was stained using \u03b1 - CD44 ( Rat ) following which the cells were permeablised using 0 . 05 % Tween20 for 15min . IRSp53 was dual stained using \u03b1 - GFP and IRSp53 antibodies both of which were generated in Rabbit . Following this , secondary antibodies against Rat ( Alexa - 568 ) and Rabbit ( Alexa - 488 ) were used . The cells then were treated with Acryloyl X - SE ( 10 mg / ml stock solution in DMSO , used 1 : 100 diluted in PBS ) for 12h at RT . The cells were washed with PBS 2\u00d7 15 min each before proceeding to gelation . For a 10 ml of monomer solution ( sod . acrylate ( \ufb01 nal concentration 8 . 6g / 100 ml ) , acrylamide ( \ufb01 nal concentration 2 . 5g / 100 ml ) , N , N \u2032 - methylenebisacrylamide ( \ufb01 nal concentra - tion 0 . 15 g / 100ml ) , NaCl ( \ufb01 nal concentration 11 . 7 g / 100ml ) were diluted in 1\u00d7 PBS . Monomer solution ( 48 \u00b5l ) was mixed with water ( 1 \u00b5l ) , TEMED ( 1 \u00b5l ) and APS ( 10 % , 1\u00b5l ) was added on to the cells . The cells were incubated at 37 \u00b0C for 30 min . The gel was incubated in the digestion buffer ( 50 mM Tris pH 8 . 0 , 1 mM EDTA , 0 . 5 % Triton X - 100 , 0 . 8M guanidine HCl , Proteinase K ( 1 : 100 , \ufb01 nal con - centration 8 units / ml , added before use ) for 6h at 37 \u00b0C . The gel was washed with double distilled water for 3 \u2013 5 times for 15min to achieve full 4\u00d7 expansion . The gel was placed on a coverslip and was imaged using \u00d7100 spinning disk microscope . Image analysis . In all cases , images were analysed with ImageJ and / or custom software written in MATLAB ( The Mathworks , Natick , Massachusetts , USA ) . The number of cells and repeats of the experiments are mentioned in the legends and \ufb01 gures . Statistical signi \ufb01 cance ( p ) was calculated by Mann \u2013 Whitney U test and two - sample Student \u2019 s t test , as reported in the legends . pH pulsing assay analysis : A semi - automated analysis was developed in MATLAB to identify newly formed endocytic vesicles in the pH pulsing assay and trace their intensity over time in pH 7 , pH 5 and RFP channels . The traces are an GTP F - Actin Arp2 / 3 complex ( Inactive ) Arp2 / 3 complex ( Active ) CDC42 Transferrin receptor Other GPI - APs Super ecliptic GFP ARF1 IRSp53 ( Active ) PICK1 ( Inactive ) IRSp53 ( Inactive ) PICK1 ( Active ) GTP GTP GTP GTP GTP GTP GTP GTP GTP GTP GTP GTP Extracellular milieu Cytoplasm GTP GTP GTP GTP GTP GTP GTP GTP a b c GTP KEY Fig . 8 Schematic depicting the proposed biphasic mechanism for CG endocytic vesicle formation . a Phase \u0399 : Characterised by the recruitment of ARF1 / GBF1 , PICK1 , ARP2 / 3 and IRSp53 but not the buildup of F - actin and CDC42 . Here , IRSp53 may be recruited by its I - BAR domain in the absence of GTP - CDC42 , keeping its SH3 domain in an intra - molecular inhibited state . PICK1 keeps ARP2 / 3 in an inactive state . b Phase \u0399\u0399 : Characterised by the recruitment of CDC42 and a sharp increase in ARF1 leading to the removal of PICK1 . This allows for the activation of ARP2 / 3 and buildup of F - actin . CDC42 binds to the CRIB domain of IRSp53 thereby activating it . The SH3 domain of IRSp53 can now bind to ARP2 / 3 activators and create F - actin . c Phase III : Characterised by endocytic vesicle formation , the presence of CDC42 , ARF1 / GBF1 and F - actin ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 14 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications average of many individual traces of all the endosomes pooled from different cells , which are compared with randomly placed spots within the cells . See S . I . for further details . Endocytic assay analysis : In all cases , images were analysed with ImageJ and custom software written in MATLAB ( The Mathworks , Natick , Massachusetts , USA ) . Each endocytic assay was performed with two technical duplicates . The number of repeats for each experiment is mentioned in its \ufb01 gure legend . For a given experiment , weighted mean for the technical duplicates was calculated as mentioned previously 14 . The total number of cells taken for analysis is mentioned in the legends and \ufb01 gures ( at least 40 \u2013 50 cells were taken from each technical duplicates ) . Subsequently , the cell - wise endocytosis distribution was normalised by weighted means of the control . This allowed data to be pooled from different days to be depicted as average and standard deviation as error bars . The statistical signi \ufb01 cance was calculated by Mann \u2013 Whitney U test . * represent p value < 0 . 05 . Co - localisation analysis : The analysis was performed by two methods . JaCoP ( Just another co - loc plugin ) . An ImageJ plugin which has multiple options by which co - localisation between two molecules can be measured 70 . The object - based overlap or Van Steensel cross - correlation function options were used . Brie \ufb02 y , images were thresholded manually and were given appropriate parameters for either of the two options . Spotter33 . It is a custom MATLAB script ( in - house ) as described previously 11 . The algorithm consists of applying a top - hat \ufb01 lter on the images , following which the structures are segmented . The threshold is manually determined for each channel . The segmented structures are then trimmed ( user determined ) until their are shape and size matched to the original structures . In a channel , each segmented particle mask \u2019 s centroid and pixel list is recorded , and the presence of a particle in that location is checked in the other channel . The number of pixels overlapped is normalied to the particle area . This value is averaged across all the particles for a given cell and reported . Residence time analysis : FR - AGS cells expressing the desired molecule tagged with a \ufb02 uorescent protein was imaged for the appropriate time 37 \u00b0C in TIRF . TagRFPt - PICK1 was imaged for 100 frames with 200 ms exposure and 500 ms interval and Nyquist criteria was satis \ufb01 ed . Spots were segmented and tracked using \u00b5 - track 71 . The residence time of TagRFPt - PICK1 spots per cell was calculated from the output of \u00b5 - track by custom MATLAB script . Brie \ufb02 y , the frame in which the spot was detected and the last frame a given spot of tracked is recorded . Spots that appeared in the \ufb01 rst and the last frame of the movie are discarded . Additionally , spots which appeared for only 1 frame are also discarded . The spots whose track end was the last frame was discared as well as the track may or may not have continued . Data availability . 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Acknowledgements We thank Ramya Purkanti , Kabir Hussain and Balaji Ramalingam for help with the analysis , Neeraj Sebastian for making TagRFPt - CDC42 , Rashmi Godbole for help with expansion microscopy , R . P . for her help with BAR domain database creation , Marcus Taylor ( UCSF / NCBS ) for mCherry - IRSp53 and SecGFP - TfR constructs , Gero Mie - senb \u04e7 ck ( University of Oxford ) for ecliptic - GPI , Paul Melan\u00e7on ( University of Alberta ) for ARF1 - mCherry , Catherine Jackson ( National Institutes of Health ) for GBF1 - mCherry , Roland Wedlich - Soeldner ( Universit\u00e4t M\u00fcnster ) for pRuby - lifeact , TagRFPt - PICK1 and GFP - PICK1 ( Harvey McMohan , MRC ) , Mike Way ( The Francis Crick Institute ) for GFP - NWASP \u0394 VCA and GFP - NWASP - VCA domain , J . Hanley ( Uni - versity of Bristol , UK ) for pIRES - EGFP - PICK KK - EE mutant and N - WASP CA domain and Manoj Matthew from CIFF ( Central Imaging and Flow cytometry Facility , NCBS ) for helping to set up the pH pulsing setup . Authors were supported by Wellcome Trust - DBT India Alliance Early Career Fellowship ( G . M . ) , NCBS - TIFR graduate student fel - lowship ( M . S . ) . J . C . Bose Fellowship and a Margadarshi Fellowship ( IA / M / 15 / 1 / 502018 ) from the Wellcome Trust - DBT Alliance ( S . M . ) , Wellcome Trust - DBT India Alliance Intermediate Fellowship and Simons Centre for Living Machines ( M . T . ) , and grants , DBT - CoE ( S . M ) , R . G . P was supported by the National Health and Medical Research Council ( NHMRC ) of Australia ( programme grant , APP1037320 and Senior Principal Research Fellowship , 569452 ) , and the Australian Research Council Centre of Excellence in Convergent Bio - Nanoscience and Technology ( CE140100036 ) , G . S . and A . D . were supported by Italian Association for Cancer Research Investigator Grant ( 10168 and 18621 to G . S . ) and European Research Council ( 268836 to G . S . ) . We acknowledge the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis at The University of Queensland . We acknowledge the CIFF ( Central Imaging and Flow cytometry Facility at National Centre for Biological Sciences , TIFR , India . We thank K . Joseph Matthew ( S . M . Laboratory ) for making the schematic in Fig . 1a and Fig . 8 . Author contributions M . S . and G . M . executed and analysed all \ufb02 uorescence microscopy experiments . M . S . and M . T . developed the analysis . R . G . P . and J . R . performed , analysed and interpreted all electron microscopy experiments . A . D . and G . S . helped with the IRSp53 knockout and IRSp53 mutant addback cell line construction . G . M . , M . S . and S . M . planned all experi - ments and wrote the manuscript with inputs from the remaining authors . Additional information Supplementary Information accompanies this paper at https : / / doi . org / 10 . 1038 / s41467 - 018 - 03955 - w . Competing interests : The authors declare no competing interests . Reprints and permission information is available online at http : / / npg . nature . com / reprintsandpermissions / Publisher ' s note : Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional af \ufb01 liations . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Creative Commons license , and indicate if changes were made . The images or other third party material in this article are included in the article \u2019 s Creative Commons license , unless indicated otherwise in a credit line to the material . If material is not included in the article \u2019 s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2018 ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / s41467 - 018 - 03955 - w 16 NATURE COMMUNICATIONS | ( 2018 ) 9 : 1835 | DOI : 10 . 1038 / s41467 - 018 - 03955 - w | www . nature . com / naturecommunications", + "bibeau2023twist": "PNAS 2023 Vol . 120 No . 4 e2208536120 https : / / doi . org / 10 . 1073 / pnas . 2208536120 1 of 12 RESEARCH ARTICLE | Significance How actin filaments respond to mechanical loads is central to understanding cellular force generation and mechanosensing . While there is consensus on the actin filament bending stiffness , reported values of the filament torsional stiffness vary by almost 2 orders of magnitude . We used magnetic tweezers and hydrodynamic flow to determine how filaments respond to applied twisting and pulling loads . Twisting causes filaments to adopt a supercoil conformation . Pulling forces inhibit supercoil formation and fragment filaments . These observations explain how contractile forces generated by myosin motors accelerate filament severing by cofilin regulatory proteins in cells . Author affiliations : a Department of Molecular Biophysics and Biochemistry , Yale University , New Haven , CT 06520 Author contributions : J . P . B . , N . G . P . , and E . M . D . L . C . designed research ; J . P . B . , N . G . P . , and N . S . performed research ; J . P . B . , N . G . P . , S . G . , N . S . , C . V . S . , W . C . , and E . M . D . L . C . analyzed data ; and J . P . B . , N . G . P . , W . C . , and E . M . D . L . C . wrote the paper . The authors declare no competing interest . This article is a PNAS Direct Submission . D . V . is a guest editor invited by the Editorial Board . Copyright \u00a9 2023 the Author ( s ) . Published by PNAS . This article is distributed under Creative Commons Attribution - NonCommercial - NoDerivatives License 4 . 0 ( CC BY - NC - ND ) . 1 To whom correspondence may be addressed . Email : enrique . delacruz @ yale . edu . This article contains supporting information online at https : / / www . pnas . org / lookup / suppl / doi : 10 . 1073 / pnas . 2208536120 / - / DCSupplemental . Published January 19 , 2023 . BIOPHYSICS AND COMPUTATIONAL BIOLOGY Twist response of actin filaments Jeffrey P . Bibeau a , Nandan G . Pandit a , Shawn Gray a , Nooshin Shatery Nejad a , Charles V . Sindelar a , Wenxiang Cao a , and Enrique M . De La Cruz a , 1 Edited by Dimitrios Vavylonis , Lehigh University , Bethlehem , PA ; received June 1 , 2022 ; accepted December 16 , 2022 by Editorial Board Member Yale E . Goldman Actin cytoskeleton force generation , sensing , and adaptation are dictated by the bending and twisting mechanics of filaments . Here , we use magnetic tweezers and microfluid - ics to twist and pull individual actin filaments and evaluate their response to applied loads . Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme . Pulling prevents plectoneme formation , which causes twisted filaments to sever . Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persis - tence length of ~ 10 \u00b5m , similar to the bending persistence length . Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces . This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics . actin | torsion | plectoneme | cofilin | severing Cells sense , respond , and adapt to internal and external forces ( 1 , 2 ) . The actin cytoskel - eton , a dynamic , branched , and cross - linked network of protein filaments ( 3 ) that behave as semiflexible polymers on cellular length scales ( 4 \u2013 6 ) , mediates many of these cellular responses . Cellular actin networks are pulled ( 7 , 8 ) , squeezed ( 9 , 10 ) , and twisted ( 11 , 12 ) during growth and remodeling and through interactions with contrac - tile and regulatory binding proteins ( 13 ) . These physical forces can stall network growth ( 14 ) , alter the filament structure ( 15 , 16 ) , modulate interactions among filaments ( 17 ) and with regulatory proteins ( 7 , 8 , 18 , 19 ) , and induce filament fragmentation ( 15 , 19 \u2013 23 ) , all of which influence network remodeling and mediate cellular \u201cmechano - sensing\u201d ( 24 , 25 ) . The capacity for actin networks to respond to force is dictated by the mechanical prop - erties of filaments . Relaxed ( i . e . , resting ) filaments are straight but helical with an intrinsic twist ( 26 ) . The forces required to twist and bend a filament scale with the filament mechan - ical properties ( 4 , 27 ) , specifically their bending and torsional stiffness , which are com - monly represented in terms of bending and twisting persistence lengths ( L B and L T ; we note these are effective persistence lengths because filaments are not homogeneous , iso - tropic materials ) . Filaments with larger persistence lengths are stiffer and require more force to deform than those with shorter persistence lengths . Similarly , stiff filaments store more elastic strain energy for any given deformation than more compliant ones . The elastic free energy ( i . e . , strain energy ) stored in the filament shape ( 16 ) can generate force and work when relaxing to the resting configuration . It can also fragment filaments ( 15 , 19 \u2013 23 , 28 ) and mediate interactions with binding partners ( 7 , 8 , 18 , 19 ) . Dissipation of elastic energy in bent filaments contributes to force generation at the leading edge of migrating cells ( 10 , 29 ) and during essential cellular processes such as endocytosis ( 9 ) . Twisted filaments are also strained . Such twisting has been implicated in symmetry break - ing ( 30 , 31 ) , network chirality ( 11 ) , and the buckling of actin networks in filopodia ( 12 ) . Quantitative knowledge of filament bending and twisting mechanics is therefore critical to reliably account for and model complex cellular behaviors . The bending mechanics of actin filaments have been extensively characterized . There is general agreement that filaments have a bending persistence length ( L B ) of ~ 10 \u00b5m ( 32 \u2013 34 ) , which can be modulated by regulatory proteins ( 35 , 36 ) and ligands ( 5 , 33 , 37 , 38 ) . A consensus on the filament twisting stiffness is lacking , with torsional persistence lengths reported from 0 . 5 to 20 \u00b5m ( 20 , 39 \u2013 44 ) . In addition , it is not known how filament twisting and bending are coupled ( 16 , 27 ) or how filaments respond to combinations of twisting , bending , and pulling forces , as experienced in cells . Here , we use a magnetic tweezers apparatus coupled with microfluidics to evaluate how single actin filaments respond to applied twisting and pulling loads . Our results and analyses provide multiple , independent determinations of the filament bending and twist - ing stiffness , demonstrate how bending and twisting are coupled , and show how this coupling is affected by pulling and filament fragmentation . These findings have D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . 2 of 12 https : / / doi . org / 10 . 1073 / pnas . 2208536120 pnas . org implications for actin cytoskeleton mechanosensing and network force generation and remodeling . Results Twisting and Pulling Actin Filaments . We developed an assay to twist actin filaments about their long axis with magnetic tweezers while simultaneously visualizing them by a TIRF microscope ( Fig . 1 ) . Short , Alexa 488 \u2013 labeled actin filament seeds were tethered to the surface of the microscope coverslip and elongated from the barbed end with purified , Alexa 647 \u2013 labeled actin monomers . These filaments were further elongated from their barbed ends with digoxigenin - conjugated actin monomers to which we attached a paramagnetic bead . The barbed - end \u2013 conjugated paramagnetic bead was twisted at a constant rate ( 0 . 31 rot s \u22121 ) with permanent magnets mounted on a stepper motor ( Fig . 1 B ) . Filament - attached beads and filaments rotated in phase with the permanent magnets ( Fig . 1 B \u2013 D and Movies S1 and S2 ) , indicating that no slipping occurs during manipulation . Filaments attached to two beads ( Fig . 1 B ) were used only to determine whether rotations were in phase with the permanent magnet ( Fig . 1 C ) ; filaments attached to a single bead were used in all subsequent experiments . Pulling forces exerted by the permanent magnet are negligible in our experimental setup ( SI Appendix , Fig . S1 ) , so they were applied with fluid flow using a microfluidic device . The force applied on the filament scales with the size of the bead ( d = 2 . 8 \u00b5m ) and the fluid velocity ( SI Appendix ) . This force is constant through - out the filament ( at a constant flow rate ) and is independent of the filament length in contrast to the much smaller and negligible forces exerted by flow on tethered filaments without conjugated beads ( 17 , 22 , 45 , 46 ) . Actin Filament Force \u2013 Extension Response . To establish the mechanical properties of actin filaments can be reliably determined with our experimental conditions , we measured the bending persistence length ( L B ) in the absence of twisting from the force \u2013 extension response of filaments undergoing thermally driven shape fluctuations ( Fig . 2 and Movie S3 ) . The pulling force required to straighten thermally bent filament scales with the bending stiffness ( L B ) . The filament end - to - end distance ( R ) , defined as the linear distance from the bead and surface attachment points , depends on the long - axis pulling force ( Fig . 2 ) . In the absence of fluid flow ( i . e . , pulling force ~ 0 ) filaments undergo thermally driven bending , so R is shorter than the filament contour length ( L ) , i . e . , R / L < 1 . At ~ 0 . 02 pN pulling force , the ratio of the filament end - to - end distance and the contour length ( R / L ) was ~ 0 . 92 ( Fig . 2 ) . The end - to - end distance approached the contour length R / L ~ 1 at Fig . 1 . Twisting actin filaments with magnetic tweezers . ( A ) Cartoon schematic of the experimental setup . Alexa 488 \u2013 labeled actin filament seeds ( green ) were attached to a Biotin - PEG - Silane surface through biotin ( yellow circles ) and neutravidin ( black diamonds ) interactions and elongated from the barbed ends with Alexa 647 \u2013 labeled actin ( red ) . Filaments were further elongated from their barbed ends with digoxigenin ( DIG ) - labeled actin ( purple ) . Paramagnetic beads ( 2 . 8 \u03bc m in diameter ; gray ) coated with DIG antibodies ( purple ) were attached to filaments at or near their barbed ends . Filament - attached beads can be rotated by a permanent magnet ( blue and red rectangles ) and pulled by buffer flow ( black arrow ) . Relative filament and bead sizes are not drawn to scale . Twisting clockwise or counterclockwise corresponds to under - or overtwisting , respectively . ( B ) Rotation of a phalloidin - decorated filament attached to two paramagnetic beads . ( C ) Cosine of the rotational angle of the second paramagnetic bead ( black trace ) and the magnet ( red trace ) indicates that the bead and magnet rotation are in phase . ( D ) Rotation of an Alexa 647 \u2013 labeled actin filament with visible attachment to paramagnetic bead indicates that the bead and filament rotation are in phase . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . PNAS 2023 Vol . 120 No . 4 e2208536120 https : / / doi . org / 10 . 1073 / pnas . 2208536120 3 of 12 > 3 pN pulling force ( Fig . 2 ) . The best fit of the force dependence of the filament end - to - end distance ( Fig . 2 ) to a worm - like chain model [ Eq . 1 ; ( 47 ) ] yields an actin filament bending persistence length ( L B ) of 10 . 7 ( \u00b11 . 0 ) \u00b5m ( Table 1 ) , consistent with previous wet lab and computational model determinations of bare filaments [ i . e . , without phalloidin or other binding partners ( 19 , 32 \u2013 35 , 42 , 48 \u2013 50 ) ] . The forces in these experiments are calculated using a bead - center distance from the surface ( d ) equal to the bead radius ( r ; d = r ) since the bead was at the surface of coverslip ( SI Appendix ) . Twisted Filaments Bend and Supercoil . Filaments undergo a series of shape transitions when continuously twisted with magnetic tweezers ( Movie S4 ) . In the absence of applied twist ( and with or without pulling loads ) , filaments bend randomly due to thermally driven forces . Applied twisting ( twist density < 0 . 8 rot \u00b5m \u22121 ) causes filaments to bend further in a nonrandom manner , provided long - axis pulling forces are weak ( \u22640 . 03 pN ; Fig . 3 A ) , and the value of R continues to shorten gradually with twisting until a critical twist density ( \u03c3 s ) is reached , at which point filaments form a looped segment . Additional twisting causes the linear , nonlooped filament segments to wind up and twist around each other , yielding an interwound , actin filament supercoil ( called a plectoneme ) like those observed with twisted DNA ( 51 , 52 ) . This transition is detected as a dramatic and abrupt reduction in R ( Fig . 3 B ) . Only a single loop ( i . e . , one plectoneme ) per filament was observed in our experiments . Plectoneme formation is reversible and relaxed ( i . e . , straight ) filaments can be recovered with untwisting ( Movie S5 ) . We note that rhodamine phalloidin \u2013 decorated actin filaments supercoil when subjected to high - intensity laser light ( 53 ) , presumably due to photo - induced torsional strain . Actin filaments are helical and can be described as having an intrinsic right - handed twist . Therefore , counterclockwise rotations increase the intrinsic twist ( \u201covertwisting\u201d ) , whereas clockwise rotations lower the intrinsic twist ( \u201cundertwisting\u201d ) . Binding of cofilin / ADF regulatory proteins , for example , also undertwists filaments ( 54 , 55 ) . Filament plectoneme formation depends on the pulling force but not on the twisting direction ( i . e . , over - ver - sus undertwisting ) , as indicated by the symmetry of the twist \u2013 extension response curves ( Fig . 3 B ) . The lack of a detectable asymmetry with over - and undertwisting is surprising given the intrinsic filament twist . However , this observed behavior likely arises from the fact that the deformations associated with plec - toneme formation are modest at the subunit level and not in the regime in which differences between the two directions could be detected ( 16 ) . Filaments adopt a plectoneme configuration at low twist densities ( ~ 0 . 2 rot \u00b5m \u22121 ) when the pulling force is low ( < 0 . 01 pN ) but require more twist ( ~ 0 . 8 rot \u00b5m \u22121 ) at higher pulling forces ( 0 . 03 pN ; Fig . 3 ) . Plectonemes did not form when the pulling force was 0 . 25 pN , even up to twist densities of ~ 1 rot \u00b5m \u22121 . The reversibility of plectoneme formation was also independent of the twisting direction . The plectoneme loop size also depends on the pulling force ( SI Appendix , Fig . S2 ) . The average loop radius was ~ 400 to 500 nm under 0 . 01 pN , whereas it was ~ 200 nm under 0 . 03 pN . The local filament curvatures at these radii are small compared with those predicted to significantly accelerate filament fragmentation ( 15 ) , consistent with these plectoneme loops being stable through - out the duration of our experiments . Filament Twist \u2013 Extension Response . The experimental data presented thus far demonstrate that twisted actin filaments bend and adopt supercoiled plectoneme structures when pulling forces are low ( < 0 . 25 pN ) . This response originates from the intrinsic filament bending and twisting mechanics and the coupling between these two deformations ( 16 , 52 , 56 ) . Fig . 2 . Actin filament force \u2013 extension response . ( A ) Representative fluorescent images of Alexa 647 \u2013 labeled actin filaments ( magenta ) conjugated to a paramagnetic bead ( cyan ) under fluid flow . No magnetic field is applied . ( B ) Force \u2013 extension curves for actin filaments of varying lengths ( colored points ) with the global best fit to Eq . 1 ( colored lines ) with pulling forces at d = r ( Methods ) . ( C ) Average force \u2013 extension curves , normalized to filament lengths , for 7 actin filaments and corresponding theory with the global best fit persistence length of 10 . 7 ( \u00b11 ) \u00b5m ( solid red line ) ( Eq . 1 ) . Theoretical force \u2013 extension curves ( Eq . 1 ) in descending order with L B = 100 , 5 , 1 , and 0 . 1 \u00b5m ( dashed red lines ) . Uncertainty bars indicate standard error of the mean ( SEM ) . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . 4 of 12 https : / / doi . org / 10 . 1073 / pnas . 2208536120 pnas . org Accordingly , the actin filament mechanical properties can be extracted from the data with appropriate theory , analysis , and modeling . A two - state model used to describe plectoneme formation in DNA ( 52 ) ( modified to include polymers with L ~ L B , such as actin filaments ; Methods ; Eq . 3 ) accounts for the twist \u2013 extension response and plectoneme formation of actin filaments over the range of pulling forces evaluated here ( Fig . 3 B ) . This model con - siders actin filament segments as semiflexible rods in either \u201cstraight\u201d or plectoneme states . Pulling forces favor the straight configuration . Twisting introduces strain energy , which is dissi - pated by bending and subsequent plectoneme formation . Prior to reaching a critical twist density ( \u03c3 s ) for plectoneme formation , twisted filaments bend to dissipate torsional strain , shortening R . Once a plectoneme has formed , applied twisting strain is dissi - pated through conversion of strained , straight state segments to more relaxed plectoneme configurations ( i . e . , straight segments shorten , while plectoneme segments elongate linearly with applied twisting loads ) . Since only the linear , nonlooped filament seg - ments contribute to the R value ( plectoneme segments do not ) , the R value decreases linearly with the applied twist until R = 0 , at which the entire filament is in a plectoneme configuration ( middle line in Eq . 3 ; Fig . 3 B ) . The applied twist at R = 0 is referred to as \u03c3 p . The best fit of the filament twist \u2013 extension data to this model ( Eq . 3 ; Fig . 3 B ) yields actin filament persistence lengths for bend - ing ( L B ) and twisting ( L T ) of 10 . 9 ( \u00b12 . 6 ) and 11 . 7 ( \u00b12 . 2 ) \u00b5m , respectively ( Table 1 ) . This value of the bending persistence length ( L B ) is comparable with the value of 10 . 7 ( \u00b11 . 0 ) \u00b5m obtained from the force \u2013 extension response ( Fig . 2 ) . Filament torsional persistence lengths an order of magnitude longer or shorter do not account well for the observed experimental data ( SI Appendix , Fig . S5 ) . We note that some twisted filaments \u201cwobbled\u201d slightly dur - ing twisting manipulations ( Movie S2 ) . To estimate the maxi - mum possible error introduced by bead wobbling in these cases , we analyzed the data assuming a larger force as expected if the bead moved away from the surface during rotation . Deviations far greater than observed for wobbling ( i . e . , one full bead height deviation , such that d = 2 r ; SI Appendix ) yield essentially iden - tical L T values ( L T = 11 . 7 \u00b1 2 . 2 \u00b5m versus L T = 11 . 4 \u00b1 2 . 1 \u00b5m for beads wobbling an entire bead height ) but ~ twofold lower L B values ( 10 . 9 \u00b1 2 . 6 \u00b5m versus 6 . 5 \u00b1 1 . 5 \u00b5m for beads wobbling an entire bead height ) . Thermally Driven , Filament Twist Fluctuations . We also determined the filament torsional stiffness by directly visualizing spontaneous , thermally driven , twist fluctuations ( Fig . 4 ) . A hollow , cylindrical magnet was positioned above the tethered filament to generate a magnetic field ( perpendicular to the surface of the sample chamber ) that held the filament orthogonally to the surface without constraining its rotation ( 57 ) ( Fig . 4 A and B and Movie S6 ) . The pulling force generated Table 1 . Actin filament bending and torsional persis - tence lengths Persistence Length ( \u00b5m ) Assay Bending ( L B ) 10 . 7 ( \u00b11 . 0 ) Force \u2013 extension , Fig . 2 10 . 9 ( \u00b12 . 6 ) Twist \u2013 extension , Fig . 3 , f at d = r Twisting ( L T ) 11 . 7 ( \u00b12 . 2 ) Twist \u2013 extension , Fig . 3 , f at d = r 12 . 9 ( \u00b12 . 4 ) Twist fluctuations , Fig . 4 8 . 2 ( \u00b10 . 2 ) Cryo - EM , refinement volume of 5 subunits , histogram fit , Fig . 5 5 . 5 ( \u00b10 . 2 ) Cryo - EM , refinement volume of 5 subunits , MLE * 4 . 4 ( \u00b12 . 7 ) Cryo - EM , refinement volume of 5 subunits , MCMC \u2020 5 . 8 ( \u00b10 . 3 ) Cryo - EM , refinement volume of 1 subunit , histogram fit 7 . 0 ( \u00b11 . 1 ) Cryo - EM , refinement volume of 1 subunit , MLE * 6 . 3 ( \u00b13 . 3 ) Cryo - EM , refinement volume of 1 subunit , MCMC \u2020 * Maximum likelihood estimation ( MLE , see Methods ) . \u2020 Markov chain Monte Carlo ( MCMC , see Methods ) . Fig . 3 . Actin filament twist \u2013 extension response and supercoiling . ( A ) Representative fluorescent images of actin filament twist \u2013 extension with 0 . 01 ( Top ) , 0 . 03 ( Middle ) , and 0 . 25 ( Bottom ) pN pulling force ( SI Appendix , Eq . S1 with d = r ) . ( Scale bar , 5 \u00b5m . ) ( B ) Twist \u2013 extension curves for actin filaments under 0 . 01 ( white circles ) , 0 . 03 ( gray circles ) , and 0 . 25 ( black circles ) pN pulling forces with the global best fit to Eq . 3 ( red lines ) . Solid and dashed red lines differentiate model before and after plectoneme formation , respectively . Rotations along the positive x - axis indicate filament overtwisting , and rotations along the negative x - axis indicate undertwisting . The complete dataset represents 50 filaments and n > 3 for each experimental condition . Uncertainty bars represent SEM . The asymmetric look of red dashed fitting lines for over - and undertwists under the same pulling force is due to the different fixed parame t ers ( 1 L ) in the fitting ( Methods ) and not because of differences in response to applied over - and undertwist . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . PNAS 2023 Vol . 120 No . 4 e2208536120 https : / / doi . org / 10 . 1073 / pnas . 2208536120 5 of 12 by the closely positioned hollow magnet maintains the filament relatively straight , thereby eliminating contributions from filament bending to the observed dynamics and allowing determination of the true filament torsional stiffness ( SI Appendix , Fig . S3 , ( 39 , 56 ) ) . Filament twisting fluctuations were monitored by tracking a smaller fluorescent marker bead attached to the paramag - netic bead at the filament barbed end ( Fig . 4 C and D and Movie S7 ) . The angular fluctuations of the marker bead reflect the cumulative rotational fluctuation of all subunits between the surface and paramagnetic bead attachment points ( L = 8 to 19 \u03bc m , ~ 365 subunits \u00b5m \u22121 ; SI Appendix , Fig . S6 ) . The rotation angle of the marker beads around the filament center axis fluctuates randomly ( Fig . 4 C and D ) . Time courses of the observed marker bead angle variance ( Fig . 4 E ) plateau at times > 1 , 000 s , indicating the entire accessible diffusive space of the filament - attached bead had been sampled ( 58 ) . The twist persis - tence length for each set of data in Fig . 4 D was determined from the angular variance calculated by directly averaging the set of data and the filament length according to Eq . 4 ( Methods ) . The averaged ( n = 5 ) value from separate experiments yields a filament torsional Fig . 4 . Direct visualization of actin filament twisting fluctuations . ( A ) Cartoon schematic of the experimental setup . A cylindrical magnet with a center hole was positioned 5 mm above the coverslip surface \u2013 anchored actin filament ( i . e . , in the z direction perpendicular to the surface ) . A DIG - coated marker bead was added to the paramagnetic bead to track rotations . No flow was applied during experiments . Filament length and bending are not to scale . ( B ) ( a ) Assay is set up by identifying a filament attached to a paramagnetic bead ( large dim bead ) with identifiable marker beads ( small bright bead ) under fluid flow . ( b ) Fluid flow is turned off . ( c ) Cylindrical magnet is lowered into position . ( d ) Filament is pulled out of the focal plane . ( e ) Focal plane is adjusted to observe the rotational fluctuations of both beads . Images were taken every 5 s . ( C ) Example images of the angular fluctuations of the filament visualized by the absolute angle of a line connecting the two beads to the x direction . ( D ) The mean - subtracted absolute angle ( SI Appendix , Eq . S25 ) of the marker beads over time . Black trace indicates the angular fluctuations from the filament tracked in ( C ) . Gray traces represent four other sample traces from different experiments performed at different times . Histogram represents the distribution of absolute angles from the black trace . The actin filament torsional persistence length of 12 . 9 ( \u00b12 . 4 ) \u03bc m is an average ( n = 5 ) of separate measurements , each was determined from the value of the variance at long times ( see Panel E ) and the filament length according to Eq . 4 . ( E ) Time - dependent variance of the traces in D . It demonstrates that the measurement time of whole filament angular fluctuation has to be long enough for the variance to reach equilibrium such that experiencing all possibilities . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . 6 of 12 https : / / doi . org / 10 . 1073 / pnas . 2208536120 pnas . org persistence length of 12 . 9 ( \u00b12 . 4 ) \u00b5m ( Table 1 ) comparable with the value of 11 . 7 ( \u00b12 . 2 ) \u00b5m determined from the filament twist \u2013 extension response and plectoneme formation ( Table 1 ) and with reported values of ~ 16 \u00b5m ( 20 ) and ~ 6 \u00b5m ( 39 ) . Single Subunit Twisting Fluctuations . We measured the filament twisting persistence length a third way , from the variance of twisting angles between subunits , as visualized by electron cryomicroscopy ( Fig . 5 ) . Alignment parameters output from the 3D structure refinement yield estimates of filament subunit orientations and hence the twisting angle between them ( 54 ) . Deviations of the observed intersubunit twist angle from the intrinsic ( average ) filament twist reflect thermally driven twist fluctuations . The distribution ( i . e . , width ) of these deviations scales with the filament torsional stiffness , such that filaments displaying a narrow distribution are less compliant in twisting than those with a broader distribution . A challenge with accurately calculating this angle distribution by cryo - EM is the low signal - to - noise ratio associated with the images . The noise introduces uncertainty in the angle measure - ments that can exceed the true intersubunit angle variance . Since the torsional stiffness and persistence length ( L T ) are determined from the distribution variance ( SI Appendix , Eq . S15 ) , and large uncertainties in individual intersubunit angle measurements ( SI Appendix , Eq . S17 ) yield a larger variance than the true vari - ance , neglecting contributions from these uncertainties causes filaments to appear more compliant than they actually are . We therefore developed an analysis method ( SI Appendix , Eqs . S17 \u2013 S21 ) that addresses the uncertainty in angle measure - ment to accurately measure L T from cryo - EM micrographs . Because each filament subunit is independently subject to ther - mally driven torsional angle fluctuations in a scale dictated by the torsional stiffness , the width of the true angular distributions \u03c3 n + 1 2 , measured across filament segments , increases linearly with the number of subunits according to \ud835\udf0e 2 obs , n + 1 = n \u0394 s L T + \ud835\udf0e 2 \ud835\udf00 ( SI Appendix , Eq . S21 ) . In contrast , the uncertainty in the esti - mated twist angle \ud835\udf0e 2 \ud835\udf00 remains constant . Our cryo - EM images confirmed this behavior ( Fig . 5 C ) , yield - ing estimates of the true intersubunit torsional variance and per - sistence length ( \u03c3 n + 1 2 and L T , respectively ; SI Appendix , Eq . S21 ) from the slope of the line relating the observed variance ( \ud835\udf0e 2 \ud835\udf00 ) to n ( Fig . 5 C ) . The intercepts of these lines reflect the contribution of noise to \ud835\udf0e 2 obs ( SI Appendix , Eq . S21 ) . The best linear fit of the n - dependent variance , obtained by Gaussian distribution fit to the angle \u03a6 histogram measured from a refinement volume of 5 subunits , to SI Appendix , Eq . S21 yields an L T of 8 . 2 ( \u00b10 . 2 ; \u00b1 indicates SDs of the fit ) comparable with the L T values of 11 . 7 ( \u00b12 . 2 ) and 12 . 9 ( \u00b12 . 4 ) \u00b5m determined by plectoneme formation ( Fig . 3 ) and thermally driven filament torsional fluctuations ( Fig . 4 ) , respectively . We include two additional analysis methods , directly averaging ( MLE ) and MCMC ( Methods ) , to inde - pendently determine n - dependent variance and thus L T to see if one is more robust than the others ( Table 1 ) . The differences among the three different methods are not significant . We also repeated the analysis to the angles measured from a refinement volume of 1 subunit ( Table 1 ) . These values are not significantly different from those from a refinement volume of 5 subunits . Twist - Induced Filament Fragmentation . Filaments often fragmented during twist \u2013 extension manipulations ( Fig . 3 ) . At 0 . 25 pN pulling force , filaments fragmented before forming a plectoneme , indicating that intersubunit bonds rupture if strain from applied twisting is not dissipated ( 15 , 19 ) . Most filaments ( 83 / 96 ) fragmented at the bead or surface attachment sites ( twist density = 1 . 2 ( \u00b10 . 6 ) rot \u00b5m \u22121 ; discussed below ) , suggesting that the weakest mechanical elements are filament attachment points . The remaining events ( 13 / 96 ) could be reliably discerned as fragmentation within the filament . This occurred at a twist density of ~ 1 . 1 ( \u00b10 . 5 ) rot \u00b5m \u22121 ( Movie S8 ) . At low pulling forces ( \u22640 . 03 pN ) , filaments adopt a plectoneme configuration , which dissipates the torsional strain from applied twisting . Accordingly , no fragmentation was observed , and all filaments formed a plectoneme under 0 . 01 pN pulling force . At a pulling force of 0 . 03 pN , some fragmentation events were observed . These occurred at the onset or during plectoneme for - mation [ twist density = 0 . 83 ( \u00b10 . 17 ) ; Fig . 6 and Movie S9 ] , with 9 / 17 filaments fragmenting exclusively within the filament . Cofilin Promotes Twist - Induced Filament Fragmentation . Filaments saturated with the actin regulatory protein , cofilin , referred to as cofilactin [ cofilin ] = 2 \u03bc M , which is saturating for this yeast isoform under our conditions ( 35 , 38 ) , did not form a plectoneme , even at low ( 0 . 03 pN ) pulling force , because they fragmented . Cofilactin filament fragmentation occurred at a lower twist density than fragmentation of bare filaments ( Fig . 6 and Movies S10 and S11 ) . The twist density dependence of the filament survival probability decay ( Fig . 6 ) indicated a midpoint of ~ 1 rot \u00b5m \u22121 for fragmentation of bare actin , while the midpoint for cofilactin filaments was significantly lower ( 0 . 64 ( \u00b10 . 28 ) and 0 . 43 ( \u00b10 . 12 ) rot \u00b5m \u22121 for overtwisting and undertwisting , respectively ; Fig . 6 and Movies S10 and S11 ) . The observed fragmentation originates from twisting strain rather than flow - mediated forces as neither untwisted bare nor cofilactin filaments fragmented on the timescales of these experiments ( SI Appendix , Fig . S4 and Movie S12 ) . Discussion Actin Filament Torsional Persistence Length is ~ 10 \u03bc m . Here , we have shown through twist \u2013 extension ( Fig . 3 ) , filament rotational fluctuations ( Fig . 4 ) , and single subunit fluctuations ( Fig . 5 ) that actin filaments have a torsional persistence length of ~ 10 \u03bc m , comparable with their bending persistence lengths ( Fig . 2 ) ( 19 , 32 \u2013 35 , 38 , 42 , 48 \u2013 50 , 59 ) . The reported torsional persistence length of actin filaments varies significantly from 0 . 5 to 20 \u00b5m ( 20 , 22 , 39 \u2013 44 ) . Our measured L T is consistent with reported values determined in optical traps ( 20 , 39 ) , fluorescent polarization microscopy ( 22 ) , and electron microscopy of filaments straightened with hydrodynamic flow ( 43 ) but differs from the shorter L T values determined with fluorescent polarization microscopy ( 41 ) , negative stain electron microscopy ( 44 ) , phosphorescence anisotropy ( 40 ) , and molecular dynamics simulations ( 42 ) . Our actin persistence lengths are about two orders of magnitude more rigid than DNA , which has an L T and L B of 100 and 50 nm , respectively ( 52 ) , although these values depend greatly on solution conditions ( e . g . , salt composition and concentration ) . The large uncertainties in the measured rotation angles of indi - vidual actin subunits could contribute to the short L T values deter - mined by electron microscopy ( 44 ) . Uncertainty in subunit rotation angles overestimates the angular fluctuations of adjacent filament subunits and yields an artificially short twisting persistence length . Using an analysis method as in this work ( Methods ) that accounts for these uncertainties in rotational angles yields a larger ( i . e . , stiffer ) torsional persistence length ( Fig . 5 ) . The discrepancy in the filament twisting persistence length values determined by fluorescence polar - ization and phosphorescence anisotropy ( 40 , 41 ) may be due to the independent movement of protein side chains or subunit domains to which the spectroscopic probe is conjugated . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . PNAS 2023 Vol . 120 No . 4 e2208536120 https : / / doi . org / 10 . 1073 / pnas . 2208536120 7 of 12 Actin Filaments Fragment at a Twist Density of ~ 1 to 2 deg sub \u22121 . In our twist \u2013 extension experiments with 0 . 25 pN pulling force , actin filaments fragmented at a twist density of 1 to 2 rot \u03bc m \u22121 ( Figs . 3 B and 7 A ) . A twist density of 1 rot \u03bc m \u22121 is equivalent to ~ 1 deg rotation per actin subunit , which corresponds to a 1 - deg change in relative twist between two laterally adjacent actin subunits and a 2 - deg change in twist between two longitudinally adjacent actin subunits . This twist density introduces only 0 . 65 and 1 . 3 k B T of strain energy ( SI Appendix , Eq . S4 ) at the lateral and longitudinal contacts of actin subunits ( Fig . 7 B ) , respectively , which is considerably less than the estimated bond energies associated with lateral ( 4 to 8 k B T ) and longitudinal ( 12 to 20 k B T ) filament contacts ( 26 ) . Why then do actin filaments fragment at such low twist den - sities ? For an actin filament to fragment , three intersubunit inter - faces\u2014two longitudinal and one lateral\u2014must rupture simultaneously ( 15 , 19 ) . The combined twist strain energy in these three bonds of a filament twisted to a density of ~ 1 deg rotation Fig . 5 . Torsional persistence length of actin filaments determined by electron cryomicroscopy . ( A ) Cartoon schematic of measured filament subunit twisting fluctuations . The top cartoon depicts an actin filament ( gray ) with the average , intrinsic twist ( \u0394\u03c6 1intrinsic ) between adjacent subunits i and i + 1 illustrated as a red curved arrow . The observed twist deviates from the intrinsic twist , either over or under , because of thermal fluctuations . The middle and bottom cartoons illustrate an undertwisted filament ( light gray ) overlaying a canonical filament with an intrinsic twist ( dark gray ) . Blue arrows illustrate the observed twist between subunits i and i + 1 ( Middle ) or i and i + 3 ( Bottom ) , which differs from the intrinsic twist by \u0394\u03c6 \u2019 n ( illustrated by black arrows ) . ( B ) Histograms of actin filament subunit twist fluctuations ( \u0394\u03c6 \u2019 n , in degrees ) estimated from cryo - EM alignment parameters ( reference volume of 5 subunits ) for n = 1 , 10 , and 50 subunits . Red lines represent fits to a normal distribution with mean zero and variance ( \ud835\udf0e 2 obs , n + 1 ) . ( C ) n dependence of the twist variance ( \ud835\udf0e 2 obs , n + 1 ) . The solid red lines represent the best fit to SI Appendix , Eq . S21 . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . 8 of 12 https : / / doi . org / 10 . 1073 / pnas . 2208536120 pnas . org per subunit is only ~ 3 . 2 k B T , more than an order of magnitude lower than the 44 k B T subunit \u22121 activation energy for fragmenta - tion ( 15 ) . Although the imposed twist strain energy does not directly overcome the activation energy for filament fragmenta - tion , it does accelerate the filament fragmentation rate constant ~ twofold ( calculated from exp ( E strain / k B T ) ; SI Appendix , Eq . S28 ) ( 15 ) . While this effect may seem small , it accounts for the rapid fragmentation of twisted filaments observed in our experiments when other factors contributing to severing are considered . Two additional factors that contribute to the rapid fragmen - tation of twisted filaments are the number of potential severing sites and the time duration of the applied twisting load . Filament severing occurs at subunit interfaces , so long filaments have more potential fragmentation sites than shorter ones . That is , the severing reaction is a microscopic process , but observed filament severing is a macroscopic process that scales with the filament length , a collective effect happening at individual sub - units . A typical filament in our experiments is > 10 \u00b5m in length ( > 3 , 700 subunits ) , which means the observed , macroscopic severing rate constant is the microscopic severing rate constant times 3 , 700 . In terms of fragmentation probability , if P is the microscopic fragmentation probability expressed in units subunit \u22121 , the macroscopic probability for fragmentation of a filament that comprised n subunits is given by 1\u2212 ( 1\u2212 P ) n ~ nP + 0 ( P 2 ) ( SI Appendix , Eq . S30 ) . The duration ( \u0394 t ) of applied twist deformation is a second crit - ical factor contributing to the observed fragmentation . Twist loads introduce strain energy and accelerate filament fragmentation according to the Arrhenius equation ( SI Appendix , Eq . S28 ) ( 15 ) from which the twofold acceleration is calculated above . However , the filament fragmentation probability ( P ) scales with the fragmen - tation rate constant ( k frag ) and the duration of the applied load ( \u0394 t ) according to P = 1\u2212 exp ( \u2212 k frag \u0394 t ) ( SI Appendix , Eq . S29 ) ( 15 ) . Therefore , fragmentation will not be significantly affected if the duration of the applied twist is short relative to the characteristic severing time ( \u0394 t \u226a 1 / k frag ) . We derived the filament survival probability as a function of time for a given rate of applied twisting strain ( SI Appendix , Eq . S31 ) . Survival curves , as a function of twist den - sity , were simulated according to SI Appendix , Eq . S31 under two different conditions : 1 ) slow rotation ( ~ 0 . 3 rot s \u22121 or 3 . 2 s for 1 rot ) of a 15 - \u03bc m filament , as carried out in our experiments , and 2 ) rapid rotation ( 2 . 2 rot s \u22121 ) of a short filament ( 0 . 1 \u03bc m ) , as previously modeled ( 15 ) ( Fig . 7 A ) . Long filaments with slow rotation break at ~ 2 rot \u03bc m \u22121 ( duration 96 s ) , whereas the short filaments with fast rotation break at 5 rot \u03bc m \u22121 ( duration 0 . 22 s ) . This behavior explains why filaments do not break under thermal twist fluctuation of ~ 1 deg sub \u22121 ( Fig . 7 A ) and suggests that filaments can undergo rather large structural changes with - out fragmenting , provided the duration of these shape changes are short . Fig . 6 . Twisted cofilactin filaments fragment more easily than twisted bare actin filaments . ( A ) Representative images of twist - induced fragmentation for undertwisted ( UT ) bare , overtwisted ( OT ) bare , undertwisted cofilin saturated , and overtwisted cofilin saturated filaments . ( Scale bar , 4 \u00b5m . ) ( B ) Survival analysis from the experiments in ( A ) at a pulling force of 0 . 03 pN . Log - rank test comparing UT bare to UT cofilin ( P < 0 . 0001 ) and OT bare and OT cofilin ( P = 0 . 0094 ) . Both log - rank tests and Gehan \u2013 Breslow \u2013 Wilcoxon tests yielded similar P values , which conclude that the observed twisting response of bare and cofilin - decorated filaments is statistically different . Fig . 7 . Modeling twist - induced fragmentation of actin filaments . ( A ) Experimental actin filament survival curves for undertwisted ( blue ) and overtwisted ( black ) bare actin filaments at 0 . 25 pN pulling force ( 2 \u00b5L min \u22121 flow rate ) and a twisting rate of \u03c9 = 0 . 3 rot s \u22121 . Data include instances where fragmentation occurs close to the bead or surface interfaces . For comparison , the plot includes simulations of filament survival curves as a function of twist density ( SI Appendix , Eq . S31 ) at the same twist rate of 0 . 3 rot s \u22121 with a length of L = 15 \u00b5m ( red trace ) , as that typical in our twist \u2013 extension experiments in this study , and a twist rate of \u03c9 = 2 . 2 rot s \u22121 with a length L = 0 . 1 \u00b5m ( gray trace ) . Inset image is an example of twist - induced fragmentation . Inset graph is the model - predicted filament torque ( SI Appendix , Eq . S9 ) . ( B ) Model - predicted twisting strain energy per subunit ( left y - axis , SI Appendix , Eq . S4 ) and the relative increase in fragmentation rate constant of strained relative to relaxed , native filaments ( right y - axis , SI Appendix , Eq . S28 ) . Dashed lines indicate the model - predicted twisting strain energy for the twist density imposed at boundaries of human cofilin clusters ( blue ) and by singly isolated bound human cofilin ( red ) . D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . PNAS 2023 Vol . 120 No . 4 e2208536120 https : / / doi . org / 10 . 1073 / pnas . 2208536120 9 of 12 Local Twist Strain Drives Filament Severing by Cofilin . The actin filament severing protein , cofilin , binds between two adjacent longitudinal actin subunits and undertwists the filament ~ 4 . 3 deg sub \u22121 ( 54 , 55 ) . The filament twist changes abruptly , within ~ 1 to 2 subunits , at boundaries between bare and cofilin - decorated segments ( 54 , 55 , 60 ) . Filaments preferentially sever at these boundaries ( 45 , 61 , 62 ) within the bare actin side of the boundary ( 60 ) . If we assume that the ~ 4 . 3 - deg twist strain spreads evenly ( 54 ) among the two subunits at the boundary such that each experiences a twist change of ~ 2 . 1 deg , a twist density of ~ 2 . 1 deg sub \u22121 introduces strain energy of ~ 2 . 9 k B T sub \u22121 ( 1 2 L Ts \ud835\udf0e 2 , SI Appendix , Eq . S4 , Fig . 7 B ) . The strain energy of this magnitude is predicted to accelerate the bare actin intrinsic severing rate constant ~ 18 - fold ( exp ( 2 . 9 ) using SI Appendix , Eq . S28 ; Fig . 7 B ) . This value agrees with estimates of a boundary severing rate constant that is ~ 10 to 25 times faster than that of bare actin ( 32 , 35 , 38 ) . It has been reported that boundary severing rate constants vary among cofilin isoforms [ e . g . , Saccharomyces cerevisiae severs more rapidly than human cofilin ( 32 , 35 , 38 , 45 , 61 , 63 , 64 ) ] . Subtle differences in cofilin - induced twist could potentially account for large variability in severing , given the quadratic dependence of the severing rate constant on twist density ( Fig . 7 B ) . We note that the filament twist is constant and does not change within bare and cofilin - decorated segments and therefore does not introduce strain between subunits in those regions as it does at boundaries where twist discontinuities exist ( 62 ) . A singly bound [ i . e . , isolated ( 65 ) ] cofilin also changes the fil - ament twist , although less than at a boundary and only at the one subunit to which it directly binds ( 55 ) . Accordingly , a singly bound cofilin severs filaments but does so more slowly than boundaries ( 32 , 38 ) . Assuming the twist induced by isolated bound cofilin is ~ 1 to 2 deg , the local twist strain should accelerate fragmentation ~ 2 to 18 - fold ( Fig . 7 B ) , consistent with the reported ~ fivefold acceleration ( 32 ) . Cofilin Renders Twisted Actin Filaments Brittle . Cofilactin filaments ( i . e . , saturated with cofilin ) break at lower twist densities than bare actin filaments ( Fig . 6 ) . Several factors can potentially contribute to this mechanical instability . A twisted cofilactin filament could store more elastic strain energy than a bare filament for a given twist load , thereby resulting in more rapid fragmentation . While conceivable , cofilactin filaments are thought to be more compliant in bending ( 16 , 19 , 34 , 42 ) and twisting ( 40 , 42 ) than bare actin filaments so they have less strain energy for an identical shape deformation . It is also possible that cofilactin filaments are more fragile and fragment more easily than bare actin filaments . While this is also conceivable , cofilin bridging interactions stabilize cofi - lactin filaments and protect them from fragmentation ( 15 , 60 ) . These bridging interactions render fully decorated cofilactin filaments comparably stable to bare actin filaments ( 61 , 62 , 65 \u2013 68 ) . A third more likely possibility is that cofilin dissociation from actin filaments transiently introduces a boundary , which frag - ments more easily under twisting ( and bending ) loads ( 15 , 19 ) . Spontaneous cofilin dissociation can introduce this effect but it would be more prominent if twisting loads weakened cofilin binding and / or accelerated cofilin dissociation , as predicted from modeling studies ( 19 ) . Pulling Accelerates Cofilactin Filament Fragmentation . Surface tethering and cross - linking constrain filament bending and twisting , which prevents the dissipation of cofilin - induced torsional strain , thereby enhancing cofilin severing activity ( 15 , 19 , 22 , 67 ) . Long - axis pulling forces on actin filaments , of magnitude comparable with those exerted by myosin motors [ 3 to 5 pN per ATP hydrolyzed ( 69 ) ] , also dampen thermally driven ( Fig . 2 ) and twist - induced filament bending ( Fig . 3 ) and dramatically accelerate filament fragmentation . This behavior supports models in which contractile forces gen - erated by myosin motors rapidly sever twisted filaments such as those with bound cofilin ( 15 , 19 , 28 ) . Recent studies show that contractile forces produced by myosin motors \u201ccatalyze\u201d cofil - in - mediated actin filament disassembly and turnover in Aplysia neurons ( 70 ) and also contribute to actin filament turnover during contractile ring constriction in S . pombe ( 71 ) , demonstrating how combinations of pulling , bending , and twisting forces can dra - matically accelerate actin filament network fragmentation and turnover in cells . Twisting a Filament Bundle . With the torsional persistence length measured in this study , it is possible to make predictions about the behavior of bundled actin filaments . We can model a perfectly cross - linked actin filament bundle as a filament with different dimensions such that the radius of the bundle cross - section ( R ) is determined by the radius of a single actin filament ( r ) and the number ( n ) of filaments comprising the bundle . The area of the cross - section of the bundle is the sum of the cross - sectional areas of each filament forming the bundle ( i . e . , \u03c0 R 2 = n \u03c0 r 2 ) . Therefore , R 2 = nr 2 , and the bundle\u2019s torsional and bending persistence lengths become L T , bundle = G ( \u03c0 R 4 / 2 ) / k B T = n 2 G ( \u03c0 r 4 / 2 ) / k B T = n 2 L T and L B , bundle = E ( \u03c0 R 4 / 4 ) / k B T = n 2 E ( \u03c0 r 4 / 4 ) / k B T = n 2 L B , where G is the shear modulus , and E is the Young\u2019s modulus of an actin filament , respectively . Therefore , a filament bundle\u2019s torsional and bending persistence lengths and corresponding strain energies scale with n 2 . This suggests that twisted bundles are a result of very large applied torques as twisting a three - filament bundle requires nine times as much torque to twist compared with a single filament . The authors of this study ( 12 ) concluded that these torsional loads on actin bundles in filopodia are driven by myosin contractility , indicating the off - axis torques generated by myosin motors ( 72 \u2013 74 ) are sufficiently strong to twist filament bundles . Materials and Methods A brief description of the experimental materials and methods used is provided here , and for more details , see SI Appendix . Protein Purification . Actin was purified from rabbit skeletal muscle and labeled on surface lysines with NHS ester derivatives of Alexa 488 , Alexa 647 , biotin , or digoxigenin ( 17 ) . Alexa 488 phalloidin was purchased from Thermo Fisher ( catalog # A12379 ) . Ca 2 + - actin monomers ( 5 \u00b5M ) were converted to Mg 2 + - actin by addition of 50 \u00b5M MgCl 2 and 0 . 2 mM EGTA and equilibrated for 5 min on ice immediately before use ( 75 ) . Saccharomyces cerevisiae cofilin with a surface - en - gineered cysteine was purified and labeled with Alexa 488 ( 61 ) . Microscope Sample Preparation . Surface functionalization and passiva - tion of microscope coverslips with 2 to 5 % Biotin - labeled PEG - Silane slides were adapted from elsewhere ( 76 ) . Microfluidic chambers were assembled as described ( 17 , 77 ) . Superparamagnetic Dynabeads\u2122 M - 270 Epoxy ( 2 . 8 \u00b5m in diameter , Thermo Fisher catalog # 14301 ) were conjugated to antidigoxigenin antibody following the company - provided protocol . Samples were prepared and experiments carried out in KMI buffer ( 10 mM fluorescence - grade imidazole pH 7 . 0 , 50 mM KCl , 2 mM MgCl 2 , 0 . 2 mM ATP , and 2 mM DTT ) supplemented with 15 mM glucose , 0 . 02 mg mL \u22121 catalase , and 0 . 1 mg mL \u22121 glucose oxidase . Actin polymerization was done as previously D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . 10 of 12 https : / / doi . org / 10 . 1073 / pnas . 2208536120 pnas . org described ( 65 ) . Filament \u2013 bead conjugation in sample chambers is described in detail in SI Appendix . Microscopy . Imaging was conducted on a Till iMic digital total internal reflection fluorescence ( TIRF ) microscope equipped with a 100\u00d7 objective ( Olympus ) and an Andor iXon897 electron - multiplying charge - coupled device ( EMCCD ) camera ( 17 , 33 , 38 ) . Filament Force \u2013 Extension . In the force \u2013 extension experiments without twist , filament end \u2010 to \u2010 end length ( R ) was measured as a function of the ten - sile force ( f ) applied with buffer flow . Images were recorded at each unique buffer flow ( tensile force ) with an acquisition rate of 1 frame s \u22121 . The positions of the paramagnetic beads were tracked with the TrackMate plugin in ImageJ ( NIH , USA ) ( 78 ) . The value of R was determined as the direct straight - line distance between the two attachment points at the bead and surface . A fila - ment contour length ( L ) was measured as R at a high flow rate to make the filament straight . The measured force dependence of R ( i . e . , force \u2013 extension curve ) was fitted to the following equation describing the force \u2013 extension behavior of semiflexible polymers ( 6 , 47 ) : [ 1 ] R = L \u2212\u0394 L = L \u239b\u239c\u239c\u239d 1 \u2212 1 2 \ufffd k B T L B f \u239b\u239c\u239c\u239d coth \ufffd L \ufffd f L B k B T \ufffd \u2212 1 L \ufffd L B k B T f \u239e\u239f\u239f\u23a0\u239e\u239f\u239f\u23a0 = \u23a7\u23aa\u23aa\u23a8\u23aa\u23aa\u23a9 L \u2192 0 \u2192 L L \u2192 \u221e \u2192 L \u239b\u239c\u239c\u239d 1 \u2212 1 2 \ufffd k B T L B f \u239e\u239f\u239f\u23a0 , where L B is the filament bending persistence length , k B is Boltzmann con - stant , and T is the room temperature ( 296 K ) . \u0394 L is the deviation of R from L of a semiflexible polymer due to thermally driven random bending , and it does not scale with L linearly . For the polymers with very short L , \u0394 L ~ 0 , the deviation per L is negligible and R ~ L , whereas with very long L , the deviation of R from L reaches the maximum per unit length 1 2 \u221a k B T L B f . This nonlinear L dependence of \u0394 L is consistent with the conclusion by another study ( 79 ) , which claims a popular force \u2013 extension equation ( 80 ) with \u0394 L L = const is overly simplified . Using Origin software ( Originlab , Northampton , MA , USA ) , experimental rep - licates were globally fitted with L B as a global fitting parameter and L unique to each individual filament dataset ( Fig . 2 B ) . In addition to globally fitting individual filament force \u2013 extension curves , we compiled and fitted data for filaments of different lengths by averaging the nor - malized filament end \u2010 to - end length ( R / L ) , which is given as follows : [ 2 ] \ufffd R L \ufffd = 1 n n \ufffd i = 1 R i L i = 1 \u2212 1 2 \ufffd k B T L B f \u239b \u239c\u239c \u239c \u239d 1 + 2 n n \ufffd i = 1 e \u2212 2 L i \ufffd f LBkBT 1 \u2212 e \u2212 2 L i \ufffd f LBkBT \u2212 \ufffd L B k B T f 1 n n \ufffd i = 1 1 L i \u239e\u239f \u239f \u23a0 , \u223c 1 \u2212 1 2 \u221a k B T L B f + k B T 2 f ( 1 L ) , because the term 2 e \u2212 2 L i \u221a f LBkBT 1 \u2212 e \u2212 2 L i \u221a f LBkBT is < 0 . 08 and can be ignored since it is \u226a 1 and more than 1 order of magnitude smaller than \u221a L B k B T f 1 L i in our experimental force and filament length ranges . Filament Twist \u2013 Extension . Filament images in the twist \u2013 extension exper - iments were recorded with an acquisition rate of 1 frame s \u22121 while twisting filament at a constant rate of 0 . 31 rot s \u22121 and applying a given tensile force ( f ) with hydrodynamic flow . The position of the paramagnetic beads was tracked with the same procedure , and filament end \u2010 to \u2010 end distance ( R ) and contour length ( L ) were determined in the same manner as those in the preceding Force \u2013 Extension section . R / L is a function of twist density , \u03c3 ( unit : rot \u03bc m \u22121 ) , and L . Filament contour lengths in our experiments range from 7 to 20 \u03bc m . Normalized R / L data were averaged by binning according to the filament twist density ( rot \u03bc m \u22121 ) with a bin size of 0 . 025 rot \u00b5m \u22121 . To describe the twist \u2013 extension behavior of actin filaments , an analytical two - state model developed for DNA ( 52 ) was modified for actin filaments . In the model , it is assumed that a filament subunit exists in one of two states when a filament is twisted : plectonemic or nonplectonemic ( linear ) . A filament with a plectoneme consists of a mixture of plectonemic and nonplectonemic regions . The fraction of plectonemic regions increases linearly with twist density and does not contribute to R / L . Eq . 3 ( SI Appendix , Eq . S10 ) gives the force depend - ence of R / L contributed from only the fraction in the nonplectonemic state ( 52 ) ( SI Appendix ) as follows : [ 3 ] R L = \u23a7\u23aa\u23aa\u23aa\u23aa\u23aa\u23aa\u23aa\u23aa\u23a8\u23aa\u23aa\u23aa\u23aa\u23aa\u23aa\u23aa\u23aa\u23a9 1 \u2212 1 2 \ufffd k B T L B f + k B T 2 f \ufffd 1 L \ufffd \u2212 \ud835\udf14 02 \ud835\udf0e 2 \ufffd 4 L B L T \ufffd fL B k B T + 1 \ufffd 2 \ufffd k B TL B 3 f , \ufffd \ud835\udf0e \ufffd < \ufffd\ufffd \ud835\udf0e s \ufffd\ufffd \ufffd\ufffd\ufffd \ud835\udf0e p \ufffd\ufffd\ufffd \u2212 \ufffd \ud835\udf0e \ufffd \ufffd\ufffd\ufffd \ud835\udf0e p \ufffd\ufffd\ufffd \u2212 \ufffd\ufffd \ud835\udf0e s \ufffd\ufffd \u239b\u239c\u239c\u239d 1 \u2212 1 2 \ufffd k B T L B f + k B T 2 f \ufffd 1 L \ufffd \u2212 \ud835\udf14 02 \ud835\udf0e s 2 \ufffd 4 L B L T \ufffd fL B k B T + 1 \ufffd 2 \ufffd k B TL B 3 f \u239e\u239f\u239f\u239f\u23a0 , \ufffd\ufffd \ud835\udf0e s \ufffd\ufffd \u2264 \ufffd \ud835\udf0e \ufffd < \ufffd\ufffd \ud835\udf0e P \ufffd\ufffd 0 , \ufffd\ufffd\ufffd \ud835\udf0e p \ufffd\ufffd\ufffd \u2264 \ufffd \ud835\udf0e \ufffd . In the equation , \u03c3 s is a special applied twist density at which filaments form plectonemes , and \u03c3 p is applied twist density when the entire filament is plec - tonemic . The fraction of subunits in the nonplectonemic conformation is given by | | | \ud835\udf0e p | | | \u2212 | \ud835\udf0e | | | | \ud835\udf0e p | | | \u2212 | | \ud835\udf0e s | | . A custom routine was written with Origin software to globally fit the exper - imental data of R / L as a function of applied twist at different pulling forces to Eq . 3 ( Fig . 3 ) with L B and L T as shared unconstrained parameters . \u03c3 s for pulling force of 0 . 01 and 0 . 03 pN were fixed parameters from averaging experimentally observed values , but the other \u03c3 s for pulling force of 0 . 25 pN and value of \u03c3 p for all forces were also unconstrained during the fitting procedure but were not shared because they depend on and vary with the pulling force . The value of ( 1 L ) was constrained during fitting to the values calculated from the actual filament contour lengths measured under a high pulling force ( to straighten ) . Design of Freely Orbiting Magnetic Tweezers . The rotational fluctuations of actin filaments were measured with freely orbiting magnetic tweezers ( 57 ) . Five cylindrical magnets ( R422 - N52 , K & J Magnetics ) arranged in series were mounted on a linear XY micrometer stage ( XR25 , Thorlabs ) and centered directly above the microscope objective while visualizing with wide - field microscopy . The procedure for conjugating antidigoxigenin paramagnetic beads to digoxigenin marker beads for these experiments is given in detail in SI Appendix . Analysis of Filament Rotational Fluctuations . In whole filament twisting fluctu - ation experiments ( Fig . 4 A ) , we monitor how the angle \u03b8 ( t ) between the two filament D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 . PNAS 2023 Vol . 120 No . 4 e2208536120 https : / / doi . org / 10 . 1073 / pnas . 2208536120 11 of 12 attachment points ( at the surface and on the bead , length L ) changes over time , and the variance ( \u03c3 \u03b8 2 ) of the angle fluctuation is given by Eq . 4 ( SI Appendix , Eq . S22 ) : [ 4 ] \ud835\udf0e 2 \ud835\udf03 = < ( \ud835\udf03 ( t ) \u2212 < \ud835\udf03 ( t ) > ) 2 > t \u2192 \u221e = L L T + \ud835\udf0e 2 \ud835\udf00 \u223c L L T . Here , \u03c3 \u03b5 2 is the variance of the uniform randomly distributed noise in a relative twist angle between two adjacent filament subunits , and we neglect it because it is much smaller than L / L T ( SI Appendix ) . Eq . 4 indicates that at times sufficiently long to sample the equilibrium distribution , indicated by plateau of the variance ( Fig . 4 E ) , the variance of the angle fluctuations at equilibrium is inversely proportional to L T , and scales with L linearly ( SI Appendix , Fig . S6 ) , which has been demonstrated for actin filaments by experimental observation ( 20 , 39 ) . Analysis of Filament Subunit Angular Fluctuations from cryo - EM Data . From the electron micrographs of actin filaments , which were classified as described previously ( 54 ) , we defined \u0394 s as the length of an actin subunit and \u03a6 as the rotational Euler angle around the filament centerline . Any rotation angle difference ( twist ) between two subunits spaced n subunits apart ( SI Appendix , Eq . S20 ) was calculated by the sum of all the observed relative twists between 2 adjacent subunits and it displays a Gaussian distribution with a variance that scales linearly with n ( SI Appendix , Eq . S21 ) . Fitting SI Appendix , Eq . S21 to the n dependence of the observed variance ( \ud835\udf0e 2 obs , n + 1 ) yields \u0394 s / L T from the slope of the best linear fit of the data and the noise variance ( \ud835\udf0e 2 \ud835\udf00 ) from the y intercept . The variance of the angle distribution was extracted from the data using three different analysis procedures . In one approach , the histogram of \u0394 \u03a6 I , obs , n + 1 for every value of n was independently fitted to a normal distribution , yielding \u03c3 2 i , obs , n + 1 . Then , \u03c3 2 i , obs , n + 1 as a function of n was fitted to SI Appendix , Eq . S21 . In addition , in maximum likelihood estimation ( MLE ) , each variance with spacing of n was directly calculated from the square of the SD as follows : [ 5 ] \ud835\udf0e 2 obs , n + 1 = \u27e8\ufffd \u0394\ufffd i , obs , n + 1 \u2212 \u27e8 \u0394 \ud835\udf03 obs , n + 1 \u27e9\ufffd 2 \u27e9 . The n dependence of \u03c3 2 i , obs , n + 1 was then fitted to SI Appendix , Eq . S21 . In the third method , we applied Bayesian inference using Markov chain Monte Carlo ( MCMC ; Metropolis \u2013 Hastings algorithm ) to sample the pos - terior probability distribution of the true variance ( 81 ) . The method was coded in language R ( www . r - project . org ) , and the most likely L T and \ud835\udf0e 2 \ud835\udf00 values were determined from the peaks of their probability distributions . These three methods of analyses were repeated for the rotation Euler angle \u03a6 determined from a refinement volume of 1 subunit instead of 5 with comparable results ( Table 1 ) ( 54 , 55 ) . 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D o w n l o a d e d fr o m h tt p s : / / www . pn a s . o r g by \" UN I V E R S I T Y O F W A S H I NG T ON L I BR A R I E S , A RC S - S E R I A L S \" on M a r c h 13 , 2024 fr o m I P a dd r e ss 205 . 175 . 106 . 171 .", + "lundmark2008gtpaseactivating": "Report The GTPase - Activating Protein GRAF1 Regulates the CLIC / GEEC Endocytic Pathway Richard Lundmark , 1 , 3 , 4 , * Gary J . Doherty , 1 , 3 Mark T . Howes , 2 Katia Cortese , 2 Yvonne Vallis , 1 Robert G . Parton , 2 and Harvey T . McMahon 1 , * 1 Medical Research Council Laboratory of Molecular Biology Hills Road Cambridge , CB2 0QH UK 2 Institute for Molecular Bioscience and Centre for Microscopy and Microanalysis University of Queensland Brisbane , Queensland 4072 Australia Summary Clathrin - independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [ 1 , 2 ] . Electron - microscopy studies have de\ufb01ned the pleio - morphic CL athrin - I ndependent C arriers ( CLICs ) and G PI - E nriched E ndocytic C ompartments ( GEECs ) as related ma - jor players in such uptake [ 3 , 4 ] . This CLIC / GEEC pathway relies upon cellular signaling and activation through small G proteins , but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking . Here we show that the Rho - GAP - domain - containing protein GRAF1 marks , and is indispensable for , a major Clathrin - indepen - dent endocytic pathway . This pathway is characterized by its ability to internalize bacterial exotoxins , GPI - linked pro - teins , and extracellular \ufb02uid . We show that GRAF1 localizes to PtdIns ( 4 , 5 ) P2 - enriched , tubular , and punctate lipid struc - tures via N - terminal BAR and PH domains . These membrane carriers are relatively devoid of caveolin1 and \ufb02otillin1 but are associated with activity of the small G protein Cdc42 . This study provides the \ufb01rst speci\ufb01c noncargo marker for CLIC / GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and mem - brane remodeling to facilitate internalization of CLIC / GEEC pathway cargoes . Results and Discussion The protein G TPase R egulator A ssociated with F ocal Adhe - sion Kinase - 1 ( GRAF1 ) is predicted to comprise an N - terminal BAR domain , a PH domain , a RhoGAP domain , a proline - rich domain , and a C - terminal SH3 domain ( Figure 1A ) . GRAF1 ex - hibits GAP activity for the small G proteins RhoA and Cdc42 and has been shown to interact with the kinases FAK and PKN b [ 5 , 6 , 14 ] . The presence of a predicted BAR domain in GRAF1 suggests that it might function in membrane sculpting [ 7 ] . GRAF1 was found to be expressed in a variety of cell lines ( Figure S1A ) , and by immunocytochemistry we found that GRAF1 was predominantly localized to pleiomorphic tubular and punctate structures in HeLa and NIH 3T3 cells ( Figures 1B \u2013 1D and Figure S1B ) . Although GRAF1 can be found primar - ily on long tubules in some cells , other cells within the same population exhibit a predominantly punctate GRAF1 localiza - tion . GRAF1 - positive tubules are disrupted at low tempera - tures , and a 37 (cid:2) C \ufb01xation was required for their integrity to be preserved ( Figure S1C ) . Upon monitoring both GRAF1 lo - calization and endocytosis of either the plasma membrane marker DiI or the \ufb02uid - phase marker dextran by confocal mi - croscopy , we found that endocytic structures extensively co - localized with GRAF1 ( Figures 1B and 1C ) . Colocalization of GRAF1 was even evident when only very early endocytic struc - tures were examined , indicative of an early endocytic role for GRAF1 - positive membranes ( Figure 1D ) . We then examined the turnover of GRAF1 - positive membranes with time by over - expressing GFP - tagged full - length GRAF1 in HeLa cells and examining its localization by using four - dimensional spin - ning - disc confocal microscopy . We found GRAF1 - positive tu - bular structures to be spectacularly dynamic ( Figure 1E and Movie S1 ) . By electron microscopy , we found that GRAF1 - labeled tubules were around 40 nm in diameter in vivo ( Figure 1G ) . We also found that overexpressed GFP - tagged GRAF1 BAR + PH protein ( missing the GAP , proline - rich , and SH3 domains ) also labeled tubular membranes that could accumulate DiI ( Figure 1F , Movie S2 , and data not shown ) . However , these tubules were much more static than those la - beled by overexpressed full - length GRAF1 , suggesting that GRAF1 BAR + PH might act in a dominant - negative manner to stabilize early endocytic tubules ( see also below ) . To examine the speci\ufb01c properties of the predicted lipid - binding region of GRAF1 , we performed lipid cosedimentation assays with puri\ufb01ed GRAF1 BAR + PH protein ( Figure 2A ) . Us - ing liposomes of varying diameter , we found that GRAF1 BAR + PH bound better to smaller ( more highly curved ) lipo - somes , consistent with the presence of a membrane - curva - ture - sensing BAR domain [ 7 ] . Furthermore , we examined the effects of mutating key lysine residues [ 7 ] in the BAR domain to glutamates ( KK131 / 132EE ) and found that this mutant was now cytoplasmically distributed ( Figure S1D ) . Expression of the BAR domain alone in cells resulted in a predominantly cy - toplasmic and sometimes punctate localization ( Figure S1E ) , suggesting that both the BAR and PH domains are necessary for tubular localization of GRAF1 . We therefore tested whether , in addition to a curvature - sensing or - generating capability , the GRAF1 BAR + PH unit has phosphoinositide - binding speci\ufb01city . GRAF1 BAR + PH protein was subjected to lipid cosedimentation assays with 10 % phosphatidylserine - con - taining liposomes with varying phosphoinositide composition ( Figure 2B ) . Greatest binding was observed for PtdIns ( 4 , 5 ) P2 - enriched liposomes . PtdIns ( 4 , 5 ) P2 is plasma - membrane en - riched [ 8 ] , consistent with our observations that GRAF1 - asso - ciated traf\ufb01cking occurs from this site . GRAF1 BAR + PH protein was also capable of generating tubules in vitro from spherical liposomes as examined by electron microscopy * Correspondence : richard . lundmark @ medchem . umu . se ( R . L . ) , hmm @ mrc - lmb . cam . ac . uk ( H . T . M . ) 3 These authors contributed equally to this work 4 Present address : Department of Medical Biochemistry and Biophysics , Umea\u02da University , 901 87 Umea\u02da , Sweden Current Biology 18 , 1802 \u2013 1808 , November 25 , 2008 \u00aa 2008 Elsevier Ltd . Open access under CC BY license . DOI 10 . 1016 / j . cub . 2008 . 10 . 044 under negative - staining conditions ( Figure 2C ) . The diameter of these tubules was around 40 nm , consistent with the ob - served diameter of GRAF1 - positive tubules found in cells ( Figure 1G ) . Taken together , these data strongly suggest that the BAR and PH domains of GRAF1 function together to pro - duce and / or stabilize endocytic tubules in vivo . To identify interacting partners for GRAF1 , we immunopre - cipitated GRAF1 from rat brain cytosol . Interestingly , GRAF1 was found in a complex with the membrane scission protein Dynamin1 ( Figure 2D and S2A ) . We con\ufb01rmed binding to both Dynamin1 and Dynamin2 by using GST - tagged GRAF1 SH3 domain as bait in pull - down experiments against puri\ufb01ed Dynamin1 , brain cytosol , or HeLa cell lysate ( Figure 2E ; also Figures S2B and S2C ) . Monomeric GRAF1 SH3 domain was found to bind to a peptide from Dynamin1 proline - rich domain with a k d of 106 m M ( Figure S2D ) . Furthermore , treatment of HeLa cells with dynasore resulted in a profound reduction of tubular endocytosis in HeLa cells and a redistribution of GRAF1 to basally located puncta ( Figure S2E ) . These data , taken together with our observation that C - terminally trun - cated versions of GRAF1 ( lacking the SH3 domain ) are found on long static tubules , suggests that the complex between Dynamin and GRAF1 might function to regulate the scission and stability of these ordinarily pleiomorphic tubulovesicular structures . Clathrin polymers are rarely found on tubular membranes by electron microscopical techniques . Indeed , GRAF1 - positive tubules , and other GRAF1 - positive structures , were devoid of Clathrin and did not colocalize with internalized or internal - izing transferrin or the transferrin receptor , which is widely used as a marker for Clathrin - mediated endocytic events ( Fig - ures S3A \u2013 S3C ) . Alongside the canonical Clathrin - mediated en - docytic routes , other prevalent endocytic pathways coexist [ 1 , 2 ] . For example , the internalization of MHC class I proteins , many GPI - linked receptors , and bacterial exotoxins rely on traf\ufb01cking compartments with heretofore unde\ufb01ned coat com - ponents . The data presented above strongly suggest that GRAF1 might function as a coat within a prevalent Clathrin - independent endocytic pathway . To characterize further the nature of GRAF1 - positive tubules , we incubated GRAF1 - over - expressing HeLa cells with cholera toxin B subunit ( CTxB ) , a marker that is used for the study of Clathrin - independent en - docytic pathways but that also enters via Clathrin - mediated routes [ 9 ] . We found that internalized CTxB colocalized with Myc - GRAF1 - positive tubules in HeLa cells ( Figure 2F ) as well as with endogenous GRAF1 - positive structures after 5 min in - cubation at 37 (cid:2) C ( Figure 2G ) . In addition , we found that GRAF1 BAR + PH overexpression ( which leads to static tubules ; Figure 1F ) halved the number of cells internalizing CTxB with - out effecting transferrin uptake ( Figures S4A \u2013 S4C ) . We then followed CTxB and transferrin internalization in real time in NIH 3T3 cells ( almost all of which are capable of binding and internalizing CTxB ; cf . HeLa cells , which often lack its glyco - lipid receptor , GM1 ) . NIH 3T3 cells were transfected with constructs encoding either GFP - tagged GRAF1 or GRAF1 BAR + PH and incubated with CTxB and transferrin on ice be - fore stimulation of endocytosis by incubation of cells with pre - warmed ( 37 (cid:2) C ) media . Imaging commenced immediately ( Figure S4D and Movie S3 ) . Newly forming dynamic GRAF1 - positive tubules were shown to contain CTxB at extremely early stages of its internalization and were unassociated with internalizing / internalized transferrin . Furthermore , the more static , elongated GRAF1 BAR + PH - positive tubules maintained the presence of CTxB , again independently of transferrin , suggesting that here the toxin is trapped in structures that can - not undergo \ufb01ssion from the plasma membrane and progress further ( data not shown ) . Although GRAF1 - positive carriers resemble the previously characterized Arf6 - dependent structures that are responsible for uptake of MHC Class I proteins [ 10 ] , we found no evidence for overlap between these pathways . Neither GRAF1 - nor GRAF1 BAR + PH - positive tubules were found to contain endo - cytosed or recycled MHC class I proteins at 5 , 15 , or 45 min af - ter stimulation of their internalization when speci\ufb01c antibodies were used , and the amount of total MHC class I protein endo - cytosis was not altered upon overexpression of the dominant - negative GRAF1 BAR + PH construct ( Figures S6A and S6B and data not shown ) . In addition , the tubular localization of GRAF1 was independent of overexpression of wild - type Arf6 or dom - inant - active Arf6 ( Arf6Q67L ; data not shown ) . In contrast , Arf6 Q67L overexpression was found to completely block the inter - nalization of MHC class I molecules ( [ 11 ] and data not shown ) . Electron - microscopy studies , focusing on the uptake of CTxB and GPI - linked receptors , have suggested that a large portion of these proteins are internalized by Cl athrin - i ndepen - dent c arriers ( CLICs ) seemingly into G PI - AP - enriched e arly e n - dosomal c ompartments ( GEECs ) , together with \ufb02uid - phase markers such as dextran [ 4 ] . Our data have shown that GRAF1 - positive structures morphologically and functionally resemble the endocytic structures of this CLIC / GEEC endo - cytic pathway [ 4 ] . We therefore examined whether GRAF1 could regulate the internalization of a model GPI - linked protein known to undergo endocytosis into the CLIC / GEEC pathway . Consistent with this , we found by using immuno - electron microscopy that GRAF1 labeled GFP - GPI - positive tubules ( Figure 3A ) . We then examined the internalization of GFP - GPI in mouse embryonic \ufb01broblasts by binding anti - GFP anti - bodies and transferrin ( as a control ) to cells on ice and subse - quently moving these cells to 37 (cid:2) C for variable chase periods . We did this in both wild - type ( data not shown ) and caveolin1 - knockout mouse embryonic \ufb01broblasts ( to exclude caveolae - associated uptake ) that we transfected with either a Myc - tagged GRAF1 or a Myc - tagged GRAF1 BAR + PH construct and examined after cytosol washout ( Figure 3B ; note that washout treatment damages GRAF1 - positive membranes but is necessary for the objective quantitative analysis of co - localization of GRAF1 with internalized GFP - GPI because cy - toplasmic GRAF1 would otherwise result in overestimation of colocalization ) . GRAF1 was found to signi\ufb01cantly colocalize with internalized GFP - GPI ( 79 % colocalization ) but not trans - ferrin after a 2 min chase ( Figures 3B and 3C and data not shown ) . A reduced level of colocalization was seen after a 10 min chase ( 47 % ) , and even lower colocalization levels were observed after a 40 min chase ( when the protein is recy - cling back to the plasma membrane ; 23 % ) . These data are consistent with our previous observations , strongly suggest - ing a role for GRAF1 function in sculpting the highly curved membranes of the CLIC / GEEC endocytic pathway . In contrast , GRAF1 BAR + PH consistently colocalized with GFP - GPI after 2 min ( 60 % ) , 10 min ( 68 % ) , and 40 min ( 75 % ) chase periods , further supporting a role for this protein as a dominant - negative protein that traps early CLIC / GEEC carriers . To char - acterize this dominant - negative effect in greater detail , we examined the effect of GRAF1 BAR + PH protein on GFP - GPI in - ternalization ( Figures 3B and 3C ) . Compared with GFP - GPI in - ternalization in the presence of GRAF1 , the total amount of GFP - GPI internalization was profoundly reduced in the pres - ence of GRAF1 BAR + PH ( to around 10 % of control levels ) , Membrane Sculpting in Clathrin - Independent Endocytosis 1803 Figure 1 . GRAF1 Tubules Are Highly Dynamic and Mark a Prevalent Endocytic Pathway ( A ) Domain architecture of GRAF1 and the site of introduced BAR domain mutations ( * ) . ( B \u2013 D ) Micrographs showing that GRAF1 - positive tubules are derived from the plasma membrane as shown by colabeling with the membrane dye DiI after 5 min ( B ) or internalized dextran after either 5 min ( C ) or 1 min ( D ) of incubation . ( E ) Overlaid maximum projections of spinning - disc confocal micrographs of HeLa cells expressing GFP - tagged GRAF1 demonstrate that GRAF1 - positive tubules are completely turned over in 10 min . Z sections were performed continuously for 10 min in 0 . 5 m m steps . The initial maximum projection ( in green ) was then merged with the \ufb01nal maximum projection ( in red ) . See also Movie S1 . ( F ) Overlaid maximum projections as in ( a ) , but for a cell overexpressing GFP - tagged GRAF1 BAR + PH . See also Movie S2 . Current Biology Vol 18 No 22 1804 ( G ) Electron micrographs prepared as described and immunolabeled for GRAF1 ( 10 nm gold particles ) . Note the GRAF1 - positive tubular structures . Dis - tance between arrowhead tips = 40 nm . Scale bars represent 10 m m . Figure 2 . The BAR and PH Domains Localize GRAF1 to Highly Curved , PtdIns ( 4 , 5 ) P2 - Enriched Membranes , and the SH3 Domain Binds Dynamin ( A ) Coomassie - stained gel of liposome cosedimentationassay showing the preference of GST - tagged GRAF1 BAR + PH protein for binding to smaller - sized liposomes derived from total brain lipids ( the average diameters of liposomes are shown ) . Pellet ( P ) and supernatant ( S ) fractions were separated by ultra - centrifugation . The graph shows quanti\ufb01cations of total band intensities for each condition normalized to binding of the non - curvature - sensitive protein Dab2 ( as a way of controlling for total lipid in each experiment ) . The error bars show 95 % con\ufb01dence intervals ( calculated by t tests ) for each condition . ( B ) Liposome cosedimentation assay as performed in ( A ) but with 0 . 8 - m m - diameter liposomes enriched with varying phosphoinositides . ( C ) Electron micrographs of negatively stained liposomes incubated in the presence or absence of GST - tagged GRAF1 BAR + PH protein . Note the protein - dependent presence of tubular structures . The scale bar represents 200 nm . ( D ) Immunoprecipitation of GRAF1 from rat brain cytosol ( via Ab2 ) reveals a GRAF1 - Dynamin1 complex as identi\ufb01ed by mass spectrometry and con\ufb01rmed by immunoblot . ( E ) Coomassie - stained gels of pull - down experiments with puri\ufb01ed Dynamin and either bead - bound GST - tagged GRAF1 / Amphiphysin SH3 domain or GRAF1 BAR + PH protein . P = pellet fraction , S = supernatant fraction . ( F and G ) Epi\ufb02uorescence micrographs of HeLa cell overexpressing Myc - tagged GRAF1 and incubated with CTxB for 5 min before \ufb01xation and staining . ( G ) Epi\ufb02uorescencemicrographsofaHeLacellincubatedwithCTxBfor5minbefore\ufb01xationandstainingforendogenousGRAF1 . Scalebarsrepresent10 m m . Membrane Sculpting in Clathrin - Independent Endocytosis 1805 Figure 3 . GRAF1 Regulates the CLIC Endocytic Pathway ( A ) Representativeelectron micrographsof 65nm ultrathin cryosections of HeLa cellstransientlytransfectedwith GFP - GPI aloneorwithboth GFP - GPI and Myc - tagged GRAF1 FL . Cells were \ufb01xed in 2 % PFA with 0 . 2 % glutaraldehyde and labeled with anti - GFP and anti - Myc antibodies . Protein A 10 nm gold was used for revealing the GFP in the single labeling ( upper image ) . As shown , GFP - GPI was found in uncoated vesicles and tubules within the cell . Double la - beling of GRAF1 ( Protein A gold 10 nm ) and GFP - GPI ( Protein A gold 15 nm ) is shown in the bottom image , where colocalization of GRAF1 and GFP - GPI is seen in an intracellular tubule . Arrowheads point to 10 nm gold particles . Due to limitations of the Tokuyasu method , it is not possible to discriminate cell surface - connected tubules from intracellular tubules . ( B ) Caveolin1knockoutMEFswere cotransfected withGFP - GPI ( green ) andeitherMyc - tagged GRAF1 ( upper row ; red ) orGRAF1 BAR + PH ( lower row ; red ) . GFP antibodies ( blue ) were bound to cells for 30 min on ice prior to induction of internalization at 37 (cid:2) C and 5 % CO 2 for the indicated times . Surface labeling was removed , and cytosol extraction followed ( see Supplemental Experimental Procedures ) . Panels on the left side of each image show GRAF1 proteins ( top ) , internalized anti - GFP ( middle ) , and a merged , triple - labeled image ( bottom ) . Arrows indicate points of colocalization . ( C ) MicrographsofCaveolin1knockoutMEFsco - overexpressingGRAF1andGFP - GPI , afteranti - GFPinternalization , acidstripping , andcytosolextraction , were processed with Volocity 3 . 7 . 0 so that the percentage colocalization could be calculated . The histogram shows the number of anti - GFP pixels that co - localize with GRAF1 relative to all anti - GFP pixels . Five to seven images across three independent experiments were taken after 2 , 10 , and 40 min of inter - nalization . Error bars indicate the standard errors of the mean . Images were also processed in Adobe Photoshop CS2 so that the number of anti - GFPpixels relative to total pixels within the image could be calculated . The histogram represents standardized values of anti - GFP pixels . Error bars indicate the stan - dard errors of the mean . Current Biology Vol 18 No 22 1806 suggesting further that this protein traps cargoes in rather static early endocytic carriers that are incapable of undergoing \ufb01ssion from the plasma membrane . Importantly , the steady - state surface levels of GFP - GPI were indistinguishable be - tween cells expressing GRAF1 and those expressing GRAF1 BAR + PH ( Figure S6C ) . CTxB has been shown to be endocytosed via caveolin and \ufb02otillin - positive structures [ 12 ] . Although we did not \ufb01nd cav - eolin1 or \ufb02otillin1 in GRAF1 - positive structures at steady state by \ufb02uorescence microscopy ( Figures S5A and S5B ) , stabiliza - tion of early GRAF1 - positive carriers by GRAF1 BAR + PH over - expression caused \ufb02otillin1 , and to a lesser extent caveolin1 , to be found in such regions with CTxB ( Figures S5C and S5D ) , suggesting that these membrane regions can communi - cate with the CLIC / GEEC pathway . Uptake through the CLIC / GEEC endocytic pathway has pre - viously been shown to be Cdc42 dependent [ 3 ] . Consistent with this , we observed that overexpressed dominant - active Cdc42 protein colocalizes with GRAF1 in basally located puncta ( Figure 3D ) . The absence of Cdc42 in long tubular structures might suggest that the role of Cdc42 in this pathway is restricted spatially and temporarily to the earlier endocytic stages or that progression is blocked through the dominant - active construct , which would be expected to have pleiotropic effects . To determine whether GRAF1 was necessary for endocyto - sis , we depleted GRAF1 levels by using siRNA . This treatment was capable of reducing the amount of GRAF1 to background levels as assessed by immunocytochemistry and immunoblot - ting of HeLa cells and their lysates , respectively ( Figure 4A ; also Figure S6D ) . GRAF1 - depleted cells were assessed for their ability to endocytose dextran ( allowing assessment of total endocytic capacity ) , both by epi\ufb02uorescence microscopy and by a quantitative \ufb02uorimetric assay ( Figures 4B and 4C ) . GRAF1 depletion resulted in a 50 % \u2013 60 % reduction of total dextran endocytosis , similar to that observed upon depletion of AP2 , the major Clathrin adaptor at the plasma membrane ( Figure 4B ) , suggesting that GRAF1 - mediated endocytosis and Clathrin - mediated endocytosis account for roughly equal amounts of volume internalization in these cells . GRAF1 - depleted cells had no observable defects in their ability to en - docytose transferrin ( Figure 4D ) , demonstrating that GRAF1 is not necessary for Clathrin - mediated endocytosis . In this study we have shown that GRAF1 is found on tubular and vesicular membranes in vivo , and that these membranes de\ufb01ne a prevalent Clathrin - independent endocytic pathway . This endocytic pathway is capable of internalizing Cholera toxin , GPI - linked proteins , and large amounts of extracellular \ufb02uid , and it corresponds ( at least in part ) to the CLIC / GEEC en - docytic pathway . Activity of Arf1 , in addition to that of its GEF ( D ) Confocal micrographs of HeLa cells co - overexpressing GFP - tagged Cdc42 L61 ( dominant - active ) and Myc - tagged GRAF1 showing colocalization in discrete puncta and short tubules . Scale bars represent 10 m m . Figure 4 . GRAF1 Is Indispensable for Clathrin - Independent Fluid - Phase Uptake in Fibroblasts ( A ) Immunoblots on HeLa celllysates transfected withacontrolsiRNA oreither oftwosiRNAs directedagainstGRAF1 mRNA ( siRNAs aandb ) . Detectionof GRAF1 and tubulin ( loading control ) was performed with speci\ufb01c antibodies on lysates obtained 48 hr after transfection . ( B ) GRAF1 - depletedcellsshowamajorreductionin\ufb02uid - phaseendocytosisasshownbythedecreaseintheuptakeofFITC - labeleddextran ( controlsiRNA ( n = 5 ) , siRNAa ( n = 8 ) , or AP2 siRNA ( n = 3 ) ) . The error bars show the standard deviation of the mean . ( C and D ) HeLa cells depleted of GRAF1 were incubated with dextran ( C ) or transferrin ( D ) for 15 min before \ufb01xation and staining . Scale bars represent 10 m m . Membrane Sculpting in Clathrin - Independent Endocytosis 1807 ARHGAP10 , is necessary for uptake via the CLIC / GEEC path - way , and Arf1 activity modulation also interferes with Cdc42 dynamics , suggesting interplay between these proteins [ 13 ] . Indeed , GRAF1 is capable of downregulating the activity of Cdc42 [ 14 ] , which is known to be necessary for the CLIC / GEEC pathway [ 3 ] and which colocalizes with GRAF1 in vivo . The protein machinery responsible for the biogenesis and pro - cessing of CLIC / GEEC endocytic membranes is largely un - known . Although Dynamin certainly plays a role in CLIC / GEEC membrane processing ( it is necessary for delivery of CTxB to the Golgi apparatus [ 4 ] ) , its precise role in the CLIC / GEEC pathway is uncertain . Recent microinjection experi - ments with antibodies directed against speci\ufb01c isoforms of Dynamin , or with siRNAs directed against these , showed that Dynamin is involved in constitutive \ufb02uid - phase uptake and suggested that it might play an important role in high - vol - ume Clathrin - independent endocytic events [ 15 ] . We build on this work by showing that there exists a tight biochemical in - teraction between GRAF1 and Dynamin . Although we impli - cate Dynamin in this pathway , precisely determining whether it functions here in an analogous manner to Dynamin at Cla - thrin - coated pits or works via a noncanonical mechanism will require further work . Our experiments lead us to suggest that GRAF1 coordinates the highest volume Clathrin - independent endocytic pathway in HeLa cells . Although several Clathrin - independent endo - cytic pathways appear to coexist ( such pathways include cav - eolin1 - and \ufb02otillin1 - positive pathways , an Arf6 - associated tu - bular uptake pathway , and the CLIC / GEEC pathway ) , the precise contributions that each of these pathways makes to constitutive endocytosis , and the interrelationships between these pathways , are uncertain . Although we have found that GRAF1 - positive membranes contain little \ufb02otillin1 or caveolin1 at steady state , we have also shown that these membranes probably transiently communicate . We further show that GRAF1 - positive membranes are not associated with internal - izing or recycled MHC class I proteins . Taken together , our data link biochemical and cell biological observations to iden - tify the \ufb01rst important modulator of membrane curvature in a highly prevalent Clathrin - independent endocytic pathway . We believe that , in the context of our \ufb01ndings , study of Cla - thrin - independent endocytic pathways will reveal new layers of complexity underlying lipid and protein cargo selection , ap - propriate intracellular traf\ufb01cking of endocytic membranes , and the mechanisms of endocytic intermediate formation by mem - brane deformation and membrane - curvature stabilization . Supplemental Data Supplemental Data include Supplemental Experimental Procedures , six \ufb01g - ures , and three movies and are available with this article online at http : / / www . current - biology . com / supplemental / S0960 - 9822 ( 08 ) 01413 - 9 . Acknowledgments Many thanks to Sew Peak - Chew and Farida Begum for assistance with mass spectrometry analysis , Marijn Ford for providing puri\ufb01ed Dynamin , and Sven Carlsson and all members of the McMahon lab for help and sup - port . We also thank the Kempe and Magn Bergvallfoundations and Medical faculty Umea\u02da University for grants . Thanks to M . Fa\u00a8llman , B Nichols , J . W . G . Andersson and S . Mayor for providing reagents . R . L was supported by a postdoctoral fellowship from the Swedish Research Council . G . D was supported by a Trinity College Cambridge Internal Graduate Studentship and Research Scholarship . The main work was founded by Medical Re - search Council UK . Received : April 7 , 2008 Revised : October 4 , 2008 Accepted : October 8 , 2008 Published online : November 24 , 2008 References 1 . Mayor , S . , and Pagano , R . E . ( 2007 ) . Pathways of clathrin - independent endocytosis . Nat . Rev . Mol . Cell Biol . 8 , 603 \u2013 612 . 2 . 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Cytoskeletal changes induced by GRAF , the GTPase regulator associated with focal adhesion kinase , are mediated by Rho . J . Cell Sci . 112 , 231 \u2013 242 . 15 . Cao , H . , Chen , J . , Awoniyi , M . , Henley , J . R . , and McNiven , M . A . ( 2007 ) . Dynamin 2 mediates \ufb02uid - phase micropinocytosis in epithelial cells . J . Cell Sci . 120 , 4167 \u2013 4177 . Current Biology Vol 18 No 22 1808", + "ezzatSpecificityAbstractionExamples2020": "H I C H A M E Z Z A T M A R I N E A G O G U (cid:1) E P A S C A L L E M A S S O N B E N O I T W E I L M A T H I E U C A S S O T T I Speci\ufb01city and Abstraction of Examples : Opposite Effects on Fixation for Creative Ideation ABSTRACT Fixation is one of the major obstacles that individuals face in creative idea generation contexts . Several studies have shown that individuals unintentionally tend to \ufb01xate to the examples they are shown in a cre - ative ideation task , even when instructed to avoid them . Most of these studies used examples formulated with high level of speci\ufb01city . However , no study has examined individuals\u2019 creative performance under an instruction to diverge from given examples , when these examples are formulated with a high level of abstraction . In the present study , we show that ( a ) instructing participants to avoid using common examples when formulated with a high level of speci\ufb01city increases \ufb01xation ; whereas ( b ) instructing participants to avoid such examples while using a more abstract level for stating these common examples \u2014 such as a cate - gorization of these examples \u2014 mitigates \ufb01xation and doubles the number of creative ideas generated . These \ufb01ndings give new insights on the key role of categorization in creative ideation contexts . Keywords : \ufb01xation effect , idea generation , examples , categorization . Creativity has been described as the capacity to generate ideas that are both novel and useful ( Amabile , 1996 ) , and constitutes one of the key cognitive skills that individuals use daily in various contexts . Accord - ing to divergent thinking scholars ( Guilford , 1959 ) , creative individuals usually exhibit high levels of idea - tional \ufb02uency , \ufb02exibility , and originality ( Runco & Acar , 2012 ) . However , the process of creative ideation is not always an easy task , and could be spontaneously and intuitively constrained by previously acquired knowledge , by individuals\u2019 own mental models or even by earlier ideas generated . Indeed , several studies have shown that these various factors could constrain one\u2019s cognitive ability to generate novel and creative ideas ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ; Jansson & Smith , 1991 ; Smith , Ward & Schumacher , 1993 ) . Psycholo - gists have labeled this phenomenon as \ufb01xation ( Cassotti , Agogu (cid:1) e , Camarda , Houd (cid:1) e & Borst , 2016 ; Jansson & Smith , 1991 ) , aka the blind mental adherence to a set of ideas ( Cassotti , Camarda , Poirel , Houd (cid:1) e & Agogu (cid:1) e , 2016 ) . One classical task to illustrate \ufb01xation is called the \u201ctwo cords problem\u201d of Norman Maier ( Maier , 1931 ) . Two cords ( tied to the ceiling ) and pair of pliers were provided to participants , who were asked to tie together the free ends of these two cords . In this experiment , most participants were \ufb01xated on the knowledge they had of pliers , and were unable to look at it alternatively by simply tying the pliers to one of the cords to form a pendulum that will swing to reach the second one . In fact , the accumulated knowledge about the typical use of pliers was spontaneously activated in individuals mind , and prevented them from seeing alternative uses ( Maier , 1931 ) , and thus impending creative thinking . Such effect , characterized in this case in a problem - solving situation , has been demonstrated to occur as well in more openly framed prob - lems , where participants need to generate a lot of different ideas to a speci\ufb01c situation ( Agogu (cid:1) e , Poirel , Pineau , Houd (cid:1) e & Cassotti , 2014 ) . More speci\ufb01cally , multiple experimental psychology studies have showed that previously acquired knowl - edge in individuals\u2019 mind is most likely to act as a mental set promoting \ufb01xation ( Adamson , 1952 ; Sio , Kotovsky & Cagan , 2015 ) . Moreover , other studies related to the role of examples on creativity have shown indeed that exposure to examples could play either a positive or negative role to modulate \ufb01xation . Smith and colleagues demonstrated that designers unintentionally tend to conform to the features of the example The Journal of Creative Behavior , Vol . 54 , Iss . 1 , pp . 115 \u2013 122 \u00a9 2018 by the Creative Education Foundation , Inc . (cid:1) DOI : 10 . 1002 / jocb . 349 115 they were shown ( Smith et al . , 1993 ) . In three experiments using a creative ideation paradigm in which sub - jects had to imagine and sketch new exemplars of experimenter - de\ufb01ned categories , the authors showed that individuals surprisingly replicate the features of the example even when they are explicitly asked to propose ideas that are different from the given example . Deepening our understanding of the role - speci\ufb01c examples may play in creative thinking , Agogu (cid:1) e and colleagues showed that two types of examples could actually have opposite effects on \ufb01xation modulation ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ) . In a creative ideation task where participants had to generate creative ideas to ensure that a hen\u2019s egg dropped from a height of ten meters does not break , the authors experimentally demonstrated that participants exposed to a common example ( within the \ufb01xation ) increased the number of solutions within the \ufb01xation ; whereas participants exposed to a creative example ( outside the \ufb01xation ) decreased the number of solutions within the \ufb01xation , and consequently increased originality of solutions . Focusing on the way instructions to be creative are delivered to participants , other studies have provided discrepant results regarding the role of warnings and constraints on \ufb01xation modulation . Chrysikou and Weisberg ( 2005 ) showed that the \ufb01xation effect can be reduced using instructions which warn subjects about the use of the \ufb02aws of an example ( Chrysikou & Weisberg , 2005 ) . On the contrary , Viswanathan and Linsey ( 2013 ) con\ufb01rmed Smith and colleagues\u2019 \ufb01ndings ( 1993 ) , and demonstrated that even when designers are presented with warnings about the undesirable example features along with the reasons for those warnings , \ufb01xation to those features is not mitigated ( Viswanathan & Linsey , 2013 ) . These discrepant results could let one think at \ufb01rst glance that there could be a certain relationship between the level of abstraction of the examples and the level of \ufb01xation mitigation . In fact , these \ufb01ndings may suggest that the more the con - straints and warnings on examples are abstract ( without clear indications about the features of the exam - ples ) , the more \ufb01xation would be mitigated . Similarly , one could also think that the more constraints on examples are speci\ufb01c ( with clear speci\ufb01cation of the features of the examples ) , the more \ufb01xation would be reinforced . In consistency with this role of speci\ufb01city and abstraction on \ufb01xation modulation in creative ideation , Baughman and Mumford ( 1995 ) demonstrated that when people are asked to combine exemplars from sep - arate categories to form a single inclusive category , which is considered an act of categorization , participants become more original ( Baughman & Mumford , 1995 ) . These results provided interesting indications about the role of abstraction for \ufb01xation mitigation and the generation of creative ideas . Moreover , Ward and col - leagues ( Ward , Patterson & Sifonis , 2004 ) con\ufb01rmed these \ufb01ndings on the role of abstraction for creativity . In their classical experiments where participants were asked to imagine life on other planets , the authors demonstrated that instructions encouraging participants to formulate the given task in more abstract ways led to more creativity than instructions encouraging participants to formulate the task in very speci\ufb01c ways . However , the in\ufb02uence of the level of abstraction on creativity has been only solely explored through the perspective of the task formulation . More focus could be placed on the relationship between the level of speci\ufb01city and abstraction , and the level of \ufb01xation mitigation by applying it to the use of examples . To assess this relationship between the level of abstraction and \ufb01xation mitigation \u2014 and in line with the abovementioned \ufb01ndings \u2014 the aim of the present study was to examine the effects of instructions warning about using either common examples formulated with a high level of speci\ufb01city , or more abstract levels of these same examples . To achieve this aim , participants were asked to solve a creative task ( i . e . , the hen\u2019s egg task ) and were provided with instructions warning about using common examples \u2014 either at a high level of speci\ufb01city or at a more abstract level \u2014 at the beginning of the task . We reasoned that if the in\ufb02uence of examples on creative ideation depends on the level of abstraction , then instructing individuals to avoid using common examples \u2014 at a high level of speci\ufb01city \u2014 should reinforce \ufb01xation ; whereas instructing individuals to avoid using common examples \u2014 by framing those examples at a more abstract level , such as a categoriza - tion of these examples \u2014 should mitigate \ufb01xation . METHOD PARTICIPANTS Seventy - \ufb01ve participants of an introductory course of innovation design were recruited for this study . To ensure that the content of the course had no in\ufb02uence on the performance of the participants , the experi - ment was made at the very beginning of the course . Participants were engineering students and professionals ( 91 % of the subjects were engineering students , while 9 % were professionals ) . Participants ( 69 % men ) were between 19 and 58 years old ( M = 25 . 6 years , SD = 6 . 9 ) . Participants were randomly assigned to one of the three experimental groups : a control group without constraints ( n = 25 , M = 24 . 84 years , SD = 5 . 51 , 18 116 Effects of Examples on Creative Ideation 21626057 , 2020 , 1 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / j o c b . 349 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 09 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e men ) , a group \u201cSpeci\ufb01c\u201d with constraints on examples ( n = 25 , M = 25 . 72 years , SD = 5 . 95 , 20 men ) and a group \u201cAbstract\u201d with constraints on categories of examples ( n = 25 , M = 26 . 36 , SD = 9 , 14 men ) . ANOVA and chi - squared analyses indicated that the mean ages ( F ( 2 , 72 ) < 1 ) and gender distributions ( v 2 = 3 . 51 , p = . 17 ) did not differ signi\ufb01cantly between the groups . Sample size was determined pre - hoc by running an a priori power analysis using G * Power 3 . 1 . 9 . 2 ( Faul , Erdfelder , Buchner & Lang , 2009 ) , revealing that a minimum of 66 participants would be needed to detect a medium effect size of 0 . 20 ( according to Cohen\u2019s effect size conventions ) , with a power ( 1 - b ) set at . 80 and an a set at . 05 . \u201d DESIGN AND PROCEDURE Each participant was given 10 minutes to perform individually a creativity task where the aim was to propose the maximum number of original solutions to \u201censure that a hen\u2019s egg dropped from a height of ten meters does not break\u201d . Individuals had to write down on a sheet of paper all the solutions they could come up with , and were prohibited to talk with each other . Not only the hen\u2019s egg task could be the appro - priate creativity task to select since it does not require speci\ufb01c knowledge and expertise from the partici - pants , but as we have mentioned earlier , previous studies of Agogu (cid:1) e and colleagues ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ) used Concept - Knowledge theory ( Hatchuel & Weil , 2002 ) , to build a cartography of solutions of the hen\u2019s egg task , distinguishing between solutions labeled \ufb01xation ( common or less novel ideas based on the most accessible knowledge ) and solutions labeled expansion ( rare or more novel ideas based on less accessi - ble knowledge outside \ufb01xation ) . In fact , over the past 5 years , the authors demonstrated that 81 % of the solutions generated by participants to this task were \ufb01xated around three categories of solutions ( which are \u201cdamping the shock\u201d , \u201cprotecting the egg\u201d , and \u201cslowing the fall\u201d ) . These three categories were considered to be inside \ufb01xation . However , only 19 % of the solutions were outside \ufb01xation ( for instance : \u201cbefore and after the fall\u201d , \u201cwith a living device\u201d , \u201cusing intrinsic properties of the environment\u201d , etc . . . ) . This database comprises a total of 716 proposed solutions from 122 students ( from the Faculty of Psychology of Paris Descartes University ) who performed the hen\u2019s egg task in two previous studies ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ; Agogu (cid:1) e , Poirel , et al . , 2014 ) , and is updated regularly with new solutions and categories of solutions if participants come up with new solutions that do not \ufb01t with the current categories . Table 1 shows a list of the range of categories of solutions of the hen\u2019s egg task . We used these works to delimitate the frontier between what is inside \ufb01xation ( simply labeled \u201c\ufb01xation\u201d according to these studies ) and outside \ufb01xation ( labeled \u201cexpansion\u201d according to these studies ) , and therefore identify the common examples ( inside \ufb01xa - tion ) for this creativity task . TABLE 1 . Categories of Solutions of the Egg\u2019s Task ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ) Categories of Examples Example of Solutions Fixation / Expansion ( ideas that are not \ufb01xation ) Damping the shock Place a mattress at the reception Fixation Protecting the egg Pack the egg with bubble wrap Fixation Slowing the fall Hang the egg to a parachute Fixation Interrupting the fall Catch the egg with a net Expansion Acting before the fall Drop the egg at a height of 11 m Expansion Acting after the fall Replace the broken egg with an unbroken one Expansion Using a living device Train an eagle to take down the egg Expansion Modifying the properties of the egg Freezing the egg Expansion Using the natural properties of the egg Drop the egg on its most robust axis Expansion Using the properties of the environment Drop the egg at zero gravity Expansion 117 Journal of Creative Behavior 21626057 , 2020 , 1 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / j o c b . 349 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 09 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e Participants were randomly assigned to one of the three groups . All participants were provided with the typical instruction of the hen\u2019s egg task : \u201cYou are a designer and your manager gives you the following problem : Ensure that a hen\u2019s egg dropped from a height of 10 m does not break\u201d . The control group was provided with an additional guideline stating that : \u201cThe evaluation of your man - ager will be based on the number of original ideas you will propose\u201d . Participants of the group \u201cSpeci\ufb01c\u201d were provided with another additional guideline , imposing con - straints on speci\ufb01c examples inside \ufb01xation . The guideline stated that : \u201cThe evaluation of your manager will be based on the number of original ideas you will propose , knowing that your solutions must not use mat - tress , nor parachute , nor bubble wrap\u201d . These three speci\ufb01c examples were precisely chosen among others , due to the fact that they were the most generated examples in each of the three categories of \ufb01xation in an existing database of participants that performed this task in the past 5 years ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ; Agogu (cid:1) e , Poirel , et al . , 2014 ) . Participants of the group \u201cAbstract\u201d were provided with another additional guideline , imposing con - straints on a more abstract level of these examples inside \ufb01xation , namely categories of these examples . The guideline stated that : \u201cThe evaluation of your manager will be based on the number of original ideas you will propose , knowing that your solutions must neither dampen the shock , nor slow the fall , nor protect the egg\u201d . These three abstract examples were considered the three categories of \ufb01xation of the present creative ideation task ( Agogu (cid:1) e , Le Masson , Dalmasso , Houd (cid:1) e & Cassotti , 2015 ; Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ; Agogu (cid:1) e , Poirel , et al . , 2014 ; Cassotti , Agogu (cid:1) e , et al . , 2016 ; Cassotti , Camarda , et al . , 2016 ; Ezzat et al . , 2017 ) . DATA ANALYSIS The creative performance of the participants of the hen\u2019s egg task was quanti\ufb01ed by measuring the num - ber of solutions participants were given inside and outside \ufb01xation ( Agogu (cid:1) e et al . , 2015 ) . This could be done using an existing cartography of solutions of the hen\u2019s egg task , representing the distribution of solu - tions across different categories . To perform this measure , two trained raters assigned each of participants\u2019 solutions to one of the ten categories of solutions . The obtained inter - rater reliability score was 92 % . Only three categories among the ten were assigned inside \ufb01xation ( damping the shock , slowing the fall , and pro - tecting the egg ) . All solutions outside these three categories were assigned outside \ufb01xation ( modifying the properties of the egg , acting before / after the fall , or using a living device , etc . . . ) . Given that creativity requires both novelty / originality and usefulness / feasibility , we applied an external rating procedure to assess feasibility . More speci\ufb01cally , two independent raters were instructed to evaluate each idea on a \ufb01ve - point rating scale ranging from 1 ( \u201cnot feasible at all\u201d ) to 5 ( \u201chighly feasible\u201d ) . The raters displayed satisfactory intra - class correlation ( ICC = 0 . 90 ) . We also computed an objective measurement of the originality of the solutions by considering the fre - quency of the responses provided by all participants . For this score , the originality of a solution was de\ufb01ned as the normalized statistical infrequency of that particular solution . A mean originality score was calculated for each participant , which could range from 0 to 1 ( 0 represented lower originality and 1 represented higher originality ) . RESULTS In order to examine whether the numbers of proposed solutions ( ideational \ufb02uency ) inside \ufb01xation ( \ufb01x - ation ) and outside \ufb01xation ( expansion ) varied according to the experimental conditions , we conducted a repeated - measures analysis of variance ( ANOVA ) with the experimental conditions ( speci\ufb01c ; control and abstract ) as a between - subjects factor and the category of solution ( \ufb01xation vs . expansion ) as a within - sub - jects factor . We used the partial eta squared ( g 2 p ) and Cohen\u2019s d to assess the effect size . The ANOVA revealed a main effect of the category of solution ( F ( 1 , 72 ) = 7 . 83 , p = . 007 , g 2 p = . 10 ) indicating that the participants provided more solutions in the \ufb01xation path than in the expansion path . This analysis also showed a main effect of the experimental conditions ( F ( 2 , 72 ) = 4 . 13 , p = . 02 , g 2 p = . 10 ) . Moreover , there was a signi\ufb01cant experimental conditions 9 category of solution interaction ( F ( 2 , 72 ) = 48 . 06 , p < . 0001 , g 2 p = . 57 , see Figure 1a ) . Critically , the experimental conditions 9 category of solu - tion interaction was still signi\ufb01cant after controlling for the feasibility scores , F ( 2 , 71 ) = 32 . 70 , p < . 0001 , g 2 p = . 48 ) . Because main effects were modulated by the two - way interactions , we focused further analysis on the lat - ter . Planned comparisons , corrected with a Holm \u2013 Bonferroni procedure , revealed no signi\ufb01cant difference 118 Effects of Examples on Creative Ideation 21626057 , 2020 , 1 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / j o c b . 349 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 09 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e between the number of solutions in the expansion path in the control group ( M = 2 . 48 , SD = 1 . 78 ) and those in the group \u201cspeci\ufb01c\u201d ( M = 2 . 56 , SD = 1 . 74 , F ( 1 , 72 ) < 1 , d = . 04 ) . However , participants of the group \u201cabstract\u201d ( M = 5 . 16 , SD = 2 . 76 ) proposed much more solutions in the expansion path compared to the control group ( M = 2 . 48 , SD = 1 . 78 ; F ( 1 , 72 ) = 19 . 48 , p corr < . 0001 , d = 1 . 15 ) , and to the group \u201cspeci - \ufb01c\u201d ( M = 2 . 56 , SD = 1 . 74 , F ( 1 , 72 ) = 18 . 34 , p corr < . 0001 , d = 1 . 13 ) . Interestingly , the participants of the group \u201cabstract\u201d ( M = 1 . 36 , SD = 1 . 15 ) proposed fewer solutions in the \ufb01xation path than did those in the control group ( M = 5 . 24 , SD = 2 . 35 ; F ( 1 , 72 ) = 38 . 43 , p corr < . 0001 , d = 2 . 1 ) , as well as did the participants of the group \u201cspeci\ufb01c\u201d ( M = 6 . 52 , SD = 2 . 8 ; F ( 1 , 72 ) = 67 . 97 , p corr < . 0001 , d = 2 . 41 ) . Additionally , the group \u201cspeci\ufb01c\u201d ( M = 6 . 52 , SD = 2 . 8 ) proposed more solutions in the \ufb01xation path than the control group ( M = 5 . 24 , SD = 2 . 35 ; F ( 1 , 72 ) = 4 . 18 , p corr = . 04 , d = 0 . 5 ) . To examine whether originality scores varied according to the experimental conditions , we conducted a one - way analysis of variance ( ANOVA ) with the experimental conditions ( speci\ufb01c ; control and abstract ) as a between - subjects factor . We used the partial eta squared ( g 2 p ) and Cohen\u2019s d to assess the effect size . The ANOVA revealed a main effect of the experimental conditions ( F ( 2 , 72 ) = 3 . 18 , p = . 047 , g 2 p = . 08 , see Fig - ure 1b ) . Planned comparisons , revealed no signi\ufb01cant difference between the originality scores of the control group ( M = 0 . 32 , SD = . 16 ) and those of the group \u201cspeci\ufb01c\u201d ( M = . 31 , SD = . 18 , F ( 1 , 72 ) < 1 , d = . 06 ) . However , the solutions proposed by the group \u201cabstract\u201d ( M = . 42 , SD = . 16 ) were more original than those provided by the control group ( M = . 32 , SD = . 16 , F ( 1 , 72 ) = 4 . 26 , p = . 04 , d = . 62 ) and the group \u201cspeci\ufb01c\u201d ( M = . 31 , SD = . 18 , F ( 1 , 72 ) = 5 . 24 , p = . 025 , d = . 65 ) . DISCUSSION In the present study , we demonstrated that according to the level of the speci\ufb01city and abstraction of common examples presented prior to a creative idea generation task , we could obtain opposite effects on \ufb01xation mitigation . We showed that ( a ) instructing participants to avoid using common examples when for - mulated with a high level of speci\ufb01city increases \ufb01xation , and therefore constrains participants\u2019 capacity to generate creative ideas ; whereas ( b ) instructing participants to avoid such examples while using a more abstract level for stating these common examples \u2014 such as a categorization of these examples \u2014 mitigates \ufb01x - ation , and consequently increases the number of creative ideas generated \u2014 and in quite a signi\ufb01cant way . More precisely , statistical results show that constraints on common examples , formulated with high level of speci\ufb01city , lightly increase the number of solutions inside the \ufb01xation path , but however have no effect on the number of solutions outside the \ufb01xation path . However , statistical analysis shows that constraints on common examples , formulated with high level of abstraction \u2014 such as a categorization of these examples \u2014 approximately reduce the number of solutions inside the \ufb01xation path by more than one third , and FIGURE 1 . ( A ) Mean number of responses according to the experimental condition ( Speci\ufb01city / Control / Abstract ) and the type of solution ( Fixation / Expansion ) . ( B ) Mean originality scores according to the experimental condition ( Speci\ufb01city / Control / Abstract ) . 119 Journal of Creative Behavior 21626057 , 2020 , 1 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / j o c b . 349 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 09 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e surprisingly double the number of solutions outside the \ufb01xation path . Thus , the magnitude of the stimula - tion effect produced by explicit warning of not using certain categories is quite surprising and requires some discussion . Theoretically , these \ufb01ndings might suggest that when constraints on examples are formulated with high level of abstraction , individuals are forced to reason outside \ufb01xation , and therefore succeed to generate novel alternatives to the common and typical categories of solutions provided to them ( outside the \ufb01xation path ) . Whereas , when constraints on examples are formulated with high level of speci\ufb01city , individuals are more affected by the speci\ufb01c examples provided to them , and follow the path of least resistance , which consist of generating alternatives to the common and typical solutions ( inside the \ufb01xation path ) . These \ufb01ndings \ufb01rst of all con\ufb01rm the studies regarding the negative role of examples for creativity ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ; Jansson & Smith , 1991 ; Smith et al . , 1993 ) , since the results of the group \u201cspeci\ufb01c\u201d showed that the introduction of a common example with high level of speci\ufb01city could highly increase \ufb01xation effect , and therefore constrains the capacity of individuals to generate creative ideas . Secondly , our \ufb01ndings add to the literature on new practical procedures to overcome \ufb01xation effects in creative ideation contexts ( Agogu (cid:1) e , Kazakc (cid:1) i , et al . , 2014 ; Linsey et al . , 2010 ; Moreno , Yang , Hern (cid:1) andez , Lin - sey & Wood , 2015 ; Zahner , Nickerson , Tversky , Corter & Ma , 2010 ) , through the appropriate use of con - straints and warnings . We demonstrate that two types of constraints could have opposite effects on \ufb01xation modulation , according to their level of speci\ufb01city and abstraction . The more constraints on inappropriate examples are abstract , the more \ufb01xation effect is mitigated . Similarly , the more the constraints on inappro - priate examples are speci\ufb01c , the more \ufb01xation effect is facilitated . Thirdly , our \ufb01ndings raised more questions than what they contribute to , especially regarding the crucial role of categorization in creative ideation contexts . First of all , the present study con\ufb01rms previous studies ( Baughman & Mumford , 1995 ; Nagai & Taura , 2009 ; Ward et al . , 2004 ) regarding the important role of abstraction for \ufb01xation mitigation . Furthermore , since we explicitly measure the number of solutions inside / outside the \ufb01xation path in the present experiments , using the statistical measurement of the variable \u201cex - pansivity\u201d ( Agogu (cid:1) e et al . , 2015 ) , our \ufb01ndings present further evidence regarding the key role that could be played by categorization for \ufb01xation modulation and creativity . Finally , from a purely managerial perspective , our \ufb01ndings are consistent with the view of the literature on expertise and categorization ( Chi , Feltovich & Glaser , 1981 ) , arguing that experts have the skills to both recognize and restructure problems ( Akin , 1990 ) , in a way in which it allow them seeing the problem from a broader and more abstract view than novices . Additionally , it gives new sights for understanding how leaders could brief their teams through initial instructions in creativity situations , in a way they could help their teams to avoid falling in the cognitive trap of \ufb01xation , and stimulate their creative performance ( Car - son & Carson , 1993 ; Chaffois , Gillier , Belkhouja & Roth , 2015 ; Ezzat et al . , 2017 ; Runco , Illies & Eisenman , 2005 ) . We show that it is not suf\ufb01cient and enough for leaders to simply impose constraints on unwanted ideas and solutions in creative projects for their teams , but leaders must have the ability to formulate these constraints in appropriate levels of abstraction , in a way that ensures \ufb01xation is majorly set aside , in order to open the way for creativity to \ufb02ourish . One possible limitation of our study was that we considered common or less novel ideas and solutions generated by participants , consisting of \u201cdamping the shock\u201d , \u201cprotecting the egg\u201d , or \u201cslowing the fall\u201d , as \ufb01xation categories . In fact , we did not measure or pretest in the present experiment whether the participants have used that techniques for similar purposes ( avoid breaking objects ) before . In line with this limitation , further experiments could consist of controlling this speci\ufb01c \ufb01xation issue using previous techniques tested on creative idea generation task ( Benedek et al . , 2014 ; Silvia , Nusbaum & Beaty , 2015 ) to assess whether individuals\u2019 responses were based on \u201cold\u201d ( responses retrieved from memory ) or \u201cnew\u201d ( responses gener - ated on the spot ) . CONCLUSION The present study demonstrates that common examples \u2014 if formulated in a high level of abstraction \u2014 can play a crucial role to help individuals overcome \ufb01xation effects occurring in creative idea generation sit - uations . Our results clearly suggest that the way the common examples are formulated prior to a creativity task \u2014 either with speci\ufb01city or abstraction \u2014 could have opposite effects on \ufb01xation . As a result , the present study provides new insights to the literature on the positive and negative role of examples in creative ideation . 120 Effects of Examples on Creative Ideation 21626057 , 2020 , 1 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / j o c b . 349 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 09 / 05 / 2024 ] . 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Hicham Ezzat , MINES ParisTech \u2013 PSL Research University Marine Agogu (cid:1) e , HEC Montr (cid:1) eal Pascal Le Masson , Benoit Weil , MINES ParisTech \u2013 PSL Research University Mathieu Cassotti , Paris Descartes University Correspondence concerning this article should be addressed to Hicham Ezzat , MINES ParisTech \u2013 PSL Research University , Center for Management Science , i3 UMR CNRS 9217 , France . E - mail : hicham . ezzat @ mines - paristech . fr ACKNOWLEDGMENT This research was \ufb01nanced by a grant from the French National Research Agency ( ANR - 13 - SOIN - 0004 - 02 ID (cid:1) e\ufb01xE ) . 122 Effects of Examples on Creative Ideation 21626057 , 2020 , 1 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / j o c b . 349 by U n i v e r s it y O f M a r y l a nd , W il e y O n li n e L i b r a r y on [ 09 / 05 / 2024 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e", + "numasawa2020fluorescent": "German Edition : DOI : 10 . 1002 / ange . 201914826 Fluorescent Probes International Edition : DOI : 10 . 1002 / anie . 201914826 A Fluorescent Probe for Rapid , High - Contrast Visualization of Folate - Receptor - Expressing Tumors In Vivo Koji Numasawa , Kenjiro Hanaoka , * Naoko Saito , Yoshifumi Yamaguchi , Takayuki Ikeno , Honami Echizen , Masahiro Yasunaga , Toru Komatsu , Tasuku Ueno , Masayuki Miura , Tetsuo Nagano , and Yasuteru Urano * Abstract : Folate receptors ( FRs ) are membrane proteins involved in folic acid uptake , and the alpha isoform ( FR - a ) is overexpressed in ovarian and endometrial cancer cells . For fluorescence imaging of FRs in vivo , the near - infrared ( NIR ) region ( 650 \u2013 900 nm ) , in which tissue penetration is high and autofluorescence is low , is optimal , but existing NIR fluores - cent probes targeting FR - a show high non - specific tissue adsorption , and require prolonged washout to visualize tumors . We have designed and synthesized a new NIR fluorescent probe , FolateSiR - 1 , utilizing a Si - rhodamine fluorophore having a carboxy group at the benzene moiety , coupled to a folate ligand moiety through a negatively charged tripeptide linker . This probe exhibits very low background fluorescence and afforded a tumor - to - background ratio ( TBR ) of up to 83 in FR - expressing tumor - bearing mice within 30 min . Thus , FolateSiR - 1 has the potential to contribute to the research in the field of biology and the clinical medicine . Introduction Folic acid , which is required in one - carbon metabolic reactions and for the synthesis of nucleotide bases , emerged as a targeting ligand for imaging of cancer tissues in the 1990s . [ 1 , 2 ] It is internalized into cells through folate receptors ( FRs ) expressed on the cell surface . The alpha isoform of folate receptor ( FR - a ) is upregulated in about 40 % of human cancers , especially in malignant tissues such as ovarian cancer , whereas normal tissues , except for the kidney , do not accumulate folic acid or its conjugates . [ 3 \u2013 5 ] Folic acid binds to FR - a with high affinity ( K d & 10 @ 9 m ) even after conjuga - tion to imaging agents , and undergoes receptor - mediated endocytosis . [ 3 ] Consequently , various folate - linked drugs and imaging agents have been developed . [ 1 \u2013 3 ] Among available imaging modalities , fluorescence imaging provides real - time images with millimeter resolution , and has attracted interest for intraoperative fluorescence imaging of tumor tissues . [ 6 ] For example , 90 \u2013 95 % of epithelial ovarian cancers over - express FR - a , and a folate - linked fluorescent dye , folate - FITC , was recently employed for intraoperative tumor - specific fluorescence imaging in patients . [ 7 ] However , FITC emits green fluorescence ( around 520 nm ) , and is unsuitable for imaging deep tissues . For this purpose , dyes emitting in the near - infrared ( NIR ) region ( 650 \u2013 900 nm ) are most useful because tissue penetration is high and autofluorescence is low , resulting in a low background signal . [ 8 , 9 ] However , existing folate - linked NIR fluorescent dyes show nonspecific adsorp - tion on tissues , and require a washout period of up to 24 h to clearly image tumors in tumor - bearing mice ( Scheme 1 ) . [ 10 \u2013 12 ] Therefore , we aimed to develop a NIR fluorescent probe suitable for rapid , high - contrast detection of FR - expressing tumors in vivo , without the need for a washout procedure . Two approaches have so far been used to improve the signal - to - background ratio ( SBR ) of fluorescent probes . One approach is to develop activatable fluorescent probes whose fluorescence signal is emitted only in response to a specific feature of the local environment , such as pH . For example , a probe targeting human epidermal growth factor receptor type 2 ( HER2 ) employed a fluorescent dye - labeled antibody that is fluorescently activated in the low pH environment of lysosomes after cellular internalization . [ 13 ] This approach provides a high tumor - to - normal - tissue signal ratio . However , endocytotic transport of the probe to lysosomes takes up to 24 h . Yang et al . designed and synthesized an off / on - type fluorescent probe for detection of FRs by utilizing a pH - sensitive acyl hydrazone linker and a dark quencher , but failed to observe fluorescence enhancement after cellular internalization . [ 14 ] They also developed a folate conjugate whose fluorescence wavelength is changed by reduction of disulfide in the probe structure during receptor - mediated [ * ] Dr . K . Numasawa , Prof . K . Hanaoka , N . Saito , T . Ikeno , H . Echizen , Dr . T . Komatsu , Dr . T . Ueno , Prof . M . Miura , Prof . Y . Urano Graduate School of Pharmaceutical Sciences , The University of Tokyo 7 - 3 - 1 Hongo , Bunkyo - ku , Tokyo 113 - 0033 ( Japan ) E - mail : khanaoka @ mol . f . u - tokyo . ac . jp uranokun @ mol . f . u - tokyo . ac . jp Prof . Y . Yamaguchi Institute of Low Temperature Science , Hokkaido University Sapporo 060 - 0819 ( Japan ) Dr . M . Yasunaga Division of Developmental Therapeutics , Exploratory Oncology Research & Clinical Trial Center , National Cancer Center 6 - 5 - 1 Kashiwanoha , Kashiwa , Chiba 277 - 8577 ( Japan ) Prof . T . Nagano Drug Discovery Initiative , The University of Tokyo 7 - 3 - 1 Hongo , Bunkyo - ku , Tokyo 113 - 0033 ( Japan ) Prof . Y . Urano Graduate School of Medicine , The University of Tokyo 7 - 3 - 1 Hongo , Bunkyo - ku , Tokyo 113 - 0033 ( Japan ) andCREST ( Japan ) Agency for Medical Research and Development ( AMED ) 1 - 7 - 1 Otemachi , Chiyoda - ku , Tokyo 100 - 0004 ( Japan ) Supporting information and the ORCID identification number ( s ) for the author ( s ) of this article can be found under : https : / / doi . org / 10 . 1002 / anie . 201914826 . Angewandte Chemie Research Articles 6015 Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 T 2020 Wiley - VCH Verlag GmbH & Co . KGaA , Weinheim endocytosis . [ 15 ] The latter probe was suitable for cellular applications , but the half - time of disulfide reduction was as long as 6 h after endocytosis . The second approach is to minimize nonspecific adsorption of the probe in vivo . Choi et al . reported that the zwitterionic heptamethine indocya - nine NIR fluorophore ZW800 - 1 exhibits low serum - protein binding , ultralow nonspecific tissue background , and rapid elimination from the body through renal filtration . [ 16 ] In tumor model applications , a tumor - to - background ratio ( defined as the contrast - to - background ratio in the tumor divided by the contrast - to - background ratio in nearby normal tissue ) of over 17 was achieved at 4 h after injection of ZW800 - 1 conjugated to cRGD . Herein , we build on this approach to design and synthesize an NIR fluorescent probe that we believe offers sufficiently high performance for practical in vivo imaging . Results and Discussion First , we needed a system to assess the occurrence of nonspecific adsorption of folate - linked NIR fluorescent dyes . For this purpose , we performed live - cell fluorescence imaging of FR - a - expressing and FR - a - non - expressing cultured cells , i . e . , KB cells ( FR + ) and OVCAR - 3 cells ( FR @ ) , with a commercially available NIR fluorescent probe , FolateR - Sense 680 ( PerkinElmer , USA ) . The expression of FRs on these cells was confirmed by immunostaining ( Supporting Information , Figure S1 ) . KB cells showed strong fluorescence at the cell membrane due to probe binding to FR - a on the cell surface , but we also observed some bright dots inside the cells , apparently due to nonspecific internalization of the probe ( Figure S2a ) . On the other hand , OVCAR - 3 cells showed many very bright dots within the cells ( Figure S2b ) . There - fore , we considered that this cellular imaging system was suitable to judge whether or not our newly synthesized folate - linked fluorescent dyes might show nonspecific adsorption when used for in vivo fluorescence imaging . In the molecular design of our folate - linked fluorescent dyes , we chose to conjugate the linker moiety to the folate glutamate moiety ( Figure 1a ) . The crystal structure of human FR - a complexed with folic acid has the folate pteroate moiety buried inside FR , whereas the glutamate moiety is exposed to the solvent and protrudes from the binding pocket entrance , so that it can be conjugated with a fluorescent dye without adversely affecting the binding to FR - a . [ 17 ] By this molecular design , we expected that fluorescent probes would show the high affinity for FR , i . e . , a K d value around 10 @ 9 m , because folate conjugates ( and folic acid ) normally bind to FR with this affinity . [ 3 ] We also employed a negatively charged peptide linker , Asp \u2013 Lys \u2013 Gly , in order to reduce the cell - membrane permeability of the folate - linked fluorescent dye , and we attached various xanthene fluorophores to the amino group of the lysine side chain in the linker ( Figure 1a and Figure S3 ) . We applied each compound to the live - cell fluorescence Scheme 1 . Imaging strategy of existing near - infrared fluorescent probes for in vivo fluorescence imaging of whole animals . Figure 1 . a ) Molecular design of fluorescent probes for detection of folate receptors . The structures of FolateSiR - 1 and FolateSiR - 2 are also shown . b ) Absorption and emission spectra of 1 m m FolateSiR - 1 in 100 m m sodium phosphate buffer at pH 7 . 4 , l ex = 652 nm . c ) Absorp - tion and emission spectra of 1 m m FolateSiR - 2 in 100 m m sodium phosphate buffer at pH 7 . 4 , l ex = 656 nm . Angewandte Chemie Research Articles 6016 www . angewandte . org T 2020 Wiley - VCH Verlag GmbH & Co . KGaA , Weinheim Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 15213773 , 2020 , 15 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / a n i e . 201914826 by U n i v e r s it y O f W a s h i ng t on , W il e y O n l i n e L i b r a r y on [ 14 / 11 / 2023 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e imaging with KB cells and OVCAR - 3 cells . As shown in Figure S3 , basically fluorescein derivative - labeled folates such as Fluorescein Folate , 2 - Me DCTM Folate , and 2 - COOH DCTM Folate showed the selective detection of FR in living cells , while rhodamine derivative - labeled folate such as TAMRA Folate and Alexa488 Folate stained the cell membrane of KB cells , but they also showed nonspecific adsorption . Among them , we fortunately found a promising NIR fluorescent probe for detecting FR , FolateSiR - 1 , along with a control compound , FolateSiR - 2 , which has very similar chemical structure to FolateSiR - 1 ( Figure 1a ) . Both Folate - SiR - 1 and FolateSiR - 2 showed similar absorption and emis - sion spectra in the NIR region , and their fluorescence quantum yields were 7 . 6 % and 5 . 1 % , respectively ( Figure 1 ) . The relatively low fluorescence quantum yields were prob - ably due to dynamic quenching between the fluorophore and the electron - rich pteroate moiety , [ 18 ] but are sufficiently high for cellular and in vivo fluorescence imaging . When we applied FolateSiR - 1 to KB cells , the cell membrane was clearly stained ; there were no bright dots inside the cells , and the fluorescence disappeared in the presence of 1 m m folic acid as a competitor ( Figure 2a ) , indicating that nonspecific endocytosis of FolateSiR - 1 did not occur . FolateSiR - 1 may also possess the low cell - membrane permeability owing to the relatively large molecular weight and the negatively charged peptide linker . Further , when we applied FolateSiR - 1 to OVCAR - 3 cells , almost no fluorescence was observed , supporting the idea that there is little nonspecific adsorption of the dye ( Figure 2b ) . On the other hand , FolateSiR - 2 stained the cell membrane of KB cells , but also exhibited many bright dots inside the cells both in the presence and absence of 1 m m folic acid ( Figure 2a ) . It also afforded some bright dots inside OVCAR - 3 cells , supporting the existence of nonspecific adsorption ( Figure 2b ) . Although folate recep - tors can normally induce cell uptake of ligands through endocytosis , the endocytosis of FolateSiR - 1 and FolateSiR - 2 through folate receptors was not observed . In short , Folate - SiR - 1 showed a high S / N ratio with little nonspecific adsorption in cellular fluorescence imaging . Next , we applied these two fluorescent probes to ex vivo mouse embryos to visualize endogenously expressed folate - binding protein 1 ( Folbp1 ) , the mouse orthologue of human FR - a . Periconceptional folate supplementation significantly reduces the risk of neural tube defects , and Folbp1 is one of the membrane proteins that mediate cellular uptake of folate ; mice lacking Folbp1 are defective in early embryonic devel - opment . [ 19 ] Maternal anti - FR antibodies are also associated with neuronal tube defects in humans . [ 20 ] In situ hybridization revealed a distinct expression pattern of Folbp1 mRNA in the neural folds prior to the initiation of neural tube closure at the cervical region and the prosencephalic / mesencephalic boun - dary . [ 21 ] Folbp1 mRNA is mainly localized to the most dorsal regions of the neural folds , where fusion takes place , and as neural fold fusion proceeds , Folbp1 mRNA expression extends to the adjacent unfused neural folds ( Figure 3a ) . However , the expression pattern of Folbp1 protein has not been reported . We firstly applied FolateSiR - 1 to embryos at day 8 . 5 postcoitum . The living embryos were stained with 20 m m FolateSiR - 1 for 30 min at 37 88 C and fluorescence imaging was quickly performed . The neural tube closing region showed strong fluorescence compared with other regions , Figure 2 . a ) Bright - field ( left ) and fluorescence ( right ) images of KB cells incubated with 5 m m FolateSiR - 1 or FolateSiR - 2 in the presence or absence of 1 m m folic acid and 0 . 5 % DMSO as a cosolvent . White arrows indicate bright dots inside cells ; l ex = 650 nm , l em = 670 \u2013 750 nm . Scale bars = 20 m m . b ) Bright - field ( left ) and fluorescence ( right ) images of OVCAR - 3 cells incubated with 5 m m FolateSiR - 1 or FolateSiR - 2 and 0 . 5 % DMSO as a cosolvent . White arrows indicate bright dots inside cells , l ex = 650 nm , l em = 670 \u2013 750 nm . Scale bars = 20 m m . Figure 3 . a ) Schematic of folate receptor ( = Folbp 1 ) expression in mouse embryo ; regions reported to show folate receptor expression are shown in gray . b ) Fluorescence image of mouse embryo incubated with 20 m m FolateSiR - 1 . Locations stained with FolateSiR - 1 are indicat - ed by white arrowheads . c ) Fluorescence image of mouse embryo incubated with 20 m m FolateSiR - 2 . Angewandte Chemie Research Articles 6017 Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 T 2020 Wiley - VCH Verlag GmbH & Co . KGaA , Weinheim www . angewandte . org 15213773 , 2020 , 15 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / a n i e . 201914826 by U n i v e r s it y O f W a s h i ng t on , W il e y O n l i n e L i b r a r y on [ 14 / 11 / 2023 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e while weak fluorescence was observed throughout the embryo ( Figure 3b ) . On the other hand , we observed strong , speckled fluorescence throughout the whole embryo when FolateSiR - 2 was loaded ( Figure 3c ) , suggesting the idea that FolateSiR - 2 binds non - specifically in the cells . We then performed a competition experiment of FolateSiR - 1 with 1 m m folic acid . The fluorescence images changed with time , probably because embryogenesis is rapid , so in this experi - ment we fixed the embryos with 4 % formaldehyde after staining with FolateSiR - 1 . We confirmed that FolateSiR - 1 was retained in the cell membrane of KB cells after the fixation process ( Figure S4a ) . Fluorescence imaging of the FolateSiR - 1 - stained , fixed embryos showed a similar pattern of strong fluorescence in the neural tube closing region to that shown in Figure 3b ( Figures S4b and S5 ) . This pattern disappeared in the presence of 1 m m folic acid ( Figure S4c ) . Thus , FolateSiR - 1 appears to stain Folbp1 - expressing regions in a folic acid - competitive manner . We further applied FolateSiR - 1 to in vivo fluorescence imaging of tumors in mouse models . FolateSiR - 1 was intra - venously injected through the tail vein into tumor - bearing mice prepared by subcutaneous inoculation of KB cells . FR - a - expressing tumors were clearly visualized with an extreme - ly low background ( TBR up to 83 ) just 30 min after injection of FolateSiR - 1 ( Figure 4a , b ) . The mice were sacrificed , and we confirmed that FolateSiR - 1 was strongly accumulated in the tumor ( fluorescence in the stomach may have been derived from the feed in this experiment ) ( Figure S6a ) . On the other hand , relatively strong fluorescence throughout the whole body was observed at the same time - point after injection of FolateSiR - 2 , resulting in a low TBR ( Figure 4c ) . Even at 6 h after injection of FolateSiR - 2 , the fluorescence was relatively strong throughout the whole body and the TBR was around 12 , which was much lower than that of FolateSiR - 1 ( Figure 4c , d ) , though tumor accumulation of FolateSiR - 2 was observed in excised tissues ( Figure S6b ) . We also prepared an FR ( @ ) - tumor model by subcutaneous injection of HT1080 cells . The absence of FR was confirmed by immunostaining ( Figure S1 ) . No fluorescence was observed when FolateSiR - 1 was injected into these mice ( Figure S7 ) . On the other hand , when we injected FolateSiR - 2 into these mice , fluorescence rapidly appeared throughout the whole body ( Figure S8 ) ; it subsequently decreased with time , but the tumor was not visualized . Moreover , when we injected 6 m m folic acid in saline into KB tumor - bearing mouse , followed by 100 m m FolateSiR - 1 in saline ( 100 m L ) , no fluorescence was observed except in the intestine , which might be due to the feed ( Figure S9 ) . These results indicate that FolateSiR - 1 binds specifically to FR - expressing tumors in these mice , affording high - contrast images unlike typical NIR fluorescent probes such as FolateSiR - 2 . To further investigate the potential applicability of FolateSiR - 1 for intraoperative tumor detection , we utilized a tissue microarray of patient - derived ovarian tumor tissues and normal tissues from regions adjacent to tumors ( Fig - ure S10 ) . FolateSiR - 1 successfully visualized ovarian tumor tissues , while exhibiting little binding to normal tissues ( Figure 5a and Figure S11 ) . Furthermore , the fluorescence image of the tissue microarray closely matched the immu - nostaining image ( Figure 5b and Figures S12 and S13 ) , confirming the ability of FolateSiR - 1 to visualize human tumors ex vivo . The selective staining of tumor tissues by FolateSiR - 1 was also observed in a specimen of tissue microarray , which contains both tumor tissues and non - tumor tissues ( vascular tissues and fibrous tissues ) ( Figure S13c ) . Conclusion In this study , we designed and synthesized a series of folate - fluorescent dye conjugates . We focused on the non - specific adsorption of typical NIR fluorescent dye such as cyanine dyes and folate conjugates to tissues inside the body when they are injected through the tail vein . We conjugated various xanthene - based fluorescent dyes to folic acid and applied them to the live - cell fluorescence imaging for selection of fluorescent probes showing low nonspecific adsorption . Among xanthene fluorescent dye - conjugated folates , we fortunately found that FolateSiR - 1 , consisting of Figure 4 . a ) Fluorescence images at different time points of KB tumor - bearing mouse injected with 100 m m FolateSiR - 1 in 100 m L saline containing 1 % DMSO as a cosolvent . Fluorescence and white light images were obtained before and 0 , 0 . 5 , 1 , 2 , 3 and 6 h after the probe injection ; l ex = 661 ( 641 \u2013 681 ) nm , l em = 700 \u2013 800 nm . T = tumor , M = muscle . Fluorescence intensity scale : gray scale 0 to 255 . b ) Time - dependent change of fluorescence intensity in tumor and non - tumor ( muscle ) areas of three mice , including the mouse in ( a ) . Error bar shows S . E . c ) Fluorescence images at different time points of KB tumor - bearing mouse injected with 100 m m FolateSiR - 2 in 100 m L saline containing 1 % DMSO as a cosolvent . Fluorescence and white - light images were obtained before and 0 , 0 . 5 , 1 , 2 , 3 and 6 h after the probe injection ; l ex = 661 ( 641 \u2013 681 ) nm , l em = 700 \u2013 800 nm . T = tumor , M = muscle . Fluorescence intensity scale : gray scale 0 to 255 . d ) Time - dependent change of fluorescence intensity in tumor and non - tumor ( muscle ) areas of three mice , including the mouse in ( c ) . Error bar shows S . E . Angewandte Chemie Research Articles 6018 www . angewandte . org T 2020 Wiley - VCH Verlag GmbH & Co . KGaA , Weinheim Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 15213773 , 2020 , 15 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / a n i e . 201914826 by U n i v e r s it y O f W a s h i ng t on , W il e y O n l i n e L i b r a r y on [ 14 / 11 / 2023 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e a Si - rhodamine fluorophore having a carboxy group at the benzene moiety , coupled to a folate ligand moiety through a negatively charged tripeptide linker ( Figure 1a ) , achieved a very low level of nonspecific tissue binding . Indeed , this probe could rapidly provide high - contrast images of tumors in a mouse model in vivo without any washout procedure , due to the very low background fluorescence . We think the low background may be due to the combination of the intrinsic low level of nonspecific binding derived from the probe design and the preference of the 2 - COOH SiR650 fluoro - phore for the intramolecularly spirocyclized , nonfluorescent form in hydrophobic environments , such as those in plasma / inner membranes . [ 22 ] The spirocyclization converts the cat - ionic xanthene moiety to neutral form , whereas in contrast , the cationic charge of cyanines and Si - rhodamines favors nonspecific adsorption . Indeed , 2 - COOH SiR650 showed a large absorbance decrease in 100 m m sodium phosphate buffer ( pH 7 . 4 ) containing 10 % fetal bovine serum , which contains many proteins ( Figure S14 ) . This idea is also supported by the observation that when FolateSiR - 1 was dissolved in mouse serum , the absorbance was significantly decreased , presumably due to nonspecific binding to proteins of mouse serum and the formation of the intramolecularly spirocyclized , nonfluorescent form ( Figure S15 ) . This is also consistent with the finding of Weissleder et al . that a neutral 2 - COOH SiR650 derivative could follow the intracellular localization of a therapeutic drug in live cells . [ 23 \u2013 25 ] Notably , these ligand - 2 - COOH SiR650 conjugates show almost no fluorescence signal at non - targeted regions inside the cells , in accordance with the concept of using background - free \u201ctame\u201d fluorescent probes [ 26 ] to obtain clear fluorescence images in live cells . Our work extends the scope of this concept to in vivo fluorescence imaging of whole animals . Indeed , the effective blocking of nonspecific adsorption on non - targeted tissues enabled us to clearly visualize tumors within less than 30 min after probe injection . During the operation , at most 5 to 10 min may be allowed as an incubation time after probe injection , and FolateSiR - 1 has a potential to be used even during the operation . A similar approach might be applied to other tumor - related membrane proteins , such as carbonic anhydrase IX , which is overexpressed in hypoxic tumor tissues . [ 27 ] Moreover , the NIR probe OTL - 38 , which is based on the cyanine fluorophore , is currently in phase II clinical trial for intraoperative imaging of ovarian cancer . [ 28 \u2013 30 ] Our new probe FolateSiR - 1 might be superior to OTL - 38 in terms of high S / N ratio , and we believe it should be suitable for precise intraoperative fluorescence detection of tiny tumors in the millimeter size range , as well as for the efficient endoscopic detection of cancer cells in abdominal dropsy immediately after surgical operation . Recently , therapeutic antibodies have been applied to the treatment of various types of cancer . However , although it was confirmed to be safe , anti - FRs antibody did not meet the primary endpoints of progression - free survival or overall survival in a clinical trial . Therefore , anti - FRs antibody \u2013 drug conjugates ( ADCs ) have been de - veloped in order to enhance the efficacy . [ 31 , 32 ] However , poor penetration into solid tumors still poses a problem for ADCs as well as naked therapeutic antibodies . On the other hand , small - molecular drug conjugates are expected to overcome this issue . [ 33 ] The binding activity and specificity for FR of FolateSiR - 1 are thought to be high as well as anti - FRs antibodies , [ 3 ] and so FolateSiR - 1 might be available as a basic scaffold for the development of new small - molecular drug conjugates to treat FR - expressing ovarian cancer . Further - more , the theranostic features of such agents could be valuable to assess the pharmacokinetic profile within the tumor tissue . Acknowledgements This work was supported in part by JSPS KAKENHI Grant Numbers JP18H04609 and JP16H05099 to K . H . and JP26110005 to Y . Y . , SENTAN , JST to K . H , and Hoansha Foundation and Daiichi Sankyo Foundation of Life Science to K . H . This work was also supported by a grant JSPS Core - to - Core program , A . Advanced Research Networks and a Grant - in - Aid for Scientific Research on Innovative Areas \u201cSingularity Biology ( No . 8007 ) \u201d ( JP19H05414 to K . H . ) of The Ministry of Education , Culture , Sports , Science , and Technology , Japan . Conflict of interest The authors declare no conflict of interest . Keywords : cancer \u00b7 chromophores \u00b7 fluorescent probes \u00b7 imaging \u00b7 vitamins Figure 5 . a ) The ovarian cancer tissue microarray was incubated with 5 m m FolateSiR - 1 and 2 . 9 m m DAPI ( nuclear stain ) in phosphate - buffered saline containing 0 . 05 % Tween 20 ( PBST ) for 2 h . Then , the tissue microarray was washed with PBST three times and the fluores - cence image was obtained . The microarray contains 37 tumor tissues and 3 normal tissues . Fluorescence images of the 3 normal tissues and 3 representative tumor tissues are shown , scale bar = 2 mm . b ) Immunostaining of folate receptors in the tissue microarray . The immunostained 3 normal tissues and 3 tumor tissues correspond to those in ( a ) , scale bar = 2 mm . Angewandte Chemie Research Articles 6019 Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 T 2020 Wiley - VCH Verlag GmbH & Co . KGaA , Weinheim www . angewandte . org 15213773 , 2020 , 15 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / a n i e . 201914826 by U n i v e r s it y O f W a s h i ng t on , W il e y O n l i n e L i b r a r y on [ 14 / 11 / 2023 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e How to cite : Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 Angew . Chem . 2020 , 132 , 6071 \u2013 6076 [ 1 ] W . Xia , P . S . Low , J . Med . Chem . 2010 , 53 , 6811 \u2013 6824 . [ 2 ] Y . G . Assaraf , C . P . Leamon , J . A . Reddy , Drug Resist . Updates 2014 , 17 , 89 \u2013 95 . [ 3 ] P . S . Low , S . A . 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Grasso , E . F . Albone , Mol . Cancer Ther . 2018 , 17 , 2665 \u2013 2675 . [ 33 ] M . P . Deonarain , G . Yahioglu , I . Stamati , A . Pomowski , J . Clarke , B . M . Edwards , S . Diez - Posada , A . C . Stewart , Anti - bodies 2018 , 7 , 16 . Manuscript received : November 20 , 2019 Revised manuscript received : December 30 , 2019 Accepted manuscript online : January 26 , 2020 Version of record online : February 26 , 2020 Angewandte Chemie Research Articles 6020 www . angewandte . org T 2020 Wiley - VCH Verlag GmbH & Co . KGaA , Weinheim Angew . Chem . Int . Ed . 2020 , 59 , 6015 \u2013 6020 15213773 , 2020 , 15 , D o w n l o a d e d fr o m h tt p s : / / on li n e li b r a r y . w il e y . c o m / do i / 10 . 1002 / a n i e . 201914826 by U n i v e r s it y O f W a s h i ng t on , W il e y O n l i n e L i b r a r y on [ 14 / 11 / 2023 ] . S ee t h e T e r m s a nd C ond iti on s ( h tt p s : / / on li n e li b r a r y . w il e y . c o m / t e r m s - a nd - c ond iti on s ) on W il e y O n li n e L i b r a r y f o r r u l e s o f u s e ; OA a r ti c l e s a r e gov e r n e d by t h e a pp li ca b l e C r ea ti v e C o mm on s L i ce n s e", + "yamada2016actin": "INTERNATIONAL JOURNAL OF ONCOLOGY 49 : 877 - 886 , 2016 Abstract . The endocytic protein dynamin participates in the formation of actin - based membrane protrusions such as podosomes , pseudopodia , and invadopodia , which facilitate cancer cell migration , invasion , and metastasis . However , the role of dynamin in the formation of actin - based membrane protrusions at the leading edge of cancer cells is unclear . In this study , we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F - actin bundles in filopodia of the lung cancer cell line H1299 . Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation . Furthermore , dynamin 2 and cortactin mostly colocalized along F - actin bundles in filopodia of serum - stimulated H1299 cells by immuno - fluorescent and immunoelectron microscopy . Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum - stimulated H1299 cells , concomitant with a loss of F - actin bundles . Expression of wild - type cortactin rescued the punctate - like localization of dynamin 2 and filopodial forma - tion . The incubation of dynamin 2 and cortactin with F - actin induced the formation of long and thick actin bundles , with these proteins colocalizing at F - actin bundles . A depolymer - ization assay revealed that dynamin 2 and cortactin increased the stability of F - actin bundles . These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F - actin bundles in filopodia . Taken together , these findings suggest that dynamin might be a possible molecular target for anticancer therapy . Introduction Cancer cell migration , invasion , and metastasis are preceded by the formation of pseudopodia such as lamellipodia and filopodia . During these cellular processes , F - actin filaments remodel into a higher order structure and then assemble an intricate cytoskeletal network within cells ( 1 ) . These dynamic three - dimensional changes are mediated by several actin - bundling and crosslinking proteins , and are essential for supporting filopodia at the leading edge of migrating cells ( 2 ) . Dynamin plays an essential role in endocytosis , partici - pating in the membrane fission process ( 3 - 5 ) . Dynamin also functions in the formation of actin - rich structures , including lamellipodia and dorsal membrane ruffles ( 6 , 7 ) , invadopodia ( 8 ) , podosomes ( 9 ) , growth cones ( 10 - 12 ) , and phagocytic cups ( 13 , 14 ) . Three dynamin isoforms exist , namely , dynamin 1 , 2 , and 3 ( 5 ) . Dynamins are characterized by a GTPase domain at the N - terminus , a bundle signaling element , a stalk domain , a phosphoinositide - binding pleckstrin homology domain , and a proline and arginine - rich domain at the C - terminus ( PRD ) ( 15 , 16 ) . The PRD interacts with different proteins that contain the Src - homology - 3 ( SH3 ) domain . Of these GTPases , dynamin 2 is ubiquitously expressed . Cortactin , an F - actin - binding protein , was first identified as an Src substrate ( 17 ) . Cortactin also participates in cancer cell migration , invasion , and metastasis by regulating actin dynamics at the leading edge of migrating cells ( 18 ) . Cortactin is composed of an N - terminal acidic domain and a six - and - a - half tandem repeats domain , which directly binds to F - actin . Cortactin also contains an \u03b1 - helix , a proline - rich region , and an SH3 domain at the C - terminus , which interacts with the PRD of several binding partners ( 19 ) . Both dynamin and cortactin are implicated in the dynamics of cancer cells , including migration , invasion , and metastasis ( 18 ) . In addition , the pharmacological inhibition of dynamin by GTPase inhibitors suppresses specific cellular processes such as the lamellipodial formation and invasion of human osteocarcinoma cells ( 20 ) and the growth of human prostate adenocarcinoma cells ( 21 ) . Actin bundling by dynamin 2 and cortactin is implicated in cell migration by stabilizing filopodia in human non - small cell lung carcinoma cells HIROSHI YAMADA 1 , 3 , TETSUYA TAKEDA 1 , 3 , HIROYUKI MICHIUE 2 , TADASHI ABE 1 , 3 and KOHJI TAKEI 1 , 3 Departments of 1 Neuroscience and 2 Physiology , Graduate School of Medicine , Dentistry and Pharmaceutical Sciences , Okayama University ; 3 CREST , Japan Science and Technology Agency , Kita - ku , Okayama 700 - 8558 , Japan Received April 6 , 2016 ; Accepted May 25 , 2016 DOI : 10 . 3892 / ijo . 2016 . 3592 Correspondence to : Dr Hiroshi Yamada , Department of Neuro - science , Graduate School of Medicine , Dentistry and Pharmaceutical Sciences , Okayama University , 2 - 5 - 1 Shikata - cho , Kita - ku , Okayama 700 - 8558 , Japan E - mail : hiroyama @ md . okayama - u . ac . jp Key words : actin , cortactin , dynamin , filopodia , migration YAMADA et al : ACTIN BUNDLING BY DYNAMIN AND CORTACTIN IN CANCER CELL MIGRATION 878 A previous study reported that dynamin 2 binds to cortactin ( 7 , 12 ) . A disruption of this protein complex can affect the shape of cancer cells ( 7 ) , organization of the F - actin network within these cells ( 22 ) , and structure of growth cones ( 11 , 12 ) . However , the role of the dynamin 2 - cortactin complex in the dynamics of the actin cytoskeleton in cancer cells is unclear . In this study , we investigated whether dynamin 2 and cortactin regulate the F - actin bundle formation in filopodia in the human non - small cell lung carcinoma cell line H1299 . Materials and methods Antibodies and reagents . Rabbit polyclonal anti - dynamin 1 ( cat . no . PA1 - 660 ; Thermo Fisher Scientific , Waltham , MA , USA ) and anti - c - myc ( cat . no . C3956 ; Sigma - Aldrich , St . Louis , MO , USA ) antibodies , and a goat polyclonal anti - dynamin 2 ( cat . no . sc - 6400 ; Santa Cruz Biotechnology , Santa Cruz , CA , USA ) antibody , were purchased . In addition , mouse monoclonal anti - \u03b2 - actin ( cat . no . A5441 , Sigma - Aldrich ) , Dynasore ( cat . no . D7693 , Sigma - Aldrich ) , anti - c - myc ( cat . no . sc - 40 ; Santa Cruz Biotechnology ) , anti - green fluorescent protein ( GFP ; cat . no . sc - 9996 , Santa Cruz Biotechnology ) , and anti - cortactin ( cat . no . 05 - 180 ; EMD Millipore , Darmstadt , Germany ) antibodies were purchased . MitMAB and Dynole 34 - 2 were purchased from Abcam Biochemicals ( Bristol , UK ) . Alexa Fluor 488 - conjugated anti - rabbit IgG , rhodamine - conjugated anti - mouse IgG , and rhodamine or Alexa Fluor 488 - labeled phalloidin were obtained from Thermo Fisher Scientific . Purified rabbit skeletal \u03b1 - actinin was purchased from Cytoskeleton , Inc . ( Denver , CO , USA ) . Goat anti - mouse IgG - and goat anti - rabbit IgG - conjugated gold particles were purchased from British BioCell International ( Cardiff , UK ) . Cell culture . The human non - small cell lung carcinoma cell line H1299 ( Cat . no . ATCC CRL - 5803 ; American Type Culture Collection , Manassas , VA , USA ) was cultured in Dulbecco ' s modified Eagle ' s medium ( DMEM , Thermo Fisher Scientific ) supplemented with 10 % fetal bovine serum ( FBS ) at 37\u02daC in an atmosphere of 5 % CO 2 . Expression and purification of dynamin 2 and cortactin wild - types and mutants . GFP - tagged dynamin 2 cloned into pEGFP - N1 was a kind gift from Dr Mark McNiven ( Mayo Clinic , Rochester , MN , USA ) ( 6 ) . His - tagged dynamin 2 produced with the Bac - to - Bac baculovirus expression system ( Thermo Fisher Scientific ) was a kind gift from Dr Hiroshi Handa ( Tokyo Institute of Technology , Tokyo , Japan ) ( 23 ) . The dynamin solution was concentrated using a Centriplus YM50 ( Thermo Fisher Scientific ) and stored at - 80\u02daC . The protein suspension ( 2 - 5 mg / ml protein ) was thawed at 37\u02daC before use . The cDNAs encoding full - length rat cortactin and its mutants were prepared by polymerase chain reaction ampli - fication using specific primers ( 12 ) . Full - length cortactin or 1 - 450aa ( Cort \u2206 SH3 ) was subcloned into the plasmid pGEX - 6p vector as Bam HI - Eco RI fragments . GST - tagged cortactin W525K was generated by mutating pGEX - 6p - cortactin with the QuickChange site - directed mutagenesis kit ( Agilent Technologies , Santa Clara , CA , USA ) . For expression in cells , full - length cortactin or Cort \u2206 SH3 was subcloned into the pEF1 myc - His vector ( Thermo Fisher Scientific ) as Eco RI - Xba I fragments . The nucleotide sequences of the constructs were verified by DNA sequence analysis . The resulting plasmid was transformed into the bacterial BL21 ( DE3 ) pLysS strain for protein expression . The expression of GST - fusion proteins was induced by 0 . 1 mM isopropyl - 1 - thio - d - galactopyranoside at 37\u02daC for 3 - 6 h in LB medium supplemented with 100 \u00b5g / ml ampicillin to A 600 = 0 . 8 . The purification of GST - fusion proteins was performed as previously described ( 24 ) , and the cleavage of the GST with PreScission protease was performed according to the manufacturer ' s instructions . The protein was purified on a MonoQ column equilibrated in 20 mM Tris - HCl and 0 . 2 M NaCl , pH 7 . 7 . The eluted protein fraction ( 1 mg / ml protein ) was stored at - 80\u02daC . For the pull - down assay , the proteins were used without cleaving GST . siRNA - mediated interference . Pre - annealed siRNAs for human dynamin 2 and cortactin , and the negative control siRNA , were synthesized and purified ( Thermo Fisher Scientific ) . The sequences for the siRNAs for human dynamin 2 were as follows : 5 ' - GGAUAUUGAGGGCAAGAAGtt - 3 ' ( sense ) , 5 ' - CUUCUUGCCCUCAAUAUCCtt - 3 ' ( antisense ) for oligo 1 ; 5 ' - GCGAAUCGUCACCACUUACtt - 3 ' ( sense ) , 5 ' - GUAAGUG GUGACGAUUCGCtc - 3 ' ( antisense ) for oligo 2 ; and 5 ' - GGAC UUACGACGGGAGAUCtt - 3 ' ( sense ) , 5 ' - GAUCUCCCGU CGUAAGUCCtt - 3 ' ( antisense ) for oligo 3 . The sequences for the siRNAs for human cortactin were as follows : CCGAAUG GAUAAGUCAGUCtt - 3 ' ( sense ) , 5 ' - AGCUGACUUAUCCAU UCGGtc - 3 ' ( antisense ) for oligo 1 ; GGUUUCGGCGGCA AAUACGtt - 3 ' ( sense ) , CGUAUUUGCCGCCGAAACCtt - 3 ' ( antisense ) for oligo 2 ; and CGAAUAUCAGUCGAAACUUtt - 3 ' ( sense ) , AAGUUUCGACUGAUAUUCGtg - 3 ' ( antisense ) for oligo 3 . A scrambled siRNA with no significant sequence homology to all mouse , rat , or human gene sequences was used as the negative control . The day before transfection , the cells were plated in 6 - well plates ( 5x10 4 cells / well ) . One hundred picomoles of the duplex siRNAs was transfected into the cells using 4 \u00b5l of Lipofectamine 2000 ( Thermo Fisher Scientific ) . After 72 h , the cells were treated differently according to experimental design . In pilot experiments , we confirmed that all three transfections of siRNA for dynamin 2 and cortactin were effective . Filopodial formation . H1299 cells were serum - starved for 16 h . Thereafter , the cells were transfected with dynamin 2 siRNAs , cortactin siRNAs , or the control siRNA , followed by incubation with DMEM supplemented with 10 % FBS for 45 min . For the rescue experiments , cortactin was silenced in H1299 cells with oligo 3 , and the cells were cultured for 24 h . The cells ( 1x10 5 / coverslip ) were then transfected with rat wild - type cortactin or cortactin W525K ( 0 . 25 \u00b5g each ) cloned into the pIRES2 - AcGFP1 expression vector ( Clontech Laboratories , Santa Clara , CA , USA ) . Thereafter , the cells were stimulated with serum for 45 min , fixed , and stained with Alexa Fluor 488 or rhodamine - labeled phalloidin for visualization of filopodia . Wound healing assay . H1299 cells were cultured to confluence on glass - bottom dishes ( 35 mm diameter ; AGC Techno Glass Co . Ltd . , Tokyo , Japan ) in DMEM supplemented with 0 . 2 % FBS INTERNATIONAL JOURNAL OF ONCOLOGY 49 : 877 - 886 , 2016 879 for 8 h . Thereafter , the cell layer was wounded with a plastic pipette tip as previously described ( 25 ) . The cells were washed with DMEM supplemented with 0 . 2 % FBS and incubated for 8 h in the presence of Dynasore , Dynole 34 - 2 or MitMAB at the indicated concentrations . For the negative control , cells were incubated with 1 % dimethyl sulfoxide ( DMSO ) . The cells were visualized by Giemsa staining , followed by the acquisi - tion of phase contrast images from \u226520 randomly selected areas per dish . Areas filled with migrating cells were analyzed with ImageJ software ( National Institutes of Health , Bethesda , MD , USA ) . Formation of in vitro F - actin bundles . For the fluorescent detection of F - actin , non - muscle actin was polymerized in F - buffer ( 10 mM Tris - HCl , 0 . 2 mM DTT , 0 . 2 mM CaCl 2 , 2 mM MgCl 2 , 50 mM KCl , and 0 . 5 mM ATP , pH 7 . 5 ) for 1 h . Thereafter , 3 . 3 \u00b5M F - actin was incubated with 5 \u00b5M dynamin 1 or 2 and cortactin for 1 h , followed by an additional 30 min with 3 \u00b5M Alexa Fluor 488 - phalloidin . The samples were spread onto glass slides and mounted , and the F - actin bundles were observed under an epifluorescent microscope . For the immunolocalization of dynamin and cortactin , F - actin bundles were incubated with dynamin 1 or 2 and cortactin for 30 min with 3 \u00b5M phalloidin to stabilize the filaments , followed by centrifugation at 5 , 000 x g for 10 min . The pellet was resuspended with 50 \u00b5l of F - buffer and then immunostained in suspension for 30 min with 1 \u00b5l of primary antibody . The mixture was centrifuged at 5 , 000 x g , and the pellet was washed with F - buffer . The samples were incubated with secondary antibodies and washed as previously done for the primary antibody . All steps were performed at room temperature . The samples were spread onto glass slides and mounted . The samples were examined under a spinning disc confocal microscope system ( CSU10 , Yokogawa Electric Co . , Tokyo , Japan ) combined with an inverted microscope ( IX - 71 , Olympus Optical Co . , Ltd . , Tokyo , Japan ) and a CoolSNAP - HQ camera ( Roper Technologies , Sarasota , FL , USA ) . The confocal system was controlled by MetaMorph software ( Molecular Devices , Sunnyvale , CA , USA ) . Images were processed using Adobe Photoshop CS3 or Illustrator CS3 software . Immunoprecipitation assay . For the immunoprecipitation assay , H1299 cells were co - transfected with GFP - tagged dynamin 2 and either myc - tagged cortactin or cortactin \u2206 SH3 . The cells were lysed with 1 % NP - 40 , 100 mM KCl , 0 . 5 mM EDTA , 10 mM NaF , and 20 mM HEPES / KOH , pH 7 . 4 , and a protease inhibitor cocktail tablet ( Roche Diagnostics , Basel , Switzerland ) . The protein complexes were immunoprecipitated from 1 mg of cell extract using either 5 \u00b5g of the polyclonal anti - myc antibodies or preimmune IgG , and then visualized by western blotting with a monoclonal anti - GFP or anti - myc antibody . Immunostaining and fluorescent microscopy . H1299 cells were fixed with 4 % paraformaldehyde and stained by immu - nofluorescence as previously described ( 12 ) . Transmission electron microscopy . Specimens were embedded for immunoelectron microscopy as previously described ( 12 ) . In brief , H1299 cells were fixed with cytoskeleton buffer ( 10 mM 2 - ( N - morpholino ) ethanesulfonic acid , 150 mM NaCl , 5 mM EGTA , 5 mM MgCl 2 , and 5 mM glucose , pH 6 . 0 ) containing 10 \u00b5g / ml phalloidin , 0 . 1 % Triton X - 100 , and 3 % formaldehyde for 1 min . The cells were then fixed for an additional 30 min without Triton X - 100 , followed by washing with 5 \u00b5g / ml phal - loidin in phosphate - buffered saline ( PBS ) . After incubation in blocking solution ( 10 \u00b5g / ml phalloidin , 2 mg / ml BSA , and 100 mM glycine in PBS ) , the specimens were incubated with a primary antibody diluted in blocking solution , washed with 5 \u00b5g / ml phalloidin in PBS , incubated with goat anti - mouse or rabbit anti - goat IgG conjugated to 10 - nm gold particles , and then fixed with 2 . 5 % glutaraldehyde and 5 \u00b5g / ml phalloidin in PBS . The specimens were post - fixed with 1 % OsO 4 in 0 . 1 M sodium cacodylate buffer for 1 h , dehydrated , and embedded in EPON 812 for ultrathin sectioning . Cross - sections were visualized under a Hitachi H - 7100 transmission electron microscope . Determination of filopodial length . For the measurement of the filopodial length , H1299 cells were fixed and stained with rhodamine - or Alexa Fluor 488 - conjugated phalloidin . Membrane protrusions supported with F - actin bundles were defined as filopodia , and digital images were acquired at 400 - 1 , 000x magnifications . Up to five filopodia for each cell were randomly selected , and their lengths were measured with ImageJ software . Statistical analysis . Data were analyzed for statistical significance using KaleidaGraph software ( version 4 . 1 ) for the Macintosh ( Synergy Software Inc . , Essex Junction , VT , USA ) . Analysis of variance and Tukey ' s honest significant difference post hoc test were applied for more than two different groups , and Student ' s t - test was applied for two different groups . P - values of < 0 . 05 and 0 . 001 were considered as statistically significant . Results Inhibition of dynamin decreases the migration of the human non - small cell lung carcinoma cell line H1299 . To determine whether dynamin 2 is involved in cell migration , the effects of dynamin inhibition on cell migration were determined by a wound healing assay . Cell migration decreased after treatment of cells with Dynasore ( 26 ) , Dynole 34 - 2 ( 27 ) , and MitMAB ( 28 ) ( Fig . 1A ) . Dynasore ( 80 \u00b5M ) inhibited cell migration by ~ 80 % compared to that of control cells , whereas Dynole 34 - 2 and MitMAB inhibited cell migration by 20 - 40 % ( Fig . 1B ) . These results indicate that dynamin is important for the migra - tion of H1299 cells . Dynamin 2 colocalizes with cortactin along F - actin bundles in filopodia of H1299 cells . Fig . 1 shows that dynamin is involved in cell migration mediated by pseudopodia . Thus , we investi - gated whether dynamin 2 participates in filopodial formation in H1299 cells . Because cortactin functions with dynamin 1 in the bundling of F - actin , which is important for the stability of filopodia in human neuroblastoma cell line SH - SY5Y ( 12 ) , dynamin 2 and cortactin were immunostained in serum - stimu - lated H1299 cells . H1299 cells formed numerous filopodia YAMADA et al : ACTIN BUNDLING BY DYNAMIN AND CORTACTIN IN CANCER CELL MIGRATION 880 after serum stimulation ( Fig . 2A ) . Furthermore , dynamin 2 and cortactin colocalized to F - actin bundles in filopodia as bright dots ( Fig . 2A ) . The negative controls showed little immunoreactivity for dynamin 2 and cortactin ( Fig . 2B ) . In addition , immunoelectron microscopy revealed that both proteins localized to F - actin bundles in filopodia ( Fig . 2C ) . These results prompted us to examine the possible inter - action of dynamin 2 and cortactin by immunoprecipitation . Exogenously expressed dynamin 2 - GFP was co - precipitated with full - length cortactin - myc using a polyclonal anti - myc anti - bodies and H1299 cell lysates ( Fig . 2D , left ) . Cort \u2206 SH3 - myc , a dynamin 2 binding deficient mutant that lacks its SH3 domain , was unable to precipitate dynamin 2 ( Fig . 2D , right ) . Taken together , these results illustrate that these proteins interact at F - actin bundles in filopodia of H1299 cells . Dynamin 2 and cortactin are required for serum - induced filopodial formation in H1299 cells . To examine the role of dynamin 2 in filopodial formation , dynamin 2 was silenced in H1299 cells by RNAi . Compared with the control , knockdown of dynamin 2 in H1299 cells with specific siRNAs reduced its level by ~ 95 % as revealed by western blotting ( Fig . 3A ) . Compared with the length of filopodia in serum - stimulated control cells ( 10 . 2\u00b10 . 5 \u00b5m ) , dynamin 2 knockdown decreased filopodial extension in silenced cells ( 4 . 7\u00b10 . 6 \u00b5m ) ( Fig . 3B and C ) . In addition , dynasore inhibited filopodial extension ( 2 . 4\u00b10 . 08 \u00b5m ) . This effect was rescued after the inhibitor was removed ( 8 . 1\u00b12 . 4 \u00b5m ) ( Fig . 3D and E ) . We also examined the effects of cortactin knockdown by RNAi on filopodial formation . Compared with the control , knockdown of cortactin reduced its level by ~ 95 % as revealed by western blotting ( Fig . 4A ) . Compared with the length of filopodia in control cells ( 10 . 2\u00b10 . 39 \u00b5m ) , cortactin knockdown also decreased filopodial extension after serum - stimulation ( 5 . 6\u00b10 . 17 \u00b5m ) ( Fig . 4B and C ) . The inhibition of filopodial formation in cortactin - silenced cells was rescued by exogenous expression of wild - type cortactin ( 10 . 8\u00b10 . 54 \u00b5m ) but not by cortactin W525K , a binding - defective mutant of dynamin 2 ( 29 ) ( Fig . 4D and E ) . In addition , the punctate - like localization of dynamin 2 along F - actin bundles reappeared in wild - type cortactin expressing cells ( Fig . 4F , right ) . These results indi - cate that dynamin 2 and cortactin are required for filopodial formation . F - actin bundling by the dynamin 2 - cortactin complex stabi - lizes F - actin . The effects of dynamin 2 and cortactin on the formation of F - actin bundles were examined in vitro . In this experiment , preformed F - actin were incubated with or without cortactin and dynamin 2 in the presence of GTP . F - actin alone appeared as uniform filaments ( Fig . 5A , actin alone ) . The addition of dynamin 2 to F - actin filaments did not cause any visible change in their distribution ( Fig . 5A , + Dyn2 ) . However , F - actin incubated with wild - type cortactin , cortactin W525K or cortactin \u2206 SH3 often formed small clusters ( Fig . 5A , + Cort WT , + Cort W525K , or + Cort \u2206 SH3 ) , consistent with a previ - ously published report ( 12 ) . The presence of both dynamin 2 Figure 1 . Dynamin GTPase inhibitors inhibit the migration of H1299 cells . ( A ) Representative images acquired by light microscopy showing cell migration in a wound healing assay . Confluent H1299 cells were wounded and then incubated for 8 h in the presence or absence of dynamin GTPase inhibitors at the indicated concentrations . For the negative control ( control 0 h ) , cells were incubated with 1 % DMSO . Scale bar , 200 \u00b5m . ( B ) Morphometric analysis of the wound area filled by migrating cells after treatment with inhibitors at the indicated concentrations . The changes were normalized to the control . Results represent the means \u00b1 SEM of three independent experiments . INTERNATIONAL JOURNAL OF ONCOLOGY 49 : 877 - 886 , 2016 881 and wild - type cortactin resulted in the formation of long and thick F - actin bundles ( Fig . 5A , + Dyn2 + Cort WT ) , which were similar to those formed by dynamin 1 and cortactin ( Fig . 5A , + Dyn1 + Cort WT ) . On the other hand , the long and thick F - actin bundles were much less evident in the presence of dynamin 2 and cortactin W525K or \u2206 SH3 ( Fig . 5A , + Dyn2 + Cort W525K or + Dyn2 + Cort \u2206 SH3 ) . To localize dynamin 2 and cortactin to F - actin bundles , the preformed F - actin bundles were used for immunofluores - cent staining . Dynamin 2 and cortactin colocalized as bright dots along F - actin bundles ( Fig . 5B , left ) . The localiza - tion of dynamin 2 and cortactin was similar to that of the dynamin 1 - cortactin complex ( Fig . 5B , right ) ( 12 ) . Lastly , we examined whether actin bundling by the dynamin 2 - cortactin complex can affect F - actin stability . To address this , the depolymerization kinetics of preformed pyrene - labeled F - actin were examined after the solution was diluted 10 - fold with buffer . In the presence of dynamin 2 and cortactin , the rate of depolymerization by dilution decreased to a level comparable to that induced by \u03b1 - actinin , an actin - crosslinking protein , indicating that dynamin 2 and cortactin stabilize F - actin bundles ( Fig . 5C ) . These results indicate that the dynamin 2 - cortactin complex stabilizes F - actin bundles in filopodia prior to cell migration . Discussion The involvement of dynamin in the dynamics of cancer cells such as cell migration , invasion , and metastasis has been reported ( 18 ) . However , the precise role of dynamin in these Figure 2 . Dynamin 2 colocalizes with cortactin along F - actin bundles in filopodia of serum - stimulated H1299 cells . ( A ) Colocalization of dynamin 2 ( Dyn2 , left ) and cortactin ( Cort , middle ) by double - immunofluorescent staining in filopodia of serum - stimulated H1299 cells . Boxed areas correspond to enlarged images shown below . Dynamin 2 - and cortactin - positive puncta were present periodically along F - actin bundles in filopodia ( arrowheads ) . Scale bar , 5 \u00b5m ( upper panels ) , 1 . 6 \u00b5m ( lower panels ) . ( B ) In the negative controls , the primary antibodies were omitted for dynamin 2 ( left ) and cortactin ( right ) . Boxed areas correspond to enlarged images shown below . Bar , 10 \u00b5m ( top and bottom panels ) , 2 . 8 \u00b5m ( middle panels ) . ( C ) Representative images acquired by immunoelectron microscopy showing the localization of dynamin 2 ( top three panels ) and cortactin ( bottom three panels ) in filopodia of serum - stimulated H1299 cells . Immunoreactive dynamin 2 and cortactin were present along F - actin bundles ( arrowheads ) . Scale bar , 20 nm . ( D ) Immunoprecipitation ( IP ) results demonstrating an in vivo interaction between dynamin 2 and cortactin . H1299 cells were co - transfected with GFP - tagged dynamin 2 ( Dyn2 - GFP ) and either myc - tagged wild - type cortactin ( Cort WT - myc , left ) or cortactin \u2206 SH3 ( Cort \u2206 SH3 - myc , right ) . The protein complexes were immunoprecipitated using a polyclonal anti - myc antibody or preimmune IgG ( IgG ) , and then visualized by western blotting ( WB ) with monoclonal anti - GFP or anti - myc antibodies . Total cell lysates ( 4 , 10 and 20 \u00b5g ) were also analyzed ( input ) . YAMADA et al : ACTIN BUNDLING BY DYNAMIN AND CORTACTIN IN CANCER CELL MIGRATION 882 cellular processes is not entirely clear . We recently reported that actin bundling by the dynamin 1 - cortactin complex is crucial for neurite extension in developing neurons ( 12 ) . In this study , we examined the possibility that a similar F - actin - bundling mechanism is involved in the migration of H1299 cells , a human non - small cell lung carcinoma cell line . We showed that cortactin and dynamin 2 mostly colocal - ized along F - actin bundles in filopodia of serum - stimulated H1299 cells ( Fig . 2 ) . Pharmacological inhibition of dynamin 2 by Dynasore , Dynole 34 - 2 or MitMAB decreased cell migra - tion ( Fig . 1 ) and filopodial formation ( Fig . 3 ) . Furthermore , filopodia were shorter in dynamin 2 - and cortactin - depleted cells than in control cells ( Figs . 3 and 4 ) . In cortactin - silenced cells , the exogenous expression of wild - type cortactin rescued the punctate - like localization of dynamin 2 and filopodial formation ( Fig . 4 ) . Both dynamin 2 and cortactin bundled Figure 3 . Knockdown of dynamin 2 decreases filopodial formation in H1299 cells . ( A ) Western blotting showing knockdown of dynamin 2 ( Dyn2 ) expression by RNAi in H1299 cells . \u03b2 - actin served as the control . Three micrograms of cell lysate from each sample was analyzed by gel electrophoresis . ( B ) F - actin was visualized in H1299 cells by Alexa Fluor 488 - phalloidin staining after knockdown of dynamin 2 . Extensive filopodial formation was observed in cells after serum stimulation ( left ) . Filopodial formation was inhibited in dynamin 2 - silenced cells ( right ) . Boxed areas correspond to enlarged images shown below . Scale bar , 20 \u00b5m ( upper panels ) , 5 \u00b5m ( lower panels ) . ( C ) Filopodial length in H1299 cells cultured in the presence or absence of serum . The cells were visual - ized by fluorescent confocal microscopy , and filopodial length was measured as described in Materials and methods . ( D ) Inhibition of filopodial formation by dynasore in serum - stimulated H1299 cells . Serum - starved cells were incubated with 240 \u00b5M dynasore for 30 min , and then stimulated with 10 % FBS for 45 min in the presence of 240 \u00b5M dynasore ( middle ) . Thereafter , dynasore was removed , and the cells were incubated in serum - containing medium for 45 min ( right ) . For the negative control , cells were cultured in the presence of 3 % DMSO ( left ) . All steps were performed at 37\u02daC . ( E ) Analysis of filopodial formation in the H1299 cells shown in ( D ) . The cells were analyzed by fluorescent confocal microscopy , and filopodial length was measured . Results in ( C ) and ( E ) represent the means \u00b1 SEM from three independent experiments . INTERNATIONAL JOURNAL OF ONCOLOGY 49 : 877 - 886 , 2016 883 Figure 4 . Knockdown of cortactin decreases filopodial formation in H1299 cells . ( A ) Western blotting showing knockdown of cortactin expression by RNAi in H1299 cells . \u03b2 - actin was used as the control . Three micrograms of cell lysate from each sample was analyzed by gel electrophoresis . ( B ) F - actin was visualized in serum - stimulated H1299 cells by Alexa Fluor 488 - phalloidin staining . Boxed areas correspond to enlarged images shown below . Similar to results from dynamin 2 - depleted cells , filopodial formation decreased in cortactin - depleted cells ( right ) . Scale bar , 20 \u00b5m ( upper panels ) , 5 \u00b5m ( lower panels ) . ( C ) Analysis of filopodial formation in H1299 cells cultured with or without serum . The samples were analyzed by fluorescent confocal microscopy , and the filopodial length was measured . ( D ) Expression of wild - type cortactin rescues filopodial formation . Cortactin - depleted H1299 cells were transfected with rat wild - type cortactin ( left ) or cortactin W525K ( right ) cloned into the pIRES2 - AcGFP1 expression vector . Boxed areas correspond to enlarged images shown ( middle panels ) . Transfected cells were identified by GFP expression ( bottom panels ) . Scale bar , 20 \u00b5m ( top and bottom panels ) , 3 . 5 \u00b5m ( middle panels ) . ( E ) Analysis of filopodial formation in H1299 cells . The samples were analyzed by fluorescent confocal microscopy , and the filopodial length was measured . Results in ( C ) and ( E ) represent the means \u00b1 SEM from three independent experiments . ( F ) Rescue of the punctate - like localization of dynamin 2 along F - actin bundles in filopodia by re - expression of cortactin in cortactin - depleted cells . Cortactin - depleted H1299 cells ( left panels ) were transfected with rat wild - type cortactin or cortactin W525K cloned into the pIRES2 - AcGFP1 expression vector ( right panels ) . The cells were immunostained with an anti - dynamin 2 antibodies . Boxed areas correspond to enlarged images shown below . Transfected cells were identified by GFP expression ( right bottom panels ) . Scale bar , 5 \u00b5m ( top and right bottom panels ) , 2 . 3 \u00b5m ( left bottom and right middle panels ) . YAMADA et al : ACTIN BUNDLING BY DYNAMIN AND CORTACTIN IN CANCER CELL MIGRATION 884 F - actin , and these proteins increased F - actin stability ( Fig . 5 ) . These results indicate that dynamin 2 and cortactin partici - pate in cancer cell migration by stabilizing F - actin bundles in filopodia . Dynamin assembles at the neck of deeply invaginated endocytic pits ( 30 ) . Upon GTP hydrolysis , however , dynamin undergoes a conformational change , resulting in the fission of endocytic pits and release of endocytic vesicles ( 31 - 33 ) . In addition , dynamin 1 forms a ring - like complex with cortactin , which switches from an open to a closed state upon GTP hydrolysis . This change promotes the bundling of F - actin filaments ( 12 ) . The mechanism of actin bundling mediated by the dynamin 2 - cortactin complex is similar to that of the dynamin 1 - cortactin complex , because dynamin 2 and cortactin also facilitated the formation of long and thick F - actin bundles to which they colocalized ( Fig . 5 ) . This mechanochemical Figure 5 . Actin bundling by dynamin 2 and cortactin stabilizes F - actin bundles . ( A ) Long F - actin bundles were formed in the presence of dynamin 2 and cortactin ( lower left ) . Preformed F - actin ( 3 . 3 \u00b5M ) was incubated with or without the indicated proteins ( 5 \u00b5M each ) . F - actin was visualized with Alexa Fluor 488 - phalloidin . Scale bar , 30 \u00b5m . ( B ) Representative images acquired by fluorescent microscopy showing the localization of dynamin and cortactin along F - actin bundles . Actin bundles were formed in vitro by incubating dynamin 2 with wild - type cortactin ( left ) or dynamin 1 and wild - type cortactin ( right ) . Protein colocalization was performed by double - immunofluorescence . Scale bar , 2 \u00b5m . ( C ) Kinetics of F - actin disassembly induced in 10 - fold diluted preformed pyrene - labeled F - actin solution with buffer . F - actin bundles disassembled in the presence of dynamin 2 and cortactin , as well as in the presence of 5 \u00b5M \u03b1 - actinin . The rate of F - actin bundle disassembly was measured by pyrene - fluorescence . INTERNATIONAL JOURNAL OF ONCOLOGY 49 : 877 - 886 , 2016 885 property may be critical for the formation of F - actin bundles in filopodia of other cell types as well ( Fig . 6 ) . Additional studies are needed to determine the precise mechanism . Dynamin associates with tumorigenesis , particularly tumor cell migration and invasion . For example , increased dynamin 2 expression potentiates the migration and invasion of pancreatic ductal cancer cells ( 25 ) , and tyrosine phosphorylated dynamin 2 promotes the growth and invasiveness of glioblastomas ( 34 ) . Thus , the involvement of dynamin in the formation of F - actin bundles might promote cancer malignancy . In conclusion , we showed that dynamin 2 and cortactin participate in the formation of F - actin bundles , which stabi - lize filopodia in migrating cancer cells . Taken together , these results suggest that dynamin might be a potential molecular target for anticancer therapy . Acknowledgements The authors thank Yuki Masuoka , Dr Shun - AI Li , and Nana Okazaki for technical assistance . This study was supported in part by grants from the Ministry of Education , Science , Sports , and Culture of Japan ( grant no . 26670201 to H . Y . ; grant no . 15K1533007 to K . T . ) , the Astellas Foundation for Research on Metabolic Disorders ( to H . Y . ) , and the Japan Foundation for Applied Enzymology ( to H . Y . ) . References 1 . Arjonen A , Kaukonen R and Ivaska J : Filopodia and adhesion in cancer cell motility . Cell Adhes Migr 5 : 421 - 430 , 2011 . 2 . Ridley AJ : Life at the leading edge . Cell 145 : 1012 - 1022 , 2011 . 3 . Takei K , Slepnev VI , Haucke V and De Camilli P : Functional partnership between amphiphysin and dynamin in clathrin - mediated endocytosis . Nat Cell Biol 1 : 33 - 39 , 1999 . 4 . Mettlen M , Pucadyil T , Ramachandran R and Schmid SL : Dissecting dynamin ' s role in clathrin - mediated endocytosis . Biochem Soc Trans 37 : 1022 - 1026 , 2009 . 5 . 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Oncogene 31 : 2691 - 2702 , 2012 .", + "xu2024myosini": "1 Myosin - I Synergizes with Arp2 / 3 Complex to Enhance Pushing Forces 1 of Branched Actin Networks 2 3 Mengqi Xu 1 \u2020 , David M . Rutkowski 2 \u2020 , Grzegorz Rebowski 1 , Malgorzata Boczkowska 1 , Luther W . Pollard 1 * , 4 Roberto Dominguez 1 * , Dimitrios Vavylonis 2 * , E . Michael Ostap 1 * 5 1 Department of Physiology , Pennsylvania Muscle Institute , Perelman School of Medicine , University of 6 Pennsylvania , Philadelphia , PA 19104 . 2 Department of Physics , Lehigh University , Bethlehem , PA . 7 Email : ostap @ pennmedicine . upenn . edu , vavylonis @ lehigh . edu , 8 droberto @ pennmedicine . upenn . edu , Luther . Pollard @ pennmedicine . upenn . edu 9 Author Contributions : \u2020 These authors contributed equally . 10 Abstract ( 149 words ) 11 Myosin - Is colocalize with Arp2 / 3 complex - nucleated actin networks at sites of membrane protrusion and 12 invagination , but the mechanisms by which myosin - I motor activity coordinates with branched actin 13 assembly to generate force are unknown . We mimicked the interplay of these proteins using the \u201ccomet 14 tail\u201d bead motility assay , where branched actin networks are nucleated by Arp2 / 3 complex on the surface 15 of beads coated with myosin - I and the WCA domain of N - WASP . We observed that myosin - I increased 16 bead movement efficiency by thinning actin networks without affecting growth rates . Remarkably , myosin - 17 I triggered symmetry breaking and comet - tail formation in dense networks resistant to spontaneous 18 fracturing . Even with arrested actin assembly , myosin - I alone could break the network . Computational 19 modeling recapitulated these observations suggesting myosin - I acts as a repulsive force shaping the 20 network ' s architecture and boosting its force - generating capacity . We propose that myosin - I leverages its 21 power stroke to amplify the forces generated by Arp2 / 3 complex - nucleated actin networks . 22 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 2 Introduction 23 Actin assembly stimulated by Arp2 / 3 complex provides pushing forces for diverse cellular 24 processes ( 1 - 8 ) , including lamellipodial protrusion , endocytosis , phagocytosis , and cell adhesion . After 25 being activated by membrane - associated nucleation promoting factors ( NPFs ) , Arp2 / 3 complex nucleates 26 new actin branches from the sides of pre - existing mother filaments , generating branched , dendritic actin 27 networks that exert pushing forces against or deform the membrane . The network geometry , assembly 28 kinetics , and mechanical properties are dynamically adapted by a set of actin binding proteins ( e . g . , capping 29 proteins , nucleation promoting factors , profilin , cofilin , and crosslinkers ) that respond to mechanical loading 30 ( 9 - 19 ) . 31 Class - I myosins ( myosin - Is ) ( 20 , 21 ) frequently colocalize with Arp2 / 3 complex - nucleated branched 32 actin networks near membranes ( 22 - 26 ) . As an actin - activated ATPase , the myosin - I motor dynamically 33 detaches and attaches from actin filaments in an ATP - dependent manner while generating force through a 34 lever arm - mediated power stroke ( 27 ) . Myosin - Is are single - headed , membrane - anchored motors that bind 35 directly to cell membranes and function in membrane deformation , cell adhesion , and intracellular trafficking 36 ( 21 ) . Although multiple cellular studies have shown that the myosin - I motor activity cooperates with dynamic 37 actin assembly by Arp2 / 3 complex at the plasma membrane , the functional outcome of this interaction 38 remains unknown . 39 To investigate the functional interaction between Arp2 / 3 complex and myosin - I in actin assembly , 40 we developed a biomimetic system using a comet - tail bead motility assay ( 9 , 12 , 28 ) . This system 41 recapitulates the colocalization of NPFs and myosin - I observed on cellular membranes by employing 42 micron - sized beads coated with both NPF and myosin - I . We investigated the impact of myosin - I on Arp2 / 3 43 complex - mediated branched actin assembly at varying network densities achieved through different 44 capping protein ( CP ) concentrations ( 12 , 14 , 29 ) . 45 Our findings revealed that myosin - I alters actin assembly kinetics by reducing Arp2 / 3 complex - 46 mediated branching at NPF - coated surfaces , resulting in a sparser actin network that exhibits enhanced 47 elongation efficiency . This effect is attributed to the pushing force exerted by the myosin - I power stroke 48 directly on surrounding actin networks , propelling actin filaments away . Furthermore , the myosin - I power 49 stroke generates sufficient force to disrupt the network and trigger actin shell breakdown . Notably , a 50 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 3 computational model at the molecular level provides mechanistic insights , suggesting that myosin - I 51 promotes force generation during Arp2 / 3 complex - mediated actin polymerization by exerting a repulsive 52 force on the branched network via its power stroke . 53 54 Results 55 Comet - tail bead motility assay 56 The comet - tail bead motility assay was used to investigate the effect of myosin - I activity on Arp2 / 3 57 complex - mediated branched actin assembly ( 9 , 28 ) . Full - length , biotinylated Drosophila Myo1d was 58 attached to the bead surface via neutravidin ( myosin - bead ; see Methods ; Fig . 1A ) alongside a GST - tagged 59 WCA domain from human N - WASP . Drosophila Myo1d was chosen as it functions optimally at 20 - 22 \u00b0C 60 ( 30 , 31 ) . Bead - bound Myo1d activity was confirmed by processive bead movement along actin filaments 61 ( Fig . 1B ; Movie S1 ) . Control - beads were made identically to the myosin - beads , except that a biotinylated 62 far - red fluorescent dye ( CF640 , see Methods ) was bound to the neutravidin ( Fig . 1A ) . Actin assembly 63 around myosin - and control - beads was imaged simultaneously by epifluorescence microscopy ( Fig . 1C ) . 64 Upon mixing of assay components , surface - bound NPFs stimulate Arp2 / 3 complex - mediated 65 branched actin assembly . Polymerization at the bead surface displaces the branched actin arrays 66 outwards , building tension in the actin network , and ultimately breaking symmetry and forming polarized 67 comet tails ( 9 , 14 , 32 - 35 ) ( Fig . 1C ) . By varying CP concentration , we created networks of varying actin 68 density that ranged from tightly packed and symmetry - breaking resistant ( dense ) to loosely woven and 69 fracture - prone ( sparse ) , allowing us to investigate Myo1d ' s influence on symmetry breaking , comet tail 70 formation , and morphology . 71 The morphology of comet tails generated by myosin - beads differed from control - beads at all 72 network densities achieved by varying CP concentrations ( Fig . 1D ; Fig . S1 ) , and these differences were 73 more pronounced at higher Myo1d densities . We identified three distinct regimes : high , intermediate , and 74 low network densities , controlled by the CP concentration ( 200 nM , 40 nM - 100 nM , and 30 nM , respectively ) 75 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 4 ( Fig . 1D ; Fig . S1 ) , and describe each regime in greater detail using the 0 . 43 myosin / 1 NPF ratio in the 76 following sections . 77 78 Myosin - I prevents comet tail formation from sparse actin networks 79 Under high CP concentrations ( 200 nM ) , actin elongation was suppressed by capping , resulting in 80 actin arrays consisting of short branches . As a result , actin comet tails grown from control - beads were 81 irregularly organized with sparse networks ( Fig . 1D ; Fig . 2A ) , consistent with previous reports ( 12 , 14 , 36 ) . 82 Myosin - beads ( 0 . 43 : 1 myosin / NPF ) failed to generate a comet tail ( Fig . 2A ; Movie S2 ) , but instead formed 83 loose actin clouds which diffused away from the bead when flow was present in the chamber . The presence 84 of myosin compromised the network cohesion , making it more prone to disruption , and inhibiting comet tail 85 formation . 86 87 Myosin - I facilitates fracturing of dense actin networks 88 At a low CP concentration ( 25 nM ) , long actin filaments emanating from bead surfaces become 89 entangled , forming dense actin networks that are highly resistant to fracture by actin polymerization forces 90 ( 12 , 14 , 34 , 36 ) . Control - beads remained encapsulated within the shell , without symmetry breaking , for > 91 15 minutes after the initiation of the polymerization ( Fig . 1D ; Fig . 2B ) . Strikingly , myosin - beads grown under 92 the same condition broke symmetry within 10 min of mixing ( Fig . 2B ; Movie S3 ) . This result suggests that 93 myosin - I may enhance the force generation during actin assembly and / or may alter actin architecture that 94 promotes network fracture and symmetry breaking . 95 96 Myosin - I induces efficient comet tail growth 97 At intermediate CP concentrations ( 40 nM - 100 nM ) , myosin - beads generated comet tails with 98 sparser actin networks compared to control - beads ( Fig . 1D ; Fig . S1 ; Fig . 3A - B ; Movie . S4 ) . Following the 99 time course of tail elongation , we found that the sparser network architecture of myosin - beads arises from 100 a 0 . 76 - fold lower actin assembly rate ( p < 0 . 0001 , n = 11 , Fig . 3E - F ) as quantified by measuring the 101 rhodamine - actin fluorescence in the comet tail over time . Remarkably , despite the lower actin assembly 102 rate , myosin comet tails in the presence of 50 nM CP elongated ( 0 . 69 \u00b1 0 . 10 \u03bcm / min ) at the same speed 103 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 5 as the control - beads ( 0 . 66 \u00b1 0 . 20 \u03bcm / min ; Fig . 3C - D ) . If we define the growth efficiency of actin comet tail 104 as the comet tail length divided by the total amount of actin incorporated , the myosin - beads exhibited a 1 . 4 - 105 fold higher growth efficiency than the control - beads ( Fig . 3G ) . 106 We quantified the amount of a fluorescently labeled Arp2 / 3 complex ( SNAP - Arp2 / 3 complex ; see 107 Methods ) in the comet tail ( Fig . 4A - C ) . Myosin - beads showed significantly reduced levels of SNAP - Arp2 / 3 108 complex ( p < 0 . 0001 , n = 33 , Fig . 4C ) in the network . This reduced Arp2 / 3 complex level also resulted in 109 higher actin to Arp2 / 3 complex ratios on myosin - beads ( Fig . 4D ) , indicating a sparse network organization 110 with fewer branch points but longer filaments ( 15 ) . Despite the reduced amount of Arp2 / 3 complex in the 111 comet tails , we observed equivalent SNAP - Arp2 / 3 complex fluorescence on the control - and myosin - bead 112 surfaces ( Fig . 4E ) , suggesting that myosin - I does not affect the loading of Arp2 / 3 complex onto the bead - 113 bound NPF . Rather , Arp2 / 3 complex is recruited to NPF - coated beads and is ready for the arrival of a 114 mother filament and G - actin to initiate branched nucleation . Taken together , myosin - beads have less dense 115 network structure as a result of reduced Arp2 / 3 complex - stimulated actin branching . 116 We note that the myosin effect on growth efficiency depends on the CP concentration ( Fig . 1D ) . 117 Under low CP conditions ( < 50 nM ) , myosin beads grew even longer tails than control - beads with sparse 118 networks , suggesting an even higher growth efficiency than the 50 nM CP case as quantified above . High 119 CP concentrations ( > 50 nM ) resulted in highly diffuse networks whose cohesion was easily compromised 120 by myosin - I , causing actin dispersal , thus making their growth efficiency difficult to assess ( 12 , 14 , 17 ) . 121 122 The myosin power stroke is required to alter network architecture 123 We examined the role of myosin - I mechanochemical activity on actin assembly by removing ATP , 124 resulting in the population of a long - lived , actin - bound , rigor state ( i . e . rigor myosin ) ( 31 ) . To maintain normal 125 actin polymerization , G - actin was pre - treated with ATP and gel filtered to eliminate free nucleotide ( see 126 Methods ) . Rigor myosin inhibited the formation of monopolar comet tails , while control - beads generated 127 comet patterns as previously observed when free ATP was present ( Fig . 5A - C ) . At 50 nM CP , rigor myosin - 128 beads formed dense actin shells , which subsequently fractured , forming multiple short tails of high network 129 density ( Fig . 5A - C ; Fig . S3C ; Movie S5 ) . Restoring myosin motor activity by adding ATP reproduced the 130 sparse comet architecture with high growth efficiency as observed before ( Fig . 5B ; Fig . S3B ) . At the two 131 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 6 extreme CP conditions ( 15 nM and 200 nM ) , rigor myosin - beads neither enhanced symmetry breaking nor 132 shed actin away ( Fig . 5C ; Fig . S3D ) . Instead , actin shells were formed around the beads as a result of the 133 strong actin - binding characteristics of rigor myosin . In addition to probing the effect of rigor myosin , we 134 coupled biotinylated , Halo - tagged , actin - binding domain ( Halo - ABD ) of \u03b1 - actinin to the beads , which binds 135 actin filaments more dynamically than rigor myosin but does not undergo a power stroke ( 31 , 37 , 38 ) . 136 HaloABD - beads behaved similarly to rigor myosin ( Fig . 5C ; Fig . S3A ; Movie . S6 ) . 137 To further elucidate the role of the myosin mechanochemistry in modulating the actin network , we 138 uncoupled the myosin actin - activated ATPase activity from the power stroke by adding calcium . Calcium 139 weakens the affinity of the lever - arm stabilizing calmodulin light chains , resulting in an inhibited power 140 stroke but preserved ATPase activity ( 27 , 39 , 40 ) . We confirmed calcium inhibition using the in vitro gliding 141 assay , where the Myo1d - powered F - actin gliding ( speed 90 . 2 \u00b1 14 . 5 nm / s ) was completely halted in the 142 presence of 100 \u00b5M free calcium ( Fig . S3E - F ) . Under these conditions , myosin - beads generated slightly 143 shorter comet tails with network densities that were similar to control - beads ( Fig . 5D - F ) . We conclude that 144 myosin power stroke is required to alter the network architecture and induce a sparse actin organization 145 with higher growth efficiency . 146 147 The myosin power stroke alone can fracture the actin shell 148 We decoupled the force generated by myosin power stroke from the force generated by actin 149 polymerization using Arp2 / 3 complex inhibitor , CK - 666 , and the polymerization inhibitor , Latrunculin B 150 ( LatB ) ( 12 , 41 ) . The goal was to arrest actin assembly around the bead prior to the fracturing of the actin 151 shell that results in symmetry breaking . Polymerization inhibitors were added ~ 100 s after the initiation of 152 polymerization . Strikingly , most of the myosin - beads fractured the actin shell and ejected the bead 15 - 20 153 min after polymerization arrest . In contrast , control - beads remained enclosed in the actin shell without 154 observable changes during a 40 min time window ( Fig . 6A ; also see Fig . S4 and Movie S7 - 8 acquired in 155 the absence of phalloidin ) . 156 Shell fracture events were quantified by defining a shell - breaking angle , \ud835\udf03 , where \ud835\udf03 = 0 , indicates 157 no detected shell fracture ; 0 \u00b0 < \ud835\udf03 < 180 \u00b0 , indicates shell fracture ; and \ud835\udf03 \u2265 180 \u00b0 indicates the bead has 158 been ejected from the shell ( Fig . 6B ) . We found 80 % of the myosin - beads were ejected from the shell and 159 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 7 an additional 11 % had fractured shells , while the corresponding control - beads showed only 16 % shell 160 fracture ( Fig . 6C ) . Inhibition of myosin motile activity by adding 10 mM ADP ( 31 ) together with Latrunculin 161 B and CK - 666 resulted in substantial reduction in shell fracture events and eliminated bead ejection ( Fig . 162 6A , C ; Fig . S4C - D ) . Finally , we performed experiments with a higher CP concentration ( 100 nM ) that results 163 in control - beads of lower actin shell densities ( Fig . 6A ) and found only 2 % shell fracture ( Fig . 6C ) , which 164 further confirmed that the shell breaking was a direct result of myosin power stroke , and is not attributed to 165 differences in the shell network density . 166 167 Simulations show myosin - I forces produce sparser actin networks and aid bead propulsion 168 We developed a filament - level computational model with an overall system size comparable to our 169 experimental setup ( Fig . 7A , see Methods , Table S1 ) . We incorporated the myosin - I power stroke as a 170 repulsive force that pushes actin away from the bead surface , and explore whether this myosin - induced 171 pushing mechanism reproduces the experimentally observed comet tail patterns . In this model , we 172 represented semiflexible actin filaments as beads connected by springs , polymerizing at their barbed ends 173 and pushing against a spherical bead according to Brownian - ratchet - type force - elongation relationship . 174 Spontaneous filament nucleation and branching at 70\u00ba angles occurs close to the bead ; elongation stops 175 by capping when filaments reach a specified length . The effect of fluid and bead - filament friction was 176 combined into a single bead friction parameter . Excluded volume interactions prevent filament crossing , 177 resulting in tensile and compressive stresses developing within a shell of branched actin filaments that 178 nucleates uniformly around an initially bare bead . By allowing filaments to break or debranch above a 179 certain tensile force or branch angle threshold , we found that these networks can crack open , leading to 180 symmetry breaking and bead propulsion ( Fig . 7B ; Movie S9 ) as we observed in the experiments ( Fig . 1C ) . 181 To account for changes in CP concentration , we varied the average filament branch length in the 182 simulations . 183 The effect of the myosin - I power stroke was modeled as constant tangential pushing forces of 184 magnitude F myo acting along every actin filament segment close to the bead , with equal and opposite force 185 on the bead ( Fig . 7A ) . With myosin - I pushing force incorporated , simulations recapitulated many of the 186 experimental findings . For the short filament scenario ( high CP concentration ) , simulated myosin - beads 187 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 8 showed a significant delay or absence of symmetry breaking due to myosin pushing short filaments away 188 from the bead surface ( Fig . S5A , C ; Movie S10 ) . For the long filament scenario ( low CP concentration ) , 189 simulations reproduced the accumulation of a dense actin shell around the beads , which significantly 190 inhibited the symmetry breaking of the control - beads ( Fig . S5B , C ; Movie S11 ) . Myosin - beads by contrast 191 promoted the growth of an asymmetric dense cloud ( Fig . S5B , C ) , similar to the experimental observations 192 in early stages prior to bead ejection ( Fig . 2B ) . We note that simulations do not account for depletion of bulk 193 actin occurring during late stages of bead ejection in experiment ( Fig . 2B ) . We also note a faster 194 accumulation of actin in the simulations compared to the experiments ( Fig . S5B ) , which suggests additional 195 factors limiting network growth not considered in the model . For the intermediate filament length scenario 196 ( i . e . intermediate CP concentration ) , simulations showed a similar elongation speed for the myosin - bead 197 compared to the control - bead , albeit with a less dense actin network ( Fig . 7D - F ; Movie S12 ) , in agreement 198 with the experimental observations ( Fig . 3A - F ) . Further , simulation predicted that actin filaments in the 199 myosin comet tail experienced less stress ( Fig . 7C ) , possibly due to the sparse actin organization , which 200 reduces stresses arising from piling interconnected actin filaments on top of each other . 201 Notably , the ratio of actin to Arp2 / 3 complex density in simulated comet tails did not increase in the 202 presence of myosin ( Fig . 7F - G ; Fig . S6A ) , as observed in experiments ( Fig . 4D ) . This fixed ratio is a 203 consequence of our fixed - filament - length assumption to mimic a certain CP concentration . This assumption 204 validates when capping rate reduces equally as actin polymerization rate in response to opposing force , as 205 reported by Li et al ( 15 , 19 ) . However , the mismatch between our simulations and the experiments suggests 206 an inequivalent effect on actin polymerization and CP capping due to the pushing force of myosin . We also 207 tested whether the presence of myosin would promote actin debranching , which could also change the 208 actin to Arp2 / 3 ratio . We found that myosin forces do not enhance debranching in our simulations ( Fig . S6 ) . 209 Indeed , less debranching occurs since comet tail stresses are smaller in the presence of myosin ( Fig . 7C ) . 210 We next dissected the force contribution from different sources ( actin polymerization or myosin 211 power stroke ) that powered the bead propulsion in our simulations . Strikingly , we found that while in control - 212 beads the symmetry breaking and net comet propulsion were exclusively powered by actin polymerization , 213 in myosin - beads , the myosin pushing forces contributed significantly to bead propulsion after symmetry 214 breaking , and had a predominant effect at high F myo ( Fig . 7H ; Fig . S7 ) . Simulated myosin forces also partly 215 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 9 reorganized the network such that a larger fraction of actin filaments was polymerizing facing more 216 perpendicular to the bead surface at larger myosin forces ( Fig . 7I ) . When actin polymerization was arrested 217 before shell breaking , simulations replicated the experimental findings that myosin pushing forces alone 218 can eject the bead out of an actin shell ( Fig . 7J ; Movie S13 ) , provided the shell is not too dense or too 219 sparse ( Fig . S8 ) 220 Overall , our simulations with the myosin - I power stroke incorporated as an effective repulsive force 221 replicate most of our experimental observations and support the experimental finding that myosin - I 222 enhances force generation during branched actin assembly to promote shell breaking and enhance the 223 efficiency of bead propulsion . 224 225 Discussion 226 Myosin - I force generation and modulation of actin network density 227 The primary finding of this study is that Myo1d synergizes with Arp2 / 3 complex to enhance the 228 pushing forces of branched actin networks , and that this force enhancement requires the myosin power 229 stroke . Importantly , we also found that Myo1d alters the actin network composition grown from NPF beads , 230 producing a less dense actin network with decreased incorporation of Arp2 / 3 complex . 231 How does myosin - I modulate Arp2 / 3 complex incorporation ? We propose a model where myosin - 232 I motor activity pushes the actin filaments away from the bead surface , reducing the accessibility of actin 233 binding sites for NPF - activated Arp2 / 3 complex to bind and nucleate new branches ( Fig . 8 ) . Alternatively , 234 the mechanochemistry of myosin - I may differently affect the incorporation of actin monomer and CP to 235 filament ends in a way that promotes actin elongation while slowing capping ( 19 , 42 ) . Additional 236 experiments that measure the actin elongation rate and capping kinetics near the NPF - coated surface are 237 required to test this hypothesis . 238 We do not favor a model in which myosin - I sterically inhibits NPFs from binding and activating 239 Arp2 / 3 complex for three reasons . First , the concentration of SNAP - Arp2 / 3 complex bound on beads is 240 unchanged in the presence of myosin ( Fig . 4E ) . Second , networks grown in the presence of calcium where 241 the myosin power stroke was uncoupled from actin binding showed similar actin densities between myosin - 242 and control - beads ( Fig . 5 D , F ) , which ruled out the possibility that the sparse actin organization was due to 243 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 10 the competition between myosin - I and Arp2 / 3 complex for actin binding sites . Finally , calculations of 244 molecular occupancy on bead surface through protein quantification verified that the steric hindrance effect 245 of myosin - I is likely negligible ( See SI for detailed quantifications ) . 246 It remains possible that force generation by myosin - I debranches actin filaments , resulting in less 247 dense network with reduced Arp2 / 3 incorporation . Mechanical forces ranging from 0 pN to 2 pN dissociate 248 actin branches from their mother filaments together with Arp2 / 3 complex ( 43 ) . A myosin - I paralog ( Myo1b ) 249 that generates such forces has been shown to dissociate branches via motor activity ( 44 ) . Although our 250 simulations disfavor this hypothesis ( Fig . S6 ) , a role for myosin - I induced debranching should be explored 251 further in the cell . 252 Finally , it will be intriguing to explore these models further by performing experiments and 253 computational modeling to probe network growth under mechanical load in the presence of myosin - I , 254 especially given previous work that demonstrates the substantial loading effects on network architecture 255 and power generation ( 15 , 19 ) . 256 257 Myosin - I Cell Biology 258 Myosin - Is connect the actin cytoskeleton to cellular membranes where they contribute to plasma 259 membrane dynamics , organelle deformation , and shaping of actin network architecture . Although the 260 molecular details of myosin - I function have been difficult to determine , recent studies suggest some 261 paralogs are powerful motors that have active roles in shaping membranes . For example , myosin - Is in 262 budding yeast have mechanochemistry suitable for generating power working with polymerizing actin to 263 drive membrane invagination during endocytosis ( 23 , 24 , 45 ) . Our current study confirms the ability of 264 myosin - I to exert substantial pushing forces capable of fracturing actin networks . This power - generating 265 capacity likely translates across species , with vertebrate Myo1c regulating actin architecture in diverse 266 cellular regions ( 46 - 48 ) and Myo1e playing key roles in processes like endocytosis ( 49 , 50 ) , phagocytosis 267 ( 26 ) , and cancer invadosome formation ( 51 , 52 ) . Taken together , these findings point to some myosin - Is 268 as dynamic players , actively shaping cellular structures and processes through their unique ability to both 269 link and manipulate membranes and the actin cytoskeleton . 270 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 11 Not all paralogs are expected to modulated the actin networks as Myo1d . Notably , some myosin 271 paralogs have substantially slower kinetics which are better suited for a motor that functions as dynamic 272 tether , providing force - dependent linkages between actin and membranes ( 22 - 24 , 45 , 48 , 53 , 54 ) . Our 273 experiments performed at low ATP concentrations ( Fig . 5 A - C ) mimic the behavior of myosins with slow 274 motility rates , and show clearly that slow kinetics can inhibit network fracturing and comet tail growth . 275 Further studies to investigate how the intrinsic kinetic properties of different myosin - I paralogs influence 276 actin polymerization and membrane dynamics will further reveal the diverse role of myosin - I function ( 23 , 277 24 , 26 , 45 , 48 , 54 ) . 278 279 Summary 280 Overall , our study provides insights into how myosin - I molecules coordinate with Arp2 / 3 complex , 281 regulating the dynamics and architecture of the branched actin network and promoting the actin - based 282 motile force generation ( Fig . 8 ) . This work sheds light on synergy between myosin motor activity and actin 283 polymerization , underscoring their collective role in driving morphological changes at the cellular membrane 284 interfaces . Future work to determine how myosin - I affects actin network architecture and polymerization 285 forces will reveal the molecular roles of this important myosin family . 286 287 Materials and Methods 288 Protein Purification 289 Actin was purified from rabbit skeletal muscle acetone powder as previously described ( 55 ) . Monomeric G - 290 actin was purified by gel filtration on Sephacryl S - 300 in G - buffer ( 2 mM Tris \u2013 HCl [ pH 8 . 0 ] , 0 . 2 mM ATP , 291 0 . 1 mM CaCl 2 , 1 mM NaN 3 and 0 . 5 mM DTT ) and used within 2 - 3 weeks . Actin was labeled with NHS - 292 Rhodamine at random surface lysine residues ( 56 ) . Full - length Drosophila Myo1d , with C - terminal FLAG - 293 Avi tags , was expressed , purified as previously described ( 57 ) , and subsequently biotinylated at the C - 294 termini Avi tag sequence via BirA biotin - protein ligase ( Avidity ) ( 31 ) . GST - tagged WCA domain of human 295 WASP protein ( GST - WCA ) was purchased from Cytoskeleton ( Cat . # VCG03 - A ) and used without further 296 purification . Arp2 / 3 complex was isolated from bovine brain as previously described ( 58 ) . SNAP - tagged 297 Arp2 / 3 complex was constructed and purified as described ( 59 ) . SNAP - tagged Arp2 / 3 complex was labeled 298 with SNAP - Surface 488 ( Biolab , Cat . # S9124S ) using commercially provided protocol . Human CapZ was 299 expressed and purified as previously described ( 60 ) . Halo - ABD was constructed and purified as described 300 in ( 61 ) . 301 302 Bead Preparation 303 Carboxylate polystyrene beads ( Polybead , 2 . 0 \u03bcm diameter , CAT . # 18327 - 10 ) were purchased from 304 Polysciences . Beads were coated with NPF and neutravidin following a previous protocol ( 28 ) with 305 modifications . Briefly , 5 - 10 \u03bcL bead slurry was washed with X buffer ( 10mM HEPES [ pH7 . 5 ] , 100 mM KCl , 306 1 mM MgCl 2 , 100 \u03bcM CaCl 2 , 1 mM ATP ) , and then incubated with 50 - 100 \u03bcl 2 . 3 \u03bcM ( 0 . 1mg / ml ) GST - WCA 307 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 12 and various concentrations of Neutravidin ( ThermoFisher , Cat . # 31000 ) ( 8 . 3 \u03bcM ( 0 . 5mg / ml ) for 308 # myosin / NPF = 0 . 28 : 1 , 16 . 7 \u03bcM ( 1mg / ml ) for # myosin / NPF = 0 . 35 : 1 , 33 . 3 \u03bcM ( 2mg / ml ) for 309 # myosin / NPF = 0 . 43 : 1 , or 83 . 3 \u03bcM ( 5mg / ml ) for # myosin / NPF = 0 . 80 : 1 ) on a slow rotator at 4 \u00b0 C for 2hr . 310 Beads were then pelleted by spinning at 16000 g for 2 min at 4 \u00b0 C , to remove the unreacted reagents , and 311 then resuspended in 200 - 400 \u03bcL 10mg / mL BSA , incubating on ice for 30 min to block the free space left on 312 bead surface . Finally , beads were washed twice and stored in 50 - 100 \u03bcL 1mg / mL BSA in X buffer for up to 313 3 days . 314 315 The NPF and neutravidin - coated beads were next split in half , with one half coupled with biotinylated 316 Drosophila Myo1d ( myosin - bead ) or biotinylated Halo - ABD , and the other half coated with Biotin - CF640 317 ( Biotium , Cat . # 80032 ) fluorescence dye ( control - bead ) . The NPF and neutravidin - coated beads were first 318 washed with M buffer ( 20 mM HEPES [ pH7 . 5 ] , 100 mM KCl , 1 mM MgCl 2 , 1 mM EGTA , 2 mM ATP ) to 319 remove the free calcium in the storage X buffer , and were then incubated with 1 \u03bcM biotinylated Drosophila 320 Myo1d , 1 \u03bcM Halo - ABD , or 1 \u03bcM Biotin - CF640 fluorescence dye for 30 min on ice . Beads were then pelleted 321 under 16000xg for 2 min at 4 \u00b0 C , to remove the free unbound reagents , and washed with 1mg / mL BSA in 322 M buffer and used immediately for motility assay on the same day after preparation . 323 324 Bead motility assay / comet tail assay 325 Unless specified otherwise , a typical motility mixture contained 4 \u03bcM actin ( 5 % Rhodamine labeled ) , 200 326 nM Arp2 / 3 complex or SNAP - Arp2 / 3 complex , 6 . 5 - 200 nM CP , 2 \u03bcM Calmodulin , and 3 \u03bcL bead slurry 327 ( 1 . 5 \u03bcL of myosin - beads or HaloABD - beads , and 1 . 5 \u03bcL of control - beads ) , mixed in 20 mM HEPES [ pH7 . 5 ] , 328 100 mM KCl , 1 mM MgCl 2 , 1 mM EGTA , 1 mM MgATP , 40 mM DTT , 10 mg / mL BSA and 0 . 2 % 329 methylcellulose , contributing to a final volume of 50 \u03bcL . The activity of SNAP - Arp2 / 3 complex is slightly 330 lower than the unlabeled complex , so we changed the CP concentration ( 40 nM ) to achieve similar tail 331 lengths , actin densities and growth efficiencies as observed for the native complex ( Fig . 4 ) . For experiments 332 with calcium present , G - actin was pre - incubated with 200 \u00b5M EGTA and 50 \u00b5M MgCl 2 for 5 min to be 333 exchanged to Mg - G - actin before using . Calcium experiments included 1 . 1 mM CaCl 2 in place of Calmodulin 334 to disrupt the myosin power stroke . Beads were first mixed with all other reagents , excluding actin , with a 335 pipette , to ensure an even distribution . The reaction was then initiated by adding actin into the system , 336 mixed thoroughly , and denoted as time 0 . Slides and coverslips were wiped with 70 % ethanol and ddH 2 O , 337 followed by a plasma cleansing for 10 min . Upon mixing , a volume of 2 . 1 \u03bcL motility mixture was carefully 338 applied between a glass slide and a coverslip ( 22 mm \u00d7 22 mm ) forming a so called ' squeeze chamber ' 339 with a height of 4 . 3 \u03bcm . The squeeze chamber was then sealed with vacuum grease and imaged 340 immediately under the microscope . Time - lapse movies were acquired of microscope fields that included 341 both myosin - and control - beads . In most cases , the actin comet tails emerging from myosin - or control - 342 beads grew with a constant speed during the first ~ 10 minutes following symmetry breaking . As the 343 reagents in the polymerization mixture depleted , tail elongation slowed and eventually stopped ~ 30 min 344 after mixing . 345 346 347 Fluorescence imaging and Data analysis 348 Fluorescence microscopy imaging was performed via Leica DMIRB epifluorescence microscope ( 100x , oil - 349 immersive objective of numerical aperture 1 . 4 ) and Metamorph ( Molecular Devices ) imaging software . 350 Movies were recorded at 25 \u00b0 C and acquired every 10 s for 30 - 60 min . Exposure time was 200 ms for most 351 experiments , and 1s to image the SNAP - Surface 488 labeled Arp2 / 3 complex . Images were analyzed and 352 quantified using Fiji software . Actin comet tail length were measured manually at each frame using the 353 segmented line draw tool ( from the end of the tail to the center of the bead ) and converted to the microns . 354 Tail growth rate and fluorescent assembly rate were calculated by fitting the first 7 to 10 data points in the 355 time courses to get the initial slope of the growth and assembly . Growth efficiency was determined by 356 dividing the tail growth rate by the fluorescent assembly rate . Image brightness and contrast were carefully 357 adjusted using Fiji and Adobe Illustrator . Unless otherwise specified , the lookup table for each pair of 358 control - and myosin - bead ( or HaloABD - bead ) were kept the same . 359 360 Statistical analysis 361 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 13 The statistical significance was calculated using paired or unpaired two - tail Student\u2019s t test in Prism v9 . 0 . 362 Further details are described in figure legends . 363 364 Modeling methods 365 We simulated actin comet tail formation at the level of individual filaments , each represented as a series of 366 segments of length \ud835\udc59 ! , following earlier work ( 62 ) . The pointed ends of actin filament branches are assumed 367 to be connected to a point element of a mother filament , at the location of Arp2 / 3 complex . Such a filament 368 representation allows us to model the effect of myosin as a tangential force acting along filament segments 369 close to the nucleating bead . We thus generalize earlier filament models that did not explicitly account for 370 filament bending mechanics ( 35 , 63 - 65 ) , earlier mechanical models that did not monitor the whole network 371 of actin filaments ( 36 , 66 - 69 ) , or modeled the full process of symmetry breaking and propulsion ( 70 ) . We 372 do not explicitly consider diffusiophoretic contributions to bead motion ( 71 ) . ( Also see SI for further details ) . 373 Forces on actin filaments 374 The position \ud835\udc93 \ud835\udc8a of point element i of actin filaments / filament branches evolves according to ( 62 ) 375 \ud835\udf01 # \ud835\udc93 \ud835\udc8a # % = \ud835\udc6d & \u2019 ( ) & * + + \ud835\udc6d & , - * # + \ud835\udc6d & . * + / - + \ud835\udc6d & - 01 / 2 # - # + \ud835\udc6d & 345 . 376 Here \ud835\udf01 is an effective filament segment drag coefficient that allows the actin network to evolve through 377 approximate quasi - static mechanical equilibrium , while also providing numerical stability . The spring force 378 is \ud835\udc6d & \u2019 ( ) & * + = \ud835\udc58 . 1 % & * , \ud835\udc51 & ( & 78 ) \u2212 \ud835\udc59 ! / \ud835\udc510 & ( & 78 ) \u2212 \ud835\udc58 . 1 % & * , \ud835\udc51 ( & : 8 ) & \u2212 \ud835\udc59 ! / \ud835\udc510 ( & : 8 ) & where ( i - 1 ) , ( i + 1 ) are neighboring point 379 elements ( if they exist ) before and after i , \ud835\udc51 & ; is the separation distance between i and j , and \ud835\udc510 & ; is the unit 380 vector from i to j . The equilibrium length is \ud835\udc59 ! , except for ( i ) uncapped barbed ends that elongate on average 381 according to \ud835\udc59 ! ( \ud835\udf0f ) = \ud835\udc59 ! ! + \ud835\udeff \ud835\udc5f ! ( 5 / \ud835\udf0f after their initiation at \ud835\udf0f = 0 , in discrete steps of half - monomer size \ud835\udeff = 382 2 . 7 nm ( see below ) and ( ii ) a short branch segment joining the mother filament point element to the daughter 383 pointed end , which has length of \ud835\udc59 , ) . * 1 < . 384 The bending force is \ud835\udc6d & , - * # = \ud835\udf05 / \ud835\udc59 . = + [ \ud835\udf15 , \ud835\udc510 & ( & 78 ) \u2219 \ud835\udc510 ( & : 8 ) & / / \ud835\udf15\ud835\udc93 & + , \ud835\udc510 ( & : 8 ) & \u2219 \ud835\udc510 ( & : > ) ( & : 8 ) / / \ud835\udf15\ud835\udc93 & + , \ud835\udc510 ( & 78 ) ( & 7 > ) \u2219 385 \ud835\udc510 & ( & 78 ) / / \ud835\udf15\ud835\udc93 & ] , \ud835\udf05 = \ud835\udc58 ? \ud835\udc47\ud835\udc59 ( is the flexural rigidity , \ud835\udc59 ( is the persistence length of the actin filament , and \ud835\udc59 . = + is 386 the average length of the two filament segments composing the angle . For the straight angle that exists 387 among the first three beads in the connection between mother and daughter filaments \ud835\udc59 . = + = \ud835\udc59 ! for 388 numerical stability . 389 An angular potential keeps Arp2 / 3 complex branches at 70 \u00b0 . The angular force is \ud835\udc6d 3 . * + / - = 390 \ud835\udf16 . * + / - , \ud835\udc50\ud835\udc5c\ud835\udc60\ud835\udf03 & ; @ \u2212 \ud835\udc50\ud835\udc5c\ud835\udc60\ud835\udf03 ! / \ud835\udf15 , \ud835\udc51 0 ; @ \u2219 \ud835\udc51 0 & ; / / \ud835\udf15\ud835\udc93 3 where \ud835\udf16 . * + / - is a spring constant , i , j , k are the indices of the point 391 elements that make up the angle , and m is one of these indices . 392 The excluded volume force is due to repulsion between two actin filaments and between actin filaments 393 and the nucleating bead . The former prevents crossing of filaments and is exerted along the direction of 394 vector \ud835\udc85 AB that connects the two closest approach points on the filament segments \u03b1 and \u03b2 ( 72 ) . It is 395 modeled as a stiff spring force with a max range of \ud835\udc51 - 01 / 2 # - # , with \ud835\udc6d A - 01 / 2 # - # , . 1 % & * = \u2212\ud835\udc6d B - 01 / 2 # - # , . 1 % & * = 396 \ud835\udc58 - 01 / , \ud835\udc51 AB \u2212 \ud835\udc51 - 01 / 2 # - # / \ud835\udc510 AB , where \ud835\udc51 AB is the minimum distance between neighboring filament segments . 397 This force is distributed to the endpoints of filament segments \u03b1 and \u03b2 ( including element i ) according to a 398 level arm rule . Though the typical diameter of actin filaments is around 7 nm , we set \ud835\udc51 - 01 / 2 # - # to be 20 nm 399 in order to mimic the effect of thermally fluctuating filaments taking up a larger volume ( which is not included 400 in our simulations ) . Additionally , since the excluded volume is modeled as a stiff spring rather than a hard - 401 core potential , some level of overlap between segments is possible . At a separation of order the filament 402 diameter , 7 nm , the excluded volume force is 22 pN . Filament segments experience a radially - oriented 403 excluded volume force with the nucleating bead if they are closer to the nucleator than R ; this force can be 404 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 14 written as \ud835\udc6d & - 01 / 2 # - # , , - . # = \ud835\udc58 - 01 / ( \ud835\udc51 & D \u2212 \ud835\udc45 ) \ud835\udc510 & D where \ud835\udc51 & D is the closest approach distance between the 405 filament segment and the nucleator bead . 406 Myosin forces are given by \ud835\udc6d & 345 = \ud835\udc39 345 \ud835\udc510 & ( & : 8 ) , where i - 1 is the neighboring actin filament point element 407 along the pointed end direction . It acts on all filament elements i closer than \ud835\udc51 345 to the nucleator bead 408 surface . The myosin force acts tangentially along the segment towards the pointed end . We do not model 409 the individual binding , lever arm motion , and unbinding of myosin ; instead the magnitude of \ud835\udc39 345 410 approximates a time and ensemble average over many binding and unbinding cycles . 411 Nucleating bead motion 412 The nucleating bead evolves through time according to : 413 \ud835\udf01 * 21 / , - . # \ud835\udc51\ud835\udc93 * 21 / , - . # \ud835\udc51\ud835\udc61 = \u2212 G ( \ud835\udc6d & - 01 / 2 # - # , , - . # + \ud835\udc6d & 345\u2019 & * ) & 414 Where the sum is over all actin segments that contact the bead through excluded volume or myosin 415 interactions , according to the interaction distances defined above . We assume the relative motion between 416 actin network and nucleating bead is dominated by frictional forces between them . We used a value of 417 \ud835\udf01 * 21 / , - . # that was large enough to prevent the rapid ejection of bare beads out of a shell at the onset of 418 symmetry breaking , a phenomenon that is not seen in our experiments . We thus monitor the relative motion 419 of the bead and actin network in the limit of quasi - static mechanical equilibrium of the actin network , 420 including implicit transient attachment and detachment of actin filaments to the bead . This approximation 421 does not account for the varying concentration of actin near the bead or the absolute motion of actin comet 422 and bead in the lab frame , which in reality would be influenced by small forces between the bead or actin 423 and the glass slide . 424 Actin barbed end polymerization rate 425 Polymerization of the barbed end of uncapped filaments occurs with the last segment of the actin filament 426 lengthening in increments of half - monomer size \u03b4 at a rate given by the polymerization rate . If the end 427 segment reaches a length of \ud835\udc59 ! , a new segment is added with initial length \ud835\udc59 ! ! . Polymerization of the barbed 428 end is attenuated by compressive forces on the spring bond connecting the barbed end point element to 429 the rest of the filament . The polymerization rate is \ud835\udc5f ( 5 / ( \ud835\udc47 ) = \ud835\udc5f ! ( 5 / exp ( \u2212\ud835\udc47 \ud835\udeff / \ud835\udc58 ? \ud835\udc47 ) , where the free filament 430 elongation rate is \ud835\udc5f ! ( 5 / and \ud835\udc47 = | \ud835\udc6d & \u2019 ( ) & * + | when \ud835\udc6d & \u2019 ( ) & * + is the compressive tension on the barbed end point 431 element i ( otherwise \ud835\udc5f ( 5 / ( \ud835\udc47 ) = \ud835\udc5f ! ( 5 / ) ( 73 , 74 ) . Because each filament segment starts with an initial length of 432 \ud835\udc59 ! ! ( for numerical stability ) , the rate \ud835\udc5f ( 5 / ( 0 ) is effectively multiplied by a factor of 1 . 12 . 433 Barbed end capping 434 Filaments polymerize until they reach a final length specified for a given simulation , \ud835\udc3f F & / ( an integer multiple 435 of \ud835\udc59 ! ) , which we vary to simulate the effect of varying CP concentration . Here we didn\u2019t study the effects of 436 a varying filament length distribution . The assumption that \ud835\udc3f F & / is independent of the force is based on prior 437 experiments ( 19 ) and the fact that the filament length added by capping protein is close to \u03b4 . According to 438 this evidence , the polymerization to capping rate ratio , as well as the average filament length , remains 439 unchanged by force for a given CP concentration . 440 Branching 441 Branching occurs from actin filament point elements that are within \ud835\udc59 ! of the nucleating bead , at rate \ud835\udc5f ! , ) . * 1 < , 442 independent of the CP concentration . The rate was chosen to approximate the timescale of symmetry 443 breaking and comet speed at intermediate CP . The segment length connecting the mother filament point 444 element to the daughter filament pointed end point element is \ud835\udc59 ! \" # $ % & . Their orientation is chosen according 445 to a uniform angle distribution in the cone around the mother filament opening towards the barbed end . To 446 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 15 maintain a discretization of the network at segment length \ud835\udc59 ! , branches cannot branch from the barbed end 447 point element of the filament . Branches nucleating from the same mother filament have no torsional 448 restriction ( i . e . daughter filaments can rotate about the axis of the mother filament without restriction ) , but 449 implicit torsional restrictions are imposed due to the dense surrounding network limiting this motion . 450 De novo nucleation 451 Nucleation of new filaments of length \ud835\udc59 ! ! occurs at a rate of \ud835\udc5f ! # - * 5 = 5 . These small filaments are introduced 452 with a uniform spherical orientation with either their pointed or barbed end touching the nucleating bead 453 surface . This slow rate of filament introduction was tuned to allow for startup of the network on timescales 454 comparable to experiment , and to allow for the buildup of a thin actin cloud around the leading bead edge 455 during actin comet propulsion , as observed in experiments . At the beginning of the simulation , 200 of these 456 de novo filaments are added to provide enough initial filaments for symmetric growth of the shell . 457 Filament fragmentation and debranching 458 For simplicity , filament fragmentation was assumed to occur above a certain tensile force threshold \ud835\udc39 \u2019 \" # ( 459 ( instead of implementing a rate of severing on as a function of tension ) . 460 For filament segments the threshold was \ud835\udc39 \u2019 \" # ( = 50 pN . This is lower than the experimental fragmentation 461 force of phalloidin - labeled actin filaments , which is several hundred pNs ( 75 ) , but we found that the shell 462 would have difficulty breaking unless we had this lower force . Once a filament segment is fragmented , the 463 filament with the newly created barbed end is left uncapped and can grow to the final length specified for 464 that simulation . Debranching occurs for branches that have a branch bond with tension greater than 465 \ud835\udc39 # - , ) . * 1 < = 30 pN or if the angle deviates by more than \ud835\udee5\ud835\udf03 # - , ) . * 1 < = 25 \u00b0 off of the 70 \u00b0 equilibrium angle . 466 The value of \ud835\udee5\ud835\udf03 # - , ) . * 1 < was chosen to allow easy debranching when the branch is bent ( similar to 467 experiments where branches were bent and pulled by fluid forces of order pN , ( 43 ) ) . The value of \ud835\udc39 # - , ) . * 1 < 468 had to be sufficiently high such that the network does not easily fall apart . 469 470 Acknowledgments 471 We thank Daniel Safer , Faviolla A . Baez - Cruz and Richard Wike for their kind help with protein purifications 472 and technical assistance on the project . We thank everyone in Ostap Lab and Dominguez Lab for their 473 valuable inputs throughout the project duration . 474 475 Funding : This work was supported by NIH grants R37 GM057247 ( EMO ) and R01 GM073791 ( RD ) . DV 476 and DMR were supported by NIH grant R35GM136372 . Portions of this research were conducted on the 477 Rockfish ( Johns Hopkins ) cluster through allocations MCB180021 and BIO230116 from the Advanced 478 Cyberinfrastructure Coordination Ecosystem : Services & Support ( ACCESS ) program , which is supported 479 by NSF grants # 2138259 , # 2138286 , # 2138307 , # 2137603 , and # 2138296 . 480 481 Author contributions : 482 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 16 Conceptualization : M . X . , D . M . R . , L . W . P . , R . D . , D . V . , E . M . O . 483 Methodology : M . X . , D . M . R . , L . W . P . 484 Formal analysis : M . X . , D . M . R . 485 Investigation : M . X . , D . M . R . , L . W . P . 486 Resources : G . R . , M . B . 487 Visualization : M . X . , D . M . R . L . W . P . , D . V . , E . M . O . 488 Supervision : L . W . P . , R . D . , D . V . , E . M . O . 489 Writing\u2014original draft : M . X . , D . M . R . , D . V . , E . M . O . 490 Writing\u2014review & editing : M . X . , D . M . R . , L . W . P . , M . B . , R . D . , D . V . , E . M . O . 491 492 Competing interests : The authors declare that they have no competing interests . 493 494 Data and materials availability : All data needed to evaluate the conclusions in the paper are present in 495 the paper and / or the Supplementary Materials . Additional data related to this paper may be requested from 496 the authors . 497 498 References 499 500 1 . L . Blanchoin , R . Boujemaa - Paterski , C . Sykes , J . Plastino , Actin dynamics , architecture , and 501 mechanics in cell motility . Physiol Rev 94 , 235 - 263 ( 2014 ) . 502 2 . K . Rottner , J . Faix , S . Bogdan , S . Linder , E . Kerkhoff , Actin assembly mechanisms at a glance . 503 Journal of Cell Science 130 , 3427 - 3435 ( 2017 ) . 504 3 . T . Svitkina , The Actin Cytoskeleton and Actin - Based Motility . Cold Spring Harb Perspect Biol 10 , 505 ( 2018 ) . 506 4 . V . Papalazarou , L . M . 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It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 21 Figure 1 . Alteration of comet tail morphology by myosin - I . ( A ) Schematic of ( left ) control - and ( right ) 662 myosin - beads coated with NPF and neutravidin , where neutravidin is further conjugated with ( control - bead ) 663 biotinylated CF640 fluorescent dye or ( myosin - bead ) biotinylated Drosophila Myo1d . ( B ) Time - lapse 664 sequence of a ( red ) myosin - bead walking on a ( green ) single - actin - filament track . Speed ~ 8 nm / s . Scale 665 bar 2 \u00b5m . ( C ) Time series of the growth of actin comet tail from symmetry breaking to generation of a 666 polarized comet tail from ( top ) control - bead and ( bottom ) myosin - bead . Scale bar 5 \u00b5m . ( D ) Phase map 667 showing representative examples of 2 \u00b5m - diameter beads coated with a range of myosin - densities in the 668 presence of 30 \u2013 200 nM CP , 4 \u00b5M ( 5 % Rhodamine labeled ) actin , and 200 nM Arp2 / 3 complex . For each 669 myosin density condition , the left column show control - beads that were acquired in the same imaging field 670 as the myosin - beads in the right column . The myosin and NPF densities were determined by SDS - PAGE 671 gel , where the NPF density is the same for group 0 . 28 : 1 , 0 . 35 : 1 and 0 . 43 : 1 , ~ 6000 um - 2 , and ~ 4000 um - 2 672 for group 0 . 80 : 1 . Images were acquired 20 - 35 min after mixing . Brightness and contrast were linearly set 673 to be the same for each control - and myosin - bead pair but different among panels at different conditions 674 for better visualization . Scale bar 5 \u00b5m . 675 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 22 676 677 Figure 2 . Disruption and fracturing of actin shells by myosin - I . ( A ) Time series of ( top ) control - beads 678 and ( bottom ) 0 . 43 : 1 myosin - beads acquired in the same imaging field in the presence of 200 nM CP 679 showing the inability of myosin - beads to form a comet tail . ( B ) Time series of ( top ) control - beads and 680 ( bottom ) 0 . 43 : 1 myosin - beads acquired in the same field in the presence of 25 nM CP showing fracturing 681 of the actin shell and comet tail growth from a myosin - bead but not the control - bead . Conditions : 4 \u00b5M actin 682 ( 5 % Rhodamine labeled ) , 200 nM Arp2 / 3 complex , 200 nM or 25 nM CP . Scale bar 5 \u00b5m . 683 A 180 s 380 s 580 s 780 s 980 s 1180 s 1380 s 116 s 216 s 316 s 416 s 516 s 616 s 716 s C on t r o l M y o s i n B C o n t r o l M y o s i n . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 23 684 685 Figure 3 . Myosin - I decreases the actin density of comet tails . ( A ) Time - series of ( top ) control - and 686 ( bottom ) 0 . 43 : 1 myosin - beads growing comet tails in the presence of 50 nM CP . The actin network growing 687 from the myosin - bead is less dense but has a similar tail length as the control . Conditions : 4 \u00b5M actin ( 5 % 688 Rhodamine labeled ) , 200 nM Arp2 / 3 complex and 50 nM CP . Scale bar 5 \u00b5m . ( B ) Comparison of the mean 689 fluorescence intensities ( Fluor . Int . / Area ) of actin comet tails grown from control - and myosin - beads , 690 captured 20 min after mixing . The solid lines connect experimental pairs ( N = 5 , n = 11 ) . ( C ) Actin comet tail 691 length as a function of time and ( D ) tail growth rates derived from the slopes of the time courses in ( C ) . ( E ) 692 Comet tail fluorescence as a function of time . Control - and myosin - bead experimental pairs are normalized 693 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 24 to the average fluorescence level of the control - beads from 1100 s \u2013 1300 s . ( F ) Rate of fluorescent actin 694 incorporation into comet tails derived from the slopes of the time courses in ( E ) . ( C , E ) Large points show 695 the averaged value at binned time interval ( every 100 sec ) . Traces are from individual beads with each 696 myosin - bead acquired with a control - bead in the same field of view ( N = 5 , n = 11 ) . Error bars are SD . ( G ) 697 Growth efficiency for control and myosin - beads . Efficiency is defined as comet tail length per unit actin 698 fluorescence intensity . Box plots ( B , D , F , G ) show median ( center line ) , interquartile range ( box ) and min - 699 max values ( whiskers ) . p values were calculated using two - tail paired t test . Each point represents a control - 700 and myosin - bead pair acquired in same field of view . See also Supplementary Fig . 701 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 25 702 703 Figure 4 . Comets grown from myosin - beads incorporate less Arp2 / 3 complex . ( A ) Representative 704 actin comet tails assembled from ( top ) control - and ( bottom ) myosin - beads showing ( magenta ) actin and 705 ( green ) SNAP - Arp2 / 3 complex . Images were acquired approximately 25 - 35 min after mixing . Brightness 706 and contrast were set differently for actin and SNAP - Arp2 / 3 complex panels for visualization . Conditions : 4 707 \u00b5M actin ( 5 % Rhodamine labeled ) , 200 nM Arp2 / 3 complex ( 80 % SNAP - Surface 488 labeled ) , 40 nM CP . 708 Scale bar is 5 \u00b5m . ( B ) Total actin fluorescence intensity , ( C ) total SNAP - Arp2 / 3 fluorescence intensity and 709 ( D ) actin to SNAP - Arp2 / 3 fluorescence ratio over the entire comet tail region . ( E ) Total SNAP - Arp2 / 3 710 fluorescence intensity on bead surface . Plot shows median ( center line ) , interquartile range ( box ) and min - 711 max values ( whiskers ) . p values were calculated using two - tail paired t test . Each point represents a pair 712 of control - and myosin - beads ( N = 2 , n = 33 ) 713 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 26 714 715 Figure 5 . The myosin power stroke is required for altering network architectures . ( A ) Time - series of 716 actin assembly around ( top ) control and ( bottom ) rigor myosin - beads at 50 nM CP . Rigor myosin heavily 717 delayed the growth of actin comet tails . ( B ) Representative images of actin comet tails grown under different 718 ATP concentrations ( top : control beads ; bottom : myosin - beads ) . Adding ATP back rescued comet tail 719 growth . Images were acquired 20 - 30 min after mixing . ( C ) Representative actin network patterns assembled 720 on control - , myosin - , rigor - myosin , and HaloABD - beads under three different CP concentrations , as 721 indicated . Images were acquired 20 \u2013 30 min after mixing . Brightness and contrast were set to the same 722 values for each panel . ( D ) Representative actin comet tails generated by ( top ) control - and ( bottom ) myosin - 723 beads in the absence and presence of 100 \u00b5M free Ca 2 + at 50 nM CP . Images were acquired approximately 724 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 27 15 - 20 min after mixing . ( E ) Length and ( F ) network density quantified by total fluorescence intensity per 725 area for control - and myosin - beads in the absence and presence of 100 \u00b5M free Ca 2 + ( N = 1 , n = 15 ) . Control - 726 and myosin - bead experimental pairs are normalized to the average fluorescence level of the control - beads . 727 Box plots ( E , F ) show median ( center line ) , interquartile range ( box ) and min - max values ( whiskers ) . p 728 values were calculated using two - tail paired t test . Conditions : 4uM actin ( 5 % Rhodamine labeled ) , 200 nM 729 Arp2 / 3 complex , 15 - 200 nM CP , 0 - 1 mM ATP , as indicated . Scale bar 5 \u00b5m . 730 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 28 731 732 Figure 6 . The myosin power stroke can fracture the actin shell . ( A ) Representative actin shells of 733 control - , myosin - beads assembled under 50 nM CP , arrested by adding 20\u03bcM ( 5 molar excess ) of 734 Latrunculin B and CK - 666 before symmetry breaking ; as well as myosin - beads assemble under the same 735 conditions but arrested with the addition of 10mM ADP to inhibit myosin power stroke . Myosin - beads 736 fractured and ejected from the actin shell , while control - and myosin - beads ( with 10mM ADP ) remained 737 enclosed in the shell . Control - bead assembled under 100 nM CP showing similar network density as the 738 myosin - beads , didn\u2019t show shell fracture or bead ejection . Image was captured approximately 40min after 739 arrest . ( B ) The extents of shell breaking was classified by shell - breaking angle \u03b8 : \u03b8 = 0 , no symmetry 740 breaking ; 0 < \u03b8 < 180 , shell fracture ; \u03b8 \u2265 180 , bead ejected . Scale bar is 5\u03bcm . ( C ) Percentage of populations 741 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 29 with different extents of shell breaking for control ( 50 nM CP ) ( n = 182 ) , myosin ( n = 158 ) , myosin ( with 10mM 742 ADP ) ( n = 89 ) , control ( 100 nM CP ) ( n = 65 ) , N = 2 . Conditions : 4\u03bcM actin ( 5 % Rhodamine labeled ) , 200 nM 743 Arp2 / 3 , 50 or 100 nM CP . 20\u03bcM ( 5 molar excess ) phalloidin and 2 mM ATP were also added to prevent 744 actin depolymerization and preserve myosin motor activity . Actin assembly was arrested 100s after mixing . 745 Scale bar is 5 \u00b5m . 746 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 30 747 748 Figure 7 . Filament level model of actin comet tail recapitulates experimental results . ( A ) Schematic 749 of model of actin polymerization around the nucleating bead . Model includes filament level nucleation , 750 branching , filament fragmentation , debranching , capping , and force exerted by implicit myosin . ( B ) 751 Timelapse of symmetry breaking event under intermediate capping condition ( branch length = 0 . 5 \u03bcm ) with 752 no myosin . Color scale indicates filament tension ( Red : tensile ; Blue : compressive ) . ( C ) Tension 753 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 31 distributions within actin comet tails formed at either control ( 0 . 0 pN ) or two myosin forces ( 0 . 2 pN or 0 . 4 754 pN ) under intermediate capping condition ( branch length = 0 . 5 \u03bcm ) . Image shows a cut through the center 755 of the comet tail . Color scale indicates filament tension ( Red : tensile ; Blue : compressive ) . ( D ) Simulated 756 timelapse of epifluorescence images under different myosin forces ( branch length = 0 . 5 \u03bcm ) . ( E ) Elongation 757 speed ( F ) actin intensity and ( G ) Arp2 / 3 complex intensity for simulated beads as a function of myosin force 758 ( branch length = 0 . 5 \u03bcm ) . ( H ) Forces acting on beads along the direction of bead propulsion due to actin 759 polymerization ( green ) and myosin pushing ( orange ) as a function of the myosin force ( branch length = 0 . 5 760 \u03bcm ) . ( I ) Orientation of filaments around the beads ( within 0 . 15 \u03bcm of the bead surface ) as a function of 761 myosin force ( branch length = 0 . 5 \u03bcm ) . ( J ) Simulated symmetry breaking after actin polymerization and 762 branching arrest . Actin was allowed to polymerize around the bead for 42 . 7 s ( 0 . 2 pN myosin force with 0 . 5 763 \u03bcm filament length ) to form a shell of intermediate thickness before halted . The time of arresting was set 764 as t = 0 s . 765 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 32 766 Figure 8 . Schematic of how myosin - I modulates actin network structure through its power - strok 767 Myosin - I Actin Arp2 / 3 Complex NPF - coated surface A B F . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 33 768 Supplementary Materials for 769 770 Myosin - I Synergizes with Arp2 / 3 Complex to Enhance Pushing 771 Forces of Branched Actin Networks 772 773 Mengqi Xu et al . 774 775 * Corresponding author . Email : ostap @ pennmedicine . upenn . edu , vavylonis @ lehigh . edu , 776 droberto @ pennmedicine . upenn . edu , Luther . Pollard @ pennmedicine . upenn . edu 777 778 779 780 781 782 This PDF file includes : 783 784 Supplementary Text 785 Figs . S1 to S8 786 Tables S1 787 Movies S1 to S13 788 789 Other Supplementary Materials for this manuscript include the following : 790 791 Movies S1 to S13 792 793 794 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 34 Supplementary Text 795 Myosin steric hindrance effect 796 We quantified the average spatial occupancy of individual components ( NPF or myosin - I ) bound 797 to bead surface for the first three myosin / NPF ratio groups ( 0 . 28 , 0 . 35 , 0 . 43 ) , all of which maintain 798 the same NPF densities ( ~ 6000 / um 2 ) . We found that individual molecular components occupied 799 similar areas in all three groups : 11 . 4x1 . 4 nm 2 / molecule for 7680 molecules in 0 . 28 : 1 800 ( myosin / NPF ) group , 11 . 1x11 . 1 nm 2 / molecule for 8100 molecules in 0 . 25 : 1 ( myosin / NPF ) group 801 and 10 . 7x10 . 7 nm 2 / molecule for 8580 molecules in 0 . 43 : 1 ( myosin / NPF ) group . Therefore , the 802 steric hindrance effect in each of the three groups should be nearly identical . In the 0 . 28 : 1 803 ( myosin / NPF ) group , both control - and myosin - beads displayed similar actin comet patterns , 804 which implies that the steric hindrance of myosin - I does not play a significant role in modifying the 805 Arp2 / 3 - medicated branching under the current conditions . 806 807 Bead gliding assay on actin filament tracks 808 F - actin was made by polymerizing 25 \u03bcM G - actin in MB buffer ( 10 mM MOPS [ pH7 . 0 ] , 25 mM 809 KCl , 1 mM EGTA , 1 mM MgCl 2 , 1 mM DTT ) with 2 mM ATP at room temperature for 1 hr , and 810 stabilized with AlexaFluor 488 Phalloidin ( ThermoFisher Cat . # A12379 ) . Myosin - beads were 811 prepared following the procedures outlined in Methods in the main text and labeled with TRITC 812 during the blocking process using 10 % TRITC - labeled BSA ( Sigma , Cat . # A2289 ) . Flow 813 chambers were made by sandwiching the plasma cleaned slides and coverslips ( 22 mm \u00d7 22 814 mm ) with two pieces of double - stick tape , forming a 5mm - wide channel that can hold a volume of 815 up to 10 \u03bcL . To immobilize single - actin - filament tracks on the coverslip , we coated the flow 816 chamber with 0 . 5 mg / mL NEM - myosin , incubated for 5 min , followed by two washes with 2 817 mg / mL casein ( 1 min for each ) to remove any unbound NEM - myosin and block the remaining free 818 spaces on the surface . Next , 5 - 10 nM AlexaFluor 488 Phalloidin stabilized F - actin was loaded 819 into the flow chamber to avoid any overlapping between the actin filaments , incubated for 2 min , 820 followed by a wash with 20uL MB buffer . Finally , 10 uL of the 50 uL bead motility mixture 821 containing 3uL TRITC - labeled myosin - bead slurry ( # myosin / NPF = 0 . 4 : 1 ) , 2 \u03bcM Calmodulin , 0 . 2 822 mg / mL glucose oxidase , 0 . 04 mg / mL catalase and 5 mg / mL glucose in MB buffer supplemented 823 with 5 mM Mg - ATP and 40 mM DTT , was loaded into the flow chamber , sealed with vacuum 824 grease and imaged immediately under the microscope . 825 826 Myosin inhibition experiments 827 828 Rigor myosin was prepared by excluding ATP from the motility mixture . Gel - filtered actin 829 monomers were incubated with 2 mM ATP for 15 minutes on ice , followed by passage through a 830 G25 Sephadex column to remove residual free nucleotides . The resulting ATP - treated actin 831 exhibited similar polymerization capability in ATP - free mixtures as to those containing 1 mM ATP . 832 Alternatively , myosin was inhibited by introducing 10 mM Mg - ADP to prolong the lifetime of the 833 myosin ADP - bound state . 834 835 Actin gliding assay 836 Coverslips were plasma - cleaned and coated with 0 . 5 % Nitrocellulose and used to make a flow 837 chamber . Flow chamber was first incubated with 0 . 1 mg / mL neutravidin for 2 min followed by 838 blocking with 2 mg / mL casein , twice , 1 min each . Then , 250 nM biotinylated Drosophila Myo1d 839 was loaded into the chamber , incubated for 2 min , and washed with 20uL MB buffer ( 10 mM 840 MOPS [ pH7 . 0 ] , 25 mM KCl , 1 mM EGTA , 1 mM MgCl 2 , 1 mM DTT ) . Next , 20 nM F - actin 841 stabilized with AlexaFluor 488 Phalloidin was loaded into the chamber , incubated for 2 min , 842 washed with 20uL MB buffer . Finally , the imaging mixture containing 8 . 4 \u03bcM Calmodulin , 1 mM 843 Mg - ATP , 40 mM DTT in 20 mM HEPES [ pH7 . 5 ] , 100 mM KCl , 1 mM MgCl 2 , 1 mM EGTA and 844 0 . 2 % methylcellulose was loaded into the chamber and observed immediately . For experiments 845 with calcium , 8 . 4 \u03bcM Calmodulin was replaced by 1 . 1 mM CaCl 2 to disrupt the myosin power - 846 stroke . 847 848 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 35 Actin arresting 849 Actin assembly was quenched by combining equal volume of motility mixture and arresting 850 solution , which contains 20 \u03bcM ( 5 molar excess ) of both Latrunculin B and CK - 666 ( together with 851 20 \u03bcM phalloidin for ' + Phalloidin ' experiments ) in 20 mM HEPES [ pH 7 . 5 ] , 100 mM KCl , 100 852 \u03bcg / ml BSA , 0 . 5 mM TCEP . Quenched motility mixture was either applied on a squeezer chamber 853 or loaded into a NEM - myosin coated flow chamber ( to make sure the observed shell - breaking 854 was not due to the squeezing effect between the slides and the coverslips ) and imaged under the 855 microscope immediately after sealing with vacuum grease . 856 857 Computational modeling 858 859 Reduction of de novo and branch nucleation rate due to crowding 860 If upon introduction to the system a de novo filament or new branch would strongly overlap with 861 other filaments or the nucleating bead , their insertion is rejected . Specifically , their introduction is 862 rejected if the closest distance between a newly added segment and an existing segment is less 863 than / 4 , or if the distance between the newly added branch and the nucleating bead is less than 864 ( \ud835\udc51 - 01 / 2 # - # / 2 + \ud835\udc45 ) / 4 . No explicit reattempts at introduction of a rejected filament are performed , 865 and the rate of branching , \ud835\udc5f ! , ) . * 1 < , and the rate of de novo introduction , \ud835\udc5f ! # - * 5 = 5 , are left 866 unchanged by a rejection event . 867 Simulation timescale 868 We wrote C + + code with force parallelization using OpenMP . For the case of Fig . 7B ( 0 . 5 \u03bcm 869 length filaments with no myosin force ) it takes 360 hours on 10 CPU cores to run the simulation to 870 260 s . The amount of time to run a simulated second increases as the simulation proceeds since 871 no filament segments ( beyond those that are severed ) are removed from the system . Simulations 872 with myosin or with shorter filaments take a shorter amount of time to run the same simulated 873 time since they have overall less filament segments at the same point in time . 874 Simulated fluorescence images 875 To generate simulated fluorescence images of actin comet tail formation we first rotate the 876 system so the net displacement of the nucleating bead occurs along the x - direction . For a given 877 frame , each actin or Arp2 / 3 complex spring bond centroid is placed in a 2d grid with gridlines 878 along the x and y axes and bin size 0 . 05 \u03bcm ( z - component is not considered ) . Actin filament 879 segments of length \ud835\udc59 ! contribute 37 actin monomers to the bin they are assigned , while shorter 880 filament segments contribute 37 \ud835\udc59 / \ud835\udc59 ! monomers , where \ud835\udc59 is the length of the segment . Arp2 / 3 881 complex bonds contribute a single Arp2 / 3 molecule to the bin they are placed in . A grayscale 882 image is constructed from the grid where each voxel in the image corresponds to a single bin and 883 the intensity corresponds to the sum of the count of monomers in each bin . Finally , a Gaussian 884 filter with standard deviation 0 . 1 \u03bcm is applied to the image to smooth the image to a comparable 885 level as in experiment . 886 Measurement of Forces 887 Forces in Figure 7H and Figure S7 are calculated by first rotating the system so the net 888 displacement of the nucleating bead occurs along the x - direction . Excluded volume forces acting 889 between actin segments and the nucleating bead are spatially divided into three regions based on 890 the closest approach point of the actin segment to the nucleating bead . Forces where the closest 891 approach point is greater than R / 4 behind the nucleating bead center in the x - direction are 892 considered back forces . Forces where the closest approach point is greater than R / 4 in front of 893 the nucleating bead center in the x - direction are considered front forces . All other forces are 894 labeled as side forces . Excluded volume forces are further subdivided into whether they are 895 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 36 caused by filament segments at their maximum length \ud835\udc59 ! ( actin excluded volume ) or filament 896 segments with length less than \ud835\udc59 ! ( actin polymerization ) . 897 Measurement of filament orientation with respect to nucleating bead 898 Filament angle distributions with respect to the nucleating bead , for filaments close to the 899 nucleating bead in Figure 7I are calculated by first rotating the system so the net displacement of 900 the nucleating bead occurs along the x - direction . Only filaments segments with length less than 901 \ud835\udc59 ! that are also in the back region ( as defined in Measurement of Forces ) and are also closer to 902 the nucleating bead surface than 0 . 15 \u03bcm are considered . For these actively growing filaments 903 we measure the angle between the filament displacement vector and the vector joining the 904 midpoint of the filament segment to the nucleating bead center . This angle is zero if the filament 905 is perpendicular to the nucleating bead surface and 90\u00b0 if the filament is lying flat on the bead 906 surface . We generate a probability distribution of these angles over approximately 70 s and when 907 the nucleating bead is moving at a relatively constant velocity . The probability of finding the angle 908 to be less than 70\u00b0 from these distributions is reported in Figure 7I . 909 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 37 910 911 Fig . S1 . Phase diagram of actin comet tails assembled under different CP concentrations 912 and myosin densities . Experimental points demonstrate control - and myosin - bead pairs 913 assembled under each specified conditions . Star \u2013 myosin - beads promote symmetry breaking ; 914 Triangle - myosin - beads have longer tails than control ; Circle \u2013 myosin - beads exhibit similar tail 915 length to control ; Square \u2013 myosin - beads have shorter tails than control ; Diamond \u2013 myosin - beads 916 shows no comet tail . For extremely low CP concentration ( e . g . 6 . 5 nM , Crossline ) , both myosin - 917 coated and control - beads displayed aster - like structure . No significant difference was observed 918 between the two types of beads . Conditions : 4 \u00b5M G - actin , 200 nM Arp2 / 3 complex , 0 - 200 nM of 919 CP as indicated in graph . 920 0 . 27 0 . 34 0 . 42 0 . 71 0 50 100 200 Myosin Density ( # myosin / NPF ) C P ( n M ) . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 38 921 Fig . S2 . Myosin - I decreases the actin density in comet tails ( supplement for Fig . 3 in the 922 main text ) . ( A ) Time - series of ( top ) control - and ( bottom ) 0 . 43 : 1 myosin - beads growing comet tails 923 in the presence of 50 nM CP . The actin network growing from the myosin - bead is less dense but 924 has a similar tail length as the control . ( B ) Actin comet tail length as a function of time ( C ) Comet 925 tail fluorescence as a function of time . The fluorescent intensity of control - and myosin - bead 926 experimental pairs are normalized to the average fluorescence level of the control - beads from 927 1100 - 1300 s . Large points show the averaged value at binned time interval ( every 100 sec ) . Traces 928 are from individual beads with each myosin - bead acquired with a control - bead in the same field of 929 view ( N = 5 , n = 11 ) . Error bars are SD . Conditions : 4 \u00b5M actin ( 5 % Rhodamine labeled ) , 200 nM 930 Arp2 / 3 complex and 50 nM CP . Scale bar 5 \u00b5m . 931 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 39 932 Fig . S3 . The myosin power - stroke is required for altering network architectures ( supplement 933 for Fig . 5 in the main text ) . ( A ) Time - series of actin assembly around ( top ) control - and ( bottom ) 934 HaloABD - bead at 50 nM CP . HaloABD of \ud835\udf36 - actinin heavily delayed the growth of actin comet tail 935 as rigor myosin . Scale bar 5 \u00b5m . ( B ) Quantification of comet tail length generated under different 936 ATP concentrations ( 0 - 1 mM ) for control - , myosin - and HaloABD - beads ( N = 2 , n > 15 ) . Error bars 937 are SD . p values were calculated using unpaired two - tail t test . ( C ) Network density quantified by 938 total fluorescence intensity per area for control - , myosin - , rigor - myosin and HaloABD - beads . 939 Normalized to the mean value of control - beads ( N = 2 , n > 11 ) . Dot plots show mean \u00b1 SD . p values 940 were calculated using unpaired two - tail t test . ( D ) Percentages of control - , myosin - , rigor - myosin 941 and HaloABD - beads that performed symmetry breaking or no symmetry breaking at 15nM CP 942 ( N = 3 , n > 23 ) . ( E ) Time collapsed images of actin filaments gliding ( with no Ca 2 + ) or swirling ( with 943 100 \u00b5M free Ca 2 + ) on a Myo1d - coated surface . Rainbow bar represents timescale over 61 frames 944 with 5 s frame interval . Scale bar 5 \u00b5m . ( F ) Quantification of actin gliding speed with and without 945 100 \u00b5M free Ca 2 + . ( G ) Total actin fluorescence intensity and ( H ) Growth efficiency for control - and 946 myosin - beads with and without the presence of 100 \u00b5M calcium ( N = 1 , n = 15 ) . Efficiency is defined 947 as comet tail length per unit actin fluorescence intensity . Box plots ( G , H ) show median ( center 948 line ) , interquartile range ( box ) and min - max values ( whiskers ) . p values were calculated using two - 949 tail paired t test . 950 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 40 951 Fig . S4 . Myosin power - stroke alone can break sparse actin shell ( w / o Phalloidin ) . ( A - B ) . 952 Time - lapse series of ( top ) control - and ( bottom ) myosin - bead that performed ( A ) shell fracture or 953 ( B ) bead ejection . Control - and myosin - beads were mixed with 4 \u03bcM actin ( 5 % Rhodamine labeled ) , 954 200 nM Arp2 / 3 , and 50 nM CP , incubated for 100s , and then actin assembly was arrested by adding 955 20\u03bcM ( 5 molar excess ) of Latrunculin B and CK - 666 . ( C ) Status of shell breaking approximately 956 50min after arrest . The extents of shell breaking was classified by shell - breaking angle \u03b8 . ( D ) 957 Percentage of different populations with different extents of shell breaking . Without Phalloidin : 958 control ( n = 141 ) , myosin ( n = 55 ) , myosin with 10mM ADP ( n = 125 ) ; With Phalloidin : control ( n = 182 ) , 959 myosin ( n = 158 ) , myosin with 10mM ADP ( n = 89 ) . Conditions : 4 \u03bcM actin ( 5 % Rhodamine labeled ) , 960 200 nM Arp2 / 3 complex , 50 nM or 100 nM CP was incubated for 100 s and arrested by adding 5 961 molar excess of Latrunculin B , CK - 666 and phalloidin ( for \u2018 + Phalloidin\u2018 experiments ) or 10mM ATP 962 ( for \u2018myosin with 10 mM ADP\u2019 experiments ) . Scale bar 5\u03bcm . 963 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 41 964 965 Figure S5 . Simulated comet tail structure and tensile / compression force distributions as a 966 function of branch length and myosin force F myo . ( A ) Simulated epifluorescence images as in 967 Figure 7D , but with filaments grow to a length of 0 . 3 \u03bcm before capping . The simulations can be 968 compared to high CP concentration experiments of Figure 2A where a comet tail fails to form for 969 myosin - beads . ( B ) Same as in panel A , but for filament length of 2 . 0 \u03bcm . The simulations mimic 970 the low CP concentration experiments of Figure 2B where myosin was found to promote symmetry 971 breaking . ( C ) State diagram for actin comet tails as a function of filament length and myosin force . 972 Images show a cut through the center of the comet tail . Color scale indicates filament tension ( red : 973 tensile ; blue : compressive ) . 974 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 42 975 976 Figure S6 . Simulated actin intensity , Arp2 / 3 intensity and Actin : Arp2 / 3 ratio across the entire 977 actin comet tail for intermediate length condition ( intermediate CP concentration ) under 978 different debranching thresholds . ( A ) Actin : Arp2 / 3 complex ratio ( calibrated to number of actin 979 monomers per Arp2 / 3 complex ) , average actin intensity , and average Arp2 / 3 complex intensity 980 from simulated epifluorescent images of comet tails formed at reference debranching conditions 981 with filament length 0 . 5 \u03bcm ( \u2248 187 subunits ) . Solid circles indicate averages calculated where the 982 Arp2 / 3 complex is removed upon debranching ; empty squares indicate averages calculated where 983 the Arp2 / 3 complex remains attached to the daughter filament upon debranching . ( B ) Same as in 984 panel A except under enhanced debranching condition ( debranching occurring at \u00b110\u00b0 away from 985 70\u00b0 equilibrium branch instead of \u00b125\u00b0 as in the reference case in A ) . The ratio varies with myosin 986 force when Arp2 / 3 complex dissociates after debranching , but in the opposite manner as compared 987 to the experimental results in Figure 4D . 988 B A Enhanced Debranching ( Debranching at \u00b110\u00b0 from equilibrium 70\u00b0 angle ) Reference Debranching ( Debranching at \u00b125\u00b0 from equilibrium 70\u00b0 angle ) Arp2 / 3 complex dissociates Arp2 / 3 complex remains Arp2 / 3 complex dissociates Arp2 / 3 complex remains . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 43 989 990 Figure S7 . Force acting on beads during comet tail elongation . Subdivision of forces acting on 991 beads along the direction of bead propulsion due to actin polymerization ( light green , gray , dark 992 green continuous ; calculated as excluded volume interactions between barbed end segment and 993 bead ) , actin excluded volume ( light green , gray , dark green dashed ; calculated as excluded volume 994 interactions on the bead from filament segments other than barbed ends ) , and myosin forces on 995 the bead ( orange line ) . The bead drag force that balances the sum of these forces is shown in 996 black . Actin forces are subdivided into whether they are located behind the bead ( light green ) , at 997 the side of the bead ( gray ) , or in front of the bead ( dark green ) . 998 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 44 999 1000 Figure S8 . Simulated expulsion of bead after halting actin polymerization / branching . 1001 Simulation snapshots at intermediate filament length ( 0 . 5 \u03bcm ) ( intermediate CP concentration ) 1002 show a cut through the bead center during the evolution of the actin network after halting actin 1003 polymerization and branching . Expulsion of the bead only occurs at intermediate cloud thickness 1004 ( Growth time = 42 . 7 s , middle row and Fig . 7J ) . Thinner shells fall apart ( Growth time = 31 . 5 s ) , 1005 while thicker shells cannot be broken by 0 . 2 pN myosin alone ( Growth time = 59 . 85 s ) . Network 1006 evolved in the presence of the same myosin force ( 0 . 2 pN ) . Histograms show filament tension 1007 distribution at t = 0 s ( time of growth arrest ) ; dashed lines indicate region containing 95 % of data , 1008 centered at zero . 1009 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 45 Table S1 . Table of simulation constants 1010 Parameter Description Value Justification R Radius of nucleating bead 0 . 75 \u03bcm Smaller but comparable to experiment , for numerical efficiency \u2206\ud835\udc61 Simulation timestep 3 . 5 \u00d7 10 : G s Allows for numerical stability of excluded volume interactions \ud835\udf01 Friction coefficient for actin segment 0 . 377 pN s \u03bcm - 1 Sufficiently high for numerical stability \ud835\udf01 * 21 / , - . # Friction coefficient for nucleating bead 2970 pN s \u03bcm - 1 Sufficiently high for bead to not eject bare from actin shell \ud835\udc59 ( Actin filament persistence length 17 . 0 \u03bcm \ud835\udc58 . 1 % & * Actin filament segment spring constant 1000 pN \u03bcm - 1 As large as allowed for numerical stability ( 62 ) \ud835\udc59 ! Actin segment spring equilibrium distance 0 . 1 \u03bcm Length of filament segment \ud835\udc59 ! ! Starting filament length 0 . 035 \u03bcm Numerical stability \ud835\udc59 , ) . * 1 < Bond length between mother point element and pointed end of daughter filament 0 . 02 \u03bcm Numerical stability \ud835\udc51 , ) . * 1 < Range of branching nucleation 0 . 1 \u03bcm Of order \ud835\udc59 ! \ud835\udf16 . * + / - Spring constant for filament branch 2 pN \u00b5m Maintains angle between 60\u00b0 - 80\u00b0 with thermal fluctuations ( 62 ) \ud835\udc51 - 01 / 2 # - # Excluded volume interaction range between filaments 0 . 02 \u03bcm Larger than 7 nm filament diameter for increased numerical stability \ud835\udc58 - 01 / 2 # - # Excluded volume interaction constant 1690 pN \u03bcm - 1 Prior computational model ( 72 ) \ud835\udc51 345 Range of myosin force 0 . 025 \u03bcm Comparable to size of myosin 1d \ud835\udc39 345 Magnitude of myosin force acting on actin filaments within range . 0 . 0 \u2013 0 . 4 pN Varied \ud835\udc5f ! ( 5 / Rate of polymerization of uncapped barbed ends 40 sub s - 1 4 \u03bcM actin solution . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 46 \ud835\udc5f ! , ) . * 1 < Rate of branching per filament point element 0 . 15 s - 1 Tuned to approximate timescale of symmetry breaking and comet speed ( intermediate CP ) \ud835\udc5f ! # - * 5 = 5 Rate of spontaneous introduction of new filaments 0 . 125 s - 1 Tuned to allow actin shell on timescales comparable to experiment \ud835\udc39 F ) . + Threshold force for tension fragmentation of filaments 50 pN Allows symmetry breaking \ud835\udc39 # - , ) . * 1 < Threshold force for tension debranching of branches 30 pN Of order \ud835\udc39 F ) . + \ud835\udee5\ud835\udf03 # - , ) . * 1 < \u03a4threshold angle deviation from 70\u00b0 for debranching 25\u00b0 Debranching by bending and pulling ( 72 ) 1011 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint 47 Movie S1 . Myosin - bead ( red ) processively moving along a ( green ) single - actin - filament track . 1012 1013 Movie S2 . Movie of control - beads and myosin - beads ( 0 . 43 : 1 # myosin / NPFs ) acquired in the 1014 same imaging field in the presence of 200 nM CP showing the inability of myosin - beads to form a 1015 comet tail . Scale bar 5 \u00b5m . 1016 1017 Movie S3 . Movie of control - beads and myosin - beads ( 0 . 43 : 1 # myosin / NPFs ) acquired in the 1018 same field in the presence of 25 nM CP showing fracturing of the actin shell and comet tail growth 1019 from a myosin - bead but not the control - bead . Scale bar 5 \u00b5m . 1020 1021 Movie S4 . Movie of control - beads and myosin - beads ( 0 . 43 : 1 # myosin / NPFs ) growing comet tails 1022 in the presence of 50 nM CP . The actin network growing from the myosin - bead is less dense but 1023 has a similar tail length as the control . Scale bar 5 \u00b5m . 1024 1025 Movie S5 . Movie of actin assembly around control - beads and rigor myosin - beads ( 0 . 43 : 1 1026 # myosin / NPFs ) at 50 nM CP . Rigor myosin heavily delayed the growth of actin comet tails . Scale 1027 bar 5 \u00b5m . 1028 1029 Movie S6 . Movie of actin assembly around control - and HaloABD - bead at 50 nM CP . HaloABD of 1030 \ud835\udefc - actinin heavily delayed the growth of actin comet tail as rigor myosin . Scale bar 5 \u00b5m . 1031 1032 Movie S7 . Movie of myosin - beads ( 0 . 43 : 1 # myosin / NPFs ) break actin shell apart acquired 11 min 1033 after actin polymerization was arrested by Latrunculin B and CK - 666 in the absence of phalloidin . 1034 Scale bar 5 \u00b5m . 1035 1036 Movie S8 . Movie of myosin - beads ( 0 . 43 : 1 # myosin / NPFs ) ejected out of actin shell acquired 24 1037 min after actin polymerization was arrested by Latrunculin B and CK - 666 n the absence of 1038 phalloidin . Scale bar 5 \u00b5m . 1039 1040 Movie S9 Simulated symmetry breaking and comet growth for intermediate length ( 0 . 5 \u03bcm ) 1041 filaments , without myosin forces ( Fig . 7B ) . Top video shows a cutaway ( filament point elements 1042 with position z > 0 are not shown ) while bottom video shows all filaments . Bead is semi - 1043 transparent . 1044 1045 Movie S10 . Movie of simulated actin comet tail formation for short filaments ( 0 . 3 \u03bcm , High 1046 CP ) , _ as a function of myosin force ( Fig . S5A ) . 1047 1048 Movie S11 . Movie of simulated actin comet tail formation for long filaments ( 2 . 0 \u03bcm , Low CP ) , as 1049 a function of myosin force ( Fig . S5B ) . 1050 1051 Movie S12 . Movie of simulated actin comet tail formation for intermediate filaments ( 0 . 5 \u03bcm , 1052 Intermediate CP ) , as a function of myosin force ( Fig . 7D ) . 1053 1054 Movie S13 . Movie of simulated symmetry breaking and bead ejection by myosin forces following 1055 arrest of actin polymerization and branching at 42 . 7 s ( Fig 7I ) . Myosin force is 0 . 2 pN throughout 1056 the simulation and filament length is 0 . 5 \u03bcm . 1057 . CC - BY - NC - ND 4 . 0 International license available under a ( which was not certified by peer review ) is the author / funder , who has granted bioRxiv a license to display the preprint in perpetuity . It is made The copyright holder for this preprint this version posted February 12 , 2024 . ; https : / / doi . org / 10 . 1101 / 2024 . 02 . 09 . 579714 doi : bioRxiv preprint", + "kosmalska2015physical": "ARTICLE Received 12 Dec 2014 | Accepted 24 Apr 2015 | Published 15 Jun 2015 Physical principles of membrane remodelling during cell mechanoadaptation Anita Joanna Kosmalska 1 , 2 , Laura Casares 1 , 2 , Alberto Elosegui - Artola 1 , Joseph Jose Thottacherry 3 , Roberto Moreno - Vicente 4 , V\u0131\u00b4ctor Gonza\u00b4lez - Tarrago\u00b4 1 , 2 , Miguel A\u00b4ngel del Pozo 4 , Satyajit Mayor 3 , Marino Arroyo 5 , Daniel Navajas 1 , 2 , 6 , Xavier Trepat 1 , 2 , 7 , Nils C . Gauthier 8 & Pere Roca - Cusachs 1 , 2 Biological processes in any physiological environment involve changes in cell shape , which must be accommodated by their physical envelope\u2014the bilayer membrane . However , the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear . Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process . This process is passive , local , almost instantaneous , before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane . We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell \u2013 substrate interface . Once formed , cells reabsorb the invaginations through an active process with duration of the order of minutes . DOI : 10 . 1038 / ncomms8292 OPEN 1 Institute for Bioengineering of Catalonia ( IBEC ) , Barcelona 08028 , Spain . 2 Department of Physiological Sciences I , University of Barcelona , Barcelona 08036 , Spain . 3 National Centre for Biological Sciences ( TIFR ) , Bangalore 560065 , India . 4 Centro Nacional de Investigaciones Cardiovasculares ( CNIC ) , Madrid 28029 , Spain . 5 LaCa ` N , Universitat Polite ` cnica de Catalunya - BarcelonaTech , Barcelona 08034 , Spain . 6 Ciber Enfermedades Respiratorias , Madrid 28029 , Spain . 7 Institucio\u00b4 Catalana de Recerca i Estudis Avanc \u00b8 ats ( ICREA ) , Barcelona 08010 , Spain . 8 Mechanobiology Institute , National University of Singapore , Singapore 117411 , Singapore . Correspondence and requests for materials should be addressed to N . C . G . ( email : mbinclg @ nus . edu . sg ) or to P . R . - C . ( email : rocacusachs @ ub . edu ) . NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications 1 & 2015 Macmillan Publishers Limited . All rights reserved . P hysiological processes in development , wound healing , breathing or any other scenario generally involve cell shape variations , which are constrained by the physical envelope of cells\u2014the plasma membrane . In any such process , the plasma membrane must adapt to often fast cell rearrangements , a requirement that is at odds with the very low membrane extensibility / compressibility given by its high stretching elastic modulus 1 , 2 . Other than simple extension and compression , the regulation of membrane area and shape therefore requires additional mechanisms , which could include active cell processes like endocytosis and exocytosis 3 \u2013 5 or the formation and \ufb02attening of membrane invaginations / evaginations , either at the micron scale as in membrane folds 6 , 7 , blebs 8 or vacuole - like dilations ( VLDs ) 9 or at the nanoscale as in caveolae 10 . However and despite extensive work on membrane mechanical interactions 11 \u2013 15 , there is no clear physical understanding of the manner in which the cell membrane responds to changes in area and shape while remaining highly con\ufb01ned by adjacent cells or substrates . Here we show that in response to changes in the area and volume of adherent cells , membrane remodelling occurs through a mechanical process that is passive , local , almost instantaneous and before any active response . This process generates invagina - tions with shapes that minimize the elastic and adhesive energy required to store both membrane area and liquid volume at the cell \u2013 substrate interface . Once formed , cells reabsorb the invagi - nations through an active process with duration of the order of minutes . Results Membrane response to changes in area and shape . To under - stand how cell membranes respond to changes in area and shape , we labelled the membrane of mouse embryonic \ufb01broblasts ( MEFs ) by transfection with a membrane \ufb02uorescent marker ( pEYFP - mem ) and seeded them on \ufb01bronectin - coated poly ( dimethylsiloxane ) ( PDMS ) membranes . We observed membrane dynamics after modifying two different cell shape parameters : cell volume ( regulated through changes in medium osmolarity ) and cell spreading area ( regulated through a custom - built biaxial stretch device , Supplementary Fig . 1 ) . After submitting cells to 6 % linear strain ( corresponding to a 12 % increase in surface ) , we noted that additional required area was obtained by \ufb02attening membrane ruf\ufb02es ( Fig . 1a and Supplementary Movie 1 ) . If stretch magnitude was increased , however , the membrane reservoir was depleted and the membrane teared within 3 min of constant stretch application ( Fig . 1a , e and Supplementary Movie 1 ) . In contrast , exposure to medium with a 50 % reduction in osmolarity for 3 min increased cell volume by 20 % , but only required an increase in plasma membrane area of 2 % ( Fig . 1c , d ) . By itself , this small increase in required area had a negligible effect on the membrane , as checked after stretching cells by 2 % ( Supplementary Fig . 2 ) . Accordingly , 50 % hypo - osmotic shock did not eliminate ruf\ufb02es ( Fig . 1b and Supplementary Movie 2 ) , and exposure to 100 % deionized water was required to eliminate membrane ruf\ufb02es through cell swelling , or to lyse the membrane ( Fig . 1b , f and Supplementary Movie 2 ) . After 3 min of stretch application , the release of stretch resulted in the accumulation of excess membrane in small membrane reservoirs of 0 . 5 \u2013 1 m m in diameter ( Fig . 2i \u2013 k ) , which extended from both the cell ventral and dorsal surfaces ( Fig . 2a , c and Supplementary Movie 3 ) . Through pEYFP - mem \ufb02uorescence quanti\ufb01cation , we calculated that these reservoirs stored approxi - mately 15 % of the total projected cell \u2013 substrate membrane area ( see methods ) , roughly matching the 12 % change in area associated with 6 % linear biaxial strain . Reservoirs were resorbed and eliminated by cells within B 2 min ( Fig . 2b ) , although their formation / resorption dynamics depended on temperature and stretch magnitude ( see Supplementary Note 1 and Supplementary Fig . 3 ) . Further , reservoirs appeared in open spaces devoid of actin \ufb01bres and focal adhesions , suggesting that membrane invaginations avoided cytoskeletal resistance ( Fig . 2d ) . Similarly , re - application of isotonic medium after 3 min of exposure to 50 % hypo - osmotic medium resulted in the formation of membrane invaginations in the cell ventral surface , which were however larger ( B 2 m m in diameter ) and with a spherical cap shape ( Fig . 2e , g and Supplementary Movie 3 ) . The invaginations were quanti\ufb01ed to store B 2 % of projected cell \u2013 substrate membrane area , thereby also matching the associated membrane requirement imposed by the osmotic shock ( Fig . 1d ) . Those invaginations were also eliminated by cells within B 3 min ( Fig . 2f ) , and their formation / resorption dynamics depended on temperature and magnitude of hypo - osmotic shock ( see Supplementary Note 1 and Supplementary Fig . 3 ) . The membrane structures were concentrated at the central part of the cell , with a less dense actin meshwork and less focal adhesions , and their formation had to displace actin \ufb01bres and disrupt adhesions ( Fig . 2h ) . This suggests that osmotically induced invaginations avoided sites of high cytoskeletal resistance like stretch - induced reservoirs , but due to their larger size also had to generate an opening through the cytoskeleton . The dynamic formation of those openings was con\ufb01rmed by time - lapse images of cells co - transfected with both membrane and actin markers ( Supplementary Fig . 4 ) . The formation of such structures ( termed VLDs ) upon increases in medium osmolarity has long been described in neurons and other cell types 9 , 16 , and has been hypothesized to constitute a mechanism to accommodate excess membrane area upon osmotic - induced cell shrinking . However , 3 - min incubation with 50 % hypo - osmotic medium only imposed a 2 % increase in membrane area ( Fig . 1d ) , which by itself had negligible effects on the membrane ( Supplementary Fig . 2 ) . Thus , other factors beyond regulation of membrane area may drive VLD formation . VLD formation is driven by water con\ufb01nement . Alternatively to being regulated by membrane area , VLDs formed after increasing osmolarity could be caused by water \ufb02ows exiting cells , which would be con\ufb01ned between cells and the substrate and thereby generate hydrostatic pressure . To test this , we seeded cells on polyacrylamide gels , through which water can \ufb02ow . In those conditions , VLDs did not form upon the change from hypo - to iso - osmotic medium ( Fig . 3a and Supplementary Movie 4 ) . This effect was due to the water - permeable properties of polyacrylamide and not by its lower stiffness , as VLDs clearly formed in softer but hydrophobic silicone elastomers ( Fig . 3a and Supplementary Movie 4 ) . Interestingly , application of stretch for 3 min and subsequent release in cells seeded on polyacrylamide gels resulted in the formation not only of reservoirs as expected , but also of VLDs ( Fig . 3b and Supplementary Movie 5 ) . This was due to the poroelastic properties of polyacrylamide gels 17 , by which gels gradually swelled when stretched for 3 min , and then gradually released water and shrank upon stretch release ( Supplementary Fig . 5 ) . Con\ufb01rming this , reservoirs but not VLDs formed on both soft silicone elastomers and polyacrylamide gels where swelling was prevented by submitting them to stretch only during a short pulse ( Fig . 3c ) . Thus , water pressure formed either through con\ufb01nement or poroelastic \ufb02ows was equivalently successful at generating VLDs . We then evaluated further the degree of water con\ufb01nement at the cell \u2013 substrate interface by submitting cells to a 3 - min 50 % hypo - osmotic shock , and then restoring iso - osmotic medium ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 2 NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications & 2015 Macmillan Publishers Limited . All rights reserved . labelled with membrane - impermeable red \ufb02uorescent dextran . We did this at a low temperature of 26 (cid:2) C , which slowed VLD formation and allowed us to distinguish the formation and resorption phases of VLD dynamics ( see Supplementary Note 1 ) . Whereas the medium surrounding cells immediately became \ufb02uorescent , the dextran - free water expelled by cells formed VLDs that had initially a very low \ufb02uorescence in the red channel ( Fig . 3d , e ) . This shows that medium in VLDs was indeed con\ufb01ned , and did not immediately mix with external medium . However , as time progressed VLDs gradually increased their \ufb02uorescence even after VLDs started decreasing in size ( Fig . 3e ) , demonstrating that water \ufb02ow and mixing was impaired but not eliminated . Thus , the water pressure driving VLD formation was not caused by a complete seal , but by a transient and dynamic con\ufb01nement generated by friction and \ufb02ow restriction at the cell \u2013 substrate interface . Consistently , cells submitted to a gradual rather than sudden osmolarity increase had suf\ufb01cient time to evacuate expelled water , preventing VLD formation ( Fig . 4b ) . Interestingly , gradual rather than abrupt de - stretch also reduced reservoir formation , leading instead to membrane accumulations at the cell edge ( Fig . 4a ) . This suggests that cells subjected to slow deformations can release membrane excess at locations where the membrane is not con\ufb01ned by a substrate , such as the cell edge . However , the increase in friction caused by fast de - stretch would prevent such long - scale rearrangements , forcing the membrane to release tension locally in reservoirs at the cell \u2013 substrate interface . Mechanism of membrane mechanical adaptation . In summary , reservoirs or VLDs were formed locally by mechanical stimuli imposing , respectively , a change in area ( through stretch ) or volume stored at the cell \u2013 substrate interface ( through osmotic shocks or poroelastic \ufb02ows , see Supplementary Note 2 ) . We then evaluated different potential mechanisms to explain how those mechanical stimuli led to the formation of membrane structures . First , reservoirs and VLDs could be mediated by caveolae \ufb02attening , reported to occur in response to both stretch and hypo - osmotic shocks 10 . However , neither reservoirs nor VLDs co - localized with caveolin during our experiments , and both types of membrane structures still formed and resorbed in caveolin 1 knockout cells ( Supplementary Fig . 6 ) . Further , reservoirs and VLDs formed and resorbed equally in caveolin 1 knockout cells reconstituted either with caveolin 1 - GFP or with an empty vector ( Supplementary Fig . 6 ) . Second , the membrane could adapt through any of the active ATP - dependent remodelling processes ( such as exo - or endocytosis ) regulating its area and shape 3 \u2013 5 . However , whereas ATP depletion inhibited reservoir and VLD resorption , it did not affect their formation ( Fig . 5a , e and Supplementary Movie 6 ) . ATP depletion also inhibited the dynamic reservoir rearrangements that occurred during their resorption by cells ( Supplementary Movie 6 ) , showing that reservoir resorption but not formation is mediated by an ATP - dependent process . Similarly , actin cytoskeleton depolymerization with cytochalasin D or a reduction in temperature slowed the resorption of both reservoirs and VLDs , but did not prevent their formation ( Supplementary Fig . 7 ) . In addition , both reservoirs and VLDs were consistently observed across different cell types from diverse species ( Supplementary Fig . 8 ) . Thus , whereas both reservoirs and VLDs resorbed through an active actin - and temperature - dependent response , they formed by a general % M e m b r ane t ea r i ng Strain ( % ) 100 80 40 6 8 10 Iso - osmotic medium Hypo - osmotic medium % Of osmolarity % M e m b r ane l ys i s 0 60 20 40 20 0 30 20 10 A r ea V o l u m e 100 80 40 0 60 20 % I n c r ea s e 60 80 6 % 10 % Strain 50 % 0 % 0 % t = 0 S t r a i n R e l a x ed t = 30 s t = 0 m i n H y po - o s m o t i c m ed i u m I s o - o s m o t i c m ed i u m t = 2 m i n Osmolarity Figure 1 | Membrane response to stretch and osmotic changes . ( a ) Cells transfected with pEYFP - mem before ( top panel ) and after ( middle and bottom panels ) applying different magnitudes of constant stretch . Yellow arrow indicates a membrane ruf\ufb02e \ufb02attened by stretch . ( b ) Cells transfected with pEYFP - mem before and after reducing medium osmolarity to either 50 or 0 % of original medium . Cells submitted to 0 % osmolarity ( de - ionized water ) for 3min sometimes rounded and \ufb02attened membrane ruf\ufb02es ( middle panel ) and sometimes underwent membrane lysis ( right panel ) . Yellow arrows indicate membrane ruf\ufb02es , which either remain or \ufb02atten after applying 50 or 0 % hypo - osmotic medium , respectively . ( c ) Confocal slice showing a cell before ( green ) and after ( red ) application of medium with 50 % osmolarity for 3min . ( d ) Corresponding quanti\ufb01cation of the increase in cell volume and required membrane area ( n \u00bc 5 cells ) . ( e ) % of cells showing membrane tearing after 3min of constant stretch application ( n \u00bc 70 cells ) . ( f ) % of cells showing membrane lysis after 3min of application of medium with different osmolarity ( n \u00bc 50 cells ) . Scale bars , 20 m m . Error bars are mean \u00b1 s . e . m . NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 ARTICLE NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications 3 & 2015 Macmillan Publishers Limited . All rights reserved . t = 3 min t = 6 min V L D f l uo r e sc en c e t = 0 min t ( min ) R e s e r v o i r f l uo r e sc en c e Iso - osmotic medium Hypo - osmotic medium t = 3 min t = 6 min t = 0 min Relaxed 6 % Strain pEYFP - mem t ( min ) 0 1 2 3 4 5 6 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 0 1 2 3 4 5 6 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 1 2 3 * * * VLDs 20 10 * * * Num per 100 \u00b5 m 2 pEYFP - mem Height ( \u00b5 m ) 3 2 1 Diameter ( \u00b5 m ) Reservoirs * * * pEYFP - mem Actin pEYFP - mem Pax Actin Paxillin pEYFP - mem Actin pEYFP - mem Pax Actin Paxillin Figure 2 | Cell membranes use different strategies to readapt to normal surface and volume . ( a ) pEYFP - mem - transfected cells before , during and after constant stretch application during 3min . ( b ) Quanti\ufb01cation of reservoir \ufb02uorescence after stretch release ( 1 : initial \ufb02uorescence , 0 : background ) . n \u00bc 100 reservoirs from 10 cells . ( c ) Confocal vertical slice from a pEYFP - mem - transfected cell before ( top ) and after ( bottom ) application of 6 % stretch for 3min . ( d ) Staining images of cells \ufb01xed immediately after stretch release showing the membrane ( pEYFP - mem transfection ) , paxillin and actin . Merged co - localization images are shown to the right . ( e ) pEYFP - mem - transfected cells before , during and after application of 50 % hypo - osmotic medium during 3min . ( f ) Quanti\ufb01cation of VLD \ufb02uorescence after re - application of iso - osmotic medium ( 1 : initial \ufb02uorescence , 0 : background ) . n \u00bc 100 VLDs from 10 cells . ( g ) Confocal images of a pEYFP - mem - transfected cell before ( top ) and after ( bottom ) application of 50 % hypo - osmotic medium for 3min . ( h ) Staining images of cells \ufb01xed immediately after re - application of iso - osmotic medium showing the membrane ( pEYFP - mem transfection ) , paxillin and actin . Merged co - localization images are shown to the right . ( i ) Quanti\ufb01cation of mean diameter of structures formed after stretch release ( reservoirs ) and re - application of iso - osmotic medium ( VLDs ) . n \u00bc 250 / 100 structures from 8 / 10 cells . ( j ) Quanti\ufb01cation of mean density of structures formed after stretch release ( reservoirs ) and re - application of iso - osmotic medium ( VLDs ) . n \u00bc 30 / 50 regions from 5 / 8 cells . ( k ) Quanti\ufb01cation of mean height of structures formed after stretch release ( reservoirs ) and re - application of iso - osmotic medium ( VLDs ) . n \u00bc 80 / 50 structures from 6 / 4 cells ( * * * P o 0 . 001 , two - tailed Student\u2019s t - test ) . We note that reservoir heights are close to the axial resolution of our confocal microscope ( 0 . 9 m m ) and thus represent upper estimates rather than accurate measurements . Scale bars are 5 m m in c , g and 20 m m in d , h . In all cases , zoomed insets ( 10 (cid:2) 6 m m 2 in c , g and 10 (cid:2) 10 m m 2 in d , h ) show a magni\ufb01cation of the area marked in the main image . Error bars are mean \u00b1 s . e . m . ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 4 NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications & 2015 Macmillan Publishers Limited . All rights reserved . passive mechanical process . Further con\ufb01rming the passive nature of reservoirs , their resorption in ATP - depleted cells could be induced by re - applying mechanical stretch ( Fig . 5a , b and Supplementary Movie 6 ) . Interestingly , whereas VLDs in ATP - depleted cells did not resorb , they gradually collapsed as water leaked from them ( Fig . 5e , f and Supplementary Movie 7 ) , leaving membrane accumulations similar to reservoirs . Those accumulations did not disappear upon re - application of hypo - osmotic medium , con\ufb01rming ( as observed in cells plated on polyacrylamide gels ) that osmotic changes per se do not directly regulate membrane invaginations . Thus , the two types of membrane invaginations could apparently convert to each other , pointing at a uni\ufb01ed framework of membrane mechanical adaptation . Given its passive nature , this framework could potentially mimic the behaviour of synthetic cell - free membrane systems . To explore this hypothesis , we adapted a theoretical approach ( see Supplementary Note 3 ) previously shown to reproduce the behaviour of passive synthetic bilayer membranes adhered to a deformable substrate 18 . In this approach , membrane invaginations are understood as structures that store excess membrane area or interstitial volume with the least energy penalty . The energy sources considered are the elastic strain energy required to stretch and bend the membrane , and the adhesion energy required to detach the membrane from the substrate . This adhesion energy includes nonspeci\ufb01c interactions ( as in the case of synthetic bilayers ) , but also speci\ufb01c bonds to the extracellular matrix mediated for instance by integrins . In the case of dorsal reservoirs substrate adhesion would not apply , but certain adhesion energy would still be required to detach the membrane from the underlying actin cortex . In this system , introducing liquid at the membrane \u2013 substrate interface results in the formation of VLD - like shallow spherical cap invaginations , which optimally store volume . In contrast , compressing the membrane results in the formation of tubular invaginations ( with much higher surface / volume ratio ) , which optimally store excess membrane area . As surface / volume requirements increase , the model provides a phase diagram with increasingly large shallow caps to store volume , increasingly long tubules to store surface , and spherical invaginations with a small connecting neck to store both ( Fig . 6a ) . If this framework applies to live cells , then the membrane reservoirs generated upon stretch release would in fact be short tubules , which should become longer for larger compressions . To test this , we seeded cells on pre - stretched membranes , and then further stretched the membrane . After 3 min , the total stretch ( between 12 and 22 % ) was released , compressing the membrane . By using this two - step approach , we prevented the membrane tearing generally observed upon high stretch ( Fig . 1 ) . As expected , releasing stretch above 12 % resulted 6 % S t r a i n pu l s e H y po - o s m o t i c m . ( 3 m i n ) 6 % S t r a i n ( 3 m i n ) R e l a x ed R e l a x ed I s o - o s m o t i c m ed i u m Dextran pEYFP - mem 0 1 2 3 4 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 Soft silicone elastomer Soft silicone elastomer Soft silicone elastomer PA gel PA gel PA gel p EY F P - m e m D e x t r an p EY F P - m e m D e x t r an p EY F P - m e m D e x t r an t ( min ) F l uo r e sc en c e t = 0 min t = 0 . 5 min t = 6 min t = 0 min t = 0 . 5 min t = 6 min 5 6 1 . 4 Figure 3 | VLD formation is driven by the con\ufb01nement of liquid \ufb02ows at the cell \u2013 substrate interface . Response of pEYFP - mem - transfected cells seeded on either poly - acrylamide ( PA ) gels or soft silicone elastomers to : ( a ) the application of 50 % hypo - osmotic medium for 3min , ( b ) the application of 6 % strain for 3min and ( c ) a fast 6 % strain pulse . Insets show zoomed views ( 10x10 m m 2 ) of membrane structures . Scale bars , 20 m m . No signi\ufb01cant differences were observed between any of the cases either in the diameter of reservoirs ( n \u00bc 150 reservoirs from 3 cells ) or in their density ( n \u00bc 30 cell regions from 3 cells ) . ( d ) Time sequence of VLD formation and resorption in pEYFP - mem - transfected cells exposed to dextran - labelled iso - osmotic media after 3min incubation with 50 % unlabelled hypo - osmotic media . ( e ) Zoomed insets ( 20 (cid:2) 20 m m 2 ) corresponding to red square in d showing the evolution of membrane and dextran \ufb02uorescence , and merged images . ( f ) Corresponding quanti\ufb01cation of pEYFP - mem and dextran relative \ufb02uorescence levels . NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 ARTICLE NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications 5 & 2015 Macmillan Publishers Limited . All rights reserved . in the formation of tubules , which became longer as stretch increased ( Fig . 6b and Supplementary Movie 8 ) . In some cases , tubules were observed to dynamically grow from reservoirs , showing that indeed reservoirs correspond to nascent membrane tubules ( Supplementary Movie 8 ) . Also as predicted by the model , VLD size , and therefore contained volume , increased with the magnitude of the hypo - osmotic shock ( Fig . 6c ) . We then explored more complex membrane deformation pathways within the phase diagram ( Fig . 7a \u2013 d ) . First , we generated VLDs in cells by decreasing and then restoring osmolarity , and then quickly re - applied hypo - osmotic medium . Cells quickly swelled and re - absorbed water in VLDs , leading to their immediate collapse ( Fig . 7e ) and con\ufb01rming that hydrostatic pressure is key to their formation and maintenance ( see Supplementary Note 2 ) . However , mere removal of hydrostatic pressure was not suf\ufb01cient to resorb membrane recruited upon VLD formation . As in ATP - depleted cells ( Fig . 5e - f ) , bright membrane accumulations ( \u2018collapsed\u2019 VLDs akin to reservoirs ) remained at VLD sites ( Fig . 7e ) . Subsequent application of stretch could then eliminate collapsed VLDs , closing the path in the phase diagram ( Fig . 7a ) . In contrast , full non - collapsed VLDs were maintained by hydrostatic pressure and only increased in diameter upon stretch ( Fig . 7f , i ) . Next , we submitted cells to both hypo - osmotic shock and stretch for 3 min and \ufb01rst restored iso - osmotic medium , leading to VLD formation as expected ( Fig . 7g ) . Upon stretch release , excess membrane did not form reservoirs or tubules but rather accumulated at the site of VLDs , as indicated by a sharp increase in pEYFP - mem \ufb02uorescence ( Fig . 7k ) . Confocal sections showed that VLDs became taller and more invaginated ( Fig . 7j ) , in agreement with the structures predicted by the model to store both volume and membrane area ( Fig . 7c ) . This latter \ufb01nding highlights that pre - existing membrane invaginations ( which are already bent and detached from the substrate ) act as seeds for further membrane storage . To con\ufb01rm this , we submitted cells to both stretch and hypo - osmotic medium and \ufb01rst released stretch , which resulted in reservoir formation ( Fig . 7h ) . When we then restored iso - osmotic medium , VLDs indeed formed at the \u2018seed\u2019 sites where reservoirs were previously located ( Fig . 7l ) . As VLD formation was now dictated by the denser reservoir network , VLDs appeared in higher number and smaller size than those formed either in the absence of stretch or before stretch release ( Fig . 7m ) . Finally , we evaluated the effect of modifying one of the key parameters of the system , adhesion energy . To this end , we treated cells with a blocking antibody against a 5 b 1 integrin , which we previously identi\ufb01ed to provide adhesion strength to \ufb01bronectin - coated substrates in the same cell type 19 . Decreasing adhesion strength reduced the density of stretch - mediated reservoirs ( Supplementary Fig . 9 ) . Similar to the case of slow stretch release ( Fig . 4 ) , this suggests that reduced friction and interaction at the membrane \u2013 substrate interface allowed cells to release excess membrane in more distant but less con\ufb01ned membrane regions . In contrast , VLDs in cells with reduced adhesion slightly increased in density , and markedly increased in diameter ( Supplementary Fig . 9 ) . This is consistent with model predictions , as a reduction in adhesion would make it energetically favourable to detach a larger membrane area to generate each VLD . Inhibition of a 5 b 1 also slowed VLD resorption , demonstrating that cells had an impaired ability to re - adhere detached membrane areas . In conclusion , the phase diagram provided by passive minimization of elastic and adhesive energies consistently predicted how the different perturbations generated membrane structures . The approximate dimensions of those structures , and their relative variations , were also correctly predicted after assuming parameter values consistent with experimental conditions ( see Supplementary Note 3 ) . Discussion Despite extensive work , the mechanisms of membrane adaptation to physical constraints have remained elusive . It was recently shown that membrane area can be stored or released upon mechanical stimulation through the assembly / disassembly of caveolae 10 . However , the estimated 0 . 3 % of membrane area contained in caveolae contrasts with membrane requirements of up to 10 % for instance in spreading cells 7 , suggesting that additional buffers are required . Such buffers can be provided in time scales from seconds to minutes by active exocytic / endocytic Iso - osmotic medium Hypo - osmotic medium Relaxed 6 % Strain 6 % Strain Hypo - osmotic medium Figure 4 | Effect of stimulus application rate on membrane structure formation . ( a ) Cell submitted to two successive steps of 6 % stretch for 3min , in which the \ufb01rst is released immediately and the second slowly ( 15s ) . ( b ) Cell submitted to two successive applications of 50 % hypo - osmotic media , in which iso - osmotic medium is restored \ufb01rst immediately and then slowly ( 1min ) . Scale bars , 20 m m . Insets show zoomed views ( 10 (cid:2) 10 m m 2 ) of membrane structures . ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 6 NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications & 2015 Macmillan Publishers Limited . All rights reserved . processes 3 \u2013 5 , which protect membrane integrity for instance in alveolar lung epithelial cells in response to both stretch 20 and osmotic changes 21 . Here we show that , before the onset of any such active process , membranes adapt almost instantaneously through a passive process minimizing membrane elastic and adhesion energies , akin to what is observed in synthetic lipid membranes 18 , 22 . This process leads to the nucleation and growth of reservoirs / tubules to accommodate membrane area fractions that can be above 10 % . The analogy between cell membranes and synthetic bilayers is surprising and has striking implications . First and addressing an unresolved issue 2 , our results suggest that if the perturbation is fast enough , membrane tension is released locally and not instantaneously transmitted across the cell ( Fig . 4 ) . The shape and size of membrane structures thus depends on both the magnitude and the dynamics of applied perturbations , which could lead for instance to the travelling membrane / cortex waves observed in cell - free surfaces 23 . Second and despite the complex molecular composition , cytoskeletal attachment and active behaviour of cell membranes 24 , we show that their mechanical adaptation can be successfully modelled by simply considering the two lipid layers . This is because the main mechanical parameter that constrains membrane deformation is the stretching modulus , which is determined by the membrane itself and not by the underlying actin cortex ( see Supplementary Note 3 ) . Finally , we note that reservoirs can store and release membrane area upon subsequent stretch cycles ( Fig . 5 ) , providing a regulatory mechanism potentially applicable to mechanical processes with time scales below the B 2 min required for active membrane resorption ( such as breathing , heart beating or muscle contraction ) . Our results also demonstrate that VLDs , which were broadly understood as membrane area containers 4 , 9 , 25 , are driven instead by hydrostatic pressure from water stored at the cell \u2013 substrate interface . Their role in processes such as cell adaptation to A T P dep l e t i on C on t r o l R e s e r v o i r f l uo r e sc en c e ATP depletion Control V L D f l uo r e sc en c e Iso - osmotic m . Hypo - osmotic medium t = 0 min t = 5 min t = 6 min t = 9 min A T P dep l e t i on C on t r o l t ( min ) t ( min ) 1 . 0 2 . 0 3 . 0 N u m be r pe r 100 \u00b5 m 2 0 . 2 0 . 6 1 . 0 D i a m e t e r ( (cid:2) m ) 5 10 15 20 t = 0 min t = 5 min t = 6 min t = 9 min Relaxed 6 % Strain 0 2 4 6 8 10 12 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 1 . 4 0 2 4 6 8 10 12 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 1 . 4 ATP depletion Control NS NS NS NS D i a m e t e r ( \u00b5 m ) N u m be r pe r 100 \u00b5 m 2 C on t r o l A T P C on t r o l A T P C on t r o l A T P C on t r o l A T P 1 . 0 2 . 0 3 . 0 Figure 5 | Membrane mechanical adaptation is a passive process followed by active recovery . ( a ) Examples of control and ATP - depleted pEYFP - mem - transfected cells before , during and after application of two 3 - min constant stretch pulses . ( b ) Quanti\ufb01cation of reservoir \ufb02uorescence after release of \ufb01rst stretch pulse and during application and release of the second pulse ( n \u00bc 50 / 70 reservoirs from 5 / 5 cells ) . The effect of ATP depletion was signi\ufb01cant ( P o 0 . 001 ) . ( c ) Quanti\ufb01cation of reservoir size in control and ATP - depleted cells ( n \u00bc 150 / 200 reservoirs from 3 / 4 cells ) . No signi\ufb01cant differences were observed , two - tailed Student\u2019s t - test . ( d ) Quanti\ufb01cation of reservoir density in control and ATP - depleted cells ( n \u00bc 50 / 50 regions from 5 / 5 cells ) . No signi\ufb01cant differences were observed . ( e ) Examples of control and ATP - depleted pEYFP - mem - transfected cells before , during and after application of 50 % hypo - osmotic medium in two 3 - min pulses . ( f ) Quanti\ufb01cation of VLD \ufb02uorescence after the \ufb01rst re - application of iso - osmotic medium and during application and release of the second pulse ( n \u00bc 35 / 40 VLDs from 3 / 3 cells ) . The effect of ATP depletion was signi\ufb01cant ( P o 0 . 001 , two - tailed Student\u2019s t - test ) . ( g ) Quanti\ufb01cation of VLD size in control and ATP - depleted cells ( n \u00bc 30 / 35 VLDs from 3 / 3 cells ) . No signi\ufb01cant differences were observed . ( h ) Quanti\ufb01cation of VLD density in control and ATP - depleted cells ( n \u00bc 20 / 25 regions from 3 / 3 cells ) . No signi\ufb01cant differences were observed . Scale bars , 20 m m . Insets show zoomed views ( 10 (cid:2) 10 m m 2 ) of membrane structures . NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 ARTICLE NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications 7 & 2015 Macmillan Publishers Limited . All rights reserved . shrinking should therefore be revisited , as cells in most physiological settings will be surrounded by permeable extracellular matrices , which would not constrain water \ufb02ow from cells . However , we show that VLDs can be equivalently formed by hydrostatic pressure arising from other sources ( such as poroelastic \ufb02ows , Fig . 3b ) , suggesting a signi\ufb01cant role in the cellular adaptation to any excess water pressure at the cell \u2013 matrix interface . The fact that increasing osmolarity leads to the immediate formation of VLDs rather than merely expelling water through the dorsal surface also con\ufb01rms that the cytoplasm exhibits limited water mobility and slow pressure redistribution 26 . Both reservoirs / tubules and VLDs form at sites of low cytoskeletal density and resorb through an actin - and ATP - dependent process . This active cell response likely involves actin polymerization to push the lamellipodium and re - stretch and \ufb02atten the cell membrane , endocytic processes to detach invaginated reservoirs or tubules , or vesiculation , that is , the detachment of membrane vesicles from tubules that has been observed both in live cells 27 and in passive bilayer systems 18 . However and regardless of the speci\ufb01c active mechanisms by which cells re - absorb membrane structures , the physical principles that drive their formation may themselves be harnessed by cells to respond to changes in cell shape arising in any instance of cell migration or deformation . Further , the biochemical activity of membrane curvature - sensitive molecules 2 , 28 , 29 could also be affected by the local curvature induced by tubules or VLDs , potentially initiating mechano - transduction cascades . Methods Cell culture and reagents . MEFs were previously described 19 , 30 and cultured in DMEM supplemented with 10 % fetal bovine serum ( FBS ) . One day before experiments , cells were transfected with the membrane - targeting plasmid pEYFP - mem ( Clontech ) or lifeact - ruby using the Neon transfection device according to the manufacturer\u2019s instructions ( Invitrogen ) . pEYFP - mem contains the N - terminal 20 amino acids of neuromodulin , which is palmytoylated post - translationally and targets the EYFP \ufb02uorophore to membranes . Cells were incubated with cytochalasin D for 30min to depolymerize the actin cytoskeleton ( 0 . 5 m M , Sigma ) and with 10mM deoxy - D - glucose plus 10mM NaN 3 ( Sigma ) to deplete ATP levels . Dextran experiments were carried out with 0 . 5mgml (cid:3) 1 of tetramethylrhodamine - labelled dextran ( 10 , 000 MW , Life technologies ) , and a 5 b 1 integrins were blocked with 10 m gml (cid:3) 1 a 5 b 1 antibody ( Merck Millipore , clone BMB5 ) . The role of caveolin 1 was analysed by using Caveolin1 Knock out MEFs reconstituted with Cav1 or IRES - GFP as a control 31 . Cav1 was cloned in the lentiviral vector pRR SIN 18 CMV IRES EGFP . To generate these stable cell lines , the infected cells were selected by sorting for GFP marker expression by \ufb02ow cytometry ( FACS ) . Cav1 expression was checked by western blot analysis and reverse transcription \u2013 quantitative PCR . Cells expressing levels of Cav1 - GFP comparable to endogenous Cav1 levels in wild - type MEFs were selected for experiments . Chinese Hamster Ovary cells were cultured in HF - 12 medium supplemented with 10 % FBS . Human keratinocytes ( HaCaT ) were cultured in DMEM supplemented with 10 % FBS . Human squamous carcinoma cells ( A431 ) were cultured in Earle\u2019s Balanced Salt Solution supplemented with 10 % FBS . Preparation of stretchable membranes . The stretchable PDMS membranes ( see schematic in Supplementary Fig . 1 ) were prepared by mixing the PDMS base and crosslinker at a 10 : 1 ratio , degassing for 1h , spinning the mixture on a 13 - cm sheet ( 500r . p . m . , 1min ) and curing at 65 (cid:2) C overnight . Once cured , PDMS membranes were peeled off and placed tightly between the rings of the stretching device ( Supplementary Fig . 1 ) . Membranes were then coated with 10 m gml (cid:3) 1 \ufb01bronectin ( Sigma ) overnight at 4 (cid:2) C , or attached to either polyacrylamide or soft silicone elastomers . To attach polyacrylamide gels to membranes 32 , gels were prepared by using a mixture of 10 % acrylamide and 0 . 3 % bis - acrylamide and polymerizing between two coverslips treated with repel - silane ( Young\u2019s modulus B 30kPa ) . Once polymerized , one coverslip was removed and the gel was pressed in contact with the PDMS membrane , which had previously been treated with 3 - aminopropyl triethoxysilane 10 % in ethanol for 1h at 65 (cid:2) C and with glutaraldehyde ( 1 , 5 % ) in PBS for 25m at room temperature . After overnight incubation at 37 (cid:2) C in a humid chamber for covalent binding , the other coverslip was removed and the gel was incubated with 10 m gml (cid:3) 1 \ufb01bronectin overnight at 4 (cid:2) C , resulting in membrane - attached gels ready for cell culture . Soft silicon elastomers ( CY 52 \u2013 276 , Dow Corning , with Young\u2019s modulus B 8kPa ( ref . 33 ) ) were prepared by mixing CyA and CyB components at a 1 : 1 ratio and curing at 80 (cid:2) C for 2h ( ref . 33 ) . The substrates were then attached to membranes following the same procedure as for polyacrylamide gels . In some experiments not involving stretch , PDMS membranes were cured directly on glass coverslips instead of placing them in the stretch system . Stretch and osmolarity experiments . Once PDMS membranes were either directly coated with \ufb01bronectin or covalently attached to \ufb01bronectin - coated polyacrylamide / soft silicone gels , cells were seeded on the membrane and allowed to spread in the incubator for 0 . 5h . Then , membranes were placed on the stretch system ( Supplementary Fig . 1 ) , consisting of a central loading post and an external ring . Vacuum was then applied through the space between the loading post and the external ring , thereby deforming and stretching the membrane . Cells spread on the membrane directly on top of the central loading post experienced an equibiaxial strain which depended on the vacuum pressure applied ( Supplementary Fig . 1 ) . The system was then mounted on the microscope stage . To modify osmolarity , cells were exposed to medium mixed with de - ionized water in which the concentrations of Ca 2 \u00fe and Mg 2 \u00fe had been corrected . 0 5 3 . 5 1 . 5 Surface storage V o l u m e s t o r age % D e s t r e t c h 6 12 t ( min ) 22 % Of destretch % Of osmolarity V L D d i a m e t e r ( \u00b5 m ) Leng t h ( \u00b5 m ) 7 6 5 4 3 2 1 5 10 15 20 25 1 2 3 4 80 40 20 60 0 Release Strain Figure 6 | Membrane mechanical adaptation is explained by minimization of the strain and adhesion energies required to generate surface and volume containers . ( a ) Phase diagram showing the predicted structures that require minimal energy to deform the membrane and detach it from the substrate in order to accommodate membrane surface area ( upon stretch release ) and liquid volume at the cell \u2013 substrate interface ( upon an increase in osmolarity ) 18 . Surface storage is achieved optimally with increasingly long tubules , whereas volume storage leads to the formation of spherical caps ( VLDs ) . When both volume and surface storage are required , spherical caps \u2018bud\u2019 and become more invaginated . ( b ) Left : time - course sequences of cell membrane regions showing the formation of either reservoirs or increasingly long tubules after releasing different stretch magnitudes . Right : Mean reservoir / tubule length ( black dots , experimental data , red line , theoretical prediction ) as a function of de - stretch magnitude ( for increasing stretch , n \u00bc 80 / 50 / 50 structures from 6 / 3 / 3 cells ) . ( c ) Left : images showing the formation of increasingly large VLDs after restoring iso - osmotic medium in cells previously exposed to different magnitudes of hypo - osmotic shocks for 3min . Right : mean VLD diameter ( black dots , experimental data , red line , theoretical prediction ) as a function of hypo - osmotic shock magnitude ( for increasing osmotic shock , 60 / 100 / 100 / 50 structures from 5 / 5 / 10 / 3 cells ) . Scale bars , 5 m m . Error bars are mean \u00b1 s . e . m . ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 8 NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications & 2015 Macmillan Publishers Limited . All rights reserved . Imaging . Cell images of \ufb02uorescently labelled cells were obtained using an upright microscope ( Nikon eclipse Ni - U ) with a water immersion objective ( (cid:2) 60 magni\ufb01cation , NA \u00bc 1 . 0 ) and an Orca Flash 4 . 0 camera ( Hamamatsu ) . To obtain three - dimensional stacks , an inverted microscope ( Nikon Eclipse Ti ) with a spinning disk confocal unit ( CSU - W1 , Yokogawa ) , a Zyla sCMOS camera ( Andor ) and a (cid:2) 60 objective was used . This objective was either of oil immersion osm . shock osm . shock + destretch t ( min ) 0 1 2 3 4 5 6 0 . 5 1 . 0 1 . 5 2 . 0 VLD diam . ( \u00b5 m ) 1 2 3 * * * * * * 2 4 6 N . VLD per 100 \u00b5 m 2 o s m . s ho ck o s m . s ho ck + D e s t r e t c h D e s t r e t c h + o s m . s ho ck * * * * * * F l uo r e sc en c e i n t en s i t y H y po - o s m o t i c m ed i u m 6 % S t r a i n R e l a x ed I s o - o s m o t i c m ed i u m H y po - o s m o t i c m ed i u m 6 % S t r a i n R e l a x ed I s o - o s m o t i c m ed i u m I m age # 3 I m age # 4 R e l a x ed I s o - o s m o t i c m ed i u m H y po - o s m o t i c m ed i u m R e l a x ed I s o - o s m o t i c m ed i u m H y po - o s m o t i c m ed i u m I m age # 4 T i m e 6 % S t r a i n 6 % S t r a i n I m age # 3 H y po t o i s o I s o t o h y po Stretch H y po t o i s o Stretch H y po t o i s o Destretch Destretch H y po t o i s o # 1 # 2 # 4 # 3 # 5 # 1 # 2 # 4 # 3 # 1 # 2 # 4 # 3 # 1 # 2 # 4 # 3 # 3 # 4 # 5 # 3 # 4 # 3 # 4 # 3 # 4 # 2 # 2 # 2 # 2 l Figure 7 | Membrane remodelling can be described through pathways along the surface / volume phase diagram . ( a \u2013 d ) Different pathways tested in the phase diagram by applying stretch and hypo - osmotic shocks ( red arrows , numbers refer to the corresponding image in the panel below ) . ( e \u2013 h ) Corresponding response of pEYFP - mem - transfected cells after applying stretch and osmotic shocks as indicated to follow the pathways . Time \ufb02ows from top to bottom . In all cases , the \ufb01rst application of stretch / hypo - osmotic shock ( second row of cells ) lasted 3min . Subsequent steps were carried out as quickly as possible to evaluate membrane response before cells had time to actively eliminate structures . ( i ) In cells submitted to hypo - osmotic shock , co - localization of membrane structures formed after \ufb01rst restoring iso - osmotic medium ( red ) and then applying stretch ( green ) . ( j ) Confocal vertical slices showing VLD shape before ( top ) and after ( bottom ) stretch release . Zoomed image to the right shows the superimposed shape prediction from the theoretical model in red . ( k ) Quanti\ufb01cation of VLD \ufb02uorescence for cells under hypo - osmotic medium after either restoring iso - osmotic medium ( blue symbols ) or restoring iso - osmotic medium and then releasing stretch application ( pink symbols , arrow indicates moment of stretch release ) . N \u00bc 100 / 50 structures from 10 / 5 cells . ( l ) In cells submitted to both hypo - osmotic shock and stretch , co - localization of membrane structures formed after \ufb01rst releasing stretch ( red ) and then restoring iso - osmotic medium ( green ) . ( m ) Quanti\ufb01cation of VLD diameter ( n \u00bc 100 / 50 / 70 structures from 10 / 3 / 3 cells , * * * P o 0 . 001 , analysis of variance ( ANOVA ) ) and density ( n \u00bc 50 / 30 / 30 regions from 8 / 3 / 3 cells , * * * P o 0 . 001 , ANOVA ) in cells submitted only to osmotic shocks or also to de - stretch ( stretch release ) before or after restoring iso - osmolarity ) . Scale bars are 5 m m in j and 20 m m elsewhere . Insets show zoomed views ( 10 (cid:2) 10 m m 2 ) of membrane structures . Error bars are mean \u00b1 s . e . m . NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 ARTICLE NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications 9 & 2015 Macmillan Publishers Limited . All rights reserved . ( NA \u00bc 1 . 42 ) or of water immersion ( NA \u00bc 1 . 0 ) for experiments involving stretch , as viscous oil droplets dragged the \ufb02exible PDMS membrane used for stretch and precluded proper focusing . Analysis of membrane structures . The evolution of membrane structures ( reservoirs and VLDs ) was analysed by measuring the time course of the pEYFP - mem \ufb02uorescence of each structure . To correct for photobleaching , this \ufb02uorescence was expressed as the fold - increase with respect to the background \ufb02uorescence of neighbouring cell regions without membrane structures , and normalized between 1 ( initial \ufb02uorescence after stretch release or osmolarity increase ) and 0 ( background cell \ufb02uorescence ) . The diameter and density of structures was also measured , and the height was obtained from confocal slices . We note , however , that reservoir heights are close to the axial resolution of our confocal microscope ( 0 . 9 m m ) and thus represent upper estimates rather than accurate measurements . The membrane fraction contained in membrane structures was estimated by comparing the average pEYFP - mem \ufb02uorescence of cell regions containing structures to the average \ufb02uorescence of structure - free zones within the same region . To ensure that we only considered the \ufb02uorescence of structures induced by stretch or osmotic shocks , the analysis was carried out in regions devoid of visible endomembrane structures before the application of stretch or osmotic shocks . In all time - lapse \ufb02uorescence images shown in \ufb01gures , contrast was adjusted in each image to correct for the effect of photobleaching and leave cell background at a uniform level . Supplementary Videos show the full time - lapse videos without this adjustment , thereby showing the effect of photobleaching . The videos also show a decrease in \ufb02uorescence upon application / release of stretch , caused by added photobleaching during the re - centering and re - focusing of cells after their movement . Cell volume and surface estimations . Changes in cell volume and required membrane surface were calculated from spinning disk confocal slices obtained in cells before and after a 3 - min 50 % hypo - osmotic treatment . Cells were \ufb01rst re - sliced in the XZ plane ( resulting in the images observed for instance in Fig . 2g ) , and membrane \ufb02uorescence images were binarized by thresholding . Cell volume was then calculated by adding the total number of pixels inside cells from all slices and multiplying by voxel size . To measure membrane surface , the cell perimeter in XZ - plane confocal slices was drawn manually . This avoided spurious increases in the estimated cell perimeter caused by the jagged cell edge resulting from binarization . We note that our area estimates do not correspond to the total membrane area , which may contain small folds not resolved by microscope images . Rather , our measurements estimate the increase in membrane area required to accommodate the global change in shape produced by cell swelling . Immunostaining . For \ufb02uorescence staining , cells were \ufb01xed with 4 % paraformaldehyde , permeabilized with 0 . 1 % Triton X - 100 and labelled \ufb01rst with primary antibodies ( 2h , room temperature ) , and then with Alexa - conjugated secondary antibodies ( Invitrogen , 2h , room temperature ) . Primary antibodies used were against paxillin ( 2 m gml (cid:3) 1 , clone 349 produced in mouse , ref . 610051 from BD Transduction Laboratories ) and Caveolin 1 ( 4 m gml (cid:3) 1 , CAV1 , polyclonal antibody produced in rabbit , ref . 610060 from BD Transduction Laboratories ) . Phalloidin - Tetramethylrhodamine B isothiocyanate ( Sigma ) was used instead of primary antibodies to label actin . 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Acknowledgements We acknowledge support from the Spanish Ministry for Economy and Competitiveness ( BFU2011 - 23111 , BFU2012 - 38146 , and FIS - PI11 - 00089 ) , a Career Integration Grant within the seventh European Community Framework Programme ( PCIG10 - GA - 2011 - 303848 ) , the European Research Council ( Grant Agreements 242993 , 240487 and 240487 ) , the Generalitat de Catalunya , Fundacio\u00b4 La Caixa , Fundacio\u00b4 la Marato\u00b4 de TV3 ( project 20133330 ) and the Mechanobiology Institute Singapore grant , initiative of the the National Research Foundation of Singapore ( to N . C . G ) . We thank F . Lolo and the members of the X . T . and P . R - C . laboratories for technical assistance and discussions . ARTICLE NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 10 NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications & 2015 Macmillan Publishers Limited . All rights reserved . Author contributions A . J . K . , M . A . , N . C . G . and P . R . - C . conceived the study , A . J . K . , S . M . , M . A . , X . T . , N . C . G . and P . R . - C . designed the experiments , A . J . K . and J . J . T . performed the experiments , A . E . - A , R . M . - V . and M . A . delP . contributed new reagents / analytical tools , A . J . K . , L . C . , V . G . - T . , D . N . and X . T developed the stretch device , M . A . carried out the theoretical modelling , and A . J . K . , M . A . , and P . R . - C . wrote the paper . Additional information Supplementary Information accompanies this paper at http : / / www . nature . com / naturecommunications Competing \ufb01nancial interests : The authors declare no competing \ufb01nancial interests . Reprints and permission information is available online at http : / / npg . nature . com / reprintsandpermissions / How to cite this article : Kosmalska , AJ . et al . Physical principles of membrane remodelling during cell mechanoadaptation . Nat . Commun . 6 : 7292 doi : 10 . 1038 / ncomms8292 ( 2015 ) . This work is licensed under a Creative Commons Attribution 4 . 0 International License . The images or other third party material in this article are included in the article\u2019s Creative Commons license , unless indicated otherwise in the credit line ; if the material is not included under the Creative Commons license , users will need to obtain permission from the license holder to reproduce the material . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / NATURE COMMUNICATIONS | DOI : 10 . 1038 / ncomms8292 ARTICLE NATURE COMMUNICATIONS | 6 : 7292 | DOI : 10 . 1038 / ncomms8292 | www . nature . com / naturecommunications 11 & 2015 Macmillan Publishers Limited . All rights reserved .", + "mueller2017load": "Article Load Adaptation of Lamellipodial Actin Networks Graphical Abstract Highlights d Lamellipodial actin density co - \ufb02uctuates with the size of the projected area d Lamellipodial actin density adapts to changes in membrane tension d Actin branch geometry prescribes adaptations in lamellipodial actin Authors Jan Mueller , Gregory Szep , Maria Nemethova , . . . , Kinneret Keren , Robert Hauschild , Michael Sixt Correspondence sixt @ ist . ac . at In Brief How do mechanical perturbations in\ufb02uence the density and the geometry of actin networks at the leading edge of migrating cells ? Mueller et al . , 2017 , Cell 171 , 1 \u2013 13 September 21 , 2017 \u00aa 2017 Elsevier Inc . http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 Article Load Adaptation of Lamellipodial Actin Networks Jan Mueller , 1 Gregory Szep , 1 Maria Nemethova , 1 Ingrid de Vries , 1 Arnon D . Lieber , 2 Christoph Winkler , 3 , 4 Karsten Kruse , 5 J . Victor Small , 6 Christian Schmeiser , 3 , 4 Kinneret Keren , 2 , 7 Robert Hauschild , 1 and Michael Sixt 1 , 8 , * 1 Institute of Science and Technology Austria ( IST Austria ) , am Campus 1 , 3400 Klosterneuburg , Austria 2 Department of Physics and Russell Berrie Nanotechnology Institute , Technion , Israel Institute of Technology , Haifa 32000 , Israel 3 RICAM , Austrian Academy of Sciences , Apostelgasse 23 , 1030 Vienna , Austria 4 Faculty of Mathematics , University of Vienna , Oskar - Morgenstern - Platz 1 , 1090 Vienna , Austria 5 NCCR Chemical Biology , Departments of Biochemistry and Theoretical Physics , University of Geneva , 30 , quai Ernest - Ansermet , 1211 Geneva , Switzerland 6 Institute of Molecular Biotechnology GmbH ( IMBA ) , Dr . Bohr - Gasse 3 , 1030 Vienna , Austria 7 Network Biology Research Laboratories , Technion , Israel Institute of Technology , Haifa 32000 , Israel 8 Lead Contact * Correspondence : sixt @ ist . ac . at http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 SUMMARY Actin \ufb01laments polymerizing against membranes power endocytosis , vesicular traf\ufb01c , and cell motility . In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces . We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated . In a steady state , migrating cell \ufb01laments assume the canonical dendritic geometry , de\ufb01ned by Arp2 / 3 - generated 70 ! branch points . Increased tension triggers a dense network with a broadened range of angles , whereas decreased tension causes a shift to a sparse con\ufb01guration dominated by \ufb01la - ments growing perpendicularly to the plasma mem - brane . We show that these responses emerge from the geometry of branched actin : when load per \ufb01lament decreases , elongation speed increases and perpendicular \ufb01laments gradually outcompete others because they polymerize the shortest dis - tance to the membrane , where they are protected from capping . This network - intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load . INTRODUCTION From endocytic processes and vesicle traf\ufb01cking to cellular locomotion and intracellular pathogen movement , the actomy - osin cytoskeleton mediates most of the mechanical responses of eukaryotic cells ( Pollard and Cooper , 2009 ) . Actomyosin net - works have two principal mechanisms of force generation : ( 1 ) polymerization can expand the network by elongating \ufb01laments against a load , and ( 2 ) myosin motors can contract the network or transport cargo along \ufb01laments . Every actin \ufb01lament is born by a nucleation event . Nucleation is catalyzed by a number of molecular machines , the most prominent being formins and the Arp2 / 3 complex . While formins achieve de novo linear nucle - ation , the Arp2 / 3 complex binds to an existing \ufb01lament and branches off a new \ufb01lament at a 70 ! angle . Elongation , the addi - tion of new monomers to the growing ( barbed ) end of the \ufb01la - ment , is mainly mediated by formins or by proteins of the VASP family . Both nucleation and elongation are enhanced at the interface to membranes , where growing \ufb01laments are shielded from capping proteins , which otherwise terminate \ufb01la - ment elongation by sealing the \ufb01lament\u2019s barbed end ( Carlsson , 2010 ; Pollard , 2007 ) . Arp2 / 3 - dependent actin polymerization is a force - sensitive process . This has been demonstrated in vitro , in minimalist re - constituted systems , whereby increasing the mechanical load on a protruding reconstituted network led to higher network den - sity ( Bieling et al . , 2016 ; De\u00b4moulin et al . , 2014 ; Parekh et al . , 2005 ) . Thus , the network structure was able to adapt to the ambient mechanical conditions and to support force generation and mechanical resilience under varying loads . It has been shown that such adaptation is partially mediated by differential force sensitivities of nucleation , elongation , and capping , leading to enhanced branching under higher loads ( Bieling et al . , 2016 ) . However , these kinetic effects were only partially able to explain the changes in network density , and it was speculated that the remaining adaptation might be mediated by spatial rearrange - ments of the network . The mechanochemical response of protruding actin networks has not yet been studied in living cells . However , indirect evi - dence suggests that the leading edge of a motile cell is able to respond to load . When the lamellipodium , the \ufb02at protrusive front of a migrating cell , encounters an elastic obstacle ( e . g . , the ver - tical cantilever of an atomic force microscope ) , it was shown to substantially increase its pushing force , until collapsing when the load became excessive . For a migrating cell encountering a barrier , this means that its front pushes harder until the obstacle is pushed away or the leading edge stalls and the cell either retracts or circumnavigates the barrier ( Heinemann et al . , 2011 ; Prass et al . , 2006 ) . In \ufb01broblasts or epithelial cells , where lamellipodia typically undergo cycles of protrusion and retraction , it has been sug - gested that during each protrusive phase the polymerizing actin \ufb01laments experience a gradually growing load , because increase Cell 171 , 1 \u2013 13 , September 21 , 2017 \u00aa 2017 Elsevier Inc . 1 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 in projected cell area is accompanied by increase in membrane tension ( the cell stretches out the rather inextensible bag of plasma membrane ) ( Diz - Mun\u02dcoz et al . , 2013 ; Gauthier et al . , 2011 ; Raucher and Sheetz , 2000 ; Sens and Plastino , 2015 ) . This growing load on the pushing \ufb01laments is accompanied by accelerated incorporation of the actin nucleating Arp2 / 3 com - plex , which might , in turn , increase actin network density and boost force generation until the \ufb01laments stall and the leading edge retracts ( Ji et al . , 2008 ; Lee et al . , 2015 ; Ryan et al . , 2012 ) . While these response patterns might result from complex mechanosensitive signaling processes , theoretical consider - ations put forward the alternative idea that branched actin net - works respond to varying polymerization kinetics by changing their geometry ( Maly and Borisy , 2001 ; Schaus et al . , 2007 ; Weichsel and Schwarz , 2010 ) . Such responses would argue that load adaptation is an emergent geometrical property of branched actin . Here , we use the lamellipodium of \ufb01sh keratocytes to quantita - tively examine how protruding actin networks respond to varying forces . We use a combination of quantitative light and electron microscopy to describe geometrical changes of lamellipodial actin upon varying load regimes and employ stochastic modeling to elucidate how the network structure geometrically adapts to counter - forces . RESULTS Temporal Fluctuations in Actin Density Arise at the Leading Edge and Are Correlated with Projected Cell Area and Protrusion Speed To quantitatively explore how lamellipodial actin reacts to changes in load we employed \ufb01sh keratocytes migrating on planar surfaces ( Mogilner and Keren , 2009 ) . We established pro - tocols to derive keratocytes from adult zebra\ufb01sh , allowing ac - cess to stable transgenesis . On serum - coated coverslips , these cells migrate with exceptional speed , persistence , and morpho - logical stability ( Keren et al . , 2008 ) . We \ufb01rst chose a correlative approach and observed single migrating keratocytes expressing the actin reporter lifeact : GFP ( Riedl et al . , 2008 ) with high spatio - temporal resolution ( Figure 1A ; Movie S1 ) . This allowed us to monitor \ufb02uctuations in actin network dynamics and density together with protrusion speed and morphological parameters ( Figures 1A \u2013 1D , S1A , and S1B ) . The projected area of a kerato - cyte typically \ufb02uctuated \u00b1 5 % around a baseline , which was consistent with earlier studies ( Keren et al . , 2008 ) ( Figure S1A ) . These limited \ufb02uctuations supported the notion that the kerato - cyte\u2019s plasma membrane does not harbor major membrane res - ervoirs , which might be mobilized upon mechanical stretch ( Lieber et al . , 2013 ) . When we measured lifeact : GFP intensity within a \" 1 - m m broad zone behind the leading front , intensity \ufb02uctuations were more substantial than area changes and varied \u00b1 20 % ( Figures 1D and S1A ) . Strikingly , the normalized , temporal \ufb02uctuations in projected cell area were tightly correlated with life - act : GFP intensities as shown by cross - correlation peaks be - tween 0 . 6 \u2013 0 . 7 at time lag zero ( Figures 1D , 1E , and S1B ) . This correlation was also observed in actin : GFP - expressing kerato - cytes , but not when the plasma membrane was uniformly labeled ( Figure S2 ) . Correlation analysis of consecutive \" 1 - m m broad lamellipodial regions showed that the time delay between correlation peaks increased with the distance between the measured regions ( Fig - ures S1C \u2013 S1E ) . Dividing the respective lag times by the speed of each cell yields distances matching those between adjacent la - mellipodial regions ( Figure S1F ) . This analysis showed that , \ufb01rst , as previously demonstrated ( Theriot and Mitchison , 1991 ; Wil - son et al . , 2010 ) keratocytes exhibited minimal retrograde \ufb02ow of actin in relation to the substrate . Second , temporal \ufb02uctua - tions of actin density arise at the leading edge and propagate rearward . We next quanti\ufb01ed protrusion speed by optical \ufb02ow analysis at each pixel along the cell front . Protrusion speed was negatively correlated with both projected cell area and lifeact : GFP intensity with a negative cross - correlation peak between # 0 . 3 and # 0 . 4 at time lag zero ( Figures 1B \u2013 1E ) . Lifeact : GFP intensities were equally co - \ufb02uctuating with area changes when corrected for the \ufb02uctuations in protrusion speed ( Figure S1A ) . Taken together , our quantitative analysis demonstrates that keratocytes migrate slower and produce denser actin networks during intervals when their projected area is larger . Experimental Manipulations of Membrane Tension Reveal a Lamellipodial Response to Altered Load Among several interpretations of these correlative data we considered that a \ufb02uctuation in projected cell area might corre - spond to changes in membrane tension ( Raucher and Sheetz , 2000 ) . To dissect the causal relationship between actin network density and membrane tension , we experimentally increased membrane tension by aspirating the trailing edge of migrating keratocytes with a micropipette ( Houk et al . , 2012 ) ( Figure 2A ; Movie S2 ) . As membrane tension equilibrates almost instanta - neously over the whole cell , this manipulation allowed us to tune lateral tension at the lamellipodial tip ( Diz - Mun\u02dcoz et al . , 2013 ) . Experiments were performed by transiently applying four different vacuum levels ranging from # 10 to # 40 mbar . In all cases , we found that aspiration caused an increase in life - act : GFP signal concomitant with a moderate decrease in protru - sion speed ( Figures 2A \u2013 2C ) . Actin density and protrusion speed were dependent on the applied vacuum ( Figures 2D and 2E ) . Following transient aspiration , the band of denser actin network traveled backward ( in the cell frame of reference ) with the actin \ufb02ow ( Figures 2A and 2C ) . Under the given parameters the cells kept protruding during aspiration , demonstrating that the in - crease in tension was below the stall force of the lamellipodium . These results suggested that lamellipodial actin responds to an increase in membrane tension by increasing network density . To experimentally decrease membrane tension we took advantage of the fact that on adhesive substrates cells occa - sionally form tethered trailing edges , which leads to a stretched morphology ( Figure 2F ) . When these tethers spontaneously detach or when we cut them with a pulsed laser , tension is released and the tether snaps forward . Here , the projected cell area shrunk within a few seconds when the cell went from a stretched to a more compact con\ufb01guration . Such high recoil ve - locities indicate the rapid drop in tension , which had previously built up in the stretched cell . To directly test if shrinkage is accompanied by changes in membrane tension , membrane 2 Cell 171 , 1 \u2013 13 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 tethers were pulled from the plasma membrane of migrating ker - atocytes using an optical trap ( Lieber et al . , 2013 ) ( Figure S3A ) . While in steady - state migrating cells there was no consistent relation between projected area and tether force , abrupt retrac - tion events were accompanied by area shrinkage concurrent with a drop in tether force and thus membrane tension ( Fig - ure S3B ) . During abrupt retraction , the previously observed cor - relation between projected cell area and actin intensity at the leading edge manifested in a sudden drop in lamellipodial life - act : GFP intensity at the moment of area shrinkage ( Figures 2F and 2G ) . The resultant abrupt change in actin density was main - tained while it steadily traveled backward ( in the cell frame of reference ) with the actin \ufb02ow , further substantiating that altered polymerization originated at the leading edge of the cell ( Figures 2F and 2H ; Movie S2 ) . Cell shrinkage was also accompanied by a rapid but very transient increase in protrusion speed ( Figure 2G ) . These results suggested that lamellipodial actin responds to a decrease in membrane tension by decreasing network density . Figure 1 . Correlative Analysis of Actin , Projected Cell Area , and Protrusions in Migrating Zebra\ufb01sh Keratocytes ( A ) Confocal imaging of lifeact : GFP - expressing keratocyte moving on a glass coverslip . ( B ) Time frame from ( A ) showing the 1 . 09 - m m wide area of lifeact : GFP intensity measurements ( left ) , a binary mask used for quantifying the area ( middle ) , and a pseudo - colored Horn - Schunck optical \ufb02ow analysis image of the leading edge ( right ) . ( C ) Fluorescence intensity and leading edge velocity maps for the time lapse of the cell shown in ( A ) . ( D ) Temporal \ufb02uctuations of cell area , lifeact : GFP intensity , and protrusion speed averaged across the analyzed region shown in red in ( B ) . ( E ) Average of temporal cross - correlation functions of 21 migrating keratocytes . Mean and SEM are shown . See also Figures S1 and S2 and Movie S1 . Cell 171 , 1 \u2013 13 , September 21 , 2017 3 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 Figure 2 . Cytoskeletal Response to Manipulations of Membrane Tension ( A ) Lifeact : GFP imaging following micropipette aspiration . ( 1 ) Migrating keratocyte before contact with the micromanipulator , ( 2 ) aspiration of membrane , and ( 3 ) release of vacuum . ( B ) Temporal\ufb02uctuationsoflifeact : GFPintensityattheleadingedgede\ufb01nedasinFigure1B , area , andprotrusionspeed . Theareabetweenthedottedlinesshows where the cell was aspirated . ( C ) Kymograph along dashed yellow line in ( A ) . ( D ) Change in lifeact : GFP signal following aspiration with four different vacuum levels . ( E ) Change in cell edge protrusion for the same cells as in ( D ) . Mean and SEM are shown for 28 aspiration events in 13 individual cells in ( D ) and ( E ) . ( legend continued on next page ) 4 Cell 171 , 1 \u2013 13 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 As our mechanical manipulations of membrane tension might have caused additional changes in the cells , we chose an inde - pendent approach to challenge our hypothesis . Migrating cells were exposed to cycles of altered osmolarity ( Diz - Mun\u02dcoz et al . , 2013 ) , by supplementing the medium with sucrose versus pure water . Monitoring lifeact : GFP at the leading edge showed that increased osmolarity ( water ef\ufb02ux \u2013 cell shrinkage ) was accom - panied by a decrease in lifeact : GFP signal and this was reversed when water was added ( water in\ufb02ux \u2013 cell swelling ) ( Figures S3C and S3D ) . Together , our data show that lamellipodial actin responds to changes in membrane tension by altering its density : density in - creases following an increase in tension and decreases following a decrease in tension . Quantitative Analysis of Lamellipodial Ultrastructure during Steady - State Migration To gain quantitative insight into lamellipodial architecture at the single \ufb01lament level we employed three - dimensional ( 3D ) elec - tron microscopy of \ufb01xed and negatively stained keratocytes ( Vinzenz et al . , 2012 ) . This approach allows tracing single \ufb01la - ments in 3D space ( Winkler et al . , 2012 ) . In keratocyte lamellipo - dia , \ufb01laments are consistently oriented with their barbed ends toward the front ( Narita et al . , 2012 ; Sivaramakrishnan and Spu - dich , 2009 ; Svitkina et al . , 1997 ) , which is in line with the polar forward growth of actin at the leading cell front ( Figures S1C \u2013 S1F ) ( Keren , 2011 ; Lai et al . , 2008 ; Theriot and Mitchison , 1991 ) . In the digitalized tomograms we de\ufb01ned a barbed end as the end of a \ufb01lament proximal to the leading edge and a pointed end as the one distal to the leading edge . Any barbed end could either represent an actively growing or a capped \ufb01la - ment . A pointed end represents ( 1 ) a nucleation event generated by Arp2 / 3 ( a branch ) , ( 2 ) a nucleation event generated by an alternative nucleation factor , or ( 3 ) a debranched or severed \ufb01la - ment ( Ydenberg et al . , 2013 , 2011 ) . Throughout the lamellipo - dium of steady - state migrating keratocytes we found that the numbers of barbed and pointed ends were balanced ( Figures 3A \u2013 3D and S4A ) . Only at the leading front , barbed ends occurred in excess , which is expected due to ongoing polymer - ization at this site . Next , we analyzed the angles , which the \ufb01la - ments assumed relative to the leading membrane and found that the distribution of their orientations peaked at \u00b1 35 ! ( with 0 ! de\ufb01ned as perpendicular to the leading membrane ) ( Fig - ure S4B ) . This con\ufb01guration predominated in keratocytes as shown by a global order parameter de\ufb01ned as ( ( Filaments 0 ! \u2013 20 ! ) # ( Filaments 30 ! \u2013 50 ! ) ) / ( ( Filaments 0 ! \u2013 20 ! ) + ( Filaments 30 ! \u2013 50 ! ) ) as a readout for network orientation ( Weichsel and Schwarz , 2010 ) ( Figures 3E and 3F ) . Together , our tomography data provide a quantitative descrip - tion of lamellipodial actin as derived from \ufb01lament con\ufb01guration . We conclude that in a steady - state migrating keratocyte , nucle - ation , capping and elongation create an expanding network with a canonical branch geometry prescribed by the 70 ! branch structure of the Arp2 / 3 complex ( Svitkina et al . , 1997 ) . Network Con\ufb01guration following Load Increase We next used trailing edge aspiration to interrogate the ultra - structural changes in lamellipodial networks polymerizing against increased load . To this end we employed correlated \ufb02uorescence live - cell imaging and electron tomography on lifeact : GFP - expressing keratocytes migrating on electron micro - scopy grids . After 4 s of # 30mbar aspiration , cells were released , \ufb01xed and prepared for electron microscopy ( Figure 4A ; Movie S3 ) . Under the given conditions where density \ufb02uctuations arise from the leading edge , the temporal growth - history of lamellipo - dial actin is spatially encoded in the network : the pre - aspiration zone ( proximal to the nucleus ) represents steady - state growth , the aspiration zone the increase in load , and the post - aspiration zone at the leading edge the phase at which the load drops again due to the release of the vacuum . In the low - magni\ufb01cation electron micrograph , the transient in - crease in density during aspiration was clearly visible as a darker zone running parallel to the leading edge ( Figure 4A ) . This was consistent with the lifeact : GFP live cell imaging ( Figures 2A \u2013 2C ) . Electron tomography with automated tracking of actin \ufb01la - ments allowed us to trace the temporal evolution of the load response at the single \ufb01lament level . The pre - aspiration zone resembled a steady - state lamellipodium with a canonical 70 ! branched network . Within an 800nm broad aspiration zone ( cor - responding to a 4 - s aspiration time ) \ufb01lament density was mark - edly increased . At the beginning of the aspiration zone ( distal from the leading front , hence earlier in time ) a transient drop in the number of barbed ends and a gradual increase in pointed ends created a mismatch between \ufb01lament birth and death , along with the increase in \ufb01lament density ( Figures 4C and 4D ) . This increase in \ufb01lament density was also accompanied by an in - crease in the 0 ! \u2013 20 ! and 50 ! \u2013 70 ! fraction of \ufb01lament angles , whereas \ufb01laments at intermediate angles of 20 ! \u2013 50 ! increased only moderately ( Figures 4E and S4C ) . At the leading front , \ufb01la - ment density decreased to reach values comparable to levels before aspiration ( Figure 4D ) and \ufb01lament angles reverted to the canonical branch - pattern ( Figures 4E , S4C , and S4D ) . These data show that an increase in membrane tension causes an in - crease in network density and a change in geometry , with \ufb01la - ments growing at steeper angles toward the plasma membrane at higher membrane tensions . Network Con\ufb01guration following Load Decrease We followed the same correlative visualization strategy to analyze the network following a decrease in load . Similar to the laser cutting used in Figures 2F \u2013 2H rapid cell shrinkage could also be triggered by mechanically detaching one side of a cell with a micropipette ( Figure 5A ; Movie S4 ) . This maneuver al - lowed suf\ufb01cient experimental control to \ufb01x the cell seconds after ( F ) Lifeact : GFP signal following fast cell shrinkage . ( 1 ) Keratocyte exhibiting stretched morphology , while its trailing edge is tethered to the substrate . ( 2 ) The trailing edge is cut with a pulsed laser ( yellow arrowhead ) . ( G ) Temporal \ufb02uctuations of leading edge lifeact : GFP intensity , area , and protrusion speed . The rapid retraction is marked with a dotted line . ( H ) Kymograph along dashed yellow line in ( F ) . See also Figure S3 and Movie S2 . Cell 171 , 1 \u2013 13 , September 21 , 2017 5 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 ( legend on next page ) 6 Cell 171 , 1 \u2013 13 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 shrinkage . Analogous to the increase in actin density following aspiration , the decrease in actin density in the time window of rapid shrinkage was clearly visible as a transition zone in life - act : GFP signal as well as phalloidin staining ( Figure 5A ) . Both sig - nals showed a sharp decrease indicating the drop in \ufb01lament density . This was substantiated by low - magni\ufb01cation views of the same cell after extraction and negative stain , which showed a sharp decrease in electron density , overlapping with the \ufb02uo - rescent imaging ( Figures 5A and S6C ) . Quanti\ufb01cation of manu - ally as well as automatically traced \ufb01laments together with auto - mated image segmentation showed a drop in \ufb01lament numbers in the transition zone ( Figures 5C , 5D , S5 , and S6 ) . As expected , Figure 3 . Electron Tomography of Migrating Wild - Type Keratocytes ( A ) Overview electron micrograph of migrating keratocyte with acquired tomogram montage marked in red . ( B ) 5 . 5 - nm slice of a negatively stained tomogram of the actin network behind the leading edge . ( C ) Automated tracking results of the same region with \ufb01laments shown in green , barbed ends in red , and pointed ends in blue . ( D ) Normalized densities of \ufb01laments , barbed ends , and pointed ends in106 - nm - wide bins of four averaged tomogram montages . Graph shows mean and SEM . ( E ) Scheme showing the \ufb01lament angle bins used for calculating the global order parameter . ( F ) Histogram of combined \ufb01lament length growing at indicated angle toward the cell membrane ( black ) is shown together with a global order parameter ( blue ) in 212 - nm distance bins de\ufb01ned as ( ( Filaments 0 ! \u2013 20 ! ) # ( Filaments 30 ! \u2013 50 ! ) ) / ( ( Filaments 0 ! \u2013 20 ! ) + ( Filaments 30 ! \u2013 50 ! ) ) . See also Figures S4A and S4B . Figure 4 . Correlated Live Microscopy - Electron Tomography of Ultrastructural Changes in Networks with Increasing Filament Density ( A ) Amigrating keratocyteexpressinglifeact : GFPwas aspirated attherearwithamicropipette , \ufb01xed within z 3s , and prepared forelectron microscopy . Thecell shifted out of focus because of the manipulating procedure . A transient increase in actin density can be seen in the low - magni\ufb01cation electron micrograph on the right . ( B ) 5 . 5 - nm tomogram slice of the region marked by a red box in ( A ) . The rear of the cell is toward the left side of the picture , and the cell front is seen on the right . Regions of steady - state density , increased density , and decreased density are marked with black , blue , and red throughout the whole \ufb01gure . ( C ) Filament tracks of the lamellipodium shown in ( B ) , with actin \ufb01laments shown in green , barbed ends in red , and pointed ends in blue . ( D ) Filament numbers and densities of barbed and pointed ends in 106 - nm - wide spatial bins throughout the lamellipodium shown in ( B ) and ( C ) . ( E ) Histogram showing \ufb01lament angles to the cell edge in 212 - nm distance bins . See also Figures S4C and S4D and Movie S3 . Cell 171 , 1 \u2013 13 , September 21 , 2017 7 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 the pre - transition zone ( prior to mechanical manipulation ) showed the quantitative signature of the steady - state lamellipo - dium . The onset of the transition zone was marked by a transient increase of barbed ends , which preceded a drop in pointed ends ( Figure 5D ) . Here , the resultant gap between putative capping and nucleation events explained the concomitant drop in \ufb01la - ment density . The transition zone was followed by a leveling of barbed and pointed ends and a continuous recovery of the network toward the cell front . Notably , the transition zone was characterized by radically changed network geometry . When we quanti\ufb01ed and parame - terized \ufb01lament angles depending on their position in the network we found that at the onset of cell shrinkage the canon - ical \u00b1 35 ! dominated network abruptly changed into a con\ufb01gu - ration dominated by \ufb01laments growing perpendicularly ( 0 ! ) to the membrane . Toward the cell front the network again recov - ered gradually to canonical ( Figures 5E and S6D ) . The change in network geometry was due to selective elimination of \ufb01la - ments : in the transition zone the rate of \ufb01lament survival strictly depended on \ufb01lament orientation : \ufb01laments at a higher angle were preferentially eliminated , whereas low angle \ufb01laments had a higher rate of survival ( Figures S5E and S6D ) . These data show that a decrease in membrane tension causes a decrease in network density and a change in geometry , with more \ufb01laments growing perpendicularly to the plasma membrane . Figure 5 . Correlated Live Microscopy - Electron Tomography of Ultrastructural Changes in Networks with Decreasing Filament Density ( A ) Migrating keratocyte manipulated with a microneedle to induce a rapid decrease in projected cell area with an accompanying decrease in lifeact : GFP signal , \ufb01xed within z 3 s , and prepared for electron microscopy . A rapid decrease in the lifeact : GFP signal is preserved in the \ufb01xed lifeact : GFP sample and the low - magni\ufb01cation electron micrograph . ( B ) 5 . 5 - nm tomogram slice showing the region marked with a red box in ( A ) . The cell edge is seen on the right side , and the region of lower density is distin - guishable toward the middle of the micrograph . Region of steady - state network density and decreased density are marked in black and red throughout the whole \ufb01gure . ( C ) Filament tracks of the lamellipodium shown in ( B ) , with actin \ufb01laments shown in green , barbed ends in red , and pointed ends in blue . ( D ) Filament numbers and densities of barbed and pointed ends in 106 - nm - wide spatial bins throughout the lamellipodium shown in ( B ) and ( C ) . ( E ) Histogram showing \ufb01lament densities growing at the indicated angle from the membrane in 212 - nm distance bins . An order parameter ( blue ) in 212 nm is de\ufb01ned like in Figure 3 . See also Figures S5 and S6 and Movie S4 . 8 Cell 171 , 1 \u2013 13 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 A Stochastic 2D Model of Actin - Based Protrusion Recapitulates Light and Electron Microscopy Data To gain a quantitative understanding of the experimental data we built on earlier work by Maly and Borisy ( 2001 ) and Weichsel and Schwarz ( 2010 ) and developed a stochastic model of lamellipo - dial network growth . We formulated a two - dimensional ( 2D ) model taking into account \ufb01lament elongation and Arp2 / 3 - medi - ated ( 70 ! ) nucleation at the leading front . Elongation is termi - nated by capping whenever \ufb01laments detach from the mem - brane and thereby leave the zone in which they are protected from capping ( by elongation factors like VASP or formins ) . Fila - ments push the membrane forward according to a force - velocity relation based on thermal \ufb02uctuations as described in Dickinson ( 2009 ) and Mogilner and Oster ( 1996 , 2003 ) ( for details see STAR Methods and Tables S1 and S2 ) . Here , network geometry dic - tates that increased protrusion velocity ( as caused by decreased load ) rapidly decreases \ufb01lament density in an angle - dependent manner : A \ufb01lament growing at an angle 4 has to polymerize with \u00f0 1 = cos 4 \u00de the speed of the advancing membrane in order to keep contact with the membrane . Hence , with increasing pro - trusion speed \ufb01laments growing at higher angles will lag behind the leading edge and therefore detach from the membrane where they are protected from capping ( Figures 6A , 6B , S7D , and S7E ) . Consequently , higher angle \ufb01laments will be capped at higher rates and the family of low angle \ufb01laments ( and their accompanying \u00b1 70 ! side branches ) will outcompete the \u00b1 35 ! network . Slowing down the membrane has the opposite effect : More \ufb01laments at higher angles are able to catch up with the membrane , leading to a denser network consisting of \ufb01laments growing at a broad range of angles . The stochastic simulation is able to reproduce the correlative electron tomography \ufb01ndings of \ufb01lament orientation and density by using a sharp decrease from 300 pN as force input . 300 pN were taken as a realistic force reported for steady - state migrating \ufb01sh keratocytes ( Lieber et al . , 2013 ) . Together , our stochastic simulation faithfully repro - duced both kinetics and geometry of network parameters upon externally induced changes in load ( Figures 6C , 6D , S7A , and S7B ; Tables S1 and S2 ; Movie S5 ) . Next we wanted to test if the stochastic simulation can pro - duce the co - \ufb02uctuations of protrusion speed , actin density and projected area as we observed them in our light microscopy ex - periments in unperturbed migrating keratocytes ( Figure 1 ) . The amount of decrease in tension necessary to obtain the correct response at the \ufb01lament level in the correlated experiments was used as a scaling factor to relate membrane tension to pro - jected area . The experimental decrease and increase of mem - brane tension ( Figure 2 ) could be recapitulated by simulating representative protrusion events of 10 s with increasing or decreasing tension ( Figure S7C ) . By using the measured tempo - ral \ufb02uctuations in projected cell area ( Figure 1D ) with the ob - tained scaling factor as a proxy for tension we could recapitulate the positive correlation of membrane tension and actin density as well as the negative correlations of membrane tension , actin density , and protrusion speed ( Figures 7A and 7B ) . This showed that our \ufb01lament - level model is able to recapitu - late lamellipodial dynamics as we observed them at the cellular scale and using \ufb02uorescent markers . Our model predicts that keratocytes migrating on an adhesive substrate in 2D exhibit a canonically 70 ! branched network in their lamellipodia the vast majority of the time . This is consistent with the measurements on steady state migrating keratocytes . However , with a low fre - quency , periods of perpendicular dominated networks ( with an order parameter above 0 ) during phases of high protrusion speed are predicted ( Figure 7C ) . DISCUSSION We demonstrate that the lamellipodial actin network undergoes profound structural changes when \ufb01laments pushing against the membrane experience varying load . These changes happen at short time - scales and can be a sheer consequence of the geometricalandkineticpropertiesofArp2 / 3 - nucleatedbranched networks . The key - ingredients of the mechanism we propose are the force - velocity relation determining \ufb01lament elongation rates , the geometry of protruding networks , and the protection from capping near the membrane . Filaments polymerize faster when growing against decreased loads ( here , the load is due to mem - brane tension ) . With decreasing load the expanding network reaches a speed , where \ufb01laments growing perpendicularly to the membrane gradually \u2018\u2018outrun\u2019\u2019 \ufb01laments pushing at steeper angles because these have to travel a longer distance . Hence , these steeper - angle \ufb01laments are eliminated , because they lose contact with the membrane , where the polymerizing factors ( VASP and formins ) protect them from capping proteins . If the perpendicular \ufb01laments originate from \u2018\u2018noise\u2019\u2019 in the angular dis - tributions of a purely Arp2 / 3 - nucleated network or if they repre - sent a family nucleated by other factors like formins remains open , as \u2018\u2018non - branch\u2019\u2019 pointed ends in the tomograms might not only represent Arp2 / 3 - independent \ufb01laments but could also be Arp2 / 3 nucleated , but later severed or unbranched . Whereas this proposed mechanism will operate in any Arp2 / 3 - nucleated process like endocytosis , phagocytosis , vesicle traf\ufb01cking , intracellular pathogen transport , dendritic spine for - mation , etc . , its role in cell motility is most intuitive . Unlike in the idealized keratocyte system , cells migrating in physiological environments will almost never experience mechanically isotropic environments . First , a cell usually pushes against an in - terstitium with inhomogeneous viscoelastic features , and here it has been shown that lamellipodia can increase their force when counter - resistance increases locally ( Heinemann et al . , 2011 ; Prass et al . , 2006 ) . Second , the adhesiveness of the substrate is often variable ( e . g . , in any 3D \ufb01brillar environment ) . As the actin network slides back in areas of the cell , which are less coupled to the substrate , the load experienced by \ufb01laments polymerizing against the leading membrane will be reduced . It has been shown that such local inhomogeneities can be compensated by adaptations in polymerization speed , which \u2018\u2018\ufb01ll up the space\u2019\u2019 resulting from retrograde slippage of the network ( Barnhart et al . , 2011 ; Graziano and Weiner , 2014 ; Renkawitz et al . , 2009 ) . Thus , for a cell migrating through a complex 3D environment , the local adaptations we describe serve to keep the cell edge and its actin network coherent . This geometrical adaptation might provide another remarkable feature : wherever two adjacent regions of the network show differential \ufb02ow speed , the faster network will be less crosslinked to the neighboring , slower , regions , because it is less interconnected by branches . This might Cell 171 , 1 \u2013 13 , September 21 , 2017 9 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 Figure 6 . Stochastic Model Recapitulating Light and Electron Microscopy Results ( A ) The critical angle at which \ufb01laments are not able to keep up with the advancing membrane , 4 c , depends on the maximal polymerization rate , set here as 450nm / s , and theangleof the\ufb01lament towardthemembrane , 4 ( black line ) . A drop inexternal force leads toatransient increase inprotrusion speed leading toa decreasein 4 c andasubsequent , angle - dependentdecreasein\ufb01laments ( redline ) . Forceincreasecausesadecreaseinprotrusionspeed , leadingtoanincrease in 4 c and \ufb01lament density ( blue line ) . ( B ) Scheme of \ufb01laments growing at steady state at intermediate load ( middle , gray arrow ) . Following an increase in load ( top , blue arrow ) \ufb01lament density in - creasesandtheangledistributionbroadens , whereasatadecreaseinload ( bottom , redarrow ) \ufb01lamentsatahigherangle 4 i arepreferentiallycappedanddensity decreases . ( C ) Visualization ofmodelresultswith\ufb01laments showningreen , barbed endsinred , and pointedends inblue . Visualizationof modelresultsinaregime withforce increase ( top ) and decrease ( bottom ) , D F , at around 900 nm . Subsequent panels are aligned next to the respective force regimes . ( D ) Filamentnumbers , barbed , andpointedenddensityquanti\ufb01edforthethreeconditionslikeinFigures3 , 4 , and5 . Intheunperturbedsituationwith\ufb01xedexternal force , F , the network assumes a steady state with balanced \ufb01lament density , branching , and capping . After a decrease in F , capping increases , nucleation decreases , and the network is thinned out , whereas force increase causes a nucleation peak and an increase in \ufb01lament density . For all graphs , mean and SD for an average of 20 runs are shown . See also Figure S7 , Tables S1 and S2 , Movie S5 , and the STAR Methods . 10 Cell 171 , 1 \u2013 13 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 potentially allow the two networks to slide relative to each other and might explain how lamellipodia or actin cortices can harbor differentially \ufb02owing populations of actin as shown previously ( Ponti et al . , 2004 ; Vitriol et al . , 2015 ) . In our work we used manipulations of membrane tension as a proxy for altered load . For the protruding lamellipodium mem - brane tension likely plays the same mechanical role as an external load . However , lateral membrane tension cannot act locally as it equilibrates instantaneously across the cell surface ( Diz - Mun\u02dcoz et al . , 2013 ; Sens and Plastino , 2015 ) . Based on this potential signaling function it has been proposed that membrane tension acts as a global long - range regulator of cell polarity . Whenever lamellipodial actin stretches the leading membrane it suppresses polymerization at the sides and the trailing edge . Here , polymerization will stall under high mem - brane tension as it is not boosted by the biochemical enhancers driving the lamellipodium ( Houk et al . , 2012 ) . This mechanism to suppress competing protrusions has a pure mechanical compo - nent ( \ufb01laments stall under high load ) but is also complemented by a signaling mechanism , where membrane tension triggers mTORC2 signaling , which in turn dampens polymerization ( Diz - Mun\u02dcoz et al . , 2010 ) . Although this mechanism seems to coun - teract the geometrical adaptations we describe , it likely acts at much higher force regimes , where \ufb01laments ultimately fail to protrude the membrane . We think that the geometrical rules we describe here represent the most basic regulatory framework of Arp2 / 3 - mediated actin polymerization . They are a fundamental feature of the network structure , which demonstrates that mechanosensitivity is inherently built into the architecture of branched networks . Additional layers of regulation by chemoattractant - sensing and mechanosensitive signaling pathways will act on top of this basic Figure 7 . The Stochastic Model Recapitulates Cell Migration Parameters of Fish Keratocytes ( A ) Example of temporal \ufb02uctuations in lifeact : GFP intensity , area , and protrusion speed for the cell shown in Figure 1 compared to the migration parameters produced by a simulation run with the projected cell area as scaled tension input . ( B ) Average of temporal cross - correlation functions of the simulations obtained with the projected cell area of the 21 migrating keratocytes shown in Figure 1 . Temporal correlation peaks at time lag zero between lifeact : GFP intensity , projected cell area , and protrusion match the ones observed in vivo . Mean and SEM are shown . ( C ) Histogramoftheorderparametersobtainedforthe21steady - statemigratingkeratocytes . Thestochasticmodelpredictscellsmigratingwithapredominantly negative order parameter , with only occasional switches to positive order parameter values as cells undergo rapid shrinkage and a transient burst in protru - sion speed . See also Figure S7 . Cell 171 , 1 \u2013 13 , September 21 , 2017 11 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 mechanism and might add modulations e . g . , by altering the \ufb01la - ment distribution via different nucleators and elongators . STAR + METHODS Detailed methods are provided in the online version of this paper and include the following : d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Zebra\ufb01sh d METHOD DETAILS B Keratocyte preparation B mRNA injection B Transfection B Membrane dye B Live cell imaging B Micromanipulators B Osmotic treatment B Tether pulling experiments and force measurements B Image analysis B Electron tomography B Actin \ufb01lament tracking and analysis B Correlated light and electron microscopy B Stochastic Simulation d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental Information includes seven \ufb01gures , two tables , and \ufb01ve movies and can be found with this article online at http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 . AUTHOR CONTRIBUTIONS Conceptualization : J . M . and M . S . ; Methodology : J . M . , G . S . , A . D . L . , K . Keren , and R . H . ; Software : C . W . , K . Kruse , and C . S . ; Formal Analysis : J . M . , G . S . , and R . H . ; Investigation : J . M . , M . N . , I . d . V . , and A . D . L . ; Writing \u2013 Original Draft : J . M . and M . S . ; Writing \u2013 Review & Editing : all authors ; Visualization : J . M . and G . S . ; Project Administration : M . S . ; Funding Acquisition : M . S . ACKNOWLEDGMENTS Wethankthescienti\ufb01csupportfacilitiesofISTAustriaandBiocenterViennafor technical support and CP Heisenberg for \ufb01sh lines . This work was supported by the European Research Council ( ERC StG 281556 ) , a grant from the Aus - trian Science Foundation ( FWF ) ( to M . S . ) , and a grant from Vienna Science and Technology Fund ( WWTF ) ( No . LS13 - 029 to C . S . and M . S . ) . Research in the lab of K . Keren was supported by a grant from the United States - Israel Binational Science Foundation ( No . 2013275 with Alex Mogilner ) . 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Ydenberg , C . A . , Padrick , S . B . , Sweeney , M . O . , Gandhi , M . , Sokolova , O . , and Goode , B . L . ( 2013 ) . GMF severs actin - Arp2 / 3 complex branch junctions by a co\ufb01lin - like mechanism . Curr . Biol . 23 , 1037 \u2013 1045 . Cell 171 , 1 \u2013 13 , September 21 , 2017 13 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 STAR + METHODS KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be ful\ufb01lled by the Lead Contact , Michael Sixt ( sixt @ ist . ac . at ) . EXPERIMENTAL MODEL AND SUBJECT DETAILS Zebra\ufb01sh Wild - type zebra\ufb01sh lines AB and TL and the transgenic line Tg ( actb1 : lifeact - GFP ) ( Behrndt et al . , 2012 ) were housed and bred in the IST Austria \ufb01sh facility . Zebra\ufb01sh were maintained at 28 ! C and embryos collected according to standard \ufb01sh laboratory protocol . Animals were sacri\ufb01ced for experiments at 6 months to about 1 . 5 years of age regardless of sex . REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals , Peptides , and Recombinant Proteins Dulbecco\u2019s Modi\ufb01ed Eagle\u2019s Medium GIBCO Cat # 10566 - 016 L - 15 Leibovitz medium Sigma Cat # L1518 PCR puri\ufb01cation kit Qiagen Cat # 28106 SP6 mMessage mMachine kit Invitrogen Cat # AM1340 Fugene 6 Roche Cat # 11814443001 Cell Mask Orange Molecular Probes Cat # C10045 PLL ( 20 ) - g [ 3 . 5 ] - PEG ( 2 ) / PEG ( 3 . 4 ) - RGD Surface Solutions N / A PLL - PEG - RGD Surface Solutions N / A Experimental Models : Organisms / Strains Zebra\ufb01sh line : Tg ( actb1 : lifeact - GFP ) Gift from Heisenberg Laboratory ( IST Austria ) Behrndt et al . , 2012 N / A Zebra\ufb01sh line : wt AB Gift from Heisenberg Laboratory ( IST Austria ) N / A Zebra\ufb01sh line : wt TL Gift from Heisenberg Laboratory ( IST Austria ) N / A Recombinant DNA pEGFP - actin Clontech Cat # 6084 - 1 zf actin : GFP This paper N / A Software and Algorithms MATLAB 2015b The MathWorks https : / / ch . mathworks . com / products / matlab Fiji 1 . 50d Schindelin et al . , 2012 https : / / \ufb01ji . sc / Ilastik 1 . 1 . 5 The ilastik Team http : / / ilastik . org / index . html SerialEM 3 . x Mastronarde , 2005 http : / / bio3d . colorado . edu / ftp / SerialEM / IMOD 4 . 7 Kremer et al . , 1996 http : / / bio3d . colorado . edu / imod / Python 2 . 7 Python Software Foundation https : / / www . python . org / download / releases / 2 . 7 / Prism 5 . 0b GraphPad https : / / www . graphpad . com / scienti\ufb01c - software / prism / LabView National Institutes http : / / www . ni . com / en - us / shop . html NIS - Elements AR 3 . 2 Nikon Instruments https : / / www . nikoninstruments . com / en _ EU / Products / Software / NIS - Elements - Advanced - Research Stochastical Simulation \u2013 Code repository Authors of this study https : / / github . com / gszep / lamellipodium Other 200mesh HF15 Gold \ufb01nder grids Maxtaform Cat # G245A 200mesh hexagonal gold grids Agar Scienti\ufb01c Cat # AGG2450A e1 Cell 171 , 1 \u2013 13 . e1 \u2013 e6 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 Primary keratocyte cultures for these experiments were prepared from the Central American cichlid Hypsophrys Nicaraguensis as described previously ( Lieber et al . , 2013 ) . METHOD DETAILS Keratocyte preparation Keratocytes were prepared from adult zebra\ufb01sh scales ( plucked from sacri\ufb01ced animals ) and washed three times with Dulbecco\u2019s Modi\ufb01ed Eagle\u2019s Medium ( DMEM ) ( GIBCO ) . Scales were incubated in START medium ( Small et al . , 1995 ) at room temperature for one or two days to allow keratocytes to migrate off in a monolayer . The monolayer was then washed three times in PBS , incubated for 40min with Running Buffer ( Small et al . , 1995 ) with 1mM EGTA and the scales removed . The remaining adherent cells were washed three times with PBS , trypsinized for 2min with 0 . 25 % Trypsin - EDTA ( GIBCO ) at room temperature , resuspended in the same volume of trypsin inhibitor ( Sigma ) and transferred to a coverslip or electron microscopy grid . After allowing the cells to adhere for 40min the medium was changed to START medium , the cells were incubated for another 40min before live cell imaging or \ufb01xation for electron microscopy . mRNA injection To produce synthetic mRNA , zf actin : GFP was linearized by double strand cutting with NotI , puri\ufb01ed with a PCR puri\ufb01cation kit ( Qiagen ) and mRNA was synthesized using the SP6 mMessage mMachine Kit ( Ambion ) . 50pg of mRNA were injected in freshly har - vested embryos . Zebra\ufb01sh embryonic keratocytes were prepared from these embryos 1 day post fertilization as described elsewhere ( Lou et al . , 2015 ) . Transfection The plasmid used for transfection was pEGFP - actin ( Clontech ) . Transfection was performed by mixing 100 m l OPTIMEM , 6 m l Fugene 6 ( Roche ) and 1 m g plasmid DNA for 30min and adding it to the cells in L - 15 Leibovitz medium ( Sigma ) for 5h . Then the medium was changed to START medium again and the transfected cells were re - plated onto coverslips as described above . Membrane dye As a membrane dye for negative controls CellMask Orange plasma membrane stain ( Molecular probes ) was used according to the manufacturer\u2019s protocol for live , adherent cell cultures with the following modi\ufb01cations : Keratocytes seeded on glass coverslips were incubated at room temperature for 15min , washed \ufb01ve times with PBS and imaged immediately in START medium . Live cell imaging All live cell imaging was performed at room temperature in START medium . Confocal microscopy was performed with an inverted microscope ( Zeiss ) , equipped with a Spinning disk system ( Yokogawa X1 , iXon897 , Andor ) , a C - Apochromat 63x / 1 . 2 Water Objective ( Zeiss ) , a motorized stage and 488nm and 561nm lasers . While imaging with this microscope , laser ablations of the trailing edge were performed with a 355nm laser cutter . For wide\ufb01eld imaging an inverted wide\ufb01eld microscope ( Nikon ) , equipped with 60x / 1 . 4 and 100x / 1 . 4 Apochromat Oil Objectives , a motorized stage and a light source with \ufb02exible excitation bands ( Lumencor ) was used . Micro - pipette aspiration experiments were performed on an upright confocal microscope ( SP5 , Leica ) , equipped with HCX 63x / 1 . 4 Apo - chromat Oil Objective ( Leica ) , a motorized stage and a 488nm laser line . The time interval was 0 . 2 s - 1 . 0 s . Micromanipulators Micropipettes were produced from 1 . 0mm diameter glass capillaries as described before ( Ma\u0131\u02c6tre et al . , 2012 ) . For cell manipulations negative pressures from 10mbar to 40mbar were used for aspirating the cell membrane and 0 - 2mbar of positive pressure to release the cell again . For correlated live and electron microscopy cell membrane was aspirated with 30mbar for about 5 s and extracted and \ufb01xed about 3 s after release from the micropipette . The same micromanipulator system was used to induce rapid cell shrinking by carefully detaching part of the cell from the underlying substrate . In this case , the cell was \ufb01xed on the grid immediately afterward and prepared for electron microscopy as described below . Osmotic treatment For experiments with changing osmotic pressure , lifeact : GFP expressing keratocytes were subjected to repeated additions of 50 m l 0 . 2M sucrose in START medium and 50 m l double distilled water to a starting volume of 300 m l START medium . Tether pulling experiments and force measurements Primary keratocyte cultures for these experiments were prepared from the Central American cichlid Hypsophrys Nicaraguensis as described previously ( Lieber et al . , 2013 ) . One day old cultures were replated and cultured at room temperature in Leibovitz\u2019s L - 15 media ( GIBCO BRL , Grand Island , NY ) , supplemented with 14 . 2 mM HEPES pH 7 . 4 , 10 % Fetal Bovine Serum ( Invitrogen , Grand Island , NY ) , and 1 % antibiotic - antimycotic ( GIBCO BRL ) . Tether force measurements were carried out as in ( Lieber et al . , 2013 ) with a laser tweezers system ( PALM microtweezers , Carl Zeiss MicroImaging GmbH , Jena , Germany ) using a 63 3 1 . 2 NA water immersion Cell 171 , 1 \u2013 13 . e1 \u2013 e6 , September 21 , 2017 e2 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 objective and a motorized stage ( Ludl Electronic Products , Hawthorne , NY ) on an inverted microscope ( Axiovert 200M , Zeiss , Jena , Germany ) . Trapping was done with a 3W 1064nm Nd : YAG laser focused to a diffraction limited spot , and imaging by bright \ufb01eld was done simultaneously . Tether force measurements on cells were done by attaching a Concanavalin A coated bead to the rear part of the cell body and pulling it away by moving the stage . The measured tether force was corrected for the contribution of the dynamic friction due to stage movement , when relevant , as in ( Lieber et al . , 2013 ) . A membrane tether was held continuously during sponta - neous instances of abrupt rear retraction , providing a read out of the changes in the tether force that accompany the large changes in the projected cell area during the retraction event . Membrane tension values T were calculated from the tether force F T using , T = \u00f0 F 2 T = 8 p 2 B \u00de , where B = 0 : 14 pN , m m is the measured bending modulus of the membrane in keratocytes ( Lieber et al . , 2013 ) . Image analysis Time - lapse images of migrating keratocytes were analyzed in Fiji by \ufb01rst de\ufb01ning the projected cell area by applying a Gaussian blur to the raw image stack and then threshold it to obtain a stack of binary images . The same binary image was used to de\ufb01ne a 1 . 09 m m wide region along the leading edge as the lamellipodial region of interest . An optical \ufb02ow analysis based on the Horn - Schunck method was then used to calculate the optical \ufb02ow between the binary mask frames . Both the original image stack and the optical \ufb02ow data were stabilized using a custom - made image - stabilizing MATLAB algorithm . The resulting stabilized stacks were further analyzed by skeletonizing the 1 . 09 m m wide lamellipodium region for each stack slice and plotting both the gray value of the original image and the amount of shift determined by the optical \ufb02ow analysis along the whole length of the leading edge for every time step . A custom - made MATLAB program was used for this step . The resulting 2D heatmaps of lifeact : GFP , actin : GFP or membrane dye intensity and protrusion were averaged along the length of the leading edge and plotted against time and the whole images were correlated to each other and the projected area . For cross correlation analysis the \ufb01rst derivative of the lifeact : GFP , actin : GFP or membrane dye intensity and protrusion heatmaps as well as the projected cell area were taken and the cross covariance function of MATLAB was used normalized so that the auto - covariance at zero lag equaled 1 . For analysis of signal \ufb02ow within the lamellipo - dium 1 . 09 m m wide bands were eroded from the leading edge in consecutive steps and the resulting bands were used to quantify the lifeact : GFP intensity in adjacent parts further removed from the leading edge . The resulting heatmaps were correlated to each other using the same procedure as described above and the resulting peaks were multiplied by the speed of the respective cell to obtain the distance lag . The calculated distance was plotted against the known distance between the eroded bands . Electron tomography Cells were grown on 200mesh HF15 Gold \ufb01nder grids ( Maxtaform ) and 200mesh hexagonal gold grids ( Agar Scienti\ufb01c ) coated with 2 % Formvar and a 2nm carbon layer and incubated with 0 . 5mg / ml PLL ( 20 ) - g [ 3 . 5 ] - PEG ( 2 ) / PEG ( 3 . 4 ) - RGD ( Surface Solutions ) for 50min . Extraction and \ufb01xation was performed using 0 . 25 % glutaraldehyde and 0 . 5 % Triton X - 100 in Cytoskeleton Buffer ( 10mM MES buffer , 150mM NaCl , 5mM EGTA , 5mM glucose , 5mM MgCl2 , pH 6 . 1 ) for 1min at room temperature . The grids were then trans - ferred to 2 % glutaraldehyde in Cytoskeleton buffer for 10min and kept in another dish with 2 % glutaraldehyde in Cytoskeleton buffer at 4 ! C until staining for electron microscopy . For staining , grids were carefully blotted with \ufb01lter paper from the bottom and imme - diately stained with 70 m l 4 % sodium silicotungstate supplemented with BSA - gold \ufb01ducials from a gold stock as described before ( Vinzenz et al . , 2012 ) . Double axis tilt series with typical tilt angles from # 65 ! to + 65 ! and 1 ! increments following the Saxton scheme were acquired on an FEI Tecnai G20 transmission electron microscope operated at 200kV equipped with a Eagle 4k HS CCD camera ( Gatan ) . The automated acquisition of double axes tilt series was driven by SerialEM 3 . x ( Mastronarde , 2005 ) . The defocus was set to # 5 m m for all acquisitions and the primary on - screen magni\ufb01cation was 25 , 000x . For montage tomograms montages of 2x2 or 3x2 images were acquired for each tilt angle . Back - projection of the tilt series was performed using the IMOD package 4 . 7 with gold particle \ufb01ducials as markers and high - pass \ufb01ltering of the \ufb01nal reconstruction ( Kremer et al . , 1996 ) . A typical tomogram comprised 50 - 70 stacks of 0 . 92nm and an area of 1 . 9 m m x 1 . 9 m m . The montage tomograms had dimensions of 3 . 1 m m x 3 . 1 m m and 4 . 0 m m x 3 . 1 m m , respectively . Actin \ufb01lament tracking and analysis Filaments were quanti\ufb01ed in three different ways : Manual tracking of the region shown in Figure S6B using 3dmod software . Auto - mated tracking using a MATLAB - based tracking algorithm ( Winkler et al . , 2012 ) . Computerized segmentation of actin \ufb01laments by the pixel classi\ufb01cation function of ilastik 1 . 1 was used to quantify actin \ufb01lament density in the correlated tomograms ( Summarized in Fig - ures S6A \u2013 S6C ) ( Kreshuk et al . , 2011 ) . The manually and automatically tracked \ufb01laments were further analyzed in the following way . In the tomogram montages the re - corded \ufb01laments were divided in 106 . 15nm wide distance bins spanning from the leading edge up to 2 . 8 m m toward the back of the lamellipodium ( shown in Figures S4D and S6E ) . For the single tomograms of actin \ufb01lament density steps two regions of 300nm each before and after the step were used . In automated tomograms \ufb01laments shorter than 55nm were excluded from analysis . Filament numbers indicate the number of individual \ufb01laments crossing a plane in the middle of the respective analysis region . Pointed and barbed ends were de\ufb01ned as ends of \ufb01laments furthest and closest from the leading edge , which was clearly visible in every e3 Cell 171 , 1 \u2013 13 . e1 \u2013 e6 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 case , respectively . Filament angles were de\ufb01ned as the angle subtended between a \ufb01lament and the leading edge in the plane of the \ufb02at lamellipodium , with 0 ! meaning a \ufb01lament growing perpendicularly toward the leading edge . Angles were weighted by the total length of the \ufb01laments growing in that direction . The density in the different angle bins was taken as number of \ufb01laments growing at that angle normalized by the size of the angle bin . As a global measure of \ufb01lament architecture an order parameter was de\ufb01ned as : \u00f0 Filaments # 10 ! to + 10 ! \u00de # \u00f0 Filaments # 40 ! to # 20 ! + Filaments + 20 ! to + 40 ! \u00de = 2 \u00f0 Filaments # 10 ! to + 10 ! \u00de + \u00f0 Filaments # 40 ! to # 20 ! + Filaments + 20 ! to + 40 ! \u00de = 2 ( Weichsel and Schwarz , 2010 ) . Correlated light and electron microscopy For correlated light and electron microscopy 200mesh HF15 Gold Finder grids were coated with 0 . 5mg / ml PLL - PEG - RGD ( Surface Solutions ) for 50min . Then keratocytes were allowed to adhere for z 1h , imaged directly on the grid with an inverted confocal spinning disk microscope and either aspirated for about 4 s and then released or partly detached from the grid using micromanipulators as described above . Immediately after release from the micropipette or induction of a rapid shrinking event the cells were \ufb01xed by taking off the medium and adding the extraction and \ufb01xation buffers . To \ufb01nd the correlated cells back , the grids were \ufb01rst completely map - ped by using the navigator map function of SerialEM on an FEI Tecnai G20 transmission electron microscope . Once the relevant grid square was found , high magni\ufb01cation montages and tomograms of the transition zones were acquired . Stochastic Simulation Stochastic 2D models of actin - based lamellipodial protrusion have been proposed in the past to show the self - organization of orien - tation patterns ( Maly and Borisy , 2001 ; Schaus et al . , 2007 ; Weichsel and Schwarz , 2010 ) . These models considered network steady state analysis and simulations with \ufb01xed velocity . We build on the published models to include transient responses of leading edge velocity and network organization due to changing external forces exerted on the \ufb01laments . This simulation was implemented in py - thon 2 . 7 and has been made publicly available at https : / / github . com / gszep / lamellipodium . We formulate a 2D model , where each actin \ufb01lament is rigid and immobile . The \ufb01rst assumption can be justi\ufb01ed by electron tomo - grams , where mostly straight \ufb01laments are observed . Immobility of \ufb01laments has to be understood relative to each other , which still permits a coordinated retrograde \ufb02ow . Thus the state of the network close to the leading edge at time t can be described by the set F \u00f0 t \u00de with N \u00f0 t \u00de number of growing barbed ends , each having 2D position vectors x \u00f0 t \u00de = \u00f0 x \u00f0 t \u00de ; y \u00f0 t \u00de\u00de and constant orientations u . F \u00f0 t \u00de = n \u00f0 x \u00f0 t \u00de ; u \u00de 1 ; . ; \u00f0 x \u00f0 t \u00de ; u \u00de N \u00f0 t \u00de o ( 1 ) u encodes the direction and magnitude j u j = d of elongation with the addition of one actin monomer . The plasma membrane is modeled as a \ufb02at edge parallel to the y - axis at position p \u00f0 t \u00de along the x - axis . With all \ufb01laments only pushing from one side , the resul - tant protrusion velocity v \u00f0 t \u00de is positive in the x - direction . Periodic boundary conditions are imposed in the interval 0 % y \u00f0 t \u00de < L along the y - axis . We shall describe elongation , branching , and capping events at a barbed end at position x \u2013 dropping time variable for conve - nience \u2013 at time t as Poisson processes with rates l \u00f0 x ; t \u00de ; b \u00f0 x ; t \u00de and k \u00f0 x ; t \u00de respectively . This means that in a suf\ufb01ciently small time step from t to t + D t , the \ufb01lament will experience one of these events given by rate u \u00f0 x ; t \u00de with probability u \u00f0 x ; t \u00de D t . We introduce a useful notation n \u00f0 x ; t j u \u00de which is a Boolean variable that tells us whether an event with rate u \u00f0 x ; t \u00de happened or not at a barbed end at position x and time t . Elongation happens in the strip of width w behind the leading edge position p \u00f0 t \u00de with the rate l 0 and zero otherwise . This assump - tion is motivated by the observation that most growth happens at the leading edge . l \u00f0 x ; t \u00de = ! l 0 p \u00f0 t \u00de # w < x \u00f0 t \u00de < p \u00f0 t \u00de 0 elsewhere ( 2 ) An elongation event for a particular barbed end means x \u00f0 t + D t \u00de = x \u00f0 t \u00de + u . There are N \u00f0 t \u00de barbed ends , meaning we have to check events at each one of them . Branching occurs at the leading edge with a rate that depends on the linear density of nucleation factors a \u00f0 t \u00de such as Arp2 / 3 at the leading edge ( Weichsel and Schwarz , 2010 ) . Let a 0 be the rate of activation that leads to branching . b \u00f0 x ; t \u00de = ! a 0 a \u00f0 t \u00de p \u00f0 t \u00de # w < x \u00f0 t \u00de < p \u00f0 t \u00de 0 elsewhere Consider the \ufb01nite availability of nucleation factors and activation being the rate - limiting step in branching new \ufb01laments . Let D \u00f0 t \u00de be the linear density of barbed ends along the leading edge , which increases with branching rate a 0 a \u00f0 t \u00de and decreases with possibly time dependent capping rate k \u00f0 t \u00de : In addition , we know that there is a \ufb01nite positive rate of arrival b 0 of nucleation factors at the lead - ing edge coming from a reservoir . Finally , nucleation factors are depleted when branching occurs , with a rate a 0 D \u00f0 t \u00de depending of barbed end concentration . Cell 171 , 1 \u2013 13 . e1 \u2013 e6 , September 21 , 2017 e4 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 v t D \u00f0 t \u00de = a 0 a \u00f0 t \u00de D \u00f0 t \u00de # k \u00f0 t \u00de D \u00f0 t \u00de v t a \u00f0 t \u00de = b 0 # a 0 a \u00f0 t \u00de D \u00f0 t \u00de Assuming that the arrival of nucleation factors at the leading edge is faster than branching or capping events , we can employ the quasi - steady state assumption ( Manhart et al . , 2015 ) , setting v t a \u00f0 t \u00de = 0 . Rearranging for the concentration a \u00f0 t \u00de ; we can \ufb01nally write : b \u00f0 x ; t \u00de = 8 > < > : b 0 D \u00f0 t \u00de p \u00f0 t \u00de # w < x \u00f0 t \u00de < p \u00f0 t \u00de 0 elsewhere ( 3 ) Branching add a new \ufb01lament \u00f0 x 0 \u00f0 t + D t \u00de ; u 0 \u00de to the frontier at the position of the barbed end x 0 \u00f0 t + D t \u00de = x \u00f0 t \u00de of the mother \ufb01lament . The orientation is rotated by a random branching angle q sampled from a distribution . This can be written as a rotation matrix R \u00f0 q \u00de acting on the mother \ufb01lament orientation u 0 = R \u00f0 q \u00de u . The branching angle distribution B \u00f0 q j m ; s \u00de is the sum of two Gaussians with respective means at \u00b1 m and standard deviations s taken from electron microscopy data . The daughter \ufb01lament is required to be oriented toward the leading edge , meaning that the random choice might have to be repeated , until this requirement is ful\ufb01lled . This model neglects that there is a preferred distance between branches , due to the helical structure of actin \ufb01laments ( Vinzenz et al . , 2012 ) . Capping irreversibly removes a barbed end from the frontier . We assume there is a low capping rate k 0 within width w behind the leading edge , and a high rate k 1 further behind , leading to \ufb01laments being immediately capped outside this zone . k \u00f0 x ; t \u00de = 8 < : k 0 p \u00f0 t \u00de # w < x \u00f0 t \u00de < p \u00f0 t \u00de k 1 x \u00f0 t \u00de < p \u00f0 t \u00de # w 0 elsewhere ( 4 ) Actin \ufb01laments polymerizing against the plasma membrane exert a force that in turn sets the protrusion velocity . It has to balance the restricting force due to membrane tension . We assume an equal distribution of the total force among the pushing \ufb01laments and the absence of retrograde \ufb02ow as observed in keratocytes migrating on serum - coated glass coverslips . We use a force - velocity relation based on thermal \ufb02uctuations of the membrane and / or the \ufb01laments , as is appropriate for the Brownian ratchet model ( Lee et al . , 2001 ; Mogilner and Oster , 1996 ) of polymerization , but also for other established models like the tethered ratchet or end - tracking motors ( Dickinson , 2009 ) . This leads to the following rule for updating the position of the leading edge from small time steps : p \u00f0 t + D t \u00de = p \u00f0 t \u00de + v \u00f0 t \u00de D t v \u00f0 t \u00de = l 0 d e # d F \u00f0 t \u00de k B T D \u00f0 t \u00de ( 5 ) Here the maximum possible speed is equal to the maximal polymerization speed l 0 d of unobstructed \ufb01laments . By F \u00f0 t \u00de we denote the membrane force per leading edge length , D \u00f0 t \u00de the linear density of barbed ends and k B T is the thermal energy . The experiments with a rapid decrease of membrane tension and subsequent return to a steady protruding state are approximated by a stepwise decrease D F in force density at time t for a duration D t , followed by a stepwise increase back to original force den - sity F 0 . F \u00f0 t \u00de = ! F 0 # D F t < t < t + D t F 0 elsewhere ( 6 ) The initial barbed end positions are sampled from a uniform distribution within the polymerization zone . The orientations are sampled from a uniform distribution along a half circle of radius d . The density D \u00f0 t \u00de is updated before each time step and stored for the sampling of branching events and for updating the velocity of the leading edge . Then we iterate through all binding sites in the frontier , checking \ufb01rst for capping events , then branching , and \ufb01nally elongation . Finally the position of the membrane is updated , along with the elapsed time . The process is repeated until \ufb01nal simulation time T 0 . First the simulation is run for the above parameters except we set D F = 0 . The initialization is uniform . Beyond a time T 0 \" 10s a steady state in both \ufb01lament density and angular distribution with respect to the membrane is reached . Using the \ufb01nal state of a pre - vious run as an initial state for the next run , we simulate 20 runs each of time T 0 = 10 s . By recording all the history of barbed end move - ment during a run , we collect statistics at the end of each run . This way we end up with 20 data points per statistic , of which we return the mean and standard deviation to produce wild - type regime shown in Figures 6B \u2013 6D ( middle ) , Figures S7A \u2013 7C ( middle ) and Movie S5 ( \ufb01rst part ) . e5 Cell 171 , 1 \u2013 13 . e1 \u2013 e6 , September 21 , 2017 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 This unperturbed wild - type steady state with orientation peaks at \u00b1 35 ! is used as an initial condition for every run of the perturbed simulation . Setting D F = \u00b1 270pN m m # 1 and executing another 20 runs , collecting statistics at the end of each run obtains the results shown in Figures 6B \u2013 6D ( top and bottom ) , Figures S7A \u2013 7C ( top and bottom ) and Movie S5 ( second and third part ) . To analyze if our stochastic model would be able to reproduce the correlations shown in Figure 1 , we used the measured changes in projected cell area as input . Using the correlated cells shown in Figures 5 and S3 with known decreases in projected cell area and changes in network parameters , we scaled the changes in projected cell area by normalizing to the maximum value , subtracting a constant value and setting the mean to 300pN . The mean force of the \ufb01rst 2 s was used as constant tension for 5 s to let the simulation reach a steady state , before the force changes scaled from the projected area changes were used as input . The \ufb02uctuations in pro - jected area from the 21 migrating keratocytes used for Figure 1 were then used as force input and the resulting output parameters averaged and cross - correlated to generate Figure 7 . QUANTIFICATION AND STATISTICAL ANALYSIS MATLAB ( MathWorks ) and Prism ( GraphPad ) were used for statistical analysis . For live - cell imaging 21 and 7 individual cells were used for analysis of lifeact : GFP and membrane dye \ufb02uctuations , respectively . For membrane aspiration experiments 28 aspiration events in 13 individual cells were performed . For tomography experiments 4 wild - type tomography montages were used as well as 7 correlated shrinking events in 3 individual cells and one correlated aspiration event . Statistical parameters for individual exper - iments can be found within the \ufb01gure legends . A paired t test was used for changes in median angle at the density step ( Figure S5D ) . Pearson correlation was used for correlation analysis of measured distances from the leading edge to calculated distances ( Fig - ure S1F ) and for angle to density decrease analysis ( Figure S5E ) . A p value of < 0 . 05 was considered statistically signi\ufb01cant . Cell 171 , 1 \u2013 13 . e1 \u2013 e6 , September 21 , 2017 e6 Please cite this article in press as : Mueller et al . , Load Adaptation of Lamellipodial Actin Networks , Cell ( 2017 ) , http : / / dx . doi . org / 10 . 1016 / j . cell . 2017 . 07 . 051 Supplemental Figures Figure S1 . Details of Quanti\ufb01cation of Live Cell Imaging Parameters , Related to Figure 1 ( A ) Temporal \ufb02uctuations of lifeact : GFP signal , projected cell area and protrusion speed of the cell shown in Figure 1 are shown normalized from the maximum value to zero . The lifeact : GFP signal multiplied by the instantaneous speed is shown normalized from the maximum to zero as well . ( legend continued on next page ) ( B ) Temporal crosscorrelation functions of Area \u2013 lifeact : GFP intensity , Area - protrusion speed and lifeact : GFP intensity \u2013 Protrusion speed for 21 individual cells are shown in black . Averaged cross - correlation functions from a spline \ufb01t to the combined single - cell cross - correlation functions are shown in purple ( Area \u2013 lifeact : GFP intensity ) , green ( Area \u2013 Protrusion speed ) and yellow ( lifeact : GFP intensity \u2013 Protrusion speed ) . ( C ) Confocal images of a migrating keratocyte expressing lifeact : GFP were analyzed using consecutive 1 . 09 m m wide regions spanning the lamellipodium . ( D ) Resulting lifeact : GFP intensity maps are plotted against time . ( E ) Temporal cross correlation functions of region \u20181\u2019 at the very front of the cell to consecutive regions are plotted as a function of time lag . ( F ) Time lag between peaks of the curves shown in H multiplied by cell velocity and resulting distances are plotted against distance between analyzed regions . Measured mean and s . d . of twelve cells are shown together with a linear regression ( Pearson constant = 0 . 9997 , p = 0 . 0003 ) . ( legend on next page ) Figure S2 . Live Cell Imaging Controls with a Membrane Marker and actin : GFP , Related to Figure 1 ( A ) Example images of migrating keratocytes incubated with the membrane dye CellMask . ( B and C ) Temporal \ufb02uctuations of the resulting intensity maps are shown normalized as inFigure 1D ( B ) and as in Figure S1A ( C ) . Neither an ampli\ufb01cation of area \ufb02uctuations nor a correlation could be observed . ( D ) Resulting temporal cross correlation functions for eight individual cells are shown in black and averaged cross correlation in dark blue . The averaged cross correlation as mean with s . e . m . is shown in dark blue on the right . ( E ) Zebra\ufb01sh keratocytes transfected with actin : GFP were analyzed in the same way as shown before for lifeact : GFP . ( F ) Temporal cross correlation analysis area \u2013 GFP signal intensity showing peaks at time lag zero for transfected cells expressing actin : GFP and lifeact : GFP . Additionally cells generated from actin : GFP microinjected Zebra\ufb01sh embryos were analyzed and showed apositive cross correlation coef\ufb01cient at time lag zero . Plot shows averaged cross correlation and s . e . m . for seven cells for both actin : GFP transfection and mRNA injection . FigureS3 . MembraneTensionMeasurementbyTetherPullingandMembraneTensionManipulationbyChangingOsmoticPressure , Related to Figure 2 ( A ) Amigratingkeratocytewasimagedwithbright\ufb01eldmicroscopyandsimultaneouslyacoatedbeadcontrolledbyalasertweezerwasusedtopullamembrane tether . ( B ) The measured tether force decrease correlated with rapid decreases in projected cell area . ( C ) A lifeact : GFP expressing keratocyte was imaged by confocal microscopy and subjected to rapid changes in osmotic pressure of the medium by alternating additions of 0 . 2M sucrose and pure water . ( D ) lifeact : GFP signal is negatively correlated with the osmolarity of the medium . When the pressure increases upon addition of 0 . 2M sucrose , lifeact : GFP signal drops , which is reversible upon restoring a lower osmotic pressure by addition of pure water . Figure S4 . Actin Network Parameters for Individual Tomogram Montages of Keratocyte Lamellipodia , Related to Figures 3 and 4 ( A ) Filament density , barbed and pointed ends for individual cells shown in Figure 3 . Data for individual lamellipodia are shown in black together with average . ( B ) Filamentdensitiesgrowingattheindicatedanglefromthecellmembranein212nmdistancebinsofthesteadystatelamellipodiashowninFigure3 . Meanand s . e . m . are shown . ( C ) Filamentdensitiesgrowing attheindicated anglefromthecellmembranein212nmdistancebinsofthecorrelatedtomogramshowninFigure4 . Thedensities are shown as in Figure 4 beginning from toward the rear of the cell on the left until the leading edge on the right . ( D ) 5 . 5nm slice of a negatively stained electron tomogram montage showing the region marked by a red box in Figure 4A ( left ) and corresponding automatically tracked \ufb01laments shown color - coded according to their angle from the leading edge ( right ) . The spatial bins used for quanti\ufb01cation of network parameters in Figures 4 and S4C are also shown on the right . Figure S5 . Changes in Filament Parameters at a Decrease in Filament Density , Related to Figure 5 ( A and B ) Network structure analysis of seven lamellipodia tomograms in three correlated cells revealed consistent features at the point , where the actin density decreased due to the manipulation by the micropipette . Overview electron micrographs and 5 . 5nm tomogram slices are shown for all regions . ( C ) Averages of \ufb01lament density , barbed and pointed ends and median angle from the cell edge showed the consistent changes at the density step . ( D ) Mediananglesofallanalyzedregionsareplottedfor300nmspacebinsbeforeandafterthedecreaseinactin\ufb01lamentdensity ( right ) . ( Pairedttest , p = 0 . 0249 ) . ( E ) Normalized ratio of \ufb01laments after density step to \ufb01laments before density step is plotted against the \ufb01lament angle from the cell edge together with a linear regression curve . ( Pearson constant , r = ! 0 . 936 ) Mean and s . e . m . are shown on all graphs . ( legend on next page ) Figure S6 . Quanti\ufb01cation of Correlated Live Microscopy - Electron Tomography , Related to Figure 5 ( A ) A 5 . 5nm slice of the tomogram montage used for Figure 5 ( left ) is shown together with automated tracking results ( middle and right ) . ( B ) Close - upofregioncontainingthedropin\ufb01lamentdensityandchangeinactinarchitecturewith\ufb01lamentsshowningreen , barbedendsinredandpointedends in blue . Manually tracked \ufb01laments , barbed and pointed ends of a region including the density step are shown . ( C ) Filament density quanti\ufb01ed by manual and automated tracking , ilastik image analysis and the lifeact : GFP signal of the correlated cell shown in Figure 5 . ( D ) Filamentdensitiesin212nmdistancebinsgrowingatdifferentanglestowardthemembrane . ThedensitiesareshownasinFigure5beginningfromtowardthe rear of the cell on the left until the leading edge on the right . ( E ) 5 . 5nmsliceofnegativelystainedelectrontomogrammontageshowingregionmarkedwithredboxinFigure5A ( left ) andcorresponding automaticallytracked \ufb01laments shown color - coded according to their angle from the leading edge ( right ) . Additionally , the spatial bins used for quanti\ufb01cation of network parameters in Figures 5 and S6D are displayed on the left . The cell edge is seen on the right side . Figure S7 . Details of Stochastic Simulation , Related to Figures 6 and 7 The graphs in A \u2013 C are aligned as in Figure 6 . ( A ) Thetemporalchangesin\ufb01lamentsinthreedifferentanglebinsareshown . Thenumberof\ufb01lamentsintheindicatedanglebinsisconstantatgrowthatconstant externalforce . Intheforcedecreasescenario\ufb01lamentsgrowingathigheranglesarecappedpreferentiallyandthenetworkthinsout . Whenforceincreaseisused as an input , \ufb01laments in all angle bins increase . ( B ) Histograms of \ufb01lament density ( black ) and order parameter as de\ufb01ned in Figure 3 ( blue ) . Without perturbation the network architecture is dominated by \u00b1 35 \" peaks and the order parameter is consistently negative . Histograms of \ufb01lament density before ( black ) and after ( red ) decrease in F and order parameter ( blue ) showbiasedelimination of\ufb01laments growing athigher angle . This leads toachange from the \u00b1 35 \" architecture toonedominated by straight , 0 \" , \ufb01laments and a transiently positive order parameter . Filaments of all angles increase upon increase in force , which is also re\ufb02ected in a negative order parameter . ( C ) The migration parameters force ( the input parameter ) , actin density and protrusion speed \ufb02uctuate around steady state values during the unperturbed simulation . The force decrease causes an increase in protrusion speed and a decrease in actin density , with the reciprocal situation observed for the force increase . ( D ) Capping , elongation and branching rates used for the stochastic model . ( E ) Underlying lamellipodial feedback loop : Force , F , is set as an external parameter and decreases velocity , v , which is in turn increased by increased \ufb01lament density , D , as suggested by the elastic ratchet model of actin polymerization . Actin branching is modeled as a zeroth order process and therefore actin density decreases branching rate , b . The parts of the feedback loop linking velocity , v , to the \ufb01lament density , D , shown in this study are marked in red : Velocity , v , increasescappingrate , k , inanangle - dependentmannerandleadstoadecreasein\ufb01lamentdensity , D . Forallgraphsmeanands . d . foranaverageof20runsare shown , for details see STAR Methods .", + "zwettler2020molecular": "ARTICLE Molecular resolution imaging by post - labeling expansion single - molecule localization microscopy ( Ex - SMLM ) Fabian U . Zwettler 1 , Sebastian Reinhard 1 , Davide Gambarotto 2 , Toby D . M . Bell 3 , Virginie Hamel 2 \u2709 , Paul Guichard 2 \u2709 & Markus Sauer 1 \u2709 Expansion microscopy ( ExM ) enables super - resolution \ufb02 uorescence imaging of physically expanded biological samples with conventional microscopes . By combining ExM with single - molecule localization microscopy ( SMLM ) it is potentially possible to approach the resolution of electron microscopy . However , current attempts to combine both methods remained challenging because of protein and \ufb02 uorophore loss during digestion or denaturation , gela - tion , and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers . Here we show that re - embedding of expanded hydrogels enables d STORM imaging of expanded samples and demonstrate that post - labeling ExM resolves the current limitations of super - resolution microscopy . Using microtubules as a reference structure and centrioles , we demonstrate that post - labeling Ex - SMLM preserves ultrastructural details , improves the labeling ef \ufb01 ciency and reduces the positional error arising from linking \ufb02 uorophores into the gel thus paving the way for super - resolution imaging of immunolabeled endogenous proteins with true molecular resolution . https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 OPEN 1 Department of Biotechnology and Biophysics , Biocenter , University of W\u00fcrzburg , Am Hubland , 97074 W\u00fcrzburg , Germany . 2 Department of Cell Biology , Sciences III , University of Geneva , Geneva , Switzerland . 3 School of Chemistry , Monash University , Clayton , VIC 3800 , Australia . \u2709 email : virginie . hamel @ unige . ch ; paul . guichard @ unige . ch ; m . sauer @ uni - wuerzburg . de NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 1 1 2 3 4 5 6 7 89 0 ( ) : , ; B y linking a \ufb02 uorophore or a protein of interest into a dense , cross - linked network of a swellable polyelectrolyte hydrogel , biological specimen can be physically expanded allowing for magni \ufb01 ed imaging with subdiffraction - resolution on conventional microscopes . Since its introduction in 2015 1 , expansion microscopy ( ExM ) has shown impressive results including the magni \ufb01 ed visualization of pre - or postexpansion labeled proteins and RNAs with \ufb02 uorescent proteins ( FPs ) , antibodies , and oligonucleotides , respectively , in isolated organelles , cells , pathogens , tissues , and human clinical specimen 2 \u2013 4 . In addition , various protocols have been developed to anchor proteins or RNA into charged poly - acrylamide hydrogels 5 \u2013 8 . Using 2 . 5 % ( w / w ) acrylamide and 8 . 55 % sodium acrylate with 0 . 15 % ( w / w ) of the cross - linker N - N \u2032 - methylenebisacrylamide accomplishes a ~ 4 . 5x linear expansion of biological specimens . Decreasing the cross - linker concentration usually permits higher gel expansion factors of up to 10x but also increases proportionally the linkage error de \ufb01 ned by the af \ufb01 nity reagent , linker and \ufb02 uorophore and leads to greater gel instability 9 . It is also possible to expand samples in series enabling gel expansion factors of 20x and higher with a demonstration of 53x expansion of microtubules 10 . However , \ufb02 uorescence imaging of such greatly enlarged samples is complicated by the dilution of \ufb02 uorescent labels and dramatic increase in the physical separation between the \ufb02 uorophore and its target due to the linkage error . Nevertheless , ExM with lower expansion factors enables confocal diffraction - limited \ufb02 uorescence imaging with spatial resolutions comparable to that of super - resolution microscopy methods 11 , 12 . To further enhance the resolution , ExM has been combined with structured illumination microscopy ( SIM ) 13 , 14 and stimu - lated emission depletion ( STED ) microscopy 2 , 15 . By careful optimization of the expansion protocol U - ExM demonstrated that even ultrastructural details of multiprotein complexes such as centrioles can be truthfully preserved 2 . Combining ExM with SMLM methods ( Ex - SMLM ) can then potentially further improve the spatial resolution to enable true molecular resolution and bridge the gap to the electron microscopy regime . However , despite these apparent advantages , attempts to combine ExM with SMLM have remained rare and unoptimized due to several challenges 5 , 16 . There are two major determinants that control the resolution of SMLM , the localization precision and the localiza - tion density 11 , 12 . The localization precision remains unaltered by sample expansion and therefore allows achieving an improved resolution depending on the expansion factor . The localization density is arguably the more important determinant for SMLM on expanded samples . According to information theory , the required density of \ufb02 uorescent probes has to be suf \ufb01 ciently high to satisfy the Nyquist \u2013 Shannon sampling theorem 17 . At its most basic level , the theorem states that the mean distance between neighboring localized \ufb02 uorophores ( the sampling interval ) must be at least twice as \ufb01 ne as the desired resolution . In real samples , however , the relationship between localization density and reso - lution is far more complex 18 . Empirically , it seems that for a given resolution the distance between neighboring localizations should be signi \ufb01 cantly less than that indicated by a naive application of the Nyquist limit 19 . These considerations illustrate the challenges Ex - SMLM is confronted with . First , the \ufb02 uorophore density is considerably diluted in expanded samples 9 , 10 , which often results in unclear views of biological structures and complicates SMLM data interpretation . For example , a 4x expansion in three dimensions effectively lowers the labeling density 64 - fold . Second , addition of a thiol - containing phosphate - buffered saline ( PBS ) photoswitch - ing buffer as required for d STORM 20 , 21 to a swellable polyelec - trolyte hydrogel with hydrophilic ionic side groups results in substantial shrinking of the gel in the worst case down to its initial size . Finally , ExM protocols use free - radical polymerization to form polymers . However , free radicals also have the potential to react with the \ufb02 uorophores which can irreversibly destroy them 1 , 3 , 5 . Therefore , the \ufb02 uorophore density will be further diluted in ExM protocols that use pre - expansion labeling and consequently reduce the structural resolution . The extent of irreversible \ufb02 uorophore destruction during gelation varies across \ufb02 uorophores . Unfortunately , the best suited dyes for d STORM , the carbocyanine dyes Cy5 and Alexa Fluor 647 19 , 20 , are almost completely destroyed during gelation 1 , 3 , 5 Here , post - expansion labeling approaches ( post - labeling ExM ) offer acceptable solutions 2 , 7 , 8 , though they require preservation of protein epi - topes during expansion . An additional bene \ufb01 t of post - labeling ExM is improved epitope accessibility for antibodies and a reduction of the linkage error proportional to the expansion factor compared to pre - labeling ExM 2 . For instance , after 4x expansion , the immunolabeling linkage error of 17 . 5 nm de \ufb01 ned by the primary and secondary antibodies 22 , would reduce to 4 . 4 nm , which is the size of a tubulin monomer 23 . Combining SMLM with post - labeling ExM reduces the linkage error by the expansion factor and could thus enable \ufb02 uorescence imaging with molecular resolution . Here , we set out to develop post - labeling Ex - SMLM with organic \ufb02 uor - ophores with minimal linkage error . Results Re - embedding of expanded samples enables Ex - SMLM in photoswitching buffer . A major problem of Ex - SMLM is the shrinking of the expanded hydrogels in photoswitching buffer due to ionic interactions between ions of the buffer and the ionic side groups of the gel . Therefore , we re - embedded expanded charged hydrogels in an uncharged polyacrylamide gel as recently introduced for ExM of RNA 6 . We started using pre - labeling ExM in combination with standard immunostaining using unmodi \ufb01 ed primary and \ufb02 uorophore labeled secondary antibodies to realize Ex - SMLM ( Supplementary Fig . 1 ) . We used microtubules as reference structure to investigate the expansion factor , spatial resolution , structural distortions , and the labeling density . Microtubules are assembled from \u03b1 \u00df tubulin hetero - dimers , which stack head - to - tail into polar proto \ufb01 laments with a periodicity of 8 nm , with ~ 13 proto \ufb01 laments associating laterally in parallel to form a hollow , polar cylinder ( Fig . 1a ) 23 , 24 . As previously measured by transmission electron microscopy ( TEM ) , microtubules are hollow tubes with an outer diameter of 25 nm and 60 nm , respectively , after immunostaining with pri - mary and secondary antibodies 22 . This results in a linkage error de \ufb01 ned by the size of the primary and secondary antibody of 17 . 5 nm ( Fig . 1a ) . We used the proExM protocol , in which proteins are directly anchored to the hydrogel using the succinimidyl ester of 6 - ( ( acryloyl ) amino ) hexanoic acid ( AcX ) 5 . To minimize \ufb02 uorophore loss during gelation in pre - labeling ExM methods , we used the rhodamine derivative Alexa Fluor 532 , which retains ~ 50 % of its pre - gelation brightness after expansion 1 , 3 , 5 . To prevent shrinking of the hydrogel upon addition of photoswitching buffer , expanded hydrogels were re - embedded in acrylamide for serial staining of the expanded specimen 6 . Hydrogels were incubated twice in 10 % AA , 0 . 15 % bis - acrylamide , 0 . 05 % APS , 0 . 05 % TEMED in 5 mM Tris ( pH 8 . 9 ) for 30 min each and subsequently transferred onto coverslips functionalized with acrydite via glass silanization to minimize lateral drift of expanded samples ( Supplementary Fig . 1 ) . After polymerization of the re - embedding gel , hydrogels were immersed in photoswitching buffer containing 100 mM mercaptoethylamine ( MEA ) in PBS . The expansion factor was determined by comparing the post - expansion and post re - embedding \ufb02 uorescence images with ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 2 NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications pre - expansion \ufb02 uorescence images . The results showed a low distortion introduced by the re - embedding process and a reduction in gel size of ~ 20 % from ~ 3 . 9x before to ~ 3 . 1x after re - embedding ( Supplementary Figs . 2 and 3 ) . A caveat of imaging expanded samples is that super - resolution imaging methods , and in particular SMLM , are most effective when used on thin samples located within a few micrometers above the coverslip surface . However , expanded specimen can be easily located several tens of micrometers above the coverslip . In addition , expanded specimens are transparent because they consist largely of water . Hence , the use of oil - immersion objectives and total internal re \ufb02 ection \ufb02 uorescence ( TIRF ) microscopy as used in most SMLM applications to achieve a higher signal - to - background ratio is in this case not the best choice . Therefore , we decided to use a water - immersion objective and epi - illumination in all SMLM experi - ments . The corresponding d STORM images of pre - labeled expanded microtubules showed homogeneously labeled \ufb01 laments with some labeling gaps re \ufb02 ecting \ufb02 uorophore and protein loss during polymerization and enzymatic digestion , respectively ( Fig . 1b , c ) . In addition , we imaged unexpanded microtubules by d STORM under identical experimental conditions ( Fig . 1f , g ) . To examine the achieved spatial resolution , cross - sectional pro \ufb01 les of selected microtubule areas are often consulted 21 . If the two - dimensional ( 2D ) projection of the \ufb02 uorescence intensity distribution measured from microtubule \ufb01 laments show a bimodal distribution , the peak - to - peak distance can then be \ufb01 tted with a sum of two Gaussians and used as an estimate of the spatial resolution . To ensure an objective evaluation and comparison of the spatial resolution achieved , we developed \u201c Line Pro \ufb01 ler \u201d , an automated image processing software . Line Pro \ufb01 ler automatically evaluates potential regions of interest along \ufb01 lamentous structures to generate cross - sectional pro \ufb01 les that can be \ufb01 t by a sum of two Gaussians to determine the peak - to - peak distance between the sidewalls of the \ufb01 lamentous structure ( Supplementary Fig . 4 ) . In order to compare the experimentally measured peak - to - peak distances of different expansion protocols , we simulated trans - verse pro \ufb01 les of unexpanded and expanded microtubules using a cylindrical distribution function to describe the hollow annular structure of microtubules ( Fig . 2a and Supplementary Fig . 5 ) similar to the approach used recently for iterative expansion 10 . The resulting peak - to - peak distances were used to determine the molecular expansion factor of expanded immunolabeled microtubules considering the in \ufb02 uence of the label size on the broadening of the microtubule diameter ( Supplementary Table 1 and Supplementary Note 1 ) . \u03b1 - tubulin \u03b2 - tubulin 8 nm sab sab pab pab a b c proExM AI532 137 . 1 \u00b1 10 . 1 nm pre post 36 . 2 \u00b1 5 . 4 nm Unexpanded AI532 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 1 . 0 0 . 5 I n t en s i t y [ a . u . ] 0 . 0 \u2013 400 \u2013 200 \u2013 200 400 0 Distance [ nm ] 120 130 140 150 160 Peak - to - peak distance [ nm ] 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 1 . 0 0 . 5 I n t en s i t y [ a . u . ] 0 . 0 \u2013 200 200 0 Distance [ nm ] 20 25 30 35 40 45 5055 Peak - to - peak distance [ nm ] g f d e h i Fig . 1 Re - embedding enables Ex - d STORM . a Model of microtubules with an outer diameter of 25 nm stained with conventional primary ( pab ) and \ufb02 uorescently labeled secondary IgG antibodies ( sab ) results in a total diameter of 60 nm with a linkage error ( de \ufb01 ned by the size of the primary and secondary antibody ) of 17 . 5 nm 22 . b d STORM image of pre - labeled proExM expanded and re - embedded Cos - 7 cells stained with primary antibodies against \u03b1 - tubulin and secondary Alexa Fluor 532 conjugated antibodies ( Al532 ) . The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion ( pre - labeled ) . c Zoom in on highlighted region in ( b ) . d Averaged cross - sectional pro \ufb01 le of nine microtubule segments with a total length of 29 . 1 \u00b5 m ( segment lengths range from 2 . 1 - 4 . 5 \u00b5 m ) measured in two cells from 1 expanded sample . e Histogram of peak - to - peak distances with normalized normal distribution curve ( red ) determined by bi - Gaussian \ufb01 tting of the data analyzed in ( c ) with an average distance of 137 . 1 \u00b1 10 . 1 nm ( mean \u00b1 sd ) . The data were obtained from n = 9 microtubule segments in 2 cells from 1 expanded sample . f Unexpanded Cos - 7 cells labeled with an anti \u03b1 - tubulin primary antibody and Alexa Fluor 532 ( Al532 ) conjugated IgG secondary antibodies . The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded . g Zoom in of the white boxed region in ( f ) . h Average intensity pro \ufb01 le of 35 microtubule segments with a length between 1 . 1 and 5 . 8 \u00b5 m ( mean = 2 . 0 \u00b5 m ) and a total length of 69 . 6 \u00b5 m analyzed in 12 d STORM images . i Histogram of peak - to - peak distances with normalized normal distribution curve ( red ) determined by bi - Gaussian \ufb01 tting of cross - sectional pro \ufb01 les of the analyzed microtubule segments in ( h ) with a mean peak - to - peak distance of 36 . 2 \u00b1 5 . 4 nm ( mean \u00b1 sd ) . The data were obtained from n = 35 microtubule segments in 12 cells and 3 independent experiments . Scale bars , 2 \u00b5 m ( b , f ) , 500 nm ( c , g ) . NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 ARTICLE NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 3 Pre - labeling Ex - SMLM . d STORM images of unexpanded and expanded microtubules clearly showed a bimodal signal distribution along the \ufb01 laments , similar to that of previous super - resolution microscopy studies ( Fig . 1c , d and 1g , h ) 21 , 25 . When the cross - sectional pro \ufb01 le of unexpanded microtubules was \ufb01 t with a sum of two Gaussians , the peak - to - peak distance between the sidewalls showed a mean value of 36 . 2 \u00b1 5 . 4 nm ( mean \u00b1 s . d . ) analyzed over several microtubule \ufb01 lament segments ( Fig . 1i ) . This value is expected for the projection of a 25 nm inner diameter cylinder that has been broadened by primary and secondary antibodies on both sides by 17 . 5 nm 22 ( Fig . 1a ) and corresponds well to the simulated peak - to - peak distance of 32 . 0 nm for unexpanded microtubules ( Fig . 2a and Supplementary Note 1 ) . The mean peak - to - peak dis - tance of proExM treated and expanded microtubules was deter - mined to 137 . 1 \u00b1 10 . 1 nm ( mean \u00b1 s . d . ) ( Fig . 1e ) . This value corresponds to an expansion factor of 3 . 1x determined from a b c g pre post pre post Antibodies unexpanded ( 32 . 0 nm ) Antibodies pre - labeling ( proExM / GA ) ( 143 . 0 nm ) DNA Cy5 unexpanded ( 41 . 5 nm ) DNA Cy5 expanded ( 202 . 0 nm ) DNA AI532 expanded ( 226 . 5 nm ) 1 . 0 0 . 5 0 . 0 \u2013 150 \u2013 100 \u2013 50 0 Distance [ nm ] I n t en s i t y [ a . u . ] 50 100 150 f j k 133 . 8 \u00b1 13 . 2 nm 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 1 . 0 0 . 5 I n t en s i t y [ a . u . ] 0 . 0 \u2013 400 \u2013 200 \u2013 200 400 0 Distance [ nm ] 120 110 100 130140150 170 160 Peak - to - peak distance [ nm ] d e 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 1 . 0 0 . 5 I n t en s i t y [ a . u . ] 0 . 0 \u2013 400 \u2013 200 \u2013 200 400 0 Distance [ nm ] 195 180 165 210 225 240 Peak - to - peak distance [ nm ] 133 . 8 \u00b1 13 . 2 nm 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 1 . 0 0 . 5 I n t e n s i t y [ a . u . ] 0 . 0 \u2013 200 200 0 Distance [ nm ] Peak - to - peak distance [ nm ] h i Antibody - AI532 ( GA ) Unexpanded DNA - Cy5 DNA - Cy5 DNA - AI532 30 35 40 45 50 55 o n 201 . 0 \u00b1 12 . 9 nm p q 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 1 . 0 0 . 5 I n t en s i t y [ a . u . ] 0 . 0 \u2013 400 \u2013 200 \u2013 200 400 0 Distance [ nm ] 210 195 180 225 240 255 270 Peak - to - peak distance [ nm ] 226 . 7 \u00b1 15 . 3 nm l m Fig . 2 Pre - labeling Ex - d STORM . a Simulated intensity pro \ufb01 les using a cylindrical distribution function to describe unexpanded or 3 . 2x expanded immunostained microtubules ( labeled with IgG antibodies or DNA modi \ufb01 ed IgG antibodies pre - expansion ) and resulting peak - to - peak distances of the cross - sectional pro \ufb01 les . b d STORM image of expanded and re - embedded \u03b1 - and \u03b2 - tubulin pre - labeled with secondary Alexa Fluor 532 IgG antibodies ( Al532 ) using the MA - NHS / GA method 6 , i . e . antibodies are cross - linked with glutaraldehyde ( GA ) into the hydrogel ( Antibody - Al532 ( GA ) ) . c Zoom in of white boxed region in ( b ) . d Averaged cross - sectional pro \ufb01 le of 8 microtubule segments with a length between 1 . 5 \u2013 6 . 4 \u00b5 m and 28 . 6 \u00b5 m in total measured in 4 expanded cells . e Histogram of peak - to - peak distance distribution with normalized normal curve ( red ) of microtubule segments analyzed in ( d ) at n = 8 microtubule segments in 4 cells from 1 expansion experiment with a mean distance of 133 . 8 \u00b1 13 . 2 nm ( mean \u00b1 sd ) . f Unexpanded d STORM image of ssDNA - Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre - expansion ( DNA - Cy5 protocol ) . g Magni \ufb01 ed view of white boxed region in ( f ) . h Average cross - sectional pro \ufb01 le of 7 microtubule segments with a length between 1 . 0 \u2013 1 . 8 \u00b5 m and 8 . 7 \u00b5 m in total . i Histogram of peak - to - peak distances with normalized normal distribution curve ( red ) of the data analyzed in ( h ) along n = 7 microtubule segments in 2 cells from 1 experiment with a mean distance of 43 . 9 \u00b1 3 . 7 nm ( mean \u00b1 sd ) . j Expanded d STORM image of microtubules labeled with \u03b1 - tubulin and dsDNA ( DNA - Al532 ) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with \ufb02 uorophores pre - expansion into the hydrogel ( original ExM trifunctional label concept ) 1 . k Zoom - in of white boxed region in ( j ) . l Average intensity pro \ufb01 le of 26 microtubule segments with a length of 2 . 4 \u2013 10 . 7 \u00b5 m and 118 . 6 \u00b5 m in total . m Histogram of peak - to - peak distances with normalized normal distribution curve ( red ) determined from n = 26 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 226 . 7\u00b1 15 . 3 nm ( mean \u00b1 sd ) . n d STORM image of \u03b1 - and \u03b2 - tubulin expanded according to the DNA - Cy5 protocol strategy with labels at Cy5 - bearing oligonucleotides introduced post - re - embedding . o Zoom in of white boxed region in ( n ) . p Average intensity pro \ufb01 le of 15 microtubule segments with a length between 1 . 6 \u2013 25 . 1 \u00b5 m and a total length of 126 . 0 \u00b5 m in 1 expanded sample . q Histogram of peak - to - peak distances with normalized normal distribution curve ( red ) determined by \ufb01 tting the cross - sectional pro \ufb01 les analyzed in ( p ) along n = 22 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 201 . 0 \u00b1 12 . 9 nm ( mean \u00b1 sd ) . The small logos in the upper left corner symbolize the labeling method , e . g . pre - and post - immunolabeled with or without DNA - linker , respectively . Scale bars , 2 \u00b5 m ( b , f , j , n ) , 500 nm ( c , g , k , o ) . ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 4 NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications simulation of expanded microtubules pre - labeled with primary and secondary IgG antibodies ( Supplementary Table 1 ) . We next used the post - stain linker - group functionalization method ( MA - NHS / GA method ) 7 as an alternative pre - labeling Ex - SMLM method . Here , the entire sample was functionalized with polymer - linking groups after conventional immunostaining . The resulting d STORM images showed a peak - to - peak distance between the microtubule sidewalls of 133 . 8 \u00b1 13 . 2 nm ( mean \u00b1 s . d . ) ( Fig . 2b \u2013 e ) corresponding to a simulated expansion factor of 3 . 0x ( Supplementary Table 1 ) . The determined peak - to - peak distance is in good agreement with the peak - to - peak distance determined from proExM expanded microtubules ( Fig . 1b \u2013 e ) . Variations in the measured peak - to - peak distances can be well explained by varying initial expansion factors of hydrogels which are typically in the range of ~ 4 . 0 \u2013 4 . 5x for the used ExM gel composition . Considering a ~ 20 % reduction in gel size caused by re - embedding of the hydrogel , an ultimate expansion factor of ~ 3 . 1 \u2013 3 . 6x can be expected which \ufb01 ts well with the determined molecular expansion factors . Next , we tested the original ExM protocol with trifunctional DNA - modi \ufb01 ed secondary antibodies 1 , which can be labeled with dye - functionalized complementary oligonucleotides that contain an acrydite linker modi \ufb01 cation . Alternatively , anti - bodies can be modi \ufb01 ed with a single stranded DNA that is incorporated directly into the hydrogel . Antisense dye - labeled oligonucleotides can then be hybridized after re - embedding of the hydrogel , which enables the use of \ufb02 uorophores that would not survive the radical polymerization process . Since the linkage error is mainly determined by the IgG antibodies and the 40 bases long DNA strand ( Supplementary Table 2 ) both methods still belong to the pre - labeling Ex - SMLM method ( Supplementary Fig . 1 ) . First , we tested the approach on unexpanded microtubules and obtained peak - to - peak distances of 43 . 9 \u00b1 3 . 7 nm ( mean \u00b1 s . d . ) ( Fig . 2f \u2013 i ) and 37 . 0 \u00b1 4 . 8 nm ( mean \u00b1 s . d . ) ( Supplementary Fig . 6 ) for labeling with Cy5 - and Alexa Fluor 532 - modi \ufb01 ed oligonu - cleotides , respectively . These values are in good agreement with the theoretically expected value of 41 . 5 nm for immunolabeling with 42 bases long trifunctional oligonucleotide - modi \ufb01 ed sec - ondary antibodies ( Fig . 2a ) . Due to the additional modi \ufb01 cation of the secondary antibodies , the peak - to - peak distances should be a few nanometers larger than the value measured for standard immunolabeled microtubules of 36 . 2 \u00b1 5 . 4 nm ( mean \u00b1 s . d . ) ( Fig . 1f \u2013 i ) . If the oligonucleotide - modi \ufb01 ed secondary antibodies are labeled with complementary Alexa Fluor 532 - modi \ufb01 ed oligonucleotides prior to expansion , we measured a peak - to - peak distance of 226 . 7 \u00b1 15 . 3 nm ( mean \u00b1 s . d . ) from d STORM images ( Fig . 2j \u2013 m ) . Since Cy5 does not survive gelation 1 , 3 , 5 , we performed an additional experiment labeling the oligonucleotide - modi \ufb01 ed secondary anti - bodies after expansion with complementary Cy5 - modi \ufb01 ed oligo - nucleotides , performed d STORM imaging in photoswitching buffer and determined a slightly shorter peak - to - peak distance of 201 . 0 \u00b1 9 . 3 nm ( mean \u00b1 s . d . ) ( Fig . 2n \u2013 q ) . Both values are in excellent agreement with the theoretical peak - to - peak distance of 226 . 5 nm and 202 nm , respectively ( Fig . 2a ) , simulated for 3 . 2x expanded microtubules taking into account the length of the 42 base pair trifunctional oligonucleotide , the position of \ufb02 uoro - phores within the DNA strand and its spatial orientation ( Supplementary Fig . 7 and Supplementary Note 1 ) . The slightly shorter peak - to - peak distance measured in the Cy5 - experiment where the dye - labeled complementary strand was hybridized after expansion can be explained most likely by coiling of the single - stranded trifunctional oligonucleotide during gelation ( Supple - mentary Fig . 7 ) . These results indicate that Ex - SMLM can resolve linker differences of 42 DNA base pairs ( corresponding to ~ 14 . 3 nm ) and , interestingly conformational differences between single and double - stranded DNA linkers . Noteworthy is that the total size of an expanded sample is not only determined by the biomolecule of interest , e . g . microtubules , but also by the \ufb02 uorescent probe , e . g . primary and secondary antibodies , used to label the biomolecule of interest . Unexpanded , microtubules labeled with primary and secondary IgG antibodies exhibit a diameter of ~ 60 nm with a linkage error ( de \ufb01 ned by the size of the primary and secondary antibody ) of 17 . 5 nm 22 . For example , for 3 . 3x expansion this translates into a microtubule diameter of 3 . 3 \u00d7 25 nm = 82 . 5 nm whereas the diameter of the immunolabeled microtubule is substantially broadened to ~ 198 nm because of the linkage error of 3 . 3 \u00d7 17 . 5 nm = 57 . 75 nm introduced by the primary and secondary antibody that has to be added to both sides of the microtubule ( Supplementary Fig . 5 ) . In other words , even though SMLM achieves high localization precisions 12 , 13 , a linkage error of > 50 nm undoes much , or even all , of the gain in resolution . Pre - versus post - labeling Ex - SMLM . In order to reduce the linkage error , we next explored post - labeling Ex - SMLM . It has been shown that the \ufb02 uorescence signals from some genetically encoded FPs as well as conventional \ufb02 uorescently labeled sec - ondary antibodies and streptavidin that are directly anchored to the gel are at least partially preserved by proExM even when subjected to the strong nonspeci \ufb01 c proteolytic digestion used in the original ExM protocol 1 , 5 . Therefore , we anticipated that pro - tein epitopes might survive the proExM protocol 26 . To compare the labeling density of pre - and post - labeling Ex - SMLM we immunostained microtubules with Alexa Fluor 532 before and additionally after expansion . Intriguingly , combining pre - with post - labeling resulted in a substantial shortening of the average peak - to - peak distance of the sidewalls of microtubules to 79 . 5 \u00b1 6 . 6 nm ( mean \u00b1 s . d . ) determined from d STORM images ( Fig . 3 ) . We speculated that the protease digestion step may destroy a large fraction of the pre - labeled antibody complexes but to our surprise , the majority of tubulin epitopes survives this critical step . Toge - ther with the increased accessibility of tubulin epitopes for pri - mary antibodies and primary antibody epitopes for secondary antibodies after expansion this results in peak - to - peak distances undistinguishable from solely post - labeled microtubules . To examine more quantitatively epitope survival and increased epitope accessibilities , we simulated the cross - sectional pro \ufb01 les expected for pre - and post - labeled microtubules . Here we assumed a ~ 10 - fold signal dilution ( 3 . 2 2 ) for the 2D projection of the \ufb02 uorescence signals of 3 . 2x expanded pre - labeled antibodies ( Fig . 3e and Supplementary Fig . 5 ) . Hence , the cross - sectional microtubule pro \ufb01 les show the superposition of the pro \ufb01 le calculated for the 3 . 2x expansion of 25 nm diameter microtubules post - immunolabeled and 60 nm diameter micro - tubules pre - immunolabeled . The resulting superposition pro \ufb01 le shows a peak - to - peak distance of 79 . 5 nm ( Fig . 3e ) emphasizing the advantage of post - labeling Ex - SMLM . Post - labeling Ex - SMLM using the proExM protocol 5 provides an improved labeling ef \ufb01 ciency and a reduced linkage error . In fact , the immunolabeling linkage error of ~ 58 nm for pre - labeling reduces to ~ 5 nm for post - labeling considering a 3 . 2x expansion factor and thus improves the effective achievable resolution ( Supplementary Fig . 8 ) . Therefore , d STORM images of Alexa Fluor 532 labeled microtubules clearly revealed the hollow cylinder of micro - tubules ( Fig . 3c ) using a water - immersion objective and epi - illumination , similar to recently published results obtained by DNA - PAINT TIRF microscopy and experimental point spread function \ufb01 tting 27 . The average distance between the sidewalls of NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 ARTICLE NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 5 the xz - projection of a 6 . 5 \u00b5m long microtubule \ufb01 lament was determined to 81 . 2 nm ( Fig . 3g \u2013 h ) highlighting the high spatial resolution of pre - labeling 3D Ex - d STORM . Post - labeling Ex - SMLM of centrioles . Motivated by the results , we set out to explore the molecular organization of centriole organelles by Ex - SMLM . We used isolated Chlamydomona s cen - trioles , which have a characteristic 9 - fold microtubule triplet - based symmetry , forming a polarized cylinder ~ 500 nm long and ~ 220 nm wide 28 ( Supplementary Fig . 9 ) . Recently 2 , U - ExM has been developed as an extension of ExM that allows for near - native expansion of organelles and multiprotein complexes and visuali - zation of preserved ultrastructures by optical microscopy . When combined with STED microscopy , details of the ultrastructural organization of isolated centrioles such as the 9 - fold symmetry and centriolar chirality could be visualized 2 . Advantageously , U - ExM uses post - labeling to improve the epitope accessibility after expansion . Here , we used U - ExM treated centrioles in combination with post - labeling with Alexa Fluor 647 secondary antibodies to enable d STORM imaging , which has previously be impossible due to shrinking of expanded hydrogels in photoswitching buffer . Therefore , samples were re - embedded and transferred onto coverslips functionalized with acrydite via glass silanization to minimize lateral drift . This allowed us to perform post - labeling 3D Ex - d STORM on ~ 3 . 4x expanded centrioles ( Fig . 4a \u2013 d and Supplementary Fig . 10 ) . Alternatively , we used the spontaneously blinking Si - rhodamine dye HMSiR 29 that enables SMLM in the absence of photoswitching buffer and does thus not require re - embedding . Using double - deionized water , we achieved a molecular expansion factor of ~ 4x ( Fig . 4d \u2013 f and Supplementary Fig . 9 ) . Unfortunately , since the pH of double - deionized water is below 7 . 0 , HMSiR does not exhibit optimal blinking characteristics 29 . Addition of PBS buffer , pH 7 . 4 improved the blinking characteristics of HMSiR but reduced the expansion factor to ~ 2x , which limits the spatial resolution of the SMLM experiments ( Fig . 4d , g ) . In contrast to 3D d STORM images of unexpanded centrioles ( Fig . 4h ) post - labeling 3D Ex - SMLM clearly visualized the centriole as a bundle of nine microtubule triplets . SMLM images of expanded isolated Chlamydomonas centrioles showed the 9 - fold symmetry of the procentrioles ( Fig . 4b , f ) with tubulin diameters of ~ 220 nm in agreement with previous studies 2 , 30 . Even in side views of centrioles imaged by 3D Ex - d STORM the neighboring microtubule triplets are clearly separated ( Fig . 4c ) . Furthermore , 3D Ex - d STORM allowed us to resolve ring - like sub - structures of centrioles indicating hollow cylinders of microtubule triplets ( Supplementary Fig . 11 ) . According to these results , re - embedding of the sample and d STORM in photoswitching buffer provides currently the best Ex - SMLM performance . Since microtubule triplets separated by 15 \u2013 20 nm 30 are very well resolved in the expanded images post - labeling Ex - SMLM exhibits a spatial resolution that is way below 15 \u2013 20 nm reaching the structural resolution required to resolve the molecular architecture of centrioles . Discussion Electron microscopy has been the only viable method able to reveal the ultrastructure of organelles and molecular complexes for decades because of the diffraction limit of optical microscopy . Super - resolution microscopy offers up to ~ 10x higher resolution than conventional diffraction - limited microscopy 11 , 12 . Improved super - resolution microscopy methods can now localize single emitters with a precision of a few nanometers 31 \u2013 33 , but limitations in labeling ef \ufb01 ciency and linkage error have thus far prevented the translation of high localization precision into sub - 10 - nm spatial resolution . Therefore , the spatial resolution provided by all these inventive methods is currently still too low a b e f g h d c pre 630 0 V iV i i ii ii iV / V iii z [ nm ] y x z x z x post 79 . 5 \u00b1 6 . 6 nm 81 . 2 nm 1 . 0 0 . 5 F r equen cy [ a . u ] 0 . 0 I n t en s i t y [ a . u . ] 1 . 0 0 . 5 0 . 0 \u2013 200 \u2013 100 100 200 0 Distance [ nm ] 1 . 0 0 . 5 I n t en s i t y [ a . u . ] 0 . 0 \u2013 150 \u2013 100 \u2013 50 100 50 150 0 Distance [ nm ] 70 75 80 85 90 95100 65 60Peak - to - peak distance [ nm ] Fig . 3 3D post - labeling Ex - d STORM . SMLM image of re - embedded and post - expansion labeled microtubules . a 3D d STORM image of re - embedded Cos - 7 cells expanded according to the Protein - Retention protocol ( proExM ) 4 pre - labeled with anti \u03b1 - and \u03b2 - tubulin antibodies and additionally post - labeled with anti \u03b1 - tubulin . The secondary antibodies were labeled with Alexa Fluor 532 ( proExM Al532 ) . The small logo in the upper left corner symbolizes the labeling method , e . g . pre - and post - immunolabeling with Al532 secondary antibodies . b Magni \ufb01 ed view of highlighted region in ( a ) . c xz - side view cross - sections ( white lines ) ( i ) and ( ii ) shown in ( b ) revealing the hollow structure of microtubules . d Magni \ufb01 ed view of highlighted region ( white box ) in ( b ) . Since post - labeling dominates the signal , the method is termed proExM Al532 ( post - labeled ) . e Averaged cross - sectional pro \ufb01 le ( blue ) of 11 analyzed microtubule segments along a total of 28 . 2 \u00b5 m \ufb01 lament ( 2 . 1 \u2013 5 . 5 \u00b5 m segments ) of one expanded sample . The simulated cross - sectional pro \ufb01 le for 3 . 2x proExM expanded pre - and post - labeled microtubule assuming a pre - to post - labeling ratio of 0 . 1 is shown in red . f Histogram of peak - to - peak distances with normalized normal curve ( red ) of \ufb01 tted pro \ufb01 les analyzed in ( e ) with an average distance of 79 . 5 \u00b1 6 . 6 nm ( mean \u00b1 sd ) analyzed along n = 11 microtubule segments in 2 cells from 1 expanded sample . g Image projection of the xz - axes averaged along two microtubule \ufb01 laments ( iv ) and ( v ) shown in ( b ) ( red dotted lines ) using the \u201c z projection analysis \u201d of the software \u201c Line Pro \ufb01 ler \u201d . h Cross - sectional pro \ufb01 le ( blue dots ) of the xz - projection shown in ( g ) . Using a bi - Gaussian \ufb01 t ( red ) the peak - to - peak distance is determined to 81 . 2 nm . Scale bars , 10 \u00b5 m ( a ) , 5 \u00b5 m ( b ) , 1 \u00b5 m ( c ) , 500 nm ( d ) , 100 nm ( g ) . ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 6 NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications to unravel the composition and molecular architecture of protein complexes or dense protein networks . Expansion microscopy ( ExM ) represents an alternative approach to bypass the diffrac - tion barrier . By linking a protein of interest into a cross - linked network of a swellable polyelectrolyte hydrogel , biological speci - mens can be physically expanded allowing for sub - diffraction resolution imaging on conventional microscopes 1 \u2013 10 . However , even in combination with super - resolution microscopy techni - ques , spatial resolutions below ~ 20 nm have so far proven to be very dif \ufb01 cult to achieve by ExM 16 . Here , we have shown that re - embedding of expanded hydrogels enables the use of standard photoswitching buffers and d STORM imaging of ~ 3 . 2x expanded samples . Our results demonstrate that post - labeling ExM using the proExM protocol 5 or U - ExM 2 provides solutions for the two major limiting problems of improved super - resolution micro - scopy , the labeling ef \ufb01 ciency and linkage error . First , as shown for microtubules , expansion of the sample increases the epitope accessibility and thus the labeling ef \ufb01 ciency . Comparison experiments demonstrated that post - labeling outperforms pre - labeling several times in this regard ( Fig . 3 ) . Second , post - labeling ExM reduces the linkage error proportionally to the expansion factor . Hence , post - immunolabeling of 3 . 2x expanded micro - tubules reduces the linkage error from 17 . 5 nm 22 to ~ 5 nm ( Fig . 3 ) . Since the linkage error also in \ufb02 uences the localization accuracy and thus the effective achievable resolution ( Supple - mentary Figs . 8 and 9 ) 34 , 35 our \ufb01 ndings are highly relevant . Very recently 36 , 37 , trifunctional linkers have been introduced that are inert to polymerization , digestion and denaturation , and enable direct covalent linking of target molecules and functional groups to the hydrogel . Therefore , trifunctional linkers can retain a high number of labels and \ufb02 uorescence molecules available for post - expansion imaging . However , since the target molecules are labeled with primary and secondary antibodies or enzymatic tags ( e . g . SNAP - tags ) functionalized with the trifunctional anchor before expansion , linkage errors remain . The improved labeling ef \ufb01 ciency of post - labeling Ex - d STORM in combination with small ( 1 . 5 \u00d7 2 . 5 nm ) camelid antibodies ( \u201c nanobodies \u201d ) 38 , 39 and 10 \u2212 20x expansion factors 9 , 10 can thus pave the way for true molecular resolution imaging of endogenous proteins with 1 \u2013 5 nm spatial resolution . On the other hand , at such a small length scale , distortions of the structure may occur . To realize more homogeneous gel network structures , a new gelation method based on a highly homogeneous expansion microscopy polymer composed of tetrahedron - like monomers has been introduced 40 . The new tetra - gel polymer chemistry may introduce fewer spatial errors than earlier versions , and enable molecular resolution post - labeling Ex - d STORM with reduced distortion . Nevertheless , already ~ 3x Ex - SMLM can resolve small linker length and con - formational differences between labeling approaches as shown here for oligonucleotide - functionalized secondary antibodies ( Fig . 2 ) . In addition , we have shown that post - labeling 3D Ex - d STORM exhibits excellent structure preservation and already 0 . 8 25 \u2013 75 % IQR 1 . 5IQR Mean Median ( IQR - Interquartile range ) p < 0 . 02 D i a m e t e r [ \u03bc m ] 0 . 7 0 . 6 0 . 5 0 . 4 0 . 3 AI647MEA HMSiRPBS HMSiRWater a b c d e f g h Fig . 4 Ex - SMLM of U - ExM expanded centrioles . a - c 3D d STORM image of U - ExM expanded and re - embedded Chlamydomonas centrioles stained post re - embedding with anti \u03b1 - tubulin primary antibody and Alexa Fluor 647 conjugated secondary antibodies measured in MEA buffer . b Zoom - in on highlighted region in ( a ) revealing the 9 - fold symmetry of the shown procentriole . c Side view of two mature centrioles with clearly separated triplets . The inlet shows the cross - sectional pro \ufb01 le along the centriole ( white box ) showing \ufb01 ve distinct peaks of microtubule triples ( marked with arrow heads ) . d Comparison of the diameters determined from expanded centrioles measured using different protocols ( re - embedded and labeled with Alexa Fluor 647 , and imaged in MEA photoswitching buffer , labeled with HMSiR 647 and imaged in double - deionized water or in pH optimized PBS ( 1x ) buffer with pH 7 . 4 ) . Mean values are 657 \u00b1 90 nm ( mean \u00b1 sd ) for Alexa Fluor 647 in MEA buffer ( n = 12 centrioles ) , 428 \u00b1 74 nm ( mean \u00b1 sd ) for HMSiR in PBS ( n = 7 centrioles ) , and 750 \u00b1 34 nm ( mean \u00b1 sd ) for HMSiR 647 in water ( n = 8 centrioles ) . Data from n = 2 independent experiments for each condition . Divided by the previously analyzed diameter of \u03b1 - tubulin labeled centriole expansion factors translates into ~ 3 . 4x , ~ 2 . 2x , and ~ 3 . 9x for expanded centrioles labeled with Alexa Fluor 647 in MEA buffer , HMSiR in PBS ( 1x ) , and HMSiR 647 in water , respectively . Statistical signi \ufb01 cance was assessed by one - way ANOVA : the mean values of the diameters are signi \ufb01 cantly different with p < 0 . 02 ( F = 3 . 80 ) ( * P \u2264 0 . 05 , * * P \u2264 0 . 01 ) . e \u2013 g 2D d STORM image of U - ExM expanded centrioles labeled with HMSiR 647 imaged in water ( e \u2013 f ) or PBS ( 1x ) ( g ) f Zoom - in on highlighted region in ( e ) . h 3D d STORM image of unexpanded isolated Chlamydomonas centrioles immunostained with antibodies against glutamylated tubulin and Alexa Fluor 647 conjugated secondary antibodies . Scale bars , 1 \u00b5 m ( a , e ) , 500 nm ( b , f , g ) , 1 . 5 \u00b5 m ( c ) , 250 nm ( h ) . NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 ARTICLE NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 7 3 . 4x expansion using standard protocols can provide a suf \ufb01 cient structural resolution to resolve details of the molecular archi - tecture of centrioles ( Fig . 4 and Supplementary Fig . 11 ) . Methods Reagents . Acetic acid ( A6283 , Sigma ) , Acrylamide ( AA , 40 % , A4058 , Sigma ) , Acryloyl - X , SE , 6 - ( ( acryloyl ) amino ) hexanoic Acid , Succinimidyl Ester ( A20770 , Thermo Fisher ) , Agarose ( A9539 , Sigma ) , Ammonium persulfate ( APS , A3678 , Sigma ) , Bind Silane ( GE17 - 13330 - 01 , Sigma ) , Bovine Serum Albumin ( BSA , A2153 , Sigma ) , Cysteamine hydrochloride ( MEA , 6500 , Sigma ) , Dextran sulfate ( D8906 , Sigma ) , DMEM / HAM \u2019 s F12 with L - glutamine ( Sigma , D8062 ) , Ethanol ( absolute , \u2265 99 . 8 % , 32205 , Sigma ) , Ethylenediaminetetraacetic acid ( E1644 , Sigma ) , Ethylene glycol - bis ( 2 - aminoethylether ) - N , N , N \u2032 , N \u2032 - tetraacetic acid ( EGTA , 03777 , Sigma ) , 10 % FBS ( Sigma , F7524 ) , Formaldehyde ( FA , 36 . 5 - 38 % , F8775 , Sigma ) , Glutaraldehyde ( GA , 25 % , G5882 , Sigma ) , Guanidine hydrochloride ( 50933 , Sigma ) , 2 - ( N - morpholino ) ethanesulfonic acid ( MES , M3671 , Sigma ) , N , N \u2032 - methylen - bisacrylamide ( BIS , 2 % , M1533 , Sigma ) , N , N , N \u2032 , N \u2032 - Tetramethylethylenediamine ( TEMED , T7024 , Sigma ) , Poly - D - lysine hydropromide ( P6407 , Sigma ) , Polyoxyethylene ( 20 ) sorbitan monolaurate solution ( Tween - 20 , 10 % , 93774 , Sigma ) , Potassium hydroxide ( P5958 , Sigma ) . Proteinase K ( P4850 , Sigma ) , Saline sodium citrate buffer ( SSC , 20x , 15557 , Thermo Fisher ) , Sodium acrylate ( SA , 97 - 99 % , 408220 , Sigma ) , Sodium chloride ( NaCl , S7653 , Sigma ) , Sodium dodecyl sulfate ( SDS , L3771 , Sigma ) , streptomycin ( 0 . 1 mg / ml ) ( Sigma , R8758 ) , Tris base ( T6066 , Sigma ) , Triton X - 100 Surfact - Amps Detergent Solution ( 10 % ( w / v ) , 28313 , Thermo Fisher ) , Yeast tRNA ( AM7119 , Thermo Fisher ) . Antibodies and labeling reagents . Rabbit anti \u03b1 - tubulin antibody ( ab18251 , abcam ) , Mouse anti \u03b2 - tubulin antibody ( T8328 , Sigma ) , Mouse anti poly - glutamylated tubulin , mAb ( GT335 ) ( Adipogen , AG - 20B - 0020 ) , Alexa Fluor 647 F ( ab \u2018 ) 2 of goat anti rabbit IgG ( A - 21246 , Thermo Fisher ) , Alexa Fluor 647 F ( ab \u2018 ) 2 of goat anti mouse IgG ( A - 21235 , Thermo Fisher , Al532 - Goat anti Rabbit IgG ( H + L ) ( A - 11009 , Thermo Fisher ) , Al532 - Goat anti Rabbit IgG ( H + L ) ( A - 11002 , Thermo Fisher ) , HMSiR 647 ( A208 - 01 , MoBiTec ) conjugated to goat anti rabbit IgG F ( ab \u2018 ) 2 ( SAB3700946 , Sigma ) , TetraSpeck Microspheres ( 0 . 1\u00b5m , T27279 , Thermo Fisher ) . Chlamydomonas reinhardtii centriole isolation . Centrioles were isolated from the cell wall - less Chlamydomonas reinhardtii strain CW15 by centrifugation at 600 g for 10min in 50 ml conical tubes 41 . Isolated centrioles were thawed on ice and diluted with cold K - Pipes 10mM pH 7 . 2 . Centrioles were then loaded in a 15ml Corex tube with a homemade adaptor and concentrator , and spun onto a 12 mm Poly - D - lysine coated coverslip through centrifugation at 10 , 000 g for 10 min with a JS - 13 . 1 swinging bucket rotor ( Beckman ) at 4\u00b0C . Coverslips were then processed for immunostaining and expansion microscopy . Cell culture of mammalian cells . COS - 7 monkey kidney cells ( purchased from CLS Cell Line Servie GmbH ) were cultured at 37\u00b0C and 5 % CO 2 in DMEM / HAM \u2019 s F12 medium with L - glutamine containing FBS ( 10 % ) and peni - cillin ( 100 U / ml ) and streptomycin ( 0 . 1 mg / ml ) . 20 \u2013 30 , 000 cells per well were seeded on round 18 mm high precision cover glasses ( No 1 . 5 ) in 12 - well culture plates ( Techno Plastic Products , 92012 ) and grown for 24 h prior to \ufb01 xation . Sample preparation . For \ufb01 xation , all solutions were pre - warmed to 37\u00b0C and \ufb01 xation was conducted on a heating plate set to 37 \u00b0C . Right before \ufb01 xation samples were rinsed once with pre - warmed Cytoskeleton buffer ( CB - buffer , 10 mM MES , 150 mM NaCl , 5mM EGTA , 5mM glucose and 5mM MgCl2 , pH 6 . 1 ) . Cells were then \ufb01 xed and permeabilized simultaneously incubating a primary \ufb01 xative solution of 0 . 3 % glutaraldehyde and 0 . 25 % Triton X - 100 in CB - buffer for 90s followed by a second \ufb01 xation using 2 % glutaraldehyde in CB - buffer for 10 min . Fixation was stopped by a 7min reduction step with freshly prepared 0 . 5 % NaBH 4 in PBS . Specimen were then washed three times with PBS ( 1x ) for 5 min each and treated differently depending on subsequent expansion method described below . Unless otherwise stated all incubations were carried out at room temperature in the following protocols . Immunostaining was either performed pre - gelation ( referred to as pre - labeling ) , post - expansion ( post - labeling ) or post - re - embedding ( post re - embedding labeling ) . Sequences and modi \ufb01 cations of DNA labels are listed in Supplementary Table 2 . A list of primary and secondary antibodies used for immunostaining in the corresponding Figures is provided in Supplementary Table 3 and Supplementary Table 4 with details about the expansion protocol used . Immunostaining of unexpanded Cos - 7 cells . Cells were placed in blocking buffer ( 5 % BSA in PBS ) for 1 h and then incubated for 1h with anti - alpha tubulin primary antibody solution ( ab1825 , diluted 1 : 500 , \ufb01 nal concentration c end = 2\u00b5g / ml ) diluted in blocking buffer . Samples were washed thrice in PBS ( 1x ) for 5 min each and incubated with secondary Alexa Fluor 532 IgG antibody solution in blocking buffer ( A - 11002 , diluted 1 : 200 , c end = 10 \u00b5g / ml ) for 1 h followed by three washes in PBS ( 1x ) for 10min each . ExM protocol using DNA trifunctional labels ( ExM protocol ) . After blocking with 5 % BSA in PBS for 1 h , cells were incubated with anti - alpha tubulin primary antibody ( ab1825 , diluted 1 : 500 , c end = 2 \u00b5g / ml ) in blocking buffer ( 5 % BSA in PBS ) for 1h , followed by three washes in PBS ( 1x ) for 5 min each and incubation of \u201c Antibody B \u201d DNA - labeled secondary antibodies ( 10 \u00b5g / ml ) in hybridization buffer ( 2x saline sodium citrate ( SSC ) , 10 % dextran sulfate , 1mg / ml yeast tRNA , 5 % BSA ) for 3h . Antisense DNA B1 - Alexa Fluor 532 and DNA B2 - Alexa Fluor 532 oligos were hybridized simultaneously at a total DNA concentration of 1 . 0 ng / \u00b5l for 3h in hybridization buffer . Then samples were washed three times with PBS ( 1x ) for 10min each . Gelation was performed on the lid of a 4 - well cell culture plate put on ice and covered with para \ufb01 lm that served as a \ufb02 at hydrophobic gelation surface . 18 mm cover glasses with cells facing down were placed on top of 90\u00b5l pre - chilled ExM monomer solution ( 8 . 625 % ( w / w ) SA , 20 % ( w / w ) AA , 0 . 15 % ( w / w ) BIS , 2M NaCl in PBS ) supplemented with 0 . 2 % APS and 0 . 2 % TEMED . Samples were then carefully transferred to a humidi \ufb01 ed chamber and incubated for 1 . 5 h at 37 \u00b0C for chemical crosslinking of acrylic monomers and trifunctional labels . After gelation samples were treated with 8 U Proteinase K in digestion buffer ( 50 mM Tris ( pH 8 . 0 ) , 0 . 5 % TritonX - 100 , 0 . 8M guanidine hydrochloride , 1 mM EDTA ) and then expanded in double - deionized water . Water was exchanged several times until the maximum expansion factor of the hydrogel was reached . The expansion factor was determined by measuring the diameter of the gel using a calipser . When the expansion factor did not change within three water exchanges this factor was assumed as maximum expansion of the hydrogel . Protein Retention protocol ( proExM protocol ) . Blocking and immunostaining were performed as described under \u201c Immunostaining of unexpanded Cos - 7 cells \u201d incubating anti - \u03b1 - tubulin antibody ( ab1825 , diluted 1 : 500 , c end = 2\u00b5g / ml ) and anti - \u00df - tubulin ( T8328 , diluted 1 : 200 , c end = 10 \u00b5g / ml ) simultaneously in blocking buffer as primary antibodies and Alexa Fluor 532 IgG antibodies ( A - 11002 and A - 1109 , each diluted 1 : 200 to c end = 10\u00b5g / ml ) diluted in blocking buffer as secondary antibodies . For copolymerization of amine groups into the hydrogel , cells were treated with the amine reactive agent Acryloyl X - SE ( 0 . 1mg / ml ) in PBS . The agent was freshly prepared from desiccated stock aliquots kept at \u2212 20 \u00b0C , incubated overnight in a humidi \ufb01 ed chamber , and subsequently washed twice for 15min each in PBS ( 1x ) . Hydrogel formation , Proteinase K digestion and expansion in water were performed as described under \u201c ExM protocol \u201d . After re - embedding of expanded hydrogels as described in section \u201c Bind - silane treatment and re - embedding \u201d , samples were labelled with \u03b1 - tubulin primary antibody solution ( ab1825 , diluted 1 : 500 , c end = 2 \u00b5g / ml ) in 2 % BSA for 3h at 37\u00b0C and then washed twice with 0 . 01 % Tween in PBS for 20min each and twice with PBS ( 1x ) for 10min each . Secondary antibodies were incubated for 3h at 37 \u00b0C and washed twice with 0 . 01 % Tween in PBS for 30 min each and twice with PBS ( 1x ) for 30 min followed by a washing step over night in PBS ( 1x ) . ExM protocol with glutaraldehyde linker ( ExM - GA protocol ) . Blocking and immunostaining were performed as described under \u201c Immunostaining of unex - panded Cos - 7 cells \u201d using \u03b1 - ( ab1825 , diluted 1 : 500 , c end = 2\u00b5g / ml ) and \u00df - tubulin ( T8328 , diluted 1 : 200 , c end = 10 \u00b5g / ml ) antibodies as primary antibodies and a mixture of Alexa Fluor 532 IgG secondary antibodies ( A - 11002 and A - 1109 , each diluted 1 : 200 to c end = 10\u00b5g / ml ) in blocking buffer . After washing with PBS ( 1x ) , cells were incubated with 0 . 25 % GA in PBS for 10min and washed thrice in PBS ( 1x ) for 5min each before proceeding with gelation of the samples . Gelation , digestion , and expansion was performed as described under \u201c ExM protocol \u201d . DNA label with Cy5 ( DNA - Cy5 protocol ) . Blocking and immunostaining were performed as described under \u201c ExM protocol \u201d with a mixture of primary \u03b1 - and \u00df - tubulin antibodies ( ab1825 diluted 1 : 500 with 2\u00b5g / ml and T8328 diluted 1 : 200 with 10\u00b5g / ml ) and DNA conjugated secondary antibodies \u201c Antibody B Cy5 \u201d or \u201c Antibody C Cy5 \u201d in hybridization buffer that were then directly incorporated into the hydrogel . Hydrogel formation , proteinase K digestion and expansion were performed as described under \u201c ExM protocol \u201d . After re - embedding on 24 - mm silanized round coverslips samples were incubated over night with Cy5 antisense oligos with a DNA concentration of 0 . 5ng / \u00b5l for each oligo in hybridization buffer . Ultrastructure expansion microscopy ( U - ExM ) . Twelve millimeters cover glasses with isolated 2 centrioles were placed in a solution containing 0 . 7 % FA , 1 % AA diluted in PBS ( 1x ) . Next , 35 \u00b5l of pre - chilled U - ExM monomer solution ( 19 % ( w / w ) SA , 10 % ( w / w ) AA , 0 . 1 % ( w / w ) BIS ) supplemented with 0 . 5 % APS and 0 . 5 % TEMED in PBS for 1min on a para \ufb01 lm coated plate put on ice . Gelation proceeded for 1h at 37\u00b0C in a humidi \ufb01 ed chamber . Samples were placed in denaturation buffer ( 200mM SDS , 200mM NaCl in 50mM Tris ( pH 9 . 0 ) ) for 15 min and then gels were carefully removed from the cover glasses and transferred to 1 . 5ml cen - trifuge tubes \ufb01 lled with denaturation buffer . Hydrogels were then incubated for 30 min at 95\u00b0C and then expanded in double deionized water until the maximum expansion of the gels were reached . After re - embedding on Bind - silane treated 24 - mm cover glasses , centrioles were labelled with anti alpha - tubulin primary antibodies ( ab1825 , c end = 2 \u00b5g / ml ) diluted 1 : 500 in 2 % BSA in PBS for 3 h at 37 \u00b0C , washed twice with 0 . 01 % Tween in PBS for 20 min each and twice with PBS ( 1x ) for 10min each . Next , secondary Alexa Fluor 647 F ( ab \u2018 ) 2 antibodies ( A - 21246 , ARTICLE NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 8 NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 1 : 200 , c end = 10\u00b5g / ml ) diluted in 2 % BSA were incubated 3h at 37\u00b0C followed by two washing steps in 0 . 01 % Tween in PBS for 30 min each and two washes with PBS ( 1x ) for 30min . Before imaging gels were washed once more overnight in PBS ( 1x ) . For imaging of unexpanded centrioles the primary antibody anti poly - glutamylated tubulin ( Adipogen , 1 : 500 ) was diluted in 5 % BSA in PBS and incu - bated for 1 h at room temperature , washed thrice in PBS for 5 min each , followed by incubation with secondary Alexa Fluor 647F ( ab \u2018 ) 2 antibodies ( A - 21246 , 1 : 200 , c end = 10\u00b5g / ml ) diluted in 2 % BSA for 1h . The samples were then washed twice in 0 . 01 % Tween in PBS and once in PBS for 10min each . Re - embedding of expanded hydrogels ( Re - embedding protocol ) . To avoid shrinking caused by d STORM photoswitching buffer and to prevent drifting of the hydrogel during image acquistion an uncharged acrylamide gel was crosslinked throughout the hydrogel while chemically binding it on Bind - silane treated cover glasses . Round 24 - mm cover glasses ( high precision ) were sonicated successively in double - deionized water , absolute ethanol and 5M potassium hydroxide for 20 min each and washed with deionized water between every sonication step and \ufb01 nally oven dried at 100\u00b0C . 200 \u00b5l of Bind - silane working solution ( 5 \u00b5l Bind - Silane in 8 ml absolute ethanol , 200 \u00b5l glacial acetic acid , 1 . 8ml double deionized water ) were distributed evenly on cleaned 24 - mm cover glasses and left for around 1h until the solution was fully evaporated . Cover glasses were then rinsed with doubly deio - nized water and air - dried . Glasses were prepared shortly before use . For re - embedding expanded hydrogels were placed in 6 - well cell culture plates and each sample was covered with 3ml of freshly prepared Re - embedding solution ( 10 % acrylamide , 0 . 15 % bis - acrylamide , 0 . 05 % APS , 0 . 05 % TEMED in 5 mM Tris ( pH 8 . 9 ) ) . Samples were incubated on a platform shaker twice with freshly prepared solution for 30min each . Shaking of the Re - embedding solution is crucial in this step as it brings oxygen into the solution that prevents it from gelling to early . The stirring speed should be adjusted so that the liquid is in motion but the gels are not damaged . After the second incubation , samples were transferred on silanized coverglasses while carefully removing excess solution from the hydrogels using laboratory wipes . Another coverglass that was not silanized was placed on top of the hydrogels during the following steps . The whole setup was transferred to a humidi \ufb01 ed container equipped with gas injection holes . To accelerate gelation oxygen was extracted from the container by purging the chamber with nitrogen for 15min . The samples were then incubated at 37\u00b0C for 2h . After polymerization of the re - embedding gel samples were washed at least thrice for 30 min in double deionized water . Coverglasses on top of the hydrogel come off themselves during washing or can be detached carefully after the \ufb01 rst washing steps . Re - embedded gels were then placed in imaging buffer or staining buffer depending on the sub - sequent protocol . Microscopes . Single - molecule localization microscopy ( SMLM ) image acquisition was performed on a custom - built setup with an inverted Zeiss Axio Observer Z1 ( Carl Zeiss Microscopy ) microscope equipped with a De \ufb01 nite Focus autofocusing system . For excitation of different \ufb02 uorescent molecules the setup provides three iBeam smart diode lasers with 405 nm ( 100mW output power ) , 488nm ( 200 mW output power ) and 640nm ( 150mW output power ) and a DPSS ( diode pumped solid state ) 532nm laser ( gem532 , Laserquantum ) . Lasers were \ufb01 ltered with laser clean - up \ufb01 lters according to the speci \ufb01 c wavelength and focused on the back focal plane of the objective to achieve a wide \ufb01 eld illumination . To match the aqueous refractive index of expanded samples awater - immersion objective ( LD C - Apochromat 63x / 1 . 15 W Corr M27 , Carl Zeiss Microscopy ) is implemented in the microscope . The excitation light passes a quad - band dichroic beam splitter ( Di01 - R405 / 488 / 532 / 635 - 25\u00d736 , BrightLine ) combined with a quad - band rejection \ufb01 lter ( ZET405 / 488 / 532 / 642m , Chroma ) . For recording the emission of excited \ufb02 uor - ophores the setup is equipped with two Andor Ixon Ultra 897 EMCCD ( electron - multiplying charge - coupled device ) cameras at the side port of the microscope . The software Andor Solis ( Version 4 . 28 . 30014 ) was used to control the EMCCD cameras . The \ufb02 uorescence light is parallelized through a 160 mm achromatic lens ( Thorlabs ) and can be spectrally separated by a 630 DCXR ( Chroma ) dichroic beam splitter . In this con \ufb01 guration , two different emission wavelengths can be focussed on two cameras arranged perpendicular to each other . For all d STORM measurement in this work the beam splitter was removed and the emission light was directed to one camera . Suitable emission light \ufb01 lters were placed in front of the camera depending on the detected \ufb02 uorescent wavelength . For 3D imaging , an additional achromatic cylindrical lens ( f = 250 mm , Thorlabs ) was placed in the detection path close to the imaging plane before the relay system . Rescanning confocal imaging ( RCM ) was performed on a Nikon TiE inverted microscope equipped with an RCM unit ( Confocal . nl ) that is based on the image scanning principle 42 . The setup was operated by the microscope software NIS - Elements ( version 4 . 6 ) . Mounting and SMLM image conditions . Re - embedded hydrogels immobilized on 24 - mm cover glasses were immersed in photoswitching buffer consisting of 100 mM cysteamine hydrochloride ( MEA ) in PBS with optimized pH ( adjusted with KOH ) depending on the utilized \ufb02 uorescent dye . For Alexa Fluor 647 and Cy5 \ufb02 uorophores , the pH of the imaging buffer was adjusted to pH 7 . 7 and to pH 7 . 9 when using Alexa Fluor 532 , respectively . The buffer was prepared freshly before use . The hydrogel was incubated in photoswitching buffer twice for 20 min each before imaging . U - ExM treated samples labeled with the spontaneously blinking Si - rhodamine dye HMSiR were immobilized on Poly - L - lysine ( 0 . 1 % ) coated 24 - mm high - precision cover glasses and additionally embedded in 1 % ( w / v ) Agarose . As imaging buffer , double deionized water or pH adjusted PBS buffer ( 1x , pH 7 . 4 ) was used . For unexpanded d STORM imaging samples were placed in 100 mM MEA in PBS adjusted to pH 7 . 5 ( with KOH ) for DNA - Cy5 and Streptavidin - Alexa Fluor 647 and pH 7 . 9 ( with KOH ) when using Alexa Fluor 532 . 3D d STORM calibration . To obtain 3D calibration curves , \ufb02 uorescent beads were mixed in U - ExM or ExM monomer solution for 3D image acquisitions of U - ExM or ExM samples , respectively . Therefor \ufb02 uorescent marker stock supension ( 0 . 1 \u00b5m , ~ 1 . 8 \u00d7 10 11 particles / mL , TetraSpeck Microspheres , Thermo Fisher ) was vortexed for ~ 1 min and then diluted 1 : 50 in the corresponding monomer solution . After adding TEMED and APS in the appropriate concentrations , the bead - gel solution was vortexed again for ~ 20 s , polymerized , and expanded as described under the respective expansion protocol ( omitting the digestion or denaturation step ) . The expanded gels were then transferred on poly - L - lysine ( 0 . 1 % ) coated coverslips and additionally embedded in 1 % ( w / v ) Agarose . 4 \u00b5m z - stacks of several \ufb02 uorescent markers dispersed in the hydrogel ~ 50 \u2013 400 \u00b5m above the coverslip were recorded and used to generate 3D calibration curves as described below . The software Micro - manager 1 . 4 was used for image acquisition and to control the piezo driven stage . Image processing . For 2D and 3D d STORM image reconstruction super - resolution images were analyzed , post - processed and visualized using the analysis platform SMAP ( Superresolution Microscopy Analysis Platform ) with the GPU based 3D \ufb01 tter \ufb01 t3Dcspline 27 and the ImageJ plugin ThunderSTORM 43 . The respective integrated calibration tools were used for generating 3D astigmatism calibration curves . Localizations were further corrected for drift using the cross - correlation method , \ufb01 ltered for molecules with poor precision and grouped to one localization when molecules appeared in several consecutive images . Expansion factor determination . Centriole diameters of U - ExM expanded sam - ples were determined by averaging peak - to - peak distances of two cross - sectional pro \ufb01 les that were drawn through the center of the ninefold - symmetrical \u03b1 - tubulin signal using the line pro \ufb01 le tool of Fiji 44 . Peaks were then determined by using the peak \ufb01 nder minitool implemented in the analyse software Origin ( OriginLab , Northampton , MA ) . To determine the expansion factor post - expansion and post - re - embedding , Cos - 7 cells were labeled with a - tubulin and \u00df - tubululin and expanded according to the \u201c proExM protocol \u201d . An additional post - expansion immunostaining for \u03b1 - tubulin was performed using the same primary and sec - ondary antibodies . RCM images of the same cells were acquired before gelation , after expansion and after re - embedding in different imaging buffers . Images were then registered via rigid ( similarity ) and non - rigid registration ( B - spline ) using the open source , command - line program elastix 6 . The transform parameters of the similarity transformation of pre - and post - expansion RCM images were used to determine the initial expansion factor of the sample . RCM images acquired post - re - embedding in PBS ( 1x ) and cysteamine hydrochloride as well as a d STORM image in photoswitching buffer of the same area were registered in the same way using elastix to determine the expansion factor after re - embedding in different imaging buffers . Furthermore , a deformation vector \ufb01 eld of pre - expansion and post re - embedding RCM images was created using elastix and transformix 6 . Elastix and transformix code were executed in Wolfram Mathematica 11 . 2 . Analysis of microtubule transversal pro \ufb01 les . To analyze and compare the dif - ferent expansion protocols we developed a home written software that detects \ufb01 ber like structures and automatically determines the transversal pro \ufb01 le along these structures in reconstructed SMLM images . In detail the SMLM images are \ufb01 rst convolved with a Gaussian blur compensating for noise discontinuity or holes . A thresholding algorithm 45 then converts the image from grayscale to binary . Using Lees algorithm 46 the expanded lines are reduced to one pixel width . The pixel coordinates from all still connected lines are then retrieved and tested for continuity . Points of discontinuity are used as breakpoints and all following coordinates are connected to a new line . Lines , shorter than the minimum required length are discarded . An univariate spline of degree 3 ( c - spline ) is \ufb01 tted to each line . Note that shape and gradient of the line depend on the smoothing parameter . The result is a table containing the spline coordinates and the local derivatives . Perpendicular to the derivative a line pro \ufb01 le is extracted from the original image at each coordinate point . The averaged pro \ufb01 les for each spline are \ufb01 tted with the following functions ( Eqs . ( 1 \u2013 5 ) ) : Gaussian : y \u00bc h e (cid:2)\u00f0 x (cid:2) c \u00de 2 2 w 2 \u00fe b \u00f0 1 \u00de ( where h is the intensity , c the center , b the offset , and w the variance of the distribution . Optimal for single pro \ufb01 les ) . Bi (cid:2) Gaussian : y \u00bc h 1 e (cid:2)\u00f0 x (cid:2) c 1 \u00de 2 2 w 2 1 \u00fe h 2 e (cid:2)\u00f0 x (cid:2) c 2 \u00de 2 2 w 2 2 \u00fe b \u00f0 2 \u00de NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 ARTICLE NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 9 ( optimal for pro \ufb01 les containing a dip ) . Tri (cid:2) Gaussian : y \u00bc h 1 e (cid:2)\u00f0 x (cid:2) c 1 \u00de 2 2 w 21 \u00fe h 2 e (cid:2)\u00f0 x (cid:2) c 2 \u00de 2 2 w 22 \u00fe h 3 e (cid:2)\u00f0 x (cid:2) c 3 \u00de 2 2 w 23 \u00fe b \u00f0 3 \u00de ( optimal for pro \ufb01 les exhibiting a dip and high background signal ) . Cylinder : y \u00bc h \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 r 22 (cid:2) \u00f0 x (cid:2) c \u00de 2 q (cid:2) \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 r 21 (cid:2) \u00f0 x (cid:2) c \u00de 2 q (cid:3) (cid:4) ; if x k k < r 1 h \ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03\ufb03 r 22 (cid:2) \u00f0 x (cid:2) c \u00de 2 q(cid:3) (cid:4) ; if x k k \u2265 r 1 ; x k k < r 2 0 ; else 8 > > > > > < > > > > > : \u00f0 4 \u00de ( y describes the theoretical intensity pro \ufb01 le of microtubules where r 1 and r 2 denote the inner and outer cylinder radius . The quality of the \ufb01 t strongly depends on the initial estimation of the parameters , due to the nonlinearity of the cylinder function . ) Multi (cid:2) Cylinder : y \u00bc cyl \u00f0 i 1 ; c ; 25 e x = 2 (cid:2) 2 a ; 25 e x = 2 (cid:2) a \u00de \u00fe cyl \u00f0 i 2 ; c ; 42 : 5 e x = 2 ; 42 : 5 e x = 2 \u00fe a \u00de \u00fe cyl \u00f0 i 3 ; c ; 25 e x = 2 \u00fe a ; 25 e x = 2 \u00fe 2 a \u00de \u00fe b \u00f0 5 \u00de ( includes the theoretical dimensions of microtubules leaving less degrees of freedom . Might result in a better \ufb01 t ) . Note that the \ufb01 t intensity ( h ) gives a good estimation for the relative labeling density . Using the splines \ufb01 tted to the maximum intensity projection we constructed xz - pro \ufb01 le projections of microtubules , by taking line pro \ufb01 les in each z - stack of the 3D image . Averaging the aligned line pro \ufb01 les in a layer yields the intensity values for the corresponding row of the xz - projection . Reporting summary . Further information on research design is available in the Nature Research Reporting Summary linked to this article . Data availability All data that support the \ufb01 ndings described in this study are available within the manuscript , the related supplementary information or deposited at https : / / doi . org / 10 . 6084 / m9 . \ufb01 gshare . 12415787 . v1 . Additional information is available from the corresponding authors upon reasonable request . Code availability The automated image processing software Line Pro \ufb01 ler is available at https : / / line - pro \ufb01 ler . readthedocs . io / en / latest / . Received : 14 April 2020 ; Accepted : 5 June 2020 ; References 1 . Chen , F . , Tillberg , P . W . & Boyden , E . S . Expansion microscopy . Science 347 , 543 \u2013 548 ( 2015 ) . 2 . Gambarotto , D . et al . Imaging cellular ultrastructures using expansion microscopy ( U - ExM ) . Nat . Methods 16 , 71 \u2013 74 ( 2019 ) . 3 . Wassie , A . 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Building skeleton models via 3 - D medial surface / axis thinning algorithms . Computer Vis . Graph . Image Process . 56 , 462 \u2013 478 ( 1994 ) . Acknowledgements The authors thank P . Gessner and L . Behringer - Pliess for assistance in immunocy - tochemistry and cell culture preparation . This work was supported by the German Research Foundation ( DFG , TRR 166 ReceptorLight , project A04 ) and the European Regional Development Fund ( EFRE project \u201c Center for Personalized Molecular Immu - notherapy \u201d ) . This work is supported by the European Research Council ( ERC ; StG 715289 ACCENT to P . G . ) and the Swiss National Science Foundation ( SNSF ) PP00P3 _ 187198 to P . G . Author contributions F . U . Z . , S . R . , D . G . , T . D . M . B . , V . H . , P . G . , and M . S . conceived and designed the project . M . S , V . H . , and P . G supervised the project . F . U . Z . performed all Ex - SMLM experiments . S . R . developed Line Pro \ufb01 ler and analyzed the data together with F . U . Z . , D . G . provided the centriole samples . All authors wrote and revised the \ufb01 nal manuscript . Competing interests The authors declare no competing interests . Additional information Supplementary information is available for this paper at https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 . Correspondence and requests for materials should be addressed to V . H . , P . G . or M . S . Peer review information Nature Communications thanks the anonymous reviewer ( s ) for their contribution to the peer review of this work . Peer reviewer reports are available . Reprints and permission information is available at http : / / www . nature . com / reprints Publisher \u2019 s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional af \ufb01 liations . 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To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2020 NATURE COMMUNICATIONS | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 ARTICLE NATURE COMMUNICATIONS | ( 2020 ) 11 : 3388 | https : / / doi . org / 10 . 1038 / s41467 - 020 - 17086 - 8 | www . nature . com / naturecommunications 11", + "linseyStudyDesignFixation2010": "J . S . Linsey Mechanical Engineering Department , Texas A & M University , 224 Engin . Physics , 3123 TAMU College Station , TX 77843 e - mail : jlinsey @ tamu . edu I . Tseng e - mail : iht @ andrew . cmu . edu K . Fu e - mail : katherine . fu @ gmail . com J . Cagan e - mail : cagan @ tamu . edu Mechanical Engineering Department , Carnegie Mellon , Scaife Hall 214 , 5000 Forbes Avenue , Pittsburgh , PA 15213 K . L . Wood Mechanical Engineering Department , The University of Texas at Austin e - mail : wood @ utexas . edu C . Schunn Department of Psychology , University of Pittsburg , LRDC 821 , 3939 O\u2019Hara St . , Pittsburgh , PA 15260 e - mail : schunn @ pitt . edu A Study of Design Fixation , Its Mitigation and Perception in Engineering Design Faculty The bridge between engineering design and cognitive science research is critical to understand the effectiveness of design methods as implemented by human designers . The study reported in this paper evaluates the effects of design \ufb01xation in a group of engi - neering design faculty , and also provides evidence for approaches to overcome design \ufb01xation . Three conditions are compared , a control , a \ufb01xation group whom were provided with an example solution , and a de\ufb01xation group whom were also given materials to mitigate their design \ufb01xation . Measures include indicators of design \ufb01xation and partici - pant perceptions . The study demonstrates that the engineering design faculty show sta - tistically signi\ufb01cant evidence of design \ufb01xation , but only partially perceive its effects . This study also indicates that design \ufb01xation can be mitigated . The group of participants in this study , due to their background in engineering design research and experience with student design teams , was expected to have more accurate perceptions or awareness of design \ufb01xation than the typical participant . Understanding the incongruities between participant perceptions and quantitative design outcomes are particularly of interest to researchers of design methods . For this study , clear evidence exists that designers , even those that study and teach design on a regular basis , do not know when they are being in\ufb02uenced or \ufb01xated by misleading or poor information . (cid:1) DOI : 10 . 1115 / 1 . 4001110 (cid:2) Keywords : design \ufb01xation , analogy , conceptual design 1 Introduction Engineering design was rigorously studied , arguably since Si - mon\u2019s Sciences of the Arti\ufb01cial was published in 1968 (cid:1) 1 (cid:2) . The \ufb01eld has explored formal and heuristic approaches to design re - \ufb01nement , manufacturing , generation , and computation (cid:1) 2 \u2013 4 (cid:2) . A critical part of engineering design is how designers think about the problem , how they reason about problem - relevant information , and how they are able to generate novel problem solutions . The study of such questions falls in the \ufb01eld of cognitive - based inno - vation and requires methods and knowledge from the \ufb01eld of cog - nitive psychology , integrated with process knowledge and partici - pants from the \ufb01eld of engineering design . The \ufb01eld of researchers who have actively pursued cognitive - based engineering is small but diverse , examples falling into the following \ufb01ve nonexhaus - tive categories . (cid:127) Representation\u2014the means to reason about and search for a solution to a design problem (cid:1) 5 \u2013 13 (cid:2) ; (cid:127) Fixation\u2014barriers to solution based on real and perceived constraints (cid:1) 14 \u2013 17 (cid:2) ; (cid:127) Analogy\u2014the mapping of knowledge from one domain to another supported by abstract representations (cid:1) 9 , 18 \u2013 29 (cid:2) ; (cid:127) Computational models\u2014models for cognitive simulation and generative tools (cid:1) 30 \u2013 32 (cid:2) ; (cid:127) Teams\u2014effective negotiation strategies , compatible repre - sentations , effective communication , trust among team members (cid:1) 7 , 33 \u2013 42 (cid:2) . Results in this \ufb01eld have begun to bear fruit , and the time is ripe for rigorous research efforts in this area . As such , in January 2008 , an NSF sponsored workshop was held in Knoxville , TN , as part of the CMMI Grantees Meeting . The workshop , entitled \u201cDiscussion on Individual and Team - Based Innovation , \u201d brought approximately 50 educators and re - searchers from the \ufb01eld of engineering design together for a day to learn about the current work and discuss potential directions for new research in the area of cognitive - based engineering design . As part of the workshop , participants took part in a formal cogni - tive study on the role of \ufb01xation and the use of analogies to overcome \ufb01xation . The experiment was formally developed , pi - loted , approved by the lead institution\u2019s Internal Review Board , and then run during the workshop . This paper presents the results of this study . One goal of the study was to allow participants to experience a formal and rigorous cognitive experiment . Since most of the participants had only engineering backgrounds , it was unlikely that many had participated in such studies . Another goal of the study was to advance the state of the \ufb01eld of cognitive - based engineering design by learning (cid:3) 1 (cid:4) if engineer - ing educators experience design \ufb01xation during a design problem solving exercise , (cid:3) 2 (cid:4) how design \ufb01xation can be overcome , adding to the current knowledge base of the \ufb01eld , and (cid:3) 3 (cid:4) whether the Contributed by the Design Education Committee of ASME for publication in the J OURNAL OF M ECHANICAL D ESIGN . Manuscript received June 19 , 2009 ; \ufb01nal manuscript received January 24 , 2010 ; published online April 13 , 2010 . Assoc . Editor : Janis Terpenny . Journal of Mechanical Design APRIL 2010 , Vol . 132 / 041003 - 1 Copyright \u00a9 2010 by ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm participants accurately perceive the effects of provided examples and materials to mitigate design \ufb01xation . On one hand , the study group is interesting because they have experience in design , as demonstrated by survey results showing considerable industrial projects and patents , but on the other hand , they think about the process of design through teaching courses in design and through research in the broader \ufb01eld of design . It is interesting to see whether this cohort is susceptible to aspects of \ufb01xation that have been prevalent in other studies of design students or practitioners . What follows is a discussion of the current state of the art of cognitive - based engineering relevant to this work , a presentation of the experiment , results , discussion , and insights from the study . 2 Background / Previous Work A number of studies showed that design \ufb01xation effects can occur when example solutions are introduced to participants (cid:1) 15 , 43 \u2013 47 (cid:2) . Jansson and Smith (cid:1) 15 (cid:2) were the \ufb01rst to apply an experimental approach to study engineering design \ufb01xation . They found that showing example solutions can reduce the range of design solutions generated by a designer , and that aspects of the example solution , including aspects that were shown to violate goals of the problem statement , can \ufb01nd their way into the design - ers\u2019 solutions . A number of later experiments by others used the same and similar design problems to further investigate the issue of design \ufb01xation (cid:1) 47 , 48 (cid:2) . Purcell and Gero (cid:1) 47 (cid:2) suggested that the susceptibility of a designer to \ufb01xation may depend on the discipline of the designer , and that design \ufb01xation is more likely if the example problem embodies principles that are in line with the knowledge base of that discipline . These studies , as a whole , dem - onstrate that introducing examples can cause design \ufb01xation , re - sulting in less creativity during ideation . 2 . 1 Possible Approaches to Overcoming Design Fixation . Some approaches to reducing design \ufb01xation have been identi\ufb01ed . Using the same \ufb01xating examples as Jansson and Smith , Chrysikou and Weisberg (cid:1) 48 (cid:2) found that including de\ufb01xation in - structions can negate the \ufb01xating effects of the examples . Another possible approach to breaking or mitigating design \ufb01xation , be - yond de\ufb01xation instructions , is to assist the designer in \ufb01nding a new way to frame the problem , which may lead to new and im - proved solutions . The power of analogical inspiration , as part of problem framing , is supported by empirical evidence , as well as by examples of professional designers using analogies to solve problems (cid:1) 9 , 49 \u2013 51 (cid:2) . Within the literature , a number of approaches for enhancing analogical retrieval and use have been noted . Some of these de - pend on the expertise of the participants , and some are more gen - eral \ufb01ndings . Casakin and Goldschmidt (cid:1) 49 (cid:2) found that visual analogies can improve design problem solving for both novice and expert architects . Ball et al . (cid:1) 52 (cid:2) found experts use more analogies than novices do , so experience seems to increase re - trieval frequency . Expertise also enhances the ability to retrieve high - level principles derived from sets of analogies (cid:3) schema - driven (cid:4) . Novices tended to use more case - driven analogies (cid:3) analo - gies where a speci\ufb01c concrete example was used to develop a new solution (cid:1) 29 (cid:2)(cid:4) rather than schema - driven analogies (cid:3) more general design solution derived from a number of examples (cid:1) 53 , 54 (cid:2)(cid:4) . This difference can be explained because novices have more dif\ufb01culty retrieving relevant information when needed and have more dif\ufb01 - culty mapping concepts from disparate domains due to a lack of experience (cid:1) 55 (cid:2) . Tseng et al . (cid:1) 17 (cid:2) found that the effectiveness of analogical in - spiration in design was dependent on the timing of when the in - spiring information is given , as well as how apparently similar the information is to the problem being solved . More speci\ufb01cally , information that shares similar keywords or domains can be ap - plied to problem solving , even if the information is given before the designer has begun work on the problem , while information that is relevant but does not share similarity of keywords or do - mains only affects problem solving when the designer has already begun work on the problem . Additionally , Dahl and Moreau (cid:1) 56 (cid:2) demonstrated that subjects exposed to within - domain examples employed fewer far - domain analogies in generating solutions , and that the originality of the solutions produced was increased when subjects were encouraged to use analogies extensively and given no example solutions . Marsh et al . (cid:1) 57 (cid:2) found that within - domain examples caused sub - jects to be biased toward generating solutions that had similar features to those found in the examples . These \ufb01ndings led to the expectation for our experiment that a within - domain example so - lution given to participants prior to ideation would cause design \ufb01xation , as exhibited through fewer solutions generated and the appearance of features from the example in the solutions gener - ated . 2 . 1 . 1 Breaking or Mitigating Fixation : Formal Design - by - Analogy Methods . Analogy is a likely candidate for alleviating design \ufb01xation . A few formal methods have been developed to support design - by - analogy . These include Synectics (cid:1) 58 (cid:2) , French\u2019s work on inspiration from nature (cid:1) 59 , 60 (cid:2) , Biomimetic concept gen - eration (cid:1) 26 , 27 (cid:2) , the WordTree Design - by - Analogy method (cid:1) 20 , 61 (cid:2) , and analogous design through the use of the function and \ufb02ow basis (cid:1) 24 (cid:2) . Synectics is a group idea generation method that uses four types of analogies to solve problems : personal (cid:3) be the problem (cid:4) , direct (cid:3) functional or natural (cid:4) , symbolic , and fantasy (cid:1) 58 (cid:2) . Synectics gives little guidance to designers about how to \ufb01nd successful analogies . Other methods also base analogies on the natural world . French (cid:1) 59 , 60 (cid:2) highlights powerful examples that nature provides for design . Biomimetic concept generation pro - vides a systematic tool to index biological phenomena (cid:1) 26 , 62 (cid:2) . In biomimetic concept generation , the functional requirements of the problem and the keywords are \ufb01rst derived . The keywords are then referenced and relevant entries can be found . Like biomi - metic concept generation , the WordTree method is also based on keywords . The WordTree method takes the key functions or cus - tomer needs of a design problem , and systematically rerepresents them through the intuitive knowledge of the designers and through the WordNet database , particularly with synonyms known as hypernyms and troponyms . Analogous concepts can be also identi\ufb01ed by creating abstracted functional models of concepts and comparing the similarities between their functionality . Analo - gous and nonobvious products can be explored using the func - tional and \ufb02ow basis (cid:1) 24 (cid:2) . Other database supported computation tools for design - by - analogy have been recently developed . Examples of such tools are the work by Kurtoglu and Campbell (cid:1) 63 (cid:2) , Chui and Shu (cid:1) 26 (cid:2) , and Chakrabarti et al . (cid:1) 64 (cid:2) . Each has created an automated tool to provide inspiration to designers as part of the idea generation process . Based on the function or behavior of a device , analogies from nature or other devices are provided as potential sources of inspiration to the designer . 2 . 2 The Perception of Being Fixated . One reason why de - sign \ufb01xation is dif\ufb01cult to overcome is that designers are often not conscious of the fact that they are \ufb01xated . Ward and co - workers (cid:1) 45 , 65 (cid:2) found that the examples were not constraining the subjects consciously by causing them to believe that they should produce solutions similar to the given examples , but rather subconsciously constraining them ; when participants were asked to avoid produc - ing solutions that were similar to the examples , the similarity between the examples and generated solutions did not signi\ufb01 - cantly decrease when compared with participants\u2019 solutions who were not told to avoid solutions similar to the given examples . In general , participants did not have control over their use of the knowledge gained from the examples . These results suggest that designers are unaware that they are being in\ufb02uenced by example solutions or previously generated solutions (cid:1) 44 , 45 , 65 (cid:2) . Consistent 041003 - 2 / Vol . 132 , APRIL 2010 Transactions of the ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm with this \ufb01nding , Linsey et al . (cid:1) 19 (cid:2) also observed that engineers were unaware that they were implementing prior examples to which they had been exposed . 2 . 3 Summary and Breaking New Ground . It is clear from this literature review that experiments have been deployed for studying \ufb01xation and the underlying causes within designers . We build on these studies and the associated results in this paper . Mitigating strategies for alleviating \ufb01xation have been studied , but much fertile ground has yet to be explored , especially in the do - main of engineering and for the use of analogies . It is also clear , to the best of our knowledge , that academics from engineering de - sign research have not been a focal group as part of \ufb01xation stud - ies . Our cognitive - based study here addresses these limitations , and constructs a framework on the foundation of the previous research . 3 Research Questions Design \ufb01xation is a common problem for both inexperienced and experienced designers . In this study , we seek to answer three fundamental research questions : (cid:3) 1 (cid:4) Do engineering academicians , both design researchers and educators , experience design \ufb01xation ? (cid:3) 2 (cid:4) How can design \ufb01xation be overcome or mitigated ? (cid:3) 3 (cid:4) Do the participants accurately perceive the effects of provided example solutions and of the materials to mitigate design \ufb01xation ? These three research questions , our associated hypotheses , and our mo - tivation for answering these questions are discussed in the follow - ing sections . In our study , three experimental conditions are implemented : a control , a \ufb01xation condition in which a negative example solution is presented , and a de\ufb01xation condition in which the negative example solution is given along with a list of possible solution directions to consider ; these conditions are referred to in presenting our hypotheses and de\ufb01ned in Sec . 4 . In addition , all participants \ufb01lled out a survey prior to the workshop that obtained demographic information and perceptions about the design pro - cess . 3 . 1 Evidence of Design Fixation . For this study , we explore the effects of \ufb01xation on experienced academic engineering de - signers . The group of participants for this experiment has a unique background that makes them interesting to study . All of the par - ticipants attended a workshop on the cognitive aspects of engi - neering design and developing cognitive experiments in engineer - ing design . They have clear interest in design and cognition . In addition , most of this group has experience teaching design and most are researchers in design . Therefore , this group is aware of design methods , they have spent time thinking about many of the issues related to design , particularly the \u201cfuzzy - front end , \u201d and they are likely to be aware of some of the dif\ufb01culties that design - ers have during idea generation . They are also aware of methods such as design - by - analogy and some of the short - comings of tra - ditional group brainstorming . Overcoming design \ufb01xation is a dif - \ufb01cult task . Yet , because of this group\u2019s background in design theory and methods , with their knowledge and skills , might they be able to more effectively overcome design \ufb01xation ? We there - fore seek to answer the following research question and contem - plate the following hypothesis : Research Question 1 : Do academic engineering design educa - tors show evidence of design \ufb01xation ? Hypothesis 1 : Academic engineering design educators will show evidence of design \ufb01xation . They will produce fewer total ideas when provided with example solutions and repeat ideas from provided examples as compared with the control group . 3 . 2 Overcoming / Mitigating Design Fixation . Prior research has shown that it is possible to reduce design \ufb01xation by instruct - ing participants to not focus on the negative aspects of the design and describing those short - comings (cid:1) 48 (cid:2) . This is clearly one ap - proach for reducing \ufb01xation , but based on anecdotal commentary in the design literature , it is likely that there are other approaches to mitigating \ufb01xation . Many product design books describe the bene\ufb01ts of functions , analogies , categories , and back - of - the - envelope calculations in the design process (cid:1) 66 \u2013 69 (cid:2) . In addition , analogy is noted as a tool for innovative design and a proli\ufb01cally implemented strategy by designers (cid:1) 9 , 19 , 70 (cid:2) . These observations lead to the following research question and hypothesis : Research Question 2 : What can engineers do to reduce their \ufb01xation on particular design solutions ? Can analogies , functions , categories of energy sources , and back - of - the - envelope calcula - tions assist in overcoming design \ufb01xation ? Hypothesis 2 : Design \ufb01xation can be reduced . The de\ufb01xation group will produce more ideas and repeat fewer ideas from the provided example solution than the \ufb01xation group . The de\ufb01xation group will implement more analogies than the other two condi - tions . 3 . 3 Participant Perceptions . Participants\u2019 perceptions fre - quently are not consistent with quantitative outcomes of their per - formance (cid:1) 37 , 44 , 45 (cid:2) . Unfortunately , perceptions are easily obtain - able and may be the basis that an individual or a company uses to choose to implement a particular method . For example , one of the reasons for group brainstorming\u2019s popularity , in spite of numerous studies contradicting its purported effectiveness , is that individu - als feel more productive during group brainstorming than when generating ideas alone (cid:1) 37 (cid:2) . In contrast to the participant\u2019s percep - tions of productivity , numerous studies quantitatively demonstrate a reduction in the number of ideas per person when comparing brainstorming in a group to individual brainstorming (cid:3) see Ref . (cid:1) 71 (cid:2) for a review (cid:4) . The basic group brainstorming method must be adapted to produce the quantity of results greater than the sum of the individuals (cid:1) 64 (cid:2) . The group of participants in this study has experience with design methods and is at least somewhat familiar with their short - comings . In addition , the majority of this group has taught design classes and observed their students\u2019 performance . Therefore , it is likely that the participants in this study will be much more aware of the effects of a provided example solution and additional de - \ufb01xation materials on their performance than participants who do not study design . In contrast , the prior literature indicates that participants are likely to inaccurately perceive the effects of an introduced example solution and the associated de\ufb01xation materi - als , if they are introduced . Therefore , we seek to answer the fol - lowing research question and test the related hypothesis : Research Question 3 : How well do participant perceptions of design results correspond to quantitative assessment of the results ? Hypothesis 3 : Participants will inaccurately perceive the effects of a provided example solution and associated de\ufb01xation materi - als . Results from survey questions collecting the participants\u2019 per - ceptions will be inconsistent with the quantitative metrics . 4 Experimental Method The experiment evaluates the effects of \ufb01xation on experienced academic engineering designers . To answer the research questions and hypotheses , we implement three experimental conditions : a control , a \ufb01xation , and a de\ufb01xation condition . All participants are given the same experimental procedure and documentation media . Participants in the \ufb01xation condition are provided with an ex - ample solution . Participants in the de\ufb01xation condition are also provided with the same example solution , but also with additional materials to potentially break or mitigate the design \ufb01xation (cid:3) de - tailed below in Sec . 4 . 4 (cid:4) . All participants are told that the goal of the experiment is to generate as many solutions to the design problem as possible , where a prize will be given to participants with the greatest number of solutions . This prize is an incentive for participants to devote serious effort to the design activity . All conditions end with a short post - experiment survey , which mea - sures prior exposure to the design problem , perceptions of partici - pants\u2019 performance and perceived in\ufb02uence of a provided example Journal of Mechanical Design APRIL 2010 , Vol . 132 / 041003 - 3 Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm solution . In the case of the de\ufb01xation condition , participations are asked questions regarding the de\ufb01xation materials and their per - ceptions of these materials . 4 . 1 Description of the Design Problem . All participants are provided with the same design problem . The design problem is to design a device to quickly shell peanuts for use in places like Haiti and West African countries , and is based on a real - world problem posted on ThinkCycle (cid:1) 72 (cid:2) . Participants are told that no electrical energy sources are available and are given customer needs (cid:3) Fig . 1 (cid:4) . This problem is chosen because it is a real world problem that is appropriate for an engineer , it has intrinsic incentive for solu - tions given its need - based nature , and the problem has a diverse set of available solutions . This problem has also been used in previous research on idea generation (cid:1) 35 , 73 , 74 (cid:2) . It is very unlikely that any of the participants would have extensive prior experience in solving this problem , yet shelling a peanut is a task that all of the participants have likely experienced . 4 . 2 Control Group . The control group is given the design problem as stated in Fig . 1 . They are not provided with an ex - ample solution or alternative representation of the problem . 4 . 3 Experimental Fixation Group . The \ufb01xation group is given the design problem as stated in Fig . 1 and an additional negative solution example (cid:3) Fig . 2 (cid:4) . They are not given an alterna - tive representation of the problem . The example solution uses a gasoline powered press to crush the shell , and does not separate the nut from the shell . The example solution focuses exclusively on a mechanical concept that crushes the shell and uses external fuel energy . This solution is dif\ufb01cult to control in terms of dam - aging the peanut , complex , and costly to manufacture for the West African environment . The participants all have graduate degrees in engineering , so these short - comings should be obvious to them . In addition , these particular process solutions are many of the common solutions found by participants in a prior experiment (cid:1) 35 , 73 (cid:2) . Common solutions to design problems were shown to create greater \ufb01xation (cid:3) fewer total solutions (cid:4) than unusual solu - tions (cid:1) 14 , 75 (cid:2) . 4 . 4 Experimental De\ufb01xation Group . The de\ufb01xation group is presented with the design problem , as in Fig . 1 , and also alterna - tive representations of the problem (cid:3) Fig . 3 (cid:4) . The alternative rep - resentations provide a brief functional description , useful analo - gies , a list of available energy sources , and a quick back - of - the - envelope calculation result . Some of the analogies were identi\ufb01ed using the WordTree Design - by - Analogy method with the key word of \u201cremove\u201d and \u201cshell\u201d to \ufb01nd the associated hypernyms and troponyms from WordNet (cid:1) 20 (cid:2) . 4 . 5 Participants . Fifty engineering academics expressed in - terest in attending the NSF sponsored workshop : \u201cDiscussion on Individual and Team - Based Innovation . \u201d 38 from this group \ufb01lled out the online presurvey for the workshop , and 34 actually at - tended the workshop . These thirty - four participants are randomly assigned to one of three conditions prior to the workshop , equally distributing the senior (cid:3) associate and full professors (cid:4) and junior faculty (cid:3) assistant professors (cid:4) . The study serves to demonstrate to the workshop participants an example cognitive study in engineer - ing design , while at the same time , providing useful experimental data . Based on the preworkshop survey results , which are only partially presented here , participants are faculty members (cid:3) 85 % (cid:4) , plus a few research scientists and graduate students (cid:3) 12 % (cid:4) , and federal government employees (cid:3) 3 % (cid:4) . There were no participants from industry . Almost half the participants are assistant professors (cid:3) 45 % (cid:4) ; 12 % are associate professors and 27 % are full professors . Most participants have mechanical engineering backgrounds . Most have at least 1 year of industrial experience (cid:3) 64 % (cid:4) and have consulted with industry at least once (cid:3) 79 % (cid:4) . There is also a high representation of women relative to the \ufb01eld of engineering (cid:3) 33 % females (cid:4) . A number of preregistered intended participants did not attend the beginning of the workshop so three participants were switched to different groups to compensate . Unintentionally , they were switched from the de\ufb01xation condition to a different condition , and they therefore had brie\ufb02y seen the de\ufb01xation materials . These three participants were removed from the data set . Fig . 1 Design problem description Fig . 2 Example solution provided to the participants in the \ufb01xation group 041003 - 4 / Vol . 132 , APRIL 2010 Transactions of the ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm 5 Metrics To understand the effects of design \ufb01xation and evaluate the research questions , a set of measures are employed . To quantify the degree of \ufb01xation , \ufb01ve metrics are implemented : (cid:3) 1 (cid:4) number of ideas , (cid:3) 2 (cid:4) number of times features from the example solution appear in generated concepts , (cid:3) 3 (cid:4) percentage of features from the example solution that appear at least once in participant solutions , (cid:3) 4 (cid:4) number of energy domains , and (cid:3) 5 (cid:4) percentage of the solutions that employ a gas engine . To evaluate the effects of providing de\ufb01xation materials , along at least one dimension , the number of analogies is also measured . To provide inter - rater reliability , one of the authors evaluated all of the data for each metric , while a second rater evaluated two from each condition or at least 18 % of the data . 5 . 1 Quantity of Ideas . Building from the procedure devel - oped by Shah et al . (cid:1) 76 (cid:2) , a set of procedural rules are de\ufb01ned for what constitutes a single idea , see Ref . (cid:1) 77 (cid:2) for more details . Our basic de\ufb01nition for an idea is something that solves one or more functions of the design , as de\ufb01ned by the functional basis (cid:3) a clearly de\ufb01ned and tested language for expressing design func - tions (cid:1) 78 , 79 (cid:2)(cid:4) . The total number of unique (cid:3) nonredundant , nonre - peated (cid:4) ideas is calculated for each person . Pearson\u2019s correlation coef\ufb01cient (cid:1) 80 (cid:2) was 0 . 97 , indicating that the measure is highly reliable . A high degree of Pearson\u2019s correlation indicates that if one evaluator had a higher score , then the other evaluator also tended to give a higher score . 5 . 2 Repeated Example Solution Features and Percentage of Features Used . Figure 4 illustrates the example solution pro - vided to the participants and all of the design ideas contained within it (cid:3) number of ideas (cid:4) , categorized by function . The number of times each participant employs one of the design features from the example solution is counted . This procedure results in two metrics , which indicate the degree of \ufb01xation : the total number of times a feature is repeated and the total percentage of features from the example that is implemented at least once . One of the authors evaluated all of the data , while a second rater measured two from each condition or 18 % of the data . In half of the cases , Fig . 3 De\ufb01xation materials Fig . 4 Example solution provided to participants Journal of Mechanical Design APRIL 2010 , Vol . 132 / 041003 - 5 Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm the two raters had identical scores for the number of repeated features , and their Pearson\u2019s correlation coef\ufb01cient (cid:1) 80 (cid:2) was 0 . 97 , indicating that the measure is highly reliable . Cohen\u2019s Kappa (cid:1) 80 (cid:2) is not used since the metric of interest is the relative number of repeated features across conditions , not whether or not the partici - pants use a particular feature or the absolute number of repeated features in this setting . To calculate the percentage of the features that each participant reused from the example , the number of fea - tures used at least once is divided by the total number of ideas within the example solution (cid:3) eight ideas , Fig . 4 (cid:4) . 5 . 3 Energy Domains and Percentage of Solutions Employ - ing a Gas Engine . In addition to recording the quantity of ideas , the number of energy sources used by each participant is analyzed (cid:3) Table 1 (cid:4) . These energy sources are categorized into 16 energy categories (cid:3) wind , solar , water streams , captured rain water at a height , water (cid:3) other (cid:4) , human , animal , nuclear , electrical outlet , \ufb01re , gas engine , engine (cid:3) other (cid:4) , fuel cell , \ufb02uid density difference , chemical , and genetic (cid:4) . The original tally included 18 categories , but it was found , due to the universality of gravity that the two gravity driven categories are dif\ufb01cult to measure reliably between raters , and were thus removed . The total of all energy sources used by each participant is recorded . Since the goal is to deter - mine the breath of energy sources spanned , a participant receives the same score , regardless of whether they use an energy source once or multiple times . Since the de\ufb01xation materials provided a list of energy sources to directly break \ufb01xation on the gas engine , the percentage of solutions using a gas engine is also calculated . 5 . 4 Analogies - Breaking Fixation . After 45 min of ideation , analogies were identi\ufb01ed by the participants by going back through their solutions , and circling or labeling any analogies used with a red permanent marker . The total number of unique analo - gies identi\ufb01ed by each participant is measured . Some participants circled components of their designs , some drew arrows with tex - tual descriptions , and others described the analogies they used while also circling them . Initially , all of this information is tabu - lated . If a participant circled something and wrote no textual de - scription , this analogy is counted for that participant toward the total number of analogies they used . If a participant wrote a de - scription , the analogy is counted for that participant toward their total number of analogies . If the participant both circles and writes text , it is determined if the two pertained to separate analogies . One participant stated that they believed all of their ideas were analogies since all their ideas came from things they had seen before . Although this statement that most of an engineers\u2019 design ideas come from prior exposure is true , this is not a working use of the concept of analogy , where a speci\ufb01c idea is borrowed or mapped onto the current problem ; thus , this participant\u2019s data are not included in the analogy analysis . Due to the fact that each participant\u2019s de\ufb01nition of \u201canalogy\u201d is unique , the analogies identi\ufb01ed by the participant are counted as valid analogies . This reduces the amount of biases introduced by the researchers , but there is still subjectivity due to differing par - ticipants\u2019 de\ufb01nitions . However , the participants\u2019 de\ufb01nitions of analogy , as provided in the presurvey , are surprisingly consistent across the participants . Prior studies have used indication of analogies such as \u201cdevice X is like device Y\u201d to identify analogies in concurrent think - aloud protocols or recorded team conversa - tions (cid:1) 9 , 23 , 52 (cid:2) . This study did not include concurrent think - aloud descriptions as the participants were working , nor was there any other indication of which ideas were based on analogies , there - fore , the participants\u2019 identi\ufb01cation of analogy had to be used . Since participants did not provide details on the analogies , it was not possible to further classify analogies along common dimen - sions such as analogical distance . In tabulating the analogies metric , as with all other metrics , an inter - rater agreement analysis is performed to ensure objectivity and consistency . In tallying the total number of analogies remov - ing any repeats , the raters achieved 99 % agreement and a Pear - son\u2019s Correlation of 0 . 99 . These measures indicate that the metric is highly reliable , and there is strong consistency between the two evaluators . 6 Results : Design Fixation A key outcome of this study is on understanding design \ufb01xa - tion , participants\u2019 perception of it and how to break or mitigate \ufb01xation when it occurs . Figures 5 and 6 illustrate examples of typical participant results with high and low degrees of \ufb01xation (cid:3) samples sizes are in Table 2 (cid:4) . Four measures are implemented to assess each participant\u2019s degree of \ufb01xation . From these measures , a participants\u2019 \ufb01xation may be ascertained and the hypotheses tested . The number of nonredundant ideas varies across the three con - ditions (cid:3) Fig . 7 (cid:4) . An ANOVA (cid:3) analysis of variance (cid:4) shows a statis - tically signi\ufb01cant effect across the \ufb01xation conditions (cid:3) F = 3 . 7 , p Table 1 Energy source categories Wind Anything powered by naturally occurring wind . Includes using a wind generator to generate electricity , a windmill to directly turn a mechanism , and naturally allowing the wind to blow peanut shells away . Does not include a fan or other wind source powered by another energy source . Solar Anything powered by the sun . Includes both solar panels to generate electricity and using the sun directly to heat water or roast the peanuts . Water streams Uses naturally existing water streams such as a water wheel in a river to power the system , either by generating electricity or directly powering the mechanical system . Captured rain water at a height Capturing rain water at a height to accomplish much the same as the previous category . Water (cid:3) other (cid:4) Can include the soaking of peanuts to soften them , and many other uses . Human Includes manual shelling , turning a hand crank , and any other human power . Animal Includes using animals to motivate a mechanical system , training or genetically manipulating animals to shell peanuts . Nuclear Nuclear power station to provide energy . Electrical outlet Any solution where the concept involves plugging into a wall socket for electricity . Fire Anything that uses a \ufb01re to generate power , excludes internal combustion or steam engines , but includes using a \ufb01re to roast peanuts or burn off shells . Gas engine Gasoline powered internal combustion engine . Engine (cid:3) other (cid:4) Any other type of fuel engine . Fuel cell Hydrogen or other type fuel cell . Fluid density difference Concepts based on whether something \ufb02oats or sinks , often used to sort peanuts and their shells . Chemical Any chemical process to generate power that is different than seen above , or to burn or dissolve the peanut shell off . Genetic Genetically altering the peanut itself . Does not include genetic manipulations on animals (cid:3) which should be categorized as animal (cid:4) . 041003 - 6 / Vol . 132 , APRIL 2010 Transactions of the ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm (cid:1) 0 . 04 (cid:4) . 1 A t - test shows that the control group produces more ideas than the \ufb01xation group (cid:3) t = 2 . 94 , p (cid:1) 0 . 02 (cid:4) . The other pair - wise comparisons are not statistically signi\ufb01cant . The variation in the number of nonredundant ideas indicates that the example did cause design \ufb01xation , resulting in fewer ideas being generated . The trend in this data is that the de\ufb01xation group produces more ideas on average than the \ufb01xation group , indicating that the additional materials assist in reducing their \ufb01xation . 6 . 1 Number of Example Solution Features Used . Another indicator of the degree of \ufb01xation is the number of times that the participants reuse features from the provided example solution . This metric differs across the three conditions and ranges from 1 to 43 repeated features (cid:3) Fig . 8 (cid:4) . The control group did not see the example solution , but they still may think of the same features that are present in the example solution . These data do not satisfy the assumptions for a standard ANOVA since Shapiro \u2013 Wilk\u2019s test of normality shows the data are not normally distributed and Lev - ene\u2019s test for equality of variances shows that the variances are not homogenous ; therefore , a Kruskal \u2013 Wallis ANOVA is imple - mented instead . ANOVA can be used when there are only small departures from normality , but if there are also unequal variances across the groups , a different approach is required . A Kruskal \u2013 Wallis ANOVA is analogous to a standard ANOVA , but is the nonparametric statistical equivalent and evaluates the relative ranks of the data points . Implementing a Kruskal \u2013 Wallis ANOVA , there is a statistically signi\ufb01cant difference across the three con - ditions (cid:3) H = 7 . 3 , df = 2 , p = 0 . 03 , N = 31 (cid:4) . 1 The data are not normally distributed but ANOVA is robust for departures from normality . The remaining assumptions for ANOVA are met . To con\ufb01rm the ANOVA results , a Kruskal \u2013 Wallis ANOVA is also completed on the data (cid:3) H = 5 . 7 , p (cid:1) 0 . 06 (cid:4) . Table 2 Sample size for each condition Group Sample Size Control 9 Fixation 12 De\ufb01xation 10 Fig . 5 A set of solutions showing a low degree of \ufb01xation on the provided example solution Fig . 6 A set of solutions showing a high degree of \ufb01xation on the provided example solution Fig . 7 The \ufb01xation group produced fewer ideas , on average , than either the control group or the de\ufb01xation group . Each er - ror bar is \u00b11 standard error . Journal of Mechanical Design APRIL 2010 , Vol . 132 / 041003 - 7 Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm The \u201cnumber of features\u201d data also indicate that the example solution caused \ufb01xation , as the features from the example (cid:3) the \ufb01xation condition (cid:4) are reused more often than for the control . This data indicate that the additional materials are effective in mitigat - ing or reducing the design \ufb01xations , since the de\ufb01xation condition reuses fewer features from the example solution than the \ufb01xation condition . In the case of the control group , participants generated features in their concepts corresponding to the example solution . This result is to be expected since some of the features , as shown in Fig . 4 , are perfectly acceptable solutions , whereas others , such as the gas - press are not . The control group , however , did not overly use the solutions of the provided example for the \ufb01xation condition , whereas the \ufb01xation group did offer solutions powered by gasoline or similar fuel - based systems . 6 . 2 Percentage of Features Used . The three conditions also caused the participants to implement different percentages of the features from the example solution (cid:3) Fig . 9 (cid:4) , another indicator of \ufb01xation . The number of times that the participants implemented one of the features from the example solution was counted . Again , these data do not satisfy the assumptions for a standard ANOVA . The Shapiro \u2013 Wilk\u2019s test of normality shows that the data is not normally distributed , and Levene\u2019s test for equality of variances shows that the variances are not homogenous ; therefore , a Kruskal \u2013 Wallis ANOVA is completed instead . There is a statisti - cally signi\ufb01cant difference across the three conditions (cid:3) H = 7 . 3 , df = 2 , p = 0 . 03 , N = 31 (cid:4) . A Wilcoxon\u2019s Rank - sum test shows that the \ufb01xation group incorporated more of the features from the ex - ample in their solutions than the de\ufb01xation group (cid:3) W s = 85 . 5 , n 1 = 10 , n 2 = 12 , p = 0 . 08 (cid:4) and the control (cid:3) W s = 67 . 5 , n 1 = 9 , n 2 = 12 , p = 0 . 02 (cid:4) . Again the \u201cpercentage of features\u201d measure shows that the ex - ample solution causes designer \ufb01xation . The de\ufb01xation condition is different from the \ufb01xation condition , based on statistical signi\ufb01 - cance , again showing that the de\ufb01xation materials are assisting the designers in overcoming the \ufb01xation induced by the presented example . 7 Results : Energy Sources Fixation In addition to the number of solutions , the energy source used in the design can be another indicator of design \ufb01xation . The de\ufb01xation condition contained a categorical list of energy sources , Fig . 3 . Participants in the \ufb01xation condition were given an example that is powered by a gas engine . It is expected that this example would \ufb01xate individuals on using a gas engine . Participants in this condition were given information in addition to the gas engine powered example that is intended to aid in breaking the induced \ufb01xation . Individuals in the control condition were given no ex - ample on which to \ufb01xate . Both predicted effects are observed in the results (cid:3) Fig . 10 (cid:4) . Again , these data do not meet the assump - tions for a standard ANOVA , as the data are not normally distrib - uted and the variances are not homogenous . The Kruskal \u2013 Wallis ANOVA compares the data based on the relative rank of the re - sults , but this approach is not accurate when there are a large number of equal outcomes as there are with this data set . For this data set , one - way ANOVA via randomization is implemented (cid:1) 81 , 82 (cid:2) . This approach is analogous to the other approaches , but does not make any assumptions about the distribution or the rank - ing of the data . Based on the graphical results shown in Fig . 10 , the \ufb01xation group is clearly different and distinct from the other two groups . One might expect the other two conditions not to use gas engines at all , but in this study , gas engines were occasionally used . Using a one - way ANOVA via randomization , there is statistically signi\ufb01 - cant difference across the groups (cid:3) p = 0 . 05 (cid:4) (cid:1) 83 , 84 (cid:2) . The \ufb01xation group produced a larger percentage of gas powered designs than the control group , indicating that the example solution caused \ufb01xation . The \ufb01xation group also showed a strong trend for pro - ducing a larger percentage of gas powered designs than the de\ufb01x - ation group (cid:3) t = 1 . 97 , p (cid:1) 0 . 08 (cid:4) , suggesting that the de\ufb01xation in - formation is effective in breaking or mitigating the induced \ufb01xation . Similar to the other measures of design \ufb01xation , the re - sults show that \ufb01xation is occurring , and the de\ufb01xation materials are having a statistically signi\ufb01cant impact . The total number of energy sources used in all stages of peanut shelling differed across the three conditions (cid:3) Fig . 11 (cid:4) . Again , these data do not satisfy the assumptions for a standard ANOVA (cid:3) data are not normally distributed and the variances are not homog - enous (cid:4) so a Kruskal \u2013 Wallis ANOVA is implemented . There is not a statistically signi\ufb01cant difference across the three conditions Fig . 8 The \ufb01xation group repeated , on average , features from the example solution more often than the other two groups . Each error bar is \u00b11 standard error . Fig . 9 The \ufb01xation group used , on average , a higher percent - age of the features from the example solution in their concepts . Each error bar is \u00b11 standard error . Fig . 10 The de\ufb01xation group produced more designs powered by a gas engine than individuals in the other conditions . The error bars are \u00b11 standard error . 041003 - 8 / Vol . 132 , APRIL 2010 Transactions of the ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm (cid:3) H = 3 . 28 , df = 2 , p = 0 . 194 , N = 31 (cid:4) . However , the \ufb01xation condi - tion produced , on average , fewer energy sources than the de\ufb01x - ation condition (cid:3) Wilcoxon\u2019s Rank - sum test , W s = 112 . 5 , n 1 = 10 , n 2 = 12 , p = 0 . 09 (cid:4) , suggesting that the de\ufb01xation materials are ef - fective in breaking the induced \ufb01xation . The other pair - wise com - parisons are not statistically signi\ufb01cant . Consistent with other results from the study , these results sug - gest that the de\ufb01xation materials are assisting in mitigating or reducing design \ufb01xation . The additional categories of available energy sources are assisting the designers in identifying solutions . 8 General Fixation Results Discussion The various measures related to \ufb01xation clearly illustrate that the example solution causes design \ufb01xation . This result is shown by the lower number of ideas generated , by a higher number of features from the example being used in the solutions , and by fewer energy categories being implemented in the participants\u2019 concepts . This \ufb01xation is of particular interest since these partici - pants are not novice designers . All participants have the required domain knowledge to identify short - comings in the presented ex - ample solution . Design \ufb01xation is experienced by the engineering design faculty . Providing participants with analogies and rerepresentations of the problem through categories did assist in reducing their \ufb01xation on the example solution , but it did not completely eliminate it . Participants in the control group still outperformed both the \ufb01xa - tion and the de\ufb01xation group in total number of concepts , whereas the de\ufb01xation group employed a greater diversity of energy - based solutions . 9 Results : Analogies Many designers employ analogies to inspire solutions to a given problem . The focal metric to consider in this study when examining analogies is simply the quantity identi\ufb01ed by the par - ticipants across the three conditions . The analogies were identi\ufb01ed by the participants after the 45 min of ideation by revisiting their solutions , circling and labeling any analogies used with a red marker . The number of reported analogies employed did not vary sta - tistically across the conditions (cid:3) Fig . 12 (cid:4) . Again , the data are non - normally distributed with unequal variances , requiring nonpara - metric tests . A Kruskal \u2013 Wallis ANOVA shows there is a not statistically signi\ufb01cant difference across the three conditions (cid:3) H = 1 . 4 , df = 2 , p = 0 . 5 , N = 30 (cid:4) . The de\ufb01xation group did , however , use slightly more analogies , on average , implementing many of the analogies provided in the de\ufb01xation materials in a variety of ways . 9 . 1 Analogies Results Discussion . The three conditions implemented in this study produced distinct levels of analogy use . The \ufb01xation group and the control have a similar level of analogy use . However , the de\ufb01xation group , given both the example solu - tion and the extra information , used slightly more analogies , on average , than that given just the example solution . This could be a result of the \ufb01xation caused by the example solution , or further indication that more supplemental information is better for a broader search of the design space . Many of the analogies used by the group given extra information were directly inspired by the key word list supplied in the materials . Future work , with more participants per condition , should investigate whether the ob - served trend replicates . An important obstacle to note is the subjectivity of identifying analogies . In order to remove the experimenters\u2019 interpretations and biases , a red marker was given to each participant after the ideation session , with which they were to circle and label any analogies used . This removed subjectivity from the experiment - ers\u2019 perspectives , however , not from the participant\u2019s perspective . Many participants showed signs of a broader understanding of the de\ufb01nition of analogy , often times circling mechanisms or pro - cesses that were taken directly from industry , for example , press . Since the press was being used in an identical way to that which is used in practice , it is not legitimately considered an analogy for the purposes of this study , but more an application of a technol - ogy . Despite this discrepancy , all analogies identi\ufb01ed by partici - pants were counted as analogies because omitting these would create too much uncertainty in the analysis . 10 Results from Post - Experiment Survey A post - experiment survey measures a variety of items including the following : participants\u2019 opinions about the design problem ; perceptions about the effect of the example solution and the addi - tional material ; and if they had exposure to the design problem and its solutions prior to the experiment . These measures serve to provide further insights and validity to the experimental results . The participants\u2019 overall opinions of the design problem and activity indicate that , on average , they found it interesting and seriously committed themselves to the task . The participants , on a semantic difference scale , felt that they worked hard on the activ - ity (cid:3) mean (cid:3) SD (cid:4) : 1 . 8 (cid:3) 0 . 90 (cid:4) 1 = worked hard , 5 = minimal effort (cid:4) and found the activity to be somewhat motivating but not inspiring (cid:3) mean (cid:3) SD (cid:4) : 2 . 2 (cid:3) 0 . 86 (cid:4) (cid:3) 1 = motivating and 5 = demotivating (cid:4) and 2 . 6 (cid:3) 0 . 78 (cid:4) (cid:3) 1 = inspiring and 5 = frustrating (cid:4)(cid:4) . None of these results show a mean shifted substantially to the right of the scales mid - point . For the validity of this study , it is important that the partici - pants were motivated and put in a substantial effort since in a more realistic design setting , it is expected that engineers are gen - erally well motivated to solve a given design problem . Since the presented design problem is an actual existing issue , it is possible that the participants may have had prior exposure to Fig . 11 The de\ufb01xation group used , on average , more energy sources in total than participants in the other two groups . Each error bar is \u00b11 standard error . Fig . 12 All groups used analogies in developing solutions to the design problem . The de\ufb01xation group , on average , em - ployed slightly more analogies during ideation . The error bars are \u00b11 standard error . Journal of Mechanical Design APRIL 2010 , Vol . 132 / 041003 - 9 Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm the problem and to the solutions . A total of seven participants , evenly distributed across the conditions , had prior exposure to the design problem , with four of them also having exposure to solu - tions to the design problem . These four participants believed their prior exposure had an insigni\ufb01cant to only some in\ufb02uence on their results (cid:3) the four responses were 1\u2014insigni\ufb01cant , 2\u2014minor , and 1\u2014some in\ufb02uence (cid:4) . Due to the low level of prior exposure and that the participants were evenly distributed across the conditions , we do not believe that the prior exposure affects the results of this experiment . The key outcome of the survey is the participants\u2019 perception about their performance . The \ufb01xation and the de\ufb01xation groups were asked , using a Likert scale question , if they felt the provided example solution had in\ufb02uenced them , and then if it had posi - tively or negatively in\ufb02uenced them (cid:3) Figs . 13 \u2013 15 , error bars are 1 standard error (cid:4) . Both groups felt that the provided example solu - tion had in\ufb02uenced them . The participants are recognizing the fact that they are being in\ufb02uenced by the provided design example . The \ufb01xation group tended to believe that the effect of the ex - ample solution is positive whereas the de\ufb01xation group is unsure of the in\ufb02uence . The differences between the groups are not sta - tistically signi\ufb01cant , although this lack of statistical signi\ufb01cance may re\ufb02ect the moderate group sizes of this study . The partici - pants\u2019 perceptions are in contrast to the quantitative \ufb01xation re - sults that indicate that the example is having a strong negative effect on the \ufb01xation group , meaning that the designer may not be aware of the negative in\ufb02uence . In addition , the participants\u2019 perceptions of the effects of the de\ufb01xation materials were also measured (cid:3) Table 3 (cid:4) . In this case , participants\u2019 correctly believe that the additional information is bene\ufb01ting them with a mean of only agree to somewhat agree (cid:3) 2 . 7 (cid:4) and a fairly high standard deviation (cid:3) 1 . 33 (cid:4) . This high stan - dard deviation may indicate the some of the participants are more accurate in their perceptions . The quantitative results indicate that there is a very strong positive effect in overcoming the \ufb01xation due to the provided de\ufb01xation materials . While the participant perceptions are generally accurate , in this case , they do not strongly match the quantitative results . This indicates that partici - pant perceptions are not an accurate tool for evaluating design methods . 11 Addressing the Research Questions Research Question 1 : Do academic engineering design educa - tors show evidence of design \ufb01xation ? Academic engineering design educators do show evidence of design \ufb01xation . The \ufb01xation group produced fewer ideas , reused more of the features from the example solution , and implemented fewer categories of energy sources than the control group . Design \ufb01xation is evidenced by the presence of a considerable number of solution elements that are clearly not appropriate for the context of the design problem . This group of participants has a high de - gree of knowledge and can clearly recognize the short - comings of the presented design . Qualitatively compared with undergraduate students in a previous study (cid:1) 73 (cid:2) , the design educators produced a larger number of highly novel solutions not identi\ufb01ed by the stu - dents , providing some validation of their status as design experts . It should be noted , however , that the students did produce a range of novel ideas for the design problem . Question 2 : What can engineers do to mitigate their \ufb01xation on design solutions ? Can analogies , functions , categories of energy sources , and back - of - the - envelope calculations assist in overcom - ing design \ufb01xation ? Some of the strategies that may mitigate design \ufb01xation are analogies , a functional decomposition , rerepresentation of the problem , categories of solutions (cid:3) such as energy sources (cid:4) , and Table 3 Participant perceptions of the effect of the de\ufb01xation materials Survey question De\ufb01xation group mean (cid:3) SD (cid:4) The provided additional information (cid:3) functions , analogies , calculation , energy domains (cid:4) bene\ufb01ted me . 2 . 7 = agree / somewhat agree (cid:3) 1 . 33 (cid:4) The provided additional information (cid:3) functions , analogies , calculation , energy domains (cid:4) hindered me . 5 . 1 = somewhat disagree (cid:3) 1 . 45 (cid:4) Scale : 1 = strongly agree , 2 = agree , 3 = somewhat agree , 4 = neutral , 5 = somewhat disagree , 6 = disagree , 7 = strongly disagree Fig . 13 Participants believe they were in\ufb02uenced by the pro - vided example solution Fig . 14 Participants in the \ufb01xation and the de\ufb01xation group felt the example solution had a positive in\ufb02uence or at least were unsure that the in\ufb02uence was positive or not Fig . 15 Participants were undecided if the example solution negatively in\ufb02uenced them 041003 - 10 / Vol . 132 , APRIL 2010 Transactions of the ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm back - of - the envelope calculations . Results from this study clearly indicate that design \ufb01xation can be mitigated or reduced through these means . The de\ufb01xation group did produce more ideas , on average , than the \ufb01xation group . In addition , they repeated fewer features from the example solution and implemented a greater number of different energy categories than the \ufb01xation group . The results do not indicate which materials are most effective for de - \ufb01xation , but that this set , as a whole , is effective . Future studies will need to consider particular categories of de\ufb01xation materials and strategies . Unlike previous studies on design \ufb01xation , this study directed participants toward the use of analogy to break design \ufb01xation . Participants in past studies likely implemented analogies since analogy is a common and effective design strategy , but our study is much more literal about analogy use . Our study provides further but somewhat indirect support for the importance and impact of analogical reasoning in the design process . Additional research is needed to fully understand the type of information that eliminates design \ufb01xation , including the numer - ous representations currently existing in the design literature , and how these materials may be generated for novel design problems . Design methods currently exist for functional decomposition (cid:1) 63 , 66 , 79 (cid:2) and for analogies (cid:1) 26 , 62 , 64 , 74 , 85 (cid:2) . As more ap - proaches to reducing design \ufb01xation are discovered , new design methods will need to be developed to assist designers . Research Question 3 : How well do participant perceptions of design results correspond to quantitative assessment of the re - sults ? Consistent with other studies on idea generation (cid:1) 37 (cid:2) , partici - pants\u2019 perceptions of effectiveness during idea generation do not always match the quantitative outcome . Participants in the \ufb01xation group inaccurately believe , in general , that the example solution has a positive in\ufb02uence on their idea generation process , or they are unsure as to this in\ufb02uence . It is clear from the \ufb01xation results that the example reduces the number of ideas generated , but this is not perceived by the participants . The de\ufb01xation group felt that the example solution in\ufb02uenced them , but are not sure if it was positive or negative . The de\ufb01xation group also correctly perceived that they are assisted by the addi - tional information that is provided , but they do not feel strongly about this . By contrast , the solution data demonstrate that the de\ufb01xation group is strongly supported by the additional materials . These participant perception results strongly warn against their use as an accurate measurement of ideation effectiveness . 12 Conclusions This study evaluates design \ufb01xation , the use of mitigation strat - egies , and the perception of design \ufb01xation in a group of mostly engineering design faculty . Results show that design \ufb01xation is a dif\ufb01culty encountered even by this group , indicating the strength and importance of this effect in the design process . The partici - pants\u2019 perceptions of the effects of the provided \ufb01xation example solution and de\ufb01xation materials are generally not accurate , ex - cept with respect to de\ufb01xation materials . This result is not ex - pected for a group of individuals who study design . This incon - gruity in perception presents a unique obstacle to engineering design methods research since one of the simplest measures to obtain is the users\u2019 perceptions of the method\u2019s effectiveness . Based on this study and past work , the participants\u2019 evaluation of a method are frequently inconsistent with the quantitative mea - sures and not suitable for accurate evaluation or assessment . This study compared three groups of participants : (cid:3) 1 (cid:4) a control group , which only received the design problem , (cid:3) 2 (cid:4) a \ufb01xation group , which also was provided a negative example solution , and (cid:3) 3 (cid:4) a de\ufb01xation group , which , in addition to the negative solution , also received a set of materials to mitigate or reduce \ufb01xation . The example solution caused design \ufb01xation , as demonstrated by a reduction in the number of ideas generated , a greater number of design features from the example being reused and fewer catego - ries of energy sources considered . Consistent with prior studies , design \ufb01xation can be mitigated . The unique \ufb01xation mitigation materials , which included functions , energy sources , and analo - gies , increased the number of ideas generated . It also reduced the frequency of design solutions that were highly similar to the ex - ample , and increased the number of energy categories spanned . Fixation is a commonplace during the idea generation process and warrants much further investigation . Solutions presented or the individuals\u2019 own ideas can cause \ufb01xation , thus limiting the ideas that are considered . The situations that tend to increase de - sign \ufb01xation need to be identi\ufb01ed , and more approaches for miti - gating or reducing \ufb01xation should be created . Acknowledgment The authors would like to thank all participants of the 2008 NSF Workshop : Discussion on Individual and Team - Based Inno - vation and NSF for supporting the workshop and this paper under Grant No . CMMI0740529 . 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S . , 2005 , \u201cA Behavioural Model for Representing Biological and Arti\ufb01cial Systems for In - spiring Novel Designs , \u201d Proceedings of the International Conference on Engi - neering Design . 041003 - 12 / Vol . 132 , APRIL 2010 Transactions of the ASME Downloaded 26 Mar 2012 to 130 . 49 . 139 . 178 . Redistribution subject to ASME license or copyright ; see http : / / www . asme . org / terms / Terms _ Use . cfm", + "johnston2018novel": "* Forcorrespondence : goode @ brandeis . edu \u2020 These authors contributed equally to this work Competing interests : The authors declare that no competing interests exist . Funding : See page 24 Received : 21 August 2018 Accepted : 22 October 2018 Published : 23 October 2018 Reviewing editor : Anna Akhmanova , Utrecht University , Netherlands Copyright Johnston et al . This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited . A novel mode of capping protein - regulation by twinfilin Adam B Johnston 1\u2020 , Denise M Hilton 1\u2020 , Patrick McConnell 2 , Britney Johnson 3 , Meghan T Harris 1 , Avital Simone 1 , Gaya K Amarasinghe 3 , John A Cooper 2 , Bruce L Goode 1 * 1 Department of Biology , Rosenstiel Basic Medical Science Research Center , Brandeis University , Waltham , United States ; 2 Department of Biochemistry and Molecular Biophysics , Washington University , St Louis , United states ; 3 Department of Pathology and Immunology , Washington University , St Louis , United States Abstract Cellular actin assembly is controlled at the barbed ends of actin filaments , where capping protein ( CP ) limits polymerization . Twinfilin is a conserved in vivo binding partner of CP , yet the significance of this interaction has remained a mystery . Here , we discover that the C - terminal tail of Twinfilin harbors a CP - interacting ( CPI ) motif , identifying it as a novel CPI - motif protein . Twinfilin and the CPI - motif protein CARMIL have overlapping binding sites on CP . Further , Twinfilin binds competitively with CARMIL to CP , protecting CP from barbed - end displacement by CARMIL . Twinfilin also accelerates dissociation of the CP inhibitor V - 1 , restoring CP to an active capping state . Knockdowns of Twinfilin and CP each cause similar defects in cell morphology , and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity . Together , these observations define Twinfilin as the first \u2018pro - capping\u2019 ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 001 Introduction Assembly of cellular actin structures with distinct architectural and dynamic properties requires the convergence and coordination of numerous actin assembly , stabilization , and disassembly mecha - nisms . Although our understanding of the functions and mechanisms of individual actin - binding pro - teins has grown tremendously , there is a need to consider more deeply how seemingly disparate and sometimes competing factors work together in vivo and take on new mechanistic roles within more complex mixtures . One particularly enigmatic example is the interaction of Twinfilin with Cap - ping Protein ( CP ) . These two conserved proteins directly interact with high - affinity , and yet have seemingly opposite effects on the barbed ends of actin filaments . Twinfilin is one of five proteins in the Actin Depolymerization Factor - Homology ( ADF - H ) domain family , of which ADF / Cofilin is the founding member ( Poukkula et al . , 2011 ) . Twinfilin is unique among the members of this family in containing two ADF - H domains , which are joined by a small linker region and followed by a short C - terminal tail . Initial biochemical studies categorized Twinfilin as an actin monomer sequestering factor because of its high affinity for ADP - bound G - actin and abil - ity to inhibit subunit addition to either end of the filament ( Goode et al . , 1998 ; Vartiainen et al . , 2000 ; Wahlstro\u00a8m et al . , 2001 ) . However , mouse Twinfilin was later shown to interact directly with the barbed ends of actin filaments ( Helfer et al . , 2006 ; Paavilainen et al . , 2007 ) , and more recently yeast Twinfilin was shown to accelerate depolymerization at actin filament ends ( Johnston et al . , 2015 ) . Alone , yeast Twinfilin enhanced barbed end depolymerization by 3 - fold through a processive filament end - attachment mechanism . Further , in conjunction with Srv2 / CAP ( cyclase - associated pro - tein ) , yeast Twinfilin increased the rate of pointed - end depolymerization by over 15 - fold Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 1 of 28 RESEARCH ARTICLE ( Johnston et al . , 2015 ) . More recently , it was shown that mouse Twinfilin isoforms accelerate barbed end depolymerization , similar to yeast Twinfilin , but do not induce robust pointed end depo - lymerization in conjunction with Srv2 / CAP ( Hilton et al . , 2018 ) . Collectively , these studies highlight the biological significance of Twinfilin . The conserved barbed - end effects of Twinfilin are particularly interesting given that both yeast and mammalian Twinfilins bind to CP ( Falck et al . , 2004 ; Palmgren et al . , 2001 ) . Further , a barbed - end regulatory role for Twinfilin is suggested by its localization to the tips of stereocilia and filopo - dia , and to the barbed ends of Drosophila actin bristles ( Peng et al . , 2009 ; Rzadzinska et al . , 2009 ; Wahlstro\u00a8m et al . , 2001 ) . In addition , Twinfilin localizes to endocytic actin patches in yeast , and to lamellipodia and cell - cell junctions in animal cells ( Goode et al . , 1998 ; Vartiainen et al . , 2000 ) . Twinfilin\u2019s localization to cortical actin patches in yeast is dependent on its interaction with CP ( Palmgren et al . , 2001 ) . In both yeast and mammals , this interaction is mediated by conserved sequences in the C - terminal tail region of Twinfilin ( Falck et al . , 2004 ) . Despite the high affinity of the Twinfilin - CP interaction ( K d ~ 10 nM for the yeast homologs [ Poukkula et al . , 2011 ] ) , studies have revealed no significant effects of Twinfilin on the barbed end capping activity of CP in vitro , and reciprocally , no obvious effect of CP on Twinfilin interactions with ADP - actin monomers ( Falck et al . , 2004 ) . Thus , the functional significance of the Twinfilin - CP interaction has remained highly enigmatic . CP is an obligate heterodimer , consisting of alpha and beta subunits , and binds stably to the barbed ends of actin filaments to block subunit addition and loss . CP is ubiquitous and highly eLife digest Plant and animal cells are supported by skeleton - like structures that can grow and shrink beneath the cell membrane , pushing and pulling on the edges of the cell . This scaffolding network \u2013 known as the cytoskeleton \u2013 contains long strands , or filaments , made from many identical copies of a protein called actin . The shape of the actin proteins allows them to slot together , end - to - end , and allows the strands to grow and shrink on - demand . When the strands are the correct length , the cell caps the growing ends with a protein known as Capping Protein . This helps to stabilize the cell\u2019s skeleton , preventing the strands from getting any longer , or any shorter . Proteins that interfere with the activity of Capping Protein allow the actin strands to grow or shrink . Some , like a protein called V - 1 , attach to Capping Protein and get in the way so that it cannot sit on the ends of the actin strands . Others , like CARMIL , bind to Capping Protein and change its shape , making it more likely to fall off the strands . So far , no one had found a partner that helps Capping Protein limit the growth of the actin cytoskeleton . A protein called Twinfilin often appears alongside Capping Protein , but the two proteins seemed to have no influence on each other , and had what appeared to be different roles . Whilst Capping Protein blocks growth and stabilizes actin strands , Twinfilin speeds up their disassembly at their ends . But Johnston , Hilton et al . now reveal that the two proteins actually work together . Twinfilin helps Capping Protein resist the effects of CARMIL and V - 1 , and Capping Protein puts Twinfilin at the end of the strand . Thus , when Capping Protein is finally removed by CARMIL , Twinfilin carries on with disassembling the actin strands . The tail of the Twinfilin protein looks like part of the CARMIL protein , suggesting that they might interact with Capping Protein in the same way . Attaching a fluorescent tag to the Twinfilin tail revealed that the two proteins compete to attach to the same part of the Capping Protein . When mouse cells produced extra Twinfilin , it blocked the effects of CARMIL , helping to grow the actin strands . V - 1 attaches to Capping Protein in a different place , but Twinfilin was also able to interfere with its activity . When Twinfilin attached to the CARMIL binding site , it did not directly block V - 1 binding , but it made the protein more likely to fall off . Understanding how the actin cytoskeleton moves is a key question in cell biology , but it also has applications in medicine . Twinfilin plays a role in the spread of certain blood cancer cells , and in the formation of elaborate structures in the inner ear that help us hear . Understanding how Twinfilin and Capping Protein interact could open paths to new therapies for a range of medical conditions . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 002 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 2 of 28 Research article Cell Biology conserved across eukaryotes , and has universal roles in controlling the assembly of actin networks that drive cell morphogenesis and cell motility ( Cooper and Sept , 2008 ; Hart and Cooper , 1999 ; Mejillano et al . , 2004 ; Schafer et al . , 1994 ; Schafer et al . , 1995 ) . In vitro , CP binds to the barbed ends of actin filaments with sub - nanomolar affinity , and dissociates from barbed ends very slowly ( half - life of ~ 30 min ) ( Schafer et al . , 1996 ) . Given the relatively high abundance of CP in the cytosol ( 1 \u2013 3 m M ) and the strength of its interactions with barbed ends ( Cooper and Sept , 2008 ) , it is not surprising that cells have evolved a number of regulatory mechanisms to spatiotemporally restrict CP activity . Cellular protein inhibitors of CP broadly fall into two classes : steric inhibitors and allosteric inhibi - tors . Steric inhibitors , which include V - 1 / myotrophin , bind to CP in a manner that physically obstructs its association with barbed ends ( Bhattacharya et al . , 2006 ; Kim et al . , 2007 ; Schafer et al . , 1996 ) . V - 1 is a highly abundant 13 kDa protein that binds CP with a K d ~ 40 nM and sterically blocks its abil - ity to bind barbed ends ( Bhattacharya et al . , 2006 ; Taoka et al . , 2003 ) . Notably , however , V - 1 does not catalyze dissociation of CP from barbed ends ( Bhattacharya et al . , 2006 ) . In contrast , allo - steric inhibitors induce conformational changes in CP that catalyze its dissociation from barbed ends ( \u2018uncapping\u2019 or \u2018displacing\u2019 CP ) , and also decrease but do not abolish its ability to bind barbed ends . The major class of allosteric inhibitors is the capping protein interaction ( CPI ) motif family of pro - teins ( Edwards et al . , 2014 ) . The founding and best characterized member of the CPI family is CAR - MIL ( Capping Protein , ARP2 / 3 and Myosin I linker ) , which is conserved across metazoans ( Stark et al . , 2017 ) . CARMIL catalyzes CP dissociation from barbed ends , reducing CP\u2019s affinity for barbed ends by ~ 100 fold , transforming it into a transient capper ( Fujiwara et al . , 2014 ; Stark et al . , 2017 ; Uruno et al . , 2006 ; Yang et al . , 2005 ) . CARMIL localizes to the leading - edge plasma membrane , where it promotes cell migration through direct interactions with CP ( Fujiwara et al . , 2014 ; Liang et al . , 2009 ; Stark et al . , 2017 ; Yang et al . , 2005 ) . Other proteins with CPI motifs include CD2AP , CKIP - 1 , CapZIP , CIN85 , and WASHCAP ( FAM21 ) ; their roles in regu - lating CP are less well understood . CPI - motif proteins share a common mode of interaction with CP , but are otherwise unrelated to each other ( Edwards et al . , 2014 ; Hernandez - Valladares et al . , 2010 ) . To date , binding partners of CP that antagonize its inhibitors , and thus function as \u2018pro - cap - ping\u2019 factors , have not been reported . Here , we uncover a novel role for Twinfilin in protecting CP from the negative regulatory effects of V - 1 and CARMIL , and thus promoting actin filament capping . These and other data lead us to propose important revisions to current models for the CP regulatory cycle . Results CP inhibits mTwf1 - mediated depolymerization by capping barbed ends Because CP binding proteins have been studied predominantly in mammalian systems , we focused our investigation on mouse rather than yeast CP and Twinfilin . Mutagenesis on the yeast Twinfilin tail previously identified a mutant , twf1 - 11 , that targets a cluster of positively charged residues ( R328A , K329A , R330A , R331A ) necessary for binding CP ( Falck et al . , 2004 ) . While truncations of the C - ter - minal tail in mouse Twinfilin ( mTwf1 ) also disrupt CP binding , the residues involved have not yet been defined . We therefore first sought to generate a specific mutant in mTwf1 that disrupts the interaction , analogous to yeast twf1 - 11 . An alignment of the three mouse and three human Twinfilin isoforms , along with the single Twinfilin genes expressed in S . cerevisiae and D . melanogaster ( Figure 1A ) , revealed a region that includes two of the basic residues mutated in the yeast twf1 - 11 mutant . We mutated these two residues in mTwf1 , changing them to alanines , to produce mTwf1 - 11 ( K332A , R333A ) . To quantify binding of mTwf1 to CP , we performed fluorescence anisotropy assays using a mTwf1 tail peptide ( 317 - 350 ) labeled at its N - terminus with HiLyte488 . The mTwf1 tail pep - tide displayed high affinity , concentration - dependent binding to CP a 1 b 2 , a major non - muscle iso - form of CP in mammalian cells ( Figure 1B ) . Moreover , full - length mTwf1 protein ( unlabeled ) competed with the labeled mTwf1 tail for CP binding , whereas full - length mTwf1 - 11 ( unlabeled ) did not ( Figure 1C ) . Thus , the mTwf1 - 11 mutant effectively uncouples mTwf1 binding to CP . Using mTwf1 - 11 , we addressed how CP binding affects Twinfilin\u2019s actin depolymerization activi - ties in total internal reflection fluorescence ( TIRF ) microscopy assays , by directly observing Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 3 of 28 Research article Cell Biology depolymerization at actin filament barbed ends in real time . In agreement with previous observa - tions using yeast and mouse Twinfilin ( Hilton et al . , 2018 ; Johnston et al . , 2015 ) , 1 m M mTwf1 accelerated barbed end depolymerization by 2 \u2013 3 fold compared to control reactions ( Figure 1D ) , and the addition of CP blocked this effect ( Figure 1E ) . Further , mTwf1 - 11 exhibited a similar rate ( Figure 1D ) , indicating that this mutant has wild type depolymerization activity , and thus separates Twinfilin\u2019s ability to bind CP from its ability to promote barbed - end depolymerization . Interestingly , the addition of CP was still able to block barbed - end depolymerization by mTwf1 - 11 ( Figure 1E ) . These observations suggest that CP sterically blocks mTwf1 access to barbed ends , independent of its direct interaction with mTwf1 . However , this left open the question of whether CP binding to mTwf1 might alter its mechanism of depolymerization independent of blocking the barbed end . To Figure 1 . Barbed end capping by Capping Protein inhibits Twinfilin1 - mediated depolymerization . ( A ) Mouse Twinfilin - 1 ( mTwf1 ) domain organization : ADF - H , actin depolymerization factor homology domain ; L , linker ; T , tail . Sequence alignment of tail regions of Twinfilin isoforms from different species with boxed region highlighting conservation of residues critical for binding to Capping Protein ( CP ) . mTwf1 - 11 carries a mutation in the tail region ( KR332 , 333AA ) that disrupts binding to CP . ( B ) Fluorescence anisotropy measurement of 100 nM HiLyte488 - labeled mTwf1 tail peptide mixed with increasing concentrations of the indicated CP construct . ( C ) Fluorescence anisotropy measurement of 100 nM HiLyte488 - labeled mTwf1 tail peptide incubated in the presence 1 m M CP and increasing concentrations of either mTwf1 or mTwf1 - 11 . Anisotropy values for each condition averaged from three independent experiments . ( D , E ) Rates of barbed end depolymerization ( subunits s (cid:0) 1 ) induced by 1 m M of the indicated mouse Twinfilin , in the ( D ) absence or ( E ) presence of 10 nM CP , determined from TIRF assays . Rates for each condition averaged from at least five filaments in each of two independent experiments . From left to right : ( D ) n = 19 , 26 , and 15 and mean depolymerization rates 1 . 13 , 2 . 784 and 2 . 81 subunits s (cid:0) 1 ; ( E ) n = 13 , 15 , and 20 and mean depolymerization rates 1 . 13 , 2 . 784 and 2 . 81 subunits s (cid:0) 1 . ( F ) Rates of barbed end depolymerization ( subunits s (cid:0) 1 ) induced by 1 m M mTwf1 , in the absence or presence of 1 m M of the indicated CP construct , determined from TIRF assays . Rates for each condition averaged from at least five filaments from at least one experiment . From left to right n = 21 , 25 , 6 , and 10 ; mean depolymerization rates 1 . 45 , 2 . 991 , 0 . 11 , and 3 . 58 subunits s (cid:0) 1 . ( G ) Summary of barbed end depolymerization activity of mTwf1 constructs in combination with different CP constructs determined from TIRF assays ( as in D , E , F ) . Error bars , s . e . m . * * * * p (cid:20) 0 . 0001 , n . s . p > 0 . 05 by one - way ANOVA with Tukey post hoc test . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 003 The following video is available for figure 1 : Figure 1\u2014video 1 . Supporting data for Figure 1F . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 015 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 4 of 28 Research article Cell Biology address this possibility , we utilized a CP mutant , CP aD 28 , which truncates the C - terminal tentacle of the alpha subunit , severely inhibiting capping activity ( Kim et al . , 2010 ) . Importantly , in binding assays the mTwf1 tail interacted equally well with wild - type CP and CP aD 28 , demonstrating that this mutant binds normally to mTwf1 ( Figure 1B ) . In TIRF assays , equimolar amounts of CP aD 28 did not significantly alter mTwf1 depolymerization activity ( Figure 1F ; Figure 1\u2014video 1 ; also summarized in Figure 1G ) , suggesting that while CP blocks Twinfilin access to barbed ends , Twinfilin - CP direct interaction does not alter Twinfilin depolymerization activity . The twinfilin tail competes with CARMIL CPI motif for binding to CP Given that CP binding does not affect Twinfilin\u2019s depolymerization activity , or other known activities of Twinfilin ( Falck et al . , 2004 ; Johnston et al . , 2015 ; Palmgren et al . , 2001 ) , we next considered whether Twinfilin binding might influence CP functions in the presence of known regulators of CP . We were particularly interested in how Twinfilin might impact the regulation of CP by CPI - motif pro - teins such as CARMIL , since we noticed that the C - terminal tail regions of evolutionarily diverse Twinfilins share sequence homology with the CPI motifs of several CPI family proteins ( Figure 2A ) . The consensus CPI motif is 17 - amino acids long , with some additional contacts contributed from out - side this motif , and tolerates significant divergence across the CPI - motif family ( Edwards et al . , 2014 ; Hernandez - Valladares et al . , 2010 ) . As an initial test , we used a mutant of CP , CP ( RY ) , which alters two surface residues on the beta subunit ( R15A , Y79A ) that make essential contacts with CPI - motif proteins ( Edwards et al . , 2014 ; Hernandez - Valladares et al . , 2010 ) . The CP ( RY ) mutant is insensitive to inhibition and uncapping by CARMIL and disrupts binding with at least two other CPI - motif proteins , CD2AP and WASHCAP ( FAM21 ) ( Edwards et al . , 2015 ) . In fluorescence anisotropy binding assays , we observed that the CP ( RY ) mutant has approximately 20 - fold reduced affinity for mTwf1 tail compared to wild type CP ( Figure 2B ) . These data are consistent with mTwf1 and CPI - motif proteins sharing at least partially overlapping binding sites on CP . In addition , we asked whether introducing a mutation in the mTwf1 tail peptide at a conserved residue in CPI consensus sequences would alter binding to CP ( Lys 325 in mTwf1 ; see red asterisk , sequence alignment in Figure 2A ) . In fluorescence anisotropy binding assays , we compared the abilities of wild - type and mutant ( K325A ) mTwf1 tail peptides to compete with labeled mTwf1 tail peptide for CP binding . This analysis revealed an ~ 30 fold reduction in binding affinity for the mutant ( K325A ) mTwf1 tail peptide compared to wild type peptide ( Figure 2C ) . We next asked whether the CP - binding region ( CBR ) of CARMIL1 ( residues 964 \u2013 1078 ) competes with mTwf1 tail for binding to CP . We observed that unlabeled CBR peptide competed with the fluorescent mTwf1 tail probe for CP binding ( Figure 2D ) . These results indicate that CARMIL and mTwf1 directly compete for binding CP . Next , we more narrowly defined the region of CARMIL that competes with mTwf1 by using peptides that divide the CBR into its two conserved components , the CPI motif ( 969 \u2013 1005 ) and the CARMIL - specific interaction ( CSI ) motif ( 1019 \u2013 1037 ) . The CSI makes additional contacts with CP , but is found only in CARMIL family members , and not in other CPI - motif proteins ( Edwards et al . , 2014 ) . As expected based on Twinfilin\u2019s sequence similarity to CPI motifs , only the CPI - motif peptide and not the CSI peptide competed with mTwf1 tail for CP binding ( Figure 2D ) . Together , these results suggest that Twinfilin is a divergent CPI - motif protein and has important implications for CP regulation in cells ( see Discussion ) . Twinfilin attenuates CARMIL - mediated displacement of CP from barbed ends Given that CARMIL and Twinfilin compete for binding to CP , we asked whether mTwf1 affects CAR - MIL\u2019s ability to displace CP from barbed ends . We addressed this question in pyrene actin assembly assays , where actin polymerization was initiated at time zero in the presence of CP and increasing concentrations of mTwf1 , and after 400 s CARMIL1 CBR was spiked into the reaction . CARMIL1 alone ( no mTwf1 ) strongly induced uncapping , leading to the rapid polymerization of previously - capped filament seeds ( Figure 3A ) . However , increasing concentrations of mTwf1 attenuated CAR - MIL\u2019s uncapping effects ( Figure 3A ) . These results are consistent with mTwf1 competing with CAR - MIL for binding CP , and thereby blocking uncapping . To more directly observe mTwf1 effects on CARMIL - induced uncapping of barbed ends , we used TIRF microscopy . In these experiments , we used fluorescently labeled SNAP - tagged CP ( SNAP - 649 - Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 5 of 28 Research article Cell Biology Figure 2 . Twinfilin is a Capping Protein Interaction ( CPI ) - motif protein that competes with CARMIL for binding Capping Protein . ( A ) CARMIL domain organization : PH , pleckstrin - homology domain ; L , linker ; N - cap ( N ) , LRR , leucine - rich repeat domain ; C , C - cap ; HD , helical dimerization domain ; CBR , Capping Protein binding domain , consisting of CPI , Capping Protein interaction domain , and CSI , CARMIL - specific interaction sequence ; MBD , membrane binding domain ; PRD , proline - rich domain . Alignment between the Capping Protein Interaction ( CPI ) motif consensus sequence , and the CPI regions of H . sapiens ( H . s . ) CARMIL1 ( UniProtKB Q5VZK9 . 1 ) , CARMIL2 ( UniProtKB Q6F5E8 . 2 ) , CARMIL3 ( UniProtKB Q8ND23 . 2 ) , CKIP1 UniProtKB Q53GL0 . 2 ) , CD2AP ( CBI NP _ 036252 . 1 ) , WASHCAP ( Fam21 ) ( UniProtKB Q9Y4E1 . 3 ) , CapZIP ( CBI NP _ 443094 . 3 ) , CIN85 ( UniProtKB Q96B97 . 2 ) , and the tail sequences of Twinfilin homologs from D . melanogaster ( D . m ) , C . lectularius ( C . l . ) , S . cerevisiae ( S . c . ) , O . Taurus ( O . t . ) , S . litura ( S . l . ) , D . rerio ( D . r . ) , H . sapiens ( H . s . ) , and M . musculus ( M . m . ) . Twinfilin isoforms ( D . m . Twf1 UniProtKB NP _ 650338 , C . l . Twf1 UniProtKB XP _ 014258437 . 1 , S . c . Twf1 GenBank GAX68393 . 1 , O . t . Twf1 XP _ 022917989 . 1 , S . l . Twf1 XP _ 022816377 . 1 , D . r . Twf1 AAH67638 . 1 , H . s . Twf1 UniProtKB NO _ 001229326 . 1 , and M . m . Twf1 GenBank AAH15081 . 1 ) . Amino acid color coding illustrates side chain chemistry similarities . The asterisk marks the residue we mutated in mTwf1 in panel . ( C ) The alignments were generated using the MAFFT algorithm in the DNASTAR Lasergene Suite / MegAlign Pro application ( MegAlign Pro . Version 15 . 0 . DNASTAR . Madison , WI . ) . ( B ) Fluorescence anisotropy measurement of 60 nM HiLyte488 - labeled mTwf1 tail peptide mixed with increasing concentrations of the indicated CP construct . ( C ) Fluorescence anisotropy measurement of 40 nM TAMRA - labeled mTwf1 tail peptide incubated with 1 m M CP and different concentrations of wild type and mutant mTwf1 tail peptides . ( D ) Fluorescence anisotropy measurement of 60 nM HiLyte488 - labeled mTwf1 tail peptide incubated in the presence of 240 nM CP and increasing concentrations of the indicated CARMIL fragment ( CBR , CSI , or CPI ) . CSI failed to compete with HiLyte 488 - mTwf1 tail peptide at the concentrations tested . Anisotropy values for each condition were averaged from three independent experiments . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 004 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 6 of 28 Research article Cell Biology Figure 3 . Direct interactions of Twinfilin with Capping Protein attenuate CARMIL - mediated uncapping . ( A ) Bulk fluorescence assays comparing the rates of actin assembly in the presence of 25 nM muscle Capping Protein ( CP a 1 b 1 ) and increasing concentrations of mTwf1 . To initiate uncapping , 250 nM CBR fragment of CARMIL ( see schematic , Figure 2A ) was spiked into the reaction at 400 s . Data shown are representative curves from experiments repeated three independent times . ( B ) Representative time - lapse images from TIRF microscopy assays monitoring the displacement of labeled CP from barbed ends . Filaments were first polymerized and tethered using 1 m M actin ( 10 % OG - labeled , 0 . 5 % biotin \u2013 actin ) , then capped at their barbed ends by flowing in SNAP - 649 - CP ( 100 % labeled ) . Next , 50 nM CBR fragment of CARMIL and different concentrations of mTwf1 were flowed in , and CP dissociation was monitored over time . Scale bar , 5 m m . ( C ) Quantification of the percentage of filaments retaining CP at the barbed ends in the presence of 50 nM CBR fragment of CARMIL and variable concentrations of mTwf1 , determined from TIRF reactions as in ( B ) . Control curve , buffer alone ( no CBR or mTwf1 ) . n > 45 events measured from at least two independent experiments . ( D ) Representative time - lapse images from TIRF microscopy assays monitoring CP displacement from barbed ends , analyzed as in ( B ) , except using 1 m M mTwf1 - 11 instead of mTwf1 . n > 45 events measured from at least two independent experiments . ( E ) Quantification of the percentage of filaments retaining CP at the barbed end in the presence of 50 nM CBR fragment of CARMIL and different concentrations of mTwf1 - 11 , determined from TIRF assays as in ( D ) . n > 45 events measured from at least two independent experiments . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 005 The following figure supplement is available for figure 3 : Figure supplement 1 . Supporting data for Figure 3 showing that multiple Twinfilin isoforms antagonize CARMIL uncapping of barbed ends . Figure 3 continued on next page Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 7 of 28 Research article Cell Biology CP ; 100 % labeled ) to monitor lifetimes of CP molecules on filament barbed ends ( Bombardier et al . , 2015 ) . Filaments were first polymerized to a desired length ( ~ 10 m m ) and then capped by flowing in SNAP - 649 - CP . Free CP was washed out , and then proteins of interest ( or con - trol buffer ) were flowed in . Capped filaments were identified in the field of view prior to flow - in , and then monitored after flow - in to measure the dwell time of SNAP - 649 - CP . As expected , in the absence of other factors , SNAP - 649 - CP had a long dwell time , remaining on barbed ends for tens of minutes ( Figure 3B and C ) . However , when CARMIL1 CBR was introduced , this led to the rapid dis - placement of SNAP - 649 - CP , with complete loss of CP from barbed ends by 100 s ( Figure 3B and C ) . The addition of mTwf1 with CARMIL1 CBR attenuated the uncapping effects in a concentration - dependent manner ( Figure 3B and C ) . Further , this attenuation required direct interactions between Twinfilin and CP , as mTwf1 - 11 failed to protect CP from CARMIL uncapping ( Figure 3D and E and Figure 3\u2014figure supplement 1 ) . Similar effects were observed for the other major isoform of mouse Twinfilin that is expressed in non - muscle cells , mTwf2a ( Figure 3\u2014figure supplement 1 ) ( Nevalainen et al . , 2011 ; Vartiainen et al . , 2003 ) . Twinfilin accelerates the dissociation of V - 1 from CP We next considered whether Twinfilin binding to CP might affect the activities of CP inhibitor V - 1 / myotrophin , which is distinct from CPI - motif proteins in its mode of CP interaction . Unlike CARMIL , V - 1 does not displace CP from barbed ends ; instead , it sequesters CP and blocks it from binding fila - ment ends ( Bhattacharya et al . , 2006 ; Jung et al . , 2016 ; Taoka et al . , 2003 ) . In contrast to the CARMIL binding site on CP , which partially encircles the \u2018stalk\u2019 of the CP heterodimer ( Hernandez - Valladares et al . , 2010 ; Johnson et al . , 2018 ; Zwolak et al . , 2010 ) , V - 1 interacts with CP on the opposite face , sterically blocking binding to the filament end ( Johnson et al . , 2018 ; Takeda et al . , 2010 ; Zwolak et al . , 2010 ) . To test how Twinfilin might affect the interaction of CP with V - 1 , we used pyrene - actin seeded elongation assays ( Figure 4A ) . As expected , filament seeds pre - incubated with CP and then mixed with pyrene - actin monomers displayed minimal growth , whereas the addi - tion of V - 1 restored actin assembly to uncapped levels . Somewhat to our surprise , the further addi - tion of mTwf1 suppressed V - 1\u2019s effects , restoring capping activity , while mTwf1 - 11 had no effect ( Figure 4A and B ) . These effects were unexpected given the above - mentioned differences in Twinfi - lin\u2019s predicted and V - 1\u2019s known binding sites on CP , and our observation that even high concentra - tions of V - 1 ( 1000 - fold excess to mTwf1 tail probe ) fail to compete with mTwf1 for CP binding in anisotropy assays ( Figure 4C ) . These results suggest that mTwf1 attenuates V - 1 effects on CP via an allosteric mechanism , distinct from a simple steric binding competition . In probing the mechanism further , we drew inspiration from a study by Fujiwara and colleagues , showing that CARMIL forms a transient ternary complex with V - 1 and CP , leading to accelerated dis - sociation of V - 1 from CP ( Fujiwara et al . , 2014 ) . We asked whether mTwf1 might similarly catalyze the dissociation of V - 1 from CP . In stopped - flow fluorescence assays , fluorescently labeled V - 1 ( TAMRA - V - 1 ) was first allowed to bind CP , and then mixed at time zero with an excess of unlabeled V - 1 . The resulting decrease in fluorescence reflects the spontaneous dissociation of TAMRA - V - 1 from CP ( Figure 4D ) . The rate of V - 1 dissociation from CP increased in the presence of increasing concentrations of mTwf1 , pointing to the possible formation of a transient ternary complex that destabilizes V - 1 interactions with CP ( Figure 4D and E ) . Importantly , mTwf1 - 11 failed to enhance V - 1 dissociation ( Figure 4E ) , showing that this effect depends on direct interactions between mTwf1 tail and CP . These results demonstrate that CARMIL and Twinfilin share a common function in cata - lyzing the dissociation of V - 1 from CP using their CPI motifs to bind CP , despite having different effects on the displacement of CP from barbed ends . Structural evidence for the twinfilin tail interacting with the CPI - binding site on CP Given the observed competition between mTwf1 tail peptide and the CPI motif of CARMIL for bind - ing to CP , and the similarity between mTwf1 and CARMIL in catalyzing V - 1 dissociation from CP , we Figure 3 continued DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 006 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 8 of 28 Research article Cell Biology sought structural evidence for the nature of the interaction between mTwf1 and CP . We hypothe - sized that the binding sites for mTwf1 and the CPI motif were likely overlapping . To test this hypoth - esis , we used hydrogen - deuterium exchange with mass spectrometry ( HDX - MS ) to interrogate the conformational dynamics and solvent accessibility of the backbone and sidechains of CP , free and in complex with Twf1 . Further , we compared our results to those in our recent study on the interactions of CARMIL with CP using the same approach ( Johnson et al . , 2018 ) . We tested three different forms of mTwf1 : a short tail peptide ( residues 317 \u2013 350 ) , a longer tail peptide ( residues 305 \u2013 350 ) , and full - length mTwf1 . These constructs were added to CP , either full - length alpha / beta hetero - dimer , or full - length alpha subunit with a beta subunit truncated at its C - terminus , removing the actin - binding beta tentacle . The results were essentially the same in each case . The presence of mTwf1 resulted in protection from H - D exchange at the N - terminal stalk of CP ( Figure 5A , Fig - ure 5\u2014figure supplements 1 and 2 ) . Similar effects to H - D exchange were observed upon CARMIL binding to CP ( Johnson et al . , 2018 ) ; also shown here in Figure 5B ) , which correspond well with the CPI - motif binding site defined by X - ray crystallography and solution NMR studies ( Hernandez - Figure 4 . Twinfilin\u2019s direct binding to Capping Protein accelerates the disassociation of V - 1 to promote capping of filaments . ( A , B ) Seeded elongation assays comparing the rates of actin assembly from spectrin - F - actin seeds ( grey ) in the presence of 0 . 5 m M actin ( 10 % pyrene - labeled ) , 25 nM muscle Capping Protein ( CapZ ) , 500 nM V - 1 , and variable concentrations of mTwf1 ( A ) or mTwf1 - 11 ( B ) as indicated . Data shown are representative curves from experiments performed three independent times . ( C ) Fluorescence anisotropy measurement of 100 nM HiLyte488 - labeled mTwf1 tail peptide mixed with 1 m M mouse Capping Protein ( CP ) and variable concentrations of CBR fragment of CARMIL or V - 1 . Rates for each condition averaged from three independent experiments . ( D ) Stopped - flow fluorescence assays measuring the kinetics of dissociation of 50 nM TAMRA - V - 1 from 1 m M CP upon addition at time zero of 2 . 5 m M unlabeled V - 1 and variable concentrations of mTwf1 as indicated . Apparent dissociation rates are listed for each condition . ( E ) Apparent dissociation rates of TAMRA - V - 1 for different concentrations of mTwf1 are from ( D ) ; and for 12 m M mTwf1 (cid:0) 11 = 1 . 0 \u00b1 0 . 003 s (cid:0) 1 . Anisotropy values for each condition were averaged from five independent experiments . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 007 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 9 of 28 Research article Cell Biology Valladares et al . , 2010 ; Takeda et al . , 2010 ; Zwolak et al . , 2010 ) . For mTwf1 , we also observed H - D exchange protection of a small region on CP corresponding to the V - 1 binding site ( Figure 5A and B , Figure 5\u2014figure supplements 1 and 2 ) , consistent with our results described above for the effects of mTwf1 in promoting V - 1 dissociation from CP . These structural effects are also consistent with our previous results for CARMIL , which alters the V - 1 binding site ( Johnson et al . , 2018 ) . How - ever , it is worth noting that mTwf1 - induced changes in CP conformation at the actin - binding Figure 5 . HDX - MS analysis of Twinfilin reveals effects on Capping Protein structure near the CPI motif - binding site . ( A ) A cartoon representation of a crystal structure of CP , based on PDB 3AAA ( Takeda et al . , 2010 ) . Differences in deuterium uptake induced by mTwf1 binding to CP are displayed as a color gradient ( see scale at bottom of panel ( B ) CPI domain of CARMIL overlaid on to its binding site on CP ( around the stalk ) . Representative comparisons of deuterium uptake curves for free CP ( black ) with mTwf1 bound CP ( red ) for CP alpha subunit ( upper panels ) and CP beta subunit ( lower panels ) . Error bars representing the results of t - tests between samples are shown above each time point to illustrate statistical significance . When error bars are not shown explicitly , the error is within the radius of the symbol . Data shown are representative curves from experiments repeated two independent times . ( B ) A cartoon representation of a crystal structure of CP , showing the differences in deuterium uptake induced by CBR domain of CARMIL binding to CP are displayed as a color gradient ( see scale at the bottom ) . CPI domain of CARMIL overlaid on to its binding site on CP ( around the stalk ) , V - 1 is overlaid on its binding site on CP ( barbed end binding surface ) for comparison . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 008 The following figure supplements are available for figure 5 : Figure supplement 1 . Supporting data for Figure 5 showing differential HDX results . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 009 Figure supplement 2 . Supporting data for Figure 5 showing differential HDX results . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 010 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 10 of 28 Research article Cell Biology interface were not as extensive as those induced by CARMIL , which is consistent with CARMIL , but not mTwf1 , weakening CP binding to actin at the barbed ends . Twinfilin and CP colocalize in cells and have similar knockdown phenotypes To investigate the functional relationship between Twinfilin and CP in cells , we started by asking whether mTwf1 and CP colocalize . While Twinfilin and CP have been localized individually , and are each reported to be enriched at the tips of filopodia and stereocilia , endocytic actin patches , lamelli - podia , and Drosophila bristles ( Avenarius et al . , 2017 ; Falck et al . , 2004 ; Goode et al . , 1998 ; Nevalainen et al . , 2011 ; Peng et al . , 2009 ; Rzadzinska et al . , 2009 ; Sinnar et al . , 2014 ; Vartiainen et al . , 2000 ) , to our knowledge they have never been co - imaged in vertebrate cells . To address this , we performed immunofluorescence on CP and Twinfilin in mouse B16F10 melanoma cells , co - staining the cells with Alexa 568 - phalloidin to visualize F - actin . We observed strong colocali - zation of Twinfilin and CP throughout the cell and a co - enrichment at the actin - rich leading and trail - ing edges ( Figure 6A and B ) . Further , quantitative western blotting showed that Twinfilin and CP are present at ~ 1 : 2 molar ratio in B16F10 cells ( Figure 6C , Figure 6\u2014figure supplement 1 ) . Previ - ous studies reported the concentration of CP in B16F10 cells to be ~ 1 m M ( Fujiwara et al . , 2014 ; Pollard and Borisy , 2003 ) , suggesting that mTwf1 is present at ~ 0 . 5 m M . Given the high affinity of the Twinfilin - CP interaction ( K d = 50 nM ) , these observations are consistent with mTwf1 being associ - ated with a substantial fraction of the CP in cells . The ability of Twinfilin to function as a \u2018pro - capping\u2019 factor in vitro , by antagonizing the inhibitory effects of V - 1 on CP , predicted that genetic loss of mTwf1 might at least partially phenocopy loss of CP . While a number of studies have examined how Twinfilin mutations affect whole animal develop - ment and physiology ( Iwasa and Mullins , 2007 ; Meacham et al . , 2009 ; Nevalainen et al . , 2011 ; Wahlstro\u00a8m et al . , 2001 ; Wang et al . , 2010 ; Yamada et al . , 2007 ) , we are unaware of any studies that have investigated how loss of Twf1 affects the morphology and actin organization of cultured mammalian cells . Using RNAi silencing in B16F10 cells , we separately depleted endogenous mTwf1 and CP , which was verified by both western blotting ( Figure 6E and F ) and immunostaining ( Fig - ure 6\u2014figure supplement 1 ) . Knockdown of either mTwf1 or CP led to a similar , marked increase in the density of peripheral protrusions or microspikes with a concomitant loss of lamellipodial surfaces ( Figure 6F and G ) . Similar phenotypes have been reported for CP depletion in multiple cell lines ( Edwards et al . , 2013 ; Edwards et al . , 2015 ; Mejillano et al . , 2004 ; Sinnar et al . , 2014 ) . Expres - sion of an RNAi - refractive mTwf1 construct , but not mTwf1 - 11 , rescued the defects caused by deple - tion of endogenous mTwf1 ( Figure 6F and G ; Figure 6\u2014figure supplement 1 ) , demonstrating that these cellular functions of mTwf1 critically depend on its interaction with CP . We also made the unexpected observation that knockdown of CP was accompanied by a dra - matic reduction in Twinfilin levels in cells , as seen by both western blotting ( Figure 6D ) and immuno - fluorescence ( Figure 6\u2014figure supplement 1 ) . This effect was confirmed using a second RNAi oligonucleotide that targets a different region of CP ( siCP2 , Figure 6D ) . Further , it was observed in additional cell lines besides B16F10 , including Neuro - 2A and NIH - 3T3 cells ( Figure 6\u2014figure sup - plement 1 ) . These observations support the closely intertwined relationship of CP and Twinfilin in vivo . Our results above also call into question whether the full extent of the phenotype caused by knockdown of CP ( Figure 6G ) is due to loss of CP , or instead is partly due to the accompanying loss of Twinfilin . To address this , we restored mTwf1 levels in cells depleted of CP by driving mTwf1 expression from a rescue plasmid , which was confirmed by western blotting and immunofluores - cence ( Figure 6\u2014figure supplement 1 ) . Forced expression of mTwf1 partially rescued the defects associated with CP depletion , indicating that a portion of the original defects observed after CP knockdown were likely due to the accompanying loss of mTwf1 . These observations also suggest that many previously reported phenotypes arising from CP knockouts and knockdowns should be revisited or reinterpreted with the potential loss of Twinfilin in mind . Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 11 of 28 Research article Cell Biology Figure 6 . mTwf1 and Capping Protein colocalize and have similar phenotypes in B16F10 melanoma cells . ( A ) Representative images from immunofluorescence staining showing colocalization of endogenous mTwf1 ( yellow ) and Capping Protein ( magenta ) . Scale bar , 20 m m . Close ups of boxed regions shown in Zooms ; scale bar , 4 m m . ( B ) Mander\u2019s correlation coefficient ( M1 and M2 ) values of overlap between mTwf1 and Capping Protein ( CP ) measured from cells ( n = 67 cells ) as in ( A ) . Error bars , s . e . m . ( C ) Comparison of the relative abundance of mTwf1 and Capping Protein ( CP ) in B16F10 cells measured from western blot analysis . Data averaged from four separate experiments . Error bars , s . d . n . s . p > 0 . 05 by t - test . ( D , E ) Representative western blots and quantification of cellular levels of mTwf1 ( D ) and CP ( E ) in B16F10 cells treated with siRNA against mTwf1 ( si - Twf1 ) or Figure 6 continued on next page Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 12 of 28 Research article Cell Biology Defects caused by CARMIL1 hyperactivity can be rescued by elevated twinfilin expression Finally , we tested the prediction of our biochemical observations that loss of capping activity in cells caused by overexpressed CARMIL1 should be restored by co - overexpression of Twf1 . B16F10 cells ectopically expressing CARMIL1 showed morphological defects similar to loss of CP , and ectopic mTwf1 expression rescued the defects ( Figure 7A and B ) . Importantly , ectopic expression of mTwf1 alone caused no significant change in cell morphology . These results support our biochemical obser - vations , and suggest that Twf1 promotes capping in vivo , at least in part by competing with CARMIL for CP binding and antagonizing the uncapping effects of CARMIL . Discussion Twinfilin and CP have been inextricably linked as interacting partners in yeast and animal cells for over 15 years ( Palmgren et al . , 2001 ) , yet until now it has remained a mystery what function their interaction serves . Here we discovered that Twinfilin binds to CP using an orphan CPI - like sequence in its C - terminal tail region , and through this interaction protects CP from inhibition and / or barbed end displacement by CARMIL and V - 1 . We found that Twinfilin binds to CP in a competitive manner with the CPI motif of CARMIL , interacts with a site on CP similar to that of CARMIL , and attenuates CARMIL - mediated uncapping of actin filaments . Separately , Twinfilin binding to CP also accelerates V - 1 dissociation from CP , despite Twinfilin and V - 1 having non - overlapping binding sites on CP . This might be achieved by an allosteric mechanism , given that CARMIL uses its CPI motif to induce V - 1 dissociation from CP through allosteric changes ( Fujiwara et al . , 2014 ; Johnson et al . , 2018 ) . Thus , we have demonstrated that Twinfilin promotes capping by protecting CP from interactions with V - 1 and CARMIL . This functional role for Twinfilin is further supported in vivo by our observations of : ( i ) strong colocalization of Twinfilin and CP , ( ii ) knockdowns of mTwf1 and CP that each give rise to sim - ilar defects in cell morphology , and ( iii ) over - expression of mTwf1 suppressing defects caused by CARMIL hyperactivity . Taken together , these results reveal that Twinfilin is a new member of the CPI - motif family of proteins , and the first within this group to show the ability to bind CP without reducing CP affinity for barbed ends , and antagonize the negative regulatory effects of another CPI protein . These functions of Twinfilin provide important new insights into the CP regulatory cycle . The best working model to date has been the Fujiwara model ( Fujiwara et al . , 2014 ) ( depicted here as \u2018Ear - lier Model\u2019 ; Figure 7C ) . It posits that the majority of CP in the cytosol is bound to V - 1 , in an inactive state , which then can be locally \u2018activated\u2019 by CARMIL at the leading edge . However , a caveat to this model is that it suggests CP - CARMIL complexes are the dominant capping species in the cell , despite this complex having ~ 100 fold reduced affinity for barbed ends compared to free CP . While this could potentially explain dynamic capping and uncapping near the plasma membrane , consis - tent with GFP - CP single molecule speckle analysis ( Miyoshi et al . , 2006 ) , it does not explain how cells maintain a pool of \u2018capping competent\u2019 CP further back from the leading edge , where CP is needed to cap barbed ends in stress fibers and other actin networks , and may cap barbed ends gen - erated by severing to promote filament disassembly . This model goes on to suggest that an Figure 6 continued CP ( si - CP ) or negative control ( Control ) . Band intensity for control cells was set to 1 . 0 . Data averaged from at least three separate experiments . , error bars , s . d . ( F ) Representative images showing F - actin immunofluorescence in B16F10 cells treated with siRNA against mTwf1 ( si - Twf1 ) or CP ( si - CP ) or negative control ( Control ) ; siRNA treated cells ( si - Twf1 or si - CP ) were also rescued using plasmids expressing si - resistant FL - myc - mTwf1 ( WT or mTwf1 - 11 ) . Scale bar , 20 m m . Close ups of boxed regions shown in Zooms ; scale bar , 4 m m . ( G ) Microspike density in cells treated as in ( D ) . Box and whisker plots show mean , first and third quartile , and the maximum and minimum values . Data averaged from two experiments . From Left to right : n = 45 , 53 , 51 , 24 , 24 , and 20 and mean microspike density 0 . 69 , 1 . 34 , 1 . 77 , 0 . 59 , 1 . 24 , and 1 . 01 filopodia per 10 m m of cell cortex . Error bars , s . e . m . * * * * p (cid:20) 0 . 0001 , * p (cid:20) 0 . 05 , n . s . p > 0 . 05 by one - way ANOVA with Tukey post hoc test . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 011 The following figure supplement is available for figure 6 : Figure supplement 1 . Supporting data for Figure 6 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 012 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 13 of 28 Research article Cell Biology Figure 7 . Overexpression of Twinfilin suppresses morphological defects caused by CARMIL hyperactivity . ( A ) Representative images of F - actin staining in untreated B16F10 cells ( control ) , and cells transfected with Flag - CARMIL1 , full - length ( FL ) - myc - mTwf1 , or both . Scale bar , 20 m m . ( B ) Average Microspike density in cells treated as in ( A ) . Box and whisker plots show mean , first and third quartile , and the maximum and minimum values . Data averaged from two experiments ( n = 19 \u2013 25 cells per condition ) . Data averaged from two experiments . From Left to right : n = 19 , 25 , 20 , and 25 ; mean microspike density 0 . 75 , 1 . 13 , 0 . 62 , 0 . 58 filopodia per 10 m m of cell cortex . Error bars , s . e . m . * * * p (cid:20) 0 . 001 , n . s . p > 0 . 05 by one - way ANOVA with Tukey post hoc test . ( C ) \u2018Earlier\u2019 model for CP regulatory cycle , adapted from Fujiwara and colleagues ( Fujiwara et al . , 2014 ) . Proposed steps in model : ( 1 ) V - 1 globally inhibits Capping Protein ( CP ) in the cytoplasm , ( 2 ) membrane - associated CARMIL ( at the protruding cell edge ) catalyzes dissociation of V - 1 from CP , ( 3 ) the resulting CARMIL - CP complex is partially active , binding weakly to free barbed ends to provide capping function , ( 4 ) an unknown factor or mechanism promotes dissociation of CARMIL from CP , allowing V - 1 to rebind CP and complete the cycle . ( D ) Our revised working model for the CP regulatory cycle . We propose that V - 1 functions to maintain a cytosolic reservoir of inactive CP , from which Twinfilin and CARMIL activate CP , generating two distinct forms of active CP in cells : Twinfilin - CP complexes and CARMIL - CP complexes . Twinfilin - CP complexes are fully active and support stable capping of barbed ends . In contrast , CARMIL - CP complexes have ~ 100 fold reduced affinity for barbed ends , and may therefore more transiently cap barbed ends , permitting restricted network growth at the cell membrane where CARMIL localizes . CARMIL and Twinfilin directly compete with each other for binding CP ( shown in close up of Transition state ) , which may result in the displacement of CP from Twinfilin . This would leave Twinfilin at the barbed end to catalyze depolymerization , or alternatively return filaments back to the original state of assembly . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 013 The following figure supplement is available for figure 7 : Figure supplement 1 . Structural model for a ternary complex formed by Twinfilin , Capping Protein and the barbed end of an actin filament . Figure 7 continued on next page Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 14 of 28 Research article Cell Biology unknown factor or mechanism dissociates the CP - CARMIL complex , allowing V - 1 to rebind CP , restoring it to an inactive state . In light of our results , we propose several additions and revisions to the Fujiwara model ( see \u2018Revised Model\u2019 ; Figure 7D ) . First , we suggest that Twinfilin\u2019s protective effects on CP , in particular against V - 1 , allow cells to maintain a larger pool of fully active CP ( Twinfilin - CP complexes ) in the cytosol than was previously thought . This view is supported by the relatively high abundance of Twinfilin in cells ( ~ 0 . 5 m M , compared to ~ 1 m M CP ) its high affinity for CP ( K d = 50 nM ) , and its ability to increase the rate of dissociation of V - 1 from CP . Given these observations , we propose that a sub - stantial fraction of CP is available in a fully active state , as Twinfilin - CP complexes , even in the pres - ence of a high concentration of V - 1 in the cytosol ( ~ 3 m M ) ( Fujiwara et al . , 2014 ; Pollard and Borisy , 2003 ) . Second , we propose that V - 1 functions to maintain a cytosolic reservoir of inactive CP , mobilized by Twinfilin and / or CARMIL dissociating V - 1 to generate \u2018stable capping\u2019 ( Twinfilin - CP ) in the cytosol and possibly \u2018transient capping\u2019 ( CARMIL - CP ) complexes at the plasma mem - brane , respectively . CARMIL - CP complexes at the plasma membrane could facilitate actin network growth to drive leading edge protrusion . In contrast , Twinfilin - CP complexes in the cytosol may facil - itate stable capping of barbed ends to limit network growth and promote filament disassembly and turnover . Third , we propose that the association of Twinfilin - CP complexes with barbed ends primes filaments for disassembly . Our data show that CARMIL , and / or other CPI proteins , compete with Twinfilin for binding CP . These interactions may competitively remove CP from barbed ends , leaving Twinfilin at the barbed end to processively depolymerize filaments , either alone or in combination with Srv2 / CAP ( as depicted in Figure 6D ) ( Hilton et al . , 2018 ; Johnston et al . , 2015 ) . In this man - ner , the interaction of Twinfilin with CP could serve not only to initially promote capping , and thus limit network growth , but also to position Twinfilin at barbed ends for subsequently catalyzing the disassembly of filaments . In summary , our results show that functions of mammalian Twinfilin and CP are closely inter - twined . This functional relationship is likely to extend to other species given CPI motif sequence con - servation in the Twinfilin tail region ( Figure 2A ) and the conserved nature of the Twinfilin - CP interaction . Indeed , S . cerevisiae Aim21 was recently identified as the first yeast CPI motif - containing protein , and was shown to regulate CP function at cortical actin patches ( Farrell et al . , 2017 ; Shin et al . , 2018 ) . We generated a structural model to explore the possible ternary complex formed by Twinfilin , CP , and the barbed end of an actin filament ( Figure 7\u2014figure supplement 1 ) . In this model , the Twinfilin tail is long enough to allow for simultaneous binding of Twinfilin\u2019s CPI motif to CP and Twinfilin\u2019s C - terminal ADFH domain to an actin subunit at the barbed end . Further , there are no clashes in binding between CP and Twinfilin on actin . It is worth noting that CP and Twinfilin appear to be able to associate with barbed ends individually or as a CP - Twinfilin complex , but with distinct consequences for the function and dynamics of actin networks . CP alone stably caps barbed ends , blocking subunit addition or loss , and our results suggest that CP - Twinfilin complexes may do the same . However , when Twinfilin alone associates with barbed ends , it drives processive depo - lymerization , while blocking new assembly ( Hilton et al . , 2018 ; Johnston et al . , 2015 ) . Thus , despite key differences in the nature of their associations with barbed ends , CP and Twinfilin each inhibit fila - ment growth , likely explaining why Twinfilin can replace CP in reconstituted actin motility assays in vitro ( Helfer et al . , 2006 ) . Finally , our data add to a broader emerging view that actin dynamics in vivo are controlled by a complex set of barbed end - associated factors , many of which interact with each other and / or stimu - late each other\u2019s dissociation from barbed ends . These multi - component mechanisms may allow cells to control rapid transitions at filament ends through different functional states , including ( i ) formin - bound elongation , ( ii ) paused growth by formin - CP \u2018decision complexes\u2019 , ( iii ) stable or transiently capped states by CP alone or CP - Twinfilin complexes ( Bombardier et al . , 2015 ; Shekhar et al . , 2015 ) , and ( iv ) depolymerization by Twinfilin , Cofilin , and / or Srv2 / CAP . These molecular mechanisms for regulating barbed end growth are vastly more elaborate and dynamic than once thought , and help explain the exquisite spatiotemporal control that cells have in tuning actin network dynamics . Figure 7 continued DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 . 014 Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 15 of 28 Research article Cell Biology Materials and methods Key resources table Reagent type ( species ) or resource Designation Source or reference Identifiers Additionalinformation Antibody Rabbit anti - Twinfilin PekkaLappalainen ( Univ . Helsinki ) WB ( 1 : 1000 ) IF ( 1 : 100 ) Antibody mouse anti - Capping Protein DevelopmentStudiesHybridomaBank Cat : 3F2 WB ( 1 : 2000 ) IF ( 1 : 50 ) Antibody Mouse anti - Flag SigmaAldrich Cat : F3165 WB ( 1 : 5000 ) IF ( 1 : 500 ) Antibody Rabbit anti - Myc GeneTex Cat : GTX29106 WB ( 1 : 5000 ) IF ( 1 : 500 ) Antibody Goat anti - mouse - HRP GEHealthcare Cat : 31430 WB ( 1 : 10000 ) Antibody Goat anti - rabbit - HRP GE Health care Cat : 31460 WB ( 1 : 10000 ) Antibody Donkey anti - rabbit Alexa Flour 488 Thermo Fisher Scientific Cat : A21206 IF ( 1 : 1000 ) Antibody Donkey anti - mouse Alexa Flour 488 ThermoFisherScientific Cat : A21202 IF ( 1 : 1000 ) Antibody Goat anti - rabbit Alexa Flour 633 ThermoFisherScientific Cat : A21071 IF ( 1 : 1000 ) Antibody Donkey anti - mouse Alexa Flour 647 ThermoFisherScientific Cat : A31571 IF ( 1 : 1000 ) Cell line ( M . musculus ) B16F10 ATCC CRL - 6475 Cell line ( M . musculus ) Neuro - 2A neuroblast ATCC CCL - 131 Cell line ( M . musculus ) NIH3T3filbroblast ATCC CRL - 1658 Chemicalcompound , drug NHS - XX - Biotin Merck KGaA Cat : 203188 Chemicalcompound , drug Oregon - Green - 488 iodoacetamide Invitrogen Cat : O6010 Chemicalcompound , drug Ni2 + - NTA - agarose beads Qiagen Cat : 30230 Chemicalcompound , drug tetramethylrhodamine ( TAMRA ) (cid:0) 5 - maleimide Invitrogen Cat : T6027 Chemicalcompound , drug methoxy - poly ( ethylene glycol ) - silane LaysanBioInc Chemical compound , drug biotin - poly ( ethylene glycol ) - sil Laysan Bio Inc Continued on next page Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 16 of 28 Research article Cell Biology Continued Reagent type ( species ) or resource Designation Source or reference Identifiers Additionalinformation Chemicalcompound , drug AquaMount Thermo Fisher Scientific Cat : 14 - 390 - 5 Chemicalcompound , drug Alexa Flour 568 - phalloidin Thermo Fisher Scientific Cat : A12380 IF ( 1 : 1000 ) Chemicalcompound , drug Formaldehyde37 % SigmaAldrich Cat : 252549 Commercialassayorkit LipofectamineRNAiMAX Thermo Fisher Scientific Cat : 137780 \u2013 0775 Commercialassayorkit Lipofectamine3000 Thermo Fisher Scientific Cat : L2000 - 015 Commercial assay or kit Pierce ECL Western Blotting Substratedetectionkit Thermo FisherScientific Cat : 34580 Other DMEM - Dulbecco\u2019s Modified Eagle Medium Gibco BRL Life Technologies Cat : 11995 \u2013 073 Other FBS - Fetal Bovine Serum SigmaAldrich Cat : F9423 Other 200 mM L - glutamine ThermoFisherScientific Cat : 25030 \u2013 081 Peptide , recombinantprotein N - terminal HiLyte488mTwf1Tail Anaspec Peptide , recombinantprotein CARMILCPI WatsonBioSciences Peptide , recombinantprotein CARMILCSI WatsonBioSciences Peptide , recombinantprotein mTwf1 A305 - D350 WatsonBioSciences Peptide , recombinantprotein mTwf1A305 - D350 , K325A WatsonBioSciences Peptide , recombinantprotein PreScissionprotease GEHealthcare Cat : GE27 - 0843 - 01 RecombinantDNAreagent chickenCP a 1 b 1 Soeno et al . , 1998 Soeno et al . , 1998 Plasmid RecombinantDNAreagent chicken SNAP - CP a 1 b 1 Bombardier et al . , 2015 Bombardier et al . , 2015 Plasmid RecombinantDNAreagent mouseCP a 1 b 2 Kim et al . , 2012 Plasmid RecombinantDNAreagent mouseCP a 1 D 28 Kim et al . , 2012 Plasmid Continued on next page Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 17 of 28 Research article Cell Biology Continued Reagent type ( species ) or resource Designation Source or reference Identifiers Additionalinformation RecombinantDNAreagent mouseCP a 1 b 2 R15A / Y79A Edwards et al . , 2015 Edwards et al . , 2015 Plasmid RecombinantDNAreagent humanCARMIL1CBR115 ( 964 \u2013 1078 ) Kim et al . , 2012 Plasmid RecombinantDNAreagent humanV - 1 Edwards et al . , 2015 Plasmid RecombinantDNAreagent CARMIL1 Edwards et al . , 2013 Edwards et al . , 2013 Plasmid RecombinantDNAreagent pGEX - 6p - 1 - mTwf1 Hilton et al . , 2018 Hilton et al . , 2018 Plasmid RecombinantDNAreagent pGEX - 6p - 1 - mTwf2a Hilton et al . , 2018 Hilton et al . , 2018 Plasmid Recombinant DNA reagent pGEX - 6p - 1 - mTwf1 - 11 This paper Plasmid RecombinantDNAreagent pEGFP - C1 Clontech Plasmid RecombinantDNAreagent pCMV - M1 Addgene Cat : 23007 Plasmid RecombinantDNAreagent pCMV - myc - mTwf1 This paper Plasmid RecombinantDNAreagent pCMV - myc - mTwf1 - 11 This paper Plasmid Sequence - basedreagent siTwf1 This paper siRNA ; CGUUACCA UUUCUUUCUGUUU Sequence - basedreagent siCP1 This paper siRNA ; CCUCAGCGA UCUGAUCGACUU Sequence - based reagent siCP2 This paper siRNA ; GCACGC UGAAUGAGAUCUA Sequence - based reagent control RNAi oligos ( Stealth RNAi ) Invitrogen Cat : 12935 \u2013 200 Software , algorithm Fiji / Image J Schindelin et al . , 2012 Software , algorithm NIS Elements software - Version 4 . 30 . 02 NikonInstruments Software , algorithm GraphPadPrism6 . 0 GraphPadSoftware Software , algorithm Adobe Creative Cloud Illustrator Adobe Systems Strain , strain background ( E . coli ) BL21 ( DE3 ) pLysS This paper Strain , strain background ( E . coli ) BL21 ( DE3 ) pRIL This paper Strain , strain background ( E . coli ) BL21 ( DE3 ) pRARE This paper Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 18 of 28 Research article Cell Biology Plasmids Plasmids used for expressing the following proteins were previously described : chicken CP a 1 b 1 ( Soeno et al . , 1998 ) , chicken SNAP - CP a 1 b 1 ( Bombardier et al . , 2015 ) , mouse CP a 1 b 2 ( Kim et al . , 2012 ) , mouse CP a 1 D 28 ( Kim et al . , 2012 ) , mouse CP a 1 b 2 R15A / Y79A ( Edwards et al . , 2015 ) , human CARMIL1 CBR115 ( 964 \u2013 1078 ) ( Kim et al . , 2012 ) , human V - 1 ( Edwards et al . , 2015 ) . The plasmid for over - expressing CARMIL1 in mammalian cells has been described ( Edwards et al . , 2013 ) . To generate plasmids for expressing mouse Twinfilin isoforms as glutathione - S - transferase ( GST ) - fusions in E . coli , ORFs were PCR amplified from pHAT2 - mTwf1 and pHAT2 - mTwf2a kindly provided by Pekka Lappalainen ( Univ . of Helsinki ) ( Nevalainen et al . , 2009 ) , and subcloned into the EcoRI and NotI sites of pGEX - 6p - 1 , yielding pGEX - 6p - 1 - mTwf1 and pGEX - 6p - 1 - mTwf2a . pGEX - 6p - 1 - mTwf1 - 11 ( K332A , R333A ) was generated by site - directed mutagenesis of pGEX - 6p - 1 - mTwf1 . For V - 1 fluorescence experiments , we used a previously demonstrated strategy of removing two surface cysteine residues to allow direct labeling on the single remaining cysteine ( Fujiwara et al . , 2014 ) ; this was achieved by performing site - directed mutagenesis on wild type pGEX - GST - V - 1 plasmid to introduce two mutations ( C45S , C83S ) . To generate an RNAi - refractive construct of mTwf1 for expression in cultured cells , the ORF of mTwf1 was PCR amplified from pGEX - 6p - 1 and subcloned into the HindIII and SacI sites of pEGFP - C1 ( Clontech , Mountain View , CA ) . Then , site - directed muta - genesis was used to introduce silent mutations at specific nucleotides of the ORF ( 703 , 709 , 711 , 715 ) , and the RNAi - refractive ORF was subcloned into the EcoRI and NotI sites of pCMV - M1 , a gift from Linda Wordeman ( Stumpff et al . , 2008 ) ( Addgene plasmid # 23007 ) , yielding pCMV - myc - mTwf1 . Site - directed mutagenesis was performed on pCMV - myc - mTwf1 to generate mutant pCMV - myc - mTwf1 - 11 ( K332A , R333A ) . All constructs were verified by DNA sequencing . Protein expression and purification Rabbit skeletal muscle actin ( RMA ) ( Spudich and Watt , 1971 ) , was purified from acetone powder generated from frozen ground hind leg muscle tissue of young rabbits ( PelFreez , Rogers , AR ) . Lyophilized acetone powder stored at (cid:0) 80 \u02da C was mechanically sheared in a coffee grinder , resus - pended in G - buffer ( 5 mM Tris - HCl pH 7 . 5 , 0 . 5 mM DTT , 0 . 2 mM ATP , 0 . 1 mM CaCl 2 ) , and then cleared by centrifugation for 20 min at 50 , 000 (cid:2) g . Actin was polymerized by the addition of 2 mM MgCl 2 and 50 mM NaCl and incubated overnight at 4 \u02da C . F - actin was pelleted by centrifugation for 150 min at 361 , 000 (cid:2) g , and the pellet solubilized by Dounce homogenization and dialyzed against G - buffer for 48 hr at 4 \u02da C . Monomeric actin was then precleared at 435 , 000 (cid:2) g , and loaded onto a S200 ( 16 / 60 ) gel filtration column ( GE healthcare , Marlborough , MA ) equilibrated in G - Buffer . Peak fractions containing actin were stored at 4 \u02da C . For labeling actin with biotin ( Breitsprecher et al . , 2012 ) or Oregon Green ( OG ) ( Kuhn and Pollard , 2005 ) , the F - actin pellet described above was Dounced and dialyzed against G - buffer lacking DTT . Monomeric actin was then polymerized by add - ing an equal volume of 2X labeling buffer ( 50 mM Imidazole pH 7 . 5 , 200 mM KCl , 0 . 3 mM ATP , 4 mM MgCl 2 ) . After 5 min , the actin was mixed with a 5 - fold molar excess of NHS - XX - Biotin ( Merck KGaA , Darmstadt , Germany ) or Oregon - Green - 488 iodoacetamide ( Invitrogen , Carlsbad , CA ) resus - pended in anhydrous DMF , and incubated in the dark for 15 hr at 4 \u02da C . Labeled F - actin was pelleted as above , and the pellet was rinsed briefly with G - buffer , then depolymerized by Dounce homogeni - zation , and dialyzed against G - buffer for 48 hr at 4 \u02da C . Labeled , monomeric actin was purified further on an S200 ( 16 / 60 ) gel filtration column as above . Aliquots of biotin - conjugated actin were snap fro - zen in liquid nitrogen and stored at (cid:0) 80 \u02da C . OG - 488 - actin was dialyzed for 15 hr against G - buffer with 50 % glycerol and stored at (cid:0) 20 \u02da C . For bulk actin assembly assays , RMA was fluorescently labeled with pyrenyl - iodoacetamide on cysteine 374 ( Pollard and Cooper , 1984 ; Graziano et al . , 2013 ) . An RMA pellet stored at 4 \u02da C ( pre - pared as described above ) was dialyzed against pyrene buffer ( 25 mM Tris - HCl , pH 7 . 5 , 100 mM KCl , 0 . 02 % NaN3 , 0 . 3 mM ATP , and 2 mM MgSO4 ) for 3 \u2013 4 hr and then diluted with pyrene buffer to 1 mg / ml ( 23 . 8 m M ) . A sevenfold molar excess of pyrenyl - iodoacetamide was added , the actin solu - tion was incubated overnight at 4 \u02da C , and aggregates were cleared by low - speed centrifugation . The supernatant ( containing F - actin ) was centrifuged for 3 hr at 4 \u02da C at 45 , 000 rpm in a Ti70 rotor ( Beck - man Coulter , Indianapolis , IN ) to pellet F - actin . The actin pellets were disrupted by Douncing , dia - lyzed against G - buffer for 1 \u2013 2 d , and gel filtered on a 16 / 60 S200 column . Peak fractions were pooled , aliquoted , snap frozen , and stored at (cid:0) 80 \u02da C . Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 19 of 28 Research article Cell Biology Mouse non - muscle CP a 1 b 2 was purified as described ( Graziano et al . , 2014 ) . Briefly , the expres - sion vector ( Soeno et al . , 1998 ) was transformed into E . coli strain BL21 pLysS . Cells were grown in LB to log phase , then expression was induced for 3 hr at 37 \u02da C by addition of 0 . 4 mM isopropyl - b - D - thiogalactopyranoside ( IPTG ) . Cells were collected by centrifugation , washed with 25 ml water , and resuspended in lysis buffer ( 20 mM Tris pH 8 . 0 , 1 mM EDTA , 0 . 1 % Triton X - 100 , protease inhibitors ) and lysed by lysozyme treatment and sonication . The cell lysate was clarified by centrifugation at 12 , 500 x g for 30 min at 4 \u02da C . Supernatants were loaded onto a 1 ml Q - HiTrap column ( GE Health - care ) and eluted with a 45 ml salt gradient ( 0 \u2013 500 mM KCl ) in 20 mM Tris , pH 8 . 0 . Peak fractions were pooled , concentrated using a centrifugal filter ( Centiprep , MWCO 10 kDa ; Millipore ) to 3 ml , and loaded onto a 26 / 60 Superdex 75 gel filtration column ( GE Healthcare ) equilibrated in 50 mM KCl , 20 mM Tris , pH 8 . 0 . Peak fractions were pooled and loaded onto a 5 ml Mono Q column ( GE Healthcare ) and eluted with a 30 ml salt gradient ( 0 \u2013 500 mM KCl ) in 20 mM Tris , pH 8 . 0 . Peak frac - tions were pooled , dialyzed overnight at 4 \u02da C into HEK buffer ( 20 mM HEPES , pH 7 . 4 , 1 mM EDTA , 50 mM KCl ) , aliquoted , snap - frozen in liquid N2 , and stored at (cid:0) 80 \u02da C . SNAP - 649 - CP ( CP a 1 b 1 ) was purified and labeled as described ( Bombardier et al . , 2015 ) . SNAP - CP was expressed E . coli strain BL21 pLysS . Cells were grown to log phase at 37 \u02da C , and then expres - sion was induced for 8 hr at 37 \u02da C by addition of 0 . 4 mM isopropyl - b - D - thiogalactopyranoside ( IPTG ) . Cells were collected by centrifugation , and resuspended in 20 mM Tris pH 8 . 0 , 1 mM EDTA , 0 . 1 % Triton X - 100 , protease inhibitors and lysed by lysozyme treatment and sonication . The cell lysate was centrifuged for 80 min at 60 , 000 rpm , 4 \u02da C in a Ti70 rotor ( Beckman / Coulter , Fullerton , CA ) . The supernatant was rotated with 0 . 75 ml of Ni2 + - NTA - agarose beads ( Qiagen , Valencia , CA ) . SNAP - CP was fluorescently labelled using 9 m M ( ~ 4 - fold excess ) dye adduct for 2 hr at room temperature , yielding SNAP - 649 - CP . To remove free dye , beads were washed three times with 20 mM imidazole ( pH 8 . 0 ) , 1X PBS , 1 mM DTT , 200 mM NaCl . Labeled SNAP - 649 - CP was eluted with 0 . 5 ml of 300 mM imidazole pH 8 . 0 , 50 mM Tris pH 8 . 0 , 100 mM NaCl , 1 mM DTT , 5 % glycerol , then purified by gel filtration on a Superose six column ( GE Healthcare ) equilibrated in 20 mM Hepes ( pH 7 . 5 ) , 1 mM EDTA , 150 mM KCl , 5 % glycerol . Peak fractions were pooled , concentrated , aliquoted , snap - frozen in liquid N2 , and stored at (cid:0) 80 \u02da C . For stopped - flow kinetics , fluorescence anisotropy binding and HDX - MS experiments , His - tagged - a 1 and b 2 subunits of mouse CP ( pRSFDuet - 1 , pBJ 2041 ) were co - expressed in E . coli BL21 ( DE3 ) pRIL and purified as described ( Johnson et al . , 2018 ) . For CP lacking the b tentacle , a prema - ture stop codon was introduced , so that the C - terminal residue of the mouse b 2 subunit was L243 instead of C272 ( pBJ 1891 ) . Twinfilin polypeptides were expressed as GST - fusions in E . coli strain BL21 pRARE . Cells were grown to log phase at 37 \u02da C , and then expression was induced for 16 hr at 18 \u02da C by addition of 0 . 4 mM isopropyl - b - D - thiogalactopyranoside ( IPTG ) . Cells were collected by centrifugation , washed with 25 ml water , and resuspended in 10 ml of PBS supplemented freshly with 0 . 5 mM dithiothreitol ( DTT ) , 1 mM phenylmethylsulphonyl fluoride ( PMSF ) , and a standard mixture of protease inhibitors . Cells were incubated with lysozyme ( 0 . 5 mg ml (cid:0) 1 ) on ice for 15 min and then sonicated . The cell lysate was clarified by centrifugation at 12 , 500 g for 20 min and incubated at 4 \u02da C ( rotating ) for at least 2 hr with 0 . 5 ml glutathione \u2013 agarose beads ( Sigma - Aldrich ; St . Louis , MO ) . Beads were washed three times in PBS supplemented with 1M NaCl and then washed two times in PBS . Twinfilin was cleaved from GST by incubation with PreScission Protease ( GE Healthcare ; Marlborough , MA ) over - night at 4 \u02da C ( rotating ) . Beads were pelleted , and the supernatant was concentrated to 0 . 3 ml , and then purified further by size - exclusion chromatography on a Superose12 column ( GE Healthcare ) equilibrated in HEK buffer ( 20 mM Hepes pH 7 . 5 , 1 mM EDTA , 50 mM KCl , 0 . 5 mM DTT ) . Peak frac - tions were pooled , concentrated , aliquoted , snap - frozen in liquid N2 , and stored at (cid:0) 80 \u02da C . CARMIL CBR115 and V - 1 were purified from E . coli as above for mTwf1 proteins , except the GST tag was removed from V - 1 by digestion with thrombin instead of PreScission protease . To purify and label V - 1 ( generating TAMRA - V - 1 ) for fluorescence experiments , BL21 E . coli expressing pGEX - GST - V - 1 ( C45S , C83S ) was lysed in a Microfluidizer ( Microfluidics Corp . ; Westwood , MA ) . Fusion protein was isolated on Glutathione Superflow Agarose ( Thermo Fisher Scientific ; Waltham , MA ) . The GST tag was cleaved by digestion with bovine thrombin ( MP Biomedicals ; Santa Ana , CA ) overnight at 4 \u02da C , then separated from V - 1 on a Sephacryl S - 200 HR 16 / 60 column ( GE Healthcare ) equilibrated in 25 mM HEPES pH 7 . 0 , 1 mM TCEP , 100 mM KCl , 1 mM NaN3 . Residual GST was removed by re - incubating peak fractions with Glutathione Superflow Agarose . Purified V - 1 ( C45S , C83S ) was then Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 20 of 28 Research article Cell Biology labeled with tetramethylrhodamine ( TAMRA ) (cid:0) 5 - maleimide ( Invitrogen ) overnight at 4 \u02da C . Excess TAMRA was removed by dialysis against 20 mM 3 - ( N - morpholino ) propanesulfonic acid ( MOPS ) pH 7 . 2 , 1 . 0 mM TCEP , 100 mM KCl , 1 mM NaN3 . TAMRA - V - 1 was stored at (cid:0) 70 \u02da C The mTwf1 tail peptides used for anisotropy were sourced as follows : N - terminal HiLyte488 labeled mTwf1 ( H317 - D350 ) was purchased from Anaspec ( Fremont , CA ) ; unlabeled CARMIL1 CPI ( G969 - A1005 ) , CARMIL1 CSI ( M1019 - M1037 ) , mTwf1 ( A305 - D350 ) and mTwf1 ( A305 - D350 , K325A ) , as well as N - terminal TAMRA labeled mTwf1 ( A305 - D350 ) , were purchased from WatsonBio Sciences ( Houston , TX ) . Bulk pyrene F - actin assembly assays Pyrene actin assembly assays were performed as previously described ( Chesarone - Cataldo et al . , 2011 ) , with slight modifications for monitoring uncapping . Reactions containing 2 m M G - actin ( 5 % pyrene labeled ) , 25 nM CapZ , and variable concentrations of mTwf1 were mixed to a volume of 52 m l followed by addition of 3 m l of initiation mix ( 40 mM MgCl 2 , 10 mM ATP , 1 M KCl ) . Fluorescence was monitored at excitation and emission wavelengths of 365 and 407 nm , respectively , in a fluores - cence spectrophotometer ( Photon Technology International ; Lawrenceville , NJ ) . Acquisition was paused at 400 s , and 5 m l of CARMIL CBR ( final concentration 250 nM ) was spiked into the reaction , mixed rapidly by pipetting , and measurement was resumed . For pyrene actin elongation assays ( as in Figure 4A and B ) , 5 m l of freshly mechanically sheared F - actin ( 10 m M ) was added to a mixture of the indicated proteins or control buffers , and then imme - diately mixed with 0 . 5 m M monomeric actin ( 10 % pyrene labeled ) in 60 m l reactions and monitored in a plate reader ( Infinite M200 ; Tecan , Ma\u00a8nnedorf , Switzerland ) at excitation and emission wave - lengths of 365 and 407 nm , respectively . Fluorescence anisotropy The following anisotropy experiments were performed in HEK buffer ( 20 mM HEPES pH 7 . 5 , 1 mM EDTA , 50 mM KCl , 0 . 5 mM DTT ) . Reactions were incubated at room temperature for 15 min , and anisotropy was determined by measuring polarized emission intensities at 525 nm when excited at 497 nm using a fluorescence spectrophotometer ( Photon Technology International ) . To compare mTwf1 - tail binding to wild type and mutant CP ( Figure 1B ) , HiLyte - 488 - mTwf1 tail peptide ( 100 nM ) was mixed with different concentrations of wild - type or mutant CP . To compare the abilities of full - length wild type mTwf1 and mutant mTwf1 - 11 polypeptides to compete with labeled mTwf1 - tail for binding CP ( Figure 1C ) , HiLyte - 488 - mTwf1 tail peptide ( 100 nM ) was mixed with 1 m M CP and vari - able concentrations of full - length mTwf1 polypeptides . The following anisotropy experiments were performed in the indicated buffer , incubated at room temperature for 2 min , and anisotropy was determined by measuring polarized emission intensities at 525 nm when excited at 497 nm for HiLyte - 488 , or at 582 nm when excited at 552 nm for TAMRA . To compare mTwf1 tail peptide binding to wild type CP and mutant CP ( RY ) ( Figure 2B ) , HiLyte - 488 - mTwf1 tail peptide ( 60 nM ) was mixed with different concentrations of CP or CP ( RY ) in HEK buffer containing 0 . 005 % TWEEN 20 . To compare the abilities of unlabeled wild type and mutant mTwf1 tail peptides to compete with labeled mTwf1 tail peptide for binding to CP ( Figure 2C ) , TAMRA - mTwf1 tail peptide ( A305 - D350 , 40 nM ) was mixed with 1 m M CP and varying concentrations of the unlabeled tail peptides ( mTwf1 A305 - D350 or mTwf1 A305 - D350 , K325A ) in 20 mM MOPS ( pH 7 . 2 ) , 1 mM TCEP , 100 mM KCl , 1 mM NaN 3 , 0 . 005 % TWEEN 20 . To test the abilities of different frag - ments of CARMIL to compete with mTwf1 tail peptide for binding CP , HiLyte - 488 - mTwf1 tail pep - tide ( 60 nM ) was mixed with 240 nM CP and different concentrations of mouse CARMIL1 CBR ( 964 \u2013 1078 ) , CPI ( 969 \u2013 1005 ) , or CSI ( 1019 \u2013 1037 ) in HEK buffer containing 0 . 005 % TWEEN20 . Stopped - flow fluorescence For kinetic dissociation experiments ( as in Figure 4D and E ) , an SX . 18MV stopped flow instrument with Pro - Data SX software V2 . 2 . 27 ( Applied Photophysics Ltd . , Leatherhead , UK ) was used . 100 nM TAMRA - V - 1 was preincubated with 2 m M CP a 1 b 2 . At time zero , TAMRA - V - 1 : CP complex was rapidly mixed via stopped - flow with an equal volume of a solution containing 5 m M unlabeled V - 1 , along with varied concentrations of mTwf1 or mTwf1 - 11 . Experiments were performed at 25 \u02da C in HEK buffer containing 0 . 005 % TWEEN20 . Excitation occurred at 505 nm , with emission detected using a Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 21 of 28 Research article Cell Biology 570 + nm band - pass filter . All concentrations of mTwf were performed in replicates of 5 \u2013 10 , and traces were averaged . Apparent dissociation rates were determined by fitting the averaged data ( 5 ms . - 120 s . ) to a single exponential model using Pro - Data Viewer software V4 . 2 . 27 ( Applied Photo - physics Ltd . ) . Total internal reflection fluorescence ( TIRF ) microscopy For all experiments , 24 (cid:2) 60 mm coverslips ( Fisher Scientific ; Pittsburg , PA ) were cleaned by succes - sive sonications as follows : 60 min in detergent , 20 min in 1 M KOH , 20 min in 1 M HCl min , and 60 min in ethanol . Coverslips were then washed extensively with ddH 2 O and dried in an N 2 - stream . A solution of 80 % ethanol pH 2 . 0 , 2 mg / ml methoxy - poly ( ethylene glycol ) - silane and 2 m g / ml biotin - poly ( ethylene glycol ) - silane ( Laysan Bio Inc . ; Arab , AL ) was prepared and layered on the cleaned coverslips ( 200 m l per coverslip ) . The coverslips were incubated for 16 hr at 70 \u02da C . To assemble flow cells , PEG - coated coverslips were rinsed extensively with ddH 2 O and dried in an N 2 - stream , then attached to a prepared flow chamber ( Ibidi ; Martinsried , German ) with double sided tape ( 2 . 5 cm x 2 mm x 120 m m ) and five min epoxy resin . Flow cells were prepared immediately before use by sequential incubations as follows : 3 min in HEK - BSA ( 20 mM Hepes pH 7 . 5 , 1 mM EDTA , 50 mM KCl , 1 % BSA ) , 30 s in Streptavidin ( 0 . 1 mg / ml in PBS ) , a fast rinse in HEK - BSA , and then equilibration in 1X TIRF buffer , pH 7 . 5 ( 10 mM imidazole , 50 mM KCl , 1 mM MgCl 2 , 1 mM EGTA , 0 . 2 mM ATP , 10 mM DTT , 15 mM glucose , 20 m g / ml catalase , 100 m g / ml glucose oxidase , and 0 . 5 % methylcellulose ( 4000 cP ) ) . To initiate reactions , actin monomers ( 10 % OG - labeled , 0 . 5 % biotinylated ) were diluted to 1 m M in TIRF buffer , and immediately transferred to a flow chamber . After several minutes , once the actin filaments reached an appropriate length ( approximately 10 m m ) , the reaction mixture was replaced by flow - in . For depolymerization experiments , the solution was replaced with TIRF buffer lacking actin monomers , with or without Twinfilin and / or CP polypeptides . For uncapping experi - ments , the solution was replaced with TIRF buffer lacking actin monomers , with 3 nM SNAP - 649 - CP ( 100 % labeled ) , and filaments were allowed to be capped for 3 min . Subsequently , the solution was again replaced with TIRF buffer lacking actin monomers , with or without 50 nM CARMIL CBR and / or variable concentration of Twinfilin polypeptides . Time - lapse TIRF microscopy was performed using a Nikon - Ti200 inverted microscope equipped with a 150 mW Ar - Laser ( Mellot Griot ; Carlsbad , CA ) , a 60X TIRF - objective with a N . A . of 1 . 49 ( Nikon Instruments Inc . ; New York , NY ) , and an EMCCD cam - era ( Andor Ixon ; Belfast , Northern Ireland ) . During recordings , optimal focus was maintained using the perfect focus system ( Nikon Instruments Inc ) . Images were captured every 5 s . The pixel size cor - responded to 0 . 27 m m . Filament depolymerization rates were determined by tracing filaments in ImageJ ( http : / / rsbweb . nih . gov / ij ) and measuring the change in length of individual filaments for 15 \u2013 20 min after flow - in , or until filaments disappeared . Differences in fluorescence intensity along the length of the filament provided fiduciary marks that allowed us to distinguish barbed - and pointed - ends . Filament uncap - ping was measured by monitoring the as the amount of time that SNAP - 649 - CP puncta remained associated with the barbed end of a filament after the addition of CARMIL to the reaction ( with or without Twinfilin ) and expressing it as a fraction of filaments that remained capped at a given time point . All results shown are data from at least two independent TIRF experiments . Cell culture , transfection , and RNAi silencing Mouse B16 - F10 ( CRL - 6475 ) , Neuro - 2a ( CCL - 131 ) , and NIH / 3T3 ( CRL - 1658 ) cells obtained directly from ATCC ( American Type Culture Collection ; Manassas , VA ) , where their identities were authenti - cated by short tandem repeat DNA profiling and where they were tested for mycoplasma contami - nation . Cells were used for experiments within one year . All cells were grown in DMEM ( Gibco BRL Life Technologies ; Carlsbad , CA ) supplemented with 10 % fetal bovine serum ( FBS ; Sigma ) and 200 mM L - glutamine ( Thermo Fisher Scientific ) at 37 \u02da C and 5 % CO 2 . All cell culture experiments were carried out in 6 - well dishes that were initially seeded with 100 , 000 cells . To knockdown Twinfilin - 1 or Capping Protein cells were transfected 24 hr after seed - ing with 30 pmol siRNA oligo using Lipofectamine RNAiMAX ( Thermo Fisher Scientific ) according to the manufacturer\u2019s instructions . RNAi oligos directed against the mouse Twinfilin - 1 coding region targeting ( siTwf1 ) 5\u2019 - CGUUACCAUUUCUUUCUGUUU (cid:0) 3\u2019 ; and against the Capping Protein b sub - unit coding region targeting ( siCP1 ) 5\u2019 - CCUCAGCGAUCUGAUCGACUU - 3\u2019 , or ( siCP2 ) 5\u2019 - GCACGC Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 22 of 28 Research article Cell Biology UGAAUGAGAUCUA - 3\u2019 . Cells were transfected in parallel with control RNAi oligos ( Invitrogen ) . For over expression experiments cultured cells were transfected using Lipofectamine 3000 ( Thermo Fisher Scientific ) according to the manufacturer\u2019s instructions 24 hr after seeding . For CARMIL over expression experiments , 5 m G of DNA was transfected , and for Twinfilin over expression experi - ments 1 m G of DNA was transfected . Antibodies The rabbit anti - Twinfilin was a generous gift from Pekka Lappalainen ( Univ . Helsinki ) and used a dilu - tion of 1 : 1000 for western blot detection and 1 : 100 in cultured cells . A mouse anti - Capping Protein ( Development Studies Hybridoma Bank ; Iowa City , IA ) was used at a dilution of 1 : 2000 for western blot detection and 1 : 50 in cultured cells . Mouse anti - Flag ( F3165 , Sigma ) and rabbit anti - Myc ( GTX29106 , GeneTex ; Irvine , CA ) was used at 1 : 5000 for western blot detection and 1 : 500 in cul - tured cells . Mouse and Rabbit horseradish peroxidase conjugated secondary antibodies ( GE Health - care ) were used at a dilution of 1 : 10 , 000 for western blot detection . Secondary antibodies for immunofluorescence ( Alexa Fluor 488 or 647 ) and Alexa Fluor 568 - phalloidin ( ThermoFisher ) were used at a dilution of 1 : 1000 . Immunostaining cells For cell - staining experiments , 48 hr post transfection , the cells were re - plated on 3 (cid:2) 1 (cid:2) 1 mm glass coverslip ( VWR International ) that had been acid washed and coated with Laminin ( Invitrogen ) and allowed to adhere for 3 \u2013 6 hr . Cells were fixed for 15 min with 4 % paraformaldehyde in PBS at room temperature and then permeabilized for 15 min in permeabilization solution ( 0 . 5 % Triton X - 100 and 0 . 3 M glycine in PBS ) at room temperature . Slips were then blocked in 3 % BSA dissolved in PBST ( 1X PBS and 0 . 1 % TWEEN 20 ) for 1 hr at room temperature , then incubated in primary antibody ( in PBST ) for 12 hr at 4 \u02da C . Coverslips were then washed three times with 1X PBST and incubated with secondary antibodies ( in PBST ) for 1 hr at room temperature . Slips were washed three times with PBST and two times with PBS , and subsequently mounted on to slides with AquaMount ( Thermo Fisher Scientific ) . Cells were imaged on a Nikon i - E upright confocal microscope equipped with a CSU - W1 spinning disk head ( Yokogawa , Tokyo , Japan ) , 60x oil objective ( NA 1 . 4 ; Nikon Instru - ments ) , and an Ixon 897 Ultra - CCD camera ( Andor Technology ) controlled by NIS - Elements soft - ware . Maximum intensity projections and raw fluorescence values were measured using Fiji . Western blotting To measure protein levels in cells after silencing and rescue , cells were harvest 48 hr after initial oligo transfection and incubated for 10 min at 4 \u02da C in RIPA buffer ( 50 mM Tris , pH 7 . 5 , 150 mM NaCl , 1 % NP - 40 , 0 . 5 % Na - deoxycholate , 0 . 1 % SDS , 2 mM EDTA , 50 mM NaF ) . Samples were incubated on ice for 30 min , vortexed every 10 min , then precleared by centrifugation at 20 , 800 x g for 15 min at 4 \u02da C , quantified by Bradford assay , and immunoblotted . Proteins were detected using a Pierce ECL West - ern Blotting Substrate detection kit ( Thermo Fisher Scientific ) . Bands were quantified using Image - Lab ( Biorad ) . Hydrogen deuterium exchange mass spectrometry ( HDX - MS ) HDX - MS was performed as described ( Johnson et al . , 2018 ) . CP and Twf1 samples were buffer - exchanged with 1X phosphate saline buffer ( PBS ) , pH 7 . 4 . HDX was initiated by diluting samples ( 25 m M , 2 m L ) 10 - fold with 1XPBS prepared in D 2 O buffer , or 1XPBS H 2 O buffer for samples measured for no - deuterium control . At different time intervals ( 10 , 30 , 60 , 120 , 360 , 900 , 3600 , and 14400 s ) , the labeling reaction was quenched by rapidly decreasing the pH to 2 . 5 with 30 m L of quench buffer ( 3 M urea , 1 % trifluoroacetic acid , H 2 O ) at 4 \u02da C . The protein mixture was immediately injected into a custom - built HDX sample - handling device that enabled digestion with a column containing immobi - lized pepsin ( 2 mm (cid:2) 20 mm ) at a flow rate of 100 m L / min in 0 . 1 % formic acid . The resulting peptic peptides were captured on a ZORBAX Eclipse XDB C8 column ( 2 . 1 mm (cid:2) 15 mm , Agilent ) for desalt - ing ( 3 min ) . The C8 column was then switched in - line with a Hypersil Gold C18 column ( 2 . 1 mm (cid:2) 50 mm , Thermo Fisher ) , and a linear gradient ( 4 \u2013 40 % acetonitrile , 0 . 1 % formic acid , 50 m L / min flow rate , over 5 min ) was used to separate the peptides and direct them to an LTQ - FTICR mass spec - trometer ( Thermo Fisher ) equipped with an electrospray ionization source . Valves , columns , and Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 23 of 28 Research article Cell Biology tubing for protein digestion and peptide separation were immersed in an ice - water bath to minimize back - exchange . To map the peptic peptides , the digest , in the absence of HDX , was submitted to accurate mass analysis by LC \u2013 MS / MS with the LTQ - FTICR , and the peptic peptides identified using Mascot ( Matrix Science ) . For samples that underwent HDX , raw mass spectra and peptide sets were submitted to HDX Workbench ( Pascal et al . , 2012 ) for calculation and data visualization in a fully automated fash - ion . Peptides for each run were assessed based on relative representation and statistical validation as implemented within HDX Workbench . Appropriate approach to determine statistical significance between these data is by using Tukey\u2019s multiple comparison test . A representative time point was manually selected , replicate data points from multiple samples at this time point used to conduct a one - way analysis of variance ( ANOVA ) the divergence between the means of the experiments were assessed . In instances with large differences , Tukey method was used to determine statistical signifi - cance if the resulting P value is less than 0 . 05 . In the case where there was a comparison between two experiments , a t - test was used . Only the top six peptides from each MS scan were used in the final analysis . The extent of HDX at each time point was calculated by subtracting the centroid of the isotopic distribution of the nondeuterated peptide from that of the deuterated peptide . The relative deuterium uptake was plotted versus the labeling time to yield kinetic curves ( % D vs time ) . Error bars represent the results of t - tests between samples are shown above each time point to illustrate statistical significance . For comparison between apo states and the complexes , differences in HDX for all time points were calculated . Absolute differences in perturbation values larger than 5 % D were considered significant . HDX values at 15 min time point were mapped onto the protein three - dimensional ( 3D ) structure for data visualization . Peptide digestions were optimized under HDX assay conditions , and the mass calculations included accommodation for back exchange with solvent . Acknowledgements We are grateful to Julian Eskin , Sean Guo , Silvia Jansen , M Angeles Juanes , and Daisy Leung for helpful discussions and / or comments on the manuscript . We thank Ms . S Smith and Ms . M Torres for general support and coordination . In addition , we thank Steve DelSignore for help with analysis of cultured cells , Alex Kozlov and Timothy Lohman for instrument access , assistance , and advice on stopped - flow experiments , and Marlene Mekel for assistance and advice with biochemistry experi - ments . This work was supported by a grant from the NIH ( R01 GM063691 ) to BLG , a grant from the NIH ( R35 GM118171 ) to JAC . , by Brandeis NSF MRSEC DMR - 1420382 , by grants from NIH ( P01AI120943 and R01AI123926 ) and a grant from the Department of the Defense ( Defense Threat Reduction Agency grant HDTRA1 - 16 - 1 - 0033 ; C Basler PI ) to GKA . We thank Dr . Michael L Gross for the use of the mass spectrometry facilities supported by P41GM103422 and for facilitating the mass spectrometry studies . The content of the information does not necessarily reflect the position or the policy of the federal government , and no official endorsement should be inferred . Additional information Funding Funder Grant reference number Author National Institutes of Health R01 GM063691 Bruce L Goode Defense Threat Reduction Agency HDTRA1 - 16 - 1 - 0033 Gaya K Amarasinghe National Institutes of Health R35 GM118171 John A Cooper National Science Foundation MRSEC DMR - 1420382 Bruce L Goode National Institutes of Health P01 AI120943 Gaya K Amarasinghe National Institutes of Health R01 AI123926 Gaya K Amarasinghe The funders had no role in study design , data collection and interpretation , or the decision to submit the work for publication . Johnston et al . eLife 2018 ; 7 : e41313 . DOI : https : / / doi . org / 10 . 7554 / eLife . 41313 24 of 28 Research article Cell Biology Author contributions Adam B Johnston , Conceptualization , Data curation , Formal analysis , Validation , Investigation , Writ - ing\u2014original draft ; Denise M Hilton , Conceptualization , Data curation , Formal analysis , Validation , Investigation , Writing\u2014original draft , Writing\u2014review and editing ; Patrick McConnell , Britney John - son , Data curation , Formal analysis , Validation , Writing\u2014review and editing ; Meghan T Harris , Avital Simone , Data curation , Formal analysis ; Gaya K Amarasinghe , Conceptualization , Formal analysis , Supervision , Funding acquisition , Investigation , Writing\u2014original draft , Project administration , Writ - ing\u2014review and editing ; John A Cooper , Conceptualization , Supervision , Funding acquisition , Vali - dation , Writing\u2014original draft , Project administration , Writing\u2014review and editing ; Bruce L Goode , Conceptualization , Supervision , Funding acquisition , Writing\u2014original draft , Project administration , Writing\u2014review and editing Author ORCIDs Adam B Johnston http : / / orcid . org / 0000 - 0002 - 1210 - 4929 Denise M Hilton http : / / orcid . org / 0000 - 0003 - 1577 - 1855 John A Cooper http : / / orcid . org / 0000 - 0002 - 0933 - 4571 Bruce L Goode https : / / orcid . org / 0000 - 0002 - 6443 - 5893 Decision letter and Author response Decision letter https : / / doi . org / 10 . 7554 / eLife . 41313 . 017 Author response https : / / doi . org / 10 . 7554 / eLife . 41313 . 018 Additional files Data availability All datasets associated with this article are included in the manuscript and supporting files . 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Vol . 190 No . 4 675 \u2013 691 www . jcb . org / cgi / doi / 10 . 1083 / jcb . 201002119 JCB 675 Correspondence to Robert G . Parton : r . parton @ imb . uq . edu . au M . Kirkham\u2019s present address is Department of Cell and Molecular Biology , Karolinska Institute , 17177 Stockholm , Sweden . J . Riches\u2019 present address is European Molecular Biology Laboratory , Meyerhofstrasse 1 , 69117 Heidelberg , Germany . K . Cortese\u2019s present address is Centro di Ricerca MicroSCoBio / IFOM , FIRC Institute of Molecular Oncology , Universit\u00e0 di Genova , 16126 Genova , Italy . F . Simpson\u2019s and M . M . Hill\u2019s present address is The University of Queensland , Diamentina Institute for Cancer , Immunology and Metabolic Medicine , Brisbane , Queeensland 4102 , Australia . R . Lundmark\u2019s present address is Department of Medical Biochemistry and Biophysics , Ume\u00e5 University , 901 87 Ume\u00e5 , Sweden . Abbreviations used in this paper : AA , ascorbic acid ; CIE , clathrin - independent endocytosis ; CLIC , clathrin - independent carrier ; CME , clathrin - mediated endo - cytosis ; CTxB , cholera toxin B subunit ; GPI - AP , glycosylphosphatidylinositol - anchored protein ; MEF , mouse embryonic fibroblast ; PM , plasma membrane ; RE , recycling endosome ; Tf , transferrin ; WT , wild type . Introduction Endocytosis provides a crucial and dynamic interface between the extracellular milieu and the interior of the cell . This interface is paramount for fluid and nutrient uptake , signaling regulation , lipid homeostasis , plasma membrane ( PM ) remodeling , synaptic vesicle recycling , and cellular motility ( Grande - Garc\u00eda et al . , 2007 ; Mayor and Pagano , 2007 ; Idone et al . , 2008b ; Sigismund et al . , 2008 ) . Correspondingly , the endocytic system entails a rich diversity of mechanisms coordinated across several pathways . Mounting evidence suggests that eukaryotic cells maintain up to five distinct , constitutive pinocytic pathways ( excluding A lthough the importance of clathrin - and caveolin - independent endocytic pathways has recently emerged , key aspects of these routes remain un - known . Using quantitative ultrastructural approaches , we show that clathrin - independent carriers ( CLICs ) ac - count for approximately three times the volume internal - ized by the clathrin - mediated endocytic pathway , forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts . Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature . Proteomic analysis pro - vides direct links between CLICs , cellular adhesion turn - over , and migration . Consistent with this , CLIC - mediated endocytosis of key cargo proteins , CD44 and Thy - 1 , is polarized at the leading edge of migrating fibroblasts , while transient ablation of CLICs impairs their ability to migrate . These studies provide the first quantitative ultra - structural analysis and molecular characterization of the major endocytic pathway in fibroblasts , a pathway that provides rapid membrane turnover at the leading edge of migrating cells . Clathrin - independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells Mark T . Howes , 1 , 2 Matthew Kirkham , 1 James Riches , 2 Katia Cortese , 1 , 2 Piers J . Walser , 1 Fiona Simpson , 1 Michelle M . Hill , 1 Alun Jones , 1 Richard Lundmark , 3 Margaret R . Lindsay , 1 , 2 Delia J . Hernandez - Deviez , 1 Gordana Hadzic , 4 Adam McCluskey , 4 Rumasia Bashir , 5 Libin Liu , 6 Paul Pilch , 6 Harvey McMahon , 3 Phillip J . Robinson , 7 John F . Hancock , 1 Satyajit Mayor , 8 and Robert G . Parton 1 , 2 1 The Institute for Molecular Bioscience and 2 The Centre for Microscopy and Microanalysis , The University of Queensland , Brisbane , Queensland 4072 , Australia 3 MRC Laboratory of Molecular Biology , Cambridge CB2 0QH , England , UK 4 Chemistry , School of Environmental and Life Sciences , The University of Newcastle , Callaghan , NSW 2308 , Australia 5 School of Biological and Biomedical Sciences , Durham University , Durham DH1 3HP , England , UK 6 Department of Biochemistry , Boston University School of Medicine , Boston , MA 02118 7 Children\u2019s Medical Research Institute , The University of Sydney , Sydney , NSW 2145 , Australia 8 National Centre for Biological Science ( TIFR ) , Bangalore 560 065 , India \u00a9 2010 Howes et al . This article is distributed under the terms of an Attribution \u2013 Noncommercial \u2013 Share Alike \u2013 No Mirror Sites license for the first six months after the pub - lication date ( see http : / / www . rupress . org / terms ) . After six months it is available under a Creative Commons License ( Attribution \u2013 Noncommercial \u2013 Share Alike 3 . 0 Unported license , as described at http : / / creativecommons . org / licenses / by - nc - sa / 3 . 0 / ) . T H E J O U R N A L O F C E L L B I O L O G Y D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 676 Results CLICs account for the major constitutive uptake of fluid and bulk membrane in fibroblasts Quantitation of the volume and surface area internalizing through the CLIC pathway is vital for understanding the contribution of this route to total endocytosis and PM homeostasis . Generic en - docytic markers that enter via CME , caveolae , and CIE routes , such as HRP or CTxB , have been used in conjunction with ultra - structural morphology and dynamin independence to define CLICs ( Kirkham et al . , 2005 ) . Therefore , we first used HRP - conjugated CTxB ( CTxB - HRP ) as an endocytic membrane marker with a previously characterized diaminobenzidine ( DAB ) incubation protocol for EM visualization ( Kirkham et al . , 2005 ) . This method reveals only internalized carriers and can be used to label such carriers within seconds of scission . Although CTxB - HRP is an excellent membrane marker , a multivalent toxin could theoretically affect endocytosis , particularly when combined with an incubation step at low temperature ( R\u00f6mer et al . , 2007 ) . Therefore , quantitative measurements of the very early uptake of the fluid phase marker , HRP ( at 20 mg . ml 2 1 ) , were compared with CTxB - HRP and assessed in the presence or absence of unlabeled CTxB ( Fig . 1 , A and B ) to determine the relative contribution of CME and CLICs in both wild - type ( WT ) and caveolin1 - null ( Cav1 2 / 2 ) mouse embryonic fibroblasts ( MEFs ) . These experiments allowed us to eliminate a cold step and showed that HRP and CTxB - HRP labeled identical compart - ments , whereas CTxB did not quantitatively affect endocytosis ( Fig . 1 , C \u2013 E ) . A defining characteristic of the CLIC pathway is indepen - dence of dynamin for PM scission ( Damke et al . , 1994 ; Parton et al . , 1994 ; Henley et al . , 1998 ; Lamaze et al . , 2001 ; Pelkmans et al . , 2002 ; Sabharanjak et al . , 2002 ; Kirkham et al . , 2005 ) . Therefore , we used a well - characterized small molecule inhibi - tor of dynamin , dynasore ( unpublished data ) , as well as a more potent , hydroxylated analogue of dynasore , Dyngo4a ( unpub - lished data ) to inhibit CME , caveolar endocytosis , and dynamin - dependent CIE pathways . The activity of these inhibitors in WT and Cav1 2 / 2 MEFs was confirmed by assessing the fluorescence uptake of the CME cargo protein transferrin ( Tf - 647 ) and the generic endocytic marker CTxB - 555 ( Fig . 1 F ) , as well as visu - alization of CTxB - HRP \u2013 labeled carriers by EM ( Fig . 1 G ) . Although Tf - 647 and CTxB - 555 were both efficiently internal - ized in untreated control cells , after treatment with 30 \u00b5M Dyngo4a ( or 80 \u00b5M dynasore ) only CTxB - 555 internaliza - tion occurred . In agreement with this observation , in untreated control cells , CTxB - HRP labeling was found in both CLICs ( Fig . 1 G , arrows ) and clathrin - coated vesicles ( CCVs ; Fig . 1 G , double arrowheads ) , visualized by EM . However , in the pres - ence of Dyngo4a , only CLICs , defined by ultrastructure , were active ( Fig . 1 G , arrows ) . Unlabeled clathrin - coated pits ( CCPs ) were readily observed but were surface connected ( Fig . 1 G , arrowheads ) . Together , these data show that CTxB is a useful marker for CLICs and , when combined with acute dynamin inhibition using Dyngo4a , CLICs remain the only detectably active endocytic pathway . phagocytosis and macropinocytosis ; Doherty and McMahon , 2009 ) . These are clathrin - mediated endocytosis ( CME ) , caveolae , and three noncaveolar clathrin - independent endocytic ( CIE ) routes , which are defined based on specific regulation by RhoA ( Lamaze et al . , 2001 ) , Arf6 ( Radhakrishna and Donaldson , 1997 ) , or Cdc42 ( Sabharanjak et al . , 2002 ) . Many of the cargo and regula - tors of the classical CME and caveolae pathways have been identi - fied and the dynamics of these pathways are the best understood ( for reviews see Conner and Schmid , 2003 ; Parton and Simons , 2007 ) . Comparatively , very little is known about the mechanisms governing CIE , although recent work has emphasized the crucial importance of these pathways ( Mayor and Pagano , 2007 ) . The existence of CIE pathways was first proposed from the study of plant and bacterial toxin internalization , in combi - nation with ultrastructural methods or with tools that inhibited CME ( Montesano et al . , 1982 ; Sandvig et al . , 1987 ; Ricci et al . , 2000 ) . The plant toxin ricin was used to first describe the clathrin - independent , apical endocytic pathway in endothelial cells ( Sandvig and van Deurs , 1994 ) . Bacterial toxins , such as the Helicobacter pylori VacA toxin ( Gauthier et al . , 2007 ) and chol - era toxin ( Torgersen et al . , 2001 ) provided further evidence for clathrin - independent endocytic pathways . However , a lack of specific tools to study these noncanonical routes has made it difficult to identify their guiding parameters . Ultrastructural work on the CIE pathway regulated by Cdc42 has identified the morphologically distinct clathrin - independent carriers ( CLICs ) as the primary carriers involved in uptake within this route ( Kirkham et al . , 2005 ) . Parameters currently identified for the CLIC pathway include dynamin - independent PM scission ( Sabharanjak et al . , 2002 ) , enrichment in GPI - anchored proteins ( Sabharanjak et al . , 2002 ) , reliance on Arf1 activity ( Sabharanjak et al . , 2002 ; Kumari and Mayor , 2008 ) , sensitivity to cholesterol depletion ( Kirkham et al . , 2005 ; Chadda et al . , 2007 ) , and contribution to a significant fraction of cholera toxin B subunit ( CTxB ) and fluid internalization ( Kirkham et al . , 2005 ) . CLICs arise directly from the PM and mature into the GPI - enriched early endosomal compartment ( GEEC ) , as they acquire Rab5 and EEA - 1 and , consequently , merge with early endosomes ( EEs ; Kalia et al . , 2006 ) . Ongoing work is linking cargo and regulators to these carriers , such as GTPase regulator associated with FAK ( GRAF1 ) , dysferlin , and Snx9 ( Yarar et al . , 2007 ; Hern\u00e1ndez - Deviez et al . , 2008 ; Lundmark et al . , 2008 ) . The basic parameters of these carriers , however , remain unknown . Here we characterize key parameters of the CLIC pathway . A quantitative analysis of the volume and surface area of CLICs identifies them as the major constitutive pathway involved in bulk endocytosis within fibroblasts . High resolution electron to - mography shows the complexity of these primary endocytic car - riers and , in conjunction with recruitment of classical trafficking regulators , they are realized to be intricate sorting compartments . Identification of novel CLIC cargo suggests links to cellular ad - hesion turnover and plasma membrane repair . Consistent with this , CLICs become polarized in migrating fibroblasts , turning over adhesion complex components , such as Thy - 1 and CD44 , at the leading edge . Inhibition of the CLIC pathway subsequently impairs the capacity of fibroblasts to migrate . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 677 Characterization of the CLIC pathway \u2022 Howes et al . Figure 1 . CTxB does not affect CLIC endocytosis . ( A ) Cav1 2 / 2 MEFs were incubated with HRP in the presence or absence of CTxB for 15 s . Cells were cooled and DAB reaction perfomed in the presence of ascorbic acid ( AA ) before fixation . Bars , 200 nm . ( B ) Quantitation of the number of HRP - filled carriers per cell across 10 \u2013 12 cells treated as in A . CLICs were defined by their characteristic ring - shaped morphology , CCVs were defined as coated vesicular carriers . Error bars show SEM . ( C ) Cav1 2 / 2 MEFs were grown in normal media ( plus energy ) , media containing 2 - deoxyglucose ( no energy ) , or 2 - deoxyglucose media for 1 h followed by a 1 - h washout in normal media ( recovery ) before CTxB - HRP internalization and DAB reaction . Labeled structures were counted across 10 cells . Error bars show SEM . ( D and E ) Cav1 2 / 2 MEFs were incubated with CTxB - HRP for 5 min before the DAB reaction for either 5 min at 37\u00b0C or 10 min at 4\u00b0C , followed by fixation at 37\u00b0C . All CTxB - HRP \u2013 positive structures were counted in 10 \u2013 12 cells across two independent experiments . Error bars show SEM . Bar , 200 nm . ( F ) NIH3T3s were treated for 20 min with vehicle ( Untreated ) or Dyngo4a before internalization of CTxB - 555 ( left panels ) and Tf - 647 ( right panels ) for 5 min . Cells were placed on ice and acid stripped to remove surface labeling . Cells were then bound with CTxB - 488 ( middle panels ) . Bar , 10 \u00b5m . ( G ) NIH3T3 cells were left untreated or were treated with Dyngo4a before internalization of CTxB - HRP , followed by DAB reaction . Arrows show CTxB - HRP \u2013 laden CLICs , double arrowheads show internalized , labeled CCVs , arrowheads show surface connected unlabeled CCPs . Bars , 200 nm . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 678 We next estimated the contribution of this pathway to total plasma membrane surface endocytosis . By measuring the surface - to - volume ratio ( S ( v ) ; see Materials and methods ) of a typical CLIC ( Fig . 2 F ) and the absolute volume of an average Cav1 2 / 2 MEF ( 2 , 346 . 7 \u00b1 289 . 3 \u00b5m 3 ; see Materials and meth - ods ) , the compiled membrane surface area of CLICs was calcu - lated after 15 s of internalization to be 97 . 1 \u00b1 10 . 9 \u00b5m 2 . Given that the estimated average PM surface area of a Cav1 2 / 2 MEF was found to be 4 , 375 . 7 \u00b5m 2 ( see Materials and methods ) , the equivalent of the total PM surface area could internalize through CLICs in less than 12 min ( or 17 min after most extreme correc - tion factor ; see Materials and methods ) . Together these data indicate that CLICs provide a substantial capacity of internal - ization , more than any other constituite endocytic pathway in fibroblast cells . CLICs exhibit the properties of a complex sorting compartment Aside from the 2D morphological description of tubular CLICs , there is currently no data describing the 3D attributes of the pri - mary carriers . To comprehensively visualize the ultrastructural properties of CLICs , high resolution electron tomography was performed on CTxB - HRP \u2013 laden structures ( Fig . 3 A ; Video 1 ) . As a membrane marker , CTxB - HRP provided superior delinea - tion of membrane profiles compared with free HRP at the con - centrations and very early time points used here . CTxB - HRP was internalized for only 15 s to capture the earliest morphology of the carriers . The DAB reaction was performed on live cells to stabilize the morphology of the structures before glutaraldehyde fixation , simultaneously providing the necessary contrast for high resolution tomography and maintaining a close - to - native morphology . Morphology of DAB - labeled structures was simi - lar in cells fixed before DAB reaction ( not depicted ) . Images of 3D rendering from electron densities are shown ( Fig . 3 A ) . There is clear evidence of vesicular membrane invaginations ( Fig . 3 A , arrows ) , excluded from CTxB - HRP labeling , indicating that CLICs may have a capacity to bud internal membranes . Addi - tionally , the tubular structure forms a complete ring , a facet pre - viously overlooked ( Fig . 3 A , arrowhead ) . As chemical fixatives can alter the morphology of endosomes ( Murk et al . , 2003 ) , it was also important to confirm these observations without the use of aldehyde fixation . Therefore , CTxB - HRP was internalized for 15 s , DAB reaction performed , and the samples were high pres - sure frozen ( HPF ) as described previously ( Richter et al . , 2008 ) . HPF CTxB - HRP \u2013 laden CLICs showed identical features to chemically fixed samples ( Fig . 3 B ) , with vesicular invaginations from the limiting membrane and tubular ring - shaped extensions . Rather than simple tubules or vesicles , these primary carriers have a surprising degree of complexity . Identification of novel CLIC - associated proteins To identify CLIC associated components , we next sought to en - rich CLICs away from other cellular material by density gradi - ent fractionation ( see Materials and methods ) . To increase the number of CLICs within a population of cells , the PI 3 K inhibi - tor wortmannin was used within a previously developed system Having established the constitutive nature of the path - way and lack of detectable perturbation by the used markers , we used EM - based stereology to calculate the proportion of total cytoplasmic volume ( V ( v ) ) contributed by the CLIC pathway . We used both Cav1 2 / 2 and WT MEFs to compare cells with and without caveolae , as well as a fibroblast cell line , NIH3T3 ( Fig . 2 A ) . In untreated Cav1 2 / 2 MEFs , total CTxB - labeled structures occupied 0 . 10 \u00b1 0 . 01 % of the total cytoplasmic volume after 15 s of internalization , 0 . 19 \u00b1 0 . 01 % after 1 min , and 0 . 50 \u00b1 0 . 04 % after 2 min ( Fig . 2 B , Untreated ) . WT MEFs internalized similar volumes as Cav1 2 / 2 MEFs , in - dicating that caveolae contributed an insignificant proportion of internalization at this time . Intriguingly , when treated with Dyngo4a , under conditions where uptake of Tf is completely blocked and CLICs remain the only detectably active pathway , internalized CTxB - HRP still accounted for 0 . 09 \u00b1 0 . 01 % of cytoplasmic volume after 15 s , 0 . 16 \u00b1 0 . 02 % after 1 min , and 0 . 37 \u00b1 0 . 02 % after 2 min ( Fig . 2 B , Dyngo4a ) . This indicates that 90 . 0 % of the total endocytic volume is contributed by the CLIC pathway after 15 s of uptake , 85 . 9 % after 1 min , and 74 . 0 % after 2 min . Confirmation of such high contribution of CLICs to total internalization was provided using strict ultrastructural criteria to compare the volume of tubular / ring - shaped structures ( CLICs ) and coated / vesicular structures ( CCVs ) in NIH3T3 cells that were not treated with any inhibitor ( Fig . 2 C ) . Note that previ - ous studies have shown that at the earliest times used here , Tf - HRP only labels CCVs as defined morphologically ( Kirkham et al . , 2005 ) but CTxB - HRP labels CCVs , caveolae , and CLICs . NIH3T3 cells internalized similar volumes to primary MEFs , with total CTxB - labeled structures after 2 min accounting for 0 . 47 \u00b1 0 . 014 % of cytoplasmic volume and tubular / ring ele - ments ( CLICs ) contributing 78 . 7 % ( 0 . 37 \u00b1 0 . 014 % ) of that vol - ume ( Fig . 2 C ) . This alternative method avoids the need for inhibition of the dynamin - dependent pathways and suggests that under these conditions CLICs are not a compensatory pathway induced after acute dynamin inhibition ( Damke et al . , 1995 ) . To confirm the striking contribution of the CLIC pathway to cellular internalization , V ( v ) measurements were next collected using free HRP , which cannot bind to mem - branes and induce clustering of microdomains ( Fig . 2 D ) . Again , CLICs , morphologically defined as tubules / rings , con - stituted \ue07a 74 % of all HRP internalization after 2 min ( V ( v ) 0 . 32 \u00b1 0 . 05 % ) . To further confirm these results using an alternative method for assessment of endocytic contribution , CTxB was biotinyl - ated and internalized in Cav1 2 / 2 MEFs , in the presence or ab - sence of Dyngo4a . Surface - accessible biotin was cleaved and the remaining , internalized biotin measured by Western blot using streptavidin - HRP . Using this alternative biochemcial method CTxB - biotin levels in Dyngo4a - treated cells were equivalent to 76 . 0 \u00b1 8 . 4 % of that in untreated control cells after 2 min of uptake ( Fig . 2 E ) . Agreement between morphometric and biochemcial approaches ( and with V ( v ) measurements using a specific marker of the CLIC pathway : see below ) provides important confirma - tion of the validity of the methods used and the significant mag - nitude of the CLIC pathway . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 679 Characterization of the CLIC pathway \u2022 Howes et al . Figure 2 . Quantitation of CLIC endocytosis . ( A ) Cav1 2 / 2 or wild - type ( WT ) MEFs were left untreated or were treated with Dyngo4a . CTxB - HRP was inter - nalized for 15 s , 1 min , or 2 min . Examples of CTxB - labeled structures after 15 s of uptake are shown ( inset , left panel ) . Substratum indicated by large arrowhead , grid sizes are 2 , 000 or 200 nm , examples of intersections shown by arrows . ( B ) 20 \u2013 25 cells treated as in A were used to calculate the volume fraction ( V ( v ) ) . Error bars show SEM . ( C ) NIH3T3 cells not treated with inhibitor were processed as in A and counted as in B . V ( v ) was calculated for both tubular and vesicular structures . Error bars show SEM . ( D ) Cav1 2 / 2 MEFs were incubated with HRP for 15 s , 1 min or 2 min and HRP - laden carriers counted as in B . Error bars show SEM . ( E ) CTxB was conjugated with NHS - SS - biotin and added to untreated Cav1 2 / 2 MEFs or Cav1 2 / 2 MEFs treated with Dyngo4a for 15 s or 2 min or was bound to untreated cells on ice for 10 min ( Surface + MesNa ) . Cells were placed on ice and residual surface biotin cleaved with MesNa . Western blots of cell lysates were probed with streptavidin - HRP . Chart represents the average intensity of streptavidin - HRP across three independent experiments . Residue luminescence in Surface + MesNa samples indicates level of uncleavable biotin . Error bars show SEM . ( F ) Absolute volume of CLICs was estimated from the volume fraction , V ( v ) , multiplied by the average volume of a Cav1 2 / 2 MEF , 2 , 347 \u00b5m 2 . Surface density ( S ( v ) ) was calculated from high resolution images of labeled structures using a cycloid grid as described in Materials and methods and multiplied by the absolute volume to give absolute surface area . Volume of a single carrier was calculated as described in Materials and methods . Number of CLIC budding events per minute per cell was calculated based on the absolute volume internalized by all CLICs divided by the volume of a single carrier . Volume adjustments for overprojection effects are in brackets ( see Materials and methods ) . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 680 Figure 3 . 3D morphology of CLICs . ( A ) Cav1 2 / 2 MEFs were incubated with CTxB - HRP for 20 min on ice , before internalization for 15 s . The DAB reac - tion was performed and cells were processed for electron tomography ( see Materials and methods ) . A single section of the original tomogram is shown ( left ) . Various rotations of a 3D contoured electron density render were generated ( middle ) . Enlarged sections selected from the tomogram ( right ) show internal vesicles ( arrows ) and a complete connection around the circumference of the structure ( arrowhead ) . ( B ) WT MEFs grown on sapphire discs were incubated with CTxB - HRP for 15 s before DAB reaction and high - pressure freezing . Tubular extensions ( large arrows ) are seen emanating from vesicular bulbs ( arrowheads ) in the characteristic ring - shaped CLIC morphology . CCPs without CTxB label ( double arrowheads ) indicate that they are still surface connected . Bars , 200 nm . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 681 Characterization of the CLIC pathway \u2022 Howes et al . Therefore , colocalization between dynamin and anti - GFP in GFP - GPI \u2013 expressing cells was assessed after treatment with wortmannin ( Fig . S3 ) . Colocalization after 10 min was signifi - cantly increased in wortmannin - treated cells compared with untreated controls ( 13 . 1 \u00b1 1 . 1 % untreated ; 39 . 2 \u00b1 2 . 0 % 100 nM wortmannin ; Fig . S3 , A and B ) , indicating that dynamin is re - cruited to CLICs rather than localizing to them from the PM . By EM , dynamin was also found on a small proportion of CTxB - HRP containing tubular / ring - shaped structures ( Fig . S3 C ) . Although the majority of dynamin coincides with the CHC profile , the small but specific concentration in Fraction 2 . 8 , as well as colocalization with GFP - GPI and CTxB , is consistent with the hypothesis that dynamin is recruited to CLICs , after PM scission . To verify that membranes in Fraction 2 . 8 represent bona fide CLICs , 3T3 - GPI cells were transfected with one of two markers for the CLIC pathway , Flot1 - HA and GRAF1 - myc . By Western blot analysis both endogenous and transfected Flot1 and GRAF1 were concentrated in Fraction 2 . 8 ( Fig . 4 F ) . When fractions from transfected cells were labeled with anti - HA or anti - myc antibod - ies , a significant proportion of the structures within Fraction 2 . 8 were labeled ( Fig . 4 G , arrows ; 31 . 5 \u00b1 2 . 5 % Flot - HA and 41 . 3 \u00b1 6 . 4 % Graf1 - myc positive ) . Comparatively , when Fraction 2 . 8 was labeled for endogenous Cav1 , very few of the structures were pos - itive ( 5 . 5 \u00b1 0 . 8 % ) and , notably , these smaller vesicular structures were distinct from the CLIC - like structures ( Fig . 4 G , arrowheads ) . Endogenous dynamin labeled a significant population of struc - tures within Fraction 2 . 8 ( Fig . 4 G , arrows ; Fig . S3 D ) , supporting the notion that dynamin can associate with CLICs . We next used this fractionation approach to identify novel CLIC components . The protein complement of Fraction 2 . 8 was examined after separation using 5 \u2013 15 % SDS - PAGE and the entire lane was cut into 2 - mm slices , digested with trypsin , and analyzed by liquid chromatography mass spectrometry ( LC - MS / MS ; see Materials and methods ) . This identified 80 proteins with two or more peptide hits of 99 % confidence ( key targets and links to endocytosis in Table I , full list in Table S1 ) . A significant cluster of proteins identified play a role in cellular adhesion and inter - action with the ECM . Thy - 1 , a glycosylphosphatidylinositol - anchored protein ( GPI - AP ) , is a critical component of focal adhesion disassembly through FAK phosphorylation ( Rege et al . , 2006 ) . This interaction is dependent on the GPI anchor of Thy - 1 , implicating the GPI - AP \u2013 enriched CLIC pathway as a mediator of FAK activation . Integrin - \ue062 1 is also a key component of focal adhesions and galectin3 is directly involved in clathrin - independent integrin - \ue062 1 endocytosis ( Furtak et al . , 2001 ) . Although a role for CME during integrin - \ue062 1 endocytosis and cellular motility has been established ( Palamidessi et al . , 2008 ) , it has also been found that integrin - \ue062 1 uptake can be indepen - dent from clathrin and is mediated by lipid rafts ( Vassilieva et al . , 2008 ) . Rap1 transmits outside - in integrin signals , leading to cell polarization , and its activation at the leading edge is necessary for chemokine - induced cellular migration ( Shimonaka et al . , 2003 ) . CD98 also mediates integrin signaling during cell spreading and is localized to cell \u2013 cell contacts ( Feral et al . , 2005 ) , where it acti - vates Rap1 ( Suga et al . , 2001 ) . Activation of Rap1 also leads to recruitment of CD44 to the leading edge ( Shimonaka et al . , 2003 ) , that inhibits the fusion of CLICs with EEs ( Kalia et al . , 2006 ; Fig . S1 ) . Using anti - GFP in GFP - GPI \u2013 expressing NIH3T3 cells ( 3T3 - GPIs ) as a marker for CLICs , we optimized conditions to concentrate GFP - GPI \u2013 positive membranes ( Fraction 1 . 2 ; Fig . 4 A ) . CCVs ( identified by clathrin heavy chain ; CHC ) , EEs and recycling endosomes ( REs ; identified by TfR ) , and Golgi ( identified by GM130 ) were below the detection limits within this fraction . However , this fraction also contained Cav1 ( caveolae ) and Grp78 ( ER ) . The 10 % fraction ( Fraction 1 . 2 ) was , therefore , subjected to a second , continuous Nycodenz gradient , which pro - vided a reproducible enrichment of anti - GFP \u2013 positive mem - branes ( Fraction 2 . 8 ; Fig . 4 A ) . To further characterize the enriched fraction , we examined the membranes in detail by whole - mount EM ( Fig . 4 B ) . Consistent with CTxB - HRP \u2013 laden CLICs visualized by EM in intact cells ( Kirkham et al . , 2005 ) , the predominant structures within Fraction 2 . 8 have the striking mor - phology of CLICs . A low magnification image shows the consis - tent morphology of the structures enriched within this fraction ( Fig . S2 A ) . At high magnification ( Fig . 4 C , arrows ) , the struc - tures within Fraction 2 . 8 were also consistent with tomography and HPF data ( Fig . 3 ) . Other structures displayed a clear connec - tion between tubular extensions and spherical bulbs ( Fig . 4 C , arrowheads ) . There was no evidence of contamination by other cellular compartments , such as mitochondria . Other fractions were enriched in structures distinct from those found in Fraction 2 . 8 ( Fig . S2 C ) . This system also gave optimal , but incomplete , separation of GFP - GPI \u2013 positive carriers from Cav1 and Grp78 . To quantify the membrane enrichment in Fraction 2 . 8 we ana - lyzed internalized CTxB - HRP within the fraction after 5 min of internalization ( Fig . 4 D , arrows ) . CTxB - HRP \u2013 labeled structures constituted 70 . 0 \u00b1 0 . 9 % of the membranes in the CLIC - enriched fraction as calculated from stereology ( Fig . S2 B ) . The validity of this method was further examined by ana - lyzing membranes within Fraction 2 . 8 after inhibition of CLIC endocytosis . Using two independent methods of CLIC perturba - tion , expression of a Cdc42 dominant - negative mutant ( Cdc42 - DN ; Sabharanjak et al . , 2002 ) and cholesterol depletion with methyl - \ue062 - cyclodextrin ( m \ue062 CD ) under conditions where CME is unaffected ( Kirkham et al . , 2005 ; Glebov et al . , 2006 ) , there was a substantial reduction of CTxB and anti - GFP \u2013 positive mem - branes within Fraction 2 . 8 ( Fig . 4 E ; 59 . 7 % reduction in CTxB levels in cells transfected with Cdc42 - DN compared with Cdc42 - WT ; 53 . 6 % reduction in anti - GFP antibody levels after treatment with m \ue062 CD ) . Thus , this approach shows concentra - tion of CLIC marker positive membranes within Fraction 2 . 8 and reduced accumulation of these membranes after treatments known to inhibit CLIC endocytosis . Unlike CHC and TfR , there was a small proportion of dynamin specifically concentrated in Fraction 2 . 8 ( Fig . 4 F ) . Although dynamin is not necessary for the formation of nascent CLICs ( Sabharanjak et al . , 2002 ; Kirkham et al . , 2005 ) , it has been previously noted that expression of the DynK44A mutant re - duces CTxB trafficking to the Golgi ( Le and Nabi , 2003 ; Kirkham et al . , 2005 ) , independently from clathrin ( Torgersen et al . , 2001 ) , suggesting a role in clathrin - independent CTxB uptake . As CLICs rapidly mature , however , it is difficult to assess a possible tran - sient recruitment of dynamin under steady - state conditions . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 682 Figure 4 . Biochemical enrichment of CLICs . ( A ) 3T3 - GPI cells were subjected to density fractionation as described in Materials and methods . Western blots of membrane markers are shown . Within the first gradient ( left ) anti - GFP \u2013 , Cav1 - , and Grp78 - positive membranes are present in Fraction 1 . 2 , highlighted by outline . CHC - , GM130 - , and TfR - positive membranes are below the detection limit within this fraction . Within the second gradient , anti - GFP \u2013 positive membranes concentrate within Fraction 2 . 8 , highlighted by outline . This represnts a yield of 9 . 6 \u00b1 0 . 2 % of anti - GFP \u2013 positive membranes . Cav1 concen - trates in Fraction 2 . 6 and Grp78 in Fraction 2 . 10 . ( B ) Fraction 2 . 8 was fixed and visualized by EM . Structures within Fraction 2 . 8 share similar profiles to CLICs seen within intact cells . Bar , 200 nm . ( C ) High resolution electron micrographs of Fraction 2 . 8 structures , providing examples of vesiculation ( arrows ) and a spherical bulb connected to tubular extension ( arrowheads ) . Bar , 100 nm . ( D ) NIH3T3 cells were incubated with CTxB - HRP before fractionation and the DAB reaction was performed on Fraction 2 . 8 . Bar , 200 nm . ( E ) NIH3T3 cells transfected with Cdc42 - WT or - DN were incubated with CTxB before fractionation . Western blots were probed with anti - CTxB . 3T3 - GPI cells were treated or not with m \ue062 CD before incubation with anti - GFP antibodies . Western blots of fractions were probed with anti \u2013 Rb - HRP . ( F ) Western blots of fractions from cells transfected with Flot1 - HA , GRAF1 - myc , or left untransfected . Frac - tions were probed with anti - HA , - myc , - dynamin , - Flotillin1 , or - GRAF1 antibodies as appropriate . ( G ) Fixed sampled from F of Fraction 2 . 8 were labeled for anti - HA , anti - myc , endogenous Cav1 , or dynamin . Structures labeled with Flotillin - 1 - HA , GRAF1 - myc , and dynamin ( arrows ) or Cav - 1 ( arrowheads ) are shown . Bars , 200 nm . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 683 Characterization of the CLIC pathway \u2022 Howes et al . Anti \u2013 Thy - 1 , anti - CD44 , and myoferlin colocalized with CTxB - 555 but not Tf - 647 in NIH3T3 cells after 2 min of uptake , indicat - ing they are specific cargo for the CLIC pathway . Interestingly , although only a low level of colocalization was seen between anti - GFP and Rab11 after 10 min ( 17 . 07 \u00b1 1 . 49 % ) in untreated 3T3 - GPI cells ( Fig . 5 , B and C ) , after treatment with wortman - nin there was a significant increase in their colocalization after 10 min ( 41 . 12 \u00b1 2 . 79 % ; Fig . 5 , B and C ) . As wortmannin inhib - its the maturation of CLICs , this localization is not likely to occur within REs . Additionally , there was no detectable TfR in the CLIC - enriched fraction , arguing against significant contam - ination of RE membranes within this fraction . Additional evidence for the sorting capability of CLICs was identified by assessment of the endocytic maturation of CD44 . Endocytosis of CD44 was consistently Tf negative ( Fig . 5 D ) , while it colocalized with CTxB after 2 min , with less overlap after 10 min . Persistent colocalization was also seen be - tween CD44 and dysferlin from 2 to 40 min ( Fig . 5 D ) . As dys - ferlin also remains distinct from the classical endocytic network while CD44 is linked to integrin - \ue062 1 signaling by forming special - ized lipid raft microdomains ( Lee et al . , 2008 ) . CD44 is also an important component of extracellular contact , involved in the degradation of ECM , and is involved in maintenance of polar - ization and directionally persistent migration ( Mrass et al . , 2008 ) . This also implicates CLICs as a mediator of cell adhesion and , given the proposed ability for CLICs to recycle directly to the PM , presents a rapid recycling mechanism during cellular adhe - sion turnover and motility . Supporting this , moesin directs actin - based rearrangements of lipid rafts directly through CD44 to orchestrate the topology of cell surface structures , such as filopo - dia ( Bretscher et al . , 1997 ) , and the CD44 - ERM ( ezrin - radixin - moesin ) association is a critical component of directional cellular motility ( Legg et al . , 2002 ) . We next sought to verify some key targets as novel cargo for the CLIC pathway . Verification of novel CLIC proteins Preliminary verification of some key targets was performed by comparison to internalized CTxB - 555 and Tf - 647 ( Fig . 5 A ) . Table I . Key targets identified from isolation of CLIC - positive fraction Target Location Link to endocytosis 4F2 cell surface antigen heavy chain / CD98 PM Receptor for Galectin3 and Integrin \ue062 - 1 endocytosis 78 - kD glucose - regulated protein PM / ER Associates with lipid rafts and receptor for MHCI Actin , cytoplasmic 1 Cytosol Drives endocytic carrier formation Alpha - 2 - HS - glycoprotein / Fetuin PM / E Endocytosis mediated by AnnexinA2 Annexin A2 E Binds glycoproteins during endocytosis CD109 antigen PM Cell surface GPI - AP CD44 antigen PM Hyaluronan receptor Collagen ECM ECM constituent Fructose - bisphosphate aldolase A Cytosol Mediates Snx9 - dynamin interaction Galectin - 3 PM Mediates Integrin \ue062 - 1 endocytosis Guanine nucleotide - binding protein G \ue061 ( i , o ) PM Involved in STAT3 signaling Integrin \ue062 - 1 PM Receptor for ECM Lysosome - associated membrane glycoprotein 2 E Lysosomal membrane regulator Membrane - associated progesterone receptor component 1 PM / ER Involved in cholesterol homeostasis , rapidly cleared from the PM by endocytosis Moesin PM Links CD44 to cortical actin to drive features in the PM Myoferlin ( Fer - 1 \u2013 like protein 3 ) PM Involved in Ca 2 + - promoted membrane fusion ; binds to AHNAK Neuroblast differentiation - associated protein AHNAK PM Binds Myoferlin and Dysferlin and with Annexin2 mediates membrane fusion Prolow - density lipoprotein receptor - related protein 1 ( LRP1 ) PM Contains both tyrosine and di - leucine sorting motifs ; binds saposin on cell surface Prosaposin PM / E Co - receptor with LRP1 ; binds GM1 Rac1 PM / E Induces membrane ruffling Rab - 11A E Endosome recycling Rab - 14 E Endosome recycling Rab - 7a E Late endosome fusion and motility Rab - 5A E Early endosome fusion and motility Rap - 1b PM Integrin \ue062 - 1 endocytosis and focal adhesion turnover ; mediates STAT3 Receptor expression - enhancing protein 5 PM Involved in Rab - mediated membrane trafficking Reticulon - 4 PM / ER Tubulates membranes ; binds NogoR , a GPI - AP at the PM Thy - 1 membrane glycoprotein PM GPI - AP ; regulates FAK signaling Tmp21 PM Contains KDEL sequence , traffics to the PM independently from p24 Vimentin Cytosol With Annexin A2 regulates FAK signaling E , Endosomes ; ECM , extracellular matrix ; ER , endoplasmic reticulum ; PM , plasma membrane . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 684 Figure 5 . Verification of novel CLIC cargo . ( A ) NIH3T3 cells were incubated with either anti \u2013 Thy - 1 or anti - CD44 antibodies and CTxB - 555 and Tf - 647 for 2 min . Myoferlin was detected after fixation , showing steady - state localization . Arrows indicate colocalization . Bar , 10 \u00b5m . ( B ) 3T3 - GPI cells were left untreated or were treated with 100 nM wortmannin before internalization of anti - GFP for 10 min . Arrows indicate colocalization . Bar , 10 \u00b5m . ( C ) Quantitation of B from 12 \u2013 15 cells in three independent experiments . ( D ) Quantitation of colocalization between internalized anti - CD44 antibodies and Tf - 647 , CTxB - 555 , or anti - myc antibodies for GFP - dysferlin - myc \u2013 expressing cells after 2 , 10 , and 40 min . Error bars show SEM . ( E ) Anti \u2013 CD44 - HRP was internalized into WT MEFs before DAB reaction . Arrows show anti - CD44 - HRP \u2013 positive carriers with morphology of CLICs . Arrowheads show large , tubular ring - shaped anti - CD44 - HRP \u2013 positive compartment . Bars , 200 nm . ( F ) Anti \u2013 CD44 - HRP or Tf - HRP was pulsed into Cav1 2 / 2 MEFs for 15 s , 1 min , or 2 min . Cells were fixed and processed for vertical sections . Arrows show HRP - labeled carriers . Bar , 200 nm . ( G ) Stereology measurements were captured across 20 \u2013 25 cells in three independent areas as treated in F . Error bars show SEM . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 685 Characterization of the CLIC pathway \u2022 Howes et al . To verify the polarization of CLIC endocytosis in migrat - ing fibroblasts , ultrastructure of CTxB - HRP \u2013 laden carriers was assessed after a 2 - min uptake in migrating MEFs ( Fig . 5 C , large arrow indicates direction of migration ) . Labeled CLICs were predominantly found at the leading edge ( Fig . 6 C ; 1 , 2 ) . Con - versely , an accumulation of unlabeled , surface - connected caveo - lae was seen at the trailing edge ( Fig . 6 C ; 3 , 4 insets ) . Quantitation of the number of endocytic and surface - connected structures at the leading and trailing edges confirmed this polarized distribu - tion ( Fig . 6 D ) . Both surface - connected CCPs and internalized CCVs showed no preference for the leading or trailing edge . To our knowledge , this is the first evidence for a striking spatial distinction between the CLIC pathway , caveolae , and CME , and is suggestive of a role for CLICs during dynamic adhesion turn - over specifically from the leading edge . As the CLIC pathway is polarized during migration and internalizes key adhesion components at the leading edge , we speculated that this pathway could play a critical role during cell migration . Thus , a system was developed in which CLIC - dependent uptake could be inhibited without affecting CME ( see Materials and methods ) . To optimize conditions for tran - sient ablation of the CLIC pathway , we internalized CTxB - HRP for various short times , performed DAB - mediated ablation of the labeled compartments , and then analyzed the uptake of a subsequent pulse of CTxB - 555 , Tf - 647 , and antibodies to CD44 . 2 min of CTxB - HRP internalization followed by the DAB reaction on living cells was found to give the strongest inhibition of CD44 and CTxB - 555 endocytosis without any effect on Tf - 647 uptake ( Fig . 6 E ) . Under these conditions Tf - 647 endocytosis and maturation was unaffected , even after a 40 - min pulse , indicating the endosomal network was still func - tional ( not depicted ) . The inhibition of CD44 and CTxB - 555 internalization following CTxB - HRP \u2013 based ablation persisted for at least 4 h after the ablation step , indicating this assay could be used to assess possible effects of CLIC inhibition on migration . Quantitation of fluorescence intensity shows that CTxB - HRP inactivation of the CLIC pathway resulted in an 81 . 8 \u00b1 7 . 2 % reduction in CTxB - 555 and a 93 . 8 \u00b1 2 . 6 % reduc - tion in CD44 endocytosis , whereas Tf - 647 internalization was not reduced ( increased by 28 . 6 \u00b1 24 . 8 % ) after a 4 - h incuba - tion ( Fig . 6 F ) . To test the requirement for CLIC endocytosis during mi - gration , this inactivation assay was applied to migrating fibro - blasts . The capacity of cell monolayers to migrate into a scratched wound was assessed by measuring the total distance between two cell fronts of a wound after CLIC inactivation . When CTxB - HRP was used to inactivate CLICs , without effect on other endo - cytic routes , cell monolayers had a reduced capacity to migrate into a wound after 4 h ( 15 . 8 \u00b1 1 . 7 % distance of wound closed ; Fig . 6 G ) compared with DAB only \u2013 treated cells ( 33 . 4 \u00b1 3 . 7 % closed ) . A similar effect was observed in Cav1 2 / 2 fibroblasts , ruling out the possibility that the migration defect was due to in - activation of caveolae . Together with previous studies ( Grande - Garc\u00eda et al . , 2007 ; Nishimura and Kaibuchi , 2007 ) , these findings suggest that three major endocytic routes in eukaryotic cells , CME , caveolae , and the CLIC pathway , are all important for normal directional cell migration . ( not depicted ) , this suggests that CLICs are directly sorting cargo rather than representing distinct populations of similar mecha - nisms . Confirming this , the ultrastructure of CD44 - positive car - riers , as visualized using anti \u2013 CD44 - HRP , showed identical morphology to that of CTxB - HRP \u2013 laden CLICs ( Fig . 5 E , arrows ) , whereas no label was found in the classical endosomal network . In some instances , intriguing tubular networks were seen with labeling for anti \u2013 CD44 - HRP after 40 min of internal - ization ( Fig . 5 E , arrowheads ) , possibly representing a mature compartment arisen from the CLIC pathway . The presence of anti - CD44 antibodies had no effect on the number or morphol - ogy of CTxB - HRP \u2013 laden CLICs between 2 and 40 min ( not de - picted ) , indicating that the CD44 antibodies do not influence the kinetics or ultrastructure of the pathway . Using CD44 as a novel and specific marker for CLICs , it was also possible to confirm stereology calculations using CTxB - HRP in the presence of dynamin inhibition . Anti - CD44 - HRP \u2013 conjugated antibodies or Tf - HRP was internalized in Cav1 2 / 2 MEFs and the DAB reaction was performed ( Fig . 5 , F and G ) . This method has the advantage of relying on the specificity of CD44 for the CLIC pathway and Tf - HRP for CME . We found that CD44 - positive CLICs accounted for 0 . 09 \u00b1 0 . 02 % of the total cytoplasmic volume after 15 s , 0 . 14 \u00b1 0 . 03 % after 1 min , and 0 . 34 \u00b1 0 . 01 % after 2 min ( Fig . 5 G ) . These volume estimates are in excellent agreement with those obtained using morphological criteria or using specific inhibitors of dynamin - dependent endo - cytosis ( see above ) . These data identify a range of novel CLIC cargo , confirm the significant magnitude of the pathway , and pro - vide evidence for the direct recruitment of dynamin and Rab11 to CLICs and the ability to sort cargo to distinct destinations . Polarization of CLICs during cellular migration Based on the association of novel CLIC cargo , such as Thy - 1 and CD44 with extracellular attachment , we sought to visualize CLICs within migrating cells . As regulation of adhesion compo - nents is a fundamental process during cellular migration the CLIC pathway may be either up - regulated or spatially restricted within motile cells . A scratch - wound was applied to a confluent monolayer of fibroblasts and cells were allowed to migrate into the space provided by the wound . Early endocytosis of CTxB and Tf was assessed in the presence of either internalizing anti - CD44 or anti \u2013 Thy - 1 antibodies or was followed by labeling for endogenous Cav1 ( Fig . 6 A ) . Colocalization was seen between CTxB - 555 and both CD44 and Thy - 1 at the leading edge of mi - grating cells ( arrows ) . In cells expressing GFP - GPI , anti - GFP antibody endocytosis also preferentially occurred at the leading edge ( Fig . 6 A ) , indicating polarization of CLICs rather than po - larized uptake of specific GPI - APs involved in migration , such as Thy - 1 . There was a clear spatial distinction between CTxB - 555 , Cav1 , and Tf - 647 in these cells , which was confirmed by assessing average pixel intensity across a selected field ( Fig . 6 B ) . It has been well documented that Cav1 localizes to the trailing edge during 2D migration ( Grande - Garc\u00eda et al . , 2007 ) . We also found that CTxB binding after fixation was uniform over the en - tire cell , excluding the possibility that GM1 was polarized within these cells ( not depicted ) . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 686 Figure 6 . CLICs become polarized during and are necessary for efficient cellular migration . ( A ) Confluent monolayers of WT MEFs or NIH3T3s were scratched and cells were allowed to migrate for 8 \u2013 12 h . CTxB - 555 and Tf - 647 were pulsed into migrating cells for 2 min in the presence of anti - CD44 ( WT MEF ) , anti - GFP ( GFP - GPI expressing WT MEF ) , or anti - Thy - 1 ( NIH3T3 ) antibodies or cells were labeled for Cav - 1 ( WT MEF ) . Dotted lines indicate leading edges . Arrows show colocalization between anti - CD44 or anti \u2013 Thy - 1 and CTxB - 555 but not Tf - 647 at the leading edge . Bar , 20 \u00b5m . ( B ) Quantita - tion of average pixel intensity from the leading to trailing edges for CTxB - 555 , Cav - 1 , and Tf - 647 . Inset shows the concentration of tubular , Cav - 1 , and Tf - negative CTxB - labeled CLICs at the leading edge . A rectangular area , outlined , was used to calculate the average pixel intensity ( along the y axis ) across the leading to trailing edge ( along the x axis ) for each endocytic marker . Plot profiles identify a concentration of CTxB in the leading edge and Cav - 1 in the trailing edge whereas Tf shows uniform intensity across the cell . Bar , 10 \u00b5m . ( C ) Electron micrographs of a migrating WT MEF . Large arrow indicates direction of migration . Magnifications from the leading edge and the trailing edge show representative images of CTxB - HRP \u2013 labeled CLICs D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 687 Characterization of the CLIC pathway \u2022 Howes et al . from the PM ( Kirkham et al . , 2005 ; Sabharanjak et al . , 2002 ) , such recruitment of dynamin to maturing CLICs , after the initial scission , provides important machinery for CLICs to compart - mentalize . Clearly , however , dynamin is not required for the generation of CLIC architecture , as the morphology of CLICs is identical in the presence or absence of dynamin inhibition . Recruitment of dynamin to CLICs as they mature would reconcile the direct interaction between GRAF1 and dynamin within this dynamin - independent pathway ( Hansen and Nichols , 2009 ) . Myoferlin , a novel CLIC - associated target identified here , is a member of the ferlin family of proteins implicated in mem - brane fusion ( Davis et al . , 2000 ) . We have previously shown that another ferlin family member , dysferlin , associates with the CLIC pathway ( Hern\u00e1ndez - Deviez et al . , 2008 ) . Myoferlin , like dysferlin , is involved in calcium - dependent membrane fusion after mechanical disruption to the PM ( Bernatchez et al . , 2007 ) . Recently , a clathrin - independent , GPI - enriched endocytic com - partment has been implicated in membrane repair after PM shearing ( Idone et al . , 2008a , b ) . This raises the exciting possibil - ity of links between the role of dysferlin / myoferlin in this pro - cess and the ability of CLICs to provide the necessary membrane for repair of PM lesions . Supporting this , two targets identified here , AHNAK and Annexin A2 , have been shown to bind dysfer - lin and myoferlin during membrane repair ( Huang et al . , 2007 ) . Localization of dysferlin / myoferlin to a subdomain of CLICs that does not merge with Tf - positive EEs highlights that this pathway has the capability to rapidly recycle membrane back to the PM under specific conditions , such as PM repair ( Fig . 7 B ) . Evidence for an association between CLICs and cellular migration is provided by the distinct polarization of CLICs in motile cells , as well as the impairment of migration after tran - sient ablation of the pathway . Specific internalization of Thy - 1 and CD44 through the CLIC pathway at the leading edge pro - vides an explicit role for CLICs in rapid turnover of adhesion receptors within a spatially restricted mechanism . How CLICs become polarized during migration remains an open question ; however , one possibility arises from activity of the initial regula - tor of the CLIC / GEEC pathway , Cdc42 . Cdc42 was originally described as directing cell polarity during yeast budding ( Johnson and Pringle , 1990 ) and plays an assortment of fundamental roles during eukaryotic cell polarization ( Etienne - Manneville , 2004 ) . After wounding of cell monolayers Cdc42 activity is specifically directed to the front of the cell , resultant from signaling between contacts and the ECM ( Etienne - Manneville and Hall , 2001 ) . As Cdc42 activity is necessary for formation of CLICs , this would lead to specific localization of CLICs at the leading edge of wounded monolayers . The clathrin adaptor , Numb , is also Discussion Here we identify the CLIC pathway as a high capacity route that accounts for the vast bulk of constitutive fluid uptake in fibro - blasts , implicating CLICs as key mediators of PM remodeling . Previous estimations of endosomal tubule volume using stereol - ogy have noted that tubules close to section thickness ( 55 nm ) can lead to overprojection effects ( Griffiths et al . , 1989 ) . When correction factors are applied to volume estimates CLICs would still account for over 70 % of internalization . Bulk phase early endocytosis in rat fetal fibroblasts has also been shown to be in - dependent from clathrin and occurs via a constitutive , noncave - olar pathway , which rapidly regurgitates substantial amounts of endocytic volume ( Cupers et al . , 1994 ) . We have previously shown that up to 40 % of endocytosed fluid , mainly internalized through the CLIC / GEEC pathway , is regurgitated in 5 min ( Chadda et al . , 2007 ) . Together , this suggests that the CLIC pathway in - ternalizes and returns significant portions of the PM quickly , rapidly turning over membrane during key cellular processes , such as PM repair and homeostasis . It also provides the endo - cytic system with a mechanism to remodel features of the PM quickly , critical for extracellular interactions during various processes ranging from adhesion to signaling . Such vast mem - brane flow and recycling has recently been observed from a GPI - AP \u2013 positive CIE compartment , which provides a signfi - cant fraction of the required membrane during cellular spread - ing ( Gauthier et al . , 2009 ) . Calculation of fluid uptake via clathrin - independent endo - cytosis has generally estimated the contribution of these routes to be 40 \u2013 60 % of total fluid internalization ( Sandvig et al . , 1987 ; Sabharanjak et al . , 2002 ; Cheng et al . , 2006 ) . The higher contri - bution of 74 % , calculated here , may be explained through differ - ences in the resolution , both spatial and temporal , of experimental procedures used . By calculating volume internalized based on ultrastructure rather than fluorescence this work provides a higher resolution analysis than previous studies , which were limited by the optical resolution of fluorescence microscopy . The 3D structure of CLICs identifies them as pleiomorphic , complex , multicomponent carriers . Although it is recognized that the bulk of CLIC - internalized fluid and membrane fuses with EEs , discrepancies between the ability of CLIC cargos , such as dysferlin / myoferlin and CD44 compared with GFP - GPI and CTxB , to fuse with Tf - positive endosomes strengthens the argument that some domain of CLICs traffics distinctly from the bulk ( Fig . 7 A ) . Recruitment of dynamin to CLICs , after budding from the PM , further demonstrates the sorting complexity of the route . Although dynamin is not necessary for scission of CLICs ( 1 , 2 ) and surface - connected caveolae ( 3 , 4 ) . Bar , 500 nm . ( D ) Quantitation of the number of endocytic structures at the leading and trailing edge of cells treated as in C . Budded caveolae and CCVs are positive for CTxB - HRP label , whereas caveolae and CCPs are not . Error bars show SEM . ( E ) WT MEFs were incubated with or without CTxB - HRP for 2 min . The DAB reaction was performed on live cells for 5 min . Cells were washed and allowed to grow for a further 4 h . CTxB - 555 , anti - CD44 antibodies , and Tf - 647 were added directly to cells for 2 min of uptake before acid stripping and fixation . Bar , 10 \u00b5m . ( F ) 12 \u2013 15 cells treated as in E were quantitated for average fluorescence intensity of CTxB - 555 , Tf - 647 , or goat anti \u2013 mouse - 488 for mouse \u2013 anti - CD44 . ( G ) Confluent monolayers of WT MEFs were scratched and cells were allowed to migrate into the space for 1 h . Cells were incubated with serum alone or serum with 10 \u00b5g ml 2 1 CTxB - HRP for 2 min and the DAB reaction was performed as in E . After 4 h of migration , cells were fixed and distance migrated was determined by measuring the distance of the gap between both sides of the wound at time zero and after 4 h of incubation . Error bars show SEM from three independent experiments . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 JCB \u2022 VOLUME 190 \u2022 NUMBER 4 \u2022 2010 688 and rapidly mature into a high capacity sorting station . We have shown that CLICs are a high capacity , dynamic , and complex sorting system and propose that they have the ability to sort subcompartments to distinct destinations for critical cellular processes such as membrane repair , migration , and maintenance of the PM . Materials and methods Internalization assay Cells on 12 - mm coverslips or 35 - mm dishes were serum starved for 4 h in serum - free DME ( Invitrogen ) before two times 2 - min wash in precooled CO 2 - independent media ( Invitrogen ) on ice and placed on 50 - \u00b5l drops of 10 \u00b5g . ml 2 1 CTxB - 555 ( Invitrogen ) , 5 \u00b5g . ml 2 1 Tf - 488 ( Invitrogen ) , and / or anti - GFP as desired for 20 min . For stereology , 35 - mm dishes were not in - cubated on ice but were subjected to constitutive uptake at 37\u00b0C , 5 % CO 2 for desired times . Coverslips were moved to prewarmed serum - free DME and incubated at 37\u00b0C , 5 % CO 2 for desired times . Coverslips were placed back on ice in precooled CO 2 - independent media . Two times 30 - s 0 . 5 M glycine ( pH 2 . 2 ) acid stripping was routinely performed before fixation in 2 % paraformaldehyde . For immunocytochemistry , cells were permeabi - lized in freshly made 0 . 1 % saponin for 10 min and blocked in 0 . 2 % fish skin gelatin / 0 . 2 % BSA in PBS . Coverslips were incubated with primary antibodies for 1 h at room temperature , followed by three times 10 - min wash in PBS before incubation with secondary Alexa Fluor \u2013 conjugated antibodies at 1 : 400 for 1 h . Fluorescence micrographs were captured on a confocal laser - scanning microscope ( Axiovert 200 m SP LSM 510 META ; Carl Zeiss , Inc . ) . Images were captured under oil with a 63x Plan - Apochromat objective , at excitation wavelengths of 488 , 543 , and 633 nm for GFP - tagged con - structs or Alexa Fluor 488 \u2013 , Alexa Fluor 555 \u2013 , or Alexa Fluor 647 / 660 \u2013 conjugated antibodies , respectively . Band - pass and meta filters were applied as appropriate for respective fluorophores and images were cap - tured from photomultiplier tube detectors ( Carl Zeiss , Inc . ) . Colocalization was quantitated using Volocity v3 . 7 . Images of individual cells were split into RGB channels , automatic thresholding applied , and colocalization co - efficients calculated on percentage of overlapping voxels . Antibodies used include Thy - 1 and CD44 ( Abcam ) ; CHC , GM130 , Grp78 , Cav1 , and HA ( BD ) ; myc ( Sigma - Aldrich ) ; and TfR ( Invitrogen ) . HRP - conjugated anti - CD44 was generated with Lightning - Link HRP conjugation kit ( Innova Bioscience ) as per the manufacturer\u2019s instructions . Dynamin and PI3K inhibition Cells were serum starved for 4 h in serum - free DME before incubation for 20 min with 30 \u00b5M Dyngo4a , 80 \u00b5M dynasore ( Sigma - Aldrich ) , or 100 nM wortmannin ( Roche ) . Cells were then incubated with an endocytic marker of interest in the presence of Dyngo4a and wortmannin for indi - cated periods at 37\u00b0C . Electron microscopy and DAB reaction After constitutive internalization of 10 \u00b5g . ml 2 1 CTxB - HRP ( Invitrogen ) at 37\u00b0C , cells were placed on ice and washed two times for 1 min in ice cold PBS , followed by 10 min in 1 mg . ml 2 1 DAB ( Sigma - Aldrich ) \u00b1 50 \u00b5M ascor - bic acid ( AA ) in PBS . Cells were then incubated with DAB \u00b1 50 \u00b5M AA with 0 . 012 % H 2 O 2 for 20 min , followed by fixation in 2 . 5 % glutaraldehyde ( ProSciTech ) in PBS for 1 h at room temperature . For plastic sections , cells were contrasted with 1 % osmium tetroxide and 4 % uranyl acetate . Cells were dehydrated in successive washes of 70 % , 90 % and 100 % ethanol before embedding in LX - 112 resin . For frozen sections , cells were scraped and embedded in 10 % gelatin and gelatin blocks were infused with 2 . 3 M sucrose . Blocks were frozen onto mounting pins and 55 - nm sections were collected using a UC6 ultramicrotome ( Leica ) . Tomograms of 300 - nm plastic sections were captured in 1\u00b0 increments , tilted \u00b160\u00b0 on a transmission elec - tron microscope ( model F30 ; Tecnai ) using UltraScan 4000 semi - automated software . High pressure frozen samples were essentially processed as de - scribed previously ( Richter et al . , 2008 ) . In brief , CTxB - HRP was added di - rectly to cells grown on sapphire discs and DAB reaction was performed on ice before discs were frozen in a Balzers HPM010 high pressure freezer ( BAL - TEC AG ) and stored under liquid nitrogen . Freeze substitution was performed in acetone with 0 . 7 % uranyl acetate ; 1 % osmium tetroxide at 2 90\u00b0C before warming to 0\u00b0C over 3 d and infiltrated with Embed 812 - Araldite resin . specifically localized to the leading edge where it mediates integrin - \ue062 1 endocytosis ( Nishimura and Kaibuchi , 2007 ) . It appears surprising that Numb - positive CCVs and CLICs both polarize to the leading edge during migration . One possible explanation for this is the types of cargo internalizing through each pathway . Adhesion components without a CCP sorting motif , such as CD44 and Thy - 1 , still need to be internalized for efficient migra - tion and this could occur via CLICs at the leading edge . Con - sistent with such a hypothesis , a screen for genes involved in epithelial cell migration identified that knock - down of GRAF1 , a regulator of the CLIC pathway ( Lundmark et al . , 2008 ) , reduced the capacity of these cells to migrate ( Simpson et al . , 2008 ) . This study has shown the significant magnitude of traf - fic through this largely uncharacterized sorting compartment . Rather than acting as passive carriers that shuttle from the PM to EEs , we propose that CLICs arise from the plasma membrane Figure 7 . Model of CLIC endocytosis . ( A ) ( 1 ) CLIC cargo , such as Thy - 1 , CD44 , and myoferlin are concentrated within Flotillin - 1 and cholesterol - enriched microdomains . ( 2 ) Actin and GRAF - 1 drive the initial formation of the carriers , within 15 s . ( 3 ) Recruitment of dynamin , Rab11 , and Rab5 / EEA - 1 complexes within 2 min provides the ability for these carriers to facilitate bulk membrane flow to early endosomes ( 4a ) and fast plasma membrane recycling ( 4b ) . ( B ) After abrasion to the PM an influx of Ca 2 + activates the fusogenic C2 domains of dysferlin / myoferlin , resulting in the preferential recycling of the CLIC pathway . D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023 689 Characterization of the CLIC pathway \u2022 Howes et al . S ( v ) of plasma membrane across 25 cells , from low magnification electron micrographs , multiplied by the total average volume . To capture the average volume of single CTxB - HRP \u2013 positive CLIC , high resolution electron micrographs from whole - mount profiles were used . As CTxB - HRP label ( as well as HRP and anti - CD44 - HRP label ) was only found within the tubular ring of CLICs , we assumed that the entire volume of CLICs is comprised solely within the tubule space . Therefore , the volume of a sphere based on the diameter of the inner membrane was subtracted from the volume of a sphere based on the diameter of the outer membrane of the rings . This calculated that an average CLIC had an estimated volume of 0 . 00263 \u00b1 0 . 00048 \u00b5m 3 . In comparison , the volume of a single CCV was estimated to be five times smaller ( 0 . 0005 \u00b5m 3 assuming sphere based on diameter of 100 nm ) . Mass spectrometry An Agilent 1100 Binary HPLC system ( Agilent Technologies ) was used to per - form reversed phase separation of the in - gel digests using a Vydac MS C18 300A column ( 150 mm \u00d7 2 mm ) with a particle size of 5 \u00b5m ( Vydac ) . Mass spectrometry experiments were performed on a hybrid quadrupole / linear ion trap 4000 QTRAP MS / MS system ( Applied Biosystems ) . All analyses were performed using information - dependent acquisition and the linear ion trap ( LIT ) acquisition modes . Analyst 1 . 4 . 1 software was used for data analysis . Cell migration Confluent WT MEFs grown on coverslips were scratched with a 200 - \u00b5l pipette tip . Cells were washed and allowed to recover for 4 h before inter - nalization assays . Average pixel intensity from leading to trailing edges within a selected area was calculated using the Plot Profile option in ImageJ . Plot profiles show a representative image of 5 \u2013 10 cells across three independent experiments . DAB ablation 10 \u00b5g . ml 2 1 CTxB - HRP was added to cell monolayers for 2 min at 37\u00b0C . Cells were then incubated with 10 mg . ml 2 1 DAB , 50 \u00b5M AA in DME for 2 min before incubation with 10 mg . ml 2 1 DAB , 50 \u00b5M AA , 0 . 012 % H 2 O 2 in DME for a further 2 min . Cells were washed five times with DME before incubation in normal growth media for 4 h . CTxB - 555 , Tf - 647 , and anti - CD44 antibodies were then directly added to cells for 2 min before acid stripping , fixation , permeabilization , and labeling for anti - CD44 antibodies . For quantitation of distance migrated , monolayers were imaged after DAB inactivation ( time zero ) and again after 4 h . Distance between front edges of monolayers on both sides of the wound was measured using Adobe Photoshop CS2 . Anti - CD44 - HRP and Tf - HRP failed to inhibit either clathrin - independent or - dependent endocytosis at concentrations of 10 \u2013 100 \u00b5g . ml 2 1 as used at early times in this assay . Online supplemental material Fig . S1 shows that wortmannin treatment of MEFs inhibits the fusion of CLICs with early endosomes , leading to a threefold increase in the number of CLICs per cell . Wortmannin treatment does not noticeably affect the pro - tein profiles of the CLIC - enriched fraction . Fig . S2 shows the consistent na - ture of membrane profiles in the CLIC - enriched fraction and that CTxB - HRP labels a majority of these profiles when added to cells before the fraction - ation . Fig . S3 shows the localization of dynamin to CLICs after wortmannin treatment by immunofluorescence and by ultrastructure . Dynamin also labels the neck of profiles in the CLIC - enriched fraction . Video 1 shows the tilt series of CTxB - HRP \u2013 laden CLICs after 15 s of internalization . Table S1 lists all the peptides identified from fraction 2 . 8 with two or more 99 % confi - dence hits from the LC - MS / MS . Online supplemental material is available at http : / / www . jcb . org / cgi / content / full / jcb . 201002119 / DC1 . The Institute for Molecular Bioscience is a Special Research Centre of the Aus - tralian Research Council . Confocal microscopy was performed at the Austra - lian Cancer Research Foundation ( ACRF ) / Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology , which was established with the support of the ACRF . The authors acknowledge the facilities as well as scientific and technical assistance from staff in the Australian Microscopy and Micro - analysis Facility ( AMMRF ) at the Centre for Microscopy and Microanalysis at The University of Queensland . We thank Jean Gruenberg for critical appraisal of the manuscript . This work was supported by grants from the National Health and Medi - cal Research Council of Australia ( 511005 ; R . G . Parton and J . F . Hancock ) and the Human Frontier Science Program . Submitted : 22 February 2010 Accepted : 26 July 2010 Nycodenz density fractionation NIH3T3 \u2013 GFP - GPI cells were serum starved and treated for 20 min with 100 nM wortmannin and 30 \u00b5M Dyngo4a . Anti - GFP antibodies were di - rectly added to treated cells in the presence of wortmannin and Dyngo4a for 5 min at 37\u00b0C . Cells were placed on ice and harvested by scraping in TES buffer ( 10 mM tricine , 1 mM EDTA , and 250 mM sucrose , pH 7 . 4 ) with dissolved complete protease inhibitor cocktail ( Roche ) . Cells were ho - mogenized by passaging 10 times each through 21 - and 27 - gauge nee - dles . A 17 , 000 - g spin at 4\u00b0C for 10 min was applied to sediment nonlysed cells and nuclei . Post - nuclear supernatants were mixed with an equal vol - ume 70 % Nycodenz in TES and placed on the bottom of a four - step gradi - ent , consisting of 25 % , 10 % , and 0 % Nycodenz steps . Gradients were spun at 160 , 000 g for 2 h . The 10 % fraction ( excluding the interface ) was collected and diluted 1 / 3 in TES with protease inhibitors and placed on top of a 5 \u2013 20 % continuous Nycodenz gradient . This was spun at 206 , 000 g for 2 h . Fractions were collected from the top of the gradient and TCA - precipitable samples analyzed by SDS - PAGE and LC / MS - MS . For visual - ization of Nycodenz fraction morphology by EM , 10 \u00b5l of fraction of interest was mixed with 10 \u00b5l 5 % glutaraldehyde and a 100 mesh copper EM grid was placed on top for 30 min at room temperature . Grids were washed three times 5 min with PBS , followed by five times 2 min H 2 O before staining with 1 : 9 ( by volume ) of 4 % uranyl acetate to 2 % methyl cellulose for 10 min on ice . CTxB biotinylation CTxB - 555 was biotinylated with 2 mg . ml 2 1 EZ - link Sulfo - NHS - SS - biotin ( Thermo Fisher Scientific ) for 10 min at 37\u00b0C and biotin quenched with 1 : 1 volume of 200 mM glycine for 5 min . CTxB - biotin was added to Cav1 2 / 2 MEFs either treated with 30 \u00b5M Dyngo4a or left untreated for the times indi - cated . After CTxB - biotin internalization , cells were placed on ice and stripped with three times 5 - min washes with 100 mM MesNa ( Sigma - Aldrich ) . MesNa was quenched with three times 5 - min washes with 54 mM iodo - acetamide ( Sigma - Aldrich ) . Cells were harvested in TNE ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , and 5 mM EDTA ) with complete protease inhibitors ( Roche ) and solubilized with 1 % Triton X - 100 . BCA protein assays ( Thermo Fisher Scientific ) were routinely performed to accommodate equivalent loading . Western blots were probed with streptavidin - HRP ( Invitrogen ) in 5 % skim milk , 0 . 1 % Tween 20 . Intensity of developed HRP chemiluminescent bands was determined for multiple exposures per sample in ImageJ using Gel Analyzer . Stereology Serum - starved cells were incubated directly with 10 \u00b5g . ml 2 1 CTxB - HRP , 20 mg . ml 2 1 HRP ( Sigma - Aldrich ) , 10 \u00b5g . ml 2 1 anti - CD44 - HRP antibodies or 20 \u00b5g . ml 2 1 Tf - HRP ( Invitrogen ) for desired times before being placed on ice , washed with precooled CO 2 - independent media , and processed for the DAB reaction and EPON embedding . Vertical sections , visualized at 20 , 000x on a transmission electron microscope ( model 1011 ; JEOL ) , were overlayed with a 2 , 000 - nm square lattice grid using iTEM software . The number of intersections that fell on top of the cytoplasm , excluding the nu - cleus , were recorded across 20 \u2013 25 cells and three independent areas . A 200 - nm square lattice grid was also applied and intersections that fell on top of DAB - labeled structures were recorded as above . Number of points lying over DAB - labeled structures multiplied by the grid size was com - pared , as a percentage , to points over cytoplasm by grid size to give the percentage of labeled structures to cytoplasmic volume ( V ( v ) ) . Overprojec - tion effects on endosomal tubules can lead to volume overestimation as de - scribed previously ( Griffiths et al . , 1989 ) . Based on the section thickness , average diameter , and length of CLICs , a correction factor of 0 . 65 can be applied to the V ( v ) measurements , based on graphs previously developed ( Weibel , 1979 ) . Correction factors are approximate only and give an esti - mate of possible error . Based on tomography data ( Fig . 3 ) , however , CLICs display a depth greater than section thickness ( entire volume is still within 300 - nm section , far thicker than 55 - nm sections used for volume calcula - tion ) , in which case overprojection effects become negligible . Surface density , or surface volume fraction ( S ( v ) ) , was calculated by placing a cycloid grid over high resolution images of CTxB - HRP \u2013 labeled structures and using the formula : S ( v ) = 2I / P\u2022l ( p ) , where I = total intersec - tions of cycloid grid with structure of interest , P = total points of cycloid grid lying over structure of interest , and l ( p ) = length of the cyloid in \u00b5m , which was an absolute distance of 75 nm in this study . Total cellular volume of a Cav1 2 / 2 MEF was calculated by binding CTxB - 555 to fixed cells . Confocal Z - stacks were obtained from five repre - sentative cells and total average cellular volume was calculated using Volocity v3 . 7 . 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Stereological Methods . 1 . Practical methods for biological morphometry . Academic Press Inc . , New York . Yarar , D . , C . M . Waterman - Storer , and S . L . Schmid . 2007 . SNX9 couples actin assembly to phosphoinositide signals and is required for mem - brane remodeling during endocytosis . Dev . Cell . 13 : 43 \u2013 56 . doi : 10 . 1016 / j . devcel . 2007 . 04 . 014 D o w n l oaded f r o m h tt p : / / r up r e ss . o r g / j c b / a r t i c l e - pd f / 190 / 4 / 675 / 1348109 / j c b _ 201002119 . pd f b y U n i v e r s i t y O f W a s h i ng t on u s e r on 30 J une 2023", + "ayyar2023clic": "Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection B . Vijayalakshmi Ayyar 1 , Khalil Ettayebi 1 , Wilhelm Salmen 2 , Umesh C . Karandikar 1 , Frederick H . Neill 1 , Victoria R . Tenge 1 , Sue E . Crawford 1 , Erhard Bieberich 3 , B . V . Venkataram Prasad 2 , Robert L . Atmar 1 , 4 & Mary K . Estes 1 , 4 Globally , most cases of gastroenteritis are caused by pandemic GII . 4 human norovirus ( HuNoV ) strains with no approved therapies or vaccines available . The cellular pathways that these strains exploit for cell entry and internaliza - tion are unknown . Here , using nontransformed human jejunal enteroids ( HIEs ) that recapitulate the physiology of the gastrointestinal tract , we show that infectious GII . 4 virions and virus - like particles are endocytosed using a unique combination of endosomal acidi \ufb01 cation - dependent clathrin - independent carriers ( CLIC ) , acid sphingomyelinase ( ASM ) - mediated lysosomal exocytosis , and membrane wound repair pathways . We found that besides the known interaction of the viral capsid Protruding ( P ) domain with host glycans , the Shell ( S ) domain interacts with both galectin - 3 ( gal - 3 ) and apoptosis - linked gene 2 - interacting protein X ( ALIX ) , to orchestrate GII . 4 cell entry . Recognition of the viral and cellular determinants regulating HuNoV entry provides insight into the infection process of a non - enveloped virus highlighting unique pathways and targets for developing effective therapeutics . Human noroviruses ( HuNoVs ) , non - enveloped , single - stranded , positive - sense RNA viruses , are classi \ufb01 ed into at least ten genogroups ( GI - GX ) and 48 genotypes and belong to the family Caliciviridae . HuNoVs are the leading cause of acute gastroenteritis worldwide 1 , posing a signi \ufb01 cant risk to global health . Speci \ufb01 c treatments and vac - cines are lacking , partly due to the extended time before a reliable and tractable culture system became available to cultivate HuNoV , which limited understanding of norovirus pathophysiology , drivers of host - virus interactions , and systemsto testapproaches to controlinfection . A breakthrough came when nontransformed human intestinal enteroids ( HIEs ) were proven to be a reproducible and biologically relevant system that supports the replication of multiple HuNoV strains 2 , 3 . HIEs have been successfully applied to ( 1 ) identify HuNoV strain - speci \ufb01 c growth requirements 2 , 4 \u2013 6 , ( 2 ) identify neutralizing monoclonal antibodies and improve understanding of host responses to HuNoV for vaccine development 7 \u2013 10 , and ( 3 ) monitor the environ - mental HuNoV load and the ef \ufb01 cacy of disinfection strategies 11 , 12 . HIEs recapitulate the cellular complexity , diversity , and physiology of the human gastrointestinal tract along with host restriction and genetic factors and mimic strain - speci \ufb01 c epidemiological host - virus infection patterns , making them an ideal system to dissect HuNoV infection and pathophysiology 13 . Virusentryintothecellisthe \ufb01 rststepininfection . Itisinitiatedby interactions between speci \ufb01 c motifs on both viral and host surface proteins and activates signaling cascades that destabilize the mem - branebarrier . Although virus - cellsurfaceinteractionsare complex and highly variable , the number of pathways utilized by viruses for cell entry is relatively limited based on certain key surface components . Received : 7 March 2022 Accepted : 30 January 2023 Check for updates 1 Department of Molecular Virology and Microbiology , Baylor College of Medicine , Houston , TX , USA . 2 Verna and Marrs McLean Department of Biochemistry and Molecular Biology , Baylor College of Medicine , Houston , TX , USA . 3 Department of Physiology , University of Kentucky , Lexington , KY 40506 and VAMC , Lexington , KY 40502 , USA . 4 Department of Medicine , Baylor College of Medicine , Houston , TX , USA . e - mail : mestes @ bcm . edu Nature Communications | ( 2023 ) 14 : 1148 1 1 2 3 4 5 6 7 8 9 0 ( ) : , ; 1 2 3 4 5 6 7 8 9 0 ( ) : , ; Herein , we sought to uncover entry determinants of a globally domi - nant GII . 4 HuNoV straininto physiologicallyrelevant HIEs that support virus replication . Our studies show that GII . 4 HuNoV binding to epi - thelial cells induces plasma membrane wounding that triggers cellular components to the injury site for membrane repair . Surprisingly , we foundtheviruscapsidinteractswiththeendosomalsortingcomplexes requiredforthetransport ( ESCRT ) proteinALIXandinduceslysosomal exocytosis along with other membrane repair mechanisms . GII . 4 HuNoV enters HIEs using a speci \ufb01 c mechanism driven by endosomal acidi \ufb01 cation and requires effectors of the CLIC pathway [ cholesterol , Cdc42 , ADP - ribosylation factor 1 ( Arf1 ) , and galectin - 3 ( gal - 3 ) ] . These studies demonstrate an active CLIC pathway in HIEs , with a crosstalk between CLIC - mediated endocytosis and host repair mechanisms highlighting these pathways as targets for interfering with pathogen entry in human intestinal cells . Results GII . 4 entry in jejunal HIEs requires endosomal acidi \ufb01 cation to initiate endocytosis To probe HuNoV entry into a permissive , secretor positive jejunal ( J2 ) HIE line , we used virus - like particles ( VLPs ) from pandemic GII . 4 Syd - ney / 2012 ( GII . 4 Syd VLPs ) and GII . 4 Sydney / 2012 virus . We \ufb01 rst validatedVLPsasasurrogateforvirusinthesestudiesbyshowingGII . 4 Syd VLPs inhibited virus replication of a GII . 4 stool isolate in a dose - dependent manner ( Fig . 1a ) . Several HuNoV strains are dependent on intestinal components such as bile acid ( BA ) and ceramide for cell entry and replication 6 . GII . 4 infection is enhanced by , but does not require , bile or BA for replication 2 , while initiation of endosomal acidi \ufb01 cation with BAglycochenodeoxycholic acid ( GCDCA ) is required for GII . 3 infection 6 . To understand if endosomal acidi \ufb01 cation is a requirement for GII . 4 entry , HIEs were inoculated with GII . 4 virus , and acidic compartments were labeled with LysoTracker . In GII . 4 - inocu - latedcells , asigni \ufb01 cantincreaseinLysoTracker - positiveorganelleswas seen , which was signi \ufb01 cantly reduced in the presence of neutralizing GII . 4 - speci \ufb01 c polyclonal antibody ( pAb ) , indicating that acidic orga - nelles are induced in response to virus - cell interactions ( Fig . 1b ) . Endosomal acidi \ufb01 cation studies showed that GII . 4 Syd VLPs , like virus , induced endosomal acidi \ufb01 cation that was not observed with GII . 3 VLPs , indicating strain - speci \ufb01 c differences in the ability to trigger endocytosis ( Fig . 1c ) . Moreover , GII . 4 infection in the presence of ba \ufb01 lomycin A1 , a vacuolar ATPase ( V - ATPase ) inhibitor , reduced the number of GII . 4 - induced acidic compartments ( Fig . S1a ) , completely inhibiting GII . 4 replication in a dose - dependent manner ( Fig . 1d ) . Time course experiments showed a partial inhibition of GII . 4 replication Untreated BafilomycinA1 ( 1 . 5nM ) BafilomycinA1 ( 3 . 1nM ) BafilomycinA1 ( 6 . 2nM ) BafilomycinA1 ( 12 . 5nM ) 0 2 4 6 8 G e no m e e qu i va l e n t s / w e ll 1h24h P = < 0 . 0001 * * * * d * * * * P = < 0 . 0001 P = < 0 . 0001 P = < 0 . 0001 * * * * * * * * Untreated GII . 4 VLP ( 10 4 ) GII . 4 VLP ( 10 5 ) GII . 4 VLP ( 10 6 ) 0 2 4 6 8 G e no m e e qu i va l e n t s / w e ll 1 h 24 h P = < 0 . 0001 P = 0 . 0005 P = 0 . 0085 * * * * * * * * * H u m a n p A b G II . 4 s t oo l G II . 4 s t oo l + hu m a n p A b 0 20 40 60 80 M ea n f l uo r esce n se i n t e n s i t y a b c e P = < 0 . 0001 * * * * F M 1 - 43 F X Labe l ed S y d V L P s f 10 m i n Media GII . 4 VLP GII . 3 VLP 50 \u00b5M FM1 - 43FX 10 m i n Media GII . 4 VLP GII . 3 VLP 0 50 100 150 200 M ea n f l uo r esce n se i n t e n s i t y P = < 0 . 0001 * * * * M e d i a G II . 3 V L P G C D C A G II . 4s t oo l G II . 4 V L P 0 1000 2000 3000 4000 M ea n f l uo r esce n se i n t e n s i t y P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * * * * * P = < 0 . 0001 20 \u00b5m 50 \u00b5m Human pAb GII . 4 stool GII . 4 stool + pAb Fig . 1 | GII . 4 capsid protein elicits acidi \ufb01 cation and endocytosis in HIEs . a Viral replication at 24h ( black dots ) compared to bound virus at 1h ( blue dots ) and inhibition of replication in the presence of VLPs compared to untreated at 24h . Replication was quanti \ufb01 ed using n = 2 independent HIE replicates for the 1h and n = 3 independent HIE replicates for 24h with 2 technical replicates / sample . b LysoTracker staining of acidic compartments in the presence of an anti - GII . 4 polyclonalantibody ( pAb ) , GII . 4virus , andpAbmixedwithGII . 4virusat37 o C . Right panel : Mean \ufb02 uorescenceintensitywasquanti \ufb01 edfromdifferentregionsofinterest ( ROIs ) forpAb ( bluebar , ROIs = 32 ) , GII . 4stool ( redbar , ROIs = 31 ) , GII . 4stool + pAb ( green bar , ROIs = 32 ) . c LysoTracker staining of acidic compartments induced by GII . 3 VLP ( green , ROIs = 100 ) , GCDCA ( purple , ROIs = 100 ) , GII . 4 virus ( cyan , ROIs = 101 ) , and GII . 4 VLP ( red , ROIs = 97 ) compared to media ( black , ROIs = 140 ) . d GII . 4 replication in the presence / absence of V - ATPase inhibitor ba \ufb01 lomycin A1 at 1h ( boundvirus , graydots ) andat24h ( blackdots ) . ViralGEswerequanti \ufb01 edusing n = 2independentHIEreplicatesforthe1hand n = 3independentHIEreplicatesfor 24h with 2 technical replicates / sample . e FM1 - 43FX ( green ) uptake showing GII . 4 VLP - induced endocytosis . VLP - induced endocytosis compared to media ( n = 4 HIE replicates ) . Right panel : Mean \ufb02 uorescence intensity quanti \ufb01 ed from ROI = 10 . f Time lapse microscopy showing GII . 4 VLP ( green ) endocytosis and FM1 - 43x uptake ( red ) . All the experiments were repeated independently three times with similar results . In a \u2013 e , error bars represent mean\u00b1SD with signi \ufb01 cance ( P values ) calculated using one - way ANOVA , Dunnett \u2019 s multiple comparisons test . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 2 when ba \ufb01 lomycin A1 was present only in the early stages of infection , while complete inhibition was observed when the inhibitor was pre - sent throughout the entire infection period [ 24h post infection ( h ) ] ( Fig . S1b , upper right panel ) . This was due to the presence of membrane - bound VLPs , which after ba \ufb01 lomycin A1 removal , activated endocytosis reversing inhibition of V - ATPase ( Fig . S1b , lower right panel ) . GII . 4 - induced endocytosis was con \ufb01 rmed using FM1 - 43FX epi - \ufb02 uorescence and time lapse microscopy ( Fig . 1e , f ) . FM1 - 43FX labels extracellular membranes and \ufb02 uorescent puncta are observed fol - lowing endocytosis . An upregulation of FM1 - 43FX - labeled compart - ments was observed in GII . 4 VLP - treated cells further con \ufb01 rming that unlikeGII . 3 ( whichrequiresBA ) , theGII . 4Sydcapsidaloneiscapableof triggering endocytosis upon interaction with the host cells ( Fig . 1e ) . Endocytosis experiments in the presence of ba \ufb01 lomycin A1 indicated that GII . 4 endocytosis is V - ATPase dependent ( Fig . S1c ) and required the intact VLP as the glycan - binding P domain alone did not trigger endocytosis ( Fig . S1d ) . FM1 - 43FX labeling experiments in the presence ofadditionalVLPsshowedthatfourGII . 4variants [ HoustonVirus / 2002 ( HOV ) , New Orleans / 2009 ( NO ) , Grimsby / 1995 ( GRV ) , Farmington Hills / 2002 ( FH ) ] induced endocytosis while HuNoV strains thatrequire BA ( GII . 3 , GI . 1 and GII . 17 ) did not induce endocytosis , further sup - porting the use of different cell entry mechanisms between BA - dependent and independent HuNoV strains ( Fig . S1d ) . GII . 4 uses a dynamin - independent pathway that requires mem - brane cholesterol and is in \ufb02 uenced by actin GII . 4 entry was explored further by using speci \ufb01 c inhibitors of membrane components known to be involved in endocytic path - ways ( Fig . 2a ) . Dynamin , important for both clathrin - and caveolin - mediated endocytosis , was \ufb01 rst tested considering the entry of murine norovirus ( MNV ) in RAW264 . 7 macrophages is dynamin - dependent 14 . Inhibition of dynamin with speci \ufb01 c inhibitors , dynasore and mitmab , did not alter GII . 4 infection , showing that clathrin - and caveolin - mediated endocytosis pathways are not involved in GII . 4 cell entry ( Fig . 2b ) . The ef \ufb01 cacy of dynasore and mitmab in HIEs was con \ufb01 rmed using a \ufb02 uorescently labeled , low - density lipoprotein ( LDL ) - uptake assay , where both inhibitors reduced LDL uptake ( Fig . 2c ) at the tested concentrations ( 40 and 100 \u00b5 M ) . We next targeted cholesterol , another abundant plasma mem - brane component implicated in the entry and replication of many viruses , including GII . 3 HuNoV 6 , 14 \u2013 18 . Cholesterol depletion in HIEs , by inhibitors methyl - \u03b2 - cyclodextrin ( M \u03b2 CD ) and \ufb01 lipin , reduced GII . 4 infection signi \ufb01 cantly ( Fig . 2d , Table S1 ) . In addition , a lower level of inhibition in GII . 4 replication with U18666A , an intracellular choles - terol transport inhibitor , was observed ( Fig . S2a ) compared to M \u03b2 CD and \ufb01 lipin . The activity of U18666A in HIEs was con \ufb01 rmed using \ufb01 lipin and CD63 staining , indicating defective intracellular cholesterol f Untreated M\u00dfCD ( 1mM ) Filipin ( 4\u03bcg / ml ) 10 3 10 4 10 5 10 6 G e no m e e qu i va l e n t s / w e ll 1 h 24h d * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 Untreated CytoD ( 2 . 5\u03bcM ) CytoD ( 5\u03bcM ) 10 3 10 4 10 5 10 6 G e no m i ce qu i va l e n t s / w e ll 1h24h 20 \u00b5m Untreated Mitmab ( 100 \u03bcM ) Dynasore ( 40 \u03bcM ) Dil - LDL a b h c e g T ubu li n Untreated Dynasore ( 40 \u03bcM ) Mitmab ( 100 \u03bcM ) 10 3 10 4 10 5 10 6 10 7 G e no m e e qu i va l e n t s / w e ll 1h 24h Untreated Noco ( 5\u03bcM ) Noco ( 20\u03bcM ) 10 2 10 3 10 4 10 5 10 6 10 7 G e no m e e qu i va l e n t s / w e ll 1 h 24h Untreated Genistein ( 40\u03bcM ) Genistein ( 80\u03bcM ) 10 3 10 4 10 5 10 6 G e no m e e qu i va l e n t s / w e ll 1h24h * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 U n t r ea t e d D y n as o r e ( 40 \u03bc M ) M i t m a b ( 100 \u03bc M ) 0 50 100 150 M ea nF l uo r esce n se i n t e n s i t y P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * Nocodazole ( 20 \u00b5M ) Media Genistein ( 80 \u00b5M ) 10 \u00b5m Fig . 2 | GII . 4 entry is dynamin - independent but depends on cholesterol and actinforinfection . a Schematicofmajorendocytosispathways , keyregulatorsand their inhibitors . b GII . 4 replication ( at 37 o C ) was assessed in the presence of dynamininhibitors , dynasoreandmitmab . ViralRNAreplicationwasquanti \ufb01 edat1 ( black ) and 24h ( green ) by RT - qPCR . c Validation of dynamin inhibitor activity by Dil - complexed low density lipoprotein ( Dil - LDL ) uptake ( red ) . Right panel : Quan - titation of Dil - LDL uptake in untreated ( 80 ROI ) , dynasore - treated ( 38 ROI ) and mitmab - treated ( 55 ROI ) HIEs . d GII . 4 replication in the presence of cholesterol sequestrants , M\u00dfCD and \ufb01 lipin , at 1h ( black ) and 24h ( red ) at 37 o C . e GII . 4 repli - cationinthe presenceof actindepolymerizingagent cytochalasinD ( Cyto D ) at1h ( black ) and 24h ( green ) . f GII . 4 replication in the presence of receptor tyrosine kinase ( RTK ) inhibitor genistein at 1h ( black ) and 24h ( blue ) at 37 o C . g GII . 4 replication in the presence of nocodazole , a microtubule disrupting agent , at 1h ( black ) and24h ( pink ) at37 o C . h Tubulinstaining ( red ) inthepresenceofgenistein andnocodazole . Alltheexperimentswererepeatedindependentlythreetimeswith similar results . In b , d \u2013 g viral GEs were quanti \ufb01 ed using n = 2 independent HIE replicates for the 1h and n = 3 independent HIE replicates for 24h with two tech - nical replicates / sample . In b , viralGEs werequanti \ufb01 ed using n = 3 HIE replicates . In b \u2013 g , errorbars represent mean\u00b1SDwith signi \ufb01 cancerelative to untreated control calculated using one - way ANOVA , Dunnett \u2019 s multiple comparisons test . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 3 transport ( Fig . S2b ) . Altogether , these results show that cholesterol is critical for GII . 4 replication . We next evaluated GII . 4 entry dependence on cytoskeletal com - ponentsusinginhibitorsagainstactin , receptortyrosinekinases ( RTK ) , and microtubules . Cytochalasin D , an actin - depolymerizing agent , reduced GII . 4 replication signi \ufb01 cantly ( 53 % ) ( Fig . 2e ) , whereas genis - tein ( RTK and caveolin inhibitor ) and nocodazole ( microtubule inhi - bitor ) had no effect on GII . 4 replication ( Fig . 2f , g ) . Tubulin staining con \ufb01 rmed the activities of genistein and nocodazole in HIEs ( Fig . 2h ) , suggesting that actin , but not microtubule or RTKs , is essential for GII . 4 entry . GII . 4 depends on the CLIC pathway for cell entry Inhibition of GII . 4 entry by cytochalasin D showed that viral entry was in \ufb02 uenced by actin manipulation , but this drug was cytotoxic above the concentrations tested . This led us to examine other regulators of actinmechanicsthatin \ufb02 uenceclathrin - independentendocytosis ( CIE ) . Aftercon \ufb01 rmingthat theconcentrationofinhibitorsusedinassaysdid not induce cytotoxicity ( Table S1 ) , we tested macropinocytosis and CLICendocytosis ( Fig . 3a ) , twomajorCIEpathways , focusingonCdc42 [ EIPA , ML141 , wiskostatin ( Wisko ) ] , Rac1 [ NSC23766 ( NSC ) ] , and RhoA ( CT04 ) . Inhibition of Cdc42 ( EIPA , ML141 , and Wisko ) dramatically reduced GII . 4 replication ( 86 \u2013 90 % ) ( Figs . 3b and S3a ) , whereas inhi - biting Rac1 or RhoA had no effect on GII . 4 replication . Other inhibitors of classical macropinocytosis such as smooth muscle myosin II inhi - bitor [ blebbistatin ( Bleb ) ] and PI3K inhibitor [ LY29402 ( LY ) ] , known to ef \ufb01 ciently block the entry of vaccinia virus 19 , human adenovirus type 3 20 , and respiratory syncytial virus 21 , were also tested along with inhi - bitors of epidermal growth factor receptor ( EGFR ) ( CAS879127 - 07 - 8 ) andproteinkinaseC ( PKC ) ( calphostin ) . NoneoftheseinhibitorsofCIE had any effect on GII . 4 replication ( Figs . 3b and S3b ) , emphasizing that GII . 4 entry is via a Cdc42 - speci \ufb01 c mechanism distinct from a classical macropinocytosis pathway . The ef \ufb01 cacy of inhibitors in HIEs was con \ufb01 rmed by actin staining , which detected morphological changes in treated cells ( Fig . S3c ) . Next , we evaluated GII . 4 infection in the pre - sence of an Arf1 inhibitor , Golgicide A ( GCA ) , and found that GCA signi \ufb01 cantly inhibited GII . 4 replication ( 65 \u2013 75 % ) at 24 h ( Fig . 3c ) . Taken together , the inhibitor data suggest that GII . 4 entry relies on cholesterol , Cdc42 and Arf1 , indicating a CLIC - mediated virus entry pathway . However , inhibitors can have many off - target effects , so we used a multipronged approach by evaluating the morphology of CLIC carriers , assessing GII . 4 association with gal - 3 ( a regulator of the CLIC pathway ) 22 and testing GII . 4 colocalization with known markers of the CLIC pathway . Fluorescence microscopy showed that GII . 4 induces tubularstructureformationreminiscentofCLICcarriersinVLP - treated cellswith variable lengths ( 200nm \u2013 2 \u00b5 m ) ( Fig . 3d ) , which were further con \ufb01 rmedbyelectron microscopy ( EM ) ( Fig . 3e ) . EMalsoshowedVLPs in vacuolar structures similar to those observed in mouse jejunal * * * * * P = < 0 . 0001 c e d a h b i VLP - treated ( 1 h ) Mock - treated ( 1 h ) 500 nm 500 nm 2 \u00b5m 2 \u00b5m G e no m e e qu i va l e n t s / w e ll * P = 0 . 0133 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 P = 0 . 0322 * * * * P = < 0 . 0001 g G e no m e e qu i va l e n t s / w e ll P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * 1 h 10 min Media G a l - 3 VP 1 M e r ge 10 \u00b5m VP1 Actin 5 \u00b5m f Fig . 3 | GII . 4 infection in HIEs is sensitive to effectors of CLIC pathway . a Schematic of macropinocytosis and the Clathrin Independent Carrier ( CLIC ) pathways , their effectors and inhibitors . b GII . 4 replication inthe presenceof EIPA ( Na + / H + exchanger inhibitor ) , ML141 ( Cdc42 inhibitor ) , Wiskostatin ( N - WASP inhi - bitor ) , NSC23766 ( RAC1 inhibitor ) , CT04 ( RhoI inhibitor ) , Blebbistatin ( myosin inhibitor ) , and LY29402 ( PI3K inhibitor ) at 1h ( gray ) and 24h ( orange ) . c GII . 4 replicationinthepresenceofArf1inhibitorGolgicideA ( GCAat1h ( black ) and24h ( blue ) . d GII . 4 - inducedtubularcarriersat1h ( 37 o C ) detectedusingguineapiganti - Sydney VLP polyclonal antibody ( Gp Syd - pAb ) for viral capsid ( green ) and phal - loidin for actin ( red ) . Images were taken using a ZEISS Laser Scanning Micro - scope LSM 980 with Airyscan 2 . e Electron microscopy to identify CLIC structures inGII . 4VLP - treatedHIEs . ( 1 \u2013 7structures / cell ) comparedto , mock - treatedcells ( no structures in n = 25cells ) . f Confocalmicroscopytodetect GII . 4VP1capsid ( green ) colocalizationwithgal - 3 ( red ) onthecellsurfaceat10minand1h ( 37 o C ) afterVLP treatment using anti - gal - 3 and Gp Syd - pAb ( n = 3 HIE replicates ) . g Effect of blocking GII . 4 virus - galectin - 3 ( gal - 3 ) surface interaction using anti - gal - 3 antibody on GII . 4 replication at 1h ( black ) and 24h ( red ) . h Dot blot analysis investigating GII . 4 VLP interaction withpuri \ufb01 ed gal - 3 . i Probing CLIC carriersutilized in HIEs for endocytosis with Alexa Fluor \u2122 594 conjugated cholera toxin B ( CTxB ) ( red ) and GII . 4 VLPs ( green ) with similar cargoes marked by white arrows ( n = 2 HIE repli - cates ) . Inset : Co - occurrence of CTxB and GII . 4 VLPs in similar cargos , scale = 5 \u00b5 m . All the experiments were repeated independently three times with similar results . In b , c , and g viral GEs were quanti \ufb01 ed using n = 2 independent HIE replicates for the 1h and n = 3 independent HIE replicates for 24h with two technical replicates / sample . Theerrorbarsrepresentmean\u00b1SDwith P valuescalculatedusingone - way ANOVA , Dunnett \u2019 s multiple comparisons test with comparisons at 24h relative to untreated control . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 4 enterocytes ( Fig . S3d ) 23 . Next , GII . 4association with gal - 3wasassessed using \ufb02 uorescence microscopy , gal - 3 inhibition assays , and direct binding respectively ( Figs . 3f , S3e , S3f , 3g , 3h , and 3i ) . We found that GII . 4 colocalizes with gal - 3 ( Fig . 3f ) in a time - dependent manner . In the presence of TD - 139 , a speci \ufb01 c gal - 3 inhibitor , both GII . 4 colocalization and endocytosis is rapidly ( 10 min ) inhibited suggesting the impor - tance of gal - 3 in GII . 4 entry into cells ( Figs . S3e and S3f ) . Gal - 3 is a multifunctional protein involved in many cellular processes and is foundinboththebiotinylatedsurfaceprotein ( BSP ) plasmamembrane and cytosolic fractions of cells ( Fig . S3g ) . As inhibitors are unable to differentiatebetween the differentpopulationsof gal - 3 , we speci \ufb01 cally used anti - gal - 3 antibody to target gal - 3 interactions on the plasma membrane . Our results showed that anti - gal - 3 antibody signi \ufb01 cantly inhibited GII . 4 replication ( 98 % reduction ) , further asserting the importance of gal - 3 - GII . 4 interaction in GII . 4 entry and infection ( Fig . 3g ) . Direct binding assays showed that gal - 3 bound to VLPs ( Fig . 3h ) , and to the GII . 4 capsid S domain , but not to the P domain , of VP1 of VLPs , with micromolar af \ufb01 nity ( Figs . S3h and S3i ) . Finally , we investigated GII . 4 colocalization using speci \ufb01 c markers of the CLIC pathway such as cholera toxin B ( CTxB ) , CD44 and glycosylphosphatidylinositol - anchored protein ( GPI - AP ) . All these proteins are well studied and characterized for their usage in CLIC - mediated endocytosis 24 \u2013 26 . Fluorescence microscopy showed that GII . 4 shared cargoes with CTxB ( Fig . 3i ) despite not having any effect on replication when recombinant CTxB was used as a competitor ( Fig . S3j ) . ThissuggeststhatalthoughGII . 4usesasimilarentrypathway as CTxB , the receptor / s used for each is distinct . GII . 4 colocalization with CD44 and GPI - AP further veri \ufb01 ed that GII . 4 uses CLIC - mediated endocytosis for entry ( Figs . S3k and S3l ) . ESCRT protein ALIX is critical for GII . 4 entry ESCRTs are known regulators of endocytosis . The unexpected pre - sence of conserved ESCRT binding motifs for ALIX and TSG101 in the GII . 4 capsid sequence ( Fig . S4a ) led us to further investigate if these proteinsareinvolvedinGII . 4entry . Bindingassays ( ELISAanddot - blot ) showedthatGII . 4SydVLPsinteractwithrecombinantALIXandTSG101 ( Figs . 4a , b , S4b ) . Confocal microscopy of virus - infected HIEs showed colocalization between replicating virus , and ALIX and TSG101 at 24 h , suggesting their role in GII . 4 replication ( Fig . S4c ) . Putative ESCRT bindingsequencesarepresentinboththeGII . 4capsidSandPdomains ( Fig . S4a ) ; however , ELISAdatashowedthatwhileTSG101bindstoboth the S and P domains ( Fig . 4b ) , ALIX binding is limited to the S domain only ( Fig . 4a ) . Bio - Layer Interferometry ( BLI ) analysis con \ufb01 rmed the ELISA results , demonstrating that binding af \ufb01 nity of ALIX to the S domain ( K D = 9 . 92 \u00b5 M ) and TSG101 to the S domain ( K D = 21 . 5 \u00b5 M ) and the P domain ( K D = 15 . 5 \u00b5 M ) were in the micromolar range ( Fig . 4c ) . We further probed the contribution of ALIX and TSG101 in GII . 4 entry using protein - speci \ufb01 c antibodies as blocking agents . While anti - ALIX pAb blocked GII . 4 replication completely in a dose - dependent manner , anti - TSG101 pAb had no effect on GII . 4 replication ( Figs . 4d and S4d ) . These data suggest that ALIX is accessible to GII . 4 VLPs on the membrane surface , which was further con \ufb01 rmed by Western blot detection of ALIX in the biotinylated surface protein ( BSP ) fraction of HIEswithananti - ALIXpAb ( Fig . S4e ) . Confocalmicroscopyestablished A b s o r b a n ce ( 450 n m ) A b s o r b a n ce ( 450 n m ) Untreated Anti - ALIX ab ( 80 \u03bcg ) Anti - TSG ab ( 80 \u03bcg ) 10 3 10 4 10 5 10 6 10 7 1 h24 h Media GII . 4 VLP \u0394VLP 50 \u00b5M i FM1 - 43x A b s o r b a n ce ( 450 n m ) c b a d e g h f bALIXvs GII . 4 S - Domain bTSG101 vs GII . 4 S - Domain bTSG101 vs GII . 4 P - Domain Dimer 10 m i n % Fo l d c h a ng e i n v i r a l RNA ( 24 h - 1 h ) no r m a li z e d t o G A P DH * * * P = 0 . 0003 P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * 25 \u00b5m Fig . 4 | GII . 4 interacts with ALIX and TSG101 , withALIX being criticalfor virus entry . a GII . 4VLP , SandPdomainbindingtoimmobilizedALIXbyELISA . Errorbars indicate mean\u00b1SD with three replicates . b GII . 4 VLP , S and P domain binding to immobilized TSG101byELISA . Error bars indicate mean\u00b1SDwith three replicates . c Bio - Layer Interferometry ( BLI ) to determine the binding af \ufb01 nity of GII . 4 S and P domains to biotinylated ALIX and TSG101 ( bALIX and bTSG101 ) . d Effect of blocking virus interaction with ALIX and TSG101 on viral replication using speci \ufb01 c pAbsat1h ( gray ) and 24h ( pink ) . Error barsindicate mean\u00b1SDcalculated using2 HIE replicates for 1h and 4 HIE replicates for 24h ( with two technical replicates / sample ) . e Confocal microscopy to probe GII . 4 - ALIX colocalization on the cell surface by detecting VP1 ( green ) and ALIX ( red ) using Gp Syd - pAb and rabbitanti - ALIX pAb . f ELISA to evaluate binding of GII . 4 S - and mutant \u0394 S - domains to immobilized ALIX . Error bars indicate mean\u00b1SD with 3 replicates . g Endocytosis induced by wild type GII . 4 VLP and GII . 4 VLP lacking the ALIX \u2013 binding motif ( \u0394 VLP ) usingFM1 - 43FXinHIEsat37 o C ( n = 3HIEreplicates ) . h Viralreplicationinthe presence of GII . 4 WT VLPs ( pink bars ) and \u0394 VLPs ( blue bars ) was compared to untreated ( black bar ) at 24h ( blue dots ) . 1h ( gray dots ) represents bound virus . Error bars indicate mean\u00b1SD calculated using 2 HIE replicates for 1h and 3 HIE replicatesfor24h ( eachconditionwithtwotechnicalreplicates ) . i GII . 4replication inWT J2and J2 ALIX - KD HIEmonolayersindicated bypercentfoldchange inviralRNA using the 2 \u2212 \u0394\u0394 CT method . Error bars indicate mean\u00b1SD calculated using 3 HIE replicates for each condition with two technical replicates . All the experiments wererepeatedindependentlythreetimeswithsimilarresults . P valuesfor d , h , and i , relative to untreated control were calculated using one - way ANOVA , Dunnett \u2019 s multiple comparisons test . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 5 a direct link between GII . 4 and ALIX as HIEs incubated with VLPs ( 10 min or 1h ) showed GII . 4 colocalization with ALIX at both time points , withmoreVLP - ALIXcolocalizationobservedat1hcomparedto 10min ( Fig . 4e ) . Notably , media - treated control cells exhibited no signi \ufb01 cant cell surface ALIX staining compared to VLP - treated cells , implying that GII . 4 binding induced ALIX recruitment to the cell sur - face ( Fig . 4e ) . Both ELISA and BLI experiments showed that ALIX binds the GII . 4 capsid S - domain , with a conserved \u201c LYTPL \u201d sequence ( Fig . S4a ) . This sequence was mutated ( \u0394 ) to \u201c AATAL \u201d and this \u0394 S - domain was expressed and puri \ufb01 ed from E . coli . ELISA showed that the \u0394 S - domain does not bind ALIX , thus , con \ufb01 rming the ALIX binding site ( Fig . 4f ) . To further understand the role of ALIX in entry , an ALIX mutant VLP ( \u0394 VLP ) was generated by substituting the \u201c LYTPL \u201d sequence with \u201c AATAL \u201d in GII . 4 Syd VLP . After con \ufb01 rming that the ALIX mutation did not interfere with VLP formation ( Fig . S4f ) , GII . 4 Syd VLP and \u0394 VLP were analyzed using both endocytosis and viral inhibition assays . Both assays showed that the \u0394 VLP was identical to GII . 4 Syd VLP in terms of inducing endocytosis and the ability to block GII . 4 viral replication ( Figs . 4g and 4h ) , which led us to reassess \u0394 VLP binding to ALIX . Binding assays showed that although \u0394 VLP ( unlike the \u0394 S - domain ) boundtoALIX , thebindingwasreducedcomparedtotheGII . 4SydVLP ( Figs . S4g and S4h ) . Thissuggests that , like TSG101 , there maybe more thanoneALIXbindingsitepresentontheviralcapsidinadditiontothe site present in the S - domain . To study the role of ALIX in GII . 4 infection , attempts were made to generate a J2ALIXknock - out HIE but were unsuccessful because the KO cultures did not survive . This led us to generate J2 ALIX knock - down ( J2 ALIX - KD ) HIEs expressing an ALIX - shRNA and a TurboGFP sensor ( Fig . S4i ) . The J2 ALIX - KD cells demon - strated a signi \ufb01 cant reduction in ALIX expression ( 75 % reduction ) compared to the parental J2 cultures ( Fig . S4j ) and in GII . 4 replication ( 75 % reduction ) ( Fig . 4i ) indicating an active role of ALIX in GII . 4 infection . Endolysosomal dynamics and membrane repair mechanisms regulate GII . 4 entry Endolysosomal dynamics play an important role in GII . 3 replication 6 , which led us to investigate the same for GII . 4 . This was achieved by disrupting endosomal maturation and traf \ufb01 cking using YM201636 [ FYVE - type zinc \ufb01 nger - containing phosphoinositide kinase ( PIKfyve ) inhibitor ] and vacuolin 1 ( PIKfyve and lysosomal exocytosis inhibitor ) , which resulted in up to 70 \u2013 80 % reduction in GII . 4 replication com - pared to untreated cells ( Fig . 5a , b ) . Time course experiments showed the presence of inhibitors up to 1 h reduced virus yield by 40 % , whereasupto80 % inhibitioninyieldwasobservedwhentheinhibitors were present up to 2 h or 24 h , thus , suggesting the inhibitor was effective in the early stages of GII . 4 entry ( Figs . S5a and S5b ) . Inhibition of lysosomal exocytosisby vacuolin - 1 led toa reduction in GII . 4 replication , indicating that GII . 4 might require acid sphingo - myelinase ( ASM ) translocation and surface ceramides for infection like P = < 0 . 0001 * * * * G e no m e e qu i va l e n t s / w e ll P = 0 . 0090 P = 0 . 0007 * * * P = < 0 . 0001 * * * * * * a c b d e f P = 0 . 0012 * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * C e ll s w i t h P I a nd DA P I c o - s t a i n i ng P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P ea r s on C o rr e l a t i on C o e ff i c i e n t ( P CC ) P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * P = < 0 . 0001 * * * * VP1 Gal - 3 ALIX / Gal - 3 VP1 / ALIX / Gal - 3 M ed i a V L P ( 1 h ) ALIX 10 \u00b5m 10 \u00b5m VLP - treated ( 1 h ) Media VP 1 L A M P - 1 M e r ge Fig . 5 | GII . 4 entry is sensitive to factors controlling endo - lysosomal home - ostasis and induces membrane wounding and subsequent wound repair mechanisms . a GII . 4 replication in the presence of PIKfyve inhibitor YM201636 at 1h ( black ) and24h ( red ) at37 o C . b GII . 4replicationinthepresenceofvacuolin - 1 ( a lysosomalexocytosisinhibitor ) at1h ( gray ) and24h ( purple ) . c GII . 4replicationin the presence of acid sphingomyelinase ( ASM , amitriptyline ) and neutral sphingo - myelinase ( NSM , GW4869 ) inhibitors at 1h ( black ) and 24h ( blue ) . d Cell injury determination using propidium iodide ( PI ) uptake assay . Right panel : Graph quantitating membrane injury calculated by counting number of PI spots ( red ) counterstained with DAPI ( blue ) when HIEs were incubated with VLPs ( ROI = 10 ) , VLP + Ca 2 + ( ROI = 12 ) and media ( ROI = 10 ) . Signi \ufb01 cance was calculated using two - way ANOVA , Tukey \u2019 s multiple comparisons test . e Immuno \ufb02 uorescence staining showingGII . 4 - inducedlysosomalexocytosisrepresentedbythepresenceofLAMP - 1 on the apical cell surface in media - treated cells compared to VLP - treated cells . LAMP - 1 ( red ) andVP1 ( green ) colocalizationwasdetectedusingmouseanti - LAMP - 1 mAband Gp Syd - pAb . ( n = 3 HIE replicates ) . f VP1 , ALIX and Gal - 3 colocalization in VLP - treated and media - treated cells ( 1h at 37 o C ) using confocal microscopy . VP1 ( green ) , ALIX ( red ) and gal - 3 ( white ) were detected using Gp Syd - pAb , rabbit anti - ALIX pAb and rat - anti - gal3 . Right panel : Graph showing colocalization between VP1 , gal - 3 and ALIX as estimated by Pearson Correlation Coef \ufb01 cient using EzCo - localization ( ROI = 16 , black for VLP - and ROI = 17 , blue for media - treated HIEs ) . Errorbarsindicate mean\u00b1SDand P valueswerecalculated using two - wayANOVA , \u0160 \u00edd\u00e1k \u2019 s multiple comparisons test . All the experiments were repeated indepen - dently three times with similar results . Error bars for a \u2013 c indicate mean\u00b1SD cal - culatedusing2HIEreplicatesfor1hand3HIEreplicatesfor24hwithtwotechnical replicates / sample . P values were calculated using one - way ANOVA , Dunnett \u2019 s multiple comparisons test by comparing replication at 24h to untreated control . Source data are provided as a Source Data \ufb01 le . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 6 other caliciviruses 6 , 18 . Therefore , we assayed different enzymes involved in ceramide synthesis for their effect on GII . 4 replication using inhibitorsagainst ASM ( amitriptyline ) , neutral sphingomyelinase ( NSM ; GW4869 ) , and ceramide synthase [ fumonisin 1 ( FB1 ) ] . In addi - tion , we tested if addition of exogenous sphingomyelin ( SM ) , a lipid product upstream of ceramide , has any effect on GII . 4 replication . Among the inhibitors tested , amitriptyline signi \ufb01 cantly inhibited GII . 4 replication ( up to 85 % ) whereas GW4869 , SM addition and FB1 , had no effect con \ufb01 rming that like GII . 3 , GII . 4 requires ASM activity ( Figs . 5c , S5c and S5d ) . FB1 - treated cells had reduced CD95 expression and enhanced intracellular lysosomal associated membrane protein 1 ( LAMP - 1 ) expression compared to untreated cells , which con \ufb01 rmed the activity of FB1 in HIEs ( Fig . S5e ) . Since GII . 4 does not require BA for infection , we postulated that GII . 4 - induced lysosomal exocytosis may catalyze ceramide generation as reported with feline calicivirus ( FCV ) or MNV in porcine kidney LLC - PK cells 18 . Using a ceramide - speci \ufb01 c rabbit antibody 27 , we established that GII . 4 VLPs induce surface cer - amides in HIEs by confocal microscopy ( Fig . S5f ) , con \ufb01 rming that ASM translocation and ceramide generation are associated with GII . 4 infection . Lastly , we examined cellular mechanisms associated with lysoso - mal exocytosis , ASM translocation and surface ceramide generation , which are known to be associated with endocytosis and wound repair mechanisms 28 . GII . 4 association with membrane wounding was inves - tigated using a propidium iodide ( PI ) uptake assay . PI uptake was sig - ni \ufb01 cantly increased in the presence of GII . 4 Syd VLPs ( 10min incubation ) compared to untreated cells and GII . 4 Syd VLP - treated cells with calcium , which facilitates membrane repair by early signaling 29 ( Fig . 5d ) . GII . 4 induction of membrane wounding was associated with VLP binding to cells at 4 o C ( Fig . S5g ) and requires initial VLP interaction with cell surface histo - blood group antigens ( HBGA ) , glycans known to be essential for virus infection . Thus VLP - induced membrane wounding did not occur in isogenic HIEs in which the fucosyltransferase 2 ( FUT2 ) gene was knocked out and thus do not express secretor - dependent HBGAs on their plasma membranes 4 ( Fig . S5h ) . GII . 4 - induced membrane wounding was further con \ufb01 rmed by staining the VLP - treated and untreated cells for ( 1 ) LAMP - 1 and ( 2 ) regulators of endo - lysosomal processes and wound healing mechan - isms , Rab11 and Rab14 30 , 31 . GII . 4 VP1 localized with LAMP - 1 ( Fig . 5e ) , Rab11 ( Fig . S5i ) , and Rab14 ( Fig . S5j ) , on the plasma membrane of HIEs treated with GII . 4 Syd VLPs , validating GII . 4 entry is associated with membrane wounding and healing mechanisms . Subsequently , we revisited gal - 3 and ALIX , as both are known to be coordinating factors in detecting membrane damage and repair 32 , 33 . As expected , we saw that traf \ufb01 cking of both gal - 3 and ALIX to the cell surface was induced by GII . 4 Syd VLP - treatment compared to untreated cells and a high degree of colocalization was observed between gal - 3 , ALIX , and GII . 4 VP1 ( Fig . 5f ) , consistent with the fact that GII . 4 enters the cell by compromising membrane integrity . Discussion Virusesexploitmanycellularpathwaysforcellentry . Sometimessimilar effectors in \ufb02 uence multiple endocytosis routes making it challenging to pinpoint a speci \ufb01 c entry mechanism 34 . For example , both porcine sapovirus ( PoSaV ) and feline calicivirus ( FCV ) are endocytosed via dynamin - and cholesterol - dependent clathrin - mediated endocytosis into kidney cells 35 , 36 , whereas murine norovirus ( MNV ) is endocytosed via dynamin - and cholesterol - dependent , clathrin - and caveoli - independent endocytosis into RAW264 . 7 macrophages 14 . Since most studies of virus entry are carried out in immortalized cell lines that do not fully recapitulate the properties of the original host , characteriza - tion of virus entry into HIEs offers previously unrealized insights into intestinal biology 37 . HIEs are relevant in that they are multicellular , nontransformed , physiologically active human cultures that contain the necessary susceptibility factors for human tropic HuNoV infection . Here , we report the pandemic GII . 4 virus and VLPs initiate a pre - viously uncharacterized and complex entry process into HIEs that involves carriers of a dynamin - independent , clathrin - independent endocytosis pathway , gal - 3 , the ESCRT protein ALIX , and wound repair mechanisms ( Fig . 6 ) . To our knowledge , this viral entry process into nontransformed HIE cells was not previously described , although individual components have been associated with entry of several other viruses into a range of different cell types . VLPs were used for many of our entry studies because infectious virus cannot yet be passaged inde \ufb01 nitely and current yields of virus from infected HIEs are not suf \ufb01 cient to produce puri \ufb01 ed particles for extensivebiochemicalstudies . Since1992 , whenHuNoVVLPswere \ufb01 rst produced and characterized 38 , they have been extensively docu - mented as useful surrogates for studies of virus structure 39 \u2013 42 , anti - genicity , immunogenicity , and glycan binding 43 \u2013 45 . Here , we validated that VLPs are useful for studying virus entry by documenting their effectiveness as competitors of homologous virus replication . VLPs also recapitulate virus differences as VLPs of different strains exhibit differencesinvirusentry ( Fig . S1d ) . GII . 4isrecognizedtobindtoHBGA glycans through the P domain of the capsid 46 , 47 , yet surprisingly , endocytosis into HIEs was not observed with the P domain ( dimer ) alone but wasobserved only with complete VLPs ( having 180 copies of the capsid VP1 protein ) . These results indicate that the capsid shell domain participates in virus entry into cells and add evidence to the hypothesis that cells contain an entry co - receptor in addition to HBGA glycans that serve as initial binding factors for HuNoV infection . Alternatively , it could be that avidity effects , such as multiple inter - actions linked together , are important for endocytosis . We show that GII . 4 uses the CLIC pathway for entry in a HBGA glycan - dependent manner ( Fig . 6 ) . The CLIC pathway is reported to be involved in uptake of abundant surface proteins such as CD44 and glycosylphosphatidylinositol - anchored proteins 25 , 26 , bacterial toxins ( CTxB and Shiga ) 24 , 48 , adeno - associated virus 49 and SARS - CoV - 2 50 . Use of the CLIC pathway by GII . 4 HuNoV in nontransformed HIEs is con - sistent with the CLIC pathway being operational in vivo in mouse enterocytes 23 and tubular invaginations observed in giant unilamellar vesicles treated with GII . 4 VLPs 51 . Our study shows that gal - 3 is recruited by GII . 4 at the site of the GII . 4 - membrane interface and GII . 4 - gal - 3 interactions are necessary for infection , suggesting a putative role of gal - 3 in endocytosis . Although intracellular gal - 3 and extra - cellular gal - 3 have a myriad of functions , extracellular gal - 3 is a key player in the biogenesis of CLIC structures 22 , 23 . Gal - 3 is also known for its crosslinking abilities , organizing membrane proteins / receptors and in \ufb02 uencing their function and signaling 52 . We hypothesize that , as reportedwithCD44 , monomericgal - 3interactswithGII . 4 ( boundtoits still uncharacterized receptor ) on the membrane surface leading to oligomerization ofthereceptor - GII . 4 - gal - 3 complex , and interactionof this complex with multipleglycosphingolipid head groups in the outer plasma membrane lea \ufb02 et causes membrane bending due to an avidity effect 22 andsubsequentlyleadstoendocytosis . AlthoughGII . 4VLPsare shownto beintrinsicallycapable of forming tubular structures in giant unilamellar vesicles , it is unknown if the formation of such structures alone is suf \ufb01 cient for endocytosis . Moreover , the GII . 4 - cell interaction causes recruitment of ALIX , an essential component of CLIC - mediated endocytosis 53 and an adaptor for several ESCRT - III subunits and phospholipids capable of regulating membrane curvature and tubular structure formation 54 \u2013 56 , consistent with the notion that GII . 4 triggers signaling pathways that accrue an ensemble of cellular factors with membrane modulating functions for cell entry . This leads to many interesting questions such as how does GII . 4 engage gal - 3 and ALIX , what prompts their recruitment to the mem - brane surface to induce endocytosis and is there an association between gal - 3 and ALIX ? Interaction of gal - 3 and ALIX with the nor - mally inaccessible S domain of the GII . 4 capsid suggests that the GII . 4 capsid undergoes conformational changes , which can be attributed to Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 7 the newly recognized plasticity of VLPs under certain conditions 42 , 57 . These conformational changes in the GII . 4 capsid may be due to its binding to the initial glycan receptor through the P domain , thus , exposing the S domain to participate in virus - host interaction . This suggests that the GII . 4 P domain interaction with the glycan receptor may be instrumental in triggering gal - 3 and ALIX recruitment to the cell surface , which is further supported by the fact that we see increased gal - 3 and ALIX recruitment with increased VLP binding . However , the GII . 4S domain interaction is still required with gal - 3 and ALIX to drive receptor oligomerization and subsequent endocytosis , which is not possible with P domain alone . To understand GII . 4 - triggered gal - 3 and ALIX translocation to the membrane , we investigated both endolysosomal traf \ufb01 cking and membrane wounding mechanismsdue to the well - documented role of gal - 3 and ALIX in both processes 32 , 33 . Wounding experiments demon - strated thatGII . 4 particles directlyinducemembranewounding , which requires plasma membrane glycan expression ( Figure S5h ) and leads to lysosomal exocytosis and generation of surface ceramides ( Figs . 5e and S5f ) . These events indicate that GII . 4 exploits membrane wound - ing and subsequent repair mechanisms , similar to non - enveloped adenovirus 58 and the protozoan parasite Trypanosoma cruzi in HeLa cells 59 , for infection . Membrane wounds are repaired by reducing membrane tension , lesion removal by endocytosis , or shedding of wounded areas 28 , 29 , 60 , and compelling evidence implicate the involvement of the gal - 3 , ALIX and ESCRT proteins in membrane repair mechanisms 32 , 33 , 60 . Gal - 3 is reported to recruit ALIX in response to lysosomal damage , further promoting interaction between ALIX and the ESCRT III effector CHMP4 33 , which is known for its membrane - modulating functions . Moreover , ALIX is known to regulate epithelial cell polarity and maintain the integrity of the epithelial barrier by physically interacting with F - actin by binding the Rho family of small GTPases Cdc42 and Rac1 61 , responsible for actin polymerization and endocytosis . Cdc42 - induced cell polarization is regulated by membrane recycling pathways 62 , 63 ( involving Rab11 and Rab14 ) , which are critical for both wound healing and CLIC - mediated endocytosis . This indicates that GII . 4 - cell interactions trigger a series of signaling events and cross talk between cellular components activating membrane repair and recy - cling events that culminates in endocytosis . It is known that inhibiting regulators of the CLIC pathway deters membrane repair processes 64 , 65 , further validating our results on GII . 4 entry , where inhibiting ALIX , lysosomal exocytosis , and ASM translocation inhibited GII . 4 replica - tion . Although , the role of ASM or ceramide in the CLIC pathway is unclear , considering the fact that ASM mediates traf \ufb01 cking of palmi - toylated proteins to the plasma membrane ( in \ufb02 uencing lipid raft formation ) 66 and ceramide production leads to plasma membrane repairvia endocytosis 28 , both ASM and ASM - generated ceramide seem to be essential for GII . 4 entry and infection . Our observation that endocytosis is not observed by adding ceramide exogenously 6 , con - \ufb01 rms that endocytosis requires both ASM translocation and sub - sequent conversion of membrane sphingomyelin to ceramide . Ceramides andsphingolipidsplaymultifacetedrolesasmediators of membrane dynamics by in \ufb02 uencing cellular signaling , modulating receptor conformation , and clustering , which can critically affect endocytosis 67 , 68 . GII . 4 binds HBGAs in glycosphingolipid - containing membranes similar to gal - 3 , which is a glycan sensor and has a high Gal - 3 Rab11 Rab14 ASM Cdc42 Cholesterol Membrane repair EndocytosisAdaptor for membrane modulating proteins Maintain cell polarity and epithelial barrier by interacting with Cdc42 Membrane organizer Membrane repair EndocytosisAdaptor for ALIX Membrane recycling EndocytosisCdc42 regulator Membrane recycling EndocytosisCdc42 regulator Arf6 - mediated regulation of lipid domains Membrane recycling Endocytosis Cdc42 regulator Arf6 - mediated regulation of lipid domains Regulates membrane fluidity EndocytosisRegulates receptor trafficking Regulates sphingomyelin homeostasis on the plasma membrane Lysosomal trafficking Membrane repair Endocytosis Lysosome ASM C e ll i n j u r y triggers SM Cer Membrane repair machinery Membrane reorganization and receptor clustering CLICs V - ATPase Endosomal acidification Virus / genome release Cdc42Cholesterol 4 1 3 5 6 7 8 9 2 Rab11 / Rab14 HBGA ASM HBGA HuNoVbinds to these complex glycans expressed by secretor positive individuals Regulators of CLIC pathway ESCRT Membrane repair machinery Cofactors ? Fig . 6 | GII . 4 uses a complex entry mechanism involving cellular wound repair mechanisms and CLIC - mediated endocytosis . ( 1 ) Binding of GII . 4 with its glycan receptor ( HBGAs and possibly with a still unidenti \ufb01 ed co - receptor ) on the cell surface ( 2 ) inducesplasmamembrane wounding ( 3 ) triggeringsignaling responses that direct multiple membrane repair cellular components to the injury site . ( 4 ) ASM translocation to the plasma membrane surface results in conversion of sphingomyelin ( SM ) to ceramide . ( 5 ) Ceramide formation along with other mem - brane repair processes involving gal - 3 ( glycan damage sensor ) , ALIX ( Ca 2 + sensor ) and membrane recycling processes regulated by Rab11 and Rab14 result in mem - brane reorganization and receptor clustering leading to ( 6 ) tubular carrier forma - tion due to multiple interactions of virus with host factors causing membrane bending and ( 7 ) endocytosis regulated by Cdc42 and cholesterol . ( 8 ) GII . 4 entry into the cell results in V - ATPase regulated endosomal acidi \ufb01 cation causing ( 9 ) conformational changes in the virus capsid and release of the viral genome from the endosomal compartment . Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 8 af \ufb01 nity for HBGAs 69 , 70 . Our data in isogenic HIEs lacking FUT2 - dependent HBGAs on the cell surface show that GII . 4 binding to suchHBGAs isnecessarytoinducemembrane wounding . We posit this triggers ceramide - mediated signaling and changes in membrane / receptor dynamics leading to gal - 3 recruitment to the interaction site and promoting cross - talk between GII . 4 and effectors of membrane repair and recycling . We postulate that recruited gal - 3 acts both as crosslinking membrane organizers and as a switch in immobilizing the ESCRT machinery followed by subsequent endocytosis and scission of CLIC structures . Although gal - 3 is a multifunctional protein strictly regulated by its ability to recognize exposed glycans , the exact mechanism of how it \ufb01 ts into the cell signaling process and recruits ESCRT proteins remains unclear and requires further investigation . In this manuscript , we have shown that GII . 4 HuNoV exploits unexpected entry mechanisms to enter the cell by causing membrane wounding , triggering membrane repair mechanisms and inducing endocytosis and endosomal acidi \ufb01 cation for viral infection ( Fig . 6 ) . Our data clearly highlight the strain - speci \ufb01 c similarities and differences between non - pandemic , BA - dependent HuNoV strains and the pandemic , BA - independent GII . 4 HuNoV . Based on our results , we posit that the dominance of GII . 4 over other HuNoV strains may be attributed to its superior ability to exploit constitutive endocytic pathways reserved for recycling abundant surface proteins and membrane repair , in addition to its ability to overcome the host innate response 5 . Other highly pathogenic microbes and toxins ( SARS - CoV - 2 , Shiga toxin and CTxB ) share many common features of entry into cells with GII . 4 HuNoV entry mechanisms described herein 24 , 48 , 50 . Fur - ther identi \ufb01 cation of a speci \ufb01 c receptor coupled with a detailed understanding of the role of each of the cellular factors identi \ufb01 ed here for infection will be helpful in deciphering the speci \ufb01 c cel - lular tropism of GII . 4 HuNoVs . This raises interesting questions regarding whether utilization of a particular endocytic pathway by these highly infectious pathogens is associated with their pathogenesis and whether features of the CLIC route of entry and associated membrane repair mechanisms represent new ther - apeutic targets . Methods Virus and VLPs The virus used in these studies was TCH12 - 580 , a GII / Hu / US / 2012 / GII . 4 Sydney [ P31 ] / TCH12 \u2013 580strain . Thestoolsamplewaspreparedas10 % stool \ufb01 ltrates in phosphate - buffered saline ( PBS ) with viral titer determinedbyreal - timeRT - PCR 2 . VLPswereproducedinabaculovirus expression system using open reading frame 2 ( ORF2 ) + ORF3 + untranslated region ( UTR ) sequences representing HuNoV strains ( Table 1 ) . Pharmacological inhibitors , additives , recombinant proteins , and antibodies A list of the various inhibitors , additives and recombinant proteins used in this study is provided in Table 2 . Preparation of J2 HIE monolayers Human jejunal HIE was established from tissue from a patient under - going bariatric surgery . The study protocol was approved by the Baylor College of Medicine Institutional Review Board . Preparation of HIE cultures used in the current study were described previously 2 , 3 . Brie \ufb02 y , a permissive , jejunal secretor positive ( J2 ) HIE line was used for almost all the experiments described in this work . An isogenic , genetically modi \ufb01 ed J2Fut2 knockout HIE that lacks the FUT2 enzyme , as previously described 4 was used in select membrane wounding studies . To prepare monolayers , 3D cultures of HIE were dissociated by trypsinization followed by gentle pipetting and passing the cells through a 40 \u03bc m cell strainer . Cells were pelleted and resuspended in a proliferation medium [ CMGF ( + ) containing the ROCK inhibitor Y - 27632 ( 10 \u03bc M , Sigma ) ] , and seeded in a 96 - well plate ( 100 , 000 cells per well ) . After 24 h of cell growth , differentiation medium was added to the cells and the cells were allowed to differ - entiate for 5 days with intermittent medium change . Table 1 | Norovirus VLP speci \ufb01 cations Genotype Variant GenBank Accession No . GII . 4 Sydney ( Syd ) JX459908 GII . 4 Grimsby ( GRV ) AJ004864 GII . 4 Lanzou ( HoV ) EU310927 GII . 4 Farmington Hills ( FH ) AY502023 GII . 4 New Orleans ( NO ) GU445325 GII . 4 Sydney Alix mutant NA GII . 3 TCH04 - 577 AB365435 GI . 1 Norwalk M87661https : / / www . ncbi . nlm . nih . gov / nuccore / 106043086 / GII . 17 Katrina ABD95934 Table 2 | List of inhibitors , additives and recombinant proteins Reagent Source Identi \ufb01 er Inhibitors Dynasore SCBT sc - 202592 Mitmab Tocris 4224 M \u03b2 CD Sigma C4555 Filipin Sigma SAE0087 Nystatin Sigma N6803 U18666A SCBT sc - 203306 Amitriptyline Sigma A8404 GW4869 SCBT sc - 218578 Cytochalasin D SCBT sc - 201442 Genistein SCBT sc - 3515 Nocodazole Sigma 487929 Bre \ufb01 ldin A SCBT sc - 200861 Golgicide A SCBT sc - 215103 EIPA SCBT sc - 202458 ML141 SCBT sc - 362768 NSC 23766 SCBT sc - 204823 CT04 Cytoskeleton Inc . CT04 CAS 879127 - 07 - 8 SCBT sc - 203934 Calphostin C Sigma C6303 Blebbistatin SCBT sc - 203532B Wiskostatin SCBT sc - 204399 Ba \ufb01 lomycin A1 SCBT sc - 201550A YM201636 SCBT sc - 204193 LY 294002 Sigma 440202 Vacuolin - 1 Sigma 673000 TD139 Cayman Chemical 28400 Additives / and recombinant proteins GCDCA Sigma G0759 Cholesterol Sigma C4951 His - tagged recombinant ALIX Fitzgerald 80R - 1259 His - tagged recombinant TSG101 Fitzgerald 80R - 1296 Recombinant gal - 3 Biolegend 599706 Cholera Toxin B subunit Sigma C9903 Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 9 HuNoV infection of J2 HIE monolayers HuNoV infection was performed with the J2 HIEs without the addition of bile / bile acids 2 , 3 . Brie \ufb02 y , J2 HIEs were incubated with 9\u00d710 5 GEs of GII . 4 stool \ufb01 ltrate in CMGF ( - ) for 1 h at 37 o C . The monolayers were washedtwicewithCMGF ( - ) toremoveunattachedviruses . Oneplateof infected J2 HIEs was frozen at this time point ( as binding reference for 1 h ) and the other plate of infected J2 HIEs were incubated for 24 h in the differentiation medium followed by Reverse Transcriptase Quan - titative Polymerase Chain Reaction ( RT - qPCR ) to quantitate HuNoV replication . Toxicity assessment in HIEs Pharmacological inhibitors were tested for toxicity in HIEs before testing their effect on HuNoV replication . The HIEs were incubated with the inhibitors for 24 h at 37 o C and the supernatant was subjected to LDH - Glo \u2122 cytotoxicity assay ( Promega ) according to the manu - facturer \u2019 s instructions . HIE lysate was used as positive control and untreated supernatant was used as negative control and Table S1 reports toxicity results for all inhibitors . Infection assays in the presence of VLPs or inhibitors The effect of VLPs or pharmacological inhibitors on GII . 4 entry and infection were studied by pretreating the J2 HIE monolayers with them 1 h prior to infection . Infection was carried out in the presence of inhibitor for 1 h at 37 o C . After washing and unbound virus removal , fresh media containing VLPs or inhibitors was added to the infected monolayers and incubated for 24 h until the cells were harvested for RNA isolation . RNA extraction and RT - PCR Total RNA was extracted from each well using the KingFisher Flex Puri \ufb01 cation System and MagMAX \u2122 Pathogen RNA / DNA Kit . For RT - qPCR , the primer pair COG2R / QNIF2d and probe QNIFS were used for GII . 4 with qScript XLT One - Step RT - qPCR ToughMix reagent with ROX reference dye . Reactions were performed on an Applied Biosystems StepOnePlus thermocycler using the following conditions : 50\u00b0C ( 15 min ) , 95\u00b0C ( 5min ) , followed by 40 cycles of 95\u00b0C ( 15s ) and 60\u00b0C ( 35 s ) . A standard curve based on a recombinant HuNoV HoV RNA transcript was used to quantitate viral GEs in RNA samples 2 . Western blot analysis Samples were boiled with 4X sample buffer supplemented with \u03b2 - mercaptoethanol at 95 \u00b0C for 10 min and subjected to SDS - PAGE for resolving the proteins . The proteins were transferred to nitrocellulose membrane and Western blot analysis was carried out using speci \ufb01 c antibodies against target proteins . Villin and GAPDH were used as cell control which were detected using 1 : 1000 dilution of mouse anti - villin ( # sc - 373997 , Santa Cruz Biotechnology ) and rabbit anti - GAPDH ( Cat # 10494 - 1 - AP , Proteintech ) antibodies . Dot - blot analysis Dot - blotanalysiswascarriedoutbyadding10 \u00b5 lofpuri \ufb01 edproteinson nitrocellulose membrane as dots . After air drying , the membrane was blocked overnight at 4 o C . Blocked membranes were probed with tar - get proteins and their corresponding antibodies to detect binding . Immuno \ufb02 uorescence staining and imaging of J2 HIE monolayers J2 HIE monolayers were either grown in 10 - well glass - bottom culture slides ( # 543979 , Greiner Bio - One ) or 8 - well 15 \u00b5 - slides ( # 80826 , Ibidi ) for imaging . J2 HIEs were differentiated for 5 days and infected with 1\u00d710 6 GEs GII . 4 . For studies using VLPs , the cells were treated with 1\u00d710 12 particles of VLPs for either 10 min or 1h . The cells were \ufb01 xed with 4 % paraformaldehyde ( PFA ) for 20min at room temperature , blockingwith5 % BSAin0 . 1 % TritonX - 100 ( forpermeabilization ) inPBS for 30 min at room temperature . The cellswere incubated overnight at 4 \u00b0Cwithprimaryantibodies . Allthesubsequentstepswereperformed in PBS + 0 . 1 % Triton X - 100 . HuNoV capsid protein ( VP1 ) was detected using guinea pig anti - HuNoV polyclonal Ab ( pAb ) , 1 : 1000 dilution . Gal - 3 , ALIX , TSG101 , LAMP - 1 , Rab11 , Rab14 , Flotilin - 1 , CD44 and glycosylphosphatidylinositol - anchored protein ( GPI - AP ) were detec - ted using 1 : 200 dilution of rat anti - gal - 3 ( # 125402 , Biolegend ) , rabbit anti - ALIX ( # 12422 - 1 - AP , Proteintech ) , anti - TSG101 ( # 14497 - 1 - AP , Pro - teintech ) , rabbit anti - LAMP - 1 ( # 9091 , Cell Signaling Technologies ) , rabbit Rab11 ( # 5589 , Cell Signaling Technologies ) and rabbit Rab14 ( # A12752 , ABclonal ) , mouse anti - Flotilin - 1 ( # 610821 , BD Biosciences ) , rabbit anti - CD44 ( # 15675 - 1 - AP , Proteintech ) , and rabbit anti - GPI - AP ( # 10104 - 1 - AP , Proteintech ) antibodies , respectively . Ceramide detec - tion was carried out as described previously 6 using a speci \ufb01 c rabbit anti - ceramide pAb 28 , 71 , 72 . After washing ( 3 times , 10min each ) , the cells were incubated with 1 : 500 dilution of goat anti - rat 549 ( # 612 - 142 - 120 , Rockland ) , donkey anti - mouse 549 ( # 610 - 742 - 124 , Rockland ) , donkey anti - rabbit 649 ( # 611 - 743 - 127 , Rockland ) , and anti - guinea pig 488 sec - ondary antibodies ( # 606 - 141 - 129 , Rockland ) , to visualize the viral and cellular proteins . Actin staining wasdone using 1 : 500 dilution of Alexa - 647 Phalloidin ( # A22287 , ThermoFisher Scienti \ufb01 c ) . The cells were washed three times , and nuclei were stained with 4 , 6 - diamidino - 2 - phenylindole ( DAPI ) ( 300 nM ) for 5minatroom temperature followed by subsequent Z - stack images captured using a Nikon A1 confocal microscope . TheeffectofinhibitorsonJ2HIEswastestedbyincubatingthecells withpharmacologicalinhibitorsfor24hand \ufb01 xingthecellswith4 % PFA for 20min ( for actin ) or methanol for 5min ( for tubulin ) at room temperature . Actin was stained using Alexa - 647 Phalloidin and tubulin was stained using mouse anti - tubulin antibody ( # T8203 , Sigma ) fol - lowed by using goat anti - mouse 594 antibody ( # 610 - 742 - 124 , Rockland ) as a secondary antibody . The images were captured either by Nikon A1 confocal microscope using NIS - Elements Viewer 4 . 20 , GE Healthcare DeltaVision Deconvolution Microscope using softWoRx - software Acquire Version : 7 . 2 . 1 or by using a ZEISS Laser Scanning Micro - scope LSM 980 confocal microscope using ZEISS ZEN 3 . 5 ( blue edition ) software . The images were further processed using ImageJ2 / FIJI . Speci \ufb01 city testing of commercial antibodies against GII . 4 VLP Antibodies ( gal - 3 , ALIX , LAMP - 1 , Rab11 , and Rab14 ) were incubated with 5 \u00b5 g of GII . 4 VLPs for 1h at room temperature . An antibody pull - down was performed by adding 25 \u00b5 l of PBS - washed protein A / G magnetic beads ( # 88802 , ThermoFisher Scienti \ufb01 c ) to the mixture and incubating the mixture for 30 min at room temperature . The beads were collected by placing tubes on a magnetic stand and the unbound proteins present in the supernatant were collected for analysis . The pelleted beads were washed with PBS ( 5 times ) and the bound proteins were analyzed after adding 100 \u00b5 l SDS - PAGE buffer and heating the beadsat95 o Cfor5min ( pull - downfraction ) . Thesupernatantandpull - down fractions were assessed for the presence of VLPs by SDS - PAGE , followed by Western blot analysis for the capsid VP1 protein ( Fig . S6a ) . Blocking assay to block GII . 4 - protein interaction using antibodies To test the blocking activity of anti - gal - 3 and anti - ALIX antibodies in blocking GII . 4 Syd VLP interactions with gal - 3 and ALIX , antibody - blocking assays were carried out using anti - gal - 3 and anti - ALIX anti - bodies . GII . 4 Syd VLPs ( 2 \u00b5 g ) were incubated with recombinant His - tagged gal - 3 and ALIX with or without anti - gal - 3 ( left ) and anti - ALIX ( right ) antibodies ( 2 and 10 \u00b5 g ) . A pull - down assay was carried out using Ni - NTA beads and GII . 4 VP1 was detected using guinea pig ( Gp ) Syd - pAb ( Fig . S6b ) . Epi \ufb02 uorescence microscopy for measurement of endocytosis Differentiated J2 HIE monolayers ( incubated with virus / VLPs for 10min ; ba \ufb01 lomycin for 1 h ) were incubated with 50nM LysoTracker Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 10 ( ThermoFisher Scienti \ufb01 c ) for 10min . The cells were washed twice with CMGF ( - ) and LysoTracker staining of acidic compartments in treated and untreated J2 HIEs ( mock ) was observed by epi \ufb02 uorescence microscopy using Olympus cellSens Standard Version 2 . 3 software . Endocytosis measurements were carried out using FM1 - 43FX ( Ther - moFisher Scienti \ufb01 c ) . HIE monolayers were treated media containing 10 \u03bc g / mL of FM1 - 43FX with or without VLPs for 10min at 37\u00b0C . Endocytosis was stopped by washing with prechilled PBS and the cells were \ufb01 xed in 4 % PFA for 20 min and nuclei were stained with 300nM DAPI for 5min at room temperature . Quantitation of the \ufb02 uorescence wasdoneusingJ - image . Brie \ufb02 y , everyexperimentwasrepeatedat least three times with 3 \u2013 4 images analyzed per condition in each experi - ment . Identical elliptical regions - of - interests ( ROIs ) were drawn per \ufb01 eld and mean \ufb02 uorescence intensity from these ROIs was measured . Dil - LDL assay Differentiated J2 HIE monolayers were treated with dynamin inhibitors ( 40 \u00b5 M of dynasore and 100 \u00b5 M mitmab ) or dimethyl sulfoxide ( DMSO ) for 1h at 37 o C . Dil - LDL was added to the cells ( 5 \u00b5 l / well ) in the presence of inhibitor and the cells were further incubated for 3 h at 37 o C . The cells were washed three times , \ufb01 xed with 4 % PFA for 20 min . Dil - LDL uptake was measured using epi \ufb02 uorescence microscopy . Time lapse microscopy Timelapsemicroscopywascarriedoutbyadding \ufb02 uorescentlylabeled VLPs with FM1 - 43FX to J2 HIE monolayers and subsequent Z - stack imageswerecaptured ( every 2 . 5 minforaperiod of1 hat 37 o C ) usinga GE Healthcare GE Healthcare DeltaVision Deconvolution Microscope . Image acquisition was done using softWoRx - Acquire Version : 7 . 2 . 1 software . The image stack was then converted into a movie using ImageJ AVI pluggin . Thin section electron microscopy J2 HIE monolayers were incubated with VLPs for 1h at 37 o C . The cells were washed twice with PBS and VLP - treated and untreated control cells were trypsinized and \ufb01 xed in 2 % paraformaldehyde \u2013 3 % phosphate - buffered glutaraldehyde . Thin sections were prepared and electron microscopy was carried out using a FEI Tecnai Spirit trans - mission electron microscope equipped with a 4K Eagle digital camera 73 . Af \ufb01 nity measurements using BLI BLI studies were carried out using an Octet RED96 instrument ( For - teBio ) . Biotinylation of ALIX , TSG101 and gal - 3 was carried out using EZ - Link NHC - LC - LC - biotin following the manufacturer \u2019 s instructions . Thebiotinylatedproteins , bTSG101 ( 0 . 5 \u03bc g / ml ) , bALIX ( 1 \u03bc g / ml ) , bgal - 3 ( 1 \u03bc g / ml ) and were loaded onto streptavidin biosensors ( 18 - 5019 , ForteBio ) in the running buffer ( 20mM HEPES , 150mM NaCl , 0 . 005 % surfactant Tween 20 , and 2 mg / ml BSA , pH 7 . 8 ) for 600 s . Af \ufb01 nity measurements were carried out by passing two - fold serial dilutions of recombinant GII . 4S ( 280 - 70 \u03bc M ) and P domains ( 144 - 36 \u03bc M ) over the captured bALIX , bTSG and bgal - 3 and allowing both association and dissociationfor900susingForteBiodataacquisition software Version 7 . 1 . 0 . 100 . The binding data were \ufb01 tted using the ForteBio data analysis software Version 7 . 1 . 0 . 38 ( 2 : 1 model ) by subtracting buffer blanks to calculate thebinding af \ufb01 nity ofGII . 4 SandP domainswith biotinylated cellular proteins ( ALIX , TSG , and gal - 3 ) . Elisa Direct binding of ALIX with GII . 4 domains ( S and P ) and VLP was determined by ELISA . ALIX ( 5 \u03bc g / ml ) - coated and blocked plates were incubated with equimolar concentrations of GII . 4 VLP , S and P domain for 2 h at room temperature . The plate was washed three times with PBST ( PBS with 0 . 05 % Tween 20 ) , and PBS followed by probing the interaction with guinea pig anti - HuNoV polyclonal Ab , 1 : 5000 dilution in 1 % blocking buffer . Horseradish peroxidase - labeled goatanti - guinea pig secondary antibody ( 1 : 5000 ) was used to detect the binding using tetramethylbenzidine ( TMB ) substrate . Color development was quenched using phosphoric acid and absorbance was measured at 450nm . Generation of GII . 4 \u0394 S - domain TheGII . 4 \u0394 S - domainconstructcontainingthe \u201c AATAL \u201d mutationatthe putative ALIX binding site was generated using the NEBaseChanger software and Q5 site - Directed Mutagenesis Kit . The sequence of the primers used was TGCGCTGCGTGCGAACAATGCT and GTCGCCGC CATTGCGATCAGTTTGATGGTC ( Integrated DNA Technologies ) . The mutant construct was thentransformed andculturedin Novabluecells made competent using a Mix & Go transformation kit ( Zymo Research ) . Cells were then cultured in 50ml Luria Broth media , con - taining 50 \u00b5 g / ml ampicillin , and puri \ufb01 ed using Qiagen Miniprep kit ( Qiagen ) . Puri \ufb01 ed DNA was then submitted to Genewiz for Sanger sequencing to con \ufb01 rm the successful incorporation of the AATAL mutation . His - tagged shell mutant was then expressed , puri \ufb01 ed and assessed using EM and SDS - PAGE . Generation of GII . 4 Syd \u0394 VLP To obtain mutant VLPs , the ORF1 + ORF2 + 3 \u2019 UTR nucleotide sequence of norovirus GII . 4 Sydney ( JX459908 ) was submitted to a commercial laboratory ( Epoch Life Science ) for modi \ufb01 cation . Nucleotides 5628 \u2013 5637 of the cDNA version of the genome were changed from TTGTATACAC to GCAGCTACAG . The coded amino acids 182 \u2013 185 of VP1 were thus mutated from \u201c LYTPL \u201d to \u201c AATAL \u201d . The laboratory synthesized the gene and after veri \ufb01 cation of the sequence , the insert was cloned into the bacmid pFastbac1 vector . The Protein and Monoclonal Antibody Production Core at Baylor College of Medicine recombinantly expressed VLPs by infecting High Five \u2122 ( # B85502 , ThermoFisher Scienti \ufb01 c ) insect cells with the bacmid construct . The VLPs were puri \ufb01 ed by passing through a 30 % sucrose cushion followed by a cesium chloride gradient and dialysis in sodium chloride / sodium phosphate buffer as previously described 74 . shRNA - mediated knockdown of the PDCD6IP gene A microRNA - adapted shRNA technology based on miR - 30 was used to knockdown gene expression in the J2 HIE line . Commercially available pGIPZ lentiviral constructs , comprising of shRNAs complementary to the sequence of the targeted PDCD6IP gene ( # RHS4430 - 200156283 , # RHS4430 - 200158656 , # RHS4430 - 200283070 ; Dharmacon ) , were providedasbacterialglycerolstocks . shRNAconstructswereextracted using Qiagen prep kit ( Qiagen ) . Lentiviral particles with the shRNAs were produced , using the third - generation lentivirus technology , by co - transfecting HEK293FT cells ( # R70007 , ThermoFisher Scienti \ufb01 c ) with a combina - tion of each GIPZ - shRNA lentiviral plasmid and three packaging plas - mids pMDLg / pRRE [ plasmid # 12251 ; Addgene ] , envelope plasmid pMD2 . G [ plasmid # 12259 ; Addgene ] , and pRSV - Rev [ plasmid # 12253 ; Addgene ] ) at a ratio of 3 . 5 : 2 : 1 : 1 , respectively , using polyethyleneimine HCl Max molecular weight ( MW ) 40 , 000 ( Polysciences ) . The culture supernatant was harvested 72 h , passed through a 0 . 45 - \u03bc m \ufb01 lter , concentrated by using LentiX - concentrator ( TaKaRa - Clontech ) according to the manufacturer \u2019 s protocol , and suspended in a high Wnt - 3A proliferation medium supplemented with 10 \u03bc M CHIR99021 and 10 \u03bc M Rho - associated protein kinase ( ROCK ) inhibitor Y - 27632 , and stored at \u2212 80 \u00b0C in 50 \u03bc l aliquots until further use . A cell suspension was prepared from three - dimensional ( 3D ) undifferentiated J2 HIEs cultivated as previously described . After trypsinization and pelleting of the cells at 300 \u00d7 g , the resulting cell pellet was suspended at a concentration of 3\u00d7 10 5 cells per ml in high Wnt - 3A proliferation medium supplemented with 10 \u03bc M CHIR99021 and 10 \u03bc M Y - 27632 . 8 \u03bc g / ml Polybrene and 50 \u03bc l of concentrated Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 11 shRNA lentivirus were added to 1\u00d7 10 5 cells , the mixture was plated in one well of a 48 - well plate and centrifuged for 1 h at 600\u00d7 g at room temperature . After spinoculation , the plate was incubated for 4 h at 37\u00b0C under 5 % CO 2 atmosphere to enhance transduction . The lenti - virus solution was removed by centrifugation , the cells were washed once with CMGF ( - ) medium , centrifuged again , embedded in 30 \u03bc l Matrigel plug , and incubated at 37\u00b0C and 5 % CO 2 in the presence of high Wnt - 3A medium with CHIR99021 and Y - 27632 for recovery . Five days post - transduction , the cells were treated with puromycin ( 2 \u03bc g / ml ) until mock - treated cells were completely dead . Single - cell clones were isolated by serial dilution of cells in 96 - well plates for shRNA - transduced HIEs , and deletion of the gene was con \ufb01 rmed by sequencing of genomic DNA from each single cell clone using primers ALIX - F and ALIX - R that amplify the portion of the shRNA targeted PDCD6IP gene . Wounding assays Differentiated J2 or J2FUT2KO HIEs were incubated with a mixture of VLP and PI ( 5 \u00b5 g / ml ) for 10 min at 37 o C . The cells were washed twice with CMGF ( - ) and \ufb01 xed with 4 % PFA for 20 min . After incubation , the cellswere washed with PBS and counterstained with DAPI for 5 min . PI / DAPI colocalization was measured to determine VLP - induced wounding . Statistical analysis Each experiment was performed three or more times independently with two or more HIE replicates ( represented by n ) . For infection assays , two HIE replicates for 1h time point and three independent HIE replicates for the 24h time point were used . RT - qPCR assays were carried out using two or more technical replicates for each HIE repli - cate depending on the treatment of cells . Samples with RNA levels below the limit of detection of the RT - qPCR assay were assigned a value that was one - half the limit ofdetection of the assay . All statistical analyses were performed on GraphPad Prism ( Version 8 . 4 . 3 ) ( Graph - Pad Software ) using geometric mean values . Comparison between untreated and treated groups at 24 h was performed using one - way ANOVA using data from independent HIE replicates of one indepen - dent experiment , with statistical signi \ufb01 cance ( P value ) determined using Dunnett \u2019 s multiple comparisons test . For imaging experiments , statistical signi \ufb01 cance was calculated either by one - way ANOVA , Dun - nett \u2019 s multiple comparisons test ( when comparing with control ) or using two - way ANOVA with Tukey \u2019 s or \u0160 \u00edd\u00e1k \u2019 s multiple comparisons test ( when comparing within groups ) . A P value above 0 . 05 was con - sidered non - signi \ufb01 cant . Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article . Data availability The authors declare that all data supporting the \ufb01 ndings of this study are available within the paper and in the Supplementary Information \ufb01 les . 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Norwalk virus - like particle hemagglutination by binding to h histo - blood group antigens . J . Virol . 77 , 405 \u2013 415 ( 2003 ) . Acknowledgements This research was supported by National Institutes of Health Grant P01 AI57788 , P30DK56338thatsupportstheTexasMedicalCenterDigestive Diseases Center and the Texas Children \u2019 s Hospital EM core , T32 AI055413 ( to V . R . T . ) , S10 OD028480 that supported purchasing the Zeiss Laser Scanning Microscope LSM 980 with Airyscan 2 and the RobertWelchFoundationQ1279 . Theauthorswouldliketoacknowledge the Advanced Technology Core Laboratories ( Baylor College of Medi - cine ) , speci \ufb01 cally the Integrated Microscopy Corewith funding from the NIH ( DK56338 , CA125123 , ES030285 ) , and CPRIT ( RP150578 , RP170719 ) . We thank Xiaomin Yu and Hannah Johnson for technical assistance with human intestinal enteroids and imaging and Drs . Sasirekha Ramani , Joseph Hyser , and Jeanette Criglar for their helpful suggestions . Author contributions B . V . A . and M . K . E . conceived the idea and designed the research ; B . V . A . , K . E . , W . S . , U . C . K . , F . H . N . , and V . R . T . performed experiments ; B . V . A . , K . E . , W . S . , U . C . K . , V . R . T , S . E . C . , B . V . V . P . , R . L . A . and M . K . E . analyzed data ; R . L . A . helped with statistical analysis ; E . B . contributed new reagent ; B . V . A . andM . K . E . wrote thepaper . All authors contributedtomanuscript revision and editing . Competing interests M . K . E . is named as an inventor on patents related to cloning of the Norwalkvirus genomeandHuNoVcultivationandhasreceivedresearch funding from Takeda Vaccines Business Unit ( Cambridge , MA , USA ) . R . L . A . is named as an inventor on patents related to HuNoV cultivation and has received research support from Takeda Vaccines Business Unit ( Cambridge , MA , USA ) . Thefunders hadnorolein thestudy design ; data collection , analyses , or interpretation ; manuscriptwriting , or decision to publish the results so there are no competing interests . The remaining authors declare no competing interests . Additional information Supplementary information The online version contains supplementary material available at https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z . Correspondence and requests for materials should be addressed to Mary K . Estes . Peer review information Nature Communications thanks the anon - ymous reviewer ( s ) for their contribution to the peer review of this work . Reprints and permissions information is available at http : / / www . nature . com / reprints Publisher \u2019 s note Springer Nature remains neutral with regard to jur - isdictional claims in published maps and institutional af \ufb01 liations . 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To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2023 Article https : / / doi . org / 10 . 1038 / s41467 - 023 - 36398 - z Nature Communications | ( 2023 ) 14 : 1148 14", + "vasconcelosEffectExplicitInstructions2018": "Artificial Intelligence for Engineering Design , Analysis and Manufacturing cambridge . org / aie Research Article Cite this article : Vasconcelos LA , Neroni MA , Crilly N ( 2018 ) . The effect of explicit instructions in idea generation studies . Artificial Intelligence for Engineering Design , Analysis and Manufacturing 32 , 308 \u2013 320 . https : / / doi . org / 10 . 1017 / S0890060417000658 Received : 28 February 2017 Revised : 28 October 2017 Accepted : 29 October 2017 Key words : Design fixation ; experimental instructions ; feature repetition ; inspiration ; stimuli introduction Author for correspondence : Luis A . Vasconcelos , E - mail : lalv4401 @ gmail . com \u00a9 Cambridge University Press 2018 The effect of explicit instructions in idea generation studies Luis A . Vasconcelos , Maria A . Neroni and Nathan Crilly Engineering Design Centre , Department of Engineering , University of Cambridge , Trumpington Street , Cambridge CB2 1PZ , UK Abstract In inspiration and fixation experiments , example designs are often provided along with the instructions for how participants should treat them . However , research has not reached a con - sensus about the influence of such instructions , leading to difficulties in understanding how the examples and the instructions each affect idea generation . We conducted an experiment in which 303 participants designed for the same design problem , while given different examples and instructions , which ranged from strongly encouraging copying the examples to strongly discouraging copying . Exposure to the examples affected the number and type of ideas gen - erated , whereas exposure to the instructions did not . However , instructions did affect how par - ticipants incorporated features of the examples in their ideas . Encouraged groups incorporated many features of the examples , while also incorporating structural features more than concep - tual ones . Surprisingly , the incorporation of features in discouraged groups was not different from that of groups given no instructions or even no stimulus . This indicates that concrete features may be easier to recognize and reproduce than abstract ones , and that encouraging instructions are more effective than discouraging ones , despite how strict or lenient those instructions are . The manipulation of different features also allowed us to observe how similar approaches to solving a design problem can compete for attention and how the calculation of feature repetition can be misleading depending on how common or obvious the features might be . These findings have implications for the interpretation of results from fixation stud - ies , and for the development of design tools that present stimuli to assist idea generation . Introduction Inspiration is vital to creative design and innovation ; yet , we know very little about it . Understanding more about inspiration and other cognitive aspects of design allows us to improve design processes and outcomes . It also allows us to develop intelligent tools to assist innovative design and computational models of creativity . The potential benefits of under - standing inspiration have driven the design research community to conduct many studies that seek to find out , for instance , what materials inspire designers ( Gon\u00e7alves et al . , 2014 ) , how designers achieve inspiration ( Zhao , 2013 ) , how inspiration can improve designers \u2019 per - formance ( Eckert , 1997 ) , and how to better develop computational tools to inspire designers ( T\u00f6re Yargin & Crilly , 2015 ) . Many of these studies have observed the use of external stimuli during idea generation and have reported that while stimuli can assist idea generation , they can also hinder it . One of these hindrances to creativity is described in the design literature as \u201c design fixation \u201d ( Jansson & Smith , 1991 ) , when prior knowledge of a solution causes designers to inadvertently limit their search for other solutions . Design fixation was originally studied in situations where designers were trying to address a problem and were provided with stimuli which represented possible solutions to the problem that was being addressed ( Jansson & Smith , 1991 ) . Fixation was measured by counting the solutions that were developed in response to the problem and counting the repetition of fea - tures from the stimuli . This general approach to measuring fixation has subsequently been employed in several other related studies ( e . g . , Purcell & Gero , 1996 ; Dahl & Moreau , 2002 ; Linsey et al . , 2010 ; Cardoso & Badke - Schaub , 2011 ; Youmans , 2011 a , b ; Vasconcelos et al . , 2017 ) . Although reproducing features of good designs might be beneficial or efficient , even flawed stimuli ( i . e . , stimuli with negative features ) are reproduced , when it might instead be expected that those stimuli would be identified and avoided ( Jansson & Smith , 1991 ; Linsey et al . , 2010 ; Youmans , 2011 b ) . In general , fixation research suggests that blindly copying features from stimuli is harmful to idea generation , and therefore to the design process itself . As such , some studies have incor - porated constraining textual instructions into the stimuli given to participants , to prevent them from copying their features . However , the efficacy of these instructions has varied across stud - ies : they were effective in some cases ( Chrysikou & Weisberg , 2005 ; Yilmaz et al . , 2010 ) and ineffective in others ( Jansson & Smith , 1991 ; Perttula & Sipil\u00e4 , 2007 ) . Thus , it is still uncertain https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press how textual instructions can influence the copying behavior , and this uncertainty might be attributed to different factors . For instance , individuals may tend to overlook the instructions that accompany the stimuli ( LeFevre & Dixon , 1986 ) , they may inter - pret the instructions in different ways if instructions are not explicit , or the instructions may lead them to question why they are being exposed to the stimuli and change their idea generation process accordingly . Whatever the reason might be , it is impor - tant to understand the relevance of the instructions provided to designers as part of the inspiration material . Methodologically , this would help in determining how fixation studies should be conducted and the results interpreted . More practically , it would also help in understanding how stimuli should be presented when designers are stimulated with examples , such as in those software tools that support idea generation by providing external stimuli [ for examples of such tools , see ( Chakrabarti et al . , 2005 ; Linsey & Wood , 2007 ; Vattam et al . , 2011 ) ] . To better understand the influence of instructions on idea gen - eration , we conducted an experiment in which participants responded to the same design problem , while being exposed to different example solutions , and following different instructions with respect to those examples . Inspiration , fixation , and the introduction of external stimuli Design researchers have studied many different aspects of the inspiration process , including characteristics of external stimuli or inspiration sources ( e . g . , the modality of representation used for the stimuli ) ( Linsey et al . , 2008 ; Sarkar & Chakrabarti , 2008 ; Atilola & Linsey , 2015 ; Viswanathan et al . , 2016 ) and aspects of the design process ( e . g . , time available for ideation ) ( Youmans , 2011 a ; Tsenn et al . , 2014 ; Siangliulue et al . , 2015 ) . One of the stimuli characteristics that has been studied is whether the quality of the examples ( i . e . , good or bad ) would influence feature repeti - tion , or whether those examples would be copied indiscrimi - nately . A series of studies have found that participants still repeat features from previous examples even when they are flawed ( Jansson & Smith , 1991 ; Chrysikou & Weisberg , 2005 ; Perttula & Liikkanen , 2006 ; Fu et al . , 2010 ; Linsey et al . , 2010 ; Youmans , 2011 b ; Viswanathan et al . , 2014 ) . It should be noted that in Jansson and Smith \u2019 s ( 1991 ) original paper , feature repetition was a way in which the researchers operationalized the degree to which the participants \u2019 exploration of the solution space was limited . Furthermore , the repetition of negative features ( or fea - tures that contradicted the problem brief ) was taken to be an indi - cation that this limited search was an unconscious behavior ( Youmans & Arciszewski , 2014 ) . To counteract the indiscriminate repetition of features , some studies have tried warning participants about the flaws in the examples ( Jansson & Smith , 1991 ; Chrysikou & Weisberg , 2005 ; Viswanathan et al . , 2014 ) , while others have tried instructing participants not to copy the examples ( Smith et al . , 1993 ; Perttula & Sipil\u00e4 , 2007 ; Yilmaz et al . , 2010 ) . Considering these two approaches , Chrysikou and Weisberg ( 2005 ) found that warning participants of flaws in the examples was not enough ; they had to be told to avoid repeating those flaws . Yilmaz et al . ( 2010 ) also told participants not to reproduce the examples and this led to a reduction in feature repetition . Conversely , Jansson and Smith ( 1991 ) and Perttula and Sipil\u00e4 ( 2007 ) found repetition even when participants were instructed to avoid using features of the examples provided . In summary , the published literature reports conflicting results about the influence of instructions when providing external stimuli to designers . While many variables have been manipulated in fixation experiments , and some studies have already tested the effective - ness of using textual instructions to some extent , the way the stim - uli are introduced in such experiments has not yet been studied systematically . Such stimuli introductions can typically be divided into two components : a descriptive statement on what the stimu - lus is ( e . g . , \u201c here is an example solution \u201d ) and a prescriptive instruction for how the stimulus should be used ( e . g . , \u201c don \u2019 t copy its features \u201d ) . Currently , the stimuli introductions ( i . e . , descriptions and instructions ) given to participants vary from study to study , with some reporting that the example is there to help , to illustrate previous solutions , or to show how ideas should be represented , while other studies only mention that the stimulus was provided to participants without describing how ( Vasconcelos & Crilly , 2016 ) . See Table 1 for a more detailed description of stimuli introductions as described in related studies . Variation in the way the stimuli are introduced might be attributed to a lack of agreement across studies about which \u201c real - world \u201d situation is being simulated ( e . g . , contexts in which exam - ples are accidentally seen , intentionally searched for , or already known ) . Regardless of the reason , the variation in the way copying is encouraged or discouraged makes it difficult to compare results across studies and to know the extent to which the design work is being affected by instructions . Although previous fixation research makes it hard to formulate a theory for the influence of instructions , there is one study that looked more carefully at how different sets of instructions affected feature repetition during idea generation ( Smith et al . , 1993 ) . In the experiment , participants were asked to either conform to or diverge from the example solutions provided in a creative task . Some participants ( conform group ) were told that the examples were great ideas previously created for that task and that partici - pants should create ideas like those while not copying them exactly . Other participants ( diverge group ) were told that the examples restricted people \u2019 s creativity and that participants should create ideas that were different from the examples . Finally , other participants ( standard group ) were given the same examples but without any specific instructions , and a baseline group ( BG ) was given no examples at all . When compared with the participants in the BG , the researchers found that all other groups generated more ideas containing features of the examples . Additionally , feature repetition in the diverge group did not decrease when compared with the standard group , while feature repetition in the conform group increased when compared with the standard group . Based on their results , the researchers proposed that the partic - ipants did not conform to examples because they had assumed that they should ; they conformed because they could not forget the examples that they had seen . However , the instructions used in the experiment were not strict ( i . e . , they suggested how ideas should be created , but neither forbade nor required the use of fea - tures from the examples , thus allowing participants to interpret the instructions in different ways ) . The description of the stimuli and the instruction for their use also varied between groups , mak - ing it difficult to infer the influence of each piece of information in isolation . Additionally , the experiment reports an analysis on the repetition of features that were always concrete or structural ( e . g . , parts of a physical object ) , thus inviting further investigation to be done on different types of features , such as more abstract or conceptual features ( e . g . , properties of a physical object ) . Artificial Intelligence for Engineering Design , Analysis and Manufacturing 309 https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press We believe that a different experimental setup with additional manipulations can provide further evidence to clarify the influ - ence of the textual instructions used in experimental research , thus having implications for design research methodology and for professional design practice . To investigate this , we designed and conducted a study to explore the effects of instructions in design inspiration and fixation experiments , with instructions that are ( slightly or very ) discouraging or encouraging , or that are neutral . Research methodology We conducted a single experiment ( N = 303 ) designed to examine the role of instructions in creativity studies . However , while the data were captured in a single session ( thus minimizing experi - mental variation and allowing for efficient data capture ) , the experimental manipulation and metrics were divided into two main blocks , and the corresponding analyses were intended to be performed over two different time frames . Thus , we here treat all the data as relating to two separate studies ; the details and results of which are discussed as follows . The experimental setup of study 1 is described in full , whereas study 2 follows up on that with shorter descriptions , in which only the main differ - ences from the previous study are highlighted . Both studies drew participants from the same population ( e . g . , educational back - ground ) , and used identical procedures ( e . g . , time available ) , materials ( e . g . , design problem ) , analysis ( e . g . , fixation metrics ) , and environment ( e . g . , room conditions ) . However , the stimulus manipulated in study 2 incorporated a distinct conceptual feature , thus being significantly different from that used in study 1 . Objective and hypothesis This experiment investigates how textual instructions accompany - ing external stimuli can shape the design work of the participants . We hypothesize that instructions for the incorporation of features provided along with an example design will determine the repeti - tion of the ideas that participants generate . In particular , we expect that positive or encouraging instructions will increase fea - ture repetition , whereas negative or discouraging instructions will reduce feature repetition . Additionally , we also expect to find some level of fixation in all groups exposed to the examples when compared with a BG . From an experimental perspective , we understand design fixation to be the process of generating ideas in response to a design problem under the influence of one or more external stimuli , in which the generation of ideas is constrained due to the repetition of the stimuli into the ideas generated . This would imply decreased productivity of the partic - ipants as well as increased idea repetition . Study 1 Experimental method Participants were randomly allocated to different experimental conditions . They were verbally asked to be creative and to gener - ate , individually , as many ideas as possible to a given problem . They were also instructed , via voice and text , to sketch and describe in writing their ideas on sheets of paper . Except for a BG that designed without any stimuli , all other experimental groups received a sketch of one example solution and a descrip - tion of what the sketch represented . These stimulated groups ( SGs ) received instructions for the use of features from the exam - ple , and these instructions varied with respect to how constraining or encouraging they were ( see the \u201c Materials \u201d section for the com - plete instructions ) . Finally , the participants \u2019 ideas were assessed to evaluate the influence of the instructions on the level of fixation observed . Participants One hundred and sixty - eight engineering students in their first year at the University of Cambridge ( UK ) were assigned to six experimental groups ( n = 28 ) . Participation in the experiment was part of the students \u2019 education ( but was not for credit ) , and was aimed at collecting data that could later be used to introduce Table 1 . Different means of introducing external stimuli to participants ( each instance was extracted from a fixation - related study ; changes to the original text were made to permit better comparison across rows ) Study Stimuli introduction ( or descriptive statement about the stimuli ) ( Cheng et al . , 2014 ) The examples should be analyzed using a set of instructions ( Dahl & Moreau , 2002 ) The example is provided to help participants get started and it might be useful in solving the design problem ( Gon\u00e7alves et al . , 2012 ) The example could be considered ( or not ) when participants were generating ideas ( Liikkanen & Perttula , 2008 ) The example should be used to raise thoughts ( Linsey et al . , 2010 ) The example should be considered as a solution that might be created for the design problem ( Lujun , 2011 ) The example could be referred to during ideation ( Perttula & Sipil\u00e4 , 2007 ) The examples should be used to awaken thoughts , but should not be reproduced as such ( Purcell & Gero , 1996 ) The example design illustrates what is meant by a sketch design ( Vasconcelos et al . , 2016 ) The example shows how participants should present their ideas ( Vasconcelos et al . , 2017 ) The example is a concept that illustrates one way to solve this problem ( Yilmaz et al . , 2010 ) The examples could be used to understand the method , but they should not be repeated in participants \u2019 own designs ( Youmans , 2011 a ) The example illustrates a previous design for the same task ( Youmans , 2011 b ) The example should be rebuilt as to allow familiarization with the materials in a construction set 310 Luis A . Vasconcelos et al . https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press them to the concept of design fixation . No demographic data were collected from the participants , but as first - year undergraduate students , they were broadly similar in age and design experience , drawn from a cohort with a male \u2013 female ratio of approximately 3 : 1 . Task and problem The participants were told to solve the following problem . \u201c Bicycles are a popular mode of transportation and recreation for many people . While growing up , a person might go through a series of ever - larger bikes , sometimes having several models , one after the other . However , having several bikes can be a problem for many reasons . Your task is to generate as many ideas as possi - ble to eliminate the need to have multiple bikes as people grow up . \u201d This problem was selected because it was expected to satisfy four criteria . First , it was unlikely that the participants had designed solutions for this problem before . Second , they were likely to have experienced the situation described in the problem previously ( i . e . , while growing up , they probably had multiple bikes ) , thus helping their understanding of it . Third , the problem could be solved in different ways , with several different underlying principles being applied , thus leaving enough room for creativity . Finally , the design brief held a low level of complexity , which was also expected for the ideas generated , thus being suitable for a quick experiment fitting with the constraints of the session . Procedure ( overview ) The experiment took place in a large lecture theatre with all the participants present . During the first 5 min , the participants lis - tened to an oral explanation about the activities to follow and received all the material they needed . Participants in the five SGs received the design brief , the external stimulus and instruc - tions , and blank sheets of paper , while participants in the BG received the brief without any stimulus . Then , the participants were asked to think of ideas for 3 min without committing any designs to paper ( because different participants had different materials and content , this ensured they all had enough time to read all the materials and start developing some ideas ) . Finally , for the remaining 10 min , all participants individually generated as many ideas as possible in silence , ideally including both a sketch and a written description of each idea . Materials All participants received the same design problem written on an A4 sheet , as well as blank A4 sheets to sketch and annotate their own ideas . Except for the participants in the BG , all partic - ipants received one additional sheet with an example solution , that is , an annotated sketch of a bike ( Fig . 1 ) . The example solution was preceded with the written descrip - tion : \u201c Below is an example of how you should present your ideas ( i . e . , annotated sketches ) \u201d . This description was either immedi - ately followed by an instruction regarding the use of features from the examples ( discouraging or encouraging ) or by no instruction whatsoever . The instructions for the different experi - mental groups are listed below against a code for each experi - mental group . (cid:129) SG \u2212 2 ( strictly forbidding ) : \u201c make sure you do not use features from this example in your own work \u201d ; (cid:129) SG \u2212 1 ( discouraging ) : \u201c avoid using features from this example in your own work \u201d ; (cid:129) SG0 ( neutral ) : no instruction was given ; (cid:129) SG + 1 ( encouraging ) : \u201c consider using features from this example in your own work \u201d ; (cid:129) SG + 2 ( strictly requiring ) : \u201c make sure you use features from this example in your own work \u201d . Analysis The assessment of the participants \u2019 ideas was conducted by three research collaborators , with backgrounds in design research , com - plexity science , and mechanical engineering . Initially , the three evaluators agreed on the metrics to be included in the analysis . Such metrics should be consistent with most of the existing design fixation literature and should be appropriate for testing our hypothesis . After that , the three evaluators analyzed the design work of a random experimental group together to agree on the assessment method , ultimately reaching a consensus with respect to how to interpret and assess the ideas . Finally , each of the eva - luators , who were blind to the experimental groups that they were rating , individually judged a random subset of the remaining ideas . To reduce the chances of mistakes , if any evaluator had trouble judging an idea , this idea was then discussed collectively . We considered \u201c one idea \u201d either to be a sketch or a written description ( usually both ) that presented an understandable way to solve the problem . Participants often generated more than one idea , but in some cases , all ideas could be considered as one , particularly when the idea was a bike . For instance , if the ideas could all be incorporated onto the same bike without inter - ference , then they were considered as a single idea . Conversely , if there were two or more ideas for the same bike component ( e . g . , frame , wheel , handlebar ) , then they were considered to be distinct ideas . The metrics used in the assessment were idea \u201c fluency \u201d and idea \u201c repetition \u201d . These metrics are central to many fixation stud - ies ( e . g . , Jansson & Smith , 1991 ; Purcell & Gero , 1996 ; Dahl & Moreau , 2002 ; Linsey et al . , 2010 ; Cardoso & Badke - Schaub , 2011 ; Youmans , 2011 a , b ; Vasconcelos et al . , 2017 ) and they can provide a fairly objective measurement ( i . e . , a direct count Fig . 1 . Example solution provided to the participants along with the following description : \u201c A modular bike with parts of various sizes that can be connected and swapped to fit people with very different heights . Apart from the socketing parts and expansible / contractible wheels , the angles between tubes can also be modified in specific joints . \u201d The sketch used is a modification of the ECO 07 Compactable Urban Bicycle ( Aleman , 2009 ) . Artificial Intelligence for Engineering Design , Analysis and Manufacturing 311 https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press of ideas and features ) that is consistent with testing our feature repetition hypothesis . Therefore , other design fixation metrics found in the literature , such as diversity and conceptual distance , were not included into the analysis because they are more subjec - tive ( i . e . , increased judgment is required on the part of assessors ) and are not suited to testing our research hypothesis . Because test - ing our hypothesis implied identifying and counting ideas and features without any further judgment , we did not require dupli - cate assessment from our evaluators . Idea fluency is the total number of ideas generated , also called \u201c quantity \u201d ( Shah et al . , 2003 ) or \u201c productivity \u201d ( Nijstad et al . , 2002 ) elsewhere . Idea repetition might occur at different levels ; for instance , the repetition of idea types , conceptual features , or structural features ( although other categorizations and granularity levels might also be identified ) . With respect to the idea type , we divided the ideas into two broad categories : bike ideas and non - bike ideas ; thus , by designing a bike , the participant would be repeating the idea type . With respect to their conceptual features , we also divided the ideas into two categories : modular ideas or non - modular ideas ; thus , by generating a modular idea , the par - ticipant would be repeating the conceptual feature . Finally , we examined the incorporation of structural features in the partici - pants \u2019 ideas , in terms of both the number of ideas containing any feature of the example ( i . e . , Is repetition there ? ) and the num - ber of example features present in each idea ( i . e . , How much repe - tition is there ? ) . These features were intentionally included in the example design to permit a measure of fixation inferred because of repetition . There were five structural features : swappable com - ponents to change bike size ; frame joints ( lugs ) that act as sockets for the tubes ; wheels with bendable spokes ; an hourglass - shaped frame ; and a saddle that cannot be adjusted in height directly . Eight participants ( 4 . 8 % ) either did not generate any idea or generated ideas that could not be interpreted by the evaluators ; the results from such participants were not included in our anal - ysis . The adjusted number of participants per experimental group is used in the following section . Results and discussion To deal with non - normality in the data , we used analysis of var - iance ( ANOVA ) with significance values estimated using 1000 bootstrap resamples and planned contrasts ( a non - parametric ver - sion of the regular ANOVA test ) . To identify differences between groups , we used five planned contrasts in the analysis . The first contrast compares the BG to all SGs , and aims to identify any effect from exposure to the example design ( the fixation test ) . The remaining contrasts incorporate only the SGs , and aim to identify any effect from instructions received as follows . The sec - ond contrast compares the discouraged and neutral groups ( the discouraging instructions test ) ; the third compares the encouraged and neutral groups ( the encouraging instructions test ) ; the fourth compares the two discouraged groups ( the discouraging subtlety test ) ; and the fifth contrast compares the two encouraged groups ( the encouraging subtlety test ) . Idea fluency Instructions had no effect on the number of ideas generated . A one - way ANOVA with the total number of ideas ( per partici - pant ) as the dependent variable showed a significant difference across the groups , F ( 5 , 157 ) = 4 . 37 , p = 0 . 001 , \u03b7 2 = 0 . 122 . However , planned contrasts revealed that this difference is explained by the BG generating on average more ideas than the SGs , t ( 32 . 7 ) = 3 . 64 , p = 0 . 002 , d = 0 . 840 , since no difference was found between the SGs . Table 2 shows summary statistics for these results . These results reveal that idea fluency was not influenced by how constraining or encouraging the instructions were . However , the presence of an example design affected idea genera - tion : designing without exposure to stimuli resulted in more ideas being generated , which we interpret as a beneficial isolation from examples ( Vasconcelos et al . , 2017 ) . This effect is consistent with other studies in which seeing an example caused reduction in the idea fluency ( Linsey et al . , 2010 ) , although studies have also reported an increase in the idea fluency as a result of external stimulation ( Purcell & Gero , 1992 ) or even no effect at all ( Jansson & Smith , 1991 ) . More importantly , our results are differ - ent from those found in ( Smith et al . , 1993 ) , where the number of ideas generated by a BG did not exceed that of SGs . However , we should mention that while all groups in our study were asked to present their ideas with both sketches and textual descriptions , the BG produced many ideas that were presented only in text . As a result , baseline participants spent less time sketching every idea , which allowed them more time to produce a greater number of less - elaborated ideas , as explained by the \u201c normative represen - tation effect \u201d ( Vasconcelos et al . , 2016 ) . Repetition of the idea type Instructions had no effect on the number of bike ideas generated . A one - way ANOVA with the average number of bike ideas ( per participant ) as the dependent variable showed a significant differ - ence across the groups , F ( 5 , 157 ) = 2 . 87 , p = 0 . 017 , \u03b7 2 = 0 . 084 . However , planned contrasts revealed that this difference is explained by participants in the BG generating , on average , fewer bike ideas than the SGs , t ( 31 . 4 ) = \u2212 2 . 59 , p = 0 . 025 , d = 0 . 613 , since no difference was found between the SGs . Table 3 shows summary statistics for these results . If we adopt a \u201c bike \u201d and \u201c non - bike \u201d categorization of the ideas generated , these results reveal that the type of idea generated was not influenced by the instructions . This can be seen as a surpris - ing result : by manipulating how participants were instructed to incorporate features of the example , we also anticipated an indi - rect effect on the type of ideas that they might have generated ( because all features were incorporated into a bike idea ) , but Table 2 . Summary of ideas generated per participant across groups Generated ideas BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of ideas per participant 2 . 39 ( 1 . 31 ) 1 . 50 ( 1 . 27 ) 1 . 30 ( 0 . 61 ) 1 . 48 ( 0 . 49 ) 1 . 41 ( 0 . 56 ) 1 . 54 ( 1 . 10 ) Range of ideas generated per participant 1 \u2013 5 1 \u2013 6 1 \u2013 3 1 \u2013 4 1 \u2013 4 1 \u2013 6 Total number of ideas 67 39 35 40 38 43 Total number of participants 28 26 27 27 28 28 312 Luis A . Vasconcelos et al . https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press this was not the case . Conversely , while the total number of bike ideas was roughly the same for all groups , the proportion of bike ideas generated per participant across all groups was significantly different . In fact , only 60 % of all ideas generated by the BG were bikes , whereas the SGs had a much greater proportion of bike ideas ( 89 % on average ) . In summary , bike ideas were likely to be generated irrespective of the experimental condition , but not seeing the example allowed participants from the BG to explore different areas of the solution space , again confirming the beneficial isolation effect . Accordingly , design fixation ( inferred because of repetition of the idea type ) occurred across the stimulated conditions , a result that is broadly consistent with other studies in which seeing an example caused participants to conform to certain types of solu - tions , thus reducing the breadth of ideas ( Jansson & Smith , 1991 ; Linsey et al . , 2010 ; Cardoso & Badke - Schaub , 2011 ) . Repetition of the conceptual feature Instructions had no effect on the number of modular ideas gen - erated . A one - way ANOVA with the average number of modular ideas ( per participant ) as the dependent variable showed no sig - nificant difference across the groups , F ( 5 , 157 ) = 1 . 86 , p = 0 . 104 , \u03b7 2 = 0 . 056 . Table 4 shows summary statistics for these results . If we adopt a \u201c modular \u201d and \u201c non - modular \u201d categorization of the ideas , these results reveal that the type of idea generated was not influenced by the instructions . In fact , modular ideas were extremely rare across all groups . Still , the fixation literature tells us that the SGs would generate more modular ideas , an effect sim - ilar to the repetition of the idea type ( i . e . , bikes ) . In particular , participants who were encouraged to use features from the exam - ple in their own work ( and modularity was a visible feature on the example provided ) did not act accordingly , and participants who were discouraged from copying features generated as many mod - ular ideas as the other groups . The fact that encouraged groups did not produce more modular ideas contradicts previous research ( Smith et al . , 1993 ) , where results pointed to an increase in feature repetition in encouraged groups . Additionally , previous research has shown that abstract , conceptual features can fixate designers in a similar way to concrete , structural features ( Vasconcelos et al . , 2017 ) . However , as there was no difference in the proportion of modular ideas between baseline and SGs , we speculate that the idea of modularity ( included in the example as a conceptual feature ) was not obvious enough to induce fixa - tion effects ( inferred because of repetition of the conceptual feature ) . Repetition of structural features Instructions had a significant effect on the number of ideas with features of the example provided . A one - way ANOVA with the average number of ideas that contained structural features of the example ( per participant ) as the dependent variable showed a significant difference between the groups , F ( 5 , 157 ) = 9 . 50 , p = 0 . 000 , \u03b7 2 = 0 . 232 ; a result that was also found for the average number of repeated features per participant , F ( 5 , 157 ) = 14 . 115 , p = 0 . 000 , \u03b7 2 = 0 . 310 . For the number of ideas containing features of the example solution , planned contrasts revealed a significant difference between the two encouraged and the neutral groups , t ( 76 . 0 ) = \u2212 5 . 33 , p = 0 . 001 , d = 1 . 14 , with the encouraged groups generating more ideas with features from the example . For the average number of features included in the participants \u2019 ideas , planned contrasts showed a marginally significant difference between the BG and all SGs , t ( 44 . 6 ) = \u2212 1 . 79 , p = 0 . 086 , d = 0 . 338 , and again , between the two encouraged and the neutral groups , t ( 71 . 0 ) = \u2212 6 . 47 , p = 0 . 001 , d = 1 . 29 , with the SGs repeating on aver - age more features than the baseline condition due to an increased feature repetition of the encouraged groups . Table 5 shows sum - mary statistics for these results . These results reveal that encouraging instructions influenced the incorporation of structural features from the example . On average , participants in encouraged groups incorporated more example features per idea and generated more ideas that incorpo - rated such features . Additionally , participants in discouraged groups produced results similar to the neutral group and the BG . This result is inconsistent with previous research from Chrysikou and Weisberg ( 2005 ) , as constraining instructions did not decrease repetition , but consistent with Smith et al . ( 1993 ) , as encouraging instructions did increase repetition . However , contrary to the results from Smith et al . ( 1993 ) , our dis - couraged ( but still stimulated ) groups did not significantly repeat more features than a BG , contradicting the idea that participants could not forget the example they had seen . It seems that a few structural features were naturally likely to be incorporated into the ideas generated ( supported by the similar results from the non - encouraged groups ) , with encouraged groups incorporating more features because they were instructed to do so . When com - paring the results from the repetition of structural features to the repetition of the conceptual feature used in this study , we can infer that concrete structural features are more easily copied than abstract conceptual features from examples [ similar to what has been suggested in previous studies ( Zahner et al . , 2010 ; Cheong et al . , 2014 ; Feng et al . , 2014 ) ] . This is despite Table 3 . Summary of bike and non - bike ideas generated across groups Bike ideas BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of bike ideas per participant 1 . 43 ( 0 . 84 ) 1 . 27 ( 0 . 92 ) 1 . 15 ( 0 . 36 ) 1 . 33 ( 0 . 83 ) 1 . 33 ( 0 . 55 ) 1 . 29 ( 0 . 53 ) Number of bike ideas ( and % ) 40 ( 60 % ) 33 ( 85 % ) 31 ( 89 % ) 36 ( 90 % ) 36 ( 95 % ) 36 ( 84 % ) Number of non - bike ideas ( and % ) 27 ( 40 % ) 6 ( 15 % ) 4 ( 11 % ) 4 ( 10 % ) 2 ( 5 % ) 7 ( 16 % ) Table 4 . Summary of modular and non - modular ideas generated across groups Modular ideas BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of modular ideas per participant 0 . 43 ( 0 . 50 ) 0 . 08 ( 0 . 27 ) 0 . 3 ( 0 . 47 ) 0 . 19 ( 0 . 40 ) 0 . 19 ( 0 . 40 ) 0 . 18 ( 0 . 39 ) Number of modular ideas ( and % ) 12 ( 18 % ) 2 ( 5 % ) 8 ( 23 % ) 5 ( 13 % ) 5 ( 13 % ) 5 ( 12 % ) Number of non - modular ideas ( and % ) 55 ( 82 % ) 37 ( 95 % ) 27 ( 77 % ) 35 ( 87 % ) 33 ( 87 % ) 38 ( 88 % ) Artificial Intelligence for Engineering Design , Analysis and Manufacturing 313 https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press the opportunity that conceptual features offer for inspiring solu - tions across multiple categories of solution ( e . g . , products , ser - vices ) . Alternatively , it is possible that either the conceptual feature was chosen or its incorporation into the example was not effective and thus not fully comprehended by the participants . Again , we should highlight that the BG produced more ideas but proportionally less sketches than the SGs . This puts the SGs in an unfavorable position with respect to the count of structural fea - tures incorporated , since these groups had to represent a shape of the bike in which repetition could be more easily recognized \u2013 it is difficult to identify structural repetition when the idea is represented only by text \u2013 thus possibly biasing the results . General discussion and limitations In summary , except for the repetition of structural features , the instructions manipulation had no effect on participants \u2019 idea gen - eration according to the creativity metrics used in this study . Idea fluency and the repetition of the idea type were only affected by the exposure to the example design , and the repetition of the con - ceptual feature did not seem to be affected by either instructions or exposure to the example . This last set of results conflicts with a previous study that found that a pictorial representation of mod - ularity ( as a conceptual and abstract feature ) induced fixation effects in a similar way to a more concrete bike example ( Vasconcelos et al . , 2017 ) . Additionally , looking more carefully into our data reveals that the generation of modular ideas hap - pened quite rarely and randomly across groups , and it is possible that the feature \u201c modularity \u201d , in particular , failed to inspire par - ticipants . These findings call for further investigation and exten - sion of this study , potentially by incorporating other stimuli . As such , to test whether our previous findings are robust to a differ - ent stimulus with different structural and conceptual features ( while keeping all other experimental variables the same ) , we now report an additional analysis done on a second dataset . Additionally , in study 1 , we did not check the inter - rater agree - ment , a test which is often performed for similar studies to dem - onstrate the reliability of the evaluators \u2019 subjective assessment when working individually . The assessment performed was rela - tively objective and yet involved many interactions between eva - luators ( thus , we expected a high agreement between them and we believe that such interaction allowed a reliable analysis of the idea ) . However , because there was no redundancy in the assessment , we cannot quantify how good this agreement might have been . As such , in study 2 , we now compute and report agree - ment coefficients . Study 2 Experimental method , participants , task and problem , and procedure Studies 1 and 2 share the same experimental method and drew participants from the same population . One hundred and thirty - five participants were assigned to five experimental groups ( n = 26 ) . The task , design problem , and procedure were the same as in study 1 , except for one aspect : we did not have a new base - line condition in study 2 since we already had data from a non - stimulated population to which we could compare our new SGs . Materials To allow a neat comparison of studies 1 and 2 , we chose a similar example solution to be given to the participants . While the prob - lem could be solved in many ways and with several underlying properties being applied , it was expected that developing an \u201c adaptable \u201d solution would be a common approach in this con - text . Such an approach could equally be realized by invoking a modular concept ( e . g . , by interchangeable parts ) or a telescopic concept ( e . g . , by extendable parts ) . As a result , we opted for using a telescopic bike ( Fig . 2 ) with both sketch and a textual description of a similar quality to that used in study 1 to avoid influencing the participants \u2019 perception of the ideas due to differ - ent sketch quality ( Kudrowitz et al . , 2012 ) . The description of the example solution was the same used in study 1 [ \u201c Below is an example of how you should present your ideas ( i . e . , annotated sketches ) \u201d ] , and it was followed by the same set of discouraging , encouraging , or neutral instructions regarding the use of features from the example . Analysis The assessment of the participants \u2019 ideas was conducted by the first two authors of this study , with backgrounds in design research and experimental psychology , respectively . First , the evaluators discussed the metrics of the analysis and assessed a random sample of ideas to reach a satisfactory agreement . As in study 1 , the coding scheme used here was not very subjective \u2013 the Table 5 . Summary of modular features incorporated into the participants \u2019 ideas and ideas with modular features across groups Feature repetition BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of features incorporated per idea 0 . 36 ( 0 . 62 ) 0 . 12 ( 0 . 33 ) 0 . 30 ( 0 . 47 ) 0 . 15 ( 0 . 36 ) 1 . 00 ( 1 . 14 ) 1 . 43 ( 1 . 07 ) Range of features incorporated per participant 0 \u2013 2 0 \u2013 1 0 \u2013 1 0 \u2013 1 0 \u2013 3 0 \u2013 3 Total number of ideas with features incorporated ( and % ) 8 ( 12 % ) 3 ( 8 % ) 8 ( 21 % ) 4 ( 11 % ) 16 ( 44 % ) 24 ( 57 % ) Fig . 2 . Example solution provided to the participants along with the following description : \u201c A telescoping bike with parts that can be extended or shortened to fit people with very different heights . Apart from the adjustable tubes and wheels , the angles between tubes can also be modified in specific joints . \u201d The sketch used is a modification of the Zee - K Ergonomic Bike ( Floss , 2010 ) . 314 Luis A . Vasconcelos et al . https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press evaluators spotted the corresponding features in the drawings and reported them . However , the drawings were not always clear and the handwriting was often poor . Consequently , the assessment was prone to human error when interpreting what was contained in each idea , and differences between evaluators were more likely to be attributed to that , instead of divergence in individual opi - nions . As such , the two evaluators assessed the complete set of ideas individually to reveal errors . Such errors were identified by reviewing all ratings and looking for ideas with discrepancies in many variables , which often indicated ideas with a poor repre - sentation . After clarifying such cases , we computed Cohen \u2019 s \u03ba coefficients for each metric , as shown in Table 6 . When consider - ing the high agreement coefficients obtained here and the very brief training provided to the evaluators , it is clear that there was little subjectivity when assessing the ideas and similar coeffi - cients should be expected for study 1 . While most particularities of the assessment were the same as in study 1 , including the metrics used ( i . e . , idea fluency and idea repetition ) , the change in the stimulus required a new set of struc - tural features to be present in the example design . Again , there were five features intentionally incorporated into the example : extendable components to change bike size ; frame joints ( lugs ) that rotate to adjust angles between parts ; wheels with no spokes ; an open - shaped frame ; and a cantilevered saddle that can be adjusted directly . Five participants ( 3 . 7 % ) either did not generate any idea or generated ideas that could not be interpreted by the evaluators ; the results from such participants were not included in our anal - ysis . The adjusted number of participants per experimental group is used in the following section . Results and discussion Idea fluency Instructions had no effect on the number of ideas generated . A one - way ANOVA with the total number of ideas ( per partici - pant ) as the dependent variable showed a significant difference across the groups , F ( 5 , 152 ) = 4 . 79 , p = 0 . 000 , \u03b7 2 = 0 . 136 . However , planned contrasts revealed that this difference is explained by the BG generating on average more ideas than the SGs , t ( 32 . 4 ) = 3 . 59 , p = 0 . 002 , d = 0 . 844 , since no difference was found between the SGs ( Table 7 shows summary statistics for these results ) . This set of results matches very closely with those obtained in study 1 , confirming that the manipulation of instructions had no effect on the number of ideas generated , but exposure to an example design did . Repetition of the idea type Instructions had no effect on the number of bike ideas generated . A one - way ANOVA with the average number of bike ideas ( per participant ) as the dependent variable showed a significant difference across the groups , F ( 5 , 152 ) = 2 . 44 , p = 0 . 037 , \u03b7 2 = 0 . 074 . However , planned contrasts revealed that this difference is explained by the BG generating , on average , fewer bike ideas than the SGs , t ( 31 . 4 ) = \u2212 2 . 45 , p = 0 . 029 , d = 0 . 576 , since no differ - ence was found between the SGs ( Table 8 shows summary statis - tics for these results ) . As in study 1 , the manipulation of instructions had no effect on the number of bike ideas generated , even though the instructions had explicit directions toward the use of features from the bike example and whether participants should comply with the instructions , we expected to observe an indirect effect on the number of bike ideas being generated . Repetition of the conceptual feature Instructions had an effect on the number of telescopic ideas gen - erated . A one - way ANOVA with the average number of telescopic ideas ( per participant ) as the dependent variable showed no sig - nificant difference across the groups , F ( 5 , 152 ) = 1 . 75 , p = 0 . 126 , \u03b7 2 = 0 . 055 . However , planned contrasts revealed a significant dif - ference between the two encouraged groups and the neutral group , t ( 152 ) = \u2212 2 . 27 , p = 0 . 021 , d = 0 . 560 , with the encouraged groups generating more telescopic ideas ( Table 9 shows summary statistics for these results ) . If we adopt a \u201c telescopic \u201d and \u201c non - telescopic \u201d categorization of the ideas , these results reveal that the type of idea generated was influenced by the instructions , a finding that differs from that reported in study 1 . As discussed before , it is possible that the notion of extendibility ( included in the example as a conceptual feature ) was more recognizable than modularity , thus being more easily copied . Interestingly , if we look at the number of mod - ular ideas in study 2 , a similar contrast between the two encour - aged and the neutral groups showed a marginally significant result , t ( 41 . 0 ) = 2 . 02 , p = 0 . 053 , d = 0 . 507 , but now with the encour - aged groups generating less modular ideas ( encouraged , M = 0 . 162 , SD = 0 . 334 ; neutral , M = 0 . 347 , SD = 0 . 394 ) . A possible explanation for this is that , whilst those participants who were encouraged to reproduce features from the telescopic example increased the generation of telescopic ideas , they were also diverted or pushed away from the generation of modular ideas ( these two approaches were so commonly adopted that together they represented 46 % of the ideas from the BG ) . Though research has already reported the occurrence of such phenomena ( Vasconcelos et al . , 2017 ) , this last set of results should be inter - preted with caution since they are derived after the formal analysis had been made , rather than being hypothesized and planned prior to the analysis . Repetition of structural features Instructions had a marginally significant effect on the number of ideas with features of the example provided . A one - way ANOVA with the average number of ideas that contained structural fea - tures of the example ( per participant ) as the dependent variable showed a marginally significant difference across the groups , F ( 5 , 152 ) = 2 . 01 , p = 0 . 081 , \u03b7 2 = 0 . 062 , and a significant difference for the average number of repeated features per participant , F ( 5 , 152 ) = 2 . 73 , p = 0 . 021 , \u03b7 2 = 0 . 082 . Planned contrasts revealed Table 6 . Ideation metrics used in this study and their computed agreement coefficients and interpretation Metric Cohen \u2019 s \u03ba Interpretation ( Landis & Koch , 1977 ) A bike idea 0 . 927 Almost perfect A telescopic idea 0 . 893 Almost perfect A modular idea 0 . 877 Almost perfect Extendable components 0 . 823 Almost perfect Rotating joints 0 . 768 Substantial No spokes 0 . 769 Substantial Open frame 0 . 915 Almost perfect Cantilevered saddle 0 . 852 Almost perfect Artificial Intelligence for Engineering Design , Analysis and Manufacturing 315 https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press significant differences between the encouraged groups and the neutral group for both the number of ideas containing features of the example solution , t ( 152 ) = \u2212 2 . 67 , p = 0 . 004 , d = 0 . 710 , and the number of features included in the participants \u2019 ideas , t ( 152 ) = \u2212 3 . 04 , p = 0 . 004 , d = 0 . 736 , with the encouraged groups generating more ideas with these features and repeating more of these features per idea ( Table 10 shows summary statistics for these results ) . As in study 1 , these results show that only the encouraging instructions influenced the incorporation of structural features from the example . On average , participants in encouraged groups incorporated more example features per idea and generated more ideas that incorporated such features , whereas discouraged groups produced results similar to the neutral group and the BG . While these findings are consistent with those of study 1 , here we observed considerably smaller F - statistics and effect sizes . This can be attributed to the BG generating more ideas with features of the telescopic example than of the modular one , t ( 54 ) = \u2212 3 . 59 , p = 0 . 001 , d = 0 . 960 , and incorporating more of such features into the ideas when compared with the incorporation of modular features , t ( 44 . 3 ) = \u2212 3 . 77 , p = 0 . 000 , d = 1 . 01 . Indeed , there was a general tendency of increased feature repetition in the SGs of study 2 , so we can assume that the features of the telescopic exam - ple were more likely to appear in the participants \u2019 ideas in study 2 than the modular features in study 1 . General discussion and limitations Study 2 results replicate most of what we have found in study 1 . In fact , the statistics regarding fluency and the repetition of the idea type were almost identical , indicating that these findings are robust to different stimuli . With respect to the conceptual feature , even though we have found some repetition in the encouraged groups , participants in study 2 generated on average less tele - scopic ideas [ M = 0 . 407 , SD = 0 . 460 , t ( 263 ) = 3 . 02 , p = 0 . 003 , d = 0 . 370 ] and more modular ideas [ M = 0 . 262 , SD = 0 . 396 , t ( 247 . 4 ) = \u2212 2 . 79 , p = 0 . 006 , d = 0 . 344 ] than study 1 participants ( telescopic ideas , M = 0 . 575 , SD = 0 . 448 ; modular ideas , M = 0 . 138 , SD = 0 . 318 ) . This is a surprising result , as it shows an apparent resistance toward incorporating the underlying mechanisms behind the adaptable bike to which participants were exposed . Moreover , it might be the case that this resistance was a conscious choice , as though participants deliberately tried not to conform to whatever they were shown , either because the example already pre - exhausted the solution space they could have explored ( Perttula & Liikkanen , 2006 ) , or because the participants were suspicious or exhibited demand awareness ( Page , 1981 ) . Figure 3 illustrates a selection of participants \u2019 ideas that were bikes , not bikes , modular , and telescopic . The results for structural feature repetition provide further support for the effectiveness of encouraging instructions in inspiration and fixation experiments , as shown in study 1 and reported in previous research ( Smith et al . , 1993 ) . However , the data also revealed a critical aspect of fixation studies : idea repeti - tion results will depend on the kind of feature that experimenters incorporate into the analysis . Presumably , differences in feature repetition results between stimulated and non - stimulated partici - pants are more pronounced when those features represent rare , unusual solutions to the design problem , thus being very unlikely to emerge during idea generation in non - SGs . Conversely , repeti - tion results might be similar if the features analyzed represent obvious solutions , thus being highly likely to appear in ideas with or without external stimulation [ although there is recent evi - dence to the contrary ( Viswanathan et al . , 2016 ) ] . Though the fea - tures used in both our studies are easily comparable ( i . e . , following a similar structure and referring to the same bike parts ) , we believe that the features used in study 2 were more common than those in study 1 , especially the \u201c extendable compo - nents to change bike size \u201d , which frequently appeared in the designs of all groups . This might have biased the idea repetition analysis , and therefore compromised the comparability of the two studies in this respect ( see Fig . 4 for a comparison of feature repetition results between the two studies ) . The main limitations of the studies reported here are the dura - tion of the generation session , the pool of participants chosen , and the design problem used . The idea generation session in this study was 10 min long , which can be considered short when compared with other fixation studies in which generation sessions typically lasted for 30 or 60 min ( Vasconcelos & Crilly , 2016 ) . In addition , the research suggests that novel ideas tend to occur later in the idea generation session ( Kudrowitz & Dippo , 2013 ) ; thus , the short session adopted for this study might have contributed to inflated fixation scores ( when considering fluency and the repetition of the respective idea types ) . However , it is also Table 7 . Summary of ideas generated per participant across groups Generated ideas BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of ideas per participant 2 . 39 ( 1 . 31 ) 1 . 29 ( 0 . 46 ) 1 . 56 ( 1 . 45 ) 1 . 44 ( 0 . 65 ) 1 . 38 ( 0 . 64 ) 1 . 62 ( 0 . 85 ) Range of ideas generated per participant 1 \u2013 5 1 \u2013 2 1 \u2013 8 1 \u2013 3 1 \u2013 3 1 \u2013 4 Total number of ideas 67 36 39 36 36 42 Total number of participants 28 28 25 25 26 26 Table 8 . Summary of bike and non - bike ideas generated across groups Bike ideas BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of bike ideas per participant 1 . 43 ( 0 . 84 ) 0 . 929 ( 0 . 22 ) 0 . 962 ( 0 . 14 ) 0 . 867 ( 0 . 33 ) 0 . 904 ( 0 . 25 ) 0 . 936 ( 0 . 18 ) Number of bike ideas ( and % ) 40 ( 60 % ) 33 ( 92 % ) 33 ( 85 % ) 31 ( 86 % ) 30 ( 84 % ) 37 ( 88 % ) Number of non - bike ideas ( and % ) 27 ( 40 % ) 3 ( 8 % ) 6 ( 15 % ) 5 ( 14 % ) 6 ( 16 % ) 5 ( 12 % ) 316 Luis A . Vasconcelos et al . https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press possible that 10 min of ideation might have been too short for fixation to take place ( or to be measured at least ) , although we believe that this is unlikely , as research has already shown that having extra time helps to diminish fixation effects ( Tseng et al . , 2008 ) . The participants in our studies were undergraduate students and the generation session was part of an ongoing engi - neering course . This might have resulted in a more diligent par - ticipant behavior when compared with other studies in which participants and experimenters did not have a student \u2013 lecturer relationship . As a result , the setup adopted for this study might have contributed to the extent to which participants adhered to the instructions . Additionally , the behavior of design students ( or novices ) and practitioners ( or experts ) differs , both with respect to the design process in general ( for a review , see Dinar et al . , 2015 ) , and design fixation in particular ( for a review , see Vasconcelos & Crilly , 2016 ) . Thus , our results might have been different had our participants been more experienced . The design problem used in this study was chosen partly because it was unli - kely that the participants had designed solutions to it before . As such , it is possible that using familiar problems will produce dif - ferent results , although research has demonstrated that fixation effects can be observed with both familiar and unfamiliar prob - lems ( Jansson & Smith , 1991 ) . Finally , although it is not necessarily a limitation of this type of work , but a characteristic of it , the evaluation method used in these studies can be considered subjective to some extent [ yet , consensual assessment is still the standard method in idea genera - tion and design fixation studies ( see Vasconcelos & Crilly , 2016 ) ] . While new methods are available today , such as eye - tracking and neuroimaging , they either require additional techniques for trian - gulation or are still very recent and their potential for providing more objective , meaningful data is yet to be developed ( Editorial board of IJDCI , 2013 ) . With the aim to achieve higher objectivity levels when analyzing data from idea generation experiments , two recent studies had their participants designing in a digital environment , such as computer games ( Neroni et al . , 2017 ) and computer - aided design tools ( Zhang et al . , 2017 ) . Apart from allowing more objective and comparable anal - yses of the design activity , these approaches may enable a series of benefits , such as using design problems and materials that are not easily manipulated in classroom settings , unobtrusive data capture throughout the design task , compatibility with other digital tools for data capture , and better control over the format and quality of the output generated . These are exciting opportunities not just for future work in design fixation ; they show themselves as new methodological paradigms to study human cognition more broadly . Conclusion and future work We have tested the influence of instructions on idea generation . More specifically , we analyzed how discouraging , encouraging , and neutral instructions may affect the number of ideas generated and the incorporation of the example or its parts into the partic - ipants \u2019 ideas . The instructions used were provided along with an external stimulus and its description . It is important to differenti - ate the descriptions from instructions because in this study we have controlled the former but manipulated the latter . We found that instructions shaped the idea generation of our partic - ipants to some extent . When encouraged or required to use features from the example , participants consistently copied struc - tural features but some failed to copy a more abstract conceptual feature . When discouraged or forbidden from using features from the example , however , most participants failed to reduce the num - ber of features copied . This result allows us to infer that more con - crete features are easier to recognize \u2013 and thus reproduce \u2013 than more abstract features such as modularity . However , this may reflect a peculiarity of the modularity feature , since an equivalent approach , such as extendibility , produced slightly different results . Additionally , the results might indicate that positive instructions are more effective than negative ones , irrespective of how flexible or strict the instructions are . This can tell us how to frame future instructions , whether that is with respect to experimental stimuli in research , illustrative cases in design education , and inspira - tional examples in design practice . Regardless of the instructions given to the participants , using different conceptual and structural features between studies allowed us to explore how two alternative but equivalent approaches to solving a design problem can compete for atten - tion . When participants complied with the encouraging instruc - tions and generated more telescopic ideas , they also decreased the number of modular ideas they generated \u2013 we call this design diversion . Two other observations were made when analyzing the datasets . The first is that the telescopic bike conditions generated more modular ideas and less telescopic ideas than the modular bike conditions , and we interpret this as the participants deliber - ately exploring an alternative solution space . The second is that feature repetition calculations can be misleading depending on Table 9 . Summary of telescopic and non - telescopic ideas generated across groups Telescopic ideas BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number of telescopic ideas per participant 0 . 43 ( 0 . 50 ) 0 . 08 ( 0 . 27 ) 0 . 3 ( 0 . 47 ) 0 . 19 ( 0 . 40 ) 0 . 19 ( 0 . 40 ) 0 . 18 ( 0 . 39 ) Number of telescopic ideas ( and % ) 19 ( 28 % ) 16 ( 44 % ) 7 ( 18 % ) 8 ( 22 % ) 15 ( 42 % ) 19 ( 45 % ) Number of non - telescopic ideas ( and % ) 48 ( 72 % ) 20 ( 56 % ) 32 ( 82 % ) 28 ( 78 % ) 21 ( 58 % ) 23 ( 55 % ) Table 10 . Summary of telescopic features incorporated into the participants \u2019 ideas and ideas with telescopic features across groups Feature repetition BG SG \u2212 2 SG \u2212 1 SG0 SG + 1 SG + 2 Mean ( and SD ) for the number features incorporated per idea 1 . 21 ( 1 . 03 ) 1 . 43 ( 1 . 55 ) 1 . 00 ( 1 . 19 ) 0 . 84 ( 1 . 07 ) 1 . 65 ( 1 . 60 ) 2 . 08 ( 1 . 72 ) Range of features incorporated per participant 0 \u2013 4 0 \u2013 6 0 \u2013 4 0 \u2013 4 0 \u2013 8 0 \u2013 5 Total number of ideas with features incorporated ( and % ) 24 ( 36 % ) 23 ( 64 % ) 15 ( 38 % ) 13 ( 36 % ) 23 ( 64 % ) 26 ( 62 % ) Artificial Intelligence for Engineering Design , Analysis and Manufacturing 317 https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press how common or obvious the features might be ( e . g . , the bike has a saddle vs . the bike has wings ) , but also on their level of granularity ( e . g . , the idea is a transportation mode , it is a bike , it is a modular bike , the pedals are made of modular - blocks , and so on ) , with repetition results likely varying according to each level . As such , future studies should reflect on both the commonness and the granularity level of the features under consideration and could report results according to these categories of features ( e . g . , a quadrant chart of features , having commonness and granularity as two orthogonal continuums ) . Nevertheless , all these potential findings need support from more data and are thus open for future investigation . Again , irrespective of how discouraging the instructions were , stimulated participants still exhibited fixation effects due to their exposure to the example design ( i . e . , they generated fewer ideas and proportionally more bike ideas than the BG ) . This result is in line with many other design fixation experiments in which par - ticipants become stuck on a specific idea type . However , it is important to emphasize that in this study the description of the stimulus itself could also be causing fixation as the stimulus was presented to the participants as \u201c an example of how they should present their ideas \u201d . Thus , perhaps there is an implicit suggestion for the participants to produce ideas that are similar to the example , i . e . , a bike . Another possible explanation is that in inspiration and fixation studies , the examples overwhelm the par - ticipants \u2019 interpretation of the problem , to the extent that the examples themselves become part of the participants \u2019 problem representation . Future studies could investigate such possibilities Fig . 3 . Some of the participants \u2019 ideas : ( a ) bike idea , ( b ) non - bike idea , ( c ) modular idea , and ( d ) telescopic idea . Note that these examples are not representative of the design work of any particular experimental group . Fig . 4 . Bar chart showing the average number of ideas with features of the example and an average number of example features per idea across groups ( averages are given per participant ; error bars indicate standard deviation ) . 318 Luis A . Vasconcelos et al . https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press and complement our understanding about stimuli introduction by manipulating the descriptions provided to designers and by testing how examples interfere with problem representation . It is also important to consider here and in similar future studies , that copying and repeating features from examples do not neces - sarily imply fixation . Fixation might be inferred when participants are discouraged from copying ( or maybe when they are given no guidance at all ) , and yet they still copy , otherwise , they may only be complying with the descriptions and instructions in the experi - ments . Similarly , we should be cautious when referring to \u201c idea repetition \u201d for those participants who are given no stimulus but spontaneously generate ideas that incorporate features that also happen to be in the example provided to others . Finally , when considering design practice , design teams and project managers can benefit from being aware of the design fixa - tion knowledge and from the findings discussed in this paper , applying such knowledge depending on the scope and stage of the design project . For instance , the idea of design diversion might be considered by project managers for strategically remov - ing complementary areas of the solution space that are not of interest . The results about the efficacy of instructions and the abstraction of stimuli reported here are also relevant to how inspirational stimuli should be framed when presented to the designers . This is particularly important for the development and implementation of computer - aided design tools that provide designers with external stimuli . Much has been researched on how such software tools might be structured and interacted with , and what form the inspirational stimuli should take ( Shneiderman , 2000 ; T\u00f6re Yargin & Crilly , 2015 ) . However , it is also important to understand how those stimuli should be intro - duced , whether by description , instruction , or both . Our findings show that using positive instructions ( either requiring or encoura - ging ) in inspiration tools can guarantee that designers will incor - porate features from a given stimulus , and that choosing a concrete stimulus will increase feature incorporation even further . However , should designers be steered toward or away from the repetition of structural features , directed to identify conceptual features , or simply be left alone to interpret and respond as they see fit ? By developing a better understanding of how stimuli instructions influence idea generation , we will move closer to answering such questions and thereby become more capable of supporting creative design . Acknowledgments . This work was supported by the CAPES Foundation , Ministry of Education of Brazil ( grant number BEX 11468 / 13 - 0 ) and the UK \u2019 s Engineering and Physical Sciences Research Council ( grant number EP / K008196 / 1 ) . Research data supporting this publication are available from the University of Cambridge data repository : https : / / doi . org / 10 . 17863 / CAM . 7571 References Aleman V ( 2009 ) ECO 07 \u2013 Compactable Urban Bicycle , accessed February 10 , 2017 . Available at https : / / www . behance . net / gallery / 293563 / ECO - 07 - Compactable - Urban - Bicycle . Atilola O and Linsey J ( 2015 ) Representing analogies to influence fixation and creativity : a study comparing computer - aided design , photographs , and sketches . 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Vasconcelos has recently completed his PhD in Engineering Design at the University of Cambridge , where he was also a member of the Design Practice research group at the Engineering Design Centre . With a back - ground in design research and methodology , his research explores new paths in design inspiration and fixation studies . He is interested in the design process at both macro and micro levels . Dr Maria A . Neroni is a Research Associate at the Design Practice Group in the Cambridge Engineering Design Centre . With a background in experi - mental psychology , cognitive neuroscience and education , her current research focuses on developing novel experimental paradigms to investigate creativity in engineering design . Dr Nathan Crilly is a Senior Lecturer in Engineering Design at the University of Cambridge . He leads the Design Practice research group at the Engineering Design Centre . His research interests lie in design , creativity , and communica - tion . He and his research group employ an interdisciplinary approach to study - ing the development of products , the properties they exhibit , and how people respond to them . 320 Luis A . Vasconcelos et al . https : / / doi . org / 10 . 1017 / S0890060417000658 Published online by Cambridge University Press", + "soriano-castell2017rock1": "1 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x www . nature . com / scientificreports ROCK1 is a novel Rac1 effector to regulate tubular endocytic membrane formation during clathrin - independent endocytosis David Soriano - Castell 1 , Albert Chavero 1 , Carles Rentero 1 , Marta Bosch 1 , Maite Vidal - Quadras 1 , Albert Pol 1 , 2 , Carlos Enrich 1 & Francesc Tebar 1 Clathrin - dependent and - independent pathways contribute for \u03b2 1 - integrin endocytosis . This study defines a tubular membrane clathrin - independent endocytic network , induced with the calmodulin inhibitor W13 , for \u03b2 1 - integrin internalization . This pathway is dependent on increased phosphatidylinositol 4 , 5 - bisphosphate ( PI ( 4 , 5 ) P 2 ) levels and dynamin activity at the plasma membrane . Exogenous addition of PI ( 4 , 5 ) P 2 or phosphatidylinositol - 4 - phosphate 5 - kinase ( PIP5K ) expression mimicked W13 - generated - tubules which are inhibited by active Rac1 . Therefore , the molecular mechanisms downstream of Rac1 , that controls this plasma membrane tubulation , were analyzed biochemically and by the expression of different Rac1 mutants . The results indicate that phospholipase C and ROCK1 are the main Rac1 effectors that impair plasma membrane invagination and tubule formation , essentially by decreasing PI ( 4 , 5 ) P 2 levels and promoting cortical actomyosin assembly respectively . Interestingly , among the plethora of proteins that participate in membrane remodeling , this study revealed that ROCK1 , the well - known downstream RhoA effector , has an important role in Rac1 regulation of actomyosin at the cell cortex . This study provides new insights into Rac1 functioning on plasma membrane dynamics combining phosphatidylinositides and cytoskeleton regulation . Endocytosis is an essential process for eukaryotic cells to internalize growth factors , hormones , and nutrients from the plasma membrane ( PM ) or extracellular fluid 1 \u2013 4 . The internalization routes can be classified into clathrin - dependent endocytosis ( CDE ) and clathrin - independent endocytosis pathways ( CIE ) 2 , 5 \u2013 9 . CIE pathways include different ways of internalization which show high complexity , though all generally share their association with PM microdomains enriched in cholesterol and glycosphingolipids 10 \u2013 13 . The molecular machinery that reg - ulates these different routes is only now beginning to emerge . In comparison with CDE pathways , the morpho - logical features of membrane carriers generated by CIE pathways range from small vesicles to membrane tubular networks of different size and extension 9 , 11 , 14 \u2013 16 . Several laboratories , including ours , have recently described the existence of PM tubular networks belong - ing to CIE pathways , with tubules of tens of micrometers in length 11 , 15 , 17 . Major histocompatibility complex I ( MHCI ) has been reported to be internalized via Arf6 - dependent , clathrin and caveolae - independent endo - cytosis 18 , and both MHCI and Arf6 were detected in tubules that lack the CDE marker transferrin 15 . Induced non - clathrin - mediated tubular membrane invaginations have also been reported for the uptake of Cholera and Shiga toxins , and the simian SV40 virus 16 , 17 , 19 . Formation of these tubules seems to require an intact microtubule network 15 , 16 . Moreover , we have demonstrated the involvement of Rac1 , calmodulin ( CaM ) , and phosphatidy - linositol 4 , 5 - bisphosphate ( PI ( 4 , 5 ) P 2 ) in this process 15 . While expression of the constitutively active Rac1 mutant Rac1 G12V completely abolishes membrane tubules , the dominant negative mutant Rac1 T17N triggers the formation . The same phenotype is generated by phosphatidylinositol 4 - phosphate - 5 - kinase ( PIP5K ) overexpression or by treatment with the CaM inhibitor N - ( 4 - aminobutyl ) - 5 - chloro - 2 - naphthalenesulfonamide ( W13 ) , which increase PI ( 4 , 5 ) P 2 levels at the PM 15 . Involvement of PI ( 4 , 5 ) P 2 in the initiation of endocytic events is determined by its 1 Departament de Biomedicina , Unitat de Biologia Cel\u00b7lular , Centre de Recerca Biom\u00e8dica CELLEX , Institut d\u2019Investigacions Biom\u00e8diques August Pi i Sunyer ( IDIBAPS ) , Facultat de Medicina , Universitat de Barcelona , Casanova 143 , 08036 , Barcelona , Spain . 2 Instituci\u00f3 Catalana de Recerca i Estudis Avan\u00e7ats ( ICREA ) , 08010 , Barcelona , Spain . Correspondence and requests for materials should be addressed to F . T . ( email : tebar @ ub . edu ) Received : 7 November 2016 Accepted : 23 June 2017 Published online : 31 July 2017 OPEN www . nature . com / scientificreports / 2 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x ability to bind and recruit several membrane - bending proteins such as dynamin or BAR - domain containing pro - teins , but also by its role in actin dynamics at the cell surface 20 \u2013 25 . Afterwards , the decrease of PI ( 4 , 5 ) P 2 by specific phosphatases and / or phospholipases , such as synaptojanin or phospholipase C ( PLC ) , is important to promote pinch - off of the plasma membrane and the consequent endocytic vesicle production 26 \u2013 28 . The small GTPases , Rac1 , RhoA , and Cdc42 , are implicated in the regulation of several CIE pathways . Rac1 and RhoA control interleukin - 2 receptor ( IL - 2R ) uptake 29 , 30 , and Rac1 regulates macropinocytosis with Cdc42 , which is also required during clathrin - independent carrier ( CLIC ) and GPI - enriched endocytic com - partment ( GEEC ) endocytosis 31 \u2013 34 . Several CIE pathways also require Pak1 , Pak2 , or cortactin activity , which are Rac1 actin - related targets 30 , 35 , suggesting that Rac1 - dependent actin polymerization plays a key role during these events . The PI ( 4 , 5 ) P 2 - binding protein dynamin , as well as cortactin , have been reported to be important actin - modulating and membrane - remodeling factors during both CDE and CIE 35 \u2013 37 . Therefore , cortactin and dynamin may be acting downstream of Rac1 to regulate the endocytic tubules formation . Moreover , recent stud - ies have identified myosins regulating endocytosis 38 \u2013 41 , and it has been shown that an increased assembly of actomyosin networks at the PM antagonizes membrane invagination and endocytosis 42 , 43 . Actomyosin is mainly regulated by RhoA through its effector ROCK1 ( rho associated coiled - coil containing protein kinase 1 ) , but also by Rac1 , and these two GTPases usually have opposite effects in several cellular processes 44 . The possible con - tribution of Rac1 - dependent actomyosin regulation to CIE has not been investigated in depth , and nor has its contribution to tubule regulation . Actually , Rac1 could control tubule outcomes by regulating PI ( 4 , 5 ) P 2 levels ( via PLC activity ) and cytoskeleton dynamics ( through actin polymerization and myosin activation ) 45 \u2013 51 . In the present study we demonstrate that increased PI ( 4 , 5 ) P 2 levels trigger dynamin - dependent endocytic tubules formation and enhance \u03b2 1 - integrin internalization , and that this process can be neutralized by Rac1 activation . We show that Rac1 regulates PM endocytic tubule formation by controlling PI ( 4 , 5 ) P 2 levels , actin dynamics and myosin activation through activation of PLC , cortactin and ROCK1 , respectively . Importantly , the results reveal ROCK1 as a new Rac1 effector and here we propose a novel Rac1 - dependent ROCK1 activation pathway to regulate membrane dynamics . Results and Discussion Integrin internalization via a clathrin - independent , Rac1 - regulated endocytic pathway . We have previously shown that Rac1 activity can regulate the formation of membrane tubular structures , with the dominant negative Rac1 increasing and the constitutively active mutant reducing the percentage of cells present - ing tubules 15 . These tubular membrane structures , which are also induced after treatment with the calmodulin inhibitor W13 , transported clathrin independent endocytic cargoes like MHCI 15 . Since Rac1 activity can control integrin trafficking , and vice versa 52 \u2013 56 , we have examined whether integrins were also present in these endocytic tubules and if the presence of such tubules affects integrin transport to early endosomes ( EEs ) . COS1 cells were incubated with an antibody that recognizes the \u03b2 1 - integrin ectodomain and treated then with W13 for 10 min - utes at 37 \u00b0C , before fixing and immunostainning cells with anti - EEA1 antibody . The images in Fig . 1a show the presence of \u03b2 1 - integrin ( red ) in W13 - induced tubules , visualized with the expressed membrane marker GFP - mem ( green ) . Whereas \u03b2 1 - integrin was clearly detected in EEA1 - positive endosomes of control cells ( Fig . 1b ) , those cells that contained extensive tubulation ( W13 treated ) showed low \u03b2 1 - integrin labeling in EEs ( Fig . 1c ) . Considering the presence in tubules as internalized molecules , cells exhibiting tubules showed increase in the total internalized \u03b2 1 - integrin after treatment with W13 at different time points ( 5 , 10 and 20 min ) compared with control cells ( Fig . 1d ) . In these settings , the effect of W13 - induced tubules on transferrin internalization , a well - established cargo of the CDE route , was also analyzed ( Fig . 1e ) . Transferrin was not observed in W13 - tubules and increased transferrin internalization was observed only at 20 minutes in W13 - induced tubules com - pared to control cells , which could be explained by the previously reported effect of W13 inhibiting sorting from early endosomes 57 \u2013 60 and consequently transferrin recycling that at later time points contributes to the uptake measurements . Likewise , this could be the reason for increased \u03b2 1 - integrin internalization at later time points in W13 - treated cells not presenting tubules . To corroborate that clathrin did not participate in W13 - induced tubule formation , clathrin expression was inhibited by siRNA knockdown ( Fig . 1f ) . Figure 1f shows that clathrin down - regulation did not modify the extend of W13 - induced tubules and , in agreement with the inhibition of recycling , W13 treatment accumulated transferrin in EEs vesicles at the cell periphery in contrast to its perinuclear locali - zation observed in control cells . These results indicate that induced tubular endocytic membrane structures are a cellular port of entry important for \u03b2 1 - integrin internalization in a CIE pathway . The effect of W13 on \u03b2 1 - integrin internalization was simultaneously analyzed in cells expressing the consti - tutively active Rac1 mutant ( GFP - Rac1 G12V ) . Image quantification showed that Rac1 G12V expression completely abrogated the W13 - increased \u03b2 1 - integrin internalization at all - time points analyzed ( Fig . 1d ) , indicating that active Rac1 blocks tubule formation instead of promoting tubule fission . Besides , the results imply that Rac1 is a \u03b2 1 - integrin internalization regulator , and suggest that it may regulate integrin turnover through CIE . Induction of dynamin - dependent tubules in CIE pathway by increased PI ( 4 , 5 ) P 2 . Next , clathrin - independent endocytic tubules were further characterized and we focused on the molecular mechanisms activated by Rac1 and its regulation . We and others reported that membrane tubules are induced after increasing PI ( 4 , 5 ) P 2 levels by overexpression of PIP5K 15 , 61 . In fact , W13 - induced PM tubules appear to depend on PIP5K activity 15 . The presence of PI ( 4 , 5 ) P 2 in W13 - tubules was confirmed by immunostaining with an anti - PI ( 4 , 5 ) P 2 antibody ( Fig . 2a ) . Moreover , tubule induction by PI ( 4 , 5 ) P 2 increase was supported by a dose response curve with exogenous diC8 - PI ( 4 , 5 ) P 2 ( previously conjugated with the neomycin carrier for its transmembrane delivery ) 62 , 63 ( Fig . 2b ) . Addition of 50 \u00b5 M of diC8 - PI ( 4 , 5 ) P 2 increased the percentage of cells with tubules up to approximately 35 % compared to 12 % observed by the neomycin carrier in control cells ( Fig . 2c , d ) . Similar ratio elicited by W13 treatment was observed by overexpression of PIP5K or diC8 - PI ( 4 , 5 ) P 2 incubation ( Fig . 2e ) . In addition , these www . nature . com / scientificreports / 3 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x Figure 1 . W13 - induced PM tubules provide an internalization pathway for \u03b2 1 - integrin . ( a ) COS1 cells grown on coverslips expressing the membrane marker GFP - mem were incubated with an anti - \u03b2 1 - integrin rat antibody for 30 minutes at 4 \u00b0C to avoid endocytosis , followed by incubation for 10 minutes at 37 \u00b0C to allow internalization in the presence of W13 ( 20 min , 4 . 5 \u00b5 g / ml ) . After fixation , \u03b2 1 - integrin was detected with an Alexa - 555 labeled anti - rat antibody , and images were acquired with a confocal microscope ( Leica TCS SP5 ) . The higher magnification insets show \u03b2 1 - integrin localization in W13 - induced tubules ( green arrowheads ) . ( b , c ) Following the same procedure explained as in ( a ) , \u03b2 1 - integrin was detected with an Alexa - 647 labeled anti - rat antibody and EEA1 with a specific antibody and the secondary Alexa - 555 anti - mouse in untreated ( b ) or W13 - treated cells ( c ) . Insets show \u03b2 1 - integrin in EEA1 - positive endosomes ( red arrowheads ) ( b ) or in tubules ( green arrowheads ) ( c ) ( bars , 10 \u00b5 m ) . ( d , e ) Quantification of internalized \u03b2 1 - integrin ( d ) and transferrin ( e ) , as explained in the Materials and Methods , in COS1 cells expressing GFP - mem or GFP - Rac1 G12V for the indicated conditions ( W13t , cells presenting tubules ; W13nt , cells without tubules ) . Mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments are shown . Statistical significance between different www . nature . com / scientificreports / 4 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x different experimental conditions similarly increased both the number of tubules per cell ( Fig . 2e ) , and PI ( 4 , 5 ) P 2 levels detected by immunofluorescence compared to control cells ( Fig . 2f ) . These results demonstrated a direct relationship between increased PI ( 4 , 5 ) P 2 levels and tubule development . Therefore , W13 - treatment could be used to increase PI ( 4 , 5 ) P 2 levels and tubulation at the PM . It is known that increased PI ( 4 , 5 ) P 2 are necessary for endocytosis to proceed because they recruit several PI ( 4 , 5 ) P 2 - binding proteins , including adaptor proteins , BAR - domain containing proteins , and dynamin ( among others ) 20 . Although dynamin has an important role in the scission of endocytic vesicles from the PM , it has also been involved in membrane deformation and tubular membrane organization 24 , 64 \u2013 68 . Therefore , we investigated the role of dynamin in these PI ( 4 , 5 ) P 2 - induced tubules . Dynamin action was inhibited by dominant negative mutant expression ( dyn K44A ; Fig . 3a ) , treatment with a specific inhibitor ( dynasore ; Fig . 3a ) , or by siRNA knock - down ( Figs 3b and 2f ) . In each of these experimental settings , W13 - induced tubules were prevented , indicating that this tubular endocytic pathway is dynamin - dependent . Dynamin was necessary to initiate tubule formation , but an additional role of dynamin in the fission of tubules cannot be discarded . Dynamin participates in membrane invagination in combination with BAR - domain containing proteins 43 , 67 . In W13 - induced tubules we have also observed the presence of PACSIN2 , an F - BAR - domain protein that binds dynamin , PI ( 4 , 5 ) P 2 and Rac1 69 , 70 . Interestingly , although PACSIN2 interacts with dynamin , it does not bind or colocalize with clathrin 71 . Moreover , in agreement with the report by Kreuk et al . 69 , we showed that expression of the active Rac1 mutant inhibited the presence of PACSIN2 - positive tubules in COS1 cells after W13 treat - ment ( Supplementary Fig . S1 ) . In addition , it has been reported that PACSIN2 regulates caveolae biogenesis and endocytosis in cholesterol rich and plasma membrane ordered domains 69 , 72 , 73 , where active Rac1 is located 52 , 74 . Actually , we have previously shown that cyclodext rin , a PM cholesterol chelator , inhibited W13 - tubule forma - tion 15 . Therefore , we analyzed whether the specific PI ( 4 , 5 ) P 2 increase elicited by W13 treatment was respon - sible for tubulation in these domains . Tubules were inhibited in cells expressing a PI ( 4 , 5 ) P 2 - phosphatase specifically targeted to PM ordered domains by the 10 N - terminal amino acids of Lck ( L10 - GFP - Inp54p ) 75 . Otherwise , no effect was observed with the phosphatase dead mutant ( L10 - GFP - Inp54p D281A ) ( Fig . 3c ) . Together , these results show that increased PI ( 4 , 5 ) P 2 levels in specific PM domains , where clathrin - independent and dynamin - dependent endocytosis takes place , are probably responsible for tubule formation . In agreement with the localization of Rac1 in ordered domains 74 , 76 , tubular endocytic membranes present in control cells , or elicited by W13 treatment , were inhibited by active Rac1 , as well as tubules induced by PIP5K overexpression or by the addition of exogenous diC8 - PI ( 4 , 5 ) P 2 ( Fig . 3d ) . These results , together with the fact that Rac1 G12V expressing cells showed reduced PI ( 4 , 5 ) P 2 immunostaining in W13 - treated cells ( Supplementary Fig . S2 ) , prompted us to study the role of Rac1 effectors in tubulation . The role of PLC - regulated PI ( 4 , 5 ) P 2 levels on tubule inhibition by active Rac1 . Rac1 can modu - late PI ( 4 , 5 ) P 2 levels at the PM by activating PLC 77 , 78 , which hydrolyzes PI ( 4 , 5 ) P 2 generating diacylglycerol ( DAG ) and inositol trisphosphate ( IP 3 ) . Then , we analyzed PLC involvement by two strategies : ( i ) inhibition of PLC activ - ity with its inhibitor U73122 , and ii ) expression of GFP fusion proteins for two previously described constitutively active ( GTP - bound ) , but PLC - deficient , Rac1 mutants ( Rac1 G12V - F37A and Rac1 G12V - W56A ) 77 . Quantification of tubule formation in COS1 cells demonstrates that U73122 impaired the tubule inhibition produced by GFP - Rac1 G12V expression ( Fig . 3e ) . Moreover , the expression of both Rac1 G12V - F37A and Rac1 G12V - W56A did not inhibit tubules in untreated cells ( Fig . 3d ) . These results strongly suggest that PLC plays an important role in Rac1 - dependent tubule inhibition . To further analyze PLC activity involvement in the Rac1 - dependent inhibition of tubule formation , we assessed the effect of Rac1 G12V - F37A and Rac1 G12V - W56A expression on PI ( 4 , 5 ) P 2 - induced tubulation , either by W13 - treatment or PIP5K - overexpression . As expected , W13 and PIP5K induced a similar percentage of cells presenting tubules in control and Rac1 G12V - F37A expressing cells ( Fig . 3f ) , confirming the involvement of PLC activity . However , the expression of the Rac1 G12V - W56A mutant was still able to block tubule formation ( Fig . 3f ) . Together , these results suggest that , although PLC plays a key role in tubule inhibition by active Rac1 , addi - tional mechanism contributes to the inhibition , as revealed through the use of the PLC - deficient mutants . Since Rac1 G12V - F37A mutant is not able to translocate cortactin to the plasma membrane or interact with ROCK1 ( two important factors for cortical actomyosin regulation ) 79 , 80 , the Rac1 G12V - W56A mutant was therefore considered a valuable tool for studying the role of cytoskeleton in the PLC - independent tubular - inhibitory effect of active Rac1 . conditions and the corresponding control was determined by the two - way ANOVA , * p < 0 . 05 , * * * p < 0 . 001 . Statistical significance in integrin internalization assay ( d ) between W13t and W13nt at 5 , 10 and 20 minutes were * p < 0 . 05 , * * * p < 0 . 001 , * * * p < 0 . 001 , respectively . ( f ) COS1 cells co - transfected with GFP - mem and a specific clathrin heavy chain siRNA or a non - specific siRNA as a control ( 72 h ) were incubated with transferrin - TRITC during 15 minutes at 37 \u00b0C in the presence or absence of W13 ( 4 . 5 \u00b5 g / ml ) . After fixation , clathrin was detected with an anti - mouse antibody ( clone \u00d7 22 ) and the corresponding Alexa - 647 secondary antibody . Confocal images were acquired with a confocal microscope ( Leica TCS SP5 ) through the corresponding channels ( bars , 10 \u00b5 m ) . Downregulation of clathrin expression by its specific siRNA is shown by western blotting using a rabbit polyclonal antibody Graph shows the percentage of cells presenting tubules ( > 5 tubules / cell ) in the indicated conditions . Mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments are shown . Statistical significance between W13 - treatment and the corresponding control was determined by the paired Student\u2019s t - test , * * p < 0 . 01 . www . nature . com / scientificreports / 5 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x Figure 2 . Increased levels of PI ( 4 , 5 ) P 2 induces membrane tubules at the PM . ( a ) COS1 cells expressing Cherry - Mem grown on coverslips were incubated with W13 ( 20 min , 4 . 5 \u00b5 g / ml ) . After fixation ( PFA 4 % , 15 min at 37 \u00b0C ) , endogenous PI ( 4 , 5 ) P 2 was detected with a specific antibody and the corresponding anti - mouse Alexa - 488 labeled secondary antibody . The images were acquired with a confocal microscope ( Leica TCS SP5 ) and magnification insets show the presence of PI ( 4 , 5 ) P 2 in W13 - induced membrane tubules . ( b ) The percentage of cells presenting more than 5 tubules was determined after 20 min of diC8 - PI ( 4 , 5 ) P 2 dose - response at the indicated concentrations . ( c ) The percentage of cells with tubules after 20 min incubation with 50 \u00b5 M diC8 - PI ( 4 , 5 ) P 2 or neomycin control . ( d ) Confocal image of cherry - mem depicted tubules in cells treated with diC8 - PI ( 4 , 5 ) P 2 as in ( c ) ( bars , 10 \u00b5 m ) . ( e ) In Cherry - Mem expressing cells , the percentage of cells presenting tubules and the number of tubules per cell were determined in different experimental conditions : W13 ( 20 min , 4 . 5 \u00b5 g / ml ) , diC8 - PI ( 4 , 5 ) P 2 ( 20 min , 50 \u00b5 M ) or GFP - PIP5K expression . Statistical significance between the different conditions and the control ( NT , non - treatment ) was determined by the unpaired Student\u2019s t - test , * * p < 0 . 01 ( n = 100 cells per condition ) . Representative confocal images acquired through the red channel and insets displaying cells presenting cherry - Mem decorated tubules are shown ( bars , 5 \u00b5 m ) . ( f ) In the same experimental conditions as in ( e ) , the intracellular levels of PI ( 4 , 5 ) P 2 were determined by immunofluorescence performed as in ( a ) . Graph shows the percentage of fluorescence ( a . u . , arbitrary units ) of each condition vs NT , mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments . Statistical significance between the different conditions and the NT was determined by the paired Student\u2019s t - test , * p < 0 . 05 . Representative z - stacks confocal projection images are shown ( bars , 10 \u00b5 m ) . www . nature . com / scientificreports / 6 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x Cortactin - dependent actin polymerization inhibits tubular endocytic membrane structures downstream of active Rac1 . Active Rac1 is important to control actin polymerization ( F - actin ) at the PM and F - actin depolymerizing agents are known to generate membrane tubules in many cell types 81 , 82 . Thus , active Rac1 , by increasing F - actin at the cell cortex , could inhibit PM invagination and consequently tubule formation . It has been reported that Rac1 G12V - F37A mutant is defective in cortical actin generation 79 , 83 , 84 . Therefore , it is plausible that the different tubule inhibition response observed with both active mutants in this study ( F37A and W56A ) may be related to their different abilities to regulate actin polymerization . Figure 3 . PI ( 4 , 5 ) P 2 - induced tubulation is dynamin - dependent and its inhibition by active Rac1 involves PLC activity . ( a , b ) In the presence or absence of W13 ( 20 min , 4 . 5 \u00b5 g / ml ) , the percentage of cells with tubules was determined after dynasore treatment ( 30 min , 150 \u00b5 M ) or dynamin K44A expression ( 24 h ) ( a ) and in cells transfected with a specific dynamin siRNA or a non - targeting siRNA as a control ( 48 h ) ( b ) . Downregulation of dynamin expression by its specific siRNA is shown by western blotting . ( c ) The percentage of cells with tubules was determined in cells expressing Cherry - Mem alone or co - expressed with L10 - GFP Inp54p or L10 - GFP Inp54p D281A , in the presence or absence of W13 ( 20 min , 4 . 5 \u00b5 g / ml ) . ( d ) The percentage of cells with tubules was determined in 1 - hour starved cells expressing Cherry - Mem or Cherry - Rac1 G12V in the presence or absence of PI ( 4 , 5 ) P 2 ( 20 min , 50 \u00b5 M ) , W13 ( 20 min , 4 . 5 \u00b5 g / ml ) , or GFP - PIP5K expression . ( e , f ) The percentage of cells displaying tubules among the starved cells expressing GFP - mem , GFP - Rac1 G12V , or active Rac1 mutants ( F37A and W56A ) after the treatment with the PLC - inhibitor U73122 ( 20 min , 5 \u00b5 M ) ( e ) , W13 ( 20 min , 4 . 5 \u00b5 g / ml ) , or PIP5K overexpression ( f ) . Mean values \u00b1 SEM from three independent experiments are shown in all cases . Statistical significance between different conditions and the corresponding controls were determined by Student\u2019s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . www . nature . com / scientificreports / 7 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x To determine the effect of these mutants on actin polymerization , F - actin was detected in Vero cells express - ing the GFP - Rac1 G12V , GFP - Rac1 G12V - F37A , or GFP - Rac1 G12V - W56A using conjugated phalloidin - TRITC . Vero cells , which also showed W13 - induced PM tubulation 15 , were used instead of COS1 to improve the visualization of actin cytoskeleton . Fluorescence confocal images showed that GFP - Rac1 G12V and GFP - Rac1 G12V - W56A modified the F - actin pattern by severely reducing stress fibers and increasing cortical F - actin . The effect of G12V was stronger than the G12V - W56A mutant . By contrast , GFP - Rac1 G12V - F37A mutant did not affect the actin organiza - tion ( Fig . 4a ) . Figure 4 . Actin cytoskeleton is involved in Rac1 - dependent tubule inhibition . ( a ) Vero cells expressing GFP - Rac1 G12V , GFP - Rac1 G12V - F37A , or GFP - Rac1 G12V - W56A were grown on coverslips , and F - actin was visualized by confocal microscopy using phalloidin - TRITC ( 15 min , 0 . 04 U / ml ) ( bars , 10 \u00b5 m ) . ( b ) The percentage of COS1 cells presenting tubules was determined in starved cells expressing GFP - Rac1 G12V or GFP - Rac1 G12V - W56A and treated with the F - actin depolymerizing agent latrunculin A ( LatA ; 20 min , 200 nM ) in the presence or absence of W13 . ( c , d ) Percentage of cells with tubules expressing GFP - mem after pre - incubation with nocodazole ( 10 min , 30 \u00b5 M ) ( c ) or the dynein inhibitor EHNA ( 6 hours , 1 mM ) ( d ) and incubation with W13 for 20 min . Mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments are shown in all cases . Statistical significance between W13 - treatment and the corresponding control were determined by Student\u2019s t - test , * p < 0 . 05 , * * p < 0 . 01 . ( e ) In cells expressing Venus - Rac1wt and treated with W13 , tubulin was detected by immunofluorescence using a mouse anti - \u03b2 - tubulin antibody and a secondary anti - mouse - Alexa594 , and F - actin was detected using SiR actin ( SC006 ) . Representative confocal images and STED images from the selected areas are shown ( bars , 10 \u00b5 m ) . www . nature . com / scientificreports / 8 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x To establish a possible connection between the increased cortical F - actin and the tubule inhibition produced by the active Rac1 mutants ( Rac1 G12V and Rac1 G12V - W56A ) , actin filaments were disrupted using the depolym - erizing agent Latrunculin A ( LatA ) in W13 - treated cells . Actin depolymerization decreased the tubule forma - tion inhibition by Rac1 G12V and completely eliminated the inhibitory effect of Rac1 G12V - W56A ( Fig . 4b ) . These results indicate that inhibition of membrane tubulation by Rac1 depends on actin polymerization , and that actin cytoskeleton is not necessary for membrane invagination and elongation to proceed . Actually , consider - ing the critical role of microtubules ( MTs ) for the stabilization of W13 - induced tubules described previously 15 , and further analyzed here using the MT depolymerizing agent nocodazole ( Fig . 4c ) , and the recently described role of dyneins for the stabilization and elongation of PM tubular structures 16 , the general dynein inhibitor erythro - 9 - [ 3 - ( 2 - hydroxynonyl ) ] adenine ( EHNA ) impaired W13 - induced tubulation in COS1 cells ( Fig . 4d ) . In addition , \u03b2 - tubulin and F - actin staining in W13 - induced tubules cells expressing Venus - Rac1wt , showed some association of these tubules with MTs but not with F - actin ( Fig . 4e ) . Although only occasional coincidence of MTs with tubules was observed , STED and confocal microscopy images revealed highly similar pattern and direction - ality between both networks ( Figs 4e and S4a ) , which is consistent with the dependency of W13 - induced tubules on the integrity of MTs ( Fig . 4c ) . Accordingly , nocodazole also inhibited \u03b2 1 - intergrin internalization elicited by W13 treatment in cells presenting tubules ( Supplementary Fig . S4b ) . These results indicate a key role of dyneins and MTs in PI ( 4 , 5 ) P 2 - induced membrane elongation towards the cell center . In summary , actin cytoskeleton is unnecessary for tubule elongation ( operated by MTs and dyneins ) and Rac1 - driven actin polymerization is critical to inhibit basal and PI ( 4 , 5 ) P 2 - induced membrane invagination . However , the results obtained with LatA cannot rule out a role of actin polymerization in PM invagination scission . Rac1 G12V - W56A and Rac1 G12V - F37A have differential effects on cortical F - actin , which may explain the differences in tubule formation inhibition by each mutant . Indeed , it has been described that active Rac1 F37A is not able to translocate cortactin , an actin polymerizing protein , to the PM 79 . To address if Rac1 G12V - W56A translocates cort - actin to the PM to inhibit PI ( 4 , 5 ) P 2 - induced tubule formation , we analyzed the location of endogenous cortac - tin in Rac1 G12V , Rac1 G12V - F37A and Rac1 G12V - W56A expressing Vero cells by immunofluorescence ( Fig . 5a ) . These images showed that Rac1 G12V and Rac1 G12V - W56A mutants translocate cortactin to the cell periphery ( being again more clear in Rac1 G12V expressing cells ) , and Rac1 G12V - F37A does not . Additionally , the involvement of cortactin in Rac1 G12V - W56A - dependent tubule inhibition was examined by overexpression of a dominant negative mutant ( cort - actin \u0394 PHSH3 ) 85 . While expression of the wild type cortactin showed no effect , cortactin \u0394 PHSH3 expression restored W13 - induced tubules in Rac1 G12V - W56A expressing cells ( Fig . 5b ) . The same result was obtained by slencing cort - actin expression through siRNA transfection in cells expressing Rac1 G12V - W56A and treated with W13 ( Fig . 5c ) , demonstrating that Rac1 needs a functional cortactin to prevent PI ( 4 , 5 ) P 2 - induced tubulation . ROCK1 activity inhibits endocytic tubule formation downstream Rac1 . In vitro yeast two - hybrid experiments demonstrated that active Rac1 interacted with ROCK1 , whereas the active Rac1 F37A mutant was defective in such interaction 80 , though the functionality of this association has not been reported yet . ROCK1 is a Ser / Thr kinase that is activated after RhoA - GTP binding to its Rho - binding domain ( RBD ) due to the release of its autoinhibitory conformation 86 . To analyze the interaction between ROCK1 and the different Rac1 G12V mutants , we performed a pull - down assay incubating lysates from GFP - Rac1 G12V , GFP - Rac1 G12V - F37A , or GFP - Rac1 G12V - W56A expressing cells with GST - ROCK1 725 \u2013 1024 immobilized on Sepharose beads . This ROCK1 fragment contains the RBD and a N - terminal portion of its kinase domain that facilitates a proper conformation for RBD / Rho - GTP binding 87 . Western blot analysis showed that Rac1 G12V and Rac1 G12V - W56A were both pulled down by GST - ROCK1 725 \u2013 1024 , whereas Rac1 G12V - F37A did not ( Fig . 6a ) . Co - immunoprecipitation experiments showing interaction between Rac1 G12V - W56A and endogenous ROCK1 were performed as well , although , these results were not consistently reproduced probably due to weak and transient Rac1 - ROCK1 interaction and have not been included in this report . In addition , expression of Rac1 G12V induced the recruitment and co - localization of ROCK1 at the PM ( Fig . 6b ) . After binding to Rac1 - GTP , ROCK1 may be activated and become functional in PM domains where Rac1 is present . Supporting the hypothesis that ROCK1 is an effector involved in Rac1 - dependent tubule formation inhibi - tion , the specific ROCK1 inhibitor Y27632 impaired the inhibition of Rac1 G12V - W56A ( Fig . 6c ) . To further confirm ROCK1 involvement in tubule inhibition by the active Rac1 mutant , we silenced ROCK1 expression by siRNA in Rac1 G12V - W56A expressing COS1 cells . Downregulation of ROCK1 completely restored the tubules induced by W13 ( Fig . 6d ) . Although ROCK1 could inhibit tubulation by recruiting cortactin to the PM , this possibility was ruled out using Y27632 in Rac1 G12V - and Rac1 G12V - W56A - transfected cells . Cortactin translocation appears to be independent of ROCK1 activity ( Supplementary Fig . S3 ) , in agreement with others authors 79 , 84 , 88 , 89 . These results indicate that , in addition to cortactin translocation and actin polymerization at the PM , active Rac1impairs tubu - lation via ROCK1 activity . However , ROCK1 is a well - known RhoA effector 44 , 90 , and to date no functional relationship has been described with other GTPases . In order to exclude RhoA as the upstream activator of ROCK1 responsible for the inhibition of PI ( 4 , 5 ) P 2 - induced tubule formation , the outcome of RhoA activity on W13 - induced tubules was examined in COS1 cells by expressing the constitutively active ( GFP - RhoA G14V ) and inactive ( GFP - RhoA T19N ) RhoA mutants . The expression of GFP - RhoA T19N did not modify the percentage of cells presenting tubules neither in control nor W13 - treated cells ( Fig . 6e ) . In contrast , when we expressed GFP - RhoA G14V ( expected to activate ROCK1 ) , an important and significant increase in tubule - presenting cells was observed after W13 - treatment , instead of inhibition ( Fig . 6e ) . RhoA and Rac1 are mutual antagonists 44 , 91 , and the observed active RhoA - induced tubulation might be a consequence of endogenous Rac1 inhibition . Moreover , while GFP - Rac1 was present in W13 - induced tubules , GFP - RhoA G14V was almost absent ( Fig . 6f ) . Then , RhoA - induced ROCK1 activation takes www . nature . com / scientificreports / 9 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x place in different sites , precluding inhibition of tubule formation by RhoA activity . Together , these data suggest that Rac1 / ROCK1 , and not RhoA / ROCK1 , plays a key role in the inhibition of the endocytic tubule formation . Rac1 / ROCK1 - dependent actomyosin assembly inhibits tubulation . Although there is no reported or conclusive role for ROCK1 as an effector of active Rac1 , this protein controls actomyosin downstream of active RhoA . It is feasible , therefore , that by controlling myosin activation , Rac1 / ROCK1 interplay could stabilize actin polymerization at the specific sites where tubules should be induced . In turn , this may inhibit tubule formation by mechanical hindrance or by membrane tension increase . It is known that phosphorylation of myosin light chain protein ( MLC ) is critical for the interaction between myosin and F - actin , and hence for actomyosin generation 92 . Accordingly , MLC phosphatase ( MLCP ) dephosphorylates MLC and impairs actomyosin formation 93 . In fact , Figure 5 . Cortactin is involved in Rac1 - dependent tubule inhibition . ( a ) Vero cells grown on coverslips were transfected with GFP - Rac1 G12V , GFP - Rac1 G12V - F37A , or GFP - Rac1 G12V - W56A . After fixation , immunofluorescence was performed to detect endogenous cortactin using an antibody and the corresponding Alexa - 555 anti - mouse secondary antibody . Magnification insets show the presence or absence of cortactin at the PM ( bars , 10 \u00b5 m ) . ( b ) Percentage of COS1 cells presenting tubules , after W13 treatment , in cells transfected with cherry - Rac1 G12V - W56A alone or co - transfected with full length cortactin or with a cortactin mutant lacking the PH and SH3 domains , acting as a dominant negative . ( c ) The percentage of cells presenting more than 5 tubules per cell was quantified in untreated ( NT ) and W13 - treated cells co - transfected with GFP - Mem or GFP - Rac1 G12V - W56A and the non - specific ( NS ) or cortactin siRNAs ( 48 h ) . Downregulation of cortactin expression by its specific siRNA is shown by western blotting . The W13 - induced tubules and cortactin downregulation in GFP - Rac1 G12V - W56A and cortactin siRNA - transfected cells is shown by immunofluorescence as in ( a ) ( bars , 10 \u00b5 m ) . Mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments are shown in all cases . Statistical significance between different conditions was determined by Student\u2019s t - test , * p < 0 . 05 , * * p < 0 . 01 . www . nature . com / scientificreports / 10 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x phosphorylation of the MLCP regulatory subunit MYPT1 by ROCK1 results in its inhibition 94 \u2013 96 . Given that ROCK1 activity may promote actomyosin , we hypothesized that actomyosin induced via Rac1 / ROCK1 97 , could be responsible for tubule inhibition . To investigate this hypothesis , myosin IIA localization was analyzed by immunofluorescence in untreated or Y27632 - treated Vero cells expressing GFP - Rac1 G12V , GFP - Rac1 G12V - F37A , or GFP - Rac1 G12V - W56A . Figure 7 shows that while GFP - Rac1 G12V - F37A did not significantly affect myosin IIA localization ( Fig . 7a ) , expression of GFP - Rac1 G12V or GFP - Rac1 G12V - W56A inhibited myosin IIA stress - fiber localization and enhanced its presence at Figure 6 . Rac1 / ROCK1 pathway prevents tubulation without altering PM localization of cortactin . ( a ) Lysates of COS1 cells expressing GFP - Rac1 G12V , GFP - Rac1 G12V - F37A , GFP - Rac1 G12V - W56A or the empty GFP - C1 vector ( EV ) were incubated with immobilized GST - ROCK1 725 \u2013 1024 in glutathione Sepharose beads , as described in the Materials and Methods . GFP - Fusion proteins present in the input lysates and in the bound fraction were detected by western blotting , using an anti - GFP antibody . A representative western blotting is shown ( n = 3 ) . ( b ) Vero cells were co - transfected with cherry - Rac1 G12V and GFP - ROCK1 . Confocal image insets show regions with high co - localization . ( c , d ) COS1 cells were transfected with GFP - Rac1 G12V - W56A and treated with or without W13 ( 20 min , 4 . 5 \u00b5 g / ml ) . The percentage of cells presenting tubules was determined after the inhibition of ROCK1 activity with Y27632 ( c ) or after the inhibition of ROCK1 expression by transfection with a specific siRNA ( d ) . Downregulation of ROCK1 expression by its specific siRNA is shown by western blotting . ( e ) The percentage of cells presenting tubules was determined in untreated and W13 - treated COS1 cells expressing GFP - RhoA T19N , GFP - RhoA G14V , or GFP - mem as a control . Mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments are shown in all cases . Statistical significance between different conditions was determined by Student\u2019s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( f ) Vero cells co - transfected with Cherry - mem and GFP - Rac1 ( left panel ) or GFP - RhoA G14V ( right panel ) were treated with W13 . Images and insets show localization of GFP - Rac1 , but not of GFP - RhoA G14V , in the cherry - mem tubules . All images were acquired using the Leica TCS SP5 confocal microscope ( bars , 10 \u00b5 m ) . www . nature . com / scientificreports / 11 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x Figure 7 . ROCK1 participates in Rac1 induction of actomyosin , and myosin activity is required for Rac1 - mediated tubule inhibition . ( a , b , c ) By immunofluorescence , myosin IIA and F - actin were detected in starved Vero cells expressing GFP - Rac1 G12V - F37A ( a ) , GFP - Rac1 G12V ( b ) , or GFP - Rac1 G12V - W56A ( c ) after treatment with Y27632 ( 30 min , 25 \u00b5 M ) . The magnification insets show GFP - Rac1 , phalloidin - A555 , and myosin - IIA - A647 in transfected ( 1 ) and non - transfected cells ( 2 ) . In the cells expressing GFP - Rac1 G12V and GFP - Rac1 G12V - W56A , the images show the loss of stress fibers ( and consequently their staining with myosin II ) plus myosin recruitment to cortical F - actin ( arrow heads ) , which is reduced after treatment with Y27632 ( bars , 5 \u00b5 m ) . ( d ) The percentage of cells presenting tubules was determined in the presence or absence of the myosin inhibitor blebbistatin ( 30 min , 50 \u00b5 M ) . Mean values \u00b1 standard error of the mean ( SEM ) from three independent experiments is shown . Statistical significance between different conditions was determined by Student\u2019s t - test , * p < 0 . 05 . www . nature . com / scientificreports / 12 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x the cell periphery colocalizing with cortical actin ( Fig . 7b , c ) , being this effect more evident in GFP - Rac1 G12V than in GFP - Rac1 G12V - W56A expressing cells . Y27632 treatment inhibited the presence of myosin IIA in both stress fibers and cell periphery in all cells regardless whether they expressed the active Rac1 mutants ( Fig . 7 ) , consistent with the restitution of the W13 - induced tubules after Y27632 treatment in GFP - Rac1 G12V - W56A cells ( Fig . 6c ) . The participation of ROCK1 in myosin IIA localization at the leading edge of wound migrating cells has been previously demonstrated 98 and the results presented here further support the involvement of ROCK1 in Rac1 induction of actomyosin . Finally , to clarify the role of myosin activity in tubule inhibition by Rac1 , we pre - incubated cells expressing GFP - Rac1 G12V - W56A with the general myosin inhibitor blebbistatin before W13 - treatment . In this experiment , blebbistatin effectively restored the W13 - induced tubules in cells expressing GFP - Rac1 G12V - W56A ( Fig . 7d ) . In con - clusion , our data establishes ROCK1 , for the first time , as a novel downstream effector of Rac1 involved in the control of membrane dynamics via myosin regulation . Proposed model of the molecular machinery implicated in the dynamics of PI ( 4 , 5 ) P 2 - induced endocytic tubulation . Taken together , these data support the model summarized in Fig . 8 . The regula - tion of PI ( 4 , 5 ) P 2 levels in cholesterol rich PM ordered domains is crucial for membrane invagination , elonga - tion and fission , enabling the correct progression of CIE ( used for \u03b2 1 - integrin internalization ) . When PI ( 4 , 5 ) Figure 8 . Molecular machinery implicated in the biogenesis and inhibition of PI ( 4 , 5 ) P 2 - induced endocytic tubulation . An increase of PI ( 4 , 5 ) P 2 in PM ordered domains could induce the recruitment of several PI ( 4 , 5 ) P 2 - binding proteins to generate an incipient membrane deformation ( 1 ) . When Rac1 activity is low ( 2 ) , the invagination can be elongated by dyneins toward the center of the cell along microtubules . PI ( 4 , 5 ) P 2 accumulation , as well as the high degree of membrane curvature , could lead to the recruitment of dynamin or BAR - domain proteins , which in turn , could propagate and stabilize the tubule . By contrast , when Rac1 activity is high , tubulation process could be inhibited by either PLC activation ( reducing PIP 2 levels ) or cytoskeleton regulation ( inducing cortical actomyosin at the PM ) ( 3 ) . Rac1 appears to stimulate cortactin PM translocation and ROCK1 activity , thereby triggering cortical actin polymerization and association with myosin ( i . e . , actomyosin ) . The resulting over - activation of local actomyosin networks could potentially impede tubulation in one of two ways : ( i ) by forming a local barrier to increasing PM tension or by causing a steric hindrance that impedes the recruitment of tubulating proteins ( 4 ) ; or ( ii ) by generating mechanical forces needed to pinch off membrane invaginations more efficiently ( 5 ) . www . nature . com / scientificreports / 13 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x P 2 levels increase due to CaM inhibition , PIP5K overexpression , exogenous diC8 - PI ( 4 , 5 ) P 2 administration or Rac1 inhibition , a long tubular plasma membrane network is formed ( Fig . 8 , points 1 and 2 ) . This membrane process requires dynamin , dynein and MTs ( point 2 ) . The results presented above demonstrated that activation of Rac1 ( overexpression of Rac1 G12V ) inhibits the PI ( 4 , 5 ) P 2 - dependent tubular PM network formation by two main molecular mechanisms : [ i ] reducing PI ( 4 , 5 ) P 2 levels through PLC activation ; and [ ii ] inducing cortical F - actin mesh ( via cortactin ) and actomyosin ( via ROCK1 ) formation . Rac1 - mediated cortactin recruitment is insufficient for tubule inhibition , and requires actomyosin formation ( myosin activation ) . Rac1 - induced actomyosin forma - tion prevents PI ( 4 , 5 ) P 2 - dependent tubule establishment either by an actively actin - dependent tubule scission process ( Fig . 8 , point 5 ) or by generating a cortical actomyosin network that produces a local mechanical barrier or increases PM tension to impede membrane invagination ( Fig . 8 , point 4 ) . For the first time , we identify ROCK1 as a novel downstream effector of Rac1 acting in a RhoA - independent manner to regulate membrane dynamics during a tubular CIE . Both Rac1 and RhoA GTPases stimulate acto - myosin formation , but at different times and locations within the cell , and it is possible that both proteins share or compete for ROCK1 . These results suggest that Rac1 activation at the leading edge of migrating cells may be important to stabilize \u03b2 1 - integrin in the newly generated adhesions , preventing its internalization and turnover , and therefore facilitating cell movement as a result . Material and Methods Reagents and Antibodies . W13 hydrochloride , U73122 and latrunculin A were from Calbiochem ( Merck Millipore ) . Y27632 , blebbistatin , neomycin and dynasore were from Sigma - Aldrich . DiC8 - PI ( 4 , 5 ) P 2 was from Echelon Biosciences . Primary antibodies used were as follows : rabbit polyclonal anti - GFP and mouse anti - actin ( Abcam ) , mouse monoclonal anti - cortactin ( Upstate ) , rabbit polyclonal anti - PACSIN2 ( Abgent ) , rat monoclonal anti - \u03b2 1 - Integrin ( AIIB2 ) ( Damsky , C . H . , Developmental Studies Hybridoma Bank ) , mouse monoclonal anti - PI ( 4 , 5 ) P 2 ( Echelon Biosciences ) , mouse monoclonal anti - early endosomal antigen1 ( EEA1 ) ( BD Transduction Laboratories ) , rabbit polyclonal anti - Myosin Heavy Chain IIA ( Biolegend Inc . ) , rab - bit polyclonal anti - Clathrin heavy chain ( CHC ) antibody ( # PA5 - 25804 ) and transferrin - Alexa546 ( # T23364 ) from ThermoFisher Scientific , and mouse monoclonal anti - CHC antibody clone \u00d7 22 ( # MA1 - 065 , Affinity BioReagents ) . Phalloidin conjugated with TRITC or Alexa - 350 , secondary Alexa - labeled antibodies and ProtA - HRP were from Molecular Probes ( Invitrogen - Life Technologies ) . SiR actin ( SC006 ) was from Spirochrome . Secondary HRP - labeled antibodies , SDS \u2013 polyacrylamide gel electrophoresis ( PAGE ) and molecular weight markers were from Bio - Rad . Glutathione - Sepharose beads were purchased from GE Healthcare . Human ROCK1 ( 4390824 ) siRNA was from Ambion , Human dynamin ( s12097 ) siRNA was from Santa Cruz Biotechnologies , Human cortactin ( CTTN , GS2017 ) and Human Clathrin heavy chain ( CLTC , GS1213 ) Flexitube Gene Solution siRNAs were from Qiagen . Cell culture . African green monkey kidney fibroblast COS1 or Vero cells were grown in Dulbecco\u2019s modified Eagle\u2019s medium ( DMEM ) supplemented with 5 % ( v / v ) or 10 % fetal calf serum ( FCS ) respectively , pyruvic acid , antibiotics and glutamine . DMEM and FCS were purchased from Biological Industries . Plasmids and transfection . Plasmid encoding the constitutively active Rac1 mutant ( Rac1 G12V ) was kindly pro - vided by Dr Michiyuki Matsuda ( University of Kyoto ) 99 and subcloned into living color vectors ( Clontech ) . Rac1 F37A point mutation was introduced into pEGFPC1 - Rac1 G12V by polymerase chain reaction ( PCR ) with the following primers : 5 \u2032 - CAGATTCACCGGTTTTCCATCTACCATAACATTGGCAGAATAATTGTCAGCGACAGTAGGG - 3 \u2032 and 5 \u2032 - GGACGAGCTGTACAAGTCCCTCATGCAGGCCATCAAGTG - 3 \u2032 , using BsrGI and BstXI restriction sites ; Rac1 W56A point mutation was introduced into pEGFPC1 - Rac1 G12V by polymerase chain reaction ( PCR ) with the following primers : 5 \u2032 - CAATTATTCTGCCAATGTTATGGTAGATGGAAAGCCAGTGAATCTGGGCTTAGCG GATACAGCTGG - 3 \u2032 and 5 \u2032 - CAGTCACGATGAATTCTTACAACAGCAGGC - 3 \u2032 , using BstXI and EcoRI restric - tion sites . Both mutants were subcloned into mCherry vector ( Clontech ) . GFP - and Cherry - mem are fusion proteins that contain the N - terminal amino acids of GAP - 43 and a GFP and mCherry fluorescent protein , respec - tively . The GAP - 43 fragment contains a signal for post - translational palmitoylation of cysteines 3 and 4 that tar - gets fusion protein to cellular membranes . Plasmid encoding dominant negative dynamin ( dynamin - K44A ) was obtained from ATCC ( MBA - 93 ) . L10 - GFP - Inp54p and L10 - GFP - Inp54p D281A were cloned from plasmids pro - vided by Dr Tobias Meyer through Addgene ( # 20155 and # 20156 respectively ) 100 . Firstly , a peptide containing the N - terminal 10 residues of Lck ( L10 ) was fused to the N - terminal GFP encoding sequence ( L10 - EGFP - C1 ) ; then , vectors from addgene were digested with EcoRI and BamHI and inserted into L10 - EGFP - C1 vector . Cortactin - WT and Cortactin - \u0394 PHSH3 were kindly provided by Dr . Thomas Parsons . Plasmid encoding GST - ROCK1 725 \u2013 1024 was generated by PCR using the primers 5 \u2032 - GACCGGTGGATCCCGGGCTGTATTAGCTTTCTTTCTATC - 3 \u2032 and 5 \u2032 - CACATGGTCCTGCTGGAGTTCGTG - 3 \u2032 with pECFP - ROCK1 , kindly provided by Dr Gareth Jones 101 , as a tem - plate . The resulting PCR product was introduced into CFP - N1 vector using XhoI and XmaI restriction sites , and then it was subcloned into pGEX - 4T - 2 for the expression in bacteria using BamHI and XmaI restriction sites . GFP - RhoA constitutively active ( G14V ) and dominant negative ( T19N ) were kindly provided by Michael Way ( Cancer Research UK , London , UK ) . COS1 and Vero cells were transfected with DNA using Effectene ( QIAGEN ) or GenJet ( Signagen ) , and transfected with combined DNA and siRNA transfection using RNAiMAX ( Invitrogen - Life Technologies ) . Cells were used for experiments 24 h after DNA transient expression or 48 \u2013 72 h after siRNA transfection . Immunofluorescence staining . Cells grown on coverslips were fixed with freshly prepared 4 % paraform - aldehyde ( PFA ) in cytoskeleton buffer ( CB ; 10 mM MES pH6 . 1 , 138 mM KCl , 3 mM MgCl2 , 2 mM EGTA ) at 37 \u00b0C for 15 min and permeabilized with 0 . 5 % Triton X - 100 in CB at room temperature for 3 min . After 5 - min incubation with blocking solution ( TBST , 2 % BSA ) , coverslips were incubated with the primary antibody diluted www . nature . com / scientificreports / 14 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x in blocking solution for 50 min at room temperature , washed intensively and then incubated with the adequate secondary antibodies labeled with Alexa488 , Alexa555 or Alexa647 . After staining , the coverslips were mounted in Mowiol ( Calbiochem , Merck ) . The images were acquired using a Leica TCS SP5 laser scanning confocal microscope ( Leica Microsystems Heidelberg GmbH ) equipped with DMI6000 inverted microscope , Argon ( 458 / 476 / 488 / 514 ) , diode pumped solid state ( 561 nm ) and HeNe ( 633 ) lasers . GFP , TRITC or Alexa - 555 and Alexa - 647 images were acquired sequentially using 488 , 561 and 633 laser lines , acoustic optical beam splitter ( AOBS ) as beam splitter , and emission detection ranges 500 \u2013 555 , 571 \u2013 625 and 640 \u2013 700 nm , respectively . STED confocal images were acquired using a Leica TCS SP8 . Final analysis of all images was performed using IMAGEJ software . \u03b2 1 - integrin internalization analysis . COS1 cells grown on coverslips were tempered to 4 \u00b0C to defuse endocytosis and then were incubated with anti - \u03b2 1 - integrin antibody and transferrin - TRITC for 30 min at 4 \u00b0C . After washing the unbound antibody and transferrin excess with PBS , cells were incubated at 37 \u00b0C for 5 , 10 and 20 min under the corresponding treatment . Cells were washed twice with PBS at 4 \u00b0C and then were subjected to a surface acid wash ( 0 . 5 % glacial acetic acid , 0 . 5 M NaCl , pH 3 . 0 ) at 4 \u00b0C for 2 min . After fixation with freshly prepared 4 % PFA at 37 \u00b0C for 15 min , immunostaining of the antigen - antibody complexes was performed as described above . Images were acquired along the Z - axis , in order to cover the whole cell , using a Leica TCS SP5 laser scanning confocal microscope ( Leica Microsystems Heidelberg GmbH ) equipped with DMI6000 inverted microscope . To determine the amount of internalized \u03b2 1 - integrin and transferrin , fluorescence intensity was normalized per cell area . Pull - down assay . 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Spatiotemporal activation of Rac1 for engulfment of apoptotic cells . Proceedings of the National Academy of Sciences of the United States of America 105 , 9198 \u2013 9203 , doi : 10 . 1073 / pnas . 0803677105 ( 2008 ) . 100 . Suh , B . C . , Inoue , T . , Meyer , T . & Hille , B . Rapid chemically induced changes of PtdIns ( 4 , 5 ) P2 gate KCNQ ion channels . Science 314 , 1454 \u2013 1457 , doi : 10 . 1126 / science . 1131163 ( 2006 ) . 101 . Shea , K . F . , Wells , C . M . , Garner , A . P . & Jones , G . E . ROCK1 and LIMK2 interact in spread but not blebbing cancer cells . PloS one 3 , e3398 , doi : 10 . 1371 / journal . pone . 0003398 ( 2008 ) . Acknowledgements This research was supported by grants BFU2012 \u2013 38259 and BFU2015 \u2013 66785 - P from Ministerio de Economia y Competitividad of Spain to F . T . ; Consolider - Ingenio from Ministerio de Innovaci\u00f3n , Ciencia y Tecnolog\u00eda of Spain to C . E . and A . P . ; BFU2011 \u2013 23745 to A . P . ( MICINN ) . D . S - C . and A . C . were recipient of FI fellowship www . nature . com / scientificreports / 17 SCIEntIFIC REPORts | 7 : 6866 | DOI : 10 . 1038 / s41598 - 017 - 07130 - x ( Generalitat de Catalunya ) . We thank Maria Calvo , Anna Bosch and Elisenda Coll for assistance in the confocal imaging ( Unitat Microscopia \u00d2ptica Avan\u00e7ada , Centres Cient\u00edfics i Tecnol\u00f2gics , Universitat de Barcelona ) ; Timo Zimmermann , Raquel Garc\u00eda , Xavier Sanjuan and Arrate Mallabiabarrena for assistance in STED microscopy ( Advanced Light Microscopy Unit at the Centre for Genomic Regulation ( CRG ) in Barcelona ) and Maria Molinos for technical assistance . We are also grateful to Salvador Soriano for their comments and critical reading of the manuscript . Author Contributions D . S . - C . , A . C . and M . V . - Q . conducted most experiments and analyzed data . C . R . and M . B . assisted with experimental design , analyses and interpretation data . A . P . and C . E . helped with reagents , equipment , and discussion of results . D . S . - C . and F . T . wrote the manuscript . All authors contributed to critical revising the manuscript . Additional Information Supplementary information accompanies this paper at doi : 10 . 1038 / s41598 - 017 - 07130 - x Competing Interests : The authors declare that they have no competing interests . Publisher ' s note : Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Cre - ative Commons license , and indicate if changes were made . The images or other third party material in this article are included in the article\u2019s Creative Commons license , unless indicated otherwise in a credit line to the material . If material is not included in the article\u2019s Creative Commons license and your intended use is not per - mitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / . \u00a9 The Author ( s ) 2017", + "jin2022branched_supp": "1 Supplementary information Branched actin networks are organized for asymmetric force production during clathrin - mediated endocytosis in mammalian cells Meiyan Jin 1 , Cyna Shirazinejad 1 , 2 , Bowen Wang 3 , Amy Yan 1 , Johannes Sch\u00f6neberg 1 , 4 , Srigokul Upadhyayula 1 , Ke Xu 3 , David G . Drubin 1 * 1 Department of Molecular and Cell Biology , University of California , Berkeley , CA 94720 2 Biophysics Graduate Group , University of California Berkeley ; Berkeley , CA , 94720 3 Department of Chemistry , University of California , Berkeley , CA 94720 4 Current address : Department of Pharmacology , and Department of Chemistry and Biochemistry , University of California , San Diego , CA 92093 These authors contributed equally : Meiyan Jin , Cyna Shirazinejad Correspondence to DGD ( drubin @ berkeley . edu ) 2 Supplementary Fig . 1 : Genome - edited iPSCs show dynamic CME sites . a Schematic model of CME . Mammalian CME proteins can be grouped into several modules , including the coat , WASP and Myosin / actin nucleation promoting factor ( NPF ) , actin and scission modules . Actin networks provide pulling forces to invaginate the membrane against membrane tension . b Immunoblot analysis of cell extracts from the WT ( WTC ) and genome - edited ( AP2M1 - tagRFP - T / DNM2 - tagGFP2 / ARPC3 - HaloTag ; ADA ) human iPSCs . The labeled proteins were detected with tag ( CGY ) FP , HaloTag , and GAPDH ( loading control ) antisera respectively . N = 1 . Uncropped and unprocessed scans of the blots are provided in the Source Data file . c Kymograph of representative CME sites of double - edited ( AP2M1 - tagRFP - 3 T / DNM2 - tagGFP2 ; AD ) and triple - edited ( AP2M1 - tagRFP - T / DNM2 - tagGFP2 / ARPC3 - HaloTag ; ADA ) cells . Scale bar : 5\u00b5m . 4 Supplementary Fig . 2 : Actin assembles at different types of CME sites . a Montage of a representative ARPC3 positive CME plaque from a TIRF movie of triple - edited ( AP2M1 - tagRFP - T / DNM2 - tagGFP2 / ARPC3 - HaloTag ; ADA ) human iPSCs ( Supplementary Movie 2 ) . b Montage of a representative ARPC3 positive splitting CME site from a TIRF movie of triple - edited ( AP2M1 - tagRFP - T / DNM2 - tagGFP2 / ARPC3 - HaloTag ; ADA ) human iPSCs ( Supplementary Movie 2 ) . 5 6 Supplementary Fig . 3 : Filtering methods for selection of CME sites . a 2 - D histogram of the first two principal components ( PCs ) of AP2 and DNM2 dynamic features . Of the 121 , 349 total tracks detected by CMEanalysis , ~ 49 % were valids ( as defined previously 30 ) , ~ 31 % had detection gaps , ~ 15 % were persistent events , and ~ 5 % were split / merges . Only valid tracks ( N = 59 , 239 ) , which appear and disappear during the course of movie acquisition , were used to generate filtering methods and for subsequent analysis . The shaded underlay represents simulated data points in principal component space and their individual probabilities of belonging to the nearest cluster center . Cluster 0 shows data points in the DNM2 - positive cluster , which contains 10 . 02 % of total valid tracks . Clusters 1 - 4 represented 24 . 60 % , 10 . 43 % , 17 . 03 % , and 37 . 91 % of valid tracks , respectively . b Cohort plots of the shortest AP2 events ( < 40 seconds ) from each cluster . Cluster 0 represents DNM2 - positive events where a strong DNM2 signal is detected . Data are presented as mean values + / - 1 / 4 standard deviation . c DNM2 - positive events are sorted by the number of DNM2 peaks using a peak - detection scheme . Representative intensity vs time plots of a single - peaked event ( top ) , non - peak event ( bottom left ) and a multi - peaked event ( bottom right ) . Percentage of the number of the events in each class is shown next to the plot . d Single - peaked DNM2 events ( N = 2 , 538 ) , hereon named CME sites , are grouped into lifetime cohorts and aligned to the peak of the DNM2 channel . Percentage of the number of the CME sites in each cohort is shown next to the plot . Data are presented as mean values + / - 1 / 4 standard deviation . 7 8 Supplementary Fig . 4 : Computational analysis reveals actin network assembly at the late stage of CME . a We generated a randomized data set by pairing ARPC3 images with AP2 and DNM2 images from an unrelated movie . b The fraction of CME sites without actin assembly ( Negative ) , with early actin assembly ( ARPC3 signal disappears before DNM2 signal peak ) , and with late actin assembly ( ARPC3 signal overlaps with DNM2 signal peak ) were calculated for real data movies and randomized control movies ( a ) . In the randomized dataset , we detected early \u201cassembly\u201d of actin in a similar fraction of CME events as in the real data set , indicating that presence of actin detected early in CME is very likely an artificial association caused when nearby actin structures overlap with CME sites by chance . Mean and standard deviation are shown on the graph . Source data are provided in the Source Data file . 9 10 Supplementary Fig . 5 : AP2 - ARPC3 separation is not due to imaging artifacts . a Montage from a TIRF movie of a multi - fluorescence bead ( Supplementary Movie 4 ) . Size of field of view : 2\u00b5m x 2\u00b5m . Intervals : 1sec . b A boxplot of inter - channel distances ( note : y - axis is base - 10 log scale ) between centroids of either CME sites or beads to highlight that the observed separation between marked proteins exceeds the measured chromatic aberration . Beads were compared between the same pairs of channels as used to image the tagged proteins . Box plot elements : center line , median ; box limits , upper and lower quartiles ; whiskers , 1 . 5x interquartile range ; points , outliers . Source data are provided in the Source Data file . c A heat map graph of distance between AP2 and ARPC3 signals before scission , and average AP2 frame to frame displacement within 6 seconds before scission . Over 95 % of the CME events present larger AP2 - ARPC3 separation than AP2 displacement . N = 1 , 385 . 11 Supplementary Fig . 6 : Arp2 / 3 - mediated actin assembly facilitates CME . a Intensity vs time plots of averaged ARPC3 negative ( top ) and positive ( bottom ) CME sites in ADA cells after 1 - 5min treatment with 2 % DMSO ( solid lines ) or 100 \u03bcM CK666 ( dashed lines ) . Events were aligned to the frames showing the maximum DNM2 intensity . Error bar : \u00bc standard deviation . ARPC negative CME sites : DMSO : N = 402 , CK666 : N = 261 , ARPC3 positive CME sites : DMSO : N = 1 , 006 , CK666 : N = 580 . b CK666 - treated cells have longer CME lifetimes than DMSO - treated control cells . p - value from two - sided Kolmogorov - Smirnov test : 1 . 83e - 3 . Source data are provided in the Source Data file . 12 Supplementary Fig . 7 : Dynamics of N - WASP at CME sites . a Immunoblot analysis of cell extracts from the control and genome - edited ( AP2M1 - tagRFP - T / DNM2 - tagGFP2 / HaloTag - WASL ; ADW ) human iPSCs . The labeled proteins were detected with HaloTag and GAPDH ( loading control ) antisera respectively . N = 1 . Uncropped and unprocessed scans of the blots are provided in the Source Data file . b Histogram of N - WASP lifetime at CME sites . The lifetime is measured from the first frame of the N - WASP signal to the presumed scission time ( the peak of DNM2 signal ) . c Intensity vs time plots of cohorts of N - WASP positive CME sites in ADW cells . Events are grouped into cohorts by the lifetimes of AP2 and aligned to the frames showing the maximum DNM2 intensity . Data are presented as mean values + / - 1 / 4 standard deviation . N = 1 , 199 . Source data are provided in the Source Data file .", + "numasawa2020fluorescent_supp": "Supporting Information A Fluorescent Probe for Rapid , High - Contrast Visualization of Folate - Receptor - Expressing Tumors In Vivo Koji Numasawa , Kenjiro Hanaoka , * Naoko Saito , Yoshifumi Yamaguchi , Takayuki Ikeno , Honami Echizen , Masahiro Yasunaga , Toru Komatsu , Tasuku Ueno , Masayuki Miura , Tetsuo Nagano , and Yasuteru Urano * anie _ 201914826 _ sm _ miscellaneous _ information . pdf S2 Materials and general information Reagents and solvents were of the best grade available , supplied by Tokyo Chemical Industries , Co . , Ltd . , Wako Pure Chemical Industries , Ltd . , Sigma - Aldrich Co . , LLC . , Kanto Chemical Co . , Inc . , Watanabe Chemical Industries Ltd . , Carbosynth Ltd . , Novabiochem or Thermo Fisher Scientific Inc . , and were used without further purification . Silica gel column chromatography was performed on Silica gel 60 ( Spherical ) ( Kanto Chmical Co . ) . Saline was purchased from Otsuka Pharmaceutical Co . Ltd . . Mice ( BALB / cAJcl - nu / nu ) were purchased from CLEA Japan . 1 H NMR and 13 C NMR spectra were recorded on a JEOL INM - LA300 ( 300 MHz ) , JNM - LA400 or ECZ - 400S instrument ( 400 MHz ) ; \u03b4 values are in ppm relative to tetramethylsilane ( TMS ) . Mass spectra ( MS ) were measured with a JEOL JMS - T100LC AccuTOF ( ESI ) . UV - vis spectra were obtained on a UV - 2550 ( Shimadzu ) . Fluorescence spectroscopic studies were performed with a Hitachi F - 7000 ( Tokyo , Japan ) ; the slit widths were 2 . 5 nm for excitation and 2 . 5 nm for emission , and the photomultiplier voltage was 700 V . Fluorescence quantum yields of SiR - based probes were measured with a Quantaurus - QY ( Hamamatsu ) . HPLC analysis was performed on an Inertsil ODS - 3 column ( GL Science Inc . ; 4 . 6 mm \u00d7 250 mm ) using a system composed of a pump ( PU - 980 , JASCO ) and a detector ( MD - 2015 , JASCO ) . HPLC purifications were performed on an Inertsil ODS - 3 column ( GL Science Inc . , 10 . 0 mm\u00d7 250 nm ) using a system composed of a pump ( PU - 2080 , JASCO ) and a detector ( MD - 2015 , JASCO ) . MPLC purifications were performed on a Yamazen Smart Flash EPCLC AI - 5805 ( Tokyo , Japan ) . UV - Vis Absorption and Fluorescence Spectroscopy UV - Visible spectra were obtained on a Shimadzu UV - 2550 . Fluorescence spectroscopic studies were performed with a Hitachi F7000 with a slit width of 2 . 5 nm for excitation and emission . The photomultiplier voltage was 700 V . Absolute quantum yields ( \u03a6 ) were measured with a Hamamatsu Photonics Quantaurus QY . Relative fluorescence quantum yields were obtained by comparing the area under the emission spectra of the test and standard samples according to the following equation . \u03a6 x / \u03a6 st = [ A st / A x ] [ n x2 / n st2 ] [ D x / D st ] where st : standard ; x : sample ; A : absorbance at the excitation wavelength ; n : refractive index ; D : area under the fluorescence spectra on an energy scale . S3 Preparation of Cultured Cells Cells of the human oral epidermoid carcinoma cell line KB ( a subline of HeLa ) were cultured in Eagle ' s minimal essential medium ( EMEM ) ( FUJIFILM Wako Pure Chemical Corp . , Japan ) , supplemented with 10 % ( v / v ) fetal bovine serum ( Invitrogen ) and penicillin ( 100 units / mL ) - streptomycin ( 100 \u03bcg / mL ) mixture ( Invitrogen ) . Epithelial ovarian cancer cell line OVCAR - 3 cells ( ATCC , USA ) were cultured in RPMI 1640 medium ( Invitrogen ) , supplemented with 20 % ( v / v ) fetal bovine serum ( Invitrogen ) and penicillin ( 100 units / mL ) - streptomycin ( 100 \u03bcg / mL ) ( Invitrogen ) . Human fibrosarcoma cell line HT1080 cells ( Health Science Research Resource Bank , Osaka , Japan ) were cultured in Dulbecco\u2019s modified Eagle\u2019s medium ( DMEM ) ( Invitrogen ) , supplemented with 10 % ( v / v ) fetal bovine serum ( Invitrogen ) and penicillin ( 100 units / mL ) - streptomycin ( 100 \u03bcg / mL ) ( Invitrogen ) . All cell lines were cultured at 37\u00b0C in a humidified incubator containing 5 % CO 2 in air . Confocal Fluorescence Imaging of Cultured Cells A confocal imaging system ( TCS - SP5 ; Leica ) equipped with a white - light laser was used . Fluorescence images were captured with excitation and emission wavelengths of 490 and 510 - 540 nm for fluorescein folate , Alexa 488 folate and Alexa 488 - conjugated IgG , 555 and 575 - 600 nm for TAMRA folate , and 650 and 670 - 750 nm for FolateSiR - 1 and FolateSiR - 2 . Immunofluorescence Staining of Cultured Cells KB cells , OVCAR - 3 cells and HT1080 cells were fixed with 4 % formaldehyde in phosphate - buffered saline ( PBS ) solution for 20 min . The medium was removed and the cells were washed with PBS three times , then incubated for 1 hr in PBS containing 0 . 1 % Triton X - 100 and 1 % bovine serum albumin ( BSA ) for blocking . Immunostaining was carried out for 1 h at room temperature with anti - folate binding protein antibody ( Abcam , Inc . ) at 1 : 100 dilution in PBS containing 0 . 1 % Triton X - 100 and 1 % BSA . Immunodetection was performed using Alexa Fluor \u00ae 488 - conjugated AffiniPure goat anti - mouse IgG ( Jackson Immuno Research Laboratories , Inc . ) at 1 : 500 dilution in PBS containing 0 . 1 % Triton X - 100 and 1 % BSA . Fluorescence images were captured with TCS - SP5 confocal S4 laser scanning microscope ( Leica ) . Fluorescence Imaging of Cultured KB Cells Cells were plated onto a 35 - mm poly - L - lysine - coated glass - bottomed dish ( Matsunami Glass Ind . , Ltd . ) in EMEM supplemented with 10 % ( v / v ) fetal bovine serum , 1 % penicillin and 1 % streptomycin . Before loading dyes , the medium was removed and the cells were washed with Hank ' s balanced salt solution ( HBSS ) three times . The cells were incubated with 5 \u03bcM folate - conjugated fluorescent dye in EMEM containing 0 . 3 % DMSO as a cosolvent at 37\u00b0C for 30 min , then the medium was removed and the cells were washed with HBSS three times . The cells were observed in HBSS . When cells were fixed with 4 % formaldehyde in PBS , the dish was kept on ice for 15 min . The medium was warmed to 37\u00b0C before all procedures . Fluorescence Imaging of Cultured OVCAR - 3 Cells Cells were plated onto a 35 - mm poly - L - lysine - coated glass - bottomed dish ( Matsunami Glass Ind . , Ltd . ) in RPMI 1640 medium supplemented with 20 % ( v / v ) fetal bovine serum , 1 % penicillin and 1 % streptomycin . Before dye loading , the medium was removed and the cells were washed with HBSS three times . The cells were incubated with 5 \u03bcM folate - conjugated fluorescent dye in RPMI 1640 containing 0 . 3 % DMSO as a cosolvent at 37\u00b0C for 30 min , then the medium was removed and the cells were washed with HBSS three times . The medium was warmed to 37\u00b0C before all procedures . Ex vivo Fluorescence Imaging of Mouse Embryos All procedures were approved by the Animal Care and Use Committee of the University of Tokyo . Imaging of embryos was done as described previously with slight modifications . SR1 Briefly , before the embryos were dissected , all media and dishes were prewarmed to 37\u00b0C . Then , about 300 \u03bcL of 2 % low - melting agarose was poured into a glass - bottomed dish ( thickness of 100 \u03bcm ; Matsunami Glass Ind . , Ltd . ) , and holes for receiving embryos were made at least 30 min before filling the dish with medium . Pregnant mice were killed at 8 . 5 d postcoitum by cervical dislocation , and the uterus was removed and placed in custom - made DMEM without phenol red or folic acid . S5 Embryos in their yolk sac were removed from the uterus and transferred into folic acid - free DMEM containing 10 % fetal bovine serum , and the yolk sac and amnion were carefully removed while the embryo was kept warm . The dissected embryos were cultured with FolateSiR - 1 ( 10 \u03bcM or 20 \u03bcM ) or FolateSiR - 2 ( 20 \u03bcM ) in DMEM without folic acid or in DMEM containing 1 mM folic acid for the competitive assay for 30 minutes in a humidified incubator at 37\u00b0C under 5 % CO 2 , and then washed twice with DMEM without folic acid . For live - imaging analysis , the embryos were immediately transferred to agarose - containing glass - bottomed dishes filled with folic acid - free DMEM containing 50 % centrifuged rat serum ( Charles River ) . Observation was done with an inverted confocal microscope ( TCS - SP5 ; Leica ) equipped with a galvo stage and a resonant scanner for fast scanning , using an HCX PL FLUOTAR 10\u00d7 0 . 3 NA dry objective ( Leica ) . During imaging , the dishes were kept in a humidified cell culture incubator with a continuous supply of 5 % CO 2 / air at 37\u00b0C ( Tokai Hit Company ) . For static observation , stained embryos were fixed with 4 % paraformaldehyde on ice for 10 min immediately after culture , then transferred to HBSS and observed . During imaging , the embryos were placed in holes in the agarose as described above . FolateSiR - 1 or FolateSiR - 2 was excited with a 633 - nm laser ( 20 \u2013 40 % power ) with the resonant scanner ( 8 , 000 Hz ; Leica ) , and the emission and bright - field signals were detected with photomultiplier tubes . The thickness of the z slices was 4 \u03bcm , and acquired images were processed to create maximum intensity projections . In Vivo Fluorescence Imaging of Mouse Models All procedures were approved by the Animal Care and Use Committee of the University of Tokyo . Tumor model mice were prepared as follows . BALB / cAJcl mice ( 7 - 8 weeks , \u2640 ) were inoculated with 3 - 5\u00d710 6 KB cells or 1\u00d710 7 HT1080 cells into the root of the forefoot at 7 - 10 days before fluorescence imaging . After intravenous injection into the tail of 100 \u03bcM FolateSiR - 1 or FolateSiR - 2 in 100 \u03bcL saline containing 1 % DMSO as a cosolvent , bright - field and fluorescence images were captured at different time points with a Maestro In - vivo Imaging System ( CRi Inc . , Woburn , MA ) equipped with an excitation filter 661 ( 641 - 681 nm ) , emission filter 704 ( 684 - 729 nm ) , using a bright - field setting . The tumor - bearing mice were carried back from the in vivo imaging system to the cage after the fluorescence image was captured at each time point . S6 Tissue Microarray - based Binding Analysis Frozen tissue array slides of ovary tumors ( Catalog No . : T6235183 - 5 , Lot No . : B705061 ) were purchased from BioChain Institute , Inc . ( Newark , CA , USA ) . For immunostaining folate receptors , the procedure was as follows . The slides were fixed with 4 % paraformaldehyde ( Wako ) at room temperature for 10 min , and washed with PBS containing 0 . 05 % Tween 20 ( PBST ) for 3 min three times . Endogenous peroxidases were blocked in 3 % H 2 O 2 for 20 min , and the slides were washed with PBST for 3 min three times . The slides were then blocked with 5 % skim milk ( Becton Dickinson ) in PBST at room temperature for 30 min , and incubated with mouse anti - folate binding protein antibody ( Abcam ) in PBST with 5 % skim milk at room temperature for 1 h . Then , the slides were washed with PBST three times , and treated with EnVision + System - HRP Labeled Polymer Anti - mouse ( Dako ) . DAB staining was performed at room temperature for 5 min with DAB chromogen substrate ( K3468 , Dako ) . The slide was dipped in water for 3 min , then subjected to hematoxylin staining at room temperature for 5 min , incubated again in water for 10 min , and washed with ethanol ( 2 min , 2 min and 3 min ) and xylene ( 3 min , 3 min and 3 min ) . MOUNT - QUICK ( Daido Sangyo Co . , Ltd . ) was used as a mounting agent , and immunostaining was observed with a VS120 virtual slide microscope ( Olympus , Inc . ) . For the fluorescence imaging of folate receptors with FolateSiR - 1 , the procedure was as follows . The slide was dipped in PBS at room temperature for 10 min , washed with PBST for 3 min three times , and blocked with 5 % skim milk in PBST at room temperature for 30 min . Then 5 \u03bcM FolateSiR - 1 and 2 . 9 \u03bcM DAPI ( nuclear stain ) in PBST containing 5 % skim milk and 0 . 1 % DMSO as a cosolvent were added and the slide was incubated at room temperature for 2 h , and washed with PBST three times . Fluoromount - G ( SouthernBiotech ) was used as a mounting agent , and fluorescence imaging was performed with a VS120 virtual slide microscope ( Olympus , Inc . ) equipped with a blue filter for DAPI , and a Cy5 filter for FolateSiR - 1 . The data were analyzed using the OlyVIA program ( Olympus , Inc . ) . S7 Synthesis and characterization of compounds Scheme S1 . Synthesis of 2 , 5 - diCOOH SiR650 Synthesis of N , N , N\u2019 , N\u2019 - tetramethyldiamino - Si - xanthone The compound was synthesized according to reference SR2 . Synthesis of 1 , 1 ' - ( 4 - bromo - 1 , 3 - phenylene ) bis ( 4 - methyl - 2 , 6 , 7 - trioxabicyclo [ 2 . 2 . 2 ] octane ) The compound was synthesized according to reference SR3 . S8 Synthesis of 2 , 5 - diCOOH SiR650 1 , 1 ' - ( 4 - Bromo - 1 , 3 - phenylene ) bis ( 4 - methyl - 2 , 6 , 7 - trioxabicyclo [ 2 . 2 . 2 ] octane ) ( 406 mg , 0 . 983 mmol ) and anhydrous THF ( 10 mL ) were added to a flame - dried flask flushed with argon . The solution was cooled to \u2013 78\u00b0C , and 1 M sec - BuLi solution in cyclohexane / n - hexane mixture ( 0 . 98 mL , 0 . 98 mmol ) was added to it . The mixture was stirred for 1 hr and N , N , N\u2019 , N\u2019 - tetramethyldiamino - Si - xanthone ( 106 mg , 0 . 327 mmol ) in anhydrous THF ( 10 mL ) was slowly added at the same temperature . The whole was warmed to room temperature and stirred for 3 . 5 h . AcOH ( 5 . 0 mL ) was added , and the mixture was evaporated to dryness . The residue was dissolved in 6 N HCl aq . . The resulting solution was refluxed overnight , allowed to cool to room temperature , and evaporated to dryness . The residue was purified by RP - HPLC to obtain 2 , 5 - diCOOH SiR650 ( 54 . 3 mg , 0 . 115 mmol , 35 % yield ) . 1 H NMR ( 300 MHz , CD 3 OD ) : \u03b4 = 0 . 58 ( s , 3H ) , 0 . 66 ( s , 3H ) , 3 . 30 ( s , 12H ) , 6 . 77 ( dd , J = 9 . 5 Hz , 2 . 9 Hz , 2H ) , 6 . 97 ( d , J = 9 . 5 Hz , 2H ) , 7 . 34 ( d , J = 2 . 9 Hz , 2H ) , 7 . 83 ( s , 1H ) , 8 . 30 ( s , 2H ) ; HRMS ( ESI + ) : Calcd for [ M ] + 473 . 1897 , Found , 473 . 1937 ( + 4 . 1 mmu ) ; HPLC analysis : eluent : A / B = 95 / 5 to 0 / 100 , 20 min , linear gradient ; solvent A : H 2 O , 0 . 1 % TFA ; solvent B : acetonitrile / H 2 O = 80 / 20 , 0 . 1 % TFA ; flow rate , 1 . 0 mL / min ; detection wavelength 650 nm . S9 Scheme S2 . Synthesis of 2 - Me - 5 - COOH SiR650 Synthesis of tert - butyl 3 - bromo - 4 - methylbenzoate 3 - Bromo - 4 - methylbenzoic acid ( 4 . 07 g , 18 . 9 mmol ) , Boc 2 O ( 11 . 2 g , 51 . 2 mmol ) and DMAP ( 62 . 5 mg , 5 . 12 mmol ) were dissolved in anhydrous THF ( 68 mL ) . The solution was refluxed overnight , then allowed to cool to room temperature , and evaporated to dryness . The residue was dissolved in ethyl acetate . The resulting solution was washed with sat . NaHCO 3 aq . and brine , dried over anhydrous Na 2 SO 4 , and filtered . The filtrate was evaporated to dryness , and the residue was purified by silica gel column chromatography ( n - hexane ) to afford tert - butyl 3 - bromo - 4 - methylbenzoate ( 3 . 47 g , 12 . 8 mmol , 68 % yield ) . 1 H NMR ( 300 MHz , CDCl 3 ) : \u03b4 = 1 . 58 ( s , 9H ) , 2 . 44 ( s , 3H ) , 7 . 27 ( d , J = 8 . 1 Hz , 1H ) , 7 . 81 ( dd , J = 8 . 1 Hz , 1 . 5 Hz , 1H ) , 8 . 12 ( d , J = 1 . 5 Hz , 1H ) ; 13 C NMR ( 75 MHz , CDCl 3 ) : \u03b4 = 23 . 1 , 28 . 1 , 81 . 4 , 124 . 6 , 128 . 2 , 130 . 5 , 131 . 3 , 133 . 2 , 142 . 6 , 164 . 5 ; HRMS ( ESI \u2012 ) : Calcd for [ M \u2012 t Bu ] \u2012 212 . 9551 , Found , 212 . 9553 ( + 0 . 2 mmu ) . Synthesis of 2 - Me - 5 - COOH SiR650 S10 tert - Butyl 3 - bromo - 4 - methylbenzoate ( 627 mg , 2 . 31 mmol ) and anhydrous THF ( 5 . 0 mL ) were added to a flame - dried flask flushed with argon . The solution was cooled to \u2012 78 \u02daC , and 1 M sec - BuLi in cyclohexane / n - hexane mixture ( 2 . 0 mL , 2 . 0 mmol ) was added to it . The mixture was stirred for 10 min at the same temperature , and N , N , N\u2019 , N\u2019 - tetramethyldiamino - Si - xanthone ( 72 . 3 mg , 0 . 223 mmol ) in anhydrous THF ( 12 . 0 mL ) was slowly added . The reaction mixture was warmed to room temperature and stirred for 2 . 5 h , then 2 N HCl aq . was added . Stirring was continued for 20 min , and the mixture was extracted with CH 2 Cl 2 . The organic layer was washed with brine , dried over anhydrous Na 2 SO 4 and evaporated to dryness . The residue was dissolved in TFA . The solution was stirred at room temperature for 2 h , then evaporated , and the residue was purified to give 2 - Me - 5 - COOH SiR650 ( 53 . 5 mg , 0 . 121 mmol , 54 % yield ) . 1 H NMR ( 300 MHz , CD 3 OD ) : \u03b4 = 0 . 61 ( s , 3H ) , 0 . 63 ( s , 3H ) , 2 . 11 ( s , 3H ) , 3 . 35 ( s , 12H ) , 6 . 78 ( dd , J = 9 . 9 Hz , 2 . 6 Hz , 2H ) , 7 . 03 ( d , J = 9 . 5 Hz , 2H ) , 7 . 37 ( d , J = 2 . 9 Hz , 2H ) , 7 . 54 ( d , J = 8 . 1 Hz , 1H ) , 7 . 73 ( s , 1H ) , 8 . 11 ( d , J = 8 . 1 Hz , 1H ) ; 13 C NMR ( 100 MHz , CD 3 OD ) : \u03b4 = \u20121 . 3 , \u20121 . 1 , 19 . 6 , 40 . 9 , 115 . 4 , 122 . 4 , 128 . 3 , 129 . 8 , 131 . 2 , 131 . 3 , 131 . 7 , 140 . 4 , 142 . 0 , 142 . 6 , 149 . 6 , 155 . 9 , 169 . 0 , 169 . 1 ; HRMS ( ESI + ) : Calcd for [ M ] + 455 . 2155 , Found , 455 . 2131 ( \u20122 . 3 mmu ) ; HPLC analysis : eluent : A / B = 95 / 5 to 0 / 100 , 20 min , linear gradient ; solvent A : H 2 O , 0 . 1 % TFA ; solvent B : acetonitrile / H 2 O = 80 / 20 , 0 . 1 % TFA ; flow rate , 1 . 0 mL / min ; detection wavelength 650 nm . S11 Synthesis of 2 - Me - 4 - COOH dichloro - TokyoMagenta ( DCTM ) The compound was synthesized according to reference SR4 . Synthesis of 2 , 4 - diCOOH DCTM The compound was synthesized according to reference SR5 . S12 Scheme S3 . Synthesis of Folate - peptide . Synthesis of Folate - peptide Folate - peptide was synthesized on an automatic peptide synthesizer using the standard protocol for fluorenylmethoxycarbonyl ( Fmoc ) solid - phase synthesis with 2 - chlorotrityl chloride resin ( 302 mg , 1 . 42 mmol / g reaction site , 0 . 428 mmol ) ( Novabiochem ) . O - ( 7 - Azabenzotriazol - 1 - yl ) - N , N , N ' , N ' - tetramethyluronium hexafluorophosphate ( HATU ) and N , N - diisopropylethylamine ( DIPEA ) were used as coupling reagents and DMF S13 and N - methylpyrrolidone ( NMP ) were used as solvents . Fmoc - Gly - OH , Fmoc - Lys ( Boc ) - OH , Fmoc - Asp ( O t Bu ) - OH and Fmoc - Glu - O t Bu were used as Fmoc amino acids . After the construction of the peptide moiety of Folate - peptide on the solid - phase , the Fmoc group at the N - terminus was deprotected with 40 % piperidine in DMF . Then , N - trifluoroacetyl pteroic acid ( 368 mg , 0 . 902 mmol ) was coupled to the peptide on the solid phase by adding a mixture of HOBt , WSCD\u2027HCl , Et 3 N and DMSO . The trifluoroacetyl group was deprotected with 2 % hydrazine in DMF . Folate - peptide was cleaved with 2 mL of TFA / triisopropylsilane / H 2 O ( 95 / 2 . 5 / 2 . 5 v / v / v ) for 90 min . The solution was evaporated to dryness and the residue was purified by RP - HPLC to afford a brown solid ( 42 . 1 mg , 12 % ) . HRMS ( ESI + ) : Calcd for [ M + H ] + 742 . 2909 Found , 742 . 2862 ( \u2012 4 . 7 mmu ) . HPLC analysis : eluent , A / B = 99 / 1 \uf0e0 70 / 30 , 10 min \uf0e0 0 / 100 , 35 min ; A : H 2 O containing 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 286 nm : Retention time was 12 . 0 min . Scheme S4 . Synthesis of FolateSiR - 1 S14 Synthesis of FolateSiR - 1 NHS ( 17 . 9 mg , 150 \u03bcmol ) and WSCD\u2027HCl ( 31 . 0 mg , 162 \u03bcmol ) were added to a solution of 2 , 5 - diCOOH SiR ( 27 . 9 mg , 47 . 0 \u03bcmol ) in THF ( 3 mL ) . The mixture was stirred at 35 \u02da C for 3 h , then evaporated to dryness , and the residue was partially purified by RP - HPLC to obtain crude 2 , 5 - diCOOH SiR650 succinimidyl ester ( 15 . 4 mg ) . DIPEA ( 57 \u03bcL , 0 . 327 mmol ) was added to a solution of the crude 2 , 5 - diCOOHSiR succinimidyl ester ( 7 . 2 mg ) and Folate - peptide ( 10 . 1 mg , 0 . 012 mmol ) in THF ( 1 . 5 mL ) . The mixture was stirred at room temperature overnight , then evaporated to dryness , and the residue was purified by RP - HPLC to obtain FolateSiR - 1 ( 2 . 7 mg , 2 . 0 \u03bcmol , 4 % yield in 2 steps ) . HRMS ( ESI + ) : Calcd for [ M ] + , 1196 . 4621 , Found , 1196 . 4621 ( 0 . 0 mmu ) . HPLC analysis : eluent , A / B = 80 / 20 \uf0e0 0 / 100 , 20 min ; A : H 2 O containing 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 650 nm : Retention time was 11 . 3 min . S15 Scheme S5 . Synthesis of FolateSiR - 2 Synthesis of FolateSiR - 2 NHS ( 45 . 7 mg , 0 . 398 mmol ) and WSCD\u2027HCl ( 38 . 0 mg , 0 . 199 mmol ) were added to a solution of 2 - Me - 5 - COOH SiR650 ( 9 . 7 mg , 17 . 5 \u03bcmol ) in THF ( 3 . 0 mL ) . The mixture was stirred at room temperature overnight , then evaporated to dryness , and the residue was partially purified by RP - HPLC to obtain crude 2 - Me - 5 - COOH SiR650 succinimidyl ester ( 5 . 4 mg ) . DIPEA ( 17 \u03bcL , 0 . 098 mmol ) was added to a solution of the crude 2 - Me - 5 - COOH SiR650 succinimidyl ester ( 6 . 5 mg ) and Folate - peptide ( 5 . 7 mg , 6 . 7 \u03bcmol ) in DMF ( 3 . 0 mL ) . The mixture was stirred at room temperature overnight , then evaporated to dryness , and the residue was purified by RP - HPLC to obtain FolateSiR - 2 ( 1 . 0 mg , 0 . 78 \u03bcmol , 4 % yield in 2 steps ) . HRMS ( ESI + ) : Calcd for [ M ] + , S16 1166 . 4880 , Found , 1166 . 4881 ( + 0 . 1 mmu ) . HPLC analysis : eluent , A / B = 80 / 20 \uf0e0 0 / 100 , 20 min ; A : H 2 O containing 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 650 nm : Retention time was 13 . 2 min . Scheme S6 . Synthesis of 2 - Me DCTM folate Synthesis of 2 - Me DCTM folate 2 - Me - 4 - COOH DCTM ( 11 mg , 24 \uf06d mol ) , N - hydroxysuccinimide ( NHS ) ( 8 . 4 mg , 73 \uf06d mol ) and WSCD\u2027HCl ( 14 . 0 mg , 73 \uf06d mol ) were dissolved in THF ( 1 . 0 mL ) . The mixture was stirred at 35 \u02daC for 5 h , then evaporated to dryness , and the residue was partially purified by RP - HPLC to obtain crude 2 - Me - 4 - COOH DCTM succinimidyl ester ( 4 . 8 mg ) . The crude product and Folate - peptide ( 18 mg , 24 \uf06d mol ) were dissolved in DMF , then DIPEA ( 10 S17 \uf06d L , 57 \uf06d mol ) was added , and the reaction mixture was stirred at 35 \u02daC for 2 days . The solution was evaporated to dryness and the residue was purified by RP - HPLC to obtain 2 - Me DCTM folate ( 0 . 12 mg , 0 . 10 \uf06d mol , 4 % yield in 2 steps ) . HRMS ( ESI + ) : Calcd for [ M + H ] + , 1180 . 3155 , Found , 1180 . 3183 ( + 2 . 8 mmu ) . HPLC analysis : eluent , A / B = 30 / 70 \uf0e0 0 / 100 , 20 min ; A : H 2 O containing 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 470 nm : Retention time was 14 . 8 min . Scheme S7 . Synthesis of 2 - COOH DCTM folate Synthesis of 2 - COOH DCTM Folate 2 , 4 - diCOOH DCTM ( 11 mg , 23 \uf06d mol ) , NHS ( ( 8 . 1 mg , 70 \uf06d mol ) and WSCD\u2027HCl ( 14 . 8 mg , 77 \uf06d mol ) were S18 dissolved in THF ( 1 . 0 mL ) . The mixture was stirred at 35 \u02daC overnight , then evaporated to dryness , and the residue was partially purified by RP - HPLC to obtain crude 2 , 4 - diCOOH DCTM succinimidyl ester ( 4 . 1 mg ) . The crude product and Folate - peptide ( 15 mg , 20 \uf06d mol ) were dissolved in DMF ( 2 . 0 mL ) , then DIPEA ( 10 \uf06d L , 57 \uf06d mol ) was added . The reaction mixture was stirred at 35 \u02daC for 2 days , then evaporated to dryness , and the residue was purified by RP - HPLC to obtain 2 - COOH DCTM folate ( 0 . 63 mg , 0 . 52 \uf06d mol , 23 % yield ) . HRMS ( ESI + ) : Calculated for [ M + H ] + , 1210 . 2896 , Found , 1210 . 2902 ( + 0 . 6 mmu ) . HPLC analysis : eluent , A / B = 70 / 30 \uf0e0 0 / 100 , 20 min ; A : H 2 O containing 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 283 nm : Retention time was 13 . 1 min . Scheme S8 . Synthesis of Fluorescein folate Fluorescein folate DIPEA ( 5 \u03bcL , 0 . 028 mmol ) was added to a solution of 5 - carboxyfluorescein succinimidyl ester ( 5 . 4 mg , 0 . 011 mmol ) ( Tokyo Chemical Industry Co . , Ltd . , Japan ) and Folate - peptide ( 14 . 5 mg , 0 . 019 mmol ) in DMF ( 500 \u03bcL ) . S19 The mixture was stirred at room temperature overnight , then evaporated , and the residue was purified by HPLC to give fluorescein folate ( 1 . 5 mg , 1 . 3 \u03bcmol , 12 % yield ) . HRMS ( ESI - ) : Calcd for [ M - H ] - 1098 . 3230 , Found , 1098 . 3190 ( \u20124 . 0 mmu ) . HPLC analysis : eluent : A / B = 80 / 20 , 6 min , 80 / 20 to 20 / 80 , 20 min , linear gradient ; solvent A : H 2 O , 0 . 1 % TFA ; solvent B : acetonitrile / H 2 O = 80 / 20 , 0 . 1 % TFA ; flow rate , 1 . 0 mL / min ; detection wavelength 450 nm : Retention time was 27 . 0 min . Scheme S9 . Synthesis of Alexa488 folate Alexa 488 folate DIPEA ( 5 \u03bcL , 0 . 028 mmol ) was added to a solution of Alexa 488 succinimidyl ester isomer mixture ( Invitrogen , Carlsbad , CA ) ( 4 . 5 mg , 7 . 1 \u03bcmol ) and Folate - peptide ( 11 . 5 mg , 0 . 015 mmol ) in DMF ( 500 \u03bcL ) . The mixture was stirred at room temperature overnight , then evaporated to dryness , and the residue was purified by RP - HPLC to obtain Alexa 488 folate ( 2 . 1 mg , 1 . 7 \u03bcmol , 24 % yield ) . HRMS ( ESI \u2012 ) : Calcd for [ M \u2012 H ] \u2012 1256 . 2686 , Found , 1256 . 2711 ( + 2 . 5 mmu ) . HPLC analysis : eluent , A / B = 80 / 20 , 6 min ; 80 / 20 \uf0e0 20 / 80 , 20 min ; A : H 2 O containing S20 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 490 nm : Retention time was 22 . 4 min . Scheme S10 . Synthesis of TAMRA folate TAMRA folate DIPEA ( 5 \u03bcL , 0 . 028 mmol ) was added to a solution of 5 - carboxytetramethylrhodamine succinimidyl ester ( Invitrogen , Carlsbad , CA ) ( 5 . 6 mg , 0 . 011 mmol ) and Folate - peptide ( 12 . 1 mg , 0 . 016 mmol ) in DMF ( 0 . 5 mL ) . The mixture was stirred at room temperature overnight , then evaporated to dryness , and the residue was purified by RP - HPLC to obtain TAMRA folate ( 2 . 5 mg , 2 . 1 \u03bcmol , 20 % yield ) . HRMS ( ESI + ) : Calcd for [ M ] + 1154 . 4332 , Found , 1154 . 4315 ( \u20121 . 7 mmu ) . HPLC analysis : eluent , A / B = 80 / 20 , 6 min ; 80 / 20 \uf0e0 20 / 80 , 20 min ; A : H 2 O containing 0 . 1 % TFA ( v / v ) , B : CH 3 CN / H 2 O = 80 / 20 containing 0 . 1 % TFA ( v / v ) ; 1 . 0 mL / min flow rate ; Detection at 547 nm : Retention time was 27 . 0 min . S21 Scheme S11 . Synthesis of 2 - COOH SiR650 Synthesis of tert - butyl 2 - bromobenzoate 2 - Bromobenzoic acid ( 5 . 65 g , 28 . 3 mmol ) , Boc 2 O ( 8 . 65 g , 39 . 5 mmol ) and N , N - dimethyl - 4 - aminopyridine ( DMAP ) ( 765 mg , 6 . 27 mmol ) were dissolved in dry THF ( 40 mL ) . The solution was refluxed overnight , then allowed to cool to room temperature , and evaporated to dryness . The residue was dissolved in ethyl acetate . This solution was washed with saturated NaHCO 3 aq . and brine , dried over Na 2 SO 4 , filtered and evaporated to dryness . The resulting crude oil was purified by silica gel column chromatography ( EtOAc / n - hexane = 1 / 10 ) to afford tert - butyl 2 - bromobenzoate ( 4 . 89 g , 19 . 0 mmol , y . 67 % ) . 1 H NMR ( 400 MHz , CDCl 3 ) : \u03b4 = 1 . 61 ( s , 9H ) , 7 . 25 - 7 . 29 ( m , 1H ) , 7 . 33 ( td , J = 7 . 4 , 1 . 3 Hz , 1H ) , 7 . 61 ( dd , J = 8 . 0 Hz , 1 . 2 Hz , 1H ) , 7 . 68 ( dd , J = 7 . 8 Hz , 2 . 0 Hz , 2H ) ; 13 C NMR ( 100 MHz , CDCl 3 ) : \u03b4 = 28 . 1 , 82 . 5 , 120 . 9 , 127 . 0 , 130 . 7 , 131 . 8 , 134 . 0 , 134 . 3 , 165 . 7 ; HRMS ( ESI + ) : Calcd for [ M \u2012 t Bu ] + 200 . 9551 , Found , 200 . 9537 ( \u20121 . 4 mmu ) . Synthesis of 2 - COOH SiR650 tert - Butyl 2 - bromobenzoate ( 186 mg , 0 . 723 mmol ) and anhydrous THF ( 5 . 0 mL ) were added to a flame - dried S22 flask that had been flushed with argon . The solution was cooled to \u2013 78 \u00b0C , 1 M sec - BuLi ( 0 . 3 mmol ) was added , and the mixture was stirred for 10 min . At the same temperature , Si - xanthone ( 33 . 5 mg , 0 . 103 mmol ) dissolved in anhydrous THF ( 5 . 0 mL ) was slowly added . The mixture was warmed to room temperature and stirred for 2 . 5 h , then 2 N HCl aq . ( 10 mL ) was added to it . Stirring was continued for 20 min , then the mixture was extracted with CH 2 Cl 2 . The organic layer was washed with brine , dried over Na 2 SO 4 and evaporated to dryness . The residue was dissolved in TFA ( 1 . 5 mL ) . The solution was stirred for 2 h , then evaporated to dryness , and the residue was purified by HPLC to give pure 2 - COOH SiR650 ( 6 . 3 mg , 0 . 012 mol , 11 % yield ) . 1 H NMR ( 300 MHz , CD 3 COCD 3 ) : \u03b4 = 0 . 11 ( s , 3H ) , 0 . 21 ( s , 3H ) , 2 . 61 ( s , 12H ) , 6 . 31 ( dd , J = 9 . 2 Hz , 2 . 6 Hz , 1H ) , 6 . 40 ( d , J = 9 . 5 Hz , 2H ) , 6 . 83 ( d , d , J = 2 . 9 Hz , 2H ) 6 . 87 ( d , J = 7 . 2 Hz , 1H ) , 7 . 21 ( t , J = 7 . 3 Hz , 1H ) , 7 . 32 ( m 1H ) , 7 . 54 ( d , J = 7 . 3 Hz , 1H ) ; HRMS ( ESI + ) : Calcd for [ M ] + 429 . 1998 , Found , 429 . 2006 ( + 0 . 8 mmu ) ; HPLC analysis : eluent : A / B = 80 / 20 to 0 / 100 , 20 min , linear gradient ; solvent A : H 2 O , 0 . 1 % TFA ; solvent B : acetonitrile / H 2 O = 80 / 20 , 0 . 1 % TFA ; flow rate , 1 . 0 mL / min ; detection wavelength 650 nm . S23 Figure S1 . Immunostaining images of KB cells ( a ) , OVCAR - 3 cells ( b ) , and HT1080 cells ( c ) . Cells were fixed with 4 % PFA / PBS for 20 min , labeled with human anti - folate binding protein antibody for 1 hr , washed , and labeled with second antibody conjugated with Alexa Fluor \u24c7 488 for 1 hr . Ex . 488 nm ; Em . 500 - 535 nm . Scale bars : 20 \u03bcm . Figure S2 . Fluorescence images of KB cells ( FR + ) ( a ) and OVCAR - 3 cells ( FR\u2012 ) ( b ) obtained with a commercially available near - infrared fluorescent probe , FolateRSense ( PerkinElmer Inc . ) . White arrows indicate bright dots inside cells . Ex . 650 nm , Em . 670 - 750 nm . Scale bars : 20 \u03bcm . 0 100 KB cells b Fluorescence Bright field a OVCAR - 3 cells Fluorescence Bright field 0 100 c HT1080 cells Fluorescence Bright field a 5 50 0 150 KB cells ( FR + ) OVCAR - 3 cells ( FR \u2013 ) Bright field Fluorescence image Bright field Fluorescence image b S24 Figure S3 . ( a ) Fluorescence images of KB cells ( left ) and OVCAR - 3 cells ( right ) incubated with fluorophore - labeled folates . Emission : 490 nm ( Fluorescein Folate , Alexa488 Folate ) , 555 nm ( TAMRA Folate ) and 595 nm ( 2 - Me DCTM Folate , 2 - COOH DCTM Folate ) . Excitation : 510 - 540 nm ( Fluorescein Folate , Alexa488 Folate ) , 575 - 600 nm ( TAMRA Folate ) and 610 - 650 nm ( 2 - Me DCTM Folate , 2 - COOH DCTM Folate ) . Scale bars : 20 \u03bcm . ( b ) Photophysical properties of fluorophore - labeled folates . a Photophysical properties were measured in 0 . 1 N NaOH aq . with fluorescein in 0 . 1 N NaOH aq . ( \uf046 fl = 0 . 85 ) as a standard . b Photophysical properties were measured in 100 mM sodium phosphate buffer ( pH 7 . 4 ) with fluorescein in 0 . 1 N NaOH aq . ( \uf046 fl = Fluorophore Folate moiety Negatively charged linker Fluorescein Folate Fluorophore = KB cells OVCAR - 3 cells Fluorescence Alexa488 Folate KB cells OVCAR - 3 cells Fluorescence 0 100 5 100 TAMRA Folate KB cells OVCAR - 3 cells Fluorescence 5 100 2 - Me DCTM Folate KB cells OVCAR - 3 cells Fluorescence 5 30 2 - COOH DCTM Folate KB cells OVCAR - 3 cells Fluorescence 5 30 a b Dye Abs max ( nm ) Em max ( nm ) \uf046 fl Fluorescein Folate a Alexa488 Folate b TAMRA Folate c 2 - Me DCTM Folate d 2 - COOH DCTM Folate d 498 523 0 . 25 496 520 0 . 19 558 587 0 . 069 598 611 0 . 12 596 612 0 . 17 S25 0 . 85 ) as a standard . c Photophysical properties were measured in 100 mM sodium phosphate buffer ( pH 7 . 4 ) with Rhodamine B in EtOH ( \uf046 fl = 0 . 65 ) as a standard . d Photophysical properties were measured in 100 mM sodium phosphate buffer ( pH 9 . 0 ) with 2 - Me TokyoMagenta in 100 mM sodium phosphate buffer ( pH 9 . 0 ) ( \uf046 fl = 0 . 42 ) as a standard . Figure S4 . ( a ) KB cells were incubated with FolateSiR - 1 , then fixed with 4 % formaldehyde , and bright - field and fluorescence images were obtained . ( b ) Fluorescence image of mouse embryo incubated with 10 \u03bcM FolateSiR - 1 a 20 \u03bcm 20 \u03bcm 5 50 Fluorescence image Bright field b 50 \u03bcm 50 \u03bcm Closure Caudal Rostral 0 80 Magnified image Fluorescence image c 50 \u03bcm 50 \u03bcm Closure Rostral Caudal 0 80 Magnified image Fluorescence image Strong surface signal in closing neural tube S26 in DMEM containing 10 % rat IC - serum . Then , the mouse embryo was fixed with 4 % formaldehyde . Locations stained with FolateSiR - 1 are indicated by white arrowheads . ( c ) Fluorescence image of mouse embryo incubated with 10 \u03bcM FolateSiR - 1 in the presence of 1 mM folic acid , and then fixed with 4 % formaldehyde . Figure S5 . ( a ) Fluorescence images of mouse embryos incubated with 10 \uf06d M FolateSiR - 1 and fixed with 4 % formaldehyde . The right images are magnifications of the indicated portions of the left images . The regions stained with FolateSiR - 1 are indicated by white arrowheads . The cells indicated by yellow arrows appear to be dead cells . Scale bars : 50 \uf06d m . ( b , c ) Bright - field ( left ) and fluorescence ( right ) images of mouse embryos fixed with 4 % formaldehyde in the absence ( b ) or presence ( c ) of 1 mM folic acid . These data indicate that the autofluorescence of the fixed embryos with or without 1 mM folic acid is sufficiently low compared with those in ( a ) . Scale bars : 50 \uf06d m . S27 Figure S6 . ( a ) White - light and fluorescence images of extracted organs of the mouse in Figure 4a . ( b ) White - light and fluorescence images of extracted organs of the mouse in Figure 4c . a White light Fluorescence Stomach Tumor b White light Fluorescence Kidney Tumor S28 Figure S7 . In vivo fluorescence imaging of HT1080 tumor - bearing mice injected with 100 \uf06d M FolateSiR - 1 in 100 \uf06d L saline ( n = 3 ) . ( a ) Time - lapse white - light ( top ) and fluorescence ( bottom ) images of a mouse . The images were obtained before the probe injection and 0 , 0 . 5 , 1 , 2 , 3 and 6 h after the probe injection . Ex . / Em . = 661 / 700 - 800 nm . T : Tumor ; M : Muscle . Fluorescence intensity scale : gray scale 0 to 255 . ( b ) The time - dependent fluorescence intensity changes of tumor and non - tumor ( muscle ) areas of three mice . Error bar shows S . E . ( c ) White - light ( top ) and fluorescence ( bottom ) images of extracted mouse organs . S29 Figure S8 . In vivo fluorescence imaging of HT1080 tumor - bearing mice injected with 100 \uf06d M FolateSiR - 2 in 100 \uf06d L saline ( n = 3 ) . ( a ) Time - lapse white - light ( top ) and fluorescence ( bottom ) images of a mouse . The images were obtained before the probe injection and 0 , 0 . 5 , 1 , 2 , 3 and 6 h after the probe injection . Ex . / Em . = 661 / 700 - 800 nm . T : Tumor ; M : Muscle . Fluorescence intensity scale : gray scale 0 to 255 . ( b ) The time - dependent fluorescence intensity changes in tumor and non - tumor ( muscle ) areas of three mice . Error bar shows S . E . ( c ) White - light ( top ) and fluorescence ( bottom ) images of extracted mouse organs . S30 Figure S9 . In vivo fluorescence imaging of KB tumor - bearing mice injected with 6 mM folic acid in 100 \uf06d L saline , then with 100 \uf06d M FolateSiR - 1 in 100 \uf06d L saline ( n = 3 ) . ( a ) Time - lapse white - light ( top ) and fluorescence ( bottom ) images of a mouse . The images were obtained before the probe injection and 0 , 0 . 5 , 1 , 2 , 3 and 6 h after the probe injection . Ex . / Em . = 661 / 700 - 800 nm . T : Tumor ; M : Muscle . Fluorescence intensity scale : gray scale 0 to 255 . ( b ) The time - dependent fluorescence intensity changes of tumor and non - tumor ( muscle ) areas of three mice . Error bar shows S . E . ( c ) White - light ( top ) and fluorescence ( bottom ) images of extracted mouse organs . S31 Position Age Sex Organ Pathological Diagnosis Tumor history Tumor size ( cm ) Differentiation A1 50 F Ovary Adenocarcinoma 1 year 4\u00d72\u00d71 . 5 \u2012 A2 A 45 F Ovary Adenocarcinoma 14 days 11\u00d78\u00d76 Poor A3 62 F Ovary Mucous cystadenocarcinoma 20 days 6\u00d74\u00d74 \u2012 A4 69 F Ovary Serous adenocarcinoma 1 month diameter 1 . 5 \u2012 A5 49 F Ovary Serous adenocarcinoma \u2012 \u2012 Poor A6 53 F Ovary Serous adenocarcinoma \u2012 \u2012 Moderate A7 80 F Ovary Serous adenocarcinoma \u2012 \u2012 Poor A8 54 F Ovary Serous cystadenocarcinoma 2 months 6\u00d76\u00d75 Poor B1 53 F Ovary Mucous adenocarcinoma 1 month 5\u00d74\u00d73 \u2012 B2 40 F Ovary Adenocarcinoma , signet ring cell carcinoma 2 months 2 . 5\u00d72 . 5\u00d72 Poor B3 55 F Ovary Clear cell adenocarcinoma 3 months diameter 13 Poor B4 67 F Ovary Serous cystadenocarcinoma 2 months diameter 2 Poor B5 59 F Ovary Serous cystadenocarcinoma 1 month 7\u00d75\u00d72 . 8 Poor B6 74 F Ovary Cystadenocarcinoma , serous & papillary 20 days 5\u00d73 . 5\u00d72 Moderate B7 71 F Ovary Endometrioid carcinoma \u2012 \u2012 Moderate B8 49 F Ovary Endometrioid carcinoma 20 days 10\u00d78\u00d74 . 5 Moderate C1 40 F Ovary Mucinous cystodenocarcinoma \u2012 \u2012 Moderate C2 42 F Ovary Poorly differentiated carcinoma \u2012 \u2012 Poor C3 52 F Ovary Serous carcinoma \u2012 \u2012 Poor C4 54 F Ovary Serous cystoadenocarcinoma \u2012 \u2012 \u2012 C5 60 F Ovary Serous cystoadenocarcinoma , papillary \u2012 \u2012 \u2012 C6 50 F Ovary Serous papillary cystoadenocarcinoma \u2012 \u2012 Moderate C7 42 F Ovary Signet ring cell carcinoma 2 months 6\u00d74\u00d74 \u2012 C8 67 F Ovary Transitional cell carcinoma 2 months 8\u00d76\u00d73 Poor D1 47 F Ovary Transitional epithelial cell Carcinoma 20 days 12 . 5\u00d710\u00d7 3 . 5 Poor D2 20 F Ovary Yolk sac cystadenoma \u2012 \u2012 \u2012 D3 26 F Ovary Leimyoma 2 months 8\u00d76 . 5\u00d74 . 5 \u2012 S32 D4 45 F Ovary Malignancy , Brenner\u2019s tumor 3 years diameter 10 \u2012 D5 21 F Ovary Mature teratoma 6 years 18\u00d716\u00d75 Moderate D6 39 F Ovary Krukenberg\u2019s tumor \u2012 \u2012 \u2012 D7 25 F Ovary Cystic mature teratoma 29 days 8\u00d77\u00d76 \u2012 D8 18 F Ovary Dysgerminoma 1 month 18\u00d712\u00d78 Poor E1 26 F Ovary Dysgerminoma \u2012 \u2012 \u2012 E2 42 F Ovary Endodermal sinus tumor 3 months 15\u00d712\u00d78 Poor E3 60 F Ovary Thecoma 6 months 9\u00d76 . 5\u00d76 \u2012 E4 20 F Ovary Thecoma 1 week 12\u00d78\u00d76 \u2012 E5 32 F Ovary Thecoma 3 months 8\u00d77 \u2012 E6 54 F Ovary Normal E7 40 F Ovary Normal E8 56 F Ovary Normal Figure S10 . Components of the frozen human ovary tumor tissue array ( Catalog No . : T6235183 - 5 ; Lot No . : B705061 ) . Figure S11 . Fluorescence staining of the tissue microarray described in Figure S8 with FolateSiR - 1 ( pink ) and DAPI ( Blue ) . Scale bar represents 2 mm . S33 Figure S12 . Immunostaining of folate receptors in the tissue microarray described in Figure S8 . Scale bar represents 2 mm . Figure S13 . ( a ) The magnified fluorescence images of C4 , B8 , C3 and A5 specimens in Figure S11 are shown . Red color indicates the fluorescence of FolateSiR - 1 . All specimens showed the fluorescence signal , though the A5 A5 A5 A5 B8 B8 C3 C3 C4 C4 a b c Magnified image S34 fluorescence intensities were weak in C3 and A5 specimens . ( b ) The magnified folate receptor - immunostaining images of C4 , B8 , C3 and A5 in Figure S12 are shown . All specimens were immunostained , though the sample condition of B8 , C3 and A5 were not good . ( c ) The magnified fluorescence image ( left ) and the magnified immunostaining image ( right ) of C4 specimen in ( a ) and ( b ) are shown . Arrows indicate the boundary between tumor tissues and non - tumor tissues ( vascular tissues and fibrous tissues ) . The tumor tissues are selectively stained by the fluorescent probe , FolateSiR - 1 . Figure S14 . Absorption spectra of 1 \u03bc M 2 - COOH SiR650 in 100 mM sodium phosphate buffer at pH 7 . 4 with or without 10 % fetal bovine serum ( FBS ) . Figure S15 . Absorbance spectra of 1 \u03bc M FolateSiR - 1 in phosphate buffered saline ( PBS ) containing 0 . 1 % DMSO with or without 10 % FBS ( Biowest , Cat . No . : S1810 - 500 ) , or in mouse serum ( FUJIFILM Wako Pure Chemical Corp . , Cat . No . : 146 - 06551 ) . - 0 . 02 0 0 . 02 0 . 04 0 . 06 0 . 08 0 . 1 0 . 12 500 550 600 650 700 PBS PBS + 10 % FBS mouse serum A b s o r ban c e Wavelength ( nm ) S35 Supporting References SR1 ) Yamaguchi , Y . , Shinotsuka , N . , Nonomura , K . , Takemoto , K . , Kuida , K . , Yosida , H . , Miura , M . Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure . J . Cell Biol . 195 , 1047 - 1060 ( 2011 ) . SR 2 ) Koide , Y . , Urano , Y . , Hanaoka , K . , Terai , T . & Nagano , T . Evolution of group 14 rhodamines as platforms for near - infrared fluorescence probes utilizing photoinduced electron transfer . ACS Chem . Biol . 6 , 600 - 608 ( 2011 ) . SR3 ) Butkevich , A . N . et al . Fluorescent rhodamines and fluorogenic carbopyronines for super - resolution STED microscopy in living cells . Angew . Chem . Int . Ed . 55 , 3290 - 3294 ( 2016 ) . SR4 ) Egawa , T . et al . Red fluorescent probe for monitoring the dynamics of cytoplasmic calcium ions . Angew . Chem . Int . Ed . 52 , 3874 - 3877 ( 2013 ) . SR5 ) Hirabayashi , K . et al . Synthesis of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell - membrane permeability . Data in Brief 12 , 351 - 357 ( 2017 ) ." +} \ No newline at end of file diff --git a/full_texts.json b/full_texts.json new file mode 100644 index 0000000000000000000000000000000000000000..1a081acc70ae72a6b286d8041e92eb99bc7de6d8 --- /dev/null +++ b/full_texts.json @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:c70feb8d542a37f6141ffeb3c2c4ecc69a8874832dd2fc9f791da40f41aa72e4 +size 21361268 diff --git a/silver_data/094f398870c97d7f5df296849920e252b2416b9f.pdf.txt b/silver_data/094f398870c97d7f5df296849920e252b2416b9f.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..7b95b41b2aa198b6c5bad96df726e8de55636637 --- /dev/null +++ b/silver_data/094f398870c97d7f5df296849920e252b2416b9f.pdf.txt @@ -0,0 +1 @@ +Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal Andrew J . Muinonen - Martin 1 , 2 , 3 , Olivia Susanto 1 , Qifeng Zhang 4 , Elizabeth Smethurst 4 , William J . Faller 1 , Douwe M . Veltman 1 , Gabriela Kalna 1 , Colin Lindsay 1 , 5 , Dorothy C . Bennett 6 , Owen J . Sansom 1 , Robert Herd 7 , Robert Jones 1 , 5 , Laura M . Machesky 1 , Michael J . O . Wakelam 4 , David A . Knecht 8 , Robert H . Insall 1 * 1 CRUK Beatson Institute , Glasgow , United Kingdom , 2 York Teaching Hospital NHS Foundation Trust , York , United Kingdom , 3 The Leeds Teaching Hospitals NHS Trust , Leeds , United Kingdom , 4 The Babraham Institute , Cambridge , United Kingdom , 5 Beatson West of Scotland Cancer Centre , Glasgow , United Kingdom , 6 Molecular Cell Sciences Research Centre , St . George’s , University of London , London , United Kingdom , 7 Alan Lyell Centre for Dermatology , Glasgow , United Kingdom , 8 Department of Molecular and Cell Biology , University of Connecticut , Storrs , Connecticut , United States of America Abstract The high mortality of melanoma is caused by rapid spread of cancer cells , which occurs unusually early in tumour evolution . Unlike most solid tumours , thickness rather than cytological markers or differentiation is the best guide to metastatic potential . Multiple stimuli that drive melanoma cell migration have been described , but it is not clear which are responsible for invasion , nor if chemotactic gradients exist in real tumours . In a chamber - based assay for melanoma dispersal , we find that cells migrate efficiently away from one another , even in initially homogeneous medium . This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition . The principal chemoattractant , unexpectedly active across all tumour stages , is the lipid agonist lysophosphatidic acid ( LPA ) acting through the LPA receptor LPAR1 . LPA induces chemotaxis of remarkable accuracy , and is both necessary and sufficient for chemotaxis and invasion in 2 - D and 3 - D assays . Growth factors , often described as tumour attractants , cause negligible chemotaxis themselves , but potentiate chemotaxis to LPA . Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward - facing gradients . We measure LPA gradients across the margins of melanomas in vivo , confirming the physiological importance of our results . We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards . Cells create their own gradients by acting as a sink , breaking down locally present LPA , and thus forming a gradient that is low in the tumour and high in the surrounding areas . The key step is not acquisition of sensitivity to the chemoattractant , but rather the tumour growing to break down enough LPA to form a gradient . Thus the stimulus that drives cell dispersal is not the presence of LPA itself , but the self - generated , outward - directed gradient . Citation : Muinonen - Martin AJ , Susanto O , Zhang Q , Smethurst E , Faller WJ , et al . ( 2014 ) Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal . PLoS Biol 12 ( 10 ) : e1001966 . doi : 10 . 1371 / journal . pbio . 1001966 Academic Editor : Andre Levchenko , Yale University , United States of America Received February 24 , 2014 ; Accepted September 5 , 2014 ; Published October 14 , 2014 Copyright : (cid:2) 2014 Muinonen - Martin et al . This is an open - access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use , distribution , and reproduction in any medium , provided the original author and source are credited . Funding : This research was funded by Cancer Research UK core grants to RI , LM , and OS ; by the Wellcome Trust ( fellowship 095186 / Z / 10 / Z to AMM and programme grant to DB ) ; and by the BBSRC ( core grant to MJOW ) . The funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript . Competing Interests : The authors have declared that no competing interests exist . Abbreviations : EGF , epidermal growth factor ; FBS , fetal bovine serum ; LPA , lysophosphatidic acid ; PDGF , platelet - derived growth factor ; RGP , radial growth phase ; SCF , stem cell factor ; SEM , standard error of the mean ; VGP , vertical growth phase . * Email : R . Insall @ beatson . gla . ac . uk Introduction Melanoma Metastasis Melanoma is an unusually aggressive cancer , which often metastasizes early during tumour development [ 1 ] . Tumours that have not clinically metastasized are frequently curable , but patients are far less likely to survive if tumours have metastasized before they are surgically removed , and metastasis is the principal cause of cancer mortality [ 2 ] . The most influential prognostic factor in predicting metastasis and survival is the thickness of the tumour ( the ‘‘Breslow depth’’ ) [ 3 ] . There is a dramatic increase in the risk of metastasis with only millimeter increases in Breslow depth [ 3 ] . This characteristic is unlike most solid tumours , in which the cytological morphology of the tumour cells and the individual genes mutated in the cancer are more important than size alone . Metastasis is therefore an important , and under - medicated , potential target for cancer therapy [ 4 , 5 ] . Melanocyte Migration during Development One principal reason behind the aggressiveness of melanoma derives from the developmental history of melanocytes , the pigment producing cells in the skin that mutate to form melanomas . During mammalian development melanoblasts , the melanocyte precursors , emerge from a restricted location at the neural crest , and migrate rapidly from there throughout the developing dermis , before maturing into melanocytes on the basement membrane of the epidermis [ 6 ] . Thus a substantial level of cell migration is required for even skin pigmentation . Even in adults—for example following treatment for vitiligo—melanocytes can spread significant distances from the hair follicles to PLOS Biology | www . plosbiology . org 1 October 2014 | Volume 12 | Issue 10 | e1001966 repopulate the surrounding skin . The melanocyte lineage is thus inherently migratory . However , several questions about melanoma progression remain unanswered . The first is what drives melanomas to change from the relatively benign radial growth phase ( RGP ) to the far more invasive vertical growth phase ( VGP ) ( see schematic diagram in Figure 1A ) . In RGP melanomas , cells only spread horizontally along the basement membrane , compared to VGP melanoma cells , which are also capable of spreading both upwards into the epidermis ( Pagetoid spread ) and downwards , into and through the dermis ( invasion ) . This spread raises the related question , of what drives cells to migrate away from the primary tumour . Simple , random migration is an extremely inefficient way of dispersing cells and also unlikely to drive cells to invade through matrix and basement membranes . Chemotaxis—cell migration directed by gradients of soluble signalling molecules—is implicated as an important driver of metastasis by a wide range of data [ 7 , 8 ] , and is considered necessary to drive efficient invasion . In breast cancer , for example , some tumour cells migrate towards epidermal growth factor ( EGF ) [ 9 ] . However , EGF gradients have only been inferred in vivo , never measured , and their sources are usually unclear . In the case of breast cancer , the EGF is thought to be secreted by macrophages recruited in a paracrine loop by the tumour [ 10 ] , but for other attractants and cell types the sources of chemotactic signals are not known . In the melanoma literature , most chemotaxis is attributed to growth factors such as platelet - derived growth factor ( PDGF ) and EGF [ 11 ] and the CXCR4 ligand SDF - 1 [ 12 ] , though a wide variety of potential attractants have been discussed [ 13 ] . Gradients of growth factor or SDF - 1 have not been identified in vivo , they can only be inferred from the cells’ behaviour or pattern of responses in vitro . Chemotaxis and Invasion Assays Chemotaxis assays are typically performed in transwell cham - bers , in which cells are grown on one side of a membrane filter and potential attractants are added to the other side . Chemotaxis is assayed by the number of cells observed on the far side of the filter after a fixed interval . These assays are subject to a wide range of artifacts . Cells’ behaviour during chemotaxis cannot be studied , which makes it extremely difficult to distinguish chemotaxis from directionless changes in migratory behaviour ( i . e . , chemokinesis [ 14 ] ) . Potential attractants form extremely steep and rather short - lived concentration gradients , unlike the physiological conditions the assay aims to reproduce . More seriously still , conditions either side of the filter may be discretely different ; cells may grow , survive , or adhere better on one side of the filter than the other , giving changes in the numbers of cells that can be artifactually interpreted as chemotaxis . Direct viewing chambers , such as Dunn , Zigmond , or Insall chambers , are more laborious to use but yield a far higher quality of data , with fewer artifacts [ 15 – 17 ] . In work described here , we use direct - viewing chambers to identify lysophosphatidic acid ( LPA ) as a far more potent chemoattractant for melanoma cells than other previously described attractants . We have developed and refined two direct - viewing assays to assess mechanisms of cell dispersal and chemotaxis , allowing us to distinguish chemotactic from chemokinetic and contact - driven responses under defined conditions that minimize artifacts . Furthermore , the use of direct - viewing chambers makes compar - ison of attractants’ relative efficiencies practical . The Source of Attractant Gradients In Vivo The suggested role of chemoattractants in cancer dispersal— whether growth factors , chemokines , or LPA—raises the crucial question of how gradients are generated . Chemotaxis will only work with signals that are presented as gradients—homogeneous signals contain no directional information—and the steeper the gradient , the more efficient the chemotaxis . Chemical gradients are typically effective over distances of less than a millimetre— limits on the efficiency of diffusion make larger gradients impractical [ 18 ] . Thus for a gradient to be formed there must be a gradient source that is close to the tumour . Alternatively , local gradients may be formed from signals that are widely produced , but are absorbed or broken down locally . This local depletion mechanism is potentially just as effective as local production , but less often invoked . In the cancer literature , only localised sources are typically invoked , for example individual macrophages within the vasculature attracting cancer cells within the tumour [ 10 ] . If cells that are responding to a stimulus are also responsible for breaking it down , the result is a self - generated gradient . Under these conditions the gradient is always oriented away from the current location of the cells . One such example has been shown during the development of the zebrafish lateral line primordium [ 19 – 21 ] , in which a dummy receptor locally absorbs an SDF - 1 stimulus to set up a gradient that is detected by a different receptor . In this work we find that melanoma cells self - generate chemotactic gradients from unlocalised , exogenous LPA . These gradients tend to direct cells to disperse outwards from tumours , thus directly promoting metastasis . Furthermore , we measure LPA gradients across real melanomas in vivo . Since melanomas of sufficient size both generate their own LPA gradients and respond to them , chemotaxis - steered spread of melanomas is almost inevitable . Results Density - Dependent Outward Migration of Tumour Cell To examine the signals that drive the spread of melanoma cells , we set up 2 - D assays for tumour cell spread using a direct - viewing chemotaxis chamber that allows detailed analysis of cell migration [ 15 ] . The chamber contains two wells , connected by a bridge that allows diffusion of attractants but not flow . Both cells were homogeneously filled with complete medium , but cultured melanoma cells [ 22 ] were only seeded in one well , at a range of different densities . Author Summary Melanoma is feared because it spreads very rapidly when tumours are relatively small . It is not known why this metastasis is so efficient and aggressive . In particular , it is not known what drives melanoma cells to start to migrate out from the tumour . Here , we have studied the chemical signals that guide the migration of melanoma cells . We find that a component of serum , lysophosphatidic acid ( LPA ) , functions as a remarkably strong attractant for all of the melanoma cells that we examined . We also observe that melanoma cells rapidly break down LPA . We conclude that melanomas create their own gradients of LPA , with low LPA in the tumour and high LPA outside . Since melanoma cells are attracted by LPA , this LPA gradient around the melanomas serves as a signal that drives the tumour cells out into the surrounding skin and blood vessels . Finally , we show that such gradients exist in a mouse model of melanoma . Self - generated LPA gradients are therefore an intriguing new driver for melanoma dispersal . Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 2 October 2014 | Volume 12 | Issue 10 | e1001966 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 3 October 2014 | Volume 12 | Issue 10 | e1001966 Our initial results were surprising : Cells consistently spread outwards from the well in which they started , even in uniform medium without an externally applied gradient ( Figure 1B ; Movie S1 ) . This effect was density - dependent ; cells plated at 2 6 10 3 or 6 6 10 3 cells / well barely migrated , while 2 6 10 4 cells / well migrated up to 350 m m in 24 hours ( Figure 1C and 1D ) . This behaviour strikingly resembles the behaviour of real melanomas , in which the chance of metastasis is more correlated with tumour thickness than any other parameter [ 3 ] . This type of density - dependent spreading requires individual cells ( or small clusters of cells ) to migrate away from the bulk population . This dispersal occurred in our assays ; cells moved directly away from the well they resided in with unprecedented accuracy ( Movie S1 ) . This directed , non - random migration can only occur if the moving cells perceive a directional cue from the bulk population of the cells to spread . We therefore analyzed the nature of the signal that was directing cells away from the bulk population . The most probable signalling mechanisms are contact inhibition of migration [ 23 ] or chemotaxis . We therefore examined these potential mechanisms in turn . Contact inhibition ( of migration , as opposed to the more frequently described contact inhibition of growth ) is an effective mechanism for short - range dispersal in which cell : cell contact directs cells away from one another . It has been shown in other neural crest - derived cell types [ 24 ] . However we found no evidence to suggest it drives cell dispersal in our assays . Movie S2 shows one example in which cells spread both individually and while contacting one another . Some cells steer accurately outwards through multiple cycles of new pseudopods independently of cell : cell contact . Others continue to migrate outwards when contacting the cell in front , where contact inhibition predicts these cells should reverse into the space behind them . Analysis of the paths of individual cells ( Figure S1 ) shows that cell - cell contact is not steering cells ; the paths of cells that are contacting others , have recently contacted others , and are not in contact are strikingly similar . The one apparent example of contact inhibition ( Movie S2 , cell 2 ) changed the cell’s direction but did not improve its outward accuracy . Thus while these cells may experience contact inhibition , we considered chemotaxis as the most likely mechanism steering them away from the main population . Cells could generate chemotactic gradients to drive dispersal by either of two mechanisms . They could secrete an autocrine chemorepellent and migrate away from it . We have previously shown this to be a key driver of Entamoeba pathogenesis [ 25 ] , in which chemotaxis away from ethanol generated by the amoebas themselves causes cells to migrate from the lumen of the gut into the walls of the gut and eventually the liver of the patient . Alternatively , the melanoma cells could locally break down or consume a chemoattractant that is produced externally , but spatially homogeneously [ 26 , 27 ] , as seen in the zebrafish lateral line primordium [ 19 , 21 ] . In either case , dense populations of cells create a gradient that consistently directs migration away from themselves . We considered that homogeneous attractants would most likely derive from the serum added to full medium . To find if dispersal used a repellent or a consumed attractant , we compared cell dispersal in serum - free and normal medium . Cells in serum - free medium are healthy and motile in control movies , but do not migrate away from one another ( Figure 1E ) , demonstrating that the cells do not secrete chemorepellents . We also compared cells moving out of fresh medium into serum - free and full medium . Cells dispersed far more efficiently into the rich medium ( Figure 1F ) , implying that they are driven by attractants in fresh medium rather than an inhibitor whose production depends on serum . To test whether consumption of a component of serum produces a positive chemotaxis response , we compared migration in uniform serum to an assay in which cells are exposed to a gradient between serum - free medium and medium supplemented with 10 % serum ( Movie S3 ) . We found that both assays produced similar directed migratory responses ; cells migrated towards the opposite well with or without a preformed serum gradient ( Figure 1G ) . This finding further supports the concept that the outward migration is driven by positive chemotaxis , most likely towards a chemoattractant globally present in the serum but depleted around the cells . We tested this hypothesis using a more traditional chemotaxis assay , in which cells are spread homogeneously over the field at the start of the assay , giving the cells the opportunity to move in any direction [ 14 ] . We loaded cells into the chamber in complete medium that had been conditioned by melanoma cells for 48 hours , then replaced the medium in one well with fresh medium containing 10 % serum . The cells migrated towards the well containing fresh medium very efficiently ( Figure 2A and 2B ) , showing that an attractant in fresh medium is consumed by the melanoma cells . We confirmed that chemoattractants are present in normal serum by exposing melanoma cells—again homogeneously seeded in the chemotaxis chamber—to exogenous gradients of serum . In homogeneous serum - free medium the cells were healthy , and migrated , but randomly ( Figure 2C ) . When a gradient of serum was applied , the cells migrated towards the higher concentrations with unprecedented precision ( Figure 2D ) ; their paths are overwhelmingly oriented up - gradient , in a manner more usually associated with neutrophils and Dictyostelium [ 28 ] than cancer cells , which typically chemotax less accurately [ 29 ] . The high chemotactic index was maintained throughout a sustained period , with narrow and accurate confidence interval , and strongly significant Rayleigh test [ 30 ] for directional migration ( Figure 2E ) . Thus serum contains a remarkably potent chemoattractant for melanoma cells . We therefore conclude that melanoma dispersal across the chamber is driven by positive chemotaxis towards an attractant that is present in serum . The attractant is broken down by the cells themselves into a gradient that efficiently disperses cells . Figure 1 . Density - dependent dispersal of melanoma cells . ( A ) Schematic showing the stages of melanoma spread . ( B ) WM239A metastatic melanoma cells dispersing in uniform medium . 2 6 10 4 cells were introduced into one reservoir of an Insall chamber containing complete medium with 10 % FBS throughout , and observed by time - lapse phase contrast microscopy . See Movie S1 . The left side of each image shows the reservoir containing cells , while the right side is the viewing bridge of the chamber . ( C – D ) Migration is density - dependent . WM1158 metastatic melanoma cells were seeded at different densities in full medium with 10 % FBS , and observed as before . At 2 6 10 4 cells / well and above , peak migration distances increase sharply , as confirmed by the distance at 17 hours ( D ; graph shows mean 6 SEM ) . ( E ) Migration is not driven by production of a repellent . 2 6 10 4 WM1158 cells were introduced into a chamber in minimal medium without serum and observed at 17 hours as before . Cells survive and adhere , but do not disperse . ( F ) Migration is not driven by production of a serum - derived repellent . 2 6 10 4 WM1158 cells were introduced into a chamber in minimal medium without serum and observed at 17 hours as before . Cells disperse less efficiently in conditioned medium than in fresh medium . ( G ) Migration mediated by chemotaxis up a serum gradient is similar to density - induced migration . Left panel : 2 6 10 4 WM1158 cells were introduced into a chamber in the presence of a gradient from 0 % FBS around the cells to 10 % in the opposite reservoir [ 15 ] . The cells rapidly migrate towards the well containing serum . Right panel : similar assay with 10 % serum in both reservoirs . Panels taken from Movies S3 and S1 , respectively . doi : 10 . 1371 / journal . pbio . 1001966 . g001 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 4 October 2014 | Volume 12 | Issue 10 | e1001966 Chemotaxis during Tumour Progression One potential explanation for cancer cells becoming metastatic is that they evolve chemotactic competence as the tumours develop [ 13 , 31 , 32 ] , and thus move from unsteered to steered migration . We therefore examined the ability of a panel of cell lines isolated from different tumour stages and selected for physiologically appropriate behaviour ( Figure 3A ) [ 22 ] . Surpris - ingly , all the lines we examined responded chemotactically to serum gradients ( Figure 3B ) . Cells from metastases were more motile than cells from earlier stages ( Figure 3C ) ; highly invasive ( VGP ) cells were slightly more accurate , but not significantly faster than the biologically earlier , RGP cells . Cells from more advanced Figure 2 . Dispersal is due to a chemoattractant present in serum . All panels show data from melanoma cells migrating in chemotaxis chambers as described [ 15 ] . ( A – B ) Cells migrate from conditioned medium towards fresh medium . WM1158 cells were randomly attached to a coverslip and assembled in a chamber in 48 hour WM1158 cell conditioned medium . The medium in one chamber was replaced with fresh medium , while the other was left alone . Tracks of individual cells are shown as coloured lines ( A ) . Cells move towards the fresh medium , as shown by the spider plot ( B ) showing all cell tracks . ( C – D ) Example images showing WM239A metastatic melanoma cells after 21 hours in serum - free medium ( C ) and a 0 % – 10 % FBS gradient ( D ) . Coloured paths show centroid tracks from time 0 . ( E ) Quantitative analysis of chemotactic responses . ‘‘Spider’’ plots ( large panels ) , rose plots , mean chemotactic index , and Rayleigh test for directionality are shown for cells in serum - free medium and a 0 % – 10 % FBS gradient ( n . 100 cells in three independent experiments for both conditions ) . Spider plots show strong chemotaxis in FBS gradients ; in serum - free medium only random movement is seen . Rose plots show overall movement from 6 – 12 hours ; the proportion of total cells in each sector is shown on a log scale , with red lines representing the 95 % confidence interval . The majority of cells in the FBS gradient move in the direction of the chemoattractant . Rayleigh tests statistically confirmed this highly significant unimodal directionality . Graphs of chemotactic index were generated from the same data . doi : 10 . 1371 / journal . pbio . 1001966 . g002 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 5 October 2014 | Volume 12 | Issue 10 | e1001966 tumours responded more robustly , but the progression from nonmetastatic to metastatic was not marked by the cells newly acquiring responsiveness—all lines examined were chemotactic enough to spread away from the tumour efficiently in the presence of an appropriate gradient . Several lines of data suggest that genetic and epigenetic changes during progression from RGP to VGP increase cells’ ability to survive [ 33 ] ; our data imply that it is cell survival , rather than chemotactic sensitivity , that defines the difference . The increase in migratory ability could modulate cells’ ability to escape from a primary tumour , but our principal conclusion is that melanoma cells from all stages are chemotactic . Identifying the Chemoattractant in Serum There are multiple reports of chemotaxis driving metastasis of melanoma and other tumour cells , in particular breast cancer . Published accounts of chemotactic invasion most often describe growth factors as the attractants—for example EGF for solid tumours [ 34 ] , and EGF , hepatocyte growth factor ( HGF ) , and stem cell factor ( SCF ) / KitL for melanoma [ 13 ] . However these attractants were often identified in transwell chambers , which as earlier discussed are subject to a range of artifacts , in particular false positive . For example , the positive well might promote survival , growth , or adhesion of cells that move randomly across the membrane . Our direct - viewing chambers provide a far more rigorous analysis . We therefore tested a broad range of attractants in our assays . To our surprise , no growth factor acted as an attractant to any measurable degree ( Figure 4A ) ; steep or shallow gradients gave no obvious movement upgradient , and no significant chemotactic index towards any growth factor tested ( Figure 4B ) . We therefore conclude that the chemotaxis towards serum we observed was unlikely to be towards growth factors . This does not , of course , demonstrate that melanoma cells are never chemotactic towards growth factors ; but it clearly shows the surprising and efficient chemotaxis towards serum observed earlier is mediated by another molecule . EGF and PDGF did increase cells’ speed ( Figure 4C ) , but they did not provide directional specificity . They therefore acted as chemokines , regulating overall cell behaviour , rather than as chemoattractants that could steer the cells . The striking accuracy of chemotaxis demonstrated by melano - ma cells towards serum was more reminiscent of neutrophil chemotaxis towards formyl peptides , or Dictyostelium towards cAMP , which signal through G - protein coupled receptors ( GPCRs ) rather than growth factor receptors like EGFR and PDGFR . We therefore investigated SDF - 1 , the ligand for the GPCR CXCR4 , which has been associated with poor prognosis and malignancy of melanoma [ 35 ] ; but again , it was not measurably attractive to cells in our assays ( Figure 4B , compare with strong response to serum ) . However , LPA , another well - known component of serum that signals through GPCRs , was strikingly attractive to melanoma cells . A gradient from 0 to 1 m M LPA across the chamber ( consistent with the approximate concentration of LPA in serum ; see below ) induced chemotaxis almost as effectively as 0 % – 10 % serum ( Figure 4D ) , yielding a comparable chemotactic index ( Figure 4E ) . This was a surprise : LPA is more typically described as an inflammatory mitogen , acting on haematopoietic cells such as macrophages . It appears frequently in the cancer literature , but more often as a mitogen and chemokine for cancer cells , acting via autotaxin , which catalyzes the production of LPA from lysophos - phatidylcholine [ 36 ] . However in our assays the chemotaxis of melanoma to LPA was again remarkably accurate compared with the weaker chemotaxis typically seen in cancer cells [ 37 ] . LPA Is the Dominant Attractant in Serum in 2 - D and 3 - D Assays To examine whether LPA was the principal attractive component of serum , we assayed chemotaxis in the presence of the antagonist Ki16425 , which specifically inhibits binding to LPA receptors 1 and 3 [ 38 ] . The effects were again remarkably clear . 10 m M Ki16425 blocked cell spread in our original , density - dependent assay ( Movie S4 ) and chemotaxis towards 10 % serum ( Figure 5A ; Movie S5 ) , reducing the chemotactic index from more than + 0 . 4 to zero ( Figure 5B ) . Ki16425 - treated cells were obviously healthy , and moved similarly to untreated cells , with similar track lengths , showing that the treatment was not making the cells nonspecifically sick or non - motile . Knockdown of LPAR1 by siRNA had a similar effect ( Figure S2A ) , showing that LPAR1 is the key receptor for this process , and 10 m M Ki16425 also blocked chemotaxis towards pure LPA ( Figure S2B ) . Again , LPA chemotaxis is not tumour stage - specific ; Ki16425 blocked chemotaxis in all cell lines from all stages of cancer progression ( Figure 5C ) . RGP and VGP cell lines were completely inhibited , and the highly motile metastatic lines were substantially inhibited . The residual chemotaxis in the presence of inhibitor could represent either incomplete inhibition by the antagonist , or a small amount of chemotaxis to another agent . From these data , we conclude that LPA is overwhelmingly the dominant chemoattrac - tant in serum for all lines examined . While chamber - based assays are optimized to allow accurate and detailed recording , they provide a 2 - D view of a process that more often happens in 3 - D tissues in vivo [ 39 ] . We therefore examined the role of LPA in a widely used organotypic tumour cell invasion model [ 40 ] . In this system melanoma cells are added to the top of a plug of collagen in which fibroblasts are growing , and over time they migrate vertically downwards into the 3 - D matrix . During the course of the assay , the collagen plug is set so only its bottom face contacts the medium , at which point malignant melanoma cells invade downwards [ 41 ] . We hypoth - esized that the melanoma cells were driven by a self - generated LPA gradient as in Figure 1B , once fresh LPA could only be supplied from the bottom . This hypothesis is supported by assays in which the collagen plugs remain submerged , and no invasion is seen ( Figure S3 ) , further rejecting contact inhibition of migration as a mechanism of invasion . When the gels were treated with Ki16425 , the melanoma cells did not invade downwards into the gel ( despite comparable numbers of cells at the end , showing no change in growth or survival ) . Quantitative analysis confirms that Ki16425 strongly inhibited invasion in both cell lines that were invasive in this assay ( Figure 5D and 5E ) . Thus LPA is a dominant steering system for 3 - D organotypic assays , as well as for 2 - D chamber assays . Melanoma Cells Break down LPA to Form Outward - Facing Gradients Our earlier data ( Figures 1B and 2A , in particular ) showed that melanoma cells disperse by depleting a chemoattractant from serum . We therefore tested whether melanoma cells are able to deplete LPA from their surroundings . Full medium with and without serum was incubated with different densities of melanoma cells for different times , then LPA was extracted from the conditioned medium and analyzed by mass spectrometry [ 42 ] . This confirms that the melanoma cells effectively break down LPA ; the conditioned medium was depleted in a density - dependent manner ( Figure 6A ) and in a timescale that correlates with the medium conditioning experiments in Figure 2A and 2B . One advantage of using mass spectrometry is the identification of Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 6 October 2014 | Volume 12 | Issue 10 | e1001966 Figure 3 . Chemotaxis of cells from different melanoma stages . ( A ) Chemotaxis of a panel of six cell lines from different melanoma stages ( RGP , green ; VGP , purple ; metastatic , red ) up a 0 % – 10 % FBS gradient was measured as above ( n $ 45 cells per cell line ) . ( B ) Chemotactic index of cells from different stages . Data from ( A ) were collated by melanoma stage . Chemotaxis improves as the stage of melanoma progresses , although even the earliest RGP cells show clear chemotaxis . ( C ) Speeds of cells from different stages . Data from ( A ) were collated by melanoma stage . Metastatic lines are conspicuously faster ( p - values from unpaired t - tests ) , although again the speed of RGP and VGP cells is still relatively high for non - haematopoietic cells . doi : 10 . 1371 / journal . pbio . 1001966 . g003 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 7 October 2014 | Volume 12 | Issue 10 | e1001966 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 8 October 2014 | Volume 12 | Issue 10 | e1001966 molecular subspecies . The biological activity of LPA is known to vary with its structure [ 43 , 44 ] . In particular , there is a strong correlation between biological activity and the degree of poly - unsaturation , and also acyl chain length [ 45 ] . Melanoma cells broke down the biologically active species more rapidly than the others ( Figure 6B ) , ensuring that the most active species also formed the steepest gradients . The Role of Growth Factors The results we have obtained conflict with the established dogma that growth factors are primary melanoma chemoattrac - tants [ 13 ] . To reconcile these accounts with our data , we examined the role of growth factors during chemotaxis towards LPA . As shown previously ( Figure 4C ) , EGF and ( particularly ) PDGF increased the basal speed of cells . Gradients of EGF and PDGF , and mixtures of both , enhanced the accuracy of chemotaxis to LPA ( Figure 7 ) ; LPA , EGF , and PDGF together in serum - free minimal medium were as effective as 10 % serum . Most tellingly , however , when cells were presented with LPA and growth factor gradients oriented in opposite directions , they chemotaxed towards the LPA not the growth factors ; if anything they migrated towards the LPA with enhanced efficiency ( Figure 7B , bottom two lines ) . Thus when examined in the high levels of detail afforded by our chambers , the growth factors are potentially important accessory factors that increase cell speed and efficiency of chemotaxis , but they do not themselves act as chemoattractants . These results are reminiscent of observations of development in vivo , in which the growth factor SCF promotes migration but not direction of melanoblast migration [ 46 ] . It is possible that the melanoma chemotaxis to growth factors observed in other work [ 13 ] is due to changes in speed alone , which as discussed earlier can cause a false positive in transwell assays . It has also been shown that growth factors can cause cancer cells to secrete LPA [ 47 ] , which could also provide an element of indirect chemotaxis in many types of assay . LPA Gradients in Tumours In Vivo We have clearly shown that LPA is a potent chemoattractant for melanoma cells of all biological stages . To determine whether this chemotaxis was an important driver of melanoma chemotaxis in vivo , we investigated whether the tissue surrounding real melanomas contained LPA gradients that would direct cells out of tumours . Mice that are heterozygotes for the driver mutation Braf V600E ( the most prevalent driver of human melanomas ) and deletion of the tumour suppressor PTEN develop sporadic melanomas ( Figure 8A ) genetically and cytologically comparable to human tumours ( Figure 8B ) . We took punch biopsies from the tissue in and across melanomas ( Figure 8C ) from several mice , extracted total lipids , and examined LPA levels using mass spectrometry . In all non - ulcerated melanomas we examined , LPA levels were low inside the tumour , higher at the edges , and higher still in the tissues immediately outside the tumour ( Figure 8D ) . Cells at the edges of the tumour are therefore experiencing an outward - oriented LPA gradient tending to drive them out into surrounding tissues and vasculature . We further examined the LPA species in the tissue . Forms that are strongly associated with signalling , in particular 18 : 2 - LPA and 20 : 4 - LPA [ 48 ] , formed the steepest gradients ( Figure 8E ) , while gradients of non - signalling forms such as 18 : 0 - LPA were flatter . This finding further supports the idea that the gradients of LPA are specifically produced as signals targeted at LPA receptors . This study is , to our knowledge , the first time a chemotactic gradient has been directly measured around tumours in vivo . There are a number of situations where the presence of a gradient has been inferred from cellular behaviour , most prominently in the paracrine loops shown by Segall and others [ 10 ] . However , such gradients must by definition be local and tend to be transient . The gradients we observe in melanomas are clear , large - scale , and provide a convincing driver for cell dispersal , and one highly plausible explanation of why melanomas above a certain size , and hence Breslow thickness , always tend to be metastatic . Discussion In this work , we have shown that LPA is a potent chemoat - tractant for melanoma cells in general , and that outward - oriented gradients of LPA are self - generated by melanoma cells . Because self - generated gradients are always oriented away from tumours , this combination provides a plausible mechanism for driving tumour cell dispersal . We do not exclude other mechanisms ; it has for example been proposed that LPA regulates cadherin levels [ 49 ] , which would not be visible in our assays . Growth factor chemotaxis may be visible under the appropriate conditions ( though , as discussed previously , many data are from transwell assays , which are artifact - prone and unreliable ) . Likewise , we do not exclude other mechanisms than chemotaxis . Contact inhibition of migration occurs in many cell types derived from the neural crest and so is probably found in melanoma , and defects in cell growth and survival in inappropriate locations are of course important factors . But the mechanism we have found that overwhelmingly dominates the dispersal in our assays is robust and is apparently active in a high proportion of melanomas . It is therefore likely to be a particularly important mediator of tumour cell dispersal . We hypothesize that similar mechanisms will be common in cancer metastasis . The Source of LPA The source of LPA around melanomas is unknown . In many tumours , including melanoma , expression of autotaxin and thus autocrine production of LPA has been associated with tumour progression [ 50 ] . This LPA production appears to be a mechanism for promoting melanoma growth , rather than driving chemotaxis and invasion . LPA generated by the tumour itself would be found Figure 4 . Identification of LPA , rather than growth factors , as the principal attractant in serum . ( A ) WM239A cells were exposed to gradients of low ( light ) and high ( dark ) concentrations of several growth factors and the chemokine SDF - 1 in combination with SFM . Spider and Rose plots with Rayleigh tests are shown ( n . 40 cells for each condition ) . Concentrations tested were EGF ( 6 . 25 and 25 ng / ml ) , PDGF ( 25 and 100 ng / ml ) , HGF ( 10 and 30 ng / ml ) , SCF ( 10 and 100 ng / ml ) , and SDF - 1 ( 100 and 300 ng / ml ) . None shows obvious chemotaxis . ( B ) Quantification of data from ( A ) . Serum gradients promote strong chemotaxis ( p , 0 . 0001 , unpaired t - test ) , but gradients of all growth factors tested show no significant chemotactic index ( p $ 0 . 40 ) . ( C ) Growth factors enhance cell speed . Data quantitated from the cells in Figure 3A . Directionless cell speed was measured by totalling the distance moved between time points . EGF and PDGF stimulate cells in serum - free minimal medium to speeds comparable with serum gradients . Single asterisk : Different from SFM alone , p , 0 . 001 , unpaired t - test ; double asterisk : p , 0 . 0001 ) . ( D ) LPA and serum drive comparably efficient chemotaxis . WM239A cells were examined in a chamber responding to 0 % – 10 % FBS and 0 – 1 m M LPA . The spider plot shows similar cellular responses to the two gradients . ( E ) Quantitative analysis of chemotaxis towards LPA and serum . Chemotactic index was calculated from three experiments including that shown in ( D ) . Cells respond comparably to both conditions . Bars show SEM . doi : 10 . 1371 / journal . pbio . 1001966 . g004 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 9 October 2014 | Volume 12 | Issue 10 | e1001966 Figure 5 . LPA responses are essential for serum chemotaxis in 2 - D and 3 - D assays . ( A ) LPA receptor antagonist Ki16425 blocks chemotaxis to serum . Chemotaxis of WM239A cells was compared with and without 10 m M Ki16425 . Inhibitor - treated cells showed no chemotaxis despite essentially normal random migration . ( B ) Quantitative analysis of Ki16425 activity . Data from three experiments , including the one in ( A ) . The chemotactic index of inhibitor - treated cells is essentially zero . ( C ) Melanoma cell lines from all stages chemotaxing up a 10 % serum gradient with and without Ki16425 . Colours represent melanoma stage . In RGP and VGP cells , chemotaxis is totally blocked , while in metastatic lines it is substantially inhibited . Bars show SEM . ( D – E ) 3 - D organotypic assays . The cell lines WM98 - 1 and WM1158 are shown 6 Ki16425 . LPA receptor antagonist greatly inhibits invasion . In ( D ) , invasion index is calculated as the percentage of total cells on the organotypic matrix that invaded beyond , 30 m m as a ratio of cells on top of the matrix ( n . 1 , 000 cells per condition ) . ( E ) shows haematoxylin and eosin - stained vertical sections through gels , showing downward invasion of melanoma cells . doi : 10 . 1371 / journal . pbio . 1001966 . g005 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 10 October 2014 | Volume 12 | Issue 10 | e1001966 at a higher level in the tumour than outside it , which would oppose outward dispersal and thus metastasis . Rather , we find that the melanoma cells in culture and in tissues break down externally generated LPA , making outward - facing gradients . LPA is there - fore more likely to be generated through inflammatory process - es—haematopoietic cells , in particular , are a principal source of LPA in tissues [ 51 ] —or by inducing LPA production from stromal cells . In metastatic breast cancer xenografts , expression of LPA receptor promotes cell growth and metastasis , but the LPA is made locally by platelets , which are in turn recruited by many tumours [ 52 ] . Platelets are also a rich source of growth factors [ 53 ] . Our data therefore implicate inflammation in initiating melanoma spread . This finding has important implications for therapy . Interventions that promote inflammation without remov - ing the entire tumour could be extremely dangerous—diagnostic punch biopsies , in particular , could promote a wave of metastasis Figure 6 . Melanoma cells preferentially break down signalling forms of LPA . ( A ) LPA concentration over 48 hours during condition - ing of media , both with and without 10 % FBS by melanoma cells ( WM239A ) . FBS conditioned media demonstrates density - dependent depletion of LPA as measured by mass spectrometry . LPA remained negligible throughout 48 hours of serum - free conditioning by the same cells . Representative graph . ( B ) Analysis of LPA subspecies during melanoma cell conditioning demonstrates bioactive isoforms were depleted more rapidly by melanoma cells in both samples . Two representative graphs are shown to illustrate quantitative variability but qualitative consistency . doi : 10 . 1371 / journal . pbio . 1001966 . g006 Figure 7 . Growth factors potentiate LPA chemotaxis . ( A ) Growth factors enhance cells’ response to LPA gradients . Figure shows plots of the WM239A paths chemotaxing in gradients of LPA , LPA + EGF + PDGF , and conflicting gradients of LPA versus EGF + PDGF . ( B ) Chemotactic indices of cells in ( A ) and other conditions . Growth factor gradients if anything increase the efficiency of LPA chemotaxis , even when applied in a gradient in the opposite direction . Bars show SEM . doi : 10 . 1371 / journal . pbio . 1001966 . g007 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 11 October 2014 | Volume 12 | Issue 10 | e1001966 in response to LPA released by inflammation . From a therapeutic perspective , data from epidemiological studies suggests the anti - inflammatory drug aspirin can protect against metastasis [ 54 ] . The increased speed of the metastatic cells may be important , but may also be an artifact of selection . It remains unclear whether the increased speed of migration is clinically important , or whether Figure 8 . LPA gradients across melanomas invivo . ( A ) TYR : : CreER T2 BRAF V600E / + PTEN lox / + mice , a genetically appropriate melanoma model , were treated with tamoxifen as described in [ 56 ] , grown until melanomas spontaneously developed . Dashed box shows the region used for the samples shown in Figure 1C . ( B ) Haematoxylin and eosin - stained biopsies of murine melanomas demonstrating the dispersal of cells from a representative tumour from Figure 5A , with cells spreading directly away from the tumour . Upper image 2 . 5 6 magnification ; lower image 20 6 magnification from dashed box above , showing melanoma cells invading toward the muscle layer ( D , dermis ; M , muscle layer ) . ( C ) Biopsies from mouse melanomas . Several sites in a linear distribution were biopsied using a 6 mm punch biopsy tool within 5 minutes of the mouse being sacrificed and immediately frozen in liquid nitrogen . The positions of biopsies used for LPA measurement are indicated ( too few distant samples were obtained for a significant measurement ) . Bar shows 5 mm . ( D ) LPA concentration gradients across the margin of a melanoma . Four melanomas were sampled at three sites in a line as shown in ( A ) ( A , tumour body ; B , tumour edge ; C , skin surrounding tumour ) . Total LPA per mg tissue was quantified by mass spectrometry after weighing the tissue specimens and extracting the LPA . Outward - directed gradients of LPA were found across the margin of all the melanomas tested . Bars show SEM . ( E ) Analysis of LPA subspecies . 18 : 2 - LPA , 20 : 4 - LPA and 22 : 6 - LPA show a clearer gradient than 16 : 0 - LPA , which is though to be less active as a signalling molecule . doi : 10 . 1371 / journal . pbio . 1001966 . g008 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 12 October 2014 | Volume 12 | Issue 10 | e1001966 the fastest strains will metastasize earlier , and thus be the first to be identified . Our data suggest that even less invasive cells move rapidly and accurately enough to metastasize , but our assays may miss factors that retard cell migration . We have shown that cultured melanoma cells from throughout tumour evolution are chemotactic towards LPA in transwell assays . A recent paper has reported the opposite , that LPA is a chemorepellent for B16 cells [ 37 ] . This seems a cell - line specific effect , as these highly derived and divergent cells do not express the LPAR1 and LPAR3 receptors , which are usually highly expressed and dominate LPA chemotaxis in our assays ( Figure S2A ) . We have found that melanomas generate their own chemotactic gradients from homogeneous LPA that is exogenously provided . LPA chemotaxis is an essential feature driving melanoma invasion in 3 - D organotypic assays . We have also shown that real tumours create a chemotactic gradient of LPA in vivo . Taken together , these lines of evidence suggest a model of chemotaxis towards self - generated LPA gradients is a major driving force for melanoma dispersal ( Figure 9 ) . One unforeseen advantage of this model is that it also provides a simple unifying explanation for upward or pagetoid spread , which is a hallmark of the invasive VGP stage melanoma . In Vivo Models We have measured actual LPA gradients in animals with experimentally induced melanomas . We have also shown that all the melanoma cells we tested perform chemotaxis towards LPA gradients , in both 2 - D and 3 - D assays . It is thus reasonable to conclude that LPA gradients are sufficient signals to mediate melanoma cell dispersal . To test whether LPA is necessary for melanoma metastasis in vivo will be very difficult . Our hypothesis is that LPA gradients drive intravasation from the tumour towards local blood vessels . Many widely used metastasis assays , for example tail - vein injection , completely miss this step . Slower assays , for example subcutaneously injected xenografts , metastasize impractically slowly , and to nonphysiological targets , in particular the lymph nodes . Pharmacological approaches , for example blockade of the LPA signalling system by LPA antagonists , are confounded by the importance of LPA to the vascular and haematopoietic systems . A mouse model of melanoma that metastasizes through a physiological route and can be crossed with inducible LPA receptor knockouts does not currently exist ; when it is developed , such a model will be the ideal system for testing our model in vivo . The Gradient , Not the Signal , Is the Information The most important message from this work is that it is the gradient of LPA—not the presence of LPA per se —that contains the information . LPA is a very prevalent molecule . It is present at high levels in serum , and may be generated within tumours by cancer cells or exogenously by , for example , platelet activation . Interestingly , cells ahead of the main group do not respond even when an external gradient is applied ( in Movie S3 , for example ) . Presumably these cells reach a region where LPA levels are homogeneously high , at which point there is little or no guidance information available to them . Likewise , if too few cells are used in the spread cell assay , no chemotaxis is observed , suggesting that LPA breakdown is important even in classical chamber assays . We suspect that LPA is not a chemoattractant for low densities of cells , because they cannot break it down rapidly enough to form an appropriate local gradient . In our invasion assays , LPA becomes an attractant when—counterintuitively— cells are present at high enough densities to break down most of it . This means that the LPA gradient is self - generated by the melanoma . Self - generated gradients are currently highly topical . Recent papers showing the detailed roles of the CXCR4 and CXCR7 receptors ( which respond to and deplete SDF - 1 , respectively ) during the formation of the zebrafish lateral line have caused a spike in interest , but other methods whereby cells drive creation of attractant gradients then respond to them occur in multiple systems , especially during embryonic development [ 26 , 27 , 55 , 56 ] . More generally self - generation provides a means whereby cells can maintain a directional cue over distances that are far too large for premade gradients . Furthermore , with externally formed gradi - ents , the information that specifies the gradient must come from somewhere else . If an external gradient attracts cells during development , the secret to understanding the process lies with understanding where and by whom the attractant is being made . Self - generated gradients are different ; there is no need for external information . The gradient is generated as an emergent property of the interaction between the cells and their environment . Thisconclusion is perhaps the most interesting feature of this work . In LPA chemotaxis during melanoma metastasis , there is no need for any other cell type to set up a local gradient . The melanoma cells first generate a gradient—once the tumour is thick enough—and then respond to it by migrating away . Thus the melanoma drives its own metastasis . Methods Ethics All mice used were control cohorts from other studies . Before they were humanely killed , all mice had reached the primary or secondary end - points of their designated study . Cell Lines All melanoma cell lines used are listed by biological stage of derivation and were transferred from the Wellcome Trust Functional Genomics Cell Bank ( Biomedical Sciences Research Centre , St . George’s , University of London ) . Cells were maintained in Roswell Park Memorial Institute ( RPMI , Invitrogen ) 1640 medium , supplemented with 10 % fetal bovine serum ( FBS ) ( PAA Labs ) , 2 mM L - Glutamine ( Gibco , Invitrogen ) , and 1 % penicillin and streptomycin ( Gibco , Invitrogen ) . siRNA constructs were obtained from QIAGEN and transfected as per instructions . WM239A cells were challenged twice with siRNA , 48 hours apart , then used in the assay 48 hours after the second transfection . Insall Chamber Chemotaxis Assay Insall chambers were manufactured and used as described [ 15 ] . The chambers were drilled in advance with a 1 . 3 mm drill bit using an overhead drill press . During drilling , the chamber was secured in a small machine vice sitting inside a V - block at 45 u and a hole was drilled into each ‘‘rabbit ear’’ of the outer well to allow reverse filling . Cells were starved in PBS for 12 hours then seeded at a density of 5 . 5 6 10 4 cells / ml in CGM . Each cover slip was coated with 2 ml of the seeding suspension . After seeding cells , the six - well dish was shaken in the x and then y planes for 5 seconds each and placed in a CO 2 incubator at 37 u C on top of a shock absorbent base to prevent vibration induced patterns of cell accumulation . VALAP sealant ( vaseline , lanolin , and paraffin ) was prepared by combining the three components together in a weight ratio 1 : 1 : 1 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 13 October 2014 | Volume 12 | Issue 10 | e1001966 and melting at 100 u C on a heat block . A fine artist’s paint brush was used to apply the VALAP . Cover slips were treated with human fibronectin ( BD Biosci - ences ) 1 mg / ml throughout , generating an adsorbed concentra - tion of 4 . 17 m g / cm 2 in the range of 1 – 5 m g / cm 2 as suggested by the manufacturer . Following fibronectin coverslips were passivated with 0 . 5 % ( w / v ) heat - treated BSA solution in PBS . Chemoattractants were added to serum - free RPMI medium as required . Addition of 5 mM HEPES to the media in the sealed chamber is essential to buffer the pH of the media throughout the experiment . LPA ( Sigma ) was dissolved in a 1 : 1 ratio of distilled water : absolute ethanol to generate a 1 mM stock solution and stored at 2 20 u C . To use this as a chemoattractant , BSA was diluted to a final concentration of 0 . 05 % ( w / v ) to SFM - H ( SFM - HB ) and then 1 m l LPA was added to 1 ml to generate a 1 m M LPA solution . EGF ( Peprotech ) , PDGF , BB Homodimer ( Calbio - chem ) , HGF / Scatter Factor ( Peprotech ) , and SDF - 1 a / CXCL12 ( Peprotech ) were dissolved in PBS to a stock concentration of 10 – 100 m g / ml , stored at 2 20 u C and used as indicated . Ki16425 ( Cambridge Bio ) was stored in absolute ethanol at a stock concentration of 10 mM as per the manufacturer’s instructions . In Insall chamber assays , cells were pre - incubated for 5 minutes with a 10 m M solution before combining with reagents in the chamber at the same concentration . Time - lapse Microscopy We used a Nikon TE2000 - E inverted time - lapse microscope equipped with a motorised stage ( Prior ) and Perfect Focus System ( PFS ) to prevent focal drift due to thermal fluctuations . The entire microscope was enclosed in a plexiglass box , humidified and maintained at 37 u C with 5 % CO 2 . The Insall chamber experiments did not require the addition of supplementary CO 2 . Our microscope system was driven by Metamorph software ( Molecular Devices ) and the x , y positions were manually selected and pre - loaded . Image Processing Images were processed using ImageJ ( http : / / rsb . info . nih . gov / ij / ) , if necessary using the Image stabilizer plugin ( http : / / www . kangli . org / code / Image _ Stabilizer . html ) to correct for drift . Cells were tracked using MtrackJ ( http : / / www . imagescience . org / meijering / software / mtrackj / ) to follow the path of the cell nucleus For consistency , we attempted to track a minimum of 40 cells in every chamber assay ; in most cases this was sufficient to ensure statistical significance . The following criteria were used for deciding which cells to track : cells that moved more than 1 cell length in 24 hours ; cells that tracked continuously until the end of the experiment or until the cell migrated off the bridge or rounded up in preparation for mitosis ; cells were excluded that migrated onto the bridge during the experiment ; avoided tracking post - mitotic cells . Figure 9 . Schematic model of self - generated LPA gradients in melanoma . Like all schematics , this model is intended to clarify the underlying mechanism rather than as a detailed description . In small tumours , the rate of LPA breakdown is insignificant compared to the rates of synthesis and diffusion . Thus although the concentration of LPA is high , there is no gradient , and thus no directional signal . As the tumour becomes thicker—corresponding to an increased Breslow depth—the concentration of LPA at the centre of the tumour drops as the rate of breakdown increases and the distance that LPA must diffuse increases . This generates an LPA gradient that is low inside the tumour and high outside , driving cells to migrate out from the tumour into the surroundings . doi : 10 . 1371 / journal . pbio . 1001966 . g009 Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 14 October 2014 | Volume 12 | Issue 10 | e1001966 We developed an Excel spreadsheet ( written by DMV and AJM - M ) to facilitate the processing , analysis , and quantification . This spreadsheet automatically produces spider plots , speed , and chemotaxis index data over time . A time window was selected ( e . g . , 6 – 12 hours for melanoma cells ) and values zeroed within this window to produce end - point data . Chemotaxis index ( cos h ) plots are presented as mean 6 standard error of the mean ( SEM ) . Cos h is a function of the distance migrated in the direction of the gradient divided by the euclidian distance ( the linear distance between the start and end position of the cell ) . These data were also processed in the Circstat toolbox for MATLAB by GK [ 30 ] . This process generated rose and polar plots with 95 % confidence intervals and a Rayleigh test . Conditioned Media Preparation for Chemotaxis Assays Conditioned media were generated as follows . A sub - confluent 10 cm petri dish of WM239A cells was washed 3 6 with PBS then cells were split in a 1 : 5 ratio into five new 10 cm petri dishes and combined with fresh CGM to a final volume of 10 ml . Conditioned medium was then harvested from one dish per time - point , staggered between 0 – 48 hours ( Marked T0 , T6 , etc . ) . All 10 ml was aliquoted into 10 6 1 ml eppendorf tubes . The samples were immediately frozen on dry ice before storing at 2 80 u C . The cells in each dish were then counted . When needed aliquots of conditioned media were thawed at 37 u C and centrifuged for 10 min using a lab top centrifuge , then filtering with a sterile 0 . 2 m m filter . Organotypic Invasion Assay Collagen gels were prepared by combining 2 mg / ml rat tail collagen solution , 10 6 Minimum Essential Medium ( MEM , Invitrogen ) , and 0 . 22 M NaOH in a ratio 8 : 1 : 1 . The pH was finely adjusted to pH 7 . 2 with the 0 . 22M NaOH . One volume of FBS containing 7 . 5 6 10 5 primary human skin fibroblasts ( passage 5 – 7 ) was immediately combined with 10 ml of the gel mixture on ice . After pipetting well , 2 . 5 ml of the gel and cell mixture was added to each 35 mm petri dish . The gels were then placed in a humidified incubator with 5 % CO 2 to set for 15 – 30 minutes . A further 1 ml MEM was added to each petri dish and the gels were carefully detached to enable gel contraction in the same incubator . The media was changed every 3 days . After 6 – 7 days the gels measured approximately 1 . 5 cm in diameter and were transferred to a 24 - well dish ready for tumour cell seeding . 1 – 2 6 10 5 tumour cells were then counted and allowed to seed on the surface of each gel . The gel was carefully transferred with forceps to an elevated stainless steel grid ( Sigma , screens for CD - 1 , size : 40 mesh ) and placed in a 6 cm petri dish and this was denoted day 0 . CGM was added to cover the grid and was then carefully aspirated to leave a meniscus around the base of each gel , thereby generating an air - liquid interface . Three gels were loaded onto each grid and the medium was changed three times weekly . In experiments using Ki16425 , the gels with adherent cells were pre - incubated for 5 minutes with 10 m M Ki16425 in the CGM before raising the gels to the air - liquid interface . 10 m M Ki16425 was maintained in the CGM throughout the experiment with thrice weekly media changes as before . A typical experiment lasted 7 – 12 days . At the end of the invasion assay , each gel was divided into two with a scalpel , fixed in 4 % formaldehyde at 4 u C and sectioned before being stained with haematoxylin and eosin . Murine Melanoma Tissue We used the inducible Tyr : : CreER T2 BRAF V600E / + PTEN lox / 2 melanoma model [ 57 ] , in which the melanomas were all generated in mixed background mice from 6 – 12 weeks of age . Animals were treated with 2 mg tamoxifen topically to shaved back skin daily for 5 days . There was no discernable phenotype until naevi or primary melanomas started developing 6 – 8 weeks after induction predominantly on the treated area . Typical grooming behaviour spread the tamoxifen to other parts of the skin and / or was ingested leading to activation in other cutaneous regions . All mice used were control cohorts from other studies . Before they were killed , all mice had reached the primary or secondary end - points of their designated study . Suitable mice were identified with at least one and up to four tumours , ideally located on the back . The smallest tumour size was 4 6 4 mm to enable at least two areas to be sampled . Skin containing the tumours was rapidly dissected off the back and pinned slightly taut to paper overlying a corkboard . Sterile Punch Biopsy tools ( Stiefel ) were used to punch circular samples from the tumour and surrounding skin . The size of punch biopsy depended on the tumour size and varied from 3 – 6 mm in diameter . Samples were taken at various locations across the tumour and were coded as follows : within the tumour ( A ) , across the margin ( B ) , 5 mm from the margin ( C ) , and 10 mm from the margin ( D ) . Samples were immediately snap frozen in liquid nitrogen and transferred to a 2 80 u C freezer for storage . Control samples of normal appearing skin in the same melanoma model activated with tamoxifen were used to calculate the basal level of LPA . Each section of mouse skin underwent a series of nine punch biopsies ( A , B , and C in three replicate series ) . Liquid Chromatography - Mass Spectrometry Mice and human melanoma / skin samples ( 1 – 20 mg ) were pulverised after thoroughly cooling with liquid nitrogen . The pulverised powder was suspended in 750 m l water then used for LPA extraction . For cell culture media samples , 750 m l of cell culture media was used for LPA extraction . Media or tissue samples were spiked with 50 ng of 17 : 0 - LPA as an internal standard before extraction . LPA was extracted with 1 ml n - butanol three times at room temperature . The combined LPA extract was dried under vacuum with SpeedVac ( Thermo ) and re - dissolved in 60 m l chloroform / methanol / water 2 : 5 : 1 . 14 m l was injected for liquid - chromatogra - phy with tandem mass spectrometry ( LC - MS / MS ) analysis . For LC - MS / MS analysis , we used a Thermo Orbitrap Elite system ( Thermo Fisher ) hyphenated with a five - channel online degasser , four - pump , column oven , and autosampler with cooler Shimadzu Prominence HPLC system ( Shimadzu ) for lipids analysis . High resolution / accurate mass and tandem MS were used for molecular species identification and quantification . The identity of the lipid subspecies was further confirmed by reference to appropriate lipids standards . All the solvents used for lipid extraction and LC - MS / MS analysis were LC - MS grade from Fisher Scientific . The final amount of LPA ( ng ) is presented as a concentration per 750 ml of conditioned media analysed or per mg tissue . The data are represented graphically plotting mean 6 SEM for the concentration of LPA versus conditioning time ( for conditioned media samples ) ; and distance from tumour margin ( for tumour samples ) . Samples were normalised to position ‘‘A’’ for compar - ison between tissue samples . Supporting Information Figure S1 Paths of cells with and without cell : cell contacts . Distances are shown in microns . Cells that are contacting one or more other cells are represented as red dots . Cells that are moving without cell : cell contacts are represented as Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 15 October 2014 | Volume 12 | Issue 10 | e1001966 green dots . There is no visible difference in directional accuracy or speed between the cells with and without contacts . ( PDF ) Figure S2 Inhibition of LPA chemotaxis . ( A ) Serum chemotaxis is blocked by siRNA inhibition of LPAR1 . Assays were performed exactly as in Figure 1G , using WM239A cells that had been transfected with a non - silencing RNA ( left ) or siRNA against LPA Receptor 1 ( LPAR1 ; Qiagen flexitube GeneSolution , catalogue number GS1902 ; right ) . ( B ) LPA chemotaxis is blocked by LPA receptor antagonists . WM1158 cells were assayed as described for Figure 4D , in the presence of the LPAR1 / 3 antagonist Ki16425 ( right panel ) or a comparable amount of ethanol vehicle ( left panel ) . ( PDF ) Figure S3 3 - D organotypic assay performed while collagen plugs remained submerged in medium . The cell line WM98 - 1 that is highly chemotactic towards serum in 3 - D organotypic assays , fails to perform chemotaxis if the gels are kept submerged throughout the 14 day assay period , despite growing on top of the plug . ( PDF ) Movie S1 Outward migration of densely packed mela - noma cells in the absence of a gradient . Both wells are filled with complete medium containing 10 % FBS , but WM239A cells are only inoculated in the left well . See Figure 1A for details . Time stamps and scale bar are shown for reference . ( MOV ) Movie S2 Outward migration is not dependent on contact inhibition . See Figure 1D for details . WM1158 cells chemotax equally effectively whether or not they are contacting their neighbours . Time stamps and scale bar are shown for reference . ( MOV ) Movie S3 Outward migration up a serum gradient . WM239A cells are inoculated in the left well in medium without serum , then the right well was filled with medium containing 10 % FBS . See Figure 1G for details . Time stamps and scale bar are shown for reference . ( MOV ) Movie S4 Effects of the LPA inhibitor Ki16425 on density - dependent dispersal . The movie shows two experi - ments , without ( left ) and with ( right ) 10 m M Ki16425 . In each case both wells contains medium with 10 % FBS as in Movie S1 . Cells were introduced into the left lane at time zero . Time stamps and scale bar are shown for reference . ( MOV ) Movie S5 Effects of the LPA inhibitor Ki16425 on serum chemotaxis . The movie shows two experiments , without ( left ) and with ( right ) 10 m M Ki16425 . WM239A cells were spread evenly on coverslips in chemotaxis chambers . In each case the left hand well contains medium without serum and the right hand well contains medium with 10 % FBS . See Figure 4A for details . Time stamps and scale bar are shown for reference . ( MOV ) Acknowledgments We are very grateful to Mike Edward , Jean Quinn , and Paul Timpson for assistance with organotypic assays , and the Beatson Advanced Imaging Resource ( BAIR ) for microscopy . Author Contributions The author ( s ) have made the following declarations about their contributions : Conceived and designed the experiments : AMM DCB OJS RH RJ LMM MJOW DAK RHI . Performed the experiments : AMM OS QZ ES WF CL . Analyzed the data : AMM QZ OS GK MJOW DAK RHI . Contributed reagents / materials / analysis tools : WF CL DMV . Wrote the paper : AMM DCB LMM MJOW DAK RHI . References 1 . Balch CM , Gershenwald JE , Soong SJ , Thompson JF , Atkins MB , et al . 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Self - generated LPA Gradients Drive Melanoma Dispersal PLOS Biology | www . plosbiology . org 17 October 2014 | Volume 12 | Issue 10 | e1001966 \ No newline at end of file diff --git a/silver_data/0ae99bbe0a038ea33aa764b4af08cca36201f90d.pdf.txt b/silver_data/0ae99bbe0a038ea33aa764b4af08cca36201f90d.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..490c660ae2fae31edae6094d16d22bd525f68fdd --- /dev/null +++ b/silver_data/0ae99bbe0a038ea33aa764b4af08cca36201f90d.pdf.txt @@ -0,0 +1 @@ +ORIGINAL PAPER Relational Reasoning and Its Manifestations in the Educational Context : a Systematic Review of the Literature Denis Dumas & Patricia A . Alexander & Emily M . Grossnickle # Springer Science + Business Media New York 2013 Abstract Relational reasoning , the ability to discern meaningful patterns within otherwise unconnected information , is regarded as central to human learning and cognition and as particularly critical for those functioning in today ’ s information age . However , the literature on this foundational ability is currently housed within a range of domains of inquiry , where divergent terminology and methodologies are commonplace . This dispersion has made it difficult to harness the power of existing work to inform future research or guide educational practice . In order to address this lack of consolidation , a systematic review of relational reasoning was undertaken . Specifically , 109 empirical studies dealing with relational rea - soning in general or one of four manifestations ( i . e . , analogy , anomaly , antinomy , and antithesis ) were analyzed . Resulting data revealed trends across fields of inquiry , including a degree of conceptual ambiguity , conceptual and operational misalignment , and a lack of ecological validity in certain research paradigms . There were also particular forms and measures of relational reasoning that were more commonly investigated , as well as certain domains that were more often studied . Implications for how future research can examine relational reasoning as a multidimensional construct within educational contexts are also discussed . Keywords Relational reasoning . Analogy . Anomaly . Antinomy . Antithesis It operates … uniting in a moment what in nature the whole breadth of space and time keeps separate . William James ( 1890 ) , The Principles of Psychology , p . 346 Educ Psychol Rev DOI 10 . 1007 / s10648 - 013 - 9224 - 4 Electronic supplementary material The online version of this article ( doi : 10 . 1007 / s10648 - 013 - 9224 - 4 ) contains supplementary material , which is available to authorized users . D . Dumas ( * ) : P . A . Alexander : E . M . Grossnickle Department of Human Development and Quantitative Methodology , University of Maryland , College Park , MD 20742 - 1131 , USA e - mail : ddumas @ umd . edu In his Principles of Psychology ( 1890 ) , William James describes a foundational human mental ability used to uncover relations of “ differences and similarity ” ( p . 346 ) between mental representations . James argues that without such an ability , we would be trapped in a world populated only by isolated stimuli , unable to connect the objects of our perception across time and space . For James , this ability necessarily serves as a starting point for human thinking and learning , as well as a mark of expertise in any domain . While the world has changed dramatically in the 120 years since James ’ s writing , such a mental ability has not diminished in significance . Rather , this ability , termed relational reasoning — which can be simply conceived as the ability to recognize or derive meaningful relations between and among pieces of information that would otherwise appear unrelated ( Alexander and the Disciplined Reading and Learning Research Laboratory 2012 ) — may be of even greater importance given the deluge of sensory information confronting members of post - industrial societies . In effect , the particular confluence of forces that currently shape the educational land - scape may contribute to the relevance of relational reasoning in the modern context . For example , today ’ s students are constantly inundated with information , much of it available in milliseconds via the Internet , from the moment they wake up in the morning to the moment they fall asleep at night . While recent research calls into question the ability of students to meaningfully deal with this deluge of information ( e . g . , Strømsø and Bråten 2010 ; List et al . 2012 ) , the availability of information continues to grow at an exponential rate ( Bohn and Short 2009 ) . At the same time , despite having near - constant access to the ocean of information available via hypermedia , students in the USA have repeatedly lagged behind other countries in international standardized assessments ; a situation that has been much maligned both in the popular media ( PBS Newshour Online 2010 ) and in scholarly writing ( Schmidt et al . 1999 ) . This apparent disconnect between the spate of information confronting students and their concomitant ability to organize and use that information effectively to guide their actions and build their knowledge — which some have suggested may be partially explained by differences in instructional emphasis on relational reasoning ( Richland et al . 2007 , 2010 ) — begs two questions . First , what cognitive abilities are most useful to students in attempting to derive coherent representations or informational patterns out of this informational torrent ? Second , how can these abilities be brought to bear on a range of academic problems to facilitate learning and development ? Granted these questions are large enough to encompass the careers of many dedicated educational psychologists , and fully answering either is well beyond the scope of this review . Nonetheless , the search for answers to these significant enigmas fueled this system - atic examination of the literatures relevant to relational reasoning . Specifically , relational reasoning is one cognitive ability that has been put forward as potentially important for those who are part of today ’ s post - industrial societies continually awash in information . This is because , when incorporated into the intellectual habits of students , relational reasoning may prove enabling by supporting the consolidation and coherence of otherwise fragmented information into meaningful and applicable units . For example , accessing information through search engines like Google or Bing can encourage students to deal with information in a piecemeal or superficial way ( Fast and Campbell 2004 ; Griffiths and Brophy 2005 ) , while relational reasoning may counteract this tendency by allowing learners to process material more deeply and to develop more principled knowledge out of seemingly isolated data ( Van Gog et al . 2004 ) . Today , relational reasoning is generally regarded as central to human cognition ( Hofstadter 2001 ) and education ( e . g . , Richland and McDonough 2010 ) . It has been identified as an Educ Psychol Rev important ability associated with early learning ( Goswami and Mead 1992 ) as well as a hallmark of expert performance , especially in scientific domains ( Dunbar 1995 ) . The empirical study of relational reasoning has emerged as a burgeoning field in recent years , especially in the field of cognitive neuroscience ( Krawczyk 2012 ) . There is also a growing interest in relational reasoning by those in the educational research community ( Aubusson et al . 2006 ) , and the construct holds an increasingly important place in educational practice ( Stephens 2006 ) . At the present time , research on the construct of relational reasoning appears to have reached a critical point : one where clarity and theoretical focus will likely fuel innovative programs of study , while vagueness and obscurity on the part of the field will likely result in a fizzling out of rigorous scientific interest in the construct ( Knowlton et al . 2012 ) . However , because relational reasoning has been studied in a variety of divergent research paradigms ( e . g . , neuroscience , cognitive psychology , child development , and educational psychology ) , achieving theoretical clarity is not a simple task . In essence , the diversity of research perspectives in the literature on relational reasoning potentially contributes to conceptual and methodological confusion ( e . g . , Tzuriel and George 2009 ; Volle et al . 2010 ) . A literature review featuring a close examination of definitions and methodologies would , therefore , be an important step in producing clarity and organization on the part of the field and supporting the communication of researchers across diverse research perspec - tives . However , to our knowledge , a review that seeks to examine the currently existing literature on relational reasoning as it pertains to learning and development has not been attempted . So , because of relational reasoning ’ s potential importance to the educational context of today , as well as the pivotal moment that the field finds itself in , we determined that a systematic review of the extant empirical literature on relational reasoning as it pertains to learning and development was warranted . While a review of literature on relational reasoning has recently been published in the field of cognitive neuroscience ( Krawczyk 2012 ) and a handbook chapter on the topic has also recently appeared ( Holyoak 2012 ) , the present review differs from these publications in several significant ways . For one , those prior examinations of relational reasoning were more specialized in terms of the arena of inquiry ( i . e . , neuroscience ; Krawczyk 2012 ) , or were intended to summarize particular programs of research ( i . e . , Holyoak 2012 ) . In addition , these prior reviews were centered on one particular form of relational reasoning ( i . e . , analogical reasoning ) and did not broadly consider interdisciplinary publications or multiple manifestations of relational reasoning . In contrast to those prior reviews , we set out to gather literature from diverse fields of inquiry and bring them together to be analyzed , as well as include publications representing four particular manifestations of relational reason - ing ( i . e . , analogy , anomaly , antinomy , and antithesis ) . Another characteristic of the prior reviews was their more traditional ( i . e . , non - systematic ) approach to literature identification . In this investigation , we systematically gathered literature using an online database by means of theoretically derived search terms . Further , neither of the previous reviews examined the literature with particular regard for academic outcomes . In the present review , in light of our concern for educational relevance , that is what we endeavored to do . The overarching objective of this review was to derive cross - disciplinary principles about the conceptual and operational nature of relational reasoning that could be used to guide the formation of theoretical models and the conduct of empirical research in the future , as well as potentially influence educational practice . Such a simple statement of overarching intention masks the challenge we faced in unearthing the relevant literature on relational reasoning that was the basis of our systematic analysis ; a literature nested within quite varied domains of inquiry ( e . g . , cognitive science or developmental psychology ) , where alternative terminology and diverse methodologies were evident . Nonetheless , we regarded this Educ Psychol Rev challenge as worthwhile . Through the identification and consolidation of this cross - disciplinary research , we were able to closely examine the included studies with an eye toward identifying any salient definitional and methodological patterns . Only by the iden - tification of these patterns were we able to delve even more deeply in this body of work and to specifically link relational reasoning ability to performance in an academic domain . Coincidentally , we were able to distill guiding principles about relational reasoning only by engaging in a manner of relational reasoning . Before we offer the details of our search efforts , overview the findings of this systematic search , and share the resulting relational reasoning principles , we first consider it informative to survey the theoretical roots of the construct of relational reasoning as defined herein . Conceptual Frame Relational reasoning has long been regarded as essential to human mental life ( e . g . , Hofstadter 2001 ; James 1890 ; Sternberg 1977 ) . As noted , James ( 1890 ) described the ability as fundamental to human thinking and learning , as well as to expert cognitive performance in any domain . Contemporaneously , Gestalt theorists like Wertheimer ( 1900 ) , whose work posits an ability to grasp the relations between discrete elements as part and parcel with perception , also described a human ability to discern relations . Later in the twentieth century , in The Abilities of Man ( 1927 ) , Charles Spearman described a model of human cognition largely driven by the ability to “ bring to mind any relations that essentially hold ” ( p . 165 ) between two ideas or concepts . His belief in the importance of this human cognitive function was so strong that he bemoaned what he saw as the neglect of the cognition of relations by his contemporaries . Using an idiomatic analogy , Spearman wrote that this could “ only be explained by their not seeing the forest for the trees ! ” ( p . 166 ) . This strong belief in the importance of reasoning by relations carried forward to his students , John Raven and Raymond Cattell , both of whom created tests of intelligence that required individuals to reason with complex relations ( Cattell 1940 ; Raven 1941 ) . Even today , the Raven ’ s Progressive Matrices ( RPM ) is a commonly used set of stimuli in empirical research studying relational reasoning ( e . g . , Baldo et al . 2010 ) . Other researchers in the field of intelligence like Sternberg ( 1977 ) also posited an ability to recognize and reason with relations as fundamental to human cognition and argued that individual differences in this ability may be important in explaining differences in intelligence . Also in the early twentieth century , in his book Judgment and Reasoning in the Child ( 1928 / 1966 ) , Jean Piaget presented an argument for the centrality of reasoning with relations to cognitive development . His claim that “ the fertility of reasoning is due to our unlimited capacity for constructing new relations ” ( p . 196 ) was evidence of the crucial role in which he cast this ability . Modern developmentalists like Goswami ( 1992 ) pursued inquiries into relational reasoning and described it as a foundational capacity used for early learning in many domains . Specifically , focusing the developmental lens on the ability to attend to and reason with relations has led to fruitful research on topics such as object permanence in infants ( Baillargeon and Graber 1987 ) , preschool reading ( Goswami and Bryant 1992 ) , the effects of preschoolers ’ socioeconomic status ( White and Caropreso 1989 ) , and adolescent brain development ( Dumontheil et al . 2010 ) . Cognitive scientists have likewise engaged in the detailed study of relational reasoning . For example , in their now - classic study , Chase and Simon ( 1973 ) observed chess masters chunking chess pieces using their strategic relations to other pieces on the board . Since that Educ Psychol Rev time , cognitive scientists have sought to understand the mechanisms underlying the human ability to recognize and reason with relations ( e . g . , Gentner 1983 ) . This field has examined these underlying mechanisms by means of diverse methodologies such as computational models ( Hummel and Holyoak 2005 ) , brain - imaging ( Waltz et al . 1999 ) , text - based studies ( Lee and Holyoak 2008 ) , and naturalistic observations ( Dunbar 1995 ) . The resulting body of research has led to a greater understanding of how humans reason relationally , as well as the role this construct plays in other mental functions like memory ( Kokinov and Petrov 2001 ) and perception ( Mitchell 1993 ) . Hofstadter ( 2001 ) describes relational reasoning as essential to all human thought when he depicts it as “ the very blue that fills the whole sky of cognition ” ( p . 499 ) . Historically , relational reasoning has been described as a singular ability , whose archetypal use was in reasoning with analogies ( Goswami 1992 ; James 1890 ) . Re - cently however , researchers have suggested that relational reasoning may not be a unitary construct , but that the term may refer to a family of relations that can arise when otherwise disparate information or representations are encountered . For example , Alexander and the Disciplined Reading and Learning Research Laboratory 2012 have suggested that at least four forms of relational reasoning merit further examination : analogy , anomaly , antinomy , and antithesis . An analogy involves the recognition of relational similarity between two seemingly disparate ideas , objects , or events ( Hesse 1959 ) . Analogic reasoning has been shown to be a vital ability for experts in scientific domains ( Dunbar 2001 ) , as well as instrumental in knowledge transfer ( Gentner et al . 2003 ) . Further , the ability to reason by analogy has been empirically connected to learning in many domains including mathematics ( Alexander et al . 1997 ) , reading ( Goswami and Mead 1992 ) , and science ( Braasch and Goldman 2010 ) . Analogical thought may also be essential for understanding language and organizing memory ( Hofstadter 2001 ) . An anomaly can be any occurrence or object that is unusual or unexpected — an aberration or digression from an established pattern ( Chinn and Brewer 1993 ) . The detection of anomalous sentences has been used for nearly a century as a test for intelligence , especially in children ( Binet et al . 1913 ) . It has been shown that attending to anomalies is critical for conceptual change ( Chinn and Malhotra 2002 ) and that it is principally during the process of anomaly resolution that individuals come to understand their own implicit assumptions and logical mistakes ( Darden 1995 ) . Some theorists , like Thomas Kuhn ( 1996 ) , have argued that anomaly detection is the principal way in which science advances . Further , anomalous thinking may be imperative for evaluating inconsistencies while dealing with multiple texts ( Afflerbach and Cho 2009 ) . Another form of relational reasoning , antinomy , allows the thinker to understand what something is by ascertaining what it is not . An antinomy is a paradoxical situation in which a domain of study has come to accept two or more ideas that appear contradictory ( Sorensen 2003 ) . By extension , antinomy can also include the ability to reason with mutual exclusivity among categories and recognize and resolve the paradox that arises should they be brought together . That is , the ideas or concepts being related appear incompatible as when theoretical models or conceptual categories are determined to be ontologically distinct ( Chi and Roscoe 2002 ; Opfer and Gelman 2011 ; Slotta and Chi 2006 ) . For example , as Cole and Wertschb ( 1996 ) pointed out , an antinomy can arise when concurrently considering the theoretical arguments of Piaget and Vygotsky . Specifically , the idea that human development can be simultaneously driven by both the individual child and by social processes may involve an antinomy - based paradox . The identification and consideration of antinomies like this one have led to productive discussion and even discovery in fields like mathematics ( Russell and Educ Psychol Rev Lackey 1973 ) , reading ( Mosenthal 1988 ) , psycholinguistics ( Shaumyan 2006 ) , and intelli - gence ( Gardner 1995 ) . The last of the four forms of relational reasoning presented in this review is antithesis , a directly oppositional relation between two mental representations ( Kreezer and Dallenbach 1929 ) . The ability to conceptualize antithetical opposition is important for argument and persuasion ( Chinn and Anderson 1998 ; Kuhn and Udell 2007 ) and conceptual change with refutation text ( Broughton et al . 2010 ) . Antithetical relations may also be one of the primary ways in which human language and thought are organized ( De Saussure 2011 ; Kjeldergaard and Higa 1962 ; Marková 1987 ) . Despite the long - standing acknowledgment of and the empirical evidence for relational reasoning ’ s importance to students ’ learning and development , there remain significant questions about the conceptualization and operationalization of relational reasoning forwarded in the extant literature . For instance , it is not yet known whether definitions of relational reasoning need greater specification at the general level , or if the particular manifestations of relational reasoning need to be more fully explicated and their interrela - tions explored . Further , the perceived need for theoretical and methodological clarity by those interested in the study of relational reasoning — especially given its potential impor - tance in the current educational climate — requires the careful examination of the currently existing cross - disciplinary writing on the construct . For these reasons , we undertook a systematic review of the literature to address these unresolved issues and to address three main research questions : 1 . How has relational reasoning been broadly conceptualized and examined in the litera - ture pertaining to human learning and development ? 2 . How have particular manifestations of relational reasoning ( i . e . , analogy , anomaly , antinomy , and antithesis ) been conceptualized and operationalized in the extant literature ? 3 . How has relational reasoning been found to relate to academic performance in various educational domains ? Methods Search Parameters For this systematic review , all searches for literature were conducted in the PsycInfo database using a title and abstract search and were limited to peer - reviewed publications in the English language . In an effort to address our first research question , we used the specific search terms “ relational thinking , ” which yielded an initial pool 15 total studies , and “ relational reasoning , ” which yielded 54 documents . These searches were not limited by year , which allowed us to be as exhaustive as possible . In order to address our second research question , we searched for terms representing manifestations of relational reasoning . For these searches , we limited results to the last 5 years ( 2007 – 2012 ) . This is because we were specifically interested in the current literature on these forms of relational reasoning . Searches were conducted for the following terms : “ analogy ” ( n = 114 ) , “ analogical reasoning ” ( n = 41 ) , “ anomaly ” ( n = 138 ) , “ anomalous ” ( n = 53 ) , “ antinomy ” ( n = 2 ) , “ ontological categories ” ( n = 2 ) , “ refutation ” ( n = 7 ) , “ opposite ” ( n = 141 ) , “ antithesis ” ( n = 2 ) , and “ antithetical ” ( n = 2 ) . These terms were selected based on the manifestations of relational reasoning forwarded in previous theoretical work by Alexander Educ Psychol Rev and the Disciplined Reading and Learning Research Laboratory 2012 and were used in an effort to capture empirical publications representing these forms of relational reasoning effectively , even if the authors of these publications did not use language explicitly describ - ing their work as manifestations of the construct . Inclusion Criteria Publications were only included if they were empirical , used human subjects , and featured participants engaged in relational reasoning . These criteria excluded non - empirical publica - tions such as theoretical treatises , reviews of the literature , and scholarly rebuttals . Further , these criteria meant that the large subfield that endeavors to examine relational reasoning using computational models was not included in this review ( e . g . , Morrison et al . 2011 ; Sebag and Rouveirol 2000 ; Taylor and Hummel 2009 ) . Additionally , in order to more closely examine relational reasoning and its mani - festations within the field of education , studies were only included if they represented research pertaining to learning and development . This meant that studies representing diverse fields like psychotherapy ( e . g . , Billow 2003 ) , relationship psychology ( e . g . , Martin 1991 ) , or marketing and business ( e . g . , Siddiqi 2012 ) were not included in the review . This criterion ensured that the lens of our review was firmly focused on those empirical studies that pertained in some way to learning and development , and not on other fields . Also excluded were studies in which the authors used a relational process to present their findings , but participants ’ relational reasoning was explicitly not examined . For example , researchers in diverse fields reported finding antithetical or opposite effects with two different experimental conditions ( e . g . , Depino et al . 2011 ) . Further , researchers made analogical references to their findings vis - à - vis what has been established in the literature . Similarly , researchers reported finding anomalies in their own research ( e . g . , Summers and Duxbury 2012 ; Walther 2010 ) or endeavored to discuss antinomies that have arisen in their fields ( e . g . , Angers 2010 ) . In all these aforementioned cases , however , there was no effort to examine antithetical , analogical , anomalous , or antinomous processing in study participants . Search Results Of the initial pool of 571 publications generated by our search terms , 462 were excluded based on the aforementioned criteria , leaving a total of 109 publications to be systematically analyzed for this review . Our final counts for included studies corresponding to each of our search terms were as follows : “ relational thinking ” ( n = 2 ) , “ relational reasoning ” ( n = 29 ) , “ analogy ” ( n = 32 ) , “ analogical reasoning ” ( n = 22 ) , “ anomaly ” ( n = 11 ) , “ anomalous ” ( n = 5 ) , “ antinomy ” ( n = 1 ) , “ refutation ” ( n = 2 ) , and “ opposite ” ( n = 5 ) . Several search terms —“ ontological categories , ” “ antithesis , ” and “ antithetical ”— did not yield any studies that met the inclusion criteria . These included studies were organized into two comprehensive tables : one table for studies pertaining to relational reasoning in general and our first research question , and another for studies pertaining to the specific manifestations of relational reasoning and our second research question . Studies in each table ( see supplementary material ) were analyzed along five central aspects : academic domains examined , definition of the applicable term stated or implied , number and type of participants studied , measures used , and outcomes reported . Educ Psychol Rev Definition Coding Scheme We first coded all included studies based on whether or not a definition was presented . Only definitions of either relational reasoning or one of the four manifestations of the construct included in this review were considered . Definitions were coded as either ( a ) explicit or ( b ) implicit . An article was coded as having an explicit definition if a definition was expressly stated . For example , Kroger et al . ( 2002 ) forwarded an explicit definition of relational reasoning as “ the ability to represent and integrate complex relationships among stimuli ” ( p . 477 ) . Because our inclusion criteria required studies to feature participants reasoning relation - ally , all included studies necessarily featured some measure or task designed to document relational reasoning . As such , all included studies that did not have an explicit definition implicitly defined the construct by the measure or task that was used to elicit relational reasoning in participants . For example , although she did not explicitly state a definition of anomaly , Filik ( 2008 ) implicitly defined the construct with her task : verbal semantic anomalies like “ the mouse picked up the dynamite ” ( p . 1038 ) . Because this stimulus was designed around a conceptualization of anomaly as something unexpected or unusual and given to participants in order to engage them in reasoning with anomalies , the task represented an implicit definition of the construct . To establish the reliability of our coding scheme , the first and third author coded a subset ( 17 . 4 % ) of the 109 included studies with a very high level ( 94 . 7 % ) of exact agreement . For the one case in which the coders disagreed , the discrepancy was resolved through discussion . The first author coded the remaining studies . Additional Study Classifications In addition to the definition , academic domain studied , measures used , number and type of participants , and outcomes reported for each of the included studies were determined and entered into the tables . In the table pertaining to particular manifestations of relational reasoning , the form of relational reasoning being examined was also entered into the table . The form of relational reasoning that corresponded to each study was determined based on the search term that yielded the study . For example , because we theoretically identified refutation texts as an instance of antithesis , any studies that were identified by the search term “ refutation ” ( e . g . , Broughton and Sinatra 2010 ) were classified as antithesis in the table . If a study examined relational reasoning relevant to a specific field of study ( e . g . , mathematics or biology ) , that domain was entered into the designated column . Simply examining a particular form of relational reasoning within an academic class or major ( e . g . , psychology students ) did not warrant designation as an academic domain unless the researchers were specifically interested in examining the implications of relational reasoning in the specific domain . If a study linked relational reasoning ability to more than one academic domain , all of the domains were indicated in the table . When no specific domain was deemed relevant , a study was designated as domain general in the table . Any measures or tasks used to engage participants in relational reasoning were entered in the corresponding section of the tables . Similarly , for the participant category , the number and type of participants used in the study were recorded . Any information provided by the author pertaining to the age , grade level , status as disabled , or enrollment in a course of the participants was entered . The outcome of each study was also entered into the table . Outcomes were only entered into the table if they pertained directly to the construct of relational reasoning . For example , Birney and Halford ( 2002a ) included findings concerning psychometric properties of their measure . Educ Psychol Rev Because these findings did not directly pertain to relational reasoning , they were not entered into the table . Specifically , outcomes were only entered if they pertained to : ( a ) the physiolog - ical processes associated with relational reasoning ; ( b ) group or individual differences in relational reasoning based on age , grade level , status as disabled , or other associated cognitive traits such as fluid intelligence ; ( c ) differences in relational reasoning based on the mode of presentation or contextual frame of the relational stimuli ; or ( d ) performance differences on academic tasks based on individual differences in relational reasoning . Results and Discussion Relational Reasoning in General Our first research question concerned the way in which relational reasoning in general has been conceptualized and examined in the literature pertaining to human learning and development . The literature analyzed in the effort to answer this question was found using the search terms “ relational thinking ” and “ relational reasoning ” because here we are interested in publications examining relational reasoning itself as a general construct , and not its specific manifestations . Findings of the included studies are summarized according to the following six aspects : ( a ) domain specificity , ( b ) definitions and conceptualizations of relational reasoning , ( c ) measures used , ( d ) methodologies and ( e ) participants , and ( f ) domain - general outcomes . Domain Specificity The current literature on relational reasoning in general is dominated by research examining the construct outside of any academic domain . Of the 31 studies that pertained to relational reasoning in general , only a small fraction ( 9 . 6 % ) of studies examined relational reasoning in a way that pertained to a specific academic domain . Of those studies that were classified as domain specific , 66 % examined relational reasoning in the domain of mathematics and 33 % in the domain of biology . Definitions and Conceptualizations Within the general relational reasoning literature , there were two trends in the conceptual - ization and definition of the core construct . The first pertained to the conceptualization of relational reasoning in terms of deductive reasoning , and the second dealt with the charac - teristics of construct definitions . Deductive vis - à - vis Relational Reasoning When examining the 31 studies relevant to this category , a distinction emerged between two contrasting conceptualizations of the construct . Specifically , the majority of reviewed studies ( 67 . 7 % ) conceptualized relational reasoning similarly to what was presented in the conceptual framing of this review , that is , as an ability to attend to and reason about multiple relations between mental representations ( Crone et al . 2009 ; Krawczyk et al . 2011 ) . In contrast , a subset of the examined studies ( 32 . 3 % ) defined relational reasoning as a specific kind of deductive reasoning in which given premises are relational in nature ( e . g . , Acredolo and Horobin 1987 ; Prado et al . 2010 ) . For example , Acredolo and Horobin ( 1987 ) defined relational reasoning as “ deductive reasoning about the relations between Educ Psychol Rev objects ” ( p . 1 ) . This definition of relational reasoning can be operationalized by the following problem posed by Van der Henst and Schaeken ( 2005 , p . 1 ) : A is to the left of B B is to the left of C D is in front of A E is in front of C What is the relation between D and E ? This frequent characterization of relational reasoning as a form of deductive reasoning is a conceptualization that pertains more precisely to reasoning about the physical positioning of some object or symbol in relation to another , or about the ordering of objects based on some specified property ( Munnelly et al . 2010 ) . As our interest in learning and development seems to necessitate and as the conceptual frame presented in the beginning of this review suggests , our interest in relational reasoning stems from its potential to be broadly applicable and widely used by learners . Thus , while we do not argue that deductive reasoning as represented in the review studies is a specific case of relational reasoning , we do not regard it as equivalent to the general construct . Despite the fact that relational – deductive reasoning has a long history with the oldest representative publication being Morgan and Carrington ’ s 1944 study , the broader concep - tualization has an even longer history reaching back at least to William James . The only study we found that used both types of conceptualizations simultaneously was by Waltz et al . ( 1999 ) . However , the principal focus of their study was the role that the prefrontal cortex plays in the integration of relational complexity , and not the differences or similarities between the two conceptualizations . The variable of relational complexity was operationalized from both a relational – deductive task and a task frequently used by those who conceptualize the construct more broadly ( i . e . , the Raven ’ s Progressive Matrices ) by counting the number of relations that a participant would have to consider to correctly solve the problem . This methodology allowed Waltz and colleagues to quantify relational com - plexity across the different types of tasks , but did not facilitate a comparison between the different relations found in each . The results of Waltz et al . ( 1999 ) showed that as relational complexity increases , so does task difficulty for both types of tasks . The researchers also showed that patients with damage to the prefrontal area of the brain had a much steeper fall in accuracy on both tasks as relational complexity increased then healthy controls . Thus , their results may suggest that these two conceptualizations of relational reasoning are , as we contend , associated . It is important to state that we recognize the significance of the recent research on deductive reasoning , especially given the strides that this field has made in uncovering how complex thought happens in the brain ( e . g . , Mason et al . 2010 ) . Nonetheless , for the remainder of this review , findings and discussion will incorporate only those publications that define relational reasoning in the broader sense as pertaining to recognizing and deriving meaningful relations between multiple mental representations . Characteristics of Definitions Among these 21 publications examining relational rea - soning in general , there was a lack of explicit definitions . Most of the included studies ( 61 . 9 % ) defined the construct of relational reasoning implicitly ( e . g . , Baldo et al . 2010 ; Scruggs et al . 1994 ) , with a minority ( 38 . 1 % ) explicitly stating a definition ( e . g . , Crone et al . 2009 ) . Notably , those studies that defined the construct implicitly used a wide variety of measures and tasks to do so . The measure that was most relied upon to implicitly define relational reasoning in the included studies was Educ Psychol Rev RPM . However , only 23 . 1 % of those studies coded as implicit utilized the RPM . The diversity of measures used in the literature pertaining to relational reasoning in general is further explicated in the measures section of this review . All of the definitions of relational reasoning put forth in these included studies — whether implicit or explicit — posited a broadly applicable ability to consider relations between concepts . Because of this , the current conceptualization of relational reasoning found in the literature seems to be compatible with the foundational ability that theorists like James and Spearman first described . For example , Crone et al . ( 2009 ) defined relational reasoning broadly as “ the ability to consider relationships between multiple mental representations ” ( p . 55 ) . As with the definitions of James and Spearman , this conceptualization portrays relational reasoning as a potentially funda - mental construct with wide - ranging implications . Based on our analysis of the general definitions of relational reasoning populating the literature , we offer a summary definition of this general construct in Table 1 , along with literature - derived definitions for more particular manifestations of relational reasoning . Further , the wide applicability of this conceptualization seems to support the argument forwarded by Alexander and the Disciplined Reading and Learning Research Laboratory 2012 that relational reasoning may not be a unitary construct , but may refer to a variety of relations that can be identified between pieces of otherwise discrete information . This work suggests that at least four forms of relational reasoning may be important for further study : analogy , anomaly , antinomy , and antithesis . Table 1 Literature - based definitions for relational reasoning and its manifestations Generalconstruct / Particularform Definition Example citations Relationalreasoning The ability to discern meaningful patterns within otherwise unconnected information Alexander and the Disciplined Reading and Learning Research Laboratory 2012 ; Crone et al . 2009 ; Holyoak 2012 ; Krawczyk et al . 2011 Analogy The identification of a structural similarity between two or more concepts , objects , or situations Bostrom 2008 ; Trey and Khan 2008 ; Leech et al . 2007 ; Watson and Chatterjee 2012 Anomaly The ability to discern an abnormality , digression , or deviation from an established pattern Ferguson and Sanford 2008 ; Filik and Leuthold 2008 ; Schulz et al . 2008 ; Trickett et al . 2009 Antinomy The ability to recognize incompatibilities or paradoxes within an informational stream Cole and Wertschb 1996 ; Gardner 1995 ; Mosenthal 1988 ; Russell and Lackey 1973 ; Shaumyan 2006 ; Sorensen 2003 Antithesis The identification of a directly oppositional relation between two ideas , concepts , or objects Baker , et al . 2010 ; Bianchi et al . 2011a , b ; Heit and Nicholson 2010 Educ Psychol Rev Measures Our analysis of the empirical literature dealing generically with relational reasoning revealed a noticeable mismatch between conceptualization and operationalization . That is to say , despite its broad conceptualization , relational reasoning was often operationalized narrowly . More to the point , many of the articles we examined tended to operationalize the construct solely by means of a measure of analogical thinking and most often via one commonly used instrument . Specifically , 33 % of the 21 included studies in this portion of the review utilized the RPM ( e . g . , Eslinger et al . 2009 ; Baldo et al . 2010 ) or some instrument for which researcher - developed items closely resembled the RPM ( e . g . , Krawczyk et al . 2011 ) . One possible reason for the aforementioned pattern is that alternative measures with adequate psychometric properties may not be readily available to those invested in the study of relational reasoning . The creator of the RPM , John Raven , was a student of Charles Spearman . Like his mentor , Raven argued for the centrality of relational reasoning to human intelligence . However , Raven ’ s instrument has come to be seen as wholly analogical in nature ( Bunge et al . 2005 ) . Although research using the RPM has been fruitful for the field , an over reliance raises several concerns . For one , it constrains the measurement of the broad construct of relational reasoning to one particular manifestation ( i . e . , analogy ) . Further , there is the issue of separating the construct of relational reasoning from fluid intelligence and general cognitive function — the trait which the RPM is currently marketed and widely used in clinical settings to assess ( e . g . , Warren et al . 2004 ) . Other commonly used measures in this literature include relational match - to - sample tasks ( used by 9 . 5 % of the 21 included studies ) where participants are asked to identify a given pattern and then select its analog ( Son et al . 2011 ) , verbal four - term analogies in the form of A : B : : C : D ( 14 . 3 % ; e . g . , Wendelken et al . 2008 ) and four - term pictorial analogies , also in the form A : B : : C : D ( 23 . 8 % ; e . g . , Krawczyk et al . 2008 ) . Each of these tasks is different from the RPM , but they all share the same focus on analogy as the form of relational reasoning being tested . Methodology More than half ( 57 . 1 % ) of the 21 included studies came from a cognitive neuroscience paradigm ( e . g . , Kroger et al . 2002 ; Wendelken et al . 2008 ) and , as such , used neuroscientific methods , especially functional magnetic resonance imaging ( fMRI ) , to examine relational reasoning in participants . This large body of work means that much is known about the cortical substrate of relational reasoning ( for a review of these findings , see Krawczyk 2012 ) . However , although brain imaging was the most common methodology used in this body of literature , there were studies incorporating methodologies closer to the educational context . These include research conducted in schools ( e . g . , Farrington - Flint et al . 2007 ; Son et al . 2011 ) , in which relational reasoning ability was empirically connected to educational outcomes and inferences were made about the best way to promote learners ’ relational reasoning . Other methodologies present in this literature included cognitive tasks and measures given in a laboratory setting ( e . g . , Birney and Halford 2002b ) and semi - structured interviews ( Stephens 2006 ) . Participants One apparent strength of the literature on generic relational reasoning is the many different populations that have been studied . The construct of relational reasoning has been examined in preschoolers ( Son et al . 2011 ) , elementary - school students ( Crone et al . 2009 ) , Educ Psychol Rev adolescents ( Eslinger et al . 2009 ) , students with intellectual disability ( Scruggs et al . 1994 ) , healthy adults ( Krawczyk et al . 2011 ) , men with Klinefelter ’ s syndrome ( Fales et al . 2003 ) , patients with brain injuries ( Waltz et al . 1999 ) , and stroke patients ( Baldo et al . 2010 ) . This diversity of samples can allow the field to make inferences on the development of this construct , as well as the ways that particular students may struggle with relational reasoning tasks based on their age and neurological condition . Domain - General Outcomes While those outcomes reported in the literature pertaining to relational reasoning in general that concerned a specific academic domain are examined in a later section of this review , outcomes concerning relational reasoning outside of any particular academic domain are discussed here . Because much of the research on relational reasoning in general has been conducted using neuroscientific methodologies , many of the outcomes reported center around the neural activation and brain physiology that is associated with the construct . For example , Krawczyk et al . ( 2008 ) found that intact pre - frontal cortex is necessary for participants to achieve a high level of accuracy on a relational reasoning task . Further , some studies reported findings that were both physiological and developmental in nature . For instance , the results of Crone et al . ( 2009 ) indicated that the development of rostrolateral prefrontal cortex is specifically implicated in children ’ s emerging ability to reason relation - ally . Reported outcomes such as this suggest that neuroscientific methods may be useful for answering questions about the development of relational reasoning ability in children and adolescents . Also present in the included literature were findings concerning the properties of rela - tional reasoning tasks , especially the elements of the task to which task - difficulty can be attributed . Studies that presented these findings generally focused on elements of tasks like relational complexity ( Birney et al . 2006 ; Waltz et al . 1999 ) or the salience of distracters ( Krawczyk et al . 2008 ) . By specifically examining the elements of a task that contribute to task - difficulty , these studies may allow the field to make more specific inferences about the source of variability in participants when reasoning relationally . Although the research on generic relational reasoning provides an important window into the role that the construct plays in learning and development , the picture may be incomplete until the research on the particular manifestations of relational reasoning ( i . e . , analogy , anomaly , antinomy , and antithesis ) are incorporated . This larger body of research — when brought together — brings to bear much more knowledge about relational reasoning and its importance in learning and development . Particular Manifestations of Relational Reasoning Our second research question for this review concerns the way in which the particular manifestations of relational reasoning ( i . e . , analogy , anomaly , antinomy , and antithesis ) have been conceptualized and operationalized in the extant literature pertaining to human learning and development . Because the literature analyzed to answer this second research question was procured using search terms that theoretically corresponded to each particular form of relational reasoning , the findings in this section of the review will be presented as corre - sponding to the form of relational reasoning the search terms represent . Similar to relational reasoning in general , findings about this literature are analyzed around six aspects of the included studies : ( a ) domain specificity , ( b ) definitions and conceptualizations , ( c ) measures used , ( d ) , participants , ( e ) methodologies , and ( f ) domain - general outcomes . Within the Educ Psychol Rev sections for each of the particular manifestations of relational reasoning , findings are organized under these six headings . Analogy The first form of relational reasoning analyzed in this review is analogy . The literature about this form of relational reasoning was identified using the search terms “ analogy ” and “ analogical reasoning . ” Domain Specificity Of the 54 studies that pertained to analogy , the majority ( 70 . 4 % ) examined the construct in a domain general way . This high proportion of studies classified as domain general suggests that the bulk of research conducted on analogical reasoning is concerned with the construct as it operates outside of any specified domain . A minority ( 29 . 6 % ) of the included studies examined analogical reasoning in a particular domain . However , domain specificity exists in a much larger proportion in the literature pertaining to analogy than it does for relational reasoning in general . Of those studies classified as domain specific , 12 . 5 % examined analogy in the domain of chemistry , 18 . 8 % in physics , and 6 . 3 % in meteorology . Combined , 37 . 5 % of the domain - specific studies examined analogy in a scientific domain . Further , 25 % examined analogy in the domain of reading , 12 . 5 % in the domain of writing , and 31 . 25 % in the domain of mathematics . These findings suggest that the interest in domain specific inquiry on analogical reasoning is not limited to one or two domains , but rather to a variety of domains in which analogical reasoning may play a role . Some of the included publications presented domain - specific findings pertaining to more than one academic domain ; these studies were included in the proportions for each of their domains . A total of 12 . 5 % of the studies classified as domain specific featured two domains of interest . For example , Klein et al . ( 2007 ) examined the role of analogical reasoning both in students writing and in their understanding of physics concepts . This study is represented in both the proportion of studies featuring the domain of writing and the proportion of studies featuring the domain of physics . Definitions and Conceptualizations In contrast to what was found in the literature pertaining to relational reasoning in general , the majority ( 57 . 4 % ) of the 54 studies pertaining to analogy featured an explicit definition ( e . g . , Ball et al . 2010 ; Cho et al . 2010 ; Thibaut et al . 2010 ) . The literature on analogy also differed from the literature on relational reasoning in general in that , while the literature on relational reasoning in general contained a rift between competing conceptualizations of the construct , the literature pertaining to analogy shared a common definitional focus . Indeed , all of the included studies on analogy forwarded a definition — implicit or explicit — that emphasized the identification of a deep structural similarity between two or more concepts , objects , or situations ( e . g . , Bostrom 2008 ; Trey and Khan 2008 ) . For example , Leech et al . ( 2007 ) defined analogical reasoning as an ability to judge “ the similarity between relations and structures of relations ” ( p . 897 ) . At the same time , Watson and Chatterjee ( 2012 ) argue that , “ two objects , concepts , or even real - world situations are analogous if they are dissimilar on the surface but share some higher - order structural similarities ” ( p . 2831 ) . As is apparent across these two quotes , a conceptualization of analogical reasoning as the recognition of and reasoning with the relation of similarity is a common thread that binds the literature together . Nonetheless , the field is full of investigations into different kinds of similarity . For example , some of the included studies focused on similarity based on category membership ( e . g . , Green et al . 2008 ) , others focused on the similarity of physical Educ Psychol Rev processes ( e . g . , Braasch and Goldman 2010 ) , and still others focused on perceptual or visual similarity ( e . g . , Holliway 2007 ) . Although these diverse conceptualizations of the relation of similarity are qualitatively different in their approach , this does not necessarily create a problem for the classification of each as analogy . This is because an analogy is not defined by the aspects of ideas that are similar , but rather by the presence of similarity itself ( Goswami 1992 ) . This relatively broad conceptualization of what constitutes an analogy has enabled the field to identify this form of relational reasoning at work in some perhaps unexpected places . For instance , an important minority of included studies framed two forms of morphological similarity between words — rime ( e . g . , spoil and boil ) and rhyme ( e . g . , light and bite ) — as analogies ( e . g . , Schiff and Ravid 2007 ) . This identification of these two forms of morpho - logical similarity as analogy has shed light on the way these forms of similarity are used in beginning reading ( e . g . , Farrington - Flint and Wood 2007 ) . Measures The most commonly used measures in the literature pertaining to analogy are verbal or pictorial four - term analogies in the form of A : B : : C : D ( e . g . , Maguire et al . 2012 ; Tzuriel 2007 ) . A significant proportion ( 40 . 7 % ) of the 54 included studies incorporated this kind of task . This prevalence of four - term analogies probably reflects the historic popularity of this form , as well the relative ease with which they can be created and presented to participants . However , other forms of analogy were also present in the literature . These forms included scene analogies ( e . g . , Gordon and Moser 2007 ) in which participants were asked to identify analogous elements between two illustrated scenarios , visual – spatial analogies similar to Raven ’ s Progressive Matrices ( e . g . , Tunteler and Resing 2010 ) , analo - gies presented in text ( e . g . , Braasch and Goldman 2010 ; Leech et al . 2007 ) , and analogies in student writing ( Holliway 2007 ) . Methodologies In contrast to the literature pertaining to relational reasoning in general — which leaned heavily on neuroscientific methodologies — there was no single dominant methodology present in the literature on analogy . The methodologies in this literature included eye - tracking ( e . g . , Vakil et al . 2011 ) , brain - imaging ( e . g . , Volle et al . 2010 ; Krawczyk et al . 2010 , 2011 ) , semi - structured interviews ( e . g . , Bostrom 2008 ) , large - scale psychometric studies ( e . g . , Ullstadius et al . 2008 ) , classroom intervention ( e . g . , Tzuriel and George 2009 ) , and think alouds ( e . g . , Braasch and Goldman 2010 ) . Participants The body of literature pertaining to analogy also encompasses an impressive diversity of participants . In the past 5 years , analogical reasoning has been studied in patients with brain damage ( e . g . , Schmidt et al . 2012 ) , students with intellectual disability ( e . g . , Denaes 2012 ; Vakil et al . 2011 ) , children with autism ( e . g . , Morsanyi and Holyoak 2010 ) , preschoolers ( e . g . , Stevenson et al . 2011 ) , elementary school students ( e . g . , Savage et al . 2011 ) , adolescents ( e . g . , Bellocchi and Ritchie 2011 ) , undergraduates ( e . g . , Braasch and Goldman 2010 ) , preservice teachers ( e . g . , James and Scharmann 2007 ) , active duty soldiers ( e . g . , Ullstadius et al . 2008 ) , and students with specific language impairment ( e . g . , Leroy et al . 2012 ) , among others . Domain - General Outcomes Because of the diversity of methodologies represented in the research pertaining to analogical reasoning , there are a wide variety of domain - general findings reported in this literature . The included studies present findings concerned with neural activation and brain physiology ( e . g . , Volle et al . 2010 ) , the mode of presentation of an analogical task ( e . g . , Stevenson et al . 2011 ) , differences in analogical reasoning based on Educ Psychol Rev age or status as disabled ( e . g . , Schiff and Ravid 2007 ) , accounts of cognitive systems that explain participants ’ processing of analogies ( e . g . , Green et al . 2008 ) , psychometric data of analogical reasoning assessments ( e . g . , Ullstadius et al . 2008 ) , and differences in the cognitive processing of analogies based on the relations present ( e . g . , Green et al . 2012 ) . For example , the results of Volle et al . ( 2010 ) suggest that the dorsomedial sub - region of rostrolateral pre - frontal cortex is specifically active during the initial mapping stages of analogical reasoning , further explicating the neural substrate of relational thought . Stevenson et al . ( 2011 ) found no significant differences in participants ’ performance across paper or computer presentation conditions of analogy tasks , alleviating any qualms in the field about presenting measures on a computer screen . Schiff and Ravid ( 2007 ) found that adult dyslexics performed similarly to typically developing third grade students on a morpholog - ical analogies task , providing a new lens on the reading deficit associated with dyslexia . Green et al . ( 2008 ) argue that four - term analogies are processed in the mind through the activation of micro - categories associated with each of the terms , using data to support their theoretical position . Ullstadius et al . ( 2008 ) present a factor analysis of an analogical reasoning measure , allowing the field to further understand the distinctions between sets of analogies based on their terms . Green et al . ( 2012 ) show that the greater the semantic distance between the terms in a four - term analogy , the greater the frontopolar cortex activation required to correctly solve the analogy , suggesting that semantic distance between terms of comparison may be associated with the difficulty of analogical reasoning in a given situation . Anomaly The second manifestation of relational reasoning to be examined in this review is anomaly . The literature analyzed here was produced using the search terms “ anomaly ” and “ anomalous . ” Domain Specificity Of the 16 studies pertaining to anomaly , 56 . 3 % examined the construct in a domain - general way , while 43 . 7 % studied anomaly in a specific academic domain . Of the four manifestations of relational reasoning reviewed , this represented the highest pro - portion of domain specificity . Of the studies classified as domain - specific , 71 . 4 % examined anomaly in the domain of reading , 14 . 2 % in meteorology , and 14 . 2 % in mathematics . Definitions and Conceptualizations There was a lack of explicit definitions in the literature pertaining to anomaly . Specifically , only a quarter ( 25 % ) of the 16 studies featured an explicit definition . The remainder ( 75 % ) defined the construct implicitly through their use of certain measures and tasks ( e . g . , Mishra et al . 2011 ; Sanford et al . 2011 ) . However , all explicit and implicit definitions coalesced around the idea of anomaly as an ability to attend to and reason with something unusual , unexpected , or that digresses from an established pattern ( e . g . , Schulz et al . 2008 ) . For example , Trickett et al . ( 2009 ) defined an anomaly as “ any phenomenon that deviates from a common form , that displays inconsistency with what is expected , or that is generally considered ‘ odd ’ or ‘ peculiar ’ in some way ” ( p . 711 ) . This definition of anomaly speaks to the importance not only of the unanticipated nature of the anomaly itself but also to the context in which the anomaly is presented . Because an anomaly is something unusual or unexpected , what is usual or expected becomes vital to its definition . Consequently , the literature on anomaly contains a subset of studies specifically concerned with manipulating the context in which an anomaly is presented , in an effort to negate its status as an anomaly , Educ Psychol Rev and turn it into something expected and quotidian ( e . g . , Ferguson and Sanford 2008 ; Filik and Leuthold 2008 ) . Measures Half ( 50 % ) of the 16 studies used verbal semantic anomalies as their principal measure ( e . g . , Faustmann et al . 2007 ; Filik 2008 ; Weber and Lavric 2008 ) . Verbal semantic anomalies are short passages or sentences that contain unusual information . For example , this short passage , taken from Filik and Leuthold ( 2008 ) , can be presented as nonanomalous : Terry was very annoyed at the traffic jam on his way to work . He glared at the [ truck ] and carried on down the road . Or anomalous : Terry was very annoyed at the traffic jam on his way to work . He picked up the [ truck ] and carried on down the road . It can also be presented in a fictional context , negating the anomaly : The Incredible Hulk was annoyed that there was a lot of traffic in his way . He picked up the [ truck ] and carried on down the road . Verbal semantic anomalies that describe something unusual , unexpected , or , in this case , physically impossible are far and away the most widely used measure in the literature pertaining to anomaly . This popularity of verbal semantic anomalies dates back to Binet et al . ( 1913 ) , who used a child ’ s ability to identify anomalies in absurd phrases as a test of intelligence . However , other measures were also utilized . These include mathematical problems solved incorrectly ( Chen et al . 2007 ) , inconsistent information presented in narrative texts ( Stewart et al . 2009 ) , and video of impossible human movements ( Kosugi et al . 2009 ) . Methodologies Similar to the literature pertaining to relational reasoning in general , the literature analyzed here leaned heavily on neuroscientific methods , with a significant proportion ( 43 . 7 % ) of the 16 studies incorporating either event - related potentials ( ERP ; e . g . , Sanford et al . 2011 ; Vissers et al . 2010 ) or fMRI ( Ahrens et al . 2007 ) . This emphasis on neuroscientific methods , especially the use of ERP , could have to do with the predicable effect — the N400 — that is elicited by verbal semantic anomalies in ERP studies . The N400 effect refers to a potential in the negative direction , observed approximated 400 ms after the stimulus onset , that is closely associated with anomaly . This clear denotation of an anomaly by the N400 effect has enabled researchers to closely study how people reason with verbal semantic anomalies and , as has already been discussed , how altering the context of the phrase can negate the anomaly and the N400 effect that goes with it . However , other methodologies are also used to make this form of relational reasoning evident in participants . For instance , eye tracking ( e . g . , Bohan and Sanford 2008 ) can operate much the same way as ERP in that it allows researchers to observe when participants identify the unexpected information in a verbal semantic anomaly . As another way to denote a participant ’ s noticing of an anomaly , read time can also be used , especially for studies incorporating slightly longer anomalous passages ( e . g . , Stewart et al . 2009 ) . A major strength of each of these methodologies is that they do not rely on participants themselves to report the existence of an anomaly , but are able to tap the consequences ( i . e . , N400 , eye - gaze patterns , and extended read time ) of an anomaly being present in stimuli . This may allow researchers to observe processes in the resolution of an anomaly that operate below the level of consciousness . Educ Psychol Rev Further , researchers like Trickett et al . ( 2009 ) have used naturalistic observation of scientific experts to explain how anomalies are attended to and resolved by professional scientists . This naturalistic observation method , termed in vivo research , was first popular - ized by Dunbar ( 1995 ) and has been shown to be effective at capturing relational reasoning happening in the minds of scientists . Participants In contrast to the bodies of literature pertaining to either relational reasoning in general or analogy , the research on anomaly relied heavily on the use of undergraduate students as participants . Three quarters ( 75 % ) of the 16 studies used undergraduates ( e . g . , Bohan and Sanford 2008 ; Chen et al . 2007 ; Sanford et al . 2011 ) . However , there are some publications in this body of literature that do incorporate participants other than undergrad - uates . For example , studies have been done with intellectually disabled adults ( Chang et al . 2012 ) , expert scientists ( Trickett et al . 2009 ) , older adults ( Faustmann et al . 2007 ) , bilinguals ( Weber and Lavric 2008 ) , infants ( Kosugi et al . 2009 ) , and preschoolers ( Schulz et al . 2008 ) . Domain - General Outcomes The domain - general outcomes reported in the included studies that pertained to anomaly included differences in cognitive processing of anomalies based on the context in which the anomaly is presented ( e . g . , Filik and Leuthold 2008 ) , the role of affect in the processing of anomalies ( e . g . , Vissers et al . 2010 ) , and reasoning with anomalies across the lifespan ( Faustmann et al . 2007 ) . For example , Filik and Leuthold ( 2008 ) found that presenting a verbal semantic anomaly in a fictional context could negate the N400 effect associated with the recognition of something anomalous , showing that the categorization of a given stimulus as an anomaly crucially depends not only on its status as unexpected but also on what , in a given context , is expected . Vissers et al . ( 2010 ) observed that the ERP effect for reasoning with an anomaly was different for participants who had just watched a happy film clip than for those who watched a sad one , showing that mood can interact with cognitive processes during the resolution of anomalies . Faustmann et al . ( 2007 ) found that age did not significantly affect the strength of the N400 effect for their sample of older adults in their 1950s , 1960s , and 1970s , providing evidence for the idea that the ability to attend to anomalies remains generally intact in older populations . Antinomy The third form of relational reasoning to be analyzed in this review is antinomy . Empirical publications studying participants ’ reasoning with antinomies were sought with the search terms “ antinomy , ” “ antinomous , ” and “ ontological categories . ” The first two search terms were chosen in the same way as the terms in our previous two searches . They are forms of the word used to represent this particular manifestation of relational reasoning . However , because of the extreme paucity of studies yielded by searches for these two terms , we expanded our search and chose the third term on a theoretical basis . Because an antinomy necessitates a paradoxical situation where two mutually exclusive categories have been brought together ( Gardner 1995 ; Sorensen 2003 ) and ontological categories are metaphysical classes of being in which all entities can be organized ( Chi and Slotta 1993 ) , we hypothesize that antinomies may arise with some frequency as learners grapple with the ontology of certain incompatible ideas and concepts . Further , previous research incorporating ontological categories ( e . g . , Slotta and Chi 2006 ) suggests that these classifications may be important for learning and conceptual change . This prompted us to include the term “ ontological categories ” with the hope that it would yield empirical research Educ Psychol Rev pertaining to learning in which participants are engaged in reasoning with antinomies . Unfortunately , this was not the case . All told , only one study representing antinomy met the criteria for inclusion in this review . This single study by Tanca et al . ( 2010 ) focused on strategies used by participants to resolve perceptual antinomies . These perceptual antinomies involved visual illusions formed by the specific placement of color and line in a figure . Although these perceptual antinomies differ from the more conceptual antinomies that are likely of greater importance for human learning and development , the strategies used by participants to resolve them may speak to strategies that students could use to resolve an antinomy between two or more conceptual categories they encounter in a learning environment . For example , Tanca et al . argued that participants resolve perceptual antinomies through a process of visually deconstructing the figure into component parts and then , when the properties of these components have been discerned , reconnecting the components into a whole . Indeed , this process seems to parallel the way in which conceptual antinomies recognized by scholars in a given field are resolved . In these publications ( e . g . , Cole and Wertschb 1996 ; Matte - Blanco 1988 ; Mosenthal 1988 ) , we observe scholars attempting to resolve antinomies through a process of first attending to the antinomy in a broad sense and then carefully deconstructing its elements and conceptually resituating them in the larger argument . Finally , when the antinomy has been resolved , the concept that was , at the outset , logically undermined by a paradox is reconstructed so as to make reasonable sense . While it is apparent that participants ’ ability to reason with antinomies is understudied in the field , it is nonetheless an important topic for examination for two reasons . First , the literature does include instances of experts reasoning by antinomy ( e . g . , Angers 2010 ) , and the ability to deal with seeming incompatibilities or paradoxes has been shown to be basic to conceptual development in classification - based sciences ( Opfer and Gelman 2011 ; Slotta and Chi 2006 ) . Such trends suggest that antinomous reasoning may play an essential , albeit it understudied , role in the development of expertise . Second , the limited body of research on antinomy , in the face of growing interest in relational reasoning more generally , begs the question as to whether it is an opportune time to raise awareness about this particular form of relational reasoning as a catalyst for further study . Antithesis The final manifestation of relational reasoning examined in this review is antithesis . The literature about this form of relational reasoning was identified using the search terms “ antithesis , ” “ antithetical , ” “ opposite , ” and “ refutation . ” The first two search terms were used because they are forms of the term used to represent this manifestation of relational reasoning , while the third was used because it is an often - used synonym for the first . The fourth search term was chosen on a theoretical basis . Specifically , because an antithesis involves a relation of direct opposition ( Kreezer and Dallenbach 1929 ) , we hypothesized that the task of directly refuting a given concept may be an important application of antithetical reasoning in the learning context . Domain Specificity Of the seven included studies that pertained to antithesis , 71 . 4 % examined the construct in a domain - general way and 28 . 6 % examined the construct in a specific academic domain . Of the studies classified as domain - specific , all of them involved two domains . Specifically , all featured the domain of reading due to the use of refutation text , and within those text - based studies , 50 % dealt with the domain of astronomy and 50 % the domain of physics . These proportions are not additive because each study that Educ Psychol Rev incorporated two domains is included in the proportion corresponding to both of those domains . For example , Diakidoy et al . ( 2011 ) featured students reading a text designed to refute misconceptions in the domain of physics . Thus , this study is included in the pro - portions for both the reading and the physics domains . Definitions and Conceptualizations None of the seven included publications in this analysis featured an explicit definition of antithesis . However , all of the implicit definitions present in this body of literature forwarded a conceptualization of antithesis as a directly oppositional relationship between two ideas or objects ( e . g . , Baker et al . 2010 ; Bianchi et al . 2011b ) . This agreement by the field means that there are no competing conceptualizations of what antithetical reasoning could be , as there was with relational reasoning in general . So , divergence in this body of literature comes mainly from other aspects of the studies , such as measures used to elicit antithetical reasoning . Measures The most common stimulus used in these publications to make participants engage in antithetical reasoning was verbal opposites like “ tall ” and “ short ” which partic - ipants place on scales of polarity ( e . g . , Bianchi et al . 2011a ) . The use of such tasks allowed researchers to make inferences about the way that the relation of opposition is used to organize our language and our thoughts ( Bianchi et al . 2011b ) . Interestingly , in a publication by Baker et al . ( 2010 ) , verbal opposites were incorporated into the design of a card game that was used to test the antithetical reasoning ability of children of different ages . This illustrates the potential use of verbal opposites to answer developmental questions . While 71 . 4 % of the seven included studies used these verbal opposites , some studies also used refutation texts designed to directly counter scientific misconceptions ( e . g . , Broughton and Sinatra 2010 ) , as well as semi - structured interviews concerning the oppositional nature of everyday concepts ( Fischer et al . 2008 ) . Methodologies Those studies included in this analysis that used refutation texts as their principle measure also used a reading based methodology in which students read the refutation text itself , as well as a control — usually an expository text of the kind often encountered by students in science classrooms ( Broughton and Sinatra 2010 ) . This research paradigm may be able to be conceptualized as a comparison between a refutation text that is designed to directly support relational reasoning and an expository text that is not . Sinatra and Broughton ( 2011 ) offer a recent review of the findings of this interesting line of research . Apart from these studies and the semi - structured interviews already described , the principal methodology utilized by this literature was the use of questionnaires concerning the oppositional nature of different words or concepts ( e . g . , Bianchi et al . 2011a ; Heit and Nicholson 2010 ) . Participants The majority ( 71 . 4 % ) of the seven studies in this section used undergraduate students as participants ( e . g . , Diakidoy et al . 2011 ; Heit and Nicholson 2010 ) . However , some participants drawn from populations other than undergraduate students were also present in this literature , namely preschoolers ( Baker et al . 2010 ) and older adults ( Fischer et al . 2008 ) . Domain - General Outcomes The domain - general findings presented in the literature pertaining to antithesis included investigations into the polarity of opposites ( e . g . , Bianchi et al . 2011b ) and age - related differences in antithetical thinking ( Baker et al . 2010 ; Fischer et al . 2008 ) . For example , Bianchi et al . ( 2011b ) found that their participants conceptualized a Educ Psychol Rev variety of types of opposites , suggesting that these multiple instances of opposition may support the idea that antithetical thinking is fundamental in the organization of human language and thought . In their investigation of age - related differences in antithetical think - ing , Baker et al . ( 2010 ) found a steep developmental trajectory in the ability to conceptualize opposites between the ages of 3 and 5 . On the opposite end of the lifespan , Fischer et al . ( 2008 ) observed that an awareness of antitheses continues to be highly prevalent in human thought after the age of 80 . Academic Outcomes The third and final research question in this review concerns the domain - specific educational outcomes to which relational reasoning has been empirically linked . To answer this question , we treated all of the studies included in this review as one body of literature . That is , all of the included publications representing particular manifestations of relational reasoning as well as that on relational reasoning in general were combined and taken to signify a broadly conceptualized construct of relational reasoning ability in diverse contexts . Then , this literature was organized around the academic domain empirically linked to relational reasoning ability . This decision to target domain - specific studies of relational reasoning was predicated on our goal to identify and examine those studies that linked relational reasoning ability to academic success in a particular educational context . The targeting of domain - specific studies meant that many studies included in the review were not featured in this particular analysis . For example , most publications from the field of cognitive neuroscience — despite the importance of their findings in our understanding of the neural and cognitive mecha - nisms of relational reasoning — were not featured here . About a quarter ( 25 . 7 % ) of the 109 studies included in this review empirically linked relational reasoning ability to success in a particular academic domain . Of these studies , 7 . 1 % examined relational reasoning in the domain of astronomy , 3 . 5 % in biology , 14 . 3 % in physics , 7 . 1 % in chemistry , and 7 . 1 % in meteorology . Combined , 39 . 1 % of the domain - specific studies examined relational reasoning in a scientific domain . Further , 28 . 6 % of the studies linked relational reasoning ability to the domain of mathematics , 39 . 3 % to reading , and 7 . 1 % to writing . Because some studies examined relational reasoning ability in two academic domains and these studies are included in the percentages corresponding to both of those domains , the percentages presented above are not additive . In elaborating further on the educational outcomes of this research , we organize discussion around the broadly conceived academic domains of mathematics , science , reading , and writing . Mathematics The studies that linked relational reasoning to mathematics learning or achievement focused on a variety of different aspects of the domain , including the correlated development of relational reasoning and mathematical ability ( Farrington - Flint et al . 2007 ) , the importance of mathematics instruction that incorporates relational reasoning ( Tzuriel and George 2009 ) , and cultural differences in cognitive supports provided for students engaging in relational mathematics learning ( Richland et al . 2007 ) . This suggests that relational reasoning ability has been empirically linked to diverse aspects of students ’ mathematics learning and achievement . For example , in their study , Farrington - Flint et al . ( 2007 ) found that the development of relational reasoning ability was an important contributor to elementary school students ’ Educ Psychol Rev ability to solve addition problems . This implies that relational reasoning ability may play a role in early mathematical operations . Indeed , Tzuriel and George ( 2009 ) found that third - grade students who took part in a classroom intervention designed to improve their relational reasoning had significantly higher mathematics ability than a control group at post test . This study utilized a standardized mathematics test that included addition , subtraction , some multiplication , and other early mathematical concepts , showing that relational reasoning ability may be fundamental for achievement in a variety of early mathematical operations . In a related study , Tzuriel and Shamir ( 2010 ) found that third - grade peer - tutors trained to teach kindergartners how to solve a relational reasoning task as well as mathematic problems showed significantly more improvement on mathematics ability than a control group who only worked on mathematics . Interestingly , these effects were found for both the tutors and the tutees , suggesting that relational reasoning ability is important for mathematics even at the kindergarten level . These findings also imply that relational reasoning may be a powerful instructional tool as well as a useful learning strategy . In an inquiry about the role of relational reasoning in mathematical pedagogy , Richland et al . ( 2007 ) analyzed videotaped instruction gathered through the Trends in International Mathematics and Science Study ( TIMSS ) . In their study , they coded the video for the cognitive support that mathematics teachers in Hong Kong , Japan , and the USA gave to relational learning . Their findings revealed that , while American teachers presented a comparable amount of relational instances as their Asian counterparts , they offered signif - icantly less cognitive support to help students reason through those situations . By presenting this finding as a partial explanation for the US ’ low score on the TIMMS as compared to Hong Kong and Japan , Richland and her colleagues pointed to a possibly fruitful paradigm in the study of relational reasoning : one where instructional support for relational reasoning , rather than the reasoning per se , is the focus of the investigation . In a follow - up study , Richland et al . ( 2010 ) found Chinese preschoolers outperform their American counterparts on a relational reasoning task even when they both have adequate knowledge of the relations at hand . Richland and colleagues point to executive functioning and a lack of explicit cognitive support for relational reasoning in the classroom as possible sources of this deficit in American children . In a later study with undergraduates in a Graduate Records Examination preparation class , Richland and McDonough ( 2010 ) found that providing cues to support relational reasoning with an instructional analogy led to increased ability at post test , as compared to teaching the same analogy and solution strategies but without the cognitive support . These findings suggest that specific teaching strategies , like visually aligning the source and target problems , may be essential if students are to reap the full benefit of relational reasoning in the mathematics classroom . However , Stephens ( 2006 ) found that many pre - service teachers do not report providing instruction about relational reasoning as a priority , despite their awareness of its utility . This finding may suggest a need to further present the importance of relational reasoning , as well as some applicable cognitive supports for its use in mathematics learning , to educational practitioners . Science Publications that empirically linked relational reasoning to achievement in scientific do - mains highlighted the advantageous use of relational thinking in science instruction ( e . g . , Braasch and Goldman 2010 ; Zheng et al . 2008 ) , text comprehension ( Broughton and Sinatra 2010 ) , and the expert performance of the professional scientist ( Trickett et al . 2009 ) . For example , in their study of 11th - grade chemistry students , Bellocchi and Ritchie ( 2011 ) Educ Psychol Rev observed that relational thinking allowed the classroom to transition into a scientific discourse from a discussion of everyday topics that were analogically related to the subject of study . Notably , Bellochi and Ritchie ’ s ( 2011 ) study represents a sociocultural view on relational reasoning and illustrates the potential for analogical thought to move a collective classroom discourse toward the understanding of a scientific concept . Multiple included studies have found that engaging students in relational reasoning about scientific topics by embedding an analogy in science instruction is a powerful tool for improving student performance ( Braasch and Goldman 2010 ; Zheng et al . 2008 ; Trey and Khan 2008 ; Zheng et al . 2008 ) . For example , Braasch and Goldman found that participants who read an expository text about weather systems , in which an analogy between weather patterns associated with El Niño and letting air out of a tire was included , had fewer misconceptions at post - test than a control group . Similarly , Trey and Khan ( 2008 ) found that dynamic computer based analogies — in this case an animated scale representing a chemical equation — enhanced high school students ’ knowledge of unobservable scientific phenomenon . Zheng et al . ( 2008 ) found a similar effect with elementary school students learning about electrical circuits using a computer interface that presented the material through the analog of water moving through pipes . Further , in their study of students with intellectual disability , Scruggs et al . ( 1994 ) found that participants were more likely to retain facts about animals if they were presented in a relational context , implying that supports for relational thinking may be useful for the study of science in the special education classroom . Publications investigating the effect of refutation texts also report important findings for science education ( Broughton and Sinatra 2010 ; Diakidoy et al . 2011 ) . This line of research has provided evidence that students who read refutation texts had fewer misconceptions about the given topic at post - test than those that read expository text , even if they spent less time reading ( Broughton and Sinatra 2010 ) . This research paradigm demonstrates how supporting students ’ relational reasoning in science learning may lead to increased achievement . It has also been shown that receiving training about pedagogy that incorporates relational reasoning — especially the analogy - based teaching of scientific concepts — can improve the teaching performance of pre - service teachers in the science classroom ( James and Scharmann 2007 ) . This finding suggests that , as in mathematics , relational reasoning can be fruitfully integrated into both teaching and learning in the science classroom . Further , Trickett et al . ( 2009 ) found that strategies for the resolution of anomalies strategies differ between experts and novices , suggesting that this form of relational reasoning continues to develop in professional scientists . Also , these strat - egies can differ in key ways based on the subject of study and the context of the problem at hand . These findings suggest that relational reasoning is not only impor - tant for the learning and teaching of scientific concepts but also for expert scientific practice in the professional setting . Collectively , this body of literature may imply that relational thinking is evident at multiple levels of scientific pursuit : learning , teaching , and expert performance . Reading Relational reasoning has been empirically linked to reading ability principally through early word reading ( Ehri et al . 2009 ; Savage et al . 2011 ) and the reading of unusual or anomalous sentences ( Ivanova et al . 2012 ) . For example , the group of studies that focuses on the importance of relational reasoning in early reading acquisition ( e . g . , Savage et al . 2011 ) conceptualize words sharing the morphological relation of rime Educ Psychol Rev ( e . g . , spoil and boil ) or rhyme ( e . g . , light and bite ) as analogically related . Reasoning with morphological relations like these may be a useful way to expand students ’ phonemic awareness and ability to decode words ( Ehri et al . 2009 ) . Further , a deficit in this relational ability may partially explain reading difficulties associated with dyslexia ( Schiff and Ravid 2007 ) . The study of participants ’ processing of verbal semantic anomalies also demon - strates ways in which relational reasoning ability can be linked to reading ( Ivanova et al . 2012 ) . For instance , passages containing inconsistent information about spatial relations or character traits reliably take participants longer to read — presumably because they must process and attempt to resolve the anomaly ( Stewart et al . 2009 ) . Further , those participants that fail to detect the anomaly in the first place generally do so because of shallow semantic processing ( Bohan and Sanford 2008 ) . Interest - ingly , it has been shown that bilingual participants reading in their second language are more likely to attend to an anomaly , and they experience a larger N400 effect upon noticing it than those reading in their first language , perhaps because they rely more heavily on a lexico - semantic network in their second language than in their first ( Weber and Lavric 2008 ) . A recent theoretical paper by Alexander et al . ( 2012 ) also points to relational reasoning as an important ability associated with reading . This work points out that relational reasoning may help students to deeply process information from multiple sources and form an integrated knowledge base out of isolated pieces of information . This potential capacity of relational reasoning — perhaps of heightened importance in the particular technological environment of the twenty - first century — still needs to be empirically explored . Writing Writing has been implicated both as a domain in which relational reasoning ability plays an important role in achievement ( Holliway 2007 ) and as an exercise useful in eliciting deeper level relational thinking in students ( Klein et al . 2007 ) . For example , in his study of elementary school students ’ writing , Holliway ( 2007 ) asked students to compose descrip - tions of abstract shapes . He found that these students used analogies , operationalized as metaphors or similes , to bridge the mental representations of writer and reader . This finding may suggest that instances of relational reasoning in students ’ writing may signify a more fully formed picture of the goals of their writing , as well as a deeper understanding of their chosen topic . In their study with undergraduate students , Klein et al . ( 2007 ) found that writing may also facilitate relational reasoning about a given topic . Participants were asked to describe commonalities between two scientific demonstrations : Some did this through speaking only , while others composed a short descriptive passage . The students in the writing condition showed greater learning about the topic at post test , than those who did not write , and those participants who had a comparatively small working memory span benefited most from the writing exercise . Notably , the positive effect of writing was mediated by an increase in mapping of and reasoning with salient relations in that condition . This finding suggests that success in relational reasoning may rely on a careful consideration of the relations at hand . Consequently , the number of relations that a student can meaningfully consider at a time may affect the extent to which that student can employ relational reasoning in a given scenario . Moreover , the exercise of writing may encourage students to engage in relational reasoning in the first place . Educ Psychol Rev Implications and Conclusions Nearly 120 years ago , James ( 1890 ) described the power and importance of relational reasoning as being able to unite in a moment “ what the whole breadth of space and time keeps separate . ” This quote was chosen as the epigraph to this review not only because it illustrates the capacity of relational reasoning to connect and bring together concepts and ideas divided temporally or spatially in our experience but because it also describes an intrinsic goal of this literature review as a whole . Part of the purpose of this review was to gather publications from a variety of fields incorporating diverse methodologies and presenting their evidence from often - divergent points of view and to bring them together as one body of work to be examined . This heterogeneous literature is united by its distinct inclusion of instances of relational reasoning in participants and can be unified by the evidence about the construct found within each piece of work . Despite the many differences in terminology and methodologies , the body of literature presented in this review has been brought together based on the inclusion of one of the theoretically identified manifestations of relational reasoning : analogy , anomaly , antinomy , and antithesis . For exam - ple , literature with such diverse foci as the cortical substrate of analogical reasoning ( e . g . , Krawczyk et al . 2010 , 2011 ) and the comprehension of refutation texts in science learning ( Broughton and Sinatra 2010 ) were included in this review because a manifestation of relational reasoning — as well as a possible applicability of findings to human learning and development — was evident in each . We do not claim that the four manifestations of relational reasoning examined in this review constitute an exhaustive list . Nor do we claim to have identified every conceivable field of study in which relational reasoning can be made evident . Although the literature we analyzed in this review was a systematically gathered and comprehensive body of work based on our search parameters , in the future , additional forms of relational reasoning may be identified , or one of the already identified manifestations may be found to be applicable to yet another context . Because of this , we hold that there is still much work to be done in the study of this potentially pervasive mental ability . We offer four underlying concepts identified in our analysis of the literature that we believe will be crucial to the study of relational reasoning going forward . These implications for the study of relational reasoning and its manifestations include : ( a ) the importance of incorporating a diversity of methodologies , participants , and measures ; ( b ) the idea that relational reasoning necessarily involves relations between relations ; ( c ) the potential for the identification of further instances of the construct at work ; and ( d ) the characterization of relational reasoning as including a family of manifestations representing various relations that can arise when ideas or information is brought together . A Diversity of Methodologies , Participants , and Measures One identifiable thread that was present throughout the literature included in this review was an overreliance on particular methodologies , types of participants , and measures . Wherever this occurred , findings were less generalizable to the larger population , easily communicated to researchers working in different fields , and informative for theoretical discussion about the construct of relational reasoning . Methodologies For example , the heavy reliance of neuroscientific methods like fMRI and ERP in the literature pertaining to relational reasoning in general as well as anomaly possibly comes Educ Psychol Rev at the expense of ecological validity and the ability of the field to describe the construct at work in diverse contexts . In contrast , the research on analogy , which incorporated a wider variety of methodologies , afforded researchers the ability to make inferences about how analogical reasoning happens in many different circumstances . It should be noted , however , that although the bodies of literature representing the neuroscience of relational reasoning and the construct ’ s importance for the educational context are still largely separate , there is a clear opportunity to incorporate these findings . Knowledge of how learners ’ ability to reason with relations happens in the brain could inform inquiries into how the construct manifests itself in education , while a greater awareness of relational reasoning ’ s role in the learning process could help frame attempts to explain this construct from a cognitive neuroscience perspective . Further , the use of exploratory qualitative methods may be an important first step by those interested in examining understudied manifestations of relational reasoning like antinomy and antithesis . For example , the evidence exists to support the idea that experts in many domains engage with antinomies ( e . g . , Cole and Wertschb 1996 ; Matte - Blanco 1988 ; Mosenthal 1988 ) . Also , theoretical arguments claim that antithesis may be a relation of fundamental importance for the organization of human cognition and language ( De Saussure 2011 ; Kjeldergaard and Higa 1962 ; Marková 1987 ) . Qualitative methods used to ascertain just how pervasive these forms of relational reasoning may be in human thought , and identifying key examples of them in action , may be an integral stage in any line of inquiry . Participants Another constraining factor identified in the literature we examined was the overreliance on undergraduate students as participants in experiments , especially in the literature on anomaly and antithesis . This dependence on undergraduates may be due in part to the research questions asked in this literature , which often focused on the modes of presentation and context of the relational stimuli being presented , and often did not require a variety of different types of participants to be addressed . Because most researchers in these fields seem to be currently interested in altering the nature of their measures and tasks , while keeping the participants relatively compara - ble , undergraduates are suitable for most experiments . However , this observation about the field could signal an opportunity to more frequently ask research questions — such as those regularly being asked by researchers working with relational reasoning in general or analogy — concerning lifespan development , or the ability of those with intellectual disability , that motivate the use of a variety of participants . In the literature pertaining to analogy , the diversity of participants may allow the field to make important inferences about the development and use of analogical reasoning . For instance , Vakil et al . ( 2011 ) found that adults with intellectual disabil - ity generally performed worse than typically developing children on an analogy task even when the children and adults were matched for mental age . In contrast , Morsanyi and Holyoak ( 2010 ) found that analogical reasoning was largely intact in participants with autism . These findings provide an opportunity for insight not only into the differing abilities of these two populations and the effects of the disorders they represent but also into the cognitive and neural roots of analogical reasoning itself . They also indicate a chance to make similar comparisons not only within other fields of research but also between bodies of literature pertaining to different mani - festations of relational reasoning . Educ Psychol Rev Measures Further , an overreliance on a particular measure or task to operationalize relational reasoning may also limit the scope of this research . For example , in the literature pertaining to anomaly , the predominance of research using verbal semantic anomalies meant that other potentially anomalous circumstances were underexamined . Importantly , the dependence on a particular measure or task can cause a mismatch between conceptualizations and defini - tions of relational reasoning and their operationalizations . For instance , while relational reasoning in general is frequently defined as a broadly applicable construct used for the integration of multiple relations , it has been operationalized all too often as roughly equivalent only to analogy . Our analysis of the literature reveals that this mismatch is driven in large part by the overreliance on a few measures and tasks — namely Raven ’ s Progressive Matrices and four - term verbal analogies — by those working in this line of inquiry . This identification of the dominance of analogical reasoning in the literature on relational reasoning may be an opportunity for the field to contemplate ways to diversify the forms of relational reasoning being studied by identifying or creating measures designed to study other manifestations of the construct in participants . Finally , the use of methodologies , measures , and participants that allow researchers to make conclusions about how relational reasoning and its manifestations may be linked to educational outcomes should be a priority for the field going forward . The observation that only about a quarter ( 25 . 7 % ) of all the included studies empirically linked relational reasoning ability to success in a particular academic domain illustrates the need for more of this type of study to be conducted . It may be that uncovering relational reasoning ’ s contribution to students ’ success in the educational context will promote future strides in this line of inquiry . Relations Between Relations Throughout the literature included in this review , the nature of the relations under study was of special interest . Each of the four forms of relational reasoning examined in this review ( i . e . , analogy , anomaly , antinomy , and antithesis ) was characterized and differentiated from the other forms by the fundamental relation that arose between the sets of information being analyzed . For example , analogies are defined on the basis of a similarity relation , whereas anomalies are distinguished by their underlying discrep - ancy , antinomies by incompatibility , and antitheses by an inherent oppositional rela - tion . These essential relations that define each form of relational reasoning and reflected in the research - based definitions summarized in Table 1 are indicative of the foundational mapping that occurs between clusters of information shaped through a series of initial associative processes . These definitive relations ( i . e . , similarity , discrepancy , incompatibility , and opposition ) that give each mode of reasoning its characteristic nature , however , are not the only manner of associations required to reason relationally . There are incalculable relations that are formed within some given set of information , as well as across sets of ideas , concepts , or objects . The variation between such relations is captured within theories of reasoning , such as Sternberg ’ s ( 1977 ) componential theory , and their differing complexity or level of abstraction has been marked by such labels as lower - order and higher - order relations ( Goswami 1992 ) . The more local associations centered within an informational set would correspond to the components of encoding and inferring in Sternberg ’ s ( 1977 ) componential theory and would generally fit within the category of lower - order relations ( Goswami 1992 ) . Educ Psychol Rev Conversely , relations mapped across sets of information and between lower - order relations previously drawn would be classified as higher - order relations . Each of the definitive relations that characterizes the forms of relational reasoning discussed in this review ( i . e . , similarity , discrepancy , incompatibility , and opposition ) are relations mapped across sets , or between lower - order relations , and as such are higher - order or relations between relations . Crucially , without a between - set mapping of a higher - order relation , relational reasoning as we have defined it does not occur . In addition , because the higher - order relations that can be mapped between sets of information or between lower - order relations can differ , the characterization of the higher - order relation that is mapped determines which form of relational reasoning is being utilized in a given situation . The distinction between higher - and lower - order relations can also be useful in interpreting findings in the relational reasoning literature . For instance , in their study of science instruction , Trey and Khan ( 2008 ) demonstrated that it was students ’ comprehension of the higher - order analogical relation of similarity between the balanced nature of a chemical equation and the balance of a scale that allowed them to understand the specific lower - order relations involved . Namely , students were able to reason that just as a scale does , each quantity present on one side of a balanced chemical equation must have its equal on the other side . This observation also implies that inquiries about the interplay between lower - order and higher - order relations could be potentially fruitful for uncovering how relational reasoning unfolds in the minds of students . The Identification of Further Instances of Relational Reasoning A conscious effort to broaden our current understanding of relational reasoning and its manifestations by identifying further instances of the construct at work can be deeply beneficial to both the study of relational reasoning itself and the field in which relational thought is identified . For example , the identification of morphological relations between words such as rime and rhyme as instances of analogy present in the work of Savage et al . ( 2011 ) has been shown to be fruitful not only for the study of analogical reasoning but also to the understanding of early reading acquisition . Similarly , the identification of incorrectly solved mathematical problems by Chen et al . ( 2007 ) as instances of anomaly allows for a program of research that examines relational thought , mathematical reasoning — and the interrelations between them — simultaneously . The identification of further instances of understudied manifestations of relational rea - soning like antinomy may be especially important going forward . While researchers in various fields may showcase their reasoning with antinomies in their scholarly writing , the identification of further instances of antinomy , especially in students and novice practitioners of a given discipline , may be particularly beneficial in the future . Relational Reasoning as a Multidimensional Construct One basic idea that has been foundational to the perspective of this review is that a broad conceptualization of relational reasoning as the ability to recognize or derive meaningful relations between and among pieces of information should include a family of relations that can arise when otherwise disparate information is encountered . In this review , we presented four possible manifestations of relational reasoning that may merit further study . As the field progresses , we perceive a need to keep the broad conceptualization of relational reasoning in mind and to continue to theoretically discuss , as well as empirically investigate the Educ Psychol Rev manifestations of relational reasoning , their interconnections , and their unique implications in the educational context . For example , this review has identified a need to further explicate how each of these forms of relational reasoning can operate in concert to produce the phenomenon of complex relational thought . The beginnings of this effort are recognizable in the investigations of Dunbar ( 1995 ) and Trickett et al . ( 2009 ) into the reasoning of expert scientists . Because their naturalistic observation method allows them to observe the reasoning of scientists as it happens in vivo , the inter - related uses of the different forms of relational reasoning are potentially made evident . But there are still many questions left to answer . For instance , are certain forms of relational reasoning more pervasive in a given domain than other forms ? Are certain strategies more or less adaptive for students given the form of relational reasoning at hand ? How do students integrate the diverse relations they may encounter in a complex learning situation ? How can relational techniques be used to improve the learning of all students ? Each of these questions deserves careful consideration and extensive empirical study , and it may be years until an answer to any of them can be put forward . However , we believe that the identification of a new and potentially fruitful line of inquiry is itself a step forward for the field — a step that would never have been possible without the examination of the four forms of relational reasoning present in this review . While most of the studies included in this review that presented outcomes related to educational success were correlational in nature and , thus , do not permit recommendations for educational practice ( Reinhart et al . 2013 ) , there are some aspects about the applicability of relational reasoning and its manifestations to the educational context that warrant con - sideration . For instance , it has been demonstrated that students ’ ability to reason relationally is predictive of success in a variety of academic domains and that the predictive relation differs depending on the form of relational reasoning and the academic domain being examined ( e . g . , Farrington - Flint et al . 2007 ; Schiff and Ravid 2007 ) . Also , researchers who have endeavored to forge interventions around relational reasoning ( e . g . , Richland and McDonough 2010 ; Tzuriel and George 2009 ) have achieved some demonstrable success . 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Educ Psychol Rev \ No newline at end of file diff --git a/silver_data/0c385f0423f496ca5ae0a80027a951e177386f50.pdf.txt b/silver_data/0c385f0423f496ca5ae0a80027a951e177386f50.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..9578616109dab653bca79fab8ac8e156e7473fc1 --- /dev/null +++ b/silver_data/0c385f0423f496ca5ae0a80027a951e177386f50.pdf.txt @@ -0,0 +1 @@ +Coordination - Artifacts Suiting : When Plans are in the Midst of Ordering Systems Ilaria Redaelli Università della Svizzera italiana G . Buffi , 9 Lugano , Switzerland ilaria . redaelli @ usi . ch Antonella Carassa Università della Svizzera italiana G . Buffi , 9 Lugano , Switzerland antonella . carassa @ usi . ch ABSTRACT This paper addresses the received understanding of the status of plans in cooperative work—that is , artefacts that anticipate future ways of performing activities , to challenge the received understanding of the plan’s capacity to anticipate interdependencies as an immutable feature of plans . We propose conceptualizing the plan’s capacity to anticipate interdependencies at work as an emergent , distributed and artifact - mediated activity that might be uncovered studying plans , planning and the plans application as work objects and activities occurring within a multiplicity of coordinative artifacts and protocols . This way we can expand our knowledge on how the plans anticipation is maintained in changing environments to support work coordination and we might support designers of computer - supported cooperative work ( CSCW ) ’ efforts for the design of CSCW systems . Author Keywords Artifacts ; Anticipation ; Coordination ; Ordering Systems ; Plans ; Planning ACM Classification Keywords H . 5 . 3 [ Group and organization interfaces ] : Computer supported cooperative work ; theory and models INTRODUCTION The objective of this paper is to contribute to the understanding of the role of plans in supporting coordination at work by proposing an approach that focuses on how the plan’s capacity to anticipate interdependencies at work is maintained in face of changes in surroundings to ensure stability in the coordination of different stakeholders . Several computer - supported - cooperative work ( CSCW ) scholars [ 7 ; 13 ; 15 ; 29 ; 43 ; 16 ] have examined how the plans’ executors deal with the anticipated courses of events that plans set up in real settings . This stream of research has significantly contributed to our understanding of how plans are used within work settings . Previous researchers have always taken as an object of inquiry plans as “static artifacts” that one person develops and another one has to use or , said in other words , as artifacts whose capacity to define in advance interdependencies at work can never be changed by the plan executors . But what happens when plans change over time ? Is it possible to conceptualize the plans capacity to anticipate interdependencies among work activities as an emergent , distributed and artifact - mediated activity that still ensure stability in the coordination of complex work activities ? This is the research question that inspired this paper and that we answer focusing on how plans are used in combination with other artifacts , thus approaching the study of plans in a way that still needs to be exploited by CSCW scholars . In particular , we take the concept of “ordering system” developed by Schmidt and Wagner [ 44 ] as particularly inspiring in that it promotes the necessity to study specialized coordinative practices as essentially linked with coordinative artifacts and to understand practices and artifacts designed to address very specific coordinative issues as interrelated . This way , the study of work must include the understanding of the complex interconnections actors establish to interrelate specific objects and practices to ensure order at work . We propose the term “coordination - artifacts suiting” to focus on the set of local practices and coordinative artifacts that permit individuals to keep plans on track— that is , to ensure the usability of plans over time , even if changes in surroundings occur . In order to develop our argument , we first present and comment on coordination - artifacts studies , then we examine coordination dynamics studies in airport and traffic control settings , and finally we focus on research on plans and planning in CSCW . We then present the ethnographic study used as a meaningful case and we conclude by providing insights into the design of CSCW systems . ARTIFACTS AND WORK COORDINATION The preoccupation with coordination that is at the basis of CSCW research has fostered the study of how artifacts contribute to the realization of cooperative work . Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from Permissions @ acm . org . CSCW ' 15 , March 14 - 18 , 2015 , Vancouver , BC , Canada . Copyright 2015 ACM 978 - 1 - 4503 - 2922 - 4 / 15 / 03… $ 15 . 00 . http : / / dx . doi . org / 10 . 1145 / 2675133 . 2675136 Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 165 Exemplar in this sense is Schmidt and Simone’s work [ 42 ] . Drawing on Strauss’ work [ 49 ] , these scholars defined cooperative work as the kind of work “constituted by multiple actors who are interdependent in their work in that they interact through changing the state of the common field . ” They further defined articulation work as the means to “curb the distributed nature of complexly interdependent activities . ” Based on these definitions , they identified coordination mechanisms as the “protocols encompassing a set of explicit conventions and prescribed procedures and supported by distinct artifacts with a standardized format , that stipulate and mediate the articulation of distributed activities so as to reduce the complexity of articulating distributed activities of cooperative ensembles” ( pp . 16 - 21 ) . The role of the artifacts in the coordination mechanism is thus “to provide persistence to the precomputations of the protocol and to make them publicly available” ( p . 35 ) . Several CSCW scholars have examined interrelated material artifacts vis - à - vis coordinative practices . Among others [ 2 ] , Schimidt and Wagner [ 44 ] explored the fundamental concept of “coordination mechanisms” and developed the concept of “ordering systems” to address clusters of coordinative protocols and concomitant coordinative artifacts . In their study , they sought to overcome the biases that the study of coordination through the study of coordination mechanisms could engender [ 45 ; pp . 17 - 21 ] as well as outline an approach that “supports the analyst in embracing the motley of coordinative practices required in highly complex cooperative work settings” [ 25 ; p . 23 ] . Other scholars have instead focused on the role of “boundary objects” [ 11 ; 48 ] as material artifacts that mediate the relationship among different cooperating groups . Such studies , focusing on how objects mediate the relationship among different communities of practices , developed the concepts of “prototypes” [ 51 ] , “intermediary objects” [ 9 ] , “boundary negotiating artifacts” [ 30 ] , boundary - object trimming [ 8 ] and boundary objects as punctuated crystallizations [ 31 ] . These studies have not only highlighted the functions of boundary objects in facilitating the crossing , pushing , and establishing of boundaries [ 30 ; p . 333 ] and the role of objects in knowledge dynamics [ 9 ] ; but they have also—and most relevantly—introduced the study of how boundary objects are created and used within information flows . In other words , they have discussed the processes by which such objects are made progressively intelligible to members of the receiving communities . We draw on such conceptualizations and put ourselves within the stream of research interested in the study of the creation and management of coordination artifacts for the understanding of plans as coordination mechanisms , focusing on the change and crystallization of artifacts in instable environments , instead of the processes involved in the standardization of their informational content . Among the studies quoted above , we find particularly inspiring Bossen et al . ’s study [ 8 ] on boundary - object trimming in that it highlights the kind of articulation work necessary to “keep - on - course boundary - objects whose structural form and informational content need to be maintained and updated during their use” to ensure the smooth execution of work and stresses “the dynamic and directional aspects of work” ( p . 101 ) . ARTIFACTS AND WORK COORDINATION IN FLIGHT AND AIRPORT MANAGEMENT Several studies investigated how coordination is achieved in the management of airport activities and flight execution since these activities are highly distributed in space and need to be coordinated in time following the trajectories [ 50 ] of the flights execution . The Lancaster group’ s studies [ among others 4 . ; 26 ; 27 ] strongly impacted the CSCW community for their focus on how to link the ethnographic study of the setting with the development of software design while other research provided insights into the functioning of the flight management and particularly how flight strips are used by air traffic controllers . Such studies investigated the coordinative functions of flight strips wondering how to replace the papered versions of flight strips by software [ 5 ; 6 ; 17 ; 25 ; 33 ; 34 ] . They highlighted different aspects of the flight strip use , such as their being aids to memory [ 33 ] ; their supporting peripheral awareness among colleagues within the room [ 34 ] ; their being the means by which flight scheduling is achieved when they are used in conjunction with other equipment , namely the radar and the radio connection with pilots [ 24 ] . Information provided by strips , radar and radio with the controllers’ knowledge of the kind of reasons that might determine planes not being in the place indicated at the time indicated allow to foresee their position in the next future thus “solving the sequencing problem as a matter of moment - to - moment problem , ” more than as a matter of following procedurally defined rules [ 24 ; p . 142 ] . Other research projects [ 5 ; 17 ; 22 ; 23 ] focused on the use of boundary objects between the air traffic control and the flight control tower and between the airport control tower and other ground services . Halverson , [ 22 ] and Halverson and Ackerman [ 23 ] showed that artifacts work as intermediary objects when they recontextualize expert knowledge across organizational boundaries and work as memory aids in that they crystallize previous solutions to routine problems . Similarly , Berndtsson and Normark [ 5 ] analyzed the coordinative function of a closed - circuit television - system that transmits the view of the flight strip rack between adjacent sectors , such as the apron tower and the air traffic control centers . They noticed that such a system worked as an “awareness mediator” that supported coordination among services reducing the necessity to draw on other form of communication , such as telephone calls . Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 166 Last but not least , it is possible to identify studies that focused on the activities necessary to the execution of flights that occur on the ground [ 21 ; 54 ; 55 ] . Goodwin and Goodwin [ 21 ] showed that to see a plane in a manner that is relevant for the task at hand ( i . e . to load baggage ) it is necessary to situate it within organizational networks and that this is possible only drawing on an heterogeneous collection of technological artifacts and a social organization that allows the transformation of individual expertise into an element of the working culture of the operation room . Suchman [ 54 ] studied the activity of the operators of an operations room who have to participate in the ground traffic control . She noticed that operators coordinate pilots and operators on the ramp by means of “embodied articulation” in that they craft messages and their timing to such stakeholders in different ways to achieve the orderly movement of planes given what video cameras allow them to see as regarding the movements of planes on the ground . Suchman [ 55 ] also showed how the interplay between the structure of the workplace , the assigned location of operators within the room and their interactions allow for the continuous transformation of restricted and public areas thus supporting both joint activities and different structure of attention among the operators . Another of Suchman’s concerns was the understanding of how “prescriptive devices , ” such as airline schedules , operate [ 56 ; p . 23 ] . She noticed that “any normative prescript requires , for its routine enactment , the management of inevitable contingencies of actual events . ” Schedules thus are both resources for the actors’ organizing of their activities and regimes created elsewhere to which participants’ activities are accountable [ 56 ; p . 28 ] . We consider Suchman [ 56 ] and Harper and Hughes’s [ 24 ] studies particularly relevant for the analysis of how the stand and gate allocation plan are set up and changed in that they face the problem of how prescriptive devices are enacted . We will refer to these papers in the following sections . PLANS , PLANNING AND RE - PLANNING To date , several CSCW scholars have focused on how plans and planning are used in work settings focusing alternately on planning as a process and plans as artifacts . Highlighting that actions are inevitably situated in that they depend in essential ways upon the material and social circumstances of their occurrence , Suchman [ 53 ] , recognized that the efficacy of plans inevitably depends on their relationship with the circumstances in which actions occur . Plans , in fact , cannot represent all the changing circumstances of their occurrence ; thus , they should be considered “weak resources” for the execution of actions as their usefulness depends only on their bringing “past experience and projected outcome to bear on our present actions” [ 53 ; p . 185 ] . This implies that — in order to get the work done well , it is necessary to deviate from the plan . Schmidt [ 43 ] strongly criticized Suchman’s conceptualization of plans by demonstrating that plans might or might not determine our actions . The role of plans , in fact , depends on the extent to which it is possible to identify and model interdependencies in advance . In addition , he asserted that Suchman had underestimated the normative dimension of plans as constructs that provide criteria for whether or not a particular action is correctly executed [ 46 ; p . 366 ] . Bardram [ 1 ] suggested that plans could be conceptualized as planning—that is , a situated activity . Bardram’s analysis of the daily clinical work showed that hospital patients’ assistance draws on a socially constructed planning activity that is enhanced by and simultaneously shapes the work activities . As a result , plan implementation allows for the adjustment of the plan to the conditions of the specific situation . Bardram also identified the “anticipation of future ways of performing activities as the strength of plans” [ 1 ; p . 24 ] . Dourish [ 16 ; p . 52 ] highlighted the role of plans as “organizational accounting devices . ” Focusing on workflow technologies he examined how such technologies are taken to be effective for the coordination of work despite the fact that their pre - defined formal descriptions of working processes do not match with the improvised accomplishment of work [ on this topic see , among others 52 ; 10 ] . In Dourish’s opinion , workflows are so widespread because they do not act as mere coordination technologies , but instead as “organizational accounting devices”—that is , they render work activities “observable - and - reportable as being the activity they describe” [ 16 ; p . 55 ] . Said in other words : workflow helps explain work . Such a conceptualization of workflow permits them to be designed as systems that support the visualization of work thus overcoming the conceptualization of workflow as technologies that pre - specify work , in favour of their conceptualization as technologies that visualize the order of work . Rönkkö et al . [ 41 ; p . 433 ] argued the necessity to study “the way plans become an accountable matter in organization activity” to understand how they help co - ordinate the development of work . They draw on Gerson and Star’s s definition of “due process” [ 19 ] , that sustained the necessity to study the articulation and coordination process around planning documentation to understand how inconsistencies that might arise in the accomplishment of work that formal description cannot fully anticipate and provide for , might be resolved . Other researchers [ 7 ; 15 ] showed that plans do not themselves ensure that work is done in an orderly manner , but that such orderliness depends on everyday work . This means that efforts have to be made to deal with planned Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 167 deadlines [ 8 ] , to give significance to formal plans within the situated rationality of social actions [ 4 ] or to make sense of work [ 11 ] . Redaelli and Carassa [ 39 ; 40 ] instead focused on how a team sets up and modifies plans showing that plans might be set up so as to make the realization of the planned work activities feasible . If , in fact , plans might define deadlines in an inconsistent manner , they might also be designed and changed so as to promote the organizations’ capability to deal with disruptions both at the level of work coordination and at the level of the maintenance of the organizational functioning on the whole . On the other hand , Bardram and Hansen [ 3 ] investigated the relationship between plans and situated actions to understand how and why plans are changed . They suggested considering planning in hospitals as a continuous activity adjusted to the conditions of specific situations . Continuous planning is therefore necessary when it is impossible to anticipate every contingency that might arise in carrying out a series of tasks . Consequently , the distinction between the planning phase and the execution phase fades as planning never ends , even if it might be carried out following different priorities over time , such as the optimization for utilization during the planning phase and the optimization for performance during the execution phase . Similarly , Munkvold et al . [ 35 ] investigated the process of planning , showing that planning is a collective and ongoing achievement that needs to be supported as such even though this might be in detriment to the standardization of work practice that plans usually support . In summary , plans have mainly been studied as “static artifacts” whose capacity to define in advance interdependencies at work can never be changed by the plan executors . CSCW studies have thus focused on how such artifacts are used by people who have to apply or realize them . As such , plans like checklists might be useful resources that help execute the planned activities or artifacts that might fail to adequately support courses of action if particular adaptive efforts devoted to match the plan content with the real situation are not made . When the focus of attention is instead on the process of planning rather than on plans as artifacts , planning is conceptualized as the adjustment to the concrete conditions of the context [ 3 ] and the relevance of plans as tools that might support the execution of action is minimized or even dismissed . In such cases , in fact , plans are conceptualized as trajectories that are the outcome of continual articulation work [ 35 ] . This undermines the possibility to study them as interdependent . Furthermore , the studies discussed thus far have been oriented to the study of such artifacts and processes in a disconnected manner from the other artifacts that inhabit the organizational settings . Only Rönkkö et al . [ 41 ] attempted to link the study of plans with other planning documents , showing that the members’ reference to the project plan and the company - wide project models make it possible for people not to cease to orient to plans even as they change . Indeed , the combined use of such documents makes the project plan useful even when it does not work out as it contributes to the identification of deviations between the plan and the project’s reality . However , a systematic attempt to understand how plans work as coordination mechanisms when used in combination with other artifacts is still missing . All this suggests that our understanding of plans might be incomplete . RESEARCH SITE The study was carried out in the ramp control tower ( RCT ) of an Italian airport where the operators plan the use of parking areas for aircrafts ( i . e . , the stands ) to ensure a proper area will be available when planes arrive for the entire duration of handling activities on the ground . Such operators also plan the use of gates to ensure the availability of gates when boarding has to start and for the duration of passengers’ boarding . Their planning activities not only play a key role in ensuring the orderly movements of planes on ground , but also ensures the orderly execution of flights in that they support the timely execution of the planes’ assistance on the ground which is crucial for the execution of flights as scheduled . In particular , the stand and allocation plan work as coordination mechanisms in that they permit the convergence of personnel involved in assisting the plane on the ground ( bus , fuelling , and towing truck drivers ; ramp agents ; utility workers for aircraft cleaning and luggage loading and unloading ; drivers of follow - me trucks and of belt - loader vehicles ) upon the planes’ arrivals and the convergence of airport personnel and passengers at the gates . The airport it is home to both passenger and cargo flights ; thus it is active 24 hours a day meaning the RCT operators have to ensure the continuity of planning over time . They do this both by planning in advance twice a day and engaging in re - planning whenever necessary . The operators’ advance planning consists of planning before the plan is executed . The operators match stands and gates with flights when the airline companies send them the rotation lists ( i . e . , documents in which scheduled flights are assigned to planes ) . This way , the RCT operators know how long each plane will stay on the ground between the execution of subsequent flights and can therefore allocate stands and gates as necessary . The operators’ re - planning occurs during the plan’s execution and it is devoted to compensating for changes in the execution of flights or in the rotation lists . As a result , the most frequent causes of changes to plans are flight delays or arrivals ahead of time , flight cancellations or the addition of charter flights , changes in the size of aircrafts used to carry out flights , or changes in the match between flights and planes . Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 168 The RCT operators match planes with stands by considering the size of the aircrafts and the technical features of stands . Planes have different and varied sizes while stands offer different features . Some of them allow for the self - maneuvering of aircraft while others do not , and sometimes the possibility for using certain stands depends on whether adjacent stands are in use or not as access to some stands overlap with other parking areas . In addition , stands offer different capacities and can be used to park aircrafts of various dimensions , up to their maximum capacity . Similarly , gates have different capacities and might allow for boarding solely by bus , solely on foot , or both . This implies that the RCT operators have to match flights with gates while also respecting the number of passengers to board and the security requirements that might involve boarding by bus for certain flights . In addition , some stands are structurally linked with gates ; RCT operators have to take this into consideration when planning . Thus , the way stands and gates might be used depends on choices made when planning . It is also worth considering that , during the last ten years , the airport has ensured a progressive increase in air transport movements and that low - cost companies provide the majority of flights hosted by the airport . As a result , the operators have to handle aircrafts on the ground while respecting short turnaround times . The stand allocation plan is set up using software available to the RCT that represents the stand plan in the form of a Gantt’s chart for the occupation of stands over time . The operators drag and drop flight numbers from a list over to the Gantt’s chart , where icons representing the length of planes’ stay on ground appear . The software highlights potential conflicts in the stand allocation—namely , the mismatch between the dimension of the stand and those of the plane as well as the overlapping of allocations for each stand during both the plan setup and execution if changes in the execution of flights occur . The gate allocation plan is set up and printed once finished , without the support of any alarm system . As a result , the operators must pay attention to overlaps or mismatch in the use of gates . The RCT operators work in an environment full of artifacts . As the image below shows , the operators have two workspaces at their disposal for the set up and change of the gate and stand allocation plans and for the coordination of all the ground activities connected with the departure and arrival of planes . The operators sit at their workstations where one of them manages incoming flights , while the other manages departures . Some tools are available to both operators , while others are not . Big windows in front of the operators’ workspaces permit the direct view of most of the ramp and taxiway .       Taxiway and ramp RCT tower             1 2 3 4 5 6 7 8 9 10 11 12 OP 1 ( departures ) OP2 ( arrivals )  13  14 15  Entrance LEGEND Operator’s 1 workspace Operator’s 2 workspace 1 Telex 8 Radio 2 Computer 9 Stand allocation plan ( paper version ) 3 Telephone 10 Gate allocation plan ( electronic version ) 4 Gate allocation plan ( paper version ) 11 Computer 5 Radio 12 Telephone 13 Close circuit camera Shared tools 6 Strip rack 14 Printer 7 Block - notes 15 Airport map Figure 1 . Layout of the RCT The RCT uses a closed - circuit system for the surveillance of the parking areas , a monitor showing the electronic strip - rack , and software for the allocation of stands and gates . The electronic strip rack offers various information on flights , such as the estimated block - off time ( EOTB ) and the calculated take - off time ( CTOT ) for departing flights , which allows the operator to know at what time the flight is scheduled to depart and at what time the plane will actually depart . The strip rack also provides information about flight status for incoming planes . Such statuses change from inactive to active , radar , and landed , corresponding with the plane’s approach to the airport . The strip - rack also provides different evaluations of the estimated time of arrival ( ETA ) so that the closer the plane is to the airport , the more accurate and reliable the estimations of the arrival time are . Meanwhile , the software for the allocation of gates allows the operator to have an overview of the gate allocations and to monitor the number of passengers boarded for each flight . The data on the scheduled flights included in the system for the gate and stand allocation need to be manually updated when changes in the scheduled time or the numbers of planes occur . The airline companies send such information in the form of telex or e - mail messages to the operation room where the operators update the database Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 169 information . RCT also uses a block - note that lists the messages that refer to daily occurrences , such as ongoing maintenance procedures at gates , stands and aircrafts ; particular messages received by airline companies ; to - do lists ( i . e . messages to send or to receive ) that the RCT operators progressively update . The operators might also consult documents glued on furniture that provide the list of gates and stands whose allocations need to be coupled ; security protocols that specify which gates and stands can be used for extra - Schengen flights ; a big map of the airport on the wall that provides information on the technical features of stands . RESEARCH METHODS The study is an ethno - methodologically oriented ethnography [ 14 ; 18 ; 38 ] . The objective of the data collection was the study of the operators’ planning practices in allocating resources . We followed Llewellyn and Spence’s [ 32 ] conceiving practices as members’ phenomenon . Therefore , data collection was oriented to the identification of patterns of activities that are intersubjectively organized and recognized by operators as embodying ( or not ) a certain practice . Our main goal was to understand how RCT operators carry out such activities and how they make those activities “accountable” to others in order to understand how such artifacts contribute to the coordination of ground activities . We did this in accordance with the ethnomethodologically inspired studies of work that take as topic the study of work as “lived experience” [ 38 ] . Empirical materials were collected within the RCT where 18 people ( 12 males and 6 females , aged from 30 to 40 ) work . The majority of them started working in the airport ten years before the beginning of the research , while only two of them started working in the service more recently . Some of the operators are part of teams that define the organization of the ground activities , others in teaching professional courses , but each of them is equally able to manage the routine work in the RCT . The data collection was carried out by means of the direct observation of the operators’ planning and re - planning activities . Data collection lasted eight months by spending at least two days a week in the service for a total amount of more than 500 hours . The operators were observed at different times of the day and night according to the operators’ shifts to catch their planning and re - planning strategies during the occurrence of hourly , daily and seasonal variations . After one month the direct observation was accompanied by the audio - recordings of naturally occurring conversations . Due to security requirements , we were not allowed to videotape and take pictures of work activities . This , of course , makes the fine - grained analysis of how discourses relate with activities as , for example , Nevile [ 36 ] did studying the flight execution , impossible . Detailed notes were taken regarding the activities carried out by the operators and were linked with the discourses that occurred during their execution and we made drawings to fix the layout of tools and documents . Field notes and the majority of the recorded conversations were transcribed for a total amount of 223 pages of field notes and 197 pages of transcripts . We decided to transcribe all conversations occurring between the operators during planning and re - planning , but not all the ones with pilots , who transmit data regarding the kind of maintenance planes need once on the ground , and the operators on the ramp with whom they talk about the execution of ground activities . We transcribed conversations drawing on a modified orthography ( see the table 1 ) that allowed for us to keep track of the “sounds as uttered” [ 57 ] to catch the operators’ inferential work that emerges in lexical choices , in the way words and utterances are produced and sequentially organized . But it is worth specifying that we decided to use the standard numeric symbols for the representation of numbers to make the reading and the analysis of transcripts easier . Transcripts of conversations are presented here following Jordan’s [ 28 ] presentation of the ethnographic study conducted at the San Jose international Airport [ 12 ] to show the relationship between discourses and actions . We also carried out 47 unstructured ethnographic interviews [ 47 ] while observing the operators’ setting up and changing the plans . The questions were devoted to the understanding of how the operators realized the necessity to change the plans , of how they reached a solution to re - allocation problems and to the identification of the strategies they usually adopt for the plans setup . The interviews thus were carried out at the workplace making it possible to discuss the features and functions of artifacts . We interviewed all the operators with the exception of one who refused to be interviewed and audio - recorded . Field notes were verified with participants and the analysis of data was carried out triangulating all collected empirical materials . The empirical materials presented here were selected to highlight the issue of how the plans capacity to anticipate interdependencies is maintained despite contingencies that might arise in carrying out a series of tasks so as to help the coordination and development of work . PLANNING AND RE - PLANNING IN THE APRON TOWER As previously explained , both the stand and gate allocation plans are coordination mechanisms . The stand and gate allocation plans are sent to all personnel involved in providing plane assistance on the ground and to the airport’s personnel to ensure the convergence of ground personnel at stands and of airport personnel and passengers at gates . This means that changes in such plans might create disruptions in the execution of airport activities . But plans also have to compensate for changes in the execution of flights or in the airlines’ efforts to match planes and flights . The RCT operators who work to match the airport with the flight activities are bound to Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 170 deal with conflicting exigencies ; plans are at the interface of such a tension between stability and flexibility . This is why we focused on how the operators set up and modify plans . Planning Practices and the Anticipation of Events Planning for the Likely Case Scenario and Preventing Delays As Suchman noticed [ 56 ] , the RCT operators are oriented to achieving a normal order of on - time arrivals and departures as prescribed by the flight schedule . They participate in the achievement of such a goal by means of planning the allocation of stands and gates . Using Suchman’s words [ 56 ; p . 27 ] , like other operators involved in the flight execution [ 24 ] , they work “to maintain a consistent relationship between an order of events prescribed via the schedule and events at the local site . ” They do this by means of precise planning and re - planning practices . The RCT operators , in fact , are able to anticipate the occurrence of some events on the basis of their knowledge of “how things usually go . ” As a consequence , they are able to plan so as to provide resources to compensate for changes in the flight execution and to ensure the execution of the ground activities as planned . The interviews indicated that RCT operators , unlike what Bardram and Hansen noted [ 3 ] , do not plan based on the best - case scenario ( i . e . , all flights will be carried out as scheduled ) , but rather on the basis of their knowledge of how flights are usually executed . Thus , for example the operators set up the stand allocation plan , ensuring the availability of slack space , i . e . at least one free stand on the ramp , and of slack time between subsequent flights to compensate for changes in the number or timetable of flights . But the RCT operators not only plan to adapt to likely - to - occur changes in the execution of flights . In fact , they , on the basis of their knowledge of how planning the use of stands and gates impact on the timely execution of the ground activities , also plan so as to prevent the occurrence of delays in the execution of flights thus contributing to their execution as planned . They do this planning to ensure the timely execution of the handling activities—that is , by planning to avoid delays in the execution of boarding , favouring the exchanges of crews and allocating planes in order to avoid bottleneck situations . The operators avoid delays in the execution of boarding by maximizing the use of stands that allow the boarding and disembarkation on foot and matching the gate dimensions with the number of passengers to board . All of us here know that you have to exploit at maximum the stands that allow boarding and disembarkation on foot so we do not need to use busses and such procedures are carried out quicker than by bus . So , for example I assign the flights that depart first in the morning to those stands so that they will be free for subsequent flights . If I put there flights that depart late in the morning , then I block their use ( Track 5 10 / 07 / 2011 ) . With the Sxxair we have to start boarding a long time before the flight departure , so you need the space to place the passengers in ; otherwise , you board 6 passengers and then you have to stop . This way , you do not gain time . So we have to avoid assigning crowded flights to gates that have a small fingers ( Track 36 10 / 25 / 2011 ) . The operators also plan in such a way to shorten the time necessary to exchange crews ( i . e . , move from one plane to another one ) . When looking at the rotation lists and we detect an aircraft change , we try to put the two planes next to each other so the crew will not take a long time to move from one plane to the other one . You know , the low cost companies do not provide vehicles for the crews to move on the ground , so if we do not do this it might take a long time for them to walk from one side of the ramp to the opposite one , increasing the risk of a delay in the flight departure ( Track 14 10 / 07 / 2011 ) . Moreover , the operators allocate planes and gates so as to avoid overlaps in the aircrafts’ and passengers’ movement on ground . This is another thing you have to keep in mind . If I assign the plane that departs first in the morning to the gate 16 instead of 14 , I risk having to stop the boarding for stand 14 if the plane at stand 16 is ready to go . Stand 16 , in fact , is a self - manoeuvring and its exit way overlaps the path passengers use to reach stand 14 ( Track 14 10 / 07 / 2011 ) . If we cannot avoid using the stand 24 , we put there the flight that departs first because this stand has to be free as soon as possible . It is a pushback stand and it is on the exit way of all these other self - manoeuvring stands . So if it is not free , we have to push back all the flights that , as a consequence , cannot leave the stands in self - manoeuvring so we risk causing delays because we have few pushback tractors and we have to push all those planes that , early in the morning , depart almost at the same time ( Track 37 10 / 14 / 2011 ) . Such planning practices contribute to the execution of flights as planned in a significant way , even though they are not enough to compensate for changes in the flight execution . The RCT operators are therefore forced to engage in re - planning . Re - planning Practices and the Anticipation of Events Planning and re - planning are different activities in that during planning the RCT operators can repeatedly change the allocations until they are satisfied—that is , until they think that the plans allow for the optimization of the use of airport resources and the orderly movement of planes on the ground . Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 171 First , I assign stands to be sure that flights fit in them ; then I cancel everything and I start assigning the crowded flights with the gates that allow boarding on foot , and I put the international flights on the other side next to the gates dedicated to international flights ( Track 37 10 / 1472011 ) . In this way , the operators make the plans resistant to changes thus reducing the need to re - plan , which is a costly activity for planners who have to re - plan while executing other activities . Moreover , changes in the allocation of stands and gates might disrupt airport activities because , for example , passengers have to move from one gate to another ; and airport operators have to make additional efforts to re - coordinate their activities according to the change . In addition , during re - planning , it is more difficult to change plans while ensuring the optimization of resources in that they do not have the possibility to use whatever stand or gate , but only those not in use at that moment or in the near future . Yet , changes to the plans cannot always be avoided . Thus the operators have to recognize the necessity to change plans and decide how to change them in advance so as to ensure the smooth execution of flights and ground activities . The RCT operators do this according to shared discursive practices and the use of artifacts as emerging from the analysis of the naturally occurring conversations between them . The transcripts analyzed here are translated from Italian . The following fragments represent routine talk within the RCT . Transcription conventions . Falling intonation ? Rising intonation , Flat or slightly rising intonation , talk which sounds incomplete ( 2 . 0 ) , ( 0 . 5 ) Silence measured in seconds and tenths of seconds word _ word Bounded words ( xx ) Incomprehensible talk [ writing ] Description of actions occurring during the talk Pronunciation of numbers 1 One 9 8 7 5 Nine eight seven five 12 Twelve References of numbers 9 8 7 5 Flight number 12 Stand number 11 Gate number * 21 * Time ( hours and minutes ) Table 1 : Transcription conventions In the following transcript one of the two operators detects the necessity to change the stand and allocation plans and involves the colleague in the solution of the re - allocation problem . Extract 1 ( Track 18 11 / 25 / 2011 ) 1 . FRANK It’s too narrow for us here with the 4 7 0 6 [ Looking at the stand allocation plan ( electronic version ) ] 2 . TINA Even there ? [ Inserting data into the database ] 3 . FRANK It’s at 16 . 4 8 8 6 , 4 7 0 7 is it already there ? [ Looking at the stand allocation plan ( electronic version ) ] 4 . TINA * 18 * and * 22 * but it’s inactive [ Looking at the strip rack ] 5 . FRANK The 4 8 8 6 is already on ground the 4 8 8 5 [ Looking at the stand allocation plan ( electronic version ) ] 6 . TINA Yes _ yes . The 4 7 0 7 is arriving * 10 * minutes ahead of time at 16 [ Looking at the strip rack ] 7 . FRANK Okay . Let’s see , Tina , let’s see [ Looking at the stand allocation plan ( electronic version ) ] 8 . TINA Can we switch them with another flight ? [ Looking at Frank ] 9 . FRANK Not here , they’re all full . 14 18 it should go at 13 . Shall we change the gate immediately and put it at 13 ? It leaves at * 19 * and * 50 * . Can we take it easy ? Let’s change it [ Looking at the stand allocation plan ( electronic version ) ] 10 . TINA We can wait until it goes on radar and then we’ll decide [ Looking at Frank ] 11 . FRANK Okay , let’s wait until it goes radar and then we’ll decide [ Looking at Tina ] 12 . TINA Okay [ ( any record ) ] 13 . FRANK But the 13 is free . Let’s check for another gate . Mh ( 13 . 0 ) [ Looking at the software ( opening the manager of gate allocation ) ] 14 . TINA 14 , 15 [ Looking at the gate allocation plan ] 15 . FRANK No there is also the 19 [ Looking at the software ( manager of gate allocation ) ] 16 . TINA But if the passengers are already at the gate it’ll be a mess moving them [ Looking at Frank ] 17 . FRANK 16 they’re already there , you are right , then 15 [ Looking at the software ( manager of gate allocation ) ] Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 172 18 . TINA 15 is the closest [ ( any record ) ] 19 . FRANK 15 is the closest . So in the end we’ll change it [ ( any record ) ] Does a Contingency Matter ? As explained above , the stand and gate allocation plans are set up to ensure the availability of slack space and time so as to compensate for changes in the flight execution . This means that not all the contingencies require changes to the plans . The RCT operators detect the necessity to change the stand allocation plan in that its representation as a Gannt’s chart allows for the detection of the shortening of the slack time between subsequent flights even before the activation of the alarm color code in that such a representation shows time in terms of space ( extract 1 , turn 1 ) . Assessing a Viable Solution Once the operators detect the necessity to change the plans , they have to find a viable solution to the re - allocation problem . They re - allocate stands by both looking at the stand allocation plan and at the representation of the strip rack . This way , in fact , they are able to identify the free stands at disposal ( extract 1 , turns 5 and 9 ) and to assess which planes might be moved from their original allocations . Planes that are already on ground might be moved but this is a time consuming activity whose occurrence interferes with the movement of planes on the taxiway . This is the reason why the operators solve re - allocation problems by changing the allocation of flights that are going to arrive . They select the plane to move on the basis of the distance of the aircraft from the airport . Usually the farthest , the better in that it is unlikely that the re - allocation negatively impacts the execution of’ ground activities . The operators assess the planes positions looking at the strip rack and in particular focusing on the status of flights ( extract 1 , turn 4 ) . Inactive flights are the farthest while the radar flights are the closest to the ground . To Re - plan or not to Re - plan ? Drawing on the information provided by the strip rack the RCT operators can also decide to postpone the change to the plan up to the achievement of more reliable information about the time of arrival of planes ( extract 1 , turns 9 , 10 , 11 ) . Usually , in fact , the estimations of the arrival time change with the plane approaching the airport thus making the re - allocation no longer necessary . The operators , in fact , might decide to use the slack time between subsequent flights to compensate for changes in the scheduled flight time . To do this safely they check the strip - rack for the estimations of the arrival time that are more and more accurate with the time passing . In addition they might use the statuses of flights as deadlines along which to organize their re - planning activity ( extract 1 , turn 11 ) . Changing the flight status from “inactive” to “radar” is a means to balance the RCT operators’ concern of preserving the plan as it was set up as much as possible with the necessity to inform the operator on ground in time to avoid disruptions in the execution of ground activities . In particular the RCT operators consider “inactive flights” as sort of mere hypothesis of how thing will go , while they consider the status “radar” as highly reliable and as fixing the optimal deadline for re - planning . The status “radar” occurring 30 minutes before the arrival of the plane permits the change of plans and the re - coordination of ground activities . Re - allocating Gates Once the RCT operators have re - allocated stands , they re - allocate gates if necessary . Similarly with the gate re - allocation , they need to know the location of passengers ( already at the gate or not ) in order to find the less disruptive solution . They can monitor the passengers’ position looking at the software at their disposal for the allocation of gates . They , in fact , can check the foreseen time of boarding to assess the presence of passengers at the gate and , on the basis of their knowledge of the airport structure and of the stands available at that moment , decide which is the less disruptive solution in the given circumstances ( extract 1 , turns 16 , 17 ) . Departing Flight The use of artifacts not only supports re - allocation when incoming flights change their scheduled times , but also supports the identification of delays in planes leaving the stands . As the extract 2 shows , Freddy asked the colleague to check whether they might have problems at stand 31 since the plane is next to land and the plane previously assigned to that stand is still on ground . It , in fact , is on the strip rack but not ready for departure . Extract 2 ( Track 66 11 / 04 / 2011 ) 1 . FREDDY Check the 31 , does it fit ? [ Looking at strip rack ] 2 . MARK Maybe let’s say maybe [ Looking at the stand allocation plan ( electronic version ) ] 3 . FREDDY Maybe ? ( 10 . 0 ) let’s put this on ice [ Looking at Mark ] 4 . MARK If it fits ok otherwise I see that there is the yellow air at 9 that we could put at 7 that is beside . The red air ( XX ) is going to leave first , isn’t it ? it is still sixty [ Looking at the software ( boarding page surveillance ) ] The colleague shares with the colleague the developed back - up plan . He thinks that it is possible to use the stand 7 since the plane at that stand is going to leave . That plane , in fact , arrived ahead of time thus permitting the timely beginning of boarding procedure . Mark is saying Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 173 that the plane is going to leave because , looking at the boarding page surveillance he notices that only 60 passengers have yet to be to boarded . Tidying the Plans Extract 3 ( Track 37 11 / 09 / 2011 ) 1 . ALAN The Stansted is bothering me . In any case there is a stand free [ Looking at the strip rack ] 2 . TINA A stand free ? [ Looking at the stand allocation plan ( electronic version ) ] 3 . ALAN My 13 is free [ Looking at the stand allocation plan ( paper version ) ] 4 . TINA Because there is 28 to move to 13 . That won’t do there , we have to use 13 or we should move this one down . Oh no this is 14 . This one ? No because it is the first one to leave . Can’t we put something there ? This one ? Let’s move this Stansted . Oh no we can’t . Guess what we can do ? Move this one and this down there [ Moving the cursor on the stand allocation plan ( electronic version ) ] 5 . ALAN In fact I was moving it to 14 , if possible [ Looking at Tina ] 6 . TINA Yes but what do you care ? Oh yes let’s have the disembarkation , let’s do that . But at what time does that one leave though ? * 20 * and * 40 * . And this will arrive at ? * 20 * and * 45 * , no I cannot . No , why is that ? Yes it fits . Okay let’s put the 4 1 9 7 at 14 . Then here at 13 we can put another flight . Let’ s see . Otherwise we could have moved this one up and the other one down , you see ? We could have put this one down and moved this one [ Moving the cursor on the stand allocation plan ( electronic version ) ] When re - planning , the RCT operators try to optimize the use of airport resources and to maintain the possibility of changing plans over time as much as possible . This means that very often for the operators the simplest solution— finding a free stand for an unexpected plane—is not the best solution even though this means to change several allocations to solve a single re - allocation problem ( extract 3 , turns 4 , 6 ) . In addition , due to the possibility to change the plans , optimizations in the use of airport resources impossible during its set up might become viable in the course of the day . Again the use of the representations of plans and of the strip rack plays a key role in such processes . In other words , the operators always “tidy up” the plans to maintain them as optimal working tools for themselves and next colleagues . Extract 4 ( Track 1 11 / 25 / 2011 ) 1 . FRANK I’m moving the 8 7 8 6 that was at 14 to 13 [ Changing the stand allocation plan ( electronic version ) ] 2 . LISA But [ Looking at the stand allocation plan ( papered version ) ] 3 . FRANK What ? [ ( any record ) ] 4 . LISA This other one swaps . Cannot we put this one closer to the16 ? ( XX ) The crew that has to board on the 12 now is at 15 and then at 22 but now we have space here [ Looking at the stand allocation plan ( papered version ) and then pointing at the stand allocation plan ( electronic version ) ] 5 . FRANK At 12 well done [ Looking at the stand allocation plan ( electronic version ) ] 6 . LISA At 13 [ ( any record ) ] 7 . FRANK Wait isn’t it already at 13 ? [ Looking at the stand allocation plan ( electronic version ) ] 8 . LISA No here it is written at 11 . It’s departing as 4 5 6 1 [ Looking at the stand allocation plan ( paper version ) ] The papered and electronic version of the stand allocation plan , in fact , representing the changes in the plan at different speed ( the electronic version is updated as new data are received and inserted into the system , while the papered version is updated by the operators at any re - allocation ) permit both an overview of the decisions made during planning ( the papered version ) and an image of events in real time ( the electronic version ) thus allowing the review of the plan and therefore the identification of areas of improvement that re - allocation have made possible ( extract 4 , turn 4 ) . Different representations of the stand allocation plans allow for the evaluation of whether pre - planned solution are still good enough and to crosscheck whether “first glance sound solutions” are feasible or not thus avoiding mistakes in the re - allocation of stands ( extract 4 , turn 8 ) . DISCUSSION The RCT operators’ work , similarly to the work of other operators involved in flight execution , is bound by the requirements of the flight plan . Other researchers [ 24 ; 56 ] have highlighted the kinds of efforts that operators have to accomplish to execute the flight plan despite the occurrence of inevitable contingent events to enact the plan . Our work does the same , focusing on the relationship between the coordination of ground activities and the execution of flight schedules as well as on the role of plans in such a process . It shows that the stand and gate allocation plans not only are supposed to not only compensate for changes in the execution of flights , but also promote the execution of flights as expected . They Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 174 reach this objective thanks to both to planning and re - planning practices that draw on different principles . Planning is developed based on the implementation of preferred scenarios , such as the movement of aircrafts on ground , while re - planning is implemented based of “what - if scenarios”—that is , the evaluation of possible consequences determined by the change of the plan . Yet despite these differences , both planning and re - planning are oriented to the anticipation of the courses of events . Anticipation in the planning phase draws on the operators’ local knowledge [ 37 ] of how flight schedules are changed and how ground activities are executed . Anticipation is characterized by two elements : It is oriented to ensuring slack resources with which to deal with contingencies ( at least a certain amount of them ) and to the prevention of disruptions in the execution of the planned activities . Re - planning , instead , anticipates events successfully because it is carried out based on the basis of the detection of the necessity to re - plan in advance ; it is oriented to the identification of successful solutions to emergent problems and it is carried out in a timely manner in terms of the execution of the ground activities it coordinates . Such activities are possible in that RCT operators can use a set of tools to support their execution . It is worth noting is that the majority of such tools are not designed to support re - planning , but to monitor the flight execution . Nevertheless , the RCT operators use the information provided by such tools and transform them into information useful for re - planning purposes . In particular , RCT operators transform data provided by the equipment in terms of changes in the timetable of scheduled flights into data regarding the position of planes . Similarly , they exploit instruments developed to control the ground activities ( i . e . , the CCC ) and the functions provided by the software system for gate allocation to assess the position of passengers . In this way , they determine the availability of stands and gates and identify the elements on which it is possible to act so as to solve contingent problems . In addition , the operators use the information regarding flight status to fix the timing of re - planning , thereby reducing the occurrences of re - planning and avoiding disruptions in the execution of the re - planned activities . Last but not least , the different representations of the same plan support the recursive optimization of the use of airport resources and the maintenance of the possibility to change the plan over time despite the recurring changes to its content that , in turn , ensure the reliability of the plans . This means that the successful use of the stand and gate allocation plans depends on the combined use of artifacts and the deployment of local knowledge coupled with the constant monitoring of the execution of the flight plan . Anticipation is recognized as one of the features of plans supporting coordination , but researchers still need to understand how anticipation for planning purposes is achieved in changing environments . Anticipation has also been seen as the “Achilles’ heel” of plans in that they cannot foresee all contingencies ahead of time . Thus , when several changes occur , plans cannot do anything else but fail to support the execution of course of actions . Our data instead show that anticipation can be an emergent , distributed and artifact - mediated activity that , at the same time , does not undermine the possibility to determine the action of its users—namely , the RCT operators and the ground personnel . Anticipating events in the RCT is a complex set of interrelated planning and re - planning practices supported by the use of a composite set of tools . This means that anticipation emerges as the outcome of complex interconnections established by RCT operators to interrelate specific objects and practices to ensure order at work . RCT operators’ combined use of specialized coordinative artifacts is instrumental in maintaining anticipation . As a result , even if plans in the RCT change , they never cease to ensure stability in the coordination of complex work activities and they never cease to be used to pre - compute interdependencies during the allocation of stands and gates . Thus , anticipation even if depending on the use of different tools , essentially draws on the use of the representations of plans . Our study suggests that the understanding of how plans can maintain their capability to anticipate events in changing environments is possible by taking a precise analytical stance . Indeed , when we extend the analytical focus over the study of the specific purposes , plans serve to encompass the understanding of the relationship they have with all the artifacts that support the orderly accomplishment of work ; thus , we can understand how plans are kept on track despite the changes that might occur in the work conditions . This is the reason why we think that we should conceptualize the study of plans as part of ordering systems [ 44 ] —that is , clusters of artifacts and practices necessary to ensure order at work . In our case , this refers to the tools and practices necessary to coordinate the execution of flights with the accomplishment of ground activities . When we adopt such a perspective , we might understand not only how consistency is maintained across distributed and interdependent activities , such as the flight execution and ground activities but also how ordering systems might support the maintenance of one of the artifacts of the cluster . We are mulling over efforts to keep the plans on track by using the term “coordination - artifact suiting . ” We know that our definition of the kind of work necessary to prevent the deterioration of plans might be understood as echoing the concept of “boundary - object trimming” developed by Bossen et al . [ 8 ] , but we contend that these concepts are radically different . Bossen et al . ’ s study , like ours , involved a group whose core task is the articulation work necessary to keep an artifact on course , and coined the term “boundary - object trimming” to Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 175 address the kind of articulation work necessary for the maintenance of a boundary object . The term “coordination - artifact suiting” instead refers to both the participants’ articulation work and the broader “artifact’s landscape” in which single artifacts are included for the dynamic maintenance of artifacts . In addition , the term “coordination - artifacts suiting” refers to the prevention of artifacts going off use , while the concept “boundary - object trimming” refers to the activities necessary to maintain optimal working tools which according to Bossen et al . includes their orderly completion and registration . We contend that the concept of “coordination - artifact suiting” can be as used a descriptive and a heuristic concept . In fact , the term “coordination - artifact suiting” can be used to describe all the cases in which ensembles of coordinative artifacts and practices are functional to the dynamic maintenance of one or more coordination mechanisms belonging to a cluster . It can also work as a sensitizing concept in that it carries a precise methodological insight—namely , that the understanding of how coordination artifacts are updated when their information content needs to be changed during their use might require expanding the analytical focus beyond the study of a single artifact . CONCLUSION Our data suggest that anticipation , which plays a key function in the coordination of work and is often crystallized in plans , needs to be studied by focusing on the set of coordinating artifacts that always inhabit complex work settings . In this way , the utility of plans might be understood even when they need to be changed throughout the day . Contrary to the results of studies by Bardram and Hansen [ 3 ] and Munkvold et al . [ 35 ] , our data indicate that the reference to artifacts remains the basis of the possibility to progressively pre - compute interdependencies during both the planning and re - planning phases when the dynamic anticipation of events is possible . Our findings thus suggest the need to distinguish between cases in which continuous planning is associated with the possibility to anticipate events for all practical purposes from the cases in which this is not possible as it requires approaching software design in different ways . Indeed , our findings , even if not oriented to the development of software , open the question on how to design software to support anticipation as a cooperative and mediated activity . The study of plans as articulation artifacts that have highlighted the need to support users’ possibility to skip the prescriptions of the plan and monitor such deviations [ 1 ; 3 ; 42 ] cannot be applied to support RCT operators’ activities as planning in the RCT depends on the use of several tools . Thus software should be designed not to produce a single workflow system , but to make planning and re - planning possible by distributing meta - content over a cluster of technologies to permit the transformation of the information provided for planning purposes . We recognize that “awareness technologies , ” such as the strip rack , the software for the gate allocation and boarding monitoring and the CCC , thereby affording information regarding work activities carried out in related and distributed settings ( on the ground and in the flight control tower ) , play a key role in the operators’ capability to anticipate events . However , they are not enough . All the information about the timing of events and positions of stakeholders is useless if not transformed into information about the possibility to act upon objects and persons to solve allocation problems . Distributing affordances [ 20 ] such as alarm systems over several tools based on the recurrent patterns of planning and re - planning , as highlighted in the previous sections , might help operators both compute interdependencies and display aspects of their activities of relevance for colleagues . In this way , operators might be adequately supported in the identification of the need to revise the plans , in developing provisional solutions and identifying them as such , in assessing the timing of re - planning , and in evaluating the appropriateness of solutions to allocation problems as necessary to address slack resources and to prevent delays during both the planning and re - planning phases . 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Sage ( 2004 ) Framing Collaboration : Systems and Analysis CSCW 2015 , March 14 - 18 , 2015 , Vancouver , BC , Canada 178 \ No newline at end of file diff --git a/silver_data/0cfb97dd6ce7e584fe5e681e5ffbca47316cc032.pdf.txt b/silver_data/0cfb97dd6ce7e584fe5e681e5ffbca47316cc032.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..e5d8a17ca23d42a9c88bf98e58ce00ec9ba4dcf2 --- /dev/null +++ b/silver_data/0cfb97dd6ce7e584fe5e681e5ffbca47316cc032.pdf.txt @@ -0,0 +1 @@ +Frontiers in Psychology | www . frontiersin . org 1 December 2021 | Volume 12 | Article 791398 MINI REVIEW published : 15 December 2021 doi : 10 . 3389 / fpsyg . 2021 . 791398 Edited by : Lars Chittka , Queen Mary University of London , United Kingdom Reviewed by : Santiago Arango - Munoz , University of Antioquia , Colombia * Correspondence : Antonio J . Osuna - Mascaró antonio . osunamascaro @ vetmeduni . ac . at Specialty section : This article was submitted to Consciousness Research , a section of the journal Frontiers in Psychology Received : 11 October 2021 Accepted : 15 November 2021 Published : 15 December 2021 Citation : Osuna - Mascaró AJ and Auersperg AMI ( 2021 ) Current Understanding of the “Insight” Phenomenon Across Disciplines . Front . Psychol . 12 : 791398 . doi : 10 . 3389 / fpsyg . 2021 . 791398 Current Understanding of the “Insight” Phenomenon Across Disciplines Antonio J . Osuna - Mascaró * and Alice M . I . Auersperg Messerli Research Institute , University of Veterinary Medicine , Medical University of Vienna , University of Vienna , Vienna , Austria Despite countless anecdotes and the historical significance of insight as a problem solving mechanism , its nature has long remained elusive . The conscious experience of insight is notoriously difficult to trace in non - verbal animals . Although studying insight has presented a significant challenge even to neurobiology and psychology , human neuroimaging studies have cleared the theoretical landscape , as they have begun to reveal the underlying mechanisms . The study of insight in non - human animals has , in contrast , remained limited to innovative adjustments to experimental designs within the classical approach of judging cognitive processes in animals , based on task performance . This leaves no apparent possibility of ending debates from different interpretations emerging from conflicting schools of thought . We believe that comparative cognition has thus much to gain by embracing advances from neuroscience and human cognitive psychology . We will review literature on insight ( mainly human ) and discuss the consequences of these findings to comparative cognition . Keywords : insight , comparative cognition , problem solving , neuroimaging , comparative psychology INTRODUCTION A 7 years old girl is standing at a table into which psychologists have fixed a vertical transparent tube containing a small basket with a handle and a sparkly sticker inside . On the table , alongside the tubes , lie a long straight piece of pipe - cleaner and a colorful string . After inserting her finger which only reaches down about a third of the tube , the girl immediately grabs the pipe - cleaner and attempts several times to use it to press the handle of the basket against the tube wall and pull it up . The tube is too narrow and the attempts remain unsuccessful . With a hesitant movement , the colorful string is also briefly dangled into the tube before she seems to get distracted ( Isen et al . , 1987 ; Subramaniam et al . , 2009 ) . Her gaze seems lost for a moment ( Segal , 2004 ; Kohn and Smith , 2009 ) when suddenly her pupils dilate ( Salvi et al . , 2020 ) and a smile appears ( van Steenburgh et al . , 2012 ) . She expresses a drawn - out and slightly soaring “Aaahhhh ! ” and immediately grabs the pipe - cleaner , bends a little hook into one of its distal ends , inserts the hooked end of the pipe - cleaner back into the tube , hooks the handle of the basket , pulls the basket over the rim , and claims her reward with determination ( Stuyck et al . , 2021 ) . The hook bending paradigm is a so - called ill - structured innovation task in which the path to the solution is missing information about how to get from its start to its goal state ( Cutting Osuna - Mascaró and Auersperg Insight Across Disciplines Frontiers in Psychology | www . frontiersin . org 2 December 2021 | Volume 12 | Article 791398 et al . , 2014 ) . Interestingly , children that are seven or older find the entire multistep solution to this problem very suddenly rather than in an incremental way . Notably , the hook bending task has similarly been used to test tool innovation in large brained birds and apes , which show a rather ratchet - like improvement upon solving the task for the first time ( rarely failing after first success ; Weir , 2002 ; Bird and Emery , 2009a ; Laumer et al . , 2017 , 2018 ) . The moment just before the little girl tackles the problem , or what Hermann von Helmholtz referred to as a “happy idea” ( Wallas , 1926 ) , may be a familiar sentiment to most of us . Such moments of so - called insight are also a recurringly described ( and romanticized ) phenomenon in scientific history : Newton and that apple , Archimedes in the bathtub , and Poincaré stepping on the bus ; all of them have a common pattern : someone with accumulated experience escapes for a moment from the problem to be solved and suddenly finds themselves surprised ( without knowing how or why ) with the solution . INSIGHT AS A GLOBAL PHENOMENON Although there are cultural differences in the importance we attribute to insight as a source of creative output ( Rudowicz and Yue , 2000 ; Niu and Sternberg , 2006 ; Shao et al . , 2019 ) , the traditional description of the stages of the creative process is very similar in European psychology ( four stage model by Wallas , 1926 ) and Eastern philosophy ( Yoga Sutras ; Maduro , 1976 ; Shao et al . , 2019 ) . Insight itself also has an important bearing in Eastern cultures . For example , in Theravada Buddhism , the goal of vipassana meditation is to reach a sudden understanding , abhisamaya ( insight ) , which contrasts with gradually attained understanding ( anapurva ) . Both the description of the phenomenon and the way in which it is achieved , fit with the popular Western notion of insight ( Laukkonen and Slagter , 2021 ) . Although we can have reasonable confidence that insight is a global phenomenon and not a myth specific to western culture ( a WEIRD one ; Henrich et al . , 2010 ) , it still holds many mysteries regarding its mechanisms and function ( Shen et al . , 2018 ) , as well as its evolution and presence ( and level of expression ) in other species ( Call , 2013 ) . SCIENTIFIC INSIGHT Given the importance of the subjectively perceived components of insight , the phenomenon is certainly easier to study in humans than in non - human animals , both because of the possibility to report verbally ( the subject might describe the suddenness of the solution’s appearance and the emotions involved , but also specific difficulties with aspects of the task , and how close the subject believes he or she is to the solution at any given moment ) and the methodology ( because of test diversity and the relative ease of applying neuroimaging technology ) . A review by Kounios and Beeman ( 2014 ) defines insight as any sudden comprehension , realization , or problem solution that involves a reorganization of the elements of a subject’s mental representation of a stimulus , situation , or event to yield a non - obvious or nondominant interpretation . Note , however , that there are various definitions of insight with some considering it as a dynamic process , and others as an end state ( Call , 2013 ; Kounios and Beeman , 2014 ; Shen et al . , 2018 ) . Insight is further frequently linked to a number of traits ( such as an impasse or a pleasant feeling of surprise ) that may or may not be considered essential to some authors , resulting in variation in the respective definitions ( as reviewed in Kounios and Beeman , 2014 ; and the reason we are using their definition ) . While neuroscience has been hampered by some inconsistencies in definitions of insight ( see Kounios and Beeman , 2014 for examples ) , experimental evidence ( especially due to advances in neuroimaging ; e . g . , Shen et al . , 2018 ) has helped to guide research along a convergent path ( Stuyck et al . , 2021 ) , suggesting that innovation achieved through insight - like experiences can be clearly distinguished from other problem solving strategies ( van Steenburgh et al . , 2012 ) . Despite the success within neuroscience , the topic of insight and even the use of the term in animal behavior has caused significant theoretical debates in comparative cognition ( e . g . , Kacelnik , 2009 ; von Bayern et al . , 2009 ; Emery , 2013 ) . Notably , few animal studies are included the recent literature on human problem solving or neuroscience ( Shettleworth , 2012 ; Call , 2013 ) . FIRST SCIENTIFIC APPROXIMATIONS TO INSIGHT In 1925 – 1926 , Wolgang Köhler and Graham Wallas independently published two books that had long lasting effects on the general perception of problem solving : The Mentality of Apes , by Köhler , and The Art of Thoughts , by Wallas . Wallas , inspired by the ideas of Hermann von Helmholtz and Henri Poincare , proposed four stages of progression for a creative process ( Wallas , 1926 ) . Helmholtz , during a banquet held for his 70th birthday in 1891 , revealed how he had reached his best ideas ; always after first researching a problem in detail , letting it rest , and seeking a pleasant distraction . This way he was often surprised by a solution in the form of a pleasant experience . Wallas named these stages preparation ( investigative stage ) , incubation ( temporally discarding the problem from conscious thought ) , and illumination ( the sudden arrival of a new “happy idea” ) , to which he added a fourth , the verification of the solution . These four stages have been recurrently used as a framework for studying insight in the psychological literature ( Luo and Niki , 2003 ; Jung - Beeman et al . , 2004 ; Sandkühler and Bhattacharya , 2008 ; Weisberg , 2013 ) . Although Wallas’ work covers the creative process in rather broad terms , its relevance to the study of insight is remarkable , due to the close proximity and similarity in conceptualization , measures , and processes ( Shen et al . , 2017 , 2018 ) . Osuna - Mascaró and Auersperg Insight Across Disciplines Frontiers in Psychology | www . frontiersin . org 3 December 2021 | Volume 12 | Article 791398 Almost at the same time , Wolfgang Köhler , one of the pioneers of Gestalt psychology , introduced the term insight into comparative psychology ( although this way of problem solving was already described before him in non - human animals ; Turner , 1909 ; Köhler , 1925 ; Weisberg , 2006 ; Galpayage Dona and Chittka , 2020 ) . Gestalt psychologists proposed that insight depends on different mechanisms to trial and error learning , which , according to Thorndike ( 1911 ) , was the only way in which animals could solve problems ( Köhler , 1925 ; Koffka , 1935 ; Duncker , 1945 ; Wertheimer , 1959 ) . Köhler worked for years at the Casa Amarilla in Tenerife ( Canary Islands , Spain ) with seven chimpanzees , testing them in experiments where they had to find unusual methods to reach food ( see Figure 1 ) . In those experiments , Köhler found problem solving strategies that did not seem compatible with classical associative learning routines : After an unsuccessful period of trial and error , in which the chimpanzees used familiar strategies , they stopped trying . Nevertheless , after a while some of them returned with a completely different and , this time , immediately successful strategy . After their first success , the animals could immediately retrieve the correct sequence of steps on the following occasions when they faced the same problem . Köhler , at the time , described these strategies as cognitive trial and error and insight , rather than associative processes . Other Gestalt psychologists adapted Köhler’s problem solving methodology to study insight in humans . Duncker ( 1945 ) , for example , designed situations in which everyday objects had to be used in unusual ways to solve a task ( e . g . , the candle problem , see Figure 1 ; Duncker , 1945 ) . Notably , if he asked the subjects to use these objects in their usual way before the test , the success rate was reduced . Duncker and other Gestalt psychologists ( e . g . , Maier , 1930 ; Luchins , 1942 ; Scheerer , 1963 ) concluded that the repeated application of incorrectly selected knowledge could prevent the deep conceptual understanding necessary to achieve insight . This phenomenon is now known as functional fixedness ( Duncker , 1945 ) . It was , however , the British ornithologist W . H . Thorpe who coined in his book Learning and Instinct in Animals ( 1956 ) the most prevalent definition of insight in psychology today ; “ the sudden production of a new adaptive response not arrived at by trial behaviour or the solution of a problem by the sudden adaptive reorganization of experience . ” We will later explain how an over - emphasis on the absence of trial and A D E B C F FIGURE 1 | ( A ) The Crow and the Pitcher , illustrated by Milo Winter ( 1919 ; Public Domain ) . Stones must be dropped into water to have access to the liquid , or to a floating object . ( B ) String - pulling ; “Still Life with Fruit and a Goldfinch , ” Abraham Mignon ( 1660 ; Public Domain ) . Goldfinch’s detail , right side . To have access to the hanging object , the string must be pulled first ; as seem in Jacobs and Osvath ( 2015 ) . ( C ) Three - boxes experiment ; “Grande on an insecure construction” The Mentality of Apes , Köhler ( 1925 ; CC ) To get the banana , the chimpanzees must pile the boxes . ( D ) Early representation of the nine - dot problem ; Egg of Columbus , Sam Loyds Cyclopedia of Puzzles ( 1914 ; Public Domain ) . Nine dots , arranged in three parallel lines , must be linked with four connected straight lines . ( E ) Candle problem ; Duncker ( 1945 ; Public Domain ) A candle must be attached to the wall ; subjects are given a box of tacks , a candle , and matches . Problem on top , solution , below . ( F ) Compound Remote Associates Test test ; developed by Mednick and Mednick ( 1967 ) . Subjects are given the three words on top and have to find one to link with each one of them ( as the one in brackets ) . All Public Domain and Creative Commons ( CC ) images can be found in Wikimedia Commons . Osuna - Mascaró and Auersperg Insight Across Disciplines Frontiers in Psychology | www . frontiersin . org 4 December 2021 | Volume 12 | Article 791398 error learning , and a lack of attention to the “reorganization of experience , ” may have affected the interpretation of insight in comparative cognition . OUR CURRENT UNDERSTANDING OF INSIGHT Insight is often conceptualized as a process in which a subject has a sudden realization of how to solve a novel problem ( Schooler et al . , 1995 ; Sheth et al . , 2009 ) . Thereby specific elements of a subject’s mental representation of various stimuli , situations , or events are reorganized to yield a nonobvious or nondominant interpretation ( Kounios and Beeman , 2014 ) . Insight is associated with a number of characteristic phases that set it apart from other mental processes employed in problem solving , such as a distinctive subjective momentary experience of surprise and delight , the “aha” or “eureka” moment ( Bowden et al . , 2005 ) . Neuroscience typically contrasts insight with analytical reasoning within problem solving . A directly perceivable difference between the two seems to be a more or less gradual progress toward a solution in analytical thinking ( Smith and Kounios , 1996 ) , while individuals are abruptly surprised by the latter during an insightful solution ( Metcalfe and Wiebe , 1987 ) . Thus , insight is believed to depend by a large degree ( but not completely ) on unconscious mental processing , as we will see in the next sections ( Sandkühler and Bhattacharya , 2008 ; Shen et al . , 2013 , 2018 ; Weisberg , 2013 ) . Convergent Insight Process Theories The main theoretical proposals to explain insight largely differ with regards to the amount of conscious processing they describe involved in an insightful event . For example , approaches , such as the representational change theory ( also called the redistribution theory ; ( Ohlsson , 1992 , 2011 ; Knoblich et al . , 1999 ) , advocate a completely unconscious redistribution of information ( Knoblich et al . , 1999 ; Ohlsson , 2011 ) , whereas the progress monitoring theory ( or criterion for satisfactory progress theory ; MacGregor et al . , 2001 ; Chu et al . , 2007 ) proposes insight through a conscious process : searching consciously among a pool of possible solutions during which wrongful presumptions are dropped in favor of a working solution . In an attempt to find a bridge between the strengths of both previous theories , Weisberg proposed an integrated theory of insight comprising several phases : the individual would first attempt to find a solution by using strategies based on long - term memory ; if this fails , the subject would use rules of thumb or more complex heuristics to acquire information about the problem before re - confronting its long - term memory ; then , a conscious solution via a restructuring of old and new information may thereby be achieved ; and if the process reaches an impasse and new information is no longer acquired , an unconscious restructuration of knowledge would take place ( Weisberg , 2015 ) . Interestingly , the four stages of Weisberg ' s ( 2015 ) proposal bear some parallels to those suggested by Wallas in the mid twentieth century ( Wallas , 1926 ) . “Preparation” would comprise the first three phases of the integrated insight theory , while “incubation” and “illumination” could be interpreted as part of the fourth , where insight is achieved through an unconscious process ( see above , section four , to find Wallas’ proposal ) . Fixation and Impasse The fixation and impasse ( the repetition of incorrect strategies , and the following temporary withdrawal of action ) , as already described by Duncker ( 1945 ) , are likely the result of an inappropriate knowledge base ( Wiley , 1998 ) or incomplete heuristics ( Knoblich et al . , 1999 , 2001 ) . Knoblich et al . ( 1999 ) found that expertise in algebra can negatively affect insightful arithmetic problem solving . Similarly , great apes have trouble innovating a solution to a problem when the tools or objects at their disposal were previously used in a different way ( Hanus et al . , 2011 ; Ebel et al . , 2020 ) . Such “functional fixedness” may be one of the factors responsible for the fixation leading to an impasse . It is important to highlight at this point that there are no insight problems but only insight solutions : any problem solved by insight could also be solved analytically ( van Steenburgh et al . , 2012 ) , and that an impasse ( although common ) is not required for insight to occur ( MacGregor et al . , 2001 ; Ormerod et al . , 2002 ; Kounios and Beeman , 2014 ) . However , the design of a problem is highly important as it determines the nature of its solution / s . Experimental subjects in classical insight challenges , such as Duncker’s candle problem ( e . g . , Duncker , 1945 ; Knoblich et al . , 2001 ; Huang , 2017 ) , often encounter an impasse prior to the solution . This is much less common in so - called CRAT - based challenges ( a specific type of word puzzle , see Figure 1 ; e . g . , Cranford and Moss , 2012 ; Webb et al . , 2019 ) even if they are also solved by insight . This could be because classical tests often have misleading structures and / or contain elements that may provoke functional fixedness ( Duncker , 1945 ; Hanus et al . , 2011 ; Stuyck et al . , 2021 ) . Nevertheless , the scientific approach for detecting an impasse may also be problematic ( Stuyck et al . , 2021 ) : Studies that found no impasse before insightful solutions mostly relied on verbal reports ( e . g . , Webb et al . , 2019 ) , while when other methods were used an impasse was more likely to be detected ( e . g . , eye tracking , Huang , 2017 ; neurophysiological measurements , Shen et al . , 2018 ) . Incubation / Restructuring and Illumination An impasse is usually followed by an incubation / restructuring stage , which is suspected to constitute the insight’s core ( Wallas , 1926 ; Sandkühler and Bhattacharya , 2008 ; Sio and Ormerod , 2009 ; Cranford and Moss , 2012 ; Weisberg , 2013 ) . Although restructuring can of course be done consciously ( Weisberg , 2015 ) , it may also happen at a time during which a subject consciously withdraws from the problem at hand ( van Steenburgh et al . , 2012 ; Kounios and Beeman , 2014 ; Shen et al . , 2018 ) . We know that insight - like responses improve when participants take a break after reaching an impasse ( or when the task is Osuna - Mascaró and Auersperg Insight Across Disciplines Frontiers in Psychology | www . frontiersin . org 5 December 2021 | Volume 12 | Article 791398 simply removed from their sight ; Kohn and Smith , 2009 ) , regardless of the duration of the break , and particularly when the break is occupied with a different , cognitively demanding task ; Segal , 2004 ) . Human neuroimaging and electrophysiology - based studies suggest a significant function of the prefrontal cortex in the process of overcoming impasse to reach incubation ( e . g . , Qiu et al . , 2010 ; Zhao et al . , 2013 ; Seyed - Allaei et al . , 2017 ; Shen et al . , 2018 ) . The right inferior frontal gyrus plays a role in evaluating possible solutions while the left gyrus seems to control the suppression of inappropriate mental sets or dominantly activated associations ( e . g . , Jung - Beeman et al . , 2004 ; Shen et al . , 2013 , 2018 ; Wu et al . , 2015 ) . This corresponds with studies reporting brain asymmetries in insight tests . Studies using insight and priming with word hints ( where the left hemisphere typically has an advantage ; van Steenburgh et al . , 2012 ) , the left visual field ( right hemisphere ) has shown a strong advantage over the right , with primed participants finding more solutions faster ( Bowden and Beeman , 1998 ; Beeman and Bowden , 2000 ) . Studies based on event - related potentials have so far been able to identify two distinct cognitive processes involved in achieving an insightful event : the breaking down of the impasse ( allowing incubation / restructuring ) and the formation of new associations prior to the solution ( Luo and Niki , 2003 ; Luo et al . , 2011 ; Zhao et al . , 2013 ; Shen et al . , 2018 ; it is also described as the enlightenment stage by Wallas , 1926 ) . Associations that will result in a solution can take different routes ; once strong yet incorrect associations can be overcome , weaker yet correct association can be detected ( Shen et al . , 2018 ) . Interestingly , the latter is facilitated by a positive emotional state ( Isen et al . , 1987 ; Subramaniam et al . , 2009 ; van Steenburgh et al . , 2012 ) . In humans , a positive emotional state at the start of testing is associated with increased activity in the anterior cingulate cortex ( which is related to monitoring cognitive conflict ; Carter et al . , 2000 ) and an increase in insightful solutions ( Subramaniam et al . , 2009 ) . While neurobiology and cognitive psychology embrace insightful solutions achieved by associations learned in the past , comparative cognition tends to exclude associative learning from its notion of insight , which is a misconception as insight can occur through distant or weak associations ( Shettleworth , 2012 ; Call , 2013 ) . In comparative cognition , insight has occasionally been used as a default explanation upon failing to detect the typical gradual process of associative learning . A candidate for explaining how we can learn non - obvious associations is latent learning ( Tolman and Honzik , 1930 ; Tolman , 1948 ) . The nervous system can register associations without the need for positive reinforcement ( such as those that can be acquired through random exploration ) . These associations remain latent and are candidates for insightful solutions ( Thorpe , 1956 ) . Latent associations , being weak , can be adjusted more flexibly if required ( Call , 2013 ) . In contrast , strong associations can result in functional fixedness where a previous solution prevents the innovation of a new solution ( e . g . , humans , Duncker , 1945 ; great apes , Ebel et al . , 2020 ) . However , the path toward a solution can be achieved by other mechanisms . The free energy principle [ the basis of Predictive Processing Theory ( PPT ) , e . g . , Hohwy and Seth , 2020 ; Francken et al . , 2021 ] predicts that all sentient beings minimize uncertainty for energetic reasons ( Friston , 2003 ) . According to PPT , all interaction with the environment involves constant amendment between perceptual input and the internal models ( Friston et al . , 2016a ) . When the flow of input stops during an impasse , models continue to be optimized without the agent consciously perceiving it . This has been called fact - free learning or model selection and reduction ( model selection , Aragones et al . , 2005 ; model reduction , Friston et al . , 2016b ) . In the absence of new data , the only way we can optimize our generative models is by making them simpler ( Friston et al . , 2017 ) . Model reduction is a similar process to that described in the N - REM phase of sleep , where redundant connections between neurons are eliminated ( Tononi and Cirelli , 2006 ) and models are reduced in complexity in the absence of new sensory input ( Friston et al . , 2017 ) . Model reduction occurs neither only during sleep , nor only in humans . Rats that move away from exploratory or spatial foraging behavior , and enter short periods of rest , have been found to have hippocampal activity similar to what we would expect in models undergoing insight - compatible changes ( Gupta et al . , 2010 ; Pezzulo et al . , 2014 ; Friston et al . , 2017 ) . Internally generated sequences ( sequences of multi - neuron firing activity that do not reflect an ongoing behavioral sequence ) seem to be able to restructure models , not only consolidating memory but also exploring potential solutions ( Pezzulo et al . , 2014 ) . The Eureka Experience A popular event related to insight is the so - called “aha” moment , a subjective experience of surprise and delight accompanied by sudden solutions ( Bowden et al . , 2005 ; Sandkühler and Bhattacharya , 2008 ; Weisberg , 2013 ; Shen et al . , 2017 ) . This pleasant experience is probably one of the reasons why insight responses are associated with positive emotions versus analytical solutions that are negatively perceived ( Shen et al . , 2016 , 2017 ; Webb et al . , 2016 , 2019 ) . This may also contribute to a better memorization and a higher success rate of insightful responses ( e . g . , Danek et al . , 2013 ; Webb et al . , 2016 ; Salvi et al . , 2020 ; Stuyck et al . , 2021 ) . Notably , insight does not necessarily require this “aha” experience . In verbal tests , insight lacking major emotional changes has been reported ( Kounios and Beeman , 2014 ) . This may be the reason why CRAT tests do not elicit a perceivable impasse experience ( Stuyck et al . , 2021 ) . Nevertheless , the impasse may be an important contributing factor to the surprise element of the insight revelation as it fosters the perception of a metacognitive error in which we solve a problem faster than expected ( Dubey et al . , 2021 ) . The subpersonal nature of model reduction ( that is , there is no explicit inner model , hence no conscious experience of the reduction process ) could explain why the agent becomes aware at the precise instance of a new association , and not before ( Metcalfe and Wiebe , 1987 ; Friston et al . , 2017 ; Shen et al . , 2018 ) . Another proposed explanation for the relation of insight with consciousness is the asymmetrical involvement Osuna - Mascaró and Auersperg Insight Across Disciplines Frontiers in Psychology | www . frontiersin . org 6 December 2021 | Volume 12 | Article 791398 of both hemispheres and the important role of the right hemisphere in key parts of the process ( see split brain perception studies , e . g . , Gazzaniga , 1998 ; van Steenburgh et al . , 2012 ) . Furthermore , the conscious perception of the solution is plausible considering the close relationship between associative learning and consciousness ( Ginsburg and Jablonka , 2007 , 2019 ) and the essential role of consciousness for the former to occur ( e . g . , Baars et al . , 2013 ; Meuwese et al . , 2013 ; Weidemann et al . , 2016 ) . NON - HUMAN ANIMALS , PROBLEMS , AND SOLUTIONS Comparative cognition has attempted to tackle the presence of insight in animals by rating the speed of their performance on technical problem or their ability to transfer information from one task to another ( Seed and Boogert , 2013 ) . One issue with this may be that , as mentioned earlier , there are no insight problems , only insight solutions ; a problem designed to be solved by insight can also be solved by other processes ( van Steenburgh et al . , 2012 ) . Epstein et al . ( 1984 ) tried to highlight this issue in a popular paper which showed that pigeons solved seemingly complex problems spontaneously by “chaining” blocks of previously learned information . Neuroscience’s results and advances have been able to compensate a lack of theoretical consistency regarding insight . Cognitive research on animal insight , on the other hand , has been limited to the creativity of experimental designs , with no apparent chance of ending long - running debates stemming from two opposing schools of thought , cognitive psychology and behaviorism , “romantics” against “killjoys” ( Shettleworth , 2010 , 2012 ; Call , 2013 ; Starzak and Gray , 2021 ) . While we believe that the progress of comparative cognition feeds ( as a dissipative structure ) on the continued conflict between the two positions , the lack of experimental progress has kept these discussions in an impasse ( e . g . , Heinrich , 1995 ; Kacelnik , 2009 ; Chittka et al . , 2012 ; Taylor et al . , 2012 ; Emery , 2013 ; Starzak and Gray , 2021 ) . Today we know that insight is a measurable phenomenon with a physiological basis that is beginning to be revealed ( Shen et al . , 2018 ) . Moreover , it makes little sense to set the phenomenon apart from associative learning and experience ( Shettleworth , 2010 , 2012 ; Hanus et al . , 2011 ; Call , 2013 ; Shen et al . , 2018 ; Ebel et al . , 2020 ) . Insight does not mean developing de novo behaviors to solve a problem , but to find a solution by restructuring the problem , even if the agent reorganizes old experiences to apply them to a novel context . Although insight involves making the nonobvious seem obvious , and even tends to correlate with a higher success rate at problem solving ( higher successful rate , Salvi et al . , 2016 ; Webb et al . , 2016 ; but see , Stuyck et al . , 2021 ) , a successful restructuring does not necessarily imply a correct conceptualization of the full nature of the problem , and an answer obtained by insight need not necessarily be correct ( Kounios and Beeman , 2014 ) . Just as a feeling of understanding does not equate to a true understanding of the problem , we must thus be careful in equating insight with understanding or suggesting that one predicts the other . Insight may exist in animals outside humans and could even be relatively widespread in nature ( e . g . , Shettleworth , 2012 ; Pezzulo et al . , 2014 ) . Yet to proficiently tackle the phenomenon in non - verbal species is an unsolved problem in comparative cognition . While rodent studies suggest that insight does not require sophisticated cognition , the role of the prefrontal cortex in important insight stages may suggest insightful solutions are more likely to emerge in species that have highly developed and functionally equivalent brain regions ( Shettleworth , 2010 , 2012 ; Call , 2013 ; Olkowicz et al . , 2016 ; Shen et al . , 2018 ) . Methodologies , such as the priming of different brain hemispheres , related to insight ( which function similarly in non - human primates as in humans ) as well as new technologies in animal eye tracking open the door to technically challenging targeted studies in species other than our own ( Krupenye et al . , 2016 ; Shen et al . , 2018 ; Völter et al . , 2020 ; Ben - Haim et al . , 2021 ) . The crucial role of subjective experience in insight , as well as the traditional reliance on verbal reports in a large number of studies , makes it tempting to conclude that the study of insight is inaccessible in non - human animals . Nonetheless , other signatures of insight do exist ( e . g . , Kounios and Beeman , 2014 ) . Apart from EEG and fMRI studies , evidence of human insight stems also from eye tracking studies ( e . g . , Salvi , 2013 ; Salvi et al . , 2016 ; Huang , 2017 ) , grip strength ( Laukkonen et al . , 2021 ) , heart rate ( Hill and Kemp , 2018 ) , pupil dilation , and eye movement ( with pupil dilation happening only just prior to an insightful event , and an increase in microsaccade rate coinciding with analytic responses ; Salvi et al . , 2020 ) . Moreover , it has been shown repeatably that agents do not even necessarily need to solve the problem . A promising approach could be to confront an animal with a problem and then , after a period unsuccessful interaction , to suddenly show the solution and record the response ( e . g . , Kizilirmak et al . , 2016 ; Webb et al . , 2019 ) . Even the “aha” moment itself might be accessible to study in non - verbal subjects , given the expected physiological emotional response that follows it . We know that many animals show an emotional response while learning how to solve tasks ( independent from the presence of a reward ; e . g . , cows , Hagen and Broom , 2004 ; goats , Langbein et al . , 2004 ; horses , Mengoli et al . , 2014 ; dogs , McGowan et al . , 2014 ; dolphins , Clark et al . , 2013 ) . Studying insight through the presentation of a solution would thus require both a behavioral analysis ( as in traditional contrafreeloading tests or yoked experimental designs ; e . g . , Hagen and Broom , 2004 ; Rosenberger et al . , 2020 ) as well as a physiological one . Artificially altering the transparency of the path toward the solution , and altering the time spent at an apparent impasse , may allow us to predict and modify the intensity of the respective physiological ( as it would be an increased heart rate ; Hill and Kemp , 2018 ) and behavioral responses ( e . g . , in dogs , we would predict pupil dilation , tail wagging , and increased general activity ; McGowan et al . , 2014 ; Webb et al . , 2019 ; Salvi et al . , 2020 ) . Osuna - Mascaró and Auersperg Insight Across Disciplines Frontiers in Psychology | www . frontiersin . org 7 December 2021 | Volume 12 | Article 791398 CONCLUSION Insight is a measurable phenomenon in humans , and the mechanisms by which it occurs may well be accessible to species other than our own . Thanks to recent progress in neuroscience and human psychology , we are beginning to clarify the ( in some cases subtle ) differences that distinguish insight problem solving from other processes . Comparative cognition , however , has so far been limited in its approach . Performance - based setups using technical problems in both birds and mammals have produced highly interesting and suggestive , yet , ambivalent evidence on animal insight ( e . g . , Heinrich , 1995 ; Mendes et al . , 2007 ; Bird and Emery , 2009a , b ; Laumer et al . , 2017 , 2018 ; von Bayern et al . , 2018 ) . We are optimistic that accomplishments in neuroscience and human psychology over the past decade can be incorporated into and inspire future comparative cognition studies in their ongoing quest to learn about the capacity for insight in species other than our own . AUTHOR CONTRIBUTIONS AO - M wrote the first draft . AO - M and AA finished the manuscript . All authors contributed to the article and approved the submitted version . FUNDING The authors are funded by the WWTF Project CS18 - 023 and START project Y 01309 by the Austrian Science Fund ( FWF ) to AA . ACKNOWLEDGMENTS We thank Poppy J . 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This is an open - access article distributed under the terms of the Creative Commons Attribution License ( CC BY ) . The use , distribution or reproduction in other forums is permitted , provided the original author ( s ) and the copyright owner ( s ) are credited and that the original publication in this journal is cited , in accordance with accepted academic practice . No use , distribution or reproduction is permitted which does not comply with these terms . \ No newline at end of file diff --git a/silver_data/0d872a822e71d50cf4992e9b11d9cda7700a7597.pdf.txt b/silver_data/0d872a822e71d50cf4992e9b11d9cda7700a7597.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..cfe4d5f0a96b2549ef7e246dfe6fb9ea9c7f93b1 --- /dev/null +++ b/silver_data/0d872a822e71d50cf4992e9b11d9cda7700a7597.pdf.txt @@ -0,0 +1 @@ +COVID - 19 and Transportation Transportation Research Record 2023 , Vol . 2677 ( 4 ) 629 – 640 (cid:2) National Academy of Sciences : Transportation Research Board 2023 Article reuse guidelines : sagepub . com / journals - permissions DOI : 10 . 1177 / 03611981221138807 journals . sagepub . com / home / trr Durations of Dockless E - Scooter Trips Before and During the COVID - 19 Pandemic in Austin , TX : An Analysis Using Hazard - Based Duration Models Amin Azimian 1 and Junfeng Jiao 1 Abstract The pandemic arising from the 2019 coronavirus disease has significantly affected all facets of human life across the world , including economies and transportation systems , thereby changing people’s travel behaviors . This research was aimed at exploring the relationship between socio - economic factors and e - scooter trip durations before and during the pandemic . We developed a hazard - based duration approach and estimated multiple spatial and non - spatial models on the basis of 2019 and 2020 dockless e - scooter data collected from the City of Austin’s Open Data Portal . The results indicated an overall increase in e - scooter trip durations after the pandemic . Moreover , analysis of variables revealed potential changes in users’ behavior before and during the pandemic . In particular , whereas e - scooter trip durations were found to be positively associated with aggregate travel time to work before the pandemic , this trend was reversed during the pandemic . In addition , during the pan - demic , e - scooter travel time was positively correlated with the ratio of individuals with bachelor’s degrees or greater to those with associate degrees or lower . However , no specific pattern was observed before the pandemic . Lastly , the results showed the presence of disparities within the study area ; therefore , it is vital to extend e - scooter service areas to cover underserved communities . Keywords spatial data , e - scooters , COVID - 19 , statistical analysis In 2020 , the global outbreak of the 2019 coronavirus dis - ease ( COVID - 19 ) that originated from Wuhan , China infected over 91 . 5 million people and resulted in a loss of 1 . 9 million lives around the world ( 1 ) . The virus rapidly spread beyond China and reached its peak in the Middle East and Europe , with especially high cases occurring in Iran and Italy . In late March 2020 , the pandemic also consolidated in the United States with a total of 82 , 000 confirmed cases ( 2 ) . Over the past year , the pandemic has affected all facets of human life , including econo - mies and transportation operations . For example , more than 25 million people lost their jobs from February to April 2020 ( 3 ) , and the U . S . suffered a $ 50 billion loss in its real gross domestic product ( 4 ) . COVID - 19 like - wise posed significant challenges to every aspect of transportation in the U . S . A case in point is the situation in the early phase of the pandemic , during which airlines and passenger railway services experi - enced an up to 50 % drop in bookings and operations . Urban public transportation has also been tremen - dously affected because of stay - at - home orders , travel anxiety , and fear of infection , which have driven a large proportion of the population to shift from public to private modes of work ( 5 ) . Similar to other transportation systems , shared mobi - lity services in the U . S . suffered a sharp reduction in usage in March 2020 , thus forcing many businesses to 1 Urban Information Lab , University of Texas at Austin , Austin , TX Corresponding Author : Amin Azimian , amin . azimian @ austin . utexas . edu suspend system operations . One of the newest forms of shared mobility services that have recently gained popu - larity in the country is shared and dockless e - scooter ser - vices ( e . g . , Lime and Bird scooters ) , which provide a convenient and sustainable mode of transportation and allow users to travel over short and inter - distance trips . E - scooters have successfully satisfied the huge gap between supply and demand in the market and have become highly favored transit modes because of their flexibility ( 6 ) . The evaluation of data released by Transportation TPBo ( 7 ) and Department CoAT ( 8 ) indicated that cities with e - scooter sharing systems , such as Austin and Portland , have seen usage rates rise by the end of sum - mer 2020 . Specifically , these cities registered a 64 % and 46 % increase in e - scooter use , respectively , including a doubling of travel in low - income neighborhoods . During a time characterized by a reluctance to opt for transit and carsharing services , people are using e - scooters for essential trips mostly because these vehicles serve as a clean and open - air mobility option that also enables users to maintain social distancing ( 9 ) . According to sur - vey data conducted by the City of Chicago , 22 % of riders have often used e - scooters to avoid public trans - port systems , given COVID - 19 concerns . The majority of e - scooter users likely belong to the 25 - to 34 - year age group , are almost twice as likely to have a bachelor’s degree , are 1 . 7 times more likely to identify as White than average , and 1 . 3 times more likely to identify as male ( 10 ) . Similar trends are expected from other U . S . cities with e - scooter sharing services . Although the built environment factors as well as socio - demographic characteristics of e - scooter users have been well examined in different studies ( 6 , 11 , 12 ) , the extent to which they have contributed to e - scooter trip durations before and during the pandemic is unclear . Such information would assist operators and decision makers to investigates potential demographic and eco - nomic disparities across census tracts during the pan - demic , and to broaden their offering and price attractively to appeal to a broader user base . The current research contributes to the literature in several ways . First , earlier research has mostly focused on e - scooter trip frequency rather than e - scooter ridership duration . Therefore , there is scant information on the extent to which socio - economic and built environment factors contribute to such duration . Second , we investigated possible changes in trip durations before and during the pandemic . Finally , we adopted a hazard - based duration model with unstructured spatial random effect terms to account for unmeasured risk factors across census tracts in Austin . Literature Review Travel Behaviors and COVID - 19 Over the last year , many studies have explored the impact of COVID - 19 on transportation on the basis of different changes , such as those occurring for modal shares ( 13 – 19 ) , transit ridership ( 14 , 20 , 21 ) , airline operation ( 22 , 23 ) , railway systems operation ( 24 ) , daily outdoor activi - ties ( 14 , 19 , 22 , 25 – 30 ) , telework status ( 22 , 31 ) , travel distances ( 18 , 32 ) , homestay dwell time , and attitudes and risk perceptions ( 33 ) . These explorations highlighted a large decline in mobility as a result of state - level lock - downs and the fear of COVID - 19 infection . Abdullah et al . ( 34 ) conducted a factor analysis and proposed a logit model to identify the effects of socio - economic factors on modal shares for primary outdoor trips across various countries . They reported that gender , car ownership , employment status , and travel distance have affected the travel mode choices of individuals dur - ing the pandemic . Jiao and Azimian ( 35 ) explored the relationship between socio - demographic factors and changes in the number of trips made via public transpor - tation systems before and during the pandemic . The authors found that age , gender , educational status , mari - tal status , work loss , difficulty with expenses , and house - hold size were significantly associated with changes in travel behavior . Bucsky ( 17 ) analyzed changes in modal shares in Budapest , Hungary in March 2020 and uncov - ered a significant decrease in transit ridership alongside the increased use of private vehicles and bicycles . de Haas et al . ( 26 ) evaluated alterations in work and travel behaviors in the Netherlands as a result of a national lockdown in the country . Their results , which were based on work and travel data on 2 , 500 respondents , indicated that work - and education - related travel activities have decreased by 68 % amid the global outbreak . The authors also reported that the elderly population has reduced participation in outdoor activities more drastically than other population groups . Employing GPS ( global posi - tioning system ) data on changes in average travel dis - tance , Engle et al . ( 32 ) used COVID - 19 cases and population characteristics to estimate the effects of dis - ease prevalence and restriction orders on individual mobility in the U . S . They revealed that traveled distances have been substantially reduced in response to changes in perceived disease prevalence and stay - at - home directives . E - Scooter Ridership and COVID - 19 Since the emergence of e - scooter ridership in the U . S . in 2017 , various studies have attempted to examine travel behavior patterns under shared e - scooter use ( 36 – 41 ) and 630 Transportation Research Record 2677 ( 4 ) their relationship with built environment and socio - demographic factors ( 6 , 11 , 12 , 37 ) . McKenzie ( 40 ) , for instance , compared the spatiotemporal patterns reflected in dockless e - scooters trips against those of station - based bike trips in Washington DC . He reported that bikeshar - ing customers are more likely to commute to and from work compared with e - scooter users . Similarly , Mathew et al . ( 39 ) performed spatiotemporal analyses of e - scooter trips in Indianapolis and found the downtown area and university campuses as two geographic hotspots for e - scooter traffic , with most scooter activities observed between 11 a . m . and 9 p . m . Additionally , the association between e - scooter ridership and built environments and has been investigated and it has been discovered that e - scooter ridership is positively correlated with land use mix , transit accessibility , open spaces , and educational centers ( 6 , 11 , 12 ) . To the best of our knowledge , few studies ( 38 , 41 ) have attempted to analyse e - scooter services during the pandemic . One of the rare pieces of research is that of Yan et al . ( 41 ) , who conducted a spatiotemporal analysis of the interaction between e - scooter services and two existing travel modes ( i . e . , public transit and bikesharing ) in Washington DC . The authors reported that e - scooters have complemented bikesharing and transit ridership in underserved neighborhoods . A similar investigation is that conducted by Li et al . ( 38 ) , who evaluated changes in micro - mobility travel behaviors before and during the pandemic in Switzerland . Their results revealed that users have traveled longer distances using micro - mobility services during the pandemic than before this crisis . Moreover , bike and e - bike services have induced more changes compared with e - scooter services during the COVID - 19 global outbreak . Despite the insights derived from these works , however , they are limited in that they failed to assess whether changes have occurred in the relationship between e - scooter trip durations and poten - tial contributing factors ( e . g . , socio - demographic and built environment factors ) before and during the pan - demic . Table 1 shows the summary of past works . Method Non - Spatial Model Survival analysis has been extensively used in biostatis - tics ( 42 ) and social sciences ( 43 ) , and it refers to analysis of time - to - event such as time to death or time to recover from a specific disease . However , in this study , we uti - lized the e - scooter trip duration as the variable of interest for the survival analysis . It should be noted that the sur - vival approach has major advantages over traditional regression models . The traditional regression model defines a probability distribution for travel time and fit it using data , whereas the survival model specifies the probability of the outcome as a sequence of conditional events ( 44 ) . That is , owing to COVID - 19 risk perceptions and availability of other transport systems , users may consistently re - evaluate the use or e - scooters for longer / shorter trips during the pandemic . Let T be a non - negative , random variable represent - ing e - scooter trip duration . f ( t ) be the probability density and F ( t ) be the cumulative distribution function giving the probability that the e - scooter travel time of individ - ual does not exceed t . F ð t Þ ¼ P ð T ł t Þ ¼ ð t 0 f ð u Þ : du ð 1 Þ Therefore , the survival function can be written as com - plement of the cumulative distribution function , which is given as follows : S ð t Þ ¼ P ð T . t Þ ¼ ð ‘ t f ð u Þ : du ð 2 Þ where S ( t ) is the survival probability , which gives the probability that the e - scooter trip duration of an individ - ual is greater than t . In survival analysis , the hazard rate function is used to characterize the distribution of T can , and it is defined as : h ð t ij Þ ¼ lim D t ! 0 P ð t ł T \ t + D t j T ø t Þ D t ¼ f ð t Þ 1 (cid:2) F ð t Þ ¼ (cid:2) d dt log ½ S ð t Þ(cid:3) ð 3 Þ where h ( t ) is the hazard rate , and it represents the instan - taneous probability that the e - scooter trip duration ends in the interval ( t , t + dt ) , given that the trip duration has not ended before t . By integrating the above expression from 0 to t , the survival function will be derived as follows : S ð t Þ ¼ exp (cid:2) ð t 0 h ð x Þ : dx (cid:2) (cid:3) ð 4 Þ As the e - scooter trip duration is influenced by built envi - ronment and socio - demographic factors , a proportional hazard must be employed to accommodate these effects . Exponential , Weibull , Lognormal , Gompertz , and so forth are some of the common distributions that can be used to specify the hazard rate . In this study , a Weibull model is parametrized as a proportional hazard . The Weibull distribution is a generalization of the exponen - tial distribution . It is popular in survival analysis as it can accurately model the time - to - event outcomes ( 45 ) . The Weibull model is suitable for modeling data with monotone ( increasing or decreasing ) hazard rates over time , and its hazard and survival functions can be writ - ten as follows : h ð t Þ ¼ h 0 ð t Þ : exp ð b X Þ ð 5 Þ Azimian and Jiao 631 T a b l e 1 . S u mm a r y o f P a s t W o r k s F o c u s i n g o n I m p a c t s o f 2019 C o r o n a v i r u s D i s e a s e ( C O V I D - 19 ) o n T r a n s p o r t a t i o n A u t h o r ( s ) M e t h o d s K e y f i n d i n g s M o b ili t y m e a s u r e s E - s c oo t e r t r i p d u r a t i o n C h a n g e i n t r a v e l e d d i s t a n c e C h a n g e i n m o d a l s h a r e s C h a n g e i n c o n g e s t i o n C h a n g e i n pub li c t r a n s p o r t d e m a n d C h a n g e i n d a il y a c t i v i t i e s C h a n g e i n a i r li n e e m p l o y ee s R i s k p e r c e p t i o n t o w a r d t r a v e l a c t i v i t i e s T e l e w o r k s t a t u s A b d u ll a h e t a l . ( 24 ) F a c t o r a n a l y s i s a n d l o g i t m o d e l A s i g n i f i c a n t m o d a l s h i f t f r o m m o t o r b i ke t o n o n - m o t o r i z e d m o d e s o f t r a v e l w a s f o un d f o r d i s t a n c e s l e ss t h a n 5 k m . N A N A x N A N A N A N A N A N A A r e ll a n a e t a l . ( 14 ) Exp l o r a t o r y d a t a a n a l y s i s N a t i o n a l p o li c i e s a n d l o c a l d e c i s i o n s h a v e d e c r e a s e d t h e d e m a n d f o r m o t o r i z e d t r i p s a c r o ss t h e c i t i e s , d i m i n i s h i n g c o n g e s t i o n l e v e l s , r e d u c i n g t r a n s i t r i d e r s h i p , a n d c r e a t i n g a r e d u c t i o n i n t r a n s p o r t e x t e r n a li t i e s . N A N A x x x x N A N A N A B u c k s k y ( 17 ) Exp l o r a t o r y d a t a a n a l y s i s P ub li c t r a n s p o r t h a d t h e g r e a t e s t d e c li n e , w h e r e a s cyc li n g a cc o un t e d f o r t h e h i g h e s t g r o w t h . N A N A x N A N A N A N A N A N A d e H aa s e t a l . ( 26 ) Exp l o r a t o r y d a t a a n a l y s i s A pp r o x i m a t e l y 80 % o f p e o p l e r e d u c e d t h e i r a c t i v i t i e s o u t d oo r s , w i t h a s t r o n g e r d e c r e a s e f o r o l d e r p e o p l e . N A N A N A N A N A x N A N A N A G a o e t a l . ( 27 ) Exp l o r a t o r y s p a t i a l d a t a a n a l y s i s T h e t h r ee - d a y m o v i n g a v e r ag e s o f t h e m e d i a n s t a y - a t - h o m e d w e ll t i m e g e n e r a ll y a r e h i g h e r f o r p e o p l e r e s i d i n g i n t h e t o p 5 m o s t i n f e c t e d s t a t e s t h a n t h a t i n t h e l e a s t i n f e c t e d s t a t e s N A N A N A N A N A x N A N A N A S h a k i b a e i e t a l . ( 1 9 ) C h i - s qu a r e t e s t o f i n d e p e n d e n c e S h o pp i n g a n d r e c r e a t i o n a l w e r e t h e o n l y a c t i v i t i e s t h a t s i g n i f i c a n t l y d e c li n e d f r o m P h a s e 1 t o P h a s e 2 . W h il e i n t r a n s i t i o n t o P h a s e 3 , a ll a c t i v i t i e s e xp e r i e n c e d s i g n i f i c a n t r e d u c t i o n s . N A N A x N A N A x N A N A N A S h a m s h i r i p o u r e t a l . ( 30 ) Exp l o r a t o r y d a t a a n a l y s i s S i g n i f i c a n t c h a n g e s i n v a r i o u s a s p e c t s o f p e o p l e ’ s t r a v e l b e h a v i o r . N A N A N A N A N A x N A x N A W il bu r e t a l . ( 21 ) Exp l o r a t o r y d a t a a n a l y s i s F i x e d - li n e bu s r i d e r s h i p d r o pp e d b y 66 . 9 % a n d 65 . 1 % f r o m 2019 b a s e li n e s . A dd i t i o n a ll y t h e r e w a s a s i g n i f i c a n t d i ff e r e n c e i n r i d e r s h i p d e c li n e b e t w ee n t h e h i g h e s t - i n c o m e a r e a s a n d l o w e s t - i n c o m e a r e a s . N A N A N A N A x N A N A N A N A P a r a d y e t a l . ( 29 ) L o g i t m o d e l s T h e p e r c e p t i o n o f d e g r ee o f s e l f - r e s t r i c t i o n o f o t h e r s w a s a ss o c i a t e d w i t h s m a ll r e d u c t i o n s i n s h o pp i n g f r e qu e n c i e s , a n d m o d e r a t e y e t n o n - n e g li g i b l e i n c r e a s e s i n g o i n g - o u t s e l f - r e s t r i c t i o n p r o b a b ili t y f o r e a t i n g - o u t a n d l e i s u r e a c t i v i t i e s . N A N A N A N A N A x N A x N A ( c o n t i nu e d ) 632 T a b l e 1 . ( c o n t i nu e d ) A u t h o r ( s ) M e t h o d s K e y f i n d i n g s M o b ili t y m e a s u r e s E - s c oo t e r t r i p d u r a t i o n C h a n g e i n t r a v e l e d d i s t a n c e C h a n g e i n m o d a l s h a r e s C h a n g e i n c o n g e s t i o n C h a n g e i n pub li c t r a n s p o r t d e m a n d C h a n g e i n d a il y a c t i v i t i e s C h a n g e i n a i r li n e e m p l o y ee s R i s k p e r c e p t i o n t o w a r d t r a v e l a c t i v i t i e s T e l e w o r k s t a t u s J e n e li u s a n d C e b e c a u e r ( 20 ) Exp l o r a t o r y d a t a a n a l y s i s T h e d e c r e a s e i n pub li c t r a n s p o r t r i d e r s h i p ( 40 % – 60 % a c r o ss r e g i o n s ) w a s s e v e r e c o m p a r e d w i t h o t h e r t r a n s p o r t m o d e s . N A N A N A N A x N A N A N A N A B e c k a n d H e n s h e r ( 15 ) Exp l o r a t o r y d a t a a n a l y s i s R e d u c t i o n s i n o v e r a ll t r a v e l , t r a v e l b y m o d e s a n d t r a v e l f o r d i ff e r e n t pu r p o s e s . N A N A x N A N A x N A N A N A B e c k a n d H e n s h e r ( 1 5 ) Exp l o r a t o r y d a t a a n a l y s i s W o r k i n g f r o m h o m e w a s a n i m p o r t a n t s t r a t e g y i n r e d u c i n g t r a v e l a n d p r e ss u r e o n c o n s t r a i n e d t r a n s p o r t n e t w o r k s . N A N A x N A N A x N A N A N A En g l e e t a l . ( 32 ) R e g r e ss i o n m o d e l N A N A x N A N A N A N A N A x N A M o ll o y e t a l . ( 18 ) Exp l o r a t o r y d a t a a n a l y s i s T h e nu m b e r o f t r i p s p e r w o r k d a y s h r i n k up t o 60 % . T r i p d u r a t i o n r e m a i n s r e l a t i v e l y c o n s t a n t e x c e p t f o r b i cyc l e u s e , w i t h d i ff e r e n c e b e t w ee n g e n d e r s . N A x x N A N A N A N A N A N A L i e t a l . ( 38 ) D i s t a n c e d e c a y e ff e c t P a ss e n g e r s w o u l d u s e m i c r o - m o b ili t y s e r v i c e f o r t h e t r i p s w i t h l o n g e r d u r a t i o n a n d d i s t a n c e d u r i n g C O V I D - 19 t h a n t h e N o r m a l p e r i o d . x N A N A N A N A N A N A N A N A J i a o a n d A z i m i a n ( 3 5 ) M i x e d l o g i t T e l e w o r k s t a t u s w a s s i g n i f i c a n t l y a ss o c i a t e d w i t h s o c i o d e m o g r a ph i c f a c t o r s . N A N A N A N A N A N A N A N A x C h a uh a n e t a l . ( 33 ) Exp l o r a t o r y d a t a a n a l y s i s U r b a n r e s i d e n t s p e r c e i v e d h i g h e r r i s k i n s h o pp i n g , w a l k i n g , a n d b i k i n g c o m p a r e d w i t h r u r a l r e s i d e n t s . N A N A N A N A N A N A N A x N A J i a o a n d A z i m i a n ( 3 5 ) L o g i t m o d e l V a r i o u s s o c i o - e c o n o m i c a n d h e a l t h f a c t o r s w e r e s i g n i f i c a n t l y a ss o c i a t e d w i t h t r a v e l b e h a v i o r s . N A N A N A N A x N A N A N A N A S o b i e r a l s k i ( 23 ) D u r i n g p e r i o d s o f un c e r t a i n t y s h o c k s , t h e e s t i m a t e d j o b l o ss i s n e a r l y 7 % o f t h e a i r li n e w o r k f o r c e w i t h a n upp e r b o un d o f o v e r 13 % . N A N A N A N A N A N A x N A N A N o t e : N A = N o t A pp li c a b l e . 633 S ð t Þ ¼ exp (cid:2) exp ð b X Þ : t p f g ð 6 Þ where h 0 ( t ) = p . t p - 1 is a baseline hazard function ; b is the vector of unknown parameters ; X is a vector of covari - ates ; p is the shape parameter to be estimated from the data . It should be noted that , one unit change in the magnitude of a variable would change the hazard by [ exp ( b x ) 2 1 ] 3 100 % . Therefore , the positive sign of a variable is associated with increased hazard rate ( reduced travel time ) and vice versa . Spatial Model To account for spatial / zone - level unmeasured risk fac - tors ( and not spatial autocorrelation ) affecting trip dura - tion of subject i in census tract j . Equations 5 and 6 were re - written to include unstructured spatial random effect term ( u j ) , which is as follows : h ð t ij Þ ¼ h 0 ð t ij Þ : exp ð b X j + u j Þ ð 7 Þ S ð t ij Þ ¼ exp (cid:2) exp ð b X j + u j Þ : t ijp (cid:4) (cid:5) ð 8 Þ where u j is a census tract - level random effect term that captures the correlation among latent / unobserved factors ( e . g . , people’s risk perception toward COVID - 19 , atti - tudes / perceptions toward traveling , geographical charac - teristics ) , affecting e - scooter trip durations in the census tract . Data To demonstrate the proposed method , we empirically analyzed data on dockless e - scooter trip durations over the Austin metropolitan area , with the geographical units of analysis being census tracts . E - scooters appeared in Austin in 2018 with around 6 , 000 e - scooters , mostly located in central census tracts including downtown area . Currently , there are 11 , 600 devices from multiple shared mobility providers ( e . g . , Bird , Lime , etc . ) in Austin area with the rate of $ 0 . 35 / min to $ 0 . 39 / min . According to the City of Austin transportation department , 5 , 383 , 780 ( with average trip duration of 11 . 8 min ) and 1 , 588 , 741 e - scooter trips ( with average trip duration of 15 . 5 min ) were taken in 2019 and 2020 respectively . Data concerning the city’s boundaries and census tracts were collected from the Texas Department of Transportation and the Topologically Integrated Geographic Encoding and Referencing digital database , respectively . Dockless e - scooter data for 2019 and 2020 ( data points after March 11 , 2020 when the World Health Organization declared COVID - 19 a pandemic ) were obtained from the City of Austin’s Open Data Portal . The dataset contained information on individual trip duration , trip distance , trip date , beginning and end - ing times , and origin and destination census tracts . According to the Austin Transportation Department , official trip reporting metrics cover only travels that meet the following criteria : ( a ) a trip distance greater than or equal to 0 . 16 km ( 0 . 1 mi ) and less than 805 km ( 500 mi ) and ( b ) a trip duration of less than 24 h . Given the large size of the dataset and to efficiently examine the data , we extracted a portion of data through a stratified sampling method ( by census tract ) using STATA statistical soft - ware to achieve convergence as well as to ensure that each census tract ( if there were observations for this tract ) was adequately represented in the selected sample . From the U . S . Census Bureau , we collected informa - tion on census tract - level socio - economic factors , such as aggregate travel time to work , median annual household income , members of the population using public and pri - vate transport systems , number of males and females aged 10 – 17 years and 18 – 34 years , White and non - White members of the population , individuals with bachelor’s degrees or higher , and those with associate degrees or lower . Land use data were obtained from the U . S . Geological Survey , sidewalk and bus stop shapefiles were derived from Austin’s Open Data Portal , and the loca - tions of grocery stores , restaurants , and educational cen - ters were acquired from SafeGraph . Finally , we utilized ArcGIS Pro to calculate the land use entropy index , as outlined by Song et al . ( 46 ) , the percentage of sidewalks present , the distance from the centroid of each census tract to downtown , and the percentage of grocery stores , restaurants , and educational centers per census tract . We assumed that the independent variables ( i . e . , socio - economic factors , land use , percentages related to loca - tions of interests , etc . ) did not change from 2019 to 2020 . Table 2 summarizes the summary statistics of the vari - ables used in our data analysis . Results We first analyzed e - scooter trip durations using Kaplan – Meier ( K – M ) curves , which are widely employed in clini - cal and fundamental research . The K – M curve repre - sents the probability of completing an e - scooter trip at a certain time interval . As shown in Figure 1 , the horizon - tal x - axis represents trip duration expressed in minutes , and the y - axis indicates the proportion of the sample who completed a trip in t minutes or more . The compari - son of the two curves revealed that users tended to go on longer e - scooter trips in 2020 than in 2019 which is in line with those of previous studies ( 47 , 48 ) who reported an average increase of nearly 34 % during the pandemic . Considering that various factors contribute to e - scooter ridership , we estimated four Weibull survival models using STATA and used a 95 % confidence interval to assess the significance of the covariates . The results are summarized in Table 2 . 634 Transportation Research Record 2677 ( 4 ) Non - Spatial Models With regard to the 2019 non - spatial model , all the vari - ables were significant , except the ratio of White to non - White members of the population and the ratio of indi - viduals with bachelor’s degrees or higher to those having associate degrees or lower ( Table 3 ) . As for the 2020 non - spatial model , the ratio of males aged 10 – 17 years to their female counterparts and the ratio of White to non - White members of the population of this age range were non - significant . The results indicated that the aggregate travel time to work was significant in non - spatial Models 1 and 3 . Nevertheless , the findings reflected negative and positive signs in Models 1 and 2 , respectively , implying that increases in aggregate travel time , with all other variables held constant , were associated with reduced and increased hazard rates in 2019 and 2020 , respectively . In other words , before the pandemic , travels via e - scooter were longer in census tracts where residents had high aggregate travel time to work than in census tracts where residents exhibited low aggregate travel time to work . However , this trend was reversed in 2020—a contrast that seems reasonable , as people in census tracts with high aggregate travel time are likely to use personal vehi - cles to travel to work . Therefore , most of the e - scooter trips in these areas tended to be for leisure purposes—a behavior that was altered by the COVID - 19 global out - break given the limited places to visit ( 49 ) . As for median household income , its coefficients had negative signs in Models 1 and 3 ( non - spatial ) , suggesting that increases in household income , with all other vari - ables held constant , were associated with reduced hazard rates . That is , e - scooter users in high - income census tracts tended toward longer e - scooter trips before and during the pandemic . In other word , a one unit ( $ 1 , 000 ) increase in the median annual household income , while other vari - ables are held constant , will decrease the hazard rate by 0 . 5 % and 0 . 3 % respectively ( increase the travel time ) . In a similar vein , Caspi et al . ( 36 ) reported that high income is associated with low e - scooter travel . These findings , in combination , suggest that in census tracts where house - holds earn high incomes , people tend to go on fewer e - scooter trips of long durations because high earners reside in neighborhoods typified by mixed land use . Table 2 . Summary Statistics of the Variables Used in the Models Variable Mean SD Min . Max . 2019 E - scooter trip duration ( min ) 11 . 25 15 . 60 0 . 07 1 , 399 . 90 2020 E - scooter trip duration ( min ) 14 . 57 17 . 74 0 . 05 1 , 332 . 25 Aggregate travel time to work ( thousand ) 65 , 268 . 66 23 , 484 . 04 125 . 00 332 , 595 . 00 Median annual household income ( thousand ) 77 , 577 . 12 41 , 544 . 61 0 . 00 182 , 981 . 00 Ratio of Public transport users to private transport users 0 . 06 0 . 03 0 . 00 1 . 00 Ratio of male10 – 17 to female10 – 17 1 . 19 2 . 20 0 . 00 30 . 00 Ratio of male18 – 34 to female18 – 34 1 . 42 0 . 54 0 . 40 3 . 00 Ratio of white population to non - white population 2 . 32 1 . 20 0 . 10 12 . 90 Ratio of population with bachelor’s degree or more to population with associate degree or less 2 . 33 0 . 96 0 . 00 10 . 80 Sidewalk ( % ) 0 . 01 0 . 00 0 . 00 0 . 02 Grocery ( % ) 0 . 01 0 . 01 0 . 00 0 . 03 Restaurant ( % ) 0 . 04 0 . 03 0 . 00 0 . 08 Educational centers ( % ) 0 . 01 0 . 01 0 . 00 0 . 03 Distance to downtown ( thousand ) 1 , 733 . 06 1 , 322 . 09 608 . 46 2 , 6301 . 33 Land use entropy index 0 . 57 0 . 06 0 . 37 0 . 83 bus stop ( % ) 0 . 01 0 . 01 0 . 00 0 . 02 Note : SD = standard deviation ; Min . = minimum ; Max . = maximum . Number of 2019 observations = 503 , 173 ; number of 2020 observations = 682 , 134 . Figure 1 . Kaplan – Meier curves of E - scooter trip durations . Azimian and Jiao 635 With regard to the ratio of public transport users to private transport users , its positive signs in both the non - spatial models indicated that before and during the pan - demic , e - scooter travel was of low durations in census tracts with a high proportion of public transport users . This finding may be explained by noting that most tran - sit users reside within central tracts and close to business centers . They may also use e - scooters to connect to tran - sit stations ( 10 ) , which are generally short trips . Additionally , the comparison of hazard ratios indicates that an increase in the ratio of public transport users to private transport users increases the hazard rate by 28 . 9 % and 41 . 6 % in 2019 and 2020 , respectively . On the matter of the ratio of males to females aged 10 – 17 years , the positive sign of this variable in Model 1 reflected that in 2019 , the e - scooter trip durations in cen - sus tracts with a high ratio of male to female teens tended to be lower than those in census tracts with a low ratio . No specific pattern was observed in 2020 . In contrast , when it came to the ratio of males to females aged 18 – 34 years , which had negative coefficients in both the non - spatial models , the opposite interpretation could be drawn . To wit , census tracts with a high ratio of 18 - to 34 - year - old males to females were correlated with long e - scooter trips . This contrast can be explained noting that that most male teenagers use e - scooters for leisure activi - ties and riding around , whereas adult males are more likely to use such vehicles for commuting to work and doing household errands , which take more time . Concerning educational levels , census tracts with a high ratio of individuals with bachelor’s degrees or greater to those with associate degrees or lower were pre - disposed toward long e - scooter travels during the pan - demic compared with census tracts typified by a low ratio . However , no specific trend was observed before the pandemic . Such disparity may be because individuals with bachelor’s degrees or higher often relying on public transport systems ( 50 ) were likely to switch to other modes of transport , including e - scooters , during the pan - demic to avoid the risk of COVID - 19 contradiction ( 51 ) . Focusing on built environment factors , sidewalks had negative signs and hazard ratios close to zero in both the non - spatial models , suggesting that in census tracts with a considerable proportion of sidewalks , e - scooter users Table 3 . Parameter Estimates in Spatial and Non - spatial Models Variable 2019 2020 Non - spatial ( Model 1 ) Spatial ( Model 2 ) Non - spatial ( Model 3 ) Spatial ( Model 4 ) Haz . Ratio Coef . Haz . Coef . Haz . Coef . Haz . Coef . Constant 0 . 148 2 1 . 911 * 0 . 103 2 2 . 269 * 0 . 130 2 2 . 042 * 0 . 080 2 2 . 530 * Aggregate travel time to work ( thousand ) 0 . 999 2 0 . 001 * 1 . 003 0 . 0003 1 . 001 0 . 001 * 1 . 001 0 . 001 Median annual household income ( thousand ) 0 . 995 2 0 . 005 * 0 . 996 2 0 . 004 * 0 . 997 2 0 . 003 * 0 . 997 2 0 . 003 * Public transport users / Private transport users 1 . 289 0 . 254 * 1 . 166 0 . 154 1 . 416 0 . 348 * 0 . 963 2 0 . 038 Male 10 – 17 / Female 10 – 17 1 . 002 0 . 002 * 1 . 010 0 . 010 * 0 . 999 0 . 000 1 . 003 0 . 003 Male 18 – 34 / Female 18 – 34 0 . 838 2 0 . 176 * 0 . 871 2 0 . 138 * 0 . 830 2 0 . 186 * 0 . 917 - 0 . 086 White / Non - white 0 . 998 2 0 . 002 1 . 002 0 . 002 1 . 005 0 . 005 * 1 . 018 0 . 018 Bachelor / Associate 1 . 004 0 . 004 1 . 021 0 . 021 0 . 959 2 0 . 042 * 1 . 012 0 . 011 Sidewalk ( % ) 0 . 000 2 38 . 180 * 123 . 97 4 . 820 0 . 000 2 33 . 288 * 2 . 743 1 . 009 Grocery ( % ) 1078 . 04 6 . 982 * 1 . 169 0 . 156 11684 . 420 9 . 366 * 0 . 854 - 0 . 158 Restaurant ( % ) 117 . 166 4 . 763 * 252 . 32 5 . 531 * 18 . 779 2 . 933 * 20 . 662 3 . 028 Educational centers ( % ) 7 , 087 . 41 8 . 866 * 0 . 074 2 2 . 601 77 . 555 4 . 351 * 0 . 039 2 3 . 242 Distance to downtown ( thousand ) 0 . 984 2 0 . 016 * 0 . 961 2 0 . 040 * 0 . 978 2 0 . 022 * 0 . 961 2 0 . 040 * Land use entropy index 0 . 596 2 0 . 517 * 0 . 779 2 0 . 249 0 . 597 2 0 . 515 * 0 . 989 2 0 . 011 Bus stop ( % ) 0 . 001 2 6 . 707 * 0 . 008 2 4 . 872 0 . 0001 2 9 . 007 * 0 . 002 2 6 . 007 ln ( p ) 0 . 114 0 . 116 0 . 072 0 . 074 Variance NA 0 . 025 NA 0 . 030 Log likelihood 2 1 , 706 , 368 2 1 , 705 , 069 2 2 , 497 , 891 2 2 , 496 , 155 Wald chi 2 ( 14 ) 18 , 987 . 690 100 . 520 18 , 364 . 64 68 . 820 LR test versus Weibull model : chi 2 ( 3 ) NA 2599 NA 3472 Note : Haz . = hazard ; Coef . = coefficient ; LR = likelihood - ratio test ; NA = Not Applicable . Hazards ratio associated with a variable is given by the exponent of its coefficient . Hazard ratio is interpreted as the proportional change in hazard when a variable change by one unit . * Denotes variables significant at 5 % . 636 Transportation Research Record 2677 ( 4 ) tended to go on much longer trips before and during the pandemic compared with census tracts with minimal sidewalks . Similarly , distance to downtown and the land use entropy index were inversely proportional to hazard rates , indicating that the further a census tract’s distance from downtown and the greater the complexity of land use within the census tract , the higher the probability of long e - scooter trip durations before and during the pan - demic . Surprisingly , census tracts with numerous bus stops were inclined to exhibit high e - scooter trip dura - tions , possibly because of the occurrence of a complex land use mix in these areas . Additionally , points of inter - est , such as grocery stores , restaurants , and educational centers , were significant and proportional to hazard rates . Put differently , the higher the number of points of interest , the greater the likelihood of short e - scooter trips . This finding is fairly reasonable , as e - scooter users go on short trips because of increased accessibility . Spatial Models After the random effect terms were accounted for , some variables that were significant in Models 1 and 3 ( e . g . , aggregate travel time to work and the ratio of public transport users to private transport users ) were no longer significant in Models 2 and 4 . This can be explained by the significant and systematic variance among the vari - ables across space . The use of random effect terms in the models captured the variations used to estimate the fixed effect parameters in the basic models ( Models 1 and 3 ) . As a result , these variables became non - significant ( 25 ) . Although the spatial models had fewer significant vari - ables and might seem less useful for planning and prac - tice , the insight concerning the inclusion of random effect terms is valuable , as these terms captured unobserved characteristics that vary across space . This highlights the need for the further identification of meaningful factors that can be used for planning and policy purposes . Figure 2 illustrates the changes in random effect esti - mates ( Model 4 versus Model 2 ) , which were classified into two types : negative values ( red highlight in the fig - ure ) and positive values ( green highlight in the figure ) , which show areas where unobserved factors were inclined to increase and decrease e - scooter trip durations during the pandemic , respectively . Even though the map revealed an overall random pattern in the study area , we can still observe a cluster of red census tracts in eastern Austin . According to Luna et al . ( 52 ) and Tu et al . ( 53 ) , these locations are historically known as low - and middle - income communities with a large proportion of non - White inhabitants who are highly dependent on public transportation systems . It therefore makes intui - tive sense that some of the residents in these localities shifted from public transport systems to e - scooters owing to fear of disease contraction and traveled more dis - tances to reach their destinations . By contrast , a big clus - ter of green census tracts can be observed in central part , which are home to high - income families . This finding can be supported by noting that such people are inclined to be able to afford personal vehicles more than low - income individuals . They were therefore more likely to use e - scooters for leisure , but such activities were signifi - cantly reduced during the pandemic . Conclusion This study used 2019 and 2020 e - scooter data on Austin to explore the socio - economic and built environment fac - tors contributing to travel behaviors . In particular , we proposed spatial and non - spatial models to identify the factors affecting e - scooter trip durations before and dur - ing the pandemic . The results confirmed changes in e - scooter trip durations in census tracts with residents who had high aggregate travel time to work and in census tracts with a substantial proportion of individuals with bachelor’s degrees or higher . Additionally , built environ - ment factors , such as sidewalks , distance to downtown , the land use entropy index , and percentage of bus stops present , were positively associated with e - scooter trip durations before and during the pandemic . By contrast , the percentage of restaurants , grocery stores , and educa - tional centers present were negatively correlated with e - scooter trip durations during both periods . Figure 2 . Changes in random effect estimates . Azimian and Jiao 637 In general , the comparison of spatial models high - lighted a slight increase or decrease in some variables’ coefficients in the 2020 model compared with the 2019 version . Note that this finding may not explicitly indicate whether trip duration changed or remained constant dur - ing the pandemic because of the limited number of vari - ables used in the model ( Many other important variables were disregarded given data limitations ) . However , the spatial models reflect that the 2020 model has a smaller coefficient than its 2019 counterpart , suggesting that , on average , when all variables are set to zero , travel time likely increased during the pandemic ( 2020 ) compared with 2019 . This finding also aligns with Figure 1 . Concerning policy implications , as many low - income families may rely on e - scooters to avoid the risk of COVID - 19 infection , it is critical to increase the mileage of such vehicles beyond core urban areas by upgrading batteries and installing charging stations across the city . E - scooters are placed in dense downtown areas and other districts where high levels of use and revenue gen - eration are expected . A necessary requirement , therefore , is to extend e - scooter service areas to communities with considerable low to zero - vehicle households ( e . g . , eastern Austin ) , where residents are encountering difficulty in accessing the public transportation system to a greater extent than that experienced in the pre - pandemic era ( 35 , 54 ) . Policymakers can address such problems and improve equity for residents of these areas , especially e - scooter users , by expediting the provision of improved infrastructure , such sidewalks and bike lanes ( 55 ) . As with any other research , the current work is encumbered by various limitations which should be con - sidered in future works . First , to better help transporta - tion planners and decision makers understand travel patterns during the pandemic , it is essential to investigate the extent to which people turned to micro - mobility devices to avoid subways and buses . Second , the e - scooter data used in this study lack individual - level socio - demographic factors , such as age , gender , and eth - nicity . A critical measure is to assess the effects of such factors on e - scooter trip durations to better understand whether e - scooter sharing services satisfy equity goals and serve target populations . Third and finally , it is highly recommended to utilize mode - specific variables to study how e - scooters are used as alternatives to other transport modes ( public transport in particular during the pandemic ) and therefore what their role is in making transport systems more resilient . Author Contributions The authors confirm contribution to the paper as follows : study conception and design : A . Azimian ; data collection : A . Azimian ; analysis and interpretation of results : A . Azimian ; draft manuscript preparation : A . Azimian , J . Jiao . All authors reviewed the results and approved the final version of the manuscript . Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research , authorship , and / or publication of this article . Funding The authors disclosed receipt of the following financial support for the research , authorship , and / or publication of this article : This work was supported by University of Texas good system grand challenge and U . S . DOT CM2 University Transportation Center at University of Texas Austin . ORCID iD Junfeng Jiao https : / / orcid . org / 0000 - 0002 - 7272 - 8805 References 1 . Reuters , T . Coronavirus : What’s happening in Canada and Around the World on Jan . 13 . CBC , 2021 . https : / / www . cbc . ca / news / canada / coronavirus - covid19 - canada - world - january - 13 - 2021 - 1 . 5871055 . 2 . Neilson , S . , and A . Woodward . A Comprehensive Time - line of the Coronavirus Pandemic at 1 Year , From China’s First Case to the Present . Business Insider , 2020 . https : / / www . businessinsider . com / coronavirus - pandemic - timeline - history - major - events - 2020 - 3 . 3 . CBO . Monthly Budget Review for April 2020 . 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J . , eds . ) , Emerald Group Publishing Limited , Bingley , 2007 , pp . 105 – 131 . 45 . McCormick , S . Survival Analysis , Part 1 : The Weibull Model . Medium , 2018 . https : / / medium . com / utility - mac hine - learning / survival - analysis - part - 1 - the - weibull - model - 5c2 552c4356f # : ~ : text = The % 20Weibull % 20distribution % 20is % 20particularly , for % 20a % 20fleet % 20of % 20machines . 46 . Song , Y . , L . Merlin , and D . Rodriguez . Comparing Mea - sures of Urban Land Use Mix . Computers , Environment and Urban Systems , Vol . 42 , 2013 , pp . 1 – 13 . 47 . Descant , S . The Pandemic Has Shifted Where Scooters and Robots Fit In . Government Technology , 2020 . https : / / www . govtech . com / fs / transportation / the - pandemic - has - shifted - where - scooters - and - robots - fit - in . html . 48 . Winberge , M . Open to the Air and Socially Distant , E - Scooters are Trending — But They’re Still Illegal in PA . Billy Penn , 2020 . https : / / billypenn . com / 2020 / 09 / 21 / electric - scooters - trending - coronavirus - illegal - pennsylvania - septa - delays - traffic - safety - lime - bird / . 49 . Cobbs , C . COVID Likely Reduced E - scooter Use , Despite More Scooters and Larger Service Area Last Year . Streets - Blog , 2021 . https : / / chi . streetsblog . org / 2021 / 05 / 19 / covid - 19 - pandemic - likely - reduced - e - scooter - usage - despite - more - e - scooters - and - a - larger - service - area - last - year / . 50 . Clark , H . M . Who Rides Public Transportation . Report . American Public Transportation Association , Washington , D . C . , 2017 . 51 . Tirachini , A . , and O . Cats . COVID - 19 and Public Transpor - tation : Current Assessment , Prospects , and Research Needs . Journal of Public Transportation , Vol . 22 , No . 1 , 2020 , p . 1 . 52 . Luna , E . , P . Blanc , R . Taylor , J . M . Styles , and M . Moyni - han . Austin COVID - 19 Community Feedback Survey . Report . MEASURE , Austin , TX , 2020 . 53 . Tu , W . , L . Li , and R . Piltner , eds . A Study of Smart Growth Initiatives Using GIS : The Case of Austin , Texas . Proc . , National Conference on Digital Government Research , Atlanta , GA , May 15 – 18 , 2005 . 54 . Park , J . II . Impacts of Big Box Development on Minority and Low - Income Communities : Big Box Location and Spa - tial Equity in Austin . Professional Report . The University of Texas at Austin , 2008 . 55 . Reinhardt , K . , and E . Deakin . Best Practices for the Public Management of Electric Scooters . Report No . UC - ITS - 2020 - 2 . The University of California Institute of Transpor - tation Studies , Berkeley , 2020 . 640 Transportation Research Record 2677 ( 4 ) \ No newline at end of file diff --git a/silver_data/0e5c332f9a18cebc24b1f0ae4503c651625cbbdc.pdf.txt b/silver_data/0e5c332f9a18cebc24b1f0ae4503c651625cbbdc.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d4a610c1ac6a0e0d8619b53510ab15153ae9e3de --- /dev/null +++ b/silver_data/0e5c332f9a18cebc24b1f0ae4503c651625cbbdc.pdf.txt @@ -0,0 +1 @@ +Mutations in twinstar , a Drosophila Gene Encoding a Cofilin / ADF Homologue , Result in Defects in Centrosome Migration and Cytokinesis Kristin C . Gunsalus , * Silvia Bonaccorsi , * Erika Williams , * Fiammetta Verni , * Maurizio Gatti , * and Michael L . Goldberg * • Section of Genetics and Development , CorneU University , Ithaca , New York 14853 ; and ~ Centro di Genetica Evoluzionistica del CNR , and Dipartimento di Genetica e Biologia Molecolare , Universit ~ di Roma " La Sapienza " , 00185 Roma , Italy Abstract . We describe the phenotypic and molecular characterization of twinstar ( tsr ) , an essential gene in Drosophila melanogaster . Two P - element induced al - leles of tsr ( tsr l and tsr 2 ) result in late larval or pupal le - thality . Cytological examination of actively dividing tis - sues in these mutants reveals defects in cytokinesis in both mitotic ( larval neuroblast ) and meiotic ( larval tes - tis ) cells . In addition , mutant spermatocytes show de - fects in aster migration and separation during prophase / prometaphase of both meiotic divisions . We have cloned the gene affected by these mutations and shown that it codes for a 17 - kD protein in the cofilin / ADF family of small actin severing proteins . A cDNA for this gene has previously been described by Edwards et al . ( 1994 ) . Northern analysis shows that the tsr gene is expressed throughout development , and that the tsr a and tsr 2 alleles are hypomorphs that accumulate de - creased levels of tsr mRNA . These findings prompted us to examine actin behavior during male meiosis to vi - sualize the effects of decreased twinstar protein activity on actin dynamics in vivo . Strikingly , both mutants ex - hibit abnormal accumulations of F - actin . Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes . Later , during ana / telo - phase of both meiotic divisions , aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of te - lophase , in contrast with wild - type . We discuss these re - suits in terms of possible roles of the actin - based cy - toskeleton in centrosome movement and in cytokinesis . ECULATED interactions between actin and actin bind - ing proteins provide an important structural basis for many cellular processes in eukaryotes , such as cell - cell adhesion , cell outgrowth and motility , muscle con - traction , and cytokinesis . Changes in the intracellular lo - calization and organization of actin are mediated by a rep - ertoire of actin - associated proteins . These fall into several classes based on their effects on actin assembly and dy - namics in vitro : proteins with cross - linking , bundling , cap - ping , severing , polymerizing / depolymerizing , monomer se - questering , and motor activities have been described ( reviewed by Vandekerckove and Vancompernalle , 1992 ) . However , defining the role that each actin - associated pro - tein plays in vivo and how multiple activities are coordi - nated remains a challenging task . Proteins in the cofilin / actin depolymerizing factor ( ADF ) 1 family have been identified in a wide range of eukaryotic organisms , from yeast to plants to humans . These small molecular mass ( 15 - 20 kD ) actin binding proteins consti - Address correspondence to Dr . M . L . Goldberg , Section of Genetics and Development , 425 Biotechnology Building , Cornell University , Ithaca , NY 14853 . Tel . : ( 607 ) 254 - 4802 . FAX : ( 607 ) 255 - 6249 . tute one of the two known classes of actin filament sever - ing proteins ; the other is comprised of the gelsolin / ffag - min / villin - type proteins ( see Vandekerckhove and Van - compernolle , 1992 ) . In addition to severing , cofilin - like proteins also display actin filament and monomer binding activities in vitro . The properties of these proteins in vitro may be influenced by pH ( Yonezawa et al . , 1985 ; Hawkins et al . , 1993 ; Hayden et al . , 1993 ; Iida et al . , 1993 ) , the pres - ence of membrane phospholipids ( Yonezawa et al . , 1990 ) , other actin binding proteins ( Nishida et al . , 1984 ; Abe et al . , 1990 ; Maciver et al . , 1991 ) , and in some cases , by phos - phorylation of the protein itself ( Morgan et al . , 1993 ) . These and other observations hint at how the activities of cofilin / ADF proteins might be regulated intracellularly . Our current appreciation of the biochemical behavior of these molecules is not reflected by an understanding of the precise roles they play in vivo , although cofilin - like pro - teins are known to provide an essential function both in the yeast Saccharomyces cerevisiae ( Iida et al . , 1993 ; Moon et al . , 1993 ) and in the nematode Caenorhabditus elegans ( McKim et al . , 1994 ) . The yeast cofilin gene COF1 is re - quired for spore viability and growth , but the nature of the essential function or functions that are improperly per - © The Rockefeller University Press , 0021 - 9525 / 95 / | 2 / 1243 / 17 $ 2 . 00 The Journal of Cell Biology , Volume 131 , Number 5 , December 1995 1243 - 1259 1243 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m formed in the mutants is not known . In C . elegans , two slightly different cofilin - like proteins are generated by al - ternative splicing . A small deletion removing part of both transcripts is lethal ( McKim et al . , 1994 ) ; other mutations apparently affecting only one transcript are viable and re - sult in paralysis and disorganized muscle filaments ( Wa - terston et al . , 1980 ; McKim et al . , 1988 ) . Vertebrates carry at least two distinct members of this family , cofilin and ADF / destrin , which differ both in their biochemical prop - erties in vitro and in their expression patterns in different tissues and developmental stages ( Bamburg and Bray , 1987 ; Matsuzaki et al . , 1988 ; Abe et al . , 1990 ; Moriyama et al . , 1990a ) . Both porcine cofilin and destrin cDNAs can rescue the viability of S . cerevisiae coil mutant spores ( Iida et al . , 1993 ) . Thus , the biochemical differences seen in vitro between these proteins is insignificant for yeast cells , but multicellular eukaryotes may still require inde - pendent functions supplied by two or more members of the cofilin gene family . In this paper , we describe both the molecular analysis of the Drosophila gene twinstar ( tsr ) , which encodes a cofilin / ADF homologue , and the phenotypic consequences of mutations in this gene . Animals mutant for tsr die at the larval - pupal boundary and exhibit frequent failures in cy - tokinesis in both mitotic and meiotic cells . In addition , they show abnormalities in centrosome migration to the opposite ends of spermatocyte nuclei during the two mei - otic divisions . These defects appear to be related to dra - matic changes observed in the actin cytoskeleton in mu - tant spermatocytes . Materials and Methods Drosophila Stocks The twinstar I ( tsr 1 ) allele was generated in a P element screen in the labo - ratory of Dr . J . Merriam ( University of California , Los Angeles , CA ) by the transposition of the single , marked P element pLacA92 ( O ' Kane and Gehring , 1987 ) . The tsr 2 allele was identified by Dr . Trisha Wilson ( Stan - ford University , Palo Alto , CA ) by screening a different collection of mu - tations induced with the marked P element P - lacW ( Bier et al . , 1989 ) . Both alleles were maintained as heterozygotes balanced either by the sec - ond chromosome balancer CyO or by TSTL 14 , a translocation between CyO and the third chromosome balancer TM6B ( see Gatti and Goldberg , 1991 ) . The deficiency strains Df ( 2R ) or BR - 6 , Df ( 2R ) or BR11 , and Df ( 2R ) GlO - BR - 27 were constructed and kindly provided by Dr . Bruce Reed ( White - head Institute , Cambridge , MA ) . The latter deletion is associated with an inversion , In ( 2LR ) ItG " ) LBR - 27 R , constructed using autosynaptic elements ( see Gubb et al . , 1988 ) . Df ( 2R ) bwS4°is described in Simpson ( 1983 ) . Germline Transformation The 6 . 4 - kb genomic fragment shown in Fig . 7 A ( RS6 . 4 ) was subcloned from Bluescript KS II + ( Stratagene Inc . , La Jolla , CA ) into the transfor - mation vector pW8 ( Klemenz et al . , 1987 ) using the NotI and XhoI sites present in both polylinkers . This putative pW8 - tsr + construct was injected into w ; Sb e [ A2 - 3 ] / TM6 embryos , which supply the transposase required for P element transposition ( Robertson et al . , 1988 ) . pW8 carries a mini - white + gene as a selectable marker . Survivors were mated individually to Z 1 W lle4 ( Lindsley and Zimm , 1992 ) , and progeny were screened for trans - mission of the w + eye color . Several lines carrying independent insertions of pW8 - tsr ÷ were recovered . One of these lines carried a nonlethal inser - tion of pW8 - tsr ÷ on the third chromosome that was able to rescue the le - thal , mitotic , and meiotic phenotypes of both twinstar alleles when present in either one or two copies . Stocks homozygous for [ pW8 - tsr + ] and either tsr I or tsr 2 were established ; Southern analysis of genomic DNA con - firmed the presence of tsr mutant alleles and tsr ÷ transgenes in these lines ( data not shown ) . Cytology Larval Neuroblasts . Homozygous tsr larvae were selected from stocks or crosses in which mutant tsr alleles were balanced over the TSTL 14 translo - cation . Because this balancer carries the dominant larval marker Tubby ( Tb ) , homozygous mutant individuals ( phenotypically non - Tb ) can be un - ambiguously distinguished from heterozygous tsr / TSTL 14 larvae ( pbeno - typically Tb ) . Mutant and control brains were dissected , fixed , and squashed in aceto - orcein according to our usual procedure ( Gatti and Goldberg , 1991 ) . To estimate the mitotic index of tsr mutants relative to controls , we determined the average number of mitotic figures per optic field in control ( Oregon R ) and mutant brain squashes ( Gatti and Baker , 1989 ) . The optic field chosen for this analysis is the circular area defined by a phase - contrast Neofluar 100× oil - immersion Zeiss objective , using 10× oculars with the Optovar set at 1 . 25× ( Carl Zeiss , Inc . , Thornwood , NY ) . Larval Testes . To examine spermatid morphology in live material , lar - val testes were dissected in testis isolation buffer ( 183 mM KCI , 47 mM NaC1 , 10 mM Tris - HCl , pH 6 . 8 ) and gently squashed in 2 ~ zl of the same buffer under a 20 × 20 coverslip . These live preparations were immedi - ately scored by phase contrast microscopy . Two different procedures were used for cytological analysis of fixed testes . For simultaneous tubulin immunostaining and Hoechst 33258 stain - ing , testes were fixed and processed as described by Cenci et al . ( 1994 ) ; tu - bulin was detected using an anti - a - tubulin Ab raised against chick brain microtubules ( Amersham Corp . , Arlington Heights , IL ; Blose et al . , 1982 ) . For simultaneous visualization of chromatin , microtubules , and F - actin , testes squashes were prepared according to Cenci et al . ( 1994 ) , but after freezing in liquid nitrogen and removal of the coverslip , squashes were fixed for 10 min by immersion in ethanol precooled at - 20°C , fol - lowed by a 7 - min treatment with freshly made 3 . 7 % formaldehyde in PBS . Slides were then washed two times in PBS ( 5 min each ) and immersed for 30 min in PBT ( PBS + 0 . 1 % Triton - X 100 ) . After this treatment , they were incubated for 1 h with anti - ct - tubulin monoclonal Ab diluted 1 : 50 in PBS , washed two times in PBS ( 5 min each ) , and then incubated for 1 h with the secondary antibody ( sheep anti - mouse IgG , F ( ab ) 2 fragment , conju - gated with 5 ( 6 ) - carboxy - fluorescein - N - hydroxysuccinimide ester [ FLUOS ] , no . 1214616 ; Boehringer Mannheim ) diluted 1 : 15 in PBS . Tubulin immu - nostained slides , after two 5 - min washes in PBS , were incubated for 2 h at 37°C in rhodamine - labeled phalloidin ( Molecular Probes , Eugene , Ore - gon ) dissolved in PBS ( 100 I ~ l of the stock solution were vacuum dried and dissolved in 200 t ~ 1 PBS ) . After a quick wash in PBS , slides were stained with Hoechst 33258 and mounted as described by Cenci et al . ( 1994 ) . Fluorescence Microscopy Preparations stained with Hoechst 33258 and anti - tubulin Ab plus FLUOS - conjugated secondary Ab were examined with a Zeiss III photo - microscope equipped for epifluorescence with an HBO 100W mercury lamp ( Osram ) . Hoechst 33258 and FLUOS fuorescence were detected us - ing the 0 . 1 ( BP 365 / 11 , FT 395 , LP 397 ) and the 0 . 9 ( BP 450 - 490 , FT 510 , LP 420 ) Zeiss filter sets , respectively . Photomicrographs were recorded on Kodak T - MAX 100 film . Preparations stained with Hoechst 33258 , anti - tubulin Ab plus FLUOS - conjugated secondary Ab , and rhodamine - labeled phalloidin were examined with a Zeiss Axioplan microscope equipped for epifluo - rescence with an HBO 50W mercury lamp ( Osram ) and with a cooled charge - coupled device ( Photometrics Inc . , Woburn , MA ) . Hoechst 33258 , FLUOS , and rhodamine fluorescence were detected with the 0 . 1 , 10 ( BP 450 - 490 , FT 510 , LP 515 - 565 ) and 15 ( BP 546 , FT 580 , LP 590 ) Zeiss filter sets , respectively . The fluorescence signals were recorded separately as gray - scale digital images using IP Lab Spectrum software . Images were then converted to Photoshop format ( Adobe Systems , Inc . , Mountain View , CA ) , pseudocolored , and merged . Nucleic Acids Molecular techniques not specifically outlined here were performed using standard protocols ( Sambrook et al . , 1989 ) . Isolation oftwinstar Genomic and cDNAs . 0 . 5 Ixg of total genomic DNA isolated from heterozygous twinstar adults was digested with Sau3A ( for tsr 1 ) or with either EcoR1 or PstI ( for tsr2 ) . The DNA was precipitated with ethanol , dried , and then resuspended in 400 Ixl of DNA ligation buffer . Circular DNA containing one end of the P element adjacent to twinstar genomic sequences was cloned by one of two methods . For tsr ~ , the ligation was amplified with primers from the P element ends using an The Joumal of Cell Biology , Volume 131 , 1995 1244 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m inverse PCR protocol ( Ochman et al . , 1988 ) . The PCR product was di - gested with restriction enzymes unique to the PCR primers and cloned into Bluescript KS II + . For tsr 2 , the ligation mix was transformed directly into XL - 1 Blue cells by the CaC12 method and desired recombinants were selected with ampicillin . This " plasmid rescue " ( Pirotta , 1986 ) was possi - ble since the P element causing the tsr 2 mutation contains a bacterial ori - gin of replication and a selectable marker for ampicillin resistance . Wild - type genomic clones were isolated from an EMBL3 phage genomic library ( kindly supplied by J . Tamkun , University of California , Santa Cruz , CA ) using the PCR product described above as a probe . Restriction fragments were subcloned into Bluescript KS II + . The full - length twinstar eDNA was isolated from a 0 - - 4 - h embryonic library ( Brown and Kafatos , 1988 ) using genomic fragment RS6 . 4 ( Fig . 7 A ) as a probe . Sequencing of Genomic and eDNAs . Sequencing of double - stranded plas - mid DNA was performed by the dideoxy method ( Sanger et al . , 1977 ) us - ing Sequenase version 2 . 0 ( United States Biochemical Corp . , Cleveland , OH ) or the Ladderman kit ( Takara Biochemical Inc . , Berkeley , CA ) , 35S - dATP ( Amersham ) , 6 % Long Ranger ( AT Biochem , Malvern , PA ) gels , and either Bluescript primers or sequence - specific primers ( Biotech - nology Synthesis and Analytical Facility , Cornell University , Ithaca , NY ) . The twinstar eDNA and the 6 . 4 - kb genomic fragment used to rescue the tsr 1 and tsr 2 mutants ( RS6 . 4 ) were completely sequenced on both strands . Sequence analysis was performed using the GCG ( Devereux et al . , 1984 ) and MacVector / AssemblyLIGN software packages ( Kodak / IBI , New Ha - ven , CT ) . Computer searches of GenBank and EMBL were performed us - ing FASTA ( Pearson and Lipman , 1988 ) . The alignment of related pro - teins was generated with the assistance of the Clustal V program included in GCG . To obtain sequences at the 5 ' end of tsr messages , RACE ampli - fication of twinstar mRNA from wild - type ( Oregon R ) or mutant animals was performed on a Hybaid OmniGene thermal cycler using a GIBCO BRL kit and three nested primers specific to the twinstar mRNA ( GIBCO BRL , Gaithersburg , MD ) . Primer 5 ' - AGTTTATTGGCGGTCGG - 3 ' was used for reverse transcription . The resulting first strand was PCR ampli - fied using primer 5 ' - CTAGAAAACTGGTCGTAC - 3 ' . This product was reamplified using primer 5 ' - TGCAGACATCAGACACAGTFACAC - 3 ' , which was located just downstream of the first ( 1 . 5 kb ) intron to ensure that final products were not due to contaminating genomic DNA . RACE products were cloned using the pCR - Script SK ( + ) cloning kit ( Strat - agene ) . Northern Analysis oftsr mRNAs . Total RNA was isolated by a modifi - cation of the method of Chirgwin et al . ( 1979 ) . Animals were homoge - nized in modified G buffer ( 6 M guanidine thiocyanate ; 0 . 025 M sodium acetate , pH 7 . 0 ; 5 mM dithiothreitol ) plus 0 . 5 % N - lauryl sarcosinate and 0 . 5 % diethylpyrocarbonate , then extracted once with an equal volume of a 1 : 1 mixture of phenol and chloroform / isoamylalcohol ( 24 : 1 ) . After cen - trifugation , the aqueous layer was recovered and RNA was precipitated by adding 1 / 50 vol of I M acetic acid and 1 / 2 vol of ethanol . This was incu - bated at - 20°C for 3 h , and the RNA pelleted by centrifugation in a mi - crocentrifuge . The pellet was resuspended in one - half the original volume of G buffer , and the RNA was repreeipitated as before . Total RNA was resuspended in sterile DEPC - treated H20 and quantitated by absorbance at 260 nm . For the developmental Northern , poly ( A ) + RNA was isolated using PolyA - Tract System III ( Promega Corp . , Madison , WI ) . Northern sample buffer was added to samples before electrophoresis on 1 . 2 % agar - ose - formaldehyde gels as described in Sambrook et al . ( 1989 ) . RNAs were then transferred to Nylon 66 ( Genescreen ; Schleicher & Schuell , Inc . , Keene , NH ) neutral membrane by electroblotting in 0 . 1x TAE or , for the developmental Northern , to Z Probe membrane ( Bio - Rad Labs . , Rich - mond , CA ) by vacuum blotting . 300 p , l of 2 . 5 mg / ml salmon sperm DNA was added to purified random hexanucleotide - primed probes labeled us - ing ~ - [ 32p ] dCTP ( Amersham ) and the mixture boiled for 5 min before hy - bridization . Membranes were prebybridized for at least 1 b at 42°C in 50 % formamide , 5x SSPE , 2x Denhardt ' s , and 0 . 1 % SDS before overnight hy - bridization at 42°C in the same buffer . Two 45 - min washes were per - formed at 42°C in 50 % formamide , 5x SSPE , 0 . 5 % SDS followed by two 20 - min washes at 65°C in lx SSPE , 0 . 5 % SDS . Membranes were then ex - posed to Kodak X - ray film at - 70°C with intensifying screens . Membranes were hybridized with a D . melanogaster rp49 clone ( O ' Connell and Ros - bash , 1984 ) as a control for loading . Results Identification of the twinstar Locus We have previously described a screening procedure to identify mutations causing aberrations during mitosis ( Gatti and Baker , 1989 ; Gatti and Goldberg , 1991 ) . One would expect that homozygosity for mutations in many genes en - coding components of the mitotic apparatus would result in lethality in late larval ( third instar ) or pupal stages . This is because the heterozygous mother of homozygous mu - tant individuals would contribute maternally supplied prod - ucts to enable the earliest rounds of embryonic and larval mitosis to proceed . However , metamorphosis could not oc - cur because the homozygous mutant zygotic genome would be unable to direct the synthesis of these molecules in imaginal discs , histoblasts , or in the larval brain . Examina - tion of cell division in mutant larval brains would there - fore reveal potential mitotic defects . This strategy has been used with considerable success to identify a variety of mitotic mutations in Drosophila ( reviewed by Ripoll et al . , 1987 ; Glover , 1989 ; Gatti and Goldberg , 1991 ) . We identified the mutation twinstafl ( tsr 1 ) by screening a collection of recessive , larval / pupal lethal mutations for mitotic abnormalities in larval brains ; the mutation twinstar 2 ( tst a ) was recovered in a similar manner in the laboratory of Dr . M . Fuller ( Stanford University School of Medicine , Palo Alto , CA ) by screening a different collection of muta - tions ( see Materials and Methods ) . ts / and tsr 2 mutants ex - hibit similar mitotic phenotypes ( see below ) and fail to complement for both lethality and cytologically detectable mitotic defects . On the basis of several criteria , the twinstar gene defined by these two mutations is located near the border between polytene chromosome intervals 60A and 60B ( bands 60A10 - 60B1 ) . First , probes containing P element - specific sequences hybridize only to this location in tst a and tsr 2 mutant sali - vary gland chromosome squashes ( data not shown ) . Sec - ond , deficiency mapping of tsr mutations places the gene in the region 60A8 - 16 ( Fig . 1 ) . Experiments kindly performed by Dr . Bruce Reed ( Whitehead Institute , Cambridge , MA ) have established that the tsr mutations are not allelic to any of the large number of genes he has previously characterized in this interval of chromosome 2R . Thus , twinstar represents a new lethal complementation group in this region . Finally , both alleles have been reverted by transposase - mediated excision of the P elements ( see Bellen et al . , 1989 ; Gatti and Goldberg , 1991 ) , demonstrat - ing that the P element insertions in the 60A10 - 60B1 inter - val are in fact the cause of both the tsr 1 and tsr 2 mutations . The Cytological Phenotype of Twinstar Mutant Neuroblasts In larval neuroblasts of tsta / tsr 1 , tsr ~ / tsr 2 , and tsta / tsr 2 indi - viduals , the major cytological consequence of lesions in the twinstar gene is the appearance of a significant proportion of polyploid cells ( Fig . 2 ; Table I ) . Other aspects of mitosis appear to be largely unaffected . The mitotic index , a pa - rameter measuring the frequency of cells engaged in mito - sis , is nearly normal ( Table I ) . The percentages of mitotic cells that are in anaphase in mutants are very similar to wild - type . Chromosomes in mutant neuroblasts seem to be normal : we see no evidence of irregular chromosome con - densation or of chromosome breakage ( Gatti and Baker , 1989 ) . The phenotype observed in both tsr 1 and tsr 2 brains is similar to that associated with mutations in a variety of Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1245 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 1 . Deficiency mapping of the twinstar locus , twinstar fails to complement Df ( 2R ) or 8R - 6 ( 59D5 - 10 ; 60B3 - 8 ) and Df ( 2R ) or BRll ( 59F6 - 8 ; 60A8 - 16 ) ( filled bars ) , but does complement Df ( 2R ) bw TM ( 59DS - 11 ; 60A7 ) and Df ( 2R ) GlO - BR - 27 ( 59F3 ; 60A8 - 16 ) ( open bars ) . Stretches of DNA that are absent in deficiency strains are represented by solid bars , with uncertainties in deficiency break - point ends indicated by crosshatching ; the twinstar locus is thus contained within the shaded region ( 60A8 - 16 ) . The right - hand breakpoint of Df ( 2R ) or BR - 1I is shown to be distal to that of Df ( 2R ) G10 - BR - 27 based on genetic mapping against other com - plementation groups in this region ( Dr . Bruce Reed , personal communication ) . Probes containing sequences found within the P element transposons also hybridize to the region 60A10 - 60B1 in squashes of salivary gland chromosomes ( not shown ) . genes that cause defects in cytokinesis ( Gatti and Baker , 1989 ; Karess et al . , 1991 ; Castrillon and Wasserman , 1994 ; Neufeld and Rubin , 1994 ) . Successive failures in cytokine - sis would result in cells that increase their ploidy geometri - cally , giving rise to large polyploid cells such as that shown in Fig . 2 d . The normality of other mitotic parameters sug - gests that these lesions in the twinstar gene do not signifi - cantly alter other mitotic processes . An example of this specificity is shown in Fig . 2 e , which demonstrates that polyploid cells , which must already be depleted for normal tsr ÷ function , are nonetheless able to enter anaphase . Fig . 2 fshows an anaphase spindle that is multipolar due to the retention of several centrosomes in a single cell , another expected result of failures in cytokinesis . Meiotic Aberrations Associated with Mutations in twinstar Male meiosis offers a particularly suitable system for the analysis of the cytological consequences of mutations af - fecting cell division . Primary spermatocyte nuclei are ~ 25 times larger than neuroblast nuclei and exhibit compara - tively larger spindles that can be clearly detected by tubu - lin immunostaining . We have recently provided a detailed description of chromosome and microtubule organization during wild - type male meiosis , which we have been able to subdivide into cytologically well - defined stages ( Cenci et al . , 1994 ) . Because meiosis occurs in the testes of third in - Figure 2 . Mitotic defects in larval neuroblasts of tsr mu - tants . ( a ) Diploid female metaphase and ( b ) diploid anaphase from Oregon R con - trois . ( c ) Tetraploid meta - phase , ( d ) highly polyploid metaphase , ( e ) tetraploid anaphase , and ( f ) tripolar polyploid anaphase from tsta / tsr z larval brains . Bar , 5 Ixm . The Journal of Cell Biology , Volume 131 , 1995 1246 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Table I . Frequency of Polyploid Cells and Mitotic Parameters in Larval Neuroblasts of tsr Mutants No . of metaphases No . of anaphases Genotype No . of brains diploid polyploid diploid polyploid No . of cells scored % polyploid figures % anaphases Mitotic index * tsrl / tsr I 23 1190 211 233 10 1644 13 . 4 tsr2 / tsr 2 10 937 108 232 3 1280 8 . 7 tsrl / tsr 2 9 519 128 117 9 773 17 . 7 Oregon R 5 478 0 94 0 572 0 . 0 14 . 8 18 . 4 16 . 3 16 . 4 0 . 78 0 . 92 0 . 84 0 . 71 * Mitotic figures were scored every fourth optic field in each brain examined q see Materials and Methods ) . star larvae , we were able to analyze the meiotic divisions in mutant tsr larvae for potential aberrations . Improper Aster Migration during ProphaselPrometaphase of Both Meiotic Divisions . A unique phenotypic consequence of lesions in twinstar occurs in primary spermatocytes at the prophase / prometaphase transition of meiosis I , and is revealed when fixed mutant testes are stained with anti - bodies directed against tubulin . In wild - type males , cen - trosomes reside just under the plasma membrane through - out most of the primary spermatocyte stage ( see Fig . 10 below ) , during which growth and gene expression prepare the cell for subsequent morphological differentiation ( Tates , 1971 ; reviewed by Fuller , 1993 ) . Near the end of this spermatocyte maturation phase , duplicated cen - trosomes ( each containing a pair of centrioles ) migrate to - gether to the nuclear membrane , where they nucleate prominent asters ( Tates , 1971 ; Cenci , 1994 ; see also Fig . 9 c below ) . During late prophase / early prometaphase of the first meiotic division ( stage Mla ) , the two asters separate from each other and migrate around the periphery of the nuclear envelope , in preparation for the establishment of the bipolar spindle . By early prometaphase , asters are lo - cated directly opposite each other on either side of the nu - cleus , in close apposition to the nuclear membrane . Wild - type spermatocytes at the Mla stage are shown in Fig . 3 a . In tsr spermatocytes at the Mla stage , the two asters of - ten remain in close proximity to each other and fail to as - sociate with the nuclear envelope ( Fig . 3 b ) . We have named the twinstar gene after this characteristic aberrant arrangement of asters . This effect occurs in a very high proportion of tsr primary spermatocytes , approaching 100 % in tsr 1 mutants ( Table II ) , but is almost never seen in wild - type . Somewhat later , in the Mlb stage , asters in tsr mu - tants can be seen to separate slightly ( Fig . 3 c ) , but remain unassociated with the nuclear membrane . It is remarkable that subsequent to the M1 stage , the spindles in tsr mutant spermatocytes progressively resume a relatively normal position and appearance and seem in - distinguishable from wild - type throughout the remainder of the first meiotic division ( Fig . 4 ) . However , after com - Figure 3 . Aster migration in meiotic prometaphase I of tsrt / tsr I males . ( Left ) Phase contrast ; ( middle ) anti - tubu - lin staining ; ( right ) Hoechst 33258 staining of DNA . ( a ) A primary spermatocyte in the Mla stage from Oregon R controls , showing well sep - arated asters closely apposed to the nuclear envelope . ( b and c ) Primary spermatocytes in ( b ) Mla and ( c ) Mlb stages from tsrl / tsr I males . Note the delay in migration and ab - normal positioning of asters with respect to the cell nu - cleus . Stage identification was based on the presence of in - tranuclear granules originat - ing from Y loop disintegra - tion and on the relative size of spermatocyte nuclei ( for details see Cenci et al . , 1994 ) . Bar , 10 txm . Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1247 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Table I1 . Meiotic Abnormalities in tsr Mutants Asters in prometaphase 1 Asters in prometaphase 1I Spermatids * Genotype Normal Irregular * % Irregular Normal Irregular * % Irregular 1 : 1 2 : 1 3 : 1 and 4 : 1 % Irregular tsrl / tsrl 2 117 98 . 3 34 91 72 . 8 422 108 73 30 . 0 tsr2 / tsr2 12 104 89 . 6 31 56 64 . 3 602 23 5 4 . 4 Oregon R 99 2 2 . 0 98 2 2 . 0 200 0 0 0 . 0 * Irregular positioning and / or delayed migration of asters . * Ratios refer to number of nuclei per Nebenkern . 1 : 1 , regular spermatids containing one nucleus and one Nebenkern of similar size . Failures in cytokinesis are revealed by other patterns ( see text ) : 2 : 1 , two nuclei associated with one large Nebenkern . 3 : 1 and 4 : 1 , three or four nuclei associated with one large Nebenkern . pletion of meiosis I , tsr prophase / prometaphase secondary spermatocytes ( stage M6a and M6b ) exhibit the same de - fect in aster migration and positioning observed in primary spermatocytes ( Fig . 5 ) . Here again , the spindles become more normal by metaphase II and retain a wild - type struc - ture throughout the remainder of meiosis II . Although te - lophase II tsr spindle structures look normal , we often observed an aberrant cross - like configuration of meiotic spindles that overlap at the midbody ( Fig . 5 j ) . Most likely this arrangement reflects a failure of the first meiotic cy - tokinesis and the consequent occurrence of two second di - visions within the same cell ( see below ) . The analysis of hundreds of ana - telophases of tsr mu - tants showed no evidence of irregular chromosome segre - gation during either meiotic division . We never observed lagging chromosomes or daughter nuclei of different size . In addition , tsr " onion stage " spermatids exhibit a uniform nuclear size ( see below ) . As it has been demonstrated that spermatid nuclear size is proportional to chromosome content ( Gonz ~ lez et al . , 1989 ) , this observation reinforces the conclusion that tsr does not affect chromosome segre - gation . Failure of Cytokinesis during Spermatogenesis in twinstar Larvae . During both meiotic divisions in wild - type sper - matocytes , mitochondria associate lengthwise along the spindle apparatus ( see Figs . 4 and 5 ; Cenci et al . , 1994 ) , which is pinched in half during cytokinesis , ensuring an even distribution of these organelles to each of the two daughter cells . Immediately after completion of meiosis , the mitochondria associated with each hemispindle fuse and form an interlaced conglomerate called the Nebenkern . Thus , each newly formed wild - type spermatid ( referred to as the onion stage ) consists of a round , phase - light nucleus associated with a single phase - dark Nebenkern of similar size ( Fig . 5 o ; reviewed by Fuller , 1993 ) . The most obvious defect in tsr mutants is seen in sper - matids at the onion stage . In tsr mutant testes , a substan - tial fraction of onion stage spermatids contain a single Nebenkern much larger than normal , along with four nor - mal - sized nuclei ( Fig . 5 q ; Table II ) . This phenotype re - Figure 4 . First meiotic division in tsr mutants . The three panels show the same partial cyst containing primary spermatocytes at different meiotic stages . ( Left ) Phase contrast ; ( middle ) anti - tubulin staining ; ( right ) Hoechst 33258 staining of DNA . 1 and 2 , spermatocytes in early meiotic prometaphase ( Mlb stage ) showing delayed migration and abnormal positioning of asters . 3 , prometaphase ( stage M2 ) , and 4 , metaphase ( stage M3 ) spermatocytes in which the asters have almost attained a regular bipolar arrangement . 5 , a morphologically normal mid - anaphase ( stage M6b ) . Bar , 10 I - tm . The Journal of Cell Biology , Volume 131 , 1995 1248 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 5 . Second meiotic di - vision and postmeiotic stages in tsr mutants , a - f show partial secondary sper - matocyte cysts observed by phase contrast microscopy ( a - c ) and after immunostain - ing with anti - tubulin Abs ( d - ] ) . ( a and d ) Prometaphase II ( stage M7 ) spermatocytes from Oregon R controls with normally positioned as - ters . ( b and e , c and f ) The same stage from tsrl / tsr l males showing delayed mi - gration and abnormal posi - tioning of asters , g - n show meiotic cells immunostained with anti - tubulin Abs ( g - j ) or stained with Hoechst 33258 ( k - n ) . ( g and k ) A metaphase cell in the M9 stage in which the asters have almost attained a regular bi - polar arrangement . ( h and l ) An early anaphase ( stage M10a ) , and ( i and m ) an early telophase ( stage Mll ) , both with regular spindles . ( j and n ) Two late telophase II occurring in the same cyto - plasm , showing overlapped central spindles , o - r show liv - ing spermatids at the onion stage observed with phase contrast . ( o ) Regular sper - matids from Oregon R con - trols with nuclei ( white discs ) and Nebenkerns ( dark discs ) of similar sizes . ( p - r ) Sper - matids from tsr mutants showing large Nebenkerns associated with ( p ) two , ( q ) four , or ( r ) three nuclei . See text for details on the origin of these aberrant sperma - rids . Bar , 10 p , m . flects a failure of cytokinesis at both meiosis I and II , as this would prevent proper subdivision of the mitochon - drial complement into four Nebenkerns ( reviewed by Fuller , 1993 ) . We also see cells with two normal - sized nuclei in association with an intermediate - size Nebenkern ( Fig . 5 p ; Table II ) , indicating a failure of cytokinesis at only one of the meiotic divisions . Because failure of cytokinesis in the first meiotic division would lead to cells containing two second division meiotic spindles ( see Fig . 5 , j and n ) that would probably encounter further difficulties in cytokine - sis , we believe that the spermatids with one Nebenkern and two nuclei are the result of second division cytokinesis failure . Finally , we observe spermatids containing one large Nebenkern plus three nuclei that are usually located in close proximity to a cell containing a normal - size Neben - kern and a single regular nucleus ( Fig . 5 r ; Table II ) . These are most likely to arise if the four hemispindles in second - ary spermatocytes derived from cytokinesis I failures ( see again Fig . 5 , j and n ) orient in such a way that mitochon - drial fusion occurs asymmetrically , yielding a small and a large Nebenkern associated with one and three nuclei , re - spectively . Molecular Analysis of the twinstar Gene Molecular Cloning and Transcriptional Analysis of twinstar . A small stretch of DNA adjacent to the P element inser - tion in tsr t was initially cloned using an inverse PCR proto - col and then used to screen a wild - type Drosophila ge - nomic library . A 6 . 4 - kb EcoR1 - SalI subclone from this library ( RS6 . 4 ) surrounds the tst a and tsr e P element inser - tion sites ( Fig . 6 A ) . Whole genome Southern analysis con - firmed the presence of P element insertions within this fragment in both tsr mutant alleles that were absent from Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1249 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m A R t s r 1 t s r 2 X PB P X M P BP kilobases S 4 P B V tsr 2 V tsr 1 2701 ~ cgtcg ~ gacgcg ~ cacg ~ cgc ~ tgt ~ g ~ t ~ ctttgc ~ ggcg ~ tgg ~ ccc ~ c ~ gtgcg ~ t ~ c ~ g ~ cc ~ at ~ g & tt ~ gt ~ ggttgtt ~ g 2801 t4aaagaa ~ g ~ ggaa ~ ttaaaaa ~ att ~ xjta ~ aatata ~ a ~ t ~ gg ~ gaa9 ~ agoaatt ~ xJ & g ~ attg ~ : xJ ~ g ~ a ~ a ~ ta ~ a ~ gta c M A S 0 V T V S 4401 OAT ~ - AA ~ % CTACATAC ~ ~ CAAAAA ~ ATC ~ ATG ~ CATCTTTTACATTCGCGA ~ ~ ~ ~ ~ D V C K T T Y • • I K K D K K H R Y V I F Y I R D E K Q I D V * e T 4501 ~ k ~ % GA ' ~ " GCAACGCC GAGTACGAC TATC ~ TOCGGACCTGGAGAGTGCCGgtaagttccgccggttagagacacccagac V A D R N A E Y D Q F L E D I Q K C G P G E C R 4601 ccatcgaaactaacctaatttaacatctattcgcttgcacag ~ TAcGGA ~ TGTTcGATTTcGAGTA ~ ATG ~ - cCk ~ TCAp ~ O ~ CAECT ~ TGAGAGTT ~ A Y G L F D F E Y M H ~ C Q G T S E S S 4701 AAGA ~ C ~ J ~ % k ~ CTGTTCCTTAT ~ TC ~ TGGTGTc ~ GATAcTGCcAAGgtatgtgaatagccagttgccaagtaatggcgcagaactcatttcgatatca E I Q K L F L M S W C P D T A I 4801 ca ca ccca c a gGTCAAGAAGA ~ T ~ TTGTACTCCAGCTCCTTCGATGCTC TCAAGAAGTC GCTC GTGGGCGTC CAAAAGTATATACAAGC ~ C ~ T V K K K M L Y S S S F D A L K K S L V G V Q K Y I Q A T D L 4901 TTCC ~ AA ~ CCTCCCGGGAAGCC ~ AGGAGAA ~ TCCGGGCCACCG ~ CCGCCAAt aaaotgta = gagoa ~ - tgtg = a t tgoa t t % aacaaa = aagg = a tg S E A S R E A V E E K L R A T D R 5001 oa ~ gaaaoag = gatggaaaoa ~ gtat ~ tgttgagatog = = aatttgtatgagaattttotacataat ttgtatttt ~ agtagaaataaaqaatgaaata 5101 tttaa = ttgaccaagtcgcgtttccgctgtggtaaaaatctcgtaccgcagaatacactttattataaggagcaaagattacattgtacatatgatgacg Figure 6 . Molecular analysis of twinstar . ( A ) Physical map of the twinstar genomic region . The 6 . 4 - kb genomic fragment used to rescue the twinstar mutants ( RS6 . 4 ) is shown . Approximate positions of the P elements causing the ts / and tsr 2 alleles are indicated . The twin - star cDNA ( open boxes and arrow ) , when mapped onto the genomic fragment , is interrupted by one large ( 1 . 5 kb ) and two small ( 72 and 63 bp ) introns . The direction of transcription is from left to right in this diagram ; we do not know the transcriptional polarity relative to the chromosome as a whole . Restriction sites shown : EcoR1 ( R ) , XhoI ( At ) , BamH1 ( B ) , PstI ( P ) , SmaI ( M ) , SalI ( S ) . ( B ) Excerpts from the DNA sequence of RS6 . 4 , showing regions containing the P element insertion sites and the twinstar cDNA sequence . Arrow - heads mark insertion sites of the P elements causing the ts / and tsr 2 alleles . These are located 19 bp apart : the ts / insertion site occurs 5 bp within the 5 ' UTR of the cDNA , while that of tsr 2 lies just upstream , cDNA sequence is in boldface type ; coding regions are in capi - tals , with one letter amino acid codes centered beneath each codon . Genomic sequences not present in the cDNA are italicized . The cDNA carries one noncoding G at the 5 ' end ( not pictured ) . The putative polyA addition signal sequence is underlined . Since the se - quence shown corresponds to part of the sequence from the 6 . 4 kb of RS6 . 4 , this figure uses a nucleotide numbering scheme that is rela - tive to the entire fragment . Genbank accession numbers : twinstar cDNA , U24490 ; genomic fragment RS6 . 4 , U24676 . wild - type DNA and from DNA of transposase - induced re - vertants of both alleles ( data not shown ) . Probes made from RS6 . 4 hybridized in situ to polytene chromosome in - terval 60A10 - 60B1 , in good agreement with the cytoge - netic position of twinstar . Based on Northern analysis suggesting that RS6 . 4 hy - bridizes to a single species of poly ( A ) + RNA 800 - 900 bp in length ( see below ) , we reintroduced genomic fragment RS6 . 4 into the Drosophila genome via P element - medi - ated germline transformation . A single transduced copy of RS6 . 4 corrects the lethality as well as the mitotic and mei - otic phenotypes associated with mutant alleles of tsr . Thus , tsr + genetic activity is contained within the 6 . 4 kb of ge - nomic fragment RS6 . 4 . The level of the single transcript detected by RS6 . 4 is decreased in homozygous ts / and tsr 2 mutants to no more than 20 % of wild - type and is restored to wild - type levels in rescued lines ( Fig . 7 A ) . This transcript is expressed during all stages of Drosophila development , is found in both adult males and females , and appears to peak in late larval and pupal stages ( Fig . 7 B ) . On the basis of these and other data ( see below ) , we believe that this cDNA represents the twinstar transcript . We have determined the entire nucleotide sequence of RS6 . 4 and that of an homologous 750 - bp cDNA clone ( Fig . 6 B ) . The putative tsr cDNA insert contains a single long , ATG - initiated open reading frame . The nucleotide sequence flanking this putative translational start , AAA - AATG , is consistent with the Drosophila translation initi - ation consensus ( C / A ) AA ( A / C ) ATG ( Cavener , 1987 ) . A consensus signal sequence for polyA addition is located 25 bases upstream from the polyA stretch found in the cDNA . Comparison of the genomic and cDNA sequences indi - cates that the twinstar gene is interrupted by one long ( 1 . 5 kb ) and two small ( 72 bp and 63 bp ) introns ( Fig . 6 ) , which are flanked by consensus splice donor and acceptor sites The Journal of Cell Biology , Volume 131 , 1995 1250 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 7 . Northern analysis of twinstar mRNA . ( A ) Expression of twinstar in mutant and rescued lines . Lane 1 , wild type ( Ore - gon R ) ; lane 2 , tsrl / tsrt ; lane 3 , tsr2 / tsr2 ; lane 4 , tsrl / tsrl ; [ pWS - tsr + ] / [ pWS - tsr + ] ; lane 5 , tsre / tsr2 ; [ pW8 - tsr + ] / [ pWS - tsr + ] . Lanes 2 and 3 are tsr mutant strains ; lanes 4 and 5 are the same tsr mutant lines that have been rescued with wild - type genomic DNA spanning the tsr locus ( see text ) . On this blot , tsr expression in the mutants appears negligible , but we estimate from additional experiments that mutants may contain a residual level of tsr mRNA up to 20 % of wild - type . 10 ixg of total RNA isolated from third instar larvae were loaded in each lane . RNA molecular weight standards ( in kb ) are indicated at left . ( B ) Developmental expression of twin - star in wild - type animals raised at 25°C . Lanes : 0 - 24 h embryos ; first and second instar larvae ; third instar larvae ; early pupae ; late pupae ; adult males ; adult females . 5 ~ g of polyA + - selected RNA were loaded in each lane . ( C ) The same blot as in B hybridized with rp49 cDNA as a loading control . ( Mount , 1982 ) . The cDNA carries one additional non - tran - scribed G at the 5 ' end , which is thought to represent re - verse transcription of the 5 - methyl - G cap during cDNA syn - thesis ( Brown et al . , 1989 ; Ketchum et al . , 1990 ; Karess et al . , 1991 ; Rose and Weischaus , 1992 ) . No other significant open reading frames were found in RS6 . 4 , in correspon - dence with its hybridization to only a single RNA species on Northern blots . By direct sequencing of mutant DNA , we determined the precise locations of the P element insertions causing the tsr a and tsr 2 mutations to be 19 bp apart , in or very near the beginning of the tsr transcript ( Fig . 6 B ) . The longest prod - uct produced by RACE ( rapid amplification of cDNA ends ) using RNA isolated from wild - type embryos or third instar larvae extends five bases beyond the cDNA 5 ' end ( data not shown ) . This places the tsr l insertion site within the mRNA 5 ' - untranslated region ( 5 ' - UTR ) and suggests that the tsr 2 insertion point would appear to be a few nu - cleotides upstream of the start of twinstar transcription . However , we do not know precisely where tsr mRNA transcription is initiated , nor do we see an obvious TATA box . To investigate how detectable ( though reduced ) amounts of normal - sized tsr mRNA could be produced in mutant animals when at least one of the P element insertion sites appears to be located within the transcribed region ~ we also analyzed RACE products from homozygous tsr 1 and tsr 2 larvae . These products contain short ( 78 - 126 bp ) unique sequences at their 5 ' ends that appear to be spliced into the normal tsr message either at or just downstream of the P element insertion sites ( data not shown ) . We have not been able to identify the unique 5 ' regions either in the 2 kb of genomic DNA we have sequenced upstream of the insertion sites or in known sequences from the marked P elements ; however , we believe they are likely to be de - rived from within the P elements . Decreased initiation ef - ficiency or stability of these hybrid transcripts could then account for the low levels of tsr - homologous mRNAs ob - served in the mutants . Twinstar Encodes a Member of the CofilinlADF Family of Actin Associated Proteins . Conceptual translation of the open reading frame in the twinstar cDNA ( Figs . 6 B and 8 ) generates a 17 - kD polypeptide related to proteins in the cofilin / ADF family . These proteins can sever filamentous actin and also bind and sequester actin monomers , leading to depolymerization of actin filaments ( reviewed by Sun et al . , 1995 ) . A cDNA coding for the twinstar protein has also been recovered independently from a screen of a Drosoph - ila cDNA library for proteins that produce changes in cell shape when overexpressed in the fission yeast S . pombe ( Edwards et al . , 1994 ) . An alignment of the predicted twinstar protein with rep - resentative homologues from nematode , yeast , amoeba , plant , and vertebrate ( human ) species is shown in Fig . 8 . The four nonvertebrate sequences shown returned the highest scores in searches of the GenBank and EMBL da - tabases , with amino acid identities in the range of 36 - 39 % over the length of the protein when compared with twin - star . Fig . 8 highlights three regions implied in specific func - tions . Two conserved motifs found in proteins of the cofi - lin / ADF family appear to be important for actin binding based on studies with mammalian homologues , corre - sponding to twinstar residues 94 - 105 ( shaded bar ) and 112 - 118 ( filled bar ) . These regions are implicated addi - tionally in either phosphoinositide binding ( shaded bar only ) or competition with tropomyosin for binding to actin ( filled bar only ) . A third motif present in vertebrate homo - 1ogues , PXXXKKRKK ( hatched bar ) , is conserved rela - tively well in twinstar but not in homologues from other organisms . This sequence resembles the nuclear localiza - tion signal sequence of SV - 40 large T antigen , and is thought to play a role in translocation of cofilins into the nucleus under conditions of stress ( see Discussion ) . Actin Behavior in tsr Mutants The finding that twinstar encodes a Drosophila cofilin homo - logue prompted us to compare actin behavior in tsr mutant and wild - type testes ( Figs . 9 - 11 ) . We consider first F - actin distribution within primary spermatocytes through early ana - phase of the first meiotic division . In cysts of young wild - type spermatocytes ( stage $ 1 ) , actin forms a ribbon - like pat - tern that connects adjacent cells ( Fig . 9 a ) ; we believe this corresponds to an analog of the fusome , a structure that extends through the ring canals of developing oocytes ( Lin et al . , 1994 ) . These ribbons gradually disassemble , so that in mature primary spermatocytes ( stage $ 5 ) , actin is only found in a loose network of cables and spots ( Fig . 9 , b and c ) . Little or no F - actin staining is visible by the time wild - type primary spermatocytes enter prometaphase ( stage M1 ; Fig . 9 c ; see also Fig . 11 a ) or later through early ana - phase of the first meiotic division ( to stage M4b ; Fig . 11 b and data not shown ) . Testes from twinstar mutants exhibit an actin staining pattern that is similar to that of wild - type through the mature primary spermatocyte stage ( stage $ 5 ; Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1251 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 8 . Comparison of the putative twinstar protein with members of the same family of small actin - severing proteins from other or - ganisms . Sequences shown , from top down : D . melanogaster twinstar , C . elegans Unc60B ( McKim et al . , 1994 ) , S . cerevisiae COF1 ( Iida et al . , 1993 ; Moon et al . , 1993 ) , A . castellanii actophorin ( Quirk et al . , 1993 ) , L . longiflorum ADF ( Kim et al . , 1993 ) , human cofilin ( Ogawa et al . , 1990 ) , human and porcine destrin ( Moriyama et al . , 1990b ; Hawkins et al . , 1993 ) . Overline bars indicate putative func - tional domains . The region defined by the shaded bar has been implicated in actin binding based on mutational ( Moriyama et al . , 1992 ) , cross - linking ( Yonezawa et al . , 1991a ) , and peptide inhibition studies ( Yonezawa et al . , 1991b ) . This region also interacts with certain phosphoinositides ( Moriyama et al . , 1992 ; Yonezawa , et al . , 1991b ) . The vertebrate sequence in the region indicated by the filled bar , DAIKKK , is identical to residues 2 - 7 at the NH2 terminus of tropomyosin ( Cho et al . , 1990 ) and can compete with it for binding to actin ( Yonczawa et al . , 1989 ) . The hatched bar represents a putative nuclear localization signal sequence present in vertebrate homologues that is similar to that of SV40 large T antigen ( Matsuzaki et al . , 1988 ) . Fig . 10 a and data not shown ) . Immediately afterwards , however , the distribution of actin in twinstar mutants be - comes dramatically different from that in wild - type : at the $ 6 stage ( a rapid phase of transition between the end of spermatocyte growth and the onset of meiosis ) , mutant primary spermatocytes develop a single large F - actin ag - gregate in the cytoplasm of each cell ( Fig . 10 , a and c ) . This actin cluster ( which we term a type 1 aggregate ) rapidly forms near the centrosomes at the cell cortex , just at the time they first begin to nucleate microtubules ( Fig . 10 , b and c ) . This aggregate remains in the vicinity of the asters when they assume their ectopic position in mutant primary spermatocytes at prometaphase ( stage M1 ; Fig . 10 a ; see also Fig . 11 e ) . The fact that little or no F - actin staining is seen in primary spermatocytes at the $ 5 stage immediately before the formation of type 1 aggregates indicates that this type of actin cluster is not simply a remnant of a previ - ously formed actin structure in these mutant cells . At metaphase ( stage M3 ) , the actin cluster in twinstar mutant primary spermatocytes dissociates from the asters and is usually seen at the equator of the cell ( Fig . 11 J0 - During early anaphase , type 1 aggregates essentially disappear ( data not shown ) . Additional differences in actin behavior become appar - ent by late anaphase / early telophase ( stages M4c - M5 ) . In wild - type , actin becomes concentrated at the spindle mid - zone where it forms a ring - like structure that contracts as the cell progresses toward telophase ( Fig . 11 , c and d ) . In twinstar mutants , actin also concentrates at the mid - zone of the central spindle at this time ( Fig . 11 g ) . This actin fo - cus ( a type 2 aggregate ) is more prominent than the wild - type contractile ring and exhibits unusual protuberances generally oriented along the central spindle axis . Type 2 aggregates are also more stable than normal contractile rings . Contractile rings in wild - type disassemble at the end of the first meiotic division ( stage M5 / M6 ) , and wild - type secondary spermatocytes do not exhibit prominent actin structures from interphase to mid - anaphase ( stages M6 - M10b ; data not shown ) . In contrast , type 2 aggregates per - sist until metaphase II ( stage M9 ) , often even increasing in size . Sometimes , a type 2 aggregate is found associated with the metaphase II spindle in only one of the two daughter cells produced by the first meiotic division ( Fig . 11 h ) , but is more often found associated with the spindles in both daughter cells ( Fig . 11 i ) . As the second meiotic division proceeds into anaphase II , type 2 aggregates eventually disassemble ( not shown ) . A final difference between actin behavior in wild - type The Journal of Cell Biology , Volume 131 , 1995 1252 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 9 . Actin behavior in Oregon - R wild - type primary spermatocytes through prometaphase ( stages $ 1 to M1 ) . To visualize tubulin , actin and DNA , testes were immunostained with anti - tubulin Abs ( green ) and then sequentially stained with rhodamine - labeled phalloi - din ( red ) and Hoechst 33258 ( blue ) . The fluorescent signals were detected separately by a cooled charge - coupled camera and merged in pseudocolors . Yellow and orange colors indicate overlap of actin and microtubule signals . ( a ) Partial cyst containing S1 / $ 2 young pri - mary spermatocytes . F - actin staining of structures connecting adjacent cells is apparent ; we suggest these structures are fusome analogs ( see text ) . ( b ) Near mature primary spermatocytes ( stage $ 4 ) showing actin spots and cables . ( c ) Spermatocytes at stages $ 3 , $ 5 , and M1 . Note that phalloidin staining gradually disappears during these stages of development , and is absent in wild - type M1 cells . Bar , 10 ~ m . and mutant meiosis becomes apparent by late anaphase and telophase of the second meiotic division ( stages M10c - Mll ) . In wild - type , F - actin is localized in a clearly visible contractile ring that disappears after completion of the second meiotic division ( data not shown ) . At the corre - sponding stages in twinstar mutants , an F - actin containing structure ( type 3 aggregate ) assembles at the spindle mid - zone . This type 3 aggregate , like the type 2 aggregate , is more prominent than the wild - type contractile ring and has protuberances oriented along the spindle axis ( Fig . 11 j ) . Moreover , the type 3 aggregate does not disappear at the end of the second meiotic division , but persists until the on - ion stage , disassembling only during spermatid elongation ( not shown ) . We analyzed actin behavior in both tsr l and tsr 2 mutant testes , and in both cases we observed the pattern described above . The only detectable differences between the two mutant alleles are in the size and morphology of the aber - rant actin aggregates . The type 1 aggregate of tsr 2 is some - what smaller than that of tsr ~ . Similarly , the type 2 and type 3 aggregates of tsr 2 tend to be less prominent and are more regular in shape than those observed in tsr 1 . Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1253 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 10 . Actin behavior in twinstar mutant primary spermatocytes through prophase / prometaphase ( stages $ 5 to M1 ) . Pseudocolors as in Fig . 9 . ( a ) Partial cyst containing $ 5 , $ 6 and M1 mutant primary spermatocytes . Note that F - actin clusters are absent in $ 5 mature spermatocytes , but are prominent in $ 6 spermatocytes and in M1 prometaphase cells ( type 1 aggregates ) . ( b and c ) $ 6 stage primary spermatocytes visualized with anti - tubulin antibodies ( b ) show the nascent asters in a particularly favorable preparation ( arrows ) . The same field is shown in c with both anti - tubulin antibodies and rhodamine - labeled phalloidin to show association of type 1 actin aggre - gates with nascent asters . Bar , 10 ixm . Discussion Structural Features of the twinstar Protein Based on primary sequence similarity , the relatedness of the twinstar protein to known members of the cofllin / ADF family of actin severing / depolymerizing factors is unam - biguous . Furthermore , preliminary biochemical data ( Dr . S . K . Maciver , MRC , Cambridge , England , personal com - munication ) have shown that bacterially expressed twin - star protein is able to bind and sever actin filaments , and that these activities are comparable to those of other pre - viously characterized cofilins . Thus , twinstar protein be - haves in vitro in a manner that is consistent with expecta - tion . This implies that the functionality of conserved domains known to be important for actin binding and de - polymerizing activities is retained in twinstar . The structural features that govern the biochemical ac - tivities of cofilin - like proteins have not been completely characterized , nor is it clear how the different properties displayed by these proteins influence actin dynamics in different cellular environments . As one illustration , why certain cofilins show pH - dependent activity is not known , but it is thought that this feature might contribute to cy - toskeletal changes seen upon alkalinization of the cyto - plasm in response to cell activation by growth factors ( see Sun et al . , 1995 ) . Nonetheless , several conserved motifs present in cofilin - like proteins are clearly implicated in specialized functions that may influence their activities . For example , one of the actin - binding domains identified in vertebrate homologues ( Fig . 8 , filled bar ) , which is well conserved in twinstar , competes with tropomyosin for binding to actin in vitro . This region may underlie the abil - ity of vertebrate cofilins to compete for actin binding with tropomyosin , myosin , and villin headpiece in vitro ( Nishida , et al . , 1984 ; Nishida et al . , 1985 ; Pope et al . , 1994 ) . We might therefore expect to find that twinstar also displays similar activities , which could be important for its function in vivo . In addition , the actin depolymerizing activities of mam - malian cofilin and destrin / ADF , yeast cofilin , and Acan - thamoeba actophorin are specifically inhibited by certain phosphoinositides , which bind cofilin and displace it from actin ( Yonezawa et al . , 1990 , 1991b ; Iida et al , , 1993 ; Moon et al . , 1993 ; Quirk et al . , 1993 ) . Phospholipid binding may help maintain proteins near the plasma membrane and could potentially be involved in regulating their activity in vivo . One of the motifs present in cofilins that is known to bind actin is also critical for phospholipid binding ( Fig . 8 , shaded bar ) . This same motif also influences the ability of cofilin to inhibit hydrolysis of phospholipids by phospholi - pase C ( Yonezawa et al . , 1991a , b ) , an important enzyme linking phospholipid metabolism with cellular signaling cascades . We do not know whether twinstar activity is in - fluenced by phosphoinositides , but based on the wide range of organisms in which this property is retained , the possibility seems likely . Other actin - binding proteins such as profilin and gelsolin also interact specifically with cer - tain phosphoinositides ( Lassing and Lindberg , 1985 , 1988 ; Janmey and Stossel , 1987 ) . However , the interaction of co - filins with phosphoinositides appears to be of both lower affinity and specificity than that of profilin , and the rele - vance of this feature in vivo remains to be established . Finally , mammalian and avian cofilins and ADFs con - tain a motif , PXXXKKRKK ( Fig . 8 , hatched bar ) , that is very similar to the nuclear localization signal sequence of SV - 40 large T antigen ( Matsuzaki , et al . , 1988 ) . This se - The Journal of Cell Biology , Volume 131 , 1995 1254 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m Figure 11 . Actin behavior in wild - type and twinstar mutant spermatocytes from prometaphase through the completion of meiosis ( stages M1 to Mll ) . a - d are from Oregon R control testes ; e - j are from tsrt / tsr t males . Pseudocolors as in Fig . 9 . ( a ) Late prophase I ( stage Mla ) and ( b ) metaphase I ( stage M3 ) wild - type primary spermatocytes without prominent actin structures . ( c and d ) Telophase I cells at the M5 stage showing contractile actomyosin rings at the central spindle midzones . Depending upon the angle of observation , these structures either appear as bars ( c ) or exhibit a circular shape ( d ) . ( e ) Mutant tsrl / tsr I primary spermatocyte at the M1 stage , show - ing a prominent type 1 aggregate ; compare with a of this figure . ( f ) Three metaphase I figures in the M3 stage associated with actin clus - ters ( type 1 aggregates ) ; compare with b of this figure . ( g ) A telophase I ( MS stage ) showing a prominent and abnormally shaped actin ring ( type 2 aggregate ) ; compare with c and d of this figure . ( h ) Two secondary spermatocytes at late prometaphase ( M8 stage ) showing a single actin cluster associated with one of these cells . Most likely this is a type 2 aggregate that persists due to a failure in actin ring dis - assembly during telophase I . ( i ) Two metaphase II figures at the M9 stage showing a single actin aggregate . This structure is located in the same area occupied by the telophase I midbody , supporting the conclusion that it is a type 2 aggregate that originates from the actin ring . ( j ) A telophase II at the Mll stage with a clearly abnormal actin structure ( type 3 aggregate ) at the central spindle midzone . Bar , 10 Ixm . Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1255 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m quence is required in vertebrates for the translocation of cofilin into the nucleus upon heat shock or treatment with 10 % DMSO ( Nishida et al . , 1987 ; Iida et al . , 1992 ) . Trans - location is thought to be regulated by the phosphorylation state of a serine residue immediately upstream of this mo - tif ( Ohta et al . , 1989 ; see Morgan et al . , 1993 ) . Twinstar re - tains distinct homology to the nuclear localization motif , while other nonvertebrate homologues retain only weak similarities ; yeast cofilin is unable to localize to the nucleus ( Moon et al . , 1993 ) . Based on sequence similarity , it seems possible that twinstar could translocate into the nucleus ; however , it does not have a serine residue in exactly the same position relative to the signal sequence . We have no direct evidence at present to establish whether the twin - star protein can also translocate into the nucleus when Drosophila cells are stressed . Actin Behavior in twinstar Mutants Our observations provide strong evidence that actin dy - namics are significantly perturbed in the absence of tsr ac - tivity . Staining with phalloidin , which specifically binds fil - amentous actin ( Estes et al . , 1981 ) , shows that tsr testes contain large F - actin structures that are not seen in wild - type . Type 1 aggregates are first detected during the last stage of primary spermatocyte growth ( stage $ 6 ) and then grow to reach their maximum size at the onset of the first meiotic division . We hypothesize that centrosomes serve as the foci for this abnormal accumulation of actin in ma - ture primary spermatocytes because type 1 F - actin aggre - gates begin to form when nascent asters first become visi - ble , and they remain associated with asters throughout prophase / prometaphase of meiosis I ( M1 stage ) . We speculate that a nucleation center for actin addition may exist near centrosomes in tsr mutants , and that the ab - sence of twinstar activity may lead to uncontrolled actin accumulation at this site . Although phalloidin staining does not reveal the presence of F - actin in the vicinity of centrosomes in wild - type primary spermatocytes ( data not shown ) , the actin - related protein Arpl ( also known as centractin or actin - RPV ) has been found in association with centrosomes in other systems ( Clark and Meyer , 1992 ; Lees - Miller et al . , 1992 ) . Arpl forms a short F - actin - like filament that is part of the dynactin complex ( Paschal et al . , 1993 ; Schafer et al . , 1994 ) , which stimulates minus end - directed dynein - mediated vesicle motility along mi - crotubules ( Gill et al . , 1991 ; Schroer and Sheetz , 1991 ) . It is conceivable that a dynactin - like complex may normally localize to centrosomes in primary spermatocytes . Alter - natively , if in tsr mutants the normal cytoplasmic or corti - cal anchors for a dynactin - like complex were disrupted , it could be pulled to the centrosome by its associated motor activity . In either case , this type of complex might provide a centrosomal focus for aberrant actin accumulation . Unusual F - actin foci are also present in secondary sper - matocytes and spermatids of tsr mutants ( type 2 and type 3 aggregates ) , and almost certainly result from a failure to disassemble the actin structures found during telophase at the positions of the meiosis I and II contractile rings . Scru - tiny of a large number of tsr meiotic cysts clearly shows that these telophase structures are more prominent than wild - type contractile rings and exhibit protuberances ori - ented along the spindle axis . During late telophase , these irregular rings do not disassemble as do normal contractile rings , but instead grow slightly larger and change morphol - ogy , becoming compact masses of F - actin . Type 2 actin ag - gregates eventually disappear during anaphase II in sec - ondary spermatocytes , but then F - actin reassembles into a contractile ring - like structure ( type 3 aggregate ) during te - lophase II . Derivatives of this structure appear to persist as aggregates in spermatids that gradually disassemble during the elongation process . We interpret the aberrant structures in secondary sper - matocytes and spermatids as indicating that in tsr mutants , actin can assemble at the correct times and sites required for formation of the contractile ring . However , these actin assemblies are improperly organized , may be unable to function in cytokinesis , and fail to disassemble on a normal schedule . The eventual disassembly of aberrant actin ag - gregates may be mediated either by residual tsr activity in the mutants ( neither tsr l nor tsr z is a null allele ) , or by the presence of other actin depolymerizing agents . Interest - ingly , the ribbon - like actin structure in young primary spermatocytes that we interpret as the male fusome de - grades normally in twinstar mutants . This suggests that ei - ther disassembly of this structure does not depend on twinstar activity , or residual levels of twinstar protein at these earlier stages are sufficient for its disassembly . In Vivo Functions of the twinstar Protein Centrosome Migration and Separation . During both meiotic divisions in tsr mutants , in contrast with wild - type , the as - ters fail to associate with the nuclear envelope , and their migration to opposite poles is significantly delayed . That mutations in an actin binding protein result in such a phe - notype is consistent with considerable precedent indicat - ing an interaction between centrosomes and the actin cy - toskeleton . In both yeast and nematodes , it appears that the establishment of correct spindle orientations involves a tethering of microtubules emanating from the centrosomes to specific sites on the actin cytoskeleton ( Hyman and White , 1987 ; Hyman , 1989 ; Palmer et al . , 1992 ; Waddle et al . , 1994 ) . Interestingly , in S . cerevisiae mutations in Arpl homologues ( Clark and Meyer , 1994 ; Muhua et al . , 1994 ) , dynein heavy chain ( Eshel et al . , 1993 ; Li et al . , 1993 ) , or actin , as well as mutations in 13 - tubulin that cause prefer - ential loss of astral microtubules ( Palmer et al . , 1992 ) , all result in similar misorientations of the mitotic spindle . Although our data clearly show that alterations in the actin cytoskeleton can interfere with centrosome migration and separation during male meiosis in Drosophila , further studies will be required to define the molecular mechanisms underlying these events . At present , we can envisage two possibilities . It is conceivable that centrosome movements normally depend upon the tethering of astral microtubules to cortical or perinuclear actin structures . If such actin struc - tures were incorrectly assembled in tsr mutants , they may fail to capture astral microtubules , leading to defects in cen - trosome migration and separation . Alternatively , the ab - normal concentration of actin around the centrosomes of primary spermatocytes in twinstar mutants may physically obstruct normal interactions required for these processes . We have been impressed by the apparent normality of The Journal of Cell Biology , Volume 131 , 1995 1256 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m spindle morphology and chromosome separation in mu - tant testes subsequent to the aberrant migration of asters during prophase / prometaphase of meiosis I . This argues that the meiotic spindle has considerable self - regulating properties , which may be related to the extended period of time ( between 20 and 45 min ) required for bivalents to achieve a stable bipolar configuration before anaphase on - set in these cells ( Church and Lin , 1985 ) . This might pro - vide sufficient time for the asters in twinstar mutants to mi - grate eventually to their proper position so that correct chromosome segregation can occur . Final aster positioning in the mutant is likely to be mediated , at least in part , by microtubule - kinetochore attachments that may provide positional signals to restore proper aster localization . We envision that lateral forces exerted between interdigitating microtubules emanating from the two poles also partici - pate in centrosome separation . Cytokinesis . Larvae carrying mutant tsr alleles clearly exhibit frequent failures of cytokinesis both in neuroblast cells and in male meiosis . At present we have no informa - tion regarding the primary cause of cytokinesis failure in neuroblasts . However , our observations on testes suggest two possible explanations for how a reduction in twinstar activity might affect meiotic cytokinesis . The first possibility is that the cytokinesis defect arises primarily from the abnormal migration and positioning of asters . It is clear that mechanisms must exist that transmit signals from the spindle asters to the contractile apparatus , explaining how difficulties in cytokinesis could result indi - rectly from problems in centrosome positioning within the cell . A number of experiments indicate that the spindle as - ters specify the position of the cleavage furrow midway be - tween them ( reviewed by Rappaport , 1986 ) , and some evi - dence suggests that the asters may be able to dictate this position as early as prophase ( Sluder and Miller , 1990 ) , when the irregularities in twinstar aster localization are manifested . We believe this possibility to be unlikely be - cause tsr mutants correctly localize actin structures to the midzone at the same time that normal contractile rings form . Nevertheless , it remains conceivable that a putative alteration of signals emanating from the asters could affect the responsiveness of the cortical cytoplasm without af - fecting positioning of the contractile ring . The more likely possibility is that cytokinesis is dis - rupted in tsr mutants because of abnormalities in the for - mation and / or disassembly of the contractile ring . Our observations clearly show that tsr mutants exhibit mor - phologically abnormal contractile rings that fail to disas - semble during cleavage . Most current models of cytokine - sis assert that the contractile ring is an overlapping array of actin filaments of opposite polarity attached to the plasma membrane . These actin filaments interact with bi - polar myosin - II filaments , leading to constriction of the cell periphery ( reviewed in Satterwhite and Pollard , 1992 ; Fishkind and Wang , 1995 ) . One could easily imagine sev - eral scenarios by which a cofilin - like protein such as twin - star , which can modulate actin dynamics in vitro by sever - ing and depolymerizing filaments ( S . K . Maciver , personal communication ) , might play a role in cytokinesis . Such an activity might function in the recruitment of cortical actin filaments to the contractile ring ( Cao and Wang , 1990 ) , turnover of actin within the contractile ring ( Inoue , 1990 ) , or disassembly of the contractile ring during furrowing and cleavage ( Maupin and Pollard , 1986 ) . In any event , there is substantial precedent to suggest that the twinstar protein could function in concert with other proteins to promote proper organization of contractile filaments . The cofilin homologue actophorin , in the presence of the actin cross - linking protein a - actinin , promotes formation of actin bundles in vitro ( Maciver , et al . , 1991 ) . In addition , the correct assembly of the contractile myofilament lattice in the body wall muscle of C . elegans requires an activity encoded by a cofilin - like gene : in unc - 60 mutants , thin fila - ments aggregate at the ends of the cell instead of interdigi - tating with thick filaments , which appear essentially normal ( Waterston et al . , 1980 ) . Most recently , vertebrate cofilin has been found to localize to the contractile ring in tissue culture ( Nagaoka et al . , 1995 ) . Additional Functions of the twinstar Protein We believe it unlikely that twinstar function is required only for cytokinesis or correct aster migrations in prophase . It is quite possible that the phenotypes we have examined represent only those events that are most susceptible to depletion of maternal twinstar protein stores . Neither the tsr l nor the tsr 2 allele appears to represent the null state of the twinstar locus : some mRNA is expressed in mutant an - imals and these mutations do not interfere with the open reading frame encoding the protein . Other functions of twinstar might therefore be uncovered by an analysis of phenotypes associated with either true null mutations , which can in theory be isolated by transposase - promoted imprecise excision of the tsr z or tsr 2 P elements ( Bellen et al . , 1989 ) , or conditional mutations . We expect to find that twinstar protein may also be required for muscle forma - tion , or for processes such as cellular motility or the deter - mination of cell shape in Drosophila . In this light , it is in - teresting to note that when a Drosophila cDNA clone essentially identical to our twinstar cDNA is overexpressed in S . pombe , changes in cell shape are observed in associa - tion with perturbations in actin distribution ( Edwards et al . , 1994 ) . Their results , in combination with the findings described in this paper , emphasize the importance to the cell of maintaining the proper stoichiometry of cofilin with respect to other cytoskeletal components . The authors wish to thank kindly the following : Drs . Trish Wilson and Minx Fuller for donating the tsr 2 allele , Drs . K . Edwards and D . Kiehart for sharing sequence information before publication and for helpful com - ments on the protein alignment , Dr . Bruce Reed for stocks and informa - tion on complementation groups , Dr . S . K . Maciver for early communica - tion of new biochemical data , Dr . J . Merriam for making a collection of P element - induced mutations available to us , Dr . H . Lin for helpful discus - sions about the fusome , E . Mirabile for participating in early stages of our screening procedure , and Janis Werner for microinjections . This work was supported by National Institutes of Health grant 5R01GM48430 to M . L . Goldberg , by National Institutes of Health Train - ing grant 5T32GM07617 to K . C . Gunsalus , and by grants to M . Gatti from Progetto Finalizzato Ingegneria Genetica and Fondazione Istituto Pas - teur - Cenci Bolognetti . Received for publication 17 May 1995 and in revised form 31 July 1995 . References Abe , H . , T . Endo , K . Yamamoto , and T . Obinata . 1990 . Sequence of cDNAs Gunsalus et al . Centrosome Migration and Cytokinesis Require tsr + 1257 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m encoding actin depolymerizing factor and cofiliu of embryonic chicken skel - etal muscle : two functionally distinct actin - regulatory proteins exhibit high structural homology . Biochemistry 29 : 7420 - 7425 . Bamburg , J . R . , and D . Bray . 1987 . Distribution and cellular localization of ac - tin depolymerizing factor . J . Cell BioL 105 : 2817 - 2825 . Bellen , H . J . , C . J . O ' Kane , C . Wilson , U . Grossniklaus , R . K . 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Centrosome Mieration and Cvtokinesis Reauire tsr + 1259 on N o v e m be r 19 , 2017 j c b . r up r e ss . o r g D o w n l oaded f r o m \ No newline at end of file diff --git a/silver_data/0eeb914ef3ebe3cfaf9dd853c605dab9c441f531.pdf.txt b/silver_data/0eeb914ef3ebe3cfaf9dd853c605dab9c441f531.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..df09ddcb9c4cd5a03075f7ce9c3dde33dbf1f121 --- /dev/null +++ b/silver_data/0eeb914ef3ebe3cfaf9dd853c605dab9c441f531.pdf.txt @@ -0,0 +1 @@ +To Scratch or not to Scratch ? A controlled experiment comparing plugged first and unplugged first programming lessons Felienne Hermans , Efthimia Aivaloglou Delft University of Technology f . f . j . hermans @ tudelft . nl , E . Aivaloglou @ tudelft . nl ABSTRACT Programming education is in fashion : there are many methods , tools , books and apps to teach children programming . This gives rise to the question of how to teach programming . Do we teach the concepts with or without the use of a computer , also called plugged and unplugged respectively ? This paper aims to measure what method is more effective to start with : plugged or unplugged first . Specifically , we are interested in examining which method is better in terms of ( 1 ) facilitating understanding of programming concepts , ( 2 ) motivating and supporting the students’ sense of self - efficacy in programming tasks , and ( 3 ) motivating the students to explore and use programming constructs in their assignments . To this end we conduct a controlled study with 35 elementary school children , in which half of the children receive four plugged lessons and the other half receives four unplugged lessons . After this , both groups receive four weeks of Scratch lessons . The results show that after eight weeks there was no difference between the two groups in their mastering of programming concepts . However , the group that started with unplugged lessons was more confident of their ability to understand the concepts , i . e . demonstrated better self - efficacy beliefs . Furthermore , the children in the unplugged first group used a wider selection of Scratch blocks . CCS CONCEPTS • Socialandprofessionaltopics → Computingeducation ; Com - putational thinking ; K - 12 education ; KEYWORDS programming education , Scratch , unplugged ACM Reference format : Felienne Hermans , Efthimia Aivaloglou . 2017 . To Scratch or not to Scratch ? . In Proceedings of WiPSCE ’17 , Nijmegen , Netherlands , November 8 – 10 , 2017 , 8 pages . DOI : 10 . 1145 / 3137065 . 3137072 Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed forprofitorcommercialadvantageandthatcopiesbearthisnoticeandthefullcitationonthefirstpage . Copyrights for components of this work owned by others than the author ( s ) must be honored . Abstracting with credit is permitted . To copy otherwise , or republish , topostonserversortoredistributetolists , requirespriorspecificpermission and / or a fee . Request permissions from permissions @ acm . org . WiPSCE ’17 , Nijmegen , Netherlands © 2017 Copyright held by the owner / author ( s ) . Publication rights licensed to ACM . 978 - 1 - 4503 - 5428 - 8 / 17 / 11 . . . $ 15 . 00 DOI : 10 . 1145 / 3137065 . 3137072 1 INTRODUCTION Over the last decade interest in programming education has been growing . An increasing number of countries is including program - ming and computational thinking ( CT ) in the curricula of elemen - tary schools . In the UK , children as young as 5 are already intro - duced to programming concepts [ 3 ] . The introduction of programming education naturally gives rise to the question how to best teach programming and CT to children . One of the topics of these discussions is the role of the computer : should we teach with or without the computer , or plugged versus unplugged . We do believe that ultimately children need to be able to apply programming concepts using a computer , so we are not interested in comparing unplugged entirely to plugged entirely . Hence , in this paper we focus on the question whether it is better to start with plugged lessons immediately , or is it better to first use unplugged materials . Specifically , we are interested in examining which method is better in terms of ( 1 ) facilitating understanding of programming concepts , ( 2 ) motivating and supporting the students’ sense of self - efficacy in programming tasks , and ( 3 ) motivating the students to explore and use programming constructs in their assignments . Our motivation for investigating the effects the teaching meth - ods to the student’s self - efficacy beliefs is that those have been found to affect certain career entry behaviors , such as college ma - jor choices and academic performance [ 12 , 19 ] . Self - efficacy was originally presented by A . Bandura as the belief that one can suc - cessfully execute behaviors required to produce a desired outcome [ 4 ] . In education research , self - efficacy has become one of the most important motivational variables that helps explain the relationship between past performance and future results [ 13 ] . This relation - ship had been found to be strong also in middle - school students [ 5 ] and for various subject areas , including mathematics [ 16 ] and programming [ 13 , 18 , 23 ] . To address our three research questions , we run a two phase ex - periment that compares starting with unplugged lessons to starting on the computer . We teach 35 elementary school children aged 8 to 12 , separated in two random groups , for eight weeks . In the first phase , consisting of four weeks , we teach half of the children ( 17 ) with Scratch , while the other half ( 18 ) used unplugged materials only . Both the plugged and the unplugged lessons covered these concepts : loops , conditionals , procedures , broadcasts , paralleliza - tion and variables . After these four weeks , both groups receive two weeks of Scratch lessons , to practice Scratch programming in more depth . In these lessons , we repeat the concepts taught in phase one . For the unplugged group , we design one special lesson that connects the concepts as they used them unplugged to con - cepts in Scratch . After these two weeks , two more weeks follow 49 WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands Felienne Hermans , Efthimia Aivaloglou in which children create their own games in Scratch . We close the experiment with an endterm test in which we assess children’s understanding and correct use of programming concepts in Scratch . The results show that after eight weeks ( 1 ) there was no differ - ence between the two groups in their mastering of programming concepts , ( 2 ) the unplugged first group demonstrated more self - efficacy , and ( 3 ) that they use a wider vocabulary of Scratch blocks , including more blocks that were not explained in the course mate - rials . 2 EXPERIMENTAL SETUP 2 . 1 Research Questions The goal of this study is to understand whether there is a differ - ence between starting with Scratch immediately ( Plugged first ) or practicing programming concepts without a computer before ( Un - plugged first ) . We therefore answer the following three research questions : RQ1 Do the children that start with unplugged materials understand programming concepts better ? RQ2 Do the children that start with unplugged materials have more self - efficacy about programming ? RQ3 Do the children that start with unplugged materials use a smaller vocabulary of Scratch blocks ? Associated with these research questions are three null hypothe - ses , which we formulate as follows : H 1 0 Learning about programming concepts unplugged first does not impact children’s understanding of programming concepts . H 2 0 Learning about programming concepts unplugged first does not impact children’s self - efficacy about program - ming . H 3 0 Learning about programming concepts unplugged first does not impact children’s use of different Scratch blocks . The alternative hypotheses that we use in the experiment are the following : H 1 1 Learning about programming concepts unplugged first increases children’s understanding of programming con - cepts . H 2 1 Learning about programming concepts unplugged first increases children’s self - efficacy about programming . H 3 1 Learning about programming concepts unplugged first impacts children’s use of different Scratch blocks . To test the null hypotheses , we perform a controlled experiment with 35 elementary school children , randomly assigned to one of two groups : starting with Scratch programming immediately or starting with four weeks of unplugged material . 2 . 2 Subjects The subjects in our experiment are children from three grades of one school , varying in age between 8 and 12 , with an average of 10 . 0 . Figure 1 shows an overview of the ages of the 35 children in the experiment . We randomly divided the 35 children into two groups , however we balanced the genders and the grades as much as possible given the constraints we were given . 1 Figure 2 shows the division of the children and their grades over the two groups , and Figure 3 shows their gender . Figure 1 : Ages of the children Figure 2 : Grades of the children in the two groups ( 8 being the final grade of elementary school ) Figure 3 : Genders of the children in the two groups 2 . 3 Programming Concepts In the materials for both the plugged first and the unplugged first group , we teach six basic programming concepts . • Loops • Conditionals • Procedures 1 among the children were two pairs of siblings who could not be in the same group since they would work on the same laptop 50 To Scratch or not to Scratch ? WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands Table 1 : Overview of the materials of the eight week programming course Week Unplugged group Plugged group Concepts 0 Getting to know Scratch , create user accounts Getting to know Scratch , create user accounts — 1 CS Unplugged Marching Orders Scratch MOOC lesson 1 Loops and Conditionals 2 Self - Efficacy Questionnaire Self - Efficacy Questionnaire — 2 CS Unplugged Network Protocols Scratch MOOC lesson 2 Broadcasts and Procedures 3 Computing Without Computers Box Variables Scratch MOOC lesson 3 Variables and Parallelization 4 Finish materials Finish materials All 5 Intro Scratch More Scratch practice Loops , Conditions and Procedures 6 Scratch Practice Scratch Practice Broadcasts , Variables and Parallelization 7 Final Project Final Project — 8 Final Project Final Project — 8 End Term Scratch Test End Term Scratch Test — 8 Self - Efficacy Questionnaire Self - Efficacy Questionnaire — • Broadcasts • Parallelization • Variables These concepts are divided over three chapters that teach two concepts each , both for the plugged first and for the unplugged first group . 2 . 4 Lesson plan Table 1 shows an overview of our lesson plan . We start with one introductory computer lesson for all children , in which we set up wifi on their computers and create a Scratch account for all children . In addition to that , we show them a demo of how Scratch works , en - suring that also the children that will do the unplugged lessons first have context of what programming is . After that initial lesson , we divide the children into two random groups which receive different treatment . Half of the children use Scratch to learn about the six programming concepts , while the other group learns programming ‘unplugged’ . The following describes the eight weeks of lessons in more detail . 2 . 4 . 1 Week 0 . In week 0 , children set up a Scratch account , and we also show them the basic idea of Scratch : that you can control a sprite on the screen with blocks . 2 . 4 . 2 Weeks 1 to 4 . After week 1 , the groups are split and work on either plugged or unplugged materials . Initially we envisioned that the children would finish the material we provided in three weeks , but we gave them an additional week to finish all lessons , and also to provide an opportunity to children that missed a lesson due to sickness to catch up . Unplugged first For the four unplugged lessons , we use ma - terial based on CS Unplugged . 2 CS Unplugged is “a collec - tion of free learning activities that teach Computer Science through engaging games and puzzles that use cards , string , crayons and lots of running around” . Previous research has shown that CS Unplugged is an effective way of teaching programming concepts [ 21 ] . All our adapted materials are available online . 3 2 http : / / csunplugged . org / 3 link removed for double blind submission Plugged first For the four plugged lessons , we use materials developed for the first author’s online Scratch course 4 , which has been used previously by over 6000 children to date . These materials too are available . 5 2 . 4 . 3 Weeks 5 and 6 . From week 5 onwards , children in both groups use Scratch to create programs and to practice the learned concepts . The plugged first group receives materials in which the concepts that were previously taught are repeated , and practiced more . For the unplugged first group , we design a special introductory lesson , in which we explain how the programming concepts they practiced with in the unplugged lessons manifest in Scratch . An example of the connection of unplugged to Scratch concepts is shown in Figure 4 . 2 . 4 . 4 Week 7 and 8 . In weeks 7 and 8 , we give children the opportunity to create their own programs , in order to measure children’s Scratch vocabulary , and see what blocks and concepts they use when creating programs . 2 . 4 . 5 Week 8 . In week 8 , we close the course with an endterm test , in which we measure understanding of programming concepts , in Scratch , but performed on paper . The endterm consisted of mul - tiple choice questions , some testing one and some testing multiple concepts . The endterm too is available . 6 Figure 5 shows questions testing the concepts operators and variables . 2 . 5 Self - efficacy assessment To measure the students’ self - efficacy beliefs we used the self - efficacy subscale of the Motivated Strategies for Learning Ques - tionnaire ( MSLQ ) [ 17 ] . MSLQ consists of fifteen subscales designed from classic social - cognitive learning theories and is widely used as a self - report instrument for measuring student motivation and learning strategies and for subsequently predicting academic per - formance [ 6 ] . The self - efficacy scale comprises of eight statements which assess both expectancy for success in the course and self - efficacy . They include judgments about the student’s abilities to 4 link removed for double blind submission 5 link removed for double blind submission 6 Link removed for double blind 51 WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands Felienne Hermans , Efthimia Aivaloglou Figure 4 : Explanation of unplugged concepts into Scratch Figure 5 : Questions in the endterm testing the concepts op - erators and variables accomplish tasks , as well as student’s confidence in their skills to perform those tasks . Using questionnaires at the beginning ( Week 2 ) and the end ( Week 8 ) of the course , the students rated themselves on a seven point likert - scale from ‘not at all true of me’ to ‘very true of me’ . As specified in the MSLQ , students’ self - efficacy scores were computed Figure 6 : Scores per group on the endterm exercises , out of 8 possible points . Center lines indicate the medians ; boxes indicate the 25th and 75th percentiles ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th per - centiles . n = 17 , 18 sample points . T - test shows that groups do not differ significantly . by taking the average of the points given to the eight statements of each questionnaire . 3 RESULTS 3 . 1 Mastering of Programming Concepts Figure 6 shows the results of both groups on the endterm , after eight weeks of lessons . A Shapiro - Wilk test showed that both the plugged first and the unplugged first sample follow the normal distribution ( p = 0 . 125 ) with means of 4 . 29 and 3 . 73 respectively , and standard deviations of 0 . 87 and 0 . 80 . Furthermore the two groups have equal variance as demonstrated by Levene’s test . We therefore use a t - test to de - termine whether there is a difference between the samples . The test resulted in a p - value of 0 . 311 , meaning we cannot reject H 1 0 . In other words children in both groups perform similar , and there is no effect measured of the plugged first versus unplugged first treatment on their understanding of programming concepts . In addition to the total score on all questions , we also separated the results for the six programming concepts . On the individual concepts too we measured no significant differences . After eight weeks , we measure no difference in understanding of programming concepts between the plugged first and unplugged first group . 3 . 2 Self - Efficacy Figure 7 shows the aggregated self - efficacy scores of the children after eight weeks , calculated as the averages of the seven - point likert - scale replies given to the 8 self - efficacy MSLQ scale state - ments as described in Section 2 . 5 . A Shapiro - Wilk test ( p = 0 . 327 ) showed that both the plugged and the unplugged first sample follow the normal distribution with means of respectively 5 . 05 and 5 . 75 and standard deviations of 0 . 54 and 0 . 32 . Furthermore the two groups have equal variance as 52 To Scratch or not to Scratch ? WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands Figure 7 : Self - efficacy scores at the end of the course per group . Scores are calculated as the averages of the seven - point likert - scale replies given to the 8 self - efficacy MSLQ scale statements . Center lines indicate the medians ; boxes indicate the 25th and 75th percentiles ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th per - centiles . n = 18 , 17 sample points . T - test shows that groups differ significantly . Figure 8 : Self - efficacy scores at the beginning of the course per group . Scores are calculated as the averages of the seven - point likert - scale replies given to the 8 self - efficacy MSLQ scale statements . Center lines indicate the medians ; boxes indicate the 25th and 75th percentiles ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th per - centiles . n = 18 , 17 sample points . T - test shows that groups do not differ significantly . demonstrated by Levene’s test . We therefore use a t - test to deter - mine whether there is a difference between the samples . The test resulted in a p - value of 0 . 023 , meaning we must reject H 1 0 . In other words children in the unplugged first group have a significantly better sense of self - efficacy in programming . At the beginning of the course we also measured the self - efficacy , and then we measured no difference , as shown in Figure 8 . After 8 weeks , children in the unplugged first group measure significantly better in self - efficacy beliefs . Figure 9 : Distinct number of Scratch blocks per group in the end projects children built in weeks 7 and 8 . Center lines indicate the medians ; boxes indicate the 25th and 75th per - centiles ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . n = 17 , 18 sample points . T - test shows that groups differ significantly . 3 . 3 Block Vocabulary Figure 9 shows the number of distinct Scratch blocks appearing in the end projects that children worked on in weeks 7 and 8 . A Shapiro - Wilk test showed that both the plugged and the unplugged sample follow the normal distribution ( p = 0 . 087 ) with means of 9 . 47 and 14 . 22 respectively , and standard deviations of 2 . 55 and 4 . 08 . Furthermore the two groups have equal variance as demonstrated by Levene’s test . We therefore use a t - test to deter - mine whether there is a difference between the samples . The test resulted in a p - value of 0 . 047 , meaning we must reject H 3 0 . In other words children in the unplugged first group use different Scratch blocks . Specifically , they use more different blocks than the Scratch first group . Figure 10 shows the division over the categories of blocks that Scratch defines . Children in the unplugged first group use more different Motion blocks , which move the sprites over the 2d plane . They also use more Control blocks which include loops and condi - tionals , and more Looks blocks . In the projects created in weeks 7 and 8 , children in the unplugged first group use more different Scratch blocks than children in the plugged first group . 4 DISCUSSION Intheabove , wehavedescribedanexperimentinwhichwecompare programming competency , self efficacy and vocabulary of children that started with four weeks on Scratch lessons , versus four weeks of unplugged lessons . In this section , we discuss the results . 4 . 1 Classroom atmosphere A difference between the two classrooms that goes beyond mea - suring performance and efficacy is the ‘vibe’ of the classrooms , which we attribute to the presence of computers , since all other factors where equal . The groups had the same teachers and used the same physical classroom . We observed a huge difference between the plugged first group where computers where present . In this 53 WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands Felienne Hermans , Efthimia Aivaloglou Figure 10 : Distinct number of Scratch blocks per group , in 8 of the 10 different categories of blocks that Scratch defines . group , the children were more excited by the presence of comput - ers , which are not commonly used in class . This lead to increased excitement , but also meant that children were often distracted by the the games they created , playing them rather than programming . In the unplugged first group the vibe was more calm and classroom like , which could explain why children got more confidence in their mastering of concepts . This might have resulted in the fact that children in the unplugged first group could easier reach a state of flow , where a person is performing a task that is not too easy or too hard , and is completely engaged in the task [ 7 ] . Kinnunen and Simon [ 10 ] describe a similar phenomenon . They explored how students experience programming assignments in CS1 courses and described the true novice experience of encountering a difficulty as similar to being hit by lightning : an unexpected , shocking ex - perience which leaves one dazed and confused , largely affecting the student’s self - efficacy beliefs . Similar observations on the re - ciprocal relationship between performance and self - efficacy beliefs were made in [ 13 ] . Maybe the unplugged first group had a more “natural” , “paved way” to learning programing , avoiding feeling confused , encountering obstacles or dealing with the increased cog - nitive load that the Scratch web interface imposes in comparison to unplugged learning . 4 . 2 Programming and Gender Regarding the effect of gender on programming performance and orientation , a number of studies have found it to be significant . For example , strong social support and high self - efficacy have been found to be associated with strong orientation toward CS careers [ 19 ] , while Lishinski et al . found that female students adjust their self - efficacy beliefs earlier in courses , which suggests that responses to early failures could be causing them to disengage from CS [ 13 ] . In week 2 of our study , we measured no difference in self - efficacy between boys and girls . In week 8 , girls have lost more self - efficacy than boys , however this difference is not significant . We further - more find no significant differences in the performance between students of different genders . Figure 11 : Distinct number of Scratch blocks per group cov - ered and not covered by the coursework . 4 . 3 Self - efficacy and Vocabulary It is interesting that we measure an increase both in the self - efficacy of children in the unplugged first group and in the diversity of the blocks they use . We hypothesize that these two observations might be related . We think that because children have increased confidence , they feel more confident exploring different Scratch blocks . This is illustrated by Figure 11 , which shows the use of blocks by children in the two groups , divided into the blocks that we did and did not cover in the coursework , again divided over the categories of Scratch blocks . As you can see , the unplugged group uses more blocks that are not covered in the lessons , potentially indicating that they feel comfortable exploring those . 4 . 4 Threats to Validity A threat to the external validity of our evaluation concerns the representativeness of children in the experiment . To mitigate this effect we have used three different classes and divided them in two random groups , however all children are pupils of one school , which means they might not be representative of all children or even all children in this country . We plan to repeat this study with a larger group of children later this year . With respect to internal validity , one of the threats is the quality of the materials . It could be that the unplugged lessons are easier than the plugged materials , resulting in higher self - efficacy . How - ever , since the children in the unplugged first group demonstrate the same understanding of concepts , this seems not to be the case . 5 RELATED WORK There is a limited number of studies that have tried to evaluate teaching programming concepts unplugged . Thies and Vahrenhold [ 21 ] researched the CS unplugged ma - terials specifically as a method of teaching . They taught a group of 25 children aged 11 and 12 in a classroom setting , and used the CS Unplugged activities to teach half the students , and used alternative methods for the other half of the students , including traditional computer science textbooks , among which Algorithms Unplugged [ 22 ] . They find that CS unplugged is as effective as the 54 To Scratch or not to Scratch ? WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands alternative methods , since there was no significant difference in achievement between the group who learned with CS Unplugged activities and the group who learned with alternative materials . However , they do not measure subsequent behavior in a plugged programming environment like we do . A number of studies have been carried out on teaching program - ming concepts to novice programmers with block - based languages in general , and Scratch in particular . Scratch was taught in mid - dle school classes containing a total of 46 students in the study presented in [ 15 ] . Evaluating the internalization of programming concepts , it was found that students had problems with concepts related to initialization , variables and concurrency . Maloney et al . [ 14 ] taught Scratch as an extracurricular activity , in an after - school clubhouse . By analyzing the 536 students’ projects for blocks that relate to programming concepts , they found that within the least used ones are boolean operators and variables . Using the performance results from an online introductory Scratch program - ming course that was run recently , Hermans and Aivaloglou found that students over 12 years of age perform significantly better in questions related to operators and procedures [ 9 ] . Apart from projects created during courses , other works analyze the public repository of Scratch programs for indications of learn - ing of programming concepts . Yang et al . examined the learning patterns of programmers in terms of block use over their first 50 projects [ 24 ] . In [ 8 ] , the use of programming concepts was exam - ined in relation to the level of participation , the gender , and the account age of 5 thousand Scratch programmers . [ 1 ] analyzed 250 thousand Scratch projects in terms of complexity , programming concepts and code smells , bad programming practices . Seiter and Foreman [ 20 ] proposed a model for assessing computational think - ingin primaryschool studentsandappliediton 150Scratchprojects , finding that design patterns requiring understanding of paralleliza - tion , conditionals and , especially , variables were under - represented until a certain age . The relationship between performance and self - efficacy in the computing education domain is widely studied . Lishinski et al . re - cently examined the interaction of self - efficacy , intrinsic and extrin - sic goal orientation , and metacognitive strategies and their impact on students performance in a CS1 course , and found that females’ self - efficacy had a different connection to programming perfor - mance than that of their male peers [ 13 ] . In [ 18 ] it is found that , in the context of a CS1 course , self - efficacy is influenced by previous programming experience and increases as a student progresses through an introductory programming course , while self - efficacy affects course performance . The MSLQ was used in a study with 39 university students in their introductory programming course , where it was found that , of all motivational and learning strategies scales on the MSLQ , self - efficacy had the strongest correlation with the students’ course performance [ 23 ] . Examining the relationships between MSLQ scores and academic performance , [ 6 ] found that the self - efficacy scores had within the highest observed validities for grades in individual classes , along with the scores for effort regulation and time and study environment . The effects of using Scratch in the self - efficacy beliefs of stu - dents is also studied by Armoni et al . , who found that learning programming through Scratch in middle school greatly facilitated learning professional textual programming languages ( C # or Java ) in secondary school , while students with Scratch experience were observed to display higher levels of motivation and self - efficacy [ 2 ] . On university - level students , however , [ 11 ] examined the effect of introducing Scratch - based game activities at the introductory programming course and found that they contributed significantly to their C + + programming performance , but had no effect on their sense of self - efficacy towards the C + + programming language . 6 CONCLUDING REMARKS The goal of this paper is to explore the impact of starting with unplugged lessons before programming in Scratch or another pro - gramming system on the computer . To that end we have designed and executed a controlled experiment in which we randomly di - vided 35 children aged 8 to 12 into two groups . One group was taught with Scratch for the first four weeks ( plugged first ) while the other did exercises on paper for four weeks ( unplugged first ) . After these lessons , both groups used Scratch for four weeks , two weeks of repeated concepts lessons and two weeks of free program creation . We find that after these eight weeks , there is no difference in performance on the understanding of programming concepts . How - ever , the unplugged first group shows more self - efficacy and uses a wider vocabulary of Scratch blocks , including more blocks not covered by the lessons . The contributions of this paper are as follows : • Lessons teaching loops , conditionals , procedures , broad - casts , parallelization and variables in a plugged and un - plugged fashion . ( Section 2 . 4 ) • Acontrolledexperimentcomparingtheuseofthesepluggedandunpluggedlessons ( Section 2 , Section 3 ) Our current research gives rise to several directions for future work . Firstly , we want to replicate these findings on a larger scale . Furthermore , we are interested in understanding more deeply why children in the unplugged first group show improved self - efficacy beliefs . An interesting way of studying this would be to use Scratch printouts and lessons in the unplugged first group . That way we could study if it is indeed the atmosphere in the classroom without computers that influences children’s self - efficacy or whether it is due to the unplugged teaching methods . 55 WiPSCE ’17 , November 8 – 10 , 2017 , Nijmegen , Netherlands Felienne Hermans , Efthimia Aivaloglou REFERENCES [ 1 ] Efthimia Aivaloglou and Felienne Hermans . 2016 . How Kids Code and How We Know : An Exploratory Study on the Scratch Repository . 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ACM , New York , NY , USA , 211 – 220 . DOI : https : / / doi . org / 10 . 1145 / 2960310 . 2960329 [ 14 ] John H . Maloney , Kylie Peppler , Yasmin Kafai , Mitchel Resnick , and Natalie Rusk . 2008 . Programming by Choice : Urban Youth Learning Programming with Scratch . In Proceedings of the 39th SIGCSE Technical Symposium on Computer Science Education ( SIGCSE ’08 ) . ACM , 367 – 371 . DOI : https : / / doi . org / 10 . 1145 / 1352135 . 1352260 [ 15 ] Orni Meerbaum - Salant , Michal Armoni , and Mordechai ( Moti ) Ben - Ari . 2010 . Learning Computer Science Concepts with Scratch . In Proceedings of the Sixth International Workshop on Computing Education Research ( ICER ’10 ) . ACM , New York , NY , USA , 69 – 76 . DOI : https : / / doi . org / 10 . 1145 / 1839594 . 1839607 [ 16 ] F . Pajares and M . D . Miller . 1994 . Role of self - efficacy and self - concept beliefs in mathematicalproblemsolving : Apathanalysis . Journalofeducationalpsychology 2 , 86 ( 1994 ) , 193 . [ 17 ] R . Paul , S . Smith , M . L . Genthon , G . G . Martens , C . L . Hauen , G . G . Martens , M . Genthon , and P . Wren . 1991 . Technical Report No . 91 - B - 004 : A Manual for the Use of the Motivated Strategies for Learning Questionnaire ( MSLQ ) . Technical Report . The Regents of The University of Michigan . [ 18 ] Vennila Ramalingam , Deborah LaBelle , and Susan Wiedenbeck . 2004 . Self - efficacy and Mental Models in Learning to Program . In Proceedings of the 9th Annual SIGCSE Conference on Innovation and Technology in Computer Science Education ( ITiCSE ’04 ) . ACM , New York , NY , USA , 171 – 175 . DOI : https : / / doi . org / 10 . 1145 / 1007996 . 1008042 [ 19 ] Mary Beth Rosson , John M . Carroll , and Hansa Sinha . 2011 . Orientation of Undergraduates Toward Careers in the Computer and Information Sciences : Gender , Self - Efficacy and Social Support . Trans . Comput . Educ . 11 , 3 , Article 14 ( Oct . 2011 ) , 23 pages . 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No Tests Required : Comparing Traditional and Dynamic Predictors of Programming Success . In Proceedingsofthe45thACMTechnicalSymposiumonComputerScience Education ( SIGCSE ’14 ) . ACM , New York , NY , USA , 469 – 474 . DOI : https : / / doi . org / 10 . 1145 / 2538862 . 2538930 [ 24 ] Seungwon Yang , Carlotta Domeniconi , Matt Revelle , Mack Sweeney , Ben U . Gelman , Chris Beckley , and Aditya Johri . 2015 . Uncovering Trajectories of Informal Learning in Large Online Communities of Creators . In Proceedings of the Second ACM Conference on Learning @ Scale . ACM , 131 – 140 . DOI : https : / / doi . org / 10 . 1145 / 2724660 . 2724674 56 \ No newline at end of file diff --git a/silver_data/1173560e19bd2ab6e1a0f7b0edfdbf26e9b85cdd.pdf.txt b/silver_data/1173560e19bd2ab6e1a0f7b0edfdbf26e9b85cdd.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..7a80287bebad1bda626b8b6fa71a5bf285e2a6d4 --- /dev/null +++ b/silver_data/1173560e19bd2ab6e1a0f7b0edfdbf26e9b85cdd.pdf.txt @@ -0,0 +1 @@ +Neural and genetic determinants of creativity Zhaowen Liu a , 1 , Jie Zhang b , * * , 1 , Xiaohua Xie b , c , 1 , Edmund T . Rolls c , d , 1 , Jiangzhou Sun e , f , 1 , Kai Zhang g , 1 , Zeyu Jiao b , i , Qunlin Chen e , f , Junying Zhang a , * * * * , Jiang Qiu e , f , j , * * * , Jianfeng Feng b , c , h , i , k , * a School of Computer Science and Technology , Xidian University , Xi ' an 710071 , Shannxi , PR China b Institute of Science and Technology for Brain Inspired Intelligence , Fudan University , Shanghai , PR China c Department of Computer Science , University of Warwick , Coventry CV4 7AL , UK d Oxford Centre for Computational Neuroscience , Oxford UK e Key Laboratory of Cognition and Personality ( SWU ) , Ministry of Education , Chongqing , PR China f School of Psychology , Southwest University ( SWU ) , Chongqing , PR China g Department of Computer and Information Sciences , Temple University , 1801 North Broad Street , Philadelphia , PA 19122 , USA h Collaborative Innovation Center for Brain Science , Fudan University , Shanghai , 200433 , PR China i Shanghai Center for Mathematical Sciences , Shanghai , 200433 , PR China j Southwest University Branch , Collaborative Innovation Center of Assessment Toward Basic Education Quality , Beijing Normal University , Beijing 100875 , PR China k Zhongshan Hospital , Fudan University , Shanghai , 200433 , PR China A B S T R A C T Creative thinking plays a vital role in almost all aspects of human life . However , little is known about the neural and genetic mechanisms underlying creative thinking . Based on a cross - validation based predictive framework , we searched from the whole - brain connectome ( 34 , 716 functional connectivities ) and whole genome data ( 309 , 996 SNPs ) in two datasets ( all collected by Southwest University , Chongqing ) consisting of altogether 236 subjects , for a better understanding of the brain and genetic underpinning of creativity . Using the Torrance Tests of Creative Thinking score , we found that high fi gural creativity is mainly related to high functional connectivity between the executive control , attention , and memory retrieval networks ( strong top - down effects ) ; and to low functional connectivity between the default mode network , the ventral attention network , and the subcortical and primary sensory networks ( weak bottom - up processing ) in the fi rst dataset ( consisting of 138 subjects ) . High creativity also correlates signi fi cantly with mutations of genes coding for both excitatory and inhibitory neurotransmitters . Combining the brain connectome and the genomic data we can predict individuals ' creativity scores with an accuracy of 78 . 4 % , which is signi fi cantly better than prediction using single modality data ( gene or functional connectivity ) , indicating the importance of combining multi - modality data . Our neuroimaging prediction model built upon the fi rst dataset was cross - validated by a completely new dataset of 98 subjects ( r ¼ 0 . 267 , p ¼ 0 . 0078 ) with an accuracy of 64 . 6 % . In addition , the creativity – related functional connectivity network we identi fi ed in the fi rst dataset was still signi fi cantly correlated with the creativity score in the new dataset ( p < 10 (cid:2) 3 ) . In summary , our research demonstrates that strong top - down control versus weak bottom - up processes underlie creativity , which is modulated by competition between the glutamate and GABA neurotransmitter systems . Our work provides the fi rst insights into both the neural and the genetic bases of creativity . Introduction Creativity in humans is a complex cognitive behavior commonly de fi ned as the ability to generate responses that are novel , useful , and appropriate , ( Sternberg and Lubart , 1996 ) . Creative thinking plays an important role in almost all aspects of our life , most prominently in the arts , science , and engineering . Divergent thinking tests are so far the major type of psychometric instrument in creativity testing ( Gardner , 1988 ; Plucker and Runco , 1998 ) , including various verbal and fi gural tasks . Based on the divergent thinking tests , mounting evidence suggests that the default mode network and brain regions associated with cognitive control may be important for creativity . For example , regions of the default mode network , including the precuneus ( Fink et al . , 2014a , 2014b ; Jauk et al . , 2015 ; Jung et al . , 2010 ; Takeuchi et al . , 2010 ) and inferior parietal lobule ( Takeuchi et al . , 2011 ) , and executive control network ( ECN ) ( Ellamil * Corresponding author . Department of Computer Science , University of Warwick , Coventry CV4 7AL , UK . * * Corresponding author . Institute of Science and Technology for Brain Inspired Intelligence , Fudan University , Shanghai , PR China . * * * Corresponding author . Key Laboratory of Cognition and Personality ( SWU ) , Ministry of Education , Chongqing , PR China . * * * * Corresponding author . School of Computer Science and Technology , Xidian University , Xi ’ an 710071 , Shannxi , PR China . E - mail addresses : jzhang080 @ gmail . com ( J . Zhang ) , jyzhang @ mail . xidian . edu . cn ( J . Zhang ) , qiuj318 @ swu . edu . cn ( J . Qiu ) , jianfeng64 @ gmail . com ( J . Feng ) . 1 These authors contributed equally to the work . Contents lists available at ScienceDirect NeuroImage journal homepage : www . elsevier . com / locate / neuroimage https : / / doi . org / 10 . 1016 / j . neuroimage . 2018 . 02 . 067 Received 27 November 2017 ; Received in revised form 27 February 2018 ; Accepted 28 February 2018 Available online 5 March 2018 1053 - 8119 / © 2018 Elsevier Inc . All rights reserved . NeuroImage 174 ( 2018 ) 164 – 176 et al . , 2012 ; Gonen - Yaacovi et al . , 2013 ) have been implicated in neu - roimaging studies of divergent thinking tasks . The activation of the default mode network ( DMN ) in creative processes may re fl ect the spontaneous generation of candidate ideas , and / or the retrieval of long - term memory ( Beaty et al . , 2016 ) , while the control network may serve to constrain cognition to meet speci fi c goals of the tasks . As a highly complex cognitive process , creativity relies on not only the activity of separate brain regions but also the interactions between different brain regions / networks ( Bullmore and Sporns , 2009 ; Fox et al . , 2005 ; van den Heuvel and Hulshoff Pol , 2010 ) . Functional connectivity ( FC ) ( the correlation between the BOLD signal of different brain regions ) depicts the interaction between different brain regions , which is infor - mative for complex behaviors such as creativity and cognition . For instance , Beaty et al . ( 2014 ) found greater connectivity between the entire DMN and the left inferior frontal gyrus ( IFG ) within the ECN in highly creative individuals . In addition , a recent study found that the strength of connectivity between the DMN and the frontoparietal network ( FPN ) was positively related to both visual and verbal creativity using independent component analysis ( Zhu et al . , 2017 ) . Such fi ndings suggest that creative thought may bene fi t from the cooperation of the DMN and ECN . The genetic basis of divergent thinking has been previously explored mainly by using candidate gene approaches . Several brain regions in the dopaminergic ( DA ) system have been found to be involved in creativity cognition ( Flaherty , 2005 ; Heilman et al . , 2003 ; Takeuchi et al . , 2010 ) . Thus , many genes in the dopamine pathway , like D2 Dopamine Receptor ( DRD2 ) ( de Manzano et al . , 2010 ; Reuter et al . , 2006 ) , D4 Dopamine Receptor ( DRD4 ) ( Mayseless et al . , 2013 ) , Tryptophan Hydroxylase ( TPH1 ) ( Reuter et al . , 2006 ) , Dopamine Transporter 1 ( DAT1 ) , and Catechol - O - Methyltransferase ( COMT ) ( Zabelina et al . , 2016 ) , were selected as candidate genes and were found to be associated with divergent thinking . In addition , as several psychiatric illnesses are found to have genetic links to creativity ( Kyaga et al . , 2011 ; Power et al . , 2015 ) , some genes involved in the pathology of psychosis , such as neuregulin 1 ( NRG1 ) , were selected and were also found to be associated with crea - tivity ( Keri , 2009 ) . Although previous studies have explored the relationship between brain networks and creativity , most studies have focused on the regions previously implicated in creativity ( ROI - based ) , or used correlation analysis , i . e . , correlation between neuroimaging metrics and behavioral scores , to search for the neural basis of creativity ( correlation - based ) . One the one hand , the ROI - based analysis may limit the potential search to only a small part of the whole brain , thus preclude the possibility of new fi ndings ( Xia and He , 2017 ) . On the other hand , correlation - based approaches have the disadvantage of over fi tting the data and often fail to generalize to novel data ( Shen et al . , 2017 ) . In addition , although several studies have explored creativity - related candidate genes , a search for the genetic basis of creativity at a genome - wide scale , especially for divergent thinking , has not been described as far as we know . The correlation - based methods have also been widely employed in the ge - netic analysis of divergent thinking which have the disadvantage of low generalization ability . Based on all the above , in the present study we used a large sample of 236 individuals ( which we separated into two non - overlapping datasets ) in this research , which enabled statistical analyses of whole - brain FCs ( 34 , 716 FCs ) and whole - genome genes ( 309 , 996 SNPs ) that were related to creativity without a priori hypotheses , therefore not creating con - straints on the brain regions and genes to be investigated . Furthermore , we used a cross - validation based approach to identify the functional brain networks that are predictive of creativity , to ensure that the results were statistically fi rm and could be generalized to new populations . Our approach , IMuDP ( Integrating Multi - scale Data for Prediction ) , is improved from one that has been used to predict sustained attention in resting - state fMRI ( Rosenberg et al . , 2016 ) , which can now deal with multi - modal data . The prediction - based approach is held to provide fMRI - derived statistics that relate to individual behaviors with more generalizability than traditional correlation - based analysis ( Dubois and Adolphs , 2016 ) . Furthermore , to understand the genetic underpinning of creative thinking , we also searched from the whole genome the SNPs ( single nucleotide polymorphisms ) whose mutations correlate most closely with the creativity score using the same prediction - based strat - egy . Part of the aim here was to investigate how multi - modal data ( in this case fMRI and genetic data ) can be combined , and whether this helps better predictions to be made . This research provides the fi rst whole - brain functional network analysis for creativity with a corre - sponding whole - genome search for related SNPs , and is aimed to shed light on both the neural and genetic underpinnings of creative thinking . Materials and methods Data Ethics statement Both the behavioral and MRI protocols were approved by the local ethics committee of Southwest China University , Chongqing . Written informed consent was obtained from all participants prior to the study , which was approved by the Institutional Human Participants Review Board of Southwest University Imaging Center for Brain Research . The methods were conducted in accordance with approved guidelines . Participants Three hundred and fi fteen subjects were recruited in this study ( Li et al . , 2016 ) . We fi rstly excluded 63 subjects without complete behav - ioral or other demographic data . After further fMRI head movement control , 236 subjects were retained for further analysis . The retained subjects were then divided into two datasets . The fi rst dataset consisted of 138 individuals , 49 males and 89 females ( age 19 . 7 (cid:3) 1 . 2 ( mean (cid:3) sd ) ) , with both genetic and neuroimaging data , which we used to perform our multimodal prediction analysis . The second dataset consisted of 98 in - dividuals , 38 males and 60 females ( age 20 . 2 (cid:3) 1 . 3 ( mean (cid:3) sd ) ) , with only neuroimaging data , which we used as an external validation dataset to validate our prediction model generated from the fi rst dataset . All the resting - state fMRI data were collected in the Southwest Uni - versity Center for Brain Imaging , Chongqing , China , using a 3 . 0 - T Siemens Trio MRI scanner ( Siemens Medical , Erlangen , Germany ) . Each subject was required not to drink alcohol the day before the experiments , which was then con fi rmed right before the scanning by questionnaires . Resting - state fMRI In resting - state fMRI scanning , the subjects were instructed to rest without thinking about a particular topic , and not to fall asleep or close their eyes . The 8 - min scan of 242 contiguous whole - brain resting - state functional images was obtained using gradient - echo planar imaging ( EPI ) sequences with the following parameters : slices ¼ 32 , repetition time ( TR ) / echo time ( TE ) ¼ 2000 / 30 ms , fl ip angle ¼ 90 , fi eld of view ( FOV ) ¼ 220 mm (cid:4) 220 mm , and thickness / slice gap ¼ 3 / 1 mm , voxel size 3 . 4 (cid:4) 3 . 4 (cid:4) 3 mm 3 . Structural MRI A magnetization - prepared rapid gradient echo ( MPRAGE ) sequence was used to acquire high - resolution T1 - weighted anatomical images ( repetition time ¼ 1900 ms , echo time ¼ 2 . 52 ms , inversion time ¼ 900 ms , fl ip angle ¼ 90 (cid:5) , resolution matrix ¼ 256 (cid:4) 256 , sli - ces ¼ 176 , thickness ¼ 1 . 0 mm , voxel size ¼ 1 (cid:4) 1 (cid:4) 1mm 3 ) . Creativity assessment We adopted the Torrance Tests of Creative Thinking ( TTCT ) score that measures divergent thinking , which is a central aspect of creativity Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 165 ( Huang et al . , 2013 ; Sternberg and O ' HARA , 1999 ) . The TTCT contains verbal , fi gural and auditory tests ( Huang et al . , 2013 ) . The Figural TTCT is supposed to be less biased than the verbal TTCT test ( Kim , 2006 ) , and thus is the focus of this paper . The fi gural test comprises of 3 tasks : picture construction , picture completion , and lines . To be speci fi c , all the participants answer the same questions with a 10 - min time limit for each task . The fi rst activity requires subjects to draw a picture based on an ellipse shape provided on the page . The second activity requires subjects to use 10 incomplete fi gures to make an object or picture . The third ac - tivity requires subjects to draw as many as possible pictures or objects on three pages of vertical lines . Participants were told to draw as many as possible and as creatively as possible . For each task the scoring comprised three components : fl uency ( the number of meaningful and relevant responses , which is associated with the ability to generate and consider other possibilities ) , fl exibility ( the number of different categories of responses , which re fl ects the ability to shift between conceptual fi elds ) , and originality ( the degree of originality of the responses , which is associated with thinking “ outside the box ” ) ( Chavez - Eakle et al . , 2007 ) . More speci fi cally , the score of fl uency is the number of meaningful responses , the score of fl exibility is the number of different categories of response , and the score of originality is the degree of originality of the response with a four - point rating scale ranging from 0 ( “ not original ” ) to 3 ( “ highly original ” ) . Three trained raters took part in the scoring . The raters displayed high internal consistency in their ratings ( Cronbach alpha ¼ 0 . 90 ) . The cur - rent study used the total creativity scores ( sum of the fl uency , fl exibility and originality scores ) ( de Souza et al . , 2010 ) for each dimension which was highly correlated with the fl uency , fl exibility and originality scores ( details in Fig . S1 ) on the basis of the fi ndings of Heausler and Thompson ( 1988 ) , who suggested that because of the high correlations between the three subscales of the TTCT the subscales do not provide meaningfully different data ( Heausler and Thompson , 1988 ) . General intelligence assessment The participants ' intellectual ability was examined using the Com - bined Raven ' s Test ( CRT ) . As the test is a highly reliable and valid in - telligence test , it is widely used ( Tang et al . , 2012 ) . In our study , all the 138 participants completed the test with the 72 items , including the Raven ' s standard progressive matrix ( C , D , E sets ) and Raven ' s colored progressive matrix ( A , AB , B sets ) , revised by the Psychology Department of East China Normal University in 1989 . The total number of correct answers that an individual presented in 40 min was used as a psycho - metric index of individual intelligence ( Jaeggi et al . , 2008 ) . Genotyping Among all the participants in our study , 138 individuals were geno - typed by using the DNA samples extracted from their whole blood . For each sample , approximately 200 ng of genomic DNA was used to geno - type on the Human Omni - Zhonghua chips ( Illumina , San Diego , CA , USA ) . We processed all the samples following the Illumina In fi nium assay manual . Our main processing procedure are as follows : fi rstly , each sample was ampli fi ed , fragment , precipitated , and resuspended in a well - designed hybridization buffer . Hybridization of the denatured samples were done on the Human Omni - Zhonghua chip for at least 16 h at 48 (cid:5) C ; Then , the beadchips were processed for the single - base extension reac - tion , stained , and imaged on the iScan Reader ( Illumina , Inc . ) , The iScan employs a laser to excite the red and green fl uorophores and a laser - scanner to recode the high - resolution images of the light emitted by the excited fl uorophores . Next , the GenomeStudio software genotyping module ( Illumina , Inc . ) was used to analyze the raw intensity data ( ‘ idat ’ fi le ) that was transformed from the high - resolution images . It trans - formed the raw intensity data to the normalized bead intensity data and then converted the normalized intensity data into SNP genotypes . Finally , only SNPs that were genotyped in more than 98 % of samples from the 138 subjects were retained . Method FMRI data preprocessing All fMRI data were preprocessed using SPM8 ( http : / / www . fi l . ion . ucl . ac . uk / spm ) and the Data Processing Assistant for Resting - State fMRI ( DPARSF ) ( Chao - Gan and Yu - Feng , 2010 ) . We fi rst discarded the fi rst 10 EPI scans to suppress the equilibration effects and the remaining scans were slice timing corrected , realigned and normalized to a standard template ( Montreal Neurological Institute ) based on T1 images using linear transformation and resampled to 3 (cid:4) 3 (cid:4) 3 mm 3 . Next , spatial smoothing ( 8 mm Full Width Half Maximum FWHM ) , band - pass tem - poral fi ltering ( 0 . 01 – 0 . 1 Hz ) , nuisance signal removal from the ventricles and deep white matter , global mean signal removal , and 24 head motion correction parameters ( Friston et al . , 1996 ) were involved to remove the sources of spurious correlations . We also implemented additional careful volume censoring ( “ scrubbing ” ) movement correction suggested by Power et al . ( Power et al . , 2014 ) to remove the head - motion artifacts . The mean framewise displacement ( FD ) was computed with FD threshold for displacement being 0 . 2 mm . In addition to the frame corresponding to the displaced time point , 1 preceding and 2 succeeding time points were also deleted to reduce the ‘ spill - over ’ effect of head movements . Subjects with > 10 % displaced frames fl agged were excluded from the analysis as it is likely that such high - level of movement would have had an in fl uence on several volumes . SNP quality control Single - nucleotide polymorphisms ( SNPs ) with call rate < 95 % , minor allele frequency < 5 % , or deviation from the Hardy - Weinberg equilib - rium with p < 10 (cid:2) 6 , were excluded from the analysis . Individual samples showing an over - or under abundance of heterozygosity ( > 5 s d from the mean ) were excluded from the subsequent analysis . In order to prevent collinearity between SNPs , we further excluded SNPs in strong linkage disequilibrium ( R 2 > 0 : 9 ) within a window of 50 kb using the – indep command implemented in PLINK . Finally , after quality control , we ob - tained 309 , 996 SNPs in the 138 participates . Whole - brain functional network construction A 264 putative functional area template that Power and colleagues de fi ned was used to identify nodes in the whole - brain network ( Power et al . , 2011 ) . Their 264 - region network de fi nition has been shown to perform better than the 90 - parcel AAL atlas and voxel - based graph in representing some aspects of the functional organization of the brain with fMRI data ( Power et al . , 2011 ) . The 264 regional time series were extracted by averaging voxel time series within each ROI . Then , for each subject , the Pearson cross - correlation between all pairs of regional BOLD signals was calculated to re fl ect the FC between region pairs . A brain network consisting of the 264 brain regions and 34 , 716 FCs that con - nected them was constructed . Creativity - related functional brain network strength We used a procedure similar to that presented by Rosenberg et al . ( Rosenberg et al . , 2016 ) to construct the functional brain network closely related to the TTCT fi gural score and then calculate the network strength . We fi rst calculated the Pearson correlation between each of the 34 , 716 ( in the 264 nodes network ) FCs and the score with the age , sex , and the Raven ' s score being covariates of interest across all subjects . Then , the functional connectivities whose p - value was smaller than a prede fi ned threshold ( p threshold ) were selected , which were further separated into those with FCs positively - related and negatively - related to the fi gural TTCT score . Since these individual FC always share common nodes ( brain regions ) , we call a set of such connectivity links a network . For example , Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 166 the functional connectivities that were signi fi cantly pos - itively / negatively related to the TTCT score comprise the positively - related / negatively - related network , and all FCs that were signi fi cantly related to the fi gural TTCT score comprise the overall network . The sum of all FCs in the corresponding networks was de fi ned as the network strength . Creativity - related polygenic alliance In order to identify SNPs that were signi fi cantly related to the TTCT score , we used a linear regression model with the TTCT score being the phenotype and age , sex and the Raven ' s score as covariates . Similar to the identi fi cation of the creativity - related functional brain network , we chose SNPs whose mutations correlated with the TTCT score signi fi cantly ( p - value smaller than the p threshold ) , and then divided them into the positive and the negative group ( according to their correlation with the TTCT score ) . Then , the sum of the mutation states ( 0 , 1 , 2 ) of all the SNPs in the positively - related polygenic alliance , negative - related polygenic alliance , and the whole polygenic alliance , were de fi ned as the mutation strengths , respectively . Predicting creativity by brain connectome or genome A leave - one - out cross - validation approach was then adopted to pre - dict the TTCT score of novel individuals , as follows , using the network strength of the creativity - related brain networks or the mutation strength of the polygenic alliances and other covariances as predictors ( see Fig . 1 for details ) . For each modality of data , we changed the p threshold ( 0 . 0001 , 0 . 0005 , and 0 . 001 to 0 . 01 with a step of 0 . 0005 ) . For a given p threshold , we per - formed the leave - one - out cross - validation approach n times ( n ¼ number of subjects ) , each time with one subject being the test set , and the rest of the subjects were used to construct the creativity - related brain networks or the polygenic alliances . The creativity - related network strength or the mutation strength were calculated for individuals in the training set and then fed into the linear regression models for prediction . We used four kinds of prediction model with 1 . the positive network / mutation strength , 2 . negative network / mutation strength , 3 . overall network / mutation strength and 4 . the positive network / mutation strength and the negative / mutation strength , being used respectively in each model . The age , sex , and the Raven ' s score were included in all the four models as covariance terms . Then , the remaining test subject , who was not used in the construction of the prediction models , was employed to test all 4 models to achieve the best prediction of the TTCT score . The goodness of prediction was assessed by the correlation between the predicted and real TTCT score . The optimal p threshold and fi nal regression model were the ones with the best prediction . Prediction of TTCT score combining multi - modal data After we determined the optimal model and p threshold for prediction of creativity for the neuroimaging and genetic data , respectively , we then combined these two modalities using the elastic net linear regression model to predict the creativity score of individuals ( Zou and Hastie , 2005 ) . The elastic regression model was used here because the neuro - imaging and genetic data may be highly correlated . This model involved the network strength , the mutation strength , age , sex and the Raven ' s score as predictors . The network strength and the mutation strength were determined by the optimal prediction model identi fi ed in single modality data . The details of the regression model we used are as follows : Y ¼ β 0 þ X i β 1 i S i þ X j β 2 j T j þ X k β 3 k C k þ ε ( 1 ) where Y is the TTCT fi gural score , S i is the ith network strength we used in the best brain connectome prediction model , T j is the jth mutation strength we used in the best genome prediction model , C k is the kth covariance ( age , sex or Raven ' s score ) , ε is the noise term , and β 0 ; β 1 i ; and β 2 j are determined by the elastic - net approach that solves the following problem : min β 1 2 ð N (cid:2) 1 Þ X N (cid:2) 1 n ¼ 1 y ð n Þ (cid:2) β 0 (cid:2) X i β 1 i s ð n Þ i (cid:2) X j β 2 j t ð n Þ j (cid:2) X j β 3 j c ð n Þ j ! 2 þ λ P α ð β Þ ! ( 2 ) where N is the number of samples , y ( n ) , s ( n ) , t ( n ) and c ( n ) represent the TTCT score , the network strength , the mutation strength and the covariance of the nth sample , β ¼ ð β 0 ; β 11 ; ⋯ ; β 21 ; ⋯ ; β 31 ; ⋯ Þ P α ð β Þ ¼ X k ¼ 0 ; 11 ; ⋯ 21 ; ⋯ 31 ; ⋯ (cid:2) ð 1 (cid:2) α Þ 2 β 2 k þ α j β k j (cid:3) ( 3 ) Finally , a leave - one - out procedure was performed again to determine the optimal α and λ ( searching in [ 0 , 1 ] with a step of 0 . 01 ) with maximal correlation between the predicted TTCT score and the real TTCT . The fi nal α that we used was 0 . 80 and the fi nal λ was 0 . 66 . Permutation analysis To determine whether the prediction result is signi fi cantly better than random , a non - parametric permutation analysis was performed . As our prediction pipeline needs hours to run , a very large number of permu - tations time was not realizable . So , we randomly shuf fl ed the TTCT scores across all the 138 individuals 1000 times and ran our prediction pipeline for each permutation run . The null distribution for the correla - tion between the predicted scores and the real scores was obtained . Then , based on the null distribution , the p value of our prediction result was obtained . Identi fi cation of the shared creativity network and polygenic alliance underlying creativity In the leave - one - out prediction of the creativity score , each trial had identi fi ed a different set of FCs or SNPs . To fi nd the FCs or SNPs that were shared among all individuals , we selected those FCs or SNPs that appeared in each leave - one - out trial . As these FCs and SNPs are related to creativity across all individuals , they may be closely related to the creativity score . Further , the Pearson correlations between the TTCT score , the network strength of the shared creativity network , and the mutation strength of the shared polygenic score , were also calculated with the age , sex and the Raven ' s score as the covariance terms . Independent validation : using the shared creativity network We used the second dataset that consisted of 98 individuals with only fMRI data to validate our imaging prediction model . First , using all the 138 samples in group 1 , a linear model for the TTCT score was con - structed , using the network strength of the shared creativity network , sex , age , and Raven ' s score ( This network consists of links that we identi fi ed that were signi fi cantly correlated with the creativity and shared among all individuals by our predictive framework ) . Then , for each of the 98 individuals in group 2 the network strength ( i . e . the sum of the functional connectivities of the links ) of the shared creativity network identi fi ed in group 1 , with their sex , age , and Raven ' s score , were fed into the linear model to predict the TTCT score of each individual in group 2 . The Pearson correlation between the real TTCT scores and the predicted TTCT scores was calculated for individuals in group 2 to indicate the generalizability of our prediction model and its signi fi cance was deter - mined by performing non - parametric permutation analysis . Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 167 Fig . 1 . Flowchart for predictions of the individual creativity score using whole brain functional - connectivity and genome information . Step 1 : Data preprocessing : A functional network with 264 nodes is constructed with the resting state fMRI data for each individual . Different colors represent the node in different networks such as the DMN and control network etc . Whole genome SNPs were also identi fi ed for each individual . Step 2 : TTCT creativity score prediction by unimodal data . ( a ) First we set a threshold of p values for the correlation between functional connectivity / mutations of SNPS and TTCT score , and only FCs or SNPs with p < p threshold are selected . ( b ) We then use a linear regression model and leave - one - out paradigm to predict TTCT score of each individual . We change p threshold to select the optimal one ( p _ opt ) that achieves the best prediction . Step 3 : TTCT creativity score prediction by multimodal data . We integrate fMRI and genome data in an elastic network in predicting the TTCT score , using the optimal p threshold identi fi ed in Step 2 . Results : ( a ) shows the correlation between the Shared networks , shared SNPs , and TTCT score obtained in Step 2 and ( b ) shows the correlation between the predicted TTCT score and real score of the individuals . Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 168 Results Prediction of the TTCT fi gural score Using the whole brain functional connectome , the correlation be - tween the TTCT score predicted by the optimal prediction model with the real TTCT score was 0 . 424 ( p ¼ 2 : 19 (cid:4) 10 (cid:2) 7 , Fig . 2a ) . With whole genome data , the correlation between the predicted and real TTCT score was 0 . 466 ( p ¼ 8 : 70 (cid:4) 10 (cid:2) 9 , Fig . 2a ) . The mean absolute percentage errors ( MAPE ) of the two prediction models were 23 . 9 % and 22 . 5 % , respectively ( 76 . 1 % and 77 . 5 % in terms of accuracy ) . With combined neuroimaging and genomic data , the correlation between the predicted TTCT score and the real TTCT score increased to 0 . 524 ( p ¼ 4 : 33 (cid:4) 10 (cid:2) 11 ) , with the MAPE of the model being 21 . 6 % ( 78 . 4 % in terms of accuracy ) , see Fig . 2b . The absolute prediction error of the model that combined neuroimaging and genomic data is signi fi cantly smaller than the one that only used neuroimaging data or genetic data ( paired two sample t - test , p ¼ 0 . 002 and p ¼ 0 . 042 ) . The details of the fi nal prediction models ( the coef fi cients of the covariates in the linear regression model ) and the optimal p thresholds we used can be found in the Supplementary Material . In the functional neuroimaging regression analysis , the co - ef fi cients for the neuroimaging data provided in the Supplementary Material show that the coef fi cient for the average network strength made a contribution to the prediction that was signi fi cant at p ¼ 6 . 19 (cid:4) 10 (cid:2) 13 , and that the coef fi cients for age , gender , and the Raven ' s score made no signi fi cant contribution to the prediction . Thus , the predictions were being made from the neuroimaging data , and not from the covariates of age , gender , and the Raven ' s score . In addition , for the prediction model using genomic data , the negative and positive mutation strength also made the main contribution to the prediction results with coef fi cients signi fi cant at p ¼ 4 . 80 (cid:4) 10 (cid:2) 14 and p ¼ 1 . 34 (cid:4) 10 (cid:2) 7 , respectively . To ensure that our leave - one - out framework can provide results that are signi fi cantly better than random , a non - parametric permutation analysis was performed to test the signi fi cance of the correlation between our predicted TTCT scores and real scores . One thousand times of random permutation were performed and resulted in a correlation signi fi cant at p < 0 . 001 ( see Fig . S2 for more details ) . Creativity network and polygenic alliance shared among subjects The TTCT - related FCs that were shared among all subjects are listed in Table 1 , divided into positively - related ( 22 FCs ) and negatively - related groups ( 60 FCs ) . Most positively - related FCs ( 12 ) were associated with DMN , control networks and attention networks . 4 FCs connect ROIs in different task control networks and attention networks ; 3 FCs connected the frontal - parietal task control network with the visual network . Another 4 FCs contained ROIs in the DMN , with 2 FCs within the DMN and 2 FCs connecting the DMN with the visual and ventral attention network . Fig . 2 . The results of predicting an individual ' s TTCT creativity score . ( a ) The results of predicting an individual ' s TTCT creativity score using whole genome / connectome data . ( b ) The results of predicting an individual ' s TTCT creativity score combining whole - genome / connectome data . Here the scatter plot between the predicted TTCT creativity score of each individual and the real TTCT score is plotted . Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 169 Table 1 Identi fi ed functional connectivities that are correlated with the TTCT fi gural score . As the 264 ROI template gives only the functional module that each ROI belongs to ( rather than the names of exact brain region ) , in the following , we provide the names of the two functional modules for the two brain regions associated with each functional connectivity . Note that although the creativity - related functional connectivities are obtained by leave - one - out cross validation ( i . e . , the FCs common to each leave - one - out trial ) , the r and p value for the correlation are obtained using all 138 subjects . The network names used here are those used by Power et al . , 2011 ) . In the tables , ROI refers to the area in the Power et al . atlas , and the Network name is the name that they used for the network of which this ROI is a part . R refers to the correlation value of the functional connectivity , and p to its signi fi cance . ( a ) Functional connectivities that are correlated positively with the TTCT fi gural score Shared positive creativity network . ROI Network ROI Network R P 208 Salience 73 Auditory 0 . 318 1 . 70E - 04 53 Cingulo - opercular Task Control 235 Ventral attention 0 . 315 2 . 00E - 04 29 Sensory / somatomotor Hand 132 Uncertain 0 . 308 2 . 82E - 04 235 Ventral attention 264 Dorsal attention 0 . 308 2 . 85E - 04 56 Cingulo - opercular Task Control 181 Fronto - parietal Task Control 0 . 302 3 . 77E - 04 192 Fronto - parietal Task Control 161 Visual 0 . 298 4 . 55E - 04 212 Salience 225 Subcortical 0 . 292 5 . 89E - 04 235 Ventral attention 62 Auditory 0 . 290 6 . 59E - 04 212 Salience 234 Subcortical 0 . 285 8 . 14E - 04 197 Fronto - parietal Task Control 234 Subcortical 0 . 281 9 . 47E - 04 235 Ventral attention 37 Sensory / somatomotor Hand 0 . 280 1 . 01E - 03 48 Cingulo - opercular Task Control 264 Dorsal attention 0 . 276 1 . 18E - 03 81 Default mode 91 Default mode 0 . 276 1 . 19E - 03 124 Default mode 138 Ventral attention 0 . 276 1 . 20E - 03 192 Fronto - parietal Task Control 151 Visual 0 . 272 1 . 42E - 03 77 Default mode 250 Uncertain 0 . 268 1 . 64E - 03 53 Cingulo - opercular Task Control 136 Memory retrieval 0 . 267 1 . 73E - 03 186 Fronto - parietal Task Control 159 Visual 0 . 267 1 . 77E - 03 97 Default mode 149 Visual 0 . 261 2 . 20E - 03 182 Uncertain 204 Salience 0 . 258 2 . 48E - 03 81 Default mode 88 Default mode 0 . 258 2 . 52E - 03 ( b ) Functional connectivities that are correlated negatively with TTCT fi gural score Shared negative creativity network ROI Network ROI Network R P 49 Cingulo - opercular Task Control 149 Visual (cid:2) 0 . 339 5 . 77E - 05 91 Default mode 225 Subcortical (cid:2) 0 . 339 5 . 80E - 05 149 Visual 243 Cerebellar (cid:2) 0 . 332 8 . 57E - 05 216 Salience 253 Uncertain (cid:2) 0 . 328 1 . 04E - 04 91 Default mode 234 Subcortical (cid:2) 0 . 325 1 . 19E - 04 191 Fronto - parietal Task Control 227 Subcortical (cid:2) 0 . 323 1 . 32E - 04 138 Ventral attention 151 Visual (cid:2) 0 . 320 Table 1 ( continued ) ( b ) Functional connectivities that are correlated negatively with TTCT fi gural score Shared negative creativity network ROI Network ROI Network R P 1 . 54E - 04 144 Visual 30 Sensory / somatomotor Hand (cid:2) 0 . 318 1 . 76E - 04 79 Default mode 235 Ventral attention (cid:2) 0 . 317 1 . 83E - 04 230 Subcortical 253 Uncertain (cid:2) 0 . 311 2 . 46E - 04 233 Subcortical 253 Uncertain (cid:2) 0 . 310 2 . 53E - 04 136 Memory retrieval 132 Uncertain (cid:2) 0 . 309 2 . 61E - 04 142 Uncertain 183 Uncertain (cid:2) 0 . 305 3 . 27E - 04 91 Default mode 224 Subcortical (cid:2) 0 . 304 3 . 39E - 04 126 Default mode 175 Fronto - parietal Task Control (cid:2) 0 . 302 3 . 67E - 04 208 Salience 253 Uncertain (cid:2) 0 . 302 3 . 67E - 04 77 Default mode 93 Default mode (cid:2) 0 . 297 4 . 78E - 04 101 Default mode 235 Ventral attention (cid:2) 0 . 296 4 . 82E - 04 53 Cingulo - opercular Task Control 151 Visual (cid:2) 0 . 295 5 . 09E - 04 53 Cingulo - opercular Task Control 245 Cerebellar (cid:2) 0 . 295 5 . 20E - 04 227 Subcortical 253 Uncertain (cid:2) 0 . 292 5 . 98E - 04 111 Default mode 235 Ventral attention (cid:2) 0 . 290 6 . 44E - 04 135 Memory retrieval 132 Uncertain (cid:2) 0 . 289 6 . 79E - 04 53 Cingulo - opercular Task Control 158 Visual (cid:2) 0 . 288 7 . 09E - 04 220 Salience 253 Uncertain (cid:2) 0 . 288 7 . 23E - 04 138 Ventral attention 150 Visual (cid:2) 0 . 285 7 . 95E - 04 86 Default mode 235 Ventral attention (cid:2) 0 . 285 8 . 00E - 04 138 Ventral attention 173 Visual (cid:2) 0 . 283 8 . 78E - 04 191 Fronto - parietal Task Control 228 Subcortical (cid:2) 0 . 283 8 . 82E - 04 125 Default mode 30 Sensory / somatomotor Hand (cid:2) 0 . 283 8 . 95E - 04 91 Default mode 73 Auditory (cid:2) 0 . 282 9 . 37E - 04 191 Fronto - parietal Task Control 233 Subcortical (cid:2) 0 . 281 9 . 63E - 04 91 Default mode 223 Subcortical (cid:2) 0 . 280 1 . 02E - 03 93 Default mode 19 Sensory / somatomotor Hand (cid:2) 0 . 278 1 . 10E - 03 228 Subcortical 253 Uncertain (cid:2) 0 . 278 1 . 12E - 03 234 Subcortical 253 Uncertain (cid:2) 0 . 278 1 . 12E - 03 138 Ventral attention 161 Visual (cid:2) 0 . 277 1 . 16E - 03 53 Cingulo - opercular Task Control 146 Visual (cid:2) 0 . 276 1 . 19E - 03 92 Default mode 121 Default mode (cid:2) 0 . 276 1 . 22E - 03 227 Subcortical 31 Sensory / somatomotor Hand (cid:2) 0 . 273 1 . 36E - 03 67 Auditory 8 Uncertain (cid:2) 0 . 272 1 . 41E - 03 133 Memory retrieval 224 Subcortical (cid:2) 0 . 271 1 . 47E - 03 ( continued on next page ) Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 170 For the negatively - related FCs , 20 of them were associated with the DMN , with 3 FCs within the DMN . Of the other 17 FCs associated with the DMN , 5 FCs connected with the ventral attention network , 1 with the fronto - parietal task control network , 5 with the primary sensory net - works such as visual , sensory and auditory network , and another 5 with the subcortical network . In addition to default mode regions , the FCs between the control / attention networks and visual networks also correlated with creativity . There were 4 FCs connecting the cingulo - opercular task control network with the visual network and 5 FC con - necting the ventral attention network and visual network . The TTCT - related SNPs that were shared among all subjects ( poly - genic alliance ) are listed in Table 2 , with 5 positively related to TTCT and 8 negatively related . Speci fi cally , SNP rs6606905 in the GABRG3 intron region was negatively correlated with the creativity score , and the cor - responding gene encodes a receptor for the major inhibitory neuro - transmitter GABA in the brain . SNP rs4340077 ( positively correlated with the score ) is in the intron region of SHANK2 , which is an adapter protein in the postsynaptic density ( PSD ) . SHANK2 has a function in coupling glutamate receptor scaffolds to the actin cytoskeleton and intracellular signaling pathways by means of their protein - protein interaction domains ( Naisbitt et al . , 1999 ) . Rs9877993 , another SNP that positively related to TTCT , is in the intron region of FGF12 a member of the fi broblast growth factor family , which had also been found to be related to the excitatory neurotransmitter glutamate ( Turner et al . , 2012 ) . Correlation within / between the creativity network and polygenic alliance The network strengths of the FCs that were positively and negatively related to TTCT ( denoted by FC _ pos _ TTCT and FC _ neg _ TTCT , respec - tively ) correlated more signi fi cantly with the TTCT score ( r ¼ 0 . 698 , p ¼ 4 : 33 (cid:4) 10 (cid:2) 21 for positively related FCs and r ¼ (cid:2) 0 . 771 , p ¼ 4 : 33 (cid:4) 10 (cid:2) 28 for negatively related FCs ) than the FCs between any single pair of brain regions . The mutation strength of the SNPs that are positively and negatively related to fi gural TTCT ( denoted by SNP _ pos _ TTCT and SNP _ neg _ TTCT , respectively ) were also more signi fi cantly correlated with the TTCT score ( r ¼ 0 . 704 , p ¼ 5 : 60 (cid:4) 10 (cid:2) 22 for positively related SNPs , and r ¼ (cid:2) 0 . 651 , p ¼ 5 : 37 (cid:4) 10 (cid:2) 18 for negatively related SNPs ) than single SNP , see Table 1 and Fig . 3a . The network strengths of FC _ pos _ TTCT and FC _ neg _ TTCT also corre - lated more signi fi cantly with the mutation strengths of SNP _ pos _ TTCT and SNP _ neg _ TTCT than each of the single FC , with r ¼ 0 . 541 , p ¼ 1 : 30 (cid:4) 10 (cid:2) 10 between FC _ pos _ TTCT and SNP _ pos _ TTCT , r ¼ (cid:2) 0 . 403 , p ¼ 1 : 48 (cid:4) 10 (cid:2) 6 between FC _ pos _ TTCT and SNP _ neg _ TTCT , r ¼ (cid:2) 0 . 539 , p ¼ 1 : 68 (cid:4) 10 (cid:2) 11 between FC _ neg _ TTCT and SNP _ pos _ TTCT , and r ¼ 0 . 632 , p ¼ 8 : 55 (cid:4) 10 (cid:2) 17 between FC _ neg _ TTCT and SNP _ neg _ TTCT , see Fig . 3a . Independent validation results Firstly , we used all the 138 samples in the fi rst group to construct a linear prediction model using the shared creativity network . The neuro - imaging model from the fi rst group used the overall network strength of the shared creativity network with age , sex and Raven ' s score to fi t the TTCT score , which was the optimal prediction model that we obtained from our leave - one - out procedure . Then , the 98 samples from the second dataset that were not used to build the model were used to independently validate the neuroimaging prediction model . The correlation between the predicted score and the real TTCT score was 0 . 267 ( p ¼ 0 . 0078 , 95 % CI 0 . 072 to 0 . 442 ) , with the MAPE of the model being 35 . 4 % ( 64 . 6 % in terms of accuracy ) ( see Fig . S3 for more details ) . A non - parametric permutation analysis was then used to determine the signi fi cance of our independent prediction results . To achieve this , we randomly permuted the real TTCT score of the 98 samples and calculated the Pearson correlation betweenthe shifted TTCT scores and the prediction scores for 10000 times to form the null distribution . The p - value of our prediction results is 0 . 0032 . In addi - tion , the correlation between the network strengths of the FC _ pos _ TTCT and FC _ neg _ TTCT that we identi fi ed in the fi rst dataset were still signi fi - cantly correlated with the TTCT score in the second dataset with r ¼ 0 . 284 ( p ¼ 8 : 85 (cid:4) 10 (cid:2) 4 ) and r ¼ (cid:2) 0 . 311 ( p ¼ 2 : 59 (cid:4) 10 (cid:2) 4 ) . Table 1 ( continued ) ( b ) Functional connectivities that are correlated negatively with TTCT fi gural score Shared negative creativity network ROI Network ROI Network R P 218 Salience 219 Salience (cid:2) 0 . 270 1 . 54E - 03 138 Ventral attention 164 Visual (cid:2) 0 . 270 1 . 56E - 03 211 Salience 253 Uncertain (cid:2) 0 . 269 1 . 59E - 03 198 Fronto - parietal Task Control 253 Uncertain (cid:2) 0 . 268 1 . 67E - 03 77 Default mode 163 Visual (cid:2) 0 . 267 1 . 76E - 03 80 Default mode 132 Uncertain (cid:2) 0 . 267 1 . 77E - 03 237 Ventral attention 241 Ventral attention (cid:2) 0 . 267 1 . 77E - 03 175 Fronto - parietal Task Control 226 Subcortical (cid:2) 0 . 266 1 . 80E - 03 144 Visual 132 Uncertain (cid:2) 0 . 266 1 . 80E - 03 1 Uncertain 183 Uncertain (cid:2) 0 . 266 1 . 80E - 03 91 Default mode 61 Auditory (cid:2) 0 . 266 1 . 82E - 03 145 Visual 250 Uncertain (cid:2) 0 . 266 1 . 83E - 03 82 Default mode 235 Ventral attention (cid:2) 0 . 265 1 . 89E - 03 151 Visual 169 Visual (cid:2) 0 . 264 1 . 96E - 03 82 Default mode 130 Default mode (cid:2) 0 . 264 1 . 99E - 03 78 Default mode 225 Subcortical (cid:2) 0 . 263 2 . 05E - 03 161 Visual 35 Sensory / somatomotor Hand (cid:2) 0 . 262 2 . 18E - 03 133 Memory retrieval 223 Subcortical (cid:2) 0 . 260 2 . 36E - 03 Table 2 Identi fi ed SNPS whose mutations are correlated with the TTCT fi gural score . ( a ) positive correlation Shared positive polygenic alliance SNP id gene name single SNP p _ value rs4631724 COL24A1 8 . 84E - 06 rs9877993 FGF12 3 . 97E - 06 rs4455277 MB21D2 6 . 34E - 06 rs2082400 SLIT3 1 . 77E - 05 rs9486484 NA 1 . 00E - 05 rs17788018 MIR548Q 3 . 12E - 05 rs11021924 GALNT18 2 . 25E - 05 rs4340077 SHANK2 2 . 72E - 05 ( b ) negative correlation Shared negative polygenic alliance SNP id gene name single SNP p _ value rs200693221 ITGA4 1 . 01E - 06 rs77555152 CERKL 3 . 42E - 06 rs7718883 SLIT3 2 . 80E - 06 rs6984541 NA 4 . 64E - 06 rs6606905 GABRG3 2 . 75E - 05 * NA represents the SNP is not in the coding gene region . Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 171 Fig . 3 . Summary of the neural and genetic basis for fi gural creativity . ( a ) The neural and genetic basis for fi gural creativity . The shared positively / negatively - related FCs ( with the TTCT score ) are shown by red and blue on the left . Different sub - networks are represented by different colors in the left legend . The mutation of the shared positively - related / negatively - related ( with the TTCT score ) polygenic alliance are shown on the right , each row representing a SNP and each column represents an individual . Different colors denote different numbers of the minor allele . The correlations within / between the network strength of the shared positive / negative network and mutation strength of the shared positive / negative creativity polygenic alliance are shown in ( a ) , as well as their correlation with the TTCT fi gural score . ( b ) The creativity model we presented . The inner rectangle shows that both the increased top - down processing ( in red arrows ) and the decreasing bottom - up processing ( in blue ) relate to high creativity . The corresponding FCs relevant for top - down / bottom - up processing are also shown . The outer rectangle demonstrates the genetic factors contributing to high creativity . The mutation strength of the excitatory neurotransmitter related genes increases with creativity , and the mutation strength of the inhibitory neurotransmitter related genes de - creases with creativity . The competition between the two different kinds of neurotransmitters may in fl uence the brain activity and further contribute to the high creativity . Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 172 Discussion In this work , we use a cross - validation based predictive framework to search for the imaging and genetic correlates of creativity to provide more generalization than traditional correlation analyses . We found that models using the network strength of the creativity network or the mu - tation strength of the creativity polygenic alliance could predict the in - dividual ' s performance with high accuracy , indicating that markers for creativity are present in both the FC between brain regions and in the genetic sequence . Further , the prediction model that combined neuro - imaging and genetic data achieved results signi fi cantly better than pre - diction with single modality data , indicating that complementary information can be provided by different modalities , in this case func - tional neuroimaging and genetic modalities . Combining multi - modal data helped us to obtain a fuller picture of the biological mechanisms underlying creativity . We were able to validate our neuroimaging pre - diction model using a completely novel group containing 98 individuals . We used the FCs obtained from the resting - state in our analysis , which can re fl ect patterns similar to those in cognitive task activation studies ( Biswal et al . , 1995 ; Smith et al . , 2009 ; Tavor et al . , 2016 ) . The FCs in the creativity - related brain networks associated with the DMN and are among various higher level networks , such as the DMN , attention network and control network . These networks have all been implicated in creative thought processes ( Dietrich and Kanso , 2010 ; Fink et al . , 2014a , 2014b ; Jung et al . , 2010 ; Takeuchi et al . , 2010 , 2011 ) . The DMN activity is associated with internally focused mental processes , such as mind wandering , perspective - taking , episodic future thinking and autobio - graphical retrieval ( Andrews - Hanna et al . , 2014 ) . A growing number of studies reported that the DMN was recruited to generate novel ideas in both domain - general and domain - speci fi c creativity tasks ( Liu et al . , 2015b ; Saggar et al . , 2015 ) . For example , individual differences in brain FC or structural characterizations within the DMN were able to predict individual creativity ( Chen et al . , 2015 ; Takeuchi et al . , 2012 ; Wei et al . , 2014 ) . Thus , the DMN activity contributes to the spontaneous generation of candidate ideas , which was considered as an initial and vital phase during creative thinking process . The control network and attention networks , on the other hand , have been shown to be responsible for the evaluation of candidate ideas during creative cognitive processes ( Die - trich , 2004 ; Dietrich and Kanso , 2010 ) . As a further step , our fi ndings highlight the importance of the interactions among high level cognitive control networks for creative thought processes . We found that the FCs between the DMN , control network and dorsal attention network / ventral attention network / memory retrieval network correlate positively with the TTCT score . Our results suggest that creative thinking involves the free generation of possible solutions as well as selection among the produced alternatives ( Campbell , 1960 ) , that arise from the DMN and executive control networks , respectively ( Dietrich A and R Kanso , 2010 ; Beaty RE et al . , 2016 ) . More importantly , these results provide support for the hypothesis that creative thought bene fi ts from dynamic cooper - ation of the default mode and control networks to produce thoughts that are both novel and appropriate . Thus , creative ideas depend on self - generated thought but also need control by a top - down goal - directed process ( Beaty et al . , 2016 ) . Our fi ndings that the FCs between the DMN with the primary sensory networks ( visual , auditory and sensory - motor ) and ventral attention networks are negatively correlated with the individuals ' creativity score , indicate that high creativity individuals have the ability to inhibit con - spicuous task - relevant stimulation and shift toward task - irrelevant stimuli during creative thinking ( Berkowitz and Ansari , 2010 ) . This is in agreement with the fi nding that an individual ' s creativity level is positively related to increased alpha power in the frontal cortex during creative ideation , which re fl ects more internally orientated attention that is characterized by the absence of bottom - up stimulation ( Fink and Benedek , 2014 ) . In addition to describing the creativity - related neural networks in terms of the few networks used by Power et al . , 2011 ) , it is also useful to consider in more detail the brain areas related to the creativity score using a more detailed anatomical atlas such as a Brodmann parcellation ( Jacobs , 2011 ) . The MNI coordinates and Brodmann areas of the ROIs are shown in Table S1 for the functional connectivities with positive corre - lations with fi gural creativity , and in Table S2 for the functional con - nectivities with negative correlations with fi gural creativity . First , we focus on the functional connectivities with positive corre - lations with fi gural creativity shown in Table S1 ( see also Table 1 ) . It is evident from Table S1 that many ( 11 ) of the 44 areas involved in FC related to fi gural creativity involved left area BA 40 ( part of Wernicke ' s area ) , or right BA 45 , 46 and 47 ( contralateral to Broca ' s area ) . Also related to this positive correlation with fi gural connectivity are the left inferior prefrontal convexity / lateral orbitofrontal cortex areas , which may relate to processing in the nearby Broca ' s area . Increased FCs of some posterior cingulate areas ( with e . g . temporal cortex areas ) may re fl ect the involvement of these posterior cingulate areas in spatial processing ( Kravitz et al . , 2011 ) which is likely to be important for fi gural creativity . The fi ndings described here are consistent with and thus supported by a meta - analysis of activations in 24 neuroimaging studies related to crea - tivity ( Boccia et al . , 2015 ) , but the FC analyses described here take the analysis an important step further , by providing evidence on which of the functional connectivities between these areas is related to fi gural creativity . Second , we consider the functional connectivities with negative cor - relations with fi gural creativity shown in Table S2 ( see also Table 1 ) . One brain area with prominent negative correlations was the fusiform gyrus , with its functional connectivities with an inferior frontal gyrus / lateral orbitofrontal cortex region ( Power et al . ROIs 175 , 208 , 211 , and 198 ) , with the anterior cingulate cortex ( Power et al . ROIs 216 ) , and with the putamen especially marked ( Table S2 ) . The fusiform gyrus has perceptual representations , and the inferior frontal gyrus on the left includes part of Broca ' s area and also non - reward / error systems ( Rolls , 2016a , 2016b ) . Another brain area with prominent negative correlations with fi gural creativity was the auditory cortex / supramarginal gyrus ( Power et al . ROIs 138 ) in especially its functional connectivities with secondary vi - sual cortical areas ( Power et al . ROIs 164 , 150 , 173 , 151 , V3 , V4 and V5 ) . The reduced FCs between high order visual and auditory association cortical areas that is associated with high fi gural creativity might suggest that high fi gural creativity is associated with less visual / auditory bottom - up processing . Finally , the right hemisphere has been shown to play a dominant role in creative thinking ( Bhattacharya and Petsche , 2005 ; Bowden and Jung - Beeman , 2003 ; Falcone and Loder , 1984 ; Friedman and Forster , 2005 ) . It is interesting to note that although both within - hemisphere and between - hemisphere FCs correlate signi fi cantly with the fi gural crea - tivity score , the 4 FCs that connect ROIs in the task control network and attention network , and the FC between the cingular - opercular control network and the memory retrieval network , are all right - lateralized . This suggests that increased top - down control in the right hemisphere facili - tates a global thinking / context - dependent thinking style and fi gural processes crucial for fi gural creativity ( Mihov et al . , 2010 ) . The creativity of the brain may be related , at least partly , to genetic and environmental interactions ( Blunt , 2010 ) . Although the genetic causes of creativity may be diverse , many heterogeneous genes have been implicated in creativity such as those affecting DA , 5HT or GABA meta - bolism ( Blunt , 2010 ; Reuter et al . , 2006 ) , mostly belonging to neuro - transmitter systems . It has been shown that reduced task - induced deactivation in the precuneus correlates with higher creativity in diver - gent thinking ( Takeuchi et al . , 2011 ) . Considering also that individuals with a higher GABA / glutamate ratio tend to suppress ongoing neural activities of the precuneus more ef fi ciently ( Hu et al . , 2013 ) , these results indicate that the GABA / glutamate ratio may be closely related to the creativity of a brain . Our genetic results are concordant with these fi ndings by showing that mutations of genes related to both excitatory and inhibitory neu - rotransmitters are associated with creative thinking . We fi nd that Z . Liu et al . NeuroImage 174 ( 2018 ) 164 – 176 173 mutation of a SNP in GABRG3 , the receptor for GABA , is negatively related to the fi gural TTCT score , while several SNPs in genes related to glutamate ( SHANK2 , FGF12 ) correlate positively with the fi gural TTCT score . The mutation of these SNPs relevant for neurotransmitter systems may disturb the balance between the two different kinds of neurotrans - mitter , i . e . glutamate and GABA . Since these mutations are found to be signi fi cantly associated with both the creativity - related network and the fi gural TTCT score , we therefore postulate that this difference in the glutamate and GABA transmitter may have an impact on the FCs asso - ciated with regions ( including the precuneus ) of the DMN , control , and attention networks . Moreover , it is interesting to note that among all the 13 SNPs we found to be related to the fi gural TTCT score , 5 are associated with genes ( ITGA4 , SHANK2 , MIR548 , GABRG3 and FGF12 ) that have been reported to be relevant to autism ( Ander et al . , 2015 ; Buxbaum et al . , 2002 ; Correia et al . , 2009 ; Leblond et al . , 2012 ; Vaccarino et al . , 2009 ; Won et al . , 2012 ) . Autistic traits have been demonstrated to be associated with high numbers of unusual responses on the divergent thinking tasks ( Best et al . , 2015 ) . The creativity - related functional brain networks and the genetic al - liances that we used to predict the creativity score in the fi rst dataset contained many different FCs and SNPs . Although we con fi rmed that the sum of all the FCs ( strength of the networks ) were signi fi cantly correlated with creativity score in all the two dataset and the FCs or SNPs as a whole ( i . e . , their sum ) could predict the creativity score well , it should be noted that not every FC / SNP in the brain networks / genetic alliances was associated with creativity score signi fi cantly ( i . e . , survived from correc - tion for multiple comparisons ) . In our feature selection and prediction procedures , we took into ac - count factors that might have affected our prediction models . For example , for head motion , which could be a potential confounding factor for neuroimaging data ( Liu et al . , 2015a ; Yan et al . , 2013 ; Zuo et al . , 2013 ) , we performed two further motion control procedures in the 138 sample group to con fi rm that it did not confound our prediction models . Firstly , the correlation between the mean framewise displacement ( FD ) with the creativity score and our predicted score were calculated . We found that the mean FD did not correlate with the real TTCT score or the predicted score ( r ¼ 0 . 116 , p ¼ 0 . 157 and r ¼ 0 . 114 and p ¼ 0 . 183 ) . Secondly , we used the brain network with the mean FD regressed out to build a new prediction model using the network strength of the creativity - related functional brain network . The correlation between the predicted score ( r ¼ 0 . 420 ) and the prediction accuracy ( 72 . 7 % ) did not differ signi fi cantly from the results provided in the Results section . The relationship between the individuals ' TTCT fi gural creativity and their age and Raven ' s score were also analyzed . The procedure was to use age , sex and the Raven ' s score as covariates in the regression equation ( C k in eq . ( 1 ) above ) that estimated the creativity score from the FCs . This ensured that the prediction was from the FCs with the effects of age , sex and the Raven ' s score regressed out . We found that the fi gural crea - tivity score was signi fi cantly though weakly ( p ¼ 0 . 037 ) negatively related to age ( from ~ 18 to ~ 22 ) ( see Fig . S4 ) . However , the Raven ' s score and sex were not signi fi cantly related to the fi gural creativity score , indicating that the fi gural creativity prediction is not confounded by individuals ' gender or general intelligence ( see Fig . S5 ) . In the regression analysis , the coef fi cients for the neuroimaging data ( part A ) show that the coef fi cient for the average network strength made a contribution to the prediction that was signi fi cant at p ¼ 6 . 19 (cid:4) 10 (cid:2) 13 , and that the co - ef fi cients for age , gender , and the Raven ' s score made no signi fi cant contribution to the prediction . In the Supplementary Material , in the prediction model from the genetic data ( part B ) , there is a negative co - ef fi cient for the contribution of age that is signi fi cant ( p ¼ 1 . 92 (cid:4) 10 (cid:2) 5 ) , and this is of interest . In conclusion , we have for the fi rst time investigated both the neural and genetic correlates of fi gural creativity using whole - brain FCs and the whole - genome . We fi nd that we can predict fi gural connectivity signi fi - cantly better from the FCs and the genome ( 78 . 4 % ) than from the FCs alone ( 76 . 1 % ) or the genetic data alone ( 77 . 5 % ) . The FCs positively related to creativity were associated with the “ control networks ” and “ attention networks ” ( as identi fi ed by Power et al . , 2011 ) typical of top - down processes , while the FCs negatively related to creativity were between the DMN and the primary sensory networks that are associated with bottom - up processes . The implication is that fi gural creativity is associated with strong top - down control versus weak bottom - up process in the resting state . The genes associated with fi gural connectivity are involved in glutamate and GABA functionality , and an implication is that the ratio of excitatory to inhibitory synaptic function may be important in fi gural creativity . The results described here provides the fi rst insight into how a combination of neural and gene - related factors are related to creative thinking . The brain areas related to fi gural creativity included increased visual association cortex connectivity with inferior frontal gyrus areas related to Broca ' s area , and with Wernicke ' s area , which may relate to enhanced visual imagery in those with high fi gural creativity . Funding J . Feng is partially supported by the key project of Shanghai Science & Technology Innovation Plan ( No . 15JC1400101 and No . 16JC1420402 ) and the National Natural Science Foundation of China ( Grant No . 71661167002 and No . 91630314 ) . There search was also partially sup - ported by the Shanghai AI Platform for Diagnosis and Treatment of Brain Diseases ( No . 2016 - 17 ) . The research was also partially supported by Base for Introducing Talents of Discipline to Universities ( No . B18015 ) . Z . Liu and JY . Zhang are supported by National Science Foundation of China ( NSFC 11674352 and 61571341 ) , and the Fundamental Research Funds for the Central Universities of China ( No . JBZ170301 ) . J . Zhang is supported by National Science Foundation of China ( NSFC 61573107 ) , and special Funds for Major State Basic Research Projects of China ( No . 2015CB856003 ) . J . Qiu was supported by the National Natural Science Foundation of China ( NSFC 31571137 and 31771231 ) , Project of the National Defense Science and Technology Innovation Special Zone , Chang Jiang Scholars Program , National Outstanding Young People Plan , the Program for the Top Young Talents by Chongqing , Natural Science Foundation of Chongqing ( No . cstc2015jcyjA10106 ) , Fok Ying Tung Education Foundation ( No . 151023 ) , the Research Program Funds of the Collaborative Innovation Center of Assessment toward Basic Education Quality at Beijing Normal University . Acknowledgements We would like to thank Prof . Keith M . Kendrick , Prof . Fengzhu Sun , and Jessie Liu for their helpful suggestions . Appendix A . 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NeuroImage 174 ( 2018 ) 164 – 176 176 \ No newline at end of file diff --git a/silver_data/15cb96176e844422513d404f9f89f3f1d20019de.pdf.txt b/silver_data/15cb96176e844422513d404f9f89f3f1d20019de.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..59e3e5893ad35332852230d617562c25fbbbc7e9 --- /dev/null +++ b/silver_data/15cb96176e844422513d404f9f89f3f1d20019de.pdf.txt @@ -0,0 +1 @@ +Articles https : / / doi . org / 10 . 1038 / s41556 - 018 - 0220 - 2 1 Department of Pathology , School of Medical Sciences , University of New South Wales , Sydney , Australia . 2 Department of Biosciences and Nutrition , Karolinska Institutet , Huddinge , Sweden . 3 Wellcome Trust Centre for Cell - Matrix Research , Faculty of Biology , Medicine and Health , University of Manchester , Manchester , UK . 4 Centre for Genomic Regulation ( CRG ) , The Barcelona Institute of Science and Technology , Barcelona , Spain . 5 ICFO , Institut de Ciencies Fotoniques , Mediterranean Technology Park , The Barcelona Institute of Science and Technology , Castelldefels , Barcelona , Spain . 6 Perelman School of Medicine , Department of Physiology , University of Pennsylvania , Philadelphia , PA , USA . 7 These authors contributed equally : John G . Lock , Matthew C . Jones . * e - mail : john . lock @ unsw . edu . au ; staffan . stromblad @ ki . se C ell – extracellular matrix ( ECM ) attachment occurs through a range of integrin - containing adhesion complexes , includ - ing focal complexes , focal adhesions and fibrillar adhesions 1 , 2 , and modulates many processes including cell movement , prolif - eration and differentiation 3 . Although structurally and functionally varied , adhesion complexes overlap substantially in their compo - sition , sharing a 60 - protein consensus adhesome 4 . As one of the most abundant consensus adhesome proteins , talin - 1 is viewed as an indispensable contributor to integrin activation 5 and adhesion complex organization 6 . Adaptor proteins that couple integrins to F - actin , such as vinculin and paxillin , are also universally associated with adhesion complexes , reflecting the pivotal role of F - actin in adhesion complex function 7 . Cell – ECM adhesion is also critical for mitotic progression and for the transmission of spatial memory between generations 8 – 11 , a key factor controlling differentiation and tissue development 12 . Paradoxically , the importance of cell – ECM attachment during mitosis conflicts with the observed disassembly of classical adhe - sion complexes at mitotic onset 13 , because failure of adhesion complex disassembly perturbs division 14 , 15 . Furthermore , integrins implicated in mitotic adhesion , such as β 1 , function not at the adhe - sion plane but in the detached cell cortex 16 . Overall , the nature of mitotic adhesion complexes remains profoundly unclear 17 , 18 . Here , we identify a class of ‘reticular’ adhesion complex with a unique adhesome , formed by integrin α V β 5 during interphase in the absence of both talin and F - actin . Reticular adhesions persist throughout mitosis , providing the ECM anchoring that is necessary for efficient division . Thus , reticular adhesions provide a solution to the paradox of mitotic cell – ECM attachment . Results α V β 5 is the predominant integrin used by cells in long - term culture . The integrin consensus adhesome was derived from cells plated on fibronectin 4 , 19 . To study the adhesome of cells that had assembled their own ECM , we performed mass spectrometry ( MS ) analysis of adhesion complex composition in U2OS cells following 72 h of growth . Unexpectedly , the most abundant integrin subunits identified were α V and β 5 , with much lower levels of β 1 , β 3 , β 8 , α 5 and α 3 ( Fig . 1a ) . Subsequent immunofluorescence analysis con - firmed that very distinct α V β 5 - positive adhesion complexes were visible in a range of cells in long - term culture , with little α V β 3 or β 1 labelling detected in U2OS , A549 and A375 cells ( Fig . 1b and Supplementary Fig . 1a ) . Notably , α V β 5 was simultaneously detected in classical focal adhesions at the cell periphery and in reticular structures across the cell body , also visible after 24 and 48 h of cell growth ( Supplementary Fig . 1b ) . U2OS cells were then plated on the integrin α V ligand vitronectin . Confocal imaging showed β 5 associated with two different structures following 3 h of spreading : peripheral focal adhesion complexes containing talin and vinculin , and centrally distributed , punctate or reticular structures lacking these components ( Fig . 1c , d ) . Subsequently , similar α V β 5 - positive , talin - negative structures were detected in each of nine cell lines assessed , including the cancer cell lines CS1 - b5 , HeLa , MCF7 and BT549 ( Supplementary Reticular adhesions are a distinct class of cell - matrix adhesions that mediate attachment during mitosis John G . Lock 1 , 2 , 7 * , Matthew C . Jones 3 , 7 , Janet A . Askari 3 , Xiaowei Gong 2 , Anna Oddone 4 , 5 , Helene Olofsson 2 , Sara Göransson 2 , Melike Lakadamyali 5 , 6 , Martin J . Humphries 3 and Staffan Strömblad 2 * Adhesion to the extracellular matrix persists during mitosis in most cell types . However , while classical adhesion complexes , such as focal adhesions , do and must disassemble to enable mitotic rounding , the mechanisms of residual mitotic cell – extracel - lular matrix adhesion remain undefined . Here , we identify ‘reticular adhesions’ , a class of adhesion complex that is mediated by integrin α v β 5 , formed during interphase , and preserved at cell – extracellular matrix attachment sites throughout cell divi - sion . Consistent with this role , integrin β 5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations . Reticular adhesions are morphologically and dynamically distinct from classical focal adhesions . Mass spectrom - etry defines their unique composition , enriched in phosphatidylinositol - 4 , 5 - bisphosphate ( PtdIns ( 4 , 5 ) P 2 ) - binding proteins but lacking virtually all consensus adhesome components . Indeed , reticular adhesions are promoted by PtdIns ( 4 , 5 ) P 2 , and form independently of talin and F - actin . The distinct characteristics of reticular adhesions provide a solution to the problem of main - taining cell – extracellular matrix attachment during mitotic rounding and division . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1290 Articles NATuRe Cell BIology 1 VN 3 VN 10 VN Talin - positive Vitronectin concentration ( µ g ml – 1 ) g T a li n I n t eg r i n β 5 1 VN 3 VN 10 VN 1 VN 3 VN 10 VN e f Integrin β 5 Talin c Integrin β 5 Vinculin d Merge Merge mCherry Vinculin Integrin β 5 2GFP IRM h mCherry Vinculin Integrin β 5 2GFP a i Integrin α V β 5 Integrin β 1 Integrin α V β 3 b α 3 α 5 α V β 1 β 3 β 5 β 8 0 10 20 30 40 50 M ean s pe c t r a l c oun t s 600 800 1 , 000 1 , 200 M ean i n t eg r i n β 5 i n t en s i t y P = 2 . 9 × 10 – 148 Talin - negative P = 4 . 8 × 10 – 20 P = 9 . 8 × 10 – 48 P = 6 . 5 × 10 – 05 P = 0 . 0054 P = 0 . 0001 Fig . 1 | integrin α V β 5 forms novel talin - and vinculin - negative reticular adhesion structures . a , MS analysis of integrin subunits detected in adhesions isolated ( after cell removal ) from U2OS cells grown in complete medium for 3 days on tissue culture plastic . Results are mean spectral counts from n = 3 biologically independent experiments , where thin horizontal lines indicate median values . P values reflect comparison via two - sided unpaired t - testing between integrin subunits α v or β 5 and the next highest expressed subunit , β 1 . b , Confocal images of immuno - fluorescently labelled integrins α V β 3 ( LM609 ) , β 1 ( 9EG7 ) and α V β 5 ( 15F11 ) for U2OS cells plated on glass coverslips for 72 h . c – f , U2OS cells were plated for 3 h in serum - free medium on surfaces coated with 10 µ g ml − 1 vitronectin ( VN ) , except where specified otherwise . c , d , Confocal images of talin ( c ) or vinculin ( d ) immunofluorescence with that of integrin β 5 . Boxed areas are shown at higher magnification to the right . e , f , Co - labelling of talin ( e ) and β 5 ( f ) in cells grown on glass coated with 1 , 3 or 10 µ g ml − 1 VN . g , Quantified intensities of talin - positive ( blue ) or - negative ( red ) β 5 structures . Data are taken from 81 cells ( > 23 per condition ) and n = 6 , 132 adhesions derived from three biologically independent experiments . Boxplot centre and box edges indicate median and 25th or 75th percentiles , respectively . Boxplot notches approximate 95 % CIs ( see Methods for details ) . P values reflect two - sided unpaired Mann – Whitney U - testing . h , TIRF images of an mCherry - vinculin - and β 5 - 2GFP - expressing U2OS cell ( U2OS - β 5V ) . Arrows in magnified boxes highlight regions lacking vinculin signal , which fall between β 5 - positive , vinculin - negative puncta . i , Confocal and interference reflection microscopy ( IRM ) images of a U2OS - β 5V cell exemplify correlations between β 5 - positive , vinculin - negative structures and regions with close cell – substrate proximity . All images are representative of results from at least three biologically independent experiments . Scale bars , 10 µ m . Source data for a and g are available in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1291 Articles NATuRe Cell BIology Fig . 1c ) , immortalized non - transformed HME1 and RPE1 cells ( Supplementary Fig . 1d ) , and primary mouse aortic endothelial ( MAE ) and human dermal fibroblast ( HDF ) cells ( Supplementary Fig . 1e ) , indicating that formation of α V β 5 - positive , talin - neg - ative structures is characteristic of a wide range of cell types . Integrin α V and β 5 subunits co - localized ( Supplementary Fig . 2a ) , but co - labelling of β 5 with antibodies against various adhesion complex - related proteins , including consensus adhesome com - ponents , α V β 5 binding partners , cytoskeletal proteins ( including F - actin ) and phosphotyrosine , revealed no specific co - localization ( Supplementary Figs . 1c and 2b – m ) . Integrin β 5 labelling inten - sity in both talin - 1 - positive and - negative structures correlated with vitronectin concentrations ( Fig . 1E - G ) , while U2OS cells plated on laminin ( not a ligand for α V β 5 ) only formed vinculin - positive adhesion complexes ( Supplementary Fig . 2n ) . In conclu - sion , formation of the reticular structures depends on α V β 5 – ECM ligand binding . We next labelled the integrin β 5 extracellular domain without prior cell permeabilization ( Supplementary Fig . 2o ) . Strong co - localisation with integrin β 5 - 2GFP demonstrated α V β 5 plasma membrane embedding and antibody specificity . Moreover , total internal reflection ( TIRF ) imaging of live U2OS cells co - expressing β 5 - 2GFP and mCherry - vinculin ( U2OS - β 5V ) revealed central , α V β 5 - positive , vinculin - negative structures in the TIRF plane ( Fig . 1h ) . Dark intracellular regions in mCherry - vinculin signals indicated where tensioned ventral membranes arced out of the TIRF plane , leaving no cytoplasmic signal . These dark regions cor - responded to large gaps between α V β 5 - positive , vinculin - negative puncta , suggesting the latter to be attachment points that pin the ventral plasma membrane to the substrate . This hypothesis was supported by live cell interference reflection microscopy , where close cell – substrate proximity corresponded precisely with integ - rin β 5 - 2GFP signals in both vinculin - positive FAs and vinculin - negative structures ( Fig . 1i ) . Collectively , these data indicate that α V β 5 - positive , consensus adhesome component - negative reticular structures are bona fide cell – ECM adhesion complexes . These are hereafter termed ‘reticular adhesions’ . Reticular and focal adhesion complexes are morphologically and dynamically distinct . Reticular adhesions were more numer - ous than classical focal adhesion complexes at all sizes ( Fig . 2a ) , increased in size more frequently ( Fig . 2b ) and were localized further from the cell periphery ( Fig . 2c ) . There was no correla - tion between RA size and integrin β 5 clustering density , unlike the increased integrin density observed in larger FAs ( Fig . 2d ) 20 , 21 . This implies molecular - scale differences between the maturation of focal and reticular adhesions , with the latter being more homogenous . Reticular adhesions formed as small puncta , grew by net periph - eral integrin recruitment , producing ring - like or reticular structures that ultimately fragmented and disassembled , all without recruit - ing vinculin ( Fig . 2e – h , Supplementary Movie 1 and cropped region from Fig . 2h in Supplementary Movie 2 ) . Thus , reticular adhesions form de novo as a distinct class of adhesion complex . Quantitative tracking highlighted stark differences in dynam - ics between reticular adhesions and focal adhesions ( Fig . 2i – n and Supplementary Movie 3 ) : isotropic reticular adhesion growth pro - duced low displacement ( Fig . 2j ) , whereas focal adhesions elongated anisotropically and slid at high velocities , reflecting F - actin - derived forces driving asymmetric component recruitment ( Fig . 2k ) 22 , 23 . Isotropic growth and immobility in reticular adhesions suggests the absence of such directed mechanical cues 24 and complements the observed lack of F - actin . This conclusion was supported by locally disordered motion of reticular adhesion trajectories ( Fig . 2l ) . In contrast , focal adhesions moved co - linearly within dif - ferent cell lobes ( Fig . 2m ) , reflecting aligned , centripetal F - actin - derived forces 25 . The relationship between average adhesion complex velocity and lifetime revealed that , for both focal adhesions and reticular adhesions , fast movement corresponded to short life - time . Thus , fast - moving focal adhesions existed for less than half the lifespan of reticular adhesions , which were relatively static and long - lived ( Fig . 2n ) . Fluorescence recovery after photobleaching analysis revealed that , despite their increased lifetime as complexes , β 5 - 2GFP turn - over in reticular adhesions was faster and more extensive than in focal adhesions ( Fig . 3a – e and Supplementary Movie 4 ) . Conversely , variability in β 5 - 2GFP fluorescence recovery was lower in reticular adhesions ( Fig . 3f ) , suggesting relative homogeneity in molecular organization and dynamics across their lifespan , consistent with the homogeneity in integrin clustering densities ( Fig . 2d ) . In stochastic optical reconstruction microscopy ( STORM ) , both adhesion complex types displayed small internal clusters of integ - rin β 5 ( Fig . 3g ) , consistent with the integrin β 1 nanocluster orga - nization within adhesion complexes that we recently reported 26 . Minimal differences were observed between the adhesion complex types in terms of nearest - neighbour distances between nanoclusters and molecular localization counts per nanocluster ( Fig . 3h , i ) . Thus , despite the absence of consensus adhesome components ( including talin - 1 , thought to control nanoscale integrin organization 6 ) and differences in macromolecular dynamics , the molecular - scale orga - nization of integrin β 5 is virtually identical in focal adhesions and reticular adhesions . Reticular adhesions mediate cell attachment but form indepen - dently of F - actin and talin . Disruption of actin polymerization by cytochalasin D or latrunculin A before cell – ECM attachment inhibited focal adhesion , but not reticular adhesion , formation ( Fig . 4a , b , Supplementary Fig . 3a and Supplementary Movie 5 ; note the simultaneous formation of both reticular adhesions and focal adhesions within 20 – 30 min of control cell attachment ) . Cytochalasin D inhibited cell spreading , but not reticular adhe - sion numbers relative to cell area , as evidenced by matched lin - ear trends in cell area versus adhesion complex number within treated and control cells ( Fig . 4c ) . Notably , while cytochalasin D substantially reduced vinculin levels in surviving focal adhe - sions , β 5 density increased in reticular adhesions ( Fig . 4d ) . Furthermore , cytochalasin D treatment after attachment caused disassembly of focal adhesions , but retention of reticular adhe - sions ( Supplementary Fig . 3a ) . Integrin β 3 - and β 5 - negative CS1 - wt cells did not attach to vit - ronectin , while CS1 cells stably expressing β 5 ( CS1 - β 5 ) attached strongly ( Fig . 4e and Supplementary Fig . 3b ) and formed both focal and reticular adhesions . CS1 - β 5 cells treated with cytochalasin D attached approximately half as strongly as unperturbed CS1 - β 5 cells , demonstrating that reticular adhesions facilitate cell attach - ment in the absence of F - actin . This residual adhesion was blocked by competitive inhibition of α V β 5 - VN ( vitronectin ) binding using cyclic RGD ( Arg - Gly - Asp ) peptides , confirming α V β 5 specific - ity ( Fig . 4e ) . Thus , reticular adhesions forming in the absence of F - actin facilitate attachment in the absence of focal adhesions . To assess the role of talin in reticular adhesion formation , talin - 1 - null mouse embryonic stem cells ( mES talin - 1 – / – ) were trans - fected with talin - 2 siRNA . Reduction of talin limited cell spreading ( Fig . 4f – h and Supplementary Fig . 3c ) 27 and ablated focal adhe - sions ( Fig . 4f , g ) ; however , integrin β 5 was more densely concen - trated within reticular adhesions following talin - 2 knockdown ( Fig . 4f , g , i ) , similar to cells treated with cytochalasin D ( Fig . 4a – d ) . Thus , reticular adhesions can form independently of talin . On acti - vation by manganese or the talin - 1 head domain , integrin α V β 3 forms reticular - like clusters in the centre of the cell 28 , 29 . In contrast , α V β 5 clustered independently of talin or additional activation stim - uli . Furthermore , mRFP - tagged talin - 1 head or rod domains nei - ther localized to reticular adhesions nor altered α V β 5 - containing NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1292 Articles NATuRe Cell BIology reticular adhesions ( Supplementary Fig . 3d ) . Expression of enhanced green fluorescent protein ( EGFP ) - tagged integrin β 5 extracellular domain fused to the integrin β 3 tail domain also dem - onstrated localization to reticular adhesions ( Fig . 4j , k ) , identifying the β 5 extracellular domain as the key facilitator of α V β 5 clustering in reticular adhesions . Adhesion mean velocity Focal 360 – 480 min Adhesion mean velocity Reticular 360 – 480 min Integrin β 5 2GFP mCherry vinculin 360 min mCherryvinculin Integrin β 5 2GFP Merge Reticular Focal Adhesionmotion angle 180 Adhesionmotion angle 320 160 190 270 430 480 550 700 720 e f g h i j k l m n A v e r age adhe s i on li f e t i m e ( h ) pe r v e l o c i t y b i n 0 2 4 6 8 10 Av . focal lifetime ( 3 . 3 h ) Av . reticular lifetime ( 7 . 3 h ) Reticular Focal Average adhesion lifetime velocity ( 12 nm min – 1 bins ) – 5051015202530354045 M ean adhe s i on nu m be r pe r a r ea pe r c e ll a S c a l ed r e l a t i v e f r equen cy R A s v e r s u s F A s pe r a r ea b i n 0 . 8 1 . 3 1 . 8 2 . 3 2 . 8 0 1 2 3 4 5 6 b c d Adhesion area ( 0 . 4 µ m 2 bins ) Adhesion area ( 1 µ m 2 bins ) M o r pho m e t r y F o r m a t i on / D i s a ss e m b l y A dh e s i on d y n a m i cs Focal Reticular Focal Reticular 9095100105110115120125130135140 I n t eg r i n β 5 r e l a t i v e f l uo r e sc en t i n t en s i t y Focal Reticular – 10 0 10 20 30 40 50 0 2 4 6 8 10 12 14 16 18 20 P e r c en t adhe s i on popu l a t i on i n d i s t an c e t o c e ll bo r de r b i n Adhesion distance to cell border ( 2 µ m bins ) Focal Reticular 0 . 0 0 . 8 1 . 6 2 . 4 3 . 2 4 . 0 4 . 8 5 . 6 6 . 4 0 . 0 0 . 8 1 . 6 2 . 4 3 . 2 4 . 0 4 . 8 5 . 6 6 . 4 Adhesion area ( 0 . 4 µ m 2 bins ) – 180 180 – 180 156 144 132 120 108 96 84 72 60 48 36 24 12 0 Fig . 2 | Comparison of focal and reticular adhesion morphometry and dynamics . a , Histogram of focal ( blue ) and reticular ( red ) adhesions by area ( error bars represent 95 % CI ) . b , Frequency of reticular adhesions by area , represented as fold - change relative to focal adhesions . c , Percentage of adhesions versus distance from cell border ( error bars represent 95 % CI ) . d , Adhesion area versus mean integrin β 5 intensity relative to smallest focal adhesions ( error bars represent 95 % CI ) . e – g , Representative images from live imaging of mCherry - vinculin ( e ) and β 5 - 2GFP ( f ) ( merged in g ; Supplementary Movie 1 ) . h , Zoomed regions of e – g at the time points indicated ( mins ) ( Supplementary Movie 2 ) . Scale bars , 10 µ m ( e – g ) and 1 µ m ( h ) . ( i – m derived from Supplementary Movie 3 ) . i , Merged image of β 5 - 2GFP and mCherry - vinculin at a representative time point . j , k , Trajectories of reticular ( j ) and focal ( k ) adhesions colour - coded by mean velocity ( green , slow ; red , fast ) . l , m , Trajectories of reticular ( l ) and focal ( m ) adhesions colour - coded by net adhesion motion angle . Line thicknesses indicate instantaneous adhesion velocity . n , Aggregate analysis of all trajectories of average adhesion velocity versus corresponding average adhesion lifetime ( dashed lines indicate adhesion class average lifetimes ; error bars represent 95 % CI ) . Data in a – n derive from live imaging and analysis of 14 U2OS - β 5V cells ( in four biologically independent experiments ) over 12 h ( 10 min intervals ) , providing n = 30 , 123 focal adhesion and n = 91 , 898 reticular adhesion observations . Source data for a – d and n are available in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1293 Articles NATuRe Cell BIology Reticular adhesion composition is unique . We next used MS to define reticular adhesion composition . U2OS cells were treated with cytochalasin D to deplete focal adhesions , followed by ventral membrane adhesion complex isolation and pro - cessing . A total of 199 proteins were identified in the control condition , 18 of which were consensus adhesome components ( Fig . 5a and Supplementary Table 2 ) 4 . Conversely , cytochalasin D - treated samples revealed 53 proteins selectively associated with reticular adhesions , only one of which was a consensus adhesome protein ( tensin - 3 ) . Four proteins were discounted from further analysis due to exceptionally high representation in the CRAPome database 30 , leaving a reticular adhesome of 49 proteins . Of these , 41 formed a highly connected protein – protein interaction network ( Fig . 5b ) . Lower diversity in the reticular adhesome supports evidence of relative homogene - ity in both integrin clustering density ( Fig . 2d ) and dynamics ( Fig . 3f ) . Gene ontology analysis revealed enrichment of terms relating to membrane organization and endocytosis ( Fig . 5c , d ) , in contrast to the control condition that was enriched for terms related to cell adhesion and regulation of actin cytoskeleton ( Supplementary Fig . 4a , b ) . Ontology analysis was consistent with MS identification of a number of known endocytic adaptors in reticular adhesions ( Fig . 5b ) . Six candidates were validated by immunofluorescence , including NUMB , DAB2 ( Fig . 5e , f ) , EPS15L1 , HIP1R , WASL and ITSN1 ( Supplementary Fig . 4c – f ) . Despite the observation that reticular adhesions did not associ - ate with F - actin and formed following disruption of F - actin , a number of actin - binding proteins were identified in the reticu - lar adhesome and two of these ( tensin - 3 and talin - 2 ) co - local - ized with β 5 in cytochalasin - treated cells ( Fig . 5g , h ) . In contrast , the Arp2 / 3 complex component Arp3 did not localize with β 5 - positive structures ( Supplementary Fig . 4g ) and inhibition of Arp2 / 3 did not abrogate reticular adhesions , despite reducing focal adhesion intensity ( Supplementary Fig . 5a – c ) . m C he rr y v i n c u li n – p r e - b l ea c h I n t eg r i n β 5 2 G F P – p r e - b l ea c h I n t eg r i n β 5 2 G F P – po s t - b l ea c h I n t eg r i n β 5 2 G F P – po s t - r e c o v e r y I n t eg r i n β 5 – i n t en s i t y r e c o v e r y Focal Reticular d Pre - bleach Post - bleach Post - recovery Integrin β 5 2GFP a b c mCherry vinculin g Reticular : conv . Reticular : STORM Focal : conv . Focal : STORM Integrin β 5 Vinculin 1 2 1 2 3 4 3 4 1 2 3 4 i Focal Reticular 50 100 150 200 Adhesion type h N ea r e s t - ne i ghbou r d i s t an c e ( n m ) 0 50 150 250 Adhesion type M o l e c u l a r l o c a li z a t i on s pe r nano - c l u s t e r Focal Reticular I n t eg r i n d y na m i cs ( F R AP ) N ano sc a l e s t r u c t u r e 0 2 4 6 8 10 12 e f M ean pe r c en t r e c o v e r y S D o f m ean pe r c en t r e c o v e r y Time ( s ) 0 10 20 30 40 0 500 1 , 000 1 , 500 Time ( s ) 2 , 000 Reticular Focal P = 0 . 0021 0 500 1 , 000 1 , 500 2 , 000 ReticularFocal Fig . 3 | Comparison of focal and reticular adhesion integrin dynamics and nanoscale structure . a , Integrin β 5 - 2GFP and mCherry - vinculin pre - bleach , post - bleach and post - recovery ( 30 min ; Supplementary Movie 4 ) . b , c , Circles indicate focal and reticular adhesion bleach regions , supported by single channel images . Scale bars , 10 µ m ( a – c ) . d , Square regions corresponding to circles in a – c . Fifth column : colour - scaled images ( low to high values : black , red , orange , yellow , white ) of intensity recovery for focal and reticular adhesions . e , Aggregate FRAP recovery curves for n = 63 focal and n = 68 reticular adhesions ( from 15 cells across three biologically independent experiments ) . Recovery curves are displayed as mean per time point ( circles ) ± 95 % CI . Loess regression defines a smoothed fit ( lines ) ± a moving 95 % CI envelope . P values reflect comparison of Loess fitted curves assessed via two - sided unpaired Kolmogorov – Smirnov testing . f , Post - bleach recovery time versus standard deviation of recovery at each time point . g – i , Comparison of integrin β 5 nanoclustering . g , β 5 immuno - labelling and mCherry - vinculin in a U2OS cell plated on VN and imaged via confocal microscopy . Representative focal ( 1 and 2 ) and reticular adhesions ( 3 and 4 ) cropped from matched confocal and STORM images ( β 5 only , ‘royal’ look - up table intensity - scaled as in legend ) . Scale bars , 2 µ m ( 500 nm in cropped images ) . h , i , β 5 nanocluster nearest - neighbour distances ( h ) and molecular localization counts per nanocluster ( i ) based on STORM data . 216 focal and 162 reticular adhesions were assessed , including n = 5 , 530 nanoclusters across four biologically independent experiments . Boxplot centre and box edges indicate median and 25th or 75th percentiles , respectively , while whiskers indicate the median ± 1 . 5 × IQR ( interquartile range ) or the most extreme observations within these limits . Boxplot notches approximate 95 % CIs . Source data for e , f and h , i are provided in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1294 Articles NATuRe Cell BIology c – 5 15 35 55 75 95 115 135 0 100 200 300 400 500 Reticular + CytoD ( 20 µ M ) Reticular control Focal + CytoD ( 20 µ M ) Focal control Mean cell area ( µ m 2 ) M ean adhe s i on nu m be r pe r c e ll ( ± 95 % C I ) C y t o D ( 20 µ M ) D M S O Merge Merge mCherryvinculin a b Integrin β 5 2GFP Integrin β 5 2GFP 0 200 400 600 800 1 , 000 1 , 200 1 , 400 0 500 1 , 000 1 , 500 I n t eg r i n β 5 i n t en s i t y Vinculin intensity CytoD - treated adhesions ( 20 µ M ) DMSO - treated adhesions h m ES T a li n 1 – / – + C T R L s i RN A m ES T a li n 1 – / – + t a li n 2 s i RN A Talin Integrin β 5 g i Merge d Integrin β 5 Talin Merge f e 120 0 20 40 60 80 100 N o r m a li z ed c e ll nu m be r Integrin β 5 CytoD ( 20 µ M ) cRAD cRGD + + + + + + + + + + + + + + + CS1 - wt CS1 - β 5 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 0 1 2 3 4 l og 2 f o l d c hange i n m ean β 5 adhe s i on i n t en s i t y Residual talin intensity relative to control P = 0 . 01621 P = 0 . 04914 – 4 . 0 – 3 . 5 – 3 . 0 – 2 . 5 – 2 . 0 – 1 . 5 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 F o l d c hange i n m ean c e ll a r ea log 2 residual talin intensity relative to control Talin - 2 siRNA1 Talin - 2 siRNA2 Talin - 2 siRNA3 Talin - 2 siRNA1 Talin - 2 siRNA2 Talin - 2 siRNA3 P < 1 × 10 – 314 P < 1 × 10 – 314 P < 1 × 10 – 314 P = 1 . 86 × 10 – 7 P = 1 . 62 × 10 – 99 P = 1 . 78 × 10 – 166 I n t eg r i n β 5 2 G F P I n t eg r i n β 5 - β 3 t a il 2 G F P k j Vinculin Vinculin Merge Merge mCherryvinculin Fig . 4 | Reticular adhesions form in the absence of F - actin and talin . a , b , Confocal images of integrin β 5 - 2GFP and mCherry - vinculin in cells pre - treated in suspension and during spreading on VN with DMSO ( a ) or 20 µ M cytochalasin D ( CytoD ) ( b ) ( Supplementary Movie 5 ) . c , Cell area versus reticular ( red ) or focal ( blue ) adhesion number following the indicated treatments ( means ± / 95 % CI ; black lines , linear regression , 12 cells per condition , n = 7 , 018 focal and n = 4 , 570 reticular adhesions across three biologically independent experiments ) . d , Quantification of immuno - labelling intensities for vinculin and β 5 per adhesion in U2OS cells attached to VN and treated with DMSO ( blue ) or CytoD ( red ) . CytoD significantly reduces vinculin intensities but increases β 5 ( P values reflect two - sided unpaired Mann – Whitney U - testing , 2 , 533 adhesions from 22 DMSO - treated cells ; 1 , 410 adhesions from 18 CytoD - treated cells across n = 3 biologically independent experiments ) . e , Boxplots summarizing n = 6 biologically independent attachment assays using CS1 - wt ( lacking α V β 5 ) and CS1 - β 5 ( expressing α V β 5 ) cells in the presence or absence of 20 µ M CytoD and / or non - inhibitory cyclic RAD peptides and / or α V β 5 inhibitory cyclic RGD peptides . Cell attachment relative to maximum ( = 100 ) CS1 - β 5 + cRAD . Boxplot centre and box edges indicate median and quartiles while whiskers indicate median ± 1 . 5 × IQR or the most extreme observations within these limits . P values reflect two - sided unpaired t - testing with Holm – Bonferroni correction for multiple tests . f , g , Representative confocal images of mES talin - 1 – / – cells transfected with control ( f ) or talin - 2 - specific siRNAs ( g ) plated on VN and immuno - labelled against β 5 and talin - 2 . h , i , Single cell ( as in f and g ) based quantification of residual talin expression versus cell spread area ( h ) or mean β 5 intensity ( i ) in segmented adhesions standardized as fold - change relative to each internal control , summarized across n = 6 independent experiments . Linear regression P values : correspondence between residual talin levels and cell area or β 5 adhesion intensity . j , k , Confocal images ( representative of three biologically independent experiments ) of U2OS cells expressing integrin β 5 - 2GFP or integrin β 5 - β 3 - tail - 2GFP plated on VN and immuno - labeled against vinculin . Scale bars , 10 µ m . Source data for c – e and h , i are provided in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1295 Articles NATuRe Cell BIology The balance between reticular and focal adhesion complexes is shaped by PIP status . The putative reticular adhesion protein interaction network contains many components reported to bind phosphatidylinositol - 4 , 5 - bisphosphate ( PtdIns ( 4 , 5 ) P 2 ; Fig . 5b and Supplementary Table 2 ) . In five out of six cases where siRNA - mediated PIP ( phosphatidylinositol ) regulator depletion would be 60 199 18 60 53 Consensus integrin adhesome Consensus integrin adhesome U2OS steady - state adhesions Reticular adhesions ( CytoD enriched ) a b 0 2 4 6 8 10 12 Membrane organization Vesicle - mediated transport Membrane invagination Endocytosis Cell motion Regulation of defence response to virus Regulation of actin polymerization Cell migration Reticular adhesions : biological process 0 1 2 3 4 5 6 Endocytosis Huntington ' s disease Pathogenic escherichia coli infection Regulation of actin cytoskeleton Reticular adhesions : KEGG pathway c TAGLN2 MSN SPTBN1 MYH10 ACTR2 PDCD6IP ACTR3 EZR SPTAN1 ARPC2 HIP1R NCK2 HIP1 WASL SNTB2 CAP1 UTRN CLTA LAMC2 CLTC AP2B1 TNS3 EPS15L1 TFRC EPS15 ITSN1 FASN AP2A1 AP2A2 AP2M1 PRKDC PHGDH ATP1A1 TLN2 ITSN2 PICALM PIK3C2A TNS1 NUMB DAB2 PTPRF DYNC1H1 FLOT2 LDHB AHCY LTBP2 IARS SCRIB CLIC1 Direct binding of PtdIns ( 4 , 5 ) P 2 Indirect PtdIns ( 4 , 5 ) P 2 association ( via direct binding partner ) No clear PtdIns ( 4 , 5 ) P 2 association 0 . 999 0 . 411 Interaction confidence Localization CONFIRMED Integrin β 5 F - actin NUMB e d Integrin β 5 F - actin DAB2 F Merge f Merge F - actin F - actin Merge Merge Integrin β 5 Integrin β 5 GFP - Tln2 GFP - Tensin3 g h 1 – log 10 ( corrected P value ) – log 10 ( corrected P value ) Fig . 5 | Mass spectrometry reveals the distinct reticular adhesome . a , U2OS cells were plated in complete medium for 3 days on tissue culture plastic then treated with either DMSO ( steady state ) or CytoD ( reticular enriched ) and MS analysis was performed on the remaining adhesions after cell removal ( n = 3 biologically independent experiments ) . The Venn diagrams summarize overlap between proteins identified by MS analysis of ventral membrane preparations isolated from each condition overlaid with the 60 consensus fibronectin - adhesome proteins 5 . b , STRING interaction network of reticular adhesion enriched proteins with interaction confidence as indicated . PtdIns ( 4 , 5 ) P 2 binding ( direct , green ; indirect , yellow ; absent , grey ) is indicated . c , d , Gene ontology analysis of reticular adhesion enriched proteins ( CytoD - treated ) showing terms from Biological Process ( c ) and KEGG pathway analysis ( d ) significantly enriched over whole cell proteome . P values were derived from EASE scores calculated using a modified Fishers exact test with Holm – Bonferroni correction from multiple tests using the DAVID annotation system . e – h , Confocal images of U2OS cells cultured on glass coverslips for 72 h then treated with 20 µ M CytoD for 2 h and immuno - labelled against integrin β 5 and NUMB ( e ) DAB2 ( f ) or in cells transfected with EGFP - tensin - 3 ( g ) or EGFP - talin - 2 ( h ) ; along with staining of F - actin . Images in e – h are representative of three biologically independent experiments . Scale bars , 10 µ m ( main images ) and 5 µ m ( magnified regions in e and f ) . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1296 Articles NATuRe Cell BIology R A t o F A i n t en s i t y r a t i o ( Z sc o r e ) −2 0 2 4 6 Treatment ( siRNA ) PIP regulators NegCtrl PosCtrl −2 0 2 4 6 Z sc o r e Treatment ( inhibitor ) – 1 . 95 0 . 47 – 1 . 48 0 . 63 – 2 . 41 1 . 15 Neg controls PIK3C2A PIP2 regulators b c d P = 1 . 3 × 10 – 61 P = 1 . 9 × 10 – 107 P = 3 . 7 × 10 – 15 P = 6 . 7 × 10 – 33 P = 5 . 8 × 10 – 17 P = 5 . 4 × 10 – 3 P = 7 . 7 × 10 – 4 P = 1 . 7 × 10 – 3 P = 9 . 0 × 10 – 6 P = 3 . 4 × 10 – 147 P = 3 . 9 × 10 – 5 P = 4 . 6 × 10 – 37 P = 9 . 0 × 10 – 21 P = 5 . 8 × 10 – 176 P = 1 . 3 × 10 – 20 Mean RA intensity ( Z score ) Mean reticular adhesion intensity ( Z score ) Mean FA intensity ( Z score ) Mean focal adhesion intensity ( Z score ) Treatment ( siRNA ) Reticular : focal adhesion intensity ratio ( Z score ) Reticular : FA intensity ratio ( Z score ) s i G eno m e C T R L P I 4 KA s i RN A Nuclei F - actin Vinculin Integrin β5 Vinc + β 5 Merge P I K 3 C 2 A s i RN A a P I K 3 C 2 A P I P 5 K 1 C P I P 5 K 1 B P T E N P I 4 K 2 A P I 4 KA O CR L I T G B 5 I T G AV s i G E N O M E non − t a r ge t i ng c on t r o l O N - T A R G E T p l u s non − t a r ge t i ng c on t r o l SS T _ 3 C t r l _ P oo l M O C K U n t r ea t ed PIP5K1C PIP5K1B PTEN PI4K2A PI4KA OCRL PIK3C2A siGENOME non− targeting control ON - TARGETplus non− targeting control SST _ 3Ctrl _ Pool MOCK Untreated D M S O N eo _ 10 m M L Y _ 25 µ M D M S O N eo _ 10 m M L Y _ 25 µ M D M S O N eo _ 10 m M L Y _ 25 µ M Fig . 6 | Reticular versus focal adhesion balance is shaped by PiP status . a , Representative images illustrating DAPI ( nuclei ) and F - actin staining as well as localization of mCherry - vinculin and integrin β 5 - 2GFP fluorescence following treatment with control siRNA or siRNAs targeting PI4KA or PIK3C2A . Scale bars , 50 µ m . b , Boxplots summarizing single cell quantification ( from images in a ; n = 3 , 917 cells analysed , averaging 280 ± 131 ( s . d . ) per condition ) of integrin β 5 intensity ratios ( represented as Z - scores ) between reticular and focal adhesions following knockdown of various PtdIns ( 3 , 4 ) P 2 and PtdIns ( 3 , 4 , 5 ) P 3 regulators . Data are derived from two biologically independent experiments . Boxplot centre and box edges indicate median and 25th or 75th percentiles , respectively , and whiskers indicate the median ± 1 . 5 × IQR or the most extreme observations within these limits . Boxplot notches approximate 95 % CI ( see Methods for details ) . P values reflect two - sided unpaired Mann – Whitney U - testing with Holm – Bonferroni correction from multiple tests . c , Parallel coordinates plot displaying ( as Z - scores ) mean focal and reticular adhesion integrin β 5 intensities , as well as the ratio of reticular versus focal adhesion intensities , following knockdown of PtdIns ( 4 , 5 ) P 2 and PtdIns ( 3 , 4 , 5 ) P 3 regulators , based on n = 3 , 917 cells analysed , averaging 280 ± 131 ( s . d . ) per condition . Data are derived from two biologically independent experiments . d , Boxplots summarizing single cell quantification ( from images as shown in Supplementary Fig . 6b ; n = 3 , 018 cells analysed , averaging 1 , 006 ± 307 ( s . d . ) per condition ) of mean focal and reticular adhesion integrin β 5 intensity and ratio following 30 min treatment with 10 mM neomycin ( PIP2PtdIns ( 3 , 4 ) P 2 binding inhibition ) or 25 μ M LY294002 ( inhibition of PtdIns ( 3 , 4 , 5 ) P 3 generation ) . Boxplot features are as detailed above . P values reflect two - sided unpaired Mann – Whitney U - testing with Holm – Bonferroni correction from multiple tests . Data in d are derived from three biologically independent experiments . Source data for b – d are provided in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1297 Articles NATuRe Cell BIology expected to reduce PtdIns ( 4 , 5 ) P 2 levels ( PI4KA , PI4K2A , PIP5K1B , PIP5K1C and PTEN ) , a shift in β 5 - 2GFP intensity ratio was observed from reticular to focal adhesions ( Fig . 6a – c and Supplementary Fig . 6a ) . Correspondingly , depletion of PIK3C2A , which generates phosphatidylinositol - 3 , 4 , 5 - trisphosphate ( PtdIns ( 3 , 4 , 5 ) P 3 ) from PtdIns ( 4 , 5 ) P 2 , caused a relative shift from focal to reticular adhe - sions . Depletion of targets that produce PtdIns ( 4 , 5 ) P 2 reduced β 5 - 2GFP levels in both adhesion complex types ( Fig . 6c ) , yet , because the effects were more pronounced for reticular adhesions , the ratio to focal adhesions decreased . In contrast , PIK3C2A deple - tion perturbed only focal adhesions . Neomycin ( a PtdIns ( 4 , 5 ) P 2 binding inhibitor ) reduced β 5 - 2GFP intensities in reticular adhe - sions while increasing intensities in focal adhesions ( Fig . 6d and Supplementary Fig . 6b ) . Conversely , LY294002 ( a PtdIns ( 3 , 4 , 5 ) P 3 formation inhibitor ) increased reticular adhesion and reduced focal adhesion intensities . These findings indicate that focal and reticular adhesions are in equilibrium , with PtdIns ( 4 , 5 ) P 2 promoting reticu - lar adhesions and PtdIns ( 3 , 4 , 5 ) P 3 promoting focal adhesions . Reticular adhesions persist throughout division when focal adhesions disassemble . siRNA - mediated knockdown of integrin β 5 reduced cell proliferation ( Fig . 7a ) without affecting S - phase progression ( Fig . 7b ) . We therefore probed a potential role for β 5 in mitosis . Unlike classical adhesion complexes , reticular adhe - sions persisted throughout division ( Fig . 7c – i and Supplementary Movie 6 ) , remaining free of consensus adhesome components ( Supplementary Fig . 7a – e ) . In virtually all cells on purified lam - inin or fibronectin , where integrin β 1 is preferentially engaged ( Supplementary Fig . 7f , g ) , we detected no β 1 - labelled adhesion complexes during mitosis . β 1 - containing adhesion complexes were detected during mitosis only in normal human fibroblasts on fibro - nectin . In other cells , mitotic cells retained adhesion by cell – cell association . These results suggest a selective role for α V β 5 in mitotic cell attachment . The pre - mitotic footprint of the mother cell is transmitted with high precision to post - mitotic daughter cells ( Fig . 7c , d ) 31 , 32 . During the rounding phase of mitosis , this footprint was demarcated by membrane dye - labelled retraction fibres and integrin β 5 - 2GFP - labelled reticular adhesions ( Fig . 7c , f , g ) . The exquisite correspon - dence between retraction fibres ( Fig . 7h ) and reticular adhesions ( Fig . 7i ) was highlighted by 3D visualization of a similarly staged mitotic cell ( Fig . 7j – l and Supplementary Movie 7 ) . Here , retraction fibres angled down and attached precisely at sites decorated with β 5 - labelled reticular adhesions . A role for reticular adhesions in directing post - division cell spreading was also exemplified by live cell imaging ( Supplementary Movie 8 ) . Quantitative comparison of focal and reticular adhesions during division confirmed that the number and intensity of vinculin - positive focal adhesions fell to virtually zero during mitosis , while β 5 - positive reticular adhesion numbers and β 5 intensity were maintained ( Fig . 7m , n ) . As previ - ously reported , mitotic retraction fibres contain dense actin fila - ments ( Supplementary Fig . 7h , i and Supplementary Movie 9 ) . We detected weak F - actin signals in reticular adhesions at the tips of mitotic retraction fibres ( Supplementary Fig . 7h – k ) , with F - actin concentrations well below those within the retraction fibres , sug - gesting that reticular adhesions have limited coupling to F - actin fol - lowing mitotic cell rounding or that retraction fibres function via membrane tension and mediate adhesion independent of F - actin . Reticular adhesions are required for division and inter - gener - ational spatial memory transmission . A detailed comparison of reticular adhesion distributions before , during and after mitosis ( Fig . 8a – c and Supplementary Movie 10 ) indicated that the over - all geometry of central reticular adhesions remained virtually unchanged between generations , providing a potential mecha - nism for spatial memory storage . In contrast , peripheral reticular adhesions ( generally associated with mitotic retraction fibres ) underwent significant remodelling characterized by both narrow - ing and intensification of the complex ( Fig . 8d ) . Nanoscale STORM imaging confirmed that central mitotic reticular adhesions were indistinguishable in nano - organization from interphase reticular adhesions ( Fig . 3g – i versus Fig . 8d – f ) , while peripheral mitotic retraction fibre - associated reticular adhesions were linearized and condensed . This was confirmed by quantification of nanocluster nearest - neighbour distances and molecular localization counts per nanocluster ( Fig . 8e , f ) . Such molecular - scale remodelling function - ally implicates reticular adhesions in the mechanical process of cell – ECM attachment during division . Many cells exhibit a preference to divide along the major axis of the pre - mitotic mother cell , thus determining the spatial arrange - ment of daughter cells 8 – 11 . We therefore measured the residual angle between the pre - mitotic major axis and the mitotic division axis in HeLa cells ( Fig . 8g , h ) , chosen for their expression of reticular adhe - sions ( Supplementary Fig . 1b ) and their extensive mitotic char - acterization 33 . Residual angle distributions were skewed towards zero ( indicating spatial memory retention ) in control cells and β 5 - rescued cells . By contrast , mitotic axis orientation in integrin - β 5 - depleted cells was almost random relative to the pre - mitotic major axis , indicating a loss of spatial memory . Thus , reticular adhesions are required for intergenerational spatial memory transmission during division . Only 20 % of β 5 - depleted cells underwent normal division , ver - sus 75 % for controls ( Fig . 8i ) . A range of defects were observed in β 5 - depleted cells , including delayed mitosis ( often with incomplete cytokinesis ) , repeated cell rounding and re - spreading without divi - sion , and failure of cytokinesis resulting in binucleate daughter cells ( Supplementary Fig . 8a , b and Supplementary Movies 11 – 14 ) . The frequency of these errors 34 was reduced by β 5 - EGFP rescue ( Fig . 8i , Supplementary Fig . 8c , d and Supplementary Movie 15 ) . Together , these findings demonstrate that integrin β 5 - mediated reticular adhe - sions are essential for normal progression of division in HeLa cells . Discussion Here , we report the identification and characterization of a previ - ously unrecognized cellular structure , the reticular adhesion , an adhesion complex mediating cell – ECM attachment during mito - sis . Reticular adhesions form in a diverse array of cell types and are characterized by both the presence of integrin α V β 5 and the absence of consensus adhesome components . Furthermore , in con - trast to focal adhesions , reticular adhesions can form independently of F - actin and talin . Reticular adhesions persist throughout mitosis and provide a solution to the paradox of mitotic cell – ECM adhesion , which endures despite the absence of all previously known adhesion com - plexes 13 , 15 , 18 . Cell – ECM attachment is essential for spatial memory transmission between cell generations , including defining the axis of division 8 – 11 and facilitating cytokinesis 35 – 37 . So far , it has been unclear how residual adhesion is maintained during mitosis , how mitotic retraction fibres 38 are tethered to the substratum , and how re - spreading is guided thereafter . Reticular adhesions now pro - vide mechanisms underpinning all these phenomena . The unique characteristics of reticular adhesions appear suited to these roles in division . For instance , F - actin independence decouples reticu - lar adhesions from large - scale cytoskeletal remodelling during cell rounding , while the ability to interact with membrane retraction fibres is maintained . This key role for reticular adhesions in division is confirmed by integrin β 5 depletion , which causes multiple mitotic defects and disturbed spatial memory transmission . While we find an important role for α V β 5 during division , cells can also proliferate on ECM ligands not engaging α V β 5 . This implies that cells can deploy alternative adhesion receptors for mitotic anchorage . For example , integrin α 6 β 4 - positive hemides - NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1298 Articles NATuRe Cell BIology c d e g f h i – 120 min 0 min + 120 min – 120 min 0 min + 120 min – 120 min 0 min + 120 min M e m b r ane d y e m C he rr y v i n c u l i n I n t eg r i n β 5 2 G F P a j k l b m n – 20 0 20 40 60 80 100 120 – 170 – 150 – 130 – 110 – 90 – 70 – 50 – 30 – 10 10 30 50 70 90 110 130 150 170 – 170 – 150 – 130 – 110 – 90 – 70 – 50 – 30 – 10 10 30 50 70 90 110 130 150 170 Vinculin Vinculin Integrin β 5 Integrin β 5 P e r c en t o f m a x i m a l A C nu m be r pe r c e ll – 20 0 20 – 20 40 60 80 100 120 P e r c en t o f m ean A C i n t en s i t y pe r c e ll Time relative to mitotic event ( min ) Time relative to mitotic event ( min ) R e l a t i v e c e ll nu m be r Days 0 1 23 0 2 4 6 Control β 5 KD P = 0 . 1739 P = 0 . 0243 P = 1 . 06 × 10 – 5 Control β 5 KD 0 20 40 60 80 100 P e r c en t E DU po s i t i v e c e ll s P = 0 . 5337 Fig . 7 | Reticular adhesions persist during mitosis and transmit spatial memory from pre - mitotic to post - mitotic daughter cells . a , b , Proliferation of control or integrin β 5 knockdown U2OS cells over 3 days post attachment : mean ± s . d . of n = 14 replicates across three biologically independent experiments ; P values reflect two - sided unpaired t - testing relative to day zero ( a ) ; percentage of EdU - positive cells 3 days post attachment for n = 26 fields of view containing 55 – 217 cells each across three biologically independent experiments , with distribution of individual values in blue rings ( b ) . Boxplot centre and box edges indicate median and 25th or 75th percentiles , respectively , whiskers indicate median ± 1 . 5 × IQR or the most extreme observations within these limits . Boxplot notches approximate 95 % CIs . P values reflect two - sided unpaired t - testing for Control versus β 5 - KD . c , e – g , U2OS cells labelled with far - red membrane dye ( c ) and expressing mCherry - vinculin ( e ) and integrin β 5 - 2GFP ( f ) , replated on vitronectin and imaged every 10 min via spinning - disc confocal microscopy during mitosis ( Supplementary Movie 6 ) . Images show a cell 120 min before , during ( merged in g ) and 120 min after mitosis . d , Overlay of membrane labelling with cell boundaries outlined at − 120 min ( red ) , 0 min ( green ) and + 120 min ( blue ) , highlighting recovery of the pre - mitotic adhesion footprint by daughter cells . h , i , Membranous retraction filaments formed during mitosis ( h , cropped from blue region of interest in c ) overlap exactly with integrin β 5 - 2GFP - positive adhesion complexes ( i , cropped from yellow region of interest in f ) . Scale bars , 10 µ m ( c – g ) and 5 µ m ( h , i ) . ( j – l derived from Supplementary Movie 7 . ) j – l , Three alternative views ( above , j ; beside , k ; below , l ; orientation indicated by arrows in planar schematics ) of a 3D confocal - reconstructed mitotic cell showing condensed DNA ( white ) , cell membrane labelling ( red ; cut through to expose DNA ) and integrin β 5 - 2GFP labelling of reticular adhesions ( green ) . Images in c – l are representative of five biologically independent experiments . m , n , Quantification of vinculin - positive adhesion complex ( AC ) number ( m , blue ) and intensity ( n , blue ) versus β 5 - 2GFP - positive adhesion complex number ( m , red ) and intensity ( n , red ) during mitosis . Mean values from n = 5 cells are shown ± s . d . , derived from three biologically independent experiments . Source data for a , b , m and n are provided in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1299 Articles NATuRe Cell BIology mosomes persist through mitosis 39 , 40 and , despite disassembly of precursor focal adhesion complexes and loss of consensus adhesome components , residual clusters of integrin β 1 continue to decorate the cell – ECM interface in mitotic retinal pigment epithelial cells 41 . These β 1 integrin clusters differ from reticular adhesions because they are remnants of disassembled focal adhesions , while reticular adhesions represent a distinct adhesion complex population during both interphase and mitosis . Nonetheless , investigation of mitotic adhesion roles for alternative integrins , and their relationship to reticular adhesions , is now merited . The limited phenotype of β 5 knockout mice suggests redundancy of function among adhesion receptors and / or a specialized role for α V β 5 in regulating division within specific ECM environments . Both β 5 knockout 42 and overex - pression 43 in mice cause deficiencies in osteoblast / osteoclast func - tion , potentially reflecting mitotic defects related to differentiation errors 12 in cells on rigid , RGD - rich substrates . Indeed , these environ - ments may be analogous to long - term cell culture conditions , where we show that cells preferentially utilize integrin α V β 5 . As α V β 5 M - 50 min M + 30 min M - 50 and M + 30 Mitosis ( 0 min ) c a b I n t eg r i n β 5 2 G F P g Control β 5KD R e s i dua l ang l e Rescue Residual angle P r obab ili t y den s i t y 40 60 80 20 0 0 . 03 β 5KDControl Rescue 40 60 80 20 0 0 . 04 0 . 02 0 . 01 0 100 h i N o r m a l d i v i s i on pe r c en t age 40 60 80 20 0 100 Control β 5KD Rescue P = 7 . 1 × 10 – 13 P = 7 . 2 × 10 – 11 P = 0 . 278 P = 0 . 0014 P = 0 . 0103 P = 0 . 193 e f M o l e c u l a r l o c a li z a t i on pe r n ano c l u s t e r Non−retraction Retraction 0 100 200 300 N ea r e s t - ne i ghbou r d i s t an c e ( n m ) Non−retraction Retraction 0 50 100 200 82 . 4 50 . 6 48 59 W i de f i e l d d S T O R M ( non - r e t r a c t i on ) S T O R M ( r e t r a c t i on ) 150 Fig . 8 | Requirement of reticular adhesions for mitosis and post - mitotic re - spreading . a – c , Confocal images of integrin β 5 - 2GFP adhesions at three time points relative to mitosis ( – 50 min ( pre ) , 0 min , + 30 min ( post ) ) . b , c , Overlay of pre - and post - mitosis adhesions ( b ) , cropped and magnified in c , confirming the persistence of reticular adhesions throughout mitosis ( Supplementary Movie 9 ) . Images in a – c are representative of at least n = 5 biologically independent experiments . d , Left , Integrin β 5 in a representative U2OS mitotic cell plated on VN and imaged via conventional TIRF microscopy . Right , Representative central ( non - retraction , orange box ) and peripheral ( retraction , green box ) reticular adhesions cropped from matched conventional and STORM ( ‘royal’ look - up table intensity - scaled as in the legend ) images . e , f , Quantification of integrin β 5 nanocluster nearest - neighbour distances ( e ) and molecular localization counts per nanocluster ( f ) based on STORM data . In total , 95 retraction and 83 non - retraction mitotic reticular adhesions were quantified , including n = 3 , 512 nanoclusters across two biologically independent experiments . Boxplot centre and box edges indicate median and 25th or 75th percentiles , respectively , while whiskers indicate median ± 1 . 5 × IQR or the most extreme observations within these limits . Boxplot notches approximate 95 % CIs . Scale bars , 10 µ m ( a – c and d , left ) and 500 nm ( d , right ) . g – i , Comparison between control siRNA ( Control ; n = 297 cells ) and integrin β 5 knockdown ( β 5 KD ; n = 176 cells ) or post - knockdown β 5 rescue ( Rescue ; n = 195 cells ) effects on spatial memory transmission between HeLa cell generations , defined by residual angle measurement between the pre - mitotic cell major axis and the cell division axis ( data derived from two biologically independent experiments ) . Boxplots ( g , blue rings indicate individual cell measurements ) and probability density plots ( h ) indicate the distribution of residual angles . Boxplot centre and box edges indicate median and 25th or 75th percentiles , respectively , while whiskers indicate the median ± 1 . 5 × IQR or the most extreme observations within these limits . Boxplot notches approximate 95 % CIs . P values reflect two - sided unpaired Mann – Whitney U - testing . ( Based on Supplementary Fig . 8 and Supplementary Movies 10 – 14 . ) i , Plots showing the percentage of rounding cells that progressed through normal cell division in each of n = 3 biologically independent experiments . P values reflect two - sided unpaired t - testing . Source data for e – i are provided in Supplementary Table 1 . NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1300 Articles NATuRe Cell BIology is expressed at high levels in a number of proliferative diseases , this raises the possibility that it promotes disease progression by enhancing division within specific ECM environments . It is now important to determine the role of α V β 5 and reticular adhesions in vivo , within physiological and disease settings . In this context , a focal adhesion - independent role for α v β 5 in three - dimensional skin formation and tumour invasion has been reported 44 . Remarkably , reticular adhesions have remained uncharacterized , although early studies reported similar reticular α v β 5 labelling pat - terns in cells spread on vitronectin 45 . The experimental induction of morphologically comparable structures , such as through manga - nese - or talin - head - mediated activation of α V β 3 ( refs 28 , 29 ) , suggests the potential for other integrins to form similar structures given modulation of their activity state . Reticular adhesions lack not only F - actin , but virtually all consensus adhesome components . Most notably , both talin - 1 and kindlin are absent , despite being consid - ered necessary and ubiquitous integrin activators 46 . Moreover , per - turbations of talin and F - actin indicate that reticular adhesions can form independently of these proteins . Proteomic analysis of reticular adhesions identified a distinct adhesome , highly enriched in PtdIns ( 4 , 5 ) P 2 - binding proteins . These include clathrin - mediated endocytosis adaptors , such as Dab2 and Numb , previously shown to interact directly with the integrin β 5 cytoplasmic tail in vitro 47 . These data are consistent with recent evidence of integrin - mediated ECM attachment via clathrin - coated structures 48 , 49 . Indeed , integrin β 5 can localize within clath - rin plaques 48 , 50 – 52 that are postulated to associate with areas of strong adhesion 34 , 53 – 56 . It will be important to determine whether reticular adhesions associate with clathrin lattices to facilitate this adhesion and whether clathrin structures remain associated with the substra - tum during mitosis . Given that both reticular adhesions and clath - rin - coated structures can form in the absence of talin , it follows that some integrins may not depend on talin for their activation 49 . In this context , it is also notable that we observe near identical nanoscale integrin β 5 clustering between talin - 1 - positive and - negative adhe - sion complexes during interphase , despite previous suggestions that talin - 1 determines nanoscale integrin organization 6 . Thus , both in terms of integrin activation and organization , it is possible that either alternative proteins can replace talin - 1 functions in reticular adhesions , or that β 5 ligand - binding and nanoscale organization are independent of cytosolic regulators . Regardless , the composition , regulation and function of integrin - mediated adhesion complexes appear more diverse than previously recognized . In conclusion , we have defined reticular adhesions , an over - looked cellular structure and adhesion complex class . Functionally , by mediating cell – ECM attachment during mitosis , reticular adhe - sions provide a distinctive solution to the paradox of mitotic cell attachment , where classical adhesion complexes must disassemble but cells must also remain adherent . These discoveries not only delineate a specific form of adhesion complex , but also highlight areas of adhesion biology that merit further attention , including the integrins and adhesion complexes employed in vivo . Online content Any methods , additional references , Nature Research reporting summaries , source data , statements of data availability and asso - ciated accession codes are available at https : / / doi . org / 10 . 1038 / s41556 - 018 - 0220 - 2 . Received : 14 December 2017 ; Accepted : 21 September 2018 ; Published online : 22 October 2018 References 1 . Lock , J . G . , Wehrle - Haller , B . & Strömblad , S . Cell - matrix adhesion complexes : master control machinery of cell migration . Semin . 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Akisaka , T . , Yoshida , H . , Suzuki , R . , Shimizu , K . & Takama , K . Clathrin sheets on the protoplasmic surface of ventral membranes of osteoclasts in culture . J . Electron Microscop . 52 , 535 – 543 ( 2003 ) . 54 . Saffarian , S . , Cocucci , E . & Kirchhausen , T . Distinct dynamics of endocytic clathrin - coated pits and coated plaques . PLoS Biol . 7 , e1000191 ( 2009 ) . 55 . Nicol , A . & Nermut , M . A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy . Eur . J . Cell Biol . 43 , 348 – 357 ( 1987 ) . 56 . Akisaka , T . , Yoshida , H . , Suzuki , R . & Takama , K . Adhesion structures and their cytoskeleton - membrane interactions at podosomes of osteoclasts in culture . Cell Tissue Res . 331 , 625 – 641 ( 2008 ) . Acknowledgements The authors thank D . Critchley ( University of Leicester ) for providing talin - 1 - null embryonic stem cells and Z . Li for assistance with the integrin β 5 / β 3 - EGFP construct . This work was supported by grants to S . S . from the EU - FP7 - Systems Microscopy Network of Excellence ( HEALTH - F4 - 2010 - 258068 ) , EU - H2020 - Multimot ( EU grant agreement no . 634107 ) , the Strategic Research Foundation of Sweden ( SB16 - 0046 ) , the Swedish Research Council ( 521 - 2012 - 3180 ) and the Swedish Cancer Society , and by a grant to M . J . H . from Cancer Research UK ( grant no . 13329 / A21671 ) . Imaging was in part performed at the live cell imaging facility and Nikon Center of Excellence at the Department of Biosciences and Nutrition , Karolinska Institutet , supported by grants from the Knut and Alice Wallenberg Foundation , the Swedish Research Council , the Centre for Innovative Medicine and the Jonasson donation to the School of Technology and Health , Royal Institute of Technology , Sweden . The authors also acknowledge support from the Bioimaging and Bio - MS core facilities at the University of Manchester . The mass spectrometers and microscopes used in this study were purchased with grants from BBSRC , Wellcome Trust and the University of Manchester Strategic Fund . The funders had no role in study design , data collection and analysis , decision to publish or preparation of the manuscript . J . A . A . and X . G . contributed equally to this paper . Author contribution J . G . L . , M . C . J . , J . A . A . , M . L . , M . J . H . and S . S . conceived the project and devised experiments . J . G . L . and J . A . A . performed live cell imaging and related image analyses . J . G . L . , M . C . J . , J . A . A . , X . G . and H . O . performed fixed cell imaging . X . G . performed the siRNA screening for PIP regulators . M . C . J . and J . A . A . undertook experiments relating to integrin β 5 RNAi and MS analyses . A . O . performed STORM imaging and related image analyses . H . O . and S . G . performed immunoblotting . J . L . performed image analyses , statistical analyses and data visualization . J . G . L . , M . C . J . , J . A . A . , X . G . , M . L . , M . J . H . and S . S . contributed to writing the manuscript . Competing interests The authors declare no competing interests . Additional information Supplementary information is available for this paper at https : / / doi . org / 10 . 1038 / s41556 - 018 - 0220 - 2 . Reprints and permissions information is available at www . nature . com / reprints . Correspondence and requests for materials should be addressed to J . G . L . or S . S . Publisher’s note : Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations . © The Author ( s ) , under exclusive licence to Springer Nature Limited 2018 NAtuRe CeLL BiOLOGy | VOL 20 | NOVEMBER 2018 | 1290 – 1302 | www . nature . com / naturecellbiology 1302 Articles NATuRe Cell BIology Methods Cell culture , plasmid generation , transfection and stable cell generation . Cell culture . U2OS human osteosarcoma cells ( ATCC ) , HeLa human cervical carcinoma cells ( ECACC ) , MCF - 7 human breast carcinoma cells ( ATCC ) , A549 human lung carcinoma cells ( ECACC ) and A375 human melanoma cells ( ECACC ) were maintained in DMEM ( Gibco ) supplemented with 10 % FBS ( Sigma ) and 2 mM l - glutamine ( Gibco ) . U2OS - β 5V cells stably expressing integrin β 5 - 2GFP and mCherry - vinculin were maintained with the addition of 600 µ g ml − 1 geneticin ( G - 418 sulfate ; Gibco ) . H1299 human non - small lung cancer cells ( gift from B . Geiger , The Weizmann Institute of Science ) and CS - 1 wild - type hamster melanoma cells were cultured in RPMI - 1640 ( Gibco ) medium supplemented with 10 % FBS and 5 mg ml − 1 l - glutamine . CS - 1 cells stably expressing integrin β 5 ( CS1 - β 5 ) were maintained with the addition of 500 µ g ml − 1 G - 418 . BT549 ( ductal breast carcinoma , ATCC ) cells were maintained in RPMI 1640 medium containing 10 % FBS and 1 mM l - glutamine . MAE cells ( ATCC ) were grown in RPMI 1640 medium with 5 % FBS . Human hTERT immortalized retinal pigment epithelial ( ( hTERT - RPE1 ) cells ( gift from J . Mansfeld , University of Dresden ) were cultured in DMEM / F12 ( Gibco ) supplemented with 10 % FBS and 2 mM l - glutamine . Normal human dermal fibroblasts ( NHDFs ) ( Biowhittaker ) were grown in DMEM ( Gibco ) supplemented with 10 % FBS ( Sigma ) and hTERT - human microvascular endothelial cells ( HME1 ) ( ATCC ) were grown in MEGM ( Lonza ) supplemented with MEGM BulletKit ( Lonza ) . All live cells were incubated and imaged in a humidified environment at 37 °C with 5 % CO 2 . DNA plasmid generation and sourcing . For construction of integrin β 5 - 2GFP , EGFP was duplicated in a pEGFP - N1 backbone vector ( gift of P . Caswell , University of Manchester ) , then a full - length integrin β 5 cDNA ( gift of E . Ruoslahti , Burnham Institute ) was subcloned into the 2XEGFP - N1 vector using the EcoRI site of the original pEGFP - N1 vector . The mCherry - vinculin plasmid was provided by V . Small ( IMBA ) . Csk - GFP was provided by A . Imamoto ( University of Chicago ) . GFP - Tensin3 was provided by P . Caswell ( University of Manchester ) and GFP - talin2 was provided by B . Goult ( University of Kent ) . RFP - talin1 Head and Rod constructs were provided by M . Parsons ( King’s College ) . LifeAct - RubyRed was provided by R . Wedlich - Soldner ( Max - Planck Institute for Biochemistry ) . Transfection and stable cell line generation . Cells were transfected at 70 – 90 % confluence , 24 h after plating into 12 - well culture plates ( except where otherwise stated ) . For DNA plasmid transfection , 0 . 3 – 2 µ g of total DNA was mixed with 0 . 5 – 3 µ l of Lipofectamine Plus or Lipofectamine 2000 ( Thermo Fisher Scientific ) according to the manufacturer’s instructions . For RNA transfection , except where otherwise stated , 15 – 30 pmol of siRNA was transfected together with 0 . 5 – 3 µ l of RNAiMAX ( Thermo Fisher Scientific ) . Cells were typically imaged 24 – 48 h after transfection . U2OS - β 5V cells expressing integrin β 5 - 2GFP and mCherry - vinculin were established via manual single colony selection followed by selection with 600 µ g ml − 1 G - 418 . ECM surface coating . Cells were typically assayed in 96 - well glass - bottomed plates ( 0 . 17 mm optical glass ; Matrical Bioscience ) . Glass coating was performed at 37 °C for 2 h after blocking with 1 % heat - denatured bovine serum albumin ( BSA ; Sigma - Aldrich ) for 2 h at 37 °C . ECM ligand coating concentrations were 10 µ g ml − 1 except where otherwise indicated . Vitronectin and fibronectin were purified from human plasma as detailed previously 57 , 58 , while purified laminin was acquired commercially ( Sigma - Aldrich ) . Antibodies , immunofluorescence labelling and immunoblotting . Primary antibodies used for immunofluorescence and / or immunoblotting are summarized in Supplementary Table 4 and include anti - integrin α V β 5 ( 15F11 ; MAB2019Z ; Millipore ) , anti - integrin α V β 5 ( P1F6 ; Abcam ) , polyclonal ( rabbit ) anti - integrin β 5 ( ab15459 ; Abcam ) , anti - integrin β 5 ( 4708S ; Cell Signalling Technology ) , anti - integrin α V ( LM142 ; Merck Millipore ) , anti - talin2 ( 53 . 8 ; BioRad ) , anti - talin ( 8d4 ; Sigma Aldrich ) , anti - talin 1 ( TA205 ; Santa Cruz ) , anti - talin - 2 ( 68E7 ; Abcam ) , anti - integrin α V β 3 ( LM609 ; Abcam ) , anti - integrin β 3 ( AP3 ; Abcam ) , anti - integrin β 1 ( LM534 ; Millipore ) , anti - vinculin ( hVIN - 1 ; Sigma - Aldrich ) , anti - vinculin ( V9131 ; Sigma - Aldrich ) , anti - intersectin 1 ( HPA018007 ; Atlas Antibodies , Sigma - Aldrich ) , anti - NUMB ( 2733 ; Cell Signaling Technologies ) , anti - EPS15L1 ( HPA055309 ; Atlas Antibodies , Sigma - Aldrich ) , anti - HIP1 ( HPA013606 ; Atlas Antibodies , Sigma - Aldrich ) , anti - WASL ( HPA005750 ; Atlas Antibodies , Sigma - Aldrich ) , anti - DAB2 ( 12906 ; Cell Signaling Technologies ) , anti - paxillin ( 5H11 ; Sigma - Aldrich ) , anti - FAK ( BD Biosciences ) , anti - zyxin ( H - 200 ; Santa Cruz ) , anti - kindlin 2 ( ab74030 ; Abcam ) , anti - ICAP1 ( 115228 ; Abcam ) , anti - DOK1 ( HPA048561 ; Atlas Antibodies , Sigma - Aldrich ) , polyclonal ( rabbit ) anti - phosphotyrosine ( 1000 ) ( Cell Signaling ) , anti - cytokeratin ( 27988 ; Abcam ) anti - α - tubulin ( DM1A ; Thermo Fisher Scientific ) , anti - vimentin ( 8978 ; Abcam ) and anti - ARP3 ( ab49671 ; abcam ) . Anti - mouse and anti - rabbit secondary antibodies conjugated with Alexa 488 , 568 or 647 were used as appropriate ( Thermo Fisher Scientific ) . For fixed F - actin labelling , phalloidin pre - conjugated with Alexa 488 , 568 or 647 was used as appropriate ( Thermo Fisher Scientific ) . DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole dihydrochloride ; Thermo Fisher Scientific ) nucleic acid stain was used as a nuclear marker as appropriate . Immunofluorescence labelling was performed either manually or using liquid - handling robotics ( Freedom EVO , Tecan ) to minimize experimental variability , as described previously 59 . In either case , standardized procedures were used except where otherwise stated . Cells were fixed with 4 % paraformaldehyde ( PFA ; Sigma - Aldrich ) for 20 min , washed three times with phosphate - buffered saline ( PBS ) and permeabilized using 0 . 1 % TX - 100 ( Sigma - Aldrich ) for 5 min at room temperature . Cells were then blocked for 15 min with 1 % BSA in PBS ( PBS / BSA ) . Primary antibody immuno - labelling then proceeded at room temperature for 30 min . After PBS / BSA washing , secondary antibodies conjugated with either Alexa 488 , 568 or 647 fluorophores were applied for 30 min at room temperature . Finally , cells were washed three times with PBS . Immunoblotting was performed on SDS – polacrylamide gels with proteins transferred to Immobilon - P - Membranes ( Millipore ) . Membranes were probed with anti - talin - 2 mouse monoclonal ( 68E7 ; Abcam ) at 1 : 500 dilution , anti - α - tubulin ( DM1A ; Thermo Fisher Scientific ) at 1 : 500 dilution , or anti - integrin β 5 ( 4708S ; Cell Signalling Technology ) at 1 : 1 , 000 dilution . Proteins were detected using enhanced chemiluminescence ( ECL ) reagent ( Amersham Pharmacia Biotech ) . Imaging . Live - and fixed - cell imaging was primarily performed using either a Nikon Ti2 - mounted A1R confocal microscope running NIS elements software ( Nikon ) with a PlanApo VC × 60 / 1 . 4 numerical aperture ( NA ) oil - immersion objective or a Leica TCS SP5 acousto - optical beamsplitter confocal microscope using a × 63 objective ( HCX Plan Apochromat , NA 1 . 25 ) and LCS software ( Leica ) . Live fluorescence imaging during cell division was carried out with a Yokagoawa CSU - X1 spinning - disk confocal and Andor electron - multiplying charge - coupled device ( EM - CCD ) . TIRF imaging employed a Nikon Ti2 inverted microscope configured for minimal ( ~ 90 nm ) evanescence wave penetration . Live - cell imaging intervals were 0 . 5 – 5 min over 1 – 8 h with pixel resolutions between 0 . 13 and 0 . 21 µ m . Live cells were maintained in normal culture medium , absent FCS / FBS ( fetal calf serum / fetal bovine serum ) , at 37 °C and 5 % CO 2 . Live cell interference reflection microscopy ( IRM ) made use of a Zeiss LSM 510 confocal microscope and Plan - Apochromat × 63 / 1 . 4 NA oil objective , with post - sample dichroic mirror displacement allowing reflected laser light ( 561 nm ) detection . Fluorescence recovery after photobleaching ( FRAP ) analyses were performed via confocal microscopy and analysed as described previously 60 . Briefly , three sequential images were acquired of integrin β 5 - 2GFP and mCherry - vinculin in U2OS - β 5V cells before bleaching , enabling robust recovery standardization . Both reticular and focal adhesions ( 2 – 3 each per cell ) were then bleached using 35 % of maximal 488 nm laser power over 40 rapid iterations ( < 3 s per cell ) . Recovery was monitored for a total of 1 , 875 s , with intervals of 6 s for the first 120 s and intervals of 45 s thereafter . STORM was performed in U2OS cells fixed during either interphase or mitosis . Cells were labelled using rabbit polyclonal anti - integrin β 5 antibody ( ab15459 ) and with Alexa 405 – Alexa 647 double - labelled secondary . Secondary antibodies ( Jackson ImmunoResearch ) were labelled in house , as previously described 61 . A Nikon N - STORM system with Apo internal reflection fluorescence × 100 / 1 . 49 NA objective was used , with images acquired using an EM - CCD camera . Before STORM imaging , TIRF images of integrin β 5 and mCherry - vinculin were acquired , enabling diffraction - limited definition of reticular and focal adhesions using the criteria detailed in the section ‘Image analysis’ . Thereafter , 647 nm laser light excited Alexa 647 , with 405 nm light used for reactivation . Standard STORM imaging buffer was used , containing 100 mM Cysteamine MEA , 0 . 5 mg ml − 1 glucose oxidase , 40 µ g ml − 1 catalase and 5 % glucose ( all Sigma - Aldrich ) . Image analysis . Patch Morphology Analysis Dynamic software ( Digital Cell Imaging Laboratories ) was used for analysis of static ( fixed ) and dynamic ( live ) cell imaging data , except where otherwise specified . Analysis strategy and parameterization were as described previously 20 , 21 , 59 , 62 . Briefly , both cells and intracellular adhesion cohorts were segmented according to pixel intensity gradient analysis . A variety of morphological , pixel intensity and dynamic properties were then extracted for each cell and for each adhesion 21 . Relationships between each adhesion and its ( parent ) cell were maintained . Minimal adhesion size was set to 0 . 3 µ m 2 . For live cell data , adhesion tracking parameters included linear motion interpolation over a maximum of one missing time point , 3 µ m maximum adhesion step size per time point and four time point minimum track lifetime . When quantifying differences between reticular and focal adhesions , we used the absence or presence ( respectively ) of canonical adhesome components as a defining indicator . Specifically , we applied a threshold such that segmented adhesions ( delineated by integrin β 5 ) were defined as reticular if they contained less than the mean of background fluorescence values ( pixel intensities inside the cell boundary but outside segmented adhesions ) plus two standard deviations for a canonical adhesion marker ( vinculin or talin ) . Integrin β 5 - positive adhesions with greater than this value of fluorescence ( for the canonical adhesion marker ) were classed as focal adhesions . For FRAP analyses , PAD software was used to segment integrin β 5 - 2GFP - positive adhesions found in the last ( third ) pre - bleach image frame . Focal and reticular adhesions were distinguished based on mCherry - vinculin content , as described above . Identical adhesion boundaries ( from pre - bleach frame 3 ) were then used as fluorescence recovery measurement locations for all subsequent image NAtuRe CeLL BiOLOGy | www . nature . com / naturecellbiology Articles NATuRe Cell BIology frames . Adhesions judged to move during this period were excluded from further analysis . Integrin β 5 - 2GFP fluorescence recovery curves were first standardized relative to intensity fluctuations ( including non - specific photobleaching ) in non - bleached areas of the cell . Thereafter , intensity values in bleached regions were standardized per adhesion as a percentage of the mean of the three pre - bleached images . The standard deviation of percentage recovery , per time point , was also recorded . Recovery curves are displayed as mean per time point ( circles ) ± 95 % confidence intervals ( CIs ; per time point ) . Loess regression defined a smoothed fit ( line ) ± a moving 95 % CI envelope . Statistical differences between Loess fitted curves were assessed via two - sided Kolmogorov – Smirnov testing . STORM data were analysed using Insight3 software ( developed by Bo Huang , University of California ) . Localization coordinates were first precisely defined via Gaussian fitting . Next , reticular and focal adhesions were segmented and defined using conventional TIRF images of integrin β 5 and vinculin , based on the thresholding criteria detailed above . Clustering was then performed on integrin β 5 localizations within each adhesion type , revealing coordinate position and localization counts for integrin nanoclusters found within each adhesion . DBSCAN was used for clustering 63 , with epsilon ( search radius ) set to 10 nm and minimum points ( within epsilon radius ) set to 3 . Nearest - neighbour distances between nanoclusters and localization numbers per cluster were assessed for each adhesion type using R . Three - dimensional rendering and animation of confocal images was performed using NIS elements software . Additional supplementary movies were prepared in FiJi software 64 . MS analysis of the reticular adhesome . Four 10 - cm - diameter dishes per condition of U2OS cells were cultured for 48 h to 90 % confluency then treated with either DMSO or 20 µ M cytochalasin D ( Sigma - Aldrich ) for 2 h . To isolate adhesion complexes , cells were incubated with the membrane - permeable crosslinker dimethyl - 3 , 3 ′ - dithiobispropionimidate ( DTBP , Sigma - Aldrich ; 6 mM , 5 min ) . DTBP was then quenched using 1 M Tris ( pH 8 . 5 , 2 min ) , after which cells were again washed once using PBS and incubated in PBS at 4 °C . Cell bodies were then removed by a combination of cell lysis in RIPA buffer ( 50 mM Tris - HCl , pH 8 . 0 , 5 mM EDTA , 150 mM NaCl , 1 % ( wt / vol ) TX - 100 , 1 % ( wt / vol ) sodium deoxycholate ( DOC ) , 0 . 5 % ( wt / vol ) SDS ; 3 min ) and a high - pressure water wash ( 10 s ) . Protein complexes left bound to the tissue culture dish were washed twice using PBS , recovered by scraping in 200 µ l recovery solution ( 125 mM Tris - HCl , pH 6 . 8 , 1 % ( wt / vol ) SDS , 15 mM dithiothreitol ) , and incubated at 70 °C for 10 min . Each sample was subsequently precipitated from solution by addition of four volumes of − 20 °C acetone , incubated for 16 h at − 80 °C , and resuspended in reducing sample buffer . For MS , samples were separated by SDS – PAGE on a 4 – 12 % SDS Bis - Tris gel ( Thermo Fisher ) , stained for 10 min with Instant Blue ( Expedeon ) , and washed in water overnight at 4 °C . Gel pieces were excised and processed by in - gel tryptic digestion as previously described 4 . Peptides were analysed by liquid chromatography ( LC ) – tandem MS ( MS / MS ) using an UltiMate 3000 Rapid Separation LC ( RSLC , Dionex Corporation ) coupled to an Orbitrap Elite mass spectrometer ( Thermo Fisher ) . Peptides were separated on a bridged ethyl hybrid C18 analytical column ( 250 mm × 75 μ m inner diameter , 1 . 7 μ m particle size , Waters ) over a 1 h gradient from 8 to 33 % ( vol / vol ) ACN in 0 . 1 % ( vol / vol ) FA . LC – MS / MS analyses were operated in data - dependent mode to automatically select peptides for fragmentation by collision - induced dissociation ( CID ) . Quantification was performed using Progenesis LC – MS software ( Progenesis QI , Nonlinear Dynamics ; http : / / www . nonlinear . com / progenesis / qi - for - proteomics / ) . In brief , automatic alignment was used , and the resulting aggregate spectrum filtered to include + 1 , + 2 and + 3 charge states only . A . mgf file representing the aggregate spectrum was exported and searched using Mascot ( one missed cleavage , fixed modification : carbamidomethyl [ C ] ; variable modifications : oxidation [ M ] ; peptide tolerance : ± 5 ppm ; MS / MS tolerance : ± 0 . 5 Da ) , and the resulting . xml file was re - imported to assign peptides to features . Three separate experiments were performed , and abundance values for proteins identified in the analysis were used to determine which proteins were enriched over twofold following treatment with cytochalasin D . Although 53 proteins were detected in the original MS data , four were excluded from further analysis due to their high representation in the CRAPome database 30 . The putative reticular adhesome interaction network was constructed using the online STRING protein – protein interaction database ( v . 10 ) 65 including experimentally validated interactions only , with a ‘medium’ interaction confidence score ( > 0 . 4 ) . Even at higher confidence ( interaction confidence score > 0 . 7 ) , this interaction network is dense : 91 known interactions relative to 11 randomly expected ( based on proteome - wide interaction frequencies ) . Biological process - and KEGG pathway - enrichment analyses were performed using the DAVID Bioinformatics resource 66 . PIP regulator siRNA screening and drug - based perturbation of PtdIns ( 4 , 5 ) P 2 and Arp2 / 3 . U2OS - β 5V cells were treated with pooled siRNAs ( four siRNAs per target ; ON - TARGET SMART Pool plus ; Dharmacon ) via reverse transfection in the inner 60 wells of 96 - well optical glass plates . Each plate contained five negative ( untreated , mock transfected and three non - targeting siRNA controls ) and three positive targeting controls ( against EGFP , integrin α v or integrin β 5 ) . The primary screen was repeated twice , with a secondary validation assay using four siRNAs individually per target ( Dharmacon ) also repeated twice . siRNA sequences are presented in Supplementary Table 5 . To prepare the siRNA library , 1 μ l of each siRNA pool from 2 μ M stock was mixed with 30 μ l nuclease - free water and added to 96 - well glass - bottomed - plate wells , before drying at room temperature . For reverse transfection , 30 µ l of RNAiMAX was first added to 9 ml of Opti - MEM ( Thermo Fisher Scientific ) . Then , 30 µ l of this mixture was added to each well , followed by 30 min incubation . U2OS - β 5V cells ( 90 % confluent ) grown in 75 cm 2 flasks were trypsinized and resuspended with 30 ml of growth medium . A 100 µ l volume of the resulting cell suspension was added to each well and pipetted five times to disperse cells . The final siRNA concentration was 15 nM . Cells were incubated for 48 h before fixation with 4 % PFA ( 15 min ) and subsequent permeabilization with 0 . 2 % TX - 100 in PBS . Finally , cells were incubated for 1 h with DAPI and Alexa 647 - conjugated phalloidin before PBS washing three times . Cells were imaged with a Nikon A1R confocal microscope with a PlanApo VC 60X / 1 . 4 NA oil - immersion objective . Image settings were identical for all samples and repeats . Montage images were acquired and stitched in NIS elements software , enabling high - resolution acquisition of ~ 100 cells and ~ 5 , 000 adhesions per condition per experimental repeat . Image data were quantified and analysed using KNIME software . Individual cells were segmented using Voronoi tessellation based on DAPI ( nuclei ) and phalloidin ( cell body ) staining . Integrin β 5 - positive adhesions were then segmented and split using spot detection and the Wählby method 67 , respectively . Focal and reticular adhesions were defined based on mCherry - vinculin content as described above . Background - corrected intensity values were extracted per channel , for each adhesion , per cell . Mean integrin β 5 intensity values in reticular adhesions were divided by values in focal adhesions to generate the relative intensity ratio . All values were Z - score standardized using robust statistics ( median and median absolute deviation ) relative to the combination of ( three ) non - targeting siRNA controls per 96 - well plate . Resulting response distributions were plotted using R and RStudio software . U2OS - β 5V cells cultured and plated as described above , including 48 h incubation in 96 - well optical glass plates , were treated for 30 min with DMSO ( control ) , 10 mM neomycin ( PtdIns ( 4 , 5 ) P 2 binding inhibition ) or 25 μ M LY294002 ( inhibition of PtdIns ( 3 , 4 , 5 ) P 3 generation ) . For treatment with Arp2 / 3 inhibitor , U2OS cells plated onto glass coverslips and cultured for 48 h were treated for 2 h with 50 μ M CK - 666 ( Arp2 / 3 inhibitor ) or 50 μ M CK - 689 ( Arp2 / 3 inhibitor control ; inactive analogue of CK - 666 ) . Cells were then fixed , permeabilized and labelled as described above . Imaging and analysis were again performed using KNIME , as described above for siRNA screening . Talin knockdown and response analysis . Talin - 1 - null mouse embryonic stem cells ( mES talin 1 – / – ; gift from D . Critchley , University of Leicester ) were transfected using RNAiMAX ( Thermo Fisher Scientific ) according to the manufacturer’s instructions using either non - targeting control siRNA ( ON - TARGETplus non - targeting control ; 5 ′ - UGGUUUACAUGUCGACUAA - 3 ′ ) or talin - 2 - specific siRNAs designated talin - 2 siRNA1 ( 5 ′ - GCAGAAUGCUAUUAAGAAAUU - 3 ′ ) , talin - 2 siRNA2 ( 5 ′ - CCGCAAAGCUCUUGGCUGAUU - 3 ′ ) or talin - 2 siRNA3 ( 5 ′ - AAGUCAGUAUUACGUUGUUUU - 3 ′ ) . siRNAs were synthesized by GenePharma . Cells were incubated for 48 h then plated for 3 h on 10 µ g ml − 1 vitronectin . Fixation and permeabilization conditions were tuned to retain cytoplasmic talin - 2 , as described previously 59 . Briefly , labelling was performed using liquid - handling robotics ( Freedom EVO , Tecan ) to reduce experimental variability . Cells were fixed with 2 % PFA for 10 min , washed with PBS , and permeabilized using 0 . 1 % TX - 100 for 5 min at room temperature . Cells were then blocked for 15 min with 1 % PBS / BSA . Immuno - labelling followed at room temperature for 30 min , targeting integrin β 5 ( polyclonal Ab ; ab15459 , Abcam ) and talin ( pan - talin mouse monoclonal Ab ‘53 . 8’ , BioRad ) or anti - talin - 2 mouse monoclonal Ab ‘68E7’ ( Abcam ) . After 1 % PBS / BSA washing , Alexa 488 and 647 secondary antibodies were applied , targeting rabbit and mouse primary antibodies , respectively . Images of integrin β 5 and residual talin - 2 were acquired with a Nikon A1R confocal and oil - immersion objective ( PlanApo VC × 60 / 1 . 4 NA ) . Image analysis was performed using PAD software to record residual talin ( mean ) intensities per cell , mean β 5 intensities per segmented adhesion ( per cell ) and cell area . Values were scaled as fold - change relative to control siRNA . A total of 20 – 40 cells were imaged per condition in each of four experimental repeats with talin - 2 siRNA1 , or single confirmatory experiments with talin - 2 siRNA2 and 3 . Immunoblotting was performed as described above . Integrin β 5 knockdown and mitotic analysis . siRNA used for knockdown of β 5 targeted the sequence 5 ′ - GGGAUGAGGUGAUCACAUG - 3 ′ and was obtained from Dharmacon . For rescue of β 5 expression , an siRNA - resistant WT β 5 - EGFP clone was generated using the QuickChange IIXL site - directed mutagenesis kit ( Agilent Technologies ) to introduce silent mutations in the siRNA target sequence . The primers were forward 5 ′ - AGCCTATGCAGGGACGAAGTTATTACCTGGGTGGACACC - 3 ′ and reverse 5 ′ - GGTGTCCACCCAGGTAATAACTTCGTCCCTGCATAGGCT - 3 ′ ( obtained from Eurofins Genomics ) . Cells were transfected simultaneously with either non - targeting or β 5 siRNA together with either EGFP alone ( pEGFP - N1 empty vector ; gift of P . Caswell , NAtuRe CeLL BiOLOGy | www . nature . com / naturecellbiology Articles NATuRe Cell BIology University of Manchester ) or WT β 5 - GFP using Lipofectamine 2000 ( Thermo Fisher ) according to the manufacturer’s instructions . Six hours after transfection , cell cycle synchronization was initiated by adding 2 mM thymidine ( Sigma ) . After 18 h , cells were released by replating in fresh medium , and a second dose of thymidine was added 8 h later . Medium was replaced the next morning and imaging started 5 h after the second release . Images were acquired on an ASMDW live - cell imaging system ( Leica ) equipped with a Cascade II EM - CCD camera ( Photometrics ) and a × 20 / 0 . 50 NA plan Fluotar air objective . Images were collected every 10 min using Image Pro 6 . 3 software ( Media Cybernetics ) and processed using ImageJ . Mitotic alignment analysis . Before analysis of mitotic alignment , image files were computationally blinded by randomized file name encoding . Thereafter , Fiji software was used to measure the angular difference between the long axis of the mother cell before cell division , and the axis of cytokinesis . All observed cell division events were analysed . Where multiple attempts at cytokinesis were observed , the orientation of the first attempt was used for angular measurement . Data were summarized using R software . Adhesion assay . Cell adhesion assays were performed as described previously 68 . Briefly , non - tissue culture - treated , polystyrene 48 - well cluster plates ( Corning Costar Corporation ) were coated with 10 µ g ml − 1 vitronectin as detailed above , and blocked with 1 % heat - denatured BSA . A total of 5 × 10 4 CS1 - wt ( negative control ) or CS1 - β 5 cells were seeded per well and allowed to attach for 30 min under incubation conditions . Cells were treated during attachment as indicated with combinations of cytochalasin D ( 20 µ M ) and either cyclic RAD ( Arg - Ala - Asp ; non - inhibitory control ) or cyclic RGD ( competitive inhibitor of integrin β 5 - vitronectin interaction ) peptides ( 20 µ g ml − 1 ) ( provided by Merck and used as described previously 69 ) . After attachment , non - adherent cells were removed by repeated washing . Remaining cells were labelled with DAPI and imaged with a Nikon A1R confocal microscope and × 10 air objective , enabling automated cell counting via NIS elements software . Statistics and reproducibility . Except where otherwise stated , all data presented reflect at least three biologically independent experiments . For analyses based on per cell quantification and / or intracellular adhesion population analyses , exact cell and / or adhesion numbers are given in the figure legends . Statistical analyses and graphical representation were predominantly performed using R software ( v . 3 . 5 . 1 ) and RStudio ( v . 1 . 1 . 453 ) , or in some cases within Excel . All raw quantitative data and R analysis code are provided in the ‘Code availability’ section . Image analyses were predominantly automated via either commercial PAD software ( v . 6 . 3 ) or open - source KNIME software ( v . 3 . 6 . 0 ) , ensuring uniform treatment and reproducibility . A representative KNIME image analyses workflow as well as sample images and subsequent data integration workflows are provided in the ‘Code availability’ section . Manual image analyses were performed in ImageJ following computational blinding of the image data identity . All graphical data representations are provided in the figure legends , as well as statistical significance testing and correction procedures . For data visualization , boxplot notches indicate ± 1 . 58 times the interquartile range divided by the square root of the observation number , approximating the 95 % CI . Except where otherwise stated , error bars also represent 95 % CIs , estimated as described above . Statistical significance tests were all unpaired and two - tailed , and included either t - testing ( for small , parametric data sets ) or Mann – Whitney U - testing ( for large , potentially non - parametric data sets ) . Holm – Bonferroni corrections were applied to correct for multiple hypothesis testing . P values are presented numerically in each instance . Reporting Summary . Further information on research design is available in the Nature Research Reporting Summary linked to this article . Code availability . Where associated with open - source software tailored for this study , code underpinning this study is available through an associated public GitHub repository ( https : / / github . com / locusJ / Lock - et - al - NCB - 2018 - Reticular - Adhesions - Data - and - Analysis - Repository ) . This includes a custom KNIME image quantification workflow used in automated analysis of siRNA and drug perturbation screening , and an associated custom KNIME data integration workflow . Sample images from the analysis of Arp2 / 3 inhibition effects are also included . An R markdown script coding for the majority of graphical outputs and statistical significance testing is included , as is the associated HTML notebook summarizing this process and results . This R code calls the multi - sheet Excel file provided as Supplementary Table 1 , which contains all presented quantitative data . In some cases , graphical analyses were generated directly in Excel ; these are embedded within relevant sheets of this file . A file titled ‘Instructional Workflow for Data Exploration and Reproduction . pdf’ is provided within this repository , and outlines the use of included code and data . Data availability MS data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository with primary accession codes PXD008645 and PXD008680 . Source data for Fig . 4a – d and Supplementary Fig . 4a , b are provided as Supplementary Table 2 . All other quantitative data are presented in Supplementary Table 1 , while sample images from screening analyses are provided via the GitHub repository described under ‘Code availability’ . All additional data supporting the findings of this study are available from the corresponding authors on reasonable request . References 57 . Smilenov , L . , Forsberg , E . , Zeligman , I . , Sparrman , M . & Johansson , S . Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography . FEBS Lett . 302 , 227 – 230 ( 1992 ) . 58 . Yatohgo , T . , Izumi , M . , Kashiwagi , H . & Hayashi , M . Novel purification of vitronectin from human plasma by heparin affinity chromatography . Cell Struct . Funct . 13 , 281 – 292 ( 1988 ) . 59 . Kiss , A . et al . Non - monotonic cellular responses to heterogeneity in talin protein expression - level . Integr . Biol . 7 , 1171 – 1185 ( 2015 ) . 60 . Li , Z . et al . Integrin - mediated cell attachment induces a PAK4 - dependent feedback loop regulating cell adhesion through modified integrin α v β 5 clustering and turnover . Mol . Biol . Cell . 21 , 3317 – 3329 ( 2010 ) . 61 . Bates , M . , Huang , B . , Dempsey , G . T . & Zhuang , X . Multicolor super - resolution imaging with photo - switchable fluorescent probes . Science 317 , 1749 – 1753 ( 2007 ) . 62 . Shafqat - Abbasi , H . et al . An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes . eLife 5 , e11384 ( 2016 ) . 63 . Ester , M . , Kriegel , H . - P . , Sander , J . & Xu , X . A density - based algorithm for discovering clusters in large spatial databases with noise . Proceedings of 2nd International Conference on Knowledge Discovery and Data Mining KDD - 96 , 226 – 231 ( 1996 ) . 64 . Schindelin , J . et al . Fiji : an open source platform for biological image analysis . Nat . Methods 9 , 676 – 682 ( 2012 ) . 65 . Szklarczyk , D . et al . STRING v10 : protein – protein interaction networks , integrated over the tree of life . Nucleic Acids Res . 43 , D447 – D452 ( 2015 ) . 66 . Huang , D . W . , Sherman , B . T . & Lempicki , R . A . Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources . Nat . Protoc . 4 , 44 – 57 ( 2009 ) . 67 . Wählby , C . , Sintorn , I . M . , Erlandsson , F . , Borgefors , G . & Bengtsson , E . Combining intensity , edge and shape information for 2D and 3D segmentation of cell nuclei in tissue sections . J . Microsc . 215 , 67 – 76 ( 2004 ) . 68 . Yebra , M . et al . Requirement of receptor - bound urokinase - type plasminogen activator for integrin α v β 5 - directed cell migration . J . Biol . Chem . 271 , 29393 – 29399 ( 1996 ) . 69 . Strömblad , S . et al . Loss of p53 compensates for α v - integrin function in retinal neovascularization . J . Biol . Chem . 277 , 13371 – 13374 ( 2002 ) . NAtuRe CeLL BiOLOGy | www . nature . com / naturecellbiology 1 n a t u r e r e s e a r c h | r e p o r t i n g s u mm a r y A p r il 2018 Corresponding author ( s ) : Lock , Strömblad Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish . This form provides structure for consistency and transparency in reporting . For further information on Nature Research policies , see Authors & Referees and the Editorial Policy Checklist . Statistical parameters When statistical analyses are reported , confirm that the following items are present in the relevant location ( e . g . figure legend , table legend , main text , or Methods section ) . n / a Confirmed The exact sample size ( n ) for each experimental group / condition , given as a discrete number and unit of measurement An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test ( s ) used AND whether they are one - or two - sided Only common tests should be described solely by name ; describe more complex techniques in the Methods section . A description of all covariates tested A description of any assumptions or corrections , such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency ( e . g . means ) or other basic estimates ( e . g . regression coefficient ) AND variation ( e . g . standard deviation ) or associated estimates of uncertainty ( e . g . confidence intervals ) For null hypothesis testing , the test statistic ( e . g . F , t , r ) with confidence intervals , effect sizes , degrees of freedom and P value noted Give P values as exact values whenever suitable . For Bayesian analysis , information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs , identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes ( e . g . Cohen ' s d , Pearson ' s r ) , indicating how they were calculated Clearly defined error bars State explicitly what error bars represent ( e . g . SD , SE , CI ) Our web collection on statistics for biologists may be useful . Software and code Policy information about availability of computer code Data collection NIS elements software ( Nikon ) , LCS software ( Leica ) and LSM ( Zeiss ) . Data analysis Patch Morphology Analysis Dynamic software ( Digital Cell Imaging Laboratories , Belgium ) was used for analysis of static ( fixed ) and dynamic ( live ) cell imaging data in most cases . FIJI was used for analysis of mitotic axis alignment . KNIME ( v3 . 6 . 0 ) was used for analysis for siRNA - as well as PIP and Arp2 / 3 drug - responses . NIS elements was used for 3D / 4D image data rendering . R ( v3 . 5 . 1 ) was used for statistical data analyses and visualisation via RStudio ( v1 . 1 . 453 ) , although some analyses and graphical outputs were generated directly within Excel . STORM data were analysed using Insight3 software . Mass spectrometry quantification was performed using Progenesis LC - MS software ( Progenesis QI , Nonlinear Dynamics , Newcastle , UK ) . R code as well as Knime image analysis and data integration workflows are available via the GitHub repository : https : / / github . com / locusJ / Lock - et - al - NCB - 2018 - Reticular - Adhesions - Data - and - Analysis - Repository This repository also includes all quantitative source data ( as presented in Supplementary Table 1 ) and sample image data from Arp2 / 3 drug inhibition studies ( for use in Knime image analysis workflows ) . Instructions as to the content and use of this repository are included in an associated . pdf file " Instructional Workflow for Data Exploration and Reproduction . pdf " . For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature , software must be made available to editors / reviewers upon request . We strongly encourage code deposition in a community repository ( e . g . GitHub ) . See the Nature Research guidelines for submitting code & software for further information . 2 n a t u r e r e s e a r c h | r e p o r t i n g s u mm a r y A p r il 2018 Data Policy information about availability of data All manuscripts must include a data availability statement . This statement should provide the following information , where applicable : - Accession codes , unique identifiers , or web links for publicly available datasets - A list of figures that have associated raw data - A description of any restrictions on data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD008645 and PXD008680 . Quantitative data underlying graphical figure panels are available via the GitHub repository : https : / / github . com / locusJ / Lock - et - al - NCB - 2018 - Reticular - Adhesions - Data - and - Analysis - Repository in the file " Supplementary Table 1 Statistics Source Data . xlsx " Figures with associated raw quantitative data include : 1A ; 1H ; 2A ; 2B ; 2C ; 2D ; 2N ; 2S ; 2T ; 2V ; 2W ; 3C ; 3D ; 3E ; 3H ; 3I ; 5B ; 5C ; 5D ; 6A ; 6B ; 6M ; 6N ; 7E ; 7F ; 8A ; 8B ; 8C ; Supp F5C ; Supp F6A . The GitHub repository also contains sample image data from Arp2 / 3 drug inhibition studies ( for use in Knime image analysis workflows ) . Instructions as to the content and use of this repository are included in an associated . pdf file " Instructional Workflow for Data Exploration and Reproduction . pdf " . Field - specific reporting Please select the best fit for your research . If you are not sure , read the appropriate sections before making your selection . Life sciences Behavioural & social sciences Ecological , evolutionary & environmental sciences For a reference copy of the document with all sections , see nature . com / authors / policies / ReportingSummary - flat . pdf Life sciences study design All studies must disclose on these points even when the disclosure is negative . Sample size Reticular adhesions are previously uncharacterised , in terms of their morphology , dynamics , content and functional influence . Therefore , no prior knowledge existed to guide prediction of sample sizes that might optimally mediate comparisons between reticular and classical adhesion characteristics . For this reason , sample sizes were based on standards in the field ( such as for FRAP analysis ) , or based on the number of cells / adhesions accessible through automated imaging and analysis ( where sample number typically exceeds current gold standards ) . At no time were the results of statistical comparisons ( e . g . Mann - Whitney U test ) used to motivate additional experiments aiming to achieve statistical significance . Data exclusions As described in text , 4 proteins detected in raw mass spectrometry data were excluded due to over - representation in a database of common contaminant proteins ( CRAPome database , Mellacheruvu et al . , 2013 ) . Replication All experiments were successfully replicated . The number of replicates is specified for each experiment . Randomization Sample randomization was not relevant to this study because analysed populations were assigned to classes based on objective measures of identity ( e . g . reticular versus classical adhesions defined by consistent quantitative criteria ) . Blinding Analysis of mitotic division axis alignment was manual and therefore computationally blinded . All other analyses were automated and therefore non - blinded . Reporting for specific materials , systems and methods Materials & experimental systems n / a Involved in the study Unique biological materials Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Methods n / a Involved in the study ChIP - seq Flow cytometry MRI - based neuroimaging 3 n a t u r e r e s e a r c h | r e p o r t i n g s u mm a r y A p r il 2018 Antibodies Antibodies used Primary antibodies used are described in Supplementary Table 4 . Anti - mouse and anti - rabbit secondary antibodies conjugated with Alexa 488 , 568 or 647 were used as appropriate ( Thermo Fisher Scientific ) . For fixed F - actin labelling , phalloidin pre - conjugated with Alexa 488 , 568 or 647 was used as appropriate ( Thermo Fisher Scientific ) . DAPI ( 4’ , 6 - Diamidino - 2 - Phenylindole , Dihydrochloride ; Thermo Fisher Scientific ) nucleic acid stain was used as a nuclear marker as appropriate . Validation Details per Ab provided in Supplementary Table 4 . Eukaryotic cell lines Policy information about cell lines Cell line source ( s ) All cell lines were acquired directly from either ATCC or ECACC , with the exception of H1299 , CS - 1 and talin 1 null mouse embryonic stem cells . Authentication Cell lines acquired directly from either ATCC or ECACC have been authenticated by the provider . H1299 , CS - 1 and talin 1 null mouse embryonic stem cells were not authenticated . Mycoplasma contamination All cell lines were tested and found to be negative for mycoplasma Commonly misidentified lines ( See ICLAC register ) No cell lines used in this study were found in the database of commonly misidentified cell lines ( v8 . 0 ) that is maintained by ICLAC and NCBI Biosample \ No newline at end of file diff --git a/silver_data/15e10fbd7b44765c7fc8952043552c023a3b8bfd.pdf.txt b/silver_data/15e10fbd7b44765c7fc8952043552c023a3b8bfd.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..9af05427ee1069ce717824275a8c49f05e5dbad6 --- /dev/null +++ b/silver_data/15e10fbd7b44765c7fc8952043552c023a3b8bfd.pdf.txt @@ -0,0 +1 @@ +輪講 担当：水野隼輔 Neural Correlates of Creativity in Analogical Reasoning Adam E . Green ( Georgetown University ) , David J . M . Kraemer ( University of Pennsylvania ) Jonathan A . Fugelsang ( University of Waterloo ) , Jeremy R . Gray ( Yale University ) , Kevin N . Dunbar ( University of Maryland ) ＜ Journal of Experimental Psychology 掲載＞ 1 Abstract  analogical mapping では脳の前頭極が関与している  認知的な研究では意味的距離を analogical mapping における創造性の主な決定要因とみ なす  （意味的に）より離れた類推は一般的により創造的  本研究では analogical mapping の意味的距離を変化させる類推生成課題を実施  fMRI を用いて類推課題生成中の脳活動を測定  潜在的意味解析から定量的に導き出した尺度を使用 2 Introduction 類推は創造性における重要な認知過程である ( Barnett & Ceci , 2002 ; Boden , 2003 ; Bowdle & Gentner , 2005 ; Costello & Keane , 2000 ; Green , Fugelsang , Kraemer , & Dunbar , 2008 ; Green , Kraemer , Fuselsang , Gray , & Dunbar , 2010 ; Holyoak & Thagard , 1995 ; Mayer , 1999 ; Sternberg , 1977 )  創造性は特定の課題の制約に合った，今までにないものの生成  創造性の定義は類推を特徴づける２つの主な特徴を備えている  新規性，制約 ( constraint )  類推（ blizzard is to snowflake as army is to ? ）  一見，類似していないようにみえる状況や表現の間の新規な繋がりを構成  [ blizzard （吹雪） : snowflake （雪片） ] と [ army （陸軍） : soldier （兵士） ]  特定の制約に適合しなければならない  洞察（ひらめき）によって，類推と創造性の繋がりは実世界の例によって支持されて いる  August Kekule は蛇が自らの尻尾を噛むという夢を見た後，ベンゼン環の炭素―炭 素を発明  James Crocker はホテルの部屋の伸びるシャワーヘッドからハッブル望遠鏡の位置 制御機能を発明 輪講 担当：水野隼輔  類推と創造性の関連は一般的ではない  ある類推は創造的，ある類推は全く創造的ではない  類推における創造性の主な要因  意味的特徴が比較的近いか遠いか ( Barnett & Ceci , 2002 ; Boden , 2003 ; Bowdle & Gentner , 2005 ; Green et al . , 2008 , Green et al . , 2010 ; Holyoak & Thagard , , 1995 )  項目間の意味的距離は創造性の新規性と関連  創造的な類推  表面的には類似していない関係表現のより深くにある類似性を表現  [ kitten （子猫） : cat （猫） ] と [ spark （火の粉） : fire （火） ] ←表面的には類似して いない  [ kitten : cat ] と [ puppy （子犬） : dog （犬） ] ←表面的に類似  項目間の意味的距離の変化が類推における創造性に関連  意味的距離を定量化可能なパラメータとして実験することによって立証  創造的な類推の重要性が一般的に認識されている ( Boden , 2003 ; Dahl & Moreau , 2002 ; Dunbar & Blanchette , 2001 ; Holyoak & Thagard , 1995 )  類推の脳機能イメージ研究において創造性に直接関連するパラメータの分析はさ れていない  類推解の生成における意味的距離の変化における脳イメージング研究を行った  脳イメージング研究  前頭極が類推に関連 ( Bunge , Wendelken , Badre , & Wagner , 2005 ; Cho et al . , 2009 ; Geake & Hansen , 2005 ; Green , Fugelsang , Kraemer , Shamosh , & Dunbar , 2006 ; Green et al . , 2010 ; Hampshire , Thompson , Duncan , & Owen , 2011 ; Volle , Gilbert , Benoit , & Burgess , 2010 ; Wendelken , Nakhabenko , Donohue , Carter , & Bunge , 2008 )  特に，左前頭前野皮質内の限局した部位は mapping の構成要素に関与 ( Green , Fugelsang , Kraemer , et al . , 2006 ; Green et al . , 2010 ) 3 Method 3 . 1 Participants  native English speakers 23 人（男 12 人，平均年齢 22 . 2 歳，右利き）  倫理委員会の承認を受け，実験前にインフォームドコンセントをとり同意書 に記入 3 . 2 Stimuli and Procedure  参加者は fMRI の稼働中に 80 の類推トライアルを行った  ３つの語と１つの？から成る類推問題を見て、類推して？を埋める（ Figure1 ） 輪講 担当：水野隼輔  解を生成したらボタンを押す  正解の単語は次の画面で？の代わりに画面に登場し、参加者が生成した語が 現れた語と同じかまたは近いか判断  項目間の意味的距離の値は潜在的意味解析で算出  潜在的な意味解析アプリケーション ( http : / / lsa . colorado . edu )  値はパラメトリック分析で用いた  84 人の独立した評価者が創造性に対して全ての類推刺激を７段階評価  この類推解はどの程度創造的か？  1 = 創造性が最も低い， 7 = 創造性が最も高い  評価者は難易度も評価  この類推解はどの程度難しいか？  1 = 最も難しくない， 7 = 最も難しい  これらの評価値をその後の fMRI 分析のためのパラメトリック回帰として使用  意味的距離が大きいほど類推的な創造性は大きくなると予想  予想と一致し意味的距離と創造性の平均評価に相関あり ( r = . 92 , p < . 001 )  意味的距離と難易度の平均評価に相関あり ( r = . 39 , p < . 001 )  刺激の統制  同じ基準語を使用  比較的距離が短い…左 [ blindness : sight ] ，右 [ deafness : ? ]  比較的距離が長い…左 [ blindness : sight ] ，右 [ poverty : ? ]  提示刺激の順番  同じ基準語のペアを持つ２つの刺激が連続して提示されない  上の条件を満たし , ランダムで表示  90 % 以上の評価者が了解した類推項目を使用 3 . 3 fMRI Data Acquisition and Analysis  データは 1 . 5 テスラのスキャナーで採取  機能的画像データは SPM99 （統計ソフト）における、一般線形モデルを用い て分析  コントラスト画像は、条件間と、各条件とベースライン間の比較のために、 voxelwise t contrast 分析を介する  トライアル開始からボタンを押す（解生成）までの時間を解生成事象として モデル化  パラメトリック分析  創造性評価に対する難易度評価，意味的距離の関係を分析 輪講 担当：水野隼輔 4 Result 4 . 1 Behavioral Findings  応答の精度…正解したトライアルの割合  応答の精度は 91 . 75 % ± 4 . 93  応答の精度と意味適距離の値にはわずかに相関あり ( r = - . 22 , p = . 05 )  エラートライアルは分析から除外  エラートライアル…生成した解が正解のワードと同じでない , 類似して いないもの  応答時間…刺激提示から解生成時のボタンを押すまでの時間  平均応答時間は 5 , 662 ms ± 645 ms ( SE = 68 ms )  応答時間は意味的距離と相関あり ( r = . 35 , p = . 002 ) 4 . 2 fMRI Findings  類推解生成中に前頭極が意味的距離の増加に伴って活性化 ( Figure2 )  意味的距離と ROI 内で正の変調を実証 ( t ( 22 ) _ 6 . 81 , SVC p _ . 05 )  評価された創造性と前頭極 ROI 内で正の変調を実証 ( t ( 22 ) = 6 . 85 , SVC p = . 05 )  難易度を統制するため，応答時間，精度から難易度を評価した  Generation task > Rest ( Figure 3 )  前頭極皮質と STG における活動だけでなく，全帯状回，左の腹部前頭野を 含む，複雑な言葉の推論に関連した領域が明らかになった 5 Discussion  類推は解を生成する創造性に焦点を合わせることができる  類推解生成中の意味的距離と左前頭極皮質の活動に関連がある  以前に analogical mapping に関与していることを示した前頭極 ROI 内 ( Green , Fugelsang , Kraemer , et al . , 2006 ; Green et al . , 2010 ) で、有意な予測因子であることを証明  課題の難易度ではなく analogical mapping の意味的距離が ROI 内の活動を変調する 5 . 1 Role of Frontopolar Cortex in Analogical Reasoning ( 類推における前頭極皮質の役 割 )  前頭極皮質は創造的な類推において重要な役割を果たしている（情報の統合に特化 している）  類推に寄与する他の部位と比較して，前頭極皮質は優先的に類推の relational integration component として採用される 輪講 担当：水野隼輔  本研究では前頭極皮質に焦点を当てた  しかしながら，パラメトリック分析ではいくつかの脳領域の活動が明らかにな った  複数の脳領域が創造的な推論に寄与することはほぼ確実である  パラメトリック分析による STG ( superior temporal gyrus （ 上側頭回 ） ) の出現は，この領域が 創造的な推論に関与している有力な候補であることを示す  STG は単一の語の理解と関連して，優先的に文章や物語を理解（読んだり聞いたり）するのに必 要とされる  洞察問題解決の「なるほど」と思う瞬間に， STG の活動が上昇している ( Jung - Beeman et al . , 2004 )  STG の活動は創造性と意味的距離が遠い類推と関連しているかもしれない  本研究では， STG の活動は前頭極皮質よりも早くピークに達している ( figure 4 )  この知見は前頭極皮質の方が STG よりも潜伏時間が長いという先行研究に一 致している ( Crone et al . , 2009 )  このことから， STG は言語理解機能に携わっているであろう 5 . 2 Relating Semantic Distance to Creativity （創造性と意味的距離の関連）  予想と一致して analogical mapping における創造性の主観評価は意味的距離と相関 あり  得られた知見が過去の研究成果と一致  意味的距離が analogical mapping における創造性の主要な決定要因となる  主観評価としての創造性が前頭極皮質の ROI 内での有意なモジュレータ（変調器）  奇抜なアイディアは，これらが使用可能で課題の制約を満たしている場合に限り真 に創造的 ( Mayer , 1999 )  意味的に遠いアイディアが一般的に奇抜  しかし，必ずしも適切でないため創造性を特徴づけるのには不十分  このパラダイムにおける解の生成は定義された推論課題の制約の範囲内  このパラダイムにおける創出は創造課題の妥当性を満たしている 5 . 3 Dissociating Semantic Distance From Difficulty （難易度から意味的距離の解離）  本結果では，意味的距離が創造性に関連していることを支持する  意味的距離が神経レベルで課題の難易度から解離している  応答時間，精度，難易度評価は ROI での活動とは無関係（先行研究から） 5 . 4 G enerating Solutions Via Creative Analogy （創造的類推による解の生成）  過去の脳ベースの類推研究での評価を調査  C が D であるように、 A が B であるというのは真ですか？偽ですか？ 輪講 担当：水野隼輔  管理が容易  analogical mapping とそのサブプロセスの神経的なメカニズムに非常に有益  しかし，これらの類推は（少なくともあからさまには）何か新しいものの生 成には関与しない  新しいものの生成は創造性の基本的な要素 ( Mayer , 1999 )  類推の脳イメージング研究では， A が B なら C なら？という型であった ( Wendelken et al . , 2008 )  類推のパラメータとして創造性を調査せず  本研究では類推における脳ベースでの創造性の調査である 6 Conclusions  意味的距離を操作することによって，類推における創造性関連因子を研究するため の新たなパラダイムが示された  我々が得たデータは，創造的な類推で意味的に遠い解の生成を支持している神経メ カニズムとして前頭極の活動の増加を暗示する 輪講 担当：水野隼輔 輪講 担当：水野隼輔 \ No newline at end of file diff --git a/silver_data/1a246194455fec917cecd715d0a40983b4b98b7a.pdf.txt b/silver_data/1a246194455fec917cecd715d0a40983b4b98b7a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..bbd34d1ddd5909bab33d4b8429f37428072290ae --- /dev/null +++ b/silver_data/1a246194455fec917cecd715d0a40983b4b98b7a.pdf.txt @@ -0,0 +1 @@ +DESIGN PROTOTYPING OF SYSTEMS Camburn , Bradley Adam ( 1 , 2 ) ; Arlitt , Ryan ( 1 ) ; Perez , K . Blake ( 1 ) ; Anderson , David ( 1 ) ; Choo , Pui Kun ( 1 ) ; Lim , Terry ( 2 ) ; Gilmour , Adam ( 2 ) ; Wood , Kristin ( 1 ) 1 : Singapore University of Technology and Design , Singapore ; 2 : Gilmour Space Technologies , Singapore Abstract In recent years , groundbreaking work in design science has identified that prototyping is one of the most critical factors leading to successful development . Many decisions regarding the detail of a design and the allocation of resources are made during design prototyping . Extant studies provide foundational insights in strategic prototyping . This work explores prototypes for developing services and systems that are complex . A framework is proposed to visualise strategic prototyping to search design spaces that span multiple domains . We define three phases of system prototyping : partitioning , search , and implementation . The framework illustrates the relationship between individual techniques and associated cost versus performance outcome . This new framework is supported through two commercial development case studies that demonstrate the approach . The first is a subsystem from a hybrid launch vehicle development effort at Gilmour Space Technologies , the second is a service centre design case from the SUTD - MIT International Design Centre . Keywords : Design costing , Complexity , Design methods , Systems Engineering ( SE ) , Product - Service Systems ( PSS ) Contact : Dr . Bradley Adam Camburn Singapore University of Technology and Design SUTD - MIT International Design Centre Singapore bradley _ camburn @ sutd . edu . sg 21 ST INTERNATIONAL CONFERENCE ON ENGINEERING DESIGN , ICED17 21 - 25 AUGUST 2017 , THE UNIVERSITY OF BRITISH COLUMBIA , VANCOUVER , CANADA Please cite this paper as : Surnames , Initials : Title of paper . In : Proceedings of the 21 st International Conference on Engineering Design ( ICED17 ) , Vol . 3 : Product , Services and Systems Design , Vancouver , Canada , 21 . - 25 . 08 . 2017 . 211 ICED17 1 INTRODUCTION Prototyping is one of the most critical factors in successful design ( Otto and Wood 2001 ) . Many decisions regarding the detail of a design and the allocation of resources are made during the prototyping phase of design ( Gero 1990 ) . Previous research has identified strategies for planning individual prototypes ( Bradley Camburn et al . 2015 , Bradley A . Camburn et al . 2015 ) . However complex products , services , or systems often require multiple streams of prototyping efforts to be co - ordinated . This paper provides a graphical framework for visualizing complex search strategies . In this work , we define three phases of systems prototyping : partitioning , search , and implementation . The framework illustrates a relationship between techniques used in each these phases with associated cost and performance outcome . This approach to model systems prototyping efforts is then demonstrated through two case studies in systems design . One is for an integrated medical service facility . The second is for one subsystem of a commercial hybrid launch vehicle from Gilmour Space Technologies . This work explores the following research questions : 1 . What are the relationships between prototyping techniques , cost , and performance ? 2 . Can traditional ( and more complex ) prototyping strategies be viewed in a graphical way ? 3 . Is the given mapping approach applicable to both service and system prototyping efforts ? A model of the way that designers prototype is presented . This is supported by quantitative cost and performance based models for individual prototyping techniques , and validated through systems design case studies . The modeling approach is provided to visualize the embodiment of systems prototyping and the relationship between multiple prototypes in a design effort . This tool , alongside the empirically validated cost and performance models provides a platform for comparing various possible ‘search strategies’ to explore the space of possible designs . 2 PARTITIONING For the design of systems , it is often necessary to segment the design problem in some way ( Reed Doke 1990 , Drezner and Huang 2009 ) . With what are referred to as ‘wicked problems’ solutions are typically multi - faceted or complex and it is infeasible to develop the entire design in one effort . This segmentation can be implemented in various ways . By key function ( group ) , by key subsystem , or by domain are typical approaches , Figure 1 . Performance and cost targets are set for each partition . Once the design team is confident that each partitioned solution will meet performance the system is re - integrated ( if applicable ) then deployed . Figure 1 : Partitioning of a system , there are numerous approaches to partitioning a complex system . Three common approaches are either by function , subsystem , or domain . 3 SEARCH TECHNIQUES There are two core techniques for searching the design space of solutions for each partition of the design problem , via testing with prototypes . These involves either the iterative , temporally sequential , testing of overlapping design concepts , or the parallel testing of multiple concepts at a single point in time ( Dahan and Mendelson 2001 , Thomke and Bell 2001 ) . Other strategies have been presented ( Hannah et al . 2008 , da Silva and Kaminski 2016 ) , there work herein helps to quantify differences and to explore how single prototypes fit in a larger problem scheme . 212 ICED17 Figure 2 : The most common techniques for searching a design space are iteration ( left ) and parallel testing ( right ) Expanding work from ( Bradley Adam Camburn et al . 2015 ) we extract empirically a new model of the effect of iterative testing on performance . Based on additional data from the empirical study of a design challenge , reported in this previous work . We also extract a new model of the impact of parallel testing on performance . A key insight is the distinct difference between the gradual performance increase of iteration and the discontinuous performance leaps associated with parallel concept testing . Equations 1 and 2 , below , depict the expected performance of iteration and parallel testing . Π ∝ Π 0 e ∆∙n ( 1 ) where 𝛱 is design performance , 𝛱 0 is the initial performance , n is the number of iterations on a single concept , ∆ is the gradient of the local design space Π ∝ max i ∈ m [ Π i ] ( 2 ) where 𝛱 is design performance , 𝛱 𝑖 is the performance of a concept i , m is the set of distinct design concepts tested . Figure 3 , below , visually depicts the performance results of iteration and parallel testing from the results of the experiment reported in previous work ( Bradley Adam Camburn et al . 2015 ) . An exponential equation of best fit , to the iteration performance results , had the highest R 2 value as compared to linear , or polynomial equation fitting results ( R 2 = 0 . 91 ) computed using all test data in the given study . These results reiterate the critical insight that parallel prototyping allows for large , discontinuous leaps in performance . The sample data was taken from a study in which participants produced a simple transport mechanism . Performance is measured by distance . Figure 3 : Empirical data for iteration versus parallel testing , performance effects . Values are averaged across all teams . For the parallel concepts , values are average for the first test only . + / - 1 standard error shown . Consider the following example . If a hypothetical team applied parallel testing of four different concepts , then they will roughly achieve a performance of 31 ft . Conversely , if the design team chose a single concept and constructed four iterations , the performance could be as high as 38 ft . or as low as 12 ft . , depending on which concept was chosen initially . This exercise highlights the risk of an iterative single - concept testing strategy . Note that , these given numerical values are one example only . The constants of performance likely vary by context . Qualitative observation of these experiments suggests 0 10 20 30 40 50 60 70 80 90 100 1 3 5 7 9 11 13 15 17 19 21 P e r f r o m a n c e ( d i s t a n c e , f t . ) Iterations - > 0 10 20 30 40 50 A B C D p e r f o r m a n c e ( d i s t a n c e , f t . ) Parallel Concepts 213 ICED17 that when an ineffective concept is chosen , multiple iterations will not recover performance until a new concept is adopted . Table 1 : Example equations for performance ( units are of distance , d , in this case measured in feet ) using iteration , and parallel testing data . Iteration Π ( d ) = Π 0 e 0 . 05∗n Parallel Π ( d ) = max [ 21 , 10 , 25 , 31 ] 4 IMPLEMENTATION TECHNIQUES There are numerous prototyping strategies that can be applied to reduce cost ( Gordon and Bieman 1995 , Buchenau and Suri 2000 , Moe et al . 2004 , Dutson and Wood 2005 , Drezner and Huang 2009 , Bradley Camburn et al . 2015 ) . Herein , key techniques have ben synthesized to form three conceptually distinct cost reduction techniques . These are scaling , the reduction of cost by reducing the size or functional order of a design ; isolation , the selection of a particular key subsystem to be prototyped , in isolation from the rest of the system ; and abstraction , the representation of a meta - function without consideration of internal functionality . Figure 4 : Three core techniques for implementing a single prototype test embodiment are scaling ( left ) , isolation ( middle ) , and abstraction ( right ) . Equation 3 , which is based on expanded analysis from ( Bradley Adam Camburn et al . 2015 ) , demonstrates that these three techniques result in a co - operative , proportional reduction of prototype cost . ℂ 𝑟 ∝ ℂ 𝑆 𝑐 ∙𝐼 𝑠 ∙𝐴 𝑏 ( 3 ) where ℂ is the full system cost , ℂ 𝑟 is the reduced implementation cost of a prototype , and 𝑆 𝑐 is the scaling factor 𝐼 𝑠 is the isolation factor 𝐴 𝑏 is the abstraction factor . A key insight is that multiple techniques can be applied and the cost of a prototype can be radically reduced . Hybrid techniques reduce cost of the overall prototyping effort . They enable a greater number of iterations or parallel tests . Figure 5 graphically depicts the interaction of scaling and isolation quantitatively . The results are based on empirical data from ( Bradley Adam Camburn et al . 2015 ) . The empirical data fits this equation closely , ( R 2 value > 0 . 9 ) based on the given data set . Exact values for the constants are to be considered with caution as they are likely highly dependent on the specific design task , they are reported in Table 2 for completeness . Figure 5 : Average prototype cost given scaling versus isolation . Area of bubble is proportional to average cost of prototypes in that category across all teams ( e . g . 0 , 0 is a full prototype , 1 , 1 is a scaled and isolated subsystem ) . Red outline shows + 1 standard deviation . S c a li n g - > Isolation - > 214 ICED17 The impact factors also seem intuitive ; as abstracted models can be extremely low fidelity ( e . g . a folded piece of paper ) . Conversely , an isolated prototype is typically of near full functional and may only result in a marginal cost reduction . Table 2 : Sample factors values extracted Abstraction , Ab 7 . 4 Scaling , Sc 3 . 2 Isolation , Is 1 . 6 5 SEARCH STRATEGIES When designers develop new products , services , and systems they may often employ complex search strategies that cannot be simply captured with the terms ‘iteration’ and ‘parallel testing’ as they are a of multiple techniques . This section explores the parameterization of designs into a topological vector space ( Figure 6 ) , and how this may aid in the visualization and analysis of complex prototyping strategies . A prototyping strategy can be seen as a search effort in a topological space ( Figure 7 ) . In reality , actual design spaces may contain hundreds of variables and be impractical to model in this manner . The example is given for visualistion . A prototyping effort can be represented as in ( Figure 8 ) with simplified maps showing the testing sequence . The wireframe strategy is presented in case studies below as it is infeasible to represent complex spaces graphically . This kind of segmentation is common in set - based design ( Schäfer and Sorensen 2010 , Yannou et al . 2013 ) . This approach helps to visualize the impact of lean strategies ( Ward et al . 1995 , Ward and Sobek II 2014 ) , and complements such approaches by highlighting a critical inter - relationship of parallel designs . They allow the team to map a design space , i . e . reduce local uncertainty accumulatively . 𝐷⃗⃗ = { 𝐿 1 𝐿 2 } 5 Figure 6 : Illustration on parametric design model of staircase . ( left ) simple staircase of two steps between the landings . ( center ) parametric representation of the design , L 1 is the first step height , L 2 is the second step height . ( right ) Topological map to visualize user preference Figure 7 : Figurative representation of hypothetical prototyping strategies . Numbers indicate test sequence . ( left ) ‘Proof - of - Concept , Alpha , Beta’ testing . ( center ) mixed strategy ( right ) multiple , diverse , low fidelity parallel tests . Figure 8 : Simplification of the strategies shown in Figure 8 , ( left ) ‘Proof - of - Concept , Alpha , Beta’ testing . ( center ) A mixed strategy . ( right ) Parallel tests . 215 ICED17 Search strategies have a direct relationship with cost and performance . As depicted in Figures 7 and 8 a higher fidelity model allows for clearer understanding of the design performance in the local space , but this information comes at a higher cost . By contrast , low fidelity models afford less information about the local design space , but may be critical in understanding global design space topology . As a key insight for designers , higher fidelity models should come later in design efforts to reduce overall cost , once the global performance topology is better understood . In summary , to maximize value ( Equation 4 ) , or the ratio of final performance to total cost , it is beneficial to explore many conceptually diverse low fidelity prototypes early in the design effort . 𝑉 ∝ max i , j ∈ n , m [ Π 𝑖 , 𝑗 ] ∑ ℂ𝑖 [ 𝑆𝑐∙𝐼𝑠∙𝐴𝑏 ] 𝑖 , 𝑗 𝑛 , 𝑚 𝑖 , 𝑗 = 1 ( 4 ) where 𝑉 is the value of a prototyping effort , 𝛱 𝑖 , 𝑗 is the performance of iteration j of concept i . n is the number of iterations ( of each concept ) and m is the set of concepts , ℂ 𝑖 is the cost of a full system of concept i , and [ 𝑆 𝑐 ∙ 𝐼 𝑠 ∙ 𝐴 𝑏 ] 𝑖 , 𝑗 is the reduction factor for each iteration j of each concept i The above modeling approach can clearly capture traditional approaches such as ‘proof of concept’ , ‘alpha’ , and ‘beta’ ( Figures 7 & 8 - left ) . It also could be used to understand why hackathons are effective in rapid solution development , as participants often rapidly produce many distinct low - fidelity prototypes to explore a wide space . This modeling approach extends previous work by the authors and extends this perspective of prototyping to incorporate systems development . 6 CASE STUDY : MEDICAL CENTRE To evaluate the effectiveness of the proposed modeling approach a service case study is supplied . The design effort was partitioned by function ( Figure 9 ) . We report the strategy employed for two key system functions . Service prototyping is a well - explored research topic ( Passera et al . 2012 ) . This work aims to demonstrate that a similar core approach can be applicable to both service and system design prototyping . The design team was working to redesign a series of working service centers for improved user experience . Figure 9 : Overview of medical center system prototyping strategy , partitioned by function . One key function was to provide visitors with a mechanism to reduce front - end loading at reception . The design solution was a pre - registration app . A series of iterations and low fidelity parallel tests were employed . Note , each prototype shown in Figures 10 and 11 is coded to the overall strategy map provided in Figure 9 . r . 1 : initial pre - screening concept r . 2 : app and control station detail r . 3 . 1 : user testing , interface one r . 3 . 2 : user testing , interface two r . 4 : low fidelity paper prototypes , final app Figure 10 : Search strategy for patient registration function . The relationship between perceived service quality and the waiting area layout was also explored . Numerous mockup prototypes supported the overall service prototype as well as scenario action cards for participants . The final prototyping effort involved deploying a prototype in the facility itself . A key 216 ICED17 insight of this effort was to discover the difficulty of way - finding in the facility through low - cost user testing . This insight was not initially reported by users , and allowed for valuable design enhancements . Notice that the effort includes a mixture of parallel and iterative testing with gradually higher fidelity . w . 1 : initial layout concept w . 2 : detailed layout concept w . 3 . 1 : first layout , mockup w . 3 . 2 : second layout , mockup w . 4 . 1 : first layout , user test w . 4 . 2 : second layout , user test Figure 11 : Search strategy for waiting area function . 7 CASE STUDY : HYBRID PROPULSION SYSTEM A second case study is the development of a launch vehicle using hybrid propulsion . This complex system requires numerous prototypes and is sequentially partitioned by domain , then subsystem , and again by domain ( Figure 12 ) . The prototyping strategies for several key subsystems are reported . In this case , the design team was working to develop a custom additive manufacturing capability . Figure 12 : Overview of hybrid propulsion system prototyping strategy , partitioned by subsystem and then by domain . We report the strategies employed for a small subset of the critical fuel generation system . One of the most critical systems in this launch system was a multi - material 3D printer . This printer produces a key component of the engines and is not available commercially . Figure 13 depicts a sequential design and testing strategy for the Fused - Deposition - Modeling ( FDM ) extruder head , which is a key subsystem . The alternating use of low cost CAD models , and high fidelity physical models allows for reduced cost . There were nearly twenty iterations of the extruder , three are shown . e . 1 : initial design , CAD e . 2 : initial design , functional e . 3 : mid - point design , CAD e . 4 : mid - point design , functional e . 5 : final design , CAD e . 6 : final design test results - > failed print structural warp improved print thermal analysis final print Figure 13 : Search strategy for FDM extruder sub - function Figure 14 shows prototypes from another key subsystem , the X - Y - Z motion stage . Two radically distinct stage concepts were compared using low fidelity models before iterating on a higher fidelity model of the chosen stage type . propulsionstructureaerodynamicsfuelavionicsguidancenavigationground control launch e . 2 plastic wax oxidiser extrudersoftwarestageelectronics stageextrudersoftwareelectronics e . 1 e . 4 e . 3 e . 6 e . 5 s . 1 s . 2 s . 3 s . 4 x . 2 x . 1 x . 3 x . 5 . 1 x . 4 . 1 x . 5 . 2 x . 4 . 2 217 ICED17 s . 1 : H bridge XY stage mockup s . 2 : stacked XY stage s . 3 : 2 - rod z axis , integrated stage s . 4 : 3 - rod z axis , final stage design Figure 14 : Search strategy for FDM x - y - z motion stage Figure 15 highlights prototypes from the secondary emulsion extrusion system . Since this was a relatively new concept , several low fidelity tests were employed to evaluate the design space . These allowed the designers to identify the valve as a critical subsystem . Two competing valve designs were developed and tested in CAD and then in physical prototypes before the simpler butterfly valve design was selected . x . 1 : material characterization x . 2 : tube flow characterization x . 3 scale extruder model x . 4 . 1 : ball valve design CAD x . 5 . 1 : ball valve physical test x . 4 . 2 : butterfly valve design CAD x . 5 . 1 : butterfly valve physical test Figure 15 : Search Strategy for emulsion extruder sub function Figure 16 shows the final , integrated , system design alongside a sample multi - material print . This print sample will be employed in the launch system . It is critical to note that only a demonstrative subset of the total prototyping effort has been shown here to highlight several exemplar strategies . It is critical that a system prototyping effort include each subsystem at some level of testing , or there may be unexpected behaviors in the final design . Figure 16 : Integrated multi - phase engine printer . ( left ) Final system CAD . ( right ) Final system physical construction . 8 DISCUSSION The paper presents a number of observations on the relationship of prototyping techniques ( iteration , parallel testing , scaling , isolation , and abstraction ) in the form of representative equations . The provides an initial response to the first research question , “What are the relationships between prototyping techniques , cost , and performance ? ” A simple graphical representation of a sample design is given , and used to illustrate prototyping strategies . This map example is then revised as a simplified linear model for compact representation of prototyping strategies . This provides preliminary response to the second research question , “Can traditional ( and more complex ) prototyping strategies be viewed in a graphical way ? ” 218 ICED17 Finally , two case studies are supplied in which the given tool is used to help build a relationship between a large number of prototypes explored in these exemple design efforts . This provides a first answer to the third research question 3 , “Is the given mapping approach applicable to both service and system prototyping efforts ? ” These results open additional questions for future consideration . The following observation is made , higher fidelity prototypes allow for better insight into the design performance in the local space , but capturing this information is costly . In contrast , low fidelity prototypes provide less information on the performance in the local design space but the global design space topology is widely explored . We may draw analogies to searching the hypothetically depicted spaces and formal search algorithms . Design of experiments literature provides insight into potentially innovative design prototyping search strategies to handle this tradeoff between information cost and value in the context of engineered systems . Established , quantitative search strategies that typically rely on a well - established set of design and response variables ( e . g . , Latin hypercube sampling ) , could be adapted to more holistic design concept testing . In such methods iterative experiment informs the next experiment such that information value per trial is high . Computational search algorithms also balance this tradeoff . For example , simulated annealing ( Kirkpatrick 1984 ) places a high value on divergent solutions . It begins with a high probability of accepting a divergent solution and decreases this probability as the number of iterations increases . For example , rather than directly iterating the design team may explore many distinct low fidelity concepts early on and then converge toward a series of higher fidelity directed tests around a well - performing concept . This is well matched to the concept of Lean manufacturing that encourages extensive information gathering early on in the design process ( Ward et al . 1995 ) . Particle swarm optimization uses many simultaneous agents that share information to guide search locally and globally , this for example may also be analogous to hackathons in which multiple agents explore the design space using many low fidelity design prototypes . Again it is easy to visualize this strategy using the given visual approach . One risk of low fidelity prototyping is that the design team may walk past a so - called “activity cliff” . These are regions of sharp sudden gradient in performance . A recurrent observation in drug design studies . Wherein small changes in chemical structure lead to large discontinuities in activity or other properties . These discontinuities introduce challenges in predicting how changes to a drug’s structure will alter its behavior . A variety of techniques have been developed to detect these discontinuities in order to produce a mapping between the structure and behavior spaces ( Stumpfe et al . 2013 ) including random forests ( Guha 2012 ) support vector machines ( Heikamp et al . 2012 ) , and particle swarm optimization ( Namasivayam and Bajorath 2012 ) . Future work could explore engaging such methods with prototypes to identify activity cliffs . However , the challenges of directly adapting search algorithms is that the design variables may be inherently unknown . This means that quantitative search strategies are only applicable by analogy . In summary , there is an opportunity to draw analogies to other means of searching design spaces and adapt them to the current approach . Prototyping is a framework into which many potential search strategies can be injected . REFERENCES Buchenau , M . and Suri , J . F . ( 2000 ) , “Experience prototyping” , in Proceedings of the 3rd conference on Designing interactive systems : processes , practices , methods , and techniques , ACM , 424 - 433 . Camburn , B . , Dunlap , B . , Gurjar , T . , Hamon , C . , Green , M . , Jensen , D . , Crawford , R . , Otto , K . and Wood , K . ( 2015 ) , “A Systematic Method for Design Prototyping” , Journal of Mechanical Design , 137 ( 8 ) . Camburn , B . A . , Jensen , D . , Crawford , R . , Otto , K . and Wood , K . ( 2015 ) , “Evaluation of a Strategic Method to Improve Prototype Performance with Reduced Cost and Fabrication Time” , in DS 80 - 4 Proceedings of the 20th International Conference on Engineering Design ( ICED 15 ) Vol 4 : Design for X , Design to X , Milan , Italy , 27 - 30 . 07 . 15 . Camburn , B . A . , Sng , K . H . E . , Perez , K . B . , Otto , K . , Jensen , D . , Crawford , R . and Wood , K . L . ( 2015 ) , “The Way Makers Prototype : Principles of DIY Design” , in International Design Engineering Technical Conferences and Computers and Information in Engineering Conference , Boston , MA . da Silva , G . C . and Kaminski , P . C . ( 2016 ) , “Selection of virtual and physical prototypes in the product development process” , The International Journal of Advanced Manufacturing Technology , 84 ( 5 - 8 ) , 1513 - 1530 . Dahan , E . and Mendelson , H . ( 2001 ) , “An extreme - value model of concept testing” , Management Science , 47 ( 1 ) , 102 - 116 . Drezner , J . A . and Huang , M . ( 2009 ) , On Prototyping : Lessons from RAND Research , RAND . 219 ICED17 Dutson , A . J . and Wood , K . L . ( 2005 ) , “Using rapid prototypes for functional evaluation of evolutionary product designs” , Rapid Prototyping Journal , 11 ( 3 ) , 125 - 131 . Gero , J . S . ( 1990 ) , “Design Prototypes : A Knowledge Representation Schema for Design” , AI Magazine , 11 ( 4 ) . Gordon , V . S . and Bieman , J . M . ( 1995 ) , “Rapid prototyping : lessons learned” , IEEE software , 12 ( 1 ) , 85 - 95 . Guha , R . ( 2012 ) , “Exploring Uncharted Territories - Predicting Activity Cliffs in Structure - Activity Landscapes” , Journal of chemical information and modeling , 52 ( 8 ) , 2181 . Hannah , R . , Michaelraj , A . and Summers , J . D . ( 2008 ) , “A proposed taxonomy for physical prototypes : structure and validation” , in ASME 2008 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference , American Society of Mechanical Engineers , 231 - 243 . Heikamp , K . , Hu , X . , Yan , A . and Bajorath , J . r . ( 2012 ) , “Prediction of activity cliffs using support vector machines” , Journal of chemical information and modeling , 52 ( 9 ) , 2354 - 2365 . Kirkpatrick , S . ( 1984 ) , “Optimization by simulated annealing : Quantitative studies” , Journal of statistical physics , 34 ( 5 ) , 975 - 986 . Moe , R . , Jensen , D . D . and Wood , K . L . ( 2004 ) , “Prototype partitioning based on requirement flexibility” , in ASME - IDETC , American Society of Mechanical Engineers , 65 - 77 . Namasivayam , V . and Bajorath , J . r . ( 2012 ) , “Searching for coordinated activity cliffs using particle swarm optimization” , Journal of chemical information and modeling , 52 ( 4 ) , 927 - 934 . Otto , K . and Wood , K . ( 2001 ) , “Product design : techniques in reverse engineering and new product design” . Passera , S . , Kärkkäinen , H . and Maila , R . ( 2012 ) , “When , how , why prototyping ? A practical framework for service development” , in ISPIM Conference Proceedings , The International Society for Professional Innovation Management ( ISPIM ) , 1 . Reed Doke , E . ( 1990 ) , “An industry survey of emerging prototyping methodologies” , Information & Management , 18 ( 4 ) , 169 - 176 . Schäfer , H . and Sorensen , D . J . ( 2010 ) , “Creating options while designing prototypes : value management in the automobile industry” , Journal of Manufacturing Technology Management , 21 ( 6 ) , 721 - 742 . Stumpfe , D . , Hu , Y . , Dimova , D . and Bajorath , J . r . ( 2013 ) , “Recent progress in understanding activity cliffs and their utility in medicinal chemistry : miniperspective” , Journal of medicinal chemistry , 57 ( 1 ) , 18 - 28 . Thomke , S . and Bell , D . E . ( 2001 ) , “Sequential testing in product development” , Management Science , 47 ( 2 ) , 308 - 323 . Ward , A . , Liker , J . K . , Cristiano , J . J . and Sobek , D . K . ( 1995 ) , “The second Toyota paradox : How delaying decisions can make better cars faster” , Sloan Management Review , 36 ( 3 ) , 43 . Ward , A . C . and Sobek II , D . K . ( 2014 ) , Lean product and process development , Lean Enterprise Institute . Yannou , B . , Yvars , P . - A . , Hoyle , C . and Chen , W . ( 2013 ) , “Set - based design by simulation of usage scenario coverage” , Journal of Engineering Design , 24 ( 8 ) , 575 - 603 . ACKNOWLEDGEMENTS This work is supported by the Singapore University of Technology and Design and the SUTD - MIT International Design Centre ( https : / / idc . sutd . edu . sg / ) . This material is also based in part on research sponsored by the United Sates Air Force Academy under agreement number FA7000 - 12 - 2 - 2005 . The US Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation thereon . The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements , either expressed or implied , of the United States Air Force Academy or the US government . The authors would like to thank Gilmour Space Technologies for providing documentation of the exemplar design of a hybrid propulsion system . 220 \ No newline at end of file diff --git a/silver_data/1ee8242352b43f5e58a35ed5f376ac5f1f040c40.pdf.txt b/silver_data/1ee8242352b43f5e58a35ed5f376ac5f1f040c40.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..8c82d81c9ccc5f484a05471332fbbfe2ab495b54 --- /dev/null +++ b/silver_data/1ee8242352b43f5e58a35ed5f376ac5f1f040c40.pdf.txt @@ -0,0 +1 @@ +Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery C . P . Hofstetter * , E . J . Schwarz † , D . Hess * , J . Widenfalk * , A . El Manira * , Darwin J . Prockop †‡ , and L . Olson * * Department of Neuroscience , Karolinska Institutet , S - 171 77 Stockholm , Sweden ; and † Center for Gene Therapy , Tulane University Health Sciences Center , New Orleans , LA 70112 Contributed by Darwin J . Prockop , December 17 , 2001 Marrow stromal cells ( MSC ) can be expanded rapidly in vitro and differentiated into multiple mesodermal cell types . In addition , differentiation into neuron - like cells expressing markers typical for mature neurons has been reported . To analyze whether such cells , exposed to differentiation media , could develop electrophysiolog - ical properties characteristic of neurons , we performed whole - cell recordings . Neuron - like MSC , however , lacked voltage - gated ion channels necessary for generation of action potentials . We then delivered MSC into the injured spinal cord to study the fate of transplanted MSC and possible effects on functional outcome in animals rendered paraplegic . MSC given 1 week after injury led to significantly larger numbers of surviving cells than immediate treatment and significant improvements of gait . Histology 5 weeks after spinal cord injury revealed that MSC were tightly associated with longitudinally arranged immature astrocytes and formed bundles bridging the epicenter of the injury . Robust bundles of neurofilament - positive fibers and some 5 - hydroxytryptamine - positive fibers were found mainly at the interface between graft and scar tissue . MSC constitute an easily accessible , easily expand - able source of cells that may prove useful in the establishment of spinal cord repair protocols . M ost , if not all , central nervous system neurons in mammals have the intrinsic capacity to regenerate a lost axon . The failure of axons to regenerate after spinal cord injury ( SCI ) has been attributed to growth - inhibitory molecules ( 1 ) , lack of appropriate trophic support ( 2 , 3 ) , and reactions of the immune system ( 4 ) . Glial cells produce growth - inhibiting molecules , such as Nogo ( 5 , 6 ) , MAG ( 7 ) , tenascin ( 8 ) , and chondroitin sulfate proteoglycans ( 9 ) . Up - regulation of neurotrophic factors after SCI is limited ( 3 ) . The role of the immune system in SCI is complex but may include nerve growth inhibitory as well as stimulatory events ( 4 , 10 ) . Davies et al . ( 11 ) demonstrated long - distance regeneration of adult dorsal root ganglion cells , transplanted into spinal cord pathways , if a micrografting tech - nique was used to avoid local scarring . However , axon outgrowth was terminated or reversed upon contact with scar tissue . One approach to overcome some of the growth - inhibiting properties of the injured spinal cord is to transplant cells with protective and (cid:1) or reparative properties to the site of injury . Recently , mouse embryonic stem cells , delivered into the injured spinal cord , were shown to differentiate into neurons , astrocytes , and oligodendrocytes and to improve motor function ( 12 ) . However , recovery was limited . Also , the use of embryonic stem cells may not be generally accepted and heterologous transplantation may elicit graft rejection . Marrow stromal cells ( MSC ) constitute an alternative source of pluripotent stem cells . Under physiological conditions , they are believed to maintain the architecture of bone marrow and regulate hematopoiesis with the help of different cell - adhesion molecules and the secretion of cytokines , respectively ( 13 ) . MSC grown out of bone marrow cell suspen - sions by their selective attachment to tissue culture plastic can be expanded efficiently ( 14 , 15 ) and manipulated genetically ( 16 ) . MSC are referred to as mesenchymal stem cells because they are capable of differentiating into multiple mesodermal tissues , including bone ( 17 ) , cartilage ( 18 ) , fat ( 17 ) , and muscle ( 19 ) . In addition , differentiation into neuron - like cells expressing neu - ronal markers has been reported ( 20 – 22 ) , suggesting that MSC may be capable of overcoming germ layer commitment . Impor - tantly , MSC can migrate along known migration pathways when injected into corpus striatum of rats ( 14 ) . MSC migrated throughout forebrain and cerebellum , integrated into central nervous system cytoarchitecture , and expressed markers typical of mature astrocytes and neurons after injection into the lateral ventricle of neonatal mice ( 23 ) . We examined whether MSC expressing neuronal markers in vitro exhibited physiological properties characteristic of neurons . We then delivered MSC into the injured spinal cord to monitor survival , spread and differentiation of such cells , and their possible effects on the motor behavior of rats rendered para - plegic by SCI . Methods Primary Marrow Stromal Cell Cultures . MSC were collected from femurs and tibias of adult male Lewis rats ( Harlan Breeders , Indianapolis ) ( 16 ) . Rats were euthanized with a mixture of 70 % CO 2 and 30 % O 2 . Tibias and femurs were placed on ice in MEM with alpha modification ( (cid:1) - MEM ; GIBCO (cid:1) BRL ) containing 20 % FCS ( Atlanta Biologicals , Norcross , GA ) , 2 mM L - glutamine ( GIBCO (cid:1) BRL ) , 100 units (cid:1) ml penicillin , 100 (cid:2) g (cid:1) ml streptomycin , and 25 ng (cid:1) ml amphotericin B ( penicillin , strep - tomycin , and amphotericin ; GIBCO (cid:1) BRL ) . Epiphyses of femurs and tibias were removed , and the marrow was flushed out by using a syringe filled with medium . Bone marrow was filtered through a 70 - (cid:2) m nylon mesh and plated in 75 - cm 2 flasks . About 24 h after plating , supernatant containing nonadherent cells was removed and fresh medium was added . After the cells had grown to near confluency , they were passaged two to five times by being detached ( 0 . 25 % trypsin (cid:1) 1 mM EDTA for 5 min ) and replated at a density of (cid:1) 5 , 000 cells (cid:1) cm 2 . Preparation of the Retroviral Vector , Production of Viral Particles , and Genetic Marking of MSC . A retroviral construct encoding green fluorescent protein ( GFP ) as an expression marker and amino - glycoside phosphotransferase as a neomycin ( G418 ) selectable marker was prepared by using the LXSN vector ( CLONTECH ) ( 24 ) . Phoenix amphotropic packaging cells ( 25 ) ( ATCC ) were transfected with the LXSN - GFP plasmid by using calcium phosphate precipitation . Viral supernatants were collected 48 h after the start of the transfection , filtered through a 0 . 45 - (cid:2) m filter , and stored at (cid:2) 80°C for further use . Phoenix packaging cells were analyzed at the time of viral harvest for GFP expres - sion . One day before the infection of MSC with GFP - retrovirus , about 100 , 000 MSC were plated in 21 . 0 - cm 2 plates . At the time Abbreviations : MSC , marrow stromal cells ; 5 - HT , 5 - hydroxytryptamine ; SCI , spinal cord injury ; NF , neurofilament ; GFP , greenfluorescentprotein ; IR , immunoreactivity ; GFAP , glial fibrillary acidic protein . ‡ To whom reprint requests should be addressed . E - mail : dprocko @ tulane . edu . The publication costs of this article were defrayed in part by page charge payment . This article must therefore be hereby marked “ advertisement ” in accordance with 18 U . S . C . § 1734 solely to indicate this fact . www . pnas . org (cid:1) cgi (cid:1) doi (cid:1) 10 . 1073 (cid:1) pnas . 042678299 PNAS (cid:2) February 19 , 2002 (cid:2) vol . 99 (cid:2) no . 4 (cid:2) 2199 – 2204 M E D I C A L S C I E N C E S of infection , defined as day 1 , 2 . 5 ml of complete medium containing 20 % heat - inactivated FCS was added to the cells in the presence of 500 (cid:2) l of viral supernatant and 8 (cid:2) g (cid:1) ml polybrene ( Sigma ) . On day 2 , the infection procedure was repeated . On day 3 , fresh complete medium was added with 20 % FCS ( not heat - inactivated ) . On day 4 , cells were split 1 : 3 in 55 . 0 - cm 2 plates in complete medium containing 200 (cid:2) g (cid:1) ml G418 ( Sigma ) for a selection period of 14 – 21 days . MSC that had stably integrated the transgene survived and were expanded for experiments by passaging cells three to nine times . Attempts at Differentiation of MSC Toward a Neuronal Fate . MSC were plated as described ( 20 ) at a density of 2 , 500 cells (cid:1) cm 2 . On the following day , the medium was replaced with preinduction medium consisting of DMEM ( Sigma ) , 20 % FCS , and 10 ng (cid:1) ml basic fibroblast growth factor . After 24 h , the preinduction medium was removed , the cells were washed twice with PBS , and neuronal induction medium containing DMEM supplemented with 2 % DMSO and 200 (cid:2) m butylated hydroxyanisole ( 20 ) was added . In later experiments , we used DMEM with 5 mM 2 - mercaptoethanol ( 20 ) as an alternative neuronal induction medium for the same incubation times . Electrophysiological Recordings of Neuron - Like MSC . Whole - cell recordings were made of MSC exhibiting possible neuronal morphologies such as rounded cell bodies and distinct processes with growth cone - like terminal expansions . Such differentiated cells will be referred to as neuron - like MSC . Whole - cell record - ings were obtained by using a patch - clamp amplifier ( Axopatch 200 A ; Axon Instruments ) . The recordings had a series resistance ranging from 4 to 10 M (cid:3) that was compensated for electronically by 75 – 85 % . The resting membrane potential was assessed in current clamp mode . The residual capacity was removed , but the linear leak was not subtracted . To investigate the existence of voltage - gated channels , neuron - like MSC were clamped at (cid:2) 120 mV and currents were evoked by 100 - ms depolarizing voltage steps to (cid:4) 30 mV . The cells were perfused through a gravity - driven microperfusion system with the nozzle positioned close to the recorded cell . The control solution was used at room temperature and contained 140 mM NaCl , 4 mM KCl , 1 . 8 mM CaCl 2 , 1 mM MgCl 2 , 23 mM sucrose , and 10 mM Hepes . The pH was adjusted to 7 . 40 ; the osmolarity , to 310 mosM . Recordings were made with pipettes of 3 – 7 M (cid:3) filled with a solution containing 4 mM NaCl , 140 mM KCl , 0 . 5 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes , and 5 mM EGTA . The pH was adjusted to 7 . 40 ; the osmolarity , to 305 mosM . Membrane currents and voltages were controlled with appropriate software ( PCLAMP ; Axon Instruments ) . Current and voltage signals were sampled at 10 kHz . MSC Transplantation into the Injured Spinal Cord . A total of 38 adult female Lewis rats ( Charles River Breeding Laboratories ) weight - ing 250 – 260 g received a standardized contusion of the spinal cord and MSC treatment immediately or 1 week after injury . Laminectomy was performed at T9 under halothane anesthesia ( Fluothane ; AstraZeneca So¨derta¨lje , Sweden ) . The impact rod of the NYU device ( 26 ) was centered above T9 and dropped from a height of 25 mm . MSC grown under normal culture conditions were detached and resuspended with (cid:1) - MEM to a final concentration of 30 , 000 viable cells (cid:1) (cid:2) l as determined by trypan blue dye exclusion . Immediately , or 7 days after injury , animals received 5 (cid:2) l of a MSC suspension or (cid:1) - MEM delivered into the injury center and two 2 . 5 - (cid:2) l deposits , one 2 - mm cranial , and the other 2 - mm caudal of the central injection . A total of 300 , 000 cells or vehicle thus was delivered at a rate of 0 . 5 (cid:2) l per minute by means of a stereotaxic frame and a glass pipette with a tip diameter of 100 (cid:2) m configured to a 10 - (cid:2) l Hamilton syringe . Muscle and skin were sutured separately . Urinary bladders were emptied manually five times per day for the first week and twice daily thereafter . Antibiotics ( Borgal ; Hoechst Pharmaceuticals ) were given to prevent urinary tract infection . Two independent experiments with time - matched controls were carried out . A total of 16 rats received MSC ( n (cid:5) 8 ) or cell culture medium ( n (cid:5) 8 ) immediately after injury . A second group ( n (cid:5) 22 ) was treated with MSC ( n (cid:5) 12 ) or vehicle ( n (cid:5) 10 ) 1 week after injury . In both groups , behavior was assessed on a weekly basis , and histological examinations were carried out on animals eutha - nized 5 weeks after injury . All experiments had been approved by the Animal Research Committee of Stockholm . Behavioral Testing . Hindlimb motor function was assessed by using the open - field BBB scoring system ( 27 ) . Individual rats were placed on an open field ( 75 (cid:6) 125 cm ) and observed for 4 min by two observers . Hindlimb function was scored from 0 to 21 ( flaccid paralysis to normal gait ) . The test was carried out 1 day postoperatively and once every week up to the fifth week after SCI . Cell Quantification . GFP - positive cell profiles containing a distinct nucleus were counted in serial sections . Cell numbers were calculated according to the formula of Abercrombie ( 28 ) . Immunocytochemistry . Tissues and cells were processed for indi - rect immunocytochemistry ( 29 ) . Animals were deeply anesthe - tized ( Pentobarbital ) and perfused intracardially with 50 ml of Tyrode’s solution containing 0 . 1 ml of Heparin , followed by 200 ml of fixative ( 4 % paraformaldehyde (cid:1) 0 . 4 % picric acid in PBS ) . Spinal cords were dissected , postfixed in similar fixative for 1 hour , transferred to 10 % sucrose solution , frozen , and cut in a cryostat at 14 - (cid:2) m thickness . Longitudinal sections were col - lected from 18 - mm - long spinal cord segments containing the injury and injection sites and thaw - mounted on gelatin - coated slides . MSC were grown in chamber slides ( Lab - Tek ; Nunc ) and fixed with 4 % paraformaldehyde for 10 min . Antisera raised in goats against fibronectin ( Calbiochem ) and GFP ( Rockland , Gilbertsville , PA ) or in rabbits against nestin ( kindly provided by Urban Lendahl , Karolinska Institute ) , laminin ( Sigma ) , GFAP ( Sigma ) , neurofilament ( NF ; Dahl , Chesnut Hill , MA ) ( 30 ) , PGP 9 . 5 ( Biogenesis , Bournemouth , U . K . ) , GFP ( Molecular Probes ) , and 5 - hydroxytryptamine ( 5 - HT ) ( Sigma ) as well as mouse mAbs to vimentin ( Dako ) , NeuN ( Chemicon ) , NF - 200 ( Sigma ) , and Map - 2 ( Sigma ) were used . Secondary antisera were conju - gated with FITC , rhodamine , or Cy5 ( Jackson ImmunoRe - search ) . Optimal dilutions were established for all primary and secondary antibodies . Controls included omitting the primary antibody . Slides were evaluated by using epifluorescence and confocal microscopy ( Radiance 2100 ; Bio - Rad ) . Statistical Analysis . Comparisons of cell survival were made by using an unpaired t test . Between - group comparisons for behav - ior were carried out by using the Mann – Whitney U test . Signif - icance levels were designated P (cid:7) 0 . 05 , P (cid:7) 0 . 01 , or P (cid:7) 0 . 001 . All values are given as mean (cid:8) SEM . Results Labeling and Characterization of MSC inVitro . Reliable detection by fluorescence microscopy of marrow stromal cells in culture and after transplantation was achieved by transducing MSC with a retrovirus encoding GFP . After a selection time of 14 days in medium supplemented with a cell - toxic concentration of G418 , only cells that had permanently integrated plasmids containing a neomycin selectable marker survived . The successful labeling of all surviving cells was confirmed by fluorescence microscopy . Genetically labeled MSC did not alter their morphology com - pared with native MSC . All analyzed cells ( n (cid:9) 500 ) were positive for fibronectin , vimentin , and laminin ( Fig . 1 A – C and 2200 (cid:2) www . pnas . org (cid:1) cgi (cid:1) doi (cid:1) 10 . 1073 (cid:1) pnas . 042678299 Hofstetter et al . Table 1 ) . In areas of high cell density , fibronectin - immunoreac - tive filaments were deposited extensively in the extracellular space ( Fig . 1 A ) . Immunoreactivity ( IR ) for the mesodermal - intermediate filament vimentin was dense in cellular processes and present in the form of a filamentous meshwork in cell bodies ( Fig . 1 B ) . A distinct subpopulation of MSC ( 37 . 5 % (cid:8) 1 . 2 , n (cid:5) 2 , 777 ) was nestin - IR ( Fig . 1 D ) . MSC were negative for the neuron - specific markers NeuN , NF , Map - 2 , and PGP 9 . 5 . Electrophysiological Properties of Neuron - Like MSC . In one experi - ment , we induced MSC with medium containing 2 % DMSO and 200 (cid:2) M butylated hydroxyanisole for 48 h ( 20 ) . However , patch - clamp recordings of these neuron - like MSC were not possible . This could have been a result of changes in the cell membrane caused by dramatic changes in the osmolarity from 646 to 310 mosM when the differentiation medium was replaced by the extracellular solution . In a second experiment , we differentiated MSC with medium containing 5 mM 2 - mercaptoethanol for 48 h . This treatment was compatible with whole - cell recordings . The resting membrane potential was (cid:2) 11 . 4 (cid:8) 8 . 7 mV ( n (cid:5) 8 ) . It was not possible to induce action potentials in any cell by application of depolarizing current ( Fig . 2 A ) . By using the voltage step protocol to activate voltage - gated currents , no inward currents could be elicited , indicating that the MSC did not express functional sodium channels . An outward currentamplitudewasfoundtobe203 (cid:8) 194 . 5pA ( n (cid:5) 8 ) ( Fig . 2 B ) . The low amplitude of the outward current and the presence of unavoidable leaks associated with the recording and leak currents make it very unlikely that the observed current represents a voltage - gated , outward potassium current . Hence , MSC differen - tiated by 2 - mercaptoethanol did not show typical neuronal prop - erties such as action potentials or voltage - gated Na (cid:4) and K (cid:4) currents and , therefore , are not mature neurons . Delayed Implantation of MSC into the Injured Spinal Cord Improves Functional Recovery . Immediate MSC treatment did not improve locomotor function , as revealed by BBB scoring ( Fig . 3 A ) . Delayed implantation led to significantly improved BBB scores ( 9 . 2 (cid:8) 0 . 5 ) compared with sham - grafted animals ( 7 . 9 (cid:8) 0 . 1 ) ( Mann – Whitney U test ; P (cid:5) 0 . 013 ) ( Fig . 3 B ) . Five weeks after injury , control animals could not support their body weight with their hindlimbs ( n (cid:5) 10 ) , whereas seven animals of the treatment Fig . 1 . Appearance of MSC in culture . ( A ) All MSC display fibronectin - IR during culture conditions . Extensive deposition of fibronectin is observed in thecellclusterinthelowerrightcorner . Vimentin - IR ( B ) andlaminin - IR ( C ) are expressedbyallMSC . ( D ) Nestin - IRisdetectedinasubsetofMSCwithdifferent morphologies . In C and D , the GFP cell marker ( green ) is shown together with laminin or nestin ( red ) . ( Bars (cid:5) 25 (cid:2) m . ) Fig . 2 . Electrophysiological properties of a neuron - like MSC . ( A ) Membrane potential of a neuron - like MSC at rest and during hyperpolarization and depolarization . Note that no action potentials were elicitable even after hyperpolarization to reactivate possible voltage - gated ion channels . ( B ) Volt - age - gatedcurrentselicitedviaavoltagecommandsteppingfrom (cid:2) 120mVto 30 mV . Neither voltage - gated Na (cid:4) channels nor voltage - gated K (cid:4) channels are present . Fig . 3 . ( A ) Analysis of locomotor recovery as measured by BBB scores . Animals treated with MSC immediately after SCI do not differ from control animals . ( B ) Delayed MSC treatment significantly improved locomotor recov - ery . * , P (cid:5) 0 . 013 . Data represent means (cid:8) SEM . Arrowheads indicate the treatment time . Table 1 . IR markers of MSC in vitro and after spinal cord implantation Marker MSC in vitro Implanted MSC Fibronectin (cid:4)(cid:4)(cid:4) (cid:4)(cid:4) Vimentin (cid:4)(cid:4) (cid:2) Laminin (cid:4) (cid:2) Nestin (cid:4)(cid:4) ( 37 . 5 % (cid:8) 1 . 2 ) (cid:2) NeuN (cid:2) (cid:4) NF (cid:2) (cid:2) GFAP (cid:2) (cid:2) Cell shape Flat Spindle - shaped (cid:2) , No signal ; (cid:4)(cid:4)(cid:4) , strong signal . Hofstetter et al . PNAS (cid:2) February 19 , 2002 (cid:2) vol . 99 (cid:2) no . 4 (cid:2) 2201 M E D I C A L S C I E N C E S group ( n (cid:5) 12 ) could lift their trunks and two of them regained stepping patterns with bilateral weight support and frequent forelimb – hindlimb coordination assessed as 13 on the BBB scale . Cryostat sections were examined 5 weeks after SCI . MSC were detected reliably by their GFP labeling , which was abun - dant in the whole cell body . Cell counts revealed significantly larger numbers of cells ( 2 , 966 (cid:8) 681 , n (cid:5) 8 ) in animals treated 1 week after injury than in animals treated immediately ( 518 (cid:8) Fig . 4 . One - week - delayedtransplantationofMSCafterSCI . ( A ) MSCformbundlesbridgingtheepicenterofthelesionvisualizedbythetransgenicGFPmarker . Arrows indicate the location of the impact injury . ( B ) Nestin - immunoreactive immature astrocytes with longitudinally aligned processes ( red ) are found within MSC bundles ( green ) . ( C ) GFAP - IR ( red ) marks astrocytic processes penetrating the grafted cell aggregates ( green ) . ( D ) 5 - HT - positive fibers ( red ) are present among the implanted MSC ( green ) . ( E ) Robust NF - IR nerve fiber bundles ( red ) are found at the interface between MSC and host tissue . ( B , C , and E ) Asterisks indicate macrophages . [ Bars (cid:5) 250 (cid:2) m ( A ) and 25 (cid:2) m ( B – E ) . ] 2202 (cid:2) www . pnas . org (cid:1) cgi (cid:1) doi (cid:1) 10 . 1073 (cid:1) pnas . 042678299 Hofstetter et al . 106 , n (cid:5) 8 ) ( unpaired t test ; P (cid:5) 0 . 0052 ) . MSC infused immediately after SCI were found mainly in the periphery of the injury zone , whereas MSC transplanted 1 week after SCI were found in the whole lesion zone ( Fig . 4 A ) . Implanted MSC exhibited a bipolar morphology with long processes extending along the axis of the spinal cord . MSC formed bundles , which were arranged mainly along the long axis of the spinal cord , and provided bridges across the epicenter of the lesion area , which was filled with debris and macrophages . All implanted MSC expressed fibronectin - IR and a weak but distinct NeuN - IR ( ref . 31 , Fig . 5 , and Table 1 ) . Interestingly , implanted MSC had lost detectable nestin - IR ( Fig . 4 B and Fig . 6 ) as well as vimentin and laminin - IR ( Table 1 ) . Nestin and GFAP antibodies revealed the presence of two different kinds of glial cells in the injured spinal cord . GFAP and nestin - positive reactive astrocytes delineated the margin of the epicenter of the lesion with their tightly interwoven processes . In animals that had received a MSC infusion , astrocytic processes reached into the epicenter by penetrating MSC - bundles ( Fig . 4 C ) . A second population of cells was nestin - positive but GFAP - negative and , thus , similar to immature astrocytes ( 32 ) . These cells had migrated into the epicenter of the injury . In animals treated with MSC , immature astrocytes populated the MSC bundles and extended their delicate processes along the engrafted cells ( Fig . 4 B and Fig . 5 A – C ) . NF - positive fibers were preferentially found at the inter - face between MSC bundles and scar tissue ( Fig . 4 E and Fig . 5 D – F ) . Some of the nerve fibers associated with the implant - ed cells were identified as 5 - HT - positive ( Fig . 4 D ) . The intra - spinal MSC did not display GFAP , NF , MAP - 2 , or PGP 9 . 5 immunoreactivity . Discussion Our results further characterize MSC and demonstrate that MSC treatment can improve recovery of animals rendered paraplegic by SCI . Engrafted MSC form bundles and guide regenerating neuropil through the spinal cord lesion . MSC grown out of marrow cell suspensions by selective attach - ment to tissue culture plastic initially are not homogeneous but become morphologically more homogeneous with time in culture ( 33 ) by depletion of hematopoietic cells . However , the relationship between hematopoietic stem cells and marrow stromal cells is the topic of intensive research ( 34 ) . When neonatal mice incapable of developing cells of the myeloid and lymphoid lineages were trans - planted with whole bone marrow fractions from healthy donors , bone marrow - derived cells expressing NeuN and NSE were found within the central nervous system ( 35 ) . In a similar experiment , adult irradiated mice were infused with a whole bone marrow fraction . Cells engrafted in the olfactory bulb were immunoreactive for NeuN , NF , and Tuj1 ( 36 ) . However , these experiments did not determine whether the bone marrow - derived cells detected in the brain were derived from mesenchymal or hematopoietic stem cells . For our experiments , we used long - term MSC cultures containing mesenchymal stem cells selected on the basis of adherence to plastic . With the exception of a weak NeuN - IR seen in intraspinal MSC , neither neuronal induction in vitro nor environmental cues Fig . 5 . MSCbundlesguidehostneuropil . ( A – C ) Beyondtheastrocyticscarsurroundingtheepicenterofthelesion ( A ) , nestin - positive ( blue ) andGFAP - negative immatureastrocytes ( B ) arefoundcloselyassociatedwithtransplantedMSC ( C ) . ( D – F ) Neurofilament - IRfibers ( red ) arefoundincloserelationshipwithnestin - IR fibers ( blue ) mainly in the periphery of MSC bundles . ( Bars (cid:5) 25 (cid:2) m . ) Fig . 6 . ( A and B ) FiveweeksafterSCI , MSCexpressadistinctnuclearNeuN - IR . ( C ) All MSC are fibronectin - positive . [ Bar (cid:5) 10 (cid:2) m ( A ) and 25 (cid:2) m ( B and C ) . ] Hofstetter et al . PNAS (cid:2) February 19 , 2002 (cid:2) vol . 99 (cid:2) no . 4 (cid:2) 2203 M E D I C A L S C I E N C E S provided by the injured spinal cord in vivo could differentiate the MSC toward a neuronal fate , suggesting that cells with more developed neuronal characteristics detected by other groups are of hematopoietic origin . By using confocal microscopy , reliable colocalization of mark - ers expressed by the GFP - labeled cells was possible . In culture , conditions all MSC displayed fibronectin , vimentin , and laminin - IR . Interestingly , a subpopulation of MSC expressed nestin , which is expressed during early stages of neurogenesis ( 37 ) and myogenesis ( 38 ) . Attempts were made to differentiate marrow stromal cells toward a neuronal fate . In addition to the two induction media published here , we tested media containing retinoic acid , brain - derived neurotrophic factor ( 21 ) , nerve growth factor , NT - 3 , glial cell line - derived neurotrophic factor ( GDNF ) , and forskolin ( unpublished data ) . However , none of these supplements improved the neuronal differentiation of MSC . Considering that differentiation of MSC into myoblasts has been reported ( 19 ) , it could be possible that the nestin - positive subpopulation of MSC differentiates spontaneously into an early myoblastic state during culture conditions . Five weeks after transplantation into the injured spinal cord , MSC had down - regulated vimentin , laminin , and nestin and begun to express a weak nuclear NeuN - IR , indicating that they had been instructed by environmental cues present in the injured spinal cord . Importantly , transplanted MSC formed bundles bridging the epicenter of the lesion filled with debris and macrophages . Regenerating host neuropil was associated with MSC aggre - gates , and , thus , a degree of cellular organization had been reestablished in the injury zone . Immature astrocytes , defined as nestin - positive and GFAP - negative cells , which are formed from stem cells in response to injury ( 32 ) , populated the MSC bundles . These cells might help promote nerve fiber outgrowth by offer - ing a growth - permissive surface . Growth of nerve fibers on the surface of astrocytes has been observed in other studies in which peripheral nerves ( 39 ) or fibroblasts secreting NGF ( 40 ) were implanted . Another explanation for the guidance of nerve fibers might be the abundant expression of N - cadherin ( 41 ) , which is known to enhance neurite extension ( 42 ) , on the surface of MSC . Importantly , we identified 5 - HT - positive nerve fibers along the MSC bundles . The 5 - HT - system of the spinal cord has been shown to be important in functional recovery after SCI ( 43 , 44 ) , and the apparent regeneration of 5 - HT elicited by MSC thus may be contributing to the observed improvement of behavioral recovery . From a clinical standpoint , it is perhaps particularly encouraging that delayed MSC treatment enhanced survival of grafted cells and exerted a beneficial effect on functional recov - ery . MSC infused immediately after SCI encounter a hostile environment characterized by ischemia , necrosis , and the pres - ence of potentially toxic compounds such as oxygen radicals and lytic enzymes . However , 12 h after SCI , maximal tissue loss is reached , leading to the next phase characterized by reactive gliosis , invasion of inflammatory cells , and reparative attempts such as up - regulation of basic fibroblast growth factor . Our data are indicating that this later phase of SCI is providing a more habitable environment for infused cells . 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Biotechnol . 18 , 415 – 419 . 43 . Bregman , B . S . , Kunkel - Bagden , E . , Reier , P . J . , Dai , H . N . , McAtee , M . & Gao , D . ( 1993 ) Exp . Neurol . 123 , 3 – 16 . 44 . Nygren , L . G . , Fuxe , K . , Jonsson , G . & Olson , L . ( 1974 ) Brain Res . 78 , 377 – 394 . 2204 (cid:2) www . pnas . org (cid:1) cgi (cid:1) doi (cid:1) 10 . 1073 (cid:1) pnas . 042678299 Hofstetter et al . \ No newline at end of file diff --git a/silver_data/1fdf08fd8401b60187ac93afce842d68629ec61d.pdf.txt b/silver_data/1fdf08fd8401b60187ac93afce842d68629ec61d.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..0554e454a4d8147a924c34d78a1a72478415d729 --- /dev/null +++ b/silver_data/1fdf08fd8401b60187ac93afce842d68629ec61d.pdf.txt @@ -0,0 +1 @@ +Cell volume change through water efflux impacts cell stiffness and stem cell fate Ming Guo a , 1 , Adrian F . Pegoraro a , Angelo Mao a , b , Enhua H . Zhou c , 2 , Praveen R . Arany d , e , Yulong Han a , f , Dylan T . Burnette g , Mikkel H . Jensen a , h , Karen E . Kasza a , i , Jeffrey R . Moore j , Frederick C . Mackintosh k , l , m , Jeffrey J . Fredberg c , David J . Mooney a , b , Jennifer Lippincott - Schwartz n , 3 , and David A . Weitz a , o , 3 a John A . Paulson School of Engineering and Applied Sciences , Harvard University , Cambridge , MA 02138 ; b Wyss Institute for Biologically Inspired Engineering , Harvard University , Cambridge , MA 02138 ; c Harvard T . H . Chan School of Public Health , Boston , MA 02115 ; d Department of Oral Biology , University at Buffalo , Buffalo , NY 14214 ; e Department of Biomedical Engineering , University at Buffalo , Buffalo , NY 14214 ; f Biomedical Engineering and Biomechanics Center , Xi ’ an Jiaotong University , Xi ’ an 710049 , China ; g Department of Cell and Developmental Biology , Vanderbilt University School of Medicine , Nashville , TN 37232 ; h Department of Physics and Astronomy , California State University , Sacramento , CA 95819 ; i Department of Mechanical Engineering , Columbia University , New York , NY 10027 ; j Department of Biological Sciences , University of Massachusetts at Lowell , Lowell , MA 01854 ; k Department of Physics and Astronomy , VU University , 1081 HV Amsterdam , The Netherlands ; l Department of Chemical and Biomolecular Engineering , Rice University , Houston , TX 77030 ; m Center for Theoretical Biophysics , Rice University , Houston , TX 77030 ; n Howard Hughes Medical Institute , Janelia Research Campus , Ashburn , VA 20147 ; and o Department of Physics , Harvard University , Cambridge , MA 02138 Contributed by Jennifer Lippincott - Schwartz , August 26 , 2017 ( sent for review March 29 , 2017 ; reviewed by Daniel A . Fletcher and Valerie M . Weaver ) Cells alter their mechanical properties in response to their local microenvironment ; this plays a role in determining cell function and can even influence stem cell fate . Here , we identify a robust and unified relationship between cell stiffness and cell volume . As a cell spreads on a substrate , its volume decreases , while its stiffness concomitantly increases . We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations , in - cluding varying substrate stiffness , cell spread area , and external osmotic pressure . The reduction of cell volume is a result of water efflux , which leads to a corresponding increase in intracellular molecular crowding . Furthermore , we find that changes in cell volume , and hence stiffness , alter stem - cell differentiation , regard - less of the method by which these are induced . These observations reveal a surprising , previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology . cell volume | cell mechanics | molecular crowding | gene expression | stem cell fate C ell volume is a highly regulated property that affects myriad functions ( 1 , 2 ) . It changes over the course of the cell life cycle , increasing as the cell plasma membrane grows and the amount of protein , DNA , and other intracellular material in - creases ( 3 ) . However , it can also change on a much more rapid time scale , as , for example , on cell migration through confined spaces ( 4 , 5 ) ; in this case , the volume change is a result of water transport out of the cell . This causes increased concentration of intracellular material and molecular crowding , having numerous important consequences ( 6 , 7 ) . Alternately , the volume of a cell can be directly changed through application of an external osmotic pressure . This forces water out of the cell , which also decreases cell volume , increases the concentration of intracellular material , and intensifies molecular crowding . Application of an external osmotic pressure to reduce cell volume also has other pronounced manifestations : For example , it leads to a significant change in cell mechanics , resulting in an increase in stiffness ( 8 ) ; it also impacts folding and transport of proteins ( 9 ) , as well as condensation of chromatin ( 10 ) . These dramatic effects of osmotic - induced volume change on cell behavior raise the question of whether cells ever change their volume through water efflux under isotonic condi - tions , perhaps to modulate their mechanics and behavior through changes in molecular crowding . Here , we demonstrate that when cells are cultured under the same isotonic conditions , but under stiffer extracellular environ - ments , they reduce their cell volume through water efflux out of the cell , and this has a large and significant impact on cell me - chanics and cell physiology . Specifically , as a cell spreads out on a stiff substrate , its volume decreases , and the cell behaves in a similar manner to that observed for cells under external osmotic pressure : Both the cortical and cytoplasmic stiffness increase as the volume decreases ; the nuclear volume also decreases as cell volume decreases . Moreover , stem - cell differentiation is also strongly impacted by cell volume changes . These results suggest that cells in different environments can change their volume through water efflux . This leads to changes in molecular crowding and impacts the mechanics , physiology , and behavior of the cell , having far - reaching consequences on cell fate . Results Bulk and Shear Moduli of Cells Increase as Cell Volume Decreases Under External Osmotic Compression . To directly probe the consequences of changes in cell volume , we control the external osmotic pres - sure through the addition of 300 - Da polyethylene glycol ( PEG 300 ) ( 8 , 11 ) . We measure the cell volume by fluorescently labeling Significance Cell volume is thought to be a well - controlled cellular charac - teristic , increasing as a cell grows , while macromolecular den - sity is maintained . We report that cell volume can also change in response to external physical cues , leading to water influx / efflux , which causes significant changes in subcellular macro - molecular density . This is observed when cells spread out on a substrate : Cells reduce their volume and increase their molec - ular crowding due to an accompanying water efflux . Exploring this phenomenon further , we removed water from mesen - chymal stem cells through osmotic pressure and found this was sufficient to alter their differentiation pathway . Based on these results , we suggest cells chart different differentiation and behavioral pathways by sensing / altering their cytoplasmic volume and density through changes in water influx / efflux . Author contributions : M . G . , J . L . - S . , and D . A . W . designed research ; M . G . , A . F . P . , A . M . , E . H . Z . , P . R . A . , Y . H . , D . T . B . , and K . E . K . performed research ; M . G . , A . M . , E . H . Z . , P . R . A . , D . T . B . , M . H . J . , K . E . K . , J . R . M . , and D . J . M . contributed new reagents / analytic tools ; M . G . , A . F . P . , Y . H . , F . C . M . , J . J . F . , and D . A . W . analyzed data ; and M . G . , A . F . P . , J . L . - S . , and D . A . W . wrote the paper . Reviewers : D . A . F . , University of California , Berkeley ; and V . M . W . , University of California , San Francisco . The authors declare no conflict of interest . 1 Present address : Department of Mechanical Engineering , Massachusetts Institute of Technology , Cambridge , MA 02139 . 2 Present address : Ophthalmology , Novartis Institutes of BioMedical Research , Cambridge , MA 02139 . 3 To whom correspondence may be addressed . Email : lippincottschwartzj @ janelia . hhmi . org or weitz @ seas . harvard . edu . Thisarticlecontainssupportinginformationonlineatwww . pnas . org / lookup / suppl / doi : 10 . 1073 / pnas . 1705179114 / - / DCSupplemental . E8618 – E8627 | PNAS | Published online September 25 , 2017 www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1705179114 the cytoplasm and cell surface and use confocal microscopy to identify the boundaries of the cell in 3D ( 12 , 13 ) . The measured volumes have been shown to be consistent with those measured by atomic force microscopy ( AFM ) ( 8 ) . We further validate our confocal measurements by comparing them to those made by using high - resolution structured illumination microscopy ( SIM ) on the same sample ; the measured cell volumes are consistent for both techniques , indicating a measurement uncertainty of < 10 % ( Fig . S1 ) . Because cell volume naturally changes during the cell cycle , we make all our observations after starving cells overnight ; however , measurements made under standard culture conditions exhibit the same behavior , albeit with increased variability . To explore the dependence of cell volume on external osmotic pressure , we culture A7 cells on glass substrates and add increasing concentrations of PEG 300 to the medium ; PEG 300 does not penetrate the cell membrane and thus increases the external os - motic pressure . Since the osmotic pressure imbalance across the cell cortex is negligible ( 14 ) , the external osmotic pressure must be matched by the internal osmotic pressure , which is controlled by the concentration of ions and small proteins . Thus , to compensate for the increase of external osmotic pressure , the cell must increase its internal osmolyte concentration either by ion influx or water efflux . We find that the cell volume decreases with increasing ex - ternal osmotic pressure ( Fig . S2 ) . This must be due to water efflux , because the cell volume recovers its original value upon removal of the external osmotic pressure . Cells eventually reach a minimum volume under extreme osmotic compression ( Fig . S2 ) ; this reflects the volume of the total intracellular material , as well as any os - motically inactive water required to hydrate ions and proteins . This behavior is reminiscent of an ideal gas system with an excluded volume , where the pressure is purely entropic in origin . Indeed , the relationship between osmotic pressure P and cell volume V is consistent with P = Nk B T / ( V − V min ) , where N is the number of intracellular osmolytes , k B is the Boltzmann constant , T is the absolute temperature , and V min is the minimum volume ( 8 ) . To confirm that the decrease in cell volume is due to water efflux , we use the Bradford assay to measure the total protein content per cell before and after osmotic compression and find no significant difference ( SI Materials and Methods ) . As cell volume decreases , it becomes increasingly difficult to remove additional water and further shrink the cell because the concentration of intracellular ions and other materials increases . The resistance of water leaving a cell is the osmotic bulk modulus and is defined as B = − V d P / d V ∼ − V Δ P / Δ V . By varying the osmotic pressure and measuring the resultant volume of A7 cells cultured on a glass substrate , we measure B and find that it in - creases as cell volume decreases , as shown by the points at the top in Fig . 1 A . Using the expression for the volume dependence of P , we can predict the behavior of the bulk modulus , B = Nk B TV / ( V − V min ) 2 . This functional form provides an excellent fit to the data , as shown by the dashed line through the points at the top in Fig . 1 A . The value of V min determined by the fit is 2 , 053 ± 30 μ m 3 ; this agrees with the minimum volume measured when the cells are exposed to extreme osmotic compression to remove all osmotically active water , ∼ 2 , 100 μ m 3 . The fit also provides a value for N , and this gives a concentration of ∼ 200 mM , which is consistent with the known salt concentration within a cell ( 15 ) . The excellent fitting with a constant N suggests that the total amounts of ions and proteins remain approximately constant during osmotic compression . While the osmotic pres - sure balance is largely controlled by ion concentration , the concentration of large proteins and organelles also increases as free water leaves the cell . It is the volume of these proteins and organelles ( including the nucleus ) that predominantly deter - mines V min . Moreover , as the cell approaches its minimum vol - ume , molecular crowding must increase within the cell , and this might lead to additional changes in cell behavior . Among many cell properties , cortical stiffness has been shown to depend on volume through molecular crowding as cells are osmotically compressed ( 8 ) . To measure changes in cortical stiff - ness , we use optical magnetic twisting cytometry ( OMTC ) to de - termine the cortical shear modulus , G , of A7 cells , as shown in Fig . S3 ( 8 , 16 , 17 ) ; the results obtained with OMTC are in quantitative agreement with those obtained by using AFM ( 18 , 19 ) . The cor - tical shear modulus and the volume both change rapidly , but si - multaneously , as the external osmotic pressure is increased , as shown in Fig . 1 B ; moreover , the cortical shear modulus recovers its initial value immediately upon removal of the external osmotic pressure ( Fig . S2 B ) . Interestingly , the cell cortical stiffness has the same trend as the bulk modulus and is described by exactly the same functional form as shown by the points and dashed line , respectively , in the middle of Fig . 1 A . The value of V min obtained by fitting the functional form to the data , 2 , 004 ± 41 μ m 3 , agrees well with that obtained from the fit to the bulk modulus . However , the value of the shear modulus is consistently three orders of magnitude less than that of the bulk modulus . The similar Fig . 1 . Mechanical properties of A7 cells on glass as a function of cell volume under external osmotic pressures . ( A ) Osmotic bulk modulus ( gray triangles ) , cortical shear modulus ( red triangles ) , and cytoplasmic shear modulus ( open triangles ) increase as cell volume is decreased upon external osmotic compression . The dashed line through gray triangles represents the least - squares fit using the functional form , B = Nk B TV / ( V − V min ) 2 ( R 2 = 0 . 99 ) . Dashed lines through the cortical and cytoplasmic moduli are exactly the same fit , simply scaled by a factor of 500 and 100 , 000 , respectively . ( B ) Cortical shear modulus of a single A7 cell , measured by OMTC , increases immediately after the application of an osmotic compression with 0 . 26 M PEG 300 ; this is concurrent with a decrease in cell volume after osmotic compression . Guo et al . PNAS | Published online September 25 , 2017 | E8619 C E LL B I O L O G Y P H Y S I C S P N A S P L U S dependence of the shear and bulk moduli on cell volume implies that molecular crowding has the dominant effect on both . The major contribution of the shear modulus of the cell comes from the cortical stiffness . However , the cell is a highly hetero - geneous structure , and the interior is much softer ; the cytoplasm of the cell is a weak elastic gel with a shear modulus that is three orders of magnitude lower than that of the cortex ( 17 , 20 ) . To measure cytoplasmic stiffness , we microinject PEG - coated poly - styrene beads into A7 cells and use laser tweezers to determine the cytoplasmic shear modulus , as shown in Fig . S3 ( Materials and Methods ) . The intracellular shear modulus increases as cell vol - ume decreases , following an identical trend as both the cortical shear and bulk moduli , as shown by the lower points in Fig . 1 A . The cytoplasmic shear modulus clearly increases as the cell vol - ume is further decreased , but its value is too large to be measured with the laser tweezers . Over the range accessible , the cytoplasmic shear modulus behavior has the same functional form as do the other two moduli , as shown by the dashed line at the bottom of Fig . 1 A ; however , its value is a further three orders of magnitude less than that of the cortical shear modulus . Cells Reduce Their Volume When Cultured on Stiffer Substrates . Cortical stiffness is a key physical property of cell mechanics , and its value typically decreases as the stiffness of the substrate on which the cells are grown decreases ( 21 ) . Cell morphology can vary markedly with substrate stiffness , and this could also impact molecular crowding , even under isotonic conditions . To investigate this possibility , we culture A7 cells on 100 - μ m - thick polyacrylamide ( PA ) gels coated with collagen I ; by varying gel composition , we tune the shear modulus of the gels from 0 . 1 to 10 kPa , matching the physiological elasticities of natural tissues ( Table S1 ) . To probe cell morphology , we measure the cell spread area ; it in - creases significantly for cells cultured on stiffer substrates ( 21 – 24 ) , as shown by the confocal images in Fig . 2 A , Upper , and as sum - marized in Fig . 2 B . Surprisingly , however , cell volume is not conserved : As substrate rigidity increases , cell volume decreases . On the most rigid substrates , the volume decrease is as much as ∼ 40 % compared with cells grown on the softest substrates where their volume is a maximum , as shown in Fig . 2 C . Other mam - malian cell types , including HeLa , NIH 3T3 , mouse mesenchymal stem cell ( mMSCs ) , and primary human airway smooth muscle cells , show similar behavior ( Fig . 2 C ) . This confirms the generality of the dependence of cell volume on substrate stiffness . Thus , even without changes in external osmotic pressure , the cell volume can change . Restricting Spread Area Increases Cell Volume . To further explore the nature of the change in cell volume with substrate stiffness , we investigate the role of spread area . We culture A7 cells on a glass substrate , but control the spread area by restricting their adhesion . We micropattern collagen “ islands ” of varying size on a glass substrate to limit the adhesion area of the cells ( 25 ) . Interestingly , we find that cells with limited spread area have a larger height compared with cells with unrestricted spread area ( Fig . 3 A ) . Moreover , the cell volume also increases as the spread area de - creases , as shown in Fig . 3 B . The same effect is observed when we grow cells on micropatterned softer substrates : If the cell spread area is restricted to be less than that of a freely spreading cell on the substrate , the volume increases . Remarkably , cell volume ex - hibits the same dependence on spread area regardless of how the area is attained , either through controlling the adhesive area on a stiff substrate or through varying substrate stiffness , as shown by the comparison of the blue and gray points in Fig . 3 C . Cell Volume Reduction Is Due to Intracellular Water Efflux . To de - termine whether the reduction in cell volume under isotonic conditions is due to changes in protein content or to water efflux , we monitor a single trypsinized cell as it spreads on a rigid sub - strate . The cell changes from its initial rounded state to a fully spread state in ∼ 20 min . During this time period , the cell volume decreases concomitantly with increasing spread area , as shown in Fig . 3 D . The relationship between volume and spread area is identical to that of cells cultured on confined areas or on substrates of varying rigidities , as shown by the red × symbols in Fig . 3 C . Water can leave the cell within a minute ( 8 , 26 ) , while changes in amount of intracellular materials due to growth can typically take hours for mammalian cells ( 3 , 27 ) . Thus , these results suggest that volume reduction under isotonic conditions is most likely con - trolled through water exchange , albeit at isotonic osmotic pressure . To confirm that cell volume variation upon spreading is due to water efflux and not protein loss , we measure the total protein content per cell for cells cultured on both stiff and soft substrates and find no significant difference ( SI Materials and Methods ) . Consistent with this , when we apply extreme osmotic pressure to cells by exchanging culture medium with pure PEG , thereby extracting all osmotically active water from the cells , we find that the resultant minimum volume , V min , is independent of substrate stiffness ( Fig . 4 ) . Since V min approximately reflects the amount of intracellular material and bound water that is osmotically inactive Fig . 2 . Morphology and volume of adherent cells change with increasing substrate stiffness . ( A ) Top and side views of fixed A7 cells on a stiff ( shear modulus of 10 kPa ) and a soft ( shear modulus of 1 , 200 Pa ) PA gel substrate coated with collagen I . The actin cortex ( green ) and nucleus ( blue ) are la - beled . ( B ) The projected cell area increases with increasing substrate stiff - ness . ( C ) Cell volume markedly decreases with increasing substrate stiffness . Error bars represent the SD ( n > 200 individual cells ) . HASM , human airway smooth muscle . E8620 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1705179114 Guo et al . ( 8 ) , these data support the view that variation of cell volume re - sults from exchange of free intracellular water . Ion Channels and the Actomyosin Cytoskeleton Play a Role in Cell Volume Reduction During Spreading . The efflux of water during cell spreading under isotonic conditions must have a different origin than the efflux of water during osmotic compression . In both cases , the osmotic pressure is balanced across the cell membrane . Under osmotic compression , the total amount of material , in - cluding ions and proteins , remains approximately constant ; the internal osmotic pressure increases as a result of increasing in - tracellular osmolyte concentration through water efflux . During Fig . 3 . Cell volume of A7 cells increases when the cell spread area is decreased by growing cells on micropatterned collagen islands . Error bars represent the SD . * P < 0 . 05 ; * * P < 0 . 01 . ( A ) Shown are 3D images of A7 cells on micropatterned islands of different sizes on glass . Cells are labeled with cell tracker green . ( Scale bars , 20 μ m . ) ( B ) Cell volume decreases with increasing cell spread area on glass . ( C ) Cell volume plotted as a function of the projected area , for cells on substrates with different stiffnesses ( gray circles ; n > 200 ) , cells on a glass substrate but with different available spread area ( blue squares ; n > 200 ) , and a dynamically spreading cell ( red crosses ; n = 3 ) . ( D ) Variation of cell spread area and volume as a single cell dynamically attaches on a stiff substrate ( n = 3 ) . ( E ) Schematic illustration of cell volume decrease through water efflux , as cells spread out or are osmotically compressed . Fig . 4 . Cell morphology and volume under drug treatment and osmotic compression . ( A ) The 3D morphology of control cells and cells with ATP depletion and under extreme osmotic compression , on stiff and soft substrates . Cytoplasm ( green ) and nucleus ( yellow ) are labeled . ( Scale bars : 20 μ m . ) ( B ) Cells without active contraction ( blebbistatin - treated and ATP - depleted ) and under extreme osmotic compression do not exhibit a volume dependence with substrate stiffness ; cells with choloride channels - inhibited ( NPPB treated ) exhibit a weaker volume dependence with substrate stiffness . The control data of A7 cells is same as in Fig . 2 C . Error bars represent the SD ( n > 200 individual cells ) . Guo et al . PNAS | Published online September 25 , 2017 | E8621 C E LL B I O L O G Y P H Y S I C S P N A S P L U S cell spreading , cell volume reduction occurs under isotonic con - ditions ; for water to leave the cell , the total amount of osmolytes must change . Since the amount of protein per cell remains con - stant , it is instead likely the reduction of osmolytes for cells on stiff substrates is due to the exchange of ions with the surroundings . During cell spreading , cytoskeletal tension increases , and this has been tied to the increase of ion channel activity ( 28 – 30 ) . To test the role of ion channel activity on cell volume variation , we inhibit chloride ion channels by 0 . 1 mM 5 - nitro - 2 - ( 3 - phenylpropylamino ) - benzoic acid ( NPPB ) after cells fully spread . The decrease in cell volume with increasing substrate stiffness is significantly sup - pressed when ion channels are blocked , as shown by the green open triangles in Fig . 4 B . This indicates that cell volume reduction under isotonic conditions requires the activity of ion channels to change the total amount of internal osmolytes and hence ensure that the osmotic pressure remains balanced . To further test if active cell processes play a role in the re - duction of cell volume , we treat cells with 10 μ M blebbistatin to inhibit myosin II motor activity and thus directly reduce cyto - skeletal tension . Blebbistatin treatment prevents cells from de - creasing their volume on stiff substrates , as shown by the blue triangles in Fig . 4 B . The same increase in volume is observed when we inhibit general motor activity by depleting ATP using 2 mM sodium azide and 10 mM 2 - deoxyglucose , as shown by the pink triangles in Fig . 4 B . These results further demonstrate that active cellular processes are involved in volume decrease under isotonic conditions and that cells must actively control the osmolyte concentration using ion channels to affect water efflux and change their volume . Cell Moduli Demonstrate a Universal Dependence on Cell Volume . Cell cortical stiffness increases with substrate stiffness as the cells increase their spread area under isotonic conditions ; cell cortical stiffness also increases as we change the spread area for fixed substrate stiffness . However , cell stiffness also increases for fixed substrate stiffness and fixed spread area when the osmotic pres - sure is increased , thereby decreasing cell volume . We therefore hypothesize that cell volume change is the common descriptor underlying the cell stiffness change observed in all these cases . To investigate this hypothesis , we plot cortical stiffness as a function of volume : Cell stiffness decreases with increasing volume as cells are grown on softer substrates ( Fig . 5 A ) . Cell stiffness decreases in a similar fashion with increasing volume as cells are grown on a substrate with fixed stiffness , but with varying adhesive areas ( Fig . 5 B ) . Similarly , cell stiffness increases as cells grown on a soft substrate are compressed by an external osmotic pressure ( Fig . 5 C ) and when cells grown on a stiff substrate and a highly con - strained area are compressed through osmotic pressure ( Fig . 5 D ) . Remarkably , when we plot all these data on a single graph ( Fig . 5 E ) , they all overlay and exhibit a universal dependency between cell stiffness and cell volume across all perturbations . Thus , cell volume change is indeed a common descriptor of cell stiffness Fig . 5 . Relationship of cell cortical stiffness and cell volume . ( A – D ) Dependence of cell cortex shear modulus of A7 cells on their volume , under different conditions , including cells cultured on substrates of varying stiffnesses ( A ) , on a stiff substrate with micropatterns of varying sizes ( B ) , on a soft substrate with a shear modulus of 100 Pa with addition of increasing amount of osmotic pressure ( C ) , and on a glass substrate with small micropatterns limiting cell spreading and with the addition of osmotic pressures ( D ) . ( E ) Cell cortical shear modulus scales with cell volume , as shown for cells growing on substrates of varying stiffness ( gray circles ) , on a glass substrate with restricted available spread area using micropatterns ( blue squares ) , on a soft substrate with osmotic compression ( shear modulus of 100 Pa , green upside down triangles ) , on an unpatterned ( red triangles ) and a micropatterned glass substrate ( yellow dia - monds ) with osmotic compression , and on a glass substrate with 10 μ M blebbistatin treatment ( cyan pentagon ) or depleted of ATP ( black triangle ) . Solid line shows the power - law fitting of the data , scales as V − 2 . Dashed line shows fitting to G ∝ k B TV / ( V − V min ) 2 Error bars represent the SD ( n > 200 individual cells ) . osm . comp . , osmotic compression ; pat . , patterned . E8622 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1705179114 Guo et al . change . The data shown here are all obtained for a single cell type , A7 ; however , similar scaling behavior between cell stiffness and cell volume is observed for each cell type , although they are shifted in amplitude and volume ( Fig . S4 ) . Interestingly , if we restrict the data to those cell volumes physiologically accessible without application of external osmotic pressure , where cells solely respond to either changes in substrate stiffness or spread area , the behavior is consistent with G ∝ 1 / V 2 , as shown by the solid line in Fig . 5 E . Intriguingly , this is very similar to the behavior of biopolymer networks reconstituted from either actin or vimentin , where the shear modulus is approxi - mately proportional to the square of the concentration ( 31 – 34 ) . This would be expected if the change in the shear modulus is due solely to the water exchange responsible for the volume change . If the data for all measured volumes are included , the full behavior is well described by G ∝ k B TV / ( V − V min ) 2 , as shown by the dashed line in Fig . 5 E . While there is no model that predicts this behavior for the shear modulus , it is nevertheless identical in functional form to the behavior of the bulk modulus over the same range of volumes , B ∝ k B TV / ( V − V min ) 2 . This functional form for the bulk modulus is a direct consequence of the measured P – V relationship of the cell , which reflects the effects of increased molecular crowding as water is drawn from the cell . Thus , our results suggest that a similar crowding phenomenon is also responsible for the change in the cortical shear modulus under various perturbations that we tested here . Similarly , both osmotic bulk modulus and cytoplasmic shear modulus across multiple perturbations are also observed to be universally dependent on cell volume ( Fig . S5 ) , as they do under osmotic compression shown in Fig . 1 A . To explore the generality of the correlation between cell stiff - ness and cell volume , we also include the data with actomyosin contraction inhibited through addition of blebbistatin ; we find that cortical stiffness and volume remain exactly on the same func - tional curve , as shown by the cyan pentagon in Fig . 5 E . Similarly , when ATP is completely depleted , the data exhibit the same be - havior , as shown by the black triangle in Fig . 5 E . Interestingly , not only for isolated cells , similar behavior is also observed for cells in a 2D monolayer . We grow epithelia MCF10 - A cells into a monolayer , but with different cell densities , and measure the corresponding cell volume and cortical shear modulus ; we find that cell volume decreases as the density of cells increases ( Fig . S4 B ) . Cell volume and stiffness again remain correlated : As cell density increases , cell volume decreases , and cortical stiffness in - creases in a fashion consistent with a 1 / V 2 dependence . These results imply that cell volume plays role in determining cell me - chanics , even for cells in confluent layers . Nuclear Volume Tracks Cell Volume . The nuclear envelope , like the cell membrane , is selectively permeable and allows water ex - change ; therefore , we wonder if water efflux from the cell ex - tends to the nucleus . To test this , we fluorescently label cell nuclei and use 3D confocal microscopy to measure their volume . We find that cell nuclear volume tracks cell volume both for cells grown under isotonic conditions on different substrates and also for cells whose volume is changed through external osmotic pressure ( 12 ) , as shown in Fig . 6 . This suggests that the cell nucleus also becomes more crowded as cell volume decreases , and this has a direct consequence on the degree of motion within the nucleus . To illustrate this , we measure fluctuating motion of GFP - tagged histone H2B , which is widely used to report on positional fluctuations of chromatin ( 35 , 36 ) ; we calculate their mean - squared displacement and find a marked reduction in the level of fluctuations as molecular crowding is increased through an external osmotic pressure ( 9 , 20 , 37 ) ( Fig . 6 B ) . Stem - Cell Fate Can Be Directed by Changing Cell Volume . Of the many properties of cells that vary with cell stiffness ( 38 ) , cell spread area ( 39 ) , and substrate stiffness ( 40 ) , one of the most consequential is stem - cell differentiation . We therefore in - vestigate the impact of cell volume on stem - cell differentiation , since cell volume is intrinsically related to cell stiffness , as well as the other parameters like molecular crowding . To do so , we externally impose changes in cell volume through osmotic com - pression ; this results in changes of cell stiffness , but not spread area or substrate properties . Moreover , we confirm that this does not change the tension within the cell , as measured by the trac - tion force microscopy ( TFM ) ( ref . 41 ; Fig . S6 ) . We grow mMSCs on two different PA gel substrates , one a stiff substrate with a shear modulus of 7 kPa and the other a soft substrate with a shear modulus of 0 . 2 kPa . On a soft substrate , cells typically have a larger volume than cells on a stiff substrate ; thus , we apply additional osmotic pressure by adding 0 . 1 M PEG 300 ( + 100 mOsm ) to the medium so that the volume of cells grown on the soft gel matches that of the cells grown on the stiff gel ( Fig . 7 D and E ) . We find that when volumes are matched , cell stiffnesses are also matched ( Fig . 7 F ) . We expose the cells to bipotential differentiation medium that is supportive of both osteogenic and adipogenic fates ( SI Materials and Methods ) . After 1 wk of culture , we observe substantially increased osteo - genic differentiation , as indicated by alkaline phosphatase ( ALP ) activity on the rigid substrate , compared with unperturbed cells on soft substrate , as expected ( 40 ) . Unexpectedly , osmotically compressed cells grown on the soft substrate also exhibit en - hanced ALP activity , suggesting preferential osteogenic differ - entiation ( Fig . 7 A and B ) . This is confirmed by Western analysis of the expression of osteogenic biomarkers runt - related tran - scription factor 2 ( RUNX2 ) and bone sialoprotein ( BSP ) , as shown in Fig . 7 C . As a contrapositive test , we use hypotonic conditions ( − 80 mOsm ) to swell cells grown on a stiff substrate , such that both cell volume and nuclear volume match those of cells grown on a soft substrate ( Fig . 7 J and K ) . We again find that when volumes are matched , cell stiffnesses are matched ( Fig . 7 L ) . In this case , we observe substantial adipogenic differentiation as shown by in situ staining of neutral lipid accumulation [ Oil Red O ( ORO ) ] and the expression of adipogenic biomarker peroxisome proliferator - activated re - ceptor gamma ( PPAR - γ ) ( Fig . 7 G – I ) . The results indicate that we can influence stem - cell differentiation either toward osteogenic or adipogenic fates by changing their volume ; this suggests that the intranuclear and possibly intracellular crowding affects stem - cell fate . Stem - Cell Fate Affects Cell Volume . In the absence of strong chemical cues , physical properties such as substrate stiffness or external osmotic pressure affect stem - cell differentiation . How - ever , chemical signals can often override these physical cues . While we have shown that physical signals change cell volume , we wonder if chemical cues also change cell volume during dif - ferentiation . Thus , we grow mMSCs on a soft PA gel , which would bias the cells toward adipogenic differentiation ; however , we add supplements ( β - glycerol phosphate , ascorbic acid , and dexamethasone ) to the medium ( details in Materials and Meth - ods ) to direct the cells toward osteogenic differentiation . The mMSCs undergo osteogenic differentiation under these condi - tions . Interestingly , we find that cell volume decreases during this process and , surprisingly , even precedes osteogenic differentia - tion , as shown in Fig . 7 M . Conversely , we find that cell volume increases when we chemically induce adipogenesis using dexa - methasone alone for mMSCs cultured on stiff substrates , as shown in Fig . 7 N . These results suggest that cell volume and stem - cell differentiation are strongly correlated . Discussion The data presented here establish the critical importance of cell volume and molecular crowding in determining cell properties , including cell stiffness and intracellular dynamics . Unlike a Guo et al . PNAS | Published online September 25 , 2017 | E8623 C E LL B I O L O G Y P H Y S I C S P N A S P L U S growing and dividing cell , where volume change is associated with an increase of intracellular protein and other materials , we show that changes in cell volume can alternately be directly as - sociated with changes in intracellular water content while levels of proteins and other materials remain constant : Upon increase of substrate stiffness or cell spread area , cells respond by de - creasing their water content and hence increasing their stiffness . While actomyosin contractility is essential for this adaptation , cell contraction itself cannot mechanically change cell volume : The stress generated by the cytoskeleton is too weak ( 42 ) ( ∼ 1 – 10 kPa , as shown in Fig . S6 ) to withstand or induce any osmotic pressure difference since pressures are on the order of megapascals ( 8 ) ; therefore , cytoskeletal forces cannot squeeze water out of cells . Instead , it is most likely that contractile tension of the cyto - skeleton increases activity of ion channels , which , in turn , affects intercellular water content and hence cell volume ( 28 , 29 ) ; this is consistent with our finding that cell volume change is suppressed as we inhibit the activity of specific ion channels and ATP - dependent processes . Surprisingly , both the cortical shear modu - lus and the cell bulk modulus exhibit exactly the same functional form with cell volume , as shown by Fig . S5 . The origin of this behavior for the bulk modulus can be understood as a conse - quence of molecular crowding ; however , the underlying origin of the behavior for the cortical shear modulus is unclear , although the behavior in the physiologically accessible volumes is consistent with the concentration dependence of reconstituted networks , and hence with water content . Our results also indicate that changing protein concentration has major implications for cell physiology , typified by the effect cell volume change has on stem - cell differentiation . Changes in intracellular water content will change intracellular molecular crowding and cellular dynamics ( Fig . 6 and Fig . S7 ) . This will un - doubtedly cause significant variations in many internal physiological processes , such as protein folding and binding kinetics ( 7 , 43 – 45 ) , structural rearrangements and transport phenomena ( 8 , 9 , 44 ) , and protein expression patterns ( 2 , 6 , 46 ) . Moreover , similar effects extend into the nucleus since the volume of the cell nucleus decreases as cell volume decreases ( Fig . 6 A ) . This will change the intranuclear molecular crowding , and possibly lamin concentration , which has been suggested to affect chromatin structure , mobility , and therefore transcription and gene expression patterns ( 10 , 47 ) . Thus , cell volume and cell nuclear volume , which are defined fea - tures of the cell , may change in physiological conditions such as under tissue compression and osmotic variance in the body , there - fore significantly impacting myriad cellular processes , such as sig - naling , protein dynamics , and even stem - cell differentiation . Materials and Methods Cell Culture and Pharmacological Interventions . Cell culture protocol and pharmacological interventions are described in SI Materials and Methods . Cells are synchronized to avoid cell size growth and volume change during cell cycle . CellLabelingandImmunofluorescence . Cellsforconfocalimagingareplatedat a density of 20 cells per mm 2 . Live cells are fluorescently labeled with Cell - Tracker , CellMask plasma membrane stains ( Invitrogen ) , and DRAQ5 ( Cell Signaling Technology ) to label cytoplasm , membrane , and cell nucleus , re - spectively . The cells are imaged and sectioned at 0 . 15 - μ m intervals by using excitation from a 633 - or 543 - nm laser or a 488 - nm line of an argon laser and a 63 × , 1 . 2 - NA water immersion objective on a laser scanning confocal mi - croscope ( TCS SP5 ; Leica ) . To label actin cortex and get a better resolution of cell boundary and cell size , living cells cultured on substrates are fixed , and their actin structures are labeled by using phalloidin . Fabrication of PA Gel Substrate and Micropatterned Islands . The fabrication of PA gel substrates with different stiffness and micropatterned islands follows standard procedures ( 24 ) and is described in SI Materials and Methods . The3DVolumeMeasurement . Stainedcellsare observedbyusing a63 × / 1 . 2 - NA water immersion lens on a Leica TSC SP5 . Cells that we observe are randomly selected . Optical cross - sections are recorded at 0 . 15 - μ m z - axis intervals to show intracellular , nuclear , and cortical fluorescence . By using theoretical point spread function , a stack of gray - level images ( 8 bits ) are subjected to deconvolution before 3D visualization . The 3D visualization is carried out by using ImageJ and AMIRA software . The volume is calculated by counting voxel number , after thresholding the stack . The confocal measurement has been previously compared with AFM ; results from two techniques agree . More details are in SI Materials and Methods . Super - resolution SIM imaging of fixed cells is performed on a microscope ( ELYRA SIM ; Carl Zeiss ) with an Apochromat 63 × / 1 . 4 - NA oil objective lens ( Fig . S1 ) . We use a voxel size of 40 × 40 × 110 nm ; the cell height and cell volume measurements from confocal and SIM do not show statistical dif - ference , as shown in Fig . S1 . Osmotic Compression . Hyperosmotic stress is applied by adding PEG 300 to isotonic culture medium . Cells arethen allowedto equilibrate in PEGsolution for 10 min at 37 °C and 5 % CO 2 , before measurements . The cell size de - creases within 20 s and maintains the small volume for hours , as shown in Movie S1 . Cortical Stiffness Measurements . The mechanical properties of the cell cortex are probed by using OMTC , which is a high - throughput tool for measuring adherent cell mechanics with high temporal and spatial resolution ( 16 , 48 , 49 ) . Measurements are done at 37 °C . Measurements of cortical stiffness using OMTC agree with values obtained by other methods such as AFM ( 50 ) . Frequency - dependent shear moduli of A7 cells in isotonic medium are shown in Fig . S3 . For convenience , in this work , we use cortexshearmodulus measured at Fig . 6 . Cell nuclear volume and nuclear dynamics change with cell volume . ( A ) Nucleus volume always directly tracks cell volume , decreasing proportionally with increasing substrate stiffness , spread area , and osmotic pressure ; the ratio between nuclear volume and cell volume remains approximately constant for each tested cell type . ( B ) Mean square displacement of GFP - tagged Histone in MCF - 10A cell nuclei , reflecting positional fluctuation of chromatin , significantly reduces under external osmotic compression through application of 0 . 1 M PEG 300 . osm . comp . , osmotic compression ; sub . , substrate . E8624 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1705179114 Guo et al . a fixed frequency , 0 . 75 Hz , for comparison under different microenvironmental conditions . More details are in SI Materials and Methods . Cytoplasmic Material Properties Measured Using Optical Tweezers . To directly measurethemicromechanicalproperties ofthecytoplasm , weperformactive microrheology measurements using optical tweezers to impose a sinusoidal oscillation on a 500 - nm - diameter probe particle microinjected in a cell , as described ( 17 , 20 ) . The trap stiffness we use is calibrated as 0 . 05 pN / nm . Trapped beads are oscillated across a frequency range of 0 . 3 – 70 Hz ; the frequency - dependent shear modulus of the cytoplasm is shown in Fig . S3 . The data plotted in the lower part of Fig . 1 A and Fig . S5are G ′ measured at 10 Hz under different conditions . More details are in SI Materials and Methods . Stem Cell Culture and Differentiation . The clonally derived murine bone marrow mesenchymal stem cell line originally from BALB / c mice ( D1s ) are purchased from American Type Cell Culture and are maintained in standard Fig . 7 . Cell volume affects differentiation of mMSCs . ( A – F ) Osteogenesis . ( A ) In situ staining of mMSC for ALP ( black ) and nucleus ( DAPI , blue ) after 1 wk of culture in the presence of combined osteogenic and adipogenic chemical supplements shows increased osteogenesis on the stiff substrate ( shear modulus of 7 kPa ) and the soft substrate ( shear modulus of 200 Pa ) with osmotic compression ( with 0 . 1 M PEG 300 , additionally to the medium ) , compared with the control on soft substrate without additional osmotic pressure . ( B ) Mean percentages of mMSC osteogenesis . Error bars , SEM ( n = 3 samples ) . * P < 0 . 05 . ( C ) Western analysis of osteogenic protein expression ( RUNX2 and BSP ) in mMSCs after 3 d of culture . ( C – F ) Cell volume ( D ) , nucleus volume ( E ) , and cortex shear modulus ( F ) measured with confocal microscopy and OMTC , for three experimental conditions ( n > 50 individual cells ) . * P < 0 . 05 . ( G – L ) Adipogenesis . ( G ) In situ staining of mMSC for fat lipids ( red ) after 2 wk of culture in the presence of combined osteogenic and adipogenic chemical supplements shows enhanced adipogenesis on stiff substrate with application of hypotonic pressure ( with the addition of 30 % DI water ) , compared with the control . ( H ) Mean percentages of mMSC adipogenesis . Error bars , SEM ( n = 3 samples ) . * P < 0 . 05 . ( I ) Western analysis of adipogenesis protein expression ( PPAR - γ ) in mMSCs after 1 wk of culture . ( J – L ) Cell volume ( J ) , nucleus volume ( K ) , and cortex shear modulus ( L ) measured with confocal microscopy and OMTC , for three experimental conditions ( n > 50 individual cells ) . * P < 0 . 05 . ( M ) mMSCs are exposed to osteogenesis medium for 10 d . The ratio of cells expressing high level of ALP , as measured by using Fast Blue staining , as described in SI Materials and Methods , is counted in three independent samples fixed each day . Volume of cells is observed each day as well . As the ratio of differentiated cells increases , cell volume decreases correspondingly ( n = 3 samples ; error bars represent SD ) . ( N ) mMSCs are exposed to adipogenesis medium for 2 wk . The ratio of cells with clear fat lipid accumulation , as visualized by ORO staining , as described in SI Materials and Methods , is counted in three independent samples fixed every 2 d . Volume of cells is measured at the same time as well . As the ratio of differentiated cells increases , cell volume increases correspondingly ( n = 3 samples ; error bars represent SD ) . ( Magnification : A and G , 400 × . ) Guo et al . PNAS | Published online September 25 , 2017 | E8625 C E LL B I O L O G Y P H Y S I C S P N A S P L U S DMEM and supplemented with 10 % FBS and 1 % penicillin / streptomycin . Theyareculturedatnomorethan80 % confluencyatnogreaterthanpassage 25 in serum - supplemented DMEM . For experiments , cells are trypsinized and platedoncollagenI - coatedsoftandstiffsubstratesatadensityof20cellspermm 2 . We then let cells fully attach and spread for 1 h , before applying osmotic pressure and adding bipotential differentiation medium . In the compression sample , weadd 0 . 1 M PEG 300 ( 100mOsm ) tothemedium , so that the volume and stiffness of cells on the soft gel match those of the cells grown on the stiff gel , as measured with confocal and OMTC . Similarly , in the swelling sample on glass , after plating cells , we apply hypotonic pressure by adding 30 % deion - ized water intoculture medium . To induce differentiation ( 51 ) , mMSC cultures are supplemented with 10 mM β - glycerol phosphate ( Sigma ) , 50 μ g / mL ascorbic acid ( Sigma ) , and 0 . 1 μ M dexamethasone ( Sigma ) , as dexamethasone alone has demonstrated the ability to induce adipogenesis of D1 in vitro ( 52 ) . Culture medium with additional osmotic pressures and supplements are ex - changed every 3 d . Western Analysis of MSC Lineage Specification . For Western blots , after 3 d ( osteogenesis , as shown in Fig . 7 A – F ) or 1 wk ( adipogenesis , as shown in Fig . 7 G – L ) of culture , cells are lysed in RIPA buffer ( Sigma - Aldrich ) with Com - plete Mini protease inhibitor ( Roche ) . Lysates are centrifuged at 16 , 000 × g at 4 °C for 20 min , and total protein is estimated with a Bradford assay ( BCA ; Thermo Scientific Inc . ) . Protein lysates are separated in precast Tris – glycine or – acetate gels and transferred onto nitrocellulose membranes ( both Invi - trogen ) . Blots are incubated with primary antibodies at 4 °C overnight . Following washes , blots are incubated with appropriate species - specific secondary antibodies ( Jackson ImmunoResearch Laboratories ) and chem - iluminescence ( Thermo Scientific Inc . ) is detected by films ( Kodak MR ; Sigma - Aldrich ) . Images of Western blots are quantified by using ImageJ software . Actin bands are scanned to normalize for loading differences between sam - ples . Antibodies we use for immunoblotting are mouse anti - RUNX2 antibody , mouse anti - BSP antibody , and mouse anti – PPAR - γ antibody ( Abcam ) , mouse anti - actin antibody ( Chemicon ) , and goat anti - mouse secondary antibody ( Jackson Immunoresearch Laboratories ) . Immunostaining . After 1 wk ( osteogenesis , as shown in Fig . 7 A – F ) or 2 wk ( adipogenesis , as shown in Fig . 7 G – L ) of culture , cells are fixed with 4 % paraformaldehyde and 0 . 1 % Triton X - 100 in PBS . For osteogenesis exami - nation , ALP activity is visualized by Fast Blue staining [ 200 μ g / mL naphtol - AS - MX - phosphate ( NAMP ) combined with 200 μ g / mL Fast Blue salt ] in alkaline buffer ( 100 mM Tris · HCl , 100 mM NaCl , 0 . 1 % Tween - 20 , and 50 mM MgCl 2 , pH 8 . 2 ) , as shown in Fig . 7 A . For adipogenesis examination , fat lipid accu - mulation is visualized by ORO ( 600 μ g / mL in isopropyl alcohol for 2 h at 25 °C ) staining , as shown in Fig . 7 G . Nuclei are visualized with 2 . 7 mM DAPI in PBS . The density of ALP - expressing cells and ORO - positive cells are calculated by counting the number of cells in more than three randomly selected fields ( 10 × magnification ) , and normalizing to the total number of cells detected by DAPI staining , in each individual sample . TFM . Cell contractility is measured using the TFM technique according to described methods ( 41 , 53 , 54 ) . More details are in SI Materials and Methods . ACKNOWLEDGMENTS . We thank J . P . Butler , F . Deng , and A . E . Ehrlicher for helpful discussions . This work was supported by NIH Grants P01GM096971 , P01HL120839 , and R01EB014703 ; Harvard Materials Research Science and Engineering Center Grant DMR - 1420570 ; and NSF Grant DMR - 1310266 . M . H . J . and J . R . 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PNAS | Published online September 25 , 2017 | E8627 C E LL B I O L O G Y P H Y S I C S P N A S P L U S \ No newline at end of file diff --git a/silver_data/202ced7b69db15430080b0875cb01845eb8cccde.pdf.txt b/silver_data/202ced7b69db15430080b0875cb01845eb8cccde.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..6984bb8513e3f85af0f271f48ee4678b20920699 --- /dev/null +++ b/silver_data/202ced7b69db15430080b0875cb01845eb8cccde.pdf.txt @@ -0,0 +1 @@ +ORIGINAL RESEARCH published : 13 April 2022 doi : 10 . 3389 / fmicb . 2022 . 856757 Frontiers in Microbiology | www . frontiersin . org 1 April 2022 | Volume 13 | Article 856757 Edited by : Axel Cloeckaert , Institut National de recherche pour l’agriculture , l’alimentation et l’environnement ( INRAE ) , France Reviewed by : Antoni Soriano - Arandes , Vall d’Hebron University Hospital , Spain Mark Parcells , University of Delaware , United States Silvia Spoto , Policlinico Universitario Campus Bio - Medico , Italy Zhenhai Zhang , Southern Medical University , China * Correspondence : Ai - e Li 565082471 @ qq . com Xiang - Rong Qin 490161337 @ qq . com Xue - jie Yu yuxuejie @ whu . edu . cn Specialty section : This article was submitted to Infectious Agents and Disease , a section of the journal Frontiers in Microbiology Received : 17 January 2022 Accepted : 14 March 2022 Published : 13 April 2022 Citation : Li D , Li A - e , Li Z - q , Bao Y , Liu T , Qin X - R and Yu X - j ( 2022 ) SARS - CoV - 2 Delta Variant in Jingmen City , Hubei Province , China , 2021 : Children Susceptible and Vaccination Breakthrough Infection . Front . Microbiol . 13 : 856757 . doi : 10 . 3389 / fmicb . 2022 . 856757 SARS - CoV - 2 Delta Variant in Jingmen City , Hubei Province , China , 2021 : Children Susceptible and Vaccination Breakthrough Infection Dan Li 1 , Ai - e Li 2 * , Zhu - qing Li 3 , Yu Bao 3 , Tian Liu 4 , Xiang - Rong Qin 5 * and Xue - jie Yu 1 * 1 State Key Laboratory of Virology , School of Public Health , Wuhan University , Wuhan , China , 2 Jingmen Municipal Health Commission , Jingmen , China , 3 Jingmen Center for Disease Control and Prevention , Jingmen , China , 4 Jingzhou Center for Disease Control and Prevention , Jingzhou , China , 5 Department of Clinical Laboratory , The Second Hospital of Shandong University , Jinan , China Background : The delta variant ( B . 1 . 617 . 2 ) of SARS - CoV - 2 was the dominant viral strain causing COVID - 19 in China , 2021 . We reported a SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China . Methods : The data of epidemiological , clinical , laboratorial , and vaccination of COVID - 19 cases were collected through field investigation and analyzed . Results : During the outbreak from 4 to 20 August 2021 , 58 cases of the SARS - CoV - 2 delta variant ( B . 1 . 617 . 2 ) were identified with 15 ( 25 . 9 % ) asymptomatic and 43 ( 74 . 1 % ) symptomatic ( mild and moderate ) patients . The mean serial interval was 2 . 6 days ( standard deviation : 2 . 0 , 95 % CI : 1 . 9 – 3 . 6 ) . The median age of the patients was 39 years ( ranging from 1 to 60 years ) with the high proportion in children ( 19 . 0 % ) . The secondary attack rate was 9 . 8 % higher from parents to their children ( < 18 years ) ( 46 . 2 % , 95 % CI : 14 . 8 – 77 . 5 % ) than that between spouses ( 36 . 4 % , 95 % CI : 14 . 5 – 58 . 2 % ) , but no significant difference was observed ( p > 0 . 05 ) . Approximately half ( 27 ; 46 . 6 % ) of cases were vaccine breakthrough infections . In vaccine breakthrough cases ( fully vaccinated ) , viral loads decreased 1 . 9 – 3 . 4 - folds ( p < 0 . 05 ) , duration of viral shedding shortened 5 days ( p < 0 . 05 ) , and the risk of becoming symptomatic from asymptomatic decreased 33 % ( 95 % CI : 5 – 53 % ) ( aged ≥ 12 years ) than those in unvaccinated infections . Conclusions : Children are highly susceptible to the SARS - CoV - 2 delta variant in the COVID - 19 outbreak in Jingmen City in 2021 . Inactivated vaccine derived from wide - type strain can effectively reduce the viral load , duration of viral shedding , and clinical severity in vaccine breakthrough cases . Our study indicates that protective measures that include full vaccination against COVID - 19 , especially in children , should be strengthened . Keywords : SARS - CoV - 2 , delta variant ( B . 1 . 617 . 2 ) , children , breakthrough infection , household transmission Li et al . Delta Variant in Hubei , China INTRODUCTION Since the pandemic of coronavirus disease 2019 ( COVID - 19 ) was reported in Wuhan in December 2019 ( Li Q . et al . , 2020 ) , 416 million COVID - 19 confirmed cases and over 5 million deaths were reported globally as of 17 February 2022 [ WHO Coronavirus ( COVID - 19 ) Dashboard , 2022 ] . Currently , pediatric cases are on the rise composing 18 . 9 % of all COVID - 19 cases with nearly 7 million additional cases since the first week of September 2021 in the US ( Children and COVID - 19 : State - Level Data Report , 2022 ) . According to the definition of the World Health Organization ( WHO ) , SARS - CoV - 2 variants of concern have signature substitutions in key amino acids of the immunodominant spike protein which can affect virus characteristics with one or more of the following changes at a degree of global public health significance : increased transmissibility or detrimental change in COVID - 19 epidemiology ; increase in virulence or change in clinical disease presentation ; and decrease in the effectiveness of public health and social measures or available diagnostics , vaccines , and therapeutics ( Tracking SARS - CoV - 2 Variants , 2022 ) . Several SARS - CoV - 2 variants of concern have emerged in the past 2 years including the alpha variant ( B . 1 . 1 . 7 ) , beta variant ( B . 1 . 351 ) , gamma variant ( P . 1 ) , delta variant ( B . 617 . 2 ) , lambda variant ( C . 37 ) , and omicron variant ( B . 1 . 1 . 529 ) ( Faria et al . , 2021 ; Tegally et al . , 2021 ; Vaidyanathan , 2021 ; Volz et al . , 2021 ; Tracking SARS - CoV - 2 Variants , 2022 ) . The delta variant was first identified in October 2020 in India and rapidly spread to more than 200 countries , areas , or territories worldwide ( Vaidyanathan , 2021 ; World Health Organization , 2021 ; Tracking of Variants , 2022 ; Tracking SARS - CoV - 2 Variants , 2022 ) . Based on the data from India and the UK , the delta variant was about 50 – 60 % more transmissible than the alpha variant ( Dhar et al . , 2021 ; Public Health England , 2021 ) . In China , the first local transmission caused by the SARS - CoV - 2 delta variant emerged in Guangzhou City in May 2021 ( Zhang et al . , 2021 ) . Since then , the SARS - CoV - 2 delta variant outbreaks have been reported in several cities and most of them infected < 100 persons . However , there have been few publications related to SARS - CoV - 2 delta variant outbreak in China , especially Hubei Province which was the epicenter of the first wave of the COVID - 19 pandemic . Jingmen City in the middle of Hubei Province , a prefectural - level municipality with a population of 2 . 9 million , experienced the first wave of the COVID - 19 pandemic with 928 confirmed cases between January and March 2020 . The second COVID - 19 outbreak caused by the delta variant occurred in August 2021 . This outbreak gave us an opportunity to systematically analyze the characteristics of epidemiology , transmission dynamic , household transmission , and vaccination in the outbreak caused by the SARS - CoV - 2 delta variant in Jingmen City , China . METHODS Study Design and Data Sources A retrospective observational study was conducted to explore the epidemiological , clinical , laboratorial , and effectiveness of vaccination in the SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China , in August 2021 . The relevant data were obtained by filed investigation and from official information system . Case Definition The definitions of confirmed case , asymptomatic infection , close contact , and disease severity ( mild , moderate , and severe ) were all defined according to the China’s national guidelines for the prevention and control of COVID - 19 ( the eighth version ) and the guidelines for the diagnosis and treatment of COVID - 19 ( the eighth revised version ) as described in Table 1 ( National Health Commission of the People’s Republic of China , 2021a , b ) . A COVID - 19 vaccine breakthrough case was defined as a fully vaccinated case who received two doses of vaccine ≥ 14 days before the last exposure to a person with COVID - 19 . Transmission Dynamic The serial interval is the time between the onset of illness in the primary case and secondary case . The basic reproductive number ( R 0 ) is defined as the expected number of additional cases that one case will generate without intervention and control measures . The effective reproduction number ( R t ) is an indicator of real - time transmissibility of disease , which is often used to evaluate intervention and control measures . The incubation period is defined as the period of time between infection and illness onset . Household Transmission A household was defined as two or more individuals living in a family in this study . Household contacts were defined as the individuals living in the same family for at least 1 day or night with the household index starting from 4 days before the onset or sampling of the index case ( Li et al . , 2021 ) . For asymptomatic infections , onset was defined as the date of specimen collection for the first positive SARS - CoV - 2 reverse transcription polymerase chain reaction ( RT - PCR ) . A household secondary attack rate ( SAR ) was defined as the number of secondary symptomatic cases and asymptomatic cases in the household within 14 days after the index case was identified divided by the total number of contacts in the household . Effect of Vaccination The COVID - 19 vaccination history of each patient was obtained from the electronic health information system and field investigation . The vaccines used in Jingmen City were inactivated virions of SARS - CoV - 2 wide - type strain and produced by Beijing Institute of Biological Products Co . , Ltd , Wuhan Institute of Biological Products Co . , Ltd , and Sinovac Life Science Co . , Ltd . The effect of inactivated SARS - CoV - 2 vaccines against the delta variant was evaluated by comparing the clinical severity , RT - PCR cycle threshold ( Ct ) value , and duration of viral shedding of vaccinated patients with breakthrough infection or partially vaccinated to the unvaccinated . Asymptomatic case was considered as effective vaccination when evaluating the impact of the vaccine on clinical symptoms . Vaccine status was categorized as the unvaccinated , partly vaccinated , and fully vaccinated Frontiers in Microbiology | www . frontiersin . org 2 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China TABLE 1 | Case definition in the SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China in August 2021 . Terms Definition Confirmed case An individual who had positive PCR result of SARS - CoV - 2 and COVID - 19 related clinical symptoms ( National Health Commission of the People’s Republic of China , 2021a ) . Asymptomatic infection An individual who had positive PCR result of SARS - CoV - 2 without clinical symptom ( National Health Commission of the People’s Republic of China , 2021a ) . Close contact An individual who was in contact without effective protection with someone with COVID - 19 starting 4 days before the infected person developed symptoms or the sampling date if they did not have symptoms such as living , eating , working , traveling , and attending social activities ( National Health Commission of the People’s Republic of China , 2021a ) . Mild COVID - 19 case A confirmed case with mild clinical symptoms , but without signs of pneumonia on chest imaging ( National Health Commission of the People’s Republic of China , 2021b ) . Moderate COVID - 19 case A confirmed case with fever or respiratory symptoms , and imaging manifestations of pneumonia ( National Health Commission of the People’s Republic of China , 2021b ) . Severe COVID - 19 adult case A confirmed case who met at least one of the following criteria : respiratory rate ≥ 30 times / min ; oxygenation index ≤ 300 mmHg ; resting oxygen saturation ≤ 93 % ; clinical symptoms were progressively aggravated and the lesion on lung had significantly progressed over 50 % on chest imaging within 24 – 48h ( National Health Commission of the People’s Republic of China , 2021b ) . Severe COVID - 19 child case A confirmed case who met at least one of the following criteria : persistent high fever for more than 3 days ; shortness of breath excluded the effects of fever and crying ( respiratory rate in different ages : < 2 months : ≥ 60 times / min ; 2 – 12 months : ≥ 50 times / min ; 1 – 5 years : ≥ 40 times / min ; > 5 years : ≥ 30 times / min ) ; resting oxygen saturation ≤ 93 % ; assisted breathing ; lethargy and convulsions ; refusing food or feeding difficulties and dehydration ( National Health Commission of the People’s Republic of China , 2021b ) . based on the fact that it takes 14 days to develop an effective protection against SARS - CoV - 2 after vaccination ( National Health Commission of the People’s Republic of China , 2021b ) . The unvaccinated individuals were either not vaccinated before the last exposure to a person with COVD - 19 or received one dose of vaccine < 14 days before the last exposure . Partially vaccinated referred to individuals who received one dose of vaccine ≥ 14 days or received two doses of vaccine < 14 days before the last exposure . Fully vaccinated referred to individuals who received two doses of vaccine ≥ 14 days before the last exposure . Laboratory Testing The SARS - CoV - 2 testing was performed according to the national guideline ( National Health Commission of the People’s Republic of China , 2021a ) . Nasopharyngeal swab samples were obtained from individuals , and RNA was extracted and tested by real - time RT - PCR with SARS - CoV - 2 - specific primers and probes aimed at the gene of the open reading frame ( ORF ) and nucleocapsid ( N ) protein . Cycle threshold ( Ct ) value < 37 was defined as positive , and persons with the Ct value between 37 and 40 were resampled and tested to confirm . Viral load was estimated by the Ct value of real - time RT - PCR . A difference of 1 Ct unit was approximately equal to a factor of 2 in the number of virus per sample ( Levine - Tiefenbrun et al . , 2021 ) . High - throughput sequencing was performed for SARS - CoV - 2 - positive specimens using MiniSeq high - output kit and Misq v2 Reagent Kit ( Illumina , US ) by the Hubei Provincial Center for Disease Control and Prevention . The whole genome sequence was compared with the nucleotide sequences in the GenBank and GISAID database . Statistical Analysis The analysis of the serial interval , R 0 , R t , and incubation period , was performed with R software 4 . 0 . 5 . The package of “ R 0 ” and “ EpiEstim ” was used for the estimation of R 0 and R t , respectively . Categorical variables were compared with the chi - square test or the Fisher’s exact test , and continuous variables were compared with the Mann – Whitney U - test using SPSS 25 . 0 as appropriate . Two - sided p < 0 . 05 and 95 % confidence intervals ( CIs ) not including 1 value were considered as statistically significant . Ethics Approval and Consent Institutional review board approval was waived as a part of a public health outbreak investigation in China . The data for analysis in our study were anonymous . RESULTS Outbreak Description On 4 August 2021 , a 33 - year - old male who had illness onset 2 days ago was diagnosed as COVID - 19 in Jingmen City , Hubei Province , China . The patient was a migrant worker and had worked in a construction filed in an area with COVID - 19 before return to Jingmen City 5 days ago . After he was confirmed to be infected with SARS - CoV - 2 , another three co - workers who returned to Jinmen City together with the index patient were also tested for SARS - CoV - 2 and two of his co - workers were also SARS - CoV - 2 - positive . After discovering the three patients , totally 4 , 436 close contacts were identified in Jingmen City , and all of them were tested for SARS - CoV - 2 with RT - PCR from 4 to 20 August 2021 . The PCR results showed that another 55 individuals to be SARS - CoV - 2 - positive . In total , 58 cases were identified in this COVID - 19 epidemic in Jingmen City ( Figure 1 ) . DNA sequencing showed that the SARS - CoV - 2 strains from the cases were delta variant . The median age of the cases was 39 years old ranging from 1 to 60 with 11 ( 19 . 0 % ) children ( ≤ 18 years ) , and the male and female ratio was 1 . 9 : 1 ( 38 men and 20 women ) . According to the clinical Frontiers in Microbiology | www . frontiersin . org 3 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China FIGURE 1 | Epidemic curve of COVID - 19 cases in Jingmen City , Hubei Province , China in August , 2021 . severity , 25 . 9 % ( 15 / 58 ) of patients were asymptomatic , and 74 . 1 % ( 43 / 58 ) of patients had clinical symptoms with 41 . 3 % ( 24 / 58 ) mild and 32 . 8 % ( 19 / 58 ) moderate . The asymptomatic proportion was 27 . 3 % ( 3 / 11 ) for children and 25 . 5 % ( 12 / 47 ) for adults ( Fisher’s exact : p > 0 . 05 ) . The mean interval time between illness onset and diagnosis was 2 days ( 95 % CI : 1 . 7 – 2 . 4 ) . The median Ct value of the cases for the first real - time RT - PCR result was 26 . 3 ( inter quartile range , IQR : 22 . 7 – 31 . 0 ) for the N gene and 27 . 35 ( IQR : 22 . 0 – 32 . 23 ) for the ORF gene ( Table 2 ) . Transmission Dynamics Based on the epidemiological data of 58 infector – infectee pairs , the transmissibility parameters were calculated . The mean serial interval was 2 . 6 days ( standard deviation : 2 . 0 , 95 % CI : 1 . 9 – 3 . 6 ) and mean incubation period was 4 . 0 days ( 95 % CI : 2 . 0 – 4 . 8 ) . The evaluated R 0 was 2 . 8 ( 95 % CI : 1 . 6 – 4 . 8 ) using the maximum likelihood method ( the coefficient of determination R 2 = 0 . 96 ) . Then , 9 days after the initial case was reported , the effective reproduction number ( R t ) decreased to 1 on 13 August , and fluctuated close to 1 from 13 to 20 August . No new case was reported since 20 August ( Figure 2 ) . Close Contacts Tracing In total , 4 , 436 close contacts were traced and quarantined , and 55 ( 1 . 2 % , 95 % CI : 0 . 9 – 1 . 6 % ) were found to be secondarily infected . The highest SAR occurred in the household setting ( 17 / 68 , 25 % , 95 % CI : 14 . 4 – 35 . 6 % ) , followed by 2 . 2 % ( 23 / 1 , 025 , 95 % CI : 1 . 3 – 3 . 2 % ) in the workplace , and 0 . 5 % ( 15 / 3 , 343 , 95 % CI : 0 . 2 – 0 . 7 % ) in other places , with significant differences ( p < 0 . 05 ) ( Figure 3 ) . Household Transmission Among all 58 COVID - 19 cases , 44 cases belonged to 27 households and 14 cases lived alone or in working dormitories . In 27 households with COVID - 19 cases , there were 68 household contacts with the median number of persons per household of three , which ranged from 2 to 7 . For the 27 households with COVID - 19 , 11 households ( 39 . 3 % ) had 17 secondary cases . The SAR was 25 . 0 % ( 17 / 68 ) ( 95 % CI : 14 . 4 – 35 . 6 % ) among the household contacts . Although the SAR was 9 . 8 % higher between parents and children ( < 18 years ) ( 46 . 2 % , 95 % CI : 14 . 8 – 77 . 5 % ) than that between spouses ( 36 . 4 % , 95 % CI : 14 . 5 – 58 . 2 % ) , no statistical difference was observed ( p > 0 . 05 ) ( Figure 4 ) . Among the 11 households with secondary transmission , the SAR was 60 . 7 % ( 17 / 28 , 95 % CI : 41 . 4 – 80 . 0 % ) , and 3 ( 27 . 3 % ) out of 11 households had a 100 % SAR . The average number of secondarily infected individuals was 2 , which ranged from 2 to 6 , and the median serial interval was 2 . 0 days ( standard deviation : 2 . 2 , 95 % CI : 1 . 5 – 4 . 0 ) . The median Ct values for the N gene and ORF1ab gene were 23 . 1 ( 21 . 7 – 26 . 4 ) and 23 . 2 ( 21 . 1 – 27 . 9 ) in households with secondary transmission and 27 . 1 ( 22 . 4 – 29 . 1 ) and 29 . 1 ( 23 . 0 – 31 . 2 ) in household without secondary transmission , which represented an increase of 4 . 0 – 5 . 9 - folds in viral load in household indexes with secondary transmission compared to those without secondary transmission ( p < 0 . 05 ) , which indicated a high viral load might contribute to secondary transmission . There was no statistically difference in the age , gender , and clinical severity between the household indexes with secondary transmission and those without secondary transmission ( p > 0 . 05 ) ( Table 3 ) . The Effect of Vaccination Among the 58 COVID - 19 cases , 27 ( 46 . 6 % ) were vaccine breakthrough cases ( fully vaccinated with inactivated virion vaccine of wide - type strain ) . The first - time PCR results showed that the median Ct values of both N gene and ORF1ab gene in vaccine breakthrough cases were higher than that in unvaccinated cases ( Table 4 ) , which represents a 1 . 9 – 3 . 4 - fold decrease of viral load in vaccine breakthrough cases compared to the unvaccinated cases ( p < 0 . 05 ) , while no statistical difference was observed in partially vaccinated cases compared to the unvaccinated cases ( p > 0 . 05 ) . The duration Frontiers in Microbiology | www . frontiersin . org 4 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China TABLE 2 | Epidemiological characteristics of the SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China in August 2021 . Characteristic Delta variant positive persons ( N = 58 ) n ( % ) * Age group , years 39 ( 1 – 60 ) Median age ( range ) , years ≤ 18 11 ( 19 . 0 ) 19 – 29 5 ( 8 . 6 ) 30 – 39 15 ( 25 . 9 ) 40 – 49 11 ( 19 . 0 ) 50 – 59 14 ( 24 . 1 ) ≥ 60 2 ( 3 . 4 ) Gender Female 20 ( 34 . 5 ) Male 38 ( 65 . 5 ) Occupation Worker 32 ( 55 . 2 ) Preschool children # 8 ( 13 . 8 ) Student 3 ( 5 . 2 ) Teacher 1 ( 1 . 7 ) Others 15 ( 25 . 9 ) District A District where the index cases located 51 ( 87 . 9 ) B District near A District 7 ( 12 . 1 ) Exposure history Returned from an endemic area 3 ( 5 . 2 ) Household contact 17 ( 29 . 3 ) Social contact 38 ( 65 . 5 ) Clinical severity Asymptomatic 15 ( 25 . 9 ) Mild 24 ( 41 . 4 ) Moderate 19 ( 32 . 8 ) Severe and Critical 0 ( 0 ) Vaccination status Unvaccinated 23 ( 39 . 7 ) Partial vaccinated 8 ( 13 . 8 ) Fully vaccinated 27 ( 46 . 6 ) Median Ct value of RT - PCR ( IQR ) N gene 26 . 7 ( 22 . 9 – 31 . 0 ) ORF1ab gene 27 . 4 ( 22 . 0 – 32 . 2 ) * n ( % ) , number ( percentage ) unless otherwise specified ; Ct , cycle threshold ; IQR , inter quartile range . # Preschool children , the children who did not attend kindergarten or school . of viral shedding was 5 days shorter in vaccine breakthrough cases ( median : 19 days ; IQR : 15 – 23 days ) than that in unvaccinated cases ( median : 24 days ; IQR : 18 – 30 days ) ( p < 0 . 05 ) ( Table 4 ) . The risk of progressing from asymptomatic to symptomatic decreased 33 % ( 95 % CI : 5 – 53 % ) in fully vaccinated patients than that in unvaccinated patients aged ≥ 12 years ( Table 5 ) . TABLE 3 | Characteristics of household indexes in the SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China in August 2021 . Characteristics of household indexes With secondary transmission ( n = 11 ) Without secondary transmission ( n = 16 ) p - value * Median age ( rang ) , years 42 ( 35 – 50 ) 43 ( 30 – 51 ) > 0 . 05 Gender Female 1 5 > 0 . 05 Male 10 11 Clinical severity Symptomatic cases 8 10 > 0 . 05 Asymptomatic infections 3 6 RT - PCR median Ct value ( IQR ) N gene 23 . 1 ( 21 . 7 – 26 . 4 ) 27 . 1 ( 22 . 4 – 29 . 1 ) < 0 . 05 ORF1ab gene 23 . 2 ( 21 . 1 – 27 . 9 ) 29 . 1 ( 23 . 0 – 31 . 2 ) < 0 . 05 Ct , cycle threshold ; IQR , inter quartile range . * Categorical variables ( gender and clinical severity ) were compared by the Fisher’s exact test , andcontinuousvariables ( medianageandRT - PCRmedianCtvalue ) werecompared by Mann – Whitney U - test . DISCUSSION The SARS - CoV - 2 delta variant outbreak in Jingmen City in August 2021 was eliminated in 17 days with 58 cases and the proportion of infected children was relatively high ( 19 % ) in this outbreak . In the first wave of COVID - 19 pandemic caused by wide - type SARS - CoV - 2 in Jingmen City in 2020 , the proportion of infected children was only 0 . 5 % ( 5 / 928 ) . The previous study showed that children appeared to be less infected by the wide - type strain of SARS - CoV - 2 ( infection rate varied from 0 . 9 – 7 . 9 % ) than adults based on a large scale COVID - 19 - related epidemiological studies ( Mehta et al . , 2020 ) . A meta - analysis based on 213 published literatures of household transmission showed that the SAR of other strains of SARS - CoV - 2 in pediatric household contacts was lower than in adult household contacts ( RR , 0 . 62 ; 95 % CI , 0 . 42 – 0 . 91 ) ( Zhu et al . , 2021 ) . We found that all pediatric cases were between ages 1 and 10 years in this outbreak and did not get vaccinated . Vaccination might be a confounding factor to affect the susceptibility in children under 12 years to delta variant . However , there was no vaccine available during wide - type strain epidemic in China . Children under 12 years were not vaccinated both in the previous wide - type strain epidemic and this delta variant outbreak in Jingmen City . Therefore , it could be ruled out that the vaccine affects the susceptibility of children to the wide - type strain outbreaks and this outbreak . Children are more susceptible to SARS - CoV - 2 delta variant in Jingmen City in 2021 than wide - type strain in 2019 – 2020 , which could be caused by different exposure opportunity and / or viral type . Frontiers in Microbiology | www . frontiersin . org 5 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China FIGURE 2 | The effective reproduction number ( R t ) of the SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China in August 2021 . FIGURE 3 | SAR occurred in the different settings in Jingmen City , Hubei Province , China in August 2021 . * * * p < 0 . 001 . The SAR between parents and children ( < 18 years ) was much higher ( 9 . 8 % ) than that between spouses in our study . However , no statistical difference was observed ( p > 0 . 05 ) , which was most likely caused by the small sample size . The higher SAR in children than parents in the household could be influenced by the mode of interactions between the adults and children ( as in preparation of food , for instance ) , or more greatly associated with a particular type of interaction , or vaccination status and not an inherent increased susceptibility of children . Whether children are more susceptible than adults to SARS - CoV - 2 delta variant needs to be further investigated . As of 4 February 2022 , a total of 8 . 6 billion doses of COVID - 19 vaccine have been administered globally including 3 . 0 billion in China ( WHO , 2022 ) . COVID - 19 vaccine breakthrough infections with varied proportion have been observed ( Abu - Raddad et al . , 2021 ; Bergwerk et al . , 2021 ) . In this study , we systematically evaluated the effect of vaccination derived from the wide - type strain in viral loads , duration of viral shedding , and clinical severity in the outbreak of SARS - CoV - 2 delta variant in Jingmen City in 2021 . Our findings showed that full vaccination with inactivated COVID - 19 vaccines derived from the wide - type strain had partial cross - protection against the SARS - CoV - 2 delta Frontiers in Microbiology | www . frontiersin . org 6 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China FIGURE 4 | SAR ( 95 % CI ) in households in the SARS - CoV - 2 delta variant outbreak in Jingmen City , Hubei Province , China in August 2021 . * Others included parents , siblings , and grandparents . TABLE 4 | The viral load of the SARS - CoV - 2 delta variant infected patients in different vaccination status in Jingmen City , Hubei Province , China in August 2021 . Characteristics Unvaccinated ( IQR ) Fully vaccinated ( IQR ) * p - value ( fully vaccinated vs . unvaccinated ) Partially vaccinated ( IQR ) * p - value ( partially vaccinated vs . unvaccinated ) RT - PCR median Ct value of N gene 25 . 1 ( 23 . 0 – 32 . 7 ) 27 . 0 ( 21 . 1 – 29 . 8 ) < 0 . 05 28 . 0 ( 25 . 2 – 30 . 2 ) > 0 . 05 RT - PCR median Ct value of ORF lab gene 25 ( 23 . 0 – 32 . 6 ) 28 . 4 ( 21 . 0 – 31 . 1 ) < 0 . 05 28 . 9 ( 25 . 5 – 31 . 6 ) > 0 . 05 Median duration of viral shedding , days 24 ( 18 – 30 ) 19 ( 15 – 23 ) < 0 . 05 19 ( 14 – 38 ) < 0 . 05 QR , inter quartile range . * Continuous variables were compared by Mann – Whitney U - test . variant ( B . 1 . 617 . 2 ) . In vaccine breakthrough cases , the SARS - CoV - 2 delta variant viral load in the first positive PCR test and duration of viral shedding decreased 1 . 9 – 3 . 4 - folds and shortened 5 days , respectively , compared with those in unvaccinated patients . The previous study in Israel ( n = 3 , 776 ) showed a decrease of 2 . 8 – 4 . 5 - folds in SARS - CoV - 2 viral load in BNT162b2 messenger RNA vaccine breakthrough patients compared with those of the unvaccinated ( Levine - Tiefenbrun et al . , 2021 ) . Our study showed that inactivated SARS - CoV - 2 vaccine derived from the wide - type strain also had the similar function in reducing the viral load in the vaccine breakthrough infections of SARS - CoV - 2 delta variant . A study in the US between January and August 2021 showed that symptomatic delta variant breakthrough cases had longer duration of viral shedding compared with non - delta variants ( Siedner et al . , 2022 ) . However , to our knowledge , few published studies explored the difference of the viral shedding duration between the vaccine breakthrough infections and the unvaccinated for the individuals who both infected with SARS - CoV - 2 delta variant . This study added the knowledge to this aspect . Vaccine breakthrough patients aged ≥ 12 years had a 33 % reduced risk in progressing from asymptomatic to symptomatic compared with that in unvaccinated cases in this outbreak . A SARS - CoV - 2 delta variant outbreak that occurred in Guangzhou City , China , in May and June 2021 showed that the function of inactive full vaccination was 59 . 0 % vaccination effectiveness against infection , 70 . 2 % against moderate COVID - 19 , 100 % against severe by case - control study , 51 . 8 % against infection , 60 . 4 % against symptomatic infection , 78 . 4 % against pneumonia , and 100 % against severe or critical illness by retrospective cohort study ( Li et al . , 2021 ; Kang et al . , 2022 ) . A previous study in the UK showed that the BNT162b2 and ChAdOx1 nCoV - 19 vaccines were 88 . 0 and 67 . 0 % effectiveness against the SARS - COV - 2 delta variant ( Lopez Bernal et al . , 2021 ) . In our study , the mean serial interval was 2 . 6 days , which was similar to what was observed in the SARS - CoV - 2 delta variant outbreak in Guangdong Province ( 2 . 3 days ) , but significantly shorter than what was observed from the wide - type strain of SARS - CoV - 2 outbreak in Wuhan City ( early estimation : 7 . 5 Frontiers in Microbiology | www . frontiersin . org 7 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China TABLE 5 | Clinical severity of delta variant infected patients in different vaccination status in Jingmen City , Hubei Province in August 2021 . Outcome Unvaccinated Partially vaccinated Fully vaccinated n n RR ( 95 % CI ) * Reduced risk % ( 95 % CI ) n RR ( 95 % CI ) * Reduced risk % ( 95 % CI ) The COVID - 19 infection ( N = 58 ) Symptomatic 19 7 1 . 35 ( 0 . 21 – 8 . 68 ) − 35 ( − 768 to 79 ) 17 0 . 66 ( 0 . 41 – 1 . 07 ) 34 ( − 7 to 59 ) Moderate 8 5 1 . 59 ( 0 . 50 – 5 . 09 ) − 59 ( − 409 to 50 ) 6 0 . 87 ( 0 . 53 – 1 . 43 ) 13 ( − 43 to 47 ) Mild 11 2 0 . 83 ( 0 . 22 – 3 . 18 ) 17 ( − 218 to 78 ) 11 0 . 78 ( 0 . 49 – 1 . 24 ) 22 ( − 24 to 51 ) Asymptomatic 4 1 Ref . 10 Ref . – Age ≥ 12 years ( N = 47 ) Symptomatic 11 7 0 . 78 ( 0 . 17 – 3 . 49 ) 22 ( − 249 to 83 ) 17 0 . 67 ( 0 . 47 – 0 . 95 ) 33 ( 5 – 53 ) Moderate 5 5 1 ( 0 . 22 – 4 . 56 ) 0 ( − 356 to 78 ) 6 0 . 6 ( 0 . 34 – 1 . 062 ) 40 ( − 6 to 66 ) Mild 6 2 0 . 5 ( 0 . 08 – 3 . 13 ) 50 ( − 213 to 92 ) 11 0 . 71 ( 0 . 48 – 1 . 06 ) 29 ( − 6 to 52 ) Asymptomatic 1 1 Ref . – 10 Ref . Ref . * Reduced risk compared with the unvaccinated ; Ct , cycle threshold ; IQR , inter quartile range ; RR , risk ratio ; 95 % CI , 95 % confidence interval ; – , not available . days ) ( Li Q . et al . , 2020 ) , Jingzhou City ( 5 . 8 days ) ( Liu et al . , 2020 ) , Hubei Province ( 4 . 0 days ) ( Du et al . , 2020 ) , and Hunan Province ( 5 . 5 days ) in 2020 ( Hu et al . , 2021 ) . The shorter serial interval of SARS - CoV - 2 delta variant implies control measures such as contact tracing and quarantining which should be conducted as soon as possible to stop further transmission . The effective reproduction number ( R t ) was an indicator of real - time transmissibility of disease and the effect of interventions . In this epidemic , R t decreased to about 1 , 9 days after the first case was reported , which was similar to the delta variant outbreak in Guangzhou City ( Zhang et al . , 2021 ) . A series of rigorous intervention measures were used to contain this outbreak , which includes active case finding , rapid contacts tracing and quarantining , three times of nucleic acid test for the whole population in the city , lockdown of affected area , community management , and travel restrictions . The quick decline of R t indicated that the control measures worked well . High transmissibility in households was another feature in this outbreak . The study of household SARs caused by the SARS - CoV - 2 delta variant ( B . 1 . 617 . 2 ) was limited ( Dougherty et al . , 2021 ) . In the previous studies of epidemics caused by the wide - type of SARS - CoV - 2 or other related variants , household SARs varied from 3 to 32 % in China and the US ( Jing et al . , 2020 ; Lewis et al . , 2020 ; Li W . et al . , 2020 ; Wu et al . , 2020 ; Cerami et al . , 2021 ) . Compared with outbreaks caused by wide - type strain of SARS - CoV - 2 , the overall household SAR in this outbreak ( 25 % ) was higher than that in Guangzhou City , China ( 12 . 4 – 17 . 1 % ) and lower than that in the US households ( 29 – 32 % ) and Zhuhai City , China ( 32 % ) ( Jing et al . , 2020 ; Lewis et al . , 2020 ; Wu et al . , 2020 ; Cerami et al . , 2021 ) . However , high SAR ( 60 . 7 % ) was observed among households with secondary transmission including 100 % SAR in 3 households in this outbreak , which was similar to SARS - CoV - 2 delta variant ( B . 1 . 617 . 2 ) outbreak in Oklahoma , US ( SARs among households with secondary transmission ranged from 80 to 100 % ) ( Dougherty et al . , 2021 ) . The viral load in index patients with household secondary transmission was much higher ( 4 . 0 – 5 . 9 - folds ) than those index patients without household secondary transmission . Factors such as household contacts without protective measures cohabitation with the index case and household crowding may increase the risk of secondary infection ( Lewis et al . , 2020 ; Wu et al . , 2020 ; Cerami et al . , 2021 ) . In our study , although the primary infections were sent to the designated hospitals as soon as possible , precautionary practices such as mask use and increased social distance basically were not observed in households , which probably increased the odds of household transmission ( Wu et al . , 2020 ) . Our study has several limitations . First , recall bias could not be fully avoided in the investigation of cases and their contacts despite the in - depth investigation . Second , the sample size in this outbreak is small , which may affect the statistical test . Third , the valuation of vaccination effectiveness by respective cohort study , case – control , or subgroup analyses was limited by insufficient data such as few cases , no severe and critical case , and unavailable data for the vaccination status of all close contacts in this study . Fourth , although the hospitalization is an important index to evaluate the effectiveness of vaccination , we could not use it because all individuals infected with SARS - CoV - 2 were hospitalized even if asymptomatic in China . In conclusion , our findings showed that children are highly susceptible to the SARS - CoV - 2 delta variant ( B . 1 . 617 . 2 ) , and the transmission could easily happen from parents to their children . Inactivated COVID - 19 vaccine derived from wide - type strain of SARS - CoV - 2 showed protection against the SARS - CoV - 2 delta variant in reducing viral load , duration of viral shedding , and disease severity in vaccine breakthrough cases Frontiers in Microbiology | www . frontiersin . org 8 April 2022 | Volume 13 | Article 856757 Li et al . Delta Variant in Hubei , China compared with those in unvaccinated cases . Protective measures against COVID - 19 among children should be strengthened , and increased full vaccination efforts should be taken not only in adults , but also in children to reduce the disease severity in individuals and prevent the spread of the virus in the community . DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article / supplementary material , further inquiries can be directed to the corresponding author / s . ETHICS STATEMENT Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements . Written informed consent from the participants’ legal guardian / next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements . AUTHOR CONTRIBUTIONS DL contributed to field investigation , analysis , and manuscript drafting . A - eL contributed to study design and supervision . Z - qL and YB contributed to field investigation . TL contributed to field investigation and revised the manuscript . X - RQ contributed to supervision . X - jY contributed to study design , revised the manuscript , and supervised the study . All authors contributed to the article and approved the submitted version . FUNDING This work was supported by the National Natural Science Funds of China ( grant number : 81971939 ) , the Natural Science Foundation of Shandong Province of China ( ZR202102210339 ) , and Jingmen Science and Technology Plan Project ( grant number : 2020YFYB114 ) . ACKNOWLEDGMENTS We thank Local and Provincial Health Sectors and Local Public Security Bureau for their supports in this investigation . REFERENCES Abu - Raddad , L . J . , Chemaitelly , H . , Ayoub , H . H . , Yassine , H . M . , Benslimane , F . M . , Al Khatib , H . A . , et al . ( 2021 ) . Association of prior SARS - CoV - 2 infection with risk of Breakthrough infection following mRNA vaccination in Qatar . JAMA 326 , 1930 – 1939 . doi : 10 . 1001 / jama . 2021 . 19623 Bergwerk , M . , Gonen , T . , Lustig , Y . , Amit , S . , Lipsitch , M . , Cohen , C . , et al . ( 2021 ) . Covid - 19 breakthrough infections in vaccinated health care workers . N . Engl . J . 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( 2021 ) CORONAVIRUS disease ( COVID - 19 ) Weekly Epidemiological Update and Weekly Operational Update . Available online at : https : / / www . who . int / publications / m / item / weekly - epidemiological - update - on - covid - 19 - 8 - june - 2021 ( accessed September 18 , 2021 ) . Wu , J . , Huang , Y . , Tu , C . , Bi , C . , Chen , Z . , Luo , L . , et al . ( 2020 ) . Household transmission of SARS - CoV - 2 , Zhuhai , China , 2020 . Clin . Infect . Dis . 71 , 2099 – 2108 . doi : 10 . 1093 / cid / ciaa557 Zhang , M . , Xiao , J . , Deng , A . , Zhang , Y . , Zhuang , Y . , Hu , T . , et al . ( 2021 ) . Transmission dynamics of an outbreak of the COVID - 19 delta variant B . 1 . 617 . 2 — Guangdong Province , China . China CDC Weekly 3 , 584 – 586 . doi : 10 . 46234 / ccdcw2021 . 151 Zhu , Y . , Bloxham , C . J . , Hulme , K . D . , Sinclair , J . E . , Tong , Z . W . M . , Steele , L . E . , et al . ( 2021 ) . A meta - analysis on the role of children in severe acute respiratory syndrome coronavirus 2 in household transmission clusters . Clin . Infect . Dis . 72 , e1146 – e1153 . doi : 10 . 1093 / cid / ciaa1825 Conflict of Interest : The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . Publisher’s Note : All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations , or those of the publisher , the editors and the reviewers . Any product that may be evaluated in this article , or claim that may be made by its manufacturer , is not guaranteed or endorsed by the publisher . Copyright © 2022 Li , Li , Li , Bao , Liu , Qin and Yu . This is an open - access article distributed under the terms of the Creative Commons Attribution License ( CC BY ) . The use , distribution or reproduction in other forums is permitted , provided the original author ( s ) and the copyright owner ( s ) are credited and that the original publication in this journal is cited , in accordance with accepted academic practice . No use , distribution or reproduction is permitted which does not comply with these terms . Frontiers in Microbiology | www . frontiersin . org 10 April 2022 | Volume 13 | Article 856757 \ No newline at end of file diff --git a/silver_data/24bcc24414af2341b97ef265a53b564b65acbaf3.pdf.txt b/silver_data/24bcc24414af2341b97ef265a53b564b65acbaf3.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..b2944d44b37ce10fe8243217514f931d69c6284f --- /dev/null +++ b/silver_data/24bcc24414af2341b97ef265a53b564b65acbaf3.pdf.txt @@ -0,0 +1 @@ +Biochem . J . ( 1988 ) 256 , 283 - 290 ( Printed in Great Britain ) Inhibitory effect of a marine - sponge toxin , okadaic acid , on protein phosphatases Specificity and kinetics Corinna BIALOJAN and Akira TAKAI * II . Physiologisches Institut , Universitat Heidelberg , Im Neuenheimer Feld 326 , 6900 Heidelberg , Federal Republic of Germany The inhibitory effect of a marine - sponge toxin , okadaic acid , was examined on type 1 , type 2A , type 2B and type 2C protein phosphatases as well as on a polycation - modulated ( PCM ) phosphatase . Of the protein phosphatases examined , the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited . For the phosphorylated myosin light - chain ( PMLC ) phosphatase activity of the enzyme , the concentration of okadaic acid required to obtain 50 % inhibition ( ID50 ) was about 1 nm . The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited ( ID50 0 . 1 - 0 . 5 , aM ) . The PMCL phosphatase activity of type 2B phosphatase ( calcineurin ) was inhibited to a lesser extent ( ID50 4 - 5 / tM ) . Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p - nitrophenyl phosphate phosphatase activity of calcineurin . The following phosphatases were not affected by up to 10 fM - okadaic acid : type 2C phosphatase , phosphotyrosyl phosphatase , inositol 1 , 4 , 5 - trisphosphate phosphatase , acid phosphatases and alkaline phosphatases . Thus okadaic acid had a relatively high specificity for type 2A , type 1 and PCM phosphatases . Kinetic studies showed that okadaic acid acts as a non - competitive or mixed inhibitor on the okadaic acid - sensitive enzymes . INTRODUCTION Okadaic acid ( C44H66013 ) is a monocarboxylic acid extracted from common black sponges of the genus Halichondria . The chemical structure has been deter - mined by Tachibana et al . ( 1981 ) . Recently we reported that micromolar concentrations of this substance strongly inhibit the phosphorylated myosin light - chain ( PMLC ) phosphatase activity of smooth - muscle extract ( Takai et al . , 1987 ; Bialojan et al . , 1987 , 1988 ) . Okadaic acid similarly inhibited a purified polycation - modulated ( PCM ) phosphatase from bovine aorta ( DiSalvo et al . , 1984 ) . So far , two endogenous phosphatase inhibitors , termed inhibitor 1 and inhibitor 2 , have been isolated ( Huang & Glinsmann , 1976 ) . According to Cohen ' s classification ( Ingebritsen & Cohen , 1983 ) , our PCM phosphatase is a type 2 phosphatase , since it is not affected by these endogenous inhibitors ( DiSalvo et al . , 1984 ) . To our knowledge , okadaic acid is the first substance that has been shown to inhibit type 2 phos - phatase in micromolar concentrations . Besides inhibitor 1 and inhibitor 2 , only rather non - specific phosphatase inhibitors such as NaF ( Morgan et al . , 1976 ) or vanadate ( Searle , 1983 ) have been described . In the present experi - ments we further studied the inhibitory effect of okadaic acid on type 1 , type 2A , type 2B and type 2C phosphatases as well as PCM phosphatase . The results show that okadaic acid has a relatively high specificity for type 2A and type 1 phosphatases , and that it acts as a non - competitive or mixed inhibitor for the okadaic acid - sensitive enzymes . EXPERIMENTAL Materials All chemicals and reagents were purchased from Sigma Chemical Co . [ y - 32P ] ATP was obtained from NEN . Okadaic acid , isolated from the black sponge Halichondria okadai , was generously given by Dr . Y . Tsukitani ( Fujisawa Pharmaceutical Co . , Tokyo , Japan ) . Preparation of proteins Phosphorylatable myosin light chains were prepared from bovine myocardium by the method of Cummins & Lambert ( 1986 ) . Myosin light - chain kinase was purifed from chicken gizzards as described by Ngai et al . ( 1984 ) . Phosphorylase b and phosphorylase kinase from rabbit skeletal muscle as well as acid phosphatase and alkaline phosphatase were obtained from Sigma Chemical Co . Calmodulin from pig brain was purchased from Boeh - ringer Mannheim . Calcineurin ( = type 2B phosphatase ; Klee & Krinks , 1978 ; Stewart et al . , 1982 ) from bovine brain was kindly provided by Professor C . Klee , National Institutes of Health , Bethesda , MD , U . S . A . The catalytic subunits of type 1 phosphatase and type 2A phosphatase ( type 2A , ) from rabbit skeletal muscle ( Tung et al . , 1984 ) as well as type 2C phosphatase from rabbit liver ( McGowan & Cohen , 1987 ) were generously given by Dr . G . Mieskes , G6ttingen , Germany . Aortic PCM phos - phatase was purified as described previously ( DiSalvo et al . , 1984 ) , involving chromatography on DEAE - Sepha - cel , polylysine - agarose , heparin - agarose and a Mono Q Vol . 256 Abbreviations used : PCM phosphatase , polycation - modulated phosphatase ; PMLC , phosphorylated myosin light chain . * To whom correspondence and reprint requests should be addressed . 283 C . Bialojan and A . Takai HR 5 / 5 anion - exchange column with the Pharmacia f . p . l . c . system . SDS / polyacrylamide - gel electrophoresis of the purified enzyme revealed three polypeptides corre - sponding to 72 , 53 and 35 kDa , confirming the previous report ( DiSalvo et al . , 1985 ) . According to Cohen ' s classification of protein phosphatases ( Ingebritsen & Cohen , 1983 ) this PCM phosphatase is a type 2A phosphatase because it is not affected by inhibitor 1 or inhibitor 2 ( DiSalvo et al . , 1984 ) and has a 5 - fold higher PMLC phosphatase activity than phosphorylase a phos - phatase activity ( see the Results section ) . Enzyme assays All enzymes were tested at 30 ' C . The assays were made in a buffer containing 20 mM - Tris / HCl , 0 . 5 mM - dithiothreitol and 1 mg of bovine serum albumin / ml , pH 7 . 4 . The reaction mixtures for the assays of phos - phorylase a phosphatase activity contained 5 mM - caffeine in addition . For standard assays , either 32P - labelled PMLC or 32P - labelled phosphorylase a was used as substrate . Reactions were started by injecting the 32P - labelled substrate into the reaction mixture , and stopped by addition of ice - cold trichloroacetic acid ( 20 % , w / v ) and 6 mg of bovine serum albumin / ml . Assessment of released Pi was performed by standard procedures ( Di - Salvo et al . , 1982 ) . The enzyme activity of type 1 phosphatase was measured in buffer containing 2 mM - MnCl2 . For the assay of type 2C phosphatase 11 mm - magnesium acetate was included in the buffer . The activity of calcineurin was measured with either p - nitrophenyl phosphate or PMLC as substrate . The reaction mixtures contained 40 mM - Tris / HCl , 0 . 1 M - KCI , 0 . 1 mg of bovine serum albumin / ml , 6 mM - MgCl2 , 0 . 25 mM - dithiothreitol , 0 . 1 sM - calmodulin and 0 . 1 mm - CaCl2 , pH 8 . 0 . p - Nitrophenyl phosphate phosphatase activity of calcineurin was measured by recording the initial rate of increase in absorbance at 400 nm . In the dose - inhibition analyses , the concentration of enzymes was such that less than 5 % of the radioactive substrate was consumed during the reaction time of 3 - 10 min . The consumption of p - nitrophenyl phosphate was less than 1 % of the initial concentration . In the enzyme - kinetic studies , we allowed consumption of the substrate to a maximum of 15 % of the initial concentra - tion , so as to make the measurements at the lower substrate concentration range sufficiently accurate . The effect of accumulating product ( dephosphorylated pro - tein and Pi ) was examined for the protein phosphatases as follows . The rate of dephosphorylation was measured with different concentrations of substrate ( 1 uM - , 2 # M - and 5 # uM - PMLC ; 2 / tM - , 5 / M - and 10 # M - phosphorylase a ) . For this purpose , the reaction was allowed to proceed until about 5 % of the substrate added was depleted . The rate was not significantly changed when the reaction was started in the presence of dephosphorylated products ( myosin light chains or phosphorylase b plus Pi ) equiva - lent to 15 % of the initial substrate concentration . Therefore the effect of accumulating product appeared to be negligible under the present experimental conditions . The effect of depletion ofsubstrate can be mathematically corrected ( see below ) . Alkaline phosphatases ( from bovine intestinal mucosa and pig placenta ) and acid phosphatases ( from milk and potato ) were tested by standard procedures in alkaline and citrate buffer ( Sigma kit ) respectively . p - Nitrophenol phosphate ( 0 . 1 M ) was used as a substrate and the rate of decrease in the absorbance at 400 nm was measured . Dr . E . Ulug and Dr . S . Courtneidge from the Euro - pean Molecular Biology Laboratory , Heidelberg , Ger - many , kindly performed the phosphotyrosine - specific phosphatase assays . The assays were carried out by incubating crude cell lysates from mouse NIH 3T3 cells with various 32P - labelled phosphotyrosine - containing substrates such as enolase , poly ( Glu - Tyr ) and ' in - vitro ' - autophosphorylated cell lysates . Assessment of inositol trisphosphate phosphatase was performed in Professor A . P . Somlyo ' s and Professor Y . E . Goldman ' s laboratories in Philadelphia , PA , U . S . A . , together with Dr . J . Walker from the National Institute for Medical Research , London N . W . 7 , U . K . Both a crude membrane and a cytosolic fraction of rabbit main pulmonary artery were incubated with [ 3H ] inositol trisphosphate , and the initial rate of break - down ofinositol trisphosphate was measured as described by Walker et al . ( 1987 ) . Okadaic acid was dissolved in pure dimethyl sulph - oxide and added to the experimental solution . Control solutions contained the same concentration ( 0 . 5 % , v / v ) of dimethyl sulphoxide . Protein was measured by the method of Lowry et al . ( 1951 ) . Analysis of kinetic data When an enzyme reaction is monitored over an ex - tended time , the velocity of the reaction progressively slows down as a result of depletion of substrate and accumulation of product . In the present experiments , accumulation of product was limited , so that its effect on the reaction velocity could be neglected ( see above ) . The slowing down resulting from depletion of substrate was corrected for by the following mathematical procedure . Let v ( s ) be the initial steady - state velocity at a given substrate concentration s ( = [ S ] ) . When the reaction obeys the Michaelis - Menten kinetics : v ( s ) = VK + s Km , + s ( 1 ) where Km is the Michaelis constant and V is the maximal velocity ofthe reaction . The value ofv ( s ) can be estimated by measuring the mean rate of the increase of product , which is equal to the decrease of substrate , i . e . : t13 ( s ) = s - s ( t ) t ( 2 ) where O ( s ) is the estimate of v ( s ) and s ( t ) is the concentration of substrate remaining in the reaction mixture at time t [ s ( 0 ) = s ] . Because of depletion of substrate , v ( s ) < v ( s ) . We define the correction factor , f , by the following equation : v ( s ) = i ( s ) / f ( 3 ) Inserting eqns . ( 1 ) and ( 2 ) into eqn . ( 3 ) , and rearranging , we have : ( Km + s ) [ s - s ( t ) ] s - V - t The integrated form of eqn . ( 1 ) is : Yt = s - s ( t ) + Kmln ( s ) ) ( 4 ) ( 5 ) ( see Cornish - Bowden , 1977 ; Dixon & Webb , 1979 ) . 1988 284 Inhibition of protein phosphatase by okadaic acid Eliminating V - t in eqn . ( 4 ) by eqn . ( 5 ) , and rearranging , we obtain : s f = J ( S , Km , Pp ) = Kl ( 6 ) s In ( 1 - p ) Km P 95 % confidence limits . The other numerical data were described as means + S . E . M . , and differences were assessed by the Student ' s t test . The dose - inhibition relations were fitted by the linear least - squares method to the linear form of the Hill function : ln [ ( 100 - E ) / EJ = h * lnI + lnK where p = [ s - s ( t ) ] / s . The enzyme - kinetic results pre - sented have been corrected for substrate depletion by using this correction factor ( see also eqn . 3 ) . As for the present data , the values off were not less than 0 . 9 , i . e . 0 . 9 < f < 1 . Dose - inhibition relation In the following description , the apparent values of Km and V in the presence of inhibitor ( okadaic acid ) are denoted as Km ' and V ' respectively . In general , the concentration of inhibitor required to obtain 50 % inhibition ( ID50 ) is a function of substrate concentration . This fact is important when the values of ID50 are compared . The function can be mathematically derived if the dependence of initial steady - state velocity on the substrate concentration is described in the form of the Michaelis - Menten equation both in the absence and in the presence of inhibitor , and if the dose - inhibition relation is given in the form of the Hill function ( see Bialojan et al . , 1988 ) . The following equations are relevant to the present results . ( i ) When Km ' > Km and V ' < V ( mixed inhibition ) : ID50 ( s ) = K2 ( 7 ) where ID50 ( s ) is the value of ID50 at a given substrate concentration s , h is the Hill coefficient , and K1 and K2 are the constants that give the upper and lower limits of ID50 ( s ) respectively . The values of K1 and K2 are experi - mentally determined by using the relations : K1 = ( I - l I * I ( 8 ) and K2 = Km V ' ) ( 9 ) ( ii ) When Kin = Km and V ' < V ( non - competitive inhibition ) : ID50 = K1 = K2 ( constant ) ( 10 ) ( see eqns . 7 , 8 and 9 ) . The results presented have been corrected for a higher extent of substrate utilization at lower inhibitor concen - trations by using the correction factor described above ( see eqns . 3 and 6 ) . The increase of the values of h produced by the correction procedure was less than 0 . 05 in the present results . Statistics The kinetic constants of enzyme reactions were deter - mined by the direct - linear - plot method ( Eisenthal & Cornish - Bowden , 1974 ) , and evaluation of their dif - ference by a non - parametric method ( Hollander & Wolfe , 1973 ) was made in accordance with Porter & Trager ( 1977 ) with the use of computer programs . The values of the constants obtained were presented with where E is the percentage enzyme activity , I is the concentration of inhibitor , h is the Hill coefficient and KA is the association constant . The values in the range 10 % < E < 90 % were used for the fitting . The values of ID50 and Hill coefficient were compared by a method of co - variance analysis ( Snedecor & Cochran , 1980 ) . Very similar values of ID50 and Hill coefficient were obtained when the fitting was made by a non - linear least - squares method ( Snedecor & Cochran , 1980 ) . In every case , differences were evaluated as statistically significant when a two - sided probability of less than 0 . 05 was obtained . RESULTS Dose - inhibition relations Fig . l ( a ) shows the dose - inhibition relations for the PMLC phosphatase activity of various types of phos - phatase . For type 2AC , PCM and type 1 phosphatases the 1001 a ) Cm ^ CU 0 ~ o > . c . 5 a . 50 - o 10 - 11 10 - ' ° 10 - 9 10 - 8 10 - 7 10 - 6 10 - 5 [ Okadaic acid ] ( M ) 100 - W Q n CO 0 + , 50 - 0 . o 0 . CQ 0 ( L 0 ( b ) 10 _ 11 10 - 10 lo - 9 10 - 8 1o - 7 10 - 6 10 - 5 [ Okadaic acid ] ( M ) Fig . 1 . Effect of okadaic acid on protein phosphatases The inhibitory effect of okadaic acid on various types of protein phosphatase was examined with PMLC ( 4 uM ) ( a ) and phosphorylase a ( 1O / M ) ( b ) as substrates . 0 , Type 2AC phosphatase ; * , PCM phosphatase ; El , type I phosphatase ; * , type 2B phosphatase ; A , type 2C phos - phatase . Tris buffer , pH 7 . 4 , was used . The enzyme activi - ties are given as percentages of the control values ( see Table 1 ) . The values of ID50 and Hill coefficients are given in Table 1 . See the text for further explanation . Vertical bars indicate S . E . M . ( n = 4 - 6 ) . Vol . 256 285 C . Bialojan and A . Takai Table 1 . Parameters of the dose - inhibition relations Enzyme activities were measured with 4 1tM - PMLC , 10 ftM - phosphorylase a or 5 mM - p - nitrophenyl phosphate as substrate . The control activities are the average of 12 - 16 values . See the text for explanation . Control activity ( units / mg of ID50 Phosphatase Substrate protein ) ( nM ) h Type 2A . Type 2A . PCMPCMType 1 Type 1 Type 2B Type 2B PMLC Phosphorylase a PMLC Phosphorylase a PMLC Phosphorylase a PMLC p - Nitrophenyl phosphate 8800 + 160 1750 + 50 1550 + 120 300 + 23 47 . 2 + 3 . 9 22 . 6 + 2 . 0 1 . 4 + 0 . 2 62 . 1 + 8 . 1 1 . 2 1 . 6 205723152724530 3600 0 . 84 0 . 84 0 . 95 0 . 97 0 . 80 0 . 82 0 . 97 1 . 04 100 - mo - 0 c LO U o . QO . _ - Q _ as > a ) 50 - a CD o m - L Q Um Z O . , m QL a 0 - 1 1 _ 50 40 30 * 20 If 10 4 \ \ 16 - 8 16 - 7 16 - 6 16 - 5 16 - 4 [ Okadaic acid ] ( M ) 0 Fig . 2 . Inhibition of the p - nitrophenyl phosphate phosphatase activity of calcineurin by okadaic acid The p - nitrophenyl phosphate phosphatase activity of calci - neurin was measured in Tris buffer , pH 8 . 0 . The activity is presented as a percentage of the control activity ( see Table 1 ) . The values of ID50 and Hill coefficient are given in Table 1 . See the text for further explanation . Vertical bars indicate S . E . M . ( n = 4 - 5 ) . dose - inhibition relations were also examined for their phosphorylase a phosphatase activity ( Fig . lb ) . Para - meters of the inhibition curves are given in Table 1 . When 4 uM - PMLC was used as substrate , the catalytic subunit of the skeletal - muscle type 2A phosphatase ( type 2AJ ) was most potently inhibited ( ID50 1 . 2 nM ) . Okadaic acid also strongly inhibited PCM phosphatase ( ID50 205 nM ) and type 1 phosphatase ( ID50 315 nM ) . Calci - neurin ( type 2B phosphatase ) was inhibited to a lesser extent ( ID50 4350 nM ) . Type 2C phosphatase was not affected by up to 10 # , M - okadaic acid ( Fig . la ; for the dependence of ID50 on the substrate concentration see below . ) When 10 / gM - phosphorylase a was used as substrate , results were similar , except for PCM phosphatase , for which the value of ID50 ( 72 nM ) was considerably smaller than that for its PMLC phosphatase activity ( Table 1 ) . This seems to be related to the enzyme - kinetic nature of the phosphorylase a phosphatase activity of PCM phos - phatase ( see below ) . 80 60 - 40 20 ( a ) Km / [ PMLC ] ( b ) 10 20 Km / [ phosphorylase a ] 30 Fig . 3 . Enzyme kinetics of the inhibition of type 2AC phosphatase by okadaic acid The dependence of initial steady - state velocity , v , on substrate concentration was examined for the PMLC phosphatase activity ( a ) and phosphorylase a phosphatase activity ( b ) of type 2AC phosphatase . 0 , Control ; * , okadaic acid ( a , 5 nM ; b , 2 nM ) . The values are normalized to the kinetic constants determined by the direct - linear - plot method ( Table 2 ) . See the text for further explanation . 1988 286 Inhibition of protein phosphatase by okadaic acid ( a ) 4 - 1 . 0 - 0 . 5 0 0 . 5 Kmn / [ PM LC ] 1 . 0 1 . 5 - 1 . 0 - 0 . 5 0 0 . 5 1 . 0 1 . 5 Km / [ phosphorylase a ] Fig . 4 . Enzyme kinetics of the inhibition of PCM phosphatase by okadaic acid The experiments are similar to those illustrated in Fig . 3 . The PMLC phosphatase activity ( a ) and phosphorylase a phosphatase activity ( b ) of PCM phosphatase were meas - ured . 0 , Control ; * , okadaic acid ( a , 300 nM ; b , 100 nM ) . The values are normalized to the kinetic constants deter - mined by the direct - linear - plot method ( Table 2 ) . In ( b ) the values of Km and V at higher substrate concentrations ( 10 - 80 , UM ; broken lines ) were both different from those at lower concentrations ( 0 . 5 - 5 , M ; continuous lines ) ; the constants for the substrate concentration range 0 . 5 - 5 / SM were used for normalization . See the text for further explanation . As for phosphatase 2B , the effect of okadaic acid was also tested for its p - nitrophenyl phosphate phosphatase activity in Tris buffer , pH 8 . 0 ( Fig . 2 ) . The control activity ( Table 1 ) was similar to the value [ 120 units ( nmol of Pi / min ) / mg of protein ] obtained in Professor C . Klee ' s laboratory ( personal communication ) . The dose - inhibition relation was similar to that obtained for the PMLC phosphatase activity of the enzyme in Tris buffer , pH 7 . 5 ( see also Table 1 ) . When the PMLC phosphatase activity was measured in Tris buffer , pH 8 . 0 , the control activity ( 1 . 4 units / mg of protein ) was lower than that obtained in Tris buffer , pH 7 . 5 , but the values of ID50 ( 5130 nM ) and Hill coefficient ( 0 . 95 ) were not significantly changed . The values of the Hill coefficient h tended to be smaller than 1 ( Table 1 ) , although the data were corrected for a higher extent of substrate utilization at lower inhibitor concentrations ( see the Experimental section ) . Enzyme kinetics Figs . 3 , 4 and 5 show the results of the enzyme - kinetic study . The data are presented as double - reciprocal plots . The values are normalized to the kinetic constants determined by the direct - linear - plot method ( Table 2 ) . Type 2A , phosphatase . For type 2A , phosphatase ( catalytic subunit of the skeletal - muscle type 2A phos - phatase ) the double - reciprocal plots were linear over the substrate concentration range for both PMLC phos - phatase activity and phosphorylase a phosphatase activity of the enzyme . For the PMLC phosphatase activity the values of Km and V were estimated , as de - scribed in the Experimental section , to be 3 . 8 , UM and 17200 units / mg of protein respectively ( Table 2 ) . These were significantly changed to 12 mm ( = Km ' ; P < 0 . 008 ) and 8550 units / mg of protein ( = V ' ; P < 0 . 016 ) by 5 nm - okadaic acid , indicating that okadaic acid acts as a mixed inhibitor . From the values of V ' / V ( 0 . 50 ) and Km ' / Km ( 3 . 2 ) , the upper and lower limits of the change of ID50 produced by changing the substrate concentration were estimated , by using eqns . ( 8 ) and ( 9 ) , to be 5 nM ( = K1 ) and 0 . 7 nm ( = K2 ) respectively . For the phosphorylase a phosphatase activity the Km was 8 . 2 - fold larger whereas the V was 2 . 3 - fold smaller than those for the PMLC phosphatase activity ( Table 2 ) . This qualitatively agrees with the previous report by Ingebritsen & Cohen ( 1983 ) . Okadaic acid ( 2 nM ) signifi - cantly decreased the value of V whereas it did not affect the value of Km , i . e . the inhibition was non - competitive . As expected from eqn . ( 10 ) , the value of ID50 of the dose - inhibition relation was not altered when the sub - strate concentration was changed from 10 / M to 2 UM and 20 , u / M . PCM phosphatase . When PMLC was used as substrate , the double - reciprocal plot was linear over the concen - tration range ( 0 . 5 - 10 / tM ) of PMLC examined ( Fig . 4a ) . Both Km and V were significantly ( P < 0 . 002 ) changed by 300 nM - okadaic acid ( Table 2 ) , indicating that okadaic acid acts as a mixed inhibitor . From the change of the kinetic constants , the lower and upper limits of the variation of ID50 produced by changing the substrate concentration were estimated , by using eqns . ( 7 ) and ( 8 ) , to be 60 nm and 460 nm respectively . For the phosphorylase a phosphatase activity of PCM phosphatase the double - reciprocal plot had two com - ponents ( Fig . 4b ) , one below and one above a substrate concentration of about 8 / LM ( Km / [ phosphorylase a ] = 0 . 14 - 0 . 18 ) . The values of Km and V above 8 / M substrate were both significantly different ( P < 0 . 002 ) from those below this critical concentration . In the lower substrate concentration range ( 0 . 5 - 5 / tM ) both Km and V were changed by 100 nM - okadaic acid ( P < 0 . 002 ) , i . e . the inhibition was a mixed type . In the higher concentration range ( 10 - 80 , uM ) V was decreased ( P < 0 . 002 ) whereas Km was not significantly changed by okadaic acid , i . e . the inhibition was a non - competitive type . As a consequence the dependence of ID50 on the substrate concentration was somewhat complicated ( Fig . 6 ) . In the range 0 . 5 - 8 1tM the ID50 increased , as expected from eqn . ( 7 ) ( K1 = 50 nM and K2 = 115 nM ) , as the concentration of Vol . 256 287 C . Bialojan and A . Takai ( a ) ( b ) 0 10 20 Km / ! [ PMLC ] 0 10 20 Km / [ PMLC ] Fig . 5 . Enzyme kinetics of the inhibition of type 1 phosphatase and calcineurin by okadaic acid The experiments are similar to those illustrated in Fig . 3 . The initial steady - state velocity , v , of the dephosphorylation of PMLC ( 0 . 5 - 5 laM ) by type 1 phosphatase ( a ) and calcineurin ( b ) was measured . 0 , Control ; * , okadaic acid ( a , 315 nm ; b , 5000 nM ) . See Table 2 for the kinetic constants . See the text for further explanation . Table 2 . Kinetic constants of the phosphatase inhibition by okadaic acid The values of Km and V determined by the direct - linear - plot method for the results shown in Figs . 3 , 4 and 5 . They are presented with the 95 % confidence limits ( in parentheses ) . [ S ] denotes the substrate concentration . [ Okadaic Range of [ S ] acid ] Km Phosphatase Substrate ( / SM ) ( nm ) ( # M ) V ( units / mg of protein ) Type 2A . PMLC 0 . 5 - 5 0 3 . 8 ( 3 . 4 - 4 . 2 ) 17200 ( 16200 - 18200 ) Type 2A . PMLC 0 . 5 - 5 5 12 ( 10 - 15 ) 8550 ( 7240 - 10800 ) Type 2A . Phosphorylase a 1 - 30 0 31 ( 28 - 36 ) 7350 ( 6720 - 7930 ) Type 2A . Phosphorylase a 1 - 30 2 32 ( 28 - 38 ) 2590 ( 2300 - 2920 ) PCM PMLC 0 . 5 - 10 0 0 . 6 ( 0 . 5 - 0 . 7 ) 1830 ( 1790 - 1910 ) PCM PMLC 0 . 5 - 10 300 2 . 0 ( 1 . 6 - 2 . 6 ) 1100 ( 990 - 1250 ) PCM Phosphorylase a 1 - 5 0 1 . 4 ( 1 . 0 - 1 . 7 ) 340 ( 310 - 365 ) PCM Phosphorylase a 1 - 5 100 2 . 2 ( 1 . 8 - 2 . 6 ) 180 ( 165 - 195 ) PCM Phosphorylase a 10 - 80 0 14 ( 11 - 16 ) 710 ( 650 - 770 ) PCM Phosphorylase a 10 - 80 100 12 ( 9 - 16 ) 290 ( 250 - 320 ) Type 1 PMLC 0 . 5 - 5 0 11 ( 9 - 13 ) 177 ( 155 - 207 ) Type 1 PMLC 0 . 5 - 5 315 10 ( 7 - 14 ) 68 ( 50 - 83 ) Type 2B PMLC 0 . 5 - 5 0 7 . 9 ( 5 . 9 - 9 . 1 ) 4 . 2 ( 3 . 7 - 4 . 7 ) Type 2B PMLC 0 . 5 - 5 5000 6 . 5 ( 3 . 4 - 9 . 3 ) 2 . 1 ( 1 . 1 - 2 . 6 ) phosphorylase a concentration was increased , whereas it abruptly decreased to a constant value when the phosphorylase a concentration exceeded 8 ^ M as ex - pected from eqn . ( 10 ) ( K1 = K2 = 64 nM ) . At the critical concentration ( 8 - 10 , lM ) the co - variance analysis of the dose - inhibition relation gave a significantly larger re - sidual variance ( P < 0 . 01 ) than for both sides of this concentration . [ Note the relatively large S . E . M . values in the dose - response relation for this enzyme activity ( Fig . lb ) . ] The range of variation of ID50 produced by changing substrate concentration was estimated to be 50 - 100 nm ( Fig . 6 ) . This falls within the variation range of ID50 estimated for the PMLC phosphatase activity of the enzyme ( see above ) . The control value of V was 5 . 4 - fold higher for the PMLC phosphatase activity than for the phosphorylase a phosphatase activity of the enzyme ( Table 2 ) . In this respect the substrate specificity of our PCM phosphatase has a similarity to that of type 2A phosphatase ( cf . Ingebritsen & Cohen , 1983 ) . Type 1 phosphatase and calcineurin . For these types of enzyme , kinetic studies were made with PMLC as sub - strate ( Fig . 5 ) . The values of Km were one order of magnitude larger whereas the values of V expressed in terms of the specific activity were considerably smaller than those of the PMLC phosphatase activity of PCM phosphatase ( Table 2 ) . In the range of substrate concen - 1988 288 Inhibition of protein phosphatase by okadaic acid 100 90 1 - 1 2 80 2 0 70 - 50 A qI 0 o L0 0 0 6 10 20 30 40 50 60 [ Phosphorylase ] ( # M ) Fig . 6 . Dependence of ID50 on substrate concentration The effect of okadaic acid on the phosphorylase a activity of PCM phosphatase was examined with various concen - trations ( 1 - 60 uM ) of phosphorylase a . The values of ID50 of the dose - inhibition relation were plotted against the concentration of phosphorylase a . The curve and hori - zontal line show the relation expected from eqns . ( 7 ) and ( 10 ) ( see the Experimental section ) ; the kinetic constants for the lower and higher concentration ranges of phosphoryl - ase a given in Table 2 were used for computation . The Hill coefficient ( h = 0 . 96 - 0 . 98 ) was not significantly changed when the substrate concentration was varied . See the text for further detail . trations examined the inhibition was non - competitive for both enzymes ; okadaic acid changed only the values of V ( P < 0 . 02 ) . In accordance with eqn . ( 10 ) , we found that the values of ID50 of the dose - inhibition relations were not significantly altered when the substrate concentration was changed from 4 m to 1 / SM and 10 / LM . Effect on other phosphatases Neither cytosolic nor particulate - bound inositol tris - phosphate phosphatase from smooth muscle was in - hibited by up to 50 # uM - okadaic acid . No effect was found with tyrosine - specific phosphatase ( s ) from cell lysates . Alkaline phosphatases ( from bovine intestinal mucosa and pig placenta ) and acid phosphatases ( from milk and potato ) tested with p - nitrophenyl phosphate as substrate were not affected by 10 1tM - okadaic acid . However , a three - subunit phosphatase isolated from dog heart ( Kranias & DiSalvo , 1986 ) was strongly inhibited by okadaic acid ( E . G . Kranias & J . DiSalvo , personal communication ) . The enzyme expressed phosphatase activity against phospholamban and phosphorylase a ; both activities were completely inhibited by okadaic acid in micromolar concentration . DISCUSSION In previous papers we have reported that micromolar concentrations of okadaic acid potently inhibit the PMLC phosphatase activity of smooth - muscle extract and of the aortic PCM phosphatase ( Takai et al . , 1987 ; Bialojan et al . , 1987 , 1988 ) . The present results show that okadaic acid inhibits type 2A , type 1 and type 2B phosphatases as well as the PCM phosphatase . On the other hand , the following phosphatases are not affected by up to 10 / tM - okadaic acid : type 2C phosphatase , phosphotyrosyl phosphatase , inositol trisphosphate phosphatase , acid phosphatases and alkaline phosphat - ases ( the present work ) . We have also shown that okadaic acid does not affect the Ca2 + + calmodulin - dependent activity of myosin light - chain kinase or phos - phodiesterase ( Takai et al . , 1987 ) . Thus the inhibitory action of okadaic acid appears to be specific for some restricted types of protein phosphatase . The type of substrate has little effect on the inhibitory action ofokadaic acid . Essentially similar dose - inhibition relations are obtained for the PMLC phosphatase activity and the phosphorylase a phosphatase activity of type 2A . , PCM and type 1 phosphatases ( Fig . 1 ) . The p - nitrophenyl phosphate phosphatase activity of type 2B phosphatase ( calcineurin ) has nearly the same suscepti - bility to okadaic acid as its PMLC phosphatase activity ( see also Fig . 2 ) . These results support the idea that the inhibitory action of okadaic acid is enzyme - directed . The affinity of okadaic acid differs remarkably among the okadaic acid - sensitive phosphatases . The catalytic subunit of skeletal - muscle type 2A phosphatase ( type 2AJ ) is about 200 times more strongly inhibited by okadaic acid than is that of type 1 phosphatase , although there is a striking sequence homology between these catalytic subunits ( Berndt et al . , 1987 ) . The three - dimen - sional conformation of the enzyme molecule may be an important factor for the binding of okadaic acid . The aortic PCM phosphatase is a polymolecular enzyme consisting of three subunits , and belongs to the category of type 2A phosphatases ( see the Experimental section ) . This phosphatase is considerably less susceptible to okadaic acid than is type 2A , phosphatase ( Fig . 1 ) . The reason for this difference is not clear from the present results . It may imply that the aortic PCM phosphatase has a different catalytic subunit from that of the skeletal - muscle type 2A phosphatase . Another possibility is that the non - catalytic subunits make the catalytic subunit less accessible to okadaic acid . The present kinetic study has shown that okadaic acid acts as a non - competitive or mixed inhibitor of the okadaic acid - sensitive enzymes , suggesting that the bind - ing site for okadaic acid is different from that for the substrate . However , the reaction mechanism is likely to be more complicated . The Hill coefficients of dose - inhibition relations tend to be smaller than 1 even after the correction for the higher extent of substrate consump - tion at lower inhibitor concentrations ( see the Experi - mental section ) . When the Hill coefficient is not 1 , the inhibition is not ' linear ' according to Cleland ' s ( 1963 ) classification of enzyme inhibition ( see Bialojan et al . , 1988 ) . In the model of ordinary ( linear ) inhibition , the constants K1 and K2 denote the dissociation constants of inhibitor for free enzyme and enzyme - substrate respect - ively ( see Dixon & Webb , 1979 ) . If this scheme qualita - tively applies for the present results concerning the PMLC phosphatase activity of the PCM phosphatase , where the Hill coefficient ( 0 . 95 ) was close to 1 , the larger value of K1 than that of K2 may suggest that the affinity of okadaic acid for the phosphatase is considerably diminished when the substrate binds to the enzyme . The kinetic data give an explanation for the compli - cated nature of the dependence of ID50 observed for the phosphorylase a phosphatase activity of the PCM phos - phatase ( see the Results section ) . The kinetics rather abruptly change at a critical concentration , and the affinity of okadaic acid for the enzyme apparently becomes unstable at this concentration . This fact must be noted especially when phosphorylase a is used as a substrate of PCM phosphatase for studies of inhibitors . Vol . 256 289 60 - v - - , C . Bialojan and A . Takai The phenomenon is not observed for the phosphorylase a phosphatase activity of type 2A , phosphatase . There - fore the change of kinetics is likely to be due to a property of the PCM phosphatase rather than to that of phosphorylase a . To our knowledge , okadaic acid is the first - described potent substance that inhibits type 2 phosphatases as well as type 1 phosphatase . The inhibition of phosphatase is reversible , as judged from its effects on myosin light - chain phosphorylation ( Bialojan et al . , 1988 ) . Therefore okadaic acid may serve as a unique tool for analysing phosphorylation - regulated systems . We thank Professor J . C . Ruegg for continuous encouragement , Professor J . DiSalvo for helpful discussion and Dr . J . Leah and Dr . E . Ball for improving the manuscript . Invaluable technical assistance of Ms . M . Troschka is also gratefully acknowledged . This work was supported by the Deutsche Forschungsgemeinschaft . REFERENCES Berndt , N . , Campbell , D . G . , Caudwell , F . B . , Cohen , P . , da Cruz e Silva , E . F . , da Cruz e Silva , 0 . B . & Cohen , P . T . W . ( 1987 ) FEBS Lett . 223 , 340 - 346 Bialojan , C . , Takai , A . & Riiegg , J . C . ( 1987 ) Adv . Protein Phosphatases 4 , 253 - 267 Bialojan , C . , Riiegg , J . C . & Takai , A . ( 1988 ) J . Physiol . ( London ) 398 , 81 - 95 Cleland , W . W . ( 1963 ) Biochim . Biophys . Acta 67 , 173 - 187 Cornish - Bowden , A . ( 1977 ) Principles of Enzyme Kinetics , pp . 143 - 144 , Butterworth and Co . , London Cummins , P . & Lambert , S . J . ( 1986 ) Circ . Res . 58 , 846 - 858 DiSalvo , J . , Jiang , M . J . , Vandenheede , J . R . & Merlevede , W . ( 1982 ) Biochem . Biophys . Res . Commun . 108 , 534 - 540 DiSalvo , J . , Gifford , D . & Kokkinakis , A . ( 1984 ) Proc . Soc . Exp . Biol . Med . 177 , 24 - 32 DiSalvo , J . , Gifford , D . & Kokkinakis , A . ( 1985 ) Adv . Protein Phosphatases 1 , 327 - 345 Dixon , M . & Webb , E . C . ( 1979 ) The Enzymes , 3rd edn . , pp . 65 - 67 and 339 - 341 , Longman Group , London Eisenthal , R . & Cornish - Bowden , A . ( 1974 ) Biochem . J . 139 , 715 - 720 Hollander , M . & Wolfe , D . A . ( 1973 ) Non - Parametric Statisti - cal Methods , pp . 27 - 38 , 209 - 217 and 269 - 271 , John Wiley and Sons , New York Huang , F . L . & Glinsmann , W . H . ( 1976 ) Eur . J . Biochem . 70 , 419 - 426 Ingebritsen , T . S . & Cohen , P . ( 1983 ) Eur . J . Biochem . 132 , 255 - 261 Klee , C . B . & Krinks , M . H . ( 1978 ) Biochemistry 17 , 120 - 126 Kranias , E . G . & DiSalvo , J . ( 1986 ) J . Biol . Chem . 261 , 10029 - 10032 Lowry , 0 . H . , Rosebrough , N . J . , Farr , A . L . & Randall , R . J . ( 1951 ) J . Biol . Chem . 193 , 265 - 275 McGowan , C . H . & Cohen , P . ( 1987 ) Eur . J . Biochem . 166 , 713 - 722 Morgan , M . , Perry , S . V . & Ottaway , J . ( 1976 ) Biochem . J . 157 , 687 - 697 Ngai , P . K . , Carruthers , C . A . & Walsh , M . P . ( 1984 ) Biochem . J . 218 , 863 - 870 Porter , W . R . & Trager , W . F . ( 1977 ) Biochem . J . 161 , 293 - 302 Searle , B . M . ( 1983 ) Circ . Res . 53 , 186 - 191 Snedecor , G . W . & Cochran , W . G . ( 1980 ) Statistical Methods , 7th edn . , pp . 143 - 160 , 175 - 191 , 385 - 388 and 407 - 409 , Iowa State University Press , Ames Stewart , A . A . , Ingebritsen , T . S . , Manalan , A . , Klee , C . & Cohen , P . ( 1982 ) FEBS Lett . 137 , 80 - 84 Tachibana , Y . , Scheuer , P . J . , Tsukitani , Y . , Kikuchi , H . , VanEngen , D . , Clardy , J . , Giopichand , Y . & Schmitz , F . J . ( 1981 ) J . Am . Chem . Soc . 103 , 2469 - 2471 Takai , A . , Bialojan , C . , Troschka , M . & Ruegg , J . C . ( 1987 ) FEBS Lett . 217 , 81 - 84 Tung , H . Y . L . , Resink , T . J . , Shenorikar , S . & Cohen , P . ( 1984 ) Eur . J . Biochem . 138 , 635 - 641 Walker , J . W . , Somlyo , A . V . , Goldman , Y . E . , Somlyo , A . P . & Trentham , D . R . ( 1987 ) Nature ( London ) 327 , 249 - 252 Received 31 December 1987 / 20 June 1988 ; accepted 27 July 1988 1988 290 \ No newline at end of file diff --git a/silver_data/299add361f7113dbfbe9cf8ab9b7e3c2afb3653d.pdf.txt b/silver_data/299add361f7113dbfbe9cf8ab9b7e3c2afb3653d.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..6732b7b38e702eb520172d48bc21c95060ab47c7 --- /dev/null +++ b/silver_data/299add361f7113dbfbe9cf8ab9b7e3c2afb3653d.pdf.txt @@ -0,0 +1 @@ +u n i v e r s i t y o f c o p e n h a g e n Dynamics of membrane nanotubes coated with I - BAR Farhangibarooji , Younes ; Rørvig - Lund , Andreas ; Semsey , Szabolcs ; S . Reihani , S . Nader ; Bendix , Pól Martin Published in : Scientific Reports DOI : 10 . 1038 / srep30054 Publication date : 2016 Document version Publisher ' s PDF , also known as Version of record Citation for published version ( APA ) : Farhangibarooji , Y . , Rørvig - Lund , A . , Semsey , S . , S . Reihani , S . N . , & Bendix , P . M . ( 2016 ) . Dynamics of membrane nanotubes coated with I - BAR . Scientific Reports , 6 , [ 30054 ] . https : / / doi . org / 10 . 1038 / srep30054 Download date : 28 . aug . . 2019 1 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 www . nature . com / scientificreports Dynamics of membrane nanotubes coated with I - BAR Younes F . Barooji 1 , Andreas Rørvig - Lund 1 , Szabolcs Semsey 1 , S . Nader S . Reihani 2 & Poul M . Bendix 1 Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53 - MIM protein family . In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present . Here , we show that the IM domain ( also called I - BAR domain ) from the protein ABBA , forms semi - flexible nanotubes protruding into Giant Unilamellar lipid Vesicles ( GUVs ) . By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes . I - BAR decorated tubes were quantified to have a diameter of ~ 50 nm and exhibit no stiffening relative to protein free tubes of the same diameter . At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV . Bleaching experiments of the fluorescently tagged I - BAR confirmed that the mobility of the tubes correlates with the mobility of the I - BAR on the GUV membrane . Finally , at low density of I - BAR the protein upconcentrates within tubes protruding into the GUVs . This implies that I - BAR exhibits strong preference for negatively curved membranes . Membrane shaping constantly takes place in cellular processes such as endocytosis 1 , cell division 2 and in tubular protrusions like filopodia 3 – 5 and Tunneling NanoTubes ( TNT’s ) 6 . These deformed and highly curved membrane structures facilitate a host of cellular processes like intercellular transport and extracellular sensing . Essential players involved in membrane deformation are proteins from the superfamily of BAR domain proteins which contain membrane binding domains with specific curvatures thus enabling the cell to create positive and negative membrane curvatures for a number of processes 7 . A family of proteins containing convex shaped dimeric I - BAR domains ( also called IM domains ) facilitate formation of oppositely curved membranes which are found inside tubular structures like filopodia and invadopodia 8 , 9 . The I - BAR containing protein ABBA has been associated with membrane protrusions in radial glial cells where it localizes to the inner surface in curved membrane ruf - fles connecting the membrane with the cortical actin cytoskeleton 10 . Deletion of ABBA in cells leads to reduced lamellipodium dynamics thus suggesting that ABBA has an important role in sensing and deformation of the ruffled membrane shape at the front of the lamellipodium . Overexpression of ABBA and other I - BAR containing proteins in cells leads to filopodia formation which shows that I - BAR domains are involved in curvature genera - tion . The observation of membrane deformation in cells by I - BARs is directly consistent with findings that I - BAR domains have the ability to deform membranes in vitro , in particular negatively charged membranes containing phosphatidylserine and phosphoinositide lipids ( PIP ) 9 . A number of different BAR domain proteins have been studied with respect to mechanical effects on mem - branes 7 and also on curvature sensing and curvature induction 11 , but remarkably little information exists regarding the mechanical effect of I - BAR domains on membranes and how the domains are recruited to certain membrane regions . However , recently the I - BAR domain from IRSp53 was found to be highly sensitive to negative membrane curvatures 12 and another investigation has shown that IRSp53 induces shape instability in membranes in a tension dependent manner 13 by a similar mechanism as was found previously with N - BAR domains 14 , 15 . Also , physical char - acteristics , such as tube width , have been investigated by cryo - electron microscopy ( cryo - EM ) in fixed samples using multilamellar vesicles tubulated by different types of I - BAR domains . These experiments have revealed that different I - BAR domains produce tubules ranging from ca . 40 nm to 80 nm in diameter 9 . The dynamics of the interaction between membrane tubes and I - BAR domains can provide interesting information regarding possible mechanical effects of the protein inside the membrane tube . Protein sorting 1 Niels Bohr Institute , University of Copenhagen , Blegdamsvej 17 , 2100 Copenhagen , Denmark . 2 Department of Physics , Sharif University of Technology , Teheran 11365 - 9161 , Iran . Correspondence and requests for materials should be addressed to P . M . B . ( email : bendix @ nbi . dk ) r eceived : 23 March 2016 a ccepted : 29 June 2016 P ublished : 21 July 2016 OPEN www . nature . com / scientificreports / 2 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 as well as protein oligomerization and resulting stiffening of tubes has been previously reported experimentally for F - BAR domains 11 , 16 . Membrane curvature sensing by I - BAR proteins can potentially play an important role in the recruitment of proteins to high curvature regions . At higher membrane densities protein induced stiffening could play a role in stabilization of tubular membrane structures like filopodia 3 , tunneling nanotubes connecting cells 17 and membrane ruffles at the leading edge of lamellipodia 10 . However , possible stiffening effects have so far not been studied with I - BAR proteins despite its presence in stiff cellular membrane tubes where the stiffening is often attributed to stiff polymers like F - actin inside the tube 18 . Filopodia , induced by expression of C . elegans I - BAR proteins , were reported to appear stiffer than filopodia which were formed by expression of the vertebrate I - BAR domains from MIM and IRSp53 9 . However , no quantification of filopodia stiffness was provided , and moreover , the contribution from filopodial actin cannot be neglected in cellular systems . Experimentally , tubulation and curvature sensing by BAR domain proteins has been studied in vitro where the protein has been added to the outside of spherical membrane vesicles . The width of tubules formed by BAR domains binding to multilamellar lipid vesicles have been quantified by using electron microscopy 8 , 16 , 19 . Another approach is to employ optical trapping together with micropipette aspiration to pull tubes , with specific curva - tures , out of GUVs which has allowed investigation of curvature sensing by various BAR domain proteins 11 , 20 . The effects of I - BAR domains on membranes remain poorly studied in reconstituted in vitro systems , most likely due to the natural association of the protein with the inner surface of membranes . To mimic the cellular settings the protein needs to be encapsulated in closed spherical membranes 12 , 21 . However , in the context of exploring the local effects of I - BARs , addition of I - BAR domains to the outside of GUVs can be argued to be equally relevant since the protein , at a molecular scale , essentially faces a flat GUV at the outer surface as well . Addition of I - BAR to the outside results in formation of inwards pointing tubes which cannot readily be held fixed by optical twee - zers and therefore a different approach is needed to study such tubes . To extract the isolated mechanical effect of I - BARs on membrane tubes , protruding into GUVs , it is neces - sary to carry out a dynamic study of the membrane nanotubes which allows extraction of tube stiffness as well as tube diameter of freely fluctuating tubes . This could answer the question concerning possible stiffening due to oligomerization of I - BAR domains inside membrane nanotubes , an effect which was reported for F - BAR domains bound on the outside of membrane nanotubes 11 , 16 . Here we present a new strategy which can provide both the stiffness and width of nanotubes connected to a GUV , but fluctuating in three dimensions thus resembling intracellular nanotubes which are free to move in the cytoplasm , but might be connected to a larger membrane compartment . Parallel measurements of stiffness and width of unilamellar nanotubes , fluctuating in three dimensions , have to our knowledge not been investigated previously . This assay allows quantification of the physical properties of I - BAR coated nanotubes penetrating into GUVs and the effect of I - BAR can be isolated by direct comparison with protein free nanotubes of the same diameter . By performing a fluctuation analysis of the nanotubes which diffuse transiently into the focus of the microscope we extract both their stiffness and radius . We quantify the width and stiffness of I - BAR coated mem - brane nanotubes fluctuating inside GUVs by adding the protein to the outside of the GUVs . The tube radius is found by performing parallel intensity and fluctuation analysis of nanotubes coated with I - BAR domains at the inner surface of the tubes . By comparing the persistence length of protein coated tubes with bare tubes which are formed spontaneously in GUVs , we investigate whether I - BAR domains have an effect on the stiffness and width of the membrane nanotubes . Finally , we show how I - BAR domains exhibit strong membrane curvature sensing of negative membrane curvatures only at low membrane density of I - BAR . Results Proteins containing I - BAR domains induce shape instabilities of GUVs at a critical density and critical membrane tension 13 , 15 . At low tension I - BAR domains can induce shape changes at low protein density whereas at high ten - sion a higher protein density is needed to deform the membrane 15 . Here we minimize the membrane tension by incubating the GUVs with the I - BAR domain from ABBA in a slightly hyperosmotic buffer ( see Materials and Methods ) and by minimizing adhesion of the GUVs to the glass substrate . To minimize surface adhesion and consequent increase in membrane tension we passivate the glass surface using α - casein before adding the GUVs to the imaging chamber . GUVs incubated with the I - BAR domain from ABBA exhibited membrane protrusions pointing into the lumen of the vesicle as shown in Fig . 1 . Radial intensity plots for the membrane and protein are plotted in Fig . 1C , D at high and low membrane density of I - BAR , respectively . At high density the protein is nearly immobile ( see Figure S1 ) presumably due to crowding effects whereas at low membrane density the pro - tein recovers quickly following bleaching . The protein density used in Fig . 1D is not sufficient for tubulation , but instead the tubes were formed by a spontaneous process during the GUV - electroformation and the protein is seen to diffuse into these preformed tubes . The single tubes are imaged by confocal microscopy by imaging both the YFP tagged I - BAR as well as the Texas Red labeled lipid TR - DHPE . The measured intensity level from YFP scales linearly with membrane density of I - BAR . All intensities from YFP , presented in this work , have been normalized with the same intensity corre - sponding to the membrane density at which the protein was found to be immobile on the membrane . Tubes had variable lengths , but individual segments which were transiently within the quasi two - dimensional focal plane were used to quantify the membrane and protein intensity associated with the tubes as shown in Fig . 2 . We ana - lyzed the intensity of tube segments within GUVs by using an adaptive filament algorithm which adapts a curve to the shape of the filament and allows extraction of the intensity profile . Only clearly visible tube segments of length ~ 3 μ m or more were quantified by this method ( see Fig . 2A , B ) . The intensity along the tube was plotted and the maximum intensity corresponds to the part of the tube which is closest to the focus of the microscope ( see Fig . 2C ) . Tubes which formed spontaneously within GUVs during the electroformation were analyzed and the maximum intensity of a number of tube segments was collected for each GUV . Histograms of tube intensi - ties from two GUVs containing tubes of different widths are shown in Fig . 2D . The narrow distribution of tube www . nature . com / scientificreports / 3 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 intensities in each GUV shows that all tube diameters are similar in each GUV . The diameter of a tube connected to the GUV membrane is set by the membrane tension and the bending rigidity of the bilayer 22 and we note that a low membrane tension is a requirement for the existence of tubes as shown in Figures S3 and S4 . The membrane tension is constant for all tubes in each GUV and hence results in uniformly sized tubes , but might vary among different GUVs due to small changes in osmotic pressure and differences in adhesion to the glass substrate . This causes tubes to have different widths in different GUVs as shown by the example in Fig . 2D . As shown in Fig . 2E the intensity distribution of both the protein ( green circles ) and membrane ( red squares ) signals on tubes , from the same GUV , are normally distributed around a mean value which indicates a uniform density of protein on tubes with similar diameters . Figure 1 . I - BAR domains from ABBA can form tubes pointing into Giant Unilamellar lipid Vesicles ( GUVs ) at high density or bind to existing tubes at lower density . ( A , B ) Schematic depiction showing I - BAR binding to tubes pointing inwards into the lumen of a GUV . ( C ) Incubation of a GUV with 2 . 3 μ M I - BAR . Graph shows radial intensity plot of TR - DHPE signal ( red ) and YFP labeled I - BAR signal ( green ) from a GUV containing a number of inward pointing tubes as shown by the confocal images . ( D ) Incubation of a GUV with 290 nM I - BAR . Graph shows radial intensity of TR - DHPE signal ( red ) and YFP labeled I - BAR signal ( green ) from a GUV containing a number of inward pointing tubes as shown by the confocal images . Each intensity plotted in ( C , D ) is the average of all pixels with the same distance to the center of the GUV . The YFP intensities in ( C , D ) are normalized by the same constant . Membrane composition is DOPC : DOPS : TR - DHPE 59 . 7 : 40 : 0 . 3 . www . nature . com / scientificreports / 4 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 Figure 2 . Examples of how intensities of tube segments are recorded and quantified . Tube segments transiently diffuse into focus of the confocal microscope . An adaptive filament algorithm 41 was used to track the filaments and extract the intensity while they are in focus . By recording a number of images several different tube segments can be analyzed . ( A ) A GUV displaying a number of tube segments which are in focus ( right panel ) . Left panel shows the fitted curves ( red curves ) which overlap with the tube segments which are in focus . The GUV membrane can similarly be quantified using the same strategy . Scale bar , 10 μ m . ( B ) Enlarged image of a tube from ( A ) with the fitted curve as an overlay ( red curve ) . ( C ) Intensity profile along the red curve in www . nature . com / scientificreports / 5 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 The spontaneously formed tubes in protein free GUVs were used to calibrate the stiffness and width of the tubes using a similar strategy as we used in ref . 23 . The shape of the tube is tracked and the correlation of tangent vectors along the tube is quantified as shown in Fig . 3A . The correlation between tangent vectors separated by a distance , x , along a tube with persistence length L P is given by 23 , 24 < ⋅ + > = − t s t s x x L ( ) ( ) exp ( / ) ( 1 ) P where < > denotes the average performed over the variable s . Since the tubes in each GUV have similar intensity and hence similar width we can sample a number of tube segments diffusing transiently into the confocal plane of the microscope and analyze their shapes . The average correlation of a number of tube segments from a single GUV is plotted Fig . 3B . The slope of the graph in Fig . 3B gives the average persistence length of the tubes in the GUV . Theoretically , the persistence length , L p , of a tube is linearly dependent on its radius , R tube22 κπ = L R K T 2 / ( 2 ) tube P B where K T B is the thermal energy , κ is the bending rigidity of the membrane . We used the value from ref . 13 where κ = 23 K T B was measured for a similar lipid mixture as used here . The factor of two , in eq . 2 , stems from the fact that we only detect tube segments which are within the two dimensional space of the microscope focus 25 . Therefore , by calculating the persistence length for tubes in individual GUVs we directly obtain R tube . The persis - tence length of a number of tubes are plotted in Fig . 3C ( red circles ) as a function of R tube ( top axis ) . Moreover , in the bottom axis in Fig . 3C we also plot the tube intensity originating from the membrane dye ( see Fig . 2A , B ) , normalized by the intensity on the GUV membrane . This intensity ratio should scale linearly with the tube radius since the measured intensity from the membrane dye is a function of the tube area . The linear relation in Fig . 3C between L P and intensity confirms that the tube stiffness is well described by eq . 2 . To investigate whether I - BAR coated tubes exhibit any stiffening relative to protein free tubes we plot the persistence length of I - BAR coated tubes together with the persistence length of protein free tubes in Fig . 3C . The tubes coated with ABBA are represented by green squares and can be seen to have similar stiffness as the protein free tubes ( red circles ) of the same width . However , the I - BAR coated tubes span a restricted range of radii rang - ing from 25 nm to 35 nm in radius . The radius of I - BAR coated tubes approaches 25 nm as the density of I - BAR on the membrane increases which is shown in Fig . 3D where tube radius is plotted versus intensity of YFP labeled I - BAR . These results indicate that tubes formed by I - BAR obtain a specific curvature dictated by the convex shape of the membrane binding surface of the I - BAR domain . To further characterize the stiffness of the I - BAR coated tubes we used an alternative approach to measure the stiffness . By analyzing short membrane tubes fixed on the GUV membrane allowed us to image the same short tubes ( L = 3 – 5 μ m ) for longer times , see Fig . 4 . The measured positions of the tube can be compared with simula - tions in which tubes are modeled as stiff or semi - flexible rods with a fixed anchor point on the GUV membrane . We construct the probability distribution function for finding the tip at different locations near the GUV mem - brane as a function of the ratio r = L P / L as derived in ref . 26 Considering a tube fixed on the GUV membrane we define a coordinate system with x pointing inwards and normal to the GUV membrane , y is orthogonal to x but lies parallel within the focal plane of the microscope and finally θ is the angle between x and the tangent to a point on the tube . The probability of finding a point on the tube at a location given by x , y , θ is 26 θ π θ θ θ = −  × − − + − { } G x y L L L L L L x L y L ( , , ) 18 exp 2 exp 6 [ ( cos ) ( sin ) ] ( 3 ) P P P 3 3 7 2 3 2 2 Eq . 3 gives the probability that the tip of a tube with length L and persistence length L P is located at a position ( x , y , θ ) . Numerically , integrating out the angle θ gives a two - dimensional probability distribution for the location of the tip . In our data the whole tube is labeled and we can therefore not track the tip only . However , eq . 3 applies to any point on the tube and therefore , following integration with respect to θ , we integrate with respect to L to obtain the probability density plot for the entire tube , see Fig . 4E . The calculations presented in Fig . 4E show that stiff rods , with a ratio of r = 20 , fluctuate slightly with their free end ( Fig . 4E , top image ) whereas semi - flexible tubes with r ~ 1 have a wider probability distribution , see Fig . 4E , middle and bottom images . A cross - section of the probability distribution ( indicated by the lines in the images in Fig . 4 ) is plotted in the lowest panel of Fig . 4E . The probability density of an experimental tube can be obtained by integrating the intensity from a number of images recorded of the same fluctuating tube which is fixed on the GUV membrane . In Fig . 4A – D we show examples of short tubes connected to the GUV membrane through ( B ) . The portion of the tube which is closest to the microscope focus corresponds to the maximum intensity value and is used in the following quantification of tube intensities . ( D ) Distribution of the maximum intensities from a number of tube segments from two different GUVs ( images of GUV1 and GUV2 are shown to the right ) . The number of tube segments are N seg = 31 ( GUV1 ) and N seg = 33 ( GUV2 ) . ( E ) Distribution of tube intensities from a GUV containing YFP labeled I - BAR coated tubes ( [ I - BAR ] = 2 . 9 μ M ) . The red squares represent maximum tube intensities from the membrane channel ( TR - DHPE ) and the green circles corresponds to the equivalent signal from the YFP labeled I - BAR ( N seg = 64 ) . To the right are shown confocal images of the YFP labeled I - BAR ( green ) and membrane ( red ) . Membrane composition is DOPC : DOPS : TR - DHPE 59 . 7 : 40 : 0 . 3 . www . nature . com / scientificreports / 6 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 a fixed anchor point . The top panels show a single image of the tubes whereas the middle and bottom images represent the maximum intensity projection and average of a time series of images , respectively . The maximum intensity projection reveals all the positions that the tube has visited whereas the average of all the images in a time series is proportional to the probability density for the localization of the tube and can be compared to the analytical predictions presented in Fig . 4E . The cross sectional profiles are plotted in Fig . 4A – D and have a Full Width at Half Maximum ( FWHM ) of ~ 0 . 2 . The theoretical predictions in Fig . 4E also reveal a FWHM of ~ 0 . 2 for tubes having r = 1 . The persistence length thereby has approximately the same magnitude as the length of the tubes presented in Fig . 4 which means that the tubes are semi - flexible . Since , the approaches used in Figs 3 and 4 to find the persistence length of tubes give similar results , we conclude that the persistence length of I - BAR coated tubes is L P ~ 4 μ m at high I - BAR density at which the protein becomes immobile on the membrane . The density of protein , however , affects the mobility of the protein as well as the mobility of the tube junctions with the GUV membrane . A high membrane density of I - BAR leads to very slow and only partial recovery of the intensity following a bleaching experiment as shown in Figure S1 and in the inset of Fig . 3D . Lowering the protein density below a certain threshold , results in high mobility of the protein which recovers after approximately 10 s , see Figure S1B and inset of Fig . 3D . A similar behavior is found for the junctions between the GUV and tubes . In absence of I - BAR or at low density of I - BAR the tubes connected to the GUV membrane move freely around the GUV ( see Fig . 5A ) whereas at higher density of I - BAR the tube - GUV junction becomes immobile as seen in Fig . 4 and Figure S2 . Lower membrane densities of I - BAR on the GUV membrane results in two - dimensional diffusion of I - BAR on the membrane . Interestingly , when low concentrations ( 290 nM ) of I - BAR are added to a chamber containing GUVs , with preexisting inwards pointing tubes , we see strong preference of the protein for binding to the inte - rior of the tubes , see Fig . 5 . By quantifying the rotationally averaged radial intensity of YFP labeled I - BAR and TR - DHPE from the GUV center and out to the GUV membrane we find quite distinct relative intensities at low and high density of I - BAR , respectively . For high concentrations ( > 1 μ M ) of I - BAR the relative ratio between the protein and membrane signal is close to 1 both inside and on the GUV membrane ( see Fig . 5B ) and hence the Figure 3 . Membrane nanotubes coated with I - BAR domains have uniform widths with well - defined stiffness . ( A ) Quantification of tubular shape by finding local tangent vectors is performed on tube segments which are transiently within the focal plane of the microscope . Scale bar , 10 μ m . ( B ) By analyzing the shape of < N seg > = 92 tube segments from each GUV the average correlation between tangent vectors along the tube segments is found . The slope of the graph equals the persistence length . ( C ) Persistence lengths versus the intensity ratio between the tube membrane and the GUV membrane . The corresponding radius of the nanotube is found from eq . 2 and plotted in the top axis . Each data point is obtained by analyzing , on average , the shape of 29 tube segments from each GUV . Green data points represent tubes which contain I - BAR bound to the interior of the tubes and red data points represent spontaneous tubes formed in GUVs in absence of I - BAR . Tubes from a total of 59 GUVs were analyzed and the intensity distributions had an average width of 17 . 0 % of the mean . ( D ) Tubule width as a function of intensity of YFP labeled I - BAR . Inset shows the relative recovery after 15s , following photo bleaching of spot on the GUV membrane , as a function of intensity of YFP labeled I - BAR on the GUV membrane . The inset graph shows binned data from 21 FRAP experiments , error bars denote the standard deviation . Membrane composition DOPC : DOPS : TR - DHPE 59 . 7 : 40 : 0 . 3 . www . nature . com / scientificreports / 7 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 density of I - BAR is the same on the GUV membrane as inside the tubes . However , at low membrane density of I - BAR we find the same ratio to be ~ 7 fold higher within the tubes protruding the GUV , relative to on the less curved GUV membrane ( see Fig . 5D ) . The ratio between the protein and membrane signals in the tubes and on the GUV can be expressed as a ‘Sorting’ parameter as follows = ( ) ( ) Sorting ( 4 ) I I tube I I GUV prot mem prot mem where I prot and I mem are the intensities from the YFP labeled protein and Texas Red labeled membrane , respec - tively . The Sorting values for the data in Fig . 5B , D are plotted in Fig . 5C , E and the data reveal that no sorting ( Sorting ≈ 1 ) occurs in Fig . 5B whereas in Fig . 5D the density of protein is 7 times higher ( Sorting ≈ 7 ) on the tubes than on the GUV membrane . Since the tubes are all protruding into the GUVs these results strongly support that I - BAR domains can sense negative membrane curvatures . Finally , we note that the range of concentrations of I - BARs used here are similar to the concentrations used in other studies where membrane deformation and membrane curvature sensing by different types of BAR domains , has been investigated in in vitro assays 11 – 13 , 15 , 27 , 28 . Figure 4 . Short membrane nanotubes coated with high density of I - BAR are semi - flexible with L p ~ L and do not move laterally on the GUV membrane . ( A ) From top to bottom panel , ( i ) example of a tube fixed on the GUV membrane ( ii ) the corresponding maximum projection of the intensity of a series of images of the same tube ( iii ) the corresponding average intensity from the entire time series and ( iv ) line profile parallel to the GUV membrane at a distance corresponding to L / 3 from the GUV membrane . ( B – D ) More examples of short tubes plotted in the same sequence as in ( A ) . ( E ) Theoretical calculation of the probability of a fluctuating rod which is fixed at one end . Top image shows a rod which has L p / L = 20 , in the middle image L p / L = 1 . 7 and in the bottom image L p / L = 0 . 7 . x and y axes range from 0 to L and – L to L , respectively . The graph below the images corresponds to line profiles along the depicted lines in the three images , L p / L = 20 ( blue ) , L p / L = 1 . 7 ( green ) and L p / L = 0 . 7 ( red ) . All images in ( A – D ) present intensities from YFP labeled I - BAR . Membrane composition DOPC : DOPS : TR - DHPE 59 . 7 : 40 : 0 . 3 . The figure shows representative examples of the ( N short > 100 ) short tubes observed in this work . All scale bars are 2 μ m . www . nature . com / scientificreports / 8 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 Discussion I - BAR induced membrane tubes are being studied due to their similarity with protrusive structures like filopodia and invadopodia 3 , 29 . Although , filopodia - like structures can be induced by overexpression of I - BAR contain - ing proteins 8 , 9 the role of I - BAR domains in the formation of protrusive membrane structures remains unclear . Filopodia have been found to contain F - actin which polymerizes towards the tip and F - actin bundles have been shown to play a major role in formation and function of filopodia 3 , 18 , 30 . I - BAR containing proteins can have actin binding domains and hence could be synergistically involved in formation of filopodia together with F - actin as suggested in ref . 8 . The tubulating activity of I - BAR domains from ABBA and other I - BAR domains 9 , 13 in model membrane systems containing high density of negatively charged lipids , clearly suggests that I - BAR domains play an important role in the formation and stabilization of cellular membrane morphologies like filopodia and membrane ruffles . Here we have shown that binding of the I - BAR domain from ABBA forms visible tubes protruding into GUVs when the I - BAR domains are added to the outer side of the GUVs at a high concentration . The tubes diffuse transiently into the focal plane of the microscope and are analyzed with respect to their intensity and shape . By exploiting the linear relation between persistence length and the width of a tube together with the fact that tube intensity scales linearly with tube radius , we can directly infer the tube width from the persistence length and Figure 5 . Membrane curvature sensing by I - BAR depends on protein density on the membrane . ( A ) Overlay of the membrane ( red ) and YFP labeled I - BAR ( green ) showing higher intensity of the protein inside the highly curved tube than on the nearly flat GUV membrane . The arrow shows an example of a tube connected with the GUV membrane . In subsequent images the tube disappears due to high mobility of the tube at low protein density . Protein concentration in solution is 290 nM . Scale bar , 10 μ m . ( B ) Radial intensity of the membrane and YFP signal in a GUV incubated with YFP labeled I - BAR [ I - BAR ] = 2 . 3 μ M . ( C ) Protein sorting as a function of distance from the center of the GUV . Sorting is defined as the ratio between the protein density on the tube and on the GUV membrane which can be calculated by using eq . 4 . ( D ) Example of radial intensity at lower density of [ I - BAR ] = 290 nM . ( E ) Corresponding sorting of the signals presented in ( D ) . Each intensity plotted in ( B , D ) is the average of all pixels with the same distance to the center of the GUV . Confocal images in ( B , D ) show the overlay of the protein ( green ) and membrane ( red ) . Membrane composition DOPC : DOPS : TR - DHPE 59 . 7 : 40 : 0 . 3 . Data were recorded for 15 GUVs incubated with 290 nM of YFP labeled I - BAR and all showed similar level of sorting . At micromolar concentrations of the protein we never detected any sorting ( N = 59 GUVs ) . www . nature . com / scientificreports / 9 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 intensity . At high density of protein on the membrane , the tubes are fixed to the GUV membrane and the pro - tein exhibits minimal mobility on the membrane . Surprisingly , we find no stiffening effect on membrane tubes even at the high membrane density of I - BAR at which the protein mobility is nearly absent ( see Figure S1 and inset of Fig . 3D ) . Previous work has shown that the F - BAR domain from Syndapin 1 induced weak stiffening of membrane tubes 11 whereas another F - BAR protein ( hFBP17 ) 16 induced significant stiffening of membrane tubes by forming an oligomerized lattice around the tube . Also , N - BAR decorated membrane tubes have been shown to exhibit stiffening with a persistence length of 9 . 1 μ m 16 which is quite significant considering the thin tubules formed by N - BAR ( ~ 20 nm in diameter ) 16 . However , in all studies which reported persistence length measure - ments of membrane nanotubes , the tube width and possible multilamellarity , was not measured despite the linear dependence between persistence length and tube radius ( see . Eq . 2 ) . Comparing the measured stiffness of I - BAR coated tubes with protein free tubes we see that I - BAR produces tubes of a certain diameter of ~ 50 nm which is consistent with previous data from ref . 9 where diameters were measured using electron microscopy of multilamellar lipid vesicles which had been mixed with I - BAR . This suggests that membranes adapt to the molecular curvature of I - BAR domains . Moreover , since many preexisting tubes are found in the GUVs this means that I - BAR either constricts or expands preexisting tubes if they are wider or narrower than 50 nm ; a similar effect was recently measured for the I - BAR from IRSp53 12 in a different assay . The importance of considering the lamellarity and width of the tubes is evident from the known dependence of tube width and lamellarity on tube stiffness 23 . Tubes having several bilayers with a small separation between each bilayer may appear unilamellar in bright field microscopy which has been used for analyzing the stiffness of tubes 16 . However , fluorescence microscopy of tubes can be used for both size calibration and to detect possible multilamellarity 23 . The persistence lengths presented in Fig . 3C are plotted versus radius and therefore the effect of the protein on the tube stiffness can be isolated from the effect of tube width . The experimental settings mimic the environ - ment that tubes experience in cells where tubes are diffusing in three dimensions , but with one end fixed to some larger membrane structure . Consequently , the tubes studied here are constantly undergoing thermal motion which causes the protein conformation on the tube to be constantly exposed to stress and potential rearrange - ments through bending fluctuations leading to high local membrane curvatures . The absence of any stiffening effects of the protein on the tube is surprising considering the lack of recovery of the protein following bleaching ( Fig . 3D , inset and Figure S1 ) . Previous work has also shown very slow recovery and only partial recovery fol - lowing bleaching of fluorescently labeled F - BAR 27 or I - BAR 28 which indicated protein oligomerization . The low protein mobility together with the absence of stiffening observed here could suggest that I - BARs interact through flexible linkages . Membrane curvature sensing experiments are typically performed using optical tweezers for pulling out high curvature membrane tubes from a GUV which is exposed to a solution containing the protein . Recently , a differ - ent approach was used where the I - BAR domain from the IRSp53 protein was first encapsulated within the lumen of GUVs followed by extrusion of tubes by an optical trap . This allowed the I - BAR domains to sense membrane curvature from inside of the GUV 12 . The I - BAR domains from ABBA used here could not be encapsulated using standard electro - formation protocols and therefore we measured the membrane curvature sensing by I - BAR inside membrane tubes protruding into the GUVs thus allowing the protein to be added to the outside of the GUVs . These results revealed a strong preference of I - BAR for the negatively curved membrane inside tubes which are unrestricted by optical tweezers ( see Fig . 5 ) . The mechanism of membrane curvature sensing by BAR and I - BAR domains has been ascribed to both the geometry of the dimer 31 as well to amphipathic helices which can insert into a curved membrane . The I - BAR domain from MIM ( MIM - IMD ) was found to sense positive curvatures on small lipid vesicles and consequently it was concluded that the I - BAR domain in MIM is not critical in membrane curvature sensing 32 . However , here we find that the closely related I - BAR domain from ABBA does indeed sense negative membrane curvature . The I - BAR domain from ABBA also contains a membrane inserting amphipathic helix which could assist binding to flat or positively curved membranes , but is unlikely to contribute to curvature sensing inside negatively curved nanotubes . Therefore , we expect the membrane curvature sensing presented in Fig . 5 to be induced by the shape of the convex binding side of I - BAR . This is consistent with the negative membrane curvature sensing reported for IRSp53 I - BAR domain which binds to membranes electrostatically without insertion of an amphipathic helix 12 . In conclusion , these results indicate that the I - BAR domain of ABBA could be an important regulator of protein density by sensing membrane curvature in e . g . membrane ruffles found in glial cells . Membrane protein density and membrane tension have recently been proposed as master regulators of a number of cellular processes which involve a change in membrane morphology and membrane area 15 , 33 – 35 . The membrane shaping process is naturally more energy expensive at high membrane tension and hence the deforma - tion carried out by proteins is counteracted by high tension 13 – 15 . N - BAR domains are well known to be curvature sensitive at low density whereas at high density they facilitate membrane deformation . Recently , it was shown how membrane tension could control the shape instability of GUVs induced by N - BAR domain of endophilin 15 as well as for the I - BAR domain from IRSp53 13 . A transition density for shape instability was quantified as a function of tension thus clearly demonstrating the interplay between tension and protein density in the membrane shaping function of BAR domains . Here , we observe a similar effect of tension and concentration on shape instability , but with the I - BAR domain from ABBA . GUVs tubulate efficiently under low tension , as shown in Supplementary Figure S3 by exposing GUVs to a solution with high osmolarity delivered by a micropipette . Also , we find that existing tubes can be reversibly retracted into the GUV membrane after varying the membrane tension by succes - sively aspirating a GUV into a micropipette , as presented in Figure S4 . The spontaneous tubes observed in our experiments in absence of I - BAR appear consistently in a fraction of the GUVs and their tube radius varies according to the data in Fig . 3C ( circles ) . These tubes could result from www . nature . com / scientificreports / 10 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 the fact that experiments are carried out in a hyperosmotic buffer . Also , we note that highly charged lipids are exquisitely sensitive to local pH variations and these tubes could be due to lipid flip - flop between the inner and outer leaflet of the GUV caused by slight changes in pH across the membrane . A pH gradient has previously been shown to induce membrane deformations in GUVs containing high percentage of charged lipids 36 . Finally , we note that the electroformation process is not understood despite its wide use for making GUVs . A compositional asymmetry between the leaflets caused by some artefact from the electroformation process cannot be ruled out . The quantitative assay developed here to investigate the properties of I - BAR coated membrane nanotubes was tested on a simple binary lipid mixture . Tube formation by IRSp53 was recently shown to be significantly enhanced by including 5 % of phosphatidylinositol bisphosphate ( PI ( 4 , 5 ) P 2 ) in the GUV membrane compared with GUV membranes containing no PI ( 4 , 5 ) P 2 , but containing an overall identical lipid charge density 13 . Also , I - BARs have been shown to cluster PI ( 4 , 5 ) P 2 lipids and reduce their diffusion 28 which indicates strong binding between the I - BAR and the PI ( 4 , 5 ) P 2 lipids . In future work the physical properties of I - BAR coated tubes should be investigated with a composition containing other important lipids like DOPE , cholesterol as well as phospho - inositide lipids . Conclusion By employing a new method for quantifying the size and properties of freely fluctuating lipid nanotubes we have here shown how I - BARs deform membranes into semi - flexible tubes that have a narrow size distribution . The diameter of the tubes approaches 50 nm at high membrane density of I - BARs . No tube stiffening could be attrib - uted to the binding of I - BAR and tubes formed by I - BAR were found to be semi - flexible ( L p ~ 4 μ m ) in accordance with the stiffness expected for tubes of similar diameter . I - BAR was found to be sensitive to membrane curvature with a ~ 7 fold increase in density inside high curvature nanotubes relative to the density on the nearly flat GUV membrane . The coupling of cellular shape , lateral membrane tension and membrane mechanics is emerging as the main regulator of cellular function , but needs to be further explored since it seems to involve a complicated interplay between membrane tension , protein activity and also lateral protein density . Here we have demonstrated a new strategy for quantifying the stiffness and width of unilamellar lipid nanotubes in three dimensions . This assay can be further used to reveal physical properties of membrane nanotubes interacting with other proteins or for investigating the stiffness of nanotubes with different lipid compositions in the vicinity of critical phase transition temperatures 37 . Materials and Methods Microscopy . Confocal fluorescence microscope ( Leica SP5 ) equipped with a water immersion objective ( Leica , PL APO NA : 1 . 2 , 63 × ) and photomultiplier tubes for detection was used to record all fluorescent images . Excitation of YFP was performed at λ = 488 nm and TR - DHPE was excited at λ = 594 nm and the emitted light was collected at 495 – 570 nm and 612 – 744 nm for the two fluorophores , respectively . FRAP measurements were performed by choosing a region on the GUV membrane and briefly exposing it to high laser power from the 488 nm laser line . Since membrane nanotubes were acquired using confocal scanning microscopy which is a sequential scanning of a two dimensional space we carried out a control to check if sequential acquisition of the area affected the quantification of the persistence lengths . As shown in Figure S5 we measure similar persistence lengths using a sequential scanning technique as when using wide field excitation with camera detection . This can be rationalized by considering the time it takes to scan a tube segment of 5 – 10 μ m in length : with a scanning frequency of 400Hz and pixel size of 160 nm it takes less than 100 ms to scan the largest tube segments in our data . The diffusion length scale during the same time interval of a tube segment having a diameter of 50 nm and length of 10 μ m is < 200 nm 38 , 39 and hence this is on the same scale as our pixel width . Since the time scale of bending of these nanotubes is on the order of 1s 38 we conclude that persistence lengths can be accurately quantified by sequential confocal scanning microscopy which requires ~ 100 ms to record the shape of a nanotube segment . Since the imaging depth of the confocal microscope is ~ 0 . 5 μ m we can obtain proper two dimensional images of the tube segments ; the tubes are stiff on a 0 . 5 μ m length scale and hence if they bend in the axial direction they will disappear from the imaging plane . Electroformation . GUVs were formed by using a Nanion Vesicle Prep Pro electroformation device ; 50 nmo - les ( 50·10 − 9 moles ) of lipid mixture dissolved in 25 μ L of chloroform was dropcast on a conducting ITO - coated glass slide and placed in a vacuum chamber for ~ 1 h to remove residual chloroform solvent . To facilitate hydration 275 μ L of a 333 mM sucrose solution was then added to the dry lipid film and confined by a d = 18 mm O - ring . We found that adding 1mM of TRIZMA - base to the hydration medium resulted in higher number of spon - taneous tubes protruding into the GUVs . This method was used for forming the protein free tubes in Fig . 3C . The chamber was sandwiched with a second ITO - slide . Unless stated otherwise all GUVs were prepared from a lipid / dye mixture composed of DOPC ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine , Avanti polar lipids ) , DOPS lipids ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphoserine , Avanti Polar Lipids ) and 1 , 2 - dihexadecanoyl - sn - glycero - 3 - p hosphoethanolamine - Texas Red was purchased from Invitrogen . Unless noted otherwise the lipids were mixed with the following stoichiometry : DOPC : DOPS : TR - DHPE 59 . 7 : 40 : 0 . 3 . After 2 hours of standard electroforma - tion ( 10 Hz , 1 V , 55 °C ) a high yield of GUVs could be transferred to an eppendorf tube and used immediately or stored overnight at 4 °C in the dark . Sample preparation . Experiments were conducted on cover slips with a thickness of 170 μ m which were cleaned in repeated rounds in detergent and alcohol baths under ultrasonication . The glass was treated with 2 g / L α - casein ( from Sigma Aldrich ) suspended in 20 mM Tris and 2mM EDTA by incubating for 30 min followed by thorough rinsing with a phosphate buffered ( pH 7 . 2 ) solution containing 50 mM NaCl and 295 mM sucrose . The circular glass was inserted into a chamber with a teflon inset which confines the solution . The passivation of the www . nature . com / scientificreports / 11 Scientific RepoRts | 6 : 30054 | DOI : 10 . 1038 / srep30054 surface minimized the adhesion of the vesicles to the glass . GUVs were mixed with protein at the desired protein concentration and incubated for 30 min in a 1 mL plastic tube placed on a rotating wheel to prevent sedimentation of the GUVs . The GUVs were suspended in a PBS buffer containing 50 mM NaCl and 295mM sucrose and hence the solution had a higher osmolarity than the solution inside the GUVs . Micropipette experiments . Glass micropipettes were fabricated using a micropipette puller ( P - 97 Flaming / Brown Micropipette Puller , Sutter Instruments ) and microforged to a diameter of 8 μ m using Micro Forge MF - 900 , Narishige co . ltd Japan . The pipettes contained a solid filament on the inner surface of the glass to facilitate easy back - filling of the pipette with either protein or buffer solution . Micropipettes were controlled using a standard micromanipulator ( Narishige co . ltd Japan ) attached to a two dimensional piezo stage ( PI 731 . 20 , Physik Instrumente , Germany ) to facilitate precise positioning of the pipette . Protein expression and purification . The plasmids encoding the hexahistidine - tagged I - BAR domains were a kind gift from Pekka Lappalainen ( University of Helsinki ) . The region containing the T7 promoter and the ABBA I - BAR domain was PCR amplified using the primer 5 ′ - TTTTGAATTCTAATACGACTCACTATAGGGAATTGT - 3 ′ and the primer 5 ′ - TCTCACCGGTAGAACCCTTCAGGTCTTTGATCACCT - 3 ′ . The amplified DNA fragment was digested with Eco RI and Age I and inserted between the same sites of plasmid pSEM3073 40 . The resulting plasmid encoded the I - BAR domain fused to an N - terminal His - tag and a C - terminal YFP - tag . The fusion protein was expressed in Escherichia coli BL21 ( DE3 ) cells . Harvested cells were resuspended in Lysis I buffer ( 10 mM sodium phosphate , pH 8 . 0 , 0 . 5 mg / ml lysozyme ) and stored on ice for 30 min . Equal volume of Lysis II buffer ( 10 mM sodium phosphate , pH 8 . 0 , 2 M NaCl , 20 % glycerol , 0 . 1 % Triton X - 100 ) was added and incubated for 30 min on ice . The cell debris was removed by centrifugation . Addition of 3 % Ni - NTA slurry ( Qiagen ) to the solu - tion was followed by 1 h incubation at 4 °C . A Poly - Prep Chromatography Column ( BIO - RAD ) was used to col - lect the protein bound to Ni - NTA agarose from the mixture . Twenty column volumes of washing buffer ( 10 mM sodium phosphate , pH 8 . 0 , 1 M NaCl , and 10 % glycerol ) was allowed to flow through the column . Proteins were eluted by four column volumes of elution buffer ( 10 mM sodium phosphate , pH 8 . 0 , 1 M NaCl , 50 % glycerol , 250 mM imidazole ) . The imidazole and the residual Triton X - 100 was removed by dialysis , using the final storage buffer ( 20 mM HEPES , 150 mM NaCl , and 1 mM TCEP ( Tris ( 2 - carboxyethyl ) phosphine ) . The purified protein was aliquoted and stored at − 80 °C . Data analysis . Images were recorded using Leica Application Suite and exported images were analyzed using custom made software in Matlab ( The MathWorks , Inc . , Natick , Massachusetts , United States ) and using an ImageJ plugin called Jfilament 41 for extracting the intensity of tube segments . Tube persistence lengths were calculated based on the method described in ref . 23 . Intensities of tube segments and the GUV membrane were found by using an adaptive filament algorithm 41 which fits a curve to the maximum intensity along a segment . Quantification of protein sorting in Fig . 5 was performed using Matlab by calculating the radial , rotationally averaged , intensity from the center of the GUV to the GUV membrane for both the membrane and protein channel . The center of the GUV was found by fitting a circle to the GUV . The ratio between the intensities directly gives the Sorting according to eq . 4 . Intensities from YFP labeled I - BAR in Fig . 3C , D were quantified using the Jfilament adaptive algorithm 41 to fit a curve to the relevant GUV membrane and extract the average GUV inten - sity . Normalized YFP - intensities used throughout the paper refer to the same normalization constant which was the highest YFP intensity measured on the GUV membranes . In the sorting experiments in Figs 1D and 5D we used higher laser excitation to visualize the low density of YFP labeled I - BAR . The relative ratio between the excitation powers used in the paper was quantified by measuring reflection signals from the glass / water interface at different laser powers and this allowed us to use the same normalization of the YFP signal in all figures . References 1 . Doherty , G . J . & McMahon , H . T . Mechanisms of endocytosis . Annu . Rev . Biochem . 78 , 857 – 902 ( 2009 ) . 2 . Scholey , J . M . , Brust - Mascher , I . & Mogilner , A . Cell division . Nature 422 , 746 – 752 ( 2003 ) . 3 . Leijsne , N . , Oddershede , L . B . & Bendix , P . M . An updated view at actin dynamics within filopodia . Cytoskeleton 72 , 71 – 79 ( 2015 ) . 4 . Mattila , P . K . & Lappalainen , P . Filopodia : molecular architecture and cellular functions . Nat . Rev . Mol . Cell Biol . 9 , 446 – 454 ( 2008 ) . 5 . Lowery , L . A . & Van Vactor , D . 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Nanotechnology 25 , 505101 ( 2014 ) . 38 . Powers , T . R . Dynamics of filaments and membranes in a viscous fluid . Rev . Mod . Phys . 82 , 1607 – 1631 ( 2010 ) . 39 . Tirado , M . M . , Martínez , C . L . & Torre , J . C . Comparison of theories for the translational and rotational diffusion coefficients of rod like macromolecules . Application to short DNA fragments . J . Chem . Phys . 81 , 2047 – 2052 ( 1984 ) . 40 . Mitarai , N . , Jensen , M . & Semsey , X . Coupled Positive and Negative Feedbacks Produce Diverse Gene Expression Patterns in Colonies . mBio 6 , e00059 – 00015 ( 2015 ) . 41 . Smith , M . B . et al . Segmentation and tracking of cytoskeletal filaments using open active contours . Cytoskeleton 67 , 693 – 705 ( 2010 ) . Acknowledgements This work was supported by the Villum Kann Rasmussen Foundation , the Danish Council for Independent Research ( grant ID : DFF – 4181 - 00196 ) , C - mol ( DNRF Centers of Excellence ) and University of Copenhagen . We are grateful to Prof . Lene B . Oddershede for support and fruitful discussions . We thank P . Lappalainen , University of Helsinki , for kindly providing the plasmids for the I - BAR proteins . Author Contributions Y . F . B . , A . R . - L . and P . M . B . performed research , Y . F . B . analyzed all persistence lengths data . S . S . expressed and purified the I - BAR . S . N . S . R . and P . M . B . supervised the project . P . M . B . quantified all tube intensities and wrote the first draft . All authors critically read the manuscript . Additional Information Supplementary information accompanies this paper at http : / / www . nature . com / srep Competing financial interests : The authors declare no competing financial interests . How to cite this article : Barooji , Y . F . et al . Dynamics of membrane nanotubes coated with I - BAR . Sci . Rep . 6 , 30054 ; doi : 10 . 1038 / srep30054 ( 2016 ) . This work is licensed under a Creative Commons Attribution 4 . 0 International License . The images or other third party material in this article are included in the article’s Creative Commons license , unless indicated otherwise in the credit line ; if the material is not included under the Creative Commons license , users will need to obtain permission from the license holder to reproduce the material . To view a copy of this license , visit http : / / creativecommons . org / licenses / by / 4 . 0 / \ No newline at end of file diff --git a/silver_data/2abc08bb719a85ed05507ba9a2c74217148ccd5a.pdf.txt b/silver_data/2abc08bb719a85ed05507ba9a2c74217148ccd5a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..da51301097fc0fefdccaa2a6b50e3da7ffe10d6f --- /dev/null +++ b/silver_data/2abc08bb719a85ed05507ba9a2c74217148ccd5a.pdf.txt @@ -0,0 +1 @@ +Peptides 37 ( 2012 ) 273 – 284 Contents lists available at SciVerse ScienceDirect Peptides j ourna l ho me pa ge : www . elsevier . com / locate / peptides Protein transduction in human cells is enhanced by cell - penetrating peptides fused with an endosomolytic HA2 sequence Ji - Sing Liou a , b , Betty Revon Liu a , b , Adam L . Martin c , Yue - Wern Huang d , ∗ , Huey - Jenn Chiang b , Han - Jung Lee a , ∗∗ a Department of Natural Resources and Environmental Studies , National Dong Hwa University , No . 1 , Sec . 2 , Da - Hsueh Road , Shoufeng , Hualien 97401 , Taiwan b Graduate Institute of Biotechnology , National Dong Hwa University , No . 1 , Sec . 2 , Da - Hsueh Road , Shoufeng , Hualien 97401 , Taiwan c Department of Biological Sciences and the cDNA Resources Center , Missouri University of Science and Technology , 206 Schrenk Hall , 400 West 11th Street , Rolla , MO 65409 - 1120 , USA d Department of Biological Sciences , Missouri University of Science and Technology , 105 Schrenk Hall , 400 West 11th Street , Rolla , MO 65409 - 1120 , USA a r t i c l e i n f o Article history : Received 15 June 2012 Received in revised form 21 July 2012 Accepted 23 July 2012 Available online 31 July 2012 Keywords : Cell - penetrating peptide Cytotoxicity Endosomal escape Hemagglutinin - 2 Membrane fusion a b s t r a c t Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell - penetrating peptides ( CPPs ) and their cargoes . However , a major limitation of endocytic pathway is entrapment of the CPP - cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity . Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin - 2 ( HA2 ) remarkably enhances the cytosolic delivery of proteins in human A549 cells . To determine the endosome - disruptive effects , recombinant DNA plasmids containing coding sequences of HA2 , CPPs and red fluorescent proteins ( RFPs ) were constructed . The fusion proteins were purified from plasmid - transformed Escherichia coli , and their effects on protein transduction were examined using live cell imaging and flow cytometry . Our data indicate that endocytosis is the major route for cellular internalization of CPP - HA2 - tagged RFP . Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP - mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm . Furthermore , incorporating the HA2 fusion peptide of the CPP - HA2 fusion protein improved cytosolic uptake without causing cytotoxicity . These findings strongly suggest that the CPP - HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs . © 2012 Elsevier Inc . All rights reserved . 1 . Introduction While many small molecules , such as ions , sugars and amino acids , permeate cells through carriers and channels in the plasma membrane , this mode of entry is generally unavailable for macro - molecules , such as proteins , DNAs and RNAs . In order to develop highly efficient strategies for the controlled cellular delivery of Abbreviations : BFP , blue fluorescent protein ; CPP , cell - penetrating peptide ; CytD , cytochalasin D ; EEA1 , early endosome antigen 1 protein ; GFP , green fluorescent protein ; FITC , fluorescein isothiocyanate ; HA , hemagglutinin ; HA2 , hemagglutinin - 2 ; MTT , 1 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 3 , 5 - diphenylformazan ; 6His , hexa - histidine ; NLS , nuclear localization signal ; PBS , phosphate buffered saline ; PTD , protein transduction domain ; QD , quantum dot ; R9 , nona - arginine ; RFP , red fluorescent protein ; SUMO , small ubiquitin - related modifier ; TAT , transactivator of transcription . ∗ Corresponding author . Tel . : + 1 573 341 6589 ; fax : + 1 573 341 4821 . ∗∗ Corresponding author . Tel . : + 886 3 8633642 ; fax : + 886 3 8633260 . E - mail addresses : huangy @ mst . edu ( Y . - W . Huang ) , hjlee @ mail . ndhu . edu . tw ( H . - J . Lee ) . bioactive macromolecules with therapeutic potential , several non - viral carrier systems , including liposomes , polycationic carriers , nanomaterials and peptides , have been developed . Cell - penetrating peptides ( CPPs ) , also called protein transduction domains ( PTDs ) , are a group of short , highly basic peptides that are able to pene - trate the cell membrane either alone or carried with cargoes [ 9 ] . In 1988 , two groups independently discovered that the transactiva - tor of transcription ( TAT ) protein of the human immunodeficiency virus type 1 ( HIV - 1 ) can penetrate cells and activate viral genome replication [ 16 , 21 ] . The process by which TAT protein and other CPPs cross the cell membrane and deliver macromolecule cargoes is referred to as protein transduction [ 11 , 31 , 50 ] . CPPs can be cate - gorized into three groups based upon their composition [ 35 ] . The first group is comprised of protein - derived peptides , such as PTD of TAT and antennapedia . The second group includes chimeric pep - tides , such as transportan , that contain two or more motifs from different peptides [ 41 ] . The last group is comprised of synthetic peptides , such as polyarginines [ 17 ] . Although protein transduction has been widely used as a research tool , and twenty clinical trials are now testing 0196 - 9781 / $ – see front matter © 2012 Elsevier Inc . All rights reserved . http : / / dx . doi . org / 10 . 1016 / j . peptides . 2012 . 07 . 019 274 J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 CPP - mediated delivery of drug conjugates in patients with a variety of diseases [ 48 ] , the mechanisms of CPP - mediated cellu - lar uptake and the subsequent intracellular trafficking are still under extensive investigation . Presently , it is believed that most CPPs utilize multiple pathways for cellular entry [ 35 , 37 , 43 , 48 ] . The two major uptake mechanisms of CPPs are direct membrane translocation and endocytosis . Direct membrane translocation , also called direct cell penetration , encompasses a variety of energy - independent pathways , including pore formation , inverted micelle formation , carpet - like model and membrane thinning model [ 35 , 48 ] . Endocytosis is divided into two major categories ; phago - cytosis involves the uptake of large particles while pinocytosis involves solute uptake [ 23 , 35 ] . Pinocytosis can be further divided into macropinocytosis , clathrin - dependent , caveolin - dependent and clathrin / caveolin - independent pathways [ 6 ] . Numerous fac - tors , including the experimental conditions and physicochemical properties of CPPs and their cargoes , appear to influence the route of cellular uptake [ 35 , 48 ] . Insofar as endocytosis is one of the primary mechanisms for cel - lular delivery mediated by CPPs , the fate of endocytosed cargoes is of paramount importance . Endocytosed cargoes often become trapped in organelles , such as vesicles , endosomes , lysosomes and macropinosomes [ 42 ] , where they may be degraded by hydrolytic enzymes . Thus , escape from endocytic vesicles into the cytoplasm is essential to preserve biological activity of endocytosed cargoes and becomes a limiting factor in the usage of CPPs to deliver bioactive molecules [ 11 , 35 , 43 , 48 , 51 ] . Recently , several endosome - disruptive peptides ( membrane destabilizing / fusion peptides ) were derived from certain pathogens , including viruses and bacterial toxins [ 37 ] . These membrane - disrupting peptides are triggered by endosomal acidification and promote endosomal escape [ 12 , 39 , 51 ] . The human influenza virus is an enveloped virus that contains two major envelope glycoproteins , hemagglutinin ( HA ) and neu - raminidase [ 58 ] . HA is composed of two subunits : hemagglutinin - 1 responsible for binding to cells and hemagglutinin - 2 ( HA2 ) respon - sible for endosomal escape . The N - terminal domain of the HA2 subunit possesses 23 amino acids ( GLFGAIAGFIENGWEGMIDG - WYG ) , a relatively hydrophobic region referred to as fusion peptide [ 7 , 56 ] . This fusion peptide domain is buried in the HA trimer in its resting conformation . Acidification in the endosome trig - gers an irreversible conformational change of HA2 , exposing the fusion peptide and allowing it to insert itself into endosomal membranes . Subsequent formation of a fusion pore results in membrane fusion and leading to transfer of viral genome into the cytosol [ 7 ] . The INF7 ( GLFEAIEGFIENGWEGMIDGWYG ) pep - tide , a glutamic acid - enriched HA2 analog , was identified as a more potent endosome membrane - destabilizing peptide [ 40 ] . In this peptide , two glutamic acid moieties ( underlined in peptide sequence ) were introduced into the original HA2 fusion peptide to extend the (cid:2) - helix structure , thereby increasing pH sensitivity [ 14 ] . HA2 was used to enhance CPP - mediated endosomal escape of cargoes [ 13 , 18 , 28 – 30 , 36 , 45 , 46 , 51 , 61 , 62 ] . Certain delivered molecules are intended to target nucleus . Molecules can enter the nucleus from the cytoplasm by both passive diffusion and active transport mechanisms [ 10 ] . Small molecules less than 10 nm in diameter or 50 – 60 kDa in size can diffuse through nuclear pore complexes [ 50 ] . Most protein molecules are transported by energy - dependent transport mecha - nisms mediated by nuclear localization signals ( NLS ) . These signals are recognized by importin family proteins that mediate the trans - port across the nuclear envelope with the participation of Ran - GTP [ 10 ] . The goals in the present study were to ( 1 ) compare the trans - duction efficiency of CPP - , HA2 - and / or NLS - tagged red fluorescent proteins ( RFPs ) and ( 2 ) determine the uptake mechanisms and subcellular localization of these proteins . To achieve these goals , Table 1 Sequences of tags . Tag Amino acid sequence ( single letter code ) HA2 ( INF7 ) GLFEAIEGFIENGWEGMIDGWYG 6His HHHHHH NLS PKKKRKV R9 RRRRRRRRR we first constructed a series of novel DNA plasmids containing coding sequences of CPP , HA2 and / or NLS fused RFP . These plas - mids were expressed in bacteria , and the transduction efficiency of these fusion proteins was determined in human lung cancer A549 cells using live cell imaging and flow cytometry . To elucidate the uptake mechanisms , pharmacological and physical inhibitors were used to block cellular uptake processes . We found that uptake of CPP - HA2 - tagged RFP involves energy - dependent endocytosis . This study with constructed CPP - containing plasmids reveals valuable mechanistic insights into how these CPPs cause endosomal escape and provides a basis for the design of optimized delivery agents . 2 . Materials and methods 2 . 1 . Plasmid construction The mCherry plasmid ( kindly provided by Dr . Roger Y . Tsien , University of California , San Diego , CA , USA ) is a prokaryotic expression vector that encodes a hexa - histidine ( 6His ) - tagged monomeric RFP sequence ( Table 1 ) [ 44 ] . The pR9 - mCherry plasmid containing a nona - arginine ( R9 , a CPP ) and HA tag ( YPYDVPDYA ) fused RFP coding region under the control of the T7 promoter was described previously [ 54 ] . This HA tag does not appear to interfere with the bioactivity of recombinant fusion proteins and facilitates the detection , isolation and purification of many protein fusions [ 47 ] . The pR9 - HA2 - mCherry plasmid consisting of an R9 and HA2 ( INF7 ) fused RFP coding sequence was constructed by the annealing and digestion of two overlapping primers ( HA2 - U [ 5 (cid:3) - TTAGATCTAGGCCTATTCGAGGCAATAGAAGGTTTCATAGAAAATGG - TTGGGA - 3 (cid:3) , with the Bgl II site underlined ] and HA2 - D [ 5 (cid:3) - TTGGATCCCCGTACCAACCGTCTATCATTCCCTCCCAACCATTTTCTAT - GAAA - 3 (cid:3) , with the Bam HI site underlined ] ) cloned into the Bam HI site of the pR9 - mCherry plasmid . The pR9 - NLS - mCherry plasmid containing an R9 and NLS fused RFP was generated using two overlapping 5 (cid:3) phosphorylated primers ( NLS - U [ 5 (cid:3) - GATCCCAAGAAGAAGAGGAAAGTC - 3 (cid:3) ] and NLS - D [ 5 (cid:3) - GATCGACTTTCCTCTTCTTCTTGG - 3 (cid:3) ] ) cloned into the Bam HI site of the pR9 - mCherry plasmid . The pR9 - HA2 - NLS - mCherry plasmid consisting of an R9 , HA2 and NLS fused RFP was constructed by cloning the NLS - U / D fragment at the Bam HI site of the pR9 - HA2 - mCherry plasmid . All constructs were confirmed by DNA sequencing . 2 . 2 . Protein purification and characterization Protein expression and purification were as described pre - viously with modifications [ 3 , 27 ] . All plasmid constructs were transformed into Escherichia coli KRX strain ( Promega , Madison , WI , USA ) . Bacteria were grown to OD 600 = 0 . 4 and then induced with 0 . 1 % ( w / v ) rhamnose ( Sigma - Aldrich , St . Louis , MO , USA ) overnight at 37 ◦ C . Expressed proteins were purified by metal chela - tion chromatography . The binding ( 5 mM imidazole , 0 . 5 M NaCl , 20 mM Tris – HCl , pH 7 . 9 ) and wash ( 80 mM imidazole , 0 . 5 M NaCl , 20 mM Tris – HCl , pH 7 . 9 ) buffers were used during purification . Purified proteins were concentrated as well as dialyzed using Ami - con Ultra - 4 centrifugal filter devices ( Millipore , Billerica , MA , USA ) and quantified by a protein assay kit ( Bio - Rad , Hercule , CA , USA ) . J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 275 Luminescent images of proteins were captured using the Typhoon FLA 9000 Biomolecular Imager ( GE Healthcare , Piscataway , NJ , USA ) with the excitation wavelength at 532 nm for RFPs [ 32 ] . Emission spectra of RFPs were evaluated using an EnSpire 2300 Multilabel Reader ( PerkinElmer , Waltham , MA , USA ) with excitation at 488 nm and emission varying from 510 to 700 nm . 2 . 3 . Protein transduction , subcellular colocalization and mechanistic assays Human lung carcinoma A549 cells ( American Type Culture Col - lection , Manassas , VA , USA ; CCL - 185 ) were cultured in Roswell Park Memorial Institute ( RPMI ) 1640 medium ( Gibco , Invitrogen , Carlsbad , CA , USA ) containing phenol red supplemented with 10 % ( v / v ) bovine serum ( Gibco ) , as previously described [ 54 ] . To deter - mine the optimal concentration for protein transduction , cells were seeded at a density of 1 × 10 5 cells per 35 - mm petri dish and then incubated overnight in 1 ml of growth medium . Cells were washed twice with 1 ml of phosphate buffered saline ( PBS ) and then treated with various proteins at the final concentrations of 1 , 5 , 10 , 30 and 60 (cid:3) M in RPMI 1640 medium with neither phenol red nor serum at 37 ◦ C for 1 h . Cells were washed five times with PBS to remove free proteins . To determine subcellular colocalization , cells were treated with 30 (cid:3) M of various proteins for different durations ( 30 min , 1 , 3 , 6 , 12 , 18 and 24 h ) in RPMI medium with neither phenol red nor serum at 37 ◦ C . Cells were washed with PBS for five times to remove free proteins followed by staining with fluorescent organelle - specific trackers [ 27 ] . Hoechst 33342 , LysoSensor Green DND - 153 ( Invit - rogen ) and fluorescein isothiocyanate ( FITC ) mouse anti - human early endosome antigen 1 protein ( EEA1 ) antibody ( BD Biosciences , Franklin Lakes , NJ , USA ) were utilized to visualize nuclei , lyso - somes and early endosomes , respectively . Fluorescent images were detected using a BD Pathway 435 System ( BD Biosciences ) . To conduct energy - dependent uptake experiments , cells were incubated for 30 min at 4 ◦ C at which temperature energy - dependent molecular movement in the cell membrane is essentially arrested [ 49 ] . Cells were then treated with 30 (cid:3) M of R9 - HA2 - mCherry at 4 ◦ C for 6 h followed by staining with Hoechst 33342 for 40 min and LysoSensor trackers for 30 min . The influence of modulators that inhibit or enhance uptake processes was studied in cells treated without or with cytochalasin D ( CytD ) , nocodazole [ 33 ] or chloroquine ( Sigma – Aldrich ) [ 60 ] . Cells were pretreated in the absence or presence of 10 (cid:3) M of CytD , 10 (cid:3) M of nocodazole or 25 (cid:3) M of chloroquine at 37 ◦ C for 30 min . These cells were then treated with 30 (cid:3) M of R9 - HA2 - mCherry in the absence or presence of CytD or nocodazole at 37 ◦ C for 6 h , or chloroquine for 12 h fol - lowed by staining with Hoechst 33342 and LysoSensor trackers . To test any synergistic effect of the HA2 and chloroquine combination [ 57 ] , cells were pretreated in the absence or presence of chloro - quine at 37 ◦ C for 30 min . These cells were then treated with 30 (cid:3) M of R9 - mCherry or R9 - HA2 - mCherry in the absence or presence of chloroquine for 12 h . To assess the CPP - HA2 - mediated cargo delivery , cells were treated with collagen - fluorescein ( Sigma – Aldrich ) [ 23 ] , R9 - HA2 - mCherry or R9 - HA2 - mCherry / collagen - fluorescein complexes at various ratios . Fluorescent images were detected using a BD Path - way 435 System ( BD Biosciences ) . 2 . 4 . Confocal and fluorescent microscopy Fluorescent and bright - field live cell images were recorded using a BD Pathway 435 System ( BD Biosciences ) equipped with the Olympus 20 × and 60 × oil objectives ( Olympus , Tokyo , Japan ) [ 27 ] . This system includes both confocal and fluorescent microscopy sets . Excitation filters were set at 377 / 50 nm , 482 / 35 nm and 543 / 22 nm for blue , green and red fluorescence , respectively . Emis - sion filters were set at 435LP ( long - pass ) , 536 / 40 nm and 593 / 40 nm for blue ( BFP ) , green fluorescent protein ( GFP ) and RFP channels , respectively . Transmitted light with the 536 / 40 nm emission filter was used to observe cell morphology as bright - field images . 2 . 5 . Flow cytometric analysis A549 cells were seeded at a density of 1 × 10 5 cells per well in 24 - well plates and then incubated overnight in 500 (cid:3) l / well of cul - ture medium . Cells were treated with five proteins for designated durations ( 1 , 5 , 10 , 30 , 60 min , 3 , 6 , 12 , 18 and 24 h ) in RPMI medium with neither phenol red nor serum at 37 ◦ C and then washed five times with PBS . Cells were analyzed using a Cytomics FC500 Flow Cytometer ( Beckman Coulter , Fullerton , CA , USA ) , as previously described [ 24 ] . For RFP detection , excitation was set at 488 nm and emission at 615 nm with a FL3 filter . Results are reported as the percentage of the total cell population . 2 . 6 . Cytotoxicity assay A549 cells were plated at a density of 1 × 10 4 cells in 96 - well plates and incubated overnight in 200 (cid:3) l / well of growth medium . Cells were treated with 1 , 5 , 10 , 30 and 60 (cid:3) M of five proteins in RPMI medium without phenol red nor serum , washed with PBS , and cultured in RPMI medium without serum at 37 ◦ C for 24 h . Cells were treated with PBS as a negative control and 70 % alcohol as a positive control . Cytotoxicity was assessed by the ability of the cells to reduce 1 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 3 , 5 - diphenylformazan ( MTT ) [ 23 , 54 ] . MTT absorbance was measured at 570 nm wave - length using a Model 680 Microplate Reader ( Bio - Rad ) . 2 . 7 . Statistical analysis Data are expressed as mean ± standard deviation . Mean val - ues and standard deviations were calculated from at least three independent experiments carried out in triplicates in each group . Statistical comparisons between the control and treated groups were performed by the Student t - test , using levels of statistical significance of P < 0 . 05 ( * , † , ‡ ) and 0 . 01 ( * * , †† , ‡‡ ) , as indicated . 3 . Results 3 . 1 . RFP - fusion protein analysis To examine the endosomolytic effect of HA2 , we constructed several CPP - containing RFP expression plasmids ( Fig . 1 ) . The mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry , and R9 - HA2 - NLS - mCherry proteins were overexpressed and purified in E . coli KRX strain transformed with the relevant plasmids . These plasmids produced 6His - tagged RFP alone or in - frame fused RFPs with a combination of R9 , HA2 and NLS ( Table 1 ) under the control of the T7 promoter . SDS - PAGE analysis of purified mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry and R9 - HA2 - NLS - mCherry fusion proteins was detected by a luminescent imager ( Fig . 2A ) or by staining with Coomassie brilliant blue ( Fig . 2B ) . The five purified recombinant RFPs have calculated molec - ular masses of 30 . 6 – 36 . 4 kDa . Luminescent scan revealed emission spectra of the fluorescent RFPs ( Fig . 3 ) . All RFPs had maximal emis - sion at about 610 nm upon excitation at 488 nm , indicating that the fluorescent property of the RFP - fusion proteins is not compromised by the addition of peptide tags to the original RFP . 276 J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 D pR9 - NLS - mCherry 6His T7 R9 mCher ry NLS C pR9 - HA 2 - mCherr y 6His T7 R9 mC her ry HA2 B pR9 - m Cherry 6His T7 R9 mC her ry A mCherry 6His T7 mCherry E pR9 - HA2 - NLS - mChe rry 6His T7 R9 mCher ry NLS HA2 Fig . 1 . Schematic structure of DNA plasmids . ( A ) The mCherry plasmid . This is the original bacterial expression cassette containing the coding region of a hexa - histidine ( 6His ) - tagged monomeric RFP under the control of the T7 promoter . ( B ) The pR9 - mCherry plasmid . This plasmid contains a nona - arginine ( R9 ) tagged RFP coding sequence . ( C ) The pR9 - HA2 - mCherry plasmid . This plasmid contains an R9 and HA2 ( INF7 ) tagged RFP coding sequence . ( D ) The pR9 - NLS - mCherry plasmid . This plasmid contains an R9 and nuclear localization signal ( NLS ) tagged RFP cod - ing sequence . ( E ) The pR9 - HA2 - NLS - mCherry plasmid . This plasmid contains an R9 , HA2 and NLS tagged RFP coding sequence . 3 . 2 . Protein transduction efficiency of RFPs To determine the optimal concentration for protein transduc - tion , human A549 cells were treated with various concentrations of mCherry , R9 - mCherry or R9 - HA2 - mCherry proteins for 1 h . No flu - orescent signal was detected by flow cytometric analysis in the cells treated with mCherry ( Fig . 4 ) . Red fluorescent signal was observed in the cells treated with R9 - mCherry at the concentration of 60 (cid:3) M , which is consistent with our previous results [ 2 , 24 , 34 , 54 ] . Remark - ably , the cells treated with ≥ 1 (cid:3) M of R9 - HA2 - mCherry exhibited significantly higher fluorescence intensity than those treated with equivalent concentrations of mCherry or R9 - mCherry . The inten - sity of the fluorescent signal in R9 - HA2 - mCherry treated cells was concentration - dependent . Measurements of protein transduction were obtained by confocal imaging . A549 cells were treated with a series of concentrations of mCherry ( Fig . 5A ) , R9 - mCherry ( Fig . 5B ) or R9 - HA2 - mCherry ( Fig . 5C ) proteins for 30 min , stained with Hoechst 33342 and then imaged . The imaging data were in good accordance with the above results of flow cytometric analysis , con - firming that HA2 tag is able to remarkably increase CPP - mediated protein transduction activity . After 30 min , R9 - HA2 - mCherry was primarily located around the inner perimembraneous area . Accord - ing to Figures 4 and 5 , 30 (cid:3) M of fluorescent proteins were chosen as the optimal concentration for subsequent experiments due to more clear and consistent images . 3 . 3 . Time course analysis of CPP - HA2 - mediated protein transduction To understand the kinetics of protein transduction , cells were treated with mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry or R9 - HA2 - NLS - mCherry fluorescent proteins , and cellular uptake of proteins was measured at various time points by flow cytometry . The cells treated with R9 - HA2 - mCherry at 5 min showed higher red fluorescent intensity than the cells treated with the other four proteins ( Fig . 6A ) . The fraction of the Fig . 2 . SDS - PAGE analysis . ( A ) Luminescent photography . ( B ) Coomassie brilliant blue stain . Purified mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry and R9 - HA2 - NLS - mCherry proteins are in lanes 1 – 5 , respectively . Red fluorescent ( F3401 , Fluorescent Low Molecular Weight Markers , Sigma ) and standard protein markers ( SM0671 , Fermentas , Glen Bumie , MD , USA ) are displayed on the left and right lanes M , respectively . ( For interpretation of the references to color in this figure legend , the reader is referred to the web version of the article . ) Fig . 3 . Luminescent emission spectra of RFPs . Five purified mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry and R9 - HA2 - NLS - mCherry proteins were eval - uated by divergent emission scan for their optical absorption using an EnSpire 2300 Multilabel Reader ( PerkinElmer ) . J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 277 Fig . 4 . Flow cytometric analysis of protein transduction by RFPs . A549 cells were treated with 1 , 5 , 10 , 30 and 60 (cid:3) M of mCherry , R9 - mCherry or R9 - HA2 - mCherry for 1 h and analyzed using a Cytomics FC500 flow cytometer ( Beckman Coulter ) . Data are presented as mean ± standard deviation from three independent experiments . Significant differences were determined at P < 0 . 05 ( * ) and P < 0 . 01 ( * * ) . Fig . 5 . Confocal microscopy of protein transduction of RFPs . Cells were treated with 1 , 5 , 10 , 30 and 60 (cid:3) M of mCherry ( A ) , R9 - mCherry ( B ) or R9 - HA2 - mCherry ( C ) for 30 min and stained with Hoechst 33342 . Images were recorded using a BD pathway system at a magnification of 200 × . RFP and BFP channels in the microscope are used to reveal the distribution of fluorescent proteins and nuclei , respectively . Overlap between fluorescent proteins and nuclei exhibits purple color in merged RFP and BFP images . 278 J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 Fig . 6 . Time course analysis of CPP - HA2 - mediated protein transduction . Cells were treated with 30 (cid:3) M of mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry or R9 - HA2 - NLS - mCherry for short ( 1 , 5 , 10 , 30 and 60 min ) ( A ) and long ( 1 , 3 , 6 , 12 , 18 and 24 h ) ( B ) periods of time and then analyzed by a flow cytometer . Data are presented as mean ± standard deviation from three independent experiments . R9 - HA2 - mCherry treated cells with fluorescence continued to increase over time . At 1 h , the R9 - HA2 - mCherry group showed 80 times higher protein uptake than the other groups . When the time point was extended to 18 h , the uptake of R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry and R9 - HA2 - NLS - mCherry fluorescent proteins reached about 90 % of all cells ( Fig . 6B ) . These results confirm that HA2 peptide accelerates endosomolytic activity to increase CPP - mediated protein transduction . Moreover , NLS tags seemed to inhibit HA2 enhancement of protein transduction . To study the endosomolytic ability of HA2 , cells were treated with mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry or R9 - HA2 - NLS - mCherry proteins for 1 , 3 , 6 , 12 , 18 and 24 h , stained with organelle - specific green fluorescent markers and then analyzed by confocal microscopy . No red fluorescence could be detected in the cells treated with mCherry at any time ( Fig . 7A ) . In contrast , the cells treated with R9 - mCherry ( Fig . 7B ) and R9 - NLS - mCherry ( data not shown ) proteins exhibited a time - dependent increase in red fluorescence in the cytoplasm , a few yellow / orangey spots in lysosomes but not the nuclei . Strikingly , the cells treated with R9 - HA2 - mCherry displayed red fluorescence in plasma mem - brane at 1 h , around the inner perimembraneous area at 3 h and throughout the cytosol after 6 h ( Fig . 7C ) . Results from the cells treated with R9 - HA2 - NLS - mCherry ( data not shown ) were simi - lar to those of R9 - HA2 - mCherry treatment , although the uptake was lower . Further , we noticed some yellow / orangey punctate for - mations within the cells treated with R9 - HA2 - mCherry after 6 h ( Fig . 7C and D ) and R9 - HA2 - NLS - mCherry after 12 h ( Fig . 7D ) , sug - gesting that these proteins were associated with lysosomes and endosomes . Together , these results suggest that the pathway of cel - lular internalization of R9 - HA2 - mCherry and R9 - HA2 - NLSmCherry involves endocytosis . The fusogenic HA2 peptide dramatically enhanced CPP - mediated protein transduction apparently by help - ing the escape of RFPs from endosomes into the cytoplasm . 3 . 4 . Energy - dependent mechanism of CPP - HA2 - mediated protein transduction To reveal the mechanistic aspect of R9 - HA2 - mCherry internal - ization , cells were treated with R9 - HA2 - mCherry at 4 ◦ C or 37 ◦ C ( as a control ) for 6 h ( Fig . 8A ) . Low temperature incubation inhib - ited cellular uptake of R9 - HA2 - mCherry ( Fig . 8A and B ) . These data are in agreement with the results in Fig . 7C and suggest that pro - tein transduction of R9 - HA2 - mCherry involves energy - dependent endocytosis . To examine the molecular aspect of endocytic processes , cells were treated with R9 - mCherry or R9 - HA2 - mCherry in the absence or presence of CytD , nocodazole or chloroquine . Both endo - cytic inhibitors , CytD and nocodazole , disrupted cellular uptake of R9 - HA2 - mCherry ( Fig . 8B ) . The lysosomotropic agent chloro - quine further enhanced the cellular uptake of R9 - HA2 - mCherry compared to that of R9 - mCherry ( Fig . 8C and D ) . However , the com - bination of chloroquine and HA2 fusion peptide did not produce a synergistic increase of protein transduction ( Fig . 8D ) . 3 . 5 . MTT - based cell viability assay To assess cytotoxicity caused by CPPs tagged with HA2 and / or NLS in eukaryotes , A549 cells were treated with mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry or R9 - HA2 - NLS - mCherry proteins for 24 h and then analyzed by the MTT reduction assay ( Fig . 9 ) . Cells treated with PBS and 70 % alcohol were used as negative and positive controls , respectively . Cells treated with 1 , 5 , 10 , 30 and 60 (cid:3) M of any of RFP - fusion proteins did not display cytotoxicity . 3 . 6 . CPP - HA2 - based cargo delivery To assess that the CPP - HA2 - mediated protein transduction is applicable in cargo delivery , collagen - fluorescein previously used as a fluorescence - labeled protein cargo in our transdermal study [ 23 ] was served as one of examples . A549 cells were treated with collagen - fluorescein alone , R9 - HA2 - mCherry or R9 - HA2 - mCherry / collagen - fluorescein complexes and analyzed by confocal microscopy . No green fluorescence could be detected in the cells treated with collagen - fluorescein ( Fig . 10 ) . In contrast , the cells treated with R9 - HA2 - mCherry and R9 - HA2 - mCherry / collagen - fluorescein complexes in different ratios displayed red and red / green fluorescence , respectively . These results demonstrated J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 279 Fig . 7 . Subcellular colocalization analysis of CPP - HA2 - mediated protein transduction . Cells were treated with 30 (cid:3) M of mCherry for 24 h ( A ) , or R9 - mCherry ( B ) or R9 - HA2 - mCherry ( C ) for 1 , 3 , 6 , 12 , 18 and 24 h and then stained with Hoechst 33342 and LysoSensor Green DND - 153 . Cells were treated with 30 (cid:3) M of R9 - HA2 - mCherry for 6 h , R9 - mCherry or R9 - HA2 - NLS - mCherry for 12 h and then stained with Hoechst 33342 and FITC - labeled anti - EEA1 antibody ( D ) . Images were recorded with a BD pathway system at a magnification of 600 × . BFP ( blue ) , GFP ( green ) and RFP ( red ) channels in the microscope are used to reveal the distribution of nuclei , lysosomes / endosomes and R9 - HA2 - mCherry , respectively . Overlap between R9 - HA2 - mCherry ( RFP channel ) and nuclei ( BFP channel ) exhibits purple color in merged RFP and BFP images . Overlap between R9 - HA2 - mCherry ( RFP channel ) and lysosomes / endosomes ( GFP channel ) exhibits yellow color in merged RFP and GFP images . ( For interpretation of the references to color in this figure legend , the reader is referred to the web version of the article . ) 280 J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 Fig . 7 . ( Continued ) . J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 281 Fig . 8 . Effects of endocytic modulators on CPP - HA2 - mediated protein transduction . ( A ) Cells were treated with 30 (cid:3) M of R9 - HA2 - mCherry in the absence ( control ) or presence of low temperature ( 4 ◦ C ) , CytD , or nocodazole followed by staining with Hoechst 33342 and LysoSensor Green DND - 153 trackers . ( B ) Data of cells treated with R9 - HA2 - mCherry in the absence or presence of endocytic inhibitors are presented as mean ± standard deviation from three independent experiments . ( C ) Cells were treated with R9 - mCherry or R9 - HA2 - mCherry in the absence ( – ) or presence ( + ) of chloroquine , followed by staining with Hoechst and LysoSensor trackers . Images were recorded using a BD pathway system at a magnification of 600 × . BFP ( blue ) , GFP ( green ) and RFP ( red ) channels in the microscope are used to reveal the distribution of nuclei , lysosomes and R9 - HA2 - mCherry , respectively . ( D ) Cells were treated with R9 - mCherry or R9 - HA2 - mCherry in the absence or presence of chloroquine . Data are presented as mean ± standard deviation from three independent experiments . Significant differences were set at P < 0 . 05 ( * ) and P < 0 . 01 ( * * ) between R9 - mCherry ( control ) and others , P < 0 . 05 ( † ) and P < 0 . 01 ( †† ) between R9 - mCherry / chloroquine ( control ) and R9 - HA2 - mCherry / chloroquine , or P < 0 . 05 ( ‡ ) and P < 0 . 01 ( ‡‡ ) between R9 - HA2 - mCherry ( control ) and R9 - HA2 - mCherry / chloroquine . ( For interpretation of the references to color in this figure legend , the reader is referred to the web version of the article . ) Fig . 9 . MTT - based cell viability assay . A549 cells were treated with 1 , 5 , 10 , 30 and 60 (cid:3) M of mCherry , R9 - mCherry , R9 - HA2 - mCherry , R9 - NLS - mCherry or R9 - HA2 - NLS - mCherry for 24 h . Cells treated with 70 % alcohol ( EtOH ) and PBS ( control ) served as positive and negative controls , respectively . Cell viability was determined by the ability of the cells to reduce MTT . Data are presented as mean ± standard deviation from three independent experiments . Significant differences were determined at P < 0 . 05 ( * ) and P < 0 . 01 ( * * ) . 282 J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 Fig . 10 . CPP - HA2 - based cargo delivery . A549 cells were treated with collagen - fluorescein alone , R9 - HA2 - mCherry or R9 - HA2 - mCherry / collagen - fluorescein complexes in ratios of 3 / 1 and 6 / 1 . Images were recorded using a BD pathway system at a magnification of 200 × . RFP and GFP channels in the microscope are used to reveal the distribution of CPP - HA2 fusion protein and collagen - fluorescein , respectively . Overlap between R9 - HA2 - mCherry ( RFP channel ) and collagen - fluorescein ( GFP channel ) exhibits yellow color in merged RFP and GFP images . that the CPP - HA2 - mediated protein transduction system is indeed applicable in cargo delivery into cells . 4 . Discussion In this report , we demonstrate that the endosomolytic HA2 tag increases cellular uptake , accelerates endosomal escape and pro - motes even cytosolic distribution of endocytosed CPP - containing RFPs in human A549 cells . We constructed a series of plasmids con - taining coding sequences of CPP , HA2 and / or NLS fused RFP . These plasmids were expressed in bacteria , and the uptake of the puri - fied proteins was measured in A549 cells . Live cell imaging and flow cytometry revealed mechanistic details of cellular uptake and endosomolytic activity . Our results indicate that ( 1 ) endocytosis is the major route for cellular uptake of CPP - HA2 - tagged RFP , ( 2 ) the endosomolytic HA2 peptide promotes the escape of RFPs from endosomes into the cytoplasm and ( 3 ) incorporating the HA2 fusion peptide of the CPP - HA2 fusion protein improves cytosolic uptake . Recent studies indicated that poor intracellular trafficking and limited endosomal release are major factors reducing the efficiency of protein transduction [ 11 , 35 , 43 , 48 , 51 ] . CPP - cargo trapped in endosomes or macropinosomes can be released into the cytosol by membrane perturbation , such as that induced by the INF7 peptide ( a glutamic acid - enriched HA2 analog ) [ 13 , 18 , 28 , 36 , 45 , 46 , 61 , 62 ] . Contrarily , a recent report showed that the glutamate - rich HA2 analog E5 tags can lyse endosomes , while HA2 - protein ( E5 - TAT - mCherry ) conjugates may remain associated with lysed endosomes [ 30 ] . In this case , HA2 causes the retention of its fused protein inside endosomes without the diffusion into the cytoplasm even after endosomal lysis takes place [ 30 ] . Our results show that R9 - HA2 - mCherry has the highest protein uptake efficiency among five RFPs examined ( Figs . 4 – 6 ) . Subcellular colocalization and mechanistic studies of CPP - mediated protein internalization indicate that the HA2 tag promotes the escape of RFPs from endosomes into the cyto - plasm ( Figs . 7 and 8 ) . Together , we conclude that the incorporation of fusogenic HA2 facilitates endosomal disruption and increases cellular internalization of R9 - HA2 - mCherry . Studies have used the vacuolating toxin VacA to depict a typical endocytosis timeline . The process requires dynamic F - actin struc - tures on early endosomes for trafficking to late endosomes in HeLa cells [ 19 , 20 ] . The pinocytosis of VacA involves four steps : ( 1 ) bind - ing of VacA to the cell by a Rac - dependent process , ( 2 ) accumulation of VacA into early endosomes within 10 min in a Cdc42 - dependent nonmacropinocytic manner , ( 3 ) enrichment of VacA in the early endosomes within 30 min and ( 4 ) transfer of VacA from early to late endosomes within 120 min . According to reports on VacA and related compounds [ 15 , 19 , 20 , 22 ] , our studies of kinetics , molecu - lar mechanisms of uptake and subcellular localization suggest that endosomal disruption caused by HA2 of R9 - HA2 - mCherry occurs within 0 . 5 – 2 h ( Figs . 6 – 8 ) . Our study informed quick endosomal release of R9 - HA2 - mCherry compared to the other four proteins ( Fig . 6 ) . Rapid escape from endosomes may preserve biological activity of delivered cargoes . A report of N - stearylated NLS sug - gested an enhancement of the CPP - mediated transfection activity by overcoming limitations of cell membrane and nuclear pores [ 53 ] . Contrarily , our limited data from the constructs with the NLS tag seemed to suggest a possible interference of the NLS tag with the HA2 - enhanced protein transduction at the relatively early time points . The details of this NLS interference remained to be eluci - dated . Despite the broad acceptance of CPPs as molecular carriers , the cellular uptake mechanism of CPPs and CPP - cargo is still under vigorous investigation [ 35 , 37 , 43 , 48 ] . The internalization of R9 - HA2 - mCherry was inhibited by incubation at 4 ◦ C and by CytD as well as nocodazole , indicating that the intracellular delivery of R9 - HA2 - mCherry involves energy - dependent endocytosis ( Fig . 8B ) . Chloroquine , a lysosomotropic agent that prevents endosomal acidification and the degradation by lysosomal enzymes , is often used to improve the efficiency of the gene delivery in laboratory studies [ 37 , 60 ] . The combination of chloroquine and INF7 - SGSC peptide caused a synergistic increase of polylysine - mediated DNA J . - S . Liou et al . / Peptides 37 ( 2012 ) 273 – 284 283 transfection activity in human embryonic kidney ( HEK ) 293 cells [ 57 ] . Our data indicate that chloroquine increases the cellular uptake of R9 - HA2 - mCherry ( Fig . 8C and D ) in a non - synergistic manner . CPPs are very attractive , noncytotoxic candidates for affect - ing the delivery of therapeutic macromolecules , such as proteins and nucleic acids [ 12 , 42 , 48 , 51 ] . The safety of most CPPs has been confirmed in a detailed metabolic analysis [ 26 ] . Our data demon - strate that CPPs do not compromise membrane integrity nor result in cytotoxicity of A549 cells as indicated by trypan blue [ 1 , 2 , 4 , 8 , 23 , 54 , 55 ] , MTT [ 5 , 23 , 55 ] , MTS [ 59 ] and sulforhodamine B ( SRB ) [ 24 , 27 , 32 , 33 ] assays . Previous studies used CPPs at 0 . 1 (cid:3) M for 2 h [ 36 ] , 1 – 20 (cid:3) M for 3 – 24 h [ 28 , 62 ] , 20 – 50 (cid:3) M for 1 – 2 h [ 38 ] or 0 – 100 (cid:3) M for 6 – 24 h [ 45 , 46 ] for fusion protein treat - ment . Further , the usages of CPP concentrations at or less than 100 (cid:3) M did not cause cytotoxicity [ 25 ] . Together , these findings suggest the HA2 fusion peptide as an effective and safe trig - ger to enhance protein transduction activity . However , the HA2 fusion peptide located at the N - terminus of CPP - fusion proteins has been reported to be cytotoxic [ 28 , 38 , 45 , 62 ] . HA2 - TAT [ 45 , 62 ] , E5 - TAT [ 28 ] ( 5 (cid:3) M ) and N - E5L [ 38 ] ( 100 (cid:3) M ) caused cytotoxic - ity , while HA2 - p53 - R9 [ 36 ] ( 0 . 1 (cid:3) M ) and stearylated INF7 [ 13 ] did not . Further , chemically synthesized HA2 - R9 peptide with HA2 at the N - terminus exerted a certain degree of cytotoxicity at high concentrations in eukaryotes ( manuscript in preparation ) . Due to data limitation , the true effect of HA2 located at either N - or C - terminus of fusion protein remains unknown . It is impor - tant to note that the E5 - TAT - mCherry construct was successfully expressed in E . coli by blocking the N - terminus of the fusion pro - tein with a cleavable SUMO ( small ubiquitin - related modifier ) tag [ 30 ] . This SUMO tag can be removed by the SUMO protease to obtain an N - terminal glycine residue of the E5 sequence after purification of E5 - TAT - mCherry protein . We therefore conjectured that protein with HA2 tagged at the extreme N - terminus may be membrane - lytic or membrane - active [ 36 , 52 ] . It is possible that strong lytic ( including endosomolytic ) activities of HA2 have been reduced by addition of tags to the N - terminus of HA2 . This is a big advantage of our CPP - HA2 - tagged proteins with increased plasma membrane association and possibly greater endocytic uptake , because other HA2 - fusion proteins have shown much higher toxicity [ 28 , 38 , 45 , 62 ] . In order to ascertain whether RFP - fusion proteins cause cytotox - icity during protein transduction , we performed the MTT toxicity test in human A549 cells ( Fig . 9 ) . None to very minimal cytotox - icity was observed with any of the five RFPs at concentrations up to 30 (cid:3) M . When collagen - fluorescein was used as a cargo , our results demonstrated that the CPP - HA2 - mediated protein trans - duction system is applicable in cargo delivery into cells ( Fig . 10 ) . These results suggest that CPP tagged with a HA2 fusion peptide at the C - terminus is a relatively safe design for protein transduction promoting agents . 5 . Conclusion Endocytosis is the major route for cellular uptake of CPP - HA2 - tagged RFP . The fusogenic HA2 tag facilitates the release of endocytosed RFPs from endosomes into the cytoplasm resulting in a diffuse cytosolic distribution . Remarkably , incor - porating the HA2 fusion peptide of the CPP - HA2 fusion protein improved cytosolic uptake without causing cytotoxicity . R9 - HA2 - mCherry was capable of delivering collagen - fluorescein into cells . Collectively , these results indicate that the CPP - HA2 tag could be an efficient and safe carrier of biologically active molecules . Acknowledgements We thank Dr . Roger Y . Tsien for provision of the mCherry plas - mid . We are grateful to Dr . Robert S . Aronstam ( Missouri University of Science and Technology , USA ) for editing the manuscript . This work was supported by Award Number R15EB009530 from the National Institutes of Health ( Y . - W . H . ) , Postdoctoral Fellowship NSC 101 - 2811 - B - 259 - 001 ( B . R . L . ) and Grant Number NSC 101 - 2320 - B - 259 - 002 - MY3 from the National Science Council of Taiwan ( H . - J . L . ) . References [ 1 ] Chang M , Chou JC , Lee HJ . Cellular internalization of fluorescent proteins via arginine - rich intracellular delivery peptide in plant cells . Plant Cell Physiol 2005 ; 46 : 482 – 8 . [ 2 ] Chang M , Chou JC , Chen CP , Liu BR , Lee HJ . Noncovalent protein transduction in plant cells by macropinocytosis . New Phytol 2007 ; 174 : 46 – 56 . [ 3 ] Chang M , Hsu HY , Lee HJ . Dye - free protein molecular weight markers . Elec - trophoresis 2005 ; 26 : 3062 – 8 . 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Organelle - targeted delivery of biological macromolecules using the protein transduction domain : potential applications for peptide aptamer delivery into the nucleus . J Mol Biol 2008 ; 380 : 777 – 82 . \ No newline at end of file diff --git a/silver_data/2d5431c5e0402971a6284ce39e1e605a14a41efd.pdf.txt b/silver_data/2d5431c5e0402971a6284ce39e1e605a14a41efd.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..928f26674eb9f2dd181ec948fae11d3500011907 --- /dev/null +++ b/silver_data/2d5431c5e0402971a6284ce39e1e605a14a41efd.pdf.txt @@ -0,0 +1 @@ +Electronic copy available at : http : / / ssrn . com / abstract = 2480861 An Ounce of Common Sense Is Worth a Pound of Theory Ernest N . Biktimirov * Faculty of Business Brock University 500 Glenridge Ave St . Catharines , Ontario , Canada L2S 3A1 905 . 688 . 5550 x 3843 ebiktimirov @ brocku . ca Published in Advances in Financial Education 1 , Fall 2003 , 1 - 12 Citation information : Biktimirov E . N . ( 2003 ) . “An ounce of common sense is worth a pound of theory , ” Advances in Financial Education 1 , Fall , 1 - 12 . ABSTRACT This paper proposes the use of proverbs in the teaching of finance . Familiar proverbs facilitate teaching by appealing to students’ prior knowledge , by presenting ideas in a concise and original way , and by making the learning process more enjoyable and efficient . The paper presents more than five dozen proverbs accompanied by their related financial concepts , which are organized into twelve thematic groups . Strategies for the effective use of proverbs in the classroom are suggested as well . * I thank Barbara Sainty , Robert Welch , and an anonymous referee for helpful comments . Electronic copy available at : http : / / ssrn . com / abstract = 2480861 2 An Ounce of Common Sense Is Worth a Pound of Theory INTRODUCTION Teaching can be more efficient when an instructor can relate new concepts to the existing experience of students . Students will also retain the new material better if it is presented concisely , from an original point of view , and in an entertaining way . This paper proposes the use of proverbs in the teaching of finance . The introduction of proverbs into financial instruction can “kill several birds with one stone . ” First , familiar proverbs will appeal to students’ prior knowledge and will help them to understand abstract financial concepts easier . Second , using proverbs to present ideas in a concise and original way will serve as an efficient explanation tool and will improve retention . Third , proverbs with their wit , humor , and original point of view will bring entertainment and excitement into the classroom , making the learning process more enjoyable and efficient . Instructors have offered analogies , metaphors , and examples to explain financial concepts . For instance , Forbes [ 1991 ] suggests using relations between American and Canadian dollars to illustrate the time value of money concept . Mann [ 1996 ] uses air conditioners and heaters to explain calls and puts . Gilkeson and Lamb [ 2000 ] propose using the baseball card and Beanie Baby collectibles markets to describe equity and emerging markets , respectively . Finally , Kane [ 1999 ] shows how examples from popular music can be used to teach principal - agent problems . Instructors have also addressed the incorporation of humor in the teaching of finance to make education more attractive . For example , Becker [ 1993 ] suggests using a David Letterman type “Top Ten List” in a finance class , and Ardalan [ 1998 ] demonstrates the use of entertaining metaphors in an introductory finance course . Taken together , an examination of financial education literature shows a continual search for new ways to teach finance . This paper contributes to this literature by offering the use of proverbs as an innovative teaching tool to relate financial concepts to students’ prior knowledge . Proverbs also present complex concepts in a concise and original way and bring excitement to the classroom . 3 The paper is organized as follows . The next section describes the teaching benefits of proverbs , presents the results of empirical research , and suggests strategies for the effective use of proverbs in the classroom . The following section contains proverbs , accompanied by the appropriate financial concept they illustrate , and classifies them into twelve subsections . The final section provides a summary and conclusions . PROVERBS AS A TEACHING TOOL The Oxford English Dictionary defines a proverb as “a concise sentence , often metaphorical or alliterative in form , which is held to express some truth ascertained by experience or observation and familiar to all . ” Being a colorful part of any language , proverbs have been extensively applied in the teaching of English and other languages . The use of proverbs has been limited outside of language instruction . The few examples of applying proverbs in other areas of teaching include the use proverbs as a tool to promote reflection among science teachers [ Nichols , Tippins and Wieseman , 1997 ] and a quiz on the chemical interpretation of proverbs [ Ibanez , 2002 ] . Proverbs , however , can provide a number of teaching benefits , which are discussed in the next subsection . Teaching Benefits of Proverbs This subsection uses several examples to illustrate the teaching benefits of proverbs . Suppose an instructor needs to explain the principle of diversification . To illustrate this concept , an instructor can use the proverb “don’t put all your eggs in one basket . ” The sense of diversification by the spreading of investments across several assets is clearly conveyed by the proverb . Using this proverb will help an instructor to achieve a number of teaching objectives . First , this well - known proverb , which advises to not risk everything on one venture , will appeal to students’ prior knowledge and will help them to relate the concept of diversification to their life experience . Second , a concise and familiar proverb associated with a financial concept facilitates the retention of the concept by students . Also , the terse and pithy nature of proverbs makes them an indispensable explanation tool that can summarize the main idea of a half - hour lecture . Finally , the proverb makes the learning process more exciting by providing an original 4 point of view on an otherwise abstract financial principle . The metaphorical analogy provided by the proverb spices up the material . The advantage of presenting a financial concept from a different perspective , or by using an interesting analogy , can be especially beneficial when students have problems understanding that concept . For example , students frequently have difficulty accepting that costs that occurred in the past and cannot be recovered ( sunk costs ) should be ignored in the capital budgeting analysis . To clarify this concept for students , an instructor can illustrate it with the proverb “it’s no use crying over spilled milk . ” This familiar expression will present the concept of sunk costs from a different point of view , one that may be more easily understood by students . Students appreciate it when instructors explain new concepts in an original way . For instance , Csikszentmihalyi and McCormack [ 1995 , p . 6 ] present a student’s description of exemplary teaching : “Mr . C . is such a fantastic teacher because he has a special way of thinking that catches your attention . He makes brains go , he makes brains think , and he says things in a way that you just can’t forget them . ” The use of proverbs can serve as an indispensable teaching tool for capturing students’ attention and conveying new ideas in a unique and memorable way . The following subsection provides support for the teaching benefits of proverbs based on the results of empirical educational research . Empirical Educational Research A large body of literature examines the effect of analogies , metaphors and humor on learning . The study by Moreno and Di Vesta ( 1994 ) is especially of interest here because it examines the effect of proverbs , which are referred to as adages in their paper , on learning . Specifically , the authors analyze the effect of proverbs on college students’ recall and text comprehension . They compare the learning outcomes of the proverb titles , chosen to represent the main idea of each passage , with the learning effects of general titles . For example , they study the effect of the proverb title “A bird in the hand is worth two in the bush” compared to the impact of the general title “The effects of delay of gratification , ” and the proverb heading “A stitch in time saves nine” is contrasted with the general heading “Some theories on timing of experiences in development . ” 5 Moreno and Di Vesta ( 1994 ) find that proverbs facilitate comprehension and retention of new material . Moreover , the elaboration of a proverb and the explanation of its relation to the topic further enhanced the learning benefits . While mixed results have been found , a growing body of research supports the view that college students benefit from the use of analogies in the classroom . For example , Halpern , Hansen and Riefer [ 1990 ] and Newby , Ertmer and Stepich [ 1995 ] find that analogies have a positive impact on college students’ comprehension and retention . Baker and Lawson [ 2001 ] report not only an improvement in student comprehension , but also positive student evaluations of analogy - based instruction . The empirical research on the role of metaphors in learning has produced similar results . For instance , Pearson , Raphael , TePaske and Hyser [ 1981 ] and Reynolds and Schwartz [ 1983 ] show that the use of metaphors facilitates student recall of new information . Certain proverbs also have a dash of humor in them , which assists the learning process and makes it more enjoyable . Researchers have found a positive relationship between teachers’ use of humor and learning [ Ziv , 1988 ] , student perceptions of their learning [ Gorham and Christophel , 1990 ; Wanzer and Frymier , 1999 ] , and teacher evaluations [ Bryant , Comisky , Crane and Zillmann , 1980 ] . In addition , some studies also show that humor that is relevant to the lecture material facilitates retention [ Kaplan and Pascoe , 1977 ; Desberg , Henschel , Marhsall and McGhee , 1981 ] . Furthermore , researchers also argue that humor promotes a more enjoyable classroom environment [ Korobkin , 1988 ; Neuliep , 1991 ; Desberg et al . , 1981 ] . Taken together , empirical educational research provides evidence that innovative teaching tools , such as proverbs , analogies , metaphors , and humor , facilitate comprehension and retention of new material , and make the learning process more enjoyable . The simple addition of proverbs into instruction , however , may have only a marginal impact or no effect at all . To achieve all the teaching benefits of using proverbs , it is necessary to follow several teaching strategies that are discussed in the next subsection . 6 Teaching Strategies for Using Proverbs in the Classroom In this subsection , I present several teaching strategies for the effective use of proverbs in the classroom . These strategies originate from my personal teaching experience and from the recommendations of educational literature . The first and most important prerequisite for the successful use of proverbs in teaching is the students’ understanding of the meaning of proverbs and their relations to financial concepts . If students do not understand the interpretation of a proverb , they will not be able to relate it to their prior knowledge and to a new financial topic . This requirement becomes especially important given a growing number of foreign students enrolled in finance courses who may not be familiar with many English proverbs . Explanation of proverbs will not only help their understanding , but will also facilitate their recall [ Honeck , 1973 ; Honeck , Riechmann and Hoffman , 1975 ] . Also , even if students recognize a proverb , the instructor has to ensure that they can establish connections between the proverb and its financial concept . As mentioned earlier , the elaboration of a proverb and the explanation of its relation to new material improve comprehension and retention compared to using proverbs without any interpretation [ Moreno and Di Vesta , 1994 ] . Second , I suggest starting the explanation of a new financial concept with a proverb and then continuing it with the discussion of the concept itself . In this case , students will have a point of reference to which they will be able to relate the new material . For example , an instructor can begin a lecture on the risk - return tradeoff with the proverb “nothing ventured , nothing gained . ” This familiar proverb , which implies that higher expected returns are associated with larger risks , will appeal to students’ prior knowledge and will help them to follow the instructor’s explanation . Third , I recommend using all relevant proverbs when more than one proverb can illustrate the same financial concept . For example , both “a door must be shut or open” and “you cannot have your cake and eat it too” can be used to explain to students the concept of mutually exclusive alternatives . If students are not familiar with a proverb , have difficulty with connecting the proverb to a financial concept , or the proverb does not appeal to their prior knowledge , a second proverb may offer another perspective and , therefore , avoid these problems . Dupin and 7 Johsua [ 1989 ] and Spiro , Feltovich , Coulson , and Anderson [ 1989 ] provide the same recommendation based on their research of the use of analogies for instruction , and Cameron [ 2002 ] offers similar advice based on her study of the use of metaphor in learning science . Anecdotally , I have followed these teaching strategies and have had a positive experience using proverbs in my finance courses . Each proverb attracted students’ attention , added a new dimension to the discussion of a financial concept and created a friendlier atmosphere in the classroom . On many occasions students told me that proverbs helped them grasp the essence of financial concepts . PROVERBS AND THEIR FINANCIAL APPLICATIONS This section presents 62 proverbs accompanied by their related financial concepts . The great majority of the presented proverbs are selected from Simpson and Speake [ 1998 ] . Each proverb is classified into one of twelve subsections . The first eleven subsections refer to different financial areas , and the last subsection contains proverbs that can be used to convey an instructor’s advice to students about learning , or serve as captivating introductory statements . For easy reference , all subsections , except the last one , and proverbs are listed in alphabetical order by first word . Proverbs appear in italics , and if more than one proverb can be used to illustrate a financial concept , the alternative proverbs are separated by “and . ” To save space , descriptions of the financial concepts being illustrated are kept to a minimum . Depending on the level of instruction , a teacher can provide as much additional explanation as necessary . Agency Relationships If you would be well served , serve yourself suggests that someone , hired to act on behalf of another person , may not always act in the best interests of that person . Such conflict is called an agency problem . 8 Capital Budgeting A door must either be shut or open and you cannot have your cake and eat it too define mutually exclusive alternatives . It is no use crying over spilled milk highlights the irrelevance of sunk costs in capital budgeting decisions . Know which side one’s bread is buttered on emphasizes the importance of identifying where a company’s profits come from in a performance analysis of different divisions of the same company . Separate the wheat from the chaff and separate the sheep from the goats describe the objective of capital budgeting analysis , which is to separate good investment opportunities from bad ones . Derivative Securities Kill two birds with one stone describes financial instruments that achieve two objectives at the same time . For example , convertible bonds allow investors to attain both the safety of bonds and the upward potential of stocks . One man’s loss is another man’s gain illustrates the concept of a zero - sum game , under which investor’s profits can come only from someone else’s losses , for example , options and futures markets’ payoffs . Financial Planning and Forecasting A chain is no stronger than its weakest link warns that the accuracy of forecasted numbers is limited by the precision of the inputs used in the analysis . Better the devil you know than the devil you don’t know and forewarned is forearmed emphasize the importance of financial planning and forecasting . Don’t count your chickens before they are hatched cautions that forecasted numbers are only our best estimates , and that they should never be considered as actual numbers . 9 Garbage in , garbage out advises that even a sophisticated analysis can produce grossly false conclusions if the original numbers used in the analysis were seriously in error . Hindsight is better than foresight suggests that , due to uncertainty , forecasted numbers will never be as accurate as the actual numbers , which can be observed only after the event has taken place . Hope for the best and prepare for the worst illustrates scenario analysis , which requires the forecasting of the best and worst possible outcomes . Personal Investing Don’t bite off more than you can chew , and if you don’t like the heat , get out of the kitchen , and stretch your arm no further than your sleeve will reach advise investors to not undertake investments that exceed their risk tolerance . He laughs best who laughs last stresses that in long - term horizon investments short - term performance is irrelevant . If you can’t beat them , join them advises an investor who cannot outperform the market to receive market returns simply by investing in market index funds . Make hay while the sun shines , and strike while the iron is hot , and the early bird catches the worm recommend decisive action in capital markets . Since security prices adjust quickly to new information in efficient capital markets , an investor should react promptly to new information to have a better chance of receiving above - average returns . One man’s meat is another man’s poison illustrates that what is appropriate for one person’s portfolio can be unsuitable for another person’s portfolio . For example , risky investments with the potential of high returns , which are suitable for a retirement portfolio of a young person , can be a disastrous investment for a person who is near retirement . Rome was not built in a day conveys that it takes time to achieve a large financial goal . There is a time and place for everything states that different investments are appropriate for different situations . There is no such thing as bad weather , only the wrong clothes can be paraphrased as “there is no such thing as a bad market , only the wrong investments . ” The paraphrased form of the proverb suggests that investors can earn superior returns in both rising and declining markets . 10 Indeed , short selling and put options give investors an opportunity to profit in a falling market as well . You pays your money and you takes your chances advises an investor that paying for investment advice does not guarantee above - average returns . Portfolio Management A stitch in time saves nine illustrates the importance of a regular portfolio rebalancing . Cut your coat according to your cloth emphasizes that each portfolio should be constructed according to its specific objectives and constraints . Fight fire with fire suggests that risky securities ( e . g . , options , futures ) can be used to reduce the risk of a portfolio . Keep no more cats than will catch mice advises limiting the number of assets in a portfolio only to those that are required to achieve an investment objective , for example , diversification benefits . Risk and Return Don’t put all your eggs in one basket explains the principle of diversification , according to which investing in more than one asset can reduce risk . Nothing is certain but the unforeseen , and nothing is permanent but change , and the unexpected always happens state that the total return will always include both expected and unexpected portions , no matter how sophisticated the analysis used to derive the expected return . Nothing ventured , nothing gained and there is no such thing as a free lunch describe the fundamental relation between risk and return , which is that an investor should be willing to take a larger amount of risk to have higher expected returns . You can’t find gold in a coalmine implies that high returns cannot be found among low risk investments . 11 Security Valuation All that glitters is not gold and you can’t tell a book by its cover can be used to explain two points . First , just because a company has sound fundamentals and an experienced management team does not necessarily mean it is a profitable investment . Investors must compare their value estimates with the current market price of the stock . As a result , good companies can be poor investments and average firms can provide great value . Second , the proverbs point out that an investment should not be evaluated simply based on its return or risk alone . Only if an investment provides an adequate compensation for its risk can it be considered as an attractive investment . Beauty is in the eye of the beholder illustrates that an investment that looks unattractive for one investor can look appealing to another . It is the market consensus that determines securities prices . The worth of a thing is what it will bring introduces the fundamental theory of valuation , which is that the value of any financial asset can be determined as the present value of all expected cash flows . Stock Price Behavior A rising tide lifts all boats suggests that a rising stock market can raise the prices of all stocks . The bigger they are , the harder they fall implies that sometimes stocks that have experienced spectacular returns in one period are the ones that are hit the hardest in the following period . The darkest hour is just before the dawn and when things are at the worst they begin to mend suggest that financial markets are often at their worst just before they begin to improve , providing a great buying opportunity for disciplined investors . 12 Technical Analysis Coming events cast their shadows before illustrates one of the main differences between efficient market hypothesis and technical analysis . While under the efficient market hypothesis stock prices react quickly to new information , under technical analysis stock prices adjust slowly . As a result , technical analysts can benefit from gradual price adjustments by trading as soon as prices begin to change . History repeats itself provides the rationale behind technical analysis , which is based on the assumption that prices move in trends that repeat themselves over time . One swallow does not make a summer notes that technical analysts should not focus only on one or two indicators , but should develop their estimates using a consensus of numerous technical indicators . Straws tell which way the wind blows describes how small changes in stock price or trading volume behavior can trigger technical trading rules signaling large future stock price changes . Time Value of Money Better an egg today than a hen tomorrow and time is money emphasize the time value of money , which is that a dollar today is more valuable than a dollar tomorrow . Money makes money , and great oaks from little acorns grow , and little and often fills the purse illustrate the power of compounding , which is the principle of keeping earned interest in the investment to earn more interest , resulting in considerable growth over long time periods . General Teaching Proverbs In addition to the above proverbs illustrating financial concepts , proverbs can also be used to convey instructional advice to students regarding learning or simply serve as intriguing introductory statements . This subsection presents several of these types of proverbs . 13 For want of a nail the shoe was lost and the devil is in the details advise students to pay attention to details when answering questions . Nothing is certain but death and taxes provides a humorous introduction to the topic of taxes . There is more than one way to skin a cat serves as an introduction to a discussion of several techniques that address the same problem , for example , different capital budgeting techniques or various methods for assessing a project’s risk . We must learn to walk before we can run introduces the order of instructional topics . For example , one must first learn the present value of future cash flows to be able to value stocks and bonds . SUMMARY AND CONCLUSIONS Instructors have suggested analogies , metaphors , examples , and humor to teach finance to students . This paper contributes to the financial education literature by offering proverbs as an innovative teaching tool to explain financial concepts . This paper presents more than five dozen proverbs accompanied by their related financial concepts . Each proverb is classified into one of twelve groups to make it easier for instructors to use the suggested proverbs . Eleven of these groups represent different financial areas , while the last group refers to general teaching proverbs . The use of proverbs in finance instruction provides several benefits . First , familiar proverbs will appeal to students’ prior knowledge and will help them to understand abstract financial concepts easier . Second , proverbs , presenting ideas in a concise and original way , will serve as an effective explanation tool and will improve retention . Third , proverbs with their wit , humor , and original point of view will bring entertainment and excitement into the classroom , making the learning process more enjoyable and efficient . To apply proverbs effectively in the classroom , I recommend several teaching strategies in this paper . First , an instructor has to ensure that students understand the interpretation of a proverb and its relation to a financial concept . Second , I suggest starting with a proverb and then following it with the explanation of the financial concept itself . Finally , I recommend using all relevant proverbs when more than one proverb can illustrate the same financial concept . 14 While this paper presents a relatively large number of proverbs , this list , by no means , is all - inclusive . Instructors are encouraged to introduce new proverbs , some of which maybe used primarily in their regions , into their teaching . Also , a growing number of business schools offer special programs for homogeneous groups of international students and executives both in North America and overseas . Since many English proverbs have equivalents in other languages , I suggest to instructors that they use related foreign proverbs whenever possible . While identifying equivalent foreign proverbs and understanding them will require additional work on the part of the instructor , the benefits will greatly outweigh the costs . As a rule , international students do not expect English - speaking instructors to know foreign proverbs , or , especially , to use them for explaining new material . Therefore , the use of foreign proverbs in the classroom pleasantly surprises students and shows that the instructors have some knowledge of the culture of their students . I personally used Chinese proverbs in my teaching to groups of students from China , and , in my opinion , the positive teaching effect of proverbs was even larger than that for groups of domestic students . Proverbs have the potential to become an effective teaching instrument in the toolbox of finance instructors , and make both teaching and learning processes more efficient , exciting and enjoyable for everyone . 15 REFERENCES Ardalan , K . “On the Use of Entertaining Metaphors in the Introductory Finance Course , ” Financial Practice and Education , 8 ( Spring / Summer , 1998 ) , 108 - 119 . Baker , W . P . and A . E . Lawson . “Complex Instructional Analogies and Theoretical Concept Acquisition in College Genetics , ” Science Education , 85 ( November , 2001 ) , 665 - 683 . Becker , M . W . “Top Ten Lists in Finance Class , ” Financial Practice and Education , 3 ( Fall , 1993 ) , 109 - 111 . Bryant , J . , P . W . Comisky , J . S . Crane , and D . Zillmann . “Relationship between College Teachers’ Use of Humor in the Classroom and Students’ Evaluations of Their Teachers , ” Journal of Educational Psychology , 72 ( August , 1980 ) , 511 - 519 . Cameron , L . “Metaphors in the Learning of Science : A Discourse Focus , ” British Educational Research Journal , 28 ( No . 5 , 2002 ) , 673 - 688 . Csikszentmihalyi , M . and J . McCormack . “The Influence of Teachers , ” in K . Ryan and J . M . Cooper ( Eds . ) Kaleidoscope : Readings in Education ( Boston , 1995 ) , 7th Edition , Houghton Mifflin Co . , 2 - 8 . Desberg , P . , D . Henschel , C . Marhsall , and P . McGhee . “The Effect of Humor on Retention of Lecture Material , ” Paper presented at the annual meeting of the American Psychological Association . ( Montreal , Canada , September 1 - 5 , 1981 ) , ( ERIC Document Reproduction Service No . ED 223118 ) . Dupin , J . J . and S . Johsua . “Analogies and “Modeling Analogies” in Teaching : Some Examples in Basic Electricity , ” Science Education , 73 ( April , 1989 ) , 207 - 224 . Forbes , S . W . “A Note on Teaching the Time Value of Money , ” Financial Practice and Education , 1 ( Spring , 1991 ) , 91 . Gilkeson , J . H . and R . P . Lamb . “From Beanie Babies to Baseball Cards : A Financial Application of Collectibles Markets , ” Journal of Financial Education , 26 ( Spring , 2000 ) , 14 - 21 . Gorham , J . and D . M . Christophel . “The Relationship of Teachers’ Use of Humor in the Classroom to Immediacy and Student Learning , ” Communication Education , 39 ( January , 1990 ) , 46 - 62 . 16 Halpern , D . F . , C . Hansen , and D . Riefer . “Analogies as an Aid to Understanding and Memory , ” Journal of Educational Psychology , 82 ( June , 1990 ) , 298 - 305 . Honeck , R . P . “Interpretive versus Structural Effects on Semantic Memory , ” Journal of Verbal Learning and Verbal Behavior , 12 ( August , 1973 ) , 448 - 455 . Honeck , R . P . , P . Riechmann , and R . R . Hoffman . “Semantic Memory for Metaphor : The Conceptual Base Hypothesis , ” Memory and Cognition , 3 ( July , 1975 ) , 409 - 415 . Ibanez , J . G . “Using Proverbs in Chemistry , ” Journal of Chemical Education , 79 ( April , 2002 ) , 454 - 455 . Kane , S . “Teaching Principal - Agent Problems Using Examples from Popular Music , ” Financial Practice and Education , 9 ( Spring / Summer , 1999 ) , 116 - 120 . Kaplan , R . M . and G . C . Pascoe . “Humorous Lectures and Humorous Examples : Some Effects upon Comprehension and Retention , ” Journal of Educational Psychology , 69 ( February , 1977 ) , 61 - 65 . Korobkin , D . “Humor in the Classroom : Considerations and Strategies , ” College Teaching , 36 ( Fall , 1988 ) , 154 - 158 . Mann , S . V . “Calls are Like Air Conditioners ; Puts are Like Heaters , ” Journal of Financial Education , 22 ( Spring , 1996 ) , 61 - 64 . Moreno , V . and F . J . Di Vesta . “Analogies ( Adages ) as Aids for Comprehending Structural Relations in Text , ” Contemporary Educational Psychology , 19 ( April , 1994 ) , 179 - 198 . Neuliep , J . W . “An Examination of the Content of High School Teachers’ Humor in the Classroom and the Development of an Inductively Derived Taxonomy of Classroom Humor , ” Communication Education , 40 ( October , 1991 ) , 343 - 355 . Newby , T . J . , P . A . Ertmer , and D . A . Stepich . “Instructional Analogies and the Learning of Concepts , ” Educational Technology Research and Development , 43 ( No . 1 , 1995 ) , 5 - 18 . Nichols , S . E . , D . Tippins , and K . Wieseman . “A Toolkit for Developing Critically Reflective Science Teachers , ” Journal of Science Teacher Education , 8 ( May , 1997 ) , 77 - 106 . Pearson , P . D . , T . E . Raphael , N . TePaske , and C . Hyser . “The Function of Metaphor in Children’s Recall of Expository Passages , ” Journal of Reading Behavior , 13 ( Fall , 1981 ) , 249 - 261 . Reynolds , R . E . and R . M . Schwartz . “Relation of Metaphoric Processing to Comprehension and Memory , ” Journal of Educational Psychology , 75 ( June , 1983 ) , 450 - 459 . 17 Simpson , J . and J . Speake . The Concise Oxford Dictionary of Proverbs ( Oxford , 1998 ) , 3rd Edition , Oxford University Press . Spiro , R . J . , P . J . Feltovich , R . L . Coulson , and D . K . Anderson . “Multiple Analogies for Complex Concepts : Antidotes for Analogy - Induced Misconception in Advanced Knowledge Acquisition . In S . Vosniadou and A . Ortony ( Eds . ) Similarity and Analogical Reasoning ( Cambridge , 1989 ) , Cambridge University Press , 498 - 531 . Wanzer , M . B . and A . B . Frymier . “The Relationship between Student Perceptions of Instructor Humor and Students’ Reports on Learning , ” Communication Education , 48 ( January , 1999 ) , 48 - 62 . Ziv , A . “Teaching and Learning with Humor : Experiment and Replication , ” Journal of Experimental Education , 57 ( Fall , 1988 ) , 5 - 15 \ No newline at end of file diff --git a/silver_data/32bc9393383df8df9dd6dc7ae098f5ad47069801.pdf.txt b/silver_data/32bc9393383df8df9dd6dc7ae098f5ad47069801.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..0c4adcce7bf8cdce4738f2793e168356c388693c --- /dev/null +++ b/silver_data/32bc9393383df8df9dd6dc7ae098f5ad47069801.pdf.txt @@ -0,0 +1 @@ +1 Voltage - dependent volume regulation controls epithelial cell extrusion and morphology Saranne J . Mitchell # * , Carlos Pardo - Pastor * , Thomas A . Zangle† , and Jody Rosenblatt * * The Randall Centre for Cell & Molecular Biophysics , School of Basic & Medical Biosciences , & School of Cancer and Pharmaceutical Sciences , King’s College London , London , UK # Department of Biomedical Engineering , University of Utah , Salt Lake City , Utah , USA †Department of Chemical Engineering , University of Utah , Salt Lake City , Utah , USA 2 ABSTRACT : Epithelial cells work collectively to provide a protective barrier , yet also turn over rapidly by cell death and divi - sion . If the number of dying cells does not match those dividing , the barrier would vanish , or tumors can form . Mechanical forces and the stretch - activated ion channel ( SAC ) Piezo1 link both processes ; stretch promotes cell division and crowding triggers cell death by initiating live cell extrusion 1 , 2 . However , it was not clear how particular cells within a crowded region are selected for extrusion . Here , we show that individual cells transiently shrink via water loss before they extrude . Artificially inducing cell shrinkage by increasing extracellular osmolarity is suffi - cient to induce cell extrusion . Pre - extrusion cell shrinkage requires the voltage - gated potassium channels Kv1 . 1 and Kv1 . 2 and the chloride channel SWELL1 , upstream of Piezo1 . Activation of these voltage - gated channels requires the mechano - sensitive Epithelial Sodium Channel , ENaC , acting as the earliest crowd - sensing step . Imaging with a voltage dye indicated that epithelial cells lose membrane potential as they become crowded and smaller , yet those selected for extrusion are markedly more depolarized than their neighbours . Loss of any of these channels in crowded conditions causes epithelial buckling , highlighting an important role for voltage and water regulation in controlling epithelial shape as well as extrusion . Thus , ENaC causes cells with similar mem - brane potentials to slowly shrink with compression but those with reduced membrane potentials to be eliminated by extrusion , suggesting a chief driver of cell death stems from insufficient energy to maintain cell membrane potential . MAIN : Cell extrusion promotes cell death to maintain homeostatic epithelial cell numbers , using a mechanism con - served from sea sponge to humans . During extrusion , a live cell is ejected apically by coordinated basolateral actomyosin contraction of the extruding cell and its neighbors 1 . After extrusion , the cell dies due to lack of sur - vival signaling . We previously discovered that Piezo1 activates live cell extrusion ( LCE ) in response to crowding to maintain constant epithelial cell numbers 1 , 2 . Crowding activation of Piezo1 triggers a canonical pathway that also extrudes cells triggered to die by apoptosis . To extrude , cells produce the lipid Sphingosine 1 - Phosphate ( S1P ) , which binds the G - protein - coupled receptor S1P 2 to activate Rho - mediated actomyosin contraction need - ed for extrusion 2 - 4 . As crowding - induced cell extrusion drives most epithelia cell death , an important outstanding question is which cells within a crowded epithelial field are chosen for extrusion ? In sparse epithelia , topological defects within a normal hexagonal - packed epithelial promote outlier cells to extrude 5 , 6 . Additionally , C . elegans and mammalian cells with DNA damage or replicative stress are selected for extrusion 6 , 7 . However , as most cell extrusions occur in crowded regions , where cells do not divide or have obvious topological defects , what marks most cells for extrusion has remained a mystery . 3 One possible model is Darwinian , where cells that are fitter remain in epithelia while weaker ones are marked for extrusion and death 8 . One possible measure of cell fitness in this model is cell mass . Cells with higher dry cell mass might be fitter in terms of protein and macromolecule production and maintenance . To test this possibility , we adapted quantitative phase imaging ( QPI ) to analyze dry mass changes over time in Madin - Darby Canine Kidney II ( MDCKII ) epithelial monolayers ( see methods ) 9 , 10 . However , QPI revealed that dry mass does not change prior to cell extrusion , unlike the clear mass increase before a cell divides ( Supplemental Fig . 1A - B and video 1 , representing 31 cells from 12 total cell islands ) , disfavoring cell mass change as a factor for selecting LCE . While investigating cell mass , we noted a consistent , transient increase in cell - cell junction brightness by phase microscopy that lasts ~ 6 . 5 minutes before a cell extrudes ( Fig . 1A , B , and Supplemental Video 2 ) . To readily assay the transient phase brightness , we developed the ‘lightning assay’ that relies on intensity thresholding of junctional brightness surrounding cells before they extrude ( Fig . 1C & D ) . By contrast , less than 0 . 03 % of cells Supplementary Figure 1 . Quantitative phase imaging ( QPI ) shows unaltered dry mass in cells that will extrude . A , As QPI requires a reference area without cells throughout imaging , we grew MDCKII monolayers on confined 100µm islands within a 35 mm dish by plasma treating patterned areas , as in this example . Scale bar = 100µm . B , QPI stills from Supplemental video 1 , representing 31 other cells from 12 QPI islands that similarly show dry mass before cell extrusion , where blue is low cell mass and red is higher mass . Note mass increases after cell extrusion , as the density gets higher with cell contraction ( h : mm ) . C , By contrast , another cell , in red box , increases mass before dividing . Scale bar = 50µm . 0 2 . 5 pg / μm 2 MICROPATTERN MONOLAYER A B QPI : EXTRUSION C QPI : DIVISION CYTOKINESIS 0 : 00 0 : 46 G2 0 : 00 1 : 30 4 filmed ( 8 in 750 ) exhibit junctional brightness without extruding ( Fig . 1E ) . Increased phase brightness might indi - cate a decrease in cell volume or shape . To test if transient volume loss accounts for the phase - brightness before extrusion , we expressed cytoplasmic GFP mosaically in MDCKII cells and measured cell volume throughout extrusion by confocal timelapse microscopy . We find that live cells experience a transient area and volume loss before extruding that corresponds to the junctional brightness measured in our lightning assay ( Fig . 1D , F & G ) . Given this direct correlation between phase brightness and cell volume , we used the lightning assay for mea - suring Homeostatic Early Shrinkage ( HES ) before LCE . Importantly , we find that HES before extrusion occurs in only crowded regions , as measured by cell density ; the few cells that shrink without extruding do so in uncrowded epithelial regions ( Fig . 1H ) . Thus , crowding followed by single cell shrinkage appear to initiate cell extrusion . A 57 . 000 min 57 . 000 min 58 . 000 min 58 . 000 min 67 . 000 min 67 . 000 min 96 . 000 min 96 . 000 min 164 . 000 min 164 . 000 min B D LIGHTNING ASSAY P ha s e Shrink Before Shrink Relax Extrusion LIGHTNING ASSAY - 3 - 2 - 1 0 1 2 3 0 2 4 6 Time ( Min ) N O R M A L I Z E D A R EA BE T W EE N C E LL S E C CELL VOLUME BEFORE EXTRUSION 0 . 6 0 . 8 1 . 0 1 . 2 N O R M A L I Z E D V O L U M E ns Baseline Shrink Max Relax Max Extrusion H 1 . 2 ns CELL AREA BEFORE EXTRUSION Baseline Shrink Max Relax Max Extrusion 0 . 6 0 . 8 1 . 0 N O R M A L I Z E D A R EA N o E x t r u s i on 0 20 40 60 80 DENSITY - DEPENDENCE No shrink F Shrink Relax Extrude 0 20 40 60 KINETICS V eh i c l e G s M T x 4 B l ebb i s t a t i n 0 5 10 15 20 25 H ES / 10 00 C e ll s LIGHTNING ASSAY SHRINK V . EXTRUSION No Extrusion - 0 . 2 0 . 0 0 . 2 0 . 4 60 80 90 % O F C E LL S T H A T S HR I N K Extrusion G I 70 LIVE CELL EXTRUSION EVE N T DUR A T I O N ( m i n s ) C E LL S PE R 100 mm 2 N o E x t r u s i on E x t r u s i on Shrink Y 27632 T h r e s ho l d a r ea Figure 1 : Live cells shrink briefly before extruding in dense regions . A , Stills from a phase timelapse movie show junctional lightning ( white arrowhead ) around a cell before it extrudes . Scale bar = 10µm B , Kinetics of live cell extrusion denoting average time ( in minutes ) cells experience shrink , relax , and from shrink to extrusion , extrusion , mean ± SEM . C , Lightning assay , showing representative stills from time - lapse phase microscopy where white space is thresholded ( red , bottom ) at key points during extrusion , quantified in ( D ) as mean ± SEM of normalized white area around cell , where t0 is shrink ; n = 10 cells . E , Percent cells that shrink before extruding ± SEM and P from unpaired T - test using n = 10 biolog - ical replicates . F , Normalized area mean ± SEM of cells at each defined step : baseline , shrink maximum , relax maximum and extrusion ; n = 15 cells ; p values from two - way ANOVA with Dunnett’s multiple comparisons test . G , Normalized mean volume ± SEM of cells at baseline , shrink maximum , relax maximum , and onset of extrusion ; n = 5 cells with p values from two - way ANOVA with Dunnett’s multiple comparisons test . H , Regional density - dependence ofcell shrinkage ( red ) with or without extrusion , compared to no shrink ( black ) ; as mean ± SEM , n = 3 with one - way ANOVA with Dunnett’s multiple comparisons test . I , Mean rate of HES with inhibitors of cell extrusion ± SEM ; n = 3 , using one way ANOVA with Dunnett’s multiple comparisons test . For all graphs , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 or * P = 0 . 0124 . 5 While ~ 70 % of extruding cells undergo HES , it was not clear why the remaining 30 % do not shrink . One pos - sibility is that this minor population of non - shrinking extrusions were apoptotic , which accounts for ~ 20 - 30 % of extruding cells at steady state in cultured epithelia 1 . Combining a fluorescent reporter of the cleaved caspase - 3 apoptotic marker with the lightning assay , we find cell shrinkage rarely occurs ( ~ 3 % ) before apoptotic extrusion ( Supplemental Fig 2A - B , Supplemental Video 3 ) . Thus , HES typically precedes LCE but not apoptotic extrusion . Transient cell volume loss could be due to myosin II contraction , which occurs before apoptotic extrusion 11 , 12 . To investigate if myosin contraction is important for HES , we inhibited ROCK or myosin II and measured cell shrink - age with our lightning assay . We found blocking myosin II contraction increased the number of cells shrinking by ~ 23X compared to untreated controls ( Fig . 1I ) , suggesting HES occurs independently from myosin contractility . Additionally , HES did not cause blebbing , typically associated with myosin II contraction 13 , 14 , further ruling out a role for myosin contraction in HES . As HES occurs in crowded regions , we investigated if Piezo1 , the SAC required for crowding - induced live cell extrusion 2 , might instead control transient cell volume loss . However , we find that the SAC inhibitor GsMTx4 does not block HES , suggesting Piezo1 acts downstream of HES ( Fig . 1I ) . This was surprising , as our previous studies suggested Piezo1 as the earliest mechano - sensor to trigger extru - sion in response to crowding 1 . Having ruled out dry mass , contractility , and early extrusion regulators as controllers of HES , we next investi - gated if water efflux may regulate the transient volume loss cells experience before extrusion . Cellular water regulation is critical for controlled epithelial cell death 7 , 15 , 16 and could account for the volume changes seen before LCE . To first test if water loss is sufficient to initiate LCE , we incubated MDCKII monolayers in media containing increasing osmolarities ( hypotonic to hypertonic ) for ten minutes before returning to isotonic , control medium to mimic the duration of shrinkage before extrusion ( Supplemental Video 4 & 5 , Fig . 2A ) . While hypotonic medium A 0 . 000 min 0 . 000 min 0 . 000 min 0 . 000 min 42 . 000 min 42 . 000 min 42 . 000 min 42 . 000 min 52 . 000 min 52 . 000 min 52 . 000 min 52 . 000 min 77 . 000 min 77 . 000 min 77 . 000 min 77 . 000 min P ha s e C a s pa s e Apoptotic eviL 0 20 40 60 80 100 % EX T RUD I N G C E LL S T H A T S HR I N K B C on t r o l DC P I B DC P I B + z VA D - f m k 0 10 20 30 40 50 HOMEOSTATIC EXTRUSION E x t r u s i on s / 10 , 000 c e ll s NS C APOPTOTIC EXTRUSION SHRINK Supplemental Figure 2 . HES does not occur before apoptotic cell extrusion . A , Stills from a phase ( top ) timelapse and fluorescent apoptosis ( Cell Event - caspase ) marker ( bottom ) before apoptotic extrusion . Scale = 10µm . B , Mean percentage of cells ± SEM that shrink by lightning assay before extruding . n = 5 ; * * * * P < 0 . 0001 by an unpaired T - test . C , Mean extrusion rate ± SEM in monolayers treated with SWELL1 inhibitor ±zVAD - FMK ; n = 3 ; where * P = 0 . 0104 values are from one - way ANOVA with Sidak’s multiple comparisons test . 6 had no impact on cell extrusion ( Fig . 2A ) , 15 - 20 % hypertonicity dramatically increased both cell shrinkage and LCE rates ( Fig . 2A & B ) , with higher concentrations causing so much extrusion that it destroyed the monolayer ( data not shown ) . Although increasing hypertonicity can provoke Apoptotic Volume Decrease ( AVD ) 12 , 17 , the lack of fluorescence associated with cleaved caspase - 3 / 7 apoptotic cell probe indicated that hypertonic treatment 0 5 10 15 20 - 40 - 30 - 20 - 10 0 % C e ll V o l u m e C hange ( ( V - V o ) / V o ) * 100 Time ( min ) B I s o 0 10 20 30 40 50 E x t r u s i on / 1000 C e ll s A D C CELLS EXTRUDED Voltage - gated K + channel Cl - Channel Piezo1 ( SAC ) Osmolarity : - 35 % - 30 % - 25 % - 20 % - 15 % - 10 % - 5 % 0 % 5 % 10 % 15 % 20 % 0 5 10 15 20 E x t r u s i on / 1000 C e ll s I s o t on i c Hypo Hyper I s o H y pe r H y pe r Live Apoptotic Iso Hyper - 40 - 30 - 20 - 10 0 % C e ll V o l u m e C hange ( ( V - V o ) / V o ) * 100 CELLS MAINTAINED 0 5 10 15 20 Time ( min ) Iso Hyper Ca 2 + - activated K + channel D M S O 4 A P A Z I I B X T R A M 34 A P A M I N D C P I B + Z V A D - F M K C C C A C FT R G s M T x 4 0 10 20 30 40 E x t r u s i on / 1000 C e ll s 10min Hyper / Hypo 100minIso HOMEOSTATIC EXTRUSION INHIBITORS Figure 2 : Water loss is suf - ficient to induce live cell extrusion . A , Mean MDCKII extrusion rate ± SEM follow - ing incubation in media with increasing osmolarity for 100’ , scored by immunostaining for actin and DNA , with n = 3 ex - periments ; where * * P = 0 . 0068 ; * * * * P < 0 . 0001 by one way ANOVA with Dunnett’s multiple comparisons test . B , Mean ex - trusion rates ±SEM in normal medium or 100’ following 10 minute 20 % hypertonic shock , where apoptotic extrusion is scored by immunostaining with cleaved caspase - 3 anti - body ; n = 3 experiments ; and * P = 0 . 0105 is from an unpaired T - test . C , Volume changes ( measured by confocal sec - tioning of mosaically expressed GFP ) over time in cells shifted from isotonic to 20 % hypertonic media that extrude ( left ) or do not ( right ) , n = 5 . D , Number of extrusions in MDCKII monolay - ers incubated with volume reg - ulating inhibitors and scored by immunostaining for actin and DNA ; n = 3 ; where * * * * P < 0 . 0001 by one way ANOVA with Dun - nett’s multiple comparisons test . Inhibitor key describes volume regulating channel fam - ily inhibitor target by assigned color and icon . 7 Control 20 % Hyper E CELL EXTRUSION RATES A B C on t r o l D M S O A Z I 4 A P D C P I B + z VA D - f m k C C C a C FT R G s M T X 4 0 10 20 30 EX T RU S I O N / 1000 C E LL S ns CELL SHRINKAGE D M S O D M S O A z i 4 A P D C P I B C C C a C FT R G s M T x 4 z VA D - f m k - 100 0 100 200 300 400 % CH A N G E PEAK A R E A HOMEOSTATIC CELL SHRINKAGE Ctrl Kv1 . 1 Kv1 . 2 Swell1 CELL SHRINKAGE - 75 - 50 - 25 0 25 50 75 100 % CH A N G E PEAK A R EA 0 50 100 150 EX T RU S I O N / 1 0000 C E LL S D F C siRNA : Ctrl Kv1 . 1 Kv1 . 2 Ctrl Kv1 . 1 Kv1 . 2 C e ll s t ha t E x t r ude C t r l 4 A P D C P I B K v 1 . 1 K v 1 . 2 S w e ll 1 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 20 30 40 50 H ES PE R 1000 C E LL S siRNA LIGHTNING ASSAY OVER TIME 0 . 8 1 . 0 1 . 2 1 . 4 1 . 6 Time ( min ) N O R M A L I Z E D A R EA B / N C E LL S Voltage - Gated K + Cl - Channel Apoptosis Extrusion Control INHIBITORS HYPERTONIC - INDUCED CELL EXTRUSION Cells that remain in monolayer siRNA : Swell1 zVAD - fmk + Swell1 zVAD - fmk + 10min Hyper / Hypo 100minIso 10minHyper 10minHyper 10min Hyper / Hypo 100minIso Hypertonic shock 7 . 5 10 12 . 5 15 Figure 3 : Homeostatic live cell extrusion requires K + voltage - gated and Cl - channel activation . A , Mean extrusion rate ± SEM from monolayers treated with inhibitors only during the 10’ 20 % hypertonic treatment n = 3 ; P values from two - way ANOVA with Dunnett’s multiple comparisons test . B , Adapted lightning assay for a crowded epithelial field following hypertonic treatment , quantified over time . C , Mean percent peak change in area surrounding cells ( from adapted lightning assay ) ± SEM with inhibitors during 20 % hypertonic challenge , n = 6 ; with P values from a two - way ANOVA with Dunnett’s multiple comparisons test . Channel nhibitor key ( for B & C ) . D , Mean extrusion rate ± SEM of MDCKIIs with siRNA - knock - downs ± 20 % hypertonic challenge , compared to controls ; n = 4 ; P values from two - way ANOVA with Dunnett’s multiple comparisons test . E , Percent change of peak area ( from adapted lightning assay ) ± SEM during hypertonic challenge of siRNA - knockdowns , compared to controls , n = 6 ; with P values from one - way ANOVA with Dunnett’s multiple comparisons test . F , Mean rate of HES ± SEM when treated with 4AP , DCPIB , or with Kv1 . 1 , Kv1 . 2 or SWELL1 siRNA knockdowns , compared to control background cells or those that shrink before extruding ; n = 3 ; where P values are from two - way ANOVA with Dunnett’s multiple comparisons test . For all graphs , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 0100 , * P < 0 . 05 8 induces only LCE ( Fig . 2B ) . Further , the pan caspase inhibitor zVAD - fmk does not prevent 20 % hypertonic shrink - induced extrusions ( Supplemental Fig . 3A & BB ) , ruling out AVD as a mechanism for hypertonic - induced extrusion . These findings further support that HES controls live but not apoptotic cell extrusion . Interestingly , although the entire monolayer was treated with 20 % hypertonic solution , not all cells shrunk equally . Only cells that shrank 20±3 % as measured by cytoplasmic GFP volume extrude , whereas those shrinking 11±2 . 5 % do not ( Fig . C2C ) . Thus , experimentally reducing cell volume ~ 20 % is sufficient to trigger individual cells to extrude , which we refer to as osmotic - induced cell extrusion ( OICE ) . Since potassium ( K + ) and chloride ( Cl - ) efflux drive water loss during AVD 23 , we investigated if they control HES and extrusion . Using a panel of K + and Cl - channel inhibitors , we assayed for those that block homeostatic ex - trusion rates by timelapse microscopy and immunostained samples . The inhibitor carrier , dimethyl sulfoxide ( DMSO ) , can change water permeability of cells 18 , however it did not decrease the basal rate of extrusion , ruling out nonspecific effects ( Fig . 2D and A3A ) . Although most inhibitors were not toxic , the Cl - channel SWELL1 is essential for cell survival 19 , requiring the addition of caspase inhibitor zVAD - fmk to its inhibitor DCPIB for all our DMSOSKI - V2 SKI - II S1P / S1P2 INHIBITOR KINETICS DURING SHRINK C on t r o l D M S O D C P I B + z VA D - f m k A 0 10 20 30 40 E x t r u s i on / 1000 c e ll s ns HYPERTONIC INDUCED EXTRUSION Voltage - gated K + channels Ca 2 + - activated K + channels Cl - Channel channels Contractility Extrusion C on t r o l C on t r o l D M S O A pa m i n T R A M 34 I BX Y 27632 H 1152 B l ebb i s t a t i n S k I - II SK I - V 5 z VA D - f m k 0 10 20 30 E x t r u s i on / 1000 c e ll s ns ns EXTRUSIONS INHIBITING ONLY DURING HYPERTONIC B 4 AP A z i A pa m i n T R A M 34 I BX Y 27632 B l ebb i s t a t i n C C C a C FT R S k I - II SK I - V 5 z VA D - f m k G s M T x 4 F SAC INHIBITOR KINETICS DURING SHRINK E 4AP KINETICS DURING SHRINK 0 . 5 1 . 0 1 . 5 Time ( min ) 4APDMSO 7 . 5 10 15 Hypertonic Shock 12 . 5 C DCPIB KINETICS DURING SHRINK 0 . 5 1 . 0 1 . 5 N o r m a li z ed A r ea B e t w een C e ll s DMSODCPIB 17 . 5 Hypertonic Shock D DMSOGsMTx4 Targets 10minHyper + Inh 100minIso 10minHyper + Inh 100minIso + Inh 7 . 5 10 15 12 . 5 7 . 5 10 15 12 . 5 7 . 5 10 15 12 . 5 Time ( min ) Time ( min ) Time ( min ) Hypertonic Shock Hypertonic Shock Supplemental Figure 3 . Ion channel inhibitors impact on extrusion and shrink . A , Mean extrusion rate ± SEM in MD - CKII monolayers treated with inhibitors before , during , and after hypertonic shock ; n = 3 ; where * * * * P = 0 . 0001 by one way ANOVA with Dunnett’s multiple comparisons test . B , Mean extrusion rate ± SEM in monolayers treated with inhibitors only during hypertonic shock . n = 3 experiments where * * P = 0 . 0024 is from two - way ANOVA with Dunnett’s multiple comparisons test . Inhibitor key for A and B describes ion channel family inhibitor target by color icon . Representative “Lightning assays” where increased space around the cells indicates cell shrinkage in the presences of : 4AP ( KV1 . 1 & KV1 . 2 inhibitor ) ( C ) , DCPIB ( SWELL1 inhibitor ) ( D ) , SAC inhibitor GsMTx4 ( E ) , or S1P / S1P 2 inhibitors ( F ) , compared to DMSO controls before and during hypertonic media incubation ( mins ) . 9 assays ( Fig 2D , Supplementary Fig 2C ) . Remarkably , all inhibitors tested significantly reduced extrusion rates during normal homeostatic turnover to levels seen when SAC channels are inhibited with GSMTx4 ( Fig . 2D and Supplementary Fig 3A ) . Interestingly , 4 - AP , an inhibitor of voltage - gated Kv1 . 1 and Kv1 . 2 channels , also blocks apoptotic extrusion 20 , suggesting a conserved activator for all cell extrusions ( Fig 2D , Supplementary Fig 3A ) . Other channels are implicated in pleotropic diseases , like Kv11 . 1 in carcinomas 21 , 22 and heart arrhythmias 23 and the cystic fibrosis transmembrane receptor ( CFTR ) in cystic fibrosis 24 , suggesting possible new roles for misreg - ulated extrusion in these diseases . While these findings were surprising and suggest interesting future studies , here , we focus solely on those that control the HES step . To determine which K + and Cl - channels , if any , were needed for HES , we first tested which inhibitors blocked extrusions when present only during the 10 - minute hypertonic treatment of OICE . Timelapse phase microscopy revealed that only early inhibition of SACs , Kv channels , and Cl - channels stopped extrusion ( Fig . 3A and Supple - mentary Fig . 3B ) . Even though GsMTx4 does not stop cell shrinkage , it inhibits cell extrusion when inhibited only in the first 10 - minutes , signifying its role early during extrusion pathway , yet downstream of cell shrinkage ( Fig . 1I , 2D ) . Thus , we next investigated if the voltage - gated K + and Cl - channels required in early stages of extrusion regulate both osmotic - induced and homeostatic shrinkage before extrusion . To identify which ion channels regulate HES , we first adapted our lightning assay ( Fig 1D & E ) to measure increas - es in light intensity of all cell - cell junctions within a crowded field in response to a 20 % hypertonic shock , ( Fig . 3B , normalized to isotonic medium ) , as a proxy for cell volume loss . This assay identified only DCPIB ( combined with zVAD - fmk ) and 4 - AP , which target the chloride channel SWELL 1 and the voltage - gated channels Kv1 . 1 , Kv1 . 2 , respectively , as inhibitors of both hypertonic - induced cell shrinkage and extrusion ( Fig . 3A & C & Supplementary Fig . 3A - DD ) . Immunostaining showed that all three channels localize to the cell apex , with Kv1 . 1 co - localizing with ZO - 1 at tight and tri - cellular junctions , regions known to act as tension sensors 25 ( Supplementary Fig . 4A ) . We used siRNA - mediated knockdown to test the roles of Kv1 . 1 , Kv1 . 2 , and SWELL 1 , the targets of 4 - AP and DCPIB , in both OICE and HES ( Suppl . Fig . 4B & C ) . We found that knockdown of any of these channels prevent - ed HES and OICE ( Fig . 3D - F ) , suggesting that they regulate both cell shrinkage and extrusion during normal cell turnover . The fact that Kv1 . 1 , Kv1 . 2 are voltage - gated channels suggested that membrane depolarization could trigger both HES and LCE 26 . Although most frequently studied in neuronal cells , electrical membrane potentials are critical for all cells , requiring between 30 - 70 % of all the energy consumed within a cell , depending on the cell type 27 . Na + / K + ATPases maintain polarized ions with intracellular K + and Cl - and extracellular Na + and organic 10 A B C - 4 : 10 4 : 10 7 : 00 - 7 : 00 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 preshrinkno shrink Ctrl Ctrl Kv1 . 1 / 2 ENaC 0 10 20 30 D i BA C 4 ( 3 ) no r m a li z ed ns DEPOLARIZATION D - 40 - 20 0 20 100200300400500 % C hange P ea k A r ea siRNA : Iso Hyper C t r l αβ γ ENaC C t r l αβ γ ENaC siRNA : 0 20 40 60 E x t r u s i on s / 1 , 000 C e ll s siRNA : Iso Hyper C t r l αβ γ ENaC C t r l αβ γ ENaC V eh i c l e 0’ 27’ 53’ KV 1 . 1 / 2 I nh i b i t o r PHASE DiBAC 4 ( 3 ) PHASE PHASE DiBAC 4 ( 3 ) E N a C i nh i b i t o r 0’ 27’ 53’ 0’ 27’ 53’ 14’ 14’ 14’ DiBAC 4 ( 3 ) E Extrusion ION CHANNEL LOSS DISRUPTS WATER REGULATION AND EXTRUSION J LOSS OF CHANNEL & BUCKLING 0 5 10 15 20 B u ck l e s P e r F i e l d o f V i e w C t r l K v 1 . 1 K v 1 . 2 S W E LL1 + z V A D - F M K E N a C α siRNA : E N a C β E N a C γ siCtrl MAINTAINS CONSTANT CELL NUMBER S AND SHAPE FUNCTIONAL EXTRUSION Monolayer height D a r k e r E qua l B r i gh t e r 0 20 40 60 80 % EX T RUD I N G C E LL S C O M PA R E D T O N E I G H B O R S EXTRUDING V . NEIGHBOR POTENTIAL 0 20 40 60 80 0 10000 20000 Cells / Field D I BA C 3 ( 4 ) ( a . u . ) Crowded Sparse CROWDING V . MEMBRANE POTENTIAL F H G PHASEDiBAC 4 ( 3 ) 0 : 00 0 : 00 0 : 00 siENaC NO WATER REGULATION BUCKLED VOLTAGE REGULATION OF VOLUME DETERMINES CELL SIZE AND EXTRUSION > 17 % cell shrink activates extrusion Piezo1S1P / S1P 2 Na + K + Na + K + K + K + K + K + K + Cl - Cl - Cl - K + Na + K + K + Cl - K + Na + Na + Cl - Cl - Na + Kv1 . 1 Swell ENaC H 2 O cell extrudes SIMILAR POTENTIAL CELLS CROWDING TOGETHER DIFFERENTIAL POTENTIAL SINGLE CELL CROWDING crowding tension alleviated Na + Na + Na + K + K + Cl - Cl - Cl - K + Na + Na + K + K + K + K + K + K + Cl - Cl - Cl - crowding tension alleviated crowding activates Na + entry via ENaC activates Kv1 . 1 / Kv1 . 2 Swell1 and H 2 O egress depolarization I K SUBCONFLUENT CONFLUENT CROWDED ns D i BA C 4 ( 3 ) no r m a li z ed Time ( min ) Shrink Extrusion Maintained in monolayer Monolayer height DEPOLARIZATION 11 anions to create these membrane potentials . While individual cells create membrane potentials , epithelial cells tethered together with tight , adherens , and gap junctions , work collectively to maintain a transepithelial potential difference of between 1 – 46mV , with a net negative charge on the apical side 28 , 29 . Interestingly , imposing a direct current that reinforces this polarity ( from basal to apical ) enhances and even repairs impaired epithelial cell - cell junctions , whereas reversing the current ( from apical to basal ) causes cells to contract and detach from each other 30 , reminiscent of the cell shrinkage and junctional lightning between cells before extrusion or with hyper - tonic treatment ( Figs . 1F & 3C ) . Since voltage - gated channels control cell shrinkage and experimentally depolarizing a whole epithelial monolay - er produces similar shrinkage of all cells , we next investigated if cells depolarize their membranes before they shrink . To visualize membrane potential , we filmed MDCKII monolayers with the slow - response potential - sensitive probe DiBAC3 ( 4 ) , which increases fluorescence with membrane depolarization31 - 33 . Unlike excitable tissues that rapidly depolarize in an all or nothing reversible manner , we found that MDCKII cells within the monolayer have different membrane potentials ; only some cells become DiBAC3 ( 4 ) - brighter , depolarizing as they crowd and maintaining this depolarized state ( Fig . 4A & B ) . This shows that despite epithelial cells being connected with gap junctions , they are not fully electrically coupled . This also supports our previous finding that cells become Figure 4 : ENaC - dependent depolarization activates cellular shrinkage and extrusion . A , Membrane depolarization , measured by DiBAC 3 ( 4 ) fluorescence ( greater depolarization with higher fluorescence ) , in sub - confluent , confluent , and crowded MDCKII monolayers . Scale bar = 50µm . B , Density - dependence of membrane potential , as measured by DiBAC 3 ( 4 ) fluorescence , n = 3 . C , Timelapse ( mins ) measuring membrane depolarization before homeostatic extrusion ( top panel , red arrow indicating a cell before extrusion ) , in the presence of 4AP ( KV1 . 1 / 2 inhibitor , middle panel ) , and with amiloride ( ENaC inhibitor , bottom panel ) . Scale bars = 10µm . D , Mean cells ± SEM with darker , equal , or brighter DiBAC 3 ( 4 ) staining , com - pared to surrounding neighbouring cells before extruding ; n = 3 experiments ; P values from one - way ANOVA with Tukey’s multiple comparisons test . E , Normalized DiBAC 3 ( 4 ) fluorescence , as mean ± SEM from cells that undergo HES / extrusion ( blue ) or not ( red ) over time , n = 6 . F , Mean membrane polarization status ± SEM , as measured by DiBAC 3 ( 4 ) fluorescence of cells before they extrude compared to non - extruding cells in background or when Kv1 . 1 / Kv1 . 2 or ENaC are inhibited with 4AP or amiloride ; n = 3 experiments ; P values from one - way ANOVA with Tukey’s multiple comparisons test . G , Peak mean percent area change ± SEM ( by adapted lightning assay ) of siRNA - mediated ENaC - a , b , or g knockdown in MDCKs with and without 20 % hypertonic challenge , compared to non - targeted controls , n = 3 for each siRNA , with P values from a two - way ANOVA with Dunnett’s multiple comparisons test . H , Mean extrusion rates ± SEM of ENaC - a , b , or g siRNA - mediated knockdown with and without 20 % hypertonic challenge , compared to control , n = 3 ; with P values from two - way ANOVA with Dunnett’s multiple comparisons test . I , Representative volume view of control and ion channel knocked down monolayers , with alpha color bit coded bar indicating depth 0 . 0 - 16 . 0µm ( blue - pink ) . Schematics below depict how functional extrusion maintains monolayer shape whereas disrupting water regulation leads to buckling . J , Mean number of epithelial buckles per field ± SEM 48 hours post siRNA - treatment compared to non - target controls ; n = 3 ; with P values from two - way ANOVA with Dunnett’s multiple comparisons test . K , Model for how ENaC / Kv1 . 1 / Kv1 . 2 / Swell1 - dependent water regulation controls cell size and extrusion , depending on cellular membrane potential . Left : If one cell is more depolarized than its neighboring cells , crowd activation of ENaC will preferentially activate voltage - gated potassium channels in this cell , causing shrink . If this shrink reaches a threshold of ~ 20 % , it will activate the extrusion pathway . Cell elimination by extrusion will alleviate crowding pressure in the monolayer and maintain constant densities . Right : Crowd activation of ENaC in cells with equiva - lent membrane potentials cause moderate voltage - gated potassium - dependent water loss across all cells , resulting in slight cell shrinkage but not enough to activate cell extrusion . Here , slight shrinkage also helps accommodate overall crowding of the monolayer . In this way , crowding can set up competition to eliminate cells with the lowest energy that cannot main - tain a sufficient membrane potential , a significant energy demand for the cell . For all graphs , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 0100 . 12 smaller in regions of crowding1 , 34 and suggests that individual cell volume / size may be governed by existing Na + - K + pumps or ATP available to maintain resting membrane potential35 . Importantly , we found that before cells shrink and extrude , they depolarize significantly , becoming DiBAC3 ( 4 ) - brighter ( Fig . 4C - E , Supplemental Video 7 ) , whereas cells that do not shrink , do not depolarize , linking shrinkage to depolarization ( Fig . 4E & F ) . Strikingly , most cells that depolarize before shrinking and extruding have markedly more positive membrane potentials than the cells directly neighboring them ( Fig . 4D and Supplemental Video 6 ) . Thus , as cells within a monolayer experience crowding , those with similar membrane potentials remain in the field and shrink slightly whereas those with strikingly lower potentials are eliminated by extrusion . From our measurements of thresholds s i C t r l s i K v 1 . 1 s i C t r l s i K v 1 . 2 s i C t r l s i S W E LL1 0 . 00 0 . 05 0 . 10 0 . 15 0 . 20 0 . 90 0 . 95 1 . 00 1 . 05 1 . 10 F o l d C hange 48 Hours Post Transfection A B qPCR OF KNOCKDOWNS C ION CHANNEL LOCALIZATION XZ XZ ENaC β ENaC γ ZO1 MERGE ZO1 MERGE 20μm 20μm 20μm 20μm XZ Kv1 . 1 20μm Kv1 . 2 20μm SWELL1 20μm XZ XZ ENaC α XZ D s i C t r l s i E N a C α - 0 . 2 0 . 0 0 . 2 0 . 8 0 . 9 1 . 0 1 . 1 1 . 2 F o l d C hange 48 Hours Post Transfection s i C t r l s i C t r l s i E N a C β s i E N a C γ qPCR OF KNOCKDOWNS S . Figure 4 . HES required ion chanel location and siRNA knock down validation . A , Confocal representative projections and xz images of Kv1 . 1 / 1 . 2 , SWELL1 or ENaC a , b , or g ( magenta ) with apical tricellular junction protein ZO - 1 ( green ) . XY Scale bar = 20 m m . B , Table of primer sequences used to quantify siRNA - mediated knockdown for each channel and control GAPDH by qPCR . C , Scatter plots showing mRNA fold change ( 2 - ∆∆Ct ) at 48 hours post transfection ; n = 3 ; where * * * * P = 0 . 0001 from an unpaired T - Test . 13 for shrinkage , the difference in whether a cell extrudes or not may be due to whether the cell shrinks enough ( 20±3 % ) to elicit extrusion . Membrane depolarization before voltage - gated dependent cell shrinkage suggests that another channel acti - vates Kv1 . 1 and Kv1 . 2 . One attractive candidate is the Epithelial Sodium Channel ( ENaC ) —a highly conserved , mechanically activated , apically localized ( Supplementary Fig . 4D ) ion channel that causes membrane depolar - ization via Na + entry 36 , 37 . Inhibiting ENaC with amiloride or knocking down any of its subunits ( a , b , or g , Supple - mentary Figs . 4 B & C ) prevented both HES and OICE ( Fig . 4G & H ) . Importantly , ENaC inhibition with amiloride also prevented depolarization of all epithelial cells in response to hypertonic challenge or during steady state turnover ( Fig . 4C & F ) . Because inhibiting Kv1 . 1 and Kv1 . 2 with 4 - AP does not prevent membrane depolarization ( Fig . 4C & F and Supplementary Fig . 5C ) , we surmise that ENaC triggers membrane depolarization , which , in turn , activates the voltage - gated potassium channels Kv1 . 1 and Kv1 . 2 . If ENaC , Kv1 . 1 , Kv1 . 2 , or SWELL1 control epithelial cell shrinkage and extrusion , their loss should change epithelial morphology . We find that deregulating water and extrusion from knockdown of any of these chan - nels , in fact , causes epithelial buckling ( Fig 4 I & J ) . Compared to control monolayers that shrink and extrude in crowded areas 34 ( Fig1 . H ) , those that lack any one of these channels do not undergo HES or OICE ( Figs . 3D - F and Supplementary Figs . 5A , B , & D ) but instead form progressive buckles as cells crowd ( Supplemental Video 8 ) . Thus , cell shrinkage and extrusion alleviate crowding of epithelia sheets to maintain constant numbers and morphology ; when epithelia cannot regulate their volume , they can become buckled ( see schematic below Fig . A B - 5 : 00 - 2 : 30 0 : 00 2 : 30 5 : 00 - 7 : 00 0 . 5 1 . 0 1 . 5 2 . 0 D i BA C 3 ( 4 ) no r m a li z ed DEPOLARIZATION CONTROL SHRINK ENaC inh . Kv1 . 1 / 2 inhib minutes C C e ll s T ha t E x t r ude B a ck g r ound A m il o r i de E N a C _ E N a C _ E N a C _ 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 20 30 40 50 H ES pe r 1000 C e ll s siRNA D V eh A m i 0 10 20 30 E x t r u s i on s / 1 , 000 C e ll s ISO HYPER V eh A m i - 100 0 100 200 300 400 500 600 % C hange P ea k A r ea ISO HYPER V eh A m i V eh A m i Supplemental Figure 5 . Inhibition of ENaC on membrane potential and extrusion . A , Mean extrusion rate ± SEM of MDCKIIs treated with amiloride , compared to DMSO controls . n = 4 homeostatic ( isotonic ) and n = 3 for hypertonic challenges , where * * P < 0 . 0100 by an unpaired t - test . B , Mean percent peak change in area ± SEM of cells ( by lightning assay ) with amiloride during isotonic or 20 % hypertonic challenge , compared to DMSO controls , n = 3 for homeostatic ( isotonic ) and n = 6 for hypertonic challenge , where * * P = 0 . 0036 derives from an unpaired t - test . C , Rep - resentative depolarization of MDCKII cell over time ( mm : ss ) treated with DMSO control , Kv1 . 1 / 1 . 2 inhibitor 4AP , or ENaC inhibitor amiloride . D , Mean rate of HES ± SEM ( by lightning assay ) in non - extruding cells when treated with amiloride , or with ENaC - a , b or g siRNA , compared to cells that shrink before extruding ; n = 3 where * * * * P < 0 . 0001 is derived from two - way ANOVA with Dunnett’s multiple comparisons test . 14 4K ) . Presumably , in wild type tissues , cells must decrease their plasma membranes by endocytosis or other mechanisms to become smaller overall . Decreasing only cell volume without surface area may instead lead to cell pseudo - stratification with crowding . Thus , we suggest that water movement and pressure is sufficient to control epithelial cell numbers and morphologies . Potentially , the set points for water regulation in different epi - thelia could govern whether tissues become buckled like the gut , kidney , and lungs 38 , 39 or pseudostratified , like neuroepithelia 40 and some adenomas 41 , 42 , reinforcing the role of hydrodynamics in morphogenesis 43 While several models could explain our findings , we favor the following model for how crowding , Na + , K + , and Cl - control epithelial cell volume and turnover ( Fig . 4K ) . As epithelial cells divide and accumulate , they cause crowd - ing within a layer , which activates the tension sensitive sodium channel ENaC that depolarizes cells . Membrane depolarization by ENaC activates Kv1 . 1 and Kv1 . 2 that with Swell1 drive water egress and cell shrinkage . Cells with similar membrane potentials will experience depolarization but only slight water loss and shrinkage within a crowded field . However , ENaC - dependent depolarization in a cell that is already more depolarized than its neigh - bors will reach the threshold for Kv activation more readily . Moreover , mechanical forces can reduce the voltage needed to activate Kv channels and increase their maximal current 44 , 45 . Considering this , ENaC - Kv coupling will be more efficient in a crowded , partially depolarized cell than in its neighbours , further amplifying the initial differ - ence . Thus , a cell under mechanical strain would shrink more readily and significantly , with those shrinking > 17 % of their original volumes triggering extrusion . Cell extrusion would also serve to alleviate crowding and ENaC activation in nearby cells , inhibiting the axis and preventing excessive extrusion . Additionally , the differential in membrane potentials between a cell targeted for extrusion and its neighbors could set up a directional current , which has been shown to activate migration during wound healing 46 . While our study revealed a new role for water regulation and membrane potential in regulating epithelial cell ex - trusion and morphology , it also opened several new questions for further investigation . For instance , shrinkage seems to amplify the crowding experienced in a cell that will extrude , but we still lack information as to how tran - sient cell shrinkage activates Piezo1 to trigger S1P signaling vesicles , essential to extrusion 2 - 4 . Further , while we identified potassium and chloride channels essential for HES , it is likely that aquaporins may work in conjunction with these channels to coordinate water loss ; identifying which will be a future goal . Our findings also revealed the importance of other ion channels in regulating extrusion and it will be important to define what roles they play in controlling extrusion . For example , malfunction of ENaC and CFTR are known drivers of Cystic Fibro - sis 24 , 47 , 48 and mutations or misregulation of ENaC , KV1 . 1 , and Kv1 . 2 are indicators of poor prognosis in a variety of cancers 49 , 50 . As epithelial cell extrusion is a fundamental mechanism for epithelial cell turnover , misregulation of extrusion may contribute to disease etiologies arising from mutations in these channels . 15 Our study identifies early voltage and hydrodynamic regulators as the earliest step controlling homeostatic , crowding - induced cell extrusion , a central driver of epithelial cell death . The channels identified here may be specific for kidney epithelia , yet similar channels expressed in different epithelia 51 may analogously tune tissues to enable bending and specific threshold densities for activating cell shrinkage and extrusion , depending upon organ function and shape . METHODS & MATERIALS Cell culture MDCK II cells ( tested for Mycoplasma but not authenticated , gift from K . Matlin , University of Chicago , Chicago , IL ) were cultured in Dulbecco’s minimum essential medium ( DMEM ) high glucose with 10 % FBS ( Thermofisher ) and 100 μg / ml penicillin / streptomycin ( Invitrogen ) at 5 % CO2 , 37°C . Osmolarity solutions To test induction of live cell extrusion a range of solution osmolarities were produced . Since MDCKII cells are cultured in DMEM , D - Mannitol ( Sigma - aldrich , M4125 - 1kg ) or nuclease - free water ( Ambion , AM9937 ) were add - ed to DMEM to create hyper or hypotonic media , respectively . Initial DMEM osmolarity were measured with a freezing point osmometer ( Gonotec , Osmomat 3000 ) , ranged 334 - 368 mOsm / kg . Solutions were tested bi - week - ly and each time a new batch was prepared . Immunostaining Cells were fixed with 4 % formaldehyde in PBS at room temperature for 20 min , rinsed three times in PBS , perme - abilised for 5 min with PBS containing 0 . 5 % Triton X - 100 , and blocked in AbDil ( PBS 5 % BSA ) . Coverslips were then incubated in primary antibody ( in PBS 1 % BSA ) overnight at 4 ° C , washed three times with PBS , and incu - bated in secondary fluorescently - conjugated antibodies . Antibodies used at 1 : 200 unless otherwise specified : rabbit Piezo1 ( Novus , NBP1 - 78446 ) ; mouse S1P ( Santa Cruz , CA sc - 48356 ) ; rabbit KCNA1 ( Alomone Labs , APC - 161 ) ; rabbit KCNA2 ( Alomone Labs , APC - 010 ) ; rabbit LRRC8A ( Alomone labs , AAC - 001 ) ; mouse ZO1 ( In - vitrogen , 33 - 9100 ) ; rabbit ENaC antibodies SCNNA1 ( Invitrogen , PA1 - 920A ) , SCNNB1 ( Invitrogen , PA5 - 28909 ) , and SCNNG1 ( Invitrogen PA5 - 77797 ) . Alexa Fluor 488 , 568 and 647 goat anti – mouse and anti – rabbit IgG were used as secondary antibodies ( Invitrogen ) . F - actin was stained using either conjugated 488 or 568 Phalloidin ( 66 μ M ) at 1 : 500 and DNA was detected with 1μg / ml DAPI ( Themofisher ) in all fixed cell experiments . Experimental setups and Quantification QPI Micropatterning MDCK II cells for QPI acquisition relies on having nearby cell - free areas for measurement of cell mass . To achieve this , we grew monolayers on small patterned circles within a dish by adhering a silicone laser 16 cut 100 micromesh disk ( Micromesh Array , MMA - 0500 - 100 - 08 - 01 ) on to a non - tissue culture treated 35 mm dish ( Ibidi , 81151 ) . The dishes were plasma treated with mesh in place using a chamber ( Harrick Plasma , Cleaner PD - 32G ) applied in a vacuum ( Agilent Technologies , IDP - 3 Dry Scroll Vacuum pump ) for 10 minutes to create cell adherent coating islands with cell free background ( Supplemental Figure 1A ) . Immediately following plasma treatment , the silicone mesh was aseptically removed and MDCKII cells were seeded at 128 , 000 cell density per well in a microscopy imaging 35 mm dish ( Ibidi , 81151 ) and incubated at 37 ° C for 6 hours in DMEM . Before filming , excess cells were removed from un - patterned areas by gently washed twice with DMEM and grown an - other 48 hours . To image , cells were placed in and on - stage incubator for QPI and islands showing the entire cell island bound - ary and encompassing empty space were filmed . A minimum of two images of empty space were used for back - ground correction . Images were acquired every 2 minutes for 10 hours at 37 ° C , 5 % CO 2 and 88 % humidity . Dry mass was then calculated by subtracting the reference images from the island images to correct background . Next background was flattened by subtracting the background average image to flatten the island images . The phase shift was then converted to dry mass using previous established methods 1 , 2 in Matlab ( version R2022a ) . Extrusions were quantified from 12 separate islands . Volume quantification MDCK II cells were plated at 28 , 000 cell density per well in a microscopy imaging 35 mm dish ( Ibidi , 81156 ) and incubated at 37 ° C overnight or until 60 % confluent . Once 60 % confluent , cells were transfected following manu - facturer’s protocol with Lipofectamine 3000 ( Thermofisher , L3000001 ) with Cytoplasmic GFP plasmid ( addgene , pEGFP - N1 CODE ) for 18 hours . Transfection medium was removed , and cells rinsed twice with 1xPBS and in - cubated in full DMEM until cell to cell junctions were mature ( 72 hours ) . To image , cells were stained with Deep Red Cell Mask ( Thermofisher , 1 . 5 : 1000 ) for 30 minutes and Hoechst ( Invitrogen , 1 : 1000 ) in PBS for 10 minutes at 37 ° C , washed twice with PBS , and then incubated in DMEM media in an enclosed incubated stage at 37°C with 5 % CO 2 ( Oko labs ) and 0 . 4 μ m Z - slices were captured imaged using a spinning disk microscope ( Nikon , Ti2 ) for up to 10 hours every 10 seconds . Images were analysed using a threshold macro ( Nikon Elements AR , 5 . 41 . 02 ) to quantify the volume changes of cells expressing cytoplasmic GFP with cell mask membrane - stained boundaries to highlight extrusions . The volume data was normalized to baseline volume before junctional chang - es and extrusion in Excel ( Microsoft ) and graphed and analysed using GraphPad Prism 9 . 4 . 1 . Serial osmolarity treatment of MDCKII 0 . 53 x10 5 cells were grown to confluence ( ~ 100 hours ) on glass round coverslips 22x55mm ( Academy , 400 - 05 - 21 ) . Epithelial monolayers were incubated with isotonic DMEM for 10 minutes , before treating another 10 minutes in hyper or hypotonic media to induce cell shrinking or swelling , respectively , then replaced in isotonic DMEM for 17 120 mins . Cells were either filmed ( see below ) or fixed and stained for quantifying extrusions . Experiment cell density were analysed with bright spots macro in NIS Elements General Analysis ( Nikon Elements AR , 5 . 41 . 02 ) using DNA staining to determine cell density per field and phalloidin and DNA to identify extrusions . Live Hypertonic Shock For live imaging following hypertonic shock , MDCK II cells were plated on an 8 well slide ( Ibidi , IB - 80801 ) at a seeding density of 10 , 000 cells per well and incubated for ~ 72 hours until monolayers were confluent with mature cell - cell adhesions . Cells were stained with Hoechst ( 1 : 1000 ) in PBS for 10 minutes at 37 ° C , washed twice with PBS , and incubated in isotonic DMEM media ( + / - inhibitors ) , placed in a microscope stage incubator ( 37 ° C , 5 % CO 2 , Okolabs ) , and imaged every 10 seconds for 2 . 5 hours using a widefield microscope ( Nikon , Ti2 specifica - tions below ) . For live cleaved caspase 3 staining , cells were incubated as per manufactures instructions ( 1 : 200 , Incucyte® Caspase - 3 / 7 Dyes ) before treatment and imaging . All experiments consist of three phases : Baseline : 0 - 10minutes - cells incubated in isotonic medium ( + / - inhibitors , see table below or siRNA knockdown Suppl . Fig 3 ) . Hypertonic Challenge - 10 - 20 minutes : imaging is paused , isotonic medium is syphoned off and replaced with 20 % hypertonic medium + / - inhibitors . Imaging promptly restarted to capture shrinkage . Effects on Extrusion - 20minutes - end of imaging : imaging is paused and 20 % hypertonic medium + / - inhibitors is replaced with isotonic medium + / - inhibitors and timelapse imaging resumed to capture extrusions over the next 2hrs . INHIBITOR TABLE Inhibitor Concentration Target 4AP 1mM Voltage - Gated Potassium Channels , e . g . K V 1 . 1 , K V 1 . 2 Azimilide Dihydrochloride ( Azi ) 0 . 5 μ M Voltage Gated Potassium Channel K v 11 . 1 ( hERG ) E4031 1 μ M Voltage Gated Potassium Channel K v 11 . 1 ( hERG ) Apamin 10 μ M Small Conductance Potassium Channel ( SK ) Tram 34 30 μ M Intermediate Conductance Potassium Channel ( IK ) Iberiotoxin 100nM Big Conductance Potassium Channel ( BK ) GdCl 3 10 μ M Stretch Activated ion channels GsMTx4 10 μ M Piezo1 , TRPC1 , and TRPC6 CaCC inh - A01 10 μ M Calcium activated Chloride channel CFTR ( inh ) - 172 5 μ M Cystic Fibrosis Transmembrane Regulator ( CFTR ) DCPIB 20 μ M SWELL1 / LRRC8A Blebbistatin 100 μ M Myosin II Inhibitor Y27632 50 μ M Rho kinase ( ROCK ) H1152 5nM Rho kinase ( ROCK ) SKI - II 30 μ M Sphingosine Kinase SkI V2 4 μ M Pan S1P receptor JTE - 013 10 μ M S1P 2 receptor 18 zVAD - fmk 100 μ M Pan caspase Experiments were then quantified for extrusion rates per 1 , 000 or 10 , 000 cells over timelapse video identified by phase microscopy or % shrinkage by Lightning Assays described below . siRNA knock down 4 - siRNA smart pools ( Horizon Discovery , L - 006210 - 00 - 0010 ( Kv1 . 1 ) , L - 006212 - 00 - 0010 ( Kv1 . 2 ) , L - 026211 - 01 - 0010 ( SWELL1 ) , L - 006504 - 00 - 0010 ENaC α , L - 006505 - 00 - 0010 ( ENaC β ) or L - 006507 - 01 - 0010 ( ENaC γ ) or D - 001810 - 01 - 20 ( non - targeting control ) ) were prepared to in DNase RNase free water to a 100 μ M stock . MDCK II cells were seeded in tandem one set on a 6 well plate ( Thermofisher , 140675 ) at 28 , 000 cell density ( for qPCR analysis ) and the other on a 8 well slide ( Ibidi , IB - 80801 ) at 10 , 000 cell density ( for live cell imaging ) or on coverslips seeded with 53 , 000 MDCK II cells per well on a 24 well plate ( Thermofisher , 142475 ) for extrusion and buckling quantification ( described below ) , and grown overnight until 60 % confluent . Cells were then transfected with RNAi Max kit ( Thermo Fisher , 13778150 ) and 1 μ M siRNA for 24h before re - placing with fresh DMEM for 48h . Cell knockdowns plated for RT - qPCR analyses were lysed for RNA extraction using RNAeasy kit ( Qiagen , 74104 ) , following the manufacturer’s instructions . One μg of RNA was purified with 1 μL of 10x DNAse I Reaction Buffer , 1 μL of DNAse I Amplification and RNase - free water in a final volume of 10 μL . The samples were incubated for 10 min at 37°C then enzyme was deactivated with 1 μL of 0 . 5M EDTA for 10 min at 75°C . Samples were stored at - 20°C . or directly processed by RT - qPCR using Brilliant III Ultra - Fast SYBR Green QRT - PCR Master Mix ( Agilent Technologies ) , using primers designed with SnapGene ® ( version 6 . 1 . 1 ) below and produced by Sigma Aldrich ( see Supplemental Fig 3B for each ) . Reactions were analysed with ViiA 7 Real - Time PCR System ( Thermofisher ) using the following cycle conditions : 50°C for 10 minutes , 95°C for 3 minutes , followed by 40 cycles at 95°C for 15 seconds and 60°C for 30 seconds . Results were normalized to Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) expression and graphed and statistically analysed with GraphPad Prism 9 . 4 . 1 . Extrusion rates were quantified per 1 , 000 cells using timelapse phase microscopy identified and % shrinkage using Lightning Assay , described below . Target Forward P rimer R evers e P rimer GAPDH TTCCACGGCACAGTCAAG ACTCAGCACCAGCATCAC Kv1 . 1 Kv1 . 2 Swell 1 ENaC ᵦ ENaC ɑ ENaC ᵧ TCTTCAAACTCTCCCGCCAC GGAGCTAACGTGGAGCAACT GGCAGTCGTCTCCATGACAA GTTGTTTACCCCCTCCTGG GCCTGTAGCAACTTCTGGTTCAAG CCTGCTCCCCCTCTTTCTTG ACCACTCCTGGATGGCATTG AGGCGCCTACGATCAAAGAG GCGGTACCACTCCCTGACTG TCAGAGGCCTGGCACACAG GGCCTGGGATTGGCTTTAGT CTCCCTCTGTCACGATGGTC TABLE OF SEQUENCES The Lightning Assay 19 To expedite analysis of cell shrinkage following modulation of different channels , we used the ‘lightning assay’ . To do this , areas of interest were cropped out of larger phase microscopy timelapse movies . Junction intensity changes were collected by applying a simple threshold macro based on white light detection before , during , and briefly after extrusion . The threshold was set to capture the area changes around the cells in the frames before homeostatic or experimental cell shrinkage . The same threshold was applied to all frames of movie until com - pletion of cell extrusion . This same method was used for both single cell and whole regions of crowding in an 85 μ m 2 ( 400 by 400 - pixels ) area . Data were then normalized in Microsoft Excel ( version 16 . 67 ) using an average of 10 frames before lightning and analysing peak percentage change , and then graphed and statistically analysed using GraphPad Prism 9 . 4 . 1 . Buckling MDCK II cells were seeded at 0 . 53 x10 5 on glass round coverslips 22x55mm ( Academy , 400 - 05 - 21 ) for fixed imaging or seeded at 1 . 28 x10 5 cells per well in a 35 mm dish for live imaging . After seeding cells were grown overnight , and transfected with either non - targeting control , KV1 . 1 , KV1 . 2 , SWELL1 , ENaC - α , β , or γ siRNA , as described above . After transfection coverslip plated epithelial cells were incubated for cover 48 hours at 37 ° C , then coverslips were fixed and immuno - stained . siRNA - treated MDCK II cells were imaged using a spinning disk confocal microscope with 0 . 4 μ m thick sections and a maximum height of 32 μ m . After transfection of epithelial cells , plated on 35mm dishes , were incubated 24 hours then live imaging was completed using the widefield microscope . Phase images were captured every 10 minutes for 10 or more hours . For both live and fixed buckle analysis , buckles were scored as mounds with more than 3 connected cells within each field and analysed in Graph Pad Prism 9 . 4 . 1 . Depolarization MDCK II cells were seeded at 128 , 000 cells per well in a 35 mm dish and grown ~ 72 hours to confluence and junctional maturity . Once mature , monolayers were stained with DiBAC 4 ( 3 ) following protocol entitled “Track - ing transmembrane voltage using DiBAC 4 ( 3 ) fluorescent dye ( PDF ) ” at : https : / / ase . tufts . edu / biology / labs / levin / resources / protocols . htm ) . Monolayers were then treated with DMSO ( vehicle ) , 4 - AP , or amiloride and imaged ( phase and GFP settings ) every 10 seconds for a minimum of 2 . 5h . The images were analysed using Nikon Elements AR , 5 . 41 . 02 , using a region of interest ( ROI ) over any cell that was maintained or extruded . ROI mean intensity of DiBAC 4 ( 3 ) in each cell over time was normalized in Excel using 10 baseline frames before shrink and depolarization over time was graphed in GraphPad Prism 9 . 4 . 1 . For neighbour voltage differentials , the cells immediately in contact with an extruding cell were classified as brighter , equal , or darker than the extruding cell , based on DiBAC 4 ( 3 ) intensities . Cell counts where then plotted and analysed in Graph Pad Prism 9 . 4 . 1 20 Microscopy QPI Time - lapse QPI and brightfield images were collected on an Olympus IX83 inverted microscope ( Olympus Corp ) using a 40x NA 0 . 75 objective . Samples were illuminated using red LED light ( 623nm , Thorlabs , DC2200 ) for 120ms exposure a QWLSI wavefront sensing camera - ( Phasics SID4 - 4MP ) , driven by Micro Manager open - source microscopy software . Samples were incubated with a stage - top incubator ( Okolabs ) set at 37 ° C tempera - ture with 5 % CO 2 gas and 95 % humidity . Widefield Timelapse phase and fluorescence images were captured on Nikon Eclipse Ti2 using a Plan Fluor 20x Ph1 DLL NA = 0 . 50 objective with a Photometrics Iris 15 16 - bit camera and a Cool LED pE - 4000 lamp driven by NIS Ele - ments ( Nikon , version 5 . 30 . 02 ) . Spinning disk Images were captured on a Nikon Eclipse Ti2 using 20x , 40x plan fluor 0 . 75 air or 60X or 100X plan fluor 1 . 40 oil objectives with an iXon 888 Andor 16 - bit camera , a Yokogawa CSU - W1 confocal spinning disk unit and Toptica photonics laser driven by NIS Elements ( Nikon , 5 . 21 . 03 ) . Cell staining with phalloidin and Hoechst were quanti - fied for extrusions per 1 , 000 or 10 , 000 cells using Nikon Elements Software . Statistical analysis For statistical analysis , all experiments were repeated on at least three separate days to capture variation in the biological replicates . Data were analysed using statistical software GraphPad Prism 9 . 4 . 1 to measure normality by Shapiro - Wilk test and significance by unpaired t - tests , Two - Way ANOVA , or One - way ANOVA with Dunnett’s multiple comparisons , Tukey’s or Sidak’s corrections as described in figure legends . To reduce bias , we imaged random fields within the middle or crowded areas of glass coverslips for quantification or the center of an 8 well dish ( Ibidi 80806 ) for live cell shrink experiments . We excluded low - density epithelia ( fields with less than 2000 cells ) as these regions are not crowded enough to elicit extrusion . Graphs were generated using GraphPad Prism 9 . 4 . 1 . Figure layouts and models were created in Adobe Illustrator ( 26 . 3 . 1 ) . 1 Pradeep , S . & Zangle , T . A . Quantitative phase velocimetry measures bulk intracellular transport of cell mass during the cell cycle . Sci Rep 12 , 6074 ( 2022 ) . https : / / doi . org : 10 . 1038 / s41598 - 022 - 10000 - w 2 Zangle , T . A . & Teitell , M . A . Live - cell mass profiling : an emerging approach in quantitative biophysics . Nat Methods 11 , 1221 - 1228 ( 2014 ) . https : / / doi . org : 10 . 1038 / nmeth . 3175 21 ACKNOWLEDGEMENTS We especially thank Michael Redd for his typical in - depth questioning that made us revise models and improve the manuscript and also to Alexandra Berr , Dustin Bagley , John Fadul , Tobias Russell , Faith Fore , Laura Akin - tche , and Lily Gates for helpful comments on our manuscript . J . R . is the recipient of a National Institute of Health R01GM102169 , a Howard Hughes Faculty Scholar Award ( 55108560 ) , a Cancer Research UK Programme Grant ( DRCNPG - May21 \ 100007 ) , an Academy of Medical Sciences Professorship ( APR2 \ 1007 ) , and a Wellcome Trust Investigator Award ( 221908 / Z / 20 / Z ) C . P . - P . is the recipient of a Long - Term Fellowship ( LT000654 / 2019 - L ) from the Human Frontier Science Program organization and a Marie Skłodowska - Curie Fellowship ( 898067 ) from the European Union’s Horizon 2020 research and innovation program . 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C , Lightning assay , showing representative stills from time - lapse phase microscopy where white space is thresholded ( red , bottom ) at key points during extrusion , quantified in ( D ) as mean ± SEM of normalized white area around cell , where t0 is shrink ; n = 10 cells . E , Percent cells that shrink before extruding ± SEM and P from unpaired T - test using n = 10 biological replicates . F , Normalized area mean ± SEM of cells at each defined step : baseline , shrink maximum , relax maximum and extrusion ; n = 15 cells ; p values from two - way ANOVA with Dun - nett’s multiple comparisons test . G , Normalized mean volume ± SEM of cells at baseline , shrink maximum , relax maximum , and onset of extrusion ; n = 5 cells with p values from two - way ANOVA with Dunnett’s multiple com - parisons test . H , Regional density - dependence ofcell shrinkage ( red ) with or without extrusion , compared to no shrink ( black ) ; as mean ± SEM , n = 3 with one - way ANOVA with Dunnett’s multiple comparisons test . I , Mean rate of HES with inhibitors of cell extrusion ± SEM ; n = 3 , using one way ANOVA with Dunnett’s multiple comparisons test . For all graphs , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 or * P = 0 . 0124 . Figure 2 : Water loss is sufficient to induce live cell extrusion . A , Mean ± SEM MDCKII extrusion rate fol - lowing incubation in media with increasing osmolarity for 100’ , scored by immunostaining for actin and DNA , with n = 3 experiments ; where * * P = 0 . 0068 ; * * * * P < 0 . 0001 by one way ANOVA with Dunnett’s multiple comparisons test . B , Mean ± SEM extrusion rates in isotonic medium or 100’ following 10 minute 20 % hypertonic shock , where apoptotic extrusion is scored by immunostaining with cleaved caspase - 3 antibody ; n = 3 experiments ; and * P = 0 . 0105 is from an unpaired T - test . C , Volume changes ( measured by confocal sectioning of mosaically ex - pressed cytoplasmic GFP ) over time in cells shifted from isotonic to 20 % hypertonic media that extrude ( left ) or do not ( right ) , n = 5 . D , Number of extrusions in MDCKII monolayers incubated with volume regulating inhibitors and scored by immunostaining for actin and DNA ; n = 3 ; where * * * * P < 0 . 0001 by one way ANOVA with Dunnett’s multiple comparisons test . Inhibitor key describes volume regulating channel family inhibitor target by assigned 26 color and icon . Figure 3 : Homeostatic live cell extrusion requires K + voltage - gated and Cl - channel activation . A , Mean ± SEM extrusion rates from monolayers treated with inhibitors only during the 10 minutes ofof 20 % hypertonic treatment n = 3 ; P values from two - way ANOVA with Dunnett’s multiple comparisons test . B , Adapted lightning assay for a crowded epithelial field following hypertonic treatment , quantified over time . C , Mean ±SEM percent peak change in area surrounding cells ( from adapted lightning assay ) with inhibitors during 20 % hypertonic challenge , n = 6 ; with P values from a two - way ANOVA with Dunnett’s multiple comparisons test . Channel nhibitor key ( for B & C ) . D , Mean ± SEM extrusion rateof MDCKIIs with siRNA - knockdowns ± 20 % hypertonic challenge , compared to controls ; n = 4 ; P values from two - way ANOVA with Dunnett’s multiple comparisons test . E , Percent change of peak area ( from adapted lightning assay ) ± SEM during hypertonic challenge of siRNA - knockdowns , compared to controls , n = 6 ; with P values from one - way ANOVA with Dunnett’s multiple comparisons test . F , Mean ± SEM rate of HESwhen treated with 4AP , DCPIB , or with Kv1 . 1 , Kv1 . 2 or SWELL1 siRNA knockdowns , compared to control background cells or those that shrink before extruding ; n = 3 ; where P values are from two - way ANOVA with Dunnett’s multiple comparisons test . For all graphs , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 0100 , * P < 0 . 05 Figure 4 : ENaC - dependent depolarization activates cellular shrinkage and extrusion . A , Membrane depo - larization , measured by DiBAC 3 ( 4 ) fluorescence ( greater depolarization with higher fluorescence ) , in sub - con - fluent , confluent , and crowded MDCKII monolayers . Scale bar = 50µm . B , Density - dependence of membrane potential , as measured by DiBAC 3 ( 4 ) fluorescence , n = 3 . C , Timelapse ( mins ) measuring membrane depolar - ization before homeostatic extrusion ( top panel , red arrow indicating a cell before extrusion ) , in the presence of 4AP ( KV1 . 1 / 2 inhibitor , middle panel ) , or amiloride ( ENaC inhibitor , bottom panel ) . D , Mean ± SEM cells with darker , equal , or brighter DiBAC 3 ( 4 ) signal before extruding , compared to neighbouring non - extruding cells ; n = 3 experiments ; P values from one - way ANOVA with Tukey’s multiple comparisons test . E , Normalized DiBAC 3 ( 4 ) fluorescence , as mean ± SEM from cells that undergo HES / extrusion ( blue ) or not ( red ) over time , n = 6 . F , Mean ± SEM membrane polarization status , as measured by DiBAC 3 ( 4 ) fluorescence of cells before they extrude com - pared to non - extruding cells in background or when Kv1 . 1 / Kv1 . 2 or ENaC are inhibited with 4AP or amiloride ; n = 3 experiments ; P values from one - way ANOVA with Tukey’s multiple comparisons test . G , Peak mean ± SEM percent area change ( by adapted lightning assay ) of siRNA - mediated ENaC - a , b , or g knockdown in MDCKs with and without 20 % hypertonic challenge , compared to non - targeted controls , n = 3 for each siRNA , with P values from a two - way ANOVA with Dunnett’s multiple comparisons test . H , Mean ± SEM extrusion rates of ENaC - a , b , or g siRNA - mediated knockdown with and without 20 % hypertonic challenge , compared to control , n = 3 ; with P values from two - way ANOVA with Dunnett’s multiple comparisons test . I , Representative volume view of control 27 and ion channel knocked down monolayers , with alpha color bit coded bar indicating depth 0 . 0 - 16 . 0mm ( blue - pink ) . Schematics below depict how functional extrusion maintains monolayer shape whereas disrupting water regulation leads to buckling . J , Mean ± SEM number of epithelial buckles per field48 hours post siRNA - treatment compared to non - target controls ; n3 ; with P values from two - way ANOVA with Dunnett’s multiple comparisons test . K , Model for how ENaC / Kv1 . 1 / Kv1 . 2 / Swell1 - dependent water regulation controls cell size and extrusion , depending on cellular membrane potential . Left : If one cell is more depolarized than its neighboring cells , crowd activation of ENaC will preferentially activate voltage - gated potassium channels in this cell , causing shrink . If this shrink reaches a threshold of ~ 20 % , it will activate the extrusion pathway . Cell elimination by extrusion will alleviate crowding pressure in the monolayer and maintain constant densities . Right : Crowd activation of ENaC in cells with equivalent membrane potentials cause moderate voltage - gated potassium - dependent water loss across all cells , resulting in slight cell shrinkage but not enough to activate cell extrusion . Here , slight shrinkage also helps accommodate overall crowding of the monolayer . In this way , crowding can set up competition to eliminate cells with the lowest energy that cannot maintain a sufficient membrane potential , a significant energy demand for the cell . For all graphs , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 0100 . SUPPLEMENTAL FIGURES S . Figure 1 . Quantitative phase imaging ( QPI ) shows unaltered dry mass in cells that will extrude . A , As QPI requires a reference area without cells throughout imaging , we grew MDCKII monolayers on confined 100mm islands within a 35 mm dish by plasma treating patterned areas , as in this example . Scale bar = 100mm . B , QPI stills from Supplemental video 1 , representing 31 other cells from 12 QPI islands that similarly show dry mass before cell extrusion , where blue is low cell mass and red is higher mass . Note mass increases after cell extrusion , as the density gets higher with cell contraction ( h : mm ) . C , By contrast , another cell , in red box , increas - es mass before dividing . Scale bar = 50mm . S . Figure 2 . HES does not occur before apoptotic cell extrusion . A , Stills from a phase ( top ) timelapse and fluorescent apoptosis marker ( Cell Event - caspase 3 / 7 , bottom ) before apoptotic extrusion . B , Mean ± SEM percentage of cells that shrink by lightning assay before extruding . n = 5 ; * * * * P < 0 . 0001 by an unpaired T - test . C , Mean ± SEM extrusion rates in monolayers treated with SWELL1 inhibitor DCPIB ± pan - caspase inhibitor zVAD - FMK ; n = 3 ; where * P = 0 . 0104 values are from one - way ANOVA with Sidak’s multiple comparisons test . S . Figure 3 . Ion channel inhibitors impact on extrusion and shrink . A , Mean ± SEM extrusion rates in MD - CKII monolayers treated with inhibitors before , during , and after hypertonic shock ; n = 3 ; where * * * * P = 0 . 0001 by one way ANOVA with Dunnett’s multiple comparisons test . B , Mean ± SEM extrusion rates in monolayers treated with inhibitors only during hypertonic shock . n = 3 experiments where * * P = 0 . 0024 is from two - way ANOVA with Dunnett’s multiple comparisons test . Inhibitor key for A and B describes ion channel family inhibitor target by 28 color icon . Representative “Lightning assays” where increased space around the cells indicates cell shrinkage in the presences of : 4AP ( KV1 . 1 & KV1 . 2 inhibitor ) ( C ) , DCPIB ( SWELL1 inhibitor ) ( D ) , SAC inhibitor GsMTx4 ( E ) , or S1P / S1P 2 inhibitors ( F ) , compared to DMSO controls before and during hypertonic media incubation ( mins ) . S . Figure 4 . HES required ion chanel location and siRNA knock down validation . A , D Confocal representative projections and xz images of Kv1 . 1 / 1 . 2 , SWELL1 ( A ) or ENaC a , b , or g ( D ) ( ma - genta ) with apical tricellular junction protein ZO - 1 ( green ) . XY Scale bar = 20 m m . B , Table of primer sequences used to quantify siRNA - mediated knockdown for each channel and control GAPDH by qPCR . C , E Mean ± SEM mRNA fold change ( 2 - ∆∆Ct ) at 48 hours post transfection ; n = 3 ; where * * * * P = 0 . 0001 from an unpaired T - Test . S . Figure 5 . Inhibition of ENaC on membrane potential and extrusion . A , Mean extrusion rate ± SEM of MDCKIIs treated with amiloride , compared to DMSO controls . n = 4 homeostatic ( isotonic ) and n = 3 for hypertonic challenges , where * * P < 0 . 0100 by an unpaired t - test . B , Mean percent peak change in area ± SEM of cells ( by lightning assay ) with amiloride during isotonic or 20 % hypertonic challenge , compared to DMSO controls , n = 3 for homeostatic ( isotonic ) and n = 6 for hypertonic challenge , where * * P = 0 . 0036 derives from an unpaired t - test . C , Representative depolarization of MDCKII cell over time ( mm : ss ) treated with DMSO control , Kv1 . 1 / 1 . 2 inhibitor 4AP , or ENaC inhibitor amiloride . D , Mean rate of HES ± SEM ( by lightning assay ) in non - extruding cells when treated with amiloride , or with ENaC - a , b or g siRNA , compared to cells that shrink before extruding ; n = 3 where * * * * P < 0 . 0001 is derived from two - way ANOVA with Dunnett’s multiple com - parisons test . Supplemental Videos S Video 1 : MDCKII dry mass changes before extrusion . Images taken every 2 minutes using QPI adapted tech - nique shown in parula color map . S Video 2 : Phase timelapse ( hh : mm ) of MDCKII cell show junctional lightning before extrusion . S Video 3 : Phase and cleaved caspase 3 ( green ) timelapse of apoptotic MDCKII cell that does not show junc - tional lightning before extruding . S Video 4 : Phase microscopy timelapse ( hh : mm ) of MDCKII monolayer in isotonic media . S Video 5 : Phase microscopy timelapse ( hh : mm ) of MDCKII cells after ten - minute incubation in 20 % hypertonic media undergo increased rate of extrusion . S Video 6 : Phase and DiBAC 4 ( 3 ) ( green ) timelapse ( mm : ss ) showing depolarization of MDCKs before extrusion . S Video 7 : Phase and DiBAC 4 ( 3 ) ( green ) timelapse ( mm : ss ) showing differential depolarization as MDCKs crowd ( red arrows ) and extrude ( white arrows ) . Scale bar ( white ) 25mm . S Video 8 : Representative phase timelapse ( mm : ss ) of MDCKII monolayers where Swell1 is knocked down , which buckle over time , with scale bar 25mm . \ No newline at end of file diff --git a/silver_data/34d7a07c493ca6336c92156806a2947e115caadc.pdf.txt b/silver_data/34d7a07c493ca6336c92156806a2947e115caadc.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..42273cd6c43f8c5579922ccbd506a5d5cc7ba459 --- /dev/null +++ b/silver_data/34d7a07c493ca6336c92156806a2947e115caadc.pdf.txt @@ -0,0 +1 @@ +Proceedings of the Second Workshop on Statistical Machine Translation , pages 228 – 231 , Prague , June 2007 . c (cid:13) 2007 Association for Computational Linguistics Meteor : An Automatic Metric for MT Evaluation with High Levels of Correlation with Human Judgments Alon Lavie and Abhaya Agarwal Language Technologies Institute Carnegie Mellon University Pittsburgh , PA , 15213 , USA { alavie , abhayaa } @ cs . cmu . edu Abstract Meteor is an automatic metric for Ma - chine Translation evaluation which has been demonstrated to have high levels of corre - lation with human judgments of translation quality , significantly outperforming the more commonly used Bleu metric . It is one of several automatic metrics used in this year’s shared task within the ACL WMT - 07 work - shop . This paper recaps the technical de - tails underlying the metric and describes re - cent improvements in the metric . The latest release includes improved metric parameters and extends the metric to support evalua - tion of MT output in Spanish , French and German , in addition to English . 1 Introduction Automatic Metrics for MT evaluation have been re - ceiving significant attention in recent years . Evalu - ating an MT system using such automatic metrics is much faster , easier and cheaper compared to human evaluations , which require trained bilingual evalua - tors . Automatic metrics are useful for comparing the performance of different systems on a common translation task , and can be applied on a frequent and ongoing basis during MT system development . The most commonly used MT evaluation metric in recent years has been IBM’s Bleu metric ( Papineni et al . , 2002 ) . Bleu is fast and easy to run , and it can be used as a target function in parameter op - timization training procedures that are commonly used in state - of - the - art statistical MT systems ( Och , 2003 ) . Various researchers have noted , however , var - ious weaknesses in the metric . Most notably , Bleu does not produce very reliable sentence - level scores . Meteor , as well as several other proposed metrics such as GTM ( Melamed et al . , 2003 ) , TER ( Snover et al . , 2006 ) and CDER ( Leusch et al . , 2006 ) aim to address some of these weaknesses . Meteor , initially proposed and released in 2004 ( Lavie et al . , 2004 ) was explicitly designed to im - prove correlation with human judgments of MT qual - ity at the segment level . Previous publications on Meteor ( Lavie et al . , 2004 ; Banerjee and Lavie , 2005 ) have described the details underlying the met - ric and have extensively compared its performance with Bleu and several other MT evaluation met - rics . This paper recaps the technical details underly - ing Meteor and describes recent improvements in the metric . The latest release extends Meteor to support evaluation of MT output in Spanish , French and German , in addition to English . Furthermore , several parameters within the metric have been opti - mized on language - specific training data . We present experimental results that demonstrate the improve - ments in correlations with human judgments that re - sult from these parameter tunings . 2 The Meteor Metric Meteor evaluates a translation by computing a score based on explicit word - to - word matches be - tween the translation and a given reference trans - lation . If more than one reference translation is available , the translation is scored against each refer - ence independently , and the best scoring pair is used . Given a pair of strings to be compared , Meteor cre - ates a word alignment between the two strings . An alignment is mapping between words , such that ev - ery word in each string maps to at most one word in the other string . This alignment is incrementally produced by a sequence of word - mapping modules . The “exact” module maps two words if they are ex - actly the same . The “porter stem” module maps two words if they are the same after they are stemmed us - ing the Porter stemmer . The “WN synonymy” mod - ule maps two words if they are considered synonyms , based on the fact that they both belong to the same “synset” in WordNet . The word - mapping modules initially identify all 228 possible word matches between the pair of strings . We then identify the largest subset of these word mappings such that the resulting set constitutes an alignment as defined above . If more than one maxi - mal cardinality alignment is found , Meteor selects the alignment for which the word order in the two strings is most similar ( the mapping that has the least number of “crossing” unigram mappings ) . The order in which the modules are run reflects word - matching preferences . The default ordering is to first apply the “exact” mapping module , followed by “porter stemming” and then “WN synonymy” . Once a final alignment has been produced between the system translation and the reference translation , the Meteor score for this pairing is computed as follows . Based on the number of mapped unigrams found between the two strings ( m ) , the total num - ber of unigrams in the translation ( t ) and the total number of unigrams in the reference ( r ) , we calcu - late unigram precision P = m / t and unigram recall R = m / r . We then compute a parameterized har - monic mean of P and R ( van Rijsbergen , 1979 ) : F mean = P · R α · P + ( 1 − α ) · R Precision , recall and Fmean are based on single - word matches . To take into account the extent to which the matched unigrams in the two strings are in the same word order , Meteor computes a penalty for a given alignment as follows . First , the sequence of matched unigrams between the two strings is di - vided into the fewest possible number of “chunks” such that the matched unigrams in each chunk are adjacent ( in both strings ) and in identical word or - der . The number of chunks ( ch ) and the number of matches ( m ) is then used to calculate a fragmenta - tion fraction : frag = ch / m . The penalty is then computed as : Pen = γ · frag β The value of γ determines the maximum penalty ( 0 ≤ γ ≤ 1 ) . The value of β determines the functional relation between fragmentation and the penalty . Finally , the Meteor score for the align - ment between the two strings is calculated as : score = ( 1 − Pen ) · F mean In all previous versions of Meteor , the values of the three parameters mentioned above were set to be : α = 0 . 9 , β = 3 . 0 and γ = 0 . 5 , based on experimen - tation performed in early 2004 . In the latest release , we tuned these parameters to optimize correlation with human judgments based on more extensive ex - perimentation , as reported in section 4 . 3 Meteor Implementations for Spanish , French and German We have recently expanded the implementation of Meteor to support evaluation of translations in Spanish , French and German , in addition to English . Two main language - specific issues required adapta - tion within the metric : ( 1 ) language - specific word - matching modules ; and ( 2 ) language - specific param - eter tuning . The word - matching component within the English version of Meteor uses stemming and synonymy modules in constructing a word - to - word alignment between translation and reference . The re - sources used for stemming and synonymy detection for English are the Porter Stemmer ( Porter , 2001 ) and English WordNet ( Miller and Fellbaum , 2007 ) . In order to construct instances of Meteor for Span - ish , French and German , we created new language - specific “stemming” modules . We use the freely available Perl implementation packages for Porter stemmers for the three languages ( Humphrey , 2007 ) . Unfortunately , we have so far been unable to obtain freely available WordNet resources for these three languages . Meteor versions for Spanish , French and German therefore currently include only “exact” and “stemming” matching modules . We are investi - gating the possibility of developing new synonymy modules for the various languages based on alterna - tive methods , which could then be used in place of WordNet . The second main language - specific issue which required adaptation is the tuning of the three parameters within Meteor , described in section 4 . 4 Optimizing Metric Parameters The original version of Meteor ( Banerjee and Lavie , 2005 ) has instantiated values for three pa - rameters in the metric : one for controlling the rela - tive weight of precision and recall in computing the Fmean score ( α ) ; one governing the shape of the penalty as a function of fragmentation ( β ) and one for the relative weight assigned to the fragmenta - tion penalty ( γ ) . In all versions of Meteor to date , these parameters were instantiated with the values α = 0 . 9 , β = 3 . 0 and γ = 0 . 5 , based on early data ex - perimentation . We recently conducted a more thor - ough investigation aimed at tuning these parameters based on several available data sets , with the goal of finding parameter settings that maximize correlation with human judgments . Human judgments come in the form of “adequacy” and “fluency” quantitative scores . In our experiments , we looked at optimizing parameters for each of these human judgment types separately , as well as optimizing parameters for the sum of adequacy and fluency . Parameter adapta - 229 Corpus Judgments Systems NIST 2003 Ara - to - Eng 3978 6 NIST 2004 Ara - to - Eng 347 5 WMT - 06 Eng - to - Fre 729 4 WMT - 06 Eng - to - Ger 756 5 WMT - 06 Eng - to - Spa 1201 7 Table 1 : Corpus Statistics for Various Languages tion is also an issue in the newly created Meteor instances for other languages . We suspected that parameters that were optimized to maximize corre - lation with human judgments for English would not necessarily be optimal for other languages . 4 . 1 Data For English , we used the NIST 2003 Arabic - to - English MT evaluation data for training and the NIST 2004 Arabic - to - English evaluation data for testing . For Spanish , German and French we used the evaluation data provided by the shared task at last year’s WMT workshop . Sizes of various corpora are shown in Table 1 . Some , but not all , of these data sets have multiple human judgments per translation hypothesis . To partially address human bias issues , we normalize the human judgments , which trans - forms the raw judgment scores so that they have sim - ilar distributions . We use the normalization method described in ( Blatz et al . , 2003 ) . Multiple judgments are combined into a single number by taking their average . 4 . 2 Methodology We performed a “hill climbing” search to find the parameters that achieve maximum correlation with human judgments on the training set . We use Pear - son’s correlation coefficient as our measure of corre - lation . We followed a “leave one out” training proce - dure in order to avoid over - fitting . When n systems were available for a particular language , we train the parameters n times , leaving one system out in each training , and pooling the segments from all other systems . The final parameter values are calculated as the mean of the n sets of trained parameters that were obtained . When evaluating a set of parameters on test data , we compute segment - level correlation with human judgments for each of the systems in the test set and then report the mean over all systems . 4 . 3 Results 4 . 3 . 1 Optimizing for Adequacy and Fluency We trained parameters to obtain maximum cor - relation with normalized adequacy and fluency judg - Adequacy Fluency Sum α 0 . 82 0 . 78 0 . 81 β 1 . 0 0 . 75 0 . 83 γ 0 . 21 0 . 38 0 . 28 Table 2 : Optimal Values of Tuned Parameters for Different Criteria for English Adequacy Fluency Sum Original 0 . 6123 0 . 4355 0 . 5704 Adequacy 0 . 6171 0 . 4354 0 . 5729 Fluency 0 . 6191 0 . 4502 0 . 5818 Sum 0 . 6191 0 . 4425 0 . 5778 Table 3 : Pearson Correlation with Human Judg - ments on Test Data for English ments separately and also trained for maximal corre - lation with the sum of the two . The resulting optimal parameter values on the training corpus are shown in Table 2 . Pearson correlations with human judgments on the test set are shown in Table 3 . The optimal parameter values found are somewhat different than our previous metric parameters ( lower values for all three parameters ) . The new parame - ters result in moderate but noticeable improvements in correlation with human judgments on both train - ing and testing data . Tests for statistical significance using bootstrap sampling indicate that the differ - ences in correlation levels are all significant at the 95 % level . Another interesting observation is that precision receives slightly more “weight” when op - timizing correlation with fluency judgments ( versus when optimizing correlation with adequacy ) . Recall , however , is still given more weight than precision . Another interesting observation is that the value of γ is higher for fluency optimization . Since the frag - mentation penalty reflects word - ordering , which is closely related to fluency , these results are consistent with our expectations . When optimizing correlation with the sum of adequacy and fluency , optimal val - ues fall in between the values found for adequacy and fluency . 4 . 3 . 2 Parameters for Other Languages Similar to English , we trained parameters for Spanish , French and German on the available WMT - 06 training data . We optimized for maximum corre - lation with human judgments of adequacy , fluency and for the sum of the two . Resulting parameters are shown in Table 4 . 3 . 2 . For all three languages , the parameters that were found to be optimal were quite different than those that were found for English , and using the language - specific optimal parameters re - 230 Adequacy Fluency Sum French : α 0 . 86 0 . 74 0 . 76 β 0 . 5 0 . 5 0 . 5 γ 1 . 0 1 . 0 1 . 0 German : α 0 . 95 0 . 95 0 . 95 β 0 . 5 0 . 5 0 . 5 γ 0 . 6 0 . 8 0 . 75 Spanish : α 0 . 95 0 . 62 0 . 95 β 1 . 0 1 . 0 1 . 0 γ 0 . 9 1 . 0 0 . 98 Table 4 : Tuned Parameters for Different Languages sults in significant gains in Pearson correlation levels with human judgments on the training data ( com - pared with those obtained using the English opti - mal parameters ) 1 . Note that the training sets used for these optimizations are comparatively very small , and that we currently do not have unseen test data to evaluate the parameters for these three languages . Further validation will need to be performed once ad - ditional data becomes available . 5 Conclusions In this paper we described newly developed language - specific instances of the Meteor metric and the process of optimizing metric parameters for different human measures of translation quality and for different languages . Our evaluations demonstrate that parameter tuning improves correlation with hu - man judgments . The stability of the optimized pa - rameters on different data sets remains to be inves - tigated for languages other than English . We are currently exploring broadening the set of features used in Meteor to include syntax - based features and alternative notions of synonymy . The latest re - lease of Meteor is freely available on our website at : http : / / www . cs . cmu . edu / ~ alavie / METEOR / Acknowledgements The work reported in this paper was supported by NSF Grant IIS - 0534932 . References Satanjeev Banerjee and Alon Lavie . 2005 . ME - TEOR : An Automatic Metric for MT Evalua - tion with Improved Correlation with Human Judg - ments . In Proceedings of the ACL Workshop on Intrinsic and Extrinsic Evaluation Measures 1 Detailed tables are not included for lack of space . for Machine Translation and / or Summarization , pages 65 – 72 , Ann Arbor , Michigan , June . John Blatz , Erin Fitzgerald , George Foster , Simona Gandrabur , Cyril Goutte , Alex Kulesza , Alberto Sanchis , and Nicola Ueffing . 2003 . Confidence Es - timation for Machine Translation . Technical Re - port Natural Language Engineering Workshop Fi - nal Report , Johns Hopkins University . Marvin Humphrey . 2007 . Perl In - terface to Snowball Stemmers . http : / / search . cpan . org / creamyg / Lingua - Stem - Snowball - 0 . 941 / lib / Lingua / Stem / Snowball . pm . Alon Lavie , Kenji Sagae , and Shyamsundar Jayara - man . 2004 . The Significance of Recall in Auto - matic Metrics for MT Evaluation . In Proceedings of the 6th Conference of the Association for Ma - chine Translation in the Americas ( AMTA - 2004 ) , pages 134 – 143 , Washington , DC , September . Gregor Leusch , Nicola Ueffing , and Hermann Ney . 2006 . CDER : Efficient MT Evaluation Using Block Movements . In Proceedings of the Thir - teenth Conference of the European Chapter of the Association for Computational Linguistics . I . Dan Melamed , Ryan Green , and Joseph Turian . 2003 . Precision and Recall of Machine Transla - tion . In Proceedings of the HLT - NAACL 2003 Conference : Short Papers , pages 61 – 63 , Edmon - ton , Alberta . George Miller and Christiane Fellbaum . 2007 . Word - Net . http : / / wordnet . princeton . edu / . Franz Josef Och . 2003 . Minimum Error Rate Train - ing for Statistical Machine Translation . In Pro - ceedings of the 41st Annual Meeting of the Asso - ciation for Computational Linguistics . Kishore Papineni , Salim Roukos , Todd Ward , and Wei - Jing Zhu . 2002 . BLEU : a Method for Auto - matic Evaluation of Machine Translation . In Pro - ceedings of 40th Annual Meeting of the Association for Computational Linguistics ( ACL ) , pages 311 – 318 , Philadelphia , PA , July . Martin Porter . 2001 . The Porter Stem - ming Algorithm . http : / / www . tartarus . org / mar - tin / PorterStemmer / index . html . Matthew Snover , Bonnie Dorr , Richard Schwartz , Linnea Micciulla , and John Makhoul . 2006 . A Study of Translation Edit Rate with Targeted Hu - man Annotation . In Proceedings of the 7th Confer - ence of the Association for Machine Translation in the Americas ( AMTA - 2006 ) , pages 223 – 231 , Cam - bridge , MA , August . C . van Rijsbergen , 1979 . Information Retrieval . Butterworths , London , UK , 2nd edition . 231 \ No newline at end of file diff --git a/silver_data/351fdc720975271c3b04fc8dc86658bebb88aea7.pdf.txt b/silver_data/351fdc720975271c3b04fc8dc86658bebb88aea7.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..bca49ea4f6a0a32b542cc187a73116a7c4e9fa3d --- /dev/null +++ b/silver_data/351fdc720975271c3b04fc8dc86658bebb88aea7.pdf.txt @@ -0,0 +1 @@ +Confidential . Do not distribute . Pre - embargo material . STUDY Incidence Estimate of Nonmelanoma Skin Cancer in the United States , 2006 Howard W . Rogers , MD , PhD ; Martin A . Weinstock , MD , PhD ; Ashlynne R . Harris ; Michael R . Hinckley , MD ; Steven R . Feldman , MD ; Alan B . Fleischer , MD ; Brett M . Coldiron , MD Objectives : To estimate the incidence of nonmela - noma skin cancer ( NMSC ) in the US population in 2006 and secondarily to indicate trends in numbers of proce - dures for skin cancer treatment . Design : A descriptive analysis of population - based claims and US Census Bureau data combined with a population - based cross - sectional survey using multiple US govern - ment data sets , including the Centers for Medicare and Medicaid Services Fee - for - Service Physicians Claims da - tabases , to calculate totals of skin cancer procedures per - formed for Medicare beneficiaries in 1992 and from 1996 to 2006 and related parameters . The National Ambula - tory Medical Care Service database was used to estimate NMSC - related office visits . We combined these to esti - mate totals of new skin cancer diagnoses and affected in - dividuals in the overall US population . Results : The total number of procedures for skin can - cer in the Medicare fee - for - service population increased by 76 . 9 % from 1 158 298 in 1992 to 2 048 517 in 2006 . The age - adjusted procedure rate per year per 100 000 ben - eficiaries increased from 3514 in 1992 to 6075 in 2006 . From 2002 to 2006 ( the years for which the databases allow procedure linkage to patient demographics and di - agnoses ) , the number of procedures for NMSC in the Medicare population increased by 16 . 0 % . In this pe - riod , the number of procedures per affected patient in - creased by 1 . 5 % , and the number of persons with at least 1 procedure increased by 14 . 3 % . We estimate the total number of NMSCs in the US population in 2006 at 3 507 693 and the total number of persons in the United States treated for NMSC at 2 152 500 . Conclusions : The number of skin cancers in Medicare beneficiaries increased dramatically over the years 1992 to 2006 , due mainly to an increase in the number of af - fected individuals . Using nationally representative data - bases , we provide evidence of much higher overall to - tals of skin cancer diagnoses and patients in the US population than previous estimates . These data give the most complete evaluation to date of the underrecog - nized epidemic of skin cancer in the United States . Arch Dermatol . 2010 ; 146 ( 3 ) : 283 - 287 N ONMELANOMA SKIN CAN - cer ( NMSC ) is the most common malignancy in the United States , with substantial associated morbidity and cost , as well as relatively smaller but significant mortality . The most recent , peer - reviewed , published , na - tional incidence estimate is from 1994 . This study estimated 900 000 to 1 200 000 NMSCs in that year , approximately equal - ing all other cases of human malignan - cies combined . 1 , 2 However , the exact in - cidence of NMSC is unknown because the condition is not typically reported to can - cer registries . Several studies have documented trends in skin cancer incidence in the US popu - lation . 3 - 10 A study of 3 counties in New Mexico described increases in age - adjusted NMSC rates from 71 . 8 per 100 000 persons in 1972 to 150 . 4 per 100 000 persons in 1999 , a 109 % in - crease . 6 Evaluation of the Southeastern Ari - zona Skin Cancer Registry demonstrated a leveling off of NMSC incidence with a decrease in squamous cell carcinoma ( SCC ) rates . 10 Another study of age - adjusted rates for NMSC in New Hamp - shire showed a 235 % increase in females and a 350 % increase in males for SCC , and an 80 % increases in basal cell carcinoma ( BCC ) incidence from 1979 to 1993 . 4 In - creases in skin cancer incidence have also been documented in multiple areas out - side the United States . 11 - 15 Understanding skin cancer incidence and treatment is important for planning prevention strategies and allocating re - sourcesfortreatment . Medicareclaimsdata Author Affiliations : Advanced Dermatology , Norwich , Connecticut ( Dr Rogers ) ; Departments of Dermatology and Community Health , Brown Medical School , Providence , Rhode Island ( Dr Weinstock ) ; Alpert Medical School at Brown University , Providence ( Ms Harris ) ; Department of Dermatology , Wake Forest University School of Medicine , Winston - Salem , North Carolina ( Drs Hinckley , Feldman , and Fleischer ) ; and Department of Dermatology , University of Cincinnati Hospital , Cincinnati , Ohio ( Dr Coldiron ) . ( REPRINTED ) ARCH DERMATOL / VOL 146 ( NO . 3 ) , MAR 2010 WWW . ARCHDERMATOL . COM 283 ©2010 American Medical Association . All rights reserved . Confidential . Do not distribute . Pre - embargo material . from 1992 to 1995 showed that the cost per affected pa - tient per year of NMSC was only 5 % to 10 % that of many other cancers . 16 For example , in 1992 , the costs per af - fected patient of stomach , lung , breast , melanoma , and NMSC were $ 9010 , $ 5609 , $ 1718 , $ 1130 , and $ 431 , re - spectively . However , the large number of cases made NMSC the fifth most costly cancer to treat overall in the Medicare population , with NMSC accounting for over 4 . 5 % of all Medicare cancer costs and over 0 . 7 % of the Medicare budget for physician services . 16 , 17 Further - more , these costs increased 41 % between 1992 and 1995 . 16 The primary purpose of this study is to estimate 2006 NMSC incidence in the United States using national popu - lation - based data sources . METHODS DATA SOURCES Our analyses were based primarily on 2 distinct Medicare da - tabases and on national survey data . The Medicare physician / supplier procedure summary master file ( hereinafter , Total Claims Data Set ) was analyzed for the years 1992 and 1996 to 2006 ( available years ) . 18 For our primary approach to the es - timation of NMSC , the 2006 Total Claims Data Set was used to provide total numbers of approved fee - for - service Medicare claims categorized by Current Procedural Terminology ( CPT ) procedure code number . 19 However , the Total Claims Data Set does not contain information relating to patient age or Inter - national Classification of Diseases , Ninth Revision , Clinical Modi - fication ( ICD - 9 - CM ) diagnosis , associated with each proce - dure code . 20 The Medicare Limited Data Set Standard Analytic File5 % SamplePhysicianSupplierData ( hereinafter , 5 % Sample Data Set ) was available for 2002 to 2006 . 21 This nationally sampled Medicare database contains information on claims filed for approved procedures with their associated ICD - 9 - CM di - agnosis codes , patient age stratification , and counts of unique persons receiving the services . Hence , the 5 % Sample Data Set allowed estimation of the proportion of procedures for skin can - cer that were for NMSC , the proportion of procedures that were conducted on enrollees older than 65 years , and the mean num - ber of procedures per enrollee with any procedures . The National Ambulatory Medical Care Survey ( NAMCS ) is a cross - sectional survey system of ambulatory - based physi - cians wherein participating physicians complete a question - naire for patient visits during a random 1 - week period of the year . 22 These visit observations are then used to provide a na - tional estimate of physician visits and limited characteristics of these visits for that year . The NAMCS allowed estimation of the proportion of visits for NMSC in the United States that were conducted in the population older than 65 years . ESTIMATION OF THE TOTAL NUMBER OF NMSCs IN 2006 For this study , we define NMSC incidence in 2 ways : as newly diagnosed NMSCs and as persons with a newly diagnosed NMSC , with the latter as our primary definition , although we present both . The number of skin cancers in the fee - for - service Medicare population was estimated in this study as the total of approved skin cancer treatment procedures ( malig - nant destructions , malignant excisions , and Mohs micro - graphic surgical procedures ) for that year from the Total Claims Data Set . Thus , the crude number of skin cancers for a given year was estimated by adding the numbers of approved claims for skin cancer procedure code series ( 11600 - 11606 , 11620 - 11626 , and 11640 - 11646 for malignant excisions ; 17260 - 17266 , 17270 - 17276 , and 17280 - 17286 for malignant destructions , 17304 for Mohs surgical procedures ) . The total specific to NMSC was determined by multiplying the esti - mated crude number of skin cancers by the proportion of skin cancer procedure code claims associated with the ICD - 9 - CM diagnoses for invasive nonmelanoma cutaneous malignancy ( 173 . 0 - 173 . 9 ) and in situ malignancy ( 232 . 0 - 232 . 9 ) from the 5 % Sample Data Set . The number of procedures per affected individual and the number of unique persons that underwent at least 1 procedure were also derived from the 5 % Sample Data Set . Based on our ICD - 9 - CM code definition of NMSC , almost all of the skin cancers measured in this study were keratino - cyte carcinomas ( ie , BCC , invasive SCC , or SCC in situ ) . However , other varieties of skin cancer are also included in our totals , such as Merkel cell carcinoma , adnexal carcinomas , and malignant melanoma in situ . These cancers are relatively uncommon compared with BCC and SCC , and because of the imprecise nature of ICD - 9 - CM coding , we cannot separate procedures for these diagnoses . Excluded from our count were some forms of NMSC , such as cutaneous lymphoma and genital skin cancers that have separate ICD - 9 - CM codes . Therefore , although some malignant melanomas in situ are included in our estimates , and some NMSCs are excluded , the overall number of keratinocyte carcinomas is so much larger that these inclusions and exclusions should have a small effect on our overall estimate . For example , analysis of the Surveil - lance , Epidemiology , and End Results ( SEER ) database for 2006 estimates 49710 new US cases of malignant melanoma in situ ( 1 . 4 % of our total NMSC estimate ) . 23 For this article , we will use the common but admittedly imprecise term NMSC . The number of NMSCs in the Medicare population 65 years or older was established from the Total Claims Data Set and the 5 % Sample Data Set . The proportion of the entire US popu - lation ( (cid:2) 65 years ) covered under Medicare was derived from the Center for Medicare and Medicaid Services 2007 Trustee’s report and US census data , allowing estimation of the number of NMSCs in the entire population segment that was 65 years or older . 24 , 25 The proportion of total office visits for NMSC ICD - 9 - CM codes ( 173 . 0 - 173 . 9 and 232 . 0 - 232 . 9 ) that were for the segment of the population that was 65 years or older in 2006 was obtained from the NAMCS . The number of NMSCs in the US population ( (cid:2) 65 years old ) was then divided by the pro - portion of office visits for NMSC in that group , allowing esti - mation of the total number of skin procedures for NMSC in the United States . The total number of persons in the United States diagnosed as having NMSC in that year was calculated fromtheskincancerproceduretotalsandthenumberofNMSCsperaffectedMedicarepatient . More detailed representation of the calculation described in this section is available at the Skin Cancer Center Web site . 26 AGE ADJUSTMENT OF NMSC RATES The age - adjusted NMSC Medicare Part B Fee - for - Service pro - cedure rate was calculated , standardized to the year 2006 . The Total Claims Data Set for 1992 and 1996 to 2006 does not con - tain age - stratified data . Therefore , crude skin cancer proce - durerateswerederivedusingthisdatabase , butage - specificrates could not be determined . The 5 % Sample Data Set is age strati - fied in 5 - year intervals and allowed calculation of age - adjusted procedure rates . The methods and calculations deriv - ing age - adjusted procedure rates are available at the Skin Cancer Center Web site . 27 ( REPRINTED ) ARCH DERMATOL / VOL 146 ( NO . 3 ) , MAR 2010 WWW . ARCHDERMATOL . COM 284 ©2010 American Medical Association . All rights reserved . Confidential . Do not distribute . Pre - embargo material . RESULTS The total number of fee - for - service Medicare skin can - cer procedures increased over the 15 years of the study ( 1992 , 1996 - 2006 ) from 1 158 298 to 2 048 517 , with an overall increase of 77 . 0 % ( Table 1 ) . The age - adjusted rate of skin cancer procedures increased from 3514 per 100 000 beneficiaries in 1992 to 6075 in 2006 ( Table 1 ) . Using the diagnosis - specific 5 % Sample Data Set , the over - all NMSC - specific procedure rate increased by 16 . 0 % from 2002 to 2006 , whereas the average number of proce - dures per beneficiary increased by 1 . 5 % from 1 . 61 to 1 . 63 ( Table 2 ) . Extending this analysis , the number of per - sons with at least 1 procedure for NMSC increased by 14 . 3 % from 2002 to 2006 . Using the Total Claims Data Set and 5 % Sample Data Set , the following calculations were performed . In 2006 , 93 . 7 % or 1 919 460 of the approved procedures were specific for NMSC . The 2006 total NMSC procedures for fee - for - service Medicare patients 65 years or older , as a proportion of such procedures for fee - for - service Medicare patients at all ages , were 83 . 5 % ( or 1 604 477 procedures ) . The percentage of the US population 65 years or older enrolled in fee - for - service Medicare was 73 . 8 % . Thus , the number of procedures for NMSC in 2006 in the United States in persons 65 years or older can be estimated at 2 174 383 . The NAMCS estimates of office visits for NMSC diagnoses in the US population in 2006 are 4 003 887 , of which 2 482 801 ( 62 . 0 % ) were ascribed to patients 65 years or older . Combining the Total Claims Data Set , 5 % Sample Data Set , and NAMCS , we estimate the total number of NMSCs treated in the United States in 2006 at 3 507 693 . From the 5 % Sample Data Set , the ratio of skin cancers treated per affected patient in 2006 is 1 . 63 . If this ratio is extended to the US population , then the number of persons in the United States who were treated for NMSC in 2006 can be estimated at 2 152 500 . COMMENT Nonmelanoma skin cancer is the most common malig - nancy in the United States and is not reported in most registries ; hence , the exact incidence is unknown . Based on the limited studies available , skin cancer rates seem to be climbing rapidly in the US population . The data pre - sented herein from national databases indicate that the incidence of skin cancer in the United States has sub - stantially increased from 1992 through 2006 and is now about double the last published estimate from 1994 . 1 In evaluating increases in the US incidence of NMSC since 1994 , we considered the relatively direct estimate described in the “Methods” section , and the alternative of estimating change in incidence over the 12 - year pe - riod and applying that proportion increase to the pub - lished 1994 data . We elected the former because the pub - lished 1994 incidence was itself a projection from a variety of sources and subject to considerable error . Estimation of trends from published data was limited by the very small number of published reports . Estimation by regression modeling of NAMCS data was ultimately rejected be - cause the small sampling frame and year - to - year varia - tion in visit frequency limited the reliability of our con - clusions . This study’s strengths are that it is current and based on Medicare and NAMCS nationally representative data sets . In particular , the Medicare data incorporate a huge sample size of all covered individuals and provide strong estimative power . There are some important limitations in this study’s estimates of NMSC incidence . This study assumes that 1 NMSC treatment procedure equals 1 incident NMSC . Given that a physician should not bill for a skin cancer procedure without first obtaining a pathologic diagno - sis of skin cancer , the number of skin cancer procedures is a reasonable proxy for the number of skin cancers . On the one hand , retreatment of a skin cancer owing to posi - tive histologic margins or recurrence would result in over - estimation in this model . On the other hand , biopsies that remove an entire lesion would not be captured by the pro - cedure codes we used , which would result in underes - timation . Reimbursement denial of a skin cancer claim by Medicare or treatment of a cancer by a nonsurgical modality ( such as radiation or topical chemotherapy ) would also result in underestimation . Another limita - tion of this study is that in calculating the total number Table 1 . Number of Procedures and of Age - Adjusted Procedures for All Skin Cancers in the Medicare Fee - for - Service Population Year Total Skin Cancer Procedures Age - Adjusted Procedure Rate per 100000 Beneficiaries ProcedurestoTreatNMSC , % 1992 1158298 3514 NA 1996 1377741 4136 NA 1997 1450746 4400 NA 1998 1473728 4521 NA 1999 1497444 4647 NA 2000 1577165 4947 NA 2001 1694913 5173 NA 2002 1785136 5312 92 . 6 2003 1834443 5322 93 . 8 2004 1905121 5477 92 . 7 2005 2007826 5772 92 . 4 2006 2048517 6075 93 . 7 Abbreviations : NA , not available ; NMSC , nonmelanoma skin cancer . Table 2 . Nonmelanoma Skin Cancer ( NMSC ) Specific Statistics for the Medicare Fee - for - Service Population Year No . Total NMSC Procedures ServicesperAffectedPerson AffectedPersons 2002 1653260 1 . 61 1030220 2003 1720560 1 . 61 1067620 2004 1765900 1 . 61 1098700 2005 1854480 1 . 62 1147600 2006 1918340 1 . 63 1177400 ( REPRINTED ) ARCH DERMATOL / VOL 146 ( NO . 3 ) , MAR 2010 WWW . ARCHDERMATOL . COM 285 ©2010 American Medical Association . All rights reserved . Confidential . Do not distribute . Pre - embargo material . of NMSCs and the numbers of affected persons , mul - tiple other assumptions are required . The calculations as - sume that the portion of the US population that is older than 65 years and not covered under Medicare fee - for - service has the same rate of skin cancer as the portion that is covered under Medicare ; that the ratio of office visits for a diagnosis of NMSC to the number of proce - dures for NMSC as defined by the NAMCS is approxi - mately the same in those 65 years or older or younger than 65 years ; and that the rate of NMSCs per affected individual is the same in the US population as a whole as for the Medicare fee - for - service population . We found little similar literature published in the past 15years . Themostrecentpeer - reviewedestimateofNMSC incidence was from 1994 . 1 That study was a projection to 1994 from population - based estimates of NMSC from the 1970s , based on trends from studies of distinct regions of North America . The estimate of 900000 to 1 . 2 million new cases of NMSC in the United States in 1994 has been car - ried forward to the present day and is cited by the Ameri - can Cancer Society and National Cancer Institute , de - spitethepresumedincreasesinincidencesincethattime . 2 , 28 Although the present report has substantial limitations , it provides a much stronger NMSC estimate than has pre - viously been published . Nevertheless , this effort under - scores the need for a system of sentinel registries for NMSC to allow for better monitoring of our efforts to combat the most common cancer in the United States . Having accurate estimates of NMSC incidence is im - portant in establishing the public health burden of this condition . However , other components beyond total num - bers must be considered . Mortality from NMSC is low but not zero . Morbidity and decreased quality of life are magnified and take on increased importance in com - mon conditions that underscore the importance of bet - ter defining and decreasing morbidity due to NMSC . Last , the dollar cost of NMSC treatment is also still poorly de - fined . 29 However , further study on the cost of skin can - cer treatment should allow better estimation of the costs to the health care system . 30 There is an epidemic of NMSC in the United States , as illustrated by comparison with the previously pub - lished estimates and the 4 . 2 % yearly average increase in cases in the Medicare population from 1992 to 2006 . To date , educational programs emphasizing sun protection have mainly been disappointing in slowing skin cancer rates . 31 In the face of ongoing increases in skin cancer incidence , continued national research and programs on treatment , education , and prevention are critical . Accepted for Publication : November 22 , 2009 . Correspondence : Howard W . Rogers , MD , PhD , Ad - vanced Dermatology , 111 Salem Turnpike , Ste 7 , Nor - wich , CT 06360 ( rogershoward @ sbcglobal . net ) . Author Contributions : Drs Rogers and Weinstock had full access to all of the data in the study and take respon - sibility for the integrity of the data and the accuracy of the data analysis . Study concept and design : Rogers , Wein - stock , Feldman , and Coldiron . Acquisition of data : Wein - stock , Hinckley , Feldman , Fleischer , and Coldiron . Analy - sis and interpretation of data : Rogers , Weinstock , Harris , Harris , Feldman , and Fleischer . Drafting of the manu - script : Rogers , Weinstock , Harris , and Coldiron . Criti - cal revision of the manuscript for important intellectual con - tent : Rogers , Weinstock , Harris , Hinckley , Feldman , and Fleischer . Statistical analysis : Rogers , Harris , Feldman , and Fleischer . Obtained funding : Coldiron . Administra - tive , technical , and material support : Rogers , Harris , Hinck - ley , and Coldiron . Study supervision : Rogers , Wein - stock , Feldman , and Coldiron . Financial Disclosure : Dr Feldman has received grant sup - port from Galderma , Connetics Corp , Astellas , Abbott Laboratories , Warner Chilcott , Centocor , Amgen , Bio - genidec , Coria , Pharmaderm , the National Psoriasis Foun - dation , Ortho Pharmaceuticals , Aventis Pharmaceuti - cals , Roche Dermatology , 3M , and Bristol - Myers Squibb Dermatology ; has received research support from Pho - tomedex , Genentech , and Novartis ; has been a speaker for Galderma , Connetics Corp , Abbott Laboratories , Warner Chilcott , Centocor , Amgen , Genentech , Biogen - idec , 3M , Bristol - Myers Squibb Dermatology , and Novar - tis ; has been a consultant for Galderma , Connetics Corp , Abbott Laboratories , Warner Chilcott , Centocor , Am - gen , Photomedex , Genentech , Biogenidec , and Bristol - Myers Squibb Dermatology ; has received stock options from Photomedex ; and has received research grants from the Dermatology Foundation and the American Society for Dermatologic Surgery . Dr Fleisher has been a mem - ber of the advisory boards for Allergan , Amgen , Astel - las , Galderma , GlaxoSmithKline , Ortho Dermatologics , and Stiefel ; has been a consultant for Allergen , Astellas , Asubio , Combe , Galderma , Gerson Lehrman , Intendis , Kikaku America International , Merz , Novartis , Serentis , and Taisho ; has been an investigator for 3M , Abbott Labo - ratories , Amgen , Astellas , Asubio , Biogen , BioCryst , Dow , Centocor , Coria , Galderma , GlaxoSmithKline , Genen - tech , Intendis , Medicis , Novartis , Ortho Dermatologics , Pfizer , Serentis , Stiefel , and Taisho ; and has been a mem - ber of the speakers bureau for Amgen , Astellas , Gal - derma , Intendis , Medicis , Novartis , Stiefel , and Upsher - Smith . Funding / Support : Dr Weinstock received support from the Department of Veterans Affairs Office of Research and Development Co - operative Studies Program and from grants R01CA106592 , R01CA106807 , and R25CA087972 from the National Institutes of Health . Additional Information : This study does not report data from studies involving human participants ; therefore , for - mal review and approval , or formal review and waiver , by an appropriate institutional review board or ethics com - mittee have not been described in the “Methods” section . Additional Contributions : Christopher Hogan , PhD , pro - vided statistical analysis . REFERENCES 1 . MillerDL , WeinstockMA . NonmelanomaskincancerintheUnitedStates : incidence . J Am Acad Dermatol . 1994 ; 30 ( 5 , pt 1 ) : 774 - 778 . 2 . AmericanCancerSocietyhomepage . http : / / www . cancer . org / docroot / home / index . asp . Accessed September 27 , 2009 . 3 . Christenson LJ , Borrowman TA , Vachon CM , et al . Incidence of basal cell and squamouscellcarcinomasin apopulation youngerthan 40 years . JAMA . 2005 ; 294 ( 6 ) : 681 - 690 . 4 . KaragasMR , GreenbergER , SpencerSK , StukelTA , MottLA ; NewHampshireSkin ( REPRINTED ) ARCH DERMATOL / VOL 146 ( NO . 3 ) , MAR 2010 WWW . ARCHDERMATOL . COM 286 ©2010 American Medical Association . All rights reserved . Confidential . Do not distribute . Pre - embargo material . Cancer Study Group . Increase in incidence rates of basal cell and squamous cell skin cancer in New Hampshire , USA . Int J Cancer . 1999 ; 81 ( 4 ) : 555 - 559 . 5 . Glass AG , Hoover RN . The emerging epidemic of melanoma and squamous cell skin cancer . JAMA . 1989 ; 262 ( 15 ) : 2097 - 2100 . 6 . Athas WF , Hunt WC , Key CR . Changes in nonmelanoma skin cancer incidence between1977 - 1978and1998 - 1999inNorthcentralNewMexico . CancerEpide - miol Biomarkers Prev . 2003 ; 12 ( 10 ) : 1105 - 1108 . 7 . Swetter SM , Boldrick JC , Jung SY , Egbert BM , Harvell JD . Increasing incidence of lentigo maligna melanoma subtypes : northern California and national trends 1990 - 2000 . J Invest Dermatol . 2005 ; 125 ( 4 ) : 685 - 691 . 8 . InsingaRP , ReitherEN , RemingtonPL , Stephenson - VineL . Trendsinmalignant melanoma incidence and mortality in Wisconsin , 1979 - 1997 . WMJ . 2001 ; 100 ( 6 ) : 27 - 31 . 9 . Jemal A , Devesa SS , Hartge P , Tucker MA . Recent trends in cutaneous mela - noma incidence among whites in the United States . J Natl Cancer Inst . 2001 ; 93 ( 9 ) : 678 - 683 . 10 . Harris RB , Griffith K , Moon TE . Trends in the incidence of nonmelanoma skin cancers in southeastern Arizona , 1985 - 1996 . J Am Acad Dermatol . 2001 ; 45 ( 4 ) : 528 - 536 . 11 . Brewster DH , Bhatti LA , Inglis JH , Nairn ER , Doherty VR . Recent trends in inci - dence of nonmelanoma skin cancers in the East of Scotland , 1992 - 2003 . Br J Dermatol . 2007 ; 156 ( 6 ) : 1295 - 1300 . 12 . Staples MP , Elwood M , Burton RC , Williams JL , Marks R , Giles GG . Non mela - noma skin cancer in Australia : the 2002 national survey and trends since 1985 . Med J Aust . 2006 ; 184 ( 1 ) : 6 - 10 . 13 . Hayes RC , Leonfellner S , Pilgrim W , Liu J , Keeling DN . Incidence of nonmela - noma skin cancer in New Brunswick , Canada , 1992 to 2001 . J Cutan Med Surg . 2007 ; 11 ( 2 ) : 45 - 52 . 14 . Demers AA , Nugent Z , Mihalcioiu C , Wiseman MC , Kliewer EV . Trends of non - melanomaskincancerfrom1960through2000inaCanadianpopulation . JAm Acad Dermatol . 2005 ; 53 ( 2 ) : 320 - 328 . 15 . Wassberg C , Thörn M , Johansson AM , Bergström R , Berne B , Ringborg U . Increasing incidence rates of squamous cell carcinoma of the skin in Sweden . Acta Derm Venereol . 2001 ; 81 ( 4 ) : 268 - 272 . 16 . Housman TS , Feldman SR , Williford PM , et al . Skin cancer is among the most costly of all cancers to treat for the Medicare population . J Am Acad Dermatol . 2003 ; 48 ( 3 ) : 425 - 429 . 17 . JosephAK , MarkTL , MuellerC . Theperiodprevalenceandcostsoftreatingnon - melanoma skin cancers in patients over 65 years of age covered by Medicare . Dermatol Surg . 2001 ; 27 ( 11 ) : 955 - 959 . 18 . Physician / Supplier Procedure Summary Master File . Centers for Medicare and Medicaid Services Web site . http : / / www . cms . hhs . gov / NonIdentifiableDataFiles / 06 _ PhysicianSupplierProcedureSummaryMasterFile . asp . Accessed Septem - ber 27 , 2009 . 19 . Overview . Centers for Medicare and Medicaid Services Web site . http : / / www . cms . hhs . gov / MedHCPCSGeninfo / . Accessed September 27 , 2009 . 20 . ICD - 9 - CM OfficialGuidelinesforCodingandReporting . CentersforDiseaseCon - trol and Prevention Web site . http : / / www . cdc . gov / nchs / data / icd9 / icdguide . pdf . Accessed September 27 , 2009 . 21 . Standard Analytical Files : Limited Data Sets . Centers for Medicare and Medicaid Services Web site . http : / / www . cms . hhs . gov / LimitedDataSets / 12 _ StandardAnalyticalFiles . asp . Accessed September 27 , 2009 . 22 . Ambulatory health care data . Centers for Disease Control and Prevention Web site . http : / / www . cdc . gov / nchs / ahcd . htm . Accessed September 27 , 2009 . 23 . JemalA , SiegelR , WardE , etal . Cancerstatistics , 2006 . CACancerJClin . 2008 ; 58 ( 2 ) : 71 - 96 . 24 . 2007 annual report of the boards of trustees of the federal hospital insurance and federal supplementary medical insurance trust funds . Centers for Medicare and Medicaid Services Web site . http : / / www . cms . hhs . gov / ReportsTrustFunds / downloads / tr2007 . pdf . Accessed September 27 , 2009 . 25 . Populationestimates . USCensusBureauWebsite . http : / / www . census . gov / popest / national / asrh / NC - EST2007 - sa . html . Accessed September 27 , 2009 . 26 . Method A . The Skin Cancer Center Web site . http : / / www . theskincancercenter . net / images / Method % 20A . pdf . Accessed September 27 , 2009 . 27 . Method B . The Skin Cancer Center Web site . http : / / www . theskincancercenter . net / images / Method % 20B . pdf . Accessed September 27 , 2009 . 28 . Skin cancer . The National Cancer Institute Web site . http : / / www . cancer . gov / cancertopics / types / skin . Accessed September 27 , 2009 . 29 . Bickers DR , Lim HW , Margolis D , et al ; American Academy of Dermatology Association ; Society for Investigative Dermatology . The burden of skin dis - eases : 2004 a joint project of the American Academy of Dermatology Associa - tion and the Society for Investigative Dermatology . J Am Acad Dermatol . 2006 ; 55 ( 3 ) : 490 - 500 . 30 . Rogers HW , Coldiron BM . A relative value unit - based cost comparison of treat - ment modalities for nonmelanoma skin cancer : effect of the loss of the Mohs multiple surgery reduction exemption . J Am Acad Dermatol . 2009 ; 61 ( 1 ) : 96 - 103 . 31 . Weinstock MA . The struggle for primary prevention of skin cancer . Am J Prev Med . 2008 ; 34 ( 2 ) : 171 - 172 . ( REPRINTED ) ARCH DERMATOL / VOL 146 ( NO . 3 ) , MAR 2010 WWW . ARCHDERMATOL . COM 287 ©2010 American Medical Association . All rights reserved . \ No newline at end of file diff --git a/silver_data/36f5c65ab3e65bea72d5d6bb329a26641c74d5c5.pdf.txt b/silver_data/36f5c65ab3e65bea72d5d6bb329a26641c74d5c5.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..aadc0ffd6af59ccba08f8f8de40f212527e99b4b --- /dev/null +++ b/silver_data/36f5c65ab3e65bea72d5d6bb329a26641c74d5c5.pdf.txt @@ -0,0 +1 @@ +Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 56 Information Needs of Physicians and Surgeons of Jammu & Kashmir S . M . Shafi * Dr . Mudassir Ashraf Wani * * Abstract Purpose : The paper attempts to explore the information needs of physicians and surgeons of Jammu & Kashmir Design / Methodology / Approach : The survey is conducted among physicians and surgeons working at primary , secondary and tertiary health care institutions by administrating a questionnaire employing stratified random sampling . Findings : The information needs of the physicians and surgeons vary with practice location , affiliation and specialization . Most of the physicians and surgeons need patient data / patient care information , latest medical knowledge , information about new drugs and medical products / equipments . Originality / Value : The identification of information needs of physicians and surgeons is a maiden attempt for the state of J & K . This study may encourage and foster further research for effectiveness and better performance of medical libraries and information managers in the State . Keywords : Medical Practitioners , Physicians , Surgeons , Information Needs , Jammu & Kashmir . Paper Type : Research Introduction nformation need is an area which involves exact and accurate study of information requirements among different stakeholders of a profession . The advances in user and behavioural studies , living patterns , standards and styles add varied dimensions to information needs and its seeking behaviour . However , literature shows it’s beginning in the 20 th century as an area of study . A number of papers pertaining to information seeking behaviour of scientists and technologists have been presented at the Royal Society Scientific Information Conference since 1948 ( Wilson , 1999 ) . Later , during 1960s and 1970s , the works of Line , Brittain , Paisley , Lin , Garvey , Herner and others focusing on information requirement of Social Scientists , popularly known as INFROSS appeared in Annual Review of Information Technology ( Goodall , 2005 ) . These works motivated scholars to work on similar lines . Similarly , the studies of Dervin , Ellis , Kuhlthau , Wilson , Ingwersen and others have outlined the theoretical perspectives on the area and devised several useful models for the improvement of the existing state of the information user studies ( Wilson , 1999 ) . The research on the information needs of medical practitioners is a relatively new endeavour . Forsythe states , “identifying the information * Head , Department of Library and Information Science . University of Kashmir . Jammu and Kashmir . India . 190 006 . shafi _ sm @ rediffmail . com * * Librarian . Entrepreneur Development Institute ( EDI ) , Pampore . Jammu and Kashmir . I Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 57 needs of Physicians was recognized as a significant problem as early as 1972” ( Forsythe , Buchanan , Osheroff & Miller , 1992 ) . Earlier study by Strasser ( 1978 ) , a Library and Information Science student at the State University of New York , on practicing physicians opened the area for extensive research . Since its publication in 1978 , numerous studies have been carried out about information needs of physicians and nurses . However , no such work has been carried out in J & K , a small and geographically isolated state of India . Literature Review A sizeable number of studies have been carried out from time to time to study the information needs of medical practitioners since 1972 . Covell , Uman and Manning ( 1985 ) report that the physicians require information about patient management , medical specialists and information highly specific to the individual patient’s problem . Sub - specialists most frequently require information related to other specialties . Woolf and Benson ( 1989 ) carried a survey at Johns Hopkins Hospital which reveals that the faculty more often requires information for research and preparation of conferences and rounds . The most frequently required information by house staff is about differential diagnosis and treatment recommendations . Internists involved with ambulatory patient care need instant information about the treatment of specific conditions , the diagnosis of physical findings or symptoms and drug information . Osheroff , Forsythe , Buchanan , Bankowitz , Blumenfeld and Miller ( 1991 ) have identified the physician’s information requests and reveal that on average , five clinical questions are raised for each patient discussed . Out of them 74 % requests concern patient care . Dee & Blazek ( 1993 ) report that 75 % of rural physician’s requests regarding patient - care ( clinical ) information needs relate to treatment , 14 . 7 % relate to diagnosis , 8 . 3 % relate to etiology and 2 . 1 % to the psychological aspects of disease . Later , Lundeen narrowed the information needs of physicians into two categories : research or patient care ( Lundeen , Tenopir & Wermager , 1994 ) . But Ocheibi and Buba ( 2003 ) reveal that the doctors in Maiduguri , Nigeria need specific medical information to enhance their knowledge on a day - to - day basis . Gonzalez , Dawes , Sanchez - Mateos , Riesgo - Fruetes , Escortell - Mayer , et al . ( 2007 ) show that the most frequent questions asked by primary care physicians in Spain relate to diagnosis ( 53 % ) and treatment ( 26 % ) . Shabi , Kutetyi , Odewale and Shabi ( 2008 ) found that the information needs of family physicians in Nigeria focus on new developments in area of specialization ( 87 . 3 % ) ; drug information ( 74 . 2 % ) ; government regulations on health care ( 70 . 2 % ) ; and routine patient care ( 65 . 9 % ) . Problem Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 58 As health is a phenomenon of global concern , the information needs of medical professionals need to be explored so that adequate infrastructure is provided to them and barriers that thwart their information seeking behaviours can be removed depending upon the results and suggestions in order to provide the best to the patient health and medical education especially for the state of Jammu and Kashmir that has remained under stress because of the prevailing political conflict . Objectives The main objectives set forth for the study are :  To identify different information needs of the physicians and surgeons .  To assess the context and diversity in information needs of physicians and surgeons . Scope The study attempts to identify the information needs of physicians and surgeons working in J & K at primary , secondary and tertiary health care institutions encompassing 3567 medical colleges / associations / health institutions in public and 51 registered private nursing homes bedsides several solo practitioners . ( Appendix - I ) Methodology A stratified random sample of 226 physicians and surgeons of major medical fields and sub - fields was drawn using the sample size determination formula . The practitioners on the basis of their work roles were first divided into different strata . The strata were selected from the seven main working environments as given in the scope . A questionnaire was drafted , tested and later distributed . The data was collected personally from practitioners ( 4th of January 2009 to 31st of August 2009 ) . The survey was previously piloted among several groups of physicians and surgeons . Findings and Discussion Different professional activities give rise to various information needs . Variations do exist in the information needs among the physicians and surgeons as their scope and operation of practice differs .  Information Needs ( Institution wise ) The information needs of physicians and surgeons vary at each location in view of nature of work , patient type and treatment level like primary , secondary or tertiary care . Every individual practitioner has unique information needs . These perceived and actual needs also change with time , place and clinical case load . Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 59 The physicians and surgeons of medical institutions and hospitals require data about patients and latest medical knowledge to improve their practice . Majority of them ( 83 - 100 % ) want updates on information pertaining to drugs and medical products / equipments . Information about hospital administration is needed more by physicians / surgeons ( 47 - 70 % ) of SKIMS , its associated Medical College and ASCOMS compared to those of working in other institutions ( 11 - 28 % ) . Similarly , the information regarding latest research findings and subjects being taught in the institutions is required more by physicians / surgeons ( 60 - 100 % ) of SKIMS / its associated Medical College , GMC ( K / J ) / its associated hospitals / GDC ( K / J ) and ASCOMS compared to those working in District , Sub - District Hospitals , Dispensaries , Health centres , Ayurvedic cum Unani Hospitals / Dispensaries , Private nursing homes and solo settings ( 12 - 25 % ) . ( Table 1 ) Thus , it can be concluded that physicians and surgeons in different practice settings require patient data / patient care information , latest medical knowledge , information about new drugs and medical products / equipments . Gorman ( 2001 ) has made similar conclusion for rural and non - rural primary care physicians having equal information needs . However , practitioners working in teaching hospitals [ SKIMS and associated Medical College , GMC ( K / J ) / its associated hospitals and GDC ( K / J ) and ASCOMS ] in addition to above requirements need information about latest research , subjects taught and hospital administration .  Information Needs ( Designation Wise ) Physicians and surgeons play various roles like teaching , clinical practice and administration . Their information needs also vary with their roles in the profession . The practitioners in teaching and clinical practice need lesser information about population statistics ( 5 - 13 % ) , logistics and social influences ( 42 - 53 % ) compared to those in administration wherein all the practitioners need information about population statistics and many require information about logistics and social influences ( 53 % ) . The practitioners in teaching and clinical practice require more information on drugs ( 100 % ) , new medical products / equipments ( 73 - 76 % ) and lab / imaging procedures / techniques ( 36 - 43 % ) compared to those in administration wherein majority of these professionals ( 83 . 5 % ) demand information regarding drugs , new medical products / equipments ( 49 % ) and lab / imaging procedures / techniques ( 21 % ) . Information pertaining to latest research findings and government regulations related to healthcare is required more by practitioners in teaching ( 53 - 60 % ) compared to those in clinical practice and administration ( 23 . 5 - 42 % ) . Similarly , the practitioners ( 84 % ) in teaching state that more information is required about the subjects being taught in the institutions compared to those in Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 60 clinical practice and administration ( 5 - 17 % ) . Practitioners in clinical practice ( 41 % ) need more information on disaster management as compared to those in teaching and administration ( 8 - 33 % ) . The practitioners in teaching ( 5 % ) and clinical practice ( 11 % ) require less information regarding hospital / health administration compared to those in administration ( 65 % ) . ( Table 2 ) It shows that physicians and surgeons of all categories need patient care information and current medical information in their respective specialized field . However , practitioners in teaching and clinical practice require more information about drugs , new medical products / equipments and lab / imaging procedures / techniques compared to those in administration , while as practitioners in administration require more information regarding hospital / health administration and practitioners in teaching require information more about subjects they teach , latest research findings and government regulations related to healthcare . Woolf and Benson ( 1989 ) has drawn similar conclusion reporting that both the faculty and house staff in Internal Medicine and Pediatrics at an academic medical center frequently requires treatment recommendations and differential diagnosis . However , the information needs of house staff differs significantly in several categories from those of faculty physicians . House staff frequently needs information for patient care . Faculty need information more frequently on activities unrelated to patient care .  Information Needs ( Specialization Perspective ) The specialization in medicine is evolving with the growth in knowledge and technology , and resources are diversifying at an alarming rate . Consequent upon such developments , new specialties are sprouting and physicians and surgeons need various types of information to carry forward their mission . Therefore , the professional information needs are specific to a particular discipline . Practitioners in various specialized fields need information regarding patient data / patient care information ( 100 % ) , latest medical knowledge ( 100 % ) , drug information ( 60 - 100 % ) and logistic information ( 43 - 70 % ) . Practitioners in General medicine ( 40 % ) , General surgery ( 20 % ) , Obstetrics & Gynaecology ( 13 % ) , Pediatrics & Neonatology ( 20 % ) , Psychiatry ( 21 % ) , Community Medicine & Public Health or Social & Preventive Medicine ( SPM ) ( 100 % ) and Hospital Administration ( 20 % ) need information about population statistics while as the practitioners in other medical fields do not need such type of information . The practitioners in Otorhinolaryngology or ENT ( ear , nose & throat ) , Toxicology & Forensic Medicine , Community Medicine & Public Health and Hospital Administration ( 36 - 53 % ) report less information requirements about medical products and equipments compared to Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 61 those in other specialized fields ( 100 % ) except in Psychiatry . Physicians / surgeons in Orthopedics , Physical Medicine & Rehabilitation , and Orhinolaryngology / ENT report to need information pertaining to lab / imaging procedures / techniques . Information about latest research findings is reported more by practitioners in General medicine and General surgery ( 80 % ) compared to those in other specialized fields ( 21 - 44 % ) . Information regarding subjects being taught in the institutions is needed more by practitioners in General medicine , General surgery , and Community Medicine & Public Health / SPM ( 60 - 67 % ) compared to those in other specialized fields ( 20 - 36 % ) . Hospital administrators ( 100 % ) report requirement of information more about hospital / health administration compared to those in other specialized fields ( 14 - 36 % ) ( Table 3 ) . The above studies show that the physicians / surgeons in the entire specialized fields need patient data / patient care information , latest medical knowledge and drug information . The results found are however , similar to those in other published studies . Gorman ( 2001 ) affirms that primary care physicians in Family Medicine , Internal Medicine , and Pediatrics have similar information needs . The results are corroborated by the study of Lakshmi , Rao , Gore and Bhaskaran ( 2003 ) which shows that pediatricians , general physicians , dermatologists , gynecologists and other practitioners need information about drug product availability / identification , contraindications / safety , adverse drug reactions , choice of drugs , banned drug information and use of drug during pregnancy . Similarly , the neurologists at National Institute of Neurological Disorders and Stroke ( Bethesda ) also came to similar conclusion and report that they need the patient ' s medical history and information about various laboratory test results for diagnosis e . g . , blood or urine tests , skin or tissue sampling , electroencephalogram or EEG , electrical studies of the eyes , brain scans , measurement of enzyme activity , and DNA analysis ( National Institute of Neurological… . . , 2009 ) . Bauer ( 2009 ) also came up with similar findings while reporting that urologists at Regional Medical Centers in Michigan require patient’s health history , information about medications ( if they are taking ) and diagnostic test results e . g . , blood or urine test , x - rays etc . The above studies show that the physicians / surgeons in the entire specialized fields need patient data / patient care and drug information . Conclusion Physician and surgeon role and related tasks undertaken in the course of daily practice prompt specific Information needs . The environment is characterized by specialty , practice type and setting etc . The information needs of Physicians and surgeons vary at each place of work in view of nature of work , patient type , and treatment like primary , Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 62 secondary or tertiary care . These information needs change with their roles in the profession like teaching , clinical practice and administration . The practitioners of all categories require updates on latest medical knowledge , information regarding new drugs and medical products / equipments . Besides , practitioners in teaching need information more about subjects being taught in the institutions , latest research papers and hospital / health administration while as practitioners in clinical practice need patient data / patient care information depending upon the type of ailment . However , practitioners performing administrative roles need information more about hospital / health administration and also to some extent information pertaining to latest research papers and patient data / patient care . Those in lab . technology require patient data , and information about lab / imaging procedures / techniques . Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 63 Table 1 : Diversity in Information Needs ( Institution Wise ) Information needs P a t i e n t d a t a / p a t i e n t c a r e i n f o r m a t i o n P o pu l a t i o n s t a t i s t i c s L a t e s t m e d i c a l k n o w l e d g e D r u g i n f o r m a t i o n L o g i s t i c i n f o r m a t i o n S o c i a l i n f l u e n c e s N e w m e d i c a l p r o d u c t s a nd e qu i p m e n t s L a b . / i m a g i n g d i a g n o s i s / p r o c e du r e s / t e c hn i qu e s R e s e a r c h S ub j e c t D i s a s t e r m a n a g e m e n t H o s p i t a l / h e a l t h a d m i n i s t r a t i o n G o v t . r e g u l a t i o n s o n h e a l t h c a r e SKIMS / associated Medical College 17 17 ( 100 . 0 ) 4 ( 23 . 5 ) 17 ( 100 . 0 ) 17 ( 100 . 0 ) 14 ( 82 . 4 ) 5 ( 29 . 4 ) 15 ( 88 . 2 ) 10 ( 58 . 8 ) 16 ( 94 . 1 ) 17 ( 100 . 0 ) 3 ( 17 . 6 ) 8 ( 47 . 1 ) 12 ( 70 . 5 ) GMC / associated hospitals / GDC in Kashmir region 18 18 ( 100 . 0 ) 2 ( 11 . 1 ) 18 ( 100 . 0 ) 15 ( 83 . 3 ) 13 ( 72 . 2 ) 13 ( 72 . 2 ) 13 ( 72 . 2 ) 2 ( 11 . 1 ) 15 ( 83 . 3 ) 17 ( 94 . 4 ) 7 ( 38 . 9 ) 3 ( 16 . 7 ) 10 ( 55 . 5 ) GMC / associated hospitals / GDC in Jammu region 9 9 ( 100 . 0 ) 1 ( 11 . 1 ) 9 ( 100 . 0 ) 9 ( 100 . 0 ) 5 ( 55 . 6 ) 5 ( 55 . 6 ) 8 ( 88 . 9 ) 2 ( 22 . 2 ) 6 ( 66 . 7 ) 6 ( 66 . 7 ) 2 ( 22 . 2 ) 1 ( 11 . 1 ) 6 ( 66 . 7 ) District / Sub - district hospitals / dispensaries / health centres in the State 122 122 ( 100 . 0 ) 18 ( 14 . 8 ) 122 ( 100 . 0 ) 112 ( 91 . 8 ) 62 ( 50 . 8 ) 89 ( 73 . 0 ) 68 ( 55 . 7 ) 42 ( 34 . 4 ) 30 ( 24 . 6 ) 22 ( 18 . 0 ) 40 ( 32 . 8 ) 20 ( 16 . 4 ) 44 ( 36 . 0 ) cont … Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 64 Information needs P a t i e n t d a t a / p a t i e n t c a r e i n f o r m a t i o n P o pu l a t i o n s t a t i s t i c s L a t e s t m e d i c a l k n o w l e d g e D r u g i n f o r m a t i o n L o g i s t i c i n f o r m a t i o n S o c i a l i n f l u e n c e s N e w m e d i c a l p r o du c t s a nd e qu i p m e n t s L a b . / i m a g i n g d i a g n o s i s / p r o c e du r e s / t e c hn i qu e s R e s e a r c h S ub j e c t D i s a s t e r m a n a g e m e n t H o s p i t a l / h e a l t h a d m i n i s t r a t i o n G o v t . r e g u l a t i o n s o n h e a l t h c a r e Ayurvedic cum Unani hospitals / dispensaries in the State 25 25 ( 100 . 0 ) 3 ( 12 . 0 ) 25 ( 100 . 0 ) 25 ( 100 . 0 ) 7 ( 28 . 0 ) 5 ( 20 . 0 ) 12 ( 48 . 0 ) 4 ( 16 . 0 ) 3 ( 12 . 0 ) 3 ( 12 . 0 ) 2 ( 8 . 0 ) 7 ( 28 . 0 ) 5 ( 20 . 0 ) ASCOMS 10 10 ( 100 . 0 ) 4 ( 40 . 0 ) 10 ( 100 . 0 ) 9 ( 90 . 0 ) 6 ( 60 . 0 ) 4 ( 40 . 0 ) 9 ( 90 . 0 ) 4 ( 40 . 0 ) 6 ( 60 . 0 ) 9 ( 90 . 0 ) 2 ( 20 . 0 ) 7 ( 70 . 0 ) 5 ( 50 . 0 ) Private nursing homes / solo practitioners in the state 25 25 ( 100 . 0 ) 1 ( 4 . 0 ) 25 ( 100 . 0 ) 25 ( 100 . 0 ) 9 ( 36 . 0 ) 13 ( 52 . 0 ) 22 ( 88 . 0 ) 10 ( 40 . 0 ) 6 ( 24 . 0 ) 4 ( 16 . 0 ) 5 ( 20 . 0 ) 3 ( 12 . 0 ) 12 ( 48 . 0 ) Figures in parentheses indicate percentage cont … Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 65 Table 2 : Diversity in Information Needs ( Designation Wise ) Information needs P a t i e n t D a t a / P a t i e n t c a r e i n f o r m a t i o n P o pu l a t i o n S t a t i s t i c s L a t e s t M e d i c a l k n o w l e d g e D r u g i n f o r m a t i o n L o g i s t i c i n f o r m a t i o n S o c i a l i n f l u e n c e s N e w m e d i c a l P r o d u c t s a nd E qu i p m e n t s L a b . / i m a g i n g d i a g n o s i s / p r o c e du r e s / t e c hn i q u e s R e s e a r c h S ub j e c t D i s a s t e r M a n a g e m e n t H o s p i t a l / h e a l t h a d m i n i s t r a t i o n G o v t . r e g u l a t i o n s o n h e a l t h c a r e Teaching * 75 75 ( 100 . 0 ) 10 ( 13 . 3 ) 75 ( 100 . 0 ) 75 ( 100 . 0 ) 37 ( 49 . 3 ) 32 ( 42 . 6 ) 55 ( 73 . 3 ) 32 ( 42 . 6 ) 45 ( 60 . 0 ) 63 ( 84 . 0 ) 6 ( 8 . 0 ) 4 ( 5 . 4 ) 40 ( 53 . 3 ) Clinical Practice * * 66 66 ( 100 . 0 ) 3 ( 4 . 5 ) 66 ( 100 . 0 ) 66 ( 100 . 0 ) 35 ( 53 . 0 ) 28 ( 42 . 4 ) 50 ( 75 . 7 ) 24 ( 36 . 3 ) 19 ( 28 . 7 ) 11 ( 16 . 6 ) 27 ( 40 . 9 ) 7 ( 10 . 6 ) 18 ( 27 . 2 ) Administration * * * 85 85 ( 100 . 0 ) 20 ( 23 . 5 ) 85 ( 100 . 0 ) 71 ( 83 . 0 ) 45 ( 52 . 9 ) 45 ( 52 . 9 ) 42 ( 49 . 4 ) 18 ( 21 . 1 ) 20 ( 23 . 5 ) 4 ( 4 . 7 ) 28 ( 32 . 9 ) 55 ( 64 . 7 ) 36 ( 42 . 3 ) Figures in parentheses indicate percentage * Principals , Professors , Associate Professors , Additional Professors , Assistant Professors , Demonstrators , Lecturers and Health Educators . * * Consultants , Residents , Physician Specialists , General Physicians , Surgeon specialists , Asstt . Surgeon and Dental surgeons . * * * Registrars , Directors / Dy . / Asst . Directors , CMOs , BMOs , Medical superintendent and Hospital administrators . Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 66 Table 3 : Diversity of Information Needs ( Specialization Wise Distribution ) Information needs P a t i e n t D a t a / P a t i e n t c a r e i n f o r m a t i o n P o pu l a t i o n S t a t i s t i c s L a t e s t M e d i c a l k n o w l e d g e D r u g i n f o r m a t i o n L o g i s t i c i n f o r m a t i o n S o c i a l i n f l u e n c e s N e w m e d i c a l P r o du c t s a nd E qu i p m e n t s L a b o r a t o r y / i m a g i n g P r o c e du r e s / t e c hn i qu e s R e s e a r c h S ub j e c t D i s a s t e r M a n a g e m e n t H o s p i t a l / h e a l t h a d m i n i s t r a t i o n G o v t . r e g u l a t i o n s o n h e a l t h c a r e General Medicine 15 15 ( 100 . 0 ) 6 ( 40 . 0 ) 15 ( 100 . 0 ) 15 ( 100 . 0 ) 7 ( 46 . 7 ) 5 ( 33 . 3 ) 12 ( 80 . 0 ) 9 ( 60 . 0 ) 12 ( 80 . 0 ) 10 ( 66 . 7 ) 9 ( 60 . 0 ) 5 ( 33 . 3 ) 8 ( 53 . 3 ) General Surgery 15 15 ( 100 . 0 ) 3 ( 20 . 0 ) 15 ( 100 . 0 ) 12 ( 80 . 0 ) 8 ( 53 . 3 ) 5 ( 33 . 3 ) 15 ( 100 . 0 ) 8 ( 53 . 3 ) 12 ( 80 . 0 ) 9 ( 60 . 0 ) 8 ( 53 . 3 ) 5 ( 33 . 3 ) 9 ( 60 . 0 ) Obstetrics and Gynaecology 15 15 ( 100 . 0 ) 2 ( 13 . 3 ) 15 ( 100 . 0 ) 15 ( 100 . 0 ) 7 ( 46 . 7 ) 8 ( 53 . 3 ) 15 ( 100 . 0 ) 7 ( 46 . 7 ) 4 ( 26 . 7 ) 5 ( 33 . 3 ) 4 ( 26 . 7 ) 3 ( 20 . 0 ) 8 ( 53 . 3 ) Pediatrics and Neonatology 15 15 ( 100 . 0 ) 3 ( 20 . 0 ) 15 ( 100 . 0 ) 15 ( 100 . 0 ) 9 ( 60 . 0 ) 6 ( 40 . 0 ) 10 ( 66 . 7 ) 4 ( 26 . 7 ) 5 ( 33 . 3 ) 3 ( 20 . 0 ) 3 ( 20 . 0 ) 4 ( 26 . 7 ) 7 ( 46 . 7 ) Orthopedics 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 14 ( 100 . 0 ) 6 ( 42 . 9 ) 5 ( 35 . 7 ) 12 ( 85 . 7 ) - - 6 ( 42 . 9 ) 4 ( 28 . 6 ) 3 ( 21 . 4 ) 5 ( 35 . 7 ) 6 ( 42 . 9 ) Physical Medicine and Rehabilitation 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 11 ( 78 . 6 ) 8 ( 57 . 1 ) 4 ( 28 . 6 ) 11 ( 78 . 6 ) - - 4 ( 28 . 6 ) 3 ( 21 . 4 ) 4 ( 28 . 6 ) 4 ( 28 . 6 ) 7 ( 50 . 0 ) Otorhinolaryngology ( ENT ) 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 14 ( 100 . 0 ) 6 ( 42 . 9 ) 5 ( 35 . 7 ) 6 ( 42 . 9 ) - - - - 4 ( 28 . 6 ) - - 3 ( 21 . 4 ) 7 ( 50 . 0 ) Ophthalmology 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 14 ( 100 . 0 ) 7 ( 50 . 0 ) 7 ( 50 . 0 ) 12 ( 85 . 7 ) 4 ( 28 . 6 ) 5 ( 35 . 7 ) 5 ( 35 . 7 ) 3 ( 21 . 4 ) 3 ( 21 . 4 ) 6 ( 42 . 8 ) cont … Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 67 Information needs P a t i e n t D a t a / P a t i e n t c a r e i n f o r m a t i o n P o pu l a t i o n S t a t i s t i c s L a t e s t M e d i c a l k n o w l e d g e D r u g i n f o r m a t i o n L o g i s t i c i n f o r m a t i o n S o c i a l i n f l u e n c e s N e w m e d i c a l P r o du c t s a nd E qu i p m e n t s L a b o r a t o r y / i m a g i n g P r o c e du r e s / t e c hn i qu e s R e s e a r c h S ub j e c t D i s a s t e r M a n a g e m e n t H o s p i t a l / h e a l t h a d m i n i s t r a t i o n G o v t . r e g u l a t i o n s o n h e a l t h c a r e Burns and Plastic Surgery 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 14 ( 100 . 0 ) 9 ( 64 . 3 ) 6 ( 42 . 9 ) 9 ( 64 . 3 ) 5 ( 35 . 7 ) 3 ( 21 . 4 ) 3 ( 21 . 4 ) 7 ( 50 . 0 ) 2 ( 14 . 3 ) 8 ( 57 . 1 ) Dermatology and Venerology 15 15 ( 100 . 0 ) - - 15 ( 100 . 0 ) 15 ( 100 . 0 ) 8 ( 53 . 3 ) 5 ( 33 . 3 ) 8 ( 53 . 3 ) 4 ( 26 . 7 ) 3 ( 20 . 0 ) 5 ( 33 . 3 ) - - 3 ( 20 . 0 ) 8 ( 53 . 3 ) Dental Surgery 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 14 ( 100 . 0 ) 7 ( 50 . 0 ) 4 ( 28 . 6 ) 12 ( 85 . 7 ) 5 ( 35 . 7 ) 2 ( 14 . 3 ) 5 ( 35 . 7 ) - - 5 ( 35 . 7 ) 3 ( 21 . 4 ) Psychiatry 14 14 ( 100 . 0 ) 3 ( 21 . 4 ) 14 ( 100 . 0 ) 14 ( 100 . 0 ) 7 ( 50 . 0 ) 10 ( 71 . 4 ) - - 4 ( 28 . 6 ) 6 ( 42 . 9 ) 4 ( 28 . 6 ) 3 ( 21 . 4 ) 3 ( 21 . 4 ) 3 ( 21 . 4 ) Anaesthesiology 15 15 ( 100 . 0 ) - - 15 ( 100 . 0 ) 12 ( 80 . 0 ) 8 ( 53 . 3 ) 6 ( 40 . 0 ) 10 ( 66 . 7 ) 4 ( 26 . 7 ) 4 ( 26 . 7 ) 4 ( 26 . 7 ) - - 4 ( 26 . 7 ) 2 ( 13 . 3 ) Toxicology and Forensic Medicine 14 14 ( 100 . 0 ) - - 14 ( 100 . 0 ) 14 ( 100 . 0 ) 7 ( 50 . 0 ) 12 ( 85 . 7 ) 6 ( 42 . 9 ) 8 ( 57 . 1 ) - - 3 ( 21 . 4 ) 7 ( 50 . 0 ) 2 ( 14 . 3 ) 3 ( 21 . 4 ) Community Med . & Public Health ( SPM ) 14 14 ( 100 . 0 ) 14 ( 100 . 0 ) 14 ( 100 . 0 ) 13 ( 92 . 9 ) 6 ( 42 . 9 ) 11 ( 78 . 6 ) 5 ( 35 . 7 ) 9 ( 64 . 3 ) 5 ( 35 . 7 ) 9 ( 64 . 3 ) 6 ( 42 . 9 ) 5 ( 35 . 7 ) 5 ( 35 . 7 ) Hospital Administration 10 10 ( 100 . 0 ) 2 ( 20 . 0 ) 10 ( 100 . 0 ) 6 ( 60 . 0 ) 7 ( 70 . 0 ) 6 ( 60 . 0 ) 4 ( 40 . 0 ) 3 ( 30 . 0 ) 3 ( 30 . 0 ) 2 ( 20 . 0 ) 4 ( 40 . 0 ) 10 ( 100 . 0 ) 4 ( 40 . 0 ) Figures in parentheses indicate percentage cont … Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 68 Appendix - I 1 . Sher - i - Kashmir Institute of Medical Sciences ( SKIMS ) and associated Medical College and Hospital , Srinagar . 2 . Government Medical College ( GMC ) , Associated Hospitals / Government Dental College ( GDC ) in Kashmir region . 3 . Government Medical College ( GMC ) , Associated Hospitals / Government Dental College ( GDC ) in Jammu region . 4 . District , Sub - district Hospitals , Dispensaries and Health Centres in the State . 5 . Ayurvedic cum Unani Hospitals and Dispensaries in the State . 6 . Acharya Shri Chandra College of Medical Sciences ( ASCOMS ) , Jammu , and 7 . Private Nursing Homes / Solo practitioners in the State . SKIMS and ASCOMS are tertiary health care institutions . GMCs / Associated hospitals & GDCs are secondary health care institutions while District / Sub - district hospitals / dispensaries / health centres , Ayurvedic cum Unani hospitals / dispensaries and Private nursing homes are primary health care institutions . References Balen , Robert M . , Jewesson , Peter J . & Cochran , Gary ( 2004 ) . Pharmacist computer skills and needs assessment survey . Journal of Medical Internet Research , 6 ( 1 ) . Retrieved September 17 , 2009 , from http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC1550586 / ? tool = pubmed Cogdill , Keith W . ( 2003 ) . Information needs and information seeking in primary care : A study of nurse practitioners . J . Med . Libr . Assoc . , 19 ( 2 ) , 203 - 215 . Retrieved May 17 , 2005 , from http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC153161 / pdf / i00 25 - 7338 - 091 - 02 - 0203 . pdf Covell , D . G . , Uman , G . C . & Manning , P . R . ( 1985 ) . Information needs in office practice : Are they being met ? Annals of Internet Medicine , 103 ( 4 ) , 596 - 599 . Retrieved June 8 , 2005 , from http : / / www . welch . jhu . edu / about / covell . pdf Dee , C . & Blazek , R . ( 1993 ) . Information needs of the rural physician : a descriptive study . Bull . Med . Libr . Assoc . , 81 ( 3 ) , 259 - 264 . Retrieved September , 27 , 2005 , from http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC225785 / pdf / mla b00112 - 0017 . pdf Dorsch , J . L . , & Pifalo , V . ( 1997 , October ) . Information needs of rural health practitioners : A retrospective use study . Bull . Med . Libr . Assoc . , 85 ( 4 ) , 341 - 347 . Retrieved March 17 , 2009 , from Information Needs of Physicians Shafi & Wani TRIM 7 ( 1 ) Jan - June 2011 69 http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC226290 / pdf / mla b00097 - 0031 . pdf Forsythe , D . E . , Buchanan , B . G . , Osheroff , J . A . , & Miller , R . A . ( 1992 ) . Expanding the concept of medical information : An observational study of physicians ' information needs . Computers and Biomedical Research , 25 , 181 - 200 . Goodall , George R . ( 2005 ) . Successive approximations : The story of information behaviour research . Retrieved June 27 , 2005 , from http : / / www . deregulo . com / facetation / pdfs / successiveApproxim ations . pdf Gonzalez , A . I . , Dawes , M . , Sanchez - Mateos , J . , Riesgo - Fruetes R . , Escortell - Mayer , E . , et al . ( 2007 ) . Information needs and information - seeking behavior of primary care physicians . Ann . Fam . Med . , 5 , 345 - 352 . Retrieved September 25 , 2009 , from http : / / www . annfammed . org / cgi / reprint / 5 / 4 / 345 Lundeen , G . W . , Tenopir , C . , & Wermager , P . ( 1994 , April ) . Information needs of rural health care practitioners in Hawaii . Bull . Med . Libr . Assoc . , 82 ( 2 ) , 197 - 205 . Retrieved May 21 , 2006 , from http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC225898 / pdf / mla b00107 - 0079 . pdf Osheroff , J . A . , Forsythe , D . E . , Buchanan , B . G , Bankowitz , R . A . , Blumenfeld , N . H . & Miller , R . A . ( 1991 ) . Physician’s information needs : Analysis of questions posed during clinical teaching . Ann . Intern . Med . , 114 ( 7 ) , 576 - 81 . Retrieved March , 9 , 2006 , from http : / / www . ncbi . nlm . nih . gov / pubmed / 2001091 Shabi , O . M . , Kutetyi , E . A . , Odewale , M . A . & Shabi , I . N . ( 2008 ) . Information needs of family physicians in Nigeria . S . A . Fam . Pract . , 50 ( 5 ) , 52 . Retrieved November 14 , 2008 , from http : / / www . safpj . co . za / index . php / safpj / article / viewFile / 915 / 12 56 Strasser , T . C . ( 1978 ) . The information needs of practicing physicians in northeastern New York State . Bull . Med . Libr . Assoc . , 66 ( 2 ) , 200 - 209 . Retrieved May 17 , 2005 , from http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC199446 / pdf / mla b00126 - 0048 . pdf Wilson , T . D . ( 1999 ) . Models in information behaviour research . Journal of Documentation , 55 ( 3 ) , 249 – 270 Woolf , S . H . & Benson , D . A . ( 1989 ) . The medical information needs of internists and pediatricians at an academic and medical centre . Bull . Med . Libr . Assoc . , 77 ( 4 ) , 372 - 380 . Retrieved April 23 , 2006 , from http : / / www . ncbi . nlm . nih . gov / pmc / articles / PMC227491 / pdf / mla b00044 - 0066 . pdf \ No newline at end of file diff --git a/silver_data/3857979caaca0e8805b7e6327deb1a2b784fcff4.pdf.txt b/silver_data/3857979caaca0e8805b7e6327deb1a2b784fcff4.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..724f4070dee4562696ec90fce78ce181ebfcb3a5 --- /dev/null +++ b/silver_data/3857979caaca0e8805b7e6327deb1a2b784fcff4.pdf.txt @@ -0,0 +1 @@ +INTERNATIONAL CONFERENCE ON ENGINEERING DESIGN , ICED11 15 - 18 AUGUST 2011 , TECHNICAL UNIVERSITY OF DENMARK THE PSYCHOLOGICAL EXPERIENCE OF USER OBSERVATION Elizabeth Gerber Northwestern University ABSTRACT While scholars have studied what design practices accomplish , few have considered the psychological experience of practicing design and the implications for completing their work . An eighteen - month ethnographic study of a high - tech firm examined the psychological experience of design work , specifically how people experience user observation . The study finds that when people observe others in context , they engage in a state of exploration . Regular observation supports curiosity , or the tendency to focus attention and seek answers to unanswered questions . Focused attention fosters commitment to the design problem . Results suggest how design work practices can be designed to help employees to reduce anxiety about the unknown , an inherent feature of the design process . Keywords : Psychology of design ; user observation ; design practice ; design cognition ; job design 1 INTRODUCTION Scholars describe design as a learning process [ 1 - 3 ] . People construct new knowledge through user observation that yield insights ; insights support frameworks , which inspire ideas that lead to innovative solutions [ 3 ] . Through this process , people construct knowledge [ 4 ] , moving back and forth from the analytic phase of design , which focuses on finding and discovery , and to the synthetic phase , which focuses on invention and making [ 1 ] . Building on Kolb’s experiential learning theory , Beckman and Berry [ 3 ] describe knowledge creation through the design process as movement between concrete experiences and abstract conceptualization , reflective observation , and active experimentation . Kolb’s experiential learning theory [ 5 ] focuses on how knowledge is created by transforming experiences - when a person carries out a particular action in a particular setting , reflects on the effects of that action , attempts to understand those effects , and then modifies actions to accommodate new ideas . Inductive and deductive practices support the construction of new knowledge that designers use to shape the environment in ways that did not previously exist . Given that design is a learning process , scholars have studied what knowledge is gained when people engage in popular design practices such as user observation [ 6 , 7 ] , brainstorming [ 8 , 9 ] , sketching [ 10 - 12 ] design documentation [ 4 ] and low - fidelity prototyping [ 11 , 13 , 14 ] , yet few scholars have considered how people feel when engaging in these popular design practices . Even fewer have investigated how people feel when engaged in design practices in a work context , as opposed to a laboratory study . Understanding what work is accomplished and how workers feel about the work is critical for organizations that rely on motivated and satisfied workers to complete the work outcomes necessary for success [ 15 , 16 ] . This paper explores the experiences of a thirty - five member team at a large high - tech firm as they practiced user observation to design and develop globally - distributed digital products . More broadly , this study is concerned with how people psychologically experience the construction of knowledge while enacting design work practices . When people construct new knowledge , they initially experience uncertainty , or a state of being in doubt , because the final outcomes are not yet known [ 17 ] . The experience of uncertainty is mediated by perceptions of control and fear of failure . Peoples’ experience of uncertainty depends on their perception of their ability to control the uncertain conditions [ 18 ] . In uncertain conditions which promote high control , individuals experience increased intrinsic motivation , greater interest , less pressure and tension , more creativity , more cognitive flexibility , better conceptual learning , higher self - esteem , more trust , and greater persistence [ 19 , 20 ] . In these environments , they are more likely to be proactive and take action in the face of setbacks [ 20 ] . In contrast , in uncertain environments that promote low control , they are less likely to experience these positive outcomes and engage in productive creative work in the face of setbacks [ 21 ] . Design scholars find that designers embrace the uncertainty to create new solutions not yet identified by others [ 22 ] , but what they do to embrace the uncertainty is not clear . More recently , researchers propose that designers adopt design practices such as low - fidelity prototyping to promote control in the face of uncertainty [ 23 ] . When prototyping , practitioners break larger tasks into modest size tasks , allowing them to take frequent action . By taking frequent action on manageable tasks , practitioners experience small wins by observing their impact and attributing success to their actions . This paper similarly employs a psychological lens to understand the how people feel when observing others in context and how this practice may help to reduce anxiety about the unknown while designing . 2 METHODS The mid - range theory is grounded in an eighteen - month ethnographic study of 35 member team in a large software firm , “Big Tech , ” and in pertinent behavioral science . Big Tech’s product development efforts are multi - national and the firm’s products and services are sold throughout the developed world . This study focused on the “Green Team , ” a team charged with working with a diverse set of teams throughout the company to apply a design process which emphasized user observation as a way of quickly realizing insights about users to inform design decisions . The Green Team initially formed in September 2004 due to senior management concerns about product usability and development time . For the first three months , I observed the members of the corporate strategy team researching and preparing for the development of the team . A design consultancy firm specializing in the user - centered design process taught a six - member team through active hands - on coaching with the expected outcome of innovation , a process called , participant innovation [ 23 ] . The hands on nature of participant innovation stood in contrast to an innovation consulting engagement typically delivered by traditional management consulting firms in which consultants either present power point decks on process or manage work independently of the client so the client is less actively involved in learning new skills . After this initial three - month trial and training period , the board officially approved the creation of the team to use the user - centered design process to positively impact the company’s revenue generating products , processes and services . I spent the remaining 15 months of my study observing the Team develop its 35 member team , solicit projects upon which to apply their design process , and coach internal teams through their user observation based design process to drive innovation throughout Big Tech . During the observation period , the team worked on eight distinct projects , however no products made it to market during the observation period . While the team’s stated goal was to create at least 3 user - centered designs to positively impact revenue in the first year , by the end of the observation , no products had been introduced to the market . However , during user - testing products , users described products as more user - friendly than existing products offered by the firm . In this way , the user observation practice did influence the products pre - production , however market impact was not unknown . Despite not meeting the stated goal , the Team remained committed to enacting the user - centered design process . 2 . 1 Research Design As is typical with grounded mid - range theory building , which are inspired by an empirical phenomenon and then blend theoretical research to formulate a theory { Merton , 1957 # 486 } , I initiated this study with open data collection , rather than specific hypotheses about what was to be found [ 4 ] . After initial data collection , I searched for emerging patterns and followed the most interesting and promising patterns [ 5 ] . I collected data from the time the team was introduced to the design process and observed them applying the process to over 15 products and services . Primarily , case studies are conducted retroactively , relying on reflection . The advantage to this research approach is the ability to collect real - time longitudinal data ; the disadvantage is that biased is introduced through participant observation [ 6 ] . I observed the Team one day a week over the 18 - month period ( September 2004 - March 2006 ) , writing detailed transcripts of conversations I heard and interactions I observed between the Team’s members , its users , clients , partners , and stakeholders . A typical visit entailed informal and formal conversations with team members as well as observation of formal and informal meetings about design projects throughout the day . I watched the teamwork on projects at different stages of development and asked questions about how and why they were using various work practices such as user observation , prototyping , and brainstorming . Throughout the observation , I cultivated relationships with all team members – discussing the development of the team and adoption of design practices . I was welcomed to observe all meetings throughout the 18 - month period except for three meetings pertaining to employee compensation ; moreover , the company provided me with space in the Team office area , allowing me to observe the naturally occurring team interactions , work practices , and day - to - day activity . The office area had an open office plan with making it easy to observe most interactions and the outcomes that resulted from their work . Throughout the observation period , I understood the majority of the technical content discussed by the team as I had worked in a related industry prior to becoming a researcher . I began this study with a broad research question : “What is the psychological experience of practicing design ? ” I chose qualitative methods to explore this question because I did not want to unnecessarily constrain my emergent framework by precisely identifying and operationalizing variables before data collection began . Following guidelines for inductive research [ 7 ] , I wrote descriptive accounts of my observations of team members both engaging in the practice of user observation and discussing observations with colleagues until major themes emerged such as , curiosity , control , and uncertainty . Following guidelines for developing well - grounded theory [ 7 ] , I clustered these phenomena into larger conceptual categories determining strength of the evidence by frequency of occurance . Simultaneously , I read pertinent literature to understand existing theory and to uncover related phenomena . Moving between inductive and deductive thinking , I began to develop my emerging conceptual framework , linking the work practice of user observation to the observed phenomena . This iterative process allowed me to develop initial inferences about the psychological experience of enacting user observation . I continued to validate my theory against the data by reviewing all relevant data and compiling evidence and evaluating the strength of my evidence to inform whether I should modify or abandon my inferences based on insubstantial evidence . Following Strauss and Corbin’s [ 7 ] guidelines , I flip - flopped between data and theory to construct an evidence - supported theory of how people experience control in the face of uncertainty through the enactment of the user observation practice . 2 . 2 Data Throughout the study , I used qualitative methods to gather data from multiple sources to inform my inferences about the informants’ experiences of observing users in context . By gathering data from multiple sources , I had multiple measures of the same phenomenon , avoiding the potential problem of construct validity within a single case [ 24 ] . The framework was strongly supported by the convergence of multiple , independent observations . These sources can be divided into six categories : observed meetings , observed strategic off - sites , observations of employees observing users in context , semi structured interviews , team generated materials , and externally generated materials related to the practice of user observation . When my data collection ended , I had accumulated 484 research hours . 3 DESIGN PRACTICE : USER OBSERVATION Design researchers have identified user observation , or the observation of people in context , as a critical activity in the design process [ 25 ] . User observation allows practitioners to inform ideas based on real human activity in context . When observing users , practitioners create new knowledge about the most appropriate solution for the identified problem at hand [ 26 ] . Researchers distinguish user observation from user testing which is concerned with ideas before they are fully formed rather than testing ideas ready for production . With user observation , individuals construct new knowledge quickly by observing users in their actual context rather than bringing them into an artificial laboratory to discuss their habits and preferences . The aim is to spend time with users rather than in time in isolation of users generating ideas [ 26 ] These insights are used to inform the next steps of their design process . For practitioners , much is understood about the role of user observation in helping individuals and teams make decisions about future directions throughout the development process . However , few academic studies consider the individual’s psychological experience of rapidly realizing insights and constructing new knowledge through user observation . Consistent with the research on user observation , Green Team members described the usefulness of user observation in gaining information to inform the next steps in their design process . One team member described user observation as “the source of innovation” in their design process because demonstrated explicit and implicit user needs . 4 PSYCHOLOGICAL EXPERIENCE OF USER OBSERVATION Drawing on psychological research , I present a mid - range theory to explain how the practice of user observation allows practitioners to remain committed to the design process despite uncertainty about final outcomes . I propose that curiosity is triggered through regular exploration supported by the user observation . Regular exploration exposed practitioners to new information and alerted them to the presence of gaps in their knowledge . Exposed to gaps in their knowledge , individuals were motivated to use the work practices to continue to explore , seeking knowledge in order to close these knowledge gaps . I propose that by using user observation to satiate curiosity , the practitioners’ anxiety about the unknown was reduced . The curiosity mechanism is largely informed by Loewenstein and Litman’s conception of curiosity . According to Loewenstein [ 27 ] , curiosity is a form of cognitively induced deprivation . This deprivation arises when an individual perceives a gap in his or her understanding of a situation or when “one’s informational reference point in a particular domain becomes elevated above one’s current level of knowledge” [ 27 : 87 ] . In this state , individuals are motivated by a desire to know , see , or experience . According to Litman [ 28 ] , this desire motivates “exploratory behavior directed towards the acquisition of new information” ( 793 ) . People are motivated to act in ways that close the information gap to experience the pleasurable anticipation of closing the gap [ 29 ] . 1 Practitioners of the user observation practice exemplified this knowledge gap conception of curiosity . 2 Practitioners often initiated a project using the user observation practice . Through this practice , practitioners routinely perceived gaps in their understanding of human behavior and used the practices to gather information to close these knowledge gaps . Nancy , a member of a four - person team , enthusiastically described her first experience of identifying a knowledge gap and seeking knowledge to close the gap through the user observation work practice . “The first time … we observed a team meeting in their conference room . We watched the team as they interacted / collaborated about product and content issues . We watched what they did and…recorded with videos and photos . We were flies on the wall this time . So we came in not knowing anything . We were excited to be there , though throughout the observation we constantly came up with questions we just wrote down . This first meeting left us curious with lots of questions that we later asked in interviews . This experience generated a lot of questions for us that we needed and wanted to answer to understand our ‘users’ . We felt the need to know more . ” In the description of her experience observing users in context , Nancy described her transition from “not knowing anything” to seeking answers to her team’s newly formed questions . She described the first observation as raising questions , or identifying knowledge gaps . These questions motivated her team to return to the site to conduct follow - up interviews to answer their questions or to close the knowledge gap . Nancy’s team “needed and wanted” to gather more information . This behavior supports the knowledge gap theory of curiosity in which individuals are motivated and gain pleasure closing knowledge gaps [ 28 ] . Nancy and her team’s initial exposure to unknown information through the user observation practice triggered an initial devotion to the practice . Nancy then expected the work practices by which the questions were raised and knowledge gaps were identified to also close the gaps by providing answers to her questions . For this reason , I propose that Nancy’s reliance on the user observation practice to both open and close knowledge gaps reduced her anxiety about the unknown . The work practice served as a means for both realizing and satiating curiosity . Loewenstein posits that people are likely to expose themselves to curiosity - inducing situations if they feel they will gain pleasure from the experience of gaining information and satiating their curiosity [ 27 ] . He describes this voluntary exposure to curiosity - inducing situations as a gamble because in these situations , it is not necessarily clear that once curiosity is piqued , new information will be identified to satiate one’s 1 A major distinction between Loewenstein’s ( 1994 ) and Litman’s ( 2005 ) model is that Loewenstein proposes that curiosity is motivated by an adversiveness to not having information whereas Litman suggests that people derive pleasure from seeking information to satiate curiosity . Litman’s claim is most consistent with my data . curiosity . In this way , Nancy and her team gambled with their approach . Through the practice of user observation , Nancy and her team could have only identified gaps in knowledge and not been able to generate solutions to close the gap . As was regularly the case with the user observation practice , the payoff was greater than the knowledge deprivation initially experienced during the initial observation . Researchers explain this need for closure as motivated by a desire to reduce cognitive demands allocated to searching for the answer . The need may be heightened when the perceived benefits of closure are positive ( such as under time pressure to meet a deadline or when the process is arduous ) and weakened when the perceived benefits of closure are negative ( such as fear of making a costly judgmental mistake ) [ 30 ] . 4 . 1 Curiosity Inducing Stimuli Awareness of information is an antecedent to experiencing curiosity [ 27 ] . Exploration supports an awareness of new information [ 17 ] . During data collection , the user observation practice supported regular exploratory behavior , continually exposing practitioners to new information . For example , during user observation , practitioners discovered how people , other than themselves , felt and behaved in a natural context . The user observation practice also revealed new information and made practitioners aware of the information they do not know . Practitioners were exposed to knowledge gaps through regular exploration , combating the tendency for them to become overconfident and underestimate the extent of the gaps in their knowledge [ e . g . 31 , 32 ] . Researchers propose several conditions under which individuals are exposed to curiosity - inducing situations . In these situations , curiosity inducing stimuli alert people to the existence of an information gap [ 27 , 33 ] . My data suggest that the user observation practice supported two of these identified conditions and alerted practitioners to the existence of an information gap . The first condition occurred when an individual was posed a question that made him aware of the information he was missing . And the second condition occurred when an unexpected event occurred and unexpected information was revealed [ 27 , 33 ] . In my data , these conditions regularly occurred through exploration . 4 . 1 . 1 Awareness of Missing Information Next , I offer specific examples in which the user observation practice exposed practitioners to each of the curiosity - inducing situations described above , although in reality , the conditions more often occurred simultaneously . In the first condition , questions are raised making individuals aware of the information they are missing [ 27 , 33 ] . Unable to answer a question , individuals become aware of their knowledge gap and seek information to fill it . Curiosity researcher , Berlyne [ 34 ] referred to these questions as “thematic probes . ” When practicing user observation , the Green Team developed “thematic probes , ” raising specific questions about how people behaved and how related products and services were designed to fit their needs . The questions made practitioners aware of missing information and motivated them to seek information to answer the questions . During a project related to improving the subway navigation for first time users , the Green Team members gathered information through user observation in context . The team spent the day in teams of three observing passengers locating , entering , travelling on , and leaving the subway . Armed with new information about the situation , the Green Team returned to their office to post their observations from the subway system . The participants noted their observations on post - it notes and stuck them on the walls of the room in which they were brainstorming ideas . Based on these insights , team members began to generate ideas . With each additional insight and idea posted , participants became aware of the breadth of possible ideas by raising new questions . Over the course of one hour , the participants generated approximately one hundred ideas by building off of each other’s ideas . The brainstorms elicited ideas the participants called “wild” and “surprising” ideas . The user observation combined with the brainstorming practice allowed my informants to answer outstanding questions as well as generate new questions to be answered . In this example , curiosity was strengthened by the public nature of the work practices . By getting people to publicly commit their ideas , individuals become more engaged and curious in the outcome [ 35 , 36 ] . When individuals make hypotheses and receive accurate feedback to confirm or deny their hypotheses , curiosity increases [ 27 ] . If individuals did not receive accurate feedback , individuals would not experience the satisfaction of reducing cognitive deprivation . Members of the Green Team often made hypotheses about how people would behave and then tested their hypotheses using the user observation practice . When observing elderly at the shopping mall , Greg , a Green Team member , described his satisfaction with the experience of testing hypotheses and receiving feedback about his ideas . “We went to the shopping center… [ it was ] eye - opening . Eye - opening . I remember it was hot out there , and I was talking with people , strangers that I never thought I’d ever talk to . [ He laughs . ] And I was amazed how much information they would share . So , I had elderly people , and my hypothesis was how can you make it a better shopping experience for elderly people ? Well , you get onramps for their wheelchairs and stuff , and easier parking , and I couldn’t be further from the truth . These guys are not going there for shopping , they’re going there to hang out at a classy place . So , what you got to do , you got to make it a more classy place to hang out . It’s not about shopping , it’s really about , you know , little yellow benches and shade trees and stuff where people can hang out and spend their time and buy an occasional coffee . They’re not there to go to Bloomingdale’s only . We even saw people , you know , the body language . They would have their hands stuck , you know , pushed into their belts so they couldn’t even touch anything there . Kind of solely window - shopping , but don’t buy anything . [ He laughs . ] Eye - opening . I was thinking , wow…I thought I knew it all and you learn something new . ” In this excerpt , Greg described his excitement of asking questions and seeking answers for why people behave as they do . Rather than being embarrassed about making false assumptions , Greg proudly acknowledged that his assumptions about how and why people behave the way they do was inaccurate . His excitement was around the process of discovering new information in response to a question being raised rather than knowing all of the answers . In this way , curiosity - inducing situations , as fostered through the user observation practice , supported pleasurable experiences . Empirical research supports this finding . When an individual is confronted with unexpected , new information , he or she often experiences pleasantness followed by curiosity [ 37 ] . 4 . 1 . 2 Violation of Expectations A second way in which curiosity is induced is when individuals experience a violation of expectations . This violation of expectations makes people aware of the information they are missing - leading them to search for explanations to make sense of their environment [ 33 ] . Drawing on work by cognitive psychologists , Hastie [ 33 ] explains that unexpected events are processed differently than expected events and that the unexpected events will be recalled more easily than expected events [ 38 ] . This is because unexpected events will be linked to relevant scripts , or knowledge structures [ 39 ] . These links are connected with distinctive tags which are shown to be more easily remembered on recognition tests [ 33 ] . Frequently when enacting user observation , practitioners experienced curiosity when their expectations were violated . User observation routinely set up situations for practitioners , who may have been familiar with a situation , to have their expectations violated . Before observing users in the field , team members publicly stated all of the things they thought they knew about the project to be completed . By making their assumptions about the project explicit , they identified the personal bias of their assumptions . Through this activity , the team members aimed to clear their minds of their expectations for what they’d observe so that they could observe “as if they were seeing something for the first time . ” In this way , participants were more likely to have their expectations violated when they were observing unfamiliar topics . Practitioners were encouraged to pretend as if they were from another planet visiting earth for the first time . The logic behind this advice was that if a participant had little knowledge of human behavior , they would have fewer schemas upon which to rely and their awareness of new information would increase . When overconfident , individuals underestimate the extent of what they do not know [ e . g . 31 , 32 ] . With this approach , the practice of user observation made the mundane seem novel even to practitioners who were intimately familiar with the situation being observed . For example , when the Green Team observed users in a familiar setting , they were continually surprised by how users behaved . After spending the morning observing people use the subway ticket purchasing system in a metropolitan center , members of the Green Team collaborated with an internal team at Tech Co . to generate ideas about how to improve this experience for subway users . Together , they shared their findings from the user observation practice . Examples of observations recorded in the notebooks they carried included : “Travelers with luggage – hard to keep an eye on luggage and look for money , purse , grab change , etc . , ” “Pick - up window is too small . Ouch ! Hands get stuck in ticket drawer , ” “Exit Turnstile – suit case a problem . It’s too narrow – more time required to get through turnstile , ” and “Ticket machine asking for ‘Credit Card or ATM’ but machine takes ATM only . ” Each observation illustrated the ways in which the participants’ expectations were violated . This violation of expectations led the participants to identify gaps in their knowledge about how people engaged with the ticketing purchasing system . They generated questions about how people engaged with a system that was difficult to use . Although participants initially predicted the observation site would be uninteresting as many were familiar with the location and didn’t perceive the ticket machine as problematic , the site became a subject of great interest during the three - hour observation period . They were quickly captivated by their initial observation that first time subway users were challenged when purchasing tickets . They remained in the subway , observing users and interviewing subway facility managers to learn more about how and why the system worked as it did – or to close the gaps in their knowledge . The time passed quickly and when they were asked to return to the office to generate ideas about how they could improve current conditions , some were hesitant to leave the observation site because they felt they could still learn more about why people behaved the way they did . The data suggest that the presumed familiarity with the observation site enhanced the sense of expectations being violated . Research finds that individuals experience surprise , or a sharp increase in neural stimulation [ 40 ] when their anticipation of an experience differs form the actual experience [ 41 ] . This situation may be characterized by a high degree of pleasantness and self - assurance [ 37 ] . Members of the Green Team frequently reported being pleasantly surprised by unexpected events that occurred when practicing user observation . When observing a man using an electric ticket machine , Mary , a member of the Green Team , was surprised when she saw that the man’s hand was too large to retrieve his change from the return bin . Mary excitedly motioned for her observation partner to come to the machine to observe the man’s behavior and the machine’s design . Mary was surprised by this unexpected event so much so that she became solely focused on this unexpected situation . Research suggests that when a familiar schema for how things are supposed to work is broken , individuals try to make sense of the situation [ 33 , 42 ] . The need for closure is motivated by a desire for the eschewal of ambiguity [ 30 ] . When making sense of a situation , surprise serves to void ongoing emotion and cognition so individuals can respond to the stimulus situation [ 40 ] . Empirical research finds surprise often leads to curiosity because surprise clears the nervous system of ongoing activity in order to explain the stimulus situation [ 40 ] . This finding supports a Green Team manager’s observation about the role of user observation in stimulating interest in the methodology . Despite the costly nature of the practice , the Green Team manager described the practice of user observation as being critical to fueling people’s devotion to the methodology because the process made them aware of “how blind they are to the world around them” and inspired people to open their eyes and look more closely at what they take for granted . As a long time member of Tech Co . , he believed that he and his colleagues were so familiar with the internal perceptions of Tech Co . ’s products that exposure to external perceptions of Tech Co . ’s products routinely surprised them . He argued that the “magic” of the user observation practice came from paying attention to unexpected user behavior and being intrigued enough to want to learn more . Another informant described surprise leading to curiosity when he saw “old things in new ways . ” According to him , “It [ the observation practice ] awakens people that this is the world we live in , which we are often ignorant of . ” The informant’s observation is supported by empirical research that surprise is associated with exploration , curiosity , and knowledge - seeking [ 43 , 44 ] . 4 . 2 Seeking out Curiosity Inducing Experiences Voluntary exposure to curiosity inducing experiences is described as a type of gamble [ 27 ] . Before exploring , a person must guess the likelihood that his or her curiosity will be satiated . If a person expects to be left in an aversive state , exposing him or herself to the curiosity inducing experience may not be worth the gamble . If a person expects to experience a satisfying closure , exposing him or herself to the curiosity inducing experience may be worth his or her while . When people voluntarily seek out seek out experiences that initially create information gaps , they have a high expectation that they will be able to derive pleasure from satisfying their curiosity [ 27 ] . During a semi - structured interview about how and why the informant engaged in user observation , a Green Team member explained , “…by conducting generative end - user research , the information … will feed into the design in a way that it provides value add above and beyond what you think you know . ” The assumption was that the practice of observing people in context would inevitably reveal knowledge gaps and lead to satisfying discoveries . In this way , user observation strongly supported both knowledge seeking and fulfilling behavior described by Loewenstein [ 27 ] . Cialdini [ 45 ] offers the example of mystery and cliffhanger novels as voluntary exposures to curiosity inducing experiences . Mysteries are powerful because they “initiate the wonder , ” creating a need for closure and then creating the opportunity for closure . “All of us have heard of the famous ‘Aha ! ’ experience , ” Cialdini notes , “Well , the ‘Aha ! ’ experience becomes much more satisfying when it is preceded by the ‘Huh ? ’ experience . ” The process of setting up and resolving mysteries is similar to the process of scientific investigation : people identify a question or pose a mystery , generate hypotheses , and test the implications or consider alternatives to explain the mystery , collect evidence or provide clues to inform the proper explanation for the mystery , and derive a satisfactory resolution or resolve the mystery [ 45 : 24 - 27 ] . During a training workshop for user - centered software products , Green team members explained to attendees that they should expect to be extremely confused when first observing people in context – as if they were “visiting an alien planet . ” Further , they explained after iterating through the practice , they should expect to experience distinct and satisfying moments of insight and control over what was previously perceived to be an ambiguous problem . 4 . 3 Continuous Curiosity The data suggest that the Green Team’s curiosity was typically sustained throughout a project by the quick iterations between the work practices , allowing participants to gather new information and adjust their informational reference point . Curiosity was often captured upon initial initiation of the observation work practice . The Green Team experienced curiosity as generated by the violated experiences realized when enacting the process . When questions raised by the novel experiences were not immediately resolved , Green Team members repeated the work practices to find explanations for the questions initially raised . Project typically began with team members leaving the Tech Co . ’s campus to visit unfamiliar users in their work environments . This practice of leaving Tech Co . ’s campus violated the normative expectation that all “work” be completed on campus . As described earlier , by leaving the office , the Green Team members were more likely to encounter unexpected situations because they were in an unfamiliar context observing the routines of people they didn’t know . This initial violation of expectations was sustained during the actual observation . Practitioners continued to have their expectations violated as they observed either the familiar in an unfamiliar context , or the unfamiliar in a familiar context . In either case , the contrast of the familiar with the unfamiliar heightened what they did not know . Throughout an observation , members of the Green Team asked themselves questions like , “Why do people do what they do ? ” and “How could their life be improved ? ” highlighting specific knowledge that they were missing . Observations revealed novelty in human behavior that may have been previously viewed as mundane . The first observation teased participants about what they didn’t know , identifying knowledge gaps and causing them to continue to observe in order to gather more knowledge . Subsequent observations gave practitioners at Tech Co . the sense that they were making progress to close their knowledge gaps . For example , David’s experience collecting new information changed the perceived size of his information set and led to continuous curiosity . David , a Green Team member , described his experience of observing colleagues struggling to get useful feedback on the graphic designs they created . The experience raised questions for David about how people gave feedback , leading him to further observe how people gave feedback to his colleagues . He observed people printing out hard copies of the graphic designs and sticking post - it notes on the printouts . He reported being surprised by this behavior because he expected them to make their edits directly on the soft copy . He reported becoming curious about why people behaved in this way . In other words , he identified a gap in his knowledge about why people behaved in the way they did . This gap in knowledge led to further observations in which he learned that the people giving feedback liked the affordances of post - it notes . This initial curiosity led to his interest in Tech Co . ’s technical capability to make a tool that made giving feedback to Tech Co . ’s user interface design work easier . Although not formally sponsored by Tech Co . , David continued to work on the project . He began prototyping ideas for how to get feedback on graphic designs quickly . He searched for ways in which other systems elicited feedback and observed how people used a popular web site called “Hot or Not” to vote on people they thought were attractive . He also observed how people used sticky notes to make their comments on hardcopy written drafts . In response to this knowledge gathering experience , he and a colleague made several prototypes to test possible solutions . One prototype he made allowed individuals to make extensive comments on line – similar to the way in which sticky notes were used to offer comments on written drafts . Another prototype allowed individuals to just give a positive or negative rating of their idea - similar to the way he observed users voting for their preferences on the “Hot or Not” website . David described his extreme interest in manipulating components of the prototype to see which design would most successfully satiate the need . He tried many ways , eager to figure out which method would be most effective . In this example , David experienced multiple curiosity stimulating conditions including a violation of expectations and question raising [ 27 , 33 ] . David’s behavior is supported by empirical research that suggests that as people gain knowledge , rather than becoming less curious due to fewer gaps , people may become more curious [ 27 ] . Later in the year , David reported observing watching senior management printing out reports from their computers and saving them in paper files rather than filing the reports in folders on their computers . He reported being surprised by their paper based system because Tech Co . emphasized paperless work processes for employees . Although the user observation practice was typically scheduled at the beginning of a project , people were encouraged to always be observing people’s behavior and in particular , people were encouraged to be attuned to unexpected behaviors . In this case , David was not formally “observing” senior management , however this surprising behavior caught his attention . He had identified a knowledge gap : he knew his company advocated paperless work , yet senior management often printed out hard copies of their online reports . Noticing this behavior , he continued to observe how senior management and others interacted with online and offline interfaces . At the time of writing , this Green Team member continued to collect data and make notes about people’s use of online printouts , building his knowledge about this type of behavior – a behavior previously unfamiliar to him . Despite repetitive engagement with the methodology over the course of a year and half , the Green Team remained committed to the process for knowledge seeking and satiation . Their interest in the process was sustained when coaching internal team members and learning how they interpreted the work practices . In one example , the Green Team accompanied a team of Tech Co . developers in Beijing onto the streets to observe how individuals use transportation software . Upon their return to the office , both the Chinese developers and the Green Team reported their findings . The Chinese developers reported intriguing findings about the people they observed on the streets of Beijing , and the Green Team , who were coaching the Chinese developers through the methodology , reported their surprise about the Chinese developers’ approach to the practice . The Green Team members who were culturally American were surprised by how easily and comfortably the Chinese developers approached strangers on the street to ask them about their use of transportation software . The team members revealed a cultural knowledge gap between how they expected the developers to behave versus how they behaved . In this case , the Green Team members shared new insights about what they previously conceived of the user observation practice , a practice about which they thought they knew a great amount . They shared new insights about the cultural differences in approach behavior and suggested ways that the Green Team could learn about how other cultures initiate conversations with strangers . The knowledge gap revealed the extent to which the Green Team members assumed all cultures behaved similarly around strangers . After realizing their assumption , the Green Team was motivated to close the knowledge gap by learning more about cultural differences in this domain . After this experience , the Green Team demonstrated increased curiosity and attention to noticing different cultures’ comfort interacting with unfamiliar people and places . Through teaching the user observation practice to others , the team increased their commitment to the process as a way of overcoming uncertainty . 5 DISCUSSION This paper offers an explanation for how individuals manage the uncertainty inherent when designing – or creating new knowledge and shaping the environment in new ways . This paper proposes that individuals who engage in user observation encourages regular exploration . Regular exploration gave practitioners the opportunity to become aware of information – specifically providing opportunities for knowledge gaps to be created and filled . The information - gap perspective of curiosity purports that individuals seek out information that closes the knowledge gap [ 27 ] and that the elimination of the gap in knowledge is inherently satisfying [ 28 ] . Individuals continually realized and satiated their curiosity through interactions with users . In this way , individuals managed the uncertainty of the design process . This lens is unique in the design literature supports a growing stream of research that considers studying the psychological experience of enacting design practices in a work context [ 21 ] . When the design process is viewed as a learning process in an organizational context and from the perspective of the practitioners , questions are raised and how the practices are experienced and additional outcomes are realized – such as reducing anxiety and commitment to the design process in the face of uncertainty . This study suggests that when enacting the user observation work practice , user feedback and decision - making may not be the sole effectiveness outcome . While user observation may not always be resource efficient , as it may demand costly resources to enact , it may be effective at accomplishing other organizational objectives such as commitment to a design process and giving control in a highly ambiguous situation . Identifying outcomes beyond the traditional effectiveness outcomes is consistent with Sutton and Hargadon’s [ 46 ] study of brainstorming that found six additional consequences of practicing brainstorming not previously examined in the experimental literature . In addition to the expected outcome of generating ideas , brainstorming supported the organization’s memory of solutions , provided skill variety , supported an attitude of wisdom in and outside the session , created a status auction that maintained a focus on designing products , and impressed clients and generated income . Together , this research contributes to a growing body of work that explores how psychological outcomes are supported at work [ 22 ] and also highlights the importance of studying work practices in an organizational context , rather than in a laboratory setting . Finally , it extends research on the user observation practice beyond a consideration the role of user observation in eliciting user feedback and in decision making . By extending the research on this design critical activity , user observation , we can better understand the design process at large . 5 . 1 Implications for Design Management This research study suggests ways in which individuals can manage design work in uncertain conditions . The design process , like other innovation processes , does not systematically result in marketplace innovation , and when it does , this feedback comes long after the design process has been enacted . Although employees may initially be persuaded to pursue an innovation process because of successful implementation of the process in other organizations and interest in doing “something new , ” this motivation may not be sustainable [ 47 ] . Over time , employees may express uncertainty about the effectiveness of the innovation process if they do not benefit from the day - to - day enactment of their work practices . As first intimated by Hackman and Oldman’s work on job redesign [ 16 ] , managers may actively design employee work experiences by using behavioral science theory to evoke cognitive , emotional , and behavioral reactions from employees . User observation is an important and useful work practice that delivers immediate knowledge to employees who are tackling challenges with great uncertainty . Increased knowledge about work increases worker satisfaction and motivation , reducing likelihood for costly worker turnover [ 48 ] . Managers may adopt and design work practices , such as user observation , to which employees are committed so they experience intermediate benefits before formal outcomes are realized . 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Administrative Science Quarterly , 1980 , 25 ( 2 ) , pp226 - 251 . [ 42 ] Weick , K . E . , The Social Psychology of Organizing . ( Addison - Wesley , Reading , MA , 1979 ) . [ 43 ] Silvia , P . J . , What is interesting ? Exploring the appraisal structure of interest . Emotion , 2005 , 5 , pp89 - 102 . [ 44 ] Turner , S . A . and Silvia , P . J . , Must Interesting Things be Pleasant ? A Test of Competing Appraisal Structures . Emotion , 2006 , 6 ( 4 ) , pp670 - - 674 . [ 45 ] Cialdini , R . , What ' s the best secret device for engaging student interest ? The answer is in the title . Journal of Social & Clinical Psychology , 2005 , 24 ( 22 - 29 ) . [ 46 ] Sutton , R . I . and Hargadon , A . , Brainstorming Groups in Context : Effectiveness in a Product Design Firm . Adminstrative Science Quarterly , 1996 , 41 ( 4 ) , pp685 - 718 . [ 47 ] Abrahamson , E . , Management Fashion . Academy of Management Review , 1996 , 21 ( 1 ) , pp254 - 285 . [ 48 ] Hackman , J . R . and Oldham , G . R . , Work redesign . ( Addison - Wesley , Reading , Mass , 1980 ) . Contact : Elizabeth Gerber Northwestern University Segal Design Institute Mechanical Engineering Evanston , IL 60201 USA Tel 847 467 0607 Fax 847 491 2603 Email egerber @ northwestern . edu URL www . creativeactionlab . com Elizabeth is a professor of Mechanical Engineering in the Segal Design Institute , Mechanical Engineering Department in the McCormick School of Engineering at Northwestern University . She researches creative and innovation work and designs technologies to increase performance . \ No newline at end of file diff --git a/silver_data/39bcb98db9a9c9da344007d72c55ee428bdc7930.pdf.txt b/silver_data/39bcb98db9a9c9da344007d72c55ee428bdc7930.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..9d62fc1d02f5fd1d72376fa429fc8b30711995a5 --- /dev/null +++ b/silver_data/39bcb98db9a9c9da344007d72c55ee428bdc7930.pdf.txt @@ -0,0 +1 @@ +In - cell structures of a conserved supramolecular array at the mitochondria - cytoskeleton interface in mammalian sperm Miguel Ricardo Leung 1 , 2 , Riccardo Zenezini Chiozzi 3 , 4 , Marc C . Roelofs 1 , Johannes F . Hevler 3 , 4 , Ravi Teja Ravi 1 , Paula Maitan 5 , 6 , Min Zhang 7 , Heiko Henning 5 , Elizabeth G . Bromfield 7 , 8 , Stuart C . Howes 1 , Bart M . Gadella 7 , Albert J . R . Heck 3 , 4 , and Tzviya Zeev - Ben - Mordehai 1 , 2 , * 1 Cryo - Electron Microscopy , Bijvoet Centre for Biomolecular Research , Utrecht University , 3584 CH Utrecht , The Netherlands 2 The Division of Structural Biology , Wellcome Centre for Human Genetics , The University of Oxford , Oxford OX3 7BN , United Kingdom 3 Biomolecular Mass Spectrometry & Proteomics , Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences , Utrecht University , 3584 CH Utrecht , The Netherlands 4 Netherlands Proteomics Centre , 3584 CH Utrecht , The Netherlands 5 Department of Clinical Sciences , Faculty of Veterinary Medicine , Utrecht University 3584 CM Utrecht , The Netherlands 6 Veterinary Department , Universidade Federal de Viçosa , 36570 - 900 Viçosa , Minas Gerais , Brazil 7 Department of Farm & Animal Health and Biomolecular Health Sciences , Faculty of Veterinary Medicine , Utrecht University , 3584 CM Utrecht , The Netherlands 8 Priority Research Centre for Reproductive Science , Faculty of Science , The University of Newcastle , Callaghan , Australia , 2308 * correspondence to : z . zeev @ uu . nl Summary 1 Mitochondria - cytoskeleton interactions modulate cellular 2 physiology by regulating mitochondrial transport , position - 3 ing , and immobilization . However , there is very little struc - 4 tural information defining mitochondria - cytoskeleton inter - 5 faces in any cell type . Here , we use cryo - focused ion beam 6 milling - enabled cryo - electron tomography to image mam - 7 malian sperm , where mitochondria wrap around the ciliary 8 cytoskeleton . We find that mitochondria are tethered to their 9 neighbors through inter - mitochondrial linkers and are an - 10 chored to the cytoskeleton through ordered arrays on the 11 outer mitochondrial membrane . We use subtomogram aver - 12 aging to resolve in - cell structures of these arrays from three 13 mammalian species , revealing they are conserved across 14 species despite variations in mitochondrial dimensions and 15 cristae organization . We find that the arrays consist of 16 boat - shaped particles anchored on a network of membrane 17 pores whose arrangement and dimensions are consistent 18 with voltage dependent anion channels . Proteomics and in - 19 cell cross - linking mass spectrometry suggest that the con - 20 served arrays are composed of glycerol kinase - like proteins . 21 Ordered supramolecular assemblies may serve to stabilize 22 similar contact sites in other cell types where mitochondria 23 need to be immobilized in specific subcellular environments , 24 such as in muscles and neurons . 25 sperm | mitochondria - cytoskeleton contact | cryo - electron tomography | cryo - 26 FIB milling | cross - linking mass spectrometry | subtomogram averaging 27 Introduction 28 In many cell types , mitochondria collectively form a dy - 29 namic network whose members divide , fuse , and commu - 30 nicate with one another ( Glancy et al . , 2015 ; Viana et al . , 31 2020 ; Vincent et al . , 2017 ) . Through interactions with the cy - 32 toskeleton , mitochondria are transported – sometimes across 33 large distances – and positioned in response to dynamic stim - 34 uli ( Fenton et al . , 2021 ; Moore and Holzbaur , 2018 ) . Inter - 35 actions with the cytoskeleton can also restrain mitochondria 36 to specific subcellular locations . In neurons , axonal mito - 37 chondria can be immobilized by interactions with the micro - 38 tubule or actin cytoskeletons ( Chen and Sheng , 2013 ; Gut - 39 nick et al . , 2019 ; Kang et al . , 2008 ) . In cardiac and skele - 40 tal muscle , mitochondrial distribution is regulated by inter - 41 actions with myofibrils and intermediate filaments ( Milner et 42 al . , 2000 ; Stone et al . , 2007 ) . However , despite the preva - 43 lence of inter - mitochondria and mitochondria - cytoskeleton 44 interactions and their integral roles in cellular function , there 45 is very little information on the molecular architectures of 46 these interaction sites in any cell type . 47 One of the most striking mitochondrial configurations oc - 48 curs in amniote sperm , where mitochondria are arranged in a 49 spiral around the axoneme , defining a region called the mid - 50 piece ( Fawcett , 1970 , 1975 ) . Mitochondria are among the 51 few organelles retained in sperm throughout their maturation , 52 during which they otherwise lose most of their cytoplasm and 53 organelles en route to becoming highly streamlined cells spe - 54 cialized for finding and fusing with the egg . The extensive 55 mitochondrial sheath in amniote sperm may be an adaptation 56 needed to power the large , long flagellum in these lineages . 57 Variations in midpiece morphometry affect sperm motility 58 and competitiveness ( Firman and Simmons , 2010 ; Fisher et 59 al . , 2016 ) , and different species rely on energy from mito - 60 chondrial respiration to different extents ( Marin et al . , 2003 ; 61 Tourmente et al . , 2015 ) , warranting comparative studies of 62 mitochondrial structure across species . 63 The core of the midpiece is the ciliary cytoskeleton , 64 composed of the microtubule - based axoneme and acces - 65 sory elements called outer dense fibers ( ODFs ) . A poorly - 66 characterized network of cytoskeletal filaments called the 67 submitochondrial reticulum lies between the ODFs and the 68 mitochondria . The submitochondrial reticulum co - purifies 69 with the outer mitochondrial membrane ( OMM ) , suggesting 70 that they are intimately associated ( Olson and Winfrey , 1986 , 71 1990 ) . Mitochondria wrap around the cytoskeleton and are 72 in turn ensheathed by the plasma membrane . As a conse - 73 quence of this arrangement , each mitochondrion has three 74 distinct surfaces ( Olson and Winfrey , 1992 ) – one facing the 75 axoneme , one facing the plasma membrane , and one facing 76 neighboring mitochondria . Thin - section electron microscopy 77 ( EM ) ( Olson and Winfrey , 1992 ) and freeze - fracture EM 78 ( Friend and Heuser , 1981 ) suggest that each surface is char - 79 acterized by a unique membrane protein profile . Notwith - 80 standing the insight gained from these methods , such tech - 81 niques require harsh sample preparation steps that can dis - 82 Leung et al . | February 12 , 2021 | 1 – 17 tort fine cellular structure and limit achievable resolution ( Al - 83 Amoudi et al . , 2004 ) . As such , the molecular landscape of 84 inter - mitochondrial and mitochondrial - cytoskeleton contacts 85 in the sperm midpiece remains largely unexplored . 86 Assembly of the mitochondrial sheath occurs late in 87 spermiogenesis and involves an intricately choreographed se - 88 ries of events ( Ho and Wey , 2007 ; Otani et al . , 1988 ) . Ini - 89 tially , spherical mitochondria are broadly distributed in the 90 cytoplasm . Mitochondria are then recruited to the flagellum , 91 where they form ordered rows along the flagellar axis . Fi - 92 nally , mitochondria elongate and twist around the axoneme . 93 While our understanding of the molecular details of these 94 processes is cursory at best , studies on gene - disrupted mice 95 have implicated a number of proteins in mitochondrial sheath 96 morphogenesis . For instance , mice expressing mutant forms 97 of kinesin light chain 3 ( KLC3 ) have malformed midpieces , 98 hinting at a role for microtubule - based transport ( Zhang et 99 al . , 2012 ) . Another example are the voltage dependent anion 100 channels ( VDACs ) , which are highly abundant proteins that 101 mediate transport of metabolites , ions , and nucleotides like 102 ATP across the OMM ( Colombini , 2012 ) . Male mice lacking 103 VDAC3 are infertile and their sperm cells have disorganized 104 mitochondrial sheaths ( Sampson et al . , 2001 ) , so VDACs 105 may also have unappreciated roles in mitochondrial traf - 106 ficking ; indeed , KLC3 binds mitochondria through VDAC2 107 ( Zhang et al . , 2012 ) . Similarly , disrupting sperm - specific iso - 108 forms of glycerol kinase leads to gaps in the mitochondrial 109 sheath despite proper initial alignment of spherical mitochon - 110 dria ( Chen et al . , 2017b ; Shimada et al . , 2019 ) . Mice lacking 111 spermatogenesis - associated protein 19 ( SPATA19 ) ( Mi et al . , 112 2015 ) or glutathione peroxidase 4 ( GPX4 ) ( Imai et al . , 2009 ; 113 Schneider et al . , 2009 ) also have structurally abnormal mito - 114 chondria . 115 Here , we use cryo - focused ion beam ( cryo - FIB ) milling - 116 enabled cryo - electron tomography ( cryo - ET ) to image the 117 mitochondrial sheath in mature sperm from three mammalian 118 species . We take advantage of the uniquely multi - scale 119 capabilities of cryo - ET to unveil new aspects both of the 120 overall organization of the mitochondrial sheath and of the 121 molecular structures important for its assembly . We find 122 that mitochondria are tethered to their neighbors through 123 inter - mitochondrial linkers and to the underlying cytoskele - 124 ton through conserved protein arrays on the OMM . Subto - 125 mogram averaging revealed that these arrays are anchored 126 on a lattice of OMM pores whose arrangement and dimen - 127 sions are consistent with VDACs . Proteomics and in - cell 128 cross - linking mass spectrometry suggest that the arrays con - 129 sist of glycerol kinase ( GK ) - like proteins . Our data thus show 130 that although mitochondrial dimensions and cristae architec - 131 ture vary across species , the architecture of the mitochondria - 132 cytoskeleton interface is conserved at the molecular level . 133 Results 134 Mitochondrial dimensions and cristae organization 135 vary across species . We imaged the mitochondrial sheath 136 in mature sperm from three mammalian species , namely the 137 pig ( Sus scrofa ) , the horse ( Equus caballus ) , and the mouse 138 ( Mus musculus ) ( Fig . 1 ) . These species differ in terms of 139 sperm size , motility patterns , and metabolism . To visualize 140 the overall organization of the mitochondrial sheath , we im - 141 aged whole sperm with a Volta phase plate ( VPP ) ( Danev et 142 al . , 2014 ; Fukuda et al . , 2015 ) . Neural - network based seg - 143 mentation ( Chen et al . , 2017a ) of the mitochondrial mem - 144 brane allowed us to visualize mitochondrial organization in 145 three dimensions ( Fig . 1a - f ) . 146 To investigate variations in mitochondrial width along the 147 midpiece , we first measured the width of each mitochondrion 148 at multiple points along its length . We then divided mito - 149 chondria into groups based on their positions along the mid - 150 piece , as measured by their distance from the head ( Fig . S1 ) . 151 The midpiece is ~ 10 µm long in both pig and horse sperm , 152 but ~ 20 µm long in mouse sperm , so each group represents 153 ~ 2 µm in the pig and the horse and ~ 4 µm in the mouse . We 154 found that mouse sperm mitochondria are ~ 1 . 5 times wider 155 than pig and horse sperm mitochondria overall ( Fig . S1a ) . 156 In all three species studied , most mitochondria in the mid - 157 dle ( ~ 60 % ) of the midpiece are crescent - shaped tubes ( Fig . 158 1d - f ) with consistent widths along their lengths ( Fig . S1b ) . 159 Mitochondria at the proximal end of the midpiece are larger 160 than their more distal counterparts ( Fig . 1a - c , S1a ) . More - 161 over , proximal mitochondria have more variable shapes , evi - 162 denced by greater variation in their widths at different point 163 along their lengths ( Fig . S1b ) . Because mitochondria wrap 164 around the axoneme , variations in mitochondrial dimensions 165 both across species and along the proximodistal axis of the 166 flagellum affect the overall diameter and rigidity of the mid - 167 piece , likely fine - tuning the hydrodynamics of sperm motil - 168 ity . 169 To visualize the internal organization of sperm mitochon - 170 dria in a near - native state , we imaged sperm thinned by cryo - 171 FIB milling ( Fig . 1g - l ) . This revealed unexpected diversity in 172 the internal ultrastructure of mitochondria across mammalian 173 species , especially in terms of cristae morphology . Horse 174 sperm mitochondria have an expanded intermembrane space 175 and a condensed matrix ( Fig . 1i - j ) . Mouse sperm mitochon - 176 dria have an expanded matrix , with a narrow intermembrane 177 space and thin cristae ( Fig . 1k - l ) . Pig sperm mitochondrial 178 morphology is intermediate ( Fig . 1g - h ) , and although the mi - 179 tochondrial matrix was dense , we could identify individual 180 complexes that resembled ATP synthase on cristae of FIB - 181 milled mitochondria ( Fig . S2a - b ) , which was confirmed by 182 subtomogram averaging ( Fig . S2b’ ) . 183 Inter - species differences in cristae morphology correlate 184 with measurements of matrix volume relative to mitochon - 185 drial volume ( Fig . S2d ) . In this regard , horse sperm mito - 186 chondria resemble “condensed” mitochondria , which corre - 187 late with higher rates of oxidative activity in a number of 188 different cell types , including developing germ cells , neu - 189 rons , and liver ( Hackenbrock , 1968 ; De Martino et al . , 1979 ; 190 Perkins and Ellisman , 2011 ) . Indeed , horse sperm are de - 191 pendent on oxidative phosphorylation ( Davila et al . , 2016 ) , 192 whereas pig ( Marin et al . , 2003 ) and mouse sperm ( Mukai 193 and Okuno , 2004 ; Odet et al . , 2013 ) are thought to rely 194 largely on a glycolytic mechanisms . 195 2 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface Fig . 1 . Mitochondrial dimensions and cristae organization vary across species . ( a - f ) Slices through Volta phase plate cryo - tomograms ( left ) and corresponding three - dimensional segmentations ( right ) of mitochondria from the start ( a - c ) or middle ( d - f ) of the midpiece from pig ( a , d ) , horse ( b , e ) , and mouse ( c , f ) sperm . ( g - l ) Slices through cryo - tomograms of FIB - milled pig ( g , h ) , horse ( i , j ) , and mouse ( k , l ) sperm midpieces . Right panels show digital zooms of the regions boxed out in the left panels . The outer mitochondrial membrane is traced in green , the inner mitochondrial membrane in yellow , and the plasma membrane in blue . Arrowheads indicate inter - mitochondrial linker complexes . Labels : nuc – nucleus , sc – segmented columns , m – mitochondria , odf – outer dense fibers , dc – distal centriole , ax – axoneme , mtd – microtubule doublets , cpa – central pair apparatus , pm – plasma membrane . Scale bars : ( a - l ) left panels – 250 nm , ( g - l ) right panels – 100 nm . Inter - mitochondrial junctions are associated with 196 linker complexes . Mitochondria are closely packed within 197 the mitochondrial sheath , but it is unclear whether or how 198 individual organelles communicate with their neighbors . To 199 address this , we imaged inter - mitochondrial junctions cap - 200 tured in FIB - milled sperm lamellae . We observed trans - 201 mitochondrial cristae alignment in mouse sperm ( Fig . 1k - 202 l ) , but not in pig or in horse sperm ( Fig . 1g - j ) . Trans - 203 mitochondrial cristae alignment has also been observed in 204 muscle tissue of various organisms , and is proposed to medi - 205 ate electrochemical coupling between adjacent mitochondria 206 ( Picard et al . , 2015 ) . To our knowledge , this is the first time 207 this phenomenon has been observed in mature sperm from 208 any lineage . It is particularly curious , however , that trans - 209 mitochondrial cristae alignment in sperm is species - specific . 210 We found that inter - mitochondrial junctions are charac - 211 terized by novel inter - mitochondrial linker complexes in all 212 three species ( arrowheads in Fig . 1g - l , Fig . S2c ) . These 213 inter - mitochondrial linkers span the 8 - nm distance between 214 the outer membranes of neighboring mitochondria . In mouse 215 sperm , these linkers are specifically associated with sites of 216 trans - mitochondrial cristae alignment ( Fig . 1k - l ) ; in the pig 217 and in the horse , they are positioned at regularly spaced 218 intervals along inter - mitochondrial junctions ( Fig . 1h - j ) . 219 Electron - dense inter - mitochondrial junctions were also seen 220 in cardiomyocytes by classical EM ( Duvert et al . , 1985 ; 221 Huang et al . , 2013 ; Picard et al . , 2015 ) . Thus , it is plausi - 222 ble that the as - yet - unidentified linker complexes we visual - 223 ize here represent a more general structural mechanism for 224 orchestrating inter - mitochondrial communication in various 225 cell types . 226 Ordered protein arrays at the mitochondria - cytoskele - 227 ton interface are conserved across species . To deter - 228 mine how mitochondria interact with the flagellar cytoskele - 229 ton , we imaged the mitochondria - cytoskeleton interface in 230 cryo - FIB milled lamellae ( Fig . 2 ) . We found that the 231 axoneme - facing surface of the OMM is characterized by 232 an ordered protein array that is absent from the plasma 233 membrane - facing surface ( Fig . 2a ) . These arrays are present 234 Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 3 Fig . 2 . Ordered protein arrays on the outer mitochondrial membrane directly interact with the submitochondrial reticulum . ( a ) Slice through a cryo - tomogram of a FIB - milled horse sperm midpiece , showing mitochondria ( mito ) , the submitochondrial reticulum ( smr ) outer dense fibers ( odf ) , microtubule doublets ( mtd ) , and the central pair apparatus ( cpa ) . Note how individual complexes ( like the radial spoke , rs ) are visible in the raw tomogram . The ordered protein array is only found on the axoneme - facing surface ( yellow ) of midpiece mitochondria , and not on the plasma membrane - facing surface ( red ) . ( b , c ) Slices through a cryo - tomogram of a FIB - milled horse sperm midpiece showing how the array directly interacts with the submitochondrial reticulum to anchor mitochondria to the ciliary cytoskeleton ( arrowheads ) . In right panels , the outer mitochondrial membrane is traced in green , the inner mitochondrial membrane in yellow , and the plasma membrane in blue . Scale bars : ( a ) left – 250 nm , insets – 100 nm ; ( b , c ) 100 nm . in all three species and along the entire midpiece ( Fig . S3a - f ) 235 and resemble the particle rows seen on the axoneme - facing 236 surface of the OMM in guinea pig sperm ( Friend and Heuser , 237 1981 ) and in mouse sperm ( Woolley et al . , 2005 ) by freeze - 238 fracture EM . We observed direct interactions between the ar - 239 rays and cytoskeletal filaments surrounding the ODFs ( Fig . 240 2b - c ) , indicating that these arrays tether mitochondria to the 241 midpiece cytoskeleton . 242 We then aligned and averaged sub - volumes containing 243 the protein arrays and the underlying OMM ( Fig . 3 , Ta - 244 ble S1 ) . Our averages revealed ~ 22 - nm - long two - fold - 245 symmetric boat - shaped structures connected via four densi - 246 ties to a porous membrane ( Fig . 3 , Fig . S3g - i ) . Each boat - 247 shaped particle rises ~ 5 nm above the membrane and consists 248 of two tilde - shaped densities arranged end - to - end . The boat - 249 shaped structures form rows in which each particle is related 250 to its closest neighbors by a ~ 10 nm translation perpendicu - 251 lar to the particle long axis and a ~ 6 nm shift along this axis , 252 yielding a center - to - center spacing of ~ 12 nm ( Fig . 3d - f ) . 253 Each row is oriented ~ 120° to the long axis of the flagellum 254 and adjacent rows are spaced ~ 12 nm apart , forming exten - 255 sive arrays on the axoneme - facing surface of the OMM ( Fig . 256 3g ) . Remarkably , the averages we obtained from the three 257 species were highly similar , both in terms of individual parti - 258 cle dimensions and in terms of their supramolecular arrange - 259 ment ( Fig . 3 , Fig . S3 ) . This conservation suggests that these 260 arrays are a crucial structural element of the mitochondrial 261 sheath . 262 Our averages revealed that the OMM underlying the pro - 263 tein arrays is studded with ~ 3 - 4 nm pores arranged in a 264 pseudo - lattice with a center - to - center spacing of ~ 5 nm . 265 ( Fig . 3a - c , Fig . S3g - i ) . These pore sizes are consistent 266 with the diameters of the voltage dependent anion channels 267 ( VDACs ) , which are known to form ordered arrays in the 268 4 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface Fig . 3 . Ordered protein arrays at the mitochondria - cytoskeleton interface are conserved across species . ( a - c ) Subtomogram averages of the protein arrays and underlying outer mitochondrial membrane ( OMM ) after applying twofold symmetry ( note that density is black ) . ( d - f ) Isosurface renderings of the subtomogram averages in ( a - c ) with boat - shaped particles in grey and the OMM in green . ( g ) Left panel : Segmentation of the tomogram shown in Figure 2a , with the OMM in green , the IMM in yellow , microtubule doublets in blue , and the cpa in pink . Subtomogram averages of boat - shaped particles are colored grey and plotted back into their positions and orientations in the tomogram . Right panel : Rotated and zoomed - in view of the axoneme - facing surface of a mitochondrion . The axoneme is oriented horizontally , so the ladder - like arrays are oriented ~ 120° to the flagellar long axis , and individual particles within the array are oriented ~ 60° to this axis . Scale bars : ( a - c ) 10 nm . OMM ( Gonçalves et al . , 2007 ; Guo and Mannella , 1993 ; 269 Hoogenboom et al . , 2007 ; Mannella , 1982 ) . Indeed , our 270 label - free quantitative proteomics experiments show that the 271 most abundant OMM proteins in pig sperm are VDAC2 and 272 VDAC3 ( Table S2 ) . Furthermore , the lattice dimensions in 273 our averages closely match those of VDAC in purified Neu - 274 rospora OMM ( Guo and Mannella , 1993 ; Mannella , 1998 ) . 275 The lattice can be modeled by fitting multiple copies of the 276 VDAC2 crystal structure ( Schredelseker et al . , 2014 ) ( Fig . 277 S4a ) . We oriented VDAC2 in the membrane plane based 278 on its known topology ( Bayrhuber et al . , 2008 ; Tomasello 279 et al . , 2013 ) ; however , at the current resolution , we cannot 280 determine the orientation around the pore axis . Thus , in our 281 model , each boat - shaped particle stretches across 8 VDAC 282 molecules ( Fig . 3 ) . 283 Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 5 Fig . 4 . Modelling the outer mitochondrial membrane ( OMM ) array as glycerol kinase - like ( GK ) proteins anchored on voltage dependent anion channels ( VDACs ) . ( a ) The VDAC2 / VDAC3 interactome derived from in - cell XL - MS of pig sperm . Protein nodes are colored according to their known subcellular localizations ( yellow – mitochondria , blue – head , grey – cytoskeleton / unknown ) . Gray spheres indicate the phosphate groups of a simulated lipid bilayer which was structurally aligned based on the simulation for monomeric mouse VDAC1 ( PDB 4C69 ) obtained from the MemProtMD server ( Newport et al . , 2019 ) . ( b ) Modeling the OMM array as GK - like proteins anchored on VDACs . A GK - like dimer - of - dimers homology model ( red and green ) and VDAC homology models ( yellow ) were fitted into the pig subtomogram average map ( white ) . ( c ) The positions of cross - linked Lys residues ( red circles ) are consistent with GK and VDAC orientation assignments in our model . 6 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface Glycerol kinase - like proteins are probable con - 284 stituents of the conserved arrays at the mitochondria – 285 cytoskeleton interface . To search for possible constituents 286 of the protein arrays on the VDAC lattice , we used in - cell 287 cross - linking mass spectrometry ( XL - MS ) ( Fasci et al . , 2018 ; 288 Liu et al . , 2018 ) to find potential VDAC2 / VDAC3 interac - 289 tion partners on the OMM ( Fig . 4 ) . We treated pig sperm 290 cells with the cross - linker disuccinimidyl sulfoxide ( DSSO ) , 291 which covalently links free lysines that are within ~ 3 nm 292 ( C α - C α ) of each other . To increase confidence , we screened 293 for cross - links identified with at least two cross - link spectral 294 matches ( CSMs ) ( see Materials and Methods for details ) . 295 We first screened candidate proteins based on their known 296 subcellular localizations ( Fig . 4a ) . VDAC2 / VDAC3 cross - 297 linked to mitochondria - associated proteins as well as to 298 sperm head - associated proteins . This is consistent with im - 299 munofluorescence studies localizing VDAC2 / VDAC3 both to 300 the midpiece and to the acrosome , a large vesicle capping 301 the anterior sperm nucleus ( Hinsch et al . , 2004 ; Kwon et al . , 302 2013 ) . Of the proteins in the mitochondria - associated inter - 303 action hub , three proteins are particularly noteworthy because 304 they are known to localize to the OMM and because their 305 disruption results in dysplasia of the mitochondrial sheath : 306 spermatogenesis - associated protein 19 ( SPATA19 ) ( Mi et al . , 307 2015 ) , glutathione peroxidase 4 ( GPX4 ) ( Imai et al . , 2009 ; 308 Schneider et al . , 2009 ) , and glycerol kinase ( GK ) ( Chen et 309 al . , 2017b ; Shimada et al . , 2019 ) . 310 To distinguish among these candidates , we compared the 311 location of the cross - links with the known topology of VDAC 312 in the OMM ( Bayrhuber et al . , 2008 ; Tomasello et al . , 2013 ) . 313 GPX4 would interact on the side facing the intermembrane 314 space , whereas SPATA19 and GK would interact on the cy - 315 toplasmic face . Both SPATA19 and GK are highly abundant 316 ( Table S2 ) , as would be expected for proteins forming exten - 317 sive arrays . Assuming an average protein density of ~ 1 . 43 318 g / cm 3 ( Quillin and Matthews , 2000 ) , which corresponds to 319 ~ 0 . 861 Da / Å 3 , we estimate that each boat - shaped particle in 320 the array has a molecular weight of ~ 250 kDa . SPATA19 is 321 a small protein with an estimated molecular weight of ~ 18 322 kDa . To fit into our EM densities , it must either be present 323 in multiple copies or form a complex with other proteins . In 324 contrast , GK has an estimated molecular weight of ~ 60 kDa 325 and is known to form S - shaped dimers ( ~ 120 kDa ) that are 326 conserved from bacteria ( Bystrom et al . , 1999 ; Fukuda et 327 al . , 2016 ) to eukaryotes ( Balogun et al . , 2019 ; Schnick et al . , 328 2009 ) . 329 To build a GK - VDAC model based on our subtomogram 330 average , we used rigid - body fitting to place two GK dimers 331 end - to - end into a boat - shaped density ( Fig . S4b , Fig . 4b ) . 332 These fits defined a clear orientation for GK , with the N - 333 termini pointing upwards and the C - terminal helices facing 334 the OMM ( Fig . 4b ) . To validate our fits , we mapped the 335 cross - linked lysines onto the resulting model ( Fig . 4c ) . All 336 cross - links were between the cytosolic face of VDAC2 and 337 the OMM - facing surface of GK , which is consistent with 338 the orientation expected from our fits . Assigning GK - like 339 proteins as constituents of the ordered OMM arrays at the 340 mitochondria - cytoskeleton interface is also supported by re - 341 cent genetic studies . Sperm from mice lacking sperm - specific 342 GK isoforms , which do not show glycerol kinase activity 343 in vitro ( Pan et al . , 1999 ) , have disorganized mitochondrial 344 sheaths ( Chen et al . , 2017b ; Shimada et al . , 2019 ) . In these 345 mice , spherical mitochondria properly align along the flagel - 346 lum but fail to properly elongate and coil around the ODFs 347 ( Shimada et al . , 2019 ) . This phenotype is consistent with our 348 data showing direct links between GK protein arrays and the 349 submitochondrial reticulum ( Fig . 2b - c ) . 350 Discussion 351 In this study , we used cryo - FIB milling - enabled cryo - 352 ET to image the sperm mitochondrial sheath in three mam - 353 malian species . Our data reveal that overall mitochondrial di - 354 mensions are remarkably consistent in sperm from the same 355 species ( Fig . 1 , S1 ) . This contrasts with other mitochondria - 356 rich tissues such as skeletal muscle , where there are massive 357 variations in mitochondrial size and morphology within indi - 358 vidual cells ( Vincent et al . , 2019 ) . In addition , we did not ob - 359 serve mitochondrial nanotunnels in any of the species we ex - 360 amined , in contrast to their relative abundance in muscle tis - 361 sue ( Vincent et al . , 2017 , 2019 ) . Our data also show that mi - 362 tochondrial dimensions and cristae architecture vary across 363 species ( Fig . 1 ) , providing possible structural bases for in - 364 terspecific differences in mitochondrial energetics . Further 365 comparative studies of how mitochondrial structure varies 366 with sperm metabolism will undoubtedly contribute to our 367 broader understanding of how mitochondrial form relates to 368 function . 369 Our data show that , despite this diversity , the molecular 370 underpinnings of mitochondrial sheath architecture are con - 371 served , at least in mammals . Specifically , we identified novel 372 inter - mitochondrial linkers that tether adjacent mitochondria 373 ( Fig . 1 , S2 ) and arrays of boat - shaped particles that anchor 374 mitochondria to the cytoskeleton ( Fig . 2 , 3 ) . In - cell subtomo - 375 gram averaging and in - cell XL - MS suggest that these arrays 376 consist of GK - like proteins anchored on VDAC lattices in the 377 OMM ( Fig . 4 ) . Given that VDACs are ubiquitous OMM pro - 378 teins , our findings motivate further efforts to explore whether 379 they also regulate mitochondria - cytoskeleton interactions in 380 other cell types . 381 The OMM arrays may function to regulate the precise 382 elongation and coiling of mitochondria , contributing to the 383 striking consistency within the mitochondrial sheath . In ma - 384 ture sperm , these arrays may help maintain the integrity of 385 mitochondria - cytoskeleton contacts , stabilizing them against 386 shear stresses during sperm motility and hyperactivation . 387 However , it is unclear what determines the organization of 388 these arrays in the first place . Our averages do not hint at di - 389 rect interactions between boat - shaped particles . Instead , their 390 spacing may be defined by the organization of the underlying 391 VDAC lattice . Another intriguing possibility is that the ar - 392 rays are organized by their cytoskeletal binding partners ; the 393 periodicity of relevant motifs on the submitochondrial retic - 394 ulum could dictate the spacing of the OMM arrays . 395 To our knowledge , this is the first time such assemblies 396 Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 7 have been visualized at any organelle - cytoskeleton interface 397 in any cell type . Defining whether similar arrays are present 398 in other differentiated cell types – and whether they use a sim - 399 ilar pool of protein components – is an area ripe for study . In 400 striated muscle , proper mitochondrial positioning is critical 401 for muscle function and depends on direct associations be - 402 tween mitochondria and intermediate filaments ( Konieczny 403 et al . , 2008 ; Milner et al . , 2000 ) . Similarly , in skin cells , 404 mitochondrial organization depends on keratin ( Steen et al . , 405 2020 ) . The structural bases for these associations are un - 406 known , but cryo - ET and in - cell XL - MS may prove useful in 407 these contexts as well . 408 Acknowledgements 409 The authors thank Dr . M Vanevic for excellent compu - 410 tational support ; Dr . D Vasishtan for providing scripts that 411 greatly facilitated subtomogram averaging ; Ingr . CTWM 412 Schneijdenberg and JD Meeldijk for managing and maintain - 413 ing the Utrecht University EM Square facility ; Stal Schep 414 ( Tull en het Waal , The Netherlands ) for providing horse se - 415 men ; MW Haaker and M Houweling for providing mouse 416 reproductive tracts ; Prof . F Förster and Prof . A Akhmanova 417 for critical reading of the manuscript ; and Prof . EY Jones for 418 insightful discussions . The authors also thank the Henriques 419 Lab for the publicly - available L A T E X template . This work 420 benefitted from access to the Netherlands Center for Electron 421 Nanoscopy ( NeCEN ) with support from operators Dr . RS 422 Dillard and Dr . CA Diebolder and IT support from B Alewi - 423 jnse . RZC , JFH , and AJRH acknowledge support from NWO 424 funding the Netherlands Proteomics Centre through the X - 425 omics Road Map program ( project 184 . 034 . 019 ) . This work 426 was funded by NWO Start - Up Grant 740 . 018 . 007 to TZ , and 427 MRL is supported by a Clarendon Fund - Nuffield Department 428 of Medicine Prize Studentship . 429 Author Contributions 430 PM , MZ , HH , EGB , and BMG provided sperm samples . 431 MRL , MCR , and RTR prepared samples for cryo - ET . MRL 432 performed cryo - FIB milling . MRL , MCR , RTR , SCH , and 433 TZ collected cryo - ET data . MRL and MCR processed cryo - 434 ET data . MRL , MCR , and TZ analyzed cryo - ET data . RZC 435 and JFH performed all proteomics and cross - linking mass 436 spectrometry experiments along with corresponding struc - 437 tural modelling under the supervision of AJRH . MRL and 438 TZ wrote the manuscript , and all authors contributed to revi - 439 sions . 440 Declaration of Interests 441 The authors declare no competing interests . 442 Materials and Methods 443 Sperm collection and preparation . Pig sperm samples 444 were purchased from an artificial insemination company 445 ( Varkens KI Nederland ) , stored at 18°C , and prepared for 446 imaging within 1 day of delivery . Sperm were layered onto a 447 discontinuous gradient consisting of 4 mL of 35 % Percoll® 448 ( GE Healthcare ) underlaid with 2 mL of 70 % Percoll® , both 449 in HEPES - buffered saline ( HBS : 20 mM HEPES , 137 mM 450 NaCl , 10 mM glucose , 2 . 5 mM KCl , 0 . 1 % kanamycin , pH 451 7 . 6 ) and centrifuged at 750 g for 15 min at RT ( Harrison et 452 al . , 1993 ) . Pelleted cells were washed once in phosphate - 453 buffered saline ( PBS : 137 mM NaCl , 3 mM KCl , 8 mM 454 Na 2 HPO 4 , 1 . 5 mM KH 2 PO 4 , pH 7 . 4 ) , resuspended in PBS 455 and counted with a hemocytometer . 456 Horse semen was collected from mature Warmblood stal - 457 lions using a Hanover artificial vagina in the presence of a 458 teaser mare . After collection , semen was filtered through 459 gauze to remove gel fraction and large debris , then trans - 460 ported to the laboratory at 37°C and kept at room tempera - 461 ture until further processing . Semen was diluted in INRA96 462 ( IMV Technologies ) to obtain a sperm concentration of 30 463 x 10 6 cells / mL . After this , sperm were centrifuged through 464 a discontinuous Percoll gradient as described above for pig 465 sperm for 10 min at 300 g followed by 10 min at 750 g ( Har - 466 rison et al . , 1993 ) . The remaining pellet was resuspended in 467 1 mL of PBS and centrifuged again for 5 min at 750 g . 468 Mouse sperm were collected from the cauda epididymis 469 of adult male C75BL / 6 mice as described in ( Hutcheon et 470 al . , 2017 ) . Briefly , male mice were culled as described in 471 ( Mederacke et al . , 2015 ) and the cauda epididymides were 472 dissected with the vas deferens attached and placed in a 473 500 µL droplet of modified Biggers , Whitten , and Whitting - 474 ham media ( BWW : 20 mM HEPES , 91 . 5 mM NaCl , 4 . 6 475 mM KCl , 1 . 7 mM D - glucose , 0 . 27 mM sodium pyruvate , 476 44 mM sodium lactate , 5 U / mL penicillin , and 5 µg / mL 477 streptomycin , adjusted to pH 7 . 4 and an osmolarity of 300 478 mOsm / kg ) . To retrieve the mature cauda spermatozoa from 479 the epididymides , forceps were used to first gently push the 480 stored sperm from the vas deferens , after which two incisions 481 were made with a razor blade in the cauda . Spermatozoa 482 were allowed to swim out of the cauda into the BWW over a 483 period of 15 min at 37°C , after which the tissue was removed 484 and sperm were loaded onto a 27 % Percoll density gradient 485 and washed by centrifugation at 400 g for 15 min . The pellet 486 consisting of an enriched sperm population was resuspended 487 in BWW and again centrifuged at 400 g for 2 min to remove 488 excess Percoll . 489 Cryo - EM grid preparation . Typically , 3 µL of a suspen - 490 sion containing either 2 - 3 x 10 6 cells / mL ( for whole cell to - 491 mography ) or 20 - 30 x 10 6 cells / mL ( for cryo - FIB milling ) 492 was pipetted onto a glow - discharged Quantifoil R 2 / 1 200 - 493 mesh holey carbon grid . One µL of a suspension of BSA - 494 conjugated gold beads ( Aurion ) was added , and the grids then 495 blotted manually from the back ( opposite the side of cell de - 496 position ) for ~ 3 s ( for whole cell tomography ) or for ~ 5 - 6 s 497 ( for cryo - FIB milling ) using a manual plunge - freezer ( MPI 498 Martinsreid ) . Grids were immediately plunged into a liquid 499 ethane - propane mix ( 37 % ethane ) ( Tivol et al . , 2008 ) cooled 500 to liquid nitrogen temperature . Grids were stored under liq - 501 uid nitrogen until imaging . 502 8 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface Cryo - focused ion beam milling . Grids were mounted into 503 modified Autogrids ( ThermoFisher ) for mechanical support . 504 Clipped grids were loaded into an Aquilos ( ThermoFisher ) 505 dual - beam cryo - focused ion beam / scanning electron micro - 506 scope ( cryo - FIB / SEM ) . All SEM imaging was performed at 507 2 kV and 13 pA , whereas FIB imaging for targeting was per - 508 formed at 30 kV and 10 pA . Milling was typically performed 509 with a stage tilt of 18° , so lamellae were inclined 11° rela - 510 tive to the grid . Each lamella was milled in four stages : an 511 initial rough mill at 1 nA beam current , an intermediate mill 512 at 300 pA , a fine mill at 100 pA , and a polishing step at 30 513 pA . Lamellae were milled with the wedge pre - milling tech - 514 nique described in ( Schaffer et al . , 2017 ) and with expansion 515 segments as described in ( Wolff et al . , 2019 ) . 516 Tilt series acquisition . Tilt series were acquired on ei - 517 ther a Talos Arctica ( ThermoFisher ) operating at 200 kV 518 or a Titan Krios ( ThermoFisher ) operating at 300 kV , both 519 equipped with a post - column energy filter ( Gatan ) in zero - 520 loss imaging mode with a 20 - eV energy - selecting slit . All 521 images were recorded on a K2 Summit direct electron detec - 522 tor ( Gatan ) in either counting or super - resolution mode with 523 dose - fractionation . Tilt series were collected using SerialEM 524 ( Mastronarde , 2005 ) at a target defocus of between - 4 and - 6 525 µm ( conventional defocus - contrast ) or between - 0 . 5 and - 1 . 5 526 µm ( for tilt series acquired with the Volta phase plate ) . Tilt 527 series were typically recorded using either strict or grouped 528 dose - symmetric schemes , either spanning ± 56° in 2° incre - 529 ments or ± 54° in 3° increments , with total dose limited to 530 ~ 100 e - / Å 2 . 531 Tomogram reconstruction . Frames were aligned either 532 post - acquisition using Motioncor2 1 . 2 . 1 ( Zheng et al . , 2017 ) 533 or on - the - fly using Warp ( Tegunov and Cramer , 2019 ) . 534 Frames were usually collected in counting mode ; when 535 super - resolution frames were used , they were binned 2X dur - 536 ing motion correction . Tomograms were reconstructed in 537 IMOD ( Kremer et al . , 1996 ) using weighted back - projection , 538 with a SIRT - like filter ( Zeng , 2012 ) applied for visualization 539 and segmentation . Defocus - contrast tomograms were CTF - 540 corrected in IMOD using ctfphaseflip while VPP tomograms 541 were left uncorrected . 542 Tomogram segmentation . Segmentation was generally 543 performed semi - automatically using the neural network - 544 based workflow implemented in the TomoSeg package in 545 EMAN 2 . 21 ( Chen et al . , 2017 ) . Microtubules , however , 546 were traced manually in IMOD . Segmentation was then man - 547 ually refined in Chimera 1 . 12 ( Pettersen et al . , 2004 ) or in 548 ChimeraX ( Goddard et al . , 2018 ) . Visualization was per - 549 formed in ChimeraX . 550 Subtomogram averaging of ATP synthase and outer 551 mitochondrial membrane arrays . Subtomogram averag - 552 ing with missing wedge compensation was performed using 553 PEET 1 . 13 . 0 ( Heumann et al . , 2011 ; Nicastro et al . , 2006 ) . 554 Resolution was estimated using the Fourier shell correlation 555 ( FSC ) at a cut - off of 0 . 5 ( Nicastro et al . , 2006 ) . Alignments 556 were generally performed first on binned data , after which 557 aligned positions and orientations were transferred to less - 558 binned data using scripts generously provided by Dr . Daven 559 Vasishtan . Details of acquisition parameters and particle 560 numbers are summarized in Table S1 . 561 Alignment strategies for these complexes were designed 562 to take advantage of their defined orientations relative to the 563 membrane plane . Particles were picked manually and their 564 initial orientations were defined using stalkInit . Initial refer - 565 ences were either a randomly chosen particle ( for ladder - like 566 arrays ) or an average of all particles after roughly aligning 567 them based on their initial orientations ( for ATP synthase ) . 568 Independent alignments using independent initial references 569 were performed for datasets from different species . Align - 570 ments allowed for large rotational search ranges around the 571 particle long axis ( defined as the y - axis , perpendicular to the 572 membrane plane ) , with limited search ranges around the x - 573 and z - axes ( the membrane plane ) . 574 All initial alignments were performed without symmetry . 575 After visual inspection of the maps , twofold symmetry was 576 applied for ladder - like arrays . Symmetrization involved us - 577 ing the aligned positions from the unsymmetrized runs as 578 seed points and rotating particles around the axis of symme - 579 try to generate virtual particles . A symmetrized volume was 580 generated by averaging all particles and virtual particles and 581 used as a reference for a final , restricted alignment . 582 Plotbacks were generated in IMOD by first running cre - 583 ateAlignedModel to generate model files reflecting updated 584 particle positions and orientations after alignment . The rel - 585 evant subtomogram average was then thresholded for visu - 586 alization and saved as an isosurface model , which was then 587 placed back into the tomograms using clonemodel . 588 Measurements and quantification . All measurements of 589 mitochondrial width were performed in IMOD on Volta 590 phase plate tomograms filtered with a SIRT - like filter . Mi - 591 tochondrial width was measured in the non - missing wedge 592 direction at five points along the length of each mitochon - 593 drion . Only mitochondria that were entirely in the field of 594 view were included in the measurements . Tomograms and 595 corresponding measurements were then grouped based on 596 their locations relative to the connecting piece , which were 597 determined based on low - magnification images used for tar - 598 geting during data acquisition . 599 Internal mitochondrial ultrastructure was quantified from 600 tomograms from cryo - FIB milled lamellae . The volume oc - 601 cupied by the matrix ( V matrix , the volume enclosed by the 602 IMM ) was measured relative to the volume occupied by the 603 entire mitochondrion ( V mito , the volume enclosed by the 604 OMM ) . Mesh volumes were extracted from segmentations 605 using imodinfo . Because neural network - based segmentation 606 often resulted in gaps , mitochondrial membranes were seg - 607 mented manually in IMOD for quantification . Only slices in 608 which both the IMM and OMM were clearly defined were 609 used for segmentation . 610 Cross - linking , lysis , digestion and peptide fractiona - 611 tion . All proteomics and cross - linking mass spectrometry ex - 612 Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 9 periments were performed on Percoll - washed pig sperm pre - 613 pared as described above . For cross - linking , approximately 614 300 x 106 cells were used from 3 different animals . Briefly , 615 pelleted sperm cells were resuspended in 540 µL of PBS 616 supplemented with disuccinimidyl sulfoxide ( DSSO , Thermo 617 Fisher Scientific ) to a final concentration of 1 mM . The reac - 618 tion mix was incubated for 30 min at 25°C with 700 rpm 619 shaking in a ThemoMixer C ( Eppendorf ) and subsequently 620 quenched for 20 min by adding Tris - HCl ( final concentra - 621 tion 50 mM ) . Cross - linked cells were spun down at 13 800g 622 for 10 min at 4°C , after which the supernatant was removed . 623 Cells were then lysed according to a protocol modified from 624 ( Potel et al . , 2018 ) . Cells were resuspended in 1 mL of ly - 625 sis buffer ( 100 mM Tris - HCl pH 8 . 5 , 7 M Urea , 1 % Triton 626 X - 100 , 5 mM TCEP , 30 mM CAA , 10 U / ml DNase I , 1 mM 627 MgCl2 , 1 % benzonase ( Merck Millipore , Darmstadt , Ger - 628 many ) , 1 mM sodium orthovanadate , phosphoSTOP phos - 629 phatases inhibitors , and cOmpleteTM Mini EDTA - free pro - 630 tease inhibitors ) . Cells were sonicated on ice for 2 min us - 631 ing an ultrasonic processor ( UP100H , Hielscher ) at 80 % am - 632 plitude . The proteins were then precipitated according to 633 ( Wessel and Flügge , 1984 ) and the dried protein pellet re - 634 suspended in digestion buffer ( 100 mM Tris - HCl pH 8 . 5 , 1 % 635 sodium deoxycholate ( Sigma - Aldrich ) , 5 mM TCEP , and 30 636 mM CAA ) . Trypsin and Lys - C proteases were added to a 1 : 25 637 and 1 : 100 ratio ( w / w ) respectively and protein digestion per - 638 formed overnight at 37°C . The final peptide mixtures were 639 desalted with solid - phase extraction C18 columns ( Sep - Pak , 640 Waters ) and fractionated with an Agilent 1200 HPLC pump 641 system ( Agilent ) coupled to a strong cation exchange ( SCX ) 642 separation column ( Luna SCX 5 µm – 100 Å particles , 50 x 643 2mm , Phenomenex ) , resulting in 25 fractions . 644 Liquid chromatography with mass spectrometry . Ap - 645 proximately 1000 ng of peptides from each biological repli - 646 cate before SCX fractionation were first injected onto an Ag - 647 ilent 1290 Infinity UHPLC system ( Agilent ) on a 50 - cm an - 648 alytical column packed with C18 beads ( Dr Maisch Reprosil 649 C18 , 3 µm ) coupled online to an Orbitrap HF - X ( Thermo 650 Fisher Scientific ) . For this classical bottom - up analysis , we 651 used the following LC - MS / MS parameters : after 5 min of 652 loading with 100 % buffer A ( H2O with 0 . 1 % formic acid ) , 653 peptides were eluted at 300 nL / min with a 95 - min gradient 654 from 13 % to 40 % of buffer B ( 80 % acetonitrile and 20 % 655 H2O with 0 . 1 % formic acid ) . For MS acquisition we used an 656 MS1 Orbitrap scan at 60 , 000 resolution , automatic gain con - 657 trol ( AGC ) target of 3 x 106 ions and maximum inject time 658 of 20 ms from 375 to 1600 m / z ; the 15 most intense ions 659 were submitted to MS2 Orbitrap scan at 30 , 000 resolution , 660 AGC target of 1 x 105 ions and maximum inject time of 50 661 ms ( isolation window of 1 . 4 m / z , NCE at 27 % and dynamic 662 exclusion of 16 seconds ) . The SCX fractions were analyzed 663 with same Agilent HPLC and the same nano - column cou - 664 pled on - line to an Orbitrap Lumos mass spectrometer ( Ther - 665 moFisher Scientific ) . For these runs , we used a gradient from 666 6 % to 39 % buffer B over 100 min with specific MS settings 667 for DSSO cross - links : survey MS1 Orbitrap scan at 60 , 000 668 resolution from 375 to 1500 , AGC target of 4 x 105 ions and 669 maximum inject time of 50 ms ; MS2 Orbitrap scan at 30 , 000 670 resolution , AGC target of 5 x 104 ions , and maximum in - 671 ject time of 100 ms for detection of DSSO signature peaks 672 ( difference in mass of 37 . 972 Da ) . The four ions with this 673 specific difference were analyzed with a MS3 Ion Trap scans 674 at AGC target of 2 x 104 ions , maximum inject time of 150 675 ms for sequencing selected signature peaks ( representing the 676 individual peptides ) . 677 Mass spectrometry data processing . The 3 raw files ob - 678 tained with classical bottom - up approach were analyzed with 679 MaxQuant version 1 . 6 . 17 with all the automatic settings 680 adding Deamidation ( N ) as dynamic modification against the 681 Sus scrofa reference proteome ( Uniprot version of 08 / 2020 682 with 49 , 795 entries ) . With this search , we were able to 683 calculate intensity - based absolute quantification ( iBAQ ) val - 684 ues and created a smaller FASTA file to use for analysis of 685 cross - linking experiments ( table as Supplementary informa - 686 tion ) . Raw files for cross - linked cells were analyzed with 687 Proteome Discoverer software suite version 2 . 4 . 1 . 15 ( Ther - 688 moFisher Scientific ) with the incorporated XlinkX node for 689 analysis of cross - linked peptides as described in ( Klykov et 690 al . , 2018 ) . Data were searched against the smaller FASTA 691 created in house with “MS2 _ MS3 acquisition strategy” . For 692 the XlinkX search , we selected full tryptic digestion with 3 693 maximum missed cleavages , 10 ppm error for MS1 , 20 ppm 694 for MS2 , and 0 . 5 Da for MS3 in Ion Trap . For modifications , 695 we used static Carbamidomethyl ( C ) and dynamic Oxidation 696 ( M ) , Deamidation ( N ) and Met - loss ( protein N - term ) . The 697 crosslinked peptides were accepted with a minimum score of 698 40 , minimum score difference of 4 and maximum FDR rate 699 set to 5 % ; further standard settings were used . 700 Interactome analysis , homology modelling , and 701 cross - link mapping . The interaction map for VDAC pro - 702 teins was generated in R ( Grant et al . , 2006 ) using the igraph 703 package ( v 1 . 2 . 4 . 2 ) . Only cross - links with at least two cross - 704 link spectral matches ( CSMs ) were used for network genera - 705 tion . Homology models of GK and VDAC2 were generated 706 in Robetta ( Kim et al . , 2004 ) and fitted into subtomogram 707 average maps by rigid body fitting in Chimera X . Cross - links 708 were mapped onto the resulting models using ChimeraX . 709 Data availability . Subtomogram average maps have been 710 deposited to the Electron Microscopy Data Bank ( EMDB ) 711 with the following accession numbers : EMDB - 12354 , 712 12355 , 12356 , and 12357 . The model of putative glycerol 713 kinase - like proteins anchored on a VDAC array has been de - 714 posited to the Protein Data Bank ( PDB ) with the accession 715 number PDB ID 7NIE . 716 10 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface Fig . S1 . Mitochondrial dimensions are consistent within species but vary across species and spatially along the midpiece . ( a ) Plotting the average width of mitochondria from different regions of the midpiece shows that mouse sperm mitochondria are larger than pig and horse sperm mitochondria . Note also that , in all three species , mitochondria at the proximal end of the midpiece are larger than those in more distal parts . ( b ) Mitochondrial width was measured at five points along the length of each mitochondrion . Plotting the coefficient of variation from different regions along the midpiece shows that mitochondria at the start of the midpiece have more variable widths along their lengths . In ( a ) , n indicates the number of mitochondria analyzed . In both ( a ) and ( b ) , solid lines represent the median and dotted lines represent the first and third quartiles . Fig . S2 . Cryo - focused ion beam ( cryo - FIB ) milling reveals the internal organization of sperm mitochondria . ( a ) Slice through a cryo - tomogram of FIB - milled pig sperm mitochondria close to the connecting piece . ( b ) ATP synthase can be directly identified on cristae based on its characteristic shape , which is confirmed by subtomogram averaging ( b’ ) . ( c ) Novel inter - mitochondrial linkers tether neighboring mitochondria to each other ( arrowheads in left panel ) . ( d ) Quantifying the volume enclosed by the mitochondrial matrix relative to the volume enclosed by the whole mitochondrion reveals that pig and horse sperm mitochondria have more expanded cristae and more condensed matrices than mouse sperm mitochondria . Lines represent mean ± standard deviation . Scale bars : ( a ) 250 nm , ( b - c ) 100 nm . Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 11 Fig . S3 . The particles forming the ordered arrays at the mitochondria - cytoskeleton interface are two - fold symmetric . ( a - f ) Slices through cryo - tomograms of FIB - milled pig ( a , b ) , horse ( c , d ) , and mouse ( e , f ) mitochondria . Right panels show digital zooms of the regions boxed out in the left panels , with arrowheads indicating arrays . ( g - i ) Subtomogram averages of the arrays and the outer mitochondrial membrane ( OMM ) without ( left ) and with ( right ) twofold symmetry . Scale bars : ( a - f ) 250 nm , ( g - i ) 10 nm . 12 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface Fig . S4 . Fitting crystal structures of glycerol kinase ( GK and voltage dependent anion channels ( VDACs ) into the pig subtomo - gram average map . ( a ) The crystal structure of VDAC2 from zebrafish ( PDB 4BUM ) is shown in grey , fitted into the cryo - ET averaged map ( green ) . ( b ) Two copies of a crystal structure of GK ( pink and blue ) from Trypanosoma brucei ( PDB 5GN6 ) fitted into the cryo - ET averaged map ( grey ) . On the right , the GK crystal structure is shown filtered to 30Å resolution . Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 13 Table S1 . Image acquisition and processing metrics for subtomogram averaging of mitochondrial protein complexes in mam - malian sperm . Parameter Species Sus scrofa Equus caballus Mus musculus ATP synthase sample type lamellae - - number of cells used 3 - - microscope ( accelerating voltage ) Arctica ( 200 kV ) - - pixel size ( Å ) 4 . 34 - - symmetry C1 - - number of particles 209 - - estimated resolution ( Å ) 38 - - outer mitochondrial membrane arrays sample type lamellae lamellae lamellae number of cells used 3 3 8 microscope ( accelerating voltage ) Arctica ( 200 kV ) Arctica ( 200 kV ) Arctica ( 200 kV ) pixel size ( Å ) 4 . 34 5 . 66 5 . 66 symmetry C1 / C2 C1 / C2 C1 / C2 number of particles 268 / 536 962 / 1924 972 / 1944 estimated resolution ( Å ) 39 / 35 38 / 33 38 / 22 14 | Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface T a b l e S 2 . Top 35 m o s t a bund a n t ou t e r m i t o c hond r i a l m e m b r a n e p r o t e i n s i d e n t i fi e d i n t h e p i g s p e r m p r o t e o m e . H u m a n h o m o l og g e n e n a m e * H u m a n h o m o l o g p r o t e i n n a m e * i B A Q un i qu e p e p t i d e s s e qu e n ce c ov er ag e ( % ) M W ( k D a ) VDA C 2 V o lt a g e - d e p e nd e n t a n i on - s e l ec ti v e c h a nn e l p r o t e i n 2 9 . 42 17 70 . 4 31 . 6 VDA C 3 V o lt a g e - d e p e nd e n t a n i on - s e l ec ti v e c h a nn e l p r o t e i n 3 9 . 19 13 62 . 2 30 . 6 SP A T A 18 M it o c hond r i a - ea ti ng p r o t e i n 8 . 78 30 48 63 . 3 U B A 52 U b i qu iti n - 60 S r i bo s o m a l p r o t e i n L 40 8 . 73 6 43 14 . 7 GK G l y ce r o l k i n a s e 8 . 73 10 58 . 6 57 . 7 C Y B 5 B C y t o c h r o m e b5 t yp e B 8 . 72 5 50 . 4 13 . 9 C I S D 1 C DG S H i r on - s u l f u r do m a i n - c on t a i n i ng p r o t e i n 1 8 . 72 5 50 . 5 12 . 8 SP A T A 19 S p e r m a t og e n e s i s - a ss o c i a t e d p r o t e i n 19 , m it o c hond r i a l 8 . 63 8 62 . 3 18 . 0 VDA C 1 V o lt a g e - d e p e nd e n t a n i on - s e l ec ti v e c h a nn e l p r o t e i n 1 8 . 62 2 7 . 4 30 . 7 F UND C 2 F UN 14 do m a i n - c on t a i n i ng p r o t e i n 2 8 . 58 10 58 . 2 20 . 6 HK 1 H e xok i n a s e - 1 8 . 54 48 52 . 3 102 . 6 HADH B T r i f un c ti on a l e n z y m e s ubun it b e t a , m it o c hond r i a l 8 . 49 24 66 . 2 49 . 4 M AOA A m i n e ox i d a s e [ fl a v i n - c on t a i n i ng ] A 8 . 4 32 38 . 9 102 . 4 A C S L 6 L ong - c h a i n - f a t t y - ac i d – C o A li g a s e 6 8 . 02 40 71 . 3 77 . 8 C Y B 5 R 3 NADH - c y t o c h r o m e b5 r e du c t a s e 3 7 . 99 13 59 . 2 30 . 8 GK G l y ce r o l k i n a s e 7 . 98 3 43 . 9 57 . 6 P H B 2 P r oh i b iti n - 2 7 . 98 12 43 . 1 33 . 4 S E P T I N 4 S e p ti n - 4 7 . 83 18 50 53 . 1 S H 3 G L B 1 E ndoph ili n - B 1 7 . 64 13 42 . 6 44 . 1 N M E 1 N u c l e o s i d e d i ph o s ph a t e k i n a s e A 7 . 55 5 60 . 5 17 . 1 T O MM 34 M it o c hond r i a l i m po r t r ece p t o r s ubun it T O M 34 7 . 51 14 37 . 9 34 . 6 BR I 3 B P BR I 3 - b i nd i ng p r o t e i n 7 . 5 4 16 . 1 27 . 0 S A MM 50 S o r ti ng a nd a ss e m b l y m ac h i n e r y c o m pon e n t 50 ho m o l og 7 . 47 17 41 . 5 51 . 5 P GA M 5 S e r i n e / t h r e on i n e - p r o t e i n pho s ph a t a s e P GA M 5 , m it o c hond r i a l 7 . 46 12 40 . 3 31 . 9 M T X 2 M e t a x i n - 2 7 . 45 5 27 . 1 32 . 1 LET M D 1 LET M 1 do m a i n - c on t a i n i ng p r o t e i n 1 7 . 35 10 37 . 8 39 . 8 VA T 1 S yn a p ti c v e s i c l e m e m b r a n e p r o t e i n VA T - 1 ho m o l og 7 . 29 11 41 42 . 7 DNA J C 11 D n a J ho m o l og s ub f a m il y C m e m b e r 11 7 . 22 15 33 . 9 57 . 3 C OA S Y B i f un c ti on a l c o e n z y m e A s yn t h a s e 7 . 21 13 31 61 . 7 S YN J 2 B P S yn a p t o j a n i n - 2 - b i nd i ng p r o t e i n 7 . 17 4 28 . 3 15 . 8 T O MM 40 M it o c hond r i a l i m po r t r ece p t o r s ubun it T O M 40 ho m o l og 7 . 13 6 32 . 3 34 . 7 T O MM 22 M it o c hond r i a l i m po r t r ece p t o r s ubun it T O M 22 ho m o l og 7 . 1 2 22 . 7 15 . 4 C P T 1 B C a r n iti n e O - p a l m it oy lt r a n s f e r a s e 1 , m u s c l e i s o f o r m 7 . 05 13 21 . 4 84 . 0 V PS 13 A V ac uo l a r p r o t e i n s o r ti ng - a ss o c i a t e d p r o t e i n 13 A 6 . 87 50 21 . 8 347 . 9 M FF M it o c hond r i a l fi ss i on f ac t o r 6 . 79 4 26 . 6 27 . 0 Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 15 References 717 Al - Amoudi , A . , Chang , J . - J . , Leforestier , A . , McDowall , A . , Salamin , L . 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Methods 14 , 331 – 332 . 1049 1050 Leung et al . | supramolecular assemblies at the mitochondria - cytoskeleton interface 17 \ No newline at end of file diff --git a/silver_data/3bd60f4eb15af626baff7b87cc1ffa6cb64167ac.pdf.txt b/silver_data/3bd60f4eb15af626baff7b87cc1ffa6cb64167ac.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..66ccf6d2c46d19ddd1f3881ebf87e9378e5f42f4 --- /dev/null +++ b/silver_data/3bd60f4eb15af626baff7b87cc1ffa6cb64167ac.pdf.txt @@ -0,0 +1 @@ +AnAlysis nAture methods | VOL . 14 NO . 7 | JULY 2017 | 673 Chromosome conformation capture ( 3C ) and fluorescence in situ hybridization ( Fish ) are two widely used technologies that provide distinct readouts of 3d chromosome organization . While both technologies can assay locus - specific organization , how to integrate views from 3C , or genome - wide hi - C , and Fish is far from solved . Contact frequency , measured by hi - C , and spatial distance , measured by Fish , are often assumed to quantify the same phenomena and used interchangeably . here , however , we demonstrate that contact frequency is distinct from average spatial distance , both in polymer simulations and in experimental data . Performing a systematic analysis of the technologies , we show that this distinction can create a seemingly paradoxical relationship between 3C and Fish , both in minimal polymer models with dynamic looping interactions and in loop - extrusion simulations . together , our results indicate that cross - validation of hi - C and Fish should be carefully designed , and that jointly considering contact frequency and spatial distance is crucial for fully understanding chromosome organization . While genomes are often considered as one - dimensional sequences , they are also physically organized in three dimensions inside the cell nucleus , with far - reaching consequences 1 – 4 . One of the many important implications relates to gene regulation : regions of regulatory DNA are often very far away in the linear genomic sequence from the genes they regulate , yet their regula - tory interactions are presumed to rely on direct encounters in three - dimensional space . Current methods provide indirect , yet complementary , readouts of the high - resolution chromosomal dynamics underlying these processes . Below we focus on two widely used techniques for assaying chromosomes , DNA - FISH and chromosome conformation capture ( 3C ) . 3C techniques have become popular for their high - throughput ability to connect spatial information to the genomic sequence 2 – 5 . 3C - based approaches use crosslinking and ligation to capture the information that two genomic loci are spatially proximal . Moreover , 3C techniques are readily generalized to the genome - wide scale , and are then usually termed Hi - C 6 ; here we refer to the contact frequency obtained from Hi - C and 3C interchangeably . Importantly , while 3C records whether two loci were in contact in some fraction of cells in the population , it does not record where in the nucleus this contact occurred . Also , 3C is usually performed on large populations of cells . This is advantageous in that 3C can assay both very frequent and very rare events , as the large population allows a large dynamic range . However , informa - tion regarding cell - to - cell variability is not available in popula - tion - average maps of chromosomal contact frequencies 7 , 8 . FISH technologies are appreciated for their ability to specifi - cally determine the spatial position of sets of chromosomal loci by imaging 9 . FISH is based on optically labeled probes that hybridize to complementary regions of chromosomes . Importantly , as an imaging - based approach , FISH is intrinsically able to probe cell - to - cell variability and directly record spatial position inside the nucleus . Many different labeling approaches have been consid - ered , including labeling pairs of loci ( two - locus FISH ) 10 as well as labeling larger contiguous regions 11 and even whole - chromosome painting 12 – 14 . High - throughput 15 , 16 and super - resolution 17 – 19 FISH approaches are currently in development . Still , obtaining high - resolution pairwise distance distributions for all pairs of loci—that is , constructing a pairwise distance map similar to a genome - wide Hi - C contact map—currently remains out of reach . For further experimental background on the connection between FISH and Hi - C , see ref . 20 . In studies that rely primarily on 3C - based approaches , FISH is often performed on a subset of loci as a means of validation . Typically , for loci at increasing genomic separations , their aver - age FISH spatial distance increases and 3C contact frequency decreases 6 , 21 – 23 . Additionally , for a limited set of tested pairs of loci , it was found that loci in the same A / B compartment contact each other more frequently and are on average closer 6 ; similar findings are obtained for loci in the same topologically associat - ing domain ( TAD ) 24 , 25 . Moreover , spatial distance and contact frequency are largely correlated at the ~ 300 kb – 10 Mb scale 26 . However , this does not seem to be strictly the case for all loci 8 , and such assessments can even lead to seemingly paradoxical observa - tions when comparing FISH and 3C 20 , 27 . Because of the stochastic and variable nature of chromosome folding in vivo , polymer models provide a useful framework Fish - ing for captured contacts : towards reconciling Fish and 3C Geoffrey Fudenberg 1 , 2 & Maxim Imakaev 1 1 Center for the 3D Structure and Physics of the Genome , and Institute for Medical Engineering and Science ( IMES ) , Massachusetts Institute of Technology , Cambridge , Massachusetts , USA . 2 Current address : Gladstone Institutes , University of California , San Francisco , California , USA . Correspondence should be addressed to G . F . ( geoff . fudenberg @ gmail . com ) or M . I . ( mimakaev @ gmail . com ) . Received 14 NovembeR 2016 ; accepted 3 may 2017 ; published oNliNe 12 JuNe 2017 ; doi : 10 . 1038 / Nmeth . 4329 674 | VOL . 14 NO . 7 | JULY 2017 | nAture methods AnAlysis for interpreting 3C or FISH data 8 , 28 . Modeling efforts naturally started with homopolymer models , which assume the chemi - cal equivalence of all monomers ( i . e . , that there is no sequence specificity for chromosomal interactions or folding 28 ) . While chromosomes in vivo are generally better described by models that include locus - specific folding 8 , 28 , homopolymer models are highly studied in the physics literature and are often more amena - ble to analytical understanding . In the majority of homopolymer models , the further apart two monomers are along the polymer chain , the further apart they are in space , and the less frequently they are in contact ; this leads to an often - useful , but potentially misleading , heuristic that the two quantities are directly related ( Supplementary Note ) . As measuring contact frequency was largely unachievable in polymer systems prior to the develop - ment of 3C for chromosomes , the concordance or discordance of contact frequency and spatial distance has received relatively little attention in the polymer physics literature . Here we demonstrate how 3C and FISH generally probe differ - ent aspects of spatial chromosome organization . We first illustrate how spatial distance and contact frequency can display a seem - ingly paradoxical relationship in currently available experimental data , focusing on the simplest two - locus labeling approach for FISH , as it is most directly comparable to 3C . We then study the connection between contact frequency and average spatial dis - tance in a simple polymer model ; these simulations show that a minimal assumption , introduction of a single dynamic loop between two loci , can break the typical relationship between con - tact frequency and average spatial distance . We then consider pol - ymer simulations with loop extrusion , and find that this process can also affect contact frequency and spatial distances to greatly different degrees . Together our results show how seemingly para - doxical relationships between contact frequency and spatial dis - tance can easily emerge in experimental data for physical reasons , demonstrate that cross - validation of Hi - C and FISH must be very carefully considered , and argue that joint consideration of contact frequency and spatial distance will underlie further understand - ing of chromosome organization in vivo . results3C and Fish probe different aspects of spatial organization To investigate the connection between 3C and FISH , we focused on the simplest case of both methods , in which each method probes the relationship between a pair of loci ( Fig . 1a , b ) . A com - mon design for FISH experiments involves labeling a pair of genomic loci to directly visualize their distances in a population of cells ( Fig . 1a ) . This experimental design allows the measure - ment of the probability density function ( PDF ) of spatial distances between a pair of loci ( Fig . 1c ) . Results from such experiments are often shown as cumulative distribution functions ( CDFs , Fig . 1d ) as these do not require binning or density - estimation steps to obtain relatively smooth curves for limited numbers of cells . In contrast with FISH , 3C experiments capture rare contacts that occur when the loci are closer than the capture radius imposed by crosslinking and ligation ( Fig . 1b ) . Roughly , 3C measures the inte - gral of the spatial distance PDF up to the capture radius ( Fig . 1c ) , or , equivalently , the value of the CDF at the capture radius ( Fig . 1d ) 20 . Since such small distances are relatively rare , imaging many cells is certainly a requirement for directly comparing 3C contact probabilities with distances measured by FISH . We further examined the connection between 3C and FISH by considering recent publicly available Hi - C data 23 . The authors of this publication performed high - resolution Hi - C experiments and reported several thousands of CTCF - mediated peaks , termed loops , in the Hi - C data . As a validation of the loops by FISH , they report CDF FISH plots for four pairs of ‘loop’ and ‘control’ loci at matched genomic separations ( for example , peak1 – loop and peak1 – control , re - plotted in Supplementary Fig . 1 ) for the same cell type . As part of the validation , the authors reported that for each of the loop – control pairs of loci , the median spatial distance changed concordantly with the Hi - C signal 23 . While this holds , we also found that this was not always the case when we compared loops and controls from different pairs . Indeed , we found a seem - ingly paradoxical relationship between peak4 – loop and peak3 – control ( Fig . 2 ) : peak4 – loop has higher contact frequency despite being further away on average than peak3 – control . Nevertheless , the change in the value of the CDF at small distances actually was in agreement with measurements from Hi - C , suggesting that this short - range behavior of the CDF is more closely connected with contact frequency 21 . A similar situation is observed for peak4 – loop and peak2 – control . In contrast , for all control – control pairs of loci , the median spatial distance changed concordantly with the Hi - C signal . We note that seemingly paradoxical pairs involved comparisons between a loop and a control . Together , these observations suggest that locus - specific chro - mosome organization in vivo can be an important reason why average spatial distance and contact frequency could behave diver - gently . They additionally argue that to reconcile this divergence and cross - validate observations from 3C , it will be necessary to obtain the full spatial distance distribution from FISH , includ - ing very short distances . Given the currently limited availability of high - resolution matched experimental Hi - C and FISH data , we turned to polymer models to study the relationship between Fluorescence in situ hybridization ( FISH ) Chromosome conformation capture ( 3C ) R P ( R ) Spatial distance , R ( nm ) CD F 1 . 0 0 . 0 0 . 5 Contactprobability P D F 1 . 0 0 . 0 0 . 5 Median spatial distance Contactprobability 3C Capturedistance FISH R a b c d Figure 1 | Illustrated relationship between 3C and FISH . ( a ) FISH obtains information for all cells in a population to build up a full distribution of pairwise distances between labeled loci . ( b ) 3C - based approaches ( including 4C , 5C and Hi - C ) capture contacts from the small fraction of cells in which two loci are within the capture radius . ( c , d ) Illustration of a PDF and CDF of pairwise spatial distance , R , between two loci for a large population of cells . Vertical black lines show the capture distance and the median spatial distance . nAture methods | VOL . 14 NO . 7 | JULY 2017 | 675 AnAlysis spatial distance and contact frequency , where these two quantities can be unambiguously calculated for any desired pairs of loci from the same set of conformations . simulations can reconcile contact frequency and spatial distances To understand the minimal set of assumptions that can decouple contact frequency from average spatial distance , we investigated both of these quantities in equilibrium polymer simulations of a sin - gle dynamic chromatin loop ( Fig . 3 ) . Following past work in which we investigated the effect of a fixed chromatin loop 29 , we modeled chromatin as a semi - flexible polymer fiber with excluded volume interactions ( Online Methods , Supplementary Note ) . We per - formed simulations using OpenMM 30 , 31 , and calculated simulated contact maps ( Fig . 3a ) , spatial distance distributions ( Fig . 3b , c ) , and average spatial distance maps ( Supplementary Fig . 2b ) from the simulated ensemble of conformations . We imposed the dynamic looping interaction using a short - ranged attractive force . Monomers at the base of the 25 - kb dynamic loop interacted with attractive energy ( 4 kT unless noted ) when they were closer than a distance of 2 monomer diameters ; for other monomers the attractive part of the potential was set to be negligibly small ( 0 . 1 kT ) . This pairwise interaction poten - tial could arise from direct molecular interactions , and the two monomers involved in the dynamic looping interaction can be thought of as hard spheres that stick to some degree upon coming into contact , following a stochastic encounter in 3D ( reviewed in refs . 8 , 32 ) . In our simulations , the dynamic looping interac - tion is clearly visible in the contact frequency map , but is faint in a map of average spatial distances ( Supplementary Fig . 2a , b ) . Interestingly , the PDF of spatial distances for monomers at the base of the dynamic loop and control monomers ( Fig . 3b ) appeared quite similar , apart from a sharp peak at short distances for the monomers at the loop base . For typically considered polymer systems of indistinguish - able monomers , mean spatial distance and contact probability are generally inversely related ( Supplementary Note ) . However , our simulations demonstrate that even a minimal modification , introduction of a single dynamic loop , changes this typical behav - ior ( Fig . 4 ) . While a comparison between control loci of separa - tion 15 kb or 25 kb displays the typical monotonic behavior over all genomic separations ( Fig . 4b ) , an apparent paradox emerges when comparing the control loci separated by 15 kb with the 25 - kb dynamic loop ( Fig . 4c ) . While the 25 - kb dynamic loop is further apart on average , it displays a higher contact frequency . This behavior can emerge because contacts are rare events , and therefore contact frequency can increase many - fold without large changes in the average distance ( Supplementary Fig . 2d , f ) . Similar behavior emerges in simulations for a range of param - eter values of chromatin stiffness , chromatin density , dynamic loop attraction strength , and loop size ( Supplementary Fig . 3 ) . Consideration of dynamic loop models as equilibrium ensembles of conformations provides further support for the widespread possibility of seemingly paradoxical pairs ( Supplementary Note ) . Together , our simulations show how , even in a particularly simple case , seemingly paradoxical relationships can emerge between spatial distance and contact frequency , arguing for caution when designing comparisons between FISH and 3C . simulations illustrate how experimental limitations could affect validation of a dynamic loop We next investigated how possible experimental limitations to either FISH or 3C can impact our ability to ascertain the presence of this simulated looping interaction . In FISH experiments , assaying a finite number of cells both imposes uncertainty on the PDF and makes the probability of rare events difficult to estimate . Consistently , it is more difficult to reliably detect changes in contact frequency than median spa - tial distance in simulations when assaying a limited number of conformations ( Supplementary Fig . 4 ) . As contact frequencies are often quite low , this indicates that consistent validations of Hi - C by FISH would require assaying orders - of - magnitude - larger numbers of cells than is typical in FISH experiments . For FISH , additional uncertainty can be imposed by factors including probe size , chromatin movement during denaturation and hybridization , background noise , and ambiguities arising 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 S pa t i a l d i s t 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 0 . 00 0 . 05 0 . 10 0 . 15 0 . 20 P ( < 300 n m ) 0 . 00 0 . 05 0 . 10 0 . 15 0 . 20 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 l og 10 ( H i - C ) 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 Spatial distance ( µ m ) Spatial distance ( µ m ) C u m u l a t i v e p r obab ili t y C u m u l a t i v e p r obab ili t y a b P ea k 3 – c on t r o l P ea k 4 – c on t r o l P ea k 3 – c on t r o l P ea k 4 – l oop 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 Peak3 – control Peak4 – control Peak3 – control Peak4 – loop S pa t i a l d i s t P ( < 300 n m ) l og 10 ( H i - C ) Figure 2 | Experimental data demonstrate the complex relationship between Hi - C and FISH . ( a ) Comparison between a pair of typical loci shows that increased spatial distance as determined by FISH coincides with decreased Hi - C counts . ( b ) Comparison between a pair of loop loci and a pair of control loci shows a seemingly paradoxical increase in spatial distance as determined by FISH accompanied by increased Hi - C counts . FISH and Hi - C data re - plotted from ref . 23 for GM12878 cells ( supplementary table 1 ) . Horizontal gray line intersects the median spatial distance , and vertical gray line intersects the probability of an observation less than 300 nm , P ( < 300 nm ) . Bar plots show median spatial distance , P ( < 300 nm ) , and log 10 ( corrected Hi - C counts ) . 676 | VOL . 14 NO . 7 | JULY 2017 | nAture methods AnAlysis from the presence of homologous chromosomes ( reviewed in ref . 20 ) . In simulations , we considered how the first two factors might affect spatial distance distributions of loop and control loci ( Supplementary Fig . 5 ) . To simulate the impact of probe size , we considered the pairwise distributions between centroids of cho - sen regions , rather than an exact pair of monomers . To simulate uncertainty or perturbation of relative distances due to chromatin movement during the FISH protocol , we simulated probe localiza - tion uncertainty by adding Gaussian noise to each set of simulated probe distances . We find that even a small uncertainty or impreci - sion in the spatial localization of probes during FISH makes the existence of a dynamic looping interaction much more difficult to ascertain , whereas larger probe size had a relatively smaller impact on the spatial distributions ( Supplementary Fig . 5 ) . In a 3C experiment , whether two loci in close spatial prox - imity in a given cell are recorded as a contact depends on the effective capture radius . The capture radius can be influenced by a number of factors , including restriction efficiency , restric - tion frequency , and the details of crosslinking , which may depend on the particular complement of DNA - associated proteins at a given genomic locus 33 – 35 . Our simulations show that a larger contact radius for simulated 3C can also obscure the exist - ence of a dynamic looping interaction ( Supplementary Fig . 5 ) . Additional measurement noise may also come from library complexity , sequencing depth , and ligations in solution 23 , 33 , 35 – 37 , which could all obscure the detection of looping interactions in 3C - based methods ( reviewed in ref . 36 ) . Together , these simulated perturbations to the idealized FISH and 3C protocols illustrate how considering many experi - mental details will be required to fully reconcile observations from FISH and Hi - C . loop - extrusion simulations can display divergent contact frequency and spatial distance After considering spatial distance and contact frequency in this minimal model , we then considered their relationship in simula - tions of loop extrusion with locus - specific boundary elements , recently proposed by us 38 and others 39 , 40 as explaining key aspects of interphase chromosome organization . Loop extrusion is a potential mechanism for the formation of TADs and loops in mammalian Hi - C maps , making it a subject of recent interest 41 – 45 ( for review , see ref . 1 ) . To consider genomic scales similar to those of the published 23 loop - control pairs considered above , we simu - lated a genomic region containing several TADs of 210 – 870 kb , with parameters of the chromatin fiber as defined in our previous study of interphase loop extrusion 38 . For each loop at the cor - ner of every TAD , we considered a matched control at the same genomic separation , but offset by 100 kb . 0 . 00 0 . 01 0 . 02 C on t a c t f r equen cy C on t r o l 1 5 C on t r o l 2 5 S pa t i a l d i s t an c e 0 4 8 12 0 . 00 0 . 04 0 . 08 C on t r o l 2 5 L oo p 2 5 0 4 8 12 b 0 5 10 15 20 25 30 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 5 10 15 20 25 30 – 6 . 4 – 4 . 8 – 3 . 2 – 1 . 6 0 . 0 log ( contact probability ) CD F Spatial distance ( monomers ) e a c d Control 25 Loop 25 Control 15 Control 25 Loop 25 Control 15 Control 15 Figure 4 | Simulations clarify the complex relationship between contact frequency and median spatial distance . ( a ) Contact frequency map for a polymer with a specifically interacting dynamic 25 - kb loop , as in Figure 3 , indicated as a dashed arc between two loop bases in orange . Three locations for simulated FISH ( control 15 , red square ; control 25 , yellow square , and loop 25 , blue circle ) are indicated on the contact map ; control 15 and control 25 indicate regions without specific interactions between monomers . ( b , c ) CDFs for indicated loci ; in each , vertical gray line intersects contact frequency ( here , the probability of distance ≤ 3 ) and horizontal gray line intersects median spatial distance . ( d , e ) Changes in contact frequency for indicated loci and median spatial distance for indicated locus pairs ( supplementary table 2 ) . Note that loop 25 has a higher contact frequency , but larger median spatial distance , than control 15 . c 0 . 00 0 . 25 0 . 50 0 . 75 1 . 00 0 5 10 15 20 25 30 CD F Spatial distance , d ( monomers ) Control 25 Loop 25 b 0 . 000 0 . 002 0 . 004 0 . 006 0 . 008 0 . 010 0 . 012 – 6 . 4 – 4 . 8 – 3 . 2 – 1 . 6 0 . 0 log ( contact probability ) P D F a Control 25 Loop 25 Control 25 Loop 25 Spatial distance , d ( monomers ) 0 5 10 15 20 25 30 Figure 3 | Simulations demonstrate the effect of introducing a single dynamic loop . ( a ) Contact frequency map for a polymer with a specifically interacting dynamic 25 - kb loop , indicated as a dashed arc between two loop bases in orange . Two locations for simulated FISH ( control 25 , yellow square , and loop 25 , blue circle ) are indicated on the contact map . Loop 25 indicates the loci at the loop bases , and control 25 indicates an equally spaced pair of control loci , without any specific interactions . ( b ) PDFs of spatial distances for the loop and control pairs of loci ( colors as in a ) . Here and in the next panel , vertical gray lines show capture distance . ( c ) CDFs for indicated loci ; horizontal gray line intersects median distance . nAture methods | VOL . 14 NO . 7 | JULY 2017 | 677 AnAlysis The dynamics of loop extrusion are governed by key parameters : processivity , separation , and extrusion speed 38 , 46 . In simulations , we found that certain combinations of these parameters led to loop – control pairs with seemingly paradoxical relationships ( Fig . 5 ) . Such relationships could emerge because , much as in the mini - mal dynamic loop model considered above , certain regimes of loop extrusion can greatly increase contact frequency between subsequent boundary elements while minimally altering aver - age spatial distance ( Supplementary Fig . 6 ) . In our simulations , the number of seemingly paradoxical pairs increased with larger separation , and also for slower loop extrusion ( Supplementary Fig . 6 ) . Interestingly , the best - fitting parameters from the sweep in our previous study 38 did not produce seemingly paradoxi - cal pairs for the considered TAD sizes . However , simply making loop extrusion slower was sufficient to create seemingly para - doxical pairs , while having little effect on the simulated contact map ( Supplementary Fig . 7 ) . This indicates that perturbing loop - extrusion dynamics could alter average FISH distances while having little effect on Hi - C contact maps , and thus using Hi - C alone to infer mechanisms of chromosomal folding may be insufficient . disCussion Our results illustrate that while median spatial distance and contact frequency are often inversely proportional , they are far from equivalent . Indeed , our simulations show that a relatively minor perturbation—the existence of a dynamic looping interac - tion between two loci—clearly breaks the equivalence between these two quantities . In particular , we show that there is great freedom to make large shifts in contact frequency with small shifts to median spatial distance , since contacts between distal chromosomal loci are generally rare events . We then show that loop extrusion can similarly break the typical correspondence between median spatial distance and contact frequency . Together , our simulations demonstrate that our expectation in vivo should be a nontrivial relationship between contact frequency and spatial distance , and that Hi - C and FISH data together will be necessary to better understand chromosome organization . Given these factors , 3C experiments cannot be simply validated ( or invalidated ) by FISH without carefully considering technical details of the two methods . Indeed , efforts to integrate results from these technologies will need to carefully address unknowns of the 3C capture radius and FISH localization uncertainty , in addition to assaying sufficiently large numbers of cells to populate the small - distance portion of the FISH distribution . While here we limit ourselves to considering the relatively simple compari - son between 3C and two - locus FISH , many other comparisons would be valuable in future work , including how to best integrate information obtained from contiguously stained regions 11 , 17 with Hi - C experiments . In addition to the implications for validation , our results also caution against certain modeling approaches . In particular , our results show how a common strategy of simply transforming ensemble average 3C contact frequencies into spatial distances ultimately leads to inconsistent models of chromosomal organiza - tion ( reviewed in ref . 8 ) . Finally , our results also demonstrate how polymer modeling can in principle be used to reconcile FISH spatial distances and 3C or Hi - C contact frequencies . This is because both quantities are readily calculable from an ensemble of simulated polymer con - formations . One of the central goals of the recently formed 4DN consortium is to systematically compare Hi - C and high - through - put high - resolution imaging data and understand any potential discrepancies ( https : / / doi . org / 10 . 1101 / 103499 ) . As these matched data become available , systematically comparing polymer models to both Hi - C and imaging data will be an essential step toward understanding principles of chromosomal organization . methods Methods , including statements of data availability and any associated accession codes and references , are available in the online version of the paper . Note : Any Supplementary Information and Source Data files are available in the online version of the paper . ACknoWledgments The authors thank A . Goloborodko for thoughtful comments regarding the statistical mechanics analogy of re - weighting loop conformations , G . Nir for helpful discussions regarding imaging , and anonymous reviewers for thoughtful and detailed feedback . The authors also thank J . Dekker and other members of the UMass – MIT Center for 3D Structure and Physics of the Genome for helpful – 4 . 8 – 3 . 2 – 1 . 6 0 . 0 0 20 40 60 80 100 120 140 160 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 Control 210 kb Control 660 kb 0 20 40 60 80 100 120 140 160 Control 210 kb Loop 660 kb 0 . 00 0 . 01 0 . 02 0 . 03 01020304050 0 . 00 0 . 02 0 . 04 0 . 06 010203040 Spatial distance ( monomers ) Control 210 Control 660 Loop 660 log ( contact probability ) CD F C on t r o l 210 C on t r o l 660 C on t r o l 210 Loop 660 C on t a c t f r equen cy S pa t i a l d i s t an c e a b c d e Figure 5 | Loop - extrusion simulations can display divergent contact frequency and spatial distance . ( a ) Illustration of loop - extrusion dynamics : loop - extruding factors translocate along the chromatin fiber , forming progressively larger loops , until dissociating or becoming halted at a boundary element ( red hexagons ) . ( b ) Region of a simulated contact frequency map at 6 - kb resolution , showing TADs of sizes 210 kb and 660 kb for the loop - extrusion parameters processivity 240 kb , separation 480 kb and relative velocity 20 , 000 . Three locations for simulated FISH ( control 220 , red ; control 660 , yellow ; and loop 660 , blue ) are indicated on the contact map . ( c , d ) CDFs for indicated loci ; in each , vertical gray line intersects contact frequency ( here , probability of distance ≤ 10 ) and horizontal gray line intersects median spatial distance . ( e ) Changes in contact frequency for indicated loci , and median spatial distance for indicated locus pairs ( supplementary table 3 ) . Note that loop 660 has a higher contact frequency , but larger median spatial distance , than control 220 . 678 | VOL . 14 NO . 7 | JULY 2017 | nAture methods AnAlysis feedback . Finally , the authors thank L . Mirny for comments on earlier drafts of this paper and for supporting their independent work . This work was supported by NSF 1504942 Physics of Chromosomes ( PI : L . Mirny ) and U54 DK107980 3D Structure and Physics of the Genome ( PIs : J . Dekker and L . Mirny ) . During revisions , G . F . was supported by the San Simeon Fund ( PI : K . Pollard ) . Author ContriButions G . F . and M . I . jointly conceived of the study , performed analyses , and wrote the manuscript , and are listed in alphabetical order on the first page . ComPeting FinAnCiAl interests The authors declare no competing financial interests . reprints and permissions information is available online at http : / / www . nature . com / reprints / index . html . 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Non - equilibrium chromosome looping via molecular slip - links . Preprint at https : / / arxiv . org / abs / 1612 . 07256 ( 2016 ) . 46 . Goloborodko , A . , Marko , J . F . & Mirny , L . A . Chromosome compaction by active loop extrusion . Biophys . J . 110 , 2162 – 2168 ( 2016 ) . nAture methods doi : 10 . 1038 / nmeth . 4329 online methods Polymer simulation overview . Polymer models were simulated with OpenMM 30 , 31 , a high - performance GPU - assisted molecular dynamics software ( https : / / simtk . org / home / openmm ) . We used an in - house openmm - polymer library to implement simulations ( pub - licly available http : / / bitbucket . org / mirnylab / openmm - polymer ) . Dynamic loop simulations . Dynamic loop simulations were per - formed as in the work of Doyle et al . , 2014 29 , albeit with a dynami - cally interacting loop rather than a static loop , implemented as described below . Following Doyle et al . , we modeled chromatin as a semi - flexible polymer fiber with excluded volume interactions , where spherical monomers of 15 - nm diameter represent ~ 500 bp , or approximately three nucleosomes . Adjacent monomers were connected by harmonic bonds with a potential U = 25 * ( r – 1 ) 2 ( here and below , energy is in units of kT ) . The stiffness of the fiber was modeled by a three point interaction term , with the potential U = k * ( 1 – cos ( α ) ) , where α is an angle between neighboring bonds and k is a parameter controlling stiffness , here set to 1 kT . To model the dynamic loop considered in the present work , we used a Lennard – Jones ( LJ ) potential U = 4 ε ij * ( 1 / r 12 – 1 / r 6 ) where ε ij was set to 4 kT for the monomers at the base of the dynamic loop ( i , j ) , and was set to negligibly small otherwise ( ε = 0 . 1 kT ) . We ini - tialized our simulations as a system of 8 compact rings ( see ref . 47 ) , and used periodic boundary conditions to achieve a density of 0 . 10 ( in the middle of the estimated range for mammalian cells 48 ) . We then simulated 50 runs of this system using Langevin dynamics , for 10e8 time steps . For the fiber lengths considered here , polymer simulations reached equilibrium in less than 1e7 time steps ; this was confirmed by observing that monomer dis - placement saturates after about 5e6 time steps . Conformations were saved every 1e5 time steps and an equilibrium ensemble of 900 conformations obtained after the initial equilibration was used for our analysis . To obtain simulated contact maps , we first found all contacts within each polymer conformation , and then aggregated these contacts for all pairs of monomers . Following ref . 29 , a con - tact was defined as two monomers being at a distance less than 3 monomer diameters . To obtain simulated FISH distributions , we calculated a list of spatial distances for a chosen set of loci , and built a histogram of distances starting at 0 in bins of 0 . 1 mono - mers . To display PDFs this histogram was then smoothed with a moving average window with a size of 0 . 7 monomers . Loop - extrusion simulations . Loop - extrusion simulations were performed as in ref . 38 with slight modification to the sizes and number of TADs , and an upgraded loop - extruding factor ( LEF ) simulation engine . To better span the genomic size range of TADs and loops probed by FISH in ref . 23 , we considered simulations of a 6 - Mb region , with 10 , 000 monomers , 12 TADs , and monomers representing 600 bp as in ref . 38 . These TADs were separated by 11 boundary elements that stalled loop extrusion , positioned at monomers : 750 , 1 , 550 , 1 , 900 , 3 , 000 , 3 , 650 , 4 , 300 , 5 , 750 , 6 , 550 , 6 , 900 , 7 , 550 , 9 , 000 . We used the same fiber stiffness and the same volume density as in the best - fitting model in ref . 38 : density of 0 . 2 and stiffness of 2 ; where values of parameters are as defined as previously . In the upgraded the loop - extruding factor ( LEF ) simulation engine , simulations do not need to be re - initialized between each subsequent step of loop extrusion . Instead , simulations are done in blocks of 100 loop - extrusion steps . At the start of each block , all LEF - mediated bonds that would occur in the next 100 loop - extrusion steps were initialized , and all but current ( step = 0 ) bonds were given a strength of zero . Current bonds were given the same strengths as previously . Langevin dynamics ( LD ) was then advanced by a certain number of LD timesteps , reflecting the relative velocity of loop extrusion ( 1 , 000 , 5 , 000 , 20 , 000 used in this manuscript ) . After that , strengths of the bonds were adjusted such that only bonds that exist at step = 1 of loop extrusion have nonzero strengths . This allowed us to avoid restarting simulations between subsequent extrusion steps , and do so only every 100 extrusion steps . The new engine lead to significant performance improvement , as it eliminated the necessity to frequently restart simulations . The reason for this updated scheme , with blocks of 100 LEF steps , is that OpenMM does not allow addition or removal of bonds once a simulation is initialized , but does allow for changing bond strengths . The new engine also allowed us to advance loop extrusion by only one step at a time ( 4 steps were used previously to decrease the number of restarts ) . This addi - tionally allows higher - fidelity simulations of loop extrusion , with less abrupt motion of the polymer after updating the positions of bonds imposed by loop extrusion . The new loop - extrusion engine was added to the example folder of the openmmlib pack - age ( http : / / bitbucket . org / mirnylab / openmm - polymer ) . We performed simulations for three different loop - extrusion speeds : 1 , 000 , 5 , 000 , and 20 , 000 LD steps per LEF step . Simulations with 1000 LD steps were run for 2 , 000 , 000 blocks of LD ; simula - tions with 5 , 000 steps were run for 500 , 000 blocks of LD , and simulations with 20 , 000 steps were run for 250 , 000 blocks of LD . We obtained every 20th block for the simulation with 20 steps , and every 5th block for other simulations , yielding 100 , 000 total conformation for simulations with 1 , 000 and 5 , 000 LD steps per LEF step , and 50 , 000 conformations for 20 , 000 LD steps . Contact maps were built using capture radius of 10 monomers . Simulated FISH distributions were calculated as above . Experimental data . FISH CDFs corresponding to ref . 23 were obtained from https : / / groups . google . com / forum / # ! topic / 3d - genomics / from their UPDATED spreadsheet . Published publicly available Hi - C data 23 , GEO accession GSE63525 , was re - proc - essed , filtered , and iteratively corrected using hiclib , https : / / bit - bucket . org / mirnylab / hiclib 49 . Data availability . This manuscript used publicly available data as indicated above . Polymer simulation code relevant for this study is publicly available in the example folder of the openmmlib package ( http : / / bitbucket . org / mirnylab / openmm - polymer ) . 47 . Imakaev , M . V . , Tchourine , K . M . , Nechaev , S . K . & Mirny , L . A . Effects of topological constraints on globular polymers . Soft Matter 11 , 665 – 671 ( 2015 ) . 48 . Halverson , J . D . , Smrek , J . , Kremer , K . & Grosberg , A . Y . From a melt of rings to chromosome territories : the role of topological constraints in genome folding . Rep . Prog . Phys . 77 , 022601 ( 2014 ) . 49 . Imakaev , M . et al . Iterative correction of Hi - C data reveals hallmarks of chromosome organization . Nat . Methods 9 , 999 – 1003 ( 2012 ) . \ No newline at end of file diff --git a/silver_data/40f5199102f43437b1461c134faa32ffd72c9eed.pdf.txt b/silver_data/40f5199102f43437b1461c134faa32ffd72c9eed.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..95c65240c62aba78576bd634ce0802bfe4aa4c75 --- /dev/null +++ b/silver_data/40f5199102f43437b1461c134faa32ffd72c9eed.pdf.txt @@ -0,0 +1 @@ +Cellular and behavioral characterization of Pcdh19 mutant mice : subtle molecular changes , increased exploratory behavior and an impact of social environment . Natalia Galindo - Riera 1 , 3 , Sylvia Adriana Newbold 1 , Monika Sledziowska 1 , 4 , Jessica Griffiths 1 , 5 , 6 , Erik Mire 2 , Isabel Martinez - Garay 1 , * 1 Division of Neurosciences , School of Biosciences , Cardiff University , Cardiff CF10 3AX , UK 2 Hodge Centre for Neuropsychiatric Immunology , Neuroscience and Mental Health Research Institute , Division of Psychological Medicine and Clinical Neurosciences , School of Medicine , Cardiff University , Cardiff CF24 4HQ , UK 3 Current address : Department of Medical Biochemistry and Biophysics , Karolinska Institute , 171 77 Stockholm , Sweden 4 Current address : School of Biosciences , University of Birmingham , Birmingham , B15 2TT , UK 5 Current address : School of Pharmacy , University of Reading , Reading , RG6 6LA , UK 6 Current address : Institute of Biomedical and Clinical Sciences , Medical School , University of Exeter , Exeter , EX4 4PS , UK * Corresponding author : Isabel Martinez - Garay , Division of Neurosciences , School of Biosciences , Cardiff University , The Sir Martin Evans Building , Museum Avenue , Cardiff CF10 3AX . Email : martinezgarayi @ cardiff . ac . uk . Tel : + 44 ( 0 ) 2922510029 . Running Title : Anatomical and behavioural characterization of Pcdh19 mutant mice ABSTRACT Mutations in the X - linked cell adhesion protein PCDH19 lead to seizures , cognitive impairment and other behavioral comorbidities when present in a mosaic pattern . Neither the molecular mechanisms underpinning this disorder , nor the function of PCDH19 itself are well understood . By combining RNA in situ hybridization with immunohistochemistry and analyzing single cell RNAseq datasets , we provide a first account of the subtypes of neurons expressing Pcdh19 / PCDH19 , both in the mouse and the human cortex . Our quantitative analysis of the Pcdh19 mutant mouse reveals subtle changes in cortical layer composition , with no major alterations of the main axonal tracts . However , Pcdh19 mutant animals , particularly females , display preweaning behavioral changes , including reduced anxiety and increased exploratory behavior . Our experiments also reveal an effect of the social environment on the behavior of wild - type littermates of Pcdh19 mutant mice when compared with wild - type animals not housed with mutants . This is a second case of a mutated X - linked gene encoding a membrane protein expressed in the developing cortex impacting the behavior of co - housed wild - type littermates . KEYWORDS : cortical lamination , impact of mutant littermates , neuronal subtypes , open field , single cell RNAseq INTRODUCTION PCDH19 is one of several genes located on the X chromosome known to impact neurodevelopment and behavior . Mutations in this gene were identified in patients suffering from EIEE9 ( Epileptic Encephalopathy , Early Infantile , 9 , OMIM # 300088 ) , also known as Girls Clustering Epilepsy ( GCE ) , over a decade ago ( Dibbens et al . 2008 ) . Since then , more than 140 mutations have been described ( Kolc et al . 2018 ) , consolidating PCDH19 as the second most relevant gene in epilepsy after SCNA1 ( Depienne and Leguern 2012 ; Duszyc et al . 2015 ) . The pathogenicity of PCDH19 mutations is dependent on cellular mosaicism and therefore the disorder follows an unusual inheritance , manifesting in heterozygous females and in males with somatic mutations ( Depienne et al . 2009 ; Terracciano et al . 2016 ) . Affected patients develop symptoms during early infancy , often within their first year of life , and display clustered seizures , varying degrees of cognitive impairment and other comorbidities , including autism spectrum disorder , attention deficits and obsessive - compulsive features ( Kolc et al . 2020 ) . PCDH19 codes for protocadherin 19 , a calcium - dependent cell - cell adhesion molecule of the cadherin superfamily . This delta 2 protocadherin has 6 extracellular cadherin repeats , a single transmembrane domain and a cytoplasmic tail with two conserved motives of unknown function ( CM1 and CM2 , Wolverton and Lalande 2001 ) . In addition , a WRC interacting receptor sequence ( WIRS ) downstream of CM2 allows PCDH19 to interact with the WAVE regulatory complex , enhancing its Rac1 - mediated activation ( Chen et al . 2014 ) . PCDH19 is involved in different processes , ranging from neurulation and organization of the optic tectum in zebrafish ( Emond et al . 2009 ; Cooper et al . 2015 ) to neurogenesis and regulation of GABAergic transmission in mammals ( Fujitani et al . 2017 ; Bassani et al . 2018 ; Homan et al . 2018 ; Lv et al . 2019 ) . In addition , PCDH19 is involved in gene expression regulation with estrogen receptor alpha ( Pham et al . 2017 ) and mutations in PCDH19 lead to a deficiency of the neurosteroid allopregnanolone and of other neuroactive steroids ( Tan et al . 2015 ; Trivisano et al . 2017 ) . To date , two different Pcdh19 knockout ( KO ) mouse models have been developed to explore the function of PCDH19 . The first , produced by Taconic Biosciences , has the first three exons of the gene replaced by a beta galactosidase and neomycin ( LacZ - neo ) resistance cassette ( Pederick et al . 2016 ) . The second model retains exons 2 and 3 , with a Lacz - neo selection cassette replacing exon 1 , which encodes the entire extracellular and transmembrane domains ( Hayashi et al . 2017 ) . Lack of Pcdh19 mRNA and protein was confirmed for the Taconic mouse ( Pederick et al . 2016 ) and no major anatomical defects were reported in either of the two models . However , increased neuronal migration has been described ( Pederick et al . 2016 ) , as well as behavioral alterations ( Hayashi et al . 2017 ; Lim et al . 2019 ) . In addition , heterozygous females display a striking segregation of Pcdh19 expressing and non - expressing progenitors in the developing cortex and altered electrocorticogram traces ( Pederick et al . 2018 ) . Although no major abnormalities in cortical architecture have been reported in either KO mouse model , no detailed , quantitative analysis has been carried out yet . Similarly , while RNA in situ hybridization revealed strongest Pcdh19 expression in layers II / III and V ( a ) in mice ( Pederick et al . 2016 ; Hayashi et al . 2017 ) , the neuronal subtypes expressing Pcdh19 have not been characterized , possibly due to the difficulty of labeling PCDH19 expressing cells with current antibodies . Here we report on the identity of Pcdh19 expressing neurons in the mouse and human cortex . We also uncover subtle alterations in cortical neuronal distribution in the cortex of Taconic Pcdh19 mutant animals , as well as robust differences in the behavior of heterozygous females , including an impact of mutant animals on the behavior of their wild type littermates . MATERIAL AND METHODS Experimental Animals Animals were housed under a 12h light / dark cycle with ad libitum access to water and food , and controlled temperature and humidity . All experiments using mice were conducted at Cardiff University , approved by the local ethical boards and carried out following the directions of the UK Animal Scientific Procedures Act ( update 1986 ) . C57BL6 / J wild - type ( WT ) animals were purchased from Charles River Laboratories and the Pcdh19 knock - out ( KO ) line ( TF2108 ) was acquired from Taconic Biosciences . Experimental matings for anatomical and cellular characterization , as well as for behavioral analysis were set up using wild type males and Pcdh19 heterozygous ( HET ) females to produce litters with WT males and females , KO males and HET females . Analysis of single cell RNAseq datasets Gene expression matrices and metadata were downloaded from https : / / portal . brain - map . org / atlases - and - data / rnaseq . Analysis and visualization were carried out using R v . 3 . 6 . 3 , assisted by RStudio v . 1 . 2 . 1335 . Raw counts were normalised to account for library size ( total sum of counts per cell ) and transformed to counts per million ( CPM ) using R package scater v . 1 . 16 . 2 . Violin plots were generated with R packages gridExtra v . 2 . 3 and ggplot2 v . 3 . 3 . 1 . River plots were made with R packages gridExtra v . 2 . 3 , ggplot2 v . 3 . 3 . 1 and ggforce v . 0 . 3 . 2 . Tissue Processing Animals were perfused with PBS followed by 4 % paraformaldehyde ( PFA ) in PBS . After perfusion , brains were extracted and post - fixed in PFA 4 % overnight at 4 C . For RNA ISH , brains were then cryoprotected in 30 % sucrose in PBS before embedding in OCT compound ( Tissue - Tek ) prior to freezing . Samples were stored at - 80 C until sectioning . 12 or 20 μm sections were cut with a cryostat ( CM3050 , Leica Systems ) and stored at - 80 C until use . For immunostaining , fixed brains were briefly washed in PBS and embedded in 4 % low melting point agarose . 50 μm sections were cut with a vibrating microtome ( VT1000S , Leica Systems ) and stored in PBS with 0 . 05 % Sodium Azide at 4 C until use . RNA in situ Hybridization and Immunohistochemistry The probe to detect Pcdh19 has been described before ( Gaitan and Bouchard 2006 ) . Its sequence was amplified using primers Pcdh19e1 - F , 5’ - CACCAAGCAGAAGATTGACCGAG - 3’ and Pcdh19e1 - R , 5’ - GCCTCCCATCCACAAGAATAGTG - 3’ and cloned into pCRII - Blunt - TOPO ( Invitrogen ) . This plasmid was then used to generate digoxigenin - labeled sense and antisense probes . Thawed sections were post - fixed in 4 % PFA , endogenous peroxidases were quenched with 3 % hydrogen peroxidase and slices were then acetylated in a 0 . 25 % acetic anhydride solution . Pre - hybridization took place in pre - warmed hybridization buffer ( 50 % formamide , 0 . 1 % Tween - 20 , 0 . 25 % CHAPS , 250 μg / ml yeast tRNA , 500 μg / ml herring sperm , 5x Denhardts , 5x SSC , 50 μg / ml heparin , 2 . 5 mM EDTA ) for 1h at 65 C . Slices were hybridized with the denatured sense or antisense probes overnight at 65 C in a humidified chamber . The next day , slides were washed with 0 . 2x SSC ( GIBCO ) and PBST , and then blocked in ISH blocking solution ( 10 % DS and 0 . 1 % TritonX - 100 in PBS ) for 20 min at RT . After blocking , brain slices were incubated in primary antibody for 1 h at RT ; washed in PBST and incubated in secondary antibody for 1 h at RT . Antibodies used are described below . Slides were then washed in PBST , equilibrated in TN buffer ( 150 mM NaCl and 100 mM Tris pH = 7 . 5 in water ) and incubated for 30 min in 1 : 2000 HRP - coupled anti - DIG antibody ( Sigma - Aldrich , 11207733910 ) . Following the incubation , tissue was rinsed in TNT ( TN + 0 . 5 % Tween ) and immersed in Cy3 - Tyramide ( TSATM Plus Cy3 Fluorescence kit , Perkin - Elmer , NEL744001KT ) in a 1 in 50 dilution dissolved in the amplification diluent . Slides were then washed , counterstained with DAPI and mounted in DAKO . Immunohistochemistry Antigen retrieval was performed for stainings with antibodies against RORB , SATB2 , Pvalb and CR , with the tissue either immersed in a 10 mM citrate buffer pH = 6 , at 95 C for 5 min ( RORB and SATB2 ) or 10 min ( Pvalb , CR ) before blocking . 50 μm coronal sections were blocked ( 4 % BSA , 3 % donkey serum , 0 . 1 % Triton X - 100 in PBS ) at RT for 1 h . The tissue was then incubated in primary antibody diluted in blocking solution overnight at 4 C . Primary antibodies used for immunostaining were as follows : anti - CUX1 rabbit polyclonal ( 1 : 200 ; Proteintech , 11733 or Santa Cruz Biotechnology , sc - 13024 ) , anti - CTIP2 rat monoclonal ( 1 : 250 ; Abcam , ab18465 ) , anti - SATB2 mouse monoclonal ( 1 : 400 ; Abcam , ab51502 ) , anti - RORB rabbit polyclonal ( 1 : 200 ; Proteintech , 17635 - 1AP ) , anti - TBR1 rabbit polyclonal ( 1 : 350 ; Abcam , ab31940 ) , anti - Pvalb rabbit polyclonal ( 1 : 10000 or 1 : 500 for ISH ; Swant , PV27 ) , anti - CB rabbit polyclonal ( 1 : 5000 ; Swant , CB38 ) , anti - CR mouse polyclonal ( 1 : 1000 ; Merck , AB5054 ) , anti - SST rat monoclonal ( 1 : 200 ; Merck , MAB354 ) , anti - L1CAM rat monoclonal ( 1 : 500 , Merck , MAB5272 ) , anti - Neuropilin1 goat polyclonal ( 1 : 300 , R & D Systems , AF566 ) . Slices were then rinsed in PBS and incubated with secondary antibodies coupled to fluorochromes ( Alexa Fluor range , Thermo Fisher Scientific ) for 1 h at RT . Nuclei were counterstained with DAPI for 10 min , washed again in PBS and mounted with DAKO mounting medium . Image acquisition and analysis Images were acquired using a confocal laser scanning microscope ( LSM 780 , Carl Zeiss ) and ZEN Black software ( version 2 . 0 , Carl Zeiss ) . Image analysis was conducted with ImageJ Fiji software ( Schindelin et al . 2012 ) . For quantification , the cortical wall was divided into ten horizontal bins of equal width . The number of marker positive cells in each bin was quantified and is shown as mean percentage relative to the total number of cells in all ten bins , ± standard error of the mean ( SEM ) . Behavioral analysis Behavioral tests were conducted at P21 ( pre - weaning ) and in young adults ( P60 and over ) . Two different WT controls were tested : WT littermates of the mutant animals ( mixed genotyped housing mice , MGH ) and animals from pure WT litters ( single genotype housed mice , SGH ) . The WT parents of the SGH animals were derived from the Pcdh19 colony . Mice habituated to the new environment by taking them to the behavioral room 30 min prior to the tests . Mice were handled with open hands to reduce anxiety levels and a maximum of one behavioral test was performed per day . Open field Open field behavioral analysis was performed on two consecutive days , using the first day to habituate the mice to the new environment . Mice were allowed to explore freely , in the dark , for 20 min , in an open field arena ( 40 cm x 40 cm ) . Spontaneous locomotion was recorded using a computer - linked video camera ( The Imaging Source ) located above the arena and an infrared illumination box ( Tracksys ) located underneath the arena . The EthoVision XT software ( Noldus ) was used to analyze total distance travelled , distance travelled in intervals of 5 min and time spent in the center of the arena . The center of the arena was defined as the area separated from the wall by 5 cm or more . Elevated plus maze Each mouse was left to explore freely for 5 min in a maze consisting of 4 perpendicular arms ( 40 cm x 7 cm ) : two open arms ( 1 cm high ) and two closed arms ( 16 cm high ) , in a well - lit room . Behavior was recorded using a computer - linked video camera ( The Imaging Source ) located above the maze . Total time spent in the open arms was measured using EthoVision XT software . Social interaction At P21 , test pups were habituated to the arena for 3 min . Subsequently , WT females in estrous , unfamiliar to the pups , were added to the cage and both mice were allowed to interact with each other for another 3 min in a well - lit room . The interaction between the pups and the females was recorded using a computer - linked video camera ( The Imaging Source ) located above the arena . Videos were manually scored , and interaction recorded when both mice were within 2 cm of each other , not including tail - tail interactions . At P60 , only female mice were tested for social interaction . In this case the unknown WT females were not required to be in oestrus . To determine which females were in oestrus , vaginal smears were stained with Giemsa solution ( Polysciences inc . ) ( Caligioni 2009 ) prior to the experiment . 24 - hour activity P60 experimental mice were placed in individual clear boxes ( 40 cm x 24 cm x 18 cm ) and let to roam free for 24 h with ad libitum access to food and water and their normal 12 h light / dark cycle . Three infrared beams traversed each cage at the bottom . Data were analyzed using the MED - PC® IV software suite and extracted using the MPC2XL program . The number of beams breaks in 24 h and in 1 - hour slots , as well as the total number of beam breaks during the light and dark periods were analyzed . Experimental design and Statistical analysis For all experiments , individual animals were considered the experimental unit and data obtained from each animal was averaged if more than one quantification was performed ( for example when analysing several brain slices from the same animal ) . Experimenters were blind to the genotype of the animals until all quantification or scoring was completed . Statistical analysis was performed using IBM SPSS Statistics® 25 software ( cortical lamination analysis ) or R software ( behavior ) , version 3 . 6 . 2 . ( R Core Team 2019 ) . Normality of the data was tested using the Shapiro - Wilk test and homogeneity of variance was assessed with Levene’s test . If either assumption was violated an appropriate non - parametric test was used . For comparison of more than two groups , analysis of variance ( ANOVA ) was used for normal data and Kruskal - Wallis if the assumption of normality was not met . If only the assumption of homogeneity of variance was not met , a Welch’s ANOVA test was used . Post - hoc test following ANOVA was adjusted according to Tukey’s HSD or , in the case of the social interaction analysis , Dunnet’s test . Kruskal - Wallis was followed by Dunn’s correction and Welch’s ANOVA was followed by Games - Howell correction . All statistical data are presented as mean ± SEM . RESULTS Pcdh19 is expressed by different subtypes of cortical projection neurons and interneurons Previous RNA in situ hybridization ( ISH ) studies have shown two main areas of Pcdh19 expression in the cortex , corresponding to the upper regions of layer V ( layer Va ) and II / III ( Hertel and Redies 2010 ; Pederick et al . 2016 ) . However , a detailed analysis of the cortical neuronal subtypes expressing Pcdh19 , an important consideration given the cellular diversity of the cortex , is still lacking . ISH against Pcdh19 was combined with immunohistochemistry ( IHC ) against several cortical markers for principal neurons and interneurons in the somatosensory cortex at postnatal days 10 ( P10 ) and P20 , respectively ( Fig . 1 A - D ) . At P10 , Pcdh19 + cells were found to co - express markers for layer IV neurons ( RORB , Fig . 1A ) , callosal projection neurons ( SATB2 , Fig . 1B ) , corticospinal neurons ( CTIP2 , Fig . 1B ) , and corticothalamic neurons ( TBR1 , Fig . 1C ) . Strongest co - expression was seen in SATB2 + neurons , whereas RORB + cells showed weaker expression and in a smaller proportion of cells . CTIP2 + neurons with strong Pcdh19 expression tended to be located in the upper half of layer V , whereas TBR1 + cells co - expressing Pcdh19 did so at generally lower levels . At P20 , interneurons were identified co - expressing Pcdh19 with Parvalbumin in layers II / III and V ( Fig . 1D ) , as well as double positive cells for Calbindin and Pcdh19 ( data not shown ) . This approach is limited to available antibodies that withstand the harsh treatments needed for ISH . In addition , recent studies using single cell transcriptomics on mouse and human cortical tissue have revealed numerous subtypes of excitatory and inhibitory neurons that cannot be readily identified through immunostaining . In view of these limitations , we then turned to publicly available datasets of cortical single cell RNA expression and re - analyzed them to assess Pcdh19 expression in the mouse adult cortex ( Fig . 1 E , F ) . We chose the dataset published by Tasic et al in 2018 ( Tasic et al . 2018 ) , which includes 23 , 822 single - cell transcriptomes with cluster - assigned identity isolated from the primary visual ( VISp ) and anterior lateral motor ( ALM ) cortices of adult mice ( Mouse – V1 & ALM – SMART - seq , available at https : / / portal . brain - map . org / atlases - and - data / rnaseq ) . This study currently provides the most comprehensive classification of cortical cellular types to date with 133 cell types , of which 56 are glutamatergic , 61 GABAergic and 16 non - neuronal . In agreement with our P10 and P20 results , scRNAseq data shows that Pcdh19 expression is maintained in both excitatory and interneuronal cortical populations in the adult that co - express the markers of our ISH analysis ( Fig . 1E , F ) . Figure 1 . Pcdh19 is expressed by excitatory and inhibitory neurons in the mouse cortex . ( A - D ) Confocal micrographs of P10 ( A - C ) and P20 ( D ) cortical slices hybridized with an RNA probe against Pcdh19 ( red ) and antibodies against ( A ) RORB ( green ) , ( B ) SATB2 and CTIP2 ( green and blue , respectively ) , ( C ) TBR1 ( green ) and ( D ) Parvalbumin ( Pvalb , green ) . The left panel shows the entire cortical wall with boxes indicating the regions enlarged in the right panels . White arrowheads point to double positive cells , empty arrowheads point to single positive cells ( Pcdh19 negative ) . Scale bars : left panels , 100 μm ; right panels , 50 μm . ( E , F ) Violin plots representing gene expression and distribution for Pcdh19 and the markers used in ( A - D ) in the different excitatory ( E ) and interneuronal ( F ) subtypes defined in the Tasic et al 2018 study ( Allen Brain Atlas Mouse V1 & ALM Dataset ) . Dots indicate the median value of the cluster in CPM . CPM values are displayed on log10 scale . Pcdh19 expression is cluster - specific in all layers and , in general terms , within projection neurons Pcdh19 expression is most consistent in layer V cells , particularly in those projecting through the pyramidal tract ( PT ) and in the newly defined near - projecting population ( NP ) ( Fig . 1E ) . Intratelencephalically projecting neurons in layer V show lower expression levels in the primary visual cortex , but much higher levels in the anterior motor cortex , with the two L5 IT ALM Tmem163 subpopulations ( Dmrtb1 and Arhgap25 ) displaying the strongest expression of all characterized neuronal subtypes . Interestingly , one particular L5 IT ALM cluster is defined by Pcdh19 expression ( L5 IT ALM Gkn1 Pcdh19 ) , although it does not exhibit the highest expression levels for this gene and is also not included in any of the homology clusters with human neurons ( see below ) . Expression in layer II / III is lower on average , but some neuronal clusters ( L2 / 3 IT VISp Rrad , L2 / 3 IT VISp Adamts2 and L2 / 3 ALM Sla ) present equivalent expression levels as layer V cells , both in the visual and the motor cortex . Expression of Pcdh19 in layer VI neurons is negligible in the visual cortex , but stronger in corticothalamic and near - projecting cells in the anterior motor cortex . Expression in layer VIb follows a similar pattern ( Fig . 1E ) . Therefore , expression of Pcdh19 in excitatory neurons shows regional differences between brain areas , with generally higher expression in the anterior lateral motor cortex than in the primary visual cortex . As in the case of projection neurons , Pcdh19 expression in interneurons is strongly cluster - dependent . More specifically , strongest average expression is found in the Sncg and Pvalb subclasses and the Sst - Chodl type ; however , there is considerable variation and several Sst clusters also express Pcdh19 widely ( Fig . 1F ) . Sncg neurons are Vip + , Cck + multipolar or basket cells located mainly in upper layers , and 3 out of their 4 subtypes have consistent Pcdh19 expression . Only one other cluster of Vip interneurons , corresponding to bipolar or multipolar cells , also shows relevant Pcdh19 expression ( Vip Rspo4 Rxfp1 Chat ) . Within the Pvalb subclass , Pcdh19 is expressed by Chandelier cells ( Pvalb Vipr2 ) and several subtypes of basket cells ( Pvalb Gpr149 Islr , Pvalb Reln Tac1 and Pvalb Tpbg ) . Finally , within the Sst subclass , Pcdh19 expression is strongest in some subtypes of upper layer basket cells ( Sst Tac1 Htr1d , Sst Tac1 Tacr3 and Sst Nr2f2 Necab1 ) and Martinotti cells ( Sst Calb2 Necab1 and Sst Calb2 Pdlim5 ) , and in the long - range projecting population ( Sst - Chodl ) . In summary , our analysis demonstrates that mouse Pcdh19 is expressed by a heterogeneous neuronal population that includes a wide variety of cortical projection neurons and interneurons , with strong variation between neuronal subtypes . Expression in non - neuronal cells is very low ( data not shown ) . Human PCDH19 is also expressed in excitatory and inhibitory neurons The expression profile of human PCDH19 and a comparison with its murine counterpart is relevant with regards to the use of Pcdh19 mutant mice as a model to investigate the pathophysiology of the disorder caused by mutations in the human gene . Human cortical neurons have also been characterized at the transcriptional level using single cell RNAseq ( Lake et al . 2016 ; Hodge et al . 2019 ) . We therefore chose to analyze a dataset obtained from predominantly neuronal nuclei of the human middle temporal gyrus ( MTG ; Human – MTG – SMART - seq , available at https : / / portal . brain - map . org / atlases - and - data / rnaseq ) because it has been systematically compared to the mouse data described above ( Hodge et al 2019 ) . That comparison yielded 32 homologous neuronal clusters , allowing a meaningful comparison of the expression of PCDH19 between species ( Suppl . Fig . 1 ) . In this human dataset , PCDH19 expression in excitatory neurons is more prevalent in layer V neurons projecting through the pyramidal tract , whilst all other excitatory clusters show lower expression levels ( Fig . 2A ) . In contrast , different interneuronal types display strong PCDH19 expression , including several VIP and Parvalbumin clusters , as well as Chandelier cells ( Fig . 2A ) . When expression is compared between species across the 32 homology clusters , only Chandelier cells and layer V PT projecting neurons show strong expression in both human and mouse ( Fig . 2B ) . In addition , there is also strong expression in subpopulations of bipolar or multipolar interneurons in both species ( cluster VIP4 , mouse subtype Vip Rspo4 Rxtp1 Chat ; human subtype Inh L1 - 3 VIP - GGH ) , although the pooling of several subtypes into cluster VIP4 dilutes the overall expression . In general , PCDH19 expression in human neurons seems to be restricted to fewer cell types , while in the mouse , higher levels of expression are found in most excitatory clusters and in inhibitory clusters Pvalb2 , Sst2 , Sst3 , Sst4 , Sst5 , Sst Chodl and Vip Sncg . In human , neurons in clusters Lamp5 Lhx6 and VIP2 show higher expression levels than their murine counterparts . Figure 2 . PCDH19 is expressed by excitatory and inhibitory neurons in the human cortex . ( A ) Gene expression and distribution of PCDH19 in the Allen Brain Atlas human MTG dataset , represented by violin plots . ( B ) Comparison of gene expression and distribution between mouse and human neurons in the 32 homology clusters defined by Hodge et al in their 2019 study . For each cluster , human data are shown on the left ( black outline ) and mouse data on the right ( grey outline ) . Dots indicate the median value of the cluster in CPM . CPM values are displayed on log10 scale . As the mouse data presented above show that Pcdh19 expression vary between brain regions ( anterior lateral cortex and primary visual cortex in this case ) , we extended our analysis to a second human dataset from the Allen Brain Atlas , obtained from several different brain areas ( middle temporal gyrus , anterior cingulate gyrus , primary visual cortex , primary motor cortex , primary somatosensory cortex and primary auditory cortex ) that is also publicly available ( Human – Multiple Cortical Areas – SMART - seq , available at https : / / portal . brain - map . org / atlases - and - data / rnaseq ) . This dataset , which also includes the MTG data , comprises 49 , 417 cell nuclei , compared with 15 , 928 for the middle temporal gyrus alone . It has therefore allowed the definition of 56 excitatory and 54 inhibitory subtypes , as opposed to 23 and 41 , respectively , in the MTG dataset ( Suppl . Fig . 2 shows the correspondence between the clusters in the two datasets ) . In addition to analyzing expression levels across the whole dataset , using the metadata of individual nuclei , we separated the expression of PCDH19 in the different excitatory and inhibitory clusters by brain region ( Suppl . Fig . 3 and 4 ) . Our analysis confirmed that , as a whole , within excitatory neurons strongest and most consistent PCDH19 expression is found in FEZF2 subtypes in layer V or layers III - V , but it also revealed strong expression of PCDH19 in several other excitatory neuronal subtypes spanning layers II - V , particularly Exc L3 RORB CARTPT , Exc L3 - 4 RORB FOLH1B , Exc L4 - 5 RORB LCN15 and Exc L5 RORB SNHG7 ( Suppl . Fig . 3 ) . In addition , low expression is evident in many other excitatory neurons of layers III - VI , although several layer IV clusters , particularly in sensory regions , tend to express much lower levels of PCDH19 , as was the case in the mouse . Regarding interneurons , PCDH19 expression is highest in the L3 - 6 VIP KCTD13 subtype , with strong expression in all cells . In addition , PCDH19 is also relatively highly expressed in several other VIP , LAMP5 , SST and PVALB subpopulations ( Suppl . Fig . 4 ) . A comparison between different brain regions reveals that , in general , PCDH19 is expressed in each cluster at similar levels across areas . However , there are some exceptions , like L1 VIP PCDH20 interneurons , which show much higher PCDH19 expression in the visual cortex ( V1C ) than in somatosensory areas ( S1lm and S1ul ) or L1 - 2 VIP RPL41P3 , with higher PCDH19 expression in motor areas ( Suppl . Fig . 4 ) . Overall , although in a direct comparison of the 32 homology clusters PCDH19 expression seemed to be more widespread in mouse than in human neurons , this was probably due to the particular brain region sampled . The larger and more diverse human sample allows a more fine - grained neuronal classification , revealing more prevalent expression of PCDH19 among excitatory and inhibitory neurons and suggesting some regional variations between cortical areas in the human brain , as seen also in the mouse brain . No obvious defects in axonal tracts in Pcdh19 mutant animals Our results indicate that Pcdh19 is expressed in cortical projection neurons that project through the corpus callosum ( layer II - III and some layer V neurons ) , as well as in neurons projecting outside the cortex , mainly through the pyramidal tract ( layer V PT neurons ) . Although several members of the cadherin superfamily , including delta protocadherins 7 , 10 , 17 and 18 , have been shown to play a role in axonal outgrowth ( Uemura et al . 2007 ; Piper et al . 2008 ; Hayashi et al . 2014 ) , fasciculation ( Williams et al . 2011 ; Hayashi et al . 2014 ) and arborization ( Biswas et al . 2014 ) , it is not known whether mutations in Pcdh19 have an impact on any of these processes . We therefore conducted a general characterization of axonal tracts in Taconic Pcdh19 WT , KO and HET animals by immunostaining against the cell adhesion molecule L1CAM ( Fig . 3A ) . No differences were apparent between genotypes in the major axonal tracts , including the internal capsule , stria terminalis , fimbria or corpus callosum . Next , we analyzed the corpus callosum in more detail by labelling dorsally located axons with Neuropilin - 1 , which allows the analysis of topographical organization at the midline . Again , the dorso - ventral extension of the corpus callosum and the dorsal restriction of Neuropilin - 1 expressing axons was similar between all genotypes ( Fig . 3B - D ) ( D - V extension : Kruskal - Wallis , H = 2 . 86 , P = 0 . 2635 , df = 2 ; dorsal restriction : Kruskal - Wallis , H = 0 . 07 , P = 0 . 9783 , df = 2 ) . Thus , our results revealed no major abnormalities in the main axonal tracts , although they do not preclude the existence of more subtle defects that would require a more detailed analysis to be revealed . Figure 3 . No major anomalies in the main axonal tracts in Pcdh19 mouse mutants . ( A ) Confocal micrographs of P0 - P1 mouse hemispheres stained with anti - L1CAM ( red ) . Nuclei were counterstained with DAPI ( blue ) . ( B ) Confocal micrographs of the corpus callosum of P0 - P1 mice stained with anti - L1CAM ( red ) , anti - Neuropilin - 1 ( green ) and counterstained with DAPI ( blue ) . ( C ) Quantification of the dorso - ventral extension of the corpus callosum in WT and mutant animals . ( D ) Quantification of the dorsal restriction of Neuropilin - 1 positive axons in WT and mutant animals . All results are indicated as mean ± SEM . 2 images per brain , obtained from four animals originating from three different litters were analyzed for each condition . Cx , cortex ; Hip , hippocampus ; Th , thalamus , fi , fimbria ; st , striatum ; ic , internal capsule ; Cg , cingulate cortex ; cc , corpus callosum ; hc , hippocampal commissure . Scale bars : 200 µ m ( A ) and 50 µ m ( B ) . Cortical lamination is largely preserved in Pcdh19 mutant animals Although no major lamination defects have been described in Pcdh19 mutant animals ( Pederick et al . 2016 ; Hayashi et al . 2017 ) , no detailed quantitative studies have been performed so far that could reveal subtle changes in layer composition . Given that Pcdh19 is expressed in projection neurons and interneurons , we performed an analysis with markers for both neuronal populations . We first selected cortical markers for different types of projection neurons ( CUX1 , SATB2 , RORB , CTIP2 and TBR1 ) and performed immunohistochemistry in the somatosensory cortex ( SSC ) at P10 , once radial migration is completed ( Fig . 4A ) . For each marker , we determined the proportion of positive cells , as well as their distribution within 10 bins covering the whole width of the cortical plate ( Fig . 4J ) . In accordance with previous reports ( Pederick et al . 2016 ) , we found no differences in cortical width between genotypes ( WT = 1321 . 09 ± 21 . 59 μm , HET = 1354 . 23 ± 33 . 78 μm and KO = 1305 . 41 ± 30 . 62 μm ; Fig . 4B ) . The proportion of positive neurons for all five examined markers was also unaltered ( Fig . 4C ) . CUX1 + cells made up approximately one fifth of all DAPI + cells ( WT = 21 . 45 ± 1 . 2 % , HET = 22 . 36 ± 1 . 65 % , KO = 24 . 82 ± 2 . 13 % ) and SATB2 + cells represented more than half of all cells ( WT = 59 . 08 ± 0 . 92 % , HET = 58 . 08 ± 3 . 62 % , KO = 57 . 82 ± 2 . 74 % ) . The proportion of RORB + cells seemed lower in KO brains , especially when compared to WT ( WT = 27 . 73 ± 1 . 75 % , HET = 24 . 43 ±2 . 5 % , KO = 18 . 98 ± 3 . 7 % ) , but statistical analysis revealed this difference was not significant ( one way ANOVA , main effect of genotype F ( 2 , 9 ) = 2 . 39 , P = 0 . 1468 ) . CTIP2 + cells represented about 13 % in all three genotypes ( WT = 13 . 33 ± 0 . 46 % , HET = 13 . 82 ± 1 . 32 % , KO = 13 . 61 ± 1 . 14 % ) and TBR1 + cells added up to approximately one third of all cells ( WT = 33 . 65 ± 2 . 92 % , HET = 34 . 03 ± 2 . 64 % , KO = 38 . 51 ± 1 . 7 % ) . Furthermore , the distribution of SATB2 + , RORB + and CTIP2 + neurons between the 10 bins was unchanged ( Fig . 4D - G ) . However , we detected some subtle deviations in the distribution of CUX1 + and TBR1 + neurons , in both cases regarding bin 5 ( Fig . 4D , E , H - J ) . PCDH19 - HET animals showed a significant 2 . 4 - fold reduction in the percentage of CUX1 + neurons in this bin compared to wild types ( WT = 2 . 08 ± 0 . 18 % , HET = 0 . 86 ± 0 . 27 % , KO = 1 . 14 ± 0 . 32 % ; one way ANOVA , main effect of genotype F ( 2 , 9 ) = 5 . 81 , P = 0 . 0239 ; Tukey : q ( 1 , 9 ) = 4 . 60 , P = 0 . 0245 HET vs WT ) . Conversely , the percentage of TBR1 + cells in this bin was increased in HET brains when compared to KO brains ( WT = 4 . 97 ± 0 . 71 % , HET = 7 . 46 ± 0 . 35 % and KO = 4 . 34 ± 0 . 41 % ; Kruskal - Wallis , H = 8 . 0 , P = 0 . 0048 , df = 2 ; post - hoc Dunn : Z = 2 . 75 , P = 0 . 0181 HET vs KO ) . Figure 4 . Minor changes in the distribution of cortical excitatory neurons in Pcdh19 mutant animals . ( A ) Diagram indicating the position of the cortical area analyzed ( primary somatosensory cortex , SSp ) . ( B ) Quantification of cortical width at P10 in Pcdh19 WT , HET and KO animals . ( C ) Relative percentages of the different cortical markers examined with respect to total DAPI + cells . ( D ) Representative confocal micrographs of immunohistochemistry with anti - CUX1 ( red ) and anti - CTIP2 ( green ) antibodies on WT , HET and KO tissue . ( E ) Quantification of the percentage of marker + cells in each of 10 equal bins spanning the cortical wall . ( F ) Representative confocal micrographs of immunohistochemistry with anti - RORB ( red ) and anti - SATB2 ( green ) antibodies on WT , HET and KO tissue . ( G ) Distribution of marker + cells in each of 10 equal bins spanning the cortical wall , shown as percentage . ( H ) Representative confocal micrographs of immunohistochemistry with anti - TBR1 ( red ) antibodies on WT , HET and KO tissue . Nuclei are counterstained with DAPI ( blue ) . ( I ) Quantification of the percentage of marker + cells in each of 10 equal bins spanning the cortical wall . ( J ) Confocal micrograph of the SSp counterstained with DAPI ( blue ) , indicating the correspondence between cortical layers and the bins used for quantification . All results are indicated as mean ± SEM . A minimum of 3 images per brain , obtained from four animals originating from three different litters were analyzed for each condition . * P < 0 . 05 . Scale bars : 200 μm . To complete our analysis on cortical composition and lamination , we stained the SSC with four different interneuronal markers ( SST , PVALB , CB , and CR ) in P20 brains . As before , cortical thickness showed no difference between genotypes ( WT = 1457 . 18 ± 32 . 71 μm , HET = 1402 . 97 ± 42 . 92 μm and KO = 1387 . 02 ± 9 . 88 μm ; Fig . 5A ) and we did not detect any significant changes in the proportion of marker positive neurons ( Fig . 5B ) . The most abundant type was CB + cells ( WT = 19 . 11 ± 1 . 04 % , HET = 16 . 20 ± 1 . 21 % , KO = 18 . 77 ± 0 . 20 % ) , whereas PVALB + , SST + and CR + accounted for less than 5 % of DAPI + cells each ( PVALB : WT = 2 . 99 ± 0 . 28 % , HET = 3 . 7 ± 0 . 2 % , KO = 3 . 15 ± 0 . 21 % ; SST : WT = 2 . 22 ± 0 . 24 % , HET = 1 . 33 ± 0 . 11 % , KO = 1 . 61 ± 0 . 33 % ; CR : WT = 1 . 61 ± 0 . 42 % , HET = 1 . 14 ± 0 . 04 % , KO = 0 . 98 ± 0 . 1 % ) . Because the absolute number of neurons counted for those three markers in the SSC was very low , we extended our analysis to include the adjacent motor cortex and compared the number of neurons positive for each marker , rather than the percentage over DAPI . No differences in the number of neurons were found in this analysis ( Fig . 5C ) . Regarding cellular distribution in the SSC , no differences were apparent for CR + and PVALB + cells . However , we detected small but significant changes in the distribution of CB + and SST + interneurons between HET and KO brains ( Fig . 5D - G ) . HET brains showed a 1 . 5 - fold increase in the percentage of CB + cells in bin 7 when compared to KO brains ( WT = 2 . 17 ± 0 . 17 % , HET = 2 . 95 ± 0 . 26 % , KO = 1 . 98 ± 0 . 26 % ; one way ANOVA , main effect of genotype F ( 2 , 9 ) = 5 . 04 , P = 0 . 034 ; Tukey : q ( 1 , 9 ) = 4 . 23 , P = 0 . 0366 HET vs KO ) , and KO brains displayed a 1 . 7 - fold increase over HET brains in the percentage of SST + cells in bin 5 ( WT = 11 . 21 ± 1 . 34 % , HET = 10 . 58 ± 1 . 62 % , KO = 17 . 71 ± 2 . 12 ; one way ANOVA , main effect of genotype F ( 2 , 9 ) = 5 . 26 , P = 0 . 0307 ; Tukey : q ( 1 , 9 ) = 4 . 14 , P = 0 . 0403 HET vs KO ) . In summary , the relative neuronal proportions and distribution are mostly normal in the SSC of Pcdh19 mutant animals , although subtle differences in distribution cannot be ruled out for particular neuronal types . Figure 5 . Subtle changes in the distribution of inhibitory neurons in the cortex of Pcdh19 mutant animals . ( A ) Quantification of cortical width at P20 in Pcdh19 WT , HET and KO animals . ( B ) Relative percentages of the different cortical markers examined with respect to total DAPI + cells in the somatosensory cortex . ( C ) Absolute numbers of marker + cells in the somatosensory cortex ( SSC ) and adjacent motor cortex ( MC ) . ( D ) Representative confocal micrographs of immunohistochemistry with anti - Calbindin ( CB , red ) and anti - Somatostatin ( SST , green ) antibodies on WT , HET and KO tissue . Inserts : high magnification of SST + cells . Nuclei were counterstained with DAPI ( blue ) . ( E ) Quantification of the percentage of marker + cells in each of 10 equal bins spanning the cortical wall . ( F ) Representative confocal micrographs of immunohistochemistry with anti - Parvalbumin ( Pvalb , red ) and anti - Calretinin ( CR , green ) antibodies on WT , HET and KO tissue . ( G ) Distribution of marker + cells in each of 10 equal bins spanning the cortical wall , shown as percentage . All results are indicated as mean ± SEM . A minimum of 3 images per brain , obtained from four animals originating from three different litters were analyzed for each condition . * P < 0 . 05 . Scale bars : 200 μm ; insets : 50 μm . Altered behavior in Pcdh19 mutant animals and their littermates While correct cortical lamination is obviously important for proper brain function , the lack of any major lamination defects in the cortex of Pcdh19 mutant animals does not preclude connectivity defects due to PCDH19 mosaicism that could become apparent at the behavioral level . As part of our characterization of the Taconic Pcdh19 mutant mice , we also carried out a series of tests to determine whether these animals present any behavioral alterations . The paradigms included open field to evaluate general locomotor activity , anxiety and exploratory behavior , elevated plus maze to measure anxiety , and a social interaction test . We assessed animals at preweaning age and as adults , to account for any developmental effects . In addition to the WT littermates that Pcdh19 mutant animals were housed with , we included a further control of single genotype housed WT animals ( WT SGH ) ( Fig . 6A ) . Indeed , we note that a previous study on the X - linked ASD - related gene Nlgn3 , also a membrane protein expressed in the developing cerebral cortex , revealed that housing conditions impact the behavior of wild - type animals when housed together with mutant animals ( Kalbassi et al . 2017 ) . The parents of the animals used to analyze behavior in the single genotype housed WT condition originated from our Pcdh19 colony and behavior was analyzed separately for male and female mice . Differences in male behavior were evident at P21 ( Fig . 6B - E ) . Overall distance travelled during the 20 min open field paradigm was significantly different between genotypes ( one way ANOVA , main effect of genotype F ( 2 , 72 ) = 5 . 02 , P = 0 . 0091 ) , with mixed genotype housed WT animals ( WT MGH ) showing a 23 % increase compared to WT SGH ( Tukey : q ( 1 , 72 ) = 4 . 48 , P = 0 . 0063 ; Fig . 6B ) . An analysis by 5 - minute slots showed that the increased distance travelled by WT MGH males was due to increased activity during the first 5 minutes ( Kruskal - Wallis , H = 9 . 35 , P = 0 . 0093 , df = 2 ; post - hoc Dunn : Z = 3 . 01 , P = 0 . 0079 WT MGH vs WT SGH ; Fig . 6C ) . This effect disappeared thereafter ( Fig . 6C ) and also when animals were tested again at ³ P60 ( Total distance : one way ANOVA , main effect of genotype F ( 2 , 68 ) = 1 . 13 , P = 0 . 329 ; First 5 minutes : one way ANOVA , main effect of genotype F ( 2 , 68 ) = 1 . 31 , P = 0 . 2759 , Fig . 6B and Suppl . Fig . 5A ) . In accordance with these results , spontaneous activity ( number of beam breaks ) over a 24h period in adult male mice did not differ significantly between conditions ( Suppl . Fig . 5B , C ) , neither when analyzed in total ( one way ANOVA , main effect of genotype F ( 2 , 34 ) = 0 . 48 , P = 0 . 621 ) , nor in the light ( one way ANOVA , main effect of genotype F ( 2 , 34 ) = 3 . 03 , P = 0 . 0615 ) or dark periods ( one way ANOVA , main effect of genotype F ( 2 , 34 ) = 0 . 31 , P = 0 . 733 ) . There were , however , differences at individual timepoints ( 19 : 00 , 20 : 00 , 8 : 00 and 10 : 00 , Suppl . Fig . 5C ) . KO animals were less active during the start of the dark phase ( 19 : 00 , Kruskal - Wallis , H = 16 . 08 , P = 0 . 0003 , df = 2 ; post - hoc Dunn : Z = 4 . 01 , P = 0 . 0002 KO vs WT MGH ; 20 : 00 , one way ANOVA , main effect of genotype F ( 2 , 34 ) = 5 . 18 , P = 0 . 0109 ; post hoc Tukey : q ( 1 , 34 ) = 4 . 42 , P = 0 . 0099 HET vs KO vs WT SGH ) and more active at 10 : 00 ( 10 : 00 , Kruskal - Wallis , H = 10 . 78 , P = 0 . 0046 , df = 2 ; post - hoc Dunn : Z = 3 . 11 , P = 0 . 0056 KO vs WT MGH ; Z = 2 . 62 , P = 0 . 0267 KO vs WT SGH ) . WT MGH males were less active at 8 : 00 during the light period ( 8 : 00 , Kruskal - Wallis , H = 7 . 17 , P = 0 . 0277 , df = 2 ; post - hoc Dunn : Z = 2 . 51 , P = 0 . 0361 WT MGH vs KO ) . Nevertheless , those differences do not seem to point to an overall activity defect and might be due to a smaller number of animals being tested . Figure 6 . Behavioral alterations in Pcdh19 KO males and their WT littermates . ( A ) Schematic of the behavioral experiments carried out . ( B ) Total distance travelled by males during the 20 minutes of the open field test at P21 and P60 . ( C ) Distance travelled in the open field by P21 males , split in 5 - minute intervals . ( D ) Total time spent by males in the open arms of the elevated plus maze during the 5 - minute test at P21 and P60 . ( E ) Time spent by P21 males interacting with a non - familiar female in oestrus . The total duration of the test was 5 minutes . Numbers of tested animals were : 26 WT SGH , 18 WT MGH , 31 KO at P21 and 24 WT SGH , 18 WT MGH , 29 KO at P60 . Open field results correspond to the second day of testing at each age . Results are indicated as mean ± SEM , * P < 0 . 05 ; * * P < 0 . 01 . WT SGH , single genotype housed WT animals ; WT MGH , mixed genotype housed animals . To investigate whether the increased distance travelled by pre - weaned mixed genotype housed WT animals in the first 5 minutes of the open field could be due to increased anxiety , we analyzed the time spent in the center of the arena . No differences were found between the three conditions , neither at P21 ( Kruskal - Wallis , H = 2 . 76 , P = 0 . 2518 , df = 2 ) , nor at P60 ( Kruskal - Wallis , H = 3 . 58 , P = 0 . 1671 , df = 2 , Suppl . Fig . 5D ) . The results of the elevated plus maze confirmed the lack of differences at P21 ( Kruskal - Wallis , H = 4 . 57 , P = 0 . 1016 , df = 2 , Fig . 6D ) . However , this was not the case for adult animals , as adult KO males spent over 40 % more time in the open arms than their WT MGH littermates and WT SGH controls , indicating reduced anxiety ( one way ANOVA , main effect of genotype F ( 2 , 68 ) = 6 . 88 , P = 0 . 0019 ; Tukey : q ( 1 , 68 ) = 4 . 10 , P = 0 . 0138 KO vs WT MGH and q ( 1 , 68 ) = 4 . 68 , P = 0 . 0042 KO vs WT SGH ; Fig . 6D ) . Interestingly , we also detected a difference in social behavior at P21 ( one way ANOVA , main effect of genotype F ( 2 , 72 ) = 3 . 39 , P = 0 . 039 , Fig . 6E ) . In this case , WT MGH males spent significantly less time ( 19 % decrease ) interacting with an unfamiliar female in estrous than single - genotype housed WT males ( Dunnett : q ( 1 , 72 ) = 2 . 37 , P = 0 . 0382 WT MGH vs WT SGH ) . KO males also showed a trend towards reduced interaction ( 14 % decrease ) , but this difference was not significant ( Dunnett : q ( 1 , 72 ) = 2 . 07 , P = 0 . 0771 HET vs WT SGH ) . Changes in behavior were more pronounced in female mice than in their male counterparts . We found again differences in the total distance travelled during the open field test at P21 ( one way ANOVA , main effect of genotype F ( 2 , 69 ) = 9 . 54 , P = 0 . 0002 , Fig . 7A ) , with HET and WT MGH females displaying an increase of 35 % and 19 % , respectively , when compared with single - genotype housed controls ( Tukey : q ( 1 , 69 ) = 6 . 17 , P = 0 . 0001 for HET vs WT SGH and q ( 1 , 69 ) = 3 . 55 , P = 0 . 0382 for WT MGH vs WT SGH ) . Unlike in males , this effect was maintained at P60 , but only in HET females , which travelled on average 19 % more distance than WT SGH animals ( one way ANOVA , main effect of genotype F ( 2 , 69 ) = 3 . 99 , P = 0 . 0229 ; Tukey : q ( 1 , 69 ) = 3 . 87 , P = 0 . 0214 for HET vs WT SGH , Fig . 7A ) . Analysis by 5 - minute intervals showed that the increase in total distance was mainly due to increased activity during the first 5 minutes in the open field arena both at preweaning age and in adults ( P21 : Kruskal - Wallis , H = 21 . 86 , P < 0 . 0001 , df = 2 ; P60 : one way ANOVA , main effect of genotype F ( 2 , 69 ) = 17 . 95 , P < 0 . 0001 , Fig . 7D - E ) . This effect was significant at both ages for HET females and their WT siblings when compared with single - genotype housed females , with increases of 95 % ( HET ) and 54 % ( WT MGH ) at P21 , and 53 % ( HET ) and 49 % ( WT MGH ) in adult animals ( P21 : Dunn : Z = 4 . 61 , P < 0 . 0001 HET vs WT SGH and Z = 3 . 12 , P = 0 . 0055 WT MGH vs WT SGH ; P60 : Tukey : q ( 1 , 69 ) = 7 . 38 , P < 0 . 0001 HET vs WT SGH and q ( 1 , 69 ) = 7 . 43 , P < 0 . 0001 for WT MGH vs WT SGH , Fig . 7D - E ) . HET females also travelled a significantly longer distance than WT SGH females during the second 5 - minute interval at P21 , although the increase ( 25 % ) was much smaller in this case ( one way ANOVA , main effect of genotype F ( 2 , 69 ) = 3 . 29 , P = 0 . 0432 ; Tukey : q ( 1 , 69 ) = 3 . 58 , P = 0 . 0359 HET vs WT SGH ) . Similarly to male mice , the spontaneous activity over 24h , measured as the number of beam breaks , was not altered for any of the three experimental groups in the light ( one way ANOVA , main effect of genotype F ( 2 , 36 ) = 2 . 29 , P = 0 . 1159 ) , dark ( one way ANOVA , main effect of genotype F ( 2 , 36 ) = 1 . 10 , P = 0 . 3429 ) or total periods ( one way ANOVA , main effect of genotype F ( 2 , 36 ) = 1 . 08 , P = 0 . 3512 ) ( Suppl . Fig . 6A , B ) . Again , isolated differences were evident at two timepoints during the dark phase ( 22 : 00 , one way ANOVA , main effect of genotype F ( 2 , 36 ) = 3 . 84 , P = 0 . 0309 , Tukey : q ( 1 , 69 ) = 3 . 65 , P = 0 . 0364 WT MGH vs WT SGH ; 4 : 00 , Welch’s ANOVA W ( 2 , 23 . 61 ) = 8 . 52 , P = 0 . 0016 , Dunnett T3 : t ( 2 , 18 . 32 ) = 3 . 83 , P = 0 . 0036 WT MGH vs HET ; Dunnett T3 : t ( 2 , 23 . 41 ) = 2 . 65 , P = 0 . 0417 WT MHG vs WT SGH , Suppl . Fig . 6B ) , but no overall changes in activity were apparent in this test . Figure 7 . Behavioral alterations in Pcdh19 HET females and their WT littermates . ( A ) Total distance travelled by females during the 20 minutes of the open field test at P21 and P60 . ( B ) Total time spent by females in the open arms of the elevated plus maze during the 5 - minute test at P21 and P60 . ( C ) Time spent by females interacting with a non - familiar female at P21 and P60 . The total duration of the test was 5 minutes . ( D ) Distance travelled in the open field by P21 females , split in 5 - minute intervals . ( E ) Distance travelled in the open field by P60 females , split in 5 - minute intervals . Numbers of tested animals were : 22 WT SGH , 29 WT MGH , 21 HET at P21 and P60 . Open field results correspond to the second day of testing at each age . Results are indicated as mean ± SEM , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . WT SGH , single genotype housed WT animals ; WT MGH , mixed genotype housed animals . Since the increase in distance travelled during the first 5 minutes in the open field test does not seem to be caused by overall hyperactivity of HET animals and their WT siblings , we again analyzed anxiety - related behaviors in these animals . There were no differences in the time spent in the center of the open field arena for any of the conditions at P21 ( Kruskal - Wallis , H = 4 . 68 , P = 0 . 0962 , df = 2 ) or P60 ( Kruskal - Wallis , H = 4 . 09 , P = 0 . 1296 , df = 2 ; Suppl . Fig . 6C ) , but , similar to the results obtained with male animals , HET females spent significantly more time in the open arms of the elevated plus maze than any of the WT females at P21 ( Kruskal - Wallis , H = 20 . 94 , P < 0 . 0001 , df = 2 ; Dunn : Z = 3 . 19 , P = 0 . 042 between HET and WT SGH and Z = 4 . 49 , P < 0 . 0001 between WT MGH and WT SGH ) and P60 ( one way ANOVA , main effect of genotype F ( 2 , 69 ) = 5 . 95 , P = 0 . 0041 ; Tukey : q ( 1 , 69 ) = 4 . 67 , P = 0 . 0043 for HET vs WT SGH and q ( 1 , 69 ) = 3 . 72 , P = 0 . 0281 between HET and WT MGH ) . The increases against WT SGH and WT MGH amounted to 76 % and 103 % at preweaning age and 60 % and 39 % in adults , indicating reduced anxiety , as seen also for adult male KO animals ( Fig . 7B ) . As in the case of male mice , the social interaction test revealed differences between single and mixed genotype housed WT females ( Fig . 7C ) . However , this effect was only present in adult animals , with WT MGH females spending 15 % less time interacting with an unfamiliar female in estrous ( One way ANOVA , main effect of genotype F ( 2 , 69 ) = 3 . 38 , P = 0 . 0398 ; Dunnett : q ( 1 , 69 ) = 2 . 32 , P = 0 . 0432 WT MHG vs WT SGH ) . Overall , we found significant behavioral differences between wild type and mutant animals that were generally more pronounced in HET females than in KO males . Importantly , we also uncovered an effect of housing on the behavior of WT animals . DISCUSSION Although recent studies have shed light into the different functions of PCDH19 ( Pederick et al . 2016 ; Hayashi et al . 2017 ; Pham et al . 2017 ; Bassani et al . 2018 ; Homan et al . 2018 ; Pederick et al . 2018 , reviewed in Gerosa et al . 2019 ; Gécz and Thomas 2020 ) , just exactly how mutations in this cell adhesion protein lead to the distinct symptoms associated with EIEE9 is still not well understood . Here we present a detailed analysis of neuronal sub - types expressing Pcdh19 / PCDH19 in the cortex of mice and humans . Our study reveals that Pcdh19 / PCDH19 is not only expressed in pyramidal neurons , but also in different types of interneurons , and that , in general , higher expression is limited to specific subpopulations in both cases . Our analysis also rules out major anomalies in the main axonal tracts and provides a quantitative assessment of cortical composition and lamination . Despite the lack of major lamination defects , out data suggest the possibility of subtle defects in layer composition that could contribute to the pathophysiology of EIEE9 . Indeed , mutant animals display behavioral alterations in the open field ( females ) and elevated plus maze tests ( males and females ) . Importantly , and as previously revealed with the analysis of Nlgn3 mutants ( Kalbassi et al . 2017 ) , the Pcdh19 mutation affects the behavior of wild - type littermates when housed in the same cage . Hitherto , the characterization of the neuronal populations expressing PCDH19 has been hindered by the lack of specific antibodies that perform satisfactorily in immunohistochemistry analyses . In addition , as PCDH19 is likely distributed in both axons and dendrites ( Pederick et al . 2016 ; Hayashi et al . 2017 ; Bassani et al . 2018 ) , the unambiguous identification of cell bodies expressing PCDH19 is a challenging objective , as is the case for most membrane proteins in the cortex . To overcome this challenge , we focused on the expression of Pcdh19 mRNA , which is detected in the cell soma and allows a better assessment of co - expression with other neuronal markers , which tend to be either nuclear or cytoplasmic . Although mRNA and protein expression are not necessarily correlated , available data show a good match between the regions with strongest mRNA and protein signals ( Hayashi et al . 2017 ; Pederick et al . 2018 ) . Our ISH / IHC combination approach provides experimental evidence for the expression of Pcdh19 by different neuronal types across cortical layers , but it is inherently restricted to a particular brain region ( in this case the SSC ) and a few neuronal markers . However , the availability of datasets from scRNAseq studies , mainly carried out by the Allen Brain Atlas ( https : / / portal . brain - map . org / atlases - and - data / rnaseq ) , allowed us to conduct a much more in depth expression analysis , not only in the mouse , but also in the human brain . Our analysis of the mouse dataset published by Tasic et al in 2018 ( Tasic et al . 2018 ) confirmed that Pcdh19 is expressed by a majority of excitatory neurons in Layer V , projecting both intra - and extra - cortically , as well as by certain subtypes of Layer II / III projection neurons , in agreement with the ISH data . Expression in layer IV is minimal , and in layers VI and VIb it is mostly restricted to a couple of neuronal subtypes in the ALM . In interneurons , expression is widespread in the Sncg cluster , subtype specific in the Vip , Sst and Pvalb clusters , and very low in the Lamp5 and Serpinf1 clusters . These results demonstrate that while Pcdh19 is expressed by a variety of excitatory and inhibitory neurons , expression remains specific for particular subtypes . This subtype specificity would suggest a role for PCDH19 in the establishment of neuronal circuits . Human PCDH19 follows a similar pattern , with expression in both excitatory and inhibitory neuronal types . We initially chose the MTG dataset to be able to establish a direct comparison between mouse and human neurons ( Hodge et al . 2019 ) . This comparison revealed shared high expression in Layer V excitatory neurons projecting through the pyramidal tract and in Chandelier cells . It also suggested that Pcdh19 expression in mouse seems to be more widespread than in human , especially regarding excitatory neurons . However , PCDH19 expression became much more apparent in the analysis of the Multiple Cortical Areas dataset that expanded the excitatory neuronal subtypes from 23 to 56 . This probably reflects the splitting of subtypes that contained different PCDH19 expressing populations into new subtypes in which PCDH19 expressing cells cluster together . Nevertheless , expression in human excitatory neurons still seems to be more graded , with many more subtypes showing intermediate expression levels than in mouse . A similar picture emerges regarding expression in interneurons . High PCDH19 expression can be found in subtypes of LAMP5 , VIP , SST and PVALB interneurons , although most subtypes in humans seem to display some level of expression . One main difference between mouse and human , though , is the much lower expression of PCDH19 in long range projecting interneurons in humans ( Inh L3 - 6 SST NPY in the MTG dataset , Inh L6 SST NPY in the Multiple Cortical Areas dataset , Sst Chodl in the mouse dataset ) . The functional relevance of Pcdh19 / PCDH19 expression in particular neuronal subtypes will need to be established experimentally , but we expect our results to provide a framework to support those functional studies in the future , not least because of regional differences in the expression of this gene within neuronal subtypes . Although two different Pcdh19 KO models have been generated and published to date ( Pederick et al . 2016 ; Hayashi et al . 2017 ) , no detailed quantitative characterization of cortical composition and lamination has been reported . We have quantified 5 excitatory and 4 inhibitory markers , looking at overall abundance , as well as distribution throughout the cortical plate in the somatosensory cortex . In the case of parvalbumin , somatostatin and calretinin , the adjacent motor cortex was included in the study , due to the very low numbers of positive cells , whereas calbindin expressing neurons were much more abundant , especially in the upper layers , reflecting a population of excitatory neurons that also express this marker ( van Brederode et al . 1991 ) . Our analysis reveals no differences in the abundance of the different neuronal types and confirms the lack of major lamination defects ( Pederick et al . 2016 ; Hayashi et al . 2017 ) . However , our quantitative approach exposes more subtle changes in the distribution of certain neuronal types , suggesting the possibility of a slightly altered composition of particular layers or sublayers . The origin of these differences is unknown , but one possibility is that they could arise as a consequence of altered neurogenesis , since PCDH19 has been shown to play a role in this process ( Fujitani et al . 2017 ; Homan et al . 2018 ; Lv et al . 2019 ) . It is also important to consider that we carried out our analysis mainly in the SSC , but given that Pcdh19 expression varies between cortical regions , it is possible that different areas might be affected in different ways by a total or partial loss of PCDH19 . Reports of focal cortical dysplasia in EIEE9 patients ( Kurian et al . 2018 ; Pederick et al . 2018 ) and focal areas of disorganization in ASD patients ( Stoner et al . 2014 ) seem to support this possibility . Despite the involvement of other delta protocadherins in the development of axonal tracts ( Uemura et al . 2007 ; Piper et al . 2008 ; Biswas et al . 2014 ; Hayashi et al . 2014 ) , our data do not support a major role of PCDH19 in this process . We did not detect any alterations in the main axonal tracts in the brain after staining for the axonal protein L1CAM , and a more detailed analysis of the corpus callosum also showed no differences in its dorso - ventral extension or the dorsal restriction of Neuropilin - 1 expressing axons . This is in agreement with the lack of defects found by Hayashi et al in the projection of axons through this particular tract ( Hayashi et al . 2017 ) . More subtle defects in specific tracts would require much deeper analyses to be revealed . Regardless of any anatomical alterations , assessing behavior allows a relevant functional assessment of the consequences of Pcdh19 loss . Our analysis differed from the previously carried out with Pcdh19 mutants ( Hayashi et al . 2017 ; Lim et al . 2019 ) . First , in addition to testing adult animals , we also tested animals at a much younger age ( pre - weaning , P21 ) , as EIEE9 is a developmental disorder and therefore it is relevant to determine when any behavioral changes begin . Second , we added a second cohort of control animals : wild - type single genotype housed mice , that have only been exposed to other WT animals during their life . An effect of WT littermates on the behavior of mutant animals was shown by Yang et al when they demonstrated that raising less sociable BTBR T + tf / J mice with highly sociable C57Bl6 / J animals improved BTBR T + tf / J sociability ( Yang et al . 2011 ) . However , the impact of social environment on the behavior of WT littermates has only recently been demonstrated in a study with mice mutant for Nlgn3 , an X - linked cell adhesion protein that has been implicated in ASD ( Kalbassi et al . 2017 ) . Therefore , this is further evidence to suggest that mutant mice can alter the behavior of their WT littermates . In line with a previous study ( Hayashi et al . 2017 ) , changes in behavior were more apparent in HET females than in their KO male siblings . Pcdh19 KO males only showed increased time spent in the open arms of the EPM , indicating reduced anxiety , when tested as adults . This same behavior was displayed by young Pcdh19 HET females ( P21 ) , which maintained it into adulthood . However , HET females also exhibited increased exploratory behavior , or maybe hypersensitivity to new environments , from a young age , as demonstrated by their consistently higher distance travelled during the first 5 minutes in the open field at P21 and P60 . It is important to consider that animals were placed into the open field arena 4 times in total , as they were tested on two consecutive days at both ages . Although habituation to the environment would be expected in this situation , the increased distance travelled during the first 5 minutes was apparent in all 4 trials , indicating a robust behavioral response . These results also suggest that behavioral changes in Pcdh19 heterozygous animals start early in life , validating them as a good model for a developmental condition . Open field and EPM tests were also performed in the study by Hayashi et al ( Hayashi et al . 2017 ) . They found no differences in the EPM , but whether this is due to experimental design or differences in the mouse model used is difficult to ascertain . Regarding the open field test , Hayashi et al found no differences in total distance or time in the center when the test was conducted at 11 - 12 weeks of age . However , when they repeated the test 23 weeks later , Pcdh19 HET females spent significantly more time in the center of the open field arena , suggesting reduced anxiety . Although our animals did not display such behavior , they were tested around P60 , which would be in agreement with the data from their first open field test . In addition , the results of our EPM test also indicate reduced anxiety in our animals , which could therefore represent a behavioral characteristic of Pcdh19 mutant animals . Because no specific analysis of the first 5 minutes was carried out in that study , it is difficult to assess whether their animals exhibited increased activity during that period . Nevertheless , the fact that WT females display the same behavioral phenotype as their HET siblings indicates an effect of the social environment that can only be detected through the inclusion of single genotype housed WT animals . Interestingly , this effect was also present in young males , with WT MGH travelling a higher distance in the first 5 minutes of the open field test than KO or WT SGH males . However , unlike in the female population , this behavior disappeared in adulthood . Because adult male and female animals are housed separately , it is tempting to speculate that this effect of the social environment is somehow mediated by the HET females , although other causes , like a maternal effect , cannot be ruled out based on our experiments . One of the comorbidities of EIEE9 patients is ASD ( Kolc et al . 2020 ) , and changes in PCDH19 have also been linked to ASD cases ( Piton et al . 2010 ; Harssel et al . 2013 ) . Indeed , a recent behavioral study with the Taconic Pcdh19 ko mouse model has revealed social interaction deficits in the three chamber test in KO males and HET females , as well as increased repetitive behavior in males ( females were not tested ) ( Lim et al . 2019 ) . In our analysis , we also found differences in social behavior , but , interestingly , only in WT MGH animals . Both males and females spent less time interacting with a strange female at P21 and P60 , respectively , than WT SGH animals , in what appears to be another example of the effect of the environment on mouse behavior . Since males were not tested at P60 , because at that age it becomes a measure of courtship behavior rather than simple social interaction and as such is not comparable to the P21 behavior , we don’t know if this phenotype would be maintained into adulthood or if , similar to the results of the open field , it would revert to normal with age . The fact that HET and KO animals did not differ in their behavior from their WT littermates is in contradiction with the results from Lim et al , although different tests were carried out in both studies , making a direct comparison difficult . In summary , our behavioral characterization of the Pcdh19 Taconic mouse model reveals a stronger effect of Pcdh19 mutation in HET females than in KO males and a significant effect of the social environment on the behavior of WT littermates , as previously described for Nlgn3 mutant animals ( Kalbassi et al . 2017 ) . This effect should be taken into consideration for the design of future behavioral experiments , as failure to do so could result in misinterpretation of data and missed behavioral phenotypes . FUNDING This work was supported by the Life Science Research Network Wales , an initiative funded through the Welsh Government’s Ser Cymru program ( fellowships to N . G - R . and J . G . , initial fellowship to IM - G ) , the Wellcome Trust ( Seed Award 109643 / Z / 15 / Z to I . M - G . , fellowship 204021 / Z / 16 / A to S . A . N . ) , Cardiff University ( Grant 501100000866 to M . 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TBR2 coordinates neurogenesis expansion and precise microcircuit organization via Protocadherin 19 in the mammalian cortex . Nat Comms . 10 : 1 – 15 . Pederick DT , Homan CC , Jaehne EJ , Piltz SG , Haines BP , Baune BT , Jolly LA , Hughes JN , Gécz J , Thomas PQ . 2016 . Pcdh19 Loss - of - Function Increases Neuronal Migration In Vitro but is Dispensable for Brain Development in Mice . Sci Rep . 6 : 26765 . Pederick DT , Richards KL , Piltz SG , Kumar R , Mincheva - Tasheva S , Mandelstam SA , Dale RC , Scheffer IE , Gécz J , Petrou S , Hughes JN , Thomas PQ . 2018 . Abnormal Cell Sorting Underlies the Unique X - Linked Inheritance of PCDH19 Epilepsy . Neuron . 97 : 59 – 66 . e5 . Pham DH , Tan CC , Homan CC , Kolc KL , Corbett MA , McAninch D , Fox AH , Thomas PQ , Kumar R , Gécz J . 2017 . Protocadherin 19 ( PCDH19 ) interacts with paraspeckle protein NONO to co - regulate gene expression with estrogen receptor alpha ( ERα ) . Hum Mol Genet . 26 : 2042 – 2052 . 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Autism Res . 4 : 17 – 27 . \ No newline at end of file diff --git a/silver_data/439eff117a56ddc9b70eac8787455ac031a51d7d.pdf.txt b/silver_data/439eff117a56ddc9b70eac8787455ac031a51d7d.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..963ffd9655936d699711f2ef0c8ba43521e1eda4 --- /dev/null +++ b/silver_data/439eff117a56ddc9b70eac8787455ac031a51d7d.pdf.txt @@ -0,0 +1 @@ +COMMENTARY Increasing COVID - 19 vaccine acceptance among the general population while maintaining autonomy Dorcas Serwaa a , Emmanuel Lamptey a , and Ephraim Kumi Senkyire b a Institute of Life and Earth Sciences ( Including Health and Agriculture ) , Pan African University , University of Ibadan , Ibadan , Nigeria ; b Department of Nursing , Ga West Municipal Hospital , Ghana Health Service , Accra - Ghana ABSTRACT The accelerated dissemination of coronavirus disease , its effects on the general public and healthcare system have been unparalleled . COVID - 19 vaccination is critical for reducing the alarming incidence of the infection , promoting herd immunity , preventing associated mortality and morbidity , and maintaining public health and safety . Through the development of COVID - 19 vaccines , many people are keen to protect themselves against the virus ; however , the willingness for vaccination especially in Africa , is far below what is required to stop the ongoing COVID - 19 pandemic . As a result , there is an immediate need to implement plans aimed at increasing population vaccine willingness . The slippery slope is whether sanctions , bans and restriction should be imposed on hesitant persons , since transmission of the epidemic can be prevented through stringent enforcement of the control barriers , while eradicating the disease would necessitate vaccination . This commentary provides recommendations about how to increase population vaccine acceptance while maintaining autonomy . ARTICLE HISTORY Received 5 April 2021 Accepted 21 April 2021 KEYWORDS Autonomy ; COVID - 19 ; vaccination ; vaccine acceptance ; vaccine hesitancy Introduction The superspreading nature and impact of coronavirus disease ( COVID - 19 ) on the general population and healthcare system have been unprecedented . 1 The pandemic has attained rapid global transmission partly owing to the high number of asymp - tomatic carriers interacting in a world interconnected by supply chain , communications technology and travel . 2 Movements to workplaces , urbanization , daily human connection , the mingling of families in communities and international travels intensify the transmissibility of the virus . 3 Such undertakings pose a challenge for mitigating the spread and predispose close contacts of COVID - 19 patients to the infection . Recent report revealed that the family household infection rate is estimated between 16 . 3 % and 30 % . 4 The aged , as well as the elderly , with certain underlying medical condition , are at high risk of serious infection from the virus , which , and can result in hospitalization , admission to intensive care units , intubation and even death . The threats posed by COVID - 19 are immense , but everyone must remain vigilant , follow the mandatory protection precautions and most preeminently , get vaccinated as this would keep the pandemic under control while still shielding individuals and families in the community from the deadly virus . COVID - 19 vaccine hesitancy COVID - 19 vaccination is critical for reducing the alarming incidence of the infection , promoting herd immunity , prevent - ing associated mortality and morbidity , and maintaining public health and safety . 5 Numerous persons are eager to defend themselves against the virus now that the COVID - 19 vaccines have been developed and made available ; however , certain families and individuals may deliberately opt not to be immu - nized . Vaccine hesitancy is known to be caused by a number of factors , especially when new vaccines are being produced . These factors include misconceptions regarding the need for vaccines , vaccine protection and efficacy , negative health effects , lack of confidence in the health system , and lack of community awareness about vaccine - preventable diseases . 6 – 8 Although the World Health Organization approved COVID - 19 vaccines have undergone stringent testing and clinical trials to demonstrate their safety and success in con - trolling the pandemic , a report from the United Kingdom stated that one of every five persons was unlikely to take the COVID - 19 vaccines for a number of reasons . 9 Summarily , a study that assessed hesitancy to a COVID - 19 vaccine in Cameroon and its global implication among 2512 participants shockingly reported vaccine hesitancy rate of 84 . 6 % citing communication and media environment , perception of phar - maceutical industry , reliability and / or source of vaccine and cost as influencing factors . 10 Another study investigating the level of willingness for COVID - 19 vaccination in the Democratic Republic of Congo ( DRC ) reported that , of the 4131 responses , 2310 ( 55 . 9 % ) indicated their willingness to be vaccinated and income status , COVID - 19 status , COVID - 19 community vaccine acceptance and acknowledging the exis - tence of COVID - 19 were associated with an increased will - ingness to be vaccinated . 11 CONTACT Serwaa Dorcas dserwaa0327 @ stu . ui . edu . ng Institute of Life and Earth Sciences ( Including Health and Agriculture ) , University of Ibadan , Olatunde Runsewe Hall , Ibadan , Nigeria HUMAN VACCINES & IMMUNOTHERAPEUTICS 2021 , VOL . 17 , NO . 12 , 5139 – 5141 https : / / doi . org / 10 . 1080 / 21645515 . 2021 . 1922265 © 2022 Taylor & Francis Group , LLC The slippery slope between COVID - 19 vaccine autonomy and uptake enforcement Every person has the right to confidently refuse vaccination and this decision must be understood and respected . Therefore , managing COVID - 19 reluctant individuals and their families’ actions may trigger conflict between honoring and promoting a person’s decision - making autonomy and upholding of professional duty to encourage evidence - based decisions , aimed at achieving optimal outcome for both families and individuals . The slippery slope is whether sanc - tions , bans and restriction be imposed on people that resist COVID - 19 vaccination , since it is already commonly recog - nized that the transmission of the epidemic can be prevented through stringent enforcement of the control barriers , while eradicating the disease would necessitate the uptake of a vaccine . The willingness for vaccination especially in Africa , is far below what is required to stop the ongoing COVID - 19 pandemic , as a result , there is an immediate need to implement plans to increase population vaccine willingness . Recommendations to increase population vaccine acceptance Educational campaigns Outreach campaigns aimed at improving awareness and dis - pelling some of the vaccine’s well - known stereotypes must be extended to build more optimistic perceptions and decisions toward the vaccine use . An educational strategy of this kind should include factual information , discuss various vaccination issues , and provide answer to questions regarding COVID - 19 vaccine misconception . People must be persuaded that the system for developing , testing and regulating vaccine demands that every vaccine shows both safety and efficacy prior to licensure , and therefore the safety of the current COVID - 19 is well assured and monitored . The campaigns should also emphasize the benefits of the vac - cinations to the recipient , immediate family , neighbors , society and the world at large . The advertisement should appeal to the emotions and fears of individuals but not to scare anyone into taking a specific action . It must also be greared towards protecting the general population’s vulnerability and effective enough to encourage vaccination . If the morbidities , mortalities , and realities associated with the virus are intensely communicated , the public would recognize the growing burden and be willing to take the vaccines . Strategic communication Health communication must reach all communities , especially the most vulnerable , educating them about the safety of the vaccines and the potency to prevent future infections and deaths . Communication about the vaccine can be promoted through community van mobilization , radios , televisions , leaflets and tele - phone hotlines , to enlighten the general public about vaccine - related information like accessibility , availability , efficacy , known side effects , benefits , protocols and triage decisions . Respected community - based organizations and non - governmental agencies , such as the Red Cross , Faith leaders and religious leaders , which are widely regarded as neutral , are critical in fostering interest in COVID - 19 vaccine uptake . Health professionals’ ‘self - vaccination’ strategy Health professionals’ self - vaccination strategy can influence families and individuals who refuse COVID - 19 vaccination . Vaccine acceptance depends on public trust and confidence in the safety , efficacy of the vaccines and immunization . The recommendation of healthcare providers and trust in the healthcare system are most often cited explanation for general public approval for vaccination . 8 , 12 This implies that the public relies on the expertise judgment and willingness of health professionals to view these results accurately and in the best interests of the public . Therfore , the uptake of COVID - 19 vaccine by healthcare providers in the presence of their patients will build trust and affirms the safety of the vaccine . Healthcare professionals ‘patients’ vaccination’ strategy Health professionals should prescribe the COVID - 19 vaccines for those unwilling to vaccinate . The key reasons for healthcare professionals prescribing vaccinations to their patients include a belief in the benefits of vaccines and a sense of responsibility to encourage protection and vaccination . One research found that recommending vaccines to patients requires a trusting doctor – patient partnership . 13 Healthcare professionals have the responsibility of continuously engaging COVID - 19 hesi - tant families and individuals during the provision of healthcare services and public health education sessions in an attempt to modifying their behavior to reconsider vaccination . Healthcare providers must be capable of articulating the safety and efficacy of the vaccine to their clients and also refrain from suggesting that refusing vaccination may be safer . Recent study on acceptance of a pandemic influenza vac - cine : a systematic review of surveys of the general public , reported that respondents who received positive advice from a primary care physician for vaccination were more likely to accept the pandemic vaccine , and those who did not receive positive advice were more likely to decline the vaccine . 12 Safety information must be presented in a non - confrontational dia - logue . Opel et al . 14 reported that hesitant families do accept vaccination after their healthcare practitioners have provided a rationale for vaccine administration . ‘COVID - 19 vaccination passports’ or proof of COVID - 19 vaccination The introduction of ‘COVID - 19 vaccination passports’ or proof of COVID - 19 vaccination , however this topic is highly debatable . Travel restrictions are public health emergency mea - sures to reduce the spread of pandemics and other highly infectious diseases like influenza , yellow fever and polio . The revised International Health Regulations ( IHR ) in 2005 expanded the scope of internationally important diseases from three ( cholera , plague and yellow fever ) to include all “events which may constitute public health emergencies of international concern” . 15 5140 D . SERWAA ET AL . In the yellow fever antecedent , prior to the availability of a vaccine in 1937 , the key tactic used in an attempt to limit its dissemination was to rely on old - fashioned methods of isolation and quarantine . 16 Vaccine entry requirement became the key disease prevention mechanism of the post - vaccination period . Vaccine entry requirement are provisions for travelers to present evidence of vaccination for specific diseases in order to enter a country . These conditions extend to all travelers or visitors coming from areas where the disease is at risk of spread . Likewise , issuing ‘COVID - 19 vaccination passports’ or proof of COVID - 19 vaccination could mean that persons would be allowed to travel freely outside their home country provided they have evidence of immunization . This will promote the resump - tion of air transport and help in the revitalization of national economies . However , there are several arguments as to whether it is ethically permissible to impose such practice on people who refuse vaccination . This will depend on factors like the level of vaccination in the general population , vaccine supply , the nature of restriction and how the restrictions are operationalized . COVID - 19 vaccine certificates The general public should be required to have COVID - 19 vac - cine certificates to gain access to some essential public services and places within the country . Just as children must show - up their immunity certificate against certain diseases before they can be enrolled at a new school and also similar to ‘no mask no entry’ advocacy . This will reopen the economy , make people adapt to the new normal , remind others to get their shots and also improve the safety of staffs and customers in such public places . The pre - pandemic freedom for individuals who refuse to vaccinate against the COVID - 19 should not affect others nega - tively or cause more serious cases of coronavirus disease . It is therefore justifiable to restrict vaccine refusers to reduce onward transmission . A section of the population will accept vaccination if given the choice between immunization and restric - tions at essential public services . However , these restrictions must be conducted in a manner consistent with established health guidelines and prior official notification must be provided to all and sundry involved . In situations where there is limited ability to restrict hesitant people , as is the case of a hospital environment , established organizational policies should be followed and all necessary services must be rendered regardless . Conclusion The COVID - 19 vaccines can contribute to the control of the deadly pandemic , although the vaccines may not be fully protec - tive . Refusal of the vaccine will trigger re - emergence of the pan - demic , which can be harmful to individuals and the general populace . Hence , public health authorities , health providers and all stakeholders involved in the fight against this pandemic have a professional responsibility to provide scientifically proven infor - mation in managing encounters with non - vaccinating families and individuals . The approach should identify the basis of people’s decisions , address concerns and execute the recommended actions to encourage vaccine uptake while upholding ethical responsibilities . Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed . ORCID Dorcas Serwaa http : / / orcid . org / 0000 - 0001 - 8583 - 4415 Emmanuel Lamptey http : / / orcid . org / 0000 - 0002 - 7822 - 7593 Ephraim Kumi Senkyire http : / / orcid . org / 0000 - 0003 - 0789 - 701X References 1 . Lango MN . How did we get here ? Short history of COVID - 19 and other coronavirus - related epidemics . Head Neck . 2020 ; 42 ( 7 ) : 1535 – 38 . doi : 10 . 1002 / hed . 26275 . 2 . Bai Y , Yao L , Wei T , Tian F , Jin D - Y , Chen L , Wang M . Presumed asymptomatic carrier transmission of COVID - 19 . Jama . 2020 ; 323 ( 14 ) : 1406 – 07 . doi : 10 . 1001 / jama . 2020 . 2565 . 3 . Heesterbeek H , Anderson RM , Andreasen V , Bansal S , De Angelis D , Dye C , Eames KT , Edmunds WJ , Frost SD , Funk S . et al . Modeling infectious disease dynamics in the complex land - scape of global health . 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Hum Vaccin Immunother . 2018 ; 14 ( 7 ) : 1599 – 609 . doi : 10 . 1080 / 21645515 . 2018 . 1459252 . 9 . McDonnell A How many Britons are willing to take a coronavirus vaccine ? [ Internet ] . YouGov ; 2020 . https : / / yougov . co . uk / topics / health / articles - reports / 2020 / 11 / 16 / how - many - britons - are - willing - take - coronavirus - vacc 10 . Dinga JN , Sinda LK , Titanji VPK Assessment of vaccine hesitancy to a COVID - 19 vaccine in Cameroonian adults and its global implication ; 2021 . 11 . Ditekemena JD , Nkamba DM , Mavoko AM , Hypolite M , Siewe Fodjo JN , Luhata C , Obimpeh M , Van Hees S , Nachega JB , Colebunders R . COVID - 19 vaccine acceptance in the Democratic Republic of Congo : a cross - sectional survey . Vaccines . 2021 ; 9 ( 2 ) : 153 . 12 . Nguyen T , Henningsen KH , Brehaut JC , Hoe E , Wilson K . Acceptance of a pandemic influenza vaccine : a systematic review of surveys of the general public . Infect Drug Resist . 2011 ; 4 : 197 . doi : 10 . 2147 / IDR . S23174 . 13 . Yaqub O , Castle - Clarke S , Sevdalis N , Chataway J . Attitudes to vaccination : a critical review . Soc Sci Med . 2014 ; 112 : 1 – 11 . doi : 10 . 1016 / j . socscimed . 2014 . 04 . 018 . 14 . Opel DJ , Heritage J , Taylor JA , Mangione - Smith R , Salas HS , DeVere V , Zhou C , Robinson JD . The architecture of provider - parent vaccine discussions at health supervision visits . Pediatrics . 2013 ; 132 ( 6 ) : 1037 – 46 . doi : 10 . 1542 / peds . 2013 - 2037 . 15 . Schlagenhauf P , Patel D , Rodriguez - Morales A , Gautret P , Grobusch MP , Leder K . Variants , vaccines and vaccination pass - ports : challenges and chances for travel medicine in 2021 . Travel Med Infect Dis . 2021 ; 40 : 101996 . doi : 10 . 1016 / j . tmaid . 2021 . 101996 . 16 . Vanderslott S , Marks T . Travel restrictions as a disease control measure : lessons from yellow fever . Glob Public Health [ Internet ] . 2021 ; 16 ( 3 ) : 340 – 53 . doi : 10 . 1080 / 17441692 . 2020 . 1805786 . HUMAN VACCINES & IMMUNOTHERAPEUTICS 5141 \ No newline at end of file diff --git a/silver_data/44f3dc42a1a07549ec0d094c5e3853da9809c6d9.pdf.txt b/silver_data/44f3dc42a1a07549ec0d094c5e3853da9809c6d9.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d5fc070b7f1272713ca303941f99c232b5a1dc8b --- /dev/null +++ b/silver_data/44f3dc42a1a07549ec0d094c5e3853da9809c6d9.pdf.txt @@ -0,0 +1 @@ +COMMENTARY 2100 Journal of Investigative Dermatology ( 2007 ) , Volume 127 We now can return to the theme of bioengineering stem cells to form hair follicles . With the advent of stem cell biology , we anticipate that many attempts to engineer hair follicles will be made in the near future . The epider - mal stem cells or progenitor cells may derive from many sources : embryonic stem cells , engineered embryonic stem cells or cell lines , interfollicular epider - mal stem cells , bulge stem cells , or even bone marrow stem cells . Because the cells may have different competences , the “hair follicles” built with them may be different . In the future , we must ask whether they fulfill all the criteria of a bona fide hair follicle . Work that has had partial success is nonetheless valuable because it helps in the analysis of distinct events during hair - follicle morphogen - esis . The results here highlight the chal - lenge faced by all who aspire to engineer human organs , including hair follicles . ACKNOWLEDGMENT CMC and GC are supported by grants from the National Institutes of Health . CONFLICT OF INTEREST The authors state no conflict of interest . 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In : The Mouse in Biomedical Research ( Fox J , Barthold S , Davisson M , Newcomer C , Quimby F , Smith A , eds ) , 2nd edn , Vol 3 , Academic Press : Amsterdam Reynolds AJ , Jahoda CA ( 2004 ) Cultured human and rat tooth papilla cells induce hair follicle regeneration and fiber growth . Differentiation 72 : 566 – 75 Sengel P ( 1976 ) Morphogenesis of Skin . Cambridge University Press : Cambridge , UK Sokolov VE , Chernova OF ( 2001 ) Skin appendages of mammals ( in Russian ) . GEDS : Moscow Stenn KS , Cotsarelis G ( 2005 ) Bioengineering the hair follicle : fringe benefits of stem cell technology . Curr Opin Biotechnol 16 : 493 – 7 Weinberg WC , Goodman LV , George C , Morgan DL , Ledbetter S , Yuspa SH et al . ( 1993 ) Reconstitution of hair follicle development in vivo : determination of follicle formation , hair growth , and hair quality by dermal cells . J Invest Dermatol 100 : 229 – 36 Wu P , Hou L , Plikus M , Hughes M . , Scehnet J , Suksaweang S , et al . ( 2004 ) Evo - devo of amniote integuments and appendages . Int J Dev Biol 48 : 249 – 70 Yue Z , Jiang TX , Widelitz RB , Chuong CM ( 2006 ) Wnt 3a gradient converts radial to bilateral feather symmetry via topological arrangement of epithelia . Proc Natl Acad Sci USA 103 : 951 – 5 Zheng Y , Du X , Wang W , Boucher M , Parimoo S , Stenn K ( 2005 ) Organogenesis from dissociated cells : Generation of mature cycling hair follicles from skin - derived cells . J Invest Dermatol 124 : 867 – 76 See related article on pg 2116 Merkel Cell Carcinoma : More Deaths but Still No Pathway to Blame Bianca Lemos 1 and Paul Nghiem 1 Merkel cell carcinoma ( MCC ) is a neuroendocrine skin cancer with a rising incidence ( 1500 U . S . cases per year ) that now exceeds that of cutaneous T - cell lymphoma and a mortality ( 33 % ) exceeding that of melanoma . Despite this impact , little is known about its biology . Recent studies have shown that Ras / MAP kinase activity is absent and possibly detrimental to this cancer . This makes MCC distinct from other UV - induced skin cancers and highlights the question of what drives this malignancy . Journal of Investigative Dermatology ( 2007 ) 127 , 2100 – 2103 . doi : 10 . 1038 / sj . jid . 5700925 Merkel cell carcinoma ( MCC ) is an aggressive neuroendocrine carcinoma of the skin associated with ultraviolet exposure . Although uncommon , its incidence is increasing and has in fact tripled in the past 15 years ( Hodgson , 2005 ) . The rise in incidence is due in part to improved diagnosis through the 1 University of Washington Dermatology / Medicine , Fred Hutchinson Cancer Research Center , Seattle Cancer Care Alliance , Seattle , Washington 98109 , USA . Correspondence : Dr Paul Nghiem , 815 Mercer Street , Seattle , Washington 98109 , USA . E - mail : pnghiem @ u . washington . edu COMMENTARY www . jidonline . org 2101 routine use of cytokeratin - 20 staining but is also due to an aging population with extensive sun exposure . The National Cancer Data Base , a registry that captures approximately 75 % of all cancer cases in the United States , recorded 986 cases of MCC in 2004 ( most recent data available ) . According to multiple national databases , there has been a 5 – 10 % annual increase in MCC diagnoses beginning in the early 1990s ( Agelli and Clegg , 2003 ; National Cancer Data Base data provided by Jerri Linn Phillips ) . Taking these factors into account , there are now approximately 1 , 500 cases of MCC per year in the United States . This exceeds the inci - dence of cutaneous T - cell lymphoma , a well - known and frequently studied can - cer , whose incidence has been stable since the early 1980s ( Weinstock and Gardstein , 1999 ) . MCC is also one of the most lethal skin cancers , with a 33 % mortality ( Hodgson , 2005 ) —it now kills more patients than cutaneous T - cell lymphoma and a number similar to that for chronic myelogenous leukemia ( Figure 1 ) ( American Cancer Society , 2006 ; Weinstock and Gardstein , 1999 ) . Despite the mortality and increasing incidence , virtually nothing is known about the molecular basis of MCC . Several studies of oncogenic pathways have been carried out in MCC but have not elucidated a significant role for these pathways in this cancer ( Table 1 ) . Van Gele et al . ( 2000 ) revealed a lack of p53 mutations in 12 of 15 cases of MCC . Three of the five p53 mutations found in three tumors were of the sig - nature UV type , consistent with MCC’s known association with UV exposure . The lack of p53 mutations in 80 % of MCCs , however , suggests that p53 is not typically involved in this cancer . The same group ( Van Gele et al . , 2001 ) also looked at the role of the tumor suppres - sor PTEN . Although loss of heterozygos - ity ( loss of one allele ) was seen in 9 of 21 cases ( mostly involving loss of the entire arm of chromosome 10 ) , only a single mutation was seen in the remain - ing allele of one MCC case . Thus , inacti - vation of PTEN ( by loss of one allele and disruption of the second ) does not play a frequent role in MCC oncogenesis . It is possible , however , that loss of a second PTEN allele would be via an epigenetic silencing mechanism such as methyla - tion , which has not been examined . The Wnt pathway and its role in MCC have also been evaluated by looking for nuclear accumulation of β - catenin and for mutations in β - catenin and three other related genes . In a series of 12 MCC cases , Liu et al . ( 2007 ) found elevated β - catenin accumulation in only one tumor and no mutations in any tumors and concluded that the Wnt pathway is not implicated in MCC . Several studies have reported on c - Kit expression in MCC tumor samples . Most recently , Swick et al . ( 2007 ) deter - mined that although eight of nine MCC tumors were positive for c - Kit by immu - nohistochemical staining , no activat - ing mutations were present in the four exons commonly found to have muta - tions in this gene . MAP kinase signaling , a common feature of many epithelial cancers , is the most studied oncogenic pathway in MCC ( Figure 2 ) . Popp et al . ( 2002 ) looked for activating mutations in exons 1 and 2 of the H - , K - , and N - ras genes in six MCC cell lines but found none . Figure 1 . Annual incidence and mortality for three cancers in the United States . MCC incidence is similar to that of CTCL , and MCC deaths are similar to those of CML . Abbreviations : MCC , Merkel cell carcinoma ; CTCL , cutaneous T cell lymphoma ; CML , chronic myelogenous leukemia . ( Data from American Cancer Society , 2006 ; Weinstock and Gardstein , 1999 . ) Table 1 . Cancer - associated pathways and genes studied in MCC oncogenesis Cancer - associated pathway / gene Likely relevant Summary of findings Reference p53 – No mutations found in 12 of 15 samples Van Gele et al . , 2000 Ras – No activating mutations in H - ras , K - ras , or N - ras found in six MCC cell lines Popp et al . , 2002 B - Raf V600E – No mutations in 46 MCCs Houben et al . , 2006 MAP kinase activity – MAP kinase silenced in 42 / 44 MCCs Houben et al . , 2006 Wnt – No mutations in β - catenin , APC , AXIN1 , or AXIN2 in 12 MCC tumors Liu et al . , 2007 c - Kit – No activating mutations in nine MCC tumors Swick et al . , 2007 PTEN ? No mutations in 20 of 21 samples but loss of heterozygosity for region in 43 % Van Gele et al . , 2001 bcl - 2 + High expression in 15 of 20 MCC tumors ; bcl - 2 antisense decreases tumor size in xenograft model Kennedy et al . , 1996 ; Pletten - berg et al . , 1996 ; Schlagbauer - Wadl et al . , 2000 | The MAP kinase pathway is silent in 42 of 44 MCC tumors . COMMENTARY 2102 Journal of Investigative Dermatology ( 2007 ) , Volume 127 Recently , Houben et al . ( 2006 ) evaluat - ed 46 MCC tumors for the characteristic activating B - Raf mutation ( V600E ) found in 43 % of melanomas , and all tumors were negative . Further , the authors showed that the MAP kinase pathway was silent ( no phospho - ERK staining ) in 42 of 44 cases , and Raf kinase inhibitor protein was increased in all 20 tumors evaluated . Taken together , the studies strongly suggest no role for MAP kinase pathway activation in MCC , a pathway heavily implicated in the etiology of melanoma , squamous cell carcinoma , and many other cancers ( Schubbert et al . , 2007 ) . The sole potentially positive finding in MCC is that of bcl - 2 . Its expression was seen in 15 of 20 MCC tumors in two stud - ies ( Kennedy et al . , 1996 ; Plettenberg et al . , 1996 ) . A separate study determined that decreasing bcl - 2 expression in vivo by systemic antisense oligonucleotide ( oblimersen / Genasense ) administration in a SCID mouse / human tumor xeno - graft model resulted in tumor shrinkage ( Schlagbauer - Wadl et al . , 2000 ) . The expression of this anti - apoptosis protein is a common finding in many cancers and suggests one of its mechanisms to avoid cell death ; however , it does not illuminate the promitotic pathways that drive MCC . In this issue of the Journal of Investigative Dermatology , Houben et al . ( 2007 ) expand on their findings noted above , that the MAP kinase path - way is silent in 42 of 44 MCC tumors . Using an MCC cell line ( UISO ) that , like primary tumors , shows no evidence of MAP kinase activity , they tested wheth - er activating this pathway could result in MCC cell death . Indeed , inducible expression of c - Raf - 1 kinase in the UISO MCC cell line resulted in morphologic changes and apoptosis . Rounding up and detachment of cells , development of long cytoplasmic extensions , and loss of actin stress fibers were seen begin - ning at 10 hours after activation of the pathway . Further , they demonstrated that active Raf kinase induced apopto - sis in UISO cells in a time - and dose - dependent manner . This appears to be via a classic apoptotic pathway because it was blocked by a caspase inhibitor ( Z - VAD - FMK ) . The fact that a MEK kinase inbibitor ( U0126 ) also blocked Raf’s ability to induce apoptosis suggests that the Raf effect is indeed mediated via the predicted MAP kinase pathway ( Houben et al . , 2007 ) . A limitation of the study is that it was performed in a single MCC cell line . Few reagents for MCC are available to study , and of the available cell lines , only one ( UISO ) had preservation of the MCC in vivo characteristic of silencing of the MAP kinase pathway . The lack of cell death observed in the study from Raf overexpression in other cell lines positive for MAP kinase activity ( three melanoma lines and two other MCC lines ) argues for some level of specific - ity for the cell - death effect to cells that are silent for MAP kinase activity . The striking lack of knowledge of what drives MCC oncogenesis is an important impediment because no effec - tive targeted therapies exist . Molecular therapies used in other malignancies are now being extended to clinical tri - als in MCC . Phase II trials of imatinib ( Gleevec ) and oblimersen ( Genasense ) are currently under way in MCC . The tyrosine kinase inhibitor imatinib , ini - tially designed to target the Bcr – Abl translocation in chronic myelogenous leukemia , is being explored because of the c - Kit positivity of MCC . Recent data from Swick et al . ( 2007 ) suggest that its effectiveness may be limited , however , given that no c - Kit - activating muta - tions were seen in their series . The bcl - 2 inhibitor oblimersen is also in a phase II trial for MCC , but no results of its effi - cacy in humans with MCC have been published . The present study by Houben et al . ( 2007 ) provides data for the possible use of MAP kinase pathway – activating agents in MCC . Use of the Raf - 1 activa - tor ZM336372 did indeed induce ERK phosphylation in UISO MCC cells that are normally silent for MAP kinase sig - naling . A separate study using carcinoid tumor cells ( another neuroendocrine carcinoma ) demonstrated that in vitro treatment with ZM336372 inhibited cel - lular proliferation ( Van Gompel et al . , 2005 ) . The data suggest that pharmaco - logic activation of this normally silent pathway is a potential target for treat - ment of certain neuroendocrine tumors . Even though we know essentially nothing about what promitotic signal - ing mechanisms drive MCC , there is a glimmer of hope that recent knowledge about what does not drive it may be brought to bear in the fight against MCC and , potentially , other neuroendocrine cancers . ACKNOWLEDGMENT This work was supported by Harvard / NCI Skin Cancer SPORE , the University of Washington Merkel cell carcinoma patient fund , and NIH K02 - AR050993 . CONFLICT OF INTEREST The authors state no conflict of interest . REFERENCES Agelli M , Clegg LX ( 2003 ) Epidemiology of primary Merkel cell carcinoma in the United States . Figure 2 . The MAP kinase pathway . Text boxes annotate study findings of relevance in MCC . Double box indicates findings from study in this issue . ( Adapted from Houben et al . , 2006 . ) COMMENTARY www . jidonline . org 2103 J Am Acad Dermatol 49 : 832 – 41 American Cancer Society ( 2006 ) Cancer Facts and Figures 2006 . < http : / / www . cancer . org / docroot / STT / content / STT _ 1x _ Cancer _ Facts _ _ Figures _ 2006 . as P > Hodgson NC ( 2005 ) Merkel cell carcinoma : changing incidence trends . J Surg Oncol 89 : 1 – 4 Houben R , Michel B , Vetter - Kauczok CS , Pfohler C , Laetsch B , Wolter MD et al . ( 2006 ) Absence of classical MAP kinase pathway signalling in Merkel cell carcinoma . J Invest Dermatol 126 : 1135 – 42 Houben R , Ortmann , S , Schrama D , Herold MJ , Berberich I , Reichardt HM et al . ( 2007 ) Activation of the MAP kinase pathway induces apoptosis in the Merkel cell carcinoma cell line UISO . J Invest Dermatol 127 : 2116 – 22 Kennedy MM , Blessing K , King G , Kerr KM ( 1996 ) Expression of bcl - 2 and p53 in Merkel cell carcinoma . An immunohistochemical study . Am J Dermatopathol 18 : 273 – 7 Liu S , Daa T , Kashima K , Kondoh Y , Yokoyama S ( 2007 ) The Wnt - signaling pathway is not implicated in tumorigenesis of Merkel cell carcinoma . J Cutan Pathol 34 : 22 – 6 Plettenberg A , Pammer J , Tschachler E ( 1996 ) Merkel cells and Merkel cell carcinoma express the BCL - 2 proto - oncogene . Exp Dermatol 5 : 183 – 8 Popp S , Waltering S , Herbst C , Moll I , Boukamp P ( 2002 ) UV - B - type mutations and chromosomal imbalances indicate common pathways for the development of Merkel and skin squamous cell carcinomas . Int J Cancer 99 : 352 – 60 Schlagbauer - Wadl H , Klosner G , Heere - Ress E , Waltering S , Moll I , Wolff K , et al . ( 2000 ) Bcl - 2 antisense oligonucleotides ( G3139 ) inhibit Merkel cell carcinoma growth in SCID mice . J Invest Dermatol 114 : 725 – 30 Schubbert S , Shannon K , Bollag G ( 2007 ) Hyperactive Ras in developmental disorders and cancer . Nat Rev Cancer 7 : 295 – 308 Swick BL , Ravdel L , Fitzpatrick JE , Robinson WA ( 2007 ) Merkel cell carcinoma : evaluation of KIT ( CD117 ) expression and failure to demonstrate activating mutations in the C - KIT proto - oncogene—implications for treatment with imatinib mesylate . J Cutan Pathol 34 : 324 – 9 Van Gele M , Kaghad M , Leonard JH , Van Roy N , Naeyaert JM , Geerts ML et al . ( 2000 ) Mutation analysis of P73 and TP53 in Merkel cell carcinoma . Br J Cancer 82 : 823 – 6 Van Gele M , Leonard JH , Van Roy N , Cook AL , De Paepe A , Speleman F ( 2001 ) Frequent allelic loss at 10q23 but low incidence of PTEN mutations in Merkel cell carcinoma . Int J Cancer 92 : 409 – 13 Van Gompel JJ , Kunnimalaiyaan M , Holen K , Chen H ( 2005 ) ZM336372 , a Raf - 1 activator , suppresses growth and neuroendocrine hormone levels in carcinoid tumor cells . Mol Cancer Ther 4 : 910 – 7 Weinstock MA , Gardstein B ( 1999 ) Twenty - year trends in the reported incidence of mycosis fungoides and associated mortality . Am J Public Health 89 : 1240 – 4 See related article on pg 2236 How Different Wavelengths of the Ultraviolet Spectrum Contribute to Skin Carcinogenesis : The Role of Cellular Damage Responses Thomas M . Rünger 1 The carcinogenic properties of ultraviolet ( UV ) light are mediated by its ability to generate DNA damage . Cellular responses to UV - induced DNA damage pro - foundly modulate the carcinogenic effects of UV exposures , and these responses are wavelength dependent . However , the exact contributions of different wave - lengths of UV light to DNA damage , cellular damage responses , mutation , and skin carcinogenesis are incompletely understood . Given that UV - induced apop - tosis is a protective cellular response to UV that prevents survival of damaged cells , inhibition of UVB - induced apoptosis by adding UVA , as reported by Ibuki et al . in this issue , may be a mechanism by which UVA augments UVB - mediated mutation and skin cancer formation . Journal of Investigative Dermatology ( 2007 ) 127 , 2103 – 2105 . doi : 10 . 1038 / sj . jid . 5700988 Photocarcinogenesis is most common - ly thought to be the result of a chain of events that involve formation of DNA damage and subsequent mutation for - mation following exposure to ultra - violet light ( UV ) ( Figure 1 ) ( Rünger , 2003 ) . Several cellular defense mech - anisms reduce the likelihood that one occurrence in this cascade leads to the next ( Figure 1 ) . Many of these protec - tive responses can be induced by UV , thereby providing additional protec - tion against subsequent exposures . All of these responses are mediated by a tightly controlled network of signaling pathways , many of which involve the tumor suppressor p53 . UV - induced mutations of p53 with subsequent dis - ruption of cellular damage responses are a hallmark of most sun - induced cutaneous squamous - cell carcino - mas ( Brash et al . , 1996 ) . This further underlines the importance of these damage responses for the prevention of photocarcinogenesis . The melanin microparasol that cov - ers the nucleus of basal keratinocytes reduces formation of DNA damage with exposure of the skin to UV . Once DNA damage is formed , most of it is repaired and does not give rise to mutations . This is exemplified by the genodermatosis xeroderma pigmen - tosum , in which the relative inability to repair UV - induced DNA damage results in UV hypermutability in cells and an increased frequency of skin cancers in UV - exposed areas of affect - ed patients . Another mechanism that inhibits mutation formation at sites of DNA damage is a G1 / S cell – cycle arrest that prevents cells from rep - licating damaged DNA , a situation particularly prone to the introduction of mutations ( Decraene et al . , 2001 ) . DNA polymerase η , which is mutated in xeroderma pigmentosum variant , is a translesion DNA polymerase spe - 1 Department of Dermatology , Boston University School of Medicine , Boston , Massachusetts , USA Correspondence : Dr Thomas Rünger , Department of Dermatology , Boston University School of Medi - cine , 609 Albany Street , Boston , Massachusetts 02118 , USA . E - mail : truenger @ bu . edu | UVA radiation inhibits UVB - induced apoptosis in mouse epidermal cells . \ No newline at end of file diff --git a/silver_data/47508e871afa8ab50970e6e39e901c837dbf453d.pdf.txt b/silver_data/47508e871afa8ab50970e6e39e901c837dbf453d.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..ee4c8a915dd281fd4322c4f569be37e9c2d88a5f --- /dev/null +++ b/silver_data/47508e871afa8ab50970e6e39e901c837dbf453d.pdf.txt @@ -0,0 +1 @@ +LETTERS Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling Oliver Daumke 1 * { , Richard Lundmark 1 * { , Yvonne Vallis 1 , Sascha Martens 1 , P . Jonathan G . Butler 1 & Harvey T . McMahon 1 The ability to actively remodel membranes in response to nucleo - tide hydrolysis has largely been attributed to GTPases of the dynamin superfamily , and these have been extensively studied 1 . Epsin homology ( EH ) - domain - containing proteins ( EHDs / RME - 1 / pincher ) comprise a less - well - characterized class of highly con - served eukaryotic ATPases implicated in clathrin - independent endocytosis 2 , and recycling from endosomes 3 , 4 . Here we show that EHDs share many common features with the dynamin super - family , such as a low affinity for nucleotides , the ability to tubulate liposomes in vitro , oligomerization around lipid tubules in ring - like structures and stimulated nucleotide hydrolysis in response to lipid binding . We present the structure of EHD2 , bound to a non - hydrolysable ATP analogue , and provide evidence consistent with a role for EHDs in nucleotide - dependent membrane remodelling in vivo . The nucleotide - binding domain is involved in dimeriza - tion , which creates a highly curved membrane - binding region in the dimer . Oligomerization of dimers occurs on another interface of the nucleotide - binding domain , and this allows us to model the EHD oligomer . We discuss the functional implications of the EHD2 structure for understanding membrane deformation . EHDs comprise a highly conserved eukaryotic protein family with four members ( EHD1 – 4 ) in mammals and a single member in Caenorhabditis elegans , Drosophila melanogaster and in many eukaryotic parasites , such as the genera Plasmodium , Leishmania and Entamoeba . The proteins have a molecular mass of approxi - mately 60 kDa and contain an amino - terminal G - domain , followed by a helical domain and a carboxy - terminal EH - domain ( Fig . 1a ; in plant homologues the EH - domain is N - terminal ) . The EH - domain is known to interact with Asn - Pro - Phe ( NPF ) motifs in proteins involved in endocytosis . Overexpressed EHDs can be found on tubules inside cells and EHD family members ( including RME - 1 / EHD1 and pincher ) have been shown to regulate exit from the endocytic recycling compartment , TrkA - receptor - mediated macro - pinocytosis and other trafficking pathways 2 – 12 . Here we explore the structure and function of mouse EHD2 as a model for the EHD family . Mouse full - length EHD2 was expressed in bacteria and purified to homogeneity ( Supplementary Fig . 1 ) . The purified protein was nucleotide - free , as judged by HPLC analysis , and was dimeric in analytical velocity centrifugation ( Supplementary Fig . 2 ) . It was pre - viously reported that EHDs—despite havinga predicted G - domain— bind to adenine nucleotides 13 . We confirmed these results by using isothermal titration calorimetry : the affinities for ATP - c - S and ADP were 13 m M and 50 m M , respectively ( Fig . 1b ) . Nucleotide binding was Mg 2 1 - dependent ( data not shown ) . A mutation in the GKT motif in the phosphate - binding ( P - ) loop , T72A , prevented binding to ATP - c - S in a manner similar to that of equivalent mutations in other GTPases . No binding signal was observed for GTP - c - S ( Fig . 1b ) . We still refer to the N - terminal domain as a G - domain because of sequence and fold similarity ( see below ) to other G - domains . We investigated membrane - binding properties of EHD2 and found effi - cient binding to liposomes of brain - derived lipids ( Folch extract ) and to 100 % anionic phosphatidyl - serine ( PtdSer ) liposomes ( Fig . 1c ) . However , using synthetic liposomes containing different phosphatidyl inositols ( PtdIns ) and more stringent conditions ( only 10 % PtdSer , Fig . 1c ) , we observed preferential binding to liposomes containing PtdIns ( 4 , 5 ) bisphosphate , PtdIns ( 4 , 5 ) P 2 . The consequence of membrane binding was analysed by electron microscopy , and we found that EHD2 deforms PtdSer liposomes in a nucleotide - independent manner into 20 - nm diameter tubules and oligomerizes in ring - like structures around these tubules ( Fig . 1d , and Supplementary Fig . 3 ) . Nucleotide independence of liposome tubulation in vitro was also observed for dynamin 1 . No noticeable tubule fission or alteration in tubule diameter was found for EHD2 in the presence of ATP . Frequently , we observed a complex network of connected tubules that had an extensive surface area , implying that there is considerable fusion occurring between liposomes ( Supple - mentary Fig . 3 ) . We also saw a few instances where EHD2 oligomeric rings were of variable diameter ( Fig . 1d , right panel ) suggesting that the interface used for EHD2 oligomerization is rather flexible . Folch liposomes were also tubulated by EHD2 in a nucleotide - independent manner ( data not shown ) . However , with synthetic liposomes con - taining only 2 . 5 % PtdIns ( 4 , 5 ) P 2 and in the absence of PtdSer , the amount of EHD binding was reduced , and in this less - favourable binding condition we only observed tubulation in the presence of ATP - c - S and ADP , but not in the absence of nucleotides ( Supple - mentary Fig . 3 ) . When enhanced green fluorescent protein ( EGFP ) - tagged EHD2 was overexpressed in HeLa cells it marked punctate and tubular structures ( Fig . 1e , endogenous EHD2 in various cell lines shows a similar peripheral distribution 5 , 14 ) . By total internal reflection fluor - escence microscopy , those structures were mainly found close to the plasma membrane ( data not shown ) , consistent with the observed PtdIns ( 4 , 5 ) P 2 specificity . Although the nucleotide - free T72A mutant bound to Folch liposomes in vitro ( Fig . 1c ) , it showed a cytoplasmic distribution when overexpressed in vivo ( Fig . 1e ) , indicating that nucleotide binding is required for oligomerization in vivo , in agree - ment with previous results 3 . The effect of membrane binding on the ATPase activity of EHD2 was monitored under multiple - turnover conditions ( tenfold excess of ATP over EHD2 ) , in the presence and absence of Folch liposomes ( Fig . 1f , and Supplementary Fig . 4 ) . The intrinsic / background * These authors contributed equally to this work . 1 MRC Laboratory of Molecular Biology , Hills Road , Cambridge , CB2 0QH , UK . { Present addresses : Max - Delbru¨ck - Centrum fu¨r Molekulare Medizin ( MDC ) , Robert - Ro¨ssle - Str . 10 , 13092 Berlin , Germany ( O . D . ) ; Umea˚ University , Department of Medical Biochemistry and Biophysics , 90187 Umea˚ , Sweden ( R . L . ) . doi : 10 . 1038 / nature06173 1 Nature ©2007 PublishingGroup ATPase activity is extremely slow ( k obs 5 0 . 7 h 2 1 ) but is stimulated by liposome binding and can be maximally activated eightfold in the presence of saturating Folch or PtdSer liposomes ( k obs 5 5 . 6 h 2 1 ) . In contrast to GBP1 ( ref . 15 ) , we did not observe hydrolysis to nucleo - side mono - phosphate . GTP was not hydrolysed in the presence or absence of Folch liposomes , and the T72A mutant did not show membrane - stimulated ATPase activity . EHD2 displays a 600 - fold slower stimulated nucleotide hydrolysis than dynamin , which hydro - lyses GTP under our standard conditions ( see Methods ) with a k cat of < 1 s 2 1 . To obtain structural insights , we solved the crystal structure of EHD2 in the presence of the non - hydrolysable ATP analogue AMP - PNP ( see Methods , Table 1 and Supplementary Table 1 for the statistics ) . The nucleotide - binding domain of EHD2 possesses a typical G - domain fold with a central b - sheet surrounded by a - helices ( Fig . 2a , b , and Supplementary Fig . 5 ) . An AMP - PNP molecule occu - pies the canonical nucleotide - binding site . In comparison to the Ras - like G - domain , EHD2 contains an insertion of two additional b - strands in the switch I region , which are also present in the G - domain of dynamin 16 ( Fig . 2b ) . Surprisingly , switch I and switch II are not well ordered in this dimeric protein ( Fig . 2a ) . Residues 110 – 130 , which are distal to switch I , are disordered and contain predicted EH - domain binding motifs , KPFxxxNPF . In agreement with our biochemical analysis , EHD2 crystallizes as a dimer , and the dimer axis corresponds to a crystallographic two - fold axis ( Fig . 2a ) . Dimerization is mediated by a highly conserved , mostly hydrophobic interface of approximately 2 , 100 A˚ 2 in the G - domain ( Supplementary Fig . 6 ) . At the centre of the interface , the entirely conserved W238 in helix a 6 is buried in a hydrophobic pocket , and mutations of this residue render the protein insoluble ( data not shown ) . This novel interface is highly conserved among EHD family members and involves a different face of the G - domain to the dimer interfaces from the structurally characterized GBP1 and bacterial dynamin - like protein ( BDLP ) dimers 15 , 17 . The helical domain is composed of helix a 1 and a 2 from the N - terminal region ( residues 18 – 55 , which follow disordered residues 1 – 18 ) and helices a 8 to a 12 ( residues 285 – 400 ) following the G - domain ( Fig . 2a , b ) . Helix a 8 is the organizing scaffold against which most of the other helices fold . It also has extensive contacts with the G - domain . The dimeric G - domain , together with the helical region , adopts a scissor shape , in which the membrane is proposed to bind between the blades ( see later ) . Following the middle domain , a 40 - residue linker connects the helical domain with the C - terminal EH - domain ( residues 443 – 543 ) . The EH - domain of EHD2 is similar to the previously determined second EH - domain of Eps15 solved by NMR studies 18 , 19 , with a root mean squared deviation of 1 . 5 A˚ for the main - chain atoms ( Fig . 2c ) . It is built of two closely packed perpen - dicular EF hands , which are connected by a short b - sheet . We included a Ca 2 1 - ion in the second EF hand , which is ligated by four oxygens from acidic side chains and one main - chain carbonyl oxygen b Molar ratio ( nucleotides : EHD2 ) ∆∆ H p e r m o l e o f nu c l e o t i d e s ( ca l m o l – 1 ) ADP c 0 4 2 0 6 1 , 000 2 , 000 3 , 000 0 20 40 60 80 100 180 160 140 120 100 80 60 40 20 0 200 Time ( min ) PtdIns ( 4 ) P P PtdIns ( 3 ) P PtdIns S S S P P PtdIns ( 5 ) P S P S P S S P P S P S P Folch PtdSer P S P S 1 56 Helical G - domain Helical EH - domain Linker a d Folch P S 285 400 440 543 EHD2 T72A ATP - γ - S K D = 13 µ M K D = 50 µ M T72A - ATP - γ - S GTP - γ - S PtdIns ( 3 , 5 ) P 2 PtdIns ( 4 , 5 ) P 2 PtdIns ( 3 , 4 , 5 ) P 3 PtdIns ( 3 , 4 ) P 2 No lipids Wild type T72A e f A T P r e m a i n i n g ( µ M ) 50 nm 50 nm 500 nm Figure 1 | EHD2 shares common properties with the dynamin superfamily . a , Domain structure of EHD proteins ( numbering from mouse EHD2 amino acids ) . b , EHD2 binds to adenine nucleotides , as determined by isothermal titration calorimetry . For clarity , DD H 5 D H n 2 D H 1 is plotted . c , Coomassie - stained gels of liposome co - sedimentation assays in the presence of 1mM ATP - c - S using 0 . 8 - m m - filtered Folch , 100 % PtdSer or synthetic liposomes containing 10 % of the indicated PtdIns ( plus 70 % Ptd choline , 10 % PtdSer , 10 % cholesterol ) . S , supernatant ; P , pelleted fraction . d , Electron micrographs of negatively stained PtdSer liposomes in the absence ( left panel ) or presence ( middle , right panels ) of EHD2 and 1mM ATP - c - S . The right panel shows an intermediate in the tubulation process , surrounded by EHD2 rings of variable diameter . e , Amino - terminally EGFP - tagged EHD2 wildtype and the T72A mutant were overexpressed in HeLa cells . Confocal images were acquired close to the basal cell surface ; the scale bar is 10 m m . f , Nucleotide hydrolysis by EHD2 was measured by HPLC . Intrinsic reactions in absence of lipids are open symbols ( mean 6 s . d . , n 5 2 ) and stimulated reactions are filled symbols ( mean 6 s . d . , n 5 3 ) . Whereas the intrinsic ATP reaction was eightfold - stimulated by Folch lipids ( open versus filled circles ) , GTP hydrolysis was not stimulated ( open versus filled triangles ) . The nucleotide - free T72A mutant did not show stimulation of ATP hydrolysis ( open versus filled squares ) . Table 1 | X - ray refinement statistics Resolution ( A˚ ) 20 – 3 . 1 Number of reflections 12 , 091 R work / R free ( % ) 23 . 6 / 27 . 6 Number of atoms Protein 3 , 765 Ligand / ion 33 Water 6 B - factorsProtein 61 A˚ 2 Ligand / ion 50 A˚ 2 Water 51 . 5 A˚ 2 Root mean squared deviations Bond lengths ( A˚ ) 0 . 019 Bond angles ( u ) 1 . 119 LETTERS NATURE 2 Nature ©2007 PublishingGroup ( Supplementary Fig . 7a ) . The EH - domain is localized on top of the G - domain ( the top - site position ) with a buried interface of 1 , 600 A˚ 2 . Eighteen disordered residues connecting the EH - domain to the helical domain mean that it is ambiguous which EH - domain con - nects to which helical domain . We assign the EH - domains to oppos - ing monomers ( red Elt - domain belongs to the red helical domain in Fig . 2a ) because the last visible residue of the linker from the helical domain is closer to the opposite EH - domain ( distance 29 A˚ ) than to the superjacent EH - domain ( distance of 34 A˚ ) , and , in this latter case , the linker would have to wind around the EH - domain and thus one would expect to see some parts of this in the structure ( Fig . 2a ) . Unexpectedly , the peptide - binding sites of both EH - domains are occupied by a GPF motif ( residues 420 – 422 ) from the linker region ( Fig . 2a and c ) . The GPF motif adopts a similar conformation to an NPF - containing peptide bound to the EH2 domain of Eps15 ( ref . 18 ) , involving a tight turn with F422 projecting into a hydrophobic pocket that is lined by W490 at its base ( Fig . 2c and Supplementary Fig . 7a ) . R536 in the C - terminal tail of the EH - domain points into the active site of the G - domain below and forms a hydrogen bond to D222 from the NKxD motif , which in other GTPases is responsible for specific recognition of the guanosine base ( see Supplementary Fig . 8 for details ) . Furthermore , M223 sterically prevents binding of GTP , thus explaining the ATP specificity of EHD2 . To test if a polybasic stretch close to the tip of the helical domain was involved in lipid binding , we monitored lipid binding of K327D , K328D and also F322A ( Fig . 3a ) . These mutants showed reduced liposome binding and ATPase activity in the presence of Folch lipo - somes ( Fig . 3b , c ) . In vivo , F322A , and every lysine to aspartate mutation of K324 , K327 , K328 , K329 , led to a cytoplasmic distri - bution of the protein ( Fig . 3d , and data not shown ) . Thus , the lipid interaction site of EHD2 is composed of two closely apposing lipid - binding sites in the dimer , leading to a highly curved mem - brane - binding interface ( Fig . 3a , and Supplementary Fig . 9 ) . Under stringent binding conditions , EHD2 indeed showed a binding pre - ference for very small liposomes , consistent with this curvature , but only in the nucleotide - free form ( Supplementary Fig . 9 ) . In the nucleotide - bound form , EHD binding was not curvature - sensitive , and this is probably due to oligomer formation along an axis per - pendicular to the high curvature ( see below ) . In EHD2 , the phosphate groups of the AMP - PNP molecule are occluded from the exterior by a ‘phosphate cap’ that is formed by switch I and the P - loop , which would not allow the insertion of a catalytic residue in trans into the catalytic site ( Supplementary Fig . 10 ) . We searched for in cis catalytic residues and found that the switch I mutant T94A bound to ATP - c - S with nearly wild - type affin - ity and oligomerized around PtdSer liposomes , but did not show any membrane - stimulated ATPase activity ( Fig . 3e ) . This is consistent with a catalytic function for T94 . When overexpressed in HeLa cells , the T94A mutant labelled extensive tubular structures with essen - tially no punctate staining ( compare wild type and T94A in Fig . 3d , f and g , and Supplementary Movies 1 – 4 ) , suggesting that ATP hydro - lysis is involved in the break - down of tubular structures in vivo . We previously observed severely inhibited GTP hydrolysis and an extens - ive tubulation phenotype ( like that found with EHD2 T94A ) with dynamin 1 when T65 in switch I was mutated to alanine 20 . We ana - lysed another mutant in switch II , in which a glutamine was intro - duced close to the catalytic site . I157Q hydrolysed ATP faster than wild type , even in theabsence of membranes andtubulated liposomes ( Fig . 3e ) . When overexpressed in HeLa cells this protein labelled only very short tubules and puncta ( Fig . 3f , g ) . Many of these puncta were highly mobile as determined by live - cell microscopy ( Supplementary Movies 5 , 6 ) . Thus , there is a clear correlation between the measured ATP hydrolysis rates in vitro and the extent of tubule formation in vivo . The ATPase rates might be further stimulated in vivo , by binding partners or modifications . Altogether , these results are consistent with a role of EHD2 ATP hydrolysis in membrane remodelling and / or the scission of membranes in vivo . A highly conserved surface patch encompasses switch I , switch II and the surrounding area of the EHD2 dimer ( Fig . 4a ) . GBP1 and BDLP use this same interface , with the same relative orientation of the G - domains , for dimerization 15 , 17 . Thus , the EHD2 dimer may further oligomerize into the observed rings ( Fig . 1d ) using this sec - ond G - domain interface . This oligomerization probably leads to the ordering of residues in the switch regions , leading to increased nuc - leotide hydrolysis . Four single mutations of conserved surface - exposed residues in this interface did not affect lipid binding or oligomerization on liposomes but greatly reduced the liposome - stimulated ATPase reaction ( Fig . 4b , Supplementary Fig . 11a ) . The most drastic mutant , K193D , was further tested in vivo and showed extensive tubulation , whereas the E91Q mutant , which had the mild - est effect on the stimulated ATPase reaction , was indistinguishable from wild type in vivo ( Supplementary Fig . 11b ) . These results are Gly Pro Phe Met Val Switch I EH - domain G - domain Helical domain Switch II GPF motif N C C Lipidbinding Disordered loop with NPF motif N N C Ras P - loop Switch I Switch II Switch II Switch I P - loop GPF motif G - domain Helicaldomain EH - domain N C Dynamin P - loop Switch II Switch I c EHD2 b a AMP - PNP EHD2 EH Eps15 EH2 C N Ca 2 + Ca 2 + α 13 α 14 α 2 α 9 α 9 α 11 α 8 α 4 α 5 α 6 α 3 α 7 α 12a α 12b α 1a α 11 α 6 α 8 α 16 β 2 B β 2 A M g 2 + NPF peptide bound to Eps15 EH2 EHD2GPFpeptide Ca 2 + β 2B β 3 β 3 β 1 β 1 β 4 β 5 β 6 β 4 β 5 β 6 β 2 β 2B β 2A β 3 β 1 β 4 β 5 β 6 α 7 α 8 α 12 α 1a α 2 α 2 α 3 α 4 α 1 α 1b α 13 β E1 β E2 α 14 α 16 α 15 C α B α 1 α 2 α 3 α A α 4 α C ′ α C α 5 α 5 α 9 α 11 α 10 α 3 α E α 4 α 5 α 6 β 2 β 2 β 2A Figure 2 | ThestructureofEHD2 . a , Ribbon - typepresentationof the EHD2 dimer ( PDB code 2QPT ) . One molecule is coloured according to the secondary structure ( helices in red , b - strands in green ) and the other according to the domain structure ( see Fig . 1a ) . GPF and NPF motifs are indicated . b , Topology plot of EHD2 ( circles represent a - helices , triangles represent b - strands ) . EHD2 has a dynamin - related switchI extension when compared with the classical Ras - like G - domain ( boxed ) and the dynamin G - domain . c , Left panel , electrostatic surface representation of the EHD2 EH - domain ( atneutralpH : red , negative ; blue , positive ) . F422 ispenetrating in the non - chargedpeptide - bindingpocket . Right panel , close superposition of the EHD2 EH - domain ( dark - green ) with the second EH - domain of Eps15 ( orange ) bound to an NPF - containing peptide ( PDB code 1FF1 ) 19 . NATURE LETTERS 3 Nature ©2007 PublishingGroup c d a F322A Wild type T94A I157Q f 0 0 0 . 9 ± 0 . 1 0 . 66 ± 0 . 02 8 . 8 ± 0 . 8 0 . 64 ± 0 . 03 5 . 4 ± 0 . 2 8 . 8 ± 0 . 7 + + + 40 80 120 160 200 20 40 60 80 100 Time ( min ) K327 K327 K324 K324 K328K329 b Folch liposomes No liposomes F322 F322 Cells expressing a low and medium level of protein ( per cent of total ) Onlypuncta Wild type T94Al157Q Wild type T94A l157Q Puncta and tubules Only tubules Onlypuncta Puncta and tubules Only tubules 12 88 0 86 0 14 0 0 99 0 100 1 0 100 0 0 0 100 P S P S S P S P K327 Wild type F322A e K D for ATP - γ - S , stoichiometry Intrinsic rate ( h – 1 ) Stimulatedrate ( h – 1 ) Tubulation in vitro 13 µM , n = 0 . 96 21 µM , n = 0 . 93 Not measured g K328D Cells expressing a high level of protein ( per cent of total ) A T P r e m a i n i n g ( µ M ) Figure 3 | Membrane binding and the role of ATP hydrolysis . a , Putative membrane - binding site with residues tested for membrane binding in ball - and - stick representation . The highly curved membrane interaction site of EHD2 is indicated . b , Sedimentation assays in the presence ( upper panel ) and absence ( lower panel ) of Folch liposomes using wild - type EHD2 and mutants , as in Fig . 1c . c , Nucleotide hydrolysis of lipid binding mutants as described in Fig . 1f . The wild - type protein had a k obs of 1 . 6h 2 1 ( open circles , intrinsic ; filled circles , lipid stimulated ) . The F322A mutant ( open versus filled inverted triangles ) showed a 40 % decrease in the stimulated ATPase reaction ( k obs 5 3 . 0h 2 1 ) , and the K328D mutant ( open versus filled triangles ) showed a 75 % reduced rate ( k obs 5 1 . 6h 2 1 ) , whereas for the K327D mutant ( open versus filleddiamonds ) stimulation was barely visible . d , EGFP - tagged F322A mutant showed a completely cytoplasmic distribution when overexpressed in HeLa cells . Scale bar , 10 m m . e , Affinity to ATP - c - S was measured as in Fig . 1b . Nucleotide hydrolysis was measured as described in Fig . 1f ( values represent mean 6 s . e . m . ; n 5 2 , intrinsic ; n 5 3 , stimulated reactions ) . In vitro tubulation activity of PtdSer liposomes was analysed as described in Fig . 1d . f , Confocal images of HeLa cells overexpressing the indicated mutants . Scale bar , 10 m m . g , Quantification of the overexpression phenotypes from Fig . 3f . For each construct , three independent experiments with < 50 cells per experiment were analysed . Intrinsic rate ( h – 1 ) Wild type E91Q R167E K193D D198R EHD2 ∆ EH EHD2 0 . 66 ± 0 . 02 0 . 50 ± 0 . 04 1 . 8 ± 0 . 1 0 . 45 ± 0 . 02 0 . 53 ± 0 . 02 0 . 24 ± 0 . 03 0 . 71 ± 0 . 03 5 . 4 ± 0 . 2 1 . 7 ± 0 . 1 2 . 0 ± 0 . 2 0 . 25 ± 0 . 03 1 . 0 ± 0 . 1 0 . 13 ± 0 . 03 0 . 72 ± 0 . 03 8 . 0 3 . 4 1 . 1 – 1 . 9 – 1 + + + + + – – F122A / F128A E91 R167 K193 D198 EH - domain Disordered top - site linker AMP - PNP Disordered NPF loop in G - domain b G - domain Helical domain EH - domain c Phosphatecap Disordered NPF loop in G - domain AMP - PNP Lipid - binding sites EH1 EH2 EH1 EH2 90° a Disordered NPF loop Dimertwo - fold axis 90° 90° Stimulatedrate ( h – 1 ) Fold stimulation Tubulation in vitro Top View of top Figure 4 | The EHD2 oligomer . a , Surface conservation plot of the EHD2 dimer ( in the same orientation as in Fig . 2a , using the alignment in Supplementary Fig . 5 ) with conserved residues in purple and non - conserved residuesincyan . Conservedsurface - exposedresiduesmutatedinthefollowing experiments are indicated . The relative orientation of the dimers in Fig . 4c is alsoindicatedbyarrows . b , Nucleotidehydrolysis ( asinFig . 1f ) formutantsin theproposedoligomerizationinterface . NotethatmutantR167E ( inswitchII ) showed an increased basal ATPase activity , which was not further stimulated by Folch liposomes . Tubulation of PtdSer liposomes was assayed by negative - stainelectronmicroscopyasinFig . 1d . c , ModeloftheEHD2oligomer . Upper panel , top view of the proposed oligomer , limited to four EHD2 dimers , in which the two - fold axis of the dimers is indicated by the black oval . Only the blue – cyandimerhasitsEH - domainsincludedsothattheproposedmovement of the EH - domains from the top - site linker to the side - site of the G - domain canbebettervisualized ( seearrowsformovements ) . TheAMP - PNPmolecules face each other in a head - to - head fashion diagonally across each dimer interface ( seedottedbox ) , asseenintheGBP1andBDLPdimers . Lowerpanel , sideward view of the proposed oligomer ; the membrane - binding sites are facing all in the same direction towards the putative membrane interface . Movies are available at http : / / www . endocytosis . org / EHDs / . LETTERS NATURE 4 Nature ©2007 PublishingGroup consistent with this interface being involved in oligomerization - dependent reorientation of residues responsible for ATP hydrolysis . Using the dimeric GBP1 . GDP . AlF 3 G - domain structure 15 as a tem - plate and taking into account the observed 20 - nm ring of the EHD2 coat ( Fig . 1d ) , we predicted the arrangement of the EHD2 dimers within the oligomer , in which the nucleotides of two opposing EHD monomers are facing each other in a head - to - head fashion ( see Methods and Fig . 4c ) . The predicted oligomer has a compact struc - ture with a high degree of shape - complementarity between the oli - gomerizing dimers ( Supplementary coordinates ) . Furthermore , the membrane - binding sites are pointing in the same direction towards the putative membrane interface ( Fig . 4c , Supplementary Fig . 12a ) , and the thickness of the oligomeric ring agrees well with the thickness of the rings observed by electron microscopy ( < 10 nm , Fig . 1d ) . In the predicted oligomer , the highly curved membrane interface of the EHD2 dimer is oriented perpendicular to the direction of the tubule curvature ( Supplementary Fig . 12b , c ) . Furthermore , the disordered surface loop at the side of the G - domain containing the two con - served PF motifs ( NPF and KPF ) comes into the vicinity of the EH - domain linker ( Fig . 4c and Supplementary Fig . 7b ) . Thus , we speculated that the EH - domain might switch position from the observed top site in the dimer in solution to the side - site position of the opposing dimer , during oligomerization ( Fig . 4c ) . Inter - estingly , only a side - site NPF peptide , and not the top - site GPF peptide , bound with a measurable affinity ( < 130 m M ) to the isolated EH - domain of EHD2 ( Supplementary Fig . 7c ) . Furthermore , in agreement with our prediction we observed that a deletion mutant of the EH - domain ( D EH ) or a double mutant of the two side - site xPF motifs ( F122A / F128A ) did not show any membrane - stimulated ATPase activity ( Fig . 4b ) , and we did not find any regular oligomers on liposomes in electron microscopy studies for these mutants ( see Supplementary Fig . 7 for further discussion ) . On the basis of the conservation of structural and mechanistic elements we propose to expand the dynamin superfamily to include other multidomain large G - domain proteins such as EHDs and BDLPs . The structure outlined above , the architecture of the mem - brane interaction site and the proposed oligomer provide a frame - work to understand membrane remodelling for the EHD family , and perhaps for dynamin superfamily members . The EHD2 dimer inter - acts with membranes via ionic interactions mediated by a highly curved interface and we predict that this interaction results in the insertions of V321 and F322 into the hydrophobic phase of the lipid bilayer . Both the curved interface and the hydrophobic residue insertions ( see synaptotagmin 21 ) will result in the bending of the membrane towards the EHD2 dimer ( buckling ) . The membrane curvature imposed by our proposed oligomer would be perpendic - ular to the curvature imposed by the concave membrane - binding face of the EHD2 dimer ( Supplementary Fig . 12b ) . This would cause considerable curvature stress in the lipid bilayer and would thus facilitate the lipid rearrangement required for the formation of intermediate stages towards membrane fission / fusion 22 . Finally we observe that nucleotide hydrolysis is most probably leading to mem - brane scission in vivo , and thus we would argue that conformational changes induced by nucleotide hydrolysis are transmitted through helix 8 to the membrane - binding interface leading to further mem - brane destabilization . METHODS SUMMARY Isothermal titration calorimetry measurements were performed at 10 u C in 20mM HEPES ( pH7 . 5 ) , 300mM NaCl , 2mM MgCl 2 . Liposome binding assays were performed using 0 . 33mgml 2 1 of 0 . 8 - m m - filtered liposomes as described previously ( www . endocytosis . org ) . For electron microscopic studies , 2 . 5 m M EHD2 in 20mM HEPES ( pH7 . 5 ) , 150mM NaCl , 1mM MgCl 2 was incubated for 15min at 25 u C in the presence of 1mM nucleotide and 0 . 05mgml 2 1 ( final concentration ) oftheindicated0 . 8 - m m - filteredliposomes . Sampleswerespotted on carbon - coated copper grids ( Canemco and Marivac ) and negatively stained with 2 % uranyl acetate . Full Methods and any associated references are available in the online version of the paper at www . nature . com / nature . Received 21 June ; accepted 15 August 2007 . Published online 3 October 2007 . 1 . Praefcke , G . J . & McMahon , H . T . The dynamin superfamily : universal membrane tubulation and fission molecules ? Nature Rev . Mol . Cell Biol . 5 , 133 – 147 ( 2004 ) . 2 . Shao , Y . et al . Pincher , a pinocytic chaperone for nerve growth factor / TrkA signaling endosomes . J . Cell Biol . 157 , 679 – 691 ( 2002 ) . 3 . Grant , B . et al . Evidence that RME - 1 , a conserved C . elegans EH - domain protein , functions in endocytic recycling . Nature Cell Biol . 3 , 573 – 579 ( 2001 ) . 4 . Caplan , S . etal . AtubularEHD1 - containingcompartmentinvolvedintherecycling of major histocompatibility complex class I molecules to the plasma membrane . EMBO J . 21 , 2557 – 2567 ( 2002 ) . 5 . Blume , J . J . , Halbach , A . , Behrendt , D . , Paulsson , M . & Plomann , M . EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids . Exp . Cell Res . 313 , 219 – 231 ( 2007 ) . 6 . George , M . et al . Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C . elegans . BMC Cell Biol . 8 , 3 ( 2007 ) . 7 . Jovic , M . , Naslavsky , N . , Rapaport , D . , Horowitz , M . & Caplan , S . EHD1regulates b 1 integrin endosomal transport : effects on focal adhesions , cell spreading and migration . J . Cell Sci . 120 , 802 – 814 ( 2007 ) . 8 . Lin , S . X . , Grant , B . , Hirsh , D . & Maxfield , F . R . Rme - 1regulatesthedistributionand function of the endocytic recycling compartment in mammalian cells . Nature Cell Biol . 3 , 567 – 572 ( 2001 ) . 9 . Park , S . Y . et al . EHD2 interacts with the insulin - responsive glucose transporter ( GLUT4 ) in rat adipocytes and may participate in insulin - induced GLUT4 recruitment . Biochemistry 43 , 7552 – 7562 ( 2004 ) . 10 . Rotem - Yehudar , R . , Galperin , E . & Horowitz , M . Associationofinsulin - likegrowth factor 1 receptor with EHD1 and SNAP29 . J . Biol . Chem . 276 , 33054 – 33060 ( 2001 ) . 11 . Valdez , G . et al . Pincher - mediated macroendocytosis underlies retrograde signaling by neurotrophin receptors . J . Neurosci . 25 , 5236 – 5247 ( 2005 ) . 12 . Braun , A . etal . EHDproteinsassociatewithsyndapinIandIIandsuchinteractions play a crucial role in endosomal recycling . Mol . Biol . Cell 16 , 3642 – 3658 ( 2005 ) . 13 . Lee , D . W . etal . ATPbindingregulatesoligomerizationandendosomeassociation of RME - 1 family proteins . J . Biol . Chem . 280 , 17213 – 17220 ( 2005 ) . 14 . Guilherme , A . et al . EHD2 and thenovel EHdomain binding protein EHBP1 couple endocytosis to the actin cytoskeleton . J . Biol . Chem . 279 , 10593 – 10605 ( 2004 ) . 15 . Ghosh , A . , Praefcke , G . J . , Renault , L . , Wittinghofer , A . & Herrmann , C . How guanylate - binding proteins achieve assembly - stimulated processive cleavage of GTP to GMP . Nature 440 , 101 – 104 ( 2006 ) . 16 . Reubold , T . F . etal . Crystalstructure oftheGTPasedomainofratdynamin 1 . Proc . Natl Acad . Sci . USA 102 , 13093 – 13098 ( 2005 ) . 17 . Low , H . H . & Lowe , J . A bacterial dynamin - like protein . Nature 444 , 766 – 769 ( 2006 ) . 18 . de Beer , T . , Carter , R . E . , Lobel - Rice , K . E . , Sorkin , A . & Overduin , M . Structure and Asn - Pro - Phe binding pocket of the Eps15 homology domain . Science 281 , 1357 – 1360 ( 1998 ) . 19 . deBeer , T . etal . Molecular mechanism ofNPFrecognitionbyEHdomains . Nature Struct . Biol . 7 , 1018 – 1022 ( 2000 ) . 20 . Marks , B . etal . GTPaseactivityofdynaminandresultingconformationchangeare essential for endocytosis . Nature 410 , 231 – 235 ( 2001 ) . 21 . Martens , S . , Kozlov , M . M . & McMahon , H . T . How synaptotagmin promotes membrane fusion . Science 316 , 1205 – 1208 ( 2007 ) . 22 . Zimmerberg , J . & Kozlov , M . M . How proteins produce cellular membrane curvature . Nature Rev . Mol . Cell Biol . 7 , 9 – 19 ( 2006 ) . Supplementary Information is linked to the online version of the paper at www . nature . com / nature . Acknowledgements Long - term fellowships supported O . D . ( The International Human Frontier Science Program Organization ) , R . L . ( Swedish Research Council ) and S . M . ( EMBO ) . We thank M . Plomann for providing the complementary DNAs for mammalian EHDs , and the ESRF beam staff in Grenoble for their support . The authors declare no competing financial interests . Author Information The atomic coordinates of mouse EHD2 have been deposited in the Protein Data Bank ( PDB ) with the accession number 2QPT . Reprints and permissions information is available at www . nature . com / reprints . The authors declare no competing financial interests . Correspondence and requests for materials should be addressed to H . T . McM . ( hmm @ mrc - lmb . cam . ac . uk ) or O . D . ( oliver . daumke @ mdc - berlin . de ) . NATURE LETTERS 5 Nature ©2007 PublishingGroup METHODS Protein expression and structure determination . Mouse EHD2 full - length protein , the D EH - domain construct ( amino acids 1 – 404 ) and all mutants were expressed as N - terminal His - fusions followed by a PreScission cleavage site in Escherichia coli BL21 DE3 Rosetta ( Novagen ) from a modified pET28 vector . Bacterial cultures in TB medium were induced at a OD 600 of 0 . 2 with 40 m M IPTG , and grown overnight at 18 u C . Bacteria were lysed in 50mM HEPES ( pH7 . 5 ) , 400mM NaCl , 25mM imidazole , 2 . 5mM b - mercaptoethanol , 500 m M Pefablock SC ( Boehringer Ingelheim ) using an Emulsiflex homogenizer ( Avestin ) . After centrifugation at 100 , 000g for 45min at 4 u C , the soluble extract was applied to a NiNTA - column ( Qiagen ) equilibrated with lysis buffer . The column was extensively washed with 20mM HEPES ( pH7 . 5 ) , 700mM NaCl , 30mM imidazole , 2 . 5mM b - mercaptoethanol , 1mM ATP , 10mM KCl , and shortly with 20mM HEPES ( pH7 . 5 ) , 300mM NaCl , 25mM imidazole , 2 . 5mM b - mercaptoethanol . Bound protein was eluted with 20mM HEPES ( pH7 . 5 ) , 300mM NaCl , 100mM imidazole , 2 . 5mM b - mercaptoethanol , and dialysed overnight at 4 u C against 20mM HEPES ( pH 7 . 5 ) , 300mM NaCl , 2 . 5mM b - mercaptoethanol in the presence of 250 m g PreScission protease to cleave the His - tag . The protein was re - applied to a NiNTA column to which it bound under these buffer conditions in the absence of the His - tag . The column was extensively washed with 20mM HEPES , 300mM NaCl , 2 . 5mM b - mercaptoethanol , and the protein finally eluted with 20mM HEPES , 300mM NaCl , 2 . 5mM b - mercaptoethanol , 25mM imidazole , concentrated and further purified using a Sephadex200 size - exclusion column ( two conse - cutive runs for proteins used for the ATPase assays ) . Typical yields were 4mg of purified EHD2 / 1 bacterial culture . At 300mM NaCl , we could concentrate the protein to 40mgml 2 1 , but at lower salt concentration we observed some pre - cipitation at this protein concentration . The protein was partially stabilized by 1mM MgCl 2 . ATPase assays . Multiple turnover ATPase assays were performed in 20mM HEPES ( pH7 . 5 ) , 135mM NaCl , 15mM KCl , 1mM MgCl 2 at 30 u C with 10 m M EHD2 ( or mutants ) as enzyme and 100 m M ATP as substrate , in the absence or presence of 1mgml 2 1 sonicated Folch ( Sigma - Aldrich ) liposomes , with an average diameter of 135nm , as determined by dynamic light scattering . ReactionswerestartedbytheadditionoftheproteintothefinalreactionmixandnucleotidehydrolysiswasfollowedusingstandardHPLCmeasurement 23 . Initial ratesweredeterminedbyapplyingalinearfittodatapointsupto40 % nucleotide hydrolysis . For the dynamin reaction , 1 m M of protein with 1mM of GTP as substrate has been used . Crystallization and structure determination . For crystallization , a selenomethionine - substituted point mutant Q410A was prepared , as described 24 . This mutant showed identical biochemical properties as the wild - type protein but displayed less degradation in the linker region when incubated over longer periods of time . The protein was concentrated to 40mgml 2 1 and supplemented with 4mM MgCl 2 , 2mM AMP - PNP ( Sigma - Aldrich ; both final concentrations ) . The hanging - drop vapour - diffusion method was used for crys - tallization . Protein solution ( 2 m l ) was mixed with an equal volume of reservoir solution containing 3 % PEG2000 MME , 50mM MES ( pH6 . 4 ) , 4mM MgCl 2 . Crystals appeared after one week at 4 u C and had dimensions of < 0 . 2 3 0 . 2 3 0 . 05mm 3 . For flash - freezing in liquid nitrogen , they were first transferred for 10s into 50mM MES ( pH6 . 4 ) , 75mM NaCl , 4mM MgCl 2 , 2mM AMP - PNP , 14 % MPD before incubation in the final cryo - solution containing 50mM MES ( pH6 . 4 ) , 75mM NaCl , 4mM MgCl 2 , 2mM AMP - PNP , 27 % MPD . No crystals were obtained in the presence of ADP or in nucleotide - free conditions . One data set at the selenium peak wavelength was collected from a single crystal at the ESRF beamline ID14 - EH4 ( see Supplementary Table 1 ) and pro - cessed and scaled using the Xds program suite 25 . Crystals belonged to the mono - clinic crystal system and contained one molecule in the asymmetric unit . Thirteen out of sixteen selenium atoms were found from SHELXD 26 using the anomalous signal of the data set . Selenium sites were refined and initial phases were calculated using the program SHARP 27 . In the resulting electron density , themainchainwasclearlytraceable , andaninitialmodelcouldbebuiltusingthe XtalView package 28 . The model was refined using Refmac5 ( ref . 29 ) with 3 TLS groups ( Table 1 ) . The asymmetric unit contains 477 amino acids , one AMP - PNP , onemagnesium , onecalciumandfivewatermoleculesandhasanexcellent geometry with all residues in the favoured and most favoured region of the Ramachandran plot as judged by the program Procheck 30 . Ribbon plots were prepared using the program Molscript 31 and rendered with Raster3D 32 . Surface conservationplotswerepreparedusingtheConSurfserver 33 andCcp4molecular graphics 34 . ElectronpotentialmapsweregeneratedusingCcp4moleculargraph - ics . AllothersurfacerepresentationswerepreparedusingPymol 35 . Topredictthe arrangement of the EHD2 dimer in the oligomer , two EHD2 dimers were super - imposed with one of the two monomers of the GBP1 . GDP . AlF 3 - dimer ( PDB code 2B92 ) using Swisspdb viewer 36 . The EHD2 dimers were manually re - aligned to avoid amino acid clashes such that the two - fold axis between the oligomerizing EHD2 monomers was maintained . A high degree of shape com - plementarity between the EHD2 dimers in the resulting tetramer supported this approach ( Supplementary Fig . 11 ) . Furthermore , the lipid - binding sites of both EHD2dimersareexpectedtocontactthemembrane , andthisrestraintisfulfilled in the tetramer . To obtain a 20nm ring , an 18 u tilt was introduced between the dimers . We refrained from energy - minimising of this structure because major conformational changes in the interface are expected to take place on oligomer - ization ( ordering of switchI and switchII ) and the resolution of the structure is not appropriate for an accurate prediction . The programs Superpose and PdbsetfromCcp4 ( ref . 37 ) wereusedtogeneratetheoligomerfromthetetramer . PDB coordinates of the proposed oligomer are found in the Supplementary Materials . Additional movies of the EHD structure will be posted on http : / / www . endocytosis . org / EHDs / . Ultracentrifugation . Sedimentation velocity experiments were performed in a Beckman Optima XLA ultracentrifuge , using an An - 60Ti rotor . Centrifugation was at 50 , 000revolutions min 2 1 and 5 u C at an EHD2 concentration of 15 m M , with scans as fast as possible ( , 1 . 5min intervals ) . The data were analysed using DCDT 1 v . 2 ( refs 38 , 39 ) , with the partial specific volume for the protein ( from the amino acid composition ) and solvent density and viscosity calculated using Sednterp 40 . Selected scans ( at equal , , 15min intervals ) , of g ( s 20 , w ) ( the amount of material sedimenting between s 20 , w and s 20 , w 1 d s ) and also the residuals for fitting the data with DCDT 1 , were plotted with the program Profit v . 5 . 6 . 7 ( Quantum soft ) . Cell biology . Amino - terminal EGFP - tagged EHD2 and all mutants were over - expressed in HeLa cells from the pEGFP - C3 vector ( Clontech ) . HeLa cells were grown in DMEM containing 10 % fetal bovine serum and transfected using Genejuice ( Novagen ) for transient protein expression . Twenty four hours after transfection , cells were fixed for 20min at 37 u C in 3 . 2 % paraformaldehyde and mounted . All confocal images were taken sequentially using a BioRad Radiance system and LaserSharp software ( Biorad ) . For real - time microscopy , transfected cells on glass - bottom Petri dishes ( WillCo Wells BV ) were washed with 25mM HEPES ( pH7 . 5 ) , 125mM NaCl , 5mM KCl , 10mM D - glucose , 1mM MgCl 2 , 2mM CaCl 2 , and epifluorescence images were taken using an Olympus IX70 microscope ( Southhall ) and Argon laser ( Melles Griot ) with a Princeton in - struments ( Trenton ) - cooled I - PentaMAX camera with MetaMorph software ( Universal imaging ) . 23 . Lenzen , C . , Cool , R . H . & Wittinghofer , A . Analysis of intrinsic and CDC25 - stimulated guanine nucleotide exchange of p21ras - nucleotide complexes by fluorescence measurements . Methods Enzymol . 255 , 95 – 109 ( 1995 ) . 24 . Van Duyne , G . D . , Standaert , R . F . , Karplus , P . A . , Schreiber , S . L . & Clardy , J . Atomic structures of the human immunophilin FKBP - 12 complexes with FK506 and rapamycin . J . Mol . Biol . 229 , 105 – 124 ( 1993 ) . 25 . Kabsch , W . Automatic processing of rotation diffraction data from crystals of initiallyunknownsymmetryandcellvonstants . J . ofAppl . Crystallogr . 26 , 795 – 800 ( 1993 ) . 26 . Sheldrick , G . M . & Schneider , T . R . SHELXL : High - resolution refinement . Methods Enzymol . 277 , 319 – 343 ( 1997 ) . 27 . de la Fortelle , E . & Bricogne , G . in Methods in Enzymology ( eds Carter , C . W . Jr & Sweet , R . M . ) 472 – 494 ( 1997 ) . 28 . McRee , D . E . XtalView / Xfit—A versatile program for manipulating atomic coordinates and electron density . J . Struct . Biol . 125 , 156 – 65 ( 1999 ) . 29 . Murshudov , G . N . , Vagin , A . A . & Dodson , E . J . Refinement of macromolecular structuresbythemaximum - likelihoodmethod . ActaCrystallogr . D 53 , 240 ( 1997 ) . 30 . Laskowski , R . A . , Macarthur , M . W . , Moss , D . S . & Thornton , J . M . Procheck—a program to check the stereochemical quality of protein structures . J . Appl . Crystallogr . 26 , 283 – 291 ( 1993 ) . 31 . Kraulis , P . J . Molscript—a program to produce both detailed and schematic plots of protein structures . J . Appl . Crystallogr . 24 , 946 – 950 ( 1991 ) . 32 . Merritt , E . A . & Murphy , M . E . Raster3DVersion2 . 0 . Aprogramforphotorealistic molecular graphics . Acta Crystallogr . D 50 , 869 – 73 ( 1994 ) . 33 . Landau , M . et al . ConSurf 2005 : the projection of evolutionary conservation scores of residues on protein structures . Nucleic Acids Res . 33 , W299 – W302 ( 2005 ) . 34 . Potterton , E . , McNicholas , S . , Krissinel , E . , Cowtan , K . & Noble , M . The CCP4 molecular graphics project . Acta Crystallogr . D 58 , 1955 – 1957 ( 2002 ) . 35 . DeLano , W . L . ThePyMOLMolecularGraphicsSystem ( DeLanoScientific , PaloAlto , California , USA , 2002 ) . 36 . Guex , N . & Peitsch , M . C . SWISS - MODEL and the Swiss - PdbViewer : An environment for comparative protein modeling . Electrophor . 18 , 2714 – 2723 ( 1997 ) . 37 . Collaborative Computational Project . The CCP4 suite : programs for protein crystallography . Acta Crystallogr . D 50 , 760 ( 1994 ) . 38 . Philo , J . S . A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions . Analyt . Biochem . 279 , 151 – 163 ( 2000 ) . doi : 10 . 1038 / nature06173 Nature ©2007 PublishingGroup 39 . Philo , J . S . Improved methods for fitting sedimentation coefficient distributions derived by time - derivative techniques . Analyt . Biochem . 354 , 238 – 246 ( 2006 ) . 40 . Laue , T . M . , Shah , B . D . , Ridgeway , T . M . & Pelletier , S . L . in Analytical UltracentrifugationinBiochemistryandPolymerScience ( edsHarding , S . E . , Rowe , A . J . & Horton , J . C . ) 90 – 125 ( Roy . Soc . of Chem . , Cambridge , UK , 1992 ) . doi : 10 . 1038 / nature06173 Nature ©2007 PublishingGroup \ No newline at end of file diff --git a/silver_data/475c39bba02f4f649123ec1545fb1d6a0a31fcc8.pdf.txt b/silver_data/475c39bba02f4f649123ec1545fb1d6a0a31fcc8.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..2e4ea5c4d3aa1336920d482a3ae58e6d4b8a277d --- /dev/null +++ b/silver_data/475c39bba02f4f649123ec1545fb1d6a0a31fcc8.pdf.txt @@ -0,0 +1 @@ +1 Pixelated microfluidics for drug screening on tumour spheroids and ex vivo microdissected primary tissue Dina Dorrigiv , a , b Pierre - Alexandre Goyette , b Amélie St - Georges - Robillard , a , c Anne - Marie Mes - Masson , a , d and Thomas Gervais a , b , c * a . Centre de recherche du Centre hospitalier de l’Université de Montréal , ( CRCHUM ) and Institut du cancer de Montréal , Montreal , QC H2X 0A9 , Canada b . Institute of Biomedical Engineering , Polytechnique Montréal , Montreal , QC H3T 1J4 , Canada c . Department of Engineering Physics , Polytechnique Montréal , Montreal , QC H3T 1J4 , Canada d . Department of Medicine , Université de Montréal , Montreal , QC H3T 1J4 , Canada * Corresponding author : thomas . gervais @ polymtl . ca Abstract Anti - cancer drugs have the lowest success rate of approval in drug development programs . Thus , preclinical assays that closely predict the clinical responses to drugs are of utmost importance in both clinical oncology and pharmaceutical research . 3D tumour models preserve the tumoural architecture and are cost - , labour - , and time - efficient . However , the short - term longevity , limited throughput , and limitations to live imaging of these models have so far driven researchers towards simpler , less realistic tumour models such as monolayer cell cultures . Here , we present a static open - space microfluidic drug screening platform that enables the formation , culture , and multiplexed delivery of several reagents to various 3D tumour models , namely cancer cell line spheroids and ex vivo primary tumour fragments . Our platform utilizes an open - space microfluidic technology , a pixelated chemical display , which creates fluidic “pixels” of biochemical reagents that stream over tumour models in a contact - free fashion . Up to 9 different treatment conditions can be tested over 144 samples in a single experiment . We provide a proof - of - concept application by staining fixed and live tumour models with multiple cellular dyes . Furthermore , we demonstrate that the various responses of the tumour models to biological stimuli can be assessed using the proposed drug screening platform . The platform is amenable to various 3D tumour models , such as tumour organoids . Upscaling of the microfluidic platform to larger areas can lead to higher throughputs , and thus will have a significant impact on developing treatments for cancer . Introduction A major impediment to cancer treatment is predicting the response of patients to anti - cancer drugs as they have an extremely low clinical approval rate in drug development programs . [ 1 , 2 ] Improving preclinical models to predict the response of patients to treatments can improve drug precision and effectiveness , spare patients from exposure to unnecessary toxicities , accelerate the drug development process , and ultimately reduce healthcare costs . [ 3 ] , [ 4 ] Various predictive preclinical tumour models are available to researchers . Preclinical tumour models include 2D monolayer cultures of cancer cells , 3D tumour models such as cancer cell line spheroids , tumour organoids , ex vivo cultured tumour fragments , and in vivo models , from the simplest to the most complex , respectively . Monolayer cell cultures are easy to replicate but lack the 3D tumour structure and the interactions between the cancer cells and the tumour microenvironment . [ 5 ] In vivo models are the gold standard of preclinical models , but their production is time - consuming and labour intensive . They may also fail to predict the clinical efficacy of drugs due to species differences . [ 6 ] 3D tumour models can bridge the gap between 2D and in vivo models : unlike 2D 2 monolayers , 3D tumour models mimic the tumoral architecture and are human - derived and easier to work with than in vivo models . [ 7 - 12 ] Three main groups of 3D tumour models exist . In order of increasing complexity and in vivo relevance , they are cell line spheroids , tumour organoids , and ex vivo cultured tumour explants . They can be selected according to the purpose and requirements of a given study . [ 11 ] [ 13 ] Drawbacks of the various 3D tumour models include limitations of live imaging and interfacing with histopathology , and the generally low throughput and low viability of tissue , especially for ex vivo tumour explants . The most advanced live imaging methods , such as confocal and multiphoton microscopy , are well known to have severe limitations in 3D biology , notably their limited light penetration depth in live tissue and cost . [ 14 ] In addition , the universally recognized standard for primary tissue - based clinical decision - making is histopathology [ i . e . , the practice of preserving tumour tissues in paraffin or a freezing medium and dissecting them into thin ( 5 - 10 µm ) slices ] . [ 15 ] To overcome the limitation of live imaging and increase clinical relevance , in particular taking into account routine clinical pathology , it would be advantageous for 3D tumour models to be compatible with standard histopathology practice . Various techniques have been developed for culture and drug screening on 3D tumour models and preparing them for histopathological analyses . The most conventional technique is culturing tumour models in plastic well plates . Samples are subjected to reagents manually or using robotic liquid handlers in wells . Sample manipulation using pipettes , whether manually or using pipetting robots , imposes risks such as aspirating or shearing the sample while changing the medium . In addition , removing the samples out of the wells for further histopathology processing is tedious . Moreover , plastic well plates are not optimal for preserving the viability and metabolic activity of fragile 3D tumour models , such as ex vivo tumour explants . Our group has previously studied the ex vivo survival of tumour tissue explants and has shown that tumour tissue slices cultured in non - perfused well plates start to die in two days due to insufficient oxygen supply . [ 16 ] Microfluidics can palliate this problem by introducing chips for high throughput processing of micron - sized tumour models . [ 5 , 17 ] The drawback of most microfluidic chips is that they require sample entrapment in closed microchannels , and are not amenable to surface - based work environments such as Petri dishes . [ 18 ] Perifusion - based microfluidic devices have been developed to preserve the viability of larger tissue explants over a longer time . [ 19 , 20 ] It is important to differentiate between perfusion and perifusion . [ 21 ] Tumour tissues are dense structures with permeabilities that are orders of magnitude below the permeability of flow channels . [ 22 , 23 ] Creating convective flow inside tumour models ( i . e . , perfusion ) is not feasible unless flow around them is prevented . In most cases , when samples are small ( < 1 mm ) , perifusion is sufficient to avoid any form of starvation , anoxia and necrosis in tissue . [ 24 ] Perifusion - based devices , while presenting a technical breakthrough , have extremely limited throughputs . [ 25 , 26 ] New culture platforms that can improve survival and high throughput drug screening on 3D tumour models , while remaining fully compatible with gold standard tissue analysis , are promising avenues to improve pre - clinical drug testing . In this article , we present a platform that bridges the concepts behind well plates and perifusion - based microfluidics . The platform uses open - space microfluidic laminar flow confinement to stream reagents within self - contained fluidic pixels in which a large number of various 3D tumour models can be placed . The pixels reagent content can be modulated over time with specific frequencies . Open - space microfluidic systems are channel - free and contact - free fluidic processors that deliver reagents directly over the sample . [ 27 ] Pioneering open - space microfluidic systems have been used for various purposes including single cell analysis , [ 28 - 30 ] perifusion - based culture of brain slices , [ 31 ] localized immunohistochemistry , [ 32 ] and imaging mass spectrometry . [ 33 ] The open - space microfluidics system here , which we call the Pixelated Chemical Display ( PCD ) , has been used for various processes over flat 2D surfaces , such as immunoassays . [ 34 ] Here , for the first time , we have utilized the PCD for multiplexed reagent screening over 3D tumour models . To this end , we investigated the stability of the PCD when working over a large number of 3D biological samples deposited in a custom - built microwells array . To provide proof of concept evidence for the applicability of the platform across a whole spectrum of 3D tumour models , we 3 first worked with the simplest 3D tumour models , spheroids , and later with the most complex 3D models , ex vivo tumour explants . Using sequences of cellular dyes , we performed crosstalk - free tissue staining on both 3D tumour models . We have also adapted our previously published paraffin - embedding lithography to transfer all samples simultaneously to a paraffin or optimal cutting temperature ( OCT ) compound block while preserving their spatial orientation . Finally , we investigate the feasibility of using this method to study signalling pathways and cell fate in microdissected tumour tissues . The opportunities and challenges of the method are discussed with respect to competing methodologies such as robotic liquid handlers and closed microfluidic chips . Results and discussion Design and fabrication of the pixelated chemical display drug screening platform The PCD operates based on the hydrodynamic confinement of a stream of fluid in another miscible fluid through recirculation . [ 18 , 35 ] It comprises a blunt tip with multiple apertures and is installed in close vicinity of an immersed substrate . The PCD and the immersed substrate form a Hele - Shaw cell , a quasi - 2D flow that can be precisely computed using potential flow theory . [ 36 , 37 ] During its operation over the substrate , fluid streams are expelled through the injection apertures and re - collected through the aspiration apertures . As a result of the convective recirculation , fluid streams leaving the PCD form well - defined patterns over the substrate without mixing . [ 38 ] Fluidic “pixels” are created when a fluid stream injected above the surface is confined by neighbouring identical fluid streams , forming a repeating flow unit with translational symmetries . [ 39 ] By modulating the design of the PCDs and the injection and aspiration flow rates , different sizes , numbers , and patterns of fluidic pixels can be achieved . Our group has previously demonstrated theoretically and experimentally the operation of up to 144 fluidic pixels ( 12 x 12 ) and demonstrated that the number of active pixels and their reagent content can be modulated without altering the stability of the system . [ 35 ] , [ 39 ] Based on our previous findings , we adapted a 9 - pixel PCD for tissue culture and drug screening . Each pixel is 36 mm 2 ( 6 x 6 mm 2 ) such that the resulting array fits within a paraffin cassette for later embedding ( Figure 1 a , b ) . For this work , each group of 3 pixels was connected to the same reagent flask to create experimental triplicates . Three different conditions were tested in each experiment . We designed and micromachined a microwell array to keep the tumour models in place at the PCD interface during the experiment . The microwell array features 9 groups of 16 microwells for a total of 144 microwells on a polymethylmethacrylate ( PMMA ) slab ( Figure 1 b ) . Each microwell group is covered by an independent fluidic pixel when the PCD is aligned over the microwell array . The PMMA slab also features a flat surface on the side of the microwell array to safely install the PCD and test its operation prior to biological experiments over tumour models ( Figure 1 b ) . 3D printed holder assembly parts ( Figure 1 c ) were fabricated to securely hold the PCD and the PMMA slab together , to stabilize the PCD , and ensure its alignment over the microwell array ( Figure 1 d ) . 4 Pressure pump - operated fluidic lines Syringe pumps were previously used to operate PCDs [ 34 , 39 ] as they offer precise and simple control , and are commonly used in microfluidic systems . [ 40 ] However , syringe pumps , even high precision ones , have relatively high minimum working flow rates , and require frequent recharging of reagents in syringes . [ 41 ] To avoid these limitations , we used pressure pumps in this work . Pressure pumps enable pressurizing of a wide range of flask sizes ( from microliters to litres capacity ) and thus allow for longer - run experiments . More importantly , a single pressure pump can be used to pressurize several reagent flasks , whereas each syringe pump is dedicated to a single syringe . Different flow rates can be achieved in different fluidic lines pressurized by one pump by controlling the hydraulic resistance of the tubes ( i . e . , by using different sizes of tubing ) . In this work , two pressure pumps were used to operate the PCD : one for the injection groups and one for the aspiration . Similar to our previous works , we used 3D - printed manifolds to deliver fluids from one pump into all the pixels sharing the same reagents . [ 39 ] Tubes connecting the manifolds to the PCD were used as precision hydraulic resistors to match the flow rate from all apertures . Four precision flowmeters were installed on the fluidic lines to measure the flow rate for the four injection and aspiration groups . A closed - loop control system with a feedback loop control ( a . k . a . , proportional – integral – derivative [ PID ] controller ) was developed to control the pressure - driven flows . The PID controller Figure 1 PCD components . a ) PCD tip and b ) tubes connected to the PCD , c ) micromachined microwell array featuring 144 microwells , d ) holder assembly parts : holder foundation ( left ) and a bracket to stabilize the PCD once installed over the microwells ( right ) , and e ) schematic of the fully assembled PCD drug screening platform . Scale bar = 1 cm . 5 estimates the deviation between the target and the measured injection flow rates and regulates the pressure to reduce deviations in real time . Moreover , we added medical three - way stopcock valves on the fluidic lines to enable on - demand reagent switching between the various reagent flasks ( e . g . , priming reagents such as ethanol and isopropanol , culture medium , biochemicals , and cellular dyes ) . Switch valves enable us to add or remove reagent flasks without interrupting the system ( Supplementary Figure S1 ) . We refer to the PCD , microwell array , pumps , and fluidic lines complex as the PCD drug screening platform . Finite element simulations Our group has previously studied the mass transport , stability , and reconfigurability of PCDs using 2D convection - diffusion finite - element methods and has demonstrated that stochastic errors such as minor pressure , flowrate changes , or clogging of one aperture do not impact the PCD ’s operation . [ 39 ] Here , we conducted numerical simulations to gain insight into the quality , crosstalk , and stability of fluidic pixels when the PCD is working over 3D structures , such as tumour models . We added a secondary set of simulations to predict the convective - diffusive transport of diluted species in microwells and inside tumour models . We used experimental geometrical and operational parameters : flow rates were selected based on the minimum flow rate that we could achieve with the pressure pumps to have sharp and stable pixels while minimizing reagent consumption . To visualize the pixel formation and crosstalk , we modelled a PCD that functioned in a chequerboard pattern : five injection apertures inject a concentrated solution , and four injection apertures inject a zero - concentration solution ( Figure 2 a ) . Simulation results suggest that the presence of microwells and tumour models does not disturb the pixel shapes , similarly to working over flat impermeable surfaces [ Figure 2 a ( i ) ] : stable crosstalk - free pixels are formed over the microwell array [ Figure 2 a ( ii ) ] . Moreover , we investigated the impact of the tumour model positioning in microwells on fluidic pixels . We modelled a scenario in which random tumour models were partially sticking out of the wells and touched the PCD . We observed that regardless of the tumour model positioning in the well , the pixels are stable ( Figure 2 b ) . We then evaluated the shear stress induced on the cells by the flow and observed that the maximum shear stress imposed by the PCD is 0 . 0021 Pa , which is 500 times less than the physiologically safe shear stress regime for sensitive cells ( ~ 1 Pa ) . [ 42 ] By visualizing velocity fields , we observed that there is no convective flow inside the tumour models [ Figure 2 c ] , showing the diffusion dominant transfer of species inside tumour models . Next , we measured the amount of time required to reach a constant concentration of injected species inside the tumour models with zero initial concentration . Simulation results predict that the system reaches a steady state in less than 20 minutes . The time to reach this state was set as the transition time in the experiments ; upon the change of a reagent flask , reagents were streamed for 20 minutes before starting the experiment countdown . Overall , our simulations predict that the PCD provides excellent control over the fluidic pixels , and locally perifuses the tumour models . Finally , to model the spheroid formation assay ( i . e . , the static culture of 500 µm - tumour models in the microwell array ) , we used passive diffusion and Michaelis - Menten kinetics parameters for oxygen and glucose consumption by cancer cell lines . [ 24 ] The numerical model predicts that spheroids have access to sufficient levels of oxygen and glucose over 24 hours ( i . e . , the typical medium refreshment interval ) ( Figure 2 d ) . 6 High - throughput formation of cancer cell line spheroids is possible in the microwell array Cancer cell line spheroids are self - formed spherical cell aggregates formed from one or more cancer cell lines . [ 43 ] Spheroids are the simplest and most often used 3D tumour models . The commonly used technique to form spheroids is to seed a high - density suspension of cells on a non - adherent surface . Spheroids will form if the cell - cell adhesion forces are greater than the cell - surface adhesion forces . [ 44 ] To meet our claim about the amenability of PCD to work with different tumour models , we optimized the surface modification technique and cell seeding densities based on previous findings to form spheroids directly in the microwell array . [ 45 - 47 ] We further formed spheroids from squamous cell carcinoma and colorectal cancer cell lines to test the practicality of the approach . Figure 3 shows that we can form uniform spheroids in the microwell array in 48 hours . Spheroids were later subjected to the PCD for dynamic cellular staining . Figure 2 Numerical simulations of the PCD operation over tumour models . a ) Fluidic pixels formed over a flat surface a ( i ) are comparable to those formed over the microwell array a ( ii ) ; b ) The positioning of the MDTs or spheroids in the microwells does not impact the operation of the PCD ; c ) arrow plots to visualize the distribution of velocity field in the numerical model suggest the lack of free flow inside tumour models ; d ( i ) Oxygen and d ( ii ) glucose consumption profile in tumour models cultured in the microwell array without perifusion . Tissues of up to 500 µm in diameter can survive in microwells for over 24 hours , as the oxygen and glucose concentration stays above typical K m values ( normalized ) for cancer cells . 7 The PCD drug screening platform enables dynamic multiplexed staining of spheroids To test the performance , stability , and precision of the drug screening platform , we used it to stain spheroids formed in the microwell array . Two phases of reagent streaming over spheroids were used to test the stability of the PCD over subsequent changes of reagents , and to verify its potential for dynamic reagent screening . The PCD streamed culture medium for 20 minutes over the microwell array containing spheroids . Subsequently and without interrupting the system , the culture medium was replaced by three cellular dyes that were streamed in the 9 pixels of the PCD for 2 hours . We then switched the reagent flasks , subjecting spheroids to a second dye . The second part of reagent streaming went on for 3 hours , and cellular dyes were swapped with PBS 1X to purge the dyes ( Figure 4 a ) . Spheroids were imaged using fluorescence microscopy . The results show crosstalk - free staining of spheroids with the colours of interest ( Figure 4 b ) . We further assessed the fluorescent intensity ( FI ) per unit area of spheroids for different channels and demonstrated that spheroids subjected to a dye for 3 hours have a higher FI than spheroids subjected to the same dye for 2 hours ( Figure 4 c ) . Figure 3 . Formation of uniform and compact spheroids of colon cancer cell line HCT - 116 in the microwell array over time . Scale bar = 100 µm . 8 The PCD drug screening platform can process fragile ex vivo tumour tissue explants Ex vivo tumour tissue explants provide an excellent tumour model since they are readily available from biopsy or surgery , do not require disintegration , and mirror the individual’s tumo ur features including the histological and gene expression profiles . [ 17 , 48 ] However , they are the least frequently used 3D tumour model due to the frailty of the tissue and limited throughput . Our group has devised a methodology in which tumour tissues are dissected into sub - millimetre - sized fragments and cultivated on microfluidic chips . [ 49 ] The micro - dissected tumour tissue ( MDT ) methodology yields many MDTs from a small primary tissue and maximizes tissue viability . [ 17 ] Its applications are restricted by the fact that limitations are imposed due to its closed microfluidic chips . To free the system from these limitations , we investigated the application of the PCD drug screening platform to MDTs . Fresh or formalin - fixed MDTs produced from xenografted tumour tissue were deposited in the microwell array and subjected to the PCD . The PCD streamed cellular dyes over the MDT . Images of whole MDTs captured using the fluorescent microscope showed crosstalk - free staining of MDTs with the intended colours , similar to what was observed for spheroids ( Supplementary Figure S2 ) . Tumour tissue microarray Fluorescence microscopy captured the mean fluorescence emission of tumour model structures but did not allow us to examine the distribution of reagents throughout the tumour models . To demonstrate this capability in our Figure 4 Crosstalk - free multiplexed staining of tumour models using the PCD . The PCD is used to stain HCT - 116 spheroids 48 hours after cell seeding . Spheroids are exposed spheroids are subjected to 3 different cellular dyes streaming at the 9 pixels of the PCD for two hours . Then , the reagents were switched so that a different dye was streamed at each pixel for 3 hours . Spheroids were imaged after rinsing out the dyes using an inverted fluorescent microscope . The staining protocol ( a ) , micrograph of stained spheroids ( b ) , and quantification of Fluorescent Intensity ( FI ) of spheroids for each channel ( c ) . Longer incubation with fluorophores results in higher fluorescent emission of spheroids . Blue : Hoechst , green : Celltracker™ Green , red : Celltracker™ Red . Scale bar = 100 µm . 9 system , we developed a methodology to take tumour models out of the microwells and directly embed them in a freezing medium or paraffin . It is also essential to keep the arrangement and orientation of tumour models as they were in the microwells to be able to correlate them with the treatment conditions ( in each fluidic pixel ) that they have been exposed to . For this , we adapted a technique previously described by Jones and Calabresi [ 50 ] to first embed the tumour models in a hydrogel in their microwells . Then , the hydrogel block containing the tumour models is de - moulded from the microwell array and re - embedded in the OCT compound . The OCT blocks were then sectioned into 5 µm - thick slices , and slices were used for further histopathological staining and analysis ( Figure 5 ) . It is noteworthy that this protocol makes the PCD drug screening platform compatible with standard histopathology practice . Supplementary Figure S2 shows cryosections of MDTs that were stained using the PCD for different durations and that underwent the agarose and OCT embedding protocol . The PCD drug screening platform enables the tracking of biological responses in tumour models After demonstrating that the PCD platform is capable of forming crosstalk - free fluidic pixels over various tumour models , we sought to examine the ability of the technology to follow various biological responses in tumour models . For this , we assessed the response of Nuclear factor kappa B ( NF - κB ) transcription factors in MDTs to a cytokine ( tumour necrosis factor [ TNF ] ) stimulation . The NF - κB transcription factor is reported to play a role in tumour angiogenesis and invasiveness and is a possible target to improve the clinical diagnosis and prognosis . [ 51 ] NF - κB resides in the cytoplasm of every cell and is translocated to the nucleus when activated by various stimuli such as cytokines , viruses , and free radicals . [ 52 ] TNF is a proinflammatory cytokine that is known to activate NF - κB . [ 53 ] Real - time monitoring of nuclear translocation of Nf - κB proteins in previous studies revealed a rapid increase in the nuclear signal of sub - units of NF - κB that peaks a few minutes after the exposure , followed by a decline in the nuclear signal of proteins . [ 54 , 55 ] With this in mind , we used the PCD to expose MDTs produced from cell line xenografts to TNF for 0 , 30 minutes , or 240 minutes by progressively switching on TNF delivery in certain pixels by replacing neutral culture medium with a TNF solution . We evaluated the nuclear signal of p65 , an NF - κB subunit , by immunofluorescence ( IF ) staining . As expected , the quantification of the IF staining showed an increase in the Figure 5 The protocol developed to remove the tumour models from microwells while preserving their address for further histopathology analyses . Tumour models are embedded in agarose and removed from the microwell array . Microwell groups exposed to different fluidic pixels are separated , and tumour models that have been subjected to the same treatment condition are regrouped in an OCT block . OCT blocks are sectioned to 5 µm sections to visualize the tissue core and for further immunostaining . 10 nuclear signal of p65 in MDTs that have been subjected to TNF stimulus for 30 minutes compared to the control group . The p65 nuclear signal dropped in the MDTs that were treated for 240 minutes ( Figure 6 ) . This is also expected since p65 translocation is known to be reversible . [ 53 ] To further validate the results , we performed parallel experiments on MDTs produced from the same xenograft that were cultured on chips . The on - chip MDT treatment experiment is a repeat of a protocol previously published by our laboratory for other cell line xenograft MDTs . [ 17 ] Similar responses were also observed in MDTs on chips ( Figure 6 ) . 2D cell cultures of the same cell line treated with TNF showed similar results ( Supplementary Figure S3 ) , substantiating the results seen in MDTs . The results showcase that the PCD drug screening platform can reflect the response of tumour samples to biological stimuli . Material and methods Design and fabrication of parts We followed our previously published methodology to design and fabricate PCDs , manifolds , and the holder assembly . [ 39 ] Briefly , all parts except PCDs and manifolds were designed in Fusion 360 ( Autodesk Inc . , CA , USA ) software . PCDs and manifolds were designed using script - assisted CAD in Catia V5 ( Dassault Systèmes , France ) Figure 6 The PCD drug screening platform can recreate the on - chip response of tumour models to a cytokine . Xenograft cell line MDTs ( TOV 21G ) were treated with a TNF solution for different durations using the PCD and on - chip ( a ) , and the change in the nuclear translocation of p65 was quantified ( b - c ) . Red : p65 and blue : DAPI . Scale bar = 20 µm for zoomed - in MDTs , and 100 µm for whole MDTs . N = 3 . 11 previously developed by our group . [ 34 ] The parts were 3D printed using a stereolithography 3D printer ( PICO2 HD , Asiga , Australia ) . The resin used was Pro3dure GR - 1 black ( P3GR1BLK - 1L , Pro3dure medical GmbH , Iserlohn , Germany ) . After the printing , the excess resin was cleaned by sonication of the parts in an isopropanol bath , then post - cured by UV exposure ( Flash UV Curing Chamber , Asiga ) . To assemble the PCD , 1 / 16” ( RK - 06419 - 01 , Masterflex Tygon , Cole - Parmer , Quebec , Canada ) and 1 / 32” ( RK - 06420 - 01 , Masterflex Tygon , Cole - Parmer ) tubing was plugged and glued using a UV - sensitive resin . Polycarbonate three - way stopcock valves ( RK - 30600 - 02 , Cole - Parmer ) were installed on the fluidic lines . Glue and screws were used to assemble the parts of the holder assembly . The microwell array was micromachined using an MDX - 40A milling machine ( Roland DGA , Irvine , CA , USA ) on a 1 / 8” PMMA slab ( 8560K239 , McMaster - Carr , Elmhurst , IL , USA ) . The holder assembly was fixed over the PMMA slab . Polydimethylsiloxane ( PDMS ; Sylgard® 184 silicone elastomer kit , Dow Corning , Midland , MI , USA ) was used to seal the holder assembly - PMMA slab interface and form a liquid - tight environment inside the holder assembly . Flow rates were controlled using AF1 microfluidic pressure pumps and MFS4 microfluidic flow sensors ( Elveflow , Paris , France ) . Microfluidic chips for drug testing on MDTs were fabricated using our previously published protocol . [ 56 ] System operation The operation of the system was controlled using custom Python ( Python software foundation ) and LabView codes ( National Instrument , Austin , TX , USA ) . A flow rate of 0 . 5 µl / s per aperture was used for all reagent streaming over live tissue . The aspiration to injection flowrate ratio was kept at 1 . 4 . To prepare the system , isopropanol was first streamed at 1 µL / s per aperture in all of the injection and aspiration tubes for at least 15 minutes to wet , prime , and sterilize the fluidic lines . The system was further infused for 5 minutes with PBS 1X ( PBS 10X ; 3072318 , Wisent Inc . , Saint - Bruno - de - Montarville , Canada ) in all lines to purge isopropanol . Next , without pausing the pumps , the PCD was installed in the holder assembly over the immersed flat surface beside the microwell array . The experimental flow rates for injection and aspiration were administered , and the PCD was gently slid over the microwell array . PBS 1X for formalin fixed tissue , or neutral culture medium for live tissue , was administered for 20 minutes to rinse the microwells . Next , the reagents of interest were put in place . For formalin - fixed tissue staining experiments , Sytox ™ Green ( S7020 , Thermo Fisher Scientific , Waltham , MA , USA ) , Nuclear Mask Red ( H10326 , Thermo Fisher Scientific ) , and DAPI ( D1306 , Thermo Fisher Scientific ) were used . 10 mM solution of DAPI in PBS 1X was prepared and aliquoted . Dye solutions were diluted at a 1 : 500 ratio in PBS 1X . For live tissue staining experiments , Celltracke r™ Green ( CMFDA ; C2925 , Thermo Fisher Scientific ) , Celltracker ™ Red ( CMRA ; C34551 , Thermo Fisher Scientific ) , and Hoechst ( Hoechst 33342 , H3570 , Thermo Fisher Scientific ) were used . Dye solutions were diluted at a 1 : 500 ratio in the culture medium . At the end of the experiment , PBS 1X was injected for at least 10 minutes to rinse the fluidic lines and tumour models from the reagents . Experiments were performed on a microscope stage and at room temperature . Injection reagent flasks were put in a water bath at 40 ˚C . Heating the reagent helps to prevent bubble formation in the fluidic lines and over the microwell array . Finite element methodology We used COMSOL Multiphysics© software v . 5 . 6 ( COMSOL Inc , Burlington , MA , USA ) to simulate the convection and diffusion of species under the PCD and within tissue models . Passive diffusion of oxygen and glucose in the static culture of tumour models in between the medium changes was also modelled for the spheroid formation assay . The geometry of the model was drawn using built - in COMSOL drawing tools . The dimensions of the model can be found in Supplementary Table S1 . All simulations were conducted at a constant biological temperature ( 37 °C ) . We used a time - dependent solver to model the PCD tumour model system and the spheroid formation assay . For the PCD tumour model system , Fick’s second law of diffusion and Navier - Stokes equation for laminar flow were applied using the “ transport of diluted species in porous medium ” module . The injection apertures were considered 12 as inflows ( i . e . , source ) , injecting species with two interchangeably varying concentrations ( i . e . , concentrated or zero concentration solutions ) : starting from the top right side of the PCD tip , every other pixel received the concentrated solution , resulting in 5 pixels streaming the concentrated solution and 4 pixels streaming the zero - concentration solution . The aspiration apertures were considered as outflows ( i . e . , sink ) . The aspiration to injection flow rate ratio was optimized to yield sharp pixels at the experimental flow rates and was kept constant throughout the simulation . The operational parameters of the model can be found in Supplementary Table S2 . All the liquid compartments of the model had the physical properties ( i . e . , density and viscosity ) of water at 37 ˚C . The porosity and hydraulic permeability are extremely low for the tumour model compartment . [ 22 , 23 ] With this in mind , we assumed the tumour models non - porous and used the diffusion coefficient of glucose in water to model the transport of concentrated solution in the tissue models . For the spheroid formation assay , the transport of diluted species module was used to model the passive uptake of glucose and oxygen by tumour models . We first simulated oxygen transfer within the tumour models in the spheroid formation assay . We considered a constant oxygen concentration at the medium - air interface over the microwell array . PMMA is not gas - permeable , thus we imposed no - flux ( Neumann ) boundary conditions at the bottom and walls of the microwells . For glucose , we assumed continuity boundary conditions at the medium - tumour model interface . We used Michaelis – Menten ( MM ) kinetics to model cancer cells’ glucose and oxygen consumptio n rates in the spheroid formation assay . The average Michaelis – Menten uptake kinetics found in the literature [ 24 , 49 , 57 ] imply high consumption rates in the abundance of nutrients and decreased consumption rates when nutrients are depleted . The Michaelis – Menten constants refer to concentration thresholds , below which the normal cell metabolism is impacted . [ 58 ] We evaluated the minimum concentration of oxygen and glucose in the core of tissues of 500 µm in diameter . Tissue uptake and diffusion parameters are provided in Supplementary Table S2 . Cancer cell lines xenograft tumour production A human carcinoma cell line derived from an ovarian cancer tumour TOV21G ( RRID : CVCL _ 3613 ) was used to produce mouse xenografts . Ovarian cancer cells were grown as monolayers ( 2D culture ) in OSE medium ( 316 - 030 - CL , Wisent Inc . ) supplemented with 10 % fetal bovine serum ( FBS ; Gibco™ , Thermo Fisher Scientific ) , 55 mg / L gentamicin ( Gibco™ , Thermo Fisher Scientific ) and 0 . 6 mg / L amphotericin B ( Gibco™ , Thermo Fisher Scientific ) . After reaching confluency , cells were detached with 0 . 25 % trypsin - EDTA solution ( Life Technologies , California , USA ) , and cell suspensions ( 1 000 000 cells ) were mixed with Matrigel ( BD Biosciences , Franklin Lakes , NJ , USA ) at a 1 : 1 ratio and subcutaneously injected into the flank of immunodeficient NOD . Cg - Rag1tm1Mom Il2rgtm1Wjl / SzJ female ( Charles River , Wilmington , MA , USA ) . Xenograft tumours were harvested once they reached a volume between 1 500 and 2 000 mm 3 . All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the CRCHUM and approved by the Animal Ethics Committee ( the Comité Institutionnel de Protection des Animaux ) . MDT production from cell line xenograft tumours We used our previously published method [ 17 , 56 ] for the production of MDTs . Briefly , a tissue chopper ( McIlwain , Ted Pella , Redding , CA , USA ) was used to cut the xenograft into 350 µm - thick tissue slices . Tissue slices were kept in Hank’s Balanced Saline Solution ( HBSS , 311 - 516 - CL , Wisent Inc . ) supplemented with serum and antibiotics . Tissue slices were further punched into MDTs using a 500 µm diameter tissue punch ( Zivic Instruments , Pittsburgh , PA , USA ) and kept in HBSS supplemented with antibiotics . 13 Microwell preparation and MDT loading in the microwell array Similar to PDMS devices [ 49 ] , the microwell arrays were wetted and rendered hydrophilic by plasma treatment and rinsed with 100 % ethanol . They were then sterilized by soaking in 70 % ethanol for 15 minutes and prepared by incubation with a triblock copolymer ( Pluronic® F - 108 , Sigma - Aldrich , St . Louis , MO , USA ) overnight ( at least 16 hours ) at 37 °C in a 5 % CO 2 incubator . The microwell arrays were then rinsed with PBS 1X three times to purge the Pluronic® F - 108 solution . We adapted the previously published method of our laboratory to load the MDTs in the microwells . [ 17 , 56 ] Briefly , the overlay liquid over the microwells was removed . 16 MDTs were picked using a 20 µL pipette and emptied over a microwell group . MDTs were diverted towards empty microwells using the pipette tip where they would fall in the microwells . In the case of more than one MDT falling in a microwell , the extra MDTs were pipetted out of the well and transferred to empty wells . This process was repeated for all 9 microwell groups . Spheroid formation assay We used a human squamous cell carcinoma FaDu ( RRID : CVCL _ 1218 ) and a human colon cancer cell line HCT - 116 ( RRID : CVCL _ 0291 ) for spheroid formation experiments . Cells were grown as monolayers ( 2D culture ) in Dulbecco ' s Modified Eagle Me dium ( DMEM ; 11965118 , Gibco™ , Thermo Fisher Scientific ) supplemented with serum and antibiotics . After reaching confluency , cells were detached and cell suspensions of 2 000 000 cells in 1 ml of culture medium were prepared . 400 µL of cell suspension were seeded over each microwell array , and the cell suspension was replenished three times to exchange the liquid in the microwells with the cell suspension . 15 minutes after the cell seeding , the cell suspension over the microwell array was removed by drawing 400 µL of the cell suspension and adding 400 µL of medium to remove the floating cells over the microwell array . Medium was changed every 24 hours by adding 400 µL of fresh medium near one corner of the microwell array , removing 400 µL from the opposite corner , and repeating the process three times . OCT embedding protocol Following fresh tissue experiments , some tumour models underwent formalin fixation in the microwell arrays . 400 µL of formalin was added near one corner of the microwell array and removed from the opposite corner and repeated three times . The tumour models were incubated in formalin for 40 minutes and formalin was rinsed by three washes with PBS 1X . For agarose embedding , an 8 % solution of agarose ( Ultrapure ™ Low Melting Point Agarose ; 16520100 , Thermo Fisher Scientific ) in PBS 1 % was prepared by dissolving 8 g of agarose in 100 ml PBS 1X and microwaving the solution for 80 seconds ( 4 cycles of 20 seconds ) or until agarose powder was completely dissolved . The agarose in PBS solution was then cooled down to 62 ˚C . 400 µL of agarose solution was discharged and removed three times over the microwell array using a positive displacement pipette . The microwell array was placed in an oven at 60 ˚C for 30 minutes to ensure agarose permeates in the microwells and tissues . The microwell arrays were further cooled at 4 ˚C for 30 minutes , and the agarose layer was peeled of f gently . If tumour models were left in the microwells after the removal of the agarose , a needle was used to remove the tumour models from the wells and add them to the agarose tissue array . The agarose block was cut to separate the microwell groups , and microwells subjected to the same treatment condition were placed in the same plastic mold , ensuring that tissues were touching the bottom of the plastic mold . OCT was poured gently over the agarose block to prevent bubble formation . Plastic molds were placed on a flat and levelled surface in dry ice and cooled down for 20 minutes for OCT to solidify . Each OCT block was sliced into 5 µm - thick sections using a cryostat , and each section was placed on a TOMO® hydrophilic adhesion slide ( Matsunami , Bellingham , WA , USA ) . 14 Histopathological staining OCT sections underwent hematoxylin and eosin ( H & E ) staining as well as IF staining to assess the expression of p65 protein ( Anti - NFkB p65 protein ; SC - 8008 , Santa Cruz , Texas , USA ) and DAPI in the tumour models . IF staining was performed using the BenchMark XT automated stainer ( Ventana Medical System Inc . , Tucson , AZ ) . Antigen retrieval was carried out with Cell Conditioning 1 ( # 950 - 123 , Ventana Medical System Inc ) for 90 minutes for all primary antibodies . Mouse anti - p65 ( 1 : 200 ) antibody was automatically dispensed . The slides were incubated at 37 °C for 60 minutes and secondary antibodies were incubated at room temperature on the bench . We used our lab oratory’s protocol to quantify the TNF response in 2D culture . [ 59 ] Briefly , cells were seeded onto coverslips at 20 000 cells / well in 24 - well plates . After 24 h , cells were incubated with TNF solution for 5 minutes or 2 hours . Cells were fixed with formalin for 15 min utes at room temperature , washed using PBS 1X , permeabilized with 0 . 25 % Trito n ( Triton™ X - 100 solution ; 93443 , Sigma - Aldrich ) , and incubated with mouse anti - p65 ( 1 : 400 ) overnight . Primary antibody was detected by incubation with secondary antibody for 60 minutes . Coverslips were mounted onto slides using Prolong Gold® anti - fade rea gent with DAPI ( 14209 S , Life Technologies Inc . ) . All sections were scanned with a 20x / 0 . 75 NA objective with a resolution of 0 . 3225 μm ( bx61vs , Olympus , Toronto , Ontario ) . Tumour model treatment with TNF For cytokine stimulation experiments , MDTs were exposed to a neutral culture medium or to a 20 ng / ml of TNF solution ( Recombinant TNF alpha human ; 300 - 01A , PeproTech , Thermo Fisher Scientific ) in culture medium for either 30 minutes or 240 minutes . The TNF treatment using the PCD lasted 300 minutes . First , the PCD streamed neutral culture medium at every pixel for 20 minutes . Then , we streamed TNF in one group ( 3 pixels ) while the two remaining groups ( 6 pixels ) received a neutral culture medium . At 230 minutes , TNF streaming was started in a second group . For the next 50 minutes , TNF was streaming at 6 pixels , all the while the control group on the same 3 pixels received culture medium . At 280 minutes , we swapped all reagent flasks for PBS 1X , and PBS 1X was streamed for 20 minutes to rinse the tumour models . The PCD was then removed , and the immersion liquid over the microwell array was withdrawn . Tumour models underwent OCT embedding for further histopathology processes . We followed our group’s protocol fo r MDT treatment on - chip . [ 17 ] Quantification of immunofluorescent staining To measure the FI of tumour models stained using the PCD , an open - source image processing software ( Fiji ) was used . [ 60 ] At least 3 spheroids were randomly selected in each fluidic pixel , and the corrected FI per area ( subtracting the background FI from tissue FI ) was calculated for each fluorescent channel . The average corrected FI per area of the 3 pixels subjected to the same treatment was compared between the 2 - and 3 - hour incubation time for each channel . To quantify protein expressions using immunofluorescent staining , we used VisiomorphDP software ( VisioPharm , Hørsholm , Denmark ) [ 40 , 41 ] . Briefly , the tissue core surface area was detected through the DAPI channel . The nuclear signal of p65 was quantified by dividing the surface area of p65 - positive nuclei by the total surface area of the nuclei . We used a similar approach for quantifying p65 translocation in 2D culture of the cells . [ 61 ] Statistical analysis Statistical analyses were performed in GraphPad Prism version 8 . 0 ( San Diego , CA , USA ) using the non - parametric one - way ANOVA Kruskal – Wallis test and post hoc Dunn’s test , because the data were not normally distributed according to the D’Agostino and Pearson omnibus normality test . For TNF treatment experiment , a minimum of 15 MDTs were analyzed for each condition , and e xperiments were repeated three times ( N = 3 ) . All data are 15 reported as the mean ± standard error of the mean ( SEM ) unless otherwise stated . The reported p - values were generated using a post hoc test ( Dunn’s test ) . Conclusions The need to improve the predictive power of in vitro and ex vivo model systems to maximize the chances of success in clinical trials has made 3D tumour models , such as microdissected tissue and cancer cell line spheroids , attractive in preclinical settings . [ 62 , 63 ] Thus , it is essential to implement tools and techniques to automate drug screening on 3D tumour model systems and make them compatible with clinical practices . To address this , we introduced a drug screening platform for automated simultaneous streaming of up to 9 reagents on 144 tumour models . We used human cancer cell line xenograft and spheroid models to validate the potential of the PCD drug screening platform for multiplexed and dynamic streaming of biochemicals over tumour models . Microtissues processed in the drug screening platform can directly be transferred to an embedding medium and undergo various endpoint measurements ( i . e . , immunohistochemistry , immunofluorescence , and H & E ) . These measurements are standard protocols in clinical and pharmaceutical practices and allow the monitoring of multiple biological pathways . Furthermore , because the platform is amenable to different 3D tumour models , it allows the co - culture of spheroids , organoids , and ex vivo tumor tissue explants . In turn , this enables comparing treatment efficacy on various tumour models . The main drawback of the PCD arises from the continuous streaming of reagents . Even though flow rates are extremely low , streaming over several hours consumes a considerable amount of reagents , and thus limits applications in cases where reagents are extremely expensive ( e . g . , recombinant protein drugs ) . However , a highly parallel drug screening assay using the PCD would probably even be worthwhile despite the high reagent consumption . We have reported a low number of large pixels ( 9 × 6 mm 2 ) in this article . However , PCDs of up to 144 × 1 mm 2 pixels have been produced routinely in our laboratory with successive reagent changes as fast as 1 change per 30 s . This makes the PCD drug screening platform appealing for highly parallel and dynamic assays . The reconfigurable sizes and numbers of pixels along with the fast reagent change will speed up the throughput . Compared to traditional well plate - based approaches [ 64 ] and their downsized microfluidic counterparts such as InSphero GravityTRAP™ , [ 65 ] idenTx™ , [ 66 ] and Organoplate® [ 67 ] , our platform does not require the manual delivery of reagents to microtissues or the use of robotic liquid handlers . This should greatly reduce the time and cost required to perform the experiments . Moreover , the possibility of the direct transfer of tumour models to an embedding medium reduces the potential tissue damage and makes our platform more efficient compared to previous approaches where each individual sample is transferred from well plates to an embedding medium . Author contributions Conceptualization : D . D . , P . - A . G . , A . - M . M . - M . and T . G . ; Data curation , D . D . ; Formal analysis , D . D . ; Funding acquisition , A . - M . M . - M . and T . G . ; Investigation , D . D . , A . - M . M . - M . and T . G . ; Methodology , D . D . , P . - A . G . , A . S . R . , A . - M . M . - M . and T . G . ; Project administration , A . - M . M . - M . and T . G . ; Resources : A . - M . M . - M . and T . G . ; Supervision , A . - M . M . - M . and T . G . ; Validation , D . D . , A . S . R . , P . - A . G . , A . - M . M . - M . and T . G . ; Visualization , D . D . , and A . S . R . ; Writing — original draft , D . D . and T . G . ; Writing — review and editing , A . S . R . , and T . G . All authors have read and agreed to the published version of the manuscript . Conflicts of interest T . G . is the co - founder and Chief Technological Officer of MISO Chip Inc . , a company operating in the field of ex vivo tissue culture . 16 Acknowledgements We acknowledge Kim Leclerc - Desaulniers for technical assistance with the animal work . We thank the CRCHUM Microfluidics Core Facility supported by the TransMedTech Institute and its main financial partner , the Canada First Research Excellence Fund and namely Jennifer Kendall - Dupont and Benjamin Péant for performing tissue dissection and useful scientific and technical discussions . We thank Liliane Meunier and Véronique Barrès of the CRCHUM Molecular Pathology Core Facility for performing the OCT block sectioning and slide scanning . We acknowledge Jacqueline Chung for manuscript editing . T . G . acknowledges CMC Microsystems . References 1 . 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Smalley , K . S . , et al . , Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases . Molecular cancer therapeutics , 2006 . 5 ( 5 ) : p . 1136 - 1144 . 65 . Falkenberg , N . , et al . , Three‐dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer . Cancer medicine , 2016 . 5 ( 4 ) : p . 703 - 710 . 66 . Teh , J . L . , et al . , Rapid spheroid assays in a 3 - dimensional cell culture chip . BMC research notes , 2021 . 14 ( 1 ) : p . 1 - 6 . 67 . Lanz , H . L . , et al . , Therapy response testing of breast cancer in a 3D high - throughput perfused microfluidic platform . BMC cancer , 2017 . 17 ( 1 ) : p . 1 - 11 . 19 Supplementary material Supplementary figures Figure S1 : Fluidic connections in the PCD drug screening platform . The use of valves allows for switching between the streaming of various reagents and the flowrate sensors allow for control and validation that the platform is working correctly . OD ; outside diameter Figure S2 : Micrographs of MDTs stained with various cellular dyes using the PCD . Images taken from the tumour model cores that have been treated with cellular dyes for different amounts of time show that core cells are not stained in the shorter treatment durations . 20 Figure S3 : time - dependent treatment of 2D culture of TOV21G cells with TNF shows a response similar to MDTs treated on - chip or using the PCD . This further validates the potential of the PCD drug screening platform to predict the response of 3D tumour models to stimuli . Supplementary tables Table S1 dimensions Parameter Value ( µm ) Microwell array well height 900 well width 700 well length 700 Distance between microwells 1000 PCD Gap between the PCD and the microwell array 100 Aperture diameter 200 Distance between apertures ( i . e . , pixel size ) 5000 Number of aspiration apertures 16 Number of injection apertures 9 Tissue Tissue diameter 450 21 Table S2 tissue uptake parameters , diffusion properties , the PCD working condition Parameter value Diffusion / Reaction Parameters Diffusion constant of glucose ( cm 2 / s ) Tissue 2 . 7x10 - 6 1 , 2 Medium 9 . 27x10 - 5 1 Agar 5 % Same as water 3 , 4 Diffusion constant of oxygen ( cm 2 / s ) Tissue 1 . 8x10 - 5 Medium 2 . 6x10 - 5 Agar 5 % 2x10 - 5 4 PDMS 3 . 4x10 - 5 5 , 6 Diffusion constant of Carboplatin / Paclitaxel ( cm 2 / s ) Could we use glucose properties ? 7 , 8 Saturation concentration ( mM ) Oxygen Tissue 1 . 02 medium 0 . 21 Agar 5 % 0 . 21colagen 9 PDMS 1 . 43 Glucose 11 Oxygen partition coefficient ( relative solubility of oxygen ) PDMS - Medium 0 . 15 Medium - Tissue 4 . 8 Maximum cellular uptake rate ( mM / S ) Oxygen 2 . 07 Glucose 1 . 09 Michaelis - Menten constant ( mM ) Oxygen 4 . 63x10 - 3 Glucose 4x10 - 2 PCD working conditions Injection pressure ( Pa ) 4 Aspiration pressure ( Pa ) 4 Injection / Aspiration flowrate ( nL / s ) 100 \ No newline at end of file diff --git a/silver_data/4800eb429c06a9951220fb5e0d1cfa1a2d155018.pdf.txt b/silver_data/4800eb429c06a9951220fb5e0d1cfa1a2d155018.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..02741973cac7390ce22a54ed01123843c853f9c8 --- /dev/null +++ b/silver_data/4800eb429c06a9951220fb5e0d1cfa1a2d155018.pdf.txt @@ -0,0 +1 @@ +C H A P T E R F O U R Live - Cell Imaging of Clathrin Coats Comert Kural * , † and Tom Kirchhausen * , † Contents 1 . Introduction 60 2 . Dynamics of Clathrin Assembly 61 3 . Limits of Fluorescence Microscopy 62 4 . TIRF Microscopy 63 5 . How to Optimize Your TIRF System 64 6 . Spinning - Disk Confocal Microscopy 65 7 . Spherical Aberration Correction Applied to Spinning - Disk Confocal Microscopy 66 8 . Getting Around the Diffraction Limit 67 9 . Use of 2 D Spinning - Disk Confocal Microscope to Study Clathrin - Mediated Endocytosis at the Ventral and Dorsal Surfaces of a Cell 69 10 . Use of TIRF Microscopy to Study Clathrin - Mediated Endocytosis 70 11 . The Third Dimension 71 12 . 3 D Tracking in Spinning - Disk Imaging 72 13 . Use of 3 D Tracking to Monitor Clathrin - Mediated Entry of Reovirus Particles at the Apical Surface of Polarized Cells 74 14 . Using the Optimum Pixel and Step Sizes in z - Stacks 76 15 . Conclusion 76 Acknowledgments 77 References 77 Abstract We compare the use of two - dimensional total internal reflection fluorescence microscopy with a rapid , simple - to - implement method for three - dimensional ( 3 D ) imaging using spinning - disk confocal microscopy suitable for reliable 3 D tracking of clathrin - coated endocytic and endosomal carriers . These carriers contain about 20 EGFP ( enhanced green fluorescent protein ) equivalents of a chimeric fluorescent protein ( either clathrin light chain or one of the clathrin adaptor subunits ) . Under tissue culture conditions , the clathrin - containing Methods in Enzymology , Volume 505 # 2012 Elsevier Inc . ISSN 0076 - 6879 , DOI : 10 . 1016 / B978 - 0 - 12 - 388448 - 0 . 00012 - 7 All rights reserved . * Department of Cell Biology , Harvard Medical School , Boston , Massachusetts , USA { Immune Disease Institute and Program in Cellular and Molecular Medicine at Children’s Hospital , Boston , Massachusetts , USA 59 carriers correspond to a variable number of relatively sparse , diffraction - lim - ited , fluorescent objects that can be identified with a spatial precision of (cid:1) 30 nm or better and a temporal resolution of < 1 s . The applicability of these approaches to mammalian cells in culture allows investigators detailed monitoring of the composition dynamics of the clathrin - containing carriers which can then be used to study in living cells the molecular mechanisms required for the formation and traffic of clathrin - coated pits and vesicles . 1 . Introduction Cells require ordered movement of proteins and lipids from one membrane - bound compartment to another , while maintaining the organi - zation , function , and heterogeneity of the donor and acceptor membranes . A number of molecular assemblies ( e . g . , those based on clathrin , COPI , or COPII coatomers ) have evolved to deform and invaginate membrane patches , which after pinching and scission become carriers of membrane traffic . Clathrin - coated pits and vesicles were the first membrane - traffic system to be recognized and analyzed in detail , because of the distinctive morphology of budding coated pits , the ease with which coated vesicles could be purified , and the importance of clathrin - coated structures for receptor - mediated endocytosis . Clathrin - coated vesicles are the most prominent form of traffic from the plasma membrane to endosomes ( endo - cytosis ) , a pathway by which ligands such as hormones , transferrin , immu - noglobulins , LDL ( low - density lipoprotein ) , viruses , and their receptors enter cells . They are also important for traffic between endosomes and the trans - Golgi network ( TGN ; Bonifacino and Traub , 2003 ; Brodsky et al . , 2001 ; Duncan and Payne , 2005 ; Hirst and Robinson , 1998 ; Kirchhausen , 2000 ; McMahon and Mills , 2004 ; Robinson , 2004 ; Traub , 2005 ; Ungewickell and Hinrichsen , 2007 ) . They have become a paradigm for efforts to understand molecular mechanisms of other modes of vesicular transport ( Harrison and Kirchhausen , 2010 ; Lee and Goldberg , 2010 ) . Cellular , biochemical , and high - resolution structural approaches have defined the molecular properties of clathrin and many of its associated proteins ( Brett and Traub , 2006 ; Edeling et al . , 2006 ; Gaidarov and Keen , 2005 ; Keyel et al . , 2006 ; McMahon and Mills , 2004 ; Miwako and Schmid , 2006 ; Motley et al . , 2006 ; Owen et al . , 2004 ) . In our own work , we have combined crystal structures with electron cryomicroscopy to image a clathrin coat at 8 A˚ resolution ( Fotin et al . , 2004b ) , a coat in association with a fragment of auxilin at 12 A˚ resolution ( Fotin et al . , 2004a ) , and a coat associated with both the auxilin fragment and a specifically bound Hsc70 ( Xing et al . , 2010 ) . But biochemical and structural approaches , however powerful , can only provide snapshots or ensemble - averaged information 60 Comert Kural and Tom Kirchhausen about the properties of objects within a heterogeneous population . They are not sufficient to resolve important steps in coated vesicle formation and uncoating . To achieve temporal resolution in the context of a cell , many groups have turned to advanced imaging methods to study the localization of components during a given step , the order in which components are incorporated or released , and the way composition of an assembling vesicle affects its behavior ( Ehrlich et al . , 2004 ; Gaidarov et al . , 1999 ; Kaksonen et al . , 2005 ; Keyel et al . , 2004 ; Le Clainche et al . , 2007 ; Loerke et al . , 2009 ; Merrifield et al . , 2002 , 2005 ; Mettlen et al . , 2009 ; Newpher et al . , 2005 ; Rappoport et al . , 2005 , 2006 ; Saffarian and Kirchhausen , 2008 ; Yarar et al . , 2005 ; Zoncu et al . , 2007 ) . By analyzing assembly and disassembly of individual molecular complexes with fluorescence microscopy , one can link in vitro reconstitution studies , in which molecular concentrations and other external conditions can be fixed , thereby circumventing the hard - to - control complexities of an intact cell , with results from live - cell imaging , in which essentially identical detection schemes follow the same processes in their complete biological context . This review highlights our use of total internal reflection fluorescence ( TIRF ) and spinning - disk confocal imaging of living cells to investigate the dynamics of clathrin coat formation . Depending on the acquisition mode ( TIRF or spinning - disk confocal microscopy ) , the temporal resolution ranges between 10 and 100 ms . The required signal is typically emitted by 2 – 5 fluorescent molecules . Under carefully controlled TIRF conditions , it is also possible to record the signal from a single EGFP molecule . The spatial precision attained under these circumstances is 10 – 30 nm along the x - , y - , and z - axis ( Kural et al . , submitted ; Saffarian and Kirchhausen , 2008 ) . The signal - to - noise ratio ( SNR ) and resolution depend , in part , on such issues as detector response and particle tracking . We concentrate here primarily on the contributions of imaging modalities . 2 . Dynamics of Clathrin Assembly Our current picture of coated pit formation derives primarily from analysis by live - cell imaging of coated pits and vesicles at the plasma mem - brane ( Fig . 4 . 1 ) . The most important structural components of the assembly are clathrin and the AP - 2 ( a - b 2 - m 2 - s 2 ) heterotetrameric adaptor complex . A number of accessory proteins associate with coated pits at specific stages of assembly / disassembly ( Henne et al . , 2010 ; Reider et al . , 2009 ; Toshima et al . , 2006 ; Traub , 2009 ) . Eps15 , epsin , FCHo1 / 2 , and intersectin form an inter - acting complex that appears to be localized at the rim of a coated pit ( Henne et al . , 2010 ; Reider et al . , 2009 ; Saffarian et al . , 2009 ; Tebar et al . , 1996 ; Traub and Wendland , 2010 ) . This “rim complex” accumulates during early stages Live - Cell Imaging of Clathrin Coats 61 of coated pit assembly , but its components are excluded from a budded coated vesicle . Dynamin , the GTPase that drives membrane scission , accu - mulates both gradually during pit assembly and in a burst following clathrin lattice completion ( Ehrlich et al . , 2004 ; Loerke et al . , 2009 ; Macia et al . , 2006 ; Rappoport and Simon , 2003 ) . Auxilin and Hsc70 arrive following scission , to direct uncoating ( Lee et al . , 2006 ; Massol et al . , 2006 ) . Hip1R , which binds clathrin light chains , recruits actin , required in some instances for coated vesicle maturation and budding ( Ferguson et al . , 2009 ; Merrifield et al . , 2002 , 2004 ; Saffarian et al . , 2009 ) . 3 . Limits of Fluorescence Microscopy Even though fluorescence microscopy techniques enable real - time recordings from living cells and , hence , yield temporal information about cellular processes , spatial resolution is lost in most cases . Like most of the biological machinery , clathrin - coated structures ( (cid:1) 100 nm in diameter ) are smaller than the wavelength of the visible light ( (cid:1) 500 nm ) , resulting in a Initiation Growth / completion / scission Uncoating Clathrin coat Rim proteins Rim proteins + LCs / Hip1 / Hip1R F - BAR ( endophilin , FBP17 , . . . ) CortactinArp2 / 3 N - WASP Actin 100 n m Dynamin AuxilinHsc70 ( eps15 , epsin , FCHo1 / 2 , SIG11a ) Figure 4 . 1 Coated pit formation proceeds by sequential addition of clathrin triskelions to an initial nucleus , generating a sharply curved coat ; adaptor - mediated interactions with membrane - bound proteins ( and lipids ) deform the underlying membrane ; dynamin mediates scission when the deformation has created a suitably narrow neck ; auxilin , which arrives immediately following scission , recruits the uncoating ATPase , Hsc70 . Under conditions of membrane tension ( hyposmolarity , cell stretching , apical membranes of polarized cells , elongated cargo ) , coated pit maturation requires the formation of short - branched actin filaments ; by contrast , actin polymerization does not generally accompany the assembly or budding of coated pits in membranes without tension . Continuous line , plasma membrane ; dashed stripe , a clathrin coat ( clathrin plus AP - 2 adaptor ) ; red rods , rim proteins ( Eps15 , epsin , FHCo1 / 2 , SIG1a ) ; gray lines , short - branched actin polymers plus the Arp2 / 3 complex , cortactin , N - Wasp ; green bar dynamin ; blue bar , F - BAR containing proteins ; green dots , uncoating ATP Hsc70 ; red lines , auxilin . We use stable and transient expression of recombinant , fluorescently - tagged constructs to follow the dynamic behavior of plasma membrane structures containing different combinations of clathrin , AP - 2 , auxilin , Arp2 / 3 complex , cortactin , dynamin , etc . We mostly use TIRF and spinning disc confocal microscopy to obtain live - cell imaging data . 62 Comert Kural and Tom Kirchhausen convolution of a punctate image with the point - spread function ( PSF ) of the microscope ( see below ) . Resolution of two identical fluorophores is limited by the Rayleigh criterion ; two point sources that are closer than (cid:1) l / 2 ( l is the wavelength of the emitted light ) cannot be resolved by conventional light microscopy . The accuracy of localizing a point source can be substantially better , however , depending on the SNR . Other important limitations of fluorescence microscopy are photo - bleaching and phototoxicity . Organic fluorophores and fluorescent proteins can only emit a limited number of photons before ceasing to fluoresce . This phenomenon , also called photobleaching , results in a loss in signal as the sample is illuminated for long periods of time . Extensive illumination also creates toxic oxygen radicals . These factors necessitate using smart ways of illumination that can obtain high SNR without high levels of exposure . We start by outlining some of the microscopy techniques that enable cell biologists to obtain high - quality images with as little laser exposure as possible . Most of these methods are designed to confine the fluorescence excitation to a volume of interest , while leaving the rest of the specimen in the dark , to minimize background fluorescence and to avoid damage outside the illuminated region . 4 . TIRF Microscopy TIRF is based on the property of electromagnetic radiation that when it travels from a medium of high refractive index ( n ) to one of lower refractive index , it diverges from its original path toward the interface . This phenomenon , refraction , can result in a mirror effect ( or total internal reflection ) when the incidence angle exceeds a critical value ( Fig . 4 . 2A ) . In this case , the reflected beam preserves most of its energy but a small portion of it is dissipated as an evanescent field at the interface . This quickly fading portion of light penetrates a few hundred nanometers ( depending on wave - length , incidence angle , and the difference between the refractive indices ) into the low - index medium , which becomes an ideal volume of illumination for fluorescence microscopy . In this geometry , fluorophores that are within a few hundred nanometers of the interface can be excited , while others that lie deeper within the sample are not illuminated ( Axelrod , 1989 ) . Because the plasma membranes of cells plated on glass coverslips are in contact with the glass – water interface ( n ¼ 1 . 51 and 1 . 33 , respectively ) , TIRF microscopy is an excellent imaging technique for recording clathrin - coated pit formation at the adherent surface of the plasma membrane . In this configuration , TIRF illumination excites the fluorescently tagged components of the clathrin machinery that interact with the plasma membrane ( high signal ) , but does not excite those that diffuse in the cytoplasm ( low background ; Fig . 4 . 3 ) . Live - Cell Imaging of Clathrin Coats 63 5 . How to Optimize Your TIRF System Objective - type and prism - type TIRF systems are the most widespread fluorescence microscopy techniques that use total internal reflection . Objective - type TIRF is generally preferred because water immersion objectives with lower numerical apertures used in prism type cannot collect a n 2 n 1 n 2 n 1 n 2 Evanescent Field Evanescent Field Penetrationdepth Penetrationdepth n 1 n 2 n 1 b c d a A B Fluorescent beads Wide field SNR TIRF SNR SNR SNR Wide fieldTIRF 28 200 N u m b e r o f s po t s 100 0 10 15 20 25 24 20 16 12 8 b c d q i < q c q i = q c q i > q c q i > > q c q i q i q i q i Figure 4 . 2 TIRF illumination . ( A ) Schematic representation of ray paths at different angles of incidence as they reach the glass / sample interphase ( panels a – d ) ; total internal reflection conditions are achieved in panels c and d . The evanescence field depth depends on the incidence angle . ( B ) The images are of diffraction - limited fluorescent beads imaged using (cid:1) 4 ms exposures with the same light source under total internal reflection ( panels a and c ) or widefield illumination ( panels b ) conditions . Panels b and c are color - coded representations of the SNR . Panel d highlights the substantial increase in SNR of the images acquired using TIRF microscopy . Spinning disk Spinning disk Spinning disk TIRF / spinning disk Clathrin / adaptor Reovirus Vesicle stomatitis virus Cell migration Figure 4 . 3 Schematic representation of the imaging strategies used to follow the dynamics of clathrin - coat assembly in different regions of the cell . The drawing indicates the location of various regions imaged in different cell types : apical surface of polarized cells ( red box ) , leading edge of migrating cells ( yellow box ) , dorsal surface of nonpolarized cells ( blue box ) , and adherent surface ( black box ) . 64 Comert Kural and Tom Kirchhausen as many photons . However , in both arrangements , it is possible to increase image quality by tweaking some of the parameters such as incidence angle and intensity of the excitation beam . Researchers generally use fluorescent bead samples for aligning their TIRF setups . The ideal bead sample prepared for TIRF imaging should be composed of a number of beads affixed to the coverslip and others that diffuse in the solution . In order to attain total internal reflection , the researcher should increase the incidence angle of the excitation beam to the point that the beads diffusing in the solution can no longer be imaged . Beads can be irreversibly immobilized to the coverslips by heating at 100 (cid:3) C ( Nugent - Glandorf and Perkins , 2004 ) . SNR of imaged fluorescent spots can be used as a measure to find the optimum TIRF configuration . SNR of a diffraction - limited spot can be calculated according to the formula I = ffiffiffiffiffiffiffiffiffiffiffiffi I þ b 2 p where I is the signal collected at the brightest pixel and b is the standard deviation of the background . For instance , if the peak intensity of the PSF is 300 and the background fluctua - tions have a standard deviation of 10 , then the SNR will be 300 / ( 300 þ 10 2 ) 1 / 2 ¼ 15 . The first term in the noise ( the denominator ) is called the shot noise ( also known as “photon” or “quantum” noise ) which is inherent to systems following Poisson distribution and cannot be eliminated . Whereas the second term represents the noise stemming from the fluctuations of the background which is a combination of camera noise and out - of - focus signal coming from the diffusing fluorophores . The researcher can determine the optimum values of incidence angle and excitation intensity by searching for the highest SNR of immobilized beads ( Fig . 4 . 2B ) . In live - cell experiments , however , target molecules labeled with fluo - rescent proteins are not as bright as beads . In this low - signal regime , instead of using super - bright fluorescent bead samples , researchers may prefer single organic fluorophores immobilized on coverslip . In order to boost photo - stability of organic fluorophores , oxygen scavenging and reducing agents such as glucose oxidase ( Yildiz et al . , 2003 ) and trolox ( Rasnik et al . , 2006 ) can be used . 6 . Spinning - Disk Confocal Microscopy This modality , also called Nipkow type or Nipkow disk confocal fluorescence microscopy , uses a rapidly rotating ( spinning ) disk decorated with thousands of microlenses and pinholes , which focus the excitation beam onto a thin slab of the sample and filter out - of - focus fluorescence , respec - tively . In contrast to scanning confocal systems , spinning - disk microscopes record pulses of emission from many regions at once , scanning over and over to accumulate signal . The image can be recorded with a standard CCD camera to facilitate high rates of data acquisition . In cell biology , spinning - disk confocal microscopy is a more general technique than TIRF microscopy , because fluorescent objects that are microns away from the coverslip can be Live - Cell Imaging of Clathrin Coats 65 observed . ( In TIRF microscopy , this distance is only a few hundred nan - ometers . ) Thus , spinning - disk microscopy is well suited for imaging processes in the cytoplasm and at the nonadherent surface of the plasma membrane ( Fig . 4 . 3 ) . In some cases , spinning - disk imaging is preferable to TIRF , even at the ventral surface of cells , since it does not require any corrections for variations in intensity of illumination as a function of depth . 7 . Spherical Aberration Correction Applied to Spinning - Disk Confocal Microscopy Even though high NA ( numerical aperture ) objectives are necessary for obtaining better signal , they have their drawbacks as well . One of such problems occur due to the fact that light rays passing through the peripheral regions of the lens are refracted more than the ones closer to the optical axis ( Fig . 4 . 4A ) . This results in longitudinally separated focal points which Perfect lens A Spherical aberration Moving lenses Objective lens No correction SAC z y x z y x SAC unit P l a n A po 100 ´ / 1 . 40 B C Tube lens Figure 4 . 4 Spherical aberration . ( A ) Schematic representation of ray paths in the absence and presence of spherical aberration . ( B ) Representation of the optical hard - ware used to correct spherical aberration . ( C ) Fluorescence image of two beads of different size acquired in three dimensions using the spinning - disk confocal micro - scope , in the absence ( no correction ) and presence ( SAC ) of spherical aberration correction . Note the increase in fluorescence intensity and decrease in the axial elon - gation of the diffraction - limited bead . 66 Comert Kural and Tom Kirchhausen reduce spatial resolution . In optical microscopy , this phenomenon is called spherical aberration ( SA ) . If there is a refractive index mismatch between the cover glass and media , the SA increases with the distance traversed the medium . In order to circumvent this problem , commercially available spherical aberration correction ( SAC ) units can be installed to the emission path of the microscope . The working principle of SAC units is quite similar to correction collars attached to some objectives which are used to compensate the aberrations stemming from the variations in cover glass thickness . Just like the correction collar , the SAC unit contains a motile lens doublet . However , the position of the doublet is computer controlled ; hence , the built - in calibration protocols of SAC units enable to find the best correction possible automatically . Another advantage of computer control is that SAC can dynamically correct for SA at varying penetration depths during a z - acquisition ( Fig . 4 . 4B , C ) . During the calibration process , the SAC will determine the correct setting for minimal SA at Z 0 , it will then determine the setting for minimal SA at the deepest penetration depth Z d . During the 3D acquisition , the SAC will dynamically move the correction lens relative to the z - position , therefore producing a 3D image that corrects SA at every plane in the series , as opposed to the correction collar that corrects for a single plane . 8 . Getting Around the Diffraction Limit Single light emitting fluorophores focused on a flat surface by using a lens with a circular aperture appear as an Airy function ( generally referred as PSF ) . The Airy disk , the bright region at the center of Airy function , has a width of (cid:1) l / 2NA where NA is the numerical aperture of the lens ( for oil - immersion objectives , NA is generally between 1 . 4 and 1 . 65 ) . Determining the actual position of the fluorescent object in this extended “blob” of signal depends on localizing the center point of the Airy disk with high fidelity . A method called fluorescence imaging with one nanometer accuracy ( FIONA ) , developed by the Selvin laboratory , has proved to be an effective way to locate a single fluorescent object with a precision of one nanometer in x and y ( Kural et al . , 2005 ; Yildiz et al . , 2003 ) . FIONA relies on the observation that the Airy disk can be represented to good approximation by a two - dimensional ( 2D ) Gaussian function ; if the SNR is high enough , then the centroid of the Gaussian can be determined to about 1 nm . The standard error of the mean ( SEM ) of the centroid can be used to represent the accuracy ; it is given by ( Thompson et al . , 2002 ) : Live - Cell Imaging of Clathrin Coats 67 s m i ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi s i 2 N þ a 2 = 12 N þ 8 p s i 4 b 2 a 2 N 2 (cid:3) (cid:4) s ð 4 : 1 Þ where s is the standard deviation of the Gaussian fit , N is the number of photons collected , a is the effective pixel size of the CCD camera ( i . e . , pixel size of the CCD chip divided by the total magnification ) , and b is the standard deviation of the background ( which includes the camera noise together with the fluctuations of the background fluorescence ) . According to this equation , the most straightforward ways to increase the localization accuracy are ( 1 ) to maximize the total number of collected photons and ( 2 ) to minimize the standard deviation of the background . There are multiple ways to increase the number of collected photons . The easiest is to increase the excitation power ( or exposure time per imaging cycle ) . The experimenter must , however , consider that high power and long exposure increase the rate of photobleaching and photo - toxicity . A more efficient but expensive way is to use a high NA lens . Especially in TIRF microscopy systems , numerical apertures up to 1 . 65 can be obtained by switching to oil - immersion lenses designed for sap - phire coverslips ( n (cid:4) 1 . 8 ) . The background fluctuations can be minimized by filtering out the out - of - focus signal , for which both TIRF and spinning - disk systems are quite effective . In TIRF microscopy , the exper - imenter can increase the incidence angle of the excitation beam to reduce the penetration depth of the evanescent field and thus excite fewer fluorophores in the solution . For an objective type TIRF system , the back aperture of the objective sets a limit to the maximum incidence angle . Using advanced CCD cameras with low readout noise is another way to diminish fluctuations of the background . Binning CCD chips is an alternative approach to reduce the noise factor , as well as an efficient way to increase frame rate . With the advent of FIONA and related techniques for localizing point sources ( e . g . , SHRIMP , SHREC , STORM , and PALM , some of which rely on specialized fluorophores and specialized excitation regimes ) , new possibilities have emerged for using fluorescence microscopy to obtain high temporal and spatial information about processes in living cells ( Betzig et al . , 2006 ; Churchman et al . , 2005 ; Gordon et al . , 2004 ; Rust et al . , 2006 ) . These techniques necessitate advanced computational and analytical tools to make use of the raw data . To illustrate these points , we will briefly describe some of the applications in which 2D imaging techniques have been used to study clathrin - mediated endocytosis at the ventral ( adherent ) and dorsal ( free ) surfaces of living cells ( Fig . 4 . 3 ) . 68 Comert Kural and Tom Kirchhausen 9 . Use of 2D Spinning - Disk Confocal Microscope to Study Clathrin - Mediated Endocytosis at the Ventral and Dorsal Surfaces of a Cell Clathrin interacts with lipid bilayers through its adaptor proteins . The major clathrin adaptor present solely at the plasma membrane is AP2 . When imaged in the fluorescence microscope , labeled AP2 gives less - crowded images than does clathrin itself , because clathrin decorates endosomes and TGN compartments as well as the plasma membrane . For this reason , mam - malian cells stably expressing AP2 adaptors tagged with fluorescent proteins are the systems of choice for studying clathrin - mediated endocytosis in real time . The high imaging rates and low background noise offered by spinning - disk confocal microscope enabled Ehrlich et al . ( 2004 ) to resolve the complete lifetime of endocytic clathrin coats at the adherent ( ventral ) surface BSC1 cells ( Fig . 4 . 5 ) . Their work revealed a nucleation - growth mechanism , which proceeds at a steady rate that is much slower than the uncoating reaction . Confocal imaging can penetrate several microns into the specimen , but at the expense of increased SA . Cureton et al . used a spinning - disk confocal microscope to characterize clathrin - mediated endocytosis of vesicular sto - matitis virus ( VSV ) particles at the free ( dorsal ) surface of BSC1 cells . Their results showed that virus - containing clathrin coats are larger and longer lived than the virus - free clathrin coats at the same dorsal surface ( Fig . 4 . 6 ; Initiation Maturation LCa 00 100 B A 50 12 s 18 s 42 s 36 s 6 s 30 s 24 s 0 s 10 20 30 40 Time ( s ) % f l uo r e s ce n ce σ 2 Uncoating Figure 4 . 5 Recruitment of clathrin and AP2 in endocytic coated pits . ( A ) Arrow heads point to an example of clathrin LCa - mRFP recruitment during the formation of a canonical endocytic clathrin - coated pit . ( B ) Plot of the fluorescence intensity normal - ized to the highest value before uncoating of a subset of 28 coated pits containing s 2 - EGFP and LCa - mRFP , each with a lifetime of 42 s . The data were obtained using a spinning - disk confocal microscope from three BSC1 cells stably expressing s 2 - EGFP and transiently expressing LCa - mRFP . ( Reproduced from Fig . 4 of Ehrlich et al . , 2004 ; from Fig . 4A and E in Ehrlich et al . , 2004 . ) Live - Cell Imaging of Clathrin Coats 69 Cureton et al . , 2009 ) . The elongated virus particle stalls completion of the coat , which must be rescued by actin - driven membrane deformation . 10 . Use of TIRF Microscopy to Study Clathrin - Mediated Endocytosis The evanescent field formed upon total internal reflection decays exponentially according to formula I x z ð Þ ¼ I 0 x e (cid:5) z = d ð Þ ð 4 : 2 Þ where I x 0 is the excitation intensity at the coverslip – medium interface , z is the distance from the interface , and d is the penetration depth . For an object that carries a fixed number of fluorescent particles , a similar equation would also work for fluorescence emission . That is , I m ( z ) ¼ I m 0 e ( (cid:5) z / d ) , where I m 0 is the emission intensity at the interface . By using this relationship between z and emission intensity , it is in principle possible to map the changes in the z - position of an object which is moving within the evanescent field . In practice , clathrin - coated structures initiate , mature , and uncoat during the observation period , and in each one of these phases , the coat has different levels of fluorescent components . For objects , such as clathrin - coated 300 A B − VSV + VSV − VSV + VSV 200 100 σ 2 LCa s 2 LCa 0 150 50 200 100 0 0 30 Time ( s ) % f l uo r e s ce n ce % f l uo r e s ce n ce 60 0 30 Time ( s ) % LCa lifetime 60 0 25 50 75100 % LCa lifetime 25 n = 16 n = 23 50 75 100 Figure 4 . 6 Live - cell imaging of clathrin - dependent endocytosis of single VSV parti - cles . Plot of the kinetics of fluorescent AP - 2 ( tagged with s 2 - EGFP ) and clathrin ( tagged with Tom - LCa ) recruitment to endocytic clathrin - coated pits in BSC - 1 cells associated or not associated with the internalization of VSV particles . ( A ) Fluorescence intensities were plotted relative to the time of clathrin detection and are expressed as a % of the average maximum clathrin observed in all pits lacking virus . ( B ) Plot of the average kinetics of AP - 2 and clathrin recruitment to coated pits containing or not containing virus . Average fluorescence intensity and time are expressed as a % relative to clathrin - coated vesicles lacking virus observed in the same cells ( from Fig . 1 in Cureton et al . 2009 ) . 70 Comert Kural and Tom Kirchhausen structures , for which variation in fluorescence is part of the phenomenon being studied , it is essential , in order to map the z - position , to determine whether the variation in the emission signal stems from movement in z or changes in the number of fluorescent components . To overcome this ambiguity , Saffarian et al . coupled widefield illumination with TIRF microscopy on BSC1 cells stably expressing fluorescently labeled AP2 and clathrin ( Saffarian and Kirchhausen , 2008 ) . Making use of the uniform intensity of widefield illumination along the axial direction , they could quantify the total emitted signal , regardless of the position . In this case , the distance of the coat from the interface at time point t can be calculated as z t ð Þ ¼ (cid:5) d (cid:6) log I WF F TIRF t ð Þ I 0TIRF F WF t ð Þ (cid:3) (cid:4) ð 4 : 3 Þ where I WF is the intensity of the wide - field illumination , I TIRF0 is the intensity of the TIRF illumination at the interface , F WF ( t ) is the fluorescence signal collected at the wide - field channel at time point t , and F TIRF ( t ) is the fluorescence signal collected at the TIRF channel at time point t . Saffarian et al . extended this analysis to two different fluorophores recorded at the same time ; they named the technique Differential Nano - metry ( DiNa ) . The DiNa analysis of coats carrying clathrin labeled with Tomato and AP2 adaptors labeled with EGFP showed that the center of fluorescence of adaptors is separated from that of clathrin , indicating an asymmetric distribution of adaptors within the clathrin coat ( Saffarian and Kirchhausen , 2008 ; Fig . 4 . 7 ) . 11 . The Third Dimension 2D time - lapse data acquisition has generally been the dominant high - resolution mode for fluorescence imaging experiments with living cells . This restriction has applied to a variety of illumination and recording modalities , for example , widefield , confocal , and TIRF microscopy . Although powerful for describing the localization of the fluorescent object and for resolving the time - dependence of the fluorescence intensity associated with a tagged protein , lipid , or organelle , 2D data fail to represent the intrinsically rich behavior of objects within the three - dimensional ( 3D ) context of a cell . For instance , efforts to follow AP1 - and AP3 - containing clathrin carriers have not been successful because rapid movement , particularly along the direction of view ( Z ) , has not permitted one to distinguish initiation of assembly or dissociation of the clathrin coat from passage into or out of the imaging plane . In order to circumvent this problem , we have developed a 3D analysis technique Live - Cell Imaging of Clathrin Coats 71 optimized for 3D time - lapse images taken with a spinning - disk confocal microscope . The method makes use of the fact that the 3D PSF of a diffraction - limited spot can be imaged at different planes in the axial dimension and its position can be estimated from the distribution of integrated intensities collected from each plane . 12 . 3D Tracking in Spinning - Disk Imaging A spinning - disk confocal microscope equipped with a piezo - driven stage can image up to 8 planes ( in the axial dimension ) per second . This rapid imaging makes it possible to determine the 3D trajectory of an intracellular object . The accuracy of trajectories depend on localizing each spot in x , y , and z with high precision at each time point . The FIONA procedure enables one to extract the x and y positions of fluorescent diffraction - limited objects from 2D movies , such as those taken with TIRF microscopy . In the case of 3D time - lapse movies acquired with a spinning - disk microscope , the projection of the brightest signals along the 45 30 − 30 − 45 0 . 0 0 . 2 0 . 4 Time fraction Δ z ( n m ) 0 . 6 0 . 8 1 . 0 15 0 − 15 Figure 4 . 7 DiNa analysis applied to the recruitment of AP2 and clathrin during the formation of endocytic clathrin - coated pits . The experiment was done using the total internal reflection and widefield illumination signals of coated pits and vesicles forming on the ventral surface of BSC1 cells . AP2 was tagged by stable expression of s2 - EGFP and clathrin by transient expression of Tom - LCa . D z calculated for the difference in the average axial position between clathrin LCa - Tomato and AP - 2 s 2 - EGFP from the individual pits selected for DiNa analysis plotted as a function of the fraction of their respective lifetimes . Values outside the red dotted lines represent meaningful data in excess of 1 . 5 times the expected calculated resolution of D z ( from Fig . 5E in Saffarian and Kirchhausen , 2008 ) . 72 Comert Kural and Tom Kirchhausen axial direction ( maximum projection ) can be used to project the 3D posi - tion onto the x – y plane for each z - stack and hence to obtain a 2D projected trajectory , to which FIONA can then be applied . Determining the z - position needs some extra analytical tools that can make use of the information embedded within the z - stacks . If the distance between the consecutive planes is chosen appropriately , then the 3D PSF of fluorescent objects can be imaged on multiple planes in the z - stack . The intensity measured on each plane is related to distance from the actual position of the object in z . In other words , if an object is between planes n and n þ 1 , but closer to plane n , then the fluorescence intensity in plane n will be higher than the intensity in plane n þ 1 . Thus , one can determine the 3D PSF along z ; by calculating the centroid of the distribution , the position of the object can be estimated . The 3D tracking routine that we have programmed accepts data as a time - series of z - stacks , each plane in the stack being a recorded optical section . The required z - spacing is about one - fourth to one - third of the full height of the 3D PSF ( in the examples described here , D z (cid:1) 250 – 450 nm ) . In order to perform automated tracking , single fluorescent spots are detected in the maximum projection image of a stack by using a local maximum - finding algorithm performed after 2D Gaussian and Laplacian filtering . x and y positions of the spots are then obtained in a window of 7 (cid:6) 7 pixels by FIONA . For each spot , the 7 (cid:6) 7 pixel window is then extended to each plane in the z - stack , and integrated intensities for each plane are calculated . The centroid of the intensity distribution in z is then calculated as C z ¼ P i ¼ 1 n z i I i / P i ¼ 1 n I i , where n is the number of planes in the z - stack , z i is the coordinate of the plane on the z - axis , I i is the integrated signal of that plane . The integrated intensity of the plane with the minimum signal is the threshold subtracted from all planes . By using this approach , biological molecules such as EGFP - tagged AP1 coats can be localized with (cid:1) 20 nm accuracy in z . In living cells , we have followed AP1 - and AP3 - containing endosomal carriers from assembly to uncoating , despite quite rapid motion in all three dimensions , well displaced from either cell surface . These structures have lifetimes and sizes ( the latter as estimated from maximum fluorescence intensity ) comparable to those of endocytic , clathrin - coated carriers ( Kural et al . , submitted ) . The analysis can be performed on hundreds of fluorescent objects simultaneously . It is therefore possible to classify objects in a single cell according to their position and movement . One such example is classifica - tion of the clathrin - coated structures forming at the ventral and dorsal surface of migrating U373 astrocytes ( Fig . 4 . 3 ) . By tracking AP2 puncta in 3D , we have found that AP2 carriers are absent from the ventral surface during migration and that they reappear when cellular migration ceases . The abundant AP2 structures that formed on the dorsal surface tended to initiate in the region close to the leading edge . The AP2 objects in this Live - Cell Imaging of Clathrin Coats 73 region moved with a characteristic centripetal retrograde flow that was inversely related to the migration rate of the cell ( Fig . 4 . 8 ) . 13 . Use of 3D Tracking to Monitor Clathrin - Mediated Entry of Reovirus Particles at the Apical Surface of Polarized Cells Clathrin - mediated internalization of VSV particles can easily be traced by using spinning - disk confocal systems at the free surface of nonpolarized BSC - 1 cells ( Cureton et al . , 2009 , 2010 ) . Unlike the dorsal membrane of nonpolarized cells , however , the apical membrane of polarized cells has a dome - shaped profile . The absence of a flat region at the top of a polarized cell means that one cannot monitor virus entry in a single optical plane . If the recording is restricted to 2D , many virus particles disappear from view because they move out of the confocal plane . To study virus entry using polarized MDCK ( Madin - Darby canine kidney ) cells , we therefore used rapid 3D imaging as outlined above to visualize a larger part of the apical plasma membrane ( Fig . 4 . 3 ) . In particular , we determined the 3D trajectory of reovirus particles . 3D movies were recorded from the apical surface of polarized cells , with z - stacks of 3 – 5 consecutive optical planes spaced at 0 . 35 m m . A 2D movie was then obtained by generating a maximum intensity Z ( nm ) Migration Time ( s ) 100 Z po s i t i on ( n m ) 600 800 1000 1200 1400 1600 120 140 160 180 200 220 Time ( s ) 100 Z po s i t i on ( n m ) 600 800 1000 1200 1400 1600 120 140 160 180 200 220 Retrograde flow 2 2 1 1 1120 930 740 550 360 170 Figure 4 . 8 Formation of endocytic clathrin and AP2 - containing coated pits and vesicles on the dorsal surface of the leading lamellipodium of migrating U373 astro - cytes . The image shows tracings color - coded for the z - position of AP2 fluorescent spots tagged with s 2 - EGFP . The axial information was obtained by tracking in 3D the temporal position of individual AP2 spots imaged by spinning - disk confocal micro - scopy . Insets display representative tracings corresponding to the location along the z - axis of two AP2 - containing objects . 74 Comert Kural and Tom Kirchhausen z - projection for each time point . The colocalization of the viral particles and AP2 - GFP was analyzed from the resulting 2D movies , and the position of the virus particles in the three dimensions was then extracted from the raw z - stack . As also found in nonpolarized BSC - 1 cell , reovirus particles were internalized by clathrin - mediated endocytosis . Virions were captured by a coated pit and the intensity of the AP2 - GFP signal increased steadily as the clathrin - coated pit formed . An abrupt disappearance of fluorescence was then observed . Our 3D imaging method allowed us to visualize the budding of the coated pit from the plasma membrane and the displacement of virus - containing vesicles as uncoating of AP2 - GFP occurred ( Fig . 4 . 9 ) . Soon after the recruitment of AP2 - GFP had reached a plateau , we detected a rapid displacement of the coated pits in the z - dimension , away from the plasma membrane . After budding of the clathrin - coated vesicles , uncoating occurred . We measured the average displacement of a virus - containing Reovirus Z 100 Clathrin coat 80 A P 2 - G F P s i g n a l ( a u ) 60 40 40 30 50 20 20 Time ( s ) 10 0 600 100 200 300 Z - d i s p l a c e m e n t ( n m ) 400 500 0 0 Figure 4 . 9 Real - time tracking of the internalization of a reovirus particle by an endocytic AP2 - containing coated carrier . Top panel : schematic representation of the experimental outcome representing the internalization of a fluorescent reovirus particle at the apical surface of a polarized MDCK cell stably expressing s 2 - EGFP . Bottom panel : plot of z - displacement of reovirus and the AP2 - containing pit during coated pit formation . The z - position of the virion displays an abrupt displacement toward the cytoplasm at the onset of AP2 uncoating . The data was obtained using 3D spinning - disk confocal microscopy . Live - Cell Imaging of Clathrin Coats 75 coated vesicles before uncoating and found that on average the vesicles moved (cid:1) 500 nm away from the plasma membrane . Similar values were found for coated vesicles that did not contain virus particles . 14 . Using the Optimum Pixel and Step Sizes in z - Stacks As shown in Eq . ( 4 . 1 ) , SEM of the location in x and y is a function of effective pixel size . For larger pixel sizes ( under low magnification ) , the PSF converges into a 2D delta function . In this case , the pixelation noise becomes dominant due to the fact that it is not possible to pinpoint the position where the photon arrived in the pixel . For that reason , using pixel sizes larger than the standard deviation of the PSF is not recommended . In the other extreme , for smaller size pixels ( under high magnification ) , the maximum signal reduces dramatically ( a magnification (cid:5) 2 ) and camera read - out noise becomes stronger since the spot spreads over a larger number of pixels ( Thompson et al . , 2002 ) . For systems with intrinsically high magnifi - cation , binning the CCD pixels ( 2 (cid:6) 2 ) can be a good alternative to reduce the readout noise . Binning also increases CCD frame rate and by doing so enables faster imaging . In localizing the center of 3D PSF in the axial dimension , using small steps to acquire z - stacks may increase spatial precision slightly . On the other hand , using larger steps to scan the same volume speeds up the acquisition and reduces exposure per imaging cycle which in return slows down photobleaching ( Fig . 4 . 10 ) . The researcher should consider these factors and find the optimum imaging parameters to be able to perform 3D tracking for his / her specific application . For tracking objects that move with slower pace , z - stacks can be acquired with longer time intervals to minimize photobleaching and elongate total time of imaging . To be able to track fast - moving objects , the researcher has to reduce the time interval between z - stacks to increase temporal resolution . Scanning the volume with larger step sizes can prevent excessive photobleaching . 15 . Conclusion The strategies presented here have broad potential for investigating the dynamics of processes involving assembly , translocation , and disassembly of a small and variable number of diffraction - limited , fluorescent molecules at the ventral plasma membrane and throughout the volume of a cell . 76 Comert Kural and Tom Kirchhausen ACKNOWLEDGMENTS We gratefully acknowledge David Cureton and Steeve Boulant for sharing their data shown in Figs . 4 . 2 and 4 . 9 , respectively , and Eric Marino for maintaining the imaging resources used here . 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Myosin V walks hand - over - hand : Single fluorophore imaging with 1 . 5 - nm localization . Science 300 , 2061 – 2065 . Zoncu , R . , Perera , R . M . , Sebastian , R . , Nakatsu , F . , Chen , H . , Balla , T . , Ayala , G . , Toomre , D . , and De Camilli , P . V . ( 2007 ) . Loss of endocytic clathrin - coated pits upon acute depletion of phosphatidylinositol 4 , 5 - bisphosphate . Proc . Natl . Acad . Sci . USA 104 , 3793 – 3798 . 80 Comert Kural and Tom Kirchhausen \ No newline at end of file diff --git a/silver_data/499b98c1bccb209763eaf353dcfebb286e2167d4.pdf.txt b/silver_data/499b98c1bccb209763eaf353dcfebb286e2167d4.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..ed26ecfa92686e457e3355b7a52396ff9bc41a1e --- /dev/null +++ b/silver_data/499b98c1bccb209763eaf353dcfebb286e2167d4.pdf.txt @@ -0,0 +1 @@ +Portrait of L . S . " ygotsky at age . 35 tIL , . ; J ( ~ " . ' ; ! ! ! , ; > - : ; ) . ' 0 ' . • ( r alae c . / } - . , ; ( £a - t : ifj , , 14 ' • " Lt£ ! . , a ~ pi / ( 4 I ~ ~ ~ ~ . jui . J ~ 1I ' I ' ll ~ J ~ I , . ff ! , / UU ( . . 9t ; ~ t ' . ; ' 1 . 1 . a . II ' £td ! lL . v . jt 4 ? 4 ( . { t ~ . f / kp ! - . kd4 It / ~ t , u ! . . . ! LiJ - ·£ < " 1 . f ! . tu4 . - , > c kIP / { fL . tOUdd { 1t4 - k , UdA . . y . z : tU ~ d - - IcJf1 \ h Utr / 4 . . iA · ( nm - fLUfUf . t . ~ . / t < L - ~ k ~ 2 cfh ~ & 1 . ( / te ~ ; we cR , u : t feU ~ t . 1 / a . « J ~ . . LiAUL . Jb ' . J , J ~ M ~ ~ ~ . ~ J ; - O , ft - ~ . 2 . , £ ' 04C . ~ ak ; Z ~ . t« . Ju ~ At ( 4 a HflM / lAJ4 ~ J , . J . J . ' i ~ dA . . ' ~ ' frit4 ~ . ~ JlA ! , P Uje£ : J Jz ) ott - - I·f· ~ Fragment of Vygotsky ' s note first suggesting mediation as the basis of higher psychological processes . ( " NB . The essence of the instrumental method resides in the functionally different use of two stimuli , which differen . . tially determine behavior ; from this results the mastery of one ' s own psychological operations . Always assuming two stimuli , we HUlSt answer the following ~ questions : 1 . How does one remember stimulus 81 with the aid of stimulus 82 ( where SI is the object and S2 is the instru . . nlent ) . 2 . How is attention directed to 51 with the aid of 82 . . 3 . How is a word associated with s . retrieved via 82 and so on . " ) L . S . VYGOTSKY " Mind in Society / The Development : 7 of Higher Psychological Processes Edited by . Michael Cole Vera John - Steiner Sylvia Scribner Ellen Souberman Harvard University Press Cambridge , Massachusetts London , England NewlY ) ' B F 3 I I . VCf3 1978 c . . ~ Copyright © 1978 by the President and Fellows of Harvard College All right reserved Second printing , 1979 Printed in the United States of America Library of Congress Cataloging in Publication Data Vygotskii , Lev Semenovich , 1896 - 1934 . Mind in society . Includes index . 1 . Cognition . 2 . Cognition in children . I . Cole , Michael , 1938 - rr , Title . BF31I . V93 1978 155 . 4 ' 13 77 - 26023 ISBN 0 - 674 - 57628 - 4 To the memory of Alexander Romanovich Luria Editors ' Preface Lev Semyonovich Vygotsky has figured prominently in American psychology since the publication in 1962 of his monograph Thought and Language . Five years ago , at the urging of Vygotsky ' s student Alexander Luria , we agreed to edit a collection of Vygotsky ' s essays which would reflect the general theoretical enterprise of which the study of the rela tion between thought and language was one important aspect . Luria made available to us rough translations of two of Vygotsky ' s works . The first , " Tool and Symbol in Children ' s Development ' ) ( 1930 ) , had never been published . The second was a translation of a monograph entitled The History , of the Development of Higher Psychological Functions , which appeared in the second volume of Vygotsky ' s writings published in Moscow in 1960 . A cursory study of these essays quickly convinced us that the scope of Vygotsky ' s work reached considerably beyond Thought and Language . Furthermore , we came to believe that the image of Vygotsky as a sort of early neobehaviorist of cognitive development - an impression held by many of our colleagues - was strongly belied by these two works . We have constructed the first four chapters of this volume from " Tool and Symbol . " The fifth chapter summarizes the major theoretical and methodological points made in " Tool and Symbol " and applies them to a classic problem in cognitive psychology , the nature of choice reac tion . This chapter was taken from section 3 of The History of the De velopment of Higher Psychological Functions . Chapters 6 and 8 ( learn ing and development , and the developmental precursors of writing ) are from a posthumously published collection of essays entitled Mental De velopment of Children and the Process of Learning ( 1935 ) . Chapter 7 , ix EditMS ' Preiace on play , is based on a lecture delivered at the Leningrad Pedagogical Institute in 1933 and published in Voprosi Psikhologii ( Problems of Psy chology ) in 1966 . Complete references are given in the list of Vygotsky ' s works that follows the text of this volume . At several places we have inserted material from additional sources in order to more fully explicate the meaning of the text . In most cases these importations are from sections of The History of the Development of Higher Psychological Functions other than the one included here ; the rest are taken from other essays which appear in either the 1956 or the 1960volumes of collected works . In a few cases passages have been taken from the work of Vygotsky ' s students or collaborators which provide concrete examples of experimental procedures or results which the orig inal text describes with extreme brevity . References to these sources are given in the notes . In putting separate essays together we have taken significant lib erties . The reader will encounter here not a literal translation of Vygot sky but rather our edited translation of Vygotsky , from which we have omitted material that seemed redundant and to which we have added material that seemed to make his points clearer . As other editors have noted , Vygotsky ' s style is extremely difficult . He wrote copiously and many of his manuscripts have never been properly edited . In addition , during frequent periods of illness he would dictate his papers - a prac tice which resulted in repetitions and dense or elliptical prose . Gaps in the original manuscripts make them even less accessible now than they might have been at the time they were written . Because proper refer ences were rarely given , we have supplied our best guess as to the exact sources to which Vygotsky referred . The process of tracking down and reading these sources has itself proved a very rewarding enterprise ; many of his contemporaries were fascinatingly modern in important respects . We realize that in tampering with the original we may have distorted history ; however , we hope that by stating out procedures and by adhering as closely as possible to the principles and content of the work , we have not distorted Vygotsky ' s meaning . We owe a special debt to the late Alexander R . Luria for providing an initial translation of much of the material included in chapters 1 - 5 , for tirelessly tracking down references and expanding upon details of experiments , and for reading our manuscript . Chapters 6 and 7 were translated by Martin Lopez - Morillas . Chapter 5 and parts of chapters 1 - 5 were translated by Michael Cole . We wish to thank James Wertsch for his assistance in translating and interpreting especially difficult pas sages . Editors ' Preface xi The editing of these writings has occupied us for several years . Working in separate locations , educated in differing intellectual tradi tions , each team of editors found certain material of special interest . Since there is not one but many issues to be illuminated by such a com plex body of thought , we have written two essays reflecting various aspects of " reading Vygotsky . " Vera John - Steiner Ellen Souberman University of New Mexico Michael Cole Sylvia Scribner The Rockefeller University Contents Introduction 1 Michael Cole and Sylvia Scribner Biographical Note on L . S . Vygotsky 15 Basic Theory and Data 1 . Tool and Symbol in Child Development 19 2 . The Development of Perception and Attention 31 3 . Mastery of Memory and Thinking 38 4 . Internalization of Higher Psychological Functions 52 5 . Problems of Method 58 Educational Implications 6 . Interaction between Learning and Development 79 7 . The Role of Play in Development 92 8 . The Prehistory of Written Language 105 Afterword 121 Vera [ ohn - Steiner and Ellen Souberman Notes 135 Vygotsky ' s Works 141 Index 153 The spider carries out operations reminiscent of a weaver and the boxes which bees build in the sky could disgrace the work of many architects . But even the worst architect differs from the most able bee from the very outset in that before he builds a box out of boards he has already constructed it in his head . At the end of the work process he obtains a result which already existed in his mind before he began to build . The architect not only changes the form given to him by nature , within the constraints imposed by nature , he also carries out a purpose of his own which defines the means and the character of the activity to which he must subordinate his will . Karl Marx , Capital It is precisely the alterationof nature by men ; t not nature as such , which is the most essential and immediate basis of human thought . Friedrich Engels , Dialectics of Nature Introduction MICHAEL COLE AND SYLVIA SCRIBNER Educated as a lawyer and philologist , Lev S . Vygotsky had already made several contributions to literary criticism when he began his career as a psychologist following the Russian Revolution in 1917 . He was a student in the heyday of Wilhelm Wundt , the founder of experi mental psychology , and William James , the American pragmatist . His scientific contemporaries included Ivan Pavlov , Vladimir Bekhterev , and John B . Watson , popularizers of stimulus - response theories of behavior , as well as Wertheimer , Kohler , Koffka , and Lewin , the found ers of the Gestalt psychology movement . The reader might expect , then , that Vygotsky ' s work will prove to be primarily of historical interest - perhaps as a glimpse of the way in which modem psychology ' s founding fathers influenced Soviet psychology in postrevolutionary Russia . These essays are certainly of interest from the perspective of intellectual history , but they are not historical relics . Rather , we offer them as a contribution to quandaries and discussions in contemporary psychology . In order to understand how the ideas in this volume can retain their relevance across the reaches of time and culture that separate us from Vygotsky , we have repeatedly found ourselves reflecting upon the state of European psychology which provided the initial setting for Vygotsky ' s theories . We have also found it helpful to examine the condition of psychology and society in postrevolutionary Russia , since they were the source of the immediate problems facing Vygotsky as well as a source of inspiration as he and his colleagues sought to develop a Marxist theory of human intellectual functioning . 1 Introduction NINETEENTH - CENTURY BEGINNINGS Until the latter half of the nineteenth century the study of man ' s nature was the province of philosophy . The intellectual descendants of John Locke in England had developed his empiricist explanation of mind , which emphasized the origin of ideas from environmentally produced sensations . The major problem of psychological analysis for these British empiricists was to describe the laws of association by which simple sensations combine to produce complex ideas . On the continent the followers of Immanuel Kant argued that ideas of space and time and concepts of quantity , quality , and relation originate in the human mind and cannot be decomposed into simpler elements . Neither side budged from its armchair . Both of these philosophical traditions were operating under the assumption , dating from the work of Rene Descartes , that the scientific study of man could apply only to his physical body . To philosophy was assigned the study of his soul . While the conflict between these two approaches reaches down to the present day , in the 1860s the terms of this discussion were changed irrevocably by the almost simultaneous publication of three ' books . Most famous was Darwin ' s Origin of Species , which argued the essential continuity of man and other animals . One immediate consequence of this assertion was an effort by many scholars to establish discontinuities that set human adults off from their lower relatives ( both ontogenetically and phylogenetically ) . The second book was Gustav Fechner ' s Die Psuchophqsik , which provided a detailed , mathematically sophisticated description of the relation between changes in specifiable physical events and verbalizable " psychic " responses . Fechner claimed no less than an objective , quantitative description of the contents of the human mind . The third book was a slim volume entitled Reflexes of the Brain , written by a Moscow physician , I . M . Sechenov . Sechenov , who had studied with some of Europe ' s leading physiologists , had advanced understanding of simple sensory - motor reflexes by using techniques that isolated nerve - muscle preparations from the living organism . Sechenov was convinced that the processes he observed in the isolated tissue of frogs were the same in principle as those that take place in the central nervous systems of intact organisms , including humans . If responses of leg muscles could be accounted for by processes of inhibition and exci tation , might not the same laws apply to the operations of the human cerebral cortex ? Although he lacked direct evidence for these specula tions , Sechenov ' s ideas suggested the physiological basis for linking . - the natural scientific study of animals with the heretofore philosophical Introduction 3 study of humans . The tsar ' s censor seemed to understand the revolu tionary , materialist implications of Sechenov ' s thesis ; he banned pub lication of the book for as long as he could . When the book appeared , it bore a dedication to Charles Darwin . These books by Darwin , Fechner , and Sechenov can be viewed as essential constituents of psychological thought at the end of the nine - · teenth century . Darwin linked animals and humans in a single con ceptual system regulated by natural laws ; Fechner provided an example of what a natural law describing the relationship between physical events and human mental functioning might look like ; Sechenov , extrap olating from muscle twitches in frogs , proposed a physiological theory of how such mental processes worked within the normally functioning individual . None of these authors considered themselves ( or were considered by their contemporaries ) to be psychologists . But they pro vided the central questions with which the young science of psychology became concerned in the second half of the century : What are the relationships between animal and human behavior ? Environmental and mental events ? Physiological and psychological processes ? Various schools of psychology attacked one or another of these questions , providing partial answers within theoretically limited perspectives . The first such school was established by Wilhelm Wundt in 1880 . Wundt took as his task the description of the contents of human con sciousness and their relation to external stimulation . His method con sisted of analyzing various states of consciousness into their constituent elements , which he defined as simple sensations . On a priori grounds , he ruled out such sensations as " feelings of awareness " or " perception of relations " as elements of consciousness , considering these phenomena to be " nothing more than " the by - product of faulty methods of obser vation ( introspection ) . Indeed , Wundt propounded the explicit view that complex mental functions , or as they were then known , " higher psycho logical processes " ( voluntary remembering and deductive reasoning , for example ) , could not in principle be studied by experimental psycholo gists . They could only be investigated , he maintained , by historical studies of cultural products such as folktales , customs , and language . By the beginning of World War I introspective studies of human conscious processes came under attack from two directions . In the United States and Russia psychologists discontented with the contro versies surrounding the correct introspective descriptions of sensations , and with the sterility of the research this position had produced , re nounced the study of consciousness in favor of the study of behavior . Exploiting the potential suggested by Pavlov ' s study of conditioned Introduction 4 reflexes ( which built upon Sechenov ) and Darwin ' s assertion of the continuity of man and beast , they opened up many areas of animal and human behavior to scientific study . In one important respect , however , they agreed with their introspective antagonists : their basic strategy was to identify the simple building blocks of human activity ( substi tuting stimulus - response bonds for sensations ) and then to specify the rules by which these elements combined to produce more complex phenomena . This strategy led to a concentration on processes shared by animals and humans and , again , to a neglect of higher processes thought , language , and volitional behavior . The second line of attack on descriptions of the contents of consciousness came from a group of psychologists who objected to the one point upon which Wundt and the behaviorists agreed : the appropriateness of analyzing psychological processes into their basic constituents . This movement , which came to be known as Gestalt psychology , demonstrated that many intellectual phenomena ( Kohler ' s studies with anthropoid apes were an example ) and perceptual phenomena ( Wertheimer ' s studies of apparent movement of flickering lights , for example ) could not be accounted for in terms of either the basic elements of consciousness postulated by Wundt or simple stimulus - response theories of behavior . The Gestalt psychologists . rejected , In principle , the possibility of accounting for complex processes in terms of simple ones . Such , in great brevity , was the situation in European psychology when Vygotsky first appeared on the scene . The situation was not very different in Russia . POSTREVOLUTIONARY PSYCHOLOGY IN RUSSIA In the early decades of the twentieth century psychology in Russia , as in Europe , was torn between contending schools , each of which offered partial explanations of a limited range of phenomena . In 1923 at the first all - Russian psychoneurological congress K . N . Kornilov initiated the first major organizational and intellectual shift in psychology follow ing the revolution . At that time the prestigious Institute of Psychology in Moscow was headed by G . I . Chelpanov , an adherent of Wundt ' s introspective psychology and a foe of behaviorism . ( He had published the sixth edition of his book , The Mind of Man , a critique of materialist theories of the mind , in 1917 , just before the revolution . . ) Chelpanov assigned a restricted role to Marxism in psychology , asserting it could help explain the social organization of consciousness but not the prop - , . erties of indivi ~ ual consciousness . In a talk entitled " Contemporary Introduction 5 Psychology and Marxism " Kornilov criticized Chelpanov both for the idealistic basis of his psychological theory and for the restricted role he assigned to Marxism in psychology . Kornilov , who caned his own ap proach reactology , sought to subsume all branches of psychology within a Marxist framework that used behavioral reactions as the basic data . Kornilov ' s critique of Chelpanov in 1923 won the day . Chelpanov : was removed as director of the Institute of Psychology and was replaced by Kornilov , who immediately brought together a corps of young scientists dedicated to formulating and promoting a behavioral , Marxist theory of psychology . Vygotsky must have produced quite a sensation one year later at the second psychoneurological meeting when he gave a talk entitled " Consciousness as an Object of the Psychology of Be havior . " Whatever else one extracted from Kornilov ' s reactological approach , it quite clearly did not feature the role of consciousness in human activity , nor did it accord the concept of consciousness a role in psychological science . " Vygotsky was dissenting from newly established authority . He was not , however , promoting a return to the position advocated by Chelpanov . In his initial speech and a series of subsequent publications , he made it clear that in his view none of the existing schools of psychol ogy provided a firm foundation for establishing a unified theory of human psychological processes . Borrowing a phrase from his German contemporaries , he often referred to the " crisis in psychology " and set himself the task of achieving a synthesis of contending views on a completely new theoretical basis . For Vygotsky ' s Gestalt contemporaries , a crisis existed because established theories ( primarily Wundt ' s and Watsonian behaviorism ) could not , in their view , explain complex perceptual and problem solving behaviors . For Vygotsky , the crisis went much deeper . He shared the Gestalt psychologists ' dissatisfaction with psychological analysis that began by reducing all phenomena to a set of psychological " atoms . " But he felt that the Gestalt psychologists failed to move beyond the description of complex phenomena to the explanation . of them . Even if one were to accept the Gestalt criticisms of previous approaches , a crisis would still exist because psychology would remain split into two irreconcilable halves : a " natural science " branch that could explain elementary sensory and reflex processes , and a " mental science " half that could describe emergent properties of higher psychological proc esses . What Vygotsky sought was a comprehensive approach that would make possible description and explanation of higher psychological functions in terms acceptable to natural science . To Vygotsky , explana - Introduction 6 tion meant a great deal . It included identification of the brain mechan isms underlying a particular function ; it included a detailed explication of their developmental history to establish the relation between simple and complex forms of what appeared to be the " same behavior ; and , importantly , it included specification of the societal context in which the behavior developed . Vygotsky ' s goals were extremely ambitious , perhaps unreasonably so . He did not achieve these goals ( as he was well aware ) . But he did succeed in providing us with an astute and prescient analysis of modern psychology . A major reason for the continued relevance of Vygotsky ' s work is that in 1924 and the following decade he constructed a penetrating critique of the notion that an understanding of the higher psychological functions in humans can be found by a multiplication and complication of principles derived from animal psychology , in particular those prin ciples that represent the mechanical combination of stimulus - response laws . At the same time he provided a devastating critique of theories which claim that the properties of adult intellectual functions arise from maturation alone , or are in any way preformed in the child and simply waiting for an opportunity to manifest themselves . In stressing the social origins of language and thinking , Vygotsky was following the lead of influential French sociologists , but to our knowledge he was the first modem psychologist to suggest the mechan isms by which culture becomes a part of each person ' s nature . Insisting that psychological functions are a product of the brain ' s activity , he became an early advocate of combining experimental cognitive psychol ogy with neurology and physiology . Finally , by claiming that all of these should be understood in terms of a Marxist theory of the history of human society ) he laid the foundation for a unified behavioral science . MARXIST THEORETICAL FRAMEWORK Contrary to the stereotype of Soviet scholars scurrying to make their theories conform to the Politburo ' s most recent interpretation of Marxism , Vygotsky clearly viewed Marxist thought as a valuable scien tific resource from very early in his career . " A psychologically relevant application of dialectical and historical materialism ' > would be one accurate summary of Vygotsky ' s sociocultural theory of higher mental processes . Vygotsky saw in the methods and principles of dialectical materi alism a solution to key scientific paradoxes facing his contemporaries . A central tenet of this method is that all phenomena be studied as processes Introduction 7 in motion and in change . In terms of the subject matter of psychology , the scientist ' s task is to reconstruct the origin and course of development of behavior and consciousness . Not only does every phenomenon have its history , but this history is characterized by changes both qualitative ( changes in form and structure and basic characteristics ) and quanti tative . Vygotsky applied this line of reasoning to explain the transforma - : tion of elementary psychological processes into complex ones . The schism between natural scientific studies of elementary processes and speculative reflection on cultural forms of behavior might be bridged by tracing the qualitative changes in behavior occuring in the course of development . Thus , when Vygotsky speaks of his approach as " develop menta } , " this is not to be confused with a theory of child development . The developmental method , in Vygotsky ' s view , is the central method of psychological science . Marx ' s theory of society ( known as historical materialism ) also played a fundamental role in Vygotsky ' s thinking . According to Marx , historical changes in society and material life produce changes in " human nature " ( consciousness and behavior ) . Although this general proposition had been echoed by others , Vygotsky was the first to attempt to relate it to concrete psychological questions . In this effort he creatively elaborated on Engels ' concept of human labor and tool use as the means ' by which man changes nature and , in so doing , transforms himself . In chapters 1 through 4 below , Vygotsky exploits the concept of a tool in a fashien that finds its direct antecedents in Engels : " The specialization of the hand - this implies the tool , and the tool implies specific human activity , the transforming reaction of man on nature " ; 2 " the animal merely uses external nature , and brings about changes in it simply by his presence ; man , by his changes , makes it serve his ends , masters it . This is the final , essential distinction between man and other animals " ( p . 291 ) . Vygotsky brilliantly extended this concept of media tion in human - environment interaction to the use of signs as well as tools . Like tool systems , sign systems ( language , writing , number systems ) are created by societies over the course of human history and change with the form of society and the level of its cultural development . Vygotsky believed that the internalization of culturally produced sign systems brings about behavioral transformations and forms the bridge between early and later forms of individual development . Thus for Vygotsky , in the tradition of Marx and Engels , the mechanism of individual developmental change is rooted in society and culture . In later chapters ( especially chapter 5 ) Vygotsky generalizes his conception of the origin of higher psychological functions in a way that Introduction 8 reveals the close relationship between their fundamentally mediated nature and the dialectical , materialist conception of historical change . Citations of Marxist classics were sometimes used to excess by certain Soviet psychologists as they sought a means for building a Marx ist psychology from the chaos of competing schools of thought . Yet in unpublished notes Vygotsky repudiated the " quotation method " of relating Marxism to psychology and made explicit the way in which he . . thought its basic methodological principles might contribute to theory building in psychology : I don ' t want to discover the nature of mind by patching together a lot of quotations . I want to find out how science has to be built , to approach the study of the mind having learned the whole of Marx ' s method . . . . In order to create such an enabling theory - method in the generally accepted scientific manner , it is necessary to discover the essence of the given area of phenomena , the laws according to which they change , their qualitative and quantitative characteristics , their causes . It is necessary to formulate the categories and concepts that are specifically relevant to them - in other words , to create one ' s own Capital . The whole of Capital is written according to the following method : Marx analyzes a single living " cell " of capitalist society - for example , the nature of value . Within this cell he discovers the structure of the entire system and all of its economic institutions . He says that to a layman this analysis may seem a murky tangle of tiny details . Indeed , there may be tiny details , but they are exactly those which are essential to " micro anatomy . " Anyone who could discover what a " psychological " cell is the mechanism producing even a single response - - would thereby find the key to psychology as a whole . [ from unpublished notebooks ] A . careful reading of this manuscript provides convincing proof of both Vygotsky ' s sincerity and the fruitfulness of the framework he developed . THE INTELLECTIJAL AND SOCIAL SETTING Developmental and historical approaches to the study of human nature were not unique to Vygotsky in the Soviet Union in the 1920s . Within psychology , an older colleague , P . P . Blonsky , had already adopted the position that an understanding of complex mental functions requires developmental analysis , " From Blonsky Vygotsky adopted the notion that " behavior can be understood only as the history of behavior . " Blonsky was also an early advocate of the view that the technological activities of people were a Jeey to understanding their psychological makeup , a view that Vygotsky exploited in great detail . Introduction 9 Vygotsky and many other Soviet theorists of the day were also heavily influenced by the work of western European sociologists and anthropologists , like Thurnwald and Levy - Bruhl . t who were interested in the history of mental processes as reconstructed from anthropological evidence of the intellectual activity of primitive peoples . The scant references in this book are a pale reflection of the extent of Vygotsky ' s interest in the development of mental processes understood historically . This aspect of his work received special attention in a publication titled Studies in the History of Behavior published jointly with A . R . Luria in 1930 . It served as the impetus for Luria ' s two expeditions to Central Asia in 1931 and 1932 , the results of which were published long after Vygotsky ' s death , " This historical emphasis was also popular in Soviet linguistics , where interest centered on the problem of the origin of language and its influence on the development of thought . Discussions in linguistics dealt with concepts similar to Vygotsky ' s and also similar to the work of Sapir and Whorf , who were then becoming influential in the United States . While an acquaintance with academic issues of the 1930s is helpful to understanding Vygotsky ' s approach to human cognition , a considera tion of sociopolitical conditions during this time in the Soviet Union is essential as well . Vygotsky worked within a society that put a premium on science and had high hopes for the ability of science to solve the pressing economic and social problems of the Soviet people . Psycho logical theory could not be pursued apart from the practical demands made on scientists by the government , and the broad spectrum of Vygot sky ' s work clearly shows his concern with producing a psychology that would have relevance for education and medical practice . For Vygotsky , the need to carryon theoretical work in an applied context posed no contradiction whatsoever . He had begun his career as a teacher of litera ture , and many of his early articles had dealt with problems of educa tional practice , especially education of the mentally and physically handicapped . He had been a founder of the Institute of Defectology in Moscow , with which he was associated throughout his working life . In such medical problems as congenital blindness , aphasia , and severe mental retardation Vygotsky saw opportunities both for understanding the mental processes of all people and for establishing programs of treatment and remediation . Thus , it was consistent with his general theoretical view that his work should be carried out in a society that sought the elimination of illiteracy and the founding of educational programs to maximize the potential of individual children . Vygotsky ' s participation in the debates surrounding the formulation Introduction 10 of a Marxist psychology embroiled him in fierce disputes in the late 1920s and early 1930s . In these discussions ideology , psychology , and policy were intricately intertwined , as different groups vied for the right to represent psychology . With Kornilov ' s ouster from the Institute of Psychology in 1930 , Vygotsky and his students were for a brief time in the ascendancy , but he was never recognized as the official leader . In the years just prior to his death Vygotsky lectured and wrote extensively on problems of education , often using the term " pedol ogy , " which roughly translates as " educational psychology . " In general he was scornful of pedology that emphasized tests of intellectual ability patterned after the IQ tests then gaining prominence in western Europe and the United States . It was his ambition to reform pedology along the lines suggested in chapter 6 in this volume , but his ambition far exceeded his grasp . Vygotsky was mistakenly accused of advocating mass psychological testing and criticized as a " Great Russian chauvin ist " for suggesting that nonliterate peoples ( such as those living in nonindustrialized sections of central Asia ) had not yet developed the intellectual capacities associated with modern civilization . Two years following his death the Central Committee of the Communist Party issued a decree halting all psychological testing in the Soviet Union . At the same time all leading psychological journals ceased publication for almost twenty years . A period of intellectual ferment and experi mentation was at an end . But by no means did Vygotsky ' s ideas die with him . Even before his death he and his students established a laboratory in Kharkov headed by A . N . Leontiev ( currently Dean of the Psychology Faculty at Mos cow University ) and later by A . V . Zaporozhets ( now Director of the Institute of Preschool Education ) . Luria completed his medical train ing in the latter half of the 1930s and went on to carry out his world famous pioneering work in developmental and neuropsychology . Many of Vygotsky ' s former students hold leading positions in the Institute of Defectology and the Institute of Psychology within the Soviet Acad emy of Pedagogical Sciences , as well as university departments of psy chology such as that at Moscow University . As inspection of any compendium of Soviet psychological research will show , Vygotsky continued and continues to influence research in a wide variety of basic and applied areas related to cognitive processes , their development and dissolution . His ideas have not gone unchal lenged , even - by his students , but they remain a living part of Soviet psychological thought . • Introduction 11 VYGOTSKYS USE OF THE EXPERIMENTAL METHOD Vygotsky ' s references in the text to experiments conducted in his laboratory sometimes leave readers with a sense of unease . He pre sents almost no raw data and ' summaries are quite general . Where are the statistical tests that record whether or not observations reflect " real " effects ? What do these studies prove ? Do they in fact lend any support to Vygotsky ' s general theories , or is he , in spite of his dis claimers , conducting psychology in a speculative manner without sub jecting his central propositions to empirical test ? Those steeped in the methodology of experimental psychology as practiced in most American laboratories may be inclined to withhold the term " experiment " from Vygotsky ' s studies and consider them to be little more than interesting demonstrations or pilot studies . And so , in many respects , they were . We have found it useful to keep in mind the nature of the manu scripts that are the basis of this book . They do not constitute a report of a series of research studies from which general propositions are ex trapolated . Rather , in these writings Vygotsky was concerned with pre senting the basic principles of his theory and method . He drew upon the very limited pool of empirical work available to him in order to illus trate and support these principles . The description of specific studies is schematic and findings are often given as general conclusions rather than as raw data . Some of the studies referred to have been published in greater detail by his students and a few are available in English . " Most studies , however , were conducted by students as pilot investiga tions and were never prepared for publication . Vygotsky ' s laboratory existed for only a decade and his death from tuberculosis was expected at any time . The implications of his theory were so many and varied , and time was so short , that all energy was concentrated on opening up new lines of investigation rather than pursuing any particular line to the fullest . That task remained for Vygotsky ' s students and their suc cessors , who adopted his views in varying ways , incorporating them into new lines of research . " However , the style of experimentation in these essays represents more than a response to the urgent conditions in which they were conducted . Vygotsky ' s concept of the experiment differed from that of American psychology , and understanding this difference is important for an appreciation of Vygotsky ' s contribution to contempo rary cognitive psychology . As every student of an introductory experimental course knows , the purpose of an experiment as conventionally presented is to deter - Introduction 12 mine the conditions controlling behavior . Methodology follows from this objective : the experimental hypothesis predicts aspects of the stimu lus materials or task that will determine particular aspects of the re sponse ; the experimenter seeks maximum control over materials , task , and response in order to test the prediction . Quantification of responses provides the basis for comparison across experiments and for drawing inferences about cause - and - effect relationships . The experiment , in short , is designed to produce a certain performance under conditions that maximize its interpretability . For Vygotsky , the object of experimentation is quite different . The principles of his basic approach ( presented in chapter 5 of this volume ) do not stem from a purely methodological critique of established ex perimental practices ; they How from ' his theory of the nature of higher psychological processes and the task of scientific explanation in psy chology . If higher psychological processes arise and undergo changes in the course of learning and development , psychology will only fully understand them by determining their origin and mapping their history . At first sight it would appear that such a task precludes the experimental method and requires study of individual behavior over long periods of time , But Vygotsky believed ( and ingeniously demonstrated ) that the experiment could serve an important role by making visible processes that are ordinarily hidden beneath the surface of habitual behavior . He wrote that in a properly conceived experiment the investigator could create processes that " telescope the actual course of development of a given function . " He called this method of investigation the " experi mental - genetic " method , a term he shared with Heinz Werner , an out standing contemporary whose developmental , comparative approach to psychology was well - known to Vygotsky . To serve as an effective means of studying " the course of develop ment of process , " the experiment must provide maximum opportunity for the subject to engage in a variety of activities that can be observed , not just rigidly controlled . One technique Vygotsky effectively used for this purpose was to introduce obstacles or difficulties into the task that disrupted routine methods of problem solving . For example , in study ing children ' s communication and the function of egocentric speech Vygotsky set up a task situation that required children to engage in co operative activity with others who did not share their language ( foreign speaking or deaf children ) . Another method was to provide alternative routes to problem solving , including a variety of materials ( Vygotsky called them " external aids " ) that could be used in different ways to satisfy the demands of the ta ; k . By careful observation of the uses made Introduction 13 of these external aids by children at different ages under different condi tions of task difficulty , Vygotsky sought to reconstruct the series of changes in intellectual operations that normally unfold during the course of the child ' s biographical development . A third technique was to set a task before the child that exceeded his knowledge and abilities , in order to discover the rudimentary beginnings of new skills . This pro - . cedure is well illustrated in studies on writing ( chapter 7 ) , in which young toddlers were provided with pencil and paper and asked to make representations of events , thus disclosing to the investigator the child ' s earliest understanding of the nature of graphic symbolism . With all these procedures the critical data furnished by the experi ment is not performance level as such but the methods by which the per formance is achieved . The contrast between conventional experimental work ( focusing on performance ) and Vygotsky ' s work ( focusing on process ) has its contemporary expression in recent studies on children ' s memory by American investigators . Many studies ( including a number of our own ) have presented children of various ages with lists of words to be remembered and have analyzed such performance measures as number of words recalled and the order of recall . From these indicators the investigators have sought to make inferences about whether or not , and to what extent , young ~ hildren engage in organizing activities as a memory strategy . On the other hand , John Flavell and his colleagues , using procedures very much like those of Vygotsky ' s students , provided children the materials to be remembered , and instructed them to do whatever they wanted to help them remember . They then observed children ' s attempts at classifying the items , the kinds of grouping they made , and other indices of children ' s tendency to use organizational strategies in remembering . As with Vygotsky , the central question is : What are the children doing ? How are they trying to satisfy task demands ? In this connection we would like to clarify a basic concept of Vygotsky ' s theoretical approach and experimental method that we be lieve has been widely misinterpreted . In several places in the text Vygot sky , in referring to the structure of behavior , uses a term that we have translated as " mediated . " Occasionally this term is accompanied by a fig ure depicting a stimulus , a response , and a " mediating link " between them ( for example , S - X - R ) . The same term , and virtually the same dia gram , were introduced into American learning theory in the late 1930s and became very popular in the 1950s as attempts were made to extend stimulus - response theories of learning to complex human behavior , especially language . It is important to keep in mind that Vygotsky was Introduction 14 not a stimulus - response learning theorist and did not intend his idea of mediated behavior to be thought of in this context . What he did intend to convey by this notion was that in higher forms of human be havior , the individual actively modifies the stimulus situation as a part of the process of responding to it . It was the entire structure of this activity which produced the behavior that Vygotsky attempted to de note by the term " mediating . " Several implications follow from Vygotsky ' s theoretical approach and method of experimentation . One is that experimental results will be qualitative as well as quantitative in nature . Detailed descriptions , based on careful observation , will constitute an important part of experimental findings . To some , such findings may seem merely anec dotal ; Vygotsky maintained that if carried out objectively and with scientific rigor , such observations have the status of validated fact . Another consequence of this new approach to experimentation is to break down some of the barriers that are traditionally erected be . . tween " laboratory " and " field . " Experimental interventions and obser . . vations may often be as well or better executed in play , school , and clinical settings than in the psychologist ' s laboratory . The sensitive ob servations and imaginative interventions reported in this book attest to this possibility . Finally , an experimental method that seeks to trace the history of the development of psychological functions sits more comfortably than the classical method alongside other methods in the social sciences con cerned with history - including the history of culture and society as well as the history of the child . To Vygotsky , anthropological and sociological studies were partners with observation and experiment in the grand enterprise of accounting for the progress of human conscious ness and intellect . Biographical Note on L . S . Vygotsky Lev Semyonovitch Vygotsky was born November 5 , 1896 , in the town of Orsha , northeast of Minsk in Byelorussia . In 1913 he completed gymnasium in Gomel with a gold medal . In 1917 , after graduating from Moscow University with a specialization in literature , he began his literary research . From 1917 to 1923 Vygotsky taught literature and psychology in a school in Gomel , where he also directed the theater section of the adult education center and gave many speeches and lectures on problems of literature and science . During this period Vygotsky founded ' the literary journal Verask . Here he published his flrst literary research , later reissued as The Psychology of Art . He also founded a psychological laboratory in the Teacher Training Institute , where he gave a course on psychology , the contents of which were later published in Pedagogi cal Psychology . In 1924 Vygotsky moved to Moscow and began to work first at the Institute of Psychology and then in the Institute of Defectology , which he founded . At the same time he directed a department for the educa tion of physically defective and mentally retarded children in Nar compros ( Peoples Committee on Education ) , and taught courses in the Krupskaya Academy of Communist Education , the Second Moscow State University ( later the Moscow State Pedagogical Institute ) , and the Hertzen Pedagogical Institute in Leningrad . Between 1925 and 1934 Vygotsky gathered around him a large group of young scientists work ing in the areas of psychology , defectology , and mental abnormality , An interest in medicine led Vygotsky simultaneously to undertake medi cal training , first in the medical institute in Moscow and later in Kharkov , 15 Biographical Note 16 where he gave a psychology course in the Ukrainian Psychoneurologi cal Academy . Not long before his death Vygotsky was invited to head the department of psychology in the All - Union Institute of Experi mental Medicine . He died of tuberculosis June 11 , 1934 . A . R . Luria Part One / Mind in Society Basic Theory and Data Tool and Symbol in Child Development The primary purpose of this book is to characterize the uniquely human aspects of behavior , and to offer hypotheses about the way these traits have been formed in the course of human history and the way they develop over an individual ' s lifetime . This analysis will be concerned with three fundamental issues : ( 1 ) What is the relation between human beings and their environment , both physical and social ? ( 2 ) What new forms of activity were responsi ble for establishing labor as the fundamental means of relating humans to nature and what are the psychological consequences of these forms of activity ? ( 3 ) What is the nature of the relationship between the use of tools and the development of speech ? None of these questions has been fully treated by scholars concerned with understanding animal I and human psychology . Karl Stumpf , a prominent German psychologist in the early years of the twentieth century , based his studies on a set of premises completely different from those I will employ here . ! He compared the study of children to the study of botany , and stressed the botanical character of development , which he associated with maturation of the whole or ganism . The fact is that maturation per se is a secondary factor in the de velopment of the most complex , unique forms of human behavior . The development of these behaviors is characterized by complicated , quali tative transformations of one form of behavior into another ( or , as Hegel would phrase it , a transformation of quantity into quality ) . The conception of maturation as a passive process cannot adequately de scribe these complex phenomena . Nevertheless , as A . Gesell has aptly 19 Mind in Society . 20 pointed out , in our approaches to development we continue to use the botanical analogy in our description of child development ( for example , we say that the early education of children takes place in a " kinder garten " ) . 2 Recently several psychologists have suggested that this botan ical model must be abandoned . In response to this kind of criticism , modern psychology has ascended the ladder of science by adopting zoological models as the basis for a new general approach to understanding the development of children . Once the captive of botany , child psychology is now mes merized by zoology . The observations on which these newer models draw come almost entirely from the animal kingdom , and answers to questions about children are sought in experiments carried out on animals . Both the results of experiments with animals and the proce dures used to obtain these results are finding their way from the animal laboratory into the nursery . This convergence of child and animal psychology has contributed _ significantly to the study of the biological basis of human behavior . Many links between child and animal behavior , particularly in the study of elementary psychological processes , have been established . But a paradox has now emerged . When the botanical model was fashionable , psychologists emphasized the unique character of higher psychological functions and the difficulty of studying them by experimental means . But this zoological approach to the higher intellectual processes - those processes that are uniquely human - has led psychologists to interpret the higher intellectual functions as a direct continuation of correspond ing processes in animals . This style of theorizing is particularly apparent in the analysis of practical intelligence in children , the most important aspect of which concerns the child ' s use of tools . PRACTICAL INTELLIGENCE IN ANIMALS AND CHILDREN The work of Wolfgang Kohler is particularly significant in the study of practical intelligence . " He conducted many experiments with apes during World War I , and occasionally compared some of his observa tions of chimpanzees > behavior with particular kinds of responses in children . This direct analogy between practical intelligence in the child and similar response by apes became the guiding principle of experi mental work in the field . K . Buhler ' s research * also sought to establish similarities between child and ape . " He studied the way in which . young children grasp ob - Tool and Symbol in Child Development 21 jects , their ability to make detours while pursuing a goal , and the manner in which they use pr ! niitive tools : ·These observations , as well as his experiment in which a youngchild i ; ' " asked to remove a ring from a stick , illustrate an approach akin to Kohler ' s . Buhler interpreted the manifestations of practical intelligence in children as being of exactly the same type as those we are familiar with in chimpanzees . Indeed , ' there is a phase in the life of the child that Buhler designated the " chimpanzee age " ( p . 48 ) . One ten - month - old infant whom he studied was able to pull a string to obtain a cookie that was attached to it . The ability to remove a ring from a post by lifting it rather than trying to pull it sideways did not appear until the middle of the second year . " Although these experiments were interpreted as support for the analogy between the child and apes , they also led Buhler to the important dis covery , which will be explicated in later sections , that the beginnings of practical intelligence in the child ( he termed it " technical thinking " ) , as well as the actions of the chimpanzee , are independent of speech . Charlotte Buhler ' s detailed observations of infants during their first year of life gave further support to this conclusion . " She found the first manifestations of practical intelligence took place at the very young age of six months , However , it is not only tool use that develops at this point in a child ' s history but also systematic movement and perception , the brain and hands - in fact , the child ' s entire organism . Consequently , the child ' s system of activity is determined at each specific stage both by the child ' s degree of organic development and by his or her degree of mastery in the use of tools . K . Buhler established the developmentallyimportant principle that the beginnings of intelligent speech are preceded by technical thinking , and technical thinking comprises the initial phase of cognitive develop ment . His lead in emphasizing the chimpanzee - like features of children ' s behavior has been followed by many others . It is in extrapolating this idea that the dangers of zoological models and analogies between human and animal behaviors find their clearest expression . The pitfalls are slight in research that focuses on the preverbal period in the child ' s development , as Buhler ' s did . However , he drew a questionable conclu sion from his work with very young children when he stated , " The achievements of the chimpanzee are quite independent of language and in the case of man , even in later life , technical thinking , or think ing in terms of tools , is far less closely bound up with language and concepts than other forms of thinking . " ? Buhler proceeded from the assumption that the relationship be tween practical intelligence and speech that characterizes the ten - Mind in Society . 2 . 2 month - old child remains intact throughout her lifetime . This analysis postulating the independence of intelligent action from speech runs contrary to our own Bndings , which reveal the integration of speech and practical thinking in the course of development . Shapiro and Gerke offer an important analysis of the development of practical thinking in children based upon experiments modeled after Kohler ' s problem - solving studies with chimpanzees . " They theorize that children ' s practical thinking is similar to adult thought in certain respects and different in others , and emphasize the dominant role of social experience in human development . In their view , social experience exerts its effect through imitation ; . when the child imitates the way adults use tools and objects , she masters the very principle involved in a particular activity . They suggest that repeated actions pile up , one upon another , as in a multi - exposure photograph ; the common traits become clear and the differences become blurred . The result is a crys talized scheme , a definite principle of activity . The child , as she be comes more experienced , acquires a greater number of models that she understands . These models represent , as it were , a refined cumula tive design of all similar actions ; at the same time , they are also a rough blueprint for possible types of action in the future . However , Shapiro and Gerke ' s notion of adaptation is too firmly linked to a mechanical conception of repetition . For them , social ex perience serves only to furnish the child with motor schemas ; they do not take into account the changes occurring in the internal structure of the child ' s intellectual operations . In their descriptions of children ' s problem solving , the authors are forced to note the " specific role ful filled by speech " in the practical and adaptive efforts of the growing child . But their description of this role is a strange one . " Speech , " they say , " replaces and compensates for real adaptation ; it does not serve as a bridge leading to past experience but to a purely social adaptation which is achieved via the experimenter . " This analysis does not allow for the contribution speech makes to the development of a new struc tural organization of practical activity . Guillaume and Meyerson offer a different conclusion regarding the role of speech in the inception of uniquely human forms of behavior . " : From their extremely interesting experiments on tool use among apes , they concluded that the methods used by apes to accomplish a given task are similar in principle and coincide on certain essential points to those used by people suffering from aphasia ( that is , individuals who are deprived of speechl . e ' Their findings support my assumption that Tool and Symbol in Child Development 23 speech plays an essential role in the organization of higher psychological functions . 10 These experimental examples bring us full circle to the beginning of our review of psychological theories regarding child development . Buhler ' s experiments indicate that the practical activity of the young child prior to speech development is identical to that of the ape , and ' Guillaume and Meyerson suggest that the ape ' s behavior is akin to that observed in people who are deprived of speech . Both of these lines of work focus our attention on the importance of understanding the practi cal activity of children at the age when they are just beginning to speak . My own work as well as that of my collaborators is directed at these same problems . But our premises differ from those of previous investi gators . Our primary concern is to describe and specify the development of those forms of practical intelligence that are specifically human . RELATION BETWEEN SPEECH AND TOOL USE In his classic experiments with apes Kohler demonstrated the futility of attempting to develop even the most elementary sign and symbolic operations in animals . He concluded that tool use among apes is independent of symbolic activity . Further attempts to cultivate productive speech in the ape have also produced negative results . These experiments showed once more that the purposive behavior of the ani mal is independent of any speech or sign - using activity . The study of tool use in isolation from sign use is common in re search work on the natural history of practical intellect , and psychol ogists who studied the . development of symbolic processes in the child have followed the same procedure . Consequently , the origin and de velopment of speech , as well as all other sign - using activity , were treated as independent of the organization of the child ' s practical activity . Psychologists preferred to study the development of sign use as an example of pure intellect and not as the product of the child ' s develop mental history . They often attributed sign use to the child ' s spontaneous discovery of the relation between signs and their meanings . As W . Stern stated , recognition of the fact that verbal signs have meaning constitutes " the greatest discovery in the child ' s life . " 11 A number of authors fix this happy " moment " at the juncture of the child ' s first and second year , regarding it as the product of the child ' s mental activity . De tailed examination of the development of speech and other forms of sign use was assumed to be unnecessary . Instead , it has routinely been as - Mind in Society 24 sumed that the child ' s mind contains all stages of furore intellectual development ; they exist in complete form , awaiting the proper moment to emerge . Not only were speech and practical intelligence assumed to have different origins , but their joint participation in common operations was considered to be of no basic psychological importance ( as in the work of Shapiro and Gerke ) . Even when speech and the use of tools were closely linked in one operation , they were still studied as separate processes belonging to two completely different classes of phenomena . At best , their simultaneous occurrence was considered a consequence of accidental , external factors . The students of practical intelligence as well as those who study speech development often fail to recognize the interweaving of these two functions . Consequently , the children ' s adaptive behavior and sign using activity are treated as parallel phenomena - a view that leads to Piaget ' s concept of " egocentric " speech . P He did not attribute an Important role to speech in the organization of the child ' s activities , nor did he stress its communicative functions , although he was obliged to admit its practical importance . Although practical intelligence and sign use can operate inde pendently of each other in young children , the dialectical unity of these systems in the human adult is the very essence of complex human be havior . Our analysis accords symbolic activity a specific organizing function that penetrates the process of tool use and produces funda mentally new forms of behavior . SOCIAL INTERACTION AND THE TRANSFORMAnON OF PRACTICAL ACTIVITY Based on the discussion in the previous section , and illustrated by experimental work to be described later , the following conclusion may be made : the most significant moment in the course of intellectual de velopment , which gives birth to the purely human forms of practical and abstract intelligence , occurs when speech and practical activity , two previously completely independent lines of development , converge . Although children ' s use of tools during their preverbal period is com parable to that of apes , as soon as speech and the use of signs are incorporated into any action , the action becomes transformed and or ganized along entirely new lines . The specifically human use of tools is thus realized , going beyond the more limited use of tools possible among the higher animals . Tool and Symbol in Child Development 25 Prior to mastering his own behavior , the child begins to master his surroundings with the help of speech . This produces new relations with the environment in addition ' to the new organization of behavior itself . The creation of these uniquely human forms of behavior later produce the intellect and become the basis of productive work : the specifically human fonn of the use of tools . Observations of children in an experimental situation similar to that of Kohler ' s apes show that the children not only a . ¢Jn attempting to achieve a goal but also speak . As a rule this speech arises spontane ously and continues almost without interruption throughout the experi ment . It increases and is more persistent every time the situation be comes more complicated and the goal more difficult to attain . Attempts to block it ( as the experiments of my collaborator R . E . Levina have shown ) are either futile or lead the child to " freeze up . " Levina posed practical problems for four - and five - year - old children such as obtaining a piece of candy from a cupboard . The candy was placed out of reach so the child could not obtain it directly . As the child got more and more involved in trying to obtain the candy , " egocentric " speech began to manifest itself as part of her active striving . At first this speech consisted of a description and analysis of the situation , but it gradually took on a " planful " character , reflecting possible paths to solution of the problem . Finally , it was included as part of the solution . For example , a four - and - a - half - year - old girl was asked to get candy from a cupboard with a stool and a stick as possible tools . Levina ' s description reads as follows : ( Stands on a stool , quietly looking , feeling along a shelf with stick . ) " On the stool . " ( Glances at experimenter . Puts stick in other hand . ) " Is that really the candy ? " ( Hesitates . ) cCI can get it from that other stool , stand and get it . " ( Gets second stool . ) " No , that doesn ' t get it . I could use the stick . " ( Takes stick , knocks at the candy . ) C ' It will move now . " ( Knocks candy . ) celt moved , I couldn ' t get it with the stool , but the , but the stick worked . " 13 In such circumstances it seems both natural and necessary for children to speak while they act ; in our research we have f ~ und that speech not only accompanies practical activity but also plays a specific role in carrying it out . Our experiments demonstrate two important facts : ( 1 ) A child ' s speech is as important as the role of action in attaining the goal . Children not only speak about what they are doing ; their speech and action are part of one and the same complex psychological function , directed toward the solution of the problem at hand . ( 2 ) The more complex the action demanded by the situation and Mind in Society 26 the less direct its solution , the greater the importance played by speech in the operation as a whole . Sometimes speech becomes of such . vital importance that , if not permitted to use it , young children cannot ac complish the given task . These observations lead me to the conclusion that children solve practical tasks with the help of their speech , as well as their eyes and hands . This unity of perception , speech , and action , which ultimately produces internalization of the visual field , constitutes the central sub ject matter for any analysis of the origin of uniquely human forms of behavior . To develop the first of these two points , we must ask : What is it that , really distinguishes the actions of the speaking child from the actions of an ape when solving practical problems ? The first thing that strikes the experimenter is the incomparably greater freedom of children ' s operations , their greater independence from the structure of the concrete , visual situation . Children , with the aid of speech , create greater possibilities than apes can accomplish through action . One important manifestation of this greater flexibility is that the child is able to ignore the direct line between actor and goal . Instead , he engages in a number of preliminary acts , using what we speak of as . IDstrumental , or mediated ( indirect ) , methods . In the process of solving a task the child is able to include sti ; muli that do not lie within the immediate visual field . Using words ( one class of such stimuli ) to create a specific plan , the child achieves a much broader range of activity , applying as tools not only those objects that lie near at hand , but searching for and preparing such stimuli as can be useful in the solution of the task , and planning future actions . Second , the practical operations of a child who can speak become much less impulsive and spontaneous than those of the ape . The ape typically makes a series of uncontrolled attempts to solve the given problem . In contrast , the child who uses speech divides the activity into two consecutive parts . She plans how to solve the problem through speech and then carries out the prepared solution through overt ac tivity . Direct manipulation is replaced by a complex psychological process through which - inner motivation and intentions , postponed in time , stimulate their own development and realization . This new kind of psychological structure is absent in apes , even in rudimentary forms . Finally , it is decisively important that speech not only facilitates the child ' s effective manipulation of objects . but also controls the child ' s own behavior . Thus , with the help of speech children , unlike apes , acquire the capacity to be both the subjects and objects of their own behavior . \ Tool and Symbol in Child Development 27 Experimental investigation of the egocentric speech of children en gaged in various activities such as that illustrated by Levina produced the second fact of great importance demonstrated by our experiments : the relative amount of egocentric speech , as measured by Piaget ' s meth ods , increases in relation to the difficulty of the child ' s task . > On the basis of these experiments my collaborators and I developed the hypothesis that children ' s egocentric speech should be regarded as the transitional form between external and internal speech . Functionally , egocentric speech is the basis for inner speech , while in its external form it is embedded in communicative speech . One way to increase the production of egocentric speech is to complicate a task in such a way that the child cannot make direct use of tools for its solution . When faced with such a challenge , the children ' s emotional use of language increases as well as their efforts to achieve a less automatic , more intelligent solution . They search verbally for a new plan , and their utterances reveal the close connection between ego centric and socialized speech . This is best seen when the experimenter leaves the room or fails to answer the children ' s appeals for help . Upon being deprived of the opportunity to engage in social speech , children immediately switch over to egocentric speech . While the interrelationship of these two functions of language is apparent in this setting , it is important to remember that egocentric speech is linked to children ' s social speech by many transitional forms . The first significant illustration of the link between these two language functions occurs when children find that they are unable to solve a prob lem by themselves . They then turn to an adult und verbally describe the method that they cannot carry out by themselves . The greatest change in children ' s capacity to use language as a problem - solving tool takes place somewhat later in their development , when socialized speech ( which has previously been used to address an adult ) is turned inuiard . Instead of appealing to the adult , children appeal to themselves ; lan guage thus takes on an intrapersonal function in addition to its inter personal use . When children develop a method of behavior for guid ing themselves that had previously been used in relation to another person , when they organize their own activities according to a social form of behavior , they succeed in applying a social attitude to them selves . The history of the process of the internalization of social speech is also the history of the socialization of children ' s practical intellect . The relation between speech and action is a dynamic one in the course of children ' s development . The structural relation can shift even during an experiment . The crucial change occurs as follows : At an Mind in Society 28 early stage speech accompanies the child ' s actions and reflects the vicissitudes of problem solving in a disrupted and chaotic form . At a later stage speech moves more and more toward the starting point of the process , so that it comes to precede action . It functions then as an aid to a plan that has been conceived but not yet realized in behavior . An interesting analogy can be found in children ' s speech while drawing ( see also chapter 8 ) . Young children name their drawings only after they have completed them ; they need to see them before they can decide what they are . As children get older they can decide in advance what they are going to draw . This displacement of the naming process signifies a change in the function of speech . Initially speech follows actions , is provoked by and dominated by activity . At a later stage , however , when speech is moved to the starting point of an activity , a new relation be tween word and action emerges . Now speech guides , determines , and dominates the course of action ; the planning function of speech comes into being in addition to the already existing function of language to reflect the external world . " Just as a mold gives shape to a substance , words can shape an activity into a structure . However , that structure may be changed or reshaped when children learn touse Ianguage in ways that allow them to go beyond previous experiences when planning future action . In contrast to the notion of sudden discovery popularized by Stern , we envisage verbal , intellectual activity as a series of stages in which the emotional and communicative functions of speech are expanded by the addition of the planning function . As a result the child acquires the abil ity to engage in complex operations extending over time . Unlike the ape , which Kohler tells us is " the slave of its own visual field , " children acquire an independence with respect to their concrete surroundings ; they cease to act in the immediately given and evident space . Once children learn how to use the planning function of their language effectively , their psychological field changes radically . A view of the future is now an integral part of their approaches to their surroundings . In subsequent chapters , I will - describe the developmental course of some of these central psychological functions in greater detail . To summarize what has been said thus far in this section : The specifically human capacity for language enables children to provide for auxiliary tools in the solution of difficult tasks , to overcome impulsive action , to plan a solution to a problem prior to its execution , and to master their own behavior . Signs and words serve children first and foremost as a means of social contact with other people . The cognitive and communicative functions of language then become the basis of a Tool and Symbol in Child Development 29 new and superior form of activity in children , distinguishing them from animals . The changes I have described do not occur in a one - dimensional , even fashion . Our research has shown that very small children solve problems using unique mixtures of processes . In contrast with adults , who react differently to objects and to people , young children are likely to fuse action and speech when responding to both objects and social beings . This fusion of activity is analagous to syncretism in perception , which has been described by many developmental psychologists . The unevenness I am speaking of is seen quite clearly in a situation where small children , when unable to solve the task before them easily , combine direct attempts to obtain the desired end with a reliance upon emotional speech . At times speech expresses the children ' s desires , while at other times it serves as a substitute for actually achieving the goal . The ' Child may attempt to solve the task through verbal formulations and by appeals to the experimenter for help . This mixture of diverse forms of activity was at first bewildering ; but further observations drew our attention to a sequence of actions that clarify the meaning of the children ' s behavior in such circumstances , For example , after completing a number of intelligent and interrelated actions that should help him solve a particular problem successfully , the child suddenly , upon meeting a difficulty , ceases all attempts and turns for help to the experi menter . Any obstacle to the child ' s efforts at solving the problem may interrupt his activity . The child ' s verbal appeal to another person is an effort to fill the hiatus his activity has revealed . By asking a question , the child indicates that he has , in fact , formulated a plan to solve the task before him , but is unable to perform all the necessary operations . Through repeated experiences of this type , children learn covertly ( mentally ) to plan their activities . At the same time they enlist the assist ance of another person in accordance with the requirements of the problem posed for them . The child ' s ability to control another person ' s behavior becomes a necessary part of the child ' s practical activity . Initially this problem solving in conjunction with another person is not differentiated with respect to the roles played by the child and his helper ; it is a general , syncretic whole . We have more than once ob served that in the course of solving a task , children get confused because they begin to merge the logic of what they are doing with the logic of the same problem as it has to be solved with the cooperation of another person . Sometimes syncretic action manifests itself when children realize the hopelessness of their direct efforts to solve a problem . As in the example from Levina ' s work , children address the objects of their atten - Mind in Society 30 tion equally with words and sticks , demonstrating the fundamental and inseparable tie between speech and action in the child ' s activity ; this unity becomes particularly clear when compared with the separation of these processes in adults . In summary , children confronted with a problem that is slightly too complicated for them exhibit . . a complex variety of responses including direct attempts at attaining the goal , the use of tools , speech directed toward the person conducting the experiment or speech that simply accompanies the action , and direct , verbal appeals to the object of attention itself . If analyzed dynamically , this alloy of speech and action has a very specific function in the history of the child ' s development ; it also demon strates the logic of its own genesis . From the very first days of the child ' s development his activities acquire a meaning of their own in a system of social behavior and , being directed towards a definite purpose , are re fracted through the prism of the child ' s environment . The path from object to child and from child to object passes through another person . This complex human structure is the product of a developmental process deeply rooted in the links between individual and social history . 2 The Development of Perception and Attention The linkage between tool use and speech affects several psycho logical functions , in particular perception , sensory - motor operations , and attention , each of which is part of a dynamic system of behavior . Experimental - developmental research indicates that the connections and relations among functions constitute systems that change as radically in the course of a child ' s development as do the individual functions themselves . Considering each function in turn , I will examine how speech introduces qualitative changes in both its form and its relation to other functions . Kohler ' s work emphasized the importance of the structure of the visual field in organizing the ape ' s practical behavior . The entire process of problem solving is essentially determined by perception . In this respect Kohler had ample grounds for believing that these animals are bound by their sensory field to a much greater extent than adult humans . They are incapable of modifying their sensory field by means of volun tary effort , Indeed , it would probably be useful to view as a general law the dependence of all natural forms of perception on the structure of the sensory field . However , a child ' s perception , because it is human , does not develop as a direct continuation and further perfection of the forms of animal perception , not even of those animals that stand nearest to humankind . Experiments conducted to clarify this problem led us to discover some basic laws that characterize the higher human forms of perception . The first set of experiments concerned developmental stages of picture perception in children . Similar experiments describing specific aspects of young children ' s perception and its dependence On higher 31 Mind in Society 32 psychological mechanisms had been carried out earlier by Binet and analyzed in detail by Stern . ' Both authors found that the way small children describe pictures differs at successive developmental stages . A two - year - old usually limits his description to separate objects within the picture . Older children describe actions and indicate the complex rela tions among the separate objects within the picture . Stern inferred from these observations that a stage when children perceive separate objects precedes the stage when they perceive actions and relations in addition to objects , that is , when they perceive the picture as a whole . However , many psychological observations suggest that the child ' s perceptual processes are initially fused and only later become more differentiated . We resolved the contradiction between these two positions through an experiment replicating Stern ' s study of children ' s descriptions of pictures , in which we asked children to communicate the contents of a picture without using speech . We suggested that the description be made in pantomime . The two - year - old child , who according to Stem ' s schema is still at the separate " object " stage of development , perceived the dynamic features of the picture and reproduced them with ease through pantomime . What Stem regarded as a characteristic of the child ' s perceptual skills proved to be a product of the limitations of her language development or , in other words , a feature of her verbalized perception . A series of related observations revealed that labeling is the primary function of speech used by young children . Labeling enables the child to choose a specific object , to single it out from the entire situation he is perceiving . Simultaneously , however , the child embellishes his first words with very expressive gestures , which compensate for his diffi culties in communicating meaningfully through language . By means of words children single out separate elements , thereby overcoming the natural structure of the sensory field and forming new ( artifically introduced and dynamic ) structural centers . The·child begins to perceive the world not only through his eyes but also through his speech . As a result , the immediacy of " natural " perception is supplanted by a complex mediated process ; as such , speech becomes an essential part of the child ' s cognitive development . Later , the intellectual mechanisms related to speech acquire a new function ; verbalized perception in the child is no longer limited to labeling . At this next stage of development , speech acquires a synthe sizing function , which in turn is instrumental in achieving more complex forms of cognitive perception . These changes give human perception an The Development of Perception and Attention 33 entirely new character , quite distinct from the analogous processes in higher animals . The role of language in perception is striking because of the oppos ing tendencies implicit in the nature of visual perception and language . The independent elements in a visual field are simultaneously perceived ; in this sense , visual perception is integral . Speech , on the other hand , requires sequential processing . Each element is separately labeled and then connected in a sentence structure , making speech essentially analytical . Our research has shown that even at very early stages of develop ment , language and perception are linked . In the solution of nonverbal tasks , even if a problem is solved without a sound being uttered , lan guage plays a role in the outcome . These findings substantiate the thesis of psychological linguistics as formulated many years ago by A . Poteb nya , who argued for the inevitable interdependence between human thought and language , " A special feature of human perception - which arises at . a very young age - is the perception of real objects . This is something for which there is no analogy in animal perception . By this term I mean that I do not see the world simply in color and shape but also as a world with sense and meaning . I do not merely see something round and black with two hands ; I see a clock and I can distinguish one hand from the other . Some brain - injured patients say , when they see a clock , that they are seeing something round and white with two thin steel strips , but they do not know it is a clock ; such people have lost their real relationship with objects . These observations suggest that all human perception consists of categorized rather than isolated perceptions . The developmental transition to qualitatively new forms of behavior is not confined to changes in perception alone . Perception is part of a dynamic system of behavior ; hence , the relation between transforma tions of perceptual processes and transformations in other intellectual activities is of primary importance . This point is illustrated by our studies on choice behavior , which show the changing relation between percep tion and motor action in young children . STUDIES OF CHOICE BEHAVIOR IN CHILDREN We requested four - and five - year - old children to press one of five keys on a keyboard as they identified each one of a series of picture stimuli assigned to each key . Because this task exceeds the capabilities Mind in Society 34 of the children , it causes serious difficulties and more intensive efforts to solve the problem . Perhaps the most remarkable result is that the entire process of selection by the child is external , and concentrated in the motor sphere , thus allowing the experimenter to observe the very nature of the choice process itself in the child ' s movements . The child does her selecting while carrying out whatever movements the choice requires . The structure of the child ' s decision does not in the least resemble the adult process . Adults make a preliminary decision internally and subsequently carry out the choice in the for ~ of a single movement that executes the plan . The child ' s choice resembles a somewhat delayed selection among his own movements . Vascillations in perception are directly reflected in the structure of movement . The child ' s movements are replete with diffuse gropings that interrupt and succeed one another . A mere glance at the chart tracing the child ' s movements is sufficient to convince one of the basic motor nature of the process . The main difference between the choice processes in the child and in the adult is that for the child the series of tentative movements consti tute the selection process . The child does not choose the stimulus ( the necessary key ) as the starting point for the consequent movement but rather selects the movement , using the instruction as a guide to check the results . Thus , the child resolves her choice not through a direct process of visual perception but through movement , hesitating between two stimuli , her fingers hovering above and moving from one key to an other , going half - way and then coming back . When the child transfers her attention to a new location , thereby creating a new focus in the \ dynamic structure of perception , her hand obediently moves toward this new center , in unison with the eye . In short , movement is not separated from perception : the processes coincide almost exactly . In the behavior of the higher animals , visual perception forms part of a more complex whole in a similar way . The ape does not perceive the visual situation passively ; a complex behavioral structure consisting of reflexive , affective , motor , and intellectual factors is directed toward acquiring the object that attracts it . The ape ' s movements constitute an immediate dynamic continuation of its perception . In human children , this early , diffusely structured response undergoes a fundamental change as soon as a more complex psychological function is utilized in the choice process . The natural process present in animals is then transformed into a higher psychological operation . Subsequent to the experiment described above we attempted to sim plify the task of selection - by marking each key with a corresponding The Development of Perception and Attention 35 sign to serve as an additional stimulus that could direct and organize the choice process . The child was asked , upon the appearance of a target stimulus , to press the key marked with the corresponding sign . As early as age five or six the child is able to fulfill this task easily . The addition of this new ingredient radically changes the structure of the choice process . The elementary , " natural " operation is replaced by a new and more complicated one . The simpler task evokes a more complexly struc tured response . When the child attends to the auxiliary sign in order to find the key corresponding to the given stimulus , he no longer exhibits those motor impulses that arise directly from perception . There are no uncertain groping movements in the air such as we observed in the earlier choice reaction when auxiliary aids were not used . The use of auxiliary signs breaks up the fusion of the sensory field and the motor system and thus makes new kinds of behavior possible . A " functional barrier " is created between the initial and final moments of the choice response ; the direct impulse to move is shunted by pre liminary circuits . The child who formerly solved the problem impulsively now solves it through an internally established connection between the stimulus and the corresponding auxiliary sign . The movement that previously had been the choice now serves only to fulfill the prepared operation . The system of signs restructures the whole psychological process and enables the child to master her movement . It reconstructs the choice process on a totally new basis . Movement detaches itself from direct perception and comes under the control of sign functions included in the choice response . This development represents a fundamental break with the natural history of behavior and initiates the transition from the primitive behavior of animals to the higher intellectual activ ities of humans . Attention should be given first place among the major functions in the psychological structure underlying the use of tools . Beginning with Kohler , scholars have noted that the ability or inability to direct one ' s attention is an essential determinant of the success or failure of any practical operation . However , the difference between the practical intel ligence of children and animals is that children are capable of recon structing their perception and thus freeing themselves from the given structure of the field . With the help of the indicative function of words , the child begins to master his attention " creating new structural centers in the perceived situation . As K . Ko£fka so aptly put it , the child is able to determine for herself the «center of gravity " of her perceptual field ; her behavior is not regulated solely by the salience of individual ele - Mind in Society 36 ments within it . The child evaluates the relative importance of these elements , singling out new CCfigures " from the background and thus widening the possibilities for controlling her activities . " In addition to reorganizing the visual - spatial field , the child , with the help of speech , creates a time field that is just as perceptible and real to him as the visual one . The speaking child has the ability to direct his attention in a dynamic way . He can view changes in his immediate situation from the point of view of past activities , and he can act in the present from the . viewpoint of the future . For the ape , the task ' is unsolvable unless the goal and the object needed to reach it are both simultaneously in view . For the child , this gap is easily overcome by verbally controlling her attention and thereby reorganizing her perceptual field . The ape will perceive a stick one moment , but cease to pay attention to it after its visual field has changed and the goal comes into view . The ape must see his stick in order to pay attention to it ; the child may pay attention in order to see . Thus , the child ' s field of attention embraces not one but a whole series of potential perceptual fields that form successive , dynamic structures over time . The transition from the simultaneous structure of the visual field to the successive structure of the dynamic field of atten tion is achieved through the reconstruction of the separate activities that are a part of the required operations . When this occurs , we can say that the field of attention has detached itself from the perceptual field and unfolded itself in time , as one component of a dynamic series of psycho logical activities . The possibility of combining elements of the past and present visual fields ( for instance , tool and goal ) in one field of attention leads in turn to a basic reconstruction of another vital function , memory . ( See chapter 3 . ) Through verbal formulations of past situations and activities , the child frees himself from the limitations of direct recall ; he succeeds in synthesizing the past and present to suit his purposes . The changes that occur in memory are similar to those that occur in the child ' s perceptual field where centers of gravity are shifted and figure and ground rela tionship are altered . The child ' s memory not only makes fragments of the past more available , but also results in a new method of uniting the elements of past experience with the present . Created with the help of speech , the time field for action extends both forward and backward . Future activity that can be included in an ongoing activity is represented by signs . As in the case of memory and attention , the inclusion of signs in temporal perception does not lead to a simple lengthening of the operation in time ; rather , it creates the The Development of Perception and Attention 37 conditions for the development of a single system that includes effective elements of the past , present , and future . This emerging psychological system in the child now encompasses two new functions : intentions and symbolic representations of purposeful action . This change in the structure of the child ' s behavior is related to basic alterations in the child ' s needs and motivations . When Lindner compared the methods by which deaf children solved tasks to the methods used by Kohler ' s ape , he noted that the motives guiding the ape and those guiding the child to achieve mastery of a goal were not the same . " The " instinctive " urges predominating in the animal become secondary in the child . New motives , socially rooted and intense , pro vide the child with direction . K . Lewin described these motives as Quasi - Beduerinisse ( quasi - needs ) and argued that their inclusion in any given task leads to the reorganization of the child ' s whole affective and voluntary system . " He believed that with the development of these quasi needs , the child ' s emotional thrust is shifted from a preoccupation with the outcome to the nature of the solution . In essence , the " task " ( Aufgabe ) in experiments with apes exists only in the eyes of the experimenter ; as far as the animal is concerned there exists only the bait and the obstacles standing in his way . The child , however , strives to solve the given prob lem and thus has an entirely different purpose . Because he is able to form quasi - needs , the child is capable of breaking the operation into its separate parts , each of which becomes an independent problem that he formulates for himself with the help of speech . In his excellent analysis of the psychology of purposeful activity , Lewin gives a clear - cut definition of voluntary activity as a product of the historical - cultural development of behavior and as a unique feature of human psychology . The fact that man displays extraordinary freedom with respect to even the most senseless intention is astounding in itself , he asserts . This freedom is incomparably less characteristic of children and probably of nonliterate humans , too . There is reason to believe that voluntary activity , more than highly developed intellect , distinguishes humans from the animals which stand closest to them . Mastery of Memory and Thinking In the light of what my collaborators and I had learned about the functions of speech in reorganizing perception and creating new relations among psychological functions , we undertook a broad study of other forms of sign - using activity in children in all its concrete manifestations ( drawing pictures , writing , reading , using number systems , and - so on ) . We also considered whether other operations not related to practical intellect would show the same laws of development we had discovered when analyzing practical intellect . Several series of experiments carried out by my colleagues and myself dealt with these problems , and now , based on the data we ob tained from them , we are able to describe in schematic form the basic laws that characterize the structure and development of the child ' s sign operations . These will be presented through a discussion of memory , which is exceptionally appropriate for study of the changes that signs introduce into basic psychological functions because it clearly reveals the social origin of signs as well as their crucial role in the individual ' s development , SOCIAL ORIGINS OF INDIRECT ( MEDIATED ) MEMORY A comparative investigation of human memory reveals that , even at the earliest stages of social development , there are two , principally different , types of memory . One , dominating in the behavior of non literate peoples , is characterized by the nonmediated impression of materials , by the retention of actual experiences as the basis of mnemonic ( memory ) traces . We call this natural memory , and it is clearly iIIus - 38 Maste1 ' Y of Memory and Thinking 39 trated in E . R . jaensch ' s studies of eidetic imagery . ' This kind of memory is very close to perception , because it arises out of the direct influence of external stimuli upon human beings . From the point of view of struc ture , the entire process is characterized by a quality of immediacy . Natural memory is not the only kind , however , even in the case of nonIiterate men and women . On the contrary , other types of memory belonging to a completely different developmental line coexist with natural memory . The use of notched sticks and knots . s the beginnings of writing and simple memory aids all demonstrate that even at early stages of historical development humans went beyond the limits of the psychological functions given to them by nature and proceeded to a new culturally - elaborated organization oftheir behavior . Comparative analy sis shows that such activity is absent in even the highest species of animals ; we believe that these sign operations are the product of specific conditions of social development . Even such comparatively simple operations as tying a knot or mark ing a stick as a reminder change the psychological structure of the memory process . They extend the operation of memory beyond the biological dimensions of the human nervous system and permit it to in corporate artificial , or self - generated , stimuli , which we call signs . This merger , unique to human beings , signifies an entirely new fonn of behavior . The essential difference between it and the elementary func tions is to be found in the structure of the stimulus - response relations of each . The central characteristic of elementary functions is that they arc totally and directly determined by stimulation from the environment . For higher functions , the central feature is self - generated stimulation , that is , the creation and use of artificial stimuli which become the immediate causes of behavior , STRUCTURE OF SIGN OPERATIONS Every elementary form of behavior presupposes a direct reaction to the task set before the organism ( which can be expressed by the simple S ) R formula ) . But the structure of sign operations re quires an intermediate link between the stimulus and the response . This intermediate link is a second order stimulus ( sign ) that is drawn into the operation where it fulfills a special function ; it creates a new relation between Sand R . The term " drawn into " indicates that an individual must be actively engaged in establishing such a link . This sign also pos sesses the important characteristic of reverse action ( that is , it operates on the individual , not the environment ) . Mind in Society 40 Consequently , the simple stimulus - response process is replaced by a complex , mediated act , which we picture as : s x Figure 1 R In this new process the direct impulse to react is inhibited , and an auxiliary stimulus that facilitates the completion of the operation by indirect means is incorporated . Careful studies demonstrate that this type of organization is basic to all higher psychological processes , although in much more sophisti cated forms than that shown above . The intermediate link in this formula is not simply a method of improving the previously existing operation , nor is it a mere additional link in an S - R chain . Because this auxiliary stimulus possesses the specific function of reverse action , it transfers the psychological operation to higher and qualitatively new forms and per mits humans , by the aid of extrinsic stimuli , to control their behavior from the outside . The use of signs leads humans to a specific structure of behavior that breaks away from biological development and creates new forms of a culturally - based psychological process . EARLY SIGN OPERATIONS IN CHILDREN The following experiments , conducted under A . N . Leontiev in our laboratories , demonstrate with particular clarity the role of signs in voluntary attention and memory . " Children were asked to playa game in which they were to answer a set of questions without using certain words in their answers . As a rule each child was presented three or ' four tasks differing in the constraints placed upon answers and the kinds of potential stimulus aids the child could use . In each task the child was asked eighteen questions , seven of which had to do with color ( for example , " What color is . . . ? " ) . The child was asked to answer each question promptly using a single word . The initial task was conducted in exactly this fashion . With the second task , we began to introduce additional rules that the child had to follow in order to succeed . For example , there were two color names the child MQ8tery of Memory and Thinking 41 was forbidden to use , and no color name could be used twice . The third task had the same rules as the second , but the child was given nine colored cards as aids to playing the game ( " these cards can help you to win " ) . The fourth task was like the third and was used in cases where the child either failed to use the color cards or began to do so only late in the third task . Before and after each task we asked the child questions . to determine if she remembered and understood the instructions . A set of questions for a typical task is the following ( in this case green and yellow are the forbidden colors ) : ( 1 ) Have you a playmate ? ( 2 ) What color is your shirt ? ( 3 ) Did you ever go in a train ? ( 4 ) What color are the railway - carriages ? ( 5 ) Do you want to be big ? ( 6 ) Were you ever at the theater ? ( 7 ) Do you like to play in the room ? ( 8 ) What color is the floor ? ( 9 ) And the walls ? ( 10 ) Can you write ? ( 11 ) Have you seen lilac ? ( 12 ) What color is lilac ? ( 13 ) Do you like sweet things ? ( 14 ) Were you ever in the country ? ( 15 ) What colors can leaves be ? ( 16 ) Can you swim ? ( 17 ) What is your favorite color ? ( 18 ) What does one do with a pencil ? For the third and fourth tasks the following color cards were pro vided as aids : black , white , red , blue , yellow , green , lilac , brown , and gray . The results for thirty subjects ranging in age from five to twenty seven years are summarized in table 1 , which contains the average number of errors on tasks 2 and 3 and the difference between the two tasks . Looking first at the data from task 2 , we see a slight decrease in errors from ages five to thirteen and a sharp drop in adulthood . For task 3 the sharpest drop occurs between the Hve - tc - six and eight - to - nine year - old groups . The difference between tasks 2 and 3 is small for both Table 1 . Errors on forbidden colors task . Number of Errors ( average ) Age subjects Task 2 Task 3 Difference 5 - 6 7 3 . 9 3 . 6 0 . 3 8 - 9 7 3 . 3 1 . 5 1 . 8 10 - 13 8 3 . 1 0 . 3 2 . 8 22 - 27 8 1 . 4 0 . 6 0 . 8 the preschool children and the adults . The difference is largest for the school - age children . The processes that give rise to the summary figures are most readily revealed by looking at transcripts representative of children in the difler - Mind in Society 42 ent groups . The preschool children ( age five to six years ) were generally unable to discover how to use the auxiliary color cards and had a great deal of trouble with both tasks . Even when we tried to explain to them how the color cards could help them , children at this age were incapable of using these external stimuli in order to organize their own behavior . The following transcript is from a five - year - old boy ; Task 4 . Forbidden colors : blue and red ( with cards ) . 2 . What color are houses ? 3 . Is the sun shining brightly ? 4 . What color is the sky ? 8 . What colors are tomatoes ? 9 . And what color are exercise books ? 12 . What color are balls ? 13 . Do you live in the town ? Do you think you have won ? What must you not do if you want to win ? And what else ? Red [ without looking at forbidden colors ] . Yes . White [ without looking at card ; but after replying , searches for white card ] . Here it is ! [ Picks it up and keeps it in his hand . ] Red . [ Glances at cards . ] White - like this ! [ pointing to white card ] . White [ looking at card ] . No . Don ' t know - s - yes . Mustn ' t say red or blue . Mustn ' t say the same word twice . This transcript suggests that the " aids " actually hindered this child . His repeated use of " white " as a response occurred when his attention was fixed on the white card . The aids are only an accidental feature of the situation for him . Still , there is no doubt that preschool children sometimes demonstrate precursors of the use of external signs . From this point of view certain cases are of special interest . For example , after we suggested to a child that he use the cards to carry out his task ( " take the cards , they will help you to win " ) , he searched for the forbidden colors and put all such cards out of his sight , as if trying to prevent himself from naming them . In spite of their apparent variety , methods for using the cards can be reduced to two basic types . First the child may put forbidden colors out of sight , display the remainder , and , as he answers the questions , place the colors already named to one side . This is the less effective but Mastery of Memory and Thinking 43 the earliest method used . The card in this case serves only to register the named color . Initially , children often do not turn to the cards before they answer the question about color , and only after it is named do they search among the cards , turn over , move , or put away the one named . This is undoubtedly the simplest act of memorization with the help of external means . It is only later that the conditions of the experiment . bestow a new , second function on the cards . Before naming a color the child makes a selection with the help of the cards . It makes no difference whether the child looks at the cards so far unused or whether she attends to the colors she has already named . In either case the cards are inter posed in the process and serve as a means of regulating her activity . The preliminary hiding of forbidden colors , which is a distinguishing char acteristic of the first method for using the cards , does not yet lead to the complete substitution of a less mature operation by a more complex one ; it represents merely a step in that direction . Its occurrence is explained partly by the greater simplicity of this operation in mastering memory and partly by a " magical " attitude toward various potential problem solving aids that children frequently display . The following examples from a thirteen - year - old schoolgirl illus trate these points : Task 2 . Forbidden colors : green and yellow ( without cards ) . 1 . Have you playmates ? 2 . What color is your blouse ? 3 . Have you been in a train ? 4 . What color are railway carriages ? 5 . Do you want to be a big girl ? 6 . Were you ever in a theater ? 7 . Do you like to play in the room ? 8 . What color is the Hoor ? 9 . And the walls ? 10 . Can you write ? 11 . Have you seen lilac ? 12 . What color is lilac ? 13 . Do you like sweets ? 14 . Were you ever in the country ? Yes . Gray . Yes . Gray . [ Notices that she has re peated the same ~ color twice , Iaughs . ] Yes . Yes . Yes . Gray . [ Hesitates . ] Again - I repeated it . White . Yes . Yes . Lilac color . Yes . Yes . Mind in Society 44 Task 2 . Forbidden colors : green and yellow ( without cards ) - - - cont . 15 . And what color were the leaves ? Green - no , shouldn ' t have said green - brown , red , sometimes . 16 . Can you swim ? 17 . What is your favorite color ? 18 . What do you do with a pencil ? What do you think , did you win or lose ? What should you not have said ? And what else ? Yes . Yellow ! I can ' t ! [ Throws up hands behind head . ] Write . Lost . Green and yellow . Shouldn ' t repeat . Task 3 . Forbidden colors : blue and red ( with cards ) . The subject puts forbidden colors to one side and spreads out the remainder in a row before her . 1 . Do you go for walks in the street ? 2 . What color are the houses ? 3 . Is the sun shining brightly ? 4 . What color is the sky ? 5 . Do you like candy ? 6 . Have you seen a rose ? 7 . Do you like vegetables ? 8 . What color are tomatoes ? 9 . And exercise books ? 10 . Have you any toys ? 11 . Do you play ball ? 12 . And what color are balls ? 13 . Do you live in the town ? 14 . Did you see the demonstration ? 15 . What color are flags ? 16 . Have you any books ? 17 . What colors are their covers ? 18 . When does it get dark ? Yes . Gray . [ After answering , looks at the cards and turned over the gray one . ] Brightly . White . [ First looks at card and then turns it over . ] Yes . Yes . Yes . Green . [ Turns over card . ] Yellow . [ Turns over card . ] No . Yes . Gray [ without glancing at cards ; after answering , glances and notices mistake ] . Yes . Yes . Black . [ First looks at cards and then turns one over . ] Yes . Lilac [ turning over card ] . At night . Mastery of Memory and Thinking 45 Our results as reflected in the transcripts and table 1 indicate three basic stages in the development of mediated remembering . At the first stage ( preschool age ) the child is not capable of mastering his behavior by organizing special stimuli . The colored cards that might help the child in his task do not increase to any considerable extent the effective ness of this operation . Although they act as stimuli , they do not acquire . an instrumental function . The second stage of development is charac terized by a sharp difference in the indices in both of the main tasks . The introduction of cards as a system of auxiliary , external stimuli raises the effectiveness of the child ' s activity considerably . At this stage the external sign predominates . The auxiliary stimulus is a psychological instrument acting from the outside . At the third stage ( among adults ) the diHerence between their performance in the two tasks decreases and their coefficients become more nearly equal , but now on a new and higher basis . This does not mean that the behavior of adults again becomes direct and natural . At this higher stage of development behav ior remains mediated . But now we see that in the third task the auxiliary stimuli are emancipated from primary external forms . What takes place is what we have called internalization ; the external sign that school children require has been transformed into an internal sign produced by the adult as a means of remembering . This series of tasks applied to people of different ages shows how the external forms of mediated behavior develop . THE NATURAL mSTORY OF SIGN OPERATIONS Although the indirect ( or mediated ) aspect of psychological opera tions is an essential feature of higher mental processes , it would be a great mistake , as I pointed out with respect to the beginnings of speech , to believe that indirect operations appear as the result of a pure logic . They are not invented or discovered by the child in the form of a sudden insight or lightning - quick guess ( the so - called " aha " reaction ) . The child does not suddenly and irrevocably deduce the relation between the sign and the method for using it . Nor does she intuitively develop an abstract attitude derived , so to speak , from " the depths of the child ' s own mind . " This metaphysical view , according to which inherent psycho logical schemata exist prior to any experience , leads inevitably to an a priori conception of higher psychological functions . Our research has led us to quite different conclusions . We have found that sign operations appear as a result of a complex and prolonged process subject to all the basic laws of psychological evolution . This Mind in Society 46 means that sign - using activity in children is neither simply invented nor passed down by adults ; rather it arises from something that is originally not a sign operation and becomes one only after a series of qualitative transformations . Each of these transformations provides the conditions for the next stage and is itself conditioned by the preceding one ; thus , transformations are linked like stages of a single process , and are his torical in nature . In this respect , the higher psychological functions are no exception to the general rule that applies to elementary processes ; they , too , are subject to the fundamental law of development which knows no exceptions , and appear in the general course of the child ' s psychological development as the outcome of the same dialectical process , not as something introduced from without or from within . If we include this history of higher psychological functions as a factor in psychological development , we must arrive at a new concept of development itself . Within a general process of development , two qualitatively diHerent lines of development , differing in origin , can be distinguished : the elementary processes , which are of biological origin , on the one hand , and the higher psychological functions , of sociocultural origin , on the other . The history of child behavior is born from the inter weaving of these tuio lines . The history of the development of the higher psychological functions is impossible without a study of their prehistory , their biological roots , and their organic disposition . The developmental roots of two fundamental , cultural forms of behavior arise during infancy : the use of tools and human speech . This alone places infancy at the center of the prehistory of cultural development . The potential for complex sign operations is embedded in the earliest stages of individual development . However , observations show that between the initial level ( elementary behavior ) and the higher levels ( mediated . forms of behavior ) many transitional psychological sys tems occur . In the history of behavior these transitional systems lie be tween the biologically given and the culturally acquired . We refer to this process as the natural history of the sign . Another experimental paradigm designed to study mediated memo rizing provides the opportunity to observe this natural history of the sign . N . G . Morozova presented children with words to remember and . auxiliary pictures that could be used as mediators , " She found that during the preschool years the idea of purposefully using the auxiliary picture ( sign ) asa means of memorizing is still foreign to the child . Even if the child did tum to the auxiliary picture in order to memorize a given word , it was not necessarily easy for him to execute the reverse operation . At this stage the learner does not usually recall the primary stimulus Mastery of Memory and Thinking 47 when being shown - the auxiliary stimulus . Rather , the sign evokes a new associative or syncretic series represented by the following scheme : A ~ X Figure 2 The operation has not yet progressed to the more advanced level which is mediated in form using culturally elaborated features . In contrast with figure 2 , the usual scheme for mediated memorizing can be represented by the following : Figure 3 During the process represented by figure 2 , Y may lead to a whole series of new associations , among which the subject may arrive at the starting point A . However , this sequence is still devoid of its purposeful and instrumental character . In the second scheme , the word ' s auxiliary sign , X , possesses the quality of reverse action , so that the subject can reliably retrieve A . The steps leading from the scheme in figure 2 to the scheme in figure 3 cap be illustrated by the following examples taken from the work of my students . L . V . Zankov demonstrated that younger children , particularly between the ages of four and six , must rely on meaningful , ready - made links between the " reminder " sign and the word to be re membered . " If meaningless figures were presented as memory aids , the children would ~ often refuse to make use of them ; they would make no attempt to make up connections between the picture cue and the word they were supposed to remember . Rather , they would attempt to turn these figures into direct copies of the to - be - remembered word . For example , the figure Q , presented as a reminder of the word " bucket , " was turned upside down by the children and served to remind them of the word only when the figure D really began to resemble a bucket . Similarly , the figure t : = : : : 1 became the sign of the word " bench " only when turned upside down ( r : : : = : : : : J ) . In all these Mind in Society 48 cases , children linked the figures to the word stimuli by changing the meaning of the sign instead of using the mediating link offered by the experimenter . The introduction of these meaningless figures encouraged the children to engage in active mnemonic activity instead of relying on already formed links , but it also led them to treat the sign stimulus as the direct representation of the object to be remembered . When this proved impossible , the child refused to memorize . A similar phenomenon is apparent in u . C . Yussevich ' s unpublished study with small children . The auxiliary stimuli , which were pictures that bore no direct relation to the word presented , were rarely used as signs . The child looked at the picture and tried to see in it the object she had to remember . For example , when asked to remember the word " sun " with the help of a picture showing an axe , one child did it very easily ; she pointed to a small yellow spot in the drawing and said , " There it is , the sun . " This child replaced potentially complex instrumental memorization by a search for a direct representation of the stimulus ( akin to an eidetic image ) . The child sought an eidetic - like representation in the auxiliary sign . In both the Zankov and Yussevich examples , the child reproduced the required word through a process of direct repre sentation rather than mediated symbolization . The laws describing the role of sign oper ~ tions at this stage of de velopment are completely different from the laws describing how the child links up a word with a sign in fully developed sign operations . Children in the experiments just described illustrate a stage of de velopment between the elementary and the completely instrumental process from which fully mediated operations will later develop . Leontiev ' s work on the development of sign operations in memory provides examples supporting the theoretical points discussed above as well as later stages in the development of sign operations in memory . P He gave a set of twenty words for recall to children of different ages and levels of mental ability . The materials were presented in three ways . First , the words were simply spoken at intervals of about three seconds and the child was told to recall them , In a second task the child was given a set of twenty pictures and told to lise them to help recall the words . The pictures were not replicas of the words but were associated with them . In the third series twenty pictures bearing no obvious rela tion to the to - be - remembered words were used . The basic questions in this research were to what extent can children convert their remem bering into a mediated activity using pictures as auxiliary memory aids and how does their success depend upon the different degrees of diffi culty represented by the two , potentially mediated , series . As we might expect , the results differed depending upon the group Mastery of Memory and Thinking 49 of children and the difficulty of the recall task . Normal children ( ten to twelve years of age ) recalled twice as many words when the pictures were available as memory aids as they did without them . They were able to make use of both picture series equally well . Mildly retarded children of the same age benefited little , if at all , from the presence of the pictures ; and for severely retarded children , the auxiliary stimuli . actually interfered with performance . The original transcripts from this study clearly show intermediate levels of functioning in which the child attends to the auxiliary picture stimulus and even associates it with the word to be recalled but cannot integrate the stimulus into his system of remembering . Thus , one child selected a picture of an onion to recall the word " dinner . " When asked why she chose the picture , she gave the perfectly satisfactory answer , " Because I eat an onion . " However , she was unable to recall the word " dinner " during the experiment . This example shows that the ability to form elementary associations is not sufficient to ensure that the associa tive relation will fulfill the instrumental function necessary to produce recall . This kind of evidence leads us to conclude that the development of mediated psychological functions ( in this case , mediated memory ) represents a special line of development that does not wholly coincide with the development of elementary processes . I should mention also that the addition of pictures as memory aids did not facilitate recall of adults . The reason for the " failure " is directly opposite to the reasons underlying the failure of memory aids to affect the severely retarded children . In the case of adults , the process of mediated memorizing is so fully developed that it occurs even in the absence of special external aids . MEMORY AND THINKING Remembering activities do not simply change as the child grows older ; the role of these activities in the system of psychological functions also changes , Nonmediated memory takes place in the context of psy chological operations that may have nothing at all in common with the psychological operations that accompany mediated remembering ; con sequently , experimental results may make it appear that some psycho logical functions are replaced by others . In other words , with a change in developmental level there occurs a change not so much in the struc ture of a single function ( which , for example , we may call memory ) as in the character of those functions with the aid of which remembering takes place ; what changes is the interfunctional relations that connect memory with other functions . Mind in Society 50 The memory of older children is not only different from the memory of younger children ; it also plays a different role in the older child ' s cognitive activity . Memory in early childhood is one of the central psychological functions upon which all the other functions are built . OUf analyses suggest that thinking in the very young child is in many respects determined by his memory , and is certainly not the same thing as the thinking of the more mature child . For the very young child , to think means to remember ; at no time after very early childhood do we see such a close connection between these two psychological func tions . I will give three examples . The first is the definition of concepts in children , which are based on their recollections . If you ask a child to tell you what a snail is , he will say that it is little , it slithers , and it sticks out its foot ; if you ask him to tell you what a grandmother is , he is likely to reply , " She has a soft lap . " In both cases the child gives a very clear summary of the impressions which the topic has made upon him and which he recollects ~ The content of the thinking act in the child when defining such concepts is determined not so much· by the logicai struc ture of the concept itself as by the child ' s concrete recollections . It is syncretic in ch ~ racter and reflects the fact that the child ' s thinking de pends first of allan his memory . Another example is the development of visual concepts in very young children . Investigations of children ' s thinking when they are re quired to transpose a relation learned with one set of stimuli to a similar set have shown that their transfer is nothing more than remembering with respect to isolated instances . Their general representations of the world are based on the recall of concrete instances and do not yet possess the ch ' aracter of an abstraction . " The last example concerns the analysis of word meaning . Investi gations in this area show that the connections underlying words are fundamentally different in the young child and in the adult . Children ' s concepts relate to a series of examples and are constructed in a manner similar to the way we represent family names . To name words for them is not so much to indicate familiar concepts as to name familiar families or whole groups of visual things connected by visual ties . In this way the experience of the child and the " unmediated " influence of the child ' s experience are documented in his memory and directly deter mine the entire structure of the young child ' s thought . All these facts suggest that , from the point of view of psychological development , memory rather than abstract thought is the definitive Mastery of Memory and Thinking 51 characteristic of the early stages of cognitive development . However , in the course of development a transformation occurs , especially in adoles cence . Investigations of memory at this age have shown that toward the end of childhood the interfunctional relations involving memory reverse their direction . For the young child , to think means to recall ; but for the adolescent , to recall means to think . Her memory is so " logicalized " that remembering is reduced to establishing and finding logical relations ; recognizing consists in discovering that element which the task indicates has to be found . This logicalization is indicative of how relations among cognitive functions change in the course of development . At the transitional age all ideas and concepts , all mental structures , cease to be organized accord ing to family types and become organized as abstract concepts . There can be no doubt that to remember an item when thinking in concepts is a completely different task from thinking in complexes , although the processes are compatible with each other . " Therefore , the development of children ' s memory must be studied not only with respect to changes happening within memory itself , but also with respect to the relation between memory and other functions . When a human being ties a knot in her handkerchief as a reminder , she is , in essence , constructing the process of memorizing by forcing an external object to remind her of something ; she transforms remembering into an external activity . This fact alone is enough to demonstrate the fundamental characteristic of the higher forms of behavior . In the ele mentary form something is remembered ; in the higher form humans remember something . In the first case a temporary link is formed owing to the simultaneous occurrence of two stimuli that affect the organism ; in the second case humans personally create a temporary link through an artificial combination of stimuli . The very essence of human memory consists in the fact that human beings actively remember with the help of signs . It may be said that the basic characteristic of human behavior in general is that humans personally influence their relations with the environment and through that environment personally change their behavior , subjugating it to their control . . It has been remarked that the very essence of civilization consists of purposely building monuments so as not to forget . In both the knot and the monument we have manifestations of the most funda mental and characteristic feature distinguishing human from animal memory . 4 Internalization of Higher Psychological Functions When comparing the principles regulating unconditioned and con ditioned reflexes , Pavlov uses the example of a telephone call . One possi bility is for the call to connect two points directly via a special line . This corresponds to an unconditioned reflex . The other possibility is for the phone call to be relayed through a special , central station with the help of temporary and limitlessly variable connections . This corresponds to a conditioned reflex . The cerebral cortex , as the organ that closes the conditioned reflex circuit , plays the role of such a central station . The fundamental message of our analysis of the processes that underlie the creation of signs ( signalization ) may be expressed by a more generalized form of the same metaphor . Let us take the case of tying a knot as a reminder or drawing lots as a means of decision making . There is no doubt that in both cases a temporary conditioned connection is formed , that is , a connection of Pavlov ' s second type . But if we wish to grasp the essentials of what is happening here , we are forced to take into consideration not only the function of the telephone mechanism but also of the operator who plugged in and thus connected the line . In our example , the connection was established by the person who tied the knot . This feature distinguishes the higher forms of behavior { rom the lower . The invention and use of signs as auxiliary means of solving a given psychological problem ( to remember , compare something , report , choose , and so on ) is analogous to the invention and use of tools in one psychological respect . The sign acts as an instrument of psychological activity in a manner analggous to the ' role of a tool in labor . But this analogy , like any other , does not imply the identity of these similar 52 Internalization of Higher Psychological Functions 53 concepts . We should not expect to find many similarities with tools in those means of adaptation we call signs . What ' s more , in addition to the similar and common feature shared by the two kinds of activity , we see very essential differences . Here we want to be as precise as possible . Leaning for support on the term ' s figurative meaning , some psychologists have used the word ' " tool " when referring to the indirect function of an object as the means for accomplishing some activity . Expressions such as " the tongue is the tool of thought " or " aides de memoire " are usually bereft of any definite content and hardly mean more than what they really are : simple metaphors and more colorful ways of expressing the fact that certain objects or operations play an auxiliary role in psychological activity . On the other hand , there have been many attempts to invest such expressions with a literal meaning , to equate the sign with the tool . By erasing the fundamental distinction between them , this approach loses the specific characteristics of each type of activity and leaves us with one general psychological form of determination . This is the position adopted by Dewey , one of pragmatism ' s representatives . He defines the tongue as the tool of tools , transposing Aristotle ' s definition of the human hand to speech . I wish it to be clear that the analogy between sign and tool that I propose is diHerent from either of the approaches just discussed . The uncertain , indistinct meaning that is usually read into the figurative use of the word " tool " in no way eases the researcher ' s task . His task is to uncover the real relationship , not the figurative one , that exists be tween behavior and its auxiliary means . Should we conceive of thought or memory as being analogous to external activity ? Do the " means of activity " simply play the indefinite role of supporting the psychological process that leans on them ? What is the nature of this support ? What in general does it mean to be a " means " of thought or of memory ? Psychol ogists who so enjoy using these fuzzy expressions furnish us with no answer to these questions . But the position of those psychologists who treat such expressions literally turns out to be even fuzzier . Concepts that have a psychological aspect but do not actuaIly belong to psychology - such as " technique " are psychologized without any grounds whatsoever . Equating psycho logical and nonpsychological phenomena is possible only if one ignores the essence of each form of activity , as well as the differences between their historic roles and nature . Distinctions between tools as a means of labor , of mastering nature , and language as a means of social intercourse Mind in Society 54 become dissolved in the general concept of artifacts or artificial adapta tions . We seek to understand the behavioral role of the sign in all its uniqueness . This goal has motivated our empirical studies of how both tool and sign use are mutually linked and yet separate in the child ' s cultural development . We have adopted three conditions as a starting point for this work . The first pertains to the analogy and common points of the two types of activity , the second clarifies their basic differences , and the third attempts to demonstrate the real psychological link exist ing between the one and the other , or at least to hint at its existence . As we have already noted , the basic analogy between sign and tool rests on the mediating function that characterizes each of them . They may , therefore , from the psychological perspective , be subsumed under the same category . We can express the logical relationship between the use of signs and of tools using the schema in figure 4 , which shows each concept subsumed under the more general concept of indirect ( mediated ) activity . I Mediated activity I / " " I Sign I I Tool I Figure 4 That concept , quite justly , was invested with the broadest general meaning by Hegel , who saw in it a characteristic feature of human reason : " Reason , " he wrote , " is just as cunning as she is powerful . Her cunning consists principally in her mediating activity which , by causing objects to act and react on each other in accordance with their own nature , in this way , without any direct interference in the process , car ries out reasons ' intentions . " ! Marx cites that definition when speaking of working tools , to show that man " uses the mechanical , physical , and chemical properties of objects so as to make them act as forces that affect other objects in order to fulfill his personal goals . " 2 This analysis provides a sound basis for assigning the use of signs to the category of mediated activity , for the essence of sign use consists in man ' s affecting behavior through signs . In both cases the indirect ( mediated ) function comes to the forefront . I shall not define further the relation of these jointly subsumed concepts to each other , or their rela tion to the more generic concept of mediated activity . I should only Internalization of High ~ Psychological Functions 55 like to note that neither can , under any circumstance , be considered iso morphic with respect to the functions they perform , nor can they be seen as fully exhausting the concept of mediated activity . A host of other mediated activities might be named ; cognitive activity is not limited to the use of tools or signs . On the purely logical plane of the relation between the two con - . cepts , our schema represents the two means of adaptation as diverging lines of mediated activity . This divergence is the basis for our second point . A . most essential difference between sign and tool , and the basis for the real divergence of the two lines , is the different ways that they orient human behavior . The tool ' s function is to serve as the conductor of human influence on the object of activity ; it is externally oriented ; it must lead to changes in objects . It is a means by which human external activity is aimed at mastering , and triumphing over , nature . The sign , on the other hand , changes nothing in the object of a psychological opera tion . It is a means of internal activity aimed at mastering oneself ; the sign is internally oriented . These activities are so different from each other that the nature of the means they use cannot be the same in both cases . Finally , the third point pertains to the real tie . between these activi ties and , hence , to the real tie of their development in phylo - and onto genesis . The mastering of nature and the mastering of behavior are mutually linked , just as man ' s alteration of nature alters man ' s own nature . In phylogenesis we can reconstruct this link through fragmentary but convincing documentary evidence , while in ontogenesis we can trace it experimentally . One thing is already certain . Just as the first use of tools refutes the notion that development represents the mere unfolding of the child ' s organically predetermined system of activity , so the first use of signs demonstrates that there cannot be a single organically predetermined internal system of activity that exists for each psychological function . The use of artificial means , the transition to mediated activity , funda mentally changes all psychological operations just as the use of tools limitlessly broadens the range of activities within which the new psychological functions may operate . In this context , we can use the term higher psychological function , or higher behavior as referring to the combination of tool and sign in psychological activity . Several phases in the use of sign operations have been described thus far . In the initial phase reliance upon external signs is crucial to the child ' s effort . But through development these operations undergo radi cal changes : the entire operation of mediated activity ( for example , Mind in Society 56 memorizing ) begins to take place as a purely internal process . Para doxically , late stages of the child ' s behavior appear to be the same as early stages of memorizing , which were characterized by a direct process . The very young child does not rely upon external means ; rather he uses a " natural , ' " eidetic " approach . Judging only from external appearances , it seems that the older child has simply begun to memorize more and better ; that she has somehow perfected and developed her old methods of memorizing . At the highest levels she appears to have abandoned any reliance upon signs . However , this appearance is only illusory . Development , as often happens , proceeds here not in a circle but in , a spiral , passing through the same point at each new revolution while advancing to a higher level . We call the internal reconstruction of an external operation in ternalization . A good example of this process may be found in the development of pointing . Initially , this gesture is nothing more than an unsuccessful attempt to grasp something , a movement aimed at a certain object which designates forthcoming activity . The child attempts to grasp an object placed beyond his reach ; his hands , stretched toward that object , remain poised in the air . His fingers make grasping move ments . At this initial stage pointi . ng is represented by the child ' s move ment , which seems to be pointing to an object - that and nothing more . When the mother comes to the child ' s aid and realizes his move ment indicates something , the situation changes fundamentally . Pointing becomes a gesture for others . The child ' s unsuccessful attempt engenders a reaction not from the object he seeks but from another person . Conse quently , the primary meaning of that unsuccessful grasping movement is established by others . Only later , when the child can link his unsuc cessful grasping movement to the objective situation as a whole , does he begin to understand this movement as pointing . At this juncture there occurs a change in that movement ' s function : from an object - oriented movement it becomes a movement aimed at another person , a means of establishing relations . The grasping movement changes to the act of pointing . As a result of this change , the movement itself is then physi cally simplified , and what results is the form of pointing that we may call a true gesture . It becomes a true gesture only after it objectively manifests all the functions of pointing for others and is understood by others as such a gesture . Its meaning and functions are created at first by all objective situation and then by people who surround the child . As the above description of pointing illustrates , the process of internalization consists of a - series of transformations : ( a ) An operation that initially represents an external activity is Internalization of Higher Psychological Functions 57 reconstructed and begins to occur internally . Of particular importance to the development of higher mental processes is the transformation of sign - using activity , the history and characteristics of which are illus trated by the development of practical intelligence , voluntary attention , and memory . ( b ) An interpersonal process is transformed into an intrapersonal one . Every function in the child ' s cultural development appears twice : first , on the social level , and later , on the individual level ; first , between people ( interpsychological ) , and then inside the child ( intrapsychologi cal ) . This applies equally to voluntary attention , to logical memory , and to the formation of concepts . All the higher functions originate as actual relations between human individuals . ( c ) The transformation of an interpersonal process into an intraper sonal one is the result of a long series of developmental events . The process being transformed continues to exist and to change as an external form of activity for a long time before definitively turning inward . For many functions , the stage of external signs lasts forever , that is , it is their final stage of development . Other functions develop further and gradu ally become inner functions . However , they take on the character of inner processes only as a result of a prolonged development . Their transfer inward is linked with changes in the laws governing their ac tivity ; they are incorporated into a new system with its own laws . The internalization of cultural forms of behavior involves the re construction of psychological activity on the basis of sign operations . Psychological processes as they appear in animals actually cease to exist ; they are incorporated into this system of behavior and are culturally reconstituted and developed to form a new psychological entity . The use of external signs is also radically reconstructed . The developmental changes in sign operations are akin to those that occur in language . Aspects of external or communicative speech as well as egocentric speech turn " inward " to become the basis of inner speech . The internalization of socially rooted and historically developed ac tivities is the distinguishing feature of human psychology , the basis of the qualitative leap from animal to human psychology . As yet , the barest outline of this process is known . 5 Problems of Method In general , any fundamentally new approach to a scientific problem inevitably leads to new methods of investigation and analysis . The in vention of new methods that are adequate to the new ways in which problems are posed requires far more than a simple modification of previously accepted methods . Contemporary psychological experimen tation is no exception in this respect ; its methods have always reflected the ways in which fundamental psychological problems were viewed and solved . Therefore , our criticism of current views concerning the essential nature and development of psychological processes must inevitably result in a reexamination of methods of research . Despite great diversity in procedural details , virtually all psycho logical experiments rely on what We shall term a stimulus - response framework . By this we mean that no matter what psychological process is under discussion , the psychologist seeks to confront the subject with some kind of stimulus situation designed to influence him in a particular way , and then the psychologist examines and analyzes the response ( s ) elicited by that stimulating situation . After all , the very essence of experimentation is to evoke the phenomenon under study in an artificial ( and thereby controllable ) way and to study the variations in response that occur in conjunction with various changes in the stimulus . On the surface it may appear that various schools of psychology could not possibly agree on this methodology . The objective psychology of Watson , Bekhterev , and others , for example , was constructed in opposition to the subjective theories of Wundt and the Wiirzburg school . But closer examination of the differences between schools of psychology reveals that those differences arise out of the theoretical interpretation 58 Problems of Method 59 psychologists want to assign to the consequences of various stimulating environments and not out of variations in the general methodological approach within which observations are made . Reliance on a stimulus - response framework is an obvious feature of those schools of psychology whose theories as well as experiments are based on stimulus - response interpretations of behavior . Pavlovian theory , for example , has utilized the notion of cortical excitation incited by various stimuli to explain how connections are formed in the brain that enable the organism to learn to respond to hitherto neutral stimuli . It may be less obvious that exactly the same framework applies to intro spective psychology as well , since the framework and the theory do not seem to coincide . However , taking Wundt as an example , we find that the stimulus - response framework provided the context within which the experimenter - theorist could obtain descriptions of the processes presumed to have been elicited by the stimulus . The adoption of a stimulus - response framework by introspective psychology in the 1880s was a revolutionary step forward for psychology because it brought psychology closer to the method and spirit of the natural sciences and prepared the way for the objective psychological approaches that followed . But to claim that both introspective and ob jective psychology share a common methodological framework does not in any way imply that there are no important differences between them . I am emphasizing their common methodological framework be cause its recognition helps us to appreciate the fact that introspective psychology was rooted in the firm soil of natural sciences and that psychological processes have long been understood within a reactive context . It is also important to realize that the experimental method was first formulated by introspective psychologists in that area of psychophysics and psychophysiology that dealt with the simplest psychological phe nomena , phenomena that could plausibly be interpreted as directly and uniquely linked to external agents . Wundt , for example , saw the very essence of psychological method as the systematic alteration of the stimuli that generate a change in the psychological process linked to them . He sought the maximally objective way to record the external manifestations of these internal processes , which is what he believed the subject ' s introspective reports to be . At the same time , it is important to keep in mind that for Wundt the stimulus and response functioned only to set up the framework within which the important events , psychological processes , could be studied in a reliable and controlled way . Introspective reports of these Mind in Society 60 processes remained the paramount evidence concerning their nature an interpretation not shared by later investigators . Our description of the basic framework of psychological experi mentation as practiced by Wundt implies limitations on its application : such experimentation was considered adequate only to the study of elementary processes of a psychophysiological character . The higher psychological functions did not allow study in this form and thus remained a closed book as far as experimental psychology was con cerned . If we recall the kinds of experimentation on the cognitive de velopment of children that characterized the research reviewed in earlier chapters of this book , we can easily understand why previous investi gators concentrated on elementary psychological functions ; this limita tion is a built - in feature of the experimental method as it was generally accepted in psychology . Wundt understood and accepted this fact , which is why he eschewed experimental studies of higher psychological functions . From the foregoing it should be clear that a stimulus - response framework for constructing experimental observations cannot serve as the basis for the adequate study of the higher , specifically human forms of behavior . At best it can only help us to record the existence of the lower , subordinated ' forms , which do not capture the essence of the higher forms . Using current methods , we can only determine quanti tative variation in the complexity of stimuli and in the responses of different animals and humans at different stages of development . It is my belief , based upon a dialectical materialist approach to the analysis of human history , that human behavior differs qualitatively from animal behavior to the same extent that the adaptability and his torical development of humans differ from the adaptability and de velopment of animals . The psychological development of humans is part of the general historical development of our species and must be so understood . Acceptance of this proposition means that we must find a new methodology for psychological experimentation . The keystone of OUf method , which I will try to describe analyti cally in the following sections , follows directly from the contrast Engels drew between naturalistic and dialectical approaches to the under standing of human history . Naturalism in historical analysis , according to Engels , manifests itself in the assumption that only nature affects human beings and only natural conditions determine historical develop ment . The dialectical approach , while admitting the influence of nature on man , asserts that man , in turn , affects nature and creates through his changes in nature new natural conditions for his existence . ' This posi - Problems of Method 61 tion is the keystone of our approach to the study and interpretation of man ' s higher psychological functions and serves as the basis for the new methods of experimentation and analysis that we advocate . All stimulus - response methods share the inadequacy that Engels ascribes to naturalistic approaches to history . Both see the relation between human behavior and nature as unidirectionally reactive . My collaborators and I , however , believe that human behavior comes to have that " transforming reaction on nature " which Engels attributed to tools . We must , then , seek methods adequate to our conception . In conjunction with new methods , we also need a new analytic framework . I have emphasized that a basic goal of our research is to provide an analysis of the higher forms of behavior , but the situation in contempo rary psychology is such that the problem of analysis itself must be dis cussed if our approach is to be generalized beyond the specific examples presented . Three principles form the basis of our approach to the analysis of higher psychological functions . Analyzing process , not objects . The first principle leads us to dis tinguish between the analysis of an object and of a process . As Ko£fka put it , psychological analysis has almost always treated the processes it analyzes as stable , fixed objects . The task of analysis consisted in breaking these forms down into their components . Psychological anal ysis of objects should be contrasted with the analysis of processes , which requires a dynamic display of the main points making up the processes ' history . Consequently , developmental psychology , not experimental psychology , provides the new approach to analysis that we need . Like Werner , we are advocating the developmental approach as an essential addition to experimental psychology . f Any psychological process , whether the development of thought or voluntary behavior , is a process undergoing changes right before one ' s eyes . The development in ques tion can be limited to only a few seconds , or even fractions of seconds ( as is the case in normal perception ) . It can also ( as in the case of complex mental processes ) last many days and even weeks . Under certain condi tions it becomes possible to trace this development . Werner ' s work furnishes one example of how a developmental viewpoint may be applied to experimental research . Using such an approach , one can , under laboratory conditions , provoke development . Our method may be called experimental - developmental in the sense that it artificially provokes or creates a process of psychological development . This approach is equally appropriate to the basic aim of Mind in Society 62 dynamic analysis . If we replace object analysis by process analysis , then the basic task of research obviously becomes a reconstruction of each stage in the development of th ~ process : the process must be turned back to its initial stages . Explanation versus description . In associationistic and introspective psychology , analysis is essentially description and not explanation as we understand it . Mere description does not reveal the actual causal dynamic relations that underlie phenomena . K . Lewin contrasts phenomenological analysis , which is based on external features ( phenotypes ) , with what he calls genotypic analysis , wherein a phenomenon is explained on the basis of its origin rather than its outer appearance . " The difference between these two points of view can be elucidated by any biological example . A whale , from the point of view of its outer appearance , stands closer to the fish family than to the mammal , but in its biological nature it is closer to a cow or a deer than to a pike or a shark . Following Lewin , we can apply this distinction between the phenotypic ( descriptive ) and genotypic ( explanatory ) viewpoints to psychology . By a developmental study of a problem , I mean the disclosure of its genesis , its causal dynamic basis . By pheno typic I mean the analysis that begins directly with an object ' s current features and manifestations . It is possible to furnish many examples from psychology where serious errors have been committed because these viewpoints have been confused . In our study of the development of speech , we have emphasized the importance of the distinction between phenotypic and genotypic similarities . In their external , descriptive aspects , the first manifestation of speech in the one - and - a - half to two - year - old child are similar to adult speech . On the basis of this similarity , such serious researchers as Stern come to the conclusion that in essence the eighteen - month - old child is already conscious of the relation between sign and meaning . " In other words , he classes together phenomena that have absolutely nothing in common from the developmental point of view . On the other hand , ego centric speech - which in its outer manifestations differs from internal speech in essential ways - must be classed together with internal speech from the developmental point of view . Our research on young children ' s speech brings us to the basic principle formulated by Lewin : two phenotypically identical or similar processes may be radically different from each other in their causal dynamic aspects and vice versa ; two processes that are very close in their causal - dynamic nature may be very different phenotypically . Problems of Method 63 I have said that the phenotypic approach categorizes processes ac cording to their external similarities . Marx commented on the pheno typic appr ~ ach in a most general form when he stated that " if the essence of objects coincided with the form of their outer manifestations , then every science would be superfluous - c - an extremely reasonable observation . " If every object was phenotypically and genotypically ' equivalent ( that is , if the true principles of its construction and opera tion were expressed by its outer manifestation ) , then everyday experi ence would fully suffice to replace scientific analysis . Everything we saw would be the subject of our scientific knowledge . In reality , psychology teaches us at every step that though two types of activity can have the same external manifestation , whether in origin or essence , their nature may differ most profoundly . In such cases special means of scientific analysis are necessary in order to lay bare internal differences that are hidden by external similarities . It is the task of analysis to reveal these relations . In that sense , real scientific analysis differs radically from subjective , introspective analysis , which by its very nature cannot hope to go beyond pure description . The kind of objective analysis we advocate seeks to lay bare the essence rather than the perceived characteristics of psychological phenomena . For example , we are not interested in a description of the immedi ate experience elicited by a flashing light as it is revealed to us by intro spective analysis ; rather we seek to understand the real links between the external stimuli and internal responses that underlie the higher form of behavior named by introspective descriptions . Thus , psychological analysis in our sense rejects nominal descriptions and seeks instead to determine causal - dynamic relations . However , such explanation would also be impossible if we ignored the external manifestations of things . By necessity , objective analysis includes a scientific explanation of both external manifestations and the process under study . Analysis is not limited to a developmental perspective . It does not repudiate the expla nation of current phenotypical idiosyncrasies , but rather subordinates " them to the discovery of their actual origin . The problem of " fossilizedbehavior , " The third principle under lying our analytic approach is based on the fact that in psychology we often meet with processes that have already died away , that is , processes that have gone through a very long stage of historical development and have become fossilized . These fossilized forms of behavior are most easily found in the so - called automated or mechanized psychological processes which , owing to their ancient origins , are now being repeated Mind in Society 64 for the millionth time and have become mechanized . They have lost their original appearance , and their outer appearance tells us nothing whatsoever about their internal nature . Their automatic character ' cre ates great difficulties for psychological analysis . The processes that have traditionally been referred to as voluntary and involuntary attention provide an elementary example that demon strates how essentially different processes acquire outer similarity as a result of this automation . Developmentally speaking , these two pro cesses differ very profoundly . But in experimental psychology it is con sidered a fact , as formulated by Titchener , that voluntary attention , once established , functions just like involuntary attention . " In Titchener ' s terms , " secondary " attention constantly changes into " primary " attention . Having described and contrasted the two types of attention , Titchener then says , " There exists , however , a third stage in the development of attention , and it consists in nothing less than a return to the first stage . " The last and highest stage in the development of any process may demonstrate a purely phenotypic similarity with the first or primary stages , and if we take a phenotypic approach , it is impossible to dis tinguish between higher and lower forms of this process . The only way to study this third and highest stage in the development of attention is to understand it in all its idiosyncrasies and differences . In short , we need to understand its origin . It follows , then , that we need to concentrate not on the product of development but on the very process by which higher forms are established . To do so the researcher is often forced to alter the automatic , mechanized , fossilized character of the higher form of behavior and to turn it back to its source through the experiment . This is the aim of dynamic analysis . Inactive , rudimentary functions stand not as the living remnants of biological evolution but as those of the historical development of be havior . Consequently , the study of rudimentary functions must be the point of departure for evolving a historical perspective in psychological experiments . It is here that the past and the present are fused and the present is seen in the light of history . Here r ~ e find ourselves simultane ously on two planes : that which is and that which was . The fossilized form is the end of the thread that ties the present to the past , the higher stages of development to the primary ones . The concept of a historically based psychology is misunderstood by most researchers who study child development . For them , to study something historically means , by definition , to study some past event . Hence , they naively imagine an insurmountable barrier between his toric study and study of present - day behavioral forms . To study some - Problems of Method 65 thing historically means to study it in the process of change ; that is the dialectical method ' s basic demand . To encompass in research the process of a given thing ' s development in all its phases and changes - from birth to death - s - fundamentally means to discover its nature , its essence , for " it is only in movement that a body shows what it is . " Thus , the historical study of behavior is not an auxiliary aspect of theoretical study , but rather forms its very base . As P . P . Blonsky has stated , " Behavior can be understood only as the history of behavior . " ? The search for method becomes one of the most important prob lems of the entire enterprise of understanding the uniquely human forms of psychological activity . In this case , the method is simultaneously pre requisite and product , the tool and the result of the study . In summary , then , the aim of psychological analysis and its essen tial factors are as follows : ( 1 ) process analysis as opposed to object anal ysis ; ( 2 ) analysis that reveals real , causal or dynamic relations as op posed to enumeration of a process ' s outer features , that is , explanatory , not descriptive , analysis ; and ( 3 ) developmental analysis that returns to the source and reconstructs all the points in the development of a given structure . The result of development will be neither a purely psycho logical structure such as descriptive psychology considers the result to be , nor a simple sum of elementary processes such as associationistic psychology saw it , but a qualitatively new form that appears in the process of development . THE PSYCHOLOGY OF COMPLEX ·CHOICE RESPONSES In order to illustrate the contrasting approaches to psychological analysis , I will discuss in some detail two different analyses of one task . In the task I have chosen , the subject is presented one or more stimuli ( visually or auditorily as a rule ) . TIle required response differs according to the number of stimuli and the interests of the investigator : some approaches seek to break the reaction down into a series of elementary processes whose durations can be added and subtracted to establish the laws of their combination ; others seek to describe the emotional reaction of the subject as he responds to the stimulus . In either case , the subjects ' introspective analyses of their responses are used as basic data . In these experiments the inadequacies of prior formulations provide useful illus trations of our basic analytic principles . " It is also characteristic of these analyses that complex and simple responses are distinguished primarily by the quantitative complexity of the stimuli : a simple reaction is said to occur when a single stimulus is Mind in Society 66 presented , and the complexity of the response is said to increase with an increasing number of stimuli . An essential presumption in this line of thinking is that the complexity of the task is identical to the com plexity of the subject ' s internal response . This identity is clearly expressed in the algebraic formulas com monly used in the analysis of responses to such tasks . If we present a single stimulus , we can write an equation in which the complex reac tion is equivalent to a simple reaction ( sensory recognition ) : R t = R , where R , is the response time for the total , complex reaction and R s is the response time for a single recognition reaction . If we present two or more stimuli , from which the subject must select one , this equation be comes : R , = R , + D , where P is the time taken to discriminate between the target stimulus and the remainder . Using these two equations , we could establish the time required both for a simple reaction and for the discriminative reaction . If we complicate the task by requiring the sub ject to choose a different response for each stimulus ( for example , press the left - hand key for stimulus A and the right - hand key for stimulus B ) , we obtain the classical choice reaction formula : R t = R , + D + C , where C is the time required to choose the correct movement , for ex ample , to press the key corresponding to the stimulus presented . A verbal description of the theory underlying this set of formulas would be the following : the discrimination response is a simple reaction plus discrimination ; the choice reaction is a simple reaction plus dis crimination plus choice . The higher , more complex response is Seen as the arithmetic sum of its elementary components . Proponents of this analytic approach apply it quite widely . Thus , for example , Cattell believes that by subtracting the time needed to comprehend and name a word from the time needed to comprehend , translate a word into another language , and name it , we can obtain a pure measure of the translation process . " In short , even higher processes such as speech comprehension and production can be analyzed by these methods . A more mechanical notion of the complex , higher forms of behavior would be hard to imagine . However , this analytic approach has been shown to lead to a variety of difficulties . The most basic , empirical observation that contradicts this theory comes from Titchener , who pointed out that the time to execute a carefully prepared choice reaction may be equal to the reaction time for a simple , sensory response . By the logic of the analysis summa rized in the equations given above , this state of affairs is impossible . In our view , the basic premise underlying this entire line of analysis is incorrect . It is not true that a complex reaction consists of a chain of , Problem« of Method 67 separate processes which may be arbitrarily added and subtracted . Any such reaction reflects processes that depend upon the entire process of learning at every level of practice . This mechanical analysis substi tutes relations existing between stimuli for the real relations under lying the process of choosing . This kind of substitution reflects a general intellectualism in psychology which seeks to understand psychological processes in the manipulations that make up the experiment itself ; , experimental procedures become surrogates for psychological processes . While various scholars have demonstrated the inadequacy of psy chological analysis based upon a mechanical decomposition of responses into their elements , these critics face the problem that their introspective analyses of complex reactions must be restricted to description : the description of external responses is replaced by the description of inter nal feelings . In either case , we are restricted to phenotypical psychologi cal analysis . Introspective analysis in which highly trained observers are in structed to note every aspect of their own conscious experience cannot carry us very far . A curious result of this work , as Ach put it in discussing choice reaction studies , has been the discovery that there are no con scious feelings of choice in the choice reaction . to Titchener emphasized that one must keep in mind the fact that the names given to a complex or simple reaction ( for example , " differentiation " or " choice " ) refer to the external conditions of the task . We do not differentiate in the diHer entiation reaction and we do not choose in the choice reaction . This kind of analysis broke the identity between experimental pro cedures and psychological processes . Process names like " choosing " and " differentiating " were treated as leftovers from a previous era of psy chology when experimentation was still unknown : introspective ob servers were trained to make a clear distinction between process names and their conscious experience in order to circumvent this problem . These introspective studies resulted in the conclusion that a situation which seems to require choice processes furnishes no grounds for speak ing of a psychological choice response ; talk of such responses was replaced by a description of the subjects ' feelings during the experiment . But no one could provide evidence that these feelings were an integral part of the particular response process . It seems more likely that they are only one of its components , and require explanation themselves ; we are led to conclude that introspection is often unable to provide an accurate description , let alone a correct explanation ) for even the sub jective aspect of the response . For the same reasons , the frequent discrepancies among the introspective descriptions of various observers Mind in Society 68 which plague this area of research might be expected . It should be clear that introspective analysis cannot provide a real causal or dynamic explanation of a process ; for that to occur , we must give up reliance on phenotypic appearances and move to a developmental viewpoint . Research on complex reactions also illustrates psychology ' s reliance on the analysis of processes only after they have become fossilized . This point was noted by Titchener , who remarked that researchers have concentrated on the reaction time of the responses they study , not on the learning processes or the content of the reaction itself . This same conclusion is seen clearly in the standard practice of discarding the data from early sessions when the response is being established . Uni formity was sought , so that it was never possible to grasp the process in flight ; instead , researchers routinely discarded the critical time when a reaction appears and when its functional links are established and adjusted . Such practices lead us to characterize the responses as " fossil ized . " They reflect the fact that these psychologists were not interested in complex reactions as a process of development . This approach is also a major cause of the confusions which arose concerning complex and simple reactions that have surface similarities . It might be said that complex reactions have been studied postmortem . Another perspective on this issue can be gained from comparing complex reactions with reflexes , which are psychologically different in many respects . One point of comparison will suffice for purposes of illustration . It is well known that the latent period for a complex re action is longer than the latent period for a reflex . But Wundt long ago established that the latent period of a complex reaction decreases with ' practice . As a result , the latency of the complex reaction and the simple reflex become equivalent . The most important differences between a complex reaction and a reflex are usually most apparent when " the reaction is in its early stages ; as practice proceeds , the differences become more and more obscured . Therefore , the differences between these two forms of behavior should be sought in the analysis of their development . But instead of increasing the discernible differences be tween them , investigations of well - practiced choice reactions and reflexes hide these differences . The preparatory trials demanded by standard experimental methods often last for several long sessions . When these data are then discarded or ignored , the researcher is left with an automa tized reaction that has lost its developmental difference from a reflex and has acquired a surface , phenotypical similarity to it . These factors have led to our assertion that previous researchers have studied reactions in psychological experiments only after they have become fossilized . Problems of Method 69 This discussion of traditional analyses of complex reaction defines , albeit negatively , the basic tasks confronting us . In order to obtain the kind of causal - dynamic analysis we have been advocating , we will have to shift the focus of our research . A CAUSAL - DYNAMIC STUDY OF CHOICE REACTIONS Obviously , the early sessions during which a reaction is formed are of crucial concern because only data from this period will reveal the reaction ' s true origin and its links to other processes . Through an objective study of the entire history of the reaction , we can obtain an integrated explanation of both its internal and surface manifestations . Thus , we will want to study the reaction as it appears initially , as it takes shape , and after it is firmly formed , constantly keeping in mind the dynamic flow of the entire process of its development . From my previous discussion , another part of the task is clear : the complex reaction must be studied as a living process , not as an object . We must transform the reaction back to its source if we encounter it in automatized fonn . When we examine the experimental procedures used in complex reactions , we find that all are restricted to meaningless connections between stimuli and responses . The subject is presented several stimuli to which he must respond in different ways : neither the relations between the stimuli and the required responses nor the sequence in which the stimuli are presented have any significance from the subject ' s point of view . When a motor response , such as a key press , is required , subjects may make the movement in any way they like . These conven tions render the relations among the elements of the problem mechanical in principle and place the procedures on a plane with the research on memory that uses nonsense stimuli . This analogy between choice reaction and memory studies can be extended by considering the similarity of the role of repetition in the two tasks . Although no one has dwelt on a study of the practice trials in choice reaction studies , it is safe to conclude that if the reaction is formed through repeated training ( or training plus written or oral instruction ) , it has been learned by rote , just as learning the connection between two nonsense syllables is a rate process . If simple reactions were involved and the subject was given extensive explanation ahead of time so that the relation between stimulus and response were meaningful ( for example , push key number 1 when I say " one , ' push key number 2 when I say " two " ) , we would be dealing with already existing links . In Mind in Society 70 neither case could we study the process of organizing the reaction , during which its underlying links would be discoverable . To make all of this clear , let us trace the stages through which the choice reaction moves , first in experiments with adults and then with children . If we set up a relatively simple choice reaction , say , pressing a button with the left hand when a red stimulus is shown and pressing with the right hand when a green stimulus is shown , adults quickly acquire a stable response . Suppose , however , we increase the number . of stimuli and responses to five or six and diversify the responses so that the subject has to respond not only with both hands , but sometimes pressing a button and sometimes simply by moving a finger . With this larger number of stimulus - response pairings , the task is considerably more difficult . Suppose further that instead of a lengthy pretraining period in which the subject is allowed to learn the stimulus - response relations , we give only minimal instructions . Faced with this situation , adults often refused even to attempt to deal with the problem , objecting that they could not remember what to do . Even after the session started , they , kept repeating the instructions to themselves , asked about aspects of the task they had forgotten , and generally sought to master the entire system of relations as a whole before they settled down to the task as it is usually conceived . However , if we placed additional stimuli on the response buttons and keys in a manner analogous to the procedures in previously de scribed memory studies , the adults immediately used these auxiliary means to remember the necessary stimulus - response relations . Among young children , a different picture emerged . We first pre sented the problem as we did with adults , by asking the child to make a number of different responses to different stimuli . Unlike the adults , children six to eight years of age often started right into the task after listening to the instructions and attempted to follow them without the slightest hesitation . As soon as the experiment began , most children found themselves in great difficulty . If a child recalled one or two of the required relations and responded correctly to those stimuli , he would naively ask about the remaining stimuli , treating each of them in isola tion from each other . This behavior contrasted with that of the adults who generally failed to deal effectively with the individual stimuli until all the necessary relations were mastered . We view this behavior on the part of the children as evidence that they are in the stage of responding to the task in a natural or primitive manner because they rely on unmedi ated memory for the task elements . The fact that children would unhesi - Pl ' oblems of Method 71 tatingly accept the challenge of establishing a complex choice response to as many as ten stimuli suggests that they do . not yet know their own capacities and limitations . They operate with complex tasks in the same way they operate with simple ones . The child ' s behavior also differs from adult behavior when we introduce auxiliary stimuli , although we can discern the beginnings of the restructuring that characterize the adult . First , we introduce auxiliary stimuli that bear a clear relation to the primary stimuli with which we began . For example , if the primary stimulus was a horse , in response to which the child was supposed to press a key with his left index finger , we pasted a picture of a sleigh on that key . On the key corresponding to a loaf of bread we pasted a picture of a knife . In this case , the child understands that sleigh goes with horse , the knife with - bread , and so on . Choice reactions are smoothly estab lished from the outset . Furthermore , it does not matter how many stimuli and responses are involved ; the qualitative features of responding re main the same . The child quickly works out a rule for the problem ' s solution and makes his choice on the basis of this rule . It would be incorrect , however , to assume that the child has mas tered a mediated system of behavior in its full , adult form . We need only to change the relations among the primary and auxiliary stimuli to discover the limits of the child ' s response system . If we pair the stimuli in a different way ( say , horse with knife , bread with sleigh ) the child will no longer use the auxiliary stimuli in a proper way . The child recalls only that horse helped to find sleigh in some way . He reveals by his responses that he had been using the conventional association of horse and sleigh to guide the choice , but had not mastered the internal logic of using one stimulus to mediate the response to another . If we continue our experiment long enough , we will begin to see changes in the way the child responds . In the first stage of responding to arbitrarily related stimuli , the child has insufficient experience with the task to organize his behavior effectively . He uses experience naively , But in the course of the experiment , he gains experience necessary for restructuring his behavior . Just as naive physical knowledge is acquired as the child operates with objects , knowledge of psychological operations is acquired as the child strives to carry out the choice reaction task . As he attempts to recall which stimuli are linked to which responses , the child begins to learn what remembering in this situation consists of and begins to use one or another of the auxiliary stimuli effectively . The child begins to realize that certain relations among the stimuli and auxiliary pictures produce correct choice responses , while others do not . He soon Mind in Society 72 begins to object to the arrangement of pictures , asking that the pictures on the keys be arranged to fit the primary stimuli that are associated with the key . When told to press the bread key in response to the horse picture , the child answers 4 : ' No , I want the sleigh key . " This shows that the child is accumulating experience which is changing the structure of his own memorizing . Having naively comprehended what the memorizing operations require , the child moves to the following stage . If presented with primary and auxiliary stimuli in an arrangement that seems haphazard , the child will ask to put them in a special order , thus personally establishing a specific relation between them . At this point the child is showing that he knows that certain signs will help to achieve certain operations . In short , he is beginning to memorize through the use of signs . Once this happens , the child no longer experiences difficulties in creating relations and using them . Given some pairing of primary and auxiliary stimuli , the child is no longer restricted to using already avail able relations ( such as horse - sleigh ) but can create relations of his own . This may be called the stage of external sign use . It is characterized " by the independent formation of new relations in the child ' s internal opera tions using externally presented signs . Now the child is organizing external stimuli to carry out its responses . This fundamental stage is then followed by the stage at which the child begins to organize stimuli of an internal nature . These changes are manifested in the course of the choice reaction experiment . After considerable practice in the choice experiment , the reaction time begins to grow shorter and shorter . If the reaction time to a particular stimulus had been 500 milliseconds or more , it reduces to a mere 200 milliseconds . The longer reaction time reflected the fact that the child was using external means to carry out the operations of remembering which key to push . Gradually , the child casts off the external stimuli , no longer paying attention to them . The response to the external auxiliary stimuli is replaced by a response to internally produced stimuli . In its most developed form , this internal operation co . nsists of the child grasping the very structure of the process , learning to understand the laws according to which external signs must be used . When this stage is reached , the child will say , " I don ' t need pictures anymore . I ' ll do it myself . " CHARACTERISTICS OF THE NEW MEmOD I have attempted to demonstrate that the course of child develop ment is characterized by a radical alteration in the very structure of Problems of Method 13 behavior ; at each new stage the child changes not only her response but carries out that response in new ways , drawing on new " instruments ' of behavior and replacing one psychological function by another . Psy chological operations that were achieved through direct forms of adapta tion at early stages are later accomplished through indirect means . The growing complexity of children ' s behavior is reflected in the changed . means they use to fulfill new tasks and the corresponding reconstruction of their psychological processes . . Our concept of development implies a rejection of the frequently held view that cognitive development results from the gradual accumu lation of separate changes . We believe that child development is a complex dialectical process characterized by periodicity , unevenness in ' the development of different functions , metamorphosis or qualitative transfo ; rmation of one form into another , intertwining of external and internal factors , and adaptive processes which overcome impediments that the child encounters . Steeped in the notion of evolutionary change , most workers in child psychology ignore those turning points , those spasmodic and revolutionary changes that are so frequent in the history of child development . To the naive mind , revolution and evolution seem incompatible and historic development continues only so long as it follows a straight line . Where upheavals occur , where the historical fabric is ruptured , the naive mind sees only catastrophe , gaps , and dis continuity . History seems to stop dead , until it once again takes the direct , linear path of development . Scientific thought , on the contrary , sees revolution and evolution as two forms of development that are mutually related and mutually presuppose each other . Leaps in the child ' s development are seen by the scientific mind as no more than a moment in the general line of development . As I have repeatedly emphasized , an essential mechanism of the reconstructive processes that take place during a child ' s development is the creation and use of a number of artificial stimuli . These play an auxiliary role that permits human beings to master their own behavior , at first by external means and later by more complex inner operations . Our approach to the study of cognitive functioning does not require the experimenter to furnish subjects with ready - made , external or arti ficial means in order that they may successfully complete the given task . The experiment is equally valid if , instead of giving children artificial means , the experimenter waits until they spontaneously apply some new auxiliary method or symbol that they then incorporate into their operations . The specific area to which we apply this approach is not important . Mind in Society 74 We might study the development of memorizing in children by making available to them new means for solving the given task and then observ ing the degree and character of their problem - solving efforts . We might use this method to study how children organize their active attention with the aid of external means . We might trace the develop ment of arithmetic skills in young children by making them manipulate objects and apply methods either suggested to them or " invented " by them . What is crucial is that in all these cases we must adhere to one principle . We study not only the final effect of the operation , but its specific psychological structure . In all these cases , the psychological structure of the development appears with much greater richness and variety than in the classic method of the simple stimulus - response ex periment . Although stimulus - response methodology makes it extremely easy to ascertain subjects ' responses , it proves , useless when our objective is to discover the means and methods that subjects use to organize their own behavior . Our approach to the study of these processes is to use what we call the functional method of double stimulation . The task facing the child in the experimental context is , as a rule , beyond his present capabilities and cannot be solved by existing skills . In such cases a neutral object is placed near the child , and frequently we are able to observe how the neutral stimulus is drawn into the situation and takes on the function of a sign . Thus , the child actively incorporates these neutral objects into the task of problem solving . We might say that when difficulties arise , neutral stimuli take on the function of a sign and from that point on the operation ' s structure assumes an essentially different character . By using this approach , we do not limit ourselves to the usual method of offering the subject simple stimuli to which we expect a direct response . Rather , we simultaneously offer a second series of stimuli that have a special function . In this way , we are able to study the process of accomplishing a task by the aid of specific auxiliary means ; thus we are - also able to discover the inner structure and development of the higher psychological processes . The method of double stimulation elicits manifestations of the crucial processes in the behavior of people of all ages . Tying a knot as a reminder , in both children and adults , is but one example of a pervasive regulatory principle of human behavior , that of signification , wherein people create temporary links and give significance to previously neutral stimuli in the context of their problem - solving efforts . We regard our method as important because it helps to objectify inner psychological processes ; stimulus - response methods are objective , Problems of Method 15 but they are limited to the study of external responses that are usually in the subject ' s repertoire to begin with . We believe that our approach to objectifying inner psychological processes is much more adequate , where the goals of psychological research are concerned , than the method of studying preexisting , objective responses . ' ! Only the objecti fication of the inner process guarantees access to specific forms of higher behavior as opposed to subordinate forms . Part Two / Mind in Society Educational Implications 6 Interaction between Learning and Development The problems encountered in the psychological analysis of teaching cannot be correctly resolved or even formulated without addressing the relation between learning and development in school - age children . Yet it is the most unclear of all the basic issues on which the application of child development theories to educational processes depends . Needless to say , the lack of theoretical clarity does not mean that the issue is removed altogether from current research efforts into learning ; not one study can avoid this central theoretical issue . But the relation between learning and development remains methodologically unclear because concrete research studies have embodied theoretically vague , critically unevaluated , and sometimes internally contradictory postulates , prem ises , and peculiar solutions to the problem of this fundamental relation ship ; and these , of course , result in a variety of errors . Essentially , all current conceptions of the relation between develop ment and learning in children can be reduced to three major theoretical positions . The first centers on the assumption that processes of child develop ment are independent of learning . Learning is considered a purely external process that is not actively involved in development . It merely utilizes the achievements of development rather than providing an impetus for modifying its course . In experimental investigations of the development of thinking in school children , it has been assumed that processes such as deduction and understanding , evolution of notions about the world , interpretation of physical causality , and mastery of logical forms of thought and ab stract logic all occur by themselves , without any . influence from school 79 Mind in Society 80 learning . An example of such a theory is Piaget ' s extremely complex and interesting theoretical principles , which also shape the experimental methodology he employs . The questions Piaget uses in the course of his " clinical conversations " with children clearly illustrate his approach . When a five - year - old is asked " why doesn ' t the sun fall ? " it is assumed that the child has neither a ready answer for such a question nor the general capabilities for generating one . The point of asking questions that are so far beyond the reach of the child ' s intellectual skills is to eliminate the influence of previous experience and knowledge . The experimenter seeks to obtain the tendencies of children ' s thinking in " pure " form , entirely independent of learning . ' Similarly , the classics of psychological literature , such as the works by Binet and others , assume that development is always a prerequisite for learning and that if a child ' s mental functions ( intellectual operations ) have not matured to the extent that he is capable of learning a particular subject , then no instruction will prove useful . They especially feared premature instruction , the teaching of a subject before the child was ready for it . All effort was concentrated on finding the lower threshold of learning ability , the age at which a particular kind of learning first becomes possible . Because this approach is based on the premise that learning trails behind development , that development always outruns learning , it precludes the notion that learning may playa role in the course of the development or maturation of those functions activated in the course of learning . Development or maturation is viewed as a precondition of learning but never the result of it . To summarize this position : Learning forms a superstructure over development , leaving the latter essentially unaltered . The second major theoretical position is that learning is develop ment . This identity is the essence of a group of theories that are quite diverse in origin . One such theory is based on the concept of reflex , an essentially old notion that has been extensively revived recently . Whether reading , writing , or arithmetic is being considered , development is viewed ' as the mastery of conditioned reflexes ; that is , the process of learning is com pletely and inseparably blended with the process of development . This notion was elaborated by James , who reduced the learning process to habit formation and identified the learning process with development . ' " Reflex theories have at least one thing in common with theories such as Piaget ' s : in both , development is conceived of as the elaboration and substitution of innate responses . As James expressed it , " Education , Interaction between Learning and Development 81 in short , cannot be better described than by calling it the organization of acquired habits of conduct and tendencies to behavior . " 2 Develop ment itself is reduced primarily to the accumulation of all possible responses . Any acquired response is considered either a more complex form of or a substitute for the innate response . But despite the similarity between the first and second theoretical positions , there is a major difference in their assumptions about the temporal relationship between learning and developmental processes . Theorists who hold the first view assert that developmental cycles pre cede learning cycles ; maturation precedes learning and instruction must lag behind mental growth . For the second group of theorists , both processes occur simultaneously ; learning and development coincide at all points in the same way that two ' identical geometrical figures coincide when superimposed . The third theoretical position on the relation between learning and development attempts to overcome the extremes of the other two by simply combining them . A clear example of this approach is Koflka ' s theory , in which development is based on two inherently different but related processes , each of which influences the other , " On the one hand is maturation , which depends directly on the development of the nervous system ; on the other hand is learning , which itself is also a developmental process . _ Three aspects of this theory are new . First , as we already noted , is the combination of two seemingly opposite viewpoints , each of which has been encountered separately in the history of science . The very fact that these two viewpoints can be combined into one theory indicates that they are not opposing and mutually exclusive but have something essential in common . Also new is the idea that the two processes that make up development are mutually dependent and interactive . Of course , the nature of the interaction is left virtually unexplored in Koflka ' s work , which is limited solely to very general remarks regarding the relation between these two processes . It is clear that for Koffka the process of maturation prepares and makes possible a specific process of learning . The learning process then stimulates and pushes forward the maturation process . The third and most important new aspect of thiS ' theory is the expanded role it ascribes to learning in child developmentl This emphasis leads us directly to an old pedagogical problem , that of formal discipline and the problem of transfer . Pedagogical movements that have emphasized formal discipline and urged the teaching of classical languages , ancient civilizations , and mathematics have assumed that regardless of the irrelevance of these Mind in Society 82 particular subjects for daily living , they were of the greatest value for the pupil ' s mental development . A variety of studies have called into question the soundness of this idea . It has been shown that learning in one area has very little influence on overall development . For example , reflex theorists Woodworth and Thorndike found that adults who , after special exercises , had achieved considerable success in determining the length of short lines , had made virtually no progress in their ability to determine the length of long lines . These same adults were successfully trained to estimate the size of a given two - dimensional figure , but this training did not make them successful in estimating the size of a series of other two - dimensional figures of various sizes and shapes . According to Thorndike , theoreticians in psychology and education believe that every particular response acquisition directly enhances overall ability in equal measure . ' Teachers believed and acted on the basis of the theory that the mind is a complex of abilities - powers of observation , attention , memory , thinking , and so forth - and that any improvement in any specific ability results in a general improvement in all abilities . According to this theory , if the student increased the atten tion he paid to Latin grammar , he would increase his abilities to focus attention on any task . The words " accuracy , " " quick - wittedness , ' " ability to reason , " " memory , ' " power of observation , " " attention , " " concentra tion , " and so forth are said to denote actual fundamental capabilities that vary in accordance with the material with which they operate ; these basic abilities are substantially modified by studying particular subjects , and they retain these modifications when they tum to other areas . There fore , if someone learns to do any single thing well , he will also be able to do other entirely unrelated things well as a result of some secret connection . It is assumed that mental capabilities function indepen dently of the material with which they operate , and that the development of one ability entails the development of others . Thorndike himself opposed this point of view . Through a variety of studies he showed that particular forms of activity , such as spelling , are dependent on the maste ! y of specific skills and material necessary for the performance of that particular task . The development of one particu lar capability seldom means the development of others . Thorndike argued that specialization of abilities is even greater than superficial observation may indicate . For example , if , out of a hundred individuals we choose ten who display the ability to detect spelling errors or to measure lengths , it is unlikely that these ten will display better abilities regarding , for example , the ' estimation of the weight of objects . In the Interaction between Learning and Development 83 same way , speed and accuracy in adding numbers are entirely unrelated to speed and accuracy in being able to think up antonyms . This research shows that the mind is not a complex network of general capabilities such as observation , attention , memory , judgment , and so forth , but a set of specific capabilities , each of which is , to some extent , independent of the others and is developed independently . Learning is more than the acquisition of the ability to think ; it is the acquisition of many specialized abilities for thinking about a variety of things . Learning does not alter our overall ability to focus attention but rather develops various abilities to focus attention on a variety of things . According to this view , special training affects overall development only when its elements , material , and processes are similar across specific domains , habit governs us . This leads to the conclusion that because each activity depends on the material with which it operates , the development of consciousness is the development of a set of particular , independent capabilities or of a set of particular habits . Improvement of one function of consciousness or one aspect of its activity can affect the development of another only to the extent that there are elements common to both functions or activities . Developmental theorists such as KoHka and the Gestalt School - who hold to the third theoretical position outlined earlier - oppose Thorn dike ' s point of view . They assert that the influence of learning is never specific . From their study of structural principles , they argue that the learning process can never be reduced simply to the formation of skills but embodies an intellectual order that makes it possible to transfer general principles discovered in solving one task to a variety of other tasks . From this point of view , the child , while learning a particular operation , acquires the ability to create structures of a certain type , regardless of the diverse materials with which she is working and regard less of the particular elements involved . Thus , Koffka does not conceive of learning as limited to a process of habit and skill acquisition . The relationship he posits between learning and development is not that of an identity but of a more complex relationship . According to Thorndike , learning and development coincide at all points , but for Koffka , develop ment is always a larger set than learning . Schematically , the relationship between the two processes could be depicted by two concentric circles , the smaller symbolizing the learning process and the larger the develop mental process evoked by learning . Once a child has learned to perform an operation , he thus assimilates some structural principle whose sphere of application is other than just Mind in Society 84 the operations of the type on whose basis the principle was assimilated . Consequently , in making one step in learning , a child makes two steps in development , that is , learning and development do not coincide . This , concept is the essential aspect of the third group of theories we have discussed . ZONE OF PROXIMAL DEVELOPMENT : A NEW APPROACH Although we reject all three theoretical positions discussed above , analyzing them leads us to a more adequate view of the relation between learning and development . The question to be framed in arriving at a solution to this problem is complex . It consists of two separate issues : first , the general relation between learning and development ; and second , the specific features of this relationship when children reach school age . That children ' s learning begins long before they attend school is the starting point of this discussion . Any learning a child encounters in school always has a previous history . For example , children begin to study arithmetic in school , but long beforehand they have had some experience with quantity - they have had to deal with operations of division , addition , subtraction , and determination of size . Consequently , children have their own preschool arithmetic , which only myopic psychologists could ignore . It goes without saying that learning as it occurs in the preschool years differs markedly from school learning , which is concerned with the assimilation of the fundamentals of scientific knowledge . But even when , in the period of her first questions , a child assimilates the names of objects in her environment , she is learning . Indeed , can it be doubted that children learn speech from adults ; or that , through asking questions and giving answers , children acquire a variety of information ; or that , through imitating adults and through being instructed about how to act , children develop an entire repository of skillsi ' ll . earning and devel opment are interrelated from the child ' s very first day of life . 1 KoHka , attempting to clarify the laws of child learning and their relation to mental development , concentrates his attention on the sim plest learning processes , those that occur in the preschool years . His error is that , while seeing a similarity between preschool andschool learning , he fails to discern the difference - he does not see the specifically new elements that school learning introduces . He and others assume that the difference between preschool and school learning consists of non - Interaction between Learning and Development 85 systematic learning in one case and systematic learning in the other . But " systematicness " is not the only issue ; there is also the fact that school learning introduces something fundamentally new into the child ' s development . In order to elaborate the dimensions of schoolleaming , we will describe a new and exceptionally important concept without which the issue cannot be resolved : the zone of proximal development . A well known and empirically established fact is that learning should be matched in some manner with the child ' s developmental level . For example , it has been established that the teaching of reading , writ ing , and arithmetic should be initiated at a specific age level . Only recently , however , has attention been directed to the fact that we cannot limit ourselves merely to determining developmental levels if we wish to discover the actual relations of the developmental process to learning capabilities . We must determine at least two developmental levels . The first level can be called the actual developmental level , that is , the level of development of a child ' s mental functions that has been established as a result of certain already completed developmental cycles . When we determine a child ' s mental age by using tests , we are almost always dealing with the actual developmental level . In studies of children ' s mental development it is generally assumed that only those things that children can do on their own are indicative of mental abilities . We give children a battery of tests or a variety of tasks of varying degrees of difficulty , and we judge the extent of their mental develop ment on the basis of how they solve them and at what level of difficulty . On the other hand , if we offer leading questions or show how the problem is to be solved and the child then solves it , or if the teacher initiates the solution and the child completes it or solves it in collaboration with other children - in short , if the child barely misses an independent solution of the problem - the solution is not regarded as indicative of his mental development . This " truth " was familiar and reinforced by com mon sense . Over a decade even the profoundest thinkers never ques tioned the assumption ; they never entertained the notion that what children can do with the assistance of others might be in some sense even more indicative of their mental development than what they can do alone . Let us take a simple example . Suppose I investigate two children upon entrance into school , both of whom are ten years old chronologi cally and eight years old in terms of mental development . Can I say that they are the same age mentally ? Of course . What does this mean ? It means that they can independently deal with tasks up to the degree of difficulty that has been standardized for the eight - year - old level . If I Mind in Society 86 stop at this point , people would imagine that the subsequent course of mental development and of school learning for these children will be the same , because it depends on their intellect . Of course , there may be other factors , for example , if one child was sick for half a year while the other was never absent from school ; but generally speaking , the fate of these children should be the same . Now imagine that I do not terminate my study at this point , but only begin it . These children seem I to be capable of handling problems up to an eight - year - old ' s level , but not beyond that . Suppose that I show them various ways of dealing with the problem . Different experimenters might employ different modes of demonstration in different cases : some might run through an entire dem onstration and ask the children to repeat it , others might initiate the solution and ask the child to finish it , or offer leading questions . In short , in some way or another I propose that the children solve the problem with my assistance . Under these circumstances it turns out that the first child can deal with problems up to a twelve - year - old ' s level , the second up to a nine - year - old ' s . Now , are these children mentally the same ? When it was first shown that the capability of children with equal levels of l mental development to learn under a teacher ' s guidance varied to a high degree , it became apparent that those children were not mentally the same age and that the subsequent course of their learning would obviously be different . This difference between twelve and eight , or between nine and eight , is what we call the zone of proximal develop ment . It is the distance between the actual developmental level as de termined by independent problem solving and the level of potential development as determined through problem solving under adult guidance or in collaboration tvith more capable peers . If we naively ask what the actual developmental level is , or , to put it more simply , what more independent problem solving reveals , the most common answer would be that a child ' s actual developmental level defines functions that have already matured , that is , the end products of development . If a child can do such - and - such independently , it means that the functions for such - and - such have matured in her . What , then , is defined by the zone of proximal development , as determined through problems that children cannot solve independently but only with ~ s­ sistance ? The zone of proximal development defines those functions that have not yet matured but are in the process of maturation , functions that will mature tomorrow but are currently in an embryonic state . These I functions could be termed the " buds " or " flowers " of development rather than the " fruits ' of development . The actual developmental level characterizes mental development retrospectively , while the zone of Interaction between Learning and Development 81 proximal development characterizes mental development prospectively . The zone of proximal development furnishes psychologists and educators with a tool through which the internal course of development can be understood . By using this method we can take account of not only the cycles and maturation processes that have already been com pleted but also those processes that are currently in a state of formation , that are just beginning to mature and develop . Thus , the zone of proximal development permits us to delineate the child ' s immediate future and his . dynamic developmental state , allowing not only for what already has been achieved developmentally but also for what is in the course of maturing . The two children in our example displayed the same mental age from the viewpoint of developmental cycles already completed , but the developmental dynamics of the two were entirely different . The state of a child ' s mental development can be determined only by clarify ing its two levels : the actual developmental level and the zone of proximal development . I will discuss one study of preschool children to demonstrate that what is in the zone of proximal development today will be the actual developmental level tomorrow - that is , what a child can do with as sistance today she will be able to do by herself tomorrow . The American researcher Dorothea McCarthy showed that among children between the ages of three and five there are two groups of functions : those the children already possess , and those they can perform under guidance , in groups , and in collaboration with one another but which they have not mastered independently . McCarthy ' s study dem onstrated that this second group of functions is at the actual develop mental level of Hve - to - seven - year - olds . What her subjects could do only under guidance , in collaboration , and in groups at the age of three - to - five years they could do independently when they reached the age of five - to seven years . " Thus , if we were to determine only mental age - that is , only functions that have matured - we would have but a summary of completed development , while ! f we determine the maturing functions , we can predict what will happen to these children between five and seven , provided the same developmental conditions are maintained . The zone of proximal development can become a powerful concept in devel opmental research , one that can markedly enhance the effectiveness and utility of the application of diagnostics of mental development to educational problems . A full understanding of the concept of the zone of proximal development must result in reevaluation of the role of imitation in learn ing . An unshakable tenet of classical psychology is that only the inde - Mind in Society 88 pendent activity of children , not their imitative activity , indicates their level of mental development . This view is expressed in all current testing systems . In evaluating mental development , consideration is given to only those solutions to test problems which the child reaches without the assistance of others , without demonstrations , and without leading questions . Imitation and learning are thought of as purely mechanical processes . But recently psychologists have shown that a person can imitate only that which is within her developmental level . For example , if a child is having difficulty With a problem in arithmetic and the teacher solves it on the blackboard , the child may grasp the solution in an instant . But if the teacher were to solve a problem in higher mathematics , the child would not be able to understand the solution no matter how many times she imitated it . Animal psychologists , and in particular Kohler , have dealt with this question of imitation quite well . " Kohler ' s experiments sought to deter mine whether primates are capable of graphic thought . The principal question was whether primates solved problems independently or whether they merely imitated solutions they had seen performed earlier , for example , watching other animals or humans use sticks and other tools and then imitating them . Kohler ' s special experiments , designed to determine what primates could imitate , reveal that primates can use imitation to solve only those problems that are of the same degree of difficulty as those they can solve alone . However , Kohler failed to take account of an important fact , namely , that primates cannot be taught ( in the human sense of the word ) through imitation , nor can their intellect be developed , because they have no zone of proximal development . A primate can learn a great deal through training by using its mechanical and mental skills , but it cannot be made more intelligent , that is , it cannot be taught to solve a variety of more advanced problems inde pendently . For this reason animals are incapable of learning in the human sense of the term ; human learning presupposes a specific social nature and a process by which children grow into the intellectual life of those around them . \ Children can imitate a variety of actions that go well beyond the limits of their own capabilities . Using imitation , children are capable of doing much more in collective activity or under the guidance of adults . This fact , which seems to be of little significance in itself , is of fundamental importance in that it demands a radical alteration of the entire doctrine concerning the relation between learning and develop ment in children . One direct ' consequence is a change in conclusions that may be drawn from diagnostic tests of development . Interaction between Learning and Development 89 Formerly , it was believed that by using tests , we determine the mental development level with which education should reckon and whose limits it should not exceed . This procedure oriented learning toward yesterday ' s development , toward developmental stages already completed . The error of this view was discovered earlier in practice than in theory . It is demonstrated most clearly in the teaching of mentally retarded children . Studies have established that mentally retarded children are not very capable of abstract thinking . From this the pedagogy of the special school drew the seemingly correct con clusion that all teaching of such children should be based on the use of concrete , look - and - do methods . And yet a considerable amount of experience with this method resulted in profound disillusionment . It turned out that a teaching system based solely on concreteness one that eliminated from teaching everything associated with - abstract thinking - not only failed to help retarded children overcome their innate handicaps but also reinforced their handicaps by accustoming children exclusively to concrete thinking and thus suppressing the rudiments of any abstract thought that such children still have . Pre cisely because retarded children , when left to themselves , will never achieve well - elaborated forms of abstract thought ) the school should make every effort to push them in that direction and to develop in them what is intrinsically lacking in their own development . In the current practices of special schools for retarded children , we can ob serve a beneficial shift away from this concept of concreteness , one that restores look - and - do methods to their proper role . Concreteness is now seen as necessary and unavoidable only as a stepping stone for develop ing abstract thinking - as a means , not as an end in itself . - Similarly , in normal children , learning which is oriented toward de velopmental levels that have already been reached is ineffective from the viewpoint of a child ' s overall development . It does not aim for a new stage of the developmental process but rather lags behind this process . Thus , the notion of a zone of proximal . development enables us to propound a new formula , namely that the only " good learning " is that which is in advance of development . The acquisition of language can provide a paradigm for the entire problem of the relation between learning and development . Language arises initially as a means of communication between the child and the people in his environment , Only subsequently , upon conversion to internal speech , does it come to organize the child ' s thought , that is , become an internal . mental function . Piaget and others have shown that reasoning occurs in a children ' s group as an argument intended Mind in Society 90 to prove one ' s own point of view before it occurs as an internal activity whose distinctive feature is that the child begins to perceive and check the basis of his thoughts . Such observations prompted Piaget to con clude that communication produces the need for checking and confirm ing thoughts , a process that is characteristic of adult thought . " In the same way that internal speech and reflective thought arise from the ' interactions between the child and persons in her environment , these interactions provide the source of development of a child ' s voluntary behavior . Piaget has shown that cooperation provides the basis for the development of a child ' s moral judgment . Earlier research established that a child first becomes able to subordinate her behavior to rules in group play and only later does voluntary self - regulation of behavior arise as an internal function . These individual examples illustrate a general developmental law for the higher mental functions that we feel can be applied in its en tirety to children ' s learning processes . We propose that ~ essential feature of learning is that it creates the zone of proximal development ; that is , learning awakens a variety of internal developmental processes that are able to operate only when the child is interacting with people in his environment and in cooperation with his peers ~ Once these pro cesses are internalized , they become part of the child ' s independent developmental achievement . From this point of view , learning is not development ; however , properly organized learning results in mental development and sets in motion a variety of developmental processes that would be impossible apart from learning . Thus , learning is a necessary and universal aspect of the process of developing culturally organized , specifically human , psychological functions . To summarize , the most essential feature of our hypothesis is the notion that developmental processes do not coincide with learning processes . Rather , the developmental process lags behind the learning process ; this sequence then results in zones of proximal development . Our analysis alters the traditional view < that at the moment a child assimilates the meaning of a word , or masters an operation such as addition or written language , her developmental processes are basically completed . In fact , they have only just begun at that moment . The major consequence of analyzing the educational process in this manner is to show that the initial mastery of , for example , the four arithmetic operations provides the basis for the subsequent development of a variety of highly complex internal processes in children ' s thinking . Our hypothesis establishes the unity but not the identity of learning Interaction between Learning and Development 91 processes and internal developmental processes . It presupposes that the one is converted into the other . Therefore , it becomes an important con cern of psychological research to show how external knowledge and abilities in children become internalized . Any investigation explores some sphere of reality . An aim of the psychological analysis of development is to describe the internal rela tions of the intellectual processes awakened by school learning . In this respect , such analysis will be directed inward and is analogous to the use of x - rays . If successful , it should reveal to the teacher how develop mental processes stimulated by the course of school learning are carried through inside the head of each individual child . The revelation of this internal , subterranean developmental network of school subjects is a task of primary importance for psychological and educational analysis . A second essential feature of our hypothesis is the notion that , although learning is directly related to the course of child development , the two are never accomplished in equal measure or in parallel . De velopment in children never follows school learning the way a shadow follows the object that casts it . In actuality , there are highly complex dynamic relations between developmental and learning processes that cannot be encompassed by an unchanging hypothetical formulation . Each school subject has its own specific relation to the course of child development , a relation that varies as the child goes from one stage to another . This leads us directly to a reexamination of the prob lem of formal discipline , that is , to the significance of each particular subject from the viewpoint of overall mental development . Clearly , the problem cannot be solved by using anyone formula ; extensive and highly diverse concrete research based on the concept of the zone of proximal development is necessary to resolve the issue . 7 The Role of Play in Development To define playas an activity that gives pleasure to the child is inaccurate for two reasons . First , many activities give the child much keener experiences of pleasure than play , for example , sucking a paci fier , even though the child is not being satiated . And second , there are games in which the activity itself is not pleasurable , for example , games , predominantly at the end of preschool and the beginning of school age , that give pleasure only if the child finds the result interesting . Sporting games ( not only athletic sports , but other games that can be won or lost ) are very often accompanied by displeasure when the outcome is unfavorable to the child . But while - pleasure cannot be regarded as the defining characteristic of play , it seems to me that theories which ignore the fact that play fulfills children ' s needs result in a pedantic intellectualization of play . In speak ing of child development in more general terms , many theorists mis takenly disregard the child ' s needs - understood in the broadest sense to include everything that is a motive for action . We often describe a child ' s development as the development of his intellectual functions ; every child stands before us as a theoretician who , characterized by a higher or lower level of intellectual development , moves from one stage to another . But if we ignore the child ' s needs , and the incentives which are effective in getting him to act , we will never be able to under stand his advance from one developmental stage to the next , because every advance is connected with a marked change in motives , inclina tions , and incentives . That which is of the greatest interest to the infant has almost ceased to interest the toddler . The maturing of needs is a dominant issue in this discussion because it is impossible to ignore the 92 The Role of Play in Development 93 fact that the child satisfies certain needs in play . If we do not under stand the special character of these needs , we cannot understand the uniqueness of playas a form of activity . A very young child tends to gratify her desires immediately ; nor mally the interval between a desire and its fulfillment is extremely short . No one has met a child under three years old who wants to do something a few days in the future . However , at the preschool age , a great many unrealizable tendencies and desires emerge . It is my belief that if needs that could not be realized immediately did not develop during the school years , there would be no play , because play seems to be invented at the point when the child begins to experience unrealizable tendencies . Sup pose that a very young ( perhaps two - and - a - half - year - old ) child wants something - for example , to occupy her mother ' s role . She wants it at once . If she cannot have it , she may throw a temper tantrum , but she can usually be sidetracked and pacified so that she forgets her desire . Toward the beginning of preschool age , when desires that cannot be immediately gratified or forgotten make their appearance and the tend ency to immediate fulfillment of desires , characteristic of the preceding stage , is retained , the child ' s behavior changes . To resolve this tension , the preschool child enters an imaginary , illusory world in which the unrealizable desires can be realized , and this world is what we call play . Imagination is a new psychological process for the child ; it is not present in the consciousness of the very young child , is totally absent in animals , and represents a specifically human form of conscious activity . Like all functions of consciousness , it originally arises from action . The old adage that child ' s play is imagination in action must be reversed : we can say that imagination in adolescents and school children is play without action . From this perspective it is clear that the pleasure derived from preschool play is controlled by different motives than simple sucking on a pacifier . This is not to say that play arises as the result of every unsatisfied desire ( as when , for example , the child wants to ride in the cab , but the wish is not immediately gratified , so the child goes into her room and pretends she is riding in a cab ) . It rarely happens in just this way . Nor does the presence of such generalized emotions in play mean that the child herself understands the motives giving rise to the game . In this respect play differs substantially from work and other forms of activity . Thus , in establishing criteria for distinguishing a child ' s play from other forms of activity , we conclude that in play a child creates an imaginary situation . This is not a new idea , in the sense that imaginary Mind in Society 94 situations in play have always been recognized ; but they were previ ously regarded as only one example of play activities . The imaginary situation was not considered the defining characteristic of play in general but was treated as an attribute of specific subcategories of play . I find previous ideas unsatisfactory in three respects . First , if play is understood as symbolic , there is the danger that . it might come to be viewed as an activity akin to algebra ; that is , play , like algebra , might be considered a system of signs that generalize reality , with no char acteristics that I consider specific to play . The child would be seen as an unsuccessful algebraist who cannot yet write the symbols but can depict them in action . I believe that play is not symbolic action in the proper sense of the term , so it becomes essential to show the role of motivation in play . Second , this argument stressing the importance of cognitive processes neglects not only the motivation for , but also the circumstances of , the child ' s activity . And third , previous approaches do not help us to understand the role of play in later development . If all play is really the realization in play fonn of tendencies that cannot be immediately gratified , then elements of imaginary situations will automatically be a part of the emotional tone of play itself . Con sider the child ' s activity during play . What does a child ' s behavior in an imaginary situation mean ? We know that the development of playing games with rules begins in the late preschool period and develops during school age . A number of investigators , although not belonging to the camp of dialectical materialists , have approached this issue along the lines recommended by Marx when he said that " the anatomy of man is the key to the anatomy of the ape . " They have begun their exami nation of early play in the light of later rule - based play and have con cluded from this that play involving an imaginary situation is , in fact , rule - based play . One could go even further and propose that there is no such thing as play without rules . The imaginary situation of any form of play already contains rules of behavior , although it may not be a game with formulated rules laid down in advance . The child imagines himself to be the mother and the doll to be the child , so he must obey the rules of maternal behavior . Sully early noted that , remarkably , young chil dren could make the play situation and reality coincide . ' He described a case where two sisters , aged five and seven , said to each other , " Let ' s play sisters . " They were playing at reality . In certain cases , I have found it easy to elicit such play in children . It is very easy , for example , to have a child play at being a chiljl while the mother is playing the role of mother , that is , playing at what is actually true . The vital difference , The Role of Play in Development 95 as Sully describes it , is that the child in playing tries to be what she thinks a sister should be . In life the child behaves without thinking that she is her sister ' s sister . In the game of sisters playing at " sisters , " however , they are both concerned with displaying their sisterhood ; the fact that two sisters decided to play sisters induces them both to acquire rules of behavior . Only actions that fit these rules are acceptable to the play situation : they dress alike , talk alike , in short , they enact whatever emphasizes their relationship as sisters vis - a - vis adults and strangers . The elder , holding the younger by the hand , may keep telling her about other people : " That is theirs , not ours . " This means : " My sister and I act the same , we are treated the same , but others are treated differently . " In this example the emphasis is on the sameness of everything that is connected with the child ' s concept of a sister ; as a result of playing , the child comes to understand that sisters possess a different relationship to each other than to other people . What passes unnoticed by the child in real life becomes a rule of behavior in play . What would remain if play were structured in such a way that there were no imaginary situation ? The rules would remain . Whenever there is an imaginary situation in play , there are rules - not rules that are formulated in advance and change during the course of the game but ones that stem from an imaginary situation . Therefore , the notion that a child can behave in an imaginary situation without rules is simply inaccurate . If the child is playing the role of a mother , then she has rules of maternal behavior . The role the child fulfills , and her relation to the object ( if the object has changed its meaning ) , will always stem from the rules . At first it seemed that the investigator ' s only task in analyzing play was to disclose the hidden rules in all play , but it has been demonstrated that the so - called pure games with rules are essentially games with imaginary situations . Just as the imaginary situation has to contain rules of behavior , so every game with rules contains an imaginary situation . For example , playing chess creates an imaginary situation . Why ? Be cause the knight , king , queen , and so forth can only move In specified ways ; because covering and taking pieces are purely chess concepts . Although in the chess game there is no direct substitute for real - life relationships , it is a kind of imaginary situation nevertheless . The sim plest game with rules immediately turns into an imaginary situation in the sense that as soon as the game is regulated by certain rules , a number of possibilities for action are ruled out . Just as we were able to show at the beginning that every imaginary situation contains rules in a concealed form , we have also demonstrated Mind in Society 96 the reverse - that every game with rules contains an imaginary situa tion in a concealed form . The development from games with an overt imaginary situation and covert rules to games with overt rules and a covert imaginary situation outlines the evolution of children ' s play . ACTION AND MEANING IN PLAY The influence of play on a child ' s development is enormous . Play in an imaginary situation is essentially impossible for a child under three in that it is a novel form of behavior liberating the child from constraints . To a considerable extent the behavior of a very young child - and to an absolute extent , that of an infant - s - is determined by the conditions in which the activity takes place , as the experiments of Lewin and others have shown . s For example , Lewin ' s demonstration of the great difficulty a small child has in realizing that he must first turn his back to a stone in order to sit on it illustrates the extent to which a very young child is bound in every action by situational constraints . It is hard to imagine a greater contrast to Lewin ' s experiments showing the situa tional constraints on activity than what we observe in play . It is here that the child learns to act in a cognitive , rather than an externally · visual , realm by relying on internal tendencies and motives and not on incentives supplied by external things . A study by Lewin on the moti vating nature of things for a very young child concludes that things dictate to the child what he must do : a door demands to be opened and closed , a staircase to be climbed , a bell to be rung . In short , things have such an inherent motivating force with respect to a very young child ' s actions and so extensively determine the child ' s behavior that Lewin arrived at the notion of creating a psychological topology : he expressed mathematically the trajectory of the child ' s movement in a field accord ing to the distribution of things with varying attracting or repelling forces . The root of situational constraints upon a child lies in a central fact of consciousness characteristic of early childhood : the union of motives and perception . At this age perception is generally not an independent but rather an integrated feature of a motor reaction . Every perception is a stimulus to activity . Since a situation is communicated psychologically through perception , and since perception is not separated from motiva tional and motor activity , it is understandable that with her conscious ness so structured , the child is constrained by the situation in which she finds herself . But in play , things lose their determining force . The child sees one The Role of Play in Development 97 thing but acts differently in relation to what he sees . Thus , a condition is reached in which the child begins to act independently of what he sees . Certain brain - damaged patients lose the ability to act indepen dently of what they see . In considering such patients one can appreciate that the freedom of action adults and more mature children enjoy is not acquired in a Hash but has to go through a long process of development . Action in an imaginary situation teaches the child to guide her behavior not only by immediate perception of objects or by the situation immediately affecting her but also by the meaning of this situation . Experiments and day - to - day observation clearly show that it is impos sible for very young children to separate the field of meaning from the visual field because there is such intimate fusion between meaning and what is seen . Even a child of two years , when asked to repeat the sen tence " Tanya is standing up " when Tanya is sitting in front of her , will change it to " Tanya is sitting down . " In certain diseases , exactly the same situation is encountered . Goldstein and Gelb described a number of patients who were unable to state something that was not true . " Gelb has data on one patient who was left - handed and incapable of writing the sentence " I can write well with my right hand . " When looking out of the window on a fine day he was unable to repeat " The weather is nasty today , " but would say " The weather is fine . " Often we find that a patient with a speech disturbance is incapable of repeating senseless phrases , for example , " Snow is black , " while other phrases equally difficult in their grammatical and semantic construction can be repeated . This tie between perception and meaning can be seen in the process of children ' s speech development . You say to the child , " clock , " and he starts looking for the clock . The word originally signi fies a particular spatiallocation . A divergence between the fields of meaning and vision first occurs at preschool age . In play thought is separated from objects and action arises from ideas rather than from things : a piece of wood begins to be a doll and a stick becomes a horse . Action according to rules begins to be determined by ideas and not by objects themselves . This is such a reversal of the child ' s relation to the real , immediate , concrete situation that it is hard to underestimate its full significance . The child does not do this all at once because it is terribly difficult for a child to sever thought ( the meaning of a word ) from object . Play provides a transitional stage in this direction whenever an object ( for example , a stick ) becomes a pivot for severing the meaning of horse from a real horse . The child cannot as yet detach thought from object . The child ' s weakness is that in order to imagine a horse , he needs Mind in Society 98 to define his action by means of using " the - horse - in - the - stick " as the pivot . But all the same , the basic structure determining the child ' s relation to reality is radically changed at this crucial point , because the structure of his perceptions changes . As I discussed in earlier chapters , a special feature of human per ception ( one arising at a very early age ) is the so - called perception of real objects , that is , the perception of not only colors and shapes , but also meaning . This is something to which there is no analogy in animal per ception . Humans do not merely see something round and black with two hands ; they see a clock and can distinguish one thing from another . Thus , the structure of human perception could be figuratively expressed as a ratio in which the object is the numerator and the meaning is the denominator ( object / meaning ) . This ratio symbolizes the idea that all human perception is made up of generalized rather than isolated per ceptions . For the child the object dominates in the object / meaning ratio and meaning is subordinated to it . At the crucial moment when a stick becomes the pivot for detaching the meaning of horse from a real horse , this ratio is inverted and meaning predominates , giving meaning / object . This is not to say that properties of things as such have no meaning . Any stick can be a horse but , for example , a postcard cannot be a horse for a child . Goethe ' s contention that in play any thing can be anything for a child is incorrect . Of course , for adults who can make conscious use of symbols , a postcard can be a horse . If I want to show the location of something , I can put down a match and sa . y , " This is a horse . " That would be enough . For a child it cannot be a horse because one must use a stick ; because of the lack of free substitution , the child ' s activity is play and not symbolism . A symbol is a sign , but the stick does not func tion as the sign of a horse for the child , who retains the properties of things but changes their meaning . Their meaning , in play , becomes the central point and objects are moved from a dominant to a subordinate position . The child at play operates with meanings detached from their usual objects and actions ; however , a highly interesting contradiction arises in which he fuses real actions and real objects . This characterizes the transitional nature of play ; it is a stage between the purely situa tional constraints of early childhood and adult thought , which can be totally free of real situations . When the stick becomes the pivot for detaching the meaning of " horse " from a real horse , the - child makes one object influence another semantically . He cannot detach meaning from an object , or a word from The Role of Play in Development 99 an object , except by finding a pivot in something else . Transfer of meanings is facilitated by the fact that the child accepts a word as the property of a thing ; he sees not the word but the thing it designates . For a child , the word " horse " applied to the stick means " there is a horse , " because mentally he sees the object standing behind the word . A vital transitional stage toward operating with meanings occurs when a child first acts With meanings as with objects ( as when he acts with the stick as though it were a horse ) . Later he carries out these acts con sciously . This change is seen , too , in the fact that before a child has acquired grammatical and written language , he knows how to do things but does not know that he knows . He does not master these activities voluntarily . In playa child spontaneously makes use of his ability to separate meaning from an object without knowing he is doing it , just as he does not know he is speaking in prose but talks without paying attention to the words . Thus , through play the child achieves a functional definition of concepts or objects , and words become parts of a thing . The creation of an imaginary situation is not a fortuitous fact in a child ' s life , but is rather the first manifestation of the child ' s emancipa tion from situational constraints . The primary paradox of play is that the child operates with an alienated meaning in a real situation . The second paradox is that in play she adopts the line of least resistance - she does what she most feels like doing because play is connected with pleasure - and at the same time she learns to follow the line of greatest resist ance by subordinating herself to rules and thereby renouncing what she wants , since subjection to rules and renunciation of impulsive action constitute the path to maximum pleasure in play . Play continually creates demands on the child to act against im mediate impulse . At every step the child is faced with a conflict between the rules of the game and what he would do if he could suddenly act spontaneously . In the game he acts counter to the way he wants to act . A child ' s greatest self - control occurs in play . He achieves the maxi mum display of willpower when he renounces an immediate attraction in the game ( such as candy , which by the rules of the game he is for bidden to eat because it represents something inedible ) . Ordinarily a childexperiences subordination to rules in the renunciation of something he wants , but here subordination to a rule and renunciation of action on immediate impulse are the means to maximum pleasure . Thus , the essential attribute of play is a rule that has become a desire , Spinoza ' s notions of " an idea which has become a desire , a con cept which has turned into a passion " finds its prototype in play , which is the realm of spontaneity and freedom . To carry out the rule is a source Mind in Society 100 of pleasure . The rule wins because it is the strongest impulse . Such a rule is an internal nile , a rule of self - restraint and self - determination , as Piaget says , and not a rule the child obeys like a physical law . In short , play gives a child a new form of desires . It teaches her to desire by relat ing her desires to a fictitious " I , " to her role in the game and its rules . In this way a child ' s greatest achievements are possible in play , achieve ments that tomorrow will become her basic level of real action and morality . SEPARATING ACTION AND MEANING Now we can say the same thing about the child ' s activity that we said about objects . Just as we had the obje ~ t ratio , we also have meanmg th action t ' Wh . domi I . d I t e . ra 10 . ereas action ominates ear y IP . eve opmen , meamng this structure is inverted ; meaning becomes the numerator , while action takes the place of the denominator . In a child of preschool age , action is initially dominant over meaning and is incompletely understood . The child is able to do more than he can understand . But it is at this age that an action structure first arises in which meaning is the determinant , although meaning must influence the child ' s behavior within constraints provided by structural features of the action . Children , in playing at eating from a plate , have been shown to perform actions with their hands reminiscent of real eating , while all actions that did not designate eating were impossible . Throw ing one ' s hands back instead of stretching them toward the plate turned out to be impossible , for such an action would have a destructive effect on the game . A child does not behave in a purely symbolic fashion in play ; rather he wishes and realizes his wishes by letting the basic cate gories of reality pass through his experience . The child , in wishing , carries out his wishes . In thinking , he acts . Internal and external action are inseparable : imagination , interpretation , and will are the internal processes carried by external action . What was said about detaching meaning from objects applies equally well to the child ' s own actions . A child who stamps on the ground and imagines herself riding a horse has h bv i d h action . meaning t ere y inverte t e ratio to · meaning action The developmental history of the relation between meaning and action is analogous to the development history of the meaning / object relation . In order to detach . the meaning of the action from the real action ( riding a horse , without the opportunity to do so ) , the child re - The Role of Play in Development 101 quires a pivot in the form of an action to replace the real one . While action begins as the numerator of the acti ~ n structure , now the meaning structure is inverted and meaning becomes the numerator . Action retreats to second place and becomes the pivot ; meaning is again de tached from action by means of a different action . This is another exam ple of the way in which human behavior comes to depend upon opera tions based on meanings where the motive that initiates the behavior is sharply separated from fulfillment . The separation of meaning from objects and action has different consequences , however . Just as operat ing with the meaning of things leads to abstract thought , we find that the development of will , the ability to make conscious choices , occurs when the child operates with the meaning of actions . In play , an action replaces another action just as an object replaces another object . How does the child float from one object to another , from one action to another ? This is accomplished by movement in the field of meaning which subordinates all real objects and actions to itself . Behavior is not bound by the immediate perceptual field . This movement in the field of meaning predominates in play . On the one hand , it represents move ment in an abstract field ( which thus makes an appearance in play prior to the appearance of voluntary operation with meanings ) . On the other hand , the method of movement is situational and concrete . ( It is an affective , not a logical change ) . In other words , the field of meaning appears , but action within it occurs just as in reality . Herein lies the main developmental contradiction of play . CONCLUSION I would like to close this discussion of play by first showing that play is not the predominant feature of childhood but it is a leading factor in development . Second , I want to demonstrate the significance of the change from predominance of the imaginary situation to predomi nance of rules in the development of play itself . And third , I want to point out internal transformations in the child ' s development brought about by play , How does play relate to developrnentf In fundamental , everyday situations a child ' s behavior is the opposite of his behavior in play . In play , action is subordinated to meaning , but in real life , of course , action dominates meaning . Therefore , to consider playas the prototype of a child ' s everyday activity and its predominant form is completely in correct . Mind in Society 102 This is the main flaw in Koffka ' s theory . He considers playas the child ' s other world . " Everything that concerns a child is play reality , while everything that concerns an adult is serious reality . A given object has one meaning in play and another outside of it . In a child ' s world the logic of wishes and of satisfying urges dominates , and not real logic . The illusory nature of play is transferred to life . This would all be true if play were indeed the predominant fonn of a child ' s activity . But it is difficult to accept the insane picture that comes to mind if the form of activity we have been speaking of were to become the predominant form of a child ' s everyday activity , even if only partially transferred to real life . Koffka gives a number of examples to show how a child transfers a situation from play into life . But the ubiquitous transference of play behavior to real life could only be regarded as an unhealthy symptom . To behave in a real situation as in an illusory one is the first sign of delirium . Play behavior in real life is normally seen only in the type of game when children begin to play at what they are in fact doing , evi dently creating associations that facilitate the execution of an unpleasant action ( as when children who do not want to go to bed say , " Let ' s play that it ' s nighttime and we have to go to sleep " ) . Thus , it seems to me that play is not the predominant type of activity at preschool age . Only theories which maintain that a child does not have to satisfy the basic requirements of life but . can live in search of pleasure could possibly suggest that a child ' s world is a play world . Looking at the matter from the opposite perspective , could one suppose that a child ' s behavior is always guided by meaning , that a preschooler ' s behavior is so arid that he never behaves spontaneously simply because he thinks he should behave otherwise ? This strict subordination to rules is quite impossible in life , but ill : play it does become possible : thus , play creates a zone of proximal development of the child . In playa child always behaves beyond his average age , above his daily behavior ; in play it is as though he were a head taller than himself . As in the focus of a magnifying glass , play contains all de velopmental tendencies in a condensed form and is itself a major source of development . Though the play - development relationship can be compared to the instruction - development relationship , play provides a much wider back ground for changes in needs and consciousness . Action in the imagina tive sphere , in an imaginary situation , the creation of voluntary inten tions , and the formation of . real - life plans and volitional motives - all appear in play and make it the highest level of preschool development . The Role of Play in Development 103 The child moves forward essentially through play activity . Only in this sense can play be considered a leading activity that determines the child ' s development . How does play change ? It is remarkable that the child starts with an imaginary situation that initially is so very close to the real one . A reproduction of the real situation takes place . For example , a child play ing with a doll repeats almost exactly what his mother does with him . This means that in the original situation rules operate in a condensed and compressed form . There is very little of the imaginary . It is an imaginary situation , but it is only comprehensible in the light of a real situation that has just occurred . Play is more nearly recollection of some thing that has actually happened than imagination . It is more memory in action than a novel imaginary situation . As play develops , we see a movement toward the conscious realiza tion of its purpose . It is incorrect to conceive of playas activity without purpose . In athletic games one can win or lose ; in a race one can come in first , second , or last . In short , the purpose decides the game and justifies the activity . Purpose , as the ultimate goal , determines the child ' s affective attitude to play . When running a race , a child can be highly agitated or distressed and little pleasure may remain because she finds it physi cally painful to run , and if she is overtaken she will experience little functional pleasure . In sports the purpose of the game is one of its domi nant features , without which there would be no point - like examining a piece of candy , putting it into one ' s mouth , chewing it , and then spitting it out . In such play , the object , which is to win , is recognized in advance . At the end of development , rules emerge , and the more rigid they are the greater the demands on the child ' s application , the greater the regulation of the child ' s activity , the more tense and acute play becomes . Simply running around without purpose or rules is boring and does not appeal to children . Consequently , a complex of originally unde veloped features comes to the fore at the end of play development features that had been secondary or incidental in the beginning occupy a central position . at the end , and vice versa . In one sense a child at play is free to determine his own actions . But in another sense this is an illusory freedom , for his actions are in fact subordinated to the meanings of things , and he acts accordingly . \ From the point of view of development , creating an imaginary situation can be regarded as a means of developing abstract thought . The corresponding development of rules leads to actions on the basis Mind in Society 104 of which the division between work and play becomes possible , a division encountered at school age as a fundamental fact . As figuratively expressed by one investigator , play for a child under three is a serious game , just as it is for an adolescent , although , of course , in a different sense of the word ; serious play for a very young child means that she plays without separating the imaginary situation from the real one . For the school child , play becomes a more limited form of activity , predominantly of the athletic type , which fills a specific role in the school child ' s development but lacks the significance of play for the preschooler . At school age play does not die away but permeates the attitude toward reality . It has its own inner continuation in ' school instruction and work ( compulsory activity based on rules ) . It is the es sence of play that a new relation is created between the field of mean ing and the visual field - that is , between situations in thought and real situations . Superficially , play bears little resemblance to the complex , medi ated form of thought and volition it leads to . Only a profound internal analysis makes it possible to determine its course of change and its role in development . 8 The Prehistory of Written Language Until now , writing has occupied too narrow a place in school prac tice as compared to the enormous role that it plays in children ' s cultural development . The teaching of writing has been conceived in narrowly practical terms . Children are taught to trace out letters and make words out of them , but they are not taught written language . The mechanics of reading what is written are so emphasized that they overshadow written language as such . Something similar has happened in teaching spoken language to deaf - mutes . Attention has been concentrated entirely on correct produc tion of particular letters and distinct articulation of them . In this case , teachers of deaf - mutes have not discerned spoken language behind these pronunciation techniques , and the result has been dead speech . This situation is to be explained primarily by historical factors : specifically , by the fact that practical pedagogy , despite the existence of many methods for teaching reading and writing , has yet to work out an effective , scientific procedure for teaching children written language . Unlike the teaching of spoken language , into which children grow of their own accord , teaching of written language is based on artificial training . Such training requires an enormous amount of attention and effort on the part of teacher and pupil and thus becomes something self - contained , relegating living written language to the background . Instead of being founded on the needs of children as they naturally develop and on their own activity , writing is given to them from without , from the teacher ' s hands . This situation recalls the development of a technical skill such as piano - playing : the pupil develops finger dexterity 105 Mind in Society 106 and learns to strike the keys while reading music , but he is in no way involved in the essence of the music itself . Such one - sided enthusiasm for the mechanics of writing has had an impact not only on the practice of teaching but on the theoretical state ment of the problem as well . Up to this point , psychology has conceived of writing as a complicated motor skill . It has paid remarkably little attention to the question of written language as such , that is , a particular system of symbols and signs whose mastery heralds a critical tuming point in the entire cultural development of the child . A feature of this system is that it is second - order symbolism , which gradually becomes direct symbolism . This means that written language consists of a system of signs that designate the sounds and words of spoken language , which , in turn , are signs for real entities and relations . Gradually this intermediate link , spoken language , disappears , and written language is converted into a system of signs that directly sym bolize the entities and relations between them . It seems clear that mastery of such a complex sign system cannot be accomplished in a purely mechanical and external manner ; rather it is the culmination of a long process of development of complex behavioral functions in the child . Only by understanding the entire history of sign development in the child and the place of writing in it can we approach a correct solution of the psychology of writing . The developmental history of written language , however , poses enormous difficulties for research . As far as we can judge from the avail able material , it does not follow a single direct line in which something like a clear continuity of forms is maintained . Instead , it offers the most unexpected metamorphoses , that is , transformations of particular forms of written language into others . To quote Baldwin ' s apt expression re garding the development of things , it is as much involution as evolution . ' This means that , together with processes of development , forward motion , and appearance of new forms , we can discern processes of curtailment , disappearance , and reverse development of old forms at each step . The developmental history of written language among chil dren is full of such discontinuities . Its line of development seems to disappear altogether ; then suddenly , as if from nowhere , a new line begins , and at first it seems that there is absolutely no continuity between the old and the new . But only a naive view of development as a purely evolutionary process involving nothing but the gradual accumulation of small changes and the gradual conversion of one form into another can conceal from us the true nature of these processes . This revolutionary type of development is in no way new for science in general ; it is new The Prehistory of Written Language 107 only for child psychology . Therefore , despite a few daring attempts , child psychology does not have a cogent view of the development of written language as a historical process , as a unified process of develop ment . The first task of a scientific investigation is to reveal this prehistory of children ' s written language , to show what leads children to writing , through what important points this prehistorical development passes , and in what relationship it stands to school learning . At the present time , in spite of a variety of research studies , we are in no position to write a coherent or complete history of written language in children . We can only discern the most important points in this development and discuss its major changes . This history begins with the appearance of the gesture as a visual sign for the child . GESTURES AND VISUAL SIGNS The gesture is the initial visual sign that contains the child ' s future writing as an acorn contains a future oak . Gestures , it has been correctly said , are writing in air , and written signs frequently are simply gestures that have been fixed . Wurth pointed out the link between gesture and pictorial or picto graphic writing in discussing the development of writing in human history . P He showed that figurative gestures often simply denote the reproduction of a graphic sign ; on the other hand , signs are often the fixation of gestures . An indicating line employed in pictographic writing denotes the index finger in fixed position . All these symbolic designations in pictorial writing , according to Wurth , can be explained only by derivation from gesture language , even if they subsequently become detached from it and can function independently . There are two other domains in which gestures are linked to the origin of written signs . The first concerns children ' s scribbles . We have observed in experiments on drawing that children frequently switch to dramatization , depicting by gestures what they should show on the drawing ; the pencil - marks are only a supplement to this gestural repre sentation . I could cite many instances . A child who has to depict running begins by depicting the motion with her fingers , and she regards the resultant marks and dots on paper as a representation of running . When she goes on to depict jumping , her hand begins to make movements depicting jumps ; what appears on paper remains the same . In general , we are inclined to view children ' s first drawings and scribbles rather as gestures than as drawing in the true sense of the word . We are also Mind in Society 108 inclined to ascribe to the same phenomenon the experimentally demon strated fact that , in drawing complex objects , children do not render their parts but rather general qualities , such as an impression of round ness and so forth . When a child depicts a cylindrical can as a closed curve that resembles - a circle , she thus depicts something round . This developmental phase coincides nicely with the general motor set that characterizes children of this age and governs the entire style and nature of their first drawings . Children behave in the same way in depicting concepts that are at all complex or abstract . Children do not draw , they indicate , and the pencil merely fixes the indicatory gesture . When asked to draw good weather , a child will indicate the bottom of the page by making a horizontal motion of the hand , explaining , " This is the earth , " and then , after a number of confused upward hatchwise motions , " And this is good weather . " We have had the occasion to verify more pre cisely , in experiments , the kinship between gestural depiction and depiction by drawing , and have obtained symbolic and graphic depiction through gestures in flve - year - olds . DEVELOPMENT OF SYMBOLISM IN PLAY The second realm that links gestures and written language is children ' s games . For children some objects can readily denote others , replacing them and becoming signs for them , and the degree of similarity between a plaything and the object it denotes is unimportant . What is most important is the utilization of the plaything and the possibility of executing a representational gesture with it . This is the key to the entire symbolic function of children ' s play . A pile of clothes or piece of wood becomes a baby in a game because the same gestures that depict holding a baby in one ' s hands or feeding a baby can apply to them : The child ' s self - motion , his own gestures , are what assign the function of sign to the object and give it meaning . All symbolic representational activity is full of such indicatory gestures ; for instance , a stick becomes a riding - horse for a child because it can be placed between the legs and a gesture can be employed that communicates that the stick designates a horse in this instance . From this point of view , therefore , children ' s symbolic play can be understood as a very complex system of " speech " through gestures that communicate and indicate the meaning of playthings . It is only on the basis of these indicatory gestures that playthings themselves gradually acquire their meaning - s - justcas drawing , while initially supported by gesture , becomes an independent sign . The Prehistory of Written Language 109 We attempted experimentally to establish this particular special stage of object writing in children . We conducted play experiments in which , in a joking manner , we began to designate things and people involved in the play by familiar objects . For example , a book off to one side designated a house , keys meant children , a pencil meant a nurse maid , a pocket watch a drugstore , a knife a doctor , an inkwell cover a horse - drawn carriage , and so forth . Then the children were given a simple story through figurative gestures involving these objects . They could read it with great ease . For example , a doctor arrives at a house in a carriage , knocks at the door , the nursemaid opens , he examines the children , he writes a prescription and leaves , the nursemaid goes to the drugstore , comes back , and administers medicine to the children . Most three - year - olds can read this symbolic notation with great ease . Four - or - five - year - olds can read more complex notation : a man is walking in the forest and is attacked by a wolf , which bites him ; the man extricates himself by running , a doctor gives him aid , and he goes to the drugstore and then home ; a hunter sets out for the forest to kill the wolf . What is noteworthy is that perceptual similarity of objects plays no noticeable part in the understanding of the symbolic notation . All that matters is that the objects admit the appropriate gesture and can func tion as a point of application for it . Hence , things with which this gestural structure cannot be performed are absolutely rejected by chil dren . For example , in this game , which is conducted at a table and which involves small items on the table , children will absolutely refuse to play if we take their fingers , put them on a book , and say , " Now , as a joke , these will be children . " They object that there is no such game . Fingers are too connected with their own bodies for them to be an object for a corresponding indicatory gesture . In the same way , a piece of furniture in the room or one of the people in the game cannot become involved . The object itself performs a substitution function : a pencil substitutes for a nursemaid or a watch for a drugstore , but only the relevant gesture endows them with this meaning . However , under the influence of this gesture , older children begin to make one exceptionally important discovery - that objects can indicate the things they denote as well as substitute for them . For example , when we put down a book with a dark cover and say that this will be a forest , a child will spontaneously add , " Yes , it ' s a forest because it ' s black and dark . " She thus isolates one of the features of the object , which for her is an indication of the fact that the book is supposed to mean a forest . In the same way , when a metal inkwell cover denotes a carriage , a child will point and say , " This is the seat . " When a pocket watch is to denote a drugstore , one child Mind in Society 110 might point to the numbers on the face and say , " This is medicine in the drugstore " ; another might point to the ring and say , " This is the en trance . " Referring to a bottle that is playing the part of a wolf , a child will point to the neck and say , " And this is his mouth . " If the experi menter asks , pointing to the stopper , " And what is this ? " the child an swers , " He ' s caught the stopper and is holding it in his teeth . " In all these examples we see the same thing , namely , that the cus tomary structure of things is modified under the impact of the new meaning it has acquired . In response to the fact that a watch denotes a drugstore , a feature of the watch is isolated and assumes the function of a new sign or indication of how the watch denotes a drugstore , either through the feature of medicine or of the entrance . The customary structure of things ( stopper in a bottle ) begins to be reflected in the new structure ( wolf holds stopper in teeth ) , and this structural modification becomes so strong that in a number of experiments we sometimes in stilled a particular symbolic meaning of an object in the children . For example , a pocket watch denoted a drugstore in all our play sessions . whereas other objects changed meaning rapidly and frequently . In taking up a new game , we would put down the same watch and explain , in accordance with the new procedures , " Now this is a bakery . " One child immediately placed a pen edgewise across the watch , dividing it in half , and , indicating one half , said , " All right , here is the drugstore , and here is the bakery . " The old meaning thus became independent and functioned as a means for a new one . We could also discern this acqui sition of independent meaning outside the immediate game ; if a knife fell , a child would exclaim , " The doctor has fallen . " Thus , the object acquires a sign function with a developmental history of its own that is now independent of the child ' s gesture . This is second - order symbolism , and because it develops in play , we see make - believe playas a major contributor to the development of written language - a system of second order symbolism . As in play , so too in drawing , representation of meaning initially arises as first - order symbolism , As we have already pointed out , the first drawings arise from gestures of the ( pencil - equipped ) hand , and the gesture constitutes the first representation of meaning . Only later on does the graphic representation begin independently to denote some object . The nature of this relationship is that the marks already made on paper are given an appropriate name . H . Hetzer undertook to study experimentally how symbolic repre sentation of things - so important in learning to write - develops in three to - six - year - old children , " Her experiments involved four basic series . The The Pl ' ehisto . , y of Written Language 111 first investigated the function of symbols in children ' s play . Children were to portray , in play , a father or mother doing what they do in the course of a day . During this game a make - believe interpretation of par ticular objects was given , making it possible for the researcher to trace the symbolic function assigned to things during the game . The second series involved building materials , and the third involved drawing with colored pencils , Particular attention in both these experiments was paid to the point at which the appropriate meaning was named . The fourth series undertook to investigate , in the form of a game of post office , the extent to which children can perceive purely arbitrary combinations of signs . The game used pieces of paper of various colors to denote different types of mail : telegrams , newspapers , money orders , packages , letters , postcards , and so forth . Thus , the experiments explicitly related these different forms of activity , whose only common feature is that a sym bolic function is involved in all of them , and attempted to link them all with the development of written language , as we did in our experiments . Hetzer was able to show clearly which symbolic meanings arise in play via figurative gestures and which via words . Children ' s egocentric language was widely manifest in these games . Whereas some children depicted everything by using movements and mimicry , not employing speech as a symbolic resource at all , for other children actions were accompanied by speech : the child both spoke and acted . For a third group , purely verbal expression not supported by any activity began to predominate . Finally , a fourth group of children did not play at all , and speech became the sole mode of representation , with mimicry and gestures receding into the background . The percentage of purely play actions decreased with age , while speech gradually predominated . The most important conclusion drawn from this developmental investigation , as the author says , is that the difference in play activity between three year - olds and six - year - olds is not in the perception of symbols but in the mode in which various forms of representation are used . In our opinion , this is a highly important conclusion ; it indicates that symbolic representation in play is essentially a particular form of speech at an earlier stage , one which leads directly to written language . As development proceeds , the general process of naming shifts farther and farther toward the beginning of the process , and thus the process itself is tantamount to the writing of a word that has just been named . Even a three - year - old understands the representational function of a toy construction , while a four - year - old names his creations even before he begins to construct them . Similarly , we see in drawing that a three - year - old is still unaware of the symbolic meaning of a drawing ; it Mind in Society 112 is only around age seven that all children master this completely . At the same time , our analysis of children ' s drawings definitely shows that , from the psychological point of view , we should regard such drawings as a particular kind of child speech . DEVELOPMENT OF SYMBOLISM IN DRAWING K . Buhler correctly notes that drawing begins in children when spoken speech has already made great progress and has become habit ual . " Subsequently , he says , speech predominates in general and shapes the greater part of inner life in accordance with its laws . This includes drawing . Children initially draw from memory . If asked to draw their mother sitting opposite them or some object before them , they draw without ever looking at the original - not what they see but what they know . Often children ' s drawings not only disregard but also directly contradict the actual perception of the object . We find what Buhler calls " x - ray drawings . " A child will draw a clothed figure , but at the same time will include his legs , stomach , wallet in his pocket , and even the money in the wallet - that is , things he knows about but which cannot be seen in the case in question . In drawing a figure in profile , a child will add a second eye or will include a second leg on a horseman in profile . Finally , very important parts of the object will be omitted ; for instance , a child will draw legs that grow straight out of the head , omitting the neck and torso , or will combine individual parts of a figure . As Sully showed , children do not strive for representation ; they are much more symbolists than naturalists and are in no way concerned with complete and exact similarity , desiring only the most superficial indi cations . " We cannot assume that children know people no better than they depict them ; rather they try more to name and designate than to represent . A child ' s memory does not yield a simple depiction of repre sentational images at this age . Rather , it yields predispositions to judg ments that are invested with speech or capable of being so invested . We see that when a child unburdens his repository of memory in drawing , he does so in the mode of speech - telling a story . A major feature of this mode is a certain degree of abstraction , which any verbal representation necessarily entails . Thus we see that drawing is graphic speech that arises on the basi . s of verbal speech . The schemes that distinguish children ' s first drawings are reminiscent in this sense of verbal concepts that communicate only the ~ ssential features of objects . This gives us The Prehistory of Written Language 113 grounds for regarding children ' s drawing as a preliminary stage in the development of written language . The further development of children ' s drawing , however , is not something self - understood and purely mechanical . There is a critical moment in going from simple mark - making on paper to the use of pencil - marks as signs that depict or mean something . All psychologists ' agree that the child must discover that the lines he makes can signify something . Sully illustrates this discovery using the example of a child who haphazardly drew a spiral line , without any meaning , suddenly grasped a certain similarity , and joyfully exclaimed , " Smoke , smoke ! " Although this process of recognizing what is drawn is encountered in early childhood , it is still not equivalent to the discovery of symbolic function , as observations have shown . Initially , even if a child perceives a similarity in a drawing , he takes the drawing to be an object that is similar or of the same kind , not as a representation or symbol of the object . When a girl who was shown a drawing of her doll exclaimed , " A doll just like mine ! " it is possible that she had in mind another object just like hers . According to Hetzer , there is no evidence that forces us to assume that assimilation of the drawing to an object means at the same time an understanding that the drawing is a representation of the object . For the girl , the drawing is not a representation of a doll but another doll just like hers . Proof of this is ' provided by the fact that for a long time children relate to drawings as if they were objects . For example , when a drawing shows a boy with his back to the observer , the child will turn the sheet over to try to see the face . Even among Hve - year - olds we always observed that , in response to the question , ' Where is his face and nose ? " children would turn the drawing over , and only then would answer , " It ' s not there , its ' s not drawn . " We feel that Hetzer is most justified in asserting that primary symbolic representation should be ascribed to speech , and that it is on the basis of speech that all the other sign systems are created . Indeed , the continuing shift toward the beginning in the moment of naming a drawing is also evidence of the strong impact of speech on the develop ment of children ' s drawing . We have had the opportunity of observing experimentally how children ' s drawing becomes real written language by giving them the task of symbolically depicting some more or less complex phrase . What was most clear in these experiments was a tendency on the part of school - age children to change from purely pictographic to ideographic Mind in Society 114 writing , that is , to represent individual relations and meaning by ab stract symbolic signs . We observed this dominance of speech over writ ing in one school child who wrote each word of the phrase in question as a separate drawing . For example , the phrase " I do not see the sheep , but they are there " was recorded as follows : a figure of a person ( " ] " ) , the same figure with its eyes covered ( " don ' t see " ) , two sheep ( " the sheep " ) , an index finger and several trees behind which the sheep can be seen ( " but they are there " ) , The phrase " I respect you " was rendered as follows : a head ( " I " ) , two human figures , one of which has his hat in hand ( " respect " ) and another head ( " you : " ) . Thus , we see how the drawing obediently follows the phrase and how spoken language intrudes into children ' s drawings . In this process , the children frequently had to make genuine discoveries in inventing an appropriate mode of representation , and we were able to see that this is decisive in the development of writing and drawing in children . SYMBOLISM IN WRITING In connection with our general research , Luria undertook to create this moment of discovery of the symbolics of writing so as to be able to study it systematically . " In his experiments children who were as yet unable to write were confronted with the task of making some simple fonn of notation . The children were told to remember a certain number of phrases that greatly exceeded their natural memory capacity . When each child became convinced that he would not be able to remember them all , he was given a sheet of paper and asked to mark down or record the words presented in some fashion . Frequently , the children were bewildered by this suggestion , saying that they could not write , but the experimenter furnished the child with a certain procedure and examined the extent to which the child was able to master it and extent to which the pencil - marks ceased to be simple playthings and became symbols for recalling the appropriate phrases . In the three - to - four - year - old stage , the child ' s notations are of no assistance in remembering the phrases ; in recalling them , the child does not look at the paper . But we occasionally encountered some seem ingly astonishing cases that were sharply at variance with this general observation . In these cases , the child also makes meaningless and un differentiated squiggles and lines , but when he reproduces phrases it seems as though he is reading them ; he refers to certain specific marks and can repeatedly indicate , without error , which marks denote which phrase . An entirely new relationship to these marks and a self - reinforc - The Prehistory of Written Language 115 ing motor activity arise : for the first time the marks become mnemo technic symbols . For example , the children place individual marks on different parts of the page in such a way as to associate a certain phrase with each mark . A characteristic kind of topography arises - one mark in one comer means a cow , while another farther up means a chimney sweep . Thus the marks are primitive indicatory signs for memory purposes . We are fully justified in seeing the first precursor of future writing in this mnemotechnic stage . Children gradually transform these undif ferentiated marks . Indicatory signs and symbolizing marks and scribbles are replaced by little figures and pictures , and these in turn give way to signs . Experiments have made it possible not only to describe the very moment of discovery itself but also to follow how the process occurs as a function of certain factors . For example , the content and forms intro duced into the phrases in question first break down the meaningless nature of the notation . If we introduce quantity into the material , we can readily evoke a notation that reflects this quantity , even in four and - Bve - year - olds . ( It was the need for recording quantity , perhaps , that historically first gave rise to writing . ) In the same way , the introduction of color and form are conducive to the child ' s discovery of the principle of writing . For example , phrases such as " like black , " " black smoke from a chimney , " " there is white snow in winter , " " a mouse with a long tail , ' or " Lyalya has two eyes and one nose " rapidly cause the child to change over from writing that functions as indicatory gesture to writing that contains the rudiments of representation . It is easy to see that the written signs are entirely first - order symbols at this point , directly denoting objects or actions , and the child has yet to reach second - order symbolism , which involves the creation of written signs for the spoken symbols of words . For this the child must make a basic discovery - namely that one can draw not only things but also speech . It was only this discovery that led humanity to the brilliant method of writing by words and letters ; the same thing leads children to letter writing . From the pedagogical point of view , this transition should be arranged by shifting the child ' s activity from drawing things to drawing speech . It is difficult to specify how this shift takes place , since the appropriate research has yet to lead to definite conclusions , and the generally accepted methods of teaching writing do not permit the observation of it . One thing only is certain - that the written language of children develops in this fashion , shifting from drawings of things to drawing of words . Various methods of teaching writing perform this in various ways . Many of them employ auxiliary gestures as a means of Mind in Society 116 uniting the written and spoken symbol ; others employ drawings that depict the appropriate objects . The entire secret of teaching written language is to prepare and organize this natural transition appropriately . As soon as it is achieved , the child has mastered the principle of written language and then it remains only to perfect this method . Given the current state of psychological knowledge , our notion that make - believe play , drawing , and writing can be viewed as different moments in an essentially unified process of development of written language will appear to be very much overstated . The discontinuities and jumps from one mode of activity to another are too great for the relationship to seem evident . But experiments and psychological analysis lead us to this very conclusion . They show that , however complex the process of development of written language may seem , or however erratic , disjointed , and confused it may appear superficially , there is in fact a unified historical line that leads to the highest forms of written language . This higher form , which we will mention only in passing , involves the reversion of written language from second - order symbolism to first - order symbolism . As second - order symbols , written symbols function as designations for verbal ones . Understanding of written language is first effected through spoken language , but gradually this path is curtailed and spoken language disappears as the intermediate link . To judge from all the available evidence , written language be comes direct symbolism that is perceived in the same way as spoken language . We need only try to imagine the enormous changes in the cultural development of children that occur as a result of mastery of written language and the· ability to read - and of thus becoming aware of everything that human genius has created in the realm of the written word . PRACTICAL IMPLICATIONS An overview of the entire developmental history of written lan guage in children leads us naturally to three exceptionally important practical conclusions . The first is that , from our point of view , it would be natural to transfer the teaching of writing to the preschool years . Indeed , if younger children are capable of discovering the symbolic function of writing , as Hetzer ' s experiments have shown , then the teaching of writing should be made the responsibility of preschool education , In deed , we see a variety of , circumstances which indicate that in the The Prehistory of Written Language 111 Soviet Union the teaching of writing clearly comes too late from the psychological point of view . At the same time , we know that the teach ing of reading and writing generally begins at age six in most European and American countries . Hetzer ' s research indicates that eighty percent of three - year - oIds can master an arbitrary combination of sign and meaning , while almost all six - year - olds are capable of this operation . On the basis of her ob servations , one may conclude that development between three and six involves not so much mastery of arbitrary signs as it involves progress in attention and memory . Therefore , Hetzer favors beginning to teach reading at earlier ages . To be sure , she disregards the fact that writing is second - order symbolism , whereas what she studied was first - order symbolism . Burt reports that although compulsory schooling begins at age five in England , children between three and five are allowed into school if there is room and are taught the alphabet , " The great majority of chil dren can read at four - and - a - half . Montessori is particularly in favor of teaching reading and writing at an earlier age . " In the course of game situations , generally through preparatory exercises , all the children in her kindergartens in Italy begin to write at four and can read as well as first - graders at age five . But Montessori ' s example best shows that the situation is much more complex than it may appear at first glance . If we temporarily ignore the correctness and beauty of the letters her children draw and focus on the content of what they write , we find messages like the following : " Happy Easter to Engineer Talani and Headmistress Montes sori . Best wishes to the director , the teacher , and to Doctor Montessori . Children ' s House , Via Campania , " and so forth . We do not deny the possibility of teaching reading and writing to preschool children ; we even regard it as desirable that a younger child enter school if he is able to read and write . But the teaching should be organized in such a way that reading and writing are necessary for something . If they are used only to write official greetings to the staff or whatever the teacher thinks up ( and clearly suggests to them ) , then the exercise will be purely mechanical and may soon bore the child ; his activity will not be manifest in his writing and his - budding personality will not grow . Reading and writing must be something the child needs . Here we have the most vivid example of the basic contradiction that appears in the teaching of writing not only in Montessori ' s school but in most other schools as well , namely , that writing is taught as a motor skill and not Mind in Society 118 as a complex cultural activity . Therefore , the issue of teaching writing in the preschool years necessarily entails a second requirement : writing must be " relevant to life " - in the same way that we require a " relevant " arithmetic . A second conclusion , then , is that writing should be meaningful for children , that an intrinsic need should be aroused in them , and that writing should be incorporated into a task that is necessary and relevant for life . Only then can we be certain that it will develop not as a matter of hand and finger habits but as a really new and complex form of speech . The third point that we are trying to advance as a practical conclu sion is the requirement that writing be taught naturally . In this respect , Montessori has done a great deal . She has shown that the motor aspect of this activity can indeed be engaged in in the course of children ' s play , and that writing should be " cultivated " rather than " imposed . " She offers a well - motivated approach to the development of writing . Following this path , a child approaches writing as a natural moment in her development , and not as training from without . Montessori has shown that kindergarten is the appropriate setting for teaching reading and writing , and this means that the best method is one in which chil dren do not learn to read and write but in which both these skills are found in play situations . For this it is necessary that letters become elements of children ' s life in the same way , for instance , that speech is . In the same way as children learn to speak , they should be able to learn to read and write . Natural methods of teaching reading and writing involve appropriate operations on the child ' s environment . Reading and writing should become necessary for her in her play . But what Mon tessori has done as regards the motor aspects of this skill should now be done in relation to the internal aspect of written language and its func tional assimilation . Of course , it is also necessary to bring the child to an inner understanding of writing and to arrange that writing will be organized development rather than learning . For this we can indicate only an extremely general approach : in the same way that manual labor and mastery of line - drawing are preparatory exercises for Montessori in developing writing skills , drawing and play should be preparatory stages in the development of children ' s written language . Educators should organize all these actions and the entire complex process of transition from one mode of written language to another . They should follow it through its critical moments up to the discovery of the fact that The Prehistory of Written Language 119 one can draw not only objects but also speech . If we wished to sum marize all these practical requirements and express them as a single one , we could say that children should be taught written language , not just the writing of letters . The great basic idea that the world is not to be viewed as a complex of fully fashioned objects , but as a complex of processes , in which appar ently stable objects , no less than the images of them inside our heads ( our concepts ) , are undergoing incessant changes . . . In the eyes of dialectical philosophy , nothing is established for all time , nothing is absolute or sacred . On everything and in everything it sees the stamp of inevitable decline ; nothing can resist it save the un ceasingprocess of formation and destruction , the unending ascent from lower to the higher - a process of which that philosophy itself is only a simple reflection within the thinking brain . Friedrich Engels , Ludwig Feuerbach Afterword VERA JOHN - STEINER AND ELLEN SOUBERMA ~ In this essay we hope to highlight several of Vygotsky ' s major theoretical assumptions , in particular those that could be the source of contemporary psychological research . After working for several years with the manuscripts and lectures that make up this volume , we came to recognize that Vygotsky ' s theory was primarily inductive , constructed midstream as he explored diverse phenomena such as memory , inner speech , and play . Our purpose is to explore in a systematic way those concepts that have had the greatest impact on us personally and intel lectually while editing Vygotsky ' s manuscripts . and preparing this work . As readers , we discovered that the consequences of internalizing Vygotsky ' s ideas have a dynamic of their own . At first , an increasing familiarity with his ideas helps one go beyond the polarities of contem porary psychological writings ; he offers a model for new psychological thought and research to those who are dissatisfied with the tension be tween traditional behaviorists and nativists . To some readers Vygotsky may seem to represent an intermediary position ; but a careful reading reveals his emphasis on the complex transformations that constitute human growth , the understanding of which requires active participation on the part of the reader . To Vygotsky , development was not merely a slow accumulation of unitary changes , but rather , as he wrote , " a complex dialectical process , characterized by periodicity , unevenness . in the development of dif ferent functions , metamorphosis or qualitative transformation of one form into another , intertwining of external and internal factors , and 121 Afterw01 ' d 122 adaptive processes " ( chapter 5 ) . And indeed , in this sense , his views of the history of the individt ) al ~ , Jhe - history _ Qf _ cultur _ e _ were similar . In both cases Vygotsky rejects the concept of linear development and in corporates into his conceptualization both evolutionary and revolution ary change . The recognition of these two interrelated forms of develop ment is for him a necessary component of scientific thought . Because it is not easy to conceptualize a dialectical process of change , we found that his concepts did not make their full impact until we attempted to combine our own research with his seminal ideas . ' This process required working through , again and again , the expansion of his condensed but powerful concepts and applying them either to our work or to daily observations of human behavior . The cryptic nature of Vygotsky ' s writing , though it can be explained by the conditions of his life during his last years , forced us to search deeply for his most sig nificant concepts . In this way we isolated those ideas that were strikingly original and which , forty years after his death , still offer new and un fulfilled promise for both psychology and education . CONCEPTS OF DEVELOPMENT Each chapter of this volume deals with some aspect of developmen tal change as Vygotsky conceived it . Although he is clearly committed to a theoretical position distinct from those of his influential contem poraries - Thorndike , Piaget , Koffka - he constantly returns to and ana lyzes their thinking in order to enrich and sharpen his own . While his contemporaries also addressed the issue of development , Vygotsky ' s approach differed from theirs in that he focused upon the historically shaped and culturally transmitted psychology of human beings . His analysis also differs from that of the early behaviorists . Vygotsky wrote : In spite of the significant advances attributable to behaviorist method ology , that method nevertheless is seriously limited . The psychologist ' s most vital challenge is that of uncovering and bringing to light the hidden mechanisms underlying complex human psychology . Though the be haviorist method is objective and adequate to the study of simple re flexive acts , it clearly fails when applied to the study of complex psycho logical processes . The inner mechanisms characteristic of these processes remain hidden . The naturalistic approach to behavior in general does not take into account the qualitative difference between human history and that of animals . The experimental ramification of this kind of analysis is that human behavior is studied without regard to the general history of human development . P . . Afterword 123 In contrast , Vygotsky emphasizes a theoretical approach , and con sequently a methodology , that telescopes change . His effort in charting developmental change is , in part , to show the psychological implications of the fact that humans are active , vigorous participants in their own existence and that at each stage of development children acquire the means by which they can competently affect their world and them selves . Therefore , a crucial aspect of human mastery , beginning in in fancy , is the creation and use of auxiliary or " artificial " stimuli ; through such stimuli an immediate situation and the reaction linked to it are altered by active human intervention . These auxiliary stimuli created by humans have no inherent relation to the existing situation ; rather , humans introduce them as a means of active adaptation . Vygotsky views auxiliary stimuli as highly diverse : they include the tools of the culture into which the child is born , the language of those who relate to the child , and the ingenious means pro duced by the child himself , including the use of his own body . One of the most striking examples of this sort of tool use can be seen in the play activity of poor children who do not have access to prefabricated toys but who , nevertheless , are able to play house , train , and so on with whatever resources are available to them ~ Theoretical explorations of these activities in a developmental context are a recurrent theme of this volume , for Vygotsky sees playas the primary means of children ' s cul tural development . Piaget shares Vygotsky ' s emphasis upon an active organism . They share , as well , the ability to observe children astutely . However , Vygot sky ' s skills of observation were enhanced by his knowledge of dialectical materialism and his view of the human organism as highly plastic and of the environment as historically and culturally shifting contexts into which children are born and which they , too , will eventually change . While Piaget stresses biologically supported , universal stages of devel opment , Vygotsky ' s emphasis is on the interaction between changing social conditions and the biological substrata of behavior . He wrote that " in order to study development in children , one must begin with an understanding of the dialectical unity of two principally different lines [ the biological and the cultural ] , to adequately study this process , then , an experimenter must study both components and the Jaws which gov ern their interlacement at each stage of a child ' s development / ? Although the work of a great number of psychological theorists , including Piaget , has been characterized as interactionist , the premises of such an approach are still lacking full formulation . Some of the con cepts described in this volume constitute the basis for a more fully Afterword 124 articulated interactionist - dialectical analysis of development . One of the critical issues in any theory of development is the relation between the biological bases of behavior and the social conditions in and through which human activity takes place . A key concept Vygotsky proposed to represent this important interaction is the functional learning system . In the development of this notion he departed significantly both from the then - existing psychology and from concepts of learning strongly bound up with the study of animal behavior . Vygotsky recognized , as had others before him , that functional systems are rooted in the most basic adaptive responses of the organism , such as unconditioned and conditioned reflexes . His theoretical contri bution , however , is based on . his description of the relation among these diverse processes : They are characterized by a new integration and co - relation of their parts . The whole and its parts develop parallel to each other and to gether . We shall call the first structures elementary ; they are psycho logical wholes , conditioned chiefly by biological determinants . The latter structures which emerge in the process of cultural development are called higher structures . . . The initial stage is followed by that first structure ' s destruction , reconstruction , and transition to structures of the higher type . Unlike the direct , reactive processes , these latter structures are constructed on the basis of the use of signs and tools ; these new forma tions unite both the direct and indirect means of adaptation . s Vygotsky argued that in the course of development psychological sys tems arise which unite separate functions into new combinations and complexes . This concept was further elaborated by Luria , who states that the components and relations into which these unitary functions enter are formed during each individual ' s development and are de pendent upon the social experiences of the child . The functional sys tems of an adult , then , are shaped essentially by her prior experiences as a child , the social aspects of which are more determinative than in tra ditional cognitive theory ( including that of Piaget ) . In this theory perhaps the most fundamental characteristic of de velopmental change is the manner in which previously separate and elementary functions are integrated into new functional learning sys terns : " Higher psychological functions are not superimposed as a second story over the elementary processes ; they represent new psychological systems . " These systems are changeable and are optimally adaptive to the particular tasks confronting the child as well as to the child ' s stage of development . Even though it may appear that children are learning in a purely external manner , that is , mastering new skills , the learning of any new operation is in fa ~ t the result of , and dependent on , a child ' s Afterword 125 process of development . The formation of new functional learning sys tems includes a process akin to that of nourishment in body growth , wherein at any particular time certain nutrients are digested and assimi lated while others are rejected . An approach analogous to Vygotsky ' s has emerged from the con temporary discussions of the role of nutrition in development . Birch and , Gussow , who conducted many cross - cultural studies of physical and intellectual growth , have advanced the following interactionist theory : " The effective environment of any organism is never merely the ob jective situation in which he finds himself , but is rather the product of an interaction between his unique organismic characteristics and what ever opportunities for experience his objective surroundings may pro vide / " In a similar vein , Vygotsky argues that because the historical conditions which determine to a large extent the opportunities for human experience are constantly changing , there can be no universal schema that adequately represents the dynamic relation between in ternal and external aspects of development . Therefore , a functional learning system of one child may not be identical to that of another , though there may be similarities at certain stages of development . Here , too , Vygotsky ' s analysis is different from that of Piaget , who describes universal stages that are identical for all children as a function of age . This point of view , which alms at linking the biological substrata of development to the study of functions culturally and historically achieved , may be oversimplified and give rise to misunderstandings . Luria , Vygotsky ' s student and collaborator , sought to clarify the com plex physiological implications of this view of the cognitive evolution of Homo sapiens : The fact that in the course of history man has developed new functions does not mean that each one relies on a new group of nerve cells and that new " centers " of higher nervous functions appear like those so eagerly sought by neurologists during the last third of the nineteenth century . The development of new " functional organs " occurs through the forma tion of new functional systems , which is a means for the unlimited de velopment of cerebral activity . The human cerebral cortex , thanks to this principle , becomes an organ of civilization in which are hidden boundless possibilities , and does not require new morphological apparatuses every time history creates the need for a new function . f The focus upon socially elaborated learning in Vygotsky ' s work emerges most clearly in his studies of mediated memory . It is in the course of interaction between children and adults that young learners identify effective means for remembering - means made accessible to them by those with more highly developed memory skills . The lack of AfterwMd 126 recognition among educators of this social process , of the many ways in which an experienced learner can share his knowledge with a less ad ~ vanced learner , limits the intellectual development of many students ; their capabilities are viewed as biologically determined rather than socially facilitated . In addition to these studies of memory ( chapter 3 ) , Vygotsky explores the role of social and cultural experiences through an examination of children ' s play ( chapter 7 ) . In their play children both depend on and imaginatively transform those socially produced objects and forms of behavior made available to them in their particular environment . An ever - present theme in this volume is the Marxian con cept of a historically determined human psychology . Some of Vygotsky ' s other writings , which are still unavailable in English , develop further his fundamental hypothesis that the higher mental functions are socially formed and culturally transmitted : " If one changes the tools of thinking available to a child , his mind will have a radically different structure . " ? Through signs children are able to internalize the adaptive social means already available to them from society at large . For Vygotsky , one of the essential aspects of development is the increasing ability of children to control and direct their own behavior , a mastery made possi ble by the development of new psychological forms and functions and by the use of signs and tools in this process . At a later age children ex tend the boundaries of their understanding by integrating socially elaborated symbols ( such as social values and beliefs , the cumulative knowledge of their culture , and the scientifically expanded . concepts of reality ) into their own consciousness . ' In Thought and Language Vygotsky presents a sophisticated argu ment demonstrating that language , the very means by which reflection and elaboration of experience takes place , is a highly personal and at the same time a profoundly social human process . He sees the relation between the individual and the society as a dialectical process which , like a river and its tributaries , combines and separates the different ele ments of human life . They are never frozen polarities to him . By far the most important sign - using behavior in children ' s de velopment is human speech . Through speech children free themselves of many of the immediate constraints of their environment . They pre pare themselves for future activity ; they plan , order , and control their own behavior as well as that of others . Speech is also an excellent exam ple of sign usage which , once internalized , becomes a pervasive and profound part of the higher psychological processes ; speech acts to organize , unify , and integrate many disparate aspects of children ' s behavior , such as perception , ·memory , and problem solving ( chapter 4 ) . Afte1 ' Wo . , d 127 He offers the contemporary reader a provocative avenue for dealing with a recurrent controversial issue , the relation between overt and covert processes . Like words , tools and nonverbal signs provide learners with ways to become more efficient in their adaptive and problem - solving efforts . Vygotsky often illustrates the varied means of human adaptation with , examples drawn from nonindustrialized societies : Counting fingers was once an important cultural triumph of humankind . It served as a bridge between immediate quantitative perception and counting . Thus , the Papuas of New Guinea began to count with the pinky of their left hand , follow through with the remaining left hand fingers , then add the left hand , forearm , elbow , shoulder , right shoulder , and so on , finishing with the pinky of the right hand . When this was insufficient they often used another person ' s fingers , or their own toes , or sticks , shells , and other small portable objects . In early counting systems , we may observe in developed and active form the same process that is pres ent in rudimentary form during the development of a child ' s arithmetical reasoning . Similarly , the tying of knots as a reminder not to forget something is related to the psychology of everyday life . A person must remember something , to fulfill some request , do this or that , pick up some object . Not trusting his memory and unwilling to go by it , he often ties his hanky into a knot or uses a similar device , such as sticking a little piece of paper under the cover of his pocket watch . Later on , the knot is sup posed to remind him of what he was supposed to do . And , this device often successfully carries out that function . Here , again , is an operation that is unthinkable and impossible in the case of animals . In the very fact of the introduction of an artificial , auxiliary means of memorizing , in the active creation and use of a stim ulus as a tool for memory , we see a principally new and specifically human feature of behavior . " The use of tools and signs share some important properties ; both involve mediated activity . But they also diverge from each other : signs are internally oriented , according to Vygotsky , a means of psychological influence aimed at mastering oneself ; tools , on the other hand , are externally oriented , aimed at mastering and triumphing over nature . The distinction between signs and tools is a good example of Vygotsky ' s analytical capacity to interweave diverse and similar aspects of human experience . Some other examples are thought and language , immedi ate and mediated memory , and , on a broader scale , the biological and the cultural , the individual and the social . In a concise passage in which he describes a two - stage psychological transformation that captures the way in which the child internalizes her social experience , Vygotsky also depicts a dynamic that he believes Afterw01 ' d 128 is present throughout the entire span of a human life : " Every function in the child ' s cultural development appears twice , on two levels . First , on the social , and later on the psychological level ; first , between people as an interpsychological category , and then inside the child , as an intrapsychological category . This applies equally to voluntary attention , to logical memory and to the formation of concepts . The actual rela tions between human individuals underlie all the higher functions " ( chapter 4 ) . In the buzzing confusion that surrounds the infant during the first few months of her life , parents assist her by pointing and carrying the child close to objects and places of adaptive significance ( toys , refrigerator , cupboard , playpen ) , thus helping the child to ignore other irrelevant features of the environment ( such adult objects as books , tools , and so on ) . This socially mediated attention develops into the child ' s more independent and voluntary attention , which she will come to use to classify her surroundings . In contrast with the well - known formulation by J . B . Watson , who wrote of thought as " subvocal language , " Vygotsky , in Thought and Language , describes how the growing child internalizes social language and makes it personal and how these two aspects of cognition , first in dependent of each other , are later joined : " Up to a certain point in time the two follow different lines , independently of each other . . . At a certain point these lines meet , whereupon thought becomes verbal and speech rational " ( p . 44 ) . In this way Vygotsky demonstrates the effec tiveness of conceptualizing related functions not as an identity but as the unity of two diverse processes . We believe this conception of human growth in its many varied manifestations is of value to contemporary psychological investigations . Though Vygotsky focused much of his research energies on the study of children , to view this great Russian psychologist as primarily a student of child development would be an error ; he emphasized the study of development because he believed it to be the primary theoretical and methodological means necessary to unravel complex human processes , a view of human psychology that distinguishes him from his and our contemporaries . There was , for him , no real distinction between devel opmental psychology and basic psychological inquiry . Moreover , he recognized that an abstract theory is insuffiicent to capture the critical moments of change ; and he demonstrated that the researcher must be an astute observer of children ' s play , their efforts at learning , their responses to teaching . The ingenuity of Vygotsky ' s experiments was a product of his skill ~ nd interest as both observer and experi menter . Afterword 129 EDUCATIONAL IMPLICATIONS Throughout this volume Vygotsky explores the various temporal dimensions of human life . He never equates the historical development of humankind to the stages of individual growth , since he is opposed to the biogenetic theory of recapitulation . Rather , his concern is with the consequences of human activity as it transforms both nature and so ciety . Although the labor of men and women to improve their world is rooted in the material conditions of their era , it is also affected by their capacity to learn from the past , to imagine , and to plan for the future . These specifically human abilities are absent in newborns , but by the age of three young children may already experience the tension between desires that can be fulfilled only in the future and demands for immedi ate gratification . Through play this contradiction is explored and tem porarily resolved . And so Vygotsky places the beginnings of human imagination at the age of three : " Imagination is a new formation which is not present in the consciousness of the very young child , is totally absent in animals , and represents a specifically human form of con scious activity . Like all functions of consciousness , it originally arises from action . The old adage that child ' s play is imagination in action can be reversed : we can say that imagination in adolescents and school children is play without action " ( chapter 7 ) . In their play children project themselves into the adult activities of their culture and rehearse their future roles and values . Thus , play is in advance of development , for in this manner children begin to acquire the motivation , skills , and attitudes necessary for their social participa tion , which can be fully achieved only with the assistance of their peers and elders . During preschool and school years the conceptual abilities of chil dren are stretched through play and the use of their imagination . In the course of their varied games they acquire and invent rules , or as Vygot sky describes it , " In playa child is always above his average age , above his daily behavior , in play it is as though he were a head taller than himself " ( chapter 7 ) . While imitating their elders in culturally pat terned activities , children generate opportunities for intellectual de velopment . Initially , their games are recollections and reenactments of real situations ; but through the dynamics of their imagination and the recognition of implicit rules governing the activities they have repro duced in their games , children achieve an elementary mastery of ab stract thought . In this sense , Vygotsky argued , play leads development . Similarly , school instruction and learning is in advance of chil - Afterword 130 dren ' s cognitive development . Vygotsky proposes a parallel between play and school instruction : both create a " zone of proximal develop ment " ( chapters 6 and 7 ) , and in both contexts children elaborate socially available skills and knowledge that they will come to internal ize . While in play all aspects of children ' s lives become themes in their games , in school both the content of what is being taught as well as the role of the specially trained adult who teaches them is carefully planned and more narrowly focused . In an essay on the psychological ideas of L . S . Vygotsky , Leontiev and Luria summarize some of the specific features of classroom education : School education is qualitatively different from education in the broad sense . At school the child is faced with a particular task : to grasp the bases of scientific studies , i . e . , a system of scientific conceptions . In the process of school education the child starts off from what have become his own complex generalizations and significances ; but he does not so much proceed from them , as proceed onto a new path to gether with them , onto the path of intellectual analysis , comparison , unification , and establishment of logical relations . He reasons , follow ing the explanations given to him and then reproducing new , for him , logical operations of transition from one generalization to other generali zations . The early concepts that have been built in the child in the process of living and which were assisted by rapport with his social environment ( Vygotsky called them " everyday " or " spontaneous " con cepts , spontaneous in the sense that they are formed aside from any process specially aimed at mastering them ) are now switched to a new process , to a new specially cognitive relationship to the world , and so in this process the child ' s concepts are transformed and their structure changes . In the development of a child ' s consciousness the grasping of the bases of a science - system of concepts now takes the lead . ? In Vygotsky ' s lifetime he and Luria initiated studies aimed at examining the cognitive consequences of rapid social change and the specific impact of schooling . ! " In addition to his interest in cognitive development among nonliterate peoples , his concern encompassed other aspects of the social and educational transformations brought about by the October Revolution . These concerns occupy many con temporary educators in countries undergoing rapid modernization and urbanization . Even in the United States , where the concept of public education is two centuries old , similar issues arise because large groups of people have not yet been integrated into or benefited from mass education . Some of the issues of concern to Vygotsky that are still alive today are the length - and scope of public education , the use of standardized tests to assess the educational potential of children , and effective models of teaching and curriculum . Afterword 131 Through the concept of the zone of proximal development as ad vanced by Vygotsky during intense educational debates in the 1930s , he telescopes , from the point of view of instruction , central tenets of his cognitive theory : the transformation of an interpersonal ( social ) proc ess to an intrapersonal one ; the stages of internalization ; and the role of experienced learners . The zone of proxima ] development , he wrote , is " the distance between the [ child ' s ] actual developmental level as de termined by independent problem solving and the level of potential development as determined through problem solving under adult guid ance or in collaboration with more capable peers " ( chapter 6 ) . Many educators , recognizing that the rate of learning may vary from child to child , isolate particularly " slow learners " from their teachers as well as their peers through the use of programmed and fre quently mechanized instruction . In contrast , Vygotsky , because he views learning as a profoundly social process , emphasizes dialogue and the varied roles that language plays in instruction and in mediated cog nitive growth . The mere exposure of students to new materials through oral lectures neither allows for adult guidance nor for collaboration with peers . To implement the concept of the zone of proximal develop ment in instruction , psychologists and educators must collaborate in the analysis of the internal ( " subtefranean " ) developmental processes which are stimulated by teaching and which are needed for subsequent learning . In this theory , then , teaching represents the means through which development is advanced ; that is , the socially elaborated con tents of human knowledge and the cognitive strategies necessary for their internalization are evoked in the learners - according to their " actual developmental levels . " Vygotsky criticizes educational intervention that lags behind developed psychological processes instead of focusing upon emerging functions and capabilities . A particularly imaginative appli cation of these principles are Paolo Freire ' s literacy campaigns in Third World countries . Because he adapted his educational methods to the specific historical and cultural setting in which his students lived , they were able to combine their " spontaneous " concepts ( those based on social practice ) with those introduced by teachers in instructional settings . ' ! VYGOTSKY ' S HISTORICAL - CULTURAL APPROACH ~ Perhaps the most distinguishing theme of Vygotsky ' s writings is his emphasis on the unique qualities of OUf species , how as human beings we actively realize and change ourselves in the varied contexts - 4 of culture and history . Repeatedly in this volume Vygotsky differenti - Afterword 132 ates the adaptive capabilities of animals from those of humans . The critical factor on which this distinction is based is the historically created and culturally elaborated dimensions of human life that are absent from the social organization of animals . In the development of higher functions - that is , in the internalization of the processes of knowing - the particulars of human social existence are reflected in human cognition : an individual has the capacity to externalize and share with other members cf her social group her understanding of their shared experience . The relative immaturity of the human infant , in contrast with other species , necessitates a lengthy reliance on caretaking adults , a circumstance that creates a basic psychological contradiction for the infant " : on the one hand he is totally dependent on organisms vastly more experienced than himself , and on the other hand he reaps the benefits of a socially developed and optimal setting for learning . Al though children are dependent on lengthy nurturance and caretaking , they are active participants in their own learning within the supportive contexts of family and community . As Edward E . Berg pointed out : Just as the tools of labor change historically , so the tools ' of thinking change historically . And just as new tools of labor give rise to new social structures , new tools of thinking give rise to new mental structures . Traditionally , it was thought that such things as the family and the state always existed in more or less their present form . Likewise , one also tends to view the structure of the mind as something universal and eter nal . To Vygotsky , however , both social structures and mental structures turn out to have very definite historical roots , and are quite specific products of certain levels of tool development . P Vygotsky ' s study of human development was deeply influenced by Friedrich Engels , who stressed the critical role of labor and tools in transforming the relation between human beings and their environment . The role of tools in human development was described by En gels as follows : " The tool specifically symbolizes human activity , man ' s transformation of nature : production . " 13 Such an approach re quires an understanding of the active role of history in human psycho logical development . In The Dialectics of Nature Engels presented some key concepts that were elaborated by Vygotsky . They both criti cized psychologists and philosophers who held the view " that only nature affects man and only natural conditions determine man ' s historic development , " and emphasized that in the course of history man , too , " affects nature , changes it , - creates for himself new natural conditions of existence . " 14 Furthermore , Vygotsky argued that the effect of tool Afterword 133 use upon humans is fundamental not only because it has helped them relate more effectively to their external environment but also because tool use has had important effects upon internal and functional rela tionships within the human brain . Although Engels and Vygotsky based their theories on the limited archaeological findings available to them during the years in which they wrote , contemporary archaeologists and physical anthropologists such as the Leakeys and Sherwood Washburn have interpreted more recent findings in a manner consistent with Engels ' and Vygotsky ' s point of View . Washburn states , " It was the success of the simplest tools that started the whole trend of human evolution and led to the civilization of today . " Most likely Vygotsky would have agreed with Washburn , who views the evolution of human life from our primate ancestors as resulting in " intelligent , exploratory , playful , and vigorous primates . . . and that tools , hunting , fire , complex social speech , the human way and the brain evolved together to produce ancient man . " ' 15 These archaeo logical discoveries support Vygotsky ' s concepts of what it is to be human . The impact of Vygotsky ' s work - as that of great theoreticians everywhere - is both general and specific . Cognitive psychologists as well as educators are interested in exploring the present - day implica tions of his notions , whether they refer to play , to the genesis of scien tific concepts , or to the relation of language and thought . The men and women who were his students forty years ago still debate his ideas with the intensity and vigor due a contemporary - and we who worked as his editors found many possible , sometimes contradictory , interpreta tions of his work . But there is a powerful tlplead drawing together Vy gotsky ' s diverse and stimulating writings : it is the way in which his mind worked . His legacy in an increasingly destructive and alienating world is to offer through his theoretical formulations a powerful tool for restructuring human life with an aim toward survival . " Notes INTRODUCTION 1 . K . N . Kornilov , " Psychology and Marxism , " in K . N . Kornilov , ed . , Psychology and Marxism ( Leningrad : State Publishing House , 1925 ) , pp . 9 24 . L . S . Vygotsky , " Consciousness as a Problem in the Psychology of Be havior , " in Komilov , ed . , Psychology and Marxism , pp . 175 - 198 . See also K . N . Kornilov , " Psychology in the Light of Dialectical Materialism , " in C . Murchison , ed . , Psychologies of 1930 ( Worcester : Clark University Press , 1930 ; rpt . , New York : Arno Press , 1973 ) . 2 . Friedrich Engels , Dialectics of Nature ( New York : International Pub lishers , 1940 ) , p . 40 . 3 . P . P . Blonsky , Studies in Scientific Psychology ( Moscow : State Pub lishing House , 1911 ) . 4 . R . Thumwald , " Psychologic des primitiven Menschen , " in Handbuch der uergleichenden Psychologie ( Munich , 1922 ) . L . Levy - Bruhl , Primitive Mentality ( New York : Macmillan , 1923 ) . 5 . A . R . Luria , Cognitive Development : Its Cultural and Social Founda tions ( Cambridge : Harvard University Press , 1976 ) . 6 . Z . M . Istomina , " The Development of Voluntary Memory in Pre school Age Children , " Soviet Psychology , 13 , no . 4 ( 1975 ) : 5 - 64 . 7 . M . Cole and I . Maltzman , eds . , A Handbook of Contemporary Soviet Psychology ( New York : Basic Books , 1969 ) . A . V . Zaporozhets and D . B . Elkonin , eds . , The Psychology of Preschool Children ( Cambridge : MIT Press , 1971 ) . CHAPTER , l 1 . K . Stumpf , " Zur Methodik der Kinderpsychologie , " Zeitsch . f . pddag . Psychol . , 2 ( 1900 ) . 2 . A . Gesell , The Mental Growth of the Preschool Child ( New York : Macmillan , 1925 ; Russian ed . , Moscow - Leningrad : Gosizdat . , 1930 ) . 135 Notes to Pages 20 - 37 136 3 . W . Kohler , The Mentality of Apes ( New York : Harcourt , Brace , 1925 ) . 4 . K . Buhler , The Mental Development of the Child ( New York : Har court , Brace , 1930 ; Russion ed . , 1924 ) . 5 . This particular experiment was described by D . E . Berlyne , " Chil dren ' s Reasoning and Thinking , " in Carmichael ' s Manual of Child Psychology , 3rd ed . , Paul H . Mussen , ed . ( New York : John Wiley , 1970 ) , pp . 939 - 981 . 6 . C . Buhler , The First Year of Life ( New York : Day , 1930 ) . 7 . K . Buhler , Mental Development , pp . 49 - 51 . See also C . Buhler , First Year . The linguistic capabilities of chimpanzees are currently the subject of controversy among psychologists and linguists . It seems clear that chimpanzees are capable of more complex signing than expected at the time Buhler and Vygotsky wrote these passages . However , the inferences about cognitive and linguistic competence warranted by these observations are still hotly debated . 8 . S . A . Shapiro and E . D . Gerke , described in M . Ya . Basov , Fundamen tals of General Pedology ( Moscow - Leningrad : Gosizdat . , 1928 ) . 9 . P . Guillaume and I . Meyerson , " Recherches sur l ' usage de l ' instrument chez les singes , " Journal de Psychologie , 27 ( 1930 ) : 177 - 236 . 10 . Research on aphasia was barely begun by Vygotsky during his own lifetime . The error of this conclusion and subsequent changes in his theory regarding aphasia may be found in the work of A . R . Luria ; see Traumatic Aphasia ( The Hague : Mouton , 1970 ) . 11 . W . Stem , Psychology of Early Childhood up to the Sixth Year of Age ( New York : Holt , Rinehart and Winston , 1924 ; Russian ed . , Petrograd , 1915 ) . 12 . J . Piaget , The Language and Thought of the Child ( New York : Meridian Books , 1955 ; also International Library of Psychology , 1925 ) . The differences between Vygotsky ' s and Piaget ' s views of early language develop ment and the role of egocentric speech is treated extensively in chapter 3 of Vygotsky ' s Thought and Language ( Cambridge : MIT Press , 1962 ) and in Piaget ' s volume of essays , Six Psychological Studies ( New York : Random House , 1967 ) . 13 . See R . E . Levina , for L . S . Vygotsky ' s ideas on the planning role of speech in children , Voprosi Psikhologii , 14 ( 1938 ) : 105 - 115 . Although Levina made these observations in the late 1920s , they remain unpublished except for this brief explication . 14 . Piaget , Language and Thought , p . 110 . 15 . A fuller description of these experiments is presented in chapter 7 of Thought and Language . CHAPTER 2 1 . A . Binet , " Perception de ' enfants , " Revue Philosophique , 30 ( 1890 ) : 582 - - 611 . Stern , Psychology of Early Childhood . 2 . A . A . Potebnya , Thought and Language ( Kharkhov , 1892 ) , p . 6 . 3 . K . KoHka , The Growth of the Mind ( London : Routledge and Kegan Paul , 1924 ) . 4 . R . Lindner , Gas Taubsiumme Kind in Vergleich mit vollstandigen Kinder ( Leipzig , 1925 ) . Notes to Pages 37 - 66 137 5 . K . Lewin , Wille , Vorsatz und Beduerfniss ( Berlin : Springer , 1926 ) . CHAPTER 3 1 . E . R . Jaensch , Eidetic Imagery ( New York : Harcourt , Brace , 1930 ) . 2 . Vygotsky is referring here to the technique of using knotted rope as a mnemonic device among Peruvian Indians . No reference is given in the text , but from other manuscripts it appears that the writing of E . B . Taylor and Levy - Bruhl provided these examples . 3 . These observations are taken from an article by A . N . Leontiev , " Studies on the Cultural Development of the Child , " Journal of Genetic Psychology , 40 ( 1932 ) : 52 - 83 . 4 . A fuller description of this technique may be found in A . R . Luria , " The Development of Mental Functions in Twins , " Character and Personality , 5 ( 1937 ) : 35 - 47 . 5 . L . V . Zankov , Memory ( Moscow : Uchpedgiz . , 1949 ) . 6 . A . N . Leontiev , " The Development of Mediated Memory , " Problemi Defektologiga , no . 4 ( 1928 ) . 7 . See H . Werner , Comparative Psychology of Mental Development ( New York : Science Editions , 1961 ) , pp . 216ff . 8 . See Vygotsky , Thought and Language , chapter 6 , for a more extensive discussion of the distinction . CHAPTER 4 1 . G . Hegel , " Encyklopadie , Erster Theil . Die Logik " ( Berlin , 1840 ) , p . 382 , cited in K . Marx , Capital ( Modem Library Edition , 1936 ) . 2 . Marx , Capital , p . 199 . CHAPTER 5 1 . Engels , Dialectics of Nature , p . 172 . 2 . H . Werner , The Comparative Psychology of Mental Development ( New York : International Universities Press , 1948 ) . 3 . K . Lewin , A Dynamic Theory of Personality ( New York : McGraw Hill , 1935 ) . 4 . Stern , Psyclwlogy of Early Childhood . 5 . Exact references are not included , but in his other writings , Vygotsky quotes extensively from Capital , vol . 1 . 6 . E . Titchener , Textbook of Psychology ( Moscow , 1914 , in Russian ) . 7 . P . P . Blonsky , Essays in Scientific Psychology ( Moscow : State Publish ing House , 1921 ) . 8 . For an extended discussion of the importance of reaction time experi ments in early twentieth - century psychology , see E . G . Boring , " The Psychol ogy of Controversy , " Psychological Review , 36 ( 1929 ) : 97 - 121 . 9 . Several of Cattell ' s papers on the reaction time study are reprinted in W . Dennis , Readings in the History of Psychology ( New York : Appleton Century - Crofts , 1948 ) . Notes to Pages 67 - 121 138 10 . N . Ach Uber die Willenstatigkeit und das Denken ( 1905 ) . 11 . For an outstanding application of these ideas to the development of voluntary memory in preschool age children , see the article by Istomina in Soviet Psychology , 12 , no . 4 ( 1975 ) : 5 - - 64 . CHAPTER 6 1 . Piaget , Language and Thought . 2 . William James , Talks to Teachers ( New York : Norton , 1958 ) , pp . 36 - 37 . 3 . Koffka , Growth of the Mind . 4 . E . L . Thorndike , The Psychology of Learning ( New York : Teachers College Press , 1914 ) . 5 . Dorothea McCarthy , The Language Development of the Pre - school Child ( Minneapolis : University of Minnesota Press , 1930 ) . 6 . Kohler , Mentality of Apes . 7 . Piaget , Language and Thought . CHAPTER 7 1 . J . Sully , Studies of Childhood ( Moscow , 1904 , in Russian ) , p . 48 . 2 . Lewin , Dynamic Theory of Personality , p . 96 . 3 . See K . Goldstein , Language and Language Disorders ( New York : Greene and Stratton , 1948 ) . 4 . Koffka , Growth of the Mind , pp . 381ff . CHAPTER 8 1 . J . M . Baldwin , Mental Development in the Child and the Race ( New York , 1895 ; Russian ed . , 1912 ) . 2 . Wurth ( reference not available ) . 3 . H . Hetzer , Die Symbolische Darstelling in der fruhen Windhert , ( Vienna : Deutscher Verlag fur Jugend und Yolk , 1926 ) , p . 92 . 4 . K . Buhler , Mental Development of the Child . 5 . J . Sully , Studies of Childhood ( London , 1895 ) . 6 . A . R . Luria , " Materials on the Development of Writing in Children , " Problemi Marksistkogo Vospitaniya , I ( 1929 ) : 143 - 176 . 7 . C . Burt , Distribution of Educational Abilities ( London : P . S . King and Sons , 1917 ) . 8 . M . Montessori , Spontaneous . Activity in Education ( New York : Schocken , 1965 ) . AFTERWORD 1 . See Nan Elsasser and Vera John - Steiner , ( CAn Interactionist Approach to Advancing Literacy , " Harvard Educational Review , 47 , no . 3 ( August 1977 ) : 355 - 370 . . . Notes to Pages 122 - 126 139 2 . Translation of passage from ' ' Tool and Symbol " and Development of Higher Psychological Functions not included in this text . Vygotsky used the term " natural " widely ; see pp . 38 - 39 , above . 3 . In this volume , the editors have interpreted Vygotsky ' s use of " natural " aspects of behavior to mean biologically given features , such as reflexes present at birth . An additional interpretation of " natural " can be gained from the following passage taken from A . N . Leontiev ' s , A . R . Luria ' s , and B . M . Teplov ' s preface to Vygotsky ' s Development of Higher Psychologi cal Functions . His attempt to show that it was impossible to reduce the formation of man ' s higher mental functions to ' the process of the development of their elementary forms leads to the false division at the genetic plane and at the plane of coexistence at higher levels of development . Thus , for ex ample , memory development is presented as going through two stages : the stage of purely natural memory which terminates at a preschool age and the following stage of development of a higher , mediated memory . The development of co - existing forms of memory is treated in the same way . One form rests exclusively on biological foundations , and others are the product of the child ' s social and cultural development . This op position which appears in L . S . Vygotsky ' s writings and in the research of his collaborators justifiably was criticized in its time . It is truly with out foundation : after all , even in very young children psychological processes are formed under the influence of verbal interaction with adults and consequently are not " natural . " The young child ' s memory processes are not " natural " because they already have changed as a result of language acquisition . We can say the same with regard to cases of the preservation of a sharply distinguished " natural " eidetic memory which turns out to be subject to transformation in man . While pointing out the inadequacy of Vygotsky ' s false contrast between natural ( organic ) and higher ( cultural ) forms of mental processes , we must emphasize that this contrast in no way is implied from his general theoretical position . Although Vygotsky has been criticized for - posing this artificial duality between the natural and the cultural , as Leontiev and Luria point out , the distinction is in fact an abstraction , a vehicle for describing a very complex process . " The child ' s mental development is a continuous process of gaining active control over initially passive mental functions . To gain this control the child learns to use signs and thus converts these ' natural ' mental functions into sign - mediated , cultural functions . " Edward E . Berg , " L , S . Vygotsky ' s Theory of the Social and Historical Origins of Consciousness " ( Ph . D . Diss . , University of Wisconsin , 1970 ) , p . 164 . 4 . This passage is from the unedited translation of " Tool and Symbol . " 5 . Herbert G . Birch and Joan Dye Gussow , Disadvantaged Children : Health , Nutrition and School Failure ( New York : Harcourt , Brace and World , 1970 ) , p . 7 . 6 . A . R . Luria , " L . S . Vygotsky and the Problem of Functional Locali zation , " Soviet Psychology , 5 , no . 3 ( 1967 ) : 53 - 57 . 7 . E . Berg , " Vygotsky ' s Theory , " p . 46 . 8 . Translation of passage from Development of Higher Psychological Functions not included in text . Notes to Pages 127 - 133 140 9 . A . N . Leontiev and A . R . Luria , " The Psychological Ideas of L . S . Vygotskii , " in B . B . Wolman , ed . , Historical Roots of Contemporary Psychol ogy ( New York : Harper and Row , 1968 ) , pp . 338 - 367 . 10 . A . R . Luria , Cognitive Development : Its Cultural and Social Foun - dations ( Cambridge : Harvard University Press , 197 - 6 ) . 11 . P . Freire , Pedagogy of the Oppressed ( New York : Seabury , 1970 ) . 12 . " Vygotsky ' s Theory , " pp . 45 - 46 . 13 . K . Marx and F . Engels , Selected Works ( Moscow : 1953 ) , p . 63 . 14 . Engels , Dialectics of Nature ( New York : International Publishers , 1940 ) , p . 172 . 15 . " Tools and Human Evolution , " Scientific American , 203 , no , 3 ( 1960 ) : 63 - 75 . 16 . We would especially like to thank . Stan Steiner and Ricardo Maez for their continued support through our many years of work on this volume and for their recognition of the importance of Vygotsky ' s writings to our joint futures . Vygotsky ' s Works IN RUSSIAN 1915 " The Tragedy of Hamlet , Prince of Denmark . " Private archives of L . S . Vy gotsky . Manuscript . 1916 " Literary Remarks on Petersburg by Andrey Biely . " The New Way , 1916 , no . 47 , pp . 27 - 32 . Review of Petersburg by Andrey Biely . Chronicle , 1916 , no . 12 , pp . 327 - 328 . Review of Fumnos and Bounds by Vyacheslav Ivanov published in ( Musatet , 1916 ) . Chronicle , 1916 , no . 10 , pp . 351 - 352 . " The Tragedy of Hamlet , Prince of Denmark . " Private archives of L . S . Vy gotsky . Manuscript . 1917Review of loy Will Be ( a play ) by D . Merezhkovsky ( published in The Lights , 1916 ) . Chronicle , 1917 , no . 1 , pp . 309 - 310 . Foreword to and remarks on " The Priest " ( a poem ) by N . L . Brodsky . Chronicle , 1917 , nos . 5 - 6 , pp . 366 - 367 . 1922 " About the Methods of Teaching Literature in Secondary Schools . " Report on the District Scientific Methodological Conference , Aug . 7 , 1922 . Private archives of L . S . Vygotsky . Manuscript , 17 pp . 1923 " The Investigation of the Processes of Language Comprehension Using Mul tiple Translation of Text from One Language to Another . " Private archives of L . S . Vygotsky . Manuscript , 8 pp . 141 Vygotsky ' s Works 142 1924 Vygotsky , L . S . , ed . Problems of Education of Blind , Deaf - Dumb and Re tarded Children . Moscow : SPON NKP Publishing House , 1924 . " Methods of Reflexological and Psychological Investigation . " Report of the National Meeting of Psychoneurology , Leningrad , Jan . 2 , 1924 . In The Problems of Contemporary Psychology , 11 , 26 - 46 . Leningrad : Gov ernment Publishing House , 1926 . " Psychology and Education of Defective Children . " In Problems of Education of Blind , Deaf - Dumb and Retarded Children , pp . 5 - 30 . Moscow : SPON NKP Publishing House , 1924 . Foreword to Problems of Education of Blind , Deaf - Dumb and Retarded Chil dren . Moscow : SPON NKP Publishing House , 1924 . " The Principles of Education of Physically Defective Children . " Report of the Second Meeting of SPON , Dec . 1924 . Public Education , 1925 , no . 1 , pp . 112 - 120 . 1925 Review of The Auxiliary School by A . N . Graborov . Public Education , 1925 , no . 9 , pp . 17Q - 171 . ' Foreword to Beyond the Pleasure Principle by S . Freud . Moscow : Contempo rary Problems , 1925 . ( With A . R . Luria . ) Foreword to General and Experimental Psychology by A . F . Lasursky . Lenin grad : Government Publishing House , 1925 . " The Principles of Social Education of Deaf - Dumb Children . " Private arch . . ives of L . S . Vygotsky . Manuscript , 26 pp . The Psychology of Art . Moscow : Moscow Art Publishing House , 1965 ( 379 pp . ) ; 2nd ed . , 1968 ( 576 pp . ) . " The Conscious as a Problem of the Psychology of Behavior . " In Psychology and Marxism , I , 175 - 198 . Moscow - Leningrad : Government Publishing House , 1925 . 1926 - 1927 Graphics of Bikhovsky . Moscow : Contemporary Russia Publishing House , 1926 . " Methods of Teaching Psychology . " ( Course program . ) The State Archives of Moscow District , fo ! ' 948 , vol . I , set 613 , p . 25 . " About the Influence of Speech Rhythm on Breathing . " In Problems of Con temporary Psychology , II , 169 - 173 . Leningrad : Government Publishing House , 1926 . Pedagogical Psychology . Moscow : The Worker of Education Publishing House , 1926 . " Introspection " by Koffka . In Problems of Contemporary Psychology , pp . 17 ~ 178 . Moscow - Leningrad : Government Publishing House , 1926 . Foreword to Principles of Learning Based upon Psychology by E . L . Thorn dike ( tr , from the English ) , pp . 5 - 23 . Moscow : The Worker of Education Publishing House , 1926 . Foreword to The Practice of Experimental Psychology , Education and Psy chotechnics by R . Schulz ( tr . from the German ) , pp . 3 - 5 . Moscow : Prob lems of Labor Publishing House , 1926 . ( With A . R . Luria . ) Vygotsky ' s Works 143 " The Problem - of Dominant Reactions . " In Problems of Contemporary Psy chology , II , 100 - 123 . Leningrad : Government Publishing House , 1926 . Review of The Psyche of Proletarian Children by Otto RuIle ( Moscow Leningrad , 1926 ) . Private archives of L . S . Vygotsky . Manuscript , 3 pp . " The Biogenetic Law in Psychology and Education . " The Great Soviet Encyclopedia , 1927 , vol . VI , cols . 275 - 279 . " Defect and Supercompensation . " In Retardation , Blindness and Mutisni , pp . 51 - 76 . Moscow : Down with Illiteracy Publishing House , 1927 . " The Historical Meaning of the Crisis in Psychology . " Private archives of L . S . Vygotsky . Manuscript , 430 pp . The Manual of Experimental Psychology . Moscow : Government Publishing House , 1927 . ( With V . A . Artomov , N . A . Bernshtein , N . F . Dobrinin , and A . R . Luria . ) Readings in Psychology . Moscow - Leningrad : Government Publishing House , 1927 . ( With V . A . Artomov , N . F . Dobrinin , and A . R . Luria . ) Review of The Method of Psychological Observation of Children by M . Y . Basov ( Moscow - Leningrad : Government Publishing House , 1926 ) . Teacher of the People , 1927 , no . 1 , p . 152 . " Contemporary Psychology and Art . " Soviet Art , 1927 , no . 8 , pp . ~ ; 1928 , no . 1 , pp . 5 - 7 . 1928 " Anomalies of Cultural Development of the Child . " Report to the Department of Defectology , Institute of Education of the Second Moscow State Uni versity , April 28 , 1928 . Problems of Defectology , 1929 , no . 2 ( 8 ) , pp . 106 - 107 . " Behaviorism . " The Great Soviet Encyclopedia , 1928 , vol . III , cols . 483 - 486 . " Sick Children . " Pedagogical Encyclopedia , 1928 , vol . II , cols . 396 - 397 . " The Will and Its Disturbances . " The Great Soviet Encyclopedia , 1928 , vol . V , cols . 590 - 600 . " The Education of Blind - Deaf - Mute Children . " Pedagogical Encyclopedia , 1928 , vol . II , cols . 395 - 396 . " Report of the Conference of Methods of Psychology Teaching in Teachers ' College , " April 10 , 1928 . The State Archives of Moscow District , fol . 948 , vol . I , pp . 13 - 15 . " The Genesis of Cultural Forms of Behavior . " Lecture , Dec . 7 , 1928 . Private archives of L . S . Vygotsky . Stenography , 28 pp . " Defect and Compensation . " Pedagogical Encyclopaedia , 1928 , vol . II , cols . 391 - 392 . " The Instrumental Method in Psychology . " In The Main Problems of Pedology in the USSR , pp . 158 - 159 . Moscow , 1928 . " The Results of a Meeting . " Public Education , 1928 , no . 2 , pp . 56 - 67 . " Invalids . " Pedagogical Encyclopaedia , 1928 , vol . II , col . 396 . " The Question of the Dynamics of Children ' s Character . In Pedology and Education , pp . 99 - 119 . Moscow : The Worker of Education Publishing House , 1928 . " The Question Concerning the Duration of Childhood in the Retarded Child . " Report to the Meeting of Defectology Department by the Insti - Vygotsky ' s Works 144 tute of Pedagogics of the Second Moscow State University , Dec . 18 , 1928 . Problems of Defectology , 1929 , no . 2 ( 8 ) , p . Ill . " The Question of Multilingualism in Childhood . " Private archives of L . S . Vygotsky . Manuscript , 32 pp . " Lectures on the Psychology of Development . " Private archives of L . S . Vygotsky . Stenography , 54 pp . " The Methods of Investigating Retarded Children . " Report to the First National Conference of Auxiliary School Workers . Archives of the Institute of Defectology , Academy of Pedagogical Sciences , USSR . ' Manuscript , 1 p . " On the Intersections of Soviet and Foreign Education . " Problems of De fectology , 1928 , no . 1 , pp . 18 - 26 . " To the Memory of V . M . Bekhterev . " Public Education , 1928 , no . 2 , pp . 68 - 70 . The Pedology of School - age Children . Lectures 1 - 8 . Moscow : Extension Division of the Second Moscow State University , 1928 . " The Problem of the Cultural Development of Children . " Pedology , 1928 , no . 1 , pp . 58 - 77 . " Psychological Science in the USSR . " In The Social Sciences of the USSR ( 1917 - 1927 ) , pp . 25 - 46 . Moscow : The Worker of Education Publishing House , 1928 . " The Psychological Basis for Teaching Dumb - Mute Children . " Pedagogical Encyclopaedia , 1928 , vol . II , col . 395 . " The Psychological Basis for Teaching Blind Children . " Pedagogical Encyclo paedia , 1928 , vol . II , cols . 394 - 395 . " Psychophysiological Basis for Teaching Abnormal Children . " Pedagogical Encyclopaedia , 1928 , vol . II , cols . 392 - 393 . " The Investigation of the Development of the Difficult Child . " In Leading Problems of Pedology in the USSR , pp . 132 - 136 . Moscow , 1928 . " Abnormal and Normal Children . " Pedagogical Encyclopaedia , 1928 , vol . II , col . 398 . " The Sociopsychological Basis for Teaching the Abnormal Child . " Peda gogical Encyclopaedia , 1928 , vol . II , cols . 393 - 394 . " The Three Main Types of Abnormality . " Pedagogical Encyclopaedia , 1928 , vol . II , col . 392 . " Difficult Childhood . " Lectures 3 and 4 . Archives of the Institute of De fectology , Academy of Pedagogical Sciences , USSR . Stenography , 9 pp . " The Retarded Child . " Pedagogical Encyclopaedia , 1928 , vol . II , eols . 397 398 . - 1929 " Lectures on Abnormal Childhood . " Problems of Defectology , 1929 ( 1930 ) , no . 2 ( 8 ) , pp . 108 - 112 . " Developmental Roots of Thinking and Speech . " Natural Science and Marx ism , 1929 , no . 1 , pp . 106 - 133 . " Genius . " The Great Soviet Encyclopedia , 1929 , vol . VI , cols . 612 - 613 . " About the Plan of Research Work for the Pedology of National Minorities . " Pedology , 1929 , no . 3 , pp . 367 - 377 . Vygotsky ' s Works 145 " The Intellect of Anthropoids in the Work of W . Kohler . " Natural Science and Marxism , 1929 , no . 2 , pp . 131 - 153 . " Some Methodological Questions . " The Archives of the Academy of Peda gogical Science , USSR , fo1 . 4 , vol . I , no . 103 , pp . 51 - 52 , 73 - 74 . " The Main Postulates of the Plan for Pedagogical Research Work Concern ing Difficult Children . " Pedology , 1929 , no . 3 , pp . 333 - 342 . " The Main Problems of Contemporary Defectology . " Report to the Defee tological Section of the Institute of Education , Moscow State University . In The Works of the Second Moscow State University , I , 77 - 106 . Mos cow , 1929 . " History of the Cultural Development of the Normal and Abnormal Child . " Private archives of L . S . Vygotsky . 1929 - 1930 . Manuscript . The Pedology of Teenagers . Lectures 1 - 4 , 5 - 8 . Moscow : Extension Division of the Second Moscow State University , 1929 . Subject and Methods of Contemporary Psychology . Moscow : Extension Divi sion of the Second Moscow State University , 1929 . " The Problem of Cultural Age . " Lecture , Feb . 15 , 1929 . Private archives of L . S . Vygotsky . Stenography , 18 pp . " The Development of Active Attention during Childhood . " In Problems of Marxist Education , I , 112 - 142 . Moscow : Academy of Communist Educa tion , 1929 . Also in Selected Psychological Investigations , pp . 389 - 426 . Moscow : Academy of Pedagogical Sciences Publishing House , 1956 . Review of School Dramatic Work as the Basis for Investigation of the Child ' s Creativity by Dimitrieva , Oldenburg , and Perekrestova ( Moscow : Gov ernment Publishing House , 1929 ) . Art in the School , 1929 , no . 8 , pp . 29 - 31 . Review of Contemporary Advances in Animal Psychology by D . N . Kash karov ( Moscow : Government Publishing House , 1928 ) . Natural Science and Marxism , 1929 , no . 2 , pp . 209 - 211 . . Review of The Language of Children by C . Stern and W . Stern ( Leipzig : Barth , 1928 ) . Natural Science and Marxism , 1929 , no . 3 , pp . 185 - 192 . Review of Means of Educational Influence by S . M . Rives ( Moscow : The Worker of Education Publishing House , 1929 ) . Pedology , 1929 , no . 4 , pp . 645 - 646 . " The Structure of Interests in Adolescence and the Interests of the Teenage Worker . In Problems of Pedology of the Teenage Worker , IV , pp . 2 ~ 8 . Moscow , 1929 . 1930 " The Biological Base of Affect . " 1 Want to Know . Everything , 1930 , nos . 15 16 , pp . 480 - 481 . Foreword to materials collected by workers of the Institute of Scientific Edu cation , April 13 , 1930 . Archives of the Academy of Pedagogical Sciences , USSR , fo1 . 4 , vol . I , no . 103 , pp . 81 - 82 . " Is It Possible to Simulate Extraordinary Memory ? " I Want to Know Every thing , 1930 , no . 24 , pp . 700 - 703 . Vygotsky ' s ' Works 146 " Imagination and Creativity in Childhood . " Private archives of L . S . Vygot sky , Manuscript . Problems of Defectology , VI . L . S . Vygotsky , ed . 1930 . ( With D . I . Asbukhin and L . V . Zankov . ) Foreword to The Essay of Spiritual Development of the Child by K . Buhler . Moscow : The Worker of Education Publishing House , 1930 . " Extraordinary Memory . " I Want to Know Everything , 1930 , no . 19 , pp . 553 554 . " The Instrumental Method in Psychology . " Report in the Academy of Com munist Education . Private archives of L . S . Vygotsky . Manuscript . " The Question of Speech Development and Education of the Deaf - Mute Child . Report to the Second National Conference of School Workers . Archives of the Institute of Defectology , Academy of Pedagogical Sci ences , USSR . Manuscript , 2 pp . " The Problem of the Development of Interests in Adolescence . " Education of Workers , 1930 , nos . 7 - 8 , pp . 63 - 81 . " The Cultural Development of Abnormal and Retarded Children . " Report to the First Meeting for Investigation of Human Behavior , Moscow , Feb . 1 , 1930 . In Psychological Sciences in the USSR , pp . 195 - 196 . Moscow Leningrad : Medgiz , 1930 . " New Developments in Psychological Research . " Report to the Third National Meeting of Child Care , May 1930 . The Internat , 1930 , no . 7 , pp . 22 - 27 . " Psychological Systems . " Report to the Neurology Clinic of the First Moscow State University , Oct . 9 , 1930 . Private archives of L . S . Vygotsky . Stenog raphy . " Tool and Sign . " Private archives of L . S . Vygotsky . Manuscript . " The Connection between Labor Activity and the Intellectual Development of the Child . " Pedology , 1930 , nos . 5 - 6 , pp . 588 - 596 . " The Behavior of Man and Animals . " Private archives of L . S . Vygotsky , 1929 - 1930 . Manuscript . Foreword to Teachers " Guide to the Investigation of the Educational Process by B . R . Bekingem . Moscow : The WorJ < er of Education Publishing House , 1930 . Foreword to Investigation of the Intellect of Anthropoids by W . Kohler . Moscow : Publishing House of the Communist Academy , 1930 . . " The Problem of the Higher Intellectual Functions in the System of Psycho logical Investigation . ' Psychology and Psychophysiology of Labor , vol . 3 ( 1930 ) , no . 5 , pp . 374 - 384 . " The Mind , Consciousness , Unconsciousness . " In Elements of General Psy chology , 4th ed . , pp . 48 - 61 . Moscow : Extension Division of the Second Moscow State University , 1930 . " The Development of the Highest Patterns of Behavior in Childhood . " Report to the First Meeting of Human Behavior , Jan . 28 , 1930 . In Psychoneuro logical Sciences in the USSR , pp . 138 - 139 . Moscow - Leningrad : Medgiz , 1930 . " The Development of Consciousness in Childhood . " Private archives of L . S . Vygotsky . Stenography . Vygotsky ' s Works 147 " Sleep and Dreams . " In Elements of General Psychology , pp . 62 - 75 . Moscow : Extension Division of the Second Moscow State University , 1930 . " The Communist Reconstruction of Man . " Varnitso , 1930 , nos . 9 - 10 , pp . 36 - 44 . " Structural Psychology . " In Main Trends in Contemporary Psychology by L . S . Vygotsky and S . Gellershtein , pp . 84 - 125 . Moscow - Leningrad : Gov - ernment Publishing House , 1930 . . " Eidetics . " In Main Trends in Contemporary Psychology by L . S . Vygotsky and S . Cellershtein , pp . 178 - 205 . Moscow - Leningrad : Government Pub lishing House , 1930 . " Experimental Investigation of the Highest Processes of Behavior . " Report to the First Meeting for Studying Human Behavior , Jan . 28 , 1930 . In Psychoneurological Sciences in the USSR . Moscow - Leningrad : Medgiz , 1930 . 1931 Buhler , C . , et aI . The Social - Psychological Study of the Child During the First Year of Life . L . S . Vygotsky , ed . Moscow - Leningrad : Medgiz , 1931 . ( With A . R . Luria . ) " Report of the Reactological Discussion , . 1931 . " Archives of the Institute of General and Pedagogical Psychology , Academy of Pedagogical Sciences , USSR , fol . 82 , vol . I , pp . 5 - 15 . Stenography ( corrected by L . S . Vygot sky ) . The Diagnosis - of Development and Pedological Clinics for Difficult Chil dren . Moscow : Publishing House of the Experimental Defectology Insti tute , 1936 . " The History of the Development of Higher Psychological Functions . " In Development of Higher Psychological Functions by L . S . Vygotsky , pp . 13 - 223 . Moscow : Academy of Pedogogical Sciences , RSFSR , 1960 . " The Question of Compensatory Processes in the Development of the Re tarded Child . " Report to the Conference of the Workers of Auxiliary Schools , Leningrad , May 23 , 1931 . Private archives of L . S . Vygotsky . Stenography , 48 pp . " Problems of Pedology and Related Sciences . " Pedology , 1931 , no . 3 , pp . 52 - 58 . " The Collective as a Factor of Development in the Abnormal Child . " In Prob lems of Defectology , 1931 , nos . 1 - 2 , pp . 8 - 17 ; no . 3 , pp . 3 - 18 . " Thinking . " The Great Soviet Encyclopedia , 1931 , vol . XIX , cols . 414 - 426 . The Pedology of Teenagers . Lectures 9 - 16 . Moscow - Leningrad : Extension Division of the Second Moscow State University , 1931 . " Practical Activity and Thinking in the Development of the Child in Con nection with a Problem of Politechnism . " Private archives of L . S . Vygot sky . Manuscript , 4 pp . Foreword to Development of Memory by A . N . Leontiev . Moscow - Leningrad : Uchpedgiz , 1931 . . . Foreword to Essay on the Behavior and Education of the Deaf - Mute Child by Y . K . Zvelfel . Moscow - Leningrad : Uchpedgiz , 1931 . Vygotsky ' s Works 148 The Psychological Dictionary . Moscow : Uchpedgiz , 1931 . ( With B . E . Var shava . ) " Psychotechnics and Pedology . " Report to the Meeting of the Communist Academy , Nov . 21 , 1930 . Archives of the Institute of General and Peda gogical Psychology , Academy of Pedagogical Sciences , USSR , fo1 . 82 , vol . I , no . 3 , pp . 23 - 57 . 1932 " The Problem of Creativity in Actors . " In The Psychology of the Stage Feel ings of an Actor by P . M . jakobson , pp . 197 - 211 . Moscow : Government Publishing House , 1936 . " Toward a Psychology of Schizophrenia . " Soviet Neuropathology , Psychiatry and Psychohygiene , vol . 1 ( 1932 ) , no . 8 , pp . 352 - - 361 . " Toward a Psychology of Schizophrenia . " In Contemporary Problems of Schizophrenia , pp . 19 - 28 . Moscow : Medgiz , 1933 . " Lectures on Psychology . " Leningrad Pedagogical Institute , March - April 1932 . Archives of the Leningrad Pedagogical Institute . Stenography . Also in Development of Higher Psychological Functions , pp . 235 - 363 . Moscow : Academy of Pedagogical Sciences , RSFSR , 1960 . " Infancy . " Private archives of L . S . Vygotsky , Manuscript , 78 pp . Foreword to Education and Teaching of the Retarded Child by E . K . Gra cheva , Moscow - Leningrad : Uchpedgiz , 1932 . Foreword to Development of Memory by A . N . Leontiev . Moscow , 1932 . ( With A . N . Leontiev . ) " The Problem of Development of the Child in the Research of Arnold Gesell . " In Education and Childhood by A . Gesell , pp . 3 - 14 . Moscow - Leningrad : Uchpedgiz , 1932 . " Problem of the Speech andThinking of the Child in the Teachings of Pia get . " In Language and Thought of the Child by J . Piaget , pp . 3 - 54 . Moscow - Leningrad : Uchpedgiz , 1932 . " Early Childhood . " Lecture , Leningrad Pedagogical Institute , Dec . 15 , 1932 . Archives of the Leningrad Pedagogical Institute . Stenography , 50 pp . " Contemporary Directions in Psychology . " Report to the Communist Acad emy , June 26 , 1932 . In Development of Higher Psychological Functions by L . S . Vygotsky , pp . 458 - 481 . Moscow : Academy of Pedagogical Sciences , RSFSR , 1960 . 1933 " Introductory Lecture about Age - Psychology . " The Central House of Art Education of Children , Dec . 19 , 1933 . Archives of the Leningrad Peda gogical Institute . Stenography , 34 pp . " Dynamics of Mental Development of School Children in Connection with Education . ' " Report to the Meeting of the Department of Defectology of Bubnov Pedagogical institute , Dec . 23 , 1933 . In Mental Develop ment of Children during Education by L . S . Vygotsky , pp . 33 - 52 . Mos cow - Leningrad : Government Publishing House , 1935 . Vygotsky ' s Works 149 " Preschool Age . " Lecture , Leningrad Pedagogical Institute , Dec . 13 - 14 , 1933 . Private archives of L . S . Vygotsky . Stenography , 15 pp . " Play and Its Role in the Psychological Development of the Child . " Lecture , Leningrad Pedagogical Institute , 1933 . Problems of Psychology , 1966 , no . 6 , pp . 62 - 76 . " Questions about the Dynamics of Development of the Intellect of the Normal and Abnonnal Child . " Lecture , Bubnov Pedagogical Institute , Dec . 23 , 1933 . Private archives of L . S . Vygotsky . Stenography . " Crisis of the First Year of Life . " Lecture , Leningrad Pedagogical Institute . Archives ' of the Leningrad Pedagogical Institute . Stenography , 37 pp . " Critical Ages . " Lecture , Leningrad Pedagogical Institute , June 26 , 1933 . Archives of the Leningrad Pedagogical Institute . Manuscript , 15 pp . " The Negative Phase of Adolescence . " Lecture , Leningrad Pedagogical Insti tute , June 26 , 1933 . Archives of the Leningrad Pedagogical Institute . Manuscript , 17 pp . " Study of Schoolwork in School Children . " Report to the Leningrad Peda gogical Institute , Jan . 31 , 1933 . Private archives of L . S . Vygotsky . Stenography . " Pedological Study of the Pedagogical Process . " Report to the Experimental Defectological Institute , March 17 , 1933 . In Mental Development of Children during Education by L . S . Vygotsky , pp . 116 - 134 . Moscow Leningrad : Uchpedgiz , 1935 . " Adolescence . " Lecture , Leningrad Pedagogical Institute , June 25 , 1933 . Archives of the Leningrad Pedagogical Institute . Stenography , 19 pp . " Pedology of Preschool Age . " Lecture , Leningrad Pedagogical Institute , Jan . 31 , 1933 . Archives of the Leningrad Pedagogical Institute . Stenography , 16pp . Foreword to Difficult Children in Schoolwork by L . V . Zankov , M . S . Pevs ner , and V . F . Shmidt . Moscow - Leningrad : Uchpedgiz , 1933 . " Problems of Age : Play . " Concluding speech to the Seminar of the Leningrad Pedagogical Institute , March 23 , 1933 . Archives of the Leningrad Peda gogical Institute . Stenography , 39 pp . " Problems of Development . " Lecture , Leningrad Pedagogical Institute , Nov . 27 , 1933 . Archives of the Leningrad Pedagogical Institute . Stenography , 17pp . " The Problem of Consciousness . ' > Report after the speech of A . R . Luria on Dec . 5 and 9 , 1933 . In Psychology of Grammar , pp . 178 - 196 . Moscow : Moscow State University , 1968 . " Development of Common Sense and Scientific Ideas during School Age . " Report to the Scientific Conference , Leningrad Pedagogical Institute , May 20 , 1933 . In Mental Development of Children during Education , pp . 96 - 115 . Moscow - Leningrad : Uchpedgiz , 1935 . " Study of Emotions . " Private archives of L . S . Vygotsky , 1933 . Manuscript . 555 pp . Also " The Study of Emotions in the Light of Contemporary Psychoneurology . " Questions of Philosophy , 1970 , no . 6 , pp . 110 - 130 . See also , " Two Directions in the Comprehension of the Nature of Emo tions in Foreign Psychology in the beginning of the Twentieth Century . " Problems of Psychology , 1968 , no . 2 , pp . 149 - 156 . Vygotsky ' s Works 150 1934 " Dementia during Pick ' s Disease . " Soviet Neuropathology , Psychiatry , Psy chohygiene , vol . 3 ( 1934 ) , no . 6 , pp . 97 - 136 . ( With G . V . Birenbaum and N . V . Samukhin . ) " Development of Scientific Ideas during Childhood . " In The Development of Scientific Ideas of School Children by Zh . I . Shif , pp . 3 - 17 . Moscow Leningrad : Uchpedgiz , 1935 . " Infancy and Early Age . " Lecture , Leningrad Pedagogical Institute , Feb . 23 , 1934 . Archives of the Leningrad Pedagogical Institute . Stenography , 24pp . Thought and Language . Moscow - Leningrad : Sozekgiz , 1934 . " The Thinking of School Children . " Lecture , Leningrad Pedagogical In stitute , May 3 , 1934 . Archives of the Leningrad Pedagogical Institute . Stenography , 11 pp . Fundamentals of Pedology . Moscow : Second Moscow Medical Institute , 1934 . " Adolescence . " Lecture , Leningrad Pedagogical Institute , March 25 , 1934 . Archives of the Leningrad Pedagogical Institute . Stenography . " Problems of Age . " Private archives of L . S . Vygotsky . Manuscript , 95 pp . Also in Problems of Psychology , 1972 , no . 2 , pp . 114 - 123 . " Problems of Education and Mental Development in School Age . " I ~ Mental Development of Children during Education by L . S . Vygotsky , pp . 3 - 19 . Moscow - Leningrad : Uchpedgiz , 1935 . " Problem of Development in Structural Psychology . " In Fundamentals of Psychological Development by K . Koffka , pp . ix - Ixi , Moscow - Leningrad : Sozekgiz , 1934 . " Problem of Development and Destruction of the Higher Psychological Functions . " In Development of Higher Psychological Functions by L . S . Vygotsky , pp . 364 - 383 . Moscow : Academy of Pedagogical Sciences , RSFSR , 1960 . ( Vygotsky ' s last report , prepared one month before his death . ) " Psychology and Teaching of Localization . " In Reports of the First Ukranian Meeting of Neuropathologists and Psychiatrists , pp . 34 - 41 . Kharkov , 1934 . " Dementia during Pick ' s Disease . " Private archives of L . S . Vygotsky , 1934 . Manuscript , 4 pp . Fascism in Psychoneurology . Moscow - Leningrad : Biomedgiz , 1934 . ( With V . A . Giljarovsky et al . ) " School Age . " Private archives of D . B . Elkonin , 1934 . Manuscript , 42 pp . " School Age . " Lecture , Leningrad Pedagogical Institute , Feb . 23 , 1934 . Archives of the Leningrad Pedagogical Institute . Stenography , 61 pp . " Experimental Investigation of the Teaching of New Speech Reflexes by the Method of Attachment with Complexes . " Private archives of L . S . Vygotsky . Manuscript . 1935 " Education and Development during School Age . " Report to the National Vygotsky ' s Works lSI Conference of Preschool Education . In Mental Development of Childrei during Education , pp . 20 - 32 . Moscow - Leningrad : Uchpedgiz , 1935 . " Problem of Dementia . " In The Retarded Child , pp . 7 - 34 . Moscow - Lenin grad : Uchpedgiz , 1935 . The Retarded Child . L . S . Vygotsky , edt Moscow - Leningrad : Uchpedgiz , 1935 . Works of Various Years Pedology of Youth : Features of the Behavior of the Teenager . Lessons 6 - 9 . Moscow : Extension Division of the Faculty of Education , Second Mos cow State University . " Problem of the Cultural Development of the Child . " Private archives of L . S . Vygotsky . Manuscript , 81 pp . ' ' ' The Blind Child . " Private archives of L . S . Vygotsky . Manuscript , 3 pp . Difficult Childhood . Moscow : Extension Division of the Faculty of Education , Second Moscow State University . IN ENGLISH " The Principles of Social Education of Deaf and Dumb Children in Rus sia . " In International Conference on the Education of the Deaf , pp . 227 - 237 . London , 1925 . " The Problem of the Cultural Development of the Child . " lournal of Genetic Psychology , 1929 , vol . 36 , pp . 415 - 434 . " Thought in Schizophrenia . " Archives of Neurological Psychiatry , 1934 , vol . 31 . " Thought and Speech . " Psychiatry , 1939 , vol . 2 , pp . 29 - 54 . Rpt . in S . Saporta , ed . , Psycholinguistics : A Book of Readings , pp . 509 - 537 . New York : Holt , Rinehart and Winston , 1961 . Thought and Language . Cambridge : MIT Press and Wiley , 1962 . ( Originally published in Russian in 1934 . ) - " Psychology and Localization of Functions . " Neuropsychologia , 1965 , vol . 3 , pp . 381 - 386 . ( Originally published in Russian in 1934 . ) " Development of the Higher Mental Functions . " In A . Leontiev , A . Luria , and A . Smirnov , eds . , Psychological Research in the USSR , vol . I , pp . 11 - 46 . Progress Publishing , 1966 . ( Abridged . ) " Play and Its Role in the Mental Development of the Child . " Soviet Psychol ogy , 1967 , vol . 3 . ( Vygotsky memorial issue . Includes preface by J . S . Bruner and articles by Soviet psychologists Luria , Davydov , El ' konin , Gal ' perin , and Zaporozhetz . Article based on 1933 lecture . ) The Psychology of Art . Cambridge : MIT Press , 1971 . ( Collected writings of literary and art criticism spanning several decades . ) " Spinoza ' s Theory of the Emotions in Light of Contemporary Psychoneurol ogy . " Soviet Studies in Philosophy , 1972 , vol . 10 , pp . 362 - 382 . Ach , N . , 67 Action : repetition of , 22 ; purposeful , symbolic representations of , 37 ; - meaning ratio , 100 - - 101 ; play and , 129 . See also Gestures ; Linguistics ; Play ; Symbolism Adaptation : and children ' s adaptive be havior , 22 , 24 ; sign and tool use as means of , 53 , 55 , 123 , 124 , 127 ( see also Signs , use of ; Tools , use of ) ; and adaptability of animals , 60 , 132 ( see also Animals ) Adolescence , see Age Age : " chimpanzee , " 21 ( see also Ani mals ) " ; and practical intelligence / thought , 21 - 22 , 23 , 24 , 51 , 79 - 80 , 97 ; andspeedh , 25 - 26 , 28 , 29 , 32 , 62 ; and perception , 31 - 32 , 33 , 97 , 98 ; and choice behavior / response , 33 , 70 ; and color - task errors , 41 - 45 ; infancy , and roots of cultural behavior , 46 ; and memory , 47 - 49 , 50 , 56 , 139n . 3 ; adoles cence , 61 , 83 , 104 , 129 ; mental , 85 - 88 , 89 ; and play , 92 - 94 , 102 , 104 , 111 , 129 ; and situational constraints , 96 , 98 - 99 ; and action vs . meaning , 100 101 ; and graphic depiction , 108 ( see also Gestures ) ; and symbolic notation , 109 - 112 , 113 , 114 , 115 ; and reading , writing , 117 ; and imagination , 129 . See also Child development ; Linguis tics ; Maturation ; Tests and testing All - Union Institute of Experimental Medicine ( USSR ) , 16 Index America , see United States Analysis : developmental , 8 , 61 - 62 , 65 ( see also Behavior ) ; of process vs . objects ( experimental - developmental ) , 61 - 62 , 65 ; explanatory vs , descriptive ( genotypic vs . phenotypic ) , 62 - 63 , 65 , 67 , 68 ; aim of , 63 , 64 , 65 ; introspective , 63 , 65 - - 68 ; " fos - silized " behavior and , 63 - 65 , 68 ; of choice reaction , 6 ~ 69 ( see also Choice behavior and responses ) ; of teaching , 79 ; of educational process , 90 - 91 Animals : " study of , linked with human studies , 2 - 3 , 4 , 6 , 60 , 122 , 124 ; and use of tools , 7 , 22 , 23 , 24 , 88 ; Kohler ' s ape studies , 20 - 23 ( passim ) , 25 , 28 , 31 , 37 , 88 ; and child - ape compari sons ; 20 - 23 , 26 , 28 , 34 , 36 , 37 ; and sign use , 23 , 39 ; and linguistics , 23 , 136n . 7 ; and perception , 31 , 32 - 33 , 34 , 35 , 36 , 37 ; and attention , 36 ; and voluntary activity , 37 ; and memory aids , 39 , 51 , 127 ; and internalization , 57 ; and adaptability / adaptation , 60 , 132 ; and learning , 88 ; and imagina tion , 93 , 129 Aphasia , 9 , 22 . See also Linguistics Aristotle , 53 Attention : in problem solving ( of child ) , 35 - 36 ; role of signs in , 40 ; development of , 57 ; voluntary and involuntary « ( ' secondary , " primary " ) , 64 153 Index 154 Baldwin , ] . M . , 106 Behavior : beginning of study of , 3 - 4 ; Marxist approach to , 4 - 5 ; Vygotsky and unified science of , ~ ; animal vs . human , 6 , 23 , 25 , 124 ( see also Ani mals ) ; " developmental " approach to , 7 , 8 , 12 , 19 - 20 , 21 , 30 , 33 , 61 , 62 , 65 ; history of , 8 , 46 , 64 - 65 ; " botanical " vs . " zoological " models of , 19 - 20 , 21 ; speech and , 26 - 30 ( see also Linguis tics ) ; and voluntary activity , 37 , 90 ; outside control of ( extrinsic stimuli ) , 40 , 51 ; " higher , " 52 , 55 , 61 , 64 , 75 ; naturalistic approach to , 60 - 61 , 122 , 132 , 139nn . 2 , 3 ; " Fossilized , " 63 - 65 , 68 ( see also Analysis ) . See also Choice behavior and responses ; Medi ation concept ; Play ; Problem solving ; Stimulus - response theories Bekhterev , V . M . , 1 , 58 Berg , E . E . , 132 Binet , A . , 32 , 80 Birch , H . C . , 125 Blonsky , P . P . , 8 , 65 Botany : vs . zoology in child studies , 19 - 20 , 21 . See also Child development Buhler , C . , 21 Buhler , K . , 20 - 21 , 23 , 112 Burt , C . , 117 Capital ( Marx ) , 8 Cattell , ] . M . , 66 Chelpanov , C . 1 . , 5 ; The Mind of Man , 4 Child development : and " preformation " of intellectual functions , 6 , 24 ; vs . " developmental ' approach , 7 ( see also Behavior ) ; and commtinication , 12 , 24 , 27 , 28 , 32 , 56 , 89 , 90 , 108 ( see also Gestures ) ; and graphic sym bolism , 13 , 112 , 115 - 116 ( see also Drawing ; Symbolism ) ; botanical vs . zoological models of , 19 - 20 , 21 ; ani mal behavior and intelligence com pared to , 20 - - 21 , 23 , 25 , 26 , 28 - - 29 , 31 , 34 , 35 , 36 , 37 ; inter - and intra personal , 27 , 57 , 131 ; attention in , 35 - 36 , 40 , 57 ; history and historic study of , 46 , 64 ; as dialectical process , 73 - 75 ; relationship of learning to , 79 - 91 , 124 - 125 , 129 - 130 ; and devel opmentallevels , zone of proximal development , 84 - 91 , 102 , 130 , 131 ; mental age in , 85 - 88 , 89 ( see also Age ) ; maturing of needs in , 92 ( see also Maturation ) ; role of play in , 92 104 , 116 , 118 , 123 , 126 , 129 - 130 ( see also Play ) ; imagination in , 93 - 99 ( passim ) , 103 , 104 , 126 ; and situa tional constaints , 96 , 98 - - 99 . See also Choice behavior and responses ; Deaf ness ; Experimentation ; Linguistics ; Memory ; Perception ; Problem solv ing ; Retardation ; Signs , use of ; Thought ; Tools , use of ; Writing Choice behavior and responses : of chil dren , 33 - 37 , 70 - 72 , 101 ; and decision making , 52 ; psychology of , 65 - 69 ; causal - dynamic study of , 69 - 72 . See also Problem solving Color : in sign - operation experimenta tion , 40 - - 45 Communication ( by children ) : and " egocentric speech , " 12 , 27 ; Piaget and , 24 , 90 ; language and , 28 , 89 ( see also Linguistics ) ; gestures and , 32 , 108 , 111 ( see also Gestures ) . See also Drawing ; Writing Communist Party , Central Committee of , 10 Conditioning , see Reflexes Counting systems , 127 . See also Culture " Crisis in psychology , " 5 Culture : historical investigations of , 3 ; Vygotsky ' s theory of , 6 , 7 ; and cul tural development , 27 , 57 , 131 ; in fancy and , 46 ; written language and , 106 ( see also Linguistics ; Writing ) ; reading and , 116 ; counting systems and , 127 Darwin , C . R . , 4 ; Origin of Species , 2 - 3 Deafness , 12 , 37 , 105 Decision making , see Choice behavior and responses ; Problem solving Descartes , Rene , 2 Development , child , see Child development Developmental approach , see Behavior Dewey , J . , 53 Dialectical materialism , 6 , 8 , 94 , 120 , 123 ( see also Marx , K . ) ; and dialecti cal method / process , 46 , 60 , 65 , 73 , 121 , 122 , 126 . See also Engels , F . Dialectics of Nature , The ( Engels ) , 132 Drawing : children ' s speech and , 28 , 112 ; as sign - using activity , 38 , 108 Drawing - continued ( see also Signs , use of ) ; gestures as precursors of , 107 - 108 ( see also Ges tures ) ; " x - ray , " 112 ; development of symbolism in , 112 - 114 ; as object ( vs . representation of object ) , 113 ; impact of speech on , 113 - 114 ; as preliminary to writing , 113 - 114 , 116 , 118 ( see also Writing ) Education : Vygotsky ' s concern for , 9 - 10 , 129 - 131 ; and " pedology " ( " ed ucational psychology " ) , 10 ; and child development - learning relationship , 79 - 91 , 124 - 125 , 129 - 130 ; premature , 80 ; James ' s description of , 80 - 81 ; formal discipline in , 81 - 84 , 91 ; and Gestalt theory of learning process , 83 ; preschool vs . school , 84 ; imitation in learning , 84 , 87 - 88 ( see also Imita tion ) ; and testing , 88 - 89 ( see also Tests and testing ) ; of retarded chil dren , 89 ; and internalization of learning processes , 90 - 91 , 130 , 131 , 132 ; and teaching of spoken language , 105 ; and teaching of writing , reading , 105 ~ 115 - 119 ; compulsory , in Eng land , 117 ; public , in U . S . , 130 ; of " slow learners " ( language and ) , 131 ; Third World literacy campaigns , 131 Eidetic imagery , see Memory Empiricism , 2 , 54 Engels , F . , 7 , 60 - 61 , 633 ; Ludwig F euerbach ( quoted ) . 120 ; The Dia lectics of Nature , 132 England , 2 , 117 Europe : psychological studies in , 1 , 4 , 9 ; IQ tests in , 10 ; teaching in , 117 Experimentation : Vygotsky and , 11 - 14 ; on child development , 12 - 13 , 60 , 73 - 74 , 97 ; Kohler ' s , with apes , 20 - 23 ( passim ) , 25 , 28 , 31 , 37 , 88 ; and child - animal comparisons , 20 - - 23 , 25 , 31 ( see also Child development ) ; and speech , 27 ; and choice reaction , 33 - 37 , 70 - 72 ( see also Choice be havior and responses ) ; and early sign operations , 40 - 45 , 46 - 49 ( see also Signs , use of ) ; and experimentaI developmental approach , ·51 ; and stimulus - response framework , 58 - 61 , 69 - 72 ; problems of method in , 58 - 75 ; procedure in , 67 ; and reaction time experiments , 72 , 137n , 8 ; Piaget ' s methodology of , 80 ; and object writ ing / symbolic notation , 109 - 111 , 114 , 115 , 116 ( see also Writing ) . See also Methodology Fechner , G . , 3 ; Die Psuchophusik , 2 Flavell , J . , 13 France : and French sociologists , 6 Freire , P . , 131 Game ( s ) : color - testing , 40 - 45 ; sporting , 92 , 103 ; with rules , 95 - 96 , 99 - 100 , 103 , 104 , 129 ; symbolism in , 108 , 109 , 110 , 111 . See also Play Gelb , A . , 97 Gerke , E . D . , 22 , 24 Gesell , A . , 19 Gestalt psychology , 1 , 4 , 5 , 83 Gestures : as communication , 32 , 108 , 111 ; pointing , 56 , 128 ; as precursors of drawing / writing , 107 - 108 , 110 , 115 ; symbolic , 108 , 109 , 110 Goethe , J . , 98 Goldstein , K . , 97 Gomel ( USSR ) : Teacher Training Institute at , 15 Guillaume , P . , 22 , 23 Cussow , J . D . , 125 Handicaps , see Deafness ; Retardation Hegel , G . W . F . , 19 , 54 Hertzen Pedagogical Institute ( Lenin grad , 15 Hetzer , H " 110 , 111 , 113 , 116 , 117 Historical materialism , 6 , 7 . See also Dialectical materialism Imagination : the child and , 93 - 94 , 97 - 99 , 103 , 104 , 126 , 129 ; and games with rules , 95 - 96 Imitation : of social experience , 22 , 129 ; in learning , 84 , 87 - 88 . See also Memory Institute of Defectology ( Moscow ) , 9 , 10 , 15 Institute of Preschool Education , 10 Institute of Psychology ( Moscow ) , 4 , 5 , 10 , 15 Intelligence : tests ( IQ ) , 10 ( see also Tests and testing ) ; child - ape analogy , 20 - 23 ( see also Child development ) ; as " technical thinking , " 21 ( see also Index 155 Index 156 Intelligence - - continued Thought , thinking and ) ; and speech , 21 - 24 ( see also Linguistics ) ; percep tion and , 35 ( see also Perception ) ; sign - using activity and , 57 ( see also Signs , use of ) ; primates and , 88 Intention : in psychological system , 37 " Interactionist " approach , 12 ~ 124 , 125 Internalization : of visual field , 26 ; of child ' s social speech , 27 , 89 - 90 , 126 , 128 ( see also Linguistics ) ; in memory , 45 ; pointing as illustration of , 56 ( see also Gestures ) ; process of , 56 - 57 , 127 ; of learning processes , 90 - 91 , 130 , 131 , 132 , Introspective psychology : experimental methods of , 59 - 60 , 62 . See also Analysis Jaensch , E . B . , 39 James , W . , 1 , 80 - 81 Kant , I . , 2 Kharkov , 10 , 15 Koffka , K . , 1 , 35 , 61 , 81 , 83 , 84 , 102 , 122 Kohler , W . , 1 , 4 , 35 ; experiments with apes , 20 - 23 ( passim ) , 25 , 28 , 31 , 37 , 88 Kornilov , K . N . , 4 - 5 , 10 Labeling , 32 , 33 . See also Linguistics Language , see Linguistics Leakey family , 133 Learning , see Education ; Intelligence ; Memory ; Stimulus - response theories ; Thought , thinking and Leningrad : Hertzen Pedagogical Insti - tute in , 15 Leontiev , A . N . , 10 , 40 , 48 , 130 , 139n . 3 Levina , R . E . , 25 , 27 , 29 Levy - Bruhl , L . , 9 Lewin , K . , 1 , 37 , 62 , 96 Lindner , R . , 37 Linguistics : and language as social process , 6 , 126 ; Soviet , 9 ; and " ego centric " speech , 12 , ~ 4 , 25 , 27 , 57 , 62 , 111 ; and stimulus - response theories applied to language , 13 ; and intel1i gence - speech relationship , 21 - 23 , 24 ; and speech - tool use relationship , 22 , 23 - - 24 " 31 , 53 ; and speech - action re lationship , 22 , 25 - 30 , 36 , 111 , 112 , 113 , 126 ; animals and , 23 , 136n . 7 ; and " planful " speech , 25 - 29 ; and language as problem - solving tool , 21 , 33 ; and internalization of speech , 27 , 89 - 90 , 126 , 128 ; and verbalized per ception , 32 , 33 ; and child ' s develop ment of speech , 46 , 84 , 112 , 118 ; and word meanings for children , 50 , 90 , 98 - 99 ; and phenotypical speech , 62 ; and translation process , 66 ; and teaching of language , 105 ( see also Education ) ; and other sign systems , 113 - 114 ; and speech as sign usage , 113 , 126 ; and language as auxiliary stimulus , 123 ; language and learning , 131 . See also Culture ; Writing Locke , J . , 2 Ludwig Feuerbacb ( Engels ) , 120 Luria , A . H . , 10 , 16 , 114 , 124 , 125 , 130 , 139n . 3 ; Studies in the History of Behavior , 9 Marx , K . : and theory of human intellec tual functioning , I ; and role of Marx ism in psychology , 4 - 5 , 6 , 8 , 10 , 126 ; and historical materialism , 6 , 7 ; Capital , 8 ; quoted , 54 , 63 , 94 Maturation : as precondition of learning , 6 , 80 , 81 ; botanical vs . zoological analogies of , 19 - 20 , 21 ; and zone of proximal development , 86 - 87 ( see also Child development ) ; and matur ing of needs in child development , 92 . See also Education McCarthy , D . , 87 Mediation concept , 7 , 54 - 55 ; and " medi ated " behavior , 13 - 14 , 26 , 32 , 45 , 55 , 127 , 128 , 139n . 3 ; and nonmediated vs . mediated memory , 38 - - 39 , 40 , 45 , 46 - 50 , 70 - 72 , 125 , 127 ( see also Memory ) Memory : in child development , 13 , 36 , 40 - 51 , 56 - 57 , 70 - 72 , 103 , 125 , 126 , 139n . 3 ; mnemonic activity and , 38 , 48 , 115 ; natural ( nonmediated ) vs . use of signs ( mediated ) , 38 - 39 , 40 , 45 , 46 - 50 , 7Q - 72 , 125 , 127 ; aids to , 39 , 42 , 47 , 48 - 49 , 51 , 52 , 53 , 72 , 74 , 114 - 115 , 127 ( see also Signs , use of ) ; eidetic imagery and , 39 , 48 , 56 , 1390 . 3 ; human vs . animal , 39 , 51 , 127 ; and internalization , 45 ; age and , 47 - 49 , 50 , 56 , 139n . 3 ; and thinking , 49 - - 51 ( see also Thought , thinking Memory - continued and ) ; " logicalization " of , 51 ; civiliza tion and , 51 ; and choice reactions , 69 - 72 ( see also Choice behavior and responses ) ; and child ' s drawings , 112 ( see also Imitation ) Methodology , 12 , 58 - 75 ; and " quotation method " ( Marxism and ) , 8 ; and search for method , 65 , 74 ; stimulus response , 74 ( see also Stimulus response theories ) ; and functional method of double stimulation , 74 - 75 ; learning - development , 79 ( see also Education ) ; of Piaget , 80 ; of Vygotsky , 123 , 128 Meyerson , I . , 22 , 23 Mind of Man , The ( Chelpanov ) , 4 Mnemonic activity , see Memory Montessori , M . , 117 , 118 Morozova , N . G . , 46 Moscow : Institute of Psychology , 4 , 5 , 10 , 15 ; Institute of Defectology , 9 , 10 , 15 ; State Pedagogical Institute , 15 Moscow University , 10 , 15 Naming : ( by child ) of drawings , con structions , 28 , 111 , 112 , 113 . See also Linguistics Naturalism , see Behavior October Revolution , 130 Origin of Species ( Darwin ) , 2 Papuans , 127 Pavlov , I . P . , 1 , 3 - 4 , 52 , 59 Pedagogical Psychology , 15 Pedagogy , see Education " Pedology , " 10 . See also Education Perception : and problem solving , 26 , 31 , 34 - 35 ; syncretism in , 29 ; human vs . animal , 31 , 32 - 33 , 34 , 35 , 36 , 37 ; age and , 31 - 32 , 33 , 97 , 98 ; verbalized ( vs . " natural " ) , 32 ; role of language in , 33 , 126 ; " center of gravity " of , 35 , 36 ; as stimulus to activity , 96 ; fusion of , with meaning , 97 ; and children ' s drawings , 112 Peruvian Indians , 137 ~ . 2 Piaget , J . , 24 , 27 , 80 , 89 - 90 , 100 , 122 125 ( passim ) Planning , 129 ; and children ' s " planful " speech , 2 ~ 29 . See also Problem solving Play : and color - testing game , 40 - 45 ( see also Tests and testing ) ; vs . sporting games , 92 , 103 ; child ' s need for , 92 - 95 ; age and , 92 - 94 , 102 , 104 , Ill , 129 ; imagination and , 93 - 96 , 103 , 104 , 126 , 129 ; symbolism and , 94 , 98 , 100 , 108 - 112 ; rule - based , 94 - 96 , 99 100 , 103 , 104 , 129 ; action and mean - . ing in , 96 - 101 ; and relation to development , 101 - 104 , 116 , 118 , 123 , 126 , 129 - 130 Pointing , see Gestures Potebnya , A . A . , 33 Problem solving : Vygotsky ' s studies of , 2 ; role of speech / language in , 22 , 25 - 30 , 33 , 36 , 126 ; perception and , 26 , 31 , 35 ; by primates , 31 , 88 ; attention and , 35 - - 36 ; motives / quasi - needs in , 37 ; neutral stimuli in , 75 ; and mental age , 86 ; zone of proximal development and , 86 , 131 . See also Choice behavior and responses ; Tests and testing ; Zone of proximal development Psychology of Art , The ( Vygotsky } , 15 Psychophysik , Die ( Fechner ) , 2 Reactions , complex : vs . reflexes , 68 ; study of , 69 ; and reaction time , 72 , 137n . 8 . See also Choice behavior and responses " Reactology , " 5 . See also Behavior Reading : teaching of , 105 , 117 , 118 ; of symbolic notation , 109 , 114 - 115 ; and cultural development , 116 . See also Education ; Writing Reflexes : conditioned , 3 - 4 , 52 , 124 ; un conditioned / Ynatural , " 68 , 124 , 139n . 3 ; and learning process , 80 ( see also Education ) Reflexes of the Brain ( Sechenov ) , 2 Relationship ( s ) : intelligence - speech , 21 - 23 , 24 ; speech - tool use , 22 , 23 - 24 , 31 , 53 ; speech - action , 22 , 27 - 30 , 36 , 111 ; of language functions ( egocen tric , social ) , 27 ; of sign use and tool use , 52 - 55 ; nature - human behavior , 61 ; child development - learning , 79 91 , 12 ~ 125 ; action - meaning , 100 101 ; individual - society , 126 Retardation , 9 ; and memory aids , 49 ; and abstract thought , 89 Russia , prerevolutionary , 3 , 4 . See also Soviet Union Russian Revolution , 1 , 130 Index 157 Index 158 Sapir , E . , 9 Sechenov , I . M . , 3 , 4 ; Reflexes of the Brain , 2 Shapiro , S . A . , 22 , 24 Signs , use of : vs . practical activity , 23 , 24 ; animals and , 23 , 39 ; by children , 28 , 38 , 55 , 56 , 72 , 108 , 124 , 126 , 139n . 3 ; and social origin of signs , 38 - 39 ( see also Writing ) ; and mem ory , 39 , 72 ( see also Memory ) ; and structure of operations ( " S - R " for mula ) , 39 - 40 ; early stages of , 40 - 45 ; natural history of , 45 - 49 ; and signali zation / signification , 52 , 74 ; as psycho logical tool , 52 - 55 , 127 ; speech as basis of , 113 , 126 . See also Drawing ; Gestures Situational constraints , 96 , 98 - 99 . See also Age Soviet Academy of Pedagogical Sciences , 10 Soviet Union : psychology in , 1 , 4 , 5 , ~ 9 ; Marxism and , 6 , 8 ( see also Marx , K . ) ; linguistics in , 9 ; and halt of testing and publications , 10 ; teaching of writing in , 116 Speech , see Linguistics Spinoza , B . , 99 Stem , W . , 23 , 28 , 32 , 62 Stimulus - response theories , 1 , 4 ; Vygot - sky and , 6 , 13 - 14 ; and u . s . learning theory ( 1930s ) , 13 ; diagrams / for mulae of , 66 ; and framework of exper iments , 58 - - 61 , 69 - 72 , 139n . 2 ; and double stimulation , 74 . See also Behavior Studies in the History of Behavior ( Vy - gotsky and Luria ) , 9 Stumpf , K . , 19 Sully , J . , 94 , 95 , 112 , 113 Symbolism : graphic , child ' s understand ing of , 13 , 112 , 115 - 116 ; and symbolic activity in tool use vs . sign use , 23 , 24 ; and symbolic representation of purposeful action , 37 ; in memory aids , 48 , 115 ( see aha Memory ) ; in child ' s play , 94 , 98 , 100 , 108 - 112 ; and written / pictorial language , 106 , 107 , 116 , 117 ; of gestures , 108 , 109 , 110 ; and symbolic notation , 109 - 112 , 114 115 ; development of , in drawing , 112 - 114 ( see also Drawing ; Writing ) Teacher Training Institute ( Cornel ) , 15 Teaching , see Education " Technical thinking , " 21 . See also Intel ligence ; Thought , thinking and Tests and testing , 130 ; IQ , 10 ; psycho logical , halted in SovietUnion , 10 ; color - task , 40 - 45 ; of mental age , 85 - 86 , 88 , 89 . See also Education ; Experimentation ; Problem solving Thorndike , E . L . , 82 , 83 , 122 Thought , thinking and : social origin of , 6 ; " technical : ' 21 ( see also Intelli gence ) ; age and , 21 - 22 , 23 , 24 , 51 , 79 - 80 , 97 ; preceding speech , 21 - 22 ; and language , interdependence be tween , 33 ; memory and , 49 - 51 ( see also Memory ) ; learning and , 83 ; graphic , primates and , 88 ; abstract , 89 , 129 ; child development and , 89 - 90 , 126 ; and action vs . meaning , 100 ; imagination and , 103 , 129 ( see also Imagination ) Thought and Language ( Vygotsky ) , 126 , 128 Thurnwald , R . , 9 Titchener , E . , 64 , 66 , 67 , 68 Tools , use of , 7 , 132 - 133 ; by child , 20 - - 26 ( passim ) , 28 , 30 , 31 , 46 , 55 , 123 , 124 , 126 ; by ape , 22 , 23 , 24 , 88 ; relationship of , to speech , 22 , 23 - 24 , 31 , 53 ; and language as problem - solv ing tool , 27 , 33 ; and sign use as psychological tool , 52 - 55 , 127 Ukrainian Psychoneurological Academy , 16 United States : behavior studies begun in , 3 ; linguistic studies in , 9 ; IQ tests in , 10 ; experimental methodology in , 11 ; and stimulus - response theories of learning , 13 ; teaching of reading and writing in , 117 ; public education in , 130 Yerask ( literary journal ) , 15 Voluntary activity : defined , 37 . See also Behavior Vygotsky , Lev S . : early life , career of , 1 , 4 , 9 , 15 - 16 ; and Marxist thought , I , 6 - 8 , 10 ; as founder of unified be havioral science , ~ ; influences on , 9 , Vygotsky - continued 122 , 132 ; and concern for education , 9 - 10 , 129 - 131 ; laboratories and stu dents of , 10 , 11 , 13 , 15 , 133 ; experi mental method of and studies by , 11 - 14 , 123 - 133 ; historical - cultural approach of , 131 - 133 ; writings of : Studies in the History of Behavior ( co - author ) , 9 ; The Psychology of Art , 15 ; Thought and Language , 126 , 128 Washburn , S . , 133 Watson , ] . B . , 1 , 5 , 58 , 128 Werner , H . , 12 , 50 , 61 Wertheimer , M . , 1 , 4 Whorf , B . L . , 9 Woodsworth , R . S . , 82 Writing : as sign - using activity , 38 ( see also Signs , use of ) ; teaching of , 105 , 11 ~ 118 ; prehistory of , 10 ~ 118 ; symbolism and , 106 , 107 , 109 - 112 , 114 - 116 , 117 ; gestures and , 107 - 108 , 11 0 ~ 115 ( see also Gestures ) ; naming process and , 111 ; drawing as prelim inary to , 113 - 114 , 116 , 118 ( see also Drawing ) ; dominance of speech over , 114Wundt , W . , 1 , 3 , 58 , 68 ; and behavior ism , 4 , 5 ; and stimulus - response framework , 59 - 60 Wurth ( and studies of pictorial writing } , 107 Wiirzburg school , 58 Yussevich , U . C . , 48 Zankov , L . V . , 47 , 48 Zaporozhets , A . V . , 10 Zone of proximal development , 84 - 91 , 102 , 130 , 131 ; defined , 86 Zoology : and zoological models in be havior studies , 19 - 20 , 21 . See also Animals ; Child development Index 159 \ No newline at end of file diff --git a/silver_data/4e739b29e371df29c4d20d35005e51992cd1ed7f.pdf.txt b/silver_data/4e739b29e371df29c4d20d35005e51992cd1ed7f.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..5d3b03b4b86b0e102f08844e0bf5db8763ef57f7 --- /dev/null +++ b/silver_data/4e739b29e371df29c4d20d35005e51992cd1ed7f.pdf.txt @@ -0,0 +1 @@ +DOI : 10 . 1126 / science . 1140324 , 1753 ( 2007 ) ; 316 Science et al . Chris Bakal , Signaling Networks Regulating Cell Morphology Quantitative Morphological Signatures Define Local www . sciencemag . org ( this information is current as of November 2 , 2007 ) : The following resources related to this article are available online at http : / / www . sciencemag . org / cgi / content / full / 316 / 5832 / 1753 version of this article at : including high - resolution figures , can be found in the online Updated information and services , http : / / www . sciencemag . org / cgi / content / full / 316 / 5832 / 1753 / DC1 can be found at : Supporting Online Material found at : can be related to this article A list of selected additional articles on the Science Web sites http : / / www . sciencemag . org / cgi / content / full / 316 / 5832 / 1753 # related - content http : / / www . sciencemag . org / cgi / content / full / 316 / 5832 / 1753 # otherarticles , 13 of which can be accessed for free : cites 24 articles This article 2 article ( s ) on the ISI Web of Science . cited by This article has been http : / / www . sciencemag . org / cgi / content / full / 316 / 5832 / 1753 # otherarticles 1 articles hosted by HighWire Press ; see : cited by This article has been http : / / www . sciencemag . org / cgi / collection / cell _ biol Cell Biology : subject collections This article appears in the following http : / / www . sciencemag . org / about / permissions . dtl in whole or in part can be found at : this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS . is a Science 2007 by the American Association for the Advancement of Science ; all rights reserved . The title Copyright American Association for the Advancement of Science , 1200 New York Avenue NW , Washington , DC 20005 . ( print ISSN 0036 - 8075 ; online ISSN 1095 - 9203 ) is published weekly , except the last week in December , by the Science on N o v e m be r 2 , 2007 www . sc i en c e m ag . o r g D o w n l oaded f r o m Quantitative Morphological Signatures Define Local Signaling Networks Regulating Cell Morphology Chris Bakal , 1 , 2 , 3 * † John Aach , 1 * George Church , 1 Norbert Perrimon 1 , 2 Although classical genetic and biochemical approaches have identified hundreds of proteins that function in the dynamic remodeling of cell shape in response to upstream signals , there is currently little systems - level understanding of the organization and composition of signaling networks that regulate cell morphology . We have developed quantitative morphological profiling methods to systematically investigate the role of individual genes in the regulation of cell morphology in a fast , robust , and cost - efficient manner . We analyzed a compendium of quantitative morphological signatures and described the existence of local signaling networks that act to regulate cell protrusion , adhesion , and tension . M orphogenesis commonly relies on the spatial and temporal regulation of distinct groups of genes acting in local signaling networks . The morphology of a single cell also results from the spatio - temporally regulated activity of signaling proteins . For example , Rac - type guanosine triphosphatases ( GTPases ) promote the for - mation of protrusive lamellipodia at the lead - ing edge of motile cells , whereas Rho - type GTPases promote cortical tension and cell re - traction at the rear of the cell through the activation of the actomyosin machinery ( 1 ) . Both protrusive activity and cell body retrac - tion are tightly coupled to the assembly and disassembly of adhesive structures through Rho signaling ( 2 ) . Many signaling proteins must act both upstream and downstream of specific Rho GTPases in spatially distinct subcellular local networks to translate extra - cellular signals to changes in GTPase activa - tion and ultimately in cellular morphology . However , the components of these networks and the precise role they play in regulating cell shape remain largely unclear . We performed a genetic screen of 249 gene - overexpressionordouble - strandedRNA ( dsRNA ) treatment conditions ( TCs ) using the Drosophila BG - 2 cell line to determine the roles of genes actingin localnetworks to controldistinct aspects of cell morphology . BG - 2 cells are highly motile and exhibit many of the traits observed in mam - malian fibroblasts and epithelial cells , including the formation of integrin - based adhesions , polar - ized lamellipodia , and coordinated retraction of the cell body ( 3 , 4 ) ; but unlike many mammalian cell types , the growth of BG - 2 cells is not inhibited by contact with other cells . To analyze the morphologies of cells in each TC , we used protocol and image - processing techniques de - signed to detect clear and complete boundaries of individual cells and to quantitatively analyze the shapes of these boundaries along with the in - tensities and textures of their interiors . First , we stochastically labeled samples with green fluo - rescent protein ( GFP ) [ supporting online material ( SOM ) ] to enable individual cells to stand out in the crowded and overgrown samples , acquired images of these cells using conventional fluores - cence microscopy , and used software to identify the boundaries of individual cells ( Fig . 1A and SOM ) . Although these techniques make lower numbers of cells per sample available to sub - sequent analysis as compared to other published methods ( 5 , 6 ) , we preferred them because they yielded detailed , high - quality empirical bounda - ries instead of algorithmically determined approx - imations . For each individual cell , we computed 145 different quantitative features that reflected basic aspects of cell geometry , detailed aspects of cellular protrusions , or the distribution of GFP intensity within the cellular boundaries ( Fig . 1B and SOM ) . Altogether we analyzed 12 , 601 individual cells from our 249 TCs . To transform our 145 features into biologi - cally meaningful morphological indicators , we trained a set of neural networks ( NNs ) to use informative subsets of the features to discriminate cells from particular reference TCs from sets of other reference TCs ( Fig . 1D and SOM ) . We targeted seven TCs for NN training because they produced phenotypes that were qualitatively dis - tinctive and discernable from control cells ( SOM ) . For example , overexpression of an N - terminally truncated form of the Rho guanine nucleotide exchange factor ( RhoGEF ) SIF ( D N - SIF ) , the Drosophila ortholog of mammalian Tiam - 1 , stimulates extensive lamellipodia formation , cell spreading , and a general loss of tension , dem - onstrated by the flat and thin appearance of the D N - SIF cells ( Fig . 2 ) ( 7 ) . After training a D N - SIF NN to distinguish D N - SIF cells from the cells of our six other target TCs , we applied this NN to all 12 , 601 cells in our data set to score each of them for this distinctive morphology ( Fig . 1D ) . In ad - dition to D N - SIF , we trained NNs for TCs treated with overexpression constructs for RacV12 , RacF28L , RhoV14 , RhoF30L , CG3799 full - length , and D N - RhoGEF3 . The seven TCs selected for NN training included several for which the underlying mechanisms responsible for their phenotypes are not understood . Finally , for each NN and TC , we calculated a NN Z score ( NNZ ) , which is the variance - adjusted difference between the mean NN score of all cells in the TC and the mean NN score of all cells in our data set . Each NNZ is thus an index of the morphology of an entire TC ( SOM ) ( Fig . 1E ) . For example , for cells in which CG10188 ( a RhoGEF ) was targeted by dsRNA , the NNZ for the D N - SIF NN was 0 . 76 , which indicates that CG10188 dsRNA induces a mor - phology that is slightly more “ D N - SIF – like ” than an equal number of randomly chosen cells ( which would have a NNZ of 0 ) . In contrast , the NNZ for D N - SIF cells using the D N - SIF classi - fier is 26 . 77 . Together , the seven NNZs com - puted for each TC constituted a quantitative morphological signature ( QMS ) of the TC ( Fig . 1E ) . A QMS is thus a high - order represen - tation of the morphology of cells in a TC as a vector of seven specific quantitative similarities and dissimilarities with seven panels of refer - ence cells with distinctive phenotypes . Two - dimensional hierarchical clustering ( SOM ) of 249 QMSs revealed that TCs , and in particular RNA interference ( RNAi ) against indi - vidual genes , fell into several distinct clusters . QMSs with similar qualitative phenotypes clus - tered tightly together ( Fig . 2 ) . We define “ pheno - clusters ” as genes grouped at the highest node in the clustering for which the cluster distance metric ( an average of uncentered Pearson corre - lation coefficients ) was greater than 0 . 80 and term this a “ cluster distance cutoff ” ( CDC ) ( Fig . 2 ) ( 8 ) . A value of 0 . 80 was chosen as the CDC because smaller cutoffs resulted in groupings of visually diverse morphologies , whereas higher thresholds resulted in the segregation of visually similar morphologies into distinct clusters . A large phenocluster was composed of TCs that clustered because their QMSs have high D N - RhoGEF3 NNZs ( cluster 6 ) . All cells in this cluster were extremely roundand had very fewor no protrusions of any type ( Fig . 2 and fig . S18 ) . QMSs for p190RhoGAP , SCAR , slingshot , arma - dillo , ankyrin , Sop2 , and RhoGEF3 RNAi were clustered together . Moreover , we observed an enrichment in this cluster for RNAi against genes involved in Rap signaling ( three of six genes in the data set ) . The finding that depletion of either SCAR , the cofilin phosphatase Slingshot , or Sop2 resulted in defects in protrusion is consist - ent with the known function of these three pro - teins ( 4 , 9 – 14 ) . Gef26 and its downstream target Rap1 function in the formation of adherence junctions in Drosophila ( 15 – 17 ) and have recently been observed to be required for cell spreading and migration of Drosophila macrophages ( 18 ) . In mammalian systems , p190RhoGAP acts down - stream of integrins and adhesions to promote cell 1 Department of Genetics , Harvard Medical School , Boston , MA 02115 , USA . 2 Howard Hughes Medical Institute , Boston , MA 02215 , USA . 3 MIT Computer Science and Technology Laboratory , Cambridge , MA 02139 , USA . * These authors contributed equally to this work . † To whom correspondence should be addressed . www . sciencemag . org SCIENCE VOL 316 22 JUNE 2007 1753 REPORTS on N o v e m be r 2 , 2007 www . sc i en c e m ag . o r g D o w n l oaded f r o m spreading ( 19 ) . Thus , our method successfully identified two distinct but coupled signaling path - ways that regulate the formation of protrusions . We anticipate that Drosophila RhoGEF3 plays a critical role in the regulation of adhesion , because overexpression of a N - terminally deleted form or inhibition by dsRNA have similar QMSs . D N - RhoGEF3 is not likely to be constitutively activated ( 20 ) but may promote cell rounding by acting in a dominant - negative manner toward endogenous RhoGEF3 signaling . A second phenocluster contained a group of TCs that co - clustered with RhoF30L and had high RhoF30L NNZs ( cluster 8 ) . These cells dif - fered qualitatively from cells in the D N - RhoGEF3 phenocluster by virtue of the fact that cells in this cluster did have some visible , but poorly formed , lamellipodial protrusions ( Fig . 2 and fig . S18 ) . dsRNAs in this cluster may target genes that specifically promote the formation of lamellipo - dia , and RhoF30L ( an activated form of Rho that cycles between GTP - and GDP - bound states ) may inhibit this process . In support of this notion , dsRNAs targeting twinstar , capt , and ARC - p20 were part of this cluster , representing an enrich - ment in genes that have been identified in pre - vious screens for genes required for lamellipodia organization ( three of seven genes ) ( 10 ) . Given the fact that lamellipodia formation occurs after the formation of adhesion , protrusion , and actin - filament nucleation , this suggests that phenotypic profiling can not only simultaneously monitor the activity of coupled signaling pathways ( within phenoclusters ) but can also monitor the temporal hierarchical relationships that exist among local signaling networks . The largest phenocluster was a group of TCs that shared high D N - SIF , RacV12 , and RacF28L NNZs ( cluster 18 ) and that corresponded to large flat cells , typically with extensive lamellipodia ( Fig . 2 and fig . S18 ) . This phenotype is consistent with repeated observations of cells overexpress - ing activated Rac mutant proteins or activated RacGEFs such as SIF or Tiam - 1 ( 7 , 21 ) . QMSs for cenG1A , cenB1A , CG16728 , and CG13692 RNAi were members of this phenocluster , rep - resenting an enrichment in ArfGAPs ( four of six genes ) . Mammalian ArfGAPs such as GIT1 and Fig . 1 . Phenotypic pro - filing workflow . ( A ) Cul - tured Drosophila BG - 2 cellsweretransfectedwithplasmidsencodingGFPandeithercotransfectedwithplasmidsencodingredfluorescentprotein – tagged proteins or incu - bated in the presence of dsRNA for 4 days . Images of GFP - labeled cells were acquired by standard fluo - rescence microscopy , and individualcellimageswithclearandcompletebound - aries were selected with custom - developed software ( SOM ) . ( B ) Graphical rep - resentations of some of the features computed from each individual cell image ( SOM ) . ( C ) 145 different features relevant to cell morphology and GFP signal intensity were derived from individual cells and expressed as Z scores relative to their values over a subset of ∆ N - RhoGEF3 Classifier Phenotypes RhoF30L RacV12 RacF28L ∆ N - SIF RhoV14 CG3799 0 . 00 0 . 00 0 . 00 0 . 01 . . . 0 . 08 0 . 14 0 . 05 0 . 15 . . . 0 . 32 0 . 05 0 . 08 0 . 10 . . . 0 . 01 0 . 18 0 . 04 0 . 00 . . . 0 . 67 0 . 38 0 . 76 0 . 47 . . . 0 . 03 0 . 45 0 . 18 0 . 18 . . . 0 . 00 0 . 99 0 . 00 0 . 00 . . . QMS - 2 . 1 - 5 . 1 11 . 5 3 . 9 26 . 8 - 6 . 9 - 2 . 8 N o r m a li z ed f ea t u r e v a l ue s 145 features 1 4 3 2 S i ng l e - C e l l NN S c o r e NN Z - scores for TC 1 2 3 4 n cells Cellsin TC 1 Cell Culturing + GFP + / - dsRNA + / - Gene overexpression 2 Image Acquisition ( GFP ) 3 Cell Identification 4 Feature Extraction DNAGFPF - actin GFP Raw Image Half - Mass Boundary Process Area Drainage Area A B C D E GFP control cells which were transfected only with a construct coding for constitutive expression of GFP . ( D ) Individual cells were then scored with NNs trained to discriminate seven ref - erence TCs with distinctive morphologies ( SOM ) . ( E ) QMSs were computed for each TC as seven dimensional vectors containing the NNZs of the individual cells in the TC ( SOM ) . 22 JUNE 2007 VOL 316 SCIENCE www . sciencemag . org 1754 REPORTS on N o v e m be r 2 , 2007 www . sc i en c e m ag . o r g D o w n l oaded f r o m GIT2 promote the disassembly of integrin - based focal adhesions by binding the tyrosine - phosphorylated form of Paxillin , which in turn results in ArfGTPase activation and a concomi - tant down - regulation of Rac activity and adhesion turnover ( 22 – 24 ) . In addition to ArfGAPs , this phenocluster also contained QMSs for dsRNAs targeting paxillin ( fig . S18 ) and a - actinin , and was defined by high levels of Rac activity ( Fig . 2 ) . Based on this and sequence analysis , we suggest that CG16728 is the Drosophila ortholog of mammalian GITArfGAPs . Proteomic analysis has recently revealed that GIT1 is part of a supramolecular complex that , in addition to Paxillin , includes b 2 centaurin and gelsolin and directly binds moesin ( 25 ) . Drosophila cenB1A ( the Drosophila ortholog of b 2 centaurin ) , Gelsolin , and Moesin dsRNAwere also members of same phenocluster as CG16728 and paxillin , further demonstrating that our methodology is capable of identifying both functionally and physically coupled signaling components . Taken together , this suggests that the large , flat , and spread morphology of cells in this phenocluster was partially due to inhibiting the disassembly of adhesion and that QMSs can be used to functionally annotate genes . We have demonstrated that quantitative mor - phological profiling of single cells combined with RNAi - based genetic screening technology results in the identification of local signaling networks with spatially , temporally , and func - tionally defined characteristics that act in a hierarchical manner to regulate cell shape and migration . These methods can be used not only in the context of genetic screens but also in large - scale screens of small - molecule libraries or screens involving the overexpression of cDNAs . Because our approach is a fast and cost - effective way to query the activity of multiple signaling proteins and pathways , quantitative morpholog - ical profiling may also be useful as a diagnostic tool in the analysis of clinical samples . Further - more , akin to gene - expression data , we can now use morphological phenotypic data for computa - Rac1 cluster ( 4 ) Rac1 , Rac1 / Rac2 / MTL , GEF64C overex , L amellipodia formation ( 25 ) Arc - p20 , GEF64C , Trio , Twinstar , Kelch , Rab26 , Rab35 , Rab3 - GAP , Graf , RhoGAP68F , CSN1a , RhoF30L overex MT capture ( 3 ) APC , APC2 , Dia GFP / wild - type cluster ( 24 ) Cdc42 , Cdc42 Y32A overex , CG3799 , CG3799 overex , RhoV14 overex Rho1 cluster ( 19 ) Rho1 , RhoGAP1A , RhoGAP5A , RhoGAP93B , RhoGEF2 , RhoGEF4 1 4 6 7 8 17 18 20 242526272830 31 33 37 Cluster R a c V 12 R a c F 28L G F P C G 3799 R h o1 d s R N A R a c 1 d s RN A R ho F 30L R ho F 30L R a c F 28L R a c V 12 R ho V 14 C G 3799 Actin Polymerization Cortical Tension Tail Retraction Pebble , RacGAP50C Adhesion disassembly Twinstar , Capt , Arc - p20 SCAR , Sop2 Actin filament nucleation Adehsion complex formation RapGEFs , RapGAPs Rho1 , RhoGEF2 , RhoGEF4 Cytokinesis Fig . 2 . Identificationoflocalnetworksthatregulatedistinctaspectsofmorphology . Hierarchicalclusteringofthegenesinthedataset ( the y axis ) byhowcellsscoredon the D N - SIF , D N - RhoGEF3 , CG3799 , RacF28L , RacV12 , RhoF30L , and RhoV14 NNs ( the x axis ) isshown . WedefinephenoclustersofgenesasclusterswithaCDC > 0 . 80 , which results in 41 total clusters comprising 17 multigene clusters and 24 singletons . All multigene clusters are identified in brackets on the right - hand side of the clustergram . For some clusters , we describe prominent TCs , and the number of TCswithintheseclustersisindicatedinparentheses . Examplesofindividualcellsand theirpositionsintheclustergramareshownontheleft - handsideoftheclustergram . Based on their gene membership , a number of clusters were determined to have specialized roles in cell morphology . A complete listing of clusters is provided in table S8 . Scale bars , 10 m m . www . sciencemag . org SCIENCE VOL 316 22 JUNE 2007 1755 REPORTS on N o v e m be r 2 , 2007 www . sc i en c e m ag . o r g D o w n l oaded f r o m tional approaches that aim to model the dynamic nature of signaling networks , while the RNAi component pushes us closer to causal mechanis - tic linkages . References and Notes 1 . A . Hall , Biochem . Soc . Trans . 33 , 891 ( 2005 ) . 2 . K . A . DeMali , K . Wennerberg , K . Burridge , Curr . Opin . Cell Biol . 15 , 572 ( 2003 ) . 3 . Y . Takagi , K . Ui - Tei , T . Miyake , S . Hirohashi , Neurosci . Lett . 244 , 149 ( 1998 ) . 4 . A . Biyasheva , T . Svitkina , P . Kunda , B . Baum , G . Borisy , J . Cell Sci . 117 , 837 ( 2004 ) . 5 . Z . E . Perlman et al . , Science 306 , 1194 ( 2004 ) . 6 . A . E . Carpenter et al . , Genome Biol . 7 , R100 ( 2006 ) . 7 . M . Sone et al . , Science 275 , 543 ( 1997 ) . 8 . K . C . Gunsalus et al . , Nature 436 , 861 ( 2005 ) . 9 . J . A . Zallen et al . , J . Cell Biol . 156 , 689 ( 2002 ) . 10 . S . L . Rogers , U . Wiedemann , N . Stuurman , R . D . Vale , J . Cell Biol . 162 , 1079 ( 2003 ) . 11 . P . Kunda , G . Craig , V . Dominguez , B . Baum , Curr . Biol . 13 , 1867 ( 2003 ) . 12 . N . Zebda et al . , J . Cell Biol . 151 , 1119 ( 2000 ) . 13 . M . Nishita et al . , J . Biol . Chem . 279 , 7193 ( 2004 ) . 14 . M . D . Welch , A . H . DePace , S . Verma , A . Iwamatsu , T . J . Mitchison , J . Cell Biol . 138 , 375 ( 1997 ) . 15 . A . L . Knox , N . H . Brown , Science 295 , 1285 ( 2002 ) . 16 . H . Wang et al . , Dev . Cell 10 , 117 ( 2006 ) . 17 . S . R . Singh et al . , Dev . Growth Differ . 48 , 169 ( 2006 ) . 18 . S . Huelsmann , C . Hepper , D . Marchese , C . Knoll , R . Reuter , Development 133 , 2915 ( 2006 ) . 19 . W . T . Arthur , K . Burridge , Mol . Biol . Cell 12 , 2711 ( 2001 ) . 20 . K . Murayama et al . , J . Biol . Chem . ( 26 December 2006 ) . 21 . E . E . Sander , J . P . ten Klooster , S . van Delft , R . A . van der Kammen , J . G . Collard , J . Cell Biol . 147 , 1009 ( 1999 ) . 22 . D . J . Webb et al . , Nat . Cell Biol . 6 , 154 ( 2004 ) . 23 . M . C . Brown , L . A . Cary , J . S . Jamieson , J . A . Cooper , C . E . Turner , Mol . Biol . Cell 16 , 4316 ( 2005 ) . 24 . N . Nishiya , W . B . Kiosses , J . Han , M . H . Ginsberg , Nat . Cell Biol . 7 , 343 ( 2005 ) . 25 . M . W . Mayhew et al . , J . Proteome Res . 5 , 2417 ( 2006 ) . 26 . We thank P . Bradley and A . Kiger for contributions to the stochastic labeling method , as well as B . Mathey - Prevot , A . Friedman , R . Griffin , B . Berger , S . Altschuler , the Drosophila RNAi Screening Center , and the Harvard Research Information Technology Group . This work is supported in part by grants from the National Human Genome Research Institute Centers of Excellence in Genomic Science . N . P . is an investigator of the Howard Hughes Medical Institute . C . B . is a fellow of the Leukemia and Lymphoma Society . Supporting Online Material www . sciencemag . org / cgi / content / full / 316 / 5832 / 1753 / DC1 Materials and Methods SOM Text Figs . S1 to S19 Tables S1 to S9 References 24 January 2007 ; accepted 15 May 2007 10 . 1126 / science . 1140324 Restriction of an Extinct Retrovirus by the Human TRIM5 a Antiviral Protein Shari M . Kaiser , 1 , 2 Harmit S . Malik , 3 Michael Emerman 2 , 3 * Primate genomes contain a large number of endogenous retroviruses and encode evolutionarily dynamic proteins that provide intrinsic immunity to retroviral infections . We report here the resurrection of the core protein of a 4 - million - year - old endogenous virus from the chimpanzee genome and show that the human variant of the intrinsic immune protein TRIM5 a can actively prevent infection by this virus . However , we suggest that selective changes that have occurred in the human lineage during the acquisition of resistance to this virus , and perhaps similar viruses , may have left our species more susceptible to infection by human immunodeficiency virus type 1 ( HIV - 1 ) . A large portion of primate genomes is composed of endogenous retroviruses that can be thought of as an archaeolog - ical record of past infections . Both chimpanzee and gorilla genomes harbor more than 100 copies of Pan troglodytes endogenous retrovirus ( PtERV1 ) , whereas it is absent from the human genome ( 1 ) . Comparison of individual PtERV1 proviruses in gorilla and chimpanzee genomes suggest that this virus was active 3 to 4 million years ago , after the separation of chimpanzee and human lineages ( 1 ) . This raises an evolutionary conundrum as to why sister species , but not humans , acquired germline copies of this retro - virus even though all three species cohabited when PtERV1 was an active exogenous virus ( 1 ) . One mechanism of active restriction from retroviral infections is conferred by the TRIM5 a protein , which binds directly to the incoming retroviral capsid ( CA ) core and targets its premature disassembly or destruction ( 2 , 3 ) . Each primate species encodes a TRIM5 a with a different antiviral specificity ( 4 ) . For example , TRIM5 a encoded by rhesus macaques renders them resistant to infection by HIV - 1 , but human TRIM5 a affords no such protection ( 5 ) . Indeed , the antiviral specificity of TRIM5 a has rapidly evolved by dramatic episodes of positive selec - tion during the past 30 million years of primate evolution ( 6 ) . The branch leading to the human lineage shows one of the strongest signatures of positive selection ( 6 ) , which suggests that at least one major pathogenic retroviral assailant has challenged the human lineage in the past 4 to 5 million years . Taken together , these findings suggest that TRIM5 a evolution was shaped by a species - specific history of ancestral retroviral challenges . Although human TRIM5 a has rela - tively poor activity against retroviruses compared with the gene from other primates , it potently blocks a g - retrovirus N - MLV , which is related to PtERV1 ( 7 , 8 ) . We therefore tested the hypothesis that TRIM5 a may have protected early humans from invasion by PtERV1 . All copies of PtERV1 in the chimpanzee genome have been inactivated by accumulated detrimental mutations ( 1 ) . However , the numer - ous proviral copies of PtERV1 present in the chimpanzee genome allow us to reconstruct the ancestral sequence of the gag gene of this an - cient , extinct retrovirus in silico ( see supporting online material ) . Analysis of the reconstructed PtERV1 ancestral sequence reveals about 50 % identity with murine leukemia virus ( MLV ) , and several characteristic conserved elements are in - tact ( Fig . 1A ) . Phylogenetic analysis of chim - panzee and gorilla PtERV sequences shows that a single - source virus likely infected both chimpan - zees and gorillas because viral sequences from both species form a monophyletic group ( Fig . 1B ) . We next used site - directed mutagenesis to reconstruct the ancestral p12 and CA coding regions ( ignoring synonymous changes ) starting from one chimpanzee PtERV1 provirus cloned from the genome . We focused on CA because it is the functional target of TRIM5 a and included p12 because of functional interactions that exist between p12 and CA in other g - retroviruses ( 9 ) . Because TRIM5 a interacts with the retroviral CA only in the multimeric structure characteristic of mature retroviral particles ( 10 ) , we generated the PtERV1 capsid core in the context of an infectious virus capable of only a single round of infection . This was achieved by constructing a chimeric virus between PtERV1 and MLV that encodes a gag / pol gene expressing the recon - structed p12 and CA proteins of PtERV1 with the remainder of the viral structural proteins and enzymes of MLV ( Fig . 1C ) . Our MLV / PtERV1 chimeric virus was indeed infectious ( Fig . 1D ) , which demonstrates that regions of a 3 - to 4 - million - year - old primate endogenous retrovirus can be successfully resurrected . We tested human TRIM5 a restriction of PtERV1 by infecting cells that express an exogenous copy of human TRIM5 a . A much younger humanendogenous retrovirus , HERV - K , was also recently resurrected ( 11 , 12 ) but was not restricted by human TRIM5 a ( 12 ) . In contrast , expression of human TRIM5 a in a heterologous cell type resulted in a dramatic reduction of infectivity of the MLV / PtERV1 chimera by a factor of more than 100 compared with cells that do not express TRIM5 a ( Fig . 2A ) . These data indicate that humans possess an intrinsic immu - nity gene capable of effectively neutralizing an extinct retrovirus that never successfully fixed into the human genome . Specificity of TRIM5 a for a particular retro - viral capsid is largely determined by amino acids within the C - terminal B30 . 2 domain . Within this 1 Molecular and Cellular Biology Program , University of Washington , Seattle , WA 98195 , USA . 2 Division of Human Biology , Fred Hutchinson Cancer Research Center , Seattle , WA 98109 , USA . 3 Division of Basic Sciences , Fred Hutchinson Cancer Research Center , Seattle , WA 98109 , USA . * To whom correspondence should be addressed . E - mail : memerman @ fhcrc . org 22 JUNE 2007 VOL 316 SCIENCE www . sciencemag . org 1756 REPORTS on N o v e m be r 2 , 2007 www . sc i en c e m ag . o r g D o w n l oaded f r o m \ No newline at end of file diff --git a/silver_data/503951a3e4ce4e67f8bc4fe63c161b4c9360abfd.pdf.txt b/silver_data/503951a3e4ce4e67f8bc4fe63c161b4c9360abfd.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..4f8ef2973ac8dce5cf546738915cfb4c5a5882a0 --- /dev/null +++ b/silver_data/503951a3e4ce4e67f8bc4fe63c161b4c9360abfd.pdf.txt @@ -0,0 +1 @@ +Editorials Scoring systems : Is a fix needed ? * S coring systems , starting with the Therapeutic Intervention Scoring System ( TISS ) and Acute Physiology and Chronic Health Evaluation ( APACHE ) and later with the Simplified Acute Physiology Score and Mortality Probability Models , have been used to compare patient out - comes in intensive care units ( ICUs ) all over the world . Newer avatars of now proprietary systems have demanded our attention . The institutions have to invest heavily in terms of money , personnel , and time to implement the systems . That is one of the main reasons that APACHE II , in the public domain since its appearance in 1985 , has remained the most popular system . Dr . Combes and colleagues ( 1 ) , in this issue of Critical Care Medicine , have commented on the same . The systems evaluated in large data - bases have been used for quality assur - ance purposes and for comparing mortal - ity statistics with use of the standardized mortality ratio . Shortcomings of the scoring systems in analyzing data for one or another group of patients have been pointed out . In fact , the original APAPCHE was criticized as soon it ap - peared in print by trauma surgeons as being unrepresentative of surgical pa - tients . Subsequently , it was pointed out that APACHE II underperformed in pa - tients in coronary care units , patients un - dergoing coronary artery bypass surgery , patients with cardiogenic pulmonary edema , patients with acute respiratory distress syndrome , and for patients re - quiring prolonged ICU care . For patients undergoing coronary artery bypass sur - gery , specialized scoring systems have been developed and used with success ( 2 ) . This reviewer with his colleagues had the temerity to even develop a scoring system specifically for invasively monitored pa - tients , only to be reminded by the distin - guished Joseph Civetta that we did not need “new and improved” scoring sys - tems ( 3 , 4 ) ! He commented , “The unfor - tunate implication that hospitals with high death rates deliver poor quality care has been made in numerous newspaper articles . ” Carson and Bach ( 5 ) compared four scoring systems and concluded that except for APACHE II , all vastly underes - timated mortality in patients transferred for long - term care . Over a 6 - yr period in a French tertiary care hospital , Dr . Combes and colleagues ( 1 ) identified patients who were trans - ferred from other ICUs . These patients constituted 12 % of their total admissions and accounted for 13 % higher mortality . The transferred patients seemed to con - sume more ICU resources such as me - chanical ventilation days and diagnostic tests . The authors believe that the aca - demic medical centers are likely to be “penalized” for excess mortality . They document that the patients’ transfer from another ICU as the only fact that seemed to account for excess mortality . Inciden - tally , Zimmerman et al . ( 6 ) reported mor - tality in teaching hospitals at 18 . 4 % vs . 15 . 1 % in non – teaching hospitals , figures that are lower than the current study . In the North American context , the effect of penalization could mean any of the following : 1 ) bad report cards , 2 ) postings on the Internet , 3 ) reduced hos - pital payments , 4 ) reduced physician pay - ments , or 5 ) threatened loss of insurance contracts . Dr . Combes and colleagues ( 1 ) do not clarify whether , in France , academic medical centers compete for patients and money the same way as they do in the United States . I believe it is the mission of the tertiary care centers to care for more seriously ill patients , as such higher mor - tality rates in academic centers are to be expected . The fact that it is not reflected in Simplified Acute Physiology Score II has to do with shortcomings of the mea - suring tool . Dr . Combes and colleagues ( 1 ) recog - nize that the transferred patients had re - ceived needed care in the transferring facility . If vital signs had been stabilized and laboratory abnormalities corrected at the first institution , the Simplified Acute Physiology Score measured in the trans - ferring facility would underestimate se - verity of illness . That would influence the standardized mortality ratio . APACHE III accounts for patients’ source of admis - sion—the lead time bias . The authors suggest some solutions to the problem . Ignoring these patients or assigning the mortality to the original facility is not a good suggestion . They also doubt if sec - ond - level customization would help . Per - haps all of the systems need to emulate APACHE III for including source of ad - mission . Dr . Combes and colleagues ( 1 ) re - ported ICU mortality and not hospital mortality . The latter is usually 5 – 10 % higher , even in the published French studies ( 7 ) . They also did not record do - not - resuscitate orders at the time of ICU discharge . In general , French intensivists are handicapped by antiquated ideas about euthanasia ( 8 ) . It seems that they would finally adopt some laws similar to those in North America . Many of our pa - tients who are designated do - not - resusci - tate are transferred out of the ICU for continued palliative care and are expected to die . The authors do recognize these limitations . The question then is , do the trans - ferred patients represent a flaw of the scoring systems ? Are there other limits to data analysis , some recognized and some ill - understood ? The techniques for quan - titative analysis of ICU survival and mor - tality will always have limits . Marginal improvements in 10 – 15 % misclassifica - tion rates seem to require enormous re - sources . I think that as clinicians , it is our duty to recognize these limits . Vinod K . Puri , MD Providence Hospital and Medical Center Southfield , MI REFERENCES 1 . Combes A , Luyt CE , Trouillet JL , et al : Adverse effect on a referral intensive care unit’s per - * See also p . 705 . Key Words : scoring systems ; outcomes ; Simplified Acute Physiology Score II ; Acute Physiology and Chronic Health Evaluation Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159725 . 75514 . 11 894 Crit Care Med 2005 Vol . 33 , No . 4 formance of accepting patients transferred from another intensive care unit . Crit Care Med 2005 ; 33 : 705 – 710 2 . Higgins TL , Estafanous FG , Loop FD , et al : Stratification of morbidity and mortality out - come by preoperative risk factors in coronary artery bypass patients : A clinical severity score . JAMA 1992 ; 267 : 2344 – 2348 3 . Yeung HC , Lu MW , Martinez EG , et al : Critical care scoring system : New concept based on hemo - dynamic data . Crit Care Med 1990 ; 18 : 1347 – 1352 4 . Civetta J : New and “improved scoring” scoring systems . Crit Care Med 1990 ; 18 : 1487 – 1490 5 . Carson SS , Bach PB : Predicting mortality in patients suffering from prolonged critical ill - ness : An assessment of four severity - of - illness measures . Chest 2001 ; 120 : 928 – 933 6 . Zimmerman JE , Shortell SM , Knaus WA , et al : Value and cost of teaching hospitals : A pro - spective , multicenter , inception cohort study . Crit Care Med 1993 ; 21 : 1432 – 1442 7 . Azoulay E , Adrie C , De Lassence A , et al : De - terminants of postintensive care unit mortal - ity : A prospective multicenter study . Crit Care Med 2003 ; 31 : 428 – 432 8 . Lemaire FJ : A law for end of life care in France ? Int Care Med 2004 ; 30 : 2120 Critical illness polyneuropathy and myopathy in acute respiratory distress syndrome : More common than we realize ! * F irst reported in the early 1980s , critical illness polyneuropathy and myopathy ( CIPNM ) is a neuromuscular disorder that has been described in critically ill patients , particularly among those surviving severe sepsis and multiple organ dysfunction syn - drome ( MODS ) ( 1 – 3 ) . This disorder is man - ifested by difficulty in weaning from the ventilator , severe weakness of limb mus - cles , and reduced or absent deep tendon reflexes . Electrophysiologic testing reveals an axonal polyneuropathy with sensorimo - tor involvement and occasional myopathic altered motor unit potentials ( 4 , 5 ) . Muscle atrophy or necrosis with noticeably absent inflammatory changes is evident on muscle biopsy . The majority of CIPNM patients who survive their critical illness demon - strate improvement in neuromuscular function over several weeks to months ( 2 ) . Currently , there is no specific treatment for CIPNM ; thus , knowledge of risk factors and the implementation of preventive strategies are crucial in the care of high - risk patients . The pathogenesis and pathophysiology of CIPNM are unclear but likely a conse - quence of multiple factors , including al - terations in the microcirculation , activa - tion of proinflammatory cytokines , and changes in skeletal muscle membrane ex - citability and intracellular calcium re - lease . Disorders of proteolytic and apo - ptotic pathways may also be contributory ( 2 , 6 – 8 ) . Known risk factors for CIPNM include a high Acute Physiology and Chronic Health Evaluation ( APACHE ) III score on admission to the intensive care unit ( ICU ) and the presence of the sys - temic inflammatory response syndrome ( 9 ) . Others have reported duration rather than the severity of MODS , the presence of hyperglycemia , administration of cor - ticosteroids , duration of mechanical ven - tilation , and female gender as added in - dependent risk factors ( 4 , 10 ) . On the other hand , the use of aminoglycoside antibiotics , neuromuscular blocking agents , parenteral nutrition , or renal re - placement therapy has not been consis - tently shown to be associated with CIPNM ( 10 , 11 ) . The acute respiratory distress syn - drome ( ARDS ) is a frequent complication of severe sepsis ; thus , CIPNM would be anticipated to occur commonly in pa - tients with ARDS . Herridge et al . ( 12 ) reported a high prevalence of persistent muscle weakness and fatigue in survivors of ARDS 1 yr after ICU discharge . Others have also reported on the significant re - duction of physical activity and quality of life in survivors of ARDS compared with critically ill control patients ( 13 , 14 ) . As there was no comparison or control group of non - ARDS survivors in the study by Herridge et al . ( 12 ) , the investigators were unable to fully determine whether the neuromuscular sequelae observed were specific to ARDS survivors or typical for other severely affected critically ill patients . In this issue of Critical Care Medicine , Dr . Bercker and colleagues ( 15 ) examined the risk factors that may promote the development of CIPNM in patients with ARDS . Additionally , they assessed the di - agnostic utility of early electrophysi - ologic testing . In their retrospective study , they reviewed the charts of 50 con - secutive ARDS patients admitted to an academic referral center in Germany be - tween May 1998 and November 2001 . All patients fulfilled the ARDS criteria ( as defined by the American - European Con - sensus Conference ) ( 16 ) and were man - aged with standard therapies , including low tidal volume ventilation . Thirty - eight ( 76 % ) of the 50 patients had severe sepsis and MODS . Of the 50 ARDS patients , 27 ( 60 % ) were found to have CIPNM . Com - pared with patients who did not develop CIPNM , these patients were older ( me - dian age , 44 vs . 24 yrs , p (cid:1) . 010 ) , had higher mean daily peak blood glucose lev - els ( 166 vs . 144 mg / dL , p (cid:2) . 001 ) , spent longer days on mechanical ventilation ( median , 20 vs . 13 days , p (cid:1) . 018 ) , and had an increased ICU length of stay ( me - dian , 38 vs . 26 days , p (cid:1) . 001 ) . There was no significant difference between the two groups for the following observations : duration of sepsis ; severity of illness or MODS ; Pa O 2 / F IO 2 ratio on admission ; ad - ministration of corticosteroids , amino - glycoside antibiotics , or neuromuscular blocking agents ; and use of renal replace - ment therapy or parenteral nutrition . Although the study examined only 50 ARDS patients , it sheds light on the inci - dence and risk factors of CIPNM in this patient group . The authors showed that CIPNM is frequent in patients with ARDS and can be associated with prolonged me - chanical ventilation and ICU length of stay . The finding of hyperglycemia as an independent risk factor for CIPNM in this study is in agreement with prior observa - tions ( 4 , 17 ) . The study findings provide added justification for early electrophysi - ologic testing in critically ill patients to identify those with CIPNM who may be at risk for weaning failure . * See also p . 711 . Key Words : critical illness polyneuropathy ; critical illness myopathy ; acute respiratory distress syndrome ; hyperglycemia ; intensive care Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000160009 . 97759 . BA 895 Crit Care Med 2005 Vol . 33 , No . 4 The conclusions that can drawn from this study are limited by its retrospective design , the relatively small number of patient records reviewed , and the lack of data on muscle strength . In addition , no long - term data regarding the clinical out - come and results of neuropsychological or electrophysiologic studies after dis - charge from the ICU were provided . Fur - thermore , there were several issues that were not fully addressed in the current study : whether preexisting CIPNM was present at the time of ICU admission , whether early diagnosis and appropriate management of CIPNM could have al - tered outcome , and whether the patient’s nutritional status ( given the lack of data on how these patients were fed during their ICU stay ) played a role in the devel - opment of CIPNM . Interestingly , the au - thors point out that there was no signif - icant difference in the frequency of tracheostomy between the ARDS patients with and without CIPNM . It is increasingly clear that critical care practitioners should be aware that CIPNM may occur and can influence the course and management of disease in critically ill patients , particularly those with ARDS and severe sepsis . Thus , early electrophysiologic testing should be con - sidered in this patient population , espe - cially among the elderly and those with hyperglycemia . Prospective studies are warranted to investigate the incidence and severity of CIPNM in the critically ill and to determine whether tight glycemic control ( with intensive insulin therapy ) can minimize this complication and im - prove patient outcome . Stephen M . Pastores , MD , FCCM Memorial Sloan - Kettering Cancer Center New York , NY REFERENCES 1 . Bolton C , Gilbert J , Hahn A , et al : Polyneu - ropathy in critically ill patients . J Neurol Neurosurg Psychiatry 1984 ; 47 : 1223 – 1231 2 . Bolton CF , Gillert J , Hahn AF : Neuromuscu - lar abnormalities in critically ill patients . In - tensive Care Med 1993 ; 19 : 309 – 310 3 . Bolton CF : Sepsis and the systemic inflam - matory response syndrome : Neuromuscular manifestations . Crit Care Med 1996 ; 24 : 1408 – 1416 4 . Witt N , Zochodne D , Bolton C , et al : Periph - eral nerve function in sepsis and multiple organ failure . Chest 1991 ; 99 : 176 – 184 5 . Trojaborg W , Weimer LH , Hays AP : Electro - physiologic studies in critical illness associ - ated weakness : myopathy or neuropathy—A reappraisal . Clin Neurophysiol 2001 ; 112 : 1586 – 1593 6 . de Letter MA , van Doorn PA , Savelkoul HF , et al : Critical illness polyneuropathy and myop - athy ( CIPNM ) : Evidence for local immune ac - tivation by cytokine - expression in the muscle tissue . J Neuroimmunol 2000 ; 106 : 206 – 213 7 . Di Giovanni S , Mirabella M , D’Amico A , et al : Apoptotic features accompany acute quadri - plegic myopathy . Neurology 2000 ; 55 : 854 – 858 8 . Friedrich O , Hund E , Weber C , et al : Critical illness myopathy serum fractions affect membrane excitability and intracellular cal - cium release in mammalian skeletal muscle . J Neurol 2004 ; 251 : 53 – 65 9 . de Letter MA , Schmitz PIM , Visser LH , et al : Risk factors for the development of polyneu - ropathy and myopathy in critically ill pa - tients . Crit Care Med 2001 ; 29 : 2281 – 2286 10 . De Jonghe B , Sharshar T , Lefaucheur JP , et al : Paresis acquired in the intensive care unit : A prospective multicenter study . JAMA 2002 ; 288 : 2859 – 2867 11 . Deem S , Lee CM , Curtis JR : Acquired neuro - muscular disorders in the intensive care unit . Am J Respir Crit Care Med 2003 ; 168 : 735 – 739 12 . Herridge MS , Cheung AM , Tansey CM , et al : One - year outcomes in survivors of the acute respiratory distress syndrome . N Engl J Med 2003 ; 348 : 683 – 693 13 . Davidson TA , Caldwell ES , Curtis JR , et al : Reduced quality of life in survivors of acute respiratory distress syndrome compared with critically ill control patients . JAMA 1999 ; 281 : 354 – 360 14 . Angus DC , Musthafa AA , Clermont G , et al : Quality - adjusted survival in the first year af - ter the acute respiratory distress syndrome . Am J Respir Crit Care Med 2001 ; 163 : 1389 – 1394 15 . Bercker S , Weber - Carstens S , Deja M , et al : Critical illness polyneuropathy and myop - athy in patients with acute respiratory dis - tress syndrome . Crit Care Med 2005 ; 33 : 711 – 715 16 . Bernard GR , Artigas A , Brigham KL , et al : The American - European Consensus Confer - ence on ARDS . Definitions , mechanisms , rel - evant outcomes , and clinical trial coordina - tion . Am J Respir Crit Care Med 1994 ; 149 : 818 – 824 17 . Van den Berghe G , Wouters P , Weekers F , et al : Intensive insulin therapy in critically ill patients . N Engl J Med 2001 ; 345 : 1359 – 1367 Does nitric oxide control the counter - inflammatory response in endotoxic shock ? * I n this issue of Critical Care Med - icine , Dr . Miki and colleagues ( 1 ) , from Kansai Medical University in Osaka , Japan , present the results of a study addressing the influence of nitric oxide ( NO ) , an inflammatory factor , on the host response to bacterial endo - toxin . Endotoxin is a component of the cell wall of Gram - negative bacteria and is the primary initiator of the pathophysio - logic consequences of the sepsis - medi - ated shock response . Dr . Miki and col - leagues ( 1 ) use a mouse endotoxin challenge model to test the hypothesis that NO plays a major role in controlling the inflammatory response to endotoxin . Previous work in this area suggested that NO inhibition can enhance mortality in animals challenged with endotoxin ( 2 , 3 ) . NO is an intercellular signaling factor that is produced by innate immune cells in response to endotoxin and other in - flammatory stimuli ( 4 ) . Although it is well established that NO is indeed a proinflammatory mediator , there is also evidence to suggest that NO has a counter - inflammatory activity acting on cells of the adaptive immune system , in particular , T cells ( 5 , 6 ) . These authors show that exposure to endotoxin causes a shift in the T - cell response phenotype to what is referred as a T - helper 2 ( Th2 ) – type response , a counter - inflammatory – type adaptive immune response ( 7 ) . How - ever , when mice that lack an enzyme that is required to make NO ( inducible NO * See also p . 716 . Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159696 . 69557 . 41 896 Crit Care Med 2005 Vol . 33 , No . 4 synthase [ iNOS ] ) were given the same dose of endotoxin , they failed to observe this natural shift toward a Th2 - type adap - tive immune response . Instead , the oppo - site type of adaptive immune response , a T - helper 1 ( Th1 ) – type response was ob - served . Th1 - type immune responses are proinflammatory in nature and are asso - ciated with heightened antimicrobial im - munity ( 7 ) . Moreover , they found that iNOS - deficient mice succumb more readily to endotoxin challenge than mice that have the capacity to produce normal levels of NO , suggesting that NO defi - ciency augments the toxicity of endo - toxin . Their data support two general conclusions . First , NO plays a protective role during endotoxemia , and second , the Th2 - type response induced by endotoxin is dependent on NO . However , their find - ings also suggest that the Th2 - type re - sponse displayed by the adaptive immune system ( CD4 (cid:3) T cells ) after endotoxemia may be an important protective response during sepsis . Dr . Miki and colleagues ( 1 ) promote the idea that NO plays a role in inducing immune dysfunction after endotoxemia . However , their findings really suggest that NO protects the host from excessive inflammatory reactivity and death . I be - lieve that an increased understanding of mechanistic basis for this observation and how NO is involved in the induction of a protective Th2 - type response will surely advance our understanding of the pathologic consequences of endotoxemia and sepsis . Some possibilities that were not addressed by these authors is the potential role of the NO - producing my - eloid suppressor macrophage subset in controlling inflammatory reactivity in their model or the idea that endotoxin might activate CD4 (cid:3) CD25 (cid:3) regulatory T cells that are known to control inflam - matory responses through Th2 - type mechanisms ( 8 – 10 ) . Interestingly , CD4 (cid:3) CD25 (cid:3) regulatory T cells expresses receptors , Toll - like receptor 4 , that react specifically with endotoxin ( 11 ) . Similar conclusions concerning the protective counter - inflammatory activity of Th2 - type responses have been made in other inflammatory - driven diseases such as trauma , burn injury , colitis , and auto - immune inflammatory diseases ( 10 , 12 – 15 ) . Unfortunately , a prolonged and ex - cessive Th2 - type response can also cause generalized immune suppression and the development of opportunistic infections , as has been characterized after severe trauma , burn injury , and sepsis ( 16 , 17 ) . This suggests that maintaining immune homeostasis with respect to the balance between inflammatory - and counter - inflammatory – type responses plays a ma - jor role in maintaining a healthy immune response . Is NO a major determinant in the balance between Th1 - and Th2 - type immune responses after sepsis or injury ? The results presented in this study , al - though limited to endotoxemia , suggest that it might be one important determi - nant . Other studies also support this pos - sibility , showing that NO deficiency re - sults in a bias toward Th1 - type reactivity and reduced Th2 - type responses . In par - ticular , it was shown that iNOS - deficient mice had higher Th1 - type responses to herpes simplex virus – 1 or Leishmania in - fection ( 18 , 19 ) . Enhanced Th1 - type re - activity was also observed in iNOS - deficient mice challenged with the parasite Schistosoma mansoni ( 20 ) . However , in all these studies , it was shown that the biased Th1 - type response did not provide a protective benefit to the host . It was eventually shown that the cytokine , interleukin - 12 , was responsible in part for the skewed Th1 - type response because NO suppresses interleukin - 12 production and cells from iNOS - deficient mice produce higher levels of interleu - kin - 12 ( 21 ) . In summation , it seems that NO can be an important determinant of the bal - ance between Th1 - and Th2 - type immune responses . However , the most important contribution of NO to overall host immu - nity may be to control excessive inflam - matory responses through the subse - quent induction of a counter - inflammatory protective Th2 - type adaptive immune response . The recent report of higher mortality in patients with septic shock treated with the NO synthase inhibitor drug 546C88 ( 59 % vs . 49 % for placebo ) suggests that NO does indeed play a natural protective role in septic shock ( 22 ) . Future considerations of therapeutic strategies targeting the NO or other immune regulatory networks during sepsis and trauma need to con - sider this balance between Th1 - and Th2 - type control of inflammatory responses as an important determinant of patient out - come . James A . Lederer , MS , PhD Department of Surgery Brigham and Women’s Hospital Harvard Medical School Boston , MA REFERENCES 1 . Miki S , Takeyama N , Tanaka T : Immune dys - function in endotoxicosis : Role of nitric ox - ide produced by inducible nitric oxide syn - thase . Crit Care Med 2005 ; 33 : 716 – 720 2 . Billiar TR , Curran RD , Harbrecht BG , et al : Modulation of nitrogen oxide synthesis in vivo : NG - monomethyl - L - arginine inhibits endotoxin - induced nitrate / nitrate biosynthe - sis while promoting hepatic damage . J Leu - koc Biol 1990 ; 48 : 565 – 569 3 . Cobb JP , Natanson C , Hoffman WD , et al : N omega - amino - L - arginine , an inhibitor of ni - tric oxide synthase , raises vascular resistance but increases mortality rates in awake ca - nines challenged with endotoxin . J Exp Med 1992 ; 176 : 1175 – 1182 4 . MacMicking J , Xie QW , Nathan C : Nitric ox - ide and macrophage function . Annu Rev Im - munol 1997 ; 15 : 323 – 350 5 . van der Veen RC , Dietlin TA , Dixon Gray J , et al : Macrophage - derived nitric oxide inhibits the proliferation of activated T helper cells and is induced during antigenic stimulation of rest - ing T cells . Cell Immunol 2000 ; 199 : 43 – 49 6 . van der Veen RC : Nitric oxide and T helper cell immunity . Int Immunopharmacol 2001 ; 1 : 1491 – 1500 7 . Abbas AK , Murphy KM , Sher A : Functional diversity of helper T lymphocytes . Nature 1996 ; 383 : 787 – 793 8 . Garn H , Siese A , Stumpf S , et al : Shift toward an alternatively activated macrophage re - sponse in lungs of NO 2 - exposed rats . Am J Respir Cell Mol Biol 2003 ; 28 ( 3 ) : 386 – 396 9 . Atochina O , Daly - Engel T , Piskorska D , et al : A schistosome - expressed immunomodula - tory glycoconjugate expands peritoneal Gr1 ( (cid:3) ) macrophages that suppress naive CD4 ( (cid:3) ) T cell proliferation via an IFN - gamma and nitric oxide - dependent mecha - nism . J Immunol 2001 ; 167 : 4293 – 4302 10 . Powrie F , Read S , Mottet C , et al : Control of immune pathology by regulatory T cells . No - vartis Found Symp 2003 ; 252 : 92 – 98 ; discus - sion , 98 – 105 , 106 – 114 11 . Caramalho I , Lopes - Carvalho T , Ostler D , et al : Regulatory T cells selectively express toll - like receptors and are activated by lipopoly - saccharide . J Exp Med 2003 ; 197 : 403 – 411 12 . Munford RS , Pugin J : Normal responses to injury prevent systemic inflammation and can be immunosuppressive . Am J Respir Crit Care Med 2001 ; 163 : 316 – 321 13 . Guo Z , Kavanagh E , Zang Y , et al : Burn injury promotes antigen - driven Th2 - type re - sponses in vivo . J Immunol 2003 ; 171 : 3983 – 3990 14 . Zang Y , Dolan SM , Choileain NN , et al : Burn injury initiates a shift in superantigen - induced T cell responses and host survival . J Immunol 2004 ; 172 : 4883 – 4892 15 . Sakaguchi S : Naturally arising CD4 (cid:3) regu - latory T cells for immunologic self - tolerance and negative control of immune responses . Annu Rev Immunol 2004 ; 22 : 531 – 562 16 . Hotchkiss RS , Karl IE : The pathophysiology 897 Crit Care Med 2005 Vol . 33 , No . 4 and treatment of sepsis . N Engl J Med 2003 ; 348 : 138 – 150 17 . Lederer JA , Rodrick ML , Mannick JA : The effects of injury on the adaptive immune response . Shock 1999 ; 11 : 153 – 159 18 . Wei XQ , Charles IG , Smith A , et al : Altered immune responses in mice lacking inducible nitric oxide synthase . Nature 1995 ; 375 : 408 – 411 19 . MacLean A , Wei XQ , Huang FP , et al : Mice lacking inducible nitric - oxide synthase are more susceptible to herpes simplex virus in - fection despite enhanced Th1 cell responses . J Gen Virol 1998 ; 79 ( Pt 4 ) : 825 – 830 20 . James SL , Cheever AW , Caspar P , et al : In - ducible nitric oxide synthase - deficient mice develop enhanced type 1 cytokine - associated cellular and humoral immune responses af - ter vaccination with attenuated Schistosoma mansoni cercariae but display partially re - duced resistance . Infect Immun 1998 ; 66 : 3510 – 3518 21 . Huang FP , Niedbala W , Wei XQ , et al : Nitric oxide regulates Th1 cell development through the inhibition of IL - 12 synthesis by macrophages . Eur J Immunol 1998 ; 28 : 4062 – 4070 22 . Lopez A , Lorente JA , Steingrub J , et al : Mul - tiple - center , randomized , placebo - con - trolled , double - blind study of the nitric oxide synthase inhibitor 546C88 : Effect on survival in patients with septic shock . Crit Care Med 2004 ; 32 : 21 – 30 The quest to improve cardiac arrest survival : Overcoming the hemodynamic effects of ventilation * C ardiovascular disease claims almost 800 , 000 lives annually in the United States . Of these , about one half are due to sud - den cardiac death ( 1 ) . Survival varies largely by community but is uniformly dismal , with only 5 % surviving out - of - hospital cardiopulmonary resuscitation . These rates are disappointing given the widespread international research focus on cardiac resuscitation and the measur - able success achieved over the years with various cardiac drugs , procedures , and devices in the laboratory setting . The question is why many of these interven - tions so often work in large animal stud - ies but only work some of the time in human studies . Translational research may be our best opportunity to address this quandary . An area of escalating scientific interest and promise is in the idea of enhancing the low - flow state of cardiac resuscitation by manipulating intrathoracic pressures during cardiopulmonary resuscitation ( CPR ) . The stability of the myocardium is dependent on adequate perfusion of the coronary vessels . It has been shown that stroke volume and additionally myocar - dial perfusion are greatly diminished by the high intrathoracic pressures pro - duced with positive pressure ventilation ( 2 – 5 ) . To mitigate this problem , multiple techniques have been considered to re - duce the intrathoracic pressures during CPR : changing the chest compression - ventilation ratios ; decreasing ventilation frequency , amplitude , and duration ; aug - menting the elastic recoil of the chest to enhance the return of blood back into the chest ; and selective blockade of ventila - tion during the decompression phase of CPR . The idea is to reduce intrathoracic pressures , creating a vacuum - like nega - tive pressure state in the chest , to in - crease venous return and ultimately im - prove cardiac output and vital organ perfusion . Coronary perfusion pressure ( CPP ) is largely considered a measure of efficacy of CPR and is the calculated difference between the diastolic aortic pressure and the right atrial pressure . Previous inves - tigators have tried both direct and indi - rect methods in attempts to improve the CPP . Direct aortic arch perfusion may be accomplished by infusing volume through a balloon catheter directed to the thoracic aorta via the femoral artery ( 6 ) . Alternatively , indirect methods such as the active compression - decompression CPR device use the vacuum concept , ac - tively assisting the natural recoil of the chest wall to create a negative pressure state in the thorax to enhance myocardial flow ( 7 , 8 ) . Indirect methods are perhaps more favorable in the prehospital setting as they may be accomplished by nonin - vasive means . The inspiratory impedance threshold device ( ITD ) is a small , inexpensive , air - way adjunct to reduce intrathoracic pres - sures by preventing positive pressure ventilation during the decompression phase of CPR ( 9 ) . The device is a one - way valve that is attached to a face mask or an endotracheal tube and is used with the ventilation bag . It essentially blocks air from entering the lungs during the de - compression phase of CPR . Animal stud - ies clearly show that the impedance threshold device improves circulation during CPR by enhancing negative in - trathoracic pressures ( 10 – 13 ) . A study in this issue of Critical Care Medicine reports on the first double - blinded randomized controlled trial de - signed to evaluate clinical outcomes of an ITD ( 14 ) . The Milwaukee study provides further data for the ITD in out - of - hospital cardiac arrest patients , which previously had only been tested clinically in com - bination with active compression - decompression CPR ( 15 – 17 ) . The study compares short - term outcomes ( ICU admission , 24 - hr survival ) of patients treated with standard CPR combined with either ITD or a sham device . No - table is that the trial was terminated early for several technical reasons but not for lack of patient safety or efficacy of the device . Consequently , the study was underpowered to assess even short - term outcomes—thus , any subgroup analysis , a priori or not , must be eval - uated critically in this light . Although no significant association was found overall with use of the device , a post hoc analysis identified a significant sur - vival benefit for the ITD patients who had a documented cardiac rhythm of pulseless electrical activity at any time during the cardiac arrest . For patients with pulseless electrical activity , a group that typically has few survivors , 24 - hr survival was double with the use of the ITD vs . sham . The data are prom - ising at best . Given the small sample size and the lack of alpha - adjustment * See also p . 734 . Key Words : cardiopulmonary resuscitation ; cardio - pulmonary resuscitation impedance threshold device Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159748 . 51702 . 20 898 Crit Care Med 2005 Vol . 33 , No . 4 for multiple testing , it is difficult to eluci - date the clinical importance of this finding , which perhaps is not the most noteworthy finding of this investigation . During patient recruitment for this study , investigators made an alarming discovery regarding the way that emer - gency personnel ventilate patients in the field . Patients were ventilated at an aver - age rate of 37 breaths / min , which is more than double the American Hospital Asso - ciation recommendation ( 2 , 3 ) . Using a porcine model , investigators showed that this practice of hyperventilation adversely affected stroke volumes and coronary perfusion pressures . To moderate this , all emergency personnel went through a CPR retraining session , and a timing de - vice was used to give rescuers a prompt to ventilate at a proper rate . It is likely that this practice of hyperventilating patients is common among emergency agencies , and thus this finding has potentially global implications . The concern that we now face is whether many of our prior clinical studies of cardiac drugs and pro - cedures have been hampered by this same mechanism—by which hyperventilation has created a state of such poor coronary perfusion pressures as to prohibit ade - quate circulation of study drugs and sat - isfactory trial of these interventions . Finally , it is important to understand that the authors of this study have estab - lished significant financial ties with the patent , corporation , and manufacturer of the trial device . Relationships between industry and clinical researchers have been increasingly scrutinized in recent years . The implication is that industry - supported clinical trials may yield biased findings and violate basic research prin - ciples . We each must consider for our - selves the relative importance of any af - filiations that may present here as an appearance of a conflict of interest . Should we be adopting the use of the impedance threshold device based on these preliminary clinical results ? The devices are inexpensive and easy to use . The safety profile appears satisfactory . Animal studies have clearly shown that the ITD successfully reduces intratho - racic pressures and enhances circulation during CPR . Moreover , clinical trials combining ITD with active compression - decompression CPR also show improved coronary perfusion pressures and short - term survival outcomes . The modality ap - pears promising , but further research is necessary before adopting this new de - vice . We do not want this to be another example of “letting the horse out of the barn , ” whereby we change clinical prac - tice at the first sight of a potentially promising technology and thus lose any further window of opportunity to subject the intervention to appropriate study to elucidate its true potential . Although an - imal studies are mostly positive , and this article gives us reason to be optimistic , there is currently insufficient evidence to support a change in general clinical prac - tice . Appropriately , authors are currently conducting a further , more definitive trial to evaluate the effectiveness of this device , incorporating both a larger sam - ple size and a more clinically meaningful primary outcome of survival to hospital discharge . Should we evaluate our systems to de - termine whether our emergency person - nel are following American Hospital As - sociation guidelines for ventilation rates during CPR ? The Milwaukee experience provides compelling evidence to suggest that we should more closely monitor our emergency personnel to ensure that pa - tients are not being hyperventilated dur - ing CPR . Resuscitation researchers are now acutely aware of the detrimental ef - fect of hyperventilation on stroke volume and vital organ perfusion . Is it possible that the simple correction of this proce - dure alone could lead to important sur - vival benefits on a wide - scale basis ? Now is the time for clinical researchers to re - consider all those negative cardiac arrest trials that failed to bear out the same promise purported by their colleagues in the animal laboratory . Valerie J . De Maio , MD , MSc Department of Emergency Medicine University of North Carolina Chapel Hill , NC REFERENCES 1 . Zheng ZJ , Croft JB , Giles WH : State specific mortality from sudden cardiac death - United States , 1999 . MMWR Morbid Mortal Wkly Rep 2002 ; 51 : 123 – 126 2 . Aufderheide TP , Sigurdsson G , Pirrallo RG , et al : Hyperventilation - induced hypotension during cardiopulmonary resuscitation . Cir - culation 2004 ; 109 : 1960 – 1965 3 . Aufderheide TP , Lurie KG : Death by hyper - ventilation : A common and life - threatening problem during cardiopulmonary resuscita - tion . Crit Care Med 2004 ; 32 ( 9 suppl ) : S345 – S351 4 . Sigurdsson G , Yannopoulos D , McKnite SH , et al : Cardiorespiratory interactions and blood flow generation during cardiac arrest and other states of low blood flow . Curr Opin Crit Care 2003 ; 9 : 183 – 188 5 . Pepe PE , Raedler C , Lurie KG , et al : Emer - gency ventilatory management in hemor - rhagic states : Elemental or detrimental ? J Trauma 2003 ; 54 : 1048 – 1047 6 . Manning JE , Murphy CA Jr , Hertz CM , et al : Selective aortic arch perfusion during car - diac arrest : A new resuscitation technique . Ann Emerg Med 1992 ; 21 : 1058 – 1065 7 . Guly UM , Robertson CE : Active decompres - sion improves the haemodynamic state dur - ing cardiopulmonary resuscitation . Br Heart J 1995 ; 73 : 372 – 376 8 . Malzer R , Zeiner A , Binder M , et al : Hemo - dynamic effects of active compression - decompression after prolonged CPR . Resus - citation 1996 ; 31 : 243 – 253 9 . Lurie KG , Mulligan KA , McKnite S , et al : Optimizing standard cardiopulmonary resus - citation with an inspiratory impedance threshold valve . Chest 1998 ; 113 : 1084 – 1090 10 . Marino BS , Yannopoulos D , Sigurdsson G , et al : Spontaneous breathing through an in - spiratory impedance threshold device aug - ments cardiac index and stroke volume index in a pediatric porcine model of hemorrhagic hypovolemia . Crit Care Med 2004 ; 32 ( 9 Suppl ) : S398 – S405 11 . Yannopoulos D , Sigurdsson G , McKnite S , et al : Reducing ventilation frequency combined with an inspiratory impedance device im - proves CPR efficiency in swine model of car - diac arrest . Resuscitation 2004 ; 61 : 75 – 82 12 . Lurie KG , Barnes TA , Zielinski TM , et al : Evaluation of a prototypic inspiratory imped - ance threshold valve designed to enhance the efficiency of cardiopulmonary resuscitation . Respir Care 2003 ; 48 : 52 – 57 13 . Lurie K , Zielinski T , McKnite S , et al : Im - proving the efficiency of cardiopulmonary re - suscitation with an inspiratory impedance threshold valve . Crit Care Med 2000 ; 28 ( 11 Suppl ) : N207 – N209 14 . AufderheideTP , PirralloRG , ProvoTA , etal : Clin - ical evaluation of an inspiratory impedance threshold device during standard cardiopulmo - naryresuscitationinpatientswithout - of - hospital cardiac arrest . Crit Care Med 2005 ; 33 : 734 – 740 15 . Plaisance P , Lurie KG , Vicaut E , et al : Eval - uation of an impedance threshold device in patients receiving active compression - decompression cardiopulmonary resuscita - tion for out of hospital cardiac arrest . Resus - citation 2004 ; 61 : 265 – 271 16 . Wolcke BB , Mauer DK , Schoefmann MF , et al : Comparison of standard cardiopulmonary resuscitation versus the combination of ac - tive compression - decompression cardiopul - monary resuscitation and an inspiratory im - pedance threshold device for out - of - hospital cardiac arrest . Circulation 2003 ; 108 : 2201 – 2205 17 . Frascone RJ , Bitz D , Lurie K : Combination of active compression decompression cardio - pulmonary resuscitation and the inspiratory impedance threshold device : state of the art . Curr Opin Crit Care 2004 ; 10 : 193 – 201 899 Crit Care Med 2005 Vol . 33 , No . 4 Acute lung injury : New insights from computed tomography * C omputed tomography ( CT ) has become a superb research tool for dissecting pulmonary pathophysiology during me - chanical ventilation in patients with acute lung injury ( ALI ) . Since the first descriptions of lung morphometry in acute respiratory distress syndrome ( ARDS ) almost 2 decades ago ( 1 , 2 ) , im - provements in CT technology have both improved scan resolution and reduced scan time . Now multiple - detector , helical scanners can scan the entire thorax in (cid:2) 3 secs at a slice thickness of 1 – 1 . 5 mm . Using differences in Hounsfield density , lung regions that are normally aerated can be distinguished from lung regions that are either derecruited or overdis - tended . The Hounsfield density of each CT voxel , although displayed on a computer screen as a single pixel , is in reality an average density of all of the alveoli within the voxel’s three - dimensional volume . Within each voxel , some alveoli may be derecruited , some may be normally aer - ated , and some may be overdistended . Volume averaging effectively hides from the reader the heterogeneity of aeration that exists within each voxel . The larger the voxel , the less regional heterogeneity is apparent . Density histograms can be used by readers to calculate the percentage of lung tissue that falls within each category of aeration . Decreasing slice thickness re - duces the size of each voxel , which , in turn , reduces the number of alveoli aver - aged in each voxel . The resulting en - hanced spatial resolution permits small regions of hyperinflation to be identified that would have escaped detection if thicker slices had been used . In this issue of Critical Care Medicine , Dr . Vieira and colleagues ( 3 ) report the results of a study that is proof of this principal . They compare the sensitivity of low to high spatial resolution CT sections in the detection lung overinflation in 30 pa - tients with ALI / ARDS . In this study ( 3 ) , lung overinflation was measured using previously validated proprietary imaging software on thick ( 10 mm ) and thin ( 1 . 5 mm ) CT sections obtained at functional residual capacity at either zero end - expiratory pressure or positive end - expiratory pressure of 10 cm H 2 O . Pa - tients were anesthetized , paralyzed , and ventilated using controlled mechanical ventilation . Overinflated lung volume was measured as the end - expiratory vol - ume of lung regions with CT attenuations (cid:2)(cid:4) 900 Hounsfield units , a density pre - viously determined as the lower limit of lung attenuation in volunteers breath - holding at total lung capacity ( 4 ) . The authors demonstrate that thick slice CT significantly underestimates regional overinflation compared with thin CT sec - tions . Underestimation of overdistension occurred primarily in patients with focal loss of aeration , that is , significant radio - graphic inhomogeneity involving primar - ily the lower lobes . Although the applica - tion of positive end - expiratory pressure significantly increased overdistension only in patients with focal infiltrates , these patients represented 60 % of the population studied . During positive end - expiratory pressure of 10 cm H 2 O , the percentage of lung estimated to be over - inflated was negligible when measured using thick CT sections but was as high as 16 % when measured using thin CT sections . When one considers that the CT images were acquired at end - expiration rather than during an inspiratory - hold maneuver , this number is certainly an underestimation of the amount of lung that would be overdistended during rou - tine ventilation . Although the focus on the current study is on new technology , Vieira and colleagues are careful not to let the real significance of these observations to es - cape ; they provide strong imaging evi - dence that overinflation is indeed heter - ogeneously distributed between adjacent alveoli during mechanical ventilation of patients with acute lung injury . Further - more , they show that regional overdis - tension can occur even when plateau air - way pressures are in the previously considered “safe” range of 25 cm H 2 O . The implications of these data are tre - mendous and again beg the question : “Is there a safe plateau airway pressure ? ” In a preliminary report of a post hoc analysis of data from the ARDSnet tidal volume trial ( 5 ) , Brower and colleagues ( 6 ) investigated the effect of tidal volume reduction in patients who had plateau airway pressures in the previously consid - ered safe range of (cid:1) 32 cm H 2 O . In pa - tients from this trial who had the lowest quartile of plateau airway pressures on study day 1 , those who were randomized to receive a tidal volume 12 mL / kg of predicted body weight had plateau airway pressures (cid:1) 26 cm H 2 O and a mortality rate of 34 % . These numbers are very sim - ilar to those reported in the focal lung injury group of the present study ( 3 ) ( 25 cm H 2 O and 39 % , respectively ) . Patients in the ARDSnet trial randomized to 6 mL / kg of predicted body weight had pla - teau airway pressures (cid:1) 20 cm H 2 O and a mortality rate of only 23 % . Unfortu - nately , we do not have information on the heterogeneity of radiographic infiltration in the ARDSnet trial ( 5 ) , but using the data by Dr . Vieira and colleagues ( 3 ) and Brower et al . ( 6 ) in composite might lead one to speculate that there is not a safe plateau airway pressure below which pa - tients are spared ventilator - induced lung injury . Nor , as pointed out in a previous investigation by the authors of the current report ( 7 ) , will reliance on the pressure - volume curve reliably identify overdisten - sion since it occurs simultaneously with recruitment . Only high - resolution CT imaging can reliably detect this type of regional overdistension . In reference to the implications of these data , Dr . Vieira and coauthors ( 3 ) state that this information is of critical importance in the care of patients . How - ever , it seems a bit premature to suggest that clinicians should start rushing every ALI / ARDS patient to the CT scanner . For several reasons , the current information represents an interesting vista along our journey , not our destination , in our un - * See also p . 741 . Key Words : computed tomography ; acute lung in - jury ; acute respiratory distress syndrome Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159722 . 22115 . 44 900 Crit Care Med 2005 Vol . 33 , No . 4 derstanding of lung pathophysiology dur - ing mechanical ventilation . First , very few centers have the expertise of the authors to perform these difficult stud - ies in an accurate , precise , and safe manner . Second , just like the use of CT for the diagnosis of pulmonary embo - lism ( 8 ) , the current data will need ad - ditional validation by other centers to be generalizable to community medical centers . And finally , but most impor - tant , the clinical impact of these obser - vations is largely unknown . Although we now clearly understand the clinical relevance of ventilation with high tidal volumes and high plateau air - way pressures , we know nothing about the relevance of radiographic regional hy - perinflation during “lung - protective ven - tilation” that is visible only through the eyes of high - resolution CT imaging . De - veloping portable bedside CT density his - tograms that are used to customize both mechanical ventilation and body posi - tioning in a way that optimizes alveolar recruitment while minimizing the risk of ventilator - induced lung injury ( 9 ) will re - main a clinical science challenge for years to come . Ben deBoisblanc , MD , FCCM Section of Pulmonary / Critical Care Medicine Louisiana State University Medical School New Orleans , LA REFERENCES 1 . Gattinoni L , Presenti A , Torresin A , et al : Adult respiratory distress syndrome profiles by com - puted tomography . J Thorac Imaging 1986 ; 1 : 25 – 30 2 . Maunder RJ , Shuman WP , McHugh JW , et al : Preservation of normal lung regions in the adult respiratory distress syndrome . Analysis by computed tomography . JAMA 1986 ; 255 : 2463 – 2465 3 . Vieira SRR , Nieszkowska A , Lu Q , et al : Low spatial resolution computed tomography un - derestimates lung overinflation resulting from positive pressure ventilation . Crit Care Med 2005 ; 33 : 741 – 749 4 . Vieira SR , Puybasset L , Richecoeur J , et al : A lung computed tomographic assessment of positive end - expiratory pressure - induced lung overdistension . Am J Respir Crit Care Med 1998 ; 158 : 1571 – 1577 5 . Anonymous : Ventilation with lower tidal vol - umes as compared with traditional tidal vol - umes for acute lung injury and the acute respiratory distress syndrome . The Acute Re - spiratory Distress Syndrome Network . N Engl J Med 2000 ; 342 : 1301 – 1308 6 . Brower RG , et al : Effects of tidal volume re - duction ( VT - R ) in acute lung injury ( ALI ) pa - tients with inspiratory plateau pressures ( Pplat ) (cid:1) 32 CM H2O before VT - R . Am J Respir Crit Care Med 2003 ; 167 : A616 7 . Rouby JJ , Lu Q , Vieira S : Pressure / volume curves and lung computed tomography in acute respiratory distress syndrome . Eur Re - spir J Suppl 2003 ; 42 8 . Gulsun M , Goodman LR : CT for the diagnosis of venous thromboembolic disease . Curr Opin Pulm Med 2003 ; 9 : 367 – 373 9 . Rouby J - J , Puybasset L , Nieszkowska A : Acute respiratory distress syndrome : Lessons from computed tomography of the whole lung . Crit Care Med 2003 ; 31 ( Suppl ) : S285 – S295 Still a black box : What do we really know about the intensive care unit admission process and its consequences ? * When you choose not to decide you have already made a choice . T he purpose of intensive care medicine is to provide the fa - cilities for diagnosis , monitor - ing , prevention , and treatment of multiple organ failure in severely ill patients . This requires a multidisci - plinary team approach and the highest possible standards of nursing and medical care . Modern intensive care units ( ICUs ) offer a limited amount of highly special - ized medical services that consume a large proportion of hospital budgets for a minority of patients ( 1 ) . One of the most important challenges for the intensivist remains the allocation of scarce public resources to those patients expected to benefit from intensive care therapy . The fact that treatment—of proven value or not—is possible is not in itself justifica - tion for patients’ ICU admission . Inten - sive care therapies and invasive monitor - ing techniques have complications ( 2 , 3 ) , and inappropriate admission of low - risk patients may thus not only waste scarce resources but , more importantly , expose the patient to unnecessary risks . Never - theless , the decision to transfer a patient to the ICU may often be determined by factors other than acute medical needs and potential for benefit , eventually lead - ing to the admission of patients with little or no hope of survival ( 4 ) . In this issue of Critical Care Medicine , Dr . Garrouste - Orgeas and colleagues ( 5 ) for the French ADMISSIONREA Study Group carefully describe factors associ - ated with patients’ admission or refusal in 11 selected French ICUs . Among the pa - tient - related characteristics , dependency and a history of metastatic cancer signif - icantly influenced ICU admission deci - sions . Logistic regression analysis re - vealed a significant association of center , bed availability , and time of day with the likelihood of patient refusal ( 5 ) . In reviewing the literature , surpris - ingly little information with regard to admission policies and practices is avail - able ( 6 – 10 ) , although this may have a major effect on patients’ outcome . This lack of data is most likely due to the challenging nature of such data compila - tion and analysis . Although in 1988 a consensus panel in the United Kingdom stated , “Selection for intensive care should be based on broad concepts of prognosis derived from statistical analysis of comparable cohorts of patients backed up by sound clinical trials” ( 11 ) , the ap - plicable instruments and profound data are hardly available . In the article by Dr . Garrouste - Orgeas and colleagues ( 5 ) , the authors applied— besides demographic data and admission diagnosis—a variety of instruments at - * See also p . 750 . Key Words : intensive care unit ; admission ; triage ; process ; organization ; outcome Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159723 . 33298 . 07 901 Crit Care Med 2005 Vol . 33 , No . 4 tempting an objective characterization of all patients , thus enabling a meaningful analysis , namely the Mortality Probability Model ( MPM II 0 ) ( 12 ) , the McCabe co - morbidity score ( 13 ) , and the Omega score ( 14 , 15 ) . Nevertheless , none of the available se - verity - of - illness scoring systems ( e . g . , Acute Physiology and Chronic Health Evaluation , Simplified Acute Physiology Score , or MPM ) is capable of predicting individual patients’ outcomes reliably , and too many confounders , such as lead time bias and case mix , may have altered patients’ outcome well beyond the scope of the scoring system ( 16 , 17 ) . Yet , MPM II 0 is still the only objective measure of patients’ severity of illness that has been validated for immediate assessment and can thus be applied before or at the time of ICU admission . However , calibration and discrimination of the MPM II 0 is poor and thus precludes strong inferences ( 18 , 19 ) . Moreover , the accuracy of the MPM II 0 ( as reflected by measures of discrimi - nation and calibration ) diminishes when the case mix is varied ( 20 ) . Finally , the authors applied the MPM II 0 to the non - admitted patients as well—a group of non - ICU patients for which the MPM II 0 was neither developed nor validated . A standardized mortality rate as reported by the authors for admitted , refused , and later - admitted patients may thus be grossly misleading ( 21 ) . Considering patient - related factors as the driving force for ICU admission , chronic disease state is—besides pa - tients’ age and the severity of the acute disease—an independent predictor of outcome after intensive care ( 22 ) . Conse - quently , the authors evaluated the pa - tients’ co - morbidities by applying the Mc - Cabe score , a system originally developed in the early 1960s , which has not been validated for the intended use ( 13 ) . Pa - tients not admitted to the ICU were more frequently considered rapidly fatal , with respect to the McCabe classification ( i . e . , too sick to benefit ) , and finally exhibited a higher mortality . However , the categori - zation of patients was done by the same physician who was responsible for the triage , thereby eventually introducing bias . Among the organizational factors , a full ICU was significantly associated with refusal of patients , and for various rea - sons , outcome of refused patient was worse ( 5 ) . Although this seems intuitive , it clearly deserves closer examination . In the future , further restriction of re - sources in the healthcare systems world - wide can be expected , leading to an in - crease of bed shortage in the ICU setting . This may well be associated with ration - ing care ( 23 ) and subsequently fewer ICU admissions . The selection process re - ferred to as triage therefore assumes a pivotal role in reliably identifying those patients most likely to benefit from in - tensive care therapy . For all other pa - tients , alternative settings providing sup - portive care need to be defined . However , Wasserfallen et al . ( 24 ) have recently re - ported intentional bed closure on a sur - gical ICU without measurable effects on outcome or activity indicators . By its very nature , the triage process must include all patients already receiv - ing intensive care therapy . Thus , an in - depth analysis with a clear trade - off of risks and benefits of intensive care be - tween all patients in the ICU and the patients on request must be executed . Moreover , the availability of alternative services in the same or other hospitals , such as intermediate care facilities or other ICUs , and the potential risks and consequences of premature discharge from the ICU ( 25 , 26 ) must be acknowl - edged . Nevertheless , commonly consid - ered outcome measures may be mislead - ing because they do not necessarily reflect the quality of processes and care in complex systems such as an ICU ( 24 ) . The authors consequently grouped pa - tients referred to other institutions in the category of admitted patients but did not provide the necessary data to decide whether all patients who were treated on the referring ICU did really require inten - sive care therapy . Given the significant association of an occupied ICU and ad - mission refusal , a more in - depth analysis and detailed description of the patient cohort already cared for would have been desirable . Instruments such as the Sim - plified Therapeutic Intervention Scoring System ( TISS - 28 ) may well have served to identify the patients receiving active treatment ( i . e . , truly depending on inten - sive care therapy ) ( 27 , 28 ) . This may have also shed some light on the extreme heterogeneity with regard to refusal rates across the participating ICUs , varying from 7 % to 63 % , which has not been fully explained by the authors . It remains speculative whether specific ser - vices of these ICUs ( e . g . , case - mix , num - ber of beds , training status of the triaging physician , proportion of active treatment , or any other organizational factor ) were associated with the higher refusal rates . Patient admission and discharge re - mains a black box in intensive care med - icine . We are hardly able to define objec - tive surrogate variables for decision making , and thus , triage of patients con - tinues to be one of the most complex , accountable , and challenging obligations of the daily ICU routine . Regularly , phy - sicians are forced to decide on acceptance or denial of intensive care services for critically ill patients , with very little in - formation on hand . For the foreseeable future , ICU admis - sion practices will thus continue to be individual processes . Although recom - mendations for triage to the ICU have been issued , these are rarely followed ( 29 ) . Consequently , subjective patient as - sessment facilitated by sound medical ex - pertise , ethical experience , and responsi - bility will remain the backbone of the admission process ( 30 ) . It is hoped that more colleagues , such as the ADMISSIONREA Study Group , try to dissect their individual admission and refusal practices , thereby gaining insight in structural weaknesses and potential ar - eas for process improvement . None of this analysis will be perfect because all will inevitably suffer from a number of methodologic shortcomings , but every attempt will still serve to emphasize the principle needs and may well define fu - ture targets . Jürgen Graf , MD Medical Clinic I Department of Cardiology and Pulmonology University Hospital Aachen Aachen , Germany Uwe Janssens , MD , FESC Medical Clinic St . Antonius Hospital Eschweiler , Germany REFERENCES 1 . Kuckelt W : Notes in intensive care medicine in Europe : Intensive care medicine in Ger - many . In : Organisation and Management of Intensive Care : A Prospective Study in 12 European Countries . Reis Miranda D , Ryan DW , Schaufeli WB , et al ( Eds ) . Berlin , Springer , 1998 , pp 88 – 90 2 . Abramson NS , Wald KS , Grenvik AN , et al : Adverse occurrences in intensive care units . JAMA 1980 ; 244 : 1582 – 1584 3 . Giraud T , Dhainaut JF , Vaxelaire JF , et al : Iatrogenic complications in adult intensive care units : A prospective two - center study . Crit Care Med 1993 ; 21 : 40 – 51 4 . Vincent JL : Forgoing life support in western European intensive care units : The results of 902 Crit Care Med 2005 Vol . 33 , No . 4 an ethical questionnaire . Crit Care Med 1999 ; 27 : 1626 – 1633 5 . Garrouste - Orgeas M , Montuclard L , Timsit JF , et al : Predictors of intensive care unit refusal in French intensive care units : A mul - tiple - center study . The French ADMISSION - REA Study Group . Crit Care Med 2005 ; 33 : 750 – 755 6 . Simchen E , Sprung CL , Galai N , et al : Sur - vival of critically ill patients hospitalized in and out of intensive care units under paucity of intensive care unit beds . Crit Care Med 2004 ; 32 : 1654 – 1661 7 . Sprung CL , Geber D , Eidelman LA , et al : Evaluation of triage decisions for intensive care admission . Crit Care Med 1999 ; 27 : 1073 – 1079 8 . Metcalfe MA , Sloggett A , McPherson K : Mor - tality among appropriately referred patients refused admission to intensive - care units [ see comments ] . Lancet 1997 ; 350 : 7 – 11 9 . Hanson LC , Danis M , Lazorick S : Emergency triage to intensive care : Can we use progno - sis and patient preferences ? J Am Geriatr Soc 1994 ; 42 : 1277 – 1281 10 . Ron A , Aronne LJ , Kalb PE : The therapeutic efficacy of critical care units : Identifying sub - groups of patients who benefit . Arch Intern Med 1989 ; 149 : 338 – 341 11 . Intensive care in the United Kingdom : Re - port from the King’s Fund Panel . Anaesthe - sia 1989 ; 44 : 428 – 431 12 . Lemeshow S , Teres D , Klar J , et al : Mortality Probability Models ( MPM II ) based on an international cohort of intensive care unit patients . JAMA 1993 ; 270 : 2478 – 2486 13 . McCabe WR , Jackson GG : Gram negative bacteremia : Etiology and ecology . Arch In - tern Med 1962 ; 110 : 847 – 855 14 . Le Gall JR , Loirat P , Mathieu D , et al : The patients in management of intensive care . In : Guidelines for Better Use of Resources . Miranda DR , Williams A , Loirat P ( Eds ) . Dor - drecht , Kluwer , 1990 , pp 11 – 53 15 . Sznajder M , Leleu G , Buonamico G , et al : Esti - mation of direct cost and resource allocation in intensive care : Correlation with Omega system . Intensive Care Med 1998 ; 24 : 582 – 589 16 . Dragsted L , Jörgensen J , Jensen NH , et al : Interhospital comparisons of patient outcome from intensive care : Importance of lead - time bias . Crit Care Med 1989 ; 17 : 418 – 422 17 . Goldhill DR , Withington PS : The effect of casemix adjustment on mortality as pre - dicted by APACHE II . Intensive Care Med 1996 ; 22 : 415 – 419 18 . Arabi Y , Al Shirawi N , Memish Z , et al : As - sessment of six mortality prediction models in patients admitted with severe sepsis and septic shock to the intensive care unit : A prospective cohort study . Crit Care 2003 ; 7 : R116 – R122 19 . Zhu BP , Lemeshow S , Hosmer DW , et al : Fac - tors affecting the performance of the models in the Mortality Probability Model II system and strategies of customization : A simulation study . Crit Care Med 1996 ; 24 : 57 – 63 20 . Murphy - Filkins R , Teres D , Lemeshow S , et al : Effect of changing patient mix on the performance of an intensive care unit sever - ity - of - illness model : How to distinguish a general from a specialty intensive care unit . Crit Care Med 1996 ; 24 : 1968 – 1973 21 . Gunning K , Rowan K : Outcome data and scoring systems . BMJ 1999 ; 319 : 241 – 244 22 . Angus DC , Linde - Zwirble WT , Lidicker J , et al : Epidemiology of severe sepsis in the United States : Analysis of incidence , out - come , and associated costs of care . Crit Care Med 2004 ; 29 : 1303 – 1310 23 . Singer DE , Carr PL , Mulley AG , et al : Ration - ing intensive care – physician responses to a resource shortage . N Engl J Med 1983 ; 309 : 1155 – 1160 24 . Wasserfallen JB , Revelly JP , Moro D , et al : Can the impact of bed closure in intensive care units be reliably monitored ? Intensive Care Med 2004 ; 30 : 1134 – 1139 25 . Rosenberg AL , Watts C : Patients readmit - ted to ICUs * : A systematic review of risk factors and outcomes . Chest 2000 ; 118 : 492 – 502 26 . Goldfrad C , Rowan K : Consequences of dis - charges from intensive care at night . Lancet 2000 ; 355 : 1138 – 1142 27 . Graf J , Graf C , Janssens U : Analysis of re - source use and cost - generating factors in a German medical intensive care unit employ - ing the Therapeutic Intervention Scoring System ( TISS - 28 ) . Intensive Care Med 2002 ; 28 : 324 – 331 28 . Keenan SP , Doig GS , Martin CM , et al : As - sessing the efficiency of the admission pro - cess to a critical care unit : Does the literature allow the use of benchmarking ? Intensive Care Med 1997 ; 23 : 574 – 580 29 . Azoulay E , Pochard F , Chevret S , et al : Com - pliance with triage to intensive care recom - mendations . Crit Care Med 2001 ; 29 : 2132 – 2136 30 . Einav S , Soudry E , Levin PD , et al : Intensive care physicians’ attitudes concerning distri - bution of intensive care resources : A compar - ison of Israeli , North American and Euro - pean cohorts . Intensive Care Med 2004 ; 30 : 1140 – 1143 Another excuse bites the dust : Low tidal volume ventilation does not increase sedation use * W hile the ARDS Network’s first trial comparing tidal volume ventilations ( 6 mL / kg vs . 12 mL / kg ) in acute lung injury ( ARMA ) was enrolling , I was a pulmonary fellow and helped to managed many of our site’s recruited pa - tients . Each time a new patient entered the study , the care team ( physician , nurses , respiratory therapists ) hoped that the new patient would be randomized by the coordinating center to a 12 mL / kg tidal volume . Like any intensive care team , we might disagree on various aspects of our patients’ care , but we were always unani - mous on this point . We were very confident in our belief that patients receiving 6 mL / kg tidal volume ventilation developed worse hypoxemia , more hypercarbia , and increased tachypnea and needed lots of se - dation to maintain ventilator synchrony and comfort . When the trial was stopped early in 1999 , we assumed that the trial had ended in futility . As bedside clinicians , many of us were very surprised to learn that the trial proved that low tidal volume ventilation actually decreased mortality rate by 25 % ( 1 ) . Despite ARMA , adoption of 6 mL / kg tidal volume as the standard in patients with acute lung injury / acute respiratory distress syndrome ( ALI / ARDS ) is far from universal ( 2 – 5 ) . A recent study shows that average tidal volumes for ALI / ARDS patients have decreased only modestly since the findings of the ARMA study , from 11 . 7 mL / kg to 10 . 7 mL / kg ( 4 ) . One may posit various reasons for this slow adoption among physicians , but few are supported by recent evidence . For exam - ple , some practitioners may choose to * See also p . 766 . Key Words : acute lung injury ; acute respiratory distress syndrome ; sedation ; ARDSNet Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159693 . 52183 . 2E 903 Crit Care Med 2005 Vol . 33 , No . 4 control plateau pressures instead of tidal volumes , despite the clear benefit of us - ing 6 mL / kg tidal volume at every plateau pressure . However , the data gathered by Young et al . ( 4 ) indicate that the plateau pressures have also not changed since ARMA . Perhaps some are concerned about the initial worsening of P O 2 and P CO 2 levels when tidal volumes are low - ered ( 1 ) . This seems unlikely given that ARMA demonstrated that the initial dete - rioration in P O 2 / F IO 2 ratio is short - lived and that by the third day , low tidal vol - ume patients had lower F IO 2 compared with the high tidal volume group . Simi - larly , although low tidal volume ventila - tion causes an increase in P CO 2 initially , there is no difference in pH by day 7 . Based on my own team’s pre - ARMA thinking , I suspect that many physi - cians worry that low tidal volume ven - tilation causes increased ventilator dis - synchrony and increased sedation . In this issue of Critical Care Medicine , Dr . Kahn and colleagues ( 6 ) address the concern about sedation . Retrospectively reviewing the data from 61 ARMA pa - tients enrolled at Harborview Hospital , Seattle , from 1996 to 1999 ( 33 low tidal volume , 28 high tidal volume ) , the in - vestigators found no difference either in the proportion of patients receiving benzodiazepine sedatives or opioid an - algesics or in the dosages of these med - ications . As with any secondary subset analysis from a large trial , the Kahn report may have significant inherent flaws due to its retrospective design , small sample size , and focus on a single site where the sedation practices may not be representative of the norm . The study’s small size does not allow it to detect differences (cid:2) 4 mg / hr of mor - phine or lorazepam , a range that could be clinically significant . However , as the authors note , the study suggested increased sedative use for the high tidal volume group , making it less likely that the low power of the study caused it to miss an increase in sedation needs in the low tidal group . Despite its flaws , the study by Dr . Kahn and colleagues ( 6 ) is the best ev - idence to date that low tidal volume ventilation does not invariably increase the use of sedation . Other available lit - erature supporting the contention that low tidal volume ventilation does not increase sedation use includes pub - lished data from the San Francisco ARMA site ( 7 ) . Although not yet fully published , other data from the ARMA study also suggest that the Seattle and San Francisco findings with regard to sedation may well be true for the entire study population . Undoubtedly , the universal adoption of low tidal volume ventilation for acute lung injury patients will continue be slow . However , clinicians should be reas - sured by the growing body of evidence that low tidal volume ventilation does not result in increased sedation of ALI / ARDS patients . Another excuse against lowering ventilation volumes to 6 mL / kg no longer seems credible . Joseph A . Govert , MD Medical Intensive Care Unit Duke University Medical Center Durham , NC REFERENCES 1 . The Acute Respiratory Distress Syndrome Net - work : Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syn - drome . N Engl J Med 2000 ; 342 : 1301 – 1308 2 . Brower RG , Thompson BT , Ancukiewicz M : Clinical trial of mechanical ventilation with traditional versus lower tidal volumes in acute lung injury and acute respiratory distress syn - drome : Effects on physicians’ practices . Am J Respir Crit Care Med 2004 ; 169 : A256 3 . Weinert CR , Gross CR , Marinelli WA : Impact of randomized trial results on acute lung injury ventilator therapy in teaching hospitals . Am J Respir Crit Care Med 2003 ; 167 : 1304 – 1309 4 . Young MP , Manning HL , Wilson DL et al : Ventilation of patients with acute lung injury and acute respiratory distress syndrome : Has new evidence changed clinical practice ? Crit Care Med 2004 ; 32 : 1260 – 1265 5 . Kallet RH , Corral W , Silverman HJ , et al : Implementation of a low tidal volume ventila - tion protocol for patients with acute lung in - jury or acute respiratory distress syndrome . Respir Care 2001 ; 46 : 1024 – 1037 6 . Kahn JM , Andersson L , Karir V , et al : Low tidal volume ventilation does not increase sedation use in patients with acute lung injury . Crit Care Med 2005 ; 33 : 766 – 771 7 . Cheng IW , Eisner MD , Thompson BT , et al : Acute effects of tidal volume strategy on hemo - dynamics , fluid balance and sedation in acute lung injury . Crit Care Med 2005 ; 33 : 63 – 70 Phospholipase A 2 and acute lung injury : It’s just not that simple * T he phospholipases A 2 ( PLA 2 ) are enzymes that play a crucial role in a variety of inflamma - tory disease processes , includ - ing acute lung injury ( ALI ) and its more severe form , the acute respiratory distress syndrome ( ARDS ) ( 1 – 3 ) . For example , higher concentrations of PLA 2 are found in the bronchoalveolar lavage fluid ( BALF ) in both animals ( 3 – 5 ) and patients with ALI ( 6 , 7 ) and seem to correlate with the sever - ity of lung injury . Intratracheal administra - tion of PLA 2 induces lung injury in animal models ( 8 ) , and inhibition of PLA 2 attenu - ates oleic acid – induced lung injury in rab - bits ( 4 , 5 ) . The contribution of Dr . Nakos and colleagues ( 9 ) in this issue of Critical Care Medicine provides additional evi - dence , albeit indirect , for the role of PLA 2 in ALI and ARDS . PLA 2 are a superfamily of enzymes that hydrolyze phospholipids at the sn - 2 fatty acyl ester bond of membrane phospholip - ids , generating free fatty acids and lyso - phospholipids . PLA 2 thus play a crucial role in the induction of the host inflammatory response through the synthesis of lipid me - diators , such as platelet - activating factor , leukotrienes , and prostaglandins such as thromboxane . Platelet - activating factor is a proinflammatory phospholipid with vari - ous biological effects , including the induc - tion of leukocyte adhesion , endothelial ac - tivation , and production of cytokines and chemokines . The thromboxanes and leuko - trienes are also potent proinflammatory mediators , generated by the cyclooxygen - ase and lipoxygenase pathways , respec - tively . Thromboxane increases capillary * See also p . 772 . Key Words : acute respiratory distress syndrome ; surfactant ; phospholipase A 2 ; platelet - activating – factor acetylhydrolase ; type - II secretory phospholipase A 2 Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159694 . 64555 . F8 904 Crit Care Med 2005 Vol . 33 , No . 4 permeability , whereas the leukotrienes are potent chemoattractants . Several studies have shown that PLA 2 hydrolyze lung sur - factant , producing potentially cytotoxic by - products such as lyso - phosphatidylcholine and directly causing loss of alveolar stabil - ity and subsequent alveolar collapse through surfactant degradation ( 1 , 2 , 4 , 5 , 10 , 11 ) . PLA 2 seem to up - regulate the ex - pression of chemokines and adhesion mol - ecules in the lung vascular endothelium ( 12 ) . PLA 2 seem to play active roles in or - chestrating the host inflammatory re - sponse to lung injury , while at the same time contributing to the diffuse atelectasis and ventilation - perfusion mismatching characteristic of ALI and ARDS . Therefore , given these multiple , overlapping roles in the pathogenesis of ALI and ARDS , the PLA 2 family would appear to be an attrac - tive target for drug discovery . The PLA 2 family is classified into three main subtypes : secretory PLA 2 ( sPLA 2 ) , of which at least ten subtypes have been identified to date , cytosolic Ca 2 (cid:3) - depen - dent PLA 2 ( cPLA 2 ) , and intracellular Ca 2 (cid:3) - independent PLA 2 ( 2 ) . Although the majority of studies have focused on sPLA 2 , recent evidence implicates cPLA 2 in the pathogenesis of ALI as well ( 13 , 14 ) . Dr . Nakos and colleagues ( 9 ) extend the current body of knowledge on the role of PLA 2 in ALI and ARDS in this issue of Critical Care Medicine . Consistent with previous studies , these investigators noted that 1 ) the total PLA 2 activity in the BALF , BAL cells , and plasma in critically ill adults with ARDS is significantly higher compared with critically ill adults who did not meet criteria for either ALI or ARDS and that 2 ) total PLA 2 activity in BALF correlated inversely with Pa O 2 / F IO 2 ratio and directly with mortality . PLA 2 activity was distributed differentially in BAL subfractions . PLA 2 activity was local - ized to the very small surfactant aggre - gate in patients with ARDS , whereas PLA 2 activity was found predominately in the large surfactant aggregate of control pa - tients . As Dr . Nakos and colleagues ( 9 ) point out , the very small surfactant ag - gregate subfraction represents decom - posed surfactant with poor surface prop - erties , providing additional evidence for the role of PLA 2 in surfactant degradation in ALI and ARDS . Of potentially greater importance , these investigators provide unequivocal evidence for the presence of both sPLA 2 and cPLA 2 in the BALF and plasma of adults with ARDS by using both protein analysis and biochemical charac - terization . Although these data are de - scriptive in nature , the temporal relation - ship between BALF sPLA 2 and BALF cPLA 2 , especially when viewed in the con - text of recent animal data suggesting a cause - and - effect relationship between cPLA 2 and ALI ( 13 , 14 ) , suggests that cPLA 2 may be just as important as sPLA 2 in the development of ALI . Alveolar macrophages , the guardians of the lung , are the primary source of PLA 2 via local production and secretion into the alveolar space ( 1 , 2 ) . Alterna - tively , PLA 2 could enter the alveolar space from the pulmonary circulation due to the increased permeability of the alveo - lar - capillary membrane . The current study supports the former mechanism , as there seems to be no correlation between PLA 2 activity in the BALF and the total BALF protein ( a marker of increased al - veolar - capillary permeability ) or to the total PLA 2 activity in the plasma ( 4 ) . Dr . Nakos and colleagues ( 9 ) provide us with two potential explanations for the failure of a specific sPLA 2 inhibitor to improve survival in a recent , multiple - center , clinical trial in adults with multi - ple organ failure and ARDS ( 15 ) . First , as these investigators ( 4 ) and others ( 13 , 14 ) have shown , cPLA 2 may prove to be just as important as sPLA 2 in ALI and ARDS . Notably , the available sPLA 2 inhibitors do not affect cPLA 2 activity . Second , the in - travenous administration of PLA 2 inhibi - tors may not be sufficient to inhibit lo - cally produced enzymes such as PLA 2 . Regardless , PLA 2 seem to play instrumen - tal roles in ALI and ARDS and should remain a focus on further investigation , although once again we are offered proof that when it comes to targeting specific mediators of the inflammatory cascade in diseases such as ALI and ARDS , it is just not that simple . Derek S . Wheeler , MD Division of Critical Care Medicine Cincinnati Children’s Hospital Medical Center Cincinnati , OH REFERENCES 1 . Touqui L , Arbibe L : A role for phospholipase A 2 in ARDS pathogenesis . Mol Med Today 1999 ; 5 : 244 – 249 2 . Balsinde J , Balboa MA , Insel PA , et al : Regula - tion and inhibition of phospholipase A 2 . Annu Rev Pharmacol Toxicol 1999 ; 39 : 175 – 189 3 . Attalah HL , Wu Y , Alaoui - El - Azher M , et al : Induction of type - IIA secretory phospho - lipase A 2 in animal models of acute lung injury . Eur Respir J 2003 ; 21 : 1040 – 1045 4 . Furue S , Kuwabara K , Mikawa K , et al : Cru - cial role of group II phospholipase A 2 in oleic acid - induced acute lung injury in rabbits . Am J Respir Crit Care Med 1999 ; 160 : 1292 – 1302 5 . Furue S , Mikawa K , Nishina K , et al : Thera - peutic time - window of a group IIA phospho - lipase A 2 inhibitor in rabbit acute lung in - jury : Correlation with lung surfactant protection . Crit Care Med 2001 ; 29 : 719 – 727 6 . Kim DK , Fukuda T , Thompson BT , et al : Bronchoalveolar lavage fluid phospholipase A 2 activities are increased in human adult respiratory distress syndrome . Am J Physiol 1995 ; 269 : 109 – 118 7 . Nakos G , Kitsiouli E , Tsangas I , et al : Bron - choalveolar lavage fluid characteristics of early intermediate and late phases of ARDS : Alterations in leukocytes , proteins , PAF , and surfactant components . Intensive Care Med 1998 ; 24 : 296 – 303 8 . Durham SK , Selig WM : Phospholipase A 2 – induced pathophysiologic changes in the guinea pig lung . Am J Pathol 1990 ; 136 : 1283 – 1291 9 . Nakos G , Kitsiouli E , Hatzidaki K , et al : Phospholipases A 2 and platelet - activating – factor acetylhydrolase in patients with acute respiratory distress syndrome . Crit Care Med 2005 ; 33 : 772 – 779 10 . Arbibe L , Koumanov K , Vial D , et al : Gener - ation of lyso - phospholipids from surfactant in acute lung injury is mediated by type - II phospholipase A 2 and inhibited by a direct surfactant protein A - phospholipase A 2 pro - tein interaction . J Clin Invest 1998 ; 102 : 1152 – 1160 11 . Chabot S , Koumanov K , Lambeau G , et al : Inhibitory effects of surfactant protein A on surfactant phospholipids hydrolysis by se - creted phospholipases A 2 . J Immunol 2003 ; 171 : 995 – 1000 12 . Beck GC , Yard BA , Shulte J , et al : Secreted phospholipase A 2 induce the expression of chemokines in microvascular endothelium . Biochem Biophys Res Comm 2003 ; 300 : 731 – 737 13 . Nagase T , Uozumi N , Ishii S , et al : Acute lung injury by sepsis and acid aspiration : A key role for cytosolic phospholipase A 2 . Nat Im - munol 2000 ; 1 : 42 – 46 14 . Nagase T , Uozumi N , Aoki - Nagase T , et al : A potent inhibitor of cytosolic phospholipase A 2 , arachidonyl trifluoromethyl ketone , at - tenuates LPS - induced lung injury in mice . Am J Physiol Lung Cell Mol Physiol 2003 ; 284 : L720 – L726 15 . Abraham E , Naum C , Bandi V , et al : Efficacy and safety of LY315920Na / S - 5920 , a selective inhibitor of 14 - kDa group IIA secretory phos - pholipase A 2 , in patients with suspected sep - sis and organ failure . Crit Care Med 2003 ; 31 : 718 – 728 905 Crit Care Med 2005 Vol . 33 , No . 4 Arterial pressure , vasopressors and septic shock : Higher is not necessarily better * R esuscitation of sepsis and hy - potension begins with fluid infusion . However , in patients with septic shock , hypoten - sion persists despite vigorous fluid infu - sion . Although cardiac output increases , the marked underlying vasodilation re - sults in a persistently low blood pressure . Accordingly , vasopressor therapy is re - quired to achieve a level of arterial pres - sure that maintains organ perfusion , while avoiding excessive vasoconstriction that might further impair organ perfu - sion and potentially cardiac performance . To achieve this goal , vasopressor agents are titrated to a minimum level of mean arterial pressure ( MAP ) that is pre - sumed to be adequate for critical organ perfusion . That level has varied from 60 mm Hg , which represents the lower limit of the autoregulatory threshold below which major organ blood flow becomes pressure dependent , to much higher lev - els ( 1 – 3 ) . Indeed , levels of mean arterial pressure of 80 – 90 mm Hg have been used as end points of therapy in a number of studies ( 4 – 6 ) . One rationale for the higher end points of MAP is that sepsis , with its alterations in vascular tone , may produce alterations in normal autoregu - latory function that increase the range of pressure - dependent organ blood flow . In addition , older patients and those with underlying cardiovascular disease might require higher levels of MAP because of shifts in their normal autoregulatory curves of the type seen with chronic hy - pertension . Our group ( 7 ) previously examined the relationship between MAP and organ perfusion in patients with septic shock . In ten patients , with each patient serving as his or her own control , norepinephrine was titrated to a MAP of 65 , 75 , and 85 mm Hg . Hemodynamic variables , indexes of oxygen metabolism , splanchnic perfu - sion , skin perfusion , and urine output were monitored . No significant difference was observed in arterial lactate concen - tration , mixed venous oxygen saturation , or any index of organ perfusion between the three different levels of MAP . Cardiac index and systemic vascular resistance in - creased at the higher levels of MAP con - sistent with the higher doses of norepi - nephrine that were used . The report by Dr . Bourgoin and col - leagues ( 8 ) , in this issue of Critical Care Medicine , extends these observations . Twenty - eight patients were prospectively randomized to norepinephrine titrated to a MAP of 65 mm Hg or a MAP of 85 mm Hg . Hemodynamic variables , indexes of oxygen metabolism , and renal function variables were measured . None of their patients received dopamine , in contrast to the study of Ledoux et al . ( 7 ) . No sig - nificant differences in arterial lactate concentration , oxygen consumption , cre - atinine clearance , creatinine concentra - tion , or urine output were observed be - tween the two groups . Cardiac index and systemic vascular resistance increased as the norepinephrine was increased . Although both studies are consistent with each other , they are limited by the small patient numbers . A group of older patients with significant underlying hy - pertension and vascular disease might re - spond differently and require a higher perfusion pressure . In addition , the infu - sion periods of both studies are also rel - atively short . However , as reflected by other studies examining the effects of dif - ferent vasoactive drugs in septic shock , these periods should have been adequate to observe any effects on regional organ perfusion ( 6 , 9 , 10 ) . It is also possible that there may have a benefit from higher levels of MAP , but this seems unlikely . The lack of change in any variable over a range of 20 mm Hg in MAP suggests that the patients in the studies of Ledoux et al . ( 7 ) and Dr . Bourgoin and colleagues ( 8 ) were within their autoregulatory range . This conclusion is supported by evidence that in patients with septic shock , auto - regulation of cerebral blood flow is main - tained over a similar range of MAP ( 11 ) . These observations contrast with reports in which increases in MAP in septic shock patients from levels of 50 – 55 mm Hg to the range of 70 – 90 mm Hg were associ - ated with significant increases in urine output and improvement in renal func - tion ( 5 , 6 ) . Taken together , the data sug - gest that a MAP of 60 – 65 mm Hg may define the autoregulatory threshold , at least for renal blood flow , in patients with septic shock . Although neither study identified complications associated with the use of higher doses of norepinephrine that the end point of a MAP of 85 mm Hg re - quired , other patients may respond to similar doses with excessive vasoconstric - tion and tachyarrhythmias . Importantly , both studies involved patients who at baseline had their intravascular volume optimized and hyperdynamic profiles . Pa - tients with hypovolemia or significant cardiac dysfunction and baseline hypody - namic profiles may demonstrate different responses as vasopressors are increased to achieve higher levels of MAP . In this setting , the vasoconstriction associated with increasing MAP may result in a lower cardiac output and decreased renal blood flow ( 12 , 13 ) . In addition , these results may not be applicable to other vasopressor agents that have different he - modynamic profiles . As evidenced in the current study , the offsetting (cid:5) - and (cid:6) - ad - renergic activity of norepinephrine usu - ally results in only small increases in heart rate and modest augmentation of cardiac output ( 8 ) . Dopamine is associ - ated with greater increases in heart rate , thereby increasing the possibility of tachyarrhythmias and myocardial isch - emia ( 14 ) . Both neosynephrine and vaso - pressin have minimal intrinsic inotropic activity and have the potential to decrease cardiac output and organ perfusion as the dose is increased ( 15 ) . * See also p . 780 . Key Words : mean arterial pressure ; organ perfu - sion ; septic shock Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159695 . 20613 . 2C 906 Crit Care Med 2005 Vol . 33 , No . 4 The studies of Ledoux et al . ( 7 ) and Dr . Bourgoin and colleagues ( 8 ) should not be interpreted to indicate that 65 mm Hg should be the end point for vasopressor therapy for all patients with septic shock . These studies do , however , suggest that this is a reasonable starting point . In in - dividual patients , the goal MAP should be modified higher , or lower , as determined by measures of hemodynamic perfor - mance , tissue perfusion , and organ func - tion . Similarly , in patients in with signif - icant vascular disease , a higher level of MAP may be desired . Manuela Reisbeck , MD Mark E . Astiz , MD , FCCM Saint Vincents Catholic Medical Center of New York New York Medical College REFERENCES 1 . Johnson P : Autoregulation of blood flow . Circ Res 1986 ; 59 : 483 – 495 2 . Mueller H , Ayres S , Giannelli S , et al : Effect of isoproterenol , l - norepinephrine , and in - traaortic counterpulsation on hemodynam - ics and myocardial metabolism in shock fol - lowing acute myocardial infarction . Circ 1972 ; 16 : 335 – 350 3 . Strandgaard S , Sengupta E , MacKenzie E , et al : The lower and upper limits of autoregu - lation of cerebral blood flow . In Cerebral circulation and Metabolism . Langfitt T et al ( Eds ) . New York , Springer - Verlag , 1975 , pp 3 – 6 4 . Hayes MA , Timmins AC , Yau E , et al : Eleva - tion of systemic oxygen delivery in the treat - ment of critically ill patients . N Engl J Med 1994 ; 330 : 1717 – 1722 5 . Desjars P , Pinaud M , Bugnon D , et al : Nor - epinephrine therapy has no deleterious renal effects in human septic shock . Crit Care Med 1989 ; 17 : 426 – 429 6 . Martin C , Eon B , Akin P , et al : Renal effects of norepinephrine used to treat septic shock . Crit Care Med 1990 ; 18 : 282 – 285 7 . Ledoux D , Astiz M , Carpati C , et al : Effects of perfusion pressure on tissue perfusion in septic shock . Crit Care Med 2000 ; 28 : 2729 – 2732 8 . Bourgoin A , Leone M , Delmas A , et al : In - creasing mean arterial pressure in patients with septic shock : Effects on oxygen vari - ables and renal function . Crit Care Med 2005 ; 33 : 780 – 786 9 . Marik P , Mohedin M : The contrasting effects of dopamine and norepinephrine on systemic and splanchnic oxygen utilization in hyper - dynamic sepsis . JAMA 1994 ; 272 : 1354 – 1357 10 . Neviere R , Mathieu D , Chagnon JL , et al : The contrasting effects of dobutamine and dopa - mine on gastric mucosal perfusion in septic patients . Am J Respir Crit Care Med 1996 ; 154 : 1684 – 1688 11 . Matta B , Stow P : Sepsis - induced vasoparaly - sis does not involve the cerebral vasculature : Indirect evidence from autoregulation and carbon dioxide reactivity studies . Br J Anesth 1996 ; 76 : 790 – 794 12 . Tristani F , Cohn J : Studies in clinical shock and hypotension . VI . Renal hemodynamics before and during treatment . Circulation 1970 ; 52 : 839 – 851 13 . Gunnar R , Cruz A , Boswell J , et al : Myocar - dial infarction with shock . Hemodynamic studies and results of therapy . Circulation 1966 ; 32 : 753 – 762 14 . Mueller H , Evans R , Ayres S : Effect of dopa - mine on hemodynamics and myocardial me - tabolism in shock following myocardial in - farction in man . Circulation 1978 ; 57 : 361 – 365 15 . Landry D , Levin H , Gallant E , et al : Vasopres - sin deficiency contributes to vasodilation of septic shock . Circulation 1997 ; 95 : 1122 – 1125 Ventilator - induced lung injury : Do dynamic factors also play a role ? * T he landmark ARDS Network Study ( 1 ) comparing lower and higher tidal volume venti - lation firmly established the clinical importance of ventilator - induced lung injury ( VILI ) , with the improved outcome attributed to a lower lung strain . Rather than indicating the end of a successful bench - to - bedside program , these data signal the start of an exciting period of laboratory and clinical progress . Although most interest has focused on static variables during ventilation such as end - expiratory pressure , tidal volume , and end - inspiratory volume ( 2 ) , dynamic ventilatory variables such as inspiratory flow rate and flow profile may also cause VILI . Ventilatory strategies aimed to mini - mize VILI need to be understood in the context of normal pulmonary physiology . Increased lung volume is a potent physi - ologic stimulus for the release of surfac - tant from epithelial type II cells ( 3 ) . Sur - factant reduces surface tension at the air - liquid interface , and , as surface tension is the major factor determining lung elas - tance , the healthy lung becomes easier to inflate . However , just as an increase in lung volume with deformation of the ep - ithelium is a stimulus to surfactant se - cretion , it may also result in stress failure of the plasma membrane . Using cultured alveolar epithelial type II cells and A549 cells , the amplitude , magnitude , and rate of cell deformation are all independently cytotoxic ( 4 , 5 ) . These factors are also associated with increased vesicular lipid trafficking , with or without repair of the disrupted or wounded plasma membrane ( 6 ) . As cell wounding is a potent factor both altering proinflammatory gene ex - pression and release of growth factors , this suggests that excessive strain can lead to important changes at the blood - gas barrier before cell injury becomes ap - parent ( 7 ) . Increased amplitude and magnitude of cell deformation are analogous to static changes in tidal , end - inspiratory , or end - expiratory volume . Similarly , an increase in the rate of deformation is analogous to increased inspiratory flow or respiratory rate as a dynamic mechanism of VILI ; however , isolating the relative impor - tance of these factors can be difficult due to their inherent linkage through inspira - tory time , inspiratory to expiratory ratio , and tidal volume . In this issue of the Critical Care Medicine , Dr . Conrad and coworkers ( 8 ) report that lower respira - tory rate reduces high tidal volume VILI . Lung injury was assessed using a well - described and sensitive method for as - sessing permeability , the capillary filtra - tion coefficient , and experiments were * See also p . 835 . Key Words : ventilator - induced lung injury ; me - chanical ventilation ; inspiratory flow rate ; inspiratory flow profile ; pulmonary physiology Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159850 . 11273 . A5 907 Crit Care Med 2005 Vol . 33 , No . 4 performed in the isolated perfused rat lung , which allows control of many po - tential confounding factors . As systemic inflammatory cells and mediators are ex - cluded from this preparation , injury due to these processes could not be explored . Nevertheless , this is an important contri - bution and adds to the notion that dy - namic factors during tidal ventilation also contribute to lung injury . The clinical significance of dynamic factors in VILI is currently uncertain ; however , there is increasing evidence that respiratory rate , inspiratory flow rate , and flow profile can influence the development of VILI . Although earlier re - ports ( 9 , 10 ) of reduced injury at lower respiratory rates are consistent with the findings reported by Dr . Conrad and co - workers , in all these studies , a fixed in - spiratory to expiratory ratio meant that the decrease in respiratory rate was ac - companied by decreased inspiratory flow rate . When inspiratory flow rate is inde - pendently varied ( 11 – 13 ) , flow rather than respiratory rate is the dominant fac - tor producing lung injury . This is analo - gous to the strain rate rather than the number of deformations , although this must be a factor , as a single deformation does not appear injurious and Dr . Conrad and coworkers found a highly significant correlation between the number of breaths and injury . A series of clinical studies ( 14 – 16 ) reported increased non - linear behavior with increased flow rate , measured as an increase in viscoelastic pressure , in patients with acute respira - tory distress syndrome and as positive end - expiratory pressure was increased to 15 cm H 2 O . As surface tension at the air - liquid interface ( 17 ) and heteroge - neous distribution of ventilation account for most of the viscoelastic pressure , this suggests that the effects of increased flow rate are clinically relevant and result in greater lung strain . A second dynamic source of VILI is the inspiratory flow profile . Some clinicians use pressure - controlled ventilation as it delivers a decelerating flow profile . This leads to a lower end - inspiratory pressure than volume - controlled ventilation deliv - ered at the same tidal volume and posi - tive end - expiratory pressure , due to ear - lier dissipation of resistive pressure ; however , the elastic distending pressure is similar . Under these circumstances , there is no difference in gas exchange , hemodynamic stability , or distribution of ventilation in patients with acute lung injury ( 18 ) . Although there may be a dif - ference in nonlinear behavior favoring pressure - controlled ventilation , due to differences in the buildup of viscoelastic pressure , this is small compared with the increase found with a prolonged inspira - tion and inverse ratio ventilation . Labo - ratory and clinical studies ( 19 ) examining the significance of these differences are equivocal due to difficulties and differ - ences in study design . However , the re - cent report ( 20 ) of VILI with pressure - regulated volume control ventilation , compared with matched volume control ventilation , can be attributed to the greater peak inspiratory flow rate inher - ent in a decelerating flow profile . As is common with an important con - tribution , the data presented by Dr . Conrad and coworkers raise many fascinating is - sues . We need more carefully designed studies to better understand how dynamic ventilatory factors produce VILI and , in ad - dition to influencing the prescription of mechanical ventilation , whether there are useful pharmacologic interventions . Con - trol of carbon dioxide tension during lower tidal volume ventilation usually necessi - tates higher respiratory rates , but this could mitigate some of the beneficial ef - fects . If future clinical studies examine these issues they will need even more care - ful design , as differences in flow rate will potentially introduce confounding factors . For example , low inspiratory flow rates might lead to flow starvation , increased pa - tient - ventilator dyssynchrony , and a subse - quent increase in sedation . At higher respi - ratory rates there will be the risk of gas trapping and inadvertent positive end - expiratory pressure , so this may need to be combined with permissive hypercapnea . Despite these caveats , a growing body of elegant basic research suggests that dy - namic factors also cause VILI . As the extent of deformation injury due to rapid strain rates is similar to that with either a large amplitude or magnitude of strain , it’s time to integrate these concepts into mechanical ventilation strategies . Andrew D . Bersten , MBBS , MD Dani - Louise Bryan , PhD Flinders Medical Centre and School of Medicine Flinders University South Australia REFERENCES 1 . The Acute Respiratory Distress Syndrome Network : Ventilation with lower tidal vol - umes as compared with traditional tidal vol - umes for acute lung injury and the acute respiratory distress syndrome . N Engl J Med 2000 ; 342 : 1301 – 1308 2 . Dreyfuss D , Saumon G : Ventilator - induced lung injury : Lessons from experimental stud - ies . Am J Respir Crit Care Med 1998 ; 157 : 294 – 323 3 . Nicholas TE , Barr HA : The release of surfac - tant in the rat lung by brief periods of hy - perventilation . Respir Physiol 1983 ; 52 : 69 – 83 4 . Tschumperlein DJ , Oswari J , Margulies SS : Deformation - induced injury of alveolar epi - thelial cells . Effect of frequency , duration and amplitude . Am J Respir Crit Care Med 2000 ; 162 : 357 – 362 5 . Vlahakis NE , Schroeder MA , Pagano RE , et al : Deformation - induced lipid trafficking in alveolar epithelial cells . Am J Physiol 2001 ; 280 : L938 – L946 6 . Vlahakis NE , Schroeder MA , Pagano RE , et al : Role of deformation - induced lipid traffick - ing in the prevention of plasma membrane stress failure . Am J Respir Crit Care Med 2002 ; 166 : 1282 – 1289 7 . McNeil PL : Cell suffering and its prevention in lung . Am J Respir Crit Care Med 2003 ; 167 : 1046 – 1047 8 . Conrad SA , Zhang S , Arnold TC , et al : Pro - tective effects of low respiratory frequency in experimental ventilator - associated lung in - jury . Crit Care Med 2005 ; 33 : 835 – 840 9 . Hotchkiss JR , Blanch L , Murias G , et al : Effects of decreased respiratory frequency on ventilator - induced lung injury . Am J Respir Crit Care Med 2000 ; 161 : 463 – 468 10 . Rich PB , Douillet CD , Hurd H , et al : Effect of ventilatory rate on airway cytokine levels and lung injury . J Surg Res 2003 ; 113 : 139 – 145 11 . Rich PB , Reickert CA , Sawada S , et al : Effect of rate and inspiratory flow on ventilator - induced lung injury . J Trauma 2000 ; 49 : 903 – 911 12 . D’Angelo E , Pecchiari M , Saetta M , et al : Dependence of lung injury on inflation rate during low - volume ventilation in open - chest rabbits . J Appl Physiol 2004 ; 97 : 260 – 268 13 . Kotani M , Kotani T , Li Z , et al : Reduced inspiratory flow attenuates IL - 8 release and MAPK activation of lung overstretch . Eur Respir J 2004 ; 24 : 238 – 246 14 . D’Angelo E , Calderini E , Torri G , et al : Re - spiratory mechanics in anaesthetized para - lyzed humans : Effects of flow , volume , and time . J Appl Physiol 1989 ; 67 : 2556 – 2564 15 . Eissa NT , Ranieri VM , Corbeil C , et al : Anal - ysis of behavior of the respiratory system in ARDS patients : Effects of flow , volume and time . J Appl Physiol 1991 ; 70 : 2719 – 2729 16 . Eissa NT , Ranieri VM , Corbeil C , et al : Effect of PEEP on the mechanics of the respiratory system in ARDS patients . J Appl Physiol 1992 ; 73 : 1728 – 1735 17 . Bachofen H , Hildebrandt J , Bachofen M : Pressure - volume curves of air - and liquid - filled excised lungs—Surface tension in situ . J Appl Physiol 1970 ; 29 : 422 – 431 18 . Edibam C , Rutten AJ , Collins DV , et al : Effect of inspiratory flow pattern and inspiratory to 908 Crit Care Med 2005 Vol . 33 , No . 4 expiratory ratio on nonlinear elastic behavior in patients with acute lung injury . Am J Respir Crit Care Med 2003 ; 167 : 702 – 707 19 . Esteban A , Alia I , Gordo F , et al : Prospective randomized trial comparing pressure - con - trolled and volume - controlled ventilation in ARDS . For the Spanish Lung Failure Collab - orative Group . Chest 2000 ; 117 : 1690 – 1696 20 . Maeda Y , Fujino Y , Uchiyama A , et al : Effects of peak inspiratory flow on development of ventilator - induced lung injury in rabbits . An - esthesiology 2004 ; 101 : 722 – 728 Bubbles in the brain : What to do for arterial gas embolism ? * A rterial gas embolism ( AGE ) oc - curs in two major situations : diving while breathing com - pressed gas ( e . g . , scuba diving ) and direct entry of gas into the circula - tion during medical procedures . In div - ing , the cause is pulmonary overpressur - ization by failing to exhale or regional gas trapping during ascent , leading to alveo - lar rupture and entry of gas into pulmo - nary capillaries . Iatrogenic causes in - clude accidental air injection during cardiopulmonary bypass or angiography . Rarely , AGE can be caused by pulmonary barotrauma during mechanical ventila - tion in children and even less commonly in adults . AGE can also be caused by venous gas embolism entering the arte - rial circulation via a right - to - left shunt such as an atrial septal defect or patent foramen ovale or directly via the pulmo - nary capillary bed . Venous gas embolism commonly occurs in divers ; it can also occur due to accidental intravenous gas injection or passive entry when perform - ing surgery in tissues where venous pres - sure is subatmospheric , for example , dur - ing cesarean section , spine surgery , and cranial surgery in the sitting position . Arterial gas is distributed quantita - tively according to blood flow . Thus , the brain , with close to 20 % of cardiac out - put , receives a major portion . The spinal cord , receiving only 1 – 2 % of cardiac out - put , is almost never involved without ma - jor cerebral injury unless there has been a significant load of inert gas due to a compressed gas dive ( 1 ) . One mechanism of injury is direct vas - cular occlusion causing sudden ischemia . However , when only small amounts of arterial gas are embolized , insufficient to cause prolonged occlusion , there is a de - layed reduction in cerebral blood flow , even after the bubbles have dissipated ( 2 ) . Although the cause of this is not fully understood , leukocytes appear to be re - quired for the effect ( 3 ) , possibly due to their adherence to damaged endothelium ( 4 , 5 ) . AGE presents clinically like a stroke , with sudden onset of loss of conscious - ness , acute focal or multifocal neurologic deficit , or sudden death . There may be associated cardiac arrhythmias or evi - dence of myocardial ischemia . When AGE is due to massive or persistent venous gas embolism , there may be pulmonary edema due to capillary endothelial leak . The diagnosis is made presumptively when there is the combination of a char - acteristic presentation and a plausible cause . Few tests are helpful in managing the patient . Imaging ( e . g . , brain com - puted tomography ) reveals intravascular air inconsistently ( 6 ) . Severe AGE can cause elevation of serum creatine phos - phokinase . However , to date specific markers of AGE - induced central nervous system injury remain to be elucidated . Initial treatment , in addition to usual supportive maneuvers , consists of adminis - tration of high concentrations of oxygen ( 100 % inspired concentration if possible ) . Nitrous oxide will cause intravascular bub - bles to grow ; hence , if it is being used for general anesthesia it should immediately be discontinued in favor of oxygen . Posi - tioning of the patient should be consistent with maintenance of an adequate blood pressure and open airway and prevention of aspiration . Head - down or lateral positions are probably of little use ( 7 , 8 ) . The definitive treatment is adminis - tration of hyperbaric oxygen ( HBO ) , which acutely reduces bubble volume , fa - cilitates oxygenation of ischemic tissue , and raises the nitrogen partial pressure gradient between bubble and surround - ing tissue , thus facilitating diffusion of nitrogen out of the gas phase into solu - tion . In addition to bubble volume reduc - tion , there are other possible beneficial pharmacologic effects of HBO on cerebral ischemia , such as reduction of cerebral neutrophil sequestration ( 9 , 10 ) , excito - toxicity ( 11 , 12 ) , cyclooxygenase - 2 over - expression ( 13 ) , and apoptosis ( 14 ) . It has been suggested that the appro - priate indication for hyperbaric oxygen treatment is the presence of visible gas by brain computed tomography ( 15 ) . How - ever , a clinical response to HBO often occurs in patients with no visible air on CT ( 6 ) . Moreover , decision making fo - cused solely on removing gas fails to rec - ognize the other actions of HBO . Imaging is useful only if the diagnosis is in doubt , to exclude other possible causes such as hemorrhage . Clinical series suggest that earlier HBO treatment results in better outcome ( 16 ) , although instances of good recovery have been reported even after 24 hrs’ delay or more ( 6 ) . Severe cases can improve initially and then deteriorate ( 17 , 18 ) . Randomized trials of HBO in AGE have not been performed and are unlikely ever to be , due to the sporadic occurrence of the disease and the effectiveness of HBO , strongly supported by clinical ex - perience , meta - analysis ( 19 ) , and animal studies ( 18 , 20 ) . There is evidence that some adjuncts , such as lidocaine and per - fluorocarbon emulsions , may be effective . Since investigations on humans are difficult to do in this uncommon disease , further understanding will have to be based to a large extent on studies in an - imals . In this issue of Critical Care Med - icine , Dr . van Hulst and colleagues ( 21 ) report the latest of a series of experiments performed by their group , targeted at de - fining the mechanisms of injury and ap - propriate care in the first few hours fol - lowing AGE . In the present study , the investigators examined the effect of im - mediate and delayed HBO in anesthetized pigs after intracarotid injection of 0 . 5 mL / kg air . Using a schedule of HBO com - monly used for treatment of human AGE ( U . S . Navy Treatment Table 6 ) , two * See also p . 841 . Key Words : arterial gas embolism ; hyperbaric ox - ygenCopyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159726 . 47336 . F8 909 Crit Care Med 2005 Vol . 33 , No . 4 groups of animals received HBO , either within the first 3 mins after embolization or 1 hr later . During the 1 - hr latent pe - riod , the animals receiving delayed HBO were given 100 % oxygen and were mildly hyperventilated . End points included brain tissue P O 2 , electroencephalograph , brain glucose , glycerol , pyruvate , and lac - tate measured using microdialysis . Brain lactate increased shortly after emboliza - tion , whereas brain tissue glucose de - creased . Similar changes have been ob - served in studies of brain ischemia and traumatic injury . Brain P O 2 , which de - creased after embolization , rose signifi - cantly within a few minutes after starting HBO , both immediate and delayed . Intra - cranial pressure rose in both groups of animals but only to levels 35 – 50 % as high as untreated animals ( 22 ) . What has Dr . van Hulst and col - leagues’ study added to our knowledge ? This is the first large animal study to examine the physiologic and metabolic effects of AGE and its treatment . The study has demonstrated the effectiveness of HBO in lowering intracranial pressure and ameliorating abnormalities in cerebral metabolism , even after a 60 - min delay . What remains to be done ? Using this excellent model , fruitful studies could pursue the effects of longer delays to HBO and lower doses of air and the effec - tiveness of adjunctive agents such as li - docaine ( 23 ) , perfluorocarbons ( 24 ) , and possibly other agents . Measurement of neurotransmitters could easily be added . The further development of a survival model would allow the implementation of functional and histologic end points . Richard E . Moon , MD Departments of Anesthesiology and Medicine Center for Hyperbaric Medicine and Environmental Physiology Duke University Medical Center Durham , NC REFERENCES 1 . Neuman TS , Bove AA : Combined arterial gas embolism and decompression sickness fol - lowing no - stop dives . Undersea Biomed Res 1990 ; 17 : 429 – 436 2 . Helps SC , Parsons DW , Reilly PL , et al : The effect of gas emboli on rabbit cerebral blood flow . Stroke 1990 ; 21 : 94 – 99 3 . Helps SC , Gorman DF : Air embolism of the brain in rabbits pre - treated with mechloreth - amine . Stroke 1991 ; 22 : 351 – 354 4 . Levin LL , Stewart GJ , Lynch PR , et al : Blood and blood vessel wall changes induced by decompression sickness in dogs . J Appl Physiol 1981 ; 50 : 944 – 949 5 . Nossum V , Koteng S , Brubakk AO : Endothe - lial damage by bubbles in the pulmonary artery of the pig . Undersea Hyperb Med 1999 ; 26 : 1 – 8 6 . Benson J , Adkinson C , Collier R : Hyperbaric oxygen therapy of iatrogenic cerebral arterial gas embolism . Undersea Hyperb Med 2003 ; 30 : 117 – 126 7 . Mehlhorn U , Burke EJ , Butler BD , et al : Body position does not affect the hemodynamic response to venous air embolism in dogs . Anesth Analg 1994 ; 79 : 734 – 739 8 . Rodriguez RA , Cornel G , Weerasena NA , et al : Effect of Trendelenburg head position during cardiac deairing on cerebral microemboli in children : A randomized controlled trial . J Thorac Cardiovasc Surg 2001 ; 121 : 3 – 9 [ Er - ratum appears in J Thorac Cardiovasc Surg 2001 ; 121 : 433 ] 9 . Atochin DN , Fisher D , Demchenko IT , et al : Neutrophil sequestration and the effect of hyperbaric oxygen in a rat model of tempo - rary middle cerebral artery occlusion . Under - sea Hyperb Med 2000 ; 27 : 185 – 90 10 . Miljkovic - Lolic M , Silbergleit R , Fiskum G , et al : Neuroprotective effects of hyperbaric ox - ygen treatment in experimental focal cere - bral ischemia are associated with reduced brain leukocyte myeloperoxidase activity . Brain Res 2003 ; 971 : 90 – 94 11 . Badr AE , Yin W , Mychaskiw G , et al : Effect of hyperbaric oxygen on striatal metabolites : A microdialysis study in awake freely moving rats after MCA occlusion . Brain Res 2001 ; 916 : 85 – 90 12 . Yang ZJ , Camporesi C , Yang X , et al : Hyper - baric oxygenation mitigates focal cerebral in - jury and reduces striatal dopamine release in a rat model of transient middle cerebral ar - tery occlusion . Eur J Appl Physiol 2002 ; 87 : 101 – 107 13 . Yin W , Badr AE , Mychaskiw G , et al : Down regulation of COX - 2 is involved in hyperbaric oxygen treatment in a rat transient focal ce - rebral ischemia model . Brain Res 2002 ; 926 : 165 – 171 14 . Yin D , Zhou C , Kusaka I , et al : Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model . J Cereb Blood Flow Metab 2003 ; 23 : 855 – 864 15 . Dexter F , Hindman BJ : Recommendations for hyperbaric oxygen therapy of cerebral air embolism based on a mathematical model of bubble absorption . Anesth Analg 1997 ; 84 : 1203 – 1207 16 . Blanc P , Boussuges A , Henriette K , et al : Iatrogenic cerebral air embolism : Impor - tance of an early hyperbaric oxygenation . In - tensive Care Med 2002 ; 28 : 559 – 563 17 . Pearson RR , Goad RF : Delayed cerebral edema complicating cerebral arterial gas em - bolism : Case histories . Undersea Biomed Res 1982 ; 9 : 283 – 296 18 . Leitch DR , Greenbaum LJ Jr , Hallenbeck JM : Cerebral arterial air embolism : IV . Failure to recover with treatment , and secondary dete - rioration . Undersea Biomed Res 1984 ; 11 : 265 – 274 19 . Dutka AJ : Air or gas embolism . In : Hyper - baric Oxygen Therapy : A Critical Review . Camporesi EM , Barker AC ( Eds ) . Bethesda , MD , Undersea and Hyperbaric Medical Soci - ety , 1991 , p 1 – 10 20 . McDermott JJ , Dutka AJ , Koller WA , et al : Effects of an increased PO 2 during recom - pression therapy for the treatment of experimental cerebral arterial gas embo - lism . Undersea Biomed Res 1992 ; 19 : 403 – 413 21 . van Hulst RA , Drenthen J , Haitsma JJ , et al : Effects of hyperbaric treatment in cere - bral air embolism on intracranial pressure , brain oxygenation , and brain glucose me - tabolism in the pig . Crit Care Med 2005 ; 33 : 841 – 846 22 . van Hulst RA , Lameris TW , Hasan D , et al : Effects of cerebral air embolism on brain metabolism in pigs . Acta Neurol Scand 2003 ; 108 : 118 – 124 23 . Mitchell SJ : Lidocaine in the treatment of decompression illness : A review of the liter - ature . Undersea Hyperb Med 2001 ; 28 : 165 – 174 24 . Dromsky DM , Spiess BD , Fahlman A : Treatment of decompression sickness in swine with intravenous perfluorocarbon emulsion . Aviat Space Environ Med 2004 ; 75 : 301 – 305 910 Crit Care Med 2005 Vol . 33 , No . 4 One step forward : An advance in understanding of adrenal insufficiency in the pediatric critically ill * R elative adrenal insufficiency is a murky concept for many an endocrinologist and intensiv - ist . This is especially true in pediatrics , where data often lag those ob - tained in adults . The report published in this issue of Critical Care Medicine by Dr . Pizarro and colleagues ( 1 ) brings to light the largest prospective observational data in the pediatric intensive care unit ( PICU ) and should serve to broaden the discus - sion about adrenal insufficiency in the critically ill to include the pediatric pop - ulation . In this study , 57 patients were identi - fied with septic shock after the authors excluded any who had received etomidate or glucocorticoid therapy in the previous week . All patients underwent a 250 - (cid:7) g corticotropin stimulation test and were classified into four groups based on their baseline cortisol ( (cid:2) or (cid:2) 20 (cid:7) g / dL or 550 nmol / L ) and response to corticotropin ( (cid:1) or (cid:8) 9 (cid:7) g / dL increment or 250 nmol / L ) . Absolute or relative adrenal insufficiency was observed in a surprising 44 % of sub - jects , but this may have more to do with the lack of consistent definition across studies , detailed in Table 2 of the Pizarro study . Why this group chose their cutoff values , however , is not entirely clear . The data were remarkable in their ability to predict catecholamine - resistant shock ( p (cid:2) . 05 ) , although not mortality ( p (cid:1) . 08 ) . Whether adrenal insufficiency causes cat - echolamine - resistant shock or merely is a feature of it , like multiple organ failure , is not discernible from the data . Major pre - dictors of mortality were chronic illness and multiple organ failure . The concept of relative adrenal insuf - ficiency has gained prominence in the adult critical care world , especially after publication of a prospective , randomized control trial ( n (cid:1) 300 ) demonstrating a reduction in mortality among those with septic shock who failed a 250 - (cid:7) g tetraco - sactrin stimulation test , defined as a stimulated cortisol increment of (cid:1) 9 (cid:7) g / dL ( 2 ) . Although this study did not take into account basal cortisol concen - tration and it included for part of enroll - ment patients pretreated with etomidate , an agent know to cause adrenal suppres - sion ( 3 ) , it has contributed along with other smaller randomized control trials ( 4 – 6 ) to the establishment of treatment guidelines recommending stress dosing ( “low dose” ) of hydrocortisone for pa - tients with septic shock who fail to raise their cortisol by an increment of 9 (cid:7) g / dL in response to corticotropin stimulation ( 7 ) . In the pediatric literature , far fewer studies have been published and none have been able to demonstrate an effect on mortality of response to corticotropin stimulation or hydrocortisone therapy . The current study by Dr . Pizarro and col - leagues , therefore , helps move the field forward by confirming an association be - tween adrenal function and catechol - amine - resistant shock and suggesting an association with mortality , to be con - firmed in an appropriately powered larger study . By excluding etomidate - or ste - roid - treated patients , the authors have avoided common pitfalls in adrenal eval - uations . What remains unaddressed by this and other papers is the correct dose of corticotropin to accurately distinguish those who will benefit from steroid sup - plementation : These investigators chose to use 250 (cid:7) g ; however , it is clear that this is a pharmacologic dose , especially in small children ( median age , 27 months ) . Whether 1 (cid:7) g would produce a more revealing degree of adrenal stimulation in the PICU remains to be seen . And if the 1 - (cid:7) g test did become the standard , it is likely that new cutoff values would need to be set ( 8 ) . The study by Dr . Pizarro and col - leagues ( 1 ) , as well as the other adrenal studies cited here , all share the fact that they measured total cortisol instead of free cortisol . As is true with other hor - mones ( e . g . , thyroid hormone ) , it is the free hormone that is biologically active . Lacking a physiologic measure of circu - lating glucocorticoid action , we are left with the biochemical ability to measure free cortisol and must do so in future studies ( 9 ) . Are we ready to treat based on these data ? Unfortunately , the current body of pediatric literature addressing adrenal in - sufficiency does not yet afford us a clear answer . However , as clinicians we must practice based on the best available data . Dr . Pizarro and colleagues ( 1 ) have taken one step forward , and their data obligate us , in the setting of pediatric septic shock , to perform adrenocorticotropic hormone stimulation testing using a 250 - (cid:7) g corticotropin test and consider both the baseline as well as stimulated cortisol . As there is no significant associ - ation between outcome measures and treatment in this population , to date , there can be no formal recommendation in pediatrics to give hydrocortisone . However , it would be a reasonable ap - proach to start hydrocortisone at stress doses ( 50 – 100 mg / m 2 / day ) in the pediat - ric patient with catecholamine - requiring septic shock after sending off a 250 - (cid:7) g corticotropin stimulation test . Once re - sulted , steroids could be continued only if the cortisol increment is (cid:1) 9 (cid:7) g / dL and the patient remains critically ill . To carry this out effectively and without delaying therapy , it is important that each PICU stock corticotropin in the PICU local pharmacy . Too often , testing is deferred because of a desire to avoid a therapeutic delay . Further research is needed to clarify the multiple issues relating to the diag - nosis and treatment of adrenal insuffi - ciency in children . Without prospective randomized control trials , we can only make vague statements about ideal treat - ment plans . Dr . Pizarro and colleagues ( 1 ) have taken an important step along * See also p . 855 . Key Words : adrenal insufficiency ; septic shock ; cortisolCopyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159724 . 29534 . CF 911 Crit Care Med 2005 Vol . 33 , No . 4 this road , but we have far to go to com - plete this journey . Michael Agus , MD Pediatric Critical Care and Endocrinology Children’s Hospital Boston Harvard Medical School Boston , MA REFERENCES 1 . Pizarro CF , Troster EJ , Damiani D , et al : Ab - solute and relative adrenal insufficiency in children with septic shock . Crit Care Med 2005 ; 33 : 855 – 859 2 . Annane D , Sebille V , Charpentier C , et al : Effect of treatment with low doses of hydro - cortisone and fludrocortisone on mortality in patients with septic shock . JAMA 2002 ; 288 : 862 – 871 3 . Fragen RJ , Shanks CA , Molteni A : Effect on plasma cortisol concentrations of a single in - duction dose of etomidate or thiopentone . Lancet 1983 ; 2 : 625 – 626 4 . Yildiz O , Doganay M , Aygen B , et al : Physio - logical - dose steroid therapy in sepsis . Crit Care ( Lond ) 2002 ; 6 : 251 – 259 5 . Briegel J , Forst H , Haller M , et al : Stress doses of hydrocortisone reverse hyperdynamic sep - tic shock : A prospective , randomized , double - blind , single - center study . Crit Care Med 1999 ; 27 : 723 – 732 6 . Bollaert PE , Charpentier C , Levy B , et al : Re - versal of late septic shock with supraphysi - ologic doses of hydrocortisone . Crit Care Med 1998 ; 26 : 645 – 650 7 . Dellinger RP , Carlet JM , Masur H , et al : Sur - viving Sepsis Campaign guidelines for man - agement of severe sepsis and septic shock . Crit Care Med 2004 ; 32 : 858 – 873 [ erratum appears in Crit Care Med 2004 ; 32 : 1448 ; Note : Correc - tion of dosage error in text ] 8 . Gonzalbez J , Villabona C , Ramon J , et al : Es - tablishment of reference values for standard dose short synacthen test ( 250 microgram ) , low dose short synacthen test ( 1 microgram ) and insulin tolerance test for assessment of the hypothalamo - pituitary - adrenal axis in normal subjects . Clin Endocrinol 2000 ; 53 : 199 – 204 9 . Hamrahian AH , Oseni TS , Arafah BM : Mea - surements of serum free cortisol in critically ill patients . N Engl J Med 2004 ; 350 : 1629 – 1638 Is clinical research and ethics a zero - sum game ? * M any critical care research - ers perceive the current regulatory environment as hostile . And who could blame them for thinking so ? Clinical re - search in which informed consent is not possible came to a halt from 1993 to 1996 . Subsequent U . S . Food and Drug Administration and Health and Human Services regulations permitting a waiver of informed consent imposed numerous , and arguably unnecessary , regulatory hurdles ( 1 , 2 ) . As a result , there was a 15 % per year decline in resuscitation re - search relative to other similar clinical studies ( 3 ) . More recently , ARDSNet clin - ical trials were investigated by the Office of Human Research Protections for con - sent and design flaws , resulting in an inordinately long shutdown of the Fluid and Catheter Treatment trial ( 4 ) . Against this history , the tension between the im - perative to conduct critical care research and that to protect research participants seems like a zero - sum game . Efforts to increase research participant protections hinder scientific progress , and efforts to bolster the pace of medical discovery risk undermining participant protections . Yet , it need not be so . In this issue of Critical Care Medi - cine , Dr . Silverman and colleagues ( 5 ) , on behalf of the ARDSNet Informed Consent Working Group , present “rec - ommendations for informed consent forms for critical care clinical trials” that challenge the zero - sum view of clinical research and ethics . Their pro - posal seeks both to ensure that regula - tory requirements are fulfilled and that recognition of the decision - making rights of subjects’ understanding is en - hanced . This is achieved by a number of means . First , model language for con - sent forms for critical care trials is of - fered at a sixth - grade reading level . Sec - ond , consent forms , when possible , are to be made shorter . Third , similarities and differences between treatment in the study and in routine clinical care are to be made explicit , allowing the participants to understand what differ - ence study participation will make to them . Fourth , study procedures admin - istered with therapeutic warrant and those done solely to answer scientific ends are to be described separately , so participants understand which proce - dures may benefit them and which may only benefit society . Although the ques - tion deserves empirical study , I believe that potential participants ( or their sur - rogates ) are more likely to agree to study participation when they under - stand better to what they are being asked to consent . If this hypothesis proves true , then both participant pro - tection and scientific progress will have been advanced by these recommenda - tions . The ethical challenges facing critical care research are not limited to problems associated with informed consent forms . As modeled so admirably by Dr . Silver - man and colleagues and a recent initia - tive of the American Thoracic Society ( 5 , 5a ) , these challenges ought to be met head on by critical care researchers who map out solutions that reject the zero - sum approach . Smart solutions to these challenges will be those that enhance both participant protections and progress in clinical research . Three areas merit immediate attention . First , critical care researchers ought to draft , promulgate , and lobby for the adoption of federal regulations to protect incapable adults in research . Current Health and Human Services regulations contain subparts addressing the protec - tion of pregnant women , prisoners , and children . Regulations for the protection of incapable adults in research were first proposed in 1978 but have yet to be en - acted ( 6 ) . The absence of federal regula - tion leaves institutional review boards with an obligation to ensure that re - search involving vulnerable populations include “additional safeguards” ( 45 CFR 46 . 111 ) but without guidance as to what these safeguards ought to be when re - * See also p . 867 . Dr . Weijer’s research is supported by a Canadian Institutes of Health Research Investigator Award and Operating Grant ; he was a paid consultant to the Office of Human Research Protections with regard to the ARDSNet clinical trials . Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI : 10 . 1097 / 01 . CCM . 0000159692 . 43458 . 51 912 Crit Care Med 2005 Vol . 33 , No . 4 search involves cognitively impaired adults . In the absence of guidance , it is perhaps not surprising that institutional review boards and the Office of Human Research Protections find critical care studies deserving of special scrutiny . As Karlawish ( 7 ) concludes correctly , “ [ u ] n - til regulations exist for research that in - volves critically ill and other cognitively impaired adults , federal investigations will be inevitable , slowing and even halt - ing the progress of valuable research . ” Thus , the adoption of effective federal regulations tailored to incapable adults offers the possibility of both enhancing subject protection while allowing impor - tant studies to proceed more efficiently . Second , critical care researchers ought to identify and promulgate a systematic ap - proach to the ethical analysis of research benefits and harms . Truog ( 8 ) recently en - dorsed an approach to benefit – harm anal - ysis that I have worked on called compo - nent analysis because it “leads to a remarkable and perhaps unexpected con - clusion regarding research in the ICU . ” Critical care research is often thought to pose serious risk to participants , and this perception may hinder clinical research . Component analysis recognizes that clini - cal research often contains a mixture of therapeutic and nontherapeutic interven - tions and that these must be analyzed sep - arately ( 9 ) . Therapeutic procedures , be they study drugs or ventilator settings , must meet the standard of clinical equipoise ; that is , they must be deemed on the basis of available evidence potentially consistent with competent medical care . Nonthera - peutic procedures , such as additional blood tests or data downloaded from monitors , must pose risks judged to be minimized and reasonable in relation to the knowledge to be gained . For the critical care patient , the incremental risk posed by study partic - ipation comes only from nontherapeutic procedures . As nontherapeutic procedures in many critical care studies involve only added blood draws and the like , many stud - ies understood this way pose only minimal risk to participants . Truog ( 8 ) concludes that as a result , “much of the research performed in critical care is clearly less ethically challenging than some of the re - search performed in outpatient settings ! ” Third , critical care researchers ought to address ethical challenges posed by prehos - pital research and develop solutions that enhance patient protections and the con - duct of research . Treatment for critically ill patients often begins before the patient reaches the hospital and is administered by paramedics . Ethical and practical difficul - ties associated with prehospital research have hindered the development of evi - dence - based treatments in this setting ( 10 ) . Time constraints pose a serious challenge to obtaining meaningful consent from ei - ther the patient or a surrogate in prehos - pital research . Paramedics are required as a matter of professional practice to stabilize and transport patients expeditiously . Fur - thermore , there may be a short therapeutic window within which to institute treat - ment in the field . Novel concepts may be required to deal with these unique chal - lenges . For instance , the capacity to provide valid informed consent is usually thought of as the possession of the cognitive abili - ties to receive , understand , and rationally manipulate information and , on that basis , to express an informed choice . The role of context in capacity to consent has not been given adequate attention . Might one argue , for instance , when treatment must be ad - ministered immediately , that a patient ( and indeed the patient’s surrogate as well ) who possesses the usual requisite cognitive abil - ities is nonetheless contextually incompe - tent ? If so , the use of a consent waiver might be permitted in such circumstances . Charles Weijer , MD , PhD , FRCPC Dalhousie University Halifax , Nova Scotia , Canada REFERENCES 1 . Biros MH : Research without consent : Cur - rent status 2003 . Ann Emerg Med 2003 ; 42 : 550 – 564 2 . McRae AD , Weijer C : Lessons from everyday lives : A moral justification for acute care research . Crit Care Med 2002 ; 30 : 1146 – 1151 3 . Nichol G , Hutszti E , Rokosh J , et al : Impact of informed consent requirements on car - diac arrest research in the United States : Exception from consent or from research ? Resuscitation 2004 ; 62 : 3 – 23 4 . Steinbrook R : How best to ventilate ? Trial design and patient safety in studies of the acute respiratory distress syndrome . N Engl J Med 2003 ; 348 : 1393 – 1401 5 . Silverman HJ , Luce JM , Lanken PN , et al : Recommendations for informed consent forms for critical care clinical trials : For the NHLBI Acute Respiratory Distress Syn - drome Clinical Trials Network ( ARDSNet ) . Crit Care Med 2005 ; 33 : 867 – 882 5a . Luce JM , Cook DJ , Martin TR , et al : Amer - ican Thoracic Society . The ethical conduct of clinical research involving critically ill patients in the United States and Canada : Principles and recommendations . Am J Re - spir Crit Care Med 2004 ; 170 : 1375 – 1384 6 . Department of Health , Education , and Wel - fare : Protection of human subjects : Pro - posed regulations on research involving those institutionalized as mentally disabled . Fed Regist 1978 ; 43 : 53950 – 53956 7 . Karlawish JHT : Research involving cogni - tively impaired adults . N Engl J Med 2003 ; 348 : 1389 – 1392 8 . Truog RD : Will ethical requirements bring critical care research to a halt ? Intensive Care Med [ serial online ] . Accessed Novem - ber 4 , 2004 9 . Weijer C , Miller PB : When are research risks reasonable in relation to anticipated benefits ? Nat Med 2004 ; 10 : 570 – 573 10 . Thompson J : Ethical challenges of informed consent in prehospital research . Can J Emerg Med 2003 ; 5 : 108 – 114 913 Crit Care Med 2005 Vol . 33 , No . 4 \ No newline at end of file diff --git a/silver_data/50857cec4c59b638a0bd82a5bfec1e747200d378.pdf.txt b/silver_data/50857cec4c59b638a0bd82a5bfec1e747200d378.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..6900ccd6bbd89c57b98246d7cf37bc39993ac395 --- /dev/null +++ b/silver_data/50857cec4c59b638a0bd82a5bfec1e747200d378.pdf.txt @@ -0,0 +1 @@ +Expanded View Figures ◀ Figure EV 1 . Tamoxifen inhibits spreading , matrix attachment , and invasion in macrophages . A Time - elapsed sequential images of macrophages until cells attained maximum area , scale bar is 10 l m . B Quantification of macrophage spreading over time , values represent mean (cid:1) s . e . m . n = 15 cells per condition ( FN , fibronectin ) . C – E Quantification of cell number ratio , area , and roundness ( panels C and E n = 30 cells per condition , and numbers are relative to control – 1 kPa ) . F Representative images of macrophage attachment to 1 and 25 kPa polyacrylamide matrices , scale bar is 50 l m . G Representative images of the transwell inserts showing crystal violet - stained invading macrophages , scale bar is 50 l m . H Quantification of macrophage invasion for panel ( G ) . I Bright field images of the bottom of the lower chamber that contained the inserts showing the invading macrophages that reached these surfaces in the control and tamoxifen and GPER antagonist conditions , scale bar is 50 l m . Data information : Histogram bars represent mean (cid:1) s . e . m . ( t - test for B , C , D , and E ; and ANOVA and Tukey ’ s test for H * * * P < 0 . 001 ) . For all panels , three experimental replicates . ▸ EMBO reports Mechanoregulation in PDAC via GPER Ernesto Cortes et al EV 1 EMBO reports e 46556 | 2018 ª 2018 The Authors A B F G H I C D E Figure EV 1 . Ernesto Cortes et al Mechanoregulation in PDAC via GPER EMBO reports ª 2018 The Authors EMBO reports e 46556 | 2018 EV 2 A B C D Figure EV 2 . ExposureofPSCstoG 1 ( GPERagonistbutnottoERagonists ) changescellmorphologyfromthePSCscharacteristicelongatedspindle - shapedtoa less contractile configuration . A – D Bright field images of PSCs ( A ) , and PSCs after exposure to 1 l M of ER - a agonist / PPT ( B ) , ER - b agonist / ERB 041 ( C ) , and GPER agonist / G 1 ( D ) . Scale bar : 100 l m . EMBO reports Mechanoregulation in PDAC via GPER Ernesto Cortes et al EV 3 EMBO reports e 46556 | 2018 ª 2018 The Authors \ No newline at end of file diff --git a/silver_data/52ce9f43d109e281a163c26a84faa76f18801b40.pdf.txt b/silver_data/52ce9f43d109e281a163c26a84faa76f18801b40.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..db786e5c97f96503e3427c7986ce47b90b488a63 --- /dev/null +++ b/silver_data/52ce9f43d109e281a163c26a84faa76f18801b40.pdf.txt @@ -0,0 +1 @@ +INTRODUCTION Science educators world over are usually faced with the challenge of improving science teaching so that learners can achieve high and develop a positive attitude to science . Needless to say advancement in science for a nation is the bedrock of technological development . Thus , the government of any nation is not just interested in teaching its populace how to read and write but is interested in their scientific development which can lead to higher technological advancement . Developed nations like the United States of America , Japan , China etc . pump a lot of money into scientific innovations , which today has turned them into a nation of manufacturers rather than consumer nations to which many third world countries belong . Since science learning is so important it is necessary that science educators introduce appropriate behaviours in their classroom to enhance effective science learning and therefore better performance in science . Evidence from the result of the senior secondary school certificate examination ( SSCE ) show that between 1994 and 1998 not up 40 % of the registered candidates passed ( see Table 1 ) . The purpose of this paper therefore is to tryout a strategy which will enable students to achieve better in science and therefore be able to face the challenges of the scientific world better . Research findings already show that there is © Kamla - Raj 2010 Int J Edu Sci , 2 ( 1 ) : 21 - 28 ( 2010 ) The Effect of Analogical Reasoning and Extended Wait Time on Achievement in Biology Janet Omolayo Olajide * and Femi Adetunji Adeoye * * * Samaru College of Agriculture , Division of Agricultural Colleges , Ahmadu Bello University , Zaria , Kaduna State , Nigeria . E - mail omolajide @ gmail . com * * School of Education , National Open University of Nigeria , Lagos , Lagos State , Nigeria . E - mail feminoun2005 @ yahoo . com KEYWORDS Reasoning . Analogy . Wait Time . Biology . Traffic Light Device ABSTRACT This study investigated the effect of using analogical reasoning and extended wait time on science achievement . 95 students were used in a pretest , posttest experimental and control group design experiment . Analysis of variance ( ANOVA ) was used to analyse the result with Scheffe’s test to identify which group ( s ) showed significant difference . The result showed that achievement improved where analogy and extended wait time and analogy only groups were used . The result showed that teaching with analogy and extended wait time enhances achievement , and teachers should be trained , and retrained to use this strategy in teaching . poor performance in science among African students . The reason for this according to the proponent of the African World view thesis is that students who have high level of belief in African values and tradition perform poorly in science . This is because certain cultural factors have negative effect towards science ( Ogunniyi 1988 ; Jegede et al . 1989 ) . Dzama and Osborne ( 1999 ) said poor performance in science in deve - loping countries is not just due to the world views of students in these countries , but due to absence of supportive environment for serious science learning where science features significantly in the popular culture . Thus , if the classroom envi - ronment is to be made conducive for learning sci - ence , the teacher has to introduce a strategy which would develop students’ scientific under - standing , thinking and problem solving abilities . Over the years the inquiry method has been introduced in the teaching of science at both primary and secondary school levels in Nigeria . Inquiry which is an art of questioning , exploring and experimenting . It involves development of skills such as acquisition skills , manipulative skill , creative skills and such like ( Sund and Trowbridge 1967 ) . However this method cannot be properly practiced even at the primary level let alone at the secondary schools . This is so because of lack of equipment and even lack of proper encourage - ment of teachers . Recently in April 2009 , teachers in south - west Nigeria went on strike due to lack of proper remuneration by that government . In 22 JANET OMOLAYO OLAJIDE AND FEMI ADETUNJI ADEOYE the light of this situation educators have to think of other methods that can develop scientific under - standing of students , their thinking and problem solving abilities . Such a method would help to solve conceptual problems and result in higher achievement in science . There are over 400 national reports in the United States of America expressing growing dis - satisfaction with the quality of science teaching and learning and have called for changes in the practice of teachers and learners and policy makers ( AAAS 1989 ) . There is overwhelming consis - tency in these reports , recognizing that students should build understanding about what they are endeavoring to learn and an advocacy for chan - ges in the traditional roles of teachers . One popu - lar phrase that has been included in most of these reports is that “less is more” . This means that fewer concepts need to be studied in greater depth to enable students build deeper understanding ( Palmer 1973 ) . Thus , teaching Biology using the strategy of Analogical reasoning and extended wait time which is the main focus of this paper is going to help students achieve better in science . What is Analogy ? Analogy is a comparison of structures between domains ( Clement 1978 ) . It is a relation between parts of structures of two domains and may be viewed as a comparison statement on the grounds of structures of two domains pointing to some resemblances . The two domains are the analog and the target . The analog is the domain used as basis for structural comparison and the target is the domain to be explained . Over the years analogy has been used as a concept build - ing tools . It consists of objects , events or situa - tions that share features in common . That is to say they are in one or more ways similar . Those shared features have a significantly neural impact by causing increased cell activity which results in fast learning . The brain is an analogy processor i . e . it works by analogy and metaphors . It relates whole concept to another and looks for simi - larities , differences or relationships between them . It does not assemble thoughts and feelings from bits of data ( Silvester et al . 1999 ) . Thus , the use of analogy helps students to concretize their ideas by enriching their representation of problem situation with previously observed or observable ( but imaginable ) structures or mechanisms . Through analogies an understanding of novel situations may be constructed by comparison to more familiar domain of knowledge . Many recent cognitive approaches to instruction emphasize the importance of understanding the science . Learning begins with the learners’ science ( Wittrock 1985 ) . Thus , through the use of analogy one goes from known to the unknown , and the unfamiliar becomes familiar . What is Wait Time ? Wait time is the time the teacher waits after a question is asked , before accepting an answer , repeating the question , rephrasing it or supplying the answer ( Rowe 1969 , 1974 ) . Lake ( 1973 ) summarizes the wait time phenomenon by defining it as “the length of pause preceding any teacher utterance . By defining wait time in this way teachers are provided with direct control over relevant pauses within the course of a lesson . Wait time has been found to be a variable which could be used to determine the quality and quantity of learners discourse that occur in a science lesson . There are two types of wait time , wait time 1and ll . Wait time l is the time the teacher waits after a question . It normally begins when the teacher stops speaking and terminates when a student responds or the teacher speaks again . The sequence is as follows : Teacher asks a question and pauses . He / she calls on a student to answer the question , pauses again . The two pauses are summed up together and they constitute an instance of the first wait time ( Rowe Table 1 : SSCE Results in Science Subjects Between 1994 - 1998 Year Subject Number Number Percentage that that pass with registered passed credit 1994 Chemistry 161262 38212 23 . 7 Biology 508384 57955 11 . 4 Physics 146000 21462 14 . 7 1995 Chemistry 133188 48514 36 . 7 Biology 453353 80734 18 . 9 Physics 120768 16738 18 . 7 1996 Chemistry 144990 48514 33 . 46 Biology 506628 80734 15 . 9 Physics 132768 16738 12 . 6 1997 Chemistry 172382 40652 23 . 58 Biology 609026 96202 15 . 79 Physics 157700 14745 9 . 35 1998 Chemistry 182659 39085 21 . 39 Biology 626894 215946 34 . 4 Physics 10063 1002 9 . 95 Source : WAEC , Lagos 1999 ( Nigeria ) . 23 ANALOGICAL REASONING AND EXTENDED WAIT TIME 1969 , 1974 ) . Wait time ll is defined as the time a teacher wait after a pupil’s response to the question asked him / her by the teacher . It is calculated by taking the sum of all the students’ pause and terminates when the teacher speaks . The variable of wait time in learning science has been found to be very important in teaching . This means that if it is not properly used , effective learning cannot take place in the science class - room ( Rowe 1974 , 1986 ; Tobin 1985 ) . This is because according to Moriber ( 1971 ) , students need a period of “private thought” which help them to put ideas together during inquiry lessons . Research work by Rowe ( 1974 ) , Tobin ( 1980 ) showed that when extended wait time of 5seconds for wait time l and 3seconds for wait time ll were used for students in inquiry class certain positive behaviours were observed in both students and teachers . Such as : a ) Students increase their length of response b ) They offer more alternative responses c ) They asked more questions and interact more with other students . The above outcome showed students under - stand the lesson better and are therefore likely to achieve better and have positive attitude to science . On the part of the teacher , the effect the extended wait time had on them include : a ) Teachers’ response exhibit greater flexibility as indicated by the occurrence of fewer discourse errors . b ) The number and kind of questions for the performance of certain children seem to change . The above outcomes tend to show that the teacher’s behaviour can be positively affected . This will put them in a better position to encourage the students to achieve better in science . Thus , if wait time is properly used it will lead to those outcomes that will enhance higher achievement in science . Statement of the Problem This study aims at investigating the effect of the use of extended wait time and analogical reasoning on students’ academic achievement in Biology . Research Hypotheses The hypothesis to be tested states that : There is no significant difference in the mean academic achievements of students taught using the following methods : a ) A combination of analogical reasoning and extended wait time . b ) Analogical reasoning only . c ) A combination of traditional lecture method and extended wait time . d ) Traditional lecture method only . METHODOLOGY Research Design For this study a pretest and posttest expe - rimental control group design was adopted to determine any possible treatment effects . There were four groups , three experimental and one control groups . The subjects were members of two intact classes of Samaru and Kaduna Colleges of Agriculture randomly assigned to the three experimental and one control groups . The three experimental groups were labeled E 1 , E 2 , E 3 respectively and the control group was labeled C . Experimental group1 ( E 1 ) had treatment that involved combination of analogical reasoning plus extended wait time , experimental group ll ( E 2 ) had treatment that involved the use of analogical reasoning only . Experimental group 111 ( E 3 ) had extended wait time plus traditional lecture method of instruction . Control group ( C ) had traditional lecture method only . A pretest was administered to both the experimental and control groups . Treatments were administered to the experimental groups , but were withheld from the control group who were taught using the conventional lecture method . Thereafter a post test was administered to all four groups . The first experimental and second experimental groups had 24 subjects each while the third had 25 ; the control group had 22 subjects . Altogether 95 students were assigned to the different groups . A Flow chart of the design is illustrated below : R 1 ’ ! O 1 ’ ! X 1 ’ ! P 2 R1 = Experimental group1 ( E 1 ) R 2 ’ ! O 1 ’ ! X 2 ’ ! P 2 R2 = Experimental groupll ( E 2 ) R 3 ’ ! O 1 ’ ! X 3 ’ ! P 2 R3 = Experimental grouplll ( E 3 ) R 4 ’ ! O 1 ’ ! X 3 ’ ! P 2 R4 = Control group ( C ) X 1 = Extended wait - time and analogical reasoning X 2 = Analogical reasoning X 3 = Extended wait - time and lecture method 24 JANET OMOLAYO OLAJIDE AND FEMI ADETUNJI ADEOYE X 4 = Lecture method alone O 1 = Pretest P 2 = Posttest Illustration of the Quasi - experimental Research Design Population and Sample Population : The target population for this study was all Pre - National Diploma students of the three Colleges of Agriculture in the Division of Agricultural Colleges of Ahmadu Bello University , Zaria , Kaduna and Kabba . The three towns are located in Northern Nigeria . Zaria and Kaduna are in the present Kaduna state , while Kabba is situated in Kogi State . The total number is distributed as follows : Kabba is 23 , Kaduna 22 and Zaria 73 . Sampling : The Pre - National Diploma ( Pre - ND ) students of the Colleges of Agriculture in Zaria and Kaduna formed the subjects of this study . These two Colleges were chosen for the following reasons : First , their syllabus is similar to that of the senior secondary school ( SSS ) in Nigeria and since the results of the study had implication for teaching in the secondary school , this category of students was found to be suitable . Secondly , at the time of conducting the study , secondary schools were about going on long vacation , and it was no longer possible to use the secondary schools at the time . The Pre - ND students were found to be a suitable alternative , since the content of their syllabus was significantly similar to that of the secondary school . The proximity of the two colleges to the researcher made it less problematic for the researcher to conduct an experimental research in the colleges . The Kabba college was not included because of its distance from the researcher . The average chronological age of the Pre - National Diploma students is 20years . Candidates are admitted from the different States of Nigeria , giving the admission a wide geographical spread . The total number of students was 95 , and this comprised 73 from Samaru college of Agriculture , Zaria and 22 from kaduna College of Agriculture , Kaduna . All the students had the same entry points . The researcher used the student list to distribute the 73 students of Samaru college of Agriculture into three experimental groups of 24 , 24 , and 25 , while the 22 students in Kaduna college constituted the control group . Thus , two intact classes were used for the study . Instrumentation The instruments used for this study com - prised : 1 . Selected Biology Topics . 2 . Research Items . 3 . Multiple choice Assessment Test ( MCAT ) . Selected Biology Topics The topics to be taught during the research were selected by the researchers with consultation with two secondary school Biology teachers and two Pre - ND Biology teachers . After looking through the syllabuses the following topics were selected for teaching : 1 . Nutrition in plants with particular reference to photosynthesis 2 . Respiration in flowering plants . 3 . Transportation in plants 4 . Transpiration in plants . These topics were chosen because they are present in both Pre - ND and the senior secondary school Biology syllabuses . Further more , they are regarded as difficult topics in Biology of which analogy and extended wait time can help students to achieve better understanding of . Eight lessons were prepared altogether . Four were prepared with analogy and four without analogy . Questions were also included in between the lessons for the purpose of wait time manipulations . The lessons with analogy were taught to the experimental groups 1 and ll ( E 1 and E 2 ) . While in addition to analogical reasoning extended wait time l and ll were used for group 1 and there was no wait time manipulation for experimental group ll . The lessons without analogy were taught to experimental group lll ( E 3 ) and the control group . No variation of wait time for the control group . The optimum extended wait time’s l and ll used were 5seconds and 3seconds respectively . This is in accordance with the suggestions by Tobin ( 1980 ) . Research Items Research items include : 1 . Audiotape recorder and audio cassettes to tape the lessons . 2 . The timing light device used as signals during wait time manipulations . 3 . Teaching format – lessons prepared with and without analogical linkages . 25 ANALOGICAL REASONING AND EXTENDED WAIT TIME Multiple Choice Assessment Test ( MCAT ) The MCAT was constructed by the resear - chers . This consisted of 25 items and each had five options in the multiple choice format in order to test the knowledge of the concept taught . The questions covered all four topics . The subjects were expected to answer all the questions . The pretest and posttest was one and the same test , that means a test and retest method was used . This is quite in order because accor - ding to Tuckman ( 1978 ) from James ( 2000 ) , a pretest may increase the likelihood that an individual will do better on the subsequent posttest , particularly when it is identical to the pretest . The test gave a reliability coefficient of 0 . 79 using Kuder - Richardson coefficient of reliability . Data Collection Procedure Analogical Teaching Format For the procedure of teaching using analogy the Glynn et al . ( 1991 ) , model of Teaching with analogy ( TWA ) was adapted . It has six steps : i . Introduction of target concept ii . Recall analog concept iii . identify similar features iv . Map similar features v . Draw conclusion about concept vi . Indicate where analogy breaks down . However , for this research study this model will be compressed into four steps instead of six , by merging together steps iii and iv as it is unne - cessary for this teaching . Thus , the new steps will now be : - i . Introduction of target concept . ii . Recall analog concept . iii . Identification and mapping out similar features iv . Conclusion . The operations can be explained as follows : - i . Introduction of target concept involves giving a brief description of certain basic facts about the concepts presented to the subjects in the process of learning target concepts . ii . Recall of analog concept : This stage involves the selection of objects by the researcher of which is familiar to the learner . The analog concept is supposed to help the learner relate together concepts they previously viewed as unrelated . iii . Identification and mapping of similar features of both analog and target concepts are pre - sented to the learner . The teacher uses what is familiar to the student in the analog to the target , i . e . what the student does not know and it enhances the students understanding . iv . Drawing Conclusion : Drawing conclusion about the target concepts and indicating where analogy breaks down , i . e . pointing out the limits of the structural resemblances or similarities between target and analog concepts . Extended Wait Time Device and Wait Time Measurement A wait time of 5seconds and 3seconds were adopted from Tobin ( 1985 ) . The traffic light device used by Swift and Gooding ( 1983 ) was adapted for use in this study . Whereas the device was a voice activated relay system which operated “red and green traffic light” to signal when an appro - priate period of silence had elapsed ; the one used for the research was manually operated by trained research assistant . The light device had two switches , the first one switches off after 5 and 3 seconds depending on how it was manipulated . The four topics used for this research were taught by the researcher to remove teacher bias . The researcher adhered as much as possible to the teaching guidelines . The 4 groups designed used in this study consisted of three experimental ( E 1 , E 2 and E 3 ) and a control group © . The three experimental groups received treatment as follows : The Experimental Group 1 ( E 1 ) : The pretest was administered to them with the ( MCAT ) . They were subsequently taught all four lessons contain - ing analogy with extended wait time l and ll of 5seconds and 3 seconds , respectively . The wait time manipulation was done by a trained research assistant . Each lesson lasted for 60 minutes . The Experimental Group ll ( E 2 ) : This group was also pre tested and were taught using less - ons with analogy without wait time manipulation . The Experimental Group lll ( E 3 ) : After pre - test this group was taught using the lesson with - out analogy but with extended wait times l and ll . Control Group ( C ) : This group was pre tested . Teaching of the four lessons without ana - logy was done the conventional way with no wait time manipulation . Data Analysis The data collected from this study were ana - 26 JANET OMOLAYO OLAJIDE AND FEMI ADETUNJI ADEOYE lyzed using the analysis of variance ( ANOVA ) . This statistical method was used because more than two means were evaluated for significant diffe - rence , and the researcher was interested in deter - mining whether or not one group differed signi - ficantly from the other . Where the difference was significant , the Scheffe’s procedure was used to determine which of the teaching methods was significantly different . Only the scores of stu - dents who completed the treatment and who sat for the pre and posttest were included in the analysis . RESULTS The data analysed were obtained from the pre and post achievement tests . Analysis of variance ( ANOVA ) was used for the statistical analysis . Where the hypothesis found to be significant , Scheffe’s procedure was used to verify which one of the methods produce the main effect . To test this hypothesis the data for this hypo - thesis were first subjected to an analysis of vari - ance and the result showed that there was a significant difference as shown in table 1a . The F value of 0 . 0001 is less than the critical value of 0 . 05 . Thus , the difference observed in the achievement scores was seen to be significant at 0 . 05 level of significance . The result was then subjected to the Scheffe’s test to find out which of the method is responsible for the difference ( see Table 1b ) . The result of the Scheffe’s test showed that only the analogy plus extended wait time group and the analogy only group result showed significant difference , when compared with the traditional lecture method . Also , the traditional lecture plus extended wait time versus analogy plus extended wait time groups showed significant difference . But the mean difference between the traditional lecture and the traditional lecture plus extended wait time was not significant . All these point to the fact that analogical reasoning has a great impact on achievement in science and it really helps students’ understanding and the effect is immediate . However , since the mean score for those taught using analogy plus extended wait time method was higher than that of those using analogy only , it means that extended wait time 1 and ll of 5 and 3 seconds respectively used during questioning and answering process has a positive effect on students’ learning . The means and the standard deviation of the achievement ( see Table 1c ) . It can also be seen that the mean score of students taught using the traditional lecture method plus extended wait time was higher than those taught using the traditional lecture method Table 1a : ANOVA of the achievement of the experimental and control groups scores Source DF Sum of Square Mean Square F Value P . Value Between Group 3 368 . 77 122 . 92 7 . 61 * 0 . 0001 Within Group 84 1357 . 18 16 . 16 Total 87 1725 . 95 * Significant at P e” 0 . 05 level Table 1b : Scheffe’s test of achievement of experimental and control groups . Methods F value Remarks Traditional lecture vs Traditonal lecture + Extime 7 . 61 Not significant Traditional lecture vs Analogy only 7 . 61 Significant Traditional lecture vs Analogy + Extime 7 . 61 Significant Traditional lecture + Extime vs Analogy 7 . 61 Not significant Traditional + Extime vs Analogy + Extime 7 . 61 Significant Analogy vs Analogy + Extime 7 . 61 Not significant Table 1c : Mean score and standard deviation of the experimental and control groups in the achievement tests . Methods Mean score Standard Deviation Analogy + Extended wait - time 15 . 28 4 . 08 Analogy only 13 . 27 4 . 67 Traditional lecture + Extended wait time 11 . 77 3 . 54 Traditional lecture only 9 . 63 3 . 68 27 ANALOGICAL REASONING AND EXTENDED WAIT TIME only . This means that the extended wait time 1and ll of 5 and 3 seconds used during the questioning and answering has great positive effect on stu - dents’ learning . DISCUSSION The main aim of this study is to find out if combining extended wait time with analogical reasoning can positively affect achievement in Biology . The result showed statistical difference between the experimental and control groups and the hypothesis of no significant difference were rejected . The mean scores of the experimental and control groups showed that the achievement results of those in the combination of analogical reasoning with extended wait time and those in analogy group was significant . This shows that using analogical reasoning in teaching is very important . Lagoke ( 1993 ) showed that when analogical reasoning alone was used to teach students their performance improved as they retained concept better . Tobin ( 1980 ) said extended wait time during inquiry science teaching led to improved science achievement . According to Tobin and Caple ( 1982 ) , the use of wait time of between 3 - 5 se - conds was associated with high student achieve - ment in science . It is therefore not surprising that a combination of these two variables in teaching science resulted in better achievement . The fact that the result of the analogy group was found to be significant is in line with other previous research findings ( Gilbert 1989 ; Clement 1987 ; Lagoke 1993 ) which is why the method was recommended for teaching science . Analogical reasoning helps students to use familiar situation to explain those that are not familiar and try to make sense of it so it is important that the analogy comes from the students’ environment . It may be summarized from this study , which is on the effect of analogical reasoning and exten - ded wait time on achievement in Biology . The instructional strategies used were a combi - nation of analogical reasoning plus extended wait time , analogy only , a combination of traditional lecture method plus extended wait time , and lecture method alone . The subjects were Pre - national diploma students of colleges of Agriculture in Samaru and Kaduna . One hypothesis was tested . The result showed a significant difference in the achievements of the subjects . CONCLUSION In this study lessons were prepared with analogical linkages with which the learners are familiar and extended wait time1 of 5seconds and wait time ll of 3seconds were also prepared and administered to students . The following conclu - sions were drawn : 1 . The use of analogical reasoning and extended wait time helped to improve the quality of instructions such that students understanding of the subject matter improved and thereby improving performance . 2 . The good effect of extended wait time was seen more when used with analogical reason - ing than with the traditional lecture method . The implication of this study is that the use of a combination of analogical reasoning plus extended wait time has greatly enhanced achievement in science . RECOMMENDATION Based on the above findings the following recommendations are made : i . it is highly recommended that teachers should teach for more meaningful learning with the use of analogical reasoning and extended wait time , as this led to higher students’ achieve - ment . ii . since extended wait time provides for the student a period of “private thought” which will allow the new knowledge to be incor - porated into the existing one , science teachers should cultivate the habit of extending wait time l of 5seconds and wait time ll of 3seconds , irrespective of what ever method is being used to teach as this will help students to grasp knowledge better . iii . science educators should incorporate the use of analogy and extended wait time into the teaching methods of teachers in training , so that they can adopt it in teaching their stu - dents . REFERENCES American Association for the Advancement of Science1989 . Science For All Americans : Summary Project 2061 . Washington D . C : Author . Abdullahi A 1982 . Science Teaching in Nigeria . Ilorin : Atoto Press Limited . Ausubel DP 1968 . Educational Psychology . A Cognitive View . New York : Holt Rinehart and Winston . 28 JANET OMOLAYO OLAJIDE AND FEMI ADETUNJI ADEOYE Brown DE , Clement JJ 1989 . Overcoming misconception via analogical reasoning : Abstract transfer versus explanatory model construction . Instructional Science , 18 : 237 - 261 . Black D , Solomon J 1987 . Can pupils use taught analogies for electric current ? School Science Review , 3 : 249 - 254 . Bajah ST 1975 . Preparation of the secondary school teacher of the physical science for African environ - ment . West African Journal of Education , 19 ( 10 ) : 85 - 96 . Brooks MN 1975 . A study of locus of control and science achievement . Journal of Research in Science Teaching , 12 ( 2 ) : 175 - 181 . Black M . 1962 . Models and Metaphors . Itaca , N . Y : Cornell University Press . Clement J 1987 . Overcoming Students’ misconception in physics : The role of anchoring Intuitions and Analogical Validity ; Proceedings of the Second International Seminar on Misconceptions and Education Strategies in Science and Mathematics . 3 Ithaca . Chewprecha T , Gardner M , Sapianchai M 1980 . Compa - rison of training methods in modifying question and wait time behaviours of Thai high school chemistry teachers . Journal of Research in Science Teaching , 17 ( 5 ) : 191 - 200 . Clement J 1978 . The Role of Analogy in Scientific Thinking . Examples from a Problem Solving Interview . Massachusetts : University of Massachu - setts , Department of Physics and Astronomy . Avail - able on Microfiche . Dzama ENN , Osborne JF 1999 Poor performance in Science among African students : An alternative explanation to the African world view Thesis . Journal of Research in Science Teaching , 36 ( 3 ) : 387 - 403 . Glynn SM , Yaearny RH , Britton BK ( Eds . ) 1991 . A Teaching with Analogies Model . The Psychology of Learning Science . Hillsdale N . J : Eribaum , pp . 219 – 240 . Gilbert W 1989 . An evaluation of the use of analogy , simile and metaphor in science texts . Journal of Research in Science Teaching , 26 ( 4 ) : 315 - 327 . Gooding TC , Swift JN , Swift PR 1987 . Using wait - time feedback and supportive intervention to improve classroom discussion . The Tower Review , 4 ( 11 ) : 3 - 9 . James T 2000 . Effect of Combining Reflective Writing with Concept Mapping and Lecture method on pre - service NCE Teachers Attitude and Achievement in biology . Ph . D . Thesis , Unpublished . Zaria : Ahmadu Bello University . Jegede OJ , Olajide JO 1995 . Wait time , classroom dis - course and the influence of Sociocultural Factors in Science Teaching Science Education , 79 ( 3 ) : 233 - 295 . Jegede OJ 1989 . Toward a Philosophical basis for educa - tion of the 1990s : An African View - Point . In : DE Aerget ( Ed . ) : The History and Philosophy of Science in Science Teaching . Tallahasse FL : Florida State University , pp . 185 - 198 . Lagoke BA 1993 . Analogical linkages Between Socio - cultural Environment and Biology Concept Attain - ment by Secondary School Students Using a Con - structivist Framework . Ph . D . Thesis , Unpublished . Zaria : Ahmadu Bello University . Lake JH 1973 . The Influence of Wait Time on the Verbal Dimension of Students’ Inquiry Behaviour . PhD Thesis , Rutgers University , 34 , 6476 - A . Moriber G 1971 . Wait time in College Student . Science Education , 55 : 321 - 333 . Olajide JO , Jegede OJ 1989 . An Investigation into the Average Wait times of Nigerian Integrated Science Classroom in Inquiry Based Lessons . Nigerian Educational Forum , 11 : 295 - 298 . Ogunniyi MB 1988 . Adapting western science to traditional African culture . International Journal of Science Education , 10 : 1 - 9 . Olajide JO 1987 . Sociocultural Factors Affecting Wait time in Inquiry Based Integrated Science Classroom . M . Ed . Thesis , Unpublished . Zaria : Ahmadu Bello University . Osborne J , Freyberg P 1985 . Learning in Science : The Implication of Children’s Science . Auckland : Hein - mann . Palmer EL 1973 . A Determination of the Relative Consistency and Concordance of Student Science Interest Responsible for Utilizing Panel and Final . Ph . D . Thesis , Unpublished , Wisconsin : University of Wisconsin . Rowe MB 1986 . Wait time , slowing down may be a way of speeding up . Journal of Teacher Education , 37 : 43 - 50 . Rowe MB 1974 Wait time and rewards as instructional variables , their influences in language , logic and fate control : Part One Wait time . Journal of Research in Science Teaching , 11 ( 2 ) : 81 - 94 . Rowe MB 1969 . Science , soul and sanctions . Science and Children , 6 ( 6 ) : 11 - 13 . Sund RB , Trowbridge LW 1967 . Teaching Science by Inquiry in the Secondary Schools . Columbus , Ohio : Charles E . Merril Books Inc . Silvester D , Howard E , Kay D , Wathen A 1999 . Efficient preconditioning of the linearized navier - strokes equations for incompressible flow . Technical Report UMIACS - TR - 99 - 66 , Institute for Advanced Compu - ter Studies University of Maryland . Tobin K 1987 . The role of wait - time in higher cognitive level learning . Review of Educational Research , 57 ( 1 ) : 69 - 95 . Tobin KG , Capie W 1982 . Relationships between class - room process variables and middle science achieve - ment . Journal of Educational Psychology , 11 : 441 - 454 . Tobin K 1980 . The Effect of Extended Wait time on Science Achievement . Journal of Research in Science Teaching , 17 ( 5 ) : 469 - 475 . Wittrock MC 1985 . Learning science by generating new conceptions from old ideas . In : West Pives ( Ed . ) : Cognitive Structure and Conceptual Change . Orlando : Academic Press . Zook KB 2005 Effect of analogical processes on learning and misrepresentation . Journal of Educational Psychology Review . 3 ( 1 ) : 41 - 72 . \ No newline at end of file diff --git a/silver_data/5797da3040f670b2c2df41bb3ab99a7845d4d2c9.pdf.txt b/silver_data/5797da3040f670b2c2df41bb3ab99a7845d4d2c9.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..ee16862e56772b5cc71847e350a364fe67f4cc13 --- /dev/null +++ b/silver_data/5797da3040f670b2c2df41bb3ab99a7845d4d2c9.pdf.txt @@ -0,0 +1 @@ +a r X i v : c ond - m a t / 0602255v6 [ c ond - m a t . s o f t ] 12 D ec 2006 Dielectric relaxation of DNA aqueous solutions S . Tomi´c , ∗ S . Dolanski Babi´c , † and T . Vuleti´c Institut za fiziku , 10000 Zagreb , Croatia S . Krˇca and D . Ivankovi´c Institute Rudjer Boˇskovi´c , 10000 Zagreb , Croatia L . Gripari´c Department of Biological Chemistry , David Geffen School of Medicine , UCLA , Los Angeles , California 90095 , USA R . Podgornik Department of Physics , University of Ljubljana and J . Stefan Institute , 1000 Ljubljana , Slovenia and Laboratory of Physical and Structural Biology NICHD , National Institutes of Health , Bethesda , Maryland 20892 , USA ( Dated : October 31 , 2018 ) We report on a detailed characterization of complex dielectric response of Na - DNA aqueous solutions by means of low - frequency dielectric spectroscopy ( 40 Hz - 110 MHz ) . Results reveal two broad relaxation modes of strength 20 < ∆ ε LF < 100 and 5 < ∆ ε HF < 20 , centered at 0 . 5 kHz < ν LF < 70 kHz and 0 . 1 MHz < ν HF < 15 MHz . The characteristic length scale of the LF process , 50 < L LF < 750nm , scales with DNA concentration as c − 0 . 29 ± 0 . 04 DNA and is independent of the ionic strength in the low added salt regime . Conversely , the measured length scale of the LF process does not vary with DNA concentration but depends on the ionic strength of the added salt as I − 1 s in the high added salt regime . On the other hand , the characteristic length scale of the HF process , 3 < L HF < 50 nm , varyes with DNA concentration as c − 0 . 5 DNA for intermediate and large DNA concentrations . At low DNA concentrations and in the low added salt limit the characteristic length scale of the HF process scales as c − 0 . 33 DNA . We put these results in perspective regarding the integrity of the double stranded form of DNA at low salt conditions as well as regarding the role of different types of counterions in different regimes of dielectric dispersion . We argue that the free DNA counterions are primarily active in the HF relaxation , while the condensed counterions play a role only in the LF relaxation . We also suggest theoretical interpretations for all these length scales in the whole regime of DNA and salt concentrations and discuss their ramifications and limitations . PACS numbers : 82 . 39 . Pj 87 . 15 . He 77 . 22 . Gm I . INTRODUCTION Semiflexible polyelectrolytes are ubiquitous in biolog - ical context ranging from charged biopolymers such as DNA or filamentous F actin , and then all the way to molecular aggregates such as bacterial fd viruses or the tobacco mosaic virus . They are an essential and fun - damental component of the cellular environment and make their mark in its every structural and functional aspect [ 1 ] . Their role is not confined solely to various ( macro ) molecular assemblies in the biological milieu but are equally prevalent in colloidal systems and soft mat - ter in general [ 2 , 3 ] . It is their connectivity , stiffness and strong electrostatic interactions , that allow polyelec - trolytes to show a wide range of complex behaviors , de - pending on their concentration , their overall length and the concentration and valency of the added salt ions and ∗ URL : http : / / real - science . ifs . hr / ; Electronic address : stomic @ ifs . hr † Permanent address : Department of physics and biophysics , Med - ical School , University of Zagreb , 10000 Zagreb , Croatia . intrinsic counterions [ 4 ] . In the simplest case of monovalent counterions , poly - electrolytes are usually stretched due to electrostatic re - pulsions and therefore statistically assume a rod - like con - figuration [ 5 ] . Polyvalent counterions on the other hand can turn electrostatic repulsion into attraction [ 6 ] . This s . c . correlation effect is one of the most important fea - tures of the polyvalent counterions and has fundamental repercussions for all charged soft matter . Correlation at - traction can strongly reduce the rigidity of charged poly - mers which then collapse into highly compact states [ 7 ] . Deoxyribonucleic acid ( DNA ) is in many respects a paradigm of a stiff , highly charged polymer . The struc - tural origin of the charge is due to negatively charged phosphate groups positioned along the DNA backbone [ 8 ] . The nominal charge density of double stranded DNA amounts to one negative elementary charge per 1 . 7 ˚A or two elementary charges per base pair . When dealing with electrostatic interactions and their consequences in DNA one can usually ignore the internal chemical structure of DNA except as it transpires through the bare value of the persistence length ( see below ) . Electrostatic interactions in stiff polyelectrolytes with monovalent counterions and added salt in aqueous solu - 2 tions are standardly approached via the Poisson - Boltz - mann ( PB ) theory that combines electrostatics with sta - tistical mechanics on a simplified mean - field level [ 3 ] . On the PB level the outcome of the competition between en - tropy of mobile charges and their electrostatic interaction energy leads grosso modo to two types of effects . The first one is connected with electrostatic interaction of counterions and fixed charges on the polyelectrolyte . Positively charged counterions are attracted to the sur - face of the negatively charged DNA where they tend to accumulate in a layer of condensed counterions . This ac - cumulation can be understood within the framework of the Manning - Oosawa ( MO ) counterion condensation the - ory [ 3 ] as well as on the level of the solutions of the PB equation [ 9 ] . Within this theory the counterions accumu - late in the condensed layer around the cylindrical DNA surface only if the charge density parameter η > 1 . Here η = zl B / b , where z is the valency of the counterion , b is the linear charge spacing and l B is the Bjerrum length , defined as l B = e 20 / ( 4 πεε 0 kT ) ( 1 ) Here e 0 is elementary charge , ε 0 is permeability of vac - uum , kT is thermal energy scale and ε is the dielectric constant of the solvent . Bjerrum length is obviously de - fined as the separation between charges at which their electrostatic interaction energy equals their thermal en - ergy . In aqueous solutions l B = 7 . 1 ˚A . The charge density parameter η thus measures the relative strength of elec - trostatic interactions vs . thermal motion and is strongly dependent on the valency of the counterions . In the MO theory the counterions accumulate in the condensed layer exactly to such an extent that the effective charge den - sity parameter η is reduced to 1 [ 10 ] , i . e . the effective separation between charges is increased from b to l B . The condensed counterions in the MO theory are still assumed to be perfectly mobile . Because of counterion condensation the effective charge of DNA is reduced by a factor r = 1 − 1 / ( zη ) . It is important to note here that the concept of counterion condensation is intrinsi - cally nonlinear and is a fundamental property of highly charged polymers . This includes DNA in both its double stranded ( b = 1 . 7 ˚A , η = 4 . 2 ) as well as single stranded ( b = 4 . 3 ˚A , η = 1 . 7 ) forms . Counterion condensation for monovalent DNA salts is experimentally observed di - rectly by small - angle X - ray scattering measurements [ 11 ] . For double stranded DNA and monovalent counterions , counterion condensation occurs with r = 0 . 76 . In the MO counterion condensation theory the condensation occurs only if the salt concentration is low enough to satisfy κ − 1 ≫ a , where a is the polymer radius and κ − 1 is the Debye screening length ( see below ) [ 10 ] . Furthermore the counterion condensation strictly occurs only in the limit of vanishingly small concentration of DNA [ 9 ] . For finite DNA concentrations a more complicated model of coun - terion condensation has to be invoked [ 9 ] that is based on the solution of the PB equation in the cell model ge - ometry . The second effect of competition between entropy of mobile charges and their electrostatic interaction energy is due to the interaction of mobile ions in solution be - tween themselves and their redistribution in the field of fixed polyelectrolyte charges . This redistribution leads to screening of electrostatic interactions between fixed charges . For small enough , fixed , charge density the screening is accurately described by the Debye - H¨uckel equation , which is just a linearized form of the PB equa - tion [ 3 ] . On the Debye - H¨uckel level the screening is quan - tified by a screening or Debye length κ − 1 defined as κ 2 = 8 πl B n ( 2 ) where again l B is the Bjerrum length and n is the den - sity of added salt . For monovalent salts the Debye length in ˚ A is given numerically as 3 . 04 I − 1 / 2 s , where I s is the ionic strength in M . Indeed for monovalent salts both ef - fects , ionic screening as well as counterion condensation , coexist . In general , however , one can not invoke screen - ing effects in polyvalent salt solutions since in that case the whole PB conceptual framework breaks down and correlation , not screening , effects [ 6 ] become the salient feature of the behavior of the system . The ionic screen - ing in monovalent salt solutions , augmented by the ef - fect of thermal DNA undulations , has been shown to quantitatively describe the measured osmotic pressure of DNA solutions in a fairly wide range of concentrations [ 12 , 13 , 14 ] . Apart from being a charged polymer , DNA is also molecularly rather stiff . The flexibility of polymers is usually described via the persistence length L p . Per - sistence length is nothing but the correlation length for orientational correlations along the molecular axis of the polymer [ 1 ] . For DNA the usually accepted value is about 500 ˚A [ 10 ] . Persistence length separates two regimes of behavior : rigid chain regime for contour lengths smaller than L p and flexible chain regime for contour lengths much larger then L p . The persistence length depends on long range electrostatic interactions along the polyelec - trolyte chain . The influence of electrostatic interactions on the persistence length was first considered by Odijk [ 15 ] and independently by Skolnick and Fixman [ 16 ] . Ac - cording to the Odijk - Skolnick - Fixman ( OSF ) theory , the total persistence length can be decomposed into a struc - tural ( L 0 ) and electrostatic ( L e ) contribution as L p = L 0 + L e = L 0 + l B / ( 2 bκ ) 2 ( 3 ) where b is again the separation between charges . As - suming the MO condensation , i . e . b = l B , one gets L p = L 0 + 0 . 324 I − 1 s in ˚A . As is clear from the OSF theory , counterion condensation reduces the electrostatic contribution to the persistence length due to an increase in the effective separation between charges from b to l B . 3 The OSF result , though it can be nominally applied only at restrictive conditions , appears to work well when com - pared to experiments [ 17 ] as well as computer simulations [ 18 ] . On the other hand , in the regime of no added salt and thus weak electrostatic screening due to other chains and counterions , a semiflexible charged chain in a semidi - lute polyelectrolyte solution behaves like a random walk of correlation blobs ( see below ) with chain size R ∝ c − 0 . 25 [ 4 ] . Coming back to the electrostatic persistence length , it is worth mentioning that L e strongly depends on the valency of the counterions and in general on the details of the electrostatic interaction potential . For monovalent counterions L e is usually positive indicating an effective repulsion between monomers . It is evident that starting assumptions κ − 1 ≫ a of the MO theory ( infinitely thin polymers or highly dilute polyelectrolyte solutions ) and κ − 1 ≫ b of the OSF the - ory ( monovalent salt with low enough screening ) enforce their limited validity . It is not clear at all whether one can apply these theories in the limit where added salt concen - tration is smaller than the DNA concentration , viz . the concentration of intrinsic counterions . This fact raises further questions , even in the simplest case of DNA in monovalent salt solutions , of the general applicability of these models to predict the amount of counterion conden - sation , as well as to describe properties like fundamental length scales of DNA in solution . Due to the intrinsic length of native DNA , the concen - tration of polyelectrolyte solutions that we are dealing with in this contribution is always higher than the chain overlap concentration c * [ 4 ] . This means that we are ef - fectively always in the semidilute regime , where it has been known for a long time [ 4 , 19 ] , that a new length scale emerges describing the density correlations in the polyelectrolyte solution . This length scale is equal to the correlation length or the mesh size , describing the corre - lation volume in semidilute polyelectrolyte solutions , and is given by the de Gennes - Pfeuty - Dobrynin ( dGPD ) cor - relation length [ 4 , 20 , 21 ] that scales universally with the concentration of the polyelectrolyte , c , as ξ ∝ c − 0 . 5 ( 4 ) It is interesting that this form of scaling , first derived for uncharged polymer solutions , is preserved also in the case of polyelectrolytes with some theoretically possible adjustments only in the case of added salt effects , and is expected to be proportional to the screening length due to both free DNA counterions and added salt ions [ 4 ] . For polyelectrolyte solutions the interpretation of this scaling result is that for volumes smaller than ξ 3 the polyelec - trolyte chain is stiffened by electrostatic interactions [ 22 ] , whereas for scales larger than ξ , it behaves as a free flight chain . In an attempt to clarify several of the above issues , we have undertaken an investigation of dielectric relax - ation properties of DNA solutions that covers a broad range of DNA concentrations , as well as added salt concentrations . We have used low - frequency dielectric spectroscopy technique , widely established as a direct and non - destructive tool to probe charged entities and their structure in various bio - macromolecular systems [ 23 , 24 , 25 ] . Our aim was not only to verify the predic - tions of the various theories in the well defined conditions , but also to investigate the behavior of DNA at extreme conditions like very low and very high added salt limit . A brief report of this investigation has been published in Ref . [ 26 ] . Interpretation of our results depends on the nature of the conformation of DNA in low salt or even pure wa - ter solutions , more specifically on whether DNA is in a single stranded ( ss - ) or double stranded ( ds - ) conforma - tion . We have considered this question very carefully in what follows . Different preparation protocols for DNA solutions were adopted in order to study the issue of ds - DNA stability in pure water ( see the Materials and Methods section ) solutions . Denaturation of DNA was in particular investigated for pure water DNA solutions . These studies indicate that indeed for semidilute con - ditions the DNA double - helix was never denatured into two spatially distinguishable and well separated single strands . DNA solutions were additionally characterized by UV spectrophotometry , electrophoresis and atomic emmission spectroscopy . Our results demonstrate that DNA counterions , free as well as condensed in variable proportions , contribute to the oscillating polarization in the applied electric field and thus together determine the dielectric response of the DNA solution . The characteristic size of the relax - ation volume for the dielectric response of a semidilute DNA solution is given by one of the three fundamental length scales : the dGPD mesh size of the whole poly - electrolyte solution , the OSF salt - dependent persistence length of a single polyelectrolyte chain and the average size of the chain in the salt - free polyelectrolyte solution . The OSF prediction for the persistence length as a func - tion of added salt ionic strength is verified in the high added salt limit giving very good agreement . Our results indicate that by going from the high to the low salt limit and all the way down to the nominally pure water solu - tions , the characteristic length goes from the persistence length of the DNA in solution to a value that corresponds to the average size of the Gaussian chain composed of cor - relation blobs which scales as c − 0 . 25 DNA . Moreover , an exact condition is established that separates the high from the low added salt regime , which reads 2 I s = 0 . 4 c in , where c in is the concentration of DNA counterions , These two regimes differ in whether the added salt ions provide the screening or DNA acts as its own salt . Finally , our results confirm the theoretical prediction describing the concen - tration dependence of the mesh size of DNA solutions as ξ ∝ c − 0 . 5 DNA . In the limit of low DNA concentrations and low added salt , the semidilute solution correlation length deviates from the classical polyelectrolyte behav - ior and follows the scaling c − 0 . 33 DNA . Possible interpretations of this behavior are discussed and the appearance of lo - 4 cally fluctuating regions with exposed hydrophobic cores is suggested as the mechanism for this anomalous scaling . II . MATERIALS AND METHODS Salmon testes and calf thymus lyophilized Na - DNA threads were obtained from Sigma and Rockland , re - spectively . Low protein content was declared and ver - ified by our UV spectrophotometry measurements , for the former A 260 / A 280 = 1 . 65 − 1 . 70 and for the latter A 260 / A 280 = 1 . 87 . Inductively coupled plasma - atomic emmission spectroscopy ( ICP - AES ) elemental analysis was performed on the Na - DNA threads dissolved in pure water [ 27 ] . The results have shown that the phospho - rus and sodium contents were the same , implying that only intrinsic sodium atoms are present . Taking into account that for double stranded DNA , monomers cor - respond to base pairs of molecular weight 660 g / mol , this result implies 7 % by weight of Na + ions in DNA . This means that the concentration of intrinsic DNA counterions and the DNA concentration are related by c in [ mM ] = c DNA [ mg / mL ] × 3 µ mol / mg . Electrophore - sis measurements were performed on DNA dissolved in pure water and in NaCl electrolyte with different ionic strengths . The obtained results have shown consistently the existence of polydisperse DNA fragments , most of them in the range 2 - 20 kbp . Since the scale of 3 . 4 ˚A corresponds to one base pair , we estimate the range of contour lengths of DNA fragments to be 0 . 7 - 7 µ m . DNA solutions with different DNA concentrations and different added salt ionic strengths were prepared accord - ing to two protocols , which we describe below : I Pure water DNA solutions : Dry DNA threads were dissolved in pure water for 48 hours at 4 o C so that the solutions within concentration range 0 . 01 ≤ c DNA ≤ 15 mg / mL were obtained . The ionic strength of pure water I s ≈ 0 . 01 mM was es - timated from the measured conductivity σ = 1 . 5 µ S / cm of the pure water sample in the chamber for dielectric spectroscopy , using molar conductiv - ity of the highly diluted NaCl ( 126 . 5 Scm 2 / mol ) . An increased conductivity value of pure water , as compared to the declared one [ 27 ] , is due to manip - ulation in the laboratory environment . II DNA solutions with added salt : II1 NaCl was added to DNA water solution with a chosen c DNA ( prepared according to I ) , so that the added salt ionic strength was achieved in the range 0 . 01 mM ≤ I s ≤ 4 mM . II2 DNA solutions with the same c DNA as in II1 and with the added salt ionic strength in the range 1 mM ≤ I s ≤ 4 mM were prepared start - ing from stock DNA solutions in which DNA was dissolved in 10 mM NaCl for 48 hours at 4 o C . One of the stock solutions was dialyzed against 10mM NaCl during 24 hours at 4 o C ( II2 . 2 ) , while the other was not ( II2 . 1 ) . II3 DNA solutions with concentrations in the range 0 . 1 ≤ c DNA ≤ 1 . 25 mg / mL and with the added salt ionic strength I s = 1 mM were prepared starting from a stock DNA solution in which DNA was dissolved in 10 mM NaCl for 48 hours at 4 o C . Stock solution was dia - lyzed against 1 mM NaCl during 24 hours at 4 o C . The stock solutions were stored at 4 o C and were di - luted just before dielectric spectroscopy measurements , which were completed in a few days . UV spectrophotom - etry and electrophoresis measurements were performed within next two weeks . For longer periods the stock solu - tions were stored at - 80 o C . pH of all solutions were found to be around 7 . Similar pH values were measured for DNA solutions prepared from 10 mM Tris - EDTA buffer ( TE ) , which adjusts pH to 7 . 6 ( not discussed in this ar - ticle ) . The results of ICP - AES elemental analysis indicated that solutions of Na - DNA dissolved in pure water did not contain any additional ions except declared sodium ones . This was further confirmed by dielectric spectroscopy measurements , which gave identical results for DNA solu - tions with same DNA concentration and added salt ionic strength prepared according to the protocol II1 and II2 . Spectrophotometry measurements at 260 nm were per - formed to verify nominal DNA concentrations . The con - centration was determined assuming an extinction coef - ficient at 260 nm A 260 = 20 for 1 mg / mL , meaning that the measured absorption A 260 = 1 corresponds to 0 . 05 mg / mL [ 28 , 29 ] . Throughout this article we will refer to nominal concentrations , which we have found to be in a good agreement with the measured ones . In par - ticular , the measured concentrations for DNA solutions II2 and II3 , performed on 10 aliquots , were consistently smaller by about 20 % than the nominal ones . We in - terpret this difference to be due to water content , not taken into account by the spectrophotometry approach . Indeed , lyophilized DNA if kept at 110 o C for 30 min loses about 20 % in weight . Dielectric spectroscopy measurements [ 25 ] were per - formed at room temperature ( 25 o C ) using a set - up which consists of a home - made parallel platinum plate capaci - tive chamber and temperature control unit , in conjunc - tion with the Agilent 4294A precision impedance ana - lyzer operating in ν = 40 Hz - 110 MHz frequency range . The capacitive chamber enables reliable complex admit - tance measurements with reproducibility of 1 . 5 % of sam - ples in solution with small volume of 100 µ L and with conductivities in the range of 1 . 5 - 2000 µ S / cm . Low ac amplitudes of 50 mV were employed in order to probe the DNA response in the linear regime , once we verified that for ac signal levels in the range between 20 mV and 500 mV the result was essentially the same . Lowest ac amplitudes were not used in order to avoid extensive aver - 5 aging . Admittance was sampled at 201 frequencies at 27 points per frequency decade . At each frequency , admit - tance was sampled 10 times and averaged . In addition , three consecutive frequency sweeps were taken in order to average out the temperature variations . Total time for described measurement amounts to 60 sec . Measurement functions are the real part of the admittance G exp ( ω ) and the capacitance C exp ( ω ) , where ω = 2 πν . In addition to DNA samples , the reference sam - ples were also measured in order to minimize stray impedances , including the free ion contribution and elec - trode polarization effects , and extract the response due to DNA only [ 25 , 30 ] . Reference samples were cho - sen as NaCl solutions of different molarities , adjusted to have the real part of admittances at 100 kHz the same as DNA solutions . In comparison to these solu - tions , independent adjustment of capacitance at 1 kHz demanded the reference NaCl concentrations that differ at most by 20 % . Since the difference in capacitance be - tween reference NaCl solutions of similar molarities is approximately constant at frequencies above the influ - ence of the electrode polarization , adjusting solely the real part of admittance effectively also adjusts the ca - pacitance up to an additive term . As a result , the sub - traction of the reference response successfully eliminated influence of spurious effects down to a low frequency limit in the range 0 . 5 - 30 kHz , depending on the so - lution molarity , and up to a high frequency limit of 30 MHz . Generally speaking , electrode polarization effects are larger for higher DNA and added salt concentrations , so that the low frequency bound is shifted to higher fre - quencies . Assuming that the conductivity contribution of each entity in the solution is additive [ 2 ] , the DNA response is given by G ( ω ) = G exp ( ω ) − G ref ( ω ) and C ( ω ) = C exp ( ω ) − C ref ( ω ) , where G ref ( ω ) , C ref ( ω ) is the reference samples response . Finally , the real and imaginary parts of dieletric function are extracted using relations ε ′ ( ω ) = l S C ( ω ) ε 0 ( 5 ) ε ′′ ( ω ) = l S G ( ω ) ωε 0 ( 6 ) l / S = 0 . 1042 ± 0 . 0008 cm − 1 is the chamber con - stant , where S = 0 . 98 cm 2 is the effective electrode cross - section corresponding to the sample of 100 µ L and l = 0 . 1021 ± 0 . 0001 cm is the distance between the elec - trodes . The chamber constant was determined by mea - suring the real part of admittance at 100 kHz of a 0 . 01 M and 0 . 0005 M KCl standard solutions ( Mettler - Toledo ) . It was also corroborated by the difference in capacitance of the chamber when empty and with 100 µ L of the pure water . In the latter case , the dielectric constant of pure water ǫ w = 78 . 65 was used as a standard . Detailed analysis of the DNA response was made in terms of the complex dielectric function ε ( ω ) given by a generalization of the Debye expression known as the phenomenological Havriliak - Negami ( HN ) function [ 31 ] ε ( ω ) − ε HF = ∆ ε 1 ( 1 + ( iωτ 0 ) 1 − α ) β ( 7 ) where ∆ ε = ε 0 − ε HF is the strength of the relaxation process , ε 0 is the static dielectric constant ( ω ≪ 1 / τ 0 ) and ε HF is the high frequency dielectric constant ( ω ≫ 1 / τ 0 ) . τ 0 is the mean relaxation time , while 1 − α and β are the shape parameters which describe the symmetric broadening of the relaxation time distribution function and skewness , respectively . We worked with β = 1 since this simplified HN formulation ( also known as the Cole - Cole function ) has been widely and successfully used to describe relaxation processes in disordered systems . Measured data were analyzed by using the least squares method in the complex plane [ 32 , 33 ] . Such an approach , which takes into consideration both the real and the imaginary part of the dielectric function at the same time , strongly improves the resolution if com - pared with the method in which the real and imaginary parts are treated separately . The complex plane method proved itself to be a powerful tool to resolve reliably two close modes in frequency even if the strength of one or both modes does not exceed ∆ ε = 2 − 3 , provided that the ratio of their dielectric strengths is smaller than the ratio of their positions in frequency . III . RESULTS We now present results of the dielectric response study performed on DNA solutions prepared according to pro - tocols described in Section II . The obtained dielectric re - laxation results for salmon testes and calf thymus DNA samples were essentially the same . For the sake of sim - plicity , in the remainder of this article we will speak of DNA samples only . Fig . 1 shows the frequency dependent real and imag - inary part of the dielectric function for selected DNA concentrations of DNA solutions . The results for pure water DNA solutions ( protocol I , concentrations a1 = 2 . 5 mg / mL , a2 = 0 . 4 mg / mL , a3 = 0 . 1 mg / mL and a4 = 0 . 0125 mg / mL ) are shown in panel a ) and b ) , while the results for DNA solutions with added salt of ionic strength I s = 1 mM ( protocol II3 , concentrations b1 = 0 . 83 mg / mL , b2 = 0 . 5 mg / mL , b3 = 0 . 31 mg / mL and b4 = 0 . 125 mg / mL ) are shown in panel c ) and d ) . The observed dielectric re - sponse is complex and the data were only successfully fitted to a formula representing the sum of two HN func - tions . The full lines in Fig . 1 correspond to these fits , while the dashed lines represent single HN forms . The main features of this response , for pure water DNA so - lutions , as well as for DNA solutions with added salt , are two broad modes , whose amplitude and position in frequency depend on the DNA concentration . The pa - rameter 1 − α , which describes the symmetrical broad - ening of the relaxation time distribution function , is concentration independent and similar for both modes 6 n ( Hz ) 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 e ' ' 1 10 100 a1 a4 a3 a2 S a l m on X ' 04 . j nb / 3115 25 o C pure water DNA solutions s a l m on - DNA [ m g / m l ] a 1 C 4 2 . 5 a 2 C 12 0 . 4 a 3 C 18 0 . 1 a 4 C 27 0 . 0125 a ) e ' 1 10 100 a1 a4 a3 a2 b ) n ( Hz ) 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 e ' ' 1 10 100 b1 b4 b3 b2 D i a l DNA 30 V I _ F I T . j nb / 1615 25 o C DNA solutions with added salt DNA i n 1 m M N a C l s a l m on - DNA [ m g / m L ] b1 C 2 0 . 83 b2 C 4 0 . 5 b3 C 6 0 . 31 b4 C 9 0 . 16 d ) e ' 1 10 100 c ) b1 b4 b3 b2 FIG . 1 : Double logarithmic plot of the frequency dependence of the real ( ε ’ ) and imaginary ( ε ” ) part of the dielectric func - tion at T = 25 o C of ( a , b ) pure water DNA solutions ( protocol I ) and ( c , d ) DNA water solutions with added salt I s = 1 mM ( protocol II3 ) for representative a1 - a4 ( 2 . 5 , 0 . 4 , 0 . 1 , 0 . 0125 mg / mL ) and b1 - b4 ( 0 . 83 , 0 . 5 , 0 . 31 , 0 . 125 mg / mL ) DNA con - centrations . The full lines are fits to the sum of the two HN forms ; the dashed lines represent a single HN form . 1 − α ≈ 0 . 8 . The mode centered at higher frequencies ( 0 . 1 MHz < ν HF < 15 MHz ) is characterized by smaller dielectric strength ( 5 < ∆ ε HF < 20 ) than the mode ( 20 < ∆ ε LF < 100 ) centered at lower frequencies ( 0 . 5 kHz < ν LF < 70 kHz ) . In the remainder of this article , we will refer to these modes as the high - frequency ( HF ) and low - frequency ( LF ) mode , respectively . In what follows , we discuss possible assignments for these relaxation modes inside the framework of exist - ing theoretical approaches for polarization response of charged biopolymers in solution [ 25 , 35 , 36 , 37 , 38 ] . An applied ac field generates an oscillating flow of net charge associated with DNA counterions [ 39 ] and induces polar - ization . Since the counterion displacement is controlled by diffusion , the dielectric response is basically charac - terized by the mean relaxation time τ 0 ∝ L 2 / D in , where L is the associated length scale , and D in is the diffusion constant of counterions which is sufficiently well approx - imated by the diffusion constant of bulk ions [ 25 , 40 ] . Since we deal with Na - DNA solutions , we take the diffu - sion constant of Na + ions D in = 1 . 33 · 10 − 9 m 2 / s . Note that the equation τ 0 ∝ L 2 / D in is a scaling relationship and the proportionality constant is of order one ( see Dis - cussion , part B ) . Several length scales are theoretically expected to be associated with dielectric relaxations of polyelectrolytes in solution : the contour length , the Debye screening length , the polymer chain statistical segment length and the polymer solution mesh size . The mean relaxation time for pure water DNA solu - tions , as well as for DNA solutions with added salt , is found in the range 10 − 8 − 1 . 5 · 10 − 6 s for the HF mode and 2 · 10 − 6 − 3 · 10 − 4 s for the LF mode . The correspond - ing characteristic length for the HF mode spans the range from 3 nm to 50 nm , while the characteristic length for the LF mode varies between 50 nm and 750 nm . Both of them are thus not within the range of the contour length distribution in our samples . On the other hand , the Debye screening length , the polymer solution mesh size and the polymer statistical segment length appear as plausible candidates . Moreover , it is noteworthy that specifically the values of the characteristic length for the LF mode are close to the values expected for the DNA persistence length . A . HF mode First , we address the HF mode . In the case of pure water DNA solutions the characteristic length L HF shows the scaling L HF ∝ c − 0 . 5 DNA with respect to the DNA con - centration , all the way down to a crossover concentration c co ∼ 0 . 6 mg / mL . At that point the scaling form is then changed to L HF ∝ c − 0 . 33 DNA ( Fig . 2a ) . The observed behav - ior at high DNA concetrations conforms exactly to the de Gennes - Pfeuty - Dobrynin ( dGPD ) [ 4 , 20 , 21 ] scaling form valid for salt - free polyelectrolyte solutions [ 4 , 25 ] . At low DNA concetrations , in the regime below c co , L HF 7 c DNA ( mg / mL ) 0 . 01 0 . 1 1 10 L H F ( n m ) 10 100 DN A 2 . j nb / 0214 25 o C k - 1 ~ I s - 0 . 5 I s ( mM ) 0 . 01 0 . 1 1 10 L H F ( n m ) 10 0 . 05 mg / mL 0 . 5 mg / mL a ) c co in same as for bulk Na b ) 25 o C 0 . 8 mg / mL FIG . 2 : a ) Characteristic length of the HF mode ( L HF ) for pure water DNA solutions ( protocol I , open squares ) and for DNA solutions with added salt I s = 1 mM ( protocol II3 , full squares ) as a function of DNA concentration ( c DNA ) . The full line is a fit to the power law L HF ∝ c − 0 . 33 DNA and ∝ c − 0 . 5 DNA for c DNA smaller and larger than c co ∼ 0 . 6 mg / mL , respectively . b ) Characteristic length of the HF mode ( L HF ) for DNA so - lutions with varying added salt ( I s ) for three representative DNA concentrations : c DNA = 0 . 05 mg / mL ( diamonds , pro - tocol II1 ) , c DNA = 0 . 5 mg / mL ( full triangles , protocol II1 ; open triangles , protocol II2 . 1 ; open inverse triangles , proto - col II2 . 2 ) and c DNA = 0 . 8 mg / mL ( circles , protocol II2 . 1 ) . The full line denotes Debye screening length κ − 1 for the in - vestigated range of added salt ionic strength I s . displays an unusual behavior generally not observed in semidilute solutions . It appears as though at low DNA concentrations local conformational fluctuations partially expose the hydrophobic core of DNA so that the corre - lation length scales as c − 0 . 33 DNA , as is the case of charged chains with partially exposed hydrophobic cores [ 4 ] . In dilute solutions , but only in dilute solutions , this scaling form would be typical for the average separation between chains [ 4 ] . The observed crossover might be thought to reflect the border between dilute and semidilute solutions [ 25 , 41 ] corresponding to the crossover concentration c ∗ [ 20 ] . For the shortest fragments of 2 kbp in our DNA so - lutions , we get c ∗ of the order of 0 . 006 mg / mL , while the lowest concentration of DNA solutions is 0 . 01 mg / mL . In this manner , the interpretation of c co ∼ 0 . 6 mg / mL as the dilute - semidilute crossover concentration c ∗ is ruled out . With added 1 mM salt , the dGPD behavior of L HF remains unchanged , thus L HF ∝ c − 0 . 5 DNA , as long as the concentration of intrinsic counterions c in ( proportional to c DNA ) is larger than the concentration of added salt ions 2 I s ( Fig . 2a ) . When the concentration of intrinsic counterions becomes smaller than the added salt concen - trations , the L HF apparently shows a leveling off , with a limiting value close to the Debye length appropriate for this salt concentration . One should be cautious here since the data become much less reliable exactly at low DNA concentrations ( see error bars in Fig . 2a ) . Three sets of additional data ( Fig . 2b ) for three representative DNA concentrations with varying added salt also seem to reveal that L HF does not vary with I s in most of the mea - sured range of added salt , while the corresponding Debye screening length in the same range of added salt values decreases substantially . Unfortunately the accuracy of the data again becomes much less reliable due to the progressive merging of the HF and LF modes when one approaches the regime , where the characteristic length scale becomes apparently larger than the nominal Debye length at that salt concentration . Next we consider the behavior of dielectric strength defined as ∆ ε HF ≈ f HF · c in · α HF , where f HF is the fraction of counterions participating in the HF process , c in [ mM ] = c DNA [ mg / mL ] × 3 µ mol / mg ( as explained in Section II ) and α HF is the corresponding polarizabil - ity . The polarizability α HF is given by the scaling form α HF ∝ e 2 · L 2HF · / ( ε 0 kT ) ∝ l B · ε · L 2HF , [ 25 , 41 ] . Therefore , the fraction of counterions f HF participating in the HF process is proportional to ∆ ε HF / ( c DNA · L 2HF ) . In Fig . 3 we show the dependence of f HF on DNA concentration ( panel a ) ) and on the ionic strength of added salt ions ( panel b ) ) . The f HF data for pure water DNA solution displayed in panel a ) indicate that the fraction of counterions partici - pating in this relaxation process does not depend on the concentration of DNA . Since the HF relaxation happens at the length scale ξ which describes the density corre - lations between DNA chains , this result indicates that it is the free counterions as opposed to condensed counteri - ons , that can hop from chain to chain in the volume ξ 3 , that are the relaxation entities participating in the HF process . It is noteworthy that a similar interpretation was previously proposed by Ito et al . [ 41 ] for the relax - ation in synthetic polyelectrolytes observed in the same frequency range [ 25 ] . The data displayed in Fig . 3 b ) suggest that the frac - tion of intrinsic counterions f HF active in the HF mode remains constant when salt is added to the DNA solution as long as c DNA is substantially larger than I s . However , as soon as the concentration of added salt ions prevails over the concentration of intrinsic counterions , f HF starts to decrease . This is also discernible in the behavior of the f HF data for I s = 1 mM added salt solution shown 8 DN A 2 . j nb / 0215 c DNA ( mg / mL ) 0 . 01 0 . 1 1 10 D e H F / ( c DNA L H F 2 ) 10 - 2 10 - 1 10 0 25 o C a ) I s ( mM ) 0 . 01 0 . 1 1 10 D e H F / ( c DNA L H F 2 ) 10 - 2 10 - 1 10 0 0 . 05 mg / mL 0 . 5 mg / mL b ) 25 o C in as for bulk Na 0 . 8 mg / mL FIG . 3 : a ) Normalized dielectric strength of the HF mode ∆ ε HF / ( c DNA · L 2HF ) as a function of DNA concentration ( c DNA ) for pure water DNA solutions ( protocol I , open squares ) and for DNA solutions with added salt I s = 1 mM ( protocol II3 , full squares ) . The full line is a guide for the eye . b ) Normalized dielectric strength of the HF mode ∆ ε HF / ( c DNA · L 2HF ) vs . ionic strength of the added salt ( I s ) for three representative DNA concentrations : c DNA = 0 . 05 mg / mL ( diamonds , protocol II1 ) , c DNA = 0 . 5 mg / mL ( full triangles , protocol II1 ; open triangles , protocol II2 . 1 ; open inverse triangles , protocol II2 . 2 ) and c DNA = 0 . 8 mg / mL ( circles , protocol II2 . 1 ) . in panel a ) . A plausible suggestion would be that the salt renormalization of f HF is a consequence of screening due to added salt ions that seem to diminish the effective number of counterions that can participate in the chain - chain hopping process . B . LF mode Second , we address the LF mode . For pure water DNA solutions ( protocol I ) , the characteristic length L LF in - creases with decreasing DNA concentration in almost three decades wide concentration range ( open inverse triangles in Fig . 4a ) ) following the power law L LF ∝ c − 0 . 29 ± 0 . 04 DNA . The exponent − 0 . 29 ± 0 . 04 suggests that in this regime L LF is proportional to the average size of the polyelectrolyte chain that behaves as a random walk of correlation blobs and scales as c − 0 . 25 DNA [ 4 ] . For DNA solutions with added salt I s = 1 mM ( protocol II3 , full inverse triangles in Fig . 4a ) ) , L LF coincides with the one found for pure water solutions with high DNA concentra - tions . As soon as the concentration of intrinsic counteri - ons c in ( proportional to c DNA ) becomes smaller than the concentration of bulk ions from added salt 2 I s , L LF starts to deviate from the L LF ∝ c − 0 . 29 DNA behavior and decreases to attain value of about 500 ˚A at which it saturates . The dependence of L LF on the added salt ionic strength I s is shown in Fig . 4b ) for three DNA concentrations . The observed data can be nicely fit to the OSF behavior [ 15 , 16 ] of the form : L p = L 0 + a · I − 1 s . We get L 0 = 470 ˚A for the structural persistence length and a = 0 . 09 ˚AM . While the value of L 0 close to 500 ˚A is in accordance with standard expectations for DNA [ 10 ] , the value of the coefficient a is somewhat smaller than expected by the OSF theory a = 0 . 324 ˚ AM . It is noteworthy that the OSF model applies as long as the ionic strength of added salt is larger than the concentration of the intrinsic counterions . The data for c DNA = 0 . 05 , 0 . 5 and 0 . 8 mg / mL deviate from the OSF behavior for I s < 0 . 03 mM , I s < 0 . 3 mM and I s < 0 . 5 mM , respectively . The value of L LF in this low salt limit attains the same value as in pure water DNA solutions ( see Fig . 4a ) . In Fig . 5a ) we show dependence of f LF ∝ ∆ ε LF / ( c DNA · L 2LF ) on the DNA concentration assuming again that the polarizability α LF varies as the characteristic length squared . The data show that the fraction of counterions f LF active in the LF mode is roughly speaking indepen - dent of the DNA concentration . This is valid for the pure water DNA solution , as well as in the case of I s = 1 mM added salt solution . In our view this result together with the fact that the LF relaxation happens at the length scale of the average size of the polyelectrolyte chain sug - gests that the LF relaxation engages mostly condensed counterions along and in close vicinity of the chain . However , the data displayed in panel b ) , as well as the data for I s = 1 mM in panel a ) suggest that the fraction of counterions participating in the LF process f LF becomes larger in the case of added salt solutions , compared to the pure water case , if the concentration of added salt ions becomes larger than c DNA . This means that at least some of the free counterions join the relax - ation of the condensed counterions along the segments of the same chain . It is thus impossible to completely sepa - rate condensed counterions from free counterions in their contribution to the LF relaxation mode . This conclusion bears crucially on the assumption that the scale of polar - izability is given by L LF ( I s ) . It is noteworthy that for the HF and LF modes the addition of salt changes the effec - tive number of participating counterions in the opposite way . This might be attributed to the increased screen - ing for the interchain HF relaxation , and to the intrinsic counterion atmospheres squeezed closer to the chains due to reduced Debye length for the LF relaxation along the 9 c DNA ( mg / mL ) 0 . 01 0 . 1 1 10 L L F ( n m ) 100 1000 DN A 2 . j nb / 0216 25 o C 0 . 5mg / mL 0 . 05mg / mL Lp = 470A + 0 . 09 / I I s ( mM ) 0 . 01 0 . 1 1 10 L L F ( n m ) 10 100 1000 in same as for bulk Na 25 o C a ) b ) 0 . 8mg / mL FIG . 4 : a ) Characteristic length of the LF mode ( L LF ) for pure water DNA solutions ( protocol I , open inverse triangles ) and for DNA solutions with added salt I s = 1 mM ( pro - tocol II3 , full inverse triangles ) as a function of DNA con - centration ( c DNA ) . The full line is a fit to the power law L LF ∝ c − 0 . 29 ± 0 . 04 DNA . b ) Characteristic length of the LF mode ( L LF ) for DNA solutions with varying added salt ( I s ) for three representative DNA concentrations : c DNA = 0 . 05 mg / mL ( di - amonds , protocol II1 ) , c DNA = 0 . 5 mg / mL ( full triangles , pro - tocol II1 ; open triangles , protocol II2 . 1 ; open inverse trian - gles , protocol II2 . 2 ) and c DNA = 0 . 8 mg / mL ( circles , protocol II2 . 1 ) . The full line is a fit to the expression L p = L 0 + a · I − 1 s with L 0 = 470 ˚A and a = 0 . 09 ˚A . chain . As a final remark , we point out that our results show - ing that the HF relaxation can be attributed to the semidilute mesh size ξ , in the polyelectrolyte semidilute solution in which single chain persistence length associ - ated with the LF relaxation is always larger than ξ , con - firm the prediction of Odijk [ 19 ] that the same scaling law ξ ∝ c − 0 . 5 should also be valid for semiflexible DNA polymers . DN A 2 . j nb / 0217 c DNA ( mg / mL ) 0 . 01 0 . 1 1 10 D e L F / ( c DNA L L F 2 ) 10 - 3 10 - 2 10 - 1 25 o C a ) in as for bulk Na + 0 . 8 mg / mL 0 . 05 mg / mL I s ( mM ) 0 . 01 0 . 1 1 10 D e L F / ( c DNA L L F 2 ) 10 - 3 10 - 2 10 - 1 25 o C b ) 0 . 5 mg / mL FIG . 5 : a ) Normalized dielectric strength of the LF mode ∆ ε LF / ( c DNA · L 2LF ) as a function of DNA concentration ( c DNA ) for pure water DNA solutions ( protocol I , open inverse tri - angles ) and for DNA solutions with added salt I s = 1 mM ( protocol II3 , full inverse triangles ) . The full line is a guide for the eye . b ) Normalized dielectric strength of the LF mode ∆ ε LF / ( c DNA · L 2LF ) vs . ionic strength of the added salt ( I s ) for three representative DNA concentrations : c DNA = 0 . 05 mg / mL ( diamonds , protocol II1 ) , c DNA = 0 . 5 mg / mL ( full triangles , protocol II1 ; open triangles , protocol II2 . 1 ; open inverse triangles , protocol II2 . 2 ) and c DNA = 0 . 8 mg / mL ( circles , protocol II2 . 1 ) . IV . DISCUSSION First , let us summarize the results of dielectric spec - troscopy measurements . In the linear ac field regime , two broad relaxation modes are observed corresponding to three different time and length scales . The HF mode is centered in the frequency range between 0 . 1 MHz and 15 MHz , depending solely on the DNA concentration as long as the DNA concentration remains larger than the added salt concentration . In this regime , the characteris - tic length scale is identified with the mesh size , and varies as ξ ∝ c − 0 . 5 DNA . Our data also seem to indicate that once the added salt becomes larger than the concentration of DNA intrinsic counterions , the high frequency charac - teristic length , rather than scaling as the semidilute so - 10 lution correlation length , levels off at a value close to the corresponding Debye length . More systematic exper - iments are needed to asses the possible added salt depen - dence in this regime of salt concentrations . In the limit of low DNA concentrations and low added salt , the semidi - lute solution correlation length smoothly crosses over to a less rapid scaling as ∝ c − 0 . 33 probably reflecting the appearance of locally fluctuating regions with exposed hydrophobic cores . The LF mode is centered in the frequency range be - tween 0 . 5 kHz < ν LF < 70 kHz . In DNA solutions with added salt , the characteristic length scale of this mode corresponds to the persistence length , which varies ex - perimentally as L p ∝ I − 1 s . In the limit of low added salt , the characteristic length scale smoothly merges with the average size of the Gaussian chain composed of corre - lation blobs , which varies with DNA concentration as L LF ∝ c − 0 . 25 DNA . The dielectric data also seem to indicate that the free DNA counterions are mostly responsible for the high fre - quency relaxational mode , whereas the low frequency mode appears to be more complicated and the decou - pling of MO condensed and free DNA counterions seems to be difficult with any degree of confidence . Investigation of dielectric properties of DNA goes back to early ’60s and since then a reasonable number of pa - pers have been published [ 24 , 25 , 30 , 35 , 43 , 44 , 45 , 46 , 47 ] . In these studies , two dispersion modes associ - ated with the counterion fluctuations were found , one at very low frequencies , which depended on molecular weight ( i . e . degree of polymerization , N ) and another one in the intermediate frequency region , which showed no N dependence , but did show a pronounced concentra - tion dependence . For the sake of completeness we also mention the third relaxation in the GHz frequency range , which is due to solvent ( water ) relaxation and therefore not directly a concern of this paper . The low frequency mode was consistently associated with the DNA contour length , while the intermediate fre - quency one was often associated with the DNA statistical segment length . These interpretations were based on the theoretical models of the Mandel group [ 35 , 36 , 48 ] devel - oped for the case of a single polyelectrolyte ( limit of very dilute solutions ) . Another interpretation of the inter - mediate frequency relaxation , proposed and verified un - til now only for synthetic monodisperse polyelectrolytes , was that it was due to the counterion fluctuation along the correlation length [ 25 , 41 , 42 ] . It is worth noting the suggestion of Odijk [ 19 ] that the mesh size gives a phys - ical meaning to the statistically independent chain seg - ment length , which also scales as c − 0 . 5 [ 35 ] . The segment length was postulated to be due to the potential barriers along the chain , whose origin might be in the correlation length that measures the mean distance between contact points of the overlaping chains [ 20 ] . Our work reveals the existence of two , rather than one , distinct relaxation modes in the s . c . intermediate frequency range due to either a correlated response of counterions in the mesh of DNA chains in the solution , what we call the HF mode , or due to a response of the counterions along a single DNA chain , what we refer to as the LF mode . No previous experimental work was able to distinguish these two concentration dependent dispersions . The rea - son probably lies in the fact that the data analysis per - formed in the reported DNA dielectric spectroscopy stud - ies was not powerful enough to reveal and characterize two modes so close in frequency , where in addition one of them is small in amplitude . Indeed , work by Lee and Bone [ 47 ] hinted at two overlapping dispersions , but the authors were not able to characterize the smaller one properly . Another reason lies in the fact that none of these investigations covered so wide a range of DNA con - centrations and added salt ionic strengths , as we did in this work . A . Conformation of DNA in low salt solutions In this work we have paid special attention to the is - sue of the stability of ds - DNA helix , i . e . to the denatu - ration phase diagram [ 10 ] . The issue of DNA conforma - tion in pure water solutions is of paramount importance for proper understanding of our experiments . The ques - tion here is whether DNA at very low salt conditions is in the double stranded or single stranded form . Di - electric spectroscopy results strongly indicate that the double stranded form of DNA is stable in all pure water [ 27 ] solutions studied . Of course , a precise and defini - tive information on the polyelectrolyte intrachain confor - mation of DNA solutions would demand the small - angle neutron scattering and / or X - ray scattering experiments performed at the same conditions . First evidence for the stability of the ds - DNA form comes from the fact that the results of the dielectric spectroscopy measurements obtained in DNA solutions with added salt prepared from water ( protocol II1 ) co - incide with the results obtained on DNA solutions pre - pared from 10 mM NaCl ( protocols II2 , II3 ) ( see Fig . 2b ) , 3b ) , 4b ) and 5b ) ) . Second , we mention that Mandel [ 35 ] already reported that dielectric behavior in pure water DNA solutions was not found to differ markedly from that at low salt concentrations . Also the measured os - motic coefficient in nominally pure water conditions [ 49 ] confirms the assumption of an intact double stranded DNA form . Since the Manning charge density param - eters for ss - and ds - DNA are so different this difference should be apparent also in the measured osmotic coeffi - cient . None is detected however . In an attempt to clarify more this issue , we have mea - sured dielectric properties of pure water DNA solutions for the DNA concentration range between c DNA = 0 . 5 mg / mL and c DNA = 0 . 01 mg / mL , prepared according to protocol I , before and after the controlled denaturation protocol . Denaturation was accomplished by the heating of solutions for 20 min at temperature of 97 o C , followed 11 by quenching to 4 o C . Dielectric measurements were sub - sequently made at 25 o C . The observed results were simi - lar for all studied DNA solutions . The dielectric strength and the relaxation time of the LF mode decreased sub - stantially after the heating , while the change observed for the HF mode was much smaller ( Fig . 6 ) . For c DNA = 0 . 01 mg / mL , the LF mode was not observed at all after the denaturation , implying the dielectric strength ∆ ε < 1 ( see Fig . 6b ) ) . Notice that ∆ ε of the LF mode for this DNA concentration is about 10 . We note that L LF , which measures the average size of the DNA chain , decreased after the heating , but showed the same power law behavior L LF ∝ c − 0 . 29 ± 0 . 04 DNA as for the untreated DNA solution ( Fig . 7b ) ) . The observed change indicates that denatured ss - DNA , which is in the form of coil , is shorter and has smaller average size of the chain . Furthermore , L HF which measures the correlation length of the DNA solution , decreased after the heating and showed the power law behavior L HF ∝ c − 0 . 33 DNA in the whole DNA concentration range ( Fig . 7a ) ) . A smaller value of L HF is expected for the denatured DNA solu - tion , since such a solution should contain twice the num - ber chains . The power law behavior with the exponent - 0 . 33 , observed now also for the larger DNA concentra - tions , indicates that the hydrophobic core of DNA is fully exposed once the heating protocol was applied . All these results confirm that although untreated DNA at low salt and semidilute conditions might show locally exposed hydrophobic cores in a dynamic sense , a real / complete unzipping and separation of the strands might be accomplished only after the denatu - ration / heating protocol is applied . These observations therefore suggest that DNA in pure water solutions is not denatured into two spatially well separated single strands , but is rather in the double stranded form , lo - cally interspersed with exposed hydrophobic cores in the limit of low DNA concentrations . Furthermore , even after denaturation the two DNA strands appear to remain in relatively close proximity rather than becoming completely dissociated , an obser - vation well substantiated also by the correlation length measurements by SANS at semidilute DNA conditions [ 50 ] . In these measurements the correlation length mea - sures the characteristic distance between the hydrogen - containing ( sugar - amine base ) groups . Hammouda and Worcester [ 50 ] have recently determined that on melting of DNA in DNA / d - ethylene - glycol mixtures the correla - tion length increases from about 8 ˚A to about 12 - 15˚A , which implies that even after melting the two strands of the ds - DNA remain in very close proximity . This appears to be due to the presence of other chains in the semidi - lute solution that spatially constrain the separate strands even after melting and prevent complete dissocation [ 51 ] . The question furthermore arises if the renaturation process is fast enough to occur partially during dielectric measurements which are performed at 25 o C . In order to check this , we have repeatedly measured the response of pure water DNA solution , after the heating protocol to n ( Hz ) 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 e ' ' 1 10 100 pure water DNA solution c DNA = 0 . 1mg / mL denatured at : untreated d e n a t u r 0100 m g A ll _ t e m p E . J N B / 0404 21 . X I ' 05 97 o C 25 o C a ) n ( Hz ) 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 e ' ' 0 . 1 1 10 100 25 o C pure water DNA solution c DNA = 0 . 01mg / mL denatured at : untreated d e n a t u r a ti on B 25 . j nb / 0404 97 o C b ) FIG . 6 : Double logarithmic plot of the frequency depen - dence of the imaginary part of the dielectric function ( ε ” ) at T = 25 o C of a pure water DNA solution ( protocol I ) with DNA concentration c DNA = 0 . 1 mg / mL ( panel a ) ) and c DNA = 0 . 01 mg / mL ( panel b ) ) before ( denoted as untreated ) and after the heating to 97 o C ( denoted as denaturated , see Text ) . The full lines are fits to the sum of the two HN forms ; the dashed lines represent a single HN form . 97 o C was applied . The time span was 100 min , the first measurement was taken 3 minutes after the sample was heated from 4 o C to 25 o C . The observed response after 3 min and after 100 min was the same ( inside the error bar of 1 . 5 % ) indicating very long time constant characteriz - ing the renaturation process . This result confirmed that 12 c DNA ( mg / mL ) 0 . 01 0 . 1 1 10 L H F ( n m ) 10 100 DN A 2 . j nb / 0219 25 o C a ) c co in same as for bulk Na pure water DNA solutions untreated denatured at 97 o C c DNA ( mg / mL ) 0 . 01 0 . 1 1 10 L L F ( n m ) 100 1000 DN A 2 . j nb / 0221 in same as for bulk Na b ) denatured at 97 o C untreated 25 o C pure water DNA solutions FIG . 7 : a ) The characteristic length L HF of the HF mode before ( open squares ) and after the heating to 97 o C ( full squares ) . The full line is a fit to the power law L HF ∝ c − 0 . 33 DNA and ∝ c − 0 . 5 DNA for c DNA smaller and larger than c co ∼ 0 . 6 mg / mL , respectively . The dashed line is a fit to the power law L HF ∝ c − 0 . 33 DNA in the whole DNA concentration range . b ) The characteristic length L LF of the LF mode before ( open triangles ) and after the heating to 97 o C ( full trian - gles ) . The full line and dashed lines are fits to the power law L LF ∝ c − 0 . 29 ± 0 . 04 DNA . the observed dielectric properties are the ones of ss - DNA . Osmotic pressure results of Raspaud et al . [ 52 ] on short nucleosomal fragments of DNA also indicate that at high enough concentration , intrinsic DNA counterions prevent destabilization of ds - DNA helix in pure water solutions . These experiments show that the role of added salt ( I s ) in the ds - DNA stabilization becomes negligible in the high DNA concentration range i . e . in the limit c DNA ≫ I s . Osmotic pressure of intrinsic DNA counterions can thus be high enough to prevent denaturation of ds - DNA helix in pure water solutions . UV spectrophotometry experiments performed previ - ously by Record [ 53 ] on T4 and T7 phage DNA have also shown that ds - DNA denaturation depends not only on added salt concentration I s , but also on the con - centration of intrinsic DNA counterions , c in [ mM ] = c DNA [ mg / mL ] × 3 µ mol / mg ( see Section II ) . Moreover , ds - DNA was found to be stable at 25 o C dissolved in nom - inally pure water ( no added salt , I s → 0 ) for the concen - tration of intrinsic counterions larger than 0 . 2 mM . This again is consistent with our experiments on dielectric re - laxation of DNA solutions . Another meaningful question would be , what is the smallest c in which can still keep DNA in the double stranded form . The UV absorbance results indicated that no added salt is needed to stabilize ds - DNA in the case when the intrinsic counterion concentration , c in , is larger than 0 . 2 mM , while dielectric spectroscopy re - sults suggest it can be one order of magnitude smaller , c in > 0 . 03 mM . Notice that in the latter case c in is still larger than the estimated ion concentration of pure wa - ter ( 2 I s = 0 . 02 mM ) , i . e . c in > 2 I s . The osmotic pres - sure data indicate [ 52 ] that intrinsic counterions them - selves can stabilize ds - DNA for c in larger than 30 mM and 150 mM for two different added salt concentrations 2 I s = 4 mM and 20 mM , respectively . All these results suggest that the concentrations of intrinsic counterions and added salt themselves are less important , rather it is their ratio that defines which limit prevails . Thus ab - sorbance and osmotic pressure data both suggest that for c in / 2 I s > 10 intrinsic counterions prevail in stabiliz - ing ds - DNA helix , while dielectric results suggest that this ratio might be shifted to c in / 2 I s > 1 . Neverthe - less , the unusual scaling exponent - 0 . 33 of the semidilute correlation length found for the DNA pure water solu - tions with DNA concentrations smaller than c co ∼ 0 . 6 mg / mL seem to suggest the existence of local conforma - tional fluctuations which partially expose the hydropho - bic core of DNA . In order to verify this proposal a further comparative study , including UV spectrophotometry and dielectric spectroscopy , is planned to study in depth the conformation of DNA in pure water solutions . We are however convinced that in all our investigations DNA did not exist as two separated single coils , rather DNA was effectively always in its double stranded form . B . Ionic screening : added salt versus intrinsic DNA counterions Next we address the issue of the respective roles in ionic screening of intrinsic DNA counterions and ions from the added salt . First , we examine conditions un - der which the OSF expression for the persistence length L p = L 0 + a · I − 1 s is valid . Dielectric data show that the influence of the added salt on the persistence length is important as long as the ionic strength I s is suffi - ciently larger than the concentration of intrinsic coun - terions . Plot in Fig . 8 reveals that this condition reads 2 I s > 0 . 4 c in . In this limit where the OSF theory applies , the coefficient a should be equal 0 . 324 ˚AM ( assuming MO counterion condensation ) . However , experimentally , different values are found . Measurements of DNA elastic properties as a function of ionic strength also yielded the coefficient in the OSF expression , a = 0 . 8 ˚AM [ 17 ] , which 13 is different from the one expected from the MO counte - rion condensation theory . On the other hand , a magnetic birefringence study by Maret and Weill [ 54 ] suggested the values of a between 0 . 25 and 0 . 45 , therefore indeed close to 0 . 324 . However , the authors fitted their data to the ( 0 . 12 c in + I s ) − 1 instead to the OSF I − 1 s dependence . The first term represents the influence of free DNA counteri - ons . It is rather obvious that the OSF fit would not yield the MO value of the coefficient a . Why thus is there a dif - ference between the MO value and the measured value of the coefficient in the OSF dependence of the persistence length on the ionic strength of the added salt ? In an attempt to reconcile these various values , we have rescaled the persistence length from dielectric measure - ments as 2 . 5 · L LF → L LF in order to collapse the behav - ior from dielectric and elastic experiments onto a single curve ( Fig . 9 ) . Rescaling is justified since the expres - sion connecting L p from dielectric properties with the measured mean relaxation time is only valid as a scaling relationship . Numerical factors are less straightforward and are essentially unknown . An exact expression to - gether with an appropriate numerical coefficient , known as the Einstein - Smoluchowski formula , is only known in the dc limit , where L = √ 2 Dτ . The fit of this new rescaled version of the L LF to the OSF expression with two free parameters now gave L 0 = 530 ˚A and a = 0 . 69 ˚AM . The difference between this value of a and the one expected from the MO counte - rion condensation theory signals a different effective lin - ear charge density than the one stemming from the MO counterion condensation theory . This difference in the measured and theoretically expected value of a coefficient of the OSF fitting form for the persistence length would be consistent also with other experiments that provide a value for the effective charge density [ 14 ] . Finally , let us examine the low salt limit ( 2 I s < 0 . 4 c in ) in which we expect that the intrinsic counterions be - come dominant . The characteristic length of the LF mode varies with DNA concentration as L LF ∝ c − 0 . 29 DNA , or equivalently as c − 0 . 29 in . This value of the exponent would allow us to identify L LF with the average size of the Gaussian chain composed of correlation blobs scaling as c − 0 . 25 DNA , where electrostatic interactions are screened by other chains and counterions , and thus DNA acts as its own salt . V . SUMMARY All these results make it quite clear that for the dielec - tric properties studied in this contribution the role of free and condensed intrinsic DNA counterions can not always be separated . For the HF relaxation we are reasonably sure that it is mostly the free counterions that act as the relaxation entities in a type of hopping process between the correlated DNA chains in a semidilute polyelectrolyte solution . On the contrary , for the LF relaxation process it seems to be mostly the condensed counterions that re - Din ( 0 . 5 ) = 0 . 9e - 9 Din ( 0 . 05 ) = 1 . 7e - 9 DN A 2 . j nb / 0130 B W 2 I s / c in 0 . 1 1 10 L L F / L L F ( I s fi 0 ) 0 . 2 0 . 5 2 . 0 0 . 1 1 0 . 05 mg / mL 0 . 5 mg / mL 0 . 8 mg / mL FIG . 8 : Characteristic length of the LF mode ( L LF ) nor - malized with the value in pure water solutions ( low salt limit I s = 0 . 01 mM ) vs . added salt concentration normalized by the concentration of intrinsic counterions ( 2 I s / c in ) . Data are for three representative DNA concentrations : c DNA = 0 . 05 mg / mL ( diamonds , protocol II1 ) , c DNA = 0 . 5 mg / mL ( full triangles , protocol II1 ; open triangles , protocol II2 . 1 ; open inverse triangles , protocol II2 . 2 ) and c DNA = 0 . 8 mg / mL ( circles , protocol II2 . 1 ) . I s ( mM ) 0 . 01 0 . 1 1 10 100 1000 L L F ( n m ) 100 1000 DN A 2 . j nb / 0212 25 o C Lp = 530A + 0 . 69 / I in same as for bulk Na 0 . 5 mg / mL 0 . 05 mg / mL L P from Ref . [ 17 ] Lp = 500A + 0 . 324 / I 0 . 8 mg / mL FIG . 9 : Characteristic length of the LF mode , rescaled as 2 . 5 · L LF → L LF ( L LF data are from Fig . 4b ) ) is shown together with data from pulling of DNA ( crosses ) taken from Ref . [ 17 ] . The full line and dashed lines are fits to OSF theory with L 0 = 530 ˚A and a = 0 . 69 ˚AM and with L 0 = 500 ˚A and a = 0 . 324 ˚AM corresponding to the theoretical OSF prediction with complete MO condensation . lax along or in close proximity to an individual chain in the polyelectrolyte solution , except at large enough salt concentrations where at least some of the free counterions seem to carry on this role . For the HF relaxation , the experimentally found spa - tial correlation providing the characteristic size of the 14 relaxation process is given universally by the polyelec - trolyte semidilute solution correlation length . The LF relaxation can be characterized by two different correla - tion scales . At low salt concentration this correlation length can be identified with the average size of the polyelectrolyte chain , where electrostatic interactions are screened by other chains and counterions , thus where DNA acts as its own salt . At higher salt concentra - tions , the spatial correlations are provided by the single chain orientational correlation length , i . e . the persistence length , that depends on the salt concentration via the OSF mechanism . Both the correlation lengths for the LF relaxation seem to be telling us , that it is the single chain that is responsible for the relaxation process , in which counterions either fluctuate orientationally on the length scale of the persistence length , or they fluctuate between two ends of the average size of the chain in the solution . In conclusion , our results demonstrate that there are three fundamental length scales that determine the di - electric response of a semidilute DNA solution : the av - erage size of the polyelectrolyte chain in the regime in which DNA acts as its own salt , the OSF salt - dependent persistence length of a single polyelectrolyte chain and the dGPD semidilute solution correlation length or the mesh size of the polyelectrolyte solution . While the free DNA counterions can be identified reasonably well as the relaxation entities of the HF relaxation mode , the LF relaxation mode does not allow for such a clearcut separation between MO condensed and free counterions as relaxation entities . Our data suggest that in fact both of them contribute to various extent in different salt and DNA concentration regimes . Finally , our results confirm that although double - stranded DNA at low salt concentrations shows locally exposed hydrophobic cores in a dynamic sense , unzipping of the strands is accom - plished only after the denaturation / heating protocol is applied . But even then this unzipping of the two DNA strands is probably at most local and complete separa - tion of the strands at semidilute solutions is never really accomplished . This issue will be addressed in our further studies . Acknowledgments ICP - AES was performed by M . Ujevi´c at the Croa - tian National Institute of Public Health . We thank T . 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Phys . 105 , 5183 ( 1996 ) . \ No newline at end of file diff --git a/silver_data/586e623114dcf68a03973de6504058f70e4b79be.pdf.txt b/silver_data/586e623114dcf68a03973de6504058f70e4b79be.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..27907e279e60feadd0196feb144bfb1072b947a9 --- /dev/null +++ b/silver_data/586e623114dcf68a03973de6504058f70e4b79be.pdf.txt @@ -0,0 +1 @@ +Volume 23 December 15 , 2012 4833 MBoC | ARTICLE Calcineurin - dependent cofilin activation and increased retrograde actin flow drive 5 - HT – dependent neurite outgrowth in Aplysia bag cell neurons Xiao - Feng Zhang , Callen Hyland , David Van Goor , and Paul Forscher Department of Molecular , Cellular , and Developmental Biology , Yale University , New Haven , CT 06520 ABSTRACT Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca 2 + ; however , mechanistic roles for Ca 2 + in controlling the underlying dy - namic cytoskeletal processes have remained enigmatic . Bag cell neurons exposed to sero - tonin ( 5 - hydroxytryptamine [ 5 - HT ] ) respond with a threefold increase in neurite outgrowth rates . Outgrowth depends on phospholipase C ( PLC ) → inositol trisphosphate → Ca 2 + → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain . Calcineurin inhibitors had no effect on Ca 2 + release or basal levels of retrograde actin flow ; however , they completely suppressed 5 - HT – dependent out - growth and F - actin flow acceleration . 5 - HT treatments were accompanied by calcineurin - dependent increases in cofilin activity in the growth cone P domain . 5 - HT effects were mim - icked by direct activation of PLC , suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors . INTRODUCTION Soluble neurotropic factors play an important role in development ( Kennedy et al . , 1994 ; Ming et al . , 1997 ; Campbell and Holt , 2001 ; Briancon - Marjollet et al . , 2008 ) . However , basic cytoskeletal mecha - nisms by which soluble factors affect rates of neuronal outgrowth remain poorly understood . Serotonin ( 5 - hydroxytryptamine [ 5 - HT ] ) is a soluble ligand that can signal through G ( q ) - coupled receptors in Aplysia neurons ( Li et al . , 1995 , 2005 ; Cai et al . , 2008 ) and trigger phospholipase C ( PLC ) – and inositol trisphosphate ( IP 3 ) – dependent Ca 2 + release from intracellular endoplasmic reticulum ( ER ) stores in bag cell neuron growth cones ( Zhang and Forscher , 2009 ) . The efficacy and spatial characteristics of Ca 2 + release can be modulated by activity of the small GTPase Rac1 . Rac1 activity promotes micro - tubule assembly and microtubule - dependent ER Ca 2 + store trans - port into peripheral regions of the growth cone . Rac1 activity also promotes reactive oxygen species production , which sensitizes IP 3 - dependent Ca 2 + release ( Gordeeva et al . , 2003 ; Zhang and Forscher , 2009 ) . Here we investigate how release of Ca 2 + from intracellular stores affects actin filament dynamics involved in neurite outgrowth . We show that 5 - HT application results in increased rates of neurite out - growth , accompanied by increased rates of retrograde F - actin net - work flow . 5 - HT – evoked growth involves Ca 2 + release from IP 3 - gated stores and calcineurin ( protein phosphatase 2B ) - dependent activation of the actin - recycling protein cofilin . Of interest , the re - sulting increases in actin network flow were independent of myosin II activity , whereas increases in neurite outgrowth were myosin II dependent . RESULTS 5 - HT induces neurite outgrowth on laminin substrates Several lines of evidence suggest that laminin – integrin interactions activate Rac1 and such activity is correlated with growth cone Monitoring Editor Laurent Blanchoin CEA Grenoble This article was published online ahead of print in MBoC in Press ( http : / / www . molbiolcell . org / cgi / doi / 10 . 1091 / mbc . E12 - 10 - 0715 ) on October 24 , 2012 . Address correspondence to : Paul Forscher ( paul . forscher @ yale . edu ) . © 2012 Zhang et al . This article is distributed by The American Society for Cell Biology under license from the author ( s ) . Two months after publication it is avail - able to the public under an Attribution – Noncommercial – Share Alike 3 . 0 Unported Creative Commons License ( http : / / creativecommons . org / licenses / by - nc - sa / 3 . 0 ) . “ASCB ® , ” “The American Society for Cell Biology ® , ” and “Molecular Biology of the Cell ® ” are registered trademarks of The American Society of Cell Biology . Abbreviations used : CN - AIP , calcineurin autoinhibitory peptide ; CsA , cyclosporin A ; FSM , fluorescent speckle microscopy ; 5 - HT , serotonin ; MLCK , myosin light - chain kinase . Received : Oct 4 , 2012 Accepted : Oct 15 , 2012 4834 | X . - F . Zhang et al . Molecular Biology of the Cell that culturing neurons on laminin substrates might increase basal Rac activity to a level at which 5 - HT would elicit Ca 2 + release without the use of constitutively active Rac con - structs ( Zhang and Forscher , 2009 ) . Bag cell neurons were cultured on lami - nin substrates and neurite outgrowth as - sessed by differential interference contrast ( DIC ) time - lapse imaging for 2 h before and 6 h after 5 - HT ( 10 μM ) or vehicle addition ( Figure 1A ) . Under these conditions , 5 - HT treatment resulted in approximately three - fold increase in average neurite outgrowth rate ( Figure 1A ) . Because neurite outgrowth rates were more or less constant for up to 6 h in 5 - HT , we chose to analyze population responses before and after 1 h of 5 - HT ex - posure ( Figure 1 , B and C ) when a ∼ 3 . 5 - fold increase in neurite outgrowth rate was typi - cally observed ( Figure 1C ) . Both basal and 5 - HT – dependent out - growth rates were strongly attenuated by RGD peptide , which competitively inhibits laminin – integrin interactions ( Gruenbaum and Carew , 1999 ; Tucker et al . , 2005 ) . In contrast , the reverse sequence DGR pep - tide control had no effect . These results con - firm the specificity of the permissive role laminin plays in supporting both basal ( Turney and Bridgman , 2005 ) and 5 - HT – evoked ( Figure 1C ) neurite outgrowth . To test whether growth on laminin depended on Rac GTPase activity , we used a Rac1 - specific small - molecule inhibitor , NSC23766 ( Gao et al . , 2004 ) . NSC23766 inhibited both basal and 5 - HT – stimulated growth , consis - tent with the reported role of Rac activity in integrin function ( Figure 1C ; Kuhn et al . , 1998 ; Grabham et al . , 2003 ; Matsuo et al . , 2003 ; Laforest et al . , 2005 ) . 5 - HT increases peripheral retrograde F - actin flow rate Next we investigated actin filament dynam - ics before and during 5 - HT – evoked growth responses . Neurons were injected with ei - ther Alexa 568 – G - actin or Alexa 594 – phal - loidin at trace levels to generate F - actin speckles for kinetic analysis . Features of Al - exa 594 – phalloidin bound to F - actin or Al - exa 568 – G - actin speckles incorporated into F - actin were tracked over time using a previ - ously reported quantitative cross - correlation approach ( Ji and Danuser , 2005 ; Burnette et al . , 2007 ; Hu et al . , 2007 ) . Resulting retro - grade actin flow velocities were pseudocolor coded and corresponding vectors overlaid on images to illustrate actin translocation . Figure 2A is a representative example of Al - exa 568 – G - actin fluorescent speckle microscopy ( FSM ) data ( top ) and corresponding flow maps ( bottom ) from a growth cone before and after 5 - HT treatment . 5 - HT exposure elicited 6 . 6 ± 1 . 0 % and advance ( Kuhn et al . , 1998 ; Grabham et al . , 2003 ; Matsuo et al . , 2003 ; Laforest et al . , 2005 ) . On the basis of our previous finding that Ca 2 + release from IP 3 stores was Rac dependent , we hypothesized FIGURE 1 : 5 - HT induces neurite outgrowth on laminin substrates . ( A ) Neurite outgrowth on laminin substrates over time under control conditions ( number of neurites tested [ N ] = 48 ) or before and after addition of 10 μM 5 - HT ( N = 46 ) . Black arrow , 5 - HT or vehicle addition . Data points are averages ± SEM . ( B ) A representative example of 5 - HT ( 10 μM , 1 h ) effect on neurite outgrowth . DIC image : bar , 10 μm . ( C ) Summary of neurite outgrowth rates 1 h before and after 5 - HT ( 10 μM ) addition under these conditions : control ( number of growth cones tested [ N ] = 58 ) , RGD ( 50 μM , 1 - h pretreatment ; N = 55 ) , DGR ( 50 μM , 1 - h pretreatment ; N = 55 ) , NSC23766 ( 0 . 1 mM , 1 - h pretreatment ; N = 58 ) . * p < 0 . 001 . Values are mean ± SEM . Statistical analysis by two tailed paired t test . Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4835 of evoked outgrowth , we assessed F - actin dynamics 30 and 60 min after 5 - HT treatment . Figure 2C illustrates representative growth cone structures ( DIC ) , F - actin distributions , and corresponding actin 28 . 3 ± 2 . 7 % increases in F - actin flow rates at 5 - and 10 - min time points , respectively ( Figure 2B ) . To investigate whether growth cones maintained accelerated F - actin flow rates during prolonged periods FIGURE 2 : 5 - HT increases peripheral retrograde actin filament flow rate . ( A ) Representative G - actin FSM images ( top ) and corresponding flow maps ( bottom ) from a growth cone before and after 5 or 10 min in 5 - HT . Bar , 10 μm . Flow map colors encode speed ( see scale bar ) , and arrows indicate flow direction . ( B ) Summary of relative changes in retrograde actin flow rates in response to 5 - HT . Images acquired every 5 s with 1 - min elapsed recording time and flow rates assessed as in A . Number of growth cones tested ( N ) = 3 . * p < 0 . 01 vs . before 5 - HT addition . ( C ) DIC ( top ) , Alexa 594 – phalloidin FSM ( middle ) , and corresponding flow map ( bottom ) of a growth cone before and after 30 min in 5 - HT . Bar , 10 μm . Images acquired every 5 s with 2 - min elapsed recording time . ( D ) Summary of P domain retrograde flow rates in response to 5 - HT ( 10 μM , 30 min , and 60 min ) . Data normalized to rates before 5 - HT addition . Number of growth cones evaluated ( N ) = 25 . Values are mean ± SEM . * P < 0 . 001 vs . before 5 - HT addition . Statistical analysis by two - tailed paired t test . 4836 | X . - F . Zhang et al . Molecular Biology of the Cell Taken together , these results indicate that 5 - HT treatment triggers a persistent increase in peripheral F - actin flow that is well corre - lated with the observed accelerated rate of growth cone advance . Direct phospholipase C activation increases neurite growth and retrograde flow rates To investigate the generality of this re - sponse , we bypassed the 5 - HT receptor and directly activated PLC—the downstream ef - fector of receptor signaling through trimeric Gq proteins ( Figure 3 ) . PLC activation with m - 3M3FBS ( 25 μM ; Bae et al . , 2003 ; Li et al . , 2009 ) resulted in sustained ∼ 3 . 5 - fold in - creases in neurite outgrowth rates ( Figure 3 , A and B ) accompanied by increases in retro - grade actin flow ( Figure 3 , C and D ) essen - tially identical to those observed after 5 - HT treatments ( Figure 2 ) . 5 - HT – induced F - actin flow increases and outgrowth depend on Ca 2 + release from IP 3 - gated stores Ca 2 + is known to participate in 5 - HT function in neurons ( Dropic et al . , 2005 ; Li et al . , 2005 ; Cai et al . , 2008 ) . Previously we re - ported that 5 - HT evoked Ca 2 + release in Ap - lysia growth cones in the presence of consti - tutively active ( but not dominant negative ) Rac1 when cells were plated on PLL sub - strates ( Zhang and Forscher , 2009 ) . Given that laminin has been widely reported to increase Rac activity ( Kuhn et al . , 1998 ; Grabham et al . , 2003 ; Matsuo et al . , 2003 ; Laforest et al . , 2005 ) and the robust Rac de - pendence of the outgrowth responses un - der study ( Figure 1C ) , we tested whether 5 - HT would elicit Ca 2 + release in growth cones on laminin substrates . Supplemental Figure 1A shows that 5 - HT treatments in - deed evoked rapid and sustained Ca 2 + in - creases in the entire growth cone ( see also Supplemental Movie S2 ) . These responses depended on the PLC → IP 3 signaling cas - cade since the PLC inhibitor U73122 ( Jin et al . , 1994 ; Zhou et al . , 1999 ) or the IP 3 re - ceptor blocker xestospongin C ( XeC ; Gafni et al . , 1997 ) abolished 5 - HT – evoked Ca 2 + responses ( Supplemental Figure S1 , A and B ) . During more prolonged 5 - HT exposure ( 1 h ) , Ca 2 + levels remained elevated ∼ 22 % above baseline . These Ca 2 + changes were also inhibited by pretreatment with PLC or IP 3 receptor antagonists ( Supplemental Fig - ure 1 , C and D , and Supplemental Movie S3 ) . Finally , pretreatment with the Rac inhibitor NSC23766 abol - ished 5 - HT – dependent Ca 2 + responses ( Supplemental Figure S2 ) . Collectively , these data show that 5 - HT triggers sustained Ca 2 + in - creases in parallel with increased retrograde F - actin flow when Rac levels are sufficiently elevated . flow maps before and after 30 min in 5 - HT . Rates of growth cone advance were correlated with increased F - actin flow rates ( Figure 2C , DIC vs . flow map ; see Supplemental Movie S1 ) . On average , F - actin flow rates significantly increased by 27 . 2 ± 2 . 1 % and 29 . 3 ± 2 . 5 % after 30 - and 60 - min 5 - HT exposures , respectively ( Figure 2D ) . FIGURE 3 : PLC activator increases peripheral retrograde actin filament flow rate and promotes neurite outgrowth . ( A ) Neurite outgrowth on laminin substrates over time before and after addition of 25 μM PLC activator m - 3M3FBS ( number of neurites tested [ N ] = 32 ) . Black arrow , m - 3M3FBS addition . Data points are averages ± SEM . ( B ) Comparison of neurite outgrowth rates 1 h before and after m - 3M3FBS ( 25 μM ) addition . Data is taken from A . * p < 0 . 001 . Values are mean ± SEM . Statistical analysis by two - tailed paired t test . ( C ) Alexa 594 – phalloidin FSM ( top ) and corresponding flow map ( bottom ) from a growth cone before and after 30 min in m - 3M3FBS ( 25 μM ) . Bar , 10 μm . Flow map generated as described . Images acquired every 5 s with 2 - min elapsed recording time . ( D ) Summary of normalized P domain retrograde flow rates in response to 25 μM m - 3M3FBS . Data normalized to rates before m - 3M3FBS addition . N = 12 growth cones . * p < 0 . 001 vs . before m - 3M3FBS addition . Values are mean ± SEM . Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4837 multichannel fluorescence imaging ) . Figure 4A shows Ca 2 + levels and FSM records from a growth cone before and after a 30 - min 5 - HT treatment . Ca 2 + levels and peripheral F - actin flow rates increased in parallel by 20 . 9 and 25 . 4 % , respectively . There was a strong temporal correlation between aver - age 5 - HT – evoked Ca 2 + elevations and in - creases in F - actin flow ( Figure 4B ) . When Ca 2 + release from intracellular stores was blocked by inhibiting PLC or IP 3 using U73122 or XeC , respectively , no changes in baseline actin flow or growth rates were ob - served ; however , 5 - HT – evoked increases in F - actin flow and concomitant increases in neurite outgrowth were completely sup - pressed ( Figure 4 , C and D ) . Taken together , these results indicate that IP 3 - dependent Ca 2 + mobilization is upstream of , and neces - sary for , the increased F - actin flow rates and growth - promoting effects of 5 - HT observed . 5 - HT effect on F - actin flow is independent of myosin light - chain kinase activity Given that 5 - HT - induced increases in F - actin flow were Ca 2 + dependent and myosin II activity is known to affect F - actin flow rates ( Lin et al . , 1996 ; Medeiros et al . , 2006 ) , we investigated whether activation of myosin light - chain kinase ( MLCK ) , which is a Ca 2 + / calmodulin – dependent regulator of myosin II activity ( Kamm and Stull , 2001 ; Schmidt et al . , 2002 ) , could be the Ca 2 + effector mediat - ing flow increases . We used ML - 7 , a well - characterized MLCK inhibitor ( Ruchhoeft and Harris , 1997 ; Zhou and Cohan , 2001 ; Zhang et al . , 2003 ) effective in our system ( Zhang et al . , 2003 ) . In control experi - ments , we found that exposure to ML - 7 alone ( 15 – 20 min ) did not alter Ca 2 + lev - els ( Supplemental Figure S3A ; also see Figure 5B , inset vs . left ) ; however , F - actin flow rates decreased by ∼ 15 % ( Supple - mental Figure S3B ; also see Figure 5D , inset vs . left ) consistent with previous re - ports that MLCK activity is involved in set - ting basal F - actin flow rates ( Zhang et al . , 2003 ) . We next investigated the effect of MLCK inhibition on 5 - HT – evoked Ca 2 + release and F - actin flow . After MLCK inhi - bition , 5 - HT continued to evoke Ca 2 + release ( + 17 % ; Figure 5B ) , as well as in - creases in peripheral F - actin flow ( + 29 % ; Figure 5 , C and D ) . Indeed , statistical analysis indicated that MLCK inhibition did not significantly affect the depth of 5 - HT – evoked Ca 2 + re - sponses ( Table 1 ) or increases in F - actin flow ( Table 2 ) . In sum - mary , although MLCK plays a role in setting basal F - actin flow To directly address the relationship between Ca 2 + levels and F - actin dynamics , we performed simultaneous ratiometric Ca 2 + im - aging and F - actin FSM ( see Supplemental Movie S4 , A and B , for FIGURE 4 : 5 - HT - evoked Ca 2 + release and peripheral retrograde actin flow increases are correlated and necessary for induced neurite outgrowth . ( A ) Tandem Ca 2 + ratio imaging and actin FSM . Ca 2 + ratio image ( top ) and Alexa 594 – phalloidin FSM ( bottom ) of a growth cone before and after 30 min in 5 - HT . Bar , 10 μm . The Ca 2 + ratio is encoded by a linear pseudocolor scale . Images acquired every 10 s with 3 - min elapsed recording time . Top right , growth cone Ca 2 + response to 5 - HT over time . F o , average Ca 2 + level before 5 - HT addition . Bottom right , kymographs sampled from area of interest indicated in actin panel ( yellow arrowheads ) . Retrograde flow rate before and after 30 min of 5 - HT exposure : 4 . 88 ± 0 . 21 and 6 . 12 ± 0 . 19 μm min − 1 , respectively ( mean ± SD , N = 5 measurements ) . ( B ) Comparison of Ca 2 + levels and P domain flow rates before and after 5 - HT . The Ca 2 + ratio imaging and FSM were carried out simultaneously . Images acquired every 5 or 10 s with 2 - to 3 - min elapsed recording time . F o , average Ca 2 + level before 5 - HT addition . R o , average retrograde flow rate before 5 - HT addition . N = 14 growth cones . * p < 0 . 001 vs . before 5 - HT addition . ( C ) Retrograde flow rates in response to 5 - HT in various conditions normalized to before 5 - HT addition . N = 25 growth cones ( control ) , N = 18 ( U73122 , 2 μM , 30 - min pretreatment ) , and N = 21 ( XeC , 20 μM , 30 - min pretreatment ) . Control from Figure 2D included for comparison . * p < 0 . 001 vs . before 5 - HT addition . ( D ) Summary of neurite outgrowth 1 h before and after 5 - HT addition in various conditions . Control ( N = 58 growth cones ) , U73122 ( 2 μM , 1 - h pretreatment , N = 34 ) , and XeC ( 20 μM , 1 - h pretreatment , N = 37 ) . Control from Figure 1C is included for comparison . * p < 0 . 001 vs . before 5 - HT addition . Values are mean ± SEM . Statistical analysis was done by two - tailed paired t test . 4838 | X . - F . Zhang et al . Molecular Biology of the Cell 5 - HT effects on F - actin flow are independent of myosin II activity Previous evidence suggested that myosin II sets the maximum rate of retrograde F - actin flow in growth cones ( Lin et al . , 1996 ; Medeiros et al . , 2006 ; Burnette et al . , 2007 ) . To directly investigate the role myosin II plays in 5 - HT effects , we used blebbistatin , a specific nonmuscle myosin II ATPase inhibitor ( Straight et al . , 2003 ; Allingham et al . , 2005 ) that has been extensively char - acterized in our system ( Medeiros et al . , 2006 ) . Similar to what was previously ob - served for growth cones plated on PLL sub - strates , 10 - to 20 - min blebbistatin exposures promoted elongation of filopodial actin bundles , resulting in rearward expansion of the peripheral cytoplasmic domain ( Supple - mental Figure S4A and Supplemental Movie S5 ) . During blebbistatin treatment periph - eral F - actin flow rates typically decreased by ∼ 20 % ( Supplemental Figure 4 , A bottom , and B ) , consistent with nonmuscle myosin II playing a role in setting basal rates of retro - grade F - actin flow on laminin as previously observed on PLL substrates ( Medeiros et al . , 2006 ) . We then investigated a role for myosin II in 5 - HT – induced increases in F - actin flow . Neurons were pretreated with blebbistatin for 20 min to maximally inhibit myosin II activity ( Medeiros et al . , 2006 ) and then challenged with 5 - HT . Remarkably , after blebbistatin pretreatment , 5 - HT exposure continued to trigger increases in retrograde flow ( ∼ 25 . 5 % increase after 10 min ; Figure 6A ) at levels similar to that observed under control conditions ( Figure 6B ) that were sus - tained for up to 60 min ( Figure 6C ; kymo - graphs show flow increases of 22 . 0 and 25 . 1 % at 30 - and 60 - min time points , re - spectively ) . The average magnitude of 5 - HT – induced actin flow increases was not significantly different between control and blebbistatin - pretreated growth cones ( Figure 6D ) . These results strongly suggest that changes in myosin II activity are not in - volved in the 5 - HT – induced increases in ac - tin network flow and corresponding in - creased rates of actin filament turnover observed . We then investigated whether myosin II activity is necessary for the more global ef - fect of 5 - HT on neurite outgrowth . To be consistent with the aforementioned experiments , outgrowth rates were assessed for 1 h under control conditions and during 5 - HT exposure . We noted that in ∼ 75 % of the growth cones ( n = 90 ) , ∼ 1 . 5 h of exposure to blebbistatin alone resulted in neurite branching accompanied by enhanced rates of neurite outgrowth . Similarly , long - term blebbistatin treatment has been reported to promote neurite outgrowth in chicken retina explants , medulla , and spinal cord neurons ( Rosner et al . , 2007 ) . Given the foregoing rates , 5 - HT effects on actin network flow were essentially inde - pendent of MLCK activity . Of interest , MLCK inhibition did appear to inhibit 5 - HT – evoked neurite outgrowth ( Figure 5A ; dotted red line ) , suggest - ing that 5 - HT effects on actin dynamics versus neurite growth might have differential myosin II dependences . To test this pos - sibility , we further investigated the role of myosin II in 5 - HT – me - diated growth . FIGURE 5 : 5 - HT - induced increase in actin flow is independent of MLCK activity . ( A – D ) A representative example of growth cone responding to 5 - HT in ML - 7 background . ( A ) DIC , ( B ) Ca 2 + ratio , ( C ) Alexa 594 – phalloidin FSM , ( D ) corresponding flow map . Cells pretreated with ML - 7 ( 10 μM ) for 15 min and ML - 7 present throughout 1 h in 5 - HT . Bar , 10 μm . Data acquired every 5 s with 2 - min elapsed recording time . The Ca 2 + level is encoded by a linear pseudocolor scale as before . Yellow lines demark leading edge in C . Insets in B and D are controls before ML - 7 addition . Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4839 release ( Supplemental Figure S6 and Figure 7 , A and B ) ; however , increases in F - actin flow rate were completely suppressed ( Figure 7 , A and C ) . These results strongly suggest that 5 - HT – induced in - creases in F - actin flow depend on calcineurin activation downstream of Ca 2 + release from intracellular stores . To investigate the role of calcineurin in 5 - HT – evoked neurite out - growth , we assessed growth rates for 1 h in the presence of CN - AIP , FK - 506 , or CsA alone and after 5 - HT exposure . Calcineurin inhibi - tion had no effect on basal neurite outgrowth rates but completely blocked 5 - HT – dependent neurite outgrowth ( Figure 7D ; see also DIC in Supplemental Figure S6 ) . Taken together , these results sup - port a mechanism by which 5 - HT treatment promotes neurite out - growth through a calcineurin - dependent increase in actin network flow and turnover . 5 - HT – dependent calcineurin activation increases apCofilin1 activity Cofilin is highly expressed in neuronal growth cones , and increased cofilin activity has been implicated in neurite extension ( Meberg and Bamburg , 2000 ; Endo et al . , 2003 ; Ng and Luo , 2004 ; Tahirovic and Bradke , 2009 ) . Cofilin is inhibited by phosphorylation by LIM kinase and activated by the cofilin phosphatase Slingshot ( DesMarais et al . , 2005 ) . It has been reported that intracellular Ca 2 + elevation leads to calcineurin - dependent Slingshot activation and cofilin dephospho - rylation ( Wang et al . , 2005 ) . Therefore we investigated a potential link between calcineurin and cofilin activity in 5 - HT – induced neurite outgrowth . To generate cofilin activity probes , we identified and cloned two Aplysia californica cofilin homologues ( apCofilin1 and apCofilin2 , which share only 25 . 1 % sequence identity ) . Antibodies were generated using recombinant proteins encoding both full - length apCofilins . We successfully generated phospho - specific anti - bodies against apCofilin1 and used this antibody in combination with total anti - apCofilin1 to assess apCofilin1 activity patterns ( see Table 3 and Supplemental Figures S7 and S8 ) . To assess 5 - HT effects on the spatial profile of apCofilin1 activity , we generated ratiometric P - apCofilin1 / Total - apCofilin1 images . 5 - HT treatments significantly increased , that is , disinhibited apCofi - lin1 activity in the entire growth cone , reflected by a decrease in measured P - apCofilin1 / Total - aPcofilin1 levels ( Figure 8 , A and B ) . Pretreatment with the calcineurin inhibitor FK - 506 completely considerations , neurons were pretreated with blebbistatin for 10 min to establish maximal inhibitory effects on actin flow ( Medeiros et al . , 2006 ) and then exposed to 5 - HT for only 1 h in the continued presence of blebbistatin , at which time outgrowth rates were assessed . Blebbistatin treatment completely blocked 5 - HT – evoked neurite outgrowth ( Figure 6E ; see also DIC in C ) , consistent with effects seen after MLCK inhibition ( Figure 6E ) . Taken together , these results suggest that 5 - HT treatment leads to increases in actin network flow that are independent of myosin II activity ; however , myosin contractility appears to be essential to functionally couple increased actin network flow rates to the pro - cess of neurite advance . 5 - HT – induced increase in F - actin flow depends on calcineurin activity Calcineurin , or Ca 2 + / calmodulin – dependent protein phosphatase 2B ( PP2B ) , is enriched in growth cones and has been implicated in Ca 2 + - dependent regulation of neurite extension and filopodium dynamics ( Ferreira et al . , 1993 ; Chang et al . , 1995 ; Lautermilch and Spitzer , 2000 ; Cheng et al . , 2002 ; Arie et al . , 2009 ) . Although cy - toskeletal proteins have often been implicated in calcineurin ac - tions , to our knowledge calcineurin effects on actin dynamics have never been directly assessed . Thus we first looked at potential ef - fects of calcineurin inhibition alone on actin dynamics . To this end , we treated cells with the cell - permeable calcineurin inhibitor FK - 506 or cyclosporin A ( CsA ; Liu et al . , 1991 ; Fruman et al . , 1992 ) . The Ca 2 + levels and F - actin flow rates did not differ significantly from controls after 20 – 40 min of FK - 506 or CsA exposure alone ( Supplemental Figure S5 , A and B ) . As an alternative approach , we injected a spe - cific calcineurin inhibitor consisting of the conserved calcineurin au - toinhibitory peptide domain ( CN - AIP ; Hashimoto et al . , 1990 ; Perrino et al . , 1995 ) into cells . CN - AIP injection also had no effect on F - actin flow rates ( unpublished data ) . These observations indicate that basal levels of retrograde actin flow do not depend on calcineu - rin activity . We next investigated effects of calcineurin inhibition on 5 - HT – evoked Ca 2 + release . In CN - AIP – injected or FK - 506 – or CsA - pre - treated neurons , 5 - HT continued to evoke typical levels of Ca 2 + Control ( no pretreatment ) ML - 7 pretreatment Before 5 - HT : ( F 0 − F 0 ) / F 0 0 ± 0 0 ± 0 5 - HT 30 min : ( F 30 − F 0 ) / F 0 0 . 205 ± 0 . 023 * 0 . 201 ± 0 . 026 * 5 - HT 60 min : ( F 60 − F 0 ) / F 0 0 . 215 ± 0 . 025 * 0 . 205 ± 0 . 027 * N 17 9 Summary of ( F t − F o ) / F o recorded in the entire growth cone area quantifying Ca 2 + response to 5 - HT with or without MLCK inhibition . For MLCK inhibition , cells were pretreated with ML - 7 ( 10 μM ) for 15 – 20 min and ML - 7 was present throughout . Records acquired every 5 or 10 s with 2 - to 3 - min elapsed record - ing time . F o , average Ca 2 + level before 5 - HT addition . F 30 and F 60 , average Ca 2 + level 30 and 60 min after 5 - HT addition , respectively . N denotes number of growth cones evaluated . Values are expressed as mean ± SEM . Statistical analysis was done by two - tailed paired t test . * p < 0 . 001 vs . before 5 - HT addition . In addition , there was no significant differ - ence in the magnitude of 5 - HT responses in control vs . ML - 7 group . TABLE 1 : MLCK inhibition does not affect 5 - HT – evoked Ca 2 + increases . No pretreatment ML - 7 pretreatment Flow rate before 5 - HT 100 ± 0 100 ± 0 Flow rate 30 min after 5 - HT 127 . 2 ± 2 . 1 * 124 . 8 ± 2 . 7 * Flow rate 60 min after 5 - HT 129 . 3 ± 2 . 5 * 126 . 1 ± 2 . 7 * N 25 16 Data normalized to flow rates before 5 - HT addition ( percentage of before 5 - HT ) to quantify changes of P domain retrograde flow rates in response to 5 - HT with or without MLCK inhibition . For MLCK inhibition , cells were pretreated with ML - 7 ( 10 μM ) for 15 – 20 min and ML - 7 was present throughout . Data were acquired every 5 or 10 s with 2 - to 3 - min elapsed recording time . N denotes the number of growth cones tested . Values are expressed as mean ± SEM . Statistical analysis was done by two - tailed paired t test . * p < 0 . 001 vs . before 5 - HT addition . In addition , there was no significant differ - ence in the magnitude of 5 - HT responses in control vs . ML - 7 group . TABLE 2 : MLCK inhibition does not affect 5 - HT – evoked actin flow increases . 4840 | X . - F . Zhang et al . Molecular Biology of the Cell Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4841 The cytoskeletal mechanism of this 5 - HT growth response con - trasts with what was observed during acute transitions from nonper - missive to permissive extracellular growth substrates ( Lin and Forscher , 1995 ) or after application of apCAM - coated beads , for which increased rates of advance were correlated with decreased retrograde actin flow rates ( Suter and Forscher , 2000 , 2001 ) . The rate of retrograde flow ( V r ) in the P domain is determined by the balance of forces on actin networks ( Craig et al . , 2012 ) . Actin net - work assembly near the leading edge and nonmuscle myosin II con - tractility in the T zone generate pushing and pulling forces , f poly and f motor , respectively , which drive network flow ( Figure 9B , red arrows ; Lin et al . , 1996 ; Henson et al . , 1999 ; Mogilner and Oster , 2003 ; Medeiros et al . , 2006 ) . Constant actin polymer turnover is necessary to prevent buildup of compressive forces in the T zone ( Figure 9B ; f break ) , which resist actin filament flow ( Van Goor et al . , 2012 ) . In ad - dition to these internal forces , adhesion to extracellular substrates can generate traction forces ( Figure 9B ; f adhesion ) , which tend to op - pose retrograde flow ( Lin and Forscher , 1995 ) and are the basis of the “molecular clutch hypothesis” for regulation of neurite growth ( Mitchison and Kirschner , 1988 ; Suter and Forscher , 1998 ; Suter and Forscher , 2001 ; Schaefer et al . , 2008 ) . Adhesion and network com - pression can be modeled as viscous drags ( ε adhesion and ε break , re - spectively ) , which result in forces that scale with actin flow velocity . In summary , actin polymerization and myosin II contractility tend to increase retrograde flow rates , whereas network compression in the T zone and / or adhesion to extracelluar substrates tend to decrease it ( Figure 9C ; see Craig et al . , 2012 ) . We found that 5 - HT treatments ( or PLC activation ) resulted in acceleration of V r without a significant change in P domain width . This means that increases in actin assembly had to be matched by increases in filament turnover . In line with this finding , recent related studies from our group support a key role for actin filament turnover in determining retrograde flow rates and P domain geometry ( Van Goor et al . , 2012 ; Yang et al . , 2012 ) . The importance of F - actin turn - over for axon extension has also been reported ( Bradke and Dotti , 1999 ; Gallo et al . , 2002 ) . Cofilin activation promotes actin filament turnover , thereby re - ducing network density and decreasing ε break , which would sup - port the faster F - actin flow rates observed . Moreover , actin filament disassembly tends to increase G - actin concentration . Together these processes would increase polymerization rates and facilitate the accelerated actin treadmilling rates observed . In addition , abolished 5 - HT – dependent cofilin activation , resulting in activity patterns very similar to those of controls ( Figure 8 , A , right , and C ) . Population analysis demonstrated that 5 - HT treatment markedly in - creased cofilin activity in the distal one - third and the proximal one - third of the P domain by ∼ 15 and ∼ 22 % , respectively , compared with controls ( Figure 8D ) . However , in FK - 506 backgrounds , 5 - HT treat - ment did not cause any significant changes in cofilin activity ( Figure 8D ) . Treatment with in FK - 506 alone did not significantly change apCoflin1 activity . Taken together , these observations suggest that 5 - HT triggers calcineurin - dependent apCofilin1 activation , which supports increased rates of growth cone and neurite advance . DISCUSSION In Aplysia neurons , 5 - HT induces actin polymerization essential for synaptic remodeling associated with long - term facilitation ( Hatada et al . , 2000 ; Udo et al . , 2005 ) . Facilitation also depends on Ca 2 + re - lease from postsynaptic stores ( Li et al . , 2005 ; Cai et al . , 2008 ) . These findings suggest roles for Ca 2 + release and actin dynamics in synap - tic plasticity ; however , mechanisms by which these processes con - tribute to 5 - HT – dependent neurite outgrowth are not well under - stood . Here we describe a novel mechanism of neurite growth by which exposure to a soluble factor , 5 - HT , triggers Ca 2 + release from intracellular stores , which in turn promotes increased rates of retro - grade actin network flow accompanied by calcineurin - dependent apCofilin activation ( Figure 9A ) . Calcineurin , or PP2B , is a Ca 2 + / calmodulin - dependent serine – threonine phosphatase , which plays a role in coupling Ca 2 + signals to many neuronal responses ( Groth et al . , 2003 ; Nguyen and Di Giovanni , 2008 ; Bodmer et al . , 2011 ) and has been implicated in promoting neurite outgrowth ( Ferreira et al . , 1993 ; Chang et al . , 1995 ; Sotogaku et al . , 2007 ; Arie et al . , 2009 ; Figge et al . , 2011 ) . We found that 5 - HT continued to evoke Ca 2 + release after cal - cineurin inhibition ; however , accompanying increases in F - actin flow and neurite outgrowth were completely absent ( Figure 7 ) . Our results indicated that calcineurin activation was necessary for the observed changes in actin dynamics and raised the question of the calcineurin effector . Calcineurin - dependent activation of Slingshot phosphatase can activate cofilin ( Wang et al . , 2005 ; Pandey et al . , 2007 ; Zhao et al . , 2008 ) . In agreement , we found that 5 - HT treatments resulted in calcineurin - dependent activation of apCofilin1 in regions in which increased F - actin flow were ob - served ( Figure 8 ) . FIGURE 6 : The 5 - HT – induced increase in actin flow is independent of myosin II activity . ( A – C ) Cells were pretreated for 20 min with blebbistatin ( 60 μM ) and drug maintained throughout experiments . ( A ) Alexa 594 – phalloidin FSM ( top ) and corresponding flow map ( bottom ) from a growth cone before and after 5 and 10 min in 5 - HT . Bar , 10 μm . Flow map generated as described . ( B ) Summary of changes in retrograde F - actin flow rates in response to 5 - HT . Images acquired every 5 s with 1 - min elapsed recording time . No pretreatment ( control ) from Figure 2B is shown for comparison . N = 3 growth cones for each condition . Values are mean ± SEM . ( C ) DIC ( top ) and phalloidin – Alexa 594 FSM ( middle ) of a blebbistatin - treated growth cone before and after 30 and 60 min in 5 - HT . Bar , 10 μm . Images were acquired every 5 s with 2 - min elapsed recording time . Bottom , kymographs sampled across the P domain near yellow arrowheads showing rates of retrograde flow before and after 30 and 60 min of 5 - HT exposure . Before , 4 . 55 ± 0 . 25 ; 30 - min 5 - HT , 5 . 55 ± 0 . 27 ; 60 - min 5 - HT , 5 . 69 ± 0 . 25 μm min − 1 ( mean ± SD , five measurements each ) . ( D ) Summary of normalized P domain retrograde flow rates in response to 10 μM 5 - HT with or without myosin II inhibition . Data normalized to rates before 5 - HT addition . No pretreatment control : N = 25 growth cones from Figure 2D shown ; blebbistatin ( N = 22 , 60 μM pretreatment for 10 – 20 min ) . * P < 0 . 001 vs . before 5 - HT addition . ( E ) Summary of neurite outgrowth sampled 60 min before and 60 min after 5 - HT addition . Control conditions ( N = 58 growth cones ) , blebbistatin pretreatment ( 60 μM , 10 min , and presence throughout , N = 36 ) ; ML - 7 pretreatment ( 10 μM , 60 min , and presence throughout , N = 34 ) . Control is from Figure 1C for comparison . In blebbistatin background , data were excluded from growth cones undergoing branching . Values are mean ± SEM . Statistical analysis by two - tailed paired t test . * p < 0 . 001 . 4842 | X . - F . Zhang et al . Molecular Biology of the Cell FIGURE 7 : The 5 - HT – induced increases in actin flow rates and neurite outgrowth depend on calcineurin activation downstream of Ca 2 + release . ( A ) The Ca 2 + ratio image ( left ) and phalloidin – Alexa 594 FSM ( right ) of a growth cone before and after 30 and 60 min in 5 - HT . The cell was pretreated with FK - 506 ( 2 . 5 μM ) for 30 min . Note that FK - 506 was present throughout . Bar , 10 μm . The Ca 2 + ratio image is coded by pseudocolors in the linear scale ( see scale bar ) . Data were acquired every 10 s with 2 - min elapsed recording time . ( B ) Top , plot of Ca 2 + response to 5 - HT . Data obtained from the entire growth cone in A . F o , the average Ca 2 + level before 5 - HT addition . ( B ) Bottom , summary of F t / F o plot recorded in the entire growth cone area quantifying Ca 2 + response to 5 - HT after calcineurin inhibition . Data were acquired every 10 s with 2 - to 3 - min elapsed recording time . F o , the average Ca 2 + level before 5 - HT addition . Number of growth cones tested ( N ) = 6 ( CN - AIP , injected , 0 . 2 mM in needle ) , 8 ( FK - 506 , 2 . 5 μM pretreatment for 30 min and presence throughout ) , or 6 ( CsA , 1 μM pretreatment for 30 min and presence throughout ) . Values are expressed as mean ± SEM . Statistical analysis was done by two - tailed paired t test . * P < 0 . 005 . ( C ) Top , kymographs created from area of interest as indicated in A ( yellow arrowhead ) , showing rates of P domain retrograde flow before and after 30 and 60 min of 5 - HT exposure . Before , 3 . 22 ± 0 . 23 , vs . 30 - min 5 - HT , 3 . 24 ± 0 . 24 , vs . 60 - min 5 - HT , 3 . 21 ± 0 . 26 μm min − 1 ( mean ± SD , five measurements ) . ( C ) Bottom , summary of normalized P domain retrograde flow rates in response to 5 - HT after Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4843 increased cofilin activity leads to barbed - end production , which increases the density of actin assembly sites and promotes lead - ing - edge protrusion during cell migration in response to soluble growth factors in nonneuronal cells ( Pollard and Borisy , 2003 ; Ghosh et al . , 2004 ; Kiuchi et al . , 2007 ) and to neurotrophic factors ( BDNF , NGF , or netrin - 1 ) in embryonic DRG and retinal neuron growth cones ( Gehler et al . , 2004 ; Marsick et al . , 2010 ) . In line with these findings , we recently reported a high density of free barbed ends in a band along the leading edge of bag cell neuron growth cones that were sensitive to treatments that inhibit cofilin activity ( Van Goor et al . , 2012 ) . How can these observations be reconciled with the original mo - lecular clutch hypothesis , which predicts that accelerated neurite outgrowth is correlated with decreased rates of retrograde actin flow ? As per the foregoing discussion , retrograde flow rates depend on four parameters : 1 ) actin filament assembly , 2 ) myosin II contrac - tility , 3 ) actin filament severing / recycling , and 4 ) cell adhesion ( Figure 9 , B and C ) . Assembly and turnover facilitate retrograde flow , whereas network compression and adhesion tend to oppose it . The present results suggest that a revision of the original mole cular clutch hypothesis is in order . In particular , a less constrained multi - level slip clutch model as suggested by Giannone et al . ( 2009 ) can explain the results we observed . Indeed , with a viscous slip clutch – type model , faster flow rates alone without a change in adhesion strength ( ε adhesion ) would result in increased traction force due to increased viscous drag ( Figure 9C ) . Alternatively , ε adhesion could in - crease , but the resulting increase in drag force ( f adhesion ) would have to be matched by increased polymerization and / or motor force to account for the flow rate acceleration observed . It is also possible that flow rates are increasing in 5 - HT as a result of decreased ε adhesion . It will be of interest to measure traction forces in parallel with actin dynamics to address these outstanding issues . It should be noted that the present results are not without precedent : im - mune system dendritic cells have been shown to migrate under con - ditions of extremely low adhesion by increasing their retrograde actin flow rates ( Renkawitz et al . , 2009 ) . Myosin II activity can also promote actin turnover in growth cones ( Medeiros et al . , 2006 ) and nonneuronal cells ( Guha et al . , 2005 ; Murthy and Wadsworth , 2005 ; Haviv et al . , 2008 ) . Here we investi - gated potential roles for myosin light - chain kinase and myosin II ac - tivity in 5 - HT growth responses . Of interest , 5 - HT – dependent in - creases in network turnover and retrograde flow persisted even after MLCK inhibition or direct inhibition of myosin II ( Figures 5 and 6 , A – D , and Table 2 ) . In contrast , neurite outgrowth depended on myosin II activity ( Figures 5 , 6E , and 9 ) , as previously observed in vertebrate neurons ( Bridgman et al . , 2001 ; Turney and Bridgman , 2005 ) . Myosin II activity might regulate adhesion site dynamics and maturation ( Papusheva and Heisenberg , 2010 ) involved in generat - ing traction forces that promote neurite extension ( Zheng et al . , 1991 ; Heidemann and Buxbaum , 1994 ; Heidemann et al . , 1995 ) . Although our results are consistent with myosin II inhibition reducing point contact consolidation ( Woo and Gomez , 2006 ) , further studies involving measurement of growth cone traction force are necessary to address this outstanding issue . MATERIALS AND METHODS Cell culture and chemicals Primary culture of Aplysia bag cell neurons was as previously de - scribed ( Forscher et al . , 1987 ) . Coverslips were pretreated by 20 μg / ml poly - l - lysine ( Sigma - Aldrich , St . Louis , MO ) for 15 min , then incu - bated in a 50 μg / ml laminin ( Sigma - Aldrich ) solution for 2 h and rinsed in L15 - ASW . 5 - HT , U - 73122 , xestospongin C ( XeC ) , Gly - Arg - Gly - Asp - Ser ( RGD ) , and Ser - Asp - Gly - Arg - Gly ( DGR ) were from Sigma - Aldrich . Blebbistatin , ML - 7 , calcineurin autoinhibitory pep - tide ( CN - AIP ) , FK - 560 , and CsA were from Calbiochem ( La Jolla , CA ) . Calcium green - 1 dextran , potassium salt , 10 , 000 MW ( CG - 1 ) , Alexa Fluor 568 dextran , 10 , 000 MW ( Alexa 568 ) , Alexa Fluor 647 dextran , 10 , 000 MW ( Alexa 647 ) , Alexa Fluor 488 dextran , 10 , 000 MW ( Alexa 488 ) , Alexa Fluor 568 – rabbit skeletal muscle G - actin ( Alex 568 G - actin ) , and Alex Fluor 594 phalloidin were purchased from Molecular Probes ( Eugene , OR ) . Solutions Artificial seawater ( Na - ASW ) contained ( in mM ) 400 NaCl , 10 KCl , 15 4 - ( 2 - hydroxyethyl ) - 1 - piperazineethanesulfonic acid ( HEPES ) , 10 CaCl 2 , and 55 MgCl 2 at pH 7 . 8 . Na - AWS was supplemented with 3 mg / ml bovine serum albumin ( BSA ) , 0 . 5 mM vitamin E ( Sigma - Aldrich ) , and 1 mg / ml carnosine ( Sigma - Aldrich ) before experiments . The Ca 2 + injection buffer consisted of ( in mM ) 100 potassium aspartate and 10 HEPES at pH 7 . 4 . Microinjection Microinjection protocol as described previously ( Lin and Forscher , 1995 ) . For Ca 2 + imaging , neurons were injected with CG - 1 and Alexa 647 or Alexa 568 in Ca 2 + injection buffer ( needle concentration Name Sequence apCofilin1 MASGIKIADTVKEVYSRISLNSVKQTKLKYGVFK - FADDGASIVVETTATNADAMSYDELISGLPKD - DVRYIAYDFDFLSKDNVKTSEIVLVSWAPEKSAI - KRKMMCASTFNSLKMALAVSKNVLQGDSFDEVDS - VAALDKVGGKPLP NP1 Ac - AMASGIKIADTV - Camide PP1 Ac - AMA ( pS ) GIKIADTV - Camide ApCofilin1 contains 147 amino acids and has a predicted molecular weight of 15 . 985 kDa . Homologue - specific antibody to apCofilin1 was generated against the full - length protein of apCofilin1 ; phospho - specific anti - apCofilin1 antibody was raised against the first 11 amino acids of apCofilin1 . Ac , acetyl group added to remove positive charge of the amine group ; Camide , amide group added to remove negative charge of the carboxylic acid group ; pS , phosphory - lated serine . TABLE 3 : The full - length apCofilin1 protein sequence and peptide sequences used for homologue - specific antibody and phosphospecific antibody generation . calcineurin inhibition . Data normalized to rates before 5 - HT addition . Data were acquired every 5 or 10 s with 2 - to 3 - min elapsed recording time . Number of growth cones tested ( N ) = 10 ( CN - AIP , injected , 0 . 2 mM in needle ) , 11 ( FK - 506 , 2 . 5 μM pretreatment for 30 min and presence throughout ) , or 9 ( CsA 1 μM pretreatment for 30 min and presence throughout ) . Values are expressed as mean ± SEM . ( D ) Summary of neurite outgrowth 1 h before and after 5 - HT addition in various conditions . No pretreatment ( control ) from Figure 1C is shown for comparison . Control ( N = 58 ) , CN - AIP ( N = 45 ) , FK - 506 ( N = 52 ) , and CsA ( N = 50 ) . Values are expressed as mean ± SEM . Statistical analysis was done by two - tailed paired t test . * p < 0 . 001 . 4844 | X . - F . Zhang et al . Molecular Biology of the Cell FIGURE 8 : 5 - HT increases calcineurin - dependent cofilin activity . ( A ) Ratio images of phosphorylated vs . total cofilin of growth cones . Cells were treated with vehicle ( Na - ASW , left ) , 5 - HT ( 10 μM , middle ) for 30 min or pretreated with FK - 506 ( 2 . 5 μM ) for 30 min , followed by 30 min in 5 - HT ( 10 μM ) with the continuous presence of FK - 506 ( right ) . Dual labeling of total and phosphorylated apCofilin1 was assessed with R - α - apCofilin1 ( 1 : 1000 ) and Sh - α - P - apCofilin1 ( 1 : 100 ) primary antibodies and Alexa 488 D - α - R ( 1 : 100 ) and Alexa 594 D - α - Sh ( 1 : 100 ) secondary antibodies . Bar , 10 μm . Ratio image is coded by pseudocolors in the linear scale ( see scale bar ) . ( B , C ) Line scan analysis of the ratio of phosphorylated vs . total Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4845 ( needle concentration , 20 μM ) . For simulta - neous Ca 2 + imaging and actin dynamics , neurons were injected with CG - 1 , Alexa 647 , and Alexa Fluor 594 – phalloidin ( needle concentration , 11 . 5 mg / ml , 0 . 9 mg / ml , and 38 μM , respectively ) . Reagent or vehicle solution injections were typically ∼ 10 % of cell volume . After microinjection , cells were incubated in Na - ASW 1 h before imaging . Confocal microscopy Images were acquired using an Andor Revo - lution XD spinning disk confocal system ( Andor , Belfast , United Kingdom ) with a CSU - X1 confocal head ( Yokogawa , Tokyo , Japan ) and mounted on a Nikon TE 2000E inverted microscope with Perfect Focus ( Nikon , Melville , NY ) . Confocal images were acquired using an Andor iXon EM + 888 elec - tron - multiplying charge - coupled device ( CCD ) camera . Transillumination was pro - vided by a halogen lamp and controlled by a SmartShutter ( Sutter Instrument , Novato , CA ) . Confocal excitation was provided by an Andor Laser Combiner with three laser lines at 488 , 561 , and 647 nm . Emission wavelength was controlled using a Sutter LB10W - 2800 filter wheel outfitted with bandpass filters from Chroma Technology ( Bellows Falls , VT ) . Image acquisition and all other peripherals were controlled by iQ soft - ware ( Andor ) . A Nikon CFI Plan Apo 100 × / 1 . 4 numerical aperture ( NA ) objective was used . Ca 2 + imaging and analysis Fluorescence images of growth cones loaded with the Ca 2 + dye CG - 1 and volume tracer were obtained using the described Andor confocal microscope . Paired images with comparable intensities of CG - 1 and Alexa 568 or Alexa 647 were recorded every 10 s using 300 - to 500 - ms integration times for Ca 2 + signal ( 488 - nm laser line ) and 200 - to 300 - ms integration times for the volume signal ( 561 - or 647 - nm laser line ) . The emis - sion fluorescence filters used ( denoted as center wavelength / bandwidth ) were 535 / 40 , 605 / 40 , and 700 / 40 nm , respectively ( Chroma Technology ) . For each paired im - age , Gaussian convolution was used to re - duce noise levels , and a binary mask was also used to eliminate noise amplification outside the cell . The ratio images ( CG - 1 / volume ) were then created by dividing background - corrected intensity values of CG - 1 fluores - cence by volume fluorescence and converted into time - lapse 10 – 15 mg / ml for CG - 1 and 0 . 8 – 0 . 9 mg / ml for volume tracer ) . For actin dynamics , neurons were injected with Alex 568 – G - actin ( needle concentration , 0 . 4 mg / ml ) or Alexa Fluor 594 – phalloidin FIGURE 9 : Model . ( A ) Growth on a laminin substrate elevates basal Rac activity levels and supports 5 - HT – evoked Ca 2 + release from IP 3 - gated internal stores . Ca 2 + release leads to calcineurin - dependent cofilin activation , with increased actin filament turnover promoting faster network treadmilling . Accelerated network treadmilling can occur independent of myosin II activity ; however , 5 - HT – dependent increases in neurite outgrowth need myosin II activity . ( B , C ) Schematic and formula showing components that can define retrograde actin flow rate . apCofilin1 fluorescence in growth cones . Scattered dots represent data set from individual growth cones . Solid lines represent the population average . N , growth cones measured . ( B ) Comparison between cells treated with vehicle ( control , red ) and cells treated with 5 - HT ( 10 μM , 30min , blue ) . ( C ) In FK - 506 backgrounds ( 2 . 5 μM FK - 506 pretreatment for 30 min ) , comparison between cells treated with vehicle ( control , purple ) and cells treated with 5 - HT ( 10 μM , 30 min , green ) in the continuous presence of FK - 506 . ( D ) Quantification of average ratio of phosphorylated vs . total cofilin in sectors 1 and 3 ( see inset for schematic ) for each condition in B and C . N , growth cones measured . * p < 0 . 001 with two - tailed unpaired t test . 4846 | X . - F . Zhang et al . Molecular Biology of the Cell separated on 1 % agarose gels , and bands of the correct size ( ∼ 450 base pairs ) were cut out and purified with the QIAquick Gel Extraction Kit ( Qiagen , Valencia , CA ) . Eluted DNA was digested with Bam HI and Nco I restriction enzymes ( New England BioLabs , Ips - wich , MA ) , ligated into pET15b ( Novagen , Gibbstown , NJ ) vector digested with the same enzymes , and transformed into DH5 α bac - teria ( Invitrogen ) for amplification . Plasmids were confirmed by the Keck DNA Sequencing Facility ( Yale University , New Haven , CT ) . Antibody generation Bacterially expressed , recombinant apCofilin1 and apCofilin2 were purified and sent to Proteintech Group ( Chicago , IL ) for generation of antibodies . apCofilin1 and apCofilin2 antibodies were generated in a rabbit or a guinea pig host against full - length apCofilin1 and apCofilin2 , respectively , and were used without affinity purification . Generation of phospho - specific anti - apCofilin1 was performed by 21st Century Biochemicals ( Marlboro , MA ) from phosphorylated peptide corresponding to the first 11 amino acids of apCofilin1 ( PP1 ) . The antibody was generated in a sheep host , affinity depleted against the nonphosphorylated peptide ( NP1 ) , and affinity purified with the PP1 peptide . An attempt to generate a phospho - specific anti - apCofilin2 antibody against the first 10 amino acids of apCofi - lin2 was unsuccessful . Western blots Western blots were performed using standard methodology . Pro - tein samples were resolved by SDS – PAGE , transferred to nitrocellu - lose membranes ( Scheicher & Schuell BioScience , Dassel , Germany ) by semidry transfer ( TransBlot SD ; Bio - Rad , Hercules , CA ) , probed with the indicated primary and secondary antibodies , developed with SuperSignal West Pico Chemiluminescent Substrate ( Pierce , Rockford , IL ) , and digitally exposed using the Epi Chemi II Darkroom ( UVP Laboratory Products , Upland , CA ) . For antigen competition as - says , the primary antibody was preincubated with excess antigen for 30 min at 4°C before use ; the control was preincubated with buffer . For alkaline phosphatase treatment , the nitrocellulose membranes were incubated in CIP buffer ( 50 mM Tris - HCl , pH 7 . 9 , 100 mM NaCl , 10 mM MgCl 2 , 1 mM dithiothreitol ) at 37°C for 60 min , with or without 25 U / ml calf intestinal phosphatase ( CIP ; New England Bio - Labs ) . The membranes were washed extensively in TBS - T ( 50 mM Tris , pH 7 . 5 , 150 mM NaCl , 0 . 1 % Tween - 20 ) and then processed normally . Immunocytochemistry In a flow chamber , cells were incubated in Fix ( 4 % Formalin , 400 mM NaCl , 10 mM KCl , 15 mM HEPES , pH 7 . 8 , 10 mM CaCl 2 , 55 mM MgCl 2 , and 400 mM sucrose ) for 20 – 30 min and 1 % Triton X - 100 in Fix for 30 min before three washes with PBS - T ( 0 . 1 % Triton X - 100 , 137 mM NaCl , 2 . 7 mM KCl , 10 mM phosphate , pH 7 . 5 ) . For anti - body labeling , cells were blocked for 30 min in 5 % BSA / PBS - T , incu - bated with primary antibody for 20 – 30 min in 5 % BSA / PBS - T , washed three times in 5 % BSA / PBS - T , and incubated for 30 min to 1 h in secondary antibody diluted in 5 % BSA / PBS - T . For antigen competi - tion assays , the diluted primary antibody was incubated with 100 - to 500 - fold excess antigen for 30 min at 4°C with rotation before use . For actin visualization , Alexa 594 or Alexa 488 – phalloidin was in - cluded in the secondary antibody solution at 0 . 66 μM . Cells were washed three times in PBS - T and mounted in Mowiol media . Cofilin line scan analysis Line scans of ratio images of background - corrected intensity values of phosphorylated cofilin divided by total cofilin were used to montages for data analysis as reported previously ( Zhang and Forscher , 2009 ) . Average pixel intensity values were obtained from the entire area of the growth cones of interest . The Ca 2 + changes over time were expressed as Δ F / F o , where Δ F = F t − F o and F o is the average Ca 2 + level sampled during the 3 - to 5 - min baseline period ( before 5 - HT addition ) . Δ F / F o ( % ) levels of > 10 % are consid - ered significant . Quantification of actin dynamics by fluorescent speckle microscopy Two methods were used to visualize F - actin for FSM : 1 ) fluorescently labeled G - actin incorporated into actin filaments and 2 ) low levels of fluorescent phalloidin , which specifically binds F - actin but not G - actin . Briefly , two - channel images were acquired using 500 - to 900 - ms integration times for actin fluorescent probe and 120 ms for DIC with 5 - or 10 - s intervals as previously described ( Burnette et al . , 2007 ) . Kymography and automated speckle tracking were used to determine rates of F - actin movement . For kymographs , analysis was done as reported ( Zhang et al . , 2003 ) . For automated flow tracking , an adaptive multiframe correlation algorithm was performed , as de - scribed ( Ji and Danuser , 2005 ) . For image presentation only , the contrast of F - actin FSM images was enhanced by processing with an unsharp mask , followed by low - pass spatial filters . Tandem Ca 2 + ratio and F - actin FSM imaging Ratiometric Ca 2 + imaging was used to measure Ca 2 + levels in the growth cone at the same time as F - actin dynamics was assessed using the methods described . Cells were injected with CG - 1 , Alexa 647 – dextran , and Alex Fluor – 594 phalloidin . DIC , Ca 2 + level , vol - ume signal , and F - actin were recorded in tandem with 5 - or 10 - s sampling intervals . CG - 1 , Alex Fluor 594 – phalloidin , and Alexa 647 – dextran were excited simultaneously with 488 - , 561 - , and 647 - nm laser lines and emission monitored using 535 / 40 - , 605 / 40 - , and 700 / 40 - nm filters , respectively ( Chroma Technology ) . The F - actin dynamics was quantitatively compared and contrasted with corresponding Ca 2 + levels in the growth cone . Neurite outgrowth analysis For long - term time - lapse experiments a Zeiss Axiovert 10 micro - scope with phase contrast optics ( 10 × Achrostigmat / NA 0 . 25 ) and a CoolSnapHQ ( Photometrics , Tucson , AZ ) cooled CCD camera were used . Hardware and image acquisition were controlled with the open - source μ - Manager device adapter library ( www . micromanager . org ) through a custom Java user interface . To quantify growth cone advance 1 h before and after 5 - HT addition in culture , a Nikon Eclipse TE300 microscope equipped with a Photometrics Quantix 57 backilluminated cooled CCD camera and MetaMorph instru - mentation control software were used ( Molecular Devices , Sunny - vale , CA ) . The displacement of growth cone’s leading edge along the presumed growth axis in 1 h was used to depict neurite outgrowth . Cloning A . californica cofilin1 ( apCofilin1 ) and A . californica cofilin2 ( apCofilin2 ) Primers ( apCofilin1 for and apCofilin1 rev ; apCofilin2 for and apCofilin2 rev ) were designed to amplify apCofilin1 and apCofilin2 from cDNA while introducing Nco I sites at the initiation codon and Bam HI restriction sites after the stop codon . Extra nucleotides were included outside the restriction sites to allow efficient digestion of PCR products . PCR was performed using Herculase polymerase ( Stratagene , Santa Clara , CA ) . A cDNA library was constructed using mRNA extracted from bag cell neurons . PCR products were Volume 23 December 15 , 2012 Actin treadmilling and neurite growth | 4847 Ferreira A , Kincaid R , Kosik KS ( 1993 ) . Calcineurin is associated with the cytoskeleton of cultured neurons and has a role in the acquisition of polarity . Mol Biol Cell 4 , 1225 – 1238 . Figge C , Loers G , Schachner M , Tilling T ( 2011 ) . 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Attenua - tion of actinomyosinII contractile activity in growth cones accelerates \ No newline at end of file diff --git a/silver_data/597fdd60db83b8dadaad601e5ce18987eb674564.pdf.txt b/silver_data/597fdd60db83b8dadaad601e5ce18987eb674564.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d78eaf98b60278b51b454a16df4146960a82d467 --- /dev/null +++ b/silver_data/597fdd60db83b8dadaad601e5ce18987eb674564.pdf.txt @@ -0,0 +1 @@ +Estimating Replicability of Classifier Learning Experiments Remco R . Bouckaert 1 , 2 remco @ cs . waikato . ac . nz , rrb @ xm . co . nz 1 . Computer Science Department , University of Waikato , Hamilton , New Zealand 2 . Xtal Mountain Information Technology , Auckland , New Zealand Abstract Replicability of machine learning experi - ments measures how likely it is that the out - come of one experiment is repeated when per - formed with a different randomization of the data . In this paper , we present an estima - tor of replicability of an experiment that is efficient . More precisely , the estimator is un - biased and has lowest variance in the class of estimators formed by a linear combination of outcomes of experiments on a given data set . We gathered empirical data for comparing experiments consisting of different sampling schemes and hypothesis tests . Both factors are shown to have an impact on replicability of experiments . The data suggests that sign tests should not be used due to low replica - bility . Ranked sum tests show better perfor - mance , but the combination of a sorted runs sampling scheme with a t - test gives the most desirable performance judged on Type I and II error and replicability . 1 . Introduction Machine learning research on classifiers relies to a large extent on experimental observations . It is widely rec - ognized that there are many pitfalls in performing ex - periments [ 3 , 6 , 8 ] . But , so far , most research in this area concentrates on undesirable high levels of Type I error , the situation where the experiment indicates that one classifier outperforms another , while in real - ity it does not . An often overlooked issue with experi - mental research is that the particular randomizations used in the experiment can have a major impact on the outcome of the experiment . This effect can be so large that for some experimental designs only in 2 out of 3 cases repetition of the experiment produces the same outcome [ 2 ] . Appearing in Proceedings of the 21 st International Confer - ence on Machine Learning , Banff , Canada , 2004 . Copyright 2004 by the author . In this paper , we try to get a better insight in this issue of replicability and which factors in an exper - iment influence replicability . In order to do so , we need a practical definition of replicability and a way to measure replicability of an experiment . Once this is established we can actually perform experiments on various set - ups . In the following section , we consider a number of experimental designs . We continue in Sec - tion 3 with ways to estimate replicability and perform a theoretical analysis of their performance . Section 4 presents empirical results where we measure replica - bility for the various experimental set - ups . We finish with some concluding remarks . 2 . Machine learning experiments The problem we want to address is , given two learn - ing algorithms A and B that generate classifiers and a small data set D , how to make a decision which of the two algorithms performs best based on classification accuracy for the given data set . A general method to make such a decision is to split D into a training set D t and a test set D \ D t . Then , train algorithm A and B on D t and register the classification accuracy on the D \ D t . This way , we obtain two classification accura - cies P A and P B and the difference x = P A − P B gives an indication which algorithm performs better . A formal way to make such a decision is to apply a hy - pothesis test . However , such hypothesis test typically requires more than a single outcome x . Unfortunately , for small datasets D , we have to split D repeatedly in training and test sets to obtain multiple outcomes P A , i and P B , i with associated differences x i = P A , i − P B , i , 1 ≤ i ≤ n obtaining a sample of size n . So , an experiment has two components . Firstly , a sam - pling scheme for obtaining a sample x 1 , . . . , x n , and secondly , a hypothesis test to make a decision based on the sample . There are various ways to obtain sam - ples and to perform hypothesis tests . 2 . 1 . Sampling methods We consider six different sampling schemes . Figure 1 . Example illustrating the data used for the various sampling schemes . used when averaging over runs used when averaging over folds used when averaging over sorted runs Fold Sorted Fold Run Run 1 2 3 1 2 3 123 123 3 . 33 6 . 66 6 . 66 5 . 55 10 3 . 33 - 10 1 . 11 - 6 . 660 - 3 . 33 - 3 . 33 2 . 22 3 . 33 - 2 . 22 - 6 . 660 - 10 - 5 . 55 3 . 33 3 . 33 - 3 . 33 1 . 11 10 6 . 66 6 . 66 7 . 77 Resampling : Resampling consist of splitting the data n times in a randomly selected test set D t , i contain - ing a fraction of the data ( typically 10 % to 33 % ) and a training set D \ D t , i . The algorithms A and B learn on the training set and accuracies P A , i and P B , i , 1 ≤ i ≤ n are obtained by classifying instances on the accompanying test set giving n accuracy differences x i = P A , i − P B , i for the sample . Resampling used to be an accepted way for applying the t - test on the sam - ple x 1 , . . . , x n till it was discredited by Dietterich [ 3 ] due to its extremely high Type I error . Nadeau and Bengio [ 6 ] showed how this problem can be solved by correcting the variance . K - fold cross validation : Cross validation splits the data D into k approximately equal parts D 1 , . . . , D k , and learns on the data D \ D i , 1 ≤ i ≤ k with one part left out . The part D i left out is used as test set , giving n = k accuracy differences x i = P A , i − P B , i . Dietterich [ 3 ] observed a slightly elevated Type I error for cross validation with a t - test and its replicability is rather low [ 2 ] . Use all data : To obtain more samples , we can repeat k - fold cross validation r times with different random splits into folds for each of the runs . This gives us r × k accuracy differences . Let ˆ x i , j , 1 ≤ i ≤ r , 1 ≤ j ≤ k denote the difference in accuracy of algorithms A and B in the i th run on the j th fold . Here A and B are trained on the k − 1 remaining folds in the i th run . We obtain a sample of size n = r × k by using all of the accuracy differences x i , j ( formally by setting x i = ˆ x i mod r , ⌈ i / r ⌉ ) . Average over folds : In averaging over folds , the rec - ommended method for Weka [ 9 ] , we take the result in a repeated cross validation experiment . We obtain one sample value per run by taking the average differ - ence over all results for a single run , x i = P kj = 1 ˆ x i , j / k ( where ˆ x i , j as for the use all data scheme ) . Average over runs : Averaging over folds can be interpreted as an improved way of doing resampling . The natural extension is performing an improved way of k - fold cross validation , and instead of averaging over folds , average over runs . We obtain one sam - ple value per fold defined as the average difference x i = P ra = 1 ˆ x a , i / r . Both averaging over folds and over runs show a very high Type I error when applying a t - test [ 2 ] . Average over sorted runs : Averaging over runs combines results from different runs rather arbitrar - ily . One gets better estimates of a k - fold cross valida - tion experiment by first sorting the results for the in - dividual k - fold cross validation experiments and then taking the average . This way , the estimate for the minimum value is calculated from the minimum val - ues in all folds , the one but lowest from the one but lowest results in all folds , etc . Let ˆ x θ ( i , j ) be the j th highest value of accuracy difference ˆ x i ′ j ′ of run i . Then , the sample consisting of k values is defined by x i = P ra = 1 ˆ x θ ( a , i ) / r . Figure 1 illustrates the difference between the data used for the sampling schemes . The figure shows an example of 3x3 fold cross validation outcomes in the box at the left half ( though in practice a 10x10 fold cross validation is more appropriate ) . All the data in the box in Figure 1 is used for the ”use all data” scheme . For resampling , essentially only the first col - umn is required when performing a 2 / 3 - 1 / 3 split of training and test data . Cross validation uses only the first run , that is , the first row of a 3x3 fold cross val - idation outcome . Averaging over folds and runs is es - sentially summing over columns and rows respectively . For getting sorted means , first the results have to be sorted over folds , giving the table at the right of Fig - ure 1 . Then the means are obtained by summing over rows . 2 . 2 . Hypothesis tests In our experiment , we want to test the null hypothesis H 0 that A and B perform the same . More formally , we want to test whether the sample x 1 , . . . , x n has zero mean . There are different methods to test such hy - pothesis , all of which are based on slightly different assumptions . We consider the popular t - test , the sign test and the rank sum test , also known as Wilcoxon’s test . All these tests assume that the outcomes x i in the sample are mutually independent , an assumption that is obviously violated . These hypothesis tests follow a similar procedure . First , we calculate a statistic Z from the sample . Dif - ferent tests have different methods of calculating Z ( see below ) . Then , we calculate the probability p ( Z ) that the value Z or less is observed assuming H 0 is true . We choose a significance level α and accept H 0 if p ( Z ) is higher than α / 2 but less than 1 − α / 2 . If p ( Z ) < α / 2 , the test indicates B outperforms A and if p ( Z ) > 1 − α / 2 , the test indicates A outperforms B . Paired t - test : The assumption underlying the paired t - test is that the outcomes x i are normally dis - tributed . If this is true , then the mean can be esti - mated using ˆ m = 1 n P ni = 1 x i , the variance using ˆ σ 2 = 1 n − 1 P ni = 1 ( x i − ˆ m ) 2 . With n − 1 degrees of freedom ( df = n − 1 ) we have a statistic Z = ˆ m √ ˆ σ 2 / √ df + 1 , which is distributed according to Students t - distribution P t with df degrees of freedom . The probability that the data x 1 , . . . , x n is observed assuming the null hypoth - esis is true is obtained by finding P t ( T , df ) . Sign test : The attractiveness of the sign test is that it is simple and makes no assumptions about the under - lying distribution of the sample . Instead , it only looks at the signs of x 1 , . . . , x n and statistic Z is the num - ber of pluses . When accuracies P A , i and P B , i are the same , which occurs quite often when two algorithms perform very similarly , x i = 0 and we count this as half a plus . If the null hypothesis is true , the probability of generating a plus or a minus is 0 . 5 , in other words H 0 : p = 0 . 5 . The probability of observing Z pluses in n comparisons is P ( Z ) = P Zi = 0 (cid:0) ni (cid:1) p i ( 1 − p ) n − i , which with p = 0 . 5 is P ( Z ) = P Zi = 0 (cid:0) ni (cid:1) 12 n . Rank sum test : Like the sign test , the rank sum test makes no assumption about the underlying dis - tribution of outcomes x i . However , the rank sum test does exploit the size of the values of x i , which contains potentially valuable information . The rank sum test sorts the outcomes x i on its absolute value , giving a set of outcomes y 1 , . . . , y n , | y i | ≤ | y i + 1 | ( 1 ≤ i < n ) . When accuracies are the same ( i . e . outcomes for which x i = 0 ) they are removed from the sample , leaving n ′ items . Now , we add the ranks of outcomes that are positive , r = P n ′ i = 1 , y i > 0 i . This statistic has mean m = n ′ ( n ′ + 1 ) 4 and variance σ 2 = n ′ ( n ′ + 1 ) ( n ′ + 2 ) 24 and is approximately normally distributed . So , we use Z = r − mσ , which is normally distributed with mean 0 and variance 1 . 2 . 3 . Quality of experiments There are essentially three methods to judge the qual - ity of an experiment : • The Type I error is the probability that the conclu - sion of an experiment is there is a difference between algorithms , while in reality there is not . In theory , the Type I error equals the significance level chosen for the hypothesis test if none of the assumptions of the test are violated . In practice , the independence assump - tion is often violated resulting in an elevated Type I error . • The Type II error is the probability the conclusion of an experiment is there is no difference between algo - rithms , while in reality there is . The power is defined as 1 minus the Type II error . The power is not directly controllable like the Type I error is . However , there is a trade - off between power and Type I error and a higher power can be obtained at the cost of a higher Type I error . The exact relation between the two de - pends on the experimental design . • Replicability of an experiment is a measure of how well the outcome of an experiment can be reproduced . The most desirable experiment has a low Type I error , a high power an high replicability . In the following section we will have a closer look at replicability . 3 . Replicability In [ 2 ] , an ad hoc definition for replicability was pro - posed as follows . When an experiment is repeated ten times with different randomizations of a given data set , the experiment is deemed replicable if its outcome is the same for all ten experiments . If one or more outcomes differ , it is not replicable . An impression of the replicability of an experiment can be obtained by averaging over a large number ( say 1000 ) of data sets . This definition is useful in highlighting that repli - cability of experiments is indeed an issue in machine learning . However , the disadvantage is that replica - bility measured this way cannot be compared with re - sults for doing the experiment another number than ten times . Also , replicability defined this way would not distinguish between having 1 out of 10 outcomes being different and 5 out of 10 outcomes being differ - ent . Further , increasing the number of experiments to say 100 increases the likelihood that one of the experi - ments differ and thus decreases replicability according to the definition of [ 2 ] . A definition of replicability that does not suffer from these issues is the following . Definition : Replicability of an experiment is the prob - ability two runs of the experiment on the same data set , with the same pair of algorithms and the same method of sampling the data produces the same out - come . This definition applies both in the situation where the algorithms perform the same and when one outper - forms another . Note the difference between Type I error and replicability . When the algorithms perform the same , the Type I error expresses the probability over all data sets that a difference is found . Replica - bility only expresses that error for one data set . By defining replicability in terms of probabilities , one can compare replicability of different experiments with different experimental set - ups and number of runs . Furthermore , an experiment that produces 9 same out - comes out of 10 has a higher replicability this way than when it only produces 5 same outcomes out of 10 . Note that replicability always lies between 50 % and 100 % . Normalized replicability is replicability linearly scaled to the range 0 % to 100 % . So , if replicability is r , normalized replicability is 2 ( r − 12 ) . 3 . 1 . A simple estimator The only way to determine the replicability of an ex - periment is to measure it empirically . So , we need an estimator of replicability . A simple approach is to ob - tain pairs of runs of an experiment on a data set D and just interpret those as the outcome of Bernoulli trial with probability r that the outcomes are the same . The outcome e of an experiment on data set D is ’ac - cept’ or ’reject’ . When the outcome is ’accept’ the null hypothesis that the two learning algorithms per - form the same on D is accepted , otherwise they are not . Definition Let e = e 1 , . . . , e n ( n > 0 and n even ) be the outcomes of n experiments with different random - izations on data set D . The estimator ˆ R 1 of replica - bility r is ˆ R 1 ( e ) = P n / 2 i = 1 I ( e 2 i = e 2 i − 1 ) n / 2 where I is the indicator function , which is 1 if its ar - gument is true , and 0 otherwise . We write ˆ R 1 if it is clear from the context what the argument e of ˆ R 1 ( e ) is . Lemma 3 . 1 ˆ R 1 is an unbiased estimator of replicabil - ity r with variance r − r 2 n / 2 . Proof : The bias of ˆ R 1 is E ( ˆ R 1 ) − r . Now , E ( ˆ R 1 ) = E ( P n / 2 i = 1 I ( e 2 i = e 2 i − 1 ) n / 2 ) . Taking the constant 1 n / 2 outside the expectation gives E ( ˆ R 1 ) = 1 n / 2 E ( P n / 2 i = 1 I ( e 2 i = e 2 i − 1 ) ) . Distributing the sum results in E ( ˆ R 1 ) = 1 n / 2 P n / 2 i = 1 E ( I ( e 2 i = e 2 i − 1 ) ) . Now , E ( I ( e 2 i = e 2 i − 1 ) ) = P ( I ( e 2 i = e 2 i − 1 ) ) I ( e 2 i = e 2 i − 1 ) + P ( I ( e 2 i = e 2 i − 1 ) ) I ( e 2 i 6 = e 2 i − 1 ) . Note that P ( I ( e 2 i = e 2 i − 1 ) ) = r and P ( I ( e 2 i 6 = e 2 i − 1 ) ) = 1 − r so we get E ( I ( e 2 i = e 2 i − 1 ) ) = r · 1 + ( 1 − r ) · 0 = r . Substituting in E ( ˆ R 1 ) above gives E ( ˆ R 1 ) = 1 n / 2 P n / 2 i = 1 r = n / 2 n / 2 r = r . So , the bias of ˆ R 1 = E ( ˆ R 1 ) − r = r − r = 0 , which shows that ˆ R 1 is an unbiased estimator of r . The variance of ˆ R 1 is var ( ˆ R 1 ) = E ( ˆ R 21 ) − E ( ˆ R 1 ) 2 = P n / 2 i = 0 P ( i same pairs out of n / 2 ) ( in / 2 ) 2 − E ( ˆ R 1 ) 2 where ˆ R 1 = 1 n / 2 . From the deriva - tion above , we have E ( ˆ R 1 ) 2 = r 2 . Further , ob - serve that P ( i same pairs out of n / 2 ) follows the bi - nomial distribution with probability r . So , we have var ( ˆ R 1 ) = P n / 2 i = 0 r i ( 1 − r ) n / 2 − i (cid:0) n / 2 i (cid:1) ( in / 2 ) 2 − r 2 = 1 ( n / 2 ) 2 P n / 2 i = 0 r i ( 1 − r ) n / 2 − i (cid:0) n / 2 i (cid:1) i 2 − r 2 . Us - ing Lemma A . 1 ( see Appendix ) , P n / 2 i = 0 r i ( 1 − r ) n / 2 − i (cid:0) n / 2 i (cid:1) i 2 = ( n / 2 ) 2 r 2 − r 2 n / 2 + rn / 2 giving var ( ˆ R 1 ) = 1 ( n / 2 ) 2 ( ( n / 2 ) 2 r 2 − r 2 n / 2 + rn / 2 ) − r 2 = r − r 2 n / 2 . 3 . 2 . An advanced estimator The simple estimator ˆ R 1 uses experiment e 1 only to compare with e 2 . Since e 3 is independent of e 1 , one could compare e 1 with e 3 as well . Likewise , the pair ( e 1 , e k ) for any k > 1 could be compared and used in the estimate of replicability . In fact , we can use all pairs of outcomes and estimate replicability as the fraction of pairs with the same outcome . This defines a new estimator ˆ R 2 . Definition Let e = e 1 , . . . , e n and n as before , then we define estimator ˆ R 2 ( e ) of r as ˆ R 2 ( e ) = X 1 ≤ i < j ≤ n I ( e i = e j ) n · ( n − 1 ) / 2 ( 1 ) According to the following lemma , we can actually cal - culate ˆ R 2 directly from counting the number of ac - cepted tests out of the n experiments . So , ˆ R 2 can be calculated efficiently in linear time of the number of experiments . Lemma 3 . 2 Let e = e 1 , . . . , e n and n as before and i out of n tests be accepting the null hypothesis , then ˆ R 2 ( e ) = ˆ R 2 ( i , n ) = i · ( i − 1 ) + ( n − i ) · ( n − i − 1 ) n · ( n − 1 ) Proof : The numerator of ˆ R 2 in ( 1 ) is the number of pairs with equal outcomes . If i ( 0 ≤ i ≤ n ) tests accept the null hypothesis and the remaining n − i do not , then (cid:0) i 2 (cid:1) pairs of rejecting pairs and (cid:0) n − i 2 (cid:1) pairs of non re - jecting pairs can be formed . This gives an estimate of replicability as ˆ R 2 ( i , n ) = ( (cid:0) i 2 (cid:1) + (cid:0) n − i 2 (cid:1) ) / ( n ( n − 1 ) / 2 ) = i ( i − 1 ) + ( n − i ) ( n − i − 1 ) n ( n − 1 ) . Now , we will examine the bias and variance of ˆ R 2 . It turns out that ˆ R 2 is an unbiased estimator of replica - bility and its variance can be expressed in closed form . Theorem 3 . 1 ˆ R 2 is an unbiased estimator of replica - bility r with variance 1 n · ( n − 1 ) · ( 2 ( n − 2 ) ( n − 3 ) F ( p , 4 ) + ( 4 − 2 ( n − 3 ) ) ( n − 2 ) F ( p , 3 ) + ( n − 2 ) ( n − 3 ) + 2 ) F ( p , 2 ) − r 2 where F ( p , x ) = p x + ( 1 − p ) x and p = 1 / 2 + 1 / 2 √ 2 r − 1 . The proof that ˆ R 2 is unbiased closely follows that of Lemma 3 . 1 . The proof establishing the variance of ˆ R 2 is rather technical and is omitted here . A full proof is available in the report version of this paper . Unfortunately , the closed form expression for the vari - ance of ˆ R 2 is hard to interpret and compare with that of ˆ R 1 . Figure 2 shows the variance of ˆ R 1 and ˆ R 2 for various values of replicability r and number of experi - ments n . It shows that the variance of ˆ R 2 is equal to that of ˆ R 1 when r = 1 . This is when there is full repli - cability and in this case the variance is zero . However , for other values of r , the variance of ˆ R 2 is always be - low that of ˆ R 1 , indicating that ˆ R 2 is a more efficient estimator of replicability than ˆ R 1 . 3 . 3 . Is there a better estimator ? Is there an unbiased estimator of replicability with lower variance than ˆ R 2 based on experiments e = e 1 , . . . , e n on a single database ? We will consider the class of estimators based on linear functions of I ( e i = e j ) . Definition : Let e = e 1 , . . . , e n ( n > 0 and n even ) be the outcomes of n experiments with different ran - domizations on data set D . Then estimator ˆ R k of r is ˆ R k ( e ) = X 1 ≤ i < j ≤ n k i , j I ( e i = e j ) ( 2 ) Note that ˆ R 1 is in this class with k i , i + 1 = 1 n / 2 for odd i and k i , j = 0 otherwise . Likewise , ˆ R 2 is in this class with k i , j = 1 n ( n − 1 ) / 2 for all 1 ≤ i < j ≤ n . If we demand that ˆ R k is unbiased , we put a restriction on the coefficients k i , j expressed in the following lemma . Figure 2 . Variance of ˆ R 1 ( upper surface ) and ˆ R 2 ( lower surface ) as function of replicability r ∈ ( 0 . 5 . . . 1 . 0 ) and number of tests n ∈ ( 4 . . . 28 ) 4 8 12 16 20 24 28 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0 0 0 . 02 0 . 04 0 . 06 0 . 08 0 . 1 0 . 12 0 . 14 Lemma 3 . 3 ˆ R k is an unbiased estimator of replicabil - ity r iff X 1 ≤ i < j ≤ n k i , j = 1 ( 3 ) In the proof , we use the property that if r is the repli - cability of an experiment for a given data set , then , by definition , r is the probability two experiments pro - duce the same outcome . Now , two experiments use different independent randomizations . So , if p is the probability that the outcome of a single experiment is accept , then the replicability is the probability that two outcomes are accept ( p · p ) plus the probability that two outcomes are reject ( ( 1 − p ) · ( 1 − p ) ) . So , r = p · p + ( 1 − p ) · ( 1 − p ) , which can be solved for p giving p = 12 ± 12 √ 2 r − 1 . Proof : For ˆ R k to be an unbiased estimator of replica - bility r , we must have E ( ˆ R k ) = r . Now , E ( ˆ R k ) by def - inition of expectation is P e P ( e ) ˆ R k ( e ) . Using ( 2 ) , this equals P e P ( e ) P 1 ≤ i < j ≤ n k i , j I ( e i = e j ) . Changing the order of sums , we get P 1 ≤ i < j ≤ n P e P ( e ) k i , j I ( e i = e j ) . Note that P e P ( e ) has equal outcomes for e i and e j only with probability p 2 ( both accept ) and ( 1 − p ) 2 ( both rejects ) . So , P e P ( e ) k i , j I ( e i = e j ) = ( p 2 + ( 1 − p ) 2 ) k i , j = rk i , j . Summing over i and j gives P 1 ≤ i < j ≤ n rk i , j = r P 1 ≤ i < j ≤ n k i , j = r where the last equality follows from the condition that the estimator is unbiased . Consequently , P 1 ≤ i < j ≤ n k i , j = 1 . So , ˆ R 1 and ˆ R 2 being unbiased ( Lemma 3 . 1 and The - orem 3 . 1 ) can be proven observing ˆ R 1 and ˆ R 2 are in - stances of ˆ R k and noting that the coefficients k i , j add to 1 . Theorem 3 . 2 var ( ˆ R k ) ≥ var ( ˆ R 2 ) for any unbiased Table 1 . Type I error on Set 1 , power on Set 2 , 3 and 4 and replicability ( in percentages ) for various sampling methods ( 95 % confidence interval in brackets ) . Source 1 Source 2 Source 3 Source 4 Minimum average Test Sampling scheme Type I Power Power Power norm . replicability Rank sum Resampling 14 . 8 ( ± 0 . 4 ) 27 . 9 ( ± 0 . 6 ) 48 . 0 ( ± 0 . 3 ) 95 . 7 ( ± 0 . 3 ) 34 . 0 ( ± 0 . 5 ) test k - fold cv 11 . 0 ( ± 0 . 2 ) 23 . 2 ( ± 0 . 2 ) 45 . 8 ( ± 0 . 7 ) 97 . 5 ( ± 0 . 2 ) 46 . 0 ( ± 0 . 5 ) Use all data 55 . 2 ( ± 0 . 3 ) 71 . 5 ( ± 0 . 2 ) 88 . 0 ( ± 0 . 1 ) 100 . 0 ( ± 0 . 0 ) 61 . 8 ( ± 0 . 6 ) Average over folds 59 . 8 ( ± 0 . 5 ) 78 . 9 ( ± 0 . 4 ) 90 . 2 ( ± 0 . 2 ) 100 . 0 ( ± 0 . 0 ) 56 . 4 ( ± 0 . 7 ) Average over runs 50 . 5 ( ± 0 . 6 ) 68 . 3 ( ± 0 . 1 ) 86 . 1 ( ± 0 . 1 ) 100 . 0 ( ± 0 . 0 ) 57 . 0 ( ± 0 . 9 ) Average sorted runs 4 . 1 ( ± 0 . 1 ) 20 . 2 ( ± 0 . 3 ) 46 . 8 ( ± 0 . 5 ) 99 . 3 ( ± 0 . 1 ) 80 . 6 ( ± 0 . 2 ) Sign test Average sorted runs 5 . 0 ( ± 0 . 5 ) 21 . 2 ( ± 0 . 4 ) 48 . 6 ( ± 0 . 3 ) 99 . 1 ( ± 0 . 1 ) 75 . 2 ( ± 0 . 7 ) T - test Average sorted runs 4 . 5 ( ± 0 . 1 ) 21 . 1 ( ± 0 . 2 ) 51 . 7 ( ± 0 . 5 ) 99 . 6 ( ± 0 . 1 ) 81 . 6 ( ± 0 . 6 ) estimator ˆ R k . Proof : We determine the minimum of var ( ˆ R k ) and show that ˆ R 2 realizes the minimum . By definition , var ( ˆ R k ) equals E ( ˆ R 2 k ) − E ( ˆ R k ) 2 . Since ˆ R k is un - biased , E ( ˆ R k ) = r so var ( ˆ R k ) = E ( ˆ R 2 k ) − r 2 = P e P ( e ) ˆ R 2 k ( e ) − r 2 . At the minimum , dvar ( ˆ R k ) / dk i , j = 0 for all 1 ≤ i < j ≤ n . Taking derivatives w . r . t . k a , b for any a , b such that ( a , b ) 6 = ( 1 , 2 ) gives dvar ( ˆ R k ) / dk a , b = d P e P ( e ) ˆ R 2 k ( e ) − r 2 / dk a , b which computes as P e P ( e ) 2 ˆ R k ( e ) ( d ˆ R k ( e ) / dk a , b ) . We can write ˆ R k = P 1 ≤ i < j ≤ n , j > 2 k i , j I ( e i = e j ) + k 1 , 2 I ( e 1 = e 2 ) and use ( 3 ) to write k 1 , 2 = 1 − P 1 ≤ i < j ≤ n , j > 2 k i , j , giv - ing ˆ R k = P 1 ≤ i < j ≤ n , j > 2 k i , j I ( e i = e j ) + ( 1 − P 1 ≤ i < j ≤ n , j > 2 k i , j ) I ( e 1 = e 2 ) . So , the term d ˆ R k ( e ) / dk a , b can be written as d P 1 ≤ i < j ≤ n , j > 2 k i , j I ( e i = e j ) + ( 1 − P 1 ≤ i < j ≤ n , j > 2 k i , j ) I ( e 1 = e 2 ) / dk a , b which equals I ( e a = e b ) − I ( e 1 = e 2 ) . So dvar ( ˆ R k ) / dk a , b is P e P ( e ) 2 ˆ R k ( e ) ( I ( e a = e b ) − I ( e 1 = e 2 ) ) . We need to distinguish two cases , namely a ≤ 2 and a > 2 . If a > 2 , dvar ( ˆ R k ) / dk a , b is P e P ( e ) 2 P 1 ≤ i < j ≤ n k i , j I ( e i = e j ) ( I ( e a = e b ) − I ( e 1 = e 2 ) ) reduces to 2 k a , b ( p ( 1 − p ) 3 + p 3 ( 1 − p ) ) − 2 k 1 , 2 ( p ( 1 − p ) 3 + p 3 ( 1 − p ) ) where p = 12 + 12 √ 2 r − 1 as before . For this to equal zero , we have p = 0 or p = 1 coinciding with replicability of r = 1 , or k a , b = k 1 , 2 . Likewise , if a ≤ 2 dvar ( ˆ R k ) / dk a , b reduces to 2 k a , b ( p ( 1 − p ) 2 + p 2 ( 1 − p ) ) − 2 k 1 , 2 ( p ( 1 − p ) 2 + p 2 ( 1 − p ) ) . And again , we have r = 1 or k a , b = k 1 , 2 . So , var ( ˆ R k ) reaches an optimum at k a , b = k 1 , 2 for all a , b , which means all coefficients are equal . And since they sum to 1 , we have k a , b = 1 n ( n − 1 ) / 2 since there are n ( n − 1 ) / 2 coefficients . The optimum is a minimum , as Figure 2 shows . In summary , Theorem 3 . 2 states that ˆ R 2 is indeed an efficient ( i . e . unbiased with lowest variance ) estimator in the class of unbiased estimators ˆ R k . 4 . Empirical results First , we establish which sampling scheme results in acceptable experiments based on Type I error and power . Then , we look at factors that impact replica - bility . To measure Type I error and power , algorithm A ( naive Bayes [ 5 ] as implemented in Weka 3 . 3 [ 9 ] ) and algorithm B ( C4 . 5 [ 7 ] as implemented in Weka with default parameters ) were compared on synthetic data and UCI data sets . The synthetic data sets was generated using four data sources based on four ran - domly generated Bayesian networks ( [ 2 ] for more de - tails ) . The data sets contained 10 binary variables and 50 % class probability . Each of the data sources were used to generate 1000 data sets with 300 instances . Data source 1 has mutually independent variables , so there is no performance difference between naive Bayes and C4 . 5 , which allows us to measure the Type I error . For sources 2 , 3 and 4 , C4 . 5 outperforms naive Bayes with increasing margin ( on average 2 . 77 % , 5 . 83 % and 11 . 27 % respectively as measured on 10 . 000 instance test sets ) , which allows us to measure the power of tests . The sampling methods mentioned in Section 2 . 1 were performed 10 times with 10 folds and 10 runs at 5 % significance level . Table 1 shows the results on the synthetic data with numbers in brackets indicating a 95 % confidence inter - val . The first six rows are for the rank sum test . Note that the use all data , average over folds and over runs sampling schemes have a Type I error over 50 % , while a 5 % Type I error is desired . The resampling scheme has an elevated Type I error as has the 10 fold cross validation scheme . Only the sorted runs scheme shows an appropriate level of Type I error . This comes at the Table 2 . UCI data sets . Nr of draws of sorted 10 x 10 fold cv ( α = 5 % , 95 % intervals for ˆ R 2 within ± 3 % ) 10 + 20 + Data set 123456789 01234567890 01234567 Mean norm . ˆ R 2 Sign test 84 . 4 NB vs C45 . . 9 . . . . . 2 . 4 . . 3 . . 27 . . . . . . . 5 . 78 . 2 NB vs NN . . . . . 96 . 9 . . . . 19 . 9 . . . . . . 6 . . . 84 . 6 C45 vs NN . . . 1 . 4 . . . . . . . . . . . . 5 . . . . . . . . 90 . 4 Rank test 90 . 2 NB vs C45 . . . . . . . . . . 4 . . 3 . . . 9 . . . . . . . 7 . 87 . 6 NB vs NN . . . . . . 8 . . . . . . . . . 9 . . . . . . 8 . . . 93 . 2 C45 vs NN . . . 2 . 6 . . . . . . . . . . . . 7 . . . . . . . . 90 . 0 T test 90 . 8 NB vs C45 . . . . . . . . . . 1 . . 2 . . . 9 . . . . . . . 7 . 91 . 0 NB vs NN . . . . . . 9 . . . . . . . . . 9 . . . . . . 7 . . . 93 . 6 C45 vs NN . . . 2 . 6 . . . . . . . . . . . . 6 . . . . . . . 9 88 . 0 cost of decreased power compared to most of the other schemes . Table 1 also shows the minimum of the average repli - cability over Set 1 to 4 . It shows that resampling and 10 - fold cv has a level of replicability which is not ac - ceptable ( below 50 % ) . The schemes based on repeated cross validation do show acceptable replicability . In particular , the sorted runs scheme has a replicability of over 80 % . Results for the sign test and t - test are similar to the results for the rank sum test . Table 1 also shows Type I error and power for sorted runs with sign test and t test . Those figures are very close to the ones for the rank sum test , taking in ac - count that a slightly higher Type I error should lead to slightly better power . The replicability for sorted runs with the sign test is 75 . 2 % and with the t - test is 81 . 6 % . Compared to the 80 . 6 % for the rank sum test , the sign test performs considerably worse . This can be explained by the lack of exploiting sizes of differences in the sample by the sign test . The replicability of the t - test is only slightly better . Further experiments were performed using the sorted runs sampling scheme while varying various parame - ters of the experiment , namely • significance level ( 1 % , 2 . 5 % , 5 % and 10 % ) , • number of runs ( 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 and 100 ) , • class probability for binary data ( 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 and 0 . 5 ) , • class cardinality ( 2 , 3 and 4 ) , • different pairs of algorithms ( out of Naive Bayes , C4 . 5 , nearest neighbor , tree augmented naive Bayes , decision stump and support vector ) . Though space limitations prevent us from presenting the complete set of outcomes here , we can report that the experiments resulted in a Type I error not exceed - ing the significance level by more than 1 % with the sorted runs sampling scheme for all three tests con - sidered . Decreasing the class probability increased replicability . The explanation for this behavior can be found in realizing that learners tend to predict the majority class the more this class dominates the data . Increasing the number of runs consistently increased replicability . It appears that the sorted runs sampling scheme results in a sample for which the independence assumption is not heavily violated , so that no correc - tion in variance [ 6 ] or degrees of freedom [ 2 ] is required . Table 2 shows results for 27 data sets from the UCI repository [ 1 ] 1 using the sorted runs sampling scheme with the three different types of tests . We compared naive Bayes , C4 . 5 and nearest neighbor ( NB , C4 . 5 and NN respectively in Table 2 ) . Each algorithm was run ten times . The middle three columns show the num - ber of times that the experiment decides that the null hypothesis is acceptable ( so algorithms perform equal on a data set ) as numbered in the footnote 1 . When the null hypothesis is 0 or 10 times accepted only a dot is shown , since both situations indicate perfect repli - cability . The first observation is that replicability is an issue for non - synthetic data sets , and thus affects many machine learning researchers . Further , the sign test performs much worse than the other two tests , while the t - test shows marginally higher replicability than the rank sum test . So , not only the sampling method , but also the hypothesis test has an impact on the replicability of the experiment . 1 1 : anneal , 2 : arrhythmia , 3 : audiology , 4 : autos , 5 : balance - scale , 6 : breast - cancer , 7 : credit - rating , 8 : ecoli , 9 : German credit , 10 : glass , 11 : heart - statlog , 12 : hepati - tis , 13 : horse - colic , 14 : Hungarian heart disease , 15 : iono - sphere , 16 : iris , 17 : labor , 18 : lymphography , 19 : pima - diabetes , 20 : primary - tumor , 21 : sonar , 22 : soybean , 23 : vehicle , 24 : vote , 25 : vowel , 26 : Wisconsin breast cancer , and 27 : zoo . 5 . Conclusions We defined replicability of machine learning experi - ments in terms of probability . This has the benefit that it allows for comparison over different experimen - tal designs , unlike a previous rather ad hoc definition [ 2 ] . For example , replicability measured on n repeats of an experiment can be compared with replicability measure on 2 n repeats . Furthermore , threshold effects present in the ad hoc definition are not present in our definition . The main theoretical result of this paper is the presen - tation of an estimator for replicability that was shown to be unbiased and which has the lowest variance in its class . Using this estimator , we gathered empirical data to gain new insights in how experimental designs influence replicability and found that the hypothesis test , the sampling scheme , and the class probability impact replicability . In our experiments , replicabil - ity consistently increased with sampling methods that draw more samples from the same data set . Replica - bility appears to be an issue both with synthetic data sets as well as with UCI data sets . This indicates that machine learning researcher and data analysts should be wary when interpreting experimental results . The main practical outcome of the experiments is that judged on replicability the sorted runs sampling scheme with the widely used t - test showed superior properties compared to the sign test and performed marginally better than the rank sum test . The sorted runs scheme is based on combining accuracy estimates in a way that produces a representative sample of ac - curacy differences of learning algorithms . Surprisingly , the sorted runs sampling schemes is the only scheme out of a set of popular schemes we considered that also showed acceptable Type I errors and reasonable power for a wide range of parameters using the three hypoth - esis tests considered . Consequently , experiments based on sorted runs sampling schemes do not require vari - ance corrections [ 6 ] or calibration of degrees of freedom [ 2 ] . In summary , based on replicability , Type I error , power and theoretical considerations , we recommend using the sorted runs sampling scheme with a t - test for comparing classifiers on a small data set . One would expect that replicability ceases to be an is - sue with larger data sets . In the future , we would like to perform larger scale experiments to get a better in - sight in the relation between replicability , the number of samples taken in an experiment and data set size . This should also give a better insight in the relation between replicability and Type I and II error . In this paper , we considered machine learning experi - ments in which we choose the best of two classifiers for a given data set . In practice , more than two classifiers are available . Also , machine learning researchers rou - tinely compare algorithms over a large number of data sets . This leads to new replicability issues and mul - tiple comparison problems , issues that require further research . Acknowledgements I would like to thank the Machine Learning Group of Waikato University for stimulating discussions and the anonymous reviewers for their helpful comments . A . Appendix Lemma A . 1 For 0 ≤ p ≤ 1 , and n ≥ 2 a positive integer , P ni = 0 p i ( 1 − p ) n − i ` ni ´ = 1 P ni = 0 p i ( 1 − p ) n − i ` ni ´ i = np P n i = 0 p i ( 1 − p ) n − i ` ni ´ i 2 = n 2 p 2 − np 2 + np Proof : ( sketch ) The first equation is the binomial theorem [ 4 ] . The second follows from the observation that the term in sum is zero for i = 0 , so the range of the sum can be changed to 1 ≤ i ≤ n . Using ` ni ´ = ` n − 1 i − 1 ´ ni for i > 0 we can absorb the i at the end of the sum , and taking p outside the term in the summation , we can apply the binomial theorem . The third equation follows from a similar line of reasoning . References [ 1 ] C . L . Blake and C . J . Merz . UCI Repository of machine learning databases . Irvine , CA : University of Califor - nia , 1998 . [ 2 ] R . R . Bouckaert . Choosing between two learning algo - rithms based on calibrated tests . ICML , 51 – 58 , 2003 . [ 3 ] T . G . Dietterich . Approximate Statistical Tests for Comparing Supervised Classification Learning Algo - rithms . Neural Computation , 10 ( 7 ) 1895 – 1924 , 1998 . [ 4 ] R . L . Graham , D . E . Knuth and O . Patashnik Concrete mathematics . Addison - Wesley , 1994 . [ 5 ] G . H . John and Pat Langley . Estimating Continuous Distributions in Bayesian Classifiers . UAI , 338 – 345 , 1995 . [ 6 ] C . Nadeau and Y . Bengio . Inference for the generaliza - tion error . Advances in Neural Information Processing Systems 12 , MIT Press , 2000 . [ 7 ] R . Quinlan . C4 . 5 : Programs for Machine Learning . Morgan Kaufmann Publishers , San Mateo , CA , 1993 . [ 8 ] S . Salzberg . On Comparing Classifiers : Pitfalls to Avoid and a Recommended Approach . Data Mining and Knowledge Discovery 1 : 3 , 317 - 327 , 1997 . [ 9 ] I . H . Witten and E . Frank . Data mining : Practical ma - chine learning tools and techniques with Java imple - mentations . Morgan Kaufmann , San Francisco , 2000 . \ No newline at end of file diff --git a/silver_data/59bf262df00850638c01740084952e82ce93af00.pdf.txt b/silver_data/59bf262df00850638c01740084952e82ce93af00.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..389130a371970d488ddebf006b48ccdff7798ea8 --- /dev/null +++ b/silver_data/59bf262df00850638c01740084952e82ce93af00.pdf.txt @@ -0,0 +1 @@ +Vol . : ( 0123456789 ) 1 3 https : / / doi . org / 10 . 1007 / s00216 - 021 - 03751 - 4 REVIEW Biochemistry strategies for label‑free optical sensor biofunctionalization : advances towards real applicability Maria Soler 1 · Laura M . Lechuga 1 Received : 15 September 2021 / Revised : 18 October 2021 / Accepted : 22 October 2021 © The Author ( s ) 2021 Abstract Label - free biosensors , and especially those based on optical transducers like plasmonic or silicon photonic systems , have positioned themselves as potential alternatives for rapid and highly sensitive clinical diagnostics , on - site environmental monitoring , and for quality control in foods or other industrial applications , among others . However , most of the biosensor technology has not yet been transferred and implemented in commercial products . Among the several causes behind that , a major challenge is the lack of standardized protocols for sensor biofunctionalization . In this review , we summarize the most common methodologies for sensor surface chemical modification and bioreceptor immobilization , discussing their advantages and limitations in terms of analytical sensitivity and selectivity , reproducibility , and versatility . Special focus is placed on the suggestions of innovative strategies towards antifouling and biomimetic functional coatings to boost the applicability and reliability of optical biosensors in clinics and biomedicine . Finally , a brief overview of research directions in the area of device integration , automation , and multiplexing will give a glimpse of the future perspectives for label - free optical biosensors . Keywords Surface plasmon resonance · Silicon photonics · Antibody immobilization · Biochemical cross - linking · Antifouling coating · Lipid membrane Abbreviations OEG Oligoethylene glycol pDOPA Poly ( dopamine ) PEG Polyethylene glycol pHEMA Poly ( hydroxyethylmethacrylate ) POC Point of care SAM Self - assembled monolayer SLB Supported lipid bilayer SPR Surface plasmon resonance Introduction Label - free optical biosensors are devices able to detect , quantify , and monitor the presence of analytes of interest with high sensitivity and specificity in just a few minutes of assay directly performed at the point of need [ 1 , 2 ] . Several optical biosensor nanotechnologies , either based on nano - plasmonics ( i . e . , surface plasmon resonance ( SPR ) biosen - sors ) or silicon nanophotonics ( e . g . , ring resonators , inter - ferometers ) , have been developed and even commercialized [ 3 – 6 ] . All these sensors rely on the same physical working mechanism , the evanescent field sensing principle [ 7 , 8 ] . To explain briefly , the evanescent field is an electromagnetic field generated at the interface between a surface where light propagates ( i . e . , plasmonic surface or waveguide ) and a die - lectric media ( Fig . 1a ) . It penetrates into the dielectric media with an exponentially decaying intensity , reaching up to sev - eral hundreds of nanometers ( 100 – 1000 nm ) . The evanes - cent field is extremely sensitive to minute changes of refrac - tive index occurring in the dielectric , such as those caused by a change of composition or change of mass on the surface of plasmonic or waveguide - based sensors . The refractive index changes induce a variation in the light propagation Published in the topical collection featuring Promising Early - Career ( Bio ) Analytical Researchers with guest editors Antje J . Baeumner , María C . Moreno - Bondi , Sabine Szunerits , and Qiuquan Wang . * Maria Soler maria . soler @ icn2 . cat 1 Nanobiosensors and Bioanalytical Applications Group ( NanoB2A ) , Catalan Institute of Nanoscience and Nanotechnology ( ICN2 ) , CSIC , BIST , and CIBER - BBN , Bellaterra , 08193 Barcelona , Spain / Published online : 4 November 2021 Analytical and Bioanalytical Chemistry ( 2022 ) 414 : 5071 – 5085 1 3 parameters , which can be readily measured through spectral peaks displacements , reflectance changes , or phase varia - tions , among others . Thereby , the evanescent field can serve as a probe to detect and analyze biomolecular interactions taking place onto the sensor surface , being monitored in real time and without the need for external tags or labels ( e . g . , fluorescent or colorimetric tags ) and providing a quantita - tive analysis . These capabilities have positioned label - free optical biosensors as potential technologies for point - of - care ( POC ) testing , with appealing applications in in vitro medical diagnosis , personalized therapy , or environmental control ( safety , pollution monitoring , etc . ) [ 9 – 12 ] . In fact , over the last few years , a significant number of publications are reporting label - free optical biosensors for early diagnosis of cancer and other diseases , rapid detection of infectious bacteria or viruses , analysis and monitoring of biomarkers or drug levels in patients during therapy , etc . [ 4 , 13 ] . The performance of these biosensors often proves excellent when compared to standard clinical diagnostics ( i . e . , enzyme - linked immunosorbent assays , ELISA , or polymerase chain Fig . 1 a Illustrative scheme of an optical biosensor system based on the evanescent field sensing mechanism : light is coupled to the transducer surface generating an electro - magnetic field that penetrates evanescently into the dielectric medium where the biological interaction takes place . The biointeraction changes the refractive index of the medium , which is translated in variations of certain optical properties of the output light ( intensity , wavelength , etc . ) . b Main opti - mization parameters in sensor surface biofunctionalization : bioreceptor orientation ( top ) , grafting density ( middle ) , and antifouling coating ( bottom ) 5072 Soler M . , L . M . Lechuga 1 3 reaction tests , PCR ) in small validation studies with patient samples . But still , the adoption of biosensors in clinical or environmental settings has not been accomplished . Among the several limitations , such as system automation or signal interpretation , an important challenge is the sensor biofunc - tionalization , which refers to the preparation and chemical modification of the sensor surface for attaching the specific bioreceptor and minimizing non - specific adsorptions [ 7 , 14 ] . The label - free analysis scheme of optical biosensors imposes critical requirements to the biorecognition inter - face , as main responsible for the reliability and accuracy of the assay ( Fig . 1b ) . Two aspects must be carefully studied and optimized : the selection of the biorecognition element , which provides the affinity and specificity for the target ana - lyte ; and its immobilization on the sensor surface . The bio - receptor immobilization strategy must ensure ( i ) a uniform coverage and proper biomolecule orientation , to maximize target accessibility and detectability ; ( ii ) stability and robust - ness , allowing flow - through assays or sequential measure - ment cycles ; and ( iii ) antifouling capabilities , to avoid or minimize the undesired binding of sample matrix compo - nents ( e . g . , proteins , lipids ) to the sensor surface , which can generate false positive signals . In this review , we provide an overview of the variety of sensor biorecognition interfaces developed and demonstrated in label - free optical biosensor technologies . After a brief analysis on the selection of the biorecognition element , we describe in detail the most suc - cessful and widely used surface chemistry and cross - linking procedures as well as innovative methodologies recently pro - posed to extend the applicability of biosensors , to improve its performance , or to boost the technology transfer , com - mercialization , and implementation in the clinical practice . Finally , current and future perspectives in this area are criti - cally discussed based on recent advances in nanotechnology , materials science , and bioengineering . Selection of the biorecognition element The most common and widely employed bioreceptors are antibodies . Antibodies can be produced towards virtually any type of molecule , including proteins , peptides , small organic molecules like drugs , and even large intact cells ( e . g . , bac - teria ) . Over the years , the antibody production process has been greatly optimized and improved , ensuring reliable and reproducible collections of high - affinity monoclonal antibod - ies but also generation of novel recombinant chimeric anti - bodies [ 15 ] or nanobodies [ 16 ] , which can be easily obtained at large scales with cost - effective procedures . To face certain limitations of antibodies , such as long - term stability , other rec - ognition molecules have been proposed as alternative . It is the case , for example , of molecularly imprinted polymers ( MIPs ) or aptamers . MIPs are synthetic polymers that are templated during production to selectively bind the target molecule by a “lock and key” mechanism [ 17 ] . They exhibit extreme robust - ness and can be obtained at a low cost , making them especially attractive for biosensors employed at remote locations or under harsh conditions , but their performance in terms of affinity and specificity usually is not sufficient for clinical or biomedical applications [ 18 ] . In recent years , a novel approach for nano - sized MIPs solid - phase synthesis has been proposed , showing superior affinities [ 19 ] . Although mainly applied in electro - chemical sensors , nanoMIPs could become a potential alterna - tive to antibodies also in optical biosensors [ 20 ] . Aptamers , on the other hand , are single - stranded oligonucleotides ( DNA or RNA ) with molecular recognition capabilities based on struc - tural folding [ 21 ] . They can be chemically synthesized with the desired sequence and functionalities , therefore avoiding animal immunization and cell culture procedures , and their affinity and specificity towards target analyte can be equivalent to monoclonal antibodies . However , the design and selection process for aptamer production can be long and tedious ( i . e . , SELEX procedure ) , and often results unsatisfactory for many types of molecules , especially small organic molecules [ 22 , 23 ] . On the other hand , genomic biomarkers such as micro - RNAs have arisen as promising indicators for early and highly accurate diagnostics . The detection of single - stranded nucleic acid molecules can be straightforward via direct hybridization assays , employing synthetic DNA probes with the comple - mentary sequence . Like aptamers , these DNA probes can be chemically synthesized and purified at relatively large scales and also can incorporate specific functionalities [ 24 ] . When addressing complex applications in genomic - based diagnos - tics , such as the analysis of epigenetic DNA markers or spe - cific DNA mutations , it might be necessary to capture dou - ble - stranded DNA . For that , a few bioengineering approaches have been proposed like the formation of triple helices ( i . e . , triplex ) using hairpin - shape DNA strands , which can be easily designed with bioinformatic tools and chemically synthesized [ 24 – 26 ] . Finally , it is worth mentioning that cell receptors can also be employed as biorecognition elements , particularly useful when targeting whole intact cells , like tumor cells or viruses , for example . These receptors are commonly produced as recombinant proteins and have been applied in biosen - sors for studying cell – cell or cell - pathogen interactions [ 27 ] . Table 1 summarizes the advantages and limitations of the main biorecognition elements commonly employed in label - free optical biosensors . Bioreceptor immobilization strategies The methodology for bioreceptor immobilization depends on the type of biorecognition element ( i . e . , protein , nucleic acid , etc . ) and its functionality , but also on the target analyte 5073 Biochemistry strategies for labelfree optical sensor biofunctionalization : advances towards… 1 3 ( e . g . , nature , dimensions ) , since it will eventually determine parameters such as the optimal density that minimizes steric hindrance effects during the interaction . Given the broad variety of biorecognition elements that can be used for bio - sensing , here we focus on the two main and most widely employed types : antibodies and oligonucleotides ( i . e . , single - stranded DNA probes and aptamers ) . In the follow - ing sections , we will present and discuss a wide variety of approaches to immobilize these bioreceptors onto sensor surfaces , analyzing their advantages and limitations in terms of simplicity and efficiency , uniformity and orientation , ver - satility , and robustness . Physical and chemical adsorption The simplest approach for sensor biofunctionalization is the physical adsorption , in which bioreceptors are attached directly to the sensor surface by electrostatic and hydro - phobic forces . This scheme is employed for microwell plate functionalization in ELISA , for example . But despite being rapid and straightforward , physisorption can lead to seri - ous reproducibility and stability problems in biosensors , and importantly , it may affect the recognition activity of the bioreceptor molecules , especially for antibodies that can be denaturalized in direct contact with metallic surfaces [ 28 ] . Another easy and widely used strategy is the direct chemisorption of bioreceptors , especially those carrying a thiol - functional group ( e . g . , thiolated DNA probes ) that can directly bind to gold sensor surfaces . This procedure is efficient and rapid , resulting in a stable receptor layer . To ensure layer uniformity and bioreceptor orientation , oligonu - cleotide probes can be synthesized incorporating a vertical spacer between the recognition sequence and the functional group , which can be either a 6 - or 12 - carbon chain [ 29 ] , or a poly - Thymine ( polyT ) sequence ( e . g . , polyT 10 or polyT 15 ) [ 30 ] . The vertical spacer aids in the upright positioning of the probe especially in gold - based sensors thanks to its low affinity for metallic surfaces , but it should be avoided when analyzing samples containing RNA polyA tails , which could bind to the polyT sequence and give rise to false positive sig - nals [ 31 ] . For the case of antibodies or other protein recep - tors , terminal Cysteine residues with reactive thiol groups could be employed for direct chemisorption on gold surfaces . Otherwise , an interesting strategy is to perform antibody fragmentation by enzyme digestion or chemical reduction , releasing sulfhydryl reactive groups that can be attached to the surface [ 32 , 33 ] . In all these approaches , adjustment of the bioreceptor grafting density is commonly done by add - ing competing thiolated molecules ( e . g . , mercaptohexanol , MCH ) to act as lateral spacers . This direct competition how - ever results in a relatively low control on the receptor graft - ing density , as their interaction with gold may be influenced by not only the initial concentration , but the molecular size , electrostatic interactions , etc . An interesting approach has been recently proposed in which the bioreceptor ( antibody or DNA probe ) is linked to a polyAdenine ( polyA ) tail , which Table 1 Advantages and limitations of common bioreceptors used in label - free optical biosensors Receptor Advantages Limitations Antibodies • High affinity and specificity • Well - established production process • Wide range of targets : protein , peptide , small mol - ecule , cells , etc • Long and complex production via animal immuni - zation • High production costs • Activity loss in long - term storage • Low reusability Bioengineered antibodies • High affinity and specificity • Wide range of targets : protein , peptide , small mol - ecule , cells , etc • No animal immunization required • Scalable and cost - effective production • Need to know the sequence , or find binding regions through phage display • Storage and reusability similar to conventional antibodies Cell receptors • High affinity and specificity • In vitro production • Sequence knowledge required • Storage and reusability similar or lower than anti - bodies • Limited range of targets Molecularly imprinted polymers • High robustness and stability • Scalable and cost - effective production • Moderate - low affinity and specificity • Limited range of targets DNA strands • High affinity and specificity • High stability and reusability • Versatile production via chemical synthesis • High production costs • Limited range of targets • Limited length subject to structural conformations Aptamers • High - moderate affinity and specificity • Production via chemical synthesis • Stability and long - term storage • Reusability • Long and complex selection procedure via SELEX • Moderate range of targets • Subject to structural conformations 5074 Soler M . , L . M . Lechuga 1 3 can be directly adsorbed on gold surfaces by high - affinity electrostatic interactions , and due to its flat positioning can act also as lateral spacer [ 34 , 35 ] . The polyA - based strategy has been demonstrated and evaluated in a SPR biosensor , showing comparable results to other widely used methodolo - gies , both in terms of analytical sensitivity and selectivity . Chemical cross‑linking to functional monolayers The sensor biofunctionalization procedures that have shown the best results in terms of robustness and versatility are those based on the formation of functional chemical mon - olayers . The sensor surface is chemically modified to pro - vide a homogeneous coating that extends up to 1 or 2 nm from the sensor surface , with terminal functional groups that can be used to tether the bioreceptors via covalent cross - linking or through high - affinity molecular systems . In this way , biorecognition elements are firmly attached to the sen - sor surface in a controlled and reproducible manner , can be slightly distanced from the surface , and it also allows to coat the entire sensor surface for preventing non - specific adsorptions . For gold plasmonic sensors , two major functional chem - ical scaffolds are used : dextran - based polymers and alka - nethiol self - assembled monolayers ( SAMs ) ( Fig . 2a ) . Func - tional polysaccharides , such as carboxymethylated dextran , are known to form highly stable , compact , and hydrophilic layers on gold surfaces , containing reactive groups ( e . g . , COOH ) that can be used for bioreceptor coupling [ 36 ] . This approach has become very popular especially for SPR bio - sensors , with dextran - modified sensor chips commercially available ( CM5 sensors , Biacore , GE ) . However , the random distribution of functional groups within the polymer layer might hamper the optimum and efficient cross - linking for bioreceptor binding . On the other hand , SAMs are formed by chemisorption of short - chain organic alkanethiols , i . e . , car - bon chains with terminal sulfur - reactive groups ( - SH ) [ 37 , 38 ] . These molecules generate a densely packed and highly ordered hydrophilic layer on the sensor surface , displaying functional reactive groups at the outer end of the scaffold , therefore being available for bioreceptor cross - linking . A myriad of different alkanethiols is commercially available , being relatively easy to synthesize them incorporating differ - ent chemical end functionalities , such as carboxyl ( COOH ) , amine ( NH 2 ) , and hydroxyl ( OH ) . Furthermore , SAMs can be formed as a mixture of different alkanethiols varying the reactive / inert group ratios of the monolayer , which offers the possibility to easily control and adjust the bioreceptor graft - ing density according to the target dimensions , minimizing steric hindrance issues . In silicon - based photonic biosensors , the most com - mon materials for sensor waveguide fabrication are either silicon oxide ( SiO 2 ) or silicon nitride ( Si 3 N 4 ) and chemical modification of these surfaces is generally done via silani - zation ( Fig . 2b ) [ 39 ] . Functional alkoxysilanes can form ordered and stable monolayers in silicon - derived substrates similar to alkanethiol SAMs . However , silanization is a more complex procedure than thiol chemisorption and there is currently no consensus on the optimum parameters to achieve a stable and homogeneous monolayer . The silaniza - tion process essentially consists in three main steps : surface activation , to release reactive hydroxyl groups ( - OH ) on the substrate ; silane binding , which can be done in liquid or vapor phase ; and finally , a curing step to cross - link the silane molecules and stabilize the monolayer . For this procedure , numerous protocols have been reported with different reac - tion times , temperatures , solvents , and even atmospheric conditions . The most common protocols employ amino - functional silanes , such as the ( 3 - aminopropyl ) triethox - ysilane ( APTES ) , which provide stable films through , for instance , vapor deposition at room temperature for 4 h [ 40 ] , or vapor deposition at 150 °C for 5 min [ 41 ] , or liquid depo - sition in anhydrous toluene [ 42 ] . These examples suggest that optimum silanization can either be achieved in different ways or it needs to be particularly and carefully studied for each specific material of the sensor surface . Moreover , as in the case of alkanethiols , different functional silanes can be employed for sensor surface modification ( e . g . , COOH - terminated silanes ) , and silanization procedure might be adjusted for each case . Once the functional scaffold is formed on the sensor sur - face , the subsequent step is the anchoring of the bioreceptor molecules , which is the most delicate and critical process to achieve the highest detection sensitivity and accuracy . To achieve a highly stable and robust bioreceptor tethering on the functional layer , the primary strategy is to chemically link their reactive functional groups forming a covalent bond ( Fig . 2c , d , e ) . This reaction generally requires the activation of certain groups and / or the use of cross - linkers . The car - boxyl - amine cross - linking is probably the most well - estab - lished strategy . Generally , this reaction is carried out through a well - known carbodiimide - based chemistry , employing a mixture of EDC ( 1 - ethyl - 3 - ( 3 - dimethylaminopropyl ) carbo - diimide ) and NHS ( N - hydroxysuccinimide ) as intermediate cross - linkers , and resulting in a highly stable amide bond between the biomolecule and the scaffold [ 43 ] . This reac - tion provides very good yields and good surface coverage with a high reproducibility . Another conventional approach is the amine – amine binding , for which several cross - linking methods can be employed [ 44 , 45 ] . The simplest is the use of glutaraldehyde , a small molecule with highly reactive car - bonyl groups ( - CHO ) that condense primary amines very efficiently via reductive amination or Mannich reactions . Homobifunctional NHS ester cross - linkers , like disuccin - imidyl suberate ( DSS ) or bis ( sulfosuccinimidyl ) suberate ( BS3 ) , are however the most popular ones , offering a better 5075 Biochemistry strategies for labelfree optical sensor biofunctionalization : advances towards… 1 3 control on the cross - linking reaction as well as the incor - poration of a spacer arm that can be customized in length . Other cross - linking strategies employed for primary amine coupling are based on epoxides , isocyanates , or imidoesters , among others , that results in either amide or amidine bonds , but with certain complexity in terms of side products , reac - tion time and conditions , etc . More sophisticated cross - link - ing reactions have also been studied and employed , like the click chemistry - based methods . Although several types of click reactions have been described , the most common one is the Cu - catalyzed azide - alkyne cycloaddition ( CuAAC ) that employs Cu I catalysis to efficiently couple azide groups to terminal alkynes ( Fig . 2e ) [ 46 ] . The advantages of click chemistry are its chemoselectivity , high yields , and no pro - duction of side products ; however , it usually requires previ - ous modification of the probe with an azide - functional tag . The versatility and robustness of covalent binding strat - egies have made them one of the most widely employed approaches in antibody immobilization for biosensing . How - ever , it does not offer any control over the orientation of the biomolecules , which is mandatory to maximize analyte detection capability . It is also important to carefully opti - mize the reaction parameters ( e . g . , pH , ionic strength , tem - perature ) to ensure maximum yields and efficiency , avoiding possible electrostatic repulsions between the monolayer and the bioreceptor . Affinity and linker‑mediated immobilization A proper orientation of the bioreceptors is essential for exposing the binding sites towards the analyte , and for achieving a uniformly distributed bioreceptor layer , which Fig . 2 a Schematics of chemical scaffolds for gold - based sen - sor functionalization : dextran - based polymeric layer ( e . g . , car - boxymethyl dextran ) ( left ) and mixed alkanethiol self - assembled monolayer ( e . g . , mercaptohexadecanoic acid / mercaptoundecanol , MHDA / MUOH ) ( right ) . b Schematics of chemical scaffold for sili - con - based sensor functionalization : alkoxysilane monolayer ( e . g . , 3 - aminopropyl ( triethoxysilane ) , APTES ) . c Schematics of different examples of cross - linking strategies for amine - functional biorecep - tors . d Schematics of different examples of cross - linking strategies for thiol - functional bioreceptors . e Schematics of cross - linking strategy based on click chemistry : copper ( I ) - catalyzed alkyne - azyde cycload - dition ( CuAAC ) . Antibodies and DNA strands are only used for illus - trative purposes ; any receptor carrying the desired functionality could be employed indistinctly 5076 Soler M . , L . M . Lechuga 1 3 would ensure higher recognition efficiency , selectivity , and reproducibility . This is especially relevant for antibodies , which cannot be custom synthesized and therefore require certain biochemical strategies to be immobilized in a suit - able orientation ( Fig . 3 ) . The classic example is the Protein A or Protein G approach [ 47 ] . These two proteins—and the recombinant combination of both ( Protein A / G ) —are able to selectively capture different types of immunoglobulins through their constant region without the need for chemical manipulation of the antibodies . The affinity protein can be covalently linked to the sensor surface monolayer , and anti - bodies are captured on them , resulting in a sterically acces - sible and perfectly orientated recognition interface ( Fig . 3a ) . However , the interaction between protein A / G and antibod - ies is not particularly strong and can be disrupted with a change of pH , which limits the stability of the biolayer and the use of the biosensors for sequential measurement cycles . Another common strategy employs the biotin - avidin sys - tem . These two molecules showed one of the highest affinity interactions known in biology ( k d ≈ 10 −14 mol / L ) ; thereby , the binding of a biotinylated - Ab to an avidin layer results in a highly stable recognition interface ( Fig . 3b ) [ 48 ] . If anti - bodies are conjugated to the biotin tag through a site - specific procedure ( i . e . , through the carbohydrate moieties of the Fc region ) , the immobilization will also be oriented . Another interesting approach would be the DNA - directed immobili - zation . It consists in conjugating the antibody to a DNA oli - gonucleotide strand—ideally through the constant region— and anchor it to the sensor surface via hybridization with the complementary sequence probe , previously immobilized on the sensor surface ( Fig . 3c ) [ 49 ] . These strategies provide oriented and stable recognition interfaces , but as a drawback , they require chemical modi - fication of antibodies for tag conjugation . In this regard , an innovative methodology was reported a few years ago based on calixarenes ( e . g . , ProLinker™ ) [ 50 ] . Monolayers formed with calixarene crown ethers on the sensor surface can serve as scaffolds to directly immobilize intact antibodies through their Fc region with a high stability and in a simple and rapid procedure ( Fig . 3d ) . Nonetheless , this line has not been further investigated in recent years because the commercial product was discontinued . The design and synthesis of novel similar calixarenes could lead to a promising strategy for oriented antibody immobilization . Finally , another attractive trend is to use genetic bioengineering procedures to directly produce recombinant Fab fragments , which can be tagged Fig . 3 Antibody orientation strategies . a Affinity - based immobiliza - tion of intact antibodies on Protein A / G . b Affinity - based immobiliza - tion of biotinylated antibodies on avidin protein ( e . g . , streptavidin or neutravidin ) . c Affinity - based immobilization through DNA hybridi - zation . d Immobilization of intact antibodies on a calixarene - based linker ( e . g . , Prolinker™ ) . e Immobilization of recombinant antibody fragments , i . e . , antigen - binding region ( Fab ) 5077 Biochemistry strategies for labelfree optical sensor biofunctionalization : advances towards… 1 3 with histidine or cysteine residues or fused to larger proteins that aid in the immobilization ( Fig . 3e ) [ 51 ] . This strategy has also been combined with other approaches , like biotin - avidin system or protein - binding formats . All over , a vast library of bioreceptor immobilization strategies has been proposed , studied , and employed in label - free optical biosensors . In order to achieve maxi - mum functionality , binding capacity , and assay reliability , the attachment procedure must ensure the correct antibody orientation together with an optimum surface coverage and interface stability . Comparative studies of different immobi - lization strategies reveal that oriented receptor layers provide a higher sensitivity and binding efficiency while random ori - entation approaches are simpler , faster , and provide higher density of receptors . Eventually , the selection of the most appropriate strategy will depend on the selected application of the biosensor and its performance requirements . Towards an antifouling surface Despite all the efforts in accomplishing stable , robust , uni - form , and oriented biorecognition interfaces , there is still an unresolved challenge : eliminating non - specific adsorp - tions from sample matrix components ( e . g . , proteins , lipids , cells ) . This undesired adsorption is mainly due to long - range electrostatic and Van der Waals interactions . Most efficient mechanisms to minimize it are directed to further increase the surface hydrophilicity , generating a hydration layer ( i . e . , organized water molecules layer at the sensor coating ) , and / or alter the effective surface charges of the chemical scaffold formed on the sensor surface to reduce the ionic interactions ( Fig . 4 ) . A popular approach is to form functional monolayers incorporating oligoethylene glycol ( OEG ) moieties ( Fig . 4a ) . OEG - terminated SAMs offer a high hydrophilicity and lat - eral packing density that increases resistance to protein foul - ing [ 52 , 53 ] , which have been successfully used to minimize adsorption of proteins from blood - related fluids , but insuf - ficient to completely avoid it when dealing with undiluted blood , plasma , or serum samples ( Table 2 ) [ 54 , 58 ] . It has been shown that the antifouling capacity improves with the number of EG molecules in the SAM ; therefore , surface coatings with different polyethylene glycol ( PEG ) com - pounds have also been candidates for functional low - fouling scaffolds [ 54 , 55 ] . Commonly used compounds include lin - ear PEGs , such as silane - PEG - R or thiol - PEG - R ( where R is a functional reactive group , e . g . , COOH , NH 2 ) for silicon and gold surfaces [ 59 , 60 ] , respectively , and also polymer brushes containing PEG chains grafted to a functional back - bone , such as poly ( L - lysine ) ( PLL - PEG ) [ 61 ] . Grafted poly - mers have proven higher resistance to protein fouling ; how - ever , if grafting density and position are not well controlled , these long - chain PEGylated compounds can form disordered scaffolds where the reactive functional group for bioreceptor immobilization becomes hindered [ 59 , 62 ] . Another effective antifouling functionalization strategy is the formation of zwitterionic layers ( Fig . 4b ) . This coating Fig . 4 Antifouling strategies . a Formation of a hydration layer with hydrophilic compounds , e . g . , incorporating polyethylene glycol or oligoethylene glycol moieties . b Formation of an effective charge - balanced layer with zwitterionic compounds Table 2 Adsorption from protein solutions and undiluted blood plasma on different surface coatings * Measurements performed by SPR and / or ellipsometry Coating Fouling ( pg / mm 2 ) * Ref Fibrinogen HSA Blood plasma OEG 2 300 105 1500 – 2500 [ 54 , 55 ] OEG 6 36 0 500 [ 54 , 55 ] PEG - SAM 30 0 900 [ 55 ] pDOPA 0 - 63 [ 56 ] pHEMA 0 0 30 – 35 [ 57 ] poly ( β - peptoid ) 0 2 97 [ 57 ] 5078 Soler M . , L . M . Lechuga 1 3 is characterized by containing both cationic and anionic groups that provide an effective charge balance on the sur - face , reducing adsorptions of proteins through electrostatic interactions [ 63 ] . Zwitterionic interfaces can attract water molecules via hydrogen bonds and ionic solvation , thereby more strongly than just hydrophilic compounds . Most com - mon examples of zwitterionic monolayers are terminated in carboxybetaine ( CB ) or phosphorylcholine ( PC ) groups [ 64 , 65 ] . These zwitterionic coatings have shown an excel - lent reduction of blood - related proteins , but again they failed when challenged to undiluted plasma or other media . Simi - lar to PEG / OEG compounds , the antifouling capacity can be improved grafting zwitterionic polymer brushes , such as the poly ( carboxybetaine methacrylate ) ( pCBMA ) , which generates functionalizable interfaces with ultralow fouling properties [ 66 – 68 ] . The drawback here is that during the preparation of these polymer brushes , there is a lack of con - trol on the thickness that could extend up to several hundreds of nanometers , distancing the target detection interaction from the sensor , where the evanescent field is most sensitive . Besides PEG / OEG and zwitterionic compounds , other polymers have also been studied as antifouling scaffolds for biosensors such as polydopamine ( pDOPA ) [ 69 – 71 ] , polyacrylamide [ 72 ] or poly - ( hydroxyethyl methacrylate ) brushes ( pHEMA ) [ 73 ] , or the more recently proposed , poly ( β - peptoid ) s [ 74 , 75 ] . Peptoids are structural isomers of peptides that differ in the linking position of the func - tional sidechains , which are located in the α - nitrogen instead of the α - carbon of the backbone . This sidechain rearrange - ment increases the conformation flexibility and hydration capacity , making them ideal biomimetic compounds to form hydrophilic brushes with elevated fouling resistance . All these strategies have shown very promising results towards total resistance to protein and cell adsorptions ( Table 2 ) ; however , the bioreceptor cross - linking onto these polymer brushes is not well controlled , which limits the final sensitiv - ity of the biosensor . More studies in the design of new functional and antifoul - ing surfaces are required to allow the desired application and implementation of optical label - free biosensors for direct and reliable analysis of untreated clinical samples like blood . Many research works have studied and reported the low - fouling behavior of different materials and surface chem - istry strategies , mainly analyzing the adsorption of serum proteins like albumin , fibrinogen , or immunoglobulins , to gold sensors ( i . e . , SPR ) . However , in most cases , the fouling resistance properties have resulted insufficient when dealing with whole blood plasma samples , or when comparing dif - ferent pooled plasma or clinical specimens from different individuals . On the other hand , it has been shown that rela - tively large coatings and polymer brushes offer the highest fouling resistance but paying little attention to the hindering of bioreceptor coupling or the biointeraction distancing from the sensor surface , which would decrease the overall ana - lytical sensitivity for biosensor application . In view of this , on - going research is directed to develop innovative grafting methods ( in - solution or on - chip ) that provide a precise con - trol of the layer structure , grafting density , polymer length , etc . , and ensure highly stable attachment to the sensor sur - face [ 76 – 78 ] . Towards a biomimetic microenvironment The sensor biofunctionalization approaches described and studied so far have been mostly applied for the detection and quantification of specific molecular targets ( i . e . , proteins , nucleic acids , pathogens , etc . ) with medical or environmen - tal diagnostic purposes , essentially . Label - free optical bio - sensors , however , can also monitor the biomolecular inter - actions in real time and in their native forms ( i . e . , without fluorescent tags ) , and this capability makes them greatly useful for pharmacokinetic analysis or fundamental biol - ogy studies , such as drug - receptor interactions or cell – cell interaction , for example . In this regard , surface bioengineer - ing strategies have been described in order to provide bio - mimetic scaffolds on the sensor surface that can simulate natural biological systems , in which membrane proteins and receptors can maintain their tertiary structure and flexibility for movement , clustering , etc . Lipid membrane interfaces offer a potential solution to this challenge . Three - dimensional ( 3D ) or bidimensional ( 2D ) lipid - based structures can be deposited , arrayed , or directly grown on sensor surfaces through versatile biochem - ical engineering procedures starting from relatively low - cost phospholipid molecules , which can be synthesized with the desired properties and functionalities . Mainly , two different lipid - based approaches have been studied for optical sen - sor biofunctionalization : liposome arrays and planar lipid bilayers ( Fig . 5a and b ) . Liposomes of different sizes can be formed in an aqueous solution by spontaneous self - assem - bling of phospholipid molecules , like phosphocholine and its derivatives [ 79 ] . The hydrophilic head of the phospho - lipid can be functionalized to incorporate chemical groups , tags , or linkers to facilitate their immobilization onto the sensor surface . The most common strategies for liposome tethering on the sensor surface are based on electrostatic or covalent interactions [ 80 , 81 ] , the biotin - avidin system [ 82 , 83 ] , or by DNA hybridization ( Fig . 5c ) [ 84 , 85 ] . The latter is especially interesting for high - throughput multiplex - ing array sensors ; different liposomes can be conjugated to different oligonucleotide sequences that will hybridize to their corresponding complementary sequence previously arrayed on the sensor surface [ 86 ] . Major challenges in 3D liposome array formation are closely related to surface antifouling properties and non - specific adsorptions . The 5079 Biochemistry strategies for labelfree optical sensor biofunctionalization : advances towards… 1 3 sensor surface must be completely passivated before lipo - some tethering to ensure precise site - selective attachment , to retain the liposome structure , and to avoid the non - specific binding of receptors or targets directly onto the sensor sur - face [ 87 ] . Upon liposome immobilization , the bioreceptor is either inserted or attached to the liposome membrane . As stated before , bioreceptor attachment to the lipid external surface can be done via covalent cross - linking methods or other linker - mediated systems as described previously in “Bioreceptor immobilization strategies . ” Nonetheless , when studying drug - cell interactions or ion channel biology , bio - receptors generally are intramembrane proteins that must be inserted in their native conformation . For that , two main approaches can work : using native or synthetic liposomes . Native liposomes can be extracted from cells by techniques as solvent - assistant extraction , sonication , extrusion , freeze / thaw processes , etc . [ 88 , 89 ] . These native liposomes would exactly mimic the native cell environment , in terms of fluidity and lipid order , but they offer poor control on the amount and type of receptors , and other membrane proteins and components , that can be eventually immobilized on the sensor surface . Conveniently , synthetic liposomes can be formed by directly mixing the bioreceptor molecule with the phospholipid mixture before rehydration [ 90 ] . The use of purified compounds eliminates possible interfering mol - ecules ( e . g . , other membrane proteins ) as well as to easily modify the number of receptors per liposome according to the sensor performance requirements or assay parameters . Moreover , liposomes reconstituted from pure components are most likely to have a higher stability for in vitro studies than native liposomes , which could be degraded by enzymes during preparation . As an alternative to liposome arrays , planar lipid mem - branes can also be assembled on the sensor to mimic cell surfaces . These thin - film coatings formed by phospholipid bilayers are commonly not chemically attached but sup - ported on the sensor surface [ 91 ] . Supported lipid bilayers ( SBL ) are formed mostly by the vesicle fusion method that consists in the spontaneous rupture of small lipid vesicles in contact with the solid surface and subsequent re - assembly in a planar bilayer architecture [ 92 , 93 ] . A necessary condi - tion for the vesicle rupture and fusion process to happen is to have a highly hydrophilic surface that attracts phospho - lipid heads and repeals their hydrophobic tails , aiding in the Fig . 5 Biomimetic lipid - based biofunctionalization . a Schematic illustration of liposome - based bioreceptor immobilization . b Sche - matic illustration of bioreceptor immobilization on a planar lipid bilayer . c Representative examples of liposome tethering strategies : electrostatic interactions ( left ) , biotin / avidin system ( middle ) , and DNA - directed ( right ) . d Representative examples of planar lipid bilayer formation strategies : supported lipid bilayer ( SLB ) on hydro - philic substrate ( i . e . , glass ) ( left ) , SLB on hydrophilic coating on gold surface ( middle ) , and hybrid lipid bilayer formed by hydrophobic SAM coating on gold and single lipid layer ( right ) 5080 Soler M . , L . M . Lechuga 1 3 vesicle disruption and bilayer formation ( Fig . 5d ) . Silicon - based sensor surfaces , and especially glass , have been suc - cessfully used for SBL formation ; however , manifold factors and parameters must be carefully controlled , including lipid properties ( e . g . , vesicle size , composition , and concentra - tion ) , surface properties ( e . g . , morphology , topography , and atomic composition ) , and environmental properties ( e . g . , temperature , ionic strength , buffer composition , pH ) [ 94 ] . Formation of SLB on gold surfaces results in even more complicated , as generally vesicles adsorb on the surface and remain intact mainly due to the loss of hydrophilic behavior . Some strategies have been developed to aid in this purpose , such as the coating of gold sensors with highly hydrophilic material ( e . g . , PEGylated polymer brushes ) , or the forma - tion of hybrid bilayers , in which gold is functionalized with a hydrophobic SAM that mimics the phospholipid tail , and vesicles disrupt to form a single layer on top of the SAM ( Fig . 5d ) [ 27 ] . Overall , lipid - based biofunctionalization strategies have been widely studied and a few innovative mechanisms for liposome arraying or planar bilayer formation have been proposed . It is the case , for instance , of liposome networks ( i . e . , liposome arrays interconnected through lipid nanotubes ) [ 95 ] , solvent - assistant on - chip SLB formation [ 96 ] , or patterning of SLB patches via microcontact printing [ 97 ] , among others . Nonetheless , the appli - cation of lipid bioengineered surfaces in label - free optical bio - sensing is still in its infancy and unfortunately far from industrial scalability and potential technology transfer [ 98 ] . Conclusions and future directions An optimum sensor biofunctionalization could be the key to accelerate the technology transfer and definitive implemen - tation of powerful optical label - free biosensors in clinical and environmental analysis practices . An endless number of surface coatings , cross - linking strategies , and innovative biochemical procedures have been proposed to achieve a fully reliable and reproducible protocol for immobilizing bioreceptors in an oriented and controlled manner , pursuing maximum assay sensitivity and selectivity . Among them , the covalent binding of bioreceptors to functional chemical matrices formed onto the sensor surface is the most popular and versatile approach . Self - assembled monolayers of alka - nethiols on gold or alkoxysilanes on silicon - based sensors provide organized scaffolds for biomolecule immobilization and can be easily adapted to modify the grafting density , to add affinity tags or linkers , and to increase fouling resist - ance . The latter is , in fact , the major challenge for biosensor application in point - of - care testing . It has been shown that generating a hydration layer on the coating through incor - poration of hydrophilic moieties ( e . g . , ethylene glycol ) is necessary for reducing non - specific protein adsorptions , but so far insufficient for completely avoiding fouling from undiluted media . Research in the field proposes new alterna - tives based on polymeric materials and combinations with zwitterionic compounds that alter the effective net charge of the sensor surface to prevent as well undesired electro - static interactions . However , the large variability found in real samples composition is still a handicap to success - fully eliminate sample pretreatment processes , like dilution or filtration . Current perspectives in this area rely on the controlled growth of novel materials with desired chemical composition and distribution of functional groups to accom - plish highly ordered scaffolds , with enhanced fouling resist - ance properties , that ensure an efficient , simple , and reliable immobilization of all types of bioreceptors . On the other hand , innovation in surface biofunctionaliza - tion could also expand the applicability of optical biosensors in pharmaceutics and molecular biology studies . By exploit - ing the label - free and real - time monitoring capabilities , these systems can greatly aid in the evaluation of novel drugs , the understanding of cell interactions , etc . To that , several strategies based on lipid bilayer coatings have been suggested to create a biomimetic microenvironment onto the sensor surface in which the bioreceptors behave as they are found in native conditions , increasing the accuracy and reliability of the biological assays . These bioengineered surfaces have shown promising results in model studies , but the functionalization procedure is delicate and complex , requiring sophisticated biochemical techniques that hamper the scalability and adoption in routine experiments . Here , the integration of lab - on - a - chip microfluidic systems that facilitate in - situ preparation or process automation would sup - pose an enormous boost for the demonstration and implementa - tion of novel biomimetic sensors . It is important to remark that surface chemistry cannot move forward without the accompaniment of technology and materials development . Potential final users of optical label - free biosensors expect automated and user - friendly systems able to detect and quantify several target analytes of interest in a sample , in a few minutes , with maximum sensitivity , and at affordable prices . Sensors should be delivered ready to use , which implies the industrialization of the biofunc - tionalization procedure plus packaging methods that protect and ensure the stability and biological activity of the coating and immobilized receptors . The multiplexing capability is nowadays thought of as an engineering challenge , but also surface biofunctionalization protocols must be prepared considering an extreme reduction of reaction volumes or the need of incorporating different functional groups for site - selective immobilization of different receptors . Over - all , it becomes clear that despite the vast knowledge and the myriad of strategies developed along the last years for sensor biofunctionalization , there is still a long research way ahead in which nanotechnology , biotechnology , and materials sci - ences might play the leading role . 5081 Biochemistry strategies for labelfree optical sensor biofunctionalization : advances towards… 1 3 Funding Financial support is acknowledged from the SensCELL pro - ject ( Ref . No . PGC - 2018 – 099870 ) , funded by the Spanish Ministry of Science and Innovation , the Spanish Research Agency ( AEI ) , and the European Regional Development Fund ( ERDF ) . The ICN2 is funded by the CERCA program / Generalitat de Catalunya . The ICN2 is supported by the Severo Ochoa Centers of Excellence program , funded by AEI ( Grant No . SEV - 2017 – 0706 ) . Declarations Conflict of interest The authors declare no competing interests . 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Publisher ' s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations . 5085 Biochemistry strategies for labelfree optical sensor biofunctionalization : advances towards… \ No newline at end of file diff --git a/silver_data/5b317da1e683e80d892c6e596cf2a3b0a502fbb7.pdf.txt b/silver_data/5b317da1e683e80d892c6e596cf2a3b0a502fbb7.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d0c1855bca8b096788a64c0a0e85fd7a483f27c9 --- /dev/null +++ b/silver_data/5b317da1e683e80d892c6e596cf2a3b0a502fbb7.pdf.txt @@ -0,0 +1 @@ +PATTERNS OF BELIEF AND PATTERNED THOUGHT : RELATIONSHIPS BETWEEN RELIGIOUS FUNDAMENTALISM AND COGNITIVE RESTRUCTURING A Senior Honors Thesis By DANIEL CONOR SEYLE Submitted to the Office of Honors Progratus k . Academic Scholarships Texas ARM University In partial fulfillment of the requirements of the UNIVERSITY UNDERGRADUATE RESEARCH FELLOWS April 2000 Group : Psychology 2 PATTERNS OF BELIEF AND PATTERNED THOUGHT : RELATIONSHIPS BETWEEN RELIGIOUS FUNDAMENTALISM AND COGNITIVE RESTRUCTURING A Senior Honors Thesis By DANIEL CONOR SEYLE Submitted to the Office of Honors Programs & Academic Scholarships Texas A & M University In partial fulfillment of the requirements For the Designation of UNIVERSITY UNDERGRADUATE RESEARCH FELLOW Approved as to style and content by Steven Smith ( Fellows Advisor ) Edward A . Funkhouser ( Executive Director ) April 2000 Group : Psychology 2 ABSTRACT Patterns of Belief and Patterned Thought : Relationships Between Religious Fundamentalism and Cognitive Restructuring . ( April 2000 ) Daniel Conor Seyle Department of Psychology Texas A & M University Fellows Advisor : Dr . Steven Smith Department of Psychology Previous research on religious fundamentalism has focused on correlating fundamentalism with a number of personality variables . Religious fundamentalism has been associated with low religious quest , high right - wing authoritarianism , prejudice , and authoritarian styles of child raising . Research on cognitive variables associated with religious fundamentalism has shown that it is associated with reduced cognitive complexity and lower complexity of thinking in problem solving . The overall view which has developed is one of religious fundamentalism as a very rigid structure of belief which emphasizes traditional interpretations and ways of viewing the world . It was the hypothesis of this study that this structure of belief would interfere in the ability to solve cognitive restructuring or insight problems , as these problems require flexibility in mental representation in order to be solved . Forty - four subjects were recruited from the Psychology 107 Subject Pool and given the Altemeyer - Hunsberger religious fundamentalism scale and 10 cognitive restructuring problems . Analysis of the results using a Pearson ' s R show no significant results ( r = . 38 ) . However , when graphed the data show interesting patterns of unifomily high scores in cognitive restructuring in those who scored low in religious fundamentalism , and very high variation in restructuring scores in those who scored high in religious fundamentalism . Possible reasons for this are addressed , and directions for future research are suggested . ACKNOWLEDGEMENTS I would like to gratefully acknowledge Dr . Steven Smith for his patient help and support . TABLE OF CONTENTS ABSTRACT . Page ACNOWLEDGEMENTS . TABLE OF CONTENTS . VI INTRODUCTION . METHODS . RESULTS . DISCUSSION . . 10 CONCLUSION . REFERENCES . APPENDIX A . APPENDIX B . VITA . . 13 14 16 20 22 INTRODUCTION When anthropologists and psychologists first began to look at religious behavior in a scientific manner , the phenomenon of religious fundamentalism attracted attention . As anyone reading the newspaper can attest to , religious fundamentalism is something which can drive people to behaviors that are sometimes monstrous , sometimes beatific , and often inexplicable . As scientists began to examine this phenomenon , the immediate question raised was : is religious fundamentalism simply an extreme of religiosity ? That is , is religious fundamentalism an emergent property of simply believing very strongly in a religion , or is there something qualitatively different about fundamentalism that distinguishes it from simply an extreme of belief . Interestingly enough , both anthropologists ( e . g . Klass 1995 ) and psychologists ( e . g . Altemeyer & Hunsberger 1992 ) examining this question have come to the conclusion that there is something significant about religious fundamentalism which distinguishes it from a simple extreme of belief ( although religious fundamentalism does presuppose extreme religiosity ) . Both fields have come to similar definitions of religious fundamentalism , lending strong support to the conclusion that it is something distinguishable from other characteristics of religiosity such as strength of belief or even religious orthodoxy ( Kirkpatrick 1993 ) . The psychological definition of religious fundamentalism , as stated by Altemeyer and Hunsberger ( 1992 ) is : [ Religious fundamentalism is ] a structure of belief in which the devout believe that there is one set of religious practices handed down by a higher power in one set of religious teachings which contain the entirety of correct religious This thesis follows the style and format of the Publication Manual of the American Psychological Association . knowledge , that this truth must be followed according to past traditions , and that the followers of this teaching have a special relationship with that higher power . This definition is applicable cross - culturally . The phenomenon of religious fundamentalism is not specific to any one religion , and in has in fact been shown to have similar characteristics across religions ( Hunsberger 1996 ) . Because , as the definition states , religious fundamentalism is primarily a structure of belief with associated specific beliefs rather than a property inherent in any one set of religious beliefs , it can arise in any religion . That is , it is not so much what the devout believe that characterizes religious fundamentalism as the inflexible manner in which they believe it . Researchers have looked at many of the personality variables associated with religious fundamentalism . One of the most robust findings has been the correlation between religious fundamentalism and prejudice ( e . g . Kirkpatrick 1993 , Hunsberger 1995 ) . While there is a link between fundamentalism and racial prejudice , the strongest findings have been in the strong association of religious fundamentalism and antihomosexual prejudice ( Maret 1984 , Jackson & Esses 1997 ) . Although this issue is confounded by the fact that many religions place a moral ban against homosexuality , the prejudice felt by people high in religious fundamentalism has been shown to be in excess of expected moral outrage ( Fulton , Gorsuch , & Maynard 1999 ) . Religious fundamentalism has also been closely associated with high right - wing authoritarianism ( Altemeyer & Hunsberger 1992 , Wylie & Forest 1992 ) , a style of political belief which is similar to religious fundamentalism in its inflexibility and dismissal of alternative points of view . The association of fundamentalism and right wing authoritarianism has also been shown to be correlated with authoritarian styles of child rearing ( Danso , Hunsberger , & Pratt 1997 ) . The overall picture which is drawn of individuals high in religious fundamentalism is one of very patterned individuals who have a reduced interest in viewpoints differing from their own . When speaking of religious matters , this is not especially significant ; a part of fundamentalist belief is the belief that their religion is the whole truth , and that all other religious information is either incorrect or unnecessary . What is significant is that this same pattern of inflexibility is shown in other aspects : the prejudice against homosexuality goes beyond simple disapproval on moral grounds and into a rejection of something which the fundamentalist person sees as inherently different . In fact , an experiment which examined how two groups of people in the same congregation identified as high or low in religious fundamentalism compared on the NEO - Five Factor Personality Inventory showed similar scores across most factors but a large difference in the dimension of openness to new experiences . People high in religious fundamentalism were much less open to new experiences and novel ideas , not just in a religious context but in general ( Streyffeler & McNally 1998k This is fairly significant , as it shows that religious fundamentalism is not just an independent dimension of personality , but one which is correlated with other personality variables . The next question which was raised was if religious fundamentalism could be correlated with other less dispositional and more cognitive variables . Research on religious doubt suggested that people high in religious fundamentalism may be less likely to think in complex ways about religious matters and to engage in less critical thinking ( Hunsberger et . al 1996 ) . Further experiments examining the relationship between religious fundamentalism and cognitive complexity showed that in fact high religious fundamentalism was a reliable predictor of low cognitive complexity ( Edgington & Hutchinson 1990 ) . This fits with the earlier picture of people high in religious fundamentalism as it is consistent with the idea that a highly religiously fundamental individual is less likely to look for novel interpretations or complex solutions to problems . A finding which runs slightly contrary to this is Pancer et . al ' s ( 1995 ) study which showed that religious orthodoxy was not strongly associated with lowered complexity of thinking except with regard to religious issues . However , this study only looked at religious orthodoxy , a variable of religiosity associated with but not identical to religious fundamentalism . These characteristics ( low openness to novel ideas and interpretations and a preference for simplicity of thought in problem solving ) should indicate that people high in religious fundamentalism perform poorly on tasks which require the generation of novel ideas or tasks in which the simple interpretation of the problem will result in no solution . One set of tasks which fit this definition are restructuring or insight problems . Restructuring problems are so named because the solution depends on the ability to reconceptualize the problem , drawing the solution not from a logical process from A to B to C , but from a change in the problem space itself ( Dominowski 1995 ) . That is , they are designed so that when read , they draw upon implicit ( and incorrect ) interpretations of word meaning to create a representation of the problem in it cannot be solved . In order to solve them , it is necessary to first of all identify the initial representation as unworkable , recognize the implicit assumptions that were made , discard those assumptions , and create new interpretations which allow for a new representation in which the answer is easily found . For example , a standard restructuring task ( Dominowski 1995 ) is : " Calendars made in England do not show Lincoln ' s Birthday . Do they show the Fourth of July ? " The initial interpretation generated when this question is read often yields the incorrect answer " no . " To solve this problem correctly , it is necessary to identify the misinterpretation of " Fourth of July " as not only an American holiday but a calendar date as well . The reliance of high religious fundamentalism individuals on traditional interpretations and simple problem solving techniques would seem to make this kind of task very difficult , as the identification and discarding of implicit assumptions which is necessary to solve the problem require processes not usually associated with high religious fundamentalism . In fact , Smith , Ward , and Schumacher ( 1993 ) found that an inability to overcome deeply entrenched ideas about the shape of animals ( something which should be very evident in high religious fundamentalism individuals ) had a strong constraining effect on open - ended creative tasks . It is therefore thc hypothesis of this study that people high in religious fundamentalism will be less likely to be able to perfoim tasks requiring cognitive restructuring , as they will be less able to identify and discard implicit assumptions , and as a result , high scores on a religious fundamentalism scale will be inversely correlated v : ith performance on cognitive restructuring tasks . METHODS ~ P ' t t Participants ( N = 44 ) were recruited from the Texas A & M Psychology 107 Subject Pool . Participants were male and female undergraduates enrolled in an Introductory Psychology course who received course credit for participating . They reflected the ethnic makeup of Texas A & M ( primarily Caucasian with several African American participants ) . Materials and rocedure All participants were given the Altemeyer - Hunsberger Religious Fundamentalism Scale , an accepted measure of religious fundamentalism ( Altemeyer & Hunsberger 1992 ) . This is a Likert style self - report questionnaire composed of 20 statements scored in agreement from 1 ( completely disagree ) to 8 ( completely agree ) , giving a total composite score for religious fundamentalism ranging from 20 to 160 . Ten items are scored in reverse to avoid bias from response sets . The religious fundamentalism scale was embedded in a locus - of - control scale in order to partially disguise the focus of the experiment and help keep the responses unbiased ( see Appendix 1 ) . Participants were also given 10 cognitive restructuring tasks , including 8 verbal and 2 nonverbal tasks ( see Appendix 2 ) . Responses were identified as either exhibiting or not exhibiting restructuring , giving a score for restructuring ranging from 0 to 10 . The order of presentation of the RFS and restructuring tasks was counterbalanced , with 20 participants receiving the RFS first , and 24 receiving the restructuring tasks first . RESULTS A Pearson ' s R was generated to calculate the correlation between the scores for religious fundamentalism and restructuring . No significant correlation was found ( R = - . 046 , r = . 38 ) . There was no strong linear relationship found between religious fundamentalism and restructuring ability . There is a range limitation in that there were no individuals in the sample who scored at the extreme highs of religious fundamentalism . The highest score on the religious fundamentalism scale was l 4l , which , while high , is not comparable in degree of response to the lowest score of 26 . When the data are graphed in a dispersion pattern some patterns become visible ( see Table 1 ) . The majority of the responses fall in the middle of both religious fundamentalism and restructuring ability , accounting for the null result . However , there is very low variability in restructuring scores for those participants responding at the lowest levels of religious fundamentalism . Restructuring scores for low religious fundamentalism individuals were uniformly high . The opposite was found at the highest levels of religious fundamentalism , with a large degree of variability in restructuring found . Both the highest and the lowest scores for restructuring were found in the highest responses for religious fundamentalism . TABLE 1 Dispersion Pattern 10 4 a u n C4 0 V OX C 0 5 0 cs CJ L 0 I IV a 9 8 7 6 5 4 2 I 0 fn lf f 20 40 60 80 100 120 ] 40 160 Religious fundamentalism DISCUSSION The results of this study are difficult to apply directly to the hypothesis of the study , as the study was primarily interested in individuals very high in religious fundamentalism , and there were none in the sample . However , with the results that were found , the hypothesis was not supported . Some individuals high in religious fundamentalism were also very high in restructuring ability . These findings support those of Pancer et . al . ' s ( 1995 ) study . It is a possibility that the scales used did not accurately measure either religious fundamentalism or cognitive restructuring , but unlikely , as the Altemeyer - Hunsberger Scale is a generally accepted measure of religious fundamentalism ( Hunsberger 1996 ) and the restructuring tasks were drawn from past studies of restructuring ( e . g . Weisberg 1995 ) . The patterns which are visible at the extreme ends of the dispersion pattern raise interesting questions , although no conclusions may be drawn from them due to the small amount of data involved . Although the study did not hypothesize anything about persons low in religious fundamentalism , the uniformly high restructuring scores among very low scorers on the religious fundamentalism scale are interesting . They are especially interesting because thc scores on the scale are close to the lowest scores possible ( the lowest scores were 26 and 36 , with the lowest possible being 20 ) . In order to reach this score , the participants must consistently answer that they " completely disagree " with the fundaincntalist response . This is , in effect , a complete rejection of the fundamentalist view and not just disagreement with particular ideas . These tmdings may suggest that many people very high in restmcturing ability and therefore mental flexibility consciously reject the rigidity of the fundamentalist view , or that high restructuring ability is one of a set of personality variables incompatible with fundamentalism . This also supports Hunsberger et , al . ' s ( 1996 ) finding that individuals low in religious fundamenlalism tend to think in more complex ways . However , it is necessary to note that the highest restructuring score found in the sample fell into the area of high fundamentalism , suggesting that restructuring ability by itself is not incompatible with fundamentalism and implying other factors must be a part of the low fundamentalism scores for the other participants high in resnucturing . The very high variability shown at the opposite end of the scale is also interesflng . While the data do not support the hypothesis , the fact that both the lowest ( the only 0 ) and the highest ( the only 9 ) restructuring scores are associated with high religious fundamentalism may be significant . Whereas not too much can be read into what is really only a few data points , it is possible that this high range may reflect differences within religious fundamentalism . One possible explanation is that this could reflect a difference between individuals raised in a highly fundamental environment who had this pattern of thought ingrained from an early age and individuals who came to a fundamentalist belief as a conscious decision . Future studies in this area should work to address the questions raised by the extremes of the current data . One obvious step is to use a sample prescreened for high and low religious fundamentalism and attempt to determine if the results attained at the extremes of the current data bear out across a larger sample . Once it has been determined if these findings are real or not , potential explanations for the variation can be explored . Studies should focus on the interaction of fundamentalism and restructuring and attempt to discover what factors of are associated with both the antifundamentalist stance and the variation in restructuring ability within highly fundamental individuals . Causal designs should also be explored , to see if can be determined if it is in fact something about the fundamentalist mindset which inhibits complex thought , or if there is a self - selection process acting where those who feel more comfortable with simple answers gravitate toward a fundamental style of belief . It may even be possible to induce a mindset similar to fundamentalism in a laboratory context by adopting a very authoritarian style of running the experiment , with harsh punishments for deviations from arbitrary and inefficient rules given to the participants . CONCLUSION Religious belief and behavior is something that all of us as humans must deal with in our lives . For some people it is an afterthought , and for others the defining aspect of life . No matter what an individual ' s personal take on the issue is , he or she cannot escape being affected by religion . Because of this , any effort to understand the world or a person ' s behavior in it must take into account at least minimally the effects of religion . Religious fundamentalism especially can have profound effects upon both the devout and those who interact with them . A greater understanding of the phenomenon will lead to a greater understanding between those who are fundamentalist and those who are not , and perhaps a minute narrowing of the rift which sometimes seems to gape between people of differing beliefs . If we can learn to see what . aspects of belief are common to all and acknowledge where the differences in interpretation and belief structure arise , cooperation may be made easier . Failing that , at least we will be able to point out with some certainty where the disagreements will arise . REFERENCES Altemeyer , B . , & Hunsberger , B . ( 1992 ) . Authoritarianism , religious fundamentalism , quest , and prejudice . The International Journal for the Ps cholo of ~ R ( 2 ( 2 ) , 113 - 133 . Danso , H . , Hunsberger , B . , & Pratt , M . ( 1997 ) . The role of parental religious fundamentalism and right - wing authoritarianism in child - rearing goals and practices . The Journal for the Scientific Stud of Reli ion 36 ( 4 ) , 496 - 511 . Dominowski , R . ( 1995 ) . Productive problem solving . In S . M . Smith , T . B . Ward , & . R . A . Finke ( Eds . ) , The Creative Co nition A roach ( pp . 73 - 95 ) . Cambridge , Massachusetts : MIT Press . Edgington , T . & Hutchinson , R . ( 1990 ) . Fundamentalism as a predictor of cognitive complexity . Journal of Ps cholo and Christianit 9 ( I ) , 47 - 55 . Fulton , A . , Gorsuch , R . , Maynard , E . ( 1999 ) . Religious orientation , antihomosexual sentiment , and fundamentalism among Christians . The Journal for the Scientific Stud of Reli ion 38 ( I ) , 14 - 35 . Hunsberger , B . ( 1995 ) . Religion and prejudice : The role of religious fundamentalism , quest , and right - wing authoritarianism . Journal of Social Issues 51 ( 2 ) , 113 - 129 . Hunsberger , B . ( 1996 ) . Religious fundamentalism , right . - wing authoritarianism , and hostihty toward homosexuals in non - Christian religious groups . The International Journal for the Ps cholo of Reli ion 6 ( 1 ) , 39 - 49 . Hunsberger , B . , Alisat , S . , Pancer , S . M . , & Pratt , M . ( 1996 ) . Religious fundamentalism and religious doubts : Content , connections , and complexity of thinking . The International Journal for the Ps cholo of Reli ion 6 ( 3 ) , 201 - 220 . Jackson , L . , & Esses , V . Of scripture and ascription : The relation between religious fundamentalism and intergroup helping . Personalit and Social Ps cholo 8 11 t 23 ( 8 ) , 893 - 906 . Kirkpatrick , L . ( 1993 ) . Fundamentalism , Christian orthodoxy , and intrinsic religious orientation as predictors of discriminatoty attitudes . The Journal for the Scientific Stud of Reli ion 32 ( 3 ) . 256 - 258 . Klass , M . ( 1995 ) . Ordered Universes : A roaches to the Anthro olo of ~ Reli ion Boulder , Colorado : Westview Press , Maret , S . ( 1984 ) . Attitudes of fundamentalists toward homosexuality . Ps cholo ical Re orts 55 ( I ) 205 - 206 . Pancer , S . , Jackson , L . , Hunsberger , B . , Pratt , M . , ( 1995 ) . Religious orthodoxy and complexity of thought . about religious and nonreligious issues . Journal of Polit 63 ( 2 ) . 219 - 232 . Smith , S . M . , Ward , T . , & Schumacher , J . ( 1993 ) . Consuaining effects of examples in a creative generation task . Memo & Co nition 21 ( 6 ) , 837 - 845 . Streyffler , L . & McNally , R . ( 1998 ) . Fundamentalists and liberals : Personality characteristics of Protestant Christians . Personalit and Individual Differences 24 ( 4 ) . 579 - 580 . Weisberg , R . ( 1995 ) . Case studies of creative thinking : Reproduction versus restructuring in the real world . In S . M . Smith , T . B . Ward , & . R . A . Finke ( Eds . ) , The Creative Co nition A roach ( pp . 73 - 95 ) . Cambridge , Massachusetts : MIT Press . Wylie , L . , Forest , J . ( 1992 ) . Religious fundamentalism , right - wing authoritarianism , and prejudice . Ps cholo ical Re orts 71 ( 3 ) , 1291 - 1298 APPENDIX A Locus of control scale with embedded Altemeyer - Hunsberger religious fundamentalism scale RFS items are marked with * Numbers 4 , 6 , 12 , 16 , 18 , 27 , 32 , 40 , 44 , & 49 are scored in reverse . 1 . Personality is mainly the result of genetic makeup . Completely disagree I 2 3 4 5 6 7 8 Completely agree * 2 . God will punish most severely those who abandon his true religion . Completely disagree I 2 3 4 5 6 7 8 Completely agree 3 . Success comes through hard work , not chance . Completely disagree I 2 3 4 5 6 7 8 Completely agree * 4 . " Satan " is just a name that people give to their own bad impulses . There is really no such thing as a diabolical being which tempts us . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 5 . Something will happen to mess up any plans are made . Completely disagree I 2 3 4 5 6 7 8 Completely agree * 6 . No single book of religious writings contains all of the important truths about life Completely disagree I 2 3 4 5 6 7 8 Completely agree ' 7 . God has given mankind a complete , unfailing guide to happiness and salvation , which must be totally followed . Completely disagree I 2 3 4 5 6 7 8 Completely agree 8 . It is not possible to get ahead in life without being in the right place at the right time Completely disagree 1 2 3 4 5 6 7 8 Completely agree 9 . You cannot change your basic personality . Completely disagree I 2 3 4 5 6 7 8 Completely agree * 10 . Whenever science and sacred scripture conflict , science must be wrong . Completely disagree I 2 3 4 5 6 7 8 Completely agree 11 . Everyone has a fixed destiny . Completely disagree I 2 3 4 5 6 7 8 Completely agree * 12 . It is silly to think people can be divided into " the Good " and " the Evil . " Everyone does some good and some bad things . Completely disagree I 2 3 4 5 6 7 8 Completely agree 13 . It is never possible to make everyone happy . Completely disagree I 2 3 4 5 6 7 8 Completely agree * 14 . God ' s true followers must remember that he requires them to constantly fight Satan and Satan ' s allies on this earth . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 15 . If you set realistic goals , you can succeed no matter what . Completely disagree 1 2 3 4 5 6 7 8 Completely agree " 16 . People should encourage their children to study all religions without bias , then make up their own minds about what to believe . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 17 . Bad or good luck can really follow you around . Completely disagree 1 2 3 4 5 6 7 8 Completely agree * 18 . All of the religions in the world have flaws and wrong teachings . Completely disagree I 2 3 4 5 6 7 8 Completely agree 19 . Many people lead miserable lives because of their parents . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 20 . How you behave and who you are are the most determining factors in your interactions with other people . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 21 . Someone is a happy - go - lucky person because he / she has had an easy life . Completely disagree 1 2 3 4 5 6 7 8 Completely agree " 22 . Of the people on thc Earth , one group has a special relationship with God because it bclievcs the most of his truths and tries the hardest to follow his laws . Completely disagree 1 2 3 4 5 6 7 8 Complete ! y agree * 23 . The long established traditions in religion show the best way to honor and serve God , and should not be compromised . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 24 . If I study hard enough , I can succeed any exam . Completely disagree I 2 3 4 5 6 7 8 Completely agree 25 . Many bad things in one ' s life happen just because of bad luck . Completely disagree I 2 3 4 5 6 7 8 Completely agree 26 . Most accidents result from one ' s clumsiness and lack of skills . Completely disagree I 2 3 4 5 6 7 8 Completely agree " 27 . Religion must admit all its past failings and adapt to modern life if it is to benefit humanity Completely disagree I 2 3 4 5 6 7 8 Completely agree 28 . A person is responsible for her / his own actions , good or bad . Completely disagree I 2 3 4 5 6 7 8 Completely agree 29 . One can hardly lead a healthy social life after being rejected by peers as a kid . Completely disagree I 2 3 4 5 6 7 8 Completely agree 30 . One can overcome the painful childhood memories and diminish their impact on one ' s behavior , thinking , and emotions . Completely disagree I 2 3 4 5 6 7 8 Completely agree 31 . I can complain about politics , but that ' s about all I can do . Completely disagree I 2 3 4 5 6 7 8 Completely agree " 32 . There is no body of teachings or set of scriptures which is completely without error . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 33 . Relationships turn sour because the partners / friends don ' t do enough to make them work . Completely disagree I 2 3 4 5 6 7 8 Completely agree " 34 . When you get right down to it , there are only two kinds of people in the world : the Righteous , who will be rewarded by God , and the rest , who won ' t . Completely disagree I 2 3 4 5 6 7 8 Completely agree 35 . People get fired because they don ' t do their job properly . Completely disagree I 2 3 4 5 6 7 8 Completely agree 36 . People are out of work because they don ' t have the necessary abilities . Completely disagree I 2 3 4 5 6 7 8 Completely agree 37 . One can fail at something just because she / he is having a bad day . Completely disagree 1 2 3 4 5 6 7 8 Completely agree " 38 . There is a religion n this earth that teaches , withou1 . error , God ' s truth . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 39 . Incompetence and ignorance can explain a lot of misfortune in the world . Completely disagree 1 2 3 4 5 6 7 8 Completely agree * 40 . Different religions and philosophies have different versions of the truth , and may be equally right in their own way . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 41 . People get hurt because they don ' t pay enough attention . Completely disagree 1 2 3 4 5 6 7 8 Completely agree * 42 . The basic cause of evil in this world is Satan , who is constantly and ferociously fighting against God . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 43 . A person can change his / her personality and behavior patterns . Completely disagree 1 2 3 4 5 6 7 8 Completely agree " 44 . It is more important to be a good person than to believe in God and the right religion . Completely disagree 1 2 3 4 5 6 7 8 Completely agree * 45 . To lead the best , most meaningful life , one must belong to the one true religion . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 46 . Being in a good mood makes it easier to be liked by others . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 47 . A person cannot rise above his / her background . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 48 . Crime and violence can be abolished if people set their mind to it . Completely disagree 1 2 3 4 5 6 7 8 Completely agree " 49 . No one religion is especially close to God , nor does God favor any particular group of believers . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 50 . Sudden bursts of emotional or spiritual energy can push you huge steps ahead . Completely disagree 1 2 3 4 5 6 7 8 Completely agree 20 APPENDIX B Cognitive restructuring tasks l . A man got on to an airplane and saw that he knew the pilot . He greeted the pilot by name . Although the man was not a criminal and had broken no laws , the police arrested him immediately . Why ? 2 . An antique dealer was approached by a man offering to sell him an ancient coin . The coin is stamped with the head of the emperor on one side and the date 544 BC on the other . The dealer examined the coin and called the police . Why ? 3 . A prisoner was attempting to escape from a tower . He found a rope that was half long enough to reach the ground . He divided the rope in half and tied thc two halves together and escaped . How ? 4 . In the summer , lotus flowers double in area every 24 hours . At the beginning of the summer there is one lotus in the lake . It takes 60 days for the lake to be covered with lotus flowers . On what day is the lake halfway covered ? 5 . Is it possible to connect all nine dots below by drawing four snaight lines without lifting the pencil from the paper or retracing any line ' ? Calendars made in Great Britain do not show Lincoln ' s Birthday . Do these calendars show the Fourth of July ? Explain your answer . A man was shot in Texas . 53 bicycles were spread out around him . Why was hc shot ? 8 . Is it possible to make this triangle point dov , nwards by only shifting three numbers ? If so , which ones ? I 2 3 4 5 6 7 8 9 10 9 . A man in town married 12 different women . All are still alive , and he has never divorced any of them . Polygamy is illegal , but he hasn ' t broken any laws . How is this possible ? 10 . There was a boxing match last week scheduled to go twelve rounds . One boxer knocked out the other and the match was stopped after six rounds . It was not a kickboxing match , but no man threw a punch . How is this possible ? 22 VITA Daniel Conor Seyle 238 Walden Ct . Eureka , MO , 63025 Education : Texas A & M University , College Station , TX Expected Degree : Bachelor of Arts Major : Psychology GPR : 3 . 515 / 4 . 0 Minor : Spanish Graduation : May 2000 Honors : National Merit scholar , Only student to represent Texas A & M at Kettering Foundation / National Collegiate Honors Council press release at National Press Club October 1997 , Member of Psi Chi National Psychology Honors Society , Member of Phi Eta Sigma National Honors Society , Member of Golden Key National Honors Society , College of Liberal Arts Dean ' s List seven semesters , Lechner Hall Honors Dorm Sophomore Advisor Publications : Seyle , Conor . " N . I . F . at A & M . " ( In press ) . Hi her Education Exchange , April 2000 Interests : Psychology of religion : Religious fundamentalism , the cognitive basis of religion . Political psychology : Right wing authoritarianism , the relationship between right wing authoritarianism and attitude . Social cognition : Heuristics in social interaction . \ No newline at end of file diff --git a/silver_data/5e0dd81a3ceb4aee81f2b9f95eb6f00d1f2d1182.pdf.txt b/silver_data/5e0dd81a3ceb4aee81f2b9f95eb6f00d1f2d1182.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..73883db9db010581d4854326dc84a82d8c791107 --- /dev/null +++ b/silver_data/5e0dd81a3ceb4aee81f2b9f95eb6f00d1f2d1182.pdf.txt @@ -0,0 +1 @@ +Supporting Initial Trust in Distributed Idea Generation and Idea Evaluation Jana Schumann 1 , Patrick C . Shih 2 , David F . Redmiles 3 , Graham Horton 1 1 Department of Simulation and Graphics Otto - von - Guericke - University Magdeburg Universitätsplatz 2 , 39106 Magdeburg , Germany jana . schumann @ st . ovgu . de , graham . horton @ ovgu . de 2 College of Information Sciences and Technology Pennsylvania State University University Park , PA 16802 USA patshih @ ist . psu . edu 3 Department of Informatics University of California , Irvine Irvine , CA 92697 - 3440 USA redmiles @ ics . uci . edu ABSTRACT Previous research has shown that diversity within distributed collaborative teams can lead to innovation , but trust must exist for the open expression of innovative ideas and establishment of idea credibility . Initial trust is pivotal for distributed teams where team members have never met face - to - face and have only a very limited time to accomplish a task . Our goal is to determine if knowing specific information about other team members could enhance initial trust and improve productivity and satisfaction in idea generation and idea evaluation sessions . In an experiment , we showed that cognitive and affective trust could be successfully enhanced by presenting relevant information elements , such as domain expertise and personal hobbies , and could have positive effects on the quality and quantity of ideas in idea generation sessions as well as the satisfaction of the participants with the rating result in idea evaluation sessions . However , participants receiving personal information often misconstrue this as professional competency . We also describe gender differences observed in the idea generation sessions and discuss how to better design future systems for supporting idea generation and idea evaluation activities . Categories and Subject Descriptors H . 5 . 3 . Group and Organization Interfaces : Collaborative computing , Computer - supported cooperative work , Synchronous Interaction . General Terms Design , Experimentation , Human Factors Keywords Trust , creativity , idea generation , idea evaluation , brainstorming , TWAN schema , distributed teams , virtual teams , globalization 1 . INTRODUCTION With the spread of globalization , there is an increased need for collaboration through distributed teams and , consequently , an increased need for technological support . Distributed teams provide some advantages over traditional teams , such as taking advantage of geographically dispersed experts without having to physically relocate them , having greater flexibility , faster responsiveness , and greater diversity of perspectives [ 24 , 29 ] . However , distributed teams encounter challenges due to their distribution and communication limitations . One major issue in distributed teamwork is trust . It is especially critical for fast - forming teams where team members have never met face - to - face . Previous studies have shown that trust forms and develops over time in traditional face - to - face teams ; team members have time to assess one another based on personal interaction and shared experiences [ 15 , 45 ] . Distributed team members often do not have enough time to get the needed information about other team members . It is more difficult to determine whether a person is trustworthy or not , especially if the group’s formation is only temporary . Therefore , trust in distributed teams must be even higher than in traditional teams in order to successfully achieve a shared goal [ 17 ] . Open innovation is increasingly common in the workplace today . Crowd - sourcing and open - source software development platforms allow strangers to collectively contribute to an end product while remaining anonymous . In many instances , people may interact virtually once and never again . Our work aims to understand how we could enhance initial trust with different information elements about team members’ backgrounds , and to see how trust can be incorporated into the design process to improve productivity and satisfaction in idea generation and idea evaluation sessions . We first review prior literature on innovation and trust , and then we delve more deeply into studies that form the basis for our approach . Then , we describe our study’s goals ( and hypotheses ) followed by its design . Thereafter , we discuss the experimental results . Finally , we conclude with a discussion of the results , implications for design , limitations , and future research directions . 2 . MOTIVATION 2 . 1 Understanding the Concept of Trust Many definitions of trust have been proposed in different contexts [ 5 , 23 ] . In general , trust can be considered as the “belief that the trustee will meet the expectations of the trustor” [ 43 ] . Relatedly , researchers have observed that trust can be defined as a belief or confidence about another party’s integrity and benevolence in order to accept vulnerability [ 32 , 39 ] . According to multidimensional trust research , two dimensions of trust have been identified as important to organizations : cognitive trust and affective trust [ 30 ] . Specifically , “cognition - based trust results from deliberate assessment of each other’s characteristics and the process of weighting benefits of trusting over risks , whereas affect - based trust involves one’s emotional bonds and sincere concern for the well - being of the others” [ 21 ] . Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . GROUP’12 , October 27 – 31 , 2012 , Sanibel Island , Florida , USA . Copyright 2012 ACM 978 - 1 - 4503 - 1486 - 2 / 12 / 10 . . . $ 15 . 00 . 199 Previous research [ 22 , 23 ] has shown that distributed teams develop trust swiftly at the beginning of the project . Iacono considered initial trust in general , but did not differentiate between cognitive and affective trust [ 22 ] . In 2002 , Kanawattanachai found that distributed teams developed a higher degree of cognitive trust than affective trust [ 26 ] . That result supports the swift trust proposition of Meyerson [ 31 ] . Meyerson claimed that cognitive trust is more important than affective trust in a temporary team [ 31 ] . He described a temporary group as an analogy to a “one - night stand” . This so - called swift trust develops within “a finite time span , forming around a shared and relatively clear goal or purpose , and depending on tight and coordinated coupling of activity to achieve success” [ 31 ] . Therefore , converting the individual skills and efforts of strangers into interdependent work in a short period of time poses a major challenge to distributed team collaboration . Our work aims to aid the development of initial trust in distributed teams by building on the concept of cognitive trust and affective trust in a distributed environment . Furthermore , we want to determine whether higher initial trust influences the outcome of idea generation and idea evaluation sessions , which are both part of innovation processes . 2 . 2 Importance of Innovation Innovation is a process and several models exist to describe the different phases of that process . Herstatt defines the first phase of the innovation process as a sequence of generating and evaluating ideas [ 18 ] . Idea generation and idea evaluation are the phases where initial trust is necessary because they are part of the first phase of the innovation process . Idea generation ( or ideation ) “is the creative process of generation , developing , and communicating new ideas , where an idea is understood as a basic element of thought that can be either visual , concrete , or abstract” [ 25 ] . The result of idea generation sessions are usually a large number of ideas , however a good idea does not always appear to be a good idea at first glance . Therefore , an evaluation process that aims to select a limited number of good ideas for further development is crucial for the innovation process . Within innovation processes , two different degrees of newness can be distinguished . New - to - the - world ideas for products or services are called radical innovation , and minor adaptations of products or services are called incremental innovation [ 4 ] . Both kinds of innovation represent opposite ends of the newness spectrum [ 4 ] . More precisely , radical innovations are truly novel or unique technological solutions [ 33 ] , the development or application of new technologies , or state - of - the - art breakthroughs in technology or product category [ 11 ] . Incremental innovations are new products involving only minor or no changes in technology and are also called simple product improvements [ 11 ] . Both degrees of newness are important for innovation processes , and we hope to identify how radical and incremental ideas are affected in teams with different levels of initial trust . 2 . 3 Creativity and Trust To produce both radical and incremental innovation , creativity is necessary . Creativity is the ability to produce work that is both novel ( i . e . original , unexpected ) and appropriate ( i . e . useful , adaptive concerning task constraints ) [ 42 ] . Originality is the hallmark of creative behavior , and ideas are not considered to be creative if they are not new or unusual . Although ideas must be original in order to be called creative , they will not be implemented if they are not feasible . Hence , the usual definition of a good idea is an idea that is both highly original ( or unusual ) and highly feasible ( or useful ) . One way to enhance the creative process is by using so - called creativity techniques . More than 100 creativity techniques can be found in the literature [ 19 ] . In this paper , we focus on electronic brainstorming , a computerized version of the brainstorming technique introduced by Osborn [ 34 ] . Osborn defined brainstorming as " a creative conference for producing a list of ideas - ideas which can be subsequently evaluated and further processed " . We focused on electronic brainstorming because it is one of the most common creativity techniques . Knoll et al . [ 28 ] stated that most of the creativity techniques or idea generation techniques support an associative process with external stimuli , which are received through the five senses of the individual . Thus , brainstorming contains general rules to support a process , which is the basis for many other creativity techniques . As previous research has shown , diversity within a team can lead to innovation in collaboration [ 37 ] , but trust must exist for the open expressiveness of innovative ideas by team members . Trust has a positive characteristic leading to desirable behavior and outcomes , although negative expectations and trust can also occur during collaborations [ 1 ] . Thus , trust plays an important role in innovation , efficiency , and effectiveness of teamwork , as team members do not tend to cross - check each others’ work [ 5 , 23 ] . Moreover , low trust leads to an increase in faulty attributions regarding the source of disagreement in distributed teams [ 41 ] . In low trust environments , trust can be fragile and often fractures rapidly , and team members are more likely to question others’ intentions [ 23 ] . In this work , the main goal of supporting trust in idea generation sessions is to increase the number of radical ideas . Idea generation sessions are about risking vulnerability by writing down unusual ideas . This social effect is called evaluation apprehension . It causes participants to hold back their contributions during the process because they are afraid to be criticized by someone in the group [ 12 ] . Postmes indicated that anonymity reduces this effect [ 36 ] . On the other hand , anonymity can easily lead to social loafing . Social loafing describes the tendency of participants to expend less effort when they believe that their contributions are not needed for the group success [ 27 ] . Previous laboratory experiments showed that while participants reported less perceived level of evaluation apprehension , anonymity made no impact on the creative outcomes [ 2 , 44 ] . Collaros and Anderson are the first to study the effects of perceived team member expertise level on the idea generation outcome . They found that participants in a team full of experts generated ideas of lower originality and feasibility , and they report higher perceived inhibition in idea generation sessions [ 8 ] . Open exchange of information should be promoted since people are more likely to collaborate with individuals they trust [ 23 ] . The literature regarding the correlation of trust and creativity in face - to - face teams remains largely inconclusive . It was found that trust is beneficial to increase creativity in face - to - face teams [ 41 ] , but more recent studies showed that there is no positive impact of trust on creativity in teams [ 7 ] . Similarly , Bidault found that higher trust does not always lead to more creativity [ 3 ] . There seems to be a level of trust that maximizes the creative output . However , no previous research has focused on studying the correlation of trust and idea generation in distributed teams . 200 2 . 4 Rating Behavior and Trust A successful idea generation session typically results in a large quantity of ideas . An evaluation process that aims to select a limited number of good ideas is necessary for further development . An effective method for idea evaluation is necessary to make the right decision whether an idea is original , feasible , or both . If expertise information of team members is not made sufficiently transparent during an idea evaluation session , a lack of trust of other members’ judgment about an idea can easily develop . A lack of commitment can increase due to mistrust . Commitment is important to form a consensus about an idea within a team . The main goal of supporting trust in idea evaluation sessions is to allow the team members to arrive at a consensus while maintaining a reasonable level of satisfaction . Team members have to trust each other regarding their ability to select one or several good idea from an idea pool . There exist many approaches to evaluate ideas . One well - known technique is the SWOT analysis that evaluates ideas based on the following objectives : Strengths , Weaknesses / Limitations , Opportunities , and Threats [ 13 ] . Approaches such as the SWOT analysis are useful for comparing the advantages and disadvantages of a specific idea . To support trust in idea evaluation sessions , we borrow the basic idea of a recommender system in that users are presented with pre - populated ratings provided by other users . Research has shown that showing predictions during the rating process ( e . g . movies ) could influence the users’ rating outcomes [ 10 ] . Similar approach could also work for the evaluation of ideas and result in higher member satisfaction . A large amount of literature regarding trust and decision - making considers recommender systems , e . g . in e - commerce [ 10 ] , recommending and evaluating choices in a virtual community of use [ 20 ] . Recommender systems are useful when too much information is present . Research on decision - making has focused on trust as a variable that affects decision outcomes . In 2009 , Parayitam found that the perception of trustworthiness , when based on the competence of a person , enhances decision quality and commitment , whereas no effects on outcomes can be observed when it is based on relationships [ 35 ] . Rietzschel identified the strong tendency of people to select feasible and desirable ideas at the cost of originality as the main reason for their poor selection performance [ 38 ] . However , no publications could be found containing results about the correlation of trust and idea evaluation in distributed teams . To our knowledge , no studies have analyzed how different levels of trust can impact the idea evaluation session outcomes . In this work , we attempt to study the rating behaviors of participants with higher cognitive trust or higher affective trust . 3 . A TEMPLATE FOR SUPPORTING INITIAL TRUST 3 . 1 Support of Initial Trust The focus of this work is to find out how initial trust can be supported in a distributed team when team members only have a very limited amount of time to accomplish a task , and how trust might improve the work results in distributed teams . Therefore , the approach of the so - called first impression could be used . People make guesses on signs and signals they perceive , which is the seed of trust or distrust and also affect their subsequent behavior [ 9 , 16 ] . Signs and signals , which appear in face - to - face interaction , might or might not appear differently in computer - mediated interaction . Since members of distributed teams often do not have a prior working history and may never meet again , oral exchanges and face - to - face interaction often do not exist [ 23 ] . To enable distributed team members to form a first impression , information about their co - workers could be offered . Research has shown that the availability of information can influence trustworthiness assessments positively [ 40 ] . However , it is not entirely clear which information elements are most supportive for the assessment of team members , especially regarding teams dealing with innovation . One way to support distributed team members with the formation of trustworthiness is to provide opportunities for accumulating personal knowledge and task - relevant background information [ 21 , 26 ] . Feng claimed that , “ developing artifacts to help people to identify others who are similar to themselves or who have similar experiences may be helpful for promoting empathic attitudes that build interpersonal trust ” [ 14 ] . Jarvenpaa and Leidner found that high - performing distributed teams exchanged background and personal information and were socializing more with other members at the very beginning of their project [ 23 ] . Therefore , we aim to determine what information to provide in the beginning of a project to support initial trust in distributed idea generation and idea evaluation sessions . Furthermore , we need to know how this information is correlated to trust . 3 . 2 Information Elements Rusman introduced the TrustWorthiness ANtecedents schema ( TWAN ) as an approach to inform trustworthiness assessments in the initial phase of collaboration [ 40 ] . This is used for the design of measures to accelerate the formation of interpersonal trust . Her study determined the information elements that are important for assessment of trustworthiness . The schema of perceived trustworthiness of a trustee consists of five main categories : communality , ability , benevolence , internalized norms , and accountability . Each of these main categories can be split up in more detailed antecedents . Since our work focuses on affective trust and cognitive trust , we divided the different trustworthiness antecedents into two parts with cognitive trust referring to the main categories of communality , ability , and accountability and affective trust referring to communality , benevolence , and internalized norms . Note that internalized norms are not considered in this paper because they only refer to long - term projects and therefore cannot be integrated in the approach of this paper . Our work focuses on making impact at the start of a project , but also aims to identify how knowing different information could affect different phases of the innovation process—idea generation and idea evaluation . To refine the findings of Rusman , a set of interviews was performed to discover important information elements adapted for the intended subject pool consisting of students . Table 1 . Information Elements Personal # Expertise # Hobbies 14 Experience ( projects ) 15 Gender 13 Specific skills 15 Honorary activities 12 Specialization & interests 14 Age 11 References & awards 14 Nationality 8 Degree & years in the program 12 Taste in music 7 Companies 8 Favorite TV shows 6 Department 7 201 Overall 15 students were asked what personal and professional information they would like to know about team members they have to work with , but have never and will never meet face - to - face . In Table 1 , the summary of the results of our interviews is shown . The table lists criteria ( personal information and expertise ) that the students listed as important , and their frequency ( # ) . 3 . 3 Relation of Information Elements to Trust Information elements can have a direct relationship to the introduced trustworthiness antecedents [ 40 ] . Table 2 provides a summary of the information elements available before the collaboration activities occur and their relationship to TWAN as specified in [ 40 ] . These are factual information that can be used by the participants to know their teammates prior to team activities . In Table 3 , the information elements derived from behavior during the collaboration according to Rusman are shown . They can be perceived by the participants passively through the collaboration process . Table 2 . Available information before collaboration Information element Relation with TWAN Age Communality , Availability , Sharing Gender Communality , Availability , Sharing Nationality Communality , Availability , Sharing Hobbies Communality , Availability , Sharing Honorary activities Communality , Availability , Willingness to help , Faith in intentions , Caring , Friendliness , Commitment , Openness , Sharing Favorite TV shows Communality , Availability , Openness , Sharing , Receptivity Taste in music Communality , Availability , Openness , Sharing , Receptivity Companies , Experience ( projects ) , References & awards Communality , Self - confidence , Knowledge , Reliability Department Communality , Skills , Knowledge Degree & years in the program Communality , Reliability , Consistency , Responsibility , Persistence , Competence Specialization & Interests Communality , Consistency , Persistence , Competence , Knowledge Specific skills Communality , Knowledge , Competence , Skills Table 3 . Information derived from behavior Information element Relation with TWAN Message read by addressed person Availability , Reliability , Responsibility Suggestion / idea Competence , Willingness to help , Sharing , Openness , Commitment , Self - confidence , Persistence , Responsibility Task - status overview ( task , accepted by , deadline , status ) Competence , Reliability , Responsibility Average response Availability , Receptivity , Commitment , Consistency , Responsibility Different information elements affect trust in different ways , so the TWAN schema was divided into affective and cognitive trust . In this construct , the information elements should support affective trust and the expertise information elements should support cognitive trust . 3 . 4 System Design and Implementation Since distributed teams usually collaborate via the Internet , a web - based software tool is one possible way to implement an initial trust template for idea generation and idea evaluation sessions . The prototype consists of a client ( web browser ) and a server ( web server with database ) . The idea generation session consists of two different web pages . The start page of the idea generation session consists of the following : an overview about the different steps of the session to provide an overview for the participants , the task for the session so that the participants know what to do in the next step , information about the technology used in the task in case the participants are not familiar with it , and the profiles of two other participants , depending on the condition to which the participant was randomly assigned . The second web page of the idea generation session consists of four main parts ( see Figure 1 ) . Part ( 1 ) shows the specific task and the brainstorming rules so that the participants are reminded what they are supposed to do and how they are supposed to do it . Part ( 2 ) is a chat window that displays the contributions of the participants . Part ( 3 ) is a pre ‐ defined input mask for the entry of the participant’s contributions . Part ( 4 ) shows the profiles of the two other participants as on the start page . Figure 1 . Idea Generation Session Template The idea evaluation session also consists of two different web pages . The start page of the idea evaluation session consists of the following : an overview about the different steps of the session to provide an overview for the participants , the task for the session so that the participants know what to do in the next step , information about the technology used in the task in case the participants are not familiar with it , and the profiles of two other participants as in the idea generation session . The second web page of the idea evaluation session also consists of four main parts ( see Figure 2 ) . Part ( 1 ) shows the specific task so that the participants are reminded what they are supposed to do and how they are supposed to do it . Part ( 2 ) is the rating area that allows the participant to rate ideas by clicking on the stars . Part ( 3 ) shows the previously rated ideas by two other participants and the average rating of the idea , which represents the final group 202 rating . Part ( 4 ) shows the profiles of the two other participants as on the start page . Figure 2 . Idea Evaluation Session Template 4 . GOALS AND EXPECTATIONS As previously described , the goal of our experiment is to examine whether knowing specific information about collaborators might support trust in distributed idea generation and idea evaluation sessions . Moreover , we would like to determine how trust could impact the idea generation and idea evaluation sessions . Based on the information elements adapted from TWAN , we expect the following : Hypothesis 1a : Knowing personal information of an individual leads to higher affective trust during the distributed idea generation session . Hypothesis 1b : Knowing the expertise information of an individual leads to higher cognitive trust during the distributed idea generation session . If this expectation is satisfied , we must further determine whether an increased trust among distributed team members could affect the contributions generated during idea generation sessions . Since higher affective trust allows team members to express themselves more freely and higher cognitive trust allows team members to focus on assessing the ideas more critically , we form the following hypothesis : Hypothesis 2a : Higher affective trust during the distributed idea generation session leads to more radical ideas . Hypothesis 2b : Higher cognitive trust during the distributed idea generation session leads to more incremental ideas . Similar to the idea generation sessions , we expect to see the following as outcomes of the idea evaluation sessions : Hypothesis 3a : Knowing personal information of an individual leads to higher affective trust during the distributed idea evaluation session . Hypothesis 3b : Knowing that an individual is an expert in a specific field leads to higher cognitive trust during the distributed idea evaluation session . If this expectation is satisfied , we must further determine whether an increased trust among distributed team members could affect the rating behaviors during idea evaluation sessions . Since higher affective trust allows team members to express themselves more freely and since higher cognitive trust allows team members to focus on assessing the ideas more critically , we form the following hypotheses : Hypothesis 4a : Higher affective trust in the distributed idea evaluation session reduces consensus formation within the group . Hypothesis 4b : Higher cognitive trust in the distributed idea evaluation session induces consensus within the group . To investigate the aforementioned hypotheses , a software prototype has been designed and implemented to find out if perceived trustworthiness leads to the hypothesized results . Finally , idea generation literature has traditionally used either all male or all female groups citing important gender differences . However , to our knowledge , no idea generation studies focused on detailing such gender differences , and in studies that involve mixed - gender groups , gender is obviously not carefully controlled . In this study , we control for gender groups and hope to lead to future designs that better account for gender differences . 5 . EXPERIMENTAL DESIGN 5 . 1 Participants and Confederates Table 4 . Confederate information ( P = Personal Information ; E = Expertise Information ) Information Confederate 1 Confederate 2 P Age 27 25 Gender male female Nationality American American Hobbies playing basketball and guitar music , photography , swimming Honorary activities dean’s list , athletic department honor roll co - founder of a non - profit association TV shows How I Met Your Mother , Chuck , Seinfeld Sex and the City , The Big Bang Theory , The Simpsons Taste in music rock , indie electronic E Companies & References Google Inc . , Apple Inc . Microsoft Research Awards None Outstanding Research Award Degree M . Sc . Ph . D . Department Computer Science Social Sciences Current year in the program 3rd 5th Specialization & Interests Visualization , software engineering social networks , education Skills Java , C + + , PHP , JSP , JavaScript , Ajax experienced in quantitative and qualitative analysis We recruited 36 participants from UCI for the experiment . 18 of them were male and 18 were female . They were between the ages of 20 and 33 years . The participant pool contained undergraduate as well as graduate students from different departments . The participants were asked to complete a short questionnaire about their background . On a 5 - point Likert scale , all subjects except for one were somewhat familiar with idea generation ( mean = 3 . 77 ) and idea evaluation ( mean = 3 . 94 ) techniques . On a 7 - point Likert scale , most of the participants reported they were comfortable working with strangers ( mean = 4 . 80 ) , they occasionally have 203 online collaboration ( mean = 3 . 77 ) , and they tended to trust strangers slightly ( mean = 3 . 55 ) . Participants worked in groups of three with two of the three being confederates ( computer bots ) . One of the challenges in evaluating ideas in experimental studies is that groupthink tends to dictate the idea flow , and the ideas of participants are often triggered by the ideas generated by other participants in the same session . In this study , confederates were used in order to ensure consistent participant experiences across all experimental conditions . In the idea generation sessions , each confederate entered a predetermined list of ideas at specific time intervals . Each confederate had a list of ten ideas that were applicable to the given topic . Depending on the condition , different profile information of the confederates were shown to the participants . Under condition N , no profile information was shown except for the fact that two other participants are logged in . Under condition P and E , the confederates portrayed the profile information identified in Table 4 . 5 . 2 Independent Variables The study followed a 2 ( gender ) x 2 ( idea generation and idea evaluation sessions ) x 2 ( topic A and topic B ) x 3 ( information element conditions N , P , and E ) counterbalanced , randomly assigned , within - subjects design ( see Table 5 ) . Since there was no significant difference between topic A and topic B , we combined the results in both topic A and B in the analyses . All participants first accomplished an idea generation session and then an idea evaluation session . The ordering of the topics was counterbalanced ; participants either generated ideas about topic A in the idea generation session and then evaluated ideas about topic B in the idea evaluation session or vice versa . Topic A involved ideas for using Facebook as a platform for learning in the classroom setting , and topic B involved ideas for using the iPad2 to assist the elderly . Each participant was randomly assigned to one of three information element conditions . In condition N , the participants did not receive any information about their team members . In condition P , the participants received personal information about their team members . In condition E , the participants received the expertise information of their team members ( see Table 4 ) . Table 5 . Study design Idea Generation ( IG ) & Idea Evaluation ( IE ) Male Female Topic A = > Topic B Topic B = > Topic A Topic A = > Topic B Topic B = > Topic A N P E N P E N P E N P E 5 . 3 Procedure The study took place at a behavioral research lab . Each participant was asked to complete a demographic survey . A research staff member explained the research procedure and asked the participant to provide verbal consent . The subject then received a short training on the use of the system . Each subject logged into the system and generated ideas for 15 minutes with two confederates on the assigned topic . In the idea generation session , the participant was asked to provide a label , a short description , and the advantage for the target group for each idea . Each confederate entered a pre - populated list of ten ideas relevant to the assigned topic into the chat window at a time interval that was designed to emulate the natural thinking and typing speed that one would expect to encounter in a regular idea generation meeting . This was done to ensure that all participants encountered the same ideas across different experimental conditions . After a five - minute break , the participant was asked to fill out a questionnaire about his / her personal trust level during the idea generation activity , as well as the satisfaction with the result of the idea generation session . Then the participant was given a five - minute break . In the idea evaluation session , the participant was given 15 minutes to evaluate six pre - populated ideas that are relevant to a second topic for originality and feasibility on a five - point scale . The system also showed the ratings provided by the confederates . This was done to see if knowing either personal or expertise information about their teammates could alter the participants’ rating behaviors . After a five - minute break , the participants were asked to fill out a questionnaire about their personal trust level during the idea evaluation as well as their satisfaction with the result of the idea evaluation session . The entire study procedure lasted approximately 60 minutes . 5 . 4 Dependent Variables 5 . 4 . 1 Trust and Satisfaction To measure affective and cognitive trust of the participants we used a questionnaire adapted from the TWAN schema [ 40 ] . The cognitive trust measure asked about communality ( 1 question ) , ability ( 3 ) , and accountability ( 5 ) of the participants ( Cronbach’s alpha = 0 . 93 ) . The affective trust measure asked about communality ( 1 question ) and benevolence ( 8 ) of the participants ( Cronbach’s alpha = 0 . 87 ) . Six additional questions were asked to measure the participants’ satisfaction level ( Cronbach’s alpha = 0 . 72 ) . 5 . 4 . 2 Quality and Quantity of Ideas We measured the quantity , diversity , and quality of the ideas generated in the idea generation session . Idea quantity is defined as the number of unique ideas . Idea diversity is measured by the number of idea clusters grouped by similarity . All ideas were clustered and rated independently by two experts for originality and feasibility on a five - point scale . The quality of an idea is defined as a combination of originality and feasibility . The inter - rater agreement coefficient was calculated as shown in previous idea generation studies , which assumed that the ratings were in agreement if the expert ratings fell within one point of each other [ 12 , 38 ] . The two experts agreed on 89 . 3 % of the originality ratings and on 90 . 4 % of the feasibility ratings . Differences were reconciled by averaging the ratings . We further group ideas into radical ideas ( high originality and high feasibility ) and incremental ideas ( above average originality and above average feasibility ) . 6 . RESULTS 6 . 1 Idea Generation Table 6 summarizes the results in the idea generation sessions . To test Hypothesis 1a , 1b , 2a , and 2b , MANOVA was performed . The test showed that there is no effect of gender and topic on the trust level of the participants . The F - test showed that there is a significant effect regarding the different conditions and the trust level of participants in the idea generation session ( F [ 14 , 48 ] = 3 . 52 , p < 0 . 0006 ) . Post - hoc t - tests showed that the participants in both condition P ( t [ 22 ] = 3 . 96 , p < 0 . 0003 ) and condition E ( t [ 22 ] = 3 . 46 , p < 0 . 001 ) developed significantly higher affective trust in the idea generation session than the control group . Participants in condition E also developed significantly higher affective trust than in the control group . Similarly , participants in both condition P 204 ( t [ 22 ] = 3 . 06 , p < 0 . 003 ) and condition E ( t [ 22 ] = 2 . 73 , p < 0 . 006 ) developed significantly higher cognitive trust in the idea generation session than the control group . In regard to affective trust , it was shown that knowing of personal information leads to higher affective trust during the distributed idea generation session . It was surprising to observe that knowing the expertise level also leads to higher affective trust when its primary goal is to support cognitive trust . The same applies to cognitive trust . The results showed that knowing both personal information and expertise level enhance affective and cognitive trust in the idea generation session . Thus , knowing any information ( personal or expertise ) could enhance trust in general . As a result it can be said that the data supports Hypothesis 1a and 1b — knowing expertise and personal information could lead to higher cognitive and affective trust during the idea generation sessions . Table 6 . Idea Generation Results N P E Quantity 4 . 0 ( 0 . 48 ) b 6 . 4 ( 0 . 56 ) a 4 . 3 ( 0 . 54 ) b Diversity 3 . 5 ( 0 . 51 ) b 6 . 0 ( 0 . 48 ) a 3 . 9 ( 0 . 48 ) b Incremental Ideas 1 . 2 ( 0 . 30 ) b 2 . 3 ( 0 . 43 ) a 1 . 1 ( 0 . 29 ) b Radical Ideas 1 . 0 ( 0 . 35 ) b 1 . 6 ( 0 . 15 ) a 0 . 4 ( 0 . 15 ) c Cognitive Trust 4 . 1 ( 0 . 23 ) b 5 . 1 ( 0 . 23 ) a 5 . 1 ( 0 . 18 ) a Affective Trust 4 . 6 ( 0 . 19 ) b 5 . 5 ( 0 . 16 ) a 5 . 2 ( 0 . 17 ) a Satisfaction 4 . 3 ( 0 . 19 ) b 4 . 9 ( 0 . 12 ) a 4 . 5 ( 0 . 17 ) b * The results is displayed as Mean ( Standard Deviation ) , with superscripts denoting the following statistically significant relationships : a > b > c . The participants were also asked about their general satisfaction about the idea generation process . Post - hoc t - tests showed that participants in condition P felt significantly more satisfied than the participants in both condition E ( t [ 22 ] = 1 . 85 , p < 0 . 04 ) and in the control group ( t [ 22 ] = 2 . 55 , p < 0 . 009 ) . This shows that knowing more personal information about the other team members not only resulted in higher trust , but also induced higher satisfaction during the process . In terms of idea quantity , post - hoc t - tests showed that participants in condition P created more unique ideas than in the control group ( t [ 22 ] = 3 . 29 , p < 0 . 001 ) and in condition E with a significance of ( t [ 22 ] = 2 . 68 , p < 0 . 006 ) . Similarly , participants in condition P also created significantly more diverse ideas than in condition E ( t [ 22 ] = 3 . 06 , p < 0 . 002 ) as well as in the control group ( t [ 22 ] = 3 . 56 , p < 0 . 0009 ) . This shows that higher affective trust makes the participants open up more to others and that they are able to express their ideas more freely . This also confirms prior research that participants in a team full of experts reported higher perceived inhibition in idea generation sessions [ 8 ] . In terms of idea quality , post - hoc t - tests showed that participants in condition P tended to produce more radical ideas than in the control group ( t [ 22 ] = 1 . 54 , p < 0 . 06 ) and that the participants in the control group tended to produce more radical ideas than in condition E ( t [ 22 ] = 1 . 54 , p < 0 . 06 ) . Also , participants in condition P created more radical ideas than in condition E ( t [ 22 ] = 5 . 54 , p < 0 . 0001 ) . For incremental ideas , post - hoc t - tests showed that participants in condition P created significantly more incremental ideas than the participants in condition E ( t [ 22 ] = 2 . 26 , p < 0 . 01 ) and in the control group ( t [ 22 ] = 2 . 07 , p < 0 . 02 ) . The enhanced affective trust in condition P provides participants enough confidence to write down more interesting or unusual ideas . This result supports Hypothesis 2a but not Hypothesis 2b—higher affective trust resulted in more radical ideas , but higher cognitive trust did not produce more incremental ideas . Furthermore , it can be claimed that knowing the expertise information of the other participants has a negative influence on producing radical ideas due to the higher perceived inhibition despite the high cognitive trust . In regard to idea originality and feasibility , a significant effect of gender was found ( F ( 2 , 147 ) = 3 . 02 , p < 0 . 04 ) . Post - hoc t - tests showed that female participants on average created more feasible ideas whereas male participants on average created more original ideas ( see Table 7 ) . Table 7 . Gender Differences Female Male Originality 3 . 01 ( 0 . 78 ) b 3 . 25 ( 0 . 87 ) a Feasibility 3 . 97 ( 0 . 77 ) a 3 . 69 ( 0 . 87 ) b * The results is displayed as Mean ( Standard Deviation ) , with superscripts denoting the following statistically significant relationships : a > b . Although no prior idea generation literature focused on gender differences , existing gender in software interfaces suggested that females tend to be more risk - averse [ 6 ] . We believe that this is especially true in a distributed environment where participants do not know their remote teammates , and could lead to females proposing more feasible ideas in order to not provoke undesired responses from their teammates . 6 . 2 Idea Evaluation Table 8 summarizes the results in the idea evaluation sessions . To test Hypothesis 3a , 3b , 4a , and 4b , MANOVA was performed . The test showed that there is no effect of gender and topic on the trust level of the participants . Table 8 . Idea Evaluation Results N P E Originality 3 . 0 ( 0 . 12 ) 3 . 2 ( 0 . 18 ) 2 . 9 ( 0 . 19 ) Feasibility 3 . 8 ( 0 . 15 ) a 3 . 9 ( 0 . 14 ) a 3 . 5 ( 0 . 16 ) b Cognitive Trust 4 . 2 ( 0 . 21 ) c 4 . 8 ( 0 . 15 ) b 5 . 4 ( 0 . 12 ) a Affective Trust 4 . 5 ( 0 . 15 ) b 5 . 0 ( 0 . 21 ) a 4 . 8 ( 0 . 18 ) Satisfaction 4 . 6 ( 0 . 19 ) b 5 . 2 ( 0 . 14 ) a 5 . 1 ( 0 . 19 ) a The F - test showed that there is a significant effect regarding the different conditions and the trust level of participants in the idea evaluation session ( F [ 10 , 52 ] = 3 . 55 , p < 0 . 001 ) . Post - hoc t - tests showed that only the participants in condition P developed significantly higher affective trust in the idea evaluation session than in the control group ( t [ 21 ] = 2 . 07 , p < 0 . 02 ) . As a result it can be stated that knowing personal information leads to higher affective trust in the idea evaluation session . For cognitive trust , Participants in condition E developed higher cognitive trust than in the condition P ( t [ 22 ] = 3 . 03 , p < 0 . 003 ) , and that the participants in the condition P developed higher cognitive trust than in the control group ( t [ 22 ] = 2 . 32 , p < 0 . 01 ) . It makes sense that expertise information has a much stronger influence on cognitive trust , but this also means that personal information could also be misconstrued as domain competency during the idea evaluation process . As a result it can be said that the data supports Hypothesis 3a and 3b — knowing expertise and personal information could lead to higher cognitive and affective trust during the idea evaluation sessions . The participants were also asked about their general satisfaction about the idea evaluation process . Post - hoc t - test showed that participants in both condition P ( t [ 22 ] = 2 . 67 , p < 0 . 006 ) and 205 condition E ( t [ 22 ] = 1 . 97 , p < 0 . 03 ) felt significantly more satisfied than the participants in the control group . This shows that information about other team members in general ( both personal and expertise ) could induce higher satisfaction during the idea evaluation process . This difference , in contrast with the idea generation process in which personal information is more desirable than expertise information , is perhaps due to the generative nature of the idea generation process versus the selective nature of the idea evaluation process . Although participants seem to provide similar original ratings on the ideas presented to them in the idea evaluation sessions , no correlation was found between the ratings of the participants and those provided by the confederates . However , by comparing the average feasibility ratings of all participants in the three conditions , it was found that participants in condition E provided lower ratings than in condition P ( t [ 22 ] = 1 . 93 , p < 0 . 03 ) and in the control group ( t [ 22 ] = 1 . 68 , p < 0 . 05 ) . This means that knowing the expertise information of other team members , participants were led to judge more critically on the feasibility ratings . Our results indicate that higher affective or cognitive trust did not lead to consensus formation as there was no correlation in their rating behavior , but to better satisfaction regarding the result . Thus , the data does not support Hypothesis 4a and Hypothesis 4b —higher cognitive and affective trust levels did not exhibit correlation with consensus formation during the idea evaluation sessions . 7 . DISCUSSION AND LIMITATIONS The results of the experiment showed that topic and gender differences have little effect on trust in idea generation and idea evaluation sessions . There is , however , a significant effect of knowing information elements of participants’ profiles on their trust level and their output . The study results showed that knowing personal information leads to higher affective trust and knowing expertise information leads to higher cognitive trust , which validated our initial hypotheses . However , what surprised us was that knowing either personal or expertise information of the team members boosted both trust levels . Although cognitive trust is , by definition a type of trust that is based on the perception of domain knowledge and task competency and affective trust is based on the perception of emotional comfort , we found that our participants were not able to clearly distinguish these two dimensions of trust . Essentially , knowing any information about others , regardless of it being personal hobbies or professional knowledge could help the team members relate to and familiarize with each other and therefore lead to higher perceptions of trust . In terms of idea generation , although personal and expertise information are capable of enhancing the participants’ perception of trust overall , the personal informational elements are much more conducive ( more radical and incremental ideas ) whereas the expertise information elements are detrimental to idea generation outcomes ( less radical ideas and no more incremental ideas than the control group ) . This is consistent with the literature , as people tend to be more reserved due to evaluation apprehension when the perceived expertise level is high . In this study , we further showed that knowing personal information about the teammates could reduce that hindrance and lead to sharing more and better ideas . Another important finding is that we found gender differences in the idea generation sessions : female participants created more feasible ideas while male participants created more original ideas in the experiment . Previous literature points to the fact that females tend to be more risk - averse in unfamiliar environments [ 6 ] . Since this research focuses on inducing initial trust on distributed teams filled with strangers , this environment may be especially uncomfortable for female participants to openly share their ideas . Future studies should focus on information elements that will engender lively contributions from the female participants . Similar to idea generation sessions , the study showed that knowing personal information leads to higher affective trust and knowing expertise information leads to higher cognitive trust during the idea evaluation sessions , which validated our initial hypotheses . However , there is a difference in terms of how the information elements affected the trust level across the two phases of creativity . Due to the evaluative nature of the idea evaluation task , participants are much more cognizant of the competency level that the other team members have to offer . The expertise information elements induced higher cognitive trust than both personal information and the control groups . Participants are also much more critical at assessing the feasibility of the ideas presented to them . Our results also showed that neither type of information elements led to better consensus formation . This research has some limitations . The study was conducted with a limited pool of just 36 participants . It would be useful to perform an experiment with a larger number of participants . Computer confederates were used in order to ensure that the participants received consistent treatment across different experimental conditions . Since computer bots cannot simulate human and social behavior completely , it would be interesting to see if the results are influenced if this study is conducted with a group of real participants . Participants consisted of students , and therefore the information elements were adapted to students as well and the results may not be generalizable to professional groups . The information elements used in this study will have to be extended or replaced by other information elements that are more suitable for other settings . Furthermore , this work is based on the TWAN schema , and other researchers may have different interpretations of the proposed schema . This study lasted only about an hour . In a long - term project , the fifth category from the TWAN schema—internalized norms—could be included in the analysis . It would also be interesting to apply that approach in this paper to other distributed group work besides idea generation and idea evaluation sessions . 8 . CONCLUSION AND FUTURE WORK In this work , we determined how knowing expertise and personal information of other team members affects trust in a distributed team environment , and how trust influences idea generation and idea evaluation sessions . Our findings support previous trust research by confirming that trust influences the behavior of people in a positive manner . It also supports the two - dimensional trust research dividing trust into affective trust and cognitive trust . We provide basic research about the positive correlation of trust and distributed idea generation as well as idea evaluation . We also found indications that trust support should be carefully considered for gender groups in the innovation process , and this work can be used as a first step for further exploration and design implications . The results of this study show that information elements can be effectively used to support initial trust building in the distributed team environment . The findings of this research can be used to support the development of templates that could provide communication support for distributed teams . Higher affective trust appears to have the ability to reduce evaluation apprehension in the idea generation sessions . Since it was shown that knowing 206 personal information has the biggest influence on affective trust in the idea generation session , developers could incorporate this kind of information in designing interface layouts or group process enhancements for the purpose of an idea generation session . Likewise , interface and group process designers could incorporate expertise information elements to assist distributed idea evaluation sessions . Making the expertise information available could make people more critical about their own evaluation criteria . In general , providing any information is better than providing no information at all , as anything that could help the team members relate and share experiences could help team build trust and make their work more effective . Offering information increases team trust , which is a key factor to success in innovation processes . For future work , other ways of carrying out an idea generation or idea evaluation session could be implemented . For example , a more structured technique for creating ideas could be applied instead of using brainstorming . It would also be interesting to know what information elements are needed when participants are communicating with other mediums such as video and audio chat . Another question is whether the proposed template is scalable for a bigger group . The more information is shown the higher the cognitive load is imposed upon the participants . As another future work , a potential next step could be to create a taxonomy of information elements that are suitable for different participants , tasks , and distributed settings . This knowledge could be used for supporting collaboration , and providing group process designers the possibility to better integrate the trust factor in their processes in a structured and predictable way . Additionally , further research is necessary for understanding how systems can be better designed to encourage more creative contributions from the female participants . 9 . ACKNOWLEDGEMENTS This material is based upon work supported by the National Science Foundation under Grant Nos . 1111446 , 0943262 , and 0808783 . We thank our colleagues for their advice and feedback , especially Drs . Matthew Bietz , Gary Olson , and Filippo Lanubile , as well as the entire CRADL research group at Irvine . We also thank the anonymous reviewers for their helpful comments . 10 . 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Organizational Behavior and Human Decision Processes 99 , 1 ( January 2006 ) , 16 ‐ 33 . 208 \ No newline at end of file diff --git a/silver_data/5e67f2480e041a606a86d0f2ba6c20213dc00d52.pdf.txt b/silver_data/5e67f2480e041a606a86d0f2ba6c20213dc00d52.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d3f50fc6c2fc43844238d8f1fca44a1d2d1e1d6a --- /dev/null +++ b/silver_data/5e67f2480e041a606a86d0f2ba6c20213dc00d52.pdf.txt @@ -0,0 +1 @@ +Mission - oriented innovation policies : challenges and opportunities Mariana Mazzucato * UCL Institute for Innovation and Public Purpose , University College London , Gower Street , London WC1E 6BT , UK . e - mail : m . mazzucato @ ucl . ac . uk * Main author for correspondence . Abstract This article focuses on the broader lessons from mission - oriented programs for innovation policy— and indeed policies aimed at investment - led growth . While much has been written about case studies on missions , this has not resulted in an alternative policy making toolkit . Missions—in the least— require those tools to be just as much about market cocreating and market shaping , as they are about market fixing . The article reviews the characteristics of mission - oriented programs , ooks at key fea - tures of those programs that can provide lessons , and discusses how to choose and implement mission - oriented policies , with an example . JEL classification : B52 , O25 , O33 , O38 1 . Introduction Innovation has not only a rate but also a direction : the 21st century is becoming increasingly defined by the need to respond to major social , environmental , and economic challenges . Sometimes referred to as “grand challenges , ” these include environmental threats like climate change , demographic , health and well - being concerns , and the difficulties of generating sustainable and inclusive growth . These problems are “wicked” in the sense that they are complex , sys - temic , interconnected , and urgent , requiring insights from many perspectives . Poverty cannot be solved without at - tention to the interconnections between nutrition , health , infrastructure , and education , as well as redistributive tax policy . Grand challenge thinking is being applied both in developed and developing countries , with some of the most interesting experiments around sustainability being driven by the needs of emerging economies . Turning these challenges into concrete problems that drive innovation across multiple sectors and actors have much to learn from “mission - oriented” policies that in the past has been aimed at achieving specific objectives , whether landing a man on the moon or battling climate change ( Ergas , 1987 ; Mowery 2010 ; Mazzucato , 2014 ; Mazzucato 2017 ) . Such policies require different actors ( both public and private ) and different sectors to innovate ( going to the moon required innovation in aeronautics , robotics , textiles , and nutrition ) . At the same time , to be suc - cessful , they must enable bottom - up experimentation and learning so that the innovation process itself is nurtured through dynamic feedback loops and serendipity ( Rodrik , 2004 ) . Examples of such direction - setting policies abound , including different technology policy initiatives in the United States ( Mowery et al . , 2010 ) , France ( Foray et al . , 2009 ) , the UK ( Mowery et al . , 2010 ) , and Germany ( Cantner and Pyka , 2001 ) . Mission - oriented policies are not just about throwing funds at problems but doing so in specific ways . It is for this reason that it is useful to study how specific mission - oriented agencies and organizations have worked V C The Author ( s ) 2018 . Published by Oxford University Press on behalf of Associazione ICC . This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) , which permits unrestricted reuse , distribution , and reproduction in any medium , provided the original work is properly cited . Industrial and Corporate Change , 2018 , Vol . 27 , No . 5 , 803 – 815 doi : 10 . 1093 / icc / dty034 Original article D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 whether in the military R & D programs or in areas like health ( Sampat , 2012 ) , agriculture ( Wright , 2012 ) , or energy ( Anadon , 2012 ) . In these examples , the relevant organizations made choices on what to fund , going against the more classic position that the point of policy making is simply to level the playing field . Indeed , these agencies , and the wider programs around them , “tilted” the playing field through missions aimed at a public objective , with other poli - cies needing to be introduced to make it more profitable to move in that direction ( e . g . , the US land grant system or tax reliefs for green investments ) ( Mazzucato and Perez , 2015 ) . In this article , I focus on the broader lessons from mission - oriented programs for innovation policy—and indeed policies aimed at investment - led growth . While much has been written about case studies on missions ( Mowery et al . 2010 ) , this has not resulted in an alternative policy making toolkit . Missions—in the least—require those tools to be just as much about market cocreating and market shaping , as they are about market fixing ( Mazzucato , 2016 ) . Section 2 reviews the characteristics of mission - oriented programs ; Section 3 looks at key features of those pro - grams that can provide lessons ; Section 4 discusses how to choose and implement mission - oriented policies , with an example ; and Section 5 concludes . 2 . From technological feats to wicked problems Mission - oriented policies can be defined as systemic public policies that draw on frontier knowledge to attain specific goals , or “big science deployed to meet big problems” ( Ergas , 1987 ) . While the archetypical historical mission is NASA putting a man on the moon , contemporary missions aim to address broader challenges that require long - term commitment to the development of challenges that are as much social as technological ( Foray et al . , 2012 ) . The active role being taken by governments and transnational organizations to develop strategies for a greener economy can be seen through a mission - oriented lens—as can those being developed to create more wellbeing for an ageing popula - tion , and better jobs for modern youth ( European Commission , 2011 ) . In fact , these challenges—which can be envir - onmental , demographic , economic , or social—have entered innovation policy agendas as key justifications for action , providing strategic direction for funding policies and innovation efforts . However , as discussed in the Introduction of this special issue ( Kattel and Mazzucato , 2018 ) , societal missions are much more complex because they are less clearly defined and indeed must be co - defined by many stakeholders ( how to frame the challenge around inequality is more difficult than those around the space race ) ( Foray et al . , 2012 ) . One could add that these challenges also require big regulatory and behavioral changes at the societal / national systems level . Nelson’s work on The Moon and the Ghetto ( Nelson , 2011 ) asked the demanding question of why in - novation has resulted in such difficult feats as landing a man on the moon , and yet continues to be so terribly disor - ganized and technologically unsavvy in dealing with the more earthly problems of poverty , illiteracy , and the emergence of ghettos and slums . He argued that while politics was partly the culprit , the real problem was that a purely scientific and technological solution could not solve such problems . Even at the disciplinary level there is a greater need to combine understandings of sociology , politics , economics , and technology to solve these problems , as well as to make the conscious decision to point innovation toward them . This is exactly what a well - designed mission can achieve . The so - called Maastricht Memorandum provides a detailed analysis of the differences between old and new mission - oriented projects ( Table 1 ) . Although the memorandum specifically focuses on mission - oriented programs that tackle environmental chal - lenges , its analysis applies to other contemporary challenges ( water and food supply , energy efficiency and security , disease , demographic change , etc ) . This is because these challenges all present similar characteristics , particularly that new technological solutions to address them will require long - term commitment from both public and private agents , and increasingly those in the nonprofit sector . They will in most cases also require changes in regulation and tax policies . And the diffusion of solutions to a broad base of users requires as much attention to demand - side poli - cies as to supply side . The six characteristics of contemporary missions identified in Table 1—diffusion of technologies , economic feasi - bility , shared sense of direction , decentralized control by public agencies , development of both radical and incremen - tal innovations , and enabling complementary policies—are of pragmatic importance for the promotion and implementation of mission - oriented policies . A mission - oriented approach highlights the need to make a precise diagnosis of the technological , sectoral , or na - tional innovation system that an innovation policy wishes to transform . The alignment of different types of 804 M . Mazzucato D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 capabilities is key for the success of any mission - oriented policy . These can be described as follows ( Mazzucato and Penna , 2016a ) : • Missions should be well defined . More granular definition of the technological challenge facilitates the establish - ment of intermediate goals and deliverables , and processes of monitoring and accountability . When governance is too broad , it can become faulty , and there is a risk of being captured by vested interests . • A mission does not comprise a single R & D or innovation project , but a portfolio of such projects . Because R & D and innovation is highly uncertain , some projects will fail and others will succeed . All concerned should be able to accept failures and use them as learning experiences . Furthermore , stakeholders should not be punished because of failures derived from good - faith efforts . • Missions should result in investment across different sectors and involve different types of actors . To have highest impact , missions should embrace actors across an entire economy , not just in one sector and not just in the private or public realm . • Missions require joined up policy making , whereby the priorities are translated into concrete policy instruments and actions to be carried out by all levels of the public institutions involved . While these missions should involve a range of public institutions , it is crucial that there is a strategic division of labor among them , with well - defined responsibilities for coordination and monitoring . These considerations point to the need to adopt a pragmatic approach to defining missions . Chosen missions should be feasible , draw on existing public and private resources , be amenable to existing policy instruments , and command broad and continuous political support . Missions should create a long - term public agenda for innovation policies , address a societal demand or need , and draw on the high potential of the country’s science and technology system to develop innovations . 3 . Key lessons from mission - oriented policies Mission - oriented policies can transform the policy makers’ tool kit . The next section reviews ways in which mission thinking requires an alternative lens for policy making . 3 . 1 From picking winners to picking the willing Missions are about setting concrete directions , which of course must be picked , that is , chosen strategically . The choice is not whether to pick but how : picking directions is not the same thing as “picking winners” in the sense of picking individual firms or sectors . It is about deciding that a transformation must occur in society—and making it happen . The direction will require different missions , which provide a focusing device for the different actors and Table 1 . Characteristics of old and new mission - oriented projects Defense , nuclear , and aerospace New : Environmental technologies and societal challenges Diffusion of the results outside of the core of participants is of minor importance or actively discouraged Diffusion of the results is a central goal and is actively encouraged The mission is defined in terms of the number of technical achievements , with little regard to their economic feasibility The mission is defined in terms of economically feasible tech - nical solutions to particular societal problems The goals and the direction of technological development are defined in advance by a small group of experts The direction of technical change is influenced by a wide range of actors , including government , private firms , and consumer groups Centralized control within a government administration Decentralized control with a large number of agents involved Participation is limited to a small group of firms due to the em - phasis on a small number of radical technologies Emphasis on the development of both radical and incremental innovations to permit a large number of firms to participate Self - contained projects with little need for complementary poli - cies and scant attention paid to coherence Complementary policies vital for success and close attention paid to coherence with other goals Source : Modifiedversion ofTable5 inSoete andArundel ( 1993 : 51 ) . Mission - oriented innovation policies 805 D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 sectors to collaborate to concretely achieve it . Thus missions require picking the willing : those organizations across the economy ( in different sectors , including both the public and private sphere ) that are “willing” to engage with a societally relevant mission . Missions are a new way to frame “vertical policies . ” Industrial and innovation policies require both horizontal and vertical policies working together systemically . Traditionally , industrial strategy has often focused on ( vertical ) sectoral interventions . Until the end of the 1970s , this consisted of various measures ranging from indicative planning to outright nationalization of entire industries ( e . g . , steel , coal , shipbuilding , aerospace , and so on ) . Although certain sectors might be more suited for sector - specific strategies , there are good reasons for avoiding a sectoral approach—particularly when private lobbying interests may prevail in negotiating specific provisions with the government ( Buchanan , 2003 ) , negatively influencing the industrial strategy with indirect measures ( e . g . , tax credits ) that potentially waste public funds and create little if no additionality in terms of new investment . The patent box tax policies being adopted in many countries represents an example of these misconceived policies , since there is no reason to lower tax on monopoly profits , and it has shown to have little effect on additional research investment ( Griffith et al . , 2010 ) . A mission - oriented approach uses specific challenges to stimulate innovation across sectors . Through well - defined missions—focused on solving important societal challenges related to climate change and environmental quality , demographic changes , health and well - being , mobility issues , etc . —governments have the opportunity to determine the direction of growth by making strategic investments throughout the innovation chain and creating the potential for greater spillovers across multiple sectors , including low - tech sectors ( Foray et al . , 2012 ) . Germany’s Energiewende is an interesting case of the use of an integrated strategy that addresses several sectors and technologies in the economy and enables bottom - up learning processes . With its missions to fight climate change , phase - out nuclear power , improve energy security by substituting imported fossil fuel with renewable sources , and in - crease energy efficiency , Energiewende is providing a direction to technical change and growth across different sec - tors through targeted transformations in production , distribution , and consumption . This has allowed even a traditional sector like steel to use the “green” direction to renew itself . While the steel in - dustry in many countries remains relatively low tech and subsidized , it was the Energiewende policy that placed pres - sure on steel to lower its material content . It did so through the use of a “reuse , recycle , and repurpose” strategy ( BMUB , 2016 ) . In this sense , mission - oriented policies should be focused on ways to provide sectors with transform - ation policies—less subsidies and more focused policies that reward investment and innovation that meet a need . 3 . 2 From fixing markets to actively co - shaping Missions do not fix existing markets but create new markets . Indeed , this ambition toward transformation can be seen in the explicit remit of mission - oriented organizations . Examples below from three classic mission - oriented agencies exemplify the point : the organizations are not about fixing existing markets but creating new landscapes . • NASA : To “ [ d ] rive advances in science , technology , aeronautics , and space exploration to enhance knowledge , education , innovation , economic vitality , and stewardship of Earth” ( NASA 2014 Strategic Plan ) . • DARPA : “Creating breakthrough technologies for national security is the mission of the Defense Advanced Research Projects Agency . ” • NIH : To “seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health , lengthen life , and reduce illness and disability . ” By breaking new ground , and bringing together different players , they are a better able to attract top talent , as it is an “honour” and interesting to work for them . By actively creating new areas of growth , they are also potentially able to “crowd in” business investment by increasing business expectations about where future growth opportunities might lie ( Mazzucato and Penna , 2015a ) . This proactive approach , whereby the state leads and business follows , is different from the traditional approach where the state is at best a fixer of markets . The market fixing approach has its roots in neoclassical economic theory , which asserts that competitive markets will bring about optimal outcomes if left to their own devices . This theory jus - tifies government “intervention” in the economy only if there are explicit market failures , which might arise from the presence of positive externalities ( e . g . , public goods like basic research , which require public sector spending on sci - ence ) , negative externalities ( e . g . , pollution , which require public sector taxation ) , and incomplete information 806 M . Mazzucato D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 ( where the public sector may provide incubators or loan guarantees ) . 1 On top of this , the literature on systems of in - novation has also highlighted the presence of system failures—for example the lack of linkages between science and industry—requiring the creation of new institutions enabling those linkages ( Lundvall , 1992 ) . And yet missions exemplify a more proactive approach to policy than fixing suggests . It has required public organizations to be responsible for actively shaping and creating markets and systems , not just fixing them , and for creating wealth , not just redistributing it . In a market failure framework , ex ante analysis aims to estimate benefits and costs ( including those associated with government failures ) , while ex post analysis seeks to verify whether the estimates were correct and the market failure successfully addressed . In contrast , a mission - oriented framework , which actively cocreates new markets , requires continuous and dynamic monitoring and evaluation throughout the innovation policy process . The notion of public value becomes a more useful term than a public good , since missions may be transformative across the en - tire value chain and not be limited to narrow areas where positive and negative externalities exist . 3 . 3 From fearing failure to welcoming experimentation Systemic mission - oriented policies must be based on a sound and clear diagnosis and prognosis ( foresight ) . This requires not only the identification of missing links , failures , and bottlenecks—the weaknesses or challenges of a na - tional system of innovation—but also recognition of the system’s strengths . Foresight is necessary to scrutinize future opportunities and identify how strengths may be used to overcome weaknesses . This diagnosis should be used to de - vise concrete strategies , novel institutions , and new linkages in the innovation system ( Mazzucato , 2016 ) . In its most general form , the mission - oriented framework differentiates between public policies that target the de - velopment of specific technologies in line with state - defined goals ( “missions” ) and those that aim at the institutional development of a system of innovation ( Ergas , 1987 ) . The state must therefore be able to learn from past experiences in mission - oriented innovation policy . Systems and ecosystems of innovation ( sectoral , regional and national ) require the presence of dynamic links be - tween the different actors and institutions ( firms , financial institutions , research / education , public sector funds , and intermediary institutions ) as well as horizontal links within organizations and institutions ( Freeman , 1995 ) . What , also should be emphasized , and has not been thus far in the literature on systems of innovation , is the nature of the actual actors and institutions required for innovation - led growth ( Mazzucato , 2016 ) . To stimulate the innovation process by shaping and creating technologies , sectors , and markets , missions require dynamic relationships to be developed which create trust between actors . It is essential in this process for the lead pub - lic organizations to galvanize the interests of relevant actors and organize itself so that it has the “intelligence” to think big and formulate bold policies that create a sense of ownership among diverse public , private , and academic stake - holders . It is also crucial to be able to implement the policies by coordinating the efforts of this network of stakehold - ers through the state’s convening power , brokering of trust relationships , and the use of targeted policy instruments . Because innovation is extremely uncertain , the ability to experiment and explore is key for a successful entrepreneurial state ( Hirschman , 1967 ; Rodrik , 2004 ; Mazzucato , 2013 ) . Therefore , a crucial element in organizing the state for its entrepreneurial role is absorptive capacity or institutional learning ( Cohen and Levinthal , 1990 ; Johnson , 1992 ) . Governmental agencies learn in a process of investment , discovery , and experimentation that is part of mission - oriented initiatives . This requires , as discussed in the Introduction of this special issue , “dynamic capabilities” of the public sector . Other authors have referred to processes of experimentation and learning process as “smart specialization” ( Foray et al . , 2009 ) . 2 However , smart specialization is most commonly used in connection with a market failure framework , meaning that it is seen as a discovery process for the identification of bottlenecks , failures , and missing links ( that is , market failures or market gaps ) . Smart specialization would be more usefully employed in connection to a systemic perspective on innovation policies . Key to mission - oriented innovation is the exploration of the characteristics of innovation agencies that must be in place so that they can welcome uncertainty and build explorative capacity . Breznitz and Ornston ( 2013 ) focus on the 1 Reviews of the impact of positive externalities and incomplete information on innovation financing are provided in Hall ( 2002 ) and Hall and Lerner ( 2010 ) , and more recent evidence is reviewed in Kerr and Nanda ( 2015 ) . The role for govern - ment in the face of negative externalities ( climate change ) is laid out in Jaffe et al . ( 2005 ) . 2 See also Foray’s discussion of smart specialization as mission - oriented policy in this issue . Mission - oriented innovation policies 807 D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 role of peripheral agencies , arguing that when they become too central and well funded they lose their flexibility and ability for out of the box thinking . While the importance of flexibility is no doubt important , it is also true that some of the most important innovation agencies in Europe and the United States were not so peripheral , as can be seen by DARPA’s continued success in recent years . What seems to be more important for these organizations is a degree of political independence . Indeed , Italy’s public holding company IRI ( the Istituto per la Ricostruzione Industriale established in 1933 ) had its most successful phase before the 1970s when it was public . The key lesson is that it is not about public or private , but what kind of public and what kind of private . 3 . 4 From a focus on quantity of finance to a focus on the quality By focusing on the market making role , rather than the market fixing one of missions , it also becomes clearer why they have required public investments by mission - oriented institutions along the entire innovation chain , not only up - stream basic research . Institutions like the National Science Foundation ( NSF ) have been critical to basic research ( e . g . , NSF ) , institutions like DARPA and Advanced Research Projects Agency - Energy ( ARPA - E ) to translational re - search and institutions like Small Business Innovation Research ( SBIR ) to long - term finance for companies . Block has called this distributed network of different state actors the developmental network state ( Block and Keller , 2011 ) . Better understanding of the distribution of public agencies , their positioning across the innovation chain , and the bal - ance between directive and bottom - up interactions are a key area for future study . From 1936 to 2016 , cumulative R & D expenditure by National Institutes of Health ( NIH ) has amounted to more than $ 900 billion ( in 2015 dollars ) , and since 2004 , it has exceeded $ 30 billion per year . Perhaps unsurprisingly , re - search shows that around 75 % of the most innovative drugs on the market today ( the so - called “new molecular” entities with priority rating ) owe much of their funding to the NIH ( Angell , 2005 ) . Moreover , the share of R & D ex - penditure taken by NIH in total US federal outlays in R & D has increased year on year over the past 50years . This suggests that the surge in absolute NIH - related R & D expenditure cannot simply be conceived as resulting from a generalized and proportional increase in total R & D expenditure by the government during downturns , or to simply level the playing field . Instead , it appears as a deliberate and targeted choice on where to direct public R & D funding . Due to the short - term nature of private finance , the role of public institutions is often to provide longer lead times and the willingness to engage with high uncertainty . While in some countries this has occurred through public agen - cies , such as DARPA and NIH mentioned earlier , in others , patient finance has been provided through other institu - tions , including publicly owned development banks , otherwise known as state investment banks ( SIBs ) ( Mazzucato and Semieniuk , 2017 ) . SIBs have their historical roots in the monetary agreements of Bretton Woods and the recon - struction plans for Europe following the Second World War . The idea was to create an institution that promoted fi - nancial stability through a permanent flow of finance to fund the reconstruction plan and unleash agricultural production potential , thus preventing the deleterious effects that speculative private finance could have on postwar economic recovery ( World Bank , 2015 ) . While the traditional functions of SIBs were in infrastructure investment and countercyclical lending during reces - sion when private banks restrained credit ( thus playing a classic Keynesian role ) , they have , over time , become more active as key players in the innovation system . They have provided the patient capital for innovative firms and also focused on modern societal challenges with technological “missions . ” For example , SIBs have notably filled the vac - uum left behind by private commercial banks since the financial crisis , more than trebling their investments in clean energy projects between 2007 and 2012 ( Fried et al . , 2012 ; Mazzucato and Penna , 2016b ) . A report by Bloomberg New Energy Finance finds that in 2013 , SIBs were the largest funders of the deployment and diffusion phase of re - newable energy , outpacing investment from the private sector ( Louw , 2012 ) . Examples of “mission - oriented” invest - ments include the European Investment Bank’s e 14 . 7 billion commitment to sustainable city projects in Europe ( Griffith - Jones and Tyson , 2012 ) , the efforts of KfW to support Germany’s Energiewende policies through the green - ing and modernization of German industries and infrastructures , China Development Bank’s investments in renew - able energies , and the technology fund put in place by BNDES ( 2012 ) to channel resources toward selected technologies in Brazil ( FUNTEC ) . 3 . 5 Engagement Understanding how the definition of missions can be opened up to a wider group of stakeholders , including move - ments in civil society ( as discussed by Leadbeater in this special issue ) , is a key area of interest . Indeed , it was to a 808 M . Mazzucato D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 large extent the green movement in Germany ( including but not restricted to the Green Party ) that led to a slow cu - mulative interest in society about tackling green missions , which was subsequently represented in the Energiewende agenda . Understanding more democratic processes through which missions are defined and targeted is tied to rethinking the notion of public value . Indeed , part of building a market shaping and creating framework that can guide mission - oriented thinking beyond the market failure framework involves rethinking public value beyond the notion of the “public good . ” Too often the public good concept has been used to limit and constrain the activities of public actors , creating a static distinction between those activities for business and those for policy . This means that ambitious policies—daring to reimagine the market rather than just fixing the public good problem—have then been accused of “crowding out” private activity , whether the accused are innovation agencies , public banks , or the BBC ( Mazzucato and O’Donovan , 2016 ) . But similarly , achieving public value cannot be the work only of the public sector ; hence opening up this process to include a wider set of stakeholders—involved in the definition of missions as well as the serendipitous process of how to achieve them—will be an exciting new area of analysis linked to 21st - century innovation policy targeting grand challenges . 3 . 6 From de - risking to sharing both risks and rewards Missions require a vision about the direction in which to drive an economy , focusing investment in particular areas , not just creating the horizontal ( framework ) conditions for change . Even if this is not about “picking winners” in the classical sense , but more about “picking the willing” ( those organizations across the economy interested and willing to help achieve a mission ) , crucial choices must be made on which organizations to support , the fruits of which will create some winners , but also many losers . For example , as part of Obama’s drive to create green growth , the US Department of Energy provided guaranteed loans to two green - tech companies : Solyndra ( $ 500 million ) and Tesla Motors ( $ 465 million ) . While the latter is often glorified as a success story , the former failed miserably and became the latest example in the media of a government being inefficient and unable to pick winners ( Wood , 2012 ) . However , any venture capitalist will admit that for every winning investment ( such as Tesla ) there are many losses ( such as Solyndra ) . And these types of investments are often those that private venture capitalists are not willing to make due to their exit driven model that seeks short - term returns ( usually 3 – 5 - year cycles ) . In many sectors , venture capital ( VC ) have entered after only after decades of public investments ( e . g . , NIH in biotech , or the role of SBIR in other areas , as dis - cussed in Block and Keller , 2011 ) . And some have argued that it is precisely this short - termism that has caused prob - lems in sectors like biotechnology ( Pisano , 2006 ; Lazonick and Tulum , 2011 ) . But there is also another side of the story . If the public funds do act as public forms of VC , then there is reason to argue that the rewards should be proportional to the risks actually taken . In making its downstream investments , therefore , governments can learn from portfolio strategies of venture capitalists , structuring investments across a risk space so that lower - risk investments can help to cover the higher - risk ones . In other words , if the public sector is expected to compensate for the lack of private VC money going to early - stage innovation , it should at least be able to benefit from the wins , as private VC does . Otherwise , the funding for such investments cannot be secured . It may be desirable to allow the state to reap some of the rewards from its investments for a number of other reasons ( Mazzucato and Wray , 2015 ) . Matching this type of spending with the corresponding return would provide a meas - ure of efficiency , holding policymakers accountable ; government net spending has limits dictated by the real resource capacity of the economy ; and voters will be more willing to accept the ( inevitable ) failures if they see that those are compensated by important successes . As discussed in Mazzucato ( 2013 ) and Laplane and Mazzucato ( 2018 ) , the public sector can use a number of return - generating mechanisms for its investments , including retaining equity or royalties , retaining a golden share of the IPR , using income - contingent loans , or capping the prices ( which the tax payer pays ) of those products that em - anate , as drugs do , from public funds ( Mazzucato , 2013 ) . 3 . 7 In sum , a new approach to policy making The principles above can be summarized along four big questions , which are summarized with the provocative acro - nym R - O - A - R . Policy must ROAR to lead with an ambitious challenge , nurture organizational capabilities , new Mission - oriented innovation policies 809 D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 forms of assessment , and a better sharing of rewards so that innovation - driven growth can also result in inclusive growth ( Mazzucato , 2016 ) . • Routes and directions : How to use policy to actively set a direction of change ? How to foster more dynamic ( bottom - up ) debates about possible directions to ensure enduring democratic legitimacy ? How to choose and define particular missions concretely , but with sufficient breadth to motivate action across different sectors and actors in an economy ? • Organizations : How to build decentralized networks of explorative public organisations that can learn - by - doing and welcome trial and error , with the confidence and capability to lead and form dynamic partnerships with pri - vate and third sector partners ? How to manage and evaluate progress , learning , and adaptation ; and how to use a portfolio approach to balance inevitable failure with success ? • Assessment : How to evaluate the dynamic impact of public sector market - creating investments , going beyond the static ideas embodied in cost / benefit analysis and ideas of “crowding in” and “crowding out” based on a richer con - ception of public value creation ? How to develop new indicators and assessment tools to aid decision - making ? • Risks and rewards : How to structure new types of deals between public and private sectors so that rewards are shared as much as risks taken ? These questions provide a starting point for the new categories of thought that are needed , with many more ques - tions following in relation to application in particular contexts . 4 . Choosing and implementing mission - oriented policies Missions should be broad enough to engage the public and attract cross - sectoral investment and remain focused enough to involve industry and achieve measurable success . By setting the direction for a solution , missions do not specify how to achieve success . Rather , they stimulate the development of a range of different solutions to achieve the objective . As such , a mission can make a significant and concrete contribution to meeting an Sustainable Development Goals ( SDG ) or Societal Challenge . Figure 1 illustrates the movement from broad challenges to specific missions . Figure 1 . From challenges to missions image : RTD - A . 1 based on Mazzucato ( 2018 ) . 810 M . Mazzucato D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 For example , SDG 14 “Conserve and sustainably use the oceans , seas and marine resources for sustainable devel - opment” could be broken down into various missions , for example “A plastic - free ocean . ” This could stimulate re - search and innovation in means to clear plastic waste from oceans or in reducing use of plastics , innovation in new materials , research on health impacts from micro - plastics , behavioral research , and innovation to improve recycling or drive public engagement in cleaning up beaches . Each of these areas can be broken down into particular “projects . ” This is further analyzed in the example section of this report , as well as other illustrative examples . Missions must be chosen . Yet their success will depend on the bottom - up processes that nurture innovation while “getting there . ” A culture of experimentation and risk - taking is a crucial element in the philosophy of missions . There must be incentives to “think outside the box” to come up with new solutions to address the mission objective . This requires a portfolio approach , based on different solutions , and a broad range of different interactions . The ob - jective should be addressed by multiple actors , stimulating cross - discipline academic work , with a strong focus on the intersection between natural sciences , formal sciences , social sciences , and humanities ; collaborations across dif - ferent industries ; and new forms of partnerships between the public sector , the private sector , and civil society organ - izations . Innovation itself is often characterized by feedback effects , trial and error , and serendipity ( the search for one thing leads to the discovery of another ) —picking missions that have different possibilities for solutions will en - hance the innovation dynamic itself . How should missions be picked ? The following five criteria build on the issues raised above and are clearly set out in the European Commission report on Missions that will be framing the new Framework Programme ( FP ) Horizon program ( Mazzucato , 2018 ) : ( 1 ) Bold , inspirational with wide societal relevance Missions should engage the public . They should make clear that through ambitious , bold action at the European level , solutions will be developed that will have an impact on people’s daily lives . To do this , missions must outline exciting opportunities for bold innovation—while being connected to debates in society about what the key chal - lenges are , like sustainability , inequality , health , climate change , and increasing the quality of the welfare state . ( 2 ) A clear direction : targeted , measureable , and time - bound Missions need to be very clearly framed . While enabling long - term investments , they need a specific target that can either be formulated in binary ways ( as clearly as whether man has reached the moon and returned back safely ) or quantified ( as clearly as whether a certain percentage reduction in carbon emissions against a baseline has been reached across manufacturing ) . In addition , they will need a clear time frame within which actions should take place . This needs to be long enough to allow the process to grow , for actors to build relationships and interact , while at the same time being time - limited . Without specific targets and timing , it will not be possible to determine success ( or fail - ure ) or measure progress toward success . ( 3 ) Ambitious but realistic research and innovation actions Mission objectives should be set in an ambitious manner ( taking risks ) , centered on research and innovation activ - ities across the entire innovation chain , including the feedback effects between basic and applied research . Ambitious objectives will ensure that researchers and innovators are challenged to deliver what would otherwise not be attempted ( “additionality” in research ) . Yet , the objective should be framed to be on the one hand high - risk but also realistically feasible , at least in theory , within the given time period . Setting the technical objectives unrealistically high will result in a lack of buy - in , while setting the objective too low will not incentivise extra efforts—or provide inspiration . ( 4 ) Cross - disciplinary , cross - sectoral , and cross - actor innovation Missions should be framed in such a way as to spark activity across , and among , multiple scientific disciplines ( including social sciences and humanities ) , across different industrial sectors ( e . g . , transport , nutrition , health , and services ) , and different types of actors ( public , private , third sector , and civil society organizations ) . Missions need to be chosen to address clear challenges that stimulate the private sector to invest where it would not have otherwise invested ( “additionality” in business ) . By taking a problem focused lens and not a sectoral lens , problems related to sustainability will not just involve , for example , renewable energy , but could also involve transport , strategic design , and new digital solutions , among others . Similarly , problems related to health will not only involve innovation in pharmaceuticals but also in such areas as nutrition , artificial intelligence , mobility , and new forms of digitally enhanced public service provision . Mission - oriented innovation policies 811 D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 Missions connect all relevant actors through new forms of partnerships for codesign and cocreation by focusing on targets that require multiple sectors and actors to solve . Thus , mission - oriented innovation has the possibility of leading to system - wide transformation . ( 5 ) Multiple , bottom - up solutions Missions should not be achievable by a single development path or by a single technology . They must be open to being addressed by different types of solutions . A mission - based approach is clear on the expected outcome . However , the trajectory to reach the outcome must be based on a bottom - up approach of multiple solutions—of which some will fail or have to be adjusted along the way . An example of how the five areas above could be applied to the mission to get plastic out of the oceans is described below . ( 1 ) Bold , inspirational with wide societal relevance Every year , Europeans generate 25 million tonnes of plastic waste , of which less than 30 % is recycled . Plastic makes up 85 % of beach litter . There are two strands to tackling plastic ocean pollution . First , existing plastic pollu - tion must be removed from the ocean , and second , new ways must be found to curtail the entry of new plastic waste to the oceans . Drastically reducing the amount of plastic that enters and floats in the oceans will have a substantial impact on the health of European citizens , marine life , and the environment . This mission would be closely aligned with the objectives of the recently adopted Plastics Strategy ( European Commission , 2018 ) creating an important interaction between research and innovation activities and policy development . ( 2 ) A clear direction : targeted , measureable , and time - bound 812 M . Mazzucato D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 This mission could have a clear target to reduce the amount of plastic entering the marine environment by 90 % , and of collecting more than half of the plastic currently present in our oceans , seas , and coastal areas . This would mean stopping at least 7 . 2 million tonnes of plastic entering the marine environment and collecting at least 2 million tonnes of plastic per annum from oceans , seas , and coastal areas . A very ambitious , yet achievable timeline to reach this target would be circa 5 – 10years . ( 3 ) Ambitious but realistic research and innovation actions Research and innovation activities across the entire innovation chain would be essential to reach a plastic - free ocean . Research actions would also need to target the reduction of impact of marine litter on human and animal health . Collaboration and feedback loops between basic research ( such as chemical research on characteristics of plastic ) , applied research ( such as biotech applications in packaging design ) , and entrepreneurial innovation ( such as on - sea plastic collection stations ) will be essential . Such knowledge - based research and innovation could work in conjunction with regulatory and governance actions to see that the mission target is reached . ( 4 ) Cross - disciplinary , cross - sectoral , and cross - actor innovation Oceans are a source of life for society . Many different actors of society will need to be involved ( chemical engi - neers , marine biologists , marketing experts , environmental scientists , earth observation specialists , fishermen , citizens at large , etc . ) . These different actors will need to collaborate across sectors such as chemical , biotech , marine life , consumer goods , artificial intelligence , health , design , and waste—while incorporating cross - disciplinary research such as product design , in particular design for the food processing chain ( packaging of food ) , cosmetics , tyres , and textiles . ( 5 ) Multiple , bottom - up solutions Removing plastics from the ocean is such a large and complex exercise , that it could not be achieved by a single technological ( or policy ) solution . It will require a combination of various solutions , focusing on different facets of the problem , which will need to be coordinated to reinforce each other . Interaction between projects , and experimen - tation and risk - taking , can increase additionality . For example , an autonomous ocean plastics management station might take time to implement , but the knowledge base for this station could be used to inform a hybrid , plastics - digestion mechanism , which could be implemented first , possibly in the form of distributed nets . This might kick - start an innovative and more efficient way of overall ocean plastics removal . 5 . Conclusion : a practical approach to implementing mission - oriented innovation policies The article opened with the observation that governments are increasingly seeking economic growth that is smart ( in - novation - led ) , inclusive , and sustainable . We need to see this in the context of grand social challenges such as tackling climate change , improving public health and well - being , and adjusting to demographic changes . Missions cannot happen without new tool kits . We have discussed the need for policy itself to be seen as market making and shaping rather than just fixing , and the need for particular tools including the use of patient finance , and the ability of state actors to experiment , explore , and build capacities for learning . Successful mission - oriented policy experiments require all six factors in place . They require a more dynamic fram - ing of key questions : less about picking or not picking , and more about the institutional and organizational capacity of forming broadly defined directions , through strategic deliberation . Less about static cost – benefit metrics which so often result in accusations of “crowding out” and more about dynamic assessment criteria that can nurture and evaluate market shaping processes and capture the spillovers that are created across sectors . Mission - oriented innovation policy has a major part to play in delivering better quality growth while addressing grand challenges , but the changes in mind - set , theoretical frameworks , institutional capacities and policies required are by no means trivial . Mission - oriented innovation policy is far from being a step into the unknown . As set out in this article , there is substantial theory , evidence , case studies , and experience accumulated over many decades of successful practice . It is also important to understand the challenges associated with gathering the necessary political commitment and public legitimacy behind such ambitious policies . To reap the substantial benefits from this approach , what is needed is to abandon the ideology that often informs , and misinforms , the role that the state can play in the economy . Public , private , and third sector actors can work to - gether in new ways to cocreate and shape the markets of the future . We can learn from practical policy experiences to foster a more coherent and cohesive framework across sectors , institutions , and nations . Only in this way can Mission - oriented innovation policies 813 D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 investment - led growth help address not only the growth problem but help solve the wicked 21st - century challenges ahead . Acknowledgment This article brings together key insights from an Institute for Innovation and Public Purpose ( IIPP ) working paper originally published as Mazzucato ( 2017 ) and the European Commission report on Missions ( Mazzucato , 2018 ) . References Anadon , L . D . 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Mission - oriented innovation policies 815 D o w n l oaded f r o m h tt p s : / / a c ade m i c . oup . c o m / i cc / a r t i c l e - ab s t r a c t / 27 / 5 / 803 / 5127692 b y F UND A C A O G E T U L I O VA R G AS - R J u s e r on 20 F eb r ua r y 2019 \ No newline at end of file diff --git a/silver_data/5ec10c6607886ce82370c5fb2b6c1118b1124d5c.pdf.txt b/silver_data/5ec10c6607886ce82370c5fb2b6c1118b1124d5c.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..60a3c9df504af5191de076abcebeddd32583c751 --- /dev/null +++ b/silver_data/5ec10c6607886ce82370c5fb2b6c1118b1124d5c.pdf.txt @@ -0,0 +1 @@ +International Journal of Social Robotics https : / / doi . org / 10 . 1007 / s12369 - 022 - 00933 - 7 Design , Manufacture , and Acceptance Evaluation of APO : A Lip - syncing Social Robot Developed for Lip - reading Training Programs Alireza Esfandbod 1 · Ahmad Nourbala 1 · Zeynab Rokhi 1 · Ali F . Meghdari 1 , 2 · Alireza Taheri 1 · Minoo Alemi 1 , 3 Accepted : 27 September 2022 © The Author ( s ) , under exclusive licence to Springer Nature B . V . 2022 Abstract Lack of educational facilities for the burgeoning world population , financial barriers , and the growing tendency in favor of inclusive education have all helped channel a general inclination toward using various educational assistive technologies , e . g . , socially assistive robots . Employing social robots in diverse educational scenarios could enhance learners’ achievements by motivating them and sustaining their level of engagement . This study is devoted to manufacturing and investigating the acceptance of a novel social robot named APO , designed to improve hearing - impaired individuals’ lip - reading skills through an educational game . To accomplish the robot’s objective , we proposed and implemented a lip - syncing system on the APO social robot . The proposed robot’s potential with regard to its primary goals , tutoring and practicing lip - reading , was examined through two main experiments . The first experiment was dedicated to evaluating the clarity of the utterances articulated by the robot . The evaluation was quantified by comparing the robot’s articulation of words with a video of a human teacher lip - syncing the same words . In this inspection , due to the adults’ advanced skill in lip - reading compared to children , twenty - one adult participants were asked to identify the words lip - synced in the two scenarios ( the articulation of the robot and the video recorded from the human teacher ) . Subsequently , the number of words that participants correctly recognized from the robot and the human teacher articulations was considered a metric to evaluate the caliber of the designed lip - syncing system . The outcome of this experiment revealed that no significant differences were observed between the participants’ recognition of the robot and the human tutor’s articulation of multisyllabic words . Following the validation of the proposed articulatory system , the acceptance of the robot by a group of hearing - impaired participants , eighteen adults and sixteen children , was scrutinized in the second experiment . The adults and the children were asked to fill in two standard questionnaires , UTAUT and SAM , respectively . Our findings revealed that the robot acquired higher scores than the lip - syncing video in most of the questionnaires’ items , which could be interpreted as a greater intention of utilizing the APO robot as an assistive technology for lip - reading instruction among adults and children . Keywords Socially assistive robots · Hearing - impaired · Lip - syncing · Technology acceptance B Ali F . Meghdari meghdari @ sharif . edu B Alireza Taheri artaheri @ sharif . edu 1 Social and Cognitive Robotics Laboratory , Center of Excellence in Design , Robotics , and Automation ( CEDRA ) , Sharif University of Technology , Tehran , Iran 2 Fereshtegaan International Branch , Chancellor , Islamic Azad University , Tehran , Iran 3 Department of Humanities , West Tehran Branch , Islamic Azad University , Tehran , Iran 1 Introduction In contrast to the previous prevalent concept that consid - ered speech processing an absolute auditory phenomenon , several studies have revealed that visual speech informa - tion is complementary to encoding the speech signal due to the multimodal characteristic of speech perception [ 1 , 2 ] . For example , McGurk and MacDonald demonstrated that discrepancies between the auditory signals that individuals hear and the speakers’ visual articulation information could adversely affect auditors’ perception concerning the sound they have heard [ 3 ] . Accordingly , the audio and visual infor - mation acquired concurrently from analyzing speech and focusing on the speaker’s articulatory movements leads to 123 International Journal of Social Robotics a fuller comprehension of the speech signals [ 4 , 5 ] . This concept is in accordance with the preference of infants to concentrate on speech synchronized with articulatory ges - tures rather than improperly linked audio - visual compounds [ 6 ] . Hence , lip - reading , which is concerned with eliciting speechinformationfromthemovementsofjaws , lips , tongue , and teeth within the articulation [ 7 ] , enhances human speech perception [ 8 ] . Although lip - reading capability is considered a complementary skill to improve the auditory perception of hearing people , it plays a vital role throughout hearing - impaired communications [ 9 , 10 ] . Additionally , the lack of this expertise restricts deaf people’s interlocutors solely to individuals who are accustomed to non - verbal communica - tion channels such as Sign Language and Cued Speech . This issue leads deaf people to encounter significant barriers to various two - way communication activities such as univer - sity studies [ 11 ] . Hence , the accomplishment of deaf people’s independence throughout communication is subject to hon - ing their lip - reading skills . Lip - reading is intrinsically an arduous task . Furthermore , the resemblance between the articulatory elements’ activ - ity while pronouncing some letters , such as / g / and / k / , which are not evident on the lips , hinders the lip reading procedure [ 12 ] . Easton and Basala conducted two experi - ments to assess participants’ speech recognition capability from facial gestures . Their examination showed that hearing - impaired observers could only attain an accuracy of 17 ± 12 % for 30 one - syllable words and 21 ± 11 % for 30 multi - syllable words [ 13 ] . Therefore , lip - reading is a challenging task that requires training and practice . Generally , lip - reading teaching approaches can be classified into two main cate - gories , analytic and synthetic methods , which are sometimes deployed cooperatively . In analytical strategies , trainers are asked to analyze the speech’s constituents separately , while synthetic methods require participants to synthesize the mes - sage according to all presented clues [ 14 ] . Several studies have been carried out to determine effec - tive educational programs and investigate the potential effects of the presented methods on learners’ achievements in lip reading . Creating instructional videos [ 14 , 15 ] and devel - oping various computer - based training , test software , and games [ 16 , 17 ] are well - established techniques in this field . Most of the proposed training programs are comprised of pre - recorded sequences from tutors’ articulatory components’ activities while pronouncing letters , words , and sentences . Figure 1 depicts the content of a computer game developed for lip - reading instruction . Thanks to current demographic trends , economic factors , and the growing desire for inclusive education , a flourishing demand for various educational assistive technologies has been generated . Furthermore , literature has indicated that social interaction enhances educational achievements [ 18 , 19 ] . These premises and the evolution of robotic technol - ogy have led to the development of a new concept concerned with utilizing robots that socially interface with humans as teacher’s assistants [ 20 ] . Several studies conducted in the multidisciplinary field of Human – Robot Interaction ( HRI ) have highlighted that during interaction with physical robots , individuals’ perception and engagement levels are higher than interacting with virtual agents ( on - screen characters ) [ 21 – 23 ] . Kid and Breazeal explored and compared individu - als’ responses to a robotic character , an animated character , and a human to examine the impacts of the robot’s presence on users’ perceptions . Their study revealed that the physical robot is perceived to be more credible , informative , engaging , and enjoyable to interact with compared to the animated char - acter . Two factors of robots , physical presence and real entity , could be credited for the higher effectiveness of physical robots on users’ perception in educational scenarios com - pared to fictional animated characters [ 24 ] . Leyzberg et al . scrutinized the influences of the robot’s presence on individ - uals’ learning achievements through a robot tutoring task . The outcome of this investigation revealed that a physically - present robot leads to higher learning gains than on - screen charactersandenhancesparticipants’performanceduringthe activity [ 25 ] . In addition , the robot’s embodiment makes peo - ple more likely to follow its commands and heightens its authority [ 26 , 27 ] . As well as the potential positive impacts associated with the physical presence of social robots in edu - cation , augmenting humanoid features on social robots that do not necessarily possess a fully human - like appearance improves Human – Robot Interaction [ 28 ] . The utility of socially assistive robots in the domain of education has been the subject of several studies performed in the field of HRI [ 29 – 31 ] . The outcomes of these exam - inations illustrated that social robots’ employment within several training subjects , including English as a Foreign Lan - guage ( EFL ) [ 32 – 34 ] , Mathematics [ 35 – 37 ] , Physics [ 38 ] , and Programming [ 39 , 40 ] , can be beneficial and enhance the learners’ educational gains by keeping the participants engaged [ 41 ] . The remarkable results of social robots’ utility in the context of education could be extrapolated to teach - ing cognitive skills to individuals with special needs [ 42 , 43 ] . Taheri et al . examined the efficacy of the NAO social robot in teaching music to children who were diagnosed with Autism Spectrum Disorder ( ASD ) . The findings of this study revealed that the utility of the social robot as an assistive tool alongside the teacher throughout educational interventions considerably improves learners’ performance in information attainment compared with scenarios without the robot [ 44 , 45 ] . Due to the encouraging prospects of deploying social robots within diverse training programs , in this study , we endeavor to make use of this promising educational tech - nology to enhance hearing - impaired individuals’ lip - reading 123 International Journal of Social Robotics Fig . 1 The environment of a developed lip - reading training software [ 16 ] skills . As previously mentioned , hearing - impaired people struggle to master speech reading skills because of the ambi - guity of visual speech cues . This issue , compounded with the lack of attractive instructional tools , makes the training pro - cess tedious . Therefore , due to the aforementioned merits of physically - present robots compared to animated characters with respect to improving students’ learning performance [ 25 , 46 ] , in this study , an educational assistive robotic plat - form with capabilities that suit lip - reading training programs was designed to lessen the monotony of the instructional procedure by maintaining participants’ level of engagement during the educational scenario . One of the fundamental issues that should be considered in designing a novel robotic platform is its cost - efficiency , mak - ing the robot affordable for its users . Furthermore , the robot should possess assorted complex attributes and dynamic features to maintain participants’ engagement throughout long - term interaction [ 47 ] . Thus , there is a trade - off between the cost - efficiency aspect and the robot’s capacity to per - form diverse behaviors . These criteria , alongside the robot’s capabilities in terms of accomplishing the desired purposes , determine the appropriate type of robotic head and the required degrees of freedom that should be considered to achieve an effectual design . The social robot , APO , designed in this study is a tablet - face non - humanoid robot with two degrees of freedom intended to attract and keep learners’ attention throughout training programs by performing simple motions . The pro - posed robot’s main features include being cost - effective , easily portable , having a cute design , and possessing an LCD screen to perform lip - reading exercises during interac - tion with hearing - impaired individuals . A precise lip - syncing system with the capacity to lip - sync any utterance without being formerly registered was also designed and imple - mented on the robot to achieve APO’s instructional purposes and develop a human - like articulation . The current study comprises two experiments : evaluat - ing the proposed lip - syncing system , which determines the robot’s capacity to be employed in educational scenarios , and investigating the acceptance of the APO , which affects par - ticipants’ cognitive performance and compliance behaviors during interaction with the robot [ 48 ] . In the first experi - ment , a video of a normal - hearing individual pronouncing a set of words was first given to a group of adults , who were asked to watch , identify , and write the pronounced words . Afterward , to assess the robot’s visual articulation perfor - mance , the participants were asked to sit in front of the APO robot while it articulated the same set of words and then write the words they recognized from the robot’s articula - tion . The success of the lip - syncing system was measured by comparing the participants’ perceptions of the lip - synced words in the two scenarios . In this examination , children were excluded from the research participants because they are generally less competent in lip - reading than adults . In the second experiment , the APO robot with the approved lip - syncing capability was involved in interventions to compare the acceptance of two lip - reading training tools ( the proposed robotic platform and the silently recorded video ) . This exper - iment utilized the Unified Theory of Acceptance and Use of Technology ( UTAUT ) [ 49 ] andtheSelf - AssessmentManikin ( SAM ) questionnaires [ 50 ] to investigate the robot’s accept - ability to hearing - impaired adults and children , respectively . Figure 2 depicts the study’s contents schematically . The rest of this paper is organized as follows . Section 2 is dedicated to various aspects of designing a new domestic robotic platform , the APO social robot . In Sect . 3 , the pro - posed lip - syncing system is delineated . Section 4 addresses the approach adopted to assess the developed lip - syncing system . The intervention scenario arranged to examine the acceptance of the robot is also covered in this section . In Sects . 5 and 6 , the experiments’ results and discussion are 123 International Journal of Social Robotics Fig . 2 The schematic contents of the study presented , respectively . Section 7 is devoted to the limita - tions of the current study . Finally , in Sect . 8 , the concluding remarks are declared . 2 The “APO” Robotic Platform 2 . 1 Conceptual Design Providing individuals with a high - quality education is one of the principal concerns of developed societies . The COVID - 19 pandemic not only led to burgeoning demands for remote training programs but also demonstrated the necessity of developing assistive technologies . The use of social robots as assistive tools for educational applications has attracted growing attention among researchers due to their potential to engage students and enhance their learning achievements [ 51 ] . This section is dedicated to designing a simple non - humanoid social robot named APO for educational purposes . The primary objective of developing the APO robot was to perform the role of hearing - impaired individuals’ playmates through a lip - reading educational game . Nevertheless , APO could be utilized within wide - ranging tutoring applications outside of the original intent . Cuteness , cost - effectiveness , mobility , and practicality were the main factors in the APO robot’s design . Figures 3 and 4 depict simple initial sketches and the conceptual design of the APO robot , respectively . 2 . 2 APO robot’s Hardware Design The APO robot’s platform comprises three primary com - pounds , a tablet - face head , the robot’s upper body , and lower bodyparts . Figure5demonstratestheAPOroboticplatform’s final 3D model and exploded view . The robot includes two rotary degrees of freedom ( DOFs ) ; the first one concerns the relative rotation between the robot’s upper and lower parts with respect to the roll axis , and the second relates to the pitch rotation of the robot’s head , stated in relation to the upper body part . Hence , provided that the APO’s lower body is fixed , the robot is capable of looking at any desired points located in the robot’s surroundings . The proposed robotic platform is equipped with a camera and a microphone , consideredastheaudioandvisualinputdevices , as well as a 5 - inch tablet face and speakers , regarded as output hardware that enhances the robot’s capabilities to con - duct both verbal and non - verbal communications with users . Moreover , a Raspberry Pi computer performs the robot’s internal processing . Table 1 summarizes the robot’s features . 2 . 3 APO robot’s Software Design A Graphical User Interface ( GUI ) was developedfor theAPO robot to customize the robot’s operational system to be con - trollable by non - expert users . Through the designed GUI , the users would be able to control the APO robot’s motions , stream its camera on computers or tablets , change the robot’s facial expressions , vary the color and the brightness of LEDs , and ultimately type any utterance to be lip - synced by the robot . Figure 6 depicts the environment of the GUI devel - oped for the APO robot . 123 International Journal of Social Robotics Fig . 3 The simple initial sketches of the APO robot Fig . 4 The conceptual design of the APO robot Fig . 5 a The APO robot’s 3D and b exploded view 123 International Journal of Social Robotics Table 1 The specifications of the APO social robot Dimension 23 × 23 × 21 . 2 ( cm 3 ) Weight 2 . 7 kg Actuators 2 Servomotors Sensors Camera ( Raspberry Pi Camera Module 2 ) Microphone DOFs 2 degrees of freedom Operating section Raspberry Pi 3 Operating system ROS on Ubuntu 15 Power section 12 DC Voltage 3 Lip - syncing System 3 . 1 Lip - syncing Shapes Design Adding the lip - syncing capability to a social robot may aug - ment users’ perception of the robot’s verbal communication and enhance the potential for tutoring word pronunciation . Thischaracteristicbecomesevenmorecrucialwhentherobot is interacts with hearing - impaired individuals . Hence , a real - istic design of articulatory visual elements is consequential . In an effort to produce the most sensible lip shape design , an Iranian Sign Language ( ISL ) interpreter was hired to exag - gerate the pronunciation of the letters of the alphabet . While he was pronouncing each letter ( including vowels and con - sonants ) , several images were captured in a straight - ahead position . The most detailed image of each letter was used to design the visual parts of the robot’s mouth . Figure 7 shows the design procedure performed for each letter . Individual designs were produced in this manner for all letters of the alphabet , including consonants and vowels . Figure 8 demonstrates the designed alphabet shapes . 3 . 2 Lip Morphing Developing a dictionary comprised of several sequences for eachwordisalengthyprocessandrequiresmassivecomputer memory . Hence , developing an algorithm that can receive a word as an input , disassemble it into its constituent letters , and then smoothly morph the letters into each other would be a more efficient way . A principal consideration in develop - ing an algorithm that will morph the mouth’s elements into their equivalents in successive frames to attain natural ver - bal communication is that the deformation of the mouth’s features should be minimized . The transition problem is daunting in the animation field [ 52 ] . It should be noted that although a spectator can dis - regard flaws in drawing and schematic elements , unnatural or discrete motions are not permissible [ 53 ] . The change between the initial and final point forms the fluidity of a movement , and following the path between the initial and final points in a linear manner leads to an unnatural tran - sition [ 53 ] . Adding acceleration terms produces a dynamic tween that enhances the transition’s degree of goodness [ 54 ] . In animation jargon , easing is equivalent to morphing , which is a combination of several tools used to specify the manner in which elements transition into their corresponding elements in consecutive frames . An Easing Function is a function that determines the way that the transition from the initial point to the final point occurs with respect to terms of velocity and acceleration . Several functions can be employed to fulfill this purpose . Figure 9 shows some of these functions . Following an investigation of the diverse easing functions shown in Fig . 9 , the InOutExpo function was chosen due to its ability to naturally and smoothly transition the elements of the robot’s mouth . As Fig . 9 depicts , the velocity at the begin - ning and the end of the time interval is zero in the InOutExpo Fig . 6 The APO robot’s GUI Fig . 7 The procedure of designing the robot’s lips while pronouncing various letters 123 International Journal of Social Robotics Fig . 8 The designed lip shapes for each letter of the Persian alphabet 123 International Journal of Social Robotics easing function , which leads to an aesthetically pleasing tran - sition . The equation of this function is as follows [ 53 ] : y (cid:2) ⎧⎪⎪⎪⎨ ⎪⎪⎪⎩ 0 x (cid:2) 0 2 20 x − 11 x ∈ (cid:6) 0 , 12 (cid:7) 1 − 2 − 20 x + 9 x ∈ (cid:6) 12 , 1 (cid:8) 1 x (cid:2) 1 ( 1 ) As Fig . 8 depicts , the mouth’s elements are fundamen - tally composed of curved lines that form closed curves . An operational method of making the transition from one mouth state to another within successive frames is to divide each curve into numerous points and utilize the easing function described in Eq . ( 1 ) for each point to smooth the transition process . The subsequent challenge is to find correspond - ing points between two shapes within successive frames . Achieving a natural form of speech is subject to minimizing the articulators’ deformation ; consequently , corresponding points should be chosen to minimize the sum of the tracks followed by the points during the transition . The penalty function that describes this issue is as follows : J (cid:2) (cid:9)(cid:10) Ni (cid:2) 1 ( x i − (cid:11) x i ) 2 N 2 ( 2 ) Thus , the transition problem is simplified to minimize the above cost function . The more the number of chosen points increases , the better the transition smoothness is and the greater the computational cost accrues . Ordinarily , the term easing alludes to animation made for games and HTML applications . Qt and jQuery libraries are also utilized to implement transition functions . This study used JavaScript and HTML and benefitted from the KUTE library to implement the transition function . Also , Adobe Illustrator was used to draw the articulators’ scheme . The developed module takes any word , phrase , sentence , and time parameter as inputs and performs the lip - syncing in that time . The nature of the transition was assessed by a visual exami - nation by the sign language interpreter who cooperated with our research group . Figure 10 illustrates the way that the developed algorithm executes . 4 Methodology The current study is composed of two principal experiments . The first experiment evaluates the developed lip - syncing sys - tem by comparing the participants’ perception of a set of words articulated by the robot and a human tutor , and the second experiment investigates the acceptance of the robot through interaction with adults and children . 4 . 1 Participants To appraise the explicitness of the robot’s visual articulation performance , a group of Iranian adults studying at Fereshte - gan International Branch of the Islamic Azad University and skilled in lip - reading and sign language took part in the first experiment . The under - investigation group was composed of seven deaf individuals ( three men , four women , mean age (cid:2) 20 . 43 , standard deviation (cid:2) 1 . 72 ) , seven hard - of - hearing per - sons ( four men , three women , mean age (cid:2) 20 . 29 , standard deviation (cid:2) 1 . 80 ) , and seven normal hearing students ( four men , three women , mean age (cid:2) 20 . 14 , standard deviation (cid:2) 1 . 35 ) . Another group of 34 Iranian hearing - impaired indi - viduals participated in the second experiment . This group , composed of 16 children ( nine boys , seven girls , mean age (cid:2) 7 . 63 , standard deviation (cid:2) 1 . 15 ) and 18 adults ( ten men , eight women , mean age (cid:2) 20 . 78 , standard deviation (cid:2) 1 . 87 ) , helped explore the acceptance of the APO robot through interaction with its target groups . All participants had no pre - vious experience interacting with social robots . 4 . 2 Assessment Tools In the first experiment , two types of tests , descriptive and four - choice , were adopted . Both tests were comprised of twenty - eight questions concerned with the lip - synced words . Throughout the descriptive test , participants were asked to guess and write the articulated words , while in the four - choice test , they were required to choose the correct answer among four semi - syllable words . The collection of words was composed of fourteen one - syllable and fourteen multi - syllable words . Each of the sounds ( consonants and vowels ) was repeated more than three times throughout the set . In both trials , normalized scores , defined as the ratio of the par - ticipants’ correct responses to the total number of questions , were utilized as metrics to compare the perceptibility of the utterances lip - synced by the robot and the human teacher . Furthermore , the UTAUT [ 55 ] and SAM [ 50 ] question - naires were utilized to evaluate the adults’ and children’s acceptance of the APO robot , respectively . The UTAUT questionnaire is a well - established test developed to quan - tify the acceptance of technology by older adults . This model aims to assess the users’ intention to employ a novel technology according to performance expectancy , effort expectancy , socialinfluence , andfacilitatingconditions . Indi - vidual attributes , including gender , age , experience , and voluntariness , are the main moderating influences [ 56 ] . The UTAUT model requires some modification to fit the field of the technology it is applied in ; therefore , we employed the version modified by Heerink et al . , which is suitable in the social robotics context [ 49 ] . This modified version investigates twelve constructs , including Anxiety ( ANX ) , 123 International Journal of Social Robotics Fig . 9 Penner’s easing functions [ 53 ] Attitude Towards Technology ( ATT ) , Facilitating Condi - tions ( FC ) , Intention to Use ( ITU ) , Perceived Adaptiveness ( PAD ) , Perceived Enjoyment ( PENJ ) , Perceived Ease of Use ( PEOU ) , Perceived Sociability ( PS ) , Perceived Usefulness ( PU ) , Social Influence ( SI ) , Social Presence ( SP ) , and Trust . The second questionnaire , i . e . , the SAM model , was used to assess the robot’s acceptability by children . This test mea - sures pleasure , arousal , and dominance concerned with the social robot [ 50 ] . Both questionnaires , UTAUT and SAM , were scored via Five - Likert pictorial scales ( range : 1 – 5 ) [ 48 , 57 ] . 4 . 3 Procedure 4 . 3 . 1 Experiment one : Evaluation of the Proposed Lip - Syncing System In the study’s first phase , an educational lip - reading game was designed to evaluate the proposed lip - syncing system’s capacity for conveying messages to hearing - impaired peo - ple . The objective of this game was to compare the APO robot with human articulations . To this end , we asked a tutor working at the Fereshtegan University to lip - sync a set of Per - sian words and recorded a video while she was articulating them . The robotic platform also lip - synced the same set of words in a different succession . Subsequently , the adult par - ticipants were asked to watch the silent sequence and specify the terms they had recognized due to their greater expertise in lip - reading compared to children . Then , they were asked to take the words perception test with the APO robot in the same manner . Afterward , the students were scored accord - ing to the number of words correctly identified in each of the two lip - reading scenarios . The lip - reading games were carried out twice to provide the participants with clues about the correct answers . The first time , they were asked to note down the words they saw , while the second time , they were required to choose the correct answers from a four - choice test . Furthermore , the counterbalancing technique was used to eliminate the order effect . In this regard , half of the par - ticipants first encountered the robot and then the lip - reading video , while others underwent the reverse order . 4 . 3 . 2 Experiment Two : Investigation of the Robot’s Acceptability The second phase of the study was devoted to comparing the acceptability of the two lip - reading training programs , the lip - reading tutoring program taught by the APO robot and the one performed by the video of the tutor while she was lip - syncing the same words . The investigation was con - ducted on both adults and children . In this regard , first , the adult participants were asked to complete the UTAUT ques - tionnaire . Afterward , the acceptability test was performed through interaction with the children . The SAM question - naire was employed in this examination to quantify the children’s emotional responses . Following the meeting and playing with the APO robot . Figure 11 depicts the experi - mental setup in the two experiments . 5 Results 5 . 1 Experiment one : Evaluation of the Proposed lip - syncing System The first experiment was dedicated to comparing the partici - pants’ perception of the APO robot’s visual articulation and the video of the tutor lip - syncing the same set of words . In Fig . 10 The morphing algorithm demo while pronouncing ( / A (cid:2)(cid:3) b / ) , which means water in Persian 123 International Journal of Social Robotics Fig . 11 a Experiment one : Evaluation of the proposed lip - syncing system , b Experiment two : Investigation of the robot’s acceptability the first step of the experiment , the participants were asked to write the utterances they had recognized in the two lip - reading games , while in the second step , they were asked to choose correct answers from a four - choice questionnaire . To check the quality of the robot’s articulatory system , the participants’ normalized scores in the two lip - reading sce - narios were first statistically analyzed ( t - test ) using Minitab software . Then , the resulting p - values were utilized as met - rics to determine whether participants’ perceptions of the two lip - reading games had significant differences . Furthermore , a similarity score that states the number of words identically labeled ( correctly or incorrectly ) in the lip - reading games was also defined to measure the comparability of the robot’s lip - syncing system and the human articula - tion . In other words , the similarity score explains the number of words similarly identified in the two games , regardless of their correctness , normalized by the total words . Table 2 summarizedtheparticipants’normalizedscoresinthisexper - iment . 5 . 2 Experiment Two : Investigation of the Robot’s Acceptability In the second experiment , the acceptance of the APO robot among adults and children was investigated in comparison with the recorded lip - syncing video throughout the designed lip - reading games . Following the end of the two games , the children were asked about their preference between the two educational games to investigate the Child - Robot interaction . The SAM questionnaire was given to the children following the end of the two games , and they were asked to answer each item via Likert scores ranging from one to five . The participants’ scores concerning the robot and the recorded video , as well as the statistical analysis ( t - test ) results , are summarized in Table 3 . To scrutinize the robot’s acceptability to the adult par - ticipants , the UTAUT questionnaire was employed . Like the previous procedure , the adults were asked to declare their preference for the APO robot and the recorded video following the two games . Afterward , the partici - pants were asked to complete the UTAUT test via the Five - Likert scale . To probe into the differences between the acceptance of the robot and the recorded video , statistical analysis was performed . Table 4 presents the results concerned with the robot and the video acceptance scores . 6 Discussion 6 . 1 Experiment One : Evaluation of the Proposed Lip - syncing System Throughoutthefirstexperiment , certainwordswerecorrectly recognized in the human tutor’s video and the APO robot lip - syncing , while other items were misunderstood in lip - reading games . Interestingly , most of these misconceptions about the articulated words were the same . The statistical analysis ( t - test ) of the above results ( for an alpha of 0 . 05 ) revealed that the scores corresponding to the participants’ recognition of monosyllabic words in the lip - syncing video were significantly higher than the APO robot’s articulation ( p < 0 . 05 ) . However , their comprehension of multisyllabic words was not significantly different in the two games ( p > 0 . 05 ) . Therefore , we concluded that the robot’s artic - ulation of words composed of two or more syllables is comparable with the human tutor . However , a power analysis using G * Power 3 . 1 Software [ 58 ] revealed that determin - ing a statistically significant difference requires at least N (cid:2) 35 participants for this experiment based on the medium effect size of 0 . 5 , a power level of 0 . 8 , and a signifi - cance level of 0 . 05 . Hence , our findings , with respect to the comparability of the robot and the human tutor articu - lation of multisyllabic words , should be reported cautiously due to the limited number of participants . Additionally , the preliminary exploratory findings of the first experi - ment demonstratedthat thehard - of - hearingparticipants were more competent at both lip - reading games than the other groups . 123 International Journal of Social Robotics Table 2 The participants’ scores in the two lip - reading scenarios ( the APO robot and the human tutor ) Group Items Test type Score’s mean ( SD ) Similarity p - value Robot Video Deaf group Monosyllabic Descriptive 0 . 3163 ( 0 . 0382 ) 0 . 4184 ( 0 . 1046 ) 0 . 4538 0 . 032 Four - Choice 0 . 5408 ( 0 . 0998 ) 0 . 6531 ( 0 . 0764 ) 0 . 5936 0 . 036 Multisyllabic Descriptive 0 . 5816 ( 0 . 0961 ) 0 . 6327 ( 0 . 0643 ) 0 . 5714 0 . 266 Four - Choice 0 . 6735 ( 0 . 0382 ) 0 . 7143 ( 0 . 0583 ) 0 . 7032 0 . 147 Hard hearing group Monosyllabic Descriptive 0 . 3571 ( 0 . 0825 ) 0 . 4694 ( 0 . 1080 ) 0 . 5128 0 . 049 Four - Choice 0 . 6020 ( 0 . 0998 ) 0 . 7245 ( 0 . 0764 ) 0 . 7593 0 . 024 Multisyllabic Descriptive 0 . 6531 ( 0 . 0961 ) 0 . 6735 ( 0 . 0697 ) 0 . 7863 0 . 657 Four - Choice 0 . 7449 ( 0 . 0909 ) 0 . 7755 ( 0 . 0868 ) 0 . 8542 0 . 531 Normal - hearing group Monosyllabic Descriptive 0 . 2857 ( 0 . 0922 ) 0 . 3878 ( 0 . 0810 ) 0 . 4126 0 . 048 Four - Choice 0 . 4796 ( 0 . 1069 ) 0 . 6020 ( 0 . 0818 ) 0 . 4921 0 . 033 Multisyllabic Descriptive 0 . 5306 ( 0 . 0697 ) 0 . 6020 ( 0 . 0909 ) 0 . 5574 0 . 125 Four - Choice 0 . 6429 ( 0 . 0583 ) 0 . 6939 ( 0 . 0795 ) 0 . 6828 0 . 196 Table 3 The SAM test results and analysis comparing the children’s acceptance of the APO robot and the recorded video Item Score’s mean ( SD ) p - value Robot Video Pleasure 4 . 467 ( 0 . 443 ) 3 . 883 ( 0 . 472 ) 0 . 011 Arousal 3 . 783 ( 0 . 846 ) 3 . 100 ( 0 . 545 ) 0 . 046 Dominance 3 . 867 ( 0 . 864 ) 3 . 300 ( 0 . 706 ) 0 . 126 6 . 2 Experiment Two : Investigation of the Robot’s Acceptability A review of thechildren’s acceptanceof therobot duringtheir interaction in the study showed that the APO achieved higher scores than the recorded video on all items of the SAM ques - tionnaire . Additionally , according to the statistical analysis ( for an alpha of 0 . 05 ) , significant differences were observed for the metrics concerned with pleasure and arousal in the two scenarios ( p < 0 . 05 ) , while the dominance item showed no significant difference between the two games ( p > 0 . 05 ) . The acceptance of the robot among adults , measured via the UTAUT questionnaire , indicated that the average scores of the APO robot for the ATT , FC , ITU , PAD , PENJ , PS , PU , SI , and SP items , were higher than the recorded video , but significant differences were only observed ( p < 0 . 05 ) for the ATT , ITU , PAD , PENJ , PS , SI , and SP constructs . The robot’s significantly higher scores in the PAD and PENJ items are due to the APO robot’s enjoyability and easiness of use . The SP and PS constructs correspond to the robotic platform’s adaptability and sociability , leading to a positive image of the APO characteristics . Higher scores on the ATT and ITU items show a higher engagement level for the social robot Table 4 The UTAUT test results and analysis comparing the adults’ acceptance of the APO robot and the recorded video Item Score’s mean ( SD ) p - value Robot Video ANX 2 . 700 ( 1 . 160 ) 4 . 000 ( 0 . 816 ) 0 . 010 ATT 4 . 200 ( 0 . 789 ) 2 . 900 ( 1 . 101 ) 0 . 007 FC 3 . 400 ( 0 . 966 ) 3 . 100 ( 0 . 994 ) 0 . 503 ITU 4 . 100 ( 0 . 876 ) 3 . 100 ( 0 . 994 ) 0 . 028 PAD 3 . 500 ( 0 . 850 ) 2 . 400 ( 0 . 966 ) 0 . 015 PENJ 4 . 400 ( 0 . 699 ) 3 . 100 ( 0 . 738 ) 0 . 001 PEOU 3 . 100 ( 0 . 994 ) 3 . 700 ( 1 . 059 ) 0 . 208 PS 4 . 200 ( 0 . 919 ) 3 . 000 ( 1 . 333 ) 0 . 031 PU 4 . 000 ( 0 . 816 ) 3 . 900 ( 0 . 876 ) 0 . 795 SI 3 . 800 ( 1 . 033 ) 2 . 700 ( 1 . 160 ) 0 . 038 SP 3 . 600 ( 0 . 966 ) 2 . 700 ( 0 . 949 ) 0 . 050 Trust 3 . 700 ( 0 . 949 ) 3 . 800 ( 1 . 033 ) 0 . 824 compared to the video throughout the lip - reading training procedure . The significant difference in the SI item describes the participants’ preference to share this technology with oth - ers . The outcome of this examination should be reported with caution due to the limited number of participants calculated by the conducted power analysis . 7 Limitations and Future Work The robotic platform developed in the current study utilized an LCD screen to lip - sync the words . One of the robot’s limi - tationsisthe2Dtraitoftheproposedlip - syncingsystem . This issue complicates the participants’ perception of the APO 123 International Journal of Social Robotics robot’s utterances . Another limitation of our study was the number of volunteers . Due to the COVID - 19 pandemic , only a small number of people agreed to take part in our examina - tion . Our future work objective is to enhance the lip - syncing system to be more analogous to the articulation of a human tutor . Additionally , future studies will focus on designing an attractive collaborative game to increase hearing - impaired people’s lip - reading skills by developing a lip - reading sys - tem using CNN and RNN models to improve the lip - reading capability of the APO robot . In this way , the robot will be regarded as the playmate of learners playing the interac - tive lip - reading game . This interaction engages individuals through learning procedures and assesses their performance while lip - syncing the target words . Furthermore , comparing the lip - reading attainments through Robot - Assisted Therapy ( RAT ) sessions and conventional lip - reading instructions is another possible subject for future experiments . 8 Conclusion This study proposed a new tablet - face robotic platform , APO , that benefits from a lightweight and portable plat - form designed to enhance lip - reading training programs for hearing - impaired individuals . In this regard , a lip - syncing system based on the visual articulation of a sign language interpreter was developed and implemented on the robot to accomplish the robot’s educational objective . To assess the efficacy of the developed robotic platform’s desired objec - tive , two main experiments were conducted , evaluating the proposed lip - syncing system and investigating the accep - tance of the robot among children and adults . The first experiment’s analysis indicated that the proposed lip - syncing system performed appropriately regarding the articulation of compound words . Moreover , the exploratory outcomes of the investigation revealed that hard - of - hearing participants were more adept at comprehending lip - synced words . The outcome of the second experiment , the examination of the acceptance of the robot among both adults and children , also demonstrated that the robot scored higher acceptance than a recorded video when employed as assistive technology for a lip - reading training program . However , the reported out - comes of this study should be interpreted cautiously due to the small number of subjects , as estimated by the power anal - ysis . Acknowledgements This study was funded by the “Dr . Ali Akbar Siassi Memorial Research Grant Award” and Sharif University of Tech - nology ( Grant No . G980517 ) . We also thank Mrs . Shari Holderread for the English editing of the final manuscript . Authors’ contributions All authors contributed to the study’s concep - tionand design . Materialpreparation , data collection , andanalysis were performed by Alireza Esfandbod , Ahmad Nourbala , and Zeynab Rokhi . The first draft of the manuscript was written by Alireza Esfandbod and Zeynab Rokhi . All authors read and approved the final manuscript . Data availability All data from this project ( videos of the sessions , results of the questionnaires , scores of performances , etc . ) are available in the archive of the Social & Cognitive Robotics Laboratory . Code availability All of the codes are available in the archive of the Social & Cognitive Robotics Laboratory . In case the readers need the codes , they may contact the corresponding author . Declarations Conflictofinterest Author Alireza Taheri has received a research grant from the Sharif University of Technology ( Grant No . G980517 ) . The authors Alireza Esfandbod , Ahmad Nourbala , Zeynab Rokhi , Ali F . Meghdari , and Minoo Alemi assert that they have no conflict of interest . Ethical approval Ethical approval for the protocol of this study was provided by the Iran University of Medical Sciences ( # IR . IUMS . REC . 1395 . 95301469 ) . Consent to participate Informed consent was obtained from all indi - vidual participants included in the study . Consent for publication The authors affirm that human research par - ticipants provided informed consent for the publication of the image in Figs . 6 and 11 . All of the participants have consented to the submission of the results of this study to the journal . References 1 . DoddB ( 1979 ) Lipreadingininfants : Attentiontospeechpresented in - and out - of - synchrony . Cogn Psychol 11 ( 4 ) : 478 – 484 2 . L . D . Rosenblum ( 2008 ) Primacy of multimodal speech perception 3 . McGurk H , MacDonald J ( 1976 ) Hearing lips and seeing voices . Nature 264 ( 5588 ) : 746 – 748 4 . Sumby WH , Pollack I ( 1954 ) Visual contribution to speech intel - ligibility in noise . J Acoust Soc Am 26 ( 2 ) : 212 – 215 5 . Erber NP ( 1975 ) Auditory - visual perception of speech . J Speech Hearing Disorders 40 ( 4 ) : 481 – 492 6 . 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Venkatesh V , Thong JY , Xu X ( 2016 ) Unified theory of acceptance and use of technology : a synthesis and the road ahead . J Assoc Inf Syst 17 ( 5 ) : 328 – 376 57 . Striepe H , Donnermann M , Lein M , Lugrin B ( 2021 ) Modeling and evaluating emotion , contextual head movement and voices for a social robot storyteller . Int J Soc Robot 13 ( 3 ) : 441 – 457 58 . Faul F , Erdfelder E , Buchner A , Lang A - G ( 2009 ) Statistical power analyses using G * Power 3 . 1 : tests for correlation and regression analyses . Behav Res Methods 41 ( 4 ) : 1149 – 1160 Publisher’s Note Springer Nature remains neutral with regard to juris - dictional claims in published maps and institutional affiliations . Springer Nature or its licensor ( e . g . a society or other partner ) holds exclusive rights to this article under a publishing agreement with the author ( s ) or other rightsholder ( s ) ; author self - archiving of the accepted manuscriptversionofthisarticleissolelygovernedbythetermsofsuchpublishingagreementandapplicablelaw . Alireza Esfandbod is a Ph . D . candidate at the Mechanical Engineering Department of the Sharif University of Technol - ogy , Tehran , Iran . His research interests include Mechatronics , Robotics , Social Robotics , Pattern Recognition , Computer Vision , Artificial Intelligence , Machine Learning , and their applications in Human - Robot Interaction . Ahmad Nourbala graduated with a Mechanical Engineering M . Sc . from the Sharif Univer - sity of Technology . His research interests are Mechanical Design , Manufacturing , System Dynam - ics , and Control . Zeynab Rokhi received her M . Sc . degree in Mechanical Engineering from the Sharif University of Technology . Her research interests are Social Robotics , Computer Vision , Deep learning , Machine learning , and Human - Robot Interaction . Ali F . Meghdari is a Professor Emeritus of Mechanical Engi - neering and Robotics at Sharif University of Technology ( SUT ) in Tehran . Professor Meghdari has performed extensive research in various areas of robotics ; social and cognitive robotics , mechatronics , bio - robotics , and modeling of biomechanical sys - tems . He has been the recipient of various scholarships and awards , the latest being : the 2012 Allameh Tabatabaei distinguished profes - sorship award by the National Elites Foundation of Iran ( BMN ) , the 2001 Mechanical Engineering Distinguished Professorship Award from the Ministry of Science , Research & Technology ( MSRT ) in Iran , and the 1997 ISESCO Award in Technology from Morocco . He is the founder of the Centre ofExcellence in Design , Robotics , and Automation ( CEDRA ) , an affliate member of the Iranian Academy of Sciences ( IAS ) , a Fellow of the American Society of Mechanical Engineers ( ASME ) , and the Founder and Chancellor of Islamic Azad University - Fereshtegaan International Branch ( for students with special needs ; primarily the Deaf ) . Alireza Taheri is an Assistant Professor of Mechanical Engi - neering with an emphasis on Social and Cognitive Robotics at Sharif University of Tech - nology , Tehran , Iran . He is the Head of the Social and Cognitive Robotics Lab at Sharif University of Technology . 123 International Journal of Social Robotics Minoo Alemi received her Ph . D . in Applied Linguistics from Allameh Tabataba’i University in 2011 . She is currently an Associate Professor and Division Head of Applied Linguistics at the Islamic Azad University , West - Tehran Branch . She is the cofounder of Social Roboticsin Iran , a title she achieved as a Post - Doctoral research associate at the Social Robotics Laboratory of the Sharif University of Technology . Her areas of interest include discourse analysis , interlanguage pragmatics , materials development , and RALL . Dr . Alemi has been the recipient of various teaching and research awards from Sharif University of Technology , Allameh Tabataba’i University , Islamic Azad University , and Int . Conf . on Social Robotics ( ICSR - 2014 ) . 123 \ No newline at end of file diff --git a/silver_data/61692dbf52c6fb8185c37476a50f42a2b9f655cc.pdf.txt b/silver_data/61692dbf52c6fb8185c37476a50f42a2b9f655cc.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..87478730a8e74b74487e29667e7b87f1a6c781fe --- /dev/null +++ b/silver_data/61692dbf52c6fb8185c37476a50f42a2b9f655cc.pdf.txt @@ -0,0 +1 @@ +1 Novel features of centriole polarity and cartwheel stacking revealed by cryo - 1 tomography 2 3 4 5 Sergey Nazarov 1 , 2 , 4 , Alexandra Bezler 1 , 4 , Georgios N Hatzopoulos 1 , 4 , Veronika 6 Nemčíková Villímová 1 , Davide Demurtas 2 , Maeva Le Guennec 3 , Paul Guichard 3 and 7 Pierre Gönczy 1 8 9 10 1 Swiss Institute for Experimental Cancer Research ( ISREC ) , School of Life Sciences , 11 Swiss Federal Institute of Technology Lausanne ( EPFL ) , Switzerland 12 2 Interdisciplinary Centre for Electron Microscopy ( CIME ) , Swiss Federal Institute of 13 Technology Lausanne ( EPFL ) , Switzerland 14 3 Department of Cell Biology , University of Geneva , Sciences III , Geneva , Switzerland 15 4 These authors contributed equally to this work . 16 17 18 19 Correspondence : pierre . gonczy @ epfl . ch 20 21 22 Keywords : centriole , cartwheel , microtubules , cryo - electron tomography , 23 Trichonympha , Teranympha , SAS - 6 24 25 26 Running title : cryo - ET of centriolar cartwheel 27 28 2 Summary 29 30 Centrioles are polarized microtubule - based organelles that seed the formation of cilia , 31 and which assemble from a cartwheel containing stacked ring oligomers of SAS - 6 32 proteins . A cryo - tomography map of centrioles from the termite flagellate 33 Trichonympha spp . was obtained previously , but higher resolution analysis is likely to 34 reveal novel features . Using sub - tomogram averaging ( STA ) in T . spp . and 35 Trichonympha agilis , we delineate the architecture of centriolar microtubules , pinhead 36 and A - C - linker . Moreover , we report ~ 25 Å resolution maps of the central cartwheel , 37 revealing notably polarized cartwheel inner densities ( CID ) . Furthermore , STA of 38 centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel 39 architecture and a distinct filamentous CID . Fitting the CrSAS - 6 crystal structure into 40 the flagellate maps and analyzing cartwheels generated in vitro indicates that SAS - 6 41 rings can directly stack onto one another in two alternating configurations : with a slight 42 rotational offset and in register . Overall , improved STA maps in three flagellates 43 enabled us to unravel novel architectural features , including of centriole polarity and 44 cartwheel stacking , thus setting the stage for an accelerated elucidation of underlying 45 assembly mechanisms . 46 47 3 Introduction 48 49 Centrioles are evolutionarily conserved microtubule - based organelles that seed the 50 formation of primary cilia , as well as of motile cilia and flagella . Despite significant 51 progress in recent years , the mechanisms orchestrating centriole assembly remain 52 incompletely understood , in part because the detailed architecture of the organelle has 53 not been fully unraveled . 54 The centriole is a 9 - fold radially symmetric cylindrical organelle typically ~ 500 55 nm in length and ~ 250 nm in diameter , which is polarized along a proximal - distal axis 56 ( reviewed in Azimzadeh & Marshall , 2010 ; Gönczy & Hatzopoulos , 2019 ) . In the 57 proximal region lies a likewise symmetrical cartwheel usually ~ 100 nm in length , which 58 is critical for scaffolding the onset of centriole assembly ( reviewed in Guichard et al , 59 2018 ; Hirono , 2014 ) . In transverse view , the cartwheel is characterized by a central 60 hub from which emanate 9 spokes that extend towards peripherally located 61 microtubule triplets . 62 The SAS - 6 family of proteins is thought to constitute the principal building block 63 of the cartwheel and is essential for its formation across systems ( Culver et al , 2009 ; 64 Dammermann et al , 2004 ; Jerka - Dziadosz et al , 2010 ; Kilburn et al , 2007 ; Kleylein - 65 Sohn et al , 2007 ; Leidel et al , 2005 ; Nakazawa et al , 2007 ; Rodrigues - Martins et al , 66 2007 ; Strnad et al , 2007 ; Yabe et al , 2007 ) . SAS - 6 proteins contain an N - terminal 67 globular head domain , followed by a ~ 45 nm long coiled - coil and a C - terminal region 68 predicted to be unstructured ( Dammermann et al , 2004 ; Kitagawa et al , 2011 ; Leidel 69 et al , 2005 ; Van Breugel et al , 2011 ) . In vitro , SAS - 6 proteins readily homodimerize 70 through their coiled - coil moiety ; such homodimers can undergo higher order 71 oligomerization through an interaction between neighboring head domains ( Kitagawa 72 et al , 2011 ; Nievergelt et al , 2018 ; Van Breugel et al , 2011 ) . Ultimately , this results in 73 the formation of a SAS - 6 ring with a central hub harboring 18 juxtaposed head 74 domains , from which emanate 9 paired coiled - coils that extend peripherally . Such ring 75 oligomers are ~ 23 nm in diameter and bear striking resemblance with a transverse 76 section of the cartwheel observed in cells . Moreover , recombinant Chlamydomonas 77 reinhardtii SAS - 6 ( CrSAS - 6 ) possesses the ability not only to self - assemble into ring 78 oligomers , but also to undergo stacking of such entities , together generating a structure 79 akin to the cartwheel present in the cellular context ( Guichard et al , 2017 ) . 80 Additional features of cartwheel architecture have been unveiled through cryo - 81 electron tomography ( cryo - ET ) of centrioles purified from Trichonympha ( Guichard et 82 al , 2013 , 2012 ) . Three closely related species of these unicellular symbiotic flagellates , 83 T . campanula , T . collaris and T . sphaerica - referred to collectively as T . spp . , populate 84 4 the gut of Zootermopsis damp wood termites ( Tai et al , 2013 ) . T . spp . centrioles are 85 particularly well suited for sub - tomogram averaging ( STA ) of cryo - ET specimens 86 because they harbor an exceptionally long cartwheel - bearing region , reaching several 87 microns ( Gibbons & Grimstone , 1960 ; Guichard et al , 2013 , 2012 ) . STA of purified T . 88 spp . centrioles yielded a ~ 34 Å map ( Fourier Shell Correlation ( FSC ) criterion 0 . 143 ) , 89 which established that the cartwheel comprises stacks of ring - containing elements 90 bearing a central hub from which emanate spokes . Suggestively , a 9 - fold symmetrical 91 SAS - 6 ring generated computationally from the crystal structure of the CrSAS - 6 head 92 domain plus the first 6 heptad repeats of the coiled - coil ( CrSAS - 6 [ 6HR ] ) could be fitted 93 in the hub of this STA map ( Guichard et al , 2012 ) . However , some vertical hub 94 densities remained unaccounted for upon such fitting , raising the possibility that 95 additional components are present . In addition , the T . spp . STA map revealed 9 - fold 96 symmetrical cartwheel inner densities ( CID ) inside the hub proper , with contacts 97 between hub and CID occurring where the fitted CrSAS - 6 [ 6HR ] head domains interact 98 with one another ( Guichard et al , 2013 ) . 99 The T . spp . STA map uncovered a vertical periodicity of ~ 8 . 5 nm between 100 spoke densities emanating from the hub ( Guichard et al , 2013 , 2012 ) . Two such 101 emanating densities merge with one another as they extend towards the periphery , 102 where the vertical spacing between merged entities is hence of ~ 17 nm . There , spokes 103 abut a pinhead structure that bridges the central cartwheel with peripheral microtubule 104 triplets . The STA map also revealed the architecture of the A - C linker , which connects 105 the A - microtubule from a given triplet with the C - microtubule of the adjacent one . 106 Interestingly , both pinhead and A - C linker are polarized along the proximal - distal 107 centriole axis ( Guichard et al , 2013 ) . Given that the centrally located hub and CID were 108 not noted at the time as being polarized , this led to the suggestion that the pinhead 109 and the A - C linker might be critical for imparting polarity to the entire organelle 110 ( Guichard et al , 2013 ) . Further cryo - ET analysis of procentrioles from Chlamydomonas 111 and mammalian cells established that aspects of A - C linker architecture are 112 evolutionarily conserved , including the attachment points on the A - and C - 113 microtubules ; moreover , novel features were revealed , such as a vertical crisscross 114 pattern for the A - C linker in Chlamydomonas ( Greenan et al , 2018 ; Li et al , 2019 ) . 115 Whether the central elements of the cartwheel , including the CID , are likewise 116 conserved beyond T . spp . is unclear . 117 Considering that the earlier work in T . spp . was conducted without direct 118 electron detector and that software improvements have occurred since , we sought to 119 achieve a higher resolution map of the T . spp . cartwheel - bearing region . Moreover , to 120 explore the evolutionarily conservation of cartwheel architecture , we investigated two 121 5 other flagellates living in the gut of termites that might likewise harbor long cartwheels 122 well suited for STA . 123 124 125 Results 126 127 Exceptionally long cartwheel region in Trichonympha agilis 128 We set out to obtain a high - resolution STA map of the native cartwheel in T . spp . 129 Moreover , we likewise aimed at investigating Trichonympha agilis , a symbiotic 130 flagellate that lives in the gut of the Japanese termite Reticulitermes speratus ( Ohkuma 131 and Kudo , 1998 ) . This choice was guided by the fact that transcriptomic and genomic 132 information is being assembled in T . agilis ( Yuichi Hongoh , Tokyo Institute of 133 Technology , Japan , personal communication ) , which will be instrumental to map 134 proteins onto the STA map should sub - nanometer resolution be reached in the future . 135 As shown in Fig . 1A , T . agilis bears a large number of flagella , which stem from 136 similarly numerous centrioles inserted below the plasma membrane ( Kubai , 1973 ) . 137 Many of these flagella are tightly packed in a region called the rostrum located at the 138 cell anterior ( Fig . 1A , arrow ) . To determine the length of the cartwheel - bearing region 139 of T . agilis centrioles , cells were resin - embedded and analyzed by transmission 140 electron microscopy ( TEM ) , focusing on the rostral region . Longitudinal views 141 established that the cartwheel - bearing region is ~ 2 . 3 µ m in length on average ( Fig . 1B , 142 pink line ; SD = 0 . 36 µ m , N = 8 ) . This is less than the ~ 4 µ m observed in T . spp . ( Guichard 143 et al , 2013 ) , yet over 20 times the size of the ~ 100 nm cartwheel in centrioles of most 144 systems , including Chlamydomonas reinhardtii and Homo sapiens ( Guichard et al , 145 2017 , 2010 ; O’Toole and Dutcher , 2014 ) . In addition , we found that the centriole distal 146 region devoid of cartwheel is ~ 0 . 4 µ m in T . agilis ( Fig . 1B , white line ; SD = 0 . 07 µ m , 147 N = 6 ) , similar to its dimensions in other systems ( Guichard et al , 2013 ; Le Guennec et 148 al , 2020 ) . 149 We also analyzed transverse sections of resin - embedded T . agilis centrioles 150 using TEM . As shown in Fig . 1C , we found the characteristic features of the cartwheel - 151 bearing region , including a central hub from which emanate 9 spokes that extend 152 towards peripheral microtubule triplets . In addition , we noted the presence of the 153 pinhead and the A - C linker , as well as of the triplet base connecting these two elements 154 ( Gibbons and Grimstone , 1960 ; Vorobjev and Chentsov , 1980 ) , which is more 155 apparent in the circularized and symmetrized image ( Fig . 1C ) . 156 6 Overall , given the presence of a long cartwheel - bearing region , we conclude 157 that T . agilis also provides a suitable system to investigate the architecture of the 158 proximal part of the centriole using cryo - ET and STA . 159 160 Novel features revealed by improved STA of Trichonympha centrioles 161 Using a direct electron detector , we acquired tilt series of purified T . spp . centrioles , 162 focusing on the proximal cartwheel - bearing region ( Fig . S1A , S1B ) , followed by 163 tomogram reconstruction and STA ( Fig . 2A - E ; for all datasets see Fig . S1C - F for raw 164 tomograms , as well as Fig . S1G - J and Table S1 for processing pipeline ) . For the 165 central cartwheel , we achieved a local resolution ranging from ~ 16 Å to ~ 40 Å ( Fig . 166 S2A ) , with a global resolution of ~ 24 Å ( FSC criterion 0 . 143 ; Fig . S2B ; see Fig . S3 - S5 167 for resolution of all other STA maps , which have been deposited in EMDB ) . 168 Using line scans on 2D projections of the STA map , we determined the T . spp . 169 hub diameter to be 23 nm ( Fig . 2A , 2B ) , in line with previous work ( Guichard et al , 170 2013 , 2012 ) . Importantly , the improved resolution achieved here enabled us to uncover 171 novel features in the central cartwheel of the T . spp . centriole . Of particular interest , 172 we discovered that the position of the CID is polarized along the proximal - distal 173 centriole axis with respect to the hub and the spokes that emanate from it ( Fig . 2C , 174 2D ) . This is apparent from vertical intensity profiles of longitudinal views , which show 175 that the CID is positioned distal to the center of the hub density , which itself appears 176 to be elongated in the vertical direction ( Fig . 2C ) . Occasionally , two units can be 177 discerned within one hub density ( Fig . 2C , double peaks in light blue intensity profile 178 and corresponding dashed lines ) , a point that will be considered further below . Such 179 double units were not recognized previously , presumably owing to the lower resolution 180 map ( Guichard et al , 2013 , 2012 ) . Moreover , we found densities that vertically bridge 181 successive hub elements ( Fig . 2D , arrows ) . The polarized location of the CID unveiled 182 here is also apparent with respect to where spoke densities emerge from the hub ( Fig . 183 2E , arrow ) . In addition , we identified discontinuous densities in the center of the CID 184 ( Fig . 2B , 2D ) . 185 We likewise analyzed the central cartwheel in T . agilis observing variations 186 along the centriole axis in 2D longitudinal views ( Fig . S1D ) . Focused 3D classification 187 of sub - volumes indeed uncovered two classes , corresponding to 55 % and 45 % of 188 sub - volumes , which can occur within the same centriole ( Fig . 2F - J ; Fig . S6A - F ) . We 189 found that the central cartwheel STA map of both T . agilis classes exhibits many 190 similarities with that of T . spp . Thus , the CID is present , and spoke densities emanate 191 from a hub ~ 23 nm in diameter ( Fig . 2F , 2G ; Fig . S6A , S6B ) . Moreover , vertical 192 densities bridging successive hub elements are also present in both T . agilis classes 193 7 ( Fig . 2I , arrows ; Fig . S6D , arrows ) . Furthermore , we found that the CID is also 194 polarized along the proximal - distal centriole axis , being distal with respect to the hub 195 in both T . agilis classes , as evidenced from vertical intensity profiles ( Fig . 2H - I ; Fig . 196 S6C - D ) , as well as from the location of the CID relative to where spoke densities 197 emerge from the hub ( Fig . 2J and Fig . S6D , arrowheads ) . In the T . agilis 55 % class , 198 like in T . spp . , hub densities are elongated in the vertical direction and can be 199 sometimes discerned as two units ( Fig . 2H , double peaks in light pink intensity profile 200 and corresponding dashed lines ) . The presence of such double hub units next to the 201 CID is more apparent in the 45 % class , where they also exhibit a slight offset relative 202 to the vertical axis ( Fig . S6C , white dashed line ) , a point considered further below . In 203 addition , we found in this class that double hub units alternate with single hub units 204 that do not have a CID in their vicinity ( Fig . S6C , dashed arrow , Fig . S6D ) , an absence 205 noticeable also in raw tomograms ( Fig . S1D ) and verified in 3D top views ( Fig . S6A 206 and S6B , full circle ) . Moreover , we found that spoke densities emanating from single 207 hub units are thinner than those stemming from double hub units ( Fig . S6D ) . The 208 plausible origin of alternating double and single hub units in the 45 % T . agilis class 209 will be considered below . We noted also that the 45 % sub - volumes exhibit slight 210 variations in the spacing between double and single hub units ( Fig . S6E , 25 % and 20 % 211 sub - classes ) . 212 Taken together , our findings establish that T . spp . and T . agilis central 213 cartwheel architecture shares many features , including a polarized CID position . 214 215 Comparative STA of peripheral centriole elements in Trichonympha 216 We also investigated peripheral elements in the proximal region of T . spp . and T . agilis 217 centrioles . To this end , we extracted peripheral sub - volumes from the tomograms and 218 generated for each species three maps using STA centered either on the microtubule 219 triplet , the pinhead or the A - C linker ( Fig . 3A , 3E ) . 220 For the microtubule triplet , the resulting analysis revealed a characteristic 221 centriolar architecture . Thus , the A - microtubule bears 13 protofilaments , the B - 222 microtubule 10 protofilaments proper , with 3 extra ones shared with the A - microtubule , 223 whereas the C - microtubule exhibits a similar organization as the B - microtubule ( Fig . 224 3B , 3F ) . Moreover , we detected prominent densities corresponding to microtubule 225 inner proteins ( MIPs ) . In both species , we found a MIP located along protofilament A9 , 226 close to protofilament A10 ( Fig . 3B , 3F , empty arrowhead ) . A MIP was discovered at 227 this location in ciliary axonemes ( Nicastro et al , 2006 ) , and was also observed in 228 centrioles of T . spp . , Chlamydomonas and mammalian cells ( Greenan et al , 2020 , 229 2018 ; Guichard et al , 2013 ; Li et al , 2019 ) . In addition , we observed a MIP along 230 8 protofilament A5 in T . spp . ( Fig . 3B , chevron ) , which is visible only at low threshold in 231 T . agilis and positioned like MIP1 in Chlamydomonas centrioles and axonemes ( Li et 232 al , 2019 ; Nicastro et al , 2006 ) . In both Trichonympha species , we also detected 233 additional densities or microtubule associated proteins ( MAPs ) on the microtubule 234 exterior between the A - B and B - C inner junctions ( Fig . 3B , 3F , arrowhead ) , as 235 described also in Chlamydomonas and mammalian centrioles ( Greenan et al , 2018 ; Li 236 et al , 2019 ) . 237 For both T . spp . and T . agilis , we next conducted STA centered on the pinhead 238 or the A - C linker to uncover features in these peripheral elements . We thus found that 239 the pinhead connects to the A3 protofilament in both species ( Fig . 3C , 3G ) , in line with 240 previous observations in T . spp . and in other systems ( Greenan et al , 2018 ; Guichard 241 et al , 2013 ; Li et al , 2019 ) . Moreover , we found that the pinhead is polarized in a similar 242 manner in T . spp . and T . agilis ( Fig . 3C , 3G ) , with the two pinfeet moieties PinF1 and 243 PinF2 pointing proximally from the A3 protofilament , as reported previously for T . spp . 244 ( Guichard et al , 2013 , 2020 ) . We found also that the spacing between PinF1 and PinF2 245 elements is ~ 8 . 6 nm and ~ 7 . 9 nm in T . spp . , whereas it is ~ 8 . 4 nm and ~ 8 . 4 nm in T . 246 agilis ( Fig . 3C , 3G ) , compatible with power spectra of 2D class averages considering 247 the standard deviation of the measurements ( Fig . S2I , S3L ) . 248 Similarities between the two species are also apparent in the A - C linker that 249 bridges neighboring MT triplets . Thus , we found that the T . spp . A - C linker connects 250 protofilament A8 / A9 from one triplet with protofilaments C9 / C10 of the adjacent triplet 251 ( Fig . 3D ) , furthering the mapping of these connections compared to previous work 252 ( Guichard et al , 2013 ) . As shown in Fig . 3H , we found that the A - C linker in T . agilis 253 has a similar architecture . To generate an overview of the entire proximal region of the 254 T . agilis centriole , comprising hub , spoke , pinhead and A - microtubule , we conducted 255 STA centered on the spokes for both 55 % and 45 % classes ( Fig . S7A - H ) . Such maps 256 have a lower resolution due to the larger box size , the binning and the absence of 257 radial symmetrization , but nevertheless uncover a concerted proximal - distal polarity of 258 several elements . First , the CID is positioned distally within double hub units . Second , 259 spoke densities exhibit a slightly asymmetry in their tilt angle , with the proximal spoke 260 being more tilted . Moreover , these two polarized features present centrally are 261 connected with the polarized pinhead and A - C linker present peripherally ( Fig . S7C - D , 262 S7G - H ) . 263 Overall , we conclude that peripheral components of the cartwheel are also 264 generally conserved between the two Trichonympha species and exhibit concerted 265 polarity with central elements along the proximal - distal centriole axis . 266 267 9 Diversity of cartwheel architecture in Teranympha cartwheel 268 The gut of R . speratus termites contains another symbiotic flagellate , namely 269 Teranympha mirabilis ( Koidzumi , 1921 ; Noda et al , 2018 ) , which we found also to 270 harbor numerous centrioles and associated flagella ( Fig . S8A ) . TEM analysis of resin - 271 embedded specimens established that the cartwheel - bearing region in such centrioles 272 is ~ 1 . 1 µ m on average ( Fig . S8B , green line ; N = 9 , SD = 0 . 06 µ m ) , whereas the distal 273 region averages ~ 0 . 5 µ m ( Fig . S8B , black line ; N = 9 , SD = 0 . 08 µ m ) . Transverse 274 sections of the cartwheel - bearing region revealed the characteristic hub and spokes 275 connected through a pinhead to peripheral microtubule triplets , which are joined by an 276 A - C linker , whereas the triplet base is poorly visible ( Fig . S8C ) . 277 We conducted cryo - ET of purified T . mirabilis centrioles , acquiring tilt series of 278 the entire cartwheel - bearing region followed by tomogram reconstruction and STA . We 279 again generated separate maps for the central cartwheel , the microtubule triplet , the 280 pinhead and the A - C linker . Focused 3D classification of the central cartwheel region 281 yielded two classes representing 64 % and 36 % of sub - volumes , which can occur 282 within the same centriole ( Fig . 4A - G ; Fig . S8E ) . Similarly to the two Trichonympha 283 species , a hub ~ 23 nm in diameter is present in both T . mirabilis classes , with spoke 284 densities emanating from it ( Fig . 4A - C ) . Interestingly , we found that the architecture of 285 the central cartwheel in T . mirabilis differs slightly from that in Trichonympha . Indeed , 286 in both T . mirabilis classes , we uncovered a filamentous structure ~ 7 nm in diameter 287 present along the entire cartwheel - bearing region inside the hub , which we dubbed 288 filamentous cartwheel inner density ( fCID ) ( Fig . 4A - G ) . The fCID is also apparent in 289 transverse views of resin - embedded centrioles and symmetrized tomogram slices ( Fig . 290 S8C , S8D ) . Moreover , the fCID is consistently detected in raw tomograms ( Fig . S1E ) , 291 as well as in non - symmetrized STA comprising larger volumes centered on the spokes 292 ( Fig . S7I - P ) . 293 As shown in Figure 4D - G , the two central cartwheel T . mirabilis classes differ 294 in hub architecture , as revealed by vertical line profile intensity measurements . In the 295 64 % class , a periodicity of ~ 8 . 4 nm is apparent between hub densities , each consisting 296 seemingly of a single vertically elongated unit ( Fig . 4D , 4F ) . By contrast , in the 36 % 297 class , each hub density comprises a double unit ( Fig . 4E , 4G ) . Such double hub units 298 exhibit a peak - to - peak distance of ~ 3 . 2 nm and are separated from the adjacent double 299 hub unit by ~ 5 . 2 nm ( Fig . 4E ) . The sum of the two distances , namely 8 . 4 nm is 300 equivalent to that observed in the 64 % class . Moreover , the periodicity at the level of 301 emerging spoke densities is likewise equivalent in the two classes ( Fig . 4F , 4G ) . 302 Furthermore , as in the 45 % T . agilis class , we observed that every other double hub 303 10 unit in the 36 % T . mirabilis class exhibits a slight offset relative to the vertical axis ( Fig . 304 4E , white dashed lines ) . 305 Analysis of the peripheral STA microtubule triplet map of the T . mirabilis 306 centriole uncovered the canonical protofilament configuration for A - , B - and C - 307 microtubules ( Fig . 4H , 4I ) . As in Trichonympha , we detected additional densities on 308 the external side of the microtubules between A - B and B - C inner junctions ( Fig . 4I , 309 arrowheads ) . The previously described C - stretch that extends from protofilament C1 310 in T . spp . ( Guichard et al , 2013 ) is also observed in T . mirabilis ( Fig . 4I , chevron ) , while 311 prominent MIPs are not detected at the selected threshold . 312 The map generated by centering on the pinhead revealed a connection with 313 the A3 protofilament , with PinF1 and PinF2 being separated by 8 . 4 nm ( Fig . 4J ) , as 314 suggested also by power spectra of 2D class averages ( Fig . S4L ) . Moreover , we found 315 that the T . mirabilis A - C linker architecture is similar to that in the two Trichonympha 316 species , with anchoring to microtubules at protofilaments A9 and C9 / C10 ( Fig . 4K ) . 317 Overall , these findings indicate that T . mirabilis peripheral elements share 318 many conserved features with those in Trichonympha , as does the central cartwheel , 319 apart from the discontinuous CID being replaced by the seemingly continuous fCID in 320 T . mirabilis . 321 322 Comparative analysis of SAS - 6 ring stacking mode 323 We set out to investigate how SAS - 6 rings may fit into the central cartwheel STA maps 324 of the three species . Genomic or transcriptomic information is not available for T . spp . 325 and T . mirabilis at present . Thus , in the absence of molecular information for SAS - 6 in 326 T . spp . and T . mirabilis , as well as of structural information for T . agilis SAS - 6 , and 327 given that SAS - 6 crystal structures from a wide range of organisms exhibit extensive 328 similarities , we employed CrSAS - 6 as a model instead . We used homodimers of 329 CrSAS - 6 [ 6HR ] , the longest CrSAS - 6 fragment with a determined crystal structure , 330 which can self - assemble into 9 - fold symmetrical rings ~ 23 nm in diameter and 4 . 2 nm 331 in height ( Kitagawa et al , 2011 ) . 332 Given the presence of two units per hub density in the T . mirabilis 36 % class , 333 and also probably in both Trichonympha species , we computationally assembled 334 CrSAS - 6 [ 6HR ] into single rings , as well as into directly superimposed double rings in 335 register . We then used rigid body fitting to score the goodness of fit of both types of 336 ring assemblies with the STA maps ( Table S2 ) . Starting with the T . mirabilis 36 % 337 class , we found that whereas a single CrSAS - 6 [ 6HR ] ring fits in this map , additional 338 hub densities remain unaccounted for in this case ( Fig . 5A , arrowheads ) . By contrast , 339 the double ring configuration fills the hub density to a larger extent ( Fig . 5B ) , yielding 340 11 a better overlap score ( Table S2 ) . However , coiled - coil moieties extend slightly outside 341 the spoke densities in this case , potentially because of coiled - coil bending in vivo or 342 species - specific features . Moreover , rigid body fitting of CrSAS - 6 [ 6HR ] homodimers 343 confirmed that two directly superimposed dimers provide a better fit for the T . mirabilis 344 36 % class , a fit that also revealed a slight offset between them ( Fig . S9A ) . Next , we 345 fitted computationally assembled single and double CrSAS - 6 [ 6HR ] rings in the T . 346 mirabilis 64 % class map , finding again that head domains of double rings can be 347 readily accommodated in the hub density , although one coiled - coil moiety clearly 348 extends outside spoke densities in this case ( Fig . S9B ) Taken together , these 349 observations are compatible with the possibility that SAS - 6 rings can directly stack on 350 top of one another in vivo . 351 To explore whether direct SAS - 6 ring stacking might occur also in 352 Trichonympha , we likewise performed fitting of computationally assembled CrSAS - 353 6 [ 6HR ] single and double rings . We found again that in all cases directly superimposed 354 double rings can readily be accommodated in the hub densities with improved 355 goodness of fit compared to single rings ( Fig . 5C , 5D ; Fig . S9C , S9D ; Table S2 ) . The 356 T . agilis 45 % class , which comprises alternating double and single hub elements , 357 presents a particularly interesting case . As anticipated , double rings could be placed 358 in the elongated hub density comprising two units , but a single ring could be 359 accommodated in the thinner individual hub unit ( Fig . 5C , 5D ; Table S2 ) . 360 Prompted by the slight offset observed between two SAS - 6 ring oligomers upon 361 fitting CrSAS - 6 [ 6HR ] homodimers in the T . mirabilis 36 % class ( see Fig . S9A ) , as well 362 as the structural complementarity between superimposed SAS - 6 rings following such 363 an offset , we computationally assembled a double ring with a ~ 6 . 5° rotational offset 364 imposed between ring pairs , which allowed the two rings to come closer to one another 365 vertically by 0 . 4 nm ( Fig . S10B , compare with Fig . S10A ; Materials and Methods ) . 366 Manual fitting in the T . mirabilis 36 % class map uncovered that such an offset double 367 ring can be readily accommodated in the hub ( Fig . S10C ) . A similar conclusion was 368 drawn from fitting the offset double ring into the double hub density of the T . agilis 45 369 % class ( Fig . S10D ) . Such a slight offset between superimposed SAS - 6 rings might 370 explain the hub offset observed in the longitudinal view of the central cartwheel STA 371 in both T . mirabilis 36 % class and T . agilis 45 % class ( see Fig . 4E and Fig . S6C , 372 white dashed lines ) . Furthermore , we reasoned that such an offset double ring might 373 likewise affect the connected spoke densities . If spoke densities correspond in reality 374 to two individual spokes slightly offset from one another , as expected from an offset 375 double ring configuration , but which cannot be identified as individual units due to the 376 resolution limit , then the shape of the spoke densities should be elliptical when viewed 377 12 end on . To investigate this possibility , we unwrapped the central cartwheel maps to 378 obtain a complete view at the level of the spokes ( Fig . S10E ) . Importantly , this 379 uncovered the expected elliptical shape , revealing spoke offset in the T . mirabilis 36 380 % class ( Fig . S10F ) , as well as in both T . agilis classes ( Fig . S10G ) and in T . spp . ( Fig . 381 S10H ) . By contrast , no spoke offset was apparent in the T . mirabilis 64 % class ( Fig . 382 S10F ) . 383 Overall , these findings lead us to propose that pairs of SAS - 6 rings directly 384 stack in the cellular context and can do so with an offset ( see Discussion ) . 385 We investigated further the possibility that SAS - 6 rings directly stack onto one 386 another using a cell free assay with a recombinant CrSAS - 6 protein containing the 387 globular head domain and the entire coiled - coil ( referred to as CrSAS - 6 [ NL ] ) . It has 388 been shown previously that CrSAS - 6 [ NL ] bearing 6xHis - and S - tag moieties can self - 389 organize into stacks thought to exhibit ~ 8 . 5 nm periodicity between spokes based on 390 the analysis strictly of top views ( Guichard et al , 2017 ) . We investigated this question 391 anew , now analyzing stacking periodicity of untagged purified CrSAS - 6 [ NL ] protein . 392 Moreover , we conducted STA of side views of stacked assemblies , thus allowing us to 393 measure the underlying periodicities with a global resolution of ~ 25 Å ( Fig . 5E - I , Fig . 394 S5 ) . Importantly , we found that such in vitro assembled CrSAS - 6 [ NL ] stacks exhibit a 395 periodicity of ~ 4 . 5 nm that can each accommodate one computationally assembled 396 single ring of CrSAS - 6 [ 6HR ] ( Fig . 5G - I ) . Moreover , we found that the spokes of 397 successive in vitro assembled rings are almost in register ( Fig . S10I ) . 398 Taken together , these observations lead us to propose that direct stacking of 399 SAS - 6 rings may be an evolutionarily conserved feature of cartwheel assembly at the 400 onset of centriole biogenesis . 401 402 403 Discussion 404 405 The centriole is fundamental for numerous aspects of cell physiology and shows 406 remarkable conservation across the major eukaryotic groups of life . Understanding the 407 mechanisms that govern centriole assembly requires not only the identification of 408 essential building blocks and their regulators , but also a detailed knowledge of 409 organelle architecture . Our findings uncover conserved features in the cartwheel - 410 bearing portion of the centriole . We find that the CID located within the hub is polarized 411 along the proximal - distal centriole axis in both Trichonympha species and that a 412 potentially related fCID is present in T . mirabilis . Moreover , we establish that hub 413 densities can be fitted by directly superimposing 9 - fold symmetrical SAS - 6 double rings 414 13 that exhibit a slight rotational offset between the two rings . Furthermore , we find that 415 such an offset double ring is stacked in register with the next offset double ring . Overall , 416 our work provides novel insights into the architecture of the centriolar cartwheel at the 417 root of centriole biogenesis . 418 419 Conserved features in cartwheel architecture 420 Centriole ultrastructure began to be explored in the 1950s when TEM of resin - 421 embedded fixed specimens revealed its signature 9 - fold radial symmetry and the chiral 422 arrangement of microtubule triplets ( Fawcett & Porter , 1954 ) . More recent cryo - ET 423 analysis uncovered the native architecture of the cartwheel - bearing portion of the 424 centriole ( Guichard et al , 2013 ; Li et al , 2019 ) . Here , we set out to improve the 425 resolution previously obtained with T . spp . and to investigate whether features 426 discovered in this species are present in other flagellates harboring unusually long 427 cartwheels . 428 In the absence of fossil record or complete sequence information , it is not 429 possible to assess sequence divergence between centriolar proteins in the species 430 analyzed here , nor to date with precision the evolutionary times separating them . 431 However , an approximation is provided in the case of T . spp . and T . agilis by the 432 phylogenetic divergence between their respective hosts Zootermopsis spp . and 433 Reticulitermes speratus , which are estimated to have shared their last common 434 ancestor ~ 140 million years ago ( Bucek et al , 2019 ) . Given that Trichonympha are 435 obligate symbiotic organisms and considering that the two host species inhabit 436 different continents ( Park et al , 2006 ; Thorne et al , 1993 ) , it is reasonable to postulate 437 that T . spp . and T . agilis diverged at least this long ago . Teranympha mirabilis belongs 438 to a different genus , and based on small subunit rRNA sequences it is much more 439 distant from the Trichonympha species than they are from one another ; however , the 440 split between the two genera is thus far undated ( Carpenter and Keeling , 2007 ; Noda 441 et al , 2018 , 2009 ; Ohkuma et al , 2009 ) . Regardless of the exact evolutionary times 442 separating these flagellates , our findings , together with those of a companion 443 manuscript ( Klena et al ) , establish that the CID is conserved between distant 444 eukaryotic groups , suggesting shared evolutionary history and / or function ( Fig . 6A ) . 445 The CID exhibits a 9 - fold radial symmetry and connects with the hub approximately 446 where neighboring SAS - 6 homodimers interact with one another ( Fig . 6B ) , a 447 suggestive location raising the possibility that the CID imparts or maintains the 9 - fold 448 symmetrical SAS - 6 ring structure ( Guichard et al , 2013 ) . We discovered here that the 449 CID is polarized along the proximal - distal centriole axis , so that may also play a role in 450 imparting or maintaining organelle polarity ( Fig . 6C ) . Furthermore , the CID exhibits an 451 14 ~ 8 . 4 nm periodicity along the proximal - distal centriole axis , with no apparent continuity 452 between two superimposed CID elements . This is in contrast to the fCID , which runs 453 throughout the center of the cartwheel - bearing portion of the T . mirabilis centriole . 454 Although the fCID seems disconnected from the hub , small elements linking the two 455 can be discerned in the T . mirabilis 36 % class ( Fig . 6D , see Fig . S7N ) . Moreover , the 456 fCID might be linked with the hub through other protein segments that are small , 457 flexible or exhibit different periodicities , rendering them difficult to detect in the current 458 STA map . It will be interesting also to address whether the fCID exhibits helical 459 features . Elements related to the fCID may be present in systems other than T . 460 mirabilis . Thus , electron - dense material is discernable in the geometrical center of the 461 hub in T . spp . and less so in T . agilis ( see Fig . 2D and 2I ) , although it is discontinuous 462 and smaller in diameter than the fCID . Moreover , a density that may be related to either 463 CID or fCID is apparent inside the hub in other species , including Chlamydomonas , 464 Chytrid and several insects ( Geimer & Melkonian , 2004 ; Guichard et al , 2017 ; O’Toole 465 & Dutcher , 2014 ; Olson & Fuller , 1968 ; McNitt 1974 ; Gottardo et al , 2015 ; Uzbekov et 466 al , 2018 , Klena et al ) ( Fig . 6A ) . Determining the molecular nature of CID and fCID in 467 diverse systems is expected to help assess whether they share an evolutionary history 468 and may serve a similar function . 469 We found conserved features also in the peripheral elements of the cartwheel - 470 bearing region of the centriole . Thus , the pinhead is present in all three organisms 471 analyzed here , as is the A - C linker , although the exact arrangement of moieties within 472 these elements differs slightly . Together with the peripheral STA map in the proximal 473 region of the Chlamydomonas centriole ( Li et al , 2019 ) , these findings indicate that 474 peripheral elements have been significantly conserved across evolution . Slight 475 differences between systems are observed notably in the microtubule triplets and 476 prominent MIPs . At the resolution achieved here , we thus identified a prominent MIP 477 in both Trichonympha species next to protofilament A9 , close to protofilament A10 , 478 potentially corresponding to the A - microtubule seam ( Ichikawa et al , 2017 , 2019 ) . A 479 prominent MIP has also been observed at this position in centrioles of mammalian 480 CHO cells and Drosophila S2 cells ( Greenan et al , 2020 , 2018 ; Li et al , 2019 ) . The 481 molecular identity of several Chlamydomonas MIPs present in ciliary axonemal 482 microtubules was identified recently ( Ma et al , 2019 ) , but corresponding MIPs could 483 not be detected here using the selected threshold , perhaps because not all 484 periodicities were explored . Nevertheless , we identified one additional prominent MIP 485 next to protofilament A5 in T . spp . , which appears to be conserved in metazoan 486 centrioles ( Greenan et al , 2018 ) , and was identified as RIB72 and FAP252 in ciliary 487 axonemal microtubules ( Ma et al , 2019 ) . Given the recent spectacular progress in 488 15 assigning the molecular nature of many ciliary MIPs using high resolution cryo - EM ( Ma 489 et al , 2019 , Song et al , 2020 ) , it is likely that better resolution STA maps combined with 490 a search for different periodicities will also enable the efficient identification of centriolar 491 MIPs . 492 493 Root of proximal - distal polarity 494 The centriole is polarized along its proximal - distal axis , as evidenced for instance by 495 the presence of the cartwheel and microtubule triplets in the proximal region versus 496 microtubule doublets in the distal region . Likewise , distal and sub - distal appendages 497 are restricted to the distal region , by definition . Ultimately , proximal - distal polarity of 498 the centriole also translates into that of the axoneme that emanates from it . Prior work 499 in T . spp . raised the possibility that centriole polarity might stem from the pinhead or 500 the A - C linker , because both elements exhibit a clear proximal - distal polarity ( Guichard 501 et al , 2013 ) . Whereas it remains plausible that polarity is imparted by peripheral 502 elements in some settings , in light of the present findings an alternative view can be 503 envisaged . Indeed , the offset between double SAS - 6 rings generates an inherently 504 asymmetric structure , which correlates with the polarized location of the CID , as well 505 as with the asymmetric tilt angle of spoke densities that is also observed in distantly 506 related eukaryotic groups ( Klena et al ) . Given that central elements are present before 507 peripheral ones during organelle assembly in the canonical centriole duplication cycle , 508 it is tempting to speculate that the asymmetric properties of offset double rings and / or 509 the CID are key for imparting organelle polarity . The situation might differ during de 510 novo centriole assembly , which in human cells does not require interactions between 511 HsSAS - 6 head domains ( Wang et al , 2015 ) . In this case , peripheral elements are 512 thought to be more critical , and this may also be the case when it comes to imparting 513 organelle polarity . 514 515 Working model of SAS - 6 ring stacking 516 What are the mechanisms of SAS - 6 ring stacking ? The previous lower 517 resolution T . spp . map ( Guichard et al , 2013 ) , together with an initial analysis of in vitro 518 generated stacks of CrSAS - 6 [ NL ] ( Guichard et al , 2017 ) , led to the suggestion that the 519 periodicity between subsequent SAS - 6 ring oligomers was ~ 8 . 5 nm . The higher 520 resolution analysis performed here in three species leads us to propose instead that 521 SAS - 6 rings stack directly on top of one another , thus doubling the number of SAS - 6 522 molecules per vertical unit length . Although we cannot formally exclude that a single 523 SAS - 6 ring is present and that additional densities correspond to other unassigned 524 protein ( s ) , several pieces of evidence support the direct SAS - 6 stacking scenario . First , 525 16 two SAS - 6 rings fit without clashes within vertically elongated hub densities in 526 Trichonympha and T . mirabilis . Second , the T . agilis 45 % class contains thin hub 527 densities that can accommodate only one SAS - 6 ring , reinforcing the notion that the 528 thicker ones harbor two . Third , stacks reconstituted in a cell free assay from purified 529 CrSAS - 6 exhibit direct ring superimposition . 530 Our findings taken together lead us to propose the following working model of 531 SAS - 6 stacking in the cellular context ( Fig . 6E , 6F ) . This model entails two modes of 532 ring stacking . The first corresponds to the offset double ring configuration , with a tight 533 packing of SAS - 6 rings , which are ~ 3 . 2 nm apart for instance in the T . mirabilis 36 % 534 class . In line with our findings , directly superimposed hub densities with remarkably 535 similar spacing are observed also in Paramecium and C . reinhardtii ( Klena et al ) . In 536 the second stacking mode , two such offset double rings stack in register , being ~ 5 . 2 537 nm apart from each other in the T . mirabilis 36 % class . A similar alternating pattern is 538 observed in T . spp . and T . agilis , albeit with slightly different distances , potentially 539 owing to limitations in measurement precision . Overall , we propose that these two 540 alternating modes of SAS - 6 ring stacking represent a fundamental feature of cartwheel 541 assembly . 542 The periodicity of the basic central cartwheel unit in this working model is ~ 16 . 8 nm , 543 which corresponds to the largest conserved periodicity detected by FFT . This value is 544 consistent with the distance expected from an alternation of the two modes of SAS - 6 545 stacking ( 2x ~ 3 . 2 nm for the rings with an offset + 2x ~ 5 . 2 nm for the rings in register 546 in the case of the T . mirabilis 36 % class = ~ 16 . 8 nm ) ( Fig . 6F ) . Each SAS - 6 hub 547 element possesses one spoke emerging from it , but owing to insufficient resolution , 548 two closely superimposed spokes appear as one joint density in all cases , except for 549 the single hub element in the T . agilis 45 % class . Interestingly , we note also that hub 550 elements with accompanying spoke densities are further apart in the T . mirabilis 64 % 551 class , indicative of a potential variation in stacking mode . Perhaps the densities 552 bridging successive hub elements in this case correspond to another protein than SAS - 553 6 . One intriguing candidate to consider in this context is SAS - 6 - like , an evolutionary 554 ancient SAS - 6 related protein lacking the coiled - coil ( de Leon et al , 2013 ) . 555 556 Concluding remarks 557 In summary , conducting cryo - ET in three flagellates enabled us to uncover 558 important conservation of centriolar architecture , as well as some species specificity . 559 In the future , higher resolution analyses , including without symmetrizing , is expected 560 to reveal additional polarity and stacking features , as well as to enable one to visualize 561 17 protein domains and thus further unravel the mechanisms governing centriole 562 assembly . 563 564 565 Materials and methods 566 567 Transmission electron microscopy of Trichonympha and Teranympha cells 568 T . agilis and T . mirabilis were extracted from the hindgut of Reticulitermes speratus 569 termites ( a kind gift of Yuichi Hongoh , Tokyo University of Technology , Japan ) , as 570 described ( Guichard et al , 2015 ) , placed in a drop of 10 mM K - PIPES , and gravity - 571 sedimented for 3 - 5 min onto coverslips coated with poly - D lysine ( Sigma - Aldrich , 572 catalog number P1024 ) , until cells became in contact with the glass . Cells were fixed 573 overnight in 2 % paraformaldehyde 1 % glutaraldehyde in phosphate buffer 0 . 1 M , pH 574 7 . 4 ( with 0 . 2 % Tween - 20 for some samples ) , washed in cacodylate buffer ( 0 . 1 M , pH 575 7 . 4 ) at 4°C and post - fixed with 0 . 5 % tannic acid in cacodylate buffer ( 0 . 1 M , pH 7 . 4 ) 576 for 40 min at room temperature . After two washes in distilled water , cells were post - 577 fixed in 1 % osmium tetroxide in cacodylate buffer . Thereafter , samples were washed 578 in distilled water and dehydrated in a graded ethanol series ( 1x 50 % , 1x 70 % , 2x 96 579 % , 2x 100 % ) . Finally , samples were embedded in epon resin and polymerized at 65°C 580 overnight . 50 nm sections were cut and then stained in 1 % uranyl acetate in distilled 581 water for 10 min , followed by Reynold’s stain ( 1 . 33 g lead citrate , 1 . 76 g sodium citrate , 582 160 mM NaOH in 50 ml distilled water ( Reynolds , 1963 ) ) for 5 min . Images were 583 acquired using Tecnai Spirit at 80 kV or Tecnai F20 at 200 kV microscopes ( Thermo 584 Fischer Scientific ) . Pear - shaped T . agilis cells with long flagella covering the anterior 585 of the cell can be readily distinguished from elongated T . mirabilis cells with shorter 586 flagella arranged in rows spiraling around the cell . These distinct morphologies are 587 preserved in resin . Purified centrioles from the two species were distinguished in cryo - 588 ET based on the presence or absence of the fCID . Images of centriole cross - sections 589 from TEM and cryo - ET were circularized and symmetrized using the CentrioleJ plugin 590 ( Guichard et al , 2013 ) . 591 592 Purification of Trichonympha and Teranympha centrioles 593 Trichonympha and Teranympha cells were extracted from the hindgut of termites in 10 594 mM K - PIPES in the presence of cOmplete protease inhibitor cocktail ( 1 : 1000 ; Roche ) 595 as described ( Guichard et al , 2015 ) , with minor species - specific modifications . For the 596 purification of centrioles from mixed T . agilis and T . mirabilis populations from R . 597 speratus termites , cells were pelleted at 1000 g for 20 s and the supernatant discarded . 598 18 For enrichment of T . mirabilis centrioles , cells were sedimented on ice 3x10 min in 1 599 ml 10 mM K - PIPES ; the more fragile T . agilis cells spontaneously lysed during this 600 step and thus depleted from the resulting pellet . T . spp . ( T . collaris , T . campanula and 601 T . sphaerica ) were extracted from Zootermopsis angusticollis termites ( a kind gift from 602 Filip Husnik and Patrick Keeling , University of British Columbia , Vancouver , Canada ) , 603 which harbor the same set of Trichonympha species as Zootermopsis nevadensis 604 ( Boscaro et al , 2017 ; Tai et al , 2013 ) . After extraction , T . spp . cells were sedimented 605 on ice 3x10 min in 1 ml 10 mM K - PIPES . In all cases , centrioles were released during 606 a 20 - 40 min incubation on ice in 1 ml K - PIPES 10 mM 0 . 5 % NP - 40 plus protease 607 inhibitors . Centrioles and associated flagella were either pelleted at 500 g for 3 min or 608 the resulting supernatant spun at 1000 g for 5 min at 4° C ; both types of preparations 609 were utilized for cryo - ET . In all cases , centrioles were washed once in 1 ml 10 mM K - 610 PIPES plus protease inhibitors and pelleted at 1000 g for 5 min . The pellet was stored 611 on ice before preparing grids for cryo - ET . 612 613 Cryo - ET data acquisition 614 Grids were prepared as described previously ( Guichard et al , 2015 ) . Briefly , 4 μl of the 615 purified centriole solution were mixed with 10 nm gold beads using a 20 μl tip cut at 616 the extremity and then applied to the copper side of a glow discharged lacey holey 617 carbon grid ( carbon Cu 300 mesh , Ted Pella ) . The grid was then manually blotted from 618 the carbon side with a Whatman grade 1 filter paper and plunged into liquid ethane at 619 - 180° C with a manual plunge freezer . 620 Tilt series of centrioles that were approximately parallel to the tilt axis were 621 collected with the Tomo 4 . 0 software ( ThermoFisher Scientific ) on a Tecnai F20 TEM 622 operated at 200 kV ( ThermoFisher Scientific ) ; data were recorded with a Falcon III DD 623 camera ( ThermoFisher Scientific ) in linear mode at 29’000X magnification , 624 corresponding to a pixel size of 3 . 49 Å . The tilt series were recorded from −60° to 60° 625 using both continuous and bi - directional tilt schemes , with an increment of 2° at 2 . 5 – 626 5 µm underfocus . To allow determination of the proximal - distal axis , the blunt proximal 627 end was included in image acquisition . A total of 54 tilt series were collected ( Table 628 S1 ) . 629 Tilt series alignments using gold fiducials were performed with IMOD v4 . 9 630 ( Kremer et al , 1996 ) . The contrast transfer function ( CTF ) was estimated with 631 CTFFIND4 v1 . 8 ( Rohou and Grigorieff , 2015 ) using relion _ prepare _ subtomograms . py 632 ( Bharat and Scheres , 2016 ) . Variable SkipCTFCorrection was set to ‘True ’ to generate 633 a wedge - shaped tilt - and dose - weighted 3D CTF model . Aligned tilt series were 634 19 corrected by phase - flipping with ctfphaseflip and used for weighted back - projection 635 ( WBP ) tomogram reconstruction with IMOD . 636 637 Sub - tomogram processing 638 Areas corresponding to the central cartwheel region ( CW ) and to the peripheral 639 microtubule triplet region ( MTT ) were identified visually in individual tomograms and 640 their longitudinal axes modeled with closed contours in 3dmod . Proximal - distal polarity 641 of the centriole was assessed by the fact that the proximal end was blunt , in contrast 642 to the distal end , where the flagellum was usually present . Moreover , the clockwise 643 chirality of the microtubule triplets when viewed from the distal end provided an 644 independent means to determine proximal - distal polarity , which was registered and 645 maintained in the resulting sub - volumes . Also , reconstructions from sub - volumes re - 646 centered on the spokes with CW and polar CID density on one side , and spokes and 647 polar pinhead on the other , allowed unambiguous polarity assignment ( see Fig . S7 ) . 648 Individual model points were interpolated along the contours using the 649 addModPts from the PEET package ( Heumann et al , 2011 ) , with a step size of 3 x 85 650 Å = 252 Å corresponding to a 73 pixels shift ( see Fig . S1B ) . The interpolation step size 651 was selected to correspond to approximately three hub element , as smaller step sizes 652 could not separate cartwheel variations visible in the raw tomograms ( see Fig . S1D ) . 653 Larger step sizes were also explored to generate non - overlapping sub - volumes along 654 the proximal - distal axis of the cartwheel hub , shifted relative to each other by more 655 than half of the sub - volume size . The resulting STAs exhibited similar structural 656 features , yet were noisier due to the reduced number of sub - volumes . 657 Interpolation with a step size of 252 Å resulted in 1385 initial CW sub - volumes 658 for T . spp . , 1154 for T . agilis and 1802 for T . mirabilis . A similar approach and step size 659 was selected for extraction of MTT , resulting in 2792 initial MTT sub - volumes for T . 660 spp . , 3850 for T . agilis and 7928 for T . mirabilis ( see Fig . S1G - I ; Table S1 ) . All further 661 processing was performed with RELION v2 . 1 or v3 . 1 ( Kimanius et al , 2016 ) . Briefly , 662 2D projections of all sub - volumes along the Z axis were generated and subjected to 663 reference - free 2D classification . 2D class averages with clear CW or MTT densities 664 were selected for further data processing . At this step , the hub of both T . agilis and T . 665 mirabilis showed variations in vertical spoke and hub element spacing . Power spectra 666 of 2D class averages were used to measure the vertical spacing of the CW elements 667 ( see Fig . S2C , S3C , S3F , S4C , S4F ) or MTT ( see Fig . S2F , S3I , S7I ) . Modeling with 668 3dmod was also used to investigate the proximal - distal twist of microtubule blades . No 669 twist angle of the microtubules was observed along the proximal - distal axis of the 670 20 cartwheel - bearing region in either species , as previously reported for T . spp . ( Gibbons 671 and Grimstone , 1960 ) . 672 Selected 2D projections of CW sub - volumes were re - extracted , now as 3D sub - 673 volumes with a box size of 200 pixels ( corresponding to 80 nm , with the exception of 674 T . spp . , which corresponds to 88 nm ) and re - scaled to a pixel size of 4 Å . Initial 3D CW 675 and MTT references were built de novo with the Stochastic Gradient Descent ( SGD ) 676 algorithm from the re - extracted 3D sub - volumes . The resulting CW initial reference 677 was re - aligned with relion _ align _ symmetry to orient the visible nine - fold rotational ( C9 ) 678 axis along Z axis , and C9 symmetry applied . Similarly , the MTT initial reference 679 proximal - distal axis was re - aligned with the Z axis . Consensus 3D refinement of the 680 CW initial reference with C9 symmetry and missing wedge correction revealed a well 681 resolved CW density . Consensus 3D refinement was followed by focused 3D 682 refinement with a soft mask that included only the CW hub with the CID , as well as 683 part of the emanating spoke density , whose inclusion inside the mask was important 684 to avoid misalignment along the proximal - distal axis . Moreover , one round of focused 685 3D classification in T . agilis and T . mirabilis with a soft tight mask that included only 686 three central stacked rings revealed vertical spacing variations in 3D . 687 The T . agilis CW hub classified into two groups representing 55 % and 45 % of 688 refined sub - volumes . The 55 % class showed uniform 85 Å vertical spacing measured 689 from the 3D reconstruction in real space and from the power spectrum . Further 3D 690 classification did not yield any observable improvement in the quality of the maps as 691 judged by features and resolution . In contrast , further focused 3D classification without 692 alignment of the 45 % class revealed two sub - groups corresponding to 20 % and 25 % 693 of all refined sub - volumes , with differences in distances between alternating thin and 694 thick rings . The T . mirabilis CW hub classified into two main groups representing 64 % 695 and 36 % of refined sub - volumes . The 64 % class exhibited 85 Å vertical spacing 696 measured from the 3D reconstruction in real space and from power spectra and a 697 smooth CW hub surface . In contrast , the 36 % class revealed 85 Å vertical spacing 698 with two smaller units per hub with 42 Å spacing measured from the 3D reconstruction 699 in real space and from power spectrum . Further 3D classification did not yield any 700 observable improvement in the quality of the maps as judged by features and 701 resolution . For all 3D classes , final focused 3D refinements with local alignments were 702 performed . Analogous analyses of T . spp . revealed a single stable class with uniform 703 85 Å vertical spacing measured from the 3D reconstruction and from the power 704 spectrum . 705 To establish the polar relationship between the CID , the corresponding CW and 706 the peripheral elements ( A - microtubule , pinhead , A - C linker ) , the symmetry was 707 21 relaxed from C9 to C1 with relion _ particle _ symmetry _ expand . The resulting symmetry - 708 expanded sub - volumes were re - extracted with binning 3 and a box size of 100 pixels , 709 using the ‘re - center refined coordinates’ option approximately on the spokes center to 710 include CW density on one side and spokes and pinhead on the other . Focused 3D 711 classification of re - extracted sectors without alignment and without symmetry iterated 712 with focused 3D refinement revealed reconstructions with well - resolved connections 713 between central and peripheral elements . No structural features with a periodicity 714 different than a multiple of 85 Å were detected at this step . 715 A similar processing pipeline was applied to peripheral elements ( microtubules , 716 pinhead , A - C linker ) . Selected 2D projections of MTT sub - volumes were re - extracted 717 as binned 2 times 3D sub - volumes with a box size of 128 pixels corresponding to 89 718 nm to include microtubule triplet and emanating pinhead and A - C linker densities ( see 719 Fig . S1B ) . Initial 3D MTT references were built de novo with the SGD algorithm from 720 the re - extracted 3D sub - volumes . The resulting MTT initial reference proximal - distal 721 axis was re - aligned with the Z axis . Re - extracted MTT sub - volumes were subjected to 722 consensus 3D refinement followed by rounds of focused 3D classification and 3D 723 refinement . Refined sub - volumes were re - extracted either at the center of emanating 724 pinhead or A - C linker densities and refined separately . Local resolution distributions 725 were determined with ResMap ( Kucukelbir et al , 2014 ) and directional 3D FSC were 726 measured with 3DFSC web server ( Zi Tan et al , 2017 ) . 727 728 Rigid body fitting and computational assembly of SAS - 6 rings 729 Rigid - body fitting of CrSAS - 6 [ 6HR ] ( PDB - 3Q0X ) into T . spp . , T . agilis and T . mirabilis 730 3D STA maps was performed with ADP _ EM plugin ( Garzón et al , 2007 ) , followed by 731 symmetrical fitting in UCSF Chimera ( Pettersen et al , 2004 ) . 732 Single CrSAS - 6 [ 6HR ] rings were computationally assembled as described 733 ( Kitagawa et al , 2011 ) , while double rings were assembled by superposing two such 734 CrSAS - 6 [ 6HR ] rings using the crystallized stacks of LmSAS - 6 rings as guide ( Van 735 Breugel et al , 2011 ) . Offset double rings were assembled manually by rotating the 736 proximal ring clockwise , with the angle being estimated based on angles between fitted 737 homodimers . The vertical distance between rings in the double offset ring configuration 738 was reduced by 0 . 4 nm , and clashes were only allowed between flexible loops that are 739 not resolved in the crystal structure . CrSAS - 6 [ 6HR ] single ring , double in register rings 740 or double offset rings were fit in the map using fitmap function of ChimeraX ( Goddard 741 et al , 2018 ) . Coordinate models were initially converted to molecular maps at 742 corresponding global resolutions and then fitted into the STA maps . Calculated values 743 22 for correlation and overlap between maps are reported in Table S2 . UCSF Chimera or 744 ChimeraX were used for visualization and segmentation of 3D models . 745 746 In vitro assembly of CrSAS - 6 stacked cartwheels 747 A 6xHis - and S - tag - containing CrSAS - 6 construct harboring the N - terminal globular 748 head domain plus the entire coiled - coil ( referred to a CrSAS - 6 [ NL ] , comprising amino 749 acids 1 - 503 ) was expressed and purified as described ( Guichard et al , 2017 ) , except 750 that the tag moieties were removed by TEV protease cleavage . The resulting purified 751 CrSAS - 6 [ NL ] protein was dialyzed overnight from Tris pH 7 . 5 150 mM NaCl into 10 752 mM K - PIPES pH 7 . 2 using a mini dialysis unit at 4°C ( slide - A - lyzer , 3 . 5 K , 10 – 100 ml , 753 Pierce , catalogue number 69’550 ) . Thereafter , 5 µ l of dialyzed material was pipetted 754 onto a glow - discharged Lacey holey carbon grid ( Carbon Cu 300 mesh , Ted Pella ) , 755 blotted for 3 s ( blot force - 15 , no wait time ) and plunge - frozen by vitrification in liquid 756 nitrogen - cooled liquid ethane , using a Vitrobot MKIV ( ThermoFisher Scientific ) . 757 Tilt series were collected on a Titan Krios TEM ( ThermoFisher Scientific ) 758 operated at 300 kV and equipped with a Gatan Quantum LS energy filter ( zero loss slit 759 width 20 eV ; Gatan Inc . ) on a K2 Summit direct electron detector ( Gatan Inc . ) in 760 electron counting mode ( 110 e - / Å 2 total dose ) , at a calibrated pixel size of 2 . 71 Å . The 761 tilt series were recorded from −60° to 60° using a dose - symmetric tilt scheme ( Hagen 762 et al , 2017 ) with increments of 2° at 2 . 5 - 3 . 5 µm defocus . A total of 31 tilt series were 763 collected automatically using SerialEM ( Mastronarde , 2005 ) . 764 Tilt series alignment using gold fiducials was performed with IMOD v4 . 9 . The 765 CTF was estimated with CTFFIND4 v1 . 8 using relion _ prepare _ subtomograms . py . 766 Variable SkipCTFCorrection was set to ‘True’ to generate a wedge - shaped tilt - and 767 dose - weighted 3D CTF model . Aligned tilt series were corrected by phase - flipping with 768 ctfphaseflip and used for WBP tomograms reconstruction with IMOD . Further STA was 769 performed as described above for Trichonympha centrioles . Briefly , side views of 770 CrSAS - 6 [ NL ] stacks were identified visually in tomograms and their longitudinal axes 771 modeled with closed contours in 3dmod . Next , individual model points were added 772 along the contours using addModPts with a step size of 2 CrSAS - 6 [ NL ] rings ( 2 x 42 Å 773 = 84 Å ) to prevent misalignment of potentially merged spokes , resulting in 926 initial 774 sub - volumes ( see Fig . S1J ; Table S1 ) . Further processing was performed with RELION 775 v2 . 1 or v3 . 1 as described above . 776 777 778 Declarations 779 23 780 Ethics approval and consent to participate 781 Not applicable . 782 783 Consent for publication 784 Not applicable . 785 786 Availability of data and material 787 Sub - tomogram averages have been deposited at Electron Microscopy Data Bank 788 ( EMD - 10916 , EMD - 10918 , EMD - 10922 , EMD - 10923 , EMD - 10927 , EMD - 10928 , EMD - 789 10931 , EMD - 10932 , EMD - 10934 , EMD - 10935 , EMD - 10937 , EMD - 10938 , EMD - 10939 , 790 EMD - 10941 , EMD - 10942 ; Table S1 ) . 791 792 Competing interests 793 The authors declare that they have no competing interests . 794 795 Funding 796 This work was funded by grant from the European Research Council to PG ( AdG 797 340227 ) and the Swiss National Science Foundation ( SNSF ) to PaG 798 ( PP00P3 _ 157517 ) . 799 800 Authors ' contributions 801 SN , AB , GNH , VNV and DD collected and analyzed data . SN , AB , GNH , PG 802 contributed to experimental design and analysis , as well as writing the manuscript . 803 PaG and MLG contributed to data analysis . All authors read and approved the final 804 manuscript . 805 806 Acknowledgements 807 We are grateful to Yuichi Hongoh , Patrick Keeling and Filip Husnik for providing 808 termites . We thank Graham Knott and Stéphanie Rosset ( BioEM platform of the School 809 of Life Sciences , EPFL ) for assistance with TEM , as well as Kenneth Goddie , Lubomir 810 Kovacik and Henning Stahlberg ( Center of Nanoimaging , Biozentrum , Basel , 811 Switzerland ) with data collection on the Titan Krios . Niccolò Banterle , Graham Knott 812 and Fabian Schneider are acknowledged for their critical reading of the manuscript . 813 816 24 Figure legends 817 818 Figure 1 : Exceptionally long centriolar cartwheel in T . agilis 819 ( A ) Differential interference contrast micrograph of live T . agilis cell . The arrow points 820 to the cell anterior , where the rostrum is located ; arrowheads point to some of the 821 flagella . 822 ( B ) Transmission electron micrographs of T . agilis centriole embedded in resin - 823 longitudinal view ; the hub ( arrowhead ) is visible in the cartwheel - bearing region ( pink 824 line ) , but not in the distal region ( white line ) . 825 ( C ) Transmission electron micrographs of T . agilis centriole embedded in resin in 826 transverse view from distal end ( left ) and corresponding image circularized and 827 symmetrized with the CentrioleJ plugin ( middle ) , with schematic indicating principal 828 architectural elements ( right ) . 829 830 Figure 2 : Conserved architecture and polarity in T . spp . and T . agilis cartwheel 831 ( A , F ) Transverse 2D slices through STA of T . spp . ( A ) and T . agilis ( F ) central 832 cartwheel at indicated height , from proximal ( 0 nm ) to distal ( 8 . 7 nm ( A ) and 12 nm 833 ( F ) ) . The dashed box in the 5 . 2 nm ( A ) and 8 . 0 nm ( F ) slices indicate corresponding 834 regions shown in ( C , H ) . Schematic on top illustrates the area used to generate the 3D 835 maps of the central cartwheel . 836 ( B , G ) Transverse views of central cartwheel STA 3D map ; 9 spoke densities emanate 837 from the hub and the CID is present within the hub . The diameter of the hub is 23 . 0 ± 838 0 nm in T . spp . and 22 . 7 ± 0 . 2 in T . agilis ( both N = 3 ; here and thereafter in the figure 839 legends , N corresponds to 2D measurements from STA averages ) . An electron dense 840 structure is present inside the CID in T . spp . ( B , grey ) , which is also visible to some 841 extent before symmetrizing . Note that the coloring of elements in these and other figure 842 panels is merely illustrative and not meant to reflect underlying molecular boundaries . 843 ( C , H ) 2D longitudinal view of central cartwheel STA delineated by a dashed box in ( A , 844 F ) for T . spp . ( C ) and T . agilis ( H ) . Arrowheads denote position of line scans along the 845 vertical axis at the level of the CID ( C , purple ; H , dark pink ) , and hub ( C , light blue ; H , 846 light pink ) , with corresponding pixel intensities in arbitrary units ( AU ) . Plot profiles are 847 shifted horizontally relative to each other for better visibility . Some maxima are 848 highlighted with dashed lines ; the average distance between two CID elements is 8 . 5 849 ± 0 . 2 nm ( N = 4 ) in T . spp . ( C ) and 8 . 3 ± 0 . 5 ( N = 9 ) in T . agilis ( H ) . Note that hub densities 850 are elongated in the vertical direction , resulting in broad peak profiles where two 851 25 maxima can sometimes be discerned ( dashed light blue lines in ( C ) ; dashed light pink 852 lines in ( H ) ) . 853 ( D , I ) Longitudinal views of T . spp . ( D ) and T . agilis ( I ) central cartwheel STA . The 854 average distance between emanating spokes is 8 . 0 ± 1 . 5 nm ( N = 2 ) in T . spp . ( D ) and 855 8 . 2 ± 0 . 5 nm ( N = 9 ) in T . agilis ( I ) . Note that these values are measurements on STA 856 and are within the error of the CID periodicities reported in ( C , H ) ; the same applies for 857 other measurements hereafter . Discontinuous densities in the center of the CID ( grey ) 858 are visible in ( D , I ) ; the T . agilis volume in ( I ) is shown at a lower contour level than in 859 ( G ) . Boxed area is shown magnified in ( E , J ) . Proximal is down in this and all other 860 figure panels showing STA longitudinal views . 861 ( E , J ) The CID axis is located distal relative to axis of spoke densities , corresponding 862 to an average shift of 0 . 9 ± 0 . 7 ( N = 3 ) nm in T . spp . ( E ) and 0 . 8 ± 0 . 3 nm ( N = 9 ) in T . 863 agilis ( J ) . 864 865 Figure 3 : Architecture of peripheral elements in T . spp . and T . agilis . 866 ( A , E ) ( Top ) 2D slices through STA transverse view of microtubule triplet of T . spp . ( A ) 867 and T . agilis ( E ) , with insets showing position of pinhead ( dashed green box ) and A - C 868 linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on the pinhead 869 ( left ) or A - C linker ( right ) . Schematic on top illustrates the different areas used to 870 generate maps of the microtubule triplets ( B , F ) , pinhead ( C , G ) and A - C linker ( D , H ) . 871 ( B , F ) Transverse view of T . spp . ( B ) and T . agilis ( F ) microtubule triplet STA . 872 Microtubule protofilament numbers are indicated , as are the pinhead and A - C linker 873 ( only the C - link is visible ; the A - link lies on the edge of the volume and is thus less well 874 resolved in this STA - for better view see STA centered on A - C linker in D , H ) . 875 Prominent microtubule inner densities within the A - microtubule are highlighted ( empty 876 arrowhead next to A9 , chevron next to A5 ( B ) ) , as are additional external densities at 877 the A - B and B - C inner junctions ( black arrowheads ) . Double arrowheads point to 878 viewing direction in indicated panels . 879 ( C , G ) Longitudinal view of STA centered on the pinhead in T . spp . ( C ) and T . agilis 880 ( G ) , from the viewing point indicated in ( B , F ) . The pinfeet ( PinF1 and PinF2 ) and 881 pinbody ( PinB ) are indicated , as are microtubule protofilaments A3 and A4 . The 882 average distance between pinfeet elements is 8 . 6 ± 0 . 4 nm and 7 . 9 ± 0 . 4 nm in T . spp . 883 ( N = 3 each ; C ) and 8 . 4 ± 0 nm for each in T . agilis ( N = 3 each ; G ) . Corresponding 884 transverse views are shown below , illustrating the connection of PinF2 with 885 protofilament A3 . 886 26 ( D , H ) Longitudinal view of STA centered on the A - C linker in T . spp . ( D ) and T . agilis 887 ( H ) , from the viewing point indicated in ( B , F ) . Microtubule protofilaments A8 / 9 and 888 C9 / C10 of two adjacent triplets are indicated , as are the A - and C - links . The average 889 distance between A - and C - links is 8 . 4 ± 0 . 4 nm and 8 . 4 ± 0 . 3 nm in T . spp . ( both N = 6 ; 890 D ) as well as 8 . 4 ± 0 . 3 nm and 8 . 4 ± 0 . 9 nm in T . agilis ( N = 6 and N = 5 , respectively ; 891 H ) . Corresponding transverse views are shown below , chevrons point to connecting 892 points ; the connection of the A - link with A9 is only partially visible in the transverse 893 view at this height , as indicated by the dashed chevron ( H ) . 894 895 Figure 4 : Novel architectural features in T . mirabilis cartwheel . 896 ( A ) Transverse 2D slices through STA comprising 64 % and 36 % of sub - volumes of 897 T . mirabilis central cartwheel at indicated height , from proximal ( 0 nm ) to distal ( 8 . 0 898 nm ) . The dashed boxes in the 4 . 0 nm slices indicate region shown in D and E . Note 899 filamentous cartwheel inner densities ( fCID ) in the center of the hub . Schematic on top 900 illustrates the different areas used to generate 3D maps of the central cartwheel ( A - 901 G ) , MT triplets ( I ) , pinhead ( J ) and A - C linker ( K ) . 902 ( B , C ) Transverse views of T . mirabilis central cartwheel 3D map of 64 % ( B ) and 36 903 % ( C ) classes , with corresponding hub diameters of 23 . 5 ± 0 . 2 nm and 23 . 4 ± 0 . 5 nm 904 ( both N = 3 ) . In both classes the fCID is visible and 9 spokes emanate from the hub . 905 ( D , E ) 2D longitudinal view of T . mirabilis central region of the cartwheel STA 64 % ( D ) 906 and 36 % ( E ) classes in the region delineated by dashed boxes in ( A ) . Arrowheads 907 denote position of line scans along vertical axis at the hub level , with corresponding 908 normalized pixel intensities ( in arbitrary units ) . Some maxima are highlighted by 909 dashed lines . The average distance between hub units in the 64 % class is 8 . 4 ± 0 . 6 910 nm ( N = 8 ; D ) ; in the 36 % class , the distance between hub densities alternates between 911 3 . 2 ± 0 . 3 nm and 5 . 2 ± 0 . 3 nm and ( both N = 9 ; E ) . Dashed white line in ( E ) indicates 912 the offset between two superimposed hub units at the level of spoke densities , which 913 occurs every other hub unit pair . 914 ( F , G ) Longitudinal views of T . mirabilis cartwheel STA 64 % ( F ) and 36 % ( G ) classes . 915 The average distance between spokes is 8 . 4 ± 0 . 6 nm ( N = 8 ) in the 64 % class and 8 . 2 916 ± 1 . 5 nm ( N = 7 ) in the 36 % class . Note in both cases the continuous ~ 7 nm fCID inside 917 the hub . Note also densities bridging successive hubs vertically ( arrows ) . Asterisks 918 denote positions of individual units apparent within hub densities . Note that the fCID 919 was not always positioned in the geometrical center of the hub , suggestive of inherent 920 flexibility . 921 27 ( H ) ( top ) 2D slices through STA transverse view of microtubule triplet in T . mirabilis , 922 with insets showing pinhead ( dashed green box ) and A - C linker ( dashed red box ) . 923 ( bottom ) Longitudinal 2D slice of STA centered on the pinhead ( left ) or A - C linker 924 ( right ) . 925 ( I ) Transverse view of microtubule triplet STA in T . mirabilis . Microtubule protofilaments 926 are indicated , as are the positions of the pinhead and the C - link ( the A - link lies on the 927 edge of the volume and is thus less well resolved in this STA - for better views see 928 STA centered on A - C linker in K ) . At this contour level , MIPs are not visible . 929 Arrowheads indicate external densities at the A - B and B - C inner junctions ; chevron 930 indicates C - stretch . Double arrowheads point to viewing directions shown in ( J , K ) . 931 ( J ) Longitudinal view of T . mirabilis STA centered on the pinhead , from the viewing 932 point indicated in ( I ) . Location of pinhead consisting of pinfeet ( PinF1 and PinF2 ) and 933 pinbody ( PinB ) are indicated , as are microtubule protofilaments A3 and A4 . The 934 average distance between pinfeet elements is 8 . 4 ± 0 . 5 nm ( N = 5 ) . Corresponding 935 transverse view is shown below . 936 ( K ) Longitudinal view of T . mirabilis STA centered on the A - C linker , from the viewing 937 point indicated in ( I ) . Microtubule protofilaments A8 / A9 and C9 / C10 of two adjacent 938 triplets are indicated , as are the A - and C - links . The average distance between A - and 939 C - links is 8 . 4 ± 0 . 9 nm ( N = 6 ) and 8 . 4 ± 0 . 3 nm , respectively ( N = 5 ) . Corresponding 940 transverse views are shown below , chevrons point to connections . 941 942 Figure 5 : Direct stacking of SAS - 6 rings 943 ( A - D ) Computationally assembled CrSAS - 6 [ 6HR ] single ( A , C ) or double ring in 944 register ( ribbon diagram shown in different shades for clarity ) ( B , D ) fitted into the 3D 945 maps of the 36 % T . mirabilis class ( A , B ) and the 45 % T . agilis class ( C , D ) . Dashed 946 box shows longitudinal section through hub element ( left ) , double chevron viewing 947 point of longitudinal external views ( right ) . Extra unaccounted densities in single ring 948 fitting are indicated by arrowheads ( A , C ) . Note that each elongated hub density can 949 accommodate two tightly stacked CrSAS - 6 [ 6HR ] rings ( B , D ) , and that only one ring 950 can fit into the thin hub element in the 45 % T . agilis class ( C , D ) . Indicated distances 951 stem from measurements on the fitted models . 952 ( E ) Purified CrSAS - 6 [ NL ] protein self - organized into stacks and analyzed by cryo - ET ; 953 one longitudinal and one transverse view are highlighted by a dashed rectangle and a 954 dashed circle , respectively . 955 ( F , G ) 2D views through STA of in vitro self - assembled CrSAS - 6 [ NL ] proteins . 956 Transverse sections at the indicated heights through one assembly unit ( F ) , as well as 957 28 longitudinal view ( G ) of the area delineated by a dashed box in ( F ) ; white arrowhead 958 denotes position of line scan along the vertical axis at the level of the hub , with 959 corresponding pixel intensities in arbitrary units ( AU ) . The average distance between 960 two hub elements is 4 . 5 ± 0 . 3 nm ( N = 7 ; dashed lines ) . 961 ( H ) Longitudinal view of in vitro self - assembled CrSAS - 6 [ NL ] proteins STA 3D map . 962 Note densities bridging successive hubs vertically ( arrows ) . 963 ( I ) Two CrSAS - 6 [ HR ] single rings fitted into the 3D map of in vitro self - assembled 964 CrSAS - 6 [ NL ] proteins . Dashed box indicates longitudinal section through hub element 965 ( left ) , double chevron indicates viewing point of longitudinal external view ( right ) . Note 966 that the 3D map can accommodate rings stacked 4 . 4 nm apart ( measured on fitted 967 ring models ) , and vertical densities linking hubs are partially occupied . 968 969 Figure 6 : Working model of centriolar cartwheel architecture 970 ( A ) Occurrence of CID or fCID in species analyzed in this study ( T . spp . , T . agilis and 971 T . mirabilis , blue background ) , and in select other organisms mapped on a taxonomic 972 tree ( Tree generated using phyloTv2 based on NCBI taxonomy - 973 https : / / phylot . biobyte . de / ) . See main text for references . 974 ( B ) Schematic of cartwheel hub in T . spp . and T . agilis with 9 - fold symmetrical CID 975 connecting to the hub , equidistant between two spokes . 976 ( C ) The CID is polarized distally relative to the hub element comprising two tightly 977 superimposed ring elements in T . spp . and T . agilis . 978 ( D ) Schematic of cartwheel hub in T . mirabilis with continuous fCID along the proximal - 979 distal axis , with potential thin connections to the hub , equidistant between two spokes . 980 ( E ) Working model of cartwheel stacking , with alternation of two modes of SAS - 6 ring 981 stacking : pairs of rings are tightly superimposed with a rotational offset ( dashed line ) 982 and such offset double rings are then stacked in register ( solid line ) ; the offset direction 983 is consistent in T . spp . , T . agilis and T . mirabilis , yet may be different in other species , 984 as suggested in Paramecium ( Klena et al ) . Surface representation of SAS - 6 rings ; 985 homodimers highlighted by black contour . 986 ( F ) Consensus model of periodicities in centriolar cartwheel along the proximal - distal 987 axis . Surface representation of offset double rings tightly superimposed with ~ 3 . 2 nm 988 periodicity , in register with the next such offset double ring located ~ 5 . 2 nm away , thus 989 amounting to an overall periodicity of ~ 8 . 4 nm . Note that these values are based on 990 the T . mirabilis 36 % class , which has the most clearly resolved periodicities , yet does 991 not have a CID as shown in this composite model , with related values in other species ; 992 see text for details . The CID exhibits a related ~ 8 . 4 nm mean periodicity in the two 993 Trichonympha species . Two spoke periodicities are observed : close to the hub , pairs 994 29 of spokes are ~ 8 . 4 nm apart , whereas spoke pairs merge towards the periphery , where 995 their periodicity is hence ~ 16 . 8 nm , matching that of the pinhead and connected 996 microtubules . Note that spoke angles are merely illustrative and limited by the 997 resolution of the obtained STA maps . Note also that alternative modes of spoke 998 merging may be revealed by analyzing much larger sub - volumes and be present in 999 other species ( Klena et al ) . 1000 1001 1002 1003 Additional Data 1004 1005 Supplemental Table 1 - 2 1006 Supplemental Table Legends 1007 Table S1 1008 Statistics of Cryo - EM data collection and reconstruction and EMDB accession , 1009 see also Fig . S1 . 1010 1011 Table S2 1012 Correlation and overlap score for fitting with computationally assembled 1013 CrSAS - 6 [ 6HR ] single , double , and double rings with offset . 1014 Results of the fit in map function performed with ChimeraX by maximizing correlation 1015 as shown in Fig . 5 and Fig . S9 , Fig . S10 and described in Materials and Methods . 1016 Reported are the percentage of atoms inside the density map , the correlation , and the 1017 overlap between model and map . For each STA map , we report the gain in overlap 1018 upon fitting the double and offset double versus single ring model ; the maximum 1019 possible gain is + 100 % . 1020 1021 Supplemental Figures S1 - S10 1022 Supplemental Figure Legends 1023 Figure S1 : Cryo - EM acquisition , raw data and processing 1024 ( A ) Low magnification view of cryo - preserved purified T . spp . centriole and axoneme 1025 on carbon grid . Note that the proximal end of the centriole is blunt , and that the 1026 diameter of the centriole containing microtubule triplets is larger than that of the 1027 axoneme containing microtubule doublets , enabling one to determine the length of the 1028 30 centriole , as indicated . The black square illustrates the proximal location and the 1429 1029 x 1429 nm size of the field of view typically used for tilt series acquisition ; note that this 1030 can be less than the length of the centriole depending on the species . Dashed white 1031 rectangle : area corresponding to the tomogram shown in panel B . 1032 ( B ) 2D longitudinal view ( sum of 10 - 20 slices ) of the central cartwheel tomogram , with 1033 band - pass filter and Gaussian blur corresponding to the area with a dashed rectangle 1034 in ( A ) . Sub - volumes extracted along the longitudinal axis are ( 80 nm ) 3 for the central 1035 cartwheel ( pink squares represent one plane of the sub - volume ) and ( 89 nm ) 3 for the 1036 peripheral elements comprising microtubule triplet , pinhead and A - C linker ( blue 1037 squares represent one plane of the sub - volume ) , with a step size of 25 nm in both 1038 cases . 1039 ( C - F ) 2D longitudinal views from raw reconstructed tomograms of the central cartwheel 1040 illustrating typical sub - volumes ( sum of 10 - 20 slices of the central cartwheel tomogram , 1041 with band - pass filter and Gaussian blur ) from T . spp . ( C ) , T . agilis ( D ) , T . mirabilis ( E ) 1042 and CrSAS - 6 [ NL ] stacked assemblies ( F ) . Filled arrowhead indicate hub elements 1043 constituted of double units that can sometimes be resolved upon STA , but are not 1044 visible in this raw data ( C - E ) , empty arrowhead indicate hub elements in the T . agilis 1045 45 % class lacking the CID ( D ) . Note that classes cannot be unambiguously 1046 distinguished in the T . mirabilis raw tomograms , empty arrowheads in this case 1047 indicate hub elements with apparent variations in spacing ( E ) . Chevrons indicate 1048 resolved CrSAS - 6 [ NL ] single unit hub elements ( F ) . 1049 ( G - J ) Processing scheme with number of tomograms , initial sub - volumes extracted for 1050 cartwheel and peripheral elements ( microtubule triplet , pinhead and A - C linker ) , 1051 classification / refinement steps , and final sub - volumes used for STA in T . spp . ( G ) , T . 1052 agilis ( H ) , T . mirabilis ( I ) and CrSAS - 6 [ NL ] stacked assemblies ( J ) . See also Table S1 . 1053 1054 Figure S2 : Resolution maps of T . spp . 1055 ( A , D , G , J ) Local resolution distribution in T . spp . STA estimated with ResMap . Shown 1056 are transverse views of central cartwheel ( A ) , microtubule triplet ( D ) , pinhead ( G ) and 1057 A - C linker ( J ) . 1058 ( B , E , H , K ) Histogram and directional Fourier shell correlation ( FSC ) plots for T . spp . 1059 STA of central cartwheel ( B ) , microtubule triplet ( E ) , pinhead ( H ) and A - C linker ( K ) 1060 with global resolution at FSC 0 . 143 criterion indicated . 1061 ( C , F , I , L ) Power spectra of 2D class average with periodicities highlighted by arrows 1062 for T . spp . STA of central cartwheel ( C ) , microtubule triplet ( F ) , pinhead ( I ) and A - C 1063 linker ( L ) . The precision for the measurements in this and remaining power spectra is 1064 1 pixel , corresponding to a minimum of 0 . 4 nm ( see Table S1 ) . 1065 31 1066 Figure S3 : Resolution maps of T . agilis 1067 ( A , D , G , J , M ) Local resolution distribution in T . agilis STA estimated with ResMap . 1068 Shown are transverse views of central cartwheel 55 % class ( A ) , 45 % class ( D ) , 1069 microtubule triplet ( G ) , pinhead ( J ) and A - C linker ( M ) . 1070 ( B , E , H , K , N ) Histogram and directional FSC plots for T . agilis STA of central 1071 cartwheel 55 % class ( B ) , 45 % class ( E ) , microtubule triplet ( H ) , pinhead ( K ) and A - C 1072 linker ( N ) with global resolution at FSC 0 . 143 criterion indicated . 1073 ( C , F , I , L , O ) Power spectra of 2D class average with periodicities highlighted by 1074 arrows for T . agilis STA of central cartwheel 55 % class ( C ) , 45 % class ( F ) , microtubule 1075 triplet ( I ) , pinhead ( L ) and A - C linker ( O ) . 1076 1077 Figure S4 : Resolution maps of T . mirabilis 1078 ( A , D , G , J , M ) Local resolution distribution in T . mirabilis STA estimated with ResMap . 1079 Shown are transverse views of central cartwheel 64 % class ( A ) , 36 % class ( D ) , 1080 microtubule triplet ( G ) , pinhead ( J ) and A - C linker ( M ) . 1081 ( B , E , H , K , N ) Histogram and directional FSC plots for T . mirabilis STA of central 1082 cartwheel 64 % class ( B ) , 36 % class ( E ) , microtubule triplet ( H ) , pinhead ( K ) and A - C 1083 linker ( N ) with global resolution at FSC 0 . 143 criterion indicated . 1084 ( C , F , I , L , O ) Power spectra of 2D class average with periodicities highlighted by 1085 arrows for T . mirabilis STA of central cartwheel 64 % class ( C ) , 36 % class ( F ) , 1086 microtubule triplet ( I ) , pinhead ( L ) and A - C linker ( O ) . 1087 1088 Figure S5 : Resolution maps of CrSAS - 6 [ NL ] stacked assemblies 1089 ( A ) Local resolution distribution in CrSAS - 6 [ NL ] stacked assemblies ( generated from 1090 side - views ) STA estimated with ResMap shown in transverse views . 1091 ( B ) Histogram and directional FSC plots for CrSAS - 6 [ NL ] stacked assemblies STA with 1092 global resolution at FSC 0 . 143 criterion indicated . 1093 ( C ) Power spectra of 2D class average with periodicity highlighted by arrows for 1094 CrSAS - 6 [ NL ] stacked assemblies . 1095 1096 Figure S6 : Variation in hub architecture is some T . agilis sub - volumes 1097 ( A ) Transverse 2D slices through central cartwheel STA of T . agilis 45 % class , which 1098 comprises 25 % and 20 % sub - classes ( see E ) , at indicated height , from proximal ( 0 1099 nm ) to distal ( 18 . 0 nm ) . The pink circles mark spokes with CID ( 12 . 0 nm , dashed line ) 1100 or without CID ( 2 . 0 nm , solid line ) , as represented in ( B ) ; dashed box in the 4 . 0 nm 1101 32 slice indicates longitudinal section shown in ( C ) . Schematic on top illustrates the area 1102 used to generate the 3D maps of the central cartwheel . 1103 ( B ) Transverse views of central cartwheel STA 3D map of T . agilis 45 % class . The 1104 hub diameter is 22 . 7 nm ± 0 . 2 nm ( N = 3 ) with 9 emanating spoke densities , either at a 1105 level where the CID is present ( dashed circle ) or not ( solid circle ) , as indicated in ( A ) . 1106 ( C ) 2D longitudinal view of central cartwheel STA of T . agilis 45 % class delineated by 1107 a dashed box in ( A ) . Arrowheads denote position of line scans along the vertical axis 1108 at the level of the CID ( dark pink ) and the hub ( light pink ) , with corresponding 1109 normalized pixel intensities in arbitrary units ( AU ) . The plot profiles are shifted 1110 horizontally relative to each other for better visibility . The distance between hub 1111 densities alternates between 3 . 2 nm ( N = 1 ) and 6 . 0 ± 0 . 3 nm ( N = 2 ) ; maxima are 1112 indicated by dashed pink lines . Dashed white line indicates offset of two superimposed 1113 hub units . The average distance between two CID elements is 15 . 2 nm ( N = 1 ; dashed 1114 red line ) . The middle hub density that comprises only one unit and lacks a neighboring 1115 CID is indicated by a dashed arrow . 1116 ( D ) Longitudinal view of central cartwheel STA of T . agilis 45 % class at lower contour 1117 level than in ( B ) . Note densities bridging successive hubs vertically ( arrows ) , as well 1118 as proximal location of CID relative to the spoke density axis , resulting in a vertical 1119 offset of 1 . 2 ± 0 nm ( N = 2 ; arrowhead ) . Note also absence of CID in middle hub element 1120 comprising a single unit . 1121 ( E ) The spacing between the spoke density emanating from a double hub unit and the 1122 spoke density emanating from a single hub unit distal to it is 8 . 0 nm in the 25 % class 1123 and 7 . 1 nm in the 20 % class ( both N = 1 ) . By contrast , the spacing between the spoke 1124 density emanating from a double hub unit and the spoke density emanating from a 1125 single hub unit proximal to it is 8 . 7 nm in the 25 % class and 7 . 6 nm in the 20 % class 1126 ( both N = 1 ) . 1127 ( F ) Distribution of sub - volumes of the 55 % ( black circle ) and 45 % ( white circle ) 1128 classes along five T . agilis centrioles ; areas with neither black nor white circle could 1129 not be clearly assigned to either class . Note individual centrioles constituted of mostly 1130 one of the two classes , and others where the two classes are mixed without an 1131 apparent pattern , indicating that the distribution of 55 % and 45 % classes is not 1132 stereotyped along the centriole . 1133 1134 Figure S7 : Polar centriolar cartwheel 1135 ( A - P ) Non - symmetrized STA comprising larger volume than in Fig . 2 - 4 and centered 1136 on the spokes to jointly show the central cartwheel and peripheral elements in T . agilis 1137 ( A - D : 55 % class ; E - H : 45 % class ) and T . mirabilis ( I - L : 64 % class ; M - P : 36 % class ) . 1138 33 2D slices through STA transverse view ( A , E , I , M ) with corresponding 3D views ( B , F , 1139 J , N ) , as well as 2D longitudinal views ( C , G , K , O ) with corresponding 3D views ( D , H , 1140 L , P ) . The concerted proximal - distal polarity is visible from the central CID ( in A - H ) and 1141 the hub all the way to the pinhead . Note that proximal - distal polarity is visible also in 1142 the asymmetric spoke densities tilt angles in all cases , with a more pronounced tilt on 1143 the proximal side ( D , H , L , P ) . Note also that the unsymmetrized CID shown here 1144 resembles that in the symmetrized maps of Figure 2 , indicating that the CID exhibits 1145 bona fide 9 - fold radial symmetry ( A , E ) . Arrowhead in ( N , P ) points to a connection 1146 between fCID and hub . For representation , a Gaussian filter was applied to maps in 1147 ChimeraX . 1148 1149 Figure S8 : Very long cartwheel in T . mirabilis . 1150 ( A ) Differential interference contrast micrograph of live T . mirabilis cell . The arrow 1151 points to cell anterior , where the rostrum is located ; arrowheads point to some of the 1152 flagella . 1153 ( B ) Transmission electron micrograph of T . mirabilis centriole embedded in resin - 1154 longitudinal view . The hub ( arrowhead ) is visible in the proximal cartwheel - bearing 1155 region ( green line ) , but not in the distal region ( black line ) . 1156 ( C ) Transmission electron micrograph of T . mirabilis centriole embedded in resin in 1157 transverse view ( left ) and corresponding image circularized and symmetrized ( right ) . 1158 Note small density present inside the hub corresponding to the fCID . 1159 ( D ) Transverse view slice through cryo - electron tomogram of T . mirabilis centriole 1160 circularized and symmetrized . 1161 ( E ) Distribution of sub - volumes of the 64 % ( black circle ) and 36 % ( white circle ) 1162 classes along four T . mirabilis centrioles , proximal is left ; areas with neither black nor 1163 white circle could not be clearly assigned to either class . Note that the distribution of 1164 the two classes is not stereotyped along the centriole . 1165 1166 Figure S9 : CrSAS - 6 [ 6HR ] fitting in different cartwheel STA maps 1167 ( A ) Ribbon representation of CrSAS - 6 [ 6HR ] homodimers ( top , scale bar 5nm ) used for 1168 rigid body fitting into the 3D map from T . mirabilis 36 % class shown in side view ( left ) 1169 and end - on view magnified , with dashed line indicating offset between two 1170 superimposed homodimers ( right ) . Individual layers of homodimers are indicated in 1171 distinct colors for clarity . In this and all other panels of this figure , dashed box indicates 1172 longitudinal section through hub element ( left ) , double chevron indicates viewing 1173 direction of longitudinal external views ( right ) . 1174 34 ( B - D ) CrSAS - 6 [ 6HR ] single or double rings in register ( ribbon diagram represented in 1175 different shades for clarity ) fitted into the 3D maps from the T . mirabilis 64 % class ( B ) , 1176 T . spp . ( C ) and the T . agilis 55 % class ( D ) . Single ring fitting is shown in the top , 1177 double ring fitting at the bottom . Note unaccounted densities in the hub upon fitting of 1178 single rings in all cryo - ET maps ( arrowheads , B - D ) , whereas all maps readily 1179 accommodate double CrSAS - 6 [ 6HR ] rings at the hub element , although the coiled - coil 1180 elements extend slightly beyond the spoke densities . Note also that one coiled - coil in 1181 the T . mirabilis 64 % class sticks out of the density entirely ( B , bottom ) . 1182 1183 Figure S10 : Superimposed rings of SAS - 6 may be offset with respect to one 1184 another 1185 ( A , B ) ( left ) Ribbon representation of CrSAS - 6 [ 6HR ] homodimers computationally 1186 assembled into double rings ( rings represented in different shades for clarity ) with 1187 spokes in register ( A ) or with a 6 . 5° offset turning the proximal ring clockwise and 1188 moved 0 . 4 nm closer ( B ) ; transverse ( top ) and longitudinal view ( bottom ) . ( Right ) 1189 Magnified views of two vertically superimposed homodimers ( ribbon diagram ) , with 1190 dashed lines indicating angle between axes of the two coiled - coil axes ( top ) and 1191 surface representation of complementary interface ( bottom ) . Distances were 1192 measured on the ribbon diagram . 1193 ( C , D ) CrSAS - 6 [ 6HR ] offset double rings ( shown in B and represented in different 1194 colors for clarity ) fitted into 3D map from T . mirabilis 36 % class ( C ) and T . agilis 45 % 1195 class ( D ) , with dashed line indicating offset between double rings . Dashed box 1196 indicates longitudinal section through hub element ( left ) , double chevron viewing point 1197 of longitudinal external views ( right ) . 1198 ( E ) Schematic illustrating processing reported in ( F - H ) : after linearization of the 1199 cartwheel STA , a longitudinal section in Z - direction ( blue line ) is shown at the level of 1200 the spokes ( represented in pink ) . 1201 ( F - H ) Longitudinal section as explained in ( E ) for T . mirabilis 36 % and 64 % classes 1202 ( F ) , T . agilis 45 % and 55 % classes ( G ) and T . spp . ( H ) at the level of spoke emergence 1203 from the hub . 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T . agilis T . mirabilis C . reinhardtii centrioles in vitro CrSAS - 6 Data collection TEM F20 Krios Pixel size ( Å ) 3 . 49 2 . 71 Tilting scheme Bi - directional Dose - symmetric N tomograms 18 18 17 31 Reconstruction : cartwheel Step size for extraction ( Å ) 3 x 85 = 255 2 x 42 = 84 extracted sub - volumes 1385 1154 1802 926 sub - volumes final reconstruction 511 594 737 334 Resolution CW ( Å ) 24 23 23 15 EMDB accession EMD - 10927 EMD - 10916 EMD - 10918 EMD - 10922 EMD - 10923 EMD - 10928 Reconstruction : microtubule triplet ( MMT ) , pinhead ( PH ) , A - C linker ( AC ) Step size for extraction ( Å ) 3 x 85 = 255 na initial extracted sub - volumes : MMT , PH , AC 2792 3850 7928 na sub - volumes MMT , final 823 849 986 na Resolution MMT ( Å ) 38 40 38 na EMDB accession EMD - 10932 EMD - 10931 EMD - 10934 na sub - volumes PH , final 587 714 955 na Resolution PH ( Å ) 39 39 37 na EMDB accession EMD - 10938 EMD - 10935 EMD - 10941 na sub - volumes AC , final 1818 1512 1520 na Resolution AC ( Å ) 34 37 37 na EMDB accession EMD - 10939 EMD - 10937 EMD - 10942 na Table S1 . Map Ring model Percentage ( % ) of atoms inside contour Correlation Overlap Percentage ( % ) gain in overlap score by fitting double over single ring T . mirabilis 36 % Single 77 0 . 83 23 Double 63 0 . 71 31 + 58 Offset 69 0 . 73 27 + 87 T . mirabilis 64 % Single 93 0 . 78 17 Double 78 0 . 71 26 + 47 Offset 71 0 . 74 34 + 47 T . spp . Single 94 0 . 83 34 Double 64 0 . 65 41 + 23 Offset 63 0 . 69 45 + 33 T . agilis 55 % Single 99 0 . 86 38 Double 82 0 . 73 51 + 34 Offset 86 0 . 78 55 + 44 T . agilis 45 % ( thick ) Single 100 0 . 86 41 Double 87 0 . 79 60 + 46 Offset 78 0 . 83 64 + 56 T . agilis 45 % ( thin ) Single 93 0 . 87 33 Double 46 0 . 63 34 + 4 Offset 46 0 . 63 34 + 4 CrSAS - 6 [ NL ] in vitro Single 91 0 . 90 18 Double 91 0 . 90 35 + 97 Offset 84 0 . 88 33 + 83 Correlation and overlap are calculated with ChimeraX ( Goddard et al . , 2018 ) . Correlation value range : - 1 to 1 Table S2 . \ No newline at end of file diff --git a/silver_data/64da544dcc3a90af926cc2afe18cfbad057f9533.pdf.txt b/silver_data/64da544dcc3a90af926cc2afe18cfbad057f9533.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..a12cb1c9c90163c3cd82bf2be92a18fa1b24aac7 --- /dev/null +++ b/silver_data/64da544dcc3a90af926cc2afe18cfbad057f9533.pdf.txt @@ -0,0 +1 @@ +Predicting structural and functional sites in proteins by searching for maximum - weight cliques Franco Mascia and Elisa Cilia and Mauro Brunato and Andrea Passerini Information Engineering and Computer Science Department University of Trento - Via Sommarive , 14 I - 38100 Trento - Italy { mascia , cilia , brunato , passerini @ disi . unitn . it } Abstract Fully characterizing structural and functional sites in pro - teins is a fundamental step in understanding their roles in the cell . This extremely challenging combinatorial problem re - quires determining the number of sites in the protein and the set of residues involved in each of them . We formulate it as a distance - based supervised clustering task , where train - ing proteins are employed to learn a proper distance func - tion between residues . A partial clustering is then returned by searching for maximum - weight cliques in the resulting weighted graph representation of proteins . A novel stochas - tic local search algorithm is proposed to efficiently generate approximate solutions . Our method achieves substantial im - provements over a previous structured - output approach for metal binding site prediction . Significant improvements over the current state - of - the - art are also achieved in predicting cat - alytic sites from 3D structure in enzymes . Introduction In order to accomplish their biological function , proteins of - ten interact with different types of external molecules such as metal ions , prosthetic groups and various organic com - pounds . Metalloproteins ( Bertini , Sigel , and Sigel 2001 ) bind metal ions in order to stabilize their three - dimensional structure , induce conformational changes or assist protein function , such as electron transfer in cytochromes . Metal binding sites are characterized by the set of protein atoms directly involved in binding the ion , called ligands , and the overall geometry of the site . Furthermore , the same pro - tein often binds multiple ions , with typical numbers ranging from one to four . Enzymes are a fundamental type of pro - teins which accelerate chemical processes within a cell , by complexing with the substrate and thus lowering the activa - tion energy of the reaction . Functional residues play various roles in the catalytic process , such as donating electrons or polarizing cofactor bonds ( Bartlett et al . 2002 ) . Solely bind - ing substrates , cofactors or metals , which are often involved in enzymatic reactions , does not characterize a residue as catalytic according to the Catalytic Site Atlas ( CSA ) ( Porter , Bartlett , and Thornton 2004 ) . Being able to predict metal binding sites as well as en - zyme active sites in novel proteins is a fundamental step in Copyright c (cid:13) 2010 , Association for the Advancement of Artificial Intelligence ( www . aaai . org ) . All rights reserved . understanding their functioning . Both problems have been mostly addressed as a binary classification task at the residue level : given a protein sequence , predict for each residue whether it is involved in a metal binding site ( Passerini et al . 2006 ) , ( Shu , Zhou , and Hovmoller 2008 ) or an active site ( Tong et al . 2009 ) , ( Cilia and Passerini 2010 ) respec - tively . Most existing approaches for modeling the full metal binding geometry assume knowledge of the 3D structure of the protein ( Ebert and Altman 2008 ; Babor et al . 2008 ) and focus on detecting apo - proteins , i . e . proteins solved without the ion . A recent attempt ( Frasconi and Passerini 2008 ) to predict metal binding geometry from sequence formulates the problem as a structured - output task . The proposed solu - tion is a search algorithm greedily assigning residues to ions ( or a default nil ion if predicted as free ) guided by a scoring function trained to rank correct moves higher than incorrect ones . The algorithm is guaranteed to find the solution max - imizing the overall score , given the matroid structure of the problem . However , the scoring function is learned from ex - amples and there is no guarantee that it correctly approxi - mates the true underlying function . We take here a different viewpoint and formalize the prob - lem as a distance - based supervised clustering task ( Basu 2005 ) . Given a set of training instances , we first learn a similarity function predicting whether two residues jointly participate in a certain metal or active site . The learned sim - ilarity measure is subsequently fed to a maximum - weight clique algorithm collecting sets of residues maximizing their pairwise similarities . The algorithm has a number of desir - able features including automatic selection of the number of clusters , natural handling of overlapping clusters , and scal - ability to large datasets . Experimental results show a sub - stantial improvement over the structured - output approach for metal binding geometry prediction . Significant improve - ments over the state - of - the - art are also obtained for active site prediction from protein 3D structure , where both node and edge weights are employed in order to exploit both local predictions and spatial constraints . Problem description and formalization Given a protein sequence as a string of characters in the al - phabet of 20 amino acids , the problem consists of : detect - ing the number of binding or catalytic sites ; collecting for each site the set of protein residues involved . Metal binding Histogram of the Residue Catalytic Propensity Amino acids C a t a l y t i c P r open s i t y % A C E D G F I H K M L N Q P S R T W V Y 0 . 03 1 . 49 2 . 57 3 . 76 6 . 25 Figure 1 : Histogram of the catalytic propensities of the residues in the experimental dataset HA superfamily ( see ex - perimental section for details ) . sites tend to be rather specific in terms of possible ligands with cysteine ( C ) , histidine ( H ) , aspartic ( D ) and glutamic ( E ) acids being by far the most common ligands in tran - sition metals . Cysteines and histidines are the vast major - ity of ligands in structural sites , while aspartic and glutamic acids are quite common in proteins and their relative bind - ing frequency is thus very limited ( Passerini et al . 2006 ) . A more complex situation can be observed with alkali and alkaline - earth metals , which often bind proteins through the oxygen in backbone carbonyl groups . Catalytic propensity is even less specific , given the number of different roles that a residue can play within the active site . Figure 1 reports the catalytic propensity of the whole set of amino acids , show - ing that only few of them can be safely discarded . Previous results ( Cilia and Passerini 2010 ) on the simpler binary clas - sification task actually indicate that keeping all candidates produces slightly better results on average : the predictor oc - casionally manages to correctly predict rare amino acids as catalytic without significantly affecting precision . Concerning the number of sites , metalloproteins usually contain between one and three sites , sometimes four and oc - casionally more . The coordination number of a bound ion , i . e . the total number of its ligands , varies from one to about eight depending on the metal . Values between two and four are the most frequent for transition metals . Figure 2 shows the metal binding geometry of the equine herpes virus - 1 ( PDB code 1CHC ) , where candidate ligands in L = { C , H } not binding any ion are marked in grey . Contrarily to metal binding sites , enzymes tend to have a single catalytic site in - volving a larger number of residues , ranging from 1 to 9 in the experimental dataset we used . Multiple active sites can actually be found in some multimeric proteins , such as the 3 - isopropylmalate dehydrogenase ( PDB code 1A05 ) . Figure 3 shows the active site of cloroperoxidase T ( PDB code 1A7U and UniProtKB entry O31168 ) with seven residues corre - sponding to seven different amino acids involved . Note that proximity in sequence only partially relates to involvement in the same site , as the three - dimensional arrangement of MATVAERCPICLEDPSNYSMALPCLHAFCYVCITRWIRQNPTCPLCKVPVESVVHTIESDSEFGDQLI ZN1 ZN2 nil Figure 2 : Sequence of the equine herpes virus - 1 ( PDB code 1CHC ) . Residues composing the metal binding sites are highlighted in different colors . MPFITVGQEN STSIDLYYED HGAGQPVVLI HG F PLSGHSW 40 ERQSAALLDA GYRVITYDRR GFGQSSQPTT GYDYDTFAAD 80 LNTVLETLDL QDAVLVGF SM GTGEVARYVS SYGTARIAKV 120 AFLASLEPFL LKTDDNPDGA AP KEFFDGIV AAVKADRYAF 160 YTGFFNDFYN LDENLGTRIS EEAVRNSWNT AASGGFFAAA 200 AAPTTWYTDF RADIPRIDVP ALILHGTG D R TLPIENTARV 240 FHKALPSAEY VEVEGAP H GL LWTHAEEVNT ALLAFLAK Figure 3 : Sequence of the cloroperoxidase T ( PDB code 1A7U and UniProtKB entry O31168 ) . Residues composing the active site are highlighted in red . the protein can bring quite distant residues closer . However , additional features contribute to characterize target residues , such as conservation profile and residue neighborhood . Given these premises , we formulate the problem as a su - pervised clustering task . We provide a common formula - tion for both metal binding site and active site prediction . Slightly abusing terminology , we refer to residues involved in either type of site as ligands . While the two problems are treated as separate tasks in the experiments , they are in - deed highly correlated as metal binding sites are often part of a larger active site . We are planning to extend our work to predict a structured set of sites in order to jointly address these problems . A protein sequence is represented as the set x of its can - didate ligands , that is residues belonging to L . The output y for the sequence is a subset of the powerset of x , i . e . y ⊆ P ( x ) . Outputs for proteins in Figures 2 and 3 , for instance , would be represented as { { c 1 , c 2 , c 4 , c 5 } , { c 3 , h 1 , c 6 , c 7 } } and { f 2 , s 8 , m 2 , a 14 , p 7 , d 18 , h 6 } respectively , assuming L is equal to { C , H } for metal binding sites and the whole set of amino acids for catalytic sites . The desired output is thus a partial clustering of residues , where only predicted ligands are reported . Furthermore , at least for metal binding sites , clusters can overlap , as the same residue can simultaneously bind two ions , as happens for glutamic and aspartic acids with their two side - chain oxygen atoms . For comparison with previous approaches , experiments only deal with non - overlapping clusters , but our approach can naturally handle overlaps , as described in the next section . Distance - based supervised clustering with maximum - weight cliques A training set of labelled proteins can be easily obtained from experimentally solved protein structures and catalytic annotations , and a supervised clustering approach can thus be pursued . We opt for a distance - based supervised ap - proach ( Basu 2005 ) , where training instances are used to learn an appropriate distance ( or similarity ) measure to be later used in the clustering . The learning stage simply con - sists of training a pairwise classification function F ( x i , x j ) predicting for each pair of residues x i and x j in x whether they belong to the same site . We employ a pairwise support vector machine ( SVM ) as the underlying classification func - tion . More complex alternatives can be pursued , as will be detailed in the Discussion . Given a learned similarity function F , we represent a set x as a weighted graph , removing edges whose weight is be - low a certain threshold φ and rescaling remaining weights to be positive . A maximum - weight clique algorithm is then run on the graph in order to return a set of maximal cliques , which correspond to the predicted sites . The rationale for the approach is that given a reasonable pairwise similarity measure , the algorithm should isolate few densely connected components which correspond to the desired solution while discarding most of the nodes in the graph . The algorithm can be asked to return a single large cluster , as typical of the active site prediction task , or a set of possibly overlapping maximal cliques , as for the metal binding site case , where the number of clusters cannot be specified a priori . The maximum - weight clique clustering algorithm Maximum Clique is a paradigmatic NP - hard problem with relevant applications in many areas ; its weighted versions originate from fields such as computer vision , pattern recog - nition and robotics ( Ballard and Brown 1982 ) . A survey on recent literature on Weighted Maximum Clique algorithms can be found in ( Pullan 2008 ) . In the following we introduce our heuristic algorithm . We describe it for weighted edges only . Its extension for deal - ing with weights on both nodes and edges , as well as the case where weights are averaged on the number of nodes , is straightforward . Given a set of residues R , in the previous section we de - fined a learned symmetric similarity function F that maps each pair of residues onto a measure of likelihood that they belong to the same cluster . Given a positive threshold value φ , we define a weighted undirected graph as a triplet G φ ≡ ( R , E φ , F ) where the vertex set R is composed by the residues , the edge set E φ is defined by vertex pairs whose similarity function F is above the threshold φ E φ = (cid:8) { u , v } ⊂ R : u (cid:54) = v ∧ F ( u , v ) ≥ φ (cid:9) , and the weight of every edge e ∈ E φ is given by F ( e ) . From now on , subscript φ shall be removed for clarity . A clique in graph G is defined as a completely connected subgraph of G , i . e . , any subset R (cid:48) ⊆ R such that for every pair of nodes u , v ∈ R (cid:48) the pair { u , v } belongs to E . The Edge - Weighted Maximum Clique Problem requires to find the clique in R that maximizes the sum of weights : R (cid:48) max = arg max R (cid:48)⊆ R R (cid:48) cliquein G (cid:88) u , v ∈ R (cid:48) F ( u , v ) . Input Meaning R , E , F E Edge - weighted undirected graph Variable Meaning t Current iteration index T Prohibition period L v Last iteration when v ∈ R was added / removed ¯ R Current configuration P List of nodes that can be added to ¯ R w Clique weight v Chosen node a Action to be taken ( Add or Drop ) 1 function WMC ( R , E , F E ) 2 L v ← −∞ for v ∈ R 3 t ← 0 ; ¯ R ← ∅ ; P ← R ; w ← 0 4 repeat 5 U PDATE P ROHIBITION ( ¯ R , T ) 6 ( v , a ) ← C HOOSE N ODE ( L , ¯ R , P , T , t , E , F E ) 7 if a = Add 8 ¯ R ← ¯ R ∪ { v } 9 else 10 ¯ R ← ¯ R \ { v } 11 recompute P and w incrementally 12 L v ← t 13 if too many iterations without improvements 14 R ESTART ( ) 15 t ← t + 1 16 until termination condition is met 17 return best ¯ R found Figure 4 : The main section of WMC : the local search step is repeated and the best clique is returned ( bookkeeping opera - tions such as best configuration maintenance are not shown ) . Being a generalization of the Maximum Clique Problem , the edge - weighted version is also NP - hard . In this paper , we introduce the Reactive Local Search optimization heuristic for Weighted Maximum Clique finding ( RLS - WMC , in the following WMC for short ) , based on the RLS - MC heuristic for Maximum Clique finding ( Battiti and Protasi 2001 ) , with a novel dynamic behavior adapted from ( Battiti and Mascia 2010 ) . The reaction technique of the WMC heuristic , described below , offers an effective diversification mechanism that provides a thorough exploration of the search space , and is therefore capable of dealing with problem instances for which exhaustive enumeration is infeasible . The WMC heuristic , whose main section is shown in Fig . 4 , is a stochastic local search ( SLS ) algorithm . In SLS algorithms for the MC problem , a “current” configuration ( subset of vertices ) ¯ R ⊆ R is maintained throughout the search , being initially the empty set ( line 4 ) , and is modified by incremental moves consisting in the addition or in the re - moval of a node ( lines 10 – 13 ) . At every step the “current” configuration is required to be a clique in the original graph ( the system generally moves only within feasible solutions ) , 1 function C HOOSE N ODE ( L , ¯ R , P , T , t , E , F E ) 2 S ← (cid:110) w ∈ P : L w > t − T ∧ ∧ w maximizes future expectations (cid:111) 3 a ← Add 4 if S = ∅ 5 S ← (cid:110) w ∈ ¯ R : L i > t − T ∧ ∧ w maximizes future expectations (cid:111) 6 a ← Drop 7 Pick v ∈ S 8 return ( v , a ) Figure 5 : The C HOOSE N ODE procedure : choose the non - prohibited node having the best chance to lead to better cliques in the future ; if no nodes can be added , pick one for removal . therefore the addition move will only consider nodes that maintain the clique property , i . e . , that are connected to all nodes in ¯ R . Such set of eligible nodes is called P in Fig . 4 , and is maintained incrementally during the search . The WMC heuristic completes the generic SLS frame - work by defining the criteria by which the incremental moves are selected . In particular , a parameter T , called pro - hibition period , is set and a vector ( L v ) v ∈ R , storing the last iteration at which node v was added or removed to the cur - rent clique ¯ R , is initialized ( line 3 ) and maintained ( line 15 ) . Nodes that have been used in the last T iterations , called prohibited , are not considered for addition or removal . This mechanism , known as Tabu Search , prevents the system from getting stuck in local optima and encourages diversi - fication . The move selection routine C HOOSE N ODE , whose pur - pose is the choice of the next node to be added or removed , is outlined in Fig . 5 . Array ( L v ) is used to check prohibi - tions . Since more than one non - prohibited node is usually eligible for addition to ¯ R , other selection criteria intervene in order to maximize the chance that a large clique will be obtained , for instance by choosing the node that maximizes the average edge weight ( line 2 ) , with ties broken randomly ( line 7 ) . If no nodes are eligible for insertion in the cur - rent configuration ¯ R ( either because there are no more nodes connected to all nodes in ¯ R , or all of them are prohibited ) , then a non - prohibited node chosen within ¯ R is selected for removal ( lines 4 – 6 ) . The value of the prohibition period T is critical for the good behavior of the algorithm . Small values of T tend to be insufficient for the system to efficiently escape local op - tima , while high values highly reduce the flexibility of the search procedure by reducing the number of eligible nodes . Rather than relying on an ideal value of T as a function of the graph size and of its density , WMC determines it dynam - ically ( line 8 of Fig 4 ) by calling a function , U PDATE P RO - HIBITION , that detects anomalous situations where a change would benefit the search . To achieve this , recent configura - tions are stored in a hash table ; if a configuration is visited ( i . e . , becomes the current one ) too often , then the T param - eter is increased in order to improve the differentiation ca - pabilities of the algorithm . If , on the other hand , no config - uration is revisited for a given time , T is reduced . Further details on the dynamic adaptation of T are available in ( Bat - titi and Mascia 2010 ) . Finally , a R ESTART mechanism is provided ( lines 16 – 17 ) : if the best solution is not improved in a while , then the al - gorithm is restarted , so that new regions of the search space are visited . The RLS - WMC algorithm maintains the weight of the current configuration ¯ R by incrementally updating it at every move . For the purposes of this paper , cliques within the ex - pected size are stored along with their weight , and are post - processed in order to determine which ones represent the correct clusters . Bookkeeping operations such as the com - putation of the clique weight , storage of the visited cliques and of the best clique are not detailed here . Experimental results Predicting geometry of metal binding sites We tested our method on the task of predicting metal bind - ing sites in metalloproteins . We used the same setting de - scribed in ( Frasconi and Passerini 2008 ) , with 30 random 80 / 20 train / test splits . We encoded pairs of residues by con - catenating their features vectors , thus comparing residues according to their order in the sequence . This option was shown ( Frasconi and Passerini 2008 ) to provide better re - sults with respect to alternative approaches such as averaged pairwise comparisons , possibly because sequential ordering is relevant in characterizing sites . Pairs were labeled posi - tive if both residues bind to the same metal ion and negative otherwise , and an SVM was used as the pairwise classifier . All parameters concerning the SVM and the maximum weighted clique algorithm described below were selected by an inner - fold cross - validation on the training set of the first split and kept fixed for all remaining folds . As a result of this model selection phase , we employed a second degree poly - nomial kernel and a cost factor j = 3 outweighing error on positive with respect to negative examples . In building the weighted graph , we discarded edges having weight smaller than - 0 . 9 , and rescaled remaining weights to have positive values . The weight of each clique was averaged over the number of its nodes . The algorithm returned the set of non - overlapping solutions with at most four residues . We made no further selection of the returned solutions , except for lim - iting the number of solutions to 4 . We present here a set of measures including those re - ported in ( Frasconi and Passerini 2008 ) . Note that we are not trying to predict the identity of an ion ( e . g . the “first” zinc , the “second” iron or so ) , but only the subset of residues which jointly bind the same one . Thus , when evaluating the quality of a certain clustering , we assign each ion to the cluster containing the highest number of its true ligands ( if any ) . An equivalent approach was employed in ( Frasconi and Passerini 2008 ) . P E , R E , and F E are the precision , re - call , and F 1 of the correct assignment between a ligand and a metal ion . P S , R S , and F S are the precision , recall , and F 1 of the correct prediction of binding sites , i . e . , how many sites are entirely correctly predicted over the total number of sites in the chain . P B , R B , and F B are the precision , recall , and F 1 of the correct prediction of the bonding state of the residues in the chain , i . e . regardless of which ion they actu - ally bind . Tables 1 and 2 report the mean and standard de - viation of these performance measures averaged over the 30 splits . The breakdown of these measures for proteins bind - ing different numbers of metal ions ( i . e . from 1 to 4 ) is also reported . # sites SVM + WMC ( FrasconiandPasserini2008 ) P E R E F E P E R E F E any 79 ± 3 • 59 ± 5 • 62 ± 5 • 66 ± 5 52 ± 4 53 ± 4 1 84 ± 4 73 ± 7 73 ± 6 66 ± 7 58 ± 6 57 ± 6 2 70 ± 8 33 ± 5 42 ± 6 67 ± 7 44 ± 9 48 ± 9 3 70 ± 15 22 ± 8 32 ± 11 69 ± 19 24 ± 13 32 ± 12 4 42 ± 30 16 ± 13 23 ± 18 42 ± 31 20 ± 19 26 ± 22 P S R S F S P S R S F S any 42 ± 7 • 30 ± 7 • 31 ± 7 • 20 ± 7 17 ± 6 16 ± 6 1 50 ± 8 41 ± 9 41 ± 9 25 ± 10 22 ± 8 22 ± 8 2 25 ± 14 8 ± 7 11 ± 9 15 ± 9 7 ± 7 7 ± 7 3 23 ± 32 4 ± 7 5 ± 11 0 ± 2 0 ± 1 0 ± 2 4 9 ± 21 3 ± 6 5 ± 9 2 ± 7 1 ± 5 1 ± 5 P B R B F B P B R B F B any 88 ± 3 • 63 ± 5 67 ± 4 • 79 ± 4 64 ± 6 64 ± 4 1 84 ± 4 73 ± 7 73 ± 6 74 ± 5 68 ± 7 65 ± 6 2 92 ± 8 45 ± 6 58 ± 7 88 ± 5 60 ± 11 66 ± 10 3 100 ± 0 34 ± 12 49 ± 15 98 ± 5 38 ± 22 50 ± 20 4 67 ± 45 25 ± 18 36 ± 25 65 ± 44 32 ± 28 40 ± 31 Table 1 : Comparison on the metalloproteins dataset . The means and standard deviations are computed on the 30 ran - dom splits . A bullet indicates that the performance differ - ences are statistically significant ( p < 0 . 05 ) . # sites any 1 2 3 4 SVM + WMC 27 ± 6 • 40 ± 9 1 ± 4 0 ± 0 0 ± 0 ( FrasconiandPasserini2008 ) 14 ± 6 20 ± 8 3 ± 7 0 ± 0 0 ± 0 Table 2 : Experimental results on the metalloproteins dataset . A G is the accuracy at a chain level , i . e . , the number of entire configurations correctly predicted . A bullet indicates that the performance differences are statistically significant ( p < 0 . 05 ) . Our SVM + WMC approach achieves significant improve - ments over the previous structured - output approach in edge , site and bonding state prediction , as measured by paired Wilcoxon tests ( p < 0 . 05 ) . The most significative improvement over ( Frasconi and Passerini 2008 ) lies in the number of sites entirely correctly predicted . The overall P S , R S , and F S , is consistently better for any number of metal ions in the protein . Active sites prediction We applied our approach to the prediction of active sites in enzymes . We focused on the HA superfamily dataset ( Chea and Livesay 2007 ) , the largest dataset employed as bench - mark in the literature . Prediction of catalytic residues was previously addressed starting from either sequence or struc - tural information . We considered both settings , relying on previous state - of - the - art results by a simple support vector machine exploiting residue structural neighborhood ( Cilia and Passerini 2010 ) . The detailed description of the features employed for both sequence - based and structure - based pre - dictions can be found in this previous work . Given that most proteins contain a single active site , and the labeling found in the CSA ( Porter , Bartlett , and Thornton 2004 ) does not in - clude information on different sites , we considered a single site prediction setting . Common examples of multiple active sites are those of polymeric proteins in which a pair of spec - ular sites is found at the interface of two identical chains . We plan to extract this additional information from known 3D structures in order to fully characterize overall geometry in an extended version of the work . For sequence - based prediction , we employed a setting analogous to the metal binding site case , with pairs of residues represented as ordered pairs of feature vectors from ( Cilia and Passerini 2010 ) . Following ( Cilia and Passerini 2010 ) , we employed a linear kernel and a 6 to 1 subsampling of negative ( i . e . non - catalytic ) residues , result - ing in a 61 / 1 proportion of negative vs positive residue pairs . Following the site size distribution in training instances , we fixed the maximum size of cliques to six . For structure - based prediction , we took a slightly differ - ent approach , since we could also exploit the spatial infor - mation provided by the protein structure . We modified the maximum - weight clique algorithm in order to consider both edge and node weights . Edge weights were in this case in - verse Euclidean distances between corresponding residues , pruned for distances over 14 ˚A . This threshold was chosen according to the distribution of distances between catalytic residues in the training set . The idea of constraining can - didate solutions based on their pairwise 3D distances was actually used in the MBG prediction approach by Babor et al . ( Babor et al . 2008 ) as an initial filtering stage . However the 3D constraint is much less stringent in catalytic sites , as shown by the quite large threshold ( 14 ˚A ) we derived from data . Node weights encoded catalytic propensity as predicted by the state - of - the - art support vector machine pre - dictor described in ( Cilia and Passerini 2010 ) . Node and edge weights were normalized in order to fall within the same range of values . Experimental comparisons with the local approach in ( Cilia and Passerini 2010 ) are shown in Table 3 , where the protein - level precision , recall and F 1 measures averaged across folds are reported . ( Cilia and Passerini 2010 ) SVM + WMC P R F 1 P R F 1 seq 20 ± 4 59 ± 7 25 ± 4 22 ± 2 41 ± 4 27 ± 3 • struct 23 ± 3 65 ± 6 28 ± 3 35 ± 7 43 ± 7 34 ± 6 • Table 3 : Comparison of the results ( performance ± st . d . ) obtained in active site prediction . A bullet indicates that the performance differences are statistically significant ( p < 0 . 05 ) . The SVM + WMC approach achieves significant improve - ments at p < 0 . 05 in both sequence - based and structure - based predictions according to a paired Wilcoxon test . Note that the average protein - level F 1 of the local predictor is quite lower than the F 1 computed from average protein - level precision and recall . This happens because the local SVM produces rather unbalanced predictions , either maximizing recall with low precision or ( more rarely ) vice versa , and for a number of proteins it outputs completely wrong predic - tions . The SVM + WMC approach is much more stable and balanced in its predictions . Note also that the improvement in F 1 is not simply due to a better choice of the decision threshold with respect to the standard local approach . The best F 1 value which could be obtained with local sequence - based predictions by optimizing the threshold ( on the test set ) is just 0 . 256 . Results from the structured - based predic - tion significantly improve the current state - of - the - art thanks to an effective use of the spatial geometry information . In particular , the algorithm finds cliques that discard many of the classifier false positives . Discussion We address the problem of predicting geometry of structural and functional sites in proteins by casting it into a super - vised clustering task . We propose a novel distance - based supervised clustering approach in which the learned pair - wise distance is employed to turn instances into weighted graphs . A maximum - weight clique algorithm is executed on the graph to return a small set of densely connected compo - nents corresponding to candidate sites . Supervised cluster - ing is an active area of research and a number of different ap - proaches have been proposed in the literature ( Basu 2005 ) . We use a very simple distance learning approach based on pairwise classification of instances . The maximum - weight clique clustering algorithm is however independent of this stage , and can be easily integrated in more complex super - vised clustering approaches such as the structured - output formulation proposed in ( Finley and Joachims 2005 ) . The algorithm substantially improves over the only exist - ing approach in predicting geometry of metal binding sites from sequence alone . Focusing on small components with large overall weights , our algorithm is more robust to a pos - sibly incorrect bonding state prediction . On the other hand , the structured - output approach in ( Frasconi and Passerini 2008 ) is capable of exploiting the full relational structure of partial solutions in order to evaluate them , instead of be - ing limited to networks of pairwise interactions . Indeed , such approach is superior when bonding state information is assumed to be known . We are planning to extend our algorithm in order to deal with clique - based weights , thus combining some of the advantages of the two formulations : the ability of a structured - output approach to better model the quality of candidate solutions , and the robustness of stochastic local search strategies in dealing with a scoring function which only approximates conditions guaranteeing greedy optimality ( Frasconi and Passerini 2008 ) . Significant improvements over the state - of - the - art are also obtained in predicting active sites from 3D structure . The al - gorithm naturally handles the lack of knowledge in the num - ber of clusters , partial clusterings with many outliers and overlapping clusters . We are planning to extend it to return a structured set of solutions , such as metal binding sites as parts of wider active sites , a quite common situation in en - zymes . Acknowledgements Many thanks to Mich ` ele Sebag for very fruitful discussions . References Babor , M . ; Gerzon , S . ; Raveh , B . ; Sobolev , V . ; and Edel - man , M . 2008 . Prediction of transition metal - binding sites from apo protein structures . Proteins 70 ( 1 ) : 208 – 217 . Ballard , D . , and Brown , C . 1982 . Computer Vision . Engle - wood Cliffs : Prentice - Hall . Bartlett , G . ; Porter , C . ; Borkakoti , N . ; and Thornton , J . 2002 . Analysis of catalytic residues in enzyme active sites . J Mol Bio 2002 324 ( 1 ) : 105 – 121 . Basu , S . 2005 . Semi - supervised clustering : probabilistic models , algorithms and experiments . Ph . D . Dissertation , University of Texas at Austin . Battiti , R . , and Mascia , F . 2010 . Reactive and dynamic local search for max - clique : Engineering effective building blocks . Computers & Operations Research 37 ( 3 ) : 534 – 542 . Battiti , R . , and Protasi , M . 2001 . Reactive local search for the maximum clique problem . Algorithmica 29 ( 4 ) : 610 – 637 . Bertini , I . ; Sigel , A . ; and Sigel , H . , eds . 2001 . Handbook on Metalloproteins . Marcel Dekker , New York , 1 edition . Chea , E . , and Livesay , D . R . 2007 . How accurate and statistically robust are catalytic site predictions based on closeness centrality ? BMC Bioinformatics 8 : 153 + . Cilia , E . , and Passerini , A . 2010 . Automatic prediction of catalytic residues by modeling residue structural neighbor - hood . BMC Bioinformatics 11 ( 1 ) : 115 . Ebert , J . C . , and Altman , R . B . 2008 . Robust recognition of zinc binding sites in proteins . Protein Sci 17 ( 1 ) : 54 – 65 . Finley , T . , and Joachims , T . 2005 . Supervised clustering with support vector machines . In ICML . Frasconi , P . , and Passerini , A . 2008 . Predicting the geom - etry of metal binding sites from protein sequence . In NIPS , 465 – 472 . Passerini , A . ; Punta , M . ; Ceroni , A . ; Rost , B . ; and Fras - coni , P . 2006 . Identifying cysteines and histidines in transition - metal - binding sites using support vector ma - chines and neural networks . Proteins 65 ( 2 ) : 305 – 316 . Porter , C . T . ; Bartlett , G . J . ; and Thornton , J . M . 2004 . The catalytic site atlas : a resource of catalytic sites and residues identified in enzymes using structural data . Nucleic Acids Res 32 ( Database issue ) . Pullan , W . 2008 . Approximating the maximum vertex / edge weighted clique using local search . Journal of Heuristics 14 : 117 – 134 . Shu , N . ; Zhou , T . ; and Hovmoller , S . 2008 . Prediction of zinc - binding sites in proteins from sequence . Bioinformat - ics 24 ( 6 ) : 775 – 782 . Tong , W . ; Wei , Y . ; Murga , L . ; Ondrechen , M . ; and Williams , R . 2009 . Partial order optimum likelihood ( POOL ) : Maximum likelihood prediction of protein active site residues using 3D structure and sequence properties . PLoS Computational Biology 5 ( 1 ) : e1000266 + . \ No newline at end of file diff --git a/silver_data/64edd2c5c41e856695e1dcb950c0512c3a87edec.pdf.txt b/silver_data/64edd2c5c41e856695e1dcb950c0512c3a87edec.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..af3a518ff88b82d3341a391c1015e7d6dc4cf13f --- /dev/null +++ b/silver_data/64edd2c5c41e856695e1dcb950c0512c3a87edec.pdf.txt @@ -0,0 +1 @@ +Improving Recommendation Lists Through Topic Diversification Cai - Nicolas Ziegler 1 ∗ Sean M . McNee 2 1 Institut f¨ur Informatik , Universit¨at Freiburg Georges - K¨ohler - Allee , Geb¨aude Nr . 51 79110 Freiburg i . Br . , Germany { cziegler , lausen } @ informatik . uni - freiburg . de Joseph A . Konstan 2 Georg Lausen 1 2 GroupLens Research , Univ . of Minnesota 4 - 192 EE / CS Building , 200 Union St . SE Minneapolis , MN 55455 , USA { mcnee , konstan } @ cs . umn . edu ABSTRACT In this work we present topic diversification , a novel method designed to balance and diversify personalized recommenda - tion lists in order to reflect the user’s complete spectrum of interests . Though being detrimental to average accuracy , we show that our method improves user satisfaction with rec - ommendation lists , in particular for lists generated using the common item - based collaborative filtering algorithm . Our work builds upon prior research on recommender sys - tems , looking at properties of recommendation lists as en - tities in their own right rather than specifically focusing on the accuracy of individual recommendations . We introduce the intra - list similarity metric to assess the topical diver - sity of recommendation lists and the topic diversification approach for decreasing the intra - list similarity . We evalu - ate our method using book recommendation data , including offline analysis on 361 , 349 ratings and an online study in - volving more than 2 , 100 subjects . Categories and Subject Descriptors H . 3 . 3 [ Information Storage and Retrieval ] : Information Retrieval and Search— Information Filtering ; I . 2 . 6 [ Artifi - cial Intelligence ] : Learning— Knowledge Acquisition General Terms Algorithms , Experimentation , Human Factors , Measurement Keywords Collaborative filtering , diversification , accuracy , recommend - er systems , metrics 1 . INTRODUCTION Recommender systems [ 23 ] intend to provide people with recommendations of products they will appreciate , based on their past preferences , history of purchase , and demographic information . Many of the most successful systems make use of collaborative filtering [ 27 , 8 , 11 ] , and numerous commer - cial systems , e . g . , Amazon . com’s recommender [ 16 ] , exploit ∗ Researched while at GroupLens Research in Minneapolis . Copyright is held by the International World Wide Web Conference Com - mittee ( IW3C2 ) . Distribution of these papers is limited to classroom use , and personal use by others . WWW 2005 , May 10 - 14 , 2005 , Chiba , Japan . ACM 1 - 59593 - 046 - 9 / 05 / 0005 . these techniques to offer personalized recommendation lists to their customers . Though the accuracy of state - of - the - art collaborative fil - tering systems , i . e . , the probability that the active user 1 will appreciate the products recommended , is excellent , some im - plications affecting user satisfaction have been observed in practice . Thus , on Amazon . com ( http : / / www . amazon . com ) , many recommendations seem to be “similar” with respect to content . For instance , customers that have purchased many of Hermann Hesse’s prose may happen to obtain recom - mendation lists where all top - 5 entries contain books by that respective author only . When considering pure accu - racy , all these recommendations appear excellent since the active user clearly appreciates books written by Hermann Hesse . On the other hand , assuming that the active user has several interests other than Hermann Hesse , e . g . , his - torical novels in general and books about world travel , the recommended set of items appears poor , owing to its lack of diversity . Traditionally , recommender system projects have focused on optimizing accuracy using metrics such as precision / recall or mean absolute error . Now research has reached the point where going beyond pure accuracy and toward real user ex - perience becomes indispensable for further advances [ 10 ] . This work looks specifically at impacts of recommendation lists , regarding them as entities in their own right rather than mere aggregations of single and independent sugges - tions . 1 . 1 Contributions We address the afore - mentioned deficiencies by focusing on techniques that are centered on real user satisfaction rather than pure accuracy . The contributions we make in this paper are the following : • Topic diversification . We propose an approach to - wards balancing top - N recommendation lists accord - ing to the active user’s full range of interests . Our novel method takes into consideration both the accuracy of suggestions made , and the user’s extent of interest in specific topics . Analyses of topic diversification’s im - plications on user - based [ 11 , 22 ] and item - based [ 26 , 5 ] collaborative filtering are provided . 1 The term “active user” refers to the person for whom rec - ommendations are made . • Intra - list similarity metric . Regarding diversity as an important ingredient to user satisfaction , metrics able to measure that characteristic feature are required . We propose the intra - list similarity metric as an effi - cient means for measurement , complementing existing accuracy metrics in their efforts to capture user satis - faction . • Accuracy versus satisfaction . There have been sev - eral efforts in the past arguing that “accuracy does not tell the whole story” [ 4 , 12 ] . Nevertheless , no evidence has been given to show that some aspects of actual user satisfaction reach beyond accuracy . We close this gap and provide analysis from large - scale online and offline evaluations , matching results obtained from ac - curacy metrics against actual user satisfaction and in - vestigating interactions and deviations between both concepts . 1 . 2 Organization Our paper is organized as follows . We discuss collabora - tive filtering and its two most prominent implementations in Section 2 . The subsequent section then briefly reports on common evaluation metrics and the new intra - list similarity metric . In Section 4 , we present our method for diversify - ing lists , describing its primary motivation and algorithmic clockwork . Section 5 reports on our offline and online exper - iments with topic diversification and provides ample discus - sion of results obtained . 2 . ON COLLABORATIVE FILTERING Collaborative filtering ( CF ) still represents the most com - monly adopted technique in crafting academic and commer - cial [ 16 ] recommender systems . Its basic idea refers to mak - ing recommendations based upon ratings that users have assigned to products . Ratings can either be explicit , i . e . , by having the user state his opinion about a given product , or implicit , when the mere act of purchasing or mentioning of an item counts as an expression of appreciation . While im - plicit ratings are generally more facile to collect , their usage implies adding noise to the collected information [ 20 ] . 2 . 1 User - based Collaborative Filtering User - based CF has been explored in - depth during the last ten years [ 29 , 24 , 14 ] and represents the most popular recom - mendation algorithm [ 11 ] , owing to its compelling simplicity and excellent quality of recommendations . CF operates on a set of users A = { a 1 , a 2 , . . . , a n } , a set of products B = { b 1 , b 2 , . . . , b m } , and partial rating functions r i : B → [ − 1 , + 1 ] ⊥ for each user a i . Negative values r i ( b k ) denote utter dislike , while positive values express a i ’s liking of product b k . If ratings are implicit only , we represent them by set R i ⊆ B , equivalent to { b k ∈ B | r i ( b k ) 6 = ⊥ } . The user - based CF’s working process can be broken down into two major steps : • Neighborhood formation . Assuming a i as the ac - tive user , similarity values c ( a i , a j ) ∈ [ − 1 , + 1 ] for all a j ∈ A \ { a i } are computed , based upon the similarity of their respective rating functions r i , r j . In general , Pearson correlation [ 29 , 8 ] or cosine distance [ 11 ] are used for computing c ( a i , a j ) . The top - M most sim - ilar users a j become members of a i ’s neighborhood , clique ( a i ) ⊆ A . • Rating prediction . Taking all the products b k that a i ’s neighbors a j ∈ clique ( a i ) have rated and which are new to a i , i . e . , r i ( b k ) = ⊥ , a prediction of liking w i ( b k ) is produced . Value w i ( b k ) hereby depends on both the similarity c ( a i , a j ) of voters a j with r j ( b k ) 6 = ⊥ , as well as the ratings r j ( b k ) these neighbors a j assigned to b k . Eventually , a list P w i : { 1 , 2 , . . . , N } → B of top - N recom - mendations is computed , based upon predictions w i . Note that function P w i is injective and reflects recommendation ranking in descending order , giving highest predictions first . 2 . 2 Item - based Collaborative Filtering Item - based CF [ 13 , 26 , 5 ] has been gaining momentum over the last five years by virtue of favorable computational complexity characteristics and the ability to decouple the model computation process from actual prediction making . Specifically for cases where | A | (cid:29) | B | , item - based CF’s com - putational performance has been shown superior to user - based CF [ 26 ] . Its success also extends to many commercial recommender systems , such as Amazon . com’s [ 16 ] . As with user - based CF , recommendation making is based upon ratings r i ( b k ) that users a i ∈ A provided for products b k ∈ B . However , unlike user - based CF , similarity values c are computed for items rather than users , hence c : B × B → [ − 1 , + 1 ] . Roughly speaking , two items b k , b e are similar , i . e . , have large c ( b k , b e ) , if users who rate one of them tend to rate the other , and if users tend to assign them identical or similar ratings . Moreover , for each b k , its neighborhood clique ( b k ) ⊆ B of top - M most similar items is defined . Predictions w i ( b k ) are computed as follows : w i ( b k ) = P b e ∈ B 0 k ( c ( b k , b e ) · r i ( b e ) ) P b e ∈ B 0 k | c ( b k , b e ) | , ( 1 ) where B 0 k : = { b e | b e ∈ clique ( b k ) ∧ r i ( b k ) 6 = ⊥ } Intuitively , the approach tries to mimic real user behav - ior , having user a i judge the value of an unknown product b k by comparing the latter to known , similar items b e and considering how much a i appreciated these b e . The eventual computation of a top - N recommendation list P w i follows the user - based CF’s process , arranging rec - ommendations according to w i in descending order . 3 . EVALUATION METRICS Evaluation metrics are essential in order to judge the qual - ity and performance of recommender systems , even though they are still in their infancies . Most evaluations concentrate on accuracy measurements only and neglect other factors , e . g . , novelty and serendipity of recommendations , and the diversity of the recommended list’s items . The following sections give an outline of popular metrics . An extensive survey of accuracy metrics is provided in [ 12 ] . 3 . 1 Accuracy Metrics Accuracy metrics have been defined first and foremost for two major tasks : First , to judge the accuracy of single predictions , i . e . , how much predictions w i ( b k ) for products b k deviate from a i ’s ac - tual ratings r i ( b k ) . These metrics are particularly suited for tasks where predictions are displayed along with the prod - uct , e . g . , annotation in context [ 12 ] . Second , decision - support metrics evaluate the effective - ness of helping users to select high - quality items from the set of all products , generally supposing binary preferences . 3 . 1 . 1 Predictive Accuracy Metrics Predictive accuracy metrics measure how close predicted ratings come to true user ratings . Most prominent and widely used [ 29 , 11 , 3 , 9 ] , mean absolute error ( MAE ) represents an efficient means to measure the statistical accuracy of predic - tions w i ( b k ) for sets B i of products : | E | = P b k ∈ B i | r i ( b k ) − w i ( b k ) | | B i | ( 2 ) Related to MAE , mean squared error ( MSE ) squares the error before summing . Hence , large errors become much more pronounced than small ones . Very easy to implement , predictive accuracy metrics are inapt for evaluating the quality of top - N recommendation lists . Users only care about errors for high - rank products . On the other hand , prediction errors for low - rank products are unimportant , knowing that the user has no interest in them anyway . However , MAE and MSE account for both types of errors in exactly the same fashion . 3 . 1 . 2 Decision - Support Metrics Precision and recall , both well - known from information retrieval , do not consider predictions and their deviations from actual ratings . They rather judge how relevant a set of ranked recommendations is for the active user . Before using these metrics for cross - validation , K - folding is applied , dividing every user a i ’s rated products b k ∈ R i into K disjoint slices of preferably equal size . Hereby , K − 1 randomly chosen slices form a i ’s training set R xi . These rat - ings then define a i ’s profile from which final recommenda - tions are computed . For recommendation generation , a i ’s residual slice ( R i \ R xi ) is retained and not used for predic - tion . This slice , denoted T xi , constitutes the test set , i . e . , those products the recommenders intend to predict . Sarwar [ 25 ] presents an adapted variant of recall , record - ing the percentage of test set products b ∈ T xi occurring in recommendation list P xi with respect to the overall number of test set products | T xi | : Recall = 100 · | T xi ∩ = P xi | | T xi | ( 3 ) Symbol = P xi denotes the image of map P xi , i . e . , all items part of the recommendation list . Accordingly , precision represents the percentage of test set products b ∈ T xi occurring in P xi with respect to the size of the recommendation list : Precision = 100 · | T xi ∩ = P xi | | = P xi | ( 4 ) Breese et al . [ 3 ] introduce an interesting extension to re - call , known as weighted recall or Breese score . The approach takes into account the order of the top - N list , penalizing in - correct recommendations less severely the further down the list they occur . Penalty decreases with exponential decay . Other popular decision - support metrics include ROC [ 28 , 18 , 9 ] , the “receiver operating characteristic” . ROC mea - sures the extent to which an information filtering system is able to successfully distinguish between signal and noise . Less frequently used , NDPM [ 2 ] compares two different , weakly ordered rankings . 3 . 2 Beyond Accuracy Though accuracy metrics are an important facet of useful - ness , there are traits of user satisfaction they are unable to capture . However , non - accuracy metrics have largely been denied major research interest so far . 3 . 2 . 1 Coverage Among all non - accuracy evaluation metrics , coverage has been the most frequently used [ 11 , 19 , 9 ] . Coverage measures the percentage of elements part of the problem domain for which predictions can be made . 3 . 2 . 2 Novelty and Serendipity Some recommenders produce highly accurate results that are still useless in practice , e . g . , suggesting bananas to cus - tomers in grocery stores . Though being highly accurate , note that almost everybody likes and buys bananas . Hence , their recommending appears far too obvious and of little help to the shopper . Novelty and serendipity metrics thus measure the “non - obviousness” of recommendations made , avoiding “cherry - picking” [ 12 ] . For some simple measure of serendipity , take the average popularity of recommended items . Lower scores obtained denote higher serendipity . 3 . 3 Intra - List Similarity We present a new metric that intends to capture the diver - sity of a list . Hereby , diversity may refer to all kinds of fea - tures , e . g . , genre , author , and other discerning characteris - tics . Based upon an arbitrary function c ◦ : B × B → [ − 1 , + 1 ] measuring the similarity c ◦ ( b k , b e ) between products b k , b e according to some custom - defined criterion , we define intra - list similarity for a i ’s list P w i as follows : ILS ( P w i ) = X b k ∈ = P wi X b e ∈ = P wi , b k 6 = b e c ◦ ( b k , b e ) 2 ( 5 ) Higher scores denote lower diversity . An interesting math - ematical feature of ILS ( P w i ) we are referring to in later sec - tions is permutation - insensitivity , i . e . , let S N be the sym - metric group of all permutations on N = | P w i | symbols : ∀ σ i , σ j ∈ S N : ILS ( P w i ◦ σ i ) = ILS ( P w i ◦ σ j ) ( 6 ) Hence , simply rearranging positions of recommendations in a top - N list P w i does not affect P w i ’s intra - list similarity . 4 . TOPIC DIVERSIFICATION One major issue with accuracy metrics is their inability to capture the broader aspects of user satisfaction , hiding several blatant flaws in existing systems [ 17 ] . For instance , suggesting a list of very similar items , e . g . , with respect to the author , genre , or topic , may be of little use for the user , even though this list’s average accuracy might be high . The issue has been perceived by other researchers before , coined “portfolio effect” by Ali and van Stam [ 1 ] . We believe that item - based CF systems in particular are susceptible to that effect . Reports from the item - based TV recommender TiVo [ 1 ] , as well as personal experiences with Amazon . com’s recommender , also item - based [ 16 ] , back our conjecture . For instance , one of this paper’s authors only gets recommenda - tions for Heinlein’s books , another complained about all his suggested books being Tolkien’s writings . Reasons for negative ramifications on user satisfaction im - plied by portfolio effects are well - understood and have been studied extensively in economics , termed “law of diminishing marginal returns” [ 30 ] . The law describes effects of satura - tion that steadily decrease the incremental utility of prod - ucts p when acquired or consumed over and over again . For example , suppose you are offered your favorite drink . Let p 1 denote the price you are willing to pay for that product . Assuming your are offered a second glass of that particular drink , the amount p 2 of money you are inclined to spend will be lower , i . e . , p 1 > p 2 . Same for p 3 , p 4 , and so forth . We propose an approach we call topic diversification to deal with the problem at hand and make recommended lists more diverse and thus more useful . Our method represents an extension to existing recommender algorithms and is ap - plied on top of recommendation lists . 4 . 1 Taxonomy - based Similarity Metric Function c ∗ : 2 B × 2 B → [ − 1 , + 1 ] , quantifying the simi - larity between two product sets , forms an essential part of topic diversification . We instantiate c ∗ with our metric for taxonomy - driven filtering [ 33 ] , though other content - based similarity measures may appear likewise suitable . Our met - ric computes the similarity between product sets based upon their classification . Each product belongs to one or more classes that are hierarchically arranged in classification tax - onomies , describing the products in machine - readable ways . Classification taxonomies exist for various domains . Ama - zon . com crafts very large taxonomies for books , DVDs , CDs , electronic goods , and apparel . See Figure 1 for one sam - ple taxonomy . Moreover , all products on Amazon . com bear content descriptions relating to these domain taxonomies . Featured topics could include author , genre , and audience . 4 . 2 Topic Diversification Algorithm Algorithm 1 shows the complete topic diversification algo - rithm , a brief textual sketch is given in the next paragraphs . Function P w i ∗ denotes the new recommendation list , re - sulting from applying topic diversification . For every list en - try z ∈ [ 2 , N ] , we collect those products b from the candidate products set B i that do not occur in positions o < z in P w i ∗ and compute their similarity with set { P w i ∗ ( k ) | k ∈ [ 1 , z [ } , which contains all new recommendations preceding rank z . Sorting all products b according to c ∗ ( b ) in reverse order , we obtain the dissimilarity rank P rev c ∗ . This rank is then merged with the original recommendation rank P w i accord - ing to diversification factor Θ F , yielding final rank P w i ∗ . Factor Θ F defines the impact that dissimilarity rank P rev c ∗ exerts on the eventual overall output . Large Θ F ∈ [ 0 . 5 , 1 ] fa - vors diversification over a i ’s original relevance order , while low Θ F ∈ [ 0 , 0 . 5 [ produces recommendation lists closer to the original rank P w i . For experimental analysis , we used diversification factors Θ F ∈ [ 0 , 0 . 9 ] . Note that ordered input lists P w i must be considerably larger than the final top - N list . For our later experiments , we used top - 50 input lists for eventual top - 10 recommendations . procedure diversify ( P w i , Θ F ) { B i ← = P w i ; P w i ∗ ( 1 ) ← P w i ( 1 ) ; for z ← 2 to N do set B 0 i ← B i \ { P w i ∗ ( k ) | k ∈ [ 1 , z [ } ; ∀ b ∈ B 0 : compute c ∗ ( { b } , { P w i ∗ ( k ) | k ∈ [ 1 , z [ } ) ; compute P c ∗ : { 1 , 2 , . . . , | B 0 i | } → B 0 i using c ∗ ; for all b ∈ B 0 i do P rev − 1 c ∗ ( b ) ← | B 0 i | − P − 1 c ∗ ( b ) ; w ∗ i ( b ) ← P − 1 w i ( b ) · ( 1 − Θ F ) + P rev − 1 c ∗ ( b ) · Θ F ; end do P w i ∗ ( z ) ← min { w ∗ i ( b ) | b ∈ B 0 i } ; end do return P w i ∗ ; } Algorithm 1 : Sequential topic diversification 4 . 3 Recommendation Dependency In order to implement topic diversification , we assume that recommended products P w i ( o ) and P w i ( p ) , o , p ∈ N , along with their content descriptions , effectively do exert an impact on each other , which is commonly ignored by ex - isting approaches : usually , only relevance weight ordering o < p ⇒ w i ( P w i ( o ) ) ≥ w i ( P w i ( p ) ) must hold for recommen - dation list items , no other dependencies are assumed . In case of topic diversification , recommendation interde - pendence means that an item b ’s current dissimilarity rank with respect to preceding recommendations plays an impor - tant role and may influence the new ranking . 4 . 4 Osmotic Pressure Analogy The effect of dissimilarity bears traits similar to that of os - motic pressure and selective permeability known from molec - ular biology [ 31 ] . Steady insertion of products b o , taken from one specific area of interest d o , into the recommendation list equates to the passing of molecules from one specific substance through the cell membrane into cytoplasm . With increasing concentration of d o , owing to the membrane’s se - lective permeability , the pressure for molecules b from other substances d rises . When pressure gets sufficiently high for one given topic d p , its best products b p may “diffuse” into the recommendation list , even though their original rank P − 1 w i ( b ) might be inferior to candidates from the prevailing domain d o . Consequently , pressure for d p decreases , paving the way for another domain for which pressure peaks . Topic diversification hence resembles the membrane’s se - lective permeability , which allows cells to maintain their in - ternal composition of substances at required levels . 5 . EMPIRICAL ANALYSIS We conducted offline evaluations to understand the ram - ifications of topic diversification on accuracy metrics , and online analysis to investigate how our method affects ac - tual user satisfaction . We applied topic diversification with Θ F ∈ { 0 , 0 . 1 , 0 . 2 , . . . 0 . 9 } to lists generated by both user - based CF and item - based CF , observing effects that occur Books Science Mathematics Reference Sports Archaelogy Medicine Applied Pure Nonfiction Discrete Algebra History Astronomy Figure 1 : Fragment from the Amazon . com book taxonomy when steadily increasing Θ F and analyzing how both ap - proaches respond to diversification . 5 . 1 Dataset Design We based online and offline analyses on data we gathered from BookCrossing ( http : / / www . bookcrossing . com ) . The lat - ter community caters for book lovers exchanging books all around the world and sharing their experiences with others . 5 . 1 . 1 Data Collection In a 4 - week crawl , we collected data on 278 , 858 members of BookCrossing and 1 , 157 , 112 ratings , both implicit and explicit , referring to 271 , 379 distinct ISBNs . Invalid ISBNs were excluded from the outset . The complete BookCrossing dataset , featuring fully anon - ymized information , is available via the first author’s home - page ( http : / / www . informatik . uni - freiburg . de / ∼ cziegler ) . Next , we mined Amazon . com’s book taxonomy , compris - ing 13 , 525 distinct topics . In order to be able to apply topic diversification , we mined content information , focusing on taxonomic descriptions that relate books to taxonomy nodes from Amazon . com . Since many books on BookCrossing refer to rare , non - English books , or outdated titles not in print anymore , we were able to garner background knowledge for only 175 , 721 books . In total , 466 , 573 topic descriptors were found , giving an average of 2 . 66 topics per book . 5 . 1 . 2 Condensation Steps Owing to the BookCrossing dataset’s extreme sparsity , we decided to further condense the set in order to obtain more meaningful results from CF algorithms when computing rec - ommendations . Hence , we discarded all books missing taxo - nomic descriptions , along with all ratings referring to them . Next , we also removed book titles with fewer than 20 overall mentions . Only community members with at least 5 ratings each were kept . The resulting dataset’s dimensions were considerably more moderate , featuring 10 , 339 users , 6 , 708 books , and 361 , 349 book ratings . 5 . 2 Offline Experiments We performed offline experiments comparing precision , re - call , and intra - list similarity scores for 20 different recom - mendation list setups . Half these recommendation lists were based upon user - based CF with different degrees of diver - sification , the others on item - based CF . Note that we did not compute MAE metric values since we are dealing with implicit rather than explicit ratings . 5 . 2 . 1 Evaluation Framework Setup For cross - validation of precision and recall metrics of all 10 , 339 users , we adopted K - folding with parameter K = 4 . Hence , rating profiles R i were effectively split into training sets R xi and test sets T xi , x ∈ { 1 , . . . , 4 } , at a ratio of 3 : 1 . For each of the 41 , 356 different training sets , we computed 20 top - 10 recommendation lists . To generate the diversified lists , we computed top - 50 lists based upon pure , i . e . , non - diversified , item - based CF and pure user - based CF . The high - performance Suggest recom - mender engine 2 was used to compute these base case lists . Next , we applied the diversification algorithm to both base cases , applying Θ F factors ranging from 10 % up to 90 % . For evaluation , all lists were truncated to contain 10 books only . 5 . 2 . 2 Result Analysis We were interested in seeing how accuracy , captured by precision and recall , behaves when increasing Θ F from 0 . 1 up to 0 . 9 . Since topic diversification may make books with high predicted accuracy trickle down the list , we hypothesized that accuracy will deteriorate for Θ F → 0 . 9 . Moreover , in order to find out if our novel algorithm has any significant , positive effects on the diversity of items featured , we also applied our intra - list similarity metric . An overlap analysis for diversified lists , Θ F ≥ 0 . 1 , versus their respective non - diversified pendants indicates how many items stayed the same for increasing diversification factors . 5 . 2 . 2 . 1 Precision and Recall . First , we analyzed precision and recall scores for both non - diversified base cases , i . e . , when Θ F = 0 . Table 1 states that user - based and item - based CF exhibit almost identical accu - racy , indicated by precision values . Their recall values differ 2 Visit http : / / www - users . cs . umn . edu / ∼ karypis / suggest / . 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) P r e c i s i on Item - based CF User - based CF ( a ) 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) R e c a ll Item - based CF User - based CF ( b ) Figure 2 : Precision ( a ) and recall ( b ) for increasing Θ F Item - based CF User - based CF Precision 3 . 64 3 . 69 Recall 7 . 32 5 . 76 Table 1 : Precision / recall for non - diversified CF considerably , hinting at deviating behavior with respect to the types of users they are scoring for . Next , we analyzed the behavior of user - based and item - based CF when steadily increasing Θ F by increments of 10 % , depicted in Figure 2 . The two charts reveal that diversifica - tion has detrimental effects on both metrics and on both CF algorithms . Interestingly , corresponding precision and recall curves have almost identical shape . The loss in accuracy is more pronounced for item - based than for user - based CF . Furthermore , for either metric and either CF algorithm , the drop is most distinctive for Θ F ∈ [ 0 . 2 , 0 . 4 ] . For lower Θ F , negative impacts on accuracy are marginal . We believe this last observation due to the fact that precision and recall are permutation - insensitive , i . e . , the mere order of recommendations within a top - N list does not influence the metric value , as opposed to Breese score [ 3 , 12 ] . However , for low Θ F , the pressure that the dissimilarity rank exerts on the top - N list’s makeup is still too weak to make many new items diffuse into the top - N list . Hence , we conjecture that rather the positions of current top - N items change , which does not affect either precision or recall . 5 . 2 . 2 . 2 Intra - List Similarity . Knowing that our diversification method exerts a signif - icant , negative impact on accuracy metrics , we wanted to know how our approach affected the intra - list similarity mea - sure . Similar to the precision and recall experiments , we computed metric values for user - based and item - based CF with Θ F ∈ [ 0 , 0 . 9 ] each . Hereby , we instantiated the intra - list similarity metric function c ◦ with our taxonomy - driven metric c ∗ . Results obtained from intra - list similarity analysis are given in Figure 3 ( a ) . The topic diversification method considerably lowers the pairwise similarity between list items , thus making top - N recommendation lists more diverse . Diversification appears to affect item - based CF stronger than its user - based coun - terpart , in line with our findings about precision and recall . For lower Θ F , curves are less steep than for Θ F ∈ [ 0 . 2 , 0 . 4 ] , which also well aligns with precision and recall analysis . Again , the latter phenomenon can be explained by one of the metric’s inherent features , i . e . , like precision and recall , intra - list similarity is permutation - insensitive . 5 . 2 . 2 . 3 Original List Overlap . Figure 3 ( b ) shows the number of recommended items stay - ing the same when increasing Θ F with respect to the original list’s content . Both curves exhibit roughly linear shapes , be - ing less steep for low Θ F , though . Interestingly , for factors Θ F ≤ 0 . 4 , at most 3 recommendations change on average . 5 . 2 . 2 . 4 Conclusion . We found that diversification appears detrimental to both user - based and item - based CF along precision and recall metrics . In fact , this outcome aligns with our expectations , considering the nature of those two accuracy metrics and the way that the topic diversification method works . More - over , we found that item - based CF seems more susceptible to topic diversification than user - based CF , backed by re - sults from precision , recall and intra - list similarity metric analysis . 5 . 3 Online Experiments Offline experiments helped us in understanding the impli - cations of topic diversification on both CF algorithms . We could also observe that the effects of our approach are dif - ferent on different algorithms . However , knowing about the deficiencies of accuracy metrics , we wanted to assess actual user satisfaction for various degrees of diversification , thus necessitating an online survey . For the online study , we computed each recommendation list type anew for users in the denser BookCrossing dataset , 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) I n t r a - L i s t S i m il a r i t y Item - based CF User - based CF ( a ) 2 4 6 8 10 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) Item - based CF User - based CF O v e r l ap w i t h Θ F = 0 ( b ) Figure 3 : Intra - list similarity behavior ( a ) and overlap with original list ( b ) for increasing Θ F though without K - folding . In cooperation with BookCross - ing , we mailed all eligible users via the community mailing system , asking them to participate in our online study . Each mail contained a personal link that would direct the user to our online survey pages . In order to make sure that only the users themselves would complete their survey , links con - tained unique , encrypted access codes . During the 3 - week survey phase , 2 , 125 users participated and completed the study . 5 . 3 . 1 Survey Outline and Setup The survey consisted of several screens that would tell the prospective participant about this study’s nature and his task , show all his ratings used for making recommen - dations , and finally present a top - 10 recommendation list , asking several questions thereafter . For each book , users could state their interest on a 5 - point rating scale . Scales ranged from “not much” to “very much” , mapped to values 1 to 4 , and offered the user to indicate that he had already read the book , mapped to value 5 . In order to successfully complete the study , users were not required to rate all their top - 10 recommendations . Neutral values were assumed for non - votes instead . However , we required users to answer all further questions , concerning the list as a whole rather than its single recommendations , before submitting their results . We embedded those questions we were actually keen about knowing into ones of lesser importance , in order to conceal our intentions and not bias users . The one top - 10 recommendation list for each user was cho - sen among 12 candidate lists , either user - based CF or item - based with Θ F ∈ { 0 , 0 . 3 , 0 . 4 , 0 . 5 , 0 . 7 , 0 . 9 } each . We opted for those 12 instead of all 20 list types in order to acquire enough users completing the survey for each slot . The assignment of a specific list to the current user was done dynamically , at the time of the participant entering the survey , and in a round - robin fashion . Thus , we could guarantee that the number of users per list type was roughly identical . 5 . 3 . 2 Result Analysis For the analysis of our inter - subject survey , we were mostly interested in the following three aspects . First , the average rating users gave to their 10 single recommendations . We expected results to roughly align with scores obtained from precision and recall , owing to the very nature of these met - rics . Second , we wanted to know if users perceived their list as well - diversified , asking them to tell whether the lists re - flected rather a broad or narrow range of their reading inter - ests . Referring to the intra - list similarity metric , we expected users’ perceived range of topics , i . e . , the list’s diversity , to increase with increasing Θ F . Third , we were curious about the overall satisfaction of users with their recommendation lists in their entirety , the measure to compare performance . Both latter - mentioned questions were answered by each user on a 5 - point likert scale , higher scores denoting better performance , and we averaged the eventual results by the number of users . Statistical significance of all mean values was measured by parametric one - factor ANOVA , where p < 0 . 05 if not indicated otherwise . 5 . 3 . 2 . 1 Single - Vote Averages . Users perceived recommendations made by user - based CF systems on average as more accurate than those made by item - based CF systems , as depicted in Figure 4 ( a ) . At each featured diversification level Θ F , differences between the two CF types are statistically significant , p (cid:28) 0 . 01 . Moreover , for each algorithm , higher diversification fac - tors obviously entail lower single - vote average scores , which confirms our hypothesis stated before . The item - based CF’s cusp at Θ F ∈ [ 0 . 3 , 0 . 5 ] appears as a notable outlier , op - posed to the trend , but differences between the 3 means at Θ F ∈ [ 0 . 3 , 0 . 5 ] are not statistically significant , p > 0 . 15 . Contrarily , differences between all factors Θ F are significant for item - based CF , p (cid:28) 0 . 01 , and for user - based CF , p < 0 . 1 . Hence , topic diversification negatively correlates with pure accuracy . Besides , users perceived the performance of user - based CF as significantly better than item - based CF for all corresponding levels Θ F . 5 . 3 . 2 . 2 Covered Range . Next , we analyzed whether users actually perceived the variety - augmenting effects caused by topic diversification , illustrated before through the measurement of intra - list sim - 2 . 4 2 . 6 2 . 8 3 3 . 2 3 . 4 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) S i ng l e - V o t e A v e r age s Item - based CF User - based CF ( a ) 2 . 6 2 . 8 3 3 . 2 3 . 4 3 . 6 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) C o v e r ed R ange Item - based CF User - based CF ( b ) 3 3 . 1 3 . 2 3 . 3 3 . 4 3 . 5 3 . 6 0 10 20 30 40 50 60 70 80 90 Diversification Factor ( in % ) O v e r a l l L i s t V a l ue Item - based CF User - based CF ( c ) Figure 4 : Results for single - vote averages ( a ) , covered range of interests ( b ) , and overall satisfaction ( c ) ilarity . Users’ reactions to steadily incrementing Θ F are il - lustrated in Figure 4 ( b ) . First , between both algorithms on corresponding Θ F levels , only the difference of means at Θ F = 0 . 3 shows statistical significance . Studying the trend of user - based CF for increasing Θ F , we notice that the perceived range of reading interests covered by users’ recommendation lists also increases . Hereby , the curve’s first derivative maintains an approximately constant level , exhibiting slight peaks between Θ F ∈ [ 0 . 4 , 0 . 5 ] . Statis - tical significance holds for user - based CF between means at Θ F = 0 and Θ F > 0 . 5 , and between Θ F = 0 . 3 and Θ F = 0 . 9 . On the contrary , the item - based curve exhibits a drasti - cally different behavior . While soaring at Θ F = 0 . 3 to 3 . 186 , reaching a score almost identical to the user - based CF’s peak at Θ F = 0 . 9 , the curve barely rises for Θ F ∈ [ 0 . 4 , 0 . 9 ] , remaining rather stable and showing a slight , though in - significant , upward trend . Statistical significance was shown for Θ F = 0 with respect to all other samples taken from Θ F ∈ [ 0 . 3 , 0 . 9 ] . Hence , our online results do not perfectly align with findings obtained from offline analysis . While the intra - list similarity chart in Figure 3 indicates that diversity increases when increasing Θ F , the item - based CF chart de - fies this trend , first soaring then flattening . We conjecture that the following three factors account for these peculiari - ties : • Diversification factor impact . Our offline analysis of the intra - list similarity already suggested that the effect of topic diversification on item - based CF is much stronger than on user - based CF . Thus , the item - based CF’s user - perceived interest coverage is significantly higher at Θ F = 0 . 3 than the user - based CF’s . • Human perception . We believe that human percep - tion can capture the level of diversification inherent to a list only to some extent . Beyond that point , in - creasing diversity remains unnoticed . For the appli - cation scenario at hand , Figure 4 suggests this point around score value 3 . 2 , reached by user - based CF only at Θ F = 0 . 9 , and approximated by item - based CF al - ready at Θ F = 0 . 3 . • Interaction with accuracy . Analyzing results ob - tained , bear in mind that covered range scores are not fully independent from single - vote averages . When ac - curacy is poor , i . e . , the user feels unable to identify recommendations that are interesting to him , chances are high his discontentment will also negatively affect his diversity rating . For Θ F ∈ [ 0 . 5 , 0 . 9 ] , single - vote av - erages are remarkably low , which might explain why perceived coverage scores do not improve for increasing Θ F . However , we may conclude that users do perceive the ap - plication of topic diversification as an overly positive effect on reading interest coverage . 5 . 3 . 2 . 3 Overall List Value . The third feature variable we were evaluating , the overall value users assigned to their personal recommendation list , effectively represents the target value of our studies , mea - suring actual user satisfaction . Owing to our conjecture that user satisfaction is a mere composite of accuracy and other influential factors , such as the list’s diversity , we hypothe - sized that the application of topic diversification would in - crease satisfaction . At the same time , considering the down - ward trend of precision and recall for increasing Θ F , in ac - cordance with declining single - vote averages , we expected user satisfaction to drop off for large Θ F . Hence , we sup - posed an arc - shaped curve for both algorithms . Results for overall list value are given in Figure 4 ( c ) . Ana - lyzing user - based CF , we observe that the curve does not fol - low our hypothesis . Slightly improving at Θ F = 0 . 3 over the non - diversified case , scores drop for Θ F ∈ [ 0 . 4 , 0 . 7 ] , eventu - ally culminating in a slight but visible upturn at Θ F = 0 . 9 . While lacking reasonable explanations and being opposed to our hypothesis , the curve’s data - points de facto bear no statistical significance for p < 0 . 1 . Hence , we conclude that topic diversification has a marginal , largely negligible impact on overall user satisfaction , initial positive effects eventually being offset by declining accuracy . On the contrary , for item - based CF , results obtained look different . In compliance with our previous hypothesis , the curve’s shape roughly follows an arc , peaking at Θ F = 0 . 4 . Taking the three data - points defining the arc , we obtain sta - tistical significance for p < 0 . 1 . Since the endpoint’s score at Θ F = 0 . 9 is inferior to the non - diversified case’s , we observe that too much diversification appears detrimental , perhaps owing to substantial interactions with accuracy . Eventually , for overall list value analysis , we come to con - clude that topic diversification has no measurable effects on user - based CF , but significantly improves item - based CF performance for diversification factors Θ F around 40 % . 5 . 4 Multiple Linear Regression Results obtained from analyzing user feedback along var - ious feature axes already indicated that users’ overall satis - faction with recommendation lists not only depends on ac - curacy , but also on the range of reading interests covered . In order to more rigidly assess that indication by means of statistical methods , we applied multiple linear regression to our survey results , choosing the overall list value as depen - dent variable . As independent input variables , we provided single - vote averages and covered range , both appearing as first - order and second - order polynomials , i . e . , SVA and CR , and SVA 2 and CR 2 , respectively . We also tried several other , more complex models , without achieving significantly better model fitting . Estimate Error t - Value Pr ( > | t | ) ( const ) 3 . 27 0 . 023 139 . 56 < 2 e − 16 SVA 12 . 42 0 . 973 12 . 78 < 2 e − 16 SVA 2 - 6 . 11 0 . 976 - 6 . 26 4 . 76 e − 10 CR 19 . 19 0 . 982 19 . 54 < 2 e − 16 CR 2 - 3 . 27 0 . 966 - 3 . 39 0 . 000727 Multiple R 2 : 0 . 305 , adjusted R 2 : 0 . 303 Table 2 : Multiple linear regression results Analyzing multiple linear regression results , shown in Ta - ble 2 , confidence values Pr ( > | t | ) clearly indicate that sta - tistically significant correlations for accuracy and covered range with user satisfaction exist . Since statistical signifi - cance also holds for their respective second - order polynomi - als , i . e . , CR 2 and SVA 2 , we conclude that these relationships are non - linear and more complex , though . As a matter of fact , linear regression delivers a strong in - dication that the intrinsic utility of a list of recommended items is more than just the average value of accuracy votes for all single items , but also depends on the perceived diver - sity . 5 . 5 Limitations There are some limitations to the study , notably referring to the way topic diversification was implemented . Though the Amazon . com taxonomies were human - created , there may still be some mismatch between what the topic diversifica - tion algorithm perceives as “diversified” and what humans do . The issue is effectively inherent to the taxonomy’s struc - ture , which has been designed with browsing tasks and ease of searching rather than with interest profile generation in mind . For instance , the taxonomy features topic nodes la - belled with letters for alphabetical ordering of authors from the same genre , e . g . , Books → Fiction → . . . → Authors , A - Z → G . Hence , two Sci - Fi books from two different au - thors with the same initial of their last name would be classi - fied under the same node , while another Sci - Fi book from an author with a different last - name initial would not . Though the problem’s impact is largely marginal , owing to the rel - atively deep level of nesting where such branchings occur , the procedure appears far from intuitive . An alternative approach to further investigate the accu - racy of taxonomy - driven similarity measurement , and its limitations , would be to have humans do the clustering , e . g . , by doing card sorts or by estimating the similarity of any two books contained in the book database . The results could then be matched against the topic diversification method’s output . 6 . RELATED WORK Few efforts have addressed the problem of making top - N lists more diverse . Only considering literature on collabo - rative filtering and recommender systems in general , none have been presented before , to the best of our knowledge . However , some work related to our topic diversification approach can be found in information retrieval , specifically meta - search engines . A critical aspect of meta - search engine design is the merging of several top - N lists into one single top - N list . Intuitively , this merged top - N list should reflect the highest quality ranking possible , also known as the “rank aggregation problem” [ 6 ] . Most approaches use variations of the “linear combination of score” model ( LC ) , described by Vogt and Cottrell [ 32 ] . The LC model effectively resembles our scheme for merging the original , accuracy - based rank - ing with the current dissimilarity ranking , but is more gen - eral and does not address the diversity issue . Fagin et al . [ 7 ] propose metrics for measuring the distance between top - N lists , i . e . , inter - list similarity metrics , in order to evaluate the quality of merged ranks . Oztekin et al . [ 21 ] extend the linear combination approach by proposing rank combination models that also incorporate content - based features in order to identify the most relevant topics . More related to our idea of creating lists that represent the whole plethora of the user’s topic interests , Kummamuru et al . [ 15 ] present their clustering scheme that groups search results into clusters of related topics . The user can then conveniently browse topic folders relevant to his search in - terest . The commercially available search engine Northern Light ( http : / / www . northernlight . com ) incorporates similar functionalities . Google ( http : / / www . google . com ) uses several mechanisms to suppress top - N items too similar in content , showing them only upon the user’s explicit request . Unfor - tunately , no publications on that matter are available . 7 . CONCLUSION We presented topic diversification , an algorithmic frame - work to increase the diversity of a top - N list of recommended products . In order to show its efficiency in diversifying , we also introduced our new intra - list similarity metric . Contrasting precision and recall metrics , computed both for user - based and item - based CF and featuring different levels of diversification , with results obtained from a large - scale user survey , we showed that the user’s overall liking of recommendation lists goes beyond accuracy and involves other factors , e . g . , the users’ perceived list diversity . We were thus able to provide empirical evidence that lists are more than mere aggregations of single recommendations , but bear an intrinsic , added value . Though effects of diversification were largely marginal on user - based CF , item - based CF performance improved signif - icantly , an indication that there are some behavioral differ - ences between both CF classes . Moreover , while pure item - based CF appeared slightly inferior to pure user - based CF in overall satisfaction , diversifying item - based CF with factors Θ F ∈ [ 0 . 3 , 0 . 4 ] made item - based CF outperform user - based CF . Interestingly for Θ F ≤ 0 . 4 , no more than three items tend to change with respect to the original list , shown in Figure 3 . Small changes thus have high impact . We believe our findings especially valuable for practical application scenarios , since many commercial recommender systems , e . g . , Amazon . com [ 16 ] and TiVo [ 1 ] , are item - based , owing to the algorithm’s computational efficiency . 8 . FUTURE WORK Possible future directions branching out from our current state of research on topic diversification are rife . First , we would like to study the impact of topic diversi - fication when dealing with application domains other than books , e . g . , movies , CDs , and so forth . Results obtained may differ , owing to distinct characteristics concerning the struc - ture of genre classification inherent to these domains . For instance , Amazon . com’s classification taxonomy for books is more deeply nested , though smaller , than its movie coun - terpart [ 34 ] . Bear in mind that the structure of these tax - onomies severely affects the taxonomy - based similarity mea - sure c ∗ , which lies at the very heart of the topic diversifica - tion method . Another interesting path to follow would be to param - eterize the diversification framework with several different similarity metrics , either content - based or CF - based , hence superseding the taxonomy - based c ∗ . We strongly believe that our topic diversification approach bears particularly high relevance for recommender systems involving sequential consumption of list items . For instance , think of personalized Internet radio stations , e . g . , Yahoo’s Launch ( http : / / launch . yahoo . com ) : community members are provided with playlists , computed according to their own taste , which are sequentially processed and consumed . Con - trolling the right mix of items within these lists becomes vi - tal and even more important than for mere “random - access” recommendation lists , e . g . , book or movie lists . Suppose such an Internet radio station playing five Sisters of Mercy songs in a row . Though the active user may actually like the re - spective band , he may not want all five songs played in se - quence . Lack of diversion might thus result in the user leav - ing the system . The problem of finding the right mix for sequential con - sumption - based recommenders takes us to another future di - rection worth exploring , namely individually adjusting the right level of diversification versus accuracy tradeoff . One approach could be to have the user himself define the de - gree of diversification he likes . Another approach might in - volve learning the right parameter from the user’s behavior , e . g . , by observing which recommended items he inspects and devotes more time to , etc . Finally , we are also thinking about diversity metrics other than intra - list similarity . For instance , we envision a metric that measures the extent to which the top - N list actually reflects the user’s profile . 9 . ACKNOWLEDGEMENTS The authors would like to express their gratitude towards Ron Hornbaker , CTO of Humankind Systems and chief ar - chitect of BookCrossing , for his invaluable support . Further - more , we would like to thank all BookCrossing members par - ticipating in our online survey for devoting their time and giving us many invaluable comments . Moreover , we would like to thank John Riedl , Dan Cosley , Yongli Zhang , Paolo Massa , Zvi Topol , and Lars Schmidt - Thieme for fruitful comments and discussions . 10 . REFERENCES [ 1 ] Ali , K . , and van Stam , W . TiVo : Making show recommen - dations using a distributed collaborative filtering architec - ture . In Proceedings of the 2004 ACM SIGKDD Interna - tional Conference on Knowledge Discovery and Data Mining ( Seattle , WA , USA , 2004 ) , ACM Press , pp . 394 – 401 . [ 2 ] Balabanovi´c , M . , and Shoham , Y . Fab - content - based , collaborative recommendation . Communications of the ACM 40 , 3 ( March 1997 ) , 66 – 72 . [ 3 ] Breese , J . , Heckerman , D . , and Kadie , C . Empirical anal - ysis of predictive algorithms for collaborative filtering . 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[ 22 ] Resnick , P . , Iacovou , N . , Suchak , M . , Bergstorm , P . , and Riedl , J . GroupLens : An open architecture for col - laborative filtering of netnews . In Proceedings of the ACM 1994 Conference on Computer Supported Cooperative Work ( Chapel Hill , NC , USA , 1994 ) , ACM , pp . 175 – 186 . [ 23 ] Resnick , P . , and Varian , H . Recommender systems . Com - munications of the ACM 40 , 3 ( 1997 ) , 56 – 58 . [ 24 ] Sarwar , B . , Karypis , G . , Konstan , J . , and Riedl , J . Anal - ysis of recommendation algorithms for e - commerce . In Pro - ceedings of the 2nd ACM Conference on Electronic Com - merce ( Minneapolis , MN , USA , 2000 ) , ACM Press , pp . 158 – 167 . [ 25 ] Sarwar , B . , Karypis , G . , Konstan , J . , and Riedl , J . Ap - plication of dimensionality reduction in recommender sys - tems . In ACM WebKDD Workshop ( Boston , MA , USA , Au - gust 2000 ) . [ 26 ] Sarwar , B . , Karypis , G . , Konstan , J . , and Riedl , J . Item - based collaborative filtering recommendation algorithms . In Proceedings of the Tenth International World Wide Web Conference ( Hong Kong , China , May 2001 ) . [ 27 ] Schafer , B . , Konstan , J . , and Riedl , J . Meta - recommendation systems : User - controlled integration of di - verse recommendations . In Proceedings of the 2002 Interna - tional ACM CIKM Conference on Information and Knowl - edge Management ( 2002 ) , ACM Press , pp . 43 – 51 . [ 28 ] Schein , A . , Popescul , A . , Ungar , L . , and Pennock , D . Methods and metrics for cold - start recommendations . In Proceedings of the 25th Annual International ACM SIGIR Conference on Research and Development in Information Retrieval ( Tampere , Finland , 2002 ) , ACM Press , pp . 253 – 260 . [ 29 ] Shardanand , U . , and Maes , P . Social information filtering : Algorithms for automating “word of mouth” . In Proceedings of the ACM CHI Conference on Human Factors in Com - puting Systems ( Denver , CO , USA , May 1995 ) , ACM Press , pp . 210 – 217 . [ 30 ] Spillman , W . , and Lang , E . The Law of Diminishing Re - turns . World Book Company , Yonkers - on - Hudson , NY , USA , 1924 . [ 31 ] Tombs , M . Osmotic Pressure of Biological Macromolecules . Oxford University Press , New York , NY , USA , 1997 . [ 32 ] Vogt , C . , and Cottrell , G . Fusion via a linear combina - tion of scores . Information Retrieval 1 , 3 ( 1999 ) , 151 – 173 . [ 33 ] Ziegler , C . - N . , Lausen , G . , and Schmidt - Thieme , L . Taxonomy - driven computation of product recommendations . In Proceedings of the 2004 ACM CIKM Conference on In - formation and Knowledge Management ( Washington , D . C . , USA , November 2004 ) , ACM Press , pp . 406 – 415 . [ 34 ] Ziegler , C . - N . , Schmidt - Thieme , L . , and Lausen , G . Ex - ploiting semantic product descriptions for recommender sys - tems . In Proceedings of the 2nd ACM SIGIR Semantic Web and Information Retrieval Workshop 2004 ( Sheffield , UK , July 2004 ) . \ No newline at end of file diff --git a/silver_data/65c26f14767cbfe056bbe9b50fa2e219c85a0cb1.pdf.txt b/silver_data/65c26f14767cbfe056bbe9b50fa2e219c85a0cb1.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..f6ca7c04de384d3835cfe73a82884c2212bdfdc5 --- /dev/null +++ b/silver_data/65c26f14767cbfe056bbe9b50fa2e219c85a0cb1.pdf.txt @@ -0,0 +1 @@ +A Social Environment Model of Socio - technical Performance 1 Brian Whitworth Institute of Information and Mathematical Sciences , Massey University Auckland , New Zealand bwhitworth @ acm . org Cheickna Sylla School of Management New Jersey Institute of Technology , NJ , USA sylla @ adm . njit . edu Abstract This paper analyzes the nature of social performance , to explain how socio - technical systems ( STSs ) like chat , e - markets , social networks and wikis can succeed despite being free of charge . It defines the non - zero - sum synergy gains of cooperation and how self - interested acts can collapse the society that creates them . How physical human society dealt with this " social dilemma " then relates to the socio - technical advance . In this model society is a social environment within a world environment , so its citizens face the dual requirements of self - interest and social - interest , which can be satisfied by anchoring one demand then managing the other , e . g . competing within a social context , as in markets , or community service within an individual context of sufficiency . The latter , it is proposed , is the new social form that socio - technical systems illustrate and which could be the future of humanity . INTRODUCTION Introduction Social interaction is something people do every day , but supporting it online isn ' t easy ( Fjermestad & Hiltz , 1999 ) . Online e - commerce reduces seller costs and gives buyers more choice , yet remains a minority of all trade , growing from 2 . 7 % in 2006 to 3 . 2 % in the U . S . in 2007 ( Scheleur , 2007 ) , and still struggling to get over 5 % in 2011 . In contrast , socio - technical systems ( STSs ) like Wikipedia were lost causes that asked people to give to something for nothing gain , but today Wikipedia challenges Encyclopedia Britannica and Open Office is an alternative to Microsoft Office . The unexpected contrast between socio - technical success and e - commerce lethargy requires a new theory . The social environment model specifies the arcane role of society , and links it to modern socio - technology , by : 1 . Defining core socio - technical concepts 2 . Outlining the social dilemma inherent to social systems . 3 . Summarizing traditional socio - physical responses . 4 . Developing a social environment model . 5 . Applying the model to cases like Enron and the credit meltdown , 6 . Suggesting that socio - technical systems are the new form of an old inspiration . 1 Accepted for publication by Int . J . Networking and Virtual Organisations Page 2 The social environment model applies to traditional and modern situations , it applies consistently at any social level , from small groups to societies of millions , and is useful to anyone working with socio - technical or socio - physical systems . The socio - technical approach Sociologists see individuals as conduits of meaning that reflect external social structures , so see psychological , biological and physical explanations as faulty reductionism of social realities . They replace the determinism of biology ( Wilson , 1975 ) or psychology ( Skinner , 1948 ) with social determinism , where society writes social agendas , like communism or capitalism , upon individual tabula rasae ( blank slates ) . Yet if all individual thoughts were erased , and psychology ceased to exist , society would also cease to exist , as surely as if all its members had vanished physically . This has led to attempts to re - attach sociology to its psychological roots , e . g . Bourdieu’s habitus references the individual percepts of the social environment and Gidden ' s mental frames underlie social life ( Bone , 2005 ) . Figure 1 . Socio - technical system levels The top - down connecting of sociology to its roots matches an equally vibrant bottom - up movement in computing , which has long seen itself as more than hardware and software ( Boulding , 1956 ) . In computing today , human computer interaction ( HCI ) uses psychology ideas like attention and effort in web applications . Organizational computing ( Kuutti , 1996 ) , leads to the next socio - technical step , by general systems theory ( Bertalanffy , 1968 ) . Each system level emerges from the previous , as software data flows arise from hardware circuits ( Whitworth 2009 ) : 1 . Hardware systems , based on physical energy exchange , face problems like overheating . 2 . Software systems , based on information exchange , emerge from hardware to face problems like infinite processing loops . 3 . HCI systems , based on meaning exchange , emerge from software systems to face problems like misunderstanding or information overload . 4 . Socio - technical systems , based on community normative exchange , emerge from HCI systems to face problems like mistrust , unfairness and injustice . As the discipline of computing becomes social , the new “user” of computing is society itself . Page 3 Software can ' t exist without hardware , yet is itself more than hardware . Reducing information to hardware voltages is like describing World War II by atomic events – both difficult and pointless . Software not only described computing systems efficiently , it offered more efficient ways to design and operate it , e . g . software cache prediction revolutionized hardware chip design . Semantics emerges from information exchange in the same way ( Whitworth , 2008 ) , as Web 3 . 0 is technology designed for meaning exchange not just information exchange . Social computing suggests another emergent level — the community , i . e . Web 4 . 0 . In Figure 1 , each level comes from the previous : physical exchanges of electricity give information , information exchanges give meaning , which gives the norms , culture and identity of communities . Each level emerges from the one below it , but by its nature changes the entire system ( Whitworth 2009 ) . So engineering , computing , psychology and sociology are just different system “views” . The social level is the complex not just because it contains more lower levels but because a social unit set can form into a larger one : people form families , families form villages , villages form cities , and cities form nations . A socio - technical system ( STS ) occurs when people interact via technology to create a community , as a socio - physical system arises when they do the same physically , combining the complexity of technology and the sophistication of society . Yet the social environment model applies to any social system , regardless of architecture ( technical or physical ) . THE SOCIAL DILEMMA Social dilemmas are here proposed to be inherent to any social system , however mediated . xxx Competition Figure 2 . Individuals competing in a limited resource environment In a limited resource environment , if two beetles independently seek the same food and one wins it then the other loses out . If the beetle with the food is more likely to survive , the result is " natural selection " where systems compete for advantage . The farmer growing the food also competes with the beetles and both compete with bacteria that would also consume it . Limited resource environments reward individuals that develop competencies like strength or speed that increase success and survival . Note that these competencies exist as a web of system performance , as the diversity of nature reflects Page 4 ( Whitworth , Fjermestad , & Mahinda , 2006 ) . In Figure 2 , competition for limited resources creates an individual need for competence to succeed based on feedback from the world . Homo economicus Figure 2 suggests a homo - economicus model of society where individuals do what benefits themselves by reduced effort , increased gain , or both ( Persky , 1995 ) . Individuals seeking advantage favors the evolution of new competencies by competition . It represents Mill’s economic man , who seeks wealth , leisure , luxury and procreation above all else . Adam Smith argued that such individuals in a free market also benefit society , as if people individually produce more , so must the community ( Smith , 1776 / 1986 ) . They are rational actors who calculate their own best interests , although people may actually use heuristics—psychologically efficient versions of rational logic ( Tversky & Kahneman , 1982 ) . The gains produced when competition drives self - interested individuals can be called competence gains and the evolutionary process is one of natural selection . That free individuals act in self - interest is a defeasible rule , which game theory describes as follows . If freely acting individuals { I 1 , I 2 … } face action choices { a 1 , a 2 … } with expected individual utility outcomes { IU ( a 1 ) , IU ( a 2 ) , … } , the rule is : If IU ( a i ) > IU ( a j ) then prefer a i over a j < Rule 1 > In words : Free individuals will prefer acts expected to give more value to themselves . The concept “value” here is deliberately left vague , so it may include physical gains like food , social information tokens like money , psychological gains like appreciation , or social gains like reputation . Homo sociologicus While Rule 1 is evident in nature , social cooperation is equally common , e . g . our bodies are colonies of cells cooperating for the common good , with cancer illustrating what happens when they don’t . In the animal kingdom , only social insects like ants form massively cooperative societies as we do , but they are highly successful , accounting for at least one - third of all insect biomass . The genetics that drives their behavior evolved because individuals working together can create more value than working alone ( Ridley , 1996 ) . For ants , the unit that competes and survives is not the individual but the colony , e . g . soldier ants die protecting the colony , as without it they can ' t survive anyway . In this model , individuals combine into a community that “performs” , in evolutionary terms , based on the sum of the actions of its members ( Figure 3 ) . Hence biologists now argue for multi - level selection —evolutionary selection for groups as well as individuals ( Wilson & Sober , 1994 ) . Social cooperation changes the evolutionary reward rule— individuals still act but the acts selected are those that create value for the community , not those that create value for the individual . That socialized individuals can generate community value suggests a defeasible social alternative to game theory’s Rule 1 : if a social unit S of { I 1 , I 2 … } individuals faces social action choices { a 1 , a 2 … } with expected social utilities SU ( a 1 ) , SU ( a 2 ) , … } , then the rule is : If SU ( a i ) > SU ( a j ) then prefer a i over a j < Rule 2 > In words : Socialized individuals will prefer social acts expected to give more value to the community . Value outcomes calculated for the group as a whole , not the individual , allow social evolution . So for ants , natural selection favors acts that increase community gain not individual gain . Social acts reference the social unit , not the individual , e . g . “defend society” is a social act independent of any individual state . Social “castes” can be dedicated to social acts , like worker or soldier , as ants do . Page 5 The same can apply to human society given homo sociologicus who prefer acts that benefit the community ( Bone , 2005 ) . This is Marx’s communist man , who is politically motivated to common acts that benefit society . A psychological basis for this is Social Identity Theory ( Hogg , 1990 ) , where groups form when members share a common “identity” . If one attacks one member of such a group , all group members feel attacked and respond accordingly . Indeed most country’s defense forces work by this rule , as servicemen and women are expected to give their lives for society . While in Figure 2 individuals reap the physical consequences of their own acts , in Figure 3 society bears the physical consequences of its social acts , as productivity outcomes , which then reflect to its individual members . That these pragmatic rules , one at the individual level and one at the community level , interact to create all social dilemmas is now proposed . The prisoner’s dilemma Game theory , the systematic study of rational choices in interdependent interactions , underlies many economic , political and group decision theories . It usefully presents the essentials of social situations for analysis . In the classic “Prisoner’s Dilemma” tale two prisoners ( Bill and Bob ) face two year jail terms on circumstantial evidence for a crime they did commit . Each is separately offered a plea bargain , to testify against the other . If the other does not testify he walks free but his partner gets seven years jail . If both testify , both get six years ( one off for testifying ) . In outcome utility terms the options are : 1 . Bill and Bob stay silent , and each gets two years in jail . 2 . Bill confesses for immunity , and Bob gets seven years . 3 . Bob confesses for immunity , and Bill gets seven years . 4 . Bill and Bob both confess , and both get six years jail . Table 1 shows these outcomes as free years out of seven . If both keep quiet , or cooperate , both get five free years , but if both testify , or defect , they only get one free year each . The temptation is for one to defect and get seven free years , while the other cooperating “sucker” gets none . Working as individuals who follow Rule 1 , each prisoner concludes : Figure 3 . A community cooperating in a world environment Page 6 • Whether the other cooperates or defects doesn’t depend on my choice . • If he defects , it pays me to defect , as then I get 1 rather than 0 . • If he cooperates , it still pays me to defect , as then I get 7 rather than 5 . Table 1 . Prisoner’s dilemma — Individual outcomes Bob Years free ( Bill / Bob ) Cooperate Defect Cooperate 5 / 5 0 / 7 Bill Defect 7 / 0 1 / 1 So by Rule 1 it always pays individuals to defect . Expected outcomes for cooperating average 2 . 5 but for defecting average 4 , so defection is preferred . If both parties follow this logic , defect / defect is the equilibrium state . So individuals maximizing their profit creates the worst possible result for both . Working as a social unit and following Rule 2 gives a different result . The available social acts for the pair are mutual cooperation and mutual defection , with expected gains of 10 and 2 respectively ( Table 2 ) . If both parties follow Rule 2 mutual cooperation is the equilibrium state . Indeed if the degree of social cohesion is known , simulated agents in a prisoners dilemma situations do evolve to a cooperative equilibrium ( Dayton - Johnson , 2003 ) . It can be seen that while Rule 1 gives only 2 free years of value , Rule 2 generates 10 free years , a considerable improvement . Traditional game theory assumes rational beings calculate payoffs for themselves as individuals , but it is just as rational to calculate payoffs for the social unit as a whole ( Table 2 ) . Rule 2 is simply Rule 1 applied to the social unit instead of individual units , so is just as logical and just as pragmatic . It is egocentric to take the person as the unit of value calculation , and label alternatives to individual self - interest “irrational” , when we are ourselves a biological society of cells , and what one defines as “self” can include the community around us ( Persky , 1995 ) . it follows that both rules are equally rational and both are equally pragmatically grounded in value outcomes . Table 2 . Prisoner’s dilemma — Social outcomes Years free ( Pair ) Social Outcome Cooperate 10 Social Act Defect 2 The tragedy of the commons The “tragedy of the commons” ( Hardin , 1968 ) extends the two - person prisoner ' s dilemma concepts to many people in a group . In it , some farmers live by a common grass area , each with cows and a plot of land . If a farmer’s herd also grazes the common area it grows fat , but if over 50 % of farmers do so , the commons is overgrazed and dies off . This parallels many forest and river conservation problems . Working as individuals each farmer’s logic is : • My actions are independent of those of the other farmers . Page 7 • If ≤ 50 % graze it pays me to graze , as I get more value . • If > 50 % graze , it still pays me to graze , as I get more value initially . It always pays the farmer to graze . Take a hypothetical case where 100 farmers each get a ton of beef per month grazing their own plots , and three more tons grazing the commons , which becomes barren in three months if over 50 % of farmers graze it . Table 3 shows farmer outcomes by choice for 10 months . Using Rule 1 , the average graze benefit is 28 , while the average not - graze benefit is 10 , so graze is preferred . Destroying the commons is the equilibrium point . Table 3 . Tragedy of the commons —F armer outcomes ( 10 months ) Others Outcome 49 % graze Over 49 % graze Don’t graze 10 10 Farmer Graze 40 16 Working as a social unit , produces different outcomes . The social available to the village are by what percentage to graze the commons . Table 4 shows the expected village outcome for overgrazing is 1 , 600 tons over 10 months , the expected sustainable grazing average is 2 , 500 , making it the preferred choice . Socialized individuals following Rule 2 will not destroy the commons because it is a valuable community resource . Table 4 . Tragedy of the commons — Village outcomes ( 10 months ) Farmer Outcome Social Outcome Not overgraze 2 , 500 Social Act Overgraze 1 , 600 The social dilemma Social dilemmas like the prisoner’s dilemma and the tragedy of the commons are generic problems in social interaction ( Diekmann , 2001 ) , e . g . in the volunteer dilemma a group needs volunteers to prosper but it pays individuals to let others do it , so the group collapses . Social dilemmas arise when Rule 1 contradicts Rule 2 , i . e . when what is good for the individual is not good for the group . Let social synergy be the difference between the total utility a set of individuals produce working as a social unit compared to what they produce working independently as individuals . Synergy can be positive or negative . Trade illustrates a positive synergy and social conflict a negative synergy . If synergy is positive , it pays individuals to join the social unit while if it is negative they are better off leaving it , e . g . individuals tend to leave websites plagued by conflicts . Synergy is a property of the social interaction , not of the individuals in a society . In the prisoner’s dilemma , the synergy is the loyal friendship total ( 10 ) less the defect total ( 2 ) , i . e . 8 years . In the Page 8 tragedy of the commons , the synergy is the cooperative total ( 2 , 500 ) less the competitive total ( 1 , 600 ) , i . e . 900 tons . The social dilemma is that self - interested individuals following Rule 1 minimize synergy . In a competition , the gains individuals receive can be attributed to their acts , but synergy gains attribute mainly to the acts of others . Game theory differentiates between zero - sum and non - zero - sum games . In zero - sum games , like poker , my loss is your gain , so if you lose I gain benefit at your expense . However in non - zero - sum games , when social dilemmas occur , your loss is my loss . In zero - sum games , shrinking another’s slice of the reward “pie” increases one’s own , but in non - zero - sum games , shrinking the reward pie affects everyone . Indeed if everyone does it , everyone becomes poor , as “dog eat dog” societies evidence . Conversely , the vast wealth of modern " civilized " societies arises when enough socialized individuals increase the shared pie for all , making every slice larger . Ordinary workers today have better food , healthcare and entertainment than the richest aristocrats of the middle ages , while the aristocrats of today have more money than they know what to do with . While non - zero - sumness is an unpleasant term , the argument that increasing social synergy is the secret to modern prosperity is strong one ( Wright , 2001 ) . Modern research illustrates the benefits of synergy . Take a hypothetical case of 100 knowledge breakthroughs of equal value . Researchers following a zero - sum model , as private companies do , keep their research secret , lest others gain . If your gain is my loss , why should competitors benefit from my work ? In contrast , most academic researchers follow a non - zero sum model and give their research freely to all . In the first case , total knowledge increases by 100 units , but in the second case it increases by 1000 units , as each researcher gets 100 new ideas from others . Open research is a hundred - fold gain in knowledge synergy over keeping research secret . If the scientists of history had kept their research secret , benefits of modern science like electricity may not have occurred . Yet even today , human genome research was nearly kept secret for commercial gain . Social Instability Anti - social acts like stealing arise when individuals take from society but don ' t give anything back , i . e . aim to forever get something for nothing . This “short - circuit” of the link between social acts and synergy outputs can easily cascade as individuals with instincts for personal gain ( Rule 1 ) are only socialized to create synergy ( Rule 2 ) . If one defects they not only gain personally but also reduce the gains of others , increasing the pressure on them to defect . If another then also defects , this increases the pressure on the remainder to also defect . Indeed a common reason given for cheating is that “everyone is doing it” ( Callahan , 2004 ) . Hence a few defections can cause a social chain reaction that destabilizes the entire social system . Yet if a crime - wave “succeeds” , the social benefits it feeds on dry up , like a parasite that kills its host , i . e . the idea of getting something for nothing is a " lie " . Every social synergy has a corresponding defection , e . g . in trade , if sellers defect by false claims , shoddy products or bad warranties , then customers don’t buy . Buyers can also defect , e . g . buy an expensive ball gown , wear it to a ball , and then falsely request a refund saying it didn ' t fit . If many customers do that sellers will refuse refunds ( also defect ) , even though refunds benefit both seller ( more sales ) and buyer ( less risk ) . In such cases the end point is mutual defection and zero synergy . Is mutual synergy , despite its benefits , inherently unstable for self - interested individuals , like a ball balanced on a crest that sooner or later rolls permanently down into the valley of mutual defection ? Or can kindness cascade too , as the movie Pay it Forward suggests ? Certainly individuals alone cannot solve social dilemmas as one person cooperating in a prisoner’s dilemma is just a “sucker” . In the tragedy of the commons , the farmer who doesn ' t graze just loses out and the commons is destroyed anyway . If the choices for individuals in social dilemmas are all bad , how have we achieved synergy at all ? After thousands of years of struggle , modern civilization has Page 9 evolved to stabilize massive non - zero - sum synergies like global trade . If the path to social synergy has on both sides the cliffs of defection , how have we , alone among mammals , crossed the zero - sum barrier into the lush valley of massive social synergy ( Wright , 2001 ) ? THE ZERO - SUM BARRIER Humanity is fortunate that reason pulled us out of the dark ages but equally fortunate to have something beyond reason , so we are not only rational individual gain maximizers , e . g . people in social dilemma games are much more cooperative than game theory predicts ( Poundstone , 1992 ) . One reason that people have evolved not to rely entirely on reason is it ' s fragility facing equivocal problems , where one is either uninformed or misinformed , as is common in business ( Whitworth , Van de Walle , & Turoff , 2000 ) . Another is that kindness works : Axelrod invited programs for a simulated “survival of the fittest” online social interaction “tournament” , to see which survived . He found that none of the eight most successful programs initiated defection ( Axelrod , 1984 ) . While nasty programs succeeded at first , in time they ran out of victims and met only other nasties , while cooperative programs found allies and prospered . Indeed , mothers caring for their child at their own cost is critical to all nature . So concluding that social cooperation is " irrational " ( von Neumann & Morgenstern , 1944 ) is like deducing from the laws of physics that bumblebees can ' t fly , when in fact they do . It is time to change the laws , as human instincts know what human logic does not : that cooperation works . Social order By rule 2 social dilemmas are solved when people form into a higher social unit . If the commons farmers form a village , they can institute a cooperative grazing roster to preserve it . While game theory generally excludes such social agreements , they are critical to solving social dilemmas ( Aumann , 1998 ) . A society ' s social order is the degree its members follow common rules . In perfect social order everyone is of “one mind” , like an ordered crystal whose constituent atoms all move as one . Social anarchy in contrast is like a gas , whose atoms all move randomly according to individual exigencies . Freedom lets members of a society choose whether to act as one or in opposition , and allows creative acts against the norm . Enforcing order avoids anarchy , but also reduces freedom and creativity . Cheating increases social disorder because cheater and cheated parties act differently . Equally one does not expect to be cheated , i . e . it is behavioral uncertainty . If a whole community acts as one ( social order ) , whether by voluntarily following a religion , culture or law , or by the force of a despotic dictator , the social dilemma disappears and synergy arises , e . g . a village with a common game reserve that stops “poaching”—individuals killing the animals for personal gain—can conserve the resource that creates for example tourist income . This can be by physical barriers like fences , but whatever barriers human ingenuity throws up others can overcome , as email spam illustrates ( Whitworth & Liu , 2009 ) . However if the community declares the land sacred or poaching illegal , offenders who defy the gods or state become enemies of society , and can expect its punishment or banishment . However enforcing order , even psychologically , is blunt instrument that creates social synergy by making its members effectively “ants” , and so like ants they become all much the same , i . e . it denies individual diversity , freedom and choice . A society that “socializes” citizens to follow Rule 2 engages social evolution to synergy but simultaneously disengages individual evolution of competence and creativity . This struggle , between social and individual evolution , may underlie historical swings between the rise of sophisticated civilizations and their fall at the hands of more vigorous barbarians . Social performance , in this view , requires both individual diversity and social order . Page 10 Social hijack Centralizing control structures to create social order invites social hijack , where individuals take control of a community for their own ends as a virus can hijack a biological organism to serve its own purposes . Plato’s ideal leader was a benevolent dictator , who enforced social order to create synergy , then justly returned the gains of society to its citizens , i . e . both enforced social synergy and returned it to those who created it . Yet dictators are also the worst of leaders when they use society’s performance for their own personal luxury or power ends . Since this is unfair , they must repress individuality by police state control tactics , and indoctrinate the masses into the blind service of society by media propaganda . This makes dictatorships : 1 . Unstable . If those who create social wealth gain nothing from it , they have as Marx notes , “nothing to lose but their chains” . A privileged aristocracy living in luxury while the workers who create that luxury starve invites a grass - roots revolution . 2 . Impermanent . Kings , emperors , pharaohs , khans and other dictators eventually die , leaving a power vacuum that can cause a civil war . Royal bloodline dynasties avoid this , but inevitably over time produce incompetent or even insane offspring , whose foolish acts collapse the society . 3 . Unproductive . When a society blindly follows the whims of leader ( s ) who are isolated by wealth from the world ' s realities , it becomes incompetent and fails to generate produce from the world . Societies with absolute rulers like Burma and North Korea become poor when their rulers replace natural productivity requirements with their personal social agendas , e . g . in Zimbabwe Mugabe addressed social inequity by driving white farmers off productive farms , then gave them to cronies who looted but did not plant , grow or harvest . Equity without productivity turned Zimbabwe from the bread - basket of Africa into the basket - case of Africa . Social hijack is an evolutionary dead - end , changed only by the leader’s death or social collapse , or both . To prosper a society needs both individual competence and social synergy . Synergy is like interest paid on the capital of competence—if there is no capital there is no interest either . Synergies from social order ( Rule 2 ) can add to the competence gains produced by natural competition ( Rule 1 ) but cannot displace them . Yet neither is Rule 1 alone sufficient as it ignores synergy , e . g . most now reject the Social Darwinist argument that since nature weeds out the weak , so should society . Yet natural " fitness " involves a web of performance criteria ( Whitworth , Bañuls , Sylla , & Mahinda , 2008 ) , so nature shows extraordinary diversity and tolerance for many different life forms , and underneath its competition is a basic synergy . So the problem is how to merge these two strategies effectively . Social inventions Periodically society discovers new social forms that increase social performance . Justice — punishing unfairness—is one way society has discovered to combine social order and individual freedom . Unfairness is here not merely inequity—the unequal distribution of outcomes—but not distributing outcomes according to contribution , e . g . that fit adults idly live off society while others work to support them is considered by most to be unfair . Justice addresses the problem that while forming a social unit engages synergy , it disconnects individuals from the consequences of their acts . Studies suggest people have a natural justice perception , of whether value gained matches contribution made , and tend to avoid unfair situations ( Adams , 1965 ) . That people even prefer fairness to personal benefit ( Lind & Tyler , 1988 ) suggests they prefer situations where Rule 2 works . In contrast , chimpanzees are simple outcome maximizers , following Rule 1 entirely ( Jensen , Call , & Tomasello , 2007 ) . Selfish individuals destabilize society , but social justice changes the dynamic by punishing unfair acts . If individuals seek revenge on those who ‘wronged’ them or their family , this makes cheating less Page 11 profitable over time , as today’s defection is paid back with interest tomorrow . If a society can make unfair interactions a bad choice , selfish people will prefer mutual synergy to mutual conflict , i . e . justice aligns individual good and social good . Unfortunately , in “eye for an eye” cultures one revenge act creates another , giving endless vendetta cycles , as in the Middle East . Revenge is a primitive form of justice , but rather than individuals administering justice personally it is better for society to punish defectors by impersonal laws . The case has been made that our entire justice system of police , laws , courts and prisons aims to do precisely this - deny unfair acts ( Rawls , 2001 ) . State justice allows a society to synergize selfish individuals . Hence state justice gains must be calculated at the community level not the individual level , e . g . depression reduces individual productivity but no laws deny it as it affects people not communities . Conversely , if someone steals $ 100 and is caught penniless , a court may still sentence them to a year in jail . Yet if police , trial , and incarceration costs are over $ 100 , 000 , and the robbed get no return , where is the value ? If everyone loses , why waste money prosecuting ? The error here is to take an individual perspective to a social function . From a community perspective , $ 100 , 000 may be a small price to pay for social order . Prosecuting defectors is about changing social interaction contingencies , not about individual profit or loss , e . g . the 1980 clean up of New York crime changed the entire social environment , from one where shootings were common to one where it was safe to walk the streets . Few would argue that the increased productivity of an entire city was not worth the effort . Democracy , like justice , is another social “invention” ( Mandelbaum , 2002 ) . If a community selects leaders by vote , the power to control the community ultimately invests in the community itself . Democracies also have constitutions limiting terms of office . A dictatorship has a “centre” to hijack , but a democracy that distributes power among the people does not . This turns out to be better than trusting central elites , however benevolent , not because it is more efficient but because it allows stable evolutions , i . e . anarchy free transitions of power that avoid episodes of dynastic incompetence . Given a human history of bloody power struggles , it is always amazing to watch a government that has lost a democratic vote peacefully hand over power to a successor . Democracies combine individual freedom , social order and resistance to hijack . They produce more because free people contribute more work , more ideas and more research , and they also self - regulate more , reducing security costs ( Tyler , 1999 ) . The allied democracies overcame the Axis dictatorships in World War II because they producing more , and democratic nations have increased over time again not because democracy is “nice” , but because it is productive . THE SOCIAL ENVIRONMENT MODEL Individuals in Figure 2 compete in the world and in Figure 3 form a unified society , which combined makes society an environment within an environment ( Figure 4 ) . The social environment A social system is an “environment” to its members , as it imposes requirements on them ( laws and norms ) , and dispenses gains and losses ( salaries and taxes ) . The role of the social system is to create synergy—more value for all . It distributes social tokens , like money , which can be exchanged for world value , like food . A social system within a world environment can fail by incompetence , as a wasteful company going bankrupt . It can also fail to create synergy by allowing crime or conflict , causing instability or internal revolution . As Figure 3 shows , people in a society operate under two distinct environments , one rewarding them for competing , and the other rewarding them for cooperating . This suggests that citizens follow a hybrid of Rules 1 and 2 . Page 12 Table 5 cross - tabulates individual by community outcomes . This categorizes not just social but also biological behavior , as the first row choices are symbiosis , commensalism and predation respectively . The " selfish " Rule 1 directs an individual to choose the first row , while the " good " Rule 2 directs the individual to choose the first column . The question raised is how to optimally combine them in a way that is also cognitively possible . The utilitarian ideal of “ the greatest good for the greatest number , ” popularized by Dr Spock’s Star Trek sacrifice , seems a simple calculation but what is the “greatest good” for millions , especially over time ? Is an aircraft crash that loses lives but causes safety changes that save more future lives " good " ? The rule is hardly calculable by anyone , let alone a majority . Nor does a simple AND of Rules 1 and 2 work well . It is feasible but not optimal , as if people acted only to benefit both themselves and society , they would often not act at all . Equally some sort of weighted trade of social utility against individual utility raises complex questions , like how much social good is my individual loss worth , or how much individual good warrants a social loss ? I 1 COMMUNITY ENVIRONMENT Social acts Community utility Cooperation & Competition WORLD ENVIRONMENT Social and competencerequirements Utility Competencerequirements Competition Community Performance ( Competence + Synergy ) Anti - social acts ( crime ) Social Barriers . . . . . . . . . Other social levels I 2 Other Communities Synergy Non - social acts ( hermit ) Figure 4 . Social environment model What is needed is a combination that is simple enough for the human mind to embrace . It is proposed that people resolve such cases by cognitive anchoring , fixing one rule then applying the other ( Tversky & Kahneman , 1974 ) , as follows : If { SU ( a i ) ≥ SU ( a j ) and IU ( a i ) > IU ( a j ) } then prefer a i to a j < Rule 3a > OR If { IU ( a i ) ≥ IU ( a j ) and SU ( a i ) > SU ( a j ) } then prefer a i to a j < Rule 3b > Page 13 In words : Choose acts that : a . Don’t harm society significantly but benefit oneself OR b . Don’t harm oneself significantly but benefit society Following Rule 3a , individuals will seek opportunities provided they don’t harm society , which society usually makes clear by its own good conduct laws , i . e . one competes by the rules . Following Rule 3b , individuals will help others in society ( service ) , provided it doesn ' t involve too much personal loss . Again , it is usually clear what helps others , and whether one is able to easily do it . This rule is easier to apply than ideals of calculating the greatest good for the greatest number . Applied to Table 5 , Rule 3 produces the options synergy , opportunity and service . So while some may follow Rule 1 exclusively , to become self - interested criminals unconcerned that their acts may collapse society , and others may follow Rule 2 exclusively , willing to sacrifice themselves for society at any time , most people are free - good - citizens who espouse neither crime nor altruism . As citizens , they try to get ahead while not disobeying laws , and help others when free to do so . If they have to go to war they will , but will make every effort to personally survive . If we only followed Rule 1 crime would prevail and society would collapse , while if we only followed Rule 2 we would still be locked in under genetically bred kings or pharaohs . Our social evolutionary path seems rather a pragmatic combination of individual freedom and community cooperation , here called the free - good - citizen . Table 5 . Individual choices by self and community outcomes COMMUNITY Gain Minor effect Loss Gain Synergy Opportunity Anti - social Minor effect Service Null Malice S E L F Loss Sacrifice Self - harm Conflict Communism and capitalism In the well known political conflict of capitalism and communism , free competitive value ( Rule 1 ) is presumed the opposite of social value ( Rule 2 ) . However in the social environment model they need not conflict , as one applies to the individual and the other to the community level . Indeed they are underneath the same , as Rule 2 is simply Rule 1 applied to the social rather than the individual unit . Hence they need to combine , as a community that produces little but shares it fairly is hardly better than one that produces a lot but shares it unfairly . Given a choice between wealthy inequality or poor equality why not choose wealthy equality ? Adam Smith linked individual and public good , by suggesting the “invisible hand” of individuals in a market maximizing profits also guides the group to greater value ( Smith , 1776 / 1986 ) , as if everyone produces more so must the group . The social environment model accepts this , as competence is a world requirement that all social environments must transmit . Yet Smith’s argument for competition is not an argument against cooperation . Just as competitive sport imposes fairness by referees and penalties , so “free” markets require common good rules , e . g . the Stock Market punishes insider trading . Like playing fields , competitive environments work best when “level” to the players . Hence Page 14 economic sociologists like Granovetter argue that individual economics is always embedded in a larger social context outside any competitive framework ( Granovetter , 1985 ) . In the social environment model , Smith’s link also works the opposite way , as community infrastructures benefit competing individuals by supporting social synergy . Rule 3 combines the capitalist view of society as self - interested individuals , and the communist view of society as ant - like cooperatives , with a view of free good citizens who help both themselves and society . This is neither pure capitalism ( Rule 1 ) nor pure communism ( Rule 2 ) , but a hybrid . If pure communist societies have lower productivity , and pure capitalist societies have lower socialization , then a hybrid of competence and synergy will perform better socially . If communist countries move to “communism with a business face” , and capitalist countries move to “public good capitalism” , both will meet in the middle . In this model , social performance improves when the “invisible hand” of market competition works with the “visible hand” of public good . Social levels The vertical ellipsis in Figure 4 indicates that a social environment can be contained in another , e . g . many people can form a company , and many companies can form a stock market community . The company is a social group to its members , and also a member of the stock market social group . Both social systems add value and share it with members . Companies reward employees with pay , and stock markets reward companies with share prices that increase public investment . Equally both environments place requirements on members , as companies ask employees not to steal their product value ( stock ) , and stock markets ask companies not to steal their product value ( ratings ) by falsely reporting profits . Rule 3 can be “universalized” to the multi - environment case where S 1 contains S 2 … : If { { SU 1 ( a i ) ≥ SU 1 ( a j ) or SU 2 ( a i ) ≥ SU 2 ( a j ) … } and IU ( a i ) > IU ( a j ) } then prefer a i to a j < Rule 3 ' a > OR If { IU ( a i ) ≥ IU ( a j ) and { S 1 U ( a i ) > S 1 U ( a j ) or ( S 2 U ( a i ) > S 2 U ( a j ) … } } then prefer a i to a j < Rule 3 ' b > In words : Choose acts that don’t significantly harm higher environments but benefit oneself OR Don’t significantly harm oneself but benefit higher environments New problems arise when social dilemmas operate at higher social levels , e . g . the Enron debacle , with estimated losses of over $ 60 billion , occurred when Enron executives cheated the stock market by reporting false profits to raise their stock price . Other companies laid off staff to “compete” with Enron ' s imaginary profits of over 80 % . Within the stock market social system , Enron defected on the rule by which the stock market creates synergy , as if everyone made false claims , no one would invest . If false reporting were not illegal before Enron it would have to be made so , for the stock market to survive . The stock market had to act against Enron or collapse itself . If business did operate by a purely competitive model ( Rule 1 ) , then Enron’s innovative methods of obtaining value in the stock market environment would be a competitive advantage , as would be their paying zero U . S . tax for seven years . However the business maxim “Greed is good” does not apply to defecting on a social contract . Cheating one’s colleagues is not “competitive advantage” , as the bottom line it produces is a loss of value for society . Enron hypocritically asked its workers to serve their social environment , the company , but did not itself serve its social environment , the stock market . Gangs like the Mafia have a similar hypocrisy , demanding strict loyalty and service within their community , while as a group pillaging the community they are in . In general , a social rule that does Page 15 not apply at every level creates an inconsistency that must eventually be resolved , e . g . the U . N . is inconsistent in giving non - democratic member states democratic rights . Wildlife poaching in Africa also illustrates social levels . It is a classic tragedy of the commons situation , yet public ownership has generally been a disaster for conservation in third world countries ( Ridley , 1996 ) . Under nationalization , the government could not stop locals poaching the large animals that damaged their crops . The trend was only reversed when wild - life titles were “privatized” to local communities , like the Campfire program of Zimbabwe , where hunters purchase the right to kill game from local villages ( Ridley , 1996 p236 ) . When the village owned the animals , it looked after its resources , prevented members poaching , and wildlife numbers increased . By contrast , whales roam international oceans not owned by any country , so there is the danger we will hunt them to extinction . If a global humanity owned the whales , it would be as foolish to hunt them to extinction as for a farmer to kill all the cows in his herd . So as nations hold local power , perhaps rights should be “privatized” to nations , who would then physically prevent whale poaching in their zone . Rule 3 ' can be idealized to define categorically good acts as those that give value “all the way up” , not just for oneself , but for the community , for humanity , and even the planet we live upon . The principle that there are levels of “good” was made clear in the Nuremburg trials — where citizens following national laws were convicted of “crimes against humanity” , i . e . held to a higher standard of right and wrong . If social environments are within a world environment , is then the highest good to serve the world ? If so , does not Rule 1 also satisfy the world ? No , as in Rule 1 one satisfies oneself by meeting the world’s requirements —but what the world needs remains separate . Individuals following Rule 1 seek their own gain , not the gain of their environment . So while the natural environment has evolved , it did so unconsciously , in that the life struggling to get ahead within it had no concept of the evolution occurring on the earth . Yet extending Rule 2 , suggests that the highest good is to serve the highest level environment , not just family , tribe , nation or humanity but perhaps even the planet . However to do this , one must first survive the demands of lower more immediate environments . Perhaps the pragmatic ideal is to serve the highest environment one can conceive , while surviving the demands of lower environments one experiences . Social health Social synergy advances require social health . If “social capital” is the “… norms and networks facilitating collective action for mutual benefits ” ( Woolcock , 1998 ) , then social health is how successful those norms and networks are . Unlike ants , people must learn to socialize , e . g . with young soccer players a “cloud” of players trails the ball , as each individual tries to score a goal . Inevitably , they obstruct each other and the results are poor . Only with training do players learn positions like forward or fullback , and to engage in social acts like passing the ball . While soccer competence skills still count , teams need to cooperate to succeed . Just as one rates individual competence by testing what they can do , so one can rate community synergy by testing what it can do . In particular , to what degree will a community of free individuals support social synergy ? For example , if a group offers cheap coffee on an “honesty” system , where each person leaves 25¢ per cup , what percentage cheat , and take the coffee but leave no money ? If everyone defects and takes the coffee for free , the synergy ( and coffee ) fails . Conversely if everyone contributes , people continue to get cheap coffee . A business example is the coffee self - service model , where customers help themselves to coffee , milk and sugar . This increases serving speeds , as servers need only give customers a cup , which drastically reduces lines . However if social health is low , selfish individuals loot the offered beverage resources , which then must be kept behind the counter . Now servers must pour and apply the milk and sugar the customer wishes , which makes customer Page 16 lines much longer . Similarly , the social invention of super - markets required a degree of a social health . Traditional shopkeepers kept goods behind the counter to prevent theft . Only when most customers don’t steal can goods be put out on shelves for customer self - help , improving efficiency enormously . Social health — percentage who defect on social synergy — affects social performance . The combination of social and economic health is important to any community , as social systems in a world environment are subject to natural selection , as individuals are . Given open borders , capable people will flow into societies that offer them more value , increasing the prosperity of that country . A physical barrier like the Berlin Wall can prevent such flows , but its physical and political cost are not sustainable . Social systems , like individuals , can thus fail if they do not perform , with the Soviet Union’s collapse an example ( Diamond , 2005 ) . Social systems must generate both competence and synergy from their citizens over time to survive and prosper . Social inflation and external rectification A social environment cannot permanently insulate its members from the demands of its environment , as the outer environment ' s demands ultimately “cascade” over inner ones . Social environments that ignore the demands of their environment experience social inflation , where the value of tokens distributed to members loses external value . Monetary inflation illustrates social inflation , as money ( a social token ) loses value relative to external standards ( like a loaf of bread ) . Grade inflation occurs when professors give all students A’s regardless of competence , and the token “A grade” loses value in the University’s environment , e . g . with employers . Internally giving high grades seems to benefit everyone , as grading is easier , students are happier , and high pass rates attract more students . Yet externally it gives no value , so is unsustainable . While crime and corruption contradict society’s requirements , social inflation contradicts the requirements of its environment . It can build gradually , like a choir slowly going off - key together , but can end suddenly , in the failure of the entire social unit . In social inflation the social unit as a whole goes against its environment . Unless there is an internal rectification , eventually there must be an external rectification . World events like the great depression and the world wars illustrate external rectifications , as does the recent current credit meltdown . This world gives gains at the cost of risk , but banks and credit companies began offering loans almost regardless of risk . Internally this seemed to benefit everyone—lenders got more interest , lendees got needed money and bank popularity increased . As some banks increased lending , others followed suit to keep in the market . Finally , when companies could not recover their loans , bad debt decreased the “share” token’s value . The expected result of letting this external rectification “run its course” is the collapse of the global credit system , followed by depression or war . Knowing this , the US and other governments stepped in with billion dollar bailouts , but unless an accompanying internal rectification addresses the core problems , this will only delay the inevitable external rectification . It is a concern that when businesses leaders cheat society of billions they are removed , but when they lose even more by risk management incompetence they are not . If the same people who engineered the credit collapse still draw bonuses based on their business “skills” , no correction has been made . Just as Enron was a higher level of unethicality , so the credit collapse is a higher level of incompetence . In the social environment model , competence gains and synergy gains are both equally important to social performance . While society need not punish bank leaders for negligent risk management , it should remove them for the same reason it removes criminals—the good of society . Fiascoes like the credit crunch and Enron highlight the issue of private business and the state . When Wall St’s credit froze , by its own errors of judgment , the state stepped in to pay the $ 700 billion heating bill , quoting the public good . Similarly , when Enron ' s naughty boys , playing with the matches of cheating , nearly burnt down the entire market house , the state again stepped in , again for the public Page 17 good . However to expect state bailouts in hard times but want no state " interference " in good time is like a child who wants to be left alone but still wants its parents to pay its bills . If public good is important , then it is important all the time , not just when business is in trouble . And if in times of trouble the nation pays the piper , then in times of plenty it can call the tune . In the case of corporate cheating this means implementing fair - play public - good rules , like honest financial disclosure or that no company can pay zero tax . In the case of corporate incompetence this means replacing the incompetent by those with real skills . Any society that fails to act in its own interests in such ways invites its own collapse . Legitimacy Traditional society devotes considerable resources to denying anti - social acts : police , prison , court and legal systems all support synergy by punishing the anti - social acts that degrade it . Yet if the goal is synergy , why not seek it positively rather than negatively ? This positive social goal can be called legitimate interaction , defined as fairness plus common good ( Whitworth & deMoor , 2003 ) , e . g . freedom—that individuals own themselves—is legitimate as it is both fair to individuals and productive for society . Fairness , as earlier noted , is the antithesis of cheating , but legitimacy is not just fairness . Duels are fair but still outlawed as they harm society by killing its members . In sociology , the term legitimate applies to governments that are well justified , and not just based on coercion ( Outhwaite , 1994 ) . It is a political concept of social “rightness” beyond mere power or legality ( Barker , 1990 ) , e . g . Mill talks of the “…limits of power that can be legitimately exercised by society over the individual . ” ( p . 302 ) ( Somerville & Santoni , 1963 ) , Jefferson writes that : “… the mass of mankind has not been born with saddles on their backs , nor a favored few booted and spurred , ready to ride them legitimately … ” ( p . 246 ) ( Somerville & Santoni , 1963 ) . Human rights are essentially statements of legitimacy . As Fukuyama argues , communities ignore legitimacy do so at their peril ( Fukuyama , 1992 ) . If our social past sought to achieve synergy by denying individual self - interest , the present seems more positively focused on enabling legitimate rights . There has been a change in the social lens , from people as selfish primitives that must be controlled and repressed like wild beasts , to people as citizens whose freely offered kindness need only be kindled . Modern socio - technology , it is proposed , taps into the second world view . The golden rule Yet this positive approach also has a long social history , in the golden rule : “ Do unto others as you would they do unto you ” It has been expressed in many ways at different times and in different cultures and contexts : 1 . Rabbi Hillel’s sum of all rules : “ If you don’t like it done to you , don’t do it to others ” . 2 . Kant’s proposal : “ Act only on that maxim by which you can at the same time will that it become a universal law ” , i . e . if everyone does it , is it still successful ? 3 . Pareto’s optimality principle : “ Good actions benefit at least one other and do no harm . ” 4 . Rawl’s “ veil of ignorance ” requires state justice to be “blind” to individual needs . 5 . Harsanyi’s approach rules out immoral or anti - social acts from consideration ( Harsanyi , 1988 ) . These and other forms suggest a solid universal social principle equally applicable to information technology ( Siponen & Vartiainen , 2002 ) . Anti - social acts fail all golden rule tests , e . g . Hillel rejects stealing as one does not wish to be stolen from , Kant finds it wrong as if everyone does it , it doesn’t work , and Pareto finds it harms another . Rawls from behind his veil of ignorance cannot advocate it Page 18 without knowing who is stealing from who , and Harsanyi finds stealing an anti - social act . Rule 3 rejects stealing because overall it is a social loss , e . g . when a wallet is stolen there is not just the money lost but also disruptive losses like renewing credit cards . This generic golden rule sits above the individual economics of game theory . Kant distinguished his “categorical” imperative from “hypothetical” ones , i . e . the rule is not “Do unto others so they will do likewise unto you” . Such “deals” are merely instruments to individual benefit . Kant’s imperative in contrast is categorically “the right thing to do” , regardless of the outcome for oneself . The golden rule asks free individuals to think and act in terms higher than themselves , to hypothetically “flip” the social interaction equation to see if it still works the other way . It asks people to stand not only in the shoes of others , but of the society as a whole . Modern society shows that this is not just “nice” or " good " , it is also logically productive . It is logical because Rule 2 is just Rule 1 applied to the social instead of individual unit . It is productive because community synergy really works . Ethics , in this view , is simply pragmatics at a community level . Based on the golden rule , which advocates a good choice , the ship of human society has navigated a middle way between the social evolution dead - ends of endless selfish tribal conflicts and mindless , ant - like social conformity . FREE GOOD CITIZEN TECHOLOGY SUPPORT Socio - technology While traditional technology like word processing supports individual competence , socio - technical systems support community synergies of some sort , and also defend against anti - social defection ( Table 6 ) . If users just followed Rule 1 , systems like Wikipedia would not succeed , as why give knowledge to others for no self gain ? Conversely , if people just followed Rule 2 , these systems would not need to defend against anti - social defections , as they do . Socio - technical systems aim to both engage community synergy and put up anti - social defenses . Synergy and size Synergy gains arise from the number of participant interactions so while competence gains increase linearly with group size , synergy gains increase non - linearly . Interactions increase non - linearly with group size , so synergy becomes more important than competence for bigger groups . When the Internet allows millions to synergize then as Shirky says : “Here comes everybody” ( Shirky , 2008 ) , and the synergy values become enormous . Google illustrates the power of this approach . It began as a free service but now rivals Microsoft in influence , because if the knowledge of humanity is on the Internet , then Google is the doorway to it . This has led to new business models , based on serving the community instead of , or as well as , milking it for profit , e . g . much current research on trust frames the problem as how a seller can manipulate customers to trust them to make a sale . Yet if this were possible , such customers would soon go bankrupt buying foolish things , and so no more be customers . Even if propaganda can con a community , creating stupid customers is neither desirable nor sensible , as such a community will collapse and take the business with it . Some blame this “business” approach for the dot . com bubble collapse , which found that customers are more savvy than stupid . In contrast community based business models change the question from how to trick customers to give us money , to the more sustainable how to synergize with customers ? Instead of seeing customers as competitors , who must be kept in the dark or misled , new business models see them as partners in value creation . Socio - technical successes like eBay show that to do this one should not try to manipulate or " manage " the community , as in the saying of Lao Tse : " One should govern a state as a cook fries small fish , that is without scaling or cleaning them . " Page 19 As increasingly people use computers to socialize , socio - technology will not just support society but be society . It will support the social trend to stabilize synergy for larger groups , from hunter - gatherer tribes to mega - states like Europe , India , China and America ( Diamond , 1998 ) . At each stage , more complex social mechanisms were needed . If today’s global technologies are to enable an earth cosmopolitan , they must be designed in a spirit of service . Free good citizens A notable feature of today’s social Web is the willingness of people to help others they have not met and may not meet again , e . g . experts helping others with hardware problems on online boards . Neither Rule 1 nor its Rule 3a contextualization explain this . Yet people commonly help others in physical society too , e . g . most city people give lost visitors directions even knowing they will probably never see them again . That Rule 3b works is shown by the fact that people do willingly help others provided they don ' t lose too much themselves . That individuals in markets unconsciously help society , as Adam Smith argues , ids distinct from Rule 3b , which turns the logic of Rule 3a on its head to say that when individual needs are met , a positive urge to social value remains . Granted free people are self - motivated , they are also socially - motivated to actively help others if free to . For example , in BitTorrent systems users help each other download large files . Though the Torrent community lets individuals " download and leave” , it survives because many don’t . Even in a community of mainly opportunistic users downloading web content , people choose to help . Initiatives like SETI , and FLOSS ( Free , Libre , Open Source Software ) community sites like SourceForge and FreshMeat illustrate the same . The Creative Commons shows that people will freely give their work to others ( synergy ) , provided receivers do not copyright or sell it ( defect ) . Page 20 Socio - technical systems only succeed because free people are willing to be good citizens . The invitation to be a “small hero” , to do a small selfless act of service for a community , is taken up . The technology then adds up these many small good deeds into positive synergy . If the free - good - citizen rule did not work , socio - technical systems would not be the success they are . That such social systems are possible is an important social discovery , with implications for all humanity . We knew from history that enforcing order allows synergy . The pyramids of Egypt show what people working together can do . We also know today that markets can synergize people by individual incentives , given contextual legal systems prevent injustice . What we didn ' t know was that contented people , not subject to coercion , nor enticed by personal incentives , will freely synergize community value . We knew people could be forced to be good citizens , or enticed by reward or punishment to be so , but not that they would freely be so . Systems like Wikipedia , that throw themselves upon the goodwill of their citizens don’t just survive , they prosper . That “virtue” is both productive and can be supported by technology ( Benkler & Nissenbaum , 2006 ) is an important social discovery . This new social evolution may reflect an evolution in social health . A thousand years ago the democracies of today were not just unthinkable , but also unworkable . Instituting freedom would have quickly led to anarchy , as occurred after the French revolution . Yet today democracies work , and we find it hard to imagine why our predecessors would settle for less . Information democracy is more productive than information autocracy for the same reasons that physical democracies out - produce autocracies ( Beer & Burrows , 2007 ) . Table 6 . Socio - technical synergies and defections Aim Examples Synergy Defection Communicate Email , Chat , ListServ , IM Shared communication : People send messages they otherwise would not Spam : Spammers waste others time , giving spam filters . Learn WebCT , Moodle Blackboard , Shared learning : Students help others learn , reduce teacher bottlenecks Plagiarism : Students copy other student’s work , giving systems like Turnitin . com . Knowledge Wikipedia , Tiddlywiki Shared knowledge : Taps knowledge of the group , not just a few ”experts” Trolls : Wikipedia’s monitors and fights “trolls” who damage knowledge . Friends Facebook , Myspace Relationships : People keep in touch with friends and family Predation : Social network predators find victims , giving reporting and banishing Keeping current Digg , Del . icio . us Shared bookmarks : Social bookmarks let people see what others look at . Advocates : Who “digg” a site because of a vested interest , e . g . they own it . Play Second Life , MMORPG , Sims Shared play : An avatar experiences things impossible in reality . Bullies / Thieves : “Newbies” robbed by veterans don’t return , so need “safe” areas . Trade E - Bay , Craig’s List , Amazon Item trading : People from anywhere exchange more goods . Scams : Scammers are reduced by online reputation systems . Work Monster Work trading : People find and offer work more easily . Faking : Padded CVs and fake job offers need online reputation systems . Down - load Webdonkey , Bit - Torrent Napster , Shared down - loading : Groups share the processing load of file downloads . Piracy : Napster was in conflict with society’s copyright laws , so closed down . Media Sharing Flickr , YouTube podcasting Shared experiences : People share photos / videos with family / friends . Offensiveness : Editors remove offensive items—violence , porn , scatology… Advice Tech help boards like , AnandTech Shared technical advice : People who have solved problems can help others Confusers : People who start new tracks rather than checking old ones are scolded . Express opinions Slashdot , Boing - Boing , Blogs Shared opinions : People express and read others opinions more easily Caviling : People who “peck” new ideas to death—karma systems deselect them . Page 21 Yet socio - technical systems , which are today transforming the Web ( Kolbitsch & Maurer , 2006 ) , differ in significant ways from current physical society . By being more decentralized , more transparent and more available they become more participative . They deny all forms of social control , whether of acts ( repression ) or of information ( propaganda ) , and believe quite simply that free people will do the right thing . This is not communism as individuals are free to express themselves without social control . It is not socialism , as individuals can take value for themselves and leave if they want to . It is not anarchy , as there are anti - social defenses to oppose disorder . It is not altruism , as no - one is expected to sacrifice for the community . Socio - technical systems as a new social form based on legitimate rights , tolerance of individual freedom , community decision transparency and support for social order , suggest new strategies for physical society . For example , transparency means that the account of those who take community wealth as beneficiaries is public knowledge - as one can hardly claim that public monies accepted is private information . One wonders of the effect of a yearly " community return " , of what the community paid the citizen , as well as a tax return of what they paid the community . While an individual - community balance sheet listing medical costs as " Paid for by the State " , giving a total owing of zero , might seem a waste of time , simply making the information known might encourage individuals to give back to society with registered service groups . Transparency means that everyone in a community has a right to know how its synergy is being spent . Future directions Game theory’s specification of “rational” decision making inspires many decision strategies in business , economics and politics , but fails utterly to explain how humanity solved the social dilemma to achieve the synergies of modern civilization . This is only explained if homo - economicus , who only recognizes self - interest , exists alongside homo - sociologicus , who recognizes social good . While the first may seem " selfish " and the latter " good " , the rules embodying both their behaviors are essentially pragmatic , i . e . service is just as rational as selfishness , and indeed for large groups can give even greater rewards . The social environment model suggests that people in society recognize both these rules , and combine them by anchoring one and applying the other . Anchoring social good then applying self - interest explains the highly profitable market trade systems of the last century , where individuals seek profit under social good laws . However contented individuals could anchor individual good , and then seek community benefit . The latter is proposed to underlie the surprising successes of socio - technical systems . As technology advances minimize the effort of social acts and magnifying their effect , this forces humanity to resolve its choices . As the mutually assured destruction ( MAD ) of nuclear weapons forced nations to abandon illusions of world domination , so today ' s technology support for negative synergies like spam challenge the new illusion of profit focused market systems , that one can get something for nothing , wealth without effort . Social reward tokens like money only increase social performance up to a point . If people focus exclusively on individual profit their inevitable ideal is to get profit for nothing—Enron , World Corp and the credit crunch banks all sought perpetual profit machines . Yet just as perpetual motion contradicts physical laws so perpetual profit for no effort contradicts social laws—everyone taking from everyone else can ' t create value . Those who propagate this illusion , like pyramid profit schemers , perpetuate a lie to delude others and themselves . If online interaction becomes a “cheating culture” ( Callahan , 2004 ) it will collapse , as this is not and never has been sustainable . Inevitably , as more people find more ways to use technology for personal profit , the chances of global social collapse increase . Systems based on individual profit within a social context may be ending their useful life , as focusing on reward tokens like money distracts from the real goal of social synergy . If one offers peanuts one gets monkeys , but if one offers honey one gets wasps . The Page 22 theoretical alternative of social environment model , to forget individual profit entirely and go directly to community profit , avoids this side - effect . Instead of bribing individuals to do good with money tokens , just make it easy for them to help others . When technology makes service easy , and outcomes are immediate , the incentive to virtue is that it works . Systems that offer no individual incentives have nothing to steal . If they use no central control mechanisms there is nothing to hijack . If they are transparent then anti - social acts and attempts to hijack or misuse the system become apparent . People who do these things will be shamed . Explicitly denying individual reward diminishes propaganda that offers it . There is no need to pretend that helping society is in people’s interest if it manifestly is . So if this works online , can it work anywhere ? Can people , without central control , create synergy ? Can a transparent society , where public decisions are made transparently , create synergy ? Can systems without reward tokens , run by no - one , visible to all , work ? The Internet says they can , that we needn’t pay people to help each other , and that a community will defend itself if given the means to do so . If so , as socio - technology can learn from physical society , so society can learn from socio - technology . Glossary It may be useful to summarized some of the key terms of the social environment model : 1 . Rule 1 . Competing self - interested individuals evolve competencies ( individual evolution ) . 2 . Rule 2 . Cooperating socialized individuals evolve social synergies ( social evolution ) . 3 . Synergy . The difference between what individuals produce as a social unit vs . independently . 4 . Anti - social acts . Taking individual benefit from society without contributing to its synergy . 5 . Social dilemmas . When individual gains ( Rule 1 ) contradict social gains ( Rule 2 ) . 6 . Social instability . Social systems generating synergy are unstable to anti - social chain reactions . 7 . Social order . That all members of a social group act as one . 8 . Social freedom . That members of a social group are free to act from their own choice . 9 . Social hijack . When leaders hijack a society for their own ends , and maintain control by : a ) Repression : Forcing individuals not to follow Rule 1 . b ) Brainwashing : Convincing individuals to blindly follow Rule 2 . 10 . Social inventions . Ways to combine social synergy , world competence and evolution : a ) Justice : Punish unfair anti - social interactions by state laws , police , and sanctions . b ) Democracy : The group periodically changes its leaders by freely voting . c ) Legitimacy : The allocation of " rights " that are : i . Fair . Individual consequences match the individual contribution ( Rule 1 ) . ii . In the public good : Benefit society as a whole ( Rule 2 ) . 11 . The golden rule . That individuals can freely choose to serve an environment above themselves . 12 . Social environment model . That social units are environments within other environments . 13 . Rule 3 . That free - good - citizens combine Rules 1 and 2 by anchoring one and applying the other : a ) Rule 3a . If social laws are not broken , compete for individual advantage ( markets ) . b ) Rule 3b . If one has free time or money , give to others in the community ( service ) . 14 . Rule 3 ' . Extends Rule 3 to apply to complex , nested social structures . Page 23 15 . Rule merging . That communism and capitalism are extremes , and a hybrid is better . 16 . Social health . The percentage of individuals in a community that freely support social synergy . 17 . Social inflation . If a social unit doesn’t satisfy its environment ' s needs its social tokens lose their external value . 18 . External rectification . When the consequences of a society ' s collective incompetence eventually impact upon individual members directly . 19 . Technology . Magnifies both negative synergies , like spam , and positive synergies , like wikis . 20 . Socio - technology . A social system mediated by a technical system . 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Social capital and economic development : toward a theoretical synthesis and policy framework . Theory and Society , 27 ( 2 ) , 151 - 208 . Wright , R . ( 2001 ) . Nonzero : The logic of human destiny . New York : Vintage Books . \ No newline at end of file diff --git a/silver_data/685c62b5d2aa610f6fd078b744409124db286e95.pdf.txt b/silver_data/685c62b5d2aa610f6fd078b744409124db286e95.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..dd40a78d3f8e41299e33736c9dbfb65abf9cab6d --- /dev/null +++ b/silver_data/685c62b5d2aa610f6fd078b744409124db286e95.pdf.txt @@ -0,0 +1 @@ +Jointly published by Akadémiai Kiadó , Budapest Scientometrics , Vol . 72 , No . 1 ( 2007 ) 117 – 147 and Springer , Dordrecht DOI : 10 . 1007 / s11192 - 007 - 1700 - 5 Received November 16 , 2006 Address for correspondence : A LAN L . P ORTER Technology Policy and Assessment Center , Georgia Tech , Atlanta , GA ( USA ) E - mail : alan . porter @ isye . gatech . edu 0138 – 9130 / US $ 20 . 00 Copyright © 2007 Akadémiai Kiadó , Budapest All rights reserved Measuring researcher interdisciplinarity A LAN L . P ORTER , A LEX S . C OHEN , J . D AVID R OESSNER , M ARTY P ERREAULT * National Academies Keck Futures Initiative ( NAKFI ) , Irvine , CA ( USA ) We offer two metrics that together help gauge how interdisciplinary a body of research is . Both draw upon Web of Knowledge Subject Categories ( SCs ) as key units of analysis . We have assembled two substantial Web of Knowledge samples from which to determine how closely individual SCs relate to each other . “Integration” measures the extent to which a research article cites diverse SCs . “Specialization” considers the spread of SCs in which the body of research ( e . g . , the work of a given author in a specified time period ) is published . Pilot results for a sample of researchers show a surprising degree of interdisciplinarity . Background Research spirals inward ; science cascades outward . This dual image supports a vital realization – scientific advance requires knowledge transfer among researchers who may focus tightly on extremely circumscribed research issues . The typical research * Alan Porter is an Evaluation Consultant with the National Academies Keck Futures Initiative ( NAKFI ) , and he co - directs the Technology Policy and Assessment Center , Georgia Tech , and is Director of R & D , Search Technology . Alex Cohen , Evaluation Research Associate / Programmer , and Marty Perreault , Program Director , are staff with the U . S . NAKFI . David Roessner is the NAKFI Senior Evaluation Consultant , and also co - directs the Technology Policy and Assessment Center at Georgia Tech and is with SRI International as well . Address correspondence relating to the paper to Alan Porter : alan . porter @ isye . gatech . edu . ; correspondence pertaining to the NAKFI to Marty Perreault , the Program Director : MPerreault @ nas . edu . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 118 Scientometrics 72 ( 2007 ) investigation burrows into detail , cumulating information , probing for nuances . Once completed , the challenge shifts abruptly to communicating the research findings . Research knowledge needs to be suitably conveyed to others working on the same immediate problem – and to a wider circle of colleagues working on related problems – and beyond , to those who may gain usable knowledge for different applications from the findings . Go back for a second to the time one of us ( Porter ) was working on his dissertation in Psychology ( 1970 – 1972 ) . Most scientists ( using the term inclusively to cover social scientists , engineers , and other researchers ) published in the same half - dozen or so journals that they regularly read . Publication processes might take a couple of years . The limited quantity of information that one could digest prompted keeping close track of developments only in one’s immediate research area . One didn’t keep up with the Psychology literature ; one followed developments in the study of memory , favoring particular organisms as subjects , for certain memory forms , affected by particular stimuli & conditions , etc . The notion of a “research trail , ” narrowly tracked , made sense . At its best , this efficiently directed researcher efforts to resolving the problem at hand . At its worst , this led to inbred patterns of research leading to no lasting value . 1 The inherently opposing tendencies – focus vs . outreach – now play out in a richer context than in earlier scientific eras . Wider and faster information access is the pivotal change force . Electronic databases , the internet , and networked computers , accessed daily by essentially every scientist in the world , provide dynamic research knowledge resources . Yet , scientific norms suited to earlier eras linger . The scientific disciplines that arose in the 19 th and 20 th centuries still dominate academic life ( somewhat less so for researchers in government or industry ) . One’s professional identity , institutional home ( department ) , and external peer group ( professional societies ) are discipline - centered . However , even in the early 1970’s , the Psychology Department at UCLA did not reflect a unified field . Rather , it was home for , among others , clinical psychologists and neuroscientists who did not share the same paradigm , subject matter , or research methods . The discrepancies between disciplinary roots and research realities loom large . In the 1970’s and 1980’s interest in interdisciplinary research processes picked up momentum . The notion was to identify and overcome barriers to more effective research knowledge transfer across domains . Good insights into issues , influences , and mechanisms emerged . 2 For whatever reasons ( especially decline of research funding ) , the study of interdisciplinary research processes waned just as funding of major interdisciplinary units expanded ( e . g . , U . S . National Science Foundation Engineering Research Centers and Science & Technology Research Centers programs ) . Moving to the present , we find explosive growth in emerging research arenas , including genetics & biotechnology , information science & technology , and advanced materials and nanotechnology . Obviously these research frontiers pay little heed to 19 th A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 119 century disciplinary boundaries . How can we expedite progress in research and development ? Various parties at interest see the lowering of disciplinary barriers as vital . This has triggered renewed interest in promoting interdisciplinary research and its counterpart , interdisciplinary teaching . In particular , our own efforts derive from the National Academies Keck Futures Initiative ( NAKFI ) – a $ 40 million , 15 - year program to boost interdisciplinary research in the U . S . [ www . keckfutures . org ] . NAKFI evaluators have dual interests in measuring interdisciplinarity . At the macro scale , we seek to estimate and track the extent of interdisciplinary research ( “IDR” ) in the U . S . At the micro scale , we want to evaluate whether NAKFI conferences and seed grant programs increase the interdisciplinarity of participants’ research ( and teaching ) . But as Morillo et al . note , “we do not have appropriate indicators to measure interdisciplinarity . ” 3 This paper presents our approach to measuring how inter - disciplinary a particular body of research is – whether that of one researcher , a group of researchers , or a research domain . Premises in measuring interdisciplinarity What is interdisciplinarity ? Terminology varies and definitions abound . We apply the following definition , based on a National Academies report : 4 Interdisciplinary research ( IDR ) is a mode of research by teams or individuals that integrates • perspectives / concepts / theories and / or • tools / techniques and / or • information / data from two or more bodies of specialized knowledge or research practice . Its purpose is to advance fundamental understanding or to solve problems whose solutions are beyond the scope of a single field of research practice . Examples of bodies of specialized knowledge or research practice include : low temperature physics , molecular biology , developmental psychology , toxicology , operations research , and fluid mechanics . As Morillo et al . state : 3 “we consider ‘multidisciplinarity’ as a basic situation in which elements from different disciplines are present , whilst ‘interdisciplinarity’ is a more advanced stage of the relationship between disciplines in which integration between them is attained . ” Integration is the key concept here – distinguishing the “seamless cloth” of IDR , from the “patchwork quilt” of multidisciplinary research , and the more restricted focus of disciplinary research . We don’t emphasize the creation of new “interdisciplines” per se – i . e . , transdisciplinary research . Elsewhere we review earlier findings on IDR , explore these distinctions , and consider the variety of related causes and effects . 2 , 5 A . L . P ORTER et al . : Measuring researcher interdisciplinarity 120 Scientometrics 72 ( 2007 ) We focus first on the body of research of a given researcher . Specifically , we want to compare the outputs of Researcher A for a time period ( e . g . , 3 years ) just before participating in a NAKFI program to that of his / her work in a corresponding time period after . That said , the main exemplars of that body of research will usually be scientific papers . In most research arenas , journal articles and / or conference papers are important . In others , patents are the exemplary outputs . And in some arenas , various other forms reflect the essential outputs of research activity : reports ( sometimes confidential , sometimes in “gray literatures” ) , software , and process improvements ( perhaps kept as trade secrets ) . We acknowledge that research is not solely measured by its outputs . Interdisciplinarity can also be gauged in terms of : proposals ( e . g . , topical emphases , collaboration ) or projects ( e . g . , teaming arrangements , mixed funding sources ) . In addition , assessment may focus on other interdisciplinary professional facets : teaching ( e . g . , cross - departmental , co - teaching ) or affiliation ( e . g . , joint appointments in multiple departments , participation in organized research units ) . But for now , our attention is directed to research outputs in the form of sets of papers . We will draw heavily on citations ( referencing ) among papers ; this can be considered a form of research outcomes . Our strategy to measure interdisciplinarity ( elaborated in succeeding sections ) is based on the following premises : • Integration of knowledge not routinely found within a research field equates to greater interdisciplinarity . • Such integration can be measured by examining the spread of a paper’s references . • We devise measures by relating papers’ cited journals to their corresponding Subject Categories ( research fields , as assigned by Thomson Institute for Scientific Information – ISI – accessible through their Web of Knowledge – WoK – site ) . • The interdisciplinarity metrics take into account the inter - relatedness of Subject Categories . • Additional measures can be derived from the degree to which cited Subject Categories relate to that of the journal of the paper , and from the spread of publications across Subject Categories . Empirical aspects in measuring interdisciplinarity Many information resources are interesting candidates in gauging interdisciplinarity . We briefly note a few . Our focus on researchers leads us to begin with their CVs . Recent studies of science highlight the wealth of information to which CVs provide entrée . 6 On a pilot group of researchers from two research universities , we explored A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 121 whether some aspects , including educational background ( e . g . , whether cross - disciplinary or not ) and position , correlated with interdisciplinary publication . For example , educational background from B . S . to Doctorate to current position for 6 of the 17 researchers was cleanly disciplinary ( e . g . , all degrees and affiliation in the same field ) . For the other 11 , it was more diverse ( e . g . , degrees in Chemistry and Physics , current affiliation in Computer Science / Math department ) . Surprisingly , papers by those with relatively pure disciplinary backgrounds seemed at least as interdisciplinary . In addition , departmental affiliation aligns with the top ISI publication Subject Categories for only about 10 of 17 . So , for now , we set aside these “influence factors” as interest - ing in their own right , but not a strong indicator ( or predictor ) of IDR research output . Let’s turn to research papers . These are probably the most accessible , most clear - cut entities on which to gauge interdisciplinarity of the underlying research . Papers can be treated at various degrees of detail , including : • Bare essentials – # , co - authoring , where published ( journal ) • Key text – add information from titles , abstracts , keywords • Full text – add information from the entire paper We set aside co - authoring for now . Those who key on collaboration as the essence of IDR would likely avail themselves of co - authoring information as the primary measure . We key on integration , and note that this can be accomplished by a single author as well as by a team ; so , given our conceptualization of IDR , co - authoring becomes secondary . We considered trying to categorize papers based on their keywords or classification codes ( e . g . , as assigned by database indexers to identify what the paper addresses ) , as others have done to good effect . 7 In other applications , we have used such information to cluster papers , identify research themes inductively , define research domains , and track R & D advances . 8 , 9 We also have the capability to enrich available keyword sets by extracting noun phrases from titles and abstracts – an approach Kostoff and colleagues have used to locate papers . 10 We could also use keyword sets ( ontologies ) imported from outside sources . 11 Such approaches would offer advantages in assessment of papers and patents wherever they appear or are indexed . That said , we presently back off from these approaches because of their complexity and cost . Much work would be needed to devise a viable methodology to gauge the interdisciplinarity of sets of papers reflecting diverse research areas . In contrast , the approach we pursue is restricted to content indexed by one key information resource – ISI’s WoK . Our approach relies on the WoK , which includes the Science Citation Index , Social Science Citation Index , and the Arts and Humanities Citation Index . For a given researcher , one can search WoK in two ways : • general search – retrieves summary information on papers by that researcher , including co - authors , co - author affiliations , journal , subject A . L . P ORTER et al . : Measuring researcher interdisciplinarity 122 Scientometrics 72 ( 2007 ) category , year , and references ( first author , year , journal , volume , first page ) • citation search – retrieves summary information on each paper that CITES papers authored or co - authored by our researcher , including authors , title , journal , subject category , year , keywords , and cited references Key limitations in using WoK include : • it does not generally include conference papers and does not include patents at all [ there is a separate ISI database containing conference papers ] • coverage varies considerably among research areas • keywords are not well controlled Our pilot assessment concluded that coverage was quite good , but not uniform across a wide range of science , engineering , and bio - medical domains . We compared publications between CVs and search results in WoK . This is not as exact as it might seem as CV inclusion is not always complete and variations in citation leave some ambiguity . We deemed coverage to be excellent or very good for 12 of 17 researchers in our pilot sample . We consider coverage “good” for 4 others ( WoK containing from 65 - 85 % of their CV’s journal papers ) . We assessed coverage as “bad” for 1 Sports Medicine specialist . Medical research was quite well - covered ( 2 judged “excellent” and 1 “good” ) . Engineering is not covered as extensively by WoK , but it is acceptable – 3 judged “very good” ( Chemical Engineering , Biomedical Engineering , Industrial Engineering ) and 2 “good” ( Electrical and Mechanical Engineering ) . These pilot results caution against cross - field comparisons without explicit checking of WoK coverage . We recognize that interdisciplinarity is not a unitary construct with a clear , single indicator . At one extreme , one can conceive of researchers who always publish in the same research field . Even among such scientists , we would like to differentiate those who integrate source materials from multiple fields from those who draw on a narrower range of sources . At the other extreme , imagine researchers who publish each of their papers in a different field . Again , we might deem some of them as more interdisciplinary than others . For instance , one of the 17 pilot researchers who kindly provided CV information to us publishes in several research fields ( Table 1 shows the Subject Categories represented ) . Perusal of these fields suggests strong common threads of this polymer chemistry work among materials science , chemistry , physics , and more or less related fields . Those other fields include : crystallography , which could reasonably reflect techniques being applied ; biomedical engineering , which could be an application domain ; education , which could dovetail with teaching of the principles being researched . Such empirical findings suggest the desirability of a metric to get at how closely the research fields represented are associated . Table 1 also includes information on the Subject Categories ( “SCs” ) cited ( referenced ) by this researcher’s 116 papers ( published from 1988 – 2004 ) and the 876 A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 123 articles citing these papers ( as of Jan . 14 , 2006 ) . Note the Sums . The Sum of SCs of the papers equals 122 % of the number of papers ; that is because many journals are associated with more than one SC . The 116 papers reference 2172 items – not all of which are journal articles covered by Web of Knowledge . However , the total number of cited SCs is a bit larger ( 2250 ) because some journals are associated with multiple SCs . Table 1 . Distribution of one researcher’s 116 papers across research fields Subject Category ( SC ) # of papers published in journals associated with this SC # of cites BY these papers to papers in journals associated with this SC # of cites TO these papers by papers in journals associated with this SC CHEMISTRY , MULTIDISCIPLINARY 56 369 138 POLYMER SCIENCE 26 451 127 CHEMISTRY , PHYSICAL 14 226 268 MATERIALS SCIENCE , MULTIDISCIPLINARY 13 193 117 PHYSICS , CONDENSED MATTER 6 123 45 CHEMISTRY , ORGANIC 5 142 70 EDUCATION , SCIENTIFIC DISCIPLINES 4 27 3 CHEMISTRY , MEDICINAL 3 26 11 CRYSTALLOGRAPHY 3 38 40 CELL BIOLOGY 2 44 3 ENGINEERING , BIOMEDICAL 2 59 27 MATERIALS SCIENCE , BIOMATERIALS 2 58 27 CHEMISTRY , ANALYTICAL 1 62 61 CHEMISTRY , APPLIED 1 4 8 ELECTROCHEMISTRY 1 75 53 ENGINEERING , CHEMICAL 1 30 10 MATERIALS SCIENCE , TEXTILES 1 0 3 PHYSICS , ATOMIC , MOLECULAR & CHEMICAL 1 26 6 MULTIDISCIPLINARY SCIENCES 0 57 7 MATERIALS SCIENCE , COATINGS & FILMS 0 33 5 PHARMACOLOGY & PHARMACY 0 32 7 BIOCHEMISTRY & MOLECULAR BIOLOGY 0 29 15 PHYSICS , APPLIED 0 24 27 CHEMISTRY , INORGANIC & NUCLEAR 0 18 15 PHYSICS , MULTIDISCIPLINARY 0 12 1 MEDICINE , RESEARCH & EXPERIMENTAL 0 11 0 NEUROSCIENCES 0 10 4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY 0 9 6 BIOPHYSICS 0 2 14 PSYCHIATRY 0 3 6 Sum ( All occurrences for the 116 papers ) 142 2250 1124 A . L . P ORTER et al . : Measuring researcher interdisciplinarity 124 Scientometrics 72 ( 2007 ) Notes to table : These do include self - citations ( by authors or co - authors to their own work ) . The last column shows the SCs associated with journals that cite the 116 papers . Again , one citing journal could be associated with more than one Subject Category ( hence the tally of citing SCs – 1124 – exceeds the 876 citing articles ) . Table 1 shows all the Subject Categories in which he publishes and all of the SCs that he cites more than 5 times , or that cite his work more than 5 times . Table 1 indicates that our researcher draws on research intelligence from much the same fields in which he publishes – note that the four dominant SCs are the same , although the order changes ( he mostly references Polymer Science but publishes mostly in Multidisciplinary Chemistry ) . His work , in turn , mainly is used by these same fields ; again the same four SCs dominate the “cites to , ” but the order shifts again , with Physical Chemistry the main user of his findings . Note some of the differences too . Other Subject Categories also show up with quite different emphases . He substantially references work in Organic Chemistry and Condensed Matter Physics in which he rarely publishes , but these same SCs also heavily cite his work . He draws upon Cell Biology and Multidisciplinary Sciences , but these rarely cite his work . [ Multidisciplinary Sciences is a quite special SC as it includes the leading cross - disciplinary outlets of Science , Nature , and Proceedings of the National Academy of Sciences ( PNAS ) . ] Biophysics is an SC that cites his research in which he has not published and rarely cites . We explored classifying specific Subject Categories into broader areas ( e . g . , Engineering , Life Sciences ) . There are many different area classifications . In this pilot work , we tried 13 broad research areas . Of those , this researcher published in 7 ( Chemistry & Materials Science ; Physics ; Education ; Medical , Health & Veterinary Sciences ; Technology ; Life , Bio & Agricultural Sciences ; Engineering & Energy ) . This gives us pause – does one coherent research stream that touches into 7 broad research areas reflect more or less interdisciplinarity than another which reflects dissociated interests ? The pilot findings suggest that it would be preferable to develop similarity measures among the Subject Categories rather than try to categorize SCs into broader areas . Based on our assessment of various approaches to measure interdisciplinarity , and these pilot results , we determined to pursue use of the ISI Subject Categories as basic units of analysis . The SCs are used by ISI to classify journals . The process entails a combination of 1 . empirical information on journal - to - journal citation patterns , with 2 . editorial judgment as to journal coverage . ISI’s WoK website , under Journal Citation Reports , provides descriptions of each SC , for instance : Cell Biology Category Description : Cell Biology includes resources on all aspects of the structure and function of eukaryotic cells . The principle characteristic of resources in this category is an emphasis A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 125 on the integration at the cellular level of biochemical , molecular , genetic , physiological , and pathological information . This category considers material on specific tissues , differentiated as well as embryonic . This SC contains 155 journals as of November , 2005 ; however the cumulative thesaurus we are using ( from ISI ) contains 189 journals that have been included in Cell Biology ( e . g . , journal names change ) . The SCs depict an ever - evolving set of research fields . These slowly change over time to reflect current research practices . ISI kindly provided us a thesaurus of 244 current and recent SCs , as of early 2005 . This thesaurus associates the journals included in WoK to particular SCs . The journal set also changes over time . Each journal is associated with between 1 and 6 SCs , distributed as follows for the 10 , 550 journals in this electronic thesaurus : • 6 SCs – 1 journal is associated with this many SCs • 5 SCs – 25 journals • 4 SCs – 198 journals • 3 SCs – 877 journals • 2 SCs – 3032 journals • 1 SC – 6417 journals So , some 39 % of journals included in WoK are associated with > 1 SC . Those studying scientific process have often used SCs to classify scientific activity . Cunningham profiled British science using ISI information . 12 His analyses warn that coverage of various research areas by SCs is not uniform ( see also Table 2 ) . Table 2 . Subject category file characteristics WoK VP Files India US US Temporal coverage 2004 publication year March 30 , 2005 , publication week March 30 , May 14 , & June 11 , 2005 , publication weeks fused Publications 20 , 558 8 , 484 23 , 381 SCs ( for those publications ) 194 203 239 Cited References 410 , 669 155 , 798 596 , 194 Cited Journals ( distinct sources , not all are journals per se ) 63 , 336 33 , 291 95 , 526 SCs cited ( of 244 total SCs ) 235 236 244 SCs cited in > = 300 records 94 34 122 SCs cited in > = 100 records 142 92 181 SCs cited in > = 30 records 179 174 220 SCs cited in > = 10 records 199 215 232 SC coverage favors research domains with well - established journal sets , especially in the physical and mathematical sciences . He notes that other categorizations ( namely that of SPRU , the Science Policy Research Unit at the University of Sussex , where he was A . L . P ORTER et al . : Measuring researcher interdisciplinarity 126 Scientometrics 72 ( 2007 ) doing this research ) also cover fields unevenly . For our purposes , the SCs offer the best accessible coverage of which we are aware . Morillo and colleagues have mapped links among SCs based on shared journal assignments 3 and recently studied the relative interdisciplinarity of newly emerged versus older SCs . 13 New SCs had a greater percentage of journals assigned to multiple SCs , higher percentage of links outside their Broad Area ( they used 9 broad areas ) , stronger links , and more diverse links . Moya et al . used co - citation of SCs as the basis to map their interrelationships . 14 Boyack et al . work with journal inter - associations , using SC categorization as a validity check , in mapping science . 15 SCs hold appeal in their relative coherence . As illustrated by “Psychology , ” disciplines are not unitary . The granularity of the SCs is highly consistent with our definition of IDR with its illustrative “bodies of specialized knowledge or research practice . ” Of the 244 SCs with which we are dealing , 9 include the term , “multidisciplinary” and 3 include “interdisciplinary . ” Most of these , like “Chemistry , Multidisciplinary , ” seem to behave much like the other SCs . One special SC is “Multidisciplinary Sciences . ” Its 69 journals include Proceedings of the National Academy of Sciences , USA ; Science ; and Nature . These broad - scope journals include papers from multiple fields , but this does not imply that any given paper is inherently multi - or interdisciplinary . 5 In its interrelationships with other SCs , Multidisciplinary Sciences acts much like other high frequency biomedical SCs ( e . g . , Biochemistry ) . Here are ISI descriptions of two such multidisciplinary or interdisciplinary SCs : Mathematics , Interdisciplinary Applications [ # of journals in JCR , as of Nov . , 2005 = 52 ] Category Description : Mathematics , Interdisciplinary Applications includes resources concerned with mathematical methods whose primary focus is on a specific non - mathematics discipline such as biology , psychology , history , economics , etc . Resources that focus on specific mathematical topics such as differential equations , numerical analysis , nonlinearity , etc . , are covered in the Multidisciplinary Sciences [ # of journals in JCR , as of Nov . , 2005 = 45 ] Category Description : Multidisciplinary Sciences includes resources of a very broad or general character in the sciences . It covers the spectrum of major scientific disciplines such as Physics , Chemistry , Mathematics , Biology , etc . Nature and Science are the preeminent resources in this category and serve as typical examples . The Web site of the National Science Foundation is a good example of a web resource included in this category . Some specialized resources that have a wide range of applications in the sciences also may fall under this category . The journal Fractals – Complex Geometry Patterns and Scaling in Nature and Society would be an example of such a resource . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 127 We distinguish three different ways to consider Subject Category distribution pertinent to measuring interdisciplinarity : • Publication spread across SCs – How many different SCs are represented by the set of papers in question ? What is the frequency distribution across these SCs ? • References ( “citations by” ) spread across SCs – Either examined per paper or for the entire set of papers • Citations to the paper ( s ) spread across SCs – Either examined per paper or for the entire set of papers Reverting to our integrative definition for IDR , the distribution of references holds primacy . Diversity in publication certainly reflects diversification , but it may or may not indicate integration of intellectual content . Diversity of references cited by a paper logically seems the best gauge of intellectual integration . That is , for each given paper , examine the SCs that it references . Papers that cite more discrete SCs pertaining to some unspecified mix of substantive topics , methods , and / or concepts are presumed to be more interdisciplinary . We acknowledge that a wide spread of cited references does not unequivocally demonstrate intellectual integration . Eto notes that disciplinary scientists could collaborate , each bringing in purely disciplinary contributions . The sum of these , reflected in the spread of cited SCs could appear quite diverse , yet the collaborators may not have truly integrated this knowledge . 16 Nonetheless , we find “Integration” as reflected in diversity of cited SCs to be a compelling IDR metric . A counterbalancing perspective , to which we do not ascribe , is that IDR requires collaboration . On the other hand , we believe that much IDR does reflect cross - disciplinary collaboration . Generating a “Science Matrix” Our strategy to determine how closely SCs relate to each other is first to generate a large , representative sample of scientific publishing . We then ascertain the extent of co - citation of particular SCs [ as did Moya et al ] . 15 We have compiled three one - week samples of papers ( by searching the “most recent week” on WoK , as of March 30 , May 14 , and June 11 , 2005 , for papers with at least one U . S . author affiliation ) . We have compared results with a one - year sample of WoK papers with an author affiliation from India . For these samples we examine SCs cited within each given paper . We then calculate measures of association for the SCs across the respective sets of papers . We have explored Pearson correlation , but favor the closely related cosine measure . In text analyses , cosine generally fares slightly better than correlation . 17 Klavans and Boyack , working with co - citation measures ( for mapping of science generally ) , favor cosine adjusted for expected value based on frequencies . 18 Our resulting SCxSC cosine matrix A . L . P ORTER et al . : Measuring researcher interdisciplinarity 128 Scientometrics 72 ( 2007 ) provides the values we use in all further analyses . The cosine can be calculated from observations ( counts ) x , y as : ( ) ∑ ∑ ∑ i i i i i i i y x y x 2 2 We believe these values of SC - SC interrelationship are reasonably robust . Table 2 shows selected U . S . and India sample characteristics . The rationale is that these are reasonably representative , large science samples . Indeed , since we are basing SC - SC relationship upon co - citation of Subject Categories within papers , the fact that the total sample is not perfectly representative should not pose major problems . We do need to think about attendant issues – e . g . , the one - week samples could have clumps – special issues or uneven indexing ( e . g . , handling multiple issues of certain sources as they come available to WoK ) . WoK does not generally cover conference proceedings , but we note some such materials . We also expect SC - SC associations to evolve , so any resulting “science map” would shift over time , requiring periodic updating . Some SCs are heavily represented , while others are very lightly or not covered . Of the 244 SCs , 50 % are cited 300 or more times , and 90 % are cited at least 30 times , in the 3 - week USA sample , upon which we base our calculations . Our interests reside mainly with research in the sciences , medicine , and engineering , with less interest presently in the social sciences and little in the humanities . Coverage in the India and U . S . datasets mirrors these emphases well ( i . e . , it is thinnest in the arts and humanities ) . For instance , in matching the USA and India results , we checked for SCs included in one but not the other . Present for India , but not the U . S . - March 30 , were : Architecture , Folklore , and American Literature . Present for the U . S . - March 30 , but not for India , were : Criminology , Industrial Relations , Medieval & Renaissance Studies , and Religion . The combination of U . S . for three weeks cited all the SCs . Our immediate focus is to assess the interdisciplinarity of U . S . authors , so we use the U . S . SCxSC cosine matrix for the three sample weeks combined . We compared a range of SC intercorrelations among the US and India samples , and these generally corresponded very well . We examined correlations and cosine values . Table 3 shows cosine values for 4 high frequency SCs to give a sense of how these range for U . S . and India samples . We selected 26 SCs representing a range of research areas . We aligned the SCxSC cosine matrices , calculated using for India and for the combined 2 - week ( Mar . 30 and May 14 ) U . S . samples . The correlations of these cosine values correspond well ( averaging 0 . 96 ) . For instance , the “Biochemical research methods” correlation of 0 . 98 is based on comparing the pairs of cosine values , U . S . and India , for this SC with each of the other 243 SCs . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 129 Effectiveness in associating journals with SCs depends on the development of our thesaurus . The initial thesaurus was provided by ISI in early 2005 . Its accuracy will drift over time as ISI changes its assignments of journals to Subject Categories and their set of SCs itself evolves . Our thesaurus can continue to improve as we discover additional journal - SC matches . There are over 16 , 000 journal entries ( the thesaurus includes journals as they evolve over time ) and the format of cited journal names differs somewhat from those in the thesaurus . Currently , we have worked it to the point of diminishing returns on the three U . S . week - long searches and the India 2004 search . Table 3 . Illustrative cosine values [ based on extent of papers co - citing SCs ] Table 3A . US ( combined 3 weeks ) # Records Selected Subject Categories Multidisciplinary Sciences Biochemistry & Molecular Biology Medicine , General & Internal 6523 Multidisciplinary Sciences 1 0 . 695 0 . 187 5086 Biochemistry & Molecular Biology 0 . 695 1 0 . 141 4117 Medicine , General & Internal 0 . 187 0 . 141 1 3922 Cell Biology 0 . 641 0 . 827 0 . 131 Table 3B . India 2004 # Records Selected Subject Categories Multidisciplinary Sciences Biochemistry & Molecular Biology Medicine , General & Internal 5293 Multidisciplinary Sciences 1 0 . 579 0 . 179 4345 Biochemistry & Molecular Biology 0 . 579 1 0 . 163 2061 Medicine , General & Internal 0 . 179 0 . 163 1 1969 Cell Biology 0 . 496 0 . 732 0 . 139 We have worked through multiple iterations : • Apply our current best “Find - Replace” ( F - R ) thesaurus in VantagePoint to the cited journals from a WoK search ; apply the F - R thesaurus also to the Journal - Subject Category ( J - SC ) thesaurus . The objective is to match journal nomenclature ( that is not consistent to begin ) . • Apply the J - SC thesaurus to the WoK search results ( journals ) . • Review the journals not assigned to an SC ; use this information to enhance the F - R and J - SC thesauri . For the combined file of 23 , 381 U . S . WoK publications for three weeks in 2005 ( March 30 , May 17 , and June 11 ) , we have 95 , 526 “cited journal” names ( after A . L . P ORTER et al . : Measuring researcher interdisciplinarity 130 Scientometrics 72 ( 2007 ) cleaning ) . These include huge numbers of citations to articles in certain journals ( e . g . , PNAS , Nature , Science ) on down through single papers citing some 72 , 000 sources . It is a highly skewed distribution ! Most of the singly cited sources are not even journals . For instance , some singly cited items : • 000104 AIAA ( conference paper ) • 0092 USGS ( presumably a government report ) • 145 U Suss I Dev Stu ( possibly a thesis ) • Acta Crystallogr A 6 ( a variant on a recognized journal title ) • in press Infancy ( not yet published ) • Indian J Fisheries ( journal not included in WoK ) In general , unless there is a clear match between the “cited journal” name and a journal in our J - SC thesaurus , we leave it out . For NAKFI purposes , we have settled on use of the U . S . SCxSC interrelationships ( cosine matrix ) as we are focusing on U . S . - authored research publications . As noted above , the U . S . SCs correspond highly with those for India . We don’t think that American research is very different from research authored by others . The three weeks provide a sizeable sample to assure reasonable robustness . Table 2 gives some summary distribution characteristics for the 3 - week US sample ( 23 , 381 papers ) . For the Cited Journal SCs , all 12 with fewer than 10 occurrences fall in the Arts and Humanities . These do not pose serious concerns for NAKFI assessments . Publication SCs coverage is based on a smaller sample by a factor of 25 ( i . e . , the average U . S . journal paper cites 25 references ) . Nonetheless , the coverage is solid – 239 of 244 possible SCs are present . Of those , 222 appear in at least 10 records ; 191 in at least 30 records . Others map science in various ways , for various purposes . For instance , Klavans and Boyack are interested in an overarching map per se , to show how various fields piece together . 15 We seek to measure the degree of association among SCs , so have generated a particular form of “science map” – actually a 244 x 244 matrix of cosine values for the Subject Categories based on co - citation of SCs within papers ( Table 3a illustrates a small portion ) . The next section incorporates this SC similarity matrix in assessing interdisciplinarity . * Methods : Assessing the interdisciplinarity of sets of papers Setting out the steps in the calculations clarifies what is going on : 1 . The WoK abstract record for a given paper ( by one of the researchers under scrutiny ) lists each of its cited references . Most references are to journal articles and those are the only ones we are analyzing . * The matrix is available as a VantagePoint file for analyses . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 131 2 . For the paper itself , and for each of the paper’s references , we identify the journal in which it appeared . 3 . We then apply the dual thesauri introduced previously [ first , “Find and Replace” to match journal name abbreviation variations ; second , “Journal – SC , ” to associate journals to SCs ] . 4 . We use the combined U . S . SCxSC cosine matrix to gauge the degree of association among the resulting sets of SCs . 5 . We compute measures of interdisciplinarity based on a researcher’s set of papers . For each paper we compute interdisciplinarity measure ( s ) based on the distribution of SCs . We derive measures based on both the spread of SCs referenced , and on the SC ( or SCs ) for the journal in which the paper was published . We use the general U . S . SCxSC cosine values to measure the relatedness of the resulting SC sets . 6 . We compute measures for a given researcher based on the set of papers examined . The steps yield a set of SC instances for a given paper . Some references will generate no SCs , either because there is no journal ( e . g . , reference is to a thesis , report , or patent ) , the journal is not indexed by WoK , or the Journal - SC thesaurus has failed to capture a link that actually exists on WoK . We have extensively examined results on the U . S . and India samples to capture as many journals as feasible . Most of the remaining misses are attributable to journal name variants . We take a conservative approach , not assigning a journal to an SC unless we are quite sure that WoK does so . Note that this assignment of a reference is based on its journal . Alternative measures , in principle , could include examining its terms ( in its title , keywords , and / or abstract ) to assign its research field . This is not practical in that WoK does not provide any of that information for the “cited references . ” Note also that these data combine SC counts arising two ways : 1 ) one journal may be associated with multiple SCs , or 2 ) different SCs arise from different references . After considerable reflection , we elect not to distinguish these . Our rationale is that both mechanisms suggest inclusion of elements of the knowledge resident in that SC in the parent paper that is referencing them . Scaling issues arise . The average paper references on the order of 20 sources . Suppose one paper cites only 5 , while another cites 100 . Should we tally absolute SC instances or proportions ? A related issue concerns the form of the distributions observed . As with most bibliometrics , distributions tend to be highly skewed ( Table 1 is typical ) . One often sees very few high frequency entities and many low frequency ones . This can make categorization of “all” instances extremely costly . Our pilot findings suggest using proportions and not worrying about classifying all low frequency instances . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 132 Scientometrics 72 ( 2007 ) Experimental samples To try out these new metrics , we generated 3 separate researcher samples – A , B , and C : A . Pilot Sample : We sampled faculty from one research university seeking to span the Physical Sciences , Biomedical Sciences , Engineering , Computer Science & Math . We obtained CV’s from 14 volunteers ; then searched Web of Knowledge ( WoK ) for their career publications . To include some Medical researchers , we added 3 faculty from a medical school , for a total of 17 . B . Applicants : Researchers who applied to attend interdisciplinary NAKFI conferences on signaling , nanoscience , or genomics – a total of 20 . C . AAU : To counterpoint possible institution - specific biases of A and the interdisciplinary inclinations and topical foci of B , we added another broad sample selected from the Association of American Universities . We again sought to span the Physical Sciences , Biomedical Sciences , Engineering , Computer Science & Math . We identified candidates deliberately ranging from Assistant to full Professor . We selected 2 faculty from Brandeis ; 3 each from Brown , Caltech , Carnegie Mellon , and Columbia ; and 1 from Cornell to give us 15 . Each is from a different department , and we favored people with less - common names to facilitate WoK searching , for those with at least 5 papers since 2001 . Our main target in developing IDR indicators is to assess whether NAKFI experiences ( special conferences , seed grants ) alter the participating researchers’ interdisciplinarity . We expect to focus on time periods of about 3 years pre - and post - . So , to determine whether the indicators are sensitive enough to pick up changes , we conducted these explorations on time - slices of the researchers’ publications . Some analyses using all the indicated time periods include as large a sample as reasonable , but other results presented here focus on the more comparable 3 - year periods : A . Pilot Sample : Focus on 2002 – 2004 ; additional analyses use results for the same researchers for 1999 – 2001 B . Applicants : 3 - year periods leading up to the NAKFI conference for which they applied : 5 Signaling Conference applicants ( 2001 – 2003 ) ; 4 Nano Conference applicants ( 2002 – 2004 ) ; 8 Genomics Conference applicants ( 2003 – 2005 ) C . AAU : Focus on 2002 – 2004 ; additional analyses use results for the entire 2001 – 2005 period . In additional calculations , for Sample A we also looked at papers published in the 1999 – 2001 period . This allowed us to see the extent of variation within individuals over successive 3 - year periods . In our “max” calculations , we combined 1999 – 2004 for a A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 133 6 - year span . For Sample B , we note that 2005 publications are somewhat shortchanged as the year was not complete . For Sample C , in our “max” version , we used their publications for 2001 – 2005 ( with 2005 not complete ) . Our indicator assessments are not sensitive to these variations in time period . We exclude individuals for whom we had fewer than 3 papers with at least 3 Cited SCs each . Our measures are inherently stochastic , so drawing conclusions on interdisciplinarity on tiny individual samples seemed inadvisable . For Sample A we required individuals to meet this test for both time periods ; only 11 of 17 did so . For the 20 Sample B researchers , 17 met the criteria . And for Sample C , where we screened for those with at least 5 papers ( 2001 – 2005 ) , only one failed the criteria for the 3 - year period ( 2002 – 2004 ) . We ran our analyses on each of the three sets separately , then combined . The results presented focus on the resulting composite set of 42 ( 11 + 17 + 14 ) researchers for the most comparable , 3 - year time periods . In “max” sample calculations we show 43 researchers ; this retains the one AAU person who has sufficient papers when we take the available 2001 – 2005 span . We apologize for this bit of complexity , but that one person turns out to be an outlier – the “Purist” profiled below . In studying our measures , sometimes we want to see the fullest range sensible ; other times we want the most comparable time slicing . Paper level metrics Our primary target as an IDR indicator is Integration ( “I” ) . To what extent does a paper indicate integration of distinct research knowledge in citing multiple Subject Categories ? As with much of this exploratory development , we have tried many variations on this and additional measures . This section will also address Reach ( “R” ) . The next section , which addresses measures at the Researcher level , introduces Specialization ( “S” ) . We introduce the “I” measure with a simple computational illustration . The Appendix gives formulas and notes ongoing experimentation with those . Here we describe how we arrive at the metrics . Suppose a paper cites 4 SCs to different degrees ( Table 4 ) . These are the same 4 SCs whose degree of association using the cosine measure appears in Table 3A . Our calculations follow the 6 steps listed under Methods . Recall that some journals are associated with more than one SC . So the results below could reflect 10 references , each to a journal linked to a sole SC . Or , they might reflect 1 reference to a journal that ISI classifies in all 4 SCs , plus an additional 2 references to a journal that is linked to both Biochemistry and to Cell Biology , plus 2 more references just to Biochemistry journals . Our rationale is to pick up discrete indications ( references ) to a given body of research knowledge ( citing a given Subject Category ) , so we tally both forms together . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 134 Scientometrics 72 ( 2007 ) Table 4 . Illustration : Cited Subject Categories for calculation of integration score # of citations Cited Subject Categories ( SCs ) Multidisciplinary Sciences [ # = 1 ] Biochemistry & Molecular Biology [ # = 5 ] Medicine , General & Internal [ # = 1 ] Cell Biology [ # = 3 ] 1 Multidisciplinary Sciences 0 . 01 5 Biochemistry & Molecular Biology 0 . 05 0 . 25 1 Medicine , General & Internal 0 . 01 0 . 05 0 . 01 3 Cell Biology 0 . 03 0 . 15 0 . 03 0 . 09 10 Sum 0 . 10 0 . 45 0 . 04 0 . 09 We calculate Integration as follows : 1 . Weight cells by normalizing the frequency of citations . For instance , the Cell Biology × Biochemistry cell weight = ( 5 × 3 ) / 100 = 0 . 15 . 2 . We include the diagonal cells and the cells below the diagonal ( we do not include cells above the diagonal , to avoid double - weighting interactions among distinct cells ) . We will later adjust the normalization to reflect just the included cells ( sum = 0 . 68 ) . 3 . Multiply the cell weight by the respective cosine value ( Table 3A ) . For instance , any diagonal cell’s cosine value = 1 ( with itself ) . The cosine value for “Cell Biology” × “Biochemisty” = 0 . 827 ( based on the USA sample ) – indicating that these two Subject Categories are closely associated . The cosine value for “Cell Biology” × “Medicine , General and Internal” = 0 . 131 . [ In general , cosine values among our 244 SCs tend to be considerably closer to 0 than to 1 . ] 4 . Sum up the cells included ( 10 cells here ) ; divide by the sum of the included cell weights ( 0 . 68 ) to get the Disciplinary score . [ So if all the cited SCs were either one SC or had cosine values among themselves of 1 , this would be a purely disciplinary paper . ] 5 . Subtract this Disciplinary score from 1 to get the Integration score , “I . ” We explored using a threshold of requiring an SC to have more than a single citation , but decided it was preferable to include these as instances of knowledge being drawn in . Many papers in our sample had many single - citation SCs . Recognize that the use of the cosine measure weighting reflects our understanding that the disparity ( cognitive distance ) among SCs , not just the number of them , matters . We continue to experiment with formulations of Integration and Specialization ( see Appendix ) . We investigated an additional measure that we came to label “Reach . ” We considered combining this with Integration as a composite indicator , but decided to address it separately . We calculate Reach as follows : 1 . Identify the Publication SC ( assume this is singular , for the moment ) 2 . Identify all the Cited SCs , as above in calculating I . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 135 3 . For each Cited SC – multiply the number of times it appears by that SC’s cosine value to the Publication SC 4 . Sum those values for every Cited SC ; divide by the total number of Cited SC occurrences . 5 . Subtract this value from 1 . So , while “I” measures the extent of diversity among the Cited SCs , Reach ( “R” ) measures the degree of distinction between those Cited SCs and the Publication SC . We call it Reach as it taps into the relatedness of the cited references to the research domain of the publication journal . Is the researcher bringing in knowledge from outside that domain ? Calculating R is tricky when the Publication journal is categorized in more than one SC . As an illustration , the journal , Discrete Event Dynamic Systems—Theory and Applications , is part of the core journal set of three SCs : Automation & Control Systems ; Mathematics , Applied ; and Operations Research & Management . So we have 3 Publication SCs to match against the Cited SCs . We generated credible arguments for using : the “Furthest” reach , the “Shortest” reach , and the Average . Results were not very sensitive to the choice . More telling , the correlation between I and R within each of our 3 sample ( maximum data ) sets was 0 . 98 , 0 . 89 , and 0 . 52 . The 0 . 52 correlation increases to 0 . 76 if 1 outlier is removed ( the person with insufficient # of articles to be included in the 3 - year comparisons ) . Figure 1 plots I vs . R for the 42 researchers ( 3 - year time spans ) . The strong association between the two measures is apparent . For most purposes , this means one is largely redundant information . We prefer to focus on Integration ( I ) for its conceptual power – measuring the diversity of the referenced knowledge of a paper . We continue to explore R , particularly as a potential flag when it differs sharply from I ( e . g . , Researchers # 18 and # 20 in Figure 1 ) . The notion of drawing on research knowledge from certain Subject Categories in articles reporting to other SCs is intriguing . I ( and R ) are calculated at the Paper level . For each journal article covered by WoK that cites other journals covered by WoK , we can compute I ( and R ) . Examining the sensitivity of I values , we have decided to exclude articles for which we compute fewer than 3 Cited SC instances . We found that our coverage of the SCs is imperfect . Over time it should improve as our J - SC thesaurus gets better and better by checking which journals are missed and correcting this as experience builds . Were a given article to only reference a single journal ( linked to a single SC ) , one time , we could argue that this should be scored as purely disciplinary . In fact , that article might well reference additional intellectual resources that we do not capture . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 136 Scientometrics 72 ( 2007 ) Figure 1 . Integration vs . reach for 11 researchers These could include cites of journals missed by our thesaurus , journals not covered by WoK , conference papers not covered by WoK , and miscellaneous other sources of knowledge ( e . g . , dissertations , reports , websites ) . We see far greater danger of erring by classifying articles based on 1 or 2 references than in excluding these . Note that we do include articles that cite only a single SC , so long as they do so at least 3 times . [ Such an article would score I = 0 . ] Researcher level metrics Given that a researcher has authored ( or co - authored ) a particular set of papers , say in a given time period , how should we compute I for him or her ? An obvious choice is to take the average of the I values for each included paper . And , indeed , that is what we have determined to do . Before deciding , we did investigate distributional facets . We calculated the Standard Deviation for I for each of our researchers – reflecting the variance in Integration among the papers included . This tended to be relatively consistent across researchers . We also investigated the alternative of focusing on the 50 % highest I values for the researcher . The rationale was that a researcher might well author both disciplinary and interdisciplinary papers . Even as NAKFI seeks to encourage IDR , it does not oppose disciplinary research . So , we might ignore the more disciplinary half of one’s work and assess how far the research extends into interdisciplinarity in the other half of that body of work . We calculated this for Sample A ; it correlated 0 . 95 with I . This high association , together with conceptual complexity , caused us to drop this alternative . So , one’s I value is the average of one’s papers’ I values . We had the opportunity to show two of our pilot sample of researchers their profiling , and they found it reasonable , noting an expectation of differences by discipline . Figure 2 provides another perspective on what Integration entails . Plots of the percentage of authored papers that cite each SC are shown for three researchers as A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 137 indicative case illustrations . The steepest drop - off is for our second least integrative of the 42 researchers ( I = 0 . 18 ) – 94 % of his papers cite something from Statistics & Probability . His 15 papers cite 17 different SCs , but the proportion drops steeply – e . g . , the second – fourth most cited SCs appear in only 44 % of his papers ; beyond the sixth most cited SC , the rest are referenced in only 1 of his 15 papers . In contrast , one of our most integrative ( I = 0 . 57 ) researchers shows the most gentle slope . His 26 papers cite 51 SCs ; 96 % cite Biochemistry & Molecular Biology ; 93 % cite Cell Biology ; 93 % , Respiratory System ; etc . His 23 rd most cited Subject Category appears in 7 of his 26 papers . He draws on a width swath of research knowledge ! Our middle slope is a researcher whose 57 papers cite 68 SCs in total ; 100 % cite something from Oncology , dropping to 74 % for the second most cited SC ( Medicine , General & Internal ) . As another benchmark , his 16 th most cited SC appears in 25 % of his papers . His I = 0 . 40 is slightly below average for our 42 researchers ( see Results just ahead ) . As this illustrates , the extent of cross - fertilization of research knowledge among Subject Categories is quite remarkable . This far exceeded our expectations . Figure 2 . Relative citation frequency for 3 researchers We also measure Specialization ( “S” ) . We were attracted to this concept by Erin Leahey’s work . 7 She considers Specialization as a researcher focusing tightly on one or a few subfields , rather than spanning many . She postulates that Specialization is a key characteristic affecting research productivity ( considering that too much Specialization , as well as too little , may hamper publication ) . As we thought about Specialization , we did not take it as the antithesis of Integration , but rather as a potentially orthogonal dimension . That is , one might integrate broad swaths of knowledge in writing to a relatively singular audience , or to multiple audiences . And , conversely , one might draw in research knowledge mainly from a single field , whether writing to one or multiple audiences . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 138 Scientometrics 72 ( 2007 ) We set out to measure Specialization using WoK Subject Category information . This differs from Leahey’s data , but we adapt her formulation . We calculate S using the information on the SCs in which a researcher’s publications appear . Note that S does not use Cited SC information at all ; hence it is measured independently of I . Our formulation is : 1 . For the set of journal articles , count the number of publications in each SC . [ Again , we do not distinguish whether these derive from journals associated with single SCs vs . those associated with multiple SCs – although one might consider that as interesting data in its own right to get at IDR . ] 2 . Square the count for each SC ; then sum these . 3 . Divide that sum by the square of the sum of all the counts . For example , suppose we have 5 publications in SC1 ; 3 in SC2 ; 1 in SC3 ; and 1 in SC4 . The numerator is 25 + 9 + 1 + 1 = 36 . The denominator is ( 5 + 3 + 1 + 1 ) 2 = 100 . So , S = 0 . 36 . If all 10 publications were just in SC1 , then S = 1 . 0 . If all 10 were each in a unique SC , then S = 0 . 1 . Note that S only measures a set of papers , not individual papers . In our application , we take a researcher’s body of work for a time period ( e . g . , 3 years ) and use S to gauge the dispersion of those publications across ISI Subject Categories . [ See the Appendix for further explorations in measuring Specialization . ] Results The average levels of I and S are of interest . For the 43 researchers these are I = 0 . 43 ; S = 0 . 26 . [ If we eliminate the one researcher noted as an outlier , these shift by only 0 . 01 . ] The averages shown are based on researchers ( i . e . , we calculate each researcher’s S and I score , and take the mean of those ) . Obviously , some researchers publish far more than others . We can also calculate overall averages weighted by how many papers each authored , yielding similar results : I = 0 . 42 and S = 0 . 24 . The extent of Integration surprises us . Another measurement concern is variability . Using the maximum data sampling , the range for I is 0 . 10 to 0 . 65 across the 43 researchers ( the researcher whom we exclude in 3 - year comparisons has the I = 0 . 10 score ) . The range for S is 0 . 09 to 0 . 72 . The standard deviations are 0 . 12 for I and 0 . 13 for S . The Interquartile Range ( the middle 50 % of the values ) helps get a sense of the distribution – IQR for I is 0 . 34 to 0 . 54 ; for S , it is 0 . 18 to 0 . 31 . We can also calculate standard deviations for each individual . For example , the Integration standard deviations range from 0 . 05 to 0 . 29 . A high individual standard deviation suggests that one publishes both disciplinary and interdisciplinary ( highly integrated ) work . This might prove to be an informative measure in its own right . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 139 An intriguing question is how do Integration and Specialization relate ? As mentioned , we had hypothesized that they could well be orthogonal . We also thought in terms of a quad chart ( Table 5 ) . Figure 3 compiles the data from our three sets of researchers , using the maximum sampling , to give us 43 data points . We draw our quadrant boundaries at the mean values for S ( 0 . 26 ) and I ( 0 . 43 ) . Table 5 . Relationship between Specialization and Integration Specialization / Integration Lo Hi Hi Disciplinarian Single interdiscipline specialist Lo Grazer Renaissance integrator As Figure 3 shows , I and S are not independent ( orthogonal ) ; there is a substantial negative correlation ( - 0 . 51 ) . Researchers whose work integrates more diverse research knowledge ( high I ) tend to be less specialized , publishing in a wider span of Subject Categories ( low S ) . The Upper Left Quadrant corresponds to a “Disciplinarian” – a researcher with a sharply focused agenda . The Lower Right seems to be the locus for the interdisciplinary researcher . Note that the Upper Right Quadrant ( “Single Interdiscipline Specialist” ) is essentially empty . The Lower Left ( “Grazer” ) is reasonably populated . Figure 3 . Specialization vs . Integration A few individual examples give a sense of the interplay between Integration and Specialization . Here is a profile of one researcher with an I value right on the mean : • The “Non - Specialized , Average Integrator” – S = 0 . 19 ; I = 0 . 43 – 8 papers , all involving Physics subfields . But these 8 papers cite a total of 18 SCs ! Of those , 8 are Physics subfields ; others include Multidisciplinary Sciences ( 7 of the 8 papers ) , Physical Chemistry ( 6 of the papers ) , Materials Science , Optics , Statistics & Probability , and Math . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 140 Scientometrics 72 ( 2007 ) A sampling of more extreme cases : • 2 Disciplinarians ( Upper Left Quad ) – The “Specialist” – S = 0 . 72 ; I = 0 . 35 ( the highest S score ; note that I is not so low ) – This reflects 7 papers , of which 6 are in Neurosciences ; 1 in Physiology and 1 in Multidisciplinary Sciences . But note that his “discipline” – Neurosciences , is inherently quite interdisciplinary in nature . – The “Purist” – S = 0 . 51 ; I = 0 . 1 ; ( Our lowest I score of anyone ; one of the AAU researchers whose publication count for the narrower 3 - year period falls below our minimum of 3 , so is missing in the “N = 42 researchers” analyses ) – Based on 7 papers ( for the 4 . 5 year period ) , all in Math ( 6 ) and / or Applied Math ( 2 ) , with 1 also in the Statistics & Probability SC . These 7 papers cite 150 references that appear in 95 different sources ( i . e . , journals , with a few other forms , such as books ) . For those 95 , we can associate journals ( and , of course , not all are WoK journals ) with only 3 different SCs : Mathematics ( 70 ) , Mathematics , Applied ( 11 ) , and Computer Science , Theory & Method ( 1 ) . This is our exemplar in this sample of a disciplinary researcher ! Table 6 . The renaissance integrator # Cited SC’3s / Papers A B C D E F G H I 9 Spectroscopy 2 3 3 3 1 6 1 1 3 9 Physics , Particles & Fields 2 4 4 3 1 6 1 1 3 9 Instruments & Instrumentation 2 3 3 3 1 6 1 1 3 9 Nuclear Science & Technology 2 3 3 4 3 8 2 1 5 5 Engineering , Electrical & Electronic 1 2 2 1 2 3 Astronomy & Astrophysics 1 1 2 2 Optics 1 3 2 Physics , Atomic , Molecular & Chemical 1 3 2 Physics , Multidisciplinary 1 1 2 Physics , Condensed Matter 1 1 2 Physics , Applied 1 1 1 Chemistry , Physical 1 1 Thermodynamics 1 1 Communication 1 • Grazer ( Lower Left Quad ) – S = 0 . 18 ; I = 0 . 27 ; This value is based on 34 papers , published in 9 SCs , citing 34 SCs . His I score is relatively low because his work concentrates heavily in Cell Biology ; Biochemistry & Molecular Biology ; and Multidisciplinary Sciences , which are heavily intercorrelated ( Table 3A ) . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 141 A . L . P ORTER et al . : Measuring researcher interdisciplinarity 142 Scientometrics 72 ( 2007 ) • A Renaissance Integrator – S = 0 . 2 ; I = 0 . 63 ; reflecting 9 papers appearing in 6 different SCs ( with 19 occurrences – i . e . , each publication journal averages being in 2 SCs ) . The 9 papers combine for 88 references , consisting of 42 distinct sources ( journals or such ) . Each paper cites at least 4 different SCs , and very evenly . Table 6 shows the number of occurrences of each Cited SC ( row ) in each paper ( column ) . • The leading Integrator – S = 0 . 28 ; I = 0 . 65 ; 21 papers ( over a longer time period ) ; note the richness of the research sources drawn upon – Table 7 . This is a truncated listing showing only those Cited SC’s appearing in 9 or more of the papers . Another 20 SCs are cited , but in fewer of his papers . Our case illustration for the “Specialist” showed an I value of 0 . 35 , near the 25 th percentile level . This researcher’s 7 papers have 388 references to 73 distinct sources ( journals , etc . ) , associated with 20 different SCs . The leading recipients of citation are Neurosciences ( cited 250 times ) , Multidisciplinary Sciences ( 66 cites ) , Physiology ( 62 ) , Opthamology ( 22 ) , and Zoology ( 17 ) – all others receive 8 or fewer cites . So , this “low - Integration Specialist” really draws on quite distributed content . We also note that researchers with relatively few papers seem more apt to generate extreme I and S scores . We investigated how scores varied by major disciplinary area . In categorizing our 43 researchers , 9 of them warranted inclusion in 2 major disciplinary categories . Table 8 counts those 9 in each of these ( hence the # ’s sum to 43 + 9 ) . These are averaged over researchers ( not weighted by papers ) . The Computer Science and Math categories showed similar values , for tiny samples , so were combined . They are the one major disciplinary group that seems notably different from the others in evidencing less Integration and more Specialization . Table 8 . Breakout by major discipline ( for “Max” sample ) # Integr Spec Reach Phys Sci 7 0 . 42 0 . 22 0 . 55 Life Sci 13 0 . 42 0 . 29 0 . 50 Med Sci 11 0 . 47 0 . 25 0 . 58 CS / Math 5 0 . 29 0 . 33 0 . 43 Engr 16 0 . 46 0 . 23 0 . 59 Discussion We have derived three measures based on ISI Subject Category information for sets of research papers – Integration ( I ) , Reach ( R ) , and Specialization ( S ) . While conceptually appealing , Reach correlated so highly with Integration that we do not see it A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 143 as an independent descriptor . Integration and Specialization provide a rich , two - dimensional depiction of the nature of a body of research ( i . e . , one researcher’s publications over a given time span ) . The proposed metrics are a work in progress . We plan to continue to test them on varied and larger researcher samples . We also intend to continue comparing alternative formulations . We have examined additive models , but prefer the multiplicative ( row by column frequency ) approach presented here . We have tried exclusion of the diagonal ( self - cite ) cells , but found this conceptually unattractive . We will further compare a full matrix alternative on sets of researchers vs . our half - matrix ( Table 5 ) ; we anticipate highly correlated results , but possibly more intuitive scaling . We also plan to explore alternative ways to treat the multidisciplinary SCs . Perhaps the most remarkable finding of this exploration of new IDR metrics is the high level of interdisciplinarity indicated . Our expectations as to the degree of cross - SC citation and publication were greatly exceeded . These pilot results , for three distinct samples ( 43 researchers in total ) , show that researchers do extensively draw upon research knowledge from multiple domains . Such levels of Integration are really notable . We illustrated several profiles of individual researchers . One of those , near the relatively low , 25 th percentile of I scores ( 0 . 35 ) included 7 papers that drew upon ( cited ) research from 20 different ISI Subject Categories . Our leading Integrator ( I = 0 . 65 ) had 9 papers that drew upon research from 31 SCs . The higher I score reflects more citation of more SCs ( i . e . , a gentler slope , as illustrated for three other researchers in Figure 2 ) . The I score also incorporates use of the cosine measure to gauge how closely related such Subject Categories are , based on a good - sized national sample of articles . We aspire to compile Integration and Specialization data for reasonable samples across disciplines . Will we see notable differences across major field ( e . g . , engineering , physical science ) , discipline ( e . g . , civil vs . electrical engineering ) , and / or SC ? Having those data will benchmark research behavior . This is certainly necessary to assess “how interdisciplinary” Researcher A is . We hypothesize that different research norms , and coverage differences among SCs , will engender important differences , implying serious researcher by domain interaction effects . Also , compilation of research domain level data holds promise to track macro research behavior . Is a given research domain becoming more or less interdisciplinary over time ? Is science in general becoming more interdisciplinary ? ( The NAKFI mission concerns this . ) It would be very interesting to develop “knowledge flow” models for research areas . 19 For instance , one could look at the collective work of attendees at a NAKFI conference to discern from which SCs they draw ; where they publish ; and which SCs use their results . Application of measures based on ISI Subject Category distribution has the potential to generate highly informative tracking of cross - disciplinary knowledge flows . The sequence is A . L . P ORTER et al . : Measuring researcher interdisciplinarity 144 Scientometrics 72 ( 2007 ) • Patterns of cited SCs indicate incorporation of knowledge drawn from multiple sources • Publication SC patterns reflect the research domains that the researchers are addressing • Patterns of SCs of the journals in which articles cite those publications indicate distribution of the interdisciplinary research knowledge generated . So , for a given paper , researcher , or body of research , we can profile from whence research knowledge is drawn , where the new knowledge is presented , and which research domains pick it up [ to pursue this intriguing issue , see Boyack et al . , Figure 5 15 ] . Studies of such knowledge flows should speak to whether our conventional wisdom is well - founded . Are certain research domains foundational ? For instance , can we demonstrate that Research “A” is drawn upon by ( contributes to ) Research “B” in another SC , which in turn is actively cited by Research “C” in other SCs ? Does basic science contribute to technological development ? Do certain research domains have higher cross - domain impact than others ? If a research domain speaks just to itself , this could well be an indication of minimal benefit to science and society . 1 Future extensions hold strong appeal . Kevin Boyack suggested exploring Integration’s sensitivity to scale and examining I calculated across , as compared to within , a researcher’s papers . Given the important , but ill - understood , role of co - authoring in IDR , it could be highly informative to examine this together with Integration and Specialization . Are co - authored works more likely to be highly IDR in terms of Integration ? For papers covered by WoK we can obtain author organizational affiliations . Those give at least rough indications of discipline . Figuring out how to associate those disciplinary indicators with SCs presents a challenge . Short of that , we can compare the levels of Integration and Specialization of single - authored vs . co - authored papers by a given cadre of researchers . For instance , one of our researchers has many papers , but only one published in a journal in the Cell Biology SC . He is a co - author on that , so we wonder how much knowledge of cell biology he has internalized ? One might separate papers on which our researcher is the first author ( whether sole or not ) to better understand his or her integrative stance . We considered just focusing on single - authored papers for certain analyses , but that sacrifices too much of the research data . We nominate many issues that these new measures can help address . These include • Do more interdisciplinary papers ( higher I ) have lower or higher impact ( examine journal impact factors ) ? • Do we see increased IDR just after receiving tenure ? One might hypothesize that I increases , and S decreases , over research careers . • As we differentiate more from less interdisciplinary researchers , one could then probe : A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 145 – Where do IDR researchers come from ? [ Informal , pilot - level characterization of the Pilot Sample backgrounds as relatively disciplinary ( e . g . , B . S . through PhD all in Chemistry ) to quite multi - disciplinary did not correlate with how interdisciplinary their publications are . ] – Which research areas feature IDR ? Why so ? [ Use of these measures to compare across research fields is somewhat precarious . All of the ISI Subject Categories are not equally fine - grained . SCs vary widely in how many journals they include and their coherence . 15 Citation norms differ greatly among fields as well . And a modest number of SCs constitute what Morillo and colleagues call “horizontal” disciplines . 3 We intend to explore these implications . ] – When we’ve identified highly IDR researchers , ask them what differentiates their work ? [ Tell them their papers are more IDR ; explore with them their “secrets” in making this happen . ] – What institutional parameters nurture IDR ? We expressly invite comments and suggestions on how to improve and extend the measurement of research interdisciplinarity . * We thank Kevin Boyack , Fernanda Morillo , and Ron Kostoff for reviewing an earlier version and providing very helpful feedback . References 1 . C HUBIN , D . E . , C ONNOLLY , T . ( 1982 ) , Research trails and science policies : local and extra - local negotiations of scientific work , In : N . E LIAS et al . ( Eds ) , Scientific Establishments and Hierarchies . Sociology of the Sciences , Yearbook , Vol . 6 , Dordrecht : D . Reidel , pp . 293 – 311 . 2 . K LEIN , J . T . , P ORTER , A . L . ( 1990 ) , Preconditions for interdisciplinary research , In : P . H . B IRNBAUM - M ORE , F . A . R OSSINI , D . R . B ALDWIN ( Eds ) , International Research Management , New York : Oxford University Press , pp . 11 – 19 . 3 . M ORILLO , F . , B ORDONS , M . , G OMEZ , I . ( 2001 ) , An approach to interdisciplinarity through bibliometric indicators , Scientometrics , 51 : 203 – 222 . 4 . C OMMITTEE ON F ACILITATING I NTERDISCIPLINARY R ESEARCH , OF THE C OMMITTEE ON S CIENCE , E NGINEERING , AND P UBLIC P OLICY ( 2005 ) , Facilitating Interdisciplinary Research , Washington , D . C . : National Academies Press . 5 . P ORTER , A . L . , R OESSNER , J . D . , C OHEN , A . S . , P ERREAULT , M . ( 2006 ) , Interdisciplinary research – meaning , metrics , and nurture , Research Evaluation , 15 ( 8 ) : 187 – 195 . 6 . D IETZ , J . S . , C OMPALOV , I . , B OZEMAN , B . , O’N EIL L ANE , E . , P ARK , J . ( 2000 ) , Using the curriculum vita to study the career paths of scientists and engineers : An exploratory assessment , Scientometrics , 49 : 419 – 442 . 7 . L EAHEY , E . ( 2006 ) , Gender differences in productivity – Research specialization as a missing link , Gender & Society , 20 ( 6 ) : 754 – 780 . A . L . P ORTER et al . : Measuring researcher interdisciplinarity 146 Scientometrics 72 ( 2007 ) 8 . P ORTER , A . L . , C UNNINGHAM , S . W . ( 2005 ) , Tech Mining : Exploiting Technologies for Competitive Advantage , New York : Wiley . 9 . P ORTER , A . , K ONGTHON , A . , L U , J . ( 2002 ) , Research profiling : improving the literature review . Scientometrics , 53 : 351 – 370 . 10 . D EL R IO , J . A . , K OSTOFF , R . N . , G ARCIA , E . O . , R AMIREZ , A . M . , H UMENIK , J . A . ( 2001 ) , Citation mining : Integrating text mining and bibliometrics for reearch user profiling , Journal of the American Society for Information Science and Technology , 52 ( 13 ) : 1148 – 1156 . 11 . W ATTS , R . J . , P ORTER , A . L . ( 2005 ) , Mining conference proceedings for corporate technology knowledge management , Portland International Conference on Management of Engineering and Technology ( PICMET ) , Portland , OR . 12 . C UNNINGHAM , S . W . ( 1996 ) , The Content Evaluation of British Scientific Research , Ph . D . Thesis , University of Sussex : Science Policy Research Unit . 13 . M ORILLO , F . , B ORDONS , M . , G OMEZ , I . ( 2003 ) , Interdisciplinarity in science : A tentative typology of disciplines and research areas , Journal of the American Society for Information Science and Technology , 54 ( 13 ) : 1237 – 1249 . 14 . M OYA - A NEGON , F . , V ARGAS - Q UESADA , B . , H ERRERO - S OLANA , V . , C HINCHILLA - R ODRIGUEZ , Z . , C ORERA - A LVAREZ , E . , M UNOZ - F ERNANDEZ , F . J . ( 2004 ) , A new technique for building maps of large scientific domains based on the cocitation of classes and categories , Scientometrics , 61 : 129 – 145 . 15 . B OYACK , K . W . , K LAVANS , R . , B ORNER , K . ( 2005 ) , Mapping the backbone of science , Scientometrics , 64 : 351 – 374 . 16 . E TO , H . ( 2003 ) , Interdisciplinary information input and output of nano - technology project , Scientometrics , 58 : 5 – 33 . 17 . P ETERS , H . P . F . , B RAAM , R . R . , VAN R AAN , A . F . J . ( 1995 ) , Cognitive resemblance and citation relations in chemical engineering publications , Journal of the American Society for Information Science , 46 ( 1 ) : 9 – 21 . 18 . K LAVANS , R . , B OYACK , K . W . ( 2006 ) , Identifying a better measure of relatedness for mapping science , Journal of the American Society for Information Science and Technology , 57 ( 2 ) : 251 – 263 . 19 . P ORTER , A . L . , C HUBIN , D . E . ( 1985 ) , An indicator of interdisciplinary research , Scientometrics , 8 : 161 – 176 . A . L . P ORTER et al . : Measuring researcher interdisciplinarity Scientometrics 72 ( 2007 ) 147 Appendix : Formulas Specialization ( S ) : ( ) ( ) [ ] ( ) ( ) [ ] ∑ ∑ + + = 2 1 2 2 1 # # # # n n SC in published papers of SC in published papers of SC in published papers of SC in published papers of S K K Where the Subject Categories ( SCs 1 – n ) are those reflecting the journals in which the set of papers was published . Integration ( I ) : ( ) ( ) [ ] ( ) ⎥⎥⎦⎤ ⎢⎢⎣⎡ × − × × − = ∑ ∑ j i j i j i f f SC SC COS f f I 1 where i = row , j = column , f = frequency Where the Subject Categories ( SCs ) are those cited in a given paper . The summation is taken over the lower diagonal cells of this SC x SC matrix , inclusive of the diagonal . COS is the cosine measure of association between the two SCs , based on a national co - citation sample from Web of Science ( as described in the text ) . Reach ( R ) : ( ) ( ) [ ] ∑ − × − = p i i SC SC COS f R 1 Where f = frequency Where SC i is a given cited SC , and SC p is a publication’s SC . The summation is taken over each of the cited SCs for the given paper . COS is as above . Where a given publication is associated with multiple SCs , R is calculated for each . We explored using average R , smallest R , and largest R as the score for a given publication . We continue to experiment with alternative interdisciplinarity metrics . This is a work in progress , with some current directions as follows : • We have not actively pursued Reach due to its high correlation with Integration in these test samples . • We have modified Specialization to incorporate the cosine values among the respective SCs . Its formulation thus comes to resemble the I calculation above , except that it is calculated for a body of research , not an individual paper ; pertains to SCs instead of Cited SCs ; and is not subtracted from 1 . • We are exploring calculating I for a body of research ( set of papers ) . So , for instance , a researcher’s I value would be calculated over the entire set of Cited SCs in those papers , rather than as the average of the I values for each individual paper . • In calculating I , we are trying the full matrix , instead of the half - matrix form described here . [ This is largely for conceptual simplicity ; in our samples full - matrix I correlates extremely highly with half - matrix I ( e . g . , 0 . 99 ) . ] • We are considering whether to treat the ’horizontal’ SCs differently ; that is the dozen SCs that include ’interdisciplinary’ or ’multidisciplinary’ in their name . At this time , we don’t believe special treatment is warranted . \ No newline at end of file diff --git a/silver_data/6ad25aae5a69af733e9f898c9f92fcee74157cfa.pdf.txt b/silver_data/6ad25aae5a69af733e9f898c9f92fcee74157cfa.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d437f3e41be191173513cd366162e40dbf0431c4 --- /dev/null +++ b/silver_data/6ad25aae5a69af733e9f898c9f92fcee74157cfa.pdf.txt @@ -0,0 +1 @@ +HAL Id : jpa - 00208725 https : / / hal . archives - ouvertes . fr / jpa - 00208725 Submitted on 1 Jan 1977 HAL is a multi - disciplinary open access archive for the deposit and dissemination of sci - entific research documents , whether they are pub - lished or not . The documents may come from teaching and research institutions in France or abroad , or from public or private research centers . L’archive ouverte pluridisciplinaire HAL , est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche , publiés ou non , émanant des établissements d’enseignement et de recherche français ou étrangers , des laboratoires publics ou privés . Light scattering spectroscopy of pulydimethylsiloxane - toluene gels J . P . Munch , P . Lemaréchal , S . Candau , J . Herz To cite this version : J . P . Munch , P . Lemaréchal , S . Candau , J . Herz . Light scattering spectroscopy of pulydimethylsiloxane - toluene gels . Journal de Physique , 1977 , 38 ( 12 ) , pp . 1499 - 1509 . < 10 . 1051 / jphys : 0197700380120149900 > . < jpa - 00208725 > 1499 LIGHT SCATTERING SPECTROSCOPY OF PULYDIMETHYLSILOXANE - TOLUENE GELS J . P . MUNCH , P . LEMARÉCHAL , S . CANDAU Laboratoire d’Acoustique Moléculaire ( * ) , Université Louis - Pasteur , 4 , rue Blaise - Pascal , 67070 Strasbourg Cedex , France and J . HERZ Centre de Recherches sur les Macromolécules , C . N . R . S . , 6 , rue Boussingault , 67083 Strasbourg Cedex , France ( Reçu le 1 er juillet 1977 , accepté le 18 août 1977 ) Résumé . 2014 La fonction d’autocorrélation de la lumière diffusée a été mesurée pour des gels de polydiméthylsiloxane - toluène formés soit par gonflement de réticulats permanents , soit par dissolu - tion de macromolécules linéaires à des concentrations moyennes . Dans les deux cas le coefficient de diffusion coopératif varie avec la concentration en polymère selon une loi de puissance avec un exposant plus élevé que celui qui avait été obtenu précédemment pour des systèmes polystyrène - benzène . Par ailleurs , il est montré que les modules de compression uniaxiale n’obéissent plus à des lois d’échelle simples avec la concentration pour des réseaux gonflés par un liquide moins bon solvant que celui dans lequel a été réalisée la réticulation . Abstract . 2014 The autocorrelation function of scattered light has been measured for polydimethyl - siloxane - toluene gels formed either by swelling permanent networks or by dissolving linear macro - molecules at moderate concentrations . In both cases , the cooperative diffusion constant varies with concentration according to a power law with an exponent larger than that obtained previously for polystyrene - benzene systems . On the other hand , it is shown that uniaxial compression moduli do not obey simple scaling laws with the equilibrium concentration for networks swollen by a diluent of less quality than the solvent in which the crosslinking has been made . LE JOURNAL DE PHYSIQUE TOME 38 , DÉCEMBRE 1977 , Classification Physics Abstracts 61 . 40K201362 . 00201366 . 10 1 . Introduction . - Traditional methods for measur - ing the viscoelastic properties of gels generally depend on mechanical devices . It has been shown recently that optical mixing spectroscopy yields valuable information concerning the hydrodynamic properties of both permanent swollen networks [ 1 - 8 ] and semi - dilute or concentrated polymer solutions [ 8 - 11 ] . A theoretical model has been proposed which assumes that the light scattered from a gel arises from collective excitations of the network [ 1 , 12 , 13 ] . From this model , the correlation function of the polarized scattered light for a longitudinal fluctuation of wavevector K is predicted to have the form of an exponential decay . The decay rate is given by T = De K2 where Dp is the cooperative diffusion constant of the chains of the network . On the other hand , a corpuscular model has been proposed by Mc Adam et al . [ 2 ] and Carlson et al . [ 3 , ( * ) Equipe de Recherche Associée au C . N . R . S . 4 ] , which assumes that each macromolecule in the gel state behaves as a harmonically bound particle executing independent Brownian motion about a stationary mean . The resulting theory predicts a non - exponential intensity autocorrelation function given in terms of a chain elastic constant and the conventional translational diffusion coefficient . In previous papers , we have reported autocorre - lation measurements on benzene - swollen model net - works of polystyrene and shown that the hydro - dynamic model fits the observed data . The cooperative diffusion constant of gels has been investigated as a function of the method of synthesis of the networks and their characteristics . In a first attempt , we have assumed ideality of networks , i . e . absence of structure defects such as pendant chains , cycles , or entangle - ments , and interpreted the results obtained within the framework of the rubber elasticity theory [ 5 - 7 ] . From this analysis , we were led to the conclusion that the so - called memory term , which relates the dimension of the elastic chain in the swollen state and the reference swollen state respectively , is strongly Article published online by EDP Sciences and available at http : / / dx . doi . org / 10 . 1051 / jphys : 0197700380120149900 1500 dependent on the functionality of the cross - linking agent . Recently , we have reanalyzed the data by allowing the presence of entanglements trapped between two permanent junction points and assum - ing , according to a suggestion of de Gennes , that the average distance between the cross - links was in the first approximation identical to the dynamical screening length [ 8 ] . This assumption implies that both the cooperative diffusion constant and the extensional modulus should obey scaling laws with swelling equilibrium concentration . Such behaviour has been effectively observed for polystyrene - benzene gels . Most of the networks investigated were prepared by anionic block - copolymerization of styrene with small . amounts of divinylbenzene . In this type of network , each linear chain element is connected with two different branch points constituted by polydivinylbenzene nodules . The chain elements have the characteristic sharp molecular weight distribution of polymers prepared by anionic polymerization . A drawback of the method used here is that the actual functionality of the crosslinks is unknown . In the present paper we report measurements of the cooperative diffusion constant of a homologous series of polydimethylsiloxane ( PDMS ) networks swollen in toluene . In many respects these gels are very different from the polystyrene networks investi - gated previously : - Both the average molecular weight of the elastic chains and the functionality of the permanent cross - links are known parameters . - Branch points are formed by single pluri - functional molecules instead of nodules of appreciable dimensions as was the case for the branch points of the polystyrene networks described above . - The elastic properties of PDMS networks are very different from those of polystyrene networks since the glass transition of PDMS occurs at a tempe - rature far below room temperature ( - 120 °C ) . - In all synthesis the precursor polymer concen - tration during network formation was much higher than the equilibrium concentration of the swollen networks . Thus the networks do not contain any macropores . - The swelling degree of the PDMS networks in toluene is considerably lower than that of polysty - rene networks swollen at equilibrium in benzene . 2 . Theoretical . - The essential feature of a gel is that each macromolecule linked to the network by both chain - ends is no longer free to diffuse through the whole network structure but is confined to a region of the network compatible with the number of effective crosslinks . Two different approaches have been used to described the viscoelastic behaviour of gels . Mc Adam , et al . [ 2 ] , Carlson , et al . [ 3 , 4 ] have proposed a simple model of scattering molecules in the gel state which assumes each molecule to be harmonically bound and executing brownian motion around a stationary mean position . Upon application of the Ornstein and Uhlenbeck distribution function for such a particle [ 14 ] , Carlson and Fraser [ 3 ] have derived an expression for the non - normalized optical field autocorrelation function G ( l ) ( T ) : . where I is the average intensity of the field , D is the translational diffusion constant of the particle , K is the scattering wavevector and y ( sec - l ) the ratio of the chain elastic constant k ( dyne / cm ) to the fric - tional constant f ( dyne s / cm ) . From equation ( 1 ) it follows that the line profiles in a self - beating experiment will not be simple lorent - zian with a linear relationship between line - width and K2 . Furthermore , the initial amplitude of the normalized intensity autocorrelation function g ( 2 ) ( O ) will have a K - dependent value which is always less than 2 , in contrast to the freely diffusing particle whose g ( 2 ) ( O ) = 2 . In the derivation of equation ( 1 ) , uncorrelated Rayleigh scattering has been assumed , neglecting the presence of a large component of static scattering by the gels which is due to micro - scopic inhomogeneities . Then in a later paper [ 4 ] Wun and Carlson have included in the intensity an additional term to account for the additional static scattering which contributes to the lowering of the initial amplitude of g ( 2 ) ( - r ) . In the second model , developed by Tanaka , et al . [ 1 ] , and de Gennes [ 12 ] , the gel is considered as a continuum and the light scattering is assumed to arise from longitudinal deformation modes of the network . The deformation of the swollen network has been shown to obey a diffusion equation and the correlation function of the polarized scattered light is given by : The cooperative diffusion constant De of the chains of the network is given by : 1 " B il ) ’B wnere p ( clyne / cm - ) is tne longitudinal compressionai modulus and 0 ( dyne s / cm4 ) the frictional force per unit volume of the network as it moves with unit velocity relative to the surrounding liquid . p and 0 are given respectively by [ 12 ] : - , , vo being number density of network chains in the swollen state , 17 the viscosity of the swelling liquid and Rh hydrodynamic radius of one chain . 1501 By combining equations ( 3 ) , ( 4 ) and ( 5 ) one obtains : where Df stands for the translational diffusion coefficient for a dilute solution of free macromolecules having the hydrodynamic radius Rh . This hydrodynamic theory leads to results which are quite different from those derived from the har - monically bound particle model , since it predicts an exponential time decay of the autocorrelation function of the optical field with a linear relationship between decay rate and K2 . Uncorrelated Rayleigh scattering has been assumed so that no account has to be taken of the static scattering due to spatial non - randomness of the crosslinking . This additional component acts as a local heterodyning source . Therefore , if Io stands for the intensity of the static component ( including dust trapped in the network ) and 7g for the intensity scattered from longitudinal fluctuations , the normalized intensity autocorrelation function is given by [ 15 ] : When Io & # x3E ; I , , , equation ( 7 ) reduces to : In previous papers , we have shown that g2 ( z ) obeys equation ( 8 ) for polystyrene - benzene gels [ 7 ] . According to equation ( 6 ) , the cooperative diffusion constant depends on the dimension of the elastic chains joining two crosslinks which in tum depends on the swelling equilibrium concentration Ce . The Flory theory of swelling predicts the following dependence of Ce on the molecular weight Me of the elastic chain [ 16 ] However , this result has been derived from an expres - sion of the free energy which has been shown to be incorrect and which leads to correct results only in dilute solutions [ 17 ] . A new approach , based on scaling law theories has been recently proposed , describing static and dynamical properties of both dilute and semi - dilute solutions [ 13 , 17 , 18 ] . Semi - dilute solutions can be considered as net - , t works with a finite lifetime . The average distance between neighbouring crosslinks is given by the screening length ç which depends only on the concen - tration , according to the following scaling law [ 13 ] Therefore , the diffusion constant D , is given by In swollen networks at the equilibrium state , the average distance between neighbouring cross - links depends on the swelling equilibrium concen - tration Ce . De Gennes suggested that this distance is given as in the case of interpenetrated solutions by the screening length j . This assumption implies that : The equilibrium concentration is given in the first approximation by : where Me represents the molecular weight of the elastically effective chains connecting two junction points regardless of their nature , entanglements or permanent crosslinks . For ideal networks prepared from a precursor polymer of known molecular weight M p’ ç is equal to the radius of gyration RF of the precursor polymer . Therefore : where C * is the cross - over concentration between dilute and semi - dilute regions for a solution of macromolecules of molecular weight Mp . Combination of equations ( 12 ) and ( 13 ) leads to the following concentration dependence of Dc Then , one can predict the same exponent for both scaling laws D , , = f ( C ) and De = f ( Ce ) relative to semi - dilute solutions and swollen networks , res - pectively . This result has been verified experimentally in polystyrene gels [ 8 ] . It is also interesting to consider the concentration dependence of the compressional modulus E . In Flory’s theory , E is proportional to the number density of elastically effective chains in the dry state [ 16 ] . As a consequence , E oc Ce2 . On the other hand , scaling law theory predicts that E is proportional to the number density of elastic chains in the swollen state , resulting in the following scaling law for E [ 13 ] Such a dependence of E on Ce has been observed in polystyrene networks swollen by benzene . 1502 3 . Expérimental . - 3 . 1 PREPARATION AND CHA - RACTERISTICS OF SAMPLES . - Polydimethylsiloxane networks were obtained by the addition reaction of ( a - co ) dihydropolydimethylsiloxane precursor polymers with plurifunctional allyloxy compounds , using H2PtC’ , , as a catalyst . The method has been described earlier [ 19 ] . These crosslinking reactions were carried out in the presence of toluene at 60 OC . Triallyloxy - 1 , 2 , 3 propane , tetraallyloxyethane , and bis allyloxy - 3 dimethylallyloxy - 2 , 2 propane oxide [ 20 ] were used as 3 , 4 and 6 functional crosslinking agents . The precursor polymers were chosen in a molecular weight range between 4 500 and 17 000 . The volume swelling degree of the networks at equilibrium has been determined with an accuracy of about 5 % , using a procedure already described [ 21 ] . The experimental technique and the apparatus [ 22 , 23 ] used for unidirectional compression measurements have also been described elsewhere [ 24 ] . All compres - sion measurements were carried out on networks swollen at equilibrium in toluene at small deformation ratios 0 . 8 A 1 . In table 1 are listed the network - samples and their characteristics . Table II shows the molecular weights Mn of the linear PDMS samples used in our experiments , determined by chemical endgroup analysis . Sample 1 is a linear PDMS obtained by an anionic polyme - rization method ( 1 ) . Samples 2 - 5 are ( a - co ) hydro - genosilane polydimethylsiloxanes ( 2 ) , which have been used for the preparation of networks . TABLE 1 PDMS swollen in toluene ( a ) e ) The networks have been prepared in toluene at 70 °C at a concentration of 83 % . ( b ) Number average molecular weight of the precursor polymer determined by endgroup analysis . ( C ) Functionality of the crosslinking agent . ( a ) Data of Belkebir Mrani et al . [ 24 ] . ( E is related to the para - meter G * of the authors through the relationship E = G * q¡Õ 1 / 3 , where qio is the swelling equilibrium ratio . ) ( e ) Determined from light scattering spectroscopy . ( 1 ) The authors are grateful to Dr . S . Boileau who has prepared this high molecular weight polymer . ( 2 ) These polymers were prepared by the Silicon Division of Rhône - Poulenc . TABLE II Linear PDMS ( 1 ) The weight average molecular weight , the radius of gyration and the second virial coefficient in toluene , have been determined by conventional light scattering . We have no exact information concerning the polydispersity of the samples . However , an analysis of the autocorrelation function of the photocurrent , performed by the method of cumulants [ 25 , 26 , 15 ] showed that the polydispersity index ( ratio of weight average molecular weight to the number average molecular weight ) does not exceed 1 . 3 . Such a value of Mw / Mn is not negligible , but this point is not of great importance for the purposes of our study . 3 . 2 LIGHT SPECTROSCOPY . - The spectrometer and autocorrelator for intensity autocorrelation measure - ments have been described before [ 15 ] . The light source was an argon ion laser ( Spectra Physics Model 165 ) with a wavelength of 488 nm . The cubic shaped gel samples ( - 1 cm3 ) were put into standard glass cells containing an excess of swelling liquid . The scattered light was collected at a pre - determined angle by a lens aperture system and focused onto the sensitive part of a photomultiplier ( ITT FW 130 ) cathode . The photocurrent was analyzed after passing through an amplifier - discriminator by a 24 - channel digital autocorrelator ( Precision Devices and Systems , Ltd Malvem system 4300 ) . The data from the corre - lator were analyzed by the method of cumulants [ 25 , 26 , 15 ] which allows for a distribution of decay rates G ( F ) and makes it possible to calculate the average decay rate F and the second - order norma - lized moment ( Jl2 / f2 ) about the mean of f . We have mentioned in the theoretical section that the intensity scattered from longitudinal fluctua - tions of swollen networks must be heterodyned to some extent by the static component due to micro - scopic heterogeneities . Furthermore , no special care has been taken to eliminate dust in the reagents and the solvent used for the network synthesis ; the dust trapped in the gel will also contribute to the heterodyn - ing . As a consequence , the initial amplitude 1 gl2 @ ( 0 ) 1 of the normalized photocount clipped correlation function must be lower than that obtained for polymer solutions in an homodyne self - beating experiment , which in practice is about 1 . 75 on account of incom - plete spatial coherence . As a matter of fact , it turns out that for all the investigated gels , gk2 ( O ) 1 = 1 . 01 - 1 . 1 . These very 1503 small values of 1 gL2 ) ( O ) indicate that the heterodyne component dominates the homodyne component and hence equation ( 8 ) must apply . In order to check this last point we have measured the autocorrelation function obtained by mixing the scattered signal with an external signal . In the scattering angle range of 60 - 900 , the initial amplitude of the normalized autocorrelation function has been found to be reduced further , but the decay time is not affected . Alternatively , a two hundred - channel real time wave analyzer ( Saicor Model SAI 21 B ) was used to measure the spectrum of the photocurrent from the photomultiplier . In all cases where the experimental data were described by a single decay rate r , both wave analyzer and autocorrelator led to identical values of r within 2 % . All the measurements were performed at room temperature ( 23 OC ) . The polydimethylsiloxane net - works swollen by toluene were allowed to stand at room temperature for at least one day to allow the sample to stabilize in the cell . The stability could be checked by monitoring the intensity of the DC component . The solutions of PDMS in toluene were made dust free by centrifugation ( 15 000 rev / min . ) . For concentrations larger than 0 . 5 x 10 - 2 g . cm - 3 , the FIG . 1 . - Semi - dilute solutions of linear PDMS Mw = 6 x 106 in toluene . a ) c = 3 x 10 - 2 g . cm - 3 ; b ) c = 1 . 42x 10 - 2 g . cm - 3 ; 0 A Scattered signal alone ; 1 à Scattered signal mixed with external oscillator . solutions of sample 1 were too viscous to allow such a procédure ; therefore , the solutions were made with a previously clarified toluene . However , some dust remains in the solutions giving rise to a hetero - dyning of the scattered signal . The rate of hetero - dyning varies with both concentration and scattering angle , as illustrated in figure 1 . This point is of great practical importance since it could lead to an incorrect analysis of the concentration dependence and wave - vector dependence of the decay rate of the correlation function . For this reason we have checked the scatter - ing mode ( homodyne or heterodyne ) by mixing the scattered signal with an external oscillator , using a Michelson type interferometer . In all cases where the heterodyning from dust was only partial , we have measured the decay rate by using the Michelson geometry . Table III gives the scattering modes experimentally observed for the different concen - trations . 4 . Results . - 4 . 1 SOLUTIONS OF PDMS IN TOLUENE . - In polymer solutions of given concentration c , the shape of the correlation function for scattered light g ( i ) and the K dependence of the decay rate r depend on the following four parameters : the radius of gyration RF of the chain , the cross - over concen - tration c * , the scattering wavevector K , and the minimum wavevector Km ; n at which the relaxation time iK of a longitudinal mode of wavevector K is equal to the relaxation time T , for complete disen - tanglement of one macromolecule . FIG . 2 . - Various regimes for longitudinal fluctuations of wave vector K in solutions of polymer in a good solvent . TABLE III Scattering modes for solutions of sample 1 ( Mw = 6 x 106 ) in toluene 1504 Because of the large ranges of concentration , molecular weight and scattering wavevector investi - gated in our experiments , we have obtained data in various regimes defined by the relative values of these parameters . For the clarity of the discussion we have reproduced in figure 2 the diagram given by de Gennes [ 13 ] showing the range of concentration and momentum transfer for the predicted existence of the different types of modes . One can summarize the possible experimental situations on the following way . 4 . 1 . 1 Dilute regime c c * . - a ) Region II g ( i ) is exponential Do is the self diffusion of one chain b ) Region III g ( i ) is non exponential r is the average decay rate . One probes inner modes of a single chain . 4 . 1 . 2 Semi - dilute regime c & # x3E ; c * . - a ) Region 1 g ( il is exponential De is the cooperative diffusion constant in the gel regime b ) Région II’ g ( i ) is exponential Dfree is the diffusion constant in the disentangled regime The différence between D free and De amounts to a change of prefactors . c ) Region III’ g ( i ) is non exponential r oc K3 . One probes inner modes within a coherence length . The behaviour which is of interest for a quantitative comparaison between cooperative modes of swollen permanent networks and semi - dilute solutions is the concentration dependence of De in the region I . In order to determine accurately the concentration dependence of the diffusion constant in both regions 1 and II , one must determine the ranges of scattering wavevector where the two following conditions are fulfilled : i ) the autocorrelation function decays exponentially ; ii ) the decay rate follows a K2 depen - dence . It is not easy to characterize with accuracy the range of K values where the autocorrelation function departs from an exponential . On the other hand , the procedure which consists of detecting a departure from the K2 dependence of the decay rate obtained by fitting g ( z ) to an exponential is quite sensitive . Figure 3 shows the ranges of K values where r varies like K2 , for different polymer concen - trations . In these domains , an analysis using the method of cumulants shows that the correlation function is well described by a single exponential , except for the dilute solutions where a slight distri - bution of exponentials occurs . The average decay rate of this distribution is about the same ( within 3 % ) as the decay rate determined by force fitting a single exponential and the value of the second - order moment ( / 12 / r2 ) is about - 0 . 1 . Such a value of / 12 / r2 would indicate a polydispersity index M , , IM . - 1 . 3 . However , one must point out that the measurements have been performed at low scattering angles ( 7° 9 15° ) where the effect of the non - negligible acceptance angle for scattered light may lead to an overestimation of Mw / Mn . The solid line Kç = 1 ( where ç has been calculated from D / Do = RF / ç ) reported on figure 3 represents the transition line from regions II and 1 to regions III and III’ respectively . The experimentally observed deviation of T from the K2 dependence indicates the existence of the concentration dependent corre - FIG . 3 . - Semi - dilute solutions of linear PDMS MW = 6 x 106 in toluene . Q c = 0 . 47 x 10 - 4 g . cm - 3 ; x c = 4 . 5 x 10 - 4 g . cm - 3 ; o c = 9 x 10 - 4 g . cm - 3 ; + c = 0 . 18 x 10 - 2 g . cm - 3 ; Z c = 0 . 36 x 10 - 2 g . cm - 3 ; 0 c = 0 . 72 x 10 - 2 g . cm - 3 . 1505 lation length j ( c ) and confirms previous conventional light scattering data [ 27 ] . In the regions III and III’ , the correlation function has been found to deviate significantly from single exponential behaviour . The decay time obtained by force fitting a single exponential depends on the sampling time . On the other hand , the average decay time determined from the cumulant analysis is insensitive to a change of the sampling time . The second - order moment is of the order of magnitude of 0 . 2 . We have not attempted a rigorous analysis of the correlation function , using the exact expression given by de Gennes and Dubois - Violette [ 28 ] . Such an analysis has been performed by Adam , et al . [ 10 ] on the system polystyrene - benzene . Figure 4 shows the concentration dependence of both Do and Dc obtained from the data of figure 3 . FIG . 4 . - Semi - dilute solutions of linear PDMS samples in toluene . + Mw = 6 X 106 ; 0 Mn = 17 100 ; x Mn = 4 500 . The behaviour of the diffusion constant is quite different from that observed in other polymeric systems in many respects : - In the dilute domain , Do is independent of the concentration . - Within the experimental accuracy , the exponent of the power law De = f ( c ) is that predicted by the theory - There is quite a sharp transition between dilute and semi - dilute solutions , which defines with a fairly good accuracy the cross - over concentration c * . The concentration cé p at which Do = f ( c ) and Dc = f ( c ) intersect is found to be : The cross - over concentration calculated from ( NA : Avogadro number ) is : In figure 4 we have also plotted the diffusion constant as a function of concentration for PDMS of small molecular weights . In the dilute range , Do varies significantly with c . In the semi - dilute range the experimental points lie on the same curve De = f ( c ) as that for the high molecular weight sample . These data represent the free diffusion constant D free in the disentangled regime ( region II’ ) , since they have been obtained for a range of K values much smaller than Kmin which can be calculated from [ 13 ] : Typically for c * / c = 1 / 2 and M = 17 100 One can , therefore , conclude that for the system investigated , the diffusion constant follows the same scaling law with the concentration in both the gel and the disentangled regimes without change of the numerical value of the prefactor . 4 . 2 PDMS NETWORKS SWOLLEN IN TOLUENE TO THEIR EQUILIBRIUM STATE . - 4 . 2 . 1 Shape of the auto - correlation function . - The results reported in previous papers , related to the light spectroscopy of polystyrene networks swollen by benzene and ethyl - acetate , demonstrated the validity of the hydro - dynamic model . On the other hand , the results of Wun and Carl - son on polyacrylamide gels supported the harmoni - cally bound particle model [ 4 ] . Therefore we have proceeded to a careful investi - gation of the autocorrelation function and of the K dependence of the decay rate for PDMS networks . We have found that the autocorrelation function can be described by a single exponential with the same statistical accuracy as for dilute solutions of mono - disperse polymers . The cumulant analysis leads to a value of ( P2 / r2 ) of about 0 . 02 . The decay rate varies as K2 for scattering angles ranging from 60 to 900 . In this range , the scattered signal is fully heterodyned by the inhomogeneities present in the sample , as evidenced by experiments performed using an external oscillator . 4 . 2 . 2 The cooperative diffusion constant . - The experimental values of Dc for PDMS networks are listed in table I . Figure 5 shows the variation of Dc as a function of the polymer concentration of the gel swollen to equilibrium . The data obey approximately the same scaling law as the semi - dilute solutions , with a change of the prefactor value . However a close inspection of figure 5 shows that the data are 1506 FIG . 5 . - Plot of De versus Ce for PDMS networks swollen in toluene . + f = 3 ; 0 f = 4 ; · f = 6 . f refers to the functionality of the crosslinking agent . The straight line represents De = f ( c ) for semi - dilute solutions . rather best accounted for by fitting the data of each series of networks of given functionality separately . One obtains then three slightly shifted straight lines of slopes ranging from 0 . 9 to 1 . 4 . 2 . 3 The compressional modulus . - The data obtained by Belkebir , et al . [ 24 ] for compressional modulus E of PDMS networks swollen in toluene are listed in table I . Figure 6 shows a log - log plot of E versus the equilibrium concentration . The data can be fitted roughly with two slightly shifted straight lines , one line corresponding to the f6 networks , the other one to the f3 and f4 networks . The slope of these straight lines is about 4 . 5 . FIG . 6 . - Compressional modulus E versus ce . + f = 3 ; 01 = 4 ; tbf = 6 . 5 . Discussion . - In the following discussion , we shall summarize the main results of our study , which are quite different from those obtained on polystyrene - benzene systems in many respects , and we shall attempt a conjectural description of the topology of the networks which is able to account for the experimental observations . a ) For semi - dilute solutions of high molecular weight PDMS in toluene , the exponent of the scaling law Dc = f ( c ) is in good agreement with the predicted value , contrary to the results previously obtained in polystyrene systems . This observation may be related to the elastomeric nature of the PDMS chain , which is much more flexible than that of the polysty - rene . An alternative explanation can be inferred from the rather poor solvent quality of the toluene for PDMS , as shown by the low value of the second virial coefficient ( cf . Table II ) , which is of the same order of magnitude as that of polystyrene in cyclo - hexane at a temperature of approximately 200 above the theta temperature . In theta solvent , the coherence length is proportional to the concentration . It may be possible that the behaviour of PDMS solutions in toluene is intermediate between those of theta regime and good solvent regime , resulting in a value of the critical exponent between 0 . 67 and 1 . An unequi - vocal answer would be given by a study of self - diffusion coefficient Do as a function of molecular weight for a series of monodisperse samples in dilute solutions . Indeed , if the system PDMS - toluene obeys the good solvent scaling laws theory , then Do should vary according to MO . 6 . The exponent b of the power law Do oc M - b can also be obtained from the expo - nent a in the viscosity Mark - Houwink equation [ 1 ] oc Ma through the relationship [ 16 ] : b = ( a + 1 ) / 3 . Values of b calculated from literature viscosity data range from 0 . 57 [ 29 ] to 0 . 61 [ 30 ] for PDMS samples of high molecular weights ( & # x3E ; 20 000 ) in toluene . These results give strong evidence that in PDMS toluene systems one observes good solvent behaviour , the values of critical exponents being those predicted by the theory . On the other hand , for PDMS of low molecular weights ( 20 000 ) the measured values of b range from 0 . 5 [ 31 ] to 0 . 53 [ 24 ] , indicating poor solvent behaviour . b ) For swollen networks one observes , in the first approximation , a scaling law for Dc = f ( ce ) . This result , which confirms those previously obtained from polystyrene gels , implies that the collective modes of networks swollen at their equilibrium state are also controlled by the correlation length j associated with the equilibrium concentration Ce , There is , however , a systematic upward shift of the curve Dc = f ( ce ) relative to the networks , with respect to the De = f ( c ) curve of the semi - dilute solutions . This shift may be attributed to a différence in the equilibrium conditions : swollen networks are studied in the presence of an excess of solvent and as a conse - quence the chemical potential of the solvent within the network is the same as that of the pure solvent . On the other hand , in the semi - dilute solution the 1507 chemical potential of the solvent is lowered with respect to that of the pure solvent . Experimental investigations of self - diffusion of polymers have shown that the ratio of hydrodynamic radius to radius of gyration of a coil was quite sensitive to the quality of the solvent [ 32 ] . Such an effect could be at the origin of the observed change of the prefactor in the concentration dependence of Dc . c ) The static compressional modulus does not follow the scaling law predicted by the theory . First , let us recall the basic assumptions leading to the E oc C2 . 2 5law . The compressional modulus is pro - portional to the number of elastic chains per unit volume , that is : For good solvents ce oc Me - O’s if one assumes that all the elastic chains are mutually excluded , and therefore E oc c ; ’ 25 . Deviations from the predicted law may result from the presence of pendant chains attached by one end to the network , since these chains would not contribute to the elasticity of the network . Such an effect can be ruled out , at least partially , for the samples investigated here , since the compressional modulus of the same PDMS networks swollen by a good solvent ( heptane ) approximately obey the c2 - 21 law [ 8 ] . We should therefore look for another explanation which would probably involve the quality of the solvent . As a matter of fact one expects a larger value of the exponent of E ( c , , ) for networks swollen in a poor solvent . Let us consider for instance a gel in which we have replaced the good solvent by a theta solvent . The new swelling equilibrium ce ( in ’the presence of an excess of solvent ) will vary as Me - o . 5 , if one assumes that the elastic chains do not inter - penetrate each other . Then from equation ( 17 ) , E oc cé . However , there is some experimental evi - dence of interpenetration of chains in networks swollen by poor solvents . Neutron scattering experi - ments performed by Duplessix , et al . [ 33 ] on labelled polystyrene networks have shown that the deswelling , resulting from an interchange of a good solvent by a poor solvent is much larger than the variation of the cubic power of the radius of gyration of the elastic chains . In other words , the macroscopic deswelling of the gel exceeds that of the individual chains , resulting in an interpenetration of the equi - valent spheres of radius RF . Therefore , one could not predict anymore the variation of ce as a function of Me . The influence of the quality of the diluent on the behaviour of ce is demonstrated in the PDMS net - works by the variations of Ce as a function of the mole - cular weight Mp of the polymer precursor . In figure 7 we have plotted ce versus Mp for PDMS networks swollen in heptane and in toluene , respectively . FIG . 7 . - Plot of ce versus the molecular weight of the polymer precursor , for tetrafunctional PDMS networks swollen in to - luene ( + ) and heptane ( 0 ) . These networks have been prepared at a concentration c ; = 67 % . The lines are guides for the eye . Although Mp may differ from Me because of trapped entanglements , the relative behaviour of the two sets of data is still significant . One clearly sees that the exponent of ce = f ( Mp ) is much smaller when the diluent is toluene . It is reasonable to assume that ce = f ( Mp ) and ce = f ( M , , ) are correlated . Accord - ing to equation ( 17 ) , a smooth decrease of ce as a function of Me , would result in a sharp increase of E with ce , as observed experimentally . Furthermore , equation ( 17 ) predicts that , for a given network , the ratio Elce should not depend on the nature of the swelling diluent . We have compared the experimental values of ( E / ce ) for a series of tetrafunctional networks swollen in toluene and in heptane , respectively , and we have obtained the following relationship : ( E / ce ) toluene = ( 1 . 2 ± 0 . 1 ) ( E / ce ) heptane . The interpenetration of the chains in the PDMS networks swollen by toluene is presumably favoured by the low molecular weights of the precursor poly - mers ( 4 500 - 17 100 ) . We have seen earlier that in this range of molecular weights , the toluene behaves as a poor solvent of PDMS . Furthermore , the influence of the cross - links on the chain dimensions may also be non - negligible as discussed below . d ) Influence of the experimental conditions of preparation of the networks . Previous investigations of gel elasticity have shown that the elastic properties of swollen networks are strongly dependent on the experimental conditions of crosslinking [ 34 ] . It is generally believed that the crosslinking process exerts an influence on the chain dimensions and a reference state has been defined , in which the chain dimensions are such that they do not exert any elastic forces on the cross - links [ 34 ] . This state must depend on the experimental proce - dure of cross - linking and also on the nature of the swelling diluent . In general , the chains in a network 1508 may not actually be in the relaxed state at the swelling equilibrium . It has been assumed that the reference state refers to the conditions of the network for - mation : thus , the reference degree of swelling is equal to the state of dilution at crosslinking [ 35 ] . The influence of the concentration c ; of polymer at the gel point is illustrated in figure 8 where we have plotted the modulus E and the diffusion constant D , , as a function of c ; for a series of tetrafunctional networks prepared from a precursor polymer of molecular weight 4 500 and swollen in toluene . These results , which show a decrease of the dimensions of the elastically effective chains as the initial concen - tration ci increases , are consistent with our basic assumption which - stipulâtes that the equilibrium elastic properties are mainly accounted for by the amount of trapped entanglements rather than by FIG . 8 . - Plots of D , , , and E for tetrafunctional PDMS networks swollen in toluene , as a function of the polymer concentration at the gel point . The lines are guides for the eye . chain déformation ; the number of trapped entangle - ments presumably increases with the number of labile entanglements existing prior to crosslinking , which in turn increases with the initial concentration c ; . The amount of trapped entanglements also increases with the functionality of the crosslinking agent . However , one cannot discard the existence of déformation of the chains due to memory effects . These effects could be at the origin of the difference discussed above , between the macroscopic deswelling of the gel and the deswelling of the individual chain , when one replaces a good diluent by a poor diluent . 6 . Conclusion . - The overall conclusion from this study is that networks formed in the presence of a good diluent show a behaviour markedly dependent on the nature of the swelling agent . For PDMS networks swollen in toluene the dimensions of the elastically effective chains can no longer be identified with the correlation length relative to a semi - dilute solution at a concentration ce equal to that of swelling equilibrium , as in the case of a good diluent . Both number density of elastic chains and dynamical correlation length associated with the diffusion cons - tant De depend on the stage of dilution at cross - linking . This effect can be related to the amount of trapped entanglements . A systematic study of the influence of the nature of both the solvent of preparation and the swelling diluent on the properties of the networks swollen to equilibrium should allow a more comprehensive description of the topology of gels . Acknowledgments . - The authors wish to thank Professor H . Benoit for very helpful discussions . References [ 1 ] TANAKA , T . , HOCKER , L . and BENEDEK , G . B . , J . Chem . Phys . 59 ( 1973 ) 5151 . [ 2 ] MC ADAM , J . D . G . , KING , T . A . and KNOX , A . , Chem . Phys . Lett . 26 ( 1974 ) 6 . [ 3 ] CARLSON , F . D . and FRASER , A . , J . Mol . Biol . 89 ( 1974 ) 273 . [ 4 ] WUN , K . L . and CARLSON , F . D . , Macromolecules 8 ( 1975 ) 190 . [ 5 ] MUNCH , J . P . , CANDAU , S . , DUPLESSIX , R . , PICOT , C . and BENOIT , H . , J . Physique Lett . 35 ( 1974 ) L - 239 . [ 6 ] MUNCH , J . P . , CANDAU , S . , DUPLESSIX , R . , PICOT , C . , HERZ , J . and BENOIT , H . , J . Polym . Sci . 14 ( 1976 ) 1097 . [ 7 ] MUNCH , J . P . , CANDAU , S . and HILD , G . , J . Polym . Sci . 15 ( 1977 ) 11 . [ 8 ] MUNCH , J . P . , CANDAU , S . , HERZ , J . and HILD , G . , J . Physique 38 ( 1977 ) 971 . [ 9 ] ADAM , M . , DELSANTI , M . and JANNINK , G . , J . Physique Lett . 37 ( 1976 ) L - 53 . [ 10 ] ADAM , M . and DELSANTI , M . , Macromolecules , to be published . [ 11 ] GEISSLER , E . and HECHT , A . M . , J . Chem . Phys . 65 ( 1976 ) 103 . [ 12 ] DE GENNES , P . G . , Cours , Collège de France , 1973 , unpu - blished . [ 13 ] DE GENNES , P . G . , Macromolecules 9 ( 1976 ) 587 . [ 14 ] ORNSTEIN , L . S . and UHLENBECK , G . E . , Reprinted in Selected Papers on Noise and Stochastic Processes ( N . Wax , Ed . Dover , New York ) 1974 , p . 93 - 112 . [ 15 ] See , for instance , Photon Correlation and Light Beating Spec - troscopy , H . Z . Cummins and E . R . Pike , Ed . ( Plenum Press , New York ) 1974 . [ 16 ] FLORY , P . , Principles of Polymer Chemistry ( Cornell Un . Press , Ithaca N . Y . ) 1967 . [ 17 ] DES CLOIZEAUX , J . Physique 31 ( 1970 ) 715 . [ 18 ] DE GENNES , P . G . , Phys . Lett . 38A ( 1972 ) 339 . [ 19 ] HERZ , J . , BELKEBIR - MRANI , A . and REMPP , P . , Eur . Polym . J . 9 ( 1973 ) 1165 . [ 20 ] BELKEBIR - MRANI , A . , HERZ , J . and REMPP , P . , Makromol . Chem . 178 ( 1977 ) 485 . [ 21 ] WEISS , P . , HERZ , J . and REMPP , P . , Makromol . Chem . 141 ( 1971 ) 145 . [ 22 ] CLUFF , E . F . , CLADDING , E . K . and PARISER , R . , J . Polym . Sci . 45 ( 1960 ) 341 . [ 23 ] VAN DE KRAATS , E . J . , Thesis , Delft ( 1967 ) . 1509 [ 24 ] BELKEBIR - MRANI , A . , BEINERT , G . , HERZ , J . and REMPP , P . , Eur . Polym . J . 13 ( 1977 ) 277 . See also A . BELKEBIR - MRANI , Thesis , Strasbourg 1976 . [ 25 ] KOPPEL , D . E . , J . Chem . Phys . 57 ( 1972 ) 4814 . [ 26 ] BROWN , J . C . , PUSEY , P . N . and DIETZ , R . , J . Chem . Phys . 62 ( 1975 ) 1136 . [ 27 ] DAOUD , M . , COTTON , J . P . , FARNOUX , B . , JANNINK , G . , SARMA , G . , BENOIT , H . , DUPLESSIX , R . , PICOT , C . and DE GENNES , P . G . , Macromolecules 8 ( 1975 ) 804 . [ 28 ] DUBOIS - VIOLETTE , E . , DE GENNES , P . G . , Physics 3 ( 1967 ) 181 . [ 29 ] TAKIMOTO , H . H . , FORBES , C . T . and LANDENSCHLAGER , R . K . , J . Appl . Polym . Sci . 5 ( 1961 ) 153 . [ 30 ] VON HAUG , A . and MEYERHOFF , G . , Makromol . Chem . 53 ( 1962 ) 91 . [ 31 ] BIANCHI , U . , DALPIAZ , M . and PATRONE , E . , Makromol . Chem . 80 ( 1964 ) 112 . [ 32 ] ALLEN , G . , VASUDEVAN , P . , HAWKINS , Y . and KING , T . A . , J . Chem . Soc . Faraday Trans . II 4 ( 1977 ) 449 . [ 33 ] BENOIT , H . , DECKER - FREYSS , D . , DUPLESSIX , R . , PICOT , C . , REMPP , P . , COTTON , J . P . , FARNOUX , B . , JANNINK , G . and OBER , R . , J . Polym . Sci . A2 14 ( 1976 ) 2119 . [ 34 ] DUSEK , K . and PRINS , W . , Adv . Polym . Sci . 6 ( 1 ) ( 1969 ) 1 . [ 35 ] LUTZ , , P . , PICOT , C . , HILD , G . and REMPP , P . , Br . Polym . J . , to be published . \ No newline at end of file diff --git a/silver_data/6be599602b063b9d73a6d2ad4e315de7722df2b0.pdf.txt b/silver_data/6be599602b063b9d73a6d2ad4e315de7722df2b0.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..c04cf5ea72535c98d651d634eb8ba07de3dfcf9c --- /dev/null +++ b/silver_data/6be599602b063b9d73a6d2ad4e315de7722df2b0.pdf.txt @@ -0,0 +1 @@ +PLASMA MEMBRANE FOLDS ON THE MAST CELL SURFACE AND THEIR RELATIONSHIP TO SECRETORY ACTIVITY SUSAN JO BURWEN and BIRGIT H . SATIR From the Department of Physiology - Anatomy , University of California , Berkeley , California 94720 . Dr . Burwen ' s present address is the Cancer Research Laboratory , University of California , Berkeley , California 94720 . Dr . Satir ' s present address is the Department of Anatomy , Albert Einstein College of Medicine , Bronx , New York 10461 . ABSTRACT Changes in the surface morphology of secreting mast cells have been followed by scanning electron microscopy . Mast cells isolated from the rat peritoneal cavity have folds of plasma membrane that form snake - like ridges on their surfaces . Fold length varies considerably from cell to cell , whereas fold width and depth appear to remain relatively constant . To assess the possible relationship between secre - tory activity and surface folding , a semiquantitative method was used for measur - ing fold length in control and secreting populations . A positive correlation is found between secretion of histamine and the extent of membrane folds on the mast cell surface . The source of the membrane required for fold formation is probably secretory granule membrane incorporated into the plasma membrane as a result of exocytosis . Furthermore , a distinct cell type devoid of surface folds , designated as a raspberry - type cell , is found to occur as an integral part of a normal population of mast cells . This cell type is resistant to stimulation by polymyxin . KEY WORDS mast cell - histamine secretion membrane folds (cid:12)9 cell surface morphology (cid:12)9 scanning electron microscopy The surface features of a cell are an expression of the cell ' s response to its internal and external environment . Cell surfaces change as a function of altered physiology , for example , during cell cycle ( 18 ) , in transformed cells ( 19 ) , in response to environmental irritants ( 17 ) , etc . The experiments reported here were undertaken to determine a possible relationship between surface features and secretory activity in the rat peritoneal mast cell . The mast cell synthesizes and stores histamine , among other secretory products , and undergoes compound exocytosis when stimulated to secrete ( 6 ) . Mast cells observed in the scanning electron microscope ( SEM ) have , as characteristic surface features , folds of plasma membrane that form curved ridges in anastomosing patterns ( 3 , 9 , 23 ) , corresponding to microvillus - like projections ob - served in thin sections ( 11 , 20 ) . The folds are also visible on freeze - fracture faces of plasma mem - brane as cross - fractured areas forming snake - like patterns ( 4 ) . Previous studies of the mast cell in SEM ( 9 , 23 ) reported the presence of these folds primarily on unstimulated cells . A cell type , de - void of folds , was also described , designated as the raspberry - type cell , which was dismissed as being an artifact of the preparation ( 9 ) . When cells were exhaustively stimulated to secrete , they lost their surface folds , developed deep cavities , and par - tially collapsed , revealing sharp contours of under - lying granules . The dosages of secretagogue and incubation times used in these previous studies were many times in excess of the conditions re - 690 THE JOURNAL OF CELL BIOLOGY (cid:12)9 VOLUME 74 , 1977 " pages 690 - 697 quired to obtain 100 % histamine release ( 12 ) . In the experiments reported here , intermediate stages of secretion are obtained by using milder conditions of stimulation , i . e . , lower dosage of secretagogue , shorter incubation times , and lower temperature . Histamine release is measured to assess the secretory activity of the cells . In this way , changes in the surface of secreting mast cells can be studied relative to the secretory activity of the population . Since the mast cell is essentially nonmitotic , cell cycle is not a factor in influencing its surface features . Therefore , in this case , changes in surface features probably reflect other physiological responses , the most likely of which is secretory activity . MATERIALS AND METHODS Cell Collection and Incubation Mast cells are collected from the peritoneal cavities of exsanguinated male rats , 150 - 180 g , of the Long - Evans strain ( U . C . Berkeley colony ) by recovering phosphate - buffered saline ( PBS ) ( pH 7 . 2 ) pipetted into the cavities ( 20 ) . The cell suspensions , initially containing ~ 5 % mast cells , are enriched to > 70 % mast cells by using discontinuous Ficoll ( Pharmacia Fine Chemicals , Pisca - taway , N . J . ) gradients ( 1 ) . Mast cells derived from three to eight rats are pooled and resuspended in PBS at 180C . A controlled amount of histamine secretion is obtained by adding polymyxin B sulfate ( 4 / . tg / ml of incubation mixture ) . Samples are taken 60 s after stimulation for assay of histamine release and examination in SEM . Histamine Assay Ice - cold PBS is added to each sample at the end of an incubation period to stop secretion . The samples are centrifuged ( 200 g , 4 ~ 5 min ) and histamine determi - nations are made on each supernate and pellet pair by the method of Bergendorff and Uvn ~ is ( 2 ) . Histamine is completely extracted from each pellet by boiling in 0 . 1 N HCI for 5 min . Dilutions are made with H20 . Samples of supernate containing undiluted PBS form precipitates during the assay which do not interfere with determina - tions if removed by centrifugation or filtration before reading . Histamine release is expressed as the percent - age of histamine in the supernate ( supernatant histamine plus pellet histamine equals 100 % ) . Sample Preparation for Scanning Electron Microscopy Mast cells are fixed in suspension with ice - cold 2 % glutaraldehyde in 0 . 2 M Millonig ' s buffer ( pH 7 . 4 ) and kept at room temperature for 1 h , then washed once ( Millonig ' s buffer plus 6 % sucrose ) and postfixed for 1 h in 1 % OsO4 in Millonig ' s buffer at 4 ~ The cells are kept in suspension throughout alcohol dehydration , transferral to 100 % Freon TC ( E . I . du Pont de Nem - ours & Co . , Wilmington , Del . ) , and placement in B . E . E . M . capsule chambers ( Better Equipment for Electron Microscopy , Inc . , Bronx , N . Y . ) with Flotron - ics silver membrane filters ( Selas Corp . of America , Spring House , Pa . ) ( 0 . 45 / zm pore size ) covering one end . The cells are critical - point dried in Freon 13 ( E . I . du Pont de Nemours & Co . ) . Flotronics filters give high rates of recovery for cell suspensions ( 7 ) . Surface arti - facts caused by Flotronics filters ( 25 ) are avoided by fixation of cells before contact with them ( 7 ) . Polylysine ( 14 ) is not used for attachment of cells to glass cover slips because of the possible introduction of surface artifacts ( 16 ) . Pieces of filter containing the critical - point dried cells are glued onto specimen stubs with silver paste , coated with gold under vacuum on a tilting - rotating stage , and examined in an ETEC or Coates and Welter CWlC scanning electron microscope . Micrographs are taken with Polaroid type 55 P / N film . RESULTS Mast cells observed in SEM have folds of plasma membrane that project from their surfaces . The folds form snake - like ridges in anastomosing pat - terns ( 3 , 9 , 23 ) . In between folds , contours of underlying granules are visible . No microvilli are observed ( Fig . 1 ) . Therefore , the surface projec - tions from mast cells which were previously identi - fied as microviUi in thin sections ( 11 , 20 ) corre - spond to these folds of plasma membrane viewed in cross section . The folds appear to be fairly uniform in width on the same cell and from one cell to another , averaging ~ 0 . 1 / zm . Fold depth , as viewed at the cell edge , varies to some extent , ranging from 0 . 3 to 1 . 0 / zm with 70 - 80 % of the folds - 0 . 8 / ~ m deep . Fold length is the dimension which shows the greatest variability from one cell to another . Differences in fold length are extremely apparent from cell to cell , whereas fold width and depth tend to remain fairly constant . Variability in fold length is the factor which most accounts for the heterogeneous appearance of the mast cell in SEM with respect to surface features . The cells in Fig . 1 are arranged in a sequence of increasingly folded surfaces ( increas - ing fold length ) . Some cells lack folds entirely ( the raspberry - type cell ) ( Fig . 1 A ) and the contours of underlying secretory granules are visible . Cells with extensively folded surfaces ( Fig . 1 C ) often have the extruded contents of several granules adhering to them ( arrow ) , while underlying gran - ule contours are almost totally obscured by folds . S . J . BU ~ WEN AND B . H . SAriS Plasma Membrane Folds on the Mast Cell Surface 691 Furthermore , the complete range of cell surface appearances is present in both control and stimu - lated populations . The secretion of histamine by mast cells , under the conditions of stimulation used ( incubation in PBS at 18 ~ for 60 s in the presence of 4 / ~ g / ml polymyxin B sulfate ) , was 64 . 2 + - - 4 . 0 % ( n = 3 ) , while the corresponding histamine release by con - trol cells , incubated in the absence of polymyxin B sulfate , was 9 . 9 - - - 1 . 0 % ( n = 4 ) . To determine whether the surface features of mast cells change as a result of stimulation and secretion , a qualitative evaluation of the extent of folding was carried out as follows : Each of two preparations of cells was divided into control and stimulated populations . 50 cells from each popula - tion were randomly selected in SEM at low magni - fication ( (cid:141) 100 ) at which any surface detail is indistinguishable . Magnification was then in - creased to (cid:141) 10 , 000 , and if the cells met the criteria of being undamaged , spherical , and unob - structed , they were photographed . In this manner , 200 cells were systematically photographed . For each cell , a number was assigned on a scale of 1 to 3 , depending on the extent of folds on the cell surface . Category 1 represents cells with very few folds ( including raspberry - type cells ) , category 2 , cells with an intermediate range of folds , and category 3 , cells with extensively folded surfaces . The three cells in Fig . 1 are representative of each category . The numbers of cells falling into each category were compared in the paired control and stimulated populations ( Table I ) . The results show that more cells have extensively folded surfaces in the stimulated populations than in the control populations . To confirm the results obtained with the qualita - tive evaluation , a semiquantitative method was developed for measuring fold length per cell . If the simplifying assumption is made that fold width and depth are relatively uniform from cell to cell , then fold length becomes the variable that allows direct comparison of one mast cell to another . 100 randomly selected cells from a third preparation , divided into control and stimulated populations , FIGURE 1 The various appearances of the mast cell surface in SEM . These cells are taken from both control and stimulated populations and are arranged in order of increasingly folded surfaces . 1A : Category 1 : a rasp - berry - type cell ; 1 B : Category 2 : cell with an intermedi - ate range of folds ; 1 C : Category 3 : cell with an exten - sively folded surface . Several extruded granules are seen on the cell surface ( arrow ) . All x 8 , 750 . 692 ThE JOURNAL OF CELL BIOLOGY " VOLUME 74 , 1977 were photographed as described above , and mi - crographs were printed at a . final magnification of x 20 , 000 . Fold length measurements were car - ded out according to the method described in Fig . 2 . For each cell , a ratio of fold length in microme - ters per square micrometers of surface area was calculated , and the distributions of cells according to these ratios were compared in the paired con - trol and stimulated populations ( Fig . 3 ) . Fold length varies over a range from 0 / zm / / zm 2 surface area ( raspberry - type cells ) to - 2 . 4 / . tm / / zm 2 sur - face area , and the distribution resembles a poisson distribution ( Fig . 3 a ) . However , the proportion of TABLE I Qualitative Evaluation of Surface Folding Relative to Secretory Activity Category 1 : Category 2 : Category 3 : very few intermediate extensive Population folds folds folds no . of cells Control 11 23 16 Stimulated * 13 12 25 Control 11 31 8 Stimulated * 12 23 15 * Stimulated with polymyxin B sulfate , 4 / xg / ml , at 18 ~ for 60 s . raspberry - type cells , which varies from 6 to 12 % , is higher than would be predicted from a typical poisson distribution . There is an overall shift in distribution as a result of stimulation ( Fig . 3b ) , again demonstrating a positive correlation be - tween secretion of histamine and the appearance of membrane folds . The average length of folds per cell for the stimulated population increases - 23 % as a result of secretion of 64 % of the histamine present ( Ta - ble II ) . The relationship between the secretory activity of a population and cell size is shown in Table III . The average cell radius decreases ~ 5 % upon stimulation . However , this shrinkage alone cannot account for the above increase in mem - brane fold length ( Fig . 4 ) . At most , 0 . 07 / zm folds / / xm 2 surface area can be generated from the membrane area made available by cell shrinkage . Since the average increase in fold length from control to stimulated cell is - 0 . 3 / ~ m / / ~ m 2 surface area ( Table II ) , the 0 . 07 / zm folds / ~ m 2 can ac - count for , at most , - 23 % of the 0 . 3 / zm / p . m 2 membrane needed to generate the folds . Prolonged stimulation of cells for 15 min ( still at 18 ~ results in extensive cell damage and lysis . Cell recovery is checked by counting cells in a hemacytometer . After prolonged stimulation , - 27 % of the cells lyse and are not even recovered FmURE 2 Method for measuring fold length on mast cells . 2 A : A clear plexiglass circle with concentric rings etched on it at 1 - cm intervals is superimposed over a scanning micrograph of a mast cell originally printed at (cid:141) 20 , 000 and reproduced here at x 8 , 750 . The center point and radius of the cell are deter - mined . 2 B : A concentric circle whose radius is 80 % of the radius of the cell is drawn on the micrograph . The area falling within this circle corresponds to 20 % of the total surface area of a smooth sphere . The folds of membrane lying within this circle are traced with a map reader to obtain a linear measurement . The data for each cell are expressed as a ratio of fold length ( / zm ) to sphere surface area ( / tin2 ) . (cid:141) 8 , 750 . S . J . BURWEN AND B . H . SAaTa Plasma Membrane Folds on the Mast Cell Surface 693 o " ) J J ILl o ii 0 hi ( . 9 I ' - - Z LIJ , { . . 3 n " U . I O - 50 - o - - o CONTROL x - - x POLYMYXIN - STIMULATED x o 0 . 4 o . 8 Lz ~ . 6 z . o 2 . 4 FOLDS ( A ~ . m ) : SURFACE AREA ( , , tm 2 ) 80 o0 . . . J , , fi 60 ( , . . ) u _ o N 4o b - Z W ~ 2o w 0 - 1 . 5 1 . 4 - 2 . 2 § CONTROL I POLYMYXIN - STIMULATED | FOLDS ( Am ) : SURFACE AREA ( # m z ) FIGURE 3 The distribution of cells according to their membrane surface folds in paired control and stimulated populations . 3 a : The cells are arranged in categories of 0 . 4 - / zm increments of their ratios of fold length to sur - face area . The shift in distribution after stimulation is emphasized by the stippled vs . striped areas . 3b : The histogram presents the same data as in Fig . 3a , reduced to two categories : 0 - 1 . 3 and 1 . 4 - 2 . 2 + folds ( / zm ) / / zm 2 surface area . TABLE II Length of Folds per Surface Area population Average length of folds wn / iJ , m2 / Cell Control 1 . 3 ( n = 50 ) Stimulated * 1 . 6 ( n = 50 ) Percent Increase 23 % * Stimulated with polymyxin B sulfate , 4 / xg / ml , at 18 ~ for 60 s . for further study . Of the remaining 73 % , ~ 40 % are damaged as revealed by examination in SEM , showing collapse , partial lysis , and loss of plasma membrane integrity . In summary , upon prolonged stimulation , 27 % of the cells are not even re - covered , and 29 % are damaged , leaving only 44 % of the initial population intact . Of this 44 % , a great majority ( > 80 % ) appear to be raspberry - type cells or cells with relatively few folds ( 0 - 1 . 0 / xm / / zm 2 , Fig . 3a ) . Therefore , these remaining cells must be relatively resistant to polymyxin treatment , and the raspberry - type cell appears to be particularly insensitive to polymyxin relative to other mast cells . TABLE III Mast Cell Size as a Function of Secretory State Population Radius Area of a smooth sphere calcu - lated from ra - dius Control 4 . 34 - + 0 . 05 Stimulated * 4 . 10 - + 0 . 05 Percent Decrease 5 . 5 % Membrane available as a result of radius change p / n 2 237 211 26 n = 50 for both populations . The data are expressed as the means of the n , - + SEM . * Stimulated with polymyxin B sulfate , 4 / xg / ml , at 18 ~ for 60 s . d O . 1A . ~ m FmURE 4 Contribution of cell shrinkage , associated with secretion , to increase in membrane fold length . The average width of folds = 0 . 1 tzm , and the average depth of folds = 0 . 8 ttm ( both dimensions approximated from scanning micrographs ) . The length of fold with these dimensions which can be generated from 26 ~ m 2 mem - brane area ( Table III ) is 15 / zm ; ( ~ ' ) L = 26 / zm 2 0 . 8L + 0 . 8L + 0 . 1 ~ L 15 / J , m . 15 / zm per 211 / xm 2 surface area gives a ratio of 0 . 07 / zm / / zmL 694 THE JOURNAL OF CELL BIOLOGY ' VOLUME 74 , 1977 DISCUSSION The mast cell , observed in SEM , has folds of plasma membrane that form snake - like ridges on its surface . These folds correspond to the microvil - lus - like projections previously observed in thin sections . Other cells , observed in SEM as having similar folds , distinct from microvilli , are disso - ciated epithelial cells ( 24 ) . The mast cell folds appear to vary in length from cell to cell to a far greater extent than they vary in width and depth . Some cells lack folds entirely , whereas other cells are completely cov - ered with them . The complete range of cell surface appearances is found in both control and stimu - lated populations . However , qualitative evalua - tion of 200 cells in two separate preparations suggested that more cells have extensively folded surfaces in the stimulated populations than in the control populations . To test this observation fur - ther , a semiquantitative method was devised to compare the extent of plasma membrane folding on control vs . stimulated cells . The length of folds per cell was chosen as the variable allowing com - parison of one mast cell to another . A positive correlation between histamine secretion and fold length was found . This method tends to minimize the difference between control and stimulated cells , because observations in transmission elec - tron microscopy ( 16 ) have tentatively suggested that fold depth may actually increase with stimula - tion . Expansion of the surface area of the mast cell plasma membrane as a result of exocytosis was first reported by Kinsolving et al . ( 10 ) , using an increase in acridone - binding capacity as an indica - tor of newly available membrane surface area . The increase in plasma membrane area was re - lated to histamine secretion ( 15 ) , with 63 % hista - mine release corresponding to a 350 % plasma membrane expansion . The apparent discrepancy between those results and the results presented here is due to the fact that in the experiments of Kinsolving et al . , the internal cavities formed by compound exocytosis are included in the measure - ment . The current observations on plasma mem - brane expansion are limited to the increase in surface area visible as membrane folds on the cell surface . Cell shrinkage associated with secretion will not provide sufficient membrane for generation of folds in stimulated cells . The rapidity of the change ( within 60 s ) probably precludes de novo membrane synthesis as the source of the needed membrane . Therefore , the membrane for fold for - mation probably comes from granule membrane incorporated into plasma membrane after mem - brane fusion and exocytosis . Incorporation of vesi - cle membrane into plasma membrane after release of secretory product has been shown to occur in several secretory systems : in frog neuromuscular junction after prolonged stimulation ( 8 ) , in Tetra - hymena in which synchronous release has been induced ( 21 , 22 ) , in sperm after acrosome dis - charge ( 5 ) , and in the secretory cells of the duck salt gland ( 13 ) . The raspberry - type cell , the cell that lacks mem - brane folds , does not appear to be a damaged cell or an artifact of preparation , in contrast to views previously stated ( 9 , 23 ) . Rather , it is part of the normal mast cell population , and its proportion is higher than would be expected from a poisson distribution . This cell is resistant to damage by prolonged exposure to polymyxin under condi - tions which damage other mast cells . The rasp - berry - type cell may be harder to induce to secrete , due to its having less surface area , or having im - mature membrane which may lack necessary re - ceptor molecules for stimulation . The results sug - gest that cells with greater surface area ( exten - sively folded surfaces ) are more susceptible to stimulation , having perhaps more receptor mole - cules available . One might speculate that addi - tional receptors may be contributed by the interior surface of the granule membrane which , upon fusion , becomes the exterior surface of the plasma membrane . A positive feedback mechanism may be involved in the explosive nature of mast cell secretion at physiological temperature : stimula - tion ~ increase in folds ( receptors ) ~ further stimulation . A positive feedback effect could ac - count for previously stimulated cells being espe - cially susceptible to damage and lysis by further stimulation . Raspberry - type cells may represent different stages of mast cell maturation , during either ( a ) differentiation , or ( b ) recovery after depletion of secretory products . As for the first possibility , a precursor cell of the fully differentiated mast cell still has not been positively identified . With regard to the second possibility , the mast cell should theoretically be able to synthesize and package new product , to " recycle " , because its plasma membrane remains intact and all necessary cyto - plasmic organelles are present . However , the process of recovery after depletion has not been well studied in mast cells . Finally , the raspberry - S . J . BURWEN AND B . H . SATI ~ Plasma Membrane Folds on the Mast Cell Surface 695 type cell may represent a less mature form of a mast cell that is less susceptible to stimulation . The fact that raspberry - type cells can be se - lected for by their ability to survive prolonged stimulation ( 15 min ) may provide a means for isolating them from other cells present . Thus , problems of differentiation , recycling , membrane sites , etc . could possibly be specifically studied in these cells . The authors wish to acknowledge the assistance of Dr . Peter Satir and William Reed in the development of the semiquantitative method for measuring fold length on the mast cell surface , and Dr . J . - P . Revel for the use of the ETEC scanning microscope . This work was supported by U . S . Public Health Service grants GM 02575 , GM 21077 , and GM 24724 . Received for publication 30 August 1976 , and in revised form 26 April 1977 . REFERENCES 1 . BACH , M . K . , K . J . BLOCK , and K . F . AUSTEN . 1971 . IgE and IgGA antibody - mediated release of histamine from rat peritoneal cells . Optimum condi - tions for in vitro preparation of target cells with antibody and challenge with antigen . J . Exp . Med . 133 : 752 - 771 . 2 . BERGENDORFF , A . , and B . UVNT , S . 1972 . Storage of 5 - hydroxytryptamine in rat mast cells . Evidence for an ionic binding to carboxyl groups in a granule heparin - protein complex . Acta Physiol . Scand . 84 : 320 - 331 . 3 . BURWEN , S . J . , and B . SATIR . 1975 . Membrane surface changes during mast cell secretion . J . Cell Biol . 67 ( 2 , Pt . 2 ) : 51 a . ( Abstr . ) . 4 . BURWEN , S . J . , and B . SATIR . 1977 . A freeze - fracture study of early membrane events during mast cell secretion . J . Cell Biol . 73 : 660 - 671 . 5 . COLWIN , A . L . , and L . H . COLWIN . 1963 . Role of the gamete membranes in fertilization in Saccoglos - sus kowalevskii ( Enteropneusts ) . J . Cell Biol . 19 : 477 - 500 . 6 . DOU6LAS , W . W . 1974 . Involvement of calcium in exocytosis and the exocytosis - vesiculation sequence . Biochem . Soc . Syrup . 39 : 1 - 28 . 7 . DE HARVEN ) E . , N . LAMPEN , A . POLLIACK , A . WARFEL , and J . FOGH . 1975 . New observations on methods for preparing cell suspensions for scanning electron microscopy . Scanning Electron Micros # copy / 1975 ( Part I ) . Proceedings of the 8th Annual Scanning Electron Microscope Symposium . IIT Research Institute , Chicago . 361 - 367 . 8 . HEUSER , J . E . , and T . S . REESE . 1973 . Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junc - tion . J . Cell Biol . 57 : 315 - 344 . 9 . KESSLER , S . , and C . KUHN . 1975 . Scanning elec - tron microscopy of mast cell degranulation . Lab . Invest . 32 : 71 - 77 . 10 . KINSOLVaNG , C . R . , A . R . JOHNSON , and N . C . MORAN . 1975 . The uptake of a substituted acridone by rat mast cells in relationship to histamine release : a possible indicator of exocytosis - induced expansion of the plasma membrane . J . Pharmacol . Exp . Ther . 192 : 654 - 669 . 11 . LAGUNOFF , D . 1973 . Membrane fusion during mast cell secretion . J . Cell Biol . 57 : 252 - 259 . 12 . LAOUNOFF , D . , and H . WAN . 1974 . Temperature dependence of mast cell histamine secretion . J . Cell Biol . 61 : 809 - 811 . 13 . LEVEE , A . M . , J . A . HIC , ~ INS , and R . J . BAI ~ NETr . 1972 . Biogenesis of plasma membranes in salt glands of salt - stressed domestic ducklings : localization of acyltransferase activity . J . Cell Sci . 11 : 855 - 873 . 14 . MAZIA , D . , G . SCHATI ' EN , and W . SALE . 1975 . Adhesion of cells to surfaces coated with polylysine . Applications to electron microscopy . J . Cell Biol . 66 : 198 - 200 . 15 . MORAN , N . C . , C . R . KINSOLVlNG , and A . R . JOHNSON . 1975 . Expansion of the plasma mem - brane of rat peritoneal mast cells resulting from exocytosis . Fed . Proc . 34 : 718 . 16 . PADAWER , J . 1970 . The reaction of rat mast cells to polylysine . J . Cell Biol . 47 : 352 - 372 . 17 . PLOPPER , C . G . , D . L . DUNGWORTH , and W . S . TYLER . 1973 . Morphometric evaluation of pulmo - nary lesions in rats exposed to ozone . Am . J . Pathol . 71 : 395 - 408 . 18 . PORTER , K . R . , D . PRESCO ~ , and J . FRYE . 1973 . Changes in surface morphology of Chinese hamster ovary cells during the cell cycle . J . Cell Biol . 57 : 815 - 836 . 19 . PORTER , K . R . , G . J . TODARO , and V . FONTE . 1973 . A scanning electron microscope study of surface features of viral and spontaneous transformants of mouse Balb / 3T3 cells . J . Cell Biol . 59 : 633 - 642 . 20 . R ( SHLICH , P . , P . ANDERSON , and B . UVN ~ , S . 1971 . Electron microscope observations on compound 48 / 80 - induced degranulation in rat mast cells . Evi - dence for sequential exocytosis of storage granules . J . Cell Biol . 51 " 465 - 483 . 21 . SATIR , B . 1974 . Ultrastructural aspects of mem - brane fusion . J . Supramol . Struct . 2 : 529 - 537 . 22 . SATIR , B . 1977 . Dibucaine - induced synchronous mucocyst secretion in Tetrahymena . Cell Biol . Inter - nat . Rep . 1 : 69 - 73 . 23 . TIZARD , I . R . , and W . L . HOLMES . 1974 . Degranu - lation of sensitized rat peritoneal mast cells in re - sponse to antigen , compound 48 / 80 and polymyxin B . A scanning electron microscope study . Int . Arch . 696 THE JOURNAL OF CELL BIOLOGY " VOLUME 74 , 1977 Allergy Appl . Immunol . 46 : 867 - 879 . 24 . VIAL , J . , and K . R . PORTER . 1975 . Scanning mi - croscopy of dissociated tissue cells . J . Cell Biol . 67 : 345 - 360 . 25 . WV . S ~ BOOK , E . , B . WETZEL , G . B . CANr ~ ON , and D . BERARD . 1975 . The impact of culture conditions on the surface morphology of cells in vitro . Scan - ning Electron Microscopy / 1975 ( Part I ) . Proceed - ings of the 8th Annual Scanning Electron Micro - scope Symposium . IIT Research Institute , Chicago . 351 - 360 . S . J . BURWEN AND B . H . SATIR Plasma Membrane Folds on the Mast Cell Surface 697 \ No newline at end of file diff --git a/silver_data/70ef88e6a3781e39ee10e6e820e4c1b0676500c6.pdf.txt b/silver_data/70ef88e6a3781e39ee10e6e820e4c1b0676500c6.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..0f90c901e4debac2c373d9c9d6ffaf748aadef3e --- /dev/null +++ b/silver_data/70ef88e6a3781e39ee10e6e820e4c1b0676500c6.pdf.txt @@ -0,0 +1 @@ +© 2001 Macmillan Magazines Ltd brief communications NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http : / / cellbio . nature . com 215 Caenorhabditis elegans auxilin : a J - domain protein essential for clathrin - mediated endocytosis in vivo Tsvika Greener * † , Barth Grant†‡ , Yinhua Zhang‡ , Xufeng Wu * , Lois E . Greene * , David Hirsh‡ and Evan Eisenberg * § * Laboratory of Cell Biology , NHLBI , NIH , Bethesda , Md 20892 - 0301 ‡Columbia University College of Physicians and Surgeons , Dept . of Biochemistry and Molecular Biophysics , NY , NY 10032 †These authors contributed equally to this work § e - mail : eisenbee @ nhlbi . nih . gov The budding of clathrin - coated vesicles is essential for protein transport . After budding , clathrin must be uncoat - ed before the vesicles can fuse with other membranous structures . In vitro , the molecular chaperone Hsc70 uncoats clathrin - coated vesicles in an ATP - dependent process that requires a specific J - domain protein such as auxilin . However , there is little evidence that either Hsc70 or auxilin is essential in vivo . Here we show that C . elegans has a single auxilin homologue that is identical to mam - malian auxilin in its in vitro activity . When RNA - mediated interference ( RNAi ) is used to inhibit auxilin expression in C . elegans , oocytes show markedly reduced receptor - mediated endocytosis of yolk protein tagged with green fluorescent protein ( GFP ) . In addition , most of these worms arrest during larval development , exhibit defective distri - bution of GFP – clathrin in many cell types , and show a marked change in clathrin dynamics , as determined by fluorescence recovery after photobleaching ( FRAP ) . We conclude that auxilin is required for in vivo clathrin - mediated endocytosis and development in C . elegans . T wo mammalian J - domain proteins 1 that support uncoating of clathrin - coated vesicles by Hsc70 in vitro have been discovered — auxilin 2 , 3 , which is nerve - specific , and cyclin - G - associated kinase ( GAK ) , which is ubiquitous 4 , 5 . In addition to a carboxy - ter - minal J - domain preceded by a clathrin - binding domain and a tensin domain 6 , GAK also contains an amino - terminal kinase domain 7 . A search of the complete C . elegans genome revealed a single predict - ed protein of relative molecular mass 107 , 000 ( M r 107K ; W07A8 . 3 ) as an auxilin homologue . C . elegans auxilin shares ~ 18 % amino - acid identity with mammalian auxilin , and the M r 25K C - terminal por - tion of the molecule , which includes the J - domain , shares ~ 32 % amino - acid identity with mammalian auxilin . For in vitro studies , we used a truncated fragment of C . elegans auxilin ( CeAUX - 53 ) that contains the M r 53K C - terminal domain , including the putative clathrin - binding domain and the J - domain , as we had previously established that the equivalent fragment of mammalian auxilin retains full uncoating activity 4 . We first tested whether CeAUX - 53 polymerizes clathrin . We found that , like mam - malian auxilin 4 , CeAUX - 53 polymerizes clathrin at a roughly 1 : 1 molar ratio of CeAUX - 53 to clathrin ( Fig . 1a ) . The structure of clathrin baskets polymerized by CeAUX - 53 appears to be normal , as shown by electron microscopy ( Fig . 1b ) . We next tested whether CeAUX - 53 carries out the other princi - pal functions of mammalian auxilin in vitro , and in particular whether it acts catalytically to support uncoating of clathrin baskets by Hsc70 at pH 7 and binding of Hsc70 to clathrin baskets at pH 6 . To test uncoating , we prepared auxilin - free clathrin baskets with double - purified clathrin and an M r 58K C - terminal recombinant fragment of the brain - specific assembly protein AP - 180 . At pH 7 in the presence of ATP , CeAUX - 53 supported uncoating by Hsc70 cat - alytically rather than stoichiometrically ( Fig . 1c ) . Similarly , at pH 6 in the presence of ATP , CeAUX - 53 acted catalytically to induce binding of Hsc70 to pure clathrin baskets ( Fig . 1d ) . The latter data indicate that , like mammalian auxilin and GAK , C . elegans auxilin may support uncoating by inducing Hsc70 to bind to clathrin bas - kets . Furthermore , C . elegans auxilin acts quantitatively as well as qualitatively like mammalian auxilin and GAK in its interaction with Hsc70 and clathrin baskets in the presence of ATP 2 – 5 . We next investigated whether C . elegans auxilin is required for receptor - mediated endocytosis in vivo . We monitored endocytosis by following the uptake into growing oocytes of the C . elegans yolk protein YP170 with a C - terminal fusion to GFP ( YP170 – GFP ) 8 . YP170 is expressed at high levels in the hermaphrodite intestine and secreted into the body cavity of the worm . From there it is taken up by receptor - mediated endocytosis into the oocytes and stored in membrane - bound vesicles . Yolk uptake by C . elegans oocytes is mechanistically similar to endocytosis of low - density lipoprotein in mammalian systems 8 . Monitoring the uptake of YP170 – GFP by oocytes in C . elegans provides a new screen for C . elegans proteins that are required for receptor - mediated endocytosis . To inhibit expression of auxilin , we used RNAi with C . elegans auxilin double - stranded RNA 9 , 10 . Comparison of wild - type and auxilin ( RNAi ) worms showed that in untreated animals YP170 – GFP was found in the intestine ( where it is synthesized ) , and in the almost full - grown oocytes and embryos , with very little present in the body cavity ( Fig . 2a , c ; see also ref . 8 ) . In contrast , in auxilin ( RNAi ) worms YP170 – GFP was found in the intestine and at very high levels in the body cavity , but very little accumulated in the oocytes or embryos ( Fig . 2b , d ) . Thus , uptake of YP170 – GFP by oocytes , but not secretion from the intestine , is severely reduced in auxilin ( RNAi ) animals . These results indicate that C . elegans aux - ilin is required for receptor - mediated endocytosis . Further evidence of this requirement comes from our observa - tion that most of the progeny of auxilin ( RNAi ) worms arrest devel - opment during the L1 and L2 larval stages ( Fig . 2e , f ) . After 3 – 4 days as L1 or L2 larvae , by which time wild - type controls have already reached adulthood , these animals develop vacuolated and constipated intestines and many have a double cuticle ( Fig . 2i , j ) , indicating a moulting defect . Most of these animals eventually die at varying larval stages ; a minority of the auxilin ( RNAi ) animals eventually become necrotic , scrawny adults , some of which are ster - ile . The most obvious defect in the intestine of auxilin ( RNAi ) lar - vae , before necrosis sets in , is the accumulation of highly refractive © 2001 Macmillan Magazines Ltd brief communications NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http : / / cellbio . nature . com 216 cytoplasmic granules ( Fig . 2h ) , which are not present in wild - type animals ( Fig . 2g ) . Thus , malfunction of the intestine may be the primary cause of death for auxilin ( RNAi ) animals . One possibility is that the transport of hydrolytic enzymes to the intestinal lyso - somes from the Golgi , which is a clathrin - mediated process , is defective in these intestinal cells , leading to an inability to break down nutrients after feeding . It is interesting , with regard to the double cuticle of auxilin ( RNAi ) larvae ( Fig . 2i , j ) , that mutation of LRP - 1 , the C . elegans megalin receptor , also arrests C . elegans dur - ing larval development by preventing moulting 11 . This raises the possibility that clathrin - mediated endocytosis may be required for proper functioning of the LRP - 1 receptor . Alternatively , interfer - ence with clathrin - mediated endocytosis might cause missorting of the Kex2 homologue , a furin - like endoprotease that is required for maturation of cuticle collagens 12 . However , as yet there is no evi - dence that defects in clathrin - based transport of vesicles from the Golgi to the endosome affects the localization of Golgi - resident endoproteases . We were unable to establish the function of auxilin in the C . elegans nervous system , because RNAi is generally ineffec - tive in C . elegans neurons 13 . We next determined how inhibition of auxilin expression in C . elegans affects the distribution of clathrin . We monitored clathrin distribution in wild - type and auxilin ( RNAi ) larvae using expres - sion of a transgene encoding a C . elegans clathrin heavy chain with an N - terminal fusion to GFP ( GFP – CHC ) . Although we were unable to monitor GFP – CHC fluorescence in oocytes because transgenes are not expressed in C . elegans germ cells 14 , we did find that wild - type L1 larvae showed weakly diffuse or somewhat punc - tate GFP – CHC fluorescence in many cell types , including coelo - mocytes ( Fig . 3a ) , pharyngeal cells ( Fig . 3c ) and hypodermal cells ( Fig . 3e ) . In contrast , auxilin ( RNAi ) L1 larvae exhibited much brighter , more punctate GFP – CHC fluorescence in all somatic cell types examined ( Fig . 3b , d , f ) . The increase in the number of punc - ta in the posterior lobe of the pharynx ( compare Fig . 3e and f ) , as well as higher magnification of the puncta in the hypodermis , make this particularly clear ( compare Fig . 3g and h ) . Although the diffuse GFP – CHC staining made it somewhat difficult to quantify the number of puncta in wild - type worms , examination of several samples of the posterior lobe of the phar - ynx showed a greater than twofold increase in the number of puncta in auxilin ( RNAi ) L1 larvae compared to the wild type ( 95 – 35 compared with 36 – 15 ) . This effect could be due to an accu - mulation of clathrin - coated vesicles or to a compensatory increase in clathrin expression , although we do not yet have a functional anti - clathrin antibody that could prove the latter possibility . In this regard , whereas only ~ 40 % of auxilin ( RNAi ) worms survived beyond early larval stages , most of auxilin ( RNAi ) worms that also expressed GFP – CHC survived to adulthood . As it is very likely that worms expressing GFP – CHC possess more clathrin than wild - type worms , it is possible that this increase in clathrin would par - tially overcome the defect in endocytosis caused by the inability to recycle clathrin . It should be noted that the absence of auxilin specifically affected both GFP – CHC distribution and clathrin - mediated endocytosis . In contrast to GFP – CHC distribution , the absence of auxilin had no effect on the the distribution of two other GFP - fusion proteins , YNK - 1 – GFP , a diffuse cytosolic pro - tein 15 , and RME - 8 – GFP , a cytosolic protein that is peripherally associated with endosomes ( Y . Z . and D . H . , unpublished observa - tions ) . Furthermore , the absence of auxilin did not affect secre - tion , as shown by the fact that yolk protein – GFP is still secreted in auxilin ( RNAi ) worms , or fluid - phase endocytosis , as shown by the fact that , like wild - type worms , auxilin ( RNAi ) worms take up sol - uble GFP from the body cavity and concentrate it in the lysosomes of coelomocytes ( data not shown ) . Reasoning that the failure of clathrin uncoating in auxilin ( RNAi ) L1 larvae might affect clathrin dynamics as well as clathrin distribution , we compared clathrin exchange in the coelo - mocytes of wild - type and auxilin ( RNAi ) L1 larvae using FRAP . After photobleaching of a portion of the GFP – CHC in wild - type coelomocytes , there was marked recovery of fluorescence over a 100 80 60 40 20 0 100 80 60 40 20 0 0 0 0 0 . 05 0 . 1 0 . 15 0 . 2 0 . 16 0 . 12 0 . 08 0 . 04 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 1 1 . 5 C l a t h r i n po l y m e r i z ed ( % ) F r ee c l a t h r i n ( m M ) CeAUX - 53 ( m M ) CeAUX - 53 ( m M ) H sc 70 bound ( % ) CeAUX - 53 ( m M ) a b c d Figure 1 CeAUX - 53 polymerizes clathrin and supports Hsc70 – clathrin inter - action . a , Clathrin ( 0 . 5 m M ) was polymerized in the presence of varying concen - trations of CeAUX - 53 , and the amount of clathrin polymerized was determined by centrifugation . b , Structure of clathrin baskets polymerized by CeAUX - 53 , as determined by electron microscopy . Scale bar represents 100 nm . c , Effects of varying concentrations of CeAUX - 53 on the uncoating by 0 . 5 m M Hsc70 at pH 7 . 0 of 0 . 5 m M clathrin baskets polymerized with the M r 58K C - terminal fragment of AP180 . d , Effects of varying concentrations of CeAUX - 53 on the binding of 0 . 5 m M Hsc70 to pure clathrin baskets at pH 6 . Experiments were carried out as described 4 . © 2001 Macmillan Magazines Ltd brief communications NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http : / / cellbio . nature . com 217 3 - min period , with complete recovery after 10 min ( Fig . 4a – c ) . In contrast , there was almost no recovery of photobleached GFP – CHC puncta in auxilin ( RNAi ) coelomocytes over a 10 - min period , even though the GFP – CHC fluorescence was brighter and more punctate overall ( Fig . 4d – f ) . It is likely that clathrin exchange occurs in wild - type coelomocytes because clathrin - coated pits are a b c d e f g h i j Wild type Auxilin ( RNAi ) Figure 2 C . elegansauxilin ( RNAi ) phenotypes . L4 hermaphrodites were microinject - ed with C . elegansauxilin dsRNA . P 0 - injected animals ( parental generation ) were scored for YP170 – GFP endocytosis . In a typical experiment 12 animals were injected with aux - ilin ( RNAi ) and their F1 progeny were staged by the number of cells in the gonad and scored for developmental defects . Penetrance was 60 – 70 % ; in 10 out of 12 broods from the second day after egg laying most of the animals remained as L2 larvae for 3 days , whereas a few remained as L1 larvae . Uninjected controls were all adults after 3 days . After 6 days 42 % of the progeny had recovered and reached adulthood , where - as the remainder died as necrotic larvae . a , c , Wild - type ( P 0 ) adult hermaphrodite expressing YP170 – GFP , visualized by Nomarski optics and fluorescence microscopy . Most YP170 – GFP is found in the almost full grown - oocytes ( arrows ) and embryos . b , d , Auxilin ( RNAi ) ( P 0 ) adult hermaphrodite expressing YP170 – GFP , visualized by Nomarski optics and fluorescence microscopy . Most YP170 – GFP is found in the body cavity , not in oocytes ( arrows ) and embryos . Scale bars in a – d represent 16 m m . e , N2 adult control animal 3 days after embryogenesis . f , F1 progeny animal of an N2 hermaphrodite injected with C . elegansauxilin dsRNA 3 days after embryogenesis . This animal is still in the early L2 larval stage . Scale bars in e and f represent 100 m m . g , Wild - type control , showing normal intestinal morphology . h , Auxilin ( RNAi ) larva 3 days after embryogenesis . Abundant refractile granules are present in the intestine ( small black arrows ) , which are not found in wild - type larvae or adults . By the fourth and fifth days after embryogenesis intestines are necrotic and constipated . A vacuolat - ed cell is marked with a large white arrow . Scale bars in g and h represent 25 m m . i , Nose of wild - type control animal 3 days after embyogenesis . Scale bar represents 8 m m . j , Nose of auxilin ( RNAi ) larva 3 days after embryogenesis . Unshed cuticle in the nose is marked with large black arrows . Scale bar represents 10 m m . Coelomocytes Pharynx Posteriorpharyngealbulb Hypodermis Hypodermis a b c d e f g h i j Wild type Auxilin ( RNAi ) Figure 3 GFP – CHC fluorescence in wild - type and auxilin ( RNAi ) worms . Confocal micrographs of GFP – CHC fluorescence of various cell types in live wild - type or auxilin ( RNAi ) larvae . Micrographs of the same cell types were captured and processed under identical conditions to show relative brightness , except for c and e , which were modified with increased brightness and contrast to show detail . a , b , Coelomocytes ( macrophage - like scavenger cells ) of L1 ( first larval stage ) larvae . Each coelomocyte is bracketed . GFP – CHC tends to be concentrated in a bipolar manner in larval coelomo - cytes , and to be more evenly distributed around the cell periphery in adults . c , d , Head region of L1 larvae , showing the two lobes of the pharynx connected by a narrow isth - mus . The brightest accumulation of GFP – CHC is in punctate structures along the apical ( lumenal ) membranes of the pharynx . e , f , Higher magnification of posterior pharyngeal bulbs , showing the increase in punctate GFP – CHC fluorescence as a result of auxilin ( RNAi ) . g , h , Hypodermis of L3 larvae . i , j , Enlarged regions of panels e and f . White arrows show GFP – CHC puncta , which are brighter and larger in auxilin ( RNAi ) ani - mals . Scale bars represent 1 . 5 m m ( a , b , e , f , i , j ) , 25 m m ( c , d ) and 10 m m ( g , h ) . © 2001 Macmillan Magazines Ltd brief communications pinching off and reforming , just as occurs in mammalian cells , as has been shown by studies using GFP – clathrin 16 . In contrast , clathrin exchange may not occur in C . elegans auxilin ( RNAi ) coelomocytes because clathrin may be trapped either in clathrin - coated vesicles near the plasma membrane or in clathrin - coated pits . The latter effect might occur if Hsc70 and auxilin are involved in exchange of clathrin as planar clathrin - coated pits invaginate to form clathrin - coated vesicles , an idea that is supported by recent in vitro data indicating that Hsc70 is involved in returning clathrin to the membrane to form new clathrin - coated pits 17 . We conclude that auxilin is essential in vivo for endocytosis , probably through its function in uncoating clathrin from membranes . Future work will be required to relate this effect to the functions of other factors , such as membrane phosphoinositide levels 18 , that have also been implicated in the uncoating process . Methods Bioinformatics . Auxilin homologues in the C . elegans genome were identified using the National Center for Biotechnology Information interfaced to BLASTp BLOSUM62 . The C . elegans hypothetical protein W07A8 . 3 was compared to bovine auxilin protein S68983 by BLAST 2 PAM250 . Preparation of proteins . Hsc70 and clathrin were prepared as described 19 , 20 . The M r 58K C - terminal fragment of AP180 was prepared as a 6 · histidine ( 6His ) fusion protein from recombinant AP180 ( ref . 21 ) . Pure clathrin bas - kets and clathrin baskets made with assembly proteins were produced as described 4 , 22 . Part of a C . ele - gans complementary DNA encoding a truncated fragment of C . elegans auxilin was subcloned into pQE30 and expressed as a 6His fusion protein . The equivalent fragment of mammalian auxilin is much more easily expressed in Escerichia coli than the parent molecule and retains full uncoating activity 4 , 23 . The recombinant protein was expressed and purified as described 4 . General methods and strains . Worms were handled and cultured essentially as described 24 . All strains were grown at 20 o C unless otherwise stated . The wild - type parent for all strains was C . elegans var . Bristol strain N2 . cDNA sequencing , 5 ¢ RACE and RNAi . Two almost full - length cDNA clones , yk360c4 and yk390a4 , for the predicted C . elegans auxilin gene W07A8 . 3 were provided by Y . Kohara ( Natl Inst . Genetics , Japan ) . yk360c4 was sequenced using vector and internal sequencing primers . Two exon – intron boundries predicted by Genefinder were found to be incorrect , changing the predicted protein sequence ( see Genbank accession no . AF293972 ) . Both cDNAs contained poly ( A ) tails beginning 321 bp after the stop codon . The 5 ¢ end of the auxilin mes - sage was determined by 5 ¢ RACE ( Gibco - BRL ) , according to the manufacturer’s instructions . A single 5 ¢ RACE product was amplified by polymerase chain reaction ( PCR ) , and the product was sequenced directly . We found an SL1 trans - spliced leader starting 21 bp upstream of the predicted AUG codon , indicating the start of the mature messenger RNA . C . elegans auxilin cDNAs were used to produce dsRNA for microinjection into the pseudocoelom or intestine of L4 hermaphrodites , as described 9 , 10 . Groups of bIs1 [ vit - 2 : : GFP , rol - 6 ( d ) ] ( ref . 8 ) L4 her - maphrodites were microinjected with C . elegans auxilin dsRNA and allowed to recover for 48h before direct observation of oocytes and embryos within the P 0 by fluorescence microscopy . L4 stage N2 or bIs5 [ GFP : : CHC ] hermaphrodites were microinjected with C . elegans auxilin dsRNA , allowed to recover for 24h , and their F1 progeny were collected in single - day cohorts for analysis . Animals were immobi - lized for fluorescence microscopy using 0 . 1mgml – 1 levamisole . FRAP analyses . GFP was photobleached using a Zeiss LSM 510 confocal microscope with a · 100 1 . 4 NA objective . A defined region was photobleached at full laser power and recovery of fluorescence was monitored by scanning the whole cell at low laser power . Transgenic strains . Transgenic C . elegans strains were constructed to express a full - length C . elegans GFP – CHC fusion pro - tein under the control of the C . elegans clathrin heavy chain ( T20G5 . 1 ; ref . 8 ) promotor . The resulting plasmid , pGFP – CHC , was linearized and injected at 1 . 25ngml – 1 , together with linearized rol - 6 ( d ) marker plasmid pRF4 at 1 . 25ngml – 1 , and Pvu II - digested N2 genomic DNA at 90ngml – 1 , to create extrachromasomal transgenic lines . One of these lines was integrated into a chromosome by g - irrada - tion using standard methods 25 , 26 . All extrachromosomal and integrated lines expressed diffuse cytoplas - mic and punctate GFP – CHC in many somatic tissues , but not in germ cells . One integrated line , bIs5 [ GFP : : CHC , rol - 6 ( d ) ] was chosen for this study . RECEIVED 25 AUGUST 2000 ; REVISED 10 NOVEMBER 2000 ; ACCEPTED 10 NOVEMBER 2000 ; PUBLISHED 18 JANUARY 2000 . 1 . Kelley , W . L . Trends Biochem . Sci . 23 , 222 – 227 ( 1998 ) . 2 . Prasad , K . , Barouch , W . , Greene , L . & Eisenberg , E . J . Biol . Chem . 268 , 23758 – 23761 , ( 1993 ) . 3 . Ungewickell , E . et al . Nature 378 , 632 – 635 ( 1995 ) . 4 . Greener , T . , Zhao , X . , Nojima , H . , Eisenberg , E . & Greene , L . E . J . Biol . Chem . 275 , 1365 – 1370 ( 2000 ) . 5 . Umeda A . , Meyerholz A . , & Ungewickell , E . Eur . J . Cell Biol . 79 , 336 – 342 ( 2000 ) . 6 . Schroder , S . et al . Eur . J . Biochem . 228 , 297 – 304 ( 1995 ) . 7 . Kanaoka . Y . , Kimura , S . H . , Okazaki , I . , Ikeda M . & Nojima , H . FEBS Lett . 402 , 73 – 80 ( 1997 ) . 8 . Grant , B . & Hirsh , D . Mol . Biol . Cell 10 , 4311 – 4326 ( 1999 ) . 9 . Fire , A , Xu , S . , Montgomery , M . K . , Kostas , S . A . , Driver , S . E . & Mello , C . C . Nature 391 , 806 – 811 ( 1998 ) . 10 . Montgomery , M . K . , Xu , S . & Fire , A . Proc . Natl Acad . Sci . USA 95 , 15502 – 15507 ( 1998 ) . 11 . Yochem , J . , Tuck S . , Greenwald , I . & Han , M . Development 126 , 597 – 606 ( 1999 ) . 12 . Thacker , C . , Peters , K . Srayko , M . & Rose , A . M . Genes Dev . 9 , 956 – 971 ( 2000 ) . 13 . Tavernarakis N . , Wang S . L . , Dorovkov , M . , Ryazanov , A . & Driscoll , M . Nature Genet . 2 , 180 – 183 ( 2000 ) . 14 . Kelly , W . G . , Xu S . , Montgomery , M . K . & Fire , A . Genetics 146 , 227 – 238 ( 1997 ) . 15 . Che S . , Weil , M . M . , Etkin , L . D . , Epstein , H . F . & Kuang , J . Biochim . Biophys . Acta 1354 , 231 – 240 ( 1997 ) . NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http : / / cellbio . nature . com 218 Auxilin ( RNAi ) Wild type Initial Bleach Recovery a b c d e f Figure 4 Different dynamics of GFP – clathrin in coelomocytes from wild - type and auxilin ( RNAi ) worms . Fluorescence images of GFP – clathrin in the coelomo - cytes of wild – type ( a – c ) and auxilin ( RNAi ) ( d – f ) larvae , obtained before photobleach - ing ( a , d ) , immediately after ( b , e ) , and 10 min after ( c , f ) . GFP – clathrin in wild - type coelomocytes exhibited ~ 75 % recovery in 3 min , whereas there was no significant recovery in the auxilin ( RNAi ) coelomocytes within 10 min . Coelomocytes were all obtained from C . elegans at the L1 or L2 larval stage . The area that was photo - bleached is outlined in each figure and the coelemocyte is indicated by brackets . © 2001 Macmillan Magazines Ltd 16 . Gaidorov , I . , Santini , F . , Warren , R . A . & Keen , J . H . Nature Cell Biol . 1 , 1 – 7 ( 1999 ) . 17 . Jiang , R . Gao , B . , Prasad , K . , Greene , L . E . & Eisenberg , E . J . Biol . Chem . 275 , 8439 – 8447 ( 2000 ) . 18 . Brodin L . , Low , P . & Shupliakov , O . Curr . Opin . Neurobiol . 10 , 312 – 320 ( 2000 ) . 19 . Greene , L . E . & Eisenberg , E . J . Biol . Chem . 265 , 6682 – 6687 ( 1990 ) . 20 . Prasad , K . , Heuser , J . , Eisenberg , E . & Greene , L . J . Biol . Chem . 269 , 6931 – 6939 ( 1994 ) . 21 . Ye , W . & Lafer , E . M . J . Biol . Chem . 270 , 10933 – 10939 ( 1995 ) . 22 . Barouch , W . , Prasad , K . , Greene , L . E . & Eisenberg , E . J . Biol . Chem . 269 , 28563 – 28568 ( 1994 ) . 23 . Holstein , S . E . , Ungewickell , H . & Ungewickell , E . J . Cell Biol . 135 , 925 – 937 ( 1996 ) . 24 . Brenner , S . Genetics 77 , 71 – 94 ( 1974 ) . 25 . Fire , A . EMBO J . 5 , 2673 – 2680 ( 1986 ) . 26 . Mello , C . C . , Kramer J . M . , Stinchcomb , D . & Ambros , V . EMBO J . 10 , 3959 – 3970 ( 1991 ) . ACKNOWLEDGEMENTS We thank M . Krause for valuable assistance , Y . Xu for electron micrographs of the baskets , A . Fire for GFP plasmids , Y . Kohara for cDNA clones of C . elegans auxilin , and W . Przylecki for technical support . This work was supported in part by March of Dimes grant FY99 - 583 ( to D . H . ) . Correspondence and requests for materials should be addressed to E . E . brief communications NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http : / / cellbio . nature . com 219 \ No newline at end of file diff --git a/silver_data/71bf7a7057d829e0a0ce2bf3ae76a93d224c38e3.pdf.txt b/silver_data/71bf7a7057d829e0a0ce2bf3ae76a93d224c38e3.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..da023c65628296b906b0d217eb868fda8bbf671a --- /dev/null +++ b/silver_data/71bf7a7057d829e0a0ce2bf3ae76a93d224c38e3.pdf.txt @@ -0,0 +1 @@ +Knowledge Management Enablers , Processes , and Organizational Performance : An Integrative View and Empirical Examination HEESEOK LEE AND BYOUNGGU CHOI HEESEOK LEE is a Professor of Information Systems at the Graduate School of Man - agement . Korea Advanced Institute of Science and Technology , Korea . He received his Ph . D . in MIS from the University of Arizona , an M . S . from Korea Advanced Institute of Science and Technology , and a B . S . from Seoul National University . He was previously on the faculty at the University of Nebraska at Omaha . His research interests include knowledge management . Internet business , and IS strategy . His re - cent publications appear in Journal of Management Information Systems , Informa - tion and Management . Journal of Organizational Computing and Electronic Commerce , Expert Systems with Applications , Annals of Operations Research , Jour - nal of Sy . stems and Software , and Information Systems . BYOUNGGU CHOI is an Associate Researcher at the Carlson School of Management . University of Minnesota . He received his Ph . D . and M . S . in Management from Korea Advanced Institute of Science and Technology and his B . S . from Korea University . His research interests include Knowledge Management and Electronic Commerce . His recent publications appear in Journal of Management Information Systems . In - formation and Management , and Expert Systems with Applications . ABSTRACT : Knowledge is recognized as an important weapon for sustaining com - petitive advantage and many companies are beginning to manage organizational knowl - edge . Researchers have investigated knowledge management factors such as enablers , processes , and performance . However , most current empirical research has explored the relationships between these factors in isolation . To fill this gap . this paper devel - ops a research model that interconnects knowledge management factors . The model inciudes seven enablers : collaboration , trust , learning , centralization , formalization . T - shaped skills , and information technology support . The emphasis is on knowledge creation processes such as socialization , externalization . combination , and iniemal - ization . To establish credibility between knowledge creation and performance , orga - nizational creativity is incorporated into the model . Surveys collected from 58 firms were analyzed to test the model . The results confirmed ihe impact of trust on knowl - edge creation . The information technology support had a positive impact on knowl - edge combination only . Organizational creativity was found to be critical for improving performance : neglecting ideas can undermine a business . The results may be used as a stepping stone for further empirical research and can help formulate rohust strate - gies that involve trade - offs between knowledge management enablers . KEY WORDS AND PHRASES : knowledge - creating processes , knowledge management , knowledge management enablers , organizational creativity , organizational performance . Journal of Management tnformtUion System . - ^ / Summer 21 ) 0 ^ . Wo \ . 20 . No . I . pp . 179 - 228 . © 2003 M . E . Sharpe , Inc . 0742 - 1222 / 2003 $ 9 . 50 + 0 . 00 . 180 LEE AND CHOI IN RECENT YEARS , IT SEEMS AS THOUGH businesses that could capture the knowledge embedded in their organization would own the future . Companies that isolate knowl - edge management risk losing its benefits . It is no surprise that knowledge is overturn - ing the old rules about strategy and competition—the foundation of industrialized economics has shifted from natural resources to intellectual assets . In response , many managers and management thinkers have proclaimed an era of knowledge manage - ment . This has compelled researchers to investigate how knowledge is managed . Evi - dence is provided by a variety of studies on knowledge [ 19 , 79 , 82 ] , knowledge process [ 38 , 70 , 76 . 114J , and knowledge management architecture [ 9 , 21 . IO5 | . Companies attempting to deploy knowledge management may be confused by a variety of efforts under way that go under the name of knowledge management [ 61 ] . Many companies have tried , with mixed success , to leverage knowledge assets by centralizing knowledge management functions or by investing heavily in information technology ( IT ) [ 44 ] . It is understandable , when confronted with a new business phe - nomenon , to look to new management practices for guidance . Caught up in the gen - eral fever , many managers may assume that knowledge management can improve their companies . However , despite their best efforts , most studies have not investi - gated how companies can leverage knowledge for the improved performance . It is important to distinguish themselves through strategies . The key question is not whether to manage knowledge , but how to manage it . These strategies should be validated by the use of further empirical tests . To fill this gap , prior research has explored which factors are essential for managing knowledge effectively . One challenge is to decipher the relationships among these factors . Most studies have examined the relationships of knowledge management enablers , processes , or performance in isolation . For example , some research has fo - cused on the relationship between enablers and processes [ 6 , 43 , 114 , 124 ] ; the em - phasis of other studies is on the relationship between enablers and organizational performance [ 8 , 11 , 35 . 104 ] . Researchers and practitioners have not tried an integra - tive model . An integrative perspective of the knowledge variables ba . sed on relevant theories is a necessity . It is also noted that very few empirical studies adopt a process - oriented perspective of organizational knowledge [ 90 ] . Knowledge creation or trans - fer would benefit companies more than knowledge itself because knowledge is not primarily about facts but more about context - specific characteristics [ 115 ] . For ex - ample . Xerox systemizes knowledge creation and transfer processes through strategic communities [ 109 ] . Consequently , another challenge is to leverage a process - oriented perspective . The primary objective of this paper is to delineate an integrative view of knowledge management and provide strategic guidelines . For this purpose , this paper analyzes the previous empirical studies and attempts to fmd relationships among knowledge management factors such as enablers , processes , and organizational performance . An integrative research model is built from a process - oriented perspective and then tested empirically . KM ENABLERS . PROCESSES , AND ORGANIZATIONAL PERFORMANCE 181 Research Background and Literature Review Theoretical Background MANY RESEARCHERS HAVE EMPHASIZED three major factors for managing knowledge : enablers , processes , and organizational performance [ 9 . 21 , 851 . Knowledge manage - ment enablers { or influencing factors ) are organizational mechanisms for fostering knowl - edge consistently [ 57 ] ; they can stimulate knowledge creation , protect knowledge , and facilitate the sharing of knowledge in an organization [ 108 ] . Knowledge processes ( knowledge management activities ) can be thouglit of as a structured coordination for managing knowledge effectively [ 35 ] . Typically , knowledge proces . ses include activi - ties such as creation , sharing , storage , and usage [ 2 , 9 ] . Whereas knowledge processes represent the basic operations of knowledge [ 105 ] . enablers provide the infrastructure necessary for the organization to increase the efficiency of knowledge processes [ 96 ] . Organizational performance may be defined as the degree to which companies achieved its business objectives [ 28 ] . It may be measured in terms of organizational learning , profitability , or other financial benefits in knowledge management [ 18 , 104 [ . Without measurable success , passion from employees and managers will vanish [ 851 . There is a general recognition among academics that knowledge management is a cross - functional and multifaceted discipline . A variety of components make up knowl - edge management and the understanding of their interaction is important ; a holistic view is very useful [ 80 ] . To this end , an integrative research model is necessary ; that is , the relationships among knowledge enablers . processes , and organizational perfor - mance can he identified within the framework of systems thinking . Systems thinking theory considers probiems in their entirety 1951 . This theory is better able to describe complex and dynamic characteristics of knowledge management in a systematic fash - ion . Therefore , our integrative framework will be based on this systems thinking theory . Our primary research focus is on the relationships between knowledge enablers and organizational performance by elaborating on the significance of knowledge pro - cesses as the foundation of organizational advantage [ 79 ] . The relationship among these three components is nothing new ; it can be found in the input - process - output model by Hackerman and Morris [ 41 ] . The model assumes that the input factors affect output performances through certain kinds of interaction processes ; knowl - edge management enablers affect organizationai performance through knowledge processes . This relationship is also explained by the use of the knowledge - chain model proposed by Holsapple and Singh [ 51 ] . This model suggests that leadership estahlish enabling conditions for achieving organizational outcome through the knowledge management activities such as acquisition , generation , internalization , and extemali - zation . It means that knowledge enablers ( e . g . . leadership ) affect organizational out - come through knowledge processes . A direct relationship between knowledge processes and organizational performance is not explored yet . Because many factors influence the determination of the organiza - tional performance , attempts to trace causality to any single factor such as knowledge 182 LEE AND CHOI Enablers , Process - IntemicdiatcOutcome L OrgaoiPertor ^ tionalmance Figure I . An Integrative Research Framework for Studying Knowledge Management process may be risky . In order to understand the effect ofthe knowledge processes on organizational perfomiance , intermediate outcomes ( for example , knowledge satis - faction or organizational creativity ) may be introduced [ 18 ] . Intermediate outcotnes reflect different aspects of an organization ' s performance , both financial and nonfi - nancial . This incorporation may help confirm that enablers ultimately create business value . In sum , this paper proposes a researcb framework as sbown in Figure 1 . Previous Empirical Studies Previous empirical studies have investigated tbe relationsbips among knowledge management factors . They can be classified into four categories depending on how they identify tbe relation . ships : ( 1 ) relationships between knowledge enablers ; ( 2 ) re - lationships between knowledge enablers and process ; ( 3 ) relationsbips between knowl - edge process and organizational performance ; and ( 4 ) relationsbips among knowledge enablers . processes , and organizational performance . Tbis comparison may be high - lighted as sbown in Figure 2 . The studies under tbe first category focus on tbe relationsbips among knowledge enablers . Tbe emphasis is on the examination of tbe effect of knowledge enablers . To identify ( his effect , they have investigated various knowledge enablers such as knowl - edge management methods , structure , and culture . For example . Bennett and Gabriel [ 10 ] analyzed a number of knowledge management metbods in view of oi ^ aniza - tional structuns , culture , size , and environment . Tbe second category explores the relationships between knowledge enablers and knowledge processes . A central proposition is that knowledge enablers ( e . g . , industry characteristics or knowledge characteristics ) should influence knowledge processes ( e . g . . transfer ) . Zander and Kogut [ 124 ] proposed that the transfer of organizational capabilities be related to the characteristics of social knowiedge ; tbey analyzed the effects of the ease of codifying manufacturing capabilities on its transfer time . Appleyard [ 6 ] explored knowledge transfer patterns among various nations and in - dustries . Szulanski 1114 | investigated the relationship between four origins of sticki - ness ( characteristics of the knowledge transferred , tbe source , the recipient , and the context in wbicb the transfer takes place ) and knowledge transfer . Hansen [ 43 ] em - ployed the notion of complex knowledge to explain the role of weak ties in transfer - ring knowledge in a multiunit organization . The tbird category examines the reiationships between knowledge enablers and organizational performance . Tbe purpose of these studies is to sharpen the under - standing ofthe effects of knowledge enablers ( e . g . , knowledge management strategy ) KM ENABLERS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE ! 83 OrganizationalPerformance Zander AKogm [ 1995 ] Appleyard [ 1996 ] S7 . ulanski | 1996 ] , , Hansen 11999 ! Note : ROA = Return on Assets ROS = Return on 5ales Figure 2 . Research Models for Studying Knowledge Management on organizational performance ( e . g . , return on assets [ ROA ] or return on sales ( ROS ) ) . Bierly and Chakrabarti [ 11 ] tried to identify how knowledge management strategies affect organizational performance . They analyzed knowledge strategies of 21 U . S . pharmaceutical companies that had been categorized into explorers , exploiters , lon - ers , and innovators . Simonin [ 104 ] tested the relationships among collaborative expe - rience , know - how , and achievement of organizational performance . He proposed that the experience of a firm has to be transformed into know - how before it could improve organizational performance . The emphasis of the fourth category is on relationships among knowledge enablers . knowledge processes , and organizational performance . The primary objective of these studies is to identify and assess knowledge enablers ( e . g . , task or infrastructure capa - bilities ) and processes { e . g . , creation or their capabilities ) for improving organiza - tional performance ( e . g . , knowledge satisfaction or organizational effectiveness ) . Becerra - Fernandez and Sabherwal [ 8 ] proposed a contingency framework including two attributes of the organizational subunit ' s tasks—process or content orientation , and focused or broad domain ^ —and linked them to Nonaka ' s knowledge creation process [ 82 ] . The relationship between knowledge creation process and knowledge satisfaction was also investigated . Gold et al . [ 35 ] analyzed two relationships : one 184 LEE AND CHOI between infrastructure capabilities and organizational effectiveness , and the other between process capabilities and organizational effectiveness . Table I compares these previous studies . Synthesis of Previous Studies Synthesis of previous studies yields some observations . First , an integrative model is still missing . Although some studies investigate the relationships among knowiedge enablers . processes , or organizational performance | 8 , 35 ) , they fail to explore the relationships between enablers and processes simultaneously . If managers understand these relationships in an integrative fashion , they ean stand a better chance of improv - ing their firm ' s performance . Second , the role of knowledge management processes is not consistent . Some stud - ies recognized both knowledge enablers and processes as antecedents of organiza - tional performance [ 8 , 351 . Other sludies recognized knowledge enablers as preconditions of knowiedge processes [ 6 , 43 , 114 , 124J . Therefore , the challenge is to clarify the role of knowledge management processes [ 108 ] . Third , measuring knowledge management performance is still difficult . Some stud - ies captured the contribution by the use of knowledge management outcome mea - sures such as knowledge satisfaction [ 8 ] , whereas others adopted conventional performance measures such as ROA [ 11 , 104 ] or organizational effectiveness [ 35 ] . It would appear that the former studies take the relationship between knowledge man - agement outcome and organizational performance for granted although the relation - ship has not been validated . The results of the latter studies should be examined carefully because the direct relationship between knowledge management processes and organizational performance has not been validated yet [ 18 ] . Fourth , the knowledge transfer process has been studied extensively [ 6 , 43 , 114 , 124 ] whereas the other processes such as creation or utilization have received rela - tively little attention . In particular , some studies have suggested that knowledge cre - ation is most critical for an organization ' s long - term success [ 30 ] . Moreover , knowledge transfer has been assessed by the use of object - perspective measures such as time to transfer [ 124 ] , number of times of knowiedge transfer [ 6 ] , or percentage of trans - ferred knowledge [ 43 | . Recently , some researchers have tried to measure knowledge processes themselves [ 8 . 35 ] . For example , Becerra - Fernandez and Sabherwal [ 8 ] measured the capacity for knowledge creation by Nonaka ' s knowledge creation model , not by the use of creation output such as the number of created ideas or patents . A Research Model OUR OBJECTIVE IS NEITHER TO PROPOSE a model that delineates all of the relation - ships underlying knowledge management nor to generate a longer list of possible knowledge enablers or processes that affect organizational performance . Therefore , 185 an c a oa ca N C o o c n3 o b - OJ T3 5 CL 6oU Si a . IS 3O i en CDD ) C rax : o " o o ! tLU 0 E B i _ _ Q E c 0 o •ri 0 & • a . E CO O CO TJ « OJ O E £ LU 52 S o •5 E « ra o . o 0 g . ffl ^ . y O cQ O ^ O Q . " to x ; c — t : ; ro ( 0 O 11 ro 9 n ^ m 0 ro t . . 1 ' - . ^ o T3 ra 0 T3 CO N 0 ) TJ o to i ; 0 0 | CO 3 ° I I I 0 ) C V DI E 1 ^ c 3 Q ) ^ •— C ^ C V ^ ^ ( 1 ) CO D ) 5 ) £ 0 ) ^ - . " «1 ll E £ 2 g S I - : = a . ^ a X3 CCO & • u s t X ) ra CC o lo i on ra c •g CO Q . 186 cd a O o s 9 •I O 55 o ) 0 ffl ^ i3 X ] £ E 5 " E ^ 0 o " o ra o £ _ c - ^ ra { > 5 0 o M CO rt \ ^ • ^ i 9 ^ x : 0 ^ S o " o z : 0 i = •o 0 ^ ffl ff cs Cfl 3 o r an s i w 0 ) 0 ) ( n V o oa . ai r an s f 2 ra " O o i cj > : * . c o . 2 O ^ U - D 10 Q . 0 CO . ^ 3 . 9 - 0 ) W 0 ) ( O T5 0 •t C 0 tt . c , Q - ^ [ ^ •o ra o S r e o GJ T3 0 = ro c ^ a ) ; = P m g ra ra o a . 12 •9 ffl ^ S a ) 0 . = : CO - JJ Illlll P . 9 ? 2 m w ™ CO = CO * : o . 5 0 o Sj 2 ffl 15 JS Q . c 9 o N i 2 ^ I i 1 1 ° I o E ra — t ra o ^ o ^ < a» IS I 5 ) — - c c O « i2 tt c 1 / i < O O CC QC i9 0 " o o El 5 ^ 0 ) _ cO o g - o O 0 O . 2 oE CO 187 •g a a . - a X ) a t > u Oil •a T3 f 0 S • ro ra ( 0 ™ p J : ^ in k CO 0 " ffl « to ij L _ - - ' 3 _ ^ o c " * " 0 o ra • . E m C * - — * - CO ra • = : O 0 N C O 1 ^ O o ra o i £ " S o c H 0 S O 2 o ro of o c - S T ) 0 03 g ra N o c i a l c ea t i o o t i on , ra N t e r na X < D c " . 9 " 5 m b i n o o i on ) . CON e r na i c w Q OCO - 5 ro — : . 0 0 ra e 5 5 g Q - r " S " D 8 ii CJ P P o •a ra C c ! Z a . _ c I / : C c ^ _ ( L N0 •a c ro 0 ra 0 O0 m CC ' ct £ 0r— f ra CO w £ . y r , a 3 CO U U ) 0 o o CO ~ ' ^ ( o « ra OJ Z3 O X } COQ . ro u CD _ 3 3 to o 3 m p 0 3 3l ^ CJ a . u 188 LEE AND CHOI our model highlights a few major factors that can explain a large proportion of the variance in knowledge management . Variables Enahlers A variety of knowledge management enablers have been addressed in the literature [ 57 , 70 . 97 | . Among these enablers . organizational culture , structure , people . , and IT are incorporated into our research model . Organizational culture is the most impor - tant factor for successful knowledge management [ 15 . 20 , 21 , 35 ] . Culture defines not only what knowledge is valued , but also what knowledge must be kept inside the organization for sustained innovative advantage [ 71 ] . Organizations should establish an appropriate culture that encourages people to create and share knowledge within an organization [ 49 , 70 ] . This study focuses on collaboration , trust , and learning on the basis ofthe concept of care [ 29 ] . Care is a key enabler for organizational relation - ships [ 68 ] . When organizational relationships are fostered through care , knowledge can be created and shared . The organizational structure within an organization may encourage or inhibit knowl - edge management [ 35 , 47 , 82 ] . For example , Ichijo et al . | 57 ] insisted that firms should maintain consistency between their structures to put their knowledge to use . Our study includes two key structural factors such as centralization and formalization [ 77 [ . They are recognized as key variables underlying the structural construct . More - over , their effects on knowledge management within organizations are widely recog - nized to be potent [ 29 , 59 , 72 . 91 ] . People are at the heart of creating organizational knowledge [ 15 , 49 , 80 ] . It is people who create and share knowledge . Therefore , managing people who are willing to create and share knowiedge is important [ 85 ] . Knowledge and competence can be acquired by admitting new people with desirable skills [ 108 ] . In particular , T - shaped skills embodied in employees are most often associated with core capability [ 56 , 60 , 70 ] . T - shaped skills may enable individual specialists to have synergistic conversa - tions with one another [ 74 ] . Technology contributes to knowledge management [ 35 ] . This technology infra - structure includes IT and its capabilities [ 90 , 99 ] . IT is widely employed to connect people with reusable codified knowledge , and it facilitates conversations to create new knowledge . Among technology - related variables , this study focuses on IT sup - port [ 108 ] . ITs allow an organization to create , share , store , and use knowledge [ 70 ] . Therefore , the support of IT is essential for initiating and carrying out knowledge management . Enablers may be structured based upon a socio - technical theory [ 86 ] . This theory describes an organization from the social and technical perspectives . The two per - spectives are not unique to management information systems CMIS ) research [ 12 ] ; they are made up of two jointly independent but correlative interacting components . Organizational culture , organizational structure , and people are social enablers ; IT is KM ENABLERS , PROCESSES , AND ORGANIZATIONAL PERFORMANCE 189 a technical enabler . For the sake of clarity , we consider the impact of each knowledge enabler independently . Processes A number of studies bave addressed knowledge management processes ; they divide knowledge management into several processes . For example . Alavi and Leidner 12 ] considered four processes such as creation , storage , transfer , and application . These processes are often concurrent and not always in a linear sequence 19 ] . Among these processes , creation - related activities ( for example , creation [ 2 ] or construction [ 21 ] ) become important because knowledge creation is a strategic weapon in today ' s global marketplace ; without the constant creation of knowledge , a business is condemned to obsolescence [ 83 , 87 ] . Knowledge creation is a continuous process whereby individuals and groups within a firm and between firms share tacit and ex - plicit knowledge | 82 ] . Although a great deal has been discussed about the importance of knowledge creation , there is relatively little empirical evidence [ 90 ] . Therefore , the emphasis of this study is on knowledge creation . To explore knowledge creation , our study adopts the SECl ( socialization , externalization . combination , internalization ) model by Nonaka and Takeuchi [ 82 | for the following reasons . First , their work has become widely accepted [ 98 ] ; it has been used in many research areas such as organizational learning , new product devel - opment , and IT [ 98 , 99 ] . Second , their model includes not only knowledge creation but also knowledge transfer . The transfer of existing knowledge and the creation of new knowledge are important , and both of them should be considered in knowiedge management [ 69 ] . Their SECI model is made up of four intertwined activity modes ; socialization ( S ) , externalization ( E ) , combination ( C ) , and internalization ( I ) . Social - ization converts tacit knowledge into new tacit knowledge through social interactions among members . Externalization codifies tacit knowledge into explicit concepts . Com - bination converts explicit knowledge into more systematic sets by combining key pieces . Internalization embodies explicit knowledge into tacit knowledge . Intermediate Outcome In order to achieve a better understanding of knowledge management performance , companies should attempt to link knowledge processes with intermediate outcomes [ 18 ] . An important intermediate outcome is organizational creativity , which pro - vides a key to the understanding of organizational effectiveness and survival [ 122 ] . Our model incorporates organizational creativity because it is the seed of all innova - tion [ 5 ] and at the very heart of knowledge management [ 40 ] . Organizational cre - ativity transforms knowledge into business value . Neglecting organizational creativity can quickly undermine a business . The relationship between knowledge creation and organizational creativity bas received relatively little attention despite its high potential [ 119 ] . 190 LEE AND CHOI Organizational Performance Measuring organizational performance is not a trivial task because it strongly affects the behavior of managers and employees . The ultimate test of any business is whether it leads to measurable improvements in organizational performance . Methods for measuring organizational performance in knowledge management can be categorized into four groups : financial measures [ 11 ] , intellectual capital [ 110 ] , tangible and intangible benefits [ 104 ] , and balanced scorecard 163 ] . This study adopts a specific measure , which is developed and validated by Deshpande el al . [ 22 ] and Drew [ 25 ] . This measure can be thought of as a variation ofthe balanced scorecard method . The balanced scorecard retains fmancial performance and supplements it with measures on the drivers of future potential . In addition , it is more useful than intellectual capital or a tangible and intangible approach because it shows cause and effect links between knowledge components and organization strategies [ 63 ] . In summary , our empirical research model illustrates the relationship among vari - ables as shown in Figure 3 . In total , the model consists of 13 variables . Hypotheses Our hypotheses are largely derived from theoretical statements made in the litera - ture on knowledge management . We present our hypotheses through the following variables . Collaboration Collaboration may be defined as the degree to which people in a group actively help one another in their work [ 55 ] . Collaborative culture affects knowledge creation through increasing knowledge exchange [ 68 , 79 ] . Exchanging knowledge among different members is a prerequisite for knowledge creation . Collaborative culture fosters this type of exchange by reducing fear and increasing openness to other members . For example , Zucker et al . [ 126 ] confirmed the significance of collaborative culture in knowledge creation by examining the biotechnology industry . Collaboration between organizational members also tightens individuai differences [ 70 ] . It can help people develop a shared understanding about an organization ' s external and internal envi - ronments through supportive and reflective communication . Without shared under - standing among organizational members , little knowledge is ever created 130 , 47 ] . We do not have a priori reason to expect a diflerent relationship . HI : There is a positive relationship between collaboration and knowledge cre - ation process . Trust Trust can be defined as maintaining reciprocal faith in each other in terms of intention and behaviors [ 67 ] . Trust may facilitate open , substantive , and influential knowiedge KM EN . A , BL£RS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE 191 Knowledge Management Enablcrs S o c i a l p e r s p ec ti v e Culture •Collaboration•Trust•LeaminK Structure •Ceruralizaiion•Formalization People •T - shaped skills Knowledge Creation Knowledge Management Organizational Intermediate Outcome Perfonnance •Sociatizabon •Exiemaliuition •Combinwion •Iirternalizalion •Organizational creaiiviiy •Organizationalperformance InformationTechnology •rr Suppon Figure 3 . A Research Model exchange [ 81 , 85 ] . When their relationships are high in trust , people are more willing to participate in knowledge exchange [ 79 ] . Szulanski [ 1141 empirically found that the lack of trust among employees is one of the key barriers against knowledge ex - change . The increase in knowledge exchange brought on by mutual trust results in knowledge creation . Trust also encourages a climate conducive to better knowledge creation by alleviating the fear of risk . The presence of a high level of trust can reduce this risk [ 81 , 92 , 100 ] . Trust is also critical in a cross - functional or interorganizationa ! team because withholding information because of a lack of trust can be especially harmful to knowledge creation [ 47 , 59 ] . Therefore , we would expect the following relationship to hold true : H2 : There is a po . sitive relationship hetween trust and knowledge creation process . Learning Learning can be defined as the degree to which it is encouraged in organizations [ 55 ] . The emphasis on learning infuses an organization with new knowledge 117 ] . Learning is the acquisition of new knowledge by people who are able and willing to apply that knowledge in making decisions or influencing others [ 78 ] . Through the emphasis on learning and development , organizations can help individuals play more active roles in knowledge creation . Kanevsky and Housel [ 62 ] insisted that the amount of time spent on learning is positively related with the amount of knowledge . For successful knowledge creation , organizations should develop a deeply ingrained learning culture 192 LEE AND CHOI [ 88 ] and provide various learning means such as education , training , and mentoring [ 112 , 113 ] . For example , Nucor [ 39 ] , which has been the most innovative steel com - pany in the United States , built a knowledge creation foundation by investing in con - tinuous and multifunctional training programs . Hence , we hypothesize : H3 : There is a positive relationship between learning and knowledge creation process . Centralization Centralization refers to the locus of decision authority and control within an organi - zational entity [ 14 , 27 ] . The concentration of decision - making authority inevitably reduces creative solutions , wbereas the dispersion of power facilitates spontaneity , experimentation , and the freedom of expression , which are the lifeblood of knowl - edge creation [ 37 ] . Moreover , centralized structure hinders interdepartmental com - munication and frequent sharing of ideas [ 122 ] due to time - consuming communication channels [ 10 ] ; it also causes distortion and discontinuou . sness of ideas [ I08 | . Without a constant flow of communication and ideas , knowledge creation does not occur . A decentralized organizational structure has been found to facilitate an environment where employees participate in knowledge building process more spontaneously [ 52 ] . Participatory work environments foster knowledge creation by motivating organiza - tional members ' involvements . Therefore , decreased centralization in the form of lo - cus of authority can lead to increased creation of knowledge [ 106 , 108 . 115 ] . We advance the fourth hypothesis : H4 : There is a negative relationship between centralization and knowledge cre - ation process . Formatization Formalization refers to the degree to which decisions and working relationships are governed by formal rules , standard policies , and procedures [ 49 , 89 | . Knowledge creation requires flexibility and less emphasis on work rules [ 57 , 73 ] . The range of new ideas seems to be restricted when strict formal rules dominate an organization . Flexibility can accommodate better ways of doing things [ 37 ] . Therefore , the in - creased flexibility in an organizational structure can result in increased creation of knowledge . Knowledge creation also requires variation [ 121 ] . In order to be more adaptable when unforeseen problems arise , an organization may accommodate varia - tion in process and structure . Low formalization permits openness and variation , which encourage new ideas and behaviors [ 17 ] . Knowledge creation is also likely to be encouraged through unhindered communications and interactions [ 10 ] . Formality stifles the communication and interaction necessary to create knowledge . Lack of formal structure tends to enable organizational members to communicate and interact with one another to create knowledge 159 ] . Hence , we hypothesize : KM ENABLERS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE 193 H5 : There is a negative rvlationship between formalization and knowledge cre - ation process . T - Shaped Skills T - shaped skills are both deep ( the vertical part of the ' T " ) and broad ( the horizontal part ofthe " T ' ) ; that is , their possessors can explore particular knowledge domains and their various applications in particular products [ 70 ] . For example , persons with T - shaped skills not only have a deep knowledge of a discipline ( like ceramic materi - als engineering ) , but also know how their discipline interacts with other disciplines ( such as polymer processing ) 156 ] . People with T - shaped skills are extremely valu - able for creating knowledge because tbey can integrate diverse knowledge assets [ 70 ] . They have the ability both to combine theoretical and practical knowledge and to see how their branch of knowledge interacts with other brancbes . Therefore , they can expand their competence across several functional branch areas , and thus create new kjiowledge [ 60 , 74 ] . H6 : There is a positive relationship between the presence of the organizational members with T - shaped skills and knowledge creation process . IT Support IT support means the degree to which knowledge management is supported by the use of ITs [ 35 | . Many researchers have found that IT is a crucial element for knowl - edge creation [ 19 , 36 , 39 ] . IT affects knowledge in a variety of ways . First , IT facili - tates rapid collection , storage , and exchange of knowledge on a scale not practicable in tbe past , thereby assisting the knowledge creation process [ 92 ] . Second , a well - developed technology integrates fragmented flows of knowledge [ 35 ] . This integra - tion can eliminate barriers to communication among departments in organization . Third , IT fosters all modes of knowledge creation and is not limited to the transfer of explicit knowledge [ 90 , 91 , 99 ) . For instance , InfoTEST ' s enhanced product realiza - tion ( EPR ) project employs electronic whiteboarding and videoconferencing to en - hance exchanges of tacit knowledge [ 91 ] . Thus , we hypothesize : H7 : There is a positive relationship between IT support and knowledge creation process . Organizational Creativity Organizational creativity is the capability of creating valuable and useful products , services , ideas , or procedures by individuals working together in a complex social system [ 5 , 122 ] . Knowledge plays an important role in the ability of the organization to be creative [ 1191 . Thus , organizations with better knowledge diffusion and creat - ing mechanisms are more intelligent [ 34 | . Organizational creativity also connects and rearranges knowledge to create new , often surprising ideas that others judge to be 194 LEE AND CHOI useful [ 65 ] . Creativity is not necessarily related to tbe amount of knowledge that an employee possesses , but rather the way in which knowledge is created and shared [ 4 ] . The processes of knowledge creation unleash organizational creativity . Naturally , organizational creativity has a strong link with knowledge creation [ 119 ] . H8 : There is a positive relationship between the knowledge creation process and organizational creativity . Organizational Performance In our study , organizational peribrmance is assessed hy the use of global output mea - sures such as market share , profitability , growth rate , innovativeness . successfulness , and the size of business in comparison with key competitors [ 22 , 25 ] . In a knowl - edge - based economy , organizational creativity represents a dramatic organizational change . Robinson and Stern [ 93 ] insisted that the tangible results of corporate cre - ativity are the organizational change such as improvements ( changes to what is al - ready done ) and innovations ( entirely new activities for the company ) , Without creativity , organizations may fail to adapt to changing internal and external condi - tions | 88 [ , and thus lose their knowledge advantage . Typically , the goals of organiza - tional change include the various aspects of organizational peribrmance such as organizational effectiveness , survival , improvement , or innovation . Organizational performance can be thought of as the output of a process that encourages creativity [ 97 ] . Thus , improvements of creativity might lead to better organizational perfor - mance [ 18 , 88 , 102 ] . We hypothesize that : H9 : There is a positive relationship between organizational creativity and orga - nizational performance . Sample and Measures SAMPLES WERE RESTRICTED TO THE LISTED COMPANIES in order to include major companies in Korea . Annual Corporation Reports by Maeil Business Newspaper [ 75 ] is the source for sampling because it analyzes all listed companies in the Korea Stock Exchange . Therefore , the unit of analysis in this study is the organization . We adopted both interviews and mail surveys . Interviews were used to investigate the current detailed status of knowledge management . This investigation included knowledge management practices such as the number of communities of practice , the rate of use of tbe knowledge management system , and the cost of investment in knowledge man - agement activities . Although interview data is not analyzed statistically , they were valuable for our interpretation . After the interview , a questionnaire - based survey was conducted . Questionnaires were administered to a total of 1 , 425 middle managers in 147 organizations . Depend - ing on each individual firm ' s size , five to 15 middle managers were surveyed from each firm . Middle managers were reached through their CEOs or CIOs . A typical job title of a middle manager was department chief . Middle managers were surveyed KM ENABLERS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE 195 because they played key roles in managing knowledge . Middle managers are posi - tioned at the intersection of the vertical and horizontal flows of knowiedge . Thus , they can synthesize the tacit knowledge of both top managers and frontline employ - ees , make it explicit , and incorporate it into new products and services [ 82 | . A multiple - item method was used to construct the questionnaires . Each item was based on a six - point Likert scale , from " very low " to " very high . " Likert scales as generally used tend to underestimate the extreme positions [ 3 ] . Respondents are re - luctant to express an extreme position even if they have it . They tend to please the interviewer , appear helpful , or respond in what they perceive to be a socially accept - able answer . Resorting to a scale without a midpoint seems to help mollify this social desirability bias without changing the direction of opinion [ 32 ] . The six - point Likert scale avoids a midpoint , which prevents respondents from using a neutral default option [ 5 ] . The questionnaires were written in Korean . Research constructs were operationalized on the basis of related studies and pilot tests . The operational definitions of instruments and their related literature are sum - marized in Appendix A . Most of the research constructs have already been validated and used for other studies on knowledge management , organizational design , learn - ing , or IT management . For example , formalization items have already been vali - dated and used by Caruana et al . 114 ] and Rapert and Wren [ 89 ] . Self - reported items have been used to assess organizational performance [ 22 , 25 ] . Although these items do not pre . sent a fully balanced scorecard , they are effective for comparing business units and industries [ 25 ] . Questionnaire items for the knowledge creation process , which were used in this study , had been validated and used by Nonaka et al . 183 ] . Analysis Sample Characteristics IN TOTAL , 451 QUESTIONNAIRES FROM 63 out of 147 firms were returned ( 43 percent response rate ) . The rates from individual firms ranged from 23 to 100 percent . Due to incomplete data , 25 responses from five tirms were eliminated . Consequently , 426 responses from 58 firms were analyzed . Table 2 summarizes the respondent charac - teristics in terms of industry type , departments , total sales revenue , and number of total employees . Samples are divided into three industry types : manufacturing , service , and finan - cial business ( banking , finance , insurance ) . The majority of these firms are in the service industry . Thirty - two firms have annual total sales revenue of $ 1 billion or more , and 31 firms have 1 , 000 employees or more . As mentioned previously , samples were collected from various middle managers . Reliability and Validity Analysis Table 3 presents the results of reliability and validity tests . An analysis was performed on the 36 items that measured the components of knowledge enablers ; other analyses 196 1 - O o I ^ ffi X ) 3 3T3C . 9 5 ) ^ " - E E ^ 0 CO o = . 0 ' iS ffl O o o jr U c 1 ) - o c oa . OS r4 3T3 3 C a . O O CO to in o r ^ CM CD O ) CD T - O - 1 - T - OJ CJ OJ • * oo OJ CD cn t ^ CO CO CD 0 • ^ cu r - ; cvi lo d •r - ; C \ J O ) O CO irj CO o TT o o - B 60 i on ill Eo LO c ffl ss t h i 0 c g 1 o % o cu o * * i on O m ill c q Eoo o O c illi o e oo T— c 1 b illi o ffi o 0 ) o illi o Eoo in I li on ; • in g o 0 ) , Q— ^ c b illi o T— lilli on o cu c b illi o LO d ) g cB c CO co O b illi t a i • ^ , o O o CO COCU _ l o o s " aj o o o o o LO O o o PJ o o o Lfi o , 00 CO $ o CD . QO OO o o o 0 , 0 o CD O o o CO oo 30 , o a > o o o o o COT3CCO o o o o " CO to 198 CO C r3 fJ c " ^ Q > b H - - y I I I £3 •J3 = ^ U Q 1— • ^ CJ d CO d T— 3 I i ca o > r ^ n • ^ 1 - r - in O > Cl CO CO CJ > CO r - . . CO CO ^ CO ^ CO t ^ fv . CO CO < 6 < D < D < D j o j o ^ c o c j o c o o r CM t ~ - C 0 C \ J O C 0 O C 0 C D ' - C 0 C0 O O t O O O C nO CO • * to CD CO O CD • < * h - O CJ CO 1 - in en en CO c ) CO CO d d d < o d N CO d N . lO • - - N < p W ( N OJ 5 8 t : gy CO - ^ • * CO CO ( O a . ( 0 lii o _ q q p o o ^ ^ ^ ^ ^ O o 3CC •B S B § CD = CO ^ — r - . — CO O B C Q O X O t ; 199 ddddddddddddddddddddddddd cvj CD 1 - 1 - d d d d d d d d d d d d d d d d d d d d d d d dd Eg CVJ Lfi d in LUO CC o LL 2 c O CO E 200 . E ^ B 3 Q on iS _ c o •3 tj s ra O • * - — ' oj o • — * — fH Uj § > fc E S • " " ra o SI 02 U E . ^ in CO 1 - 1 - o O ) • < - OS iO t - r > - CO CO CO t ~ - ; d d d d > d O CR CO in CM h - o in CD N - CO h - ; CC ) ccj r ^ d d d d d •q - r - " ^ - I - CO •51 - CM " ^ CO CM r - o o CD • ^ CO r ^ r ^ r ^ CD d d d d d CO en CM CD CO CO 1— O CO CO h - CO Ln IN CO h - If ) h - h - CO d d d d d o co o CD cn j i CO CO 0 . O o c : tu ra c . 9ra ra 6 QU c ra E o t ( DQ . KM ENABLERS . PROCESSES , AND ORGANIZATIONAL PERFORMANCE 201 were performed on the 20 items for the knowledge creation processes , on the five items for organizational creativity , and on the six items for organizational perfor - mance . Cronbach ' s alpha is used for examining the reliability of the instruments . A higher cutoff value of 0 . 7 may be used because these instruments have been adopted previously [ 84 ] . All constructs had higher than 0 . 7 cutoff alpha value , ranging from 0 . 8309 to 0 . 9203 . For convergent validity , items having item - to - total correlation scores lower than 0 . 4 were dropped from further analysis . One item relating to organiza - tional perfonnance had an item - to - total correlation of less than 0 . 4 and thus was elimi - nated from further analysis . Factor analysis is used to check discriminant validity [ 64 ] . Because each variable was measured by multi - item constructs , factor analysis with varimax was adopted to check the unidimensionality among items . Items with factor loading values lower than 0 . 5 were deleted . There was one item with factor loading of lower than 0 . 5 for the knowledge creation processes . A factor analysis for the knowledge enablers and knowledge creation processes is shown in Table 4 . Relatively high values of reliabil - ity and validity imply that the instruments used in this study are adequate . All the measures used in this study are reported in Appendix B . Inter - Rater Reliability atid Agreement Atialysis Whereas the unit of analysis in this study is the organization , the questionnaire was distributed to organizational members to measure characteristics of their organiza - tions . Therefore , answers from the same organization should be aggregated and used as an organizational indicator . Given the perceptual nature of the measures and the conversion of individual responses into organizational indicators , inter - rater reliabil - ity and agreement analysis are necessary [ 118 ] . Inter - rater reliability is defined as an index of consistency , which represents consistency of variance among raters [ 66J . In contrast , agreement is defined as the interchangeability among raters , which addresses the extent to which raters make the same ratings f58 ] . The inter - rater reliability was as . sessed by the use of the interclass correlation coef - ficient ( ICC ) . Because each company was rated by a different rater and their ratings were averaged , ICC { I , k ) was appropriate . ICC ( i , k ) is calculated by one - way analy - sis of variance ( ANOVA ) [ 103 ] . James et al . f58 | developed indices appropriate for within - group agreement for a set of raters rating a single target with a single item t ^ gcn ) or multiple - item scale ( r ^ ^ y , ) - For our study , r ^ . g ^ J ^ is adopted . Table 5 summa - rizes the results of inter - rater reliability and agreement . A number of management studies suggest that ICC ranging from 0 . 512 to 0 . 991 and r„ , ^ , _ „ ranging from 0 . 69 to 0 . 96 [ 5 , 46 ] are appropriate . Our results are consistent with these ICC and r ^ . ^ , j , ranges , and thus inter - rater reliability and agreement may be guaranteed . Regression Analysis A multiple regression analysis tests our hypotheses . For each hypothesis , models were nm for each of the dependent variables separately as shown in Figure 4 . Our model is 202 - i on ra 0 o : X ra 6 s t nx e s ra o 1 ra o l e r s 42 c V eo l e d o o it e m s u _ ^ e n t f o r I r i x raEo F ac " ra " t o r 7 F ac t o r 6 a a . o ora LL u 2 oraP - o F ac i tNuO u ra , 1 - 1 o on i a b i V a r C D O C D C O C S I i n t O CO CO lO ^ C ) Q ) CO ^ ^ 1 ^ ^ t - ^ O > CO to If ) ^ ^ r ^ ( o r ^ f ^ o d d d o d d dd d d d d d d d d dd o do d d d dd d d d d d d d d d d d d d d dd d d d d d d d d d d d d d d dd dddddddddddddddd d d d d d d d d d d d d d d dd ^ ^ p ^ ^ q qq dddddodoooodddd o o o cp 203 • ^ N . t D O i - 0 > C 0 O N i - C 0 C D C D 0 > ^ r - C 0 - i - C D 0 ) a CM ^ CO O O T— CM ' " " ' — C M ^ ^ O CO ^ O O O T— O cr o o o d d d d c p c D d d d d d d c p d d dd g O O O O O Q Q OO ^ q q q q j qq d d d o d d d d c i i d d d d d d d d d dd c3 > Ol o > m o h * T— to ify o ^ CM OO CO CO CO ^ OJ ^ LO t - - C 0 m O l C D ' — CM " * — l O l f l t D i— [ ^ C O O O C O ' O O ' ^ CO ^ CO O CM C \ J O ' ^ C M O T— C M ^ O ^ T— C M C ^ J O J O ^ c p c p d c a c i i d o d o d d d d o d d d d dd s q ; q q qq c j J c a d o d o o o o o d d d c D C D d d d dd c o O ' c n i n ^ o i o c D C M c n m c M i f i i o e o e o c ' J h - o q q c o c M i - c o p c o q q - i - q q c M o e q t D e q h - oo dodddddddooddddddddd ooddddddddddddoddddd O O O O O L L L J i i l L r i r K K K H K b b b bb 204 3C oU u oa . E3 0 em •D 1 uC CN o ^ q p p qq o o o d d o o o o o o d d o o o o od p o q d d d d d d d d d d d d d d d dd d d d d d d d d d d d d d d d d d dd c o ^ c o o j c o o o ( D C N t i f t u r > ^ i f i ' — ' ^ c o i n ' ^ ' ^ d d d d d d d d d d d d d d d d d dd CnCOCOCOCOLULULJJUJUJOOOOOII ^ y ^ H O O O O O O U O O O O O O O O O O OO KM ENABLERS , PROCESSES . AND ORGANIZATIONAL PERFORMANCE 205 Table 5 . Results of Inter - Rater Reliability and Agreement Variables Knowledge creation process Knowledge management enablers Indices Socialization Extemalization Combination Internalization Collaboration Trust Learning Centralization Formalization T - shaped skills IT support Organizational creativity Organizational performance ICC ( l , k ) 0 . 6627 0 . 6468 0 . 5252 0 . 5285 0 . 6081 0 . 8037 0 . 6863 0 . 5632 0 . 6983 0 . 5236 0 . 7515 0 . 7390 0 . 8397 0 . 8138 0 . 8815 0 . 8522 0 . 8633 0 . 8691 0 . 8929 0 . 8927 0 . 8426 0 . 8393 0 . 8203 0 . 8460 0 . 8552 0 . 8601 not meaningful if the correlation between enablers and the knowledge creation pro - cess is not significant . Therefore , the knowledge creation process is considered as an aggregated variable , and its correlation is computed . We then test each hypothesis to fmd which enablers are more important for knowledge creation and which processes are more important for organizational performance . To meet the assumptions of regression analysis , we examined Ihe linearity , constant variance , and normality | 42 ] . Because the scatterplots of individual variables do not indicate any nonlinear relationships , the linearity is guaranteed . Plotting the studentized residuals against the predicted value shows that no variable violates the constant vari - ance . The result from the normal probability plot and Kolmogorov - Smimov tests indicates no violation of normality ( statistic = 0 . 050 - 0 . 096 . p > 0 . 200 ) . The overall regression model ( for fmding the relationship between the knowledge creation process and enablers ) is significant [ F = 51 . 771 , / 7 < 0 . 000 ) . R - ( 0 . 879 ) sug - gests that 87 . 9 percent of the variance is explained by seven variables . The result of the collinearity test ( VIF = 1 . 429 - 3 . 725 ) shows no multicollinearity problem . Atialysis Results TABLE 6 SUMMARIZES OUR REGRESSION RESULTS . In order to provide a better presen - tation of significant relationships . Figure 5 has been provided . Collaboration , trust , learning , and centralization are found to be relatively significant predictors for knowl - edge creation . Organizational culture variables are found to be essential for knowledge creation . Collaboration is positively related with socialization , extemalization , and intemaliza - tion , whereas it does not affect the combination mode . In particular , trust is a signifi - cant predictor of all knowledge creation modes . Centralization is negatively related with socialization , extemalization , and internalization while it is not significantly re - lated with combination . By contrast , formalization and T - shaped skills of members 206 LEE AND CHOI Knowledge Management Enahlers S o c i a l p e r s p ec ti v e Culture •Collaboration ( COL ) •Trast ( TRU ) •LcarainR ( LEA ) Structure •Centralization { CEN ) •Formalization ( FOR ) People •T shaped skills ( TSK ) Knowledge Creation Knowledge Management Organizational Process ( KCP ) Intermediate Outcome Performance •Sacialiution ( KCS ) •ExtemalizaiioD ( KCE •Combination ( KCC ) •IniemalizauoD ( KCl ) » •Organizationalcreaiivity ( OC ) ^ •Organizationalperformance ( OP ) Infomiation Technology •IT Support ( rrs ) ( a ) Between Ihe ktiowledge creation processes and knowledge management enablers KCP = a + p , COL + p , TRU + % LEA + p , CEN + % FOR + & , TSK + p , ITS + E KCS = a + p , COL + ^ ' TRLI + p , LEA + p , CEN + ft FOR + ^ TSK + p , ITS + E KCE - a + p , COL + ^ TRU + ^ LEA + p . , CEN + ft FOR + ft TSK + ft ITS + e KCC = a + ft COL + ft TRU + ft LEA + ft CEN + ft FOR + ft TSK + ft ITS + E KCl = a + ft COL + ft TRU + ft LEA + % CEN + ft FOR + ft TSK + ft ITS + e ( b ) Between organizational creativity and knowiedge creation processes • OC = a + p , KCP + e OC = a + p , KCS + P , KCE + ft KCC + ft Ka + e ( c ) Between organizational perfonnance and organizational creativity OP = a + PI OC + E Figure 4 . Regressioti Equations do not sigtiificantly affect knowledge creation . IT support is significantly related with knowledge combination only . Knowledge creation is positively related with organizational creativity , which is positively related with organizational performance . This finding confirms that an or - ganization can achieve strategic benefits of knowiedge management through effec - tive knowledge creation . Discussion Limitations THE FINDINGS OF THIS STUDY ARE INTERESTING , but they should be considered in light of its inherent limitations . First , this study presents a snapshot research that does 2G7 . 1 ^ a : c * o oo * ^ " ^ 2 c * - ^ O " 14 : O 0 = 6 o HU lit k . II II CQ . * M S 00 " * II 7 \ II 00 W « 9 ; j o T - ; 4M ro > 4w T T o JLii ciL oII P q P CVJ o d ^ d O ) LO ^ O • ^ CO O ) T OJ o T ^ o c«i CD d ~ I I n hi O ( O o d II II CQ . - ^ < o OT go CO 1 - O o m II 11 " CO go ' - O CN O " oo d ( \ j O o c ^ j o c \ i T I T I O o II II C Q . * . . 2085 o II 4901 cti II 3525 5907 O CO II II 2138 o II 2498 0 . 2 O 3 oi T II II ^ CO ( O O 1 T II II * . CQ . * > CO . * • nr ^ ^ 1 o ll a b i o O oj " X i s t ( l 3 x _ d a r n ii < u X t i on CON i ^ " c cu O in X t i on fON lo Eo LJ . CO tn tn TS CD Q . ra sz tn 208 00 U 6 cO oc Xi e oU N t - - X . ^ o ^ c Ii a ^ a ^ oc • i - i O - a < U Q . fc 00 •a oU X ) 2 5 r - _ _ O ( O •I II < D CO to S d CM II II CO . • - CM ( o d w II It CO . * - S ? " > t CO ' - ; to o W II II CM ^ • ^ o • ^ CM tl II CO . * • • ^ CO • ^ CO q 2 m CO Oi CO CM o o esi II II 00 il • - oo II ax T " O ) ao II I ' II CO . * . If — X CO • • b o CO ; i CO ^ 6 o ? ^ O Q . CO II LL o 209 oi t I 2 ! 0 LEE AND CHOI not consider feedback effects . A longitudinal study to investigate the dynamic fea - tures of knowledge management would provide further robust results . Second , it fo - cuses on relatively large and profitable firms . The results may differ in small or venture firms . Finally , tbe results are limited to Korean firms . Tbe generalizability from a Korean setting to other countries may be questionable . Implications Our results can belp managers establish distinctive strategic positions . Knowledge management strategies can be described along two dimensions to reflect knowledge management focus [ 45 ] . One dimension refers to knowledge sharing via interper - sonal interaction . The other dimension refers to the capability to help create , store , share , and use an organization ' s explicitly documented knowledge . The former is more affected by socialization , and the latter is more affected by combination [ 16 ] . Knowledge management strategists can sbarpen weak knowledge management di - mensions on the basis of enablers mentioned in our study . Table 7 highlights tbese implications . The following is a further discussion of these implications . Our findings confirm that knowledge creation is associated with cultural factors such as collaboration , trust , and learning . For instance , groups are most creative when their members collaborate ; members stop holding back when they have mutual trust [ 54 ] . Shaping cultural factors is crucial for a firm ' s ability to manage its knowledge effectively [ 15 , 20 . 35 , 71 ] . Forexample , our interview witb an executive of a confec - tionery company highlights this point . Tbe executive pointed out that their employees did not just use tbe manual or otber codified supports . It was noted that they preferred to depend on their own experiences and networking relationships . A trust - based cul - ture is the foundation for their knowledge management initiative . However , many knowledge management projects , in reality , focus on IT [ 19 , 35 , 111 ] . An organization may face difficulties in building its knowledge creating envi - ronment due to the lack of adequate culture despite its well - constructed IT [ 23 , 72 ] . Stein and Zwass [ 107 [ insisted that successful information systems should be condi - tioned by a number of cultural factors such as organizational values and appropriate learning methods . Initiating knowledge management only through IT can be a risky proposition [ 19 ] . Our analysis confirms that IT support affects combination . There are several re - sourees for a sound understanding of the impact of IT on knowledge combination [ 82 , 1001 . Tbis finding highlights tbe characteristics of knowledge combination . IT is critical for codifying explicit knowledge : it provides fast feedback for explicit knowl - edge [ 69 , 120 ] . In order to support knowledge combination , the question is not whether to deploy IT , but bow to deploy it . Interestingly , our analysis also reveals that trust affects combination . Tbis result implies tbat simply improving the IT infrastructure does not provide a competitive advantage for knowledge combination . Througb in - terviews witb executives in the disk industry in the United States . Scott [ 100 ] found that communication of even explicit knowledge is difficult without a solid founda - 211 Q . E dge £ r u c i a l c t o r s i s C I t u r a l O OTC l ap i C / ) l en t ; fc O ) ra c raE OT o l y t h r c j m en t e m anag t OT 1 2 OTC i t i a t i c ' — no l og y c a O c . 9 £ if . Q o s i t Q . p r o 0OTTD0 e s t ab li s h o eed c e r s OTCO I t u r e . 3 ' - ' f i r m ' s n s i de r i ng ooc 0 E 0 ) OT E OT ; od i f i o i t i c a l f i og y i s c r o c o 0c a t i o b ' •a l O W l i o 1 . — OT m o l e o a t i on he i n f o r m OT _ co . E m p l CO t i v e o m p f O ra o v i de e s no t pn o l u r e o i n f r . t i on ; ra c c o m b l o w l edge £ 0 OT CCu > •o 0£ : _ o o c j | a tt e ) a y c a r e f L hou l to e r s US ra co o > i no l o o 0 a t i on • o f i n f o r m . " o CO 3 n t i a 0 o Q . 01 ' i t h t h i ii na t i on I A £ o 0 OT 0 f i r m . t r u s t i n a o i t i o n ii de o o n i m u C O . X ) " S o o 01 en ra Q . Sif G tl - ( 0 jn O o CO 0OT•a i o a . - a oo o CD 212 E Q l a li z a t i o f f o r m tfl ped ro f e r en i > £ F " D0 — : a c i t - r e i nh i b i t t > . ro o l a li z a t o T3 e t a t e •Q . i ge e x 3 j noou s ( B L U J3 i t i e s , 1 i t i e s ) ; . > _ > ts t ) CO ra c tu 0 n s h i p 1 9 " 0 " o t i on CD r \ e x p l ( tu . ^ Zi LL tfl O ro ge e r e no w l ed and c o a li z a t i E o to t i on i li z a CO f e x t e r ha s i s 0 Q . E o £ 0 •o0 0C 0 . O > ^ ro Ec a li z a t ii , e x t e r n fl ) l edg o c i t k n ro c o i n ; i f g " S N 1 ^ f o r m a : ed w i t h ro s o c i ; tfl t i v e l y ro 0 c ra o l o i CT i t s t e c : e ss o r o c " s i o j a A uo 0 T3 a s i z e sz 0 ) i on i s ( r na li z a t 0 X0 " o 0 > to tu Q . t i on ro N t e r na li ff e c t e x CO c an a o E ' ^ ^ o •is Q - 00 ol •aucc o c o ro N a li . p be t w een CO a t i on 0 iz dge c r ea t i c 0 ) k no w i c ra li b i O m a y t end t c o li z a t i ra E c o a t i " TO c n and e x t e r 9 a li z a c o CD c XI E o i a c ili t a t e s o - — r ea s 0 5 li z a t i on . CD c i n t e r " a ro a . - a oU co •a a : c Ol 213 B - ^ to Q . ro o m Q ) s 11 5 ra c c ^ x > £ E ? o Ok - o N CD P 2 11 w o o g ra i n i z i O ) b X o•o 0 E 0 - i r ag o 0 0 — : > ^ ^ o ^ CO 0 . - ; to . . ro ra " > B • = 0 • . ; = : > O > CD i ro 0 ^ - . . - ' > 3 = 5 0 ra - Q •o 0c ^ ' ra O Q . O 0 0 ) o 5 : C CD ro - O ' 5 O iS O > - - . = t o t " ' J ro 0 ro . 9 - ; ^ ' " ™ • " ro I II ro - a CD 9 ra , 9 N 5 • = ro O ] O 0 ra o Q . O > X , 0 b J3 O o 214 LEE AND CHOI tion of trust . Managers should pay careful attention to the potential impact of IT on knowledge comhination with the consideration of trust in a firm . The nonsignificant findings in this study also bear some implication . Several stud - ies have come to the conclusion that formalization weakens knowledge management [ 57 , 106 ] . In contrast , our study shows no relationship between formali / ation and knowledge creation . This rather intriguing result reflects the two different aspects of formalization . According to the ambidextrous model , which is based on the distinc - tion between the initiation and implementation stages of innovation [ 26 . 94 ] , formal - ization may inhibit tacit - related activities such as socialization ( [ i = - 0 . 052 ) and externalization { p = - 0 . 1165 ) , but may encourage explicit - related activities such as combination ( P = 0 . 0018 ) and internalization ( p = O . I 152 ) . However , this interpreta - tion needs further exploration because ail [ i values are not statistically significant . In particular , a more careful investigation of externalization is of interest . Externali - zation involves the expression of tacit knowledge [ 8 ] . From this perspective , a formal organizational structure may inhihit spontaneity and freedom of expression neces - sary for externalization [ 10 ] . In our study , Ihe emphasi . s of externalization is on tacit knowledge , and thus externalization is negativeiy associated with formalization . How - ever , extemalization may also involve conversion of tacit into expiicit knowledge [ 8 ] . The formal structure can facilitate the rapid and continuous conversion of tacit into explicit knowledge [ 37 ] . If the conversion process or its technoiogy perspective of externalization is emphasized like Becerra - Femandez and Sahherwal [ 8 ] , we may speculate that fumialization can affect extemali / ation positively . Many studies suggested that T - shaped skills positively influence knowledge cre - ation [ 60 , 70 , 74 ) . However , our study shows no relationship between T - shaped skills and knowledge creation . This contradiction may reflect the importance of T - shaped management systems . T - shaped management systems attempt to break out ofthe tra - ditional corporate hierarchy and encourage people to share knowledge 144 ] . How - ever , most current formal organizational incentives encourage l - shaped skills ( the deep functional experti . se ) in isolation [ 70 [ . Without an environment in which T - shaped skills flourish , people with T - shaped skills will not attempt to create new knowledge . It implies that a crucial element of successful knowledge management is not T - shaped skills themselves , but the systematic management of these skills . It would be expected that technologies could facilitate knowledge creation . How - ever , our result shows that IT support is not significantly related with knowledge cre - ation except for combination . It seems that IT does not support all modes of knowledge creation directly . Although groupware , intranet , or videoconferencing can help col - laborative works , this technologically facilitated communication cannot replace face - to - face contact for tacit - to - tacit knowledge transfer [ 53 ] . Accessing the tacit knowledge such as knowledge inside employees ' heads is not possible simply by an intranet or a datahase | 23 ] . That is , the current state of IT may not affect socialization , externalization , or internalization directly . Our study shows that organizational creativity affects organizational performance ( P = 0 . 6338 , p < 0 . 01 ) . This resuU is in line with previous studies [ 73 , 1021 . For example , Shani et al . 1102J provided a framework linking organizational performance KM ENABLERS , PROCESSES , AND ORGANIZATIONAL PERFORMANCE 215 and organizational creativity through a field study ofthe Seagate Corporation . It im - plies that managers pay more attention to organizational creativity in order to im - prove organizational performance . Although the relationship is statistically significant , the percentage of total variation of organizational pertbrmance explained by organi - zational creativity is relatively low ( R - = 0 . 402 ) . This may reflect the creativity para - dox [ 116 ] . If creativity is encouraged and reinforced at the expense of operational behaviors , it may decrease organizational perfonnance . That is , organizational cre - ativity is valuable , but its overencouragement may not be always useful . Conclusions OUR STUDY IS OF INTEREST FROM BOTH theoretical and practical perspectives . Theoretically , a framework is proposed for empirical studies to link knowledge management enablers and processes with organizational performance . This study is probably the first to establish this integrative view of knowledge management . We adopt a process - oriented perspective of knowledge by using Nonaka ' s creation model [ 82 ] . Our framework may be used as a stepping stone for further empirical research on knowledge management . To strengthen the feasibility of this framework , we can clarify the role of knowledge creation process ( see Appendix C ) and intermediate outcome ( see Appendix D ) . From a practical point of view , the relationships among knowledge creation , orga - nizational creativity , and organizational performance may provide a clue as to how firms can adjust knowledge creation processes to sustain their performance . Further - more , managers will he better able to find which enablers are critical for knowledge creation . Because firms may not manage all modes of knowledge creation , they may need robust strategies that involve trade - offs . The current findings of this study may indicate the following avenues for further research . First , an analysis of different factors such as domain knowledge [ 101 ] or other types of knowledge process may lead to interesting implications . For example , an interesting candidate is Szulanski ' s knowledge transfer model , which is made up of four processes—initiation , implementation , ramp - up , and integration [ 114 ] . Sec - ond , our study shows which knowledge enablers can enhance a firm ' s capability to manage knowledge . Appropriate knowledge management strategies may be ahle to facilitate these enablers . Finding these strategies may be of interest . Third , what is the effect of our fmdings on electronic commerce ? Electronic commerce is changing the business world rapidly . The quality of knowledge management may determine a suc - cess template for electronic commerce . For example , Holsapple and Singh [ 50 ] pro - posed the potential benefits of applying knowledge management principles to electronic commerce . Finally , other types of performance measures may sharpen the results of our study . ROP ( return on ideas , return on information , and return on investment ) [ 73 ] or a strategy map [ 63 ] is a good alternative . Acknowledgments : This work was supported in part by a grant from KOSEF ( 98 - 0102 - 08 - 01 - 3 ) . 216 LEE AND CHOI REFERENCES 1 . Adler . 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Tru . s ' l in Organizations : Fmntiers of Theory and Research . Thou - sand Oaks , CA : Sage , 1996 , pp . 90 - 113 . 222 LEE AND CHOI Appendix A . Operational Definitions and Related Literature Variables Operational definition Related literature Collaboration Degree of active support and helps in organization Trust Degree of reciprocal faith in others ' intentions , behaviors , and skills toward organizational goats Learning Degree of opportunity , variety , satisfaction , and encouragement for learning and development in organization Centralization Degree of authority and control over decisions Formalization Degree of formal rules , procedures , and standard polices T - shaped skills Degree of understanding his or her own and others ' task areas IT support Degree of IT support for collative work , ( or communication , for searching and accessing , for simulation and prediction , and for systematic storing Knowledge Degree of socialization , extemalization , creation combination , and internalization Socialization Degree of tacit knowledge accumulation , extra - firm social information collection , intra - firm social information gathering , and transfer of tacit knowledge Extemalization Degree of creative dialogue , deductive and inductive thinking , use of metaphors , and exchanged ideas Combination Degree of acquisition and integration , synthesis and processing , and dissemination Internaiization Degree of personal experiences , simulation , and experimentation Organizational Degree of belief that organizations is actually creativity producing creative ( novel / useful ) ideas ( services / p rod ucts ) Organizational Degree of overall success , market share , performance growth rate profitability , and innovativeness in comparison with major competitors [ 54 , 67 , 85 , 100 ] [ 19 , 54 , 57 , 68 , 74 , 81 , 85 ] [ 55 , 62 , 88 , 113 ] [ 14 , 17 . 27 , 47 , 89 , 115 ] [ 14 , 34 , 89 , 106 , 115 ] [ 56 , 60 , 70 , 74 ] [ 20 , 35 , 87 , 90 , 99 ] [ 82 , 83 ] [ 82 , 83 ] [ 82 , 83 ] [ 82 , 83 ] [ 82 , 83 ] [ 5 , 34 . 40 , 65 , 119 , 122 ] [ 22 , 25 ] KM ENABLERS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE 223 Appendix B . Questionnaire ( 1 ) Knowledge management enablers Construct Items Collaboration COLI : Our organization members are satisfied by tbe degree of ( COL ; five items ) collaboration . COi - 2 ; Our organization members are supportive . C0L3 : Our organization members are beipful . C0L4 : There is a wiilingness to coliaborate across organizational units within our organization . COL5 : There is a willingness to accept responsibility for failure . Trust Our company members . . . ( TRU ; six items ) TRU1 : are generally trustworthy . TRU2 : have reciprocal faith in otber members ' intentions and bebaviors . TRU3 : have reciprocal faith in others ' ability . TRU4 : have reciprocal faith in others ' behaviors to work toward organizational goals , TRU5 : have reciprocal faith in otbers ' decision toward organizational interests than individual interests . TRU6 : bave relationships based on reciprocal faith . Learning Our company . . . ( LEA ; five items ) LEA1 : provides various formal training programs for performance of duties . LEA2 : provides opportunities for informal individual development other tban formal training such as work assignments and job rotation . LEA3 : encourages people to attend seminars , symposia , and so on . LEA4 : provides various programs sucb as clubs and community gatherings . LEA5 : members are satisfied by tbe contents of job training or self - development programs . Centralization Our company members . . . { CEN ; five items ) CEN1 : can take action without a supervisor ( R ) . CEN2 : are encouraged to make tbeir own decisions ( R ) . CEN3 : do not need to refer to someone else ( R ) . CEN4 : do not need to ask their supervisor before action ( R ) . CEN5 : can make decisions witbout approval ( R ) . Formalization In our company . . . ( FOR ; five Items ) F0R1 : there are many activities tbat are not covered by some formal procedures ( R ) . F0R2 ; contacts witb our company are on a formal or planned basis . F0R3 : rules and procedures are typically written . F0R4 : members can ignore the rules and reach informal agreements to handle some situations ( R ) . F0R5 ; members make tbeir own rules on tbe job ( R ) . 224 Lffi AND CHOI Construct Items T - shaped skills Our company members . . . ( TKS ; five items ) TSK1 : can understand not only tbeir own tasks but also others ' tasks . TSK2 : can make suggestion about otbers ' task . TSK3 : can communicate weil not only witb their department members but also witb other department members . TSK4 : are specialists in tbeir own part . TSK5 : can perform their own task effectively witbout regard to environmental changes . IT support Our company . . . ( ITS ; five items ) ITS1 : provides IT support for collaborative works regardless of time and place . ITS2 : provides IT support for communication among organization members . ITS3 : provides IT support for searching for and accessing necessary information . ITS4 : provides IT support for simulation and prediction . ITS5 : provides IT support for systematic storing . " R " indicates that the item is actually measured in a reverse fashion . ( 2 ) Knowledge creation processes * Construct Items Socialization ( KCS ; five items ) Tacit knowiedge accumulation Tacit knowledge accumulation Extra - firm social information collection Intra - firm sociai information collection Transfer of tacit knowledge Externalization ( KCE ; five items ) DialogueMetapborMetapbor DialogueDialogue Combination ( KCC ; five items ) Acquisition and integration Syntbesis and processing Our company stresses . . . KCS1 : gathering information from sales and production sites . KCS2 ; sbaring experience with suppliers and customers . KCS3 : engaging in dialogue witb competitors . KCS4 ; finding new strategies and market opportunities by wandering inside the firm . KCS5 : creating a work environment that allows peers to understand tfie craftsmanship and expertise . Our company stresses . . . KCE1 : creative and essential dialogues . KCE2 : tbe use of deductive and inductive thinking . KCE3 : tbe use of metaphors in dialogue for concept creation . KCE4 : exchanging various ideas and dialogues . KCE5 : subjective opinions . Our company stresses . . . KCC1 : planning strategies by using publisbed literature , computer simulation and forecasting , KCC2 : creating manuals and documents on products and services . KM ENABLERS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE 22 . 5 Synthesis and processing KCC3 ; buiiding databases on products and service . Synthesis and processing KCC4 : building up materials by gathering management figures and tecbnical information . Dissemination KCC5 : transmitting newly created concepts . tnternalization Our company stresses . . . ( KCI ; four items ) Personal experience KCI1 ; enactive liaisoning activities with functional ( knowledge acquisition departments by cross - functional development form real world ) teams . Experimentation { knowledge KCI2 : forming teams as a model and conducting acquisition from virtual world ) experiments , and sharing results with entire departments . Personal experience KCI3 : searching and sbaring new values and thoughts . Personal experience KCI4 : sfiaring and trying to understand management visions through communications witb fellows . 0 ) Organizational creativity Construct Items Creativity Our company . . . ( OC ; five items ) OCI : has produced many novel and useful ideas ( services / products ) . 0C2 : fosters an environment tbat is conductive to our own ability to produce novel and useful ideas ( services / products ) . OC3 : spends mucb time for producing novel and useful ideas ( services / products ) . 0C4 : considers producing novel and useful ideas ( services / products ) as important activities . 0C5 : actively produces novel and useful ideas ( services / products ) . ( 4 ) Organizational performance Construct Items Organizational Compared with key competitors , our company . . . performance OP1 : is more successful . ( OP ; five items ) 0P2 : bas a greater market sbare . 0P3 : is growing faster . 0P4 : is more profitable . 0P5 : is more innovative . Note : * Linkage between knowledge creation constructs and our questionnaire items . Questionnaire items for the knowledge creation process , wbicb were used in this study , had been validated and used by Nonaka et al . [ 83 ] . They conducted a confirma - tory factor analysis to test Nonaka ' s | 82 ] organizational knowledge creation model with data collected from 105 Japanese middle managers . Results of the study suggest that the construct of knowledge creation consists of four knowledge conversion pro - cesses ; socialization , externalization , combination , and internalization . All four knowl - edge conversion processes explain a high amount of variance in the knowledge creation 226 LEE AND CHO ! construct . Four factors constitute the process of converting tacit to tacit knowledge ; accumulation of tacit knowledge , extra - firm social information gathering activities , intra - firm social information gathering activities , and transfer of tacit knowledge from the master to the different team members . Externalization process is made up of one factor . This result differs from Nonaka ' s theory that hypothesized that metapbor and dialogue would be retained . Combination process consists of three factors that repre - sent a three - step sequence of data processing : acquisition and integration of informa - tion , synthesis and processing of information , and dissemination of information . Explicit knowledge in the organization may be converted into tacit knowledge ( inter - nalization ) in two different ways ; personal experience in which knowledge is ac - quired from real world , and simulation and experimentation in which knowledge is acquired from tbe virtual world . KM ENABLERS . PROCESSES . AND ORGANIZATIONAL PERFORMANCE 227 Appendix C . Mediating Effect of Knowledge Creation Process OUR STUDY HINTS THAT KNOWLEDGE CREATION process mediates between enablers and organizational creativity . However , some recent studies regard both knowiedge enablers and knowledge creation process as antecedents of organizational perfor - mance [ 8 , 35 ] ; that is . both of them are independent variables of organizational per - formance . Therefore , in order to test the mediating effect of knowledge creation process , the Baron and Kenny | 7 | procedure is adopted . Table Al shows this analysis result . This results in the mediation effect because tbe following three conditions hold . First , ktiowledge enablers affect knowledge creation process significantly . It has been noted that collaboration , trust , leaming . and centralization affect creation . However , tbis is not the case with formalization . T - shaped skills , and IT support ; we could not assess the mediating effect for these three enablers . Second , collaboration , trust , leaming . and centralization affect organizational creativity . Tbird . knowledge creation process affects creativity ( p = 0 . 7042 ) while the effects of the previous four enablers are re - duced . For example , in the case of collaboration , its beta value is reduced from 0 . 2144 to 0 . 1316 . In sum , we may point out that knowiedge creation process mediates be - tween the four enablers ( collaboration , trust . learning , and centralization ) and organi - zational creativity . Table Al . Mediation Analysis Result Collaboration TrustLearningCentralization PormalizationT - shaped skills IT support Knowledge creation * * * / ) < 0 . 01 . * * / j < ( ) , 05 . Knowledge creation ( beta values ) 0 . 2085 " 0 . 3525 " * 0 . 2138 " - 0 . 2030 " - 0 . 0130 0 . 0443 0 . 0611 * p < O . l . Organizational creativity ( beta values ) 0 . 2144 ' 0 . 3916 " * 0 . 2015 * - 0 . 1808 ' - 0 . 0390 0 . 1682 " 0 . 0949 Organizational creativity ( beta values ) 0 . 1316 0 . 1353 " 0 . 1291 - 0 . 1047 - 0 . 0296 0 . 1514 " 0 . 0493 0 . 7442 * " 228 LEE AND CHOI Appendix D . Mediating Effect of Intermediate Outcome IN ORDER TO VALIDATE WHETHER an intemiediate outcome is an important predictor of knowledge management or not , anotber model without organizational creativity is built to explore the direct relationship between knowledge creation and organiza - tional perfonnance . Testing tbis direct relationship indicates no significant relation - ships except for socialization ( p = 0 . 540 , p < 0 . 05 ) . This result is consistent with the previous study [ 181 . lt implies that the intermediate outcome can help build a chain of credibility between knowledge creation and organizational performance . Although not the focus of this study , it is of interest to note an altemative concurrent model in organization theory . Tbis model would posit that efficiency and bureaucratic ( or mechanistic ) structures would chain through to oiganizational performance . For example , centralization can lead to efficiency because it prevents a strategic vacuum of organizations and enables tbe development of precise control prtxredures 130 ] . In addi - tion , formalization has been found to lead to efficiency because it may facilitate the rapid and continuous transformation of ideas into superior products and services and enhance communication flow through tbeir extensive monitoring and reporting require - ments r36 | . Similarly , standardizing business practices may encourage efficiency 148 ] . Related to an interplay between creativity ( flexibility ) and efficiency , it bas been assumed tbat a firm must either focus on efficiency or flexibility [ 33 , 123 ] . That is . flexibility ( or efficiency ) can only be achieved at the cost of efficiency ( or flexibility ) . Therefore , . some researchers have concentrated on improving efficiency [ 125 ] whereas others bave focused on bow to improve flexibility and creativity [ 13 ] . However , there are now a few studies that have suggested that it is possible to be simultaneously efficient and flexible 124 , 3 ! ] . Organizations can obtain their com - petitive advantages through achieving efficiency by emphasizing control as well as flexibility ( creativity ) by creating knowledge [ 117 ] . Case studies such as Microsoft 148 ] , Unilever [ 691 , and NUMMI ( aToyota subsidiary ) [ I ] have shown this simulta - neous approach . These studies suggest that balancing between imposing discipiine for efficiency and delegating authority to encourage flexibility and creativity pro - vides tremendous benefits for organizations . In summary , . some studies insist that efficiency and flexibility are mutually exclu - sive , whereas others argue that they are perfectly compatible . Our study focuses on creativity ( flexibility ) only . The interplay between these creativity forces and efficiency forces should be further investigated in the field of knowledge management . For ex - ample . Kiogh et al . [ 69 ] indicated that knowledge management allows an organization to improve both its efficiency and flexibility ( innovation ) capabilities simultaneously . \ No newline at end of file diff --git a/silver_data/7380916d9d078d6c2ab7465ab8de904a7034ae1b.pdf.txt b/silver_data/7380916d9d078d6c2ab7465ab8de904a7034ae1b.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..542df2d944ff83189c63ea31de1b8e45956fa224 --- /dev/null +++ b/silver_data/7380916d9d078d6c2ab7465ab8de904a7034ae1b.pdf.txt @@ -0,0 +1 @@ +Cryo - electron tomography of microtubule – kinesin motor complexes Julia Cope a , Susan Gilbert b , Ivan Rayment c , David Mastronarde a , Andreas Hoenger a , * a The Boulder Laboratory for 3 - D Microscopy of Cells , University of Colorado at Boulder , Department of Molecular , Cellular , and Developmental Biology , Boulder , CO 80309 - 0347 , USA b Rensselaer Polytechnic Institute , Biology Department—JRSC 1W14 , 110 8th Street Troy , NY 12180 - 3590 , USA c Department of Biochemistry , University of Wisconsin , Madison , WI 53706 , USA a r t i c l e i n f o Article history : Received 3 November 2009 Accepted 3 December 2009 Available online 16 December 2009 Keywords : Cryo - electron microscopy Cryo - electron tomography Kinesin Microtubules Helical 3 - D reconstruction Eg5 Kar3Vik1 a b s t r a c t Microtubules complexed with molecular motors of the kinesin family or non - motor microtubule associ - ated proteins ( MAPs ) such as tau or EB1 have been the subject of cryo - electron microcopy based 3 - D studies for several years . Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3 - D reconstruction , while few were analyzed by single particle approaches or from 2 - D crystalline arrays . Helical reconstruction of microtubule – MAP or motor complexes has been extremely successful but by definition , all helical 3 - D reconstruction attempts require perfectly helical assemblies , which presents a serious limitation and confines the attempts to 15 - or 16 - protofilament microtubules , microtubule configurations that are very rare in nature . The rise of cryo - electron tomogra - phy within the last few years has now opened a new avenue towards solving 3 - D structures of microtu - bule – MAP complexes that do not form helical assemblies , most importantly for the subject here , all microtubules that exhibit a lattice seam . In addition , not all motor domains or MAPs decorate the micro - tubule surface regularly enough to match the underlying microtubule lattice , or they adopt conforma - tions that deviate from helical symmetry . Here we demonstrate the power and limitation of cryo - electron tomography using two kinesin motor domains , the monomeric Eg5 motor domain , and the hete - rodimeric Kar3Vik1 motor . We show here that tomography does not exclude the possibility of post - tomo - graphic averaging when identical sub - volumes can be extracted from tomograms and in both cases we were able to reconstruct 3 - D maps of conformations that are not possible to obtain using helical or other averaging - based methods . (cid:2) 2009 Elsevier Inc . All rights reserved . 1 . Introduction Until now , most cryo - electron microscopy 3 - D data on microtu - bules and microtubule – kinesin complexes have been produced by so - called averaging - based methods , mainly helical averaging ( DeR - osier and Moore , 1970 ; for tubulin examples , see Sosa et al . , 1997 ; Beuron and Hoenger , 2001 ; Wendt et al . , 2002 ; Skiniotis et al . , 2003 ; Kikkawa et al . , 2000 ; Hirose et al . , 2006 ; Sindelar and Down - ing , 2007 ) , but also by single particle approaches ( Li et al . , 2002 ) and 2 - D crystallography ( e . g . to solve the structure of the a b - tubu - lin dimer itself : Nogales et al . , 1998 ) . Common to all these cases , 2 - D projections of identical particles at various angles are collected , averaged and back - projected into 3 - D space . Though this could be done in real space , it is often carried out in Fourier space where phases and amplitudes can be assessed separately . The most important prerequisites for averaging - based 3 - D reconstructions are ( A ) the averaged particle must be identical in shape and confor - mation , and ( B ) their projection angles relative to each other ( so - called Euler angles ) must be determined accurately . If this is achievable , averaging - based methods are very powerful for eluci - dating molecular structures and may yield close to atomic detail ( tubulin : Nogales et al . , 1998 ) . In most biological applications , however , many structures , even small macromolecular assemblies are often too flexible and dynamic for averaging procedures . For these cases , the cur - rently most promising alternative to averaging - based 3 - D recon - struction methods is cryo - electron tomography . Initially , tomographic 3 - D reconstruction does not rely on averaging and therefore can be applied to any specimen that can be imaged in an electron microscope , no matter how complex its structure may be ( reviewed in Lucic et al . , 2005 ; Hoenger and McIntosh , 2009 ) . However , once a tomogram is completed , sub - volumes containing identical particles may be extracted and further pro - cessed by averaging ( Walz et al . , 1997 ; Taylor et al . , 2007 ; För - ster and Hegerl , 2007 ; Bartesaghi et al . , 2008 ) . To this end , we have developed our own software called PEET ( Particle Estima - tion for Electron Tomography : Mastronarde et al . , in preparation , for example , see Nicastro et al . , 2006 ) . Here , we illustrate the advantages and disadvantages of both helical reconstruction and volume averaging methods for microtubule – kinesin motor domain complexes . 1047 - 8477 / $ - see front matter (cid:2) 2009 Elsevier Inc . All rights reserved . doi : 10 . 1016 / j . jsb . 2009 . 12 . 004 * Corresponding author . Fax : + 1 303 735 0770 . E - mail address : andreas . hoenger @ colorado . edu ( A . Hoenger ) . Journal of Structural Biology 170 ( 2010 ) 257 – 265 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage : www . elsevier . com / locate / yjsbi The kinesin superfamily comprises a large and diverse group of molecular motors that interact with microtubules to perform a wide variety of essential functions in the cell including roles in intracellular transport and cell division ( reviewed in Hirokawa et al . , 2009 ) . Mitotic kinesins are a group of kinesins that are re - quired for cell division , playing a crucial role in assembly , mainte - nance and function of the mitotic spindle ( reviewed in Wittmann et al . , 2001 ) . As such , they now constitute a new focus for studies on anti - cancer drugs and therapies targeted specifically to kinesin motor function , either directly towards the motor domains ( e . g . Monastrol : Maliga et al . , 2002 , 2006 ; Cochran et al . , 2004 , 2005 ; Crevel et al . , 2004 ; Yan et al . , 2004 ; Krzysiak et al . , 2006 ) , or to other functional parts such as cargo domains and receptors . This report focuses on the motor domains of two structurally very dif - ferent kinesins with important roles in mitosis : the plus - end direc - ted kinesin - 5 Eg5 ( Kashina et al . , 1996 ; X - ray structure : Turner et al . , 2001 ) as a monomeric motor domain , and the minus - end di - rected kinesin - 14 Kar3 ( Meluh and Rose , 1990 ) as a functional het - erodimer with Vik1 ( Manning et al . , 1999 ; Barrett et al . , 2000 ) . Eg5 is a highly conserved homotetrameric kinesin with an essential role in formation of the bipolar microtubule spindle and separation of the chromosomes during mitosis ( Blangy et al . , 1995 ) . Due to its bipolar structure , it is currently believed that Eg5 motors translocate to the plus - ends of microtubules at the on - set of mitosis and begin to organize microtubules by cross - linking leading to the formation of the stable bipolar spindle seen in meta - phase ( Kapitein et al . , 2005 ) . Once the spindle is in place , Eg5 pro - vides structural support and contributes to poleward flux by sliding the microtubules apart towards the centrosomes ( Valentine et al . , 2006 ) . Structural studies are of interest to elucidate the mechanical properties of this homotetrameric kinesin and its mode of action in spindle maintenance . Kar3Vik1 is an unusual heterodimeric kinesin found in Saccha - romyces cerevisiae . Both components of the heterodimer have been individually crystallized and solved to about 2 . 5 Å resolution ( Kar3 : Gulick et al . , 1998 ; Vik1 : Allingham et al . , 2007 ) . Kar3Vik1 is unusual because it has only one motor domain , Kar3 , whose localization and activity is regulated through its association with a non - motor protein , Vik1 ( Manning et al . , 1999 ) . Kar3Vik1 is ac - tive during vegetative growth where it localizes predominantly at the spindle poles and is thought to cross - link microtubules , con - tributing to mitotic spindle assembly and stabilization ( Manning and Snyder , 2000 ; Gardner et al . , 2008 ) . Interestingly , the recently solved crystal structure of the C - terminal globular domain of Vik1 revealed that it has a fold remarkably similar to a kinesin motor domain , but does not have an active site for ATP hydrolysis ( Alling - ham et al . , 2007 ) . Despite being unable to hydrolyze ATP , Vik1 binds tightly to microtubules in vitro supporting a model where both Kar3 and Vik1 interact with the microtubule during its walk - ing cycle . Structural studies on Kar3Vik1 thus aim to identify a no - vel kinesin – microtubule interaction and may also provide insight into the mechanisms of movement of other heterodimeric kinesins that have thus far been difficult to study ( Scholey et al . , 1996 ; De Marco et al . , 2001 ; Woehlke and Schliwa , 2007 ) . Tomography is not a new method for electron microscopy , but recent technical advances on hardware and software have since strongly boosted its popularity ( Frank , 2006 ) . In particular , tomog - raphy on vitrified specimens is now realistic due to sensitive detec - tors and automated low - dose recording software . Here , we focus exclusively on cryo - electron tomography since only frozen - hy - drated specimens have the structural preservation required for molecular analysis . By using cryo - electron tomography we could resolve molecular details in kinesin – microtubule complexes that would be impossible to image in 3 - D by helical averaging . How - ever , even by volume averaging approaches the interpretable molecular details of cryo - tomography compare to the lower - reso - lution end of helically averaged maps . Hence , both methods are very complementary . Helical averaging wins the resolution race but sometimes loses important details due to averaging and sym - metry enforcement . 2 . Materials and methods 2 . 1 . Expression and purification of Eg5 and Kar3Vik1 2 . 1 . 1 . Eg5 monomer The monomeric motor domain of Eg5 was expressed and puri - fied as described by Cochran et al . ( 2004 ) . 2 . 1 . 2 . Kar3Vik1 heterodimer Heterodimers of Kar3Vik1 were expressed and purified as de - scribed by Allingham et al . ( 2006 ) . 2 . 2 . Microtubule polymerization Microtubules were polymerized in vitro from 45 l M bovine tubulin ( Cytoskeleton , Inc . , Denver , CO ) with BRB80 ( 80 mM PIPES , pH 6 . 8 , 1 mM MgCl 2 , 1 mM EGTA ) in the presence of 1 mM GTP , 10 l M paclitaxel and 8 % ( v / v ) DMSO for 30 min at 37 (cid:3) C , and al - lowed to stabilize overnight at room temperature . 2 . 3 . Sample preparation for cryo - electron microscopy Eg5 – microtubule complexes were formed in solution in the pres - ence of 2 mM AMP – PNP ( Sigma – Aldrich Corp . , St Louis , MO ) , at a final tubulin concentration of 4 . 5 l M and a final Eg5 concentration of 18 l m ( see also Krzysiak et al . , 2006 ) . Complexes were adsorbed to holey - carbon C - flat grids ( Protochips , Inc . , Raleigh , NC ) for 45 s and subsequently plunge - frozen into liquid ethane using a home - made plunge - freezing device . Kar3Vik1 – microtubule complexes were formed directly on holey - carbon C - flat grids to prevent bundling of the microtubules . Micro - tubules at a concentration of 4 . 5 l M were adsorbed onto grids for 45 s and excess liquid was blotted away . Kar3Vik1 at concentra - tions ranging from 3 . 5 l M to 4 . 5 l M was incubated with the microtubules for 2 min in the presence of 2 mM AMP – PNP and plunge - frozen as described above . Colloidal gold ( 10 nm ) was added to all samples before plunging for use as fiducial markers . 2 . 4 . Electron microscopy data collection Samples were transferred under liquid nitrogen to a Gatan - 626 cryo - holder ( Gatan , Inc . , Pleasanton , CA ) . Cryo - electron microscopy data was collected on an FEI Tecnai F20 FEG transmission electron microscope ( FEI Company , Eindhoven , The Netherlands ) operating at 200 kV . Images and tilt series were collected at a nominal mag - nification of 29 000 (cid:2) and a defocus of (cid:3) 2 . 5 l m ( Kar3Vik1 – micro - tubules complexes ) or (cid:3) 6 l m ( Eg5 – microtubule complexes ) . Single frame images ( Fig . 2A ) were taken with an electron dose of 15 electrons / Å 2 . Tilt series consisting of about 80 low - dose images , were collected by tilting the specimen between (cid:3) 60 (cid:3) and + 60 (cid:3) ( Figs . 1A , 3A , and 4A ) , or (cid:3) 70 (cid:3) and + 70 (cid:3) ( Fig . 5A ) , imaging at 1 . 5 (cid:3) increments . Images at higher tilts were recorded with a higher dose to compensate for the increase in sample thickness . SerialEM software ( Mastronarde , 2005 ) was used to automate the data acquisition and minimize exposure of the specimen to the electron beam . The total dose for each tilt series was about 100 electrons / Å 2 . Images were recorded binned by two on a 4K (cid:2) 4K Gatan Ultrascan 895 CCD camera ( Gatan , Inc . ) . With this camera , at a nominal microscope magnification of 29 000 (cid:2) the resulting pixel size corresponds to 7 . 6 Å on the specimen . 258 J . Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 2 . 5 . Tomogram reconstruction and sub - tomogram averaging Tomograms were reconstructed from tilt series data by weighted back - projection using the IMOD software package ( Kre - mer et al . , 1996 ) . Colloidal gold beads ( 10 nm ) were tracked as fiducial markers to align the stack of tilted images . To select sub - tomograms for averaging motor - decorated micro - tubules ( Figs . 1 , 3 , 5 , and 6 ) , the center of the microtubule and the trajectory along one of its protofilaments were modeled by hand every 15 – 30 nm . A program was run to fill in model points every 8 nm along the microtubule axis ( representing the length of an a b - tubulin dimer repeat ) , and to determine the twist of the micro - tubule to produce a set of initial orientations for an alignment search in the first round of averaging . The PEET software package was used to align and average repeating sub - volumes . To average the Kar3Vik1 heterodimers in between two microtu - bules ( Fig . 4 ) , sub - volumes each containing one Kar3Vik1 ‘bridge’ were modeled by hand and input into PEET for alignment and averaging . Isosurface rendering of the sub - volume averages was carried out using IMOD ( Figs . 5 and 6 ) or UCSF Chimera ( Figs . 1 , 3 , and 4 ) ( Pettersen et al . , 2004 ) . A low pass filter was applied to all aver - ages to reduce noise before rendering . 3 . Results and discussion 3 . 1 . Cryo - electron tomography versus helical 3 - D reconstruction on kinesin – microtubule complexes The first structural analyses of microtubules date back to Amos and Klug ( 1974 ) who also produced the first 3 - D reconstruction of tubulin protofilaments and model of a microtubule ( Amos and Ba - ker , 1979 ) from zinc - induced 2 - D crystalline tubulin sheets . How - ever , 3 - D cryo - EM on microtubules picked up only after the developments of cryo - EM methods ( Dubochet et al . , 1988 ) and the discovery that microtubules composed of 15 - protofilaments may form helical polymers ( Fig . 1E – I ; Wade and Chrétien , 1993 ; Fig . 1 . Comparison between cryo - electron tomography and helical 3 - D reconstruction of frozen - hydrated microtubules decorated with monomeric kinesin ( Eg5 ) motor domains . ( A ) A 4 . 5 nm slice from a tomogram of microtubules decorated with monomeric Eg5 motors . Microtubules which are slightly tilted to the image plane expose a sequence of their top , lumen , and bottom regions as they cut through the slice . The B - lattice pattern emphasized by the motor decoration shows a different angle along the Bessel (cid:3) 2 helix according to the sketch . Since this is a left - handed helix , red lines show the angular orientation of the helical path according to the top view ( towards the observer ) , while the green lines indicate the course of the helix at the bottom of the microtubule . Occasionally a lattice seam , typical for non - helical microtubules with B - lattices , can be seendirectly . The inset magnifies the framed area and reveals a bottom view of the seam , here madevisible by the motor decoration . The seam isvisible due to the shift of one protofilament by 4 nm in the axial direction relative to the other ( interruptions in green lines ) . ( B ) Cross - section through a 9 nm projection of a volume average obtained by PEET combining 218 particles along one microtubule . This microtubule is composed of 14 - protofilaments , which can be clearly seen despite the obvious loss of resolution along the z - axis ( here top to bottom ) due to anisotropy caused by the missing wedge of data . ( C ) The surface rendering of the average clearly shows the helical path of the microtubule as well as the lattice seam typical for a 14 - protofilament microtubule . ( D – F ) Helical averaging of microtubules is only possible if they are truly helical . This is the case for a 15 - protofilament microtubule ( red frame in ( F ) ) but not for the more common 13 - protofilament microtubules ( yellow frame in ( F ) ) . Fourier filtering ( D and E ) of the microtubules marked by frames reveals strikingly different 2 - D projection patterns . 13 - Protofilament microtubules ( D ) are perfectly aligned with the tubular axis while 15 - ( E – I ) , or 16 - protofilament microtubules ( Figs . 5 and 6 ) reveals a super - twisted arrangement . The supertwist is visible in the 3 - D reconstruction in ( F ) as a deviation of the protofilaments ( dotted green line ) from the microtubule axis ( solid green line ) . Kinesin motor decoration reveals a Fourier filtered image as shown in ( G ) . The helical diffraction pattern ( H ) shows the helical layerline pattern where the layerlines around Bessel (cid:3) 2 ( axial motor – tubulin dimer repeat ) and (cid:3) 4 ( axial a – b – a – b - tubulin repeat ) form a cluster of 3 – 4 strong layerlines due to the convolution of the (cid:3) 2 and (cid:3) 4 helices with the protofilament supertwist . ( I ) The 3 - D reconstruction reveals the added kinesin heads as yellow densities , marking each a b - tubulin dimer . J . Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 259 Arnal et al . , 1996 ; Beuron and Hoenger , 2001 ) while most others carry a so - called lattice seam ( Fig . 1A and C ; Mandelkow et al . , 1986 ; Kikkawa et al . , 1994 ; Sandblad et al . , 2006 ) that interrupts helical symmetry . The second boost came from the X - ray crystal structures of monomeric and dimeric ncd ( Sablin et al . , 1996 , 1998 ) as well as monomeric ( Kull et al . , 1996 ) and dimeric kine - sin - 1 ( Kozielski et al . , 1997 ) . With these data at hand , molecular docking became popular for kinesin – microtubule structures ( Sosa Fig . 2 . Cryo - electron microscopy of microtubules decorated with Kar3Vik1 heterodimers . ( A ) Frozen - hydrated microtubules decorated with a dimeric Kar3Vik1 construct reveals obvious cooperative microtubule - binding properties . The cooperative binding creates a heterogeneous decoration pattern where some microtubules show full decoration on one side while the opposing side is free of motor decoration ( red frame ) . ( Insets in A ) Crystal structures of the Vik1 motor - homology domain ( left ) , and the Kar3 motor domain ( right ) are shown for comparison . Some structural similarities such as the central b - sheet surrounded by three a helices on each side are obvious . However , despite the similarity , Vik1 lacks a nucleotide - binding site . ( B ) Magnified and contrast - enhanced region of the frame in ( A ) . The picture contrast has been inverted and boosted by an ‘‘embossing” filter to emphasize the visibility of the tubulin – Kar3Vik1 dimer complexes . Since the motors have been flushed with AMP – PNP , we believe that the domain in contact with the microtubule surface is the Kar3 - MD while the tethered domain is Vik1 - MHD . However , this remains to be determined experimentally . Fig . 3 . Dissection of microtubules partially decorated with Kar3Vik1 by cryo - electron tomography . ( A ) A 9 nm slice through a tomogram of microtubules decorated with Kar3Vik1 heterodimers reveals features similar to those discussed in Fig . 1 . Kar3Vik1 decorates microtubules in a cooperative fashion leaving empty patches besides fully decorated areas . ( B ) Three individual 4 . 5 nm sections through the top , center , and bottom of the tomogram of the microtubule boxed in ( A ) . The inset in the center panel shows an end - on view of the microtubule . While the bottom region shows strong striations every 8 nm corresponding to a fully motor - decorated surface ( green lines ) the top region reveals empty microtubule protofilaments running axially . A striking advantage of tomographic 3 - D reconstruction is observed in the center panel in that small single events such as individual missing motors ( red circles ) are revealed with much better clarity than in 2 - D projections . These events would be lost after helical averaging due to the symmetry constraints . ( C ) An isosurface representation from a PEET average of 146 particles selected from the microtubule in ( B ) gives a clear view of how the top of the microtubule is free of motor decoration while the bottom and sides of the microtubule are completely decorated by KarVik1 . 260 J . Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 Fig . 4 . Cryo - electron tomography of Kar3Vik1 dimers connecting between adjacent microtubules . ( A ) Eighteen nanometers x – z ( top ) and x – y ( bottom ) slices through a tomogram showing the cross - section through two adjacent microtubules ( top ) and the Kar3Vik1 motors extending toward each other between the two microtubules ( bottom ) . ( B ) An18 nm projection and surface rendering of an average obtained by PEET from 44 sub - volumes selected from the tomogram in ( A ) . The bridging motors appear to bend towards each other in a manner different from the regular radially extending conformations shown in Figs . 3 and 5 . Since this is a highly artificial in vitro situation , the biological significance of these bridging motors is not clear . Rather , this exemplifies the way we are able to use cryo - electron tomography and subsequent volume averaging to analyze structural details that would not be possible by other averaging methods . Fig . 5 . Cryo - electron tomography and subsequent volume averaging of fully decorated Kar3Vik1 – microtubule complexes . ( A ) Three nanometers thick tomographic x – y slice of amicrotubule decoratedwithKar3Vik1 overlaidwiththeaveraged volumeshown in ( B ) – ( E ) andFig . 6A andB . ( B – D ) Four nanometers x – y slices throughthe topand center of an average of 99 particles selected from the tomogram in ( A ) according to the planes indicated in ( E ) . The center slice in ( D ) cuts along the missing wedge , noticeable as reduced resolution compared to the x – y slice above . The central x – y slice ( C ) through the microtubule clearly shows densities corresponding to the a - and b - subunits of tubulin and to the two globular domains of Kar3 and Vik1 extending out from the microtubule . ( E and F ) End - on view ( x – z ) of the 3 - D map before ( E ) and after rotational averaging over all protofilaments ( F ) . The rotationally averaged map now looks indistinguishable from a helical reconstruction map at the same resolution . While the missing wedge effect is clearly visible before rotational averaging as the strong densities and smearing out along the z - axis , rotational averaging eliminates the missing wedge completely . J . Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 261 et al . , 1997 ) and many other macromolecular assemblies after automated procedures became available ( Volkmann and Hanein , 1999 ; Wriggers et al . , 1999 ) . Today the best resolved 3 - D data on kinesin – microtubule complexes were obtained by helical averag - ing and show 8 – 9 Å detail ( Hirose et al . , 2006 ; Sindelar and Down - ing , 2007 ; Bodey et al . , 2009 ) . The major advantages of helical reconstruction of microtubule – MAP complexes can be summarized as follows : ( A ) Helical averaging produces low - noise data with isotropic resolution , sometimes to near - atomic detail . The averaging power is very high as a large number of unit cells ( 50 , 000 and more ) can be included rapidly , yielding an excellent sig - nal to noise ratio . Diffraction ( Fig . 1G ) and Fourier filtering ( Fig . 1D , E , and G ) allow rapid assessments of the data quality . ( B ) Raw - data are obtained from single , untilted projections which strongly facilitates corrections for the contrast trans - fer function ( CTF ) and allows the application of a high elec - tron dose ( (cid:4) 40 – 100 electrons / Å 2 ; Fig . 1F ) at each image since each individual specimen is only recorded once . The major constraints of helical averaging are : ( A ) By definition , helical 3 - D reconstruction requires a perfectly helical assembly ( Fig . 1E – I ) that is often either highly artifi - cial ( e . g . in the case of kinesin – microtubule complexes the motor to tubulin ratio is much higher than under in vivo conditions ( Fig . 1I ) ) , does not reproduce native conforma - tions , or is simply impossible to obtain ( structures too flex - ible and dynamic ) . ( B ) Single molecular events such as lattice seams ( Fig . 1A and C ) or missing individual particles ( Fig . 3B ) cannot be observed in 3 - D . If these events occur , they will be lost by being aver - aged over all other events during the 3 - D reconstruction process , potentially interfering with the accuracy of the resulting 3 - D maps . The emerging alternative method to study macromolecular assemblies without the constraints associated with helical recon - struction is cryo - electron tomography ( Figs . 1A , 3A , B , 4A , and 5A ) . As illustrated in Fig . 1A , kinesin – microtubule complexes can be reconstructed in 3 - D independent of the polymer composition and can be analyzed for structural detail ( such as the lattice seam or handedness of the helical path at distinct locations ) that would not be observable by a helical averaging approach . By focusing on thin slices through the tomogram , single motor domains , and their empty spots when missing can be observed ( Fig . 3B ) . Such single events would be difficult to detect on regular cryo - micrographs that superimpose all the densities along the projection . 3 - D data from cooperative decoration events ( Figs . 2A and 3B ) or bridging motor domains between two adjacent microtubules ( Fig . 4 ) cannot be obtained by helical averaging since they do not follow any heli - cal symmetry . However , although no averaging is done with the raw - data and / or in the tomographic 3 - D reconstruction procedure , averaging may still be applied as a post - tomographic procedure , further enhancing the signal to noise ratio for particles which can be iden - tified within a tomogram at various orientations , but which are otherwise structurally reproducible enough to be averaged as en - tire volumes ( Walz et al . , 1997 ; Taylor et al . , 2007 ; Förster and He - gerl , 2007 ; Bartesaghi et al . , 2008 ) . This is much more computationally demanding and only 10 – 15 years ago would not have been realistic without the Linux clusters or multi - core com - puters that we use today . We are developing a dedicated software suite for averaging volumes selected from tomograms called PEET ( Particle Estimation for Electron Tomography : Mastronarde et al . , in preparation , for example , see Nicastro et al . , 2006 ) . With this Fig . 6 . Rotational averaging around the tubular axis effectively eliminates the missing wedge . ( A and B ) Surface renderings of the averaged 3 - D map are shown in Fig . 5A – E . Thecross - section reveals the numberof protofilaments of thismicrotubule to be 16 , which produces aperfectly helical microtubule ( Bessel order (cid:3) 2 ) withoutseams ( see ( B ) ) . ( D and E ) Surface rendered 3 - Dmap afterrotational averaging of the mapin ( A ) ( see also Fig . 5F ) . Themissing wedge effectiseliminated . Byrotationally averaging over all16 - protofilaments the asymmetric unit changes from axialslices along the microtubule axis to one a b - tubulin – motor domain complex . Hence , the theoretical maximumnumber of asymmetric units included in the average is increased from 99 to 1584 ( 16 (cid:2) 99 ) . However , our average is based on the 1242 particles with the best correlation to the reference because this subset gave the highest resolution . ( C and F ) For Fourier - shell correlation calculations , the datasets were split in half on a random basis and the two halves were correlated against each other . Fourier - shell correlation graphs obtained from the maps in A ( C ) and D ( F ) reveal resolution limits of 3 . 8 nm and 3 . 2 nm , respectively , based on the 50 % correlation criterion . For FSC calculations a dataset is split in half on a random basis and correlated against each other . Accordingly the number of asymmetric units in each group is maximally 49 before rotational averaging , and 621 afterwards . 262 J . Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 software we produced the averages presented in Figs . 1B , C , 3C , 4B , and 5A – F . Each average requires between 10 and 20 CPU hours split up over 35 processors on our Linux cluster . Typically the num - ber of particles that go into an average is much smaller than for a helical approach . This reduces the averaging power , but 3 - D parti - cles contain more structural information than 2 - D projections , which facilitates accurate alignments . The major advantages of tomographic reconstruction of micro - tubule – MAP complexes can be summarized as follows : ( A ) Cryo - tomographic 3 - D reconstruction is applicable to any random structure and requires neither symmetry nor any structurally reproducible sub - particles ( unless post - tomo - graphic averaging should be applied ) . Cryo - tomography works well on plunge - frozen isolated macromolecular assemblies ( as presented here ) as well as on vitrified sec - tions ( Norlén et al . , 2009 ) . ( B ) Extraction of thin slices from tomograms reveals relatively noise - free data that omits the convolution of projected data with lower and upper structures ( see Figs . 3B and 5B , C ; the result is somewhat similar to a confocal recording in light microscopy ) . ( C ) Single molecular events may be observed and analyzed ( e . g . missing particles in an otherwise regular array ; Fig . 3B ) . ( D ) Post - tomographic data processing such as sub - volume aver - aging is facilitated by starting off with 3 - D data rather than 2 - D projections and if successful , yields high signal to noise 3 - D data ( see Figs . 1B , C , 3C , 4B , and 5B – F ) . The major constraints of tomographic 3 - D reconstruction are : ( A ) Cryo - tomograms are composed of tilt series ( typically 80 – 100 projections ) recorded on the very same object . The res - olution in tomograms depends on the accuracy of alignment and back - projection of each projection . Deviations from the assumed tilt angles , focus variations , and technical limita - tions such as bending support films reduce the resolution in the final tomograms . ( B ) The design of electron microscope stages and grid holder geometries limit the recording range of tilt series to approx - imately (cid:3) 70 (cid:3) and + 70 (cid:3) ( rather than a full 180 (cid:3) rotation ) , leaving a missing wedge of data that results in anisotropic resolution ( see Figs . 1B , 3C , 4B , 5E , and 6A and B ) that is usu - ally lower than what is achieved with a helical approach . ( C ) Cryo - tomograms have to be collected under strict low - dose conditions and therefore individual projections are extre - mely noisy , which may interfere with the alignment and back - projection process . ( D ) While not impossible , CTF correction on tilt series is demanding due to the various focus values in images from tilted specimens ( see Xiong et al . , 2009 ) . 3 . 2 . Cryo - EM on Kar3Vik1 – microtubule complexes reveals a highly cooperative binding pattern and a one - head - down and one - head - up dimer - binding configuration Kar3Vik1 is an unusual kinesin in the sense that it comprises a typical kinesin - 14 motor domain ( Kar3 - MD ) heterodimerized with a non - motor protein , the Vik1 motor - homology domain ( Vik1 - MHD ) by a coiled - coil interaction . Conventional cryo - electron microscopy reveals a Kar3Vik1 binding property that is dominated by a cooperative process ( Fig . 2A ) . This can be seen directly as un - even decoration along microtubules where one side of some micro - tubules is completely free of motor decoration while the other side appears fully decorated . Fig . 3 shows a cryo - tomographic 3 - D reconstruction of a partially decorated microtubule . Slices through the microtubule framed in Fig . 3A reveal the top portion to be void of motors , while the bottom portion shows full decoration . The center part reveals some missing individual motors along other - wise fully decorated protofilaments ( circles in Fig . 3B ) . Due to the partial decoration in the framed area in Fig . 2A and the optimal side - view of the motors , the overall shape of the di - meric Kar3Vik1 construct is clearly visualized . Fig . 2B shows a con - trast - enhanced magnification of the framed region in Fig . 2A . Here , the three domains of the tubulin dimer , the Kar3 - MD , and the Vik1 - MHD are well separated . The tomographic analysis shown in Fig . 5B confirms this finding . The motors have been flushed with AMP – PNP , a non - hydrolyzable ATP analog used to mimic an ATP binding state . Under these conditions we found isolated Kar3 - MD to be strongly bound to microtubules while Vik1 - MHD’s bound with much lower affinity . Accordingly , we assume that the domain in contact with the microtubule surface is the Kar3 - MD while the tethered domain is Vik1 - MHD . However , this remains to be deter - mined experimentally . Both features of Kar3Vik1 , the cooperative binding process ( Wendt et al . , 2002 ) , and the binding conformation with one - head - down , one - head - up is highly reminiscent of dimeric ncd con - structs ( Sosa et al . , 1997 ; Hirose et al . , 1998 ; Wendt et al . , 2002 ; Endres et al . , 2006 ) suggesting a common pattern for minus - end directed motors and the members of the kinesin - 14 family . Other dimeric kinesin MD’s prefer to bind with both heads simulta - neously to the microtubule surface and do not display the strong cooperativity seen here and with dimeric ncd ( kinesin - 1 : Hoenger et al . , 1998 , 2000 ; Eg5 : Krzysiak et al . , 2006 ) . 3 . 3 . The power of tomography in combination with volume averaging Although tomographic 3 - D reconstruction does not rely on any kind of averaging procedure , post - tomographic averaging consti - tutes a powerful tool to further enhance the signal to noise ratio in some parts of the tomogram . However , as in every averaging ap - proach , the structures subjected to averaging have to be identical else corrupted 3 - D maps prone to misinterpretations and false con - clusions will be obtained . The advantage of post - tomographic aver - aging lies in the fact that 3 - D structures are generally more easily assessed than 2 - D projections and often show more recognizable features . The difficulty with post - tomographic averaging , however , comes from the missing wedge of data that reduces the resolution particularly in the Z - direction and the resulting elongation of struc - tures in Z potentially interferes with the 3 - D alignment process . Our data on volume averaging presented here has been exclusively produced by our software suite PEET ( initial procedure described in Nicastro et al . , 2006 ; Mastronarde et al . , in preparation ) . The mathematical and computational details of the averaging process with PEET will be published elsewhere ( Mastronarde et al . , in preparation ) . In the case of kinesin – microtubule complexes the tomogram al - lows us to carefully inspect a microtubule for lattice defects and other irregularities . Once approved , we divide the microtubule into overlapping axial segments about 130 nm long , at intervals of 8 nm , making sure to include a full a b - tubulin repeat . While the lattice seams interrupt any helical symmetry , the sequence of cir - cular segments along the microtubule axis and each individual protofilament are still identical . Once the segments have been se - lected , they are aligned according to the microtubule supertwist ( if there is one ) and averaged . The results are shown for a microtu - bule complexed with monomeric Eg5 ( Fig . 1B and C ; a total of 218 particles were averaged ) , a microtubule partially decorated with dimeric KarVik1 ( Fig . 3C ; 146 particles ) , Kar3Vik1 dimers bridging between two adjacent , parallel microtubules ( Fig . 4B ; 44 particles ) J . Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 263 and a microtubule fully decorated with Kar3Vik1 dimers ( Figs . 5A – E and 6A ; 99 particles ) . Two factors are of utmost importance here : ( i ) the usable tilt - range and ( ii ) the number of different angular views integrated . Even with a relatively large group of particles as shown in Fig . 1B and C where 218 particles have been averaged , some missing wedge effects remain visible such as a smeared - out density wall of badly separated protofilaments along the z - axis , while the x - and y - axes show very clear separations . The same is visible in Figs . 3C , 5E , and 6A . This is an intrinsic effect of any single - tilt tomogram . For microtubule reconstructions that focus on 3 - D data surrounding the lattice seams this means no rotational averaging around the tubular axis can be done ( Fig . 1B and C ) and the only rotation that partially weakens the missing wedge effect comes from an axial supertwist ( see Fig . 1E and G ) . That supertwist is completely absent in 13 - protofilament microtubules ( see Fig . 1D ) . The microtubule in Figs . 5 and 6 , however , is a helical 16 - protofilament microtubule where rotational averaging due to the helical symmetry can be applied in addition to averaging axial segments ( Figs . 5F and 6D , E ) . We have done such averaging not by imposing rotational symmetry on the original average , but by sep - arately aligning each segment of the microtubule in 16 different orientations . The same rotational averaging could be done with any tomographic reconstruction of a microtubule that does not possess a seam , but only where features along protofilaments are identical such as fully decorated motor – microtubule complexes . As shown in Figs . 5 and 6 , rotational averaging immediately in - creases the number of asymmetric units by the number of protofil - aments ( i . e . 16 (cid:2) 99 = 1584 ) . The asymmetric unit is now reduced from an entire tubular slice to a single a b - tubulin – motor domain complex . Accordingly , the Fourier - shell correlation graphs reveal a significantly increased signal to noise ratio , and an interpretable resolution ( i . e . visible detail not obscured by noise ) improvement from 3 . 8 nm to 3 . 2 nm ( Fig . 6C and F ) . 3 . 4 . Molecular details in cryo - electron tomograms Here , we demonstrate that cryo - electron tomography and heli - cal 3 - D approaches ( or any other averaging - based methods ) are highly complementary . We can conclude that cryo - electron tomography is an excellent method for any kind of assembly that lacks apparent symmetry and that precludes helical , single particle , or 2 - D crystalline averaging , such as the examples shown here in Figs . 1 , 3 , and 4 . Even without post - tomographic averaging , struc - tural details can be interpreted to about 4 nm resolution . For example , individual protofilaments are spaced 5 nm laterally and 4 nm axially , features that are clearly visible in tomographic slices ( see Fig . 3B ) , at least when not obscured by the missing wedge ef - fect . Other structural features such as lattice angles and missing domains can be assessed reliably . However , molecular docking of small molecules such as kinesin motor domains remains difficult at the current resolution limits attainable by cryo - electron tomog - raphy . Volume averaging , where applicable currently pushes the interpretable resolution close to 3 nm detail and may reach beyond 2 nm in the future . Here , features such as the two domains of the a b - tubulin dimer and the binding geometry of motor domains to microtubules can be reliably visualized ( see Fig . 5C ; and after rota - tional averaging : Figs . 5F and 6D , E ) . However , the theoretical res - olution attainable by post - tomographic averaging ultimately depends on the initial resolution that can be preserved from the raw images by tomographic reconstruction , which is the limiting factor in this case . Therefore , even for highly symmetric assemblies such as the helical 16 - protofilament motor – microtubule complex shown in Figs . 5 and 6 , reaching better than 2 nm isotropic resolu - tion via a cryo - electron tomography approach may remain out of reach for a while longer . Acknowledgments J . C . was supported by the National Institutes of Health ( NIH ) / University of Colorado Molecular Biophysics Training Program ( NIH T32 GM - 065103 ) . D . M . was supported by Grant NIH / NCRR P41 - 000592 . We would also like to thank our colleague John Heu - mann for helpful discussions and assistance with the volume aver - aging software . References Allingham , J . S . , Sproul , L . R . , Rayment , I . , Gilbert , S . P . , 2007 . 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Cope et al . / Journal of Structural Biology 170 ( 2010 ) 257 – 265 265 \ No newline at end of file diff --git a/silver_data/753efd2f83955ab28e6654ff82ddc812a9261237.pdf.txt b/silver_data/753efd2f83955ab28e6654ff82ddc812a9261237.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..0f33f3230b9578a7d99aeb99a4c21b52e9f5720e --- /dev/null +++ b/silver_data/753efd2f83955ab28e6654ff82ddc812a9261237.pdf.txt @@ -0,0 +1 @@ +OBITUARY Peter Mansfield , physicist who developed MRI , remembered p . 180 BIOLOGY Behind the scenes in the world of synthetic biology p . 178 MILITARY A history of the US agency behind the Internet and drones p . 176 MEDICINE Don’t deregulate : the market is useless at weeding out futile drugs p . 174 Commercialize early quantum technologies Masoud Mohseni , Peter Read , Hartmut Neven and colleagues at Google’s Quantum AI Laboratory set out investment opportunities on the road to the ultimate quantum machines . F rom aspects of quantum entangle - ment to chemical reactions with large molecules , many features of the world cannot be described efficiently with con - ventional computers based on binary logic . The solution , as physicist Richard Feynman realized three decades ago 1 , is to use quan - tum processors that adopt a blend of classical states simultaneously , as matter does . Many technical hurdles must be overcome for such quantum machines to be practical , however . These include noise control and improving the fidelity of operations acting on the quan - tum states that encode the information . The quantum - computing community is channelling most of its efforts towards building the ultimate machine : a digital quantum computer that tolerates noise and errors , and that in principle can be applied to any problem . In theory , such a machine — which will need large processors comprising many quantum bits , or qubits — should be able to calculate faster than a conventional computer . Such capability is at least a decade away 2 . Correcting for errors requires redun - dancy , and the number of qubits needed quickly mounts . For example , factorizing a 2 , 000 - bit number in one day , a task believed to be intractable using classical computers 3 , would take 100 million qubits , even if indi - vidual quantum operations failed just once in every 10 , 000 operations . We have yet to assemble digital quantum processors with tens of qubits . This conservative view of quantum computing gives the impression that inves - tors will benefit only in the long term . We contend that short - term returns are possi - ble with the small devices that will emerge within the next five years , even though these will lack full error correction . A lack of theoretical guarantees need not preclude success . Heuristic ‘hybrid’ methods that blend quantum and classical approaches could be the foundation for powerful future applications . The recent success of neural net - works in machine learning is a good exam - ple . In the 1990s , when the computing power required to train deep neural networks was unavailable , it was fashionable in the field to focus on ‘convex’ methods ( based on func - tions with a clear minimum solution ) that had a strong theoretical basis . Today , these methods are no match for deep learning . The underlying algorithms of neural networks Google’s cryostats reach temperatures of 10 millikelvin to run its quantum processors . E R I K L U C E R O 9 M A R C H 2 0 1 7 | V O L 5 4 3 | N AT U R E | 1 7 1 COMMENT © 2017 Macmillan Publis h ers Limited , part of Springer Nature . All rights reserved . have hardly changed , yet impressive new performance milestones are being reached , thanks to ‘Moore’s law’ . Similarly , although there is no proof today that imperfect quantum machines can com - pute fast enough to solve practical problems , that may change . The scale , fidelity and con - trollability of analog and digital quantum hardware are improving steadily . We antici - pate that , within a few years , well - controlled quantum systems may be able to perform certain tasks much faster than conventional computers based on CMOS ( com - plementary metal oxide – semiconductor ) technology . Here we highlight three commercially viable uses for early quantum - computing devices : quantum simulation , quantum - assisted optimization and quantum sampling . Faster computing speeds in these areas would be commercially advantageous in sectors from artificial intelligence to finance and health care . Capitalizing on imminent advances in quantum technologies requires that the discipline broadens its focus and that scientists work more closely with entrepre - neurs . Hardware improvements are needed to make devices reliable and controllable enough to be commercialized . Heuristic quantum algorithms need to be developed that address practical problems within the current hardware limitations . As researchers working on quantum computing at Google , we plan to provide access to our quantum processors through cloud services to facili - tate the development and benchmarking of quantum algorithms and applications across industries , delivering real benefit to society . THREE PRIORITIES If certain feasible technological improve - ments are achieved , we believe that emerging quantum processors have a good chance of carrying out the following classes of compu - tational tasks and could become commer - cially valuable within a few years . Quantum simulation . Modelling chemical reactions and materials is one of the most anticipated applications of quantum comput - ing . Instead of spending years , and hundreds of millions of dollars , making and charac - terizing a handful of materials , researchers could study millions of candidates in silico . Whether the aim is stronger polymers for aeroplanes , more - effective catalytic con - verters for cars , more - efficient materials for solar cells , better pharmaceuticals or more - breathable fabrics , faster discovery pipelines will bring enormous value . Computational materials discovery is already a large industry . Quantum com - puters promise a radical transition : from the qualitative and descriptive to the quan - titative and predictive . Chemical - reaction rates are extremely sensitive to molecu - lar energies and span a range wider than classical methods can handle . If robust algo - rithms are developed , it might be possible to simulate important materials without the overhead of full quantum error correc - tion 4 . For example , algorithms are already known ( such as the ‘quantum variational eigensolver’ approach ) that seem to be immune to qubit control errors . A variety of business models could supply quantum simulators . Laboratories might pay a subscription for access . Computing companies could act as consultants . Some businesses might exchange equity in return for quantum - assisted breakthroughs that lead to innovative material developments . Quantum - assisted optimization . A central and difficult computational task in all quantitative disciplines of physical and social sciences , and across industries , is optimization . Such problems are diffi - cult to solve with conventional computers because algorithms can navigate only slowly through the mathematical landscape of possible solutions ; good solutions may be hidden behind high barriers that are hard to overcome . The most general classical algorithms use statistical methods ( such as thermal energy distributions ) to ‘jump’ over these barriers . We believe that this type of classical sampling could be enhanced by occasionally invoking quantum phenomena such as tunnelling ( whereby quantum infor - mation is transmitted through barriers ) to find rare but high - quality solutions . For example , online recommendations and bidding strategies for advertisements use optimization algorithms to respond in the most effective way to consumers’ needs and changing markets . More - powerful protocols , based on a combination of quan - tum and classical solvers , could improve the quality of products and services in many industries . Logistics companies need to optimize their scheduling , planning and product distribution daily . Quantum - enhanced algorithms could improve patient diagnostics for health care . The quality of search or product recommendations for large information - technology companies such as ours , Microsoft , Amazon and Facebook could be enhanced . Quantum sampling . Sampling from probability distributions is widely used in statistics and machine learning . In theory , ideal quantum circuits can sample from a larger set of probability distributions than classical circuits can in the same time . Our calculations show that , for relatively small circuits involving high - fidelity quantum gates , it will be possible to sample from probability distributions that are inaccessible classically , using a circuit of just 7 × 7 qubits in layers that are around 25 deep ( ref . 5 ) . In fact , sampling from distributions with such a shallow quantum circuit is “Quantum - enhanced algorithms could improve patient diagnostics for health care” The smaller of these chips , a 6 - mm square , holds 6 qubits . E R I K L U C E R O 1 7 2 | N AT U R E | V O L 5 4 3 | 9 M A R C H 2 0 1 7 COMMENT © 2017 Macmillan Publishers Limited , part of Springer Nature . All rights reserved . © 2017 Macmillan Publishers Limited , part of Springer Nature . All rights reserved . likely to constitute the first example of ‘quantum supremacy’ . This term was coined by theoretical physicist John Preskill 6 to describe the ability of a quantum processor to perform , in a short time , a well - defined mathematical task that even the largest classical supercomputers ( such as China’s Sunway TaihuLight ) would be unable to complete within any reasonable time frame . We predict that , in a few years , an experiment achieving quantum supremacy will be performed . Among promising applications of quantum sampling are inference and pattern recognition in machine learn - ing . To facilitate experimentation across academia and industry , we plan to offer access to the quantum hardware through a cloud - computing interface . TECHNICAL HURDLES Several technological challenges must be overcome for quantum computing to be commercialized . Quantum hardware needs to be scaled up to compete with clas - sical hardware , which has been improving exponentially for decades . Qubits require quantum coherence , which leads to quan - tum entanglement , by analogy with how classical circuits require transistors with gain . Combining scaling and coherence is the big challenge of quantum systems engi - neering . It is fundamentally difficult because quantum information cannot be copied and subsystems are entangled , leading to design trade - offs that are global in nature . We think that superconducting qubits are one of the most promising hardware platforms for quantum computers . Based on standard integrated - circuit and super - conducting technologies , they are relatively easy to construct and control . And there are many possible designs that might suit different requirements for digital and analog quantum processors . High - fidelity systems of around ten qubits have been demonstrated , showing the feasibility of the engineering concepts . New technologies are emerging that could aid scalability , such as superconduct - ing bump bonds — a two - layer architecture for information - processing units and con - trol circuits . Prototype ‘quantum annealers’ of about 1 , 000 qubits are already available commercially 7 , 8 . ( These are analog quantum processors that could find good - quality solu - tions for certain optimization tasks . ) Several improvements are required for today’s imperfect quantum devices to be practical . Shallow quantum circuits need higher gate fidelities and more stability to limit decoherence . Quantum - annealing hardware needs to be improved with respect to connectivity , control precision and coher - ence times , as well as having access to alterna - tive annealing schedules 9 . BUSINESS OPPORTUNITIES A new technology can improve business in three ways : by increasing revenue , reducing costs or lowering investments in infrastructure . In the digital era , introduc - ing a new technology has an exponential impact : even a 1 % gain in product quality can help a company to achieve overwhelming growth in terms of user numbers or revenue 10 . This is the ‘superstar effect’ , which assumes close competition , transparency and an efficient market . If early quantum - computing devices can offer even a modest increase in computing speed or power , early adopters will reap the rewards . Rival companies would face high entry barriers to match the same quality of services and products , because few experts can write quantum algorithms , and busi - nesses need time to tailor new algorithms . The markets that are most open to such dis - ruptions are information - rich and digital , and involve business challenges that rely on many variables . Such markets include financial ser - vices , health care , logistics and data analytics . Making a business case requires companies to examine demand and supply . Demand can be assessed as follows . First , identify the ‘min - imal viable products’ — early quantum inno - vations with just enough core features to enter the market . Estimate whether the innovation solves an existing need ( product – market fit ) , the time it would take to commercialize the product ( speed to market ) and the market’s response ( business traction ) . For example , encryption breaking — often portrayed in the media as a ‘killer application’ for digital quantum computers — does not score highly in terms of market fit . It will one day be superseded by cryptosystems that are immune to quantum attack . And most pri - vate enterprises are uninterested in breaking encryption systems . By contrast , portfolio optimization and risk management need immediate data feedback and could benefit from quantum - enhanced models 11 . More efficient quantum - chemistry calculations would revolutionize the development of pharmaceuticals , catalytic converters , solar cells and fertilizers . Quantum - assisted optimization and inference techniques could empower new machine - learning and artificial - intelligence systems . These could improve the manage - ment of renewable power generators , and of remote - sensing and early - warning systems . The techniques would also aid dynamic pric - ing for online goods and services , as well as warehouse automation and self - driving cars . On the supply side , companies will distinguish themselves through the quality of their technology and teams . Pioneering quantum academics and entrepreneurs will have to work together . This will be challeng - ing because academic incentives are often inconsistent with those of start - up cultures and industry . Strategic partnerships can help businesses to stand out . To attract venture capitalists , the winning quantum products should have business models that require few assets , are low on manufacturing costs and clearly help customers to increase their revenues . Through the cloud , a company could benefit from using G R E G K E N D A LL - B A LL / N A T U R E Radio - frequency and microwave electronics are used at Google to make scalable control hardware . 9 M A R C H 2 0 1 7 | V O L 5 4 3 | N AT U R E | 1 7 3 COMMENT © 2017 Macmillan Publishers Limited , part of Springer N ature . All rights reserved . © 2017 Macmillan Publis h ers Limited , part of Springer Nature . All rights reserved . existing data centres when applying classi - cal solvers to simple problems , and invoke quantum processors when it matters . WHAT NOW ? The field of quantum computing will soon achieve a historic milestone — quantum supremacy . It is still unknown whether application - related algorithms will be able to deliver big increases in speed using the sorts of processors that will soon be available . But when quantum hardware becomes sufficiently power - ful , it will become possible to test this and develop new types of algorithms . Over the next decade , academia , industry and national labs should work together to develop quantum simulation and quantum machine - learning algo - rithms . We plan to support such research by offering access to Google’s quantum processors through cloud services to others that lack the necessary capital , expertise or infrastructure . ■ Masoud Mohseni is senior research scientist at Google Quantum Artificial Intelligence Laboratory in Venice , California ; Peter Read is managing director at Vitruvian Partners in London ; Hartmut Neven is director of engineering at Google Quantum Artificial Intelligence Laboratory in Venice , California ; co - authors Sergio Boixo , Vasil Denchev , Ryan Babbush , Austin Fowler , Vadim Smelyanskiy and John Martinis are all part of Google’s Quantum AI team . e - mails : mohseni @ google . com ; petermread @ gmail . com ; neven @ google . com 1 . Feynman , R . Int . J . Theor . Phys . 21 , 467 – 488 ( 1982 ) . 2 . Touzalin , A . et al . Quantum Manifesto : A New Area of Technology ( Quantum Information Processing and Communication in Europe , 2016 ) ; available at http : / / go . nature . com / 2im6rjr 3 . Fowler , A . G . , Mariantoni , M . , Martinis , J . M . & Cleland , A . N . Phys . Rev . A 86 , 032324 ( 2012 ) . 4 . Wecker , D . , Hastings , M . B . & Troyer , M . Phys . Rev . A 92 , 042303 ( 2015 ) . 5 . Boixo , S . et al . Preprint at https : / / arxiv . org / abs / 1608 . 00263 ( 2016 ) . 6 . Preskill , J . in The Theory of the Quantum World ( eds Gross , D . , Henneaux , M . & Sevrin , A . ) 63 – 80 ( World Scientific , 2011 ) . 7 . Denchev , V . S . et al . Phys . Rev . X 6 , 031015 ( 2016 ) . 8 . Boixo , S . et al . Nature Commun . 7 , 10327 ( 2016 ) . 9 . Rams , M . M . , Mohseni , M . & Del Campo , A . New J . Phys . 18 , 123034 ( 2016 ) . 10 . Brynjolfsson , E . & McAfee , A . The Second Machine Age : Work , Progress , and Prosperity in a Time of Brilliant Technologies ( Norton , 2014 ) . 11 . Rosenberg , G . et al . IEEE J . Sel . Top . Signal Process . 10 , 1053 – 1060 ( 2016 ) . The authors declare competing financial interests : see go . nature . com / 2rygdtf for details . 1 7 4 | N AT U R E | V O L 5 4 3 | 9 M A R C H 2 0 1 7 COMMENT Show drugs work before selling them Regulation makes economic sense , argue Douglas Sipp , Christopher McCabe and John E . J . Rasko . U nder US President Donald Trump , defunct economic arguments about prescription drugs are coming to the fore . His advisers contend that today’s system is a bad deal . They want to undo regulations that require companies to show that a medical product actually works before it is sold . The advisers argue that removing the burden of large , lengthy clinical trials will cut costs and reduce delays , and that the marketplace can be trusted to sort good drugs from bad ones . Although many have raised concerns about a Trump Food and Drug Administra - tion ( FDA ; see , for example , Nature http : / / doi . org / bz92 ; 2017 ) , few have debunked the economic arguments . Here we outline what the case for deregulation gets wrong . All nations should take note — weaker stand - ards for entry of drugs onto the US market will harm health everywhere . I LL U S T R A T I O N B Y D A V I D P A R K I N S © 2017 Macmillan Publishers Limited , part of Springer Nature . All rights reserved . © 2017 Macmillan Publishers Limited , part of Springer Nature . All rights reserved . Knowledge of the history is important . The 1938 US Food , Drug , and Cosmetic Act required only that drug safety be demon - strated . In 1962 , new legislation demanded that marketed drugs also go through well - controlled studies to test for therapeutic ben - efit . More than 1 , 000 medical products were subsequently withdrawn after reviews found little or no evidence of efficacy 1 . The free mar - ket that existed before 1962 revealed no con - nection between a drug’s ability to turn a profit and its clinical usefulness . The same is likely to be true of any future deregulated market . MARKET FARCES Economic arguments against the FDA’s requirements for efficacy date back to at least the early 1970s . Originally these were advanced by libertarians and neoliberal economists at think tanks such as the Ameri - can Enterprise Institute in Washington DC . Since the early 2000s , the Manhattan Insti - tute for Policy Research in New York City has added its voice . Some economists posit that regulatory agencies are systematically biased towards excessive caution , and that the burden of testing a drug’s efficacy before it comes to market outweighs the benefits . They argue that ‘bad’ drugs can be iden - tified quickly after they go on sale , whereas harms caused by the unrealized utility of ‘good’ drugs are often invisible ( see go . nature . com / 2hymtel ) . Such reasoning has led prom - inent economists , including Nobel prize - winners Milton Friedman , Gary Becker and Vernon Smith , to recommend that efficacy requirements be weakened or abandoned . An overly stringent system will err by with - holding or delaying safe and effective ‘good’ drugs from patients . Critics of existing regu - lations often point to the case of a treatment for Hunter syndrome — a rare , inherited degenerative disease in which the absence of a crucial enzyme can be fatal . Trials of the enzyme - replacement drug Elaprase ( idursul - fase ) meant that , for a year , a group of chil - dren received a placebo instead of the drug that was eventually shown to be effective 2 . Conversely , a lax regulatory system will subject patients to ‘bad’ drugs that may be toxic . The iconic example is the more than 10 , 000 birth defects caused worldwide by the drug thalidomide , a late 1950s remedy for nausea during pregnancy . Even in the past dozen years , initially promising drugs , such as torcetrapib ( for reducing cholesterol and heart - disease risk ) and semagacestat ( for improving cognition in people with Alzheimer’s disease ) , were found to cause harm only after they had been tested in large , mandatory trials — effects that were not seen in the smaller trials 3 . The most extreme proponents of deregu - lation argue that the market can serve as the sole arbiter of utility : if a medicine is selling well , it must be delivering value 4 . A more moderate view is that reliable information on efficacy can be collected after a drug goes on sale , through uncontrolled observational studies and other post hoc analyses . There is a third type of error that these arguments neglect ( see ‘The good , the bad and the useless’ ) . Untested drugs can be rea - sonably safe but provide no benefit . Unregulated markets are hopeless at sift - ing out these ‘futile’ drugs ( witness the multi - billion - dollar industries in homeo pathy and other pseudo - medicines ) , unlike the current system . In January 2017 , the FDA released a report identifying 22 products that were initially promising but disappointed in later - stage clinical trials : 14 for lack of efficacy , 1 for lack of safety , and 7 for both reasons 3 . Futile drugs , even the non - toxic ones , cause real harms . They waste money for both patients and taxpayers . Marketing useless drugs wastes industry resources that could be used in developing effective thera - pies , squanders opportunities for patients to receive beneficial medical care , engen - ders false hope in miracle cures , and leads to cynicism about the value of research . Some countries , including South Korea and Japan , have allowed cell biologics such as stem and immune cells onto the market without requiring them to show compelling evidence of efficacy . This might boost the domestic drug industry , but lowers the value of local health care . These products have not been authorized for sale in any other countries . Europe should beware too . Lower drug - quality requirements in the large US market could make firms that adhere to the higher standards in the European Union less competitive . NO FREE LUNCH Arguments for deregulation fail to recog - nize that valuable information has a cost . Drug companies cannot afford to generate reliable evidence for efficacy unless their competitors are all held to the same high standards . Efficacy requirements level the playing field and ensure that the health sec - tor receives the data needed to inform good therapeutic and economic decisions . The government , insurers , patients and others need to know whether medicines are likely to provide benefits . Patients and physicians must have access to reliable information to make educated and ethical choices . Rigorous clinical studies are still the best way to learn whether a drug works , and regulation is essential to ensure that these studies are conducted . Pre - specified end - points , controls , randomization and blind - ing cannot be discarded without sacrificing actionable clinical information 5 . Once a drug is on the market , it is hard to gather solid efficacy data . Blinding and ran - domization in clinical studies can be com - promised when money changes hands and , historically , compliance with monitoring requirements has been poor . One analysis found that only 13 % of post - market studies required by the FDA had been completed between 1990 and 1999 ( see go . nature . com / 2mayocv ) . And a survey of 20 drugs approved by the FDA in 2008 found that fewer than one - third of post - market study commitments had been fulfilled by 2013 ( ref . 6 ) . Marketed drugs are also unlikely to be withdrawn because of a lack of efficacy 7 . The FDA’s gatekeeper role makes the med - ical marketplace function . The economic benefits of good research and a healthier population will be lost without incentives to find truly effective drugs . ■ Douglas Sipp is a researcher at the RIKEN Center for Developmental Biology in Kobe , Japan , and visiting professor at Keio University School of Medicine and Global Research Institute , Tokyo . Christopher McCabe is a health economist at the University of Alberta , Edmonton , Canada . John E . J . Rasko is head of the Department of Cell and Molecular Therapies at Royal Prince Alfred Hospital in Sydney , Australia . e - mail : sipp @ cdb . riken . jp 1 . Junod , S . W . In A Quick Guide to Clinical Trials ( eds Davies , M . & Kerimani , F . ) 25 – 55 ( Bioplan , 2008 ) ; available at http : / / go . nature . com / 2kjhny4 2 . Da Silva , E . M . K . , Strufaldi , M . W . L . , Andriolo , R . B . & Silva , L . A . Cochrane Database Syst . Rev . 2 , CD008185 ( 2016 ) . 3 . US Food and Drug Administration . 22 Case Studies Where Phase 2 and Phase 3 Trials had Divergent Results ( FDA , 2017 ) ; available at http : / / go . nature . com / 2mayug4 4 . Henderson , D . R . ‘Markets Can Determine Drug Efficacy’ Forbes ( 8 July 2009 ) ; available at http : / / go . nature . com / 2kbkzps 5 . Bothwell , L . E . , Greene , J . A . , Podolsky , S . H . & Jones , D . S . N . Engl . J . Med . 374 , 2175 – 2181 ( 2016 ) . 6 . Moore , T . J . & Furberg , C . D . JAMA Intern . Med . 174 , 90 – 95 ( 2014 ) . 7 . Siramshetty , V . B . et al . Nucleic Acids Res . 44 , D1080 – D1086 ( 2016 ) . J . E . J . R . declares competing financial interests : see go . nature . com / 2jct6ei for details . 9 M A R C H 2 0 1 7 | V O L 5 4 3 | N AT U R E | 1 7 5 COMMENT THE GOOD , THE BAD AND THE USELESS Allowed on market ? Drug is harmful ( ‘bad’ drug ) Drug is safe and beneficial ( ‘good’ drug ) Drug may be safe , but is useless ( ‘futile’ drug ) Yes Patients at risk ( toxicity ) Appropriate decision False hope , wasted money No Appropriate decision Patients lose out Appropriate decision “Unregulated markets are hopeless at sifting out futile drugs . ” © 2017 Macmillan Publishers Limited , part of Springer N ature . All rights reserved . © 2017 Macmillan Publis h ers Limited , part of Springer Nature . All rights reserved . \ No newline at end of file diff --git a/silver_data/789b02e60a8f4261666f4e3ec6ae7e0cf4109788.pdf.txt b/silver_data/789b02e60a8f4261666f4e3ec6ae7e0cf4109788.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..09efc71a57c9a25e9a5651b4114b6930c91b3d17 --- /dev/null +++ b/silver_data/789b02e60a8f4261666f4e3ec6ae7e0cf4109788.pdf.txt @@ -0,0 +1 @@ +Workplace Warriors : Identifying Team Practices of Appropriation in Software Ecosystems Sebastian Draxler University of Siegen Hoelderlinstr . 3 57076 Siegen + 49 ( 0 ) 271 - 740 - 4763 sebastian . draxler @ uni - siegen . de Adrian Jung University of Siegen Hoelderlinstr . 3 57076 Siegen + 49 ( 0 ) 271 - 740 - 4036 adrian . jung @ uni - siegen . de Alexander Boden University of Siegen Hoelderlinstr . 3 57076 Siegen + 49 ( 0 ) 271 - 740 - 4763 alexander . boden @ uni - siegen . de Gunnar Stevens University of Siegen Hoelderlinstr . 3 57076 Siegen + 49 ( 0 ) 271 - 740 - 4036 gunnar . stevens @ uni - siegen . de ABSTRACT Since the 1990s , the forms of production , distribution , configuration and appropriation of software have changed fundamentally . Nowadays , software is often embedded in software ecosystems , i . e . in complex interrelations between different stakeholders who are connected by a shared technological platform . In our paper , we investigate how small software teams deal with the challenges of appropriating and configuring software in the Eclipse ecosystem for their daily work . We empirically identify three different approaches for dealing with appropriation in software ecosystems which are represented by the “ideal types” lone warrior , centralized organization , and collegial collaboration . Based on a discussion of these strategies and the underlying appropriation practices we found in the field , we suggest theoretical and practical implications for supporting appropriation in software ecosystems . Categories and Subject Descriptors J . 4 . 4 [ Computer Applications ] : Social and behavioral sciences – sociology . H5 . 3 [ Information Interfaces and Presentation ] Group and Organization Interfaces – Computer Supported Cooperative Work . General Terms Management , Human Factors . Keywords Tailorability , Appropriation , Software Engineering , Software Ecosystems . 1 . INTRODUCTION For a long time , research on software usage has been interested in aspects of customizing and tailoring single applications to the needs of end users [ 5 , 8 , 16 ] . This was well suited in times when the software market was limited and applications had a clear border . However , several trends have changed the ways of software production , distribution and configuration considerably over the last two decades : 1 . ) the establishment of the Internet as the dominating infrastructure for disseminating and marketing digital goods ; 2 . ) the spread of new business and development models in the Open Source domain , encouraging users to share software with others ; 3 . ) the establishment of software ecosystems which consist of different manufacturers and hobbyists , creating small - scale components that can be individually assembled by end users . These developments do not only affect the ways how software is developed and maintained , but also how it is appropriated by the users ( who are increasingly understood as prosumers instead of mere consumers of software ) . As the social and collaborative aspects of appropriation in software ecosystems are not well understood , we have conducted a study which investigated how small teams of software developers deal with the necessities of appropriating the software that constitutes their work tools—in this case the Eclipse IDE and its surrounding software ecosystem [ 3 , 12 ] . The paper is organized as follows : after a discussion of the related work and our methodology , we provide an overview on three different approaches of dealing with appropriation as well as the underlying practices and implications that constitute these strategies . Based on our findings , we then identify implications for the theoretic understanding of appropriation as well as for designing supportive tools for these practices . 2 . A BRIEF HISTORY OF APPROPRIATION In the 80s and 90s , the research on the social and socio - technical aspects of software use and development coined the terms customization , adaptability and tailorability . Driver of these discussions was the fact that the existing , monolithic off - the - shelf products did not accord well with the complex , heterogeneous needs of a dynamic market . While tailoring was often considered to be an individual effort , later research highlighted the collaborative aspects of these practices . A major contribution to this shift was the work of Mackay et al [ 7 ] , who conducted empirical studies within organizations to examine patterns of sharing self - created email filter rules as well as configurations of Unix desktop systems . In a similar vein , Gantt and Nardi [ 5 ] investigated into the tailoring practices of CAD users . Under their lens , collaborative efforts became visible . While tailoring has been mainly discussed from a technical perspective , recent research has broadened the understanding of the corresponding practices by analyzing the ways how users fit the technology at hand into both the pre - existing culture and into the local patterns of use and life rhythms [ 13 ] . Conceptually labeled as appropriation , this line of research highlights the link between the creative re - configuration and the re - interpretation of given features as two dialectically connected forms of end user development [ 2 , 11 ] , implying that technical concepts as tailorability ( in terms of re - configuration of software ) should be Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . CHASE ' 11 , May 21 , 2011 , Waikiki , Honolulu , HI , USA . Copyright 2011 ACM 978 - 1 - 4503 - 0576 - 1 / 11 / 05 . . . $ 10 . 00 . Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . To copy otherwise , to republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . CHASE ’11 , May 21 , 2011 , Waikiki , Honolulu , HI , USA Copyright 2011 ACM 978 - 1 - 4503 - 0576 - 1 / 11 / 05 . . . $ 10 . 00 57 supplemented by social - technical means for supporting appropriation [ 2 , 10 , 11 ] . Generally speaking , while tailoring focuses on the technical customizations of software artifacts , appropriation also considers the social - technical process of interpreting software in daily work practice . While research in this domain has predominantly focused on the appropriation of single applications so far , we want to understand how software users design their workplaces by assembling heterogeneous resources from global software ecosystems . 3 . RESEARCH METHODOLOGY From 2006 to 2009 , we participated in a publicly funded research project to develop tailorable applications for different domains ( like monitoring complex technical plants , eLearning based on business simulation and using basic groupware technology ) . The aim was to study the potential of the Eclipse platform [ 4 ] as a core technology for realizing the concept of component based tailorability [ 9 ] . Eclipse was chosen as a platform as it was considered being a leading edge technology in respect to a consequent component architecture ( “everything is a plugin” [ 4 : 83 ] ) and a vital ecosystem ( with thousands of free or commercial extensions ) . We expected these features to empower users to personalize their applications by managing individual plugin portfolios , selected form the Eclipse software ecosystem . Our research was organized following the strategy of theoretical sampling as suggested by Strauss’ Grounded Theory [ 6 ] . We started with an open - ended , qualitative study on Eclipse plug - in adoption practices , using semi - structured interviews and on - site observations . Specifically , we cooperated with five German companies with 10 to over 2000 employees that perform software development ( see table 1 for details ) . In each company , we conducted at least three semi - structured interviews of at least one hour ( altogether , we conducted 18 interviews between August 2007 and December 2008 ) . Additionally , we visited two SMEs over a period of 3 - 5 days for on - site observation . Employees Domain Empiric data A 250 Development of Multimedia and Web applications 5 day observation 5 interviews B 10 Software Development 3 interviews C 6 + free - lancers Software Development 3 interviews 3 day observation D 1200 ( 90 developers ) Insurance company + inhouse software dev . 3 interviews E > 2000 ( 30 maintenance dept . ) Research facility , Inhouse development for . Machine maint . 4 interviews Table 1 : Research Partners All interviews were audio recorded and transcribed . The participant observations were documented by means of field notes taken during the research . In addition , we also analyzed the socio - technical structure of the Eclipse ecosystem , which determines the possibilities of users for designing their personal workspace . Related work done by other researchers was investigated for the purpose of sensitizing our analytic lens . However , we tried to prevent to subsume our observations under pre - defined concepts taken from literature . Instead we focused on categorizing our data by carefully analyzing the documented practices in minute detail . In a further step , we supplemented our qualitative analysis by an online survey where we used to assess the spreading of the observed practices [ 12 ] . 4 . A CLASSIFICATION SCHEME OF TEAM PRACTICES Our research provided us with rich insights into the manifold practices of how practitioners of small software teams appropriate software in the context of the Eclipse ecosystem . In general , we observed that practitioners were in a constant struggle of innovating their working environment and keeping it up - to - date in order to perform their work . At the same time they had to prevent possible breakdowns and incompatibilities resulting from updated and new plug - ins . As common Eclipse configurations consist of a plethora of plugins ( usually maintained by 3 rd party developers ) , there was a constant need for dealing with updates of particular plug - ins , also in cases when no new plugins where to be installed . Even when developers decided to stick to one version of Eclipse as a stable working environment , APIs to shared tools like the CVS could change and enforce the update of the related plugins . Furthermore , new tasks did sometimes require new tools and therefore new plug - ins were explored and introduced . The companies we investigated expressed quite different strategies of dealing with the challenges outlined above . These approaches move along a continuum , whose ends are autonomy ( leaving the responsibility to configure and maintain Eclipse to the individual developers ) and heteronomy ( managing Eclipse configurations in a centralized manner , thus enforcing a homogeneous working environment for the whole team ) . Following Max Weber [ 15 ] , we conveyed and conceptualized these approaches into three different “ideal types” , which represent the underlying strategies of the companies for managing appropriation in practice : the lone warrior type , the centralized organization type , and the collegial collaboration type . In the next sections , we will discuss these types and illustrate their underlying practices as well as their socio - technical implications . 4 . 1 Lone Warrior The first type is conceptually constituted by a high autonomy of the team members . It refers to the freedom , but also to the individual responsibility workers have for taking care of their workplaces . There are no guidelines for setting up the individual Eclipse configurations , although the limitations of the platform may force team members to use very similar configurations for certain collaborative functions , e . g . if only one plugin is available for connecting to the version control system . If changes to the workplace configuration need to be introduced on the team level , practitioners have to justify this constriction of the individual autonomy by proving the benefits of the suggested change to each of his colleagues . Empirically , the lone warrior practice is expressed by the circumstances that collaborative appropriation is not the normal case , but occurs at most accidentally ( e . g . in response to breakdowns of the infrastructure ) . In our study , an example for such an accidental collaboration was caused by the breakdown of a shared source code repository that was caused by an update . In this exceptional case , the incompatibility forced the team to search for a solution that was then adopted by the whole workgroup ( in the specific case , a developer worked out that the problem could be fixed by using a different Eclipse plug - in . This information was spread and the plug - in was then used by everyone ) . Except of such rare situations of collective appropriation of new tools , every 58 developer decided on his own what plug - ins to install and how they should be configured . As the lone warrior culture forced team members to search for useful tools themselves and experiment with them , practitioners were often very knowledgeable about their individual workplace . On the other hand , we recognized a lack of group - consciousness regarding tools and existing tool expertise . For example , in one company no one in the group was able to tell us what tools his colleagues were using . From a management perspective , this lack of awareness can have negative effects , like the tendency to develop heterogeneous and possibly conflicting work practices , decreasing the diffusion of innovation among the team members , and finally making it more difficult to find the right person to ask for advice in case something went wrong or in case team members evaluated new tools [ 1 ] . 4 . 2 Centralized Organization This type is constituted by heteronomy that stems from a centralized organization of the Eclipse installation . In this case , the workplace is designed by a designated team member and then distributed to the team . Changes to the common installation have to be discussed with the developer in charge , who is responsible for updating the installation and dealing with the possible incompatibilities . Empirically , we observed one case of centralized organization as the opposite extreme to the lone warrior type . In this case , the team elected a tool care taker . Once a month , his duty was to configure a stable working environment that met the needs of the group and afterwards put it on a shared file system where it was accessible for all team members . He described this work as follows : “Developer 2 : [ … ] I sit down at home on my Vista partition and an up - to - date Eclipse [ configuration ] . [ … ] I have a list of plug - ins which should be included . [ . . . ] I update this at home , put it on an USB stick and bring it here . Here , I upload it to a shared drive so that everybody can copy it . ” ( Transcript from an interview with the tool care taker ) . The decisions about what should be included in the common configuration were made by the whole team during stand - up meetings . Although the decision process was collaborative , the workplace caretaker realized the common working environment on his own . He was expected to be interested and quite skilled in finding new tools as well as in being up - to - date . Team members even reported to respect him as a “primus inter pares” in this regard . Everyone else within the team relied on his skills to regularly set up an up - to - date , stable environment for the whole team . The approach of centralized organization did not just create the opportunity to benefit from the maintenance of a common and thus homogeneous workplace configuration , but also created a latent obligation for other developers not to customize their workplaces . As experimenting with new tools for personal purposes was not legitimized by the team , the amount of influence team members could bring into discussions about the configuration management was clearly limited . The reason for this was , that it is difficult to say what the concrete benefits are before one has used a new tool in practice . Hence , the central organization type had the tendency to impede individual initiatives of developers to explore new stuff during daily work . It detached innovation activities from the daily production work . This means that the centralized organization type had to solve the latent demand to innovate in different ways than the lone warrior case , which allowed easy exploration through the integration with the daily work , which was now mainly in the responsibility of one central team member . 4 . 3 Collegial Collaboration As we have outlined in the two previous sections , our observations showed that in practice there were often exceptions from the rules impeded by the different ideal approaches that the companies chose to deal with their workplace configuration . This led to the identification of a third type , which is constituted by the dialectics between autonomy and heteronomy . In this type , responsibilities are not pre - defined , but have to be clarified in a situated manner . As a result , the used working environments were often neither as homogenous as a central organization , nor as heterogeneous as in a lone warrior culture . Empirically , we found many different practices of sharing configurations and plug - ins in the team in an unplanned and ad - hoc way . These were similar to forms of learning that have been described in the literature as learning in over - the - shoulder situations [ 14 ] . Generally , sharing was mainly based on a copy - and - adapt installation practice , whereas one user configured and adapted her own Eclipse configuration individually , and afterwards shared this configuration with his colleague ( s ) in the project . “I did configure Eclipse a few times . Or rather we configured it more or less together . For example , [ within the project ] we use the CheckStyle component and some other plug - ins , whose names I forgot , because in fact my colleague did set up the original configuration . And I eventually copied the whole configuration over to my workstation . ” ( Transcript from an interview with a junior developer ) . An important type of situation was a kind of initiation rite in which experienced Eclipse users introduced new team members or novice Eclipse users to the Eclipse technology ( e . g . if a developer joined the company and / or the team ) . In such situations , senior developers would often copy their working environments to the machines of the junior developers . One observed effect in such situations was that people worked with similar configurations for a certain time . Later , the working environments would drift apart in a lone warrior manner , as everyone modified the environment for his own purposes . The result of such a working style was that the selective cooperation fostered homogenization to a certain degree , but did not enforce it in a centralized manner . At the same time , the related ad hoc collaboration did not result in a systematic diffusion of innovations . Instead , tools and experiences were typically shared in an unsystematic manner , which was also a result of very limited support by existing technology . Accordingly , we found several attempts to organize self - made infrastructures to overcome existing shortcomings in technology support . “Developer : So , if the project requires to install something new and interesting , [ . . . ] I send a request using the mailing list : ‘Who knows a good plug - in for that ? ’ Interviewer : What do you mean by ‘mailing list’ ? Who receives these emails ? Developer : [ … ] All employees receive this message and everyone who wants to comment can answer directly . [ . . . ] Some time ago , when we really wanted to use [ a certain plugin ] , I had already heard about it on the mailing list . [ … ] And I directly contacted 59 the colleague , asking where I could get it and how I could use it . ” ( Transcript from an interview with a developer ) . In this case , employees utilized the company‘s mailing list to reach every colleague and to describe what tools they were currently using in a new project , as well as experiences regarding the usage of tools . This presents a notable example of creating a proxy for over - the - shoulder situations , coping with the challenge that several employees worked at the customer’s site full time as project leaders , programmers and technology consultants . In particular , these people described that they felt better connected to their company colleagues because of this mechanism . While they considered themselves as part of the team , they also considered themselves as part of the periphery . Living in the diaspora they were afraid of being cut off from their colleagues at the headquarters , who were perceived as leaders in appropriating domain specific innovations . 5 . CONCLUSION In this paper we studied practices of appropriating software in the Eclipse ecosystem , asking how small teams of software developers manage their working environments . We distinguished between three different approaches of managing the workplace configuration . The first one was labeled the lone warrior type . It is defined by the concept of autonomy , while the second , the centralized organization type , is defined by the concept of heteronomy . Both types have in common that informal collaboration among team members is not a constitutive element . This distinguishes them from the third type of collegial collaboration , which is defined by autonomy and heteronomy as a dialectical unity . It has to be noted , that the three types are analytic categories in the sense of Max Weber’s “ideal types” [ 15 ] . In practice , their borders were blurred as we observed various forms of collegial collaboration in all cases , leading to a fined - grained balance between autonomy and heteronomy . Sharing occurred often rather unplanned in over - the - shoulder situations , in case of breakdowns , in situations where newcomers joined the team or just by accident . In these situations , the sharing of configurations and knowledge did lead to a temporal increase in awareness on what is used and homogeneity of configurations . Between such occasions , configurations usually drifted apart as users added new plug - ins or changed configurations to their needs , which led to an increased heterogeneity and a decreased awareness which could sometimes cause breakdowns in the cooperative work . We also observed attempts of overcoming existing shortcomings by realizing collegial forms of appropriation in distributed work . Examples were setting up mailing lists to share experiences about new tools in a more systematic way , or using the source code repository to also store the tools with the code . Nowadays , most tools provide tailorability functions . Eclipse as our example can be tailored by adding plug - ins , setting preferences and changing the design of the user interface . However , our research showed a clear lack of tools for supporting the various practices of collaboration with regard to appropriating / tailoring . This is especially true for tailoring artifacts in complex software ecosystems like Eclipse , since modifications are usually delivered by 3 rd parties . In such cases conditions as practicality , compatibility , stability can hardly be estimated beforehand , making the selection of tools difficult and risky . Therefore , further research is needed to explore how collegial collaboration can be supported with regard to appropriation . Such approaches should not only take into account the provisioning models represented by the three ideal types , but also the dialectical unity of autonomy and heteronomy that framed the different practices we found in the field . In doing so , they should support tool awareness as well as practices of over - the - shoulder learning . 6 . REFERENCES 1 . Boden , A . , Draxler , S . , and Wulf , V . Aneignungspraktiken von Software - Entwicklern beim Offshoring - Fallstudie eines kleinen deutschen Softwareunternehmens . Multikonferenz Wirtschaftsinformatik 2010 , Universitätsverlag Göttingen ( 2010 ) . 2 . Dourish , P . The Appropriation of Interactive Technologies : Some Lessons from Placeless Documents . Computer Supported Cooperative Work ( CSCW ) 12 , 4 ( 2003 ) , 465 - 490 . 3 . Eclipse Foundation . Eclipse Community Survey 2010 . Eclipse Foundation , 2010 . 4 . Gamma , E . and Beck , K . Contributing to Eclipse : Principles , Patterns , and Plug - Ins . Addison - Wesley Professional , 2003 . 5 . Gantt , M . and Nardi , B . A . Gardeners and gurus : patterns of cooperation among CAD users . Proceedings of the SIGCHI conference on Human factors in computing systems , ACM ( 1992 ) , 107 - 117 . 6 . Glaser , B . and Strauss , A . The Discovery of Grounded Theory : Strategies for Qualitative Research . Aldine Transaction , 1967 . 7 . Mackay , W . E . Patterns of sharing customizable software . Proceedings of the 1990 ACM conference on Computer - supported cooperative work , ACM ( 1990 ) , 209 - 221 . 8 . MacLean , A . , Carter , K . , Lövstrand , L . , and Moran , T . User - tailorable systems : pressing the issues with buttons . CHI90 Proceedings ISBN : 0 - 201 - 50932 - 6 , ( 1990 ) , 175 - 182 . 9 . Mørch , A . I . , Stevens , G . , Won , M . , Klann , M . , Dittrich , Y . , and Wulf , V . Component - based technologies for end - user development . Commun . ACM 47 , 9 ( 2004 ) , 59 - 62 . 10 . Pipek , V . From tailoring to appropriation support : Negotiating groupware usage . University of Oulu , Oulu , 2005 . 11 . Stevens , G . Understanding and Designing Appropriation Infrastructures : Artifacts as boundary objects in the continuous software development . 2009 . 12 . Stevens , G . and Draxler , S . Appropriation of the Eclipse Ecosystem : Local Integration of Global Network Production . ( 2010 ) . 13 . Stevens , G . , Pipek , V . , and Wulf , V . Appropriation Infrastructure : Supporting the Design of Usages . In End - User Development . 2009 , 50 - 69 . 14 . Twidale , M . Over the Shoulder Learning : Supporting Brief Informal Learning . Computer Supported Cooperative Work ( CSCW ) 14 , 6 ( 2005 ) , 505 - 547 . 15 . Weber , M . The Methodology Of The Social Sciences . Free Press , 1949 . 16 . Wulf , V . " Let ' s see your Search - Tool ! " - On the Collaborative use of Tailored Artifacts . Proceedings of GROUP ' 99 , ACM - Press , ( 1999 ) , 50 - 60 . 60 \ No newline at end of file diff --git a/silver_data/7911a6913e36672a476e6edcd64403c2553fa47a.pdf.txt b/silver_data/7911a6913e36672a476e6edcd64403c2553fa47a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..2b06697cdd6bffe75f0d883e48a43dd23e071d77 --- /dev/null +++ b/silver_data/7911a6913e36672a476e6edcd64403c2553fa47a.pdf.txt @@ -0,0 +1 @@ +Butler University Digital Commons @ Butler University LAS Faculty Book Reviews College of Liberal Arts & Sciences 11 - 1 - 2008 Flat Earth : The History of an Infamous Idea by Christine Garwood , Thomas Dunne Books 2008 Michael Zimmerman Follow this and additional works at : http : / / digitalcommons . butler . edu / las _ bookreviews This Book Review is brought to you for free and open access by the College of Liberal Arts & Sciences at Digital Commons @ Butler University . It has been accepted for inclusion in LAS Faculty Book Reviews by an authorized administrator of Digital Commons @ Butler University . For more information , please contact omacisaa @ butler . edu . Recommended Citation Zimmerman , Michael , " Flat Earth : The History of an Infamous Idea by Christine Garwood , Thomas Dunne Books 2008 " ( 2008 ) . LAS Faculty Book Reviews . 11 . http : / / digitalcommons . butler . edu / las _ bookreviews / 11 Because Ideas Matter . . . The faculty and staff of Butler University ' s College of Liberal Arts and Sciences presents Recommended Readings Flat Earth : The History of an Infamous Idea by Christine Garwood , Thomas Dunne Books 2008 Reviewed by Michael Zimmerman Garwood , historian of science at the Open University in England , has produced a thoroughly enjoyable and engaging first book . She examines the belief that the world is flat from a wide array of perspectives and makes a number of important points . She demonstrates quite convincingly , for example , that , contrary to what most people believe , the ancients knew the world was not flat : " the earth has been widely believed to be a globe since the fifth century BC . " Indeed , growing acceptance of a flat earth occurred in the 19th century and was largely promoted by Biblical literalists . Garwood does an impressive job of comparing those professing this belief with modern day creationists . She also makes the case that it is all but impossible to argue effectively with true believers - Alfred Russel Wallace , co - founder of the theory of natural selection with Charles Darwin , ended up in years of litigation after he accepted a challenge to demonstrate that there is curvature to the surface of the earth . Modern believers assert that the space program is a " big , giant hoax . " When , on the 25th anniversary of the first manned landing on the moon , a 1994 Washington Post poll estimated that approximately 20 million Americans thought the landing was staged on earth , it is obvious that some outrageous beliefs still hold sway . Garwood is respectful throughout , analyzing the philosophical underpinnings of those who have doubted the earth ' s rotundity . - Michael Zimmerman is Dean of the College of Liberal Arts and Sciences and professor of biology at Butler University . \ No newline at end of file diff --git a/silver_data/7b261c11533a4bdcb14f10d840660bd60fc5130a.pdf.txt b/silver_data/7b261c11533a4bdcb14f10d840660bd60fc5130a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..7ed0416d31091d86619660fd92526483375a3e27 --- /dev/null +++ b/silver_data/7b261c11533a4bdcb14f10d840660bd60fc5130a.pdf.txt @@ -0,0 +1 @@ +Nonparametric Estimation from Incomplete Observations Author ( s ) : E . L . Kaplan and Paul Meier Reviewed work ( s ) : Source : Journal of the American Statistical Association , Vol . 53 , No . 282 ( Jun . , 1958 ) , pp . 457 - 481Published by : American Statistical Association Stable URL : http : / / www . jstor . org / stable / 2281868 . Accessed : 29 / 01 / 2013 10 : 47 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use , available at . http : / / www . jstor . org / page / info / about / policies / terms . jsp . JSTOR is a not - for - profit service that helps scholars , researchers , and students discover , use , and build upon a wide range of content in a trusted digital archive . We use information technology and tools to increase productivity and facilitate new forms of scholarship . For more information about JSTOR , please contact support @ jstor . org . . American Statistical Association is collaborating with JSTOR to digitize , preserve and extend access to Journal of the American Statistical Association . http : / / www . jstor . org This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION FROM INCOMPLETE OBSERVATIONS * E . L . KAPLAN University of California Radiation Laboratory AND PAUL MEIER University of Chicago In lifetesting , medical follow - up , and other fields the observation of the time of occurrence of the event of interest ( called a death ) may be prevented for some of the items of the sample by the previous occur - rence of some other event ( called a loss ) . Losses may be either accidental or controlled , the latter resulting from a decision to terminate certain observations . In either case it is usually assumed in this paper that the lifetime ( age at death ) is independent of the potential loss time ; in practice this assumption deserves careful scrutiny . Despite the resulting incompleteness of the data , it is desired to estimate the proportion P ( t ) of items in the population whose lifetimes would exceed t ( in the absence of such losses ) , without making any assumption about the form of the function P ( t ) . The observation for each item of a suitable initial event , marking the beginning of its lifetime , is presupposed . For random samples of size N the product - limit ( PL ) estimate can be defined as follows : List and label the N observed lifetimes ( whether to death or loss ) in order of increasing magnitude , so that one has o < tjI < t . 2 ' < ? . . < tN ' . Then P ( t ) = 11r [ ( N - r ) / ( N - r + 1 ) ] , where r assumes those values for which tr ' < t and for which tr / measures the time to death . This estimate is the distribution , unrestricted as to form , which maximizes the likelihood of the observations . Other estimates that are discussed are the actuarial estimates ( which are also products , but with the number of factors usually reduced by grouping ) ; and reduced - sample ( RS ) estimates , which require that losses not be accidental , so that the limits of observation ( potential loss times ) are known even for those items whose deaths are observed . When no losses occur at ages less than t , the estimate of P ( t ) in all cases re - duces to the usual binomial estimate , namely , the observed proportion of survivors . CONTENTS 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 1 . 1 Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 1 . 2 Nonparametric estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 1 . 3 Examples of the RS and PL estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 1 . 4 Notation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 2 . The Product - Limit Estimate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 2 . 1 Definition and calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 2 . 2 Mean and variance of P ( t ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 2 . 3 Mean lifetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 3 . The Reduced - Sample Estimate vs . the PL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469 3 . 1 Alternatives to the PL estimate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469 3 . 2 Dependence of deaths and losses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470 * Prepared while the authors were at Bell Telephone Laboratories and Johns Hopkins University respectively . The work was aided by a grant from the Office of Naval Research . 457 This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 458 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JTJNE 1958 4 . Actuarial Estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 4 . 1 Estimates using only n , B , X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 4 . 2 Estimates using average ages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 5 . Maximum Likelihood Derivation of the PL . . . . . . . . . . . . . . . . . . . . 475 6 . Means and Variances . . . . . . . . . . . I . I . . . . I . . IIII . IIIIIIII . . . 476 6 . 1 The PL estimate P ( t ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 6 . 2 Covariances and mean lifetimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478 7 . Consistency ; Testing with Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479 1 . INTRODUCTION I . Formulation . In many estimation problems it is inconvenient or im - possible to make complete measurements on all members of a random sample . For example , in medical follow - up studies to determine the distribution of sur - vival times after an operation , contact with some individuals will be lost before their death , and others will die from causes it is desired to exclude from con - sideration . Similarly , observation of the life of a vacuum tube may be ended by breakage of the tube , or a need to use the test facilities for other purposes . In both examples , incomplete observations may also result from a need to get out a report within a reasonable time . The type of estimate studied here can be briefly indicated as follows . When a random sample of N values , T1 , T2 , * * * , TN of a random variable is given , the sample distribution function Ft ( t ) is naturally defined as that which assigns a probability of I / N to each of the given values , so that P ( t ) equals 1 / N times the number of sample values less than the argument t . Besides describing the sample , this F ( t ) is also a nonparametric estimate of the population distribution , in the sense indicated in 1 . 2 below . When the observations are incomplete , the corresponding estimate is still a step - function with discontinuities at the ages of observed deaths , but it can no longer be obtained as a mere description of the sample . The samples considered in this paper are incomplete in the sense that one has given , not a random sample T1 , * * * , TN of values of the random variable T itself ( called the lifetime ) , but the observed lifetime8 ti = min ( Ti , Li ) , i - = 1 , 2 , * * N . ( la ) Here the Li , called limits of observation , are constants or values of other random variables , which are assumed to be independent of the TX unless otherwise stated ( in Sections 3 . 2 and 7 ) . For each item it is known whether one has Ti < Li , t - = Ti ( a death ) ( lb ) or Li < T , , ti = Li ( a loss ) . Ordinarily the Ti and Li are so defined as to be necessarily nonnegative . The items in the sample are thus divided into two mutually exclusive classes , namely deaths and losses . A loss by definition always precludes the desired knowledge of T , . On the other hand , a death does not always preclude the knowledge of the corresponding Li , in case the limits of observation are non - random and foreseeable . Such knowledge of the Li may have value ; for example , This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 459 it makes available the reduced - sample estimate ( Section 3 ) , if one chooses to use it . The type of sample described is a generalization of the censored sample de - fined by Hald [ 17 ] , and a specialization of the situation considered by Harris , Meier , and Tukey [ 18 ] . The term death has been adopted as being at least metaphorically appropri - ate in many applications , but it can represent any event susceptible of random sampling . In particular , the roles of death and ( random ) loss may be inter - changeable . By redefining the classification of events into deaths and losses , it may be possible to approach the same data from various points of view and thus to estimate the survivorship functions P ( t ) that would be appropriate to vari - ous categories of events in the absence of the others . This is familiar enough ; see for example [ 6 ] , [ 13 ] , [ 16 ] . 1 . 2 Nonparametric estimation . Most general methods of estimation , such as maximum likelihood or minimum chi - square , may be interpreted as procedures for selecting from an admissible class of distributions one which , in a specified sense , best fits the observations . To estimate a characteristic ( or parameter ) of the true distribution one uses the value that the characteristic has for this best fitting distribution function . It seems reasonable to call an estimation pro - cedure nonparametric when the class of admissible distributions from which the best - fitting one is to be chosen is the class of all distributions . ( Wolfowitz [ 28 ] has used the termn similarly in connection with the likelihood ratio in hypothesis testing ) . With a complete sample , it is easy to see that the sample distribution referred to in 1 . 1 is the nonparametric estimate on the maximum likelihood criterion . The same result is true of the product - limit estimate for incomplete samples , as will be demonstrated in Section 5 . The most frequently used methods of parametric estimation for distributions of lifetimes are perhaps the fitting of a normal distribution to the observations or their logarithms by calculating the mean and variance , and fitting an ex - ponential distribution e - tlAdt / ll by estimating the mean life , u alone . Such as - sumptions about the form of the distribution are naturally advantageous insofar as they are correct ; the ' estimates are simple and relatively efficient , and a complete distribution is obtained even though the observations may be re - stricted in range . However , nonparametric estimates have the important func - tions of suggesting or confirming such assumptions , and of supplying the esti - mate itself in case suitable parametric assumptions are not known . An impor - tant property of these nonparametric estimates is that if the age scale is trans - formed from t to t * = f ( t ) , where f is a strictly increasing function , then the cor - responding estimated distribution functions are simply related by F * ( f ( t ) ) F ( ) 1 . 3 Examples of the RS and PL estimates . We will consider the following situation . A random sample of 100 items is put on test at the beginning of 1955 ; during the year 70 items die and 30 survive . At the end of the year , a larger sample is available and 1000 additional items are put on test . During 1956 , 15 items from the first sample and 750 from the second die , leaving 15 and 250 survivors respectively . As of the end of 1956 , it is desired to estimate the propor - tion P ( 2 ) of items in the population surviving for two years or more . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 460 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 The survival probabilities are supposed to depend on the age ( the duration of the test ) rather than on the calendar year , and hence the data are arranged as in Table 460 . TABLE 460 Samples I II Initial numbers 100 1000 Deaths in first year of age 70 750 One - year survivors 30 250 Deaths in second year of age 15 Two - year survivors 15 This particular example is such that it is easy to form an estimate P * ( 2 ) 15 / 100 = 0 . 15 from the first sample alone . This is called the reduced - sample ( RS ) estimate because it ignores the 1000 items tested only during 1956 . It is a legitimate estimate only when the reduced sample is itself a random sample ; this will be the case only when ( as assumed here ) the observation limits ( two years for the first sample , and one year for the second ) are known for all items , deaths as well as losses . In the absence of this information , one would have no basis for discriminating among the 835 deaths observed before the age of two years . One cannot simply ignore the 250 losses at age one year ; since only 15 items have survived for two years , P ( 2 ) would then be estimated as 15 / 850 - . 018 , an absurd result . The point is discussed further in 3 . 1 below . We now inquire whether the second sample , under test for only one year , can throw any light on the estimate of P ( 2 ) . Clearly it will be necessary to assume that both samples have been drawn from the same population , an assumption that the RS estimate P * ( 2 ) avoided . At any rate , the estimates of P ( 1 ) from the two samples , namely 0 . 30 and 0 . 25 , are not sufficiently different to contra - dict the assumption . By combining the two samples , the estimate P ( l ) = P * ( 1 ) = ( 30 + 250 ) / ( 100 + 1000 ) = . 255 is obtained for P ( 1 ) . ( In this case the RS has the same value as the other esti - mate to be discussed , the product - limit or PL . ) This result exhausts the useful - ness of the second sample for the present purposes ; how does it help to esti - mate P ( 2 ) ? The answer is that there are advantages to using the first ( the smaller ) sample for estimating P ( 2 ) / P ( 1 ) , the conditional probability of survival for two years given survival for one year , rather than P ( 2 ) itself . This estimate is P ( 2 ) / P ( 1 ) = 15 / 30 = 0 . 50 , whence P ( 2 ) = 0 . 255 X 0 . 50 = 0 . 127 , a very simple example of the product - limit ( PL ) estimate . The outstanding advantage of this strategy is that it works just as well if we are not privileged to know that the 750 deaths in the second sample had observation limits of one year , because these items are irrelevant to the estimation of P ( 2 ) / P ( 1 ) in any This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 461 case . Other considerations for deciding between the two estimates will be set forth in Section 3 . The discussion of the PL estimate will be continued shortly , in Sections 2 and 3 . Section 3 is equally concerned with the RS estimate , while Section 4 is devoted to the actuarial estimates . The remaining three sections consist princi - pally of mathematical derivations . Though much older , the actuarial estimates are essentially approximations to the PL ; they are products also , but typically they aim to reduce the number of factors by grouping . ( Grouping may or may not be possible for the PL itself . ) The distinguishing designation product - limit was adopted because this estimate is a limiting case of the actuarial estimates . It was proposed as early as 1912 by Bohmer [ 6 ] ( referred to by Seal [ 26 ] ) , but seems to have been lost sight of by later writers and not further investigated . 1 . 4 Notation . The survival function P ( t ) = Pr ( T > t ) , ( ic ) giving the population probability of surviving beyond t , will be used in place of the distribution function F ( t ) = 1 - P ( t ) because of its convenience where the product - limit estimate and its actuarial approximations are concerned . In addition the following functions are defined : P ; ( t ) = product - limit ( PL ) estimate of P ( t ) . P * ( t ) = reduced - sample ( RS ) estimate of P ( t ) . n ( t ) = the number of items observed and surviving at age t , when deaths ( but not losses ) at t itself are subtracted off . N ( t ) = the expectation of n ( t ) , for fixed observation limits . N ? ( t ) = the number of items having observation limits L such that L > t . In practice this function is not necessarily known . For the first reading of the paper it may be desirable to suppose that the death of one item and the loss of the same or any other item never occur at the same age , and never coincide with an age t at which any of the above functions are to be evaluated . This condition can always be met by fudging the ages a little when necessary . On the other hand , a regular user of the techniques will probably come to regard overt fudging as naive ; he will prefer to formalize his notation and record - keeping by adopting the conventions already insinuated into the definitions of death and loss , P ( t ) , and n ( t ) above . These conventions may be paraphrased by saying that deaths recorded as of an age t are treated as if they occurred slightly before t , and losses recorded as of an age t are treated as occurring slightly after t . In this way the fudging is kept conceptual , sys - tematic , and automatic . The convention that deaths precede losses in case of ambiguity is based on the following sequence of operations , which is clearly more efficient than the reverse sequence : Examine a group of items of age to , observe the number a of deaths since the last examination , and then remove ( or lose contact with ) a number X of the survivors . It may then be convenient simply to record to as the age of death or loss of the 5 + X items , especially if to is always an integral multiple of a fundamental time interval ; in fact , however , the deaths will have preceded the losses . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 462 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 The chief exception to the immediate applicability of this conventioln occurs when the losses are random but cannot affect items that have already died . Then the possible sequences of occurrence of deaths and losses between exami - nation times ( assumed to be close together ) are approximately equally likely , and a reasonable compromise is to assume that half the losses in the interval precede and half follow the deaths , as in ( 4b ) below . If the loss of an item is compatible with the possibility of its having died ( unknown to the experi - menter ) between the time when it was last examined and the time of loss , an item lost in this way is effectively lost just after it was last examined , and the convention is entirely appropriate . The disappearance of individuals subject to medical follow - up is a case in point . The remainder of the conventions concern the treatment of discontinuities in the functions listed above . Should the value assigned to n ( t ) , say , for an argu - ment t for which it is discontinuous be the right - hand limit n ( t + 0 ) = lim n ( t + h ) as h - * 0 with h > 0 , the left - hand limit n ( t - 0 ) ( the same but with h < 0 ) , or something else ? The superior expressiveness of the notation adopted is illus - trated by the relations n ( t - 0 ) - n ( t ) the number of deaths at t , ( Id ) n ( t ) - n ( t + 0 ) = the number of losses at t , or equivalently by the formula ( 3a ) : P * ( t ) n ( t ) / N ? ( t ) , which otherwise would not be valid at discontinuities . Analogous conventions are adopted for all the above - mentioned functions of t , so that P ( t ) and P ( t ) are right - continuous , N ? ( t ) is left - continuous , and n ( t ) and P * ( t ) are neither . Other advantages of the convention for P ( t ) and its estimates are the following : ( a ) According to the sequence of operations assumed above , one may record deaths as of age t al - though they actually occurred slightly earlier . ( b ) It makes P ( 0 ) = 1 if and only if no item dies at birth ( age zero ) . This is convenient and natural . One other possibility may be mentioned briefly . The assumption that P ( oo ) = 0 , so that the lifetimes are finite with probability one , is necessary only for parts of Section 7 and for the calculation of a finite mean lifetime . However , in practice there is no apparent need to contradict the assumption either . If half of a sample dies in one day and the other half is still alive after 1000 days , one should still report P ( 1000 ) ( not P ( oo ) ) = 0 . 50 , since the argument 1000 is not an arbitrary large number , but the actual duration of the test . 2 . THE PRODUCT - LIMIT ESTIMATE 2 . 1 Definition and calculation . Both the PL and the actuarial estimates of Section 4 are based on the following general procedure : ( a ) The age scale is divided into suitably chosen intervals , ( 0 , Qi ) , ( us , U2 ) , * , as described below . ( In the example of 1 . 3 , there were only two such intervals , namely ( 0 , 1 ) and ( 1 , 2 ) . ) ( b ) For each interval ( uj ; i , uj ) , one estimates pj = Pj / Pj _ , the proportion of items alive just after uj - i that survive beyond uj . ( c ) If t is a division point ( it may be introduced specially if necessary ) , the proportion P ( t ) in the population surviving beyond t is estimated by the product of the estimated pi for all intervals prior to t . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 463 If step ( a ) is left relatively arbitrary and approximations or parametric assumptions are accepted in step ( b ) , one arrives at the actuarial estimates . The PL is obtained by selecting the intervals in ( a ) so that the estimation in ( b ) is a simple binomial , without any recourse to assumptions of functional form . The condition for this is that within each interval , deaths and losses be segre - gated in a known fashion . As a beginning , it may be assumed that no interval contains both deaths and losses . Then if the number under observation just after uj - l is denoted by ni , and 3j deaths are observed in the interval ( uj - . , uj ) , the estimate is clearly pj - = ( nj - aj ) / nj = nj ' / nj , ( 2a ) where nj ' is the number under observation just after the bj deaths . However , if the interval contains only losses ( but at least one item survives throughout the interval ) , the estimate is fj = 1 . In the product of conditional probabilities formed in step ( c ) , unit factors may as well be suppressed ; and we need not be concerned with the manner in which the losses are distributed among the intervals , so long as nj and nj ' are correctly evaluated in ( 2a ) , and no losses occur at ages intermediate to the t5 deaths . The situation is illustrated by the following scheme : TABLE 463 No . of items N nli ni ' n2 n2 ' No . of deaths or losses So ai xi B2 X2 Division points u = 0 Ul U2 Here N is the initial number of items , and the braces join the numbers whose ratios are the conditional probabilities ( 2a ) . The numbers in the second line are the differences of those in the first , the X ' s counting losses and the 6 ' s deaths ; some of these could be zero . The division points uj are placed in the third line to show that the Sj deaths occur between uj - l and u ; , while u ; is located any - where among the Xj losses . The relation n / ? n ( u ; ) ? nj + 1 holds . The PL estimate is now given by P5 ( t ) = H1 [ ( nj ' / nj ) , with Uk - t , nil = n1 - Si . ( 2 b ) j = 1 If the greatest observed lifetime t * corresponds to a loss , ( 2b ) should not be used with t > t * ; in this case P ( t ) can be regarded as lying between 0 and P ( t * ) , but is not more closely defined . If it is desired to permit the entrance of items into the sample after the com - mencement of their lifetimes , this can be done by treating such entrances as " losses " that are counted negatively in Xi . The same items can of course disap - pear again at a later age and so yield ordinary losses as well . It is assumed that nothing is known of the existence of any such item that dies before it becomes available for observation ; that is , the observation is censored on the right but truncated on the left , in the terminology of Hald [ 17 ] . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 464 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 The form ( 2b ) was selected for the PL estimate to give the minimum number of elementary factors and the maximum grouping of the observations . Never - theless , the number of deaths bj in an interval can easily be as small as unity . The resulting estimate Pi , though of limited value by itself , is none the less acceptable as a component of P ( t ) . In fact , one is at liberty to take the intervals as short and as numerous as one pleases , and to regard each death as occupying an interval by itself . To specify the resulting expression , one relabels the N ages ti of death or loss in order of increasing magnitude , and denotes them by tl _ < t2 ' < t2N . Then P ( t ) = 1 [ ( N - r ) / ( N - r + 1 ) ] , ( 2c ) r where r runs through those positive integers for which tr ' < t and tr ' is ttle age of death ( not loss ) . The cancellation of like integers in numerator and denomi - nator where they occur reduces ( 2c ) to ( 2b ) . If there are no losses , everything cancels except the first denominator N and the last numerator n ( t ) , say , and the PL reduces to the usual binomial estimate n ( t ) / N . ( 2c ) shows that P ( t ) is a step - function which changes its value only at the observed ages of death , where it is discontinuous . In analyzing data on lifetimes by the multiplication of conditional probabili - ties , one of the following three procedures will usually suffice : ( 1 ) If the number of deaths is relatively small , these deaths may be arranged in order of age without grouping , and the numbers of losses in the intervening age intervals counted . The PL estimate is calculated by ( 2c ) . ( 2 ) If ( 1 ) is too time - consuming but the number of distinct ages of loss is relatively small , these ages may be arranged in order , additional division points inserted as desired , and the numbers of deaths in the resulting age intervals counted . If some of these intervals are shorter than necessary and are found to contain no deaths , they can be combined with adjacent intervals . The PL esti - mate is calculated by ( 2b ) . ( 3 ) If neither ( 1 ) nor ( 2 ) is compact enough , then division points are chosen without close consideration of the sample , deaths and losses are counted in each interval , and an actuarial approximation to the PL , such as ( 4b ) , is used . As a miniature example of case ( 1 ) , suppose that out of a sample of 8 items the following are observed : Deaths at 0 . 8 , 3 . 1 , 5 . 4 , 9 . 2 months . Losses at 1 . 0 , 2 . 7 , 7 . 0 , 12 . 1 months . The construction of the function P ( t ) then proceeds as follows : TABLE 464 Uj ni " n / ' XjP ( u1 ) 0 . 8 8 7 2 7 / 8 3 . 1 5 4 0 7 / 10 5 . 4 4 3 1 21 / 40 9 . 2 2 1 0 21 / 80 ( 12 . 1 ) 1 1 1 21 / 80 This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 465 Each value of P ( uj ) is obtained by multiplying n / ' / nj by the preceding value P ( uj - i ) . The age 12 . 1 is recorded in the last line to show the point at which P ( t ) becomes undefined ; since it is a loss time , the 12 . 1 is enclosed in parenthe - ses . It is to be inferred from the table that P ( 5 . 3 ) - 7 / 10 , for example . The third and fourth columns could be omitted since nj ' = nji - 1 ( except in the last line , which corresponds to a loss ) and Xj = n - nj + / . A rudimentary illustration of case ( 2 ) has already been given in 1 . 3 . A little more elaborate example of a similar sort with N = 100 is given in Table 465 . Here 1 . 7 , 3 . 6 , and 5 . 0 are assumed to be the only ages at which losses occur ; they are prescribed as division points . The other division points ( 1 , 2 , 3 , 4 ) are selected at pleasure , with the object of interpolating additional points on the curve of P ( t ) vs . t . NEj is the effective sample size defined in ( 2j ) below . In practice four columns headed uj , nj , nj ' , P " will suffice . From the table one infers that . 74 < P ( 2 . 5 ) < . 87 , for example . TABLE 465 Interval Factor u Uj j nj Si i i P ( u ; ) NE ; 0 - 1 1 100 3 0 97 / 100 . 97 100 1 - 1 . 7 2 97 5 20 92 / 97 . 92 100 1 . 7 - 2 3 72 4 0 68 / 72 . 87 88 2 - 3 4 68 10 0 58 / 68 . 74 83 3 - 3 . 6 5 58 9 12 49 / 58 . 63 80 3 . 6 - 4 6 37 6 0 31 / 37 . 52 73 4 - 5 . 0 7 31 15 16 16 / 31 . 27 51 2 . 2 Mean and variance of P ( t ) . The important facts here , derived in Section 6 . 1 , are that P ( t ) is consistent and of negligible bias ( unless excessive averaging is done ; see Section 3 . 1 ) , and that an asymptotic expression for its variance can be obtained . Like the estimate itself , the sample approximation to its variance proves to be independent of the limits of observation of items not actually lost . However , the variance derived from population values does depend on all the limits of observation , which are assumed to be fixed during the sampling . It has been noted that if the greatest observed lifetime t * corresponds to a loss , then for t > t * , P ( t ) is undefined though bounded by 0 and P ( t * ) . Unless the probability of this ambiguous situation is quite small , however , a non - parametric estimate of P ( t ) will not be very informative in any case . The ambiguity cannot occur unless the NO ( t ) items observable to t all die at ages less than t . The probability of this event is [ 1 - P ( t ) ] N0 ( t ) < e - N0 ( t ) P ( t ) = e - N t ) ( 2d ) This is already less than 0 . 01 when N ( t ) is only five . It is shown in Section 6 . 1 that if one can supplement the ambiguous case by ascertaining the age of death of the item lost at t * , or of one or more other randomly selected items alive at t * , and defines P ( t ) for t > t * as P ( t * ) times the survival function for the supplementary sample , then the expected value of This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 466 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1988 P ( t ) is precisely the population value P ( t ) . In practice this supplementation would often be neither feasible nor worthwhile , but with otherwise adequate data the resulting bias in one or a few samples will be too small to have any practical importance . It will be shown in Section 6 . 1 that the variance of P ( t ) is given approxi - mately by Vffi ( t ) ] p2 ( t ) E ( qjlNjpj ) ) ( 2e ) 1 , where the distinct limits of observation Lj ' are now used as the division poin - ts ; L ' k - 1 is the greatest preceding t ; and pj = 1 - qj = P ( Lj ' ) / P ( L ' j - 1 ) , with Lot = 0 , Lk ' = t . After dividing ( 2e ) by P2 ( t ) , one sees that the square of the coefficient of variation ( CV ) of P ( t ) is set equal to the sum of the squares of the CV ' s of the estimates of the pj , the usual approximation for the variance of a product . If the sample estimates are inserted in ( 2e ) one obtains ' V [ Pf ( t ) ] - p P ( t ) E [ bilnj ( ni - 6i ) ] = p P ( t ) E ( 2f ) 1 1 f ni A very similar formula was derived by Greenwood [ 15 ] and later by Irwin [ 19 ] in connection with actuarial estimates . It is easily verified that ( 2f ) remains valid when the number of intervals is reduced to those used in ( 2b ) , or ex - panded to one interval for each death as in ( 2c ) . In the latter case it may be written V [ Pfi ( t ) ] P2 ( t ) [ ( N - r ) ( N - r - F 1 ) kl , ( 2g ) where r runs through the positive integers for which t4 ' < t and t4 ' corresponds to a death . In terms of integrals ( 2e ) can be written I / [ P ( t ) ] P2 ( t ) t dP ( u ) I O N0 ( u ) P2 ( u ) ( 2h ) ( ' dP ( u ) - P2 ( t ) | dPu | - N ( u ) P ( u ) In case of ambiguity the first form should be referred to and interpreted in ac - cordance with 1 . 4 . If P ( u ) alone is discontinuous within the range of integration , it should be regarded as a ( continuous ) independent variable , so that fP ( U ) - 21 dP ( u ) I = 1 / P ( u ) . Since NO ( t ) < ? N ? ( u ) < ? N , it is clear that ( 2h ) is greater than the complete sample variance P ( t ) [ 1 - P ( t ) ] / lN but less than the reduced - sample variance P ( t ) [ 1 - P ( t ) ] / N ? ( t ) of ( 3b ) . If losses are random and the instantaneous rates of death and loss among survivors are in the ratio 1 to p at all ages , then one has E [ NO ( u ) - ' ] [ NPP ( u ) ] - ' for large samples , and ( 2h ) reduces to T [ P ( t ) ] _ [ pI - p ( t ) - P2 ( t ) ] / ( l + p ) N ( 2i ) This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 467 It is often instructive to estimate an effective sample size ` * ( t ) , which in the absence of losses would give the same variance f [ P ( t ) ] . Evidently NE ( W = P ( t ) [ 1 - P ( t ) ] / V [ P ( t ) ] ( 2j ) It can be shown that N2v ( t ) is nonincreasing as t increases , and that n ( t ) / P ( t ) < N ' 1 ( t ) < D ( t ) / [ i - P ( t ) ] , ( 2k ) where D ( t ) is the number of deaths observed at ages not exceeding t . The upper bound was proposed as an approximation to N2E ( t ) by Cornfield [ 8 ] . The lower bound corresponds to the reduced - sample variance . In Table 464 of 2 . 1 we had P ( 6 ) - 2 1 / 40 = 0 . 525 . By ( 2f ) the variance of P ( 6 ) is estimated as [ P ( 6 ) ] - ( 0 . 525 ) 2 ( 8 2 + 5 3 ) = 0 . 042 . By ( 2g ) the same result appears in the form o0 . s525 ) 2 ( 1 + l ~ + ~ 7X8 48 X 5 3 X ; 4 The effective sample size is estimated as NE ( 6 ) = ( 0 . 525 ) ( 0 . 475 ) / 0 . 042 = 6 . 0 . The bounds for N ] E ( 6 ) in ( 2k ) are 5 . 7 and 6 . 3 . Values of i9 , are also indicated in Table 465 . 2 . 3 Mean lifetime . The PL estimate lb of the mean lifetime ji is defined as the mean of the PL estimate of the distribution . It is well - known ( and easily proved by integrating by parts ) that the mean of a nonnegative random varia - ble is equal to the area under the corresponding survivorship function . Hence st = fJ ( t ) dt . Of course , if P ( t ) is not everywhere determined , , is undefined . In cases where the probability of an indeterminate result is small , P ( t ) is practically unbiased and the same is true of , i . If we " complete " P ( t ) in the example of Table 464 by following the longest observed individual ( with t ' = 12 . 1 ) to death at t = 14 . 3 we have A = ( l . 000 ) ( 0 . 8 ) + ( 0 . 875 ) ( 3 . 1 - 0 . 8 ) + ( 0 . 700 ) ( 5 . 4 - 3 . 1 ) + ( 0 . 525 ) ( 9 . 2 - 5 . 4 ) + ( 0 . 2625 ) ( 14 . 3 - 9 . 2 ) = 0 . 800 + 2 . 012 + 1 . 610 + 1 . 995 + 1 . 339 = 7 . 76 . If P ( t ) were " completed " by setting P ( t ) = 0 for the indeterminate range , the last term in the above sum would be replaced by ( 0 . 2625 ) ( 12 . 1 - 9 . 2 ) = 0 . 761 , and , u would be estimated as 7 . 18 . Of course , if the probability of an indeterminate result is high , there is no satisfactory way to estimate , u . In such cases Irwin [ 19 ] has suggested that in This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 468 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 place of estimating the mean itself , one should estimate the " mean life limited to a time L , " say / [ L ] . This is the mean of min ( Ti , L ) , with L chosen at the in - vestigator ' s convenience . Naturally , one would choose L to make the probability of an indeterminate result quite small . If one chooses to use this procedure he should give an estimate of P ( L ) along with , If we take L = 10 in our example , AI [ 1o ] = 0 . 800 + 2 . 012 + 1 . 610 + 1 . 995 + ( 0 . 2625 ) ( 10 - 9 . 2 ) = 6 . 63 , and P ( 10 ) = 0 . 2625 . In Section 6 . 2 an approximate formula is given for the variance of - u : I - 0 A2 ( t ) I dP ( t ) | C A2 ( t ) I dP ( t ) I V % - ) I I ( 21 ) o N ( t ) P ( t ) No N ( t ) P2 ( t ) where A ( t ) = ft P ( u ) du . Upon making the obvious substitutions we find , after some reduction , the following estimate of V ( , u ) : Ar 2 VW = E A72 ( 2m ) r ( N - r ) ( N - r + 1 ) where r runs over those integers for which tr corresponds to a death , and Ar = f , r Pf ( u ) du . If there are no losses , N Ar = ( ti - tr ) N i = r + l and it can be shown that V ( , u ) reduces to , ( t , - b2 / N2 . This fact , plus the impossibility of estimating the variance on the basis of only one observed death , suggests that ( 2m ) might be improved by multiplication by D / ( D - 1 ) , where D is the number of deaths observed . For our first estimate of j above we have A1 = 2 . 012 + 1 . 610 + 1 . 995 + 1 . 339 = 6 . 956 , A4 = 1 . 610 + 1 . 995 + 1 . 339 = 4 . 944 , A5 = 1 . 995 + 1 . 339 = 3 . 334 , A7 = 1 . 339 = 1 . 339 . ( Obviously the Ar and , are best calculated in reverse order . ) We then have for the estimated variance , ( 6 . 956 ) 2 ( 4 . 944 ) 2 ( 3 . 334 ) 2 ( 1 . 339 ) 2 v ( - / 0 = + ? ? = 3 . 91 7X8 4X5 3X4 1X2 The estimated standard deviation of , u is a / 3 . 91 = 1 . 98 . If the factor D / ( D - 1 ) = 4 / 3 is included , these results become 5 . 21 and 2 . 28 respectively . If one is limited to grouped data as in Table 465 it is necessary to use actuarial - type assumptions ( e . g . , the trapezoidal rule ) to estimate , and its variance . Thus , we may estimate the mean life limited to 5 time units as follows : = ( 1 / 2 ) [ ( 1 . 00 + 0 . 97 ) ( 1 . 0 - 0 . 0 ) + ( 0 . 97 + 0 . 92 ) ( 1 . 7 - 1 . 0 ) + * * * + ( 0 . 52 + 0 . 27 ) ( 5 . 0 - 4 . 0 ) ] = 3 . 76 . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 469 We observe in conjunction with this estimate that the estimated proportion surviving the limit is P ( 5 ) = 0 . 27 . 3 . THE REDUCED - SAMPLE ESTIMATE VS . THE PL 3 . 1 Alternatives to the PL estimate . The acceptable nonparametric estimation procedures known to the writers in the situation considered are variants of the PL and of the reduced - sample ( RS ) estimate defined by P * ( t ) = n ( t ) / N ? ( t ) = n ( t ) / [ n ( t ) + D ? ( t ) ( 3a ) Here N ? ( t ) = n ( t ) + D0 ( t ) is the number of items with observation limits > t ; of these , DO ( t ) die at ages < t and n ( t ) survive beyond t . A simple example of the RS estimate has been given in 1 . 3 . Berkson and Gage [ 3 ] have called it the " ad - hoc " method . If the lifetimes are independent of the observation limits , the reduced sample of N ? ( t ) items will be a random sample and P * ( t ) a simple binomial estimate , unbiased , and with the variance V [ P * ( t ) ] = P ( t ) [ 1 - P ( t ) ] / IN ? O ( t ) ( 3b ) It has already been noted that this is equal to or larger than the approximation ( 2h ) to the PL variance . As with the PL , the estimate . 00 - * = P * ( t ) dt ( 3c ) of the mean life may have to be truncated at the greatest of the observation limits . In Section 6 . 2 it is shown that the variance of , * is given exactly by V ( , * ) = 2 1 - P ( t ) - fP ( u ) dudt . In practice one substitutes P * ( t ) for P ( t ) in this formula and perhaps approxi - mates the integrals . Whereas the PL estimate has discontinuities only at observed ages of death , it is apparent from ( 3a ) that the RS generally has discontinuities at losses and observation limits also . Furthermore , suppose that one of the observation limits L is such that all ( or a sufficient number of ) the corresponding items are observed to die prior to L , while other items survive and are observed beyond L . Then the decrease in NO ( t ) ( without an equivalent decrease in n ( t ) ) will cause P * ( t ) to increase as t increases through L . Thus P * ( t ) , unlike P ( t ) and the true P ( t ) , is not necessarily monotonic decreasing . However , this appears to be a disadvantage only in a psychological sense . It does not seem advisable to avoid it , as one could , by a further reduction of the sample , basing the estimated P ( t ) for all t < a suitable L on the fixed sample of items having observation limits > L . The expression D ? ( t ) = N ? ( t ) - n ( t ) in ( 3a ) is not the total number D ( t ) of deaths observed prior to or at age t , but the ( usually smaller ) number of such deaths having observation limits > t . Since P * ( t ) is unbiased , the estimate ob - obtained by replacing D ? ( t ) by D ( t ) is too small on the average ( except in This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 470 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 special cases where D ( t ) - = DO ( t ) with probability one ) , as has been pointed out many times ( e . g . , [ 3 ] and [ 23 ] ) . Examples such as that of 1 . 3 show that the resulting bias can be very great , being in fact limited only by the necessity that the expectation be positive . Since DO ( t ) is often unknown while D ( t ) is known , an investigator who is unacquainted with the PL or one of its approxi - mations is all too likely to fall into the trap . Another insidious but instructive characteristic of the illegitimate estimate based on D ( t ) is the fact that it is simply the average of the PL estimates P ( t ) ( those that are defined at t ) obtained by regarding each item as a sample in its own right . For a sample of one item with observed lifetime ti , P ( t ) is unity for t < t < , and for t ? t is zero or undefined according as the observation ends with death or loss . This is also the RS estimate unless one insists that P * ( t ) is unde - fined for t > LI ( the observation limit of the item ) , even though it may have been estimated as zero for smaller values of t . The moral of this discussion is that arithmetic averages of independent PL estimates from very small samples will tend to be biased . On the other hand , the averaging of independent RS estimates is quite satisfactory , especially if the values of N0 ( t ) are used as weights . To avoid bias one must at least assign zero weight when N ? ( t ) = 0 , thus supplying each estimate with a limit of ob - servation ( the maximum of those attached to its items ) beyond which it is regarded as undefined , even though a value of zero may be indicated at some earlier age . To sumnmarize , the advantages of the PL over the RS are the following : ( a ) The observation limits need not be known for items observed to die . ( b ) The sampling variance of the PL is usually a little smaller . ( c ) The PL has fewer discontinuities , and its monotonicity may be comfort - ing . On the other hand , the RS has the following advantages over the PL : ( a ) It is perfectly unbiased , and hence estimates from many small samples can be combined by ( weighted ) averaging . ( b ) It facilitates the estimation of an isolated value of P ( t ) . ( c ) It may be preferred in some cases of dependence between lifetimes and observation limits ( see 3 . 2 below ) . 3 . 2 Dependence of deaths and losses . The assumption that the full lifetimes Ti are independent of the observation limits Li is sometimes violated , as a re - sult of a change in the population sampled , or the conditions leading to the event called death , or the method of sampling the population . For example , in a study of survival after an operation , a change in surgical technique five years before the data are analyzed will affect the survival times only of those with observation limit less than five years . When loss is due to unforeseen circum - stances , such as patients moving out of the state , the possibilities for depend - ence are obvious . Merrell [ 23 ] and Sartwell [ 24 ] have emphasized this point . The estimate of P ( t ) from the sample as a whole , whether PL or RS , involves arbitrary assumptions whose danger is peculiar to the dependent case and hence easily overlooked . The PL estimate in effect assumes that for items having an This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 471 observation limit L less than t , the conditional survival probability P ( t ) / P ( L ) is the same as that for items whose observation limits exceed t , while the RS estimate makes the same assumption concerning the absolute survival prob - ability P ( t ) itself . In many applications , the PL assumption may seem as plausible as any ; on the other hand , the fact that the results are expressed in terms of absolute probabilities may lead one to prefer the RS assumption ( when there is no feeling to the contrary ) , if only because its operation is more easily visualized . For example , the RS estimate P * ( t ) ( apart from sampling fluctuations ) can never fall outside the range of the true values for the popula - tions sampled , because it is a weighted average , whereas in unfavorable cases the PL estimate P ( t ) may fall outside this range . If prior observation limits are given for all the items and the sample is large enough , dependence may be inquired into by grouping the items according to these limits , making separate estimates of P ( t ) for each group , and comparing the results . If some unanticipated losses occur in advance of the a priori limits , the PL method can be used within each group . The group estimates can then be averaged with the initial numbers of items as weights to give something similar to the RS estimate , if that is desired . On the other hand , no dependence can be demonstrated if one ' s information is limited to the values of the ti = min ( Li , Ti ) and their classification as deaths or losses ; in this case the observed rates of death and loss can always be repre - sented by a model in which deaths and losses occur independently , as well as by many models in which this is not so . However , one usually has or can obtain other information that is more or less useful . What one would like is to obtain for each item the value of an auxiliary variable V ( which may be either quantitative or qualitative ) , such that the Ti and Li are more nearly independent within subsamples defined in terms of V , than they are in the sample or population as a whole . This will generally be true if V is strongly related to the cause of loss , and independent of the cause of death , or vice versa . In this case the subsamples are formed so as to reduce the variability of the Li ( or the Ti ) within them . Even if the auxiliary variable does not have the desired properties , it may be worthwhile to get an estimate of P ( t ) by a different route for the sake of comparison . Also , the auxiliary variable may be of interest for its own sake , for example , classification of the items by starting date would be aimed at detecting a temporal change in the population . As pointed out in 3 . 1 , the procedure in the general case is not satisfactory if the estimates depend on very few items . The innocuous form of dependence that results from testing with replace - ment is considered briefly in Section 7 . 4 . ACTUARIAL ESTIMATES 4 . 1 Estimates utsing only n , 6 , X . It has already been indicated ( in 2 . 1 ) that actuarial estimates , like the PL , are formed by multiplying together a sequence of estimates of conditional probabilities of survival through intervals ( 0 , uW ) , ( us , U2 ) , * - - . Unlike the PL , the actuarial estimates will generally be some - what dependent on the selection of these intervals . It remains to consider how a typical factor p of this sort can be estimated when the number of items n at the This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 472 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 beginning of the interval is known to be depleted by 6 deaths and X losses within the interval , but the order in which these occur is not known or used . The PL estimate is denoted by P . As in 2 . 1 , the estimates P ~ y P = - - ( 4a ) n n n - X would be used if all the deaths were known to precede all the losses , and if all losses preceded all deaths , respectively . Evidently p < p < p . If p is not known , another intermediate value is clearly supplied by the well - known " adjusted - observed " estimate ( cf . [ 3 ] , [ 12 ] , [ 18 ] , [ 22 ] ) n - X / 2 - ( 4b ) n - X / 2 This can be recommended for its simplicity even though n - X / 2 - n - / 2 - X n - a5 - X - ( 4c ) n - X / 2 n - a / 2 n HTere the second fraction is the corresponding estimate of the probability of not being lost ( if losses are also random events ) , and the third fraction is the ob - served probability of escaping both death and loss . The only other estimate in this section that fails to give equality here is p ( 4 ) below . The " joint risk " estimate ( cf . [ 6 ] and [ 12 ] ) is ~ n - - x - 6 / a + x ) p ( 2 ) = [ ( 4d ) n It is the maximum likelihood estimate when the losses are random , the instan - taneous event - rate for losses is a constant times that for deaths , and the only data given are the values of n , 3 , and X . The above estimates satisfy the rela - tion p < p ( 2 ) < p ( l ) < < p . One can obtain the PL estimate p from any of the foregoing by dividing the given interval into smaller intervals no one of which contains both deaths and losses . Since there are reasons ( full use of information , absence of arbitrary assumptions , essential uniqueness of the estimate ) for preferring small intervals to large , it seems reasonable to regard p as the standard to which the large - interval actuarial methods are approximations . The error incurred in an actuarial estimate of p can be attributed to two sources , namely , the sampling error of f , reflecting the fact that a is a random variable , and the discrepancy between p ( l ) and P , reflecting the unknown ar - rangement of deaths and losses within the interval . These sources of error will be compared by calculating the variance of log p in each case , on the assumption that n is large compared with a and X , and that factors of the form 1 + 0 ( n - 2 ) can be neglected . On this basis the effect of variation in 3 is indicated sufficiently by the square of the coefficient of variation of p ( % ) , namely / ( n - X / 2 ) ( n - - X / 2 ) 45 ( 2n - - _ ) - 2 ( 4e ) This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 473 To evaluate the second source of error we note first that the total range of possible variation in log P ( given 3 and X ) is by ( 4a ) log - log p _ 45X ( 2n - a - ( 4f ) However , if the arrangement is random , the extreme values may be unlikely . Therefore it will now be assumed that all permutations of the 3 deaths and X losses are equally likely ( which happens to be a consequence of the proportional event - rates leading to ( 4d ) ) . In this case log p is minus the sum of a random sample of 3 of the 3 + X numbers log ( 1 + - ) ~ , log ( 1 + - ) log ( 1 + - Sampling theory and the Euler - Maclaurin theorem lead to the approximate variance 45X ( 6 + X + 1 ) / 3 ( 2n - 5 - X ) 4 . ( 4g ) Dividing the sampling variance ( 4e ) into the square of half the range ( 4f ) of the grouping error gives BX2 ( 2n - 5 - ) - 2 ( 4h ) which may be either > 1 or < 1 . Dividing ( 4e ) into the grouping variance ( 4g ) gives X ( 5 + X + 1 ) / 3 ( 2n - a _ - X ) 2 , ( 4i ) which is always < 1 ( when n > 0 ) . The above results suggest that rather large intervals could be used if enough was known about the mechanisms of death and loss , and that the principal source of error is the probable failure of the various permutations of deaths and losses within the interval to be equally likely , or to fit any other scheme that may be assumed . Evidence on this point could be obtained by examining the permutations that actually occur . For example , this is what is done in using ( 2a ) . Otherwise one should probably require at the least that ( 4h ) be suitably small . This still assumes that grouping errors , like the sampling errors , are inde - pendent from interval to interval . If not , one is thrown back on the ultimate in conservatism , which is to assert only that f1pj < P < fljPj . 4 . 2 Estimates using average ages . Several estimates will now be considered that make use of the individual ages of death and loss , although arbitrary age intervals continue to be used . The individual ages enter via the average age A of the a deaths , and the average age A of the X losses , both measured from the be - ginning of the age interval under consideration , which is of length h . The first estimate assumes nothing about the losses , but assumes that the instantaneous death - rate is constant throughout the interval . The total ex - posure to the risk of death in the interval is then ( n - 3S - X ) h + 3A + XA , the maxi - mum - likelihood estimate of the death - rate is 3 / [ ( n - 3 - X ) h + 3A + + XA ] , and the corresponding estimate of the survival probability p is p ( 3 ) = exp ( - 3 / [ n - 3 - X + ( SA + XA ) / h ] ) ( 4j ) This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 474 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 By using p ( 8 ) , Harris , Meier , and Tukey [ 181 have been able to give an estima - tion procedure for the more general and more difficult case in which the age of death is never known exactly , but only known to fall in some interval , semi - infinite or finite , bounded by one or two observation points which vary from item to item . The PL estimate P can be derived from p ( 3 ) as well as from p ( l ) or p ( 2 ) . Al - though the limit of the product of the estimates p ( 3 ) for the subintervals is not quite unique , the average value of log p ( 3 ) corresponding to 8 deaths occurring at the same age is du n - . Jon - 6 ( l - u ) log Y ( 4k ) JO n - 6 ( t - u ) n in agreement with p - ( n - 6 ) / n . Here A ( = uh ) has been regarded as uniformly distributed between 0 and h . However , in other respects the behavior of p ( 3 ) does not parallel that of P . If the position of one or more of the deaths is shifted toward the end of the interval , p ( ' ) is increased , while P is decreased ( or else not changed ) . Again , p ( 3 ) does not necessarily agree with P in the simple case of no losses . Finally , the extreme values of p ( 3 ) , obtained by putting A = A = 0 or h , fall outside the interval p , p within which P is confined ; in fact one can have p ( 3 ) < p while P = p , or p ( 3 ) > 7p while p = p . The following estimates have been constructed with the object of using the mean ages A and A to simulate the behavior of p : p ( 4 ) = ( n - cX - 6 ) / ( n - cX ) , ( 41 ) n - ' X _ c _ n - 6 1 - c p ( 6 ) = [ l X ( 4m ) where c = 1 / 2 + ( A - A ) / h for iA - Al < h / 2 , ( 4n ) = 0 for A - A < - h / 2 , = 1 for A - A > h / 2 . The value of c is immaterial when 6 = 0 or X = 0 . These estimates are two different averages of the bounding estimates p and p , taken with weights c and 1 - c respectively . The quantity c is so defined that the appropriate one of the bounding estimates is assigned as soon as I A - - AI > h / 2 . This is intuitively reasonable , and is justified analytically by the fact that log p ( 4 ) and log p ( 5 ) have essentially the variance ( 4g ) of log P , if each death and each loss is uniformly and independently distributed in the interval . To estimate this variance , one can multiply var c var [ ( A - A ) / h ] = ( + X ) / 126X by the square of the range ( 4f ) , since ( 4f ) gives the change in log p ( 4 ) and log p ( 5 ) corresponding to a unit change in c . Experience may indicate which estimate is preferable in a given type of situation . If relevant experience is lacking , it would seem advisable to rely This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 475 primarily on adequately small intervals , and to minimize the computation time by using simple estimates . This suggests that p ( i ) be used when the deaths and losses are thought to be arranged at random within the interval , p when the interval is chosen so that most of the deaths precede most of the losses , and p when losses precede deaths . This completes the material that seems to be most important for applica - tions . The remaining three sections are devoted primarily to mathematical derivations . 5 . MAXIMUM LIKELIHOOD DERIVATION OF THE PL In accordance with 1 . 2 the method of maximum likelihood is viewed as a means for selecting the best fitting distribution from a class of admissible dis - tributions . It will now be shown that if all possible distributions are admitted , the product - limit is the one for which the sample likelihood is a maximum . Let T1 < T2 < * , < Tk denote the distinct ages of death , with 3 , deaths observed at Tj . Let Xj denote the number of losses in the interval [ T , , Tj + i ) , including any losses at Tj but none at Tj + , . ( Here j = 0 , 1 , * , Ik ; To = 0 ; Tk + I = oo . ) The ages of the Xj losses are denoted by LiW , i = 1 , 2 , * * , Xj . Only in this section , P ( t ) will represent not the " true " survivorship but rather an arbitrary one that is to be determined so as to maximize the likelihood . The likelihood may now be written L II P ( Li ( O ) ) i = O 1x \ 1 [ P ( T1 - 0 ) - P ( Ti ) ] 61I P ( LL ( 1 ) ) ( 5a ) . . . . . . . . . . . . . . * * * * * * * * * * * * [ P ( Tk - 0 ) - P ( Tk ) ] iLk PLL P ( k ) ) J . Here P ( Tj - O ) includes , whereas P ( Tj ) excludes , the probability of death at age Tj exactly . Clearly ( 5a ) will be maximized by making the P ( Li ( O ) ) and the P ( Tj - 0 ) as large , and the P ( Tj ) as small , as is consistent with the monotonicity of the function and the assumption Tj < Liti < Tj + , . This means that P ( L ? ( 0 ) ) = 1 and P ( Tj ) = P ( L ( i ) ) = P ( Tjl - 0 ) ( 5b ) for all i and j . If the common value is denoted by Pj , ( 5a ) reduces to k II ( Pjl - Pj ) OiPj i with Po = 1 . ( 5c ) j = l Now writing pj = 1 - j = Pj / Pj - i gives P = PlP2 . * * , pj and Pj , l - = PI . . . pjilqj . With these substitutions ( 5c ) becomes J II pjXs - asq8J ~ ~ ~ ~ ( 5d ) This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 476 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 where i = - = i - 1 i = k nj - EXE = N - = ( Xi + ba ) , or n ( Tj - O ) i = O i = j in the notation of 1 . 4 . In this form each factor with a fixed index j is maximized individually by the binomial estimate p = pj = ( ni - 51 ) / n ; , in agreement with the PL in ( 2a ) . If Xi , > 0 , so that the greatest observed lifetime t * corresponds to a loss , then P ( t * ) > 0 and the likelihood of the sample is independent of the values of P ( t ) for t > t * . Thus P ( t ) and the maximum likelihood estimate are indeterminate under the same circumstances , and the identity between them is complete . 6 . MEANS AND VARIANCES In this section the mean of P ( t ) and the variances of P ( t ) and of the mean - life estimates , u and * are derived . The observation limits are regarded as fixed from sample to sample . In principle , unconditional results for random loss times could be obtained by integration . 6 . 1 The PL estimate P ( t ) . Let L1 < L2 < . . . < Lk - be the distinct observa - tion limits that are less than the age t = Lk at which P ( t ) is being estimated . Let nj + , = n ( Lj + O ) be the number of survivors ' observed beyond Li ; Nj + ' = Enj + l - N0 ( L1 + O ) P ( Lj ) ; pj = 1 - qj = P ( Lj ) / P ( Lj _ 1 ) ; and 5j the number of deaths observed in the interval ( Lj - , , Lj ] , excluding nonzero values of the left end - point } , with Lo = O . Then P ( t ) P = I pj with Pj = ( nj - Sj ) / nj . ( 6a ) j - = l Let Ej denote a conditional expectation for ni , , nj ( and hence also 811 * I * , Aj - ) fixed . Then E1jf = pi , provided that n , > 0 . If the condition n , > O could be ignored , one could write EP ( t ) = E [ tl [ . . . Pk - lEkpk ] = E [ pli Pk - lpk ] = pkE [ pl * fPk - 2Ekfik - 1 ] ( 6b ) = . . . = Pkpk - _ * pI - P ( t ) , so that fi ( t ) would be an unbiased estimate . The flaw in this demonstration is the indeterminacy resulting when ( for some j ) nj + l = 0 but P ( Lj ) > 0 , an event whose probability is bounded by ( 2d ) , and which could be obviated in principle by obtaining one or more supplementary observations . In the derivations of approximate formulas that follow , any bias that P ( t ) may have is neglected . Both Greenwood [ 15 ] and Irwin [ 19 ] when giving formulas for the variance of an actuarial estimate of P ( t ) actually treat a special case in which the ac - tuarial estimator and P ( t ) coincide . Translated into our notation their com - mon argument is the following . Suppose we consider conditional variances for nj held fixed at the values Nj . I The expression Li + 0 means that losses at Li itself have been subtracted off . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 477 Then the Pi may be treated as independent quantities and E [ P2 ( t ) ] = J ( j2 + _ = p2 . . . p2 ] 1 + ) N ~ ~ ~ i N , p ~ ~ ~ ~ ~ ~ i ( = P2 ( t ) ( i + q ( ) If we ignore terms of order Nj - 2 we have as an approximation for the variance v [ P ( t ) ] P2 ( t ) I N p ( 6d ) J = l _ _ Njpj This last expression we will refer to as " Greenwood ' s formula . " The calculation of the variance with nli , * * * , nA fixed can apparently be justified only on the ground that it doesn ' t matter a great deal ; the fixed ny are unrealistic , and in - consistent with the previous assumption of a sample of fixed size and fixed limits of observation . The authors have therefore rederived ( 6d ) by means of successive conditional expectations . The procedure is to verify by induction the approximate relation E1 [ P2 ( t ) ] _ 2 ( l + 2 pj + 12 . . pk ( Ge ) i = j + l pEJ + ni which reduces to ( 6d ) when j 0 . Applying Ej to ( 6e ) has the effect of replacing j by j - 1 provided that one sets E ) Ej + ini jp EjEZj + lni ( 6f ) pj2 / Ejn , . wherein Pj / Ej + lni has been replaced by the ratio of the expectations of numer - ator and denominator . The fact that pj and Ej + lni are positively correlated im - proves the approximation . In the special case in which k = 2 ( all observation limits less than t are equal to L1 ) and any indeterminacy is resolved by one supplementary observation , the exact variance of } P ( t ) can be calculated to be V [ P ( t ) ] = P2 ( t ) - + zqq2 ( N ' 2 N ' pi - [ N ' , P ] Np ` N ' PuL N , P ] ( 6g ) + 1 2 ( [ i ' ] N2 ' ) ] ) , where N is the total sample size ; N ' - N ? ( t ) , the number of items observable to t or beyond ; and [ N ' , P ] = 1 / E ( X - 1 ) ( 6h ) with X = max ( Y , 1 ) and Y distributed binomially ( N ' , pi ) . The methods used by Stephan [ 27 ] show that [ N ' , pl ] = N ' pu + 0 ( 1 ) for N ' large . Thus the term This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 478 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1968 in square brackets is of order 1 + 0 ( 1 / N ' ) , and ( 6g ) approaches Greenwood ' s formula ( 6d ) for large N ' . 6 . 2 Covariances and mean lifetimes . The method of Irwin [ 19 ] can be used to derive the variance of - = f0 P ( t ) dt by writing 2 ) = Ef P ( u ) P ( v ) dvdu ( 6i ) = 2 f f E [ fi ( u ) P ( v ) ] dvdu . If u < v and Lh and Lk denote u and v respectively , then apart from bias due to indeterminate cases one has E [ fi ( u ) P ( v ) ] = B [ P2 ( u ) Eh + i ( ph + lph + 2 Pk ) k - Ph + lph + 2 . . . pkE [ P2 ( U ) j ( 6j ) - [ P ( v ) / P ( u ) ] E [ P2 ( u ) ] P ( u ) P ( v ) [ 1 + U ( u ) ] where by ( 2h ) and ( 2g ) toN ( t ) P ( t ) [ ( - ) Nr + 1 ] ; the summation is over deaths at ages not exceeding u , and gives the sample estimate . Thus one has approximately V ( ) - 2 P ff ( P ( v ) U ( u ) dvdu ou 0 ( 6k ) = 2f A ( u ) P ( u ) U ( u ) du = A2 ( u ) dU ( u ) , where A ( u ) = fu ? ? P ( v ) dv . The result is discussed in 2 . 3 . To obtain analogous ( but in this case exact ) results for the RS estimates P * ( t ) and , * , let u < v as before , and split off two independent subsamples con - sisting of the N ' = N ? ( v ) items observable to v or beyond , and the N " = N ? ( u ) - N ? ( v ) additional items observable to u or beyond , but not to v . Let the first subsample yield 6 ' deaths in ( 0 , u ) and e ' in ( u , v ) , while the second yields 8 " deaths in ( 0 , u ) . Also let Pi = P ( u ) and P2 = P ( v ) . Then E [ P * ( u ) P * ( v ) ] ( with u < v ) = E [ ( N ' + N " - 6 ' - " ) ( N ' ' - 6 ' ) ] / N ' ( N ' + N " ) = E [ ( N ' + N " - 6 ' - s " ) ( N ' - 6 ' ) ] P2 / P1N ' ( zV ' + N " ) 61 = [ E ( N ' - a ' ) 2 + E ( N " - 6 " ) . E ( N ' - ' ) ] P2 / P1N ' ( N ' + N " ) = [ N 2P12 + N ' Pi ( l - P1 ) + N ' N " P12 ] P2 / P1N ' ( N ' + N " ) = P ( u ) P ( v ) + P ( v ) [ 1 - P ( u ) ] / N ? ( u ) . Substituting this result in the analogue of ( 6i ) and subtracting / A2 gives V ( A * ) as in ( 3d ) . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 479 7 . CONSISTENCY ; TESTING WITH REPLACEMENT The RS estimate P * ( t ) , being binomial , is of course consistent . In view of the approximate nature of the formula for the variance of P ( t ) , however , a proof of its consistency is in order . In fact , by giving closer attention to the errors of the approximations in the variance derivation , it can be shown that P ( t ) does have the limit P ( t ) in probability provided only that NO ( t ) - - c . However , the proof is too lengthy to be given here . Of course , the consistency is obvious enough in the special case in which the number k of conditional probabilities to be estimated remains bounded as NO ( t ) - > c . Thus far the independence of deaths and losses has been assumed . The question of consistency may also be raised in the differing context of " testing with replacement . " ( Cf . [ 7 ] , [ 10 ] , [ 11 ] , [ 14 ] . ) This is the common life - testing situation in which a fixed number of items , say v of them , are always under test ; when one dies it is replaced by another . The only preassigned constants are the duration of the test , S , and the number , i , of items on test at all times . The number of losses is then fixed at v , one for each item on test at the conclu - sion of the experiment , while the number of deaths observed , and hence the total number of items , is a random variable . The first v items on test have S as their limit of observation ; if one of these dies at age t , with t < S , its successor has S - t as its limit of observation , and so on . Consistency will mean an ap - proach to P ( t ) as vS - > co , which implies that either v - * > co or cc > co ( or both ) . The RS estimate will be considered first . Let T1 , T2 , - - * , be an infinite se - quence of independent lifetimes with survivorship P ( t ) and let Pk ( t ) be the proportion of T1 , * * * , Tk that exceed t . By the strong law of large numbers , lim Pi , ( t ) = P ( t ) with probability one as k - ? cc , for any specified t . Now the RS estimate P * ( t ) ( which exists only for values of t in the interval 0 , S ) can be represented as a value of Pk ( t ) , with k a random variable . This can be done by assigning the lifetimes T1 . * , T , to the v items initially tested . If some of these items have replacements whQse limits of observation S - Ti ( i = 1 * * * , v ) are not less than t , then succeeding lifetimes T , ? 1 , T , + 2 , * * , are assigned to these replacements . The process is repeated until no more observation intervals of at least the duration t are left . To prove P * ( t ) consistent , it only remains to prove that for any M , Pr ( k > M ) - * 1 as vS - * > X . If v - > oo the result is evident . If S - ? c , then even for v = 1 , Pr ( k > M ) = Pr ( Ti + * ? TM ? S - t ) 2 Pr [ T7 < ( S - t ) / M for i 1 , . * ' MJ ( 7a ) = { 1 - P [ ( S - t ) / M ] } M . As S - + co the last expression has the limit unity and consistency is proved , pro - vided that P ( oo ) 0 ( eventual death is certain ) . When S - ? cc , v is fixed , and P ( oc ) = 0 , the consistency of the PL estimate P ( t ) follows from the relation ( cf . ( 4a ) ) n ( t ) < n ( t ) + v n ( t ) + v NO ( t ) + D ' NO ( t ) + D ' + v N This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions 480 AMERICAN STATISTICAL ASSOCIATION JOURNAL , JUNE 1958 since the number of losses is v , P * ( t ) = n ( t ) / IN ( t ) is consistent , NO ( t ) - - oo , and the number of deaths D ' having observation limits less than t is a random variable that does not depend significantly on S . P ( t ) is undoubtedly consistent also when S is fixed and v - + oo , but a rigorous proof of this has not been con - structed . ACKNOWLEDGMENT The viewpoint adopted in this paper owes much to discussions with John W . Tukey . It also incorporates a number of suggestions kindly forwarded by readers of a preliminary draft . REFERENCES [ 11 Bailey , W . G . , and Haycocks , H . W . , " A synthesis of methods of deriving measures of decrement from observed data , " Journal of the Institute of Actuaries , 73 ( 1947 ) , 179 - 212 . [ 2 ] Bartlett , M . S . , " On the statistical estimation of mean lifetime , " Philosophical Maga - zine ( Series 7 ) , 44 ( 1953 ) , 249 - 62 . [ 31 Berkson , J . , and Gage , R . P . , " Calculation of survival rates for cancer , " Proceedings of the Staff Meetings of the Mayo Clinic , 25 ( 1950 ) , 270 - 86 . [ 4 ] Berkson , J . , and Gage , R . P . , " Survival curve for cancer patients following treat - ment , " Journal of the American Statistical Association , 47 ( 1952 ) , 501 - 15 . [ 5 ] Berkson , J . , " Estimation of the interval rate in actuarial calculations : a critique of the person - years concept , " ( Summary ) Journal of the American Statistical Association 49 ( 1954 ) , 363 . [ 6 ] B6hmer , P . E . , " Theorie der unabhangigen Wahrscheinlichkeiten , " Rapports , M6moires et Proces - verbaux de Septi6me Congr6s International d ' Actuaires , Amster - dam , 2 ( 1912 ) , 327 - 43 . [ 7 ] Brown , G . W . and Flood , M . M . , " Tumbler mortality , " Journal of the American Sta - tistical Association , 42 ( 1947 ) , 562 - 74 . [ 8 ] Cornfield , J . , " Cancer illness among residents in Atlanta , Georgia , " Public Health Service Publication No . 13 , Cancer Morbidity Series No . 1 , 1950 . [ 9 ] Davis , D . J . , " An analysis of some failure data , " Journal of the American Statistical Association 47 ( 1952 ) , 113 - 150 . [ 10 ] Epstein , Benjamin , and Sobel , Milton , " Lifetesting , " Journal of the American Statisti - cal Association , 48 ( 1953 ) , 486 - 502 . [ 11 ] Epstein , Benjamin , and Sobel , Milton , " Some theorems relevant to lifetesting from an exponential distribution , " Annals of Mathematical Statistics , 25 ( 1954 ) , 373 - 81 . [ 12 ] Fix , Evelyn , " Practical implications of certain stochastic models on different methods of follow - up studies , " paper presented at 1951 annual meeting of the Western Branch , American Public Health Association , November 1 , 1951 . [ 13 ] Fix , Evelyn , and Neyman , J . , " A simple stochastic model of recovery , relapse , death and loss of patients , " Human Biology , 23 ( 1951 ) , 205 - 41 . [ 14 ] Goodman , L . A . , " Methods of measuring useful life of equipment under operational conditions , " Journal of the American Statistical Association , 48 ( 1953 ) 503 - 30 . [ 15 ] Greenwood , Major , " The natural duration of cancer , " Reports on Public Health and Medical Subjects , No . 33 ( 1926 ) , His Majesty ' s Stationery Office . [ 16 ] Greville , T . N . E . , " Mortality tables analyzed by cause of death , " The Record , American Institute of Actuaries , 37 ( 1948 ) , 283 - 94 . [ 17 ] Hald , A . , " Maximum likelihood estimation of the parameters of a normal distribution which is truncated at a known point , " Skandinavisk Aktuarietidskrift , 32 ( 1949 ) 119 - 34 . [ 18 ] Harris , T . E . , Meier , P . , and Tukey , J . W . , " Timing of the distribution of events be - tween observations , " Human Biology , 22 ( 1950 ) , 249 - 70 . [ 19 ] Irwin , J . O . , " The standard error of an estimate of expectational life , " Journal of Hygiene , 47 ( 1949 ) , 188 - 9 . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions NONPARAMETRIC ESTIMATION 481 [ 20 ] Jablon , Seymour , " Testing of survival rates as computed from life tables , " unpub - lished memorandum , June 14 , 1951 . [ 21 ] Kahn , H . A . , and Mantel , Nathan , " Variance of estimated proportions withdrawing in single decrement follow - up studies when cases are lost from observation , " unpub - lished manuscript . [ 22 ] Littell , A . S . , " Estimation of the T - year survival rate from follow - up studies over a limited period of time , " Human Biology , 24 ( 1952 ) , 87 - 116 . [ 23 ] Merrell , Margaret , " Time - specific life tables contrasted with observed survivorship , " Biometrics , 3 ( 1947 ) , 129 - 36 . [ 24 ] Sartwell , P . E . , and Merrell , M . , ' Influence of the dynamic character of chronic dis - ease on the interpretation of morbidity rates , " American Journal of Public Health , 42 ( 1952 ) , 579 - 84 . [ 25 ] Savage , I . R . , " Bibliography of nonparametric statistics and related topics , " Journal of the American Statistical Association , 48 ( 1953 ) , 844 - 906 . [ 26 ] Seal , H . L . , " The estimation of mortality and other decremental probabilities , " Skandinavissk Aktuarietidskrift , 37 ( 1954 ) , 137 - 62 . [ 27 ] Stephan , F . F . , " The expected value and variance of the reciprocal and other negative powers of a positive Bernoullian variate , " Annals of Mathematical Statistics , 16 ( 1945 ) , 50 - 61 . [ 28 ] Wolfowitz , J . , " Additive partition functions and a class of statistical hypotheses , " Annals of Mathematical Statistics , 13 ( 1942 ) , 247 - 79 . This content downloaded on Tue , 29 Jan 2013 10 : 47 : 01 AM All use subject to JSTOR Terms and Conditions \ No newline at end of file diff --git a/silver_data/7f73ad2ece14e254c745256a77736e9b20170151.pdf.txt b/silver_data/7f73ad2ece14e254c745256a77736e9b20170151.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..0e92dbdeb1c8b3e2bbc43ab963dc57e8811e3dd0 --- /dev/null +++ b/silver_data/7f73ad2ece14e254c745256a77736e9b20170151.pdf.txt @@ -0,0 +1 @@ +Inspiration Card Workshops Kim Halskov Institute of Information and Media Studies University of Aarhus Helsingforsgade 14 DK 8200 Aarhus N halskov @ cavi . dk + 45 89 42 92 41 Peter Dalsgård Institute of Information and Media Studies University of Aarhus Helsingforsgade 14 DK 8200 Aarhus N dalsgaard @ cavi . dk + 45 89 42 92 86 ABSTRACT In this paper we start from the position that sources of inspiration play an important role in the design process albeit in a frequently intangible way . We present the Inspiration Card Workshop as a collaborative method for combining findings from domain studies , represented in Domain Cards , with sources of inspiration from applications of technology , represented in Technology Cards , to create new concepts for design . We report our findings from three projects in which we have used the method and argue that the use of Inspiration Cards can successfully frame and guide workshops with disparate participants and bring various sources of inspiration into the design process . We furthermore compare the method to four related methods in the design process , namely Future Workshops , Metaphorical Design , Interaction Relabelling and Lateral Thinking . Keywords Design , workshop , inspiration , innovation . ACM Classification Keywords H5 . 2 . Information interfaces and presentation ( e . g . , HCI ) : User - centered design . INTRODUCTION Ehn [ 12 ] identified the balance between tradition and transcendence as one of the most important dilemmas in design . On the one hand , when we design , we have to take current qualifications , work organization , and work activities as points of departure ; on the other hand we also want to design something which is innovative , and which can support new activities , or support current activities in new and better ways . A variety of design techniques and approaches which address the tradition - aspect are at our disposal , including ethnographic field studies [ 3 ] , interview [ 23 ] , use of video [ 5 ] , etc . Moreover , a vast collection of techniques address the transcendence - aspect , but are ( as the term transcendence suggests ) rooted in the existing tradition or work practices , including the use of scenarios [ 8 ] , mock - ups [ 13 ] and prototyping [ 7 ] . Additionally , there are a number of design techniques that specifically support innovation , for example Future Workshops [ 18 ] , use of metaphors [ 22 ] , and interaction relabelling [ 10 ] . In this article we zero in on what we consider to be two of the important elements in innovative processes : 1 ) design materials , in our case , index cards ; 2 ) sources of inspiration . The paper is organized as follows . First , we briefly review related work , and use this as a platform for introducing the specific format that we are proposing : The Inspiration Card Workshop . In the following sections we introduce and analyze our use of the Inspiration Card Workshop in three design cases we recently conducted . In the next section we compare our approach to four other approaches to innovation in design . BACKGROUND According to Schön [ 27 , 28 ] , design is a reflective interaction ( or in his terminology “conversation” ) with materials , wherein the designer works with different media or materials , experimenting with various aspects of the design . In the case of information systems , a diverse set of design materials is being used , including video , paper documents , mock - ups , prototypes and posters . Moreover , small paper documents are commonly used as an integrated part of various design techniques . One category of small paper documents is the Post - it® , for instance used when making affinity diagrams [ 2 ] . A different kind of small paper documents are cards with pictures or text representing other kinds of design materials . In one instance of this category , Buur and Søndergaard [ 6 ] have been using what they call ‘video cards’ , with still images of video segments , and space for annotations , to be used as part of collaborative video analysis . In their approach , Buur and Søndergaard found inspiration in the work of Tuder , Muller & Dayton [ 29 ] , who have used cards to turn ideas into tangible objects . The video card game is a precursor for the use of cards in a similar way , as part of a design workshop where virtual video prototypes have been used [ 1 ] . Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . To copy otherwise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . DIS 2006 , June 26 – 28 , 2006 , University Park , Pennsylvania , USA . Copyright 2006 ACM 1 - 59593 - 341 - 7 / 06 / 0006 . . . $ 5 . 00 . Brandt and Messerter [ 4 ] have been using various kinds of cards in four different types of workshops . In addition to using cards to make video clips tangible , they have made cards with single words ( so called ‘sign - cards’ ) , which constitute a conceptual framework for the activities of the design process . In a technology game they have used LEGO - Duplo bricks with generic functions - such as ‘transfer documents’ - written on them , taking advantage of their tangibility , and the ease with which the bricks connect to one another . From the use of such tangible objects as components of design games , Brandt and Messerter [ 4 ] have made the observation that game pieces , including the various kinds of cards , ‘support different stakeholders in making design moves on a conceptual level’ [ ibid p . 129 ] , and that such design artifacts have become an intrinsic part of the dialogue , argumentation , and means of expressing design moves . Additionally , it seems evident that the objects at hand help focus the design activities . Additionally , according to Schön [ 27 ] , rather than looking for standard solutions , the designer sees the situation as something already present in his / her repertoire of paradigm cases or prototypes , despite which he / she manages to make something new by making experimental moves , which may result in something which goes beyond his / her initial expectations . One of the renowned examples from Schön’s [ 26 ] work is the story of how a group of product developers invented a new kind of paint brush , by thinking of the paint brush as a pump . In the area of information systems design , Madsen [ 22 ] has explored how metaphors may shed new light on the way in which information technology might be used by seeing a domain of applications as something different , e . g . seeing a library as a meeting place . In a later study based on three cases in which digital artists and designers worked together , Lervig and Madsen [ 21 ] addressed the way in which design materials serve both as examples pinpointing specific attributes , and as sources of inspiration that serve as jumping - off points for work in a design project . Sanders [ 25 ] has argued that inspiration plays a prominent role in experience design and points out what she sees as a clash between an information oriented approach and an inspiration oriented approach . One particular source of inspiration ( in a meta sense ) for the ideas presented in this article is the Tech Box , as reported by T . Kelley [ 19 ] in his book about innovation and creative processes at IDEO . The Tech Box [ ibid . p . 144f ] is a centrally located file cabinet filled with gadgets and materials , such as tiny switches , Aerogel , Kevlar , rubber balls that don’t bounce , super heat conducting copper heat pipes , and the like . People look into the Tech Box for inspiration , then use it for launching new projects , and for selecting items to bring to design meetings to spark innovation , etc . Conversely , people contribute their objects , which become part of the Tech Box [ ibid 144ff ] . An essential concept relevant to successful innovation processes is the concept of cross - pollination which is , in essence , the idea of bringing together hitherto unrelated elements . Consciously looking for inspiration is part of the innovation strategy discussed by Kelly [ 19 p 280 ] : “Take a trip to Akihabara , the blinking electronic hub of Tokyo” or “Looking for the future of athletics apparel ? Head to the beach . Venice Beach , that is . ” J . Foster [ 15 ] is even more radical , suggesting , in his book on generating ideas , that you do things to which you are unaccustomed , for instance : “Study Latin” , “Read a magazine that you’ve never heard of” [ ibid . p 72 ] , “Take up water - colour painting” [ ibid . 72 ] , etc . The point is not to do all these , but to do something different [ ibid p . 73 ] . An essential point made by Foster [ 15 p69 ] is that if generating new ideas primarily consists of combining old elements , a thorough familiarity with old elements is essential . CONCEPT : INSPIRATION CARDS An Inspiration Card is a 2” by 3” cardboard card on which an image , a title , a description , and a reference is printed . The card also has an empty box for comments . We work with two broad categories of inspiraration cards , Technology Cards and Domain Cards . Figure 1 : A Technology Card A Technology Card represents either a specific technology ( i . e . Motion Capture ) or an application of one or more technologies ( i . e . The I / O Brush [ 24 ] ) . For Inspiration Cards to be comprehensible , the content needs to succinctly exemplify one clear concept . As an example , the card in Figure 1 is a Technology Card representing a specific application of a technology ( in this case , one of our experiments with camera tracking combined with a video stream ) . It has a title for easy reference ( “The All - Seeing Eye” ) followed by a short description . It also contains a reference to further information on the technology described . Technology Cards are typically created by designers . They may be related to a specific design project , but they can often be reused in various other projects . We use Technology Cards as a standard format for storing information on interesting technologies that we have encountered , whether they are of our own design , or that of other designers . We have thus created a repository of Technology Cards for ongoing use in our design projects . Technology Cards can often be reused in other projects and the ones we produced are created from a pool of resources available at http : / / www . digitalexperience . dk . Figure 2 : A Domain Card Domain Cards represent information on the domains for which we design . This information may pertain to situations , people , settings , themes etc . from the domain . As is the case with Technology Cards , these work best if the concept represented is unequivocal . Figure 2 is an example of a Domain Card from a project on designing interactive exhibits related to Norse mythology . The card represents “Blood” , a recurring trope in this domain . The Domain Cards can be created both by designers , usually as a condensation of field studies and research , and by domain experts who participate in the design process . Domain Cards are typically only meaningful within the specific project for which they were created , and reuse is limited . We work with just two categories of cards due to considerations of simplicity , since the Technology and Domain Cards represent the two main areas that converge in the design process . Designers seeking to appropriate this method may wish create further categories and subsets , eg . the domain cards could be divided into People Cards , Situation Cards etc . The Inspiration Cards can be used in a number of ways : as a standard for collecting and consistently representing sources of inspiration , as a way to gain an overview of various concepts , as means of communication between designers and domain experts , etc . In the following section , we expand on a particular application of the cards , namely the Inspiration Card Workshop . METHOD : INSPIRATION CARD WORKSHOPS In an Inspiration Card Workshop , participants create design concepts by combining Technology and domain Cards . This design method is primarily used in the early stages of a design process , during which designers and their collaborators narrow down potential future designs . The method is loosely structured , informal , and has a simple set of rules . Participants The method is participatory , and usually involves designers as well as participants with knowledge of the design domain . The participants may be users or stakeholders from the domain , or have certain areas of expertise otherwise related to the domain . The method has proved most fruitful with 4 - 6 participants . In cases involving more participants , the preparation and presentation stages of the workshop can be conducted in common , with the participants splitting into groups for the combination and co - creation stages . Preparation The preparation for the workshop primarily lies in selecting and generating the Inspiration Cards . Technology Cards , primarily generated by the designers , represent technologies that may directly or indirectly be part of the design concepts . The Domain Cards may be generated by the designers based on studies of the domain , however it often makes for more involving workshops and rewarding outcomes if the participants take part in creating them . There should be multiple copies of each card , as well as a number of blank cards to be filled out at the discretion of participants . At a later point , we expand on a number of issues to consider when selecting Technology and Domain Cards . Presentation of Inspiration Cards The workshop commences with a presentation of the Technology and Domain Cards selected . Each card is presented in turn , often with the help of images or video clips , to ensure a shared understanding . In general , this takes 1 - 3 minutes per card . Designers usually present the Technology Cards and the domain participants the Domain Cards . Combination and co - creation The main phase of the workshop consists of the participants collaboratively combining the cards on posters , in order to capture design concepts . This phase is often initiated by a discussion in which the participants establish a shared understanding of the cards . There are no set rules for turn - taking , and cards may be combined in the way the participants deem productive . Participants can start by selecting themes or situations from the domain that they wish to support , or transform and then select Technology Cards as a means to this end . Although a rarer occurence , they may also take intriguing technologies as their starting points , then look for situations to which they may be applied . Any number of cards may be combined to create a design concept . The cards are affixed to poster - sized pieces of cardboard . Participants are encouraged to write descriptions and brief scenarios on the posters , for further detail . Figure 3 : Combination and co - creation The main point of the Inspiration Cards is to inspire this creative process , and as such , the cards may be used both directly ( i . e . “This specific technology may alleviate that specific problem in the domain” ) and indirectly ( i . e . “This application of technology embodies a style that we wish to reproduce in the domain” ) . To better support creativity , criticisms of design concepts are better left for later stages . Interruptions and complementary ideas are welcome in this phase , and the resulting concepts are seldom the work of a single creator , but rather a collective effort . Presentation of posters and design concepts Figure 4 : A poster with cards combined to generate and capture a design concept . After the combination and co - creation phase , the participants take a short break to step back and reflect on the resulting design concepts . In the case of a single group of participants , each poster is discussed in plenum . In the case of several groups concurrently combining and creating posters , each group presents its design concepts . The object of this phase is to ensure a common understanding of the concepts , rather than to evaluate them in terms of whether they are appropriate or realistic . Inspiration Cards in the design process The main benefit of conducting Inspiration Card Workshops is the generation of new design concepts based on domain and technology studies . The workshop sessions described in this paper were carried out in the early stages of the design process after initial field studies , but prior to mock - up sessions , prototyping and development of final products . The over - all process for the three cases is illustrated in Figure 5 : The findings from the domain and related technology studies form the input for the Inspiration Cards in a condensed form . In turn , the outcome from the Inspiration Card Workshops provides concepts that are further explored in mock - ups sessions , virtual video prototypes [ 1 ] and prototypes , before some are eventually realized as final products . In the cases we set out in this paper , two of the projects are currently in their final stages , and in both cases , design concepts that were the resultof the Inspiration Card Workshops are being developed as final products . Figure 5 contains images of the highlighted steps in the design process of the Gumlink case , from the initial domain and technology studies to the final product . Figure 5 : The Inspiration Card Workshop in the Design Process USING THE INSPIRATION CARD WORKSHOP IN THREE DESIGN PROJECTS We currently use Inspiration Cards in the ongoing research project , “Experience - Oriented Applications of Digital Technology in Knowledge Dissemination and Marketing” . The project explores the use of digital technologies in settings ranging from museums to the retail sector . The Inspiration Cards are used in various ways in the project ; in this paper we focus on Inspiration Card Workshops conducted with three of the collaborating partners in the project : 7 th Heaven , The Danish Electricity Museum , and Gumlink . These partners and their objectives in the project are highly diverse , allowing for comparative analyses of the Inspiration Card Workshop in different design situations . 7th Heaven – a centre for children’s literature 7 th Heaven is a very small organization ( two full - time employees and a number of free - lancers and subcontractors ) that organizes exhibitions related to children’s literature . They are currently building a centre for Scandinavian children’s literature , and our function in this process is the development of interactive installations in which visitors experience settings and moods from this domain . 7 th Heaven is a very democratic organization , in that the staff communicates on a daily basis and makes major decisions based on shared agreement . The staff is very accustomed to explorative and creative processes , and has a good understanding of the domain of experience centres , based on past work and research . The Danish Electricity Museum The Danish Electricity Museum is a well - established science and cultural heritage museum . It has many permanent exhibits centered about a fully functional water - power plant , and also organizes special exhibitions . Our work with the museum aims at engaging visitors by augmenting existing exhibits and developing prototypes for new interactive , collaborative installations for learning about energy production and consumption . The museum is a fairly small organization ( 10 staff members develop exhibitions and conduct tours and talks ) . The staff is heterogeneous in their various fields of expertise , however there is a high degree of communication and shared understanding . All of the staff members have a solid understanding of the museum domain , and are somewhat accustomed to creative processes with regard to exhibitions and installations Gumlink – chewing gum research and production Gumlink is a market leader in research and production of new types of chewing gum , and has 450 employees . We work with Gumlink to create interactive elements for their booth at the world’s largest sweets convention . The organization is divided into a number of branches with specific areas of expertise , and the staff is thus very heterogeneous . The Gumlink staff has some understanding of the convention domain , as they participate in a few such events annually . The staff is generally not accustomed to design processes – it is a conventional organization with functionally distinct departments . Conducting the workshops with the three partners The three workshops were set up in a similar manner , as described in Method : Inspiration Card Workshop . The following is a short account of how the workshop sessions played out : The 7 th Heaven Workshop The 7 th Heaven workshop session had five participants , two from 7 th heaven and three from the research group . 7 th Heaven took part in the creation of the Domain Cards prior to the workshop by selecting the themes of most of the cards , and supplying many of the descriptions . The 7 th Heaven staff made almost non - stop use of the Inspiration Cards . The cards were used primarily in the sense intended by us as designers , namely to combine sources of inspiration and generate new concepts . Much of the time , we could lean back , as the 7 th Heaven participants picked out , commented on , altered , and combined the cards . The participants were quick to assemble the cards on posters to capture and freeze ideas . Posters were very rapidly completed and put aside to be finished later , in order to move on with alternate ideas . The 7 th Heaven Workshop resulted in 7 concepts . The level of detail varied across these concepts ; some were fully formed ideas , including comments on implementing them , others were sketches for further thought and exploration . The Danish Electricity Museum workshop session For this workshop , the participants split into two groups , each consisting of two participants from the museum and two participants from the research group . The first of the groups went through a process very similar to that of 7 th Heaven . The participants in the second group , however , used the Inspiration Cards for two purposes . First , they lined up and categorized the cards . The categorization served as a starting point for a discussion of the ways in which they , as domain experts , perceived the museum , in contrast with the perceptions we had formed , as visitors and designers . As designers , we had created the Domain Cards based on a number of field studies at the museum , and the participants from the museum wanted to ensure that no important aspects were left out . The participants of the second groupd were thus eager to handle and reorganize the cards , and to create new ones from the blank cards , although in a different manner than intended . This discussion took up almost half of the time allotted to the combination and co - creation phase . The remaining time was spent combining the cards and creating new concepts , although due to time constraints , the second group produced fewer concepts than the first group . The workshop resulted in a number of new concepts , the majority of these originating from the first group . The concepts produced the second , analytically oriented group were generally at an earlier phase of completion than those of the first group . However , the process of the analytically oriented group yielded insight into the self - perception of the museum staff , and prompted discussion of the domain with us as designers . This outcome , though not fruitful in terms of design concepts , established a valuable common ground for furthering the design process . The Gumlink workshop session As was the case with the Danish Electricity Museum workshop , the participants in the Gumlink workshop session carried out the combination and co - creation phases in two groups , consisting , in this case , of three Gumlink participants and two participants from the research group . The processes of the two groups were fairly similar . The participants from Gumlink made less use of the Inspiration Cards than the participants in the 7 th Heaven and Danish Electricity Museum workshops . The research group , based on field studies and interviews with Gumlink staff , created the Domain Cards . Some Gumlink participants were very hesitant to use the cards , especially the Technology Cards , handling them only a few times during the workshop . The creation of posters with concepts was largely left to the research group . The participants came from different departments , and primarily used the cards to present and discuss differing views on the convention setting among themselves , or to communicate these views to us . They were less inclined to combine the cards in new ways , and instead evaluated the Technology Cards in terms of how the technologies could be applied in concrete ways . In relation to the 7 th Heaven and Danish Electricity Museum workshops , the results of the Gumlink combination and co - creation phases were thus limited in terms of new design concepts . Prior to the workshop , the research group had presented three conceptual design proposals to Gumlink . These proposals were meant as input to discussion of new concepts . However , the design concepts that were produced in the combination and co - creation phases either were very similar to these previously presented proposals , or to the concepts presented on the Technology Cards . FINDINGS FROM CONDUCTING THE WORKSHOPS Disparate participants , disparate outcomes Although the setups for the three workshops were almost identical , there were a number of differences in how they progressed , and how fruitful the outcome was with regard to the intended purpose of the workshop , i . e . the development of new design concepts . With the identical workshop setup in mind , the disparate processes and outcomes of the three sessions point towards the following factors for participants’ influence on the success of Inspiration Card Workshops , in terms of producing rich and relevant design concepts : Familiarity with fellow participants The workshop establishes a forum for creative interchange between participants . When creating new concepts , participants put themselves on the line , and risk failure by presenting ideas that other participants may reject or deride . If participants are well acquainted and have collaborated in previous projects , they have established an understanding amongst themselves , and recognize that their behaviour in experimental , creative settings is not necessarily representative of how they would act in other fora . However , if they are only slightly acquainted , as was the case with the Gumlink staff , they may be less likely to venture into unknown terrain . The nature of the organization from which participants come exerts an influence on this , as participants from hierarchical or formal organizations may feel more constrained than participants from those that are less formal . Familiarity with creative methods and processes It was evident that the workshops were fruitful when participants had previously worked with creative methods and processes . This was the case with the participants in the 7 th Heaven workshop , and to some extent with the participants in the Danish Electricity Museum workshop . These participants quickly grasped the workshop format , and were eager to use the cards as intended . The Gumlink participants were clearly used to working in a different way . They were more reluctant to accept the workshop format , and used the cards in a limited way . Insight into use domain Combining Domain and Technology Cards is a process of appropriating aspects of existing technological applications that in some way transforms the domain . This is best achieved if the participants have a firm understanding of the domain . This was very much the case for the participants from the Danish Electricity Museum . They used the Technology Cards by evaluating the ways in which they might influence practices at the museum , and in which the museum could better communicate central concepts to visitors . The concepts from the 7 th Heaven workshop were more speculative , in that the actual domain - the literature centre - was not yet built . The participants thus drew on experiences from similar contexts , and were limited in the level of detail they could reach in the design concepts . The Gumlink participants had a more limited insight into the use domain , namely the convention setting , as they only participate in sweets convention a few times a year , and had no formal fora in which to discuss it in their everyday work . Thus , they used the Domain Cards to start such discussions , and had very few comments on ways in which to appropriate the Technology Cards . Different kinds of inspiration For the preparation of the workshops we paid attention to the differences between the various sources of inspiration , which became even more evident during the workshops . Some of the cards represented applications from the same domain as the one for which we were designing , as was the case with the Danish Electricity Museum’s where the ‘ The Theremin ’ , the predecessor to the sound synthesizer , was on one of the inspiration cards . In other cases there was a larger conceptual distance between the source of inspiration and the design domain . Both technology card close to the design domain and ones with a larger conceptual distance seem to play important roles , the former making it immediately easy to acknowledge the usefulness of inspiration sources , and the latter having a greater innovative power . Some of the Technology Cards represented a collection of technologies , such as Slow Technologies ( Hallnäs & Redström 2001 ) , or a combination of complex technologies , like Khronos ( Cassinelli et al . 2005 ) , which we found did not fit so well into a process wherein large collections of inspiration cards were presented in a short time frame . Participants simply did not grasp the idea , or could not remember the information presented . We have however observed that it is valuable to have Domain Cards that represent single elements from the domain , such as Gold , as well as complex information such as The Twilight of the Gods . In addition to sources of inspiration represented by the Technology Cards , a number of cases and examples were spontaneously brought into play by workshop participants . At the 7 th Heaven workshop , reference was made to a specific science museum , which was used to explain a type of place the domain experts did not like . Reference was in fact made to a diverse set of previous cases and examples , including ‘Vin og Ølgod’ , a well known Danish pub , which was used to suggest the atmosphere of Valhalla . The art of Bill Viola was also brought into the discussion of speed , atmosphere and the style of 7 th Heaven . At the Gumlink workshop , Virgin airlines was mentioned as an argument for the potential of doing ordinary things in a special way , and was used in a discussion of the way in which technologies from the cards could be used to enhance the potential customer awareness and recollection of Gumlink . The relations between sources of inspiration and ideas Some of the ideas generated during the workshops had a very direct relation to the source of inspiration , and the creative move merely consisted of replacing a single element from the source of inspiration with an element from the design domain . At the 7 th Heaven workshop it was suggested that the letters in The Falling Letters be replaced with short pieces of text from Norse mythology , and our Gumlink partners suggested that the letters be replaced with chewing gum . Figure 6 : The Falling Letters Technology Card . Clearly , combining previously unrelated elements is a crucial aspect of innovation . At the 7 th Heaven workshop we explored the idea of combing Wodan’s Throne with the Information Table , which stimulated a discussion about making a very contemporary implementation of Wodan’s Throne . Some situations consisted of combining just two cards , as in the examples above , but in other cases several cards were simultaneously involved . For example , at the 7 th Heaven workshop , there was an extensive exchange involving several cards , including Technology Cards , Inspiration Cards and custom - made cards created by participants during the workshop . The role of cards In addition to their direct role in idea generation , the technology cards supported focus shifts in the process , and made it easier to bring new perspectives , and , by extension , new ideas , into the design process . Using cards in this way was particularly prominent in the last past of the 7 th Heaven workshop , wherein the domain experts picked up new cards and used them to introduce new subjects for discussion , when the process was not proceeding as rapidly as in the previous very intense phase . In a similar way , participants in the Gumlink workshop used the cards to try to start the process , and made it easier for people to raise their voices . As discussed above , the cards played a vital role in generating specific design ideas , but we have also observed that the design ideas that emerge are not always further elaborated , or at least documented . Moreover , some inspiration cards were not used at all , although we could not identify a clear reason . However , part of the rationale behind The Inspiration Workshop is to bring a large selection of material into the workshop , which implies that not all material will be used . COMPARISON OF THE INSPIRATION CARD WORKSHOP AND RELATED METHODS In this section we briefly introduce four related approaches to innovation in design , and use them as a platform for comparing them with The Inspiration Card Workshops . The selection of related approaches is by no means intended to be exhaustive , but rather to serve as a starting point for putting The Inspiration Card Workshops into perspective . Metaphorical design A metaphor may be defined as a concept from one linguistic category , used to describe a phenomenon normally referred to by concepts from a different linguistic category [ 22 ] . The essence of metaphorical design is to understand the product being designed by using metaphors to see it as something different , and in this way generate new perceptions , explanations , and inventions [ 22 ] and [ 26 ] . As an example , Madsen [ 22 ] describes how a library may been seen in new ways , by seeing it metaphorically as a storage for books or as a meeting place , thereby generating different ideas about which kind of information system may support activities at the library . Future workshops ‘Future workshop’ is a highly structured process originally suggested by Jungk and Müllert [ 18 ] ; F . Kensing [ 20 ] has proposed its use in systems development . As briefly summarized by Kensing and Madsen [ 20 ] , the Future workshop technique is meant to shed light on the common , problematic situation of generating visions for the future , and to discuss how these visions can be realized . Key elements of the technique include a set of specific rules such as restricting speaking time to 30 seconds during certain periods of the workshop , and not allowing critique during the fantasy phase . Moreover , the use of materials like Post - it®’s and posters is an important aspect . Interaction Relabelling and Extreme Characters ‘Interaction Relabelling’ and ‘Extreme Characters’ , which have been suggested by Djajadiningrat , Gaver , and Frens [ 10 ] , are two methods for exploring aesthetic interaction . There has been an interest in developing new kinds of interaction , which are guided not only by ease of use and efficiency , but also by richness , attractiveness and other aesthetic qualities . ‘Interaction Relabelling’ has at its core a consideration of the product one is designing in terms of an existing product . In Djajadiningrat , Gaver , and Frens [ 10 p67 ] the technique is illustrated by the example of relabelling a toy revolver as an appointment manager , generating interaction design ideas like thinking of rotating the cylinder to scroll through appointments . The idea of ‘Extreme Characters’ , as the term suggests , takes the approach of design for characters with extreme emotional attitudes . Djajadiningrat , Gaver , and Frens [ 10 p68 ] explain the approach by showing how extreme characters , such as a drug dealer and the pope , may inform the design of an appointment manager . Lateral thinking Lateral thinking , introduced by de Bono [ 11 ] includes a large collection of creativity techniques , of which we restrict our discussion to random input [ ibid 177 ] , which has at its core the selection of a randomly chosen word ( e . g . the word number three on page 89 of a dictionary ) , which acts as the starting point for idea generation . Comparison With the exception of Future Workshops , all approaches have in common multiple domains as sources from which they draw inspiration as a driving force to innovation . Lateral Thinking , as well as Interaction Relabelling and Extreme Characters argue for a large distance between the domain for which one designs , and the domain that serves as inspiration , as a large distance stimulates seeing the design task in a new way . By a similar argument , Metaphorical Design argues for a conceptual distance between the two domains , but also recommends that there be at least one bridging concept between the two domains . According to our experiences , it seems productive to include inspiration from both close and remote sources of inspiration . Future Workshops do not include sources of inspirations as such . Figure 7 : Comparison of conceptual distance between domains of inspiration and use . The various approaches also differ with respect to the number of sources of inspiration they suggest bringing into the process . Lateral Thinking , together with Interaction Relabelling and Extreme Characters , propose few sources of inspiration at a time , in contrast to the Inspiration Card Workshop , which simultaneously brings into play numerous elements . We have identified Metaphorical Design as belonging somewhere between these two extremes . Figure 8 : Comparison of the number of sources of inspiration . Metaphorical Design and Lateral Thinking primarily use language as a tool in the innovation process , whereas physical materials are essential means of supporting the design process in Inspiration Card Workshops . Extreme characters works at the conceptual level , whereas Interaction Relabelling suggests bringing mechanical devices into the process , in order to stimulate new ways of thinking about interaction . Figure 9 : Comparison of the number of materials and tools . The Future Workshop technique is a highly structured process with clearly defined phases ( critique , fantasy and implementation ) and with a number of specific rules , such as restricting speaking time to 30 second during certain periods of the first phases , and not allowing critique during the fantasy phase . On the other hand , Lateral Thinking is very much a ‘light weight’ process with minimal structure . In between , we find the other approaches with few rules and some kind of overall structure , e . g . 1 ) generating metaphors , 2 ) evaluating metaphors and 3 ) selecting and applying the metaphors in the case of metaphorical design ; or in the case of The Inspiration Card workshop , presenting sources of inspiration and domain cards , developing design concepts , and presenting design concepts . Figure 10 : Comparison of structure and rules . CONCLUSIONS AND FUTURE WORK In this paper we have presented Inspiration Cards as a means for the designer to present condensed findings from domain and technology studies . We have further elaborated on the Inspiration Card Workshop as a method for combining sources of inspiration to develop new design concepts , and reported on three design cases in which we have employed the method . Our over - all evaluation of the Inspiration Card Workshop method and the response from participants in the workshop sessions is positive . The Inspiration Cards have clearly stimulated an innovative and productive process , and we have observed that the design concepts developed generally find a suitable balance between being innovative , and realistic in terms of implementation . The method is designed to be informal , loosely structured , and simple ( eg . we only present two categories of cards ) . These factors have facilitated the involvement and engagement of participants and have been crucial with respect to the productiveness of the workshop . The findings from the design cases highlight a number of important aspects with regard to the sources of inspiration introduced in the workshop sessions , and the role of the Inspiration Cards . We recommend including sources of inspiration that vary in their conceptual distance from the use domain , in order to foster design concepts that may both fit into , and expand the domain . In our experience , the combination and co - creation phases of the workshop work well without setting up rules for roles and turn - taking . This allows participants to use the Inspiration Cards in a number of ways , such as pointing out specific ideas , framing over - all discussions , shifting focus from one aspect of the design concept to another , moving from concrete to abstract discussions etc . The experience of participants in Inspiration Card Workshops plays at least as important a role as the setup of the workshop , with regard to the generation of viable design concepts . Prior collaboration experience , insight into the use domain , and familiarity with creative methods and processes have proved to be valuable prerequisites for participants in the workshops from which we have reported . The Inspiration Card Workshop method addresses the way in which designers draw upon repertoires of prior knowledge and expertise , while respecting the discreteness of the situations they encounter , referred to by Schön [ 27 ] as a process as of reflective conversation . Fallman [ 14 ] suggests that accounts of development of new prototypes in HCI literature focus primarily on the attributes of the prototypes themselves , rather than on the vital design process by which the prototypes come about . On the one hand , The Inspiration Card Workshop method can be construed as an approach for designers to actively consider their repertoire in relation the distinct situation they face , and , on the other hand , to engage in reflective conversations between the repertoire and the situation . We continue to experiment with the workshop format in order to incorporate our findings and iteratively improve this design technique . Among other things , we are looking into ways of supporting the method technologically , both with regard to the Inspiration Card Workshop , and the creation , storing , and sharing of the inspiration cards throughout the design process . One possible avenue to pursue in this regard would be to combine a database for inspiration card storage and sharing with input devices and displays for use during the workshop , like the Video Wall presented in Jensen , Buur & Djajadiningrat [ 17 ] . However , the current “low - tech” solution has proved successful in yielding ideas and concepts . The implementation of digital support for the method might hamper the creative , explorative and collaborative processes that the current workshop format supports , by presenting entry barriers in terms of having to learn to use new technological tools . The Inspiration Cards have a range of applications in the design process , which goes beyond the workshop method presented in this paper . One such application is the use of the cards as a standard means of representing and communicating sources of technological inspiration , as well as key findings from field studies . We plan on typologically classifying the Technology and Domain Cards into subsets as our repertoire expands . With regard to Technology Cards , this will help generate an overview of state - of - the - art applications of IT , whereas a typology of Domain Cards will support comparative analyses of recurring patterns across domains . ACKNOWLEDGMENTS We would like to thank our partners from The Danish Electricity Museum , 7 th Heaven and Gumlink , as well as the students at Information Studies who participated in the design workshops . “Experience - Oriented Applications of Digital Technology in Knowledge Dissemination and Marketing” has been funded by The Danish Ministry of Science , Technology and Innovation ( The IT - corridor ) . REFERENCES 1 . Bardram , J . , Bossen C . , Lykke - Olesen , A . , Madsen , K . H . & Nielsen , R . : Virtual Video Prototyping of Pervasive Healthcare Systems . The proceedings of DIS 2002 , London : 2002 ( 167 - 177 ) . 2 . Beyer , H . and Holtzblatt , K . ( 1998 ) : Contextual Design : Defining Customer Centered Systems . San Francisco CA : Morgan Kaufman Publishers , Inc . 3 . Blomberg , J . ; Giacomi , J . ; Mosher , A . & Swenton - wall , P . ( 1993 ) : Ethnographic Field Methods and Their Relation to Design . In Schuler , D . & Namioka , A . ( ed ) : Participatory Design : Principles and Practices . Hillsdale : Lawrence Earlbaum Associates ( 123 – 155 ) 4 . Brandt , E . & Messerter , J . ( 2004 ) : Facilitating Collaboration through Design Games . In The proceedings of PDC 2004 . 5 . Brun - Cottan , F . & Wall , P . ( 1995 ) : Using Video to Re - Present the User . In The Communications of the ACM , vol 38 no 5 ( 61 - 71 ) . 6 . Buur , J . & Søndergaard A . ( 2000 ) : Video Card Game : an augmented environment for User Centred Design discussions . In Proceedings of Designing Augmented Reality DARE 2000 . Elsinore Denmark : ACM Press ( 63 - 69 ) . 7 . Bødker , S . & Grønbæk , K . Design in Action : From Prototyping by Demonstration to Cooperative Prototyping . In Greenbaum , J . and Kyng , M . ( eds . ) . Design at Work : Cooperative Design of Computer Systems . Hillsdale , NJ : Lawrence Erlbaum Associates , 1991 ( 197 - 218 ) . 8 . Caroll , J . ( Eds . ) ( 1995 ) : Scenario - Based Design : Envisioning Work and Technology in System Development . New York , NY : John Wiley and Sons . 9 . A . Cassinelli , T . Ito and M . Ishikawa . Khronos Projector . Emerging Technologies , SIGGRAPH 2005 , Los Angeles ( 2005 ) . 10 . Djajadiningrat , J . P . , Gaver , W . W . & Frens , J . W . ( 2000 ) : Interaction Relabelling and Extreme Characters : Methods for Exploring Aesthetic Interaction . Proceedings of DIS 2000 . Brooklyn , New York : ACM Press ( 66 - 71 ) . 11 . De Bono , E . ( 1993 ) : Serious Creativity . HarperCollins Publisher . 12 . Ehn , P . ( 1988 ) : Work - oriented Design of Computer Artifacts . Stockholm Sverige : Arbeitslivscentrum . 13 . Ehn , P . & Kyng , M . ( 1991 ) : Cardboard Computers : Mocking - it - up and hands - on the future . In Greenbaum , J . and Kyng , M . ( eds ) ( 1991 ) : Design at Work : Cooperative Design of Computer Systems . Hillsdale , NJ : Lawrence Erlbaum Associates ( 169 - 195 ) . 14 . Fallman , D . ( 2003 ) Design - oriented Human - Computer Interaction , Proceedings of CHI2003 , Conference on Human Factors in Computing Systems , CHI Letters , Vol . 5 , Issue No . 1 ( Fort Lauderdale , Florida , April 5 - 10 ) , New York , NY : ACM Press , pp . 225 - - 232 . 15 . Foster , J . ( 1996 ) : How to Get Ideas . San Francisco : Berrett - Koehler Publishers . 16 . Hallnäs , Lars & Redström , Johan ( 2001 ) : Slow Technology - Designing for Reflection . Personal and Ubiquitous Computing 5 ( 3 ) : 201 - 212 ( 2001 ) . 17 . Jensen , M . V . , Buur , J . & Djajadiningrat , T . ( 2005 ) : Designing the user actions in tangible interaction . In Proceedings of The Fourth Decennial Aarhus Conference 2005 . 18 . Jungk , R . & Müllert , N . ( 1987 ) : Future Workshops : How to create desirable futures . London : Institute for Social Inventions . 19 . Kelley , T . ( 2001 ) : The Art of Innovation . New York : Random House Inc . 20 . Kensing , F . & Madsen , K . H . : Generating Visions : Future Workshops and Metaphors , in Greenbaum , J . & Kyng , M . : Design at Work , Lawrence Earlbaum 1991 ( 155 - 168 ) . 21 . Lervig , M . & Madsen , KH . ( 2003 ) : Artists in the Virtual Studio . In Madsen , K . H . ( ed ) : Production Methods : Behind the Scenes of Virtual Inhabited 3D Worlds . London : Springer - Verlag , . 22 . Madsen , K . H . ( 1994 ) : A Guide to Metaphorical Design . In The Communications of the ACM , vol 37 no 12 ( 57 - 62 ) 1994 . 23 . Patton , Q . M . ( 1990 ) : Qualitative Evaluation and Research Methods . Newbury Park , CA : Sage Publications . 24 . Ryokai , K . , Marti , S . , Ishii , H . ( 2004 ) : I / O Brush : Drawing with Everyday Objects as Ink . In Proceedings of Conference on Human Factors in Computing Systems ( CHI ' 04 ) , ( Vienna , Austria , April 24 - April 29 , 2004 ) 25 . Sanders , E . ( 2005 ) : Information , Inspiration and Co - creation . The 6th International Conference of the European Academy of Design , March 29 - 31 2005 , University of the Arts , Bremen , Germany . 26 . Schön , D . ( 1979 ) . Generative Metaphor : A Perspective on Problem - setting in Social Policy . In Ortony , A . ( ed . ) : Metaphor and Thought . Cambridge : Cambridge University Press , ( 254 - 283 ) . 27 . Schön , D . ( 1983 ) : The Reflective Practitioner . New York : Basic Books . 28 . Schön , D . ( 1988 ) : Designing : Rules , types and worlds . Design studies , vol . 9 no 3 July 1988 . 29 . Tuder , L . G . , Muller , M . & Dayton , T ( 1993 ) : A C . A . R . D . game for participatory task analysis and redesign : macroscopic complement to PICTIVE . In adjunct proceeding of InterCHI 93 . Amsterdam . \ No newline at end of file diff --git a/silver_data/8620cbbcdd06e37167f4d9b6c659d4450de12378.pdf.txt b/silver_data/8620cbbcdd06e37167f4d9b6c659d4450de12378.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..bae3c1e613db96353b1f9d7f08c5334246be25ca Binary files /dev/null and b/silver_data/8620cbbcdd06e37167f4d9b6c659d4450de12378.pdf.txt differ diff --git a/silver_data/8756e1a0511ce5187395f00c212f2d05ecd3db35.pdf.txt b/silver_data/8756e1a0511ce5187395f00c212f2d05ecd3db35.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..8ce0ff6110c2fdd60dc5d21823cb97107efbf446 --- /dev/null +++ b/silver_data/8756e1a0511ce5187395f00c212f2d05ecd3db35.pdf.txt @@ -0,0 +1 @@ +A Random Graph Model for Power Law Graphs William Aiello ∗ Fan Chung †‡ Linyuan Lu § ∗ Abstract We propose a random graph model which is a special case of sparse random graphs with given degree sequences which satisfy a power law . This model involves only a small number of parameters , called log - size and log - log growth rate . These parameters capture some universal characteristics of massive graphs . Furthermore , from these parame - ters , various properties of the graph can be derived . For example , for certain ranges of the parameters , we will compute the expected distri - bution of the sizes of the connected components which almost surely occur with high probability . We will illustrate the consistency of our model with the behavior of some massive graphs derived from data in telecommunications . We will also discuss the threshold function , the giant component , and the evolution of random graphs in this model . 1 Introduction Is the World Wide Web completely connected ? If not , how big is the largest component , the second largest component , etc . ? Anyone who has “surfed” the Web for any length of time will undoubtedly come away feeling that if there are disconnected components at all , then they must be small and few in number . Is the Web too large , dynamic and structureless to answer these questions ? Probably yes , if the sizes of the largest components are required to be exact . Recently , however , some structure of the Web has come to light which may enable us to describe graph properties of the Web qualitatively . Kumar et al . [ 13 , 14 ] and Kleinberg et al . [ 12 ] have measured the degree sequences of the Web and shown that it is well approximated by a power law distribution . ∗ AT & T Labs , Florham Park , New Jersey . † University of California , San Diego . ‡ Research supported in part by NSF Grant No . DMS 98 - 01446 § University of Pennsylvania , Philadelphia . 1 That is , the number of nodes , y , of a given degree x is proportional to x − β for some constant β > 0 . This was reported independently by Albert , Barab´asi and Jeong in [ 4 , 6 , 7 ] . The power law distribution of the degree sequence appears to be a very robust property of the Web despite its dynamic nature . In fact , the power law distribution of the degree sequence may be a ubiquitous characteristic , applying to many massive real world graphs . Indeed , Abello et al . [ 1 ] have shown that the degree sequence of so called call graphs is nicely approximated by a power law distribution . Call graphs are graphs of calls handled by some subset of telephony carriers for a specific time period . In addition , Faloutsos , et al . [ 10 ] have shown that the degree sequence of the Internet router graph also follows a power law . Just as many other real world processes have been effectively modeled by appropriate random models , in this paper we propose a parsimonious random graph model for graphs with a power law degree sequence . We then derive connectivity results which hold with high probability in various regimes of our parameters . And finally , we compare the results from the model with the exact connectivity structure for some call graphs computed by Abello et al . [ 1 ] . An extended abstract of this paper has appeared in the Proceedings of the Thirtysecond Annual ACM Symposium on Theory of Computing 2000 ( see [ 2 ] ) . In this paper , we have included the complete proofs for the main theorems and several additional theorems focused on the second largest components of power graphs in various ranges . In addition , some recent references are provided ( also see [ 11 ] ) . 1 . 1 Power - Law Random Graphs The study of random graphs dates back to the work of Erd˝os and R´enyi whose seminal papers [ 8 , 9 ] laid the foundation for the theory of random graphs . There are three standard models for what we will call in this paper uniform random graphs [ 5 ] . Each has two parameters . One parameters controls the number of nodes in the graph and one controls the density , or number of edges . For example , the random graph model G ( n , e ) assigns uniform probability to all graphs with n nodes and e edges while in the random graph model G ( n , p ) each edge is chosen with probability p . Our power law random graph model also has two parameters . The two parameters only roughly delineate the size and density but they are natural and convenient for describing a power law degree sequence . The power law random graph model P ( α , β ) is described as follows . Let y be the number of nodes with degree x . P ( α , β ) assigns uniform probability to all graphs with 2 y = e α / x β ( where self loops are allowed ) . Note that α is the intercept and β is the ( negative ) slope when the degree sequence is plotted on a log - log scale . We remark that there is also an alternative power law random graph model analogous to the uniform graph model G ( n , p ) . Instead of having a fixed degree sequence , the random graph has an expected degree sequence distribution . The two models are basically asymptotically equivalent , sub - ject to bounding error estimates of the variances ( which will be further described in a subsequent paper ) . 1 . 2 Our Results Just as for the uniform random graph model where graph properties are studied for certain regimes of the density parameter and shown to hold with high probability asymptotically in the size parameter , in this paper we study the connectivity properties of P ( α , β ) as a function of the power β which hold almost surely for sufficiently large graphs . Briefly , we show that when β < 1 , the graph is almost surely connected . For 1 < β < 2 there is a giant component , i . e . , a component of size Θ ( n ) . Moreover , all smaller components are of size O ( 1 ) . For 2 < β < β 0 = 3 . 4785 there is a giant component and all smaller components are of size O ( log n ) . For β = 2 the smaller components are of size O ( log n / log log n ) . For β > β 0 the graph almost surely has no giant component . In addition we derive several results on the sizes of the second largest component . For example , we show that for β > 4 the numbers of components of given sizes can be approximated by a power law as well . 1 . 3 Previous Work Strictly speaking our model is a special case of random graphs with a given degree sequence for which there is a large literature . For example , Wormald [ 19 ] studied the connectivity of graphs whose degrees are in an interval [ r , R ] , where r ≥ 3 . HLuczak [ 15 ] considered the asymptotic behavior of the largest component of a random graph with given degree sequence as a function of the number of vertices of degree 2 . His result was further improved by Molloy and Reed [ 16 , 17 ] . They consider a random graph on n vertices with the following degree distribution . The number of vertices of degree 0 , 1 , 2 , . . . are about λ 0 n , λ 1 n , . . . respectively , where the λ ’s sum to 1 . It is shown in [ 16 ] that if Q = (cid:1) i i ( i − 2 ) λ i > 0 and the maximum degree is not too large , then such random graphs have a giant component with probability tending 3 to 1 as n goes to infinity , while if Q < 0 then all components are small with probability tending to 1 as n → ∞ . They also examined the threshold behavior of such graphs . In this paper , we will apply these techniques to deal with the special case that applies to our model . Several other papers have taken a different approach to modeling power law graphs than the one taken here [ 3 , 6 , 7 , 12 , 14 ] . The essential idea of these papers is to define a random process for growing a graph by adding nodes and edges . The intent is to show that the defined processes asymptotically yield graphs with a power law degree sequence with very high probability . While this approach is interesting and important it has several difficulties . First , the models are difficult to analyze rigorously since the transition prob - abilities are themselves dependent on the the current state . For example , [ 6 , 7 ] implicitly assume that the probability that a node has a given degree is a continuous function . The authors of [ 12 , 14 ] will offer an improved analysis in an upcoming paper [ 18 ] . Second , while the models may gener - ate graphs with power law degree sequences , it remains to be seen if they generate graphs which duplicate other structural properties of the Web , the Internet , and call graphs . For example , the model in [ 6 , 7 ] cannot generate graphs with a power law other than c / x 3 . Moreover , all the graphs can be decomposed into m disjoint trees , where m is a parameter of the model . The ( α , β ) model in [ 14 ] is able to generate graphs for which the power law for the indegree is different than the power law for the outdegree as is the case for the Web . However , to do so , the model requires that there be nodes that have only indegree and no outdegree and visa versa . While this may be appropriate for call graphs ( e . g . , customer service numbers ) it remains to be seen whether it models the Web . Thus , while the random graph gener - ation approach holds the promise of accurately predicting a wide variety a structural properties of many real world massive graphs much work remains to be done . In this paper we take a different approach . We do not attempt to answer how a graph comes to have a power law degree sequence . Rather , we take that as a given . In our model , all graphs with a given power law degree sequence are equi - probable . The goal is to derive structural properties which hold with probability asymptotically approaching 1 . Such an approach , while potentially less accurate than the detailed modelling approach above , has the advantage of being robust : the structural properties derived in this model will be true for the vast majority of graphs with the given degree sequence . Thus , we believe that this model will be an important complement to random graph generation models . The power law random graph model will be described in detail in the 4 next section . In Sections 3 and 4 , our results on connectivity will be derived . In section 5 , we discuss the sizes of the second largest components . In section 6 , we compare the results of our model to exact connectivity data for call graphs . 2 A random graph model We consider a random graph with the following degree distribution depend - ing on two given values α and β . Suppose there are y vertices of degree x where x and y satisfy log y = α − β log x In other words , we have | { v | deg ( v ) = x } | = y = e α x β Basically , α is the logarithm of the size of the graph and β can be regarded as the log - log growth rate of the graph . We note that the number of edges should be an integer . To be precise , the above expression for y should be rounded down to (cid:9) e α x β (cid:10) . If we use real numbers instead of rounding down to integers , it may cause some error terms in further computation . However , we will see that the error terms can be easily bounded . For simplicity and convenience , we will use real numbers with the understanding the actual numbers are their integer parts . Another constraint is that the sum of the degrees should be even . This can be assured by adding a vertex of degree 1 if the sum is old if needed . Furthermore , for simplicity , we here assume that there is no isolated vertices . We can deduce the following facts for our graph : ( 1 ) The maximum degree of the graph is e αβ . Note that 0 ≤ log y = α − β log x . ( 2 ) The vertices number n can be computed as follows : By summing y ( x ) for x from 1 to e αβ , we have n = e αβ (cid:2) x = 1 e α x β ≈   ζ ( β ) e α if β > 1 αe α if β = 1 e αβ 1 − β if 0 < β < 1 where ζ ( t ) = (cid:1) ∞ n = 1 1 n t is the Riemann Zeta function . 5 ( 3 ) The number of edges E can be computed as follows : E = 1 2 e αβ (cid:2) x = 1 x e α x β ≈   12 ζ ( β − 1 ) e α if β > 2 14 αe α if β = 2 12 e 2 αβ 2 − β if 0 < β < 2 ( 4 ) The differences of the real numbers in ( 1 ) - ( 3 ) and their integer parts can be estimated as follows : For the number n of vertices , the error term is at most e αβ . For β ≥ 1 , it is o ( n ) , which is a lower order term . For 0 < β < 1 , the error term for n is relatively large . In this case , we have n ≥ e α β 1 − β − e αβ = βe α β 1 − β Therefore , n has the same magnitude as e αβ 1 − β . The number E of edges can be treated in a similarly way . For β ≥ 2 , the error term of E is o ( E ) , a lower order term . For 0 < β < 2 , E has the same magnitude as in formula of item ( 3 ) . In this paper , we mainly deal with the case β > 2 . The only place that we deal with the case 0 < β < 2 is in the next section where we refer to 2 − β as a constant . By using real numbers instead of rounding down to their integer parts , we simplify the arguments without affecting the conclusions . In order to consider the random graph model , we will need to consider large n . We say that some property almost surely ( a . s . ) happens if the probability that the property holds tends to 1 as the number n of the vertices goes to infinity . Thus we consider α to be large but where β is fixed . We use the following random graph model for a given degree sequence : The model : 1 . Form a set L containing deg ( v ) distinct copies of each vertex v . 2 . Choose a random matching of the elements of L . 3 . For two vertices u and v , the number of edges joining u and v is equal to the number of edges in the matching of L joining copies of u to copies of v . We remark that the graphs that we are considering are in fact multi - graphs , possibly with loops . This model is a natural extension of the model for k - regular graphs , which is formed by combining k random matching . For references and undefined terminology , the reader is referred to [ 5 , 20 ] . We note that this random graph model is slightly different from the uniform selection model P ( α , β ) as described in section 1 . 1 . However , by using techniques in Lemma 1 of [ 17 ] , it can be shown that if a random graph 6 with a given degree sequence a . s . has property P under one of these two models , then it a . s . has property P under the other model , provided some general conditions are satisfied . 3 The connected components Molloy and Reed [ 16 ] showed that for a random graph with ( λ i + o ( 1 ) ) n vertices of degree i , where λ i are non - negative values which sum to 1 , the giant component emerges when Q = (cid:1) i ≥ 1 i ( i − 2 ) λ i > 0 , provided that the maximum degree less than n 1 / 4 − (cid:8) . They also show that almost surely there is no giant component when Q = (cid:1) i ≥ 1 i ( i − 2 ) λ i < 0 and maximum degree less than n 1 / 8 − (cid:8) . Here we compute Q for our ( α , β ) - graphs . Q = e αβ (cid:2) x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) ≈ e αβ (cid:2) x = 1 e α x β − 2 − 2 e αβ (cid:2) x = 1 e α x β − 1 ≈ ( ζ ( β − 2 ) − 2 ζ ( β − 1 ) ) e α if β > 3 Hence , we consider the value β 0 = 3 . 47875 . . . , which is a solution to ζ ( β − 2 ) − 2 ζ ( β − 1 ) = 0 If β > β 0 , we have e αβ (cid:2) x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) < 0 We first summarize the results here : 1 . When β > β 0 = 3 . 47875 . . . , the random graph a . s . has no giant component . When β < β 0 = 3 . 47875 . . . , there is a . s . a unique giant component . 2 . When 2 < β < β 0 = 3 . 47875 . . . , the second largest components are a . s . of size Θ ( log n ) . For any 2 ≤ x < Θ ( log n ) , there is almost surely a component of size x . 7 3 . When β = 2 , a . s . the second largest components are of size Θ ( log n loglog n ) . For any 2 ≤ x < Θ ( log n loglog n ) , there is almost surely a component of size x . 4 . When 1 < β < 2 , the second largest components are a . s . of size Θ ( 1 ) . The graph is a . s . not connected . 5 . When 0 < β < 1 , the graph is a . s . connected . 6 . For β = β 0 = 3 . 47875 . . . , this is a very complicated case . It corre - sponds to the double jump of random graph G ( n , p ) with p = 1 n . For β = 1 , there is a nontrivial probability for either cases that the graph is connected or disconnected . We remark that for β > 8 , Molloy and Reed’s result immediately implies that almost surely there is no giant component . When β ≤ 8 , additional analysis is needed to deal with the degree constraints . We will prove in Theorem 2 that almost surely there is no giant component when β > β 0 . In section 5 , we will deal with the range β < β 0 . We will show in Theorem 3 that almost surely there is a unique giant component when β < β 0 . Fur - thermore , we will determine the size of the second largest component within a constant factor . 4 The sizes of connected components in certain ranges for β For β > β 0 = 3 . 47875 . . . , almost surely there is no giant component . This range is of special interest since it is quite useful later for describing the distribution of small components . We will prove the following : Theorem 1 For ( α , β ) - graphs with β > 4 , the distribution of the number of connected components is as follows : 1 . For each vertex v of degree d = Ω ( 1 ) , let τ be the size of connected component containing v . Then P r ( | τ − d c 1 | > 2 λ c 1 (cid:7) dc 2 c 1 ) ≤ 2 λ 2 where λ = d (cid:8) . In other words , the vertex v a . s . belongs to a connected component of size dc 1 + O ( d 12 + (cid:8) ) , where c 1 = 2 − ζ ( β − 2 ) ζ ( β − 1 ) , c 2 = ζ ( β − 3 ) ζ ( β − 1 ) − 8 (cid:8) ζ ( β − 2 ) ζ ( β − 1 ) (cid:9) 2 are two constants , " is an arbitrary small positive number and d is a ( slowly ) increasing function of n . 2 . The number of connected components of size x is a . s . at least ( 1 + o ( 1 ) ) e α c β − 1 1 x β . and at most c 3 e α log β 2 − 1 n x β 2 + 1 where c 3 = 4 1 + β c 2 ( β − 2 ) c 1 + β 1 is a constant only depending on β . 3 . A connected component of the ( α , β ) - graph a . s . has the size at most e 2 α β + 2 α = Θ ( n 2 β + 2 log n ) In our proof we use the second moment whose convergence depends on β > 4 . In fact for β ≤ 4 the second moment diverges as the size of the graph goes to infinity so that our method no longer applies . Theorem 1 strengthens the following result ( which can be derived from Lemma 3 in [ 16 ] ) for the range of β > 4 . Theorem 2 For β > β 0 = 3 . 47875 . . . , a connected component of the ( α , β ) - graph a . s . has the size at most Ce 2 αβ α = Θ ( n 2 β log n ) where C = 16 c 21 is a constant only depending on β . The proof for Theorem 2 is by using branching process method . We here briefly describe the proof since it is needed for the proof of Theorem 1 . Pick any vertex v in our graph , expose its neighbors , and then the neighbors of its neighbors , repeating until the entire component is exposed . We expose only one vertex at each stage . At stage i , let L i be the set of vertices exposed and X i be the random variable that counts the number of vertices in L i . We mark all vertices in L i by either “live” or “dead” . A vertex in L i , whose neighbors have not been all exposed yet , is marked “live” . A vertex , whose neighbors have all been exposed , is marked “dead” . Let O i be the set of live vertices and Y i be the random variable that is the number of vertices 9 in O i . Each step we mark exact one dead vertex , so the total number of dead vertices at i - th step is i . We have X i = Y i + i . Initially we assign L 0 = O 0 = { v } . Then at stage i ≥ 1 , we do the following : 1 . If Y i − 1 = 0 , then we stop and output X i − 1 . 2 . Otherwise , randomly choose a live vertex u from O i − 1 and expose its neighbors in N u . Then mark u dead and mark each vertex live if it is in N u but not in L i − 1 . We have L i = L i − 1 ∪ N u , and O i = ( O i − 1 \ { u } ) ∪ ( N u \ L i − 1 ) . Suppose that v has degree d . Then X 1 = d + 1 , and Y 1 = d . Eventually Y i will hit 0 if i is large enough . Let τ denote the stopping time of Y , namely , Y τ = 0 . Then X τ = Y τ + τ = τ measures the size of the connected component . We first compute the expected value of Y i and then use Azuma’s Inequality [ 16 ] to prove Theorem 2 . Suppose that the vertex u is exposed at stage i . Then N u ∩ L i − 1 contains at least one vertex , which was exposed to reach u . However , N u ∩ L i − 1 may contain more than one vertex . We call them “backedges” . We note that “backedges” causes the exploration to stop more quickly , especially when the component is large . However in our case β > β 0 = 3 . 47875 . . . , the contribution of “backedges” is quite small . We denote Z i = # { N u } and W i = # { N u ∩ L i − 1 } − 1 . Z i measures the degree of the vertex exposed at stage i , while W i measures the number of “backedges” . By definition , we have Y i − Y i − 1 = Z i − 2 − W i . We have E ( Z i ) = (cid:1) e αβ x = 1 x x eαxβ E = e α E (cid:1) e αβ x = 1 x 2 − β = ζ ( β − 2 ) + O ( n 3 β − 1 ) ζ ( β − 1 ) + O ( n 2 β − 1 ) = ζ ( β − 2 ) ζ ( β − 1 ) + O ( n 3 β − 1 ) Now we will bound W i . Suppose that there are m edges exposed at stage i − 1 . Then the probability that a new neighbor is in L i − 1 is at most mE . We have E ( W i ) ≤ ∞ (cid:2) x = 1 x (cid:8) m E (cid:9) x = mE ( 1 − mE ) 2 ( ∗ ) 10 = m E + O ( ( m E ) 2 ) provided mE = o ( 1 ) . When i ≤ Ce 2 αβ α , m is at most ie αβ ≤ Ce 3 αβ α . Hence , m E = O ( n 3 β − 1 log n ) = o ( 1 ) We have E ( Y i ) = Y 1 + i (cid:2) j = 2 E ( Y j − Y j − 1 ) = d + i (cid:2) j = 2 E ( Z j − 2 − W j ) = d + ( i − 1 ) (cid:10) ζ ( β − 2 ) ζ ( β − 1 ) − 2 (cid:11) − iO ( n 3 β − 1 log n ) = d − c 1 ( i − 1 ) + io ( 1 ) Proof of Theorem 2 : Since | Y j − Y j − 1 | ≤ e αβ , by Azuma’s martingale inequality , we have P r ( | Y i − E ( Y i ) | > t ) ≤ 2 e − t 2 2 ie 2 αβ By taking i = 16 c 21 e 2 αβ log n , and t = c 1 2 i . Since E ( Y i ) + t = d − c 1 ( i − 1 ) + io ( 1 ) + c 1 2 i = − c 1 2 i + d + c 1 + io ( 1 ) < 0 We have P r ( τ > 16 c 21 e αβ log n ) = P r ( τ > i ) ≤ P r ( Y i ≥ 0 ) ≤ P r ( Y i > E ( Y i ) + t ) ≤ 2 e − t 2 2 ie 2 αβ = 2 n 2 Hence , the probability that there exists a vertex v such that v lies in a component of size greater than 16 c 21 e 2 αβ log n is at most n 2 n 2 = 2 n = o ( 1 ) . (cid:1) 11 The proof of Theorem 1 uses the methodology above as a starting point while introducing the calculation of the variance of the above random vari - ables . Proof of Theorem 1 We follow the notation and previous results of Section 4 . Under the assumption β > 4 , we consider the following : V ar ( Z i ) = e αβ (cid:2) x = 1 x 2 x e α x β E − E ( Z i ) 2 = e α E e αβ (cid:2) x = 1 x 3 − β − E ( Z i ) 2 = ζ ( β − 3 ) + O ( n 4 β − 1 ) ζ ( β − 1 ) + O ( n 2 β − 1 ) − (cid:10) ζ ( β − 2 ) ζ ( β − 1 ) (cid:11) 2 + O ( n 3 β − 1 ) = ζ ( β − 3 ) ζ ( β − 1 ) − (cid:10) ζ ( β − 2 ) ζ ( β − 1 ) (cid:11) 2 + O ( n 4 β − 1 ) = c 2 + o ( 1 ) since β > 4 . We need to compute the covariants . There are models for random graphs in which the edges are in dependently chosen . Then , Z i and Z j are indepen - dent . However , in the model based on random matchings , there is a small correlation . For example , Z i = x slightly effects the probability of Z j = y . Namely , Z j = x has slightly less chance , while Z j = y (cid:17) = x has slightly more chance . Both differences can be bounded by 1 E − 1 − 1 E ≤ 2 E 2 Hence CoV ar ( Z i , Z j ) ≤ E ( Z i ) E 2 E 2 = O ( 1 n ) if i (cid:17) = j . Now we will bound W i . Suppose that there are m edges exposed at stage i − 1 . Then the probability that a new neighbor is in L i − 1 is at most mE . We have V ar ( W i ) ≤ ∞ (cid:2) x = 1 x 3 (cid:8) m E (cid:9) x − E ( W i ) 2 = mE ( mE + 1 ) ( 1 − mE ) 3 − O ( ( m E ) 2 ) 12 = m E + O ( ( m E ) 2 ) CoV ar ( W i , W j ) ≤ (cid:12) V ar ( W i ) V ar ( W j ) ≤ m E + O ( ( m E ) 2 ) CoV ar ( Z i , W j ) ≤ (cid:12) V ar ( Z i ) V ar ( W j ) = O ( (cid:7) m E ) When i = O ( e αβ ) , m ≤ ie αβ = O ( e 2 αβ ) , we have E ( Y i ) = d + ( i − 1 ) (cid:10) ζ ( β − 2 ) ζ ( β − 1 ) − 2 (cid:11) + iO ( n 3 β − 1 ) + imE = d − ( i − 1 ) c 1 + O ( n 4 β − 1 ) = d − ( i − 1 ) c 1 + o ( 1 ) V ar ( Y i ) = V ar ( d + i (cid:2) j = 2 ( Y j − Y j − 1 ) ) = V ar ( i (cid:2) j = 2 ( Z j − W j ) ) = i (cid:2) j = 2 ( V ar ( Z j ) + V ar ( W j ) ) + (cid:2) 2 ≤ j (cid:9) = k ≤ i ( CoV ar ( Z j , Z k ) − CoV ar ( Z j , W k ) + CoV ar ( W j , W k ) ) = ic 2 + io ( 1 ) + i 2 ( O ( 1 n ) + O ( (cid:12) e ( 2 β − 1 ) α ) + O ( e ( 2 β − 1 ) α ) ) = ic 2 + io ( 1 ) + i ( O ( e ( 2 β − 12 ) α ) + O ( e ( 3 β − 1 ) α ) ) = ic 2 + io ( 1 ) Chebyshev’s inequality gives P r ( | Y i − E ( Y i ) | > λσ ) < 1 λ 2 where σ is the standard deviation of Y i , σ = √ ic 2 + o ( √ i ) Let i 1 = (cid:9) dc 1 − 2 λc 1 (cid:12) dc 2 c 1 (cid:10) and i 2 = (cid:19) dc 1 + 2 λc 1 (cid:12) dc 2 c 1 (cid:20) . We have E ( Y i 1 ) − λσ = d − ( i 1 − 1 ) c 1 + o ( 1 ) − (cid:13) λ √ c 2 i 1 + o ( √ i 1 ) (cid:14) 13 ≥ 2 λ (cid:7) dc 2 c 1 − λ (cid:7) c 2 d c 1 − o ( √ d ) = λ (cid:7) dc 2 c 1 − o ( √ d ) > 0 Hence , P r ( τ < i 1 ) ≤ P r ( Y i 1 ≤ 0 ) ≤ P r ( Y i 1 < E ( Y i 1 ) − λσ ) ≤ 1 λ 2 Similarly , E ( Y i 2 ) + λσ = d − ( i 2 − 1 ) c 1 + o ( 1 ) + (cid:13) λ √ c 2 i 2 + o ( √ i 2 ) (cid:14) ≥ − 2 λ (cid:7) dc 2 c 1 + λ (cid:7) c 2 d c 1 + o ( √ d ) = − λ (cid:7) dc 2 c 1 + o ( √ d ) < 0 Hence , P r ( τ > i 2 ) ≤ P r ( Y i 2 > 0 ) ≤ P r ( Y i 2 > E ( Y i 2 ) + λσ ) ≤ 1 λ 2 Therefore P r ( | τ − d c 1 | > 2 λ c 1 (cid:7) dc 2 c 1 ) ≤ 2 λ 2 For a fixed v and λ a slowly increasing function to infinity , above inequality implies that almost surely we have τ = dc 1 + O ( λ √ d ) . We note that almost all components generated by vertices of degree x is about the size of d c 1 . One such component can have at most about 1 c 1 vertices of degree d . Hence , the number of component of size dc 1 is at least c 1 e αβ d β . Let d = c 1 x . Then the number of components of size x is at least e αβ c β − 1 1 x β ( 1 + o ( 1 ) ) The above proof actually gives the following result . The size of every com - ponent , whose vertices have degree at most d 0 , is almost surely Cd 20 log n where C = 16 c 21 is the same constant as in Theorem 2 . Let x = Cd 2 0 log n and 14 consider the number of components of size x . A component of size x almost surely contains at least one vertex of degree greater than d 0 . For each vertex v with degree d ≥ d 0 , by part 1 , we have P r (cid:15) | τ − d c 1 | > 2 λ d c 1 (cid:7) dc 2 c 1 (cid:16) ≤ 2 λ 2 d Let λ d = c 1 Cd 20 log n 4 (cid:12) c 1 c 2 d , we have P r ( τ ≥ Cd 2 0 log n ) ≤ P r (cid:15) τ > d c 1 + 2 λ d c 1 (cid:7) dc 2 c 1 (cid:16) ≤ C 3 d d 40 log 2 n where C 3 = 32 c 2 c 31 C 2 = c 1 c 2 8 is constant depending only on β . Since there are only e α d β vertices of degree d , the number of components of size at least x is at most e αβ (cid:2) d = d 0 e α d β C 3 d d 40 log 2 n ≤ C 3 e α d 40 log 2 n ∞ (cid:2) d = d 0 1 d β − 1 ≤ C 3 e α d 40 log 2 n 2 β − 2 1 d β − 2 0 = 2 C 3 e α ( β − 2 ) d β + 2 0 log 2 n = c 3 e α log β 2 − 1 n x β 2 + 1 where c 3 = 2 C 3 ( β − 2 ) C 1 + β 2 = 4 1 + β c 2 ( β − 2 ) c 1 + β 1 . For x = e 2 α β + 2 α , the above inequality implies that the number of components of size at least x is at most o ( 1 ) . In other words , almost surely no component has size greater than e 2 α β + 2 α . This completes the proof of Theorem 1 . 5 On the size of the second largest component For β < β 0 = 3 . 47875 . . . , we consider the giant component as well as the size of the second largest component . 15 Theorem 3 For ( α , β ) - graphs with β < β 0 = 3 . 47875 . . . , the following holds : 1 . There is a unique giant component of size Θ ( n ) . 2 . When 2 < β < β 0 , almost surely the size of the second largest compo - nent is Θ ( log n ) . 3 . When β = 2 , almost surely the size of the second largest component is Θ ( log n loglog n ) . 4 . When 1 ≤ β < 2 , almost surely the size of the second largest component is Θ ( 1 ) . 5 . When 0 < β < 1 , almost surely the ( α , β ) - graph is connected . Proof : When β < β 0 , the branching process method is no longer feasible when vertices of large degrees are involved . Thus , we can not apply Azuma’s martingale inequality for bounding Y i as in the proofs of the previous sec - tions . We will modify the branching process method as follows . 1 . Choose a number x β ( to be specified later depending on β ) . 2 . Start with Y ∗ 0 live vertices and Y ∗ 0 ≥ C log n . All other vertices are unmarked . 3 . At the i - th step , we choose one live vertex u and exposed its neighbors . If the degree of u is less than or equal to x β , we proceed as in section 4 , by marking u dead and all vertices v ∈ N ( u ) live ( provided v is not marked before ) . If the degree of u is greater than x β , we will mark exactly one vertex v ∈ N ( u ) live and others dead , provided v is unmarked . In both case u is marked dead . The main idea is to show that Y ∗ i , a truncated version of Y i , is well - concentrated around E ( Y ∗ i ) . Although it is difficult to directly derive such result for Y i because of vertices of large degrees , we will be able to bound the distribution Y ∗ i . Indeed , we will show that the set of marked vertices ( live or dead ) grows to a giant component if Y ∗ 0 exceeds a certain bound . We consider the following three ranges of β . Case 1 : 2 < β < β 0 . We consider Q = 1 E (cid:1) e αβ x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) . ( Note that Q is a positive constant . ) There is a constant integer x 0 satisfying 1 E (cid:1) x 0 x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) > Q 2 . We choose δ satisfying : δ ( 1 − δ ) 2 = Q 4 . 16 If the component has more than δE edges , it must have Θ ( n ) vertices since β > 2 . So it is a giant component and we are done . We may assume that the component has no more than δE edges . We now choose x β = x 0 and apply the modified branching process . Then , Y ∗ i satisfies the following : • Y ∗ 0 ≥ (cid:19) C log n (cid:20) , where C = 130 x 20 Q is a constant only depending on β . • − 1 ≤ Y ∗ i − Y ∗ i − 1 ≤ x 0 . • Let W i be the number of “backedges” as defined in section 4 . By inequality ( * ) and the assumption that the number of edges m in the component is at most δn , we have E ( W i ) ≤ δ ( 1 − δ ) 2 = Q 4 . Hence , we have E ( Y ∗ i − Y ∗ i − 1 ) ≈ 1 E x 0 (cid:2) x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) − E ( W i ) ≥ Q 2 − Q 4 = Q 4 . By Azuma’s martingale inequality , we have P r ( Y ∗ i ≤ Qi 8 ) ≤ P r ( Y ∗ i − E ( Y ∗ i ) ≤ − Qi 8 ) < e − ( Qi / 8 ) 2 2 ix 20 = o ( n − 1 ) provided i > C log n . The above inequality implies that with probability at least 1 − o ( n − 1 ) , Y ∗ i > Qi 8 > 0 when i > (cid:19) C log n (cid:20) . Since Y ∗ i decreases at most by 1 at each step , Y ∗ i can not be zero if i ≤ (cid:19) C log n (cid:20) . So Y ∗ i > 0 for all i . In other words , a . s . the branching process will not stop . However , it is impossible to have Y ∗ n > 0 , that is a contradiction . Thus we conclude that the component must have at least δn edges . So it is a giant component . We note that if a component has more than (cid:19) C log n (cid:20) edges exposed , then almost surely it is a giant component . In particular , any vertex with degree more than (cid:19) C log n (cid:20) is almost surely in the giant component . Hence , the second component have size of at most Θ ( log n ) . Next , we will show that the second largest has size at least Θ ( log n ) . We consider the vertices v of degree x = cα , where c is some constant . There is a positive probability that all neighboring vertices of v have degree 1 . 17 In this case , we get a connected component of size x + 1 = Θ ( log n ) . The probability of this is about ( 1 ζ ( β − 1 ) ) cα Since there are e α ( cα ) β vertices of degree x , the probability that none of them has the above property is about ( 1 − 1 ζ ( β − 1 ) cα ) eα ( cα ) β ≈ e − 1 ζ ( β − 1 ) cα eα ( cα ) β = e − ( e ζ ( β − 1 ) c ) α ( cα ) β = o ( 1 ) where we have c = (cid:17) 1 if β ≥ 3 1 − 2log ( β − 2 ) if 3 > β > 2 In other words , a . s . there is a component of size cα + 1 = Θ ( log n ) . Therefore , the second largest component has size Θ ( log n ) . Moreover , the above argument holds if we replace cα by any small number . Hence , small components exhibit a continuous behavior . Case 2 : β = 2 . We choose x β = 10 α . We note that a component with more than 2 E / 3 edges must be unique . We will prove that almost surely the unique component contains all vertices with degree greater than 101 α 2 . So it contains ( 1 − o ( 1 ) ) E edges and it is the giant component . We further modify the branching process by starting from Y ∗ 0 ≥ (cid:19) 101 α 2 (cid:20) vertices . If the component has more than 2 E / 3 edges , we are done . Other - wise , the expected number of backeges is small . E ( W i ) ≤ 2 / 3 ( 1 − 2 / 3 ) 2 = 6 from inequality ( * ) . Hence , Y ∗ i satisfies : • Y ∗ 0 ≥ (cid:19) 101 α 2 (cid:20) . • − 1 ≤ Y ∗ i − Y ∗ i − 1 ≤ 10 α . • E ( Y ∗ i − Y ∗ i − 1 ) ≈ 1 E (cid:1) 10 α x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) − E ( W i ) > 10 − 2 − 6 = 2 18 By Azuma’s martingale inequality , we have P r ( Y ∗ i ≤ i ) ≤ P r ( Y ∗ i − E ( Y ∗ i ) ≤ − i < e − i 2 i ( 10 α ) 2 = o ( n − 1 ) provided i ≥ 101 α 2 . The above inequality implies that with proability at least 1 − o ( n − 1 ) , Y ∗ i ≥ i > 0 when i > (cid:19) 101 α 2 (cid:20) . Since Y ∗ i decreases at most by 1 at each step , Y ∗ i can not be zero if i ≤ (cid:19) 101 α 2 (cid:20) . So Y ∗ i > 0 for all i . In other words , a . s . the branching process will not stop . However , it is impossible to have Y ∗ n > 0 , that is a contradiction . Thus we conclude that the component must have at least 23 E edges . We note that a . s . all vertices with degree more than (cid:19) 101 α 2 (cid:20) are in the unique component with at least 23 E edges , hence the giant component . The probability that a random vertex is in the giant component is at most 1 E 101 α 2 (cid:2) x = 1 xe α x 2 ≈ 2 log α α The probability that there are 2 . 1 α log α vertices not in the giant component is at most ( 2 log α α ) 2 . 1 α log α = e − ( 2 . 1 + o ( 1 ) ) α = o ( n − 2 ) . Since there is at most n connected components , we conclude that a . s . a connected component of size greater that 2 . 1 α log α = Θ ( log n loglog n ) must be the giant component . Now we find a vertex v of degree x and x ≤ 0 . 9 α log α . The probability that all its neighbors are of degree 1 is ( 1 α ) x . The probability that no such vertex exists is at most ( 1 − ( 1 α ) x ) eαx 2 ≈ e − ( 1 α ) x eαx 2 = e − e 0 . 1 α x 2 = o ( 1 ) Hence , a . s . there is a vertex of degree x ≤ 0 . 9 α log α , which forms a connected component of size x + 1 . This proves that a . s . the second largest component has size Θ ( log n loglog n ) . Case 3 : 0 < β < 2 . We use the modified branching process by choosing x β = e ( 5 − 2 β ) α ( 6 − 2 β ) β . If a component has more than 2 E / 3 edges , it is the unqiue giant component and we are done . Otherwise , we have E ( W i ) ≤ 2 / 3 ( 1 − 2 / 3 ) 2 = 6 . 19 Hence , Y ∗ i satisfies : • Y ∗ 0 ≥ 5 C 2 e ( 2 − β ) α ( 3 − β ) β . • − 1 ≤ Y ∗ i − Y ∗ i − 1 ≤ e ( 5 − 2 β ) α ( 6 − 2 β ) β . • E ( Y ∗ i − Y ∗ i − 1 ) ≈ 1 E (cid:1) e ( 5 − 2 β ) α ( 6 − 2 β ) β x = 1 x ( x − 2 ) (cid:9) e α x β (cid:10) − E ( W i ) ≈ Ce α 2 β Here C is a constant depending only on β . By Azuma’s martingale inequality , we have P r ( Y ∗ i ≤ 1 2 Ce α 2 β i ) < P r ( Y ∗ i − E ( Y ∗ i ) ≤ − 1 2 Ce α 2 β i ) < e − ( 12 Ce α 2 β i ) 2 i ( e ( ( 5 − 2 β ) α ) / ( ( 6 − 2 β ) β ) ) 2 = o ( n − 1 ) provided i ≥ 5 C 2 e ( 2 − β ) α ( 3 − β ) β . The above inequality shows that with probability at least 1 − o ( n − 1 ) , Y ∗ i > 12 Ce α 2 β i > 0 provided i > 5 C 2 e ( 2 − β ) α ( 3 − β ) β . Since Y ∗ i decreases at most by 1 at each step , Y ∗ i can not be zero if i ≤ 5 C 2 e ( 2 − β ) α ( 3 − β ) β . So Y ∗ i > 0 for all i . In othe words , a . s . the branching processing will not stop . However , it is impossible to have Y ∗ n > 0 , that is a contradiction . So , a . s . all vertices with degree more than 5 C 2 e ( 2 − β ) α ( 3 − β ) β are in the giant component . The probability that a random vertex in the giant component is at most 1 E 5 C 2 e ( 2 − β ) α ( 3 − β ) β (cid:2) x = 1 x e α x β = Θ ( e − ( 2 − β ) α ( 3 − β ) β ) The probability that all 2 (cid:9) 3 − β 2 − β (cid:10) + 1 vertices are not in the giant vertex is at most ( Θ ( e − ( 2 − β ) α ( 3 − β ) β ) ) 2 (cid:10) 3 − β 2 − β (cid:11) + 1 = o ( n − 2 ) . Since there is at most n connected component , we conclude that a . s . a connected component of size greater that 2 (cid:9) 3 − β 2 − β (cid:10) = Θ ( 1 ) must be the giant component . For 1 < β < 2 , we fix a vertex v of degree 1 . The probability that the other vertex that connects to v is also of degree 1 is Θ ( e α e 2 αβ ) 20 Therefore the probability that no component has size of 2 is at most ( 1 − Θ ( e α e 2 αβ ) ) e α ≈ e − Θ ( e 2 α − 2 αβ ) ≈ o ( 1 ) In other words , the graph a . s . has at least one component of size 2 . For 0 < β < 1 , we want to show that the random graph is a . s . con - nected . Since the size of the possible second largest component is bounded by a constant M , all vertices of degree ≥ M are almost surely in the giant component . We only need to show the probability that there is an edge connecting two small degree vertices is small . There are only M (cid:2) x = 1 x (cid:9) e α x β (cid:10) ≈ Ce α vertices with degree less than M . For any random pair of vertices ( u , v ) , the probability that there is an edges connecting them is about 1 E = Θ ( e − 2 αβ ) Hence the probability that there is edge connecting two small degree vertices is at most (cid:2) u , v 1 E = ( Ce α ) 2 Θ ( e 2 αβ ) = o ( 1 ) Hence , every vertex is a . s . connected to a vertex with degree ≥ M , which a . s . belongs to the giant exponent . Hence , the random graph is a . s . connected . (cid:1) 6 Comparisons with realistic massive graphs Our ( α , β ) - random graph model was originally derived from massive graphs generated by long distance telephone calls . These so - called call graphs are taken over different time intervals . For the sake of simplicity , we consider all the calls made in one day . Every completed phone call is an edge in the graph . Every phone number which either originates or receives a call is a node in the graph . When a node originates a call , the edge is directed out of the node and contributes to that node’s outdegree . Likewise , when a node receives a call , the edge is directed into the node and contributes to that node’s indegree . 21 In Figure 2 , we plot the number of vertices versus the indegree for the call graph of a particular day . Let y ( i ) be the number of vertices with indegree i . For each i such that y ( i ) > 0 , a × is marked at the coordinate ( i , y ( i ) ) . As similar plot is shown in Figure 1 for the outdegree . Plots of the number of vertices versus the indegree or outdegree for the call graphs of other days are very similar . For the same call graph in Figure 3 we plot the number of connected components for each possible size . The degree sequence of the call graph does not obey perfectly the ( α , β ) - graph model . The number of vertices of a given degree does not even monotonically decrease with increasing degree . Moreover , the call graph is directed , i . e . , for each edge there is a node that originates the call and a node that receives the call . The indegree and outdegree of a node need not be the same . Clearly the ( α , β ) - random graph model does not capture all of the random behavior of the real world call graph . Nonetheless , our model does capture some of the behavior of the call graph . To see this we first estimate α and β of Figure 2 . Recall that for an ( α , β ) - graph , the number of vertices as a function of degree is given by log y = α − β log x . By approximating Figure 2 by a straight line , β can be estimated using the slope of the line to be approximately 2 . 1 . The value of e α for Figure 2 is approximately 30 × 10 6 . The total number of nodes in the call graph can be estimated by ζ ( 2 . 1 ) ∗ e α = 1 . 56 ∗ e α ≈ 47 × 10 6 For β between 2 and β 0 , the ( α , β ) - graph will have a giant component of size Θ ( n ) . In addition , a . s . , all other components are of size O ( log n ) . Moreover , for any 2 ≥ x ≥ O ( log n ) , a component of size x exists . This is qualitatively true of the distribution of component sizes of the call graph in Figure 3 1 . The one giant component contains nearly all of the nodes . The maximum size of the next largest component is indeed exponentially smaller than the size of the giant component . Also , a component of nearly every size below this maximum exists . Interestingly , the distribution of the number of components of size smaller than the giant component is nearly log - log linear . This suggests that after removing the giant component , one is left with an ( α , β ) - graph with β > 4 ( Theorem 1 yields a log - log linear relation between number of components and component size for β > 4 . ) This intuitively seems true since the greater the degree , the fewer nodes of that degree we expect to remain after deleting the giant component . This will increase the value of β for the resulting graph . 1 This data was compiled by J . Abello and A . Buchsbaum of AT & T Labs from raw phone call records using , in part , the external memory algorithm of Abello , Buchsbaum , and Westbrook [ 1 ] for computing connected components of massive graphs . 22 There are numerous questions that remain to be studied . For example , what is the effect of time scaling ? How does it correspond with the evolution of β ? What are the structural behaviors of the call graphs ? What are the correlations between the directed and undirected graphs ? It is of interest to understand the phase transition of the giant component in the realistic graph . In the other direction , the number of tiny components of size 1 is leading to many interesting questions as well . Clearly , there is much work to be done in our understanding of massive graphs . Acknowledgments . We are grateful to J . Feigenbaum , J . Abello , A . Buchs - baum , J . Reeds , and J . Westbrook for their assistance in preparing the figures and for many interesting discussions on call graphs . We are very thankful to the anonymous referees for their invaluable comments . References [ 1 ] J . Abello , A . Buchsbaum , and J . Westbrook , A functional approach to external graph algorithms , Proc . 6th European Symposium on Algorithms , pp . 332 – 343 , 1998 . [ 2 ] W . Aiello , F . Chung , L . Lu , A random graph model for massive graphs , Pro - ceedings of the Thirtysecond Annual ACM Symposium on Theory of Comput - ing , ( 2000 ) , to appear . [ 3 ] W . Aiello , F . Chung , L . Lu , Random evolution of power law graphs , manu - script . [ 4 ] R . Albert , H . Jeong and A . Barab´asi , Diameter of the World Wide Web , Nature , 401 , September 9 , 1999 . [ 5 ] N . Alon and J . H . Spencer , The Probabilistic Method , Wiley and Sons , New York , 1992 . [ 6 ] A . Barab´asi , and R . Albert , Emergence of scaling in random networks , Science , 286 , October 15 , 1999 . [ 7 ] A . Barab´asi , R . Albert , and H . Jeong Scale - free characteristics of random networks : the topology of the world wide web , Elsevier Preprint August 6 , 1999 . [ 8 ] P . Erd˝os and A . R´enyi , On the evolution of random graphs , Publ . Math . Inst . Hung . Acad . Sci . 5 ( 1960 ) , 17 – 61 . [ 9 ] P . Erd˝os and A . R´enyi , On the strength of connectedness of random graphs , Acta Math . Acad . Sci . Hungar . 12 ( 1961 ) , 261 - 267 . 23 [ 10 ] M . Faloutsos , P . Faloutsos , and C . Faloutsos , On power - law relationships of the internet topology , Proceedings of the ACM SIGCOM Conference , Cambridge , MA , 1999 . [ 11 ] Brian Hayes , Graph theory in practice : Part II , American Scientists , 88 , ( March - April , 2000 ) , 104 - 109 . [ 12 ] J . Kleinberg , S . R . Kumar , P . Raphavan , S . Rajagopalan and A . Tomkins , The web as a graph : Measurements , models and methods , Proceedings of the International Conference on Combinatorics and Computing , July 26 – 28 , 1999 . [ 13 ] S . R . Kumar , P . Raphavan , S . Rajagopalan and A . Tomkins , Trawling the web for emerging cyber communities , Proceedings of the 8th World Wide Web Conference , Edinburgh , Scotland , May 15 – 19 , 1999 . [ 14 ] S . R . Kumar , P . Raghavan , S . Rajagopalan and A . Tomkins , Extracting large - scale knowledge bases from the web , Proceedings of the 25th VLDB Conference , Edinburgh , Scotland , September 7 – 10 , 1999 . [ 15 ] Tomasz HLuczak , Sparse random graphs with a given degree sequence , Random Graphs , vol 2 ( Pozna´n , 1989 ) , 165 - 182 , Wiley , New York , 1992 . [ 16 ] Michael Molloy and Bruce Reed , A critical point for random graphs with a given degree sequence . Random Structures and Algorithms , Vol . 6 , no . 2 and 3 ( 1995 ) . 161 - 179 . [ 17 ] Michael Molloy and Bruce Reed , The size of the giant component of a random graph with a given degree sequence , Combin . Probab . Comput . 7 , no . ( 1998 ) , 295 - 305 . [ 18 ] P . Raghavan , personal communication . [ 19 ] N . C . Wormald , The asymptotic connectivity of labelled regular graphs , J . Comb . Theory ( B ) 31 ( 1981 ) , 156 - 167 . [ 20 ] N . C . Wormald , Models of random regular graphs , Surveys in Combinatorics , 1999 ( LMS Lecture Note Series 267 , Eds J . D . Lamb and D . A . Preece ) , 239 – 298 . [ 21 ] 24 1e + 00 1e + 01 1e + 02 1e + 03 1e + 04 1e + 05 Outdegree 1e + 00 1e + 01 1e + 02 1e + 03 1e + 04 1e + 05 1e + 06 1e + 07 N u m b er o f v er t i ce s 8 / 10 / 98 1e + 00 1e + 01 1e + 02 1e + 03 1e + 04 1e + 05 Indegree 1e + 00 1e + 01 1e + 02 1e + 03 1e + 04 1e + 05 1e + 06 1e + 07 N u m b er o f v er t i ce s 8 / 10 / 98 Figure 1 : The number of vertices for each possible outdegree for the call graph of a typical day . Figure 2 : The number of vertices for each possible indegree for the call graph of a typical day . 1e + 00 1e + 01 1e + 02 1e + 03 1e + 04 1e + 05 1e + 06 1e + 07 Component size 1e + 00 1e + 01 1e + 02 1e + 03 1e + 04 1e + 05 1e + 06 N u m b er o f c o m p o n e n t s 8 / 10 / 98 Figure 3 : The number of connected components for each possible component size for the call graph of a typical day . 25 \ No newline at end of file diff --git a/silver_data/886e7e5baf212c7add7725c7f75cb230ee4dbe16.pdf.txt b/silver_data/886e7e5baf212c7add7725c7f75cb230ee4dbe16.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..36a28b4d7dd7395824ae5ddd8837458541bf7cee --- /dev/null +++ b/silver_data/886e7e5baf212c7add7725c7f75cb230ee4dbe16.pdf.txt @@ -0,0 +1 @@ +Escaping Death : How Cancer Cells and Infected Cells Resist Cell - Mediated Cytotoxicity Karoliina Tuomela , Ashley R . Ambrose and Daniel M . Davis * The Lydia Becker Institute of Immunology and In fl ammation , The University of Manchester , Manchester , United Kingdom Cytotoxic lymphocytes are critical in our immune defence against cancer and infection . Cytotoxic T lymphocytes and Natural Killer cells can directly lyse malignant or infected cells in at least two ways : granule - mediated cytotoxicity , involving perforin and granzyme B , or death receptor - mediated cytotoxicity , involving the death receptor ligands , tumour necrosis factor - related apoptosis - inducing ligand ( TRAIL ) and Fas ligand ( FasL ) . In either case , a multi - step pathway is triggered to facilitate lysis , relying on active pro - death processes and signalling within the target cell . Because of this reliance on an active response from the target cell , each mechanism of cell - mediated killing can be manipulated by malignant and infected cells to evade cytolytic death . Here , we review the mechanisms of cell - mediated cytotoxicity and examine how cells may evade these cytolytic processes . This includes resistance to perforin through degradation or reduced pore formation , resistance to granzyme B through inhibition or autophagy , and resistance to death receptors through inhibition of downstream signalling or changes in protein expression . We also consider the importance of tumour necrosis factor ( TNF ) - induced cytotoxicity and resistance mechanisms against this pathway . Altogether , it is clear that target cells are not passive bystanders to cell - mediated cytotoxicity and resistance mechanisms can signi fi cantly constrain immune cell - mediated killing . Understanding these processes of immune evasion may lead to novel ideas for medical intervention . Keywords : cell - mediated cytotoxicity , cytolytic T cells , natural killer cells , lymphocytes , cancer , resistance , viral infection , immune synapse INTRODUCTION Cytotoxic lymphocytes , including cytotoxic T lymphocytes ( CTLs ) and natural killer ( NK ) cells , are able to directly lyse malignant or infected cells using multiple mechanisms . Granule - mediated cytotoxicity involves the release of lytic granules containing perforin and granzymes , while death receptor - mediated cytotoxicity utilises tumour necrosis factor - related apoptosis - inducing ligand ( TRAIL ) or Fas ligand ( FasL ) that bind death receptors on the surface of the target cell ( 1 – 3 ) . In addition to these classic cytotoxic pathways , there is increasing evidence that the tumour necrosis factor ( TNF ) pathway also signi fi cantly contributes to lymphocyte cytotoxicity ( 4 ) . Activation of these pathways can lead to cell death in several forms , including necrosis , apoptosis , necroptosis , and pyroptosis ( 5 ) . Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 1 Edited by : Michael Loran Dustin , University of Oxford , United Kingdom Reviewed by : Julian Pardo , Fundacion Agencia Aragonesa para la Investigacion y el Desarrollo , Spain Cle ´ ment Thomas , Luxembourg Institute of Health , Luxembourg * Correspondence : Daniel M . Davis daniel . davis @ manchester . ac . uk orcid . org / 0000 - 0002 - 9182 - 291X Specialty section : This article was submitted to T Cell Biology , a section of the journal Frontiers in Immunology Received : 31 January 2022 Accepted : 04 March 2022 Published : 23 March 2022 Citation : Tuomela K , Ambrose AR and Davis DM ( 2022 ) Escaping Death : How Cancer Cells and Infected Cells Resist Cell - Mediated Cytotoxicity . Front . Immunol . 13 : 867098 . doi : 10 . 3389 / fimmu . 2022 . 867098 REVIEW published : 23 March 2022 doi : 10 . 3389 / fimmu . 2022 . 867098 Lymphocyte cytotoxicity is triggered upon contact with a cancerous or infected target cell if suf fi cient activating signals are received . For CTLs , this requires initial priming by antigen presenting cells followed by T cell receptor recognition of speci fi c target cell antigens , whereas activating receptors on NK cells recognise a range of germline - encoded ligands without prior activation ( 6 , 7 ) . The area of cell - cell contact between a lymphocyte and a target cell is termed an immune synapse , on account of it being a highly organised interface involving cytoskeletal and membrane rearrangement ( 8 – 12 ) . Integration of multiple activating and inhibitory pathways within the lymphocyte determines the outcome of this interaction with a target cell ( 13 , 14 ) . Malignant or infected cells may , therefore , evade immune recognition through the downregulation of activating signals or the upregulation of inhibitory signals . These immune evasion mechanisms – for example reducing antigen recognition , triggering immune checkpoints , secreting immunosuppressive cytokines , as well as excluding immune cells from the microenvironment – have been extensively reviewed elsewhere ( 15 – 18 ) . And of course , many immunotherapies have been developed to target these types of immune escape mechanisms , such as checkpoint inhibitors , adoptive cell therapy , and cancer vaccines , all of which aim to enhance immune cell activation ( 6 , 19 ) . However , evasion of lymphocyte cytotoxicity may also occur downstream of immune cell activation . Even if a cytolytic response is triggered by the lymphocyte , target cells are not passive bystanders to lymphocyte cytotoxicity . This is because mechanisms of cell death generally rely on active pro - death signalling within the target cell ( 5 ) . Indeed , evasion of cell death and apoptosis is considered a critical hallmark of cancer ( 20 ) . Therefore , resistance to lymphocyte attack may arise through resistance to the mediators of cytotoxicity , including perforin , granzymes , and death receptor ligands . Here we review the molecular details behind cell - mediated killing and then examine our current understanding of how target cells may resist these cytotoxic processes as immune evasion strategies . MECHANISMS OF LYMPHOCYTE CYTOTOXICITY Granule - Mediated Cytotoxicity Following activation , effector cells polarise their microtubule organising centre ( MTOC ) and lytic granules towards the immune synapse , then release the contents of these granules across the synaptic cleft ( 21 – 23 ) . Granule - mediated cytotoxicity is dependent upon the release of perforin and granzymes from granules contained within cytotoxic lymphocytes ( Figure 1 ) . Perforin is a pore - forming protein that forms ring - shaped lesions capable of mediating ion fl ux as well as the uptake of larger molecules , such as granzymes ( 24 ) . Granzymes are a family of serine proteases that cleave a variety of target proteins within cells in order to induce apoptosis . Five granzymes have been identi fi ed in humans , A , B , H , K , and M , but granzyme A and B have been characterised most extensively ( 25 , 26 ) . Granule - mediated cytotoxicity can result in cell death through two mechanisms ( Figure 1 ) . The fi rst is necrotic cell death induced by rapid osmotic fl ux through perforin pores and membrane rupture , which can be observed upon exposure to high concentrations of perforin ( 27 , 28 ) . The second is apoptotic cell death induced by perforin - mediated uptake of granzyme B into the target cell . Two primary models have been proposed to account for the entry of granzyme B into target cells : direct diffusion through perforin pores in the cell membrane , or perforin - induced endosomal uptake of granzymes . Perforin pores observed by electron microscopy have been measured to be physically large enough to permit the diffusion of granzymes into the target cell cytoplasm ( 29 ) . Furthermore , intracellular granzyme B activity can be observed within minutes of adding exogenous granzyme B and perforin to target cells ( 30 ) , which is faster than has been observed for endosomal uptake of granzyme B ( 31 ) . Conversely , perforin has been shown to trigger a calcium - dependent membrane repair response that triggers the endocytosis of granzyme B , and granzyme B - positive endosomes can be observed within target cells following interaction with NK cells ( 31 – 33 ) . It is possible that these two pathways work in parallel or in different cellular contexts to facilitate the uptake of granzyme B into target cells . Recent evidence demonstrated that perforin and granzyme can also be secreted from CTLs and NK cells in complexes , termed supramolecular attack particles ( SMAPs ) , bound by the adhesive glycoprotein thrombospondin - 1 ( 34 , 35 ) . Further understanding of SMAP biology may shed light on whether or not the two models of granzyme B delivery work synergistically or independently . Once within a target cell , granzymes can trigger cell death through several pathways , and the speci fi c pathways that are activated are dependent on the identity of the granzyme . Granzyme B is the most potent member of the granzyme family and can induce apoptosis within minutes of delivery ( 30 ) . This occurs through either direct cleavage of caspases by granzyme B or via activation of the mitochondrial pathway of apoptosis . Direct cleavage of caspases , such as caspase 3 , one of the executioner caspases , is the primary mechanism by which mouse granzyme B induces apoptosis ( 25 ) . Caspase 3 cleaves several targets including inhibitor of caspase - activated DNase ( ICAD ) and gelsolin , which leads to DNA damage and cytoskeletal disruption , respectively ( 36 ) . Both human and mouse granzyme B can also trigger the mitochondrial apoptotic pathway , which is characterised by mitochondrial outer membrane permeabilization ( MOMP ) ( 37 – 39 ) . MOMP is regulated by the Bcl - 2 family , which is made up of three classes : pro - apoptotic BH3 proteins ( e . g . Bid , Bim ) , pro - apoptotic effector proteins ( e . g . Bax , Bak ) , and anti - apoptotic proteins ( e . g . Bcl - 2 ) ( 40 ) . Granzyme B triggers MOMP by cleaving Bid into its active , truncated form ( tBid ) ( 37 ) . After cleavage , tBid recruits the pore - forming effector proteins , Bax and Bak , to the mitochondrial membrane where they form pores that mediate MOMP and the release of additional apoptotic mediators , such as cytochrome c ( 40 ) . This process can be Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 2 opposed by the action of anti - apoptotic Bcl - 2 proteins , which bind and inhibit pro - apoptotic Bcl - 2 members ( 40 ) . Importantly , granzyme B can also induce degradation of these anti - apoptotic Bcl - 2 proteins , such as Mcl - 1 , leading to the release of the pro - apoptotic BH3 protein , Bim , which activates Bax / Bak and triggers apoptosis ( 38 , 41 ) . A granzyme B - mediated mitochondrial apoptotic pathway , independent of Bax / Bak , has also been identi fi ed , and occurs through cleavage of mitochondrial proteins involved in the electron transport chain and production of reactive oxygen species ( 42 – 44 ) . Granzyme B can also cleave several additional targets that contribute to cell death . For example , granzyme B can directly cleave the caspase 3 substrate , ICAD , leading to DNA damage ( 45 ) or cleave a - tubulin , causing cytoskeletal disruption during apoptosis ( 46 , 47 ) . Recently , granzyme B was also found to cleave and activate the pore - forming protein gasdermin E , leading to an alternate form of cell death , pyroptosis , through the formation of gasdermin pores in the cell membrane ( 48 , 49 ) . Pyroptosis is a more in fl ammatory form of cell death compared to apoptosis and relies on the formation of pores in the cell membrane by members of the gasdermin family ( 5 , 50 ) . Cleavage of gasdermin E by granzyme B is a potent mechanism by which cytotoxic lymphocytes can kill cancer cells and control tumour growth ( 48 ) . FIGURE 1 | Mechanisms of lymphocyte cytotoxicity . Following activation , cytotoxic effector cells can kill through granule - mediated cytotoxicity , death receptor - mediated cytotoxicity , or TNF - mediated cytotoxicity . ( A ) During granule - mediated cytotoxicity , perforin and granzymes are released from lytic granules into the synaptic cleft . Perforin forms pores in the target cell membrane . At high concentrations of perforin , osmotic fl ux through pores leads to cell swelling and necrotic cell death . Perforin can facilitate the uptake of granzyme B through direct diffusion or endocytosis . Granzyme B directly cleaves caspase 3 to induce apoptosis or triggers the mitochondrial apoptotic pathway via Bid cleavage into tBid . tBid recruits Bax / Bak leading to mitochondrial outer membrane permeabilization ( MOMP ) and apoptosis . Granzyme B may also degrade Mcl - 1 releasing Bim to activate MOMP . Granzyme B can also cleave ICAD contributing to DNA damage , a - tubulin leading to cytoskeletal degradation , or gasdermin E , which forms pores in the cell membrane to induce pyroptosis . ( B ) Ligation of death receptors ( Fas / DR4 / DR5 ) by FasL or TRAIL triggers assembly of the death - inducing signalling complex ( DISC ) composed of FADD and pro - caspase 8 / 10 . Caspase 8 / 10 induces apoptosis via direct caspase 3 cleavage or the mitochondrial apoptotic pathway via Bid cleavage . ( C ) Ligation of TNFR1 by TNF triggers the assembly of complex I ( TRADD , RIPK1 , TRAF2 , cIAP1 / 2 ) . LUBAC ubiquitinates complex I components leading to pro - survival signalling via NF - k B and MAPK pathways . In the absence of ubiquitination , RIPK1 dissociates and forms complex II with FADD and pro - caspase 8 / 10 . Cleavage of pro - caspase 8 / 10 triggers apoptosis by the same pathways as FasL / TRAIL . In the presence of insuf fi cient pro - caspase 8 , RIPK1 can also recruit RIPK3 , which activates MLKL to trigger necroptosis . Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 3 Compared to granzyme B , granzyme A is a far less ef fi cient inducer of cell death and triggers apoptosis at a slower rate ( 51 ) . Granzyme A - induced apoptosis is caspase - independent and , although its targets are not fully de fi ned , is mediated by cleavage of a variety of nuclear , mitochondrial , and cytosolic proteins ( 52 , 53 ) . Recently , granzyme A was also shown to trigger pyroptosis of cancer cells through cleavage of the pore - forming protein , gasdermin B ( 54 , 55 ) . In murine tumours , gasdermin B expression synergised with checkpoint blockade to promote tumour clearance by cytotoxic lymphocytes ( 54 ) . Likewise , in the context of infection by Shigella fl exneri , granzyme A secreted by NK cells was found to cleave gasdermin B within the infected cell ( 55 ) . Cleaved gasdermin B demonstrated microbiocidal activity by forming pores within the bacterial membrane in order to protect the host cell . Less is known about the function and role of the other granzymes ( K , H , and M ) expressed by human lymphocytes . Death Receptor - Mediated Cytotoxicity Cytotoxic lymphocytes may also kill target cells through the expression of ligands for death receptors . Two prototypical ligands have been identi fi ed that mediate apoptosis : FasL , which binds the Fas receptor , and TRAIL , which binds death receptors 4 and 5 ( DR4 / 5 ) ( 56 ) . Although FasL and TRAIL bind different receptors , both ligands trigger similar pro - apoptotic signalling ( Figure 1 ) . Both FasL and TRAIL are transmembrane proteins that belong to the TNF superfamily and can be expressed on cytotoxic immune cells upon cytokine stimulation or interaction with a target cell ( 1 , 57 ) . Upon binding of FasL or TRAIL to their respective receptors , assembly of the death - inducing signalling complex ( DISC ) is triggered . The DISC consists of the death receptor , Fas - associated death domain protein ( FADD ) , and pro - caspase 8 or 10 ( 56 ) . The DISC mediates cleavage of pro - caspase 8 / 10 to release the active caspase , which can then activate the executioner caspases 3 , 6 , and 7 . Caspase 8 can further amplify apoptotic signalling by cleaving Bid to activate the mitochondrial pathway of apoptosis , similar to granzyme B ( 56 ) . TNF - Mediated Cytotoxicity TNF is a cytokine capable of inducing both pro - survival and pro - death signalling depending on the precise cellular context . Although the receptors for TNF , TNF - R1 and TNF - R2 , belong to the same family as the receptors for FasL and TRAIL , the downstream signalling pathways are distinct ( 4 ) . Of the two receptors for TNF , only TNF - R1 is able to trigger cell death through its cytoplasmic death domain , which recruits a key adaptor protein , TNF receptor - associated death domain ( TRADD ) ( 4 ) . Conversely , both TNF - R1 and TNF - R2 contain a TNFR - associated factor ( TRAF ) binding site that recruits TRAF1 / 2 , which is involved in triggering pro - survival signalling via the NF - k B and MAPK pathways . The pro - survival and pro - death signalling pathways controlled by TNF - R1 ligation are mediated by the assembly of two signalling complexes , complex I and II , respectively ( 4 , 58 ) . Complex I , which mediates pro - survival signalling , is composed of several proteins , including receptor interacting serine / threonine protein kinase 1 ( RIPK1 ) , TRAF2 / 5 , cellular inhibitor of apoptosis 1 / 2 ( cIAP1 / 2 ) , and linear Ub chain assembly complex ( LUBAC ) . LUBAC - mediated ubiquitination of complex I components leads to the recruitment of additional kinase complexes involved in NF - k B and MAPK survival signalling ( 4 ) . Survival signalling through complex I is generally the default pathway triggered by TNF - R1 ligation . However , in certain cell states , TNF - R1 signalling can switch instead to pro - death signalling mediated by complex II , which is composed of RIPK1 , FADD , and pro - caspase 8 ( 4 , 58 , 59 ) . Assembly of complex II occurs when RIPK1 is not ubiquitinated , such as in the absence of the complex I - associated proteins cIAP1 / 2 and LUBAC ( 59 , 60 ) . Non - ubiquitinated RIPK1 dissociates from TRADD and recruits FADD and pro - caspase 8 leading to similar pro - death signalling as TRAIL / FasL ( 4 ) . When caspase 8 activation is not suf fi cient , complex II can also lead to necroptotic cell death . This occurs through autophosphorylation of RIPK1 , leading to the recruitment and autophosphorylation of RIPK3 followed by activation of mixed lineage kinase domain - like ( MLKL ) , which induces necroptosis ( 4 ) . Altogether , much remains to be understood about the signalling that regulates the varying effects of TNF and how this can change in different cell states . MECHANISMS OF RESISTANCE TO CYTOTOXICITY Overall , there is a reliance on active processes and signalling pathways within the target cell to execute CTL and NK cell killing . This implies that each distinct mechanism of cell - mediated killing can be open to an evasion strategy by the target cell . Indeed , malignant and infected cells develop a variety of mechanisms to evade cytolytic death . The existence of these resistance mechanisms is readily observed when tracking interactions between cytotoxic lymphocytes and target cells in vitro . Even when CTLs are activated during an interaction with a target cell – as indicated by a rapid increase in calcium concentration within the effector cell – the target cell does not always die ( 61 , 62 ) . In some cases , CTLs may produce a sublethal hit , which is characterised by a transient calcium fl ux , indicative of perforin pore formation , but no cell death ( 62 ) . Alternatively , in some effector - target interactions , no calcium fl ux could be observed in the target cell despite apparent activation of the effector cell , indicating that perforin did not form pores in the target cell membrane . A similar study demonstrated that cancer cells often recover even after a cytotoxic hit that triggers a large calcium fl ux , structural perturbations , and DNA damage ( 61 ) . The target cancer cells were observed to rapidly restore calcium homeostasis , recover nuclear integrity after structural damage , and even repair DNA to stop apoptosis at its later stages ( 61 ) . Clearly , not every interaction between a cytotoxic lymphocyte and its target results in death . While some of this survival could be attributed to stochastic variability , speci fi c methods by which target cells can evade cytotoxicity can have a profound impact on Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 4 the ability of cytotoxic lymphocytes to eliminate cancer cells and infected cells in vitro . Resisting Perforin Pore - Formation The formation of perforin pores in target cell membranes is the fi rst step of granule - mediated cytotoxicity and is required for the induction of both necrosis caused by osmotic fl ux and apoptosis caused by granzyme B uptake . Therefore , resistance to this initial step of granule - mediated cytotoxicity has the potential to signi fi cantly reduce lymphocyte cytotoxicity . Perforin resistance was initially identi fi ed as a characteristic of cytotoxic lymphocytes , including CTLs and NK cells , which are less easily killed by puri fi ed perforin compared to various non - cytotoxic cell lines ( 63 – 65 ) . This resistance is thought to be integral to the survival of lymphocytes when releasing their cytotoxic cargo . Resistance to perforin has also been observed in malignant cells ( 66 – 68 ) . In studies of patient - derived leukaemia and lymphoma samples , considerable variability was observed in the ability of perforin to bind and lyse cancer cells from different patients ( 66 , 67 ) . Importantly , the susceptibility of cancer cells to perforin - induced lysis closely correlated with the amount of perforin bound ( 66 ) , indicating that cancer cells may evade perforin by reducing binding . More recently , our own research has found that in vitro irradiation of cancer cells transiently reduces susceptibility to lysis by NK cells and CAR T cells by inducing resistance to perforin , possibly by preventing pore formation ( 68 ) . Our current understanding of the mechanisms that mediate perforin resistance in malignant or infected cells is predominantly derived from protective mechanisms employed by cytotoxic lymphocytes . These mechanisms of perforin resistance include altered lipid order , phosphatidylserine exposure , modulation of cell stiffness , and cathepsin B - mediated degradation ( Figure 2 ) , each of which we will now explore in detail . Lipid Order The lipid order of a membrane is a characteristic determined by several properties : lipid packing , the rotational freedom of lipids , and the thickness of the bilayer ( 69 ) . Therefore , changes to membrane composition can alter lipid order , such as increasing lipid order upon incorporation of cholesterol ( 70 ) . From the time of its discovery , perforin has been known to preferentially bind to synthetic liposomes or planar lipid bilayers composed of low - order , fl uid - phase lipids , such as 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( DOPC ) , compared to high - order , gel - phase lipids , such as dipalmitoylphosphatidylcholine ( DPPC ) ( 71 – 74 ) . This speci fi city for lipid order appears to play a critical role in protecting cytotoxic lymphocytes against self - harm by perforin since CTLs have particularly tightly packed and ordered membranes ( 72 , 73 ) . Lipid order has also been observed to particularly increase at the site of the immune synapse compared to more distal areas of the lymphocyte membrane ( 75 – 77 ) . Replacing cholesterol in CTL membranes with a disorder - prone cholesterol variant increases perforin binding and sensitises cells to pore - induced lysis , consistent with tight lipid packing being protective against perforin pore formation ( 73 ) . Alterations in lipid packing may directly affect the susceptibility of cancer cells to perforin - mediated attack . Lymphocyte - resistant breast cancer cells , for example , have been found to increase lipid order at the site of the immune synapse and , similar to observations with CTLs , replacing cholesterol with a disorder - prone variant sensitises cancer cells to perforin - induced lysis ( 73 , 75 ) . More broadly , lipid composition is often highly altered during malignancy and infection . For example , multidrug resistant cancer cells frequently exhibit increased membrane lipid order due to increased cholesterol levels ( 78 , 79 ) . Therefore , it is possible that lipid order - mediated resistance to perforin is a common feature of cancer . However , most observations have been made in vitro and further work is necessary to understand whether alterations in the lipid order of cancer cell membranes can have a signi fi cant impact on tumour control by lymphocytes in vivo . Phosphatidylserine Externalisation Apart from altering lipid order , membrane composition can affect perforin activity through other mechanisms . Phosphatidylserine is a negatively charged phospholipid that generally localises to the intracellular lea fl et of the cell membrane but can be externalised to the outer lea fl et in certain cell states ( 80 ) . In particular , externalisation of phosphatidylserine on the outer lea fl et is often used as a marker of cell death , but it also has a variety of non - apoptotic roles for intercellular signalling ( 81 ) . Importantly , phosphatidylserine can be externalised on lymphocyte membranes at the immune synapse , where it is suggested to act as a protective mechanism against perforin pore formation ( 73 , 74 , 82 ) . Atomic force microscopy has shown that perforin is able to bind to phosphatidylserine - containing planar lipid bilayers , but it forms protein aggregates rather than membrane - spanning pores ( 73 , 74 ) . In addition , perforin shows little or no lytic activity against synthetic liposomes composed of high levels of phosphatidylserine ( 68 ) . Other negatively charged membrane lipids , such as 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - ( 1 ′ - rac - glycerol ) ( DOPG ) or cholesterol sulfate , had a similar effect in preventing perforin pore formation , suggesting that the negative charge of phosphatidylserine is critical for its inhibitory effect against perforin ( 74 ) . In addition to protecting cytotoxic lymphocytes , phosphatidylserine may also be utilised by infected or malignant cells to evade attack mediated by perforin . Phosphatidylserine exposure is a common feature of cancer cells and can be further enhanced in certain circumstances , such as following anti - cancer treatment ( 68 , 83 ) . Externalisation of phosphatidylserine following the treatment of cancer cells with radiotherapy or cell cycle inhibitors was found to closely correlate with resistance to perforin and lymphocyte cytotoxicity , despite normal recognition and activation by NK cells and CAR T cells ( 68 ) . Treatment of cancer cells with radiotherapy or cell cycle inhibitors did not affect perforin binding or membrane repair responses , suggesting that the mechanism of resistance was impaired pore formation , similar to the observed effects of phosphatidylserine in synthetic lipid membranes ( 68 , 73 , 74 ) . Increased surface phosphatidylserine on malaria - infected erythrocytes also correlated with reduced Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 5 susceptibility to perforin and reduced lysis by gd T cells ( 84 ) . The extent to which phosphatidylserine externalisation affects the elimination of target cells in vivo is unclear , and hard to establish . Whether this process could be targeted therapeutically is an open question . Cell Stiffness Physical properties of cells may also in fl uence susceptibility to perforin , including cell tension or stiffness . Cell stiffness is commonly altered during malignancy , with cancer cells being relatively soft and deformable compared to healthy cells ( 85 ) . Accordingly , soft CD133 + tumour - repopulating cells were found to be resistant to perforin and take up less granzyme B following interaction with T cells ( 86 ) . These cells also evaded T cell cytotoxicity in vivo but killing could be enhanced if cells were treated with jasplakinolide , which promotes actin polymerisation and increases cancer cell stiffness . Cell stiffness can also be arti fi cially altered by culturing cells on stiff or soft hydrogels , which directly changes the stiffness of cells to mirror the underlying substrate . Reducing stiffness in this way by culturing on a soft substrate has been shown to reduce susceptibility to perforin and lymphocyte cytotoxicity ( 87 ) . It is not entirely clear why cell stiffness affects perforin lytic ability in this way , but insertion and pore formation by hydrophobic molecules , such as perforin , is known to be more energetically favourable on stiff membranes ( 87 , 88 ) . Interestingly , cytotoxic lymphocytes have been found to utilise a mechanism which may counteract the reduced activity of perforin on soft cells . By using F - actin - rich protrusions , CTLs can exert lateral force on the target cell to increase membrane tension and enhance perforin pore formation ( 87 , 89 ) . Lytic granule secretion from CTLs was observed at the base of these FIGURE 2 | Resistance to granule - mediated cytotoxicity . Activation of a cytotoxic effector cell at an immune synapse with a target cell leads to the polarisation and secretion of lytic granules containing perforin and granzyme B . Under normal circumstances , in the absence of any resistance mechanisms , the secreted perforin will form pores in the target cell membrane and allow entry of granzyme B . This process will initiate cell death through both direct cell lysis and the activation of apoptotic pathways . Target cells can employ multiple mechanisms to evade cytotoxicity . ( A ) Increased plasma membrane lipid order to reduce perforin binding . ( B ) Externalisation of phosphatidylserine to induce perforin aggregation rather than pore formation . ( C ) Reduced cell stiffness to prevent ef fi cient perforin pore formation . ( D ) Expression of Serpin B9 to directly inhibit Granzyme B activity . ( E ) Autophagy of Granzyme B to prevent activation of apoptotic pathways . ( F ) Secretion of Cathepsin B to degrade perforin . ( G ) Reduced gasdermin B and E expression or IpaH7 . 8 - mediated ubiquitination and degradation of gasdermin B can reduce pyroptosis or lysis of shigella , respectively . Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 6 protrusions so that it was spatially localised to the areas of force exertion on the target cell membrane ( 89 ) . It is interesting to note that the stiffness of a target cell can also modulate NK cell and CTL activation itself , with activation signi fi cantly reduced against target cells exhibiting a soft phenotype or grown on a soft substrate ( 90 , 91 ) . Thus , alterations in cell stiffness at the whole - cell level , such as during malignancy , or at the nanoscale , at the immune synapse , may potently manipulate the sensitivity of cancerous or infected cells to perforin . Cathepsin B The pore - forming activity of perforin may also be reduced through direct degradation . Cysteine cathepsins are lysosomal peptidases with multiple roles in regulating immune responses ( 92 ) . It was initially shown that cathepsin B , which becomes expressed on the surface of lymphocytes following degranulation , can degrade perforin in order to protect against self - harm ( 93 ) . Inhibition of this surface - bound cathepsin B using the inhibitor CA074 led to enhanced CTL death after degranulation . Seemingly in contrast to this , it was later shown that CTLs from cathepsin B - null mice were not more susceptible to death following interaction with a target cell compared to cells from wild type mice ( 94 ) . To reconcile these observations , it is possible that there is redundancy in the system , and perhaps compensatory mechanisms are augmented in cathepsin B - null mice , such as increased lipid order or phosphatidylserine exposure , as discussed previously . More recent evidence has also linked cathepsin B with cancer cell resistance against lymphocyte cytotoxicity . Khazen et al . ( 95 ) showed that melanoma cells that were resistant to CTL cytotoxicity bound less perforin and took up less granzyme B despite inducing similar levels of CTL degranulation . This resistance was mediated by increased exocytosis of lysosomes or late endosomes at the immune synapse , which facilitated the secretion of cathepsin B leading to perforin degradation . Interestingly , cathepsin B is known to be overexpressed in multiple cancers , where it is associated with poor survival and metastasis ( 96 ) . Altogether , resistance to perforin through physical or degradative inhibition is an emerging aspect of immune resistance . Many of the mechanisms employed by cytotoxic lymphocytes to protect against self - harm appear to also be exploited by malignant or infected cells to inhibit perforin activity and enhance survival . However , the contribution of perforin resistance to immune escape has not yet been extensively explored in vivo . The ability to modify cancer cells pharmacologically to increase perforin susceptibility may be a way to increase the ef fi cacy of lymphocyte cytotoxicity . Resisting Granzyme - Mediated Apoptosis In addition to perforin , granzymes are a critical component of granule - mediated cytotoxicity . Following perforin pore formation , granzymes enter the target cell and cleave a variety of targets to potently induce cell death . As a result , reduced granzyme activity within target cells may critically constrain killing by lymphocytes . Granzyme activity may be reduced in two ways : through reduced granzyme uptake as a result of perforin inhibition , as described previously , or through direct inhibition of granzyme function . The effect of reduced granzyme uptake on cytotoxicity was clearly demonstrated by the fi nding that cancer cells , which were resistant to NK cell cytotoxicity , can undergo extensive cytoskeletal remodelling , which reduces granzyme uptake ( 97 , 98 ) . Although the underlying link between cytoskeletal remodelling and reduced granzyme uptake was not identi fi ed in these studies , cytoskeletal inhibitors were found to restore granzyme levels and cytotoxicity following interaction with NK cells . Granzyme - induced cell death may also be reduced by direct inhibition of granzyme activity . Of all the proteins in the granzyme family , the mechanisms that reduce granzyme B - mediated cell death have been characterised the most . Inhibitory mechanisms that act directly on granzyme B pathways to prevent cell death include inhibition by serpin B9 , degradation through autophagy , and disruption of gasdermin - mediated pyroptosis ( Figure 2 ) . Serpin B9 Serpin B9 ( also known as serine proteinase inhibitor B9 or proteinase inhibitor 9 ) is the only endogenous granzyme B inhibitor that has been identi fi ed . It was initially discovered in cytotoxic lymphocytes where it protects against apoptosis by binding to and inhibiting granzyme B ( 99 , 100 ) . When unbound to substrate , serpin B9 exists in a semi - stable form , but it is cleaved upon binding to granzyme B causing a conformational change into its most stable form and leaving a non - functional covalently - bound serpin B9 - granzyme B complex ( 101 ) . Apart from in immune cells and at certain immune - privileged sites , such as reproductive organs and the eye , normal human tissue does not express serpin B9 ( 102 ) . However , serpin B9 expression has been observed in multiple primary cancers , including lymphoma , melanoma , colon carcinoma , breast cancer , and lung cancer , in which it generally correlates with poor prognosis ( 102 – 107 ) . Overexpression of serpin B9 in various cancer cell lines results in resistance to killing by cytotoxic lymphocytes and , critically , is associated with resistance to immune checkpoint blockade in murine melanoma as well as against radiotherapy - induced type I interferon signalling ( 104 , 108 – 112 ) . Interestingly , the resistance of serpin B9 - expressing cancer cells to cytotoxic lymphocytes is less evident at high ratios of lymphocytes to cancer cells ( 111 ) . Furthermore , it has been shown through live imaging that multiple NK cell attacks successfully kill serpin B9 - expressing target cells , while single hits are suf fi cient to kill targets which don ’ t express serpin B9 ( 113 ) . This is consistent with serpin B9 - mediated inhibition of cytotoxicity being overcome through increased granzyme B delivery via multiple lytic hits . Although serpin B9 has primarily been described as an inhibitor of lymphocyte - derived granzyme B , it has also been shown to have a broader role in mediating tumour immune escape . For example , overexpression of serpin B9 can inhibit TRAIL - , FasL - , and TNF - mediated apoptosis through directly inhibiting caspase 8 and 10 ( 108 , 114 ) . Furthermore , serpin B9 can promote tumour survival through inhibition of cancer cell - intrinsic granzyme B , which can become expressed in various malignancies ( 107 ) . Therefore , pharmacological inhibition of Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 7 serpin B9 may aid the destruction of target cancer cells through multiple pathways . Recently , inhibition of serpin B9 has been shown to slow the development of melanoma and increase the lifespan of mice with breast , kidney and lung tumours ( 107 ) . Autophagy Cancer cells may also evade cytotoxicity through autophagic pathways . Autophagy is a physiological process by which damaged or surplus proteins and organelles are degraded and recycled ( 115 ) . It has a particularly important role in preventing cell death during cellular stress , such as nutrient starvation or hypoxia . Increased autophagy is also a common feature of tumorigenesis to protect against the harsh environment often present within tumours . This also enables cancer cells to maintain their highly proliferative and metabolically active states even when the microenvironment is not conducive to it ( 115 ) . Autophagy may also contribute to tumour growth by promoting immune evasion . Hypoxia - induced autophagy , for example , has been shown to correlate with resistance to CTL and NK cell cytotoxicity ( 116 , 117 ) . Similarly , induction of autophagy as a result of genetic inactivation of the von Hippel - Lindau ( VHL ) gene reduces killing by NK cells ( 118 ) . Genome - wide CRISPR screens searching for genes that mediate resistance to CTL cytotoxicity have also identi fi ed a range of autophagy - related genes associated with cytotoxicity resistance ( 119 , 120 ) . Degradation of granzyme B may be one mechanism by which autophagy inhibits cytotoxicity . Breast cancer cells with autophagy processes stimulated by hypoxia were found to be resistant to NK cell - mediated lysis , with granzyme B localising within their autophagosomes ( 117 ) . When hypoxia - related genes were inhibited , granzyme B activity within target cells was increased ( 117 , 118 ) . However , a later study demonstrated that a major effect of autophagy in cancer cells is to inhibit TNF a and TRAIL - induced apoptosis by reducing FADD - dependent caspase - 8 activation ( 120 ) . Thus , it is possible that autophagy can act in multiple ways to inhibit lymphocyte cytotoxicity . Gasdermins An emerging aspect of resistance to lymphocyte cytotoxicity is evasion of gasdermin - induced pyroptosis . Both granzyme A and B - mediated cleavage of gasdermin B and E , respectively , have been shown to contribute to tumour control by cytotoxic lymphocytes ( 48 , 54 ) . Furthermore , granzyme A - mediated cleavage of gasdermin B also contributes to defence against bacterial infection by NK cells ( 55 ) . Several mechanisms have been identi fi ed which may cause resistance to these gasdermin - mediated cytotoxic pathways . Firstly , reduced expression of both gasdermin B and E have been identi fi ed in many cancers ( 48 , 54 ) . In the case of gasdermin E , reduced expression can occur through epigenetic silencing via hypermethylation of the promoter region ( 121 , 122 ) . Low expression of both gasdermin B and E is associated with poor survival in various cancers , including breast cancer , bladder cancer , and melanoma ( 48 , 54 ) . Reducing granzyme - induced pyroptosis through silencing of gasdermin E expression in cancer cells has been shown to contribute signi fi cantly to the escape of murine tumours from cytotoxic lymphocytes and accelerate tumour growth ( 48 ) . Similarly , resistance to gasdermin - mediated pyroptosis can occur through the expression of mutated gasdermin . One study found that 20 out of 22 gasdermin E mutations identi fi ed within cancer samples were associated with reduced pyroptosis in response to granzyme B ( 48 ) . In the context of bacterial infection , pyroptosis has been shown to be inhibited through degradation of gasdermin B ( 55 ) . Degradation was mediated by a bacterial ubiquitin ligase IpaH7 . 8 secreted by the gram - negative bacterium , Shigella fl exneri . IpaH7 . 8 was shown to ubiquitinate N - terminal gasdermin B after its cleavage by granzyme A , leading to its degradation . Expression of IpaH7 . 8 signi fi cantly constrained the bactericidal activity of NK cells ( 55 ) . In summary , target cells may evade granzyme B - mediated apoptosis through inhibition by serpin B9 or degradation by autophagy . In addition , resistance to gasdermin - mediated pyroptosis is emerging as another mechanism by which cells may evade granzyme - mediated cytotoxicity in the context of malignancy and infection . Importantly , all of these mechanisms can have effects beyond granzymes and can affect other pathways of lymphocyte cytotoxicity as well as cell survival in other contexts . Therefore , targeting these pathways may directly impact cancer and could enhance other modes of treatment , such as immune therapies . Inhibiting Death Receptor - Mediated Killing Death receptor - mediated cytotoxicity is another critical mechanism by which cytotoxic lymphocytes may eliminate target cells . Several mechanisms have been described by which cells can evade death receptor - mediated cytotoxicity . Signalling can be directly inhibited by the activity of FADD - like IL - 1 b converting enzyme ( FLICE ) - inhibitory proteins ( FLIPs ) , expression of decoy receptors , or downregulation of death receptors ( Figure 3 ) . FLICE Inhibitory Proteins ( FLIPs ) One of the best characterised families of death receptor inhibitors are the FLIPs . This family of proteins includes both viral ( v - FLIP ) and cellular ( c - FLIP ) proteins , which share high sequence homology ( 123 ) . Several FLIP splice variants are expressed in humans , but the primary forms include the short variant , c - FLIP S , and the long variant , c - FLIP L ( 123 ) . The long variant contains an additional c - terminal domain that resembles the catalytic domains of caspase 8 and 10 but without functional caspase activity ( 123 , 124 ) . Both cellular and viral FLIPs inhibit caspase 8 activity by forming heterodimers with pro - caspase 8 ( 123 , 125 ) . This sequesters pro - caspase 8 , preventing it from forming the necessary homodimers required for processing into active caspase 8 . Inhibition of pro - caspase 8 processing by FLIPs prevents apoptosis induced by TRAIL and FasL , but not by granzyme B ( 124 , 126 , 127 ) . Immune cells have been observed to exert a selective pressure on cancer cells during in vivo tumorigenesis , allowing cells that highly express FLIP to escape ( 128 ) . Indeed , high tumour expression of c - FLIP , particularly of the long variant , has been found to correlate with poor prognosis Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 8 in a range of cancers , including acute myeloid leukaemia , colorectal cancer , and non - small cell lung cancer ( 129 – 131 ) . In addition to cellular FLIPs , viral FLIPs also appear to have a role in the promotion of tumorigenesis in humans . v - FLIPs that are expressed during viral infection act to protect host cells from death receptor - induced apoptosis resulting in immune escape from T cells ( 132 , 133 ) . This viral immune escape mechanism can contribute to the process by which certain viruses are particularly oncogenic . For example , Kaposi ’ s sarcoma - associated herpesvirus ( KSHV ) - FLIP is associated with Kaposi ’ s sarcoma and certain lymphomas ( 132 ) . To complicate the picture , although c - FLIP L has primarily been described as an inhibitor of apoptosis , it can also have pro - apoptotic effects . Heterodimers formed of c - FLIP L and pro - caspase 8 have been found to retain their catalytic activity and can process other pro - caspase 8 homodimers ( 125 , 134 – 137 ) . There is evidence that whether or not c - FLIP L promotes apoptosis is highly dependent on the level of expression of both the FLIP protein and pro - caspase 8 ( 125 , 136 , 137 ) . In the presence of very high levels of c - FLIP L , and therefore high levels of heterodimers , inhibition of apoptosis occurs since the amount of pro - caspase 8 homodimers that are available to be processed is decreased . Conversely , at lower concentrations , c - FLIP L preferentially acts as a promoter of apoptosis . Overall , FLIPs may play a signi fi cant role in the aetiology of some cancers . As a result , FLIP inhibitors or drugs that reduce FLIP expression are currently under development for the treatment of cancer , but balancing the pro - and anti - apoptotic effects may be challenging ( 138 , 139 ) . Other anti - cancer treatments , such as doxorubicin , synthetic triterpenoids , and peroxisome proliferator - activated receptor - g ( PPAR g ) ligands , can also decrease FLIP expression as a side effect , therefore increasing sensitivity to death receptor - mediated cytotoxicity ( 140 – 142 ) . FIGURE 3 | Inhibiting death receptor - mediated killing . Death receptor - mediated killing is a critical method of lymphocyte cytotoxicity . However , target cells have developed multiple mechanisms to inhibit the effectiveness of these processes . ( A ) Expression of decoy receptors , including membrane bound decoy receptors 1 and 2 that lack functional death domains , thereby preventing signalling while sequestering TRAIL . Decoy receptor 2 also inhibits death receptor 5 , preventing death receptor 4 recruitment and DISC formation . Decoy receptor 3 is soluble and binds to FasL preventing it acting upon target cell Fas . ( B ) Autophagy inhibits FADD - dependent caspase - 8 activation . ( C ) Decreased expression of death receptors , such as Fas , DR4 and DR5 inhibits apoptotic pathways . ( D ) Increased expression of cFLIP sequesters pro - caspase 8 into heterodimers to prevent its cleavage to caspase 8 and the subsequent activation of apoptotic pathways . Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 9 Decoy Receptors The majority of receptors that bind TNF superfamily proteins are capable of transducing signals . However , several receptors have been identi fi ed that , despite binding the same ligands , lack cytoplasmic death domains for signalling and cannot recruit critical adaptors , such as FADD ( 143 ) . These are , in effect , decoy receptors which compete with functional death receptors for ligand binding . Physiologically , decoy receptors have been implicated in modulating in fl ammatory responses but have also been hijacked as a survival mechanism in cancer ( 144 ) . One such protein is decoy receptor 3 ( DcR3 ) , a soluble receptor which binds FasL and is overexpressed in a large proportion of primary lung , colon , oesophageal , stomach , and rectal tumours ( 145 , 146 ) . Decoy receptors 1 and 2 ( DcR1 and DcR2 ) are membrane - bound receptors which bind TRAIL and also lack a functional death domain ( 147 , 148 ) . In addition to competing with functional TRAIL receptors , DcR2 is also able to interact with TRAIL - receptor variant , DR5 , preventing the recruitment of the DR4 variant to the DISC and inhibiting caspase activation ( 149 , 150 ) . Expression of DcR1 or DcR2 has been found to correlate with tumour progression and poor prognosis in breast cancer , prostate cancer , and leukaemia ( 151 – 153 ) . However , it is unclear whether targeting decoy receptors in tumours could have off - target effects , since they can also be expressed in several normal tissues , including the spleen , lung , gastrointestinal tract , endometrium , and activated T cells ( 145 , 154 , 155 ) . Furthermore , although there is evidence from over - expression systems that decoy receptors may constrain death receptor - mediated killing , the extent to which they are harnessed by cancer cells to evade lymphocyte cytotoxicity in vivo is not known . Death Receptor Expression and Mutation Death receptor signalling can also be lost in cancer cells through reduced surface expression or through inactivating mutations in death receptors . Reduced expression of the death receptors Fas or DR4 / 5 is a common feature of cancers ( 156 – 158 ) . Loss of these receptors is associated with poor prognosis , particularly upon loss of more than one receptor or when receptor downregulation occurs in tumours with low levels of in fi ltrating CTL ( 156 , 158 ) . Interestingly , there is a weaker correlation between Fas expression and survival in colorectal tumours with high numbers of in fi ltrating CTL ( 158 ) . This may suggest that granule - dependent cytotoxicity rather than death receptor - mediated killing is the predominant pathway of cancer cell elimination when large numbers of CTL are present ( 158 ) . Loss of death receptor expression can occur through several routes , including promoter methylation ( 159 – 161 ) , histone modi fi cations ( 157 , 162 ) , promoter region mutations ( 163 ) , or reduced traf fi cking to the cell membrane ( 164 ) . Notably , oncogenic Ras mutations can strongly downregulate Fas expression through the control of several genes associated with the promoter region of Fas , as well as through hypermethylation ( 159 , 160 ) . Conversely , death receptors are often up - regulated as a side - effect of cancer treatment , and this may contribute to the overall ef fi cacy of the treatment . For example , receptors for FasL and TRAIL can be signi fi cantly up - regulated following radiotherapy and chemotherapy , leading to enhanced killing by cytotoxic lymphocytes ( 165 – 171 ) . A less frequently occurring feature of cancers that may contribute to immune escape is mutations of the death receptors themselves . Mutations affecting function , which generally localise in the cytoplasmic domains , are infrequently observed in cancers , such as gastric cancer , non - small cell lung cancer , metastatic breast cancer , non - Hodgkin ’ s lymphoma , and head and neck cancer ( 172 – 175 ) . Induced expression of these mutated receptors in vitro can reduce pro - apoptotic signalling . Overall , death receptor signalling can be inhibited at multiple stages including reduced expression or mutation of death receptors , competition for ligand binding by decoy receptors , or inhibition of downstream signalling by FLIPs . However , there are several therapeutic strategies that may enhance death receptor signalling including pharmacological inhibition and downregulation of FLIPs or increasing death receptor expression , all of which have the potential to restore the ef fi cacy of immune cytotoxicity . Inhibiting TNF - Mediated Cytotoxicity TNF is known to have both pro - survival and pro - death effects on cancer cells depending on its precise cellular context . Recently , genome - wide CRISPR screens have identi fi ed TNF signalling as a major target of resistance to lymphocyte cytotoxicity ( 120 , 176 – 179 ) . These studies identi fi ed several genes encoding proteins related to TNF signalling that either sensitise cells to lymphocyte cytotoxicity – including TNF - R1 , caspase 8 , TRADD , and RIPK1 – or promote evasion of cytotoxicity – including TRAF2 , cIAP1 , and FADD - like apoptosis regulator ( CFLAR ) , as well as multiple genes involved in the NF - k B pathway ( 120 , 176 – 179 ) . In particular , knockout of TRAF2 was shown to redirect TNF signalling from pro - survival signalling , via complex I proteins and the NF - k B pathway , to pro - death signalling , via complex II proteins ( 177 ) . Likewise , knockout or pharmacological inhibition of HOIL - 1 - interacting protein ( HOIP ) , the catalytic subunit of LUBAC involved in ubiquitination , also enhanced sensitivity to TNF by reducing the ubiquitination of pro - survival complex I proteins , which is required for TNF - mediated survival signalling ( 180 , 181 ) . Conversely , antibody blockade of TNF signi fi cantly reduced killing by both wild - type and perforin - de fi cient T cells demonstrating that TNF signalling is a major pathway of lymphocyte cytotoxicity ( 120 , 176 ) . Resistance to TNF - mediated cell death has also been suggested as one way in which autophagy can induce resistance to lymphocyte cytotoxicity . Knockout of key autophagy - related genes sensitizes cancer cells to TNF - induced death and TNF - mediated T cell cytotoxicity ( 119 , 120 ) . Autophagy can target TNF - induced cell death by modulating FADD / caspase - 8 activity ( 120 ) . Several other studies have also noted the ability of autophagy to interfere with active caspase - 8 leading to reduced susceptibility of cells to TRAIL and TNF during hepatic injury or in colon carcinoma ( 182 , 183 ) . It remains to be seen whether TNF signalling can be harnessed to successfully treat cancer patients in the clinic because its effects are highly context - dependent . This was Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 10 demonstrated by Young et al . , who found that knockout of TNF - R1 could either protect tumours against immune checkpoint blockade or sensitise tumours to it depending on whether autophagy was impaired or intact , respectively ( 120 ) . TNF has also been shown to have cancer - promoting effects in some cancer models , leading to several clinical trials of TNF antagonists demonstrating ef fi cacy in some patients ( 184 – 186 ) . One possible approach to targeting TNF signalling is through the use of SMAC ( second mitochondria - derived activator of caspases ) mimetics . These are drugs that mimic the activity of SMAC , a protein that is an endogenous inhibitor of cIAP function ( 187 ) . Through the inhibition of cIAP , SMAC mimetics have been found to sensitise cancer cells to TNF - induced cell death both in vitro and in vivo ( 120 , 188 – 190 ) . Alternatively , directly inhibiting certain complex I components , such as HOIP , may redirect signalling towards apoptosis ( 180 ) . Therefore , there is potential for sensitising cancer cells to TNF - induced cell death , but greater understanding of how its functions vary is needed . Inhibiting Apoptotic Pathways In addition to the inhibitory mechanisms against speci fi c components of lymphocyte cytotoxicity , cancer cells may exhibit more general resistance to apoptosis through alterations in apoptotic pathways . Prevention of apoptosis may occur through either down - regulation of pro - apoptotic mediators , such as caspases or pro - apoptotic Bcl - 2 family members , or up - regulation of apoptosis inhibitors , such as Inhibitor of Apoptosis Proteins ( IAPs ) or anti - apoptotic Bcl - 2 family members . These can affect both caspase - dependent and mitochondrial pathways of apoptosis , which are involved in both granule - mediated and death receptor - mediated cell death . Caspase Inhibition – Mutations and Inhibitor of Apoptosis Proteins Caspases are critical components of both granule - mediated and death receptor - mediated cytotoxicity . Death receptor - induced apoptosis relies on the activation of caspase 8 / 10 within the DISC to activate the executioner caspases , caspase 3 , 6 , and 7 . Conversely , granzyme B can directly cleave and activate executioner caspases as well as activating them through the mitochondrial pathway of apoptosis . As a result , reducing caspase activity is a common pathway by which cancers avoid apoptosis , with reduced expression or mutations reported for both initiator ( caspase 2 , 8 , and 10 ) and executioner caspases ( caspase 3 , 6 , and 7 ) in a range of cancers ( 191 ) . For example , caspase 8 is commonly mutated , particularly in cancers of neuroendocrine or lymphoid origin ( 192 ) . Loss of caspase 8 expression contributes to resistance against TRAIL - induced apoptosis ( 193 , 194 ) . In addition to alterations in expression , caspase activity can be modulated by the enhanced expression of IAPs , such as cIAP1 , survivin , and X - linked inhibitor of apoptosis protein ( XIAP ) . IAPs can bind directly to caspases preventing their activity and leading to inhibition of granzyme B and death receptor - mediated apoptosis ( 195 – 197 ) . Bcl - 2 Family The mitochondrial pathway of apoptosis , characterised by mitochondrial outer membrane permeabilization ( MOMP ) , is a critical pathway by which both granzyme B and death receptors can mediate apoptosis . This pathway is regulated by the Bcl - 2 family of proteins , which include pro - apoptotic BH3 proteins ( e . g . Bid ) , pro - apoptotic effector proteins ( e . g . Bax and Bak ) , and anti - apoptotic proteins ( e . g . Bcl - 2 and Bcl - XL ) ( 40 ) . Disruption of this pathway through either downregulation of pro - apoptotic proteins or upregulation of anti - apoptotic proteins can prevent apoptosis induced by either death receptors or granzymes . A critical mediator of MOMP is Bid , which can be cleaved by either caspase 8 following death receptor ligation or by granzyme B . Bid is responsible for recruiting additional mediators of MOMP , such as Bax and Bak ( 40 ) . As a result , loss of Bid expression in cancer cells leads to reduced sensitivity to granzyme B - induced apoptosis ( 198 , 199 ) . Likewise , loss of Bak expression , which is directly involved in permeabilization of the mitochondrial membrane , protects against apoptosis triggered by granzyme B ( 199 ) . Reduced expression of pro - apoptotic Bcl - 2 family members , such as Bid , is observed in various cancers and is associated with poor prognosis in prostate cancer and colon cancer , for example ( 200 , 201 ) . Alternatively , inhibition of MOMP can occur through overexpression of the anti - apoptotic Bcl - 2 family proteins , which inhibit the activity of apoptotic proteins , such as Bax and Bak . Overexpression of anti - apoptotic proteins , such as Bcl - 2 or Bcl - XL , reduces apoptosis induced by both granule - mediated cytotoxicity and death receptor - mediated cytotoxicity , whereas pharmacological inhibition of Bcl - 2 sensitises cells to cytotoxicity ( 196 , 197 , 202 – 206 ) . Overexpression of Bcl - 2 is a common feature of multiple cancers ( 207 ) . Overall , cancer cells frequently develop mutations or altered expression of the critical caspases and Bcl - 2 family members involved in regulating and mediating apoptosis induced by immune attack . As a result , targeting these pathways , for example by inhibition of Bcl - 2 , could be a particularly effective way of enhancing the ef fi cacy of immunotherapy in patients ( 208 ) . CONCLUSION Effective cytotoxicity by immune cells against cancerous or infected cells is a critical mechanism of controlling these disease states . A multitude of treatments , such as checkpoint inhibitors , have been developed to boost the immune system ’ s response to these diseased cells . However , these treatments are not always effective , and malignant and infected cells can still exploit mechanisms that enable them to evade the strengthened immune system . Here , we have outlined many ways in which diseased cells can evade the cytotoxic attacks of NK cells and CTLs . Many processes that enhance target cell resistance to cytotoxicity are the same processes that cytotoxic cells themselves use to prevent self - harm by their own deadly cargo . Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 11 Other pathways that inhibit cytotoxic killing , such as autophagy , pro - survival TNF signalling , and downregulation of apoptotic pathways , also convey other bene fi ts normally , but can be exploited by cancerous cells or infectious agents . It is likely that many of these mechanisms of resistance have evolved together with the cytotoxic pathways employed by lymphocytes . This may explain why lymphocytes simultaneously utilise several cytotoxic pathways that are often redundant . For example , the broad range of granzyme B targets – including apoptotic caspases , regulators of mitochondrial apoptosis , and gasdermins – reduces the likelihood that a target cell could become resistant to granzyme B - mediated cell death . This was found to be the case in the context of haematological cancer , in which NK cells and CTLs were able to kill cancer cells despite a variety of anti - apoptotic mutations that conferred multi - drug resistance ( 209 , 210 ) . These studies demonstrate that lymphocytes can overcome some resistance mechanisms by inducing death through multiple pathways . Conceptually , it can be dif fi cult to distinguish processes that have been autonomously selected to aid the survival of diseased cells that also happen to be bene fi cial in avoiding immune attack , from processes that have been adopted by these cells to speci fi cally resist cell - mediated cytotoxicity . Further research to investigate this could include studying the evolutionary development of cancers , and comparisons with mice de fi cient in speci fi c compartments of their immune system . Either way , there is great potential here to therapeutically target the processes discussed throughout this review . Targeting the ways in which diseased cells avoid death could be used alone or in combination with other therapies , including immunotherapies . Importantly , a greater understanding of these mechanisms and processes in vivo is sorely needed to indicate the most potent interventions . AUTHOR CONTRIBUTIONS KT wrote the original manuscript . KT and ARA prepared fi gures . KT , ARA , and DMD edited the manuscript . All authors contributed to the article and approved the submitted version . FUNDING This work was funded by Cancer Research UK via funding to the Cancer Research UK Manchester Institute ( to KT ; C147 / A25254 ) and a Wellcome Trust Investigator Award ( to DMD ; 110091 / Z / 15 / Z ) . REFERENCES 1 . Prager I , Watzl C . Mechanisms of Natural Killer Cell - Mediated Cellular Cytotoxicity . JLeukocBiol ( 2019 ) 105 : 1319 – 29 . doi : 10 . 1002 / JLB . MR0718 - 269R 2 . Topham NJ , Hewitt EW . Natural Killer Cell Cytotoxicity : How do They Pull the Trigger ? 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Cell Death Differ ( 2018 ) 25 : 1536 – 48 . doi : 10 . 1038 / s41418 - 018 - 0112 - 9 Con fl ict of Interest : The authors declare that the research was conducted in the absence of any commercial or fi nancial relationships that could be construed as a potential con fl ict of interest . DMD receives research funding from GSK , Continuum Life Sciences and Bristol Myers Squibb , and advises GSK , Mogrify and Bicycle Therapeutics . Publisher ’ s Note : All claims expressed in this article are solely those of the authors and do not necessarily represent those of their af fi liated organizations , or those of the publisher , the editors and the reviewers . Any product that may be evaluated in this article , or claim that may be made by its manufacturer , is not guaranteed or endorsed by the publisher . Copyright © 2022 Tuomela , Ambrose and Davis . This is an open - access article distributed under the terms of the Creative Commons Attribution License ( CC BY ) . The use , distribution or reproduction in other forums is permitted , provided the original author ( s ) and the copyright owner ( s ) are credited and that the original publication in this journal is cited , in accordance with accepted academic practice . No use , distribution or reproduction is permitted which does not comply with these terms . Tuomela et al . Resisting Cell - Mediated Cytotoxicity Frontiers in Immunology | www . frontiersin . org March 2022 | Volume 13 | Article 867098 17 \ No newline at end of file diff --git a/silver_data/904b81583cb51300752db9d8f663dae07e87a621.pdf.txt b/silver_data/904b81583cb51300752db9d8f663dae07e87a621.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..62f80b577ed1d112d414566b7ffca6f62ede89f4 --- /dev/null +++ b/silver_data/904b81583cb51300752db9d8f663dae07e87a621.pdf.txt @@ -0,0 +1 @@ +A UTO TRIZ : A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS Shuo Jiang ∗ Singapore University of Technology and Design 8 Somapah Road , Singapore , 487372 shuo _ jiang @ sutd . edu . sg Jianxi Luo Department of Systems Engineering City University of Hong Kong , Hong Kong jianxi . luo @ cityu . edu . hk A Preprint , Version : Mar 12 , 2024 A BSTRACT Researchers and innovators have made enormous efforts in developing ideation methods , such as morphological analysis and design - by - analogy , to aid engineering design ideation for problem solving and innovation . Among these , TRIZ stands out as the most well - known approach , widely applied for systematic innovation . However , the complexity of TRIZ resources and concepts , coupled with its reliance on users’ knowledge , experience , and reasoning capabilities , limits its practicability . This paper proposes AutoTRIZ , an artificial ideation tool that leverages large language models ( LLMs ) to automate and enhance the TRIZ methodology . By leveraging the broad knowledge and advanced reasoning capabilities of LLMs , AutoTRIZ offers a novel approach to design automation and interpretable ideation with artificial intelligence . We demonstrate and evaluate the effectiveness of AutoTRIZ through consistency experiments in contradiction detection and comparative studies with cases collected from TRIZ textbooks . Moreover , the proposed LLM - based framework holds the potential for extension to automate other knowledge - based ideation methods , including SCAMPER , Design Heuristics , and Design - by - Analogy , paving the way for a new era of artificial ideation for design and innovation . Keywords Innovation · Design Ideation · Problem Solving · TRIZ · Large Language Models · Artificial Intelligence 1 Introduction Intuitive or structured ideation methods such as brainstorming , morphological analysis , and mind - mapping [ 1 , 2 , 3 ] have been used to aid creative ideation of human designers for concept generation . Among these , the Theory of Inventive Problem Solving ( TRIZ ) [ 4 ] stands out as the most well - known approach , widely applied for systematic innovation . TRIZ is a knowledge - based ideation methodology that provides a structured framework for engineering problem solving by identifying and overcoming technical contradictions using inventive principles derived from a large - scale patent database . However , the complexity of TRIZ resources and concepts poses significant cognitive challenges to effectively learning and applying it . In addition , the problem - solving process in TRIZ is highly dependent on the reasoning capabilities of human users . While some researchers have employed natural language processing [ 5 , 6 , 7 ] , the effectiveness still heavily depends on the users’ proficiency with TRIZ . Large Language Models ( LLMs ) such as OpenAI’s GPT [ 8 ] and Meta’s Llama [ 9 ] have not only acquired broad knowledge but also developed emergent abilities such as in - context learning , following instructions , and step - by - step reasoning [ 10 ] . These capabilities have been applied across various domains , including medicine [ 11 ] , chemistry [ 12 ] , and mathematics [ 13 ] . Recently , researchers have evaluated the capabilities of LLMs in engineering - related tasks [ 14 , 15 ] and reported the extensive engineering knowledge within these models and their wide applicability in engineering design and manufacturing . In terms of engineering problem solving and idea generation , there has been preliminary exploration using LLMs [ 16 , 17 , 18 , 19 ] . However , the lack of transparency and limited control over ∗ Comments are welcome : shuo _ jiang @ sutd . edu . sg a r X i v : 2403 . 13002v1 [ c s . H C ] 13 M a r 2024 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS reasoning steps during ideation often lead to divergent results , requiring multiple heuristic attempts by users to achieve desired outcomes . Besides , the interpretability of generated concepts remains challenging , as users obtain only the final results without understanding the ideation reasoning process . In this work , we aim to leverage the broad knowledge and advanced reasoning capability of LLMs to automate the TRIZ method , showcasing the potential of LLMs in design automation and interpretable innovation . We have developed an LLM - based intelligent tool , AutoTRIZ , capable of artificial ideation for problem solving with TRIZ - based interpretability . AutoTRIZ begins with a problem statement from the user and automatically generates a solution report , strictly following the TRIZ thinking flow and reasoning process . In this paper , we also evaluate the effectiveness and performance of AutoTRIZ through quantitative and comparative experiments , as well as case studies involving human uses of TRIZ from TRIZ textbooks . 2 Related Work 2 . 1 TRIZ TRIZ is a knowledge - based systematic approach of inventive problem solving , developed in the 1960s by Genrich S . Altshuller and his colleagues [ 4 ] . Through a thorough analysis of over 40 , 000 patents , Altshuller and his collaborators identified repeated patterns of innovation and underlying innovative principles within these documents . By inductively analyzing these patterns , they proposed a comprehensive problem - solving framework , applying selected inventive principles for ideation . Since then , the TRIZ has been developed continually and some modern TRIZ databases rely on analysis of over 2 million patents . It has been widely applied in industries , research , and education with notable influence in many fields , such as energy , electrical , automotive industries , and mechanical engineering [ 20 ] . The TRIZ toolkit contains a series of theories and tools that cover all aspects of problem understanding and solving , including trimming method , evolution trends , and 76 standard solutions [ 4 ] . In this paper , we focus on the best - known tool , the Method of Inventive Principles , which represents the basic reasoning logic in TRIZ . Figure 1 shows the overview of its framework ( adapted from [ 21 ] ) , which contains four steps : ( 1 ) Identify the specific problem . ( 2 ) Transform the specific problem into a general problem by identifying physical contradictions . The contradic - tions involve an improving feature and a worsening feature . These features are from Altshuller’s 39 engineering parameters . ( 3 ) Search for selected inventive principles from the contradiction matrix using identified contradictions . The contradiction matrix is organized in the form of 39 - improving features and 39 - worsening features ( a 39 by 39 matrix ) with each cell entry giving the most often used principles ( from TRIZ 40 inventive principles ) that may be used to solve the problem . ( 4 ) Use the selected principles to generate solutions to the problem . Figure 1 : Four steps for problem solving using TRIZ 2 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS Although TRIZ has demonstrated its effectiveness , it still suffers drawbacks that hinder its practical applications . For instance , the complexity of TRIZ resources and concepts poses cognitive challenges to effectively learning and applying it , particularly for non - experts . Additionally , the efficacy of TRIZ is heavily constrained by the users’ reasoning capabilities and prior knowledge acquired . Recent advancements in machine learning and natural language processing , have been applied in conjunction with TRIZ [ 5 , 7 , 22 ] . These efforts aim to intellectualize the TRIZ reasoning process , thereby reducing the difficulty of use . For instance , Cascini and Russo [ 5 ] developed the PAT - ANALYZER system which can analyze patent texts and automatically extract the contradictory information underlying the innovation for the use of TRIZ . Similarly , Guarino et al . [ 7 ] proposed the PaTRIZ , combining the Bidirectional Encoder Representations from Transformers ( BERT ) and Conditional Random Field ( CRF ) for word - level patent analysis and TRIZ contradiction mining . Li et al . [ 22 ] proposed an approach that leverages natural language processing techniques to assess patent innovations according to the level of invention as defined in TRIZ . Berdyugina and Cavallucci [ 23 ] proposed a methodology for the automatic extraction of inventive information from texts for formulating an inventive problem into TRIZ engineering parameters . Their method combined a series of text - mining techniques , including topic modeling , word embedding , and clustering . Hall et al . [ 6 ] proposed an approach that uses topic modeling and unsupervised machine learning to map TRIZ inventive principles to individual patents and detect the novelty . However , most of these works focus on utilizing algorithms to improve specific steps of the TRIZ process . They still require innovators to dedicate much time and effort to extensive reasoning . Employing these methods does not directly assist users throughout the entire process from analyzing a problem to creating practical solutions . In this paper , we aim to harness LLMs to automate the entire TRIZ reasoning process and minimize the cognitive requirements of users during its application . 2 . 2 Large Language Models for Design and Innovation Over the past years , many data - driven approaches have utilized machine learning and deep learning techniques to augment design and innovation [ 24 , 25 ] . Rooted in deep learning , LLMs represent a class of transformer - based algorithms designed for processing and generating natural language text . They are trained on extremely large - scale corpora , enabling them to acquire a wide range of knowledge and capabilities , including understanding context , generating coherent text , and step - by - step reasoning [ 10 ] . Some research has already explored the application of LLMs in engineering design and innovation within specific fields , including microfluidic devices [ 26 ] , robotics [ 27 ] and the user interface of webpages [ 28 ] . However , most of these early efforts primarily utilize conversational interactions , such as those facilitated by ChatGPT , to engage in the innovation process . Meanwhile , with the development of LLMs , there has been an increase in efforts to create LLM - driven methods and tools to offer more generalized innovation assistance and directly support users in rapid ideation . For instance , several studies have harnessed LLMs for processing vast amounts of design documentation , representing designs in specific forms , and identifying user needs for product development [ 16 , 17 , 29 ] . Han et al . [ 17 ] introduced an LLM - based attribute - sentiment - guided summarization model to extract user needs from online product reviews . Qiu et al . [ 29 ] applied a transformer - based language model to distill design - related knowledge from extensive reports and documents . Moreover , Wang et al . [ 16 ] utilized LLMs to decompose conceptual design tasks into Function - Behavior - Structure ( FBS ) formats , assisting users in ideation across different aspects . Recent studies have developed tools and methodologies utilizing LLMs to aid the design process , enhance human - computer collaborative innovation , or directly produce innovative concepts for users [ 18 , 19 , 30 , 31 ] . Ding et al . [ 30 ] conducted a systematic exploration of LLMs’ potential to boost cross - domain analogical creativity . Huang et al . [ 31 ] proposed CausalMapper , a system that combines LLMs with causal mapping to reason about the connections between problems and solutions . Zhu and Luo [ 19 ] presented GPT - based models with domain - specific tuning and task - specific learning , to generate original and useful design concepts . Notably , they applied their approach to automating bio - inspired design concept generation [ 18 ] . Although these recent idea - generation methods directly leverage the reasoning capabilities of LLMs , the lack of control over LLMs may hinder their effectiveness when assisting ideation . These approaches often lead to solutions that are too divergent to meet specific needs . Managing the problem - solving process to ensure that solutions are both innovative and practical , as well as understanding the reasoning process behind generated innovative solutions , remains a challenge . In this study , we address this issue by integrating TRIZ with LLMs , presenting AutoTRIZ as a tool that follows the TRIZ reasoning steps to generate inventive solutions with interpretability . 3 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS 3 AutoTRIZ In this section , we introduce AutoTRIZ , an artificial ideation tool that automates TRIZ with LLMs . The architecture of AutoTRIZ is depicted in Figure 2 . At the core of AutoTRIZ is the utilization of LLMs to learn the reasoning process of the TRIZ methodology , which engineers often find it challenging to learn and excel in . Figure 2 : The framework of AutoTRIZ Overall , AutoTRIZ takes a problem statement prompt from the user as its initial input , and automatically generates a solution report after the reasoning process . The report includes detailed information about the reasoning process based on TRIZ and the resulting solutions to the problem . Within AutoTRIZ , we have defined a four - step reasoning flow based on the classic TRIZ workflow . The system includes an inner fixed knowledge base which consists of three segments related to TRIZ details , enabling controlled reasoning . It is noteworthy that our focus is on controlling the entire problem - solving reasoning process , while remaining open to the knowledge used in ideation . The problem - related knowledge applied during the problem - solving process is drawn from the knowledge base that the LLM has acquired through pre - training on the large - scale corpora . 3 . 1 Controlling the TRIZ Reasoning Flow To ensure that the system strictly follows the TRIZ thinking flow and reasoning process , we have configured AutoTRIZ with four modules , corresponding to the four steps in TRIZ . As depicted in Figure 2 , Modules 1 , 2 , and 4 , outlined by solid - line frames , are driven by LLMs , whereas Module 3 , outlined by a dashed - line frame , is controlled by pre - defined functions without using LLMs . Specifically , we exploit the instruction - following capabilities of LLMs for backend reasoning control . In each module that incorporates LLMs , relevant instructions are engineered into the input as system and assistant prompts . Specifically , in Module 1 , AutoTRIZ identifies the problem to be solved from user input and converts it into descriptive text . Ideally , we hope that the content entered by the user is a clear problem statement . However , user inputs may include additional information such as scenario descriptions , background details , and even some redundant information . Therefore , in this module , AutoTRIZ is designed to identify and extract information related to the problem and then reorganize it into clear and concise text . In Module 2 , AutoTRIZ receives the processed problem description and detects its engineering contradiction , which is represented by a space constructed from two out of the 39 engineering parameters . At this stage , AutoTRIZ learns all the engineering parameters based on the inner knowledge base . The outputs of this module are presented in a structured format ( i . e . , the indexes of the improving and worsening features ) . It is important to note that for the same problem statement , the identified contradiction may differ with each execution of this module . On the one hand , a single problem may encompass multiple contradictory pairs , yet our system is designed to identify only one contradiction . On the other hand , there is an inherent randomness in the content generation by LLMs . In the next section , we will conduct experimental investigations to examine the efficacy of contradiction identification and the consistency of the outputs . 4 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS Once the contradiction is identified , Module 3 searches the contradiction matrix to find the indexes of relevant inventive principles and returns their descriptions . Following this , Module 4 synthesizes the original problem description , the identified engineering contradiction , and the inventive principles recommended by the system through TRIZ , to generate the final solutions . LLMs can generate complex structured data , such as in HTML and L A TEXformats [ 32 ] . In AutoTRIZ , we harness this capability to integrate all generated content and directly produce a reader - friendly problem - solving report in a structured format . We have engineered the format template directly into module 4 , enabling it to output documents formatted in L A TEX . In practice , the template for the report generation can be adjusted as needed to suit specific requirements . 3 . 2 Learning from the Fixed Knowledge Base AutoTRIZ acquires the necessary information to learn the prior knowledge of TRIZ , enabling it to handle various types of problems . We have curated a static knowledge base , which interacts with the modules we described above , thereby empowering AutoTRIZ to master and apply the relevant knowledge . In AutoTRIZ , the internal fixed knowledge base includes three main components : ( 1 ) the TRIZ 39 Engineering Parameters [ 4 ] , ( 2 ) the TRIZ Contradiction Matrix [ 4 ] , and ( 3 ) the TRIZ 40 Inventive Principles [ 4 ] . Notably , the contradiction matrix here is identical to the traditional TRIZ contradiction matrix . The knowledge about engineering parameters and inventive principles includes titles and detailed descriptions for each entry . For example , for the first engineering parameter : [ INDEX ] 1 [ TITLE ] Weight of moving object [ DESCRIPTION ] The mass of the object in a gravitational field , essentially the force that the body exerts on its support or suspension . Similarly , for the first inventive principle : [ INDEX ] 1 [ TITLE ] Segmentation [ DESCRIPTION ] The Segmentation principle suggests contemplating the division of an object or system into smaller , independent parts . . . All engineering parameters are configured into Module 2 as assistant information . The backend LLMs learn instructions and the output parameter space through in - context learning , enabling zero - shot reasoning . Regarding inventive principles , only selected contents are delivered to the system based on the position in the contradiction matrix . This process is very similar to LLMs’ Retrieval Augmented Generation ( RAG ) . Whereas in our system , the problem - solving process involves precise searching augmented generation , effectively bridging the gap between the prior TRIZ knowledge from experts and the reasoning capabilities of LLMs derived from large - scale pre - training . Simultaneously , all solutions generated are interpretable because each solution is derived from the application of selected inventive principles . 3 . 3 System Implementation We developed a web - based tool for public users to test use AutoTRIZ , available at : https : / / www . autotriz . ai / . Figure 3 shows the user interface of the tool . Throughout the deployment of this tool and all experiments conducted in this study , we utilized GPT - 4 - Turbo ( Version : 20231106 ) as the backend LLM . However , it is important to note that since the proposed AutoTRIZ is a general framework , the backend LLM can be replaced with any other closed - source LLM ( e . g . , Claude ) or open - source LLM ( e . g . , Llama ) with minimal effort required for adapting the corresponding prompts . For the TRIZ knowledge base in AutoTRIZ , we adopt the TRIZ definitions and descriptions in an engineering design textbook [ 33 ] . Figure 3 : AutoTRIZ web - based tool 5 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS 4 Experimental Evaluation In this section , we evaluate the effectiveness of the proposed AutoTRIZ through quantitative experiments and com - parative studies . Specifically , we collected several case studies analyzed by human experts from TRIZ textbooks , constructing a case base . Then , we explored the consistency of the system in identifying engineering contradictions , as well as its overlap with human analysis . Finally , we selected a specific problem from the case base , and compared and discussed the solutions generated by AutoTRIZ against the results of human experts . 4 . 1 Constructing the TRIZ Case Base To evaluate the performance of AutoTRIZ , we first constructed a case base containing TRIZ problem - solving cases developed by human experts . Initially , we gathered several TRIZ - related textbooks , some of which are focused on general design innovation , while others are specifically about TRIZ . From 7 of these textbooks [ 4 , 33 , 34 , 35 , 36 , 21 , 37 ] , we collected 10 initial cases . The selection criteria include : ( 1 ) the content of the case contains all elements of the TRIZ reasoning process , including problem description , contradiction identification , inventive principle positioning , and solutions ; ( 2 ) the problem is defined clearly and comprehensively ; ( 3 ) the cases do not contain similar problems . All cases are stored in JSON format . The initial 10 cases cover various domains , including environmental engineering , transportation , manufacturing and material science , aerospace technology , and so on 2 . Beyond serving experimental purposes in this study , the curated case base can also store the results generated by users with AutoTRIZ . In the future , as the size of the base expands , we can explore the interaction between the reasoning module and the existing case base , enabling AutoTRIZ’s innovative capabilities to be scalable . 4 . 2 Assessing the Contradiction Identification Detecting contradictions is an essential step in the entire TRIZ problem - solving process . Accurate identification of the contradictions within a problem can effectively assist the system in recommending the appropriate inventive principles for the next step . Sometimes , LLMs may suffer from instability during inference . As a result , some LLM - based agents adopt self - consistency techniques that create several reasoning paths and then perform an ensemble on all generated answers , selecting the most consistent one through majority voting [ 38 ] . However , in our system , analyzing the same problem from different perspectives can yield different possible contradictions . Such inconsistency can sometimes increase diversity , thereby enhancing the novelty . Based on this , we retain the setting of producing a single contradiction in each entry . To assess the performance and consistency of this setting , we conducted the following experiments . For each given problem statement , we performed the analysis 100 times , resulting in 100 pairs of identified parameters ( contradictions ) . Then , we counted all results and calculated their respective proportions . In cases with high consistency , a particular contradiction could be dominant . In some cases , one parameter in the contradiction may have higher certainty than the other , leading to more dispersed results . Information entropy is utilized to assess the consistency of the results : H ( X ) = − n (cid:88) i = 1 P ( x i ) log 2 P ( x i ) where P ( x i ) represents the probability of occurrence of the outcome , and n is the number of different possible outcomes of X . Since the number of runs is 100 , the entropy value ranges from 0 to 6 . 64 , where a higher value indicates lower consistency . Furthermore , we examined the overlap between AutoTRIZ’s detection and the analysis results of human experts from textbooks , categorizing them into three scenarios : complete match , half match , and no match . It is important to note that since human expert analysis also includes subjectivity and bias , it cannot be considered a golden standard . The main purpose of this experiment is to showcase and quantitatively compare AutoTRIZ against human uses of TRIZ . Figure 4 shows the experimental results , where the bar chart of each case illustrates the top 3 detections by proportion . In the chart , green bars represent complete match , blue bars indicate half match , and yellow bars denote not match . The table in the bottom right corner shows the entropy of each case and whether the top 3 detections hit the reference from textbooks , with symbols ( ✓ , ✓ , ✗ ) indicating complete match , half match , and not match , respectively . Overall , 7 out of 10 cases match or half - match the textbook’s analysis within the top 3 detections , indicating that AutoTRIZ’s inference overlaps with the human experts’ results to a certain degree . A minority of the cases show 2 For further details on all collected cases , please refer to this GitHub repository : https : / / github . com / shuojiangcn / AutoTRIZ - DETC24 6 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS relatively higher consistency ( cases 5 , 6 , 7 , 8 ) , where the proportion of the top 1 detection is significantly higher than the other detections , including two complete match detections . For these cases , utilizing self - consistency may be beneficial to enhance performance . For other cases , the experimental results show greater diversity , indicated by higher information entropy . By examining the content of the top 3 detections of contradiction for each case , we observe that for almost all cases , one parameter is fixed while the other varies . Moreover , when using the textbook’s analysis as a reference , a pattern emerges across all cases where outputs with higher probabilities ( within the top 3 detections ) show a better match in alignment . These findings can serve as the initial benchmark for assessing the performance of AutoTRIZ’s contradiction identification . As the case base expands in the future , we can explore these patterns in a more fine - grained way with greater statistical significance . For example , we can examine the differences between various themes , leveraging techniques such as self - consistency reasoning in conjunction with the identified patterns to improve overall performance . Figure 4 : Experimental results about contradiction detection 4 . 3 Comparing AutoTRIZ and Human Expertise In this section , we select one of the collected cases ( case 7 ) to compare AutoTRIZ’s generated report with humans’ analysis results from the textbook . The reasons for choosing case 7 are two - fold : ( 1 ) This case exhibits relatively high 7 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS consistency in identifying engineering contradictions , with one dominant outcome ( refer to Figure 4 ) ; ( 2 ) The top 3 detections of contradiction are all half - match with the reference . This ensures a certain degree of reliability while allowing the distinction between the subsequent reasoning paths of AutoTRIZ and humans . The problem of case 7 is about the pneumatic transportation of metal shots through a system of plastic piping . Here is the original problem statement : We are faced with a challenge involving the pneumatic transportation of metal shots through a system of plastic piping originally intended for plastic pellets . The transition to metal shots , despite their advantages for production purposes , has led to significant wear and damage , particularly at the pipe’s elbows . This issue arises from the incompatibility between the metal shots and the existing plastic elbow design . The task is to identify and implement a solution that resolves this conflict , ensuring the system’s durability and effectiveness for transporting metal shots . In the textbook , the identified improving parameter is " speed " ( Parameter 9 ) , and the worsening parameter is " stability of the object’s composition " ( Parameter 13 ) . According to the contradiction matrix , the author selects " Mechanical Substitution " ( Principle 28 ) from the obtained inventive principles . Applying this principle , the author describes the solution as placing a magnet at the elbow to bind the metal shots to a plastic material , thereby creating a blanket of shots that absorb the energy . Figure 5 shows the problem - solving report generated by AutoTRIZ , containing the reasoning process and solutions . The same problem statement is used as the input . Firstly , we can see that AutoTRIZ simplifies the original problem statement , identifying the main issue that needs to be addressed . Regarding the identification of contradictions , AutoTRIZ diverges from human expertise . Both AutoTRIZ and the textbook’s analysis consistently recognize the " stability of the object’s composition " ( Parameter 13 ) as the worsening feature . However , concerning the improving feature , AutoTRIZ detects " Area of stationary object " ( Parameter 6 ) , while the textbook’s analysis considers it to be " speed " ( Parameter 9 ) . From the original problem statement , we understand that the key issue is to avoid wear on the plastic elbows by the metal shots to ensure durability , which clearly indicates that one of the contradictory parameters involves stability . Whereas the identification of the other parameter is not directly mentioned , leading to a variety of possible interpretations . AutoTRIZ reasons that the surface area needs improvement to withstand the impact and wear of the metal shot , while the expert asserts speed as the system’s top priority . These two analyses highlight different needs , thereby guiding subsequent innovative directions differently . In the textbook’s analysis , the author selected a single inventive principle ( 28 , ’Mechanical Substitution’ ) and created a solution by positioning a magnet at the piping’s elbow , which magnetically attaches metal shots to the plastic , forming an energy - absorbing layer . This approach represents a direct and effective innovation . However , based on the identified parameter pair , the contradiction matrix could yield four inventive principles ( i . e . , ( 28 , ’Mechanical substitution’ ) , ( 33 , ’Homogeneity’ ) , ( 1 , ’Segmentation’ ) , ( 18 , ’Mechanical vibration’ ) ) . Some principles may be challenging to apply , as the outcomes are directly influenced by the users’ reasoning ability , experience , and familiarity with TRIZ materials . This step also requires the most human effort in TRIZ . By comparison , AutoTRIZ can effectively overcome this issue . After identifying the contradiction ( Parameter 6 vs . Parameter 13 ) , AutoTRIZ identifies two inventive principles from the contradiction matrix ( i . e . , ( 2 , ’Extraction’ ) , ( 39 , ’Strong oxidants’ ) ) . For each principle , AutoTRIZ applies it and generates a corresponding solution . Both proposed solutions demonstrate feasibility and innovation . Solution 1 implements a physical alteration to prevent direct contact between the metal shots and the piping . Solution 2 , integrating ’Strong oxidants’ , involves a surface treatment to improve the piping’s durability against metal shots through a protective coating . In summary , both the textbook’s solution and the solutions automatically generated by AutoTRIZ are practical , originating from different inventive principles and leading to different approaches . It is important to note that these solutions are relatively preliminary and can serve as foundational directions for innovators to further develop and refine their designs . On this basis , we will continue to develop AutoTRIZ to produce more detailed solutions for the given problem . 8 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS Figure 5 : AutoTRIZ generated solution report for Case 7 5 Discussion By far , we have presented a new methodology that integrates LLMs and the systematic innovation method , TRIZ , to automatically generate inventive solutions for any given problem in an interpretable way . This methodology has been implemented into a web - based tool , AutoTRIZ . We have demonstrated its effectiveness and practicality through experiments and case studies . Prior studies [ 14 , 15 ] have assessed LLMs’ capabilities across a broad range of engineering - related tasks , revealing that these models ( especially GPT - series models ) hold extensive engineering knowledge , such as design and manufacturing . Therefore , in our framework , we only control the reasoning flow , without limiting the knowledge involved in the ideation process , to fully leverage the general knowledge and capabilities of LLMs . In this study , our case base of 10 problems spans multiple distinct domains , and AutoTRIZ has effectively generated inventive solutions in each case . The proposed method significantly reduces the entry barrier to TRIZ . AutoTRIZ can generate a multitude of solutions in a short period of time because it leverages the computational power and vast knowledge base of LLMs . This efficiency is further enhanced by its user - friendly interface , allowing for easy configuration and use , significantly reducing the time needed to ideate and refine problem - solving strategies . In contrast , mastering the traditional TRIZ method for professional use typically requires months of training and substantial intellectual and cognitive efforts [ 39 ] . 9 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS In the comparative study of case 7 , we observed that the problem statement contains information related to the desired direction of improvement , which is relevant to the contradiction . Such information aids in aligning AutoTRIZ’s detections with humans’ . Accordingly , as demonstrated in Figure 6 , we can incorporate multi - input configurations into the system , enabling AutoTRIZ to produce solutions that better meet user expectations . Figure 6 : The multi - input usages of AutoTRIZ Although this study focuses on automating the TRIZ reasoning process using LLMs , the proposed framework can be extended to automate other knowledge - based innovation methods . For instance , Yilmaz et al . [ 40 ] identified 77 design heuristics from over 3 , 000 design process outcomes , and suggested a subset of heuristics to designers to be selected at random has produced improved design outcomes [ 41 ] . By applying our framework to this research , one could treat the identified heuristics as an internal knowledge base for the LLM - based agent , determining how to utilize these heuristics at the backend . Moreover , to develop a more powerful tool , one could also integrate various knowledge - based idea generation methods into the reasoning modules behind LLMs , such as SCAMPER [ 42 ] , IDEO Method Cards [ 43 ] , Bio - inspired Design [ 44 ] , and Design - by - Analogy [ 45 , 46 , 47 ] . The proposed AutoTRIZ framework has several limitations . Firstly , the solutions generated by the LLMs may contain hallucinations or erroneous information . We plan to include fact - checking modules to ensure the accuracy of the solutions . Also , there is no objective mechanism to evaluate the effectiveness of generated solutions . Users must independently assess solution quality and rank them for practical use . Moreover , this study is demonstrated on a limited set of problem cases , providing only an initial insight into AutoTRIZ that might introduce some bias . In future research , we aim to apply this method to a broader and more diverse range of problems , systematically evaluating the AutoTRIZ’s performance . 6 Conclusion In this paper , we propose AutoTRIZ , an artificial ideation workflow and tool that leverages LLMs to automate the TRIZ methodology and enhance its applications . AutoTRIZ is constructed by 3 LLM - based reasoning modules and a pre - defined function module , interacting with the inner fixed knowledge base . It takes problem statements from users as initial inputs and automatically produces an interpretable solution report by following the step - by - step TRIZ reasoning process . The efficacy of this method is demonstrated and evaluated through quantitative and comparative experiments , as well as case studies involving human uses of TRIZ from TRIZ textbooks . Although this paper primarily focuses on integrating LLMs with TRIZ , the proposed framework holds the potential to be extended to other knowledge - based ideation methods , including SCAMPER , Design Heuristics , and Design - by - Analogy . Despite its current limitations , we invite interested innovators to test use AutoTRIZ at : https : / / www . autotriz . ai / . Disclosure statement No potential conflict of interest was reported by the author ( s ) . References [ 1 ] F Zwicky . 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ISSN 0142694X . doi : 10 . 1016 / j . destud . 2016 . 05 . 001 . [ 41 ] Shanna Daly , Seda Yilmaz , James L . Christian , Colleen M . Seifert , and Richard Gonzalez . Design heuristics in engineering concept generation . Journal of Engineering Education , 101 : 602 – 628 , 2012 . [ 42 ] Bob Eberle . Scamper on : Games for imagination development . Prufrock Press Inc . , 1996 . [ 43 ] IDEO . IDEO method cards : 51 ways to inspire design . William Stout , 2003 . [ 44 ] Katherine Fu , Diana Moreno , Maria Yang , and Kristin L Wood . Bio - inspired design : An overview investigating open questions from the broader field of design - by - analogy . ASME Journal of Mechanical Design , 136 : 111102 , 11 2014 . ISSN 1050 - 0472 . doi : 10 . 1115 / 1 . 4028289 . [ 45 ] Shuo Jiang , Jie Hu , Kristin L Wood , and Jianxi Luo . Data - driven design - by - analogy : State - of - the - art and future directions . ASME Journal of Mechanical Design , 144 : 020801 , 2022 . 12 A RTIFICIAL I DEATION WITH TRIZ AND L ARGE L ANGUGAE M ODELS [ 46 ] Jeremy Murphy , Katherine Fu , Kevin Otto , Maria Yang , Dan Jensen , and Kristin Wood . Function based design - by - analogy : a functional vector approach to analogical search . ASME Journal of Mechanical Design , 136 ( 10 ) : 101102 , 2014 . [ 47 ] Jonathan Hey , Julie Linsey , Alice M Agogino , and Kristin L Wood . Analogies and metaphors in creative design . International Journal of Engineering Education , 24 ( 2 ) : 283 , 2008 . 13 \ No newline at end of file diff --git a/silver_data/9bc7d16790fe7083c0880b1bd7f3aa2a7d005381.pdf.txt b/silver_data/9bc7d16790fe7083c0880b1bd7f3aa2a7d005381.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..740a513e017baf2678d76afcf50f2aae85e3030d --- /dev/null +++ b/silver_data/9bc7d16790fe7083c0880b1bd7f3aa2a7d005381.pdf.txt @@ -0,0 +1 @@ +Creativity and Machine Learning : A Survey GIORGIO FRANCESCHELLI , Alma Mater Studiorum Università di Bologna , Italy MIRCO MUSOLESI , University College London , United Kingdom , The Alan Turing Institute , United Kingdom , and Alma Mater Studiorum Università di Bologna , Italy There is a growing interest in the area of machine learning and creativity . This survey presents an overview of the history and the state of the art of computational creativity theories , key machine learning techniques ( including generative deep learning ) , and corresponding automatic evaluation methods . After presenting a critical discussion of the key contributions in this area , we outline the current research challenges and emerging opportunities in this field . 1 INTRODUCTION The connection between creativity and machines is as old as computer science itself . In 1842 , Lady Lovelace , an English mathematician and writer recognized by many as the first computer programmer , issued the so - called “Lovelace’s objection” [ 232 ] : the Analytical Engine ( the digital programmable machine proposed by Charles Babbage [ 7 ] ) “has no pretensions to originate anything , since it can only do whatever we know how to order it to perform” [ 157 ] . Indeed , in the following centuries , several projects and studies concerning the design of machines that able to “originate something” have been carried out [ 39 , 45 , 97 , 132 , 156 , 182 ] . This has led to the emergence of a specialized field in computer science , namely Computational Creativity [ 32 ] , which concerns the study of the relationship between creativity and artificial systems [ 44 , 245 ] . In this context , the adoption of Deep Learning ( DL ) techniques has led to substantial breakthroughs in the recent years . Vast computational power and very large amounts of available data are at the basis of the increasing success of deep generative models ( i . e . , generative models based on DL [ 67 ] ) . Indeed , generative deep learning technologies have been used to write newspaper articles 1 , generate human faces [ 120 ] and voices [ 141 ] , design drugs and proteins [ 115 ] , and even create artworks sold for a few hundred thousand dollars 2 . However , while it is apparent that current technologies are able to generate impressive outputs , at the same it is also possible to argue that they cannot be considered creative in general [ 20 ] . In fact , the goal of generative deep learning is to produce synthetic data that resemble real one given in input as close as possible [ 67 ] . On the other hand , creativity involves novelty and diversity [ 168 ] . While for some problems mere content generation [ 240 ] might be sufficient , for other tasks , such as , for example , in the Arts , the ability to create different ( but still valuable ) outputs is essential . Goal and contributions of the survey . The goal of this survey is to present and critically discuss the state of the art in generative deep learning from the point of view of machine creativity . Moreover , to the best of our knowledge , this is the first survey that explores how current DL models have been or can be used as a basis for both generation ( i . e . , producing creative artifacts ) and evaluation ( i . e . , recognizing creativity in artifacts ) . The contribution of this survey can be summarized as follows . After a brief overview of the meaning and definitions of creativity in Section 2 , Section 3 presents an in - depth analysis of generative deep learning through the introduction of a new taxonomy and a critical analysis of machine creativity . Then , several ML - based methodologies to evaluate creativity are presented and discussed in Section 4 . Finally , 5 concludes the paper , outlining open questions and research directions for the field . 1 www . theguardian . com / commentisfree / 2020 / sep / 08 / robot - wrote - this - article - gpt - 3 2 www . christies . com / features / a - collaboration - between - two - artists - one - human - one - a - machine - 9332 - 1 . aspx 1 a r X i v : 2104 . 02726v3 [ c s . L G ] 5 J u l 2022 Franceschelli and Musolesi Related surveys . We now provide an overview of other surveys in areas related to the present work . For readers interested in a survey in deep generative models , we recommend [ 18 , 87 ] ; for an analysis of the state of the art in evaluation in computational creativity [ 130 ] is an essential reading ; for a review concerning AI and creativity in general , we recommend [ 199 ] ; for a practical view of generative deep learning , we suggest [ 67 ] ; finally , for an in - depth examination of artistic AI works ( also in human - computer co - creations [ 85 ] ) , [ 160 ] is a very comprehensive source of information . 2 DEFINING CREATIVITY Creativity has been studied for decades , and yet , there is no agreement about its definition . More than one hundred definitions have been provided [ 4 , 229 ] , and the number is still growing . In other words , we can say that creativity is a suitcase word , i . e . , people conflate multiple meanings into it [ 161 ] . Nonetheless , some concepts are nowadays widely accepted . One of them is the possibility of studying creativity from four different perspectives : person , press , process , product , i . e . , the so - called “four P’s” . These have been studied in computational creativity as well [ 114 ] . However , the focus has traditionally been on the product dimension . Indeed , the idea is that we study creativity without considering the inner being of the creator ( person ) , the relation with the environment ( press ) or the process . Even if person , press and process are important , we focus on aspects of creative work in relation to the output ( product ) itself . For this reason , we consider Boden’s three criteria for studying machine creativity , defined as “the ability to generate ideas or artifacts that are new , surprising and valuable” [ 16 ] . Value and novelty are always considered for creativity in product ; surprise is commonly used too [ 148 ] . These concepts might be defined in different ways . We underpin our analysis on Boden’s three criteria since they have been widely adopted . Boden also suggests that three forms of creativity can be identified [ 16 ] to describe how a novel and surprising product is obtained , therefore linking process and product . The three forms of creativity are combinatorial , exploratory and transformational . Combinatorial creativity is about making unfamiliar combinations of familiar ideas . Exploratory creativity involves the exploration of the conceptual space defined by the cultural context considered . Transformational creativity involves changing that space in a way that allows for new and previously inconceivable thoughts to become possible . It is also worth noting that Boden also identified four different questions that emerge when studying computational creativity . These are referred to as Lovelace questions , because many people would respond to them by using Lovelace’s objection . The first question is whether computational ideas can help us understand how human creativity works . The second is whether computers could ever do things which at least appear to be creative . The third is whether a computer could ever appear to recognize creativity . Finally , the fourth is whether computer themselves could ever really be creative , i . e . , with the originality of the products only deriving from the machine itself [ 16 ] . While the first one is studied in Boden’s work , we will provide the reader with an overview of the techniques that can possibly be used to answer the second ( Section 3 ) and the third ( Section 4 ) . With respect to the fourth , Boden states that “it is not a scientific question as the others are , but in part a philosophical worry about “meaning” and in part a disguised request for a moral political decision” . We agree with this position , but we hope that our survey will provide the reader with elements for answering the fourth as well . 3 GENERATIVE MODELS A generative model can be defined as follows : given a dataset of observations 𝑋 , and assuming that 𝑋 has been generated according to an unknown distribution 𝑝 𝑑𝑎𝑡𝑎 , a generative model 𝑝 𝑚𝑜𝑑𝑒𝑙 is a model able to mimic 𝑝 𝑑𝑎𝑡𝑎 . By sampling from 𝑝 𝑚𝑜𝑑𝑒𝑙 , observations that appear to have been drawn from 𝑝 𝑑𝑎𝑡𝑎 can be generated [ 67 ] . Generative deep learning 2 Creativity and Machine Learning : A Survey is just the application of deep learning techniques to form 𝑝 𝑚𝑜𝑑𝑒𝑙 . However , creativity is not only the ability to perform generation . Considering the definitions presented in Section 2 , the generated observations should be considered as novel , surprising and valuable ; and the space of solutions should be exploited in a way that is combinatorial , exploratory , or even better , transformational . In this section , we aim at studying the level of creativity of existing generative deep learning models . We will analyze how the models learn their spaces of solutions , and how the observations are generated from them . A new generative deep learning taxonomy is then introduced based on the different training and sampling techniques at the basis of each method . Figure 1 provides a summary of the seven generative classes considered in this survey . Fig . 1 . A schematic view of the seven classes of generative learning methods presented in this survey . Top , left to right : Variational Autoencoder ( 3 . 1 ) , with a decoder generating x ′ given a latent vector z , and an encoder representing x into a latent distribution ; Generative Adversarial Network ( 3 . 2 ) , with a generator to produce x ′ , and a discriminator to distinguish between real x and synthetic x ′ ; Sequence prediction model ( 3 . 3 ) , with a generator to output 𝑥 one token after the other given in input previous tokens ; Transformer - based model ( 3 . 4 ) , with a Transformer outputting x one token after the other given in input previous tokens , or a masked version of x . Bottom , left to right : Diffusion model ( 3 . 5 ) , with a model to learn an error 𝜖 , which is used to incrementally reconstruct x 0 ; RL - based method ( 3 . 6 ) , with a generative model acting ( i . e . , progressively generating x ) by maximizing a given reward function ; Input - based methods ( 3 . 7 ) , with an input optimized by a given loss . The input can be a vector z given to a generative model to obtain the desired output , or directly a product x becoming the desired output . Since our focus is on machine creativity , we do not discuss the implementation details of each class of methods in detail . We instead present the core concepts at the basis of each class of methods ; some relevant examples of models ; potential applications of these creative models ; and , finally , a critical discussion evaluating the level of machine creativity considering the definitions above . As a final remark , it is worth noting that we limit our examples to the Arts ( e . g . , poems , music , or paintings ) . Indeed , generative learning can be applied to design [ 75 , 153 ] ; game content generation ( see [ 145 ] for a comprehensive survey ) ; recipes [ 166 , 238 ] ; scientific discovery [ 40 , 206 ] ; and in general to any activity , which has a non - trivial solution [ 25 ] . 3 Franceschelli and Musolesi 3 . 1 Variational Auto - Encoders 3 . 1 . 1 Core Concepts . A Variational Auto - Encoder [ 126 , 193 ] is a learning architecture composed by two models : an encoder ( or recognition model ) and a decoder ( or generative model ) . The former compresses high - dimensional input data into a lower - dimensional representation , and the latter decompresses the representation vector back to the original domain [ 67 ] . Classic autoencoders directly learn a latent representation vector . Conversely , VAEs learn a ( Gaussian ) distribution over the possible values of the latent representation , i . e . , the encoder learns the mean and the ( log of the ) variance of the distribution . The decoder then learns to reconstruct the data by taking in input a vector sampled from the distribution . VAEs are trained by optimizing two losses : the reconstruction loss and the regularization loss . The former is the log - likelihood of the real data x from the decoder given their latent vectors z , i . e . , it is the error of the decoder in reconstructing x . The latter is the KL divergence between the distribution learned by the encoder and a prior distribution , e . g . , a Gaussian . Notably , the latent vector z in input to the decoder is obtained by means of the so - called reparameterization trick , i . e . , by sampling from the distribution defined by the mean and the variance . Without it , sampling would induce noise in the gradients required for learning [ 127 ] . The mathematical derivation of the whole loss has its roots in variational inference [ 111 ] . Indeed , VAEs can be seen as an efficient and stochastic variational inference method , in which neural networks and stochastic gradient descent are used to learn an approximation ( i . e . , the encoder ) of the true posterior [ 70 ] . In VAEs , similar high - dimensional data are mapped to similar distributions . This makes possible to sample a random point z in that space , and still obtain a comprehensible reconstruction [ 67 ] . On the other hand , VAE tends to produce blurred images [ 260 ] . It may also happen that high - density regions under the prior have a low density under the approximate posterior , i . e . , these regions are not decoded to data - like samples [ 5 ] . Finally , the objective can lead to overly simplified representations without using the entire capacity , obtaining only a sub - optimal generative model [ 28 ] . 3 . 1 . 2 Examples of Models . Several models based on VAEs have been proposed [ 127 ] in the recent years . In the following , we focus on those relevant for our discussion on machine creativity . In 𝛽 - VAE [ 89 ] , a 𝛽 parameter is used to scale the magnitude of the regularization loss , which allows for a better disentanglement of the latent space [ 29 ] . Another example is VAE - GAN [ 133 ] , which merges VAE and GAN [ 77 ] ( described in the next subsection ) . This is done by treating the decoder as the generator of the GAN , thus training it by means of the GAN loss function . This leads to the generation of substantially less blurred images . Similarly , ALI ( Adversarially Learned Inference ) [ 57 ] merges VAE and GAN by asking the discriminator to distinguish between pairs of real data ( and their latent representations ) and pairs of sampled representations and synthetic data . Instead , AAE ( Adversarial Autoencoders ) [ 154 ] substitutes the regularization loss with a discriminative signal , where the discriminator has to distinguish between random latent samples and encoded latent vectors . Another way to address the problem of “sample blurriness” is with PixelVAE [ 83 ] , where the autoregressive PixelCNN [ 234 , 235 ] is used as the decoder . A different decoder architecture has been also used in [ 19 ] . To deal with sequential data like texts , where the generation requires more steps , the encoder learns to produce a latent representation of a sentence , while the RNN - based decoder learns to reproduce it word after word . However , VAE can also generate text by means of convolution and deconvolution [ 208 ] . To solve the problem of low - density regions , the authors of [ 5 ] propose an energy - based model called noise con - trastive prior ( NCP ) , trained by contrasting samples from the aggregate posterior to samples from a base prior . Finally , 4 Creativity and Machine Learning : A Survey another interesting model is VQ - VAE ( Vector Quantised - VAE ) [ 236 ] , in which the encoder outputs discrete , rather than continuous , codes , and the prior is learnt rather than being static . 3 . 1 . 3 Applications . VAEs can be used for semi - supervised classification to provide an auxiliary objective , improving the data efficiency [ 125 , 147 ] ; to perform iterative reasoning about objects in a scene [ 61 ] ; to model the latent dynamics of an environment [ 243 ] . Of course , VAEs have also been used to generate synthetic data , including for conditional generation . For example , a layered foreground - background generative model can be used to generate images based on both the latent representation and a representation of the attributes [ 251 ] . In [ 86 ] the latent space of a VAE is trained on chemical structures by means of gradient - based optimization towards certain properties ( see 3 . 7 ) . AAEs have also been applied to the same problem [ 116 ] . Finally , another interesting application of VAE is DRAW [ 80 ] . DRAW constructs scenes in an iterative way , by accumulating changes emitted by the decoder ( then given to the encoder in input ) . This allows iterative self - corrections and a more natural form of image construction . RNNs and attention mechanism are used to consider previous generations and to decide at each time - step where to focus attention , respectively . 3 . 1 . 4 Critical Discussion . Models based on VAEs can be considered as an example of exploratory creativity . The latent space is learned with the goal of representing data in the best accurate way . The random sampling performed during generation is therefore an exploration of that space : regions not seen during training can be reached as well , even though they can lead to poor generation [ 5 ] and some more complex variants may be needed , as discussed . On the other hand , there is no guarantee that the results obtained will be valuable , novel or surprising . There is no guarantee that the the generation from random sampling is of good quality , or diverse from training data . Indeed , given their characteristics , VAEs discourage novelty in a sense . Notably , diversity could in theory be achieved using VAEs and gradient - based optimization techniques , such as those presented in [ 86 ] , with novelty and surprise as target properties . We will discuss these aspects in Section 3 . 8 in more details . 3 . 2 Generative Adversarial Networks 3 . 2 . 1 Core Concepts . A Generative Adversarial Network ( GAN ) [ 77 ] is an architecture composed by two networks : a generative model and a discriminative model . The latter learns to distinguish between real samples and samples generated by the former . In parallel , the former learns to produce samples from random noise vector such that they are recognized as real by the latter . This competition drives both models to improve their methods , until the generated samples are indistinguishable from the original ones . The adversarial training allows the generator to learn to produce seemingly real samples from random noise without being exposed to data . The simplicity of the idea and the quality of results are at the basis of the success of GANs . However , few limitations exist . For instance , GAN can suffer from mode collapse , where the generator only learns to produce a small subset of the real samples [ 159 ] . In addition , the latent space of random inputs is typically not disentangled and it is necessary to introduce constraints in order to learn an interpretable representation [ 123 ] . 3 . 2 . 2 Examples of Models . The number of variants proposed is still growing 3 . An in - depth survey on GANs is [ 81 ] . Indeed , several refinements have been proposed in the past years , such as using deep convolutional networks [ 185 ] or self - attention [ 254 ] , incrementally growing the networks [ 119 ] , or by scaling the model parameters [ 23 ] . In the following , we present examples that are relevant to the the problem of machine creativity . 3 For an exhaustive and constantly updated list of GAN papers see for example : https : / / github . com / hindupuravinash / the - gan - zoo 5 Franceschelli and Musolesi The problem of non - meaningful representation has been addressed in different ways . For instance , InfoGAN [ 35 ] adds a latent code c to z . An auxiliary model learns to predict 𝑐 given the sample generated by means of it . In this way , it can learn disentangled representations in a completely unsupervised manner . Another possibility is BiGAN [ 53 ] . In order to include an inverse mapping from data to latent representation , an encoder is added to the architecture . The discriminator is then trained to distinguish between pairs of random noise and synthetic data and pairs of real data and latent encoding . It is also possible to condition the generation by means of a target content [ 176 ] , a text [ 191 ] , or even an image [ 101 ] . In order to do so , it is sufficient to use the conditional information as input for both generator and discriminator [ 162 ] . Similarly , image - to - image translation is possible also without paired datasets . CycleGAN [ 262 ] trains two generators ( from one domain to another , and vice - versa ) so that each of them produces images both from the target domain and correctly reconstructed by the counterpart . In StyleGAN [ 121 , 122 ] , the generator architecture is re - designed in order to control the image synthesis process . The style of the image is adjusted at each layer based on the latent code ( the specific intermediate code to control each layer is provided by a non - linear mapping network ) . This allows for automatic separation of high - level attributes from stochastic variations in the generated images . It also allows for mixing regularization , where two latent codes are used alternatively to guide the generator . StyleGAN - V [ 213 ] builds on top of it to learn how to produce videos by only using few frames of it . To generate longer and more realistic motions , a two - stage approach can be used as well : first , a low - resolution generator is adversarially trained on long sequences ; then , a high - resolution generator transforms a portion of the produced , low - resolution video in a high - resolution one [ 24 ] . Finally , it is also worth mentioning variants that adapt GANs to sequential tasks ( e . g . , text generation ) . Since GANs require the generator to be differentiable , they cannot generate discrete data [ 76 ] . However , several techniques have been proposed to avoid this problem . One possibility is to transform the discrete generation into a continuous one . Music can indeed be processed like an image by considering its waveform ( as in WaveGAN [ 52 ] and GANSynth [ 60 ] ) or its musical score composed of tracks and bars ( as in MuseGAN [ 54 ] ) . Music in a desired style can be obtained through conditional inputs . Another possibility is to consider a soft - argmax function as an approximation of the inference for each step [ 257 ] . TextGAN [ 258 ] uses it together with feature matching to learn the production of sentences . In place of the discriminative signal , it uses the difference between the latent feature distributions of real and of synthetic sentences learned by the discriminator . Another solution is to transform the GAN into a Reinforcement Learning ( RL ) framework ( see SeqGAN [ 253 ] ) . The generative model is the agent ; the tokens generated so far form the state ; the selection of the next token to be generated is the action to be performed ; and the discriminative signal is the reward . The REINFORCE algorithm [ 246 ] can then be used to adversarially train the generative model . Other policy gradient methods can be used as well [ 63 ] . The advantage of using RL to adapt GANs to sequential tasks is that the reward function can be composed by more signals : additional objectives can be used as well to embed particular behaviors in the generator . As suggested by ORGAN [ 82 ] , qualitative heuristics - like tonality and ratio of steps - can be used to improve music generation . On the other hand , the learning signal ( i . e . , the reward ) might be very sparse . A way to solve this issue is to use inverse RL [ 263 ] . For example , the authors of [ 210 ] use inverse RL to learn a reward function able to associate positive rewards to real state - action pairs , and non - positive rewards to synthetic state - action pairs . Notably , this can help solve mode collapse too . Another variant is LeakGAN [ 84 ] . Here , a hierarchical generator composed by a Manager and a Worker is used . The Worker produces a sentence conditioned by a goal vector provided by the Manager . The Worker and the discriminative model are trained following SeqGAN ; the Manager is trained to predict goal vectors that lead to the identification of advantageous directions in the discriminative feature space . More specifically , the Manager receives a feature vector from the discriminator , i . e . , its last convolutional layer , at each generated token . By means of this leaked 6 Creativity and Machine Learning : A Survey information and the hierarchical architecture , LeakGAN produces longer and higher - quality texts . Finally , another possibility is to use Gumbel - softmax relaxation [ 102 , 150 ] , as in RelGAN [ 173 ] . CTERM - GAN [ 15 ] builds on the latter by also conditioning the generator on an external embedding input . In addition , it uses both a syntactic discriminator to predict if a sentence is correct and a semantic discriminator to infer if a sentence is coherent with the external input . 3 . 2 . 3 Applications . GANs have been applied to a variety of practical problems in several application scenarios . They have been widely used for semi - supervised learning [ 175 ] ; for generating adversarial examples [ 249 ] to better train image classifiers [ 151 ] ; and , in general , in computer vision ( see [ 242 ] for a detailed discussion ) . The generative power of GANs has also found its place in recommender systems ( see [ 47 ] ) to generate fashion items ; in science and chemistry [ 158 , 167 ] . Of course , its ability of generating high - quality samples has been exploited in many other areas , from anime design [ 110 ] and 3D object modeling [ 248 ] to photo - realistic consequences of climate change [ 207 ] . Conditional inputs also allow to produce artistic works by controlling stylistic properties like genre [ 226 ] or influencer [ 37 ] . Finally , the most famous example of the artistic power of GAN is the collection of paintings by Obvious , a French art collective [ 241 ] ; one of their works has been sold to more than 400 , 000 dollars 4 . 3 . 2 . 4 Critical Discussion . GANs are difficult to evaluate from a machine creativity perspective . The generator does not receive the original works as input , so it samples from a conceptual space that is built only indirectly from them . In rare cases , this can also lead to a different conceptual space ( with respect to the original one ) and so to transformational creativity , but it typically leads to exploratory creativity . In fact , the goal is to learn to generate artifacts that look as much as possible like the real ones , so the loss function itself makes it working with exploratory creativity . Products are valuable ( this is , in a sense , the objective ) , but there is no guarantee that they will also be new and surprising . Nonetheless , it seems possible to extend a GAN objective to include such properties as well ( see Subsection 3 . 8 for a discussion ) . Finally , it is worth noting that GANs can be considered appreciative [ 40 ] , since the discriminative network can judge the value of artifacts . 3 . 3 Sequence Prediction Models 3 . 3 . 1 Core Concepts . A sequence prediction model is a generative model that considers generation as a sequential process . It can therefore work in an autoregressive fashion : it predicts the future outcome of the sequence ( i . e . , the next token ) from the previously observed outcomes of that sequence . It is trained to minimize the prediction error for each token in the dataset . At inference time , this simple yet effective approach only requires to produce one token after the other , feeding back to the model what have been produced so far [ 118 ] . It makes possible to learn dependencies between tokens in real data , so that the same dependencies can be exploited when generating synthetic data . However , this causes the generation to be highly dependent from real data , e . g . , there is the risk of potentially reproducing portions of the training set . 3 . 3 . 2 Examples of Models . Several models have been proposed , most of them based on recurrent neural networks ( RNN ) , and especially on long short - term memory ( LSTM ) [ 95 ] . The reason is that RNNs can have internal states based on previous computation : inputs received at earlier time steps can affect the response to the current input . However , RNNs tend to have worse performance with longer sequences [ 12 ] . LSTM is a specific RNN architecture that addresses the problem of long - term dependencies . 4 Fun fact : the sold painting is called Portrait of Edmond De Belamy because Belamy sounds like bel ami , a sort of French translation of . . . Goodfellow . 7 Franceschelli and Musolesi RNNs can be used to model joint probabilities of characters ( Char - RNN ) [ 118 ] ; words [ 181 ] ; phonemes [ 98 ] ; syllables [ 264 ] ; and even tokens from transcriptions of folk music ( Folk - RNN ) [ 222 ] . They can also receive conditional inputs like the encoding of the previous lines [ 256 ] . Richer architectures that combine models focusing on different characteristics of the text can be used to generate more complex text , e . g . , poetry based on pentameter and rhymes [ 134 ] . Finally , sequence modeling can also be combined with reinforcement learning . For instance , the authors of [ 105 ] use a Note - RNN model ( based on single notes ) trained using Deep Q - Network [ 164 ] ; as rewards , they consider both the classic loss of sequence prediction models and a reward based on rules of music theory . In this way , the model learns a set of composition rules , while still maintaining information about the transition probabilities of the training data . The advantages of adopting an RL - based approach are described in 3 . 6 . Due to the difficulties in working with long sequences , results in tasks like narrative generation are affected by a lack of coherence [ 197 ] . Many approaches have been proposed to address this problem . For instance , stories can be generated in terms of events [ 155 ] ( i . e . , tuples with subject , verb , object , and an additional wildcard ) by an encoder - decoder RNN ( also known as Sequence - to - Sequence , see [ 223 ] ) ; events are modeled by another encoder - decoder RNN . Instead of events , it is also possible to focus on entities ( i . e . , vectors representing characters ) [ 38 ] . Sequence prediction models are also used for domains not commonly modeled as sequences , like images . Image modeling can be defined in a discrete way by means of joint distribution of the pixels : the model learns to predict the next pixel given all the previously generated ones . It starts at the top left pixel , and then proceeds towards the bottom right . The two seminal architectures for sequence prediction of images are PixelRNN and PixelCNN [ 234 ] . The former is a two - dimensional RNN ( based on rows or on diagonals ) . The latter is a CNN with an additional fixed dependency range ( i . e . , the filters on the convolution are masked in order to only use information about pixels above and to the left of the current one ) . To obtain better results , gated activation units can be used in place of the rectified linear units between the masked convolutions ; conditional inputs encoding high - level image descriptions can be used as well [ 235 ] . Notably , the Gated PixelCNN architecture can also be used for other types of data : WaveNet [ 233 ] implements it to generate audio based on the waveform , possibly guiding the generation with conditional inputs . 3 . 3 . 3 Applications . As discussed , sequence prediction models have been used to learn to write poems or stories ( by predicting a character , syllable , or word after the other ) ; to compose music ( by predicting a note or a waveform after the other ) ; to draw images ( by predicting a pixel after the other ) . In general , they can be used for any sort of time series forecasting [ 144 ] . They can also be used for co - creativity , as in Creative Help [ 197 ] . Despite their simplicity , sequence prediction models are one of the most successful generative techniques . An interesting example is Sunspring . It might be considered as the first AI - scripted movie : it was generated by a Char - RNN trained on thousands of sci - fi scripts [ 160 ] . The quality of the result is demonstrated by the fact that it was able to reach the top ten at the annual Sci - Fi London Film Festival in its 48 - Hour Film Challenge 5 . 3 . 3 . 4 Critical Discussion . Sequence prediction models generate outputs that have characteristics of both exploratory and combinatorial creativity . They are based on probabilistic predictions and they are able to generate new outputs in the induced space , but they can also reuse sequences of tokens from different works , combining them together . There is no guarantee that the results will be valuable or novel ; and classic methods like RNNs lack surprise [ 26 ] . It is worth noting that the possibility of using conditional inputs and being able to work at different levels of abstraction might 5 Quite interestingly , the AI that wrote Sunspring declared that its name was Benjamin , probably in honor of Walter Benjamin , the German philosopher that , already in 1935 [ 13 ] , understood that new mechanical techniques related to art can radically change the public attitude to art and artists . 8 Creativity and Machine Learning : A Survey indirectly lead to creative outputs , but creativity should then be attributed to the higher - level component ( or human , if the input is provided by the user ) that is guiding the generation for specific elements and characteristics of the result . 3 . 4 Transformer - Based Models 3 . 4 . 1 Core Concepts . Transformer - based models are neural networks based on the Transformer architecture [ 239 ] . They are sometimes referred to as foundation models [ 17 ] , because of the leading role they have been assuming in language , vision and robotics . A Transformer is an architecture for sequential modeling that does not require recurrent or convolutional layers . Instead , it only relies on a self - attention mechanism [ 9 ] that models long - distance context without a sequential dependency . Each layer consists of multi - head attention ( i . e . , several self - attention mechanisms running in parallel ) , a feed - forward network , and residual connections . Since self - attention is agnostic to token order , a technique called positional embedding is used to capture the ordering [ 239 ] . In principle , a Transformer is nothing more than an autoregressive model : it works by predicting the current token given the previous ones ( see 3 . 3 ) . However , few fundamental differences exist . A Transformer can also be trained by means of masked modeling : some of the input tokens are randomly masked , and the model has to learn how to reconstruct them from the entire context , and not only from the previous portions [ 48 ] . The possibility of dealing with very long sequences allows for prompting . By providing a natural language prompt in input , the model is able to generate the desired output , e . g . , the answer to a question , a class between a set of classes for a given text , or a poem in a particular style [ 25 ] . This is done by simply passing the prompt in input as a text , and then leveraging the model to predict what comes next ( e . g . , the answer to a question ) . These advantages , together with the massive amount of data available , the increasing computational power and the parallelism induced by their architecture , has led Transformer - based models to become the state of the art for several tasks . Nevertheless , the compute costs of the architecture from [ 239 ] grow quadratically with the input size . 3 . 4 . 2 Examples of Models . Several Transformer - based approaches have been proposed in recent years . The design of specific Transformers for a variety of applications in presented in several surveys ( e . g . , [ 17 , 124 ] ) and books ( e . g . , [ 231 ] ) . The domain mostly influenced by Transformers is natural language processing . BERT [ 48 ] is a Transformer - based encoder trained for both predicting the next sentence ( in an autoregressive fashion ) and reconstructing masked tokens from the context . Several enhanced variations of the original model have been proposed . For example , RoBERTa [ 146 ] is able to achieve higher - quality results only by means of masked modeling with more training time and data ; ALBERT [ 131 ] obtains competitive results with less parameters by using inter - sentence coherence as an additional loss ; and DistilBERT [ 203 ] uses distillation [ 90 ] to train a smaller model to match BERT outputs . The other main approach is that used by the GPT family [ 25 , 183 , 186 ] . Here , a Transformer - based decoder is trained in an autoregressive way by additionally providing the relative task in input . After training , it can be used to perform a wide range of tasks by providing a description or few demonstrations of the task . The effectiveness of this text - to - text generative approach has then been explored by T5 [ 187 ] . Many other large language models [ 198 , 211 , 255 ] have been proposed to achieve better results by means of more parameters and computation [ 214 ] , or data [ 96 ] . Mixture of Experts [ 209 ] can be used as well in place of feed - forward network to train a larger but lighter model ( since only portions of it are used per task ) , as done by GLaM [ 56 ] . Finally , BART [ 139 ] ideally merges together a BERT - encoder ( trained by corrupting text with an arbitrary noising function ) and a GPT - decoder ( trained to reconstruct the original text autoregressively ) . Such an encoder - decoder architecture is able to achieve state - of - the - art results in machine translations , as well as in other text - to - text tasks . 9 Franceschelli and Musolesi Transformer - based models have been used in other domains as well . Few have been proposed for music generation . One of the first examples was Music Transformer [ 99 ] , which can generate one - minute music in Bach’s style with internal consistency ; another remarkable one is Musenet [ 179 ] , which is able to produce 4 - minute musical composition with a GPT - 2 architecture ; and , finally , it is worth mentioning Jukebox [ 49 ] , which can generate multiple minutes of music from raw audio by training a Sparse Transformer [ 36 ] ( i . e . , a Transformer with sparse factorization of the attention matrix in order to reduce from quadratic to linear scaling ) over the low - dimensional discrete space induced by a VQ - VAE . Conditioning is always considered by means of genre , author , or instruments . Another important application domain is video - making . ViViT [ 6 ] generates videos using classic Transformer architectures ; VidTr [ 259 ] achieves state - of - the - art performance thanks to standard deviation - based pooling method ; and VideoGPT [ 250 ] does so by learning discrete latent representations of raw video with VQ - VAE , and then training a GPT autoregressively . Transformers have been highly influential in computer vision too . The first model was Image Transformer [ 178 ] . It restricts the self - attention mechanism to attend to local neighborhoods , so that larger images can be processed . Class - conditioned generation is also supported , by passing the embedding of the relative class in input . To avoid restricting self - attention to local neighborhoods , Vision Transformer [ 55 ] divides an image into fixed - size patches , linearly embeds each of them , adds position embeddings , and then feeds the resulting sequence of vectors to a standard Transformer encoder . Masked Autoencoders ( MAE ) [ 88 ] instead uses an encoder - decoder architecture based on Transformers trained with masked image modeling ( i . e . , to reconstruct randomly masked pixels ) . A BERT adaptation to images called BEiT [ 11 ] has also been proposed . Masked image modeling has also been used together with classic autoregressive loss [ 33 ] . Conversely , Vector Quantised - GAN ( VQGAN ) [ 62 ] allows a Transformer to be based on vector quantization . A GAN learns an effective codebook of image constituents . To do so , the generator is implemented as an auto - encoder ; vector quantization is applied over the latent representation returned by the encoder . It is then possible to efficiently encode an image in a sequence corresponding to the codebook - indices of their embeddings . The Transformer is finally trained on that sequence to learn long - range interactions . These changes also allow to avoid quadratic scaling , which is intractable for high - resolution images . Finally , also DALL - E [ 184 ] takes advantage from a discrete VAE . To generate images based on an input text , it first learns a discrete image encoding ; it concatenates the input text embedding with the image encoding ; it learns autoregressively on them . DALL - E becomes then able to generate a variety of text - conditioned images . CogView implements a similar architecture [ 51 ] . Finally , Transformer - based models have also been used in multimodal setting , in which the data sources are of different types . A survey can be found in [ 224 ] . The first examples of these systems consider text and images together as output of the Transformer architecture . By aligning their latent representations , images and texts can be generated by Transformer - based decoders given a multimodal representation . For instance , CLIP [ 184 ] has an image encoder pre - trained together with a text encoder to generate a caption for an image . ALIGN [ 108 ] , based on similar mechanisms , is able to achieve remarkable performance through a training based on a noisier dataset . In [ 230 ] the authors propose a frozen language model for multimodal few - shot learning : a vision encoder is trained to represent each image as a sequence of continuous embeddings , such that the frozen language model prompted with this embedding can generate the appropriate caption . In [ 64 ] the authors present BriVL , which performs multimodal tasks by learning from weak semantic correlation data . Finally , there is a trend toward even more complex multimodal models . For example , VATT [ 3 ] learns to extract multimodal representations from video , audio and text ; instead , Gato [ 192 ] serializes all data ( e . g . , text , images , games , other RL - related tasks ) into a flat sequence of tokens that is then embedded and passed to a standard large - scale language model . 10 Creativity and Machine Learning : A Survey 3 . 4 . 3 Applications . Transformer - based large language models can be used for almost any NLP task , including text summarization , generation and interaction . In order to do so , the model can be used as a frozen model ( i . e . , by providing latent representations in input to other models ) ; can be fine - tuned for the specific objective ; can be exploited with zero - shot , one - shot or few - shot setting by prompting the task or few demonstrations in input . Transfer learning can instead be used to perform image classification by means of Transformer - based models trained on images . Other domain - specific techniques can be used as well : for instance , PlotMachines [ 190 ] learns to write narrative paragraphs not by receiving prompts , but by receiving plot outlines and representations of previous paragraphs . From a generative learning perspective , Transformers have shown impressive performance in producing long sequences of texts and music , as well as in generating images based on input text . However , they have not successfully applied to only these data sources . For instance , AlphaFold uses a Transformer architecture to predict protein structure [ 115 ] ; RecipeGPT to generate recipes [ 135 ] ; and GitHub Copilot to help write code [ 34 ] . 3 . 4 . 4 Critical Discussion . Given the fact that the Transformers can be considered as an evolution of sequence prediction models , the observations made for that class of models ( see Subsection 3 . 3 ) apply also for them . However , the inherent characteristics of their architecture allows for larger models and higher - quality outputs , which are also able to capture a variety of dependencies of text and across data sources . More in general , a broader conceptual space is induced . This means that domain - specific tasks might be addressed by means of solutions outside or at the boundary of the sub - space linked with that domain . Moreover , possibly also through a careful use of inputs ( see Subsection 3 . 7 ) , their adoption might lead to transformational creativity . As far as Boden’s criteria are concerned , there is no guarantee that outputs of Transformer architecture would be valuable , novel , or surprising . 3 . 5 Diffusion Models 3 . 5 . 1 Core Concepts . Diffusion models are a family of methods able to generate samples by gradually removing noise from a signal [ 217 ] . The most representative approach is the Denoising Diffusion Probabilistic Model ( DDPM ) [ 91 ] . An image x 0 is corrupted by gradually adding noise until obtaining an x T from a pre - defined distribution ; the model then has to reverse the process . Each timestep 𝑡 corresponds to a certain noise level ; x t can be seen as a mixture of 𝑥 0 with some noise 𝜖 whose ratio is determined by 𝑡 . The model learns a function 𝜖 𝜃 to predict the noise component of x t by minimizing the mean - squared error . x t − 1 is then obtained from a diagonal Gaussian with mean as a function of 𝜖 𝜃 ( x t , 𝑡 ) , and with variance fixed [ 91 ] or learned [ 172 ] . At inference time , a diffusion model can generate a new sample by starting from pure random noise . The generation can also be conditioned by simply modifying the noise perturbation so that it depends on the conditional information . This process is close to the one followed by score - based generative models [ 218 , 219 ] . Instead of noise , here a model is trained to learn the score , i . e . , the gradient of the log probability density with respect to real data . Samples are then obtained by using Langevin dynamics [ 244 ] . Despite the differences , they can anyway be considered diffusion models ( both of them can be seen as specific , discrete cases of Stochastic Differential Equations [ 220 ] ) . 3 . 5 . 2 Examples of Models . In order to generate higher - quality images and to allow text - to - image generation , a variety of effective methods for conditioning have been proposed . A possibility is to use classifier guidance [ 50 ] : the diffusion score ( i . e . , the added noise ) includes the gradient of the log likelihood of an auxiliary classifier model . An alternative is classifier - free guidance [ 93 ] : to avoid learning an additional model , a single neural network is used to parameterize two diffusion models , one conditional and one unconditional ; the two models are then jointly trained simply by randomly setting the class for the unconditional model . Sampling is finally performed using a linear combination of the conditional 11 Franceschelli and Musolesi and unconditional score estimates . GLIDE [ 171 ] demonstrates how classifier - free guidance can be effectively used to generate text - conditional images . In addition , it shows how diffusion models can be used for image editing by fine - tuning in order to reconstruct masked regions . Performance improvement can be obtained by means of a cascade of multiple diffusion models performing conditioning augmentation [ 92 ] . Finally , DALL - E 2 [ 188 ] generates images by conditioning with image representations . At first , it learns a prior diffusion model to generate possible CLIP image embeddings from a given text caption , i . e . , conditioned by its CLIP text embedding . Then , a diffusion decoder produces images conditioned by the image embedding . Imagen [ 200 ] uses instead a cascaded diffusion decoder , together with a frozen language model as text encoder to increase the quality of outputs . Although the approach is particularly suitable to images , applications to other data sources have been developed as well . For instance , DiffWave [ 128 ] and WaveGrad [ 34 ] use diffusion models to generate audio . They overcome the continuous - discrete dichotomy by working on waveform . Another possibility is to use an auto - encoder like MusicVAE [ 196 ] to transform the sequence into a set of continuous latent vectors , on which training a diffusion model [ 163 ] . Finally , diffusion models for video have also been proposed , based on a novel gradient - based conditioning method [ 94 ] . 3 . 5 . 3 Applications . Diffusion models have been introduced very recently and so far , they have been used to generate audio , music and video , as well as to generate and edit images conditioned on input text . We expect applications to other data sources in a near future . Indeed , they lead to higher - quality outputs with respect to the previous state - of - the - art models . In particular , DALL - E 2 has been able to produce images from textual instructions with superior fidelity and variety . 3 . 5 . 4 Critical Discussion . Diffusion models learn a mapping between real images and a Gaussian latent space . Because of this , they are an example of exploratory creativity : they randomly sample from that space , and then they possibly navigate it in the direction imposed by conditional inputs . There is no guarantee that the results will be valuable , novel or surprising , even though these approaches are able to generate outputs characterized by a high variety . As already argued , novelty and surprise may only arise due to the conditioning input ( for example , a human describing a novel combination of elements ) , i . e . , the model is not imaginative on its own . 3 . 6 Reinforcement Learning - Based Methods 3 . 6 . 1 Core Concepts . With Reinforcement Learning ( RL ) - based methods , we aim to indicate all the generative models whose training relies on maximizing a reward . These models are based on the architectures introduced so far , e . g . , they can be GANs or autoregressive models . The difference is that they are not ( only ) trained to fool the discriminative part , or to reduce prediction error . The typical framework considers the generative model as the agent ; each action causes a modification to the current product , i . e . , the state ; and the agent learns a policy that maximizes the cumulative reward . The reward can therefore be used to impose desired behavior to the generative model . The RL - based approach can be implemented for the entire training , as well as for fine - tuning a pre - trained model . 3 . 6 . 2 Examples of Models . We have already introduced a fully RL - trained model in Subsection 3 . 2 : ORGAN [ 82 ] . Here , RL is also used to provide additional learning signals , i . e . , additional rewards from specific - domain objectives . Also [ 252 ] follows this path by using rewards like fluency , coherence , meaningfulness and overall quality to generate poems . Another possibility is to use the metrics used at test time ( e . g . , BLEU or ROUGE ) [ 8 , 189 ] . Instead , RL - DUET [ 109 ] casts online music accompaniment generation as a RL problem with an ensemble of reward models , i . e . , autoregressive models trained with or without the whole context , and with or without the human - produced counterpart . In this way , 12 Creativity and Machine Learning : A Survey inter - coherence ( between human and machines ) and intra - coherence can be obtained . Finally , Intelli - Paint [ 212 ] can paint in human style by using a sequential planner that learns a painting policy to predict vectorized brushstroke parameters from the current state . RL can also be used to fine - tune a pre - trained generative model . Doodle - SDQ [ 261 ] at first learns to draw simple strokes by supervised learning ; then , it improves its generation by means of rewards about similarity , color , and line movement . Conversely , the authors of [ 225 ] suggest to consider a pre - trained LSTM language model as a policy model . Fine - tuning then aims at maximizing the probability that a given event occurs at the end of the narrative . RL Tuner [ 106 ] uses RL to fine - tune a Note - RNN to produce music that follows a set of music theory rules . To avoid forgetting note probabilities learned from data , the probability value returned by a copy of the pre - trained Note - RNN can be used as an additional reward . Sequence Tutor [ 104 ] generalizes this idea of learning a policy that trades off staying close to the data distribution while improving performance on specific metrics . Finally , also the authors of [ 129 ] suggest to fine - tune a language model by mixing up likelihood of training data and specific rewards , e . g . , repetition minimization . 3 . 6 . 3 Applications . As seen , RL - based models can be used to fully train or fine - tune generative models for different tasks ; ideally , for any task that could benefit from domain - specific objectives . This is the case of music and molecule generation [ 82 , 104 ] , but also of dialogue generation [ 140 ] and painting [ 100 ] . In addition , the sequential nature of RL can help as well in all the tasks requiring to deal with new directives during generation ( e . g . , music interaction ) . 3 . 6 . 4 Critical Discussion . The creativity evaluation of RL - based models depend on how the agent is implemented and which rewards are considered . The learned space of solutions depends on the used rewards ( and on the pre - training technique in case of fine - tuning ) . They typically contain an adversarial signal or the likelihood with respect to training data ; thus , combinatorial or exploratory creativity is obtained . However , additional rewards can have the effect of transforming that space ( see Subsection 3 . 8 ) . As far as Boden’s criteria are concerned , value is typically ensured by some qualitative domain - specific reward . Novelty and surprise might be achieved as well by means of specific rewards ; however , it is not the case of current models . Interestingly , the common presence of a value function ( e . g . , a critic network ) can make them appreciative , as requested by the creative tripod [ 40 ] . 3 . 7 Input - Based Methods 3 . 7 . 1 Core Concepts . The last class of methods we consider in our analysis is not about a different generative model . On the contrary , it is about a different way to sample results from ( pre - trained ) generative models , namely by means of its inputs . Two different approaches can be used . The first is about carefully selecting or optimizing the input to a generative model ( e . g . , the latent vector or the text prompt ) , so that to obtain the desired output . The second approach is about optimizing the input , so that it directly becomes the desired output . The used losses are based on features learned by neural networks . 3 . 7 . 2 Examples of Models . The first possibility is to carefully modify the input of a generative model , until the output matches desired properties . The main example is VQGAN - CLIP [ 46 ] . Given a text description , VQGAN [ 62 ] produces a candidate image from a random latent vector ; the vector is then optimized by minimizing the distance between the embeddings of the description and the candidate image . Both embeddings are computed using CLIP [ 184 ] . Variants can be implemented as in Wav2CLIP [ 247 ] , where an audio encoder is learned to match the CLIP encoders so that VQGAN - CLIP can be used from raw audio ; or as in music2video [ 103 ] , where videos are generated from audio a frame after the other by both minimizing distance between subsequent frames , and distance between image and music 13 Franceschelli and Musolesi segment embeddings by Wav2CLIP . Finally , image generators like VQGAN can also be exploited in other ways , i . e . , with binary - tournament genetic algorithm [ 66 ] or more complex evolution strategies [ 227 ] . Another possibility is to optimize the input so that the generated output maximizes a target neuron of an image classifier [ 170 ] . This helps generate what that neuron has learned . The desired latent vector can also be produced by an additional model [ 169 ] . The second solution is prompt tuning . Prompt tuning is about producing prompts via backpropagation ; the optimized prompts can then condition frozen language models in order to perform specific tasks without having to fine - tune them [ 137 ] . An additional model can be trained to output the desired prompt as well [ 138 ] . The third possibility is to optimize the input so that to transform it into the desired output . DeepDream [ 165 ] generates “hallucinated” images by modifying the input in order to maximize the output of a certain level from a pre - trained classifier . Also artistic style transfer is based on the same idea . Given an input image and a target image , the input image is modified by means of both style and content losses thanks to a pre - trained classifier . The content loss is minimized if the current and the original input images cause the same outputs from the hidden layers . The style loss is minimized if the current and the target images have the same pattern of correlation between feature maps in the hidden layers [ 72 ] . Control over results can be improved by considering additional losses about colour , space , and scale [ 73 ] . 3 . 7 . 3 Applications . Input - based methods can be used with any generative model to produce the desired output . With language models , they can exploit their generality in several specific tasks without fine - tuning them . With image generators , they can obtain drawings adherent to given descriptions , or high - quality but yet peculiar paintings like colourist [ 66 ] , abstract [ 227 ] or alien [ 215 ] artworks . Videos can be generated too 6 , and we believe applications to other domains are yet to come . Both types of input - based methods can be used not only to produce desired outputs or to transfer styles ; they can also be used to better analyze what it is inside the network [ 170 , 177 ] . 3 . 7 . 4 Critical Discussion . Since input - based methods are applied on pre - trained generative models , the space of solutions in which they work is the one induced by those models , i . e . , the common spaces we can derive from real data . Nonetheless , some techniques may be able to cause productions that are outside that space or at its boundaries , i . e . , to cause transformational creativity . This can especially happen if the model is general , and the output for a specific task is not only sampled from the sub - space of solutions for that task ( e . g . , with prompt tuning over a language model ) . Input - based methods are also valuable : the input optimization itself is typically guided by some sort of qualitative loss . On the other hand , they are not explicitly novel or surprising ( even though the results might seem so ) . However , nothing prevents to optimize the loss in such directions ( see 3 . 8 ) . 3 . 8 Practical Assessment of Creativity - Oriented Methods We conclude this analysis of generative models with a discussion of how these models might improve in order to increase their creativity according to Boden’s definition . A straightforward way to obtain novel and surprising outputs is to train a generative model by means of novelty and surprise objectives . This is the core idea behind CAN ( Creative Adversarial Network ) [ 58 , 204 ] . In addition to the classic discriminative signal , i . e . , a value loss , the generator is also trained to optimize a novelty loss . This is defined as the deviation from style norms , i . e . , the error related to the prediction of the style of the generated image . The sum of the two training signals helps the model learn to produce artworks that are different ( in style ) from the training data . The same approach has been used to develop a creative StyleGAN , i . e . , StyleCAN [ 107 ] . Another , very simple way to augment the training signal of a generative model with creativity - oriented 6 Also artistic abstract videos can be produced by navigating the latent space : www . jakeelwes . com / project - latentSpace . html 14 Creativity and Machine Learning : A Survey objectives is by means of RL - based methods . The choice of the reward structure is the fundamental element of the design of effective generative reinforcement learning systems . Rewards should teach the model to generate an output with a high level of novelty and surprise . An example is ORGAN [ 82 ] , where appropriate reward functions can be used . Another possibility is the development of an input - based method where the input is optimized so that to obtain a product that is valuable , novel and surprising . This may be achieved either by forcing a further exploration of the latent space ( e . g . , by means of evolutionary search [ 65 ] ) , or by defining appropriate loss functions to perform gradient descent over the input . All these methodologies are also called as active divergence [ 14 ] , since they aim to generate in ways that do not simply reproduce training data . A survey on active divergence can be found in [ 22 ] . A different approach is followed by the Composer - Audience architecture [ 26 ] . Here , in order to produce surprising artifacts , two models are considered : the Audience , a simple sequence prediction model trained on a given dataset ; the Composer , another sequence prediction model trained on a different dataset . In addition , the Composer also receives the next - token expectations from the Audience . It therefore learns when it has to follow its dataset and to diverge from expectations , i . e . , when to be surprising . For instance , it can learn to produce jokes by considering non - humorous texts to train the Audience , and humorous texts to train the Composer . Even though this approach is useful to learn how to generate valuable and surprising outputs , it is only applicable when paired datasets are available . As far as the type of creativity is concerned , there can be ways to achieve a better exploration or even transformation of the space of solutions . For instance , since CAN novelty loss is used during training , it learns to diverge from the distribution of real data . The same is true for RL - based methods with novelty and surprise rewards ( especially if training happens from scratch ) . Finally , a more explored or transformed space may be reached by RL - based methods driven by curiosity [ 27 ] . Indeed , an agent can learn to be creative and discover new patterns thanks to intrinsic rewards to measure novelty , interestingness and surprise . This can be done by training a predictive model of the growing data history , and by using its learning progress as the reward for the agent . In this way , it is motivated to make things the predictor does not yet know . If an external qualitative reward is considered as well , the agent should in theory learn to do things that are new , but still valuable [ 205 ] . The same idea can also be applied to different techniques like evolutionary strategies [ 149 ] . DeLeNoX ( Deep Learning Novelty Explorer ) [ 142 ] uses a denoising autoencoder to learn low - dimensional representations of last generated artifacts . Then , a population of candidate artifacts ( in terms of their representation ) is evolved through feasible - infeasible novelty search [ 143 ] in order to maximize distances between them , i . e . , to increase their novelty , while still considering qualitative constraints . Other evolutionary strategies might be considered as well to search the space of artifacts for novel [ 136 ] and surprising [ 79 ] results . Table 1 summarizes all the generative approaches discussed in this section , highlighting their characteristics from a machine creativity perspective . 4 CREATIVITY MEASURES In this section we present different methodologies for evaluating the creativity of artifacts generated by artificial agents . These can typically be extended to human - generated artifacts . For each of them , we explore the core concepts , the dimensions of creativity that are considered and the protocol for evaluation , and , finally , we critically assess them . The presence of several different proposals can be associated to the fact that it is not always straightforward to determine the “right” question to ask in an evaluation of a creative artifact [ 71 ] . This is also highlighted by the absence of creativity evaluation approaches in the GEM ( Generation , Evaluation , and Metrics ) Benchmark for natural language generation [ 74 ] . This is due to the inadequacy of the creative metrics proposed until now . The focus of our overview is on measures that are based on or associated to machine learning techniques . It is worth noting that some of them 15 Franceschelli and Musolesi Generative family Type of creativity Boden’s criteria Creative tips VAE Exploratory ∼∼∼ Value ∼∼∼ Novelty ∼∼∼ Surprise Creativity - oriented input - based methods GAN Exploratory ✓✓✓ Value ∼∼∼ Novelty ∼∼∼ Surprise CAN ; Creativity - oriented input - based methods Sequencepredictionmodel Combinatorial , Exploratory ∼∼∼ Value ∼∼∼ Novelty ××× Surprise Composer - Audience ; Creativity - oriented RL - based methods Transformer - basedmodels Combinatorial , Exploratory , Transformational ∼∼∼ Value ∼∼∼ Novelty ××× Surprise Creativity - oriented prompt tuning or RL - based methods Diffusion models Exploratory ∼∼∼ Value ∼∼∼ Novelty ∼∼∼ Surprise Creativity - oriented input - based methods RL - based methods Combinatorial , Exploratory , Transformational ✓✓✓ Value ∼∼∼ Novelty ∼∼∼ Surprise Intrinsic rewards ; Novelty - based rewards Input - based methods Exploratory , Transformational ✓✓✓ Value ∼∼∼ Novelty ∼∼∼ Surprise Evolutionary search ; Novelty - based optimization Table 1 . Summary of all the methods explained so far , considering their type of creativity ; the possible presence of Boden’s criteria ; and some practical tips to achieve a higher degree of creativity . might be calculated without using machine learning , but we will refer to an implementation based on the latter . For an in - depth overview about creativity measures not strictly related to machines , we refer to [ 201 ] . Table 2 reports all the evaluation methods considered in this section , highlighting the dimensions they try to capture , their applicability and limitations . We will discuss these aspects in the remainder of this section . 4 . 1 Lovelace 2 . 0 Test 4 . 1 . 1 Overview . The Lovelace Test ( LT ) [ 21 ] was proposed in 2001 as a creativity - oriented alternative to the world - famous Turing Test [ 232 ] . More formally , LT is defined as follows : Definition 4 . 1 . An artificial agent 𝐴 , designed by 𝐻 , passes LT if and only if : 1 ) 𝐴 outputs 𝑜 ; 2 ) 𝐴 ’s outputting 𝑜 is not the result of a fluke hardware error , but rather the result of processes 𝐴 can repeat ; 3 ) 𝐻 ( or someone who knows what 𝐻 knows , and has 𝐻 ’s resources ) cannot explain how 𝐴 produced 𝑜 by appeal to 𝐴 ’s architecture , knowledge - base , and core functions . LT provides several insights for understanding and quantifying machine creativity , but it is rather abstract . For these reasons , a 2 . 0 version has then been proposed [ 194 ] . The so - called Lovelace 2 . 0 Test is defined as : Definition 4 . 2 . Artificial agent 𝐴 is challenged as follows : 16 Creativity and Machine Learning : A Survey Name What evaluates How evaluates Applicability Limits Lovelace 2 . 0 Test What evalua - tors state creativity is Mean number of challenges per evaluator passed General • Require a massive human intervention Ritchie’scriteria Quality , novelty , typicality Human opinions ( then elaborated through 18 criteria ) General • Require human evaluation • Require to state thresholds • No innovation definition FACE Tuples of generativeacts Volume of acts , number of acts , quality ( through aesthetic measure ) General • Not a punctual method • Definition of aesthetic measure left to the user SPECS What we state creativity is Identification and test of standards for the creativity components General • More a framework for eva - luation method definition than a real method Creativity implication network Value , novelty Similarity between works ( considering subsequent works for value and pre - vious works for novelty ) General • No chance to correctly com - pute creativity for last works • Wrong creativity and time - positioning correlation Chef Watson ( assessment part ) Novelty , quality Bayesian surprise , smell pleasantness regression Specific ( recipes ) • Require human ratings of pleasantness DARCI Art appreciation Neural network to associate image featu - res and description Specific ( visual art ) • Not based on product • Consider just one of the creative tripod PIERRE - Evaluation part Novelty , quality Count of new combi - nations ; user ratings Specific ( recipes ) • Require user ratings over ingredients EVE’ Feelings , meanings Negative log of prediction and posterior probability General • Require a way to explain • Value only through meaning Common model of creativity for design Novelty , value , surprise K - Means on a description space and a performance space ; degree of violation of anticipated patterns Specific ( design ) • Require to define attribute - value pairs • Require to define clustering parameters Regent - Dependent creativity metric Novelty , value Bayesian surprise and synergy General • Require to define and extract features Unexpectedness Novelty , sur - prise , trans - formativity Possibility to update , and degree of violation of expectations General • Not take care of value Essential criteria of creativity Value , novelty , surprise Sum of performance va - riables , distance between artifacts and between re - al and expected artifact General • Require to define performance variables • Require to define clustering parameters Computationalmetricsforstorytelling Novelty , rarity , recreationaleffort , surprise Distance between do - minant terms , consecutive fragments / clusters of terms Specific ( storytelling ) • Require to define domination • Require to define clustering parameters Table 2 . Summary of creativity evaluation methods and their characteristics . 17 Franceschelli and Musolesi 1 ) 𝐴 must create an artifact 𝑜 of type 𝑡 ; 2 ) 𝑜 must conform to a set of constraints 𝐶 where 𝑐 𝑖 ∈ 𝐶 is any criterion expressible in natural language ; 3 ) a human evaluator ℎ , having chosen 𝑡 and 𝐶 , is satisfied that 𝑜 is a valid instance of 𝑡 and meets 𝐶 ; 4 ) a human referee 𝑟 determines the combination of 𝑡 and 𝐶 to not be unrealistic for an average human . 4 . 1 . 2 Dimensions of Creativity Considered . Since the evaluation depends on the tests performed by the human evaluators , the dimensions of creativity considered by it might vary greatly . This allows for considering value , novelty and surprise , as well as domain - specific dimensions . 4 . 1 . 3 Protocol for Evaluation . The Lovelace 2 . 0 Test can be used to quantify the creativity of an artificial agent - by means of its artificial productions - considering a set 𝐻 of human evaluators . With 𝑛 𝑖 as the first test performed by evaluator ℎ 𝑖 ∈ 𝐻 not passed by the agent , the creativity of the artificial agent can be expressed as the mean number of challenges - per - evaluator passed : (cid:205) 𝑖 ( 𝑛 𝑖 ) | 𝐻 | . 4 . 1 . 4 Critical Examination . This methodology represents an effective way to measure creativity , since it is ideally applicable to any field and it is quantitative . Even though it is not based on machine learning , the latter may be used in principle for performing ( some of ) the tests . This methodology requires considerable human intervention , and , for this reason , it cannot be used for automatic evaluation . 4 . 2 Ritchie’s Criteria 4 . 2 . 1 Overview . Ritchie’s Criteria [ 195 ] are a set of criteria for evaluating the extent to which a program has been creative ( or not ) in generating artifacts . These criteria are based on three main factors : novelty , quality and typicality . [ 195 ] contains a proposed series of criteria , but according to the authors they should only be intended as a “repertoire” . 4 . 2 . 2 Dimensions of Creativity Considered . Ritchie’s Criteria are based on three factors : quality , typicality , novelty . Quality measures how much an item is a high quality example of its genre . Typicality measures how much an item is an example of the artefact class in question . Novelty measures the dissimilarity of an item with respect to existing examples in its class . Quality and typicality are collected using human opinions about the produced artifacts or using “measurable” characteristics about , for instance , syntax and metric ( for poetry generator ) . On the other hand , novelty is intended as the sum of “untypicality” ( the opposite of typicality ) and innovation . 4 . 2 . 3 Protocol for Evaluation . The computation of the criteria is based on the analysis of the result set of produced artifacts , alongside with the inspiring set ( composed by artifacts of that field used during training and / or generation ) . It also requires the definition of quality and typicality indicators for the considered artifacts . More specifically , the proposed criteria are : the average of typicality or quality over the result set ; the proportion of items with good typicality score , which is also characterized by high quality ; the proportion of the output that falls into the category of untypical but high - valued ; the ratio between untypical high - valued items and typical high - valued items ; the proportion of the inspiring set that has been reproduced in the result set . The assessment of these criteria is performed before on the entire result set and then only on the subset not containing any item from the inspiring set . 4 . 2 . 4 Critical Examination . These measures represent a promising way of evaluating creativity , but their application does not appear as straightforward . In fact , they do not clearly specify how to measure novelty in terms of innovation . In addition , all the measures require a large number of thresholds to be set ( and results are very sensitive to such thresholds [ 180 ] ) . The criteria for the selection of these thresholds are not trivial per se . It is difficult to identify a 18 Creativity and Machine Learning : A Survey general methodology for setting their values . Finally , the collection of correct human opinions ( in terms of consistency of measurement methodology , audience selection , etc . ) is not a trivial task either . 4 . 3 FACE 4 . 3 . 1 Overview . In [ 42 ] the authors introduce FACE as a descriptive model of the creative act of artificial systems . A creative act is considered as a non - empty tuple of generative acts . FACE is designed to provide the assessors with both quantitative and qualitative evaluations . 4 . 3 . 2 Dimensions of Creativity Considered . While FACE can be used to evaluate a product as the consequence of a creative act , its focus is on the process . The qualitative evaluation is therefore left to the aesthetic function ; the dimensions considered depend in turn on how it has been defined ( or generated ) . 4 . 3 . 3 Protocol for Evaluation . More specifically , the FACE model considers a creative act as a non - empty tuple of generative acts of eight possible types : an expression of a concept , i . e . , an instance of an ( input , output ) pair produced by the system ; a method for generating expressions of a concept ; a concept ; a method for generating concepts ; an aesthetic measure ; a method for generating aesthetic measures ; an item of framing information , i . e . , an additional information or description regarding the generative act ; a method for generating framing information . It is then possible to use it in a quantitative way ( how many acts are produced ) ; in a cumulative way ( how many types of acts are considered ) ; and in a qualitative way ( by means of the defined aesthetic measure ) . 4 . 3 . 4 Critical Examination . The FACE model represents a very comprehensive set of concepts and methodologies for assessing machine creativity . However , one of the most challenging aspects of FACE is the definition of the aesthetic measure , which is not specified ; potentially , it might be defined by the system itself , counting as a potential creative act . This may award systems performing self - evaluation , i . e . , guiding their generation based on learned objectives . This in theory might mean the systems are incentivized to develop their own tastes , which is an important part of human creativity . 4 . 4 SPECS 4 . 4 . 1 Overview . The Standardized Procedure for Evaluating Creative Systems ( SPECS ) [ 112 ] is a framework for the evaluation of creative systems , which can easily be adapted to many different potential domains . The framework is based on the definition of fourteen “components” used for the evaluation of machine creativity . 4 . 4 . 2 Dimensions of Creativity Considered . The fourteen key components of SPECS are : active involvement and persis - tence ; dealing with uncertainty ; domain competence ; general intellect ; generation of results ; independence and freedom ; intention and emotional involvement ; originality ; progression and development ; social interaction and communication ; spontaneity / subconscious processing ; thinking and evaluation ; value ; variety , divergence , experimentation . However , SPECS does not constrain researchers to use all of them ; moreover , domain - specific components can be added as well . 4 . 4 . 3 Protocol for Evaluation . SPECS is composed of three steps . The first one requires to provide a definition of creativity that the system should satisfy , using the suggested components , and potentially other domain - specific ones . The second requires to specify the standards to be used to evaluate such components . Finally , the third requires to test the system against the standards and to report the results . 19 Franceschelli and Musolesi 4 . 4 . 4 Critical Examination . This is an effective framework for working with computational creativity , but it cannot be a practical evaluation method . Its effectiveness is strongly dependent on which components are considered and how they are evaluated for each specific task . In [ 113 ] the author discusses how SPECS satisfies Ritchie’s criteria and it is more comprehensive and expressive than the FACE model and the creative tripod [ 40 ] ( according to which a creative system should exhibit skills , appreciation and imagination ) in a meta - evaluation test . They also use a human opinion survey based on five criteria : correctness , usefulness , faithfulness ( as a model of creativity ) , usability ( of the methodology ) and generality . SPECS is evaluated considering music improvisation generators and it is also judged by the developers of the generative systems . In particular , the authors show that SPECS can help obtain additional insights on how a generative model works and how it can be improved . 4 . 5 Creativity Implication Network 4 . 5 . 1 Overview . A different method to quantify creativity is based on building an art graph called Creativity Implication Network [ 59 ] . Given a set of artworks , a directed graph can be defined by considering a vertex for each artwork . More specifically , an arc connecting artwork 𝑝 𝑖 to 𝑝 𝑗 is inserted if 𝑝 𝑖 has been created before 𝑝 𝑗 . A positive weight 𝑤 𝑖𝑗 quantifying the similarity score between the two artworks under consideration is associated to each arc . The creativity of an artwork is then derived by means of computations on the resulting graph . 4 . 5 . 2 Dimensions of Creativity Considered . This method captures both value and novelty . The value is defined as the influence on future artworks . The novelty is defined as the dissimilarity between the artwork and the previous ones . 4 . 5 . 3 Protocol for Evaluation . The derivation of the Creativity Implication Network requires a similarity function to compute the similarity scores ; its definition is left to the researchers , but it can be based on ML techniques ( as done in the original paper , where computer vision techniques were used ) . Given the network , the creativity of an artwork 𝑝 𝑖 depends on the similarity with the previous artworks ( higher the similarity , lower the creativity ) and with the subsequent artworks ( higher the similarity , higher the creativity ) . 4 . 5 . 4 Critical Examination . The Creativity Implication Network represents an effective way to deal with creativity of sets of artworks . It considers both value and novelty , and it allows for using automated techniques in the computation of similarity . On the other hand , two drawbacks should be highlighted . The first one is related to artworks that occupy the position of “leaves” in the graph : if there are not subsequent works in the graph , their creativity would only be based on novelty , and not on value . The second one is more subtle , and it is about time - positioning . As demonstrated by [ 59 ] , moving back an artwork has the effect of increasing its creativity ; however , this appears as conceptually wrong . As discussed in [ 16 ] , the time location of an artwork is fundamental in the quantification of its creativity . It may happen that , due to the surprise component of creativity , an artwork that appears too early might not be considered as surprising , because observers are not able to truly understand it ; on the contrary , if it appears too late , it might be considered as obvious and not surprising at all . In conclusion , even if this approach is able to correctly capture value and novelty , it is not able to capture the concept of surprise . 4 . 6 Generate - and - Test Setting 4 . 6 . 1 Overview . Generate - and - test setting [ 228 ] is a family of methods based on the separation of the generative process in two phases : generation , and evaluation . Assuming that gen ( ) denotes the generative function of the system , and a an artifact generated by the function . Then , a is evaluated by another function eval ( a ) that has to assess the 20 Creativity and Machine Learning : A Survey degree of creativity of a . If it passes the evaluation , it is outputted by the system . For example , the authors of [ 238 ] use this approach to develop a computational creativity system for generating culinary recipes and menus called Chef Watson . [ 43 ] describes an augmentation of Painting Fool [ 41 ] with DARCI [ 174 ] in a generate - and - test setting ( i . e . , by using The Painting Fool for generation , and DARCI for evaluation ) . PIERRE [ 166 ] is based as well on two models , one for generating recipes by means of a genetic algorithm , and one for evaluating them . 4 . 6 . 2 Dimensions of Creativity Considered . The dimensions of creativity considered depend on the specific implemen - tation of the evaluation function . Different evaluation functions have been designed to evaluate , for example , quality and novelty [ 166 , 238 ] and art appreciation [ 43 ] . It is worth noting that , given the generality of the approach , other evaluation functions could be designed to capture other aspects of machine creativity . 4 . 6 . 3 Protocol for Evaluation . The protocol for evaluation strictly depends on the specific implementation of the eval ( a ) function . For instance , Chef Watson [ 238 ] uses two measures : flavorfulness for quality , and Bayesian surprise for novelty . Flavorfulness is computed by means of a regression model , built on olfactory pleasantness considering its constituent ingredients . Bayesian surprise [ 10 ] is a measure of surprise in terms of the impact of a piece of data that changes a prior distribution into a posterior distribution , calculated applying Bayes’ theorem . The surprise is then the distance between posterior and prior distributions . It is worth noting that it has been demonstrated that there exists a mathematical limit in the maximization of quality and novelty when novelty is expressed in terms of Bayesian Surprise [ 237 ] . On the other hand , The Painting Fool [ 41 ] uses DARCI [ 174 ] in place of the evaluation function . DARCI is able to make associations between image features and descriptions of the images learned using a series of artificial neural networks as the basis for the appreciation . It has therefore been used as a sort of artificial art critic to complement The Painting Fool , allowing it to assess the validity of its own creations . Finally , PIERRE [ 166 ] evaluates the generated recipes again using novelty and quality . Novelty is computed by counting new combinations of used ingredients . Quality is based on two NNs that perform a regression of user ratings based on the amount of different ingredients , working at two levels of abstraction . 4 . 6 . 4 Critical Examination . The advantage of the generate - and - test setting is that a variety of evaluation functions can be defined . This allows , for instance , to evaluate the generative system by means of Boden’s three criteria , while still considering the specific characteristics of the domain of interest . On the other hand , its applicability is not general : as we have seen in the previous section , many generative systems do not follow the proposed setting ( e . g . , input - based methods use the evaluation to guide the generation , thus merging the two stages ) . 4 . 7 Atoms of EVE’ 4 . 7 . 1 Overview . Even if not strictly related to creativity , [ 30 ] proposes an approach to measure aesthetic experience called atoms of EVE’ , which is based on a probabilistic model of the world to derive expectations and explanation . The authors state that aesthetic arises in two ways : by forming E ( expectation ) while avoiding V ( violation ) ; and by forming E’ ( explanation ) while resolving V . 4 . 7 . 2 Dimensions of Creativity Considered . Even if not explicitly considered , the three grounding concepts of Atoms of EVE’ strongly intertwine with creativity . Expectation is close to value : it measures how much we are able to understand the object of interest . Violation is close to surprise : it is the unexpectedness of an object at a certain moment . Explanation is again close to value : it measures the intelligibility ( i . e . , its usefulness ) . These same considerations have been expressed 21 Franceschelli and Musolesi by [ 31 ] , where the author uses EVE’ to define a creativity measure based on feelings ( i . e . , surprise ) , computed thanks to violation , and meanings ( i . e . , value ) , computed thanks to explanation . 4 . 7 . 3 Protocol for Evaluation . Expectation is computed as the posterior probability after the occurrence of a given object : it measures how much the prior belief can help explain that object . Violation is instead computed as the unexpectedness of that object . Together with apprehension , which is the unpredictability of the next object ( before seeing it ) , violation returns the tension , one of the two fundamental measures of aesthetics . Finally , explanation measures how much the encountered violation can be explained by the posterior belief . Together with expectation , explanation returns a quantification of pleasure , the other fundamental measure of aesthetics . 4 . 7 . 4 Critical Examination . As observed before , while such a computation for surprise is common , this is not true for value . The focus on explanations provides an interesting way to mathematically define value . However , value is not only about finding meaning , but also about utility , performance and attractiveness [ 152 ] ; this is not possible through this measure . Finally , novelty is not considered at all . 4 . 8 Common Model of Creativity for Design 4 . 8 . 1 Overview . The authors of [ 153 ] propose a model to evaluate creativity in the specific domain of design . In particular , they consider creativity as a relative measure in a conceptual space of potential and existing designs . Designs are represented by attribute - value pairs ; and novelty , value and surprise can be used to capture different aspects of creativity in their space . 4 . 8 . 2 Dimensions of Creativity Considered . The model proposed by [ 153 ] considers all the three dimensions suggested by [ 16 ] , i . e . , novelty , value , and surprise . Novelty is considered as a matter of comparing objects in a descriptive space ; it is the degree of difference . Value is related to performance , i . e . , utility preferences associated with the attributes of an object . Finally , surprise is linked to violated expectations . 4 . 8 . 3 Protocol for Evaluation . The model is based on the analysis of the conceptual space of potential and existing designs defined by all the potential attribute - value pairs . Novelty is evaluated with respect to a description space , i . e . , by considering each product as the set of its descriptive attributes . Value is considered with respect to a performance space , i . e . , by considering attributes that have utility preferences associated with them . Finally , surprise is based on finding violations of patterns that is possible to anticipate in the space of both current and possible designs . The K - means clustering algorithm is used to organize the known designs by means of their attributes . Then , novelty , value and surprise measures of a new design are obtained looking at the distance to the nearest cluster centroid . 4 . 8 . 4 Critical Examination . Even if it targets explicitly the design domain , this approach is able to combine the three dimensions of creativity by Boden . Nonetheless , it is limited by the fact that artifacts have to be described through an attribute - value pair representation . In particular , a large number of features might be needed . Otherwise , we might lose aspects of the artifacts that are fundamental to correctly quantify creativity . Since it is not possible to know the fundamental features in advance , the method requires to enumerate as many features as possible . However , the risk is to define an excessive number of non - informative attributes , making the computation of the metrics too difficult . In fact , the data points become increasingly “sparse” as the dimensionality increases ; many techniques ( especially the clustering ones ) are based on distance , and they may therefore suffer from the curse of dimensionality [ 221 ] . Finally , as for classic machine learning techniques , there is the need of manually defining and extracting the chosen features 22 Creativity and Machine Learning : A Survey from unstructured data , which is a time - consuming and potentially prone - to - error activity . A possible way to overcome the problems related to feature extraction and the curse of dimensionality might be the adoption of deep learning techniques , given their effectiveness with unstructured data . 4 . 9 Regent - Dependent Creativity Metric 4 . 9 . 1 Overview . The Regent - Dependent Creativity metric [ 69 ] is based on novelty and value . The name comes from the Regent - Dependent Model , a proposed method to describe artifacts as collections of features , i . e . , through sets of pairs with a regent ( e . g . , an action or an attribute ) and a dependent ( e . g . , the specific target or value of the regent ) , labeled with a value to express the intensity of that pair in that context . 4 . 9 . 2 Dimensions of Creativity Considered . As explained above , the Regent - Dependent creativity metric considers novelty and value as dimensions of creativity . In particular , value is dependent on the associations and rules that bond artifacts in a specific context , which defines what is worth of interest , and what is not . 4 . 9 . 3 Protocol for Evaluation . Novelty and value are computed through Bayesian surprise ( see 4 . 6 . 3 ) and synergy , respectively . In particular , synergy is a metric that captures the cooperative nature of constituent elements of an artifact . It is based on modeling the artifacts as graphs . The vertices are the dependency pairs of the artifact . The edges between pairs exist only if they belong to the same set of synergy , i . e . , if they exhibit a better effect together than separately . 4 . 9 . 4 Critical Examination . In contrast with the approach described before , the Regent - Dependent metric appears to be of more general applicability . However , only novelty and value are considered , and surprise is missing . In addition , just as detailed in 4 . 8 . 4 , this method is limited by requiring the definition and the extraction of all the features to compute creativity . 4 . 10 Unexpectedness Framework 4 . 10 . 1 Overview . The authors of [ 78 ] suggest that a framework of unexpectedness ( i . e . , violation of an observer’s expectations ) can deal with novelty , surprise and domain transformation ( also called transformativity ) . Although they do not claim it can be a measure of creativity on its own , and that value should be added as well , they suggest it can become a vital component in computational creativity evaluation . 4 . 10 . 2 Dimensions of Creativity Considered . The authors of [ 78 ] suggest that unexpectedness can be used to compute three dimensions of creativity : novelty , surprise and transformativity . Indeed , novelty is about the possibility of violating observer’s expectations about the continuity of a domain ; if the current model of belief is not applicable for the current artifact , it can be considered as novel . Surprise is instead about the possibility of violating observer’s expectations about an artifact . Finally , transformativity is about the possibility of violating observer’s expectations about the conceptual space itself ( i . e . , finding that the rules governing it were not accurate ) . 4 . 10 . 3 Protocol for Evaluation . The unexpectedness framework should allow to model expectation . Notably , expectation should be linked with the socio - cultural context of the observer , since it is the observer that forms expectation , not the domain itself . In particular , an expectation is generated by a prediction about the predicted ( i . e . , the dependent variables of the artifact ) given a condition ( i . e . , a relationship between the predicted property and some other property of the object ) that applies within a scope ( i . e . , the set of possible artifacts to which the expectations apply ) . An observation that falls within that scope can then be measured for congruence with respect to that expectation . 23 Franceschelli and Musolesi 4 . 10 . 4 Critical Examination . The unexpectedness measure appears to be able to provide researchers and practitioners with a way for deriving novelty and surprise . Notably , it also captures transformativity , clarifying at the same time how simple surprise differs from it , i . e . , that surprise is related with expectations about a single artifact , while transformativity is related with expectations about the entire domain . However , it requires to define its conceptual space in terms of explicit rules , which can be violated ( and so that such a violation can be calculated ) . In addition , it does not include value in the assessment of machine creativity . 4 . 11 Essential Criteria of Creativity 4 . 11 . 1 Overview . The metric proposed by [ 152 ] is based on three components : novelty , value and surprise . It relies on the idea that a creativity metric has to be independent not only from the domain , but also from the producer . 4 . 11 . 2 Dimensions of Creativity Considered . The criteria of creativity defined by [ 152 ] cover exactly the Boden’s three criteria . In particular , novelty is intended as how different the artifact is from known artifacts in its class . Value is quantified considering how the potentially creative artifact compares in utility , performance , or attractiveness to other artifacts in its class . Finally , unexpectedness is defined as the variation from expectation developed for the next new artifact in its class . 4 . 11 . 3 Protocol for Evaluation . Novelty is calculated as the distance between the artifact of interest and the other artifacts in its class . The partition in classes is obtained by means of a clustering algorithm . Surprise is calculated considering whether or not the artifact agrees with the expected next artifact in the pattern extracted from recent artifacts . More specifically , it is calculated as the difference between the expected next artifact and the real next artifact . Such a pattern is predicted by a self - supervised neural network ; predictions are refined using reinforcement learning to correct the learned trajectory in case of sequential data . Finally , value is calculated as the weighted sum of the performance variables of the artifact . The weights depend on a co - evolutionary algorithm with a fitness function that can change over time in case the current population of artifacts changes . 4 . 11 . 4 Critical Examination . The method considers all the three Boden’s criteria ; it is not linked to a specific domain , or the producer itself ; it deals with the evolution of creativity , capturing its volatile nature at the same time . However , in our opinion , it is limited in terms of applicability by the fact it requires the definition of performance variables ( similarly to other approaches based on attribute - value pairs , see 4 . 8 . 4 ) . Moreover , the setting of the parameters of clustering algorithms at the basis of this method and the definition of distances among artifacts require human fine - tuning . 4 . 12 Computational Metrics for Storytelling 4 . 12 . 1 Overview . For the specific case of storytelling , the authors of [ 117 ] propose a set of computational metrics in order to compute evaluation of novelty , surprise , rarity and recreational effort . 4 . 12 . 2 Dimensions of Creativity Considered . Novelty and surprise are evaluated according to the standard Boden’s definition , while rarity is intended as rare combination of properties and recreational effort as the difficulty to achieve a specific result . 4 . 12 . 3 Protocol for Evaluation . Novelty is computed as the average semantic distance between the dominant terms included in the textual representation of the story , compared to the average semantic distance of the dominant terms in all stories . Surprise is computed as the average semantic distance between the consecutive fragments of each story . 24 Creativity and Machine Learning : A Survey Rarity is computed as the distance between the individual clusters of each term in each story and those in the story set . Finally , recreational effort is computed as the number of different clusters each story contains . 4 . 12 . 4 Critical Examination . Although value is not considered , the proposed metrics appear to be appropriate to evaluate novelty and surprise . Nonetheless , they suffer from two problems : they are intrinsically domain - specific and they require that all the types of clusters are defined correctly , which is very difficult to ensure in general . 5 OUTLOOK AND CONCLUSION In this survey , we have provided the reader with an overview of the state of the art at the intersection between creativity and machine learning . Firstly , we have introduced the concept of machine creativity , including key concepts and definitions . Secondly , we have described a variety of generative learning techniques , considering their potential applications and limitations . Finally , we have discussed several evaluation frameworks for quantifying machine creativity , highlighting their characteristics and the dimensions they are able to capture . Even if the field of machine creativity has witnessed a large interest in the recent years , but there are still several open challenges . First of all , an interesting direction is the exploration of creativity - oriented objective functions , to directly train models to be creative , or to navigate the induced latent space to find creative solutions . Another open problem is the definition of techniques for exploring or transforming the space of solutions . A fundamental area is the definition of novel and robust evaluation techniques for both generated and real artifacts . As discussed in 4 , deep learning might be used as a basis for the definition of metrics for machine creativity . It is worth noting that there is also an ongoing debate around the role of human versus machine evaluation [ 130 ] . Another promising research direction concerns machine interpretation of art [ 1 ] . Moreover , machine learning techniques might also be used to investigate psychological dimensions of creativity [ 2 ] . There are also foundational questions related to generative deep learning and copyright [ 68 ] . For example , it is not clear if machine - generated works could be protected by Intellectual Property , and , if they are , who should be the owner of the related rights [ 202 ] . In addition , other problems related to copyright should be considered , such as if and when training over protected work is permitted [ 216 ] . Another ongoing important debate is about authorship and the human role in creative fields in the era of AI 7 . The models and framework discussed in this work show the remarkable potential of generative learning for machine creativity . 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In InternationalConference on Artificial Neural Networks ( Munich , Germany ) . Springer , 313 – 325 . 35 \ No newline at end of file diff --git a/silver_data/a00176470d841ea1bd2c3a13dd2991c008ac2e55.pdf.txt b/silver_data/a00176470d841ea1bd2c3a13dd2991c008ac2e55.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..c08397eacc7a4a27153cc0ec5803d8a327b910a2 --- /dev/null +++ b/silver_data/a00176470d841ea1bd2c3a13dd2991c008ac2e55.pdf.txt @@ -0,0 +1 @@ +JOURNAL OF VERBAL LEARNING AND VERBAL BEHAVIOR 5 , 381 - 391 ( 1966 ) Availability Versus Accessibility of Information in Memory for Words 1 E NDEL T ULVING AND Z ENA P EARLSTONE University of Toronto , Canada The Ss learned , on a single trial , lists of words belonging to explicitly designated con - ceptual categories . Lists varied in terms of length ( 12 , 24 , and 48 words ) and number of words per category ( 1 , 2 , and 4 ) . Immediate recall was tested either in presence or absence of category names as retrieval cues . Cued recall was higher than noncued recall , the difference varying directly with list length and inversely with number of items per category . This finding was interpreted as indicating that sufficiently intact memory traces of many words not recalled under the noncued recall conditions were available in the memory storage , but not accessible for retrieval . Further analysis of the data in terms of recall of categories and recall of words within recalled categories suggested two independent retrieval processes , one concerned with the accessibility of higher - order memory units , the other with accessibility of items within higher - order units . If a person is shown a long list of familiar words and is then asked to recall the list , he can recall some words , but not all of them . It can be assumed that the person learns each single word at the time of its presentation , in the sense that the probability of recall of the word rises from a value near zero immediately before the presentation to a value near unity immediately after the presentation . The failure to recall some of the words , therefore , reflects intratrial forgetting ( Tulving , 1964 ) . 1 This research was supported by the National Science Foundation , under grant GB - 810 . It was prepared for publication during the senior author ' s tenure of a National Research Council of Canada Senior Research Fellowship at the Institute of Human Learning , University of California at Berkeley . We are grateful to the Director and the Board of Education of the Township of Scarborough , and lo the Forest Hill Collegiate Institute and its Principal , who permitted us to test many students as subjects in their schools . We are also grateful to many teachers in the high schools in Scarborough and Forest Hill , who kindly made class time avail - able for the experiment . Our special thanks goes to Dr . Howard Russell and Mr . Vernon Trott for their generous assistance . Intratrial forgetting is a descriptive label that carries no implications as to the fate of the memory traces associated with non - recalled words . It may be attributable to the decay of traces as a consequence of passage of time between the presentation and attempted recall of an item ( Brown , 1958 ) , or to the displacement of some of the items stored earlier by subsequently presented items ( Waugh and Norman , 1965 ) . In either case , failure to recall a certain item would be interpreted to mean that the trace of the item is no longer available in the memory storage at the time of recall . It is also possible , however , that intratrial forgetting represents a failure to " find " otherwise intact traces in the storage . According to an information - processing model of memory described by Feigenbaum ( 1961 ) , for instance , forgetting occurs not because information in storage is destroyed , but because learned material becomes " inaccessible in a large and growing association network . " Thus , to interpret intratrial forgetting , it is useful to draw a distinction between what information or what traces are available in the memory 381 382 TULVING AND PEARLSTONE storage and what are accessible . This dis - tinction parallels the distinction between re - tention and recall , or the distinction between trace storage and trace utilization ( Melton ; 1963 ) . The present paper is concerned with a conceptual and experimental analysis of non - recall of learned items in terms of such a distinction between availability and accessibility . It describes an experiment whose primary purpose was to explore the hypothesis that a substantial part of nonrecall of familiar words under typical experimental conditions is attributable to inaccessibility of otherwise intact memory traces . Experimental demonstrations of the dis - tinction between availability and accessibility of information require that critical experimental treatments be administered at the time of the recall test , rather than at some earlier stage in the sequence of events involved in any memory task . Only if conditions of the experiment are held constant until the beginning of the recall period can differences in observed recall scores be attributed to differences in accessibility . While scattered examples exist in the literature of experiments satisfying these requirements ( e . g . . Fox , Blick , and Bilodeau , 1964 ; Peterson and Peterson , 1962 , Exp . IV ) , there have been no systematic attempts to distinguish between availability and accessibility of mnemonic information . Experiments in which various " measures of retention , " such as unaided recall and recognition , have been compared ( e . g . , Luh , 1922 ; Postman and Rau , 1957 ) lend support to the proposition that unaided recall does not tap all of the information that is available about previously learned material , but the interpretation of data in these experiments with respect to the distinction between availability and accessibility is complicated . Unaided recall requires the S to reproduce the whole item , while in recognition the correct item is given to the S and his task is to decide whether or not it occurred in the list . To distinguish between availability and accessibility of information that is sufficient for reproduction of a given item , comparisons between recognition and recall are only partly relevant and other methods must be used . The experiment described in this paper uses one such other method . Categorized word lists were presented to Ss for learning , and recall of words was tested in the presence or absence of category names as retrieval cues . It was expected that a large proportion of words not accessible for recall under the unaided conditions would become accessible as a consequence of experimental presentation of such retrieval cues , thus indicating that sufficient information was available in the storage for the reproduction of these words , but that this information was not accessible . The results of the experiment thus were expected to clarify the nature of intratrial forgetting as defined earlier . As the results turned out , they also illuminated the retrieval processes involved in a memory task such as the one used in the experiment , and had several interesting implications for other types of experiment . M ETHOD Design Categorized word lists , consisting of ( a ) category names , and ( b ) words representing instances of categories , were presented to Ss once . Immediately after the presentation , two recall tests were given in succession . The Ss were instructed to try to re - member as many words as possible . Three independent variables were manipulated : ( a ) list length—L ( 12 , 24 , and 48 words ) , ( b ) number of words or items per category—IPC ( 1 , 2 , and 4 words ) , and ( c ) conditions of recall in the first recall test—cued recall ( CR ) and noncued recall ( NCR ) . The second recall test was always given under the conditions of CR . All possible combinations of L and IPC were used to yield nine lists . Lists are designated in terms of the values of these two variables . For in - stance , List 24 - 2 refers to a 24 - word list in which there are two items per each of 12 categories . All combinations of nine lists and two conditions of recall in the first recall test were used to yield 18 experimental conditions . Experimental conditions MEMORYFOR WORDS 383 are designated in terms of the list and recall condition . For instance , condition 12 - 4 CR refers to list 12 - 4 recalled under the conditions of cued recall . Thus , the design of the experiment was 3 (cid:1) 3 (cid:1) 2 factorial . With respect to the first recall test the independent variables were L , IPC , and recall condition ; with respect to the second recall test they were L , IPC , and recall condition of the first list . Since the second recall test was always given under identical conditions ( CR ) , experimental groups will be uniquely defined in terms of list characteristics and recall condition of the first test . For instance , Group 48 - 1 NCR designates the sample of 48 who learned List 48 - 1 and who were first tested under the conditions of noncued recall . Subjects and Experimental Groups The Ss were high - school students of both sexes from Grades 10 to 12 from a number of different schools in two school systems in the Metropolitan Toronto area . A total of 948 Ss were tested in the experiment . D ata from 19 Ss had to be discarded because of incompleteness of recall protocols . The data discussed in this report are thus based on the records from 929 Ss . The age of Ss ranged from 14 to 21 years , with a great majority ( 94 % ) of Ss being between15 and 18 years of age . The Ss were tested in groups during a regular class period . Each of nine lists was learned by Ss in four classes . Within each class , all Ss were presented with identical material under identical conditions , but half the Ss were tested first under the conditions of CR while the other half was tested first under the conditions of NCR . The second recall test of the material , as mentioned earlier , occurred under the conditions of CR for all Ss . The sizes of the 18 experimental groups , each composed of Ss from four different school classes , arranged from 48 to 56 . Lists A practice list , consisting of 24 common adjectives , n administered under the typical single - trial free - recall conditions to all Ss prior to the presentation of the experimental list . Two different sets of nine experimental lists were constructed with the aid of the Connecticut word associations norms ( Cohen , Bousfield , and Whitmarsh , 1957 ) and with the aid of norms from a recall pilot study patterned after the procedure used by Cohen el at . ( 1957 ) . Two groups of Ss under each of the 18 experimental conditions learned a list from the first set , while the other two groups learned a corresponding list from the second set . Corresponding lists in the two sets contained identical categories but different words . The words in List 48 - 1 represented 48 different categories , 40 taken from the Connecticut norms and eight from the pilot study . Twenty - four categories were selected randomly for Lists 24 - 1 and 48 - 2 . The 12 categories represented in Lists 12 - 1 and 48 - 4 in turn were selected randomly from those occurring in Lists 24 - 1 and 48 - 2 , respectively . The same general procedure was followed in the selection of categories for other lists . Words in a given category of a list in which IPC = 4 were , in the first set , usually the second , fourth , sixth , and eighth ranking words in the norms , and in the second set , the third , fifth , seventh , and ninth ranking words in the norms , but some devia - tions from this general rule occurred . Words for categories containing two items or one item were selected randomly from such sets of four words . The order of categories in a list and the order of words within categories were determined randomly . All the words within a category occurred in immediately adjacent positions . The lists presented to Ss thus consisted of a number of category names , each category name being followed by one , two , or four items appropriate to the category . For instance , List 12 - 2 in the first set was as follows : four - footed animals— cow , rat ; weapons— bomb , cannon ; crimes — treason , theft ; forms of entertainment— radio , music ; substances for flavoring food— cinnamon , pepper ; professions — engineer , lawyer . Procedure The Ss recorded their recall in specially prepared recall booklets that were distributed at the beginning of the experimental session . Instructions about Ss ' task , and about the use of recall booklets , as well as all lists were presented to Ss by means of a high - fidelity tape - recorder . The Ss were first in - formed that they were going to take " a test to find out how people remember words , " and that although E was not interested in how well each of them did individually , they should do their best in the test . The standard free - recall instructions were then given for the practice list , followed by the presenta - tion of the practice list , at the rate of 2 sec per word . Two min were given for recall . The instructions for the second part of the test , the experimental list , informed Ss that they would next hear and try to memorize a list of nouns , or " names of various things , " pairs of nouns ( in case of IPC —2 ) , or groups of four nouns ( in case of IPC = 4 ) , and that each word ( or pair of words or group of four ) would be " preceded by another word or phrase that describes the word ( words ) to be remembered , but which in itself does not have to 384 TULVING AND PEARLSTONE be remembered . " Next , an illustrative list of the kind that Ss in a particular group had to learn was given as part of the instructions . This short list contained five categories ( country in Europe , boy ' s name , city in U . S . , name of a river , and statesman of our day ) , each category being accompanied by one , two , or four names , depending on the IPC of the experimental list . The illustrative list was read and the Ss reminded that " we want you to re - member only the word ( words ) that followed each descriptive phrase , or category . " These words that Ss had just heard , but not the category names , were then read again and referred to as the part of the list Ss would have to remember . The Ss were then told the number of words , number of categories , and number of words per category in the list they were going to learn . Apart from the general instructions to recall as many words as possible , no information was given to Ss exactly what the conditions of the recall test were going to be nor were they told that there would be different recall conditions for different Ss in the same group . The duration of presentation of the list varied for different lists according to the formula : T = 3 NoC + L , where T is the total duration of presentation in seconds , NoC is the number of categories ( L / IPC ) , and L is list length . The amount of time given for recall also varied for different lists , depending on L . The Ss had 1 , 2 , or 4 min to recall lists of 12 , 24 , or 48 words , respectively . For the condition of NCR , the recall booklets contained L consecutively numbered lines . For the condition of CR , the recall booklet listed all category names that had occurred in the list , in the same order as in the input list , and each category name was followed by one , two , or four lines , depending on IPC . At the end of the first recall test of the experimental list , all Ss recalled all the words they could remember a second time under the conditions of CR . R ESULTS The mean number of correctly recalled words on the practice list for the total sample of 929 Ss was 9 . 48 ( SD = 2 . 27 ) . The breakdown of these recall scores in terms of the 18 experimental groups showed the means to range from 8 . 81 to 10 . 06 . A one - way analysis of variance of these data yielded an F ( 17 , 911 ) of 2 . 53 which is unexplainably significant at the . 01 level . Since the median correlation coefficient between practice and experimental list recall was only + . 228 for the nine CR groups and + . 284 for the nine NCR groups , possible differences in ability among the groups suggested by differences in practice - list scores probably had only a minor effect on the evaluation of the effects of experimental treatments . Recall of Words The first analysis of the data was con - cerned with the number of words recalled under various experimental conditions . The stability of these data was tested in the fol - lowing manner . In each of the 18 experi - mental groups , the Ss were randomly divided into two subgroups , the mean recall score on the first recall test computed for each sub - group , and an intraclass correlation coefficient ( McNemar , 1962 ) between the 18 resulting pairs of means calculated . This coefficient turned out to be . 997 , indicating a high degree of stability of the mean recall scores for various experimental groups . First Recall Test . Mean number of words recalled on the first recall test of the experi - mental lists is shown by filled ( CR ) and un - filled ( NCR ) circles in Fig . 1 as a function of L and IPC . An overall analysis of variance of the number of words recalled in the first recall test showed all three main effects and all three double interactions to be significant at better than the . 001 level . The triple interaction among R , L , and IPC was not significant . Recall of words was higher under the condition of cued recall than under the conditions of noncued recall for all nine lists . The smallest numerical difference between CR and NCR was found for List 12 - 4 . This was not significant by t - test ( t = 1 . 88 ) , but all other differences were significant at better than the . 01 level . As can be seen from Fig . 1 , the superiority of CR over NCR was an increasing function of list length and a decreasing function of IPC . The largest difference ( 19 . 8 items , or 126 % ) was found for List 48 - 1 . MEMORY FOR WORDS 385 F IG . 1 . Mean number of words recalled in the first recall test ( circles ) and the second recall test ( triangles ) as a function of list length and number of items per category . When we consider CR and NCR separately , we find in Fig . 1 that NCR increases with IPC at all three levels of list length , but under the conditions of CR the effect of IPC depends on L . The inverse relation between CR and IPC is quite clear for the 24 - word list and is also obvious when we compare recall for IPC = 1 with that for IPC = 4 for the other two lists , but there was no decrease in cued recall from IPC = 1 to IPC = 2 for the 12 - word and 48 - word lists . All six possible comparisons of mean recall scores between IPC = 1 and IPC = 4 yielded significant differences when tested by means of t - tests , five being significant at the . 001 level and one ( CR for the 12 - word list ) at the . 05 level . Second Recall Test The second recall test was administered to all Ss under the conditions of cued recall , where category names were available on recall sheets . For Ss in all nine CR groups the mean number of words recalled on the second test was practically identical with the mean number of words recalled on the first test . The overall mean word - recall in all nine groups on the first test was 21 . 17 , and on the second test 21 . 20 . Thus there was neither any forgetting nor " reminiscence " from the first to the second test . The mean recall scores on the second test for the NCR groups are shown by triangles in Fig . 1 . These means were significantly higher than the means on the first test for all lists except List 12 - 4 . But for none of the nine lists did the mean second test recall score in the NCR groups equal that of the CR groups , as can be seen in Fig . 1 . The second recall test was included in the design , and the data from the second test are included in this report , primarily in order to illustrate that subsequent pre - sentation of category names as retrieval cues in the NCR groups would result in an increase in the number of retrieved words . More detailed analyses of these data , however , are not warranted , since no safe assumptions can be made about availability of information in the memory storage after different treatments in the first recall test . For this 386 TULVIXG ANDPEARLSTOXE reason , data from the second recall test will be ignored in the rest of this paper . Error Data and Guessing Bias . Errors of recall were classified into three categories : repetitions of list words , noncategorical intrusions , and categorical intrusions . Errors falling into the first two classes were few in number . On the first recall test , for instance , a total of 24 repetitions and a total of 73 noncategorical intrusions were found in all 929 recall protocols . Categorical intrusions are extralist intru - sions that are members of one of the categories used in a given list . Mean numbers of such intrusions are shown in Table 1 . Three observations are of interest . First , the frequency of categorical intrusions tended to increase with IPC at all levels of L . Second , the frequency of intrusions increased with L at all levels of IPC . Third , the number of intrusions for a given list was always greater for CR than NCR , with the exception of List 12 - 4 . Since the frequency of categorical intrusions seems to be related to the treatment variables , " correct " recall scores may be somewhat inflated . The Ss may have received credit for recall even when they arrived at the correct word through a free association to the category name , remembered under the NCR conditions and explicitly given on recall sheets under the CR conditions . One might argue , therefore , that the differences in recall between the CR and NCR conditions might in fact be smaller than the data depicted in Fig . 1 indicate . The extent of such possible bias can be roughly estimated by considering the probability of occurrence of our list - words as free associations in the T ABLE 1 M EAN N UMBER OF C ATEGORICAL I NTRUSIONS IN THE FIRST RECALL TEST OF EXPERIMENTAL LISTS norms of Cohen el al . For the nine lists , such mean probability varied over the range of . 052 to . 078 , with an overall average of . 065 . To illustrate the small extent of the bias in recall scores attribut - able to guessing of words from given categories under the CR conditions , consider List 24 - 4 for which the difference between CR and NCR was the smallest of all lists . ( We will ignore List 12 - 4 for which the difference was not significant . ) The mean number of words recorded by Ss in Group 24 - 4 CR was 17 . 7 , of which 15 . 1 were correct and 2 . 6 were categorical intrusions . The same mean for Group 24 - 4 NCR was 14 . 6 , of which 13 . 4 were correct and 1 . 2 were intrusions . Thus , Ss in Group 24 - 4 CR put down , on the average , 3 . 1 more responses than Ss in Group 24 - 4 NCR . Since the average word in List 24 - 4 had occurred in the norms with the probability of . 052 , only . 16 words of the 3 . 1 " extra " words given by Group 24 - 4 CR would be expected to match the list words and credited to correct recall . The actual difference in mean correct recall between Groups 24 - 4 CR and 24 - 4 NCR , however , was over ten times as large . It is clear , therefore , that even for the list showing the smallest difference between CR and NCR the difference could not be accounted for in terms of free associations to category names , and hence must be attributed to the facilitative effect of category names as retrieval cues . Category and Words - Within - Category Recall The analysis of word - recall data in an experiment such as the present one can be regarded as a first - level analysis only . It indicates the gross effects of the variables manipulated , but it does not provide much insight into the underlying relations . Some such insight , however , can be obtained from an analysis of the data in terms of two further response measures . The first of these is referred to as category recall . This is defined in terms of the number of categories from which at least one word is recalled . The measure has been used earlier by Cohen ( 1963 ) . We designate this measure as Rc . In lists where IPC = 1 , R c is identical with the number of words recalled ( Rw ) , but in lists where IPC > 1 , the two measures do not necessarily co - vary and usually yield different values . The second measure is words - within - category recall , or words recalled per category MEMOR FOR WORDS 387 recalled . It is defined in terms of the ratio of the number of words recalled to the number of categories recalled . This measure has been referred to as “mean word recall per category " by Cohen ( 1966 ) . We designate this measure as Rw / c . In lists where IPC = 1 , Rw / c is always 1 . 00 by definition , given that S recalls at least one word from the list , but for higher levels of IPC , Rw / c can assume all values between 1 . 00 and IPC . The word - recall score ( R w ) is a simple multiplicative function of category recall score ( R c ) and words - within - category recall score ( Rw / c ) , i . e . , Rw = R c • Rw / c . The word - recall data that we considered in the two preceding sections thus reflected the effects of the independent variables on both of the two components of Rw . We will now examine the data from the first recall test with respect to the two components of R w . Table 2 shows mean R c scores for all experi - mental conditions . It can be seen that Rc varies systematically with all three inde - pendent variables . It is less under the NCR conditions than under the CR conditions for all lists , but the magnitude of this difference depends on both L and IPC . At a given level of IPC the difference is an increasing func - tion of L , and at a given level of L it is a de - creasing function of IPC . In Table 2 . the values of Rc for lists in which TPC = 1 are in parentheses to remind the reader that they are identical with the corresponding Rw values . The mean recall scores of words recalled T ABLE 2 M EAN N UMBEK OF C ATEGORIES R ECALLED ( R C ) IN THE FIRST RECALL TEST OF E XPERIMENTAL L ISTS per category recalled ( Rw / c ) are shown in Table 3 . Again the scores for lists where IPC = 1 are included for the sake of completeness , although they are always unity by the definition of the Rw / c measure . Table 3 shows that while Rw / c is system - atically related to IPC ; it seems to be inde - pendent of recall conditions and also inde - pendent of list length for lists of 24 and 48 words . When Rw / c scores are averaged over all six lists for which IPC > 1 , the overall means are identical at 2 . 32 for both CR and NCR . None of the differences in Rw / c between CR and NCR for the six lists approaches significance by t - tests . And when the data are averaged over both recall conditions and IPC levels of 2 and 4 ; the mean Rw / c for 24 - word lists is 2 . 21 and the mean for the 48 - word lists is 2 . 18 . Interpretation of Findings on Word - Recall To aid in the interpretation of some of the findings pertaining to word - recall ( Rw ) , the data on R w , R c and Rw / c are summarized in Table 4 as mean proportions of these measures relative to maximum possible scores . These proportional measures are designated as P ( Rw ) , P ( Rc ) , and P ( R W / c ) . Values of P ( Rw ) in the right - hand panel of Table 4 were obtained by dividing each of the mean R w scores by its respective L , but these scores can also be arrived at by multiplying the corresponding P ( Rc ) and T ABLE 3 M EAN NUMBER OF WORDS RECALLED PER CATEGORY R ECALLED ( R W / C ) IN THE F IRST R ECALL T EST OF EXPERMEXTAL LLSTS 388 TULVING AND PEARLSTONE P ( Rw / c ) scores given in the two left - hand panels in Table 4 . An important fact reflected in the data in Table 4 is that the relations between P ( R C ) and P ( R W / C ) on the one hand , and IPC , on the other hand , are all monotonic , for all levels of L and for both conditions of recall , while the relation between P ( R W ) and IPC is not . P ( Rc ) is always an increasing function of IPC , and P ( R W / C ) is always a decreasing function of IPC , but the relation between P ( Rw ) and IPC cannot be stated as simply . P ( R W ) is an increasing function of IPC under the conditions of NCR , while under the conditions of CR it is a decreasing function of IPC for the 24 - word list and , taking the sample means in Table 4 literally , it increases from IPC = 1 to IPC = 2 and decreases from IPC = 2 to IPC = 4 for both the 12 - word and 48 - word lists . The relations between P ( R W ) and IPC become somewhat more meaningful if we remember that any change in P ( Rw ) de - pends on changes in both P ( R c ) and P ( R w / c ) . An increase in P ( Rw ) as a function of IPC means that under certain conditions P ( Rc ) increases at a faster rate as a function of IPC than P ( R W / c ) decreases . Conversely , a decrease in P ( Rw ) as a function of IPC means that under certain conditions P ( Rw / c ) decreases at a faster rate as a function of IPC than P ( Rc ) increases . These considerations suggest that the de - crease of Rw as a function of IPC under the conditions of CR , shown in Fig . 1 , is proba - bly an artifact related to lists of limited length and to limited number of categories . The P ( Rc ) score is already so high for lists in which IPC = 1 that there can be relatively little further improvement in this measure with higher levels of IPC . Even for List 48 - 1 , P ( R C ) is so high ( . 736 , as shown in Table 4 ) that the maximum possible in - crease of . 264 in this measure which would bring it to unity would not be sufficient to ; outweigh the decrease in P ( R w / c ) from 1 . 00 to . 652 over the range of IPC values used in this experiment . This " ceiling effect " on P ( R C ) and the role it plays in determining the relations between Rw and IPC at levels of L used in this experi - ment is made explicit as a result of the break - down of the word - recall measure into its two components . Inspection of the Rw curves plotted against IPC would not readily lead to the conclusion that we are dealing with an artifactual limit imposed on the Ss : recall per - formance , since the Rw curves have a nega - tive slope . The reversals in the Rw curves plotted as a function of IPC , for 12 - word and 48 - word lists under the cued recall conditions , can also be understood in terms of the two components of Rw , in an analogous fashion , and will not be elaborated further . Order of Recall . Two further findings , having to do with order of recall under the MEMORYFOE WORDS 389 conditions of NCR , where the order of re - call was free to vary , will be briefly mentioned . The first was the tendency for the words from a given category to be recalled to - gether despite the absence of the experi - mentally presented category name . A mea - sure reflecting this trend is provided by the proportion of times that a word from a given category was followed in recall by another word from the same category . This proportion varied between . 92 and . 95 in the three lists of IPC = 2 , and between . 89 and . 97 in the three lists of IPC = 4 . The second finding concerned the order in such words were recalled within a given category . The general tendency was for the words to be recalled in the same order in which they appeared in the input list . As an illustration we only mention some data from list 48 - 2 . In those cases where the Ss recalled with words from a category the order of recall was the same as in the input list 78 % of the time ( 311 / 397 ) and reverse to that of the input list 22 % of the time ( 86 / 397 ) . These data show that even in the case of the longest list used in this study the Ss apparently retain a fair amount of information about the order in which two words from the same category occurred in the input list . D ISCUSSION The most important finding of this experi - ment was higher recall under the conditions of cued recall than under the conditions of noncued recall . Since the experimental treatment administered to the Ss in the two recall conditions was the same , both the amount of information and the organization of this information in the memory storage at the beginning of the recall test must have been identical for the CR and NCR groups . The superiority of cued recall over noncued recall thus suggests that specific informant about many words must be available in the storage , in a form sufficient for the reproduction of words , even when this infor - mation is not accessible under a given set of recall conditions . Intratrial forgetting , defined in terms of nonrecall of words learned in the input phase of a trial , thus does not necessarily reflect the loss of relevant information from the storage , but only its inaccessibility . Accessi - bility of the information clearly depends on its availability , but it also depends on re - trieval cues . While the present findings do not rule out the possibility that some infor - mation stored in memory in the course of presentation of a list decays over intratrial retention intervals or is erased by other incoming information , they do make clear that inferences about what is available in memory cannot be made on the basis of what is accessible . Retrieval cues obviously constitute an ex - tremely important factor in determining the level of recall . The presence of a single ex - perimentally manipulated retrieval cue , the category name , resulted in large increments in the number of recalled words , particularly for longer lists . It is entirely within the realm of possibility that additional and more powerful retrieval cues would produce an even greater facilitation of recall . Experi - mental work on memory has largely ignored recall conditions as an important source of variance in recall . Melton ( 1963 ) has dis - cussed three broad theoretical problems con - cerned with retrieval and utilization of traces , but only one of these—dependence of the retrieval on the completeness of rein - statement at the time of recall of the stimu - lating situation present at the time of input —involves the analytical separation of condi - tions affecting storage and those related to retrieval , and very little experimental work has been done on this problem . The analysis of recall data in the present experiment in terms of the logically definable components of word recall , namely category recall and words - within - category recall , showed that category recall was greater under the conditions of CR than NCR and that it 390 TULVING AND PEARLSTONE increased directly with the length of the list , while words - within - category recall was inde - pendent of recall conditions and remained invariant when list length increased from 24 to 48 . The latter finding confirms the data reported by Cohen ( 1966 ) who found that mean word recall per category was constant for lists of 35 , 53 , and 70 words . The fact that variations in recall condi - tions and list length have an effect on only one component of the word recall measure , but not on the other , suggests that the two components represent two independent pro - cesses of recall . One of these has to do with the accessibility of higher - order memory units into which material has been organized , while the other is concerned with the accessibility of individual items comprising the higher - order units . Accessibility of higher - order units depends on appropriate retrieval cues and on the total number of stored higher - order units ( or list length ) , while accessibility of items within higher - order units is largely independent of these variables . In the present experiment , and in other experiments with categorized word lists , the words to be memorized were organized into higher - order units by the E . This organiza - tion apparently determined the arrangement of words in the storage and their retrieval not only for Ss working under the CR conditions , but also for those working under the NCR conditions . When two or more words from a given category were recalled by the NCR subjects , almost invariably these words occurred in immediate succession . Even when the E does not impose any particular organization on the material the S has to memorize , by selecting words for inclusion in lists randomly and by presenting them without any additional descriptive labels , Ss can and do organize the words into larger units ( Tulving , 1962 , 1964 ) . Some of these subjective units ( S - units ) consist of words from meaningful conceptual categories , but others seem to be based on other principles—associative groupings , structural characteristics , and similarity of sound patterns , and still others appear to be determine idiosyncratically . It has been suggested previously ( Tulving , 1964 ) that the functions significance of S - units , whatever their nature lies in the increased accessibility of individual items constituting a unit . We do not yet know much about the mechanism underlying the retrieval of a single unit of information , before an individual word or a larger S - unit , but it appears that if an individual list - item has been stored as a part of a larger unit it does become more accessible for retrieval where other items in the same unit are accessible . Thus organization of material , whether suggested by the E or imposed by the S , seems to affect recall performance primarily on making the desired information more accessible in an otherwise limited biological retrieval system . It need not have any effect the availability of the information in the storage . R EFERENCES B ROWN , J . Some tests of the decay theory of intermediate memory . Quart . J . exp . Psychology , 1958 , 10 , 12 - 21 . C OHEN , B . H . Recall of categorized word lists exp . Psychol . , 1963 , 66 , 227 - 234 . C OHEN , B . H . Some - or - none characteristics of coding behavior . J . verb . Learn , verb . Behav . , 1966 , 5 , 182 - 187 . C OHEN , B . H . . B OUSFIELD , W . A . , AND W HITMARSH G . A . Cultural norms for verbal items in 4 categories . Tech . Rep . No . 22 , Nonr - 631 ( 00 ) 1957 . University of Connecticut . F EIGENBAUM , E . A . The simulation of verbal leaning behavior . Proc . West . Joint Computer Conf . 1961 , 19 . 121 - 132 . Fox , P . W . , BLICK , K . A . , AND B ILODEAU , E . A . Stimulation and prediction of verbal recall and misrecall . J . exp . Psychol . , 1964 , 68 , 321 - 322 . L UH , C . W . The conditions of retention . Psychol . Mlonogr . , 1922 . 31 , No . 142 . McNEMAR , Q . Psychological Statistics ( 3 rd Ed , ) New York : Wiley , 1962 . M ELTON , A . W . Implications of short - term memory for a general theory of memory . J . verb . learn verb . Behav . . 1963 , 2 , 1 - 21 . P ETERSON , L . R . . AND P ETERSON , M . J . Minimal paired - associate learning . J . exp . Psychol , I961 , 63 , 521 - 527 . MEMORY FOR WOKDS 391 POSTMAN , L . , AXD R AU , L . Retention as a function of the method of measurement . Univ . Calif . Publ . Psychol . , 1957 , 8 , 271 - 396 . TULVING . E . Subjective organization in free recall of " unrelated " words . Psychol . Rev . , 1962 , 69 : 344 - 354 . T ULVING , E . Intratrial and intertrial retention : Notes towards a theory of free recall verbal learning . Psychol . Rev . , 1964 . 71 , 219 - 237 . W AUGH , X . C , AXD NORMAN , D . A . Primary memery Psychol . Rev . , 1965 , 72 , 89 - 104 . ( Received January 18 , 1965 ) - \ No newline at end of file diff --git a/silver_data/a20c9f734a36ac854419ec157fa2a88de54d64c7.pdf.txt b/silver_data/a20c9f734a36ac854419ec157fa2a88de54d64c7.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..4d8418b6fef6a65d6f805a167b1b0818fc977d22 --- /dev/null +++ b/silver_data/a20c9f734a36ac854419ec157fa2a88de54d64c7.pdf.txt @@ -0,0 +1 @@ +Outer Doublet Related to Beat Heterogeneity Reveals Structural Polarity Direction in C h l a m y d o m o n a s Flagella HAROLD J . HOOPS and GEORGE B . WITMAN Cell Biology Group , Worcester Foundation for Experimental Biology , Shrewsbury , Massachusetts 01545 ABSTRACT Analysis of serial cross - sections of the Chlamydomonas flagellum reveals several structural asymmetries in the axoneme . One doublet lacks the outer dynein arm , has a beak - like projection in its B - tubule , and bears a two - part bridge that extends from the A - tubule of this doublet to the B - tubule of the adjacent doublet . The two doublets directly opposite the doublet lacking the arm have beak - like projections in their B - tubules . These asymmetries always occur in the same doublets from section to section , indicating that certain doublets have consistent morphological specializations . These unique doublets give the axoneme an inherent structural polarity . All three specializations are present in the proximal portion of the axoneme ; based on their frequency in random cross - sections of isolated axonemes , the two - part bridge and the beak - like projections are present in the proximal one quarter and one half of the axoneme , respectively , and the outer arm is absent from the one doublet > 90 % of the axoneme ' s length . The outer arm - less doublet of each flagellum faces the other flagellum , indicating that each axoneme has the same rotational orientation relative to the direction of its effective stroke . This strongly suggests that the direction of the effective stroke is controlled by a structural component within the axoneme . The striated fibers are associated with specific triplets in a manner suggesting that they play a role in setting up or maintaining the 180 ° rotational symmetry of the two flagella . In forward swimming Chlamydomonas , the two fagella beat with an effective stroke in approximately the same plane , but in opposite directions ( 17 , 32 , 34 ) . Although the fine structure ( 6 , ll , 14 , 30 , 32 , 39 , 40 ) , biochemistry ( 10 , 15 , 21 , 27 , 28 , 31 , 40 ) , and waveform ( 3 , 5 , 17 , 32 , 34 ) of the Chlamydo - monas flagellum have been extensively investigated , little information is available on the mechanism by which the direction of the effective stroke is established . The isolated flagellar apparatuses of Chlamydomonas appear to retain the same beat pattern as in intact cells ( 16 , 17 ) , so the direction of beat must be controlled by a structural component within the flagellar apparatus . However , it is not known whether this component is internal or external to the axoneme . Structural differences have been observed in some of the outer doublets of Chlamydomonas ( 15 , 38 ) , but it has not been determined whether these asymmetries always occur in the same doublets along the axoneme , nor whether they are oriented in a way that is related to the plane of beat . The central pair of Chlamydomonas rotates during flagellar movement ( 19 , 20 ) , so it can not give polarity to the axoneme . To determine whether the axonemes of Chlamydomonas have an intrinsic polarity related to the plane of beat , we made a detailed examination of the fine structure of the flagellar axoneme in serial thin sections having a known orientation relative to the two basal bodies . The results indi - cate that : ( a ) there are consistent morphological differences between several outer doublets in the axoneme ; ( b ) as a consequence , the axonemes do indeed have an inherent struc - tural polarity ; and ( c ) this polarity is appropriately oriented to be of functional significance in establishing beat direction . We also found that the striated fibers connecting the two basal bodies are always associated with specific triplets , suggesting that the striated fibers play a role in determining the rotational orientation of the axonemes . MATERIALS AND METHODS Chlamydomonas reinhardtii wild - type strains 1132d - and NO + were grown in 125 - ml cultures as previously described ( 40 ) . Conventional fixation proce - dures , including those using sodium cacodylate - buffered glutaraldehyde , otten caused the flagellar matrix to appear dense , thus obscuring the delicate markers used in this study . For this reason , a double glutaraldehyde fixation was employed in which the cells were first fixed at room temperature with 1 % THE JOURNAL OF CELL BIOLOGY • VOLUME 97 September 1983 902 - 908 902 © The Rockefeller University Press - 0021 - 952518310910902107 $ 1 . 00 glutaraldehyde in medium for 10 - 15 min and then with 1 % glutaraldehyde in 100 mM sodium eacodylate , pH 7 . 2 , for 50 rain . After buffer washes , the sample was embedded in 1 % agar , postfixed with 1 % OsO4 in 50 mM sodium cacodylate at room temperature , stained en bloc with 1 % aqueous uranyl acetate , dehydrated , and embedded in Epon or Epon - Araldite . Demembranated axonemes were prepared as previously described ( 3 ) , fixed with 1 % glutaralde - hyde in 100 mM sodium cacodylate , postfixed , en bloc stained , dehydrated , and embedded as described above . Serial sections were picked up on Formvar - coated or carbon - over - Formvar - coated grids , and viewed with a Philips 301 TEM . All micrographs are printed as if viewed from inside the cell . RESULTS Structural Polarity of the Axoneme Cross - sections of the Chlamydomonas axoneme frequently reveal several structures that depart from the overall rotational symmetry of the axoneme ( Fig . 1 ) . Three outer doublet mi - crotubules contain " beak - like projections " ( 38 ) that extend partway across the lumen of their B - tubules . Two of the doublets containing the projections are adjacent ; the third is on the opposite side of the axoneme . The latter doublet lacks an outer dynein arm , although the inner arm appears to be present . A previously undescribed two - part bridge extends from the A - tubule of this doublet ( n ) to the B - tubule of the adjacent doublet ( n + 1 ) . This bridge consists of two roughly parallel , wedge - shaped components , each of which spans the gap between the doublets . The inner component is ~ 14 . 5 nm long and tapers toward the A - tubule ; the outer one is ~ 12 . 5 nm long and tapers toward the B - tubule . These components appear to be ~ 4 . 0 - 5 . 0 nm wide at their centers , with the inner one usually slightly wider than the outer . In every case of isolated and in situ axonemes examined , the beak - like projections and the two - part bridge are associ - ated with the same doublets from section to section ; similarly , the outer dynein arm is always missing from the same doublet ( Figs . 2 and 3 ) . These observations show that some of the outer doublet microtubules are morphologically distinct from the others . Consequently , the axoneme has an inherent struc - tural polarity defined by the positioning of these unique doublets . All flagella cross - sections from within the flagellar collar , and out to at least 0 . 7 ~ m ( 10 sections ) distal to the flagellar collar , have the beak - like projections and the two - part bridge , and lack the above - mentioned outer arm . However , the pro - jections and the bridge do not occur over the entire length of the flagellum . In isolated axonemes , the bridge was observed in 17 of 75 randomly chosen cross - sections and always oc - curred in association with the three beak - like projections . All three beak - like structures were present in 30 cross - sections ; one or two were present in an additional 13 . A single outer arm was lacking in 69 of the cross - sections , including all of those having one or more beak - like projections . In all cross - sections , the relative positions of these structural asymmetries around the axoneme was consistent with the arrangement described above . These observations indicate that the asym - mettles have unique and regular distributions along the axo - neme : the two - part bridge and the beak - like projections are present in the proximal quarter and half of the axoneme , respectively , and the outer arm is missing from one of the doublets over at least 90 % of its length . Rotational Orientation of the Axoneme Relative to the Cell Body Because specific doublets of the Chlamydomonas axoneme have unique structural features , these features can be used as polarity markers to ascertain the rotational orientation of the flagella in situ . To determine the rotational orientation of one flagellum of a cell relative to the other , we examined serial sections that showed the origin of both flagella in the cell body ( Figs . 2 and 3 ) . In every such cell examined , the outer arm - less doublet of one flagellum faces the other flagellum - - i . e . , the two - part bridge is located on the medial side of the axoneme . No exceptions to this pattern were observed . There - fore , the two axonemes must have a consistent rotational orientation relative to one another and the cell body . Because the two flagella of Chlamydomonas exit from the cell at about a 90 * angle from one another , cross - sectional information can usually be obtained from only one flagellum of a cell . However , occasionally one flagellum is abnormally bent , or both flagella have reverse bends at their bases , so that good cross - sections are obtained of both flagella ( Fig . 3 ) . In these cases , both axonemes have all three polarity markers . In each flagellum the doublet bearing the two - part bridge and FIGURE 1 ( A ) Cross - section of the Chlamy - domonas flagellum . Three doublets have beak - like projections in their B - tubules ( ar - rows ) . One of these doublets ( long arrow ) lacks the outer arm , and a two - part bridge ( arrowhead ) extends from the A - tubule of this doublet to the B - tubule of the adjacent doub - let . x 218 , 000 . ( B ) Diagramatic representation of A . HooPs AND WITMAN Flagellar Polarity in Chlamydomonas 903 FIGURE 2 Sections 1 , 2 , 5 , and 9 from a series . Although the figures are arranged from distal to proximal to correspond with the text , each micrograph is printed in the conventional manner ( i . e . , as if viewed from inside the cell ) . Insets show the relative positions of the two flagella in each section ; the lower flagellum is the one shown at higher magnification . ( A ) In the distal portion of the flagellar collar , three doublets ( arrows ) contain beak - like projections , and the two - part bridge ( arrowhead ) is present . The doublet with the bridge attached to its A - tubule faces the other flagellum ( inset ) . Both inner and outer arms are associated with all but one doublet ( long arrow ) , which lacks the outer arm . ( B ) The next most proximal section . The polarity markers ( arrows and arrowhead ) are associated with the same doublets as in the previous section . Slightly more proximally the arms are absent ; even at this level an outer arm is lacking from one doublet ( star ) that normally has both arms . ( C ) Further proximally , all arms are absent from the doublets . Both the beak - like projections ( arrows ) and the two part bridge ( arrowhead ) are present . Peripheral links and inner links ( see text ) also occur at this level . Occasionally , as at the bottom of this micrograph , these two links together can resemble the two - part bridge . That such apparent " bridges " actually represent a peripheral and an inner link can be ascertained by examination of adjacent serial sections ( not shown ) . ( D ) In the region of the stellate structure , the two - part bridge is no longer present . Although some doublets may contain structures similar to the beak - like projections , these structures are not necessarily arranged in the " two - opposite - one " pattern seen more distally . The upper flagellum has just exited from its flagellar collar ( inset ) . x 135 , 000 ; insets , x 38 , 000 . lacking the outer arm faces the opposite flagellum , and the two adjacent doublets with the beak - like projections face away from it . These results confirm that the position of the mark - ers - - and hence , the orientation of the axoneme - - is the same in each flagellum relative to the direction of its effective stroke . Basal Body Orientation and the Attachment of the Striated Fibers The dynein arms are present part way into the flagellar collar ( Fig . 2 , a and b ) . At this level , the outer arm is missing from the one doublet , and both the beak - like projections and the two - part bridge are associated with their respective doub - lets . More proximally , arms are no longer present , but the bridge and the beak - like projections are still observed ( Fig . 2 c ) . Rod - like " peripheral links " ( 38 ) connect adjacent doub - lets in this region . An additional , previously undescribed , linking structure is present to the inside of each peripheral link ; this inner link is bowed towards the center of the axoneme and is less opaque and well defined than the periph - FIGURE 3 Sections I , 4 , and 9 from a series showing cross - sections of both flagella of a single cell . ( A ) The upper flagellum is sectioned just distal to the flagellar collar and the lower flagellum is sectioned within the collar . ( B and C ) Further distally , the presence of beak - like projections in three doublets ( arrows ) , the two - part bridge ( arrowhead ) , and the lack of an outer arm on one doublet ( long arrow ) can be seen in both flagella . Note that the doublet lacking the outer arm and bearing the two - part bridge faces the opposite flagellum , x 130 , 000 . 904 THE JOURNAL OF CELL BIOLOGY • VOLUME 97 , 1983 HOOPS AND WlTMAN Flagellar Polarity in Chlamydomonas 905 FIGURE 4 Sections through the Chlamydomonas basal body . ( A ) The distal striated fiber ( Dr ' - ) attaches to an electron dense covering ( arrow ) on three triplets ; the middle of these triplets is continuous with the doublet lacking the outer arm . x 155 , 000 . ( B ) The proximal striated fiber ( PF ) likewise attaches to specific triplets . The triplet continuous with the doublet lacking the outer arm is marked ( diamond ) . Note that the two basal bodies are not exactly in the same plane , but are offset by about one half a basal body diameter , x 115 , 000 . eral link ( Fig . 2c ) . The two - part bridge ends just distal to the stellate pattern ( Fig . 2d ) . Here and more proximally , the lumens of the B - tubules of some doublets or triplets often contain material that is morphologically similar to the beak - like projections , but the material does not necessarily occur in the same regular pattern as in the axoneme . Therefore , none of the polarity markers described above can be used to identify individual doublets or triplets in these regions . How - ever , specific microtubules can be traced in serial sections from the axoneme down into the basal body , so that the structural relationship of specific triplets to the striated fibers and other basal structures of the flagellar apparatus can be determined . The two basal bodies are connected by the distal striated fiber ( Fig . 4a ) . The filaments of this fiber insert into electron - dense structures that cover three adjacent triplets of each basal body ( Fig . 4 a ) . The middle of these triplets is continuous with the doublet that has the two - part bridge attached to its A - tubule . The proximal striated fibers also attach to specific doublets ( Fig . 4 b ) , but their attachment is more complex and will not be described here . Tangential longitudinal sections of the basal body and proximal portion of the Chlamydomonas flagellum indicate 906 THE JOURNAL OF CELL BIOLOGY . VOLUME 97 , 1983 that the triplets or doublets are not strongly twisted in this region . However , the two flagella are not in exactly the same plane ( Fig . 4 b ) , but are offset by one - half to one basal body diameter . This offset is in the clockwise direction as viewed from above ( Fig . 5 ) . As a result , the triplet that is continuous with the doublet having the two - part bridge attached to its A - tubule is no longer exactly opposite the corresponding triplet of the other basal body . DISCUSSION Both the presence of the beak - like projections in the B - tubules ( 38 ) and the lack of an outer dynein arm ( 15 ) have been previously reported in the axoneme of Chlamydomonas . In the present study , we found that each of these structural asymmetries , as well as the newly discovered two - part bridge , is associated with a specific axonemal doublet or doublets , and that these morphologically differentiated doublets occur in specific positions with respect to one another . These obser - vations indicate that the axonemes of Chlamydomonas have an inherent structural polarity . The morphological differences between doublets also indicate that the doublets almost cer - tainly differ in some of their polypeptide components . Con - D 1 : ! 4 R2 FI2 / FI4 0 FiGure 5 Diagrammatic representation of the flagellar apparatus of Chlamydomonas as seen from above . This diagram is in the correct absolute orientation . Note that the distal striated fiber ( DF ) and the proximal striated fibers ( PF ) are in a position to determine the rotational orientation of each axoneme . The relative positions of the doublets are indicated by numbers . R2 , R4 : two - and four - membered rootlets , respectively . sequently , compositional heterogeneity among the doublets must now be considered in biochemical studies of the Chla - mydomonas axoneme . In discussing specific doublets , it is convenient to have a system of nomenclature for referring to individual doublets around the axoneme . The original basis for indexing doublets depended on the plane of the central pair of microtubules ( 1 , 4 ) . However , the central pair apparently rotates in Chlamy - dornonas ( 19 , 20 ) , as in certain other cilia and flagella ( 18 , 24 , 25 , 26 , 35 ) , and thus cannot be used for this purpose . This is confirmed by the observation that the central pair of Chlamydomonas has a variable orientation at its proximal end ( Hoops and Witman , unpublished results ) . In those or - ganisms in which the doublets have been numbered relative to the plane of the central pair , the number 5 and 6 doublets are frequently connected by a bridge ( 1 , 9 , 33 ) . These same two doublets are located on the side of the axoneme that is at the leading edge during the normal effective stroke ( 9 , 33 , 36 ) , indicating that either the bridge or the direction of beat could be used as alternative devices for numbering the doub - lets . In Chlamydomonas , the situation is complicated by the fact that the two - part bridge is on the side opposite the direction of the effective stroke . We choose to use the latter as a basis for numbering the doublets of Chlamydomonas , because ( a ) the two - part bridge of Chlamydomonas may not be analogous with the 5 - 6 bridge of other organisms ( see below ) , and ( b ) an indexing system based on beat pattern has the advantage that equivalently numbered doublets are apt to have similar roles in different organisms . In forward swim - ming Chlamydomonas , the flagella beat away from one an - other during the effective stroke ( 17 , 32 , 34 ) . Therefore , if the doublets are indexed relative to the direction of the effective stroke in a manner consistent with that in other organisms , the two adjacent doublets with the beak - like structures are the number 5 and 6 doublets . The opposite doublet , also having a beak - like projection and lacking the outer ann , is the number 1 doublet . The doublets are numbered clockwise in the direction that the dynein arms point ( Fig . 5 ) . The 5 - 6 bridge present in many animal cilia and flagella is diamond - shaped in appearance ( 1 , 9 , 33 ) and thus has a different morphology than the 1 - 2 bridge of Chlamydomonas . This , plus the fact that the two types of bridges are on opposite sides of the axoneme relative to the direction of the effective stroke , suggests that they have different origins . Nevertheless , it has been proposed that the 5 - 6 bridge prevents sliding between the doublets to which it is connected ( 37 ) , and it seems likely that the 1 - 2 bridge has a similar function . To generate the approximately planar beat of Chlamydomonas , there must be either relatively little sliding or no sliding between doublets 1 and 2 , depending on the exact plane of beat relative to a plane through the 1 - 2 bridge and the center of the axoneme . The 1 - 2 bridge may thus serve to restrain or prevent sliding between those two doublets in the region where it occurs . The absence of the outer arm on doublet 1 is consistent with there being little or no sliding between doub - lets 1 and 2 . Our analysis of the rotational orientation of the flagella showed that each axoneme is oriented in the same way relative to its direction of beat . This strongly suggests that an internal , asymmetrically distributed axonemal component determines the direction of the effective stroke . This is supported by the observation that isolated , reactivated axonemes of Chlamy - domonas attached to a glass surface do not change the direc - tion of their effective stroke over many beat cycles ( 3 ) . Because the dynein arms point clockwise when the axo - nemes are viewed from base - to - tip , and the number I doublets of each axoneme face each other , the two flagella of a cell have 180 * rotational symmetry . Previously , it was suggested that the arrangement of striated fibers and microtubular root - lets of chlorophycean unicellular motile cells , including Chla - mydomonas , display 180 * rotational symmetry when viewed along the cell axis ( 7 ) . The arrangement of these components in Chlamydomonas is diagrammed in Fig . 5 ( unpublished data ) . The fact that both the flagella and the flagella - associated structures have the same type of symmetry suggests that the flagella - associated structures should interact with unique por - tions of the basal bodies . Indeed , our study revealed that the distal striated fiber always attaches to triplets 9 , 1 , and 2 of both basal bodies . Likewise , the striated proximal fibers attach to specific regions of each basal body . The information nec - essary for morphogenesis of the flagellar apparatus may there - fore be present in the basal bodies themselves . Attachment of the striated fibers to specific sites on the basal bodies probably results in the axonemes being set up or maintained in the correct rotational orientation to give the observed beat pat - tern . This is supported by the observation that a Chlamydo - monas mutant with missing or abnormal striated fibers also has abnormal rotational orientation of its axonemes ( 13 ) . Because the flagellar apparatus has 180 " rotational sym - metry , the beat envdopes of the two flagella of a swimming cell should also display 180 " rotational symmetry . Isolated , reactivated axonemes of Chlamydomonas appear to have a slight three - dimensional component in both the asymmetrical ( corresponding to forward swimming ) and symmetrical ( cor - responding to reverse swimming ) beating modes ( 3 ) . The structural symmetry of the flagellar apparatus suggests that if aplanarity is also present in the beat envelopes of flagella in situ , the swimming cell should r011 around its longitudinal axis . Indeed , under our conditions of observation , the cells do clearly roll as they swim through the media ( R . Kamiya HOOPS AND WITMAN Flagellar Polarity in Chlamydomonas 907 and G . B . Witman , manuscript submitted for publication ) . This is in contradiction to previously published reports ( 29 , 32 ) , but is predicted by current theories of how Chlamydo - monas phototaxes ( 8 ) . In many other green algae , particularly those belonging to the Ulvaphyceae , there are septations that extend all the way across the lumen of the B - tubule ( 12 ) . Like the beak - like projections in Chlamydomonas , these are located in two adjacent doublets and in the doublet on the opposite side of the axoneme ( 12 ) . Serial sections of one of these algae ( Ul - varia ; unpublished ) indicate that these doublets are in the same position relative to the effective stroke as in Chlamy - domonas . In addition , the flagella of Chlorosarcinopsis lack an outer dynein arm on one doublet ( 23 ) . The markers used in the present study therefore appear to be widespread among green algae . Doublets number l , 5 , and 6 also appear to be morpholog - ically specialized in the cilia and flagella of many different animal species . In certain animal sperm , the lumens of one or more A - tubules may contain septa or other electron - dense material . In such cases , wherever three or more doublets contain electron - dense material , these doublets include num - bers 1 , 5 , and 6 with only a single exception ( 2 , 22 ) . 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Dynein arm conformation and mechanochem - ical transduction in the eukaryotic flagellum . In Prokaryotic and Eukaryotic Flagella . W . B . Amos and J . D . Duckett , editors . Cambridge University Press , Cambridge . 203 - 223 . 40 . Witman , G . B . , J . Hummer , and G . Sander . 1978 . Chlamydomonas flageUar mutants lacking radial spokes and central tubules . Structure , composition , and function of specific axonemal components . Z Cell Biol . 76 : 729 - 747 . 908 THE JOURNAL OF CELL BIOLOGY • VOLUME 97 , 1983 \ No newline at end of file diff --git a/silver_data/a48c60b820ef6fe75dcd130bf12052f3276baf38.pdf.txt b/silver_data/a48c60b820ef6fe75dcd130bf12052f3276baf38.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..eb9aec8df902660d8c896f64983eced572b17440 --- /dev/null +++ b/silver_data/a48c60b820ef6fe75dcd130bf12052f3276baf38.pdf.txt @@ -0,0 +1 @@ +MATEMATIIKAN JA LUONNONTIETEIDEN OPETUKSEN TUTKIMUSSEURAN TUTKIMUSPÄIVÄT 2015 ANNUAL SYMPOSIUM OF THE FINNISH MATHEMATICS AND SCIENCE EDUCATION RESEARCH ASSOCIATION 2015 Harry Silfverberg & Peter Hästö ( toim . ) Matematiikan ja luonnontieteiden opetuksen tutkimusseura r . y . http : / / www . protsv . fi / mlseura / ISBN 978 - 952 - 93 - 8233 - 0 i ESIPUHE Matematiikan ja luonnontieteiden opetuksen tutkimusseura ry : n 32 . tutki - muspäivät järjestettiin Turun yliopiston opettajankoulutuslaitoksella 29 . – 30 . 10 . 2015 . Päivillä kuultiin 46 tutkijan pitäminä yhteensä 29 esitelmää matematiikan ja luonnontieteiden didaktiikan meneillään olevista tutkimuk - sista . Kutsuttuina pääpuhujina esiintyivät PhD Kristóf Fenyvesi , prof . Raine Koskimaa ja dos . Osmo Pekonen ( Jyväskylän yliopisto ) Mathematical adventures for the senses and the mind : Math - Art activities for experience - centered education of mathemat - ics , Prof . Mirjamaija Mikkilä - Erdmann ( Turun yliopisto ) Challenges of learn - ing science in higher education - Examples from preservice teacher educa - tion and medical education , PhD Jake McMullen ( Turun yliopisto ) Spontaneous focusing on quantita - tive relations and rational number development . Perinteiseen tapaan tutkimuspäivien esitelmöitsijöille tarjottiin mahdollisuut - ta tarjota tutkimusartikkelia julkaistavaksi päivien konferenssijulkaisussa . Kaikki tarjotut artikkelit vertaisarvioitiin single - blind menettelyä käyttäen . Arviointiprosessin järjestämisestä vastasivat allekirjoittaneet yhdessä , lukuun ottamatta toimittajien omia artikkeleja , joiden arviointiprosessi järjestettiin kirjoittajilta itseltään salatusti . Korjauskierrosten jälkeen 15 parasta artikkelia hyväksyttiin julkaistavaksi . Artikkelien kirjoittajat vastaavat itse tekstiensä oikeellisuudesta . Valmis teos julkaistaan avoimena verkkojulkaisuna syksyn 2016 kuluessa . Harry Silfverberg & Peter Hästö ii PREFACE The thirty - second Annual symposium of the Finnish mathematics and science education research association was held at the University of Turku , October 29 – 30 , 2015 . The invited plenary speakers were : PhD Kristóf Fenyvesi , prof . Raine Koskimaa and dos . Osmo Pekonen ( University of Jyväskylä ) Mathematical adventures for the senses and the mind : Math - Art activities for experience - centered education of mathemat - ics , Prof . Mirjamaija Mikkilä - Erdmann ( University of Turku ) Challenges of learning science in higher education - Examples from preservice teacher education and medical education , PhD Jake McMullen ( University of Turku ) Spontaneous focusing on quantitative relations and rational number development . After due changes 15 best articles were accepted for publication . The writers of the articles are themselves responsible for the correctness of the content of the article . The completed work will be published in the course of the autumn of 2016 . Harry Silfverberg & Peter Hästö iii SISÄLLYS TABLE OF CONTENTS Esipuhe / Preface ARTIKKELIT Harmoinen Sari Tieteellinen taidekokemus – museo luonnontieteiden oppimisympäristönä 1 Havinga Mirka & Portaankorva - Koivisto Päivi Kuvataiteen ja matematiikan yhteisiä ilmiöitä etsimässä 12 Joutsenlahti Jorma & Kulju Pirjo Akateeminen lukutaito matematiikassa 23 Kokkonen Tommi & Nousiainen Maija Learning physics concepts – a description in terms of relational concepts 35 Kärki Tomi Luokanopettajaopiskelijoiden geometrisista määritelmistä 48 Laherto Antti & Laherto Jussi Videovälitteistä fysiikan opetusta luokanopettajakoulutuksessa 60 Lastusaari Mika , Laakkonen Eero & Murtonen Mari Kemian LuK - opiskelijoiden lähestymistavat oppimiseen ja halu vaihtaa pääainet - ta 71 Lehtinen Antti & Hähkiöniemi Markus Complementing the guidance provided by a simulation through teacher questioning 80 Mäkelä Ari - Mikko , Ali - Löytty Simo , Joutsenlahti Jorma & Kauhanen Janne Moodlen Työpaja – vertaisarviointi osana opetusta yliopistomatematiikan ensimmäisellä peruskurssilla 90 Nieminen Pasi , Hähkiöniemi Markus , Leskinen Jarmo & Viiri Jouni Four kinds of formative assessment discussions in inquiry - based physics and mathematics teaching 100 Palkki Riikka Virheellinen esimerkki matematiikan luokkahuonekeskustelussa 111 Ratinen Ilkka , Kähkönen Anna - Leena , Lindell Anssi & de Vocht Miikka Does RRI focused science teaching help students incorporate RRI into their inquiry - based lesson plans ? 122 MALU , Turku 2015 iv Silfverberg Harry & Tuominen Anu Murtoluvun ja lukusuoran pisteen välinen vastaavuus – tyypillisimpiä virheitä luokanopettajaopiskelijoiden suorituksissa 133 Tuomela Dimitri Task potential of reversed equation solving 143 Tuominen Anu Käsityksiä murtolukujen tiheydestä 153 Viholainen Antti , Niko Kuusisto , Mervi Asikainen & Pekka E . Hirvonen Matematiikanopettajien näkemyksiä liittyen teoriaan , esimerkkeihin ja harjoitus - tehtäviiin 162 Virmajoki Anne & Helle Laura Definitions of medication calculation competence of registered nurses : an integrative review 173 Harmoinen 1 TIETEELLINEN TAIDEKOKEMUS – MUSEO LUONNONTIE - TEIDEN OPPIMISYMPÄRISTÖNÄ Sari Harmoinen Oulun yliopisto Tämä tutkimus esittelee Oulun yliopiston matematiikan ja luonnontieteiden aineenopettajaopiskelijoiden ( n = 50 ) ja Oulun Taidemuseon henkilökunnan ( n = 4 ) kokemuksia taidemuseolla toteutetuista pajoista . Tutkimuksen kohteena olivat opiskelijoiden ilmiöpohjaisissa , koulun ulkopuolisessa oppimisympäristössä , toteutetuista pajoista saadut kokemukset . Pajoihin osallistuvat koululaiset olivat iältään alakouluikäisistä aikuisiin maahanmuuttajien kotouttamisryhmään . Koke - mukset olivat hyvin antoisat . Opiskelijat saivat haastaa itseään erityisesti erilaisten oppijoiden kohtaamisessa sekä erilaisten ryhmien hallinnassa . Museo nähtiin , ei niinkään tilana vaan vuorovaikutuksen mahdollistajana . Museoväkeä kokemus kannusti erilaisen toiminnan järjestämiseen . Saatu kokemus kannustaa lisäämään vastaavaa toimintaa jatkossakin . JOHDANTO Matematiikan ja luonnontieteiden aineenopettajiksi opiskelevien pedagogiset opinnot kestävät yhden lukuvuoden . Opettajuus ja oppiminen rakentuvat kyseisen vuoden aikana osaksi tulevaa ammatti - identiteettiä . Opettajuus on opettamista , mutta myös toimijana olemista erilaisissa tilanteissa ja ympäris - töissä . Opettajien olisi hyvä tehdä oppilaidensa kanssa erilaisia vierailuja ja retkiä muun muassa yrityksiin ja tiedemuseoihin . Siksi olisikin erittäin tärkeää ja hyödyllistä harjoitella ja miettiä sellaisen järjestämiseen liittyviä asioita jo opintojen aikana . Perusopetuksen opetussuunnitelma 2014 tuo esille ilmiöpohjaisen oppimisen merkityksen , joka voi olla monelle opettajalle ja opettajaksi opiskelevalle tilaisuus lähestyä tutkittavaa asiaa monipuolisesti . Opetusharjoitteluun kuuluu perinteisesti oppituntien suunnittelua ja toteutusta harjoittelukoululla . Resurssit eivät useinkaan riitä muun opetuk - seen liittyvän harjoittelun tekemiseen . Oulun yliopiston opettajankoulutuslai - tokselle tarjoutui mahdollisuus yhteistyöhön Oulun Taidemuseon ja tiede - keskus Tietomaan kanssa . Tässä tutkimuksessa esiteltävä toiminta on ensimmäinen Oulussa aineenopettajaopiskelijoiden kanssa tehty yhteistyö . Museon henkilökunta otti yhteyttä yliopistoon syksyllä 2014 ja tarjosi opettajaopiskelijoille tilaisuutta toteuttaa koululaisryhmille toiminnallisia pajoja talvella 2015 . Pajat liittyisivät Oulun Taidemuseolla olevaan ”Check - point Leonardo” – näyttelyyn . Opiskelijoita oli varhaiskasvatuksen - , luokan - opettaja - ja aineenopettajakoulutuksen ryhmistä , mutta tässä tutkimuksessa on keskitytty vain aineenopettajaopiskelijoihin . MALU , Turku 2015 2 TEORIAA Oppilaille tulee tarjota mahdollisuus oppimiseen koulun ulkopuolella sekä heille tulisi tarjota monipuolista ja tehokasta oppimista erilaisissa oppimis - ympäristöissä ( POPS 2014 ) . Erilaiset oppimisympäristöt lisäävät valmiuksia toimia yhteiskunnan jäsenenä ( Kuuskorpi , 2012 ) . Tämän taidon harjoittelu tulisikin voida tarjota myös opettajaksi opiskeleville . Opiskelijoilla tulisi olla mahdollisuus toimia erilaisissa oppimistilanteita ja kehittää tulevaisuudessa tarvittavia työskentelytaitoja . Euroopan komission raportissakin ( 2015 ) esitettiin , että opetuksessa , erityisesti science aineiden opetuksessa , tulisi tehdä yhteistyötä kaikilla yhteiskunnan tasoilla . Oppimisympäristöille on olemassa useita erilaisia määritelmiä . Sillä voidaan tarkoittaa sosiaalista , fyysistä ja psyykkistä oppimiseen käytettävää ympäris - töä ( Nuikkinen , 2009 ) . Sen voi muodostaa lähiympäristö , ulkopuolinen toimintaympäristö ja toiminnan ilmapiiri ( Björklid , 2005 ) . Toisaalta se voi olla paikka tai yhteisö , jossa on käytössä erilaisia resursseja , joiden avulla voidaan ymmärtää moninaisia asioita ( Wilson , 1996 ) . Tai se voi muodostua sekä ympäristöstä että teknologiasta ( Dument & Istance , 2010 ) . Oppimisympäris - töllä tarkoitetaan kuitenkin usein oppimista tukevien mahdollisuuksien huomioimista sekä tilana , toimintana että vuorovaikutuksena . Kuvio 1 . Oppimisympäristön osatekijöitä Opetussuunnitelman perusteissa ( POPS 2014 ) nostetaan esille myös tulevai - suuden taidot . Niissä korostetaan muun muassa ajattelu - ja työskentelytapo - jen harjoittelua . Harjoittelulla tavoitellaan luovuuden , kriittisen ajattelun , oppimisen , kommunikoinnin ja informaation lukutaidon lisääntymistä ( Fullan & Lanworthy , 2013 ) . Näitä taitoja opettajien tulisi tulevaisuudessa opettaa myös oppilaille . Perusopetuksen opetussuunnitelman perusteet ( POPS 2014 ) tuovat myös aiempaa korostetummin esille ilmiöpohjaisen oppimisen . Usein sillä tarkoitetaan monialaisia oppimiskokonaisuuksien , joissa huomioidaan paikalliset mahdollisuudet ( European Comission , 2015 ) . Opetussuunnitelma ja pedagogiikka eivät saisi olla erillisiä toisistaan Harmoinen 3 riippumattomia elementtejä ( Osborne , 2007 ) . Olisikin tärkeää ohjata opetta - jaksi opiskelevia näkemään opetussuunnitelman erilaisia käytännön toteutu - misia . Tämä on hyvin perusteltua , koska oppilaan kokemus synnyttää oppimista sekä uudistaa hänen ajattelua ( Hyyppä , Kiviniemi , Kukkola , Latomaa & Sandelin , 2015 ) . Museo oppimisympäristönä Museoissa on ollut opetuksellista toimintaa jo pitkään , mutta toiminta ei ole kuitenkaan ollut aina kovinkaan opetuksellisesti tavoitteellista ( Tornberg & Veteläinen , 2008 ) . Museo on voinut olla oppimisen kohde , paikka tai tila oppimiselle , virikkeiden antaja , ajattelun tai oppimisprosessin stimuloija . Se on voinut tarjota tietoa , taitoa tai elämyksiä sekä toimia arvokasvatuksen tukena . Eshachin ( 2007 ) mukaan museo voi toimia non - formaalina oppimis - ympäristönä . Usein museo nähdään ikkunana syvälle omiin kokemuksiin , menneisyyden ja tulevaisuuden välissä . Tyypillisesti museovierailu on strukturoitu , oppaan vetämä luentomainen kierros , jossa oppilaiden roolina on liikkua yhtenä ryhmänä oppaan perässä ja täyttää kyselylomake ( Griffin & Symington , 1997 ) . Oppijan ja osallistujan omia kokemuksia ei hyödynnetä tai huomioida vierailun aikana . Oppiminen on konstruktivistisen käsityksen mukaan yhteisöllistä tiedon jakamista , jossa huomioidaan luovasti oppimisympäristö ja sen mahdollisuu - det . Oppimista koulun ulkopuolella tulee suunnitella ja valmistella vähintään samalla tavoin kuin muutakin opetusta , jotta sitä voitaisiin hyödyntää oppimisen resurssina ( Griffin & Symington , 1997 ; Eshach , 2007 ) . Koulun ulkopuolisella oppimisella tulee olla myös tavoitteet , mutta ne eivät välttä - mättä ole samat kuin perinteisessä luokkahuoneopetuksessa eli oppimista tulee ajatella uudella tavalla . Cox - Peterson , Marsh , Kisiel ja Melbern ( 2003 ) tekemässä tutkimuksessa nousi esille didaktisen ajattelun merkitys museovie - railun suunnittelussa . Museovierailu voi olla didaktisesti merkityksellinen oppimisympäristön eikä niinkään sisällön kannalta . Tässä tutkimuksessa tarkastellaan museota oppimisympäristönä ja selvitetään aineenopettajaopiskelijoiden kokemuksia opetuksen suunnittelusta siellä . Lisäksi selvitetään miksi museo on merkityksellinen oppimisympäristönä . Tuloksia tarkastellaan seuraavien kysymysten kautta . 1 . Millaisia tavoitteita opettajaopiskelijat asettavat omalle työskentelyl - leen Taidemuseolla ? 2 . Millaisia kokemuksia opiskelijoilla ja museon henkilökunnalla oli Taidemuseolla työskentelystä ? MALU , Turku 2015 4 TUTKIMUSASETELMA Checkpoint Leonardo – näyttelyn järjestelyssä ja pajatoiminnan toteuttami - sessa Oulussa on ollut laaja yhteistyöverkosto . Mukana on ollut Oulun Taidemuseo OMA , Oulun Tietomaa , Maaseudun sivistysliitto , Oulun yliopiston opettajankoulutuslaitos , luokanopettajaopiskelijoita sekä matema - tiikan , fysiikan , kemian , biologian ja maantieteen aineenopettajiksi opiskele - vat sekä Oulun seudun koulut alakouluista aikuisopiskelijoihin saakka . Kouluille tiedotettiin pajoista kulttuuriyhdysopettajien kautta ja Oulun Taidemuseon kautta opettajat saivat varata etukäteen oppilasryhmälleen yhden tarjolla olevasta 18 pajasta . Ryhmän tuovien opettajien ei tarvinnut valmistella tieteellisistä näkökulmista oppilaita museotoimintaan eikä heillä ollut opetus - tai ohjausvastuuta , mutta opettajien tuli olla läsnä . Aikatauluissa oli huomioitu opiskelijoiden ja koululaisten opintoaikataulut sekä siirtymiset kouluilta ja yliopistolta museolle . Opiskelijoiden ( n = 50 ) aikataulu suunnittelun ja toteutuksen suhteen oli nopea . Opiskelijat aloittivat pedagogiset opinnot viikolla 2 ja ilmiöpohjaista oppimista ja opetusta harjoiteltiin sekaryhmissä viikolla 6 . Sekaryhmissä oli 5 opiskelija pääasiallisesti eri pääaineista . Sekaryhmissä toiminta oli kolmivai - heista . Aluksi sekaryhmille annettiin kaikkia oppiaineita yhdistävä ilmiö , josta heidän tuli tehdä suunnitelma oppitunnista , jossa näkyy kaikkien oppiaineiden näkökulma . Toisessa vaiheessa opiskelijat suunnittelivat valitsemansa ilmiönpohjalta oppitunnin , sitomatta sitä mihinkään oppiainee - seen , mutta huomioivat eri oppiaineiden tarjoaman tietosisällön . Kolmannes - sa vaiheessa ryhmille annettiin kuva Leonardo da Vincin teoksesta tai luonnosvihosta . Luonnoskirjan kuva toimi inspiroijana ja ajatuksien herättä - jänä . Tämän jälkeen kävimme viikolla 7 tutustumassa opiskelijoiden kanssa Oulun Taidemuseolla näyttelyyn , jota he käyttäisivät oppilasryhmien työskentelyn inspiroijana . Näyttelyssä oli teoksia muun muassa Heinolta , Närhiseltä , Schäringiltä ja Sylvia Grace Bordanilta . Opiskelijat toteuttivat koululaispajat viikoilla 9 ja 11 . Jokaisella ryhmällä oli toteutus kaksi kertaa . Käytännön toteutukseen opiskelijoille annettiin ohjeita 20 – 25 oppilaan ryhmän vastaanottamiseen taidemuseolla sekä toiminnan ohjaamisesta . Ryhmän tuli valita näyttelystä korkeintaan kolme taideteosta ja miettiä missä järjestyksessä kohteet kierretään . Huomioitavana oli , ettei kierros ole vapaamuotoinen läpikävely , vaan sen tulee olla ohjattua . Tämän jälkeen opiskelijat siirtyivät oppilaiden kanssa työskentelemään pajoihin taidemuse - olta tai Tietomaasta valitsemaansa tilaan . Pajatyöskentelyn onnistumiseksi , opiskelijoiden tuli miettiä huolellisesti työohjeistus ja roolit työskentelyn aikana . Lisäksi opiskelijat miettivät etukäteen mitä he vaativat oppilailta , työskentelevätkö oppilaat yksin vai ryhmissä ja miten ryhmiin jakautuminen tehdään . Opiskelijoita ohjattiin myös miettimään , mikä tavoite lopputuotokselle asetetaan ja mitä tuloksella Harmoinen 5 tehdään . Tämän vaiheen suunnittelussa kannustettiin opiskelijoita pohtimaan erilaisia toteutusvaihtoehtoja , vaikka tarkoituksena olisikin käyttää niistä vain yhtä . Lisäksi valmisteluissa yritettiin ohjata opiskelijoita arvioimaan mitä asioita , tilanteita ja mahdollisuuksia työskentelyssä voisi tulla esille . AINEISTON KERÄÄMINEN Tämä tutkimus on tapaustutkimus , jossa kuvataan aineenopettajaopiskelijoi - den kanssa toteutettua toimintaa osana ainedidaktisia opintoja . Tutkimukses - sa on kartoitettu opiskelijoiden ja museohenkilökunnan kokemuksia ja mielipiteitä google forms - lomakkeella . Vastauksia on 50 opiskelijalta ja 4 henkilöltä museolta . TULOKSIA Seuraavassa esitellään opettajaopiskelijoiden asettamia tavoitteita omalle toiminnalle sekä heidän kokemuksiaan itse toiminnasta . Lisäksi esitetään museohenkilökunnan kokemuksia yhteistyöstä . Opettajaopiskelijoiden asettamia tavoitteita toiminnalle Opiskelijoiden vastauksista oli selvästi havaittavissa tavoitteiden asettelua sekä omalle toiminnalle , oppilaiden kohtaamiselle että koulun ulkopuolisen oppimisympäristön , museon osalle . Omalle työskentelylle esitetyt tavoitteet olivat tyypillisesti hyvin konkreetti - sia . ”Esitellä oppilaille veden ominaisuuksia kokeiden avulla” ( kuva 2 . ) , jossa tavoite liittyi substanssin eli tieteellisen tiedon välittämiseen . Kuvassa 2 opiskelija tutkii pajaan osallistuneen lapsen kanssa pintajännitystä elintarvi - kevärien avulla . Osalla opiskelijoista tavoitteena oli saada aikaiseksi aktiivista vuorovaikutus - ta oppilaiden kanssa , kuten seuraavassa tulee esille yhteisen ajattelun kautta : ” Pohtia yhdessä oppilaiden kanssa ” ( kuvan 3 . Opiskelija ) MALU , Turku 2015 6 Kuva 2 . Värien lumoa vedessä . Kuva 3 . Keskustelua luonnon tilasta Kuvassa 3 opiskelija pohtii eri puolilta maailmaa ottamiensa valokuvien avulla luonnon tilaa sekä ihmisen toimintojen ja valintojen vaikutusta luontoon . Keskeisenä aiheena olivat muoviroskien ympäristövaikutukset merissä . Taidetoksista pajatoiminnalle toimi erityisesti ”Merenneidon kyyneleet” – teos . Opiskelijoiden omissa tavoitteissa tuotiin esille myös arvokasvatuksellisia asioita , kuten esimerkiksi ” Opettaa pajalaisia kierrättämään ” ( Kuva 4 . ) Kuva 4 . Käytetyistä pH - papereista tehty taideteos . Kuvassa 4 on esitetty happamuuden tutkimuksissa käytetyistä pH - papeista valmistettu taideteos . Oppilaiden kohtaamiseen ja aktivointiin liittyvät tavoitteet olivat yleisesti ” Saada oppilaat oppimaan ja ajattelemaan ” ja ” Herättää oppilaiden kiinnostusta ja ajatuksia ” ( kuva 5 . ) Kuva 5 . Innostuksen siemen sytytetty Harmoinen 7 Museon tarjoamassa oppimisympäristössä haluttiin korostaa liittymäkohtia ympäristön , taiteen ja asennekasvatuksen välille , mutta tarjota ennen kaikkea mielenkiintoisia kokemuksia . Aineenopettajaopiskelijoiden kokemuksia Kyselyn alussa selvitettiin opiskelijoiden kokemuksia monivalintakysymyk - sillä ja sen jälkeen heitä pyydettiin kuvaamaan tarkemmin omia positiivisia ja haasteellisia kokemuksiaan . Taulukossa 1 on koottu opiskelijoiden kokemuk - set kysymykseen mikä oli museotyöskentelyn merkityksellisyydestä . Taulukko 1 . Opiskelijoille merkitykselliset kokemukset museolla työskente - lystä . Merkityksellinen kokemus Lukumäärä ( N = 50 ) itsensä haastaminen 2 ilmiöpohjainen oppiminen 8 erilaiset oppimisympäristöt 13 erilaisten ryhmien kohtaaminen 27 Taulukossa 1 esitettyjen merkitykselliset kokemukset olivat erilaisten ryhmien kohtaaminen ja erilaisessa oppimisympäristössä toimiminen . Opiskelijat eivät kokeneet kovinkaan merkitykselliseksi itsensä haastamista tai ilmiöpohjaista oppimista . Avointen kysymysten vastauksissa opiskelijat toivat esille seuraavia ajatuksia . Erilaisten ennakkoon epävarmuutta tuovien tilanteiden tai ryhmien kohtaa - minen ja hallinta oli myös tuottanut onnistumisen kokemuksia . ” Kaaoksen hallinta” ( opiskelija 22 ) ja ”Kuinka ryhmää hallitaan ja ohjataan” ( opiskelija 8 ) tai ” Opin ihmistuntemusta ja ryhmän hallintaa . Yliopistossa teoriapuolella olen oppinut paljonkin erilaisia asioita , mutta koin , että käytän - nössä niistä opeista ei ollut juurikaan apua . ” ( Opiskelija 30 ) ” Sain ohjata sellaista oppilasryhmää , jota todennäköisesti en kohtaa harjoitte - lussa ” ( Opiskelija 48 ) ” Onnistumisen elämys” ( opiskelija 16 ) , ”Nähdä lasten kiinnostus / riemu ilmi - öitä testatessa . ” ( opiskelija 29 ) tai ” 2 . - luokkalaiseten oivallukset pajan aikana . Aikuisopiskelijoiden kohdalla iloa tuotti se että pajasta selvittiin ja ainakin osa oppilaista kuunteli ja osallistui aktiivisesti . ” ( Opiskelija 33 ) Koska opiskelijat olivat hyvin uudenlaisessa tilanteessa , toi se mukanaan myös erilaisia haasteita . Uutta oli yleensäkin ryhmän hallinta , mutta myös MALU , Turku 2015 8 aineen hallintaan liittyvät asiat askarruttivat etukäteen . Aineenhallintaan liittyvät asiat koettiin kuitenkin onnistuneiksi . Sen sijaan suunnittelusta huolimatta saattoivat välineet loppua kesken , mutta niistäkin opiskelijat onnistuivat selviytymään hyvin . ” Oppilaiden tietotaso yllätti iloisesti . Myös neljäsluokkalaiset esittivät melko korkeatasoisia ja vaikuttavia kysymyksiä valosta ja osasivat pohtia itsenäisesti ilmiöiden teoreettista taustaa . Lyhyesti sanottuna parasta oli tuottaa " AHAA - elämyksiä " ja nähdä oppimisen iloa . ” ( Opiskelija 12 ) ” Yhdessä työssä elintarvikevärit ja maito loppuivat kesken ja piti keksiä kor - vaavia värejä . Lisäksi oheistoimintaan kuten tiskaamiseen meni paljon aikaa . ” ( Opiskelija 45 ) Opiskelijoiden yhdessä tekeminen ja kokemusten tuottaminen tulivat myös vastauksissa esille : ” Ryhmänä onnistuttiin ainakin omasta mielestä tuottamaan positiivinen ko - kemus koululaisille . ” ( Opiskelija 14 ) Vaikka erilainen oppimisympäristö oli toiseksi merkityksin kokemus , ei se kuitenkaan noussut esille avoimissa kysymyksissä . Ilmiöpohjaisesta oppimi - sesta koettiin merkitykselliseksi sen käytännön toteutuksen onnistumisen vuoksi . ” Ilmiöpohjainen oppiminen onnistui käytännössä . ” ( opiskelija 48 ) Oleelliseksi näytti nousevan tilanteen ja ryhmien hallinta . Museohenkilökunnan kokemuksia Museolla pajojen toiminnan sujumiseen ja käytännön järjestelyjen onnistumi - seen osallistui 4 henkilöä . Heidän odotuksensa liittyivät vain siihen , että opiskelijat pitävän mielenkiintoisia pajoja , koulun ulkopuolisessa oppimis - ympäristössä . Toteutumisen kokemukset toivat esille monia ennalta arvaa - mattomiakin asioita . Seuraavassa muutamia kommentteja : ” Vaati aikaa ! ” ( H3 ) ja ” Opiskelijat olivat alussa yhtä hukassa kuin me ! Mutta hienosti suoriuduttiin loppuun asti ! ” ( H1 ) Kaikkeen ei osattu varautua etukäteen ja kokemukset opiskelijoiden läsnä - olosta tuottivat hämmennystä museon henkilökunnalle ja heidän perinteiseen työnkuvaansa . Kuitenkin kokemukset olivat pääsääntöisesti positiivisia . ” Oli hienoa nähdä innostuneita opiskelijoita ” ( H4 ) ” Vilinää , huisketta ja melua . Oli mukavaa seurata , miten lapset innostuivat taiteesta ja tieteestä ! ” ( H1 ) Vaikka kokemuksien pohjalta koettiin ajoittain yhteistyö haastavaksi , haluavat he tehdä yhteistyötä jatkossakin arvosanalla 4 , 5 ( asteikolla 1 - 5 ) . Museon henkilökuntakin koki oppivansa yhteistyöstä paljon . He nostivat esille seuraavia näkökulmia : Harmoinen 9 ” Työpajat eivät varsinaisesti lisänneet työmäärääni , toivat vain työhöni uusia tehtäviä . ” ( H1 ) ” Mitä paremmin suunniteltu tapahtuma , sen helpompi on improvisoida ja rennos - ti reagoida erilaisiin ryhmiin ja tilanteisiin . ” ( H3 ) Toisaalta heidän esille tuomissaan asioissa on havaittavissa opiskelijoiden kokemuksen vähäisyyden ja luokkahuoneen ulkopuolella tapahtuvan oppimisen mukanaan tuomia asioita , kuten seuraavat kommentitkin osoittavat . ” Opiskelijoiden tulee paneutua taideteoksiin ja otettava huomioon , että pajalaiset saattavat olla ensimmäistä kertaa taidemuseossa ( , mutta paja innostaisi tulemaan toistekin ) . ” ( H2 ) ” Opiskelijoiden on hyvä harjoitella äänenkäyttöä ja ryhmän hallintaa , taidemuseon tilat ovat aika haastavat , mutta niin ovat luokkahuoneetkin . ” ( H3 ) ” Suunnittelu ja tiedon jakaminen museon ja opiskelijoiden välillä etukäteen on tärkeää . ” ( H1 ) POHDINTAA Taidemuseon pajatyöskentely oli erilainen ainedidaktisten opintojen toteut - tamistapa ja siksi siitä oli paljon opittavaa . Kerätystä palautteesta nousi esille , että kokemus oli opiskelijoille hyvin antoisa ja mielekäs ja lisäsi eri pääai - neopiskelijoiden välistä yhteistyötä . Voidaankin todeta erästä opiskelijaa lainaten , että ” projekti opetti luottamaan ryhmässä olevaan potentiaaliin sekä suunnittelus - sa että toteutuksessa ” ( Opiskelija 10 ) Tämän tutkimuksen avulla pyrittiin selvittämään aineenopettajaopiskelijoi - den kokemuksia koulun ulkopuolisessa oppimisympäristössä toimimisesta . Museolla mahdollistunut yhteistyö oli erittäin mieliin painuva ja merkittävä kokemus kaikille osapuolille ; aineenopettajaopiskelijoille ja museohenkilö - kunnalle . Vaikka toteutus oli aikataulullisesti hyvin haastava , oli se erittäin onnistunut kokonaisuus . Yhteistyötaidot ja kriittinen ajattelu sekä tavoittei - den asettaminen , sekä itselle että oppilaille , saivat aivan uuden merkityksen , kun oppiminen siirrettiin luokkahuoneen ulkopuolelle . Toivottavasti kokemus rohkaisee opiskelijoita hyödyntämään erilaisia oppimisympäristöjä tulevassa työssään . Oppiminen on muutakin kuin tiedon jakamista . Se on yhdessä kokemista , oppimisen iloa ! Vaikka toiminta voi olla välillä kaaoksen hallintaan verrattavissa oleva selviytymistilanne , tuottaa se aina merkityksel - lisiä kokemuksia . Opiskelijat asettivat omalle toiminnalleen hyvinkin konkreettisia tavoitteita , esimerkiksi asianhallintaa liittyen , mutta toteutuksen jälkeiset kokemukset MALU , Turku 2015 10 liittyivät usein aivan erilaisiin asioihin . Oleellista ei näyttäisikään olevan tällaisella työskentelyllä onnistunut asioiden opettaminen vaan käytännön asioiden onnistuneet toteutukset , erilaiset kohtaamiset ja kokemusten saamiset . Museo nähtiin oppimisympäristönä , ei niinkään tilana tai toiminta - na , vaan ennen kaikkea vuorovaikutuksen mahdollistajana . Museo ei ollut vain taiteen tai tieteen tekemisen ja havainnoin paikka , vaan luonnollisempi kohtaamisen ympäristö . Nuikkisen ( 2009 ) mukaisesti museo toimiikin sosiaalisena ja fyysisenä oppimisympäristönä . Siellä ei ole opiskelija tai sinne tuleva oppilas oppimiskulttuuriin luonnollisessa ympäristössä . Siksi siinä toimiminen tuottaa monia , kenties luokkahuoneessa huomiotta jääviä kohtaamisia . Museon näkökulmasta näytti museotoiminnan tavoitteet toteutuvan hyvin . Opiskelijat muodostivat elämyksiä sekä taiteesta ja tieteestä inspiroituneita kävijöitä . Museolle asetetut tehtävät oppimisen kohteena , virikkeiden antajana , ajattelun tai oppimisprosessin stimuloijina näyttivät toteutuvan sekä museon henkilökunnan että opiskelijoidenkin mielestä . Fullan ja Lanworthy ( 2013 ) ajatuksien mukaisesti voidaan sanoa , että museolla voidaan harjoitella ajattelun ja yhdessä toimimisen taitoja . Tieteen ja taiteen yhdistämine ei ollut kenellekään ennestään tuttua , mutta siitä jäi kuitenkin ” hyvä fiilis ” ja tunne , että ” tämän tyyppisestä toiminnasta sai hyvin paljon irti ” . Lisäksi yhteinen mielipide oli , että toteutus mahdollisti hienon kokemuksen saamiseen . Illiciä ( 1970 ) lainaten voidaan todeta , että ” Luokkahuoneopetus siirtää oppilaan kulttuurimme ulkopuolelle ja tunkee heidät ympäristöön , joka on monin verroin ulkopuolista ympäristöä alkeellisempi , luon - nottomampi ja lisäksi kuolettavan vakava . ” Oppimisympäristön valinnalla on monia , jopa ennalta arvaamattomia merkityksiä . Saamamme kokemuksen perusteella jatkammekin yhteistyötä Oulun Taidemuseon kanssa 23 . 1 . – 20 . 3 . 2016 olevien näyttelyiden ”Skills” ja ”Ihan tiloissa” innoittamina . Olemme kehittäneet niitä kohtia , joissa Check - point Leonardossa nousi esille sekä selvitämme tässä esitetyn tutkimuksen toiminnassa huomiotta jääneiden osallistujien mielipiteeseen huomiota . LÄHTEET Bjurström , P . ( 2009 ) . Att förstå skolbygnader . Väitöskirja . KTH Arkitektursko - lan . Trita - ARK . Akademisk avhändling 2004 : 2 . Stockholm : KTH Arkitek - turskolan . Cox - Peterson , A . , Marsh , D . , Kisiel , J . & Melber , L . ( 2003 ) . Investigation of guided school tours , student learning , and science reform recommenda - tions at a Museum of Natural History . Journal of Research in Science Teach - ing , 40 ( 2 ) , 200 – 218 . Dumont , H . & Istance , D . ( 2010 ) . Analysing and designing learning environments for 21 th century . Teoksessa H . Dumont , D . Istance & F . Benavides ( toim . ) Harmoinen 11 The Nature of Learning . Using Research to Inspire Practice ( s . 19 - 34 ) . Paris : OECD Publishing , Eshach , H . ( 2007 ) . Bridging In - school and Out - of - school Learning : Formal , Non - Formal , and Informal Education . Journal of Science Education and Technology , 16 ( 2 ) , 171 – 190 . European Comission ( 2015 ) . Science education for Responsible Citizenship . European Commission Research and Innovation , Brussel . EUR 26893 EN . Fullan , M . & Lanworthy , M . ( 2013 ) Toward a New End New Pedagogies for Deep Learning . Saatavilla http : / / www . newpedagogies . info . Griffin , J . & Symington , D . ( 1997 ) . Moving from task - oriented strategies on school excursions to museums . Science Education , 81 ( 6 ) , 763 – 779 . Hyyppä , H . , Kiviniemi , L . , Kukkola , J . , Latomaa , T . & Sandelin , P . ( 2015 ) . Kokemuksen tutkimuksen ulottuvuudet . ePooki . Oulun ammattikorkea - koulun tutkimus - ja kehitystyön julkaisut 9 . Hakupäivä 15 . 10 . 2015 . http : / / urn . fi / urn : nbn : fi - fe201503252008 . Illich , L . ( 1972 ) . Kouluttomaan yhteiskuntaan ( suomentanut Valpola , A . ) . Delfiinikirjat , Helsinki : Otava . Kuuskorpi , M . ( 2012 ) . Tulevaisuuden fyysinen oppimisympäristö . Turun yliopisto , väitöstutkimus . Kasvatustieteiden laitos . Turku : Pallosalama . Nuikkinen , K . ( 2009 ) . Koulurakennus ja hyvinvointi . Teoriaa ja käyttäjän kokemuksia peruskouluarkkitehtuurista . Acta Universitatis Tamperensis : 1389 . Väitöstutkimus . Kasvatustieteiden laitos . Tampere : Tampereen yliopistopainos , Juvenes Print . Opetushallitus ( 2004 ) . Perusopetuksen opetussuunnitelman perusteet 2004 . Osborne , J . ( 2007 ) . Science Education for the Twenty First Century . Eurasian Journal of Mathematics , Science & Technology Education , 3 ( 3 ) , 173 – 184 . Tornberg , L . & Venäläinen , P ( 2008 ) . Kulttuuriperinnön opetuksesta ja oppimises - ta . Teoksessa P . Venälänen ( toim . ) . Kulttuuriperintö ja oppiminen ( s . 66 - 74 ) . Suomen museoliiton julkaisuja 58 . Jyväskylä : Gummerus Kirjapaino Oy . Wilson , B . G . ( 1996 ) . What is a constructivist learning environment ? Teoksessa B . G . Wilson ( toim . ) . Constructivist Learning Environments : Case Studies in Instructional Design ( s . 3 - 8 ) . Englewood Cliffs , NJ : Educational Technology Publics . Museon henkilökunnan ottamia valokuvia MALU , Turku 2015 12 KUVATAITEEN JA MATEMATIIKAN YHTEISIÄ ILMIÖITÄ ETSIMÄSSÄ Mirka Havinga 1 & Päivi Portaankorva - Koivisto 2 1 Sydän - Laukaan koulu , Jyväskylä , 2 Helsingin yliopisto Tutkimuksemme lähtökohta on etsiä kuvataidetta ja matematiikkaa eheyttävään opetukseen oppilaiden uteliaisuutta ja kiinnostusta herättäviä , pedagogisesti hyviä ilmiöitä . Tavoiteltavassa tiedonalapohjaisessa ilmiöoppimisessa kummankin oppiaineen luonne ja tyypilliset tavat hankkia ja prosessoida tietoa säilyvät tasavertai - sina . Aineistoina ovat yläkoulun 7 . - luokalle toteutettu opetuskokeilu ja matematiikan aineenopettajaopiskelijoille suunnattu kysely ”mitä ovat matemaattiset ilmiöt” . Havaitsimme , että riittävän avoin ilmiö mahdollistaa oppilaiden ja opettajan yhteisen aidon tutkimisen ja oppimisen . Tutkittavat ilmiöt voivat liittyä käsitteisiin , jotka ovat integroitaville tieteenaloille yhteisiä , esimerkkeinä suhde ja representaatio , tai ne voivat olla dynaamisia , pikemminkin tiedonhankinnan tai tiedonrakentelun proses - seihin liittyviä kuten lajittelu ja luokittelu . JOHDANTO Uudet perusopetuksen opetussuunnitelman perusteet ( POPS 2014 ) nostavat esiin ilmiöt . Kulttuurinen moninaisuus ja kielitietoisuus , eri tiedonaloilla käytettävät kielet ja symbolijärjestelmät antavat samoille ilmiöille tuoreita näkökulmia , joita hyvin valittujen ongelma - ja tutkimustehtävien avulla voidaan tutkia tarkemmin . Näillä herätellään uteliaisuutta ja kiinnostusta , ja ohjataan oppilaita monilukutaitoisiksi . Tämä edellyttää opettajilta uudenlaisia pedagogisia toimintatapoja ja yhteistyötä , jotta oppiainerajat ylittäen voidaan tarkastella todellisen maailman ilmiöitä kokonaisuuksina . Tässä artikkelissa pohdimme , miten ilmiökeskeisyyttä voidaan toteuttaa kahden oppiaineen , kuvataiteen ja matematiikan , kesken näiden tiedonalojen luonne ja menetelmät huomioiden . Peilaamme rinnakkain Sydän - Laukaan koulun perusopetuksen seitsemännellä luokalla toteutettua eheyttävää opetuskokeilua , jossa oppiaineina olivat kuvataide ja matematiikka , sekä matematiikan opettajaopiskelijoille suunnattua kyselyä matematiikan ilmiöistä . Tarkastelemme , millaisia ovat ilmiöt , jotka ovat oppilaille kiinnos - tavia ja samalla kirkastavat eri tiedonalojen perinnettä . Pohdimme , millaisten ilmiöiden varaan monialaisia oppimiskokonaisuuksia voidaan rakentaa väljästi ja avoimesti niin , että yhteistyölle ja ajankohtaisiin tapahtumiin reagoimiselle jää tilaa , ja katsomme mahdollistaako eheyttävä aiheen käsittely erilaisten kysymysten , ratkaisujen ja toimintatapojen käyttämisen . OPETUKSEN EHEYTTÄMINEN JA KOKONAISVALTAINEN OPETUS Perusopetuksen opetussuunnitelman perusteissa 2014 ilmiöt liitetään toimintakulttuurin kehittämistä ohjaaviin periaatteisiin , oppimisympäristöi - Havinga & Portaankorva - Koivisto 13 hin ja työtapoihin . Sen lisäksi niitä tarkastellaan useimpien oppiaineiden kohdalla erikseen . Pedagogisesti tarkasteltuna on kyse opetuksen eheyttämi - sestä . Halinen ja Jääskeläinen ( 2015 ) ehdottavat , että opetusta voitaisiin eheyttää ensinnäkin oppilaan näkökulmasta . Tällöin pyrittäisiin rakentamaan opiskelukokonaisuuksia , jotka perustuisivat oppilaan omiin kokemuksiin , kysymyksiin tai kiinnostuksen kohteisiin . Toisena eheyttämisen lähtökohtana voisivat olla oppimisyhteisöä kehittävät toimintamallit , yhteisöllisyys . Kolmantena eheyttämisen keinona olisivat tiedonalalähtöiset , useammasta oppiaineesta rakennetut oppimiskokonaisuudet . Rakenteellisesti eheyttämistä voidaan toteuttaa oppiainerajat ylittäen joko rinnastamalla tai jaksottamalla opetusta . Tällöin sama ilmiö saa tukea yhtäaikaisesti useammasta tiedonalasta käsin . Eheyttävät kokonaisuudet voidaan myös käsitellä teemapäivinä tai - viikkoina , jolloin ne irrotetaan oppiaineiden muusta opetuksesta . Laajempien oppimiskokonaisuuksien rakentaminen opettajien yhteistyönä , oppiaineiden ryhmittely oppiainekoko - naisuuksiksi tai kokonaisopetus ovat esimerkkejä pitkäkestoisemmasta eheyttävästä työskentelystä . ( Halinen & Jääskeläinen , 2015 , vrt . Hurley , 2001 . ) Kuvataiteen ja matematiikan eheyttävien oppimiskokonaisuuksien taustalla on ajatus kokonaisvaltaisesta opetuksesta . Räsänen ( 2010 ) ehdottaa integroi - van kuvataideopetuksen mallia erääksi jäsennykseksi , jonka pohjalta taide - ja taitoaineita voi tarkastella muiden oppiaineiden rinnalla . Hänen mallissaan tietäminen perustuu havainnoille , tunteille , kunkin taiteen - ja taidonalan käsitejärjestelmää edustaville muodoille sekä kulttuurisille symboleille . Oppiaineen opetuksessa tavoitteena on , että havainnoista ja tunteista saatava tieto yhdistetään tekemisen kautta käsitteellistämiseen ja kulttuuriseen tietoon . Kun Räsäsen ( 2010 ) integroivan kuvataiteen mallia tarkastellaan matematiikan näkökulmasta , huomataan , että tutkiva , elämyksellinen matematiikka koostuu samoista , yhteisistä elementeistä . TUTKIVA OPPIMINEN MATEMATIIKASSA JA KUVATAITEESSA Matematiikassa tutkiva oppiminen on saanut rinnalleen useita eri nimityksiä . Portaankorva - Koivisto ( 2010 ) kuvailee sitä elämykselliseksi . Kun oppilailla on matematiikan tunnilla tilaisuus kehittää persoonallisia , merkityksellisiä ratkaisuja tehtäviin , joihin heillä ei ole ennestään valmiita ratkaisumalleja , ja jotka he itse voivat perustella toimiviksi ja päteviksi , niin he oppivat , mitä tarkoitetaan matemaattisesti erilaisilla tavoilla ratkaista jokin ongelma , ja miten näitä tapoja muodostetaan . Tällöin voidaan puhua omistajuudesta , oppilaiden omasta matematiikasta , joka kuitenkin sijoitetaan matematiikan yhteisön muovaamiin kulttuurisiin puitteisiin . ( Portaankorva - Koivisto , 2010 ; Hähkiöniemi , 2011 . ) Oppiminen kuvataiteessa tarkoittaa Räsäsen ( 2010 ) mukaan kykyä löytää ilmiöiden välisiin suhteisiin liittyviä erityisiä piirteitä , huomata , miten niitä MALU , Turku 2015 14 voitaisiin kuvittaa tai kuvata ja millaisia metaforisia merkityksiä ne pitävät sisällään . Kyse voi olla sekä tuottavasta , että tulkitsevasta toiminnasta . Usein taiteilijan tekemät havainnot ja tunteet ovat ns . hiljaista tietoa , joten taideteok - sen tulkinta ja tuottaminen vaativat hienosyistä erottelukykyä ja suhteiden ymmärtämistä , sekä symbolijärjestelmien tunnistamista ja tuntemista . Voidaankin puhua kuvataiteen tutkivasta oppimisesta . TUTKIMUKSEN TOTEUTUS JA TULOKSET Tässä artikkelissa tarkastellaan , millaisia ovat kuvataidetta ja matematiikkaa yhdistävät ilmiöt . Tutkimustehtävänämme on pohtia opetuskokeilusta saatujen kokemusten ja matematiikan opettajaopiskelijoiden kyselyn pohjalta , millaisia ovat ne kuvataidetta ja matematiikkaa yhdistävät ilmiöt , joita voitaisiin käyttää suunniteltaessa eheyttäviä oppimiskokonaisuuksia . Artikkelin lähtökohtana on kuvataiteen ja matematiikan aineenopettaja Mirka Havingan opinnäytetyöhönsä suunnittelema opetuskokeilu . Syyslukukaudel - la 2014 Sydän - Laukaan koulun 7 . luokan oppilaille suunnatut opetusta eheyttävät kokeilut pyrkivät toteuttamaan opetussuunnitelman sisältöjä ja tavoitteita ja samalla löytämään mahdollisuuksia , joissa kuvataiteen ja matematiikan oppiaineiden itsenäiset luonteet toteutuvat ( Havinga , 2015 ) . Opetuskokeilun ensimmäinen teema käsitteli henkilökohtaisten merkitysten tuottamista ja tulkintaa kokonaisluvuista . Kokonaisluvut ovat perusopetuk - sen opetussuunnitelmassa keskeinen aihealue , joka sijoittuu heti 7 . luokan syksyyn . Työskentely aloitettiin matematiikan tunnilla . Tavoitteena oli oppia ryhmätyötä , avointa ongelmanratkaisua ja matemaattista perustelemista , sekä samalla tutustua kokonaislukujen lukujoukkoon . Oppilaat työskentelivät 3 - 4 hengen ryhmissä ja heidän tuli keskustellen valikoida seuraavasta luettelosta ( 1 , - 5 , 4 , 7 , 32 , 8 , 11 , ∞ , 13 , - 10 , - 1 , 0 49 , 64 ) vähintään viisi lukua tai symbolia siten , että voivat loogisesti ja täsmällisesti perustella valintansa . Havinga & Portaankorva - Koivisto 15 Taulukko 1 . Frekvenssit ja esimerkkejä oppilaiden esittämistä lukujoukoista Kategoria f Esimerkkejä oppilaiden lukujonoista 1 ) Ilmiö itse 0 2 ) Ilmiön kuva 7 ”yksinumeroisia lukuja” ”kaikissa luvuissa on pyöreitä muotoja” 3 ) Ilmiö , kuten se näyttäytyy 3 ”peräkkäisiä lukuja” ”luvut ovat nollan ja äärettömän välissä” 4 ) Yhteys tai tulkinta kuvan ja nähdyn välillä 14 ”luvut ovat viiden numeron välein” ”edellinen luku jaetaan kahdella” ”kaikki luvut ovat positiivisia” ”yksikään ei ole kahdella jaollinen” Oppilaitten vastauksia ( taulukko 1 ) tulkittiin yhdessä heidän kanssaan neljän kategorian avulla ( vrt . Brown , 2001 ) : 1 ) ilmiö itse ( lukumäärän ymmärtämi - nen ) , 2 ) ilmiön kuva ( luku symbolina ) , 3 ) ilmiö , kuten se näyttäytyy ja 4 ) yhteys tai tulkinta kuvan ja nähdyn välillä . Suurin osa lukujoukoista sijoittui kategoriaan ”Yhteys tai tulkinta kuvan ja nähdyn välillä” . Tässä kategoriassa perustelut olivat matemaattisia . Yllättävästi mikään ryhmä ei esittänyt täysin epämatemaattisia lukujoukkoja kuten saman urheilujoukkueen pelinumeroita tms . Kuvataiteen oppitunnilla teemaa jatkettiin . Oppilaan tehtävänä oli tehdä kuva , jonka aiheena oli itse valittu kokonaisluku . Työ tehtiin 20 cm x 20 cm kokoiselle paperille piirtäen tai maalaten . Lisätehtävänä oli tehdä kuva oppilaan valitseman luvun vastaluvusta . Yhteisessä tarkastelussa käytettiin aluksi matematiikan tunnilta tuttua kategorisointia . Koosteesta ( kuva 1 ) havaitaan , miten oppilaat olivat lähestyneet valitsemaan - sa lukua hyvin monin eri tavoin . Ensimmäisessä piirroksessa on selkeästi luvun kuva ja lukumäärä , toisessa piirroksessa luvun muoto joutsenten kauloissa ja lukumäärä useammankin kerran . Monet oppilaat olivat valinneet lukuja , jotka esittivät luvuilla esitettäviä järjestelmiä kuten rahaa , kengännu - meroa , palkintoja tai lämpötila - asteikkoa . Erään oppilaan piirroksessa näkyvät sekä luku , lukumäärä , järjestysluku että luvun saduista tutut metaforiset merkitykset . MALU , Turku 2015 16 Kuva 1 . Esimerkkejä ensimmäisen teeman kuvataiteen oppilastöistä . Oppitunnilla oppilaiden työt järjestettiin lukujen suuruusjärjestyksen perusteella lukusuoralle ja pohdittiin myös vastaluvun käsitettä sekä ääretöntä , jonka eräs oppilaista oli valinnut tarkastelunsa kohteeksi . Äärettö - män pohdinta nousi useamman kerran koko luokan puheenaiheeksi . Myöhemmin opettajan pohtiessa oppilastöiden tuloksia , hän havaitsi niissä selkeästi tiedon luonteeseen kuten yksityisyyteen tai yleisyyteen liittyviä eroja . Jotkut kuvista oli helppo tulkita , jotkut odottivat tekijän avaavan niiden tausta - ajatusta . Jotkut työt esittivät myös selkeämmin yhteisesti tunnettua symbolijärjestelmää , kun taas toiset loivat niistä omia tulkintojaan . Opettaja päätyi järjestämään kuvat uudelleen koulun seinälle nelikentäksi : Yksityinen tieto – yleinen tieto ja Yhteisesti sovittu järjestys – oma mielikuva ( kuva 2 ) . Tämä tehtävä olisi sopinut hyvin myös osaksi yhteistä luokkatyöskentelyä ja keskustelua . Havinga & Portaankorva - Koivisto 17 Kuva 2 . Ensimmäisen teeman oppilastyöt järjestettynä nelikenttään Yksityinen tieto – yleinen tieto ja Yhteisesti sovittu järjestys – oma mielikuva . Havingan ( 2015 ) opetuskokeilun toisen teeman aiheena oli lajittelu ja luokittelu kuvataiteen työskentelytapana . Työskentely aloitettiin tällä kertaa kuvataiteen oppitunnilla . Tehtävä yhdisti ryhmässä tekemistä , nykytaiteen työmenetelmiä , valokuvausta , kokeilua , leikittelyä , sommittelua ja myös kuvankäsittelyohjelmaan tutustumista . Tunti aloitettiin tutustuen nykytaiteeseen ja sen työskentelymenetelmiin . Esimerkkinä toimi Antti Laitisen teos Forest Square ( www . anttilaitinen . com ) . Työssään taiteilija hajotti aarin metsää osiin eri materiaaleihin ja rakensi uuden tavan nähdä metsä . Oppilaiden tehtävänä oli hajottaa kasvi juurineen tai hedelmä eri tekstuureihin ja koota se uudestaan lajitellen ja luokitellen visuaalisesti kiinnostavaksi teokseksi . Koko prosessi valokuvattiin ja lisäksi jokainen tekstuuri mitattiin tilavuudeltaan ja punnittiin . Kuvakoosteessa ( kuva 3 ) nähdään eri työskentelyvaiheita . Kuva 3 . Esimerkkejä toisen teeman kuvataiteen oppilastöistä . MALU , Turku 2015 18 Työskentelyä jatkettiin matematiikan tunnilla , jossa mittausten perusteella laskettiin , kuinka monta grammaa kutakin tekstuuria oli verrattuna työn kokonaismassaan . Tulokset esitettiin sekä murtolukuina , desimaalilukuina että prosentteina . Lopuksi oppilaat esittivät työnsä koko luokalle ja yhdessä pohdittiin lajittelun ja luokittelun merkitystä matematiikassa . Perustuen näihin kokemuksiin kuvataidetta ja matematiikkaa integroivista ilmiöistä lähdimme selvittämään Helsingin yliopiston matematiikkaa pää - tai sivuaineenaan opiskeleville aineenopettajaopiskelijoille suunnatulla kyselyllä , mitä ovat matematiikan ilmiöt tulevien matematiikan opettajien näkemysten mukaan . Ainedidaktiikan perusteet - kurssin seminaarissa syksyllä 2015 eheyttämistä käsittelevän ryhmätapaamisen aloitukseksi opiskelijat saivat hetken pohtia aihetta itsekseen ja vastata kirjallisena avoimeen kysymykseen : Mitä mielestä - si ovat matemaattiset ilmiöt ? Opiskelijoille kerrottiin vastausten muodostavan tutkimusaineiston ja ettei lomaketta tarvitse palauttaa , mikäli ei halua osallistua tutkimukseen . Kahta opiskelijaa lukuun ottamatta ( 3 , 8 % ) kaikki palauttivat vastauksensa ( N = 51 ) . Seminaari jatkui eheyttämisteeman käsittelyllä ja opiskelijat saivat tutustua edellä esitettyihin opetuskokeiluihin vierailijana toimineen Mirka Havingan johdolla . Matematiikan opettajaopiskelijoiden vastaukset kirjoitettiin sähköiseen muotoon ja analysoitiin aineistolähtöisesti . Ensimmäisessä vaiheessa jokainen vastaus käytiin läpi etsien kuvaussanoja ( ks . esimerkki alla ) , joita aineistosta lopulta löytyi 52 kpl . # 23 En osaa antaa esimerkkiä tietystä ilmiöstä , mutta loogiseen päättelyyn voisi olla luonteva yhdistää . Arkkitehtuuri , kultaisen leikkauksen etsiminen luonnosta ja kuvataiteesta . Näistä muodostettiin taulukko ( taulukko 2 ) , jota käytettiin luokittelun apuna . Tähän taulukkoon listattiin kaiken kaikkiaan 202 poimintaa aineistosta . Havinga & Portaankorva - Koivisto 19 Taulukko 2 . Esimerkki matematiikan aineenopettajaopiskelijoiden vastausten analyysistä . Luku - määrä Kuvaussana Poiminnat aineistosta 5 mallintaminen matemaattisesti mallinnetut , tautien mallinta - minen , matemaattisesti mallintaa , voidaan mallintaa , voidaan mallintaa 7 taide taiteesta , kuvataiteesta , taide , kuvataiteesta , kuvataiteessa , Kuvataiteesta , kuvataiteessa 4 havainnot havaintoja , havaintojen välisiä eroja / yhtäläi - syyksiä , havainto , visuaalisesti havainnoitavaa Lopulta päädyttiin kolmeen lähes yhtä suureen kategoriaan :  matematiikan sisäiset ilmiöt ( 28 % )  matemaattiset ilmiöt luonnossa ( 34 % )  matemaattiset ilmiöt kulttuuriympäristössä ( 38 % ) . YHTEENVETO TULOKSISTA Kuvataidetta ja matematiikkaa eheyttävässä 7 . luokan opetuskokeilussa ( Havinga , 2015 ) etsittiin kahden teeman avulla keinoja säilyttää oppiaineiden itseisarvo ja eheyttää niitä mielekkäästi toisiinsa . Avoimet teemat ja proses - sinomainen työskentely ovat tyypillisiä kuvataiteen opetuksessa , mutta ensimmäisestä opetuskokeilun teemasta havaittiin , että avoimuuden lisääminen matematiikan opetukseen toi oppitunneille uudenlaista tutkivaa ja keskustelevaa ilmapiiriä . Kokonaislukujen tutkiminen lukujonoja muodosta - malla ja erilaisia perusteluja tutkimalla herätti oppilaita huomaamaan , miten saman lukujonon voi perustella eri tavoin , miten jotkut perustelut ovat matemaattisempia kuin toiset ja miten sääntö voidaan kirjoittaa yleiseen muotoon . Erityisesti äärettömyyteen liittyvät keskustelut olivat antoisia . Kuvataiteen oppitunnilla tehtävä johdatteli semioottiseen taidenäkemykseen . Oppitunnilla pohdittiin muun muassa sitä , millä tavoin kuvat viittaavat kulttuurissamme vallitseviin itsestäänselvyyksiin . Molemmissa tehtävissä oli erotettavissa sama ymmärtämisen polku : havaintojen tekeminen , oma työskentely , toisin näkeminen ja ymmärtäminen . Toisessa opetuskokeilun teemoista päädyttiin vastaavuuden ja representaati - on käsitteisiin , ja intuitioon toiminnan ohjaajana , jotka jälleen kerran yhdistä - vät matematiikkaa ja kuvataidetta keskenään . Matemaattinen ajattelu näyttäytyi selkeimmin juuri lajittelussa ja luokittelussa , vaikka oppilaat tunnistivatkin pikemminkin mittaukset ja laskemisen heille tutuksi matema - tiikaksi . Matematiikan tunnilla keskeinen käsitteellinen löytö oli suhteen MALU , Turku 2015 20 ymmärtäminen ”kuinka pieni kukka on suhteessa tarvitsemansa mullan määrään” . Prosenttiluvun , murtoluvun ja desimaaliluvun yhteys sen sijaan oli jo entuudestaan tuttu asia . Kiinnostavia keskusteluja heräsi myös siitä , mitä mittausten epätarkkuus saa aikaan ”näiden prosenttilukujen summaksi ei tuukaan 100 % ” . Matematiikan ilmiöitä kartoittavan kyselyn tuloksena ( ks . taulukko 3 ) eheyttäviin oppimiskokonaisuuksiin kannattaa matematiikan näkökulmasta valita ilmiöitä , jotka perustuvat matemaattisiin ominaisuuksiin , kuten rakenteet , säännönmukaisuudet , riippuvuudet ja suhteet , tai matemaattisiin prosesseihin , jotka ovat matematiikan tiedonhankinnalle ja toimintakulttuu - rille tyypillisiä , kuten looginen päättely , ongelmanratkaisu ja todistaminen . Taulukko 3 . Matematiikan aineenopettajaopiskelijoiden näkemyksiä siitä , mitä ovat matemaattiset ilmiöt . Kategoria Esimerkkejä ( lkm ) Yhteensä f % Matematiikan sisäiset ilmiöt Säännönmukaisuudet ( 9 ) , lineaarisuus , jaksollisuus , suppenevuus , äärettömyys ( 7 ) , muodot ( 6 ) , laskeminen , lukumäärä ( 6 ) , riippuvuudet ( 5 ) , looginen päättely ( 4 ) , symmetria ( 3 ) , ongelmanratkaisu ( 3 ) , rakenteet ja suhteet ( 3 ) , vektorit , avaruu - det ( 3 ) yleistäminen , yksittäistapaukset ( 3 ) jaollisuus ( 2 ) , todistaminen ( 1 ) lukujen ainutlaatuisuus ( 1 ) 56 28 % Matemaattiset ilmiöt luonnos - sa Tapahtumat , havainnot ( 17 ) , luonnon ilmiöt ( 15 ) , kemialliset ja fysikaaliset ilmiöt ( 12 ) , geometriset ilmiöt ( 10 ) , kultainen leikkaus ( 6 ) , kaaos ( 2 ) , fraktaalit ( 2 ) , tähtikuviot ( 1 ) , vuosirenkaat ( 1 ) , kasvit ( 1 ) , lumihiutaleet ( 1 ) , entropia ( 1 ) 69 34 % Matemaattiset ilmiöt kulttu - uriympäristössä Raha , talous , verotus , palkka ( 11 ) , musiikki , taide ( 10 ) , arjen ilmiöt ( 10 ) , todennäköisyydet , pelit ( 10 ) , mallit ( 8 ) , arkkitehtuuri ( 6 ) , mittaaminen , suureet ( 4 ) , rakentaminen ( 4 ) , ruoanlaitto ( 3 ) , tilastot ( 3 ) , yhteiskunnalliset ilmiöt ( 2 ) , sovellukset ( 2 ) , matkustus ( 2 ) , ammatit ( 1 ) , matematiikka historiallisena ilmiönä ( 1 ) 77 38 % Yhteensä 202 100 % Havinga & Portaankorva - Koivisto 21 Toinen kiinnostava lähestymistapa rakentaa eheyttäviä oppimiskokonaisuuk - sia on tutkia elollisessa luonnossa ja kulttuuriympäristössä ilmeneviä ilmiöitä , jotka ovat mallinnettavissa tai kuvailtavissa matematiikan keinoin . Ihminen myös itse tuottaa ympäristöön matemaattisia ilmiöitä , kuten musiikkia , arkkitehtuuria ja taidetta sekä hallitsee yhteiskunnallisia ja taloudellisia ilmiöitä matematiikan välinein . POHDINTA Kuvataidetta ja matematiikkaa eheyttäviä monitieteisiä oppimiskokonaisuuk - sia perusopetuksen 7 . - 9 . luokille suunniteltaessa tulee miettiä , miten se toteutetaan niin , että kummankin oppiaineen luonne ja tyypilliset tavat rakentaa tietoa huomioidaan tasavertaisina . Tiedonalapohjaisessa eheyttäväs - sä opetuksessa pedagogisesti hyvä ilmiö on ilmiö , jolle löytyy käsitetasolla liittymäkohtia sekä kuvataiteesta että matematiikasta , kuten opetuskokeilussa representaatio tai suhde . Lisäksi ilmiön on oltava riittävän avoin , jotta se mahdollistaa monenlaisten kysymysten , ratkaisujen ja käsittelytapojen käytön . Silloin oppilaiden tuottamat oivallukset ovat uusia opettajallekin ja oppilaiden ja opettajan yhteinen oppiminen yhdistää luokkaa ja motivoi oppimaan . Pedagogisesti hyviä ilmiöitä valittaessa matematiikan opettaja - opiskelijoiden vastauksista saatu kolmijako voi auttaa jäsentämään , onko valittu ilmiö matematiikan sisäistä eheyttämistä vai oppiaineita yhdistävää eheyttämistä . Monesti matematiikkaa ja kuvataidetta yhdistävät aiheet , jotka liittyvät luonnossa havaittaviin ilmiöihin ja rytmeihin tai ihmisen kulttuuriympäristön havainnointiin . Tutkimuksemme nostavat kuitenkin esiin ideoita myös matematiikan sisäisen logiikan pohtimiseen ja tarkasteluun . Tällaiset matemaattiset ilmiöt nostavat katseen pois laskutaidosta ja siirtävät sitä matematiikan havaitsemiseen ympärillämme . Monet käsitteet ja tiedonhan - kinnan ja - rakentelun keinot ovat yhteisiä eri oppiaineille . Eheyttämisen lähtökohta voi myös olla oppilaan oma kokemus ja yhteisölli - syys ( Halinen & Jääskeläinen , 2015 ) . Tutkiva oppiminen työtapana sopiikin hyvin monialaisiin oppimiskokonaisuuksiin . Esitellyssä opetuskokeilussa prosessin aikana seurattiin polkuja , joita yhteinen luokassa tapahtuva ihmettely nosti pintaan . Tällainen opiskelu vahvistaa ryhmän ja opettajan yhteistä päämäärää omiin havaintoihin perustuvaa oppimista kohti . Se , mitä lopulta opitaan voi olla yllättävää . Ensimmäisessä teemassa yllättävää oli , että lopulta päädyttiin käsittelemään tiedon luonteeseen liittyviä kysymyksiä . Toisessa teemassa yllätti se , että oppilaat eivät kokeneet lajittelun ja luokitte - lun liittyvän matematiikkaan . Toivomme , että esittelemämme matematiikan ilmiöiden luokittelu tukee opettajaa ja ryhmää oppimistilanteessa hahmotta - maan yllätysten laatua ja liittämään kysymyksiä osaksi kokonaisuutta . Se , miten tämän kaltainen opiskelu lisää esimerkiksi matematiikan käsitteiden MALU , Turku 2015 22 hallintaa matemaattisissa tehtävissä , on vaikeasti mitattava asia . Kiinnostavaa on kuitenkin se , miten kuvataiteen keinoin matematiikan käsitteitä voi avata ja niiden hallintaa vahvistaa liittämällä niihin omakohtaisia kokemuksia ja keskustelua . Uusi opetussuunnitelma ( 2014 ) , Marjo Räsäsen ( 2010 ) esittämä integroivan kuvataideopetuksen pedagoginen malli sekä Portaankorva - Koiviston ( 2010 ) elämyksellistä matematiikan opetusta jäsentävä malli kannustavat etsimään ilmiöiden välisiä suhteita , uusia näkökulmia , monilukutaitoa ja omaan oppimiseen liittyvää henkilökohtaista tarttumapintaa . Oppimisen pääpaino on tuoreiden näkökulmien löytymisessä ja uteliaisuuden ja kiinnostuksen herättelyssä . Eheyttävässä tiedonalalähtöisessä oppimiskokonaisuudessa opitaan matematiikkaa ja kuvataidetta , mutta sen lisäksi se haastaa oppilaita pohtimaan , mitä matematiikka tai kuvataide oikeastaan on . LÄHTEET Brown , T . ( 2001 ) . Mathematics education and language . Interpreting herme - neutics and post - structuralism . Netherlands : Kluwer Academic Publishers . Halinen , I . & Jääskeläinen , L . ( 2015 ) . Opetussuunnitelmauudistus 2016 . Sivistysnäkemys ja opetuksen eheyttäminen . Teoksessa H . Cantell . ( toim . ) Näin rakennat monialaisia oppimiskokonaisuuksia . Juva : Bookwell Oy . PS - kustannus . Havinga , M . ( 2015 ) . YHDESSÄ VAI ERIKSEEN ? Opetuskokeilu kuvataide - kasvatuksen ja matematiikan opetuksen integraatiosta perusopetuksen seitsemännellä luokalla . Taiteen maisterin opinnäytetyö . Aalto - yliopisto . Taiteiden ja suunnittelun korkeakoulu . Taiteen laitos , kuvataidekasvatus . Hurley , M . M . ( 2001 ) . Reviewing integrated science and mathematics : The search for evidence and definitions from new perspectives . School Science and Mathematics , 101 ( 5 ) , 259 - 268 . Hähkiöniemi , M . ( 2011 ) . Johdatus GeoGebra - avusteiseen tutkivaan matema - tiikkaan . Teoksessa M . Hähkiöniemi ( toim . ) GeoGebra - avusteinen tutkiva matematiikka opetusharjoittelussa : tutkimuksia opettajan ja oppilaiden toiminnasta . Jyväskylän yliopisto , opettajankoulutuslaitos . POPS . 2014 . Perusopetuksen opetussuunnitelman perusteet 2014 . Vahvistettu 22 . 12 . 2014 . Helsinki : Opetushallitus . http : / / www . oph . fi / download / 163777 _ perusopetuksen _ opetussuunnitel man _ perusteet _ 2014 . pdf Portaankorva - Koivisto , P . ( 2010 ) . Elämyksellisyyttä tavoittelemassa – narratiivinen tutkimus matematiikan opettajaksi kasvusta . Acta Universita - tis Tamperensis , 1550 . Tampereen yliopisto . Räsänen , M . ( 2010 ) . Taide , taitaminen ja tietäminen - kokonaisvaltaisen opetuksen lähtökohtia . Synnyt / Origins - verkkojulkaisu , 3 ( 2010 ) , 48 - 61 . Joutsenlahti & Kulju 23 AKATEEMINEN LUKUTAITO MATEMATIIKASSA Jorma Joutsenlahti & Pirjo Kulju Tampereen Yliopisto Judith Moschkovich on esittänyt kuvauksen akateemisesta lukutaidosta matematiikas - sa . Se koostuu kolmesta komponentista : matemaattisesta osaamisesta , matemaattisista käytänteistä ja diskursseista . Olemme kehittäneet tästä mallista oman mallin , jonka mukaan tarkastelemme Perusopetuksen opetussuunnitelman perusteiden matematii - kan osuuksia luokilla 1 - 9 . Kaikkien kolmen komponentin keskeiset sisällöt on löydettävissä matematiikan opetussuunnitelman perusteista . Pohdimme lopuksi akateemisen lukutaidon ja monilukutaidon käsitteiden suhdetta . JOHDANTO Lukutaitoa ( literacy ) on tulkittu ja tutkittu monesta eri näkökulmasta ja siihen liittyviä lähikäsitteitä on useita , esimerkiksi teknologinen lukutaito , informaa - tiolukutaito , verkkolukutaito , kuvanlukutaito , visuaalinen lukutaito ( Kupiai - nen , Kulju & Mäkinen 2015 ) . Eri näkökulmia yhdistäväksi yläkäsitteeksi on noussut monilukutaito . On syytä muistaa , että kyseessä on moninainen , paitsi lukemiseen myös kirjoittamiseen tai tuottamiseen liittyvä osaamisalue ( Kupiainen ym . 2015 ) . Käsitteen taustalla on englanninkielinen termi multiliteracy , jolla tarkoitetaan sitä , että erilaisissa sosiaalisissa konteksteissa on erilaisia tekstikäytänteitä ; nämä sosiaaliset kontekstit voivat liittyä esimerkiksi eri oppialoihin ( Kalantzis & Cope 2012 ) . Judith Moschkovich ( 2015a , 2015b ) puolestaan käyttää käsitettä akateeminen lukutaito ( academic literacy ) tarkastellessaan lukutaitoa matematiikan opiske - lussa . Tässä artikkelissa tulkitsemme Moschkovichin ( emt . ) mallia ja sovel - lamme sitä Perusopetuksen opetussuunnitelman perusteisiin matematiikassa . Lukutaidon moninaisuuden myötä monilukutaito nostettiin Perusopetuksen opetussuunnitelman perusteissa ( 2014 ) yhdeksi laaja - alaiseksi osaamiskoko - naisuudeksi . Koska monilukutaito on näin vahvasti esillä perusopetuksen opetussuunnitelman perusteissa 1 ( 2014 ) , tarkastelemme sitä seuraavassa tarkemmin ja pyrimme myöhemmin myös pohtimaan sen suhdetta Moschko - vichin akateemiseen lukutaitoon . Monilukutaito määritellään opetussuunni - telman perusteissa seuraavasti ( POPS 2014 , 20 ) : ” Monilukutaidolla tarkoitetaan erilaisten tekstien tulkitsemisen , tuottamisen ja arvottamisen taitoja , jotka auttavat oppilaita ymmärtämään monimuotoisia kult - tuurisia viestinnän muotoja sekä rakentamaan omaa identiteettiään . Monilukutai - to perustuu laaja - alaiseen käsitykseen tekstistä . Teksteillä tarkoitetaan tässä 1 Lyhennetään POPS MALU , Turku 2015 24 sanallisten , kuvallisten , auditiivisten , numeeristen ja kinesteettisten symbolijärjes - telmien sekä näiden yhdistelmien avulla ilmaistua tietoa . ” Oppilaiden monilukutaidon kehittäminen lähtee arkikielen käytöstä kohti kunkin oppiaineen tiedonalan kielen ja esitystapojen hallintaa . Monilukutai - toa opitaan käyttämällä , tulkitsemalla ja tuottamalla erilaisia tekstejä sekä yksin että yhdessä muiden kanssa ( emt . , 21 ) . Matematiikan opiskelussa oppilas kohtaa muun muassa seuraavia symbolijärjestelmiä matematiikkaan liittyvissä teksteissä : symbolikieltä , luonnollista kieltä ( yleensä äidinkieltään ) ja kuvioita . Näiden avulla matematiikan teksteissä , kuten oppikirjoissa , luodaan merkityksiä matemaattisille käsitteille . Toisaalta oppilas voi eri symbolijärjestelmiä käyttämällä ilmaista matemaattista ajatteluaan suullisesti tai kirjallisesti eli kielentää ( ks . Joutsenlahti & Kulju 2015 ) . Moschkovich kuvaa akateemisen lukutaidon matematiikassa laajemmin kuin vain semiotiikan teorian lähtökohdista ( Moschkovich 2015a , 2015b ; vrt . Kupiainen ym . 2015 , 17 ) . Hän nimittäin rakentaa matemaattisen toiminnan kuvauksen akateemisessa lukutaidossa kolmesta näkökulmasta : kognitiivises - ta , sosiokulturaalisesta ja diskursiivisesta näkökulmasta ( Moschkovich 2015a , 77 ) . Tarkastelemme seuraavassa Moschkovichin ( 2015a , 2015b ) akateemisen lukutaidon mallia ja esittelemme sen pohjalta kehittelemäämme mallia , jossa näkökulmana on oppilas ja hänen matemaattinen ajattelunsa . Tutkimustehtä - vämme on tarkastella Perusopetuksen opetussuunnitelman perusteiden ( 2014 ) matematiikan tavoitekuvauksia kaikilla luokkatasoilla mukaillun akateemisen lukutaidon näkökulmasta . AKATEEMINEN LUKUTAITO Moschkovich ( 2015a , 2015b ) kuvaa akateemisen lukutaidon matematiikassa koostuvan kolmesta toisiinsa integroituneesta komponentista : matemaattises - ta osaamisesta ( mathematical proficiency ) , matemaattisista käytänteistä ( mathematical practices ) ja matemaattisesta diskurssista ( mathematical discourse ) . Matemaattisen toiminnan tarkastelun näkökulmista matemaattinen osaami - nen edustaa kognitiivista , matemaattiset käytänteet sosiokulturaalista ja viimeksi mainittu diskursiivista näkökulmaa . Sosiokulturaalisessa viitekehyksessä oppimista tarkastellaan asteittain syvenevänä osallistumisen prosessina , jonka aikana opitaan käyttämään yhteisön materiaalisia ( esimerkiksi kynät , kompassit , tietokoneet ) ja psykolo - gisia työkaluja ( esimerkiksi käsitteet , symbolit , erilaiset kielet ) ( Rajala , Hilppö , Kumpulainen , Tissari , Krokfors & Lipponen 2010 ) . Esimerkiksi matematiikan sanallisen tehtävän ratkaisussa oppilaan toiminnan kuvaamiseen ei riitä vain kognitiivinen näkökulma , jota on muun muassa matemaattinen päättely ja proseduraalinen sujuvuus . Edellä mainittujen lisäksi oppilaalla on oltava lisäksi kompetenssia tunnistaa tekstilaji , ymmärtää lukemansa teksti ja nähdä siitä ne merkitykselliset tiedot , jotka ohjaavat ratkaisustrategioiden käyttöä Joutsenlahti & Kulju 25 ( Moschkovich 2015a ) . Toisin sanoen oppilas ensin tunnistaa esimerkiksi matematiikan sanallisen tehtävän tekstilajina ja ymmärtää sen sisällön , joka voi muodostua luonnollisesta kielestä , symbolikielestä ja kuvioista . Näin ollen hän hallitsee oppiaineelle tyypillistä diskurssia ja käyttää metakognitii - visia taitojaan ratkaistessaan tehtävää . Moschkovich ( 2015a , 2015b ) tarkastelee akateemisen lukutaitoa matematiikas - sa oppilailla , joilla matematiikan opetus on englanninkielistä , mutta oppilai - den äidinkieli on jokin muu . Kuvattu käsite sopii kuitenkin hyvin myös oppilaan äidinkielellä annetun matematiikan opetuksen kuvaukseen . Matemaattinen osaaminen Moschkovich ( 2015a ) on todennut vuonna 2001 julkaistun matemaattisen osaamisen mallin olevan edelleen pätevä kuvaamaan akateemisen lukutaidon kognitiivista aluetta . Tämä Kilpatrickin , Swaffordin ja Findellin ( 2001 ) malli esittää matemaattisen osaamisen ( mathematical proficiency ) koostuvan viidestä piirteestä : 1 . käsitteellinen ymmärtäminen , 2 . proseduraalinen sujuvuus , 3 . strateginen kompetenssi , 4 . mukautuva päättely ja 5 . yritteliäisyys ( ks . myös Joutsenlahti 2005 , 96 ) . Käsitteellinen ymmärtäminen sisältää ymmärryksen matemaattisista käsitteistä ja niiden välisistä suhteista sekä matemaattisista operaatioista ja niiden yhteyksistä matemaattisiin käsitteisiin ( Kilpatrick ym . 2001 , Joutsen - lahti 2005 ) . Käsitteellinen ymmärtäminen näkyy niistä merkityksistä , mitkä matemaattisen tehtävän ratkaisija antaa ratkaisulle ( Mitä tulos tarkoittaa tehtävänannon näkökulmasta ? ) , ratkaisuprosessille ( Miksi valitut proseduurit ja menetelmät toimivat tässä ratkaisussa ? ) ja lopulliselle vastaukselle ( Miksi vastaus on oikein tähän tehtävään ? ) ( Moschkovich 2015a ) . Proseduraalinen sujuvuus näkyy taitona käyttää matemaattisia proseduureja joustavasti , huolellisesti , tehokkaasti ja tarkoituksenmukaisesti ( Kilpatrick ym . 2001 , Joutsenlahti 2005 ) . Koulussa korostuu erityisesti aritmetiikassa mekaanisen laskemisen osuus , mutta käsitteelliseen ymmärtämiseen liittyy usein myös oleellisena osana laskennallinen osaaminen ja toisaalta myös päinvastoin ( Moschkovich 2015a ) . Strateginen kompetenssi on kykyä muotoilla , esittää ja ratkaista matemaattisia ongelmia , jotka eivät ole rutiinin - omaisia ( Kilpatrick ym . 2001 , Joutsenlahti 2005 ) . Ongelmanratkaisussa edellä kuvattu osaamisen piirre on keskeinen . Mukautuva päättely on loogista ajattelua , pohdintaa , selityksien löytymistä ja todistamista ( emt . ) . Viimeisenä mainittu matemaattisen osaamisen piirre ”yritteliäisyys” kuvastaa oppijan käsityksiä matematiikan tärkeydestä ja hyödyllisyydestä sekä omasta ahkeruudesta ja tehokkuudesta matematiikan opiskelussa ( emt . ) . Korvaamme sen tässä yhteydessä käsitteellä matematiikkakuva ( view of mathematics ) , jota muun muassa Joutsenlahti ( 2005 ) on käyttänyt . MALU , Turku 2015 26 Edellä kuvatut viisi matemaattisen osaamisen piirrettä muodostavat akatee - misen lukutaidon kognitiivisen komponentin , jossa on kuitenkin runsaasti yhtymäkohtia kahteen muuhun komponenttiin . Matemaattiset käytänteet Yhdysvalloissa Common Core State Standards 2 ( 2010 ) määrittelee kahdeksan matemaattista käytännettä ( mathematical practices ) , joita voidaan tulkita matematiikan opetuksessa esiopetuksesta lukio - opetukseen asti . Kuvatut käytänteet ohjaavat oppilaita matemaattisten ongelmien ratkaisuprosessien aloittamiseen ja hallintaan . Matemaattiset käytänteet ovat ( CCSS 2010 ) : 1 . ymmärrä ongelmat ja ole pitkäjänteinen etsiessäsi ratkaisua , 2 . päättele abstraktisesti ja kvantitatiivisesti , 3 . perustele oma ratkaisusi muille ja ole rakentavan kriittinen erilaisiin ratkaisuihin , 4 . mallinna matematiikalla , 5 . käytä asianmukaisia työkaluja strategisesti , 6 . pyri täsmällisyyteen , 7 . etsi ja käytä rakenteita ja 8 . etsi ja ilmaise säännönmukaisuuksia toistuvassa päättelyssä . Ensimmäisenä tavoitteena on annetun ongelman ymmärtäminen , jolloin matemaattisesti osaava oppilas aloittaa ongelman ratkaisun selittämällä ensin itselleen ongelman merkitykselliset piirteet ratkaisun kannalta . Oppilas käyttää tyypillisiä ongelmanratkaisumenetelmiä ( esimerkiksi analogisten ongelmien ratkaisut , erikoistapausten ratkaiseminen ensin , jne . ) johdonmu - kaisesti ja pitkäjänteisesti samalla tarkastellen ja arvioiden omaa ratkaisupro - sessiaan . Toiseksi oppilaan on tehtävä abstrakteja ja määrällisiä päätelmiä tehtävästä . Osaava oppilas ymmärtää tehtävässä annettujen lukujen tai muuttujien merkityksen ja niiden suhteet toisiinsa tehtävässä . Hänellä on taitoa kontekstualisoida tehtävä uudelleen siten , että hän osaa kuvata tehtävässä annetut tiedot ja niiden välisiä suhteita matemaattisin symbolein sekä sieventää lausekkeita ja ratkaista yhtälöitä . ( CCSS 2010 , Moschkovich 2015a . ) Kolmantena on taito kuvata ja perustella omat valinnat ja ratkaisuprosessin vaiheet sekä esittää omat perustellut näkemykset toisten oppilaiden ratkai - suista ( CCSS 2010 ) . Suomalaisessa ainedidaktisissa tutkimuksissa on tässä yhteydessä käytetty kielentämisen käsitettä ( esimerkiksi Joutsenlahti & Kulju 2012 , Joutsenlahti & Rättyä 2015 ) . Neljäntenä ja viidentenä ovat taidot mallintaa annettuja tilanteita matemaattisesti ja käyttää asianmukaisia matemaattisia työkaluja ongelmien ratkaisussa strategisesti mielekkäällä tavalla . Esimerkiksi tietotekniikan ( esimerkiksi graafiset laskimet ja tietoko - neet ) hyödyntäminen ensin kuvaajia tutkimalla ja niiden perusteella varsinai - sen ratkaisun konstruointi algebrallisin menetelmin on yläkoulussa ja lukiossa keskeinen taito . 2 Lyhennetään CCSS . Joutsenlahti & Kulju 27 Kolmessa viimeksi mainitussa tavoitteessa oppilas pyrkii aina ilmaisemaan itsensä täsmällisesti kuvatessaan ratkaisuaan tai keskustellessaan muiden oppilaiden kanssa . Ratkaisua etsiessään hän pyrkii löytämään tehtävästä selkeitä rakenteita ja hyödyntämään niitä ratkaisussaan . Toistaessaan ratkaisuprosessin yhteydessä samaa proseduuria hän pyrkii löytämään toistoista säännöllisyyttä ja keksiä yleisen tavan tehdä proseduuri lyhyemmin halutuilla arvoilla . ( CCSS 2010 , Moschkovich 2015a . ) Edellä kuvatut kahdeksan tavoiteltavaa käytännettä matemaattisen ongelman ratkaisussa kuvaavat matemaattisesti osaavan oppilaan ongelmanratkai - suprosessin toimintoja , joita ohjaa oppilaan matemaattinen ajattelua kuvaa - van matemaattisen osaamisen piirteiden tasapainoinen kehitys ( vrt . Joutsen - lahti 2005 ) . Kuvassa 1 olemme jakaneet edellä mainitut kahdeksan käytännet - tä hienojakoisemmin kymmeneksi oppilaan matemaattista toimintaa edesauttavaksi käytänteeksi , jotka auttavat erityisesti oppilaan matemaattis - ten ongelmien ratkaisussa . Matemaattisen ajattelun ilmaisu Moschkovichin ( 2015a , 2015b ) mallissa kolmantena komponenttina mainitaan diskurssi . Hän korostaa sitä , että matemaattinen diskurssi on laajempi käsite kuin matematiikan opiskelussa käytetty kieli . Kielitoimiston sanakirja määrittää diskurssin jotakin alaa koskevien vuorovaikutustapojen kokonai - suudeksi . Moschkovich ( 2015a , 83 ) määrittelee matemaattisen diskurssin vielä tarkemmin sellaiseksi kommunikaatiokompetenssiksi , joka mahdollistaa osallisuuden matemaattisiin käytänteisiin . Hänen mukaansa matemaattinen diskurssi sisältää suullisten ja kirjoitettujen tekstien lisäksi myös monia moodeja ( tai symbolijärjestelmiä ) , kuten eleitä , toimintamateriaaleja , piirroksia , taulukoita , kuvaajia , matemaattisia symbole - ja jne . Vuorovaikutukseen kuuluvat myös erilaiset rekisterit , kuten kouluma - tematiikan kieli ja toisaalta kotikieli . ( Moschkovich 2015a , 77 . ) Moschkovich ( 2015a , 83 ) korostaa , että matemaattisen diskurssin määrittelyssä tulisi välttää vastakkainasettelua formaalin matematiikan kielenkäytön , kuten käsitteiden oppikirjamääritteiden , sekä oppilaan oman arjen rekisterin välillä . Tarkasteltaessa akateemista lukutaitoa ja sen komponentteja olemme kuitenkin valinneet keskiöön matematiikkaa opiskelevan oppilaan ja diskurssin sijasta nimenneet kolmannen komponentin " matemaattisen ajattelun ilmaisuksi " ( Kuva 1 ) . Toisin sanoen matemaattisen osaamisen piirteet kuvaavat mainitun oppilaan kognitiivista potentiaalia ja oppilaalle annettujen matemaattisten käytäntei - den omaksuminen kuvaa matemaattista toimintaa opiskelu - ja ratkaisupro - sesseissa . Kuvassa 1 esitetty mallimme kolmas komponentti , matemaattisen ajattelun ilmaisu , ikään kuin ohjaa edellä mainittua toimintaa . Sen taustalla MALU , Turku 2015 28 on oppilaan matemaattinen ajattelu , joka rakentuu olemassa olevien tietojen ja taitojen rajoissa ( ks . Joutsenlahti 2005 ) . Tähän ajattelun rakentumiseen liittyy myös ajattelun ilmaisu , jossa oppilas kielentää ajatteluaan matematii - kalle tyypillisellä tavalla monipuolisesti kieliä hyödyntäen ( esim . Joutsenlahti & Rättyä 2015 ) . Matemaattista toimintaa ohjaa myös kommunikaatio muiden oppilaiden kanssa , ja tässä vuorovaikutuksessa voi myös syntyä matemaattis - ta tietoa ja ymmärrystä . Lukutaidon näkökulmasta oppilas ilmaisee matemaattista ajatteluaan luonnollisen kielen , matematiikan symbolikielen , kuviokielen ja / tai taktiili - sen 3 toiminnankielen kautta ( Joutsenlahti & Rättyä 2015 , Joutsenlahti & Kulju 2015 ) . Moschkovichin ( 2015a , 2015b ) käyttämän diskurssin piirteistä esimer - kiksi erilaiset luonnollisen kielen rekisterit voivat olla osa matemaattisen ajattelun ilmaisua . Niin ikään matemaattista ajattelua voidaan ilmaista erilaisia symbolijärjestelmiä ( tai kieliä ) käyttämällä erilaisissa teksteissä , esimerkiksi sanallisissa tehtävissä , kertomuksissa tai oppitunnin suullisissa esitystilanteissa ( Joutsenlahti , Kulju & Tuomi 2012 ) . Kuva 1 . Akateemisen lukutaidon komponentit matematiikan opiskelussa oppilaan näkökulmasta Moschkovichia ( 2015a , 2015b ) ja Joutsenlahtea mukaillen ( Joutsenlahti & Rättyä 2015 , Joutsenlahti & Kulju 2015 ) 3 tuntoaistiin perustuva ( engl . tactile ) Matemaattinen osaaminen • Käsitteellinen ymmärtäminen • Proseduraalinen sujuvuus • Strateginen kompetenssi • Mukautuva päättely • Matematiikkakuva Matemaattiset käytänteet ongelman ratkaisussa • Merkitysten ymmärtäminen • Pitkäjänteisyys • Abstrakti päättely • Ratkaisun perustelu • Rakentava kritiikki • Matematiikalla mallintaminen • Asianmukaiset työkalut • Täsmällisyys • Rakenteet • Säännönmukaisuu - det Matemaattisen ajattelun ilmaisu • Kielentäminen : • Luonnollinen kieli ( suullinen tai kirjallinen ) • Matematiikan symbolikieli • Kuviokieli • Taktiilinen toiminnankieli Joutsenlahti & Kulju 29 AKATEEMINEN LUKUTAITO MATEMATIIKAN OPETUSSUUNNI - TELMAN PERUSTEISSA Tarkastelemme akateemisen lukutaidon komponentteja Perusopetuksen opetussuunnitelman perusteissa ( 2014 ) matematiikassa luokkatasoilla 1 - 9 . Matemaattisen osaamisen viisi piirrettä ovat löydettävissä mainittujen luokkien ( luokat 1 - 2 , 3 - 6 ja 7 - 9 ) tavoitteista . Tutkimusmenetelmämme on lähinnä teoriasidonnainen sisällönanalyysi , jossa aineistona on POPS ( 2014 ) tavoitteet matematiikan osalta . Aineistoa tarkastellaan Kuvan 1 akateemista lukutaitoa kuvaavien komponenttien mukaisesti . Matemaattinen osaaminen Taulukkoon 1 olemme koonneet esimerkkejä matematiikan viidestä osaami - sen piirteestä opetussuunnitelman perusteissa eli Kuvan 1 ensimmäisestä komponentista . Esimerkiksi 1 . ja 2 . luokkien oppimisprosessin kannalta keskeisissä arvioinnin ja palautteen antamisen kohteissa neljä kognitiivista piirrettä , jotka seuraavassa on merkitty sulkuihin , näkyvät seuraavasti : 1 ) lukukäsitteen ymmärtämisessä ja lukujonotaidoissa ( käsitteellinen ymmärtä - minen ) , 2 ) kymmenjärjestelmän ymmärtämisessä ( käsitteellinen ymmärtämi - nen ) , 3 ) laskutaidon sujuvuudessa ( proseduraalinen sujuvuus ) , 4 ) kappalei - den ja kuvioiden luokittelun taidoissa ( mukautuva päättely ) ja 5 ) matematii - kan käyttämisessä ongelmanratkaisussa ( strateginen kompetenssi ) . Taulukko 1 . Esimerkkejä matemaattisen osaamisen piirteistä perusopetuksen opetussuunnitelmien ( 2014 ) matematiikan tavoitteissa ( Kirjainlyhenne T yhdistettynä lukuun viittaa opetussuunnitelman tavoitteiden indeksointiin , ks . POPS 2014 ; 134 – 135 , 260 – 261 , 428 – 429 ) Matemaattinen osaaminen lk 1 - 2 lk 3 - 6 lk 7 - 9 Käsitteel - linen ymmär - täminen T10 ohjata oppilasta ymmärtämään mittaamisen periaate ( T5 , T6 , T7 ) T2 ohjata oppilasta havaitsemaan yhteyksiä oppimiensa asioiden välillä ( T8 , T9 ) T12 tukea oppilasta laajentamaan lukukäsitteen ymmärtämistä reaalilukuihin ( T13 , T14 , T15 , T16 , T17 ) Prosedu - raalinen sujuvuus T8 ohjata oppilasta kehittämään sujuvaa peruslaskutaitoa luonnollisilla luvuilla … ( T9 , T11 , T12 ) T10 opastaa oppilasta saavuttamaan sujuva laskutaito päässä ja kirjallisesti hyödyntä - en laskutoimitusten ominaisuuksia T11 ohjata oppilasta kehittämään kykyään laskea peruslaskutoi - mituksia rationaalilu - vuilla ( T14 , T18 , T19 ) MALU , Turku 2015 30 Strategi - nen kompe - tenssi T4 ohjata oppilasta kehittämään päättely - ja ongelmanratkaisu - taitojaan T5 ohjata ja tukea oppilasta ongelman - ratkaisutaitojen kehittämisessä T5 tukea oppilasta loogista ja luovaa ajattelua vaativien matemaattisten tehtävien ratkaisemi - sessa… ( T9 ) Mukautuva päättely T12 harjaannuttaa oppilasta laatimaan vaiheittaisia toiminta - ohjeita ja toimimaan ohjeen mukaan ( T2 ) T3 ohjata oppilasta kehittämään taitoaan esittää kysymyksiä ja tehdä perusteltuja päätelmiä havainto - jensa pohjalta ( T4 , T6 , T11 , T12 , T13 , T14 ) T20 ohjata oppilasta kehittämään algoritmista ajatteluaan sekä taitojaan soveltaa matematiikkaa ja ohjelmointia ongelmien ratkaisemi - seen ( T6 , T7 , T10 ) Matema - tiikkakuva T1 tukea oppilaan innostusta ja kiinnos - tusta matematiikkaa kohtaan sekä myönteisen minäkuvan ja itseluottamuksen kehittymistä T1 pitää yllä oppilaan innostusta ja kiinnostusta matematiikkaa kohtaan sekä tukea myönteistä minäku - vaa ja itseluottamusta T1 vahvistaa oppilaan motivaatiota , myönteistä minäkuvaa ja itseluottamusta matematiikan oppijana Matemaattiset käytänteet Kuvassa 1 on lueteltu kymmenen matemaattisen ongelman ratkaisuun ja sen esittämiseen liittyvää keskeistä toimintoa , joita voi pitää käytänteinä mate - maattisissa ratkaisuprosesseissa . Kolmea ensiksi mainittua käytännettä ( ”ymmärrä annettu ongelma ja ole pitkäjänteinen etsiessäsi ratkaisua sekä päättele abstraktisti” ) ei ole suoraan löydettävissä tavoitteista luokilta 1 - 2 , 3 - 6 ja 7 - 9 . Neljäntenä mainittu käytänne ”perustele oma ratkaisusi muille” on löydettävissä esimerkiksi luokkien 3 - 6 tavoitteissa ( POPS 2014 , 260 ) : T4 kannustaa oppilasta esittämään päättelyään ja ratkaisujaan muille konkreettisin välinein , piirroksin , suullisesti ja kirjallisesti myös tieto - ja viestintäteknologiaa hyödyntäen . Lisäksi se tulee esille myös luokkien 1 - 2 ( T3 ) sekä luokkien 7 - 9 tavoitteissa ( T3 ja S1 ) ( POPS 2014 ; 135 , 375 ) . Viidentenä olevaa rakentavaa kritiikkiä ei ole erikseen mainittu , mutta se sisältyy yleisiin tavoitteisiin . Kuudentena Kuvassa 1 oleva ”matematiikan avulla mallintaminen” tulee selkeimmin esille luokkien 7 - 9 tavoitteissa ( POPS 2014 , 374 – 375 ) : T7 rohkaista oppilasta soveltamaan matematiikkaa muissakin oppiaineissa ja ympäröivässä yhteiskunnassa ja T20 ( - - ) s oveltaa matematiikkaa ja ohjelmointia ongelmien ratkaisemiseen . Asianmukaisten työkalujen käyttö tulee esille luokkien 7 - 9 tavoitteissa ( emt . , 429 ) : T9 opastaa oppilasta soveltamaan tieto - ja viestintäteknolo - giaa matematiikan opiskelussa sekä ongelmien ratkaisussa . Tässä tuodaan esille Joutsenlahti & Kulju 31 teknologian tarkoituksenmukainen käyttö osana ratkaisuprosessia , mikä tulee olemaan keskeinen taito myös lukiomatematiikassa . Kahdeksantena on kehotus täsmälliseen ilmaisuun matematiikassa . Tämäkin tulee esille vasta luokkien 7 - 9 tavoitteissa ( emt . , 429 ) : T4 kannustaa oppilasta harjaantumaan täsmälliseen matemaattiseen ilmaisuun suullisesti ja kirjallisesti . Tämä lienee perusteltu linjaus , sillä luokilla 1 - 6 matemaattisen ajattelun kehittymisen näkökulmasta on tärkeää ilmaista ajattelua ilman erillistä täsmällisyyden vaadetta . Jo pelkkä omin sanoin ilmaisu niin suullisesti ja kirjallisesti kehittää paitsi ilmaisijan ajattelua ja myös itse ilmaisua merkittä - västi . Kahta viimeistä käytännettä kuvaa POPS : ssa ( 2014 ) sisältöalue S1 ajattelun taidot . Esimerkiksi luokkien 3 - 6 ajattelun taidoissa kuvataan mate - maattisten rakenteiden ja säännönmukaisuuksien löytämisen taitoa ( emt . , 235 ) : Kehitetään oppilaiden taitoja löytää yhtäläisyyksiä , eroja ja säännönmukaisuuk - sia . Vastaava kuvaus löytyy kaikilta muiltakin luokka - asteilta . Matemaattisen ajattelun ilmaisu Matemaattisen ajattelun ilmaisun merkitys eri kielillä ( ks . Kuva 1 ) tulee selkeästi esille matematiikan osuuksissa . On huomattava , että ajattelun ilmaisu ( kielentäminen ) itsessään voi myös kehittää oppilaan omaa ajattelua eteenpäin . Tämä tukee opetussuunnitelmassa mainittua oppiaineen opetuk - sen tehtävää : ”kehittää oppilaiden loogista , täsmällistä ja luovaa matemaattis - ta ajattelua . ” ( POPS 2014 , 128 ) . Matemaattisen ajattelun ilmaiseminen Kuvan 1 neljän kielen avulla näkyy luokka - asteilla seuraavasti : 1 ) 1 . - 2 . lk : Opetus kehittää oppilaiden kykyä ilmaista matemaattista ajattelu - aan konkreettisin välinein , suullisesti , kirjallisesti ja piirtäen sekä tulkiten kuvaa” ( emt . , 128 ) 2 ) 3 . - 6 . lk : Opetus kehittää oppilaiden taitoja esittää matemaattista ajatteluaan ja ratkaisujaan eri tavoilla ja välineillä ( emt . , 234 ) ja 3 ) 7 . - 9 lk : Oppilaita ohjataan esittämään ratkaisujaan ja keskustelemaan niistä ( emt . , 428 ) . Kaikilla luokka - asteilla korostetaan oppimista vuorovaikutuksessa muun ryhmän kanssa , jolloin keskeisenä on oma argumentointitaito ja rakentavan kritiikin antamisen taito ( vrt . matemaattiset käytänteet Kuva 1 ) . Esimerkiksi luokkien 3 - 6 tavoite T4 kannustaa oppilasta esittämään päättelyään ja ratkaisujaan muille konkreettisin välinein , piirroksin , suullisesti ja kirjallisesti myös tieto - ja viestintäteknologiaa hyödyntäen ( POPS 2014 , 260 ) ja vastaavat tavoitteet muilla luokka - asteilla kuuluvat matemaattisten käytänteiden lisäksi myös matemaattisen ajattelun ilmaisun komponenttiin . MALU , Turku 2015 32 POHDINTA Olemme tässä tutkimuksessa tarkastelleet perusopetuksen opetussuunnitel - man perusteita akateemisen lukutaidon näkökulmasta matematiikassa . Akateeminen lukutaito on erittäin laaja käsite , jonka voidaan ajatella kattavan sekä osaamisen , ongelman ratkaisun käytänteet että siihen liittyvän diskurs - sin . Diskurssi puolestaan liittyy laaja - alaisesti erilaisissa konteksteissa käytyyn vuorovaikutukseen . Olemme tässä tutkimuksessa fokusoineet näkökulmaa oppilaan ajatteluun ja sen kautta saaneet esille nämä kolme näkökulmaa opetussuunnitelman perusteissa . Näin ollen opetussuunnitelma näyttää tukevan akateemisen lukutaidon muokattua malliamme matematii - kassa . Erityisen hyvin malli näyttäisi soveltuvan matemaattisen ongelmarat - kaisuprosessien kuvaukseen kaikilla perusopetuksen luokka - asteilla . Akateemisen lukutaidon malli syventää näkemystä siitä , mitä monilukutaito saattaisi merkitä , jos sitä tarkasteltaisiin tarkemmin yhden oppiaineen näkökulmasta . Matematiikan kannalta keskiöön nousee oppiaineen tietosisäl - lön oppiminen , erilaiset oppimisprosesseihin liittyvät käytänteet sekä matemaattisen ajattelun ilmaisu . Saattaa olla , että monilukutaidon käsite ei itsessään kuitenkaan riitä kuvaamaan tätä akateemista oppimisprosessin puolta kokonaisuudessaan , vaikka käsitteen määritelmissä ( kuten Luukka 2013 ; Kalantzis & Cope 2012 ) mainitaankin oppiaine - tai tieteenalakohtaiset lukutaidot ja tavat ilmaista asioita . On myös huomattava ( vrt . Kupiainen ym . 2015 ) , että opetussuunnitelmassa on esillä myös oppimisprosesseihin liittyvä näkökulma monilukutaidosta ; mainitaanhan siinä , että ”monilukutaito tukee kriittisen ajattelun ja oppimisen taitojen kehittymistä” ( POPS 2014 , 21 ) . Kaiken kaikkiaan monilukutaito tarkoittanee matematiikan kannalta sitä , että oppilas hallitsee matematiikan oppiaineelle tyypilliset tavat ilmaista asioita ; hän tunnistaa esimerkiksi oppikirjojen merkintätavat ja osaa itse tuottaa ja tulkita oppiaineeseen liitettyjä tekstejä . Näin ollen monilukutaito menee lähimmäksi mallimme kolmatta komponenttia , matemaattisen ajattelun ilmaisua . Monilukutaidon ja akateemisen lukutaidon mallin suhdetta kuvannee parhaiten se , että matemaattinen osaaminen ja käytänteet ikään kuin suodat - tuvat monilukutaidon läpi matemaattisissa ajatteluprosesseissa . LÄHTEET Common Core State Standards ( 2010 ) . 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Tainio ( toim . ) Ainedidaktinen tutkimus koulutuspoliittisen päätöksenteon perustana . Ainedidaktisia tutkimuksia 4 . ( s . 107 – 122 ) . Helsinki , Suomi : Suomen ainedidaktinen tutkimusseura . Joutsenlahti , J . & Rättyä , K . ( 2015 ) . Kielentämisen käsite ainedidaktisissa tutkimuksissa . Teoksessa M . Kauppinen , M . Rautiainen & M . Tarnanen ( toim . ) Rajaton tulevaisuus . Kohti kokonaisvaltaista oppimista . Ainedidaktiikan symposium Jyväskylässä 13 . – 14 . 2 . 2014 . Ainedidaktisia tutkimuksia 8 . ( s . 45 – 61 ) . Jyväskylä , Suomi : Suomen ainedidaktinen tutkimusseura . Kalantzis , M . & Cope , B . ( 2012 ) . Literacies . Cambridge , UK : Cambridge University Press . Kilpatrick , J . , Swafford , J . , & Findell , B . ( toim . ) ( 2001 ) . Adding it up . Washing - ton , DC , USA : National Academy Press . Kupiainen , R . , Kulju , P . & Mäkinen , M . ( 2015 ) . Mikä monilukutaito ? Teokses - sa T . Kaartinen ( toim . ) Monilukutaito kaikki kaikessa ( s . 13 – 24 ) . Tampere , Suomi : Tampereen yliopiston normaalikoulu . Luukka , M . - L . ( 2013 ) . Opetussuunnitelmat uudistuvat : tekstien lukijasta ja kirjoittajasta monilukutaituriksi . Kielikoulutuspolitiikan verkosto , 10 . 12 . 2013 . < http : / / www . kieliverkosto . fi / article / opetussuunnitelmat - uudistuvat - tekstien - lukijasta - ja - kirjoittajasta - monilukutaituriksi / > ( Luettu 12 . 2 . 2016 ) . Moschkovich , J . N . ( 2015a ) . A sociocultural approach to academic literacy in mathematics for adolescent English learners : Integrating mathematical proficiency , practices , and discourse . Teoksessa D . Molle , E . Sato , T . Boals & C . A . Hedgspeth ( toim . ) , Multilingual learners and academic literacies : Sociocultural contexts of literacy development in adolescents ( s . 75 – 104 ) . New York , USA : Routledge . Moschkovich , J . N . ( 2015b ) . Academic literacy in mathematics for English learners . Journal of Mathematical Behavior . In press . < http : / / dx . doi . org / 10 . 1016 / j . jmathb . 2015 . 01 . 005 > ( Luettu 20 . 1 . 2016 ) Perusopetuksen opetussuunnitelman perusteet 2004 . Helsinki , Suomi : Opetushallitus . < http : / / www . oph . fi / download / 139848 _ pops _ web . pdf > ( Luettu 18 . 1 . 2016 ) MALU , Turku 2015 34 Perusopetuksen opetussuunnitelman perusteet 2014 . Helsinki , Suomi : Opetushallitus . < http : / / www . oph . fi / ops2016 / perusteet > ( Luettu 18 . 1 . 2016 ) Rajala , A . , Hilppö , J . , Kumpulainen , K . , Tissari , V . , Krokfors , L . & Lipponen , L . , 2010 . Merkkejä tulevaisuuden oppimisympäristöistä . OPH Raportit ja selvi - tykset 2010 : 3 . Helsinki , Suomi : Opetushallitus . < http : / / www . oph . fi / download / 125495 _ Merkkeja _ tulevaisuuden _ oppimi symparistoista . pdf > ( Luettu 18 . 5 . 2016 ) Kokkonen & Nousiainen 35 LEARNING PHYSICS CONCEPTS – A DESCRIPTION IN TERMS OF RELATIONAL CONCEPTS Tommi Kokkonen & Maija Nousiainen University of Helsinki Learning physics is a demanding and lengthy process . One reason for this is that students’ prior knowledge is often at odds with scientific knowledge . However , the nature of students’ knowledge and the nature of its change process are not clear . Recent research suggests that one crucial aspect of learning physics is learning its complex , relational knowledge . We synthesize research from cognitive science and science education research and develop a theoretical framework for concept learning . As an example we analyse empirical data of students’ explanations of DC circuits . Their explanations had a very simple causal structure that reflects difficulties grasping the relational knowledge that ties the concepts together . Implications and importance of the theoretical work are discussed . INTRODUCTION Learning scientific concepts often requires that the learner modifies his or her prior knowledge , which can be difficult and take a long time . A well - known problem in learning physics , for example , is that learners’ concepts often differ quite substantially from scientific ones in their meaning and content ( see , e . g . , Lee & Law , 2001 ) . Even university students have naive beliefs concerning basic physic concepts . Learning physics requires learning complex knowledge system ( theory ) , which constrains and regulates the use of physics concepts . It entails learning multiple concepts and information about their co - variation , which is embed - ded in laws and models . However , students often conflate closely related concepts and use them in undifferentiated ways – examples include heat and temperature , voltage and electric current as well as weight and density ( Lee & Law , 2001 ; C . Smith , Carey , & Wiser , 1985 ) . This is often discussed from the viewpoint of concept differentiation . Furthermore , recent research has paid attention to learners’ knowledge about the relational patterns inherent in laws and models of physics . Students are often inclined towards simple linear causal chains instead of , for example , emergent patterns of complex causality ( Perkins & Grotzer , 2005 ) . Although we have gained insight about specific obstacles and biases in students learning of scientific concepts , we are still lacking description of particular cognitive mechanisms underlying concept learning and shared conception of concepts ( Rusanen , 2014 ) . One fruitful possibility could be closer integration of cognitive scientific models of concept learning into science education research . Recently , we ( Koponen & Kokkonen , 2014 ; see also , Koponen & Huttunen , 2013 ) introduced so - called systemic view of MALU , Turku 2015 36 concept learning , which leans on views about concepts as dynamic cognitive structures . Our previous studies have focused on students’ explanations , their use of different relational patterns and the change in their ability to apply these different patterns in the context of DC circuits . Likewise , our interest lies in relational knowledge . The principal focus of this paper is theoretical . We seek to extend the previous science education research by looking at the recent cognitive science literature on relational concepts , which shows that relational knowledge is a fundamental part of concepts and concept learning . In the empirical part of this paper we examine university students’ explanations of DC circuits tasks . The specific research questions are : 1 ) Based on the recent literature , what is the nature of physics concepts and the concept learning process ? 2 ) What concepts and what kind of relations university students use in their explanations about DC circuit tasks ? The theoretical results and discussion offer a step forward in understanding the nature of the concept learning process and the cognitive processes underlying it . The theoretical ideas we present allow us to discern the roles of relational aspects of concepts in university students’ understanding . In a previous study , we used interview data to analyse students’ knowledge and identified three generic relational patterns . However , interview data may cause artefacts stemming from , for example , conversational pragmatics . Consequently , in this paper we applied the analysis method to examine students’ written answers to similar tasks . Although the data only offers us a brief snapshot , together with the previous results and the theoretical ideas presented above it offers valuable information about the different possible learning outcomes . STUDENTS’ CONCEPTIONS OF DC CIRCUITS While a rather mundane topic for physics experts , concepts related to DC circuits cause great difficulties even for university students . Previous studies have discovered multiple difficulties spanning from lack of sufficient models to false beliefs to not understanding the circuit topology . Research has found out that students conflate closely related concepts such as voltage , current and energy ( Koponen & Huttunen , 2013 ; Lee & Law , 2001 ; McDermott & Shaffer , 1992 ) . For example , voltage is sometimes used interchangeably with current or voltage is thought as the amount of current stored in the battery ( Reiner , Slotta , Chi , & Resnick , 2000 ) . This kind of thinking is reflected in the naive explanations according to which something ( electricity , current , energy , etc . ) comes from the battery and is consumed by the bulbs ( McDermott & Shaffer , 1992 ; Tarciso Borges & Gilbert , 1999 ) . This Kokkonen & Nousiainen 37 also implies a difficulty of conceptualizing the circuit as a system , since the students often think that changes in one part of the circuit does not affect the other parts ( McDermott & Shaffer , 1992 ) . Often , students apply a kind of “sequential ( or local ) thinking” to circuits and think that the direction of the current and the ordering of the components matter . This is often accompanied by a belief that current is “used up” or diminished in a circuit so that , for example , bulbs connected in series would be unequal . Sometimes , even the idea of a closed circuit is lacking and students think that only a single wire is enough to light the bulb ( Tarciso Borges & Gilbert , 1999 ) . Some intuitive explanations concentrate on the number of elements in the circuit , such as “more bulbs—less current” or “less bulbs—more current” ( McDermott & Shaffer , 1992 ) . This acknowledges some kind of relation between the components and the current but nevertheless fails to recognise the different connections . More advanced explanations may exploit some quantitative formulas but fail to recognize such elements as different connections and thus arrive at false predictions about the behaviour of the circuit . Science education research has identified a range of “misconceptions” and “alternative models” . Indeed , Brown and Hammer ( 2008 ) argue that most of the research in , for example , conceptual change has taken conceptions ( such as “more bulbs – less current” ) as “units of knowledge” . Furthermore , this view is widely held by the physics education community and ample research is still published about students’ misconceptions . Contemporary research has however noted that while some of the learner’s problems might be rooted to specific concepts and theories , there might be some more general features that play a part ( Brown & Hammer , 2008 ; Perkins & Grotzer , 2005 ) . One such feature seems to be students’ knowledge of various principles underlying physics phenomena . RELATIONAL KNOWLEDGE Evidence from science education research Grasping the underlying principles is important in , for example , treating DC circuit phenomena as a system ( cf . Slotta , Chi , & Joram , 1995 ) . That is , students need to learn the concepts and relations according to which the system operates – for example , Ohm’s law . Indeed , some researchers have argued that many of students’ difficulties are rooted in their limited aware - ness of various relational patterns in laws and models of physics ( Perkins & Grotzer , 2005 ) . Instead , students tend to use simple sequential ( for example , A causes B which causes C and so forth ) patterns instead of more complex patterns such as constraint - based interaction ( Perkins & Grotzer , 2005 ) . So as well as concept specific knowledge , students are lacking generic relational patterns and modelling styles . MALU , Turku 2015 38 Rather similarly , previous studies have examined concept differentiation and the role of relational knowledge in high school and university students’ explanations of DC circuits . It seems that in the early stages learning relation - al , law - like knowledge drives the differentiation ( Koponen & Huttunen , 2013 ; Koponen & Kokkonen , 2014 ) . This means that introducing a causal relation , which connects electric current to voltage , defines each concept’s role . Thus they are unambiguously seen as different concepts . In analysing university students’ explanations , Kokkonen ( 2013 ) identified three generic relational patterns ( see Figure 1 ) and used these to analyse students’ explanations and the concept learning process . It seems that on this higher level , the key element in successful construction of the explanation models is the flexibility in using the different relational patterns ( Kokkonen & Mäntylä , under review ; see also , Kokkonen , 2013 ; Koponen & Huttunen , 2013 ) Figure 1 . Diagrams of generic relational patterns appearing in students’ explanations identified in Kokkonen & Mäntylä ( under review ; see also , Kokkonen 2013 ) In sum , research on students’ knowledge has indicated several hurdles on the path of learning scientific knowledge . In certain domains , one important issue seems to be the differentiation of closely related concepts . In the context of DC circuits one aspect of this problem is knowing how electric current and voltage relate to the actual circuits and components ( roughly put , for example current divides in parallel connections while voltage stays the same ) . Furthermore , especially in science , educational research has begun to establish the complex relational knowledge as one of the stumbling blocks . Relational Concepts Further evidence for the centrality of relational knowledge comes from research on human cognition and concepts 4 . Broadly speaking , relations are a fundamental part of our understanding of the world . For example , even our understanding of such simple concept as father is defined by the relation father bears to his offspring ( namely , begetting ) ( Gentner , 2005 ) . Thus , father 4 Concepts can be understood as ( mental ) knowledge structure about x , used in most high - level cognitive processes ( e . g . , inference , prediction etc . ) concerning x ( Machery , 2009 ) . Kokkonen & Nousiainen 39 is essentially a relational concept . If we consider a more complex case , such as central force system , it might be even more evident that relations from the basis for understanding . In the case of Earth and Moon , for example , we may say that Moon relates to Earth by the virtue of revolving around it . We may also construct a physics model of the situation , in which case the relations in question might be represented as mathematical functions ( see , e . g . , Gentner , 2005 ) . Relations and relational knowledge are important for example in categoriza - tion , analogies , explanation , concept learning , reasoning and problem solving ( Halford , Wilson , & Phillips , 2010 ) . Thus , learning of relational concepts is a fundamental question for the study of higher - level cognition . For example , analogies enable us to gain insight of unfamiliar phenomena by virtue of comparison to previously known examples . Famous examples include the water pipe analogy for electric circuits and the solar system - atom analogy . One mechanism through which analogies work is structural alignment : identifying the underlying relational pattern in the phenomena and matching it with the novel phenomena under study ( Gentner , 2005 ) . Crucial thing to notice is that the entities involved in analogical mapping need not to share resemblances – they are mapped by the virtue of their similar roles and relational structure . Mental models , abundant in science education literature , exhibit many properties of relational knowledge ( Halford et al . , 2010 ) . While there exist no rigorous definition for mental models , they are typically considered to contain structural and / or causal information about the system being modelled ( Markman , 1999 ) . Some describe them as “inter - related system of concepts” and structural analogues of the thing they represent ( Chi , 2013 ) . Hence , they serve as the vehicles for making inferences about the phenomena . Moreover , mental models enable us to predict about the system and reason about , for example , unexpected events . Relational concepts in the context of DC circuits We can also understand physics concepts in terms of relational concepts . To this end , a useful distinction is one between first and higher order relations . While the former concerns relations between objects , the latter links relations ( Gentner , 1989 ; see also , Paatz , Ryder , Schwedes , & Scott , 2004 ) . Following Paatz and others ( 2004 ) we may use this dichotomy to understand concepts related to DC circuits . We may view electric current and voltage as governed by first order relations , namely , Kirchhoff’s laws . In addition , for simple DC circuits , current and voltage are connected via Ohm’s law , a higher order relation ( as it relates relational concepts ) . Note , that such formal definitions are not necessarily part of students’ representation of the concepts . Instead , they may view electric current as something flowing in the circuit or dividing MALU , Turku 2015 40 in junctions , and voltage as something being between the battery terminals . In these cases the concepts are nonetheless associated with essentially relational rules . Consequently , concept differentiation can now be understood as learning the first order relational structures of electric current and voltage for example . That is , figuring out how current and voltage relate to different circuit elements or whether electric current is something that comes from the battery or something that is between battery’s terminals 5 . Likewise , learning the relational structure ( e . g . , Ohm’s law ) of the DC circuit system means captur - ing the higher order relations governing the relevant concepts such as r2 in Figure 1 . In sum , relations and relational concepts form a fundamental part of our understanding of the world and higher cognitive capacities . Physics concepts , for example concepts related to DC circuits can be understood in terms of relational concepts and first and higher order relations . The first order relations correspond to the more concrete representations ( i . e . perceptual features of the DC circuits , such as topology of the circuit ) . In contrast , higher order relations concern the more abstract concepts and relations between concepts . The implications for the mechanisms of concept learning will be discussed later . METHOD Participants The participants were 11 ( 7 female ) pre - service teachers participating on a physics teacher preparation course . Seven participants were majoring in physics , three in mathematics and one in statistics . All participants had experience about electric circuits from high school level and had studied the introductory physics courses at the university . The DC circuit tasks were part of the course intended to familiarize the students with working with real circuits . Data collection was done with participants consent . Procedure We asked the participants to explain the behaviour of simple DC circuits . The tasks consisted of three different sets of circuits : 1 ) different battery combina - tions and similar bulbs ( cf . D . P . Smith & van Kampen , 2011 ) , 2 ) different combinations of bulbs but similar batteries ( cf . McDermott & Shaffer , 1992 ) , and 3 ) dissimilar bulbs with similar batteries . Each set consisted of four circuits with ascending complexity designed to bring about different aspects of concepts related to DC circuits ( cf . McDermott & Shaffer , 1992 ) . The third 5 Here , is between , for example , should be understood as a relation thus making voltage a relational concept . Kokkonen & Nousiainen 41 set appears in Figure 2 . Each set of circuit tasks was presented piecewise so that first , two circuits were presented ( A - B , in Figure 2 ) , then the other two one at a time ( CD and EF ) . After completion of each set , students constructed the circuits . After the construction , we presented them with the principle supposed to be employed in the task . The students’ task was as follows ( note : the original task was in Finnish ) : The circuits shown below consist of similar bulbs and batteries . The bat - tery’s voltage is 4 . 5 V . a ) Place the bulbs in order according to their relative brightness . If any of the bulbs are equally bright , state this in your answer . Explain your an - swers . b ) Compare the voltages across the bulbs c ) Compare the magnitude of the electric currents through each bulb For the task , in which the bulbs were dissimilar ( see Figure 2 ) , the first sentence read : In the circuits below , bulbs marked with grey have a higher resistance than the white ones . The batteries are similar in each circuit . The tasks were carried out in a quiet classroom in two 1 . 5 h sessions with half of the participants participating in each session . The students were handed the question sheet on which they also wrote their answers . Figure 2 . Circuits diagram used in the third set . The bulbs marked with grey have higher resistance than the white ones . Circuits were presented piecewise so that A and B were presented first and CD and EF after that one at a time . Data analysis We analysed the written answers by the means of content analysis ( cf . Elo & Kyngäs , 2008 ) . Based on the previous studies ( Kokkonen & Mäntylä , under review ; see also Kokkonen , 2013 ) we defined a coding scheme for the explanations and conducted a deductive analysis ( Elo & Kyngäs , 2008 ) . We first identified the concepts used in the explanation and coded the relations associated with the concepts . The relations were coded according to the generic schemes that appear in Figure 1 . We also identified explanations using “rules of thumb” such as “bulbs are equally bright because they are MALU , Turku 2015 42 connected in series” . These were categorized as “simple” explanations ( cf . Kokkonen , 2013 ) . Moreover , the students’ answers to questions about the voltages and current in the circuits were also checked in order to conclude whether the students’ could differentiate between the concepts . RESULTS Overview of the answers Across the problem sets 1 , 2 and 3 , students averaged 75 % , 50 % , 38 % correct answers , respectively . The number of right answers varied from 1 to 17 out of 24 ( 39 % correct answers on average ) . In total , three students out of 11 used relations ( see Fig . 1 ) consistently ; that is , they used it in all of the problems sets . Others relied on mostly voltage or current based explanations ( i . e . , they did not use relations in their explanations ) . Some simple explanations appeared as well . The frequencies of different explanations appear in Table 1 . Table 1 . Frequencies of the different explanations in different sets of tasks ( A - C ) . Terms r1 , r2 and r3 denote different relations used in explaining ( see Figure 1 ) . S and c denote “simple” and “one concept” explanations , respec - tively . task A B C explanation s c r1 r2 r3 s c r1 r2 r3 s c r1 r2 r3 freq . 1 16 2 5 0 4 12 7 2 1 3 15 9 6 0 Concepts and relational knowledge in the explanations Simple explanations Some students used simple explanations , which are more like descriptive “rules of thumb” than explanations . In themselves , the simple explanations are not generalizable to other contexts . In a typical simple explanation the brightnesses are explained by the virtue of the connection ( parallel or series ) . As student S1 explains in the case of one bulb being connected in series with two bulbs in parallel : S1 ( Task 1c ) : ”F and G are equally bright because they are connected in parallel . You can think of F and G as one bulb so they burn as brightly as H because of the series connection . ” Explanations with relation one concept Explanations with a reference to one physics concept go beyond the descrip - tive rules of thumb but are still not very sophisticated . However , the explana - tory power is still limited . As in the excerpt below , where S2 explains that the increased resistance results in bulbs in series being dimmer than a sole bulb ( no other concepts are used ) . Kokkonen & Nousiainen 43 S2 ( Task 2a ) : ”A is the brightest and B and C are equally bright but dimmer than A because 2 bulbs in series = > twice the resistance . ” Likewise , S1 relies only on electric current when explaining the same situation : S1 ( Task 2a ) : ” [ A > B = C ] . . . Because B and C are connected in series the electric current going through them has to light up two bulbs instead of one . ” While leading to correct answers , these explanations would most likely fail when parallel connections are introduced . Explanations with relation r1 Relation r1 means that two concepts are used in the explanation so that the one causes the other or is related to it . In the excerpt below , S3 explains that the increased resistance causes the electric current to be smaller ( and thus the bulbs are dimmer ) . S3 ( Task 3a ) : ”A > B . There is a smaller electric current going through B because its resistance is bigger . “ While leading to a correct answer , this kind of explanation is bound to fail if bulbs with different resistances are connected in series , in which case one would need to consider the power consumed by the bulbs – that is , the brightness will depend on the multiplication of voltage and electric current . Explanations with relation r2 Explanations with relation r2 entail using three concepts as in Ohm’s law , for example . These explanations are quite sophisticated as they might employ Ohm’s law but might nevertheless fail when dissimilar bulbs are introduced ( as it would require introducing the Joule’s law and the concept of electric power ) . In the excerpt below S5 explains why B ( a bulb with a bigger resistance ) is brighter than A . S5 ( Task 1a ) ”The voltage of the bulbs is as big as in the batteries , be - cause the bulb is the only component . According to Ohm’s law the electric current through bulb B is bigger than through bulb A , because the resistance is the same - > B is brighter . “ Explanations with relation r3 Like relation r2 , relation r3 entails using three concepts . With relation r3 , however , two concepts are both related to a third one but not necessarily to each other . In the excerpt below , S6 compares bulbs in series ( B and C ) with bulbs in parallel ( D and E ) and notes that voltage divides B and C but current stays the same and vice - versa for D and E – thus B , C , D and E are equally bright ( a false answer ) . MALU , Turku 2015 44 S6 ( Task 2b ) : ”P = UI . In the second circuit the voltage is different over B and C and in the third the current is different - > P is the same . “ While S6 acknowledges that , both , voltage and electric current need to be considered , he / she seems to hold the false belief that batteries provide constant current . DISCUSSION As noted earlier , most students relied on rather unsophisticated explanation models – that is , their explanations had very a simple causal structure . While the simple models ( i . e . , “rules of thumb” ) may in themselves be correct descriptions of the situations , they are superficial in that they are not applicable in other contexts . The explanations with one concept or relation r1 , for example , are somewhat more elaborated but nonetheless have limited applicability as noted in the previous section . For example , in order to correctly explain the brightnesses of the bulbs in Figure 2 , one must use Ohm’s law together with Joule’s law . Hence , for many students , the learning remains incomplete and requires the learning of relational knowledge ( models and laws ) . It should be noted , however , that even with elaborated relational knowledge , the explanations might fail because student holds a false belief for example ( see the last excerpt in the previous section ) . As for these results , they align well with our previous results thus supporting the conclusion they were not interview - induced artefacts ( see Koponen & Huttunen , 2013 ; Kokkonen & Mäntylä , under review ; Kokkonen , 2013 ) . Studies with elementary and middle - school students have noted that students are predisposed towards “direct” or “linear” causality and have difficulty learning , for example , emergent and constraint - based patterns ( Chi et al . , 2012 ; Perkins & Grotzer , 2005 ) . Our results show similar findings , which imply that the difficulties persist even at the university level . These results point towards the direction that the relational nature of physics concepts causes learners great difficulties . Consequently , students’ ability to apply and modify this knowledge is vital to the construction of the correct explanation models . As relational knowledge is central to human cognition on one hand , and to physics knowledge on the other , we see it as the major stumbling block in learning and understanding physics . This way the central learning processes are those related to revision of the relational structure . This “theory revision” could happen either through refinement ( stripping down relations ) or elaboration ( adding relations to the present structure ) ( Halford et al . , 2010 ) . In a previous study concerning DC circuits , three distinctive processes elabora - tion , refinement and switch were suggested , which align with those proposed by Halford and others ( Kokkonen , 2013 ; Kokkonen & Mäntylä , under review ) . Furthermore , as suggested by Corral and Jones ( 2014 ) the learning process is Kokkonen & Nousiainen 45 affected by the relational structure . The number of relations as well as the type of relational pattern ( such as those in Figure 1 ) affects the difficulty of learning . This may be down to , for example , limitations in working memory , too complex concepts are bound to decomposition and chunking ( Halford et al . , 2010 ) . Ideas about revision of the conceptual structure are , of course , not new in science education . 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Luokanopettajaopiskelijoiden monialaisten opintojen matematiikan opintojakson määritelmätestin vastaukset ( N = 70 ) luokiteltiin aineistolähtöisen sisällönanalyysin pohjalta . Määritelmät vaihtelivat triviaaleista kuvailuista eksakteihin matemaattisiin määritelmiin . Eroa formaaleihin määritelmiin esiintyi niin terminologian käytössä kuin yläkäsitteiden ja tarkentavien ehtojen muodostamisessa . Määritelmistä löydettiin samankaltaisuutta aiemmissa tutkimuk - sissa todettuihin peruskoulun oppilaiden ja opettajaksi opiskelevien muodostamiin määritelmiin . JOHDANTO Suomessa 2010 - luvulla toteutettujen koulusaavutustutkimusten valossa suomalaisten oppilaiden geometrian osaaminen on algebran ohella muita sisältöalueita heikompaa . Vuoden 2012 PISA - tutkimuksessa ( Programme for International Student Assessment ) tila ja muoto oli suomalaisten selvästi heikoimmin osaama matematiikan osa - alue . Siinä heikkojen suoritusten osuus oli muita sisältöalueita suurempi ja erinomaisten suorittajien osuus pienin ( Kupari , Välijärvi , Andersson , Arffman , Nissinen , Puhakka , & Vettanranta , 2013 ) . Samoin TIMSS 2011 - tutkimuksen ( Trends in International Mathematics and Science Study ) mukaan geometria ja algebra olivat suoma - laisoppilaiden heikoimmat alueet , missä tehtävien osaaminen oli lähellä kansainvälistä keskiarvoa ( Kupari , Vettenranta , & Nissinen , 2012 ) . Kansainvälisestikin tarkasteltuna oppilaiden geometrisen osaamisen on todettu olevan alhaisella tasolla ja syyksi tilanteeseen on esitetty geometrian kouluopetuksessa saaman heikon aseman lisäksi sekä geometrian didaktisten käytänteiden uudistumattomuutta että opettajien oman geometrisen tietä - myksen puutteellisuutta ( Steele , 2013 ) . Tulevien opettajien käsitys geometri - asta on Browningin ja kollegoiden ( 2014 ) metatutkimuksessa todettu varsin rajalliseksi . He näkevät opettajien matemaattisen sisältötiedon kriittisenä tekijänä oppilaiden geometrisen ymmärryksen kehittymisessä ja painottavat opettajankouluttajien vastuuta tulevien opettajien tietoperustan luomisessa . Geometrian asema osana koulumatematiikkaa on Suomessa säilynyt opetus - suunnitelmien perusteella melko samanlaisena 2000 - luvulla . Ilman kattavaa tutkimusta on vaikea arvioida , miten luokkien didaktiset käytännöt ovat Kärki 49 tuona aikana muuttuneet , mutta ainakin tietotekniikka on avannut monia mahdollisuuksia opettaa geometriaa uudella tavalla ( Battista , 2002 ; Jackiw , 2010 ; Hall , 2013 ; Joglar Prieto , Sordo Juanena , & Star , 2014 ) . Syitä suomalais - ten heikkoon geometrian osaamiseen tulisikin tutkia mm . koulujen geometri - an opetusta ja työtapoja sekä oppimateriaalien laatua analysoimalla . Tämän tutkimuksen tutkimuskohteena on alakoulun opettajien geometrinen taitotieto . Tutkimuksessa rajoitutaan tarkastelemaan luokanopettaja - opiskelijoiden kirjoittamia geometrisia määritelmiä ja pohditaan niiden antamia viitteitä geometrisen käsitetiedon tasosta . GEOMETRISEN AJATTELUN KEHITTYMINEN Matemaattinen tietämys voidaan jakaa dikotomisesti konseptuaaliseen eli käsitetietoon ja proseduraaliseen eli menetelmätietoon ( Hiebert & Lefevre , 1986 ) . Proseduraalisen ja konseptuaalisen tiedon luonnetta ja roolia matema - tiikan oppimisessa on tutkittu paljon ja näiden tietotyyppien yhteydestä matemaattisen tiedon laatuun on esitetty erilaisia näkemyksiä . Tutkijoiden erilaisista tietotyyppien määrittelyistä huolimatta mielekkään matemaattisen tiedon nähdään koostuvan monimutkaisesti yhteenkietoutuneesta vastavuo - roisessa suhteessa kehittyvästä käsite - ja menetelmätiedosta . ( Baroody , Feil , & Johnson , 2007 ; Star & Stylianides , 2013 ; Rittle - Johnson & Schneider , 2015 . ) Geometrisen tiedon osalta Silfverberg kuvaa oppilaan käsitetiedon koostuvan esimerkiksi siitä , mitä oppilas tarkoittaa suorakulmiolla , suunnikkaalla tai suorakulmaisella särmiöllä , minkälaisia ominaisuuksia näillä käsitteillä hänen mielestään on ja miten käsitteet linkittyvät toisiinsa . Geometrinen menetelmä - tieto taas koostuu geometristen objektien merkintätapojen lisäksi algoritmisis - ta toimintasäännöistä kuten pituuksien , pinta - alojen ja tilavuuksien laskuta - voista sekä kuvioiden ja kappaleiden piirtämiseen liittyvistä sopimuksista . ( Silfverberg , 1999 . ) Hyödyllisen viitekehyksen geometrisen ajattelun kehittymisen tarkastelulle tarjoaa ns . van Hielen teoria ( Browning ym . , 2014 ) . Teoria perustuu hollanti - laisten Pierre van Hielen ja Dina van Hiele - Geldofin 1950 - luvulla alkaneisiin tutkimuksiin ( Fuys , Geddes , & Tischler , 1984 ) , ja sen validoinnista ja merki - tyksestä geometrian opetukselle on tehty useita tutkimuksia ( Ma , 2015 ) . Teoria sisältää hypoteesin oppilaan geometrisen ajattelun kehittymisestä viiden tason kautta . Kehitystasojen yleissisältö on laajasti hyväksytty , mutta etenkin tasojen tiukasta erillisyydestä ja hierarkiasta on esitetty väljempiä tulkintoja . Oppilaiden geometrisessa ajattelussa näyttää tapahtuvan edistystä usean tason kohdalla samanaikaisesti , kuitenkin alemman van Hielen tason piirteiden ollessa pääsääntöisesti edellä korkeampien tasojen piirteiden kehitystä . ( Silfverberg , 1999 ; Unal , Jakubowski , & Corey , 2009 . ) Erityisesti on pohdittu käsitteiden välisen luokkainkluusion ymmärtämisen ja määrittely - taidon kehittymisen sijoittumista tasojen kuvauksiin ( mm . Silfverberg , 1999 ; Silfverberg & Matsuo , 2008 ; de Villiers , 2010 ) . Seuraava van Hielen tasojen MALU , Turku 2015 50 ominaisuuksien lyhyt kuvailu perustuu alkuperäislähteisiin ( Fuys ym . , 1984 ) sekä Silfverbergin ( 1999 ) ja Van de Wallen , Karpin ja Bay - Williamsin ( 2009 ) esittämään tulkintaan : Visualisoinnin tasolla ( vH1 ) ajattelu kohdistuu yksittäisiin kuvioihin , joiden tunnistus ja luokittelu perustuu visuaalisen ulkomuodon samankaltaisuuteen . Ominaisuuksien analysoinnin tasolla ( vH2 ) kuviot nähdään ominaisuuksiensa kantajina . Kuvioluokkaan kuuluminen perustuu ominaisuuksien analysoin - tiin eikä pelkästään visuaaliseen samankaltaisuuteen , mutta ominaisuuksien keskinäisiin riippuvuussuhteisiin ei kiinnitetä huomiota . Kuvioita määritel - lessään tälle tasolle kuuluvat oppilaat todennäköisesti luettelevat kaikki kuviolle tuntemansa ominaisuudet . Ominaisuuksien järjestämisen eli epäformaalin päättelyn tasolla ( vH3 ) ajattelun kohteena ovat kuvioiden ominaisuudet , jotka järjestäytyvät loogisiksi rakenteiksi . Oppilas ymmärtää , että jotkin ominaisuudet ovat seurausta toisista ominaisuuksista , ja hän osaa tehdä lyhyitä deduktiivisia päätelmiä . Kuvio tunnistetaan geometrisen käsitteen alaan kuuluvaksi käsitteen määritelmän perusteella . Määritelmiä osataan muotoilla ilmaisten kuvioiden riittävät ja välttämättömät ominaisuudet ja näiden ominaisuuksien avulla osataan muodostaa geometrisista käsitteistä käsitehierarkioita . Formaalin päättelyn tasolla ( vH4 ) ymmärretään deduktiivisen päättelyn idea ja hallitaan deduktiivisen geometrian ajattelutapa . Aksioomasysteemin ymmärtämisen tasolla ( vH5 ) pystytään hallitsemaan ja vertailemaan eri geometrioita aksiomaattisina järjestelminä . MÄÄRITELMÄT OSANA GEOMETRISTA KÄSITETIETOA Silfverbergin ( 1999 ) tutkimuksen mukaan määrittelytaidolla ja van Hielen tasoilla on tilastollisesti erittäin merkitsevä yhteys . Silfverberg havaitsi , että ylemmillä tasoilla olevat oppilaat kykenivät alempien tasojen oppilaita paremmin määrittelemään geometrisia kuvioita ja tulkitsemaan käsitteiden välisiä suhteita matematiikan standarditulkinnan mukaisesti . Tietorakentei - den eheytyminen näkyi erityisesti kolmannelle tasolle siirryttäessä , mutta tason vH3 yleisten tulkintojen vastaisesti ei selvää muutosta käsitteiden välisten suhteiden ja kuvioiden määrittelemisen osaamisessa voitu havaita . Fischbein ( 1993 ) kutsuu geometrisia objekteja figuraalisiksi käsitteiksi ( figural concepts ) korostaakseen niiden kaksoisluonnetta . Geometristen kuvioiden ja kappaleiden luonteessa on samanaikaisesti läsnä sekä objektin konseptuaali - nen eli käsitteellinen komponentti että figuraalinen eli kuvallinen kompo - nentti . Geometrinen objekti on abstrakti idea , jonkin aksioomasysteemin mukaiseen määritelmään perustuva olio , jota ei ole olemassa reaalimaailmas - sa eikä siihen voida liittää todellisuudessa aistittavia ominaisuuksia . Geomet - risten kuvioiden piirrokset ja kappaleiden konkreettiset mallit ovat vain Kärki 51 matemaattisten käsitteiden aineellisia malleja . Toisaalta geometriseen objektiin liittyy aina visuaalinen kuva , mentaalinen representaatio tilasta tai muodosta , joka on erottamattomasti läsnä geometrisessä ajattelussa ja joka Fischbeinin mukaan tarvitaan geometrian tieteenalan luovaan kehittymispro - sessiin . Figuraalinen käsite - termi kuvastaa loogisen ja figuraalisen näkökul - man ideaalista mentaalista yhteensulautumista , joka Fischbeinin mukaan ei tapahdu luonnollisesti itsestään . Monesti oppilaiden ajattelussa kuvallinen ja käsitteellinen komponentti ovat ristiriidassa keskenään tai kuvallinen komponentti hallitsee geometrista päättelyä . Oppilaiden tulisikin olla tietoisia määritelmistä ja niiden roolista geometrisessä ajattelussa . Geometriset tehtävät tulisi suorittaa kuvan antaman informaation sijaan objektien määritelmiin perustuen . ( Fischbein 1993 . ) Malatyn ( 1994 ) antaman esimerkin mukaan ideaalisen geometrisen käsitteen ja sen kuvallisen esityksen eroa voidaan opettaa pienillekin koululaisille . Fischbeinin ( 1999 ) näkemyksen mukaan yksi geometrian didaktiikan päätehtävistä on luoda sellaisia oppimistilanteita , joissa systemaattisesti vaaditaan käsitteellisen ja kuvallisen komponentin yhteensovittamista . Silfverberg ( 1999 ) näkee tässä yhteensovit - tamisessa keskeiseksi tekijäksi visuaalisen varioinnin kyvyn eli oppilaan kyvyn mielikuvatasolla tuottaa geometrisista käsitteistä tarkoitushakuisesti erityyp - pisiä esimerkkitapauksia . Tietämys matemaattisista määritelmistä ja käsitys määrittelemisestä osana matemaattista toimintaa vaikuttaa opettajan opetukseen , didaktisiin päätök - siin ja tapaan , jolla he käsittelevät matemaattista sisältöä ( Leikin & Zazkis , 2010 ) . Opetukseen vaikuttaa siis opettajan kokonaisymmärrys käsitteestä eli käsitekuva . Tallin ja Vinnerin ( 1981 ) mukaan käsitekuvalla ( concept image ) tarkoitetaan sitä käsitteeseen liittyvää kognitiivisen järjestelmän täyttä kokonaisuutta , joka sisältää kaikki mentaaliset kuvat , käsitteeseen liitetyt ominaisuudet ja prosessit . Käsitekuva rakentuu ja muuttuu erilaisten kokemusten ja ärsykkeiden myötä . Se voi sisältää ristiriitaisia komponentteja ja siitä voi eri tilanteissa aktivoitua erilaisia osia . Henkilökohtainen käsitteen määritelmä ( personal concept definition ) tarkoittaa sanamuotoa , jota yksilö käyttää oman aktivoidun käsitekuvansa selittämiseen . Formaali käsitteen määritelmä ( formal concept definition ) on sanallinen esitysmuoto , joka on matemaattisen yhteisön laajalti hyväksymä . ( Tall & Vinner , 1981 . ) Leikin ja Zazkis ( 2010 ) esittivät Winicki - Landmanin ja Leikinin artikkeliin ( 2000 ) perustuen seuraavat tunnettujen matemaatikkojen hyväksymät periaatteet formaalille matemaattiselle määritelmälle : 1 ) Määritelmä antaa nimen uudelle käsitteelle ja nimi esiintyy määritelmässä vain kerran . 2 ) Määritelmä esittää käsitteelle välttämättömät ja riittävät ehdot . 3 ) Uuden käsitteen määrittelemiseksi voidaan käyttää vain aiemmin määriteltyjä käsitteitä tai ns . määrittelemättömiä peruskäsitteitä . 4 ) Määritelmässä esitettyjen ehtojen joukko pyritään muodostamaa usein minimaaliseksi eli ei MALU , Turku 2015 52 esitetä ehtoja , jotka loogisesti seuraavat muista ehdoista . 5 ) Määritelmäksi voidaan valita mikä tahansa ekvivalenteista muotoiluista . Matematiikan opetuksessa tulee huomioida määritelmän matemaattisen oikeellisuuden lisäksi sen didaktinen soveltuvuus ( Winicky - Landman & Leikin , 2000 ) . Konstruktivistiseen näkemykseen pohjautuen määritelmän tulee perustua oppilaan aiemmin tuntemiin käsitteisiin ja sen tulee ottaa huomioon oppilaan senhetkinen kehitystaso . Käytetyn määritelmän tulisi mahdollisimman hyvin sopia yhteen oppijan intuition kanssa ja myös esteettiset näkökohdat , kuten ilmaisun selkeys ja eleganttius , tulisi ottaa huomioon . Opettajien ja opettajaksi opiskelevien kyky ymmärtää geometrisia käsitteitä , muodostaa määritelmiä sekä kyky ymmärtää määritelmän merkitys matema - tiikassa ja määritteleminen matemaattisena prosessina on useissa tutkimuk - sissa todettu puutteelliseksi ( mm . Linchevsky , Vinner & Karsenty , 1992 ; Cunningham & Roberts , 2010 ; Leikin & Zazkis , 2010 ; Duatepe - Paksu , İ ymen & Pakmak , 2012 ; Marchis , 2012 ; Silfverberg & Joutsenlahti , 2014 ) . Marchis ( 2012 ) löysi opettajaksi opiskelevien kirjoittamista määritelmistä monia edellä mainituista formaalin matemaattisen määritelmän periaatteista poikkeavia piirteitä : 1 ) Opiskelijan kirjoittama määritelmä saattoi olla käsitteelle täysin väärä esimerkiksi siten , että ylä - ja alakäsitteet olivat vaihtaneet paikkaa . 2 ) Määritelmää ei esitetty määritelmän muodossa vaan listana määritelmään liittyviä ominaisuuksia . 3 ) Määritelmä ei sisältänyt välttämättömiä ja riittäviä ehtoja . 4 ) Määritelmä ei ollut minimaalinen . Vastaavankaltaisesti Silfverberg ja Matsuo ( 2008 ) luokittelivat peruskoulun oppilaiden antamat geometriset määritelmät naivien kuvausten lisäksi ehtojen oikeellisuuden , riittävyyden , minimaalisuuden ja oikean yläkäsitteen käyttämisen mukaan . TUTKIMUSMENETELMÄ Tämän tutkimuksen kohteena ovat luokanopettajaksi opiskelevien kirjoitta - mat geometristen käsitteiden määritelmät peruskoulussa opetettavien aineiden ja aihekokonaisuuksien monialaisten opintojen matematiikan opintojakson määritelmätestissä . Alkeisgeometrian määritelmät oli esitetty opintojakson monisteessa kolmeen taulukkoon jaoteltuna ( geometriset peruskäsitteet 13 kpl , tasokuviot 22 kpl , avaruuskappaleet 12 kpl ) . Määritel - miä oli käsitelty opintojakson yhdellä luennolla sekä kahdella pienryhmäker - ralla sekä tehtävissä , joihin kuului mm . käsitehierarkioiden piirtämistä puumallia käyttäen . Määritelmätesti kesti noin 15 – 20 minuuttia ja sen aikana kukin opiskelija kirjoitti määritelmät kahdeksalle geometriselle käsitteelle . Määritelmät kysyttiin yksi kerrallaan kirjoittamalla ko . käsitteen nimi taululle . Opiskelijal - la oli oikeus testin aikana palata aiemmin kirjoittamiinsa määritelmiin . Kysytyt käsitteet olivat kysymisjärjestyksessä jana , kulma , monikulmio , suunnikas , suorakulmio , pallo , monitahokas ja kuutio . Näiden käsitteiden luentomonisteessa esiintyneet määritelmät on esitetty Taulukossa 1 . Testin Kärki 53 jälkeen opiskelijoiden ehdottamista määritelmistä keskusteltiin ja he saivat pisteyttää toistensa vastauksia . Testiin osallistui yhteensä 70 opiskelijaa . Taulukko 1 . Geometristen objektien määritelmät luentomonisteessa objektin nimi määritelmä jana suoran osa , jolla on sekä alku - että loppupiste kulma kahden samasta pisteestä lähtevän puolisuoran väliin jäävä tason osa monikulmio suljetun itseään leikkaamattoman murtoviivan rajaama tason osa suunnikas nelikulmio , jonka vastakkaiset sivut ovat ( pareittain ) yhdensuuntaiset suorakulmio nelikulmio , jonka kaikki kulmat ovat suoria pallo avaruuskappale , jonka rajoittava pinta muodostuu niistä kolmiulottei - sen avaruuden pisteistä , jotka ovat yhtä etäällä annetusta keskipisteestä monitahokas avaruuskappale , joka on yksinomaan monikulmioiden rajoittama kuutio suorakulmainen särmiö , jonka tahkot ovat yhteneviä neliöitä Tutkimusta varten opiskelijoiden vastaukset kirjattiin sähköiseen taulukkoon ja anonymisoitiin . Määritelmiä kirjattiin yhteensä 543 . Tyhjiä vastauksia löytyi 3 kulman , 2 monikulmion , 2 suunnikkaan , 8 monitahokkaan ja 2 kuution kohdalla . Saadusta aineistosta etsittiin sisällönanalyysin keinoin vastausta tutkimuskysymykseen : Millaisia määritelmiä luokanopettajaksi opiskelevat antavat taso - ja avaruusgeometrian käsitteille ? TUTKIMUSTULOKSET Aineistolähtöisen sisällönanalyysin mukaisesti kunkin geometrisen objektin erilaiset määritelmät ryhmiteltiin ja yhdistettiin luokiksi , joita kaikilla muilla kappaleille kertyi 7 - 16 , mutta kuution kohdalla analyysissä syntyi aluksi jopa 29 luokkaa , koska riittämättömiä ehtoja tunnistettiin 14 erilaista tyyppiä . Aineiston abstrahoinnissa päädyttiin luokkia yhdistelemällä lopulta kuvaa - maan aineistossa esiintyviä formaaleista määritelmistä poikkeavia määritel - mätyyppejä yhdeksän kategorian avulla : Y yläkäsitteeseen liittyvät puutteet , T terminologiset virheet , RE riittämättömät ehdot , TE liian tiukat ehdot , YL ylimääräiset tiedot , listamääritelmät , O objektin osien luettelu , N naiivi MALU , Turku 2015 54 kuvailu , V virheellinen tulkinta , A absurdi määritelmä . Vaikka tutkimuksen tarkoituksena ei ollut kvantitatiivisesti määrittää opiskelijoiden määrittelytai - toa , voidaan kuitenkin todeta , että sellaisia minimaalisia tai ei - minimaalisia määritelmiä , jotka liittivät käsitteen alaan kuuluvaksi tarkalleen käsitteen standarditulkintojen mukaiset tapaukset , hyväksyttiin noin 37 prosenttia . Tällaisia määritelmiä annettiin suorakulmiolle 50 , suunnikkaalle 35 , pallolle 32 , janalle ja kuutiolle 24 , kulmalle 16 , monikulmiolle 10 ja monitahokkaalle 10 . Janan kohdalla terminologiset ongelmat ( T ) johtuivat yleisesti termin suora käyttämisestä kuvaamaan äärellisen mittaista suoraa viivaa . Yläkäsitteen puuttuminen ( Y ) näkyi esimerkiksi ilmaisuna ”Janalla on alku ja loppupiste” . Tyypillinen riittämätön ehto ( RE ) oli viivan suoruusominaisuuden puuttumi - nen käsitteen määritelmästä . Yksi vastaaja oli tulkinnut käsitteen selvästi väärin ( V ) määritellessään janan ”päättymättömäksi suoraksi linjaksi” . Kulman kohdalla määritelmissä ( RE ) viitattiin puolisuorien väliseen aluee - seen mainitsematta , että puolisuorilla on yhteinen alkupiste . Terminologisesti ( T ) ongelmallista oli , että puolisuorien väliin jäävän alueen tai tason osan sijaan puhuttiin koko tasosta ja puolisuorat oli joissain määritelmissä korvattu murtoviivalla . Opiskelijoiden määritelmissä havaittiin yhdenmukaisesti Silfverbergin ja Joutsenlahden ( 2014 ) tutkimuksen kanssa epästandardi tulkinta ( V ) siitä , että kulmaan kuuluu ainoastaan kulman kärkipiste ja sen lähiympäristö . Tällainen ilmaus oli esimerkiksi ”se kohta , jossa kaksi puolisuoran pistettä kohtaa” . Absurdiksi muotoiluksi luokiteltiin mm . ”kahden kohtisuoraan leikkaavan suoran muodostava kohta , joka on alle 90°” , jossa myös kohtisuoruuden ja kulman asteluvun yhteys jäi epämää - räiseksi . Monikulmion määritelmissä oli yleistä mainita , minkä nimisiä osia se sisältää ( O ) . Riittämättömien ehtojen ( RE ) kategoriassa yleisin määritelmä oli sellainen , jossa murtoviivaa ei mainittu itseään leikkaamattomaksi . Muutama jätti mainitsematta murtoviivan suljetuksi . Terminologisesti ( T ) käytettiin jälleen tason osasta nimitystä taso , murtoviivan sijaan kirjoitettiin murtovii - voista tai suorista ja tasokuvion sijaan kappaleesta . P49 : vähintään kolmen murtoviivan muodostama kuvio P68 : suljettu itseään leikkaamaton muodostelma suorista , kuviossa on löydettävissä monta kulmaa P61 : yhtenäinen kappale , joka muodostuu yhtä monesta sivusta ja sivujen rajaamista kulmista . Myös suunnikkaan kohdalla tasokuviota nimitettiin kappaleeksi ja yhtä pitkiä sivuja yhdenmuotoisiksi ( T ) . Kategoriaan RE katsottiin kuuluvaksi mm . ne määritelmät , joissa yläkäsitteenä oli käytetty monikulmiota , mutta ehdoissa ei rajattu käsitettä nelikulmioihin . Osassa määritelmistä ( Y ) yläkäsite puuttui Kärki 55 kokonaan . Kategoriaan YL luokiteltiin suunnikkaalle tyypilliset mutta ei minimaaliset määritelmät , joissa mainitaan kaksi tai kolme seuraavista ekvivalenteista ominaisuuksista : vastakkaiset sivut ovat yhdensuuntaiset , vastakkaiset sivut ovat yhtä pitkät , vastakkaiset kulmat ovat yhtä suuret . Kahdeksan opiskelijan määritelmä vastasi jotakin muuta käsitettä ( V ) , esimerkiksi puolisuunnikasta tai suuntaissärmiötä . Myös suorakulmiota kysyttäessä kuuden opiskelijan määritelmä sopi johonkin toiseen geometri - seen objektiin , esimerkiksi suunnikkaaseen . Suorakulmion määritelmästä löytyi terminologisten puutteiden ja yläkäsitteen puuttumisen lisäksi määritelmiä ( YL ) , joissa oli ylimääräisiä ehtoja : P42 : Suorakulmio on monikulmio , jonka kaikki kulmat ovat suoria sekä suunnikas , jonka vastakkaiset sivut ovat yhtä pitkiä . Pallon määritelmässä haastava termi ( T ) oli pinta , jonka sijaan käytettiin tasogeometriaan kuuluvia sanoja kehä , kaari , piiri tai jopa taso . Yksi vastaaja määritteli pallon sijaan ympyrän ( V ) . Absurdeja ( A ) tai naiiveja kuvauksia ( N ) löytyi muutamia . P12 : Pallo on avaruuskappale , joka on kolmessa tasossa joiden jokaisesta kaaren pisteestä on yhtä pitkä matka keskipisteeseen . P70 : Pallo on avaruuskappale jolla on tilavuus . Siinä ei ole yhtään kulmaa ja se on muodoltaan symmetrinen ja pyöreä . Monitahokkaan yläkäsitteeksi ( Y ) oli virheellisesti valittu mm . monikulmio , lieriö tai särmiö . Määritelmä saattoi olla vain nimestä johdettu ilmaisu ( N ) kuten ”Monitahokas koostuu monista tahkoista” tai käsitteen osien ( tahko , särmä , kärki ) luettelo ( O ) . Tässä kaksi opiskelijaa oli antanut liian tiukat ehdot vaatimalla tahkojen yhtenevyyttä . Monitahokkaalle löytyi muita käsitteitä enemmän sellaisia määrittelyjä , joiden voitiin katsoa kuvaavan ennemmin jotakin muuta käsitettä , esimerkiksi särmiötä . Opiskelijoille varmasti hyvin tutun käsitteen , kuution , kohdalla korostui riittämättömät ehdot - kategorian ( RE ) yleisyys . Useimpiin tällaisiin määritel - miin sopi vastaesimerkiksi pirunnyrkkiä muistuttava seitsemästä samanlai - sesta kuutiosta koostuva rakennelma tai suuntaissärmiö tai suorakulmainen särmiö . Terminologisesti sekoittuivat jälleen taso - ja avaruusgeometrian termit kuten sivu ja särmä . P9 : monitahokas , jonka kaikki sivut ovat yhtä pitkät ja tahot samankokoi - set . P13 : avaruuskappale , joka muodostuu tasasivuisista nelikulmioista . P27 : kuudesta nelikulmiosta koostuva kappale , jonka kaikki kulmat ovat suoria . MALU , Turku 2015 56 POHDINTA Tässä tutkimuksessa tarkasteltiin luokanopettajiksi opiskelevien geometrisille objekteille antamia määritelmiä . On syytä mainita , että tarkoituksena ei ollut mitata opiskelijoiden geometrisen käsitetiedon tasoa , eikä se tällä tutkimus - asetelmalla olisi ollut edes mahdollista . Koska opiskelijoita kehotettiin opiskelemaan määritelmiä ja niitä oli käsitelty kurssin aikana , on luultavaa , että osa oppilaiden tuottamista formaaleiksi luokitelluista määritelmistä oli ulkoa opittuja . Tutkimuksen luotettavuutta tarkasteltaessa onkin huomattava , että käytetyllä tutkimusmenetelmällä saatiin tietoa opiskelijoiden senhetkisis - tä henkilökohtaisista käsitteiden määritelmistä , joihin määritelmätestiin valmistautumisella on ollut oma vaikutuksensa . Aineistonkeruuta voidaan pitää kattavana , sillä määritelmätestiin osallistui suurin osa yksikön monia - laisten opintojen toisen vuosikurssin opiskelijoista , testissä kysyttiin useiden erilaisten geometristen objektien määritelmiä ja analysointiyksiköitä kertyi runsaasti . Analysoinnissa määritelmien kirjaaminen sähköiseen taulukkoon mahdollisti määritelmien ominaisuuksien sujuvan vertailun ja lisäsi näin tutkimuksen luotettavuutta . Tutkijatriangulaation puuttuminen heikentää luotettavuutta , mutta raportoinnissa esimerkkeinä käytetyt opiskelijoiden vastaukset antavat lukijalle mahdollisuuden tutkijan tulkintojen oikeellisuu - den arviointiin . Tutkimuksen toteutuksessa tulisi suhtautua kriittisesti luentomonisteen määritelmiin , jotka eivät täyttäne tiukimpia formaaleja muotovaatimuksia . Esimerkiksi janan kohdalla olisi syytä puhua päätepisteis - tä vektoriin viittaavien alku - ja loppupisteen sijaan . Kulman määritelmässä selkeämpi ratkaisu olisi tarkemmin ilmaista myös puolisuorien olevan osa kulmaa . Tällöin nollakulma vastaisi tilannetta , jossa puolisuorat yhtyvät . Opiskelijoiden kirjoittamista taso - ja avaruusgeometrian käsitteiden määri - telmissä löytyi samanlaisuutta aiemmissa tutkimuksissa havaittuihin opettajaksi opiskelevien ja oppilaiden tekemiin määritelmiin ( Marchis , 2012 ; Silfverberg & Matsuo 2008 ) . Tässä sisällönanalyysissä korostui opiskelijoiden terminologisen osaamisen puutteet , mm . taso - ja avaruusgeometrian termien sekoittaminen keskenään . Monesti määritelmät eivät olleet määritelmän muodossa , vaan yläkäsite puuttui tai lueteltiin käsitteen osia . Tämän voisi tulkita siten , että opiskelija ei ole ymmärtänyt määrittelemisen ideaa . Toisaalta on huomioitava , että matemaattisten käsitteiden määrittelemisen opettelemiseen ei suomalaisessa koulujärjestelmässä kiinnitetä paljon huomiota ( Silfverberg & Joutsenlahti , 2014 ) , joten määritelmien kirjoittaminen on saattanut olla vaikeaa , vaikka opiskelijan käsitekuva olisikin ollut standarditulkinnan mukainen . Naiiveja kuvailuja tai väärien käsitteiden kuvailuja ilmeni verraten vähän . Aineistossa oli tyypillisesti määritelmiä , jotka eivät olleet minimaalisia vaan sisälsivät muista ominaisuuksista johdettavia ominaisuuksia . Riittämättömät ehdot - kategorian määritelmät olivat yleisiä . On mahdotonta tietää , yrittivätkö opiskelijat mielikuvatasolla Kärki 57 koetella määritelmiensä oikeellisuutta vastaesimerkkiä etsien mutta eivät sellaista löytäneet , vai oliko kyseessä vain ulkoa opitun määritelmän väärin muistaminen . Aineistossa havaitut poikkeamat formaaleista määritelmistä voitaneen osin tulkita osoituksiksi tavoiteltua van Hielen tasoa 3 alhaisem - masta osaamisesta . Tämän tutkimus antaa viitteitä siitä , että luokanopettajak - si opiskelevien matematiikan kursseilla tulisi geometrisen käsitetiedon vahvistamisen lisäksi käsitellä määritelmän ideaa ja määrittelemistä mate - maattisena toimintana sekä pyrkiä harjaannuttamaan opiskelijat matematii - kan kielen mukaisten termien käyttöön . Lisäksi opettajaksi opiskelevien geometrisen käsitetiedon tasoa tulisi jatkossa tarkemmin analysoida . LÄHTEET Baroody , A . J . , Feil , Y . , & Johnson , A . R . ( 2007 . ) An alternative reconceptual - ization of procedural and conceptual knowledge . 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MALU , Turku 2015 60 VIDEOVÄLITTEISTÄ FYSIIKAN OPETUSTA LUOKAN - OPETTAJAKOULUTUKSESSA Antti Laherto 1 & Jussi Laherto 2 1 Helsingin yliopisto , 2 Lahden kaupunki , Möysän koulu Raportoimme ja arvioimme uudentyyppisen opetuskokeilun , joka toteutettiin Helsingin yliopiston luokanopettajakoulutuksen fysiikan didaktiikan kurssilla yhteistyössä Lahden kaupungin Möysän koulun kanssa . LO - opiskelijat suunnittelivat ja toteuttivat opetusvideoita , joissa he antoivat ohjeita kokeellisista töistä 5 - luokkalaisten fysiikan tunnille . Lahdessa oppilaat katsoivat videot , tekivät opastetut tutkimustehtävät ja videoivat lopputuloksen palautteeksi LO - opiskelijoille . Kyselytut - kimukseen vastanneet LO - opiskelijat ( n = 130 ) kokivat oppineensa monipuolisesti fysiikan käsitteistä , kokeellisuudesta , ennakkokäsityksistä ja TVT : sta . Aitojen oppilastöiden opastaminen videon välityksellä vaikutti innostavalta työtavalta , vaikka siinä koettiin puutteitakin . Analysoimme työtapaa osana LO - koulutusta ja esitämme kehitysehdotuksia . JOHDANTO Tässä artikkelissa kuvaillaan ja arvioidaan uudenlaista opetuskokeilua , joka toteutettiin Helsingin yliopiston Opettajankoulutuslaitoksella Fysiikan didaktiikan kurssilla yhteistyössä lahtelaisen Möysän koulun kanssa . Fysiikan didaktiikan perusosa - kurssi on osa LO - koulutuksen ainedidaktiikan opintoja eli perusopetuksessa opetettavien aineiden ja aihekokonaisuuksien monialaisia opintoja . Monialaiset opinnot muodostavat yhteensä 60 opinto - pisteen kokonaisuuden , joka on kolmasosa kasvatustieteen kandidaatin tutkinnosta ( Tutkintorakenne , 2012 ) . Fysiikan didaktiikan perusosa on kaikille pakollinen kurssi , jonka laajuus on 3 opintopistettä . Opetussuunnitelman ( Opinto - opas , 2015 ) mukaan kurssilla  Tutustutaan opetussuunnitelman perusteisiin  Perehdytään fysiikkaan tieteenä , fysiikan tiedon luonteeseen ja tie - don hankkimiseen ja oikeuttamiseen  Käsitellään liike - ja voima - , sähkö - ja magnetismi - , lämpö - ja ener - gia - sekä aaltoilmiöiden malleja kvalitatiivisella tasolla sekä ja pe - rehdytään näihin liittyviin oppilaiden käsityksiin .  Tutustutaan kokeelliseen työskentelyyn koululaboratoriossa .  Tutustutaan tieto - ja viestintätekniikan käyttöön fysiikan opetukses - sa . Laherto & Laherto 61 Nämä keskeiset tavoitteet ja sisällöt ovat linjassa LO - koulutuksen yleisten tavoitteiden ( Opinto - opas , 2015 ) kanssa . Esimerkiksi tieto - ja viestintäteknii - kan ( TVT ) käytön taidot on keskeinen yleistavoite läpi koko koulutuksen . Se on myös laaja - alaisen osaamisen alue uudessa perusopetuksen opetussuunni - telmassa ( OPH , 2014 ) . Tässä raportoitava opetuskokeilu liittyy useisiin tutkimuksen osoittamiin haasteisiin LO - koulutuksessa . LO - opiskelijoiden suhde teknologiaan on havaittu usein ongelmalliseksi , ja tällä on suuri vaikutus opettajien antaman teknologiakasvatuksen muotoon ja sisältöön ( Goktas , Yildirim & Yildirim , 2009 ; Kärkkäinen & Keinonen , 2010 ) . Luonnontieteiden opetuksessa kokeelli - sen työskentelyn taidot ja tutkimusperustainen opetus ( inquiry - based science education ) vaatii tietoja ja taitoja , joissa LO - opiskelijat usein kokevat epävar - muutta ( Alake - Tuenter , 2012 ) . Tämä epävarmuus linkittyy tulevien luokan - opettajien tyypillisesti kielteisiin asenteisiin fysiikkaa kohtaan ( Kapucu , 2014 ) . Näihin kaikkiin LO - koulutuksen haasteisiin pyrittiin tarttumaan seuraavassa kuvailtavalla opetuskokeilulla . Edellä mainittujen tavoitteiden lisäksi ideana oli saada LO - opiskelijat kontaktiin oikeiden oppilaiden kanssa jo osana monialaisia opintoja . Yleensä opiskelijat saavat tämän kokemuksen vasta opetusharjoitteluissa . OPETUSKOKEILU Opetuskokeilu toteutettiin syksyllä 2013 osana Fysiikan didaktiikan perusosa – kurssia , jolle ilmoittautui 145 opiskelijaa . Ensin valittua aihealuetta , ”liikeil - miöt” , käsiteltiin neljä tuntia luennoilla ja neljä tuntia opetuslaboratoriossa käyden läpi mekaniikan peruslakeihin ja vuorovaikutuksiin liittyviä keskeisiä ilmiöitä ja havaintoja . Sitten LO - opiskelijat tekivät omatoimisesti 4 - 5 hengen ryhmissä suunnitelman tai käsikirjoituksen opetusvideosta viidesluokkalaisil - le . Videon aiheena tuli olla jokin liikeilmiö , ja videon tuli sisältää lyhyen opetustuokion ( max . 3 min ) sekä siihen liittyvän tehtävänannon oppilastyös - tä . Tehtävänanto sai olla suljettu tai avoin , mutta kuitenkin selkeä ohjeistus kokeellisesta työstä tai tutkimuksesta , jonka oppilaat toteuttavat pareittain yhden oppitunnin aikana . LO - opiskelijoille vinkattiin , että tehtävänanto voi olla useampivaiheinen : siinä voi esim . pyytää oppilaita ensin tekemään ennusteen , sitten tekemään kokeen , sitten pohtimaan havaintojaan ja raportoimaan videolle . Myös kotitehtävän sai antaa . LO - opiskelijoille kerrottiin Möysän koululla käytettävissä olevat välineet , joiden lisäksi käytössä oli tavalliset koulusta ja kotoa löytyvät tarvikkeet sekä etukäteen pyydettäessä muitakin tarvikkeita . Videoiden suunnitelmat hiottiin ja videot toteutettiin alusta loppuun yhden neljän tunnin ryhmäkokoontumisen aikana . Ensin käsikirjoitukset esiteltiin ja MALU , Turku 2015 62 muut opiskelijat ja opettaja antoivat vielä kehitysideoita ennen toteuttamista . Opiskelijaryhmät kuvasivat videomateriaalin OKL : n eri tiloissa käyttäen valintansa mukaan joko OKL : n videokameroita ( opettaja antoi teknisen tuen ) tai omia älypuhelimiaan . Videot editoitiin laboratorioluokan PC : illä Windows Live Movie Maker - ohjelmalla ( opettaja antoi pikakoulutuksen ) tai muilla ohjelmilla . Lopuksi opiskelijat siirsivät valmiin videon pilvipalveluun ( Dropbox ) Möysän koulun oppilaiden katsottaviksi . Yhteensä videoita valmistui 33 kappaletta . Opiskelijat käyttivät videoissaan paljon luovuutta , huumoria ja erilaisia esitystapoja . Aihealueet kattoivat laajasti mekaniikan peruslakeihin sekä eri vuorovaikutuksiin liittyviä ilmiöitä ja havaintoja . Esitystapoina oli opettajan kerrontaa , demonstraatioita sekä havainnollistavia videoklippejä , kuvia ja kaavioita . Lahdessa hankkeeseen osallistui Möysän koulun viides luokka yhden kouluviikon ajan . Luokalla opiskeli 31 oppilasta , joista noin puolet oli tyttöjä ja puolet poikia . Fysiikan ja kemian oppiaineeseen oli varattu vain yksi vuosiviikkotunti vuosityösuunnitelmassa , mutta erikoisjärjestelyin hankkee - seen varattiin noin kuusi oppituntia yhdelle viikolle . Oppiaineen tunteja käytettiin myöhemmin toisten oppiaineiden opetukseen . Hankkeessa käsitelty mekaniikan jakso tuli käsiteltäväksi hankkeen videoiden kautta ilman ennakkoperehtymistä . Koulussa kokeilu eteni siten , että kullekin pienelle ryhmälle ( 2 - 3 oppilasta ) annettiin linkki muutamaan LO - opiskelijoiden videoon . Tehtäväksi annettiin katsoa video , tehdä videossa annettu fysiikan koetehtävä , videoida tehtävän suoritus ja laatia muut mahdolliset tehtävänantovideossa annetut tehtävät , esimerkiksi pohdintatehtävät . Tehtävän teon ja videokuvausten jälkeen oppilasryhmä editoi iPadin iMovie – sovelluksella vastausvideon ja tallensi sen YouTube – palveluun . Lopuksi opettaja koosti vastausvideoiden linkit listaksi ja tallensi listan hankkeessa käytettyyn pilvipalveluun ( Dropbox ) edelleen LO - opiskelijoille jaettavaksi . Jakson sisältöjä käsiteltiin tämän jälkeen siten , että ensin keskeiset asiat käsiteltiin oppikirjasta , sitten koko luokka yhdessä reflektoi oppimaansa katsellen tehtävänanto - ja vastausvideoita . Lopuksi oppilaille pidettiin jakson aiheista perinteinen koe . Lopulta OKL : ssa LO - opiskelijat katsoivat ja kommentoivat omia ja toistensa opetusvideoita sekä oppilaiden videoita sekä omatoimisesti että yhdessä kurssin päätöskerralla . Videoiden tekijänoikeus - ja yksityisyydensuojakysymyksiin kiinnitettiin erityistä huomiota . Kurssin aikana pääsy videoihin oli vain kokeiluun osallistuneilla opiskelijoilla , oppilailla ja opettajilla . Kurssin jälkeen LO - opiskelijoiden tekemät videot koottiin videopankiksi , jota projektiin osallistu - neet voivat hyödyntää omassa opetuksessaan ja esitelmissään . Kaikki osapuolet sitoutuivat siihen , ettei videoita anneta muille eikä julkaista ilman Laherto & Laherto 63 videontekijöiltä erikseen kysyttävää suostumusta . Opiskelijoille tarjottiin myös oikeus kieltää videonsa käyttö em . tarkoituksissa . Kurssin jälkeen Möysän koulun oppilaiden tekemät videot eivät olleet missään käytössä ilman erikseen kysyttävää lupaa , eikä oppilailla ollut pääsyä LO - opiskelijoiden tekemiin videoihin . TUTKIMUSKYSYMYS JA – MENETELMÄ Tutkimuksessa vastattiin seuraavaan tutkimuskysymykseen : Miten luokan - opettajaopiskelijat kokevat videovälitteisen kokeellisten töiden ohjaamisen osana fysiikan didaktiikkaa ? Tutkimuskysymykseen pyrittiin vastaamaan teettämällä asiaa koskeva kysely opetuskokeiluun osallistuneiden LO - opiskelijoiden parissa . Kysely toteutet - tiin kurssin Moodle - verkkoalustalla osana kurssin anonyymiä palauteky - selyä . Näin varmistettiin , että kaikki vastanneet olivat kurssin opiskelijoita , kukin heistä saattoi vastata korkeintaan kerran ja vastauksia ei voitu yhdistää yksittäisiin vastaajiin . Vastausaikaa oli kaksi viikkoa kurssin päättymisen jälkeen . LO - opiskelijoiden kokemusta videovälitteisestä opetuskokeilusta kartoitettiin yhdellä seitsemänosaisella Likert - asteikollisella kysymyksellä ja kahdella avoimella kysymyksellä . Suljettu kysymys oli seuraava : ”Kuinka paljon opit kurssin videoprojektissa ( sis . videoiden suunnittelu ja toteutus sekä oppilai - den videoiden katsominen ja analysointi ) a ) fysiikan sisällöistä ; b ) fysiikan opettamisesta ; c ) kokeellisesta työskentelystä ; d ) fysiikan luonteesta tieteenä ; e ) oppilaiden käsityksistä ja valmiuksista ; f ) opetussuunnitelman perusteista fysiikan osalta ; g ) tieto - ja viestintätekniikan käytöstä” . Jokaiseen näihin seitsemään alakysymykseen , jotka liittyivät kurssin tavoitealueisiin ( ks . Johdanto ) , vastattiin neliportaisella Likert - asteikolla ’en mitään’ , ’vain vähän’ , ’melko paljon’ tai ’hyvin paljon’ . Avoimet kysymykset puolestaan kuuluivat yksinkertaisesti ”Mikä oli hyvää videoprojektissa ? ” ja ”Mikä oli huonoa videoprojektissa ? ” . 130 opiskelijaa vastasi kyselyyn . Kurssille oli 145 opiskelijaa , joista 136 jatkoi kurssin loppuun asti ja osallistui tenttiin . Vastausprosentti oli huomattavan korkea johtuen siitä , että opiskelijoita painokkaasti pyydettiin ja he ilmeisesti myös halusivat antaa palautetta tästä uudenlaisesta opetusmuodosta . Lähes kaikki vastaajat antoivat myös sanallista palautetta kaikkiin avoimiin kysymyksiin , joten saatu aineisto oli rikasta . Vastanneiden sukupuolijakauma vastasi kurssilaisten jakaumaa ( naisia 87 % , miehiä 13 % ) . Suljettujen Likert – tyyppisten kysymysten vastaukset analysoitiin yksinker - taisella tilastollisella frekvenssianalyysillä , jonka tavoitteena oli vain antaa kokonaiskuva siitä , mihin tavoitealueisiin liittyviä asioita ja valmiuksia LO - opiskelijat kokivat oppineensa opetuskokeilussa . MALU , Turku 2015 64 Kuvaa opiskelijoiden oppimiskokemuksesta tarkennettiin analysoimalla avointen kysymysten vastauksia . Opiskelijoiden sanallisia vastauksia tarkasteltiin luokittelemalla ne aluksi tavoitealueittain . Kvalitatiivisen sisältöanalyysin ( Patton , 1990 ) tavoitteena oli löytää aineistosta teemat ja väitteet , jotka toistuvat vastauksissa ja kuvaavat aineistoa kokonaisuutena . TULOKSET Suljettujen kysymysten vastausjakaumasta ( Taulukko 1 ) nähdään , että LO - opiskelijat kokivat oppineensa opetuskokeilussa kaikilla kurssin tavoitealueil - la . Eniten – yli 90 % : n mielestä vähintään ’melko paljon’ – opiskelijat sanoivat oppineensa fysiikan opettamisesta ( b ) ja kokeellisesta työskentelystä ( c ) . Myös fysiikan sisällöistä ( a ) , oppilaiden käsityksistä ja valmiuksista ( e ) ja TVT : n käytöstä ( g ) opiskelijat kokivat oppineensa huomattavasti ( yli 80 % : n mielestä vähintään ’melko paljon’ ) . Vähiten oppimishyötyä nähtiin tavoitealueissa ’fysiikan luonne tieteenä’ ( d ) ja ’OPS : n perusteet fysiikan osalta’ ( f ) , joissa 40 - 50 % vastaajista sanoi oppineensa melko vähän tai ei mitään . Taulukko 1 . Vastausten jakauma suljettuun Likert - asteikolliseen kysymyk - seen . Vastausten lukumäärän jälkeen on esitetty sulkeissa prosenttiosuudet kaikista vastanneista ( n = 130 ) kokonaisluvuiksi pyöristettyinä . Kuinka paljon opit videoprojektissa… hyvin paljon melko paljon melko vähän en mitään a ) fysiikan sisällöistä 28 ( 22 % ) 77 ( 59 % ) 25 ( 19 % ) 0 ( 0 % ) b ) fysiikan opettamisesta 58 ( 45 % ) 62 ( 48 % ) 9 ( 7 % ) 1 ( 1 % ) c ) kokeellisesta työsken - telystä 60 ( 46 % ) 63 ( 48 % ) 63 ( 48 % ) 0 ( 0 % ) d ) fysiikan luonteesta tieteenä 15 ( 12 % ) 61 ( 47 % ) 52 ( 40 % ) 2 ( 2 % ) e ) oppilaiden käsityksistä ja valmiuksista 48 ( 38 % ) 53 ( 41 % ) 23 ( 18 % ) 4 ( 3 % ) f ) OPS : n perusteista fysiikan osalta 12 ( 9 % ) 52 ( 40 % ) 62 ( 48 % ) 3 ( 2 % ) g ) TVT : n käytöstä 48 ( 37 % ) 60 ( 46 % ) 19 ( 15 % ) 3 ( 2 % ) Opiskelijoiden vastaukset avoimiin kysymyksiin heijastelivat tätä suljettujen kysymysten tulosta ja antoivat lisäymmärrystä siitä , miten opetuskokeilu Laherto & Laherto 65 toimi eri tavoitealueilla . Seuraavassa esitämme , miten opiskelijat sanallisissa vastauksissaan kuvasivat eri tavoitealueiden toteutumista . Sitaatteja pyrittiin valitsemaan niin , että ne kuvaavat opiskelijoiden vastauksia edustavasti sekä teemaltaan että sävyltään . Sitaateista on korjattu muutamia ilmeisiä kirjoitus - virheitä . Fysiikan opettamiseen ( b ) ja kokeelliseen työskentelyyn ( c ) liittyvissä vastauksissaan opiskelijat korostivat oppineensa näitä asioita erityisen paljon siksi , että videoiden suunnittelu pakotti miettimään ja kokeilemaan opetus - ratkaisuja itse hyvin käytännöllisesti : Yritettiin pilkkoa video helposti hyödynnettäväksi ja eheäksi kokonaisuu - deksi , josta oppilaat saisivat mahdollisimman paljon irti , joten pohdittiin paljonkin fysiikan opetuksen didaktista puolta . [ … ] Sai todella miettiä , kuinka jonkun asian opettaa oppilaille , mikä oli loistavaa ! Siihen saa mahdollisuuden hämmästyttävän harvoin täällä . Videota varten sai itsekin suorittaa kokeen , mikä edisti oppimista ja herätti uusia mielenkiintoisia kysymyksiä . Opiskelijat pitivät kokeilua tehokkaana myös fysiikan sisältöjen ( a ) ja käsitteiden oppimiseen . Opiskelijoiden sanallisessa palautteessa näkyy vanha viisaus siitä , että opettaessa asiat oppii myös itse . Ymmärsin ja hahmotin fysiikan ilmiöitä paremmin kun olin itse pyrkinyt saamaan ne mahdollisimman ymmärrettävään muotoon ja havainnollista - nut niitä . Oppilaatkin hyötyvät siitä , että opettajalla on itsellään selkeä kuva opittavasta asiasta . Joutui todella pohtimaan , jotta ymmärtäisi sen ilmiön , mitkä oli tarkoitus havannollistaa . Videoprojekti innosti pohtimaan tiettyjä fysiikan sisältöjä ja niiden havain - noimista syvällisemmin . Opetuskokeilussa hyödynnettiin videokameroita , editointiohjelmia , pilvipal - veluja ja opiskelijoiden omia laitteita . Opiskelijat nostivat tämän tieto - ja viestintätekniikan ( g ) opettelun tärkeäksi ja tarkoituksenmukaiseksi osaksi kokeilua . Samalla monet huomauttivat , että LO - koulutuksessa nämä tavoitteet usein edelleen laiminlyödään . Mahtava idea , hyvin käytetty tietoteknisten laitteiden vahvuuksia hyväksi . Suurimmalta osalta meistä opiskelijoista varmasti löytyy itseltään jo puhe - lin / tabletti jolla pystyy ottamaan hyvää videokuvaa . Tämä herätti ajatte - lemaan , että niitä tosiaan voi käyttää opetuskäytössäkin . Koin myös projektin itselleni hyödylliseksi siinäkin mielessä , että teknolo - giakammoni pienentyi . MALU , Turku 2015 66 Oli hyvin virkistävää , että pääsimme käyttämään tieto - ja viestintätekniik - kaa , sillä sen käyttöä on liian vähän mukana luokanopettajan koulutukses - sa . Itselle TVT : n käyttö ei tuota ongelmia , mutta jotkut saivat vielä hyvää harjoitusta ihan perustietokoneohjelmien käytössä ja se taito ei varmasti mene koskaan hukkaan . Toisaalta monet kaipasivat enemmän ohjausta ja eksplisiittistä huomiota TVT : n käyttöopiskeluun . Ohjeistus heikohkoa videon tekemiseen . Elämäni eka videointiprojekti aiheutti kohtuullisen paljon stressiä Kukaan ryhmässämme ei tainnut osata käyttää videoeditointilaitteita kovin hyvin , ja päädyimme siksi tekemään sellaisen videon , jossa editointia ei tarvinut . Eli sitä puolta ei oppinut nytkään , kun ei ollut aikaa opetella sitä erikseen . Ehkä odotetustikin eniten sanallista opiskelijapalautetta – sekä kehuja että kehitysideoita – kirvoittivat asiat , jotka liittyivät oppilaskontaktiin , mikä olikin kokeilun erikoispiirre . Kokeilu näytti tehokkaasti avanneen opiskelijoiden silmiä oppilaiden käsityksiin ja valmiuksiin ( e ) fysiikan suhteen . [ … ] Lisäksi projektin aikana oli jatkuvasti mietittävä lasten osaamisen tasoa ja valmiuksia Etenkin palautevideo opetti huomaamaan kuinka vaikeaa lasten on pohtia fysiikan ilmiöitä . Opiskelijat arvostivat todella korkealle sitä , että saivat opetusvideoistaan palautetta ”oikeilta oppilailta” . Tämä oli opiskelijoiden mielestä poikkeuksel - lista , hyödyllistä ja innostavaa : Oppilaiden tekemien videoiden avulla pystyi reflektoimaan sitä , mikä oman ryhmän videossa oli onnistunut ja mikä ei , ja mitkä asiat oli opittu ja mitkä taas ehkä jääneet epäselväksi . Tehtiin töitä OIKEIDEN oppilaiden kanssa ja palaute omasta opettamisesta oli välitön . Erinomainen työskentelymuoto , sopisi muillekin OKL : n kurs - seille ! Oli mukavaa että yhteistyökoulun oppilaat olivat yhtä innostuneita kuin me opiskelijat ! Toisaalta , moni opiskelija oli lopulta myös pettynyt palautteen määrään ja laatuun : [ … ] oppilailta saatu palaute jäi omalla ryhmällä todella vähäiseksi . Olisi ollut mukavampi kuulla enemmän pohdintoja , mutta ei tajuttu ohjeistaa niitä kai tarpeeksi . . . Oppilaiden palaute olisi voinut olla osan kohdalta hieman parempi , kuin 10 sek . pätkä vessasta . Laherto & Laherto 67 Oppilaille tuli niin paljon tekemistä , että vastausvideo jäi vähän tyngäksi koska opettaja ei ole ehtinyt seuraamaan , mitä oppilaat tekevät ja puhuvat . Toimiva kokeellisen työn ohjeistus oli selvästikin haastava antaa videon välityksellä . Kun video oli tehty , opetustilannetta ei ollut enää mahdollista korjata tai hienosäätää . Ei pystynyt kommunikoimaan oppilaiden kanssa , ei antamaan palautetta tai neuvoa . Meidän vastauksessa oppilaat eivät juurikaan pohtineet ilmiö - tä , ja olisikin ollut kiva jos vuorovaikutusta olisi voinut jatkaa . Näistä huonoista kokemuksista huolimatta lähes kaikki opiskelijat näkivät oppilaskontaktin – videovälitteisenkin – tekijänä , joka todella motivoi poikkeuksellisella tavalla : Oli opiskelijalle erityisen motivoivaa , että kurssitehtävä tuli oikeasti oppi - laiden käyttöön : se motivoi tekemään videoista sisällöllisesti hyvän ja pakotti myös miettimään asioita didaktisesta näkökulmasta . Tähän motivoivuuteen monilla opiskelijoilla liittyi myös ajatus siitä , että opetuskokeilussa tehtiin autenttista opetusta , josta oli konkreettista hyötyä – sekä Möysän koululaisille välittömästi että opiskelijoille itselleen jatkoa ajatellen : Näennäisissä opetustuokioiden suunnittelussa jää yritys yleensä alle kyky - jen . Videoiden suunnittelussa sen sijaan keskittyi hieman enemmän kun tiesi sen menevän ns . todelliseen käyttöön . Tällaisia projekteja saisi olla muissakin aineissa , koska projektin kautta muodostuu yhteys työelämään . Omaa videota mahdollisuus käyttää omassa opetuksessa . Muiden videois - ta hyviä ideoita . Oppilailta saatu palaute ainutlaatuista . Molemminpuoli - nen hyöty . Vähiten oppimishyötyä nähtiin tavoitealueissa fysiikan luonne tieteenä ( d ) ja OPS : n perusteet fysiikan osalta ( f ) , vaikka joitain havaintoja opiskelijat niihinkin liittyen tekivät : Lisäksi opetettavaa ilmiötä tuli pohdittua oikein kunnolla , mikä syvensi ymmärrystä fysiikan luonteesta . Oli myös pakko tutustua kunnolla opetussuunnitelmaan , ja tämä oli miele - käs tilaisuus pohtia menetelmiä niiden käsittelemiseksi . JOHTOPÄÄTÖKSET Kokeilun toimivuus osana LO - koulutusta Opiskelijapalautteen perusteella tässä raportoitu videovälitteinen opetusko - keilu toteutti luonnontieteiden didaktiikalle asetettuja tavoitteita ( Opinto - MALU , Turku 2015 68 opas , 2015 ; Tutkintovaatimukset , 2012 ) monipuolisesti ja lisäksi paransi LO - opiskelijoiden mielikuvaa fysiikasta ( vrt . Kapucu , 2014 ) . Erityisesti kokeilu edisti niitä tavoitealueita , joiden toteuttamisessa on vaikeuksia perinteisin OKL : n kurssien menetelmin : TVT : n käyttötaidot sekä ymmärrys oppilaiden käsityksistä ja valmiuksista . Samalla toki huomattiin rajoitteet , joita on videovälitteisessä kontaktissa oikeisiin oppilaisiin : on hyvin hankala suunni - tella tehokasta opetusta ja kokeellisen työn ohjeistusta , jossa oppilaita ei pääse ohjaamaan paikan päällä ja tilanteen mukaan . Tähän liittyy myös mahdolli - nen eettinen ongelma ’oppilaiden kustannuksella’ oppimisesta , jota LO - opiskelijat eivät kuitenkaan nostaneet esiin : ohjauskontaktin rajallisuudesta johtuen joillekin oppilaille saattaa jäädä vääriä käsityksiä opetettavista asioista . Kokeilu osoitti kokeellisten töiden ohjauksen haastavuuden ; kuten eräs opiskelija havaitsi : oppilaiden palautevideosta kävi harvinaisen selväksi , että opettajaa tarvi - taan , pelkät kokeet ei millään riitä fysiikan tiedon omaksumiseen . Opiskelijat pitivät opetusvideoiden tekemistä otollisena tapana TVT : n käytön opiskeluun , ja moittivat usein samalla sitä , että yleensä ottaen opinnoissaan on opetusteknologiakoulutusta vähänlaisesti tarpeeseen ja tavoitteisiin nähden . Tämä tarve on todettu LO - koulutusta koskevassa tutkimuksessakin ( esim . Kärkkäinen & Keinonen , 2010 ; Goktas , Yildirim & Yildirim , 2009 ) . Tämäkin kurssipalaute jälleen vahvisti sen tiedon , että LO - opiskelijat kokevat paljon epävarmuutta fysiikan aineenhallinnassa ja toivovat tukea nimen - omaan siihen fysiikan didaktiikan kurssilta ( vrt . Alake - Tuenter , 2012 ; Kapucu , 2014 ) . Vaikka tässä raportoidun opetuskokeilun ensisijainen tavoite ei ollutkaan fysiikan käsitteellisen hallinnan vahvistaminen , opiskelijat arvioivat oppineensa videoita suunnitellessaan erityisen paljon juuri siitä . Ilmeisesti myös se , että videot menivät todelliseen opetuskäyttöön , motivoi vastuuntuntoiset opiskelijat omaksumaan fysiikan sisällöt syvällisesti . Palautteen perusteella opiskelijat todella arvostivat sitä tämän opetuskokeilun erityispiirrettä , että he pääsivät didaktiikan kurssilla kontaktiin oppilaiden kanssa ja saivat heiltä palautetta – kuitenkin suorituspaineita helpottavalla 100 km : n turvavälillä ! Tästä kontaktista tulleet lupaukset eivät kuitenkaan täysin toteutuneet : monet opiskelijat valittivat palautteen ohuudesta . Vastaisuudessa kurssilla kannattaisi varata enemmän aikaa kokemusten purkamiseen ja palautteen antamiseen . OKL : n opettajan antaman palautteen ohella tai sijasta opiskelijat ehdottivat vertaispalautteen järjestämistä . Lisäksi LO - opetuksen kannalta olisi selvästikin hyvä , jos koululaisilla olisi enemmän aikaa ja ohjausta käytettävissä kokeellisten töiden tekoon ja vastausvideoiden laatimiseen . Tämä on kuitenkin hankala toteuttaa tämän ensikokeilun opiskelija - ja videomäärillä , kuten seuraavassakin ilmenee . Laherto & Laherto 69 Kokeilun toimivuus alakoulun kannalta Vaikka Möysän koulun oppilasryhmä oli harjaantunut videokuvauksessa ja editoimisessa sekä pilvipalvelujen käytössä aiemmin , luokan opettajalla pääosa oppituntien ajasta kului teknisten neuvojen antamisessa ja ongelmien ratkaisemisessa . Oppilaat eivät ehtineet juuri saamaan tukea ja ohjausta vastausvideoiden sisällöllisiin kysymyksiin , mikä näkyi muutamien oppilas - ryhmien vastauksissa . Toisaalta , opiskelijat saivat nyt realistista palautetta lasten ohjeenluku - ja ymmärtämistaidoista ja fysikaalisten ilmiöiden ymmär - tämisen haasteista . Oppilaiden fysiikan oppimisen näkökulmasta katsottuna ennakkoperehtyminen aiheeseen olisi ehkä ollut jälkikäteen arvioituna kannattavaa . Möysän koulun opettajan havaintojen mukaan oppilaat kokivat hankkeen erittäin motivoivaksi ja virkistäväksi tavaksi työskennellä . Työskentelyn omaehtoisuus , oma keksiminen ja oivaltaminen sekä tekniikan innostava käyttö vastausvideoita laadittaessa olivat selvästi motivaatiota lisääviä seikkoja . Lisäksi hankkeeseen osallistunut opettaja sai uusia ideoita ja materiaaleja . Aikataulu oli ongelmallinen koululle johtuen OKL : n kurssin intensiivisyydes - tä . Vastausvideot tuli saada muutamassa päivässä kuvattua , editoitua ja tallennettua . Möysän koululla oppilaiden käytössä olleet taulutietokoneet , fysiikan havaintomateriaalit sekä hyvin toimiva langaton lähiverkko tukivat hankkeen onnistumista , kuten myös se , että oppilaat olivat aiemmin käyttä - neet hankkeessa hyödynnettyjä laitteita ja ohjelmistoja . LÄHTEET Alake - Tuenter , E . , Biemans , H . , Tobi , H . , Wals , A . , Oostenheert , I . & Mulder , M . ( 2012 ) . Inquiry - Based Science Education Competencies of Primary school Teachers : A literature study and critical review of the American National Science Education Standards . International Journal of Science Educa - tion , 34 ( 17 ) , 2609 - 2640 . Goktas , Y . , Yildirim , Z . & Yildirim , S . ( 2009 ) . Investigation of K - 12 Teachers’ ICT Competences and the Contributing Factors in Acquiring These Com - petences . New Educational Review , 17 ( 1 ) , 276 - 294 . Kapucu , S . ( 2014 ) . Salient beliefs of pre - service primary school teachers underlying an attitude " liking or disliking physics " . Science Education International , 25 ( 4 ) , 437 - 458 . Kärkkäinen , S . & Keinonen , T . , 2010 . Primary school teacher students’ perceptions of technology . Problems of Education in the 21st Century , 19 . 27 - 35 . MALU , Turku 2015 70 OPH , 2014 . Perusopetuksen opetussuunnitelman perusteet 2014 . Määräykset ja ohjeet 2014 : 96 . Opetushallitus . Opinto - opas , 2015 . Luokanopettajakoulutuksen ( kasvatustiede ) opinto - opas 2015 - 2016 . Helsingin yliopisto . Haettu 29 . 5 . 2015 https : / / weboodi . helsinki . fi / Tutkintorakenne , 2012 . Luokanopettajan koulutus ( pääaineena kasvatustie - de ) . Käyttäytymistieteellisen tiedekunnan koulutusten tutkintovaatimukset 2012 - 2015 . Helsingin yliopisto . Haettu 29 . 5 . 2015 http : / / www . helsinki . fi / behav / opiskelu / vaatimukset / 2012 - 2015 / luokanopettajan _ kasvatustiede . pdf Lastusaari , Laakkonen & Murtonen 71 KEMIAN LuK - OPISKELIJOIDEN LÄHESTYMISTAVAT OPPIMISEEN JA HALU VAIHTAA PÄÄAINETTA Mika Lastusaari 1 , Eero Laakkonen 2 & Mari Murtonen 2 1 Turun yliopisto , Kemian laitos , 2 Turun yliopisto , Opettajankoulutuslaitos Kemian oppiaineen ongelmana suomalaisissa yliopistoissa on ollut pääainetta vaihtavien määrän suuruus . Tässä tutkimuksessa selvitettiin erityisesti kemian opiskeluun liittyvällä lomakkeella kemian opiskelijoiden lähestymistapoja oppimiseen ja niiden yhteyttä halukkuuteen vaihtaa pääainetta . Tutkimuksen tuloksena oli , että pääaineen vaihtoa haluavilla on pinnallisempi lähestymistapa oppimiseen kuin niillä , jotka jatkavat opintojaan kemia pääaineenaan . Siirryttäessä perusopinnoista aineopintoihin vaihtohalukkaiden määrä laskee huomattavasti ja käytännöllinen syväoppimisen lähestymistapa lisääntyy . Vaihtohalukkuutta voitaisiin mahdollisesti vähentää kehittämällä opiskelijoiden syväoppimisen lähestymistapaa . JOHDANTO Turun yliopistossa aloittaa kemian pääaineopinnot vuosittain 60 opiskelijaa . Toisena opintovuonna pääaineopiskelijoiden määrä on noin 15 - 20 , joista kandidaatin tutkinnon suorittaa käytännössä jokainen . Pääainetta vaihtavien määrä on siis hyvin suuri ja tämä aiheuttaa ongelmia oppiaineelle : Ensimmäi - sen vuoden opetukseen tarvitaan paljon opetusresursseja , mutta tutkinnoista saatava rahoitushyöty laitoksen toiminnalle jää näihin nähden pieneksi . Tämä pulma ei koske pelkästään Turun yliopistoa , sillä muissakin yliopistokau - pungeissa , joissa tarjotaan lääketieteen opetusta , on samankaltainen tilanne ( esim . Ruuska , 2010 ; Nykänen , 2013 ) . Ongelma ei myöskään koske vain kemian oppiainetta , vaan myös muita aineita , esimerkiksi fysiikkaa , joiden ensimmäisten vuosien opetus toimii hyvänä valmennuksena lääketieteen opiskelijoiksi haluaville ( esim . Ruuska , 2010 ; Nykänen , 2013 ) . Yliopisto - opiskelijoita tutkittaessa on huomattu , että opiskelijoiden lähesty - mistavat oppimiseen ovat erilaisia . Jotkut opiskelijat lähestyvät oppimista pinnallisemmin yrittäen muistaa opittavia asioita ulkoa ( pintasuuntautunut oppiminen ) , kun taas toiset ovat syvällisempiä lähestymistavassaan , yrittäen ymmärtää opittavana olevia asioita ( syväsuuntautunut oppiminen ) ( esim . Lastusaari & Murtonen , 2013 ; Lindblom - Ylänne , Parpala & Postareff , 2014 ) . Syväsuuntautuneella opiskelijalla on tyypillisesti positiivisia ajatuksia opittavaa asiaa kohtaan . Hän mieltää opittavan asian merkitykselliseksi ja tuntee oppimisessa positiivista haastetta , innostusta sekä mielihyvää . Pintasuuntautuneelta oppijalta taas tällaiset positiiviset tuntemukset puuttu - vat ( Howie & Bagnall , 2013 ) . Täten lähestymistapa on yhteydessä motivaati - oon ja kiinnostukseen , jotka ovat keskeisiä tekijöitä opiskelijoiden pohtiessa pääaineen vaihtoa . MALU , Turku 2015 72 Opiskelijoiden lähestymistapoja oppimiseen on tutkittu jo 1970 - luvulta lähtien ( Marton & Säljö , 1976 ; Biggs , 1987 ; Entwistle & Ramsden , 1983 ) . Uudemmassa tutkimuksessa on huomattu yksiköiden välisten erojen lisäksi , että lähestymistavat eivät ole yksilökohtaisestikaan pysyviä , vaan vaihtelevat ajan , tilanteen ja opittavan sisällön suhteen ( Lindblom - Ylänne , Parpala & Postareff , 2014 ; Vermunt & Vermetten , 2004 ) . Opiskelijat saattavat olla hyvinkin joustavia käyttämiensä lähestymistapojen suhteen . Opiskelija saattaa vaikkapa samana päivänä toimia pintasuuntautuneesti yhdellä opintojaksolla ja syväsuuntautuneesti toisella opintojaksolla . Tähän vaikuttaa opiskelumotivaatio , esimerkiksi kuinka kiinnostavana opiskeltavaa aihetta pidetään , tai jokin muu seikka , kuten henkilökohtainen tilanne . Syväsuuntautunut oppimistapa on tutkimusten mukaan yhteydessä hyviin oppimistuloksiin , joten opiskelijoita tulisi tukea sen kehittämisessä . Tutki - musnäyttöä onkin , että syväsuuntautuneen lähestymistavan käyttöä voidaan vahvistaa opetuksessa ( esim . Baeten , Kyndt , Struyven & Dochy , 2010 ; Dolmans , Wolfhagen & Ginns , 2010 ) . Jos esimerkiksi tiedämme , että tietyn alan opiskelijoilla on tapana suuntautua tietyssä oppimistilanteessa tietyllä tavalla , tähän voidaan yrittää saada muutos opetuksen avulla . Oppimisen lähestymistapojen tutkimuksissa on pääsääntöisesti käytetty yleisiä , kaikille aloille soveltuvia lomakkeita ( esim . Lovatt ym . , 2007 ) . Kemian opiskelussa on kuitenkin erityispiirteitä , jotka eivät tule yleisissä lomakkeissa esille . Laboratorioharjoitusten runsas määrä on tyypillistä kemian opinnoille , ja niihin liittyvää oppimista ei olemassa olevilla lomakkeilla ole mahdollista tutkia . Aiemmassa tutkimuksessamme testasimme lomaketta , jossa oli otettu huomioon laboratorioharjoitukset osana oppimisprosessia ( Lastusaari & Murtonen , 2013 ) . Tulosten perusteella nimenomaan käytännöllinen sy - väoppimisen lähestymistapa laboratoriotyöskentelyssä on tyypillistä opiskelijoille , jotka menestyvät opinnoissaan ja haluavat jatkaa opintojaan . Seuraavassa tutkimuksessamme kehitimme kemian oppimisen lähestymista - poja mittaavaa lomaketta validoimalla ChemApproach - lomakkeen ( Lastusaa - ri , Laakkonen & Murtonen , 2016 ) . Tässä tutkimuksessa tavoitteena on testata kehitetyn ja validoidun lomakkeen toimivuutta Turun yliopiston kemian pääaineopiskelijoilla . Tutkimuskysymyksenä on , ovatko oppimisen lähesty - mistavat yhteydessä pääaineen vaihtohalukkuuteen . MENETELMÄT JA AINEISTO Oppimisen lähestymistapojen , eli syvä - ja pintaoppisen tutkimukseen tarkoitettuja yleisiä kyselyjä on olemassa monia , kuten Biggsin Study Process Questionnaire ( SPQ ) , Entwistlen Approaches to Study Skills Inventory for Students ( ASSIST ) ( esim . Lovatt ym . , 2007 ) ja Inventory of Learning Styles in Higher Education ( ILS ) ( Vermunt , 1994 ) . Näitä onkin käytetty useiden alojen , kuten psykologian ( Lonka & Lindblom - Ylänne 1996 ) , lääketieteen ( Dolmans ym . 2010 ) ja kemian ( Zeegers 2001 ) opiskelijoiden tutkimiseen . Erityisesti Lastusaari , Laakkonen & Murtonen 73 kemian oppimisen lähestymistapojen tutkimiseen ei kuitenkaan ole olemassa lomaketta . Tässä työssä aineisto kerättiin 17 väittämän ChemApproach - kyselylomakkeella . Lomake pohjautuu teoreettisesti edellä mainittuihin kaavakkeisiin , mutta se on suunnattu erityisesti kemian opiskeluun . Kysely - kaavake on validoitu aiemmassa tutkimuksessa ( Lastusaari , Laakkonen & Murtonen , 2016 ) . Väittämät on esitetty Taulukossa 1 . Taulukko 1 . ChemApproach - lomakkeen väittämät ja niiden jakautuminen ryhmiin . Subsurf 1 ) Monet oppimani asiat jäävät irrallisiksi , eivätkä linkity osaksi suurempaa kokonaisuutta . 2 ) Usein lukiessani luentomateriaalia en ymmärrä mihin uusi asia liittyy . 3 ) Joudun pänttäämään päähän asioita ilman , että minulla olisi tilaisuutta ymmärtää niitä . 4 ) Usein en kemian luennolla ymmärrä mihin uusi asia liittyy . Tecsurf 1 ) Alleviivaan kirjan tekstiä lukiessani tenttiin 2 ) Jaan tenttimateriaalin osiin , jotka opettelen tenttiä varten . 3 ) Lukiessani kemian tenttiin , yritän tehdä kokonaisuuksista yhteenvedon omin sanoin . 4 ) Teen omia muistiinpanoja , kun luen tenttiin . 5 ) Jotta oppisin asian paremmin , rakentelen muistisääntöjä . Actdeep 1 ) Luennon jälkeen jään usein miettimään opetettuja asioita . 2 ) Yleensä etsin ja luen kurssiin liittyvää lisämateriaalia . 3 ) Jään usein pohtimaan tieteellisten tekstien herättämiä ajatuksia ja niiden keskinäi - siä kytkentöjä . 4 ) Etsin huolellisesti perusteluja ja näyttöä muodostaakseni omat päätelmäni opittavasta asiasta . Pradeep 1 ) Harjoitustöitä on mukava tehdä . . 2 ) Olen usein ymmärtänyt kemiallisen asian vasta tehtyäni aiheesta harjoitustyön . 3 ) Tehdessäni harjoitustyötä yritän yleensä selvittää mihin siihen kuuluvat työvai - heet perustuvat . 4 ) Kemiallisen ilmiön voi oppia vasta , kun on tehnyt aiheesta kokeellisen työn . Vastaukset väittämiin kerättiin viiden pisteen Likert - asteikolla ( 1 = täysin eri mieltä – 5 = täysin samaa mieltä ) . Väittämissä keskityttiin seuraaviin yleisiin aihepiireihin : valmistautuminen kemian tenttiin , kemian luennot , kemian opiskelutavat ja harjoitustyöt . Väittämien lisäksi kaavake sisältää lisäksi kuusi taustakysymystä ( nimi , sukupuoli , pääaine , halukkuus vaihtaa pääainetta , haluttu uusi pääaine ja opiskeluvuosi ) . Keräys tehtiin harjoitustöiden ja MALU , Turku 2015 74 luentojen yhteydessä ja vastaamiseen kului n . 10 min . Tarkoituksena oli saada opiskelijoilta tietoa , jota voisi käyttää hyväksi opetuksen suunnittelussa . Aineisto kerättiin 94 perusopintovaiheen sekä 40 aineopintovaiheen kemian pääaineopiskelijalta Turun yliopistossa 2013 - 2015 . Kukin opiskelija otti osaa kyselyyn vain kerran , joten vastausten kokonaismäärä oli 134 . Näistä 54 % oli naisia ja 46 % miehiä . Kaikkia tuloksia tarkasteltiin suhteessa halukkuuteen vaihtaa pääainetta sekä opintovaiheeseen ( perus - tai aineopinnot ) . Tilastolli - set analyysit toteutettiin käyttämällä IBM SPSS Statistics v22 - sekä Mplus - ohjelmia . TULOKSET Sopivuus oletettuun faktorirakenteeseen ChemApproach - kaavaketta validoitaessa ( Lastusaari , Laakkonen & Murto - nen , 2016 ) on todettu , että väittämät jakaantuvat neljään faktoriin : subsurf ( submissive surface learning , eli lannistunut pintaoppiminen ) , tecsurf ( technical surface learning , eli erilaisin pinnallisiin opiskelutekniikoihin keskittyvä oppiminen ) , actdeep ( active deep learning , eli aktiivinen sy - väoppiminen ) ja pradeep ( practical deep learning , eli käytäntöä painottava syväsuuntautunut oppiminen ) . Näitä faktoreita kuvaavat suuntautumistyypit voidaan määritellä seuraavasti : Subsurf - opiskelija yrittää opiskella kemiaa passiivisesti ulkoa lukemalla . Hän ei joko ole kiinnostunut oppimaan tai tekemään työtä oppiakseen , tai hän ei usko omiin kykyihinsä oppia kemiaa . Tecsurf - opiskelija on aktiivisempi : hän opettelee asioita ulkoa käyttämällä aktiivisesti hyväkseen mieleenpainamistekniikoita . Actdeep - opiskelija etsii aktiivisesti opittavaan asiaan liittyvää täydentävää lisämateriaalia ja prosessoi kognitiivisesti asioita muodostaakseen kokonaiskuvan aihepiiristä , eli opiskelee syväsuuntautuneesti . Pradeep - opiskelija on myös syväsuuntautu - nut . Hän painottaa erityisesti kemian harjoitustöitä keinona oppia ja ymmär - tää kemian teoriapohjaa . Myös tässä tutkimuksessa varmistettiin konfirmato - risen faktorianalyysin ( CFA ) avulla , että kyseinen faktorimalli sopii käsiteltä - vään aineistoon . Faktoriratkaisun tulokset osoittavat mallin toimivan , sillä yhteensopivuutta mittaavien indikaattorien CFI ja TLI arvot olivat yli 0 , 9 ja RMSEA - sekä SRMR - arvot alle 0 , 08 ( Taulukko 2 ) ( Hu & Bentler , 1999 ; Little , 2010 ) . Analyysin tulosten tarkempi kuvaus on esitetty kuvassa 1 . Seuraavaksi muodostettiin summamuuttujat ja laskettiin näihin kuuluvien väittämien sisäistä konsis - tenssia mittaavat Cronbachin alfan arvot . Myös näissä saatiin hyvät arvot ( yli 0 , 6 ; taulukko 3 ) , mikä myös vahvisti neljän faktorin mallin olevan toimiva . Lastusaari , Laakkonen & Murtonen 75 Taulukko 2 . CFA - mallin sopivuus aineistoon . Suure Arvo Khi 2 - testi : Arvo 139 , 95 Vapausasteet 110 P - arvo 0 , 028 CFI 0 , 94 TLI 0 , 92 RMSEA - arvo ( 90 % CI ) 0 , 05 ( 0 , 02 ; 0 , 07 ) SRMR - arvo 0 , 08 CFI = Comparative Fit Index ; TLI = Tucker - Lewis Index ; RMSEA = Root Mean Square Error of Approximation ; SRMR = Standardized Root Mean Square Residual Kuva 1 . CFA - mallin estimointitulokset . MALU , Turku 2015 76 Taulukko 3 . Summamuuttujien tilastolliset tunnusluvut . Muuttuja Väittämien määrä Keskiarvo Keskihajonta Cronbachin  subsurf 4 2 , 35 0 , 67 0 . 78 tecsurf 5 3 , 21 0 , 81 0 , 67 actdeep 4 2 , 87 0 , 77 0 , 71 pradeep 4 3 , 23 0 , 74 0 , 65 Lähestymistavat oppimiseen perus - ja aineopintovaiheessa Aluksi tarkasteltiin opiskelijoiden lähestymistapoja opintojen eri vaiheissa . Taulukosta 4 näkyy , että muiden lähestymistapojen yleisyys on samantyyp - pistä opintojen eri vaiheissa , paitsi käytännöllisen syväoppimisen ( pradeep ) . Tämä on samansuuntainen tulos kuin aiemmassa tutkimuksessamme ( Lastusaari & Murtonen , 2013 ) . Taulukko 4 . Keskiarvot , keskihajonnat , ANOVA - tulokset ja Cohenin d - arvot * . Lähestymistapa Keskiarvo / keskihajonta ANOVA d df F p Perusopinnot ( N = 94 ) Aineopinnot ( N = 40 ) subsurf 2 , 33 / 0 , 72 2 , 40 / 0 , 52 1 , 132 0 , 29 0 , 59 0 , 11 tecsurf 3 , 21 / 0 , 85 3 , 23 / 0 , 70 1 , 133 0 , 01 0 , 91 0 , 03 actdeep 2 , 83 / 0 , 78 2 , 97 / 0 , 76 1 , 131 0 , 93 0 , 34 0 , 18 pradeep 3 , 12 / 0 , 66 3 , 50 / 0 , 84 1 , 132 4 , 12 0 , 01 0 , 53 * Raja - arvot : 0 , 2 ”pieni” ; 0 , 5 ”keskisuuri” ; 0 , 8 ”suuri” . Pääaineen vaihtohalukkuus Koko tutkimukseen vastanneista kemian pääaineopiskelijoista 59 ( 44 % ) halusi vaihtaa pääainetta . Yleisin pääaine , johon haluttiin vaihtaa , oli lääketiede ( 86 % ) ja toiseksi yleisin biokemia ( 12 % ) . Perusopintovaiheessa vaihtohalukkaita oli 57 % , kun taas aineopintovaiheen opiskelijoista enää vain viisi halusi vaihtaa pääainetta . Tämä vastaa 13 % , eli aineopintovaiheessa vaihtohalukkuus on laskenut selvästi . Seuraavaksi tutkittiin pääaineen vaihtohalukkuuden ja lähestymistapojen välistä yhteyttä . Tulosten ( taulukko 5 ) perusteella vaihtohalukkaiden ryhmässä on tilastollisesti merkittävästi enemmän lannistunutta pintasuun - tautunutta ( subsurf ) lähestymistapaa ja vähemmän aktiivista ( actdeep ) ja käytännöllistä ( pradeep ) syväsuuntautunutta lähestymistapaa kuin vaihtoha - luttomien ryhmässä . Sen sijaan teknistä pintasuuntautunutta ( tecsurf ) lähestymistapatyyppiä on kummassakin ryhmässä käytännössä yhtä paljon . Koska subsurf kuvaa lannistunutta haluttomuutta kemian opiskelussa ja actdeep sekä pradeep taas täysin päinvastaista lähestymistapaa , osoittavat tulokset , että vaihtohalukkaat eivät ole yhtä syväsuuntautuneita kemian oppimisessaan kuin vaihtohaluttomat . Vaihtohalukkaat ovat ensimmäisen Lastusaari , Laakkonen & Murtonen 77 vuoden opiskelijoita ja aineisto on kerätty 1 . vuoden syksynä , joten esimer - kiksi kiinnostuksen voidaan olettaa vaikuttavan tuloksiin voimakkaasti oppimisen lähestymistavan lisäksi . Täytyy myös ottaa huomioon , että yksikään opiskelija ei noudata puhtaasti yhtä näistä lähestymistavoista , vaan kaikilla on piirteitä useammasta lähestymistavasta . Taulukko 5 . Keskiarvot , keskihajonnat , ANOVA - tulokset ja Cohenin d - arvot * . Lähestymistapa Keskiarvo / keskihajonta ANOVA d df F p Haluaa vaihtaa ( N = 59 ) Ei halua vaihtaa ( N = 71 ) subsurf 2 , 48 / 0 , 71 2 , 22 / 0 , 60 1 , 128 5 , 24 0 , 02 0 , 40 tecsurf 3 , 19 / 0 , 86 3 , 26 / 0 , 76 1 , 129 0 , 26 0 , 61 0 , 09 actdeep 2 , 71 / 0 , 71 2 , 98 / 0 , 80 1 , 127 4 , 13 0 , 04 0 , 36 pradeep 3 , 05 / 0 , 67 3 , 38 / 0 , 76 1 , 128 6 , 78 0 , 01 0 , 46 * Raja - arvot : 0 , 2 ”pieni” ; 0 , 5 ”keskisuuri” ; 0 , 8 ”suuri” . POHDINTAA Tämän tutkimuksen tavoitteena oli selvittää , eroavatko Turun yliopistossa kemiaa pääaineenaan opiskelevien opiskelijoiden lähestymistavat oppimiseen sen perusteella haluavatko he vaihtaa pääainetta vai eivät . Aluksi tarkasteltiin opiskelijoiden lähestymistapoja opintovaiheen perusteella . Perus - ja aineopin - tojen vaiheessa olevien opiskelijoiden välillä löytyi kiinnostava ero lähesty - mistavoissa : aineopintovaiheessa opiskelevat olivat enemmän käytännöllises - ti syväorientoituneita kuin perusopintovaiheen opiskelijat . Jatkossa aineistoa tullaan keräämään samoilta opiskelijoilta useana vuonna . Tällöin saadaan selville onko kemian parissa jatkavilla alusta lähtien enemmän käytännöllistä syväorientoituneisuutta kuin opinnot lopettavilla vai johtuuko nyt havaittu ero esim . opiskelijoiden kehittymisestä opiskelun aikana tai opetuksen rakenteesta . Pääaineen vaihtohalukkuutta tutkittaessa opiskelijoiden välillä löydettiin monia eroja . Vaihtohalukkaiden ryhmästä löytyi tilastollisesti merkittävästi enemmän lannistunutta pintasuuntautunutta lähestymistapaa ja vähemmän aktiivista ja käytännöllistä syväsuuntautunutta lähestymistapaa kuin vaihtohaluttomien ryhmässä . Sen sijaan teknistä pintasuuntautunutta lähestymistapatyyppiä oli kummassakin ryhmässä käytännössä yhtä paljon . Tulokset siis osoittavat , että vaihtohalukkaat eivät ole yhtä syväsuuntautunei - ta kemian oppimisessa kuin vaihtohaluttomat . Koska erot keskiarvoissa eivät olleet tilastollisesti merkittävien erojenkaan tapauksissa kuitenkaan kovin suuria ( efektikokoa mittaavat Cohenin d - arvot 0 , 5 tai alle ) , tuntuu siltä että vaihtohalukkuutta olisi mahdollista vähentää lisäämällä opiskelijoiden syväsuuntautuneisuutta kemiaan . Aiemman tutkimuksen perusteella syväsuuntautunut lähestymistapa on yhteydessä hyvään opintomenestykseen MALU , Turku 2015 78 ja sen käyttöä voidaan vahvistaa opetuksessa ( esim . Baeten , Kyndt , Struyven & Dochy , 2010 ; Dolmans , Wolfhagen & Ginns , 2010 ) , joten kemian oppimisen suuntautumistapojen kehittäminen ja sitä kautta kiinnostuksen herättäminen voisi lisätä opinnoissa pysyjien määrää . ChemApproach - kaavake on hyvä työkalu esimerkiksi erilaisten syväsuuntautunutta oppimistapaa edistävien opetuskokeilujen tutkimuksiin jatkossa . Kiitokset Kiitämme seuraavia henkilöitä avusta aineiston keruussa : Harri Lönnberg , Helmi Neuvonen , Henri Kivelä ja Tuomas Lönnberg ( Turun yliopisto , Kemian laitos ) . Kiitämme myös Pirjo Vahvialaa and Heidi Salmentoa ( Turun yliopisto , Opettajankoulutuslaitos ) tulosten käsittelystä . Lähteet Baeten , M . , Kyndt , E . , Struyven , K . , & Dochy , F . ( 2010 ) . Using student - centred learning environments to stimulate deep approaches to learning : Factors encouraging or discouraging their effectiveness . Educational Research Re - view , 5 , 243 - 260 . Biggs , J . ( 1987 ) . Student approaches to learning and studying . Victoria : Australian Council for Educational Research . Dolmans , D . H . J . M . , Wolfhagen , I . H . A . P . , & Ginns , P . ( 2010 ) . 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On qualitative differences in learning – I : outcomes and processes . British Journal of Educational Psychology , 46 , 4 - 11 . Nykänen , S . - T . ( 2013 ) . Kemialla käydään kääntymässä . Jyväskylän ylioppilasleh - ti , 8 . 5 . 2013 . Ruuska , M . ( 2010 ) . Katoamistemppu . Ylioppilaslehti , 3 . 9 . 2010 . Vermunt , J . D . ( 1994 ) . Inventory of learning styles ( ILS ) in higher education , Tilburg University , Tilburg , the Netherlands . Vermunt , J . D . , & Vermetten , Y . J . ( 2004 ) . Patterns in student learning : Rela - tionships between learning strategies , conceptions of learning , and learning orientations . Educational Psychology Review , 16 , 359 - 384 . Zeegers , P . ( 2001 ) . Approaches to learning in science : A longitudinal study , British Journal of Educational Psychology , 71 , 115 - 132 . MALU , Turku 2015 80 COMPLEMENTING THE GUIDANCE PROVIDED BY A SIMULATION THROUGH TEACHER QUESTIONING Antti Lehtinen & Markus Hähkiöniemi University of Jyväskylä , Department of Teacher Education The interaction between the teacher and the learners when using simulations is in need of research . This study concentrates on two questioning approaches , series of probing and series of guiding questions that teachers use to guide learning with simulations . These two approaches are contrasted with non - directive and directive guidance provided by the simulation . The data was collected through screen capture videos of pre - service primary teachers teaching physics with a PhET simulation . Two cases were selected for further analysis of teacher questioning and its adaptation to the learners . Even though teachers might use the spaces for explanations created by the simulation to probe for learners’ explanations , it is possible that the guidance provided by teachers is still not based on these ideas and explanations . INTRODUCTION Computer simulations can be used as a part of inquiry - based science teaching in supporting development of hypotheses , collection of data and revising theory ( Rutten , van Joolingen , & van der Veen , 2012 ) . Unguided inquiry - based learning is ineffective while providing guidance e . g . feedback , worked examples or elicited explanations during inquiry learning benefits learners ( Alfieri , Brooks , Aldrich , & Tenenbaum , 2011 ) . The need for guidance is even greater with simulations which contain a lot of information that can be hard to perceive ( Zacharia et al . , 2015 ) . Without proper guidance , the learners have difficulties with generating hypotheses , interpreting data and regulating their inquiry learning with simulations ( de Jong & van Joolingen , 1998 ) . Research into the learning support and guidance concerning learning with simulations has been focused on the guidance provided by the simulations themselves and not the role of the teacher ( Rutten et al . , 2012 ) . This paper studies guidance provided by the teachers during learning with simulations and its interplay with guidance provided by the simulation . Key factors of successful guidance are the same for teacher - learner interaction and for guidance provided by the simulations : adaptation to the learner , fading out and support for self - regulated learning ( de Jong & Lazonder , 2014 ; van de Pol , Volman , & Beishuizen , 2010 ) . Of these three characteristics , the ability to adapt the guidance to the learners’ needs is the focus of this paper . The development of adaptive learning analytical tools which guide learners based on their learning products is still under way ( de Jong & Lazonder , 2014 ) . On the other hand , teachers can monitor and probe learners’ needs and Lehtinen & Hähkiöniemi 81 knowledge through questioning and act upon the information gained ( Ruiz - Primo , 2011 ) . Probing questions can be used to e . g . elicit hypotheses from the learners before they start to experiment or encourage learners to reflect on their actions ( Chin , 2007 ) . Teachers’ questions also can be used to guide learners or ask for factual information ( Sahin & Kulm , 2008 ) . Hähkiöniemi ( 2015 ) found that pre - service mathematics teachers who asked series of guiding questions directed learners towards an answer through a specific path whereas those who asked series of probing questions elicited learners’ thinking and directed them towards forming explanations . These two approaches for questioning bear a resemblance to directive guidance and non - directive guidance provided by simulations ( de Jong & Njoo , 1992 ) . Directive guidance steers the learners into a certain direction through e . g . hints or direct feedback . Non - directive guidance helps learners in completing certain action but doesn’t steer them into any direction . The aim of this paper is to understand how teacher guidance complements the guidance by a simulation by two questioning approaches : one emphasiz - ing probing questions and another emphasizing guiding questions . These approaches are presented through two cases from pre - service teachers who use questions differently in similar teaching situations involving simulations . METHOD Data collection The two case study pre - service teachers were selected among 33 pre - service primary teachers ( PSTs ) who were participating in a science methods course . The PSTs were assigned to teach an inquiry - based physics lesson ( length 45 mins ) to which they had to integrate a given PhET simulation . ( University of Colorado Boulder , 2016 ) These lessons were planned and taught in groups of five PSTs to learners from grades 3 to 6 . The PSTs and the learners’ guardians agreed voluntarily to take part in the research . Lehtinen , Nieminen and Viiri ( 2016 ) describes the planning and execution of the lessons . The learners used the simulations in groups of two to five with a PST guiding them . The actions with the simulations and the talk by the learners and the PST were recorded using screen capture software . The data for this paper comes from these recordings . This paper focuses on teaching physics to learners from grades 5 and 3 with the “Balancing Act” ( University of Colorado Boulder , 2016 ) simulation . This particular simulation was chosen because of the high amount of probing and guiding questions used by the PSTs with this simulation . The excerpts come from the “Game” section of the simulation where the learners were given assignments concerning balancing the seesaw . Before this section they experimented more freely with the simulation . MALU , Turku 2015 82 Data analysis Teachers’ questions were divided into four different categories : probing , guiding , factual and other questions . These categories are based on Sahin and Kulm ( 2008 ) . The shortened definitions for the categories are as follows :  Probing questions ( code 3 ) : Asking the learners to elaborate and extend their answers . The learners can also be asked about how they would solve the task at hand in a different situation or to make a hypothesis .  Guiding questions ( code 2 ) : Suggesting some procedure for the learners or otherwise aiding in the task at hand with a question . The learners can also be told to pay attention to a particular event in the simulation through questions .  Factual questions ( code 1 ) : Asking for a specific fact , a definition or an answer to an assignment . Series of factual questions are inter - preted as guiding if it is clear that the teacher has a guiding aim in mind .  Other questions ( code 0 ) : Asking non - subject related questions e . g . about classroom management . Teacher utterances were considered questions if they invited the learners to produce an oral response . The question types were coded by author 1 using Atlas . ti video analysis software from the transcribed group activities . Part of the data was also coded by author 2 . Inter - rater reliability for a sample of 101 questions ( 12 % of all the questions ) was 90 % and κ = . 832 ( 95 % CI . 733 to . 931 ) , p < . 001 . Questioning diagrams were produced in order to study how the questions appeared in series . Similar graphs have been used to study pre - service teachers’ questioning in inquiry - based mathematics lessons ( Hähkiö - niemi , 2015 ) . Transcripts were divided into event segments which were marked by a change in topic , contrast in behavior or transition to the next type of conversation or activity ( Jordan & Henderson , 1995 ) . Questions asked in the same event segment are connected by a line in the diagrams . RESULTS The cases of two female pre - service teachers ( PST A and PST B ) are presented . The case of PST A demonstrates how series of probing questions are used to elicit information and to openly invite learners from grade 3 to share their ideas . The case of PST B on the other hand shows how series of guiding questions direct the learners from grade 5 towards an answer via a pre - determined learning path . Figure 1 shows the questioning diagrams of PST A and B . Lehtinen & Hähkiöniemi 83 Figure 1 . Questioning diagrams for PST A ( upper ) and PST B ( lower ) . The episodes presented are circled . Both pre - service teachers asked series of questions including both guiding and probing questions . Yet probing questions were more prevalent in PST A’s questioning whereas guiding questions were more common in PST B’s questioning . In the following sections we analyze one questioning series from both of the teachers . The episode of pre - service teacher A – series of probing questions The assignment asks the learners to find the mass of a trash can which is in a fixed position on the seesaw 1 meter away from the fulcrum . They do this by balancing the seesaw using a brick which weighs 15 kg . The learners have placed the brick 2 meters from the fulcrum and the seesaw has balanced itself . 1 PST A : OK , now it balanced itself so how can we deduce how much does the trash can weigh ? [ probing ] 2 Learner 1 : It weighs at least more than the other one . MALU , Turku 2015 84 3 PST A : Yes , why does it weigh more ? [ probing ] 4 L 1 : Because it’s more to that direction . At the start of the episode the simulation gives the pupils information that the seesaw is balanced but it does not show the masses of the objects . In turns 1 and 3 , PST A poses probing questions which request the learners to think about the masses and reasons for them before proceeding to submit the answer . The learners have manipulated the simulation and created an interesting situation and the teacher stops the learners to think . Thus , the interplay of the guidance by the simulation and by the teacher creates a potential place for learner explanation of the phenomena . Indeed the learners articulate the qualitative idea of the relation between the masses and their distances from the fulcrum . After this qualitative idea , PST A starts to draw the learners’ attention to the quantities of the masses . 5 PST A : If it was here on the same spot , then what would it weigh ? [ probing ] 6 L 1 : 15 7 PST A : That’s right . So now that it is half as far as the other one - here is two one and zero - it’s on top of the one so how much more should it weigh than 15… its half as far [ guiding ] 8 L2 : Does this weigh 35 kilos ? 9 PST A : Why do you think it is 35 ? [ probing ] 10 L 1 : Yeah right ! 11 L 2 : I guessed . 12 L 1 : I did too . 13 L 2 : OK so let’s try 35 . ( The simulation informs them that their answer is wrong . ) In turn 5 , PST A asks learners to hypothesize about the masses in the situation they already know from their previous experiments . This probing question is based on the learners’ idea about the distances . PST A also confirms the learners’ answer in turn 7 . In this case , the guidance by the teacher comple - ments the guidance by the simulation as PST A asks them to think about the simpler situation which draws the learners’ attention to what they already know . After this , in turn 7 , PST A turns back to the quantities in the situation at hand in the simulation . Although the teacher is hinting about “half” in turn 7 , she is not pressing it much and lets the pupils guess even though they are not able to answer the probing question in turn 9 . The pupils submit 35 kg into the simulation and get the feedback that their answer is incorrect . In this case , Lehtinen & Hähkiöniemi 85 PST A lets the simulation provide the guidance by giving feedback that the answer is incorrect . After this , the learners start suggesting other values . 14 Learner 3 : What about twenty ? 15 PST A : So who thinks that why the answer - why do you think so ? [ probing ] 16 L 1 : Because well well there goes three and then one . 17 PST A : Hmm so what if the trash can would be on the same line what would it weigh ? [ probing ] 18 L 1 and L 2 : 15 . 19 PST A : Yeah and now that the trash can is half as far from the ful - crum it is half way - 20 L 1 : 30 ! 21 PST A : Try that . 22 L 2 : Are you trying 20 ? 23 L 1 : No but - yeah 20 . ( The simulation informs them that their answer is wrong . ) 24 L 2 : It not that one either - do I have to show the correct answer ? 25 L 1 : It’s 30 . 26 PST A : Now you can’t try it anymore - here comes the correct an - swer . ( The simulation informs them that the correct answer is 30 kg . ) In turn 15 , PST A again probes for reasons for learners’ answers and in turn 17 again directs their thinking towards the simpler situation . Thus , the teacher’s guidance complements the guidance by the simulation by adapting to the situation in which the learners should move from guessing to reason - ing . In turn 19 , PST A hints again about “half” but does not press for it and does not even formulate a question about it . She lets the learners to submit a wrong answer . The discussion continues after the correct answer has been shown . 27 L 1 : It would have been 30 . 28 PST A : Yeah why was it 30 ? [ probing ] 29 L 2 : Because 15 is half from 30 . 30 PST A : That’s right OK the next one . The simulation reveals the correct answer , but doesn’t provide any reasoning for it . PST A complements the guidance provided by the simulation by MALU , Turku 2015 86 probing one more time about the reasons and learners explain that the weight is half . In the episode above , the interplay of the guidance provided by the simula - tion and by the teacher gets the learners to find the reasoning for their answer . The simulation created a space for explanations and the teacher’s probing questions stopped the situation and elicited learners’ explanations . The guidance was mainly non - directive basing on learners’ own ideas . The dialogue continued based on their answers and the teacher did not steer the learners towards a pre - determined and structured learning path . The most directive guidance happened towards the end of the episode when the teacher hinted about “half” and when the simulation gave the correct answer . The episode of pre - service teacher B – series of guiding questions The assignment asks the learners from grade 5 to find a place where a weight of 40 kg balances the seesaw when a weight of 20 kg is fixed 1 m from the fulcrum . 1 PST B : Where should you put the weight of 40 kilos if the weight of 20 kilos is there ? [ factual ] ( The learners discuss where to place weight and move the weight of 40 kilos to the correct position . The simulation in - forms them that their answer was correct . ) PST B starts the episode with a factual question in turn 1 that is aimed at finding out the answer to the assignment . She doesn’t probe the learners for their reasoning before they check their answer . The teacher’s question and giving the correct answer make the guidance received thus to be directive i . e . guiding towards an answer . 2 PST B : How did you figure out that you were supposed to put it there ? [ probing ] 3 Learner 4 : This is a bit heavier than that - this is 20 kilos heavier . 4 Learner 5 : The heavier it is - 5 Learner 6 : The more in middle it should be . In turn 2 PST B probes the learners for their reasoning for their answer . The learners articulate the relation between the masses and their distances from the fulcrum qualitatively as did the learners in the previous episode . Again , an interesting situation has been created through manipulating the simulation and the teacher stops the learners to think . Thus the non - directive guidance provided by PST B complements the guidance provided by the simulation . 6 PST B : Yes , how many times is 40 kilos than 20 kilos ? [ guiding ] 7 Learners : 20 . 8 PST B : Yes and how many times ? [ guiding ] Lehtinen & Hähkiöniemi 87 9 Learner 4 : Two times . With guiding questions in turns 6 and 8 , PST B guides the learners to think about the relations of the weights . In turn 7 it is clear that the learners are having difficulties in doing so . Their initial qualitative idea is not being taken into account but instead PST B directively guides the learners based on her own strategy . 10 PST B : Yes two times heavier - well how much closer do you have the weight of 40 kilos than the weight of 20 kilos ? [ guiding ] 11 Learner 4 : One step . 12 Learner 6 : One step closer . 13 PST B : Yeah and if the other one - well how far away is this other one ? [ guiding ] 14 Learner 6 : Two steps . 15 PST B : Well and - 16 Learner 5 : Three steps all together . 17 PST B : Yes if this is the center of the balancing beam and this weight is one step this way and the other one is two steps that way but what is it… There is the distance but how - if this is two times heavier than that then how many times is this distance longer than that ? [ factual ] 18 Learner 5 : One step . 19 PST B : Ok… you can take a look at the next assignment . In turns 10 and 13 PST B asks guiding questions aimed at finding the distances of the objects from the fulcrum . She is following the same strategy as before by directing the learners towards using the ratios of the weights and their distances from the fulcrum . In turn 17 PST B asks the final factual question about the ratios . When learners still can’t give a correct answer PST B simply instructs them to move on in turn 19 . The guidance was mainly directive based on the PST B’s own strategy . The dialogue advanced on a path laid out by the teacher which consists of structuring the task with multiple guiding questions . Even though PST B used the space for explanation created by the simulation to ask a probing question , her guidance did not take these explanations into account . Instead she used guiding questions to direct the learners towards using a specific strategy . DISCUSSION Both of the episodes discussed show how the simulation created spaces for explanations . PST A used probing questions to elicit these explanations from the learners and mostly non - directively guided them based on their answers . MALU , Turku 2015 88 The teacher’s guidance complements the simulation’s guidance by using the spaces created by the simulation to provide adaptive , non - directive guidance for the learners . PST B also elicits explanations from the learners but doesn’t use these explanations to adapt the guidance . Instead her guidance is based on structuring the task by directive guiding questions that are aimed at making the learners follow a pre - determined learning path . She doesn’t deviate from this path even though the learners have difficulties following it . Questioning diagrams reveal the teachers’ emphasis on probing or guiding questions as in Hähkiöniemi’s ( 2015 ) study . However , the diagrams do not give information about how the questions are adapted to the current situa - tion . For this microanalysis is needed as done in this study . The two episodes show that even though guidance provided by teachers can complement the guidance provided by the simulation by eliciting explanations , the adaptation to the learners’ actions is not self - evident . By eliciting the learners’ knowledge and ideas through series of probing questions , the teacher gets information about the learners’ knowledge and can adapt the guidance . This type of guidance can be compared with non - directive guidance ( de Jong & Njoo , 1992 ) as it supports the learners to come up with their explanations . On the other hand series of guiding questions lead the learners through a pre - determined learning path towards the answer which can be compared with directive guidance by de Jong and Njoo . Adaptation is an essential concept in describing all kinds of guidance ( van de Pol et al . , 2010 ) Use of simulations adds a new dimension to guidance by the teacher as it needs to adapt both to the learners and to the simulation . Through adaptation the teacher acts as orchestrator who chooses when to step in to complement the guidance provided by the simulation or when to step out to let the simulation provide guidance . Even though the simulation itself may provide directive guidance toward the answer the teacher may change the nature of guidance to non - directive . Thus the guidance provided by the teacher and the simulation have synergy with one another by interacting and working in tandem to support learning ( Tabak , 2004 ) . REFERENCES Alfieri , L . , Brooks , P . J . , Aldrich , N . J . , & Tenenbaum , H . R . ( 2011 ) . Does discovery - based instruction enhance learning ? Journal of Educational Psy - chology , 103 ( 1 ) , 1 – 18 . Chin , C . ( 2007 ) . Teacher questioning in science classrooms : Approaches that stimulate productive thinking . Journal of Research in Science Teaching , 44 ( 6 ) , 815 - 843 . de Jong , T . , & Lazonder , A . W . ( 2014 ) . 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Hannula ( Eds . ) , Nordic research in mathematics education : Proceedings of NORMA14 , Turku , June 3 - 6 , 2014 ( pp . 91 - 100 ) . Turku , Finland : Finnish Research Association for Subject Didactics . Jordan , B . , & Henderson , A . ( 1995 ) . Interaction analysis : Foundations and practice . The Journal of the Learning Sciences , 4 ( 1 ) , 39 - 103 . Lehtinen , A . , Nieminen , P . , & Viiri , J . ( 2016 ) . Preservice teachers’ TPACK beliefs and attitudes toward simulations . Contemporary Issues in Technology and Teacher Education , 16 ( 2 ) , 151 – 171 . Ruiz - Primo , M . A . ( 2011 ) . Informal formative assessment : The role of instructional dialogues in assessing students’ learning . Studies in Education - al Evaluation , 37 ( 1 ) , 15 - 24 . Rutten , N . , van Joolingen , W . , & van der Veen , J . ( 2012 ) . The learning effects of computer simulations in science education . Computers & Education , 58 ( 1 ) , 136 - 153 . Sahin , A . , & Kulm , G . ( 2008 ) . Sixth grade mathematics teachers’ intentions and use of probing , guiding , and factual questions . Journal of Mathematics Teacher Education , 11 ( 3 ) , 221 - 241 . Tabak , I . ( 2004 ) . Synergy : A complement to emerging patterns of distributed scaffolding . The Journal of the Learning Sciences , 13 ( 3 ) , 305 - 335 . University of Colorado Boulder . ( 2016 ) . PhET simulations . Retrieved from http : / / phet . colorado . edu / en / simulations / van de Pol , J . , Volman , M . , & Beishuizen , J . ( 2010 ) . Scaffolding in teacher – student interaction : A decade of research . Educational Psychology Review , 22 ( 3 ) , 271 - 296 . Zacharia , Z . C . , Manoli , C . , Xenofontos , N . , de Jong , T . , Pedaste , M . , van Riesen , S . A . , . . . Tsourlidaki , E . ( 2015 ) . Identifying potential types of guid - ance for supporting student inquiry when using virtual and remote labs in science : A literature review . Educational Technology Research and Develop - ment , 63 ( 2 ) , 257 - 302 . MALU , Turku 2015 90 MOODLEN TYÖPAJA – VERTAISARVIOINTI OSANA OPE - TUSTA YLIOPISTOMATEMATIIKAN ENSIMMÄISELLÄ PERUSKURSSILLA Ari - Mikko Mäkelä , Simo Ali - Löytty , Jorma Joutsenlahti & Janne Kauhanen Tampereen teknillinen yliopisto Tässä tutkimuksessa selvitettiin ensimmäisen vuoden matematiikan opiskelijoiden ( N = 129 ) kokemuksia ja mielipiteitä vertaisarvioinnista osana matematiikan opetusta . Vertaisarviointia tehtiin Moodlen Työpaja - aktiviteetin avulla . Kullakin harjoitusvii - kolla tuli palauttaa yksi vertaisarvioitava tehtävä ja palautuksen määräajan jälkeen opiskelijat saivat vertaisarvioitavakseen kahden kanssaopiskelijan samasta tehtävästä tehdyn ratkaisun . Vertaisarvioijan tehtävänä oli antaa tehtävistä numeroarvosana sekä perustelut arvosanalle . Arvioinnit tehtiin anonyymisti . Tulosten perusteella vertaisarvioinnin laatiminen tukee oppimista , mutta palautteen saaminen ei juurikaan . Lisäksi havaittiin , että erityisesti pintasuuntautuneet mallista oppijat , jotka eivät halua opiskella matematiikkaa kovin syvällisesti , kokivat hyötyvänsä vertaisarviointimenettelystä . JOHDANTO Uusikylän ja Atjosen ( 2007 ) mukaan arviointi on tärkeä osa opetusta ja oppimista . Arvioinnin ei pitäisi olla muusta pedagogisesta toiminnasta irrallista , vaan sen tulisi olla aidosti osa oppimisprosessia . Perinteisesti arviointia on tehnyt opettaja , mutta myös opiskelijoiden arviointitaitoja ja palautteenantokykyä on mahdollista kehittää antamalla heille mahdollisuus osallistua arviointiin . Siinä missä opiskelija luottaa opettajan antamaan arviointiin suhteellisen kritiikittömästi , saattaa opiskelijoiden välisessä arvioinnissa muodostua tilanne , jossa jostakin asiasta ollaan eri mieltä . Tämä voi johtaa opiskelijan kriittisempään ajatteluun ( Sims , 1989 ) . Racen , Brownin ja Smithin ( 2005 ) mukaan mikään ei vaikuta opiskelijoiden opiskelumotivaatioon ja – tapoihin niin kuin arviointi , vaikka he ovatkin yleensä tietämättömiä opettajien arvostelumetodeista . Vertaisarviointi tutustuttaa opiskelijat arviointiin ja lisää ymmärrystä arviointiprosessista ( Hyppönen & Linden , 2009 ) . Arviointiprosessista tulee läpinäkyvää ja samalla opiskelijat oppivat , mikä tehtävässä tai työssä on tärkeää . Vertaisarvioinnin avulla opiskelijat ymmärtävät , mitä heiltä vaaditaan ja mitä heidän tulisi oppia ( Race ym . , 2005 ) . Opiskelijoiden välinen vertaisarviointi on vastavuoroisuuteen perustuvaa arviointia . Opiskelijat tekevät tehtäviä ja arvioivat toistensa tuotoksia . Esimerkiksi matematiikan tehtäviä vertaisarvioidessaan opiskelija näkee erilaisia tapoja ratkaista tehtävä ( Havola , Majander , Hakula , Alestalo & Rasila , 2011 ) . Vertaisarviointia tehdessään opiskelijan tulee tietää arviointikri - Mäkelä , Ali - Löytty , Joutsenlahti & Kauhanen 91 teerit voidakseen tehdä arviointia onnistuneesti . Nähdessään malliratkaisun ja arvioidessaan toisen opiskelijan tekemää tehtävää opiskelija pääsee kertaamaan oppimaansa ja oppii myös mahdollisesti uutta . Vaikka opiskelijat ovatkin aktiivisia toimijoita , ei opettajakaan voi siten jäädä vertaisarviointi - prosessissa toimettomaksi , vaan opettajan tulee ohjata ja ohjeistaa opiskelijoi - ta riittävästi arvioinnin laatimisessa ( Uusikylä & Atjonen , 2007 ) . Race ym . ( 2005 ) näkevät useita perusteltuja syitä käyttää vertaisarviointia opetusmetodina . Ensinnäkin vertaisarviointia tehdessään opiskelijat oppivat syvemmin opittavan asian , sillä annettujen arviointikriteerien soveltaminen kanssaopiskelijan työhön on yksi tuottavimmista tavoista kehittää ja syventää omaa ymmärrystä opittavaan asiaan . Arvion laatiminen on huomattavasti täsmällisempi ja vaativampi prosessi kuin pelkkä työn lukeminen tai kuunteleminen . Toisaalta vertaisarvioinnin avulla opiskelijat oppivat toistensa onnistumisista . Opiskelijat useimmiten tunnistavat , milloin toinen on tehnyt tehtävän paremmin . Vertaisarvioija huomaa myös herkemmin tehtävässä tehdyt virheet kuin tehtävän tekijä . Vertaisarvioinnin vaikutusta oppimistuloksiin on tutkittu eri oppiaineissa ja sitä on tutkittu sekä luokkahuoneessa että verkossakin tapahtuvana oppimis - tapahtumana . Aiemmissa tutkimuksissa vertaisarviointimenettelystä on saatu erilaisia tuloksia riippuen kontekstista , jossa sitä on kokeiltu . Esimerkiksi Crowen , Silvan ja Ceresolan ( 2015 ) tutkimuksen mukaan luokkahuoneessa tapahtuvalla vertaisarvioinnilla ei ollut kvantitatiivisiin tutkimusmenetelmiin liittyvällä kurssilla oppimistuloksiin parantavaa vaikutusta . Joidenkin tutkimusten ( katso esim . Rourke , Mendelssohn , Coleman & Allen , 2008 ; Gehringer , 2001 ) valossa varsinkin verkossa tapahtuvan vertaisarvioinnin on puolestaan havaittu tukevan oppimisprosessia mainiosti . Aalto - yliopistossa tehdyn tutkimuksen ( Havola , L . ym . , 2011 ) mukaan opiskelijat kokivat vertaisarviointimenettelyn miellyttävänä . Esimerkiksi mahdollisuus nähdä erilaisia ratkaisutapoja tehtäviin nähtiin vertaisarvioinnissa positiivisena . Tämän tutkimuksen oli tarkoitus selvittää , miten ensimmäisen vuoden opiskelijat kokevat vertaisarvioinnin oppimiskeinona yliopiston matematii - kan peruskurssilla . Erityisen kiinnostuneita oltiin siitä , pohtivatko opiskelijat tehtäviä vertaisarvioinnin ansiosta syvällisemmin ja kokevatko opiskelijat vertaisarviointiprosessin siten edesauttavan matematiikan oppimista paremmin verrattuna perinteiseen tapaan ratkaista tehtäviä . Lisäksi tarkastel - tiin erilaisten oppijoiden mielipiteiden välisiä eroavaisuuksia . TUTKIMUSMENETELMÄT Tutkimuksen toteutus Tutkimukseen liittyvä vertaisarviointikokeilu toteutettiin syksyllä 2015 Tampereen teknillisen yliopiston Matematiikka 1 – opintojaksolla , joka on MALU , Turku 2015 92 suunnattu luonnontieteellisen tiedekunnan ensimmäisen vuoden opiskelijoil - le . Opintojakson viikoittaiset laskuharjoitukset koostuivat Moodlessa tehtävistä automaattisesti tarkistettavista STACK - harjoituksista , yhdestä Moodleen palautettavasta vertaisarvioitavasta Työpajatehtävästä sekä kolmesta perinteisestä laskuharjoituksissa paikan päällä ratkaistavista tehtävistä . STACK - harjoitukset olivat Moodlessa tehtäviä laskuharjoituksia , joihin ohjelma antaa automaattisen palautteen opiskelijan vastauksen perusteella . STACK - tehtävissä parametreja voi myös satunnaistaa , joten vaikka tehtävät ovatkin kaikilla opiskelijoilla lähes samanlaisia , ei vastauksia voi suoraan kopioida kaverilta . Vertaisarvioitavat tehtävät palautettiin Työpajaan , joka on Moodlen oma vertaisarviointiin tarkoitettu aktiviteetti . Sen ansiosta suurellekin määrälle opiskelijoita voidaan jakaa nopeasti ja vaivattomasti tehtäviä vertaisarvioita - vaksi anonyymisti . Työpajassa opiskelijat työskentelevät opettajan määritte - lemän aikataulun mukaisesti . Ensin opiskelijat ratkaisivat tehtävän ja palauttivat sen kunkin harjoitusviikon sunnuntai - iltaan mennessä Työpajaan . Toisessa vaiheessa opiskelijat saivat kaksi tehtävää vertaisarvioitavakseen . Vertaisarviointi tehtiin niin ikään Moodlessa ja sen määräaika oli seuraavan viikon sunnuntai . Työpajan sulkeuduttua opiskelijat näkivät vertaisiltaan saadut arvosanat sekä opettajan kaikille opiskelijoille yhteisesti suunnatun yhteenvedon Työpajatyöskentelystä . Työpajatehtävät ja niiden vertaisarviointiin liittyvät ohjeet käytiin läpi laskuharjoituksissa tehtävän palautuksen määräajan jälkeisellä viikolla . Mallivastaus ja arviointiohjeet olivat opiskelijoiden saatavilla myös Moodles - sa . Aineiston kerääminen Matematiikka 1 – opintojaksolla oli yhteensä kuusi Työpajatehtävää vertaisar - viointeineen . Työpajatehtävien aiheina olivat muun muassa induktiotodistus , kompleksiluvun juurien hakeminen sekä funktion raja - arvon todistaminen . Opintojakson lopuksi viimeisissä laskuharjoituksissa opiskelijoille järjestettiin vertaisarviointia koskeva mielipidekysely . Kysely toteutettiin paperisena lomakkeena , johon opiskelijat vastasivat harjoitusten aikana . Kysely koostui 13 : sta Työpajatehtäviin liittyvästä Likert - väittämästä sekä kolmesta avoimesta kysymyksestä . Tutkimuksessa tarkasteltiin myös erityylisten oppijoiden kokemuksia vertaisarvioinnista . Opiskelijoiden oppimisprofiilit saatiin perustaitojen testistä , joka järjestetään TTY : llä opintojen alussa . Testin aluksi opiskelijat vastaavat matematiikan oppimista koskevaan kysymykseen , jossa heitä pyydetään valitsemaan yksi viidestä heitä parhaiten kuvaavasta vaihtoehdosta . Opiskeli - joiden oppimisprofiilit määritetään tämän kysymyksen avulla . Oppimisprofii - lit ovat : 1 ) pintasuuntautuneet mallista oppijat , 2 ) vertaisoppijat , jotka opiskelevat Mäkelä , Ali - Löytty , Joutsenlahti & Kauhanen 93 mielellään ryhmissä , 3 ) tukea tarvitsevat , 4 ) omin päin opiskelevat sekä 5 ) osaajat , jotka kokevat olevansa hyviä matematiikassa ja haluavat oppia sitä syvällises - ti . Profiileja on eritelty tarkemmin TTY : n tutkimusraportissa ( Pohjolainen , Raassina , Silius , Huikkola & Turunen , 2006 ) . Aineiston analyysi Opiskelijakyselyn tuloksia analysoitiin Likert - väittämien osalta kvantitatiivi - sen ja kolmen avoimen kysymyksen osalta kvalitatiivisen analyysin keinoin . Ensin tarkasteltiin yleisiä tuloksia kaikkien kyselyyn vastanneiden osalta . Tämän jälkeen vastaajat jaoteltiin oppimisprofiilien mukaan ja tarkasteltiin , erosivatko tiettyyn profiiliin kuuluneiden opiskelijoiden mielipiteet muiden opiskelijoiden mielipiteistä merkitsevästi . Profiilivertailussa vaihtoehdot ”täysin / osittain eri mieltä” yhdistettiin vaihtoehdoksi ”eri mieltä” ja vastaavasti ”täysin / osittain samaa mieltä” yhdistettiin vaihtoehdoksi ”samaa mieltä” . Tämä tehtiin χ 2 - testin vaatimusten vuoksi – näin saatiin kolmen suurimman profiilin osalta riittävästi frekvenssejä kumpaankin vastausvaihtoehtoon . Opiskelijakyselyn avointen kysymysten tarkoituksena oli tiedustella opiskeli - joiden mielipiteitä Työpajatyöskentelyn hyvistä ja huonoista puolista sekä kartoittaa opiskelijoiden kokonaisvaltaista suhtautumista Työpajatehtäviin ja vertaisarviointimenettelyyn . Näillä haluttiin selvittää , millaiset teemat nousevat opiskelijoiden vastauksissa eniten esille ja onko suhtautuminen vertaisarviointiin yleisesti positiivinen , neutraali vai negatiivinen . TULOKSET Yleisiä tuloksia Opiskelijakyselyn 13 Likert - väittämää ja kaikkien opiskelijoiden vastausten jakautuminen väittämissä löytyy tämän artikkelin liitteistä ( Taulukko 4 ) . Väittämän 5 tulosten perusteella opiskelijat vaikuttavat pohtivan vertaisarvi - oitavia tehtäviä normaalia enemmän . Opiskelijoiden mielipiteet Työpajatyös - kentelystä kokonaisuudessaan oppimisen näkökulmasta puolestaan vaihteli - vat suuresti . Hieman yli puolet vastaajista koki Työpajatyöskentelyn edesaut - tavan oppimista paremmin kuin perinteiset harjoituksissa tarkastettavat tehtävät ( väittämä 7 ) . Opiskelijat oppivat paremmin tehdessään itse vertaisarviointia kuin saaman - sa vertaisarvioinnin avulla . Tämä johtopäätös voidaan tehdä tarkastelemalla kyselyn ensimmäisen ja toisen väittämän tuloksia . 63 , 7 % opiskelijoista oli väittämän 1 kanssa joko osittain tai täysin samaa mieltä . 69 , 3 % opiskelijoista puolestaan oli väittämän 2 kanssa joko osittain tai täysin eri mieltä . Saatu sanallinen arviointi ei juurikaan edesauttanut oppimista , sillä opiskelijatove - rien arviointikykyyn ei varsinkaan haastavissa tehtävissä luotettu . Tämä käy ilmi kyselyn avointen kysymysten vastauksista . Lisäksi 52 , 0 % vastaajista ( N MALU , Turku 2015 94 = 100 ) oli sitä mieltä , että tehtävistä ei saatu riittävästi sanallista palautetta ( väittämä 3 ) . Profiilikohtaisia tuloksia Profiilikohtaista vertailua tehtiin vertaamalla kolmen oppimisprofiilin ( pintasuuntautuneet mallista oppijat , vertaisoppijat ja osaajat ) vastauksia muiden opiskelijoiden vastauksiin . Tukea tarvitsevia ( N = 4 ) ja omin päin opiskelevia ( N = 6 ) oli kyselyyn vastanneiden joukossa niin vähän , että näitä kahta profiilia ei erikseen tarkasteltu . Tutkimuksessa havaittiin erityisesti pintasuuntautuneiden mallista oppijoi - den ( N = 17 ) vastausten eroavan muiden opiskelijoiden vastauksista . Pintasuuntautuneet mallista oppijat oppivat matematiikkaa esimerkkien avulla – heidän kiinnostukseensa matematiikkaa kohtaan vaikuttaa enemmän koulutusohjelma kuin oma mielenkiinto . Tämän profiilin opiskelijoilla korostuu epävarmuus oman osaamisen suhteen . Heidän asenteensa oppimis - ta kohtaan ei ole kaikkein positiivisin ja oppiminen on pinnallista . Mallista oppijat onnistuvat tehtävän ratkaisemisessa katsomalla mallia opettajan esimerkeistä ( Pohjolainen & al . 2006 ) . Tilastollisesti merkitsevimmät erot havaittiin väittämissä 1 ja 6 ( Taulukko 1 ) . Valtaosa tämän profiilin opiskelijoista koki oppivansa vertaisarvioinnin laatimisen ansiosta kurssin teemoja normaalia paremmin . Muiden opiskeli - joiden vastaukset jakautuivat puolestaan paljon tasaisemmin . Saatu vertaisar - viointi sai pintasuuntautuneet oppijat myös suhtautumaan omiin vastauksiin kriittisesti ( väittämä 6 ) . Myös tässä väittämässä on tilastollisesti merkitsevä ero , kun verrataan muiden opiskelijoiden vastauksiin ( p = 0 , 006 ) . Taulukko 1 : Pintasuuntautuneiden mallista oppijoiden vastausten vertailua muihin opiskelijoihin . Väitt ämä Pintasuuntautuneet mallista oppijat Muut Eri mieltä Samaa mieltä Eri mieltä Samaa mieltä p 1 6 7 2 ( 11 , 7 % ) 3 ( 17 , 6 % ) 12 ( 70 , 6 % ) 15 ( 88 , 3 % ) 14 ( 82 , 4 % ) 5 ( 29 , 4 % ) 40 ( 48 , 2 % ) 44 ( 54 , 3 % ) 44 ( 53 , 7 % ) 43 ( 51 , 8 % ) 37 ( 45 , 7 % ) 38 ( 46 , 3 % ) 0 , 006 0 , 006 0 , 200 Väittämien 1 ja 6 tulosten perusteella ei ole yllättävää , että selvä enemmistö pintasuuntautuneista mallista oppijoista koki Työpajatyöskentelyn edesaut - tavan matematiikan oppimista . Tämä käy ilmi tarkastelemalla väittämän 7 tuloksia . Ero muihin opiskelijoihin ei tässä väittämässä kuitenkaan ole tilastollisesti merkitsevä ( p = 0 , 200 ) vaikka muiden opiskelijoiden vastaukset jakautuivat siinäkin tasaisemmin . Mäkelä , Ali - Löytty , Joutsenlahti & Kauhanen 95 Osaajien ( N = 38 ) mielipiteet vertaisarvioinnin vaikutuksesta oppimiseen eivät eronneet merkitsevästi muiden oppimisprofiilien opiskelijoiden mielipiteisiin verrattuna . Osaajat eivät nähneet vertaisarviointimenettelyä niin paljon oppimistaan edistävänä kuin pintasuuntautuneet mallista oppijat . Vertaisarviointia ei heidän osaltaan tosin tyrmättykään – esimerkiksi väittämän 7 ( katso Taulukko 4 ) kohdalla osaajien mielipiteet jakautuivat kaksiportaiseksi tiivistetyssä asteikossa täsmälleen tasan . Myös vertaisoppijoiden ( N = 33 ) vastaukset jakautuivat useimmissa väittä - missä samansuuntaisesti kuin muillakin opiskelijoilla . Ainoastaan väittämän 6 ( ”Saamani vertaisarviointi sai minut suhtautumaan kriittisesti myös omiin ratkaisuihini . ” ) kohdalla havaittiin selvä ero muihin vastaajiin : ainoastaan 32 , 5 % vertaisoppijoista oli tämän väittämän kanssa samaa mieltä , kun muista vastaajista samaa mieltä oli 61 , 2 % ( p = 0 , 008 ) . Laadullinen analyysi Opiskelijoiden sanallisten arviointien laatua vertaisarvioinneissa arvioitiin opettajan toimesta opintojakson neljännessä Työpajatehtävässä . Opettaja arvioi kaikkien opiskelijoiden arvioinnit asteikolla 0 - 5 , missä 0 vastasi arviointia , jossa ei ollut sanallista palautetta ollenkaan ja numeron 5 puoles - taan sai , jos palaute oli täsmällinen ja noudatti kiitettävästi arviointiohjeita . Tässä tarkastelussa sanallisten arviointien laatu havaittiin heikoksi – ainoas - taan 12 , 5 % opiskelijoista ( N = 96 ) sai arvion 4 tai paremman . Näitä arviointe - ja verrattiin myös opiskelijoiden opintojakson tentissä saamiin arvosanoihin . Näiden välillä havaittiin kohtalainen korrelaatio ( r = 0 , 365 , p = 0 , 000 ) . Hyvin sanallisen arvioinnin kirjoittaneet menestyivät hyvin myös tentissä . Välttäväs - ti sanallisen arvioinnin tehneiden tenttiarvosanat vaihtelivat enemmän . Kuitenkin tentissä esimerkiksi arvosanan 0 saaneista suurin osa oli tehnyt myös vertaisarvioinnin heikosti . Kyselyn viimeisen kysymyksen ( Kysymys 16 : ”Kerro vielä mielipiteesi Moodlen Työpajasta ja vertaisarvioinnista kokonaisuudessaan . ” , N = 99 . ) perusteella tulkittiin opiskelijoiden suhtautuminen Työpaja - konseptiin ja vertaisarviointiin . 42 vastauksessa ( 42 , 4 % ) Työpajaan suhtauduttiin ilmeisen positiivisesti . Neutraalisti suhtautuneita oli lähes yhtä paljon , 36 opiskelijaa ( 36 , 4 % ) . Vain hieman yli viidennes opiskelijoista ( N = 21 ; 21 , 2 % ) suhtautui Työpajaan selvästi negatiivisesti . Alla on yksi tyypillinen opiskelijan mielipi - de Työpaja - työskentelystä . Työpajat ovat ihan ok vaihtelua perinteisiin tehtäviin . Vertaisarviointi oli välillä vaikeaa , kun ei itse tiennyt saiko tehtävää ratkaista tietyllä tyylillä . ( Opiskelija 1 ) Opiskelijakyselyn ensimmäisessä avoimessa kysymyksessä tiedusteltiin Työpajatyöskentelyn positiivisia seikkoja . Opiskelijoiden vastauksissa kolme eniten esiintynyttä teemaa on koottu Taulukkoon 2 . MALU , Turku 2015 96 Selvästi eniten opiskelijoiden vastauksissa toistui tehtävien pohtiminen . Opiskelijoiden mielestä vertaisarvioitaviin tehtäviin tuli syventyä tarkemmin ennen kaikkea riittävän hyvän vertaisarvioinnin laatimiseksi . Tämän ansioista monet käyttivät tehtävän parissa enemmän aikaa ja kokivat oppivansa tehtävissä olleita aiheita paremmin . Työpajatehtävät pakottivat minut opiskelemaan asiaa paremmin . Ver - taisarviointi myös pakotti ymmärtämään tehtävän / asian paremmin , jotta pystyin arvioimaan tehtävän . ( Opiskelija 2 ) Taulukko 2 : Kolme eniten esiintynyttä teemaa opiskelijoiden vastauksissa kysyttäessä Työpajatyöskentelyn hyviä puolia . ( Kysymys 14 : ”Mitä hyvää Työpajatyöskentelyssä mielestäsi oli ? ” ) . N = 102 . Teema f ( % ) 1 . Tehtäviä oli pohdittava tarkemmin , jotta ne osasi arvioida . 2 . Muiden tekemistä hyvistä ratkaisuista ja virheistä oppi . 3 . Pakotti huolellisuuteen . Muut 28 ( 27 , 5 % ) 16 ( 15 , 7 % ) 15 ( 14 , 7 % ) 43 ( 42 , 1 % ) Monet oppivat myös nähtyään toisten opiskelijoiden tekemiä ratkaisuja . Opiskelijat havaitsivat uusia ratkaisutapoja tehtävissä . Jotkut huomasivat paremmin myös omat virheensä toisten ratkaisuja tarkastellessaan . Vertaisar - viointimenettelyn pakotti myös osan vastaajista ratkaisemaan tehtävät vaihe vaiheelta huolellisemmin . Tehtävää tehdessä tuli kiinnitettyä enemmän huomiota sanallisiin selityk - siin ja välivaiheiden selkeyteen . ( Opiskelija 3 ) Muut teemat sisältävät vähemmän mainittujen seikkojen lisäksi myös tyhjät vastaukset tähän kysymykseen . Vastauksissa mainittiin myös mallivastaukset ja arviointiohjeet Työpajan parhaimpana antina . Taulukkoon 3 on kerätty vastauksissa useimmin esiintyneet teemat kysymyk - seen 15 , jossa kysyttiin Työpajassa ilmenneitä negatiivisia puolia . Näissä vastauksissa erottui selvästi neljä teemaa . Muut vastaukset olivat pääosin joko tyhjiä tai eivät liittyneet Työpajaan , vaan opintojakson muihin järjestelyihin . Taulukko 3 : Useimmin esiintyneet teemat opiskelijoiden vastauksissa Työpajan huonoja puolia kysyttäessä . ( Kysymys 15 : ”Mitä huonoa Työpa - jatyöskentelyssä mielestäsi oli ? ” ) . N = 102 . Teema f ( % ) 1 . Vertaisarvioinnit virheellisiä tai puutteellisia . 2 . Arviointi haastavaa , jos ei itse osannut tehtävää . 3 . Työläitä ja aikaa vieviä . 4 . Sekava järjestely . Muut 21 ( 20 , 6 % ) 21 ( 20 , 6 % ) 17 ( 16 , 7 % ) 15 ( 14 , 7 % ) 28 ( 27 , 5 % ) Mäkelä , Ali - Löytty , Joutsenlahti & Kauhanen 97 Eniten vastauksissa esiintyi kritiikkiä saatuja vertaisarviointeja kohtaan . Monen opiskelijan mielestä saadut sanalliset palautteet olivat kovin lyhyitä tai ne olivat virheellisiä eivätkä noudattaneet arviointiohjeita . Samasta tehtävästä saattoi eri opiskelijoilta saada aivan erilaisen arvostelun . Osa opiskelijoista ei ollut saanut sanallista palautetta ollenkaan . Arviointien heikko laatu liittyy toiseen vastauksissa usein esiintyneeseen teemaan . Moni koki arvioinnin liian haastavaksi varsinkin , jos tehtävää ei ollut itse osannut ratkaista , mallivastauksesta ja arviointiohjeista huolimatta . Tosin monesti tehtävät saattoi ratkaista eri tavalla kuin mallivastauksessa oli esitetty – erityisesti tällöin opiskelijat kokivat vertaisarvioinnin vaikeaksi . Osa vastaajista koki Työpajatyöskentelyn työlääksi . Tämä ei johtunut niinkään itse tehtävistä tai vertaisarvioinnin laatimisesta , vaan tehtävien konvertoimisesta PDF - tiedostoksi , mikä ärsytti osaa opiskelijoista . Työpajan väljä aikataulu sai myös nuhteita . Opiskelijat eivät enää jaksaneet palata vanhoihin tehtäviin lukemaan saamaansa vertaisarviointia , kun palaute tuli vasta silloin , kun luennoilla oli jo siirrytty uuteen asiaan . POHDINTA Opiskelijakyselyn perusteella ensimmäisen vuoden opiskelijat suhtautuvat Moodlen Työpajassa tapahtuvaan vertaisarviointiprosessiin vaihtelevasti . Osalle opiskelijoista vertaisarviointi sopii ja sen koetaan edesauttavan oppimista . Vastaavasti osa opiskelijoista ei mielestään juurikaan hyötynyt Työpajatyöskentelystä . Positiivisesti käytettyyn vertaisarviointimenettelyyn suhtautuneita oli vain kaksinkertainen määrä negatiivisesti suhtautuneihin nähden . Positiivisimmin Työpajatyöskentelyyn suhtautuivat pintasuuntautu - neet mallista oppijat . Heistä 70 , 6 % koki vertaisarviointimenettelyn edesaut - tavan oppimista . Muiden oppimisprofiilien edustajien mielipiteet jakautuivat tasaisemmin , mutta mikään profiili ei kuitenkaan erottunut negatiivisella suhtautumisella . Opiskelijoiden mielestä erityisesti vertaisarvioinnin laatiminen edistää oppimista , saatu arviointi ei niinkään . Vertaisarvioidessa tehtäviin paneudu - taan tavallista huolellisemmin ja syvällisemmin . Opiskelijat kokivat , että ratkaisuja arvioidessa tulee pohtia ja perustella itselleen , onko arvioitavan ratkaisu oikein vai ei . Tämä koettiin oppimisen kannalta hedelmälliseksi , joten voidaan olettaa , että pelkkä mallivastausten ja pisteytysohjeiden analysoiminen ilman vertaisarviointia ei tuottaisi yhtä hyviä oppimistuloksia . Hyvin sanallisen vertaisarvioinnin tehneet menestyivät keskimäärin hyvin myös tentissä . Monille vertaisarvioinnin laatiminen oli kuitenkin hankalaa , mistä johtuen opiskelijoiden toisiltaan saamien sanallisten arviointien laatu oli pääosin kehnoa . Erityisesti tästä johtuen saadusta palautteesta ei saatu sitä hyötyä , joka siitä parhaimmillaan voitaisiin saada . MALU , Turku 2015 98 Vertaisarviointikokeilun voidaan sanoa onnistuneen , sillä merkittävä osa opiskelijoista koki sen positiivisena ( 42 , 4 % vastaajista ) ja myös oppimista edesauttavana ( 53 , 5 % ) . Potentiaalia vertaisarvioinnissa matematiikan oppimisen näkökulmasta siis on . Työpajatyöskentelyssä on jatkoa ajatellen kuitenkin paljon kehitettävää . Muun muassa Työpajan aikataulua tulee pohtia jatkossa tarkemmin . Esimerkiksi Havolan ym . ( 2011 ) tutkimukseen liittyvässä opetuskokeilussa opiskelijat antoivat kiitosta juuri siitä , että ratkaisuista sai välittömästi palautetta , kun tehtävä oli vielä tuoreessa muistissa . Suurin kysymys kuitenkin on , miten opiskelijat saadaan laatimaan laadukkaampia sanallisia arviointeja . Tässä suhteessa opettajilla on suuri vastuu . Vaikka tehtävät ja arviointiohjeet käytiin melko perusteellisesti laskuharjoituksissa läpi , on varsinkin mallivastauksen laatimisessa parannet - tavaa . Jos ratkaisutapoja on useita , on myös mallivastauksia syytä laatia useita versioita . Tällöin opiskelijoiden arviointityö helpottuu . LÄHTEET Crowe , J . A . , Silva , T . , Ceresola , R . ( 2015 ) . The Effect of Peer Review on Student Learning Outcomes in a Research Methods Course . Teaching Soci - ology 43 ( 3 ) , 201 - 213 . http : / / www . asanet . org / journals / TS / Jul15TSFeature . pdf [ Luettu : 12 . 11 . 2015 ] Gehringer , E . F . ( 2001 ) . Electronic Peer Review and Peer Grading in Comput - er - Science Courses . Teoksessa Proceedings of the thirty - second SIGCSE tech - nical symposium on Computer Science Education ( s . 139 - 143 ) . New York , USA : ACM . http : / / dl . acm . org / citation . cfm ? id = 364564 [ Luettu : 12 . 11 . 2015 ] Havola , L . , Majander , H . , Hakula , H . , Alestalo , P . , Rasila , A . ( 2011 ) . Aktivoi - viin opetusmenetelmiin perustuvat matematiikan opetuskokeilut Aalto - yliopistossa . Teoksessa J . Viteli & A . Östman ( toim . ) Interaktiivinen tekniikka koulutuksessa 2011 – konferenssi ( s . 5 - 9 ) . Tampere , Suomi : Tampereen yliopis - to . Hyppönen , O . & Linden , S . ( 2009 ) Opettajan käsikirja – opintojaksojen rakenteet , opetusmenetelmät ja arviointi . Espoo , Suomi : Teknillinen korkeakoulu . Race , P . , Brown , S . , Smith , B . ( 2005 ) 500 Tips On Assessment . Abingdon , Englanti : Routledge Falmer . Rourke , A . J . , Mendelssohn , J . , Coleman K . & Allen , B . ( 2008 ) . Did I mention it’s anonymous ? The triumphs and pitfalls of online peer review . Teoksessa Hello ! Where are you in the landscape of educational technology ? Proceedings ascilite Melbourne 2008 ( s . 830 - 840 ) . Melbourne , Australia . http : / / www . ascilite . org . au / conferences / melbourne08 / procs / rourke . pdf [ Luettu : 11 . 11 . 2015 ] Sims , G . ( 1989 ) . Student peer review in the classroom : A teaching and grading tool . Teoksessa Journal of Agronomic Education 18 ( s . 105 - 108 ) . Mäkelä , Ali - Löytty , Joutsenlahti & Kauhanen 99 https : / / www . agronomy . org / files / publications / jnrlse / pdfs / jnr018 / 018 - 02 - 0105 . pdf [ Luettu 11 . 11 . 2015 ] Pohjolainen , S . , Raassina , H . , Silius , K . , Huikkola , M . & Turunen , E . 2006 . TTY : n insinöörimatematiikan opiskelijoiden asenteet , taidot ja opetuksen kehittäminen . Tampereen teknillinen yliopisto . Tampere , Suomi . Uusikylä , K . , & Atjonen , P . ( 2007 ) Didaktiikan perusteet . 3 . painos . Helsinki , Suomi : Sanoma Pro . LIITTEET Taulukko 4 . Kyselyn väittämien tulokset . ( TEM = täysin eri mieltä , OEM = osittain eri mieltä , OSM = osittain samaa mieltä , TSM = täysin samaa mieltä ) . Väittämä TEM OEM OSM TSM N 1 . Opin Työpajatehtävissä käsiteltyjä aiheita paremmin tehtyäni vertaisarviointia toisten opiskelijoiden tehtävistä . 7 , 8 % 28 , 4 % 51 , 0 % 12 , 7 % 102 2 . Opin Työpajatehtävissä käsiteltyjä aiheita paremmin saamani sanallisen palautteen ansiosta . 32 , 7 % 36 , 6 % 26 , 7 % 4 , 0 % 101 3 . Sain riittävästi sanallista palautetta ratkaisuis - tani . 12 , 0 % 40 , 0 % 40 , 0 % 8 , 0 % 100 4 . Koen opiskelijatoverien antaman palautteen oppimisen kannalta tärkeäksi . 15 , 7 % 39 , 2 % 40 , 2 % 4 , 9 % 102 5 . Työpajatyöskentely pakotti tehtävien tarkempaan pohtimiseen . 9 , 9 % 17 , 8 % 36 , 6 % 35 , 6 % 101 6 . Saamani vertaisarviointi sai minut suhtautu - maan kriittisesti myös omiin ratkaisuihini . 11 , 0 % 33 , 0 % 39 , 0 % 17 , 0 % 100 7 . Mielestäni Työpajatyöskentely ei edesauttanut oppimista sen paremmin kuin perinteiset laskuharjoitustehtävät . 19 , 8 % 33 , 7 % 24 , 8 % 21 , 8 % 101 8 . Ymmärsin Työpajatehtävissä olleita aiheita paremmin nähtyäni vertaisarvioidessa muiden opiskelijoiden hyvin tehtyjä ratkaisuja . 10 , 9 % 25 , 7 % 46 , 5 % 16 , 8 % 101 9 . Opin välttämään vertaisarvioinnissa havaitsemiani opiskelijatoverien tekemiä virheitä . 8 , 1 % 35 , 4 % 51 , 5 % 5 , 1 % 99 10 . Koin Työpajatyöskentelyn kokonaisuudes - saan liian työlääksi . 18 , 2 % 54 , 5 % 21 , 2 % 6 , 1 % 99 11 . Tekisin jatkossa mieluummin yhden Työpajatehtävän kuin kaksi perinteistä tehtävää . 24 , 8 % 24 , 8 % 32 , 7 % 17 , 8 % 101 12 . Vertaisarvioitavien Työpajatehtävien osuus viikottaisista harjoituksista voisi olla jatkossa suurempi . 48 , 5 % 37 , 6 % 12 , 9 % 1 , 0 % 101 13 . Harjoittelin LaTeXilla kirjoittamista sen verran , että osaan kirjoittaa sillä matemaattista tekstiä ainakin jonkin verran . 47 , 1 % 11 , 8 % 16 , 7 % 24 , 5 % 102 MALU , Turku 2015 100 FOUR KINDS OF FORMATIVE ASSESSMENT DISCUSSIONS IN INQUIRY - BASED PHYSICS AND MATHEMATICS TEACHING Pasi Nieminen , Markus Hähkiöniemi , Jarmo Leskinen & Jouni Viiri University of Jyväskylä This exploratory case study describes on - the - fly formative assessment interactions from two physics and mathematics inquiry lessons . On - the - fly interactions are unexpected teachable moments in which the teacher tries to probe students’ under - standing and use that information to support their inquiry process . The video data revealed two dimensions which characterize teachers’ on - the - fly practices : 1 ) How quickly do teachers elicit information from students ( quick interpretation or further probing ) and 2 ) do they use that information for helping student to proceed or express students’ thinking . These acts relate to teacher guidance in the continuum of authoritative – dialogic which basically indicates how much students’ ideas are taken into account for learning . We will validate and refine our preliminary results with larger data set in the future . INTRODUCTION Inquiry - based learning has been advocated in educational research and policy in the past decade ( Rocard et al . , 2007 ) . In science education , inquiry - based learning means that developing of an understanding of scientific concepts and skills involves the use of similar practices which have been used by scientists in their studies . For instance , the processes include identifying questions and formulating hypotheses , planning and carrying out experi - ments , and developing explanations . Similarly , in mathematics education inquiry - based learning involves various processes such as exploring problems and making conjectures , planning and carrying out problem solving strategy , and evaluation of results . These parts of inquiry can be considered as competences which should be learning goals along with scientific or mathe - matical concepts . Further , the competences should be an object of assessment too as the assessment influences on what is taught and how is taught ( Harlen , 2013 ) . This study has been done in part of an international EU funded project Assess Inquiry of Science , Technology and Mathematics Education ( ASSIST - ME ; http : / / assistme . ku . dk / ) which focuses on formative and summative assessment in competence oriented inquiry - based education . In Finland we have worked with primary and upper secondary mathematics and lower secondary physics teachers . We have collected large video data from their inquiry lessons . This paper describes two cases in which one physics teacher Nieminen , Hähkiöniemi , Leskinen & Viiri 101 and one mathematics teacher implement formative assessment on - the - fly in one inquiry lesson . The essential difference between formative and summative assessment lies behind their different purposes . Summative assessment is ‘assessment of learning’ as its purpose is summarize , value and report what is learnt after a certain point of time . Instead , formative assessment is ‘assessment for learning’ as it attempts to enhance and support student learning in a moment when learning happens ( Harlen & Qualter , 2014 ) . There are different ways to implement formative assessment depending on how the data from students is collected ( e . g . , discussions , tests , reports or portfolios ) , how the feedback is given ( e . g . , on - the - fly , marking or peer - feedback ) , and how formal ( planned ) it is . On - the - fly refers to informal formative assessment interaction between a teacher and students which is not planned beforehand , but takes place spontaneously when the teacher recognizes appropriate opportunities to support students in advancing their learning ( Shavelson et al . , 2008 ) . While there are many studies about teacher - student interaction ( e . g . , Lehesvuori , Viiri , Rasku - Puttonen , Moate & Helaakoski , 2013 ) only few have studied issues related explicitly to formative assessment . One such study is reported by Ruiz - Primo and Furtak ( 2006 ) . They developed the ESRU framework which we utilized in our study . The complete ESRU cycle contains following parts : the teacher Elicits information from the student by formulating a question , the Student responds , teacher Recognizes the student ' s response , and then Uses the information collected to student learning . In this paper our aim is to understand how teachers implement formative assessment on - the - fly in inquiry - based physics and mathematics teaching . On the other hand , we investigate the affordances and constrains of ESRU framework in analyzing formative assessment on - the - fly . Based on the two cases , we characterize four different kinds of on - the - fly interactions . Our research question is : What kinds of interactions exist during on - the - fly episodes in the lessons of two teachers in terms of ESRU cycles ? METHODS The data is collected from two Finnish classes ( Table 1 ) . Maria is an experi - enced primary school teacher who teaches only mathematics for this 3 rd grade class . James is an experienced lower secondary school teacher as well . These teachers were selected among the teachers who participated voluntarily to the project . The grades and the subjects were set by the guidelines of the interna - tional project . The lessons had the same structure : Introduction , inquiry phase in student groups and summary . Lessons ( 45 min ) were videotaped using one camera and the teacher’s wireless microphone which captured teacher - student discussions . MALU , Turku 2015 102 Table 1 . Participants and data Teacher Level Students Subject Topic Maria Primary 3 th grade , 9 years , n = 23 Mathematics Division James Lower secondary 7 th grade , 13 years , n = 13 Physics Optics All on - the - fly interactions were identified . One on - the - fly episode is a conceptual or an inquiry related discussion between a teacher and a student , student group or the whole class . Episodes are initiated by the teacher or students and there is one underlying theme such as how to illustrate the path of the light ray in a particular case . Episode ends when the theme of discus - sion changes or the teacher moves to another student group . Typically , on - the - fly episode arises when students are working in groups and the teacher is circulating around the class . The ESRU codes were applied to each speaking turn ( teacher and student ) . However , one speaking turn can include more than one ESRU code . For instance , the teacher may first recognize student answer and use that information after that . ESRU codes reveal eliciting and using which are important phases when the teacher looks for information about student understanding and helps them to proceed or express their thinking . However , ESRU codes do not tell much about the quality of formative actions . In addition , the lack of some code in an episode ( incomplete ESRU cycle ; e . g . , E is missing ) does not directly mean that the episode is poor in terms of formative assessment . Thus , episodes were further examined . We carefully compared the on - the - fly - episodes to each other and searched for similarities and difference in formative assessment . As a result , we identified four different kinds of formative assessment discussions . RESULTS In the following , we present four kinds of ESRU cycles which were found from the lessons . Although all of them contain an ESRU cycle ( complete or incomplete ) , they differ depending on how the teacher elicits and interprets information from students and how he / she uses that information . Quick interpretation and helping students to proceed In many cases , the teacher got some information of students’ progress and started quickly to guide students . For example , in the mathematics lesson , a student initiated ( Si ) the following discussion . Nieminen , Hähkiöniemi , Leskinen & Viiri 103 Turn Transcription Code 1 Student : Maria , how should we do , because [ inaudible ] and eight . Divided by eight . Si 2 Maria : Mm . By eight ? R 3 Student : Yes . S 4 Maria : Are there eight of you ? U 5 Student : No . S 6 Maria : No . Read the task one more time . How many ? R 7 Student : Everyone gets eight macarons . S In this discussion , the student mentions “dividing by eight” but in the situation 8 is not the divisor . Maria ( primary teacher ) spots this and guides the student to notice that 8 is the answer to the task . In this case , Maria did not ask further questions , for example , why they are thinking about dividing by 8 . Thus , she seems to fit the students’ thinking into her own views . This can be seen also in ESRU coding as there is no eliciting in the beginning of the discussion . Sometimes there was eliciting in the beginning of the discussion but still the teacher made a quick interpretation without further probing . For example , in the following discussion James ( lower secondary teacher ) is eliciting before using . Turn Transcription Code 1 James : How is it going here ? E 2 Alison : If we understood this… S 3 James : Could you tell me how did you understand it ? E 4 Diana : So , that we have to place the object there and then look that where we can see it . S 5 James : Yes . Okay . If we think that this is Alison’s eye , so the eye can move along this line here [ shows by hand ] . Yes . Then you can… here it is said that : “sketch the observer in different places” . You can use a ruler for example . In which line segment the eye could be for instance . U In this discussion James elicits , makes quick interpretation about students’ difficulties and in turn 5 starts to guide students how to write down an observation . MALU , Turku 2015 104 Quick interpretation and helping students express their thinking Another way how the teachers continued after making a quick interpretation was helping students to express their thinking . For example , in the following episode , students provide some information about their thinking without teacher eliciting . Turn Transcription Code 1 Student 1 : Maria . Si 2 Maria : What ? – 3 Student 1 : We put that everyone gets two at first . S 4 Student 2 : Because we put them like that , we gave at first everyone always two , two , two and two . S 5 Maria : Yeah , well write here how you have done . U 6 Student 2 : Yeah . S 7 Maria : Write , write here . Draw how you have divided . Two at first , is it ? U 8 Student 2 : Mm . S 9 Maria : Okay , well , draw it . Good thoughts , good thoughts . U When Maria gets the information that students are dividing by giving away two at a time , she does not further elicit their strategy . Instead , she guides them express their thinking on paper . In the physics lesson ( an excerpt below ) James makes quick interpretation about students’ idea and helps them to express the idea . Turn Transcription Code 1 Student : We are ready . Si 2 James : You are ready . Is this it ? R 3 Student : Yeah . S 4 James : Okay . What if the object is in front of the mirror ? When can the object be seen via mirror ? E 5 Student : [ inaudible ] …from the whole distance . S 6 James : From the whole distance . Yes . Justify like in previous part . You could put down what is this illustrating . R , U 7 Student : Well , the observer’s… [ inaudible ] S 8 James : The area where the observer is looking ? Okay . Write it down too . R , U Nieminen , Hähkiöniemi , Leskinen & Viiri 105 The student informs James that they are ready . In the turn 4 James elicits information and get students’ idea how they are able to see the object ( 5 ) . After quick interpretation he helps the students to express their idea without correcting or valuing it ( 6 and 8 ) . Probing more students’ thinking and helping them to proceed There were instances in which after the first elicitation , the teacher continued to probe the students’ ideas and base the guidance on this . In the mathematics lesson Maria was discussing with a student about the problem of dividing 92 raisins to four people . Turn Transcription Code 1 Maria : 92 raisins . Quite a lot , is it ? E 2 Maria : Okay , well try . Take , for instance , a smaller number as an example . What kind of ? What number can you take ? What number ? E 3 Student : 40 . S 4 Maria : 40 ? R 5 Student : I mean 41 . S 6 Maria : 41 ? R 7 Student : 46 . S 8 Maria : 46 ? R 9 Maria : Can you say right away who many raisins for each ? E 10 Student : 46 is half and then half of that . S 11 Maria : Well yeah , start with that . Start . Mm , could be . Good thoughts . U Here the teacher is openly waiting for student’s response . The student offers many numbers and the teacher is not evaluating them . When the student says that he is thinking about half and then half of the half , the teacher only encourages the student to continue . After being patient and getting more information about the students’ thinking , the teacher fades support . We did not find this kind of discussion from the physics lesson . MALU , Turku 2015 106 Probing more students’ thinking and helping students express their thinking Sometimes after further probing , the teacher guided students to express their thinking . The mathematics lesson included the following discussion . Turn Transcription Code 1 Student 1 : Is this a division ? Si 2 Maria : Mm , well it could be that you know division already . R 3 Maria : Would you like to draw something ? E 4 Student 2 : Cause we don’t know . . . S 5 Maria : How do you know that 8 ? E 6 Student 2 : Well , I calculated it from the multiplication table . S 7 Maria : Well , write it down , all thoughts down . U 8 Student 2 : Write ? S 9 Maria : Yes , write down all your thoughts that you have been thinking . U 10 Student 2 : Do you mean drawing or should I write with letters ? S 11 Maria : Or calculation or what . How did you think , tell me ? U 12 Student 2 : So like that I was thinking that which multiplication table . S 10 Maria : Well , write the multiplication table . Write the multiplication and what . How did you think ? U 11 Student 2 : Should I put here the thoughts ? S 12 Maria : Yes . Thoughts down . Team work . Not only you . Explain to others . R In this discussion , Maria asks many times about students’ strategy . Based on the first student utterance , they were already familiar with division and using that . However , it turns out that at least one of the students is thinking about multiplication . After this , Maria guides them to express their thinking on paper . In the following excerpt from the physics lesson , James probes many times until he gets information about students’ ideas . After that he helps students to express their idea and prompts them to write it down . Nieminen , Hähkiöniemi , Leskinen & Viiri 107 Turn Transcription Code 1 James : What is going here ? E 2 Student : Well , when… if we put that… that when… S 3 James : Are you working with conclusions ? E 4 Students : Yeah S 5 James : Okay . Do your prediction and your observation differ ? E 6 Students : Yeah . S 7 James : In what way ? E 8 Student : We thought as if I sit here . Then the mirror is there , so it would be mirrored to this corner but it is in that corner . S 9 James : Hm . So , you were here . Here is the object . You sat here . And you see the image… you supposed that it is here , but is in this side . R 10 Student : Yes . S 11 James : Well . So , you can answer : Do your prediction and observation differ ? U 12 Student : Yeah . S 13 James : Yes . How does the person’s location influence the location of the image ? U 14 Student : It is mirrored like… If you look from side the object is mirrored to that corner where you sit or stay . S 15 James : To that corner ? So , you considered that the image is like in the different side from the object than your eye , but it is in the same side… R 16 Student : Yeah . S 17 James : …than your eye . Okay . Yes it is . Yes . Then just write . R The E - S - E - S - … structure continues for turns 1 to 9 where James recognizes students’ ideas . The turn 11 is evident point in which he starts to use infor - mation to help students to express their ideas . DISCUSSION In this explorative case study , we have characterized four kinds of on - the - fly formative assessment discussions ( Table 2 ) . Getting data of student learning and giving feedback are two dimensions in this characterization . These two dimensions are essential in any formative assessment ( Black & William , 2009 ) and in on - the - fly discussions ( Ruiz - Primo & Furtak , 2006 ) . We found that teachers get information about students’ learning by quick interpretation or by further probing . Feedback was given in order or to help students to take the next step or to express their current thinking . MALU , Turku 2015 108 Table 2 . Four kinds of on - the - fly formative assessment interactions 1 Quick interpretation 2 Further probing A Help to take the next step 1A 2A B Help to express thinking 1B 2B When a teacher guides students to take the next sept ( A ) this can be done after a quick interpretation ( 1A ) or after further probing ( 2A ) . In the former , the guidance is based on correspondence of the students’ and the teacher’s ideas . In the latter , guidance is based more on the student’s ideas . Thus , the former is more authoritative guidance and the latter more dialogic guidance in the sense of Mortimer and Scott ( 2003 ) and Lehesvuori et al . ( 2013 ) . The feedback can focus also on getting the students to express their current thinking ( B ) . This can be based on a quick interpretation of where the students are in their learning ( 1B ) or it can include further probing ( 2B ) . Through further probing the teacher gets more information about the students’ learning and at the same time the students get feedback to explain more . The four identified formative assessment discussion types are related to ESRU coding ( Ruiz - Primo & Furtak , 2006 ) but there are also differences . The crucial difference is that in ESRU coding , individual utterances are coded whereas the four discussion types describe longer episodes . Sometimes types 1 and 2 can be seen in ESRU coding . For example , the pattern Student initiation – Recognition – Use implies that there was no further probing . On the hand , the pattern Elicit – Student response – Elicit – Student response implies that the episode most likely contain further probing . However , ESRU coding alone cannot be used to identify whether further probing exists because often further probing happens through follow - up questions that use the infor - mation of the previous student response and are thus coded as Use . In addition , interpreting whether an episode is of type A or B , includes analyz - ing the contents of Use . Ruiz - Primo and Furtak ( 2006 ) reported the positive relation between com - plete ESRU cycles and student achievement . Complete ESRU cycles have all the elements appearing at least once in an episode . We do not question their result , but we consider that there can be formative assessment although a cycle is incomplete . Thus , if we are considering only complete ESRU cycles , we are losing those episodes where teacher makes so quick interpretation that there is not even one elicitation . In our data these kinds of episodes were initiated by students as they explained what they were doing and asked something from the teacher . In these episodes the teacher already got some Nieminen , Hähkiöniemi , Leskinen & Viiri 109 information without eliciting and was able to use it . Much in the same way also Use can be very limited or even missing if , based on the elicited infor - mation , teacher decides to fade support for the students and let them continue on their own . We do not mean that some of the four types of assessment discussions are valued better than other . We know that further probing students’ ideas benefits both the students and the teacher , but because the teacher has to orchestrate the whole classroom , it is not always possible to probe further . For example , it is sometimes better to guide many students quickly than to only guide one student after further probing . Similarly , Mortimer and Scott ( 2003 ) argue that there is place for both authoritative and dialogic teacher talk . We will continue our analysis to find how well the four types of discussions fit with several lessons from primary , lower and upper secondary classes . The categorization will be specified or extended if needed . We will study possible links between on - the - fly discussion types and other factors such as teachers , topics and phases of lesson . We are also interested in commonalities and differences between physics and mathematics lessons in terms of on - the - fly formative assessment . ACKNOWLEDGEMENTS This study was supported by the European Union’s FP7 funding program . REFERENCES Black , P . , & Wiliam , D . ( 2009 ) . Developing the theory of formative assessment . Educational Assessment , Evaluation and Accountability , 21 ( 1 ) , 5 – 31 . Harlen , W . ( 2013 ) . Assessment & inquiry - based science education : issues in policy and practice . Global Network of Science Academies . Harlen , W . , & Qualter , A . ( 2014 ) . The teaching of science in primary schools . London : Routledge . Lehesvuori , S . , Viiri , J . , Rasku - Puttonen , H . , Moate , J . , & Helaakoski , J . ( 2013 ) . Visualizing communication structures in science classrooms : Tracing cumulativity in teacher - led whole class discussions . Journal of Research in Science Teaching , 50 ( 8 ) , 912 – 939 . Mortimer , E . F . , & Scott , P . ( 2003 ) . Meaning making in science classrooms . Milton Keynes , UK : Open University Press . Rocard , M . , Csermely , P . , Jorde , D . , Lenzen , D . , Walberg - Henriksson , H . , & Hemmo , V . ( 2007 ) . Science education now : a new pedagogy for the future of Europe . European Commission . Ruiz - Primo , M . A . , & Furtak , E . M ( 2006 ) . Exploring teachers ' informal formative assessment practices and students ' understanding in the context of scientific inquiry . Journal of Research in Science Teaching , 44 ( 1 ) , 57 – 84 . MALU , Turku 2015 110 Shavelson , R . L . , Young , D . B . , Ayala , C . C , Brandon , P . R . , Furtak , E . M . , Ruiz - Primo , M . A , Tomita , M . K . , & Yin , Y . ( 2008 ) . On the impact of curriculum - embedded formative assessment on learning : a collaboration between curriculum and assessment developers . Applied Measurement in Education , 21 , 295 – 314 . Palkki 111 VIRHEELLINEN ESIMERKKI MATEMATIIKAN LUOKKA - HUONEKESKUSTELUSSA Riikka Palkki Oulun yliopisto Oikean ja virheellisen esimerkin vertailun on todettu kehittävän matematiikan osaamista . Tässä tutkimuksessa selvitetään , miten neljä seitsemännen luokan matematiikan opettajaa käsittelevät luokissaan virheellistä esimerkkiä oikein tehdyn esimerkin rinnalla , millaisia kysymyksiä he käyttävät ja millaisia virhekäsityksiä oppilailla tulee esiin . Luokissa , joissa opettaja kysyi ”miksi” - kysymyksiä ja oppilas - määrä oli pieni , oppilaat kertoivat päättelyään , ja ilmi tuli sekä tehtävään sijoitettuun virheeseen että muuten algebraan liittyviä virhekäsityksiä . Virheellinen esimerkki vaikuttaa lupaavalta keinolta päättelyn harjoittamiseen sekä virheellisten käsitysten selvittämiseen ja korjaamiseen . JOHDANTO Algebrallisessa yhtälönratkaisussa muuttujaa aletaan käsitellä molemmilla puolilla yhtälöä . Algebraan siirtyminen on tärkeä , mutta haastava käänne - kohta koulumatematiikassa ( Andrews & Sayers , 2012 ) . Suomessa algebran käsitteelliseen osaamisen on todettu olevan heikkoa , ja opettajat ovat painottaneet laskurutiineja , kun taas yhtälöiden abstrakti ja käsitteellinen puoli on jäänyt vähäiselle huomiolle . Kansainvälisessä vertailussa algebran osaaminen on ollut muita matematiikan osa - alueita heikompaa . ( Attorps , 2006 ; Hihnala , 2005 ; Kupari , Vettenranta , Nissinen , 2012 . ) Niin ikään perusteluihin ei suomalaiskouluissa kiinnitetä riittävästi huomiota ( Andrews , 2013 ) . Virheellisen esimerkin käyttö oikein tehdyn esimerkin rinnalla ( kuva 1 ) on aiemmissa tutkimuksissa todettu kehittävän algebran käsitteellistä osaamista sekä oikeiden ratkaisumenetelmien ja käsitteiden käyttöä ( Booth , Lange , Koedinger & Newton , 2013 ; Durkin & Rittle - Johnson , 2012 ) . Joustava yhtälönratkaisu – projektin ( JYR , 2015 ) eräänä osana käytettiin vastaavia tehtäviä , jotka Virpi Kostama on suomentanut ja muokannut yhdysvaltalai - sesta Contrasting cases – materiaalista ( Star et al . , 2014 ) . JYR – projektissa painotetaan käsitteellistä osaamista , keskustelua , eri ratkaisutapojen vertailua ja itsearviointia . Tämän tutkimuksen aineistona olivat neljän seitsemännen luokan opettajan ja oppilaiden keväällä 2015 käymät luokkahuonekeskustelut . Tarkoituksena oli selvittää virheellisen esimerkin mahdollisuuksia . Tutkimuskysymykset olivat :  Millaisia virhekäsityksiä virheellisen esimerkin käyttö tuo esiin ?  Miten opettajat käyttävät virheellistä esimerkkiä ? ( opettajien toi - mintatavan kuvaus , käytetyt kysymystyypit ja vastausaika ) MALU , Turku 2015 112 Kuva 1 . Oikean ja virheellisen esimerkin sisältävä tehtävä . VIRHEELLINEN ESIMERKKI MATEMATIIKAN OPETUKSESSA Algebraan siirtyminen edellyttää riittävät esitiedot muun muassa muuttuja - käsitteestä ja yhtäsuuruusmerkin uuden roolin ymmärtämisen ( Kieran , 2004 ; Näveri , 2009 ) . Yhtälönratkaisussa voi näin ollen tehdä monenlaisia virheitä . Matematiikan opettajat näkevät virheiden taustalla neljä pääsyytä : oppilaan piirteet , opettajan roolin , matemaattisen tiedon ja noudatettavat säännöt ( Gagatsis & Kyriakides , 2000 ) . Näistä tarkastellaan nyt oppilaiden matemaat - tista tietoa heidän virhekäsitystensä kautta sekä opettajan toimintaa ( roolia ) . Virheellisen esimerkin käyttö tarkoittaa tyypillisen virheen esittämistä tarkoituksella , jolloin oppilaat paikantavat virheen ja analysoivat sitä ( Star ym . , 2014 ) . Tällöin virhekäsityksiä on mahdollista korjata ja toisaalta ennalta - ehkäistä . Kuvassa 1 esiintyvä erimuotoisten termien yhdistäminen on tyypillinen muuttujiin liittyvä virhekäsitys ( Booth , Barbieri , Eyer & Paré - Blagoev , 2014 ) . Virheellisten esimerkkien käytön vaikutuksista oppilaiden matematiikan osaamiseen on joitain tutkimustuloksia . Väärän ja oikean ratkaisutavan vertailu desimaalilukujen suuruuksia arvioitaessa auttoi oppilaita käyttämään oikeita käsitteitä ja oppimaan ratkaisumenetelmiä paremmin kuin kahden oikean ratkaisutavan vertailu ( Durkin & Rittle - Johnson , 2012 ) . Väärän ja oikean ratkaisutavan käyttö tietokoneavusteisessa ympäristössä auttoi algebran käsitteellisen ymmärtämisen kehittymisessä huolimatta opettajien aluksi epäilevästä suhtautumisesta virheiden esittämi - seen ( Booth ym . , 2013 ) . Palkki 113 Opettajan asennoituminen virheisiin siirtyy helposti oppilaille , ja toisaalta opettajan matemaattinen osaaminen ei johda kykyyn puuttua virheisiin ( Tulis , 2013 ; Son , 2013 ) . On siis perusteltua kiinnittää huomio opettajan toimintaan . Tässä tutkimuksessa kuvataan virhekäsitysten lisäksi opettajan toimintaa luokassa sekä tutkitaan opettajien käyttämiä kysymystyyppejä ja oppilaille antamaa vastausaikaa . Aiemmissa tutkimuksissa opettajan esittämät avoimet kysymykset ja oppilaille annettu 3—5 sekunnin vastausaika vaikuttivat matematiikan menestykseen ja joustavuuteen positiivisesti ( Star ym . , 2015 ; Tobin , 1986 ) . TUTKIMUSMENETELMÄ JA AINEISTON ANALYYSI Tutkimukseen osallistui neljä pohjoissuomalaista luokkaa ( A , B , C ja D ) , jotka olivat tätä ennen käyttäneet Joustava yhtälönratkaisu – materiaalia 5—6 oppi - tuntia harjoitellen yhtälökäsitettä , muunnoksia ja yhtälönratkaisutaitoja . Kaik - kiaan osallistuvia opettajia oli neljä , oppilaita 74 ja keskusteluja neljä . Luokis - sa käytiin keskustelu kuvan 1 tehtävästä , jossa Kallen ratkaisu sisälsi virheel - listä termien yhdistämistä . Keskustelut videoitiin ja litteroitiin muuttaen op - pilaiden nimet . Oppilaat , joilta ei ollut lupaa puheen äänittämiseen , eivät osallistuneet . Tutkimusmenetelmänä käytettiin tapaustutkimusta monipuoli - sen ja syvällisen ymmärryksen saavuttamiseksi tutkimuskohteesta ( Creswell , 2007 ) . Taulukko 1 . Opettajien käyttämät kysymystyypit . Kysymystyyppi Esimerkkikysymys Tarkoitus 1 Avoin ”Haluaako joku esittää näkemyksensä tähän ? ” ”Miksi ratkaisu on oi - kein ? ” ”Miksi tämä tapa on parempi ? ” ”Miten sait tämän vastauksen ? ” ”Miksi tätä kutsu - taan ? ” ”Onko vastaus viisi ? ” ”Mikä on seuraava kysymys ? ” Oppilaiden omien ideoiden jakaminen , toisten ideoiden edelleen kehittäminen tai kritisointi tai algebrallinen ajattelu . Käsitteellinen ymmärtäminen ; miksi ratkaisu on oikein tai miksi ratkaisuta - pa on ollut hyvä valita . Proseduraalinen ymmärtäminen ; miten ongelma on ratkaistu , miten sääntöä on käytetty . Tiedon jäsentäminen tai vastauksena pelkkä numero tai luku . Vastaus tyyppiä ”kyllä” / ”ei” . Halutun kysymyksen tai käytöksen pariin ohjaaminen . 2 ”Miksi” 3 ”Miten” 4 ”Mitä” 5 Retorinen / valinta 6 Logistinen / hallinta MALU , Turku 2015 114 Oppilaiden virhekäsitykset etsittiin litteroinneista ja koottiin yhteen . Keskus - teluja kuvattiin tapauksittain , tapauksia vertaillen ja merkitystä pohtien ( Creswell , 2007 ) . Opettajien toimintaa kuvattiin aineistolähtöisesti luokitellen heidän puhettaan , minkä pohjalta heidän toiminnastaan muodostettiin kuvaukset ja yhteenvedot ( otsikoissa ) . Tässä yhteydessä laskettiin opettajien ( kielitieteellisten ) lauseiden osuus suhteessa oppilaiden käyttämiin lauseisiin . Toiseksi opettajien käyttämät kysymykset jaoteltiin käyttäen Starin ym . ( 2015 ) kysymystyyppejä : 1 ) Avoin , 2 ) ”Miksi” , 3 ) ”Miten” , 4 ) ”Mitä” , 5 ) Retori - nen / valinta ja 6 ) Logistinen / hallintaan liittyvä kysymys , joiden tarkemmat selitykset löytyvät taulukosta 1 . Starin ym . ( 2015 ) analyysista poiketen nyt painotettiin virheen analysointia eikä ratkaisutapojen vertailua , joten kysymystyyppi 2 sisälsi lähinnä perusteluja etsiviä kysymyksiä . Luokittelun tukena käytettiin QSR NVivo – ohjelmaa . Kysymystyyppien lukumäärä laskettiin kunkin opettajan kohdalla ja opettajien oppilaille antama vastausai - ka arvioitiin yli tai alle 3 sekuntiin . TULOKSET Luokka A – Opettaja esittää ”miksi” - kysymyksiä ja korjaa virhekäsityksiä Luokassa A oli 12 oppilasta , joita opettaja kuvasi taidoiltaan kaikentasoisiksi , ei mielellään matematiikasta keskusteleviksi , mutta melko aktiiviseksi . Kun opettaja kysyi , kumpi ratkaisuista on oikein , oppilaat valitsivat Leenan ratkaisun . Antti esitteli matemaattisena perusteluna Leenan oikean vastauk - sen sijoittamisen alkuperäiseen yhtälöön : Opettaja A Mistä sen tietää , että se on oikein ? Antti . [ 2 ”miksi” - kysymys ] Antti Senhän voi ratkaista vaikka sillee , että 45 kertaa kuus plus 90 on… [ opettaja keskeyttää ] Opettaja A Eli tämä voidaan tuonne laittaa . [ näyttää ] No se on vähän hankala laskea , mutta voithan sä laskea tuota Kallen vastauk - sella , kun se on helpompi . Laitat tuon tuonne y : n paikalle . 45 kertaa nolla on nolla ja tuolla on 90 ja tästä tullee nolla , niin siitä huomaa , että nyt ei mennytkään oikein . Tässä ”miksi” - kysymys toi esiin oikean perustelun , joskin opettaja esitteli itse tehokkaamman tavan perustella vastausta . Oppilaat löysivät virheen ja kertoivat , ettei erimuotoisia termejä saa laskea yhteen . Eräs oppilas käytti ensin sanaa ”samannimiset” sanan ”samanmuo - toiset” sijaan , minkä opettaja korjasi . Yleistä sääntöä etsittäessä Satu esitti virheellisen perustelun : ”X ja y ja jne . on muuttujia , joten ne pitää selvittää ennen kuin niitä voi yhdistää . ” Opettaja vaikutti tulkitsevan , että Satu tarkoitti , ettei myöskään samanmuotoisia termejä saisi yhdistää ja huomautti tästä . Oppilas ei mahdollisesti hahmottanut , että lauseketta voi sieventää yhtälön sisällä . Oppilas saattoi tosin tarkoittaa , että erimuotoiset termit voisi Palkki 115 yhdistää vasta , kun olisi tiedossa muuttujan paikalle sijoitettava lukuarvo . Yleisen säännön pohdintaa : Timo Siinä niinku vakiotermit saa yhdistää tai x – termejä tai y – termejä saa yhdistää . Opettaja A Roope haluais sanoa vielä eri tavalla . Roope Jos termeissä on sama vakiokirjain tai ei kirjainta ollenkaan , niin ne voidaan yhdistää . Opettaja A Joo , keskenään . Antti haluais vielä sanoa . Antti Yhdistän vain samanmuotoiset termit . Opettaja A Joo , se ois ehkä kaikista lyhin . Jos et oo kirjoittanut vielä , niin sen voit kirjoittaa . Keskustelu eteni sujuvasti oppilaalta toiselle ja oppilaat kuulivat säännön selitettynä hiukan eri tavoin . Kokonaisuudessaan luokan A opettaja esitti melko monta ”miksi” - kysymystä ja oppilaat keskustelivat . Luokan A opettaja ohjaili keskustelua ja tarvittaessa täydensi vastauksia ja korjasi virhekäsityk - siä . Opettaja sanoi 50 lausetta , kun oppilaat 16 . Tuli esiin kaksi eri virhekäsi - tystä . Luokka B – Opettaja kyselijänä ja selittäjänä Luokassa B oli 17 oppilasta . Opettaja kuvasi luokkaa taidoiltaan heikoksi , joskus matematiikasta keskustelevaksi ja melko passiiviseksi . Ratkaisun pohtiminen : Opettaja B Kumpi ratkaisusta on oikein ? [ 5 ”valinta” – kysymys ] Ilkka Kallen . Vili Ei Kallen , Leenan ! Markus Leenan . Senhän näkee heti , kun näkee , mikä y on . Ei 45 kertaa nolla plus 90 voi olla 60 nolla . [ Opettaja jatkaa selitystä pitkällä puheenvuorolla . ] Opettaja ei tarttunut väärään vastaukseen , eikä selvinnyt liittyikö tähän mahdollisesti virhekäsitys . Kohdassa b ) keskustelu kulki oppilaalta toiselle : Kosti Se on päätellyt sen . Tuukka Se jako molemmat puolet 75 : llä . Vesa Hä . Kosti Niin . Opettaja B Miksi se ei ois saanu jakkaa 75 : llä ? [ 2 ”miksi” - kysymys ] Tuukka Siitä tulee nolla . [ … ] MALU , Turku 2015 116 Tuukan esittämään virhekäsitykseen kysyttiin yksi “miksi” - kysymys , mutta ei toista , jonka avulla olisi voinut paljastua , miksi oppilas ajattelee , ettei ratkaisuksi voisi tulla nolla . Virhekäsitystä ei nyt korjattu . Oppilailta löytyi oikea vastaus , ja he osasivat perusteella , että samanmuotoisia termejä voi yhdistää . Opettaja selitti lisää itse ja kävi ainoana opettajana läpi oikean ratkaisutavan . Yleistä sääntöä pohdittiin lopuksi : Opettaja B Minkämuotoisia termejä saa yhdistää ? [ 4 ”mitä” – kysymys ] Kosti Samanmuotoisia . Opettaja B Hyvä . Ja mitä se… ? [ 4 ”mitä” – kysymys ] Kosti Sama kirjain siellä . Sama muuttuja . Opettaja B Hyvä . Tai sama kirjain . Sama kirjainosa pittää olla siellä . [ … ] Luokan B opettaja kyseli oppilailta ja selitti runsaasti itse ( 62 lausetta ) . Oppilaat keskustelivat aktiivisesti muihin luokkiin verrattuna ( 27 lausetta ) etukäteiskuvauksesta poiketen . Kolme virhekäsitystä tuli ilmi kolmella eri oppilaalla . Koulu C – Opettaja innokkaana kyselijänä ilman vastakaikua Luokassa C on 26 oppilasta , joita opettaja kuvaili melko aktiivisiksi , mutta ei mielellään matematiikasta keskusteleviksi . Luokassa oli runsaasti sekä matematiikassa heikosti että kiitettävästi menestyneitä oppilaita . Tehtävän a ) - kohdan oikea vastaus tuli välittömästi , perusteluja ei kysytty . Kohta b ) : Opettaja C Okei . Leena teki oikein . Näin taitaa olla . No , minkäs virheen se Kalle on tehnyt ? Mitä hassua Kalle on tehnyt ? Mitä vää - rää ? Jenna . [ kolmesti 4 “mitä” – kysymys ] Jenna Se yhdisti niitä erinäköisiä termejä . Opettaja C Sanotko mitä se yhdisti ? Osaatko kattoa siitä nopeasti ? Siinä saa auttaa kaveritkin . Joo ? [ 4 “mitä” ; 6 logistinen kysymys ] Jenna 45y ja 90 . Opettaja C Se laski 45 y ja 90 ja kirjoitti seuraavalle riville , että on 135y [ - ] ei [ - ] ja sehän on mahoton , ei me voida laskea y : tä ja nu - meroita . Ei voi mennä oikein . Opettaja kyseli ja selitti itse . Yleistä sääntöä etsittäessä oppilailta tuli kirjassa ollut mallimääritelmä , johon opettaja yritti saada tarkempaa selitystä : Opettaja C Mietin , että se sana samanmuotoinen on kauhean vaikea . Että jos vaikka kotona sannoo äitille , että katoppa , oonko mää yhistäny samanmuotoisia termejä , niin osaako äiti aut - taa ? Voisko siihen keksiä vielä jonkun vielä helpomman Palkki 117 säännön ? Mikko , ootko keksinyt ? [ 5 retorinen ; 1 avoin ; 5 va - linta ] Mikko E . Opettaja C Okei , no te kaikki ossaatte siis sen perusteella . No niin , sit - ten ei mittää . [ Opettaja jatkaa seuraavaan asiaan . ] Luokan C opettaja kysyi useita , avoimiakin kysymyksiä , joten hänen toimintansa viittaa innokkaaseen kyselyyn ( 43 lausetta ) . Oppilaat eivät mielellään keskustelleet matematiikasta ( 9 lausetta ) , kuten opettaja olikin etukäteen kuvaillut . Yhtään virhekäsitystä ei ilmennyt , vaikka opettaja kertoi oppilaiden kuvailleen tehtävää vaikeaksi . Luokka D – Opettaja pienryhmien ohjaajana ja malliratkaisun kokoajana Luokassa D oli 19 oppilasta , joita opettaja kuvaili melko aktiivisiksi , jonkin verran matematiikasta keskusteleviksi ja pääosin matematiikassa lahjakkaiksi . Vastaus “Leena” perusteltiin oikeaksi sijoittamalla vastaus alkuperäiseen yhtälöön . Väärää ratkaisua ei käsitelty . Yleistä sääntöä opettaja pyysi vertailemaan pienryhmissä , joiden keskusteluja ei tallennettu . Yhteenvetona opettaja totesi , että kaikki saivat oikean vastauksen . Yhtään virhekäsitystä ei siten tullut ilmi , mihin ryhmän kiitettävät matematiikan taidot saattoivat vaikuttaa . Opettaja lähinnä ohjasi pienryhmiä ja varmisti malliratkaisun ( 33 lausetta , oppilailla 4 ) . Yhteenveto Neljä seitsemättä luokkaa käsitteli virheellistä esimerkkiä oikein tehdyn esimerkin rinnalla opettajajohtoisessa keskustelussa . Kahdelta luokan A ja kolmelta luokan B oppilaalta tuli esiin yhteensä viisi eri virhekäsitystä ( taulukko 2 ) . MALU , Turku 2015 118 Taulukko 2 . Puhutut lauseet ja esiin tulleet virhekäsitykset luokittain . Luokka Puhutut lauseet Virhekäsitykset Oppi - laita Opettaja ( % kaikista ) Oppilaat ( % kaikista ) kpl Kuvaus A 12 50 ( 76 % ) 16 ( 24 % ) 3 - Termien sekoittaminen - Muuttujatermejä ei voi yhdistää ( kahdesti ) B 17 62 ( 70 % ) 27 ( 30 % ) 3 - Kallen vastaus on oikein - Nollaa ei voi saada tu - lokseksi - ”60 nolla” eli kertomerkki puuttuu sijoituksesta C D 26 19 43 ( 83 % ) 33 ( 89 % ) 9 ( 17 % ) 4 ( 11 % ) 0 0 Luokassa A opettaja toimi keskustelun ohjaajana ja tarttui helposti virheisiin . Luokassa B opettaja käytti paljon aikaa selittääkseen ja perustellakseen asioita itse , muttei käsitellyt kaikkia virheitä . Luokassa C opettaja kyseli paljon , mutta oppilaat eivät juuri ottaneet osaa keskusteluun . Luokassa D tehtävän käsittely tapahtui lähinnä pienryhmissä . Virhekäsityksiä esiintyi luokissa A ja B , joissa oppilaat käyttivät enemmän puheenvuoroja kuin luokissa C ja D ( taulukko 2 ) . Opettajien esittämät kysymystyypit painottuivat eri tavoin ( kuva 2 ) . Luokan A opettajan rooli keskustelun ohjaajana näkyy pienenä kysymysten määränä . Luokan C opettajan useat peräkkäiset kysymykset nostavat hänen esittämiensä kysymysten lukumäärää . Luokan A ja C opettajat kysyivät muutamia avoimia kysymyksiä . Luokan A ja B opettaja kysyivät käsitteellisen osaamisen selvittämiseen tähtääviä ”miksi” - kysymyksiä ( Star ym . , 2015 ) melko paljon . Luokkien C ja D opettajat eivät käyttäneet niitä juuri lainkaan eikä virhekäsityksiä tullut esiin lainkaan toisin kuin luokissa A ja B . Oppilaita oli eniten luokissa C ja D . Luokan D opettaja kysyi eniten retorisia ja logistisia kysymyksiä , mikä johtui hänen valitsemas - taan lähestymistavasta ohjata oppilaita työskentelemään pienryhmissä . Oppilaille annettu vastausaika oli kaikissa luokissa koko ajan alle kolme sekuntia eli vähemmän kuin matematiikan osaamisen kehittymiselle hyödylliseksi koettu aika ( Tobin , 1986 ) , joten sitä ei tarkasteltu tarkemmin . Palkki 119 Kuva 2 . Opettajien A , B , C ja D käyttämät kysymystyypit POHDINTA JA JOHTOPÄÄTÖKSET Kahdessa luokassa neljästä tuli esiin yhteensä viisi eri virhekäsitystä . Luokkien A ja B oppilaat paitsi korjasivat esimerkkiin sijoitettua tarkoituksel - lista lausekkeiden sieventämiseen liittyvää virhettä , myös saivat mahdolli - suuden muiden virhekäsitysten esiintuomiseen ja korjaamisen , mikä oli uutta aiempiin tutkimuksiin verrattuna ( vrt . Booth ym . , 2013 ) . Keskustelujen yhteydessä tapahtui termien korjaamista oikeaksi . Aiemmassa tutkimuksena virheellisten esimerkkien tutkiminen oli vaikuttanut positiivisesti termien oikeaan käyttöön ( Durkin & Rittle - Johson , 2012 ) . Oppilailla oli ongelmia ymmärtää muuttujan eri rooli lausekkeessa ja yhtälössä , minkä Näverikin ( 2009 , s . 153 ) on havainnut . Oppilas vaikutti ajattelevan , ettei samanmuotoi - siakaan termejä voisi yhdistää ennen kuin niihin olisi sijoitettu jokin luku , kuten lausekkeiden kohdalla tehdään . Tähän voitaisiin mahdollisesti puuttua virheellisellä esimerkillä . Luokan B melko passiivisiksi ja matemaattisiltaan taidoiltaan heikoiksi kuvatut oppilaat kykenivät hyödyntämään virheitä oppimiseen . Tämä on samansuuntaista kuin Durkinin ja Rittle - Johnsonin ( 2012 ) tutkimuksessa , mutta päinvastaista Großen ja Renklin ( 2007 ) tulokselle , jonka mukaan vain riittävät esitiedot omaavat oppilaat pystyisivät hyödyn - tämään virheellistä esimerkkiä . Virhekäsitysten pohjalta voi päätellä , että yhtälönratkaisuun tarvittavissa esitiedoissa oli puutteita , jotka nyt saatiin näkyville . Tämän tutkimuksen tulosten mukaan opettajan esittämät ”miksi” - kysymykset saivat kahden luokan oppilaita osallistumaan : pohtimaan matematiikkaa ja esittämään virheellisiäkin käsityksiään ja perustelujaan . Tämän voi ajatella mahdollistaneen perustelemisen ja käsitteellisen osaamisen kehittymisen , joille suomalaisessa opetuksessa on perinteisesti annettu pieni painoarvo ( Andrews , 2013 ; Attorps , 2006 ) . Luokkien pieni koko saattoi vaikuttaa aktiivisuuteen . Kenties luokissa , joissa ”miksi” - kysymyksiä ei MALU , Turku 2015 120 esitetty eikä virhekäsityksiä tullut ilmi ( luokat C ja D ) , ilmapiiri ei aiemmin - kaan ollut ollut perusteluita hakeva , mistä ”miksi” - kysymysten puute toimi indikaattorina . Opettajien vaikeus havaita virheitä ja puuttua niihin ( Son , 2013 ) tuli ilmi eräässä luokassa , jossa opettaja ei reagoinut kaikkiin oppilaiden virheellisiin ajatuksiin . Vastausaika ei ollut nyt relevantti tutkimuskohde . Tutkimuksessa on rajoituksensa , sillä siinä käsiteltiin vain neljän luokan yhden tehtävän analysointia , mutta tapaustutkimuksena se oli onnistunut : löydettiin uutta näkökulmaa virheellisten esimerkkien käyttöön luokkahuo - nekeskustelussa . Virheellistä esimerkkiä voi hyödyntää sekä oppimisen että opettamisen apuna . Virheellisten esimerkkien käyttö oppikirjoissa on harvinaista ( Durkin & Rittle - Johnson , 2012 ) . Tämän tutkimuksen perusteella niiden käyttöä voisi lisätä . Tulosten mukaan opettajan aktiivinen asenne ei yksin riitä luokan aktivoimiseen , vaan kysymystyypillä on merkitystä . Jatkotutkimus voisi käsitellä ”miksi” - kysymysten vaikutusta tutkimista , perusteluja ja virheitä sallivan luokkahuonekulttuurin muotoutumiseen ja käsitteellisen osaamisen kehittämiseen . LÄHDELUETTELO Andrews , P . ( 2013 ) . Finnish Mathematics Teaching from a Reform Perspec - tive : A Video - Based Case - Study Analysis . Comparative Education Review , 57 ( 2 ) , 189—211 . Andrews , P . & Sayers , J . ( 2012 ) . Teaching linear equations : Case studies from Finland , Flanders and Hungary . Journal of Mathematical Behavior , 31 , 476— 488 . Attorps , I . ( 2006 ) . Mathematics teachers’ conceptions about equations . Dissertation . University of Helsinki . Booth , J . L . , Lange , K . E . , Koedinger , K . R . & Newton , J . K . ( 2013 ) . Using example problems to improve student learning in algebra : Differentiating between correct and incorrect examples . Learning and Instruction , 25 , 24— 34 . Booth , J . L . , Barbieri , C . , Eyer , F . , & Paré - Blagoev , E . J . ( 2014 ) . Persistent and Pernicious Errors in Algebraic Problem Solving . Journal of Problem Solving , 7 ( 1 ) , 3 . Creswell , J . W . ( 2007 ) . Qualitative inquiry & Research Design . Choosing Among Five Approaches . Sage : Thousand Oaks . Durkin , K . & Rittle - Johnson , B . ( 2012 ) . The effectiveness of using incorrect examples to support learning about decimal magnitude . Learning and Instruction , 22 ( 3 ) , 206—2014 . Gagatsis , A . & Kyriakides , L . ( 2000 ) . Teachers’ Attitudes Toward Their Pupils’ Mathematical Errors . Educational Research and Evaluation , 6 ( 1 ) , 24—58 . Palkki 121 Große , C . & Renkl , A . ( 2007 ) . Finding and fixing errors in worked examples : Can this foster learning outcomes ? Learning and Instruction , 17 ( 6 ) , 612— 634 . Hihnala , K . ( 2005 ) . Laskutehtävien suorittamisesta käsitteiden ymmärtämisen . Peruskoululaisen matemaattisen ajattelun kehittyminen aritmetiikasta algebraan siirryttäessä . Väitöskirja . Jyväskylän yliopisto . JYR ( 2015 ) . Joustava yhtälönratkaisu – LUMA SUOMI – kehittämishankkeen esittely . Oulu . http : / / ouluma . fi / joustava - yhtalonratkaisu / Kieran , C . ( 2004 ) . Algebraic thinking in the early grades : what is it ? The Mathematics Teacher , 8 ( 1 ) , 139—151 . Kupari , P . , Vettenranta , J . & Nissinen , K . ( 2012 ) . Oppijalähtöistä pedagogiikkaa etsimään . Kahdeksannen luokan oppilaiden matematiikan ja luonnontieteen osaaminen . Kansainvälinen TIMSS – tutkimus Suomessa . Koulutuksen tutki - muslaitos . Jyväskylän yliopistopaino . Näveri , L . ( 2009 ) . Aritmetiikasta algebraan Muutoksia osaamisessa peruskoulun päättöluokalla 20 vuoden aikana . Väitöskirja . Helsingin yliopisto . Son , J . ( 2013 ) . How preservice teachers interpret and respond to student errors : Ratio and proportion in similar rectangles . Educational Studies in Mathematics , 84 ( 1 ) , 49—70 . Star , J . R . , Newton , K . , Pollack , C . , Kokka , K . , Rittle - Johnson , B . & Durkin , K . ( 2015 ) . Student , teacher , and instructional characteristics related to stu - dents ' gains in flexibility . Contemporary Educational Psychology , 41 , 198—208 . Star , J . R . , Pollack , C , Durkin , K . , Rittle - Johnson , B . , Lynch , K . Newton , K . & Gogolen , C . ( 2014 ) . Learning from comparison in algebra . Contemporary Educational Psychology , 40 , 41—54 . Tobin , K . ( 1986 ) . Effects of Teacher Wait Time on Discourse Characteristics in Mathematics and Language Arts Classes . American Educational Research Journal , 23 , 191—200 . Tulis , M . ( 2013 ) . Error management behavior in classrooms : Teachers ' responses to student mistakes . Teaching and Teacher Education , 33 , 56 - 68 . MALU , Turku 2015 122 DOES RRI FOCUSED SCIENCE TEACHING HELP STUDENTS INCORPORATE RRI INTO THEIR INQUIRY - BASED LESSON PLANS ? Ilkka Ratinen 1 , Anna - Leena Kähkönen 1 , Anssi Lindell 1 & Miikka de Vocht 2 1 University of Jyväskylä , 2 University of Helsinki Responsible Research and Innovation ( RRI ) has become an ever more important part of the European society as well as of scientific literacy . RRI is a transparent , interactive process by which societal actors and innovators become mutually responsive to each other with a view to the ethical acceptability , sustainability and societal desirability of the innovation process and its marketable products . But how to include RRI into inquiry - based teaching , which is a well - studied subject , but seldom carried out in practice ? To answer this question , such a science education course was designed and piloted for 12 primary student teachers within the EU - funded IRRE - SISTIBLE project . Their incorporation of RRI was compared to a reference group of 23 primary student teachers , attending a conventional science education course . Our results indicate that , even with a pre - course on RRI , it is difficult for student teachers to include RRI aspects into lesson plans . RRI aspects should be connected explicitly into teaching during the planning phase of training lessons . The biggest impact of the course was to improve student teachers’ practise of using disciplinary core ideas in designing science teaching . Student teachers’ motivation and commitment improves immensely when they are teaching real pupils rather than taking part of a regular course . INTRODUCTION There is an urgent need to incorporate RRI into science education ( EU , 2012 ) . Von Schomberg ( 2013 , 19 ) defined RRI as follows : “ Responsible Research and Innovation is a transparent , interactive process by which societal actors and innovators become mutually responsive to each other with a view to the ( ethical ) acceptability , sustainability and societal desirability of the innovation process and its marketable products ( in order to allow a proper embedding of scientific and technological advances in our society ) . ” According to Sutcliffe ( 2011 ) , RRI in science teaching considers following components : engagement , ethics , open access , science education , governance , and gender equality . What RRI and inquiry - based teaching have in common is that they both reflect the actions of a researcher – inquiry in a smaller , research - oriented context , and RRI in the larger , societal context . There is evidence that inquiry - based teaching effectively prepares pupils for future Ratinen , Kähkönen , Lindell & de Vocht 123 challenges and supports a better understanding of science and conducting science in general ( Lederman , Antink , & Bartos , 2014 ) . Pupils who participate in inquiry - based teaching achieve better learning outcomes than in traditional teaching ( Akkus , Gunelb and Handc’s , 2007 ; Minner , Levy , & Century , 2010 ) . There is evidence that primary student teachers , who familiarize themselves with inquiry - based teaching , under - stand relatively well how to implement it also in the primary science class - room ( Lehesvuori , et al . 2011 ) . In order to support pupils’ learning , teachers must be aware of the different phases ( cf . 5E model in Bybee et al . 2006 ; or for an overview of several models , Pedaste et al . , 2015 ) of inquiry - based learning . Furtak , Seidel , Iverson and Briggs ( 2012 ) point out that the pupils , who participate in inquiry - based teaching with teacher - led activities , have larger effect sizes of learning than those with student - led conditions . We recognize that there is no single right way to carry out inquiry , but it may entail different levels of openness , as Banchi and Bell ( 2008 , 27 ) have suggested :  Confirmation inquiry is useful when a teacher’s goal is to reinforce a previously introduced idea ; students are provided with a question and procedure for confirming or reinforcing a previously learned idea or practicing the specific skills of data collection and record - ing .  In structured inquiry , the question and procedure are posed by the teacher , but students generate an explanation , supported by the ev - idence they have collected .  In guided inquiry , the teacher provides students with only the re - search question , and students design the method to test both the question and any resulting explanations .  At the highest level of openness , open inquiry , students have an op - portunity to act like scientists : deriving questions , designing and carrying out investigations , and communicating their results . However , an argument against inquiry - based learning from Abrahams and Millar ( 2008 ) goes that just doing experiments does not lead to better learning outcomes . Also Hodson ( 2014 ) criticised that inquiry - based learning necessi - tates a “doing science” approach in every class . When one envisions a future that includes RRI into inquiry - based science teaching in a large scale , it is important to know how to develop student teachers’ readiness to apply RRI in their classes . It is known that reforms in schools are unlikely to happen , if teachers are not involved from the begin - ning of the reform process , and teachers’ attitudes towards a learning innovation play a ”make - or - break” role in the success of that innovation ( van Driel , Beijaard , & Verloop , 2001 ) . MALU , Turku 2015 124 The United States National Research Council’s Framework for K - 12 Science Education and the Next Generation Science Standards ( NGSS Lead States , 2013 ) aims to focus the earlier large scale of topics in science education into a limited number of Disciplinary Core Ideas ( DCI ) . The same trend can be observed in Finnish reformed national core curriculum ( POPS , 2014 ) . For example , the only physics concepts asked to be tested explicitly after grade 6 are energy , force and motion . In this core curriculum the content is not expressed as DCIs , but as rather open expressions like “studies of phenomena of light and sound” . This apparent openness and teachers’ freedom in planning their lessons may induce teaching units with vague objectives , which then may lead to difficulties in assessment . The difficulty of assessment , and its potential to reduce lessons into rote learning exercises , is recognized by Pellegrino ( 2013 ) . He writes on the new way of expressing performance expectations via abilities and core ideas , and sees it as an aide for teachers in assessing their students : “These statements move beyond vague terms such as “know” and “under - stand” to more specific statements like analyze , compare , explain , argue , represent , predict , model , etc . , in which the practices of science are wrapped around and integrated with core content . - - - The virtue of such view is that science educators are poised to better define the outcomes desired from their instructional efforts , which in turn guides the forms of assessment that can help them know whether their students are attaining the desired objectives , as well as how they might better assist them along the way . ” ( Pellegrino , 2013 p . 320 ) The NGSS and also in Finnish national curriculum emphases the students’ skills to do science and express their learning goals as skills rather than acquired knowledge . Both curricula share the goal of engaging with fewer ideas , explored in more depth , and adopting the scientific or engineering practices ( Krajcik & Merritt , 2012 ; POPS 2014 ) . The core ideas and an ability to formulate them is a great tell of how teachers understand the content or goals of what they are teaching . The difficulty is that most elementary level teacher students have difficulty in recognizing relevant scientific DCIs , because their studies do not focus on skills needed in inquiry . In this study our data comes from lesson plans ; they are a good source in that the student teachers have already become familiar with using them in previous courses , and the plans are required to include a setting of objectives and through those goals , some ideas for assessment . Should the core ideas or RRI components not appear explicitly in the lesson plans , it is seen as unlikely that they were critically thought of or planned into the lessons by the student teachers . Through this data we will be able to show if at all the student teachers use these tools efficiently in planning a lesson , and how their usage of core ideas as basis for lessons , or raising RRI issues in class , changes during Ratinen , Kähkönen , Lindell & de Vocht 125 one science education course . The aim of the study is to examine how an RRI focused science education course helps student teachers to plan a science lesson and whether their plans include the disciplinary core ideas ( DCIs ) of science and the components of RRI . METHODS Participants In total , 35 primary school student teachers in their second year of studies participated in this study . One group ( n = 12 ) took part in the EU - funded Irresistible project ( Irresistible group ) . A control group ( n = 23 ) studied with conventional lessons ( Control group ) . The student teachers signed up to the groups on a voluntary basis . The course introduced student teachers to theoretical ideas of inquiry - based learning and teaching , planning for teaching , and implementing teaching in the elementary classroom . Materials and procedures In the Irresistible group , RRI was included explicitly as RRI workshops , but in control group only as an introduction of Finnish national core curriculum objectives within 30 contact hours of small - group sessions of the course . In the science pedagogy course for Irresistible group , we emphasised that inquiry - based science lessons should include opportunities for students to engage both in inquiry as a process and RRI as a foundation . This was learned in RRI - workshops . The workshops included a variety of tasks , such as recognizing the different stakeholders in science and society from written and video materials about inventions or research , pinpointing the RRI issues which were not realized in a series of events leading to a disaster ( e . g . the asbestos industry ) , and discussing how pupils or student teachers could act in the spirit of the RRI components ( e . g . to promote equality or enhance engagement of people and science background in local policy ) . The workshops connected RRI to the activities of student teachers in their daily lives , but did not involve plans to incorporate RRI into lessons . The decision was made to support student teachers’ freedom to design their own tasks rather than emulate the exact one discussed in class . The reference course did not include RRI workshops but the aspects of RRI issues were taught as good principles of science teaching . Both groups participated in 20 hours of inquiry - based science education lessons . Before this study , the students of Irresistible group had already passed one science education course . MALU , Turku 2015 126 During the course student teachers in RRI group developed open ( Banchi & Bell , 2008 ) inquiries of different science topics to be included into a teaching module of climate change . In the Control group student teachers also developed open inquiries but the topics were from different areas of science , such as a teaching of seasons or magnetism , for example . Such an approach nicely covers the three important aspects for curriculum development nominated by Krajcik , McNeill and Reicer ( 2007 ) : unpacking national science standards , developing a learning performances approach to specifying learning goals , and aligning learning goals , instructional activities , and assessments . An important , explicit goal in the Irresistible group was to create RRI teaching in science context . In the control group , while student teachers were intro - duced to RRI components implicitly during their course , they were not specifically asked to pay attention to them during inquiry planning . Analysis Content analysis ( Neuendorf , 2001 ) of the lesson plans was used to study the student teachers’ ideas about teaching the topic of air for Grade 6 students ( aged 12 years ) . The student teachers individually wrote a lesson plan for a 45 min primary science class , both pre and post - instruction . Pre lesson plan was written after the first lesson of the science education course , and post lesson plan after the course . The DCI defined in the national curriculum ( POPS , 2014 ) were analysed from the objectives of the lesson plans . The evidence of RRI was based on the occurrence of Sutcliffe’s ( 2011 ) RRI components in the text . The experience of student teachers’ plans containing open , vague statements , led to choosing to evaluate ambiguous statements as RRI components . Lesson plans in both groups were interpret positively : when an issue that could be read as an RRI component came up , it was coded as such . For example , open access to information was composed in a lesson plan as follows : “acquisition of information from the Internet , not just from the textbook… … and making a mind map to the smartboard where the facts pupils have found are . ” Ethical issues in the lesson plans related to research and the effects of research on the environment and it was composed if there where sentences such as “…where is air [ experiment ] … …air in our everyday live . ” Engagement was composed according to the sentence which was indicated about the role of different societal actors to make science together : “applying new knowledge… …and use the kitchen of school for the experiment [ shrinking the balloon in the freezer ] . ” The analysis consisted of two researchers’ independent coding and categori - zation , which were then compared . Finally , “science education” – component was excluded because it was obviously in every lesson plan . The percentages Ratinen , Kähkönen , Lindell & de Vocht 127 were calculated in order to know how many students included the issues of DCI and RRI to their lesson plans . Hake’s gain ( Hake , 1998 ) was calculated by using following formula : 𝐺 = 𝑝𝑜𝑠𝑡 _ 𝑡𝑒𝑠𝑡 % − 𝑝𝑟𝑒 _ 𝑡𝑒𝑠𝑡 % 100 − 𝑝𝑟𝑒 _ 𝑡𝑒𝑠𝑡 % The statistical significance of DCI and RRI between groups was tested using Mann - Whitney U - test . According to U - test , there was not statistically significant differences between the Irresistible and Control groups ( p > 0 . 05 ) . The statistical significance between the Pre - and Post - test of DCI and RRI was tested using McNemar test . Test indicated the statistically significant differences between the Pre - and Post - test of DCI ( p < 0 . 001 ) . As the goal of this research is to see whether the designed course structure had any impact on the ability to plan RRI components into teaching plans , and both groups were evaluated in a more positive manner , this decision allowed us to compare differences between the groups and the overall impact of the courses . However , we do not make claims that all coded RRI compo - nents are equally seriously thought out , or furthermore that they would be realized in enacting such a plan in class settings . RESULTS Disciplinary Core Ideas in Lesson Plans Before the science education course , none of the student teachers in the Irresistible group and only one in the control group based their plans on DCIs ( Table 2 ) . The course had a good impact on this design principle : Hake’s gain in Irresistible group was 0 . 58 and in the conventional group 0 . 43 . Table 2 . The number of student teachers’ lesson plans that were based on an adequate disciplinary core ideas ( DCIs ) in science . Irresistible ( n = 12 ) Control ( n = 23 ) Pre Post Pre Post DCI G 0 7 ( 58 % ) 0 . 58 1 ( 4 % ) 10 ( 44 % ) 0 . 43 p > 0 . 05 between groups ; p < 0 . 001 between pre - and post - test The content analysis of the lesson plans revealed that students use of DCIs improved during the science education course . According to the following MALU , Turku 2015 128 quotations , student teachers commonly wrote scientific core ideas in their pre lesson plans as a learning outcomes . They also expressed these goals quite imprecisely : Student teachers Irresistible a : Exploring the properties of air . Student teacher Irresistible b : Understand what is air , its contents , and how it behaves . Student teacher Control c : Understand what is air , content and behavior . Student teacher Control d : After lesson pupils understand what is air and how they better understand it after their observa - tions . The following post - plan quotations indicate that student teachers expressed the scientific core ideas quite vaguely also after the science education course . However , in the post - lesson plans learning goals are greatly improved . Although learning goals are still expressed in a compact manner , students are now expressing science content in greater detail : Student teacher Irresistible a : Air is a mixture of gases . Student teacher Irresistible b : Air is a substance which expands and contracts on the effect of temperature changes . Student teacher Control c : Air is a substance and behaves differently in dif - ferent temperatures . Student teacher Control d : Air is a mixture of gases . However , the statements do not explicitly express what it was that the student teachers expected their pupils to learn . Summing up , before the science education course , the DCIs were expressed vaguely , but student teachers learned to formulate scientifically more coherent core ideas during the course . RRI Components in Lesson Plans The results on the RRI components were varied . Table 3 indicates that relatively more student teachers of the Irresistible group than of the control included RRI aspects in their lesson plans – this was true already before the course . Still , the improvement during the course was better ( gain 0 . 16 ) than in the control group ( gain 0 . 05 ) , but not statistically significant . Notably , RRI components of open access and ethics were the ones most often included in the lesson plans of both groups . Some components were not Ratinen , Kähkönen , Lindell & de Vocht 129 addressed by any student teachers in either group . Especially , governance , gender and equality were absent from the lesson plans . One of the RRI components , science education - understood as promoting children’s interest toward science and understanding the importance of science education - was left out of the analysis . This is because any science education must be a part of this goal . The idea that students need the metacognitive awareness of the role of science education in public policy is not feasible to us . The way we understand science education within the RRI themes is that the students learn to appreciate scientific knowledge and understand there is a need for such knowledge in the wider society . Table 3 . At least one RRI component ( other than science education ) included in student teachers’ lesson plan Irresistible ( n = 12 ) Control ( n = 23 ) Pre Post Pre Post RRI included 6 ( 50 % ) 7 ( 58 % ) 5 ( 22 % ) 6 ( 26 % ) G 0 . 16 0 . 05 p > 0 . 05 between groups ; p > 0 . 05 between pre - and post - test Student teachers most commonly include open access as an aspect of RRI as exemplary quotations below indicate . There were no variations in pre and post lesson plan between the groups , meaning that the way student teachers wrote about a particular RRI aspect was very similar with the other groups . The following quotes are examples of ideas of open access evident in the lesson plans : Student teacher Control e : Pupils work in groups and use internet for obtain data . Student teacher Irresistible f : Based on their findings , each group makes a selected output ( video , writing etc . ) that will be presented to others ( comparing results and dis - cussing further ) . Student teacher Irresistible g : Each group makes an output ( figures , videos , texts ) and presents their ideas to the other groups . The other groups evaluate the output Moreover , as the following quotations express , student teachers associated the engagement and ethical aspects of science with pupils’ everyday life . MALU , Turku 2015 130 Student teacher Control a : Consider how air influences everyday life , such as air resistance and thermal expansion of air . Student teacher Irresistible h : Important to know how to do demonstrations and examples , which explain the theory in prac - tice and everyday life . CONCLUSIONS This study exemplifies the difficulty of integrating RRI into inquiry - based science teaching - regardless of whether RRI is embedded in the student teachers’ course materials . Neither of the groups developed a good compe - tence of using the RRI components in their lesson plans , and this despite our positively and optimistic interpretation of RRI components in the plans ! A good design of a science lesson needs to be established on the DCIs - which are not clearly expressed in the national curriculum . This principle , which is not self - evident , was learned quite well in both of the groups . The student teacher course provided a working learning environment for this task . We wonder whether it is difficult for student teachers to consider both RRI and inquiry aspects simultaneously in their lesson plans . The disciplinary core ideas and the RRI components were expressed in a relatively simple way in student teachers’ lesson plans . It was evident that student teachers most commonly incorporated ethics and open access into their lesson plans . They are aspects probably associated often with their everyday life , and also the aspects that sparked most discussion in the RRI workshops . Moreover , they typically discuss ethical issues and open access in many other pedagogical courses and teacher training , and thus these are the most reinforced of the six components . The considerably greater number of Irresistible student teachers in the Table 3 including RRI themes in their pre - plans already may be due to that they had already had an applying environmental education course before this study . The main message to us is that the RRI workshops and working methods of the course need to be changed in order to better provide student teachers with skills to use RRI components in their future lessons . Our results cannot be generalized , but they reveal student teachers’ decisions of what they view as important parts to include in their lesson plans , and RRI components were often not seen as essential . We see our RRI workshops for student teachers lacking in guidance for classroom implementation : noticing suitable spots for bridging to RRI components , comparing the RRI components with the National Curriculum to reinforce its place in school lessons , practising making assessment questions on RRI topics . In making sure the teachers weren’t copying off a given model Ratinen , Kähkönen , Lindell & de Vocht 131 for an RRI task , we accidentally deprived them of any examples or even discussions of student level implementation . Their experience of self - planning the implementations of RRI components was clearly not positive enough to carry on to future lessons . Based on our results , we cautiously recommend RRI to be explicitly ad - dressed throughout primary teachers’ education , and particularly to be included as an expected part of the practise lessons planned and given to build experience of how to engage students in discussing and pondering the RRI components . 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Silfverberg & Tuominen 133 MURTOLUVUN JA LUKUSUORAN PISTEEN VÄLINEN VASTAAVUUS – TYYPILLISIMPIÄ VIRHEITÄ LUOKAN - OPETTAJAOPISKELIJOIDEN SUORITUKSISSA Harry Silfverberg & Anu Tuominen Turun yliopisto Teetimme luokanopettajaopiskelijoilla monialaisten opintojen matematiikan kurssilla testin , jolla tutkimme , miten hyvin opiskelijat osaavat käyttää murtolukujen eri representaatioita ja miten hyvin he ymma ̈ rta ̈ va ̈ t representaatioiden välisiä yhteyksiä . Aiemmissa tutkimuksissa on todettu , että perusasteen oppilaat eivät useinkaan miellä murtolukuja lukuina , joilla on vastineensa lukusuoran pisteinä , vaan ennemminkin kahden kokonaisluvun muodostamana kombinaationa kuvaten osan suhdetta kokonaisuuteen . Artikkelissa tarkastelemme , esiintyykö myös aikuisilla luokanopetta - jaopiskelijoilla vaikeutta käsitellä murtolukuja lukuina . Havainnollistaaksemme ilmiötä tarkastelemme eräitä opiskelijoiden virhesuorituksia , joita ilmeni erityisesti osioissa , jotka käsittelivät murtoluvun ja lukusuoran pisteen välistä vastaavuutta . JOHDANTO Lukualuelaajennukset yhtäältä positiivisista kokonaisluvuista positiivisiin rationaalilukuihin ja toisaalta positiivista luvuista negatiivisiin lukuihin ovat tunnetusti isoja askelia alakoulun oppilaan lukukäsitteen kehityksessä . Kouluopetuksessa oppijan lukukäsitteen kehitystä on perinteisesti tuettu käyttämällä erilaisia malleja havainnollistamaan erityyppisten lukujen luonnetta ja ominaisuuksia . Ymmärryksen syntyä autetaan käyttämällä hyväksi oman tekemisen kautta syntyvää kehollista tuntemista ja motorista muistia sekä moninaisia visuaalisia malleja ja toimintamateriaaleja . Myös matematiikka itsessään käyttää eri asioille erilaisia esitystapoja ja representaa - tioita eri tilanteissa . Esimerkiksi murto - ja sekaluvuille , jotka tässä artikkelissa ovat keskiössä , käytetään symbolimerkinnän , kuten 1 3 , 3 7 , 5 6 1 2 3 , ohella joukko - , pinta - ala - ja janamalleja havainnollistamaan lukujen osa – kokonaisuus - tulkintaa . Vastaavasti rationaalilukujen desimaalimuunnokset ja lukuja vastaavien pisteiden sijoittaminen lukusuoralle korostavat rationaalilukujen suuruusjärjestystä ja tiheyttä sekä kuulumista osaksi reaalilukujen joukkoa . Rationaalilukujen yhteys prosentteihin tulee käyttöön erityisesti tarkasteltaes - sa eri yhteyksissä tutkittavien määrien suhteita . Opetustilanteessa erilaisia representaatioita käytetään , jotta tarkasteltavat asiat ymmärrettäisiin helpommin , paremmin ja monipuolisemmin . Tosiasia kuitenkin on , että se , MALU , Turku 2015 134 mitä erilaisilla representaatioilla havainnollistetaan ja miksi niitä käytetään , on itsessäänkin oppimistehtävä sekä oppijoille että aloitteleville opettajillekin . Kouluikäisten tavallisimmat virhekäsitykset rationaaliluvuista Tunnettua on , että lukukäsitteen laajentamiseen kokonaisluvuista rationaali - lukuihin sisältyy monia oppimisen haasteita . Rationaalilukujen ymmärryksen rakentumista on tutkittu vuosikymmeniä ja ilmiötä tutkitaan aktiivisesti edelleen . Tutkimusta tehdään sekä matematiikan opettamisen näkökulmasta että oppimispsykologisena erityiskysymyksenä . Tutkimustulokset osoittavat varsin kiistattomasti , että oppimisen alkuvaiheessa  rationaaliluvuilla oletetaan varsin yleisesti olevan sellaisia kokonaislu - kujen ominaisuuksia , joita niillä ei tosiasiassa ole ( ns . whole number bias ) , kuten yksikäsitteinen suuruusjärjestyksessä seuraavan luvun olemassaolo ( Ni & Zhou , 2005 ) ;  osa – kokonaisuus – tulkinta on vallitseva tapa ymmärtää varsinkin aitoja murtolukuja eikä murtolukuja välttämättä ajatella lainkaan lu - kuina ( Hannula , 2003 ) , joilla on vastinpisteensä lukusuoralla , vaan en - nemminkin kahden luvun muodostamana kombinaationa ( Pitkethly & Hunting , 1996 , 10 ) ;  rationaalilukujen tiheyttä on vaikea ymmärtää ja oppijat tukeutuvat luonnollisten lukujen diskreettisyyteen ( Hannula ym . , 2006 ; McMullen ym . , 2014 ; Vamvakoussi & Vosniadou , 2010 ) ;  rationaalilukujen monet tulkinnat ( osa - kokonaisuus - tulkinta , suhde - tulkinta ja sitä kautta yhteys prosentteihin , tulkinta jakolaskuksi ja sitä kautta yhteys desimaalilukuihin , desimaalilukujen tulkinta murtolu - kuina , suuruuden säilyminen laventamisessa ja supistamisessa yms . ) tekevät lukualuelaajennuksesta oppijalle haasteellisen ja monitahoisen oppimiskokonaisuuden ( ks . esim . ( Kurvits , 2009 ) ) ;  rationaalilukujen ja desimaalilukujen merkintätavat poikkeavat toisis - taan , lukujen suuruusvertailu tapahtuu erilaisella proseduurilla ja aritmeettiset operaatiot poikkeavat toisistaan , josta syystä monet oppi - laat eivät ymmärrä rationaaliluvun ja desimaaliluvun esittävän samaa lukua ( Vamvakoussi & Vosniadou , 2010 ) . Aikuisten käsitykset rationaaliluvuista Rationaalilukujen ymmärryksen tutkimus on luonnollisesti kohdistunut pääosin sen ikäisiin lapsiin ja nuoriin , joille näitä asioita koulussa opetetaan . Vähäisemmälle huomiolle on tutkimuksessa jäänyt aikuisten ja erityisesti niiden , jotka näitä asioita opettavat , ts . opettajien ja opettajaksi valmistuvien lukukäsitteen hallinta . Osittain tämä johtuu siitä , että on pidetty itsestään selvänä , että aikuiset ovat omana kouluaikanaan jo oppineet riittävän hyvin alakoulussa opetettavat asiat . Kuitenkin on tehty tutkimuksia myös opettajien Silfverberg & Tuominen 135 tavasta hahmottaa rationaalilukuja . Eräät tutkimuksista antavat viitteitä siitä , että osalla alakoulun opettajista ja opettajaksi opiskelevista on samantapaisia puutteita rationaalilukujen käsittelyssä kuin oppilaillakin ( Post , Harel , Behr , & Lesh , 1988 ; Zhou , Peverly , & Xin , 2006 ; Sowder , Bedzuk , & Sowder , 1993 ; Park , Güçler , & McCrory , 2013 ) . Aikuiset tosin näyttävät käyttävän jousta - vammin eri strategioita käsitellessään lukumääräsuhteita , kuten esimerkiksi murtolukujen suuruuden vertailua ( Bonato , Fabbri , Umiltà , & Zorzi , 2007 ) ja kompensoivat näin osin rationaaliluvun käsitteen hallinnan puutteita . Luokanopettajaopiskelijoiden koulutuksensa myötä hankkimien matematii - kan taitojen tutkimus ja arviointi on monella tapaa tärkeää . Ensinnäkin opettajan omalla aineenhallinnalla ( Ball , Thames , & Phelps , 2008 ) on yhteys siihen , millaisia matematiikan tietorakenteita ja käsityksiä hänen oppilaansa muodostavat ( Hill , Rowan , & Ball , 2005 ) . Toisaalta arviointitieto palvelee koulutuksen resurssien suuntaamista kohteisiin , joissa kehittämisen tarvetta erityisesti on . Rohkaiseva havainto siitä , että tällainen diagnostinen toiminta kannattaa , saatiin muun muassa Hannula ym . ( 2010 ) tutkimuksessa , jossa todettiin , että varsin pienillä toimenpiteillä luokanopettajakoulutuksessa voidaan korjata opiskelijoiden virhekäsityksiä rationaalilukujen luonteesta . TUTKIMUSKYSYMYS Toteuttamallamme lomakekyselyllä pyrimme saamaan yleiskuvan siitä , miten hyvin luokanopettajaopiskelijat osaavat käsitellä murtolukuja käyttäen eri representaatioita ja miten hyvin he ymma ̈ rta ̈ va ̈ t representaatioiden välisiä yhteyksiä . Testin osiot käsittelivät representaatioita ja niiden välisiä muun - noksia , kun rationaalilukuja käsitellään murto - , seka - ja desimaalilukuina tai havainnollistetaan pinta - ala - mallien tai lukusuoran avulla . Tässä artikkelissa rajoitumme kuitenkin tarkastelemaan vain seuraavaa yleisluontoista tutkimuskysymystä : Millä tavoin luokanopettajaopiskelijat ymmärtävät murtolukujen ja lukusuoran pisteiden välisen vastaavuuden ? MENETELMÄ Murtolukujen representaatioiden välisiä yhteyksiä käsittelevä testi käsitti 10 osiota , joissa seitsemässä oli 2 – 4 alaosiota . Testiin vastasi 106 luokanopettaja - opiskelijaa , joista 96 vastasi kaikkiin tässä yhteydessä tarkasteltaviin osioihin . Kyselylomakkeessa ei kysytty vastaajan nimeä eikä sukupuolta . Kaikki opiskelijat olivat suorittaneet koulutusohjelmaan kuuluneen ensimmäisen matematiikan pakollisen opintojakson ( 3 op ) . Tutkimuskysymykseen vastataksemme tarkastelemme lähemmin opiskelijoi - den vastauksia kolmeen testiosioon , joilla selvitimme , kuinka hyvin vastaajat osaavat 1 ) sijoittaa murto - tai sekalukuna annetun luvun vastinpisteen luku - suoralle ( osio 5 ) , MALU , Turku 2015 136 2 ) sijoittaa pinta - alamallin avulla havainnollistetun murtoluvun vas - tinpisteen lukusuoralle ( osio 8 ) ja 3 ) ilmoittaa lukusuoralle merkityt neljä pistettä murto - tai sekaluku - muodossa ( osio 9 ) . Osiot 5 , 8 ja 9 esittelemme lähemmin luvussa Tulokset . Kohdassa 1 ) ja 2 ) hyväksymiskriteeri oikealle suoritukselle oli , että luvun sijainti vastasi likimain pisteen todellista paikkaa ja pisteiden määrittämien osavälien ( kuudes - ja kolmasosien ) pituudet olivat silmämääräisesti arvioiden oikeassa suhteessa toisiinsa nähden . Kohdassa 3 ) edellytimme oikeaan suoritukseen , että vastaukset annettiin lukusuoralle merkittyjen jakovälien mukaisesti seitsemäsosina ja lukuarvot olivat oikeita . Oikeiden suoritusten ohella nostamme esiin eräitä opiskelijoiden suorituksiin sisältyneitä virheitä , ja pohdimme , millaisia virhekäsityksiä niiden taustalla voi olla . Kahdessa ensin mainitussa osiossa 5 ja 8 lukusuoraan oli merkitty valmiiksi vain origo ( kohta 0 ) ja yksikkövälin päätepiste ( kohta 1 ) . Tällä pyrittiin jättämään opiskelijalle mahdollisimman suuri vapaus valita strategia , jolla hän määrittää murtolukuja vastaavien pisteiden sijainnit lukusuoralla . Kolmannessa osiossa 9 lukusuoralle merkittyjen seitsemäsosien avulla korostettiin osavälien ja yksikköjanojen osa – kokonaisuus - luonnetta . TULOKSET Osio 5 Osiossa annetut murto - ja sekaluvut tuli esittää lukusuoran pisteinä . Osio 5 . Sijoita luvut 2 3 , 4 3 , 5 6 ja 1 2 3 lukusuoralle . Osioon vastasi oikein 82 vastaajaa 106 : sta ( 77 , 4 % ) . Erehtymisiä sattui asetetta - essa lukuja suuruusjärjestykseen . Pisteiden sijoituksen perusteella koko aineistossa viisi vastaajaa piti lukua 5 6 ykköstä suurempana ( Kuva 1 ) ja yksi vastaaja lukua 5 6 pienempänä kuin 2 3 . Kuvat opiskelijoiden autenttisista vastauksista on painoteknisistä syistä korvattu uudelleen piirretyillä kuvilla , joissa pisteiden sijainnit vastaavat opiskelijan tekemiä merkintöjä . Silfverberg & Tuominen 137 Kuva 1 . Esimerkki opiskelijan ratkaisusta ( 5 6 > 4 3 > 1 ) . Mahdollisesti vastaaja on ajatellut , että 5 > 4 ja 6 > 3 , joten 5 6 > 4 3 . Yleisin virhe osion suorituksessa oli pisteiden merkitseminen lukusuoralle niin , että kolmasosien pituiset jakovälit olivat selvästi erimittaiset ja kuudes - osa ei ollut puolta kolmasosasta ( Kuvat 2 ja 3 ) . Kuva 2 . Opiskelijan ratkaisu , jossa kuudesosat ykkösen vasemmalla puolella ja kolmasosat ykkösen oikealla puolella ovat lukusuoralla lähes yhtä pitkät . Kuva 3 . Opiskelijan ratkaisu , jossa kuudes - ja kolmasosien suuruudet vaihtelevat . Noin neljäsosalla vastaajista eli 24 vastaajalla lukusuoralle merkittyjen pisteiden keskinäiset välit eivät noudattaneet ”tyrkyllä” ollutta kolmas - ja kuudesosien määrittämää struktuuria . Koska useimmat vastaajista oletetta - vasti sijoittivat pisteet silmämääräisesti , luokittelussa aika suuretkin epätark - kuudet sijoittelussa hyväksyttiin . Osio 8 Osiossa pinta - alamallin avulla annettu murtoluku piti esittää lukusuoran pisteenä . MALU , Turku 2015 138 Osio 8 . Merkitse väritettyä aluetta vastaava osuus lukusuoralle a ) b ) c ) Osioon vastasi oikein 77 vastaajaa 103 : sta ( 75 , 8 % ) . Erinimiset kolmas - , neljäs - ja kahdeksasosat näyttivät tuottavan joillekin opiskelijoille vaikeuksia . Kuusi vastaajaa käytti lukujen suuruussuhteiden vertailussa hyväksi murtolukujen samannimisiksi laventamista . Esimerkiksi eräs opiskelija oli jakanut osion kohdassa c ) esitetyn neljäsosaa vastaavan sektorin kahtia , joka viittaa siihen , että hän hahmottaa väritetyn alan olevan 3 8 . Tästä huolimatta hän merkitsee kohdan 3 8 lukusuoralle paik - kaan , joka vastaa ennemminkin tulkintaa 3 8 = 2 ∙ 1 4 ( vrt . Kuva 4 ) . Kuva 4 . Opiskelijan ratkaisu , jossa kolmas - , neljäs - ja kahdeksasosien suuruudet eivät ole oikeassa suhteessa . Kuva 5 puolestaan esittää toisen opiskelijan ratkaisua , joka selvästi hahmot - taa , että kuvion väritetty osa on 1 , 5 ∙ 1 4 , mutta sijoittaa silti c - kohdassa kysytyn pisteen kohtaan , joka vastaa lähinnä tulkintaa 2 ∙ 1 4 . Kuva 5 . Opiskelijan ratkaisu , jossa lukusuoralle merkittyjen pisteiden sijainnit eivät vastaa lukujen suuruussuhteita . 0 1 2 3 x a 1 , 5 4 x c 1 4 x b Silfverberg & Tuominen 139 Kummassakaan edellä esitetyssä esimerkissä lukujen hahmotetut algebralliset suuruussuhteet eivät siirry lukusuoran geometriseen malliin pisteiden välisten etäisyyksien suhteiksi . Osio 9 Osion asetelma oli käänteinen osioon 5 verrattuna . Lukusuoralle sijoitetut neljä pistettä tuli esittää murto - tai sekalukumuodossa . Osio 9 . Ilmoita lukusuoralle merkityt luvut P , Q , R ja S murtolukumuodossa sillä tarkkuudella kuin voit ne kuvasta määrittää . P = Q = R = S = Tehtävään vastauksen antaneista 96 opiskelijasta 74 ( 77 , 1 % ) liitti oikean murto - tai sekaluvun jokaiseen annetuista neljästä pisteestä . Viisi vastaajista ilmoitti pisteet P , Q ja R murto - tai sekalukuina oikein , mutta jätti negatiivisen lukuarvon S merkitsemättä . Lisäksi kolmella vastaajalla R ja S olivat molem - mat vastaamatta , mutta P ja Q oikein . Puutteet vastauksissa antavat aiheen epäillä , oliko osalla opiskelijoista käsitys , että murtolukumuotoa käytetään osa – kokonaisuus – tulkinnan mukaisesti vain lukualueella nollasta yhteen . Erityisesti luvun S negatiivisuus aiheutti joillekin vastaajille ongelmia . Hämmästyttävästi seitsemän vastaajaa merkitsi S : n arvoksi − 5 7 . Koska käytettävissä olivat vain vastauspaperin merkinnät , voi vain arvailla , johtuiko tämä siitä , että seitsemäsosat luettiin kohdasta - 1 alkaen oikealle kohtaan S saakka , vai jostain muusta . Osioon jätti ratkaisun 96 opiskelijaa , joista 74 ratkaisi tehtävän oikein . Joka tapauksessa noin joka viidennellä vastaajalla näytti olevan vaikeuksia tämän tehtävän kanssa . Koontia havainnoista Asettamaamme tutkimuskysymykseen ”Millä tavoin luokanopettajaopiskeli - jat ymmärtävät murtolukujen ja lukusuoran pisteiden välisen vastaavuuden ? ei voida vastata yksiselitteisesti . Opettajaksi opiskelevien aineenhallinta vaihtelee melkoisesti tässäkin tutkimassamme rajatussa asiayhteydessä . Tarkasteltaessa osioita yksittäin todetaan , että kuhunkin tehtävään valtaosa opiskelijoista vastasi oikein mutta noin joka neljännellä opiskelijalla oli tehtävien ratkaisuissa puutteita . Kaikkiin kolmeen osioon 5 , 8 ja 9 vastasi oikein 52 vastaajaa 96 : sta eli vähän yli puolet vastaajista . Lähes joka toiselle vastaajalle tällaisten muunnostehtävien ratkaisu näytti siis kuitenkin tuottavan ainakin jonkin asteisia vaikeuksia . Tutkimuksen näkökulmasta MALU , Turku 2015 140 tarkastellen voidaan myös todeta , että tehdyt havainnot osoittavat , että aikuisopiskelijallakin voi olla samantyyppisiä vaikeuksia rationaalilukujen käsittelyssä kuin mitä aiemmissa tutkimuksissa on todettu lapsilla . Aineiston perusteella joillekin opiskelijoille murtolukujen suuruusjärjestykseen asettaminen ei ollut itsestään selvää ja murtolukuja käsiteltiin kokonaisluku - jen kaltaisesti . Vaikka tämä joukko oli vähäinen , koulutuksessa ilmiö tulisi havaita ja siihen puuttua . Selvästi aika moni vastaaja ei hahmottanut osioissa annettujen murtolukujen keskinäistä yhteyttä , vaan näytti sijoittaneen lukuja vastaavat pisteet lukusuo - ralle yksi kerrallaan suhteuttamatta niitä keskenään . Esimerkiksi lukusuoran pisteiden virheellinen sijoittelu osoitti , että useimmat vastaajat eivät käsitel - leet osiossa 5 annettuja lukuja 2 3 , 5 6 , 1 , 4 3 ja 1 2 3 kuudesosina . Siirryttäessä erityisesti symbolisesta murtolukumerkinnästä lukusuoraesityk - seen useissa vastauksissa oli havaittavissa että , vaikka lukujen algebralliset suuruussuhteet hahmotettiin , ne eivät siirtyneet lukusuoran geometriseen malliin pisteiden välisten etäisyyksien suhteiksi . Selitys tähän voi olla se , että vastaajat sijoittavat pisteet lukusuoralle ensisijassa lukuarvojen mukaan arvioituihin kohtiin eivätkä niinkään tarkastele osavälien keskinäisiä suhteita murtoluvun osa – kokonaisuus – tulkinnan mukaisesti . Tärkeä havainto oli myös se , että monet vastaajat näyttivät osiossa 9 rajoittavan murtolukumer - kinnän vain välille nollasta yhteen . Tarkemman selvityksen ansaitsisi se , esiintyykö joillakin opiskelijoilla osion 9 pistettä S koskeneiden virheellisten ja puuttuvien vastausten mukaisesti käsitys , joiden mukaan murtoluvut ovat aina positiivisia . POHDINTA Turun yliopiston opettajankoulutuslaitoksen Turun yksikössä luokanopetta - jaksi opiskelevien valintakokeeseen kuuluu toisin kuin monissa muissa vastaavissa yksiköissä myös matematiikan ja luonnontieteiden testi . Tällä on osaltaan pyritty varmistamaan se , että luokanopettajakoulutukseen hyväksy - tyillä opiskelijoilla olisi riittävät perustaidot matemaattis - luonnontieteellisissä aineissa . Kun hakijoiden tiedossa yleensä on , että valintakokeeseen kuuluu tällainen osa , se oletettavasti ohjaa Turun yksikköön hakijoiksi niitä , joita koe ei pelota . Lisäksi yksikön opetuksessa rationaaliluvuilla ja niiden erilaisilla havainnollistamistavoilla on ollut merkittävä rooli monialaisten opintojen kaikille yhteisessä matematiikan kurssissa . Kyselyyn vastanneet opiskelijat olivat opinnoissaan siinä vaiheessa , että he olivat suorittaneet tämän kaikille yhteisen kurssin . Ennakkoon saattoi siis olettaa , että artikkelissa tarkastellut tehtävät olisivat varsin helppoja opiskelijoille . Näin toki useimmille olikin , mutta kuitenkin kussakin kolmessa tehtävässä noin joka neljännellä oli ratkaisussa jonkinasteisia puutteita . Silfverberg & Tuominen 141 Jos havaitut puutteet olisivat tulleet ilmi peruskursseihin kuuluvissa tenteis - sä , virheet olisivat laskeneet osaltaan tentistä saatavaa arvosanaa , mutta todennäköisesti tentti olisi tullut hyväksytyksi summatiivisen arvioinnin periaatteiden mukaisesti muun osoitetun osaamisen perusteella ja mitään suurempaa ongelmaa opintojen etenemiseen tästä ei olisi aiheutunut . Tutkimuksessa esiin tulleet havainnot tulevien opettajien matematiikan aineenhallinnan tasosta asettavat haasteita opettajankoulutukselle . Niistäkin opiskelijoista , joilla on vakavia puutteita matematiikan perustaidoissa ja käsitteissä , tulee koulutuksen myötä opettajia , joiden tulee osata opettaa näitä asioita oikein ja ymmärrettävästi omille oppilailleen . Toimiiko koulutus eettisesti vastuullisesti , jos se ummistaa silmänsä perusaineenhallinnan puutteilta sillä perusteella , että osaaminen on kuitenkin kohtuullisella tasolla ? Vai pitäisikö koulutuksessa noudattaa yksittäistenkin perusasioiden osaamat - tomuuden suhteen ennemminkin nolla - toleranssi – linjaa ? Tulevan luokan - opettajan on myös itse kannettava vastuunsa siitä , että osaa ja ymmärtää perusasiat , joita työssään varmuudella tulee opettamaan oppilailleen . LÄHTEET Ball , D . L . , Thames , M . H . , & Phelps , G . ( 2008 ) . Content knowledge for teaching : What makes it special ? Journal of Teacher Education , 59 ( 5 ) , 389 – 407 . Bonato , M . , Fabbri , S . , Umiltà , C . , & Zorzi , M . ( 2007 ) . The mental representation of numerical fractions : real or integer ? Journal of Experimental Psychology . Human Perception and Performance , 33 ( 6 ) , 1410 – 1419 . http : / / doi . org / 10 . 1037 / 0096 - 1523 . 33 . 6 . 1410 Hannula , M . S . ( 2003 ) . Locating fraction on a number line . In N . A . Pateman , B . J . Dougherty , & J . Zilliox ( Eds . ) , Proceedings of the 2003 Joint Meeting of the PME and PMENA ( Vol . 3 , pp . 17 – 24 ) . Havaii : CRDG , College of Education , University of Hawaii . Hannula , M . S . , Laine , A . , Pehkonen , E . , & Kaasila , R . ( 2010 ) . Rationaalilukujen tiheyden ymmärtämisen kehittyminen luokanopettajaopintojen aikana . In K . Juuti , A . Kallioniemi , P . Seitamaa - Hakkarainen , L . Tainio , & A . Uitto ( Eds . ) , Ainedidaktiikka moninaistuvassa maailmassa . Ainedidaktiikan symposium 2010 . ( pp . 57 – 69 ) . Helsinki : Helsingin yliopiston opettajankoulutuslaitos . Hannula , M . S . , Pehkonen , E . , Maijala , H . , & Soro , R . 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Teaching and Learning Fraction and Rational Numbers : The Origins and Implications of Whole Number Bias . Educational Psychologist , 40 ( 1 ) , 27 – 52 . http : / / doi . org / 10 . 1207 / s15326985ep4001 _ 3 Pitkethly , A . , & Hunting , R . ( 1996 ) . A review of recent research in the area of initial fraction concepts . Educational Studies in Mathematics , 30 ( 1 ) , 5 – 38 . http : / / doi . org / 10 . 1007 / BF00163751 Vamvakoussi , X . , & Vosniadou , S . ( 2010 ) . How Many Decimals Are There Between Two Fractions ? Aspects of Secondary School Students’ Understanding of Rational Numbers and Their Notation . Cognition and Instruction , 28 ( 2 ) , 181 – 209 . http : / / doi . org / 10 . 1080 / 07370001003676603 Tuomela 143 TASK POTENTIAL OF REVERSED EQUATION SOLVING Dimitri Tuomela University of Oulu Reversed Equation Solving ( RES ) is a task which aims to foster understanding and reasoning above rote - based learning . RES is analyzed with a framework formed by combining two earlier frameworks for analyzing the quality of mathematical tasks . The analysis suggests that conceptual emphasis and high degree of freedom in creating equations for one’s peers to solve makes Reversed Equation Solving a cognitively demanding , rewarding and mathematically rich task . INTRODUCTION Understanding the process of equation solving is an important but challeng - ing point in learning mathematics ( Andrews & Sayers , 2012 ) . Finnish teachers often emphasize routines and procedural fluency in algebra ( Attorps , 2006 ) which is connected to learners’ poor conceptual knowledge ( Hihnala , 2005 ) and inability to describe and justify their thinking ( Andrews , 2013 ) . Seeing equation solving as a set of memorized routines prevents learners from applying algebra ( Hiebert et al . , 1997 ) and building a productive disposition towards mathematics ( Andrews , 2013 ) . Apparently there is a strong need for reasoning - centered classroom practices and material in algebra . Flexible Equation Solving project aims to answer to this need as a part of a 6 - year national program ( LUMA Suomi ) . While developing material for this project the author immersed himself in reading previous research describing aspects of high - quality mathematical tasks ( e . g . Stein , Grover & Henningsen , 1996 ; Jackson , Garrison , Wilson , Gibbons & Shahan , 2013 ) and aspects of high - quality learning environments for learning mathematics ( e . g . Hiebert et al . , 1997 ; Kilpatrick , Swafford & Findell , 2001 ) . As a consequence the ap - proach of Reversed Equation Solving ( RES ) was created and it was used as a one - lesson task in the 10 - lesson pilot of Flexible Equation Solving . Briefly described RES is an intervention where learners create , share , solve and compare equations . The approach is intended to have a positive impact on conceptual understanding related to equation solving and to change answer - orientated classroom practices towards process - oriented ones . The intervention also aims to help teachers reflect on their enacted classroom practices . This study is a theoretical analysis of the task potential of RES to support the research - based development of the approach . A framework for analyzing the task potential is formed by combining a rubric for analyzing cognitive demand of any task ( Smith & Stein , 1998 ) and a framework describing MALU , Turku 2015 144 features of high - quality mathematical tasks ( Hiebert et al . , 1997 ) . This study can be seen as an initial phase of a design study ( Cobb , Jackson & Dunlap , 2015 ) seeking to establish an optimal instructional design of RES . The aim of this paper is to understand what is the task potential of RES for supporting all learners to develop understanding on mathematical ideas related to equation solving ? THE TASK AND CONTEXT Reversed Equation Solving has been developed in the context of 10 - lesson pilot of Flexible Equation Solving – project . RES is the sixth lesson in the middle of the material . RES was preceded by three conceptually oriented lessons on what are equations and transformations , and two procedurally oriented lessons . On one of those lessons all simple transformations ( multipli - cation , division , addition and subtraction on both sides and modifying an expression ) were introduced altogether as a cluster and not one by one as separate lessons which is common in books used in Finnish classrooms . RES begins by having small groups of 2 - 5 learners decide a number and marking it equal with a symbol of their choice . Hence they have created a simple equation ( e . g . 4 = t ) . The learners grow their equation with several transformations of their choice ( e . g . 6 + 4 = 6 + t ) and write these steps above the previous . Once they are satisfied , they share their equation ( with their names ) in the blackboard for everyone to solve . Gradually as they solve each other’s equations they compare their work with the creator group . The written instruction of the task on the pilot was the following . 1 . Choose together with your group members a number and mark it equal with some variable . ( For example 12 = t ) 2 . Use transformations of your choice to this equation . ( For example multiply both sides by 3 . Add 2t to each side and combine like terms . ) 3 . You have formed an equation . Go share it in the blackboard . 12 = 𝑡 12 ∙ 3 = 3 ∙ 𝑡 12 = 𝑡 36 + 2𝑡 = 3𝑡 + 2𝑡 12 ∙ 3 = 3 ∙ 𝑡 12 = 𝑡 36 + 2𝑡 = 5𝑡 36 + 2𝑡 = 3𝑡 + 2𝑡 12 ∙ 3 = 3 ∙ 𝑡 12 = 𝑡 Figure 1 . The provided example of transforming the equation . FRAMEWORK FOR ANALYZING TASK POTENTIAL Several researchers argue that high - quality mathematics instruction should be based on fewer but more demanding tasks set up in a way that it encourages high - level mathematical thinking and reasoning ( Fennema et al . , 1996 ; Stein , Grover & Henningsen , 1996 ; Jackson , Garrison , Wilson , Gibbons & Shahan , 2013 ; Henningsen & Stein , 1997 ) . Tuomela 145 Smith and Stein ( 1998 ) described a four - level ( memorization , procedures without connections , procedures with connections , doing mathematics ) rubric for analyzing the cognitive demand of tasks . Characteristics of tasks with the highest cognitive demand ( doing mathematics ) are similar with characteris - tics of high - quality mathematical tasks described by Hiebert and colleagues ( 1997 ) . They fit to each other so well that their work could be combined resulting in a 4 - dimension framework that describes characteristics of tasks which have the potential to engage students in “doing mathematics” and provide insights into the structure of mathematics . In the following table Smith and Stein’s framework will be referred to with ( S ) and Hiebert and colleagues’s framework with ( H ) . Table 1 . Framework for analyzing task potential . Makes mathematics problematic  Learners see the task as an interesting problem and see that there is something to find out , something to make sense of ( H )  Learners don’t have memorized rules for the task , nor do they perceive there is one right solution method ( H )  Complex and non - algorithmic thinking is required instead of following a prescription ( S )  Considerable cognitive effort is required and the unpredictable nature of the solution process may involve some level of anxiety ( S ) Connects with where students are  Task is accessible to all learners regardless of their mathematical ability ( H )  Opportunities to apply previous skills and knowledge arise ( H )  Students are required to access relevant knowledge and experiences and make appropriate use of them in working through the task ( S ) Leaves behind something of mathematical value  Task engages learners in reflecting important mathematical ideas in a way that leaves behind something of mathematical value ( H )  Task offers opportunities to explore mathematics and reasonable solution methods ( H )  Task requires learners to explore and understand the nature of mathematical concepts , processes , or relationships ( S )  Task requires learners to analyze the task and actively examine task constraints that may limit possible solution strategies and solutions ( S ) Demands reflection and regulation  Task creates opportunities to reflect and communicate ( H )  Task demands self - monitoring or self - regulation of one’s own cognitive processes ( S ) MALU , Turku 2015 146 ANALYZING THE TASK POTENTIAL OF RES The potential of RES for being a high - quality mathematical task is analyzed against the previously described theoretical framework . Does RES make mathematics interesting and problematic ? In RES learners create something to think about for their peers and work on each other’s creations . This collaborative element coupled with the need to “work backwards” has the potential to make the task interesting and meaningful for the learners . They have considerable freedom in picking the transformations instead of having clear instructions what to do next . Thus they cannot apply any memorized rule for the task and it is very probable that different kind of ideas emerges and groups end up having different kind of equation creation paths . Often learners focus on just getting the right answer which has been found to be related to doing procedures without connections ( Henningsen & Stein , 1997 ) . When creating equations the learners cannot focus on getting the correct answer in any other way than checking if their equation is still true with the same value of variable . This unpredictable reverse approach can initially cause anxiety , especially for learners who are used to strictly following instructions and copying the procedures from a similar example . These elements of the task very well correspond to the aspects described in the first dimension ”Makes mathematics problematic” . Does RES connect with where learners are ? Although the task may initially seem complex for the learners , they will soon notice that everyone can succeed in the task . When the initial anxiety is overcome the learners have an opportunity to gain a positive experience in doing mathematics as they notice they can do something they thought as difficult . For procedure - oriented learners it can be relieving to see that the process of creating an equation has the following simple steps “choose a transformation , choose a number ( unless modifying an expression ) , execute” . This is a straightforward advice teachers can give while leaving room for learners’ own reasoning because the way transformations and numbers are chosen effect significantly what kind of conceptual and procedural challenges come forth . The choices learners do effect on the difficulty of the task making it accessible as well as challenging enough for everyone . The nature of the task is such that it affords learners to work on a level suitable for them by deciding how they will create their equation and what kind of equations they choose to solve . All learners need to modify expressions , they need to reflect on the concept of variable and they may start thinking what kind of numbers are they familiar with . All this demands reflection on how their previous knowledge connects to equation solving and requires learners to use their existing skills in a meaningful way . Briefly , RES shows for the learners as a Tuomela 147 difficult and complex task but turns out to be an achievable task which connects to learners’ previous skills regardless of their mathematical ability . This fits well with the second category of the framework ”connects with where learners are” . Does RES leave behind something of mathematical value ? In RES learners cannot initially focus on the answer which is connected to doing procedures without conceptual reflection according to Henningsen and Stein ( 1997 ) . Instead the task has the potential to direct learners’ attention to creating equivalent equations thus forcing learners to reflect on conceptual ideas relevant in equation solving ( adapted from Kieran , 2004 ) :  seeing relations instead of calculating a numerical answer ,  letters as unknowns and variables ,  reflecting the meaning of equal sign ,  equation as a statement that has a truth - value ,  transformations as a process of changing the equation without chang - ing its truth value ,  operations and their inverses , doing and undoing . When the learners think about which transformations to use they run into important questions : Why most transformations operate on both sides whereas the transformation of modifying one expression does not ? Can we divide if we do not get whole numbers as a result ? Can variables be added or subtracted in each side ? How about multiplying or dividing by variables ? When learners are not in a hurry to get the correct answer they have time to consider these mathematically important questions . The arising mathematical questions and challenges are likely to be different from each other in different groups . This diversity provides a rich ground for learner - driven reflection . Especially considering the question “How can I make the equation as tricky as possible” leads to exploration of new mathematical ideas in a creative manner . Cuoco , Goldenberg and Mark ( 1996 ) described important generic mathemati - cal skills needed to be considered in curricular development and named them as mathematical habits of mind . They described that learners should be pattern sniffers , experimenters , describers , tinkerers , inventors , visualizers , conjecturers and guessers . As tinkerers , learners should develop the habit of taking ideas apart and putting them back together . They should want to see what happens if something is left out or if the pieces are put back in a different way . The open nature of RES creates an opportunity to explore mathematics and practice these generic mathematical habits which can be transferred and applied to multiple contexts . Next , some of these potential opportunities are described . MALU , Turku 2015 148 RES has the potential to have learners trying out different combinations of transformations and numbers to see what happens ( Experimenters ) . They may reflect what happens if they make a small change such as change the order of two transformations or use rational numbers instead of whole numbers ( Tinkerers ) . Is there a general pattern when multiplication follows addition compared to addition following multiplication ( Pattern Sniffers ) ? Learners may conjecture that addition or substraction followed by multiplica - tion or division results in an equation with brackets ( Conjecturers ) . Learners can explore mathematical structure and start generalizing : ”Multiplication and division are somehow similar as well as addition and substraction . ” They can invent creative alternatives such as “could I use pi” or “what happens if I add a second variable” ( Inventors ) . Learners can explore important relation - ships such as finding out how reverse operations are connected to the creation and solving phases ( Pattern sniffers ) . The task has the potential to have learners describing the process of creating and solving equations - what transformations did they use and why ( Describers ) . Before answering thoroughly to the question if the task leaves behind something of mathematical value it needs to be asked : what is mathematically valuable ? One way to answer to this question is to lean on Kilpatrick and colleagues’ description of mathematical proficiency comprising five inter - twined strands ( Kilpatrick , Swafford & Findell , 2001 ) : Conceptual under - standing - comprehension of mathematical concepts , operations , and relations ; Procedural fluency - skill in carrying out mathematical procedures flexibly , accurately , efficiently , and appropriately ; Strategic competence - ability to formulate , represent , and solve mathematical problems ; Adaptive reasoning - the capacity for logical thought and for reflection on , explanation of , and justification of mathematical arguments ; Productive disposition - habitual inclination to see mathematics as a sensible , useful , and worthwhile subject to be learned , coupled with a belief in the value of diligent work and in one’s own efficacy as a doer of mathematics . Developing these strands of mathematical proficiency can be considered as mathematically valuable . When implementing the task , it is likely that the need for conceptual under - standing and procedural fluency alternate in a way which provides opportu - nities to build a stronger link between conceptual and procedural knowledge of equation solving . The consecutive creation , solving and comparing phases may provide several opportunities to develop adaptive reasoning . Explora - tive nature of the task may provide opportunities to develop strategic competence through acquiring generic mathematical thinking skills . The task may look difficult but is likely to turn out into manageable task providing positive experiences in doing mathematics thus raising an opportunity to build productive disposition . Therefore , RES seems to have the potential to Tuomela 149 support the development of mathematical proficiency ( Kilpatrick , Swafford & Findell , 2001 ) . Implementing the task certainly has the potential to have students exploring mathematical structure , ideas , concepts , processes and relationships in a way that leaves behind something of mathematical value . Does RES demand reflection and regulation ? The novel approach of the task may cause an initial challenge which necessi - tates perseverance and self - encouragement from the group to start thinking . Unpredictability of the task requires groups to reflect on the task and share their thinking when making suggestions about what to do next . The learners need to collaborate and regulate their work and potentially group members will negotiate some kind of roles . The blackboard filled with student - generated equations offers an excellent chance to wonder if they are mathe - matically different or similar in structure . Are some equations more difficult than others ? If so , why ? This kind of negotiation on what counts as mathe - matically different or more sophisticated has been claimed to develop student autonomy and argumentation in mathematics ( Yackel & Cobb , 1996 ) . Especially questions like ”How can we make the equations novel and tricky ? ” lead to rich discussions and to mathematically rich reflection . When a group solves an equation from the blackboard and compares their work with the creator group it is common that different kind of approaches , mistakes and misconceptions emerge and require reflection . In these situations learners need to communicate and reflect on each others’ work in a productive manner . Definitely RES is a task which has the potential to demand reflection and regulation when it is implemented . DISCUSSION Analyzing Reversed Equation Solving against the theoretical framework shows that it has characteristics of high - quality mathematical tasks which have the potential to engage learners in “doing mathematics” and which provide insights into the structure of mathematics . These results can inform the development of an instructional approach which could support learners in the difficult transition ( Andrews & Sayers , 2012 ) from arithmetic to abstract algebra . Design research like this can partly answer to the need for more reasoning - centered classroom practices in algebra . In a broader sense this study contributes to the reflection of student - created tasks as a method for increasing student engagement and creating learning process - oriented classroom culture . Critique and implications This study is of theoretical nature and empirical data is needed to understand how elements such as classroom social culture and teacher’s role affect on the MALU , Turku 2015 150 learning opportunities created when implementing the task . Possibly RES provides more learning opportunities for learners who have previous experience in reasoning about mathematics together and discussing concepts . It needs to be studied how to support learners and teachers in using the task to maximize learning opportunities . Previous research of how to support teachers in using challenging tasks should be adapted to the development of instructional design of RES . For example Sullivan , Mousley & Zevenbergen ( 2006 ) argued the following three teacher actions to be relevant in maximizing learning opportunities in heterogeneous classrooms : Using open - ended tasks , preparing prompts to support students experiencing difficulty , and posing extension tasks to students who finish the set tasks quickly ; as well as actions that address the socio - mathematical goals by making classroom processes explicit . Stein , Grover and Henningsen ( 1996 ) found that five most important factors assisting maintenance of high - level cognitive activity were i ) task builds on students ' prior knowledge , ii ) appropriate amount of time , iii ) high - level performance modeled , iv ) sustained pressure for explanation and meaning and v ) scaffolding . Depending on the learners in the classroom different kind of timing and supportive actions from the teacher is needed during RES . It is challenging for the teacher to make these on - the - fly decisions for example how to use learners work and when to use whole - class discussion . Thus a detailed guide for using RES is needed . Suggestions for developing Reversed Equation Solving RES could be developed further by contextualizing the task as an encryption and decryption activity and describing how these skills are increasingly important as data is digitalized and needs to be safely encrypted or how these skills were in a key role in the second world war . Having a secret that needs to be encrypted works as a metaphor of transformations as the the kind of changes to the equation which doesn’t change its truth value ( the solution ) . Encryption and decryption is also similar to the conceptually important idea of operations and their inverses , doing and undoing . Considering the framework for analyzing task potential this addition would make the task more interesting ( first dimension ) and increase the potential for leaving behind mathematically valuable conceptual understanding ( third dimension ) . By adding a phrase ”When you are satisfied with your equation , find out which value of variable makes the created equation true . Prove it ! ” in the instruction sheet before sharing equations in the blackboard would give more opportunities for conceptual reflection . This way it can be checked if the learners understand what stays the same when transformations are used to create equivalent equations ( again increasing the task potential in the third dimension ) . A section in the instruction sheet for classifying the diversity of encountered equations and to reflect on ones own learning could increase the Tuomela 151 task potential in the forth dimension ( demands reflection and regulation ) . This section would include the following kind of questions : Classify what kind of equations a ) exists b ) you can create c ) you can solve . What kind of methods did you find to make the equation trickier ? What similarities and differences you found in creating and solving equations ? Did you find some common patterns ? Could your findings be generalized ? It should be considered whether the task should be used quickly after introducing transformations thus providing an engaging way to start developing intertwined procedural and conceptual knowledge or later on to launch mathematical exploration and classification of different kind of equations thus providing more room for creativity and opportunities to deepen and summarize knowledge related to equation solving . Alternatively RES could be used as a project combining the previous two approaches . Extending the task from one lesson to a longer period could give more room for mathematical exploration . CONCLUSION Conceptual emphasis and the high degree of freedom in Reversed Equation Solving may result in a cognitively demanding but mathematically rich and rewarding implementation of the task . It has potential to engage diverse learners in sharing their reasoning and developing their mathematical proficiency . Using RES as a part of a conceptually oriented set of tasks could ease transitioning from arithmetic to abstract algebra . This study only analyzed the theoretical potential of the task . Naturally the environment it is implemented in , specifically the social culture of the classroom , is likely to have a significant effect on what kind of learning opportunities emerge . It may be a challenging task for the teacher to manage because of its unpredict - ability and potentially diverse student interactions . It would be important to develop an instructional design of RES which supports teachers and learners in creating fruitful learning opportunities . REFERENCES Andrews , P . 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Tuominen 153 KÄSITYKSIÄ MURTOLUKUJEN TIHEYDESTÄ Anu Tuominen Turun yliopisto , Turun yksikkö Maailmalla on tehty paljon tutkimuksia murtolukujen suuruusvertailusta tai laskualgoritmien hallinnasta mutta vähän koskien murtolukujen tiheyden käsitteellis - tä ymmärtämistä . Tutkimuksessa ( N = 209 ) verrataan keskenään 7 . - luokkalaisten , 1 . vuoden luokanopettajaopiskelijoiden ja 1 . vuoden matematiikan pääaineopiskelijoiden vastauksia kysymykseen ”Kuinka monta murtolukua on murtolukujen 2 / 3 ja 2 / 4 välissä ? ” . Luokanopettajaopiskelijoista suurin osa löysi lukujen väliin yhden murtoluvun , seitsemäsluokkalaisista suurin osa löysi ”monta” ja matematiikan pääaineopiskelijois - ta suurin osa löysi äärettömän monta murtolukua . Vastausta perusteltiin useimmiten laventamalla annetut murtoluvut samannimisiksi . Ne vastaajat , jotka hallitsivat murtolukujen tiheys käsitteen , osasivat sujuvasti liikkua rationaalilukujen eri esitysmuotojen ; murtolukujen ja desimaalilukujen , välillä . MURTOLUKUJEN OMINAISUUDET Murtoluvut , ja laajemmin rationaaliluvut , poikkeavat merkittävästi luonnolli - sista luvuista , joihin oppilaat ovat tutustuneet koulun ensimmäisiltä luokilta asti . Luonnolliset luvut määrittävät pitkälle sen , mitä oppilaat ajattelevat yleensä luvuista , mitkä luokitellaan luvuiksi , ja mitä ominaisuuksia luvuilla on . ( Vamvakoussi , Christou , Mertens & Van Dooren , 2011 . ) Luonnollisilla luvuilla on seuraaja : luvun 17 jälkeen tulee luku 18 ja niin edelleen , ja luvut saadaan suuruusjärjestykseen luettelemalla ( Siegler , Thompson & Schneider , 2011 ) . Luonnollisista luvuista poiketen murtoluvuilla ei ole seuraajaa , eli ei voida sanoa , mikä luku tulee välittömästi esimerkiksi luvun ½ jälkeen , eikä murtolukuja voi luetella suuruusjärjestyksessä . Oppilaat ovat oppineet , että luonnollisia lukuja kerrottaessa vastaus kasvaa ja jaettaessa vastaus pienenee . Murtoluvulla kerrottaessa vastaus saattaa olla pienempi ( 1 2 ∙ 6 = 3 ) ja jaettaessa suurempi ( 4 ∶ 1 2 = 8 ) . Luonnollisilla luvuilla on yksikäsitteinen merkintätapa ilmaisemaan lukua , murtoluvuilla sama luku voidaan esittää vaikka kuinka monella tavalla ( 1 = 2 2 = 3 3 = 4 4 = ⋯ ) . ( Vamvakoussi & Vosnia - dou , 2010 ; Bailey , Siegler & Geary , 2014 ; Gallistel & Gelman , 1992 . ) Jos oppilas ei ymmärrä edellä esitettyjä eroja luonnollisten lukujen ja murtolukujen välillä , saattaa hän hyödyntää luonnollisten lukujen ominaisuuksia tiedosta - mattaan . Tätä taipumusta kutsuttiin aluksi kokonaislukujen suosimiseksi ( Ni & Zhou , 2005 ) mutta nyttemmin termi on korjattu luonnollisten lukujen MALU , Turku 2015 154 suosimiseksi ( Van Hoof , Vandewalle , Verschaffel & Van Dooren , 2015 ; Vamvakoussi , 2015 ) . Murtolukujen keskeisimmät ominaisuudet ovat suuruus ja tiheys . Luvun suuruusominaisuus yhdistää luonnollisia lukuja , rationaalilukuja ja myöskin reaalilukuja . Äärellinen määrä murtolukuja voidaan järjestää suuruusjärjes - tykseen ja sijoittaa lukusuoralle ( Gallistel & Gelman , 1992 ) . Murtolukujen suuruusvertailutehtävissä oppilaat tukeutuvat usein luonnollisten lukujen ominaisuuksiin ja järjestävät luvut joko nimittäjän tai osoittajan mukaan suuruusjärjestykseen . Harva osaa tulkita murtolukua lukuna ja kiinnittää huomiota osoittajan ja nimittäjän suhteeseen . ( Hartnett & Gelman , 1998 . ) Tiheys on suuruutta vaikeampi käsite . Vaikka oppilaat ymmärtävät että murtolukuja on erikokoisia , silti he usein mieltävät ne diskreetteinä ; murtolu - vun 1 12 jälkeen tulee ensin 2 12 , sitten 3 12 , ja niin edelleen . ( Vamvakoussi ym . , 2011 . ) Murtoluvun komponenttien välisen suhteen ymmärtäminen ja taito sijoittaa desimaalilukuja ja murtolukuja lukusuoralle ennustaa hyvää algebran hallintaa ( DeWolf , Bassok & Holyoak , 2015 ; Torbeyns , Schneider , Xin & Siegler , 2015 ) . Tutkimuksen mukaan oppilaat saattavat ajatella murtolukuja omana lukujoukkonaan ja desimaalilukuja omana lukujoukkonaan . Oppilaat eivät ymmärrä , että kyseessä ovat saman lukujoukon , rationaalilukujen , alkioiden eri esitysmuodot . Oppikirjat saattavat vieläpä tukea tätä harhaa käsittelemällä desimaaliluvut ja murtoluvut toisistaan irrallaan . ( Vamvakoussi ym . , 2011 . ) Linkkejä käsitteiden välille ei pääse muodostumaan , tai muodostaminen saattaa olla oppilaalle hankalaa , jos asiat esitetään toisistaan irrallaan ja ajallisesti etäällä . Näin tietorakennetta ei pääse muodostumaan , vaan tieto jää irrallisiksi palasiksi . ( Hiebert & Lefevre , 1986 , 3 – 4 . ) Murtolukujen suuruusvertailun ( Booth & Newton , 2012 ; DeWolf , ym . 2015 ; ) ja proseduurien ( Torbeyns , ym . , 2015 ; Bailey , ym . , 2014 ; Cramer , Post & delMas , 1999 ) sujuvuutta on tutkittu maailmalla vuosikymmenien ajan mutta murtolukujen tiheyteen liittyviä tutkimuksia on vähemmän ( Vamvakoussi & Vosniadou , 2010 , 2012 ; Gabriel , Coché , Szucs , Carette , Rey & Content , 2013 ) . Vamvakoussi ja Vosniadou ovat tutkineet eri - ikäisten koululaisten käsityksiä rationaalilukujen tiheydestä kysymällä ”Kuinka monta lukua on annetulla lukuvälillä 0 , 005 – 0 , 006 ? Annetun lukuvälin päätepisteet olivat joko murto - lukuja tai desimaalilukuja . Tyypillisesti oppilaat sijoittivat murtolukuvälille murtolukuja ja desimaalilukuvälille desimaalilukuja . Oppilaat vastasivat desimaalilukuvälitehtävässä useammin , että lukuja on annetulla lukuvälillä äärettömästi , kuin murtolukuvälitehtävässä . Tästä on tulkittavissa se , että oppilaat eivät ymmärrä murtolukujen ja desimaalilukujen esittävän samaa lukua , ja että oppilaat ajattelevat murtolukujen esiintyvän diskreetisti , ei tiheästi . ( Vamvakoussi & Vosniadou , 2010 . ) Tuominen 155 Miten seitsemäsluokkalaiset , tulevat luokanopettajat ja erityisesti matematii - kan tulevat asiantuntijat ajattelevat murtolukujen tiheydestä ? TUTKIMUS Tutkimuksessa tutkittiin , mitä ja miten varsinaissuomalaiset 7 . - luokkalaiset ( n = 74 ) , Turun yliopiston 1 . vuoden luokanopettajaopiskelijat ( n = 82 ) ja 1 . vuoden matematiikan pääaineopiskelijat ( n = 53 ) vastaavat kysymykseen ”Kuinka monta murtolukua on murtolukujen 2 4 ja 2 3 välissä” . Yläkoululaisille oli annettu valmiit vastausvaihtoehdot ( 0 , 1 tai monta ) , yliopisto - opiskelijoille ei ollut annettu valmiita vastausvaihtoehtoja . Oletuksena on , että matematiikan pääaineopiskelijat hallitsevat asian ja tavalla tai toisella ilmaisevat , että annettujen lukujen välissä on vaikka kuinka paljon murtolukuja . Luokanopettajaopiskelijat on seulottu valintakoevaihees - sa matemaattisen ja luonnontieteellisen ajattelun testin avulla , joten oletan , että myös tässä ryhmässä suurin osa hallitsee murtoluvut hyvin . Oletuksena on , että kumpikin opiskelijaryhmä menestyy yläkoululaisia paremmin . Kaikki ryhmät testattiin syksyllä juuri opintojen alettua , yliopisto - opiskelijat ensimmäisillä luennoilla ( 2014 ) ja yläkoululaiset ensimmäisillä matematiikan tunneilla ( 2013 ) ennen alakoulun kertaamisjaksoa . Yliopisto - opiskelijoiden testissä oli neljä lyhyttä tehtävää , joiden tekemiseen meni noin 5 minuuttia . Seitsemäsluokkalaisten testissä oli kymmenen tehtävää , joiden tekemiseen meni aikaa noin 15 – 20 minuuttia . Osa yläkoululaisista luovutti ja jätti testin loppuosan tehtäviä tyhjäksi ; aika ei loppunut kesken , vaan osaaminen . Oppilaiden tuli valita annetuista vastausvaihtoehdoista mielestään paras ja tämän lisäksi perustella vastauksensa . Perusteluksi kelpasi lasku , sanallinen selitys tai piirros . Näin saatiin paremmin informaatiota siitä , miten oppilas ajattelee asiasta ja myös nähtiin kuinka moni vastaajista tukeutuu esimerkiksi laventamiseen . Yliopisto - opiskelijoiden tehtäväpaperissa ei ollut valmiita vastausvaihtoehtoja , mutta heille annettiin samanlainen vastaustila vastausta ja perustelua varten . TULOKSET Suurin osa seitsemäsluokkalaisista löysi jonkun mieleisen vastausvaihtoeh - don , vain viisi oppilasta jätti kokonaan vastaamatta tehtävään ( taulukko 1 ) . Perustelu jäi kuitenkin monelta oppilaalta kokonaan tyhjäksi ( noin 53 % ) . MALU , Turku 2015 156 Taulukko 1 . Seitsemäsluokkalaisten vastausten jakautuminen perusteluineen Perustelu Tyhjä 0 1 monta YHT Tyhjä 5 4 13 17 39 Informatiivisesti tyhjä ” arvasin ” - - - 5 5 Sanallinen selitys - 5 3 1 9 Piirros - - 2 - 2 Laventaa - - 7 4 11 Erikoinen - - 3 5 8 Yhteensä 5 9 28 32 74 % 7 % 12 % 38 % 43 % 100 % Suosituin vastaus oli ”monta” . Koska perustelu jäi niin monelta tyhjäksi , on mahdotonta sanoa , onko vastaus onnekas arvaus , vai tietoon pohjautuva . Esimerkiksi osa oppilaista lavensi murtoluvut samannimisiksi saaden luvut 6 12 ja 8 12 , ja laski lukujen erotuksen , 2 12 . Erotuksesta oppilas päätteli luvun kaksi ja antoi vastaukseksi ”monta” . Toiseksi suosituin vastaus oli ”yksi” . Oppilaat perustelivat vastauksensa useimmiten laventamalla annetut murtoluvut samannimisiksi ( kuva 1 ) ja tyytyivät siihen , kun löysivät yhden sopivan murtoluvun . Kukaan ei oivaltanut , että lukujahan voitaisiin laventaa edelleen , ja näin saada yhä pienempiä osia . Laventamista hyödyntäen , diskreetilläkin käsityksellä murtoluvuista , olisi löydettävissä paljon murtolukuja annetulle lukuvälille . Kuva 1 . Oppilaan antama vastaus ja perustelu . Oppilas 25 Seitsemäsluokkalaisten perusteluista löytyi myös mielenkiintoinen virheajat - telu ( kuva 2 ) . Oppilas luettelee ensin neljäsosia ja jatkaa sitten kolmasosista . Tästä näkee hyvin sen , että oppilas ei ymmärrä , minkä kokoisia murtoluvut ovat ja mitä esimerkiksi luku 3 3 tarkoittaa . Vastaavia perusteluja oli viisi kappaletta ( noin 7 % ) , joten kyseessä ei ole yksittäinen tapaus . Tuominen 157 Kuva 2 . Oppilaan antama erikoinen perustelu vastukselle ”monta” . Oppilas 22 Yliopisto - opiskelijoille ei ollut annettu valmiita vastausvaihtoehtoja . Odote - tusti matematiikan pääaineopiskelijoista ( MatO ) suurin osa päätyi oikeaan vastaukseen ja perusteli sen sanoin ”ääretön määrä” , LO - opiskelijoiden vastaukset jakautuivat enemmän ( taulukko 2 ) . Taulukko 2 . Luokanopettajaopiskelijoiden ( LO ) ja matematiikan pääaine - opiskelijoiden ( MatO ) vastausten jakautuminen Vastaus tyhjä 0 1 2 monta ∞ ei vastausta YHT LO f 1 1 44 14 4 13 5 82 % 1 1 54 17 5 16 8 100 MatO f 2 0 4 2 0 45 0 53 % 4 0 8 4 0 85 0 101 Tutkittaessa tarkemmin LO - opiskelijoiden perusteluja vastaukselle ”monta” , useita strategioita löytyy . Kolme opiskelijaa perusteli vastauksensa kuten kuvassa ( kuva 3 ) . Vastauksesta on nähtävissä perustelun nojaaminen diskreettiin ajatteluun : 12 24 jälkeen tulee 13 24 ja niin edelleen , vaikka vastaaja päätyykin vastaukseen ”Äärettömän monta” . Kuva 3 . LO - opiskelijan perustelu . Opiskelija nojaa ratkaisussaan diskreetti - syyteen . Opiskelija 9 Luokanopettajaopiskelijoista 66 ( 80 , 4 % ) haki ratkaisua laventamalla vertail - tavat murtoluvut samannimisiksi , matematiikan opiskelijoista laventamiseen turvautui vain kuusi opiskelijaa ( 11 , 3 % ) . Vain kaksi LO - opiskelija muunsi luvut desimaalilukumuotoon ja haki ratkaisua sitä kautta ( kuva 4 ) , matema - tiikan opiskelijoista desimaalilukuesitystä hyödynsi neljä opiskelijaa ( 7 , 5 % ) ja MALU , Turku 2015 158 seitsemäsluokkalaisista kukaan ei muuntanut murtolukuja desimaaliluku - muotoon . Kuva 4 . LO - opiskelijan perustelu . Opiskelija hyödyntää murtoluvun desi - maalilukuesitystä . Opiskelija 57 Kuvan 5 ratkaisussa on havaittavissa synteettinen käsitys . Toisaalta opiskelija nojaa diskreettiin ajatteluun väittäessään , että luvun 2 4 jälkeen tulee 3 4 , ja toisaalta opiskelija muistaa ehkä kuulleensa , että murtolukuja on ääretön määrä . Kuva 5 . LO - opiskelijan perustelu . Opiskelijalla on synteettinen käsitys . Opiskelija 30 Kun liitetään seitsemäsluokkalaisten tulokset samaan taulukkoon LO - ja MatO - opiskelijoiden kanssa , huomataan samankaltaisuutta luokanopettaja - opiskelijoiden ja peruskoululaisten vastausten jakaumissa ( taulukko 3 ) . Kun sarakkeet ”0” ja ”1” , ”monta” ja ” ∞ ” , ja ”tyhjä” ja ”ei vastausta” yhdistetään keskenään , LO - opiskelijoiden ja seitsemäsluokkalaisten vastausten jakautu - minen on hyvin samansuuntaista . Seitsemäsluokkalaisista osa oli sitä mieltä , ettei annettujen lukujen väliin mahdu yhtään murtolukua . Luokanopettaja - opiskelijoista samaan ratkaisuun päätyi onneksi vain yksi opiskelija . Luokan - opettajaopiskelijoista yli puolet ehdotti vastaukseksi yhtä , vaikkei vaihtoehtoa ollut tarjolla . Heistä suurin osa perusteli vastauksensa laventamalla murtolu - vut samannimisiksi . Tuominen 159 Taulukko 3 . LO - opiskelijoiden , Mat - opiskelijoiden ja 7 . - luokkalaisten vastausten jakautuminen POHDINTA Tutkimuksessa tutkittiin miten hyvin seitsemäsluokkalaiset , 1 . vuoden luokanopettajaopiskelijat ja 1 . vuoden matematiikan pääaineopiskelijat ymmärtävät murtolukujen ( rationaalilukujen ) tiheyden . Näyttäisi siltä , että tiheyden ymmärtäminen on hyvin haastavaa , edes kaikki matematiikan pääaineopiskelijat eivät osanneet vastata oikein , vaan osa nojasi selvästi luonnollisten lukujen diskreettiin ominaisuuteen ja seuraaja - ajatteluun : 6 12 jälkeen tulee 7 12 , ja niin edelleen . Tehtävän ratkaisemiseksi seitsemäsluokkalaisten ja luokanopettajaopiskeli - joiden yleisesti käyttämä strategia oli laventaa vertailtavat luvut keskenään samannimisiksi . Oppilaista kukaan ei keksinyt jatkaa laventamista eteenpäin , vaan oppilaat lopettivat lavennettuaan kerran . Luokanopettajista kolme keksi jatkaa laventamista ja matematiikan opiskelijoista tällaista jatkuvaa laventa - mista hyödynsi kaksi opiskelijaa . Seitsemäsluokkalaisista suurin osa valitsi oikean vastausvaihtoehdon ”monta” , mutta heidän perustelunsa jäi useimmiten tyhjäksi . Tällöin vastauksesta ei ole tulkittavissa ymmärrystä murtolukujen tiheydestä . Seitsemäsluokkalaisilla havaittu erikoinen virhekäsitys ( kuva 2 ) voisi johtua tukeutumisesta luonnollisten lukujen luetteluominaisuuteen . Oppilaat ikään kuin luettelevat vähenevästi , ensin neljäsosia ja niiden loputtua , jatkavat seuraavaksi ajattelemistaan osista , tässä tapauksessa kolmasosista . Monella vastaajalla tehtävän ratkaisu meni väärin siinä , etteivät he ymmärtä - neet murtoluvun suuruutta . Jos vastaaja olisi edes pohtinut annettujen murtolukujen desimaalilukuesityksiä , olisi ratkaisu saattanut löytyä . Sujuva liikkuminen rationaalilukujen eri esitysmuotojen välillä näyttäisi olevan eduksi . Matematiikan opiskelijoista neljä perusteli ratkaisuaan nojautuen lukujen desimaalilukuesityksiin , luokanopettajaopiskelijoista näin teki kaksi Vastaus 7 . lk LO MatO f % f % f % 0 tai 1 37 50 , 0 45 54 4 7 , 5 monta tai ∞ 31 43 , 2 31 37 , 9 47 88 , 7 ei vastausta tai tyhjä 5 6 , 8 6 7 , 3 2 3 , 8 yhteensä 74 100 82 100 , 1 53 100 , 1 MALU , Turku 2015 160 opiskelijaa ja seitsemäsluokkalaisista desimaalilukuesitykseen ei turvautunut kukaan . Tästä on tulkittavissa se , että murtoluvut ja desimaaliluvut mielle - tään toisistaan irrallisina lukujoukkoina ( Vamvakoussi ym . , 2011 ) , ja annettu - jen murtolukujen väliin etsitään vain murtolukuvastauksia ( Vamvakoussi & Vosniadou , 2010 ) . Kuitenkin juuri desimaalilukuvälille oppilaat olivat aikaisemman tutkimuksen mukaan taipuvaisempia hyväksymään äärettömän määrän lukuja ( Vamvakoussi & Vosniadou 2010 ) . Kuten viime aikaisissa tutkimuksissa on todettu , taito sijoittaa sekä murtolukuja ja desimaalilukuja lukusuoralle , toisin sanoen ymmärtää näiden lukujen suuruus , ennustaa hyvää algebran hallintaa ( DeWolf , ym . , 2015 ; Torbeyns , ym . , 2015 ) , mikä onkin havaittavissa matematiikan opiskelijoiden kohdalla . On huolestuttavaa , että luokanopettajaopiskelijoilla murtolukujen tiheyskäsi - tys on lähes samalla tasolla 7 . - luokkalaisten käsitysten kanssa . Näyttää siltä , että alakoulussa hankittu käsitteellinen tieto murtoluvuista on melko pysyvää . Ne vastaajista , jotka hallitsivat murtolukujen tiheyden , osasivat myös sujuvasti liikkua esitysmuodosta toiseen . Opetuksessa tulisikin kiinnittää huomiota erityisesti murtoluvun suuruuden ja rationaalilukujen eri esitysmuotojen ; murtolukujen ja desimaalilukujen , ymmärtämiseen . Vasta murtolukujen suuruuden ymmärtämisen myötä on mahdollista ymmärtää murtolukujen tiheys . LÄHTEET Bailey , D . , Siegler , R . & Geary , D . ( 2014 ) . Early predictors of middle school fraction knowledge . Developmental Science , 17 ( 5 ) , 775 – 785 . Booth , J . & Newton , K . ( 2012 ) . Fraction : Could they really be the gatekeeper ' s doorman ? Contemporary Educational Psychology , 37 , 247 – 253 . Cramer , K . , Post , T . & delMas , R . ( 2002 ) . Initial Fraction Learning by Fourth - and Fifth - Grade Students : A Comparison of the Effects of Using Commer - cial Curricula With the Effect of Using the Rational Number Project Cur - riculum . Journal for Research in Mathematics Education , 33 ( 2 ) . 111 – 144 . DeWolf , M . , Bassok , M . & Holyoak , K . ( 2015 ) . From rational numbers to algebra : Separable contriburion of decimal magnitude and relational un - derstanding of fractions . Journal of Experimental Child Psychology , 133 , 72 – 84 . Gabriel , F . , Coché , F . , Szucs , D . , Carette , V . , Rey , B . & Content , A . ( 2013 ) . A componential view of childrenś difficulties in learning fractions . Frontier in Psychology , 4 ( 715 ) , 1 – 12 . Gallistel , C . & Gelman , R . ( 1992 ) . Preverbal and verbal counting and computa - tion . Cognition , 44 , 43 – 74 . Hartnett , P . & Gelman , R . ( 1998 ) . Early Understanding of Numbers : Paths or Barriers to the Construction of New Understanding . Learning and Instruc - tion , 8 ( 4 ) , 341 – 374 . Tuominen 161 Hiebert , J . & Lefevre , P . ( 1986 ) . Conceptual and Procedural Knowledge in Mathematics : An Introductory Analysis . Teoksessa J . Hiebert ( toim . ) Con - ceptual and Procedural Knowledge : The Case of Mathematics ( ss . 3 – 4 ) . New Jersey : Lawrence Erlbaum Associates Inc . Publishers . Ni , Y . & Zhou , Y . ( 2005 ) . Teaching and Learning Fraction and Rational Numbers : The Origins and Implications of Whole Number Bias Teaching . Educational Psychologist , 40 ( 1 ) , 27 – 52 . Siegler , R . , Thompson C . & Schneider . ( 2011 ) . An integrated theory of whole number and fractions development . Cognitive Psychology , 62 , 273 – 296 . Torbeyns , J . , Schneider , M . , Xin , Z . & Siegler , R . ( 2015 ) . Bridging the gap : Fraction understanding is central to mathematics achievement in students from three diffrerent continents . Learning and Instruction , 37 , 5 – 13 . Vamvakoussi , X . ( 2015 ) . The development of rational number knowledge : Old topic , new insights . Learning and Instruction , 37 , 50 - 55 . Vamvakoussi , X . , Christou , K . , Mertens , L . & Van Dooren , W . ( 2011 ) . What fills the gap between discrete and dence ? Greek and Flemish students ' understanding of density . Learning and Instruction , 21 , 676 – 685 . Vamvakoussi , X . & Vosniadou , S . ( 2012 ) . Bridging the Gap Between the Dense and the Discrete : The Number Line and the " Rubber Line " Bridging Analo - gy . Mathematical Thinking and Learning , 14 , 265 – 284 . Vamvakoussi , X . & Vosniadou , S . ( 2010 ) . How many decimals are there between two fractions ? Aspects of secondary school students ' understand - ing of rational numbers and their notation . Cognition and Instruction , 28 , 181 – 209 . Van Hoof , J . , Vandewalle , Verschaffel , L . & Van Dooren , W . ( 2015 ) . In search for the natural number bias in secondary school students ' interpretation of effect of arithmetical operations . Learning and Instruction , 37 , 30 – 38 . MALU , Turku 2015 162 MATEMATIIKANOPETTAJIEN NÄKEMYKSIÄ LIITTYEN TEORIAAN , ESIMERKKEIHIN JA HARJOITUSTEHTÄVIIIN Antti Viholainen , Niko Kuusisto , Mervi Asikainen & Pekka E . Hirvonen Itä - Suomen yliopisto Matemaattisen teorian , esimerkkien ja harjoitustehtävien roolit matematiikan opetuksessa ja oppimisessa voidaan nähdä eri tavoin riippuen siitä , millaisista matematiikan luonnetta koskevista näkemyksistä lähtien asiaa katsotaan . Matematiik - kanäkemykset voidaan luokitella formalismi - , skeema - , prosessi - ja sovellusorientaati - oihin . Tässä tutkimuksessa tutkittiin kyselyn avulla , mitä eri orientaatioiden mukaisia matematiikan oppimisen tavoitteita kokeneet suomalaiset peruskoulun ja lukion matematiikan opettajat ( n = 52 ) pitävät tärkeimpinä . Teorian opetuksen suhteen opettajan korostivat formalismiorientaatioon viittaavia tavoitteita , mutta esimerkkien roolin he näkivät melko monipuolisina . Harjoitustehtävien tavoitteiden suhteen opettajien vastauksissa painottui voimakkaasti laskurutiinin hankkiminen . JOHDANTO Matemaattinen teoria , sitä havainnollistavat , ainakin osittain valmiiksi ratkaistut esimerkit ja oppilaiden ratkaistavaksi tarkoitetut harjoitustehtävät ovat olennaisia elementtejä erityisesti perinteisessä matematiikan kouluope - tuksessa ja oppimateriaaleissa ( Reys , Reys & Chavez , 2004 ; Bills , Dreyfus , Mason , Tsamir , Watson & Zaslavsky , 2006 ; Viholainen , Partanen , Piiroinen , Asikainen & Hirvonen , 2015 ) . Opettajan valitsemista opetuksen käytänteistä kuitenkin riippuu , miten näitä elementtejä käytetään opetuksessa . Annetaan - ko matemaattinen teoria oppilaille valmiina ”absoluuttisena totuutena” vai saavatko he itse keksiä ja rakentaa sitä ? Käytetäänkö esimerkkejä ensisijaisesti malliratkaisuina , joita jäljittelemällä oppilailla on hyvät mahdollisuudet suoriutua harjoitustehtävistä vai onko tärkeämpää , että esimerkit syventävät oppilaiden ymmärrystä käsiteltävästä asiasta ja aktivoivat heidän omaa pohdintaansa ? Entä millä kriteereillä opetuksessa käytettävät harjoitustehtä - vät valitaan ? Nämä opetuksen käytänteisiin liittyvät valinnat riippuvat siitä , millaisiin oppimistavoitteisiin opettaja pyrkii teorian opettamisen , esimerk - kien käsittelyn ja harjoitustehtävien teettämisen kautta . MATEMATIIKKAORIENTAATIOT Opettajien matematiikan luonnetta koskevat uskomukset ovat tärkeitä , sillä opettajat välittävät niitä oppilaille joko tietoisesti tai tietämättään ( Ernest , 1989 ) . Grigutsch , Ratz ja Törner ( 1998 ) tutkivat saksalaisten matematiikan - opettajien matematiikan luonnetta koskevia uskomuksia ja näkemyksiä . Tutkimuksensa pohjalta he muodostivat neljä erilaista matematiikkaorientaa - tiota . Näitä orientaatioita ovat myöhemmin soveltaneet ja hieman muokan - Viholainen , Kuusisto , Asikainen & Hirvonen 163 neet muun muassa Felbrich , Müller ja Blömeke ( 2008 ) sekä Viholainen , Asikainen ja Hirvonen ( 2014 ) omissa tutkimuksissaan . Viholaisen ja muiden mukaan skeemaorientaatio tarkoittaa , että matematiikka nähdään erilaisten sääntöjen , kaavojen ja laskennallisten menetelmien kokoelmana . Formalis - miorientaatiossa heidän mukaansa matematiikka nähdään staattisena ak - siomaattisena järjestelmänä ja prosessiorientaatiossa ongelmanratkaisukeskei - sen näkemyksen mukaisesti dynaamisena kulttuurisena tuotoksena . Sovel - lusorientaatiossa puolestaan matematiikka nähdään menetelmänä mallintaa reaalimaailman eri ilmiöitä . Myös Ernest ( 1989 ) on aiemmin esittänyt kirjallisuudessa melko paljon käytetyn kategorisoinnin matematiikan luonnetta koskeville näkemyksille . Ernest määrittelee instrumentaalisen , platonistisen ja ongelmanratkaisukes - keisen matematiikkanäkemyksen , jotka vastaavat perusajatukseltaan olennaisesti skeema - , formalismi - ja prosessiorientaatioita . Teoreettisessa mielessä on perusteltua ajatella , että erilaiset matematiikkanäkemykset johtaisivat myös erilaisiin näkemyksiin matematiikan oppimisesta ja opetta - misesta . Beswick ( 2005 ) päättelee Ernestin luokitusta soveltaen , että mikäli matematiikalle annetaan vain välinearvo ja se nähdään ensisijaisesti sääntö - jen , kaavojen ja menetelmien kokoelmana , johtaa se siihen , että matematiikan oppimisessa ja opetuksessa korostuvat muistaminen ja menetelmien prosedu - raalinen hallinta , mutta syvällisen matemaattisen ymmärtämisen merkitys jää vähemmälle . Jos taas matematiikka nähdään formalismiorientaation tapaan staattisena aksiomaattisena järjestelmänä , on matematiikan oppimisessa keskeistä oppia tuntemaan ja ymmärtämään tämän järjestelmän sisältöä ja rakennetta juuri sellaisena kuin se on ylhäältä käsin annettu . Beswickin mukaan näissä kahdessa näkemyksessä korostuu sisältökeskeisyys matema - tiikan oppimisessa , mutta jos taas matematiikka nähdään kulttuurillisena konstruktiona , pitäisi sen johtaa matematiikan oppimisessa ja opetuksessa oppijakeskeisyyteen sisältökeskeisyyden sijaan . Tämä tarkoittaa , että oppijan omat ideat ja niiden jalostaminen ja jakaminen muille olisivat keskeisempiä tavoitteita kuin valmiiden tulosten , menetelmien tai tietorakenteiden omaksuminen . Sovellusorientaation näkökulmasta katsottuna matematiikan oppimisen keskeisenä tavoitteena tulisi puolestaan olla reaalimaailman ilmiöiden ja niitä mallintavan matematiikan riippuvuuksien syvällinen ymmärtäminen ( Viholainen ym . , 2014 ) . Käytännössä matematiikan oppimista ja opettamista koskevat näkemykset eivät pohjaudu ainakaan eksplisiittisesti matematiikan luonnetta koskeviin ns . epistemologisiin näkemyksiin . Esimerkiksi Viholainen ym . ( 2014 ) havaitsivat , että matematiikan yliopisto - opintoja aloittavilla opiskelijoilla on usein matematiikan luonteesta formalismiorientaation mukainen näkemys pelkästään staattisena järjestelmänä . Nisbet ja Warren ( 2000 ) ovat havainneet , että myös alaluokkien opettajilla on usein samanlainen näkemys . Aloittavien MALU , Turku 2015 164 matematiikan opiskelijoiden näkemykset matematiikan oppimisesta ja opettamisesta olivat kuitenkin paljon monipuolisempia : niissä tuli esiin melko tasapuolisesti kaikkiin orientaatioihin viittaavia elementtejä . Toisaalta matematiikan luonteen syvällinen pohdinta oli monille haastatelluille opiskelijoille melko hankalaa . MENETELMÄT Tässä tutkimuksessa tarkastellaan suomalaisten matematiikanopettajien näkemyksiä matemaattisen teorian opettamiseen , esimerkkien käsittelyyn ja harjoitustehtävien teettämiseen liittyvien käytänteiden harjoituttamista opetustavoitteista . Tutkimuksen tavoitteena on selvittää , mitä tavoitteita opettajat näkevät näiden opetuskäytänteiden ensisijaisesti harjoituttavan . Tavoitteiden luokittelussa viitekehyksenä käytetään edellä kuvattuja matematiikkaorientaatioita . Siten tulokset heijastavat myös tutkimukseen osallistuneiden opettajien näkemyksiä matematiikasta . Tutkimuksen aineisto kerättiin sähköistä kyselylomaketta käyttäen . Kysely - lomakkeessa esitettiin 12 matematiikan oppimisen tavoitetta . Nämä laadittiin siten , että niistä jokainen oli teoreettisesti liitettävissä johonkin neljästä matematiikkaorientaatiosta . Väittämien luokittelua orientaatioihin ei kuitenkaan esitetty kyselylomakkeessa . Väittämien sisältämät tavoitteet välittyvät myös aineistonkeruuhetkellä voimassa olleista opetussuunnitelmis - ta ( Opetushallitus , 2003 ; Opetushallitus , 2004 ) . Aluksi vastaajia pyydettiin arvioimaan , mitkä listatuista tavoitteista ovat heidän mielestään kaikkein tärkeimpiä yleisesti matematiikan oppimisen kannalta . Vastaajia pyydettiin valitsemaan viisi mielestään tärkeintä tavoitetta . Tämän jälkeen vastaajia pyydettiin arvioimaan , että missä määrin a ) matemaattisen teorian opettami - nen , b ) esimerkkien käsittely ja c ) harjoitustehtävien teettäminen harjoitutta - vat listattuja tavoitteita . Tässäkin vaiheessa vastaajia pyydettiin valitsemaan viisi tavoitetta , joita kukin näistä käytänteistä heidän mielestään eniten harjoituttaa . Vastaajia pyydettiin jokaisessa vaiheessa myös perustelemaan ja kommentoimaan valintojaan . Pyyntö vastata kyselyyn lähetettiin sähköpostitse noin 400 suomalaiselle matematiikan aineenopettajalle . Pyyntö julkaistiin Matemaattisten aineiden opettajien liiton MAOL ry : n sähköpostitse lähetettävässä viikkotiedotteessa . Tämän lisäksi koulujen opettajille lähetettiin myös henkilökohtaisia sähkö - posteja . Vastauksia saatiin yhteensä 52 kappaletta . Vastaajista 34 opetti matematiikkaa peruskoulun yläluokilla , 16 lukiossa ja kaksi opetti sekä peruskoulun yläluokilla että lukiossa . Kyselyyn vastanneet opettajat olivat keskimäärin varsin kokeneita : heidän keskimääräinen työkokemuksensa oli 29 , 5 vuotta . Tämä ei kuitenkaan ollut tarkoituksellista aineistonkeruuvaihees - sa . Viholainen , Kuusisto , Asikainen & Hirvonen 165 TULOKSET Kyselyssä vastaajille esitetyt matematiikan opetuksen tavoitteet ja aineistosta saadut vastaajien valintajakaumat esitettyjen väittämien suhteen on esitetty Taulukossa 1 . Tärkeys - sarakkeessa esitetään kuinka suuri prosentuaalinen osuus vastaajista valitsi kysytyn tavoitteen viiden matematiikan opetuksen tärkeimpänä pitämänsä tavoitteen joukkoon . Kolmessa oikeanpuolimmaises - sa sarakkeessa esitetään , kuinka suuri osuus vastaajista valitsi kunkin tavoitteen kysyttäessä teorian opettamisen , esimerkkien käsittelyn ja harjoi - tustehtävien teettämisen harjoituttamaa viittä tärkeintä tavoitetta Taulukko 1 : Kyselyyn vastanneiden ( N = 52 ) matematiikanopettajien näke - mykset matematiikan opetuksen tavoitteiden tärkeydestä sekä teorian , esimerkkien ja tehtävien tärkeimmistä tavoitteista matematiikan opetuksessa . Kukin vastaaja valitsi viisi tärkeintä tavoitetta . Tavoite Tärkeys ( % ) Teoria ( % ) Esimerkit ( % ) Tehtävät ( % ) 1 . Selkeän ja täsmällisen matemaat - tisen esitystavan omaksuminen ( Formalismi ) 46 75 77 54 2 . Kaavojen , määritelmien ja sääntöjen muistaminen sekä soveltaminen ( Skeema ) 17 69 48 42 3 . Useiden ratkaisumenetelmien keksiminen ( Prosessi ) 19 38 62 31 4 . Jokapäiväisten ongelmien ja tehtävien ratkaiseminen ( Sovellus ) 65 15 48 42 5 . Abstraktin ja loogisen ajattelun edistyminen ( Formalismi ) 62 81 48 50 6 . Laskurutiinin hankkiminen ( Skeema ) 71 21 48 92 7 . Itsenäinen ongelman tutkiminen ja ratkaisun konstruoiminen ( Prosessi ) 56 31 21 50 8 . Arkielämän ja matematiikan välisen yhteyden havaitseminen ( Sovellus ) 62 31 44 40 9 . Matematiikan kokonaisuuden ymmärtäminen ( Formalismi ) 38 67 29 13 10 . Ratkaisumallien muistaminen 2 25 35 48 MALU , Turku 2015 166 ( Skeema ) 11 . Luovuus ja uusien ideoiden keksiminen ( Prosessi ) 48 33 31 31 12 . Matematiikan soveltaminen yhteiskunnan eri aloilla ( Sovellus ) 13 13 10 6 Seuraavassa Taulukon 1 kunkin sarakkeen tuloksia analysoidaan lyhyesti , ja samalla myös analysoidaan opettajien esittämiä perusteluja eniten kannatusta saaneille tavoitteille . Analyysissa keskitytään niihin perusteluihin , joilla on ilmeinen yhteys johonkin tai joihinkin kyselyssä esitettyihin tavoitteisiin . Sen sijaan esimerkiksi muut luonnehdinnat teorian , esimerkkien ja harjoitustehtä - vien luonteesta tai roolista sivuutetaan tässä analyysissa . Suoria lainauksia on esitetty esimerkinomaisesti erityisesti niistä vastauksista , jotka tuovat esiin tai selventävät erilaisia perusteluissa esiintyviä näkökulmia . Kukin esitetty lainaus on pyritty liittämään yhteen tai useampaan kyselyssä esitettyyn tavoitteeseen . Tämä on tehty osittain tutkijan tulkinnan perusteella , sillä vastaajat perustelivat yhdellä kertaa viiden eri tavoitteen valintaa ( ks . Menetelmät - luku ) . Matematiikan opiskelun tavoitteet yleisesti Yleisesti matematiikan opetuksen suhteen opettajien useimmin viiden tärkeimmän tavoitteen joukkoon valitsema tavoite oli skeemaorientaatioon liitetty laskurutiinin hankkiminen ( tavoite 6 ) , jonka valitsi 71 % vastaajista . Myös ns . arkipäivän matematiikkaan liittyvät tavoitteet 4 ja 8 valittiin varsin usein , samoin kuin abstraktin ja loogisen ajattelun kehittyminen ( tavoite 5 ) . Sen sijaan muistamista korostavat tavoitteet 2 ja 10 saivat melko vähän kannatusta . Opettajat perustelivat laskurutiinin hankkimisen ( tavoite 6 ) tärkeyttä vetoamalla mm . ylioppilaskirjoituksiin ja siihen , että laskurutiinin hallinta helpottaa myös muiden tavoitteiden saavuttamista : Tietty laskurutiini täytyy olla , jotta kapasiteettia vapautuu myös monimut - kaisemmille ongelmille . Tavoitteita 4 ja 8 perusteltiin esimerkiksi vetoamalla siihen mitä vastaaja uskoi oppilaiden tulevassa elämässään tarvitsevan : Harva tarvii derivointia ja muita yksittäisiä juttuja lukion jälkeen , mutta arkeen ja työhön liittyvän matematiikan ymmärtäminen on tärkeetä . Myös abstraktin ja loogisen ajattelutaidon ( tavoite 5 ) kehittämisen tärkeyttä perusteltiin sen hyödyllisyydellä matematiikan ulkopuolella : Viholainen , Kuusisto , Asikainen & Hirvonen 167 Kun ajattelun taso on kyllin abstrakti , minkä tahansa asian oppiminen on helppoa . Loogisesta ajattelusta on hyötyä kaikissa asioissa elämän aikana . Se helpottaa myös muiden aineiden oppimista . Teorian opettamisen harjoittamat tavoitteet Kysyttäessä matemaattisen teorian opettamisen harjoittamia tavoitteita nousivat formalismiorientaatioon liitetyt tavoitteet 1 , 5 ja 9 varsin suosituiksi . On tosin huomattava , että tavoite 5 ( abstraktin ja loogisen ajattelun kehitty - minen ) voitaisiin perustellusti liittää myös ainakin prosessiorientaatioon . Lähes 70 % vastaajista valitsi myös kaavojen , määritelmien ja sääntöjen muistamisen sekä soveltamisen ( tavoite 2 ) vastaustensa joukkoon . Sen sijaan varsin harva vastaaja valitsi matematiikan soveltamisen yhteiskunnan eri aloilla ( tavoite 12 ) , jokapäiväisen elämän ongelmien ratkaisemisen ( tavoite 4 ) tai laskurutiinin hankkimisen ( tavoite 6 ) teorian opettamisen kautta opitta - vien tavoitteiden joukkoon . Monet vastaajat pitivät teorian käsittelyä ikään kuin johdantona , jossa esitellään tarvittavat käsitteet ja työkalut . Teoriaan liitettiin myös täsmällisyys ja matemaattiset merkintätavat : Teoriaosuudessa määritellään merkinnät , määritelmät ja käsitteiden väliset yhteydet . ( tavoitteet 1 ja 9 ) Selkeään esitystapaan ohjaa juuri teorian esittäminen esim . taululle . Teorian opettamisen nähtiin tukevan matematiikan tietorakenteen ja käsitteiden välisten suhteiden ymmärtämistä : Teoriaosuudet esittelevät myös matematiikan luonnetta loogisesti rakentu - vana ja jatkuvasti kehittyvänä tieteenä . ( tavoite 9 ) Teoriaosuudessa pitää matematiikkaa esitellä myös kokonaisuutena , jossa eri kurssien osaset tukevat toisiaan . ( tavoite 9 ) Teorian nähtiin myös tukevan ajattelutaitojen kehittymistä : Teoria selkeyttää matematiikassa tarvittavaa loogista ajattelua . ( tavoite 5 ) Toisaalta teoriaosuuksia pidettiin myös abstrakteina ja oppilaille vaikeina : Teorian ymmärtäminen vaatii usein hyvää abstraktia ajattelukykyä . ( tavoi - te 5 ) Vastauksissa myös tuotiin esiin , että teorian käsittelyn harjoittaman tavoitteet riippuvat käsittelytavasta : Teoriaosuuden esittäminen voi tarkoittaa sitä , että oppilas itse lukee kirjas - ta tai parhaimmillaan katsoo itse nettivideolta asian ennen kuin alkaa tekemään tehtäviä . [ … ] Jos kyse on opettajajohtoisesta opetuksesta , harjoit - tuvat todennäköisemmin taidot 2 , 6 , ja 10 , toisessa ääripäässä painottuvat 3 . ja 11 . MALU , Turku 2015 168 Esimerkkien käsittelyn opettamat tavoitteet Esimerkkien käsittelyn harjoittamien tavoitteiden suhteen ehdottomasti suosituin valinta vastaajien keskuudessa oli selkeän ja täsmällisen matemaat - tisen esitystavan omaksuminen ( tavoite 1 ) . Toiseksi suosituin oli useiden ratkaisumenetelmien keksiminen ( tavoite 3 ) . Muutoin eri tavoitteita valittiin esimerkkien käsittelyn suhteen melko tasaisesti : Tavoite 12 oli ainoa , jonka valitsi alle viidennes vastaajista . Esimerkkien käsittelyyn liittyvissä kommenteissaan melko monet vastaajat korostivat esimerkkien roolia erityisesti matemaattisen esitystavan oppimi - sessa : Erityisesti merkintöjen opiskeluun esimerkit ovat välttämättömiä , sillä sovittuja tapoja ei opi itse keksimällä . ( tavoite 1 ) Esimerkeillä nähtiin myös olevan tärkeä rooli oman ajattelun ja työskentelyn ”alkuunpanijana” : Esimerkkien avulla voidaan oppilaalle antaa apua , jolla päästään oman ajattelun alkuun . ( tavoite 7 ) Sen sijaan seuraavissa vastauksessa tuotiin enemmän esille esimerkkien skeemaorientaation mukaista roolia ratkaisumallien tarjoajana : Ongelmanratkaisuun annetaan malleja . Mallioppimisen kautta edesaute - taan myös laskurutiineja . ( tavoite 6 ) Ensimmäisten esimerkkien tarkoitus on usein juuri havainnollistaa esimer - kiksi uuden kaavan soveltamista . ( tavoite 2 ) Useissa vastauksissa kuitenkin korostui , että esimerkkien tehtävänä on lisätä oppilaiden ymmärrystä ja näkemystä opiskeltavasta asiasta muun muassa johdattelemalla uuteen asiaan ja tuomalla esiin erilaisia näkökulmia ja ratkaisutapoja : Esimerkeillä on hyvä valottaa erilaisia näkökulmia ja ratkaisutapoja käsi - teltävään asiaan . ( tavoitteet 3 ja 9 ) Muutamissa vastauksissa viitattiin myös siihen , että esimerkit usein käsittele - vät käytännön tai arkipäivän ongelmia ja siten havainnollistavat matematii - kan soveltamista ja merkitystä : Esimerkeissähän lähdetään selvittämään jotain ns . ongelmaa , joka liittyy monesti käytäntöön jollain tavalla . Siihen tarvitaan ratkaisukeinoja . ( tavoi - te 4 ) Esimerkeillä pystytään näyttämään , että millaisia ongelmia opituilla työka - luilla pystytään ratkaisemaan ja miten ratkaiseminen tapahtuu . ( tavoitteet 3 ja 8 ) Esimerkkien tavoitteita koskevissa perusteluissa esiintyi siis viittauksia kaikkiin orientaatioihin . Viholainen , Kuusisto , Asikainen & Hirvonen 169 Harjoitustehtävien harjoittamat tavoitteet Laskurutiinin hankkiminen ( tavoite 6 ) oli vastaajien keskuudessa ehdotto - masti suosituin valinta kysyttäessä harjoitustehtävien tekemisen harjoittamia tavoitteita . Sen valitsi peräti 92 % vastaajista . Muut tavoitteet , tavoitteita 9 ja 12 lukuun ottamatta , sen sijaan saivat varsin tasaisen vahvaa kannatusta , sillä niiden kaikkien valintaosuus oli välillä 31 - 54 % . Myös sanallisissa perusteluissaan monet opettajat korostivat harjoitustehtä - vien teon tärkeyttä laskurutiinin kehittymisen kannalta : Laskurutiinia ei voi saada muutoin kuin ratkaisemalla itse riittävän monta tehtävää . ( tavoite 6 ) Toistoilla asia jää mieleen ja rutiini muodostuu , siksi valintani kohdistui kohtiin 6 ja 10 . Muutamissa vastauksissa laskurutiinin kehittyminen nähtiin edellytyksenä muiden tavoitteiden saavuttamiselle : Tehtävillä haetaan pohjaa matematiikan osaamiselle , se edellyttää mieles - täni jonkinlaista rutiinia ja " helppojen " asioiden automatisoitumista . Tämä hoituu osittain tehtävien hinkkaamisen kautta . ( tavoite 6 ) Vastauksissa myös tuli esiin , että oppikirjoissa olevat tehtävät ovat usein pääasiassa laskurutiinin kehittymistä painottavia : Varsinaista luovuutta ja syvällisempää ajattelua vaaditaan monesti vasta vaikeimmissa tehtävissä . Helpoimmat ensimmäiset tehtävät ovat usein kaavamaisia ja laskurutiinia kehittäviä . ( tavoitteet 6 ja 11 ) Eräs opettaja epäili oppilaidensa kykyä suoriutua muunlaisista kuin laskuru - tiinia kehittävistä tehtävistä : Ilman opettajan ohjausta tehtävien ratkaiseminen on lähinnä laskurutiinin kartuttamista mallin mukaan . Oppilaat eivät opi matemaattista esitystapaa eivätkä ratkaisumenetelmiä , jollei heitä aktiivisesti ohjaa siihen . ( tavoitteet 1 , 3 ja 6 ) Harjoitustehtävien tavoitteisiin liittyvissä perusteluissa tuli siis voimakkaim - min esille skeemaorientaatio . POHDINTA Matematiikan opetuksen yleisiä tavoitteita pohtiessaan kyselyyn vastanneet opettajat valitsivat melko tasaisesti kyselyssä listattuja tavoitteita . Myös sanallisissa perusteluissa tuli esiin , että opettajat näkivät matematiikan opetuksen tavoitteet yleisellä tasolla melko monipuolisina . Teorian opettami - sen vastaajat kokivat harjoituttavan muita enemmän formalismiorientaatioon liitettyjen oppimistavoitteiden saavuttamista . Sen sijaan esimerkkien roolin vastaajat kokivat monipuolisena ja näkivät niiden harjoituttavan useanlaisia tavoitteita . Harjoitustehtävien suhteen puolestaan korostui selkeästi niiden MALU , Turku 2015 170 rooli laskurutiinin harjoituttajana . On huomioitava , että vastaajajoukko oli erityisen kokenutta , joten nuoremmille opettajille tehty kysely saattaisi tuottaa toisenlaisen tuloksen . Lisäksi on syytä huomata , että ainakin lukion matematiikan pitkässä oppimäärässä on hieman erilaiset tavoitteet kuin peruskoulun matematiikassa tai lukion matematiikan lyhyessä oppimäärässä . Mielenkiintoinen jatkotutkimuksen aihe olisikin selvittää , miten eri orientaa - tiot painottuvat eri kouluasteiden matematiikassa . Myös kansainvälinen vertailu eri maiden kesken suuremmilla otoksilla toteutettuna auttaisi näkemään maiden välisiä eroja ja yhtäläisyyksiä painotuksissa . Prosessi - ja sovellusorientaatioiden mukaisten oppimistavoitteiden voidaan ajatella olevan sopusoinnussa konstruktivististen oppimiskäsitysten kanssa ( Beswick , 2005 ) . Skeema - ja formalismiorientaatioita voidaan perustellusti kritisoida siitä , että niissä oppilaan oma luovuus ja omien ideoiden keksimi - nen eivät painotu . Lisäksi skeemaorientaatiossa asioiden syvällinen ymmär - täminen jää toissijaiseksi tavoitteeksi , ja formalismiorientaation mukaisten tavoitteiden kautta matemaattisesta tiedosta heijastuu helposti objektivistinen kuva . Niinpä voidaan nähdä ongelmallisena , jos matematiikan opetuksessa korostuvat liian voimakkaasti pelkästään skeema - tai formalismiorientaation mukaiset tavoitteet . Kuitenkin on huomattava , että ainakin osaa skeema - ja formalismiorientaatioiden mukaisista oppimistavoitteista voidaan pitää hyvinkin tärkeinä : Esimerkiksi skeemaorientaation korostama proseduraalis - ten taitojen hallinta liittyy usein läheisesti myös käsitteellisen tiedon hallin - taan ( Haapasalo & Kadijevich , 2000 ) , eikä myöskään formalismiorientaation korostamia täsmällisen matemaattisen esitystavan hallintaa ja matemaattisten tietorakenteiden ja kokonaisuuksien ymmärtämistä voida missään tapaukses - sa pitää hyödyttöminä tavoitteina . Tämän tutkimuksen valossa suomalaisessa matematiikan opetuksessa olisi syytä kiinnittää huomiota toisaalta siihen , että matemaattista teoriaa ei esitettäisi pelkästään valmiiksi annettuna objektiivisena totuutena ja toisaalta siihen , että opetuksessa käytettävät harjoitustehtävät olisivat monipuolisem - pia siten , että ne harjoituttaisivat myös muita matematiikan oppimiseen liittyviä tavoitteita kuin laskurutiinin kehittymistä . Mm . Pehkonen ( 2003 , 2013 ) on useissa yhteyksissä painottanut , että matematiikan opetuksessa tulisi rutiinitaitojen harjoittelun sijaan painottaa ajattelutaitoja ja luovuutta . Teorian käsittelyn monipuolistamiseen voisi yksi vaihtoehto olla Hollannissa kehitetty realistinen matematiikan opetus ( realistic mathematics education ) , jossa keskeisenä ajatuksena on , että oppilaat ohjatusti konstruoivat matemaattista tietoa reaalimaailman ilmiöiden pohjalta ( Gravemeier , 1994 ; Van Den Heuvel - Panhuizen , 2003 ) . 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Virmajoki & Helle 173 DEFINITIONS OF MEDICATION CALCULATION COMPE - TENCE OF REGISTERED NURSES : AN INTEGRATIVE REVIEW Anne Virmajoki & Laura Helle University of Turku The aim of this literature review was to examine definitions of medication calculation competence ( MCC ) based on studies on registered nurses . An integrative method was chosen for the review , summarising both the empirical and theoretical literature for the purpose of understanding medication calculation in terms of the competences involved . A total of 19 studies were included in the review . Based on this review , there is no shared understanding of the definition of MCC . The definitions of MCC found in the literature consist of five elements : 1 ) abstract thinking ; 2 ) medication calculation from a nursing perspective ; 3 ) mastery of mathematics ; 4 ) equipment ; and 5 ) personal qualities . MCC is a complex and multidimensional application of these elements both in connection with each other and as separate components . INTRODUCTION Medication calculation skills are essential for nurses ( Fleming , Brady & Malone , 2014 ) . Slight mistakes in calculation can lead to hazardous situations in patient care ( Grandell - Niemi , Hupli , Puukka & Leino - Kilpi , 2006 ; Newton , Moore , Harris & Pittiglio , 2010 ) . In general , the nurse’s role is to administer medication based on a doctor’s prescription . For this purpose , a nurse needs to be competent in precisely understanding and solving dosage calculation problems based on the prescription . ( Grandell - Niemi , 2005 ; McMullan , Jones & Lea , 2010 . ) Recently , according to the study by Sneck in Finland , 32 . 7 % of registered nurses failed to pass a medication calculation test on their first attempt ( Sneck , Saarnio , Isola & Boigu , 2016 ) . Moreover , the study of Sulosaari found that 83 % of Finnish nursing students failed to calculate medication calcula - tion problems correctly 100 % of the time ( Sulosaari , Huupponen , Hupli , Puukka , Torniainen & Leino - Kilpi , 2015 ) . These results are similar to the study of Grandell - Niemi ( 2005 ) a decade earlier , also in Finland , which found that both nurses and nursing students had difficulties with arithmetical computations and dosage calculations . Earlier studies clearly demonstrated that many nursing students have difficulties learning the mathematics required for professional success ( Hunter Revell & McCurry , 2013 ) . Moreover , based on previous studies , it can be seen that nurses lack both the theoretical knowledge and practical calculation skills needed for medication activities ( e . g . McMullan et al , 2010 ; Brady , Malone & Fleming , 2009 ; Grandell - Niemi et al 2006 ) . MALU , Turku 2015 174 Tilley ( 2008 ) has defined nursing competence as the application of the knowledge and skills expected for the role of a nurse in nursing practice . From this perspective , medication administration is a complex activity , involving both theoretical knowledge and medication calculation skills as equal components ( Wright , 2012 ; Fleming et al , 2014 ; Sneck et al 2016 ) . Sulosaari et al ( 2015 ) observe that the results of medication calculation tests in several studies can be difficult to interpret because the components of medication calculation have been mixed . If the definition of the study issue is unclear , it is difficult to follow what has been measured . Moreover , different terms concerning competence have been used as synonyms . This may be one reason why medication calculation in terms of competence has remained unclear . Therefore , medication calculation competence ( MCC ) needs to be diligently examined to define the indicators of the successful performance of this competence ( Wright , 2012 ; Bagnasco , Galaverna , Aleo , Grugnetti , Rosa & Sasso , 2015 ) . This paper explores the definitions of MCC of registered nurses as laid out by other researchers or research groups . The data for this definition was collected through a literature review of earlier studies from international databases . An integrative literature review was chosen as the method for the data evaluation and analysis . AIM The aim of this literature review is to explore definitions of MCC as a framework for future study and for the purposes of the future demands of medication in both nursing education and practice . The review began by searching for studies concerning medication calculation competence , skills and abilities . The question that guided the literature search was : What kind of knowledge , skills and other qualifications does a nurse need for carrying out medication calculation in nursing practice based on the earlier studies in the literature ? METHODS An integrative literature review is a method of summarising both the empirical and theoretical literature for the purpose of understanding a specific phenomenon ( Whittemore & Knafl , 2005 ) . The purpose is to review , critique and synthesise the representative literature in such a way that new perspectives on the topic are generated ( Torraco , 2005 ) . This method was chosen for scrutinising the existing research field on MCC . The review comprises five stages : 1 ) problem identification ; 2 ) literature search ; 3 ) data evaluation ; 4 ) analysis ; and 5 ) presentation ( Whittemore & Knafl , 2005 ) . Virmajoki & Helle 175 Literature search A computerised search was carried out using in the following databases : Cumulative Index of Nursing and Allied Health Literature ( CINAHL ) , Education Resource Information Centre ( ERIC ) , Finnish Medicine and Health Sciences database ( MEDIC ) , Cochrane Collaboration ( COCHRANE ) , Mathe - matics Education Database ( MATHEDUC ) , Medical Literature Analysis and Retrieval System Online ( MEDLINE ) and US National Library of Medicine National Institutes of Health ( PUBMED ) . The following key terms were used : ’competence’ or ’proficiency’ or ’skills’ or ’master’ or ’ability’ or ’knowledge’ AND ’calculation’ or ’math’ or ’numeracy’ AND ’medication’ or ’drug’ or ’drug therapy’ AND ’nursing’ . The search was limited to the years 2000 ‒ 2016 . The search included studies in Finnish and in English . Conduct of the review The literature search involved the following stages outlined in Figure 1 . At the first stage , the computerised search produced 986 references . At the second stage , 757 references were excluded based on their topic of study . The following studies were excluded : 1 ) studies that focused on medication rather than medication calculation ; 2 ) studies that focused on teaching interventions ; 3 ) studies that focused only on medication errors ; 4 ) position papers not contributing new information ; 5 ) duplicates . At the third stage , from the remaining 229 references , 189 were excluded based on their abstract . Papers were excluded for the same reasons as above . In addition , the following were excluded : 1 ) pilot studies ; 2 ) reports on medication calculation exams ; 3 ) papers that developed a specific instrument for measuring calculation skills ; 4 ) pretest - posttest interventions ; and 5 ) studies focusing on requirements in a specific context ( e . g . cancer medication ) . These three stages were conducted by one reviewer . At the fourth stage , 40 articles or dissertations were read by two independent reviewers . At this stage , the same exclusion criteria were applied as before . In addition , studies were excluded if : 1 ) MCC was not defined in any way ; 2 ) the study failed to contribute new spesific knowledge ; and 3 ) the results of the study involved students in early grades of education ( only students near graduation were included in the review ) . Finally , a total of 19 items were selected for inclusion in this review . MALU , Turku 2015 176 Figure 1 . Summary of literature search and articles reviewed at each stage . ANALYSIS OF THE REVIEWED STUDIES In the evaluation and analysis stages , the reviewed studies were first analysed based on their methods , study design and research integrity . The review included a total of 19 studies ( articles and dissertations ) or literature reviews . They were published during 2001 ‒ 2016 , and the authors came from Finland , Italy , the UK , the USA and New Zealand . A total of 5474 nurses or graduating nursing students participated in these studies , and even more ( not countable ) participated through the literature reviews . The studies were conducted using Computerized search from 7 databases : ERIC , MEDLINE , CINAHL , MEDIC , COCHRANE , PUBMED , MATHEDUC Search terms and combinations : ( mental ) competenc * or proficienc * or skill * or master * or abilit * or knowledge AND medication * or drug or drug therapy AND calculat * or math * or numerac * ( drug dosage calculation ) AND nurs * or nursing ( + Medic : “lääkelaskenta” AND osaaminen or taidot ) Limits : years 2000 - 2016 , language English or Finnish Search produced 986 references ERIC ( 19 ) , MEDLINE + CINAHL ( 546 ) , MEDIC ( 173 ) , COCHRANE ( 3 ) , PUBMED ( 235 ) , MATHEDUC ( 10 ) Title review 229 references for further analysis Deletion of 757 references based on the topic or dupli - cates Abstract review Deletion of 189 references based on the inclusion and exclusion criteria 40 references for analysis Article review Deletion of 21 references based on inclusion and exclusion criteria 19 references to review Virmajoki & Helle 177 different research methods : qualitative , quantitative and mixed methods . The studies selected included descriptive , correlational , grounded theory and ethnographic approaches . The data for the studies was collected using questionnaires ( n = 8 ) , interviews ( n = 1 ) and mixed methods ( n = 5 ) . Five review papers were included in the review . Second , using an inductive strategy of analysis , the theory and methods sections of each study were examined carefully in order to identify theoretical ( explicit and implicit ) and operational definitions of MCC relating to compe - tence , proficiency , skills , knowledge and ability in mathematics and / or medication calculation . These identified details ( one word , several words , a phrase , a sentence or a description ) were assembled together study by study as illustrated in Appendix 1 . The parts of text were registered in one table exactly as they were written in the studies so that the information did not change . Next , the elements of the definitions were coded based on five inductive codes : code 1 included all the units concerning abstract thinking ; code 2 included all the pieces of text concerning medication calculation from a nursing perspective ; code 3 included all the pieces of text concerning mathematics ; code 4 included all the pieces of text concerning equipment ; and code 5 included all the pieces of text concerning nurses’ personal qualities . As an example of the analysis , the elements of MCC identified in three studies are outlined in Appendix 1 . RESULTS The 19 studies that were included to this analysis , defined MCC by answering the study question : What kind of knowledge , skills and other qualifications does a nurse need for carrying out medication calculation in nursing practice based on the earlier studies in the literature ? The coding scheme is presented in detail in Appendix 2 . Out of the studies , 15 discussed MCC from the perspective of mathematics . Three studies examined MCC from the perspective of medication . One study examined MCC from the perspective of medication errors . All the studies agreed that the role of medication calculation was an important part of a safety - critical context and that practice - based MCC is essential for the nursing profession and needs to be highly regulated . MCC involves the need to undertake appropriate arithmetical operations and computa - tions to calculate a numerical value that falls within an appropriate degree of accuracy for the required dose or rate ( Young , Weeks & Hutton , 2013 , p . 16 ) . In addition , it was particularly challenging to find explicit definitions of MCC in the studies . The knowledge and skills required for medication calculation MALU , Turku 2015 178 tended to be described through consideration of : 1 ) mathematics tests ; 2 ) the requirements of the vocation ; 3 ) errors and difficulties in medication calcula - tion ; and 4 ) the understanding of spesific phenomena such as anxiety or conceptual understanding . We highly agree with Sulosaari’s ( Sulosaari et al 2015 ) observation : the study results are difficult to interpret because of the lack of the definitions of the components that have been measured . According to our analysis , the largest understanding between the authors was about basic mathematics skills and the importance of them as a part of MCC . Basic mathematics like addition , subtraction , multiplication and division with whole numbers , fractions and decimal numbers are the mathematical operations needed in medication calculation according to the 12 studies of this review . Moreover , mentioned in several studies , conversion between units and understanding calculation with percentages are the basis of mathematical skills in medication calculation . However , based on our analysis , mathematical tests are a narrow point of view to evaluate MCC . The tests can ignore other components of MCC such as mathematical thinking and equipment . Moreover , according to our analysis , most of the authors of the analysed studies share the understanding that drug calculation includes dosage calculations , complex dosage calculation , diluting and solutions and calcula - tions with flow rates . However , these elements were not defined or described carefully in every analysed study . Knowledge and ability to use medical devises and instruments is thoroughly a part of dosage calculation . The practical perspective of MCC emphasizes through this ability of qualifying with the equipment of medication . There seems to be a consensus between the authors of the analysed studies that both nurses and nursing students have difficulties to connect theoretical knowledge to practical skills . The greatest difference between the studies , according to our understanding , was the definitions of the abstract factors of MCC , such as conceptual thinking , problem solving , reasoning , interpretation and representation . Some studies concentrated in concerning MCC as a perspective of mathematical thinking , as our analysis condenses these identified abstract factors , and they analysed one component rigorously , but mainly without defining MCC first . In one accurate investigation of problem solving MCC was discussed as a widely recognized phenomenon . Moreover , according to the several analysed studies , there seems to be nurses’ personal qualities affecting MCC and especially affecting how the competence is performed in clinical practice . These personal qualities have been described unclearly in most of the analysed studies and this results easily in confusion and misunderstanding , if the personal factors are correlates or effecting MCC or just the background factors of nurses in the study . Virmajoki & Helle 179 Altogether , according to our analysis , there was no shared understanding of the definition of MCC . The separate elements of MCC were identified in the studies but the definition of MCC remained unclear . Based on this literature review , it appears almost impossible to deal with all the elements of MCC at the same time . For this reason , it is extremely important to define carefully what the subject of a study is . CONCLUSION Our study highlights two issues : 1 ) most authors seem to take the definition of MCC for granted as explicit theoretical definitions of MCC are scarce in the literature ; 2 ) there is no shared understanding of the definition of MCC . Our analysis indicated that the definitions of MCC found in the literature consist of five elements : 1 ) abstract thinking ; 2 ) medication calculation from a nursing perspective ; 3 ) mastery of mathematics ; 4 ) equipment ; and 5 ) personal qualities . It is important to note that different researchers and research groups stressed different elements . To our understanding , MCC is a complex and multidimensional application of several attributes both in connection with each other and as separate components . Competence in vocational mathematics is about the ability to address and handle mathematics in the context of nursing . Competence in mathematical thinking is a crucial element for understanding mathematics and being able to make progress in handling medication calculations . Personal factors enable nurses to utilise their mathematical and vocational competences in medication activity to ensure patient safety and the accuracy of medication ( Figure 2 ) . Figure 2 . Medication calculation competence VOCATIONAL MATHEMATICS MATHEMATICAL THINKING PERSONAL FACTORS MALU , Turku 2015 180 REFERENCES Brady , A - M . , Malone , A - M . & Fleming , S . ( 2009 ) . A literature review of the individual and systems factors that contribute to medication errors in nursing practice . Journal of nursing management 17 ( 6 ) , 679 – 697 . Grandell - Niemi , H . ( 2005 ) . The Medication Calculation Skills of Nursing Students and Nurses . Developing a Medication Calculation Skills Test . Dissertation . Doctor of Nursing Science . University of Turku , Finland : Painosalama OY . Hunter Revell , S . M . and McCurry , M . K . ( 2013 ) . Effective pedagogies for teaching math to nursing students : A literature review . Nurse Education Today 33 ( 11 ) , 1352 - 1356 . Newton , S . , Moore , G . , Harris , M . & Pittiglio , L . ( 2010 ) . The effect of context on Nursing Student Math Aptitude . Journal of Professional Nursing 26 ( 6 ) , 341 – 345 . Tilley , D . ( 2008 ) . 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Virmajoki & Helle 183 APPENDIX 1 . THREE EXAMPLES OF INDUCTIVE ANALYSIS OF THE STUDIES Grandell - Niemi et al . ( 2006 ) Theoretical definition Operational definition ”In this study , the term basic level ( BL ) mathematical skills was used to refer to basic arithmetic ( e . g . addition , subtraction , multiplication , division ) and higher level ( HL ) skills refer to conversions , percentages , Roman and Arabian figures , estimation and dosage calculations . ” “Pirie ( 1987 ) identified the mathematical areas essential and needed in nursing : addition , subtraction , multiplication and division with whole numbers , fractions , decimals , ratios , percentage , conversions and problem - solving . These areas are usually included in medication or dosage calculation tests . ” intravenous calculations “It is important to connect calculation competency to the ability to give medication in clinical practice . ” “It is found that level ‘A’ ( upper secondary level ) in mathematics is a good predictor of mathematical performance . ” “Educational background , mathematical training and an excellent mark ( 9 – 10 ) in mathematics ( upper secondary school ) were connected with higher test scores in mathemat - ical tests as well as students’ self - confidence with their mathematical skills . ” “Finding suggests that when the partici - pants have earlier positive experiences of success in mathematics , they also have confidence in their mathematical skills in clinical practice . ” “Estimation of a sensible amount was found difficult . It is very important that everyone dealing with medications has an idea beforehand of what might be a sensible amount . ” “Calculations of solutions belong to critical skills which are needed when ready - made doses are not available . Nurses commented spontaneously on the test that they would not perform such calculations alone in which they were unsure of their skills and would ask for help of colleagues . ” “The study results showed that a sense of sufficient skills in mathematics , earlier good mathematical performance , calculations of dosages at work and little use of ready - made doses were associated with high test scores and predicted a good performance on the MCS Test in both groups . ” From test : nurses mean score was 22 , 7 ( maximum score = 29 ) , 9 nurses ( n = 364 ) answered all problems correctly and 71 % of nurses attained a score of 86 % Bagnasco et al . ( 2015 ) Theoretical definition Methodological definition “Nurses need adequate medication calculation skills to provide safe and effective drug administration and management , which is an important component of the nursing profes - sion . ” “Drug preparation phases include procedures that are potentially subject to errors : the reconstitution of drugs , titration , preparation of solutions , and the calculations made to fraction “The hardest section for the students was the one on the multiplication of fractions” “The lack of knowledge about maths principles in students entails difficulties performing maths calculations , and urge the need to bridge the theory practice gap . ” “Of all the students , 22 % ( n ¼ 155 ) declared that they had difficulty doing MALU , Turku 2015 184 drugs . ” “Many nurses and students do not have a full understanding of all the basic mathematical concepts ( addition , subtraction , division , and multiplication ) and are unable to apply these concepts to medication dosage calculation and use formulas . ” “Nurses have major difficulties with percent - ages , fractions , and equivalences . ” “The validity of written tests , as a measure of the drug calculation skills in clinical settings , has been questioned , and the risk of supposing that poor outcomes imply poor clinical competences has also been highlighted in literature . ” “Calculating drug dosages ( whether basic calculation skills , logics , deduction , reasoning , and understanding advanced formulas ) ” “Section A focused on the basic maths skills and included the first 4 areas : Area 1 : calculations with percentages Area 2 : multiplying fractions Area 3 : calculations with fractions Area 4 : divisions and multiplications by 10 , 100 , and 1000 Section B instead focused on maths calculation skills applied to medication administration , which used medication prescriptions selected from real clinical practice settings , and included : Area 5 : Calculations with proportions Area 6 : Solving problems on how to calculate medication dosages and solution infusion speeds . ” calculations without a calculator . ” “Drug calculations require complex maths skills ( calculation skills , logics , deduction , reasoning and understanding ) . ” “Students found it very difficult to reduce fractions and transform them into decimals . ” “This showed that the most common difficulties for the students were interpretation of information , conversion of units of measure , and the conceptuali - sation of calculations . ” “Maths skills are not learned once for all , but need to be refreshed over time , and require constant exercise and application of the maths rules and principles in order to be kept up . ” Knowledge of basic maths principles “It is clear that maths skills to calculate the right dose of a drug are essential for nurses and patients ' safety is at risk if nurses are unable to make precise calculations . ” Sneck et al . ( 2016 ) Theoretical definition Operational definition “Drug calculation is also a crucial skill as one mistake in calculation can lead to a fatal medication error . ” “In the literature , medication competence is defined as a complex combination of knowledge , skills , performance , values , attitudes , and decision - making competence . ” “Medication competence does not increase linearly but involves a more complex and individual process . ” “The drug calculation exam contains three questions . To pass the exam nurses need to “The scores of male nurses in the drug calculation exam were better than female nurses ' scores . The age of respondent at the time of answering correlated significantly with the scores , i . e . younger nurses had better scores than older nurses . ” > “Age correlated with the results of drug calculation skills . Younger nurses had significantly better skills than their older colleagues . ” “Acute care unit nurses had better scores in drug calculations than nurses in the Virmajoki & Helle 185 complete 100 % of the drug calculations correctly . “ Drug calculation : Basic arithmetic and conversions , Basic dosage calculations , Complex dosage calculation , Diluting and solutions , Flow rates . other units . ” > As in the theoretical competence , also in the drug calculation results , there was a correlation with work unit . The more the nurses implement complex medication in their daily work , the better they do in the drug calculation exam . Especially in the most challenging flow rate and dilution calculations there were significant differences between work units . Acute care unit nurses mastered the most difficult calculations better than the other nurses . ” Basic arithmetic and conversions Basic dosage calculations Complex dosage calculation Diluting and solutions Flow rates “The number of attempts and the use of time could be used as triggers to identify the nurses who struggle with drug calculations , and early intervention could be introduced . ” “Medication administration is a complex procedure where theoretical knowledge and drug calculation skills act as equally important areas of competence . ” “According to this study theoretical knowledge and drug calculations skills have positive correlation . ” Explanation of the colors : 1 ) Units concerning abstract factors 2 ) Units concerning medication calculation from nursing perspective 3 ) Units concerning mathematics 4 ) Units concerning equipment 5 ) Units concerning nurses personal qualifies MALU , Turku 2015 186 APPENDIX 2 . THE UNITS , SUBCATEGORIES AND CATEGORIES OF MCC Grouping of the units Subcategory Category Units concerning abstract factors Conceptual thinking MATHEMATICAL THINKING COMPETENCE Conceptual thinking involves different processes : a combination of confusion , anxiety , motivation , self - efficacy and confidence towards problem solving ability and making sense of the increas - ingly complex problems . ( Mills 2012 ) Conceptual thinking ( Fleming et al 2014 ; McMullan et al 2009 ) Conceptual competence = understanding the medication dosage problem to be solved ( Young et al 2013 ; Macdonald et al 2013 ) Conceptual competence = Understand the elements of prescription and other word - based medication forms and to extract the numerical information necessary to set up the dosage problem correctly ( Weeks et al 2013a ; Weeks et al 2013b ) Conceptual knowledge ( Hoyles et al 2001 ) Conceptual skills : ability to convert between measurement systems and to formulate a dosage problem ( Grandell - Niemi et al 2003 ) Nurses need to be able to conceptualise clinical information for the actual task of calculation ( Sulosaari et al 2010 ) Conceptualisation ( Bagnasco et al 2015 ; Pyo 2011 ) Interpretation ( Fleming et al 2014 ; McMullan et al 2009 ) Drug preparation = finding doses from prescriptions , drug concentrations , Interpretation and representa - tion Virmajoki & Helle 187 changing dose frequencies ( Hoyles et al 2001 ) To interpret numerical information from prescription charts and ampoule labels ( Coben & Weeks 2014 ) Infusion management = coordination of infusion rates , checking concentrations ( Young et al 2013 ) Fluid - balance monitoring = measuring hourly fluid intake and output , recording , updating and interpreting fluid - balance charts ( Young et al 2013 ) Vital - signs and laboratory - data interpre - tation = measuring blood pressure , temperature etc . , recording on - time series graphs with different scales , interpreting laboratory - report data ( Young et al 2013 ) Understanding and interpreting clinical information , interpretation of information ( Bagnasco et al 2015 ) Use of formulae , tables and graphs ( Young et al 2013 ) Recognition of incidences ( blood results ) ( Young et al 2013 ) Estimation ( Young et al 2013 ; Sulosaari et al 2010 ) To perform medication calculation correctly means having good problem solving skills ( Sulosaari et al 2010 ) Problem solving involves cognitive ( knowing that and why ) and functional ( know - how and skills ) competencies ( Macdonald et al 2013 ) Problem solving is a combination of conceptualization , calculation and technical measurement . ( Weeks et al 2013a ; Weeks et al 2013b ) Problem solving Proportional reasoning > the nursing rule Reasoning MALU , Turku 2015 188 > a general and consistent procedural approach ( Hoyles et al 2001 ) Reasoning ( Bagnasco et al 2015 ) Logics and deduction ( Bagnasco et al 2015 ) Units concerning mathematics Numeracy COMPETENCE OF VOCATIONAL MATHEMATICS Numeracy ( Jones 2009 ) Numeracy = to be competent , confident and comfortable with one’s judgments on whether to use mathematics in a particular situation and if so , what mathematics to use , how to do it , what degree of accuracy is appropriate , and what the answer means in relation to context . ( Young et al 2013 ; Coben & Weeks 2014 ) Numerical abilities ( McMullan et al 2009 ) Knowledge of basic math principles ( Bagnasco et al 2015 ) Mathematical skills ( Sulosaari et al 2010 ) Basic arithmetic Addition , subtraction , multiplication , division ( Young et al 2013 ; Grandell - Niemi 2006 ) Multiplication , division by 10 , 100 , 1000 ( Bagnasco et al 2015 ) Arithmetical skills : ability to add , subtract , multiply and divide ( Grandell - Niemi et al 2003 ) Arithmetic skills ( Sneck et al 2016 ) Basic arithmetic operations : addition , subtraction , multiplication and division of whole numbers , fractions and decimals ( Sulosaari et al 2010 ; Grandell - Niemi 2006 ; Bagnasco et al 2015 ) Use of fractions and decimals and negative numbers ( Young et al 2013 ; Sulosaari et al 2010 ; Bagnasco et al 2015 ) Virmajoki & Helle 189 Roman and Arabic numbers ( Sulosaari et al 2010 ) Percentages ( Sulosaari et al 2010 , Bagnasco et al 2015 ; McMullan et al 2009 ) Understanding percentages , ratios ( Young et al 2013 ) Ratios ( Hoyles et al 2001 ) Proportions ( Bagnasco et al 2015 ) Complex math skills ( Bagnasco et al 2015 ; Coben & Weeks 2014 ) Use of the International Unit system ( Young et al 2013 ) SI - unit conversion ( Coben & Weeks 2014 ; Macdonald et al 2013 ) Unit doses ( Macdonald et al 2013 ) Sub and multiple unit doses ( Macdonald et al 2013 ) Metric conversions ( Fleming et al 2014 ) Conversion between units ( Young et al 2013 ; Bagnasco et al 2015 ) Conversions ( Sneck et al 2016 ; Sulosaari et al 2010 ; Bagnasco et al 2015 ) Transformation ( Hoyles et al 2001 ) Sub - and multiple - unit dose ( Coben & Weeks 2014 ) Units concerning medication calculation from nursing perspective Dosage calculation Tablet dosages ( Fleming et al 2014 ; Coben & Weeks 2014 ) Calculate the correct number of tablets ( Jones 2009 ; Macdonald et al 2013 ) Drug dosage calculation and administra - tion the most common application of mathematical ability and skill ( Fleming et al 2014 ) MALU , Turku 2015 190 Dosage calculation skills ( Grandell - Niemi 2003 ) Calculation competence = computation of an accurate numerical value for the dose to be administered ( Young et al 2013 ) Calculation competence = correctly apply arithmetical operations and compute an accurate numerical value within a safe and acceptable tolerance range for the prescribed medication dose ( Weeks et al 2013 ; Macdonald et al 2013 ) Basic dosage / drug calculations 100 % correctly ( Sneck et al 2016 ) Calculation of dosages ( Sulosaari et al 2010 ) Dose for a drug ( Jones 2009 ) Drug dosages ( Bagnasco et al 2015 ) Diluting and solutions ( Sneck et al 2016 ) Calculation of solutions ( Grandell - Niemi 2006 ) Solid and liquid drugs ( Sulosaari et al 2010 ; McMullan et al 2009 ) Preparing liquids ( Sulosaari et al 2010 ) Calculate the correct fluid ( Jones 2009 ) Fluid dosages ( Fleming et al 2014 ) Basic calculation with liquid medicines and injections ( Coben & Weeks 2014 ; McMullan et al 2009 ; Macdonald et al 2013 ) Drip rates ( Fleming et al 2014 ) Flow rates ( Sneck et al 2016 ) Calculating infusion time ( Sulosaari et al 2010 ) Calculate the correct volume ( Jones 2009 ) Infusion speed of solutions ( Bagnasco et al 2015 ) Calculation of infusions Virmajoki & Helle 191 Intravenous calculations ( Grandell - Niemi 2006 ) IV - infusions ( Coben & Weeks 2014 ; Macdonald et al 2013 ) Infusion rates ( McMullan et al 2009 ) BMI - calculation , body mass index calculation ( Young et al 2013 ) Appreciation of statistics , budgeting and basic bookkeeping ( Young et al 2013 ) Clinical pharmacokinetics > ADME = absorption + distribution + metabolism + excretion ( Young et al 2013 ) Pharmacological skills : knowledge of terminology , pharmacological actions and effects of medicines , delivery routes of medicines , different forms of medi - cines and pharmacokinetics ( Grandell - Niemi et al 2003 ) Pharmacological skills are a part of medication calculation skills ( Sulosaari et al 2010 ) Components spesific to the context of nursing Drug calculations for children and other specific groups ( Coben & Weeks 2014 ) Complex dosage calculations ( Sneck et al 2016 ) Complex dosage calculation > Components spesific to the context of nursing Double - checking the medication administration by two nurses : helps ensuring that calculation is correct . ( Jones 2009 ) Intravenous - drug administration which 2 nurses were involved > dialogues ( Hoyles et al 2001 ) Clinical judgment as thinking and communicating with other professionals accessing available resources ( such as using drug formularies and information Communicating MALU , Turku 2015 192 leaflets ) and e . g . patient medication timing . ( Jones 2009 ) Ask for help of colleagues if unsure of one’s skills ( Grandell - Niemi 2006 ) Units concerning equipment ( Medication ) equipment Use of medication equipment , e . g . infusion pumps ( Fleming et al 2014 ; Coben & Weeks 2014 ; McMullan et al 2009 ) Technical measurement competence = accurate measurement of the medication dose and / or rate of administration ( Young et al 2013 ) = select an appropriate medication administration vehicle . Accurately transform the calculated numerical value to the context of the measurement device and measure the correct dose of prescribed medication or administer the correct rate of prescribed IV infusion fluid . ( Weeks et al 2013a ; Macdonald et al 2013 ) Measurement ( fluid balance , vital signs , preparing / drawing up and dispensing medicines ) ( Young et al 2013 ) To perform an accurate technical measurement of a dose in an appropriate - ly selected measurement vehicle ( like a syringe of an appropriate design and volume ) ( Coben & Weeks 2014 ) Using / not using calculator ( Sulosaari et al 2015 ; McMullan et al 2009 ; Bagnasco et al 2015 ) Calculator > equipment Units concerning nurses personal qualifies Age and gender Age : the younger , the better in arithmetic ( Grandell - Niemi et al 2003 ) Younger nurses had better results in medication calculation online test than Virmajoki & Helle 193 older nurses ( Sneck et al 2016 ) Age : students over 25 years got better results in calculation tests ( Sulosaari et al 2015 ) Age : 35 years or older statistically significantly more able than younger participants to perform basic numerical calculations ( McMullan et al 2009 ) Gender : male students scored higher in all the areas of the math test ( Bagnasco et al 2015 ) Male nurses in drug calculation exam were better than female nurses’ scores ( Sneck et al 2016 ) PERSONAL FACTORS AFFECT - ING MCC Earlier math education ( e . g . long syllabus or good grade in mathematic ) ( Sulosaari et al 2015 ; Grandell - Niemi et al 2006 ) Level of basic education ( Grandell - Niemi et al 2003 ) Excellent mathematic grade in earlier education ( Grandell - Niemi et al 2001 ; Grandell - Niemi et al 2006 ) Earlier education Experience : the number of clinical practice placements ( Sulosaari et al 2015 ) Delivering medicines / calculating dosages daily ( Grandell - Niemi et al 2003 ) The more nurses implement complex medication daily , the better they do in the calculation . ( Grandell - Niemi et al 2003 ; Sneck et al 2016 ) Update medication calculation skills increases nurse’s confidence in calcula - tion and improves performance of calculation ( Grandell - Niemi et al 2003 ) Working unit : cute care nurses had better scores in medication calculation tests than nurses in other units . ( Sneck et al 2016 ) There was a correlation with work unit . Experience MALU , Turku 2015 194 ( As in the theoretical competence , also in the drug calculation results ) ( Sneck et al 2016 ) A significant relationship between the number of hours worked and perfor - mance on the medication calculation test . ( Melius 2012 ) Exercise ( Grandell - Niemi et al 2003 ) and mathematical training ( Grandell - Niemi et al 2006 ) Participation in supportive education ( Sulosaari et al 2015 ) Positive results on math skills if the tutor helps with calculation problems ( Pyo 2011 ) The number of attempts and the use of time ( Sneck et al 2016 ) Training > Experience Attitude : perceives mathematics as easy ( Sulosaari et al 2015 ) Mathematics / dosage calculations are easy ( Grandell - Niemi et al 2001 ; Grandell - Niemi et al 2003 ) Satisfied with own medication calculation skills ( Grandell - Niemi 2001 ) Believes to have adequate skills in mathematics ( Grandell - Niemi 2001 ) Positive experiences if success in mathematics > confidence in mathemati - cal skills in clinical practice ( Grandell - Niemi et al 2006 ) Perception about mathematic can have an effect on math abilities ( Pyo 2011 ) Attitudes Confidence may be a key factor that underpins competence in mathematics . Improving confidence in mathematics is crucial to reducing calculation errors ( Jones 2009 ) A nurse’s confidence in his / her Confidence Virmajoki & Helle 195 mathematics performance may be related to prior experiences , successes , and / or failures . Nurse self - efficacy for mathe - matics is defined as “perceptions of one’s performance capabilities related to math problems , math tasks and math - related course work” ( Melius 2012 ) Increasing confidence can improve math skills ( Pyo 2011 ) Self - confidence with mathematical skills ( Grandell - Niemi et al 2006 ) Anxiety ( McMullan et al 2009 ) As anxiety increases , the performance scores on a medication calculation test decreases . Mathematics anxiety is defined as “feelings of tension and anxiety that interfere with the manipulation of numbers and the solving of mathematical problems in a wide variety of ordinary life and academic situations” ( Melius 2012 ) Decreasing anxiety with math skills can improve math skills ( Pyo 2011 ) Anxiety \ No newline at end of file diff --git a/silver_data/a782e7b28d2a6479746b705ad7b5e5e807a0a961.pdf.txt b/silver_data/a782e7b28d2a6479746b705ad7b5e5e807a0a961.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..53c9c4faf4ce11ba0ee824c5b28a3a952cfe09b6 --- /dev/null +++ b/silver_data/a782e7b28d2a6479746b705ad7b5e5e807a0a961.pdf.txt @@ -0,0 +1 @@ +Contents lists available at ScienceDirect Acta Psychologica journal homepage : www . elsevier . com / locate / actpsy Foraging through multiple target categories reveals the fl exibility of visual working memory ☆ Tómas Kristjánsson ⁎ , Árni Kristjánsson Department of Psychology , University of Iceland , Iceland A R T I C L E I N F O Keywords : Foraging Visual working memory Working memory Attention Search templates Visual search A B S T R A C T A key assumption in the literature on visual attention is that templates , actively maintained in visual working memory ( VWM ) , guide visual attention . An important question therefore involves the nature and capacity of VWM . According to load theories , more than one search template can be active at the same time and capacity is determined by the total load rather than a precise number of templates . By an alternative account only one search template can be active within visual working memory at any given time , while other templates are in an accessory state – but do not a ff ect visual selection . We addressed this question by varying the number of targets and distractors in a visual foraging task for 40 targets among 40 distractors in two ways : 1 ) Fixed - distractor - number , involving two distractor types while target categories varied from one to four . 2 ) Fixed - color - number ( 7 ) , so that if the target types were two , distractors types were fi ve , while if target number increased to three , distractor types were four ( etc . ) . The two accounts make di ff ering predictions . Under the single - template ac - count , we should expect large switch costs as target types increase to two , but switch - costs should not increase much as target types increase beyond two . Load accounts predict an approximately linear increase in switch costs with increased target type number . The results were that switch costs increased roughly linearly in both conditions , in line with load accounts . The results are discussed in light of recent proposals that working memory re fl ects lingering neural activity at various sites that operate on the stimuli in each case and fi ndings showing neurally silent working memory representations . 1 . Introduction As you search for mustard and ketchup in an unfamiliar super - market , what is the optimal strategy ? You do not know which brands this super - market sells , and you cannot think of a de fi ning feature in the shape of mustard or ketchup bottles that distinguishes them from most other condiments except that mustard tends to be yellow and ketchup red . You scan the shelves searching for red and yellow , occasionally pausing as your eyes land on a red or a yellow bottle . But what is ac - tually happening as we search the shelves for the two colors ? Do we look for both colors simultaneously , or are we possibly searching for one color at a time , rapidly switching between searching for yellow and red as our eyes scan the shelves ? This question touches on many im - portant questions within the scienti fi c literature on vision and atten - tion . How do we search complex scenes ? What roles do working memory and attention play in the search process ? Do we form search images , or templates to search e ff ectively , and how do they guide our search ? Can we maintain more than one search image ( or template ) at the same time ? Can we , in other words , search for ketchup and mustard simultaneously ? To address such questions , several models of attention have been developed . Some of the most in fl uential are two stage models involving a pre - attentive parallel stage followed by an active attentive stage in - volving serial processing such as Feature - Integration Theory ( Treisman & Gelade , 1980 ) and the Guided Search model ( Wolfe , 1994 ; Wolfe , Cave , & Franzel , 1989 ) . These models are mostly based on fi ndings from single target search tasks and do not as easily account for results from search tasks involving multiple targets , such as visual foraging tasks ( Kristjánsson , Jóhannesson , & Thornton , 2014 ; Wolfe , 2013 ) . Note that the latest version of the guided search model will take data and results from foraging and other multi - target search tasks into account ( Wolfe , Cain , Ehinger , & Drew , 2015 ) . Early models of visual foraging compared human foraging with optimal foraging , that assumes that as the target yield within a parti - cular search environment , decreases below average , foragers will switch to a new foraging patch ( Charnov , 1976 ; Pyke , Pulliam , & https : / / doi . org / 10 . 1016 / j . actpsy . 2017 . 12 . 005 Received 8 September 2017 ; Received in revised form 1 November 2017 ; Accepted 7 December 2017 ☆ Supported by a grant nb . # 152427 from the Icelandic Research Fund , and the Research Fund of the University of Iceland . Con fl icts of interest : none . ⁎ Corresponding author at : Department of Psychology , School of Health Sciences , University of Iceland , 101 Reykjavík , Iceland . E - mail address : tok1 @ hi . is ( T . Kristjánsson ) . Acta Psychologica xxx ( xxxx ) xxx – xxx 0001 - 6918 / © 2017 Elsevier B . V . All rights reserved . Please cite this article as : Kristjánsson , T . , Acta Psychologica ( 2017 ) , https : / / doi . org / 10 . 1016 / j . actpsy . 2017 . 12 . 005 Charnov , 1977 ) . But optimal foraging models also apply to foraging patterns , that is , foragers should choose the closest possible target to minimize the total distance travelled while foraging ( Pyke et al . , 1977 ) . While this account is logically enticing , several studies have shown that humans are not optimal foragers ( e . g . Hutchinson , Wilke , & Todd , 2008 ; Pierce & Ollason , 1987 ) and that there are biases and fl exibilities in foraging behavior , not accounted for by optimal foraging models ( Cain , Vul , Clark , & Mitro ff , 2011 ) . 1 . 1 . Templates guide foraging Most researchers agree that during search and foraging observers use search images or templates whose content re fl ects the task goals in each case ( Bond & Kamil , 2002 ; Dukas & Kamil , 2001 ; Jackson & Li , 2004 ; Nakayama , Maljkovic , & Kristjánsson , 2004 ) . Such templates are assumed to be held in capacity limited working memory ( Awh & Jonides , 2001 ; Bundesen , 1990 ; Carlisle , Arita , Pardo , & Woodman , 2011 ; Desimone & Duncan , 1995 ; Grubert & Eimer , 2013 ; Vickery , King , & Jiang , 2005 ; Woodman , Carlisle , & Reinhart , 2013 ) , and these capacity limits may be one reason why participants do not always forage optimally . There are , however , long standing disagreements over how these templates guide attention . A fundamental question involves the number of templates that can simultaneously guide attention . According to a recent proposal , there can only be one template active in working memory at any given time ( van Moorselaar , Theeuwes , & Olivers , 2014 ; Olivers , Peters , Houtkamp , & Roelfsema , 2011 ; Ort , Fahrenfort , & Olivers , 2017 ; see also Oberauer , 2002 ) . Similarly , Huang and Pashler ( 2007 ) proposed that observers only have access to one feature value at a given moment . This idea has also been proposed in the literature on animal foraging . For example , birds are bad at dividing their attention , and have trouble searching for two categories of prey simultaneously ( Dawkins , 1971 ; Dukas , 2002 ) . Recent evidence , that mostly involves demonstrations of a cost to switching between templates , is seemingly consistent with this proposal . In Houtkamp and Roelfsema ( 2009 ) ob - servers performed an RSVP task where they searched for either one or two targets within a stream of rapidly presented items . They had great di ffi culty with performance when there were two potential targets within the stream , while when there was only one , performance im - proved greatly , suggesting that participants could only use a single template for guidance at a given time , and that any additional tem - plates increased the chances of missing targets . In Dombrowe , Donk , and Olivers ( 2011 ) observers made saccades from the left to the right between target items of one color or two di ff erent colors . Performance was impaired when targets were of two di ff erent colors and Dombrowe et al . ( 2011 ) concluded that changing or switching between attentional templates takes around 250 - 300 ms . In van Moorselaar et al . ( 2014 ) observers performed visual search while they maintained a variable number of items in visual working memory . van Moorselaar et al . ( 2014 ) found only interference from the visual working memory load when a single color was maintained in working memory , not when more colors were maintained . Based on such fi ndings , Olivers et al . ( 2011 ) proposed a model of visual working memory where only a single template is active at any given time and capable of in fl uencing ongoing visual tasks ( such as visual search or visual foraging ) . According to their proposal , more templates can be kept in visual working memory , but only one template is active and can interact with perception at any given time , and non - active templates are kept in an accessory working memory state and do not a ff ect current visual performance ( see also Huang & Pashler , 2007 ) . Other results seemingly contradict this , however . Predators who divide attention among an increasing number of di ff erent prey types decrease their ability to detect any given type ( Dukas & Ellner , 1993 ) . This decrease in performance is gradual , but does not involve a collapse in performance as load increases from one to two templates with little , or no di ff erence between two or three templates , as a single - template model predicts , since according to such models , observers must simply switch to one of the items in the accessory state , that are all in a similar state ( van Moorselaar et al . , 2014 ) . Carlisle et al . ( 2011 ) then found ERP evidence for more than one simultaneous attentional template in visual working memory , as did Grubert and Eimer ( 2015 ) . Strong counter - evidence against the idea of a single active template was provided by Beck , Hollingworth , and Luck ( 2012 ) , who reported that observers can maintain more than one active visual working memory template . Their observers searched for a target among distractors , attempting to limit attention to objects of two colors , fi nding that observers switched gaze back and forth between the two colors with no switch costs , in contrast to single - template proposals . Perhaps the strongest evidence that observers can simultaneously maintain at least two active search templates comes from recent studies on human foraging ( e . g . Jóhannesson , Kristjánsson , & Thornton , 2017 ; Jóhannesson , Thornton , Smith , Chetverikov , & Kristjánsson , 2016 ; Kristjánsson et al . , 2014 ; Kristjánsson , Thornton , & Kristjánsson , 2016 ) . In Kristjánsson et al . ( 2014 ) , participants foraged , by tapping on the screen of an iPad , for 40 targets of two types ( e . g . red and green disks ) among 40 distractors of two di ff erent types ( e . g . blue and yellow disks ) . More than 95 % of observers switched freely between the two target types during foraging trials , without large costs . Another interesting fi nding was that when intertarget times ( times between successive taps , ITTs ) were compared between when the previous target was from the same category , or from a di ff erent category , the di ff erence in ITTs ( “ switch - cost ” ) was only around 50 ms ( Kristjánsson et al . , 2016 ) . In a recent unpublished study ( Ólafsdóttir , Gestsdóttir , & Kristjánsson , 2017 ) , such switch costs were almost non - existent , and are as low as 15 to 20 ms in other studies ( Jóhannesson et al . , 2016 ; Ólafsdóttir , Kristjánsson , Gestsdóttir , Jóhannesson , & Kristjánsson , 2016 ; see Grubert & Eimer , 2015 for related fi ndings ) . Also , in a study where observers foraged with eye gaze rather than fi ngers , the switch costs between target - types were essentially zero ( Jóhannesson et al . , 2016 ) . These results show that people can switch between target categories with seemingly little switch costs , an order of magnitude lower than the 250 – 300 ms suggested by Dombrowe et al . ( 2011 ) and therefore in - volve a challenge for single - template accounts , since they must then include a mechanism for rapid switching between templates . Another intriguing question is why participants performing these foraging tasks seemingly do not seem to care whether the next target they choose during foraging is from one target category or the other . The runs during foraging for two colored targets among two distractors are typically close to random ( Kristjánsson et al . , 2014 ) . If switch costs between templates are around 250 – 300 ms this would be an extremely ine ffi cient strategy . These fi ndings therefore seem highly discrepant with the idea of a single active template , which takes time to be re - placed . They appear to be more consistent with load theories of visual working memory that assume that working memory has limited capa - city , but do not place any constraints upon the nature of the WM re - presentations , but simply impose a capacity limit ( Alvarez & Cavanagh , 2004 ; Bays & Husain , 2008 ) . In fact , as load increased in Kristjánsson et al . ( 2014 ) and observers had to forage for 2 more complex “ con - junction ” targets ( e . g . red square and green disk targets among green square and red disk targets ) they changed their strategy , tending not to switch between targets ( Jóhannesson et al . , 2017 ; Kristjánsson et al . , 2014 ) . Observers seemed , in other words to maintain two simultaneous templates that involved simple features , but were unable , or unwilling , to maintain two more demanding conjunction templates . 1 . 2 . Current goals Our aim was to directly address the question whether more than one template can be simultaneously actively maintained in visual working memory . We therefore varied the number of targets and distractors in a visual foraging task for 40 targets among 40 distractors . We varied the number of target and distractor types in two ways : 1 ) Fixed distractor T . Kristjánsson , Á . Kristjánsson Acta Psychologica xxx ( xxxx ) xxx – xxx 2 \ No newline at end of file diff --git a/silver_data/a7c76b9d6eec2b78804ab26c9e48d0f532b353b8.pdf.txt b/silver_data/a7c76b9d6eec2b78804ab26c9e48d0f532b353b8.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..bb85156ff579d138be1e844b804d04b020c0903f --- /dev/null +++ b/silver_data/a7c76b9d6eec2b78804ab26c9e48d0f532b353b8.pdf.txt @@ -0,0 +1 @@ +City , University of London Institutional Repository Citation : Cacciatori , E . ( 2012 ) . Resolving Conflict in Problem - Solving : Systems of Artefacts in the Development of New Routines . Journal of Management Studies , 49 ( 8 ) , pp . 1559 - 1585 . doi : 10 . 1111 / j . 1467 - 6486 . 2012 . 01065 . x This is the accepted version of the paper . This version of the publication may differ from the final published version . Permanent repository link : http : / / openaccess . city . ac . uk / 16067 / Link to published version : http : / / dx . doi . org / 10 . 1111 / j . 1467 - 6486 . 2012 . 01065 . x Copyright and reuse : City Research Online aims to make research outputs of City , University of London available to a wider audience . Copyright and Moral Rights remain with the author ( s ) and / or copyright holders . URLs from City Research Online may be freely distributed and linked to . City Research Online : http : / / openaccess . city . ac . uk / publications @ city . ac . uk City Research Online 1 Resolving Conflict in Problem - Solving : Systems of Artefacts in the Development of New Routines Eugenia Cacciatori D - MTEC Swiss Federal Institute of Technology Zurich Scheuchzerstrasse 7 , 8092 Zurich , Switzerland Please cite as follows : Cacciatori , E . , 2012 . Resolving Conflict in Problem ‐ Solving : Systems of Artefacts in the Development of New Routines . Journal of Management Studies , 49 ( 8 ) , pp . 1559 - 1585 . Special Issue on the Micro - level Origins of Organizational Routines and Capabilities , T . Felin , N . Foss , T . Madsen and K . Heimeriks ( Eds . ) . 2 ABSTRACT : This paper argues that , in order to understand the development process of new routines , we have to look at the emergence of systems of artefacts rather than at individual artefacts in isolation . The paper proposes a typology of artefacts understood as material objects that are the product of human activity and analyses their interactions in the case of an integrated engineering design consultancy engaged in the effort of developing a new bidding routine . The evidence from the case study shows that agents reinforce and extend the patterns of action that individual artefacts support by bundling different types of artefacts , and that in so doing , they extend the reach and influence of the community to which they belong . This study shows that the problem - solving and truce aspects of routines are worked out in the design of these systems of artefacts . 3 INTRODUCTION Research in the structuration and pragmatic traditions has changed our understanding of routines , showing that the observable , repetitive patterns of behaviour in organizations are the effortful accomplishment of mindful individuals , rather than the mindless execution of fixed responses to given stimuli ( Feldman , 2000 , 2003 ; Feldman and Pentland , 2003 ; Howard - Grenville , 2005 ; Salvato , 2009 ; Rerup and Feldman , 2011 ; Turner and Fern , this issue ) . This work sheds light on the role of agency , or the capacity of individuals to form and achieve their goals ( Giddens , 1984 ; Leonardi , 2011 ) , in how routines persist and change over time . However , we know little about how new or radically different routines are developed ( cf . Zollo and Winter , 2002 ; Rerup and Feldman , 2011 ) , the role individual agency plays in their development ( Boisot and Li , 2005 ; Felin and Foss , 2005 ) or the part played by the development process in the translation of firm experience into capabilities ( Heimeriks , Duysters and Vanhaverbeke , 2007 ) . These are central issues that need to be addressed if , as much of the strategic and organizational literature holds , differences in routines drive different performance across firms . Structuration and pragmatic approaches suggest that to understand the development of new routines , we need to move beyond a disembodied view of the individual’s actions and cognitions to examine how agency occurs and is shaped by the artefactual context in which individuals operate . The role of artefacts - the material objects produced by human activity ( cf . Gagliardi , 1992 ; Rafaeli and Pratt , 2006 ) - in routines is controversial . Some authors emphasize their role in patterning organizational behaviour by giving a voice to some actors and not others ( e . g . , Cacciatori , 2008 ; Kaplan , 2011 ) and facilitating certain courses of action while making others more difficult ( Henderson , 1991 ; D ' Adderio , 2003 , 2008 ) . Others question the wisdom of relying on the design of artefacts in order to produce change in organizational processes ( Pentland and Feldman , 2008 ) . I take a step towards reconciling these positions arguing that the relationship between artefacts and routinization is clearer if we focus the analysis on systems of heterogeneous , interacting artefacts rather than individual artefacts in isolation . Not all artefacts influence behaviour in the same way or through the 4 same mechanisms . This paper develops a framework that classifies artefacts along two dimensions . The first considers whether or not the artefact embodies a formal representation of knowledge , such as text , drawings or mathematical formulas . The second classifies them on the basis of whether they are generic , or specific to occupations . I discuss the efforts of an engineering design firm to redesign its bidding process in response to the emergence of new integrated procurement routes . The in - depth case study focuses on an Excel Workbook used to estimate costs over the entire life cycle of a building ( including design , construction , maintenance and services ) . It describes the evolution of the workbook in relation to both the bidding routine it was meant to support and artefacts , such as drawings and procedures , that operate within that routine . I show how agents purposefully craft how an occupation - specific artefact ( such as the Excel Workbook used to estimate costs ) interacts with generic artefacts ( such as bidding procedures ) and other occupation - specific artefacts ( such as technical drawings ) . By bundling different types of artefacts , agents reinforce and extend the patterns of action that each supports and extend the influence of their particular communities to other occupational groups within the organization . Analysis of the systems of artefacts reveals the interplay between different organizational processes - aimed at solving problems and aimed at establishing positions of influence and power . The task of developing an Excel Workbook may seem trivial . However , in the case study firm it turned out to be otherwise , and the company was only marginally successful in restructuring its bidding process . I analyse the areas of partial success and failure showing that it is the interactions among heterogeneous artefacts ( as opposed to the individual features of specific artefacts ) , that explains their role in the process through which individual choices are ( or are not ) institutionalized into organizational routines . The paper is organized as follows . Section 2 develops an artefacts typology based on the literature on the role of artefacts in organizations . Sections 3 and 4 describe the empirical setting and the method . Section 5 presents the empirical evidence from the case study . Section 6 discusses the implications of the case and develops a model for the development of new routines which highlights the role of artefacts in mediating individual agency . Section 7 presents the conclusions . 5 MICRO - FOUNDING ROUTINES ON ARTEFACTS Debate on the microfoundations of routines ( Felin and Foss , 2005 , 2009 ; Hodgson , 2008 ; Salvato and Rerup , 2011 ) is part of a larger and long - standing debate on the prominence of individual agency or social structure in determining individuals’ behaviour ( e . g . , Reed 2003 ) . In this larger debate , structuration theory ( Giddens , 1984 ) seeks to integrate agency and structure , arguing that structure is ‘dual’ , that is , that social structure both shapes and is constituted by people’s practices , so that individuals can and do purposefully change it . Structuration theory and closely related pragmatic approaches ( Bourdieu , 1990 ) connect agency and structure and enable the development of solid microfoundations for the notion of routines . Several studies investigate how individuals ‘knowledgeably’ use the material and immaterial resources that constitute their practice , to maintain and change routines ( e . g . , D’Adderio , 2008 ; Howard - Grenville , 2005 ; Pentland and Rueter , 1994 ; Rerup and Feldman , 2011 ; Salvato , 2009 ; Turner and Fern , this issue ) . However , artefacts are rarely the main focus of analysis , and there is no comprehensive framework that explains how the material context of work matters for routines . At the same time , a growing body of research , particularly in the pragmatic tradition , has highlighted how human activity takes place through and is mediated by manmade material objects ( e . g . , Orlikowski , 1992 , 2007 ) . I propose a two - dimensional typology of artefacts that maps onto the ‘cognitive’ and ‘truce’ aspects of routines ( Nelson and Winter , 1982 ) . It builds primarily on studies in the structuration and pragmatic traditions , but also incorporates findings from work on knowledge codification ( e . g . , Cowan , David and Foray , 2000 ; Steinmueller , 2000 ) and the symbolic value of artefacts ( e . g . , Gagliardi , 1992 ) . The first dimension of the typology distinguishes between ‘speaking’ and ‘silent’ artefacts . The former contain formal representations of knowledge in verbal , mathematical or visual form . Since the formal representation of knowledge is fundamental to its manipulation , this dimension facilitates examination of the role of artefacts in the problem - solving , cognitive aspect of routines . The second dimension distinguishes between generic artefacts and those associated with specific occupations . The trend towards multitechnology products and greater specialization ( e . g . , Granstrand , Patel and Pavitt , 1997 ) implies an 6 increasing need for collaboration among specialists . Such collaborations can be problematic due to different ways of framing problems and representing knowledge ( e . g . , Dearborn and Simon , 1958 ; Dougherty , 1992 ) . Beyond these cognitive difficulties , occupations struggle for dominance , as extensively documented in the sociology of work and professions , and some studies have begun to investigate how artefacts contribute to this struggle ( Bechky , 2003a ) . Classifying artefacts as generic or occupation specific enables analysis of their role in organizational conflict . Figure 1 provides a classification of artefacts based on these two dimensions ; the case study shows how these two dimensions interact in the process of routine development . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - INSERT FIGURE 1 ABOUT HERE - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Speaking artefacts Speaking artefacts contain textual or visual representations of knowledge . They include procedures , manuals , reports , technical drawings and virtual prototypes – which last typically combine visual and textual information in complex ways . Formal representations facilitate the manipulation of knowledge , and speaking artefacts ( e . g . , virtual prototypes ) afford easier manipulation of the knowledge they embody than do ‘silent’ artefacts ( e . g . , clay models ) . The manipulation of formal representations of knowledge is generative in the sense that it contributes to the creation of new knowledge ( Simon , 1996 ; Foray and Steinmueller , 2003 ) . The clearest example of this is in physics , where the manipulation of mathematical representations of the world led to the ‘discovery’ of phenomena whose experimental demonstration followed much later . In the context of organizations , the introduction of virtual modelling and simulation has enabled faster development of better products ( Thomke , 1998 ; Nightingale , 2000 ) . Two types of speaking artefacts have attracted particular attention : product representations , which include sketches , technical drawings and their digitalized counterparts , virtual prototypes ; and process representations , 7 which include procedures and checklists . I examine these two types of speaking artefacts separately because process representations have more direct and more studied relationships with routines . Much of the recent work on the material aspects of organizing focuses on representations of products and studies their role in mediating the joint problem - solving activities of different specialists . Product representations are examined in their function as ‘boundary objects’ . Boundary objects ( Star and Griesemer , 1989 ; Carlile , 2002 , 2004 ) are artefacts that can adapt to different contexts while simultaneously maintaining a common identity across contexts . By combing the flexibility to adapt to the needs of specific groups in ‘local’ use with rigidity to maintain consistency across sites for ‘global’ use , boundary objects enable groups with different practices and ways of framing problems to collaborate , while preserving the advantages of specialization . i While studies of boundary objects are aimed at investigating collaborative problem - solving rather than routines , they show that product representations influence the organizational processes they support . What is represented and how , determines the way the problem is constructed , and who will have a voice in solving it and at what stage ( D ' Adderio , 2003 ; Cacciatori , 2008 ; Kaplan , 2011 ; Orlikowski , 1992 ) , thereby providing ‘paths of least resistance’ that funnel organizational action ( D’Adderio , 2003 ) . The strength of this funnelling is a function of the way in which knowledge is represented . Effective problem - solving across different occupational groups requires flexible representations that can be adapted to the needs of different specialists in local use , that are easy to manipulate and that evolve in parallel with the problem - solving activity ( Bowker and Star , 1999 ; Carlile , 2004 ) . In this respect , Ewenstein and Whyte ( 2009 ) building on Knorr - Cetina ( 1999 ) prefer to distinguish between evolving ‘epistemic objects’ that leave opportunity in their structure for what is not yet known , rather than ( static ) ‘boundary objects’ . Alternatively , if the artefact per se is not sufficiently flexible , there must be scope for each occupational group to develop ‘translating’ mechanisms , that make it possible to go back and forth between different local , group - specific representations and global representations ( D’Adderio , 2001 ) . On the contrary , rigid representations are required to make distributed action converge to specific patterns . For instance , the rigidity of representations in technical drawings compared to sketches allows 8 the former to support the coordination of production activities between design and manufacturing , making them work as ‘conscription devices’ that organize distributed activities ( Henderson , 1991 ) . Organization members often need to choose between changing the artefact or changing their behaviour : the choice tends to depend on the modifiability of the artefact ( Leonardi , 2011 ) . The flexibility that makes a product representation effective for problem - solving often makes it ineffective for funnelling action towards desired sequences , and vice versa ( Henderson , 1991 ) . The differential ability of product representations to funnel action combined with the patterning ability of artefacts being limited to the actions they support ( Pentland and Feldman , 2008 ) , points to the importance of looking beyond individual artefacts to understand their role in routinization . The role of artefacts in routines has been investigated more extensively in relation to representations of processes , and especially procedures . Procedures are representations of an intended ( by some organizational actor ) routine and , therefore , are a reification of the ostensive dimension of routines ( Feldman and Pentland , 2003 ) . The relationship between procedures as representation of routines , and the routine as performed is never one to one because procedures can never fully specify action ( Pentland and Feldman , 2005 ) . Procedures are better understood as one of the resources that individuals bring to a situation in order to deal with it ( Suchman , 1987 ; Turner and Rindova , 2011 ) . D ' Adderio ( 2008 ) shows that software embedded procedures and actual behaviour go through cycles of convergence and divergence , with procedures and behaviour alternately taking the lead . Again , research on the importance of the type of representation has produced interesting and counterintuitive results . Detailed representations of processes may be used to increase accountability and constrain the actions of actors . However , they also enable the actors to understand the purpose and functioning of the system and , therefore , to devise solutions to the problems they encounter , and can so favour deviation from a rigid pattern ( Adler and Borys , 1996 ; Narduzzo , Rocco and Warglien , 2000 ) . 9 Silent artefacts Silent artefacts , which include items such as furniture , clothing and tools - ranging from hammers to stethoscopes - do not contain textual or visual representations of knowledge . They constitute the majority of the artefacts in our world . They embody and make knowledge available for use , most notably in tools and equipment , but in ways that are less directly manipulable than is possible with speaking artefacts . Their role in distributed problem solving is important , but more limited . Bechky ( 2003b ) argues that , in the context of inter - occupational problem - solving , silent artefacts work primarily by providing grounding , that is , by providing a way of cross checking meanings across occupations . Again in the context of inter - occupational collaboration , research suggests that the primary role of silent artefacts may be that of being the objective of the collaboration , as in the case of the development of a new product , in this way acting a motivators of collaboration ( Nicolini , Menger and Swan , 2011 ) . Silent artefacts embody and make available knowledge about how to carry out a task through their action affordances , that is , the actions suggested by their shape . For instance , a handle shape can suggest a lifting movement ( a jug handle ) or a downward pressure ( a door handle ) . Evidence from psychology suggests that rather than reasoning about artefacts , we associate with them experience - based action possibilities ( Cohen , 2010 ) . These associations guide most of our use of everyday objects and are at the centre of a vast field of design ( e . g . Norman , 1988 ) and information systems ( Leonardi , 2011 ) . In suggesting the actions required to achieve a goal through their action affordances , silent artefacts make certain courses of action more likely than others , thereby contributing to patterned behaviour . Bapuji and Saeed ( this issue ) provide empirical evidence of this by showing that introducing silent artefacts with more direct affordances to desired actions considerably strengthens the stability of routines . Artefacts specific to occupations Artefacts specific to occupations constitute the ‘tools of the trade’ of an occupation or a group of closely related occupations . Examples of specific silent artefacts include the stethoscope used by medical practitioners , the scalpel used by surgeons and the compass used by engineers . Examples of speaking 10 artefacts specific to occupations include accounts ledgers , and the technical drawings used by engineers and architects . The association between these artefacts and specific occupations is not necessarily one to one : both artefacts and occupations evolve . Artefacts may be developed for a specific occupation and spread to similar occupations . Many occupations are becoming increasingly specialized , with the result that occupations that ‘branch out’ share some of the tools of the originating occupation . Nevertheless , these artefacts remain sufficiently specific and essential in an instrumental sense ( e . g . , the accountant cannot work without a ledger ) , that they often represent it symbolically ( cf . Fiol and O’Connor , 2006 ; Vilnai - Yavets and Rafaeli , 2006 ) . If we look at artefacts as the essential tools of an occupation , their potential to generate conflict becomes apparent . Studies of technical change document that , by embodying and blackboxing knowledge , new equipment can move the locus of knowledge control , or make the knowledge held by certain groups obsolete or irrelevant , sparking conflict in the process . A prominent example of this is the conflict that ensued between management and workers as a consequence of the innovations in technique and equipment introduced by Taylor and Ford . In one of the few studies that investigate the ‘truce’ aspect of routines , Coriat and Dosi ( 1998 ) show the different routines that emerged in the west and in Japan , in order to achieve truce . Similarly , Barley’s ( 1986 ) famous study of the introduction of computed tomography on the social organization of hospitals and Black et al . ’s ( 2004 ) extension of that study using a simulation model , show how new , occupationally specific artefacts produced by technical change can alter divisions of labour and threaten established authority and status systems . Knowledge codification , which changes who can use knowledge and how , is often an important part of the process ( Bowker and Star , 1999 ; D’Adderio , 2003 ; Lazaric and Denise , 2005 ; Lazaric , Mangolte and Massué , 2003 ) and suggest that speaking artefacts that are occupation specific are likely to play a critical role in both problem solving and conflict . Work on professions is exploring the role of occupation - specific artefacts not linked to technological innovation in interoccupational conflict . Occupational groups strive to achieve professional status , understood as the ability to claim exclusive jurisdiction over a specific set of tasks . ‘Adjacent’ 11 occupations , that is , occupations in closely related task domains ( e . g . , doctors and nurses ) , can come into conflict over attempts to invade one another’s authority , or establish one - way dependence ( Abbot , 1988 ) . Authority over artefacts can reinforce or alter the boundaries between the tasks performed by specific groups ; for instance , in the process of producing a new machine , designers use drawings and technicians use physical prototypes to assert and enforce their individual authority ( Bechky , 2003a ) . Artefacts generic to occupations Artefacts generic to occupations are used by the members of an organization independent of their specific occupation . Office furniture is an example of a generic , silent , occupational artefact . Among speaking artefacts , organizational procedures , discussed above , are typically generic to occupations – although the definition of the procedures for carrying out a task ( e . g . , a medical protocol ) may be occupation specific . An important mechanism through which silent artefacts generic to occupations contribute to patterned action , is their value as symbols , an aspect that is explored extensively in cultural studies of organizations ( e . g . Gagliardi , 1992 ; Rafaeli and Vilnai - Yavetz , 2004 ; Heracleus and Jacobs , 2008 ) . Artefacts embody and display a particular set of values , suggesting courses of action that are coherent with the values of the organization . For instance , an ‘open door’ office policy encourages work colleagues to drop in on one another . Thus , artefacts with a strong symbolic dimension have a normative value and can be seen as part of the ostensive aspect of an organizational routine ( Pentland and Feldman , 2008 ) . However , symbolic messages need to be interpreted in order to affect behaviour , so that symbolic function of generic silent artefacts provides less stringent patterning than affordances of action . Artefacts as systems Research on routines is beginning to highlight the need to focus on the relationship among a range of different artefacts . D’Adderio ( 2008 ) refers to the need to look at how procedures enrol human and material actors , in order to study their stability . In a study of roadmapping routines , Howard - Grenville ( 2005 ) argues that routines that make use of reinforcing rather than competing sets of artefacts may be 12 more persistent . Research in several fields provide suggestions on how artefacts may work as systems and reinforce one another . Research into distributed cognition shows that coordinated action ( including distributed problem - solving ) relies on interdependent artefacts . For instance , a ship’s position is determined through a set of coordinated activities mediated by the ‘propagation of representational states across a series of representational media’ ( e . g . , photometer , sighting telescopes , navigation charts ) ( Hutchins , 1995 ) . Similarly , the revolutions in the organization of production , introduced by Taylor and Ford , would have been impossible without systems of supporting artefacts , including standardized parts . Studies of organizational memory show that individuals and groups rely on a variety of objects to support their recall efforts , and that organizational memory is stored in many distributed , complementing and partly overlapping artefacts which individuals combine to suit their needs ( Ackerman and Halverson , 2004 ; Cacciatori , 2008 ) . While these contributions document the interdependencies among the artefacts supporting human activities , they treat all artefacts as equally important . Building on a variety of theoretical approaches to the role of materiality in cross - disciplinary collaboration , Nicolini , Mengis and Swan ( 2011 ) propose a hierarchical view of artefacts . Collaboration across disciplinary boundaries is enabled by a set of taken - for - granted artefacts that remain in the background , but provide the infrastructure for other artefacts working as boundary objects to facilitate collaboration . Motivation and emotional binding among participants is instead provided by the artefacts whose development is the objective of collaboration . Social studies of science provide important insights into how artefacts can be arranged in ways that are mutually reinforcing , using the notion of ‘standardized packages’ ( Fujimura , 1992 ) . Fujimura is interested in ideas , rather than stable patterns of behaviour and in ‘facts’ becoming ‘stabilized’ , or accepted as such in science . Fujimura ( 1992 ) argues that , because of their high reconfigurability in local settings , boundary objects enable collaboration , but make fact stabilization more difficult . Boundary objects are so plastic that they allow the creation and maintenance of divergent interpretations and could serve competing scientific theories in their efforts to stabilize their own facts . Fujimura argues that to enable a particular 13 theory to dominate , collaboration across scientific communities requires a ‘standardized package’ , i . e . a ‘ gray box which combines several boundary objects [ … ] with standardised methods [ … ] in ways which further restrict and define each . Such codefinition and corestriction narrows the range of possible actions and practices but does not entirely defines them’ ( Fujimura , 1992 , p . 169 - 170 , emphasis in original ) . In Fujimura’s approach , the key boundary objects in a standardized package are immaterial objects , that is , concepts . The significance of a standardized package is its ability to provide concepts that are sufficiently general and plastic ( and , therefore , somewhat imprecise ) to be adaptable to local concerns , alongside standardized methodologies that change local practice to the extent that its results reinforce the theory . The categories developed here facilitate the analysis of the role of artefacts in the development of new routines . In order to lay the ground for the empirical exploration of how different artefacts perform in systems in relation to routinization , the next section introduce the settings of the case study . RESEARCH SETTING This paper is based on qualitative , situated research conducted as part of a three - year , in - depth case study of a large British engineering consulting and support services provider firm operating in the British building industry . The British building industry has undergone important organizational changes ( Cacciatori and Jacobides 2005 ) , two of which are particularly relevant for this study . The first is the diffusion of integrated procurement routes , including a public procurement scheme known as the Private Finance Initiative ( PFI ) , in which a single firm wins a contract covering the phases of a facility’s life cycle , from financing to maintenance , for a period of 25 to 30 years . By bundling together previously , rigidly separated procurement phases , PFI provides strong incentives for firms to integrate the knowledge bases of the construction industry occupations in order to reduce costs and provide a better service . Therefore , this industry provides an excellent setting for an examination of the development of the new organizational routines through which this integration is achieved . 14 The second noteworthy feature of this industry is the intense occupational competition among the traditional professions ( architects , engineers , quantity surveyors ) , general contractors and facilities managers . Among western countries , the cost consulting profession of quantity surveying is unique to the UK . The quantity surveyor’s remit in traditional , specialized procurement is to monitor the building contractor by estimating the cost of construction through the ‘Bill of Quantities’ ( a list of materials to be used ) . The services offered by the actors in the building industry increasingly are overlapping ( Cacciatori and Jacobides 2005 ) , and reports from professional bodies and in the trade press indicate a full - blown ‘jurisdictional war’ ( Abbott , 1988 ) . Against this background , the case study company , Design Engineering & Facilities Management ( DE & FM ) , opted to reposition itself as an ‘integrated services provider’ ii ( see Davies , 2003 ) . This involved a continuous acquisition campaign in the late 1980s and through most of the 1990s , so that by the end of the 1990s DE & FM was operating in project financing , in all engineering fields and in architecture , in cost consulting , and in the growing facilities management market . The case study focuses on DE & FM’s efforts to develop a new bidding routine for integrated PFI school projects . METHODS Within - company sampling and data gathering During the preliminary , exploratory fieldwork , I collected information on the company , its history and its politics , and identified the bidding process as the focus of the company’s innovation efforts . I then concentrated on bidding and conducted 26 interviews with individuals involved in bidding for PFI and other projects . Interviewees included directors of the various business units , project managers , technical staff at different levels of seniority ( engineers and architects ) , and support staff involved in managing the information to support bidding . I gathered as much documentation on the bidding process ( procedures , checklists , samples of bids submitted ) as possible , in order to familiarize myself with the issue and identify specific units for detailed analysis . During this phase , I identified bidding for PFI projects as the 15 area involving the most complex and most vigorously pursued efforts to develop new routines . I also noted the attention devoted to whole - life costing tools within the bidding process . In the final phase of the fieldwork , I reconstructed the evolution of whole - life costing tools and the PFI bidding process , through direct observation and information from 14 more interviews including 7 with the three cost consultants responsible for the development of whole - life costing and 7 in other parts of the company , including DE & FM Invest , the Design Division and the Facilities Management Division . I attended three meetings between representatives of the company and the whole - life costing system developers and other actors , and searched internal company documentation ( e . g . manuals , presentations , the company Intranet ) . The company ‘tutor’ ( who acted as my main contact in the company ) helped to identify the first three interviewees for my enquiry into whole - life costing ; the rest were selected using a combination of theoretical sampling and snowballing , that is , I asked interviewees to refer me to ( a ) people in other parts of the company directly involved in the development of the tool ( key informants ) and ( b ) people likely to offer alternative , controversial views . The interviews provided competing accounts of the development , role and aims of the tools being developed , which ensured multivocality . One interview , with the main developer of the whole - life costing tool , was conducted one year after the end of the main fieldwork in order to get an update on the state of the company . In addition to gathering information on the interviewee’s background , current position and role in bidding , the interviews were aimed at finding out what the interviewees considered to be important , and their perspectives on these issues . I followed Dougherty ( 1992 , p . 184 ) and conducted interviews that were ‘structured around [ … ] general questions , but unstructured regarding what the person chose to emphasize’ . The general questions varied over the phases of the fieldwork and depending on the interviewees’ specific expertise or roles . Parts of the four interviews with the main developer of the whole - life costing tools followed a different pattern and were directed to understanding the tools and their intended role in the process . I discontinued the interviewing when I reached what Glaser and Strauss ( 1967 ) call theoretical saturation , that is , when the information being provided was helpful for understanding the process , but was adding no new theoretical categories or critical issues . 16 Since the interview topics were often a source of organizational conflict , I decided not to tape the interviews to ensure that interviewees would be more open ( Rubin and Rubin , 2005 ) . My interview notes were ordered and written up as soon as possible after the interview and combined with the other material collected . When factual information was an important part of the interview ( e . g . , when reconstructing the corporate initiative on PFI bidding ) , I sent summaries to the interviewees for verification . The confidentiality agreement I had signed with the company allowed access to internal documentation related to the whole - life costing tools ( e . g . manuals , presentations , company material available on the company’s Intranet ) , a printed copy of the first version of the tool for a specific project ( whose interpretation I checked with interviewees ) , on - site access to the modified ( actual and intended ) Life Cycle Replacement ( LCR ) Model and the Service Life Planner ( SLP ) , and on - site access to a set of whole - life costing tools developed by the cost consulting division , not discussed in this paper . I wrote reports outlining the functionalities and data sources of the tools ( actual and intended ) , which featured tables of their intended used within the bidding process . The accuracy of these reports was checked by the cost consultant in charge of the project . I was also given full access to the new procedures . Data analysis I used a ‘temporal bracketing’ strategy to analyse the material collected during the fieldwork in order to develop a process model of routine evolution . Temporal bracketing enables the handling of heterogeneous data , and is coherent with the structuration lens of the study ( Langley , 1999 ) . By adopting this strategy , I was able to analyse the eclectic material at my disposal ( company archival material , interviews , the whole life costing tools themselves , meeting notes , and grey literature on the evolution of the British construction industry ) and to identify three periods in the evolution of the PFI bidding process . During the first period , the company used variations of its existing bidding routines . This period makes it possible to identify the resources that structures make available to agents . During the second period , agents used the resources at their disposal to introduce a new bidding process . In this period is therefore possible to examine how agency takes place . During the third period , the company attempted to implement the new 17 process , with very limited success – making it possible to compare and contrast areas of success and failure . I conducted iterative data analysis , involving re - reading and cross - examination of my interview notes , and comparison between the accounts given by interviewees and the story that emerged from the whole - life costing tools themselves and their development . This enabled the identification of common patterns and points of contrast . Actors’ accounts of unfolding events are important because they provide contrasting interpretations and stated motivations , which contribute to constructing the practice . I used a very simple form of thematic coding to highlight the differences in actors’ perspectives and identify typical statements , recurring issues and common terms ( Flick , 2002 ) . The interviews on the whole - life costing tools provided statements that were revealing about the interviewees’ understanding of what the process entailed and its relevant costs . I analysed the data as they were gathered , and returned to them repeatedly as the fieldwork progressed , to compare my analysis new insights . I cross - checked accounts using three different means ( Miles and Huberman , 1994 ) . First , I contrasted and compared interviews , highlighting discrepancies which were investigated through follow up questions or , where relevant , addressed in successive interviews . Second , I conducted a detailed examination of the artefacts supporting the practice of bidding . As much research in this area shows ( e . g . , Bechky , 2003 ; Bowker and Star , 1999 ) , the ‘voices’ of the objects and the stories told by their structure and characteristics are important counterpoints to interviewees’ stated motivations and aims . Third , I gathered extensive documentation from academic work and trade press on the evolution of the British construction sector . This enabled me to map correspondences and divergences in the strategic choices of professions and professional firms in the industry , against my findings in DE & FM . I stopped the process of triangulation when discrepancies were resolved or if I found evidence of persistent difference in the accounts . This enabled me to develop an articulated understanding or ‘theory’ through which I could read and reconcile the empirical phenomena I was observing , and allowed me to identify gaps and shortcomings in the conceptual schemes developed so far to account for the dynamics of the development of routines ( Eisenhardt , 1989 ) . 18 THE CASE : BIDDING FOR PFI PROJECTS IN DE & FM Producing a bid for a PFI school project required collaboration among four organizational units : DE & FM Invest , which reported directly to the CEO and represented DE & FM as the investor ; the Design Division ( housing architects and engineers ) ; the Facilities Management ( FM ) Division ; and the Cost Consulting Division ( housing the firm’s quantity surveyors ) . With the exception of the Design Division , which operated as subcontractor to the construction contractor , these organizational units were linked through individual contracts to the ‘project company’ , the legal entity created to undertake each PFI project . Efforts to develop a new bidding routine for PFI school projects took place in three phases . The first was a period of emergent change and local adaptation of traditional design routines . In the second phase , incremental changes were perceived as inadequate ; a corporate initiative was launched to develop a new bidding process and new tools and procedures were developed to support the new bidding process . In the third phase the new tools and procedures were implemented . Phase one : adapting routines and stretching artefacts DE & FM organizations members tried to adapt existing bidding routines to the different demands of PFI projects . This entailed the ‘stretching’ of artefacts used in existing routines to support novel patterns of interaction . This is apparent in the case of product representations , sketches and drawings . In preparing a PFI bid , contractors , subcontractors and facilities managers are required to give feedback on design choices at an early stage in the bidding process , the so called ‘conceptual’ stage , on the basis of sketches or , at most , conceptual drawings . However , they required detailed drawings , normally produced much later , in order to give this feedback : We keep having a lot of trouble with contractors . You see , they are used to preparing their bid on the basis of detailed design and want us to complete the design so that they can calculate construction costs . However , they only pay our variable costs for bidding activities . [ Assistant Bid Manager - DE & FM Invest ] . 19 As a result , designers were receiving feedback at the end of the bid preparation process , when time pressures were great and when changes to design were difficult and costly , and could introduce problems in other areas . For instance , the choice of a different type of fire system could involve different health and safety requirements . The difficulty of giving and receiving feedback based on drawings was combined with uncertainty ( and attendant experimentation ) about how to divide tasks among the different groups . The Director of DE & FM Invest – School Unit emphasized that setting up a bidding process meant identifying and addressing the numerous and unfamiliar interdependencies among project components . In interview , he gave examples of how new ways of dividing labour were needed for PFI projects to avoid the bidding process grinding to a halt . New arrangements were worked out during the course of every project , contributing to the feeling that the process was unstable and that bid meetings , during which key decisions on the content of the bid should have been made , were ineffective . An assistant bid manager said that : There are just too many people in the room and you cannot get anything done . We had one of these meetings just recently on the … bid . Between designers , facilities managers , cost consultants , the contractor and a few subcontractors we were about 20 – everybody was happy that they could come , but we managed to decide very little and it cost a lot of money [ DE & FM Assistant PFI Bid Manager ] . Since sketches and drawings were perceived as insufficient to structure the process in the new context , new artefacts were developed – including representations of processes generic to occupations ( i . e . , checklists ) and occupation - specific product representations ( e . g . , FM Division design requirement manuals ) . One of the new speaking artefacts was an Excel Workbook developed to estimate the total cost of a building over its lifetime . There is often a trade off between capital ( i . e . , construction ) costs and maintenance costs . For instance , a cheaper roof may require more frequent maintenance . Design decisions related to reducing total costs must be informed by these trade - offs . The Excel Workbook , or LCR Model , was a new cost estimating tool developed to address these trade - offs . It was physically located on the 20 laptop of the quantity surveyor assigned to PFI projects . The structure of the LCR Model and its links to both occupational groups and tasks are shown in Figure 2 . The solid lines identify the sections of the tool for which each organizational unit was responsible . The first section , developed by the Cost Consulting Division , estimated Capital Cost and included periodic replacement of capital items such as elevators , heating system , etc . ( i . e . , life cycle replacement costs ) . The second section , developed by the FM Division , estimated other Occupancy Costs – that is , the costs of using the building , in particular repairs , and costs such as cleaning , catering , etc . The third section , the responsibility of DE & FM Invest and not discussed here , estimated Tax and Financial Costs , and the fourth section calculated the total costs of the building over its lifetime . The dotted line separates the parts of the tools used by the different organizational units ( shown in ovals ) to carry out their tasks . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FIGURE 2 ABOUT HERE - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The Excel Workbook was a new boundary object , developed to complement the inter - occupational problem - solving taking place around drawings with cost information . Like many boundary objects , it was a patchwork representing the needs and contributions of different occupational groups ( cf . Bowker and Star , 1999 ) . Figure 2 shows that its structure reflected the boundaries between occupations and organizational units ( cf . Cacciatori , 2008 ) . It also showed a mismatch . Life cycle replacement costs are costs sustained during occupancy , not construction . They become the responsibility of the FM Division once the school is built . However , the quantity surveyors included them in their section on the basis that they were ‘just deferred capital costs’ . The mismatched structure of the LCR Model in relation to the life cycle replacement costs highlights the conflict between quantity surveyors and facilities managers over the estimation of maintenance costs . This conflict will come to fruition in Phase 3 . 21 Phase 2 : engineering systems of artefacts The way that the LCR Model represented knowledge complicated its use as boundary object . The LCR Model was too flexible : it supported local , occupation - specific representation , but did not allow reintegration into a global representation because of the different ways in which costs were expressed . Capital and replacement costs were expressed through quantity surveyors’ traditional building elements ( e . g . , roof , foundations , etc . ) , and maintenance costs were expressed as types ( e . g . , utilities , security , repairs and maintenance ) and in cost per square metre . This did not allow easy comparison among different design choices in terms of total costs . There were also problems with how each occupation - specific representation worked in terms of ease of manipulation . The LCR Model provided alternative specifications for building elements in piecemeal fashion . Also , changes to design specifications had to be copied manually into worksheets , making it difficult to compare configurations and identify the changes that had the greatest impact on costs ( see Comments box in Figure 2 ) . Following serious delays in the preparation of two consecutive draft bids for PFI school projects , the Cost Consulting Division and the corporate R & D Unit were given funding to work on improvements to whole - life cost estimation and to develop new bidding procedures . The modified LCR Model Mark , a then recently hired cost consultant in the Cost Consulting Division , was assigned to improving the whole - life costing tools . The changes he introduced are depicted in Figure 3 ( intended ) . He increased the global representation capabilities of the LCR by developing a new , occupation specific product representation , the SLP . The SLP was an Access database , in which the building elements used in the cost consultants’ classifications were extended , with some adaptation , to include facilities management costs . In Mark’s plan , data on maintenance costs would be provided by the FM Division , which potentially could estimate very accurate maintenance costs using the software that was used to dispatch repair contractors and handled billing . The SLP included several options for each building element , and changes made in one section were automatically applied to the other sections ( see Comments box in Figure 3 ) . The 22 SLP linked capital and life cycle replacement costs to other maintenance costs . However , because it required facilities managers to stop expressing their costs as costs per square metre , adaptability to local use decreased . The LCR Model and the SLP were linked . The LCR Model incorporated a copy of the SLP , so that it was possible to select building elements from a drop - down menu . This was done so that the SLP could be maintained and updated centrally , while the LCR Model was used in projects . The left - hand side of Figure 3 shows the intended new configuration of the tool . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FIGURE 3 ABOUT HERE - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Routinized behaviour relies on connections among individuals ( Feldman and Rafaeli , 2000 ; Turner and Rindova , 2011 ) , which , in turn , reflect the communications paths required to deal with the interdependencies among different parts of the product ( Henderson and Clark 1990 ) . Mark’s work on the LCR Model and the SLP shows how agents purposefully craft product representations and their connections in order to shape the communication patterns that make up routines . Mark configured the structure of the LCR Model , its relationships with the new SLP and , potentially , with the FM Division’s operational software , according to his view of the appropriate way to represent costs and the appropriate flow of information , which differed from that of facilities managers’ . In describing their method of costing buildings , cost consultants made comparisons with the approach of facilities managers : Quantity surveyors bring to the process the ability to prioritize … . FM people have a contractor not a consulting frame of mind . They provide , not optimize . [ Cost Consulting Division , Director ] Cost consultants have what could be termed a “function oriented” way of thinking about costs , in which costs depend on technical specifications of building elements , decided on the basis of the functions required of a particular area of the building ( e . g . , front vs . back office ) . The structure of the LCR Model and SLP reflected this understanding of costs , as opposed to the more operational understanding of facilities managers – that focused on ease of maintenance . 23 Development of procedures The development of new , occupation - specific product representations was complemented by the development of new generic process representations in the form of bidding procedures . The new bidding procedures were developed with input from Mark related to the newly introduced ‘value engineering meetings’ . The focus of these meetings was analysis of the interdependencies between design , construction and facilities management costs . They were chaired by a cost consultant ( bid meeting were chaired by the bid manager , and design meetings were chaired by a designer ) , and were held before the other meetings . The new procedures introduced two new organizational roles dedicated to the estimation of whole - life costs : an advisor to the construction , design and facilities management teams who also coordinated the value engineering meetings ; an advisor to the project company , a role similar to the traditional role of quantity surveyor . The development of procedures illustrates the complementarities among product and process representations in the strategies used by actors to pattern organizational behaviour . Identification of the important product features and their interdependencies through the LCR Model and SLP is implicitly normative since it suggests a particular division of labour and gives voice to certain concerns ( e . g . , costs ) and not others ( e . g . , value creation in terms of quality of teaching environment ) . As suggested by Pentland and Feldman ( 2008 ) , the LCR Model and the SLP , on their own can have only a limited influence in the sense that they can only ‘enable and constrain’ the actions that take place through them . Indeed , they only succeeded in locally patterning actions in the bidding process in Phase 3 . The division of labour implicit in each product representation needs to be reinforced and extended , which is achieved by bundling them with organizational procedures . These procedures match activities to roles , further clarify the patterns of interdependence and communication among roles , and provide a temporal sequencing of activities . Thus , product representations that work as boundary objects and process representations , act in mutually reinforcing way similar to the complementarity between concepts and methods in science ( cf . Fujimura , 1992 ) . This combination helps in addressing the trade - off in product 24 representations between the flexibility needed for problem - solving and the rigidity necessary to funnel action . Flexible product representations when combined with procedures can , as a system , exhibit sufficient rigidity to funnel action and sufficient flexibility to enable problem - solving . Phase 3 : occupation specific artefacts and organizational conflict While Phases 1 and 2 relate primarily to the role of speaking artefacts and how they represent knowledge , Phase 3 highlights the important occupation - specific nature of the artefacts . The new group of artefacts , consisting of the LCR , the SLP , and the procedures , was not adopted in the way Mark had envisaged . Mark incorporated into the LCR Model only the SLP sections related to capital and life cycle replacement costs ( see Figure 3 – Actual ) . Because of the significant resistance from the FM Division to the new procedures and its refusal to provide data on maintenance costs , Mark continued to use the facilities management classification for other facilities management costs . The interviews show that senior FM Division staff were sceptical about the new procedures and questioned the cost consultants’ contribution . An executive in the FM Division said : The QS Division consultants are very expensive . They want our data to sell them back to us , so we are starting to develop our cost model . Because Mark did not have information on what cost data the FM Division held , and was aware of the tensions within the company , he decided to continue using the original FM Division components to prevent further friction . This partially modified version of the LCR Model reduced some of the problems , and addressed the need for a flexible representation by providing the possibility to choose building elements and to update the worksheets automatically . At the same time , it required ( and obtained ) minimum collaboration from the FM Division . Mark began to use the new SLP to facilitate discussion of different design options in the value engineering meetings at the early ‘conceptual design’ stage . He commented that : Architects’ rush to produce drawings is counterproductive really . A first discussion should focus on general approaches to optimize whole life costing through the use of the SLP and the facilitation of a cost consultant . 25 A few interviewees referred to the more positive interactions during value engineering meetings , and that they were a result of the SLP and modified LCR Model and Mark’s facilitation . However , designers often did not show up at presentations on the new whole - life costing tools , and Mark was not always notified about the design and value engineering meetings which , according to the new procedures , he should be facilitating . An interview with Mark a year after the main fieldwork revealed that the situation had not changed . Therefore , while the SLP facilitated a degree of restructuring of the bidding process in relation to the conceptual design phase , and both the SLP and the ‘actual’ version of the modified LCR Model improved problem - solving , the more integrated tools were not adopted and the bidding procedures were largely ignored , so that a new overall bidding routine did not develop . Why did Mark’s efforts have only limited success in reshaping DE & FM PFI bidding routine ? Looking at the occupation specific nature of the systems of artefacts that should have supported the new routine helps to answer this question . The LCR Model and the SLP evolved from the quantity surveyors’ occupation specific tools . Although they incorporated representations of other occupations and were used as boundary objects , they were physically controlled by the quantity surveyors . Quantity surveyors had them on their laptops . They were used and manipulated during cross - occupation meetings by the quantity surveyors . By crafting the way they represented the product and worked as a system with procedures and drawings , the quantity surveyors attempted to stabilize patterns of actions that gave them control over a larger set of activities , at the expense of both facilities managers and designers . An artefact specific to an occupation ( the LCR Model ) was combined with a generic artefact ( procedures , which introduced roles associated with the artefacts ) to extend the reach of the sponsoring occupation within the overall PFI bidding process . This combination enabled the development of new tasks and new roles , which were bundled with the object and brought new activities within control of the originating group . Continued ownership of the tool by quantity surveyors was crucial because new roles as described in procedures are justified on the basis of mastery of the new tool , and because the activities carried out by other occupations are represented in the tool , which enables later claims for full responsibility of the activities . 26 In this way , quantity surveyors aimed at appropriating cost estimation tasks related to life cycle replacement and maintenance that were the contractual responsibility of facilities managers . At the conceptual design stage , the new tools were intended partially to displace drawings and , therefore , designers . This is evident in Mark’s negative evaluation of architects’ ‘rush’ to produce drawings , the introduction of value engineering meetings facilitated by a cost consultant at the conceptual design stage , and the development of two new roles connected with whole - life costing estimation . Beyond extending quantity surveyors control over a lager span of the bidding process , the way in which the SLP and its relationship with the modified LCR Model were structured implies an important change in the control over the knowledge accumulation underpinning consulting on whole - life costing . By relying on heterogeneous classifications , the original LCR Model accessed data from the various communities . However , the databases and expertise that were the source of these data remained under the control of each occupation . The introduction of the SLP changed this . The design of the LCR Model in combination with the SLP effectively decoupled maintenance and the updating of occupancy information , from application of the tool in specific projects . This separation allowed the cost consultants to accumulate expertise in whole - life costing , a profitable service and a ‘contested terrain’ ( Abbott , 1988 ) in the industry . The FM Division in DE & FM did agree to use the partially modified version of the LCR model ( Figure 3 , actual ) , despite the quantity surveyors annexing estimation of capital replacement costs already in Phase 2 . Thus , the partially modified LCR Model and the interaction patterns it sustained responded to the problem - solving needs of PFI bidding while at the same time constituting a truce in the ongoing organizational and occupational struggle . The sub - optimal problem - solving it afforded derived from the need to sustain interaction patterns in which a truce among occupations could be preserved . Conversely , the modifications Mark made to the LCR model were a considerable improvement from a problem - solving point of view , but were unacceptable in terms of a truce in the jurisdictional war between quantity surveyors , facilities managers and ( to an extent ) designers . 27 AGENCY THROUGH SYSTEMS OF ARTEFACTS IN THE DEVELOPMENT OF NEW ROUTINES Including artefacts in the analysis , rather than focusing exclusively on individual’s actions and cognitions , allows to develop a better appreciation of the role of individual agency in the development of new routines . DE & FM data show how the effort of developing a new routine was strongly dependent on the action of individuals , particularly Mark . In line with approaches that underline the dual nature of structures , the data show that these actions were not totally independent nor were they totally determined by the structures in which Mark was embedded . Mark’s action was constrained by the resources that provided by structures , especially the artefacts , frames and objectives of his profession ( cf . Kaghan and Lounsbury , 2006 ) . His actions were further constrained by the fact that other actors in the system had access to similar resources . However , Mark exercised agency by inventively modifying resources and moving them across different , but intersecting , structures in an effort to modify them , that is , to create a new routine ( cf . Sewell , 1992 ) . Material resource , in the form of systems of interacting artefacts , are therefore important mediators in the institutionalization of individual agency into new routines . Speaking artefacts mediate efforts to develop patterns of actions that address problem solving needs . Specifically , product representations mediate the process through which individual agents attempt to match novel technical interdependencies in the product with a novel pattern of interdependent , distributed actions in the organization . Examination of the occupation - specific nature of these artefacts allows a reintegration of the analysis of conflict into problem - solving , showing that by altering the way in which product representations embody knowledge , agents attempt to both address problem - solving needs and alter control over activities and over knowledge accumulation . The attendant patterns of action are reinforced and extended by bundling the representations of products with representations of process . The limited span of influence of individual artefacts in a process ( Pentland and Feldman 2008 ) is less of a limitation in their role in patterning behaviour when one looks at systems of reinforcing artefacts . The implication of these systems in terms 28 of organizational conflict and , in particular , the kind of truce these systems embody becomes instead relevant in determining whether or not a new routine emerges . The evidence in this case suggest a model in which new routines are developed when organizations face environmental demands , such as the introduction of PFI , that force them to change their products ( Nelson and Winter , 1982 ) . This entails changes in the required bodies of knowledge , or at least a readjustment of the communication patterns among specialists deriving from changes in product architecture ( Henderson and Clark , 1990 ) . In their first attempts to deal with these demands , people use their experience of similar processes and adapt them incrementally to the new circumstances ( cf . Turner and Fern , this issue ) . This entails the ‘stretching’ of the systems of artefacts that supported the previous routines to the new demands . One or more ( competing ) patterns of interdependencies in the product ( and alternative divisions of labour ) emerge . If the current systems of artefacts , and specifically the occupation - specific product representations of the group currently dominating the process , is deemed insufficient to respond to the new demands , other groups will try to make inroads into the process using their own occupation - specific product representations . For instance , in DE & FM , when drawings began to be seen as inadequate to structure the process , the cost consultants stepped in with their LCR Model , a modified bill of quantities . Since new product features and new product architectures require the management of new interdependencies , the occupation - specific objects most likely to be modified are those used for decision making involving several different groups . Intentional modifications to occupation - specific objects enable groups to exploit the opportunity offered by a new critical environmental uncertainty , to increase their centrality in the organizational process ( cf . Hinings , Hickson , Pennings et al . , 1974 ) . The modified tool becomes a boundary object because it is used to address interdependencies among the bodies of knowledge held by different groups . This modified occupation - specific artefact , sustaining a modified pattern of problem - solving and a new truce , may simply come to operate in parallel with existing ones in a partially modified routine . Alternatively , the routine , and the occupation - specific objects supporting the new routine , may enter a new and critical phase of development if there is agreement that incremental changes are inadequate . At 29 this stage , packages of mutually reinforcing artefacts are developed , typically including one or more occupation - specific product representations , which act as boundary objects , and generic process representations . The bundling of product and process representations supports problem solving while extending the control of the occupation owning the artefacts over part of the process . The occupation owning the artefacts may also try to increase its control of knowledge accumulation . To do so , the originating occupation needs first to extend its capacity for knowledge representation to enable manipulation of other groups’ knowledge . Second , the new tool must be linked to other objects in other groups in a way that feeds experience into the new artefact . Third , local , specific problem - solving must be decoupled from the processes involved in updating and revising knowledge . The success of these strategies depends on the ability of the sponsoring occupation to craft the system of artefacts in a way that addresses problem - solving demands in a way that is deemed acceptable , while at the same time supporting a division of labour that constitutes an acceptable truce . Figure 5 highlights the main features of this process , showing the interacting nature of the artefacts that sustain a routines , and how environmental changes can lead artefacts , and the occupation to which they belong , from the periphery to the core of an organizational process . This mechanism , which is similar to the one discussed by March ( 1991 ) , highlights the benefits of internal diversity and latent conflict in providing organizations with the resources to deal with environmental change . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - INSERT FIGURE 5 ABOUT HERE - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CONCLUSIONS The approach developed in this paper makes it possible to address a central and unresolved issue in the development of new routines , that is , their dual functioning as organizational memory and as truces . These aspects of routines have been examined in isolation , with more effort devoted to examining the memory and cognitive dimensions than to examining their truce dimension ( Becker , 2004 , 2005 ) . In 30 considering routines as memory , the patterning of action is expected to reflect the ‘characteristics of the information storage problem that they solve’ and in considering routines as truces , the patterning of action is expected to ‘reflect the features of the underlying problem of diverging [ … ] interests’ ( Nelson and Winter , 1982 , pp . 110 - 111 ) . Understanding how routines develop , therefore , requires an understanding of how the patterning that enables routines to fulfil the function of memory overlaps with the patterning that enables them to act as truces . This paper shows that this overlapping occurs because development of routines is mediated by the development of systems of artefacts , which , within their own structures and relationships , reproduce the structures of problem - solving and of intraorganizational conflict . The analysis in this paper points to some of the open questions related to the multilevel nature and different characterisations of routines that need to be addressed . The first is the relationship between routines and individual skills and competencies . Work that treats routines as collective entities relies on analogies between routines as organizational level constructs , and individual skills , but provides very little analysis of their links ( Felin and Foss , 2009 ; Salvato and Rerup , 2011 ) . In occupation specific speaking artefacts , some of these links start to become clearer . These artefacts embody occupational knowledge and assumptions , and the expertise of those developing them . They regulate the type and timing of the expertise exploited for problem - solving . Thus , artefacts are important links between individual skills and routines , and their role as mediators in this relationship , and in the process that transforms firm experience into capabilities , needs further study . A second and related issue is the relationship between conceptualizations of routines as dispositions and cognitive regularities on the one hand , and routines as patterns of action on the other ( Becker , 2004 ; Cohen , 2007 ; Salvato and Rerup , 2011 ) . Because they contain formal representations of knowledge , speaking artefacts , which funnel and bound action , have a direct relationship with cognition . Similarly , occupation specific artefacts embody and promote tendencies to act in certain ways in relation to organizational conflict . Reconciling these opposing conceptualizations of routines would be a promising avenue for research . This study contributes also to the growing body of research on the material mediation of inter - disciplinary problem - solving . By analysing the process of development rather than the characteristics of 31 existing boundary objects this study shows that occupational groups can gain influence by generating boundary objects that sit at the junction of , and regulate access to , areas of expertise , which then can be annexed . Examining the occupation - specific nature of boundary objects contributes to analyses aimed at reintegrating conflict into the analysis of problem solving ( Orlikowski 1992 ; Kaplan , 2011 ; Nicolini , Menges and Swan , 2011 ) , moving beyond the focus in the literature on either problem - solving ( e . g . , Carlile , 2002 ; Bechky , 2003b ) or conflict ( e . g . , Barley , 1986 ; Bechky , 2003a ) . This study has some limitations which also offer opportunities for further research . The organizational routine examined here has a strong problem - solving component and requires collaboration among occupations that are either professions or are seeking professional status . Further research could investigate the processes through which artefacts mediate the development of new routines in contexts where joint cross - occupation problem - solving is less important . Also , the role of silent artefacts ( e . g . , clay models and other physical prototypes ) in the processes of routinization , and their relationships with speaking artefacts offers further opportunity to clarify how new routines are developed . Acknowledgments : I wish to thank Michael Cohen , Jane Dutton , David Gann , Ed Steinmueller , Scott Turner and Sid Winter for feedback , comments and encouragement on the long trip that has led to this paper . Funding from the British Engineering and Physical Sciences Research Council ( EPSRC ) is gratefully acknowledged . 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Organization Science , 13 , 339 - 51 . 40 Figure 1 – A typology of artefacts 41 Figure 2 – The LCR Model before the PFI Initiative 42 Figure 3 – Whole - life costing tools after the PFI Initiative - Intended and actual configuration 43 Figure 4 – Systems of artefacts in the development of new routines 44 Appendix 1 - List of Acronyms DE & FM Design Engineering and Facilities Management ( the case study company ) FM ( s ) Facilities Manager ( s ) / Management LCR Model Life Cycle Replacement Model ( the first whole - life costing tool developed in DE & FM ) PFI Private Finance Initiative ( public procurement method for construction ) QS ( s ) Quantity Surveyor ( s ) / Surveying SLP Service Life Planner ( Access database – core of the new whole - life costing tools storing all cost data ) 45 i A wide variety of objects , material and immaterial , have been described as boundary objects . Among artefacts , most studies have focused on speaking artefacts , such as Gant charts , which provide a locus for negotiating time allocations across groups in consultancies ( Yakura , 2002 ) and many of the tools employed in project management ( Sapsed and Salter 2004 ) . Research on product representations as boundary objects has focused particularly on sketches and drawings ( Henderson , 1991 ) and virtual prototypes ( e . g . , Carlile 2002 , 2004 ) . ii The name of the company , of all people mentioned in the following and of the objects and tools discussed were disguised to maintain anonymity . A list of acronyms is available in Appendix 1 . \ No newline at end of file diff --git a/silver_data/a9d920ff753a7f1fc9125308a4096dd3e4c42fed.pdf.txt b/silver_data/a9d920ff753a7f1fc9125308a4096dd3e4c42fed.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..73685835b889c0f02455d502efc18d2f6fa23ae2 --- /dev/null +++ b/silver_data/a9d920ff753a7f1fc9125308a4096dd3e4c42fed.pdf.txt @@ -0,0 +1 @@ +109 © Blackwell Publishing Ltd and the Department of Economics , University of Oxford , 2009 . Published by Blackwell Publishing Ltd , 9600 Garsington Road , Oxford OX4 2DQ , UK and 350 Main Street , Malden , MA02148 , USA . OXFORD BULLETIN OF ECONOMICS AND STATISTICS , 72 , 1 ( 2010 ) 0305 - 9049 doi : 10 . 1111 / j . 1468 - 0084 . 2009 . 00569 . x With or Without U ? The Appropriate Test for a U - Shaped Relationship * Jo Thori Lind and Halvor Mehlum Department of Economics , University of Oslo , PB 1095 Blindern , 0317 Oslo , Norway ( e - mail : j . t . lind @ econ . uio . no ; halvor . mehlum @ econ . uio ) Abstract Nonlinear relationships are common in economic theory , and such relationships are also frequently tested empirically . We argue that the usual test of nonlinear relation - ships is fl awed and derive the appropriate test for a U - shaped relationship . Our test gives the exact necessary and suf fi cient conditions for the test of a U shape in fi nite samples in a large class of models . I . Introduction Economics is full of humps and U’s . Famous examples of non - monotone relation - ships are the ‘Laffer curve’ , the ‘Kuznets curve ( an inverted U between income and inequality , Kuznets , 1955 ) , and the ‘Environmental Kuznets curve’ ( inverted U between income and pollution , Selden and Song , 1994 ; Grossman and Krueger , 1995 ) . In growth theory , poverty traps are generated if the growth rate of per capita capital stock fi rst increases and then decreases with income ( Nelson , 1956 ) . In industrial organization it has been found that innovation is most intense at intermediate levels of competition ( Aghion et al . , 2005 ) . In political science , a fi nding is that countries with an intermediate level of democracy are more prone to war compared with both dictatorships and democracies ( Hegre et al . , 2001 ) . Finally , it has been argued that there is a hump - shaped relationship between union bargaining centralization and wage growth ( Calmfors and Drif fi ll , 1988 ) , although few econometric studies of this exist , mostly as a result of the scarcity of data . * While carrying out this research , the authors have been associated with the ESOPcentre at the Department of Economics , University of Oslo . ESOP is supported by The Research Council of Norway . The authors are grateful for comments from two anonymous referees , Kalle Moene , and seminar participants at the University of Oslo and PRIO . They also thank Dustin Chambers for making his data available . A companion STATA module utest is available from : http : / / ideas . repec . org / c / boc / bocode / s456874 . html JEL Classi fi cation numbers : C12 , C20 . 110 Bulletin For many of the examples shown , the existence of genuinely U - shaped ( or hump - shaped ) relationships have been the subject of intense debate . The debates take many forms and take issues with conceptual , empirical , and theoretical problems . There is one important common empirical question , however , that to the best of our knowledge has not been explicitly addressed anywhere in this diverse literature : namely , given the estimates of a regression model , what is the test at some level (cid:2) of the presence of a U shape ? In most empirical work trying to identify U shapes , the researcher includes a nonlinear ( usually quadratic ) term in an otherwise standard regression model . If this term is signi fi cant and , in addition , the estimated extremum point is within the data range , it is common to conclude that there is a U - shaped relationship . We argue in this paper that this criterion is too weak . The problem arises when the true relationship is convex but monotone over relevant data values . A quadratic speci fi cation may then erroneously yield an extreme point and hence a U shape . To test properly for the presence of a U shape on some interval of values , we need to test whether the relationship is decreasing at low values within this interval and increasing at high values within the interval . As the distribution of the estimated slope at any point is readily available , a test for the sign of the slope at any given point is straightforward . A test for a U shape gets more involved , however , as the null hypoth - esis is that the relationship is increasing at the left - hand side of the interval and / or is decreasing at the right - hand side . For this composite null hypothesis , standard testing methodology is no longer suitable . Fortunately , a general framework for such tests has been developed by Sasabuchi ( 1980 ) . 1 We adopt his general framework to test for the presence of a U - shaped relationship . The extension to an inverse U shape is of course trivial . Since 2001 , there have been seven articles in the American Economic Review that use regression techniques to identify a U shape or an inverted U shape in their data . All of these revert to criteria that are intuitively sound , but potentially misleading . In this paper , we will present the appropriate test and then contrast it to the tests employed by the seven articles . In addition we provide a detailed example of the test in the case of a Kuznets curve . II . A test of a U shape To allow the regression to have a U shape , the standard approach has been to include a quadratic or an inverse term in a linear model . A more general formulation is : y i = (cid:2) + (cid:3) x i + (cid:4) f ( x i ) + (cid:5) (cid:2) z i + (cid:6) i , i = 1 , . . . , n . ( 1 ) Here x is the explanatory variable of main interest ( e . g . income ) whereas y is the variable to be explained ( e . g . inequality ) , (cid:6) is an error term , and z is a vector of control variables . The known function f gives equation ( 1 ) a curvature and , depending on the 1 Surprisingly , this contribution has so far not been applied in the econometrics literature with the exception of a few studies of economic inequality starting with Dardanoni and Forcina ( 1999 ) . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 Practitioners’ corner 111 parameters (cid:4) and (cid:3) , equation ( 1 ) may be U - shaped or not . We assume that f is chosen so that the relationship has at most one extreme point . In that case the relationship is either hump - shaped , U - shaped , or monotone . In the following we focus on the case of testing for a U shape . 2 Given equation ( 1 ) and the assumption of only one extreme point , the requirement for a U shape is that the slope of the curve is negative at the start and positive at the end of a reasonably chosen interval of x - values [ x l , x h ] . The natural choice of interval is in many contexts the observed data range [ min ( x ) , max ( x ) ] . If we want to make sure that the inverse U shape is not only a marginal phenomenon the interval could also be in the interior of the domain of x . To assure at most one extreme point on [ x l , x h ] , as assumed before , we require f (cid:2) to be monotone on this interval . A U shape is then implied by the conditions (cid:3) + (cid:4) f (cid:2) ( x l ) < 0 < (cid:3) + (cid:4) f (cid:2) ( x h ) . ( 2 ) If either of these inequalities are violated the curve is not U - shaped but inversely U - shaped or monotone . To test whether the conditions in equation ( 2 ) are supported by the data , we need to test whether the combined null hypothesis H 0 : (cid:3) + (cid:4) f (cid:2) ( x l ) ≥ 0 and / or (cid:3) + (cid:4) f (cid:2) ( x h ) ≤ 0 ( 3 ) can be rejected in favour of the combined alternative hypothesis H 1 : (cid:3) + (cid:4) f (cid:2) ( x l ) < 0 and (cid:3) + (cid:4) f (cid:2) ( x h ) > 0 . ( 4 ) Owing to the linearity of the speci fi cation ( 1 ) with respect to (cid:3) and (cid:4) , the test of equation ( 3 ) vs . equation ( 4 ) is simply a test of linear restrictions on (cid:3) and (cid:4) . The dif fi culty is that the test involves a set of inequality constraints . Hence the set of ( (cid:3) , (cid:4) ) that satisfy H 1 is a sector in R 2 contained between the two lines (cid:3) + (cid:4) f (cid:2) ( x l ) = 0 and (cid:3) + (cid:4) f (cid:2) ( x h ) = 0 . The test Assuming that (cid:6) i ∼ NID ( 0 , (cid:7) 2 ) , Sasabuchi ( 1980 ) shows that a test of H 0 in equation ( 3 ) based on the likelihood ratio principle takes the form Reject H 0 at the (cid:2) level of con fi dence only if either , H L 0 or H H 0 or both can be rejected at the (cid:2) level of con fi dence , where H L 0 and H H 0 are the null hypotheses in the two standard one - sided tests H L 0 : (cid:3) + (cid:4) f (cid:2) ( x l ) ≥ 0 vs . H L 1 : (cid:3) + (cid:4) f (cid:2) ( x l ) < 0 , H H 0 : (cid:3) + (cid:4) f (cid:2) ( x h ) ≤ 0 vs . H H 1 : (cid:3) + (cid:4) f (cid:2) ( x h ) > 0 . 2 The test for an inverse U is achieved by changing the sign on y . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 112 Bulletin The rejection area is the convex cone R (cid:2) = (cid:2) ( (cid:3) , (cid:4) ) : (cid:3) + (cid:4) f (cid:2) ( x l ) (cid:3) s 11 + 2 f (cid:2) ( x l ) s 12 + f (cid:2) ( x l ) 2 s 22 < − t (cid:2) and (cid:3) + (cid:4) f (cid:2) ( x h ) (cid:3) s 11 + 2 f (cid:2) ( x h ) s 12 + f (cid:2) ( x h ) 2 s 22 > t (cid:2) (cid:4) , ( 5 ) where s 11 , s 22 and s 12 are the estimated variances of (cid:3) and (cid:4) and the covariance between them , whereas t (cid:2) is the (cid:2) - level tail probability of the t - distribution with the appropriate degrees of freedom . The test shown is also known as an intersection – union test as the null hypothesis is that the parameter vector is contained in a union of speci fi ed sets ( see , e . g . Casella and Berger , 2002 , ch . 8 . 2 . 3 ) . Berger ( 1997 ) discusses the relationship between like - lihood ratio tests and intersection – union tests and the conditions under which they coincide . The two most common speci fi cations of equation ( 1 ) is the quadratic form y i = (cid:2) + (cid:3) x i + (cid:4) x 2 i + (cid:5) (cid:2) z i + (cid:6) i ( 6 ) and the inverse form y i = (cid:2) + (cid:3) x i + (cid:4) x − 1 i + (cid:5) (cid:2) z i + (cid:6) i . ( 7 ) In the fi rst case , the presence of a U implies (cid:3) + 2 (cid:4) x l < 0 and (cid:3) + 2 (cid:4) x h > 0 . A U shape in the second implies (cid:3) − (cid:4) x − 2 l < 0 and (cid:3) − (cid:4) x − 2 h > 0 . In both cases , the test is easily carried out as two ordinary t - tests . 3 The rejection area R (cid:2) can be manipulated so that it is expressed in terms of f (cid:2) ( x l ) and f (cid:2) ( x h ) . Solving for f (cid:2) ( x l ) and f (cid:2) ( x h ) in R (cid:2) and inserting for ( ˆ (cid:3) , ˆ (cid:4) ) yields the following two criteria for rejection : f (cid:2) ( x l ) < ˆ (cid:8) l ≡ s 12 t 2 (cid:2) − ˆ (cid:3) ˆ (cid:4) − t (cid:2) (cid:5) ( s 212 − s 22 s 11 ) t 2 (cid:2) + ˆ (cid:4) 2 s 11 + ˆ (cid:3) 2 s 22 − 2 s 12 ˆ (cid:3) ˆ (cid:4) ˆ (cid:4) 2 − s 22 t 2 (cid:2) , ( 8 ) f (cid:2) ( x h ) > ˆ (cid:8) h ≡ s 12 t 2 (cid:2) − ˆ (cid:3) ˆ (cid:4) + t (cid:2) (cid:5) ( s 212 − s 22 s 11 ) t 2 (cid:2) + ˆ (cid:4) 2 s 11 + ˆ (cid:3) 2 s 22 − 2 s 12 ˆ (cid:3) ˆ (cid:4) ˆ (cid:4) 2 − s 22 t 2 (cid:2) . ( 9 ) Here ˆ (cid:8) l and ˆ (cid:8) h are the roots of the same quadratic equation . This way of formulating the criterion has a good parallel to the work of Fieller ( 1954 ) . From his work , it is known how to construct an exact con fi dence interval for the ratio of two normally distributed estimates . Note fi rst that the estimated extreme point of equation ( 1 ) is 3 A routine to perform the test in these two cases in the software package Stata is available from : http : / / ideas . repec . org / c / boc / bocode / s456874 . html . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 Practitioners’ corner 113 f (cid:2) ( ˆ x min ) = − ˆ (cid:3) ˆ (cid:4) . Following Fieller ( 1954 ) , a ( 1 − 2 (cid:2) ) con fi dence interval for − (cid:3) / (cid:4) is given by [ ˆ (cid:8) l , ˆ (cid:8) h ] as de fi ned in equations ( 8 ) and ( 9 ) . Therefore , a ( 1 − 2 (cid:2) ) con fi dence interval for ˆ x min is [ ˜ x l , ˜ x h ] where f (cid:2) ( ˜ x i ) = ˆ (cid:8) i . To perform the test of equation ( 3 ) vs . equation ( 4 ) at the (cid:2) - level of signi fi cance is then equivalent to seeing whether the ( 1 − 2 (cid:2) ) con fi dence interval for ˆ x min is inside the data range , [ ˜ x l , ˜ x h ] ⊂ [ x l , x h ] . To fi nd a con fi dence interval for ˆ x min , one could also use the delta method . For fi nite samples , however , this may be severely biased . 4 Also when using the delta method the ( 1 − 2 (cid:2) ) interval is the proper interval to use when testing for a U shape at the (cid:2) - level . The testing strategy is easily carried over to more involved estimations . First , it is readily extended to a more general formulation like y i = (cid:2) + (cid:6) Hj = 1 (cid:3) j f j ( x ) + z (cid:2) i (cid:4) + (cid:6) i , where f j is a set of known functions . 5 The appropriate test of the presence of an inverse U - shaped relationship between x and y is now H 0 : (cid:7) (cid:3) j f (cid:2) j ( x l ) ≤ 0 and / or (cid:7) (cid:3) j f (cid:2) j ( x h ) ≥ 0 vs . H 1 : (cid:7) (cid:3) j f (cid:2) j ( x l ) > 0 and (cid:7) (cid:3) j f (cid:2) j ( x h ) < 0 . ( 10 ) The test is also applicable to studies of U - shaped relationships in the full class of generalized linear models , encompassing most models of limited dependent variables . As the estimated parameters are asymptotically jointly normally distributed , the distribution of the test is asymptotic as explained before . Performance of the test To see the performance of the test , Table 1 shows the results of some Monte Carlo analyses . Two speci fi cations are employed . First , data are generated with x ∼ U [ 0 , 1 ] , (cid:6) ∼ N ( 0 , 1 / 2 ) and y = a + bx + x 2 + (cid:6) , where b is chosen to achieve the desired minimum ranging from 0 . 6 to 1 . 6 . We then regress y on x and x 2 and perform three tests : ( i ) whether the quadratic term is signi fi cant ; ( ii ) whether the quadratic term is signi fi cant and the estimated extremum point is in [ 0 , 1 ] ; and ( iii ) the test described before . The table gives the fraction of cases where we reject the absence of a U - shaped 4 This problem is clearly illustrated in Hirschberg and Lye ( 2005 ) who study different methods for construct - ing ( 1 − (cid:2) ) intervals for the extremum of a quadratic equation and give practical recommendations consistent with our suggestions . 5 The dif fi culty with this general speci fi cation is that although the relationship is decreasing at the left - hand side of the relevant interval and increasing at the right - hand side , it may not be a U - shaped relationship inside , but instead for instance an W - shaped relationship . It is then necessary to perform a joint test of the whole shape of the relationship , which is much more complicated . See Doveh , Shapiro and Feigin ( 2002 ) for some developments in this direction . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 114 Bulletin TABLE 1 Monte Carlo analysis of the performance of the test Number of observations Correctly specified model Mis - specified model Extreme point 10 50 100 500 1 , 000 10 50 100 500 1 , 000 0 . 6 ( i ) 0 . 11 0 . 18 0 . 32 0 . 91 1 . 00 0 . 12 0 . 26 0 . 45 0 . 98 1 . 00 ( ii ) 0 . 11 0 . 18 0 . 32 0 . 91 1 . 00 0 . 12 0 . 26 0 . 45 0 . 98 1 . 00 ( iii ) 0 . 10 0 . 18 0 . 30 0 . 82 0 . 97 0 . 13 0 . 30 0 . 49 0 . 98 1 . 00 0 . 8 ( i ) 0 . 11 0 . 18 0 . 31 0 . 91 1 . 00 0 . 12 0 . 26 0 . 45 0 . 98 1 . 00 ( ii ) 0 . 11 0 . 18 0 . 31 0 . 89 0 . 97 0 . 12 0 . 26 0 . 45 0 . 98 1 . 00 ( iii ) 0 . 09 0 . 11 0 . 14 0 . 36 0 . 57 0 . 11 0 . 20 0 . 31 0 . 83 0 . 98 1 ( i ) 0 . 11 0 . 19 0 . 31 0 . 91 1 . 00 0 . 12 0 . 26 0 . 44 0 . 98 1 . 00 ( ii ) 0 . 11 0 . 19 0 . 31 0 . 51 0 . 50 0 . 12 0 . 26 0 . 44 0 . 93 0 . 98 ( iii ) 0 . 07 0 . 06 0 . 05 0 . 05 0 . 05 0 . 09 0 . 13 0 . 17 0 . 45 0 . 68 1 . 2 ( i ) 0 . 11 0 . 19 0 . 32 0 . 91 1 . 00 0 . 12 0 . 25 0 . 45 0 . 98 1 . 00 ( ii ) 0 . 11 0 . 18 0 . 26 0 . 10 0 . 03 0 . 12 0 . 25 0 . 45 0 . 81 0 . 90 ( iii ) 0 . 05 0 . 02 0 . 01 0 . 00 0 . 00 0 . 08 0 . 09 0 . 11 0 . 23 0 . 36 1 . 4 ( i ) 0 . 11 0 . 18 0 . 31 0 . 91 1 . 00 0 . 13 0 . 25 0 . 45 0 . 98 1 . 00 ( ii ) 0 . 11 0 . 16 0 . 13 0 . 00 0 . 00 0 . 13 0 . 25 0 . 44 0 . 71 0 . 78 ( iii ) 0 . 04 0 . 01 0 . 00 0 . 00 0 . 00 0 . 08 0 . 07 0 . 08 0 . 13 0 . 19 1 . 6 ( i ) 0 . 12 0 . 18 0 . 32 0 . 91 1 . 00 0 . 13 0 . 25 0 . 45 0 . 99 1 . 00 ( ii ) 0 . 11 0 . 11 0 . 05 0 . 00 0 . 00 0 . 13 0 . 25 0 . 44 0 . 63 0 . 68 ( iii ) 0 . 04 0 . 00 0 . 00 0 . 00 0 . 00 0 . 08 0 . 06 0 . 07 0 . 09 0 . 12 Notes : Tests performed at the 5 % level of signi fi cance . Results are based on 10 , 000 simulations . The tests studied are : ( i ) whether the quadratic term is signi fi cant ; ( ii ) whether the quadratic term is signi fi cant and the estimated extremum point is in [ 0 , 1 ] ; and ( iii ) the test described previously . relationship . To see how the different tests perform in a mis - speci fi ed model , the right - hand part of the table shows simulations from a true model y = a + bx + 1 / x + (cid:6) , but where we still regress y on x and x 2 . Now we have 6 x ∼ U [ 0 . 5 ; 1 ] and (cid:6) ∼ N ( 0 , 1 / 2 ) . In the correctly speci fi ed model , test ( i ) has a huge rate of Type I errors , erroneously rejecting the absence of a U - shaped relationship . This indicates that this is an inappropriate test . When it is also required that the estimated extremum be in the relevant interval as in ( ii ) , the test is stronger , particularly for large samples where this point estimate becomes precise . However , the proper test ( iii ) performs better in all cases . Notice , though , that it has somewhat lower test power than test ( ii ) when there actually is a U - shaped relationship . In the mis - speci fi ed model , both tests ( i ) and ( ii ) perform badly . There is some excess probability of committing Type I errors for our test as well , but the outcomes are much better than the commonly used procedures . 6 The smaller interval for x was chosen as the asymptote y → + ∞ when x → 0 would have a too large impact on the estimates if x was allowed to be too small . In this case , all three procedures perform badly ( results available upon request ) . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 Practitioners’ corner 115 III . A comparison with applied work We have read through a large number of applied econometrics works where U shapes are identi fi ed using equation ( 6 ) . Most authors focus on the signi fi cance and sign of both ˆ (cid:3) and ˆ (cid:4) . How does the signi fi cance of these parameters relate to the test in equation ( 5 ) ? If the data range is the whole of R the signi fi cance of ˆ (cid:4) ( with right sign ) is necessary and suf fi cient for rejecting H 0 . If the data range is any subset of R , the signi fi cance of ˆ (cid:4) is still necessary but not suf fi cient . If the data range is exactly R + or R − the signi fi cance ( with the right sign ) of ˆ (cid:3) and ˆ (cid:4) respectively is necessary and suf fi cient for rejecting H 0 . If the data range is a subset of R + ( or of R − ) the individual signi fi cance of both ˆ (cid:3) and ˆ (cid:4) is necessary but not suf fi cient . If the data range is neither R , R + or R − , the necessary and suf fi cient conditions are only found using equation ( 5 ) . Obviously , a simple fi rst check is whether the estimated minimum point ( ˆ x min = − ˆ (cid:3) / ( 2ˆ (cid:4) ) ) itself is within the date range . Most works use the criterion that if both ˆ (cid:3) and ˆ (cid:4) are signi fi cant and if the implied extreme point is within the data range , they have found a U . This is a sensible criterion but it is neither suf fi cient nor necessary . It is insuf fi cient as the estimated extreme point may be too close , given the uncertainty , to an end point of the data range . It is not generally necessary as (cid:3) may be zero if the data range extends to both sides of x = 0 . A small number of works use the delta method to calculate the standard deviation of the extreme point . This method , although sound , is only reliable with a suf fi ciently large number of observations . We have in particular looked at articles in the American Economic Review . There are seven articles since 2001 that use regression techniques to identify a U shape . All use formulations similar to equation ( 6 ) . The most common procedure is the one followed by Abiad and Mody ( 2006 ) , McKinnish ( 2004 ) , and Kalemli - Ozcan , Sørensen and Yosha ( 2003 ) . They fi nd that both ˆ (cid:3) and ˆ (cid:4) in the quadratic speci fi cation have the right sign and are individually signi fi cant . Based on this , they all conclude that they have found an inverted U shape without checking whether the implied extreme point is within any relevant range . Sigman ( 2002 ) follows the same procedure , but she also calculates the extreme point and fi nds it to be within the data range . She writes , relating to ˆ (cid:3) and ˆ (cid:4) ( p . 1157 ) : ‘The GDP coef fi cients always appear to follow an inverted U shaped pattern , but are jointly statistically signi fi cant at 5 percent only in columns ( 3 ) and ( 4 ) ’ . Her testing for joint signi fi cance of ˆ (cid:3) and ˆ (cid:4) , however , do not add much when looking for a U shape . As we have argued before , the signi fi cance of ˆ (cid:4) alone is always a necessary condition in the test of a U shape . Similarly , Aghion et al . ( 2005 ) use their estimates from the quadratic speci fi ca - tion to calculate predicted value ˆ y . They fi nd that the estimated minimum is inside the x - range , but they do not explicitly assess the signi fi cance of the result . Carr , Markusen and Maskus ( 2001 ) only include (cid:4) x 2 , and not (cid:3) x , in their speci fi - cation . As their x - range includes both negative and positive values , their speci fi cation © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 116 Bulletin rules out monotonically increasing or decreasing relationships between x and y . Hence their analysis does not allow for any meaningful test of a U shape against a monotone relationship . Imbs and Wacziarg ( 2003 ) fi rst use non - parametric techniques to fi nd the relationship between per capita income x and sectoral concentration y and they fi nd a U shape . They use this technique also to construct 95 % intervals for the minimum point . They then turn to a parametric speci fi cation with x and x 2 in the right - hand side . Both coef fi cients have the right sign and are individually signi fi cant . The signi fi cance of the U shape is assessed by plotting x and ˆ y vs . the scatter plot of the data . The regression curve fi ts the data very well and based on the plot alone one can safely conclude that the U shape is signi fi cant at all conventional levels . But , the precise signi fi cance level is unclear and so is the precision of the estimated minimum point . IV . Illustration As a concrete illustration of our methodology , we use a recent study by Chambers ( 2007 ) . The paper contains a standard Kuznets regression between gross domestic product ( GDP ) and inequality . The data are an unbalanced panel of 29 countries giving a total of 232 observations . He has information on the level of inequality measured by the Gini coef fi cient , log of purchasing power parity adjusted GDP per capita , and a set of control variables ( see Chambers , 2007 , for details ) . We replicate and extend his analysis . Results from the regression analysis , test statistics from the appropriate U test , and the derived Fieller interval are reported in Table 2 . The conventional method would simply test the signi fi cance of ˆ (cid:3) and ˆ (cid:4) , which TABLE 2 Estimates of the Kuznets curve Dependent variable : Gini index log per capita GDP ( X i ) ˆ (cid:3) = 32 . 27 ( 11 . 63 ) ÅÅÅ log per capita GDP - squared ( X 2 i ) ˆ (cid:4) = − 1 . 88 ( 0 . 65 ) ÅÅÅ Slope at X l ˆ (cid:3) + 2ˆ (cid:4) X l = 8 . 15 ( 3 . 65 ) ÅÅ Slope at X h ˆ (cid:3) + 2ˆ (cid:4) X h = − 4 . 33 ( 2 . 34 ) Å Appropriate U test 1 . 85 [ 0 . 033 ] Extremum point − ˆ (cid:3) / ( 2 ˆ (cid:4) ) = 8 . 60 90 % con fi dence interval , Fieller method [ 7 . 44 , 9 . 58 ] 90 % con fi dence interval , Delta method [ 7 . 73 , 9 . 47 ] Notes : Robust standard errors in parentheses and P - values in square brackets . * * * , * * and * denote signi fi cances at the 1 , 5 , and 10 % levels . GDP , gross domestic product . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 Practitioners’ corner 117 Figure 1 . Illustrative Kuznets curve in this case yield t - values of 2 . 77 and 2 . 89 . With the appropriate U test , however , we see that the test for a positive slope at x h yields a t - value of only 1 . 85 , and hence a P - value of 0 . 032 with the required one - sided test . Figure 1 illustrates the estimated relationship , the con fi dence interval for the maxi - mum and the two lower bounds on the slopes in each endpoint . 7 We see that the turning point of the relationship is quite close to x h , and that the slope of the curve at x h is negative , but not very steep and only just signi fi cant at the 5 % level . Hence there is a signi fi cant hump - shaped relationship over the range of the data , but the signi fi cance of this relationship is weaker than what would be detected by traditional approaches . V . Conclusion In this paper , we have provided an appropriate test of a U - shaped relationship in a regression model . In the applied econometrics literature a large number of articles try to identify non - monotone relationships using regression analysis . Any of these articles hardly use the adequate formal procedures when they test for the presence of a U shape . To the best of our knowledge none of them have used the simple test that we are suggesting . Most works , nevertheless , seem to be on fairly safe ground when they claim to have found a U shape . The reason is that the de facto common practice seems to be to check two necessary conditions , namely that the second derivative has the right sign and that the extremum point is within the data range . However , only the results from the former are usually reported . This criterion will be misleading , however , if the estimated extremum point is too close to the end point of the data range . Our test gives the exact necessary and suf fi cient conditions for the test of a U shape . In addition , the interval interpretation provides a con fi dence interval for the extremum point . Final Manuscript Received : June 2009 7 We have used average values for all the controls . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 118 Bulletin References Abiad , A . and Mody , A . ( 2006 ) . ‘Financial reform : what shakes it ? what shapes it ? ’ American Economic Review , Vol . 95 , pp . 66 – 88 . Aghion , P . , Bloom , N . , Blundell , R . , Grif fi th , R . and Howitt , P . ( 2005 ) . ‘Competition and innovation : an inverted - U relationship’ , Quarterly Journal of Economics , Vol . 120 , pp . 701 – 728 . Berger , R . L . ( 1997 ) . ‘Likelihood ratio tests and intersection – union test’ , Ch . 15 in Panchapakesan S . and Balakrishnan N . ( eds ) , Advances in Statistical Decision Theory and Application , Birkh¨auser , Boston , pp . 225 – 237 . Calmfors , L . and Drif fi ll , J . ( 1988 ) . ‘Centralization of wage bargaining’ , Economic Policy , Vol . 6 , pp . 14 – 61 . Carr , D . L . , Markusen , J . R . and Maskus , K . E . ( 2001 ) . ‘Estimating the knowledge - capital model of the multinational enterprise’ , American Economic Review , Vol . 91 , pp . 693 – 708 . Casella , G . and Berger , R . L . ( 2002 ) . Statistical Inference , 2nd edn , Duxbury Press , Paci fi c Grove , CA . Chambers , D . ( 2007 ) . ‘Trading places : does past growth impact inequality ? ’ Journal of Development Economics , Vol . 82 , pp . 257 – 266 . Dardanoni , V . and Forcina , A . ( 1999 ) . ‘Inference for Lorenz curve orderings’ , Econometrics Journal , Vol . 2 , pp . 49 – 75 . Doveh , E . , Shapiro , A . and Feigin , P . D . ( 2002 ) . ‘Testing of monotonicity in parametric regression models’ , Journal of Statistical Planning and Inference , Vol . 107 , pp . 289 – 306 . Fieller , E . C . ( 1954 ) . ‘Some problems in interval estimation’ , Journal of the Royal Statistical Society , Series B , Vol . 16 , pp . 175 – 185 . Grossman , G . M . andKrueger , A . B . ( 1995 ) . ‘Economicgrowthandtheenvironment’ , QuarterlyJournal of Economics , Vol . 110 , pp . 353 – 377 . Hegre , H . , Ellingsen , T . , Gates , S . and Gleditsch , N . P . ( 2001 ) . ‘Toward a democratic civil peace ? Democracy , political change , and civil war , 1816 – 1992’ , American Political Science Review , Vol . 95 , pp . 17 – 33 . Hirschberg , J . andLye , J . ( 2005 ) . ‘Inferencesfortheextremumofquadraticregressionmodels’ , Research Paper 906 , Department of Economics , University of Melbourne . Imbs , J . and Wacziarg , R . ( 2003 ) . ‘Stages of diversi fi cation’ , American Economic Review , Vol . 93 , pp . 63 – 86 . Kalemli - Ozcan , S . , Sørensen , B . E . and Yosha , O . ( 2003 ) . ‘Risk sharing and industrial specialization : regional and international evidence’ , American Economic Review , Vol . 93 , pp . 903 – 918 . Kuznets , S . ( 1955 ) . ‘Economic growth and income inequality’ , American Economic Review , Vol . 45 , pp . 1 – 28 . McKinnish , T . G . ( 2004 ) . ‘Gender in policy and the labor market occupation , sex - integration , and divorce’ , American Economic Review , Vol . 94 , pp . 322 – 325 . Nelson , R . R . ( 1956 ) . ‘A theory of the low - level equilibrium trap in underdeveloped economies’ , American Economic Review , Vol . 46 , pp . 894 – 908 . Sasabuchi , S . ( 1980 ) . ‘A test of a multivariate normal mean with composite hypotheses determined by linear inequalities’ , Biometrika , Vol . 67 , pp . 429 – 439 . Selden , T . M . and Song , D . ( 1994 ) . ‘Environmental quality and development : is there a Kuznets curve for air pollution emissions ? ’ Journal of Environmental Economics and Management , Vol . 27 , pp . 147 – 162 . Sigman , H . ( 2002 ) . ‘Internationalspilloversandwaterqualityinrivers : docountriesfreeride ? ’ American Economic Review , Vol . 92 , pp . 1152 – 1159 . © Blackwell Publishing Ltd and the Department of Economics , University of Oxford 2009 \ No newline at end of file diff --git a/silver_data/ab0f7cb6bf3a5f5273b19a002287bbbf9cf5e7af.pdf.txt b/silver_data/ab0f7cb6bf3a5f5273b19a002287bbbf9cf5e7af.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..d02d739b3a3c163798688e08cd652c67172aed58 --- /dev/null +++ b/silver_data/ab0f7cb6bf3a5f5273b19a002287bbbf9cf5e7af.pdf.txt @@ -0,0 +1 @@ +Coupling of cytoplasm and adhesion dynamics determines cell polarization and locomotion Wolfgang Alt 1 , 2 Martin Bock 2 Christoph M¨ohl 3 July 29 , 2009 Observation of epidermal cells or cell fragments on flat adhesive substrates has revealed two distinct morphological and functional states : A non - migrating symmetric ”unpolarized state” and a migrating asymmetric ”polarized state” . They are characterized by different spatial distributions and dynamics of impor - tant molecular components as F - actin and myosin - II within the cytoplasm , and integrin receptors at the plasma membrane contacting the substratum , thereby inducing so - called focal adhesion complexes . So far , mathematical models have reduced this phenomenon either to gradients in regulatory control and signaling molecules or to different mechanics of the polymerizing and contracting actin filament system in different regions of the cell edge . Here we offer an alternative self - organizational model in order to reproduce and explain the emergence of both functional states for a certain range of dy - namical and kinetic model parameters . We apply an extended version of a two - phase , highly viscous cytoplasmic flow model with variable force balance equations at the moving edge , coupled to a 4 - state reaction - diffusion - transport system for the bound and unbound integrin receptors beneath the spreading cell or cell fragment . In particular , we use simulations of a simplified 1 - D model for a cell fragment of fixed extension to demonstrate characteristic changes in the concentration profiles for actin , myosin and doubly bound integrin , as they 1 corresponding author , eMail wolf . alt @ uni - bonn . de 2 Universit¨at Bonn , Theoretische Biologie , Kirschallee 1 - 3 , 53115 Bonn , Germany 3 Forschungszentrum J¨ulich , Institut f¨ur Bio - und Nanosysteme ( IBN ) , Institut 4 : Biomechanik ( IBN4 ) , 52425 J¨ulich , Germany a r X i v : 0 9 07 . 5078v1 [ phy s i c s . b i o - ph ] 2 9 J u l 2009 occur during transition from the symmetric stationary state to the polarized migrating state . In the latter case the substratum experiences a low magnitude ”pulling force” within the larger front region , and an opposing high magnitude ”disruptive force” at the shorter rear region . Moreover , simulations of the cor - responding 2 - D model with free boundary show characteristic undulating pro - trusions and retractions of the cell ( fragment ) edge , with local accumulation of doubly bound adhesion receptors behind it , combined with a modulated retro - grade F - actin flow . Finally , for a stationary model cell ( fragment ) of symmetric round shape , larger fluctuations in the circumferential protrusion activity and adhesion kinetics can break the radial symmetry and induce a gradual polar - ization of shape and concentration profiles , leading to continuous migration in direction of the leading front . The aim of the chapter is to show how relatively simple laws for the small - scale mechanics and kinetics of participating molecules , responsible for the en - ergy consuming steps as filament polymerization , pushing and sliding , binding and pulling on adhesion sites , can be combined into a non - linearly coupled sys - tem of hyperbolic , parabolic and elliptic differential equations that reproduce the emergent behavior of polarization and migration on the large - scale cell level . 1 Biology of cell polarization and migration Cell polarization and migration plays a central role in the development and maintenance of tissues in multicellular organisms . During ontogenesis , new tissues are formed by the coordinated division and locomotion of single cells . The polarization of a cell not only defines the direction of migration [ 30 ] but also the cell division axis [ 61 ] and thus the three - dimensional structure of tissues , organs and finally the whole organism . 1 . 1 Asymmetry of actin polymerization and substrate adhesion The coordinated development and release of focal adhesions ( FAs ) is a basic requirement for directed cell migration . Migrating cells feature pronounced adhesion dynamics and a struc - tural polarity with a clearly distinguishable frontal and rear area . Actin polymerization predominates at the cell front resulting in a protruding lamella in direction of migration . New focal adhesions composed of clustered protein complexes develop at the lower mem - brane of the lamella near the leading edge and couple the F - actin - network mechanically to extracellular matrix proteins . Simultaneously , the matured FAs residing at the opposed trailing edge are dissolved while myosin driven contraction of F - actin moves the cell body forward [ 10 , 36 ] . Arising from the process described above , the polarity of the cell can be referred to two structural asymmetries , which are key requirements for effective cell migration : asymmetry of actin polymerization and asymmetry of adhesion strength . The growth of actin filaments 2 has to predominate at the cell front for pushing the leading edge in direction of migration [ 63 , 58 ] . To move the cell body forward , it has to be released from the substrate during contraction by dissolving the FAs at the back ( rear release ) while the FAs at the cell front have to remain stable to provide a mechanical attachment for the contractile machinery pulling the cell body . In absence of rear release , traction forces could be dominated by adhesion forces and the cell would get stuck [ 54 ] . In this regard , the spatial distribution of adhesion strength and actin polymerization defines the direction of migration and could be specifically regulated due to directed cell movement . 1 . 2 Flow of actin filaments and myosin gradient The mechanisms underlying the structural polarity of migrating cells are still under discus - sion , particularly what concerns an intrinsic directionality of the cytoplasm . For example , the finding that asymmetric adhesive structures define polarization of a touching cell [ 30 ] suggests that directional flow of the actin cytoskeleton is involved in the process of cell polarization . Moreover , recent studies on fish keratocytes have revealed that polarization occurs spontaneously and is accompanied by a reorganization of the actin cytoskeleton , which finally leads to cell locomotion [ 64 ] . In these experiments , unpolarized solitary cells feature a circular shape with a radially symmetric actin distribution . Although the cells do not move , transient protrusions and retractions appear at the cell edge , and actin flows centripetally with a decreasing flow gradient from the cell edge to the center . In this ap - parently unstable state spontaneous symmetry breaking results in a faster inward flow and in an increased concentration of actin at the rear region of the cell . On the opposed front edge , the reduced inward actin flow causes protrusion and the development of a lamel - lipodium . In this polarized state , the cell starts to migrate while attaining a more or less constant shape . A similar behavior was observed with cell fragments extracted from the lamella of fish keratocytes [ 63 ] , see Fig . 1 . Since these fragments were lacking most cell organelles and the microtubule system , they were mainly limited to the actin - myosin machinery . They appeared either in an unpolarized , non - migrating state with homogeneous actin - myosin distribution or a polarized , migrating state with a rising myosin concentration from front to rear . Interestingly , polarization of fragments could be induced by mechanical stimula - tion , e . g . by shear flow or stress release leading to a transition from the stationary to the migrating state . Cell polarization is discussed to result from the local activation of GTPases by the rho - family [ 62 , 54 , 43 ] . These proteins are known to regulate actin polymerization , myosin activity and FA assembly , whereas it remains unclear how the distribution of the GTPases is controlled . Recent experiments have revealed that by inhibition of myosin activating signalling pathways the cell’s ability to polarize is reduced [ 64 ] . However , the observations of cells or cell fragments to polarize either spontaneously or due to mechanical pertur - bations , raise doubts that biochemical signalling is the primary reason for inducing and 3 Figure 1 : Cell fragment experiments ( from [ 63 ] ) , where the mechanical stress ( release ) in - duces a transition from a circular non - migrating state ( a ) to a polarized migrating state ( b , c ) . The cell fragment was labelled with fluorescent myosin II . Time scale is min : sec and length scale of bar is 2 µ m . maintaining polarity . Experiments focussing on cell mechanics rather show that direction and strength of locomotion forces are inherently connected with the retrograde F - actin flow [ 53 , 22 ] . This suggests that the mechanical action of myosin , namely to induce cytoplasmic contraction , flow and force transduction at adhesive sites , plays a key role in explaining the ubiquitously observed phenomena of cell polarization and locomotion . 2 Previous models of cytoplasm and adhesion dynamics The first detailed mathematical model coupling cytoskeleton and adhesion dynamics was developed by Lauffenburger and coworkers almost two decades ago [ 17 ] . Here , the con - tractile actin - myosin network and its interaction with bound adhesion sites is represented as a mechanical system of connected viscoelastic units of generalized Kelvin - Voigt type , which constitute the ( three ) inner segments as well as a front segment ( lamellipod ) and a tail segment ( uropod ) for a rectangular model cell of fixed width and length . A coupled system of reaction - diffusion equations for free and substrate bound integrins or adhesion receptors on the dorsal ( upper ) and ventral ( lower ) part of the model cell is solved un - der pseudo steady - state assumptions . This dynamically provides the number of adhesion bonds in both end segments , lamellipod and uropod , whereby their rupture ( dissociation ) kinetics exponentially increases with force load onto a bound adhesion site . The resulting non - linear dynamics for the local ( forward or backward ) displacement of each viscoelastic unit produces a persistent forward translocation of the whole model cell . However , this is achieved only if a front - tail asymmetry is presupposed , either by exposing more free adhesion receptors , or by assuming higher adhesion bond affinity at the front compared to the tail . The authors present a series of results on how the simulated cell migration 4 speed depends on various model parameters as , for example , cytoskeletal contractility or adhesion strength . A later variant of this model presents a more explicit study of adhesion bond disruption kinetics at the rear of the cell and already uses a four - state model for integrin binding to the cytoskeleton and to the substratum [ 51 ] . In recent years a series of more elaborate models have been developed , accounting for details of the meanwhile discovered molecular regulation mechanisms for the chemical and mechanical processes , particularly at the free boundary of a moving cell . One model type is based on spatially discrete algorithms ( cellular Pott’s model ) using the definition of local energies to determine the protrusion and retraction of boundary elements via the stochastic Metropolis rule , e . g . [ 39 , 46 , 47 ] . Another class of mechanical cell models takes into account branching and anisotropy of actin filaments in the cytoskeleton at various times and in various regions of the cell , see e . g . [ 65 , 23 , 28 ] . Besides explicit constructions of an elastically cross - linked network in the leading edge [ 48 , 32 ] biophysical models have been developed to capture different dynamics of Arp2 / 3 - induced branching and myosin - induced contraction of F - actin networks by Mogilner and coworkers [ 44 , 25 , 33 ] . The last article is particularly devoted to explain the mentioned polarization experiments with cell fragments ( see Fig . 1 ) by assuming a different actin - myosin network organization at the rear in comparison to the front region . Based on the ‘reactive flow’ model by Dembo and coworkers [ 13 , 12 , 3 ] , the so far most elaborate model extension has been presented by Oliver et al . [ 49 ] : they use full 3 - D two - phase flow equations with free ventral boundary and with two additional rapidly diffusing messenger concentrations that regulate actin network contractility and ( de - ) polymerization . Moreover , cell adhesion is modeled by a Navier - slip boundary condition at the substratum , in which only constant adhesive properties are taken into account . The analysis of this complex model , being performed in the thin film limit , is restricted to linear - stability ar - guments for ruffle generation and to local expansion analyses at the moving tip ; there , phenomenological equations for boundary mass fluxes are considered without specifying the types of molecular mechanisms for tip protrusion . Finally , quantitative estimations for the pseudopod protrusion and cell translocation speed under various sub - limit assumptions are given , which turn out to be consistent with observed values , particularly for osteoblasts , though no numerical simulations are given that would re - insure the analytic results . A sim - ilar thin - film approximation of the 2 - D equations under incompressibility assumptions for a ‘viscous polar gel’ has been used in [ 34 ] to derive explicit expressions for the advancing speed of a cell lamella , but again by predefining its polarity . Except the last one , all so - far mentioned models do not explicitly quantify the varying force field , which is applied by the cytoskeleton onto the substrate covered by a migrating cell and which has been approximately reconstructed by different inverse methods from experimental assays of cells , e . g . moving on flexible substrata [ 15 , 38 , 56 , 40 , 6 ] . A first model implementing force transduction to the substrate has been proposed by Gracheva and Othmer [ 24 ] by specifying a spatially one - dimensional system of viscoelastic equations for cytoskeleton dynamics , whose polymerization , contractility and adhesive binding is reg - 5 ulated by signalling molecules . However , they make a pseudo - steady - state assumption for the binding kinetics of myosin polymers to actin filaments , and of transmembrane integrin proteins to the substratum . Moreover , they impose artificially defined gradients from tail to front of certain regulatory proteins in order to stimulate polarized cell translocation . Recently , adhesion kinetics has also been implemented into an extended cytoplasm flow model [ 4 , 5 ] describing the F - actin dynamics in an annular domain and its coupling to lamel - lipodial protrusions and retractions [ 59 ] . Force dependent maturation of FA complexes and active polymerization of F - actin enable the simulation model to reproduce characteristics of fibroblast shape deformation and translocation . In an earlier publication we have presented another extension of the basic two - phase flow model for the 2 - dimensional viscous cytoplasm dynamics [ 2 ] by coupling the constitutive hyperbolic - elliptic equations to a system of four reaction - diffusion - transport equations for the integrins beneath the cell or cell fragment [ 35 ] . Here we propose a generalized contin - uum model , in which we couple cytoplasm and adhesion dynamics with mechanical tension and transport of the plasma membrane , in order to reproduce spontaneous and induced cell polarization leading to migration , with assembly of adhesion sites at the cell front and adhesion release at the cell’s rear end . Moreover , the model exhibits typical features of migrating cells as protrusion / retraction cycles , rearward actin flow and pulling forces at the front and a concentration of disruptive forces at the rear . In the model , the cytoplasm is described as a viscous and contractile fluid of polymers representing the actin cytoskele - ton interpenetrated by an aqueous phase . This actin filament network is preferentially assembled at the cell edge , and can be contracted by cross - linking with diffusing myosin oligomers . The moving actin filaments then couple to transmembrane adhesion proteins that are freely diffusing in the membrane or bound to the substrate on the extracellular domain . Thus , the cytoskeleton mechanically connects to the substrate through dynamic binding processes which results in force transduction and finally cell locomotion . Throughout our model presentation we rely on continuum descriptions , in which macro - scopic mass and momentum laws are combined with mesoscopic submodels for fast molec - ular kinetics due to adequate pseudo - steady - state assumptions . 3 Two - phase flow model for cytoplasm coupled to reaction , diffusion and transport for myosin - II and integrin proteins For simplicity , we restrict our model derivation and analysis to a flat two - dimensional ge - ometry , so that cells or cell fragments are assumed to be homogeneously spread on the substrate without considerable change of cell height . Thereby , three - dimensional effects around the cell nucleus ( e . g . due to cell rolling ) or along the ventral ( upper ) plasma mem - brane are neglected . In order to reproduce the main biophysical mechanisms and biochem - ical processes that enable a cell to polarize and translocate on an adhesive substratum , we nevertheless distinguish between the cytoplasm and the exterior plasma membrane : On the 6 Figure 2 : Schematic longitudinal section through a cell fragment ( or a cell lamella ) of con - stant height with involved proteins . For further explanation and model derivation see the following sections . one hand , F - actin assembly , myosin kinetics and viscous network flow take place within the cytoplasmic interior of the cell . On the other hand , integrin binding to substrate or cytoskeleton and its transport or diffusion are confined to the dorsal ( lower ) plasma membrane , where forces are transduced to or generated at the cell periphery , leading to tip protrusion or retraction . Thus , the moving cell ( fragment ) is simply represented by a time - dependent connected domain Ω ( t ) ⊂ R 2 , over which the cytoplasmic volume ex - tends with fixed constant height , and any ruffles or blebs on top of the cell are neglected . We rather assume that the cell dynamics is completely determined by a flat cytoskeleton sheet of F - actin network , see Fig . 2 . This cross - linked filament phase with volume fraction θ ( t , x ) , x ∈ Ω ( t ) is of constant thickness and connected to the upper and lower plasma membrane in such a way that also the cytosol , i . e . the solvent phase with volume fraction ( 1 − θ ( t , x ) ) , is confined to the same volume space . By suitable model simplifications , we aim to capture the self - organizational power of the cytoplasm as a two - phase fluid coupled with the reaction , transport and diffusion of a series of chemical ingredients . We explicitly model the kinetics and dynamics of only those F - actin associated proteins that induce the main bio - mechanical processes of force generation and transduction to the substratum , namely myosin oligomers and transmembrane integrin proteins . All other regulatory proteins such as e . g . Rac , Rho or the branching protein Arp2 / 3 or smaller substrate molecules , e . g . monomeric G - actin , are assumed to rapidly diffuse within the cytosol under fast regeneration , so that they attain constant reservoir concentrations serving as parameters in the model . 7 3 . 1 Mass balance and flow equations In the chosen two - dimensional continuum description , cell deformation and transloca - tion with respect to the fixed substratum is represented by movement of the cell edge Γ ( t ) = ∂ Ω ( t ) which , however , is induced by three distinguished mass flows with poten - tially different transport velocity fields on Ω , namely the mean velocities v of the F - actin cytoskeleton ( θ ) and w of the aqueous cytosol ( 1 − θ ) plus the transport velocity u of the lower dorsal plasma membrane . Thereby both v and w are averaged over the constant cell height . For the membrane area flux u we make the simplifying assumption that the lipid bilayer between the flat substratum and an adhering cell is always stretched out to its maximal extension with constant area concentration of the lipid - protein mixture , thus constituting an incompressible two - dimensional Newtonian fluid . Thus , freely diffusing transmembrane integrin proteins are additionally transported by the membrane velocity u , myosin oligomers by the cytosol velocity w , and F - actin bound myosin - II motor molecules by the cytoskeleton velocity v . The detailed mass balance equations are discussed in the following paragraphs . Mass conservation for the two phases cytoskeleton and cytosol . Cytoskeleton and cytosol in the flat two - dimensional geometry can experience counteracting flows due to local contraction , assembly or relaxation of the F - actin network , while the bulk cytoplasmic fluid with constant volume fraction 1 can be assumed to be incompressible , at least in the range of occurring pressure differences (cid:46) kPa . Compare the analogous situation of a water - filled sponge that is internally condensing without changing its shape . Then , because of the fixed height assumption , the total two - dimensional volume flux W = θ v + ( 1 − θ ) w is divergence free at any time t , yielding the first local mass conservation law ∇ · (cid:16) θ v + ( 1 − θ ) w (cid:17) = 0 . ( 1 ) Therefore , also the total cell volume ( i . e . the 2 - dimensional cell area ) is conserved over time , so that the ‘bulk’ fluid moves together with the free boundary Γ ( t ) , meaning that the total volume flux W has to fulfill the natural free boundary condition ν Γ · W = ˙Γ ( 2 ) with ν Γ denoting the exterior normal of Γ and ˙ Γ quantifying the normal speed of the cell edge . In addition to possible convection , the F - actin network can locally be assembled by filament polymerization from the pool of monomeric G - actin within the cytosol and it can be disassembled by the reverse process of severing or depolymerization . This mass exchange between the two phases is given by a second conservation law , which for the filament volume fraction θ can be written as ∂ t θ + ∇ · ( θ v ) = R ( θ ) ( 3 ) 8 with a net assembly rate R ( θ ) to be modeled . According to our simplifying assumption , the dependence on G - actin , Arp2 / 3 and possible regulatory proteins enters only via constant parameters : R ( θ ) = ( k on a g − k off ) B ( θ ) − rθ ( 4 ) with concentration of globular actin a g , polymerization rate k on and depolymerization rate k off at the filaments’ plus ( barbed ) ends , as well as another lumped disassembly rate r , see e . g . [ 42 ] . Due to relatively fast nucleation and capping of actin filaments , the relative number B of barbed ends is assumed to stay in pseudo - equilibrium with the local F - actin concentration a = θ · a max , where we suppose a maximal condensation of actin filaments in the order of a max = 800 µ M . Following [ 42 ] we write B ( θ ) = 1 ω (cid:18) ε + ν 0 θ K a / a max + θ (cid:19) ( 5 ) with capping rate ω , a basic nucleation rate ε and an induced branching rate ν 0 = ν a Arp0 , proportional to the concentration Arp0 [ µ M ] of activated Arp2 / 3 , together with a half - saturation concentration K a for its primary actin binding site . Reaction - transport - diffusion equations for myosin oligomers . Myosin - II oligomers are the most important actin binding proteins that are responsible for the generation of con - tractile forces within cross - linked F - actin networks . Thus , their spatial distribution within a polarizing or moving cell plays a key role for the cytoplasm dynamics . In a most simple way we only distinguish between freely diffusing myosin oligomers ( m f ) and those that are bound to cytoskeletal actin filaments ( m b ) and , therefore , are convected with velocity v . Since free myosin tetramers first have to attach to the actin network at one binding site , we consider this bimolecular reaction ( m f with a ) as the rate limiting step . Correspondingly we assume , that the relatively faster processes of double binding ( m b with a ) and power stroke formation are in a pseudo - steady state . Thereby the contractile stress ψ = ψ 0 m b θ with θ = a / a max in the network is determined , see equation ( 25 ) in the following section . Finally , we suppose a constant diffusivity D m for free myosin oligomers , embedded into the cytosol flow w . Under these assumptions the local mass balance equations for the corresponding myosin concentrations are : ∂ t m f = ∇ · (cid:16) D m ∇ m f − m f w (cid:17) − α m · a · m f + δ m ( a ) · m b ( 6 ) ∂ t m b = −∇ · (cid:0) m b v (cid:1) + α m · a · m f − δ m ( a ) · m b . ( 7 ) For the association rate α m we assume a constant parameter , whereas the dissociation rate is supposed to increase quadratically with increasing network concentration a because of steric inhibition or competition for binding sites : δ m ( a ) = δ 0 m (cid:18) 1 + a 2 a 2opt (cid:19) = δ 0 m (cid:18) 1 + θ 2 θ 2opt (cid:19) , ( 8 ) 9 with an optimal actin network concentration a opt = a max θ opt . Moreover , in the case of fast diffusion and no transport , the constant equilibrium concentrations m ∗ f and m ∗ b satisfy the equality m ∗ b = α m a δ m ( a ) m ∗ f , ( 9 ) so that the resulting contractile stress ψ ( θ ) becomes optimal for θ = θ opt , Fig . 5 below . Mass conserving flow of the dorsal plasma membrane . As mentioned before , we will assume that the dorsal plasma membrane beneath an adhering cell ( fragment ) is stretched and that small fluctuations can be neglected . On the contrary top side , the ventral plasma - membrane usually shows extensive wrinkles or folds , which most probably are induced by the contractile action of the cytoskeleton itself , indicating that the plasma membrane tip at the cell edge stays under a positive tension τ Γ . Before discussing the corresponding dynamics of the dorsal membrane , we state the two extreme possibilities , namely whether the membrane moves together with the cell or not : 1 . Membrane sticking to substrate : During cell translocation over the substratum the whole dorsal membrane stays fixed and no slip may occur due to relatively strong interaction forces with the substratum , e . g . via the glykocalix , so that u ≡ 0 . 2 . Membrane sticking to cell edges : The dorsal membrane is pulled along the substratum due to cytoplasm protrusions at the leading front , but with no slip around the lamellar tips due to strong membrane curvature . In this way the membrane area flux satisfies the mass conservation law together with a boundary condition analogous to ( 2 ) , namely ∇ · u = 0 on Ω ( t ) ν Γ · u = ˙Γ on Γ ( t ) . ( 10 ) In the latter case , the membrane can slip over the substratum , thereby experiencing a finite frictional drag force , see ( 40 ) below , whereas in the first case this force is infinitely large to suppress slipping – but then the membrane has to slip around the tips at moving cell edges . Clearly , a certain mixture of both possibilities is physically realizable but not considered here . Moreover , frictional drag onto the dorsal membrane can also occur at its cytoplasmic side . Whereas the relative flow of cytosol will have negligible effects , the horizontal F - actin flow develops a vertical flow profile . This profile depends on the amount of ‘Navier - slip’ in a cortical layer on top of the dorsal membrane and emerges due to frictional forces between actin filaments and membrane proteins . In analogy to the more explicit thin film approximation [ 49 ] we only consider the averaged profile velocity v . In this way we can introduce a simplifying cortical slip parameter , 0 < κ < 1 , so that the effective relative velocity between cytoskeleton and dorsal membrane is reduced to κ ( v − u ) . Thus , the 10 resulting effective velocity of cortical F - actin in a thin layer above dorsal membrane and substratum is given by v c = κ v + ( 1 − κ ) u . ( 11 ) Here the factor κ , which clearly depends on the viscous shear properties of the cytoskeleton , should be larger than 0 . 5 to reflect observations of a generally slippy behavior ( see e . g . [ 29 , 22 ] ) . Moreover , we suppose that the vertical profile , thus v c , is not changed if some of the cortical actin filaments are ( transiently ) bound to integrins in the dorsal membrane , see Fig . 3 and the following paragraph : Either those bound integrins or integrin complexes are passively pulled through the lipid - protein bilayer with relative velocity v c − u or , in case of substrate - fixed adhesion bonds , with relative velocity − u . The whole F - actin network above such a bond is slowed down by a certain local frictional force per adhesion site , F c = Φ 0 θ v c , ( 12 ) entering into the corresponding force balance law , see ( 23 ) below . In any case , the fric - tional drag onto the dorsal membrane , induced by the relative motion of singular integrin complexes , will be neglected in our model . Reaction - transport - diffusion equations for membrane integrins . The mechanical con - nection between the actin cytoskeleton and extracellular matrix proteins is provided by transmembrane integrins appearing in four different states [ 51 , 35 ] , see Fig . 3 . In the freely diffusing state f , integrins are neither coupled to the substrate nor to the actin cytoskeleton and move according to a simple diffusion - transport law within the dorsal membrane . These integrins can change their state by binding to the actin cytoskeleton ( a ) or to the substrate ( s ) . In state a , the integrins move with the cortical F - actin velocity v c , whereas integrins in state s remain stationary with respect to the substrate . Actin - or substrate - bound integrins can switch back into the freely diffusing state f by unbinding , or into the state sa by coupling to the cytoskeleton and the extracellular matrix . Cell ad - hesion occurs only in this double bound state s representing a focal adhesion ( FA ) , where the frictional force F c ( 12 ) of the moving actin - network is transduced to the substrate . The concentration of integrins c # in the different states is described by the following cou - pled system of differential equations consisting of terms for spatial movement and binding kinetics . ∂ t c f = ∇ · (cid:0) D f ∇ c f − c f u (cid:1) + α − c s + δ − c a − (cid:0) δ + 0 a + α + (cid:1) c f ( 13 ) ∂ t c a = −∇ · (cid:0) c a v c (cid:1) + δ 0 + ac f − (cid:0) δ − + γ + (cid:1) c a + γ − 0 exp (cid:0) ρ γ | F c | (cid:1) c sa ( 14 ) ∂ t c s = α + c f − (cid:0) α − + β + 0 a (cid:1) c s + β − 0 exp (cid:0) ρ β | F c | (cid:1) c sa ( 15 ) ∂ t c sa = γ + c a + β + 0 ac s − (cid:16) β − 0 exp (cid:0) ρ β | F c | (cid:1) + γ − 0 exp (cid:0) ρ γ | F c | (cid:1)(cid:17) c sa ( 16 ) The bonds of the force - transducing integrins ( c sa ) can break when they experience a me - chanical stress by the cytoskeleton , in our model given by the modulus of the force vector 11 f α + α − γ + γ − δ + δ − β − β + sa a s Figure 3 : Scheme of four states of the transmembrane adhesion protein integrin . ( f ) : un - bound and freely diffusing within the dorsal membrane , ( s ) : bound to the sub - strate , ( a ) : bound to the actin network and thus moving with the actin - flow , ( sa ) : bound to actin and substrate ( force - transducing state ) . Integrins can switch be - tween these states due to reversible binding kinetics with binding / unbinding rates α , β , γ and δ . F c defined in ( 12 ) . Then , according to the theory of Bell [ 7 , 57 ] the dissociation rates γ − and β − depend exponentially on the mechanical load | F c | , see equations ( 14 – 16 ) above , with γ − 0 and β − 0 describing the basic dissociation rates without load and the exponential coefficients ρ # = ζ # / k B T measuring potentially different rupture rates from the substrate or cytoskeleton binding site , respectively . Mass flux conditions at the free boundary . In addition to the already mentioned bulk flux conditions ( 2 ) and ( 11 ) related to the normal speed of the cell edge Γ ( t ) , we have to impose compatible boundary conditions onto the concentrations of those molecular species that are not fixed to the substratum . For the two parabolic diffusion equations ( 6 ) and ( 13 ) we impose , respectively , natural zero - flux boundary conditions onto freely diffusing myosin ( m f ) and fixed Dirichlet conditions ( c f = c 0 f ) onto freely diffusing integrin . Thereby we suppose a constant reservoir of fresh adhesion receptors expressed in the upper ventral membrane and diffusing ( or eventually being transported ) from there around the lamellar tip into the lower dorsal part of the membrane . The F - actin flux θ v cannot leave the cell , meaning that on the free boundary Γ the relative inward normal F - actin velocity always has to satisfy the inequality V = ˙Γ − ν Γ · v ≥ 0 . ( 17 ) If the strong inequality V > 0 holds at a certain boundary point , then two different modeling situations may arise for the cell edge : 12 1 . No - stick condition at the lamellar tip : Local disruption of the actin network from the edge is allowed , e . g . under suitable load conditions [ 35 ] , so there is no new F - actin production directly at plasma membrane edge . Therefore we have to impose zero - Dirichlet condition for the F - actin concentration θ = 0 if V > 0 holds at a non - sticky point x ∈ Γ . ( 18 ) 2 . Network sticking to the lamellar tip : As indicated in Fig . 4 , active polymerization of actin filaments directly at the plasma membrane is allowed either ( a ) at fluctuating free filament ends touching the tip membrane [ 43 ] , or ( b ) at filaments that are bound to clamp - motor proteins anchored in the tip membrane [ 16 ] . Both cases could occur simultaneously in a local region , but whether active polymerization with inward mass flux θV > 0 can take place depends on local force balance conditions , see Section 3 . 2 and ( 36 ) below . Figure 4 : Schematic top view onto the lamellar tip of a cell ( fragment ) showing three pos - sibilities for actin filament ends to interact with the free plasma membrane ( and corresponding molar concentrations ) : Anchored at a membrane protein , e . g . in - tegrin , ( a b ) , freely fluctuating with polymerization by a ‘brownian ratchet’ mech - anism ( a f ) , or bound to a ‘clamp - motor’ protein , e . g . WASP - complex , with in - duced polymerization ( a c ) . See equations ( 31 ) , ( 33 ) for a quantitative model . Finally , also the two hyperbolic equations ( 7 ) and ( 14 ) require zero - influx conditions , so that for the transported concentrations m b and c a we have to impose zero - Dirichlet condi - tions only in cases of V > 0 or V c > 0 , respectively . In the latter case the inward normal velocity of v c is defined in analogy to ( 17 ) , noting that under the modeling hypothesis ( 10 ) we anyway have V c = κV . 13 3 . 2 Force balance equations Two - phase flow equations for cytoskeleton and cytosol . Since cell movement usually occurs on a time scale of minutes , and cell sizes usually are in the order of tens of mi - crons , already the cytosol , with its consistency similar to an aqueous viscous fluid , has low Reynolds number . The more this is true for the cytoskeleton with consistency similar to a dense viscoelastic gel . The viscoelastic properties of the cytoplasm have been experi - mentally studied [ 18 , 19 ] and also mathematically modeled [ 11 , 49 ] , whereby the simple model of a Maxwell fluid seems to serve as a quite good description . This means , that elastic properties are mainly effective on a shorter time scale of seconds and dominated by viscous effects on a medium scale of minutes . Moreover , in contrast to a passive elastic material , the cytoplasm mostly stays under active contractile stress as exerted by myosin - II oligomers ( in the order of kPa ) , which is able to break weaker cross - links of the F - actin network e . g . by α - actinin or filamin , so that the contraction forces are equilibrated only by viscous and frictional forces . Under these assumptions , the highly viscous two - phase creeping flow model for cyto - plasm , originally proposed 25 years ago by Dembo et al . [ 14 ] and so far extensively ap - plied , see e . g . [ 1 , 26 ] , can be condensed into a pseudo - stationary linear elliptic system for the mean F - actin velocity v and the effective hydrostatic pressure p on Ω ( t ) : ∇ · µθ (cid:101) ∇ v + ∇ (cid:16) S ( θ , m b ) − p (cid:17) = Φ ( θ , c sa ) v c , ( 19 ) ∇ p = ϕθ ( v − w ) . ( 20 ) Here the generalized ( elliptic ) Stokes equation ( 19 ) involves the effective stress S ( θ , m b ) as defined in equation ( 24 ) and the symmetrized displacement rate (cid:101) ∇ v ( see e . g . [ 35 ] ) . Moreover , v c is defined in ( 11 ) , and µ , Φ denote the coefficients of bulk viscosity and substratum friction , see ( 23 ) . Because of negligible cytosol viscosity the simple Darcy law ( 20 ) suffices as a model description , where the coefficient ϕ measures the internal two - phase flow friction . From the last equation one explicitly solves for the cytosol velocity w = v − ( 1 / ϕθ ) ∇ p , so that the total volume flux is W = v + ( 1 − θ ) ( w − v ) = v − 1 − θ ϕθ ∇ p . ( 21 ) Insertion into the mass - balance equation ( 1 ) then yields the generalized ( elliptic ) Laplace equation ∇ · 1 − θ ϕθ ∇ p = ∇ · v . ( 22 ) Notice that here the ellipticity degenerates for marginal volume fractions 0 < θ < 1 , which can be relevant in cases where θ → 0 at boundary points of cytoskeleton disruption , see ( 18 ) above . 14 We remark that together with the hyperbolic mass balance equation ( 3 ) the linear elliptic system ( 19 , 22 ) constitutes generalized pseudo - stationary Navier - Stokes equations for the F - actin network as a compressible , highly viscous and reactive fluid . It is mutually coupled to the mass concentrations in equations ( 7 ) and ( 16 ) via a myosin - mediated contractile stress term appearing in S ( θ , m b ) , see eq . ( 24 ) below , and an adhesion - mediated friction coefficient Φ ( θ , c sa ) = Φ 0 c sa θ . ( 23 ) In this way the right hand side of ( 19 ) reads F v = Φ v c = c sa F c , with the frictional force F c per doubly bound integrin defined in ( 12 ) above . Tracing these model equations back to their derivation [ 12 , 2 , 35 ] provides us not only with precise biophysical conditions on v and p at the moving boundary Γ ( see next para - graph ) , but also with genuine nonlinear parameter functions being based on thermody - namical reasoning at the molecular scale : The function S in ( 19 ) represents the effective stress in the network phase , which is induced by molecular interactions between the F - actin filaments themselves as well as by thermic interactions with solvent molecules in the cytosol . It is generally expressed as the weighted negative sum S = − θP a − ( 1 − θ ) P s of corresponding pressures P a and P s which are applied to any network volume element and any cytosol element , respectively . Passive elastic stresses may be neglected since these are already relaxed on the creeping flow timescale . When finding a free binding site on filaments , previously singly bound myosin - II tetramers exert power stroke motor forces with their free myosin heads , cf . Figs . 2 and 4 . Thus , an attractive stress − P a = ψ 0 m b is applied , where the simple coefficient ψ 0 comprises binding affinity , power stroke probability and the mean applied force per stroke . On the other hand , standard Gibbs free energy arguments suggest a molecular solvent pressure P s = − σ 0 ln ( 1 − θ ) / ( 1 − θ ) , see [ 2 ] . Then we arrive at expressions for the effective stress S = S ( θ , m b ) = ψ ( θ , m b ) − σ ( θ ) ( 24 ) contractile stress ψ ( θ , m b ) = ψ 0 θm b ( 25 ) swelling pressure σ ( θ ) = σ 0 | ln ( 1 − θ ) | , ( 26 ) see Fig . 5 for corresponding plots with m b = m ∗ b in ( 9 ) . We emphasize that the effective stress function ( 24 ) sums up the contributions from the cytoskeleton and the cytosol , so that both phases are not separated anymore . However , when deriving the corresponding natural boundary conditions for the elliptic problem , then the two phases have to be considered separately again . Stress and pressure balance conditions at the free boundary . Recall that according to ( 2 ) the cell edge Γ ( t ) always moves together with the normal component of the total cytoplasm flux W as quantified in ( 21 ) . Then , from ( 17 ) also the relative inward normal 15 0 0 . 02 0 . 04 0 . 06 0 . 08 −1 0 1 2 3 4 θ : F−actin volume fraction R [ min −1 ] : assembly ψ [ kPa ] : contraction σ [ kPa ] : swelling S [ kPa ] : stress Figure 5 : Plots of F - actin model func - tions . Bold curves : net assembly rate R ( θ ) ( 4 ) and effective stress S ( θ ) = ψ ( θ ) − σ ( θ ) ( 24 ) . Light curves : contractile stress ψ de - fined in ( 41 ) and swelling pres - sure σ ( 26 ) . F - actin flux is determined as proportional to the inward normal pressure gradient on Γ ( t ) θV = θ (cid:16) ˙Γ − ν Γ · v (cid:17) = − 1 − θ ϕ ν Γ · ∇ p . ( 27 ) This explicit linear relation between free boundary speed , normal F - actin velocity and pressure gradient serves as an extra free boundary condition in addition to the set of boundary conditions that are necessary for uniquely solving the linear elliptic system ( 19 ) and ( 22 ) on the given domain Ω ( t ) . These depend on the particular model choice of boundary pressure functions at the cell edge membrane , but also on the outcome of the F - actin flow requirement V ≥ 0 in ( 17 ) . Using the general derivations in [ 2 ] , eqs . ( 58 - 62 ) , one obtains separate pressure balance conditions for each of the two phases at all free boundary points of Γ ( t ) satisfying V > 0 , cytoskeleton pressure balance − ν Γ · T a · ν Γ + θp = θP Γ a ( 28 ) and cytosol pressure balance ( 1 − θ ) p + σ ( θ ) = ( 1 − θ ) P Γ s ( 29 ) with the intrinsic stress tensor T a = µθ (cid:101) ∇ v + ψ ( θ , m b ) I . These equations mean that at those parts of the tip plasma membrane , which are exposed to the cytoskeleton network ( θ ) or to the cytosol ( 1 − θ ) , respectively , the sum of internal pressures is in balance with a certain boundary cytoskeleton pressure P Γ a or boundary cytosol pressure P Γ s at each volume element . Modeling expressions for these pressures will be given in the following paragraph . Summing up both pressure balance equations ( 28 ) and ( 29 ) yields the general Neumann - type condition for v on Γ ( t ) ν Γ · T a · ν Γ − (cid:0) σ ( θ ) + p (cid:1) + θP Γ a + ( 1 − θ ) P Γ s = 0 , ( 30 ) which now is valid without restriction , see [ 2 ] , eq . ( 58 ) , i . e . also in boundary points where the condition V = 0 holds . However , then the Dirichlet - type boundary condition ( 29 ) for p 16 has to be replaced by a corresponding inequality , and insertion of V = 0 into ( 27 ) provides a Neumann condition for p instead . In particular we have ν Γ · ∇ p = 0 , which also holds in case of disruption with V > 0 and θ = 0 . Thus , in any constellation of the mentioned conditions on Γ ( t ) , for fixed time t and given profile of all protein concentrations , we obtain a well - posed elliptic boundary value problem for v and p to be solved on Ω ( t ) ( eventually by numeric iteration and / or conjugate gradient method , cf . [ 35 ] ) . Finally , the normal speed ˙Γ of the free boundary can be calculated by using equation ( 27 ) . This is also possible in the case of network disruption at some boundary point x 0 ∈ Γ ( t ) with θ ( t , x 0 ) = 0 , since then V ( t , x 0 ) > 0 is obtained as the L’Hospital - limit of − 1 − θ ( t , x ) θ ( t , x ) ν Γ · ∇ p ( t , x ) as x → x 0 from the interior . Boundary pressure functions at the cell edge . We only consider creeping cell migration in a medium that is not under external pressure or stress . Therefore , in a purely physical model , the boundary pressures P Γ a and P Γ s along the cell edge Γ ( t ) should be set to zero . However , the edge is physically defined as the lamellar tip , where the ventral and dorsal part of the surrounding plasma membrane meet . Thus , if τ Γ denotes the scalar tension value of the dorsal plasma membrane at the cell edge ( see the following paragraph ) then this tension appears as a boundary tip pressure acting on both phases , the cytoskeleton and the cytosol . Moreover , in addition to the counteracting cytoskeleton and cytosol pressure at the left hand sides of eqs . ( 28 ) and ( 29 ) , there could be extra pressures due to active polymerization forces , which usually are generated at barbed actin filament ends that can become exposed to the tip membrane in two variants , see Fig . 4 above : • Brownian ratchet model : Assume that at a point of the lamellar tip Γ ( t ) a frac - tion a B of filaments is bound to membrane proteins in a fast pseudo - steady state equilibrium with the actual F - actin concentration a = θa max . Then from the remain - ing network with concentration a F ( a ) = a − a B ( a ) , Arp2 / 3 induced branching can occur . The barbed filament ends are more or less normally exposed to the tip mem - brane with concentration a f = a f ( a ) = α f 0 Arp0 · a F / ( K a + a F ) with suitably chosen coefficients , see also ( 5 ) . Then , due to insertion of G - actin in - between fluctuating filament and membrane there appears a free polymerization pressure p f = π f 0 · a f ( a ) ( 31 ) with some ratchet coefficient π f 0 > 0 depending on the free energy of one monomer addition , see relevant force estimations e . g . in [ 41 ] . • Clamp - motor model : Alternatively or additionally , a certain fraction of tip bound actin filaments with concentration a c = a c ( a ) < a B ( a ) can be bound to WASP - like membrane proteins , which serve as filament - end - tracking motors . By means of an en - ergy consuming polymer elongation process at the clamped end of the filament , these 17 proteins push the bound filament outward , thus leading to a clamp polymerization pressure p c = π c 0 · a c ( a ) . ( 32 ) Here again , the clamp - motor coefficient π c 0 > 0 depends on the energy of one monomer translocation , see [ 16 ] . The sum of these active polymerization pressures would induce an averaged relative normal inward mass flux θV ≥ 0 of the whole cytoskeleton , based on the particular inflows of a f - and a c - filaments satisfying θV = a f V f = a c V c . However , this flow experiences a viscous resistance from the remaining fixed filaments ( concentration a B − a c ) , with a viscosity coefficient that could increase in the presence of bound myosin molecules . Thus we get the total tip polymerization pressure P poly = p f + p c − µ Γ ( m b ) (cid:0) a B − a c (cid:1) a max V . ( 33 ) Clearly , here the relative inward velocity V is the one defined by the normal component of v in relation to ˙Γ , see ( 17 ) . For a similar resistance model based on elastic cross - linking see [ 23 ] . Finally , since this intrinsic boundary pressure P poly acts onto the cytoskeleton volume fraction , while simultaneously inducing a corresponding counter - pressure onto the cytosol volume fraction , we can state the following distribution for the boundary pressures gener - ated at the membrane θP Γ a = θ ( 1 − κ Γ ) τ Γ + P poly , ( 34 ) ( 1 − θ ) P Γ s = ( 1 − θ + θκ Γ ) τ Γ − P poly , ( 35 ) where we introduce a weight factor , 0 ≤ κ Γ < 1 , measuring the relative effect of membrane tension τ Γ onto the cytosol phase and possibly depending on the not - explicitly modeled tip geometry . Then , by substituting p from ( 29 ) into ( 30 ) we obtain the generalized Neumann - type boundary condition for v in all points of Γ ( t ) where the network is attached , a B > 0 , and where V > 0 holds : ν Γ · T a · ν Γ − σ ( θ ) − a max 1 − θ µ Γ ( m b ) (cid:16) a B − a c (cid:17) V + 1 1 − θ (cid:16) σ ( θ ) + p f + p c − θκ Γ τ Γ (cid:17) = 0 . ( 36 ) This means , that the mean inward F - actin polymerization speed V ≥ 0 on the moving cell edge Γ ( t ) is implicitly determined by solving the linear elliptic system ( 19 ) and ( 22 ) for v and p and satisfying all boundary conditions . Thereby the Neumann condition above contains all membrane protruding pressure terms in series , namely the swelling pressure σ , the polymerization pressures p f and p c induced by a Brownian ratchet or an clamp - motor mechanism , as well as a counteracting stress due to dorsal membrane tension τ Γ . 18 Global force balance at the adhesive substratum . There are two kinds of forces exerted by the migrating cell ( fragment ) onto the fixed flat substratum : the integrin - adhesion mediated active frictional force represented by the vector field F v on the right hand side of Stokes’ equation ( 19 ) , and an analogous passive frictional force F u due to motion of the dorsal plasma membrane along the substratum : F v = c sa F c = c sa Φ 0 v c , ( 37 ) F u = Φ u u , ( 38 ) with Φ as in ( 23 ) and an assumed friction constant Φ u ≥ 0 . Supposing that on the substratum no other forces are applied than these , then their integral sum has to vanish , so that the zero force balance holds : 0 = (cid:90) Ω ( t ) ( F v + F u ) . ( 39 ) Furthermore , if we assume that the dorsal membrane , viewed as a 2 - dimensional incom - pressible fluid satisfying the zero - divergence condition ( 10 ) , has relatively low viscosity , then the membrane tension τ u induced by the frictional flow can be defined according to Darcy’s law ∇ τ u = Φ u u ( 40 ) and thus determined as solution of the Laplace equation ∆ τ u = 0 with Neumann boundary condition ν Γ · ∇ τ u = 0 , see ( 10 ) . Then the zero force balance ( 39 ) together with ( 19 ) implies that the boundary tension values τ Γ = τ u | Γ ( t ) , which are uniquely determined up to a constant , necessarily fulfill the integrability condition (cid:82) Γ ( t ) ( µθ (cid:101) ∇ v + S ( θ , µ b ) − p + τ Γ ) ν Γ = 0 , which by insertion of the Neumann boundary condition ( 30 ) and by using the symmetry of (cid:101) ∇ v reduces to the equivalent necessary condition (cid:82) Γ ( t ) ( θP Γ a + ( 1 − θ ) P Γ s − τ Γ ) ν Γ = 0 . Indeed , this condition is fulfilled for the boundary pressure model functions that were chosen in the preceding paragraph , ( 34 ) and ( 35 ) , because then the integrand in the previous condition even vanishes . 4 Results of model simulations 4 . 1 Spontaneous cell polarization in the 2 - D model We have simulated the adhesive motion of a flat cell or cell fragment represented by a 2 - dimensional domain , Ω ( t ) , with moving cell edge or lamellar tip , Γ ( t ) = ∂ Ω ( t ) , under certain simplifying assumptions : 1 . The lower dorsal membrane sticks to the substratum ( u = 0 ) and there exists a small membrane tension τ Γ > 0 , constant over the whole cell edge . 19 2 . There occurs no active polymerization pressure at the cell edge ( p f = p c = 0 ) , only the similar swelling pressure σ ( θ ) and the hydrostatic pressure p can push the boundary . 3 . Disruption of the F - actin network from the lamellar tip Γ ( t ) can locally occur if the network tension exceeds a certain threshold that might depend on the fraction of membrane - bound actin filaments a B = A a K B + a . 4 . The free myosin - II concentration is at a fixed constant level m 0 f > 0 and the amount of F - actin bound myosin - II oligomers is in a pseudo - steady equilibrium m ∗ b ( a ) according to ( 9 ) , so that the contractile stress is only a function of θ = a / a max : ψ ( θ ) = ψ 0 θm ∗ b = ψ 0 α m m 0 f a max δ 0 m θ 2 1 + θ 2 / θ 2opt . ( 41 ) More details and a list of chosen parameters can be found in [ 35 ] ( Section 1 and Table 2 ) . Since unpolarized cells and cell fragments , as observed under various conditions [ 63 , 64 ] , attain a quite regular circular shape , we choose as initial condition a circle Ω ( 0 ) of radius 6 µ m . Moreover , in order to mimic the radial spreading of cells after exposure onto a flat substratum , we start with constant integrin densities and radially symmetric initial configuration for the volume fraction θ , with slightly larger values closer to the center . Due to this initial perturbation , the F - actin concentration rapidly condenses into a central region of high θ , surrounded by a lamella - kind region of low θ , see Fig . 6 ( a , b ) . Later , also the actin - and surface - bound integrin adhesion proteins c sa concentrate around this center , see Fig . 6 ( e ) . Both phenomena are supported by a strong retrograde F - actin flow , which collects actin filaments and actin bound integrins c a in radial direction from almost everywhere in the periphery . Moreover , the hydrodynamic pressure has its maximum within the center region ( data not shown ) , so that its negative outward gradient represents the squeezed flow of cytosol out of the contracting F - actin network . At the free cell edge with relatively low F - actin concentration , there occurs a meta - stable equilibration between a positive swelling pressure pushing the lamellar tip outwards and a resisting viscous network tension pulling the tip inwards . For some time , see Fig . 6 ( b ) after 5 minutes , local regions of protrusion or retraction can be observed , which point into varying directions along the cell periphery . Notice that these spatiotemporal fluctu - ations are not due to the tiny stochastic perturbations that are imposed to the F - actin polymerization rate , but represent the emergent chaotic dynamics of the cytoplasm as a reactive and contractile two - phase fluid [ 11 , 3 ] . Furthermore , behind a cyclically protrud - ing and retracting free edge we can observe a layer with slightly increased concentration of substrate - and - actin bound integrin c sa , see Fig . 6 ( e ) . This is the onset of polarization : fresh free integrin proteins are appearing at the protruding part of the edge from the upper membrane , thus also increasing c sa in this region . As a consequence , a larger frictional force of the retrograde F - actin is generated in that direction , inducing a bias into the force 20 ( a ) ( b ) ( c ) ( d ) ( e ) ( f ) 0 0 . 1 0 . 2 0 . 3 0 . 4 0 50 100 150 Figure 6 : 2 - D simulation of a spontaneously polarizing cell ( fragment ) starting with radially symmetric initial conditions : Spatial distributions shown as pseudocolor plots of F - actin volume fraction θ in ( a , b , c ) , and concentration of actin - and - surface bound integrin proteins c sa in ( d , e , f ) at three different time instants : ( a , d ) 1 min , ( b , e ) 5 min , and ( c , f ) 10 min after initialization . The width of the shown region is 22 µ m . 21 vector field transduced to the substratum . Thus , the whole cell fragment starts to move in this direction , which later becomes the leading edge of the migrating cell fragment , see Fig . 6 ( c , f ) . The emerging polarization of the cell is most clearly expressed in the c sa dis - tributions of Fig . 6 ( e , f ) , where an increasingly dense band is formed behind the leading edge and the central region of focal adhesions is slowly shifted rearwards with increasing migration speed , while becoming deformed in shape similarly to the whole cell fragment . This simulation is an example for the autonomous formation of a meta - stable unpolar - ized , almost circular state and its spontaneous transition into a polarized , migrating state of a flat cell fragment . By changing some of the model parameters we can influence the degree of this symmetry - breaking instability , but so far we were not able to reproduce the observed longer - time stability of circular cell fragments , see again [ 63 ] . One reason for this failure seems to be , that in our model simplifications we assumed a constant distribution of freely diffusing myosin - II oligomers , contradicting experimental results on clearly expressed gradients of myosin - II concentration decaying towards the cell edge , see e . g . [ 60 ] . Therefore we have started to investigate the full coupled model system by including active tip poly - merization and the kinetics for myosin - II diffusion , binding and transport , in addition to the already implemented analogous kinetics and dynamics of integrin adhesion molecules . The next section presents the so far achieved results in the most simple but nevertheless quite instructive 1 - dimensional situation . 4 . 2 Induced onset of cell polarization and migration in the 1 - D model We have simulated adhesion , polarization and migration of an idealized flat cell fragment having a fixed extension and only one degree of freedom to move , as can be observed experimentally , for example in Fig . 1 ( c ) . Thus , in a 1 - D cross - section along its moving direction , the fragment is represented by an interval Ω ( t ) = [ x b ( t ) , x b ( t ) + L ] of fixed length L , moving with body speed v b ( t ) = ˙ x b ( t ) , so that the whole kinetics and dynamics within cytoplasm and dorsal membrane can be described by the corresponding 1 - dimensional equations and conditions as in Section 3 . 2 , but now written in cell - centric coordinates , for example ˜ θ ( t , y ) = θ ( t , x b ( t ) + y ) or ˜ v ( t , y ) = v ( t , x b ( t ) + y ) − v b ( t ) . Then , assuming the following particular restrictions , we have solved a simplified system of boundary value problems : 1 . The actin network is always sticky at the lamellar tips ( no disruption ) , so that ˜ θ > 0 on the whole closed interval [ 0 , L ] . 2 . We assume active tip polymerization with a simultaneous parallel effect of the brown - ian ratchet ( π f 0 = 5 Pa / µ M ) and the clamp - motor mechanism ( π c 0 = 5 Pa / µ M ) . More - over , we suppose that the membrane - bound cortex shear viscosity strongly increases if myosin - II oligomers are bound : µ Γ ( m b ) = 0 . 1 ( 1 + 45 m b ) Pa min / µ m . For the actin - binding membrane proteins at the tip with maximal concentration A = 50 µ M and 22 ( a ) Figure 7 : Symmetric unpolarized state of a model cell ( fragment ) with length 10 µ m showing the con - centration profiles of ( a ) F - actin a , free and bound myosin - II m f and m b , the force F onto the substrate and the ef - fective cortical F - actin velocity v c ( b ) of integrin proteins in the four different states , namely substrate - and actin - bound c sa , substrate - bound c s , freely diffus - ing c f , and actin - bound c a . self - enhanced binding with dissociation constant K = 158 . 1 µ M we use the pseudo - equilibrium 2 a B ( a ) = A − (cid:112) ( A − a ) 2 + 2 ( A + a ) K 2 / a + ( K 2 / a ) 2 + K 2 / a + a and a c = 0 . 1 a B . 3 . The dorsal membrane moves together with the cell ( no slip at the tips ) , so that ˜ u ≡ 0 and v c = v = ˜ v + v b . The tip membrane tension difference is [ τ Γ ] L 0 = Φ u Lv b with the minimum always equal to a fixed positive constant τ 0 = 25 Pa . Finally , the substrate force balance ( 39 ) reads Φ u Lv b = (cid:90) L 0 Φ 0 (cid:102) c sa ˜ θ ( ˜ v + v b ) . ( 42 ) This is an implicit equation to be solved for the migration speed v b , since the Neu - mann boundary conditions and the right hand side of the elliptic equation ( 19 ) ) for ˜ v ( after eliminating the pressure ˜ p ) contain expressions that depend ( linearly ) on v b . Starting with the same unpolarized initial condition as in the previous Section 4 . 1 , we obtain a similar 1 - dimensional symmetric configuration with no cell translocation and a central F - actin plateau , a central maximum of FA ( focal adhesion ) sites , i . e . c sa - integrins , as well as a centripetal F - actin flow , see Fig . 7 . In addition , also the concentration of total myosin - II is enriched in the central region , with actin - bound myosin forming a central plateau , consistent with fluorescence pictures of unpolarized keratinocyte fragments , see 23 [ 63 ] . Moreover , the F - actin flow is highest at the boundary ( with values V ∼ 0 . 5 µ m / min ) due to the assumed active tip polymerization . In contrast to the previous 2 - D model simulations , here we find robust parameter con - stellations yielding stability of this symmetric unpolarized state : even quite strong but still subthreshold perturbations of the F - actin concentration at one side induce only transient locomotion together with shifts in most concentration profiles : after some delayed over - shooting , the cell fragment returns to its non - moving non - polarized stable state in Fig . 7 . However , if F - actin polymerization is continuously stimulated at one side ( mimicking the effect of a chemo - or haptotactic gradient ) then , not surprisingly , the cell fragment slowly polarizes and starts to persistently translocate in this direction ( data not shown ) . Similarly , in order to mimic the mechanical stimulation experiments with keratinocyte fragments as performed in [ 63 ] , we locally increased the activation ( and F - actin - binding ) of myosin - II at the left hand side for a certain time ( up to 30 sec ) , which is thought to be analogous to the experimental push by a micropipette flow pulse , since by local compression of the cytoskeleton , filament alignment and thus myosin action will be enhanced . If time span or amplitude of this myosin - pulse stays subthreshold , the model cell responses only by a transient migration as described above and asymptotically returns to its stable resting state , see the speed curve in Fig . 8 ( a ) . However , if the pulse strength exceeds a certain ( b ) Figure 8 : Plots of cell migration velocity v b over time after a perturbation of the unpolarized state in Fig . 7 by imposing an additional activation rate α + m for actin - bound myosin - II oligomers over an induction period of 0 . 5 min locally at the left cell side : ( a ) with rate α + m = 20 / min , showing a stable convergence of v b towards the unpolarized speed zero , and ( b ) with rate α + m = 50 / min , showing the convergence towards a positive polarized speed ∼ 0 . 012 µ m / min . threshold , myosin - II and F - actin is condensed at the rear , whence active tip polymerization ( V ) is drastically reduced at this side , so that the cell rapidly starts to migrate in the other direction , see Fig . 8 ( b ) . After cessation of the pulse , the migration speed is reduced , but the cell maintains its polarized locomotion state . This induced polarization , as a non - linear threshold behavior , is supported by a further positive feedback mechanism : Due to cell translocation , the focal adhesion sites ( c sa ) are successively shifted rearwards relative to 24 the cell , which stabilizes the asymmetric polarization and later increases the locomotion speed to a constant asymptotic value ( v b ∼ 0 . 12 µ m / min ) . ( a ) Figure 9 : Migrating polarized state show - ing stable concentration profiles ( a ) of F - actin , a , free and bound myosin - II , m f and m b , the trans - duced locomotion force F and the effective cortical F - Actin ve - locity v c ; ( b ) of integrin - concentrations as in Fig . 7 . Only FA integrins in the sa state are able to transduce force from the contractile actin network to the substrate . The migration speed is v b = 0 . 125 µ m / min . In this migration state the cell ( fragment ) attains characteristic concentration profiles , see Fig . 9 . Besides the already mentioned polar gradients of F - actin and myosin - II , the most impressive distribution is that of the FA sites : the c sa profile shows a characteristic broad peak behind the leading edge ( as in the 2 - D case above ) , followed by a slight decrease and a second plateau of even more condensed adhesion sites in the back part of the cell ( fragment ) which , however , rapidly decays at the very rear , see Fig . 9 ( b ) . This last phenomenon is the theoretically expected and experimentally observed rear release of adhesion sites or integrins , not induced by any directed regulatory protein , but only by the fact that the rear part experiences a steep increase of the force F transduced to the substratum , see the plot in Fig . 9 ( a ) . By ( 37 ) this is proportional to the F - actin mass flow θ v c with respect to the substratum : While in the major front part of the migrating cell ( fragment ) the F - actin flow is retrograde and the centripetally pulling force is modest in amplitude , near the trailing edge the direction of flow reverses and the force becomes very strong , now centripetally pulling off the focal adhesion sites . Thus , the reason for cell translocation is not that there is “more adhesion” or “stronger force” at the front compared to the rear , as it is asserted in some models , cf . [ 17 ] . Indeed , for vanishing passive friction Φ u due to ( 42 ) the total force integral even vanishes . The true physical reason for cell migration rather is the aforementioned asymmetry in the polarized cell state , expressed by a wide front region with modest rearward force and short rear region with strong forward force . 25 We finally investigate the dependence of migration speed on two cell physiologically important parameters , namely the adhesiveness of the substratum quantified by the relative number of available adhesion sites Adh0 , and the responsiveness of the F - actin network measured , for instance , by the concentration Arp0 of activated Arp2 / 3 proteins . These are mainly responsible for controlling F - actin polymerization by available free filament ends as they appear in the net assembly rate ( 4 ) and the ratchet polymerization pressure ( 31 ) . ( a ) ( b ) Figure 10 : Migration speed v b of a model cell in its stable polarized state , plotted over varied parameters ( a ) of adhesiveness Adh0 , relative amount of adhesion binding sites on the substratum ( e . g . fibronectin coating ) ( b ) of F - actin responsiveness expressed by Arp0 [ µ M ] , the cytoplasmic concentration of activated Arp2 / 3 complexes . The results depicted in Fig . 10 reveal the existence of optimal ranges for both parameters , consistent with earlier modeling results , see [ 17 , 24 ] , and with experimental observations , see e . g . [ 27 , 52 ] . Particularly , the migration response curve of Fig . 10 ( a ) has been performed for fixed parameter Arp0 = 10 µ M and for adhesion values 0 ≤ Adh0 ≤ 20 , where according to Table 2 the adhesiveness proportionally influences not only the two adhesion rates α + = γ + = Adh0 · α but also the passive membrane friction coefficient Φ u = Adh0 · 6 Pa · min · µ m − 2 . This is based on the idealizing assumption that there are no other relevant exterior forces that would resist locomotion relative to the substratum , than those due to friction of the dorsal membrane with respect to an adhesive coating of e . g . fibronectin or collagen . Under these hypotheses and parameter choices our model simulations predict a minimal migration speed of v b ∼ 0 . 035 µ m / min for Adh0 ∼ 1 , which increases towards the doubled speed when lowering the adhesiveness to almost zero . This surprising phenomenon is consistent with own experimental measurements of human keratinocyte polarization and migration [ 37 ] revealing slightly increased motility of polarized cells on glass compared to those on a low density fibronectin coat . The reason for this can be seen from the 26 corresponding profiles of FA concentration ( c sa ) and substrate force distribution ( F ) for the two adhesiveness values Adh0 = 0 . 01 , 0 . 2 , see Fig . 11 ( a , b ) . Due to ongoing retrograde Figure 11 : Substrate force distribution F and integrin concentrations in the four different states , plotted for varying adhesiveness parameter Adh0 with corresponding migration speed v b as in Fig . 10 ( a ) : ( a ) 0 . 01 , speed 0 . 06 µ m / min , ( b ) 0 . 2 , speed 0 . 04 µ m / min , ( c ) 3 . 0 , speed 0 . 125 µ m / min , and ( d ) 20 . 0 , speed 0 . 138 µ m / min . flow of F - actin , working against internal viscosity and drag , the polarized cell ( fragment ) gathers the few FA sites on the back side in a way that the local FA and force distributions are almost symmetric ( for almost vanishing adhesiveness Adh0 = 0 . 01 ) but still with an additional negative ( pulling ) force plateau on the front side ( though of tiny absolute value | F | ∼ 0 . 05 P a ) . For increased but still small adhesiveness ( Adh0 = 0 . 2 ) , the much more frequent FA sites start to accumulate at the rear , the asymmetry of forces is more expressed and the increased friction reduces the migration speed . On the other hand , for further increasing adhesiveness , Adh0 > 1 , the front plateau of pulling forces at the enriched FA ‘carpet’ is proportionally increased , see Fig . 11 ( c , d ) . However , the dominant reason for the non - linear speeding - up response is the prominent increase of disruptive forces | F c ( rear ) | up to values of Adh0 ∼ 2 , leading to a drastic reduction of resistive FA sites at the very rear , see the corresponding indicator curves in Fig . 12 ( a ) . Only for very large adhesiveness , Adh0 ∼ 20 , the FA sites again start to accumulate at the very rear in spite of a strong disruptive force there , as can be seen in Fig . 11 ( d ) , then causing a slight decrease in migration speed . Thus , while an adhesiveness between 5 and 12 leads to a saturated optimality in its migration speed , within the range 1 < Adh0 < 4 the cell ( fragment ) has a high sensitivity for responding to an increase of 27 Figure 12 : Variation of migration speed v b and certain indicator functions at the cell edge in dependence of ( a ) adhesiveness Adh0 and ( b ) F - actin responsiveness Arp0 [ µ M ] : Plotted are the load | F c | per FA site at the cell rear ( bold ) and tip ( light ) , and the FA concentration c sa at the rear ( bold , dashed ) and tip ( light , dashed ) . Ripples in the force curves reflect the slight stochastic noise that was added in the force equation ( 19 ) . 28 adhesiveness with an up to 4 - fold increase of speed . Carried over to the mean forward speed of competing leading lamellae in a whole cell model ( as for the 2 - D simulations in Fig . 6 ) , this could be used to explain polarization and haptotaxis of cells in spatial adhesion gradients , cf . [ 20 ] . In order to understand the analogous optimal migration performance for responsive - ness parameters in the range between 4 µ M and 10 µ M of Arp2 / 3 concentration Arp0 , see Fig . 10 ( b ) , we plot the same indicator curves for FA concentrations and substrate forces in Fig . 12 ( b ) . Again , with increasing Arp2 / 3 - induced actin polymerization there is an almost linear increase in pulling force at the tip ( due to increased polymerization velocity V there ) while the concave migration speed curve directly corresponds to the proportional curve for the disruptive forces at the rear and the resulting convex curve for the accumulated FA sites there . For larger Arp0 values the growing accumulation of FA sites again leads to a slight speed reduction . Finally , the about 50 % gain of speed for increasing responsiveness in the range 1 µ M < Arp0 < 4 µ M could again be carried over to a competing lamellar protrusion response in spatial chemotactic gradients , which are known to stimulate F - actin polymerization at the leading front . 4 . 3 Migration speed in a simplified 1 - D model In order to explore , which properties of our coupled cytoplasm - adhesion model are essential for obtaining the 1 - D simulation results in the preceding section , we simplified the model by freezing the following variables and parameters , cf . [ 45 ] : 1 . Assuming fast diffusion and F - actin binding of free myosin - II oligomers , we take the pseudo - steady state condition ( 9 ) for bound myosin - II , but with the equivalent dissociation rate δ m ( a ) = δ 0 m exp ( − 2 a / a opt ) instead of ( 8 ) , and obtain as contractility ψ ( θ ) = (cid:102) ψ 0 · θ 2 exp ( − 2 θ / θ opt ) with a coefficient (cid:102) ψ 0 analogous to ( 41 ) . 2 . Except the FA friction function Φ v we set all other friction coefficients ( ϕ , Φ u ) to zero . Thus , the cell migration speed is determined by the zero integral in ( 42 ) . 3 . All ‘exterior’ pressures or tensions at the free membrane tip ( π f 0 , π c 0 , τ Γ ) are assumed to vanish , in particular , there occurs no active polymerization ( V ≡ 0 ) . Starting with uniform FA concentration and a minor asymmetry in the distribution of actin , the system reaches a polarized and migrating state and the distributions of the various concentrations exhibit the same characteristics as in the full model ( see . Fig 9 ) . We again analyze the effect of substratum adhesiveness and F - actin responsiveness on the migration speed , see Fig . 13 . In contrast to the observations for the full model ( see Fig . 10 ) , a saturation behavior of the migration velocity emerges with increasing adhesiveness instead of an optimal range . However , the responsiveness of the F - actin - network has an optimal value as in the full model . 29 Symbol Meaning Units θ ( t , x ) Volume fraction of F - actin dimensionless a ( t , x ) Concentration of F - actin µ M m f ( t , x ) Concentration of free myosin - II µ M m b ( t , x ) Concentration of F - actin bound myosin - II µ M c f ( t , x ) Concentration of free integrin µ m − 2 c s ( t , x ) Concentration of substrate bound integrin µ m − 2 c a ( t , x ) Concentration of actin bound integrin µ m − 2 c sa ( t , x ) Concentration of substrate - and - actin bound integrin µ m − 2 R ( θ ) F - actin net polymerization rate min − 1 ψ ( θ , m b ) Contractile stress Pa σ ( θ ) Swelling pressure Pa S ( θ , m b ) Effective cytoplasmic stress Pa p ( t , x ) Effective two - phase flow pressure Pa τ u ( t , x ) Dorsal membrane tension Pa v ( t , x ) Mean F - actin velocity µ m / min v c ( t , x ) Cortical F - actin velocity µ m / min u ( t , x ) Dorsal membrane velocity µ m / min v b ( t ) Migration velocity of cell ( fragment ) body µ m / min Φ ( θ , c sa ) Adhesional friction Pa · min / µ m 2 F v Active frictional force Pa / µ m F c Local frictional force per adhesion site Pa · µ m F u Passive frictional force at dorsal membrane Pa / µ m ˙Γ Normal speed of the free boundary Γ ( t ) µ m / min V Relative inward F - actin velocity at boundary µ m / min P Γ a Boundary cytoskeleton pressure Pa P Γ s Boundary cytosol pressure Pa τ Γ Membrane tension at boundary ( lamellar tips ) Pa a B ( a ) Concentration of tip - bound F - actin µ M a c ( a ) Concentration of F - actin bound to clamp - motor proteins µ M a f ( a ) Concentration of free filaments exposed to the tip µ M µ Γ ( m b ) F - actin shear viscosity relative to a B filaments Pa · min / µ m p f Free polymerization pressure at tip membrane Pa p c Clamp - motor polymerization pressure Pa Table 1 : List of model variables and functions 30 ( a ) ( b ) Figure 13 : Migration speed v b for a simplified model cell in its stable polarized state in dependence of ( a ) adhesiveness or ( b ) F - actin responsiveness as described in Fig . 10 . Further simulations after successive variation of the frozen parameters ( see above ) reveal , that an optimal range of adhesiveness could be gained only if a passive frictional load Φ u > 0 was chosen to increase monotonically with adhesion strength Adh0 , as it was assumed in the full model , see Table 2 and Fig . 10 ( a ) . Though such a friction appears to be physically realistic , the question remains whether such an optimal velocity response is of general biological relevance . For , the response of living systems is typically guided by adaptation : Migrating cells show the capability to dissolve their focal adhesions by proteolytic cleavage of integrins [ 55 ] which could be an adaptive strategy to optimize cellular adhesiveness on various substrates . On the other hand , the adhesiveness could be raised by active segregation of extracellular matrix proteins . Nevertheless , the universal optimality curve in dependence of F - actin responsiveness , seen in both Figs . 10 ( b ) and 13 ( b ) , seems to hold for any mechanical model , which imple - ments a saturating F - actin polymerization by association of regulating proteins as Arp2 / 3 . This indicates that an optimal control of migration velocity by tuning certain chemical processes as F - actin polymerization , is more easy to be realized than tuning adhesion , and thus may have evolved as a generally effective strategy to regulate such mechanical processes as cell adhesion and locomotion . 5 Discussion and outlook to further modeling Based on a larger set of interwoven mass and force balance equations for mechano - chemical processes within the cytoplasm and the surrounding plasma membrane , the presented con - tinuum model is a simplified though already sufficiently complex version of a more com - prehensive 3 - dimensional whole - cell model . From the 1 - D and 2 - D simulation results we 31 Symbol Meaning Value Units a g Concentration of G - actin 30 µ M a max Maximal concentration of F - actin 800 µ M k on F - actin polymerization rate at plus ends 696 ( min · µ M ) − 1 k off F - actin depolymerization rate at plus ends 258 min − 1 ω F - actin capping rate 1250 min − 1 r F - actin disassembly rate 0 . 2 min − 1 ε Basal F - actin nucleation rate 0 . 75 min − 1 Arp0 Concentration of activated Arp2 / 3 complexes 10 µ M ν a Arp2 / 3 - induced nucleation rate 60 ( min · µ M ) − 1 K a Half - saturation concentration for nucleation 3 µ M m 0 f Equilibrium reservoir concentration of free myosin - II 10 µ M D m Diffusion coefficient for free myosin - II 0 . 5 µ m 2 · min − 1 α m F - actin binding rate of myosin - II 0 . 5 ( min · µ M ) − 1 δ 0 m Dissociation rate of bound myosin - II 3 min − 1 a opt Optimal F - actin concentration for myosin - II binding 40 µ M c 0 f Reservoir concentration of free integrin at lamellar tips 50 µ m − 2 D f Diffusion coefficient for free integrin 0 . 5 µ m 2 · min − 1 Adh0 Number of available substrate sites per integrin 3 dimensionless Talin F - actin association factor 0 . 0125 dimensionless Fifactor Focal complex factor ( intracellular ) 50 dimensionless Fefactor Focal adhesion factor ( extracellular ) 50 dimensionless α − Dissociation rate for substrate bound integrin α = 5 min − 1 α + Free integrin binding rate to substrate Adh0 · α min − 1 β + 0 F - actin binding rate for substrate bound integrin α · Talin ( min · µ M ) − 1 β − 0 FA dissociation of the F - actin link α / Fifactor min − 1 δ + 0 Free integrin binding rate to F - actin α · Talin ( min · µ M ) − 1 δ − Dissociation rate for F - actin bound integrin α min − 1 γ + Substrate binding rate for F - actin bound integrin Adh0 · α min − 1 γ − 0 FA dissociation rate of the substrate link α / Fefactor min − 1 ρ β Exponential FA - rupture coefficient for F - actin link 11 . 7 ( Pa · µ m ) − 1 ρ γ Exponential FA - rupture coefficient for substrate link 11 . 7 ( Pa · µ m ) − 1 A Total free F - actin binding sites on boundary Γ ( t ) 50 µ M π f 0 Ratchet polymerization pressure coefficient 0 . 0125 Pa · µ M − 1 π c 0 Clamp - motor polymerization pressure coefficient 0 . 0125 Pa · µ M − 1 κ Γ Tip curvature weight factor for membrane tension 0 . 5 dimensionless α f 0 Relative amount of exposed barbed ends per Arp2 / 3 2 dimensionless ψ 0 Contractile stress per bound myosin - II 0 . 0163 Pa · µ M − 1 e ψ 0 Strength of contractile stress ( simplified model ) 0 . 625 Pa σ 0 Strength of the swelling pressure 0 . 125 Pa µ Viscosity of the F - actin network phase 0 . 625 Pa · min κ Cortical slip parameter for the F - actin flow 0 . 5 dimensionless ϕ Drag coefficient between network and solvent 2 Pa · min · µ m − 2 Φ 0 Friction per actin - substrate bound integrin 0 . 02 Pa · min Φ u Additional friction associated to the cell body Adh0 · 6 Pa · min · µ m − 2 L Length of the cell ( fragment ) 10 µ m Table 2 : List of parameters for the 1 - D simulations , if not otherwise specified . 32 conclude that even without organizing centers ( as the cell nucleus or microtubuli , cf . [ 31 ] ) or regulating systems ( as the Rho / Rac control cascades , cf . [ 54 ] ) the F - actin cytoskeleton and its associated proteins having mechanical functions ( myosin and integrin ) constitute a self - organizing biophysical system with the ability of autonomous polarization and loco - motion . The transition form a symmetric , unpolarized stationary state into a polarized migrating state can be mechanically induced or spontaneous due to stochastic or chaotic fluctuations , depending on the ( meta - ) stability of the stationary state . Clearly , for repro - ducing the mentioned experimental data , the 2 - dimensional system offers a wider spectrum of possibilities , one of which has been elaborated by Kozlov and Mogilner [ 33 ] . They prove a defined bi - stability between the radially symmetric cell shape and a polarized , circu - larly indented shape by allowing for different anisotropic organization of the actin - myosin cytoskeleton . In comparison to this , further numerical ‘experiments’ using our coupled vis - cous flow - transport - diffusion - reaction system with free boundary should be performed to explore the capability of isotropic continuum models . Though local orientation or alignment of actin filaments will have an enforcing effect onto the dynamics , interactions and feedback between F - actin , cross - linkers , motor - proteins and adhesion complexes ( for a 1 - dimensional model see [ 21 ] ) , already the presented simulation results show that internal concentration gradients and directional flow , leading to persistent polarization , can emerge from local fluc - tuations . Thereby super - threshold pattern formation arises , not as in common excitable media by a purely reaction - diffusion feedback , rather by a spatially distributed coupling between chemically induced force generation / relaxation and mechanically induced bond formation / dissociation . More than one decade ago , Bereiter - Hahn and L¨uers [ 8 ] claimed that , based on their ex - periments and measurements with migrating keratocytes , polarization and directionality of cells are determined by local variations in actin network stiffness and hydrostatic pressure . Together with the more recent high - resolution measurements of dynamic vector field ( as F - actin velocity , forces onto pliable substrates , or directionality of FA shape deformations ) , it could soon be possible to probe and evaluate the various assumptions and hypotheses on mass and force balance conditions , which we postulated in Sections 3 . 1 and 3 . 2 . For example , the biphasic correlations between retrograde actin flow speed and traction stress detected by Gardel et al . [ 22 ] could well be resolved by the measured and simulated FA gradient away from an advancing lamellar tip , and by our central modeling assumption that the local traction force F v is proportional to the product c sa · v c of FA concentration and cortical F - actin velocity . Moreover , the distribution of flow and force , extensively discussed for the 1 - dimensional case ( in Section 4 . 2 ) , has to be quantitatively characterized for the 2 - dimensional free boundary situation ( Section 4 . 1 ) , where the overall picture is the same : Wider distribu - tion of traction forces in the leading front region ( with maximum flow speed at the tip , depending on the active polymerization pressure ) and strong disruptive counter - forces in a confined region of ‘rear release’ . The actin flow patterns of our 2 - D simulations show in principle the same characteristics as experimental data by Yam et al . [ 64 ] for fish kerato - 33 cytes . During the process of polarization , the simulated cell looses its radial symmetry of a uniform inward flow pattern , leading to a stronger inward flow in the rear region , whereby the most pronounced forces come from the two sides relative to the establishing locomotion direction , see Fig . 6 ( c ) . Indeed , traction force experiments on polarized fish keratocytes have revealed the same pattern , with major traction forces from the two flanks at the rear and weaker retrograde forces at the front [ 50 ] . Further evaluation of the presented model has compare the simulated integrin concen - tration profiles with more precise quantitative data on the temporal turnover and spatial distribution of FAs in migrating cells , as already mentioned above . For this reason , the model has to be extended to include stochastic clustering dynamics of integrins , which probably is an essential feature of FAs determining the detailed patterns of F - actin flow and traction forces . We emphasize , that our model results do not depend on the specific mechanisms of myosin - II contraction . Essential for the emergence of coupled flow and mass gradients is the bimodal ( cubic - like ) stress function S = S ( θ ) in Fig . 5 which similarly ap - pears in a modified mechanical situations as , for example , the nematode sperm motility system , see [ 9 ] . Also the experimental response curves for the migration speed of polarized cells depend - ing on adhesiveness [ 52 ] show a broad optimum range with a logarithmic slow decay for larger Adh0 values and a steep decay for lower ones , similar to the curves found in our 1 - D model , Fig . 10 , and in analogous earlier simulation results using alternative visco - elastic mechanics [ 17 , 24 ] . The deviating behavior for very low adhesiveness values hat still to be investigated with a more precise quantification of other ( weaker ) substrate - mediated forces that may come into account . An optimum curve for the protrusion speed in dependence of the barbed end density B , thus also of the concentration Arp0 , has been obtained by Mogilner and Edelstein - Keshet [ 42 ] with the aid of 1 - dimensional diffusion - reaction - transport systems , including membrane resistance and the brownian ratchet mechanism . However , they do not model retrograde actin flow or force transduction to the substratum , so that the shape of their curve ( [ 42 ] , Fig . 6 ) essentially differs from our results in Figs . 10 ( b ) and 12 ( b ) . Finally , in generalization of the model simulations by St´ephanou et al . [ 59 ] and in compar - ison to other modeling approaches mentioned before , our full 2 - dimensional model should be used for reproducing the experimentally observed translocation paths and deformation structures of single blood or tissue cells in culture , which show well - expressed phases of persistent polarization and locomotion , interrupted by events of speed reduction , contrac - tile rounding , successive re - polarization and migration . Statistical analysis , in the same spirit as having been performed for the original annual lamella model [ 4 ] , could help to reveal the microscopic mechanisms that are responsible for cell motility behavior on the macroscopic level . Acknowledgments : This joint work has been generously supported by a grant on ”Simu - lation models for cell motility” ( I / 80543 , Volkswagen - Stiftung ) . Moreover , the hospitality 34 of the Soft Matter Physics group at Leipzig University during the final preparation of this chapter is highly appreciated . 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Actin - myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility . J . Cell Biol . , 178 : 1207 – 1221 , 2007 . [ 65 ] C . Zhu and R . Skalak . A continuum model of protrusion of pseudopod in leukocytes . Biophys . J . , 54 : 1115 – 1137 , 1988 . 39 \ No newline at end of file diff --git a/silver_data/abae5393d53c47e15679d470d061a56b07e6ed61.pdf.txt b/silver_data/abae5393d53c47e15679d470d061a56b07e6ed61.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..85ea9eb0b82ad001e6ae0685b3b87bbd0052c36c --- /dev/null +++ b/silver_data/abae5393d53c47e15679d470d061a56b07e6ed61.pdf.txt @@ -0,0 +1 @@ +Grand challenges and emergent modes of convergence science Alexander M . Petersen ∗ , 1 Mohammed E . Ahmed , 2 and Ioannis Pavlidis ∗ 2 1 Department of Management of Complex Systems , Ernest and Julio Gallo Management Program , School of Engineering , University of California , Merced , CA 95343 2 Computational Physiology Laboratory , University of Houston , Houston , Texas 77204 To address complex problems , scholars are increasingly faced with challenges of integrating diverse knowledge domains . We analyzed the evolution of this convergence paradigm in the broad ecosystem of brain science , which provides a real - time testbed for evaluating two modes of cross - domain integration – subject area explo - ration via expansive learning and cross - disciplinary collaboration among domain experts . We show that research involving both modes features a 16 % citation premium relative to a mono - disciplinary baseline . Further compar - ison of research integrating neighboring versus distant research domains shows that the cross - disciplinary mode is essential for integrating across relatively large disciplinary distances . Yet we find research utilizing cross - domain subject area exploration alone – a convergence shortcut – to be growing in prevalence at roughly 3 % per year , significantly faster than the alternative cross - disciplinary mode , despite being less effective at integrating domains and markedly less impactful . By measuring shifts in the prevalence and impact of different convergence modes in the 5 - year intervals before and after 2013 , our results indicate that these counterproductive patterns may relate to competitive pressures associated with global Human Brain flagship funding initiatives . Without additional policy guidance , such Grand Challenge flagships may unintentionally incentivize such convergence shortcuts , thereby undercutting the advantages of cross - disciplinary teams in tackling challenges calling on convergence . The history of scientific development is characterized by a pattern of convergence - divergence cycles ( Roco et al 2013 ) . In convergence , originally distinct disciplines synergistically interact to address complex problems and accelerate break - through discovery ( National Research Council 2014 ) . In di - vergence , in addition to fragmentation resulting from conflict - ing social forces ( Balietti et al 2015 ) , spin - offs occur as new techniques , tools and applications spawn . The evolving fu - sion of multi - domain expertise during the present convergence cycle carries significant intellectual and organizational chal - lenges ( Bromham et al 2016 ; Fealing & eds . 2011 ; National Research Council 2005 ; Pavlidis et al 2014 ) . The core issue is that contemporary convergence takes place in the context of team science ( Milojevic 2014 ; Wuchty et al 2007 ) . Ac - cordingly , collaboration across distinct academic cultures and units faces behavioral ( Van Rijnsoever & Hessels 2011 ) and institutional barriers ( National Research Council 2014 ) . Two early successful examples of convergence are worth mentioning to draw a comparative baseline . First , the Manhat - tan Project ( MP ) , where physicists , chemists , and engineers successfully worked in the 1940s to control nuclear fission and produce the first atomic bomb , under a tightly run govern - ment program ( Hughes & Hughes 2003 ) . A half - century later ( 1990s - 2000s ) , the Human Genome Project ( HGP ) forged a multi - institutional bond integrating biologists and computer scientists , under an organizational design known as consor - tium science model whereby teams of teams organize around a well - posed central grand challenge ( Helbing 2012 ) , with a common goal to share benefits equitably within and beyond institutional boundaries ( Petersen et al 2018 ) . In 10 short years , the HGP led to the mapping and identification of the ( a ) ∗ To whom correspondence should be addressed ; E - mail : ipavlidis @ uh . edu or apetersen3 @ ucmerced . edu human genetic code , ushering civilization into the genomics era . Brain science is presently supported by major funding pro - grams that span the world over ( Grillner et al 2016 ) . In late 2013 , the United States launched the BRAIN Initiative ® ( Brain Research through Advancing Innovative Neurotech - nologies ) , a public - private effort aimed at developing new ex - perimental tools that will unlock the inner workings of brain circuits ( Jorgenson et al 2015 ) . At the same time , the Eu - ropean Union launched the Human Brain Project ( HBP ) , a 10 year funding program based on exascale computing ap - proaches , which aims to build a collaborative infrastructure for advancing knowledge in the fields of neuroscience , brain medicine , and computing ( Amunts et al 2016 ) . In 2014 , Japan launched the Brain Mapping by Integrated Neurotechnologies for Disease Studies ( Brain / MINDS ) , a program to develop in - novative technologies for elucidating primate neural circuit functions ( Okano et al 2015 ) . China followed in 2016 with the China Brain Project ( CBP ) , a 15 year program targeting the neural basis of human cognition ( Poo et al 2016 ) . Canada ( Ja - balpurwala 2016 ) , South Korea ( Jeong et al 2016 ) , and Aus - tralia ( Committee et al 2016 ) followed suit , launching their own brain programs in the late 2010s . By nature and historical precedence , convergence tends to operate on the frontier of science . In the 2010s , brain sci - ence was declared the new research frontier ( Quaglio et al 2017 ) promising health and behavioral applications ( Eyre et al 2017 ) . Intensification of brain research has been taking place against a backdrop of an increasingly globalized , intercon - nected and online scientific commons . This stands in sharp contrast to the nationally unipolar and offline backdrop of the MP and even the HGP . Moreover , the brain funding programs were designed to act as behavioral incentives in an scientific marketplace , aimed at bringing together diverse scholars and ideas . However , despite being oriented around the compelling structure - function brain problem , there were few guidelines a r X i v : 2103 . 11547v1 [ c s . D L ] 22 M a r 2021 2 on how to configure scholarly expertise to address the brain challenge . As such , these characteristics render brain research a “live experiment” in the international evolution of the con - vergence paradigm . Accordingly , we apply data - driven methods to reconstruct the brain science ecosystem as a way to capture the contempo - rary “pulse” of convergence , explored through a progressive series of research questions regarding its prevalence , anatomy and scientific impact . Given the pervasive funding champi - oning the HBS challenge , we further analyze how the trajec - tory of HBS convergence has been impacted by the ramp - up of flagship funding initiatives oriented around the world . While previous work explored the role of cross - disciplinary collab - oration in the Human Genome Project ( Petersen et al 2018 ) , here we extend that framework to differentiate between ( a ) the disciplinary diversity of the research team and ( b ) the topical diversity of their research – two alternative means of cross - domain integration . We refer to the former as disciplinary diversity and to the latter as topical diversity . We leverage ex - isting taxonomies – in the case of disciplines , using the Clas - sification of Instructional Program ( CIP ) system developed by the U . S . National Center for Education Statistics ; and for topics using Medical Subject Heading ( MeSH ) ontology de - veloped by the U . S . National Library of Medicine disciplines – to distinguish mono - domain versus cross - domain activity . Accordingly , we classify HBS research according to four in - tegration types defined by a mono - / cross - { discipline × topic } domain decomposition . In a highly competitive and open science system with mul - tiple degrees of freedom , our motivating hypothesis is that more than one operational cross - domain integration mode is likely to emerge . With this in mind , we identify five research questions ( RQ ) addressed in each figure in series . The first ( RQ1 ) regards how to define convergence , which we address by developing a typological framework , one that is general - izable to other frontiers of biomedical science , and is rele - vant to the evaluation of multiple billion - dollar HBS flagship projects around the world . The second ( RQ2 ) regards the status and impact of brain science convergence : Have HBS interfaces have developed to the point of sustaining fruitful cross - disciplinary knowledge exchange ? Does the increasing prevalence of teams adopting convergent approaches corre - late with higher scientific impact research ? RQ3 addresses whether convergence is evenly distributed across HBS sub - domains ? And what empirical combinations of distinct sub - ject areas ( knowledge ) and disciplinary expertise ( people ) are overrepresented in convergent research ? RQ4 follows by seeking to identify whether convergence is evenly distributed over time and geographic region ? And finally , RQ5 : does the propensity to pursue convergence science or does the ci - tation impact of convergence science depend on the conver - gence mode ? To address this question , we implement hierar - chical regression models that differentiate between three con - vergence modes : research involving cross - disciplinary collab - oration , cross - subject - area exploration , or both . Given the lu - crative nature of flagship funding initiatives , we hypothesize that the ramp - up of HBS flagships correlates with shifts in the prevalence and relative impact of research adopting these dif - ferent convergence modes . Our results identify timely and relevant science policy im - plications . Given contemporary emphasis around accelerat - ing breakthrough discovery ( Helbing 2012 ) by way of strate - gic research team configurations ( B¨orner et al 2010 ) , con - vergence science originators called for cross - disciplinary ap - proaches integrating distant disciplines ( National Research Council 2014 ) . Instead , our analysis reveals that HBS teams recently tend to integrate diverse topics without necessarily in - tegrating appropriate disciplinary expertise – an approach we identify as a convergence shortcut . Theoretical background This work contributes to several literature streams , including the quantitative analysis of recombinant search and innovation ( Fleming 2001 ; Fleming & Sorenson 2004 ; Youn et al 2015 ) ; cross - domain integration ( Fleming 2004 ; Leahey & Moody 2014 ; Petersen et al 2018 ) ; cross - disciplinarity as a strategic team configuration ( Cummings & Kiesler 2005 ; Petersen et al 2018 ) facilitated by division of labor across teams of special - ists and generalists ( Haeussler & Sauermann 2020 ; Melero & Palomeras 2015 ; Rotolo & Messeni Petruzzelli 2013 ; Teodor - idis 2018 ) ; and science of science Fortunato et al ( 2018 ) and science policy ( Fealing & eds . 2011 ) evaluation of conver - gence science ( National Research Council 2005 , 2014 ; Roco et al 2013 ) . Efficient long - range exploration facilitated by multi - disciplinary teams is a defining value proposition of conver - gence science ( National Research Council 2014 ) , and pro - vides a testable mechanism underlying the increased likeli - hood of large team science producing high - impact research ( Wuchty et al 2007 ) . Hence , the emergence and densification of cross - domain interfaces are likely to increase the potential for breakthrough discovery by catalyzing recombinant inno - vation ( Fleming 2001 ) , which effectively expands the solu - tion space accessible to problem - solvers . It then follows that certain configurations are likely to amplify the effectiveness of recombinant innovation . Adapting a triple - helix model of medical innovation ( Petersen et al 2016 ) , recombinant inno - vation manifests from integrating expertise around the three dimensions of supply , demand and technological capabilities : ( i ) the fundamental biology domain that supplies a theoret - ical understanding of the anatomical structure - function rela - tion , ( ii ) the health domain that identifies demand for effec - tive science - based solutions , and ( iii ) the techno - informatics domain which develops scalable products , processes and ser - vices to facilitate matching supply from ( i ) with demand from ( ii ) ( Yang et al 2021 ) . In order to overcome the challenges of selecting new strate - gies from the vast number of possible combinations , prior re - search finds that innovators are more likely to succeed by way of exploiting their own local expertise ( Fleming 2001 ) rather than individually exploring distant configurations by way of internal expansive learning ( Engestr¨om & Sannino 2010 ) . Ex - tending this argument , exploration at unchartered multidisci - plinary interfaces is likely to be more successful when inte - grating knowledge across a team of experts from different do - mains , thereby hedging against recombinant uncertainty un - 3 ` Human brain’ publication set Brain scientists’ profiles { a } & publications { p } Brain scientists’ citations per p Brain scientists’ subject areas per p < WOS API > < Scopus API > < Scopus API > < PubMed API > Technology & Information Science [ J , L ] Techniques & Equipment [ E ] Health [ C , N ] Phenomena & Processes [ G ] Anatomy & Organisms [ A , B ] Psychiatry & Psychology [ F ] Chemistry & Physics & Math Engineering & Informatics Pathology & Pharmacology Health Sciences Medical Specialty Biotechnology & Genetics Psychology Biology Neurosciences Disciplinary Clusters Topical Clusters CIP Taxonomy SA MeSH Taxonomy Regional Clusters G e o T a x o n o m y 🟥 Australasia 🟧 North America 🟦 Europe H e a l t h S c i / E n g N e u r o / B i o BRAIN / MINDS ABA CBP KBI HBP BRAIN CBRS Projects Projects Projects SA 4 SA 4 SA 4 SA 4 SA 4 SA 4 SA 1 SA 1 CIP 1 CIP 1 CIP 1 CIP 1 CIP 5 CIP 2 CIP 1 CIP 8 ≡ 𝑀 ≡ 𝑀 ≡ 𝑋SA ≡ 𝑋CIP 1 2 3 4 5 6 1 2 8 4 5 6 7 3 9 Examples of p > FIG . 1 : Data collection and classifica - tion schemes . The upper part of the figure shows the data generation mech - anism along with the resulting topical ( SA ) and disciplinary ( CIP ) clusters . The middle part of the figure shows on the world map regional clusters pertaining to three large HBS funding initiatives – North America ( NA ) , Europe ( EU ) , and Australasia ( AA ) . The lower part of the figure shows an example of how all three categorizations are operational - ized for analytic purposes . Circles rep - resent four research articles with author - ship from distinct regions . The articles feature different keyword ( SA ) or disci - plinary ( CIP ) category mixtures assigned one of two diversity measures : mono - ( M ) and cross - domain ( X ) . derlying the exploration process ( Fleming 2004 ) . A complementary argument for convergence derives from the advantage of diversity for harnessing collective intel - ligence and identifying successful hybrid strategies ( Page 2008 ) . Recent work provides additional empirical support for the competitive advantage of diversity , using cross - border mobility ( Petersen 2018 ) as an instrument for social capital disruption to identify the positive role of research topic and collaborator diversity . Data collection and notation Figure 1 shows the multiple sources combined in our study , which integrates publication and author data from Scopus , PubMed , and the Scholar Plot web app ( Majeti et al 2020 ) ( see Supplementary Information ( SI ) Appendix S1 for detailed de - scription ) . In total , our data sample spans 1945 - 2018 and con - sists of 655 , 386 publications derived from 9 , 121 distinct Sco - pus Author profiles , to which we apply the following variable definitions and subscript conventions to capture both article - and scholar - level information . At the article level , subscript p indicates publication - level information such as publication year , y p ; the number of coauthors , k p ; and the number of key - words , w p . Regarding the temporal dimension , a superscript > ( respectively , < ) indicates data belonging to the 5 - year “post” period 2014 - 2018 ( 5 - year “pre” period 2009 - 2013 ) , while N ( t ) represents the total number of articles published in year t . Regarding proxies for scientific impact , we obtained the number of citations c p , t from Scopus , which are counted through late 2019 . Since nominal citation counts suffer from systematic temporal bias , we use a normalized citation mea - sure , denoted by z p ( see Methods – Normalization of Citation Impact ) . Regarding author - level information , we use the in - dex a – e . g . we denote the academic age measured in years since a scholar’s first publication by τ a , p . To address RQ1 we classified research according to three category systems indicative of topical , disciplinary and re - gional clusters . The first category system captures research topic clusters grouped into Subject Areas ( SA ) ; counts for each article are represented by a vector with 6 elements , −→ SA p , each corresponding to top - level Medical Subject Head - ing ( MeSH ) categories implemented by PubMed , which are indicated by the letters in brackets : ( 1 ) Psychiatry & Psychol - ogy [ F ] , ( 2 ) Anatomy & Organisms [ A , B ] , ( 3 ) Phenomena & Processes [ G ] , ( 4 ) Health [ C , N ] , ( 5 ) Techniques & Equipment [ E ] , and ( 6 ) Technology & Information Science [ J , L ] ; notably , regarding the structure - function problem that is a fundamen - tal focus in much of biomedical science , category ( 2 ) repre - sents the domain of structure while ( 3 ) represents function . The variable N SA , p counts the total number of SA categories present in a given article , with min value 1 and max value 6 . The second taxonomy identifies disciplinary clusters de - termined by author departmental affiliation , which we cate - gorized according to Classification of Instructional Program ( CIP ) codes . Article - level CIP category counts are repre - 4 sented by −−→ CIP p , with 9 elements pertaining to the following categories : ( 1 ) Neurosciences , ( 2 ) Biology , ( 3 ) Psychology , ( 4 ) Biotech . & Genetics , ( 5 ) Medical Specialty , ( 6 ) Health Sciences , ( 7 ) Pathology & Pharmacology , ( 8 ) Engineering & Informatics , and ( 9 ) Chemistry & Physics & Math . The vari - able N CIP , p counts the total number of CIP categories present in a given article , with min value 1 and max value 9 ; Methods and SI Appendix S1 offer more details . The third taxonomy captures the broad regional scope of each research article team determined by each Scopus au - thor’s affiliation location , and represented by the vector −→ R p which has 4 elements representing North America , Europe , Australasia , and rest of World . See Fig . S1 for the compo - sition of SA and CIP clusters , and SI Appendix S1 for addi - tional description of how these classification systems are con - structed . Figure S2 ( Fig . S3 ) shows the frequency of each SA ( CIP ) category and the pairwise frequency of all { SA , SA } ( { CIP , CIP } ) combinations over the 10 - year period cen - tered on 2014 , along with their relative changes after 2014 ; See SI Appendix S2 - S3 for discussion of the relevant changes in SA and CIP categories after 2014 . We represent the collection of article features by (cid:126)F p ≡ { −→ SA p , −−→ CIP p , −→ R p } . As indicated in Fig . 1 , based upon the distribution of types tabulated as counts across vector ele - ments , an article is either cross - domain , representing a diverse mixture of types denoted by X ; or mono - domain , denoted by M . We use a generic operator notation to specify how arti - cles are classified as X or M , The objective criteria of the feature operator O is specified by its subscript : for example O SA ( (cid:126)F p ) yields one of two values : X SA or M ; similarly , O CIP ( (cid:126)F p ) = X CIP or M . Note that all scholars map onto a single CIP , hence solo - authored research articles are by defi - nition classified by O CIP as M . While we acknowledge that is possible for a scholar to have significant expertise in two or more domains , we do not account for this duplicity , as it is likely to occur at the margins ; hence , the home department CIP represents the scholar’s principle domain of expertise . We also classify articles featuring both X SA and X CIP as O SA & CIP ( (cid:126)F p ) = X SA & CIP ( and otherwise M ) . To complement these categorical measures , we also devel - oped a scalar measure of an article’s cross - domain diversity ( see Materials & Methods – Measuring cross - domain diver - sity for additional details ) . By way of example , consider the vector −→ SA p ( or −−→ CIP p ) which tallies the SA ( or CIP counts ) for a given article p published in year t . We apply the outer tensor product −→ SA p ⊗−→ SA p ( or −−→ CIP p ⊗−−→ CIP p ) to represent all pairwise co - occurrences in a weighted matrix D p ( (cid:126)v p ) ( where (cid:126)v p represents a generic category vector ; see SI Appendix S4 for examples of the outer tensor product ) . The sum of elements in this co - occurrence matrix are normalized to unity so that each D p ( (cid:126)v p ) contributes equally to averages computed across all articles from a given year or period . Since the off - diagonal elements represent cross - domain combinations , their relative weight given by f D , p = 1 − Tr ( D p ) ∈ [ 0 , 1 ) is a straightfor - ward Blau - like measure of variation and disparity ( Harrison & Klein 2007 ) . Results Descriptive Analysis Increasing prevalence of cross - domain science . With the continuing shift towards large team science ( Milojevic 2014 ; Pavlidis et al 2014 ; Petersen et al 2014 ; Wuchty et al 2007 ) , one might expect a similar shift in the multiplicity of domains spanned by modern research teams – but to what degree ? Fig - ure 2 ( A ) addresses RQ2 by showing the frequencies of mono - domain ( M ) research articles versus cross - domain articles ( X ) in our HBS sample . Articles were separated into above - and below - average citation impact ( z ) for each publication - year cohort ( t ) , and within each of these two subsets we calcu - lated the fraction f # ( t | z ) of articles containing combinations across # = 1 , 2 , 3 and 4 categories . The fraction of mono - domain articles is trending downward , which we observe for both research topics ( SA ) and authors’ disciplinary affiliations ( CIP ) . The decline is much more steep for SA than for CIP . Correspondingly , cross - domain articles have become increas - ingly prevalent , in particular for SA . For both SA and CIP the two - category mixtures dominate the three - and four - category mixtures in frequency , in sequence . Accordingly , in the sec - tions that follow we do not distinguish between cross - domain articles with different # . As a first indication of the comparative advantage associated with X , we observe a robust inequality f # ( t | z > 0 ) > f # ( t | z < 0 ) for cross - domain research ( # ≥ 2 ) , meaning a higher frequency of cross - domain combinations is observed among articles with higher impact . Contrariwise , in the case of mono - domain research the opposite phenomenon occurs , f 1 ( t | z > 0 ) < f 1 ( t | z < 0 ) . Taking into consideration temporal trends , these robust patterns indicate a faster depletion of impactful mono - domain articles , coincident with an increased prevalence of impact - ful research drawing upon integrative recombinant innovation . Recombinant innovation at the convergence nexus . Com - prehensive analysis of biomedical science indicates that con - vergence has largely been mediated around the integration of modern techno - informatics capabilities ( Yang et al 2021 ) . Yet within any domain , in particular HBS , the questions remains as to the development of a functional nexus that sustains and possibly even accelerates high - impact discovery by both ex - panding the number of possible functional expertise config - urations and supporting rich cross - disciplinary exchange of new knowledge and best practices . The robust inequality f # ( t | z > 0 ) > f # ( t | z < 0 ) provides support at the aggregate level , but does not lend any structural evidence . To further address RQ2 , Fig . 2 ( B ) illustrates the compo - sition of the HBS convergence nexus , showing integration of cross - disciplinary expertise across three broad yet distinct biomedical domains . Shown are the populations of HBS re - searchers by region , represented as collaboration networks compared over two non - overlapping 10 - year intervals to in - dicate dynamics . Each node represents a researcher , colored according to three disciplinary CIP superclusters : ( i ) neuro - biological sciences ( corresponding to CIP 1 - 4 ) , ( ii ) health sciences ( CIP 5 - 7 ) , and ( iii ) engineering & information sci - 5 FIG . 2 : Trends in cross - domain scholarship in Human Brain Science . ( A ) Fraction f # ( t | z ) of articles published each year t that feature a particular number ( # ) of categories . Articles are split into an above - average citation subset ( z p > 0 ) and below - average citation subset ( z p < 0 ) . Upper panel : Articles categorized by SA . Middle panel : Articles categorized by CIP ; subpanel shows data on logarithmic y - axis ; Lower panel : Articles categorized by both SA and CIP . Distinguishing frequencies by citation group indicates higher levels of cross - domain combinations among research articles with higher scientific impact – for both SA and CIP . However , cross - domain activity levels are visibly higher for SA than for CIP , indicating higher barriers to boundary - crossing arising from mixing different scholar expertise . ( B ) Snapshots of the collaboration network at 10 - year intervals indicating researcher population sizes by region , and the densification of convergence science at cross - disciplinary interfaces . Nodes ( researchers ) are sized according to the number of collaborators ( link degree ) within each time window . ences ( CIP 8 - 9 ) . Node locations are fixed to facilitate vi - sual representation of network densification . Inter - and cross - regional comparison alludes to the emergence and densifica - tion of cross - domain interfaces ( see also Fig . S4 ) . Because the network layout is determined by the underlying structure , there is a high degree of clustering by node color , emphasiz - ing both the relative sizes of the subpopulations that are well - balanced across region and time , and also the convergent inter - faces where cross - disciplinary collaboration and knowledge exchange are likely to catalyze . As such , these communities of expertise conjure the image of a P´olya urn , whereby suc - cessful configurations reinforce the adoption of similar con - figurations . The links that span disciplinary boundaries are funda - mental conduits across which scientists’ strategic affinity for exploration ( Foster et al 2015 ; Rotolo & Messeni Petruzzelli 2013 ) is effected via cross - disciplinary collaboration that brings “together distinctive components of two or more dis - ciplines” ( Nissani 1995 ; Petersen et al 2018 ) . Our analysis of cross - disciplinary collaboration indicates that the fraction of articles featuring convergent collaboration have continued to grow over the last two decades ( see Fig . S4 ) . In what follows we further distinguish between integration across neighboring ( Leahey & Moody 2014 ) and distant domains , with the latter appropriately representing convergence ( National Research Council 2005 , 2014 ; Roco et al 2013 ) . Cross - domain convergence of expertise ( CIP ) and knowl - edge ( SA ) . In the context of the bureaucratic structure - function problem , team assembly should be optimized by strategically matching scholarly expertise and research topics to address the particular demands of a particular challenge . 6 FIG . 3 : Evolution of SA boundary - crossing within and across disciplinary clusters . ( A ) SA composition of HBS research within disciplinary ( CIP ) clusters . Each subpanel represents articles published by researchers from a given CIP cluster , showing the fraction of article - level MeSH belonging to each SA , shown over 5 - year intervals across the period 1970 - 2018 . The increasing prominence of SA 5 & 6 in nearly all domains , in particular CIP 4 ( Biotech . & Genetics ) indicates the critical role of informatic capabilities in facilitating biomedical convergence science ( Yang et al 2021 ) . ( B ) Empirical CIP - SA association networks calculated for non - overlapping sets of mono - domain ( M CIP (cid:29) M SA ) and cross - domain ( X CIP (cid:29) X SA ) articles , and based upon the Broad configuration . The difference between these two bi - partite networks ( ∆ XM ) indicates the emergent research channels that are facilitated by simultaneous X CIP and X SA boundary crossing – in particular integrating SA 2 with 3 ( i . e . the structure - function nexus ) facilitated by teams combining disciplines 1 , 2 , 4 and 9 . Hence , with 9 different disciplinary ( CIP ) domains histori - cally faced with a variety of challenges , RQ3 addresses to what degree these domains differ in terms of their compo - sition of targeted SA . Fig . 3 ( A ) illustrates the evolution of topical diversity within and across each CIP cluster , reveal - ing several common patterns . First , nearly all domains show a reduction in research pertaining to structure ( SA 2 ) , with the exception of Biotechnology & Genetics , which was ori - ented around the structure - function problem from the outset . As such , this domain features a steady balance between SA 2 - 5 , while being an early adopter of techno - informatics con - cepts and methods ( SA 6 ) . Early balance around the inno - vation triple - helix ( Petersen et al 2016 ) may explain to some degree the longstanding success of the genomics revolution , as the core disciplines of biology and computing were primed for a fruitful union ( Petersen et al 2018 ) . Other HBS disci - plinary clusters are also integrating techno - informatic capa - bilities , reflecting a widespread pattern observed across all of biomedical science ( Yang et al 2021 ) . Which CIP - SA combinations are are overrepresented in boundary - crossing HBS research ? Inasmuch as mono - domain articles identify the topical boundary closely associated with individual disciplines , cross - domain articles are useful for identifying otherwise obscured boundaries that call for both X CIP and X SA in combination . We identified these novel CIP - SA relations by collecting articles that are purely mono - domain for both CIP and SA ( i . e . , those with O CIP ( (cid:126)F p ) = O SA ( (cid:126)F p ) = M ) and a complementary non - overlapping sub - set of articles that are simultaneously cross - domain for both CIP and SA ( i . e . , O SA & CIP ( (cid:126)F p ) = X SA & CIP ) . Starting with mono - domain articles , we identified the SA that are most frequently associated with each CIP category . Formally , this amounts to calculating the bi - partite network between CIP and SA , denoted by M CIP (cid:29) M SA . These 7 Subset SA M ean d i v e r s i t y ( c a t ego r i c a l c o - o cc u rr en c e ) pe r a r t i c l e , ⟨ f D ( t ) ⟩ E B North America EuropeAustralasia Subset CIP ���� ���� ���� ���� ���� ���� ���� ���� ���� 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Information Science � � � ���� ���� ���� ���� ���� ���� ���� ���� ���� All SA F C Subset CIP ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� 1 : Neurosciences 2 : Biology 3 : Psychology 4 : (cid:1) Biotech . & Genetics 5 : Medical Specialty 6 : Health Sciences 7 : Pathology & Pharmacology 8 : Eng . & Informatics 9 : Chemistry & Physics & Math Topical Diversity ( SA ) Disciplinary Diversity ( CIP ) N e i ghbo r i ng D i s t a n t � � � [ 1 , 3 ] [ 2 , 4 - 7 ] [ 1 , 3 , 5 ] [ 4 , 8 ] � � � � � � 1 [ 2 - 4 ] [ 1 - 4 ] [ 5 , 6 ] All CIP 1 2 3 4 5 6 7 8 9 A � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ���� ���� ���� ���� ���� ���� ���� ���� ���� All SA � � � � � � � � � � � � � � � � � � � � � ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� 1 2 3 4 5 6 D B r o a d G CIP CIP SA SA CIP SA M CIP ⇄ M SA ∆ XM = ( X ⇄ X ) − ( M ⇄ M ) X CIP ⇄ X SA Difference 4 C I P — SA c oup li ng Cross - domain articles Mono - domain articles 3 1 4 2 6 7 5 8 9 1 2 4 5 3 1 4 2 6 7 5 8 9 1 2 3 4 5 3 1 4 2 6 7 5 8 9 1 2 3 4 5 CIP : SA : B r o a d FIG . 4 : Evolution of CIP and SA diversity in Human Brain Science research . ( A - F ) Each (cid:104) f D ( t ) (cid:105) represents the average article diversity measured as categorical co - occurrence , by geographic region : North America ( orange ) , Europe ( blue ) , and Australasia ( red ) . Each matrix motif indicates the set of CIP or SA categories used to compute D p in Eq . ( 2 ) ; categories included in brackets are considered in union . For example , panel ( A ) calculates (cid:104) f D , CIP ( t ) (cid:105) across all 9 CIP categories ; instead , panel ( B ) is based upon counts for two super - groups , the first consisting of the union of CIP counts for categories 1 and 3 , and the second comprised of categories 2 , 4 , 5 , 6 and 7 . ( A , D ) Broad diversity is calculated using all categories considered as separate domains ; ( B , E ) Neighboring represents the shorter - distance boundary across the neuro - psychological ↔ bio - medical interface ; ( C , F ) Distant represents longer - distance convergence across the neuro - psycho - medical ↔ techno - informatic interface . CIP - SA associations are calculated by averaging the −→ SA p for mono - domain articles from each CIP category , given by (cid:104)−→ SA (cid:105) CIP . Figure 3 ( B ) highlights the most prominent CIP - SA links ( see SI Appendix S5 for more details ) . Likewise , we also calculated the bi - partite network X CIP (cid:29) X SA using the subset of X SA & CIP articles . To identify the cross - domain frontier , we calculated the net - work difference ∆ XM ≡ X CIP (cid:29) X SA − M CIP (cid:29) M SA , and plot the links with positive values – i . e . CIP - SA links that are over - represented in X CIP (cid:29) X SA relative to M CIP (cid:29) M SA . Results identify SA that are reached by way of cross - disciplinary teams . SA 2 ( Anatomy and Organisms ) and 3 ( Phenomena & Processes ) representing the structure - function problem , stand out as a potent convergence nexus accessible by teams combining disciplines 1 , 2 , 4 and 9 . A related key insight concerns the relative increase in SA integration achieved by increased CIP diversity . Figure S5 compares the average number of SA integrated by teams with varying number of distinct CIP , N CIP , p . On average , mono - disciplinary teams ( N CIP , p = 1 ) span 2 . 2 SA , whereas teams with N CIP , p = 3 span 19 % more SA , confirming that cross - disciplinary configurations are functional in achieving research breadth . Quantitative Model Trends in cross - domain activity . To address the temporal and geographic parity associated with RQ4 , we define three types of cross - domain configurations – Broad , Neighboring , and Distant – defined according to a particular combination of SA and CIP categories featured by a given article . Broad is the most generic cross - domain configuration , based upon combinations of any two or more SA ( or CIP ) categories , and represented by our operator notation as O SA ( (cid:126)F p ) = X SA ( or O CIP ( (cid:126)F p ) = X CIP , respec - tively ) . Neighboring is the X configuration that cap - tures the neuro - psychological ↔ bio - medical interface rep - resenting articles that contain MeSH from SA ( 1 ) and also from SA ( 2 , 3 or 4 ) , represented summarily as [ 1 ] × [ 2 − 4 ] ) ; and for CIP , combinations containing CIP ( 1 or 3 ) and ( 2 , 4 , 5 , 6 or 7 ) , represented as [ 1 , 3 ] × [ 2 , 4 − 7 ] . Articles featuring these configurations are repre - sented using our operator notation as O Neighboring , SA ( (cid:126)F p ) = X Neighboring , SA , O Neighboring , CIP ( (cid:126)F p ) = X Neighboring , CIP , or O Neighboring , SA & CIP ( (cid:126)F p ) = X Neighboring , SA & CIP ; alterna - tively , articles not containing the specific category combina - tions are represented by M . 8 Distant is the X configuration that captures the neuro - psycho - medical ↔ techno - informatic interface . The spe - cific set of category combinations representing this config - uration are SA [ 1 - 4 ] × [ 5 , 6 ] ; and for CIP , [ 1 , 3 , 5 ] × [ 4 , 8 ] ; as above , articles featuring ( or not featuring ) categories span - ning these categories are represented by X Distant , SA ( belong to a counterfactual set indicated by M ) , X Distant , CIP ( resp . , M ) , X Distant , SA & CIP ( resp . , M ) . By way of example , the bottom of Figure 1 illustrates an article combining SA 1 and 4 , which is thereby classified as both X SA and X Neighboring , SA ; and , an article featuring CIP 1 , 3 , 5 , 8 , which is thereby both X CIP and X Distant , CIP . To complement these categorical variables , we also devel - oped a Blau - like measure of cross - domain diversity , given by f D , p ( see Methods Measuring cross - domain diversity ) . Fig - ure 4 shows the trends in mean diversity (cid:104) f D ( t ) (cid:105) for the Broad , Neighboring , and Distant configurations . For each configuration we provide a schematic motif illustrating the combinations measured by D p ( (cid:126)v p ) , with diagonal compo - nents representing mono - domain articles ( indicated by 1 on the matrix diagonal ) and upper - diagonal elements capturing cross - domain combinations ( indicated by X ) . Comparing SA and CIP , there are higher diversity levels for SA , in addition to a prominent upward trend . In terms of CIP , Fig . 4 ( A ) indi - cates a decline in Broad diversity in recent years , with North America ( NA ) showing higher levels than Europe ( EU ) and Australasia ( AA ) ; these general patterns are also evident for Neighboring diversity , see Fig . 4 ( B ) . Distant CIP diversity shown in Fig . 4 ( C ) indicates a recent decline for AA and NA , with NA peaking around 2009 ; contrariwise , EU shows a steady increases consistent with the computational framing of the Human Brain Project . In contradistinction , all three regions show steady increase irrespective of configuration in the case of SA diversity , consistent with scholars integrating topics without integrating scholarly expertise , possibly owing to differential costs associated with each . For both Broad and Neighboring configurations , NA and EU show remarkably similar levels of SA diversity above AA ; however , in the case of Neighboring , AA appears to be catching up quickly since 2010 , see Fig . 4 ( D , E ) . In the case of Distant , all regions show steady increase that appears to be in lockstep for the entire period . See Figs . S6 - S7 and SI Appendix Text S6 for trends in SA and CIP diversity across additional configurations . Regression model – propensity for and impact of X . To address RQ5 , we constructed article - level and author - level panel data to facilitate measuring factors relating to SA and CIP diversity and shifts related to the ramp - up of HBS flagship projects circa 2013 around the globe . To address these two outcomes , we modeled two dependent variables separately : In the first model the dependent variable is the propensity for cross - domain research ( indicated by X ; depending on the focus around topics , disciplines or both , then X is specified by X SA , X CIP or X SA & CIP ) . We use a Logit specification to model the likelihood P ( X ) . In the second model the dependent variable is the article’s scientific impact , proxied by c p . Building on previous efforts ( Petersen 2018 ; Petersen et al 2018 ) , we apply a logarithmic transform to c p that facilitates removing the time - dependent trend in the location and scale of the underlying log - normal citation distribution ( Radicchi et al 2008 ) ( see Methods – Normalization of Citation Impact ) . Figure S9 shows the covariation matrix between the principal variables of interest . Model A : Quantifying the propensity for X and the role of funding . As defined , O ( (cid:126)F p ) = X or M is a two - state outcome variable with complementary likelihoods , P ( X ) + P ( M ) = 1 . Thus , we apply logistic regression to model the odds Q ≡ P ( X ) P ( M ) , measuring the propensity to adopt cross - domain configurations . We then estimate the annual growth in P ( X ) by modeling the odds as log ( Q p ) = β 0 + β y y p + (cid:126)β · (cid:126)x , where (cid:126)x represents the additional controls for confounding sources of variation , in particular increasing k p associated with the growth of team science ( Milojevic 2014 ; Wuchty et al 2007 ) . See SI Appendix Text S7 , in particular Eqns . ( S2 ) - ( S4 ) , for the full model specification ; and , Tables S1 - S3 for param - eter estimates . Summary results shown in Fig . 5 ( A ) indicate a roughly 3 % annual growth in P ( X SA ) , consistent with descriptive trends shown in Fig . 2 . In contradistinction , growth rates for P ( X CIP ) are generally smaller , indicative of the addi - tional barriers to integrating individual expertise as opposed to just combining different research topics . In the case of P ( X SA & CIP ) , the growth rate is higher for Distant , where the need for cross - disciplinary expertise cannot be short - circuited as easily as in Neighboring . A relevant dimension of RQ5 is how HBS projects have altered the propensity for X . Hence , we added an indicator variable I 2014 + which takes the value 1 for articles with y p ≥ 2014 and 0 otherwise . Figure 5 ( B ) indicates significant decline in P ( X ) for X CIP and X SA & CIP for each configu - ration on the order of - 30 % ; this result is consistent with the recent increase in f 1 ( t | z ) visible in Fig . 2 ( B ) . Model B : Quantifying the citation premium associated with X and funding . We model the normalized citation im - pact z p = α a + γ X SA I X SA , p + γ X CIP I X CIP , p + (cid:126)β · (cid:126)x , where (cid:126)x represents the additional control variables and α a repre - sents an author fixed - effect to account for unobserved time - invariant factors specific to each researcher . The primary test variables are I X SA , p and I X CIP , p , two binary factor variables with I X SA , p = 1 if O SA ( (cid:126)F p ) = X SA and 0 if O SA ( (cid:126)F p ) = M , defined similarly for CIP . To distinguish estimates by configuration , for Neighboring we specify I X Neighboring , SA and I X Neighboring , CIP , with similar notation for Distant . Full model estimates are shown in Tables S4 - S5 . Figure 5 ( C ) summarizes the model estimates – γ X SA , γ X CIP and γ X SA & CIP – quantifying the citation premium at - tributable to X . 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oobF4q4de3OvzFtZ6GtBy6cnHMvufdEKWfauO63U1pb39jcKm9Xdnb39g + qh0cdnWSK0DZJeKJ6EWjKmaRtwwynvVRREBGn3Wh8M / O7D1Rplsh7M0lpICCWbMgIGCuF1brnutjnIGNOsa9ZLAD7qnjGIASEeW8aVmtuw50DrxKvIDVUoBVWv / xBQjJBpSEctO57bmqCHJRhhNNpxc80TYGMIaZ9SyUIqoN8fs4U160ywMNE2ZIGz9XfEzkIrScisp0CzEgvezPxP6 + fmeFVkDOZZoZKsvhomHFsEjzLBg + YosTwiSVAFLO7YjICBcTYBCs2BG / 55FXSOW94bsO7u6g1r4s4yugEnaIz5KFL1ES3qIXaiKBH9Ixe0Zvz5Lw4787HorXkFDPH6A + czx + 7T5tN < / latexit > < latexit sha1 _ base64 = " djjowv + yBMUg3TY1OmMC6IvTy5c = " > AAACDnicbVDLSgMxFM3UV62vqks3wVJwVWZE0GXRjcsK9gGdYbiTptPQJDMkGaEM / QI3 / oobF4q4de3OvzFtZ6GtBy6cnHMvufdEKWfauO63U1pb39jcKm9Xdnb39g + qh0cdnWSK0DZJeKJ6EWjKmaRtwwynvVRREBGn3Wh8M / O7D1Rplsh7M0lpICCWbMgIGCuF1brnutjnIGNOsa9ZLAD7qnjGIASEeW8aVmtuw50DrxKvIDVUoBVWv / xBQjJBpSEctO57bmqCHJRhhNNpxc80TYGMIaZ9SyUIqoN8fs4U160ywMNE2ZIGz9XfEzkIrScisp0CzEgvezPxP6 + fmeFVkDOZZoZKsvhomHFsEjzLBg + YosTwiSVAFLO7YjICBcTYBCs2BG / 55FXSOW94bsO7u6g1r4s4yugEnaIz5KFL1ES3qIXaiKBH9Ixe0Zvz5Lw4787HorXkFDPH6A + czx + 7T5tN < / latexit > < latexit sha1 _ base64 = " djjowv + yBMUg3TY1OmMC6IvTy5c = " > AAACDnicbVDLSgMxFM3UV62vqks3wVJwVWZE0GXRjcsK9gGdYbiTptPQJDMkGaEM / QI3 / oobF4q4de3OvzFtZ6GtBy6cnHMvufdEKWfauO63U1pb39jcKm9Xdnb39g + qh0cdnWSK0DZJeKJ6EWjKmaRtwwynvVRREBGn3Wh8M / O7D1Rplsh7M0lpICCWbMgIGCuF1brnutjnIGNOsa9ZLAD7qnjGIASEeW8aVmtuw50DrxKvIDVUoBVWv / xBQjJBpSEctO57bmqCHJRhhNNpxc80TYGMIaZ9SyUIqoN8fs4U160ywMNE2ZIGz9XfEzkIrScisp0CzEgvezPxP6 + fmeFVkDOZZoZKsvhomHFsEjzLBg + YosTwiSVAFLO7YjICBcTYBCs2BG / 55FXSOW94bsO7u6g1r4s4yugEnaIz5KFL1ES3qIXaiKBH9Ixe0Zvz5Lw4787HorXkFDPH6A + czx + 7T5tN < / latexit > P e r c en t i n c r ea s e i n c i t a t i on s a ss o c i a t ed w i t h c p < latexit sha1 _ base64 = " uzYFjYRXHfqWWIIseHtkNlAROZk = " > AAAB6nicbVBNS8NAEJ3Ur1q / oh69LBbBU0lE0GPRi8eK9gPaUDbbTbt0swm7E6GE / gQvHhTx6i / y5r9x2 + agrQ8GHu / NMDMvTKUw6HnfTmltfWNzq7xd2dnd2z9wD49aJsk0402WyER3Qmq4FIo3UaDknVRzGoeSt8Px7cxvP3FtRKIecZLyIKZDJSLBKFrpgfXTvlv1at4cZJX4BalCgUbf / eoNEpbFXCGT1Jiu76UY5FSjYJJPK73M8JSyMR3yrqWKxtwE + fzUKTmzyoBEibalkMzV3xM5jY2ZxKHtjCmOzLI3E / / zuhlG10EuVJohV2yxKMokwYTM / iYDoTlDObGEMi3srYSNqKYMbToVG4K / / PIqaV3UfK / m319W6zdFHGU4gVM4Bx + uoA530IAmMBjCM7zCmyOdF + fd + Vi0lpxi5hj + wPn8AUsujco = < / latexit > < latexit sha1 _ base64 = " uzYFjYRXHfqWWIIseHtkNlAROZk = " > AAAB6nicbVBNS8NAEJ3Ur1q / oh69LBbBU0lE0GPRi8eK9gPaUDbbTbt0swm7E6GE / gQvHhTx6i / y5r9x2 + agrQ8GHu / NMDMvTKUw6HnfTmltfWNzq7xd2dnd2z9wD49aJsk0402WyER3Qmq4FIo3UaDknVRzGoeSt8Px7cxvP3FtRKIecZLyIKZDJSLBKFrpgfXTvlv1at4cZJX4BalCgUbf / eoNEpbFXCGT1Jiu76UY5FSjYJJPK73M8JSyMR3yrqWKxtwE + fzUKTmzyoBEibalkMzV3xM5jY2ZxKHtjCmOzLI3E / / zuhlG10EuVJohV2yxKMokwYTM / iYDoTlDObGEMi3srYSNqKYMbToVG4K / / PIqaV3UfK / m319W6zdFHGU4gVM4Bx + uoA530IAmMBjCM7zCmyOdF + fd + Vi0lpxi5hj + wPn8AUsujco = < / latexit > < latexit sha1 _ base64 = " uzYFjYRXHfqWWIIseHtkNlAROZk = " > AAAB6nicbVBNS8NAEJ3Ur1q / oh69LBbBU0lE0GPRi8eK9gPaUDbbTbt0swm7E6GE / gQvHhTx6i / y5r9x2 + agrQ8GHu / NMDMvTKUw6HnfTmltfWNzq7xd2dnd2z9wD49aJsk0402WyER3Qmq4FIo3UaDknVRzGoeSt8Px7cxvP3FtRKIecZLyIKZDJSLBKFrpgfXTvlv1at4cZJX4BalCgUbf / eoNEpbFXCGT1Jiu76UY5FSjYJJPK73M8JSyMR3yrqWKxtwE + fzUKTmzyoBEibalkMzV3xM5jY2ZxKHtjCmOzLI3E / / zuhlG10EuVJohV2yxKMokwYTM / iYDoTlDObGEMi3srYSNqKYMbToVG4K / / PIqaV3UfK / m319W6zdFHGU4gVM4Bx + uoA530IAmMBjCM7zCmyOdF + fd + Vi0lpxi5hj + wPn8AUsujco = < / latexit > < latexit sha1 _ base64 = " uzYFjYRXHfqWWIIseHtkNlAROZk = " > AAAB6nicbVBNS8NAEJ3Ur1q / oh69LBbBU0lE0GPRi8eK9gPaUDbbTbt0swm7E6GE / gQvHhTx6i / y5r9x2 + agrQ8GHu / NMDMvTKUw6HnfTmltfWNzq7xd2dnd2z9wD49aJsk0402WyER3Qmq4FIo3UaDknVRzGoeSt8Px7cxvP3FtRKIecZLyIKZDJSLBKFrpgfXTvlv1at4cZJX4BalCgUbf / eoNEpbFXCGT1Jiu76UY5FSjYJJPK73M8JSyMR3yrqWKxtwE + fzUKTmzyoBEibalkMzV3xM5jY2ZxKHtjCmOzLI3E / / zuhlG10EuVJohV2yxKMokwYTM / iYDoTlDObGEMi3srYSNqKYMbToVG4K / / PIqaV3UfK / m319W6zdFHGU4gVM4Bx + uoA530IAmMBjCM7zCmyOdF + fd + Vi0lpxi5hj + wPn8AUsujco = < / latexit > X < latexit sha1 _ base64 = " lF / jmUAuDzRzq6sLugXrAxB1reg = " > AAAB6XicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPRi8cq9gPaUDbbTbt0swm7E6GE / gMvHhTx6j / y5r9x0 + agrQ8GHu / NMDMvSKQw6LrfTmltfWNzq7xd2dnd2z + oHh61TZxqxlsslrHuBtRwKRRvoUDJu4nmNAok7wST29zvPHFtRKwecZpwP6IjJULBKFrpoVsZVGtu3Z2DrBKvIDUo0BxUv / rDmKURV8gkNabnuQn6GdUomOSzSj81PKFsQke8Z6miETd + Nr90Rs6sMiRhrG0pJHP190RGI2OmUWA7I4pjs + zl4n9eL8Xw2s + ESlLkii0WhakkGJP8bTIUmjOUU0so08LeStiYasrQhpOH4C2 / vEraF3XPrXv3l7XGTRFHGU7gFM7BgytowB00oQUMQniGV3hzJs6L8 + 58LFpLTjFzDH / gfP4A6h2M8A = = < / latexit > < latexit sha1 _ base64 = " lF / jmUAuDzRzq6sLugXrAxB1reg = " > AAAB6XicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPRi8cq9gPaUDbbTbt0swm7E6GE / gMvHhTx6j / y5r9x0 + agrQ8GHu / NMDMvSKQw6LrfTmltfWNzq7xd2dnd2z + oHh61TZxqxlsslrHuBtRwKRRvoUDJu4nmNAok7wST29zvPHFtRKwecZpwP6IjJULBKFrpoVsZVGtu3Z2DrBKvIDUo0BxUv / rDmKURV8gkNabnuQn6GdUomOSzSj81PKFsQke8Z6miETd + Nr90Rs6sMiRhrG0pJHP190RGI2OmUWA7I4pjs + zl4n9eL8Xw2s + ESlLkii0WhakkGJP8bTIUmjOUU0so08LeStiYasrQhpOH4C2 / vEraF3XPrXv3l7XGTRFHGU7gFM7BgytowB00oQUMQniGV3hzJs6L8 + 58LFpLTjFzDH / gfP4A6h2M8A = = < / latexit > < latexit sha1 _ base64 = " lF / jmUAuDzRzq6sLugXrAxB1reg = " > AAAB6XicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPRi8cq9gPaUDbbTbt0swm7E6GE / gMvHhTx6j / y5r9x0 + agrQ8GHu / NMDMvSKQw6LrfTmltfWNzq7xd2dnd2z + oHh61TZxqxlsslrHuBtRwKRRvoUDJu4nmNAok7wST29zvPHFtRKwecZpwP6IjJULBKFrpoVsZVGtu3Z2DrBKvIDUo0BxUv / rDmKURV8gkNabnuQn6GdUomOSzSj81PKFsQke8Z6miETd + Nr90Rs6sMiRhrG0pJHP190RGI2OmUWA7I4pjs + zl4n9eL8Xw2s + ESlLkii0WhakkGJP8bTIUmjOUU0so08LeStiYasrQhpOH4C2 / vEraF3XPrXv3l7XGTRFHGU7gFM7BgytowB00oQUMQniGV3hzJs6L8 + 58LFpLTjFzDH / gfP4A6h2M8A = = < / latexit > < latexit sha1 _ base64 = " lF / jmUAuDzRzq6sLugXrAxB1reg = " > AAAB6XicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPRi8cq9gPaUDbbTbt0swm7E6GE / gMvHhTx6j / y5r9x0 + agrQ8GHu / NMDMvSKQw6LrfTmltfWNzq7xd2dnd2z + oHh61TZxqxlsslrHuBtRwKRRvoUDJu4nmNAok7wST29zvPHFtRKwecZpwP6IjJULBKFrpoVsZVGtu3Z2DrBKvIDUo0BxUv / rDmKURV8gkNabnuQn6GdUomOSzSj81PKFsQke8Z6miETd + Nr90Rs6sMiRhrG0pJHP190RGI2OmUWA7I4pjs + zl4n9eL8Xw2s + ESlLkii0WhakkGJP8bTIUmjOUU0so08LeStiYasrQhpOH4C2 / vEraF3XPrXv3l7XGTRFHGU7gFM7BgytowB00oQUMQniGV3hzJs6L8 + 58LFpLTjFzDH / gfP4A6h2M8A = = < / latexit > X SA < latexit sha1 _ base64 = " rZaE / znqWYWI / ThCo2 + LLwn8AH8 = " > AAAB7nicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPVi8eK1hbaUDbbSbt0swm7G6GE / ggvHhTx6u / x5r9x0 + agrQ8GHu / NMDMvSATXxnW / ndLK6tr6RnmzsrW9s7tX3T941HGqGLZYLGLVCahGwSW2DDcCO4lCGgUC28H4JvfbT6g0j + WDmSToR3QoecgZNVZqd / rZ / dW00q / W3Lo7A1kmXkFqUKDZr371BjFLI5SGCap113MT42dUGc4ETiu9VGNC2ZgOsWuppBFqP5udOyUnVhmQMFa2pCEz9fdERiOtJ1FgOyNqRnrRy8X / vG5qwks / 4zJJDUo2XxSmgpiY5L + TAVfIjJhYQpni9lbCRlRRZmxCeQje4svL5PGs7rl17 + 681rgu4ijDERzDKXhwAQ24hSa0gMEYnuEV3pzEeXHenY95a8kpZg7hD5zPH5KOjw0 = < / latexit > < latexit sha1 _ base64 = " rZaE / znqWYWI / ThCo2 + LLwn8AH8 = " > AAAB7nicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPVi8eK1hbaUDbbSbt0swm7G6GE / ggvHhTx6u / x5r9x0 + agrQ8GHu / NMDMvSATXxnW / ndLK6tr6RnmzsrW9s7tX3T941HGqGLZYLGLVCahGwSW2DDcCO4lCGgUC28H4JvfbT6g0j + WDmSToR3QoecgZNVZqd / rZ / dW00q / W3Lo7A1kmXkFqUKDZr371BjFLI5SGCap113MT42dUGc4ETiu9VGNC2ZgOsWuppBFqP5udOyUnVhmQMFa2pCEz9fdERiOtJ1FgOyNqRnrRy8X / vG5qwks / 4zJJDUo2XxSmgpiY5L + TAVfIjJhYQpni9lbCRlRRZmxCeQje4svL5PGs7rl17 + 681rgu4ijDERzDKXhwAQ24hSa0gMEYnuEV3pzEeXHenY95a8kpZg7hD5zPH5KOjw0 = < / latexit > < latexit sha1 _ base64 = " rZaE / znqWYWI / ThCo2 + LLwn8AH8 = " > AAAB7nicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPVi8eK1hbaUDbbSbt0swm7G6GE / ggvHhTx6u / x5r9x0 + agrQ8GHu / NMDMvSATXxnW / ndLK6tr6RnmzsrW9s7tX3T941HGqGLZYLGLVCahGwSW2DDcCO4lCGgUC28H4JvfbT6g0j + WDmSToR3QoecgZNVZqd / rZ / dW00q / W3Lo7A1kmXkFqUKDZr371BjFLI5SGCap113MT42dUGc4ETiu9VGNC2ZgOsWuppBFqP5udOyUnVhmQMFa2pCEz9fdERiOtJ1FgOyNqRnrRy8X / vG5qwks / 4zJJDUo2XxSmgpiY5L + TAVfIjJhYQpni9lbCRlRRZmxCeQje4svL5PGs7rl17 + 681rgu4ijDERzDKXhwAQ24hSa0gMEYnuEV3pzEeXHenY95a8kpZg7hD5zPH5KOjw0 = < / latexit > < latexit sha1 _ base64 = " rZaE / znqWYWI / ThCo2 + LLwn8AH8 = " > AAAB7nicbVBNS8NAEJ3Ur1q / qh69LBbBU0lE0GPVi8eK1hbaUDbbSbt0swm7G6GE / ggvHhTx6u / x5r9x0 + agrQ8GHu / NMDMvSATXxnW / ndLK6tr6RnmzsrW9s7tX3T941HGqGLZYLGLVCahGwSW2DDcCO4lCGgUC28H4JvfbT6g0j + WDmSToR3QoecgZNVZqd / rZ / dW00q / W3Lo7A1kmXkFqUKDZr371BjFLI5SGCap113MT42dUGc4ETiu9VGNC2ZgOsWuppBFqP5udOyUnVhmQMFa2pCEz9fdERiOtJ1FgOyNqRnrRy8X / vG5qwks / 4zJJDUo2XxSmgpiY5L + TAVfIjJhYQpni9lbCRlRRZmxCeQje4svL5PGs7rl17 + 681rgu4ijDERzDKXhwAQ24hSa0gMEYnuEV3pzEeXHenY95a8kpZg7hD5zPH5KOjw0 = < / latexit > X SA & CIP < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > X CIP < latexit sha1 _ base64 = " Az1qfvVTHjQOO220hL7WlBeFOMU = " > AAAB73icbVBNS8NAEJ2tX7V + VT16WSyCp5KIoMdiL3qrYNtAG8pmu2mXbjZxdyOU0D / hxYMiXv073vw3btoctPXBwOO9GWbmBYng2jjONyqtrW9sbpW3Kzu7e / sH1cOjjo5TRVmbxiJWXkA0E1yytuFGMC9RjESBYN1g0sz97hNTmsfywUwT5kdkJHnIKTFW8rxB1rxrzSqDas2pO3PgVeIWpAYFWoPqV38Y0zRi0lBBtO65TmL8jCjDqWCzSj / VLCF0QkasZ6kkEdN + Nr93hs + sMsRhrGxJg + fq74mMRFpPo8B2RsSM9bKXi / 95vdSE137GZZIaJuliUZgKbGKcP4 + HXDFqxNQSQhW3t2I6JopQYyPKQ3CXX14lnYu669Td + 8ta46aIowwncArn4MIVNOAWWtAGCgKe4RXe0CN6Qe / oY9FaQsXMMfwB + vwBJuuPXw = = < / latexit > < latexit sha1 _ base64 = " Az1qfvVTHjQOO220hL7WlBeFOMU = " > AAAB73icbVBNS8NAEJ2tX7V + VT16WSyCp5KIoMdiL3qrYNtAG8pmu2mXbjZxdyOU0D / hxYMiXv073vw3btoctPXBwOO9GWbmBYng2jjONyqtrW9sbpW3Kzu7e / sH1cOjjo5TRVmbxiJWXkA0E1yytuFGMC9RjESBYN1g0sz97hNTmsfywUwT5kdkJHnIKTFW8rxB1rxrzSqDas2pO3PgVeIWpAYFWoPqV38Y0zRi0lBBtO65TmL8jCjDqWCzSj / VLCF0QkasZ6kkEdN + Nr93hs + sMsRhrGxJg + fq74mMRFpPo8B2RsSM9bKXi / 95vdSE137GZZIaJuliUZgKbGKcP4 + HXDFqxNQSQhW3t2I6JopQYyPKQ3CXX14lnYu669Td + 8ta46aIowwncArn4MIVNOAWWtAGCgKe4RXe0CN6Qe / oY9FaQsXMMfwB + vwBJuuPXw = = < / latexit > < latexit sha1 _ base64 = " Az1qfvVTHjQOO220hL7WlBeFOMU = " > AAAB73icbVBNS8NAEJ2tX7V + VT16WSyCp5KIoMdiL3qrYNtAG8pmu2mXbjZxdyOU0D / hxYMiXv073vw3btoctPXBwOO9GWbmBYng2jjONyqtrW9sbpW3Kzu7e / sH1cOjjo5TRVmbxiJWXkA0E1yytuFGMC9RjESBYN1g0sz97hNTmsfywUwT5kdkJHnIKTFW8rxB1rxrzSqDas2pO3PgVeIWpAYFWoPqV38Y0zRi0lBBtO65TmL8jCjDqWCzSj / VLCF0QkasZ6kkEdN + Nr93hs + sMsRhrGxJg + fq74mMRFpPo8B2RsSM9bKXi / 95vdSE137GZZIaJuliUZgKbGKcP4 + HXDFqxNQSQhW3t2I6JopQYyPKQ3CXX14lnYu669Td + 8ta46aIowwncArn4MIVNOAWWtAGCgKe4RXe0CN6Qe / oY9FaQsXMMfwB + vwBJuuPXw = = < / latexit > < latexit sha1 _ base64 = " Az1qfvVTHjQOO220hL7WlBeFOMU = " > AAAB73icbVBNS8NAEJ2tX7V + VT16WSyCp5KIoMdiL3qrYNtAG8pmu2mXbjZxdyOU0D / hxYMiXv073vw3btoctPXBwOO9GWbmBYng2jjONyqtrW9sbpW3Kzu7e / sH1cOjjo5TRVmbxiJWXkA0E1yytuFGMC9RjESBYN1g0sz97hNTmsfywUwT5kdkJHnIKTFW8rxB1rxrzSqDas2pO3PgVeIWpAYFWoPqV38Y0zRi0lBBtO65TmL8jCjDqWCzSj / VLCF0QkasZ6kkEdN + Nr93hs + sMsRhrGxJg + fq74mMRFpPo8B2RsSM9bKXi / 95vdSE137GZZIaJuliUZgKbGKcP4 + HXDFqxNQSQhW3t2I6JopQYyPKQ3CXX14lnYu669Td + 8ta46aIowwncArn4MIVNOAWWtAGCgKe4RXe0CN6Qe / oY9FaQsXMMfwB + vwBJuuPXw = = < / latexit > DiD : X SA & CIP ⇥ 2014 + < latexit sha1 _ base64 = " 05wG9G5nfz7zlQvjM2zileG1Kzw = " > AAACFXicbVDJSgNBEO2JW4zbqEcvjUERlDATAoqnaHLQW0SzQCaEnk4nadKz0F0jhmF + wou / 4sWDIl4Fb / 6NneWgiQ8KHu9VUVXPDQVXYFnfRmphcWl5Jb2aWVvf2Nwyt3dqKogkZVUaiEA2XKKY4D6rAgfBGqFkxHMFq7uD0siv3zOpeODfwTBkLY / 0fN7llICW2uaJA + wB4jIvn + MEN9rx7YVzWLquJNgB7jGFJ37esgvHSdvMWjlrDDxP7CnJoikqbfPL6QQ08pgPVBClmrYVQismEjgVLMk4kWIhoQPSY01NfaI3tuLxVwk + 0EoHdwOpywc8Vn9PxMRTaui5utMj0Fez3kj8z2tG0D1rxdwPI2A + nSzqRgJDgEcR4Q6XjIIYakKo5PpWTPtEEgo6yIwOwZ59eZ7U8jnbytk3hWzxchpHGu2hfXSEbHSKiugKVVAVUfSIntErejOejBfj3fiYtKaM6cwu + gPj8wfQFJ1R < / latexit > < latexit sha1 _ base64 = " 05wG9G5nfz7zlQvjM2zileG1Kzw = " > AAACFXicbVDJSgNBEO2JW4zbqEcvjUERlDATAoqnaHLQW0SzQCaEnk4nadKz0F0jhmF + wou / 4sWDIl4Fb / 6NneWgiQ8KHu9VUVXPDQVXYFnfRmphcWl5Jb2aWVvf2Nwyt3dqKogkZVUaiEA2XKKY4D6rAgfBGqFkxHMFq7uD0siv3zOpeODfwTBkLY / 0fN7llICW2uaJA + wB4jIvn + MEN9rx7YVzWLquJNgB7jGFJ37esgvHSdvMWjlrDDxP7CnJoikqbfPL6QQ08pgPVBClmrYVQismEjgVLMk4kWIhoQPSY01NfaI3tuLxVwk + 0EoHdwOpywc8Vn9PxMRTaui5utMj0Fez3kj8z2tG0D1rxdwPI2A + nSzqRgJDgEcR4Q6XjIIYakKo5PpWTPtEEgo6yIwOwZ59eZ7U8jnbytk3hWzxchpHGu2hfXSEbHSKiugKVVAVUfSIntErejOejBfj3fiYtKaM6cwu + gPj8wfQFJ1R < / latexit > < latexit sha1 _ base64 = " 05wG9G5nfz7zlQvjM2zileG1Kzw = " > AAACFXicbVDJSgNBEO2JW4zbqEcvjUERlDATAoqnaHLQW0SzQCaEnk4nadKz0F0jhmF + wou / 4sWDIl4Fb / 6NneWgiQ8KHu9VUVXPDQVXYFnfRmphcWl5Jb2aWVvf2Nwyt3dqKogkZVUaiEA2XKKY4D6rAgfBGqFkxHMFq7uD0siv3zOpeODfwTBkLY / 0fN7llICW2uaJA + wB4jIvn + MEN9rx7YVzWLquJNgB7jGFJ37esgvHSdvMWjlrDDxP7CnJoikqbfPL6QQ08pgPVBClmrYVQismEjgVLMk4kWIhoQPSY01NfaI3tuLxVwk + 0EoHdwOpywc8Vn9PxMRTaui5utMj0Fez3kj8z2tG0D1rxdwPI2A + nSzqRgJDgEcR4Q6XjIIYakKo5PpWTPtEEgo6yIwOwZ59eZ7U8jnbytk3hWzxchpHGu2hfXSEbHSKiugKVVAVUfSIntErejOejBfj3fiYtKaM6cwu + gPj8wfQFJ1R < / latexit > < latexit sha1 _ base64 = " 05wG9G5nfz7zlQvjM2zileG1Kzw = " > AAACFXicbVDJSgNBEO2JW4zbqEcvjUERlDATAoqnaHLQW0SzQCaEnk4nadKz0F0jhmF + wou / 4sWDIl4Fb / 6NneWgiQ8KHu9VUVXPDQVXYFnfRmphcWl5Jb2aWVvf2Nwyt3dqKogkZVUaiEA2XKKY4D6rAgfBGqFkxHMFq7uD0siv3zOpeODfwTBkLY / 0fN7llICW2uaJA + wB4jIvn + MEN9rx7YVzWLquJNgB7jGFJ37esgvHSdvMWjlrDDxP7CnJoikqbfPL6QQ08pgPVBClmrYVQismEjgVLMk4kWIhoQPSY01NfaI3tuLxVwk + 0EoHdwOpywc8Vn9PxMRTaui5utMj0Fez3kj8z2tG0D1rxdwPI2A + nSzqRgJDgEcR4Q6XjIIYakKo5PpWTPtEEgo6yIwOwZ59eZ7U8jnbytk3hWzxchpHGu2hfXSEbHSKiugKVVAVUfSIntErejOejBfj3fiYtKaM6cwu + gPj8wfQFJ1R < / latexit > X SA & CIP < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > < latexit sha1 _ base64 = " 7L8NuWgBLmo5sozbbMG22E2sJyA = " > AAAB83icbVBNS8NAEJ3Ur1q / qh69LBbFU0lE0GO1F71VtB / QhLLZbtqlm03Y3Qgl9G948aCIV / + MN / + NmzQHbX0w8Hhvhpl5fsyZ0rb9bZVWVtfWN8qbla3tnd296v5BR0WJJLRNIh7Jno8V5UzQtmaa014sKQ59Trv + pJn53ScqFYvEo57G1AvxSLCAEayN5PYG6cO1e9q8a80qg2rNrts50DJxClKDAq1B9csdRiQJqdCEY6X6jh1rL8VSM8LprOImisaYTPCI9g0VOKTKS / ObZ + jEKEMURNKU0ChXf0 + kOFRqGvqmM8R6rBa9TPzP6yc6uPJSJuJEU0Hmi4KEIx2hLAA0ZJISzaeGYCKZuRWRMZaYaBNTFoKz + PIy6ZzXHbvu3F / UGjdFHGU4gmM4AwcuoQG30II2EIjhGV7hzUqsF + vd + pi3lqxi5hD + wPr8AWxCkJ0 = < / latexit > 100 h i X < latexit sha1 _ base64 = " emX4RmtxzxdSTNu7KkSLsICkG8U = " > AAACDnicdVDLSgMxFM34tr6qLt0ES8HVkKmlj53oxqWC1UKnlDtpOg0mmSHJCGXoF7jxV9y4UMSta3f + jWmtoKIHLpyccy + 590Sp4MYS8u7NzS8sLi2vrBbW1jc2t4rbO5cmyTRlLZqIRLcjMExwxVqWW8HaqWYgI8GuouuTiX91w7Thibqwo5R1JcSKDzgF66ResRwQgkMBKhYMh4bHEnCoZ88YpIRe3h73iiXiV5q1auMQE79G6rVqMCWVSvMQBz6ZooRmOOsV38J + QjPJlKUCjOkEJLXdHLTlVLBxIcwMS4FeQ8w6jiqQzHTz6TljXHZKHw8S7UpZPFW / T + QgjRnJyHVKsEPz25uIf3mdzA4a3ZyrNLNM0c + PBpnANsGTbHCfa0atGDkCVHO3K6ZD0ECtS7DgQvi6FP9PLit + QPzgvFo6Op7FsYL20D46QAGqoyN0is5QC1F0i + 7RI3ry7rwH79l7 + Wyd82Yzu + gHvNcPLJybmw = = < / latexit > < latexit sha1 _ base64 = " emX4RmtxzxdSTNu7KkSLsICkG8U = " > AAACDnicdVDLSgMxFM34tr6qLt0ES8HVkKmlj53oxqWC1UKnlDtpOg0mmSHJCGXoF7jxV9y4UMSta3f + jWmtoKIHLpyccy + 590Sp4MYS8u7NzS8sLi2vrBbW1jc2t4rbO5cmyTRlLZqIRLcjMExwxVqWW8HaqWYgI8GuouuTiX91w7Thibqwo5R1JcSKDzgF66ResRwQgkMBKhYMh4bHEnCoZ88YpIRe3h73iiXiV5q1auMQE79G6rVqMCWVSvMQBz6ZooRmOOsV38J + QjPJlKUCjOkEJLXdHLTlVLBxIcwMS4FeQ8w6jiqQzHTz6TljXHZKHw8S7UpZPFW / T + QgjRnJyHVKsEPz25uIf3mdzA4a3ZyrNLNM0c + PBpnANsGTbHCfa0atGDkCVHO3K6ZD0ECtS7DgQvi6FP9PLit + QPzgvFo6Op7FsYL20D46QAGqoyN0is5QC1F0i + 7RI3ry7rwH79l7 + Wyd82Yzu + gHvNcPLJybmw = = < / latexit > < latexit sha1 _ base64 = " emX4RmtxzxdSTNu7KkSLsICkG8U = " > AAACDnicdVDLSgMxFM34tr6qLt0ES8HVkKmlj53oxqWC1UKnlDtpOg0mmSHJCGXoF7jxV9y4UMSta3f + jWmtoKIHLpyccy + 590Sp4MYS8u7NzS8sLi2vrBbW1jc2t4rbO5cmyTRlLZqIRLcjMExwxVqWW8HaqWYgI8GuouuTiX91w7Thibqwo5R1JcSKDzgF66ResRwQgkMBKhYMh4bHEnCoZ88YpIRe3h73iiXiV5q1auMQE79G6rVqMCWVSvMQBz6ZooRmOOsV38J + QjPJlKUCjOkEJLXdHLTlVLBxIcwMS4FeQ8w6jiqQzHTz6TljXHZKHw8S7UpZPFW / T + QgjRnJyHVKsEPz25uIf3mdzA4a3ZyrNLNM0c + PBpnANsGTbHCfa0atGDkCVHO3K6ZD0ECtS7DgQvi6FP9PLit + QPzgvFo6Op7FsYL20D46QAGqoyN0is5QC1F0i + 7RI3ry7rwH79l7 + Wyd82Yzu + gHvNcPLJybmw = = < / latexit > < latexit sha1 _ base64 = " emX4RmtxzxdSTNu7KkSLsICkG8U = " > AAACDnicdVDLSgMxFM34tr6qLt0ES8HVkKmlj53oxqWC1UKnlDtpOg0mmSHJCGXoF7jxV9y4UMSta3f + jWmtoKIHLpyccy + 590Sp4MYS8u7NzS8sLi2vrBbW1jc2t4rbO5cmyTRlLZqIRLcjMExwxVqWW8HaqWYgI8GuouuTiX91w7Thibqwo5R1JcSKDzgF66ResRwQgkMBKhYMh4bHEnCoZ88YpIRe3h73iiXiV5q1auMQE79G6rVqMCWVSvMQBz6ZooRmOOsV38J + QjPJlKUCjOkEJLXdHLTlVLBxIcwMS4FeQ8w6jiqQzHTz6TljXHZKHw8S7UpZPFW / T + QgjRnJyHVKsEPz25uIf3mdzA4a3ZyrNLNM0c + PBpnANsGTbHCfa0atGDkCVHO3K6ZD0ECtS7DgQvi6FP9PLit + QPzgvFo6Op7FsYL20D46QAGqoyN0is5QC1F0i + 7RI3ry7rwH79l7 + Wyd82Yzu + gHvNcPLJybmw = = < / latexit > 100 h i X + < latexit sha1 _ base64 = " 8TI3tyZzzNMd2Nljxo2iicqiRfg = " > AAACEHicdVBNSyNBEO3JqqvxY7O7Ry + NQRSEoTtEx6Oslz0qmBjIhFDTqSRNenqG7p6FMOQnePGvePGwIl49ett / szMxgoo + KHj9XhVd9aJUSesY + + dVviwtr3xdXauub2xufat9 / 9G2SWYEtkSiEtOJwKKSGltOOoWd1CDEkcLLaHJa + pd / 0FiZ6As3TbEXw0jLoRTgCqlf2 + OM0VCBHimkoZWjGGhoFs8BKgf9vHMwq / ZrdeYH / JAfHVLmNwLeCIKSHB81gyblPpujThY469eewkEishi1Ewqs7XKWul4OxkmhcFYNM4spiAmMsFtQDTHaXj4 / aEZ3C2VAh4kpSjs6V19P5BBbO42jojMGN7bvvVL8yOtmbnjcy6VOM4daPH80zBR1CS3ToQNpUDg1LQgII4tdqRiDAeGKDMsQXi6ln5N2w + fM5 + fN + smvRRyrZJvskH3CSUBOyG9yRlpEkCtyQ / 6SO + / au / XuvYfn1oq3mPlJ3sB7 / A / xtZvv < / latexit > < latexit sha1 _ base64 = " 8TI3tyZzzNMd2Nljxo2iicqiRfg = " > AAACEHicdVBNSyNBEO3JqqvxY7O7Ry + NQRSEoTtEx6Oslz0qmBjIhFDTqSRNenqG7p6FMOQnePGvePGwIl49ett / szMxgoo + KHj9XhVd9aJUSesY + + dVviwtr3xdXauub2xufat9 / 9G2SWYEtkSiEtOJwKKSGltOOoWd1CDEkcLLaHJa + pd / 0FiZ6As3TbEXw0jLoRTgCqlf2 + OM0VCBHimkoZWjGGhoFs8BKgf9vHMwq / ZrdeYH / JAfHVLmNwLeCIKSHB81gyblPpujThY469eewkEishi1Ewqs7XKWul4OxkmhcFYNM4spiAmMsFtQDTHaXj4 / aEZ3C2VAh4kpSjs6V19P5BBbO42jojMGN7bvvVL8yOtmbnjcy6VOM4daPH80zBR1CS3ToQNpUDg1LQgII4tdqRiDAeGKDMsQXi6ln5N2w + fM5 + fN + smvRRyrZJvskH3CSUBOyG9yRlpEkCtyQ / 6SO + / au / XuvYfn1oq3mPlJ3sB7 / A / xtZvv < / latexit > < latexit sha1 _ base64 = " 8TI3tyZzzNMd2Nljxo2iicqiRfg = " > AAACEHicdVBNSyNBEO3JqqvxY7O7Ry + NQRSEoTtEx6Oslz0qmBjIhFDTqSRNenqG7p6FMOQnePGvePGwIl49ett / szMxgoo + KHj9XhVd9aJUSesY + + dVviwtr3xdXauub2xufat9 / 9G2SWYEtkSiEtOJwKKSGltOOoWd1CDEkcLLaHJa + pd / 0FiZ6As3TbEXw0jLoRTgCqlf2 + OM0VCBHimkoZWjGGhoFs8BKgf9vHMwq / ZrdeYH / JAfHVLmNwLeCIKSHB81gyblPpujThY469eewkEishi1Ewqs7XKWul4OxkmhcFYNM4spiAmMsFtQDTHaXj4 / aEZ3C2VAh4kpSjs6V19P5BBbO42jojMGN7bvvVL8yOtmbnjcy6VOM4daPH80zBR1CS3ToQNpUDg1LQgII4tdqRiDAeGKDMsQXi6ln5N2w + fM5 + fN + smvRRyrZJvskH3CSUBOyG9yRlpEkCtyQ / 6SO + / au / XuvYfn1oq3mPlJ3sB7 / A / xtZvv < / latexit > < latexit sha1 _ base64 = " 8TI3tyZzzNMd2Nljxo2iicqiRfg = " > AAACEHicdVBNSyNBEO3JqqvxY7O7Ry + NQRSEoTtEx6Oslz0qmBjIhFDTqSRNenqG7p6FMOQnePGvePGwIl49ett / szMxgoo + KHj9XhVd9aJUSesY + + dVviwtr3xdXauub2xufat9 / 9G2SWYEtkSiEtOJwKKSGltOOoWd1CDEkcLLaHJa + pd / 0FiZ6As3TbEXw0jLoRTgCqlf2 + OM0VCBHimkoZWjGGhoFs8BKgf9vHMwq / ZrdeYH / JAfHVLmNwLeCIKSHB81gyblPpujThY469eewkEishi1Ewqs7XKWul4OxkmhcFYNM4spiAmMsFtQDTHaXj4 / aEZ3C2VAh4kpSjs6V19P5BBbO42jojMGN7bvvVL8yOtmbnjcy6VOM4daPH80zBR1CS3ToQNpUDg1LQgII4tdqRiDAeGKDMsQXi6ln5N2w + fM5 + fN + smvRRyrZJvskH3CSUBOyG9yRlpEkCtyQ / 6SO + / au / XuvYfn1oq3mPlJ3sB7 / A / xtZvv < / latexit > X SA & CIP < latexit sha1 _ base64 = " Wo180o2m9D5KM / N0JWykRfFqW8I = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbFU9kVQY / VXvRW0X7AdinZNNuGZpMlyQpl6c / w4kERr / 4ab / 4b03YP2vpg4PHeDDPzwoQzbVz32ymsrK6tbxQ3S1vbO7t75f2DlpapIrRJJJeqE2JNORO0aZjhtJMoiuOQ03Y4qk / 99hNVmknxaMYJDWI8ECxiBBsr + Z1e9nDdPa3fNSa9csWtujOgZeLlpAI5Gr3yV7cvSRpTYQjHWvuem5ggw8owwumk1E01TTAZ4QH1LRU4pjrIZidP0IlV + iiSypYwaKb + nshwrPU4Dm1njM1QL3pT8T / PT010FWRMJKmhgswXRSlHRqLp / 6jPFCWGjy3BRDF7KyJDrDAxNqWSDcFbfHmZtM6rnlv17i8qtZs8jiIcwTGcgQeXUINbaEATCEh4hld4c4zz4rw7H / PWgpPPHMIfOJ8 / NIGQiQ = = < / latexit > < latexit sha1 _ base64 = " Wo180o2m9D5KM / N0JWykRfFqW8I = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbFU9kVQY / VXvRW0X7AdinZNNuGZpMlyQpl6c / w4kERr / 4ab / 4b03YP2vpg4PHeDDPzwoQzbVz32ymsrK6tbxQ3S1vbO7t75f2DlpapIrRJJJeqE2JNORO0aZjhtJMoiuOQ03Y4qk / 99hNVmknxaMYJDWI8ECxiBBsr + Z1e9nDdPa3fNSa9csWtujOgZeLlpAI5Gr3yV7cvSRpTYQjHWvuem5ggw8owwumk1E01TTAZ4QH1LRU4pjrIZidP0IlV + iiSypYwaKb + nshwrPU4Dm1njM1QL3pT8T / PT010FWRMJKmhgswXRSlHRqLp / 6jPFCWGjy3BRDF7KyJDrDAxNqWSDcFbfHmZtM6rnlv17i8qtZs8jiIcwTGcgQeXUINbaEATCEh4hld4c4zz4rw7H / PWgpPPHMIfOJ8 / NIGQiQ = = < / latexit > < latexit sha1 _ base64 = " Wo180o2m9D5KM / N0JWykRfFqW8I = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbFU9kVQY / VXvRW0X7AdinZNNuGZpMlyQpl6c / w4kERr / 4ab / 4b03YP2vpg4PHeDDPzwoQzbVz32ymsrK6tbxQ3S1vbO7t75f2DlpapIrRJJJeqE2JNORO0aZjhtJMoiuOQ03Y4qk / 99hNVmknxaMYJDWI8ECxiBBsr + Z1e9nDdPa3fNSa9csWtujOgZeLlpAI5Gr3yV7cvSRpTYQjHWvuem5ggw8owwumk1E01TTAZ4QH1LRU4pjrIZidP0IlV + iiSypYwaKb + nshwrPU4Dm1njM1QL3pT8T / PT010FWRMJKmhgswXRSlHRqLp / 6jPFCWGjy3BRDF7KyJDrDAxNqWSDcFbfHmZtM6rnlv17i8qtZs8jiIcwTGcgQeXUINbaEATCEh4hld4c4zz4rw7H / PWgpPPHMIfOJ8 / NIGQiQ = = < / latexit > < latexit sha1 _ base64 = " Wo180o2m9D5KM / N0JWykRfFqW8I = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbFU9kVQY / VXvRW0X7AdinZNNuGZpMlyQpl6c / w4kERr / 4ab / 4b03YP2vpg4PHeDDPzwoQzbVz32ymsrK6tbxQ3S1vbO7t75f2DlpapIrRJJJeqE2JNORO0aZjhtJMoiuOQ03Y4qk / 99hNVmknxaMYJDWI8ECxiBBsr + Z1e9nDdPa3fNSa9csWtujOgZeLlpAI5Gr3yV7cvSRpTYQjHWvuem5ggw8owwumk1E01TTAZ4QH1LRU4pjrIZidP0IlV + iiSypYwaKb + nshwrPU4Dm1njM1QL3pT8T / PT010FWRMJKmhgswXRSlHRqLp / 6jPFCWGjy3BRDF7KyJDrDAxNqWSDcFbfHmZtM6rnlv17i8qtZs8jiIcwTGcgQeXUINbaEATCEh4hld4c4zz4rw7H / PWgpPPHMIfOJ8 / NIGQiQ = = < / latexit > 100 h i ( X + X + ) < latexit sha1 _ base64 = " lFkdFjSXXpuYZi / SxkBYzM7uwm0 = " > AAACHXicdVBNSyQxEE37seqsrqN73EtwEFyEJm0PqDdxL3t0wdGB6WGoztS0wSTdJGlhaOaPePGvePHgIh68yP6bzXwIKvqg4OW9KlL10kIK6xj7F8zNLyx + WVpeqX1dXfu2Xt / YPLN5aTi2eC5z007BohQaW044ie3CIKhU4nl6 + Wvsn1 + hsSLXp25YYFdBpsVAcHBe6tWbEWM0kaAziTSxIlNAEzN97iQZKAW9qj3apUkfpRvz3dHPXr3BQtaM2eEeZWEc77P4YEJi1oxoFLIJGmSGk179KennvFSoHZdgbSdihetWYJzgEke1pLRYAL + EDDuealBou9XkuhHd9kqfDnLjSzs6UV9PVKCsHarUdypwF / a9NxY / 8jqlGxx0K6GL0qHm048GpaQup + OoaF8Y5E4OPQFuhN + V8gswwJ0PtOZDeLmUfk7O9sKIhdGfZuPoeBbHMvlBtsgOicg + OSK / yQlpEU6uyS25J3 + Dm + AueAgep61zwWzmO3mD4Pk / fcqg9w = = < / latexit > < latexit sha1 _ base64 = " lFkdFjSXXpuYZi / SxkBYzM7uwm0 = " > AAACHXicdVBNSyQxEE37seqsrqN73EtwEFyEJm0PqDdxL3t0wdGB6WGoztS0wSTdJGlhaOaPePGvePHgIh68yP6bzXwIKvqg4OW9KlL10kIK6xj7F8zNLyx + WVpeqX1dXfu2Xt / YPLN5aTi2eC5z007BohQaW044ie3CIKhU4nl6 + Wvsn1 + hsSLXp25YYFdBpsVAcHBe6tWbEWM0kaAziTSxIlNAEzN97iQZKAW9qj3apUkfpRvz3dHPXr3BQtaM2eEeZWEc77P4YEJi1oxoFLIJGmSGk179KennvFSoHZdgbSdihetWYJzgEke1pLRYAL + EDDuealBou9XkuhHd9kqfDnLjSzs6UV9PVKCsHarUdypwF / a9NxY / 8jqlGxx0K6GL0qHm048GpaQup + OoaF8Y5E4OPQFuhN + V8gswwJ0PtOZDeLmUfk7O9sKIhdGfZuPoeBbHMvlBtsgOicg + OSK / yQlpEU6uyS25J3 + Dm + AueAgep61zwWzmO3mD4Pk / fcqg9w = = < / latexit > < latexit sha1 _ base64 = " lFkdFjSXXpuYZi / SxkBYzM7uwm0 = " > AAACHXicdVBNSyQxEE37seqsrqN73EtwEFyEJm0PqDdxL3t0wdGB6WGoztS0wSTdJGlhaOaPePGvePHgIh68yP6bzXwIKvqg4OW9KlL10kIK6xj7F8zNLyx + WVpeqX1dXfu2Xt / YPLN5aTi2eC5z007BohQaW044ie3CIKhU4nl6 + Wvsn1 + hsSLXp25YYFdBpsVAcHBe6tWbEWM0kaAziTSxIlNAEzN97iQZKAW9qj3apUkfpRvz3dHPXr3BQtaM2eEeZWEc77P4YEJi1oxoFLIJGmSGk179KennvFSoHZdgbSdihetWYJzgEke1pLRYAL + EDDuealBou9XkuhHd9kqfDnLjSzs6UV9PVKCsHarUdypwF / a9NxY / 8jqlGxx0K6GL0qHm048GpaQup + OoaF8Y5E4OPQFuhN + V8gswwJ0PtOZDeLmUfk7O9sKIhdGfZuPoeBbHMvlBtsgOicg + OSK / yQlpEU6uyS25J3 + Dm + AueAgep61zwWzmO3mD4Pk / fcqg9w = = < / latexit > < latexit sha1 _ base64 = " lFkdFjSXXpuYZi / SxkBYzM7uwm0 = " > AAACHXicdVBNSyQxEE37seqsrqN73EtwEFyEJm0PqDdxL3t0wdGB6WGoztS0wSTdJGlhaOaPePGvePHgIh68yP6bzXwIKvqg4OW9KlL10kIK6xj7F8zNLyx + WVpeqX1dXfu2Xt / YPLN5aTi2eC5z007BohQaW044ie3CIKhU4nl6 + Wvsn1 + hsSLXp25YYFdBpsVAcHBe6tWbEWM0kaAziTSxIlNAEzN97iQZKAW9qj3apUkfpRvz3dHPXr3BQtaM2eEeZWEc77P4YEJi1oxoFLIJGmSGk179KennvFSoHZdgbSdihetWYJzgEke1pLRYAL + EDDuealBou9XkuhHd9kqfDnLjSzs6UV9PVKCsHarUdypwF / a9NxY / 8jqlGxx0K6GL0qHm048GpaQup + OoaF8Y5E4OPQFuhN + V8gswwJ0PtOZDeLmUfk7O9sKIhdGfZuPoeBbHMvlBtsgOicg + OSK / yQlpEU6uyS25J3 + Dm + AueAgep61zwWzmO3mD4Pk / fcqg9w = = < / latexit > ����� ����������� ������� � � �� �� �� * * * * * * * * * * * * * * * * * * * * * * * ����� ����������� ������� - �� - �� � �� �� �� * * * * * * * * * * * * * * * * * * * * P e r c en t i n c r ea s e i n O dd s Q = P ( X ) / P ( M ) ����� ����������� ������� � � � * * * * * * * * * * * * * * * * * * * * * * * * A B P ( X SA ) / P ( M ) < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > P ( X CIP ) / P ( M ) < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > P ( X SA & CIP ) / P ( M ) < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > : 100 y < latexit sha1 _ base64 = " Z5Dsh5qQnLJf7rX8piB3P5WE2UE = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWWEp / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0eME95mNChErFgFJ3UDXyf9CKOtJ / 3qzW / 7s9BVklQkBoUaParX72BZlnCFTJJrXXDUgwn1KBgkk8rvczylLIxHfKuo4om3IaT + cpTcuaUAYm1cU8hmau / OyY0sTZPIleZUBzZZW8m / ud1M4yvw4lQaYZcscVHcSYJajK7nwyE4Qxl7ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A / 5aQaQ = = < / latexit > < latexit sha1 _ base64 = " Z5Dsh5qQnLJf7rX8piB3P5WE2UE = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWWEp / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0eME95mNChErFgFJ3UDXyf9CKOtJ / 3qzW / 7s9BVklQkBoUaParX72BZlnCFTJJrXXDUgwn1KBgkk8rvczylLIxHfKuo4om3IaT + cpTcuaUAYm1cU8hmau / OyY0sTZPIleZUBzZZW8m / ud1M4yvw4lQaYZcscVHcSYJajK7nwyE4Qxl7ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A / 5aQaQ = = < / latexit > < latexit sha1 _ base64 = " Z5Dsh5qQnLJf7rX8piB3P5WE2UE = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWWEp / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0eME95mNChErFgFJ3UDXyf9CKOtJ / 3qzW / 7s9BVklQkBoUaParX72BZlnCFTJJrXXDUgwn1KBgkk8rvczylLIxHfKuo4om3IaT + cpTcuaUAYm1cU8hmau / OyY0sTZPIleZUBzZZW8m / ud1M4yvw4lQaYZcscVHcSYJajK7nwyE4Qxl7ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A / 5aQaQ = = < / latexit > < latexit sha1 _ base64 = " Z5Dsh5qQnLJf7rX8piB3P5WE2UE = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWWEp / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0eME95mNChErFgFJ3UDXyf9CKOtJ / 3qzW / 7s9BVklQkBoUaParX72BZlnCFTJJrXXDUgwn1KBgkk8rvczylLIxHfKuo4om3IaT + cpTcuaUAYm1cU8hmau / OyY0sTZPIleZUBzZZW8m / ud1M4yvw4lQaYZcscVHcSYJajK7nwyE4Qxl7ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A / 5aQaQ = = < / latexit > 100 2014 + < latexit sha1 _ base64 = " U0suBzZV9SiXjhpufECcsCL2Ado = " > AAAB + nicbVDLSgMxFM34rPU11aWbYBEEoSSloMuiG5cV7APaYcikaRuaZIYko5Sxn + LGhSJu / RJ3 / o1pOwttPXDhcM693HtPlAhuLELf3tr6xubWdmGnuLu3f3Dol45aJk41ZU0ai1h3ImKY4Io1LbeCdRLNiIwEa0fjm5nffmDa8Fjd20nCAkmGig84JdZJoV / CCPWGREoSZlWEaxfT0C + jCpoDrhKckzLI0Qj9r14 / pqlkylJBjOlilNggI9pyKti02EsNSwgdkyHrOqqIZCbI5qdP4ZlT + nAQa1fKwrn6eyIj0piJjFynJHZklr2Z + J / XTe3gKsi4SlLLFF0sGqQC2hjOcoB9rhm1YuIIoZq7WyEdEU2odWkVXQh4 + eVV0qpWMKrgu1q5fp3HUQAn4BScAwwuQR3cggZoAgoewTN4BW / ek / fivXsfi9Y1L585Bn / gff4ADCqSjg = = < / latexit > < latexit sha1 _ base64 = " U0suBzZV9SiXjhpufECcsCL2Ado = " > AAAB + nicbVDLSgMxFM34rPU11aWbYBEEoSSloMuiG5cV7APaYcikaRuaZIYko5Sxn + LGhSJu / RJ3 / o1pOwttPXDhcM693HtPlAhuLELf3tr6xubWdmGnuLu3f3Dol45aJk41ZU0ai1h3ImKY4Io1LbeCdRLNiIwEa0fjm5nffmDa8Fjd20nCAkmGig84JdZJoV / CCPWGREoSZlWEaxfT0C + jCpoDrhKckzLI0Qj9r14 / pqlkylJBjOlilNggI9pyKti02EsNSwgdkyHrOqqIZCbI5qdP4ZlT + nAQa1fKwrn6eyIj0piJjFynJHZklr2Z + J / XTe3gKsi4SlLLFF0sGqQC2hjOcoB9rhm1YuIIoZq7WyEdEU2odWkVXQh4 + eVV0qpWMKrgu1q5fp3HUQAn4BScAwwuQR3cggZoAgoewTN4BW / ek / fivXsfi9Y1L585Bn / gff4ADCqSjg = = < / latexit > < latexit sha1 _ base64 = " U0suBzZV9SiXjhpufECcsCL2Ado = " > AAAB + nicbVDLSgMxFM34rPU11aWbYBEEoSSloMuiG5cV7APaYcikaRuaZIYko5Sxn + LGhSJu / RJ3 / o1pOwttPXDhcM693HtPlAhuLELf3tr6xubWdmGnuLu3f3Dol45aJk41ZU0ai1h3ImKY4Io1LbeCdRLNiIwEa0fjm5nffmDa8Fjd20nCAkmGig84JdZJoV / CCPWGREoSZlWEaxfT0C + jCpoDrhKckzLI0Qj9r14 / pqlkylJBjOlilNggI9pyKti02EsNSwgdkyHrOqqIZCbI5qdP4ZlT + nAQa1fKwrn6eyIj0piJjFynJHZklr2Z + J / XTe3gKsi4SlLLFF0sGqQC2hjOcoB9rhm1YuIIoZq7WyEdEU2odWkVXQh4 + eVV0qpWMKrgu1q5fp3HUQAn4BScAwwuQR3cggZoAgoewTN4BW / ek / fivXsfi9Y1L585Bn / gff4ADCqSjg = = < / latexit > < latexit sha1 _ base64 = " U0suBzZV9SiXjhpufECcsCL2Ado = " > AAAB + nicbVDLSgMxFM34rPU11aWbYBEEoSSloMuiG5cV7APaYcikaRuaZIYko5Sxn + LGhSJu / RJ3 / o1pOwttPXDhcM693HtPlAhuLELf3tr6xubWdmGnuLu3f3Dol45aJk41ZU0ai1h3ImKY4Io1LbeCdRLNiIwEa0fjm5nffmDa8Fjd20nCAkmGig84JdZJoV / CCPWGREoSZlWEaxfT0C + jCpoDrhKckzLI0Qj9r14 / pqlkylJBjOlilNggI9pyKti02EsNSwgdkyHrOqqIZCbI5qdP4ZlT + nAQa1fKwrn6eyIj0piJjFynJHZklr2Z + J / XTe3gKsi4SlLLFF0sGqQC2hjOcoB9rhm1YuIIoZq7WyEdEU2odWkVXQh4 + eVV0qpWMKrgu1q5fp3HUQAn4BScAwwuQR3cggZoAgoewTN4BW / ek / fivXsfi9Y1L585Bn / gff4ADCqSjg = = < / latexit > : ����� ����������� ������� - �� - �� - �� - �� � * * * * * * * * * * * * * * * * * FIG . 5 : Propensity for X and citation impact attributable to cross - domain activity at the article level . ( A ) Annual growth rate in the likelihood P ( X ) of research having cross - domain attributes represented generically by X . ( B ) Decreased likelihood P ( X ) af - ter 2014 . ( C ) Citation premium estimated as the percent increase in c p attributable to cross - domain mixture X , measured relative to mono - domain ( M ) research articles representing the counterfactual baseline . Calculated using a researcher fixed - effect model specification which accounts for time independent individual - specific factors ; see Tables S4 - S5 for full model estimates . Note that “Broad” corresponds to X SA , X CIP , X SA & CIP ; “Neighboring” corresponds to X Neighboring , SA , X Neighboring , CIP , X Neighboring , SA & CIP ; and “Distant” corresponds to X Distant , SA , X Distant , CIP , X Distant , SA & CIP . ( D ) Difference - in - Difference ( δ X + ) estimate of the “Flagship project effect” on the citation impact of cross - domain research . Shown are point estimates with 95 % confidence interval . Asterisks above each estimate indicate the associated p − value level : ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 . ( see SI Appendix S7B ) . Our results indicate a robust statistically significant posi - tive relationship between cross - disciplinarity ( X CIP ) and ci - tation impact , consistent with the effect size in a different case study of the genomics revolution ( Petersen et al 2018 ) , which supports the generalizability of our findings to other convergence frontiers . To be specific , we calculate a 8 . 6 % ci - tation premium for the Broad configuration ( γ X CIP = 0 . 07 ; p < 0 . 001 ) , meaning that the average cross - disciplinary publi - cation is more highly cited than the average mono - disciplinary publication . We calculate a smaller 5 . 9 % citation premium associated with X SA ( γ X SA = 0 . 05 ; p < 0 . 001 ) . Yet the effect associated with articles featuring X CIP and X SA si - multaneously is considerably larger ( 16 % citation premium ; γ X SA & CIP = 0 . 13 ; p < 0 . 001 ) , suggesting an additive effect . Comparing results for the Neighboring configuration to the baseline estimates for Broad , the citation premium is rela - tively larger for X SA ( 11 % citation premium ; γ X Neighboring , SA = 0 . 088 ; p < 0 . 001 ) and roughly the same for X CIP and X SA & CIP . This result reinforces our findings regarding the convergence “short - cut” ( when X CIP is absent ) , indicating that this approach is more successful when integrating domain knowledge across shorter distances , consistent with innova - tion theory ( Fleming 2001 ) . The configuration most representative of convergence is Distant , which compared to Broad and Neighboring fea - tures smaller effect size for X SA & CIP ( 5 . 2 % citation pre - mium ; γ X Distant , SA & CIP = 0 . 04 ; p < 0 . 001 ) . The reduc - tion in γ X Distant , SA & CIP relative to values for Broad and Neigh - boring configurations likely reflects the challenges bridging communication , methodological and theoretical gaps across the Distant neuro - psycho - medical ↔ techno - informatic inter - face . More interestingly , this configuration is distinguished by a negative X SA estimate , indicating that the convergence shortcut yields less - impactful research than mono - domain re - search . Nevertheless , it is notable that for this convergent con - figuration , there is a clear hierarchy indicating the superiority of cross - disciplinary collaboration approaches to integrating research across distant domains . As in the Article - level model , we also tested for shifts in the citation premium attributable to the advent of Flagship HBS project funding using a similar DiD approach . Figure 5 ( D ) shows the citation premium γ X SA & CIP for articles pub - lished prior to 2014 , and the difference δ X + corresponding to the added effect for articles published after 2013 . For Broad and Distant we observe δ X + < 0 , indicating a reduced ci - 10 tation premium for post - 2013 research . By way of exam - ple for the Broad configuration : whereas cross - domain arti - cles published prior to 2014 show a 19 % citation premium ( γ X SA & CIP = 0 . 15 ; p < 0 . 001 ) , those published after 2013 have just a 19 % - 11 % = 8 % citation premium ( δ X SA & CIP + = − 0 . 09 ; p < 0 . 001 ) . The reduction of the citation premium is even larger for Neighboring ( δ Neighboring , X SA & CIP + = − 0 . 16 ; p < 0 . 001 ) . Yet for Distant , we observe a different trend – research combining both X SA and X CIP simultaneously has advantage over those with just X CIP or X SA , in that order ( δ Dist . , X SA & CIP + = 0 . 04 ; p = 0 . 016 ; 95 % CI = [ . 01 , . 08 ] ) . We briefly summarize coefficient estimates for the other control variables . Consistent with prior research on cross - disciplinarity ( Petersen et al 2018 ) , we observe a positive rela - tionship between team - size and citation impact ( β k = 0 . 415 ; p < 0 . 001 ) , which translates to a (cid:104) σ (cid:105) β k ≈ 0 . 5 % increase in citations associated with a 1 % increase in team size ( since k p enters in log in our specification ) . We also observe a posi - tive relationship for topical breadth ( β w = 0 . 03 ; p < 0 . 001 ) , which translates to a much smaller (cid:104) σ (cid:105) β w ≈ 0 . 04 % increase in citations associated with a 1 % increase in the number of major MeSH headings . And finally , regarding the career life - cycle , we observe a negative relationship with increasing ca - reer age ( β τ = − 0 . 011 ; p < 0 . 001 ) consistent with prior studies ( Petersen et al 2018 ) , translating to a 100 (cid:104) σ (cid:105) β τ ≈ - 1 . 3 % decrease in c p associated with every additional career year . See Tables S4 - S5 for the full set of model parameter estimates . Behind the Numbers Further qualitative inspection of prominent research articles in this category identifies four key convergence themes asso - ciated with past or developing breakthroughs : Magnetic Resonance Imaging ( MRI ) . MRI technology has been instrumental in identifying structure - function relations in brain networks , and has reshaped brain research since the 1990s . As a method that involves both sophisticated tech - nology and core brain expertise , MRI has been a focal point for X Distant , SA & CIP scholarship . For example , ref . ( Van Dijk et al 2012 ) addresses the problem of motion , a pernicious con - founding factor that can invalidate MR brain results . Hence , this research article exemplifies how a fundamental prob - lem threatening an entire line of research acts as an attrac - tor of distant cross - disciplinary collaborations with an all - encompassing theme , including authors from CIP 5 ( med - ical specialists ) and CIP 8 ( engineers and computer scien - tists ) , while thematically spans four topical domains : SA 2 ( Anatomy & Organisms ) , SA 3 ( Phenomena & Processes ) , SA 5 ( Techniques & Equipment ) , and SA 6 ( Technology & Information Science ) . Genomics . Following the completion of the Human Genome Project ( HGP ) in the early 2000s , genomics and biotechnology methods have established a foothold in brain research . This convergent frontier made headway in solving long - standing morbidity riddles and formulating novel ther - apies , e . g . providing a deeper understanding of the genetic basis of developmental delay ( Cooper et al 2011 ) and develop - ing treatment for glioblastoma using a recombinant poliovirus ( Desjardins et al 2018 ) . Both these articles include authors from CIP 4 and 5 ; thematically , these articles cast a wide net , with the former spanning SA 1 , 3 , 4 and 5 , while the latter covers SA 2 , SA 4 and SA 5 . Robotics . In the early 2010s neurally controlled robotic prosthesis reached fruition by way of collaboration between neuroscientists ( CIP 1 ) and biotechnologists ( CIP 4 ) . A prime example of this emerging bio - mechatronics frontier is re - search on robotic arms for tetraplegics ( Hochberg et al 2012 ) , which thematically covers all SA 1 - 6 . Artificial Intelligence ( AI ) and Big Data . Following devel - opments in machine learning capabilities ( ML ) , deep AI meth - ods were brought to bear on MR data , pushing brain imaging towards more quantitative , accurate , and automated diagnostic methods . Research on brain legion segmentation using Con - volutional Neural Networks ( CNN ) ( Kamnitsas et al 2017 ) is an apt example produced by collaboration between medical specialists ( CIP 5 ) and engineers ( CIP 8 ) , and spanning SA 2 - 4 and SA 6 . Simultaneously , massive brain datasets com - bined with powerful AI engines made their appearance along with methods to control noise and ensure their validity , as ex - emplified by ref . ( Alfaro - Almagro et al 2018 ) produced by neuroscientists ( CIP 1 ) , health scientists ( CIP 6 ) , and engi - neers ( CIP 8 ) , and also featuring a nearly exhaustive topical scope ( SA 2 - 6 ) . All together , case analysis indicates X Distant , SA & CIP prod - ucts are typically characterized by significant SA integration , typically including 3 - 4 non - technical SA plus 1 - 2 technical SA . This thematic coverage exceeds the disciplinary bounds implied by the CIP set of the authors , which typically includes one non - technical CIP plus one technical CIP . Discussion In a highly competitive and open science system with mul - tiple degrees of freedom , more than one operational mode is likely to emerge . To assess the different configurations that exist , we developed an { author discipline × research topic } classification that enables examination of several operational modes and their relative scientific impact . Competing Convergence Modes : Our key result regards the identification and assessment of a prevalent conver - gence shortcut characterized by research combining differ - ent SA ( X SA ) but not integrating cross - disciplinary expertise ( M CIP ) . Assuming the HBS ecosystem to be representative of other competitive science frontiers , our results suggest that the two operational modes of convergence evolve as substi - tutes rather than complements . Trends from the last five years indicate an increasing tendency for scholars to shortcut cross - disciplinary approaches , and instead integrate by way of ex - pansive learning . This appears to be in tension with the in - tended mission of flagship HBS programs . Instead , our analy - sis provides strong evidence that the rise of expedient conver - gence tactics may be an unintended consequence of the race among teams to secure funding . In order to provide timely assessment of convergence sci - ence , we addressed our fundamental RQ1 – how to mea - sure convergence ? – by developing a generalizable framework 11 that differentiates between diversity in team expertise and re - search topics . While it is true that a widespread paradigm shift towards increasing team size has transformed the scien - tific landscape ( Milojevic 2014 ; Wuchty et al 2007 ) , this work challenges the prevalent view that larger teams are innately more adept at prosecuting cross - domain research . Indeed , convergence does not only depend on team size but also on its composition . In reality , however , research teams targeting the class of hard problems calling for convergent approaches are faced with coordination costs and other constraints associ - ated with crossing disciplinary and organizational boundaries ( Cummings & Kiesler 2005 , 2008 ; Van Rijnsoever & Hessels 2011 ) . Consequently , teams are likely to economize in disci - plinary expertise , and instead integrate cross - domain knowl - edge in part ( or in whole ) by way of polymathic generalists comfortable with the expansive learning approach . As a re - sult , a team’s composite disciplinary pedigree tend to be a subset of the topical dimensions of the problem under investi - gation . As a consistency check , we also find this convergence shortcut to be more widespread in research involving topics that are epistemically close , as represented by the Neighbor - ing configuration we analyzed . Contrariwise , in the neuro - psycho - medical ↔ techno - informatic interface , belonging to the Distant configuration , convergent cross - disciplinary col - laboration runs strong . Perhaps not by serendipity , mixed analysis further indicates that this is exactly the configuration where transformative science has long been occurring . Arguably , a certain degree of expansive learning is needed for multidisciplinary teams to operate in harmony . For exam - ple , in the case of a psychologist collaborating with a medical specialist , it would be ideal if each one knew a little bit about the other’s field , so that they establish an effective knowledge bridge . After all , this is what transforms a multidisciplinary team to a cross - disciplinary team , such that convergence be - comes operative . However , this approach is not the dominant trend in HBS ( see the Article level Model ) , and is possibly a response to the broad and longstanding paradigm promot - ing interdisciplinarity ( Nissani 1995 ) with less emphasis on cross - domain collaboration . Again using our simple exam - ple , it may be that the medical specialist prefers not to part - ner at all with psychologists in the prosecution of bi - domain research , i . e . , opting for the streamlined substitutive strategy of total replacement over the strategy of partial redundancy , which comes with the risks associated with cross - disciplinary coordination . A limitation to our framework is that we do not specify what task ( e . g . analysis , conceptualization , writing ) a given domain expert performed , and hence do not account for di - vision of labor in the teams here analyzed . Indeed , recent work provides evidence that larger teams tend to have higher levels of task specialization ( Haeussler & Sauermann 2020 ) , which thereby provides a promising avenue for future inves - tigation , i . e . , to provide additional clarity on how bureaucra - tization ( Walsh & Lee 2015 ) offsets the recombinant uncer - tainty ( Fleming 2001 ) associated with cross - disciplinary ex - ploration . Another limitation regards the nuances of HBS programs that we do not account for , e . g . different grand ob - jectives , funding levels and disciplinary framing which varies across flagships . Yet as a truly multidisciplinary ecosystem , we believe HBS provides an ideal testbed for evaluating the prominence , interactions , and impact of the constitutional as - pects of convergence ( Eyre et al 2017 ; Grillner et al 2016 ; Jorgenson et al 2015 ; Quaglio et al 2017 ) . Our results also provide clarity regarding recent efforts to evaluate the role of cross - disciplinarity in the domain of ge - nomics ( Petersen et al 2018 ) , where we used a similar scholar - oriented framework that did not incorporate the SA dimen - sions . One could argue that the cross - disciplinary citation premium reported in the genomics revolution arises simply from the genomics domain being primed for success . Indeed , Fig . 3 ( A ) shows that HBS scholars in the domain of Biotech . & Genetics discipline maintained high levels of SA diversity extending back to the 1970s . We do not observe similar pat - terns for other HBS sub - disciplines . Yet , our measurement of a ∼ 16 % citation premium for research featuring both modes ( X SA & CIP ) are remarkably similar in magnitude to the ana - log measurement of a ∼ 20 % citation premium reported in ( Petersen et al 2018 ) . Econometric Analysis : In order to accurately measure shifts in the prevalence and impact of cross - domain integration , in addition to how they depend on the convergence mode , we employed an econometric regression specification that lever - ages author fixed - effects and accounts for research team size , in addition to a battery of other CIP and SA controls . Re - garding the growth rate of HBS convergence science , Fig . 5 ( A ) indicates that research integrating topics and disciplinary expertise is growing between ∼ 2 - 4 % annually , relatively to the mono - disciplinary baseline ; however , this upward trend reversed after the ramp - up of HBS flagships , as indicated in Fig . 5 ( B ) . Our results also indicate that the citation im - pact of publications from polymathic teams ( X Neighboring , SA and X Distant , SA ) is significantly lower than the impact of publications from more balanced cross - disciplinary teams ( X Neighboring , SA & CIP and X Distant , SA & CIP ) , see Fig . 5 ( C ) . On a positive note , a difference - in - difference strategy provides support that HBS research featuring the X Distant , SA & CIP con - figuration has increased in citation impact following the ramp - up of HBS flagships , see Fig . 5 ( D ) . There are various possible explanations to consider , most prominent of which is that the cognitive and resource demands required to address grand sci - entific challenges have outgrown the capacity of even mono - disciplinary teams , let alone solo genius ( Simonton 2013 ) . Reflecting upon these results together , it is somewhat trou - bling that the polymathic trend proliferates and competes with the gold standard , that is , configurations featuring a balance of cross - disciplinary teams and diverse topics ( X SA & CIP ) . Counterproductively , flagship HBS projects appear to have in - centivized expansive research strategies manifest in a relative shift towards X SA since the ramp - up of flagship projects in 2014 . This trend may depend upon the particular flagship’s objective framing . Take for instance the US BRAIN Ini - tiative , with the expressed aim to support multi - disciplinary mapping and investigation of dynamic brain networks . As such , its corresponding research goals promote the integra - tion of Neighboring topics , where scientists with polymathic 12 tendencies may feel more emboldened to short - circuit exper - tise . In addition , there are practical pressures associated with proposal calls . Another possible explanation regarding team formation , is that it may be easier and faster for researchers to find collaborators from their own discipline when faced with the pressure to meet proposal deadlines . Additionally , fund - ing levels are not unlimited and bringing additional reputable specialists into the team comes with great financial consid - eration . Hence , a natural avenue for future investigation is to test whether other convergence - oriented funding initiatives also unwittingly amplify such suboptimal teaming strategy . Theoretical insights – expansive learning : Indeed , the poly - mathic trends described here pre - existed the flagship HBS projects , and so must have deeper roots . One hypothesis is that this trend represents an emergent scholarly behavior owing to efficient 21st century means to pursue new topics by way of expansive learning ( Engestr¨om & Sannino 2010 ) , since the learning costs associated with certain tasks characterized by explicit knowledge have markedly decreased with the advent of the internet and other means of rapid high - fidelity commu - nication . Indeed , many of the activity signals brought to the fore by this study bear the hallmarks of expansive learning . Perhaps the most telling signal is the propensity towards topi - cally diverse publications – Fig . 4 ( D - F ) , which largely stems from horizontal movements in the research focus of individual scientists rather than vertical integration among experts from different disciplines – Fig . 4 ( A - C ) . The scientific system is increasingly interconnected , as evident from the densification of collaboration networks and emergent cross - disciplinary in - terfaces – Fig . 2 ( B ) . These interfaces satisfy the conditions that are conducive to boundary crossing , especially with re - spect to research topics , which can act as structures facilitat - ing “minimum energy” expansion ( Toiviainen 2007 ) . To this point , we also assessed wether the relationship between CIP diversity and SA integration depends on wether the configu - ration represents neighboring or distant domains . Analyzing the set of X SA & CIP articles , we find that expansive integra - tion is consistently most effective in Distant configurations , e . g . teams with N CIP , p = 3 span roughly 32 % more SA than their mono - disciplinary counterparts – Fig . S5 ( B ) . Policy Implications : Consistent also with other studies in expansive learning , actions taken by participants do not nec - essarily correspond to the intentions by the interventionists ( Rasmussen & Ludvigsen 2009 ) . The participants are brain scientists in this case , and the interventionists are the fund - ing agencies and the scientific establishment at large . While the latter aim to promote research powered by true multidisci - plinary teams , the former appear to prefer to shortcut around this ideal . Policy makers and other decision - makers within the sci - entific commons are faced with the persistent challenge of efficient resource allocation , especially in the case of grand scientific challenges that foster aggressive timelines ( Stephan 2012 ) . The implicit uncertainty and risk associated with such endeavors is bound to affect reactive scholar strategies , and this interplay between incentives and behavior is just one source of complexity among many that underly the scientific system ( Fealing & eds . 2011 ) . To begin to address this issue , policies addressing the challenges of historical fragmentation in Europe offer guid - ance . European Research Council ( ERC ) funding programs have been powerful vehicles for integrating national inno - vation systems by way of supporting cross - border collabo - ration , brain - circulation and knowledge diffusion – yet with unintended outcomes that increase the burden of the chal - lenge ( Doria Arrieta et al 2017 ) . To address this fragmenta - tion , many major ERC collaborative programs require multi - national partnerships as an explicit funding criteria . Moti - vated by the effectiveness of this straightforward integration strategy , convergence programs can can include analog cross - disciplinary criteria or review assessment to address the con - vergence shortcut . Such guidelines could help to align poly - mathic vs . cross - disciplinary pathways towards more effec - tive cross - domain integration . Much like the vision for brain science – towards a more complete understanding of the emer - gent structure - function relation in an adaptive complex system – a better understanding of cross - disciplinary team assembly , among other team science considerations ( B¨orner et al 2010 ) , will be essential in other challenging frontiers calling on con - vergence . Methods Normalization of citation impact . We normalized each Scopus citation count , c p , t , by leveraging the well - known log - normal properties of citation distributions ( Radicchi et al 2008 ) . To be specific , we grouped articles by publication year y p , and removed the time - dependent trend in the location and scale of the underlying log - normal citation distribution . The normalized citation value is given by z p = ( ln ( c p , t + 1 ) − µ t ) / σ t , ( 1 ) where µ t ≡ (cid:104) ln ( c t + 1 ) (cid:105) is the mean and σ t ≡ σ [ ln ( c t + 1 ) ] is the standard deviation of the citation distribution for a given t ; we add 1 to c p , t to avoid the divergence of ln 0 associated with uncited publications – a common method which does not alter the interpretation of results . Figure S8 ( G ) shows the probability distribution P ( z p ) calculated across all p within five - year non - overlapping time periods . The resulting normalized citation measure is well - fit by the Normal N ( 0 , 1 ) distribution , independent of t , and thus is a stationary measure across time . Publications with z p > 0 are thus above the average log citation impact µ t , and since they are measured in units of standard deviation σ t , standard intuition and statistics of z - scores apply . The annual σ t value is rather stable across time , with average and standard deviation (cid:104) σ (cid:105) ± SD = 1 . 24 ± 0 . 09 over the 49 - year period 1970 - 2018 . Subject Area classification using MeSH . Each MeSH descriptor has a tree number that identifies its location within one of 16 broad categorical branches . We merged 9 of the science - oriented MeSH branches ( A , B , C , E , F , G , J , L , N ) into 6 Subject Area ( SA ) clusters ( see Fig . 1 ) . Figure S1 shows the 50 most prominent MeSH descriptors for each SA cluster . Hence , we take the set of MeSH for each p denoted 13 by (cid:126)W p , and map these MeSH to the corresponding MeSH branch ( represented by the operator O SA ) , yielding a count vector with six elements : O SA ( (cid:126)W p ) = −→ SA p . Figure S8 ( D ) shows the distribution P ( N SA ) of the number of SA per publication : 72 % of articles have two or more SA ; the mean ( median ) SA p is 2 . 1 ( 2 ) , with standard deviation 0 . 97 , and maximum 6 . Disciplinary classification using CIP . We obtained host department information from each scholar’s Scopus Pro - file . Based upon this information provided in the profile description , and in some cases using additional web search and data contained in the Scholar Plot web app ( Majeti et al 2020 ) , we manually annotated each scholar’s home department name according to National Center for Education Statistics Classification of Instructional Program ( CIP ) codes . We then merged these CIP codes into 9 broad clusters and three super - clusters ( Neuro / Biology , Health , and Science & Engineering , as indicated in Fig . 1 ) ; for a list of constituent CIP codes for each cluster see Fig . S1 ( C ) . Analogous to the notation for assigning −→ SA p , we take the set of authors for each p denoted by (cid:126)A p , and map their individual departmental affiliations to the corresponding CIP cluster ( represented by the operator O CIP ) , yielding a count vector with nine elements : O CIP ( (cid:126)A p ) = −−→ CIP p . Measuring cross - domain diversity . We developed a measure of cross - domain diversity defined according to categorical co - occurrence within individual research articles . Each article p has a count vector (cid:126)v p : for discipline categories (cid:126)v p ≡ −−→ CIP p and for topic categories (cid:126)v p ≡ −→ SA p . We then measure article co - occurrence levels by way of the normalized outer - product D p ( (cid:126)v p ) ≡ U ( (cid:126)v p ⊗ (cid:126)v p ) | | U ( (cid:126)v p ⊗ (cid:126)v p ) | | , ( 2 ) where ⊗ is the outer tensor product , U ( G ) is an operator yielding the upper - diagonal elements of the matrix G ( i . e . representing the undirected co - occurrence network among the categorical elements ) . In essence , D p ( (cid:126)v p ) captures a weighted combination of all category pairs . The resulting matrix repre - sents dyadic combinations of categories as opposed to permu - tations ( i . e . , capturing the subtle difference between an undi - rected and directed network ) . While we did not explore it further , this matrix formulation may also give rise to higher - order measures of diversity associated with the eigenvalues of the outer - product matrix . The notation | | . . . | | indicates the matrix normalization implemented by summing all matrix el - ements . The objective of this normalization scheme is to con - trol for the variation in (cid:126)v p in a systematic way . As such , this co - occurrence is a article - level measure of diversity which controls for variations in the total number of categories and different count statistics for elements belonging to −−→ CIP p and −→ SA p . Consequently , totaling D p ( (cid:126)v p ) across articles from a given publication year yields the total number of articles pub - lished in a given year , (cid:80) p | y p ∈ t | | D p , t | | = N ( t ) . We also define a categorical diversity measure for each arti - cle given by f D , p = 1 − Tr ( D p ) ∈ [ 0 , 1 ) , which corresponds to the sum of the off - diagonal elements in D . The average article diversity by publication year is denoted by (cid:104) f D ( t ) (cid:105) . In simple terms , articles featuring a single category have f D , p = 0 whereas articles featuring multiple categories have f D , p > 0 . While the result of this approach is nearly identical to the Blau index ( corresponding to 1 - −→ SA p · −→ SA p / | SA p | 2 , also referred to as the Gini - Simpson index ) , f D , p is motivated by way of dyadic co - occurrence rather than the standard formulation motivated around repeated sampling . Data accessibility : All data analyzed here are openly avail - able from Scopus and PubMed APIs . Competing Interests The authors declare that they have no competing financial interests . Author Contributions AMP performed the research , partic - ipated in the writing of the manuscript , collected , analyzed , and visualized the data ; MA developed software to collect , analyze , and visualize the data ; and IP designed the research , performed the research , and participated in the writing of the manuscript . Funding : AMP and IP acknowledge funding from NSF grant 1738163 entitled ‘From Genomics to Brain Science’ . 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Journal of the Royal Society interface 12 : 20150272 . 1 Supplementary Information : Appendices S1 - S7 , Figures S1 - S12 , and Tables S1 - S5 Grand challenges and emergent modes of convergence science Alexander M . Petersen , 1 Mohammed E . Ahmed , 2 Ioannis Pavlidis 2 1 Department of Management of Complex Systems , Ernest and Julio Gallo Management Program , School of Engineering , University of California , Merced , California 95343 2 Computational Physiology Laboratory , University of Houston , Houston , Texas 77204 S1 . Data Collection In the effort to capture a comprehensive representation of the HBS ecosystem , this work contributes to Science of Science ( Fortunato et al 2018 ) efforts , in particular where the entry point is a scholar - oriented dataset spanning a broad research domain , such as sustainability science ( Bettencourt & Kaur 2011 ) , genomics ( Petersen et al 2018 ) , computer science ( Morgan et al 2018 ) , climate change ( Petersen et al 2019 ) , physics ( Battiston et al 2019 ; Sinatra et al 2015 ) and artificial intelligence ( Frank et al 2019 ) . Here we construct a comprehensive scholar - oriented HBS dataset to facilitate measuring factors related to cross - domain activity and corresponding strategic shifts associated with HBS flagship projects . These projects are considered within their continental framework , and since they started ramping up in late 2013 , they naturally divide the timeline under examination into “post” and “pre” periods . All together , we constructed our comprehensive scholar - centric representation of the HBS ecosystem by merging data from three publication indices – Web of Science ( WOS ) , Scopus , and PubMed , as illustrated in Fig . 1 . Author keystone via Web of Science ( WOS ) . In building a scholar - centric database , one needs a keystone for developing a list of authors who have published on a particular topic . For that , we chose WOS , using the topic field query “Human Brain” ( HB ) to search its “Core Collection” over the period 1955 - 2016 . This search resulted in 224 , 201 records with distinct WOS article identifiers . From these records , we extracted the full first and last names of all authors with ≥ 5 publications , along with their affiliations . Author name disambiguation . An important challenge in constructing a scholar - centric dataset is name disambiguation of authors . Here we overcame this challenge by using curated publication sets for each scholar obtained from Scopus via their profile - oriented API , which requires an author’s full name and affiliation in order to identify their Scopus profile . Brain Science data via Scopus and PubMed . Having amassed a comprehensive set of brain science researchers , the next step was to build a database that represents the totality of their work . Since brain science is multidisciplinary , these researchers come from different domains , publishing in diverse areas that feed brain science , thus creating an ecosystem prime for cross - domain analysis . We used the full name and affiliation - location of each WOS author to query the Scopus Author Profile repository . Among these profiles , 9 , 121 contained geographic and departmental affiliation information . In order to identify HB research articles , as opposed to other content such as comments and editorials and also non - biomedical research in the physical sciences , we searched for each article DOI in MEDLINE / PubMed . We only analyzed articles annotated with Medical Subject Heading ( MeSH ) keywords , which are indicators that this research - oriented content is biomedical in nature – resulting in a HB dataset with 655 , 386 articles over the period 1945 - 2018 ; see Fig . S8 ( A ) for N ( t ) . For each research article p published in year t , we also obtained the number of Scopus citations c p , t tallied through the API download ( census ) date in November 2019 . Topical keyword classification using MeSH . Medical Subject Headings ( MeSH ) are a quasi - hierarchical biomedical ontology developed by the National Library of Medicine and implemented across articles indexed by PubMed by expert annotators to classify articles according to their topical and methodological contributions . With on average 12 MeSH per article , this ontology facilitates topic mapping and topic co - occurrence analysis at multiple levels of specificity ( Petersen et al 2016 ) . We restrict our analysis to only the “Major Topic Headings” MeSH , which are indicated in PubMed by an asterisk , and account for roughly 1 in 3 MeSH descriptors . As such , we use these publication - level MeSH to determine the topical subject area of the research 2 reported in each article . In total , we encountered 14 , 212 distinct Major Topic MeSH . Geographic Regions . We obtained geographic location data from each scholar’s Scopus Profile , associating each individual with one of 77 countries ; the top five countries represented are the United States with 5030 scholars , Germany with 1192 , UK with 1074 , China with 1049 , and Japan with 894 . These coauthors associate each p with a set of countries , which we cluster into four localized regions indexed by R : North America , corresponding to R = 1 ( United States and Canada ) ; Europe , R = 2 ( 33 European Union and non - European Union countries including Norway , Switzerland , Israel , Iceland , and Serbia ) ; Australasia , R = 3 ( Peoples Republic of China , Japan , South Korea , Australia , Taiwan , New Zealand , Singapore , Malaysia , and Thailand ) ; and World , R = 4 ( remaining countries including Brazil , India , Turkey , and South Africa , among others ) . 88 % of the publications are covered by regional clusters R = 1 , 2 , 3 . S2 . Shifts in SA and CIP portfolios in the decade of multi - national Human Brain flagship projects Figure S2 ( A ) shows the relative frequency f < R , CIP ( f > R , CIP ) by region , calculated in the 5 - year period before ( < ) and after ( > ) the HB flagship project ramp - up year 2014 . Each f R , CIP value represents the average −−→ CIP p vector calculated across all articles belonging to a particular region , and normalized to unity to facilitate comparison , i . e . (cid:80) 9 CIP = 1 f R , CIP = 1 . In both the pre - 2014 period [ 2009 - 2013 ] and post - 2014 period [ 2014 - 2018 ] , the most prominent disciplines are Neurosciences [ CIP 1 ] and Medical Specialty [ 5 ] in the North American ( NA ) and European ( EU ) regions . The Australasian ( AA ) region shows higher levels of scholars from disciplines in Engineering & Informatics [ 8 ] and Chemistry & Physics & Math [ 9 ] than their NA and EU counterparts in the pre - 2014 period . However , after 2014 we observe a realignment of AA with the remarkably similar NA and EU profiles . This realignment is achieved by decreases in Engineering & Informatics [ 8 ] and Chemistry & Physics & Math [ 9 ] , and increases in Neurosciences [ 1 ] & Medical Specialty [ 5 ] . Fig . S2 ( B ) shows these relative shifts calculated as the difference ∆ f R , CIP = f > R , CIP − f < R , CIP . Overall , there appears to be a remarkable synchrony in the direction and magnitude of ∆ f R , CIP for the NA and EU regions , primarily associated with decreases in Neurosciences [ 1 ] and Pathology & Pharmacology [ 7 ] and increases in Psychology [ 3 ] and Medical Specialty [ 5 ] . NA is the only region showing increase in both Science & Engineering domains [ CIP 8 & 9 ] . Similarly , Fig . S3 ( A ) shows the analog frequencies f < R , SA ( f > R , SA ) for each SA by region . In the pre - 2014 period , the most prominent SA categories are Anatomy & Organisms [ SA 2 ] and Health [ 4 ] , with all regions showing similar distribution profiles . The most prominent distinction in AA is a reduced prominence of Psychiatry & Psychology [ 1 ] . By and large , the profiles remain consistent in the post - 2014 period , with AA and NA experiencing prominent increases in Health [ 4 ] , and AA showing a modest increase in Psychiatry & Psychology [ 1 ] , which nevertheless does not fully compensate for the initial deficit in this category with respect to both NA and EU . Figure S3 ( B ) indicates that all regions experienced a consistent decline in research involving the structure - oriented topics associated with Anatomy & Organisms [ 2 ] , as well as the function - oriented topics associated with Phenomena & Processes [ 3 ] . The most prominent distinction between regions is for NA and EU , which both feature increases in Technology & Information Science [ 6 ] that are relatively larger than observed for AA and World , likely reflecting the technological capacity related to the tech . hubs in these regions ; another distinction relates to the Psychiatry & Psychology [ SA 1 ] which increases in EU and AA more than for NA and World ; and also for Health [ 4 ] which increases in NA and AA more than for EU and World . S3 . Levels and changes in SA and CIP co - occurrence before and after 2014 We also seek to identify which category pairs are frequently combined in articles , and to assess their frequency shifts after 2014 . To this end , we introduce a tensor - product method to readily measure SA an CIP co - occurrence statistics for the purpose of identifying particular cross - domain orientations observed in cross - domain HB science . In order to juxtapose the relative frequencies of mono - category articles separately from multi - category articles , we define a modified outer - product matrix designed purely for visualization purposes : ˜D p ( (cid:126)v p ) ≡   U ( Υ ) | | U ( Υ ) | | , if (cid:126)v p contains 2 or more categories . U ( (cid:126)v p ⊗ (cid:126)v p ) | | U ( (cid:126)v p ⊗ (cid:126)v p ) | | = DiagonalMatrix ( Sign [ (cid:126)v p ] ) , otherwise . ( S1 ) where ⊗ is the outer tensor product and ◦ indicates the element - wise or Hadamard product . Note that this definition is slightly different than D p ( (cid:126)v p ) defined in Eq . ( 2 ) . The difference occurs in the first case , for which the matrix Υ ≡ (cid:126)v p ⊗ (cid:126)v p − (cid:126)v p ◦ (cid:49) ◦ (cid:126)v p for which the diagonal elements are eliminated via subtraction , i . e . Tr ( Υ ) = 0 . Simply stated , when (cid:126)v p ( representing −→ SA p or −−→ CIP p ) has 2 or more non - zero elements then we primarily count the off - diagonal elements of the outer - product matrix and are not concerned with the relative frequencies of the on - diagonal elements . Contrari - 3 wise , in the case that there is just one category present – e . g . (cid:126)v p = { 0 , 3 , 0 , 0 , 0 , 0 } – then we track only the diagonal element , which counts the occurrence of the single category . In this second case , the resulting matrix ˜D p ( (cid:126)v p ) = DiagonalMatrix ( Sign [ (cid:126)v p ] ) has only one non - zero element , which occurs for the diagonal value ˜ D 22 = 1 ; and all other matrix elements = 0 . Note that in either case the total sum of all elements are normalized to unity , | | ˜D p ( (cid:126)v p ) | | = 1 . This normalization implies that totaling ˜D p ( (cid:126)v p ) across articles from a given publication year yields the total number of articles , N ( t ) . We then calculated the aggregate co - occurrence matrix , denoted by C < = (cid:80) y p ∈ [ 2009 − 2013 ] ˜D p , using all articles published in the pre - period . It then follows from our normalization procedure that the total across all matrix elements is proportional to the total number of articles published in a given period , i . e . | | C < | | = (cid:80) 2013 t = 2009 N ( t ) = N [ 2009 − 2013 ] . Figures S2 ( C ) and S3 ( C ) show C < CIP and C < SA , respectively . To measure relative changes , we then calculated the percent difference in each matrix element ∆ C ij = 200 ( C > ij − θC < ij ) / ( C > ij + θC < ij ) , where θ = N [ 2014 − 2018 ] / N [ 2009 − 2013 ] corrects for bias associated with differences in the number of articles published each of the pre - and post - periods . To illustrate why this correction is important , we randomized the counts contained in (cid:126)v p = −→ SA p and plot the resulting C < rand . , SA and ∆ C rand . , SA matrices in Fig . S11 . As anticipated , this randomization scheme eliminates the variation among on - diagonal elements and off - diagonal elements in panel ( A ) ; Moreover , in panel ( B ) the off - diagonal elements all show percent change values that are in the range of ± 3 % , thereby indicative of the threshold for distinguishing statistically significant percent changes in the real data . Returning to the real data and the calculation of C < CIP , the most notable results of this visualization are the consistently strong couplings between CIP category [ 1 ] and all other categories [ 2 , 3 , 4 , 5 , 6 ] ; between categories [ 1 , 2 , 3 ] and [ 5 ] ; and also between categories [ 1 , 2 , 5 ] and [ 6 ] . Also of note is the higher - order clique among [ 1 , 5 , 6 ] where each CIP is strongly coupled to each other . Contrariwise , we observe relatively weak coupling between [ 7 , 8 , 9 ] and most all other CIP . Other prominent CIP that couple by region : NA shows relatively higher coupling between [ 1 , 4 ] and [ 4 , 5 ] and [ 5 , 8 ] compared to other regions ; and EU shows relatively higher coupling between [ 1 , 9 ] and [ 2 , 9 ] . Regarding the shifts from the pre - to post - 2014 captured by ∆ C CIP , NA and EU regions show consistent increase in CIP pairs [ 4 , 7 ] and [ 4 , 9 ] , [ 3 , 8 ] and [ 2 , 7 ] ; and consistent decrease between [ 1 , 8 ] and [ 2 , 9 ] and [ 6 , 9 ] and all combinations between 5 and [ 7 , 8 , 9 ] . Notably , AA exhibits higher % change levels , following from the fact that several elements in C < CIP that are nearly 0 . Figure S3 ( C ) shows C < SA calculated by region . For all regions , the matrix elements corresponding to SA pairs [ 2 , 3 ] and [ 2 , 4 ] and [ 3 , 4 ] are relatively strong , thus forming another clique among these SA representing traditional branches of biology . Other strongly coupings are SA [ 4 ] with both [ 1 , 5 ] ; and [ 5 ] with [ 2 , 4 , 6 ] . As with C < CIP , the technologically - oriented SA are the most weakly coupled categories . Regarding the shifts from the pre - to post - 2014 captured by ∆ C SA , the most consistent increases are between SA [ 1 ] and each of [ 2 , 4 , 6 ] ; and between 4 and both [ 5 , 6 ] . Contrariwise , the most consistent decreasing coupling is between [ 3 ] and both [ 2 , 6 ] , and between [ 5 , 6 ] . The matrices for NA and EU are rather similar , with the most prominent distinction between [ 2 , 6 ] – showing a - 12 % change for NA and a + 5 % change for EU ; and also between [ 3 , 6 ] – showing also a - 12 % decrease for NA but no significant change for EU . This latter disparity is an example of where EU may be taking the lead in in - silico - oriented approaches to HB science , consistent with the framing of the Human Brain Project . The most notable distinction for AA relative to NA and EU is in the larger magnitude of shifts , representing a period of international convergence for all couplings involving SA [ 1 ] , and in particular between [ 1 , 2 ] and between [ 1 , 6 ] ; contrariwise for AA , there is a prominent decoupling between SA [ 5 , 6 ] which is consistent with the relative shifts away from these two SA to compensate for the prominent redirection towards [ 1 ] and [ 4 ] , as also indicated by Fig . S3 ( B ) . S4 . Calculation of cross - domain co - occurrence : an illustrative example of the Tensor Product Calculating f D , p begins with the outer - product between a categorical count vector , e . g . (cid:126)SA p ⊗ (cid:126)SA p , where ⊗ is the outer tensor product . The resulting matrix represents dyadic combinations of categories as opposed to permutations ( i . e . , capturing the subtle difference between an undirected and directed network ) . The subtle difference between the Blau index and f D , p arises from U ( G ) , which is imposed to capture the difference between combinations rather than permutations ( or directed versus undirected network ) . Hence , this perspective offers a new pathway to the formulation of the common Blau diversity index by way of co - occurrence rather than repeated sampling . Take for example an article p with 4 metadata entities belonging to 3 categories , (cid:126)v p = { 1 , 2 , 0 , 0 , 1 , 0 } . Calculation of the co - occurrence matrix D p ( (cid:126)v p ) using the normalized outer - product defined in Eq . ( 2 ) yields 4 D p ( (cid:126)v p ) =   111 211 0 0 111 0 0 411 0 0 211 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 111 0 0 0 0 0 0 0   with | | U ( (cid:126)v p ⊗ (cid:126)v p ) | | = 11 . The categorical diversity is calculated as the total across off - diagonal elements , f D , p = 1 − Tr ( D p ) = 5 / 11 . For completeness , consider the representation of a mono - disciplinary article with the same number of metadata entities that all fall into the second category , (cid:126)v p = { 0 , 4 , 0 , 0 , 0 , 0 } . Then D p ( (cid:126)v p ) =      0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0      with corresponding f D , p = 1 − Tr ( D p ) = 1 − 1 = 0 . What does this measure measure ? Notably , f D , p accounts for both categorical differences ( Shannon - like ) and concentration disparity ( Gini - like ) ( Harrison & Klein 2007 ) . One the first hand , articles with more variation in SA categories will correspond to larger f D , p values , as the number of non - zero off - diagonal elements is proportional to (cid:0) M p 2 (cid:1) ∼ M 2 p , where M p is the number of distinct SA present , which contributes to larger f D , p ; and on the second hand , the off - diagonal elements will be relatively larger in combination if the count values contained in SA 2 are more evenly distributed , i . e . , are not highly concentrated in just one category . S5 . Bi - partite network between CIP and SA We quantify the empirical association between CIP and SA categories by aggregating the information contained in −−→ CIP p and −→ SA p . We first applied this method to the subset of mono - domain articles comprised of p with O CIP ( (cid:126)F p ) = O SA ( (cid:126)F p ) = M . By definition , each of these article features just a single CIP , making it possible to identify the SA that are most frequently associated with mono - domain researchers from that CIP category . Formally , this amounts to calculating the bi - partite network between CIP and SA , operationalized by averaging the −→ SA p for mono - domain articles from each CIP category , given by (cid:104)−→ SA (cid:105) CIP = (cid:80) p ∈ CIP ( −→ SA p / N SA , p ) ; importantly , this definition accounts for variability in N SA by normalizing the sum of the SA counts contained in −→ SA p by N SA , p so that each article contributes equally to the average . Less prominent CIP - SA links are pruned from our Sankey chart visualization in order to emphasize the most meaningful CIP - SA relations . To this end , we remove the weakest links contained in (cid:104)−→ SA (cid:105) CIP , excluding those with value < 0 . 5 M ax [ (cid:104)−→ SA (cid:105) CIP ] . Hence , the chart labelled M CIP (cid:29) M SA in Figure 4 ( G ) shows only the most prominent CIP - SA links . For juxtaposition , we also calculated the bi - partite network using the non - overlapping subset of articles with O SA & CIP ( (cid:126)F p ) = X SA & CIP . Since these articles by construction have N CIP , p ≥ 2 , we define the average association between CIP and SA as (cid:104)−→ SA (cid:105) CIP = (cid:80) p ∈ CIP ( −→ SA p / N SA , p ) / N CIP , p , where the vector −→ SA p / N SA , p contributes to the average for all CIP present in −−→ CIP p . The bi - partite network labeled X CIP (cid:29) X SA in Figure 4 ( G ) also shows just the most prominent CIP - SA links , applying the same threshold that excludes links that have weight less than half of the most prominent weighted CIP - SA link for a given CIP . Let A ( B ) represent the matrix representation of X CIP (cid:29) X SA ( M CIP (cid:29) M SA ) – after pruning less prominent CIP - SA links . We then compute the difference between the matrices , ∆ XM ≡ C = A − B , such that positive ( negative ) elements of C 5 indicate prominent links that are relatively over - represented in cross - domain ( mono - domain ) articles . The Sankey chart labeled ∆ XM in Figure 4 ( G ) shows just the positive elements , which tend to be larger in magnitude than the ( relatively few ) negative elements . In Fig . 3 ( B ) we presented the bi - partite network of prominent CIP - SA relations for the Broad configuration . Figure S12 complements those results showing the bipartite network for both the Neighboring and Distant configurations , which provide cross - validation for the choice of CIP and SA categories they represent . S6 . Historical trends in SA & CIP diversity : 2000 - 2018 We investigate historical trends in SA & CIP diversity using the matrix D p defined in Eq . ( 2 ) , which simultaneously measures mono - dimensional and multi - dimensional features of each article . More specifically , we define f D , p = 1 − Tr ( D p ) as the fraction of the article’s co - occurrence matrix capturing combinatorial diversity . Hence , in the limiting case that the article features just a single category , then f D , p = 0 ; and when all categories are present in equal quantities then f D , p = ( d − 1 ) / ( d + 1 ) , where d is the dimension of the categorical vector (cid:126)v p . As d increases then f D , p approaches 1 . Hence , for sufficiently large d then 0 ≤ f D , p (cid:46) 1 . Figures S8 ( D , E ) show the unconditional distributions , P ( N SA ) and P ( N CIP ) , with observed values spanning across the full range d = 6 and d = 9 , respectively . As a bounded quantity , the average article - level diversity f D ( t ) = N ( t ) − 1 (cid:80) p ∈ N ( t ) f D , p is an appropriate measure of a characteristic article , where N ( t ) is the number of articles being considered from year t . However , f D ( t ) is nevertheless sensitive to bias associated with a systematic increase over time in (cid:104) N CIP , t (cid:105) and (cid:104) N SA , t (cid:105) , the average number of categories present per article per year . We address this issue by applying a temporal deflator which adjusts the annual averages to account for systematic shifts in the underlying data generating process . To be specific , we define (cid:104) f D , SA ( t ) (cid:105) = f D , SA ( t ) × [ (cid:104) ˜ µ SA (cid:105) / ˜ µ SA ( t ) ] , where ˜ µ SA ( t ) = (cid:104) N SA , t (cid:105) / σ N SA , t is the inverse coefficient of variation ( also called the signal - to - noise ratio ) with respect to the number of SA per article , represented by N SA , p ; and (cid:104) ˜ µ SA (cid:105) is the average value calculated across the roughly 3 decades of analysis . Figure S11 ( C ) shows that ˜ µ SA ( t ) is increasing steadily with time . Hence , adjusting for this secular growth is essential so that observed increases are not simply artifacts of the underlying growth in N SA , p or N CIP , p . We apply the same method to adjust for systematic shifts in (cid:104) N CIP , t (cid:105) . To illustrate the utility of this deflator method , we randomized the SA for all articles ( by randomly shuffling the counts in each −−→ CIP p or −→ SA p ) . Figure S11 ( D ) demonstrates that there is no trend in the corresponding (cid:104) f D , SA ( t ) (cid:105) , indicating that this method removes the underlying bias . Returning to the empirical data , Fig . S6 shows the evolution of disciplinary diversity captured by coauthors’ departmental affiliations . Each panel shows (cid:104) f D , CIP ( t ) (cid:105) calculated for a specified combination of categories contained in each −−→ CIP p vector , as indicated by the schematic motif provided alongside each panel . For example , Fig . S6 ( A ) calculates (cid:104) f D , CIP ( t ) (cid:105) from all 9 CIP categories considered independently , whereas Fig . S6 ( B ) collects the counts associated with the combined categories [ 1 - 4 ] and [ 5 - 9 ] and calculates the diversity based upon the fraction of D p belonging to the single off - diagonal element D 12 , p , which records the disciplinary mixing between these two supergroups . Figure S6 ( A ) is calculated using the Broad configuration , and exhibits a slow increase in CIP diversity from 1990 to the mid 2000s in North America ( NA ) and European ( EU ) regions , which stalled thereafter , and even declined in the last decade for NA and AA , but not for EU . Figure S6 ( B ) shows relatively lower levels and trends in the diversity at the intersection of super - categories [ 1 - 4 ] ( representing traditional neuro / biology departments ) and [ 5 - 9 ] ( representing all other CIP jointly ) . By way of comparison , this trend indicates that the decline in panel ( A ) is not derived from the intersection explored in panel ( B ) . Instead , Figs . S6 ( C , D ) indicate that the decline in ( A ) is attributable to declines at the individual intersections between all permutations of CIP categories 1 - 7 , and to a lesser extent between the three disciplinary subdomains : neuro / biology [ 1 - 5 ] , health [ 5 - 7 ] and science and engineering [ 8 - 9 ] . Overall , we also observe higher levels of CIP diversity in NA , followed by EU , and then followed by AA . Likewise , Figure S7 shows the evolution of research topic diversity captured by SA counts in each −→ SA p . We observe much stronger trends for SA , suggesting that scholars tend to also cross disciplines as mono - disciplinary teams rather than via cross - disciplinary collaboration . Fig . S7 ( A ) shows (cid:104) f D , SA ( t ) (cid:105) calculated for the Broad configuration which includes all SA categories . The diversity trend is increasing since 1990 for all regions , but with reduced pace since the early 2010s . Similar to our findings for CIP , we observe AA lagging the other two regions ; however , in this case of SA we do observe more similar levels of diversity between EU and NA . Figure S7 ( B ) indicates that much of the increase in SA diversity is attributable to research combining Health [ SA 4 ] and the other categories – in other words , the domain of health science appears to be a persistent driving force behind convergence trends . Supporting evidence for this observation is also captured in the hierarchical clustering of SA represented by the minimum spanning tree ( MST ) representation of the aggregate SA co - occurrence matrix ˜ D SA , p – see Fig . S1 ( B ) . By way of comparison , the analog MST representation of ˜D CIP , p in Fig . S1 ( D ) features a less prominent hierarchy across the CIP categories . We analyzed several additional SA category subsets and super - category combinations to more deeply explore the anatomy of research topic diversity . Figure S7 ( C , D ) show that increasing diversity associated with Health [ 4 ] is largely captured via the in - 6 corporation of technology - and informatics - oriented capabilities [ 5 , 6 ] – as opposed to integrating more traditional biological SA representing research domains associated with questions relating to how Anatomy & Organisms ( structure ) [ 2 ] and Phenomena & Processes ( function ) [ 3 ] relate to complex human behavior addressed by Psychiatry & Psychology [ 1 ] – as illustrated in Fig . S7 ( E ) . Similarly , a significant component of the increasing diversity captured between SA [ 4 , 5 , 6 ] derives from the increase between research that is centered around Techniques & Equipment [ 5 ] and Technology & Information Science [ 6 ] ; although this con - tribution shown in Fig . S7 ( F ) only contributes to increases in diversity until 2010 , after which there is a prominent decline . Interestingly , this is a configuration which emphasizes the leading role of AA since 2010 in combining these two areas . To fur - ther emphasize the role of Health , we exclude this category [ 4 ] from the diversity measures shown in Fig . S7 ( G ) , indicating that combinations of SA across the traditional domains of biology and the technology - oriented domains have also saturated around 2010 , and their contribution to SA diversity primarily appears when considered the biology [ 1 - 3 ] and technology - oriented [ 5 , 6 ] as super - clusters illustrated in Fig . S7 ( H ) . S7 . Panel regression : model specification We constructed article - level and author - level panel data to facilitate measuring factors relating to SA and CIP diversity and shifts related to the ramp - up of three regional HB flagship projects circa 2013 , and several others thereafter . Figure S8 shows the distribution of various article - level features ; and Figure S9 shows the covariation matrix between the principle variables of interest . We use the following operator notation to specify how we classify articles as being cross - domain ( X ) or mono - domain ( M ) . Starting with the feature vector (cid:126)F p ≡ { −→ SA p , −−→ CIP p , −→ R p } , we obtain a binary diversity classification for each article denoted by X and M . We specify the objective criteria of the feature operator O by its subscript . For example , O SA ( (cid:126)F p ) = X SA if two or more SA categories are present , otherwise the value is M ; and by analogy , O CIP ( (cid:126)F p ) = X CIP if two or more categories are present , and otherwise O CIP ( (cid:126)F p ) = M . In the case of models oriented around articles featuring X SA and X CIP simultaneously ( represented by O SA & CIP ( (cid:126)F p ) = X SA & CIP ) , we exclude the set of articles classified as X SA but not X CIP and those classified as X CIP but not X SA . Hence , in what follows , the counterfactual baseline group for X SA & CIP articles are also the subset of mono - domain articles , which facilitates comparison of effect sizes across models oriented around X CIP , X SA and X SA & CIP . A . Article - level Model A Quantifying factors associated with propensity for CIP and SA diversity In the first model , we seek to better understand the factors associated with the prevalence of CIP and SA diversity as they evolve over time , and in particular their relation to the launching of the HB flagship programs . In order to model the article - level factors ( indicated by p ) associated with cross - domain research activity we define the binary indicator variable generically denoted as I X , p . By way of example , if we are considering SA diversity , then the indicator variable I X SA , p takes the value 1 if O SA ( (cid:126)F p ) = X SA and 0 if O SA ( (cid:126)F p ) = M . We then model the 2 - state odds Q ≡ P ( O SA ( (cid:126)F p ) = X SA ) P ( O SA ( (cid:126)F p ) = M ) = P ( X SA ) P ( M ) , which represents the propensity for cross - domain research , where P ( X SA ) + P ( M ) = 1 . Likewise , in the case of CIP diversity we model the odds as Q ≡ P ( O CIP ( (cid:126)F p ) = X CIP ) P ( O CIP ( (cid:126)F p ) = M ) ; and finally , we also consider the likelihood of research featuring both types of cross - domain activity , for SA & CIP , represented as Q ≡ P ( O SA & CIP ( (cid:126) F p ) = X SA & CIP ) P ( O SA & CIP ( (cid:126)F p ) = M ) . Because all Scopus scholars map onto a single CIP , and since this model is primarily concerned with identifying factors associated with orientation towards cross - domain research , we exclude solo - authored research papers ( i . e . those with k p = 1 ) from this analysis since the likelihood for those articles is predetermined ( i . e . P ( M ) = 1 ) ; for the same reason , we also exclude articles with a single Major MeSH category ( i . e . those with w p = 1 ) . For each article we also include several covariates of I X , p : the article publication year y p ; the mean journal citation impact , calculated as the average z p for articles from journal j , denoted by z j = (cid:104) z p | journal j (cid:105) ; the natural logarithm of the total number of coauthors , ln k p ; and the natural logarithm of the total number of Major MeSH terms , ln w p . As additional controls , we also include the total number of international regions associated with the authors’ affiliations N R , p ( with min value 1 and max value 4 ) , and also the total number of categories featured by the article , N SA , p and N CIP , p . We then model the odds Q by way of a Logit regression model , specified in the case of X SA as Logit (cid:16) P ( X SA ) (cid:17) = log (cid:16) P ( X SA ) P ( M ) (cid:17) = β 0 + β y y p + β z z p + β k ln k p + β w ln w p + β N R N R , p + β N CIP N CIP , p + (cid:15) ; ( S2 ) in the case of X CIP as 7 Logit (cid:16) P ( X CIP ) (cid:17) = log (cid:16) P ( X CIP ) P ( M ) (cid:17) = β 0 + β y y p + β z z p + β k ln k p + β w ln w p + β N R N R , p + β N SA N SA , p + (cid:15) ; ( S3 ) and in the case of X SA & CIP as Logit (cid:16) P ( X SA & CIP ) (cid:17) = log (cid:16) P ( X SA & CIP ) P ( M ) (cid:17) = β 0 + β y y p + β z z p + β k ln k p + β w ln w p + β N R N R , p + (cid:15) . ( S4 ) To account for errors that are geographically correlated over time , we estimated the model using robust standard errors clustered on a regional categorical variable . The full set of parameter results are tabulated in models ( 1 ) - ( 3 ) in Tables S1 - S3 , which report the exponentiated coefficients . To be specific , the exponentiated coefficient exp ( β ) is the odds ratio , representing the factor by which Q changes for each 1 - unit increase in the corresponding independent variable , i . e . Q + 1 / Q = exp ( β ) . In real terms , 100 β ≈ 100 ( exp ( β ) − 1 ) represents the percent change in Q corresponding to a 1 - unit increase in the corresponding independent variable ( where the approximation holds for small β values ) . As a result , exp ( β ) values that are less than ( greater than ) unity indicate variables that negatively ( positively ) correlate with the likelihood P ( X ) . Quantifying shifts in propensity for CIP and SA diversity associated with the announcement of global Flagship HB projects circa 2013 In order to identify shifts in the 5 - year period after the 2013 ramp - up of HB projects worldwide , we incorporated an interaction between the pre - / post periods – indicated by I 2014 + , p , which takes the value 1 for y p ≥ 2014 and 0 otherwise – and a categorical variable specifying the region , represented by I R , p . We use the Rest of World region category ( indicated by countries colored gray in Fig . 1 ) as the baseline for regional comparison since these regions did not feature flagship HB programs on the scale of those announced in Australia , Canada , China , Japan , Europe , South Korea , and the United States . By way of example , in the case of modeling the likelihood P ( X SA ) , the interaction term is added in the second row , Logit (cid:16) P ( X SA ) (cid:17) = log (cid:16) P ( X SA ) P ( M ) (cid:17) = β 0 + β y y p + β z z p + β k ln k p + β w ln w p + β N R N R , p + β N CIP N CIP , p + γ R I R , p + γ 2014 + I 2014 + , p + δ R + ( I R , p × I 2014 + , p ) + (cid:15) . ( S5 ) To differentiate different types of model variables , β is used to identify coefficients associated with continuous variables , γ is used for indicator variables , and δ is used to indicate interactions between indicator variables . In particular , the coefficient δ R + measures the Difference - in - Difference ( DiD ) estimate of the effect of HB projects on the propensity for research teams to pursue X SA approaches . Figures S6 and S7 demonstrate that historical trends in the prevalence of cross - domain diversity satisfy the parallel trend assumption for both CIP and SA , respectively . The full set of parameter results are tabulated in models ( 4 ) - ( 6 ) in Tables S1 - S3 , and the point estimates for principal test variables are visually summarized in Fig . S10 . B . Author - level Model B In the second model , we seek to measure the relation between the two different types of article diversity – CIP and SA – and the article’s scientific impact , proxied by c p . Our approach leverages the hierarchical features of the article - level data grouped into author - specific subgroups representing HB researcher publication portfolios . As a result , model coefficients represent estimates net of author - specific time - independent factors . In other words , this fixed - effect specification yields parameter estimates that are net of the author - specific baseline α a = (cid:104) z a (cid:105) , where a is an author index . This specification identifies a clear counterfactual framework for identifying the different outcomes associated with X and M that are relevant to researcher problem identification and team - assembly strategies . First , in order to measure relative differences in citation impact within and across publication cohorts , we apply a logarithmic transform that facilitates removing the time - dependent trend in the location and scale of the underlying log - normal citation distribution ( Radicchi et al 2008 ) . As such , the normalized citation impact defined in Eq . ( 1 ) is z p ≡ ln ( 1 + c p , t ) − µ t σ t , 8 where µ t ≡ ln ( 1 + c t ) is the mean and σ t ≡ σ [ ln ( 1 + c t ) ] is the standard deviation of the log - citation distribution for articles grouped by publication year . We uniformly add 1 to each c p , t count to avoid the divergence ln 0 associated with uncited publications , a common method that does not alter the interpretation of our results . Importantly , the standard deviation σ t ≈ (cid:104) σ (cid:105) = 1 . 24 is approximately constant over the focal period of our analysis . Consequently , we are able to transform the relation between z p and a given covariates into a percent change in c p , t associated with the same covariate . More specifically , building on previous work ( Petersen 2018 ; Petersen et al 2018 ) we define the citation premium as the percent change 100∆ c p / c p associated with shift in the independent variable v . For sake of simplicity , consider the basic linear model Y ( c ) = z p = β 0 + β v v with the decomposition of differentials , ∂Y ( c ) / ∂v = ( ∂Y / ∂c ) ( ∂c / ∂v ) = β v ; it follows from the property of logarithms that ∂Y / ∂c = 1 σ t ( 1 + c ) . Calculating the percent change 100∆ c p / c p follows from rearranging the differential relations above , yielding dc p σ t ( 1 + c p ) dv = β v . Hence , when the independent variable β v is a binary indicator variable , then the shift from value 0 to 1 corresponds to dv = 1 , and so the percent change 100∆ c p / c p ≈ 100 dc p / c p ≈ 100 × σ t × β v ≈ 100 × (cid:104) σ (cid:105) × β v . By extension , when the independent variable is a scalar quantity then the percent change in c p associated with a 1 - unit increase dv is also given by 100 × (cid:104) σ (cid:105) × β v . And in the case that the scalar quantity enters in logarithm ( e . g . ln k p ) , then a 1 % increase in v corresponds to a (cid:104) σ (cid:105) × β v percent increase in c p . Quantifying the effect of cross - domain diversity on scientific impact While previous work aimed to identify the role of X CIP in the ecosystem of biology and computing researchers that cham - pioned the genomics revolution ( Petersen et al 2018 ) , here we seek to simultaneously identify the relative impact of X CIP and X SA in the emerging ecosystem of HB science . In this way , we are able to compare research strategies that leverage combi - nations of diverse researcher expertise – i . e . cross - disciplinary collaboration – to those that do not , in the ultimate pursuit of interdisciplinary knowledge and research ( Nissani 1995 ) . To this end , we model the relation between z p and X CIP & X SA by applying ordinary least - squares ( OLS ) regression to estimate the coefficients of the panel regression model implemented with researcher profile fixed effects : z a , p = α a + β k ln k p + β w ln w p + β τ τ a , p + γ X SA I X SA + γ X CIP I X CIP + ( S6 ) γ ( y p , −→ SA p , −−→ CIP p , −→ R p ) + (cid:15) a , p , where the model parameters are estimated using Huber - White robust standard errors , which account for heteroscedasticity and serial correlation within the publication set of each scholar , indexed by a . The control variables in Eq . ( S7 ) include ln k p , measuring the natural logarithm of the total number of coauthors ; ln w p is the natural logarithm of the total number of Major MeSH terms ; the career age variable τ a , p , measuring the number of years since the researcher’s first publication , capturing variation attributable to the career life cycle ; and we also include factor variables controlling for publication year and other article - level features measured by −→ SA p , −−→ CIP p , −→ R p . We exclude solo - authored research papers ( i . e . those with k p = 1 ) along with articles with a single Major MeSH category ( i . e . those with w p = 1 ) . Table S4 shows the full parameter estimates for six similar models that differ primarily in the type of cross - domain diversity included as the principle test variable , represented generically by I X . In models ( 1 ) - ( 3 ) we vary the specification of the types of SA and CIP being tested . To be specific , in model ( 1 ) we include indicators I X SA and I X CIP , where I X CIP takes the value 1 if O CIP ( (cid:126)F p ) = X CIP and 0 if O CIP ( (cid:126)F p ) = M , and similarly for I X SA . These definitions of X correspond to the Broad configuration , calculated using all CIP and SA categories , as indicated in Figures 4 ( A , D ) . According to this definition , articles combining SA ( CIP ) from any two or more categories are classified as X SA ( X CIP ) . In model ( 2 ) we use X indicators defined according to the Neighboring configuration representing shorter - distance cross - domain activity – here capturing the neurobiological - vs - bioengineering interface . In our model specification , X is represented by the binary indicator variables I X Neighboring , SA and I X Neighboring , CIP . In the case of X Neighboring , SA , this interface corresponds to articles combining at least one MeSH mapping onto SA 1 ( Psychiatry & Psychology ) and at least one MeSH mapping onto SA [ 2 ] ( Anatomy & Organisms ) , [ 3 ] ( Phenomena & Processes ) or [ 4 ] ( Health ) . In the case of X Neighboring , CIP , this interface corresponds to articles combining at least one coauthor whose department maps onto CIP [ 1 ] ( Neurosciences ) or [ 3 ] ( Psychology ) and at least one coauthor whose department maps onto CIP [ 2 ] ( Biology ) , [ 4 ] ( Biotechnology & Genetics ) or [ 5 ] ( Medical Specialty ) or [ 6 ] ( Health Sciences ) or [ 7 ] ( Pathology & Pharmacology ) . Note that all Scopus scholars map onto a single CIP , and so solo - authored research articles are by definition mono - disciplinary . In model ( 3 ) we use X indicators defined according to the Distant configuration , representing longer - distance or “Convergent” cross - domain activity – here capturing the neuro - psycho - medical - vs - techno - computational interface . In our model specifica - tion , X is represented by the binary indicator variables I X Distant , SA and I X Distant , CIP . In the case of X Distant , SA , this interface corresponds to articles combining SAs [ 1 - 4 ] ( corresponding to Psychiatry & Psychology ( mind ) , Anatomy & Organisms ( struc - ture ) , Phenomena & Processes ( function ) and Health , respectively ) and at least one MeSH mapping onto SAs [ 5 , 6 ] ( Techniques & Equipment and Technology & Information Science , respectively ) . In the case of X Neighboring , CIP , this interface corresponds to articles combining at least one coauthor whose department maps onto CIPs [ 1 , 3 , 5 ] ( Neurosciences , Psychology and Medi - cal Specialty , respectively ) and at least one coauthor whose department maps onto CIPs [ 4 , 8 ] ( Biotechnology & Genetics and 9 Engineering & Informatics , respectively ) . Likewise , Models ( 4 - 6 ) instead focus on X SA & CIP ( represented by I X SA & CIP ) ; each model corresponds to the either the Broad , Neighboring or Distant configurations defining X SA and X CIP . As such , these models test the citation premium asso - ciated with articles featuring cross - domain diversity in combination . Because we exclude the confounding subsets of articles featuring X SA but not X CIP , or vice versa , then the counterfactual to X SA & CIP in are articles that are mono - dimensional in both categories . Thus , since the counterfactual groups are similar , the the citation premium estimated by γ X SA & CIP are compa - rable with the γ X SA and γ X CIP estimated in models ( 1 - 3 ) . The full set of parameter results are reported in Table S5 , and the transformed point estimates measuring the percent increase in c p associated with each X definition are visually summarized in Fig . 5 ( C ) . Quantifying shifts in the effect of cross - domain diversity associated with the announcement of global Flagship HB projects circa 2013 We test for shifts in the citation premium attributable to the advent of global Flagship HB projects by introducing an interaction between I 2014 + , p and I X SA & CIP , as indicated by the addition of two terms into the second row , z a , p = α a + β k ln k p + β w ln w p + β τ τ a , p + γ X SA & CIP I X SA & CIP + ( S7 ) + γ 2014 + I 2014 + + δ X SA & CIP + ( I X SA & CIP × I 2014 + ) + γ ( y p , −→ SA p , −−→ CIP p , −→ R p ) + (cid:15) a , p , As before , this Difference - in - Difference approach is based upon the counterfactual comparison of articles featuring X SA & CIP to those featuring M , integrating an additional comparison between articles published after 2014 to those published before 2014 . As in the previous citation model , the model parameter γ X SA & CIP represents the citation premium attributable to research endeavors simultaneously featuring cross - domain combinations of both SA and CIP . However , in this specification γ X SA & CIP applies to articles published before 2014 . The analog estimate of the relative citation premium for articles published after 2014 is γ X SA & CIP + δ X SA & CIP + . In other words , if all other covariates are held at the average values , then the citation premium difference is given by δ X SA & CIP + , with positive ( negative ) values indicating an increase ( decrease ) in the citation premium after 2014 . The principle test variables γ X SA & CIP , δ X SA & CIP + and their sum are visually summarized in Fig . 5 ( D ) . 10 [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ] A B SA Clusters 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Information Science Psychiatry & Psychology Anatomy & Organisms Phenomena & Processes Health Techniques & Equipment Technology & Information Science 1 : Neurosciences — Neurobiology and Neurosciences 2 : Biology — Biology / Biological Sciences , Biochemistry , Biophysics and Molecular Biology , Microbiological Sciences and Immunology , Biochemistry and Immunology , Cell / Cellular Biology and Anatomical Sciences , Molecular Medicine , Ecology , Evolution , Systematics , and Population Biology 3 : Psychology — Psychology 4 : (cid:1) Biotechnology & Genetics — Biotechnology , Genetics , Biomathematics , Bioinformatics , and Computational Biology 5 : Medical Specialty — Residency Programs C 6 : Health Sciences — Health Services / Allied Health / Health Sciences , Health and Physical Education / Fitness 7 : Pathology & Pharmacology — Pharmacology and Toxicology , Physiology , Pathology and Related Sciences 8 : Engineering & Informatics — Computer and Information Sciences , Electrical and Computer Engineering , Biomedical / Medical Engineering , Mechanical Engineering , Mechatronics , Robotics , and Automation Engineering 9 : Chemistry & Physics & Math — Chemistry , Chemical Engineering , Physics , Engineering Physics , Mathematics , Statistics D CIP Clusters — Classification of Instructional Programs ( CIP ) name ������������� ��������� ��������� ���������� ��������� ����� ��������� ����������������������������������������� ������ ���������� ������������������������� ������� �������� ��������� �������������������� ����������������������� ������������������ �������������������� ������������������ ��������������� ������������ - 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Pathology & Phrama . Chem . & Phys . & Math FIG . S1 : Subject Area and Disciplinary clusters . ( A ) Principal MeSH terms comprising 6 Subject Area ( SA ) clusters . ( B ) Minimum spanning tree representation of topical hierarchy based upon SA co - occurrence within articles ; node size proportional to total number of articles featuring a particular SA . ( C ) Department CIP codes comprising 9 disciplinary clusters . ( D ) Minimum spanning tree representation of disciplinary hierarchy based upon CIP co - occurrence within articles ; node size proportional to total number of articles featuring a particular CIP . 11 x1000 x1000 Pre - 2014 Department frequency ( per publication , by region ) , f R , CIP Frequency Difference ( Post - Pre ) , ∆ f R , CIP N . A m e r i ca E u r op e A u s t r a l as i a R es t o f W o r l d A < Department ( CIP ) (cid:2) Chemistry & Physics & Math (cid:2)(cid:1) Eng . & Informatics (cid:2) Pathology & Pharmacology (cid:2) Health Sciences (cid:2) Medical Specialty (cid:2) Biotech . & Genetics (cid:2) Psychology (cid:2) Biology (cid:2) Neurosciences B Post - 2014 Department frequency ( per publication , by region ) , f R , CIP > 1 : Neurosciences 2 : Biology 3 : Psychology 4 : (cid:1) Biotech . & Genetics 5 : Medical Specialty 6 : Health Sciences 7 : Pathology & Pharmacology 8 : Eng . & Informatics 9 : Chemistry & Physics & Math �� ��� ��� ��� ��� �� ��� ��� ��� ��� - ���� - ���� �� ���� ���� % A C I P C o - O cc u rr en c e m a t r i x , P r e pe r i od ( 2009 – 2013 ) [ N e t s ha r e ] % C hange f r o m P r e t o P o s t [ % D i ff e r en c e ] B - 120 - 120 N . America Europe Australasia x1000 x1000 x1000 0 x100 0 0 1 : Neurosciences 2 : Biology 3 : Psychology 4 : (cid:1) Biotechnology & Genetics 5 : Medical Specialty 6 : Health Sciences 7 : Pathology & Pharmacology 8 : Engineering & Informatics 9 : Chemistry & Physics & Math A R T I C L E T I T L E : R e d T e x t = N o t e s ; B l u e T e x t = t o b e upd a t e d ; R e v i s e d n o t a t i o n A u t ho r O n e a , c , 1 , A u t ho r T w o b , 1 , 2 , a nd A u t ho r Th r ee a a A f fi li a t i on O ne ; b A f fi li a t i on T w o ; c A f fi li a t i on T h r e e T h i s m anu sc r i p t w a sc o m p il edon M a y 4 , 2020 A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . C o n v e r g e n ce S c i e n ce | C r o ss - d i s c i p li n a r i t y | K e y w o r d | . . . V a r i a b l e d e fin i t i on s : 1 . S c o pu s a u t h o r i nd e x , a 2 . pub li c a t i o n i nd e x , p 3 . y e a r / t i m e , t ; pub li c a t i o n y e a r y p ; N ( t ) i s t h e t o t a l nu m b e r o f a r t i c l e s pub li s h e d i n y e a r t . 4 . S c o pu s c i t a t i o n s : c p , t i nd i c a t e s t h e nu m b e r o f c i t a t i o n s , c o un t e d t h r o u g h t h e d a t a ce n s u s y e a r 2019 , f o r a n a r t i c l e pub li s h e d i n y e a r t ; n o r m a li ze d c i t a t i o n s z p ( i n v o l v i n g µ t ) a nd ‡ t ) 5 . nu m b e r o f c oa u t h o r s , k p 6 . nu m b e r o f r e g i o n s a ss o c i a t e d w i t h a pub li c a t i o n , R p 7 . T h e a r t i c l e - l e v e l c a t e go r i c a l d i v e r s i t y m e a s u r e i s c a l c u - l a t e d u s i n g D p , t h e o u t e r - p r o d u c t m a t r i x u s e d t o c o un t c o - o cc u rr e n ce s a t t h e a r t i c l e l e v e l , d r a w i n g o n t h e g e n e r i c c a t e go r i c a l v ec t o r ˛ v p = ≠ æ S A p o r ≠≠ æ C I P p 8 . Sup e r s c r i p t > ( s ub s c r i p t < ) i n d i c a t e d a t a c o ll ec t e d o v e r 5 - y e a r p e r i o d 2014 - 2018 ( 5 - y e a r p e r i o d 2009 - 2013 ) 9 . f i nd i c a t e s a g e n e r i c f r a c t i o n / f r e q u e n c y v a r i a b l e ( i . e . w i t h r a n g e [ 0 , 1 ] ) ; s ub s c r i p t i nd i c a t e s t h e v a r i a b l e c o n t e x t , e . g . f D , S A ( t ) ( f D , C I P ( t ) ) i nd i c a t e s t h e c o - o cc u rr e n ce p e r a r t i - c l e m e a s u r e d u s i n g S A ( C I P ) . 10 . C < S A , i j a nd C < C I P , i j : m a t r i ce s c o un t i n g c o - o cc u rr e n ce s ; 11 . C S A , i j a nd C C I P , i j : m a t r i ce s r e p o r t i n g % d i e r e n ce s i n c o - o cc u rr e n ce s b e t w ee n t h e 5 - y e a r p r e / p o s t p e r i o d s 12 . t h e s e t o f “ M a j o r T o p i c ” M e S H k e y w o r d s f o r a pub li c a t i o n , ˛ W p ; t h e S A o p e r a t o r c o n v e r t s a cc o r d i n g l y : O S A ( ˛ W p ) = ≠ æ S A p ; S i m il a r l y t h e s e t o f S c o pu s a u t h o r s o n a r e , ˛ A p a nd t h e C I P o p e r a t o r c o n v e r t s a s : O C I P ( ˛ A p ) = ≠≠ æ C I P p ; 13 . Sub j ec t A r e a c l u s t e r , S A ; t h e c o rr e s p o nd i n g S A v ec t o r o f a pub li c a t i o n , ≠ æ S A p 14 . D i s c i p li n a r y c l u s t e r , C I P ; t h e c o rr e s p o nd i n g C I P v ec t o r o f a pub li c a t i o n , ≠≠ æ C I P p 15 . E a c h a r t i c l e p h a s a s e t o f S A & C I P f e a t u r e s : ˛ F p © { ≠ æ S A p , ≠≠ æ C I P p } . 16 . F r o m t h e i n f o r m a t i o n c o n t a i n e d i n ˛ F p , e a c h a r t i c l e h a s h a s o n e o f t w o ( s c a l a r ) v a l u e s : c r o ss - d o m a i n ( d i v e r s e m i x t u r e ) = X , a s o pp o s e d t o m o n o - d o m a i n = M 17 . T h e t w o v a l u e s d e p e nd o n t h e o p e r a t o r a c t i n g o n ˛ F p : e . g . O C I P ( ˛ F p ) = M o r X C I P a nd O L o n g , C I P ( ˛ F p ) = M o r X L o n g , C I P 18 . R e g r e ss i o n v a r i a b l e s : i nd i c a t o r v a r i a b l e s i d e n t i fi e d u s i n g I ; s ub s c r i p t s i nd i c a t e t h e t y p e v a l u e c o rr e s p o nd i n g t o t h e v a l u e 1 , o t h e r w i s e t h e v a l u e = 0 ; E g . I X L o n g , C I P t a k e s o n t h e v a l u e 1 f o r a r t i c l e s w i t h O L o n g , C I P ( ˛ F p ) = X a nd 0 o t h e r w i s e 19 . s c h o l a r ag e a t t i m e o f pub li c a t i o n , · i , p 20 . i nd i c a t o r v a r i a b l e I 2014 + = 1 i f y p Ø 2014 a nd 0 o t h e r w i s e 21 . R e g i o n i nd i c a t o r v a r i a b l e R r = 1 i f a r t i c l e a u t h o r s a ss o - c i a t e d w i t h r e g i o n r a nd 0 o t h e r w i s e : R N A , R E U , R AA , R W o r l d ( t h i s c a t e go r y i s b a s e li n e i n T a b l e s S 1 - S 3 ) 22 . N R , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f r e g i o n s r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 4 23 . N S A , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f S A r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 6 24 . N C I P , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m b e r o f C I P r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a - t i o n s ) , i e m i n = 1 a nd m a x = 9 25 . z j i s t h e a v e r ag e z v a l u e c a l c u l a t e d a c r o ss a ll a r t i c l e s pub li s h e d i n a s p ec i fi c j o u r n a l ( i nd e x e d b y j ) S i gn i fi ca n ce S t a t e m e n t A u t ho r s m u s t s ub m i t a 120 - w o r d m a x i m u m s t a t e m en t abou t t he s i gn i fic an c e o f t he i r r e s ea r c h pape r w r i tt en a t a l e v e l unde r - s t andab l e t o an unde r g r adua t e edu c a t ed sc i en t i s t ou t s i de t he i r fi e l d o f s pe c i a li t y . T he p r i m a r y goa l o f t he s i gn i fic an c e s t a t e - m en t i s t o e x p l a i n t he r e l e v an c e o f t he w o r k i n b r oad c on t e x t t o a b r oad r eade r s h i p . T he s i gn i fic an c e s t a t e m en t appea r s i n t he pape r i t s e l f and i s r equ i r ed f o r a ll r e s ea r c h pape r s . P l ea s ep r o v i dede t a il s o f au t ho r c on t r i bu t i on s he r e . P l ea s ede c l a r ean yc o m pe t i ng i n t e r e s t s he r e . 1 A . O . ( A u t ho r O ne ) c on t r i bu t edequa ll y t o t h i s w o r k w i t h A . T . ( A u t ho r T w o ) ( r e m o v e i f no t app li c ab l e ) . 2 T o w ho m c o rr e s ponden c e s hou l dbeadd r e ss ed . E - m a il : au t ho r . t w oe m a il . c o m www . pna s . o r g / c g i / do i / 10 . 1073 / pna s . XXXXXXXXXX P N AS | M ay4 , 2020 | v o l . XXX | no . XX | 1 – 16 A R T I C L E T I T L E : R e d T e x t = N o t e s ; B l u e T e x t = t o b e upd a t e d ; R e v i s e d n o t a t i o n A u t ho r O n e a , c , 1 , A u t ho r T w o b , 1 , 2 , a nd A u t ho r Th r ee a a A f fi li a t i on O ne ; b A f fi li a t i on T w o ; c A f fi li a t i on T h r ee T h i s m anu sc r i p t w a sc o m p il edon M a y 4 , 2020 A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . C o n v e r g e n ce S c i e n ce | C r o ss - d i s c i p li n a r i t y | K e y w o r d | . . . V a r i a b l e d e fin i t i on s : 1 . S c o pu s a u t h o r i nd e x , a 2 . pub li c a t i o n i nd e x , p 3 . y e a r / t i m e , t ; pub li c a t i o n y e a r y p ; N ( t ) i s t h e t o t a l nu m b e r o f a r t i c l e s pub li s h e d i n y e a r t . 4 . S c o pu s c i t a t i o n s : c p , t i nd i c a t e s t h e nu m b e r o f c i t a t i o n s , c o un t e d t h r o u g h t h e d a t a ce n s u s y e a r 2019 , f o r a n a r t i c l e pub li s h e d i n y e a r t ; n o r m a li ze d c i t a t i o n s z p ( i n v o l v i n g µ t ) a nd ‡ t ) 5 . nu m b e r o f c oa u t h o r s , k p 6 . nu m b e r o f r e g i o n s a ss o c i a t e d w i t h a pub li c a t i o n , R p 7 . T h e a r t i c l e - l e v e l c a t e go r i c a l d i v e r s i t y m e a s u r e i s c a l c u - l a t e d u s i n g D p , t h e o u t e r - p r o du c t m a t r i x u s e d t o c o un t c o - o cc u rr e n ce s a t t h e a r t i c l e l e v e l , d r a w i n g o n t h e g e n e r i c c a t e go r i c a l v ec t o r ˛ v p = ≠ æ S A p o r ≠≠ æ C I P p 8 . Sup e r s c r i p t > ( s ub s c r i p t < ) i nd i c a t e d a t a c o ll ec t e d o v e r 5 - y e a r p e r i o d 2014 - 2018 ( 5 - y e a r p e r i o d 2009 - 2013 ) 9 . f i nd i c a t e s a g e n e r i c f r a c t i o n / f r e q u e n c y v a r i a b l e ( i . e . w i t h r a n g e [ 0 , 1 ] ) ; s ub s c r i p t i nd i c a t e s t h e v a r i a b l e c o n t e x t , e . g . f D , S A ( t ) ( f D , C I P ( t ) ) i nd i c a t e s t h e c o - o cc u rr e n ce p e r a r t i - c l e m e a s u r e d u s i n g S A ( C I P ) . 10 . C < S A , i j a nd C < C I P , i j : m a t r i ce s c o un t i n g c o - o cc u rr e n ce s ; 11 . C S A , i j a nd C C I P , i j : m a t r i ce s r e p o r t i n g % d i e r e n ce s i n c o - o cc u rr e n ce s b e t w ee n t h e 5 - y e a r p r e / p o s t p e r i o d s 12 . t h e s e t o f “ M a j o r T o p i c ” M e S H k e y w o r d s f o r a pub li c a t i o n , ˛ W p ; t h e S A o p e r a t o r c o n v e r t s a cc o r d i n g l y : O S A ( ˛ W p ) = ≠ æ S A p ; S i m il a r l y t h e s e t o f S c o pu s a u t h o r s o n a r e , ˛ A p a nd t h e C I P o p e r a t o r c o n v e r t s a s : O C I P ( ˛ A p ) = ≠≠ æ C I P p ; 13 . Sub j ec t A r e a c l u s t e r , S A ; t h e c o rr e s p o nd i n g S A v ec t o r o f a pub li c a t i o n , ≠ æ S A p 14 . D i s c i p li n a r y c l u s t e r , C I P ; t h e c o rr e s p o nd i n g C I P v ec t o r o f a pub li c a t i o n , ≠≠ æ C I P p 15 . E a c h a r t i c l e p h a s a s e t o f S A & C I P f e a t u r e s : ˛ F p © { ≠ æ S A p , ≠≠ æ C I P p } . 16 . F r o m t h e i n f o r m a t i o n c o n t a i n e d i n ˛ F p , e a c h a r t i c l e h a s h a s o n e o f t w o ( s c a l a r ) v a l u e s : c r o ss - d o m a i n ( d i v e r s e m i x t u r e ) = X , a s o pp o s e d t o m o n o - d o m a i n = M 17 . T h e t w o v a l u e s d e p e nd o n t h e o p e r a t o r a c t i n g o n ˛ F p : e . g . O C I P ( ˛ F p ) = M o r X C I P a nd O L o n g , C I P ( ˛ F p ) = M o r X L o n g , C I P 18 . R e g r e ss i o n v a r i a b l e s : i nd i c a t o r v a r i a b l e s i d e n t i fi e d u s i n g I ; s ub s c r i p t s i nd i c a t e t h e t y p e v a l u e c o rr e s p o nd i n g t o t h e v a l u e 1 , o t h e r w i s e t h e v a l u e = 0 ; E g . I X L o n g , C I P t a k e s o n t h e v a l u e 1 f o r a r t i c l e s w i t h O L o n g , C I P ( ˛ F p ) = X a nd 0 o t h e r w i s e 19 . s c h o l a r ag e a t t i m e o f pub li c a t i o n , · i , p 20 . i nd i c a t o r v a r i a b l e I 2014 + = 1 i f y p Ø 2014 a nd 0 o t h e r w i s e 21 . R e g i o n i nd i c a t o r v a r i a b l e R r = 1 i f a r t i c l e a u t h o r s a ss o - c i a t e d w i t h r e g i o n r a nd 0 o t h e r w i s e : R N A , R E U , R AA , R W o r l d ( t h i s c a t e go r y i s b a s e li n e i n T a b l e s S 1 - S 3 ) 22 . N R , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f r e g i o n s r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 4 23 . N S A , p a r t i c l e - l e v e l c o u n t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f S A r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 6 24 . N C I P , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m b e r o f C I P r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a - t i o n s ) , i e m i n = 1 a nd m a x = 9 25 . z j i s t h e a v e r ag e z v a l u e c a l c u l a t e d a c r o ss a ll a r t i c l e s pub li s h e d i n a s p ec i fi c j o u r n a l ( i nd e x e d b y j ) S i gn i fi ca n ce S t a t e m e n t A u t ho r s m u s t s ub m i t a 120 - w o r d m a x i m u m s t a t e m en t abou t t he s i gn i fic an c e o f t he i r r e s ea r c h pape r w r i tt en a t a l e v e l unde r - s t andab l e t o an unde r g r adua t e edu c a t ed sc i en t i s t ou t s i de t he i r fi e l d o f s pe c i a li t y . T he p r i m a r y goa l o f t he s i gn i fic an c e s t a t e - m en t i s t o e x p l a i n t he r e l e v an c e o f t he w o r k i n b r oad c on t e x t t o a b r oad r eade r s h i p . T he s i gn i fic an c e s t a t e m en t appea r s i n t he pape r i t s e l f and i s r equ i r ed f o r a ll r e s ea r c h pape r s . P l ea s ep r o v i dede t a il s o f au t ho r c on t r i bu t i o n s he r e . P l ea s ede c l a r ean yc o m pe t i ng i n t e r e s t s he r e . 1 A . O . ( A u t ho r O ne ) c on t r i bu t edequa ll y t o t h i s w o r k w i t h A . T . ( A u t ho r T w o ) ( r e m o v e i f no t app li c ab l e ) . 2 T o w ho m c o rr e s ponden c e s hou l dbeadd r e ss ed . E - m a il : au t ho r . t w oe m a il . c o m w ww . pna s . o r g / c g i / do i / 10 . 1073 / pna s . XXXXXXXXXX P N AS | M a y4 , 2020 | v o l . XXX | no . XX | 1 – 16 C D N . America Europe Australasia A B FIG . S2 : Temporal and regional distributions of CIP - coded author departments in human brain research . ( A ) Relative frequency of department CIP clusters in the 5 - year period before 2014 ( f < R , CIP ) and after 2014 ( f > R , CIP ) ; f values are normalized to unity within region . ( B ) Shift in CIP cluster frequencies given by the difference ∆ f R , CIP = f > R , CIP − f < R , CIP . ( C ) Disciplinary { CIP , CIP } co - occurrence in human brain science – by region . Each co - occurrence matrix C < CIP measures the frequency of a given { CIP , CIP } pair over the 5 - year pre - period 2009 - 2013 based upon publications associated with one of three broad geographic regions ; see Eqn . ( S1 ) for its definition . By construction , matrix element values C < CIP , ij are proportional to the net share of publications featuring the indicated pair . Diagonal elements measure the frequency of publications featuring only a single CIP category . Note the use of two legends , one for the mono - disciplinary diagonal elements ( gray - scale legend reported in units of 1000 publications ) and one for off - diagonal elements ( color - scale legend reported in units of 100 publications ) ; as indicated by the legend scales , mono - CIP publications occur with significantly higher frequency than multi - CIP publications . ( D ) Relative change ( post - pre period ) in the co - occurrence matrix : ∆ C CIP , ij measures the percent difference in the frequency of publications characterized by each { CIP , CIP } pair . 12 % C SA C o - O cc u rr en c e m a t r i x , P r e pe r i od ( 2009 – 201 3 ) [ N e t s ha r e ] D - 120 - 120 N . America Europe Australasia x1000 x1000 x1000 4 . 5 4 . 5 4 . 9 A R T I C L E T I T L E : R e d T e x t = N o t e s ; B l u e T e x t = t o b e upd a t e d ; R e v i s e d n o t a t i o n A u t ho r O n e a , c , 1 , A u t ho r T w o b , 1 , 2 , a nd A u t ho r Th r ee a a A f fi li a t i on O ne ; b A f fi li a t i on T w o ; c A f fi li a t i on T h r ee T h i s m anu sc r i p t w a s c o m p il ed on M a y 4 , 2020 A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . C o n v e r g e n ce S c i e n ce | C r o ss - d i s c i p li n a r i t y | K e y w o r d | . . . V a r i a b l e d e fin i t i on s : 1 . S c o pu s a u t h o r i nd e x , a 2 . pub li c a t i o n i nd e x , p 3 . y e a r / t i m e , t ; pub li c a t i o n y e a r y p ; N ( t ) i s t h e t o t a l nu m b e r o f a r t i c l e s pub li s h e d i n y e a r t . 4 . S c o pu s c i t a t i o n s : c p , t i nd i c a t e s t h e nu m b e r o f c i t a t i o n s , c o un t e d t h r o u g h t h e d a t a ce n s u s y e a r 2019 , f o r a n a r t i c l e pub li s h e d i n y e a r t ; n o r m a li ze d c i t a t i o n s z p ( i n v o l v i n g µ t ) a nd ‡ t ) 5 . nu m b e r o f c oa u t h o r s , k p 6 . nu m b e r o f r e g i o n s a ss o c i a t e d w i t h a pub li c a t i o n , R p 7 . T h e a r t i c l e - l e v e l c a t e go r i c a l d i v e r s i t y m e a s u r e i s c a l c u - l a t e d u s i n g D p , t h e o u t e r - p r o du c t m a t r i x u s e d t o c o un t c o - o cc u rr e n ce s a t t h e a r t i c l e l e v e l , d r a w i n g o n t h e g e n e r i c c a t e go r i c a l v ec t o r ˛ v p = ≠ æ S A p o r ≠≠ æ C I P p 8 . Sup e r s c r i p t > ( s ub s c r i p t < ) i nd i c a t e d a t a c o ll ec t e d o v e r 5 - y e a r p e r i o d 2014 - 2018 ( 5 - y e a r p e r i o d 2009 - 2013 ) 9 . f i nd i c a t e s a g e n e r i c f r a c t i o n / f r e q u e n c y v a r i a b l e ( i . e . w i t h r a n g e [ 0 , 1 ] ) ; s ub s c r i p t i nd i c a t e s t h e v a r i a b l e c o n t e x t , e . g . f D , S A ( t ) ( f D , C I P ( t ) ) i nd i c a t e s t h e c o - o cc u rr e n ce p e r a r t i - c l e m e a s u r e d u s i n g S A ( C I P ) . 1 0 . C < S A , i j a nd C < C I P , i j : m a t r i ce s c o un t i n g c o - o cc u rr e n ce s ; 1 1 . C < S A , i j a nd C < C I P , i j : m a t r i ce s r e p o r t i n g % d i e r e n ce s i n c o - o cc u rr e n ce s b e t w ee n t h e 5 - y e a r p r e / p o s t p e r i o d s 1 2 . t h e s e t o f “ M a j o r T o p i c ” M e S H k e y w o r d s f o r a pub li c a t i o n , ˛ W p ; t h e S A o p e r a t o r c o n v e r t s a cc o r d i n g l y : O S A ( ˛ W p ) = ≠ æ S A p ; S i m il a r l y t h e s e t o f S c o pu s a u t h o r s o n a r e , ˛ A p a nd t h e C I P o p e r a t o r c o n v e r t s a s : O C I P ( ˛ A p ) = ≠≠ æ C I P p ; 13 . Sub j ec t A r e a c l u s t e r , S A ; t h e c o rr e s p o nd i n g S A v ec t o r o f a pub li c a t i o n , ≠ æ S A p 14 . D i s c i p li n a r y c l u s t e r , C I P ; t h e c o rr e s p o nd i n g C I P v ec t o r o f a pub li c a t i o n , ≠≠ æ C I P p 15 . E a c h a r t i c l e p h a s a s e t o f S A & C I P f e a t u r e s : ˛ F p © { ≠ æ S A p , ≠≠ æ C I P p } . 16 . F r o m t h e i n f o r m a t i o n c o n t a i n e d i n ˛ F p , e a c h a r t i c l e h a s h a s o n e o f t w o ( s c a l a r ) v a l u e s : c r o ss - d o m a i n ( d i v e r s e m i x t u r e ) = X , a s o pp o s e d t o m o n o - d o m a i n = M 17 . T h e t w o v a l u e s d e p e nd o n t h e o p e r a t o r a c t i n g o n ˛ F p : e . g . O C I P ( ˛ F p ) = M o r X C I P a nd O L o n g , C I P ( ˛ F p ) = M o r X L o n g , C I P 18 . R e g r e ss i o n v a r i a b l e s : i nd i c a t o r v a r i a b l e s i d e n t i fi e d u s i n g I ; s ub s c r i p t s i nd i c a t e t h e t y p e v a l u e c o rr e s p o nd i n g t o t h e v a l u e 1 , o t h e r w i s e t h e v a l u e = 0 ; E g . I X L o n g , C I P t a k e s o n t h e v a l u e 1 f o r a r t i c l e s w i t h O L o n g , C I P ( ˛ F p ) = X a nd 0 o t h e r w i s e 19 . s c h o l a r ag e a t t i m e o f pub li c a t i o n , · i , p 20 . i nd i c a t o r v a r i a b l e I 2014 + = 1 i f y p Ø 2014 a nd 0 o t h e r w i s e 21 . R e g i o n i nd i c a t o r v a r i a b l e R r = 1 i f a r t i c l e a u t h o r s a ss o - c i a t e d w i t h r e g i o n r a nd 0 o t h e r w i s e : R N A , R E U , R AA , R W o r l d ( t h i s c a t e go r y i s b a s e li n e i n T a b l e s S 1 - S 3 ) 22 . N R , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f r e g i o n s r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 4 23 . N S A , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f S A r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 6 24 . N C I P , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m b e r o f C I P r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a - t i o n s ) , i e m i n = 1 a nd m a x = 9 25 . z j i s t h e a v e r ag e z v a l u e c a l c u l a t e d a c r o ss a ll a r t i c l e s pub li s h e d i n a s p ec i fi c j o u r n a l ( i nd e x e d b y j ) S i gn i fi ca n ce S t a t e m e n t A u t ho r s m u s t s ub m i t a 120 - w o r d m a x i m u m s t a t e m en t abou t t he s i gn i fic an c e o f t he i r r e s ea r c h pape r w r i tt en a t a l e v e l unde r - s t andab l e t o an unde r g r adua t e edu c a t ed sc i en t i s t ou t s i de t he i r fi e l d o f s pe c i a li t y . T he p r i m a r y goa l o f t he s i gn i fic an c e s t a t e - m en t i s t o e x p l a i n t he r e l e v an c e o f t he w o r k i n b r oad c on t e x t t o a b r oad r eade r s h i p . T he s i gn i fic an c e s t a t e m en t appea r s i n t he pape r i t s e l f and i s r equ i r ed f o r a ll r e s ea r c h pape r s . P l ea s ep r o v i dede t a il s o f au t ho r c on t r i bu t i on s he r e . P l ea s ede c l a r ean yc o m pe t i ng i n t e r e s t s he r e . 1 A . O . ( A u t ho r O ne ) c on t r i bu t edequa ll y t o t h i s w o r k w i t h A . T . ( A u t ho r T w o ) ( r e m o v e i f no t app li c ab l e ) . 2 T o w ho m c o rr e s ponden c e s hou l dbeadd r e ss ed . E - m a il : au t ho r . t w oe m a il . c o m w ww . pna s . o r g / c g i / do i / 10 . 1073 / pna s . XXXXXXXXXX P N AS | M ay 4 , 2020 | v o l . XXX | no . XX | 1 – 16 % C hange f r o m P r e t o P o s t [ % D i ff e r en c e ] A R T I C L E T I T L E : R e d T e x t = N o t e s ; B l u e T e x t = t o b e upd a t e d ; R e v i s e d n o t a t i o n A u t ho r O n e a , c , 1 , A u t ho r T w o b , 1 , 2 , a nd A u t ho r Th r ee a a A f fi li a t i on O ne ; b A f fi li a t i on T w o ; c A f fi li a t i on T h r ee T h i s m anu sc r i p t w a s c o m p il ed on M a y 4 , 2020 A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a p h . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . C o n v e r g e n ce S c i e n ce | C r o ss - d i s c i p li n a r i t y | K e y w o r d | . . . V a r i a b l e d e fin i t i on s : 1 . S c o pu s a u t h o r i nd e x , a 2 . pub li c a t i o n i nd e x , p 3 . y e a r / t i m e , t ; pub li c a t i o n y e a r y p ; N ( t ) i s t h e t o t a l nu m b e r o f a r t i c l e s pub li s h e d i n y e a r t . 4 . S c o pu s c i t a t i o n s : c p , t i nd i c a t e s t h e nu m b e r o f c i t a t i o n s , c o un t e d t h r o u g h t h e d a t a ce n s u s y e a r 2019 , f o r a n a r t i c l e pub li s h e d i n y e a r t ; n o r m a li ze d c i t a t i o n s z p ( i n v o l v i n g µ t ) a nd ‡ t ) 5 . nu m b e r o f c oa u t h o r s , k p 6 . nu m b e r o f r e g i o n s a ss o c i a t e d w i t h a pub li c a t i o n , R p 7 . T h e a r t i c l e - l e v e l c a t e go r i c a l d i v e r s i t y m e a s u r e i s c a l c u - l a t e d u s i n g D p , t h e o u t e r - p r o du c t m a t r i x u s e d t o c o un t c o - o cc u rr e n ce s a t t h e a r t i c l e l e v e l , d r a w i n g o n t h e g e n e r i c c a t e go r i c a l v ec t o r ˛ v p = ≠ æ S A p o r ≠≠ æ C I P p 8 . Sup e r s c r i p t > ( s ub s c r i p t < ) i nd i c a t e d a t a c o ll ec t e d o v e r 5 - y e a r p e r i o d 2014 - 2018 ( 5 - y e a r p e r i o d 2009 - 2013 ) 9 . f i nd i c a t e s a g e n e r i c f r a c t i o n / f r e q u e n c y v a r i a b l e ( i . e . w i t h r a n g e [ 0 , 1 ] ) ; s ub s c r i p t i nd i c a t e s t h e v a r i a b l e c o n t e x t , e . g . f D , S A ( t ) ( f D , C I P ( t ) ) i nd i c a t e s t h e c o - o cc u rr e n ce p e r a r t i - c l e m e a s u r e d u s i n g S A ( C I P ) . 10 . C < S A , i j a nd C < C I P , i j : m a t r i ce s c o un t i n g c o - o cc u r r e n ce s ; 11 . C S A , i j a nd C C I P , i j : m a t r i ce s r e p o r t i n g % d i e r e n ce s i n c o - o cc u rr e n ce s b e t w ee n t h e 5 - y e a r p r e / p o s t p e r i o d s 12 . t h e s e t o f “ M a j o r T o p i c ” M e S H k e y w o r d s f o r a pub li c a t i o n , ˛ W p ; t h e S A o p e r a t o r c o n v e r t s a cc o r d i n g l y : O S A ( ˛ W p ) = ≠ æ S A p ; S i m il a r l y t h e s e t o f S c o pu s a u t h o r s o n a r e , ˛ A p a nd t h e C I P o p e r a t o r c o n v e r t s a s : O C I P ( ˛ A p ) = ≠≠ æ C I P p ; 13 . Sub j ec t A r e a c l u s t e r , S A ; t h e c o rr e s p o nd i n g S A v ec t o r o f a pub li c a t i o n , ≠ æ S A p 14 . D i s c i p li n a r y c l u s t e r , C I P ; t h e c o rr e s p o nd i n g C I P v ec t o r o f a pub li c a t i o n , ≠≠ æ C I P p 15 . E a c h a r t i c l e p h a s a s e t o f S A & C I P f e a t u r e s : ˛ F p © { ≠ æ S A p , ≠≠ æ C I P p } . 16 . F r o m t h e i n f o r m a t i o n c o n t a i n e d i n ˛ F p , e a c h a r t i c l e h a s h a s o n e o f t w o ( s c a l a r ) v a l u e s : c r o ss - d o m a i n ( d i v e r s e m i x t u r e ) = X , a s o pp o s e d t o m o n o - d o m a i n = M 17 . T h e t w o v a l u e s d e p e nd o n t h e o p e r a t o r a c t i n g o n ˛ F p : e . g . O C I P ( ˛ F p ) = M o r X C I P a nd O L o n g , C I P ( ˛ F p ) = M o r X L o n g , C I P 18 . R e g r e ss i o n v a r i a b l e s : i nd i c a t o r v a r i a b l e s i d e n t i fi e d u s i n g I ; s ub s c r i p t s i nd i c a t e t h e t y p e v a l u e c o rr e s p o nd i n g t o t h e v a l u e 1 , o t h e r w i s e t h e v a l u e = 0 ; E g . I X L o n g , C I P t a k e s o n t h e v a l u e 1 f o r a r t i c l e s w i t h O L o n g , C I P ( ˛ F p ) = X a nd 0 o t h e r w i s e 19 . s c h o l a r ag e a t t i m e o f pub li c a t i o n , · i , p 20 . i nd i c a t o r v a r i a b l e I 2014 + = 1 i f y p Ø 2014 a nd 0 o t h e r w i s e 21 . R e g i o n i nd i c a t o r v a r i a b l e R r = 1 i f a r t i c l e a u t h o r s a ss o - c i a t e d w i t h r e g i o n r a nd 0 o t h e r w i s e : R N A , R E U , R AA , R W o r l d ( t h i s c a t e go r y i s b a s e li n e i n T a b l e s S 1 - S 3 ) 22 . N R , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f r e g i o n s r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 4 23 . N S A , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f S A r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 6 24 . N C I P , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m b e r o f C I P r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a - t i o n s ) , i e m i n = 1 a nd m a x = 9 25 . z j i s t h e a v e r ag e z v a l u e c a l c u l a t e d a c r o ss a ll a r t i c l e s pub li s h e d i n a s p ec i fi c j o u r n a l ( i nd e x e d b y j ) S i gn i fi ca n ce S t a t e m e n t A u t ho r s m u s t s ub m i t a 120 - w o r d m a x i m u m s t a t e m en t abou t t he s i gn i fic an c e o f t he i r r e s ea r c h pape r w r i tt en a t a l e v e l unde r - s t andab l e t o an unde r g r adua t e edu c a t ed sc i en t i s t ou t s i de t he i r fi e l d o f s pe c i a li t y . T he p r i m a r y goa l o f t he s i gn i fic an c e s t a t e - m en t i s t o e x p l a i n t he r e l e v an c e o f t he w o r k i n b r oad c on t e x t t o a b r oad r eade r s h i p . T he s i gn i fic an c e s t a t e m en t appea r s i n t he pape r i t s e l f and i s r equ i r ed f o r a ll r e s ea r c h pape r s . P l ea s ep r o v i dede t a il s o f au t ho r c on t r i bu t i on s he r e . P l ea s ede c l a r ean yc o m pe t i ng i n t e r e s t s he r e . 1 A . O . ( A u t ho r O ne ) c on t r i bu t edequa ll y t o t h i s w o r k w i t h A . T . ( A u t ho r T w o ) ( r e m o v e i f no t app li c ab l e ) . 2 T o w ho m c o rr e s ponden c e s hou l dbeadd r e ss ed . E - m a il : au t ho r . t w oe m a il . c o m www . pna s . o r g / c g i / do i / 10 . 1073 / pna s . XXXXXXXXXX P N AS | M ay 4 , 2020 | v o l . XXX | no . XX | 1 – 16 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Information Science Pre - 2014 Subject Area frequency ( per publication , by region ) , f R , SA Frequency Difference ( Post - Pre ) , ∆ f R , SA N . A m e r i ca E u r op e A u s t r a l as i a R es t o f W o r l d / M u l t i n a t i on a l A < Subject Area (cid:2) Technology & Information Science (cid:2) Techniques & Equipment (cid:2) Health (cid:2) Phenomena & Processes (cid:2) Anatomy & Organisms (cid:2) Psychiatry & Psychology �� ��� ��� ��� ��� �� ��� ��� ��� ��� - ���� - ���� �� ���� ���� B Post - 2014 Subject Area frequency ( per publication , by region ) , f R , SA > 1 : Mind [ F ] 2 : Structure [ A , B ] 3 : Function [ G ] 4 : (cid:1) Health [ C , N ] 5 : Techniques & Equipment [ E ] 6 : Technology , Industry , Informatics [ J , L ] 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Information Science FIG . S3 : Temporal and regional distributions of Subject Areas ( SA ) in human brain research . ( A ) Relative frequency of topical SA clusters in the 5 - year period before 2014 ( f < R , SA ) and after 2014 ( f > R , SA ) ; f values are normalized to unity within region . ( B ) Shift in SA cluster frequencies given by the difference ∆ f R , SA = f > R , SA − f < R , SA . ( C ) Topical { SA , SA } co - occurrence in human brain science – by region . Each co - occurrence matrix C < SA measures the frequency of a given { SA , SA } pair over the 5 - year pre - period 2009 - 2013 based upon publications associated with one of three broad geographic regions ; see Eqn . ( S1 ) for its definition . By construction , matrix element values C < SA , ij are proportional to the net share of publications featuring the indicated pair . Diagonal elements measure the frequency of publications featuring only a single SA category . Note the use of two legends , one for the mono - dimensional diagonal elements ( gray - scale legend ) and one for off - diagonal elements ( color - scale legend ) , both of which are reported in units of 1000 publications . ( D ) Dynamic co - occurrence matrix , ∆ C SA , ij , measuring the percent difference ( post - pre ) in the frequency of publications characterized by each { SA , SA } pair . 13 F r a c t i on o f a r t i c l e s [ i nd i v i dua l do m a i n s ] ���� ���� ���� ���� ���� ���� ���� ���� ���� ��� ��� ��� ��� ���� ���� ���� ���� ���� ���� Neuro / Bio & Health Neuro / Bio & Sci / Eng Health & Sci / Eng Neuro / Bio & Health & Sci / Eng + 24 % + 46 % + 49 % + 101 % F r a c t i on o f a r t i c l e s [ c on v e r gen t do m a i n c o m b i na t i on s ] (cid:2) (cid:1) Period 1 ( P1 ) = 1999 - 2008 (cid:2) (cid:1) Period 2 ( P2 ) = 2009 - 2018 A B C Neuro / Bio & Health Neuro / Bio & Sci / Eng Health & Sci / Eng Neuro / Bio & Health & Sci / Eng Neuro / Bio HealthSci / Eng P1 P2 F r a c t i on o f a r t i c l e s [ c on v e r gen t do m a i n c o m b i na t i on s ] FIG . S4 : Increasing frequency of cross - disciplinary collaboration across convergent HBS interfaces . ( A ) Fraction of articles featuring at least one coauthor belonging to a given CIP super - group ( unconditioned on CIP of remaining coauthors ) : Neuro / Bio corresponds to CIP [ 1 - 4 ] ; Health corresponds to CIP [ 5 - 7 ] ; and Sci / Eng corresponds to CIP [ 8 , 9 ] ( see Fig . 1 ) . The network inset indicates the 7 types of combinations across these 3 types . ( B ) Fraction of articles featuring combinations of scholars from two or more CIP super - groups , representing the densification of the convergent interfaces visualized in the networks shown in Fig . 2 ( B ) , computed for two periods : P 1 = [ 1999 - 2008 ] and P 2 = [ 2009 - 2018 ] . ( C ) Increased frequency of convergent domain combinations between P 1 and P 2 . For example , the most prominent convergent interface is between Neuro / Bio and Health , which was featured in 8 . 6 % of articles in P 1 and 10 . 7 % in P 2 , corresponding to a + 24 % growth in P 2 relative to P 1 . All percent increases are significant at the p < 0 . 001 level based on a two - sample two - tailed z - test comparing the proportions for P 1 and P 2 . 14 M ean # SA pe r pub . , ⟨ N S A ⟩ � � � � � � � � � � � � � # CIP per pub . , N CIP , p � � � � � � � � � � � � (cid:2)(cid:1) z p ≥ 0 Above - average citations (cid:1) (cid:2)(cid:1) z p < 0 Below - average citations # CIP per pub . , N CIP , p All publications with k p ≥ 2 and w p ≥ 2 All publications with k p ≥ 2 and w p ≥ 2 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 # CIP per pub . , N CIP , p # CIP per pub . , N CIP , p # SA pe r pub . , N S A , p � � � � � � � � � � � # CIP per pub . , N CIP , p M ean # SA pe r pub . , ⟨ N S A ⟩ Broad Neighboring Distant P ( X SA & CIP ) / P ( M ) < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > Mono - CIP baseline ( N CIP , p = 1 ) # CIP per pub . , N CIP , p Mono - CIP baseline ( N CIP , p = 1 ) (cid:2)(cid:1) z p ≥ 0 Above - average citations (cid:1) (cid:2)(cid:1) z p < 0 Below - average citations K - S Test : * * * ( p < 0 . 001 ) for NCIP = 1 , 2 , 3 � � � � � � � � � � � � FIG . S5 : Expansive topical integration facilitated by CIP diversity . The number N CIP , p of distinct CIP featured by a given article is a measure of disciplinary diversity . ( A ) Average number of SA per article , (cid:104) N SA (cid:105) , computed for articles with a given N CIP , p and conditioned on the normalized citation impact . ( B ) Average number of SA per article , (cid:104) N SA , computed for articles featuring X SA & CIP according to a given configuration ( Broad , Neighboring and Distant ) . The Distant configuration consistently corresponds to the highest levels of SA diversity . Comparing panels ( A ) and ( B ) , (cid:104) N SA (cid:105) are also consistently larger for the N CIP subsets in ( B ) featuring X SA & CIP . For both panels , the horizontal dashed red line represents the baseline for comparison , computed as the average number of SA , (cid:104) N SA (cid:105) = 2 . 2 , calculated for mono - disciplinary articles ( N CIP , p = 1 ) . All CIP 1 2 3 4 5 6 7 8 9 � � � All CIP [ 1 - 4 ] [ 5 - 9 ] M ean C I P d i v e r s i t y pe r a r t i c l e , ⟨ f D , C I P ( t ) ⟩ �� �� �� �� �� �� All SA 1 [ 2 , 3 ] [ 4 - 7 ] Subset CIP �� �� �� �� �� �� �� �� �� �� A C C D � � � � � � � � � � � � � � � � � � � � � � � � � � � � 2 3 4 5 6 7 Subset SA F �� �� �� �� �� �� �� �� �� �� 1 2 3 4 5 6 7 � � � � � � Subset CIP [ 1 - 4 ] [ 5 - 7 ] [ 8 - 9 ] � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� 1 : Neurosciences 2 : Biology 3 : Psychology 4 : (cid:1) Biotech . & Genetics 5 : Medical Specialty 6 : Health Sciences 7 : Pathology & Pharmacology 8 : Eng . & Informatics 9 : Chemistry & Physics ���� ���� ���� ���� ���� ���� ���� ���� ���� B ���� ���� ���� ���� ���� ���� ����� ����� ����� North America EuropeAustralasia FIG . S6 : Trends in cross - disciplinary ( CIP ) scholarship in human brain science . Each curve corresponds to (cid:104) f D , CIP ( t ) (cid:105) , representing the average article diversity measured as categorical CIP co - occurrence in the off - diagonal matrix elements of D CIP , p , see Eq . ( 2 ) ; each curve is calculated for articles belonging to a given geographic region , as determined by the coauthors’ regional affiliations : Australasia ( red ) , Europe ( blue ) , and North America ( orange ) . For each panel we provide a matrix motif indicating the set of focal CIP categories ; counts for categories included in brackets are considered in union . For example , whereas panel ( A ) calculates (cid:104) f D , CIP ( t ) (cid:105) across all 9 CIP categories ( each category considered separately ) ; instead , panel ( B ) calculates each D p by considering just two super - groups , the first consisting of the union of CIP counts for categories [ 1 - 4 ] , and the second comprised of categories [ 5 - 9 ] . 15 All SA 1 2 3 4 Subset SA M ean SA d i v e r s i t y pe r a r t i c l e , ⟨ f D , S A ( t ) ⟩ All SA 1 [ 2 , 3 ] [ 4 - 7 ] All SA �� �� �� �� �� �� �� �� �� �� A C C B D �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� 2 3 4 5 6 7 Subset SA F � � � � � � � � � � 4 [ 1 - 3 , 5 - 6 ] � � � � � � � � � � � � � � � � � � � � � �� �� �� �� �� �� Subset SA ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� � � � � � � � � � �� �� �� �� �� �� [ 1 - 3 ] [ 4 - 5 ] � � � [ 1 - 3 ] [ 4 - 5 ] 4 5 6 1 2 3 4 5 6 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� Subset SA E F Subset SA ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� 5 6 ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� 1 2 3 � � � � � � � � � Subset SA G H Subset SA ���� ���� ���� ���� ���� ���� ���� ���� ���� [ 1 - 3 ] [ 5 - 6 ] ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� � � � � � � � � � � � � � � � 1 2 3 5 6 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Information Science North America EuropeAustralasia FIG . S7 : Trends in cross - topical ( SA ) scholarship in human brain science . Each curve corresponds to (cid:104) f D , SA ( t ) (cid:105) , representing the average article diversity measured as categorical SA co - occurrence in the off - diagonal matrix elements of D SA , p , see Eq . ( 2 ) ; each curve is calculated for articles belonging to a given geographic region , as determined by the coauthors’ regional affiliations : Australasia ( red ) , Europe ( blue ) , and North America ( orange ) . For each panel we provide a matrix motif indicating the set of focal SA categories ; counts for categories included in brackets are considered in union . For example , whereas panel ( A ) calculates (cid:104) f D , SA ( t ) (cid:105) across all 6 SA categories ( each category considered separately ) ; instead , panel ( C ) calculates each D SA , p by considering a subset of four SA categories 1 - 4 . 16 P D F , P ( N S A ) P D F , P ( N R ) P D F , P ( w ) P D F , P ( N C I P ) � � � � � � � ��� ��� ��� ��� ��� P D F , P ( z | { t } ) Normalized citation impact , z p 1970 - 1974 1975 - 1979 1980 - 1984 2000 - 2004 1995 - 1999 2015 - 2018 1990 - 1994 2010 - 2014 1985 - 1989 2005 - 2009 C F G # of Subject Area per pub . , N SA , p # of Authors per pub . , k p P D F , P ( k ) Mean value = 2 . 1 95th percentile = 15 # of Regions per pub . , N R , p E Mean value = 1 . 2 - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� G Normalized citation impact , z p { t } = 2000 - 2001 2002 - 2003 2004 - 2005 2012 - 2013 2010 - 2011 2018 2008 - 2009 2016 - 2017 2006 - 2007 2014 - 2015 # of Major Topic MeSH per pub . , w p � � �� �� �� �� ���� ���� ���� ���� ���� ���� Mean value = 4 . 1 � � � � ����� ����� ����� � �� � �� � �� � �� � �� - � �� - � �� - � �� - � �� - � �� - � �� - � �� � For k _ p : N = 977270 ; Difference = 404 with Kp = “NotAvail N = 977674 A � � � � �� - � �� - � ����� ����� ����� � # of Department CIP per pub . , N CIP , p D Mean value = 1 . 2 ���� ���� ���� ���� �� � �� � �� � �� � �� � �� � ���� ���� ���� ���� ������������������������ - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� - � - � � � � ��� ��� ��� ��� ��� ��� B { t } = 1945 - 1964 1965 - 1969 C oun t , N ( t ) t < latexit sha1 _ base64 = " L / T44 + 5qPX8Ur81Ix / x8 / o1lvOo = " > AAAB73icdVDLSgMxFM3UV62vqks3wSK4GjK2pdNd0Y3LCvYB7VAyaaYNTTJjkhFK6U + 4caGIW3 / HnX9j2o6gogcuHM65l3vvCRPOtEHow8mtrW9sbuW3Czu7e / sHxcOjto5TRWiLxDxW3RBrypmkLcMMp91EUSxCTjvh5Grhd + 6p0iyWt2aa0EDgkWQRI9hYqdvXbCTwwAyKJeTWfd + rIojccrmSkWqtXvah56IlSiBDc1B87w9jkgoqDeFY656HEhPMsDKMcDov9FNNE0wmeER7lkosqA5my3vn8MwqQxjFypY0cKl + n5hhofVUhLZTYDPWv72F + JfXS03kBzMmk9RQSVaLopRDE8PF83DIFCWGTy3BRDF7KyRjrDAxNqKCDeHrU / g / aV + 4HnK9m0qpcZnFkQcn4BScAw / UQANcgyZoAQI4eABP4Nm5cx6dF + d11Zpzsplj8APO2yeiA5Bc < / latexit > < latexit sha1 _ base64 = " L / T44 + 5qPX8Ur81Ix / x8 / o1lvOo = " > AAAB73icdVDLSgMxFM3UV62vqks3wSK4GjK2pdNd0Y3LCvYB7VAyaaYNTTJjkhFK6U + 4caGIW3 / HnX9j2o6gogcuHM65l3vvCRPOtEHow8mtrW9sbuW3Czu7e / sHxcOjto5TRWiLxDxW3RBrypmkLcMMp91EUSxCTjvh5Grhd + 6p0iyWt2aa0EDgkWQRI9hYqdvXbCTwwAyKJeTWfd + rIojccrmSkWqtXvah56IlSiBDc1B87w9jkgoqDeFY656HEhPMsDKMcDov9FNNE0wmeER7lkosqA5my3vn8MwqQxjFypY0cKl + n5hhofVUhLZTYDPWv72F + JfXS03kBzMmk9RQSVaLopRDE8PF83DIFCWGTy3BRDF7KyRjrDAxNqKCDeHrU / g / aV + 4HnK9m0qpcZnFkQcn4BScAw / UQANcgyZoAQI4eABP4Nm5cx6dF + d11Zpzsplj8APO2yeiA5Bc < / latexit > < latexit sha1 _ base64 = " L / T44 + 5qPX8Ur81Ix / x8 / o1lvOo = " > AAAB73icdVDLSgMxFM3UV62vqks3wSK4GjK2pdNd0Y3LCvYB7VAyaaYNTTJjkhFK6U + 4caGIW3 / HnX9j2o6gogcuHM65l3vvCRPOtEHow8mtrW9sbuW3Czu7e / sHxcOjto5TRWiLxDxW3RBrypmkLcMMp91EUSxCTjvh5Grhd + 6p0iyWt2aa0EDgkWQRI9hYqdvXbCTwwAyKJeTWfd + rIojccrmSkWqtXvah56IlSiBDc1B87w9jkgoqDeFY656HEhPMsDKMcDov9FNNE0wmeER7lkosqA5my3vn8MwqQxjFypY0cKl + n5hhofVUhLZTYDPWv72F + JfXS03kBzMmk9RQSVaLopRDE8PF83DIFCWGTy3BRDF7KyRjrDAxNqKCDeHrU / g / aV + 4HnK9m0qpcZnFkQcn4BScAw / UQANcgyZoAQI4eABP4Nm5cx6dF + d11Zpzsplj8APO2yeiA5Bc < / latexit > < latexit sha1 _ base64 = " L / T44 + 5qPX8Ur81Ix / x8 / o1lvOo = " > AAAB73icdVDLSgMxFM3UV62vqks3wSK4GjK2pdNd0Y3LCvYB7VAyaaYNTTJjkhFK6U + 4caGIW3 / HnX9j2o6gogcuHM65l3vvCRPOtEHow8mtrW9sbuW3Czu7e / sHxcOjto5TRWiLxDxW3RBrypmkLcMMp91EUSxCTjvh5Grhd + 6p0iyWt2aa0EDgkWQRI9hYqdvXbCTwwAyKJeTWfd + rIojccrmSkWqtXvah56IlSiBDc1B87w9jkgoqDeFY656HEhPMsDKMcDov9FNNE0wmeER7lkosqA5my3vn8MwqQxjFypY0cKl + n5hhofVUhLZTYDPWv72F + JfXS03kBzMmk9RQSVaLopRDE8PF83DIFCWGTy3BRDF7KyRjrDAxNqKCDeHrU / g / aV + 4HnK9m0qpcZnFkQcn4BScAw / UQANcgyZoAQI4eABP4Nm5cx6dF + d11Zpzsplj8APO2yeiA5Bc < / latexit > µ t < latexit sha1 _ base64 = " 1NWQDy5spzbOWbsM1NF + wMGCnws = " > AAAB7HicdVBNSwMxEM3Wr1q / qh69BIvgadltK + 2x6MVjBbcttEvJpmkbmmSXZFYoS3 + DFw + KePUHefPfmH6Bij4YeLw3w8y8KBHcgOd9OrmNza3tnfxuYW / / 4PCoeHzSMnGqKQtoLGLdiYhhgisWAAfBOolmREaCtaPJzdxvPzBteKzuYZqwUJKR4kNOCVgp6Mm0D / 1iyXMr5at6tYaXpLIm1Qr2XW + BElqh2S9 + 9AYxTSVTQAUxput7CYQZ0cCpYLNCLzUsIXRCRqxrqSKSmTBbHDvDF1YZ4GGsbSnAC / X7REakMVMZ2U5JYGx + e3PxL6 + bwrAeZlwlKTBFl4uGqcAQ4 / nneMA1oyCmlhCqub0V0zHRhILNp2BDWH + K / yetsut7rn9XLTWuV3Hk0Rk6R5fIRzXUQLeoiQJEEUeP6Bm9OMp5cl6dt2VrzlnNnKIfcN6 / AGCmjw8 = < / latexit > < latexit sha1 _ base64 = " 1NWQDy5spzbOWbsM1NF + wMGCnws = " > AAAB7HicdVBNSwMxEM3Wr1q / qh69BIvgadltK + 2x6MVjBbcttEvJpmkbmmSXZFYoS3 + DFw + KePUHefPfmH6Bij4YeLw3w8y8KBHcgOd9OrmNza3tnfxuYW / / 4PCoeHzSMnGqKQtoLGLdiYhhgisWAAfBOolmREaCtaPJzdxvPzBteKzuYZqwUJKR4kNOCVgp6Mm0D / 1iyXMr5at6tYaXpLIm1Qr2XW + BElqh2S9 + 9AYxTSVTQAUxput7CYQZ0cCpYLNCLzUsIXRCRqxrqSKSmTBbHDvDF1YZ4GGsbSnAC / X7REakMVMZ2U5JYGx + e3PxL6 + bwrAeZlwlKTBFl4uGqcAQ4 / nneMA1oyCmlhCqub0V0zHRhILNp2BDWH + K / yetsut7rn9XLTWuV3Hk0Rk6R5fIRzXUQLeoiQJEEUeP6Bm9OMp5cl6dt2VrzlnNnKIfcN6 / AGCmjw8 = < / latexit > < latexit sha1 _ base64 = " 1NWQDy5spzbOWbsM1NF + wMGCnws = " > AAAB7HicdVBNSwMxEM3Wr1q / qh69BIvgadltK + 2x6MVjBbcttEvJpmkbmmSXZFYoS3 + DFw + KePUHefPfmH6Bij4YeLw3w8y8KBHcgOd9OrmNza3tnfxuYW / / 4PCoeHzSMnGqKQtoLGLdiYhhgisWAAfBOolmREaCtaPJzdxvPzBteKzuYZqwUJKR4kNOCVgp6Mm0D / 1iyXMr5at6tYaXpLIm1Qr2XW + BElqh2S9 + 9AYxTSVTQAUxput7CYQZ0cCpYLNCLzUsIXRCRqxrqSKSmTBbHDvDF1YZ4GGsbSnAC / X7REakMVMZ2U5JYGx + e3PxL6 + bwrAeZlwlKTBFl4uGqcAQ4 / nneMA1oyCmlhCqub0V0zHRhILNp2BDWH + K / yetsut7rn9XLTWuV3Hk0Rk6R5fIRzXUQLeoiQJEEUeP6Bm9OMp5cl6dt2VrzlnNnKIfcN6 / AGCmjw8 = < / latexit > < latexit sha1 _ base64 = " 1NWQDy5spzbOWbsM1NF + wMGCnws = " > AAAB7HicdVBNSwMxEM3Wr1q / qh69BIvgadltK + 2x6MVjBbcttEvJpmkbmmSXZFYoS3 + DFw + KePUHefPfmH6Bij4YeLw3w8y8KBHcgOd9OrmNza3tnfxuYW / / 4PCoeHzSMnGqKQtoLGLdiYhhgisWAAfBOolmREaCtaPJzdxvPzBteKzuYZqwUJKR4kNOCVgp6Mm0D / 1iyXMr5at6tYaXpLIm1Qr2XW + BElqh2S9 + 9AYxTSVTQAUxput7CYQZ0cCpYLNCLzUsIXRCRqxrqSKSmTBbHDvDF1YZ4GGsbSnAC / X7REakMVMZ2U5JYGx + e3PxL6 + bwrAeZlwlKTBFl4uGqcAQ4 / nneMA1oyCmlhCqub0V0zHRhILNp2BDWH + K / yetsut7rn9XLTWuV3Hk0Rk6R5fIRzXUQLeoiQJEEUeP6Bm9OMp5cl6dt2VrzlnNnKIfcN6 / AGCmjw8 = < / latexit > h i = 1 . 24 < latexit sha1 _ base64 = " O0WW8U44pfyZNeYz6w4BP0 / gjwI = " > AAACBnicdVBLSwMxGMzWV62vVY8iBIvgadldW7oehKIXjxXsA7qlZNO0DU2yS5IVSunJi3 / FiwdFvPobvPlvzLYVVHQgMJmZj + SbKGFUadf9sHJLyyura / n1wsbm1vaOvbvXUHEqManjmMWyFSFFGBWkrqlmpJVIgnjESDMaXWZ + 85ZIRWNxo8cJ6XA0ELRPMdJG6tqHIUNiwAgMFR1wBEM5v55Dz / FLXbvoOmdBJfDL0HVOXa9UDjLiGyUwCXeGIlig1rXfw16MU06Exgwp1fbcRHcmSGqKGZkWwlSRBOERGpC2oQJxojqT2RpTeGyUHuzH0hyh4Uz9PjFBXKkxj0ySIz1Uv71M / Mtrp7ofdCZUJKkmAs8f6qcM6hhmncAelQRrNjYEYUnNXyEeIomwNs0VTAlfm8L / ScN3PNfxrkvF6sWijjw4AEfgBHigAqrgCtRAHWBwBx7AE3i27q1H68V6nUdz1mJmH / yA9fYJUNSXug = = < / latexit > < latexit sha1 _ base64 = " O0WW8U44pfyZNeYz6w4BP0 / gjwI = " > AAACBnicdVBLSwMxGMzWV62vVY8iBIvgadldW7oehKIXjxXsA7qlZNO0DU2yS5IVSunJi3 / FiwdFvPobvPlvzLYVVHQgMJmZj + SbKGFUadf9sHJLyyura / n1wsbm1vaOvbvXUHEqManjmMWyFSFFGBWkrqlmpJVIgnjESDMaXWZ + 85ZIRWNxo8cJ6XA0ELRPMdJG6tqHIUNiwAgMFR1wBEM5v55Dz / FLXbvoOmdBJfDL0HVOXa9UDjLiGyUwCXeGIlig1rXfw16MU06Exgwp1fbcRHcmSGqKGZkWwlSRBOERGpC2oQJxojqT2RpTeGyUHuzH0hyh4Uz9PjFBXKkxj0ySIz1Uv71M / Mtrp7ofdCZUJKkmAs8f6qcM6hhmncAelQRrNjYEYUnNXyEeIomwNs0VTAlfm8L / ScN3PNfxrkvF6sWijjw4AEfgBHigAqrgCtRAHWBwBx7AE3i27q1H68V6nUdz1mJmH / yA9fYJUNSXug = = < / latexit > < latexit sha1 _ base64 = " O0WW8U44pfyZNeYz6w4BP0 / gjwI = " > AAACBnicdVBLSwMxGMzWV62vVY8iBIvgadldW7oehKIXjxXsA7qlZNO0DU2yS5IVSunJi3 / FiwdFvPobvPlvzLYVVHQgMJmZj + SbKGFUadf9sHJLyyura / n1wsbm1vaOvbvXUHEqManjmMWyFSFFGBWkrqlmpJVIgnjESDMaXWZ + 85ZIRWNxo8cJ6XA0ELRPMdJG6tqHIUNiwAgMFR1wBEM5v55Dz / FLXbvoOmdBJfDL0HVOXa9UDjLiGyUwCXeGIlig1rXfw16MU06Exgwp1fbcRHcmSGqKGZkWwlSRBOERGpC2oQJxojqT2RpTeGyUHuzH0hyh4Uz9PjFBXKkxj0ySIz1Uv71M / Mtrp7ofdCZUJKkmAs8f6qcM6hhmncAelQRrNjYEYUnNXyEeIomwNs0VTAlfm8L / ScN3PNfxrkvF6sWijjw4AEfgBHigAqrgCtRAHWBwBx7AE3i27q1H68V6nUdz1mJmH / yA9fYJUNSXug = = < / latexit > < latexit sha1 _ base64 = " O0WW8U44pfyZNeYz6w4BP0 / gjwI = " > AAACBnicdVBLSwMxGMzWV62vVY8iBIvgadldW7oehKIXjxXsA7qlZNO0DU2yS5IVSunJi3 / FiwdFvPobvPlvzLYVVHQgMJmZj + SbKGFUadf9sHJLyyura / n1wsbm1vaOvbvXUHEqManjmMWyFSFFGBWkrqlmpJVIgnjESDMaXWZ + 85ZIRWNxo8cJ6XA0ELRPMdJG6tqHIUNiwAgMFR1wBEM5v55Dz / FLXbvoOmdBJfDL0HVOXa9UDjLiGyUwCXeGIlig1rXfw16MU06Exgwp1fbcRHcmSGqKGZkWwlSRBOERGpC2oQJxojqT2RpTeGyUHuzH0hyh4Uz9PjFBXKkxj0ySIz1Uv71M / Mtrp7ofdCZUJKkmAs8f6qcM6hhmncAelQRrNjYEYUnNXyEeIomwNs0VTAlfm8L / ScN3PNfxrkvF6sWijjw4AEfgBHigAqrgCtRAHWBwBx7AE3i27q1H68V6nUdz1mJmH / yA9fYJUNSXug = = < / latexit > Log - normal distribution location and scale location scale FIG . S8 : Distributions of Article - level variables . ( A ) N ( t ) is the number of HB articles by year . ( inset ) The log - citation distribution is well described by a log - normal distribution ( see panel G ) . As such , µ t and σ t corresponding to log - transformed citation counts are appropriate measures of log - normal location and scale ; the average and standard deviation are (cid:104) σ (cid:105) ± SD = 1 . 24 ± 0 . 09 over the 49 - year period 1970 - 2018 . ( B ) P ( k ) is the probability distribution ( PDF ) of the number of coauthors per article . ( C ) P ( w ) is the PDF of the number of Major Topic MeSH “keywords” per publication , denoted by w p . ( D ) Each MeSH keyword maps onto one of the 6 SA clusters . Shown is the PDFof the number of distinct SA categories per publication , N SA , p . ( E ) Each departmental affiliation maps onto one of the 9 CIP clusters . Shown is the PDF of the number of distinct CIP categories per publication , N CIP , p . ( F ) Each Scopus Author’s affiliation maps onto one of 4 regions : Australasia , Europe , North America , and ( rest of ) World . Shown is the PDF of the number of region categories per publication , N R , p . ( G ) Probability distribution ( PDF ) of z p disaggregated by publication cohort { t } ; each green curve represents the smoothed kernel density estimate of the P ( z ) , calculated with kernel bandwith = 0 . 1 . Data are split into 5 - year periods from 1965 - 2018 , with the first panel including data from 1945 - 1964 . Each PDF shows the baseline Normal distribution N ( 0 , 1 ) , demonstrating the stability of the distribution of normalized citation impact values over time , thereby facilitating robust cross - temporal modeling . 17 { " Yp " , " Kp " , " nMeSHMain " , " Zp " , " XSAp " , " XCIPp " , " NEUROSHORTXSAp " , " NEUROSHORTXCIPp " , " NEUROLONGXSAp " , " NEUROLONGXCIPp " , " NRegp " , " NSAp " , " NCIPp " } y k z I X SA I X CIP I X N , SA I X N , CIP I X D , SA I X D , CIP N R N SA N CIP y k z I X SA I X CIP I X Neighboring , SA I X Neighboring , CIP I X Distant , SA I X Distant , CIP N R N SA N CIP w w FIG . S9 : Cross - correlation and Descriptive statistics for regression model variables . Upper - diagonal elements : bivariate histogram between row and column variables . Diagonal elements : histogram for variable indicated by the row / column labels . Lower - diagonal elements : bivariate cross - correlation coefficient : light - shaded squares indicate the Pearson’s correlation coefficient between two variables that are both continuous measures ; dark - shaded squares indicate the Cramer’s V associate between two variables that are both nominal ( categorical ) . 18 A B ����� ����������� ������� - �� - �� � �� �� �� * * * * * * * * * * * * * * * * * * ����� ����������� ������� - �� - �� - �� � �� �� �� * * * * * * * * * * * * * * * * * * * * * * C ����� ����������� ������� - �� - �� - �� � �� �� * * * * * * * * * * * * * * * * * * * * * * * ����� ����������� ������� - �� � �� �� * * * * * * * * * ����� ����������� ������� - �� - �� � �� * * * * * D E F Regression Table : reports odds ratio e ^ beta ( \ approx 1 + beta for small beta ) Here : reports basis points = 100 * Log [ e ^ beta ] = 100 * beta Log [ P / Q ] = Log Odds > > P / Q s . t . P ( X = 1 ) + Q ( X = 0 ) = 1 E . g . Log [ P / Q ] = beta _ 0 + beta _ x * X Odds for state x = 1 : e ^ ( beta _ 0 + beta _ x ) ; odds for state x = 0 : e ^ ( beta _ 0 ) ; odds ratio = Odds _ X = 1 / Odds _ X = 0 = e ^ ( beta _ x ) Eg . g if Odds Ratio = 2 . 4 > > Odds of P is 2 . 4 times greater for those with X = 1 than X = 0 Since Odds = P / Q = P / ( 1 - P ) then P = Odds / ( 1 + Odds ) Hence P ( X = 1 ) = e ^ ( beta _ 0 + beta _ x ) / [ 1 + e ^ ( beta _ 0 + beta _ x ) ] P e r c en t i n c r ea s e i n O dd s Q = P ( X ) / P ( M ) a ss o c i a t ed w i t h 1 - un i t i n c r ea s e i n ea c h c o v a r i a t e [ P o i n t e s t i m a t e w i t h 95 % c on fi den c e i n t e r v a l ] 100 k < latexit sha1 _ base64 = " qf2Huwtq2VWx0SRQj3 / c7vDa + N4 = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWaEs / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0ecJLyMKFDJWLBKDqpG / g + 6UUcaX / cr9b8uj8HWSVBQWpQoNmvfvUGmmUJV8gktdYNSzHMqUHBJJ9WepnlKWVjOuRdRxVNuA3z + cpTcuaUAYm1cU8hmau / O3KaWDtJIleZUBzZZW8m / ud1M4yvw1yoNEOu2OKjOJMENZndTwbCcIZy4ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A6l6QWw = = < / latexit > < latexit sha1 _ base64 = " qf2Huwtq2VWx0SRQj3 / c7vDa + N4 = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWaEs / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0ecJLyMKFDJWLBKDqpG / g + 6UUcaX / cr9b8uj8HWSVBQWpQoNmvfvUGmmUJV8gktdYNSzHMqUHBJJ9WepnlKWVjOuRdRxVNuA3z + cpTcuaUAYm1cU8hmau / O3KaWDtJIleZUBzZZW8m / ud1M4yvw1yoNEOu2OKjOJMENZndTwbCcIZy4ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A6l6QWw = = < / latexit > < latexit sha1 _ base64 = " qf2Huwtq2VWx0SRQj3 / c7vDa + N4 = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWaEs / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0ecJLyMKFDJWLBKDqpG / g + 6UUcaX / cr9b8uj8HWSVBQWpQoNmvfvUGmmUJV8gktdYNSzHMqUHBJJ9WepnlKWVjOuRdRxVNuA3z + cpTcuaUAYm1cU8hmau / O3KaWDtJIleZUBzZZW8m / ud1M4yvw1yoNEOu2OKjOJMENZndTwbCcIZy4ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A6l6QWw = = < / latexit > < latexit sha1 _ base64 = " qf2Huwtq2VWx0SRQj3 / c7vDa + N4 = " > AAAB8nicbVBNSwMxEJ2tX7V + VT16CRbBU9kVQY9FLx4r2Fpol5JNs21oNlmSWaEs / RlePCji1V / jzX9j2u5BWx8EHu / NTGZelEph0fe / vdLa + sbmVnm7srO7t39QPTxqW50ZxltMS206EbVcCsVbKFDyTmo4TSLJH6Px7cx / fOLGCq0ecJLyMKFDJWLBKDqpG / g + 6UUcaX / cr9b8uj8HWSVBQWpQoNmvfvUGmmUJV8gktdYNSzHMqUHBJJ9WepnlKWVjOuRdRxVNuA3z + cpTcuaUAYm1cU8hmau / O3KaWDtJIleZUBzZZW8m / ud1M4yvw1yoNEOu2OKjOJMENZndTwbCcIZy4ghlRrhdCRtRQxm6lCouhGD55FXSvqgHfj24v6w1boo4ynACp3AOAVxBA + 6gCS1goOEZXuHNQ + / Fe / c + FqUlr + g5hj / wPn8A6l6QWw = = < / latexit > 100 z < latexit sha1 _ base64 = " yMaVVTQVW4vuA0sB62YVEjv4AhM = " > AAACAXicbVBPS8MwHE3nvzn / Tb0IXoJD8DRSEfQ49OJxgtuEtZQ0S7ewNClJKsxSL34VLx4U8eq38Oa3Md160M0Hgcd775fk98KEM20Q + nYqS8srq2vV9drG5tb2Tn13r6tlqgjtEMmluguxppwJ2jHMcHqXKIrjkNNeOL4q / N49VZpJcWsmCfVjPBQsYgQbKwX1Axch6IXU4CDzpE0WF2UPeR7UG6iJpoCLxC1JA5RoB / UvbyBJGlNhCMda912UGD / DyjDCaV7zUk0TTMZ4SPuWChxT7WfTDXJ4bJUBjKSyRxg4VX9PZDjWehKHNhljM9LzXiH + 5 / VTE134GRNJaqggs4eilEMjYVEHHDBFieETSzBRzP4VkhFWmBhbWs2W4M6vvEi6p00XNd2bs0brsqyjCg7BETgBLjgHLXAN2qADCHgEz + AVvDlPzovz7nzMohWnnNkHf + B8 / gBJCZbN < / latexit > < latexit sha1 _ base64 = " yMaVVTQVW4vuA0sB62YVEjv4AhM = " > AAACAXicbVBPS8MwHE3nvzn / Tb0IXoJD8DRSEfQ49OJxgtuEtZQ0S7ewNClJKsxSL34VLx4U8eq38Oa3Md160M0Hgcd775fk98KEM20Q + nYqS8srq2vV9drG5tb2Tn13r6tlqgjtEMmluguxppwJ2jHMcHqXKIrjkNNeOL4q / N49VZpJcWsmCfVjPBQsYgQbKwX1Axch6IXU4CDzpE0WF2UPeR7UG6iJpoCLxC1JA5RoB / UvbyBJGlNhCMda912UGD / DyjDCaV7zUk0TTMZ4SPuWChxT7WfTDXJ4bJUBjKSyRxg4VX9PZDjWehKHNhljM9LzXiH + 5 / VTE134GRNJaqggs4eilEMjYVEHHDBFieETSzBRzP4VkhFWmBhbWs2W4M6vvEi6p00XNd2bs0brsqyjCg7BETgBLjgHLXAN2qADCHgEz + AVvDlPzovz7nzMohWnnNkHf + B8 / gBJCZbN < / latexit > < latexit sha1 _ base64 = " yMaVVTQVW4vuA0sB62YVEjv4AhM = " > AAACAXicbVBPS8MwHE3nvzn / Tb0IXoJD8DRSEfQ49OJxgtuEtZQ0S7ewNClJKsxSL34VLx4U8eq38Oa3Md160M0Hgcd775fk98KEM20Q + nYqS8srq2vV9drG5tb2Tn13r6tlqgjtEMmluguxppwJ2jHMcHqXKIrjkNNeOL4q / N49VZpJcWsmCfVjPBQsYgQbKwX1Axch6IXU4CDzpE0WF2UPeR7UG6iJpoCLxC1JA5RoB / UvbyBJGlNhCMda912UGD / DyjDCaV7zUk0TTMZ4SPuWChxT7WfTDXJ4bJUBjKSyRxg4VX9PZDjWehKHNhljM9LzXiH + 5 / VTE134GRNJaqggs4eilEMjYVEHHDBFieETSzBRzP4VkhFWmBhbWs2W4M6vvEi6p00XNd2bs0brsqyjCg7BETgBLjgHLXAN2qADCHgEz + AVvDlPzovz7nzMohWnnNkHf + B8 / gBJCZbN < / latexit > < latexit sha1 _ base64 = " yMaVVTQVW4vuA0sB62YVEjv4AhM = " > AAACAXicbVBPS8MwHE3nvzn / Tb0IXoJD8DRSEfQ49OJxgtuEtZQ0S7ewNClJKsxSL34VLx4U8eq38Oa3Md160M0Hgcd775fk98KEM20Q + nYqS8srq2vV9drG5tb2Tn13r6tlqgjtEMmluguxppwJ2jHMcHqXKIrjkNNeOL4q / N49VZpJcWsmCfVjPBQsYgQbKwX1Axch6IXU4CDzpE0WF2UPeR7UG6iJpoCLxC1JA5RoB / UvbyBJGlNhCMda912UGD / DyjDCaV7zUk0TTMZ4SPuWChxT7WfTDXJ4bJUBjKSyRxg4VX9PZDjWehKHNhljM9LzXiH + 5 / VTE134GRNJaqggs4eilEMjYVEHHDBFieETSzBRzP4VkhFWmBhbWs2W4M6vvEi6p00XNd2bs0brsqyjCg7BETgBLjgHLXAN2qADCHgEz + AVvDlPzovz7nzMohWnnNkHf + B8 / gBJCZbN < / latexit > 100 R AA + < latexit sha1 _ base64 = " xQNf3VC / FtFwCQ9en0HgxrlOMSo = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLpsdeOyin1AG8JkMmmHTiZhZiLUEPwVNy4Ucet / uPNvnLZZaOuBC4dz7uXee / yEM6UR + rZKS8srq2vl9crG5tb2jr2711ZxKgltkZjHsutjRTkTtKWZ5rSbSIojn9OOP7qe + J0HKhWLxb0eJ9SN8ECwkBGsjeTZBw5CsB9QrrGX3XlZo5Gf5p5dRTU0BVwkTkGqoEDTs7 / 6QUzSiApNOFaq56BEuxmWmhFO80o / VTTBZIQHtGeowBFVbja9PofHRglgGEtTQsOp + nsiw5FS48g3nRHWQzXvTcT / vF6qw0s3YyJJNRVktihMOdQxnEQBAyYp0XxsCCaSmVshGWKJiTaBVUwIzvzLi6R9VnNQzbk9r9avijjK4BAcgRPggAtQBzegCVqAgEfwDF7Bm / VkvVjv1sestWQVM / vgD6zPH + O + lDc = < / latexit > < latexit sha1 _ base64 = " xQNf3VC / FtFwCQ9en0HgxrlOMSo = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLpsdeOyin1AG8JkMmmHTiZhZiLUEPwVNy4Ucet / uPNvnLZZaOuBC4dz7uXee / yEM6UR + rZKS8srq2vl9crG5tb2jr2711ZxKgltkZjHsutjRTkTtKWZ5rSbSIojn9OOP7qe + J0HKhWLxb0eJ9SN8ECwkBGsjeTZBw5CsB9QrrGX3XlZo5Gf5p5dRTU0BVwkTkGqoEDTs7 / 6QUzSiApNOFaq56BEuxmWmhFO80o / VTTBZIQHtGeowBFVbja9PofHRglgGEtTQsOp + nsiw5FS48g3nRHWQzXvTcT / vF6qw0s3YyJJNRVktihMOdQxnEQBAyYp0XxsCCaSmVshGWKJiTaBVUwIzvzLi6R9VnNQzbk9r9avijjK4BAcgRPggAtQBzegCVqAgEfwDF7Bm / VkvVjv1sestWQVM / vgD6zPH + O + lDc = < / latexit > < latexit sha1 _ base64 = " xQNf3VC / FtFwCQ9en0HgxrlOMSo = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLpsdeOyin1AG8JkMmmHTiZhZiLUEPwVNy4Ucet / uPNvnLZZaOuBC4dz7uXee / yEM6UR + rZKS8srq2vl9crG5tb2jr2711ZxKgltkZjHsutjRTkTtKWZ5rSbSIojn9OOP7qe + J0HKhWLxb0eJ9SN8ECwkBGsjeTZBw5CsB9QrrGX3XlZo5Gf5p5dRTU0BVwkTkGqoEDTs7 / 6QUzSiApNOFaq56BEuxmWmhFO80o / VTTBZIQHtGeowBFVbja9PofHRglgGEtTQsOp + nsiw5FS48g3nRHWQzXvTcT / vF6qw0s3YyJJNRVktihMOdQxnEQBAyYp0XxsCCaSmVshGWKJiTaBVUwIzvzLi6R9VnNQzbk9r9avijjK4BAcgRPggAtQBzegCVqAgEfwDF7Bm / VkvVjv1sestWQVM / vgD6zPH + O + lDc = < / latexit > < latexit sha1 _ base64 = " xQNf3VC / FtFwCQ9en0HgxrlOMSo = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLpsdeOyin1AG8JkMmmHTiZhZiLUEPwVNy4Ucet / uPNvnLZZaOuBC4dz7uXee / yEM6UR + rZKS8srq2vl9crG5tb2jr2711ZxKgltkZjHsutjRTkTtKWZ5rSbSIojn9OOP7qe + J0HKhWLxb0eJ9SN8ECwkBGsjeTZBw5CsB9QrrGX3XlZo5Gf5p5dRTU0BVwkTkGqoEDTs7 / 6QUzSiApNOFaq56BEuxmWmhFO80o / VTTBZIQHtGeowBFVbja9PofHRglgGEtTQsOp + nsiw5FS48g3nRHWQzXvTcT / vF6qw0s3YyJJNRVktihMOdQxnEQBAyYp0XxsCCaSmVshGWKJiTaBVUwIzvzLi6R9VnNQzbk9r9avijjK4BAcgRPggAtQBzegCVqAgEfwDF7Bm / VkvVjv1sestWQVM / vgD6zPH + O + lDc = < / latexit > 100 R NA + < latexit sha1 _ base64 = " NnDRo3h3eojA + uCdnbWvMumEcXI = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLqsunElVewD2hAmk0k7dDIJMxOhhuCvuHGhiFv / w51 / 47TNQlsPXDiccy / 33uMnnCmN0LdVWlhcWl4pr1bW1jc2t + ztnZaKU0lok8Q8lh0fK8qZoE3NNKedRFIc + Zy2 / eHV2G8 / UKlYLO71KKFuhPuChYxgbSTP3nMQgr2Aco297M7Lbi7y49yzq6iGJoDzxClIFRRoePZXL4hJGlGhCcdKdR2UaDfDUjPCaV7ppYommAxxn3YNFTiiys0m1 + fw0CgBDGNpSmg4UX9PZDhSahT5pjPCeqBmvbH4n9dNdXjuZkwkqaaCTBeFKYc6huMoYMAkJZqPDMFEMnMrJAMsMdEmsIoJwZl9eZ60TmoOqjm3p9X6ZRFHGeyDA3AEHHAG6uAaNEATEPAInsEreLOerBfr3fqYtpasYmYX / IH1 + QP3ppRE < / latexit > < latexit sha1 _ base64 = " NnDRo3h3eojA + uCdnbWvMumEcXI = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLqsunElVewD2hAmk0k7dDIJMxOhhuCvuHGhiFv / w51 / 47TNQlsPXDiccy / 33uMnnCmN0LdVWlhcWl4pr1bW1jc2t + ztnZaKU0lok8Q8lh0fK8qZoE3NNKedRFIc + Zy2 / eHV2G8 / UKlYLO71KKFuhPuChYxgbSTP3nMQgr2Aco297M7Lbi7y49yzq6iGJoDzxClIFRRoePZXL4hJGlGhCcdKdR2UaDfDUjPCaV7ppYommAxxn3YNFTiiys0m1 + fw0CgBDGNpSmg4UX9PZDhSahT5pjPCeqBmvbH4n9dNdXjuZkwkqaaCTBeFKYc6huMoYMAkJZqPDMFEMnMrJAMsMdEmsIoJwZl9eZ60TmoOqjm3p9X6ZRFHGeyDA3AEHHAG6uAaNEATEPAInsEreLOerBfr3fqYtpasYmYX / IH1 + QP3ppRE < / latexit > < latexit sha1 _ base64 = " NnDRo3h3eojA + uCdnbWvMumEcXI = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLqsunElVewD2hAmk0k7dDIJMxOhhuCvuHGhiFv / w51 / 47TNQlsPXDiccy / 33uMnnCmN0LdVWlhcWl4pr1bW1jc2t + ztnZaKU0lok8Q8lh0fK8qZoE3NNKedRFIc + Zy2 / eHV2G8 / UKlYLO71KKFuhPuChYxgbSTP3nMQgr2Aco297M7Lbi7y49yzq6iGJoDzxClIFRRoePZXL4hJGlGhCcdKdR2UaDfDUjPCaV7ppYommAxxn3YNFTiiys0m1 + fw0CgBDGNpSmg4UX9PZDhSahT5pjPCeqBmvbH4n9dNdXjuZkwkqaaCTBeFKYc6huMoYMAkJZqPDMFEMnMrJAMsMdEmsIoJwZl9eZ60TmoOqjm3p9X6ZRFHGeyDA3AEHHAG6uAaNEATEPAInsEreLOerBfr3fqYtpasYmYX / IH1 + QP3ppRE < / latexit > < latexit sha1 _ base64 = " NnDRo3h3eojA + uCdnbWvMumEcXI = " > AAAB / XicbVDLSsNAFJ3UV62v + Ni5GSyCIJSJCLqsunElVewD2hAmk0k7dDIJMxOhhuCvuHGhiFv / w51 / 47TNQlsPXDiccy / 33uMnnCmN0LdVWlhcWl4pr1bW1jc2t + ztnZaKU0lok8Q8lh0fK8qZoE3NNKedRFIc + Zy2 / eHV2G8 / UKlYLO71KKFuhPuChYxgbSTP3nMQgr2Aco297M7Lbi7y49yzq6iGJoDzxClIFRRoePZXL4hJGlGhCcdKdR2UaDfDUjPCaV7ppYommAxxn3YNFTiiys0m1 + fw0CgBDGNpSmg4UX9PZDhSahT5pjPCeqBmvbH4n9dNdXjuZkwkqaaCTBeFKYc6huMoYMAkJZqPDMFEMnMrJAMsMdEmsIoJwZl9eZ60TmoOqjm3p9X6ZRFHGeyDA3AEHHAG6uAaNEATEPAInsEreLOerBfr3fqYtpasYmYX / IH1 + QP3ppRE < / latexit > 100 R EU + < latexit sha1 _ base64 = " wtfmQm7bkAbuu + qjeCX8D1ZX8QU = " > AAAB / XicbVDLSsNAFJ34rPUVHzs3g0UQhDIRQZdFEVxWMW2hDWEymbRDJ5MwMxFqCP6KGxeKuPU / 3Pk3TtsstPXAhcM593LvPUHKmdIIfVsLi0vLK6uVter6xubWtr2z21JJJgl1ScIT2QmwopwJ6mqmOe2kkuI44LQdDK / GfvuBSsUSca9HKfVi3BcsYgRrI / n2voMQ7IWUa + znd35 + 7RYnhW / XUB1NAOeJU5IaKNH07a9emJAspkITjpXqOijVXo6lZoTTotrLFE0xGeI + 7RoqcEyVl0 + uL + CRUUIYJdKU0HCi / p7IcazUKA5MZ4z1QM16Y / E / r5vp6MLLmUgzTQWZLooyDnUCx1HAkElKNB8Zgolk5lZIBlhiok1gVROCM / vyPGmd1h1Ud27Pao3LMo4KOACH4Bg44Bw0wA1oAhcQ8AiewSt4s56sF + vd + pi2LljlzB74A + vzBwh5lE8 = < / latexit > < latexit sha1 _ base64 = " wtfmQm7bkAbuu + qjeCX8D1ZX8QU = " > AAAB / XicbVDLSsNAFJ34rPUVHzs3g0UQhDIRQZdFEVxWMW2hDWEymbRDJ5MwMxFqCP6KGxeKuPU / 3Pk3TtsstPXAhcM593LvPUHKmdIIfVsLi0vLK6uVter6xubWtr2z21JJJgl1ScIT2QmwopwJ6mqmOe2kkuI44LQdDK / GfvuBSsUSca9HKfVi3BcsYgRrI / n2voMQ7IWUa + znd35 + 7RYnhW / XUB1NAOeJU5IaKNH07a9emJAspkITjpXqOijVXo6lZoTTotrLFE0xGeI + 7RoqcEyVl0 + uL + CRUUIYJdKU0HCi / p7IcazUKA5MZ4z1QM16Y / E / r5vp6MLLmUgzTQWZLooyDnUCx1HAkElKNB8Zgolk5lZIBlhiok1gVROCM / vyPGmd1h1Ud27Pao3LMo4KOACH4Bg44Bw0wA1oAhcQ8AiewSt4s56sF + vd + pi2LljlzB74A + vzBwh5lE8 = < / latexit > < latexit sha1 _ base64 = " wtfmQm7bkAbuu + qjeCX8D1ZX8QU = " > AAAB / XicbVDLSsNAFJ34rPUVHzs3g0UQhDIRQZdFEVxWMW2hDWEymbRDJ5MwMxFqCP6KGxeKuPU / 3Pk3TtsstPXAhcM593LvPUHKmdIIfVsLi0vLK6uVter6xubWtr2z21JJJgl1ScIT2QmwopwJ6mqmOe2kkuI44LQdDK / GfvuBSsUSca9HKfVi3BcsYgRrI / n2voMQ7IWUa + znd35 + 7RYnhW / XUB1NAOeJU5IaKNH07a9emJAspkITjpXqOijVXo6lZoTTotrLFE0xGeI + 7RoqcEyVl0 + uL + CRUUIYJdKU0HCi / p7IcazUKA5MZ4z1QM16Y / E / r5vp6MLLmUgzTQWZLooyDnUCx1HAkElKNB8Zgolk5lZIBlhiok1gVROCM / vyPGmd1h1Ud27Pao3LMo4KOACH4Bg44Bw0wA1oAhcQ8AiewSt4s56sF + vd + pi2LljlzB74A + vzBwh5lE8 = < / latexit > < latexit sha1 _ base64 = " wtfmQm7bkAbuu + qjeCX8D1ZX8QU = " > AAAB / XicbVDLSsNAFJ34rPUVHzs3g0UQhDIRQZdFEVxWMW2hDWEymbRDJ5MwMxFqCP6KGxeKuPU / 3Pk3TtsstPXAhcM593LvPUHKmdIIfVsLi0vLK6uVter6xubWtr2z21JJJgl1ScIT2QmwopwJ6mqmOe2kkuI44LQdDK / GfvuBSsUSca9HKfVi3BcsYgRrI / n2voMQ7IWUa + znd35 + 7RYnhW / XUB1NAOeJU5IaKNH07a9emJAspkITjpXqOijVXo6lZoTTotrLFE0xGeI + 7RoqcEyVl0 + uL + CRUUIYJdKU0HCi / p7IcazUKA5MZ4z1QM16Y / E / r5vp6MLLmUgzTQWZLooyDnUCx1HAkElKNB8Zgolk5lZIBlhiok1gVROCM / vyPGmd1h1Ud27Pao3LMo4KOACH4Bg44Bw0wA1oAhcQ8AiewSt4s56sF + vd + pi2LljlzB74A + vzBwh5lE8 = < / latexit > Odds Q = Odds Q = Odds Q = P ( X SA ) / P ( M ) < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > < latexit sha1 _ base64 = " MPKpFa5 + 2nLPMw3 / MQj5BNjWNgA = " > AAAB9XicbVBNS8NAEJ3Ur1q / qh69LBahvdREBD1WvXgRItoPaGPZbDft0s0m7G6UEvo / vHhQxKv / xZv / xm2bg7Y + GHi8N8PMPD / mTGnb / rZyS8srq2v59cLG5tb2TnF3r6GiRBJaJxGPZMvHinImaF0zzWkrlhSHPqdNf3g18ZuPVCoWiXs9iqkX4r5gASNYG + nBLbe66d3FuHLslm8q3WLJrtpToEXiZKQEGdxu8avTi0gSUqEJx0q1HTvWXoqlZoTTcaGTKBpjMsR92jZU4JAqL51ePUZHRumhIJKmhEZT9fdEikOlRqFvOkOsB2rem4j / ee1EB + deykScaCrIbFGQcKQjNIkA9ZikRPORIZhIZm5FZIAlJtoEVTAhOPMvL5LGSdWxq87taal2mcWRhwM4hDI4cAY1uAYX6kBAwjO8wpv1ZL1Y79bHrDVnZTP78AfW5w8 + qZEH < / latexit > P ( X CIP ) / P ( M ) < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > < latexit sha1 _ base64 = " kybqNcnnCCNSL9HuNqhKhNza + 7I = " > AAAB + HicbVDLSsNAFL2pr1ofjbp0M1iEdlMTEXRZ7EYXQgT7gDaEyXTSDp08mJkINfRL3LhQxK2f4s6 / cdpmoa0HLhzOuZd77 / ETzqSyrG + jsLa + sblV3C7t7O7tl82Dw7aMU0Foi8Q8Fl0fS8pZRFuKKU67iaA49Dnt + OPmzO88UiFZHD2oSULdEA8jFjCClZY8s + xUu17WvHWmtTOnelfzzIpVt + ZAq8TOSQVyOJ751R / EJA1ppAjHUvZsK1FuhoVihNNpqZ9KmmAyxkPa0zTCIZVuNj98ik61MkBBLHRFCs3V3xMZDqWchL7uDLEayWVvJv7n9VIVXLkZi5JU0YgsFgUpRypGsxTQgAlKFJ9ogolg + lZERlhgonRWJR2CvfzyKmmf122rbt9fVBrXeRxFOIYTqIINl9CAG3CgBQRSeIZXeDOejBfj3fhYtBaMfOYI / sD4 / AFMqZGK < / latexit > P ( X SA & CIP ) / P ( M ) < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > < latexit sha1 _ base64 = " i3DMguNDzxT3oCqxYymn6zm96NQ = " > AAAB / HicbVDLSsNAFJ3UV62vaJduBovSbmoigi6r3ehCiGgf0IYwmU7q0MkkzEyEEOqvuHGhiFs / xJ1 / 47TNQlsPXDiccy / 33uPHjEplWd9GYWl5ZXWtuF7a2Nza3jF399oySgQmLRyxSHR9JAmjnLQUVYx0Y0FQ6DPS8UfNid95JELSiN + rNCZuiIacBhQjpSXPLDvVrpfdXfSPmtfOuHbsVG9qnlmx6tYUcJHYOamAHI5nfvUHEU5CwhVmSMqebcXKzZBQFDMyLvUTSWKER2hIeppyFBLpZtPjx / BQKwMYREIXV3Cq / p7IUChlGvq6M0TqQc57E / E / r5eo4NzNKI8TRTieLQoSBlUEJ0nAARUEK5ZqgrCg + laIH5BAWOm8SjoEe / 7lRdI + qdtW3b49rTQu8ziKYB8cgCqwwRlogCvggBbAIAXP4BW8GU / Gi / FufMxaC0Y + UwZ / YHz + AJoCksg = < / latexit > ����� ����������� ������� � �� ��� ��� ��� ��� ��� * * * * * * * * * * * * * * * * * * * * * * * * 100 N R < latexit sha1 _ base64 = " bQOMk915bYjo1epA5FN2ITtxrMM = " > AAAB9XicbVDLSgNBEJz1GeMr6tHLYBA8hVkR9Bj04kmimAck6zI76SRDZh / M9CphyX948aCIV / / Fm3 / jJNmDJhY0FFXddHcFiZIGGft2lpZXVtfWCxvFza3tnd3S3n7DxKkWUBexinUr4AaUjKCOEhW0Eg08DBQ0g + HVxG8 + gjYyju5xlIAX8n4ke1JwtNKDy1gnAOR + duPfjf1SmVXYFHSRuDkpkxw1v / TV6cYiDSFCobgxbZcl6GVcoxQKxsVOaiDhYsj70LY04iEYL5tePabHVunSXqxtRUin6u + JjIfGjMLAdoYcB2bem4j / ee0UexdeJqMkRYjEbFEvVRRjOomAdqUGgWpkCRda2lupGHDNBdqgijYEd / 7lRdI4rbis4t6elauXeRwFckiOyAlxyTmpkmtSI3UiiCbP5JW8OU / Oi / PufMxal5x85oD8gfP5A48 + keU = < / latexit > < latexit sha1 _ base64 = " bQOMk915bYjo1epA5FN2ITtxrMM = " > AAAB9XicbVDLSgNBEJz1GeMr6tHLYBA8hVkR9Bj04kmimAck6zI76SRDZh / M9CphyX948aCIV / / Fm3 / jJNmDJhY0FFXddHcFiZIGGft2lpZXVtfWCxvFza3tnd3S3n7DxKkWUBexinUr4AaUjKCOEhW0Eg08DBQ0g + HVxG8 + gjYyju5xlIAX8n4ke1JwtNKDy1gnAOR + duPfjf1SmVXYFHSRuDkpkxw1v / TV6cYiDSFCobgxbZcl6GVcoxQKxsVOaiDhYsj70LY04iEYL5tePabHVunSXqxtRUin6u + JjIfGjMLAdoYcB2bem4j / ee0UexdeJqMkRYjEbFEvVRRjOomAdqUGgWpkCRda2lupGHDNBdqgijYEd / 7lRdI4rbis4t6elauXeRwFckiOyAlxyTmpkmtSI3UiiCbP5JW8OU / Oi / PufMxal5x85oD8gfP5A48 + keU = < / latexit > < latexit sha1 _ base64 = " bQOMk915bYjo1epA5FN2ITtxrMM = " > AAAB9XicbVDLSgNBEJz1GeMr6tHLYBA8hVkR9Bj04kmimAck6zI76SRDZh / M9CphyX948aCIV / / Fm3 / jJNmDJhY0FFXddHcFiZIGGft2lpZXVtfWCxvFza3tnd3S3n7DxKkWUBexinUr4AaUjKCOEhW0Eg08DBQ0g + HVxG8 + gjYyju5xlIAX8n4ke1JwtNKDy1gnAOR + duPfjf1SmVXYFHSRuDkpkxw1v / TV6cYiDSFCobgxbZcl6GVcoxQKxsVOaiDhYsj70LY04iEYL5tePabHVunSXqxtRUin6u + JjIfGjMLAdoYcB2bem4j / ee0UexdeJqMkRYjEbFEvVRRjOomAdqUGgWpkCRda2lupGHDNBdqgijYEd / 7lRdI4rbis4t6elauXeRwFckiOyAlxyTmpkmtSI3UiiCbP5JW8OU / Oi / PufMxal5x85oD8gfP5A48 + keU = < / latexit > < latexit sha1 _ base64 = " bQOMk915bYjo1epA5FN2ITtxrMM = " > AAAB9XicbVDLSgNBEJz1GeMr6tHLYBA8hVkR9Bj04kmimAck6zI76SRDZh / M9CphyX948aCIV / / Fm3 / jJNmDJhY0FFXddHcFiZIGGft2lpZXVtfWCxvFza3tnd3S3n7DxKkWUBexinUr4AaUjKCOEhW0Eg08DBQ0g + HVxG8 + gjYyju5xlIAX8n4ke1JwtNKDy1gnAOR + duPfjf1SmVXYFHSRuDkpkxw1v / TV6cYiDSFCobgxbZcl6GVcoxQKxsVOaiDhYsj70LY04iEYL5tePabHVunSXqxtRUin6u + JjIfGjMLAdoYcB2bem4j / ee0UexdeJqMkRYjEbFEvVRRjOomAdqUGgWpkCRda2lupGHDNBdqgijYEd / 7lRdI4rbis4t6elauXeRwFckiOyAlxyTmpkmtSI3UiiCbP5JW8OU / Oi / PufMxal5x85oD8gfP5A48 + keU = < / latexit > FIG . S10 : Summary of Logit model parameter estimates . ( A - C ) Reported are 100 β for the main covariates of interest reported in Tables S1 - S3 , quantifying the percent increase in the odds Q ≡ P ( X ) / P ( M ) associated with a one - unit increases in : ( A ) mean journal citation impact z j , p ; ( C ) ln k ; ( B ) number of coauthors , k p ; ( C ) number of major MeSH terms ( keywords ) , w p ; ( D - F ) difference - in - difference estimates ( 100 δ R + ) capturing the effect of Flagship project ramp - ups after 2013 on rates of cross - domain research – at three levels of specificity regarding the diversity range captured by X . The Broad configuration correspond to unconstrained combinations of SA and CIP ( represented by X SA , X CIP , X SA & CIP ) . The Neighboring configuration corresponds to specific set of category combinations capturing the neurobiological - vs - bioengineering interface , represented by SA [ 1 ] × [ 2 - 4 ] and CIP [ 1 , 3 ] × [ 2 , 4 - 7 ] ( and represented by X Neighboring , SA , X Neighboring , CIP , X Neighboring , SA & CIP ) . And Distant also identifies a specific set of category combinations capturing the neuro - psycho - medical - vs - techno - computational interface , represented by SA [ 1 - 4 ] × [ 5 , 6 ] and CIP [ 1 , 3 , 5 ] × [ 4 , 8 ] ( X Distant , SA , X Distant , CIP , X Distant , SA & CIP ) . Reported are percent increase in Q , a ratio representing the propensity for cross - domain research relative to mono - domain research , directly associated with the ramp - up of Brain projects in : ( D ) Australasia ; ( E ) Europe ; ( F ) North America . Shown are point estimates with 95 % confidence interval . Standard errors clustered by region to account for residuals that are correlated within regions over time . Asterisks above each estimate indicate the associated p − value level : ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 . 19 A B - 120 - 120 N . America Europe Australasia x1000 x1000 x1000 % D M ean d i v e r s i t y ( SA C o - o cc u r en c e ) pe r a r t i c l e , ⟨ f D , S A ( t ) ⟩ ⟨ f D ⟩ = 0 . 45 [ randomized SA ] N . America [ randomized SA ] Europe [ randomized SA ] Australasia [ randomized SA ] % x1000 SA C o - O cc u rr en c e m a t r i x , P r e pe r i od ( 2009 – 2013 ) [ N e t s ha r e ] % C hange f r o m P r e t o P o s t [ % D i ff e r en c e ] ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� A R T I C L E T I T L E : R e d T e x t = N o t e s ; B l u e T e x t = t o b e upd a t e d ; R e v i s e d n o t a t i o n A u t ho r O n e a , c , 1 , A u t ho r T w o b , 1 , 2 , a nd A u t ho r Th r ee a a A f fi li a t i on O ne ; b A f fi li a t i on T w o ; c A f fi li a t i on T h r ee T h i s m anu sc r i p t w a s c o m p il ed on M a y 4 , 2020 A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A b s t r ac t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . C o n v e r g e n ce S c i e n ce | C r o ss - d i s c i p li n a r i t y | K e y w o r d | . . . V a r i a b l e d e fin i t i on s : 1 . S c o pu s a u t h o r i nd e x , a 2 . pub li c a t i o n i nd e x , p 3 . y e a r / t i m e , t ; pub li c a t i o n y e a r y p ; N ( t ) i s t h e t o t a l nu m b e r o f a r t i c l e s pub li s h e d i n y e a r t . 4 . S c o pu s c i t a t i o n s : c p , t i nd i c a t e s t h e nu m b e r o f c i t a t i o n s , c o un t e d t h r o u g h t h e d a t a ce n s u s y e a r 2019 , f o r a n a r t i c l e pub li s h e d i n y e a r t ; n o r m a li ze d c i t a t i o n s z p ( i n v o l v i n g µ t ) a nd ‡ t ) 5 . nu m b e r o f c oa u t h o r s , k p 6 . nu m b e r o f r e g i o n s a ss o c i a t e d w i t h a pub li c a t i o n , R p 7 . T h e a r t i c l e - l e v e l c a t e go r i c a l d i v e r s i t y m e a s u r e i s c a l c u - l a t e d u s i n g D p , t h e o u t e r - p r o du c t m a t r i x u s e d t o c o un t c o - o cc u rr e n ce s a t t h e a r t i c l e l e v e l , d r a w i n g o n t h e g e n e r i c c a t e go r i c a l v ec t o r ˛ v p = ≠ æ S A p o r ≠≠ æ C I P p 8 . Sup e r s c r i p t > ( s ub s c r i p t < ) i nd i c a t e d a t a c o ll ec t e d o v e r 5 - y e a r p e r i o d 2014 - 2018 ( 5 - y e a r p e r i o d 2009 - 2013 ) 9 . f i nd i c a t e s a g e n e r i c f r a c t i o n / f r e q u e n c y v a r i a b l e ( i . e . w i t h r a n g e [ 0 , 1 ] ) ; s ub s c r i p t i nd i c a t e s t h e v a r i a b l e c o n t e x t , e . g . f D , S A ( t ) ( f D , C I P ( t ) ) i nd i c a t e s t h e c o - o cc u rr e n ce p e r a r t i - c l e m e a s u r e d u s i n g S A ( C I P ) . 10 . C < S A , i j a nd C < C I P , i j : m a t r i ce s c o un t i n g c o - o cc u rr e n ce s ; 11 . C < S A , i j a nd C < C I P , i j : m a t r i ce s r e p o r t i n g % d i e r e n ce s i n c o - o cc u rr e n ce s b e t w ee n t h e 5 - y e a r p r e / p o s t p e r i o d s 12 . t h e s e t o f “ M a j o r T o p i c ” M e S H k e y w o r d s f o r a pub li c a t i o n , ˛ W p ; t h e S A o p e r a t o r c o n v e r t s a cc o r d i n g l y : O S A ( ˛ W p ) = ≠ æ S A p ; S i m il a r l y t h e s e t o f S c o pu s a u t h o r s o n a r e , ˛ A p a nd t h e C I P o p e r a t o r c o n v e r t s a s : O C I P ( ˛ A p ) = ≠≠ æ C I P p ; 13 . Sub j ec t A r e a c l u s t e r , S A ; t h e c o rr e s p o nd i n g S A v ec t o r o f a pub li c a t i o n , ≠ æ S A p 14 . D i s c i p li n a r y c l u s t e r , C I P ; t h e c o rr e s p o nd i n g C I P v ec t o r o f a pub li c a t i o n , ≠≠ æ C I P p 15 . E a c h a r t i c l e p h a s a s e t o f S A & C I P f e a t u r e s : ˛ F p © { ≠ æ S A p , ≠≠ æ C I P p } . 16 . F r o m t h e i n f o r m a t i o n c o n t a i n e d i n ˛ F p , e a c h a r t i c l e h a s h a s o n e o f t w o ( s c a l a r ) v a l u e s : c r o ss - d o m a i n ( d i v e r s e m i x t u r e ) = X , a s o pp o s e d t o m o n o - d o m a i n = M 17 . T h e t w o v a l u e s d e p e nd o n t h e o p e r a t o r a c t i n g o n ˛ F p : e . g . O C I P ( ˛ F p ) = M o r X C I P a nd O L o n g , C I P ( ˛ F p ) = M o r X L o n g , C I P 18 . R e g r e ss i o n v a r i a b l e s : i nd i c a t o r v a r i a b l e s i d e n t i fi e d u s i n g I ; s ub s c r i p t s i nd i c a t e t h e t y p e v a l u e c o rr e s p o nd i n g t o t h e v a l u e 1 , o t h e r w i s e t h e v a l u e = 0 ; E g . I X L o n g , C I P t a k e s o n t h e v a l u e 1 f o r a r t i c l e s w i t h O L o n g , C I P ( ˛ F p ) = X a nd 0 o t h e r w i s e 19 . s c h o l a r ag e a t t i m e o f pub li c a t i o n , · i , p 20 . i nd i c a t o r v a r i a b l e I 2014 + = 1 i f y p Ø 2014 a nd 0 o t h e r w i s e 21 . R e g i o n i nd i c a t o r v a r i a b l e R r = 1 i f a r t i c l e a u t h o r s a ss o - c i a t e d w i t h r e g i o n r a nd 0 o t h e r w i s e : R N A , R E U , R AA , R W o r l d ( t h i s c a t e go r y i s b a s e li n e i n T a b l e s S 1 - S 3 ) 22 . N R , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f r e g i o n s r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 4 23 . N S A , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f S A r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 6 24 . N C I P , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m b e r o f C I P r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a - t i o n s ) , i e m i n = 1 a nd m a x = 9 25 . z j i s t h e a v e r ag e z v a l u e c a l c u l a t e d a c r o ss a ll a r t i c l e s pub li s h e d i n a s p ec i fi c j o u r n a l ( i nd e x e d b y j ) S i gn i fi ca n ce S t a t e m e n t A u t ho r s m u s t s ub m i t a 120 - w o r d m a x i m u m s t a t e m en t abou t t he s i gn i fic an c e o f t he i r r e s ea r c h pape r w r i tt en a t a l e v e l unde r - s t andab l e t o an unde r g r adua t e edu c a t ed sc i en t i s t ou t s i de t he i r fi e l d o f s pe c i a li t y . T he p r i m a r y goa l o f t he s i gn i fic an c e s t a t e - m en t i s t o e x p l a i n t he r e l e v an c e o f t he w o r k i n b r oad c on t e x t t o a b r oad r eade r s h i p . T he s i gn i fic an c e s t a t e m en t appea r s i n t he pape r i t s e l f and i s r equ i r ed f o r a ll r e s ea r c h pape r s . P l ea s e p r o v i de de t a il s o f au t ho r c on t r i bu t i on s he r e . P l ea s e de c l a r e an y c o m pe t i ng i n t e r e s t s he r e . 1 A . O . ( A u t ho r O ne ) c on t r i bu t edequa l l y t o t h i s w o r k w i t h A . T . ( A u t ho r T w o ) ( r e m o v e i f no t app li c ab l e ) . 2 T o w ho m c o rr e s ponden c e s hou l d b e add r e ss ed . E - m a il : au t ho r . t w oe m a il . c o m www . pna s . o r g / c g i / do i / 10 . 1073 / pna s . XXXXXXXXXX P N AS | M ay 4 , 2020 | v o l . XXX | no . XX | 1 – 16 A R T I C L E T I T L E : R e d T e x t = N o t e s ; B l u e T e x t = t o b e upd a t e d ; R e v i s e d n o t a t i o n A u t ho r O n e a , c , 1 , A u t ho r T w o b , 1 , 2 , a nd A u t ho r Th r ee a a A f fi li a t i on O ne ; b A f fi li a t i on T w o ; c A f fi li a t i on T h r ee T h i s m anu sc r i p t w a s c o m p il ed on M a y 4 , 2020 A b s t r a c t o f no m o r e t h a n 250 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A b s t r ac t o f no m o r e t h a n 2 50 w o r d s i n a s i ng l e p a r a g r a ph . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . A ss d f a s d f as d f as d f as d f . A ss d f as d f as d f as d f as d f . C o n v e r g e n ce S c i e n ce | C r o ss - d i s c i p li n a r i t y | K e y w o r d | . . . V a r i a b l e d e fin i t i on s : 1 . S c o pu s a u t h o r i nd e x , a 2 . p u b li c a t i o n i nd e x , p 3 . y e a r / t i m e , t ; pub li c a t i o n y e a r y p ; N ( t ) i s t h e t o t a l nu m b e r o f a r t i c l e s pub li s h e d i n y e a r t . 4 . S c o pu s c i t a t i o n s : c p , t i nd i c a t e s t h e nu m b e r o f c i t a t i o n s , c o un t e d t h r o u g h t h e d a t a ce n s u s y e a r 2019 , f o r a n a r t i c l e p ub li s h e d i n y e a r t ; n o r m a li ze d c i t a t i o n s z p ( i n v o l v i n g µ t ) a n d ‡ t ) 5 . n u m b e r o f c oa u t h o r s , k p 6 . n u m b e r o f r e g i o n s a ss o c i a t e d w i t h a pub li c a t i o n , R p 7 . T h e a r t i c l e - l e v e l c a t e go r i c a l d i v e r s i t y m e a s u r e i s c a l c u - l a t e d u s i n g D p , t h e o u t e r - p r o du c t m a t r i x u s e d t o c o un t c o - o cc u rr e n ce s a t t h e a r t i c l e l e v e l , d r a w i n g o n t h e g e n e r i c c a t e go r i c a l v ec t o r ˛ v p = ≠ æ S A p o r ≠≠ æ C I P p 8 . S up e r s c r i p t > ( s ub s c r i p t < ) i nd i c a t e d a t a c o ll ec t e d o v e r 5 - y e a r p e r i o d 2014 - 2018 ( 5 - y e a r p e r i o d 2009 - 2013 ) 9 . f i nd i c a t e s a g e n e r i c f r a c t i o n / f r e q u e n c y v a r i a b l e ( i . e . w i t h r a n g e [ 0 , 1 ] ) ; s ub s c r i p t i nd i c a t e s t h e v a r i a b l e c o n t e x t , e . g . f D , S A ( t ) ( f D , C I P ( t ) ) i nd i c a t e s t h e c o - o cc u rr e n ce p e r a r t i - c l e m e a s u r e d u s i n g S A ( C I P ) . 10 . C < S A , i j a nd C < C I P , i j : m a t r i ce s c o un t i n g c o - o cc u rr e n ce s ; 11 . C S A , i j a nd C C I P , i j : m a t r i ce s r e p o r t i n g % d i e r e n ce s i n c o - o cc u rr e n ce s b e t w ee n t h e 5 - y e a r p r e / p o s t p e r i o d s 12 . t h e s e t o f “ M a j o r T o p i c ” M e S H k e y w o r d s f o r a pub li c a t i o n , ˛ W p ; t h e S A o p e r a t o r c o n v e r t s a cc o r d i n g l y : O S A ( ˛ W p ) = ≠ æ S A p ; S i m il a r l y t h e s e t o f S c o pu s a u t h o r s o n a r e , ˛ A p a nd t h e C I P o p e r a t o r c o n v e r t s a s : O C I P ( ˛ A p ) = ≠≠ æ C I P p ; 13 . Sub j ec t A r e a c l u s t e r , S A ; t h e c o rr e s p o nd i n g S A v ec t o r o f a pub li c a t i o n , ≠ æ S A p 14 . D i s c i p li n a r y c l u s t e r , C I P ; t h e c o rr e s p o nd i n g C I P v ec t o r o f a pub li c a t i o n , ≠≠ æ C I P p 15 . E a c h a r t i c l e p h a s a s e t o f S A & C I P f e a t u r e s : ˛ F p © { ≠ æ S A p , ≠≠ æ C I P p } . 16 . F r o m t h e i n f o r m a t i o n c o n t a i n e d i n ˛ F p , e a c h a r t i c l e h a s h a s o n e o f t w o ( s c a l a r ) v a l u e s : c r o ss - d o m a i n ( d i v e r s e m i x t u r e ) = X , a s o pp o s e d t o m o n o - d o m a i n = M 17 . T h e t w o v a l u e s d e p e nd o n t h e o p e r a t o r a c t i n g o n ˛ F p : e . g . O C I P ( ˛ F p ) = M o r X C I P a nd O L o n g , C I P ( ˛ F p ) = M o r X L o n g , C I P 18 . R e g r e ss i o n v a r i a b l e s : i nd i c a t o r v a r i a b l e s i d e n t i fi e d u s i n g I ; s ub s c r i p t s i nd i c a t e t h e t y p e v a l u e c o rr e s p o nd i n g t o t h e v a l u e 1 , o t h e r w i s e t h e v a l u e = 0 ; E g . I X L o n g , C I P t a k e s o n t h e v a l u e 1 f o r a r t i c l e s w i t h O L o n g , C I P ( ˛ F p ) = X a nd 0 o t h e r w i s e 19 . s c h o l a r ag e a t t i m e o f pub li c a t i o n , · i , p 20 . i nd i c a t o r v a r i a b l e I 2014 + = 1 i f y p Ø 2014 a nd 0 o t h e r w i s e 21 . R e g i o n i nd i c a t o r v a r i a b l e R r = 1 i f a r t i c l e a u t h o r s a ss o - c i a t e d w i t h r e g i o n r a nd 0 o t h e r w i s e : R N A , R E U , R AA , R W o r l d ( t h i s c a t e go r y i s b a s e li n e i n T a b l e s S 1 - S 3 ) 22 . N R , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f r e g i o n s r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 4 23 . N S A , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m - b e r o f S A r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a t i o n s ) , i e m i n = 1 a nd m a x = 6 24 . N C I P , p a r t i c l e - l e v e l c o un t v a r i a b l e i nd i c a t i n g t h e t o t a l nu m b e r o f C I P r e p r e s e n t e d ( i nd e p e nd e n t o f c o n ce n t r a - t i o n s ) , i e m i n = 1 a nd m a x = 9 25 . z j i s t h e a v e r ag e z v a l u e c a l c u l a t e d a c r o ss a ll a r t i c l e s pub li s h e d i n a s p ec i fi c j o u r n a l ( i nd e x e d b y j ) S i gn i fi ca n ce S t a t e m e n t A u t ho r s m u s t s ub m i t a 120 - w o r d m a x i m u m s t a t e m en t abou t t he s i gn i fic an c e o f t he i r r e s ea r c h pape r w r i tt en a t a l e v e l unde r - s t andab l e t o an unde r g r adua t e edu c a t ed sc i en t i s t ou t s i de t he i r fi e l d o f s pe c i a li t y . T he p r i m a r y goa l o f t he s i gn i fic an c e s t a t e - m en t i s t o e x p l a i n t he r e l e v an c e o f t he w o r k i n b r oad c on t e x t t o a b r oad r eade r s h i p . T he s i gn i fic an c e s t a t e m en t appea r s i n t he pape r i t s e l f and i s r equ i r ed f o r a ll r e s ea r c h pape r s . P l ea s ep r o v i dede t a il s o f au t ho r c on t r i bu t i on s he r e . P l ea s ede c l a r ean yc o m pe t i ng i n t e r e s t s he r e . 1 A . O . ( A u t ho r O ne ) c on t r i bu t edequa ll y t o t h i s w o r k w i t h A . T . ( A u t ho r T w o ) ( r e m o v e i f no t app li c ab l e ) . 2 T o w ho m c o rr e s ponden c e s hou l dbeadd r e ss ed . E - m a il : au t ho r . t w oe m a il . c o m www . pna s . o r g / c g i / do i / 10 . 1073 / pna s . XXXXXXXXXX P N AS | M ay 4 , 2020 | v o l . XXX | no . XX | 1 – 16 C # SA pe r a r t i c l e , s i gna l - t o - no i s e r a t i o [ i n v e r s e c oe f fic i en t o f v a r i a t i on ] ���� ���� ���� ���� ���� ���� ��� ��� ��� ��� ˜ µ S A ( t ) < latexit sha1 _ base64 = " HY / uCpzMCC3PQiJtGGQWW / nGxU4 = " > AAAB / HicbVBNS8NAEN34WetXtUcvwSLUS0lE0GPVi8eK9gOaEDabTbt0swm7EyGE + Fe8eFDEqz / Em / / GbZuDtj4YeLw3w8w8P + FMgWV9Gyura + sbm5Wt6vbO7t5 + 7eCwp + JUEtolMY / lwMeKciZoFxhwOkgkxZHPad + f3Ez9 / iOVisXiAbKEuhEeCRYygkFLXq3uAOMBzZ0oLbz8 / qpowqlXa1gtawZzmdglaaASHa / 25QQxSSMqgHCs1NC2EnBzLIERTouqkyqaYDLBIzrUVOCIKjefHV + YJ1oJzDCWugSYM / X3RI4jpbLI150RhrFa9Kbif94whfDSzZlIUqCCzBeFKTchNqdJmAGTlADPNMFEMn2rScZYYgI6r6oOwV58eZn0zlq21bLvzhvt6zKOCjpCx6iJbHSB2ugWdVAXEZShZ / SK3own48V4Nz7mrStGOVNHf2B8 / gCj55S9 < / latexit > < latexit sha1 _ base64 = " HY / uCpzMCC3PQiJtGGQWW / nGxU4 = " > AAAB / HicbVBNS8NAEN34WetXtUcvwSLUS0lE0GPVi8eK9gOaEDabTbt0swm7EyGE + Fe8eFDEqz / Em / / GbZuDtj4YeLw3w8w8P + FMgWV9Gyura + sbm5Wt6vbO7t5 + 7eCwp + JUEtolMY / lwMeKciZoFxhwOkgkxZHPad + f3Ez9 / iOVisXiAbKEuhEeCRYygkFLXq3uAOMBzZ0oLbz8 / qpowqlXa1gtawZzmdglaaASHa / 25QQxSSMqgHCs1NC2EnBzLIERTouqkyqaYDLBIzrUVOCIKjefHV + YJ1oJzDCWugSYM / X3RI4jpbLI150RhrFa9Kbif94whfDSzZlIUqCCzBeFKTchNqdJmAGTlADPNMFEMn2rScZYYgI6r6oOwV58eZn0zlq21bLvzhvt6zKOCjpCx6iJbHSB2ugWdVAXEZShZ / SK3own48V4Nz7mrStGOVNHf2B8 / gCj55S9 < / latexit > < latexit sha1 _ base64 = " HY / uCpzMCC3PQiJtGGQWW / nGxU4 = " > AAAB / HicbVBNS8NAEN34WetXtUcvwSLUS0lE0GPVi8eK9gOaEDabTbt0swm7EyGE + Fe8eFDEqz / Em / / GbZuDtj4YeLw3w8w8P + FMgWV9Gyura + sbm5Wt6vbO7t5 + 7eCwp + JUEtolMY / lwMeKciZoFxhwOkgkxZHPad + f3Ez9 / iOVisXiAbKEuhEeCRYygkFLXq3uAOMBzZ0oLbz8 / qpowqlXa1gtawZzmdglaaASHa / 25QQxSSMqgHCs1NC2EnBzLIERTouqkyqaYDLBIzrUVOCIKjefHV + YJ1oJzDCWugSYM / X3RI4jpbLI150RhrFa9Kbif94whfDSzZlIUqCCzBeFKTchNqdJmAGTlADPNMFEMn2rScZYYgI6r6oOwV58eZn0zlq21bLvzhvt6zKOCjpCx6iJbHSB2ugWdVAXEZShZ / SK3own48V4Nz7mrStGOVNHf2B8 / gCj55S9 < / latexit > < latexit sha1 _ base64 = " HY / uCpzMCC3PQiJtGGQWW / nGxU4 = " > AAAB / HicbVBNS8NAEN34WetXtUcvwSLUS0lE0GPVi8eK9gOaEDabTbt0swm7EyGE + Fe8eFDEqz / Em / / GbZuDtj4YeLw3w8w8P + FMgWV9Gyura + sbm5Wt6vbO7t5 + 7eCwp + JUEtolMY / lwMeKciZoFxhwOkgkxZHPad + f3Ez9 / iOVisXiAbKEuhEeCRYygkFLXq3uAOMBzZ0oLbz8 / qpowqlXa1gtawZzmdglaaASHa / 25QQxSSMqgHCs1NC2EnBzLIERTouqkyqaYDLBIzrUVOCIKjefHV + YJ1oJzDCWugSYM / X3RI4jpbLI150RhrFa9Kbif94whfDSzZlIUqCCzBeFKTchNqdJmAGTlADPNMFEMn2rScZYYgI6r6oOwV58eZn0zlq21bLvzhvt6zKOCjpCx6iJbHSB2ugWdVAXEZShZ / SK3own48V4Nz7mrStGOVNHf2B8 / gCj55S9 < / latexit > 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Information Science N . America EuropeAustralasia FIG . S11 : Co - occurrence metrics appropriately account for secular growth in research output and MeSH annotation . Demonstration of consistent co - occurrence metrics calculated using randomized category vectors , (cid:126)v p , rand . , with vector elements shuffled such that the total counts | (cid:126)v p | for each p is conserved . ( A ) Elimination of variation among the diagonal and off - diagonal elements of the co - occurrence matrix C < SA , rand . indicate that no other significant statistical biases underly this co - occurrence calculation . ( B ) Reduction of the relative change between two shuffled co - occurence matrices C < SA , rand . and C > SA , rand . to the level of noise ; The largest off - diagonal value observed is 3 % , representing a threshold for classifying significant shifts in the corresponding real data shown in Figs . S2 ( D ) and S3 ( D ) . ( C ) Increase in the signal - to - noise ratio ˜ µ SA ( t ) = µ SA ( t ) / σ SA ( t ) , measuring the average number of SA per article ( µ SA ) , normalized by the standard deviation ( σ SA ) . Increase in the number of SA ( and MeSH ) per article is a source of secular growth that could introduce temporal bias challenging the interpretation of the results . ( D ) Accounting for this secular growth of ˜ µ SA ( t ) yields a mean diversity per article (cid:104) f D , SA , rand . which is approximately constant over time , consistent with the expected results for randomized category vectors , (cid:126)v p , rand . . 20 N e i ghbo r i ng D i s t a n t A CIP CIP SA SA CIP SA M CIP ⇄ M SA ∆ XM = ( X ⇄ X ) − ( M ⇄ M ) X CIP ⇄ X SA Difference 4 C I P — SA c oup li ng Cross - domain articles Mono - domain articles 3 1 4 2 6 7 5 8 9 1 2 4 5 3 1 4 2 6 7 5 8 9 1 2 3 4 5 3 1 4 267 5 8 9 1 2 3 4 5 B r o a d 3 1 4 2 6 7 5 8 9 1 2 3 4 5 3 1 4 2 6 7 5 8 9 1 2 3 4 5 1 4 2 6 7 5 8 9 1 2 B C 3 1 4 2 6 7 5 8 9 1 2 3 4 3 1 4 2 6 7 5 8 9 2 4 5 3 1 4 2 6 7 5 8 9 2 5 C I P — SA c oup li ng C I P — SA c oup li ng 1 : Psychiatry & Psychology 2 : Anatomy & Organisms 3 : Phenomena & Processes 4 : (cid:1) Health 5 : Techniques & Equipment 6 : Technology & Inform . Science 1 : Neurosciences 2 : Biology 3 : Psychology 4 : (cid:1) Biotech . & Genetics 5 : Medical Specialty 6 : Health Sciences 7 : Pathology & Pharmacology 8 : Eng . & Informatics 9 : Chemistry & Physics & Math CIP : SA : � � � � � � [ 1 , 3 ] [ 2 , 4 - 7 ] [ 1 , 3 , 5 ] [ 4 , 8 ] 1 2 3 4 5 6 7 8 9 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � � � � � � 1 [ 2 - 4 ] [ 1 - 4 ] [ 5 , 6 ] � � � � � � � � � � � � � � � � � � � � � 1 2 3 4 5 6 FIG . S12 : Cross - domain CIP - SA coupling . ( A ) Broad configuration ( as also shown in Fig . 3B , reproduced here to facilitate visual comparison ) . ( B ) Neighboring . ( C ) Distant . The first column illustrates mono - mono coupling , calculated using only the mono - domain articles ( M ) . For this case , the bi - partite CIP - SA networks are rather consistent across each configuration , indicating a common baseline for comparison across configurations . The second column shows the CIP - SA coupling network calculated using only the cross - domain articles ( X SA & CIP ) . The third column shows the difference between the corresponding mono - and cross - domain networks in each row . As such , comparison across any two networks in the third column corresponds to a difference - in - difference . Comparison of ∆ XM across configurations facilitates a qualitative difference - in - difference for identifying emergent cross - domain coupling particular of a given configuration . Regarding the Neighboring configuration relative to the Broad configuration , we observe over - representation of the links between CIP [ 2 , 4 - 9 ] and the Psychiatry & Psychology domain SA [ 1 ] , and to a lesser degree Engineering & Informatics CIP [ 8 ] and Biology SA [ 2 ] . Likewise , in the case of the Distant configuration , the over - represented links are CIP [ 2 , 4 , 7 ] with the Techniques & Equipment SA [ 5 ] . 21 TABLE S1 : Modeling the prevalence of cross - domain activity at the article level . Article - level analysis implemented using the logit model . The dependent variable is a binary indicator variable taking the value 1 if the article features cross - domain combinations ( represented by X SA , p or X CIP , p or X SA & CIP , p ) and 0 otherwise . Publication data included : articles published in period y p ∈ [ 1970 , 2018 ] with k p ≥ 2 and w p ≥ 2 . Robust standard errors are shown in parenthesis below each point estimate . Reported are odds ratios , exp ( β ) . ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) X SA X CIP X SA & CIP X SA X CIP X SA & CIP y 1 . 032 ∗∗∗ 1 . 009 ∗∗∗ 1 . 046 ∗∗∗ 1 . 033 ∗∗∗ 1 . 021 ∗∗∗ 1 . 061 ∗∗∗ ( 0 . 000866 ) ( 0 . 00242 ) ( 0 . 00339 ) ( 0 . 00118 ) ( 0 . 00315 ) ( 0 . 00394 ) z j 0 . 997 1 . 282 ∗∗∗ 1 . 415 ∗∗∗ 0 . 978 1 . 223 ∗ 1 . 308 ∗∗ ( 0 . 0298 ) ( 0 . 0911 ) ( 0 . 0805 ) ( 0 . 0320 ) ( 0 . 103 ) ( 0 . 126 ) ln k 0 . 885 ∗∗∗ 1 . 753 ∗∗∗ 1 . 562 ∗∗∗ 0 . 897 ∗∗∗ 1 . 821 ∗∗∗ 1 . 639 ∗∗∗ ( 0 . 0184 ) ( 0 . 109 ) ( 0 . 124 ) ( 0 . 0142 ) ( 0 . 117 ) ( 0 . 140 ) ln w 4 . 655 ∗∗∗ 0 . 933 ∗ 4 . 858 ∗∗∗ 4 . 678 ∗∗∗ 0 . 929 ∗ 4 . 896 ∗∗∗ ( 0 . 144 ) ( 0 . 0285 ) ( 0 . 217 ) ( 0 . 150 ) ( 0 . 0286 ) ( 0 . 215 ) N R 1 . 324 ∗∗∗ 7 . 810 ∗∗∗ 12 . 00 ∗∗∗ 1 . 211 ∗∗ 3 . 028 ∗∗∗ 4 . 569 ∗∗∗ ( 0 . 0807 ) ( 0 . 928 ) ( 2 . 096 ) ( 0 . 0755 ) ( 0 . 789 ) ( 0 . 898 ) N CIP 1 . 307 ∗∗∗ 1 . 294 ∗∗∗ ( 0 . 0828 ) ( 0 . 0818 ) N SA 1 . 216 ∗∗∗ 1 . 206 ∗∗∗ ( 0 . 0476 ) ( 0 . 0487 ) I 2014 + 0 . 949 0 . 754 ∗∗∗ 0 . 716 ∗∗∗ ( 0 . 0348 ) ( 0 . 0420 ) ( 0 . 0367 ) I R NA 0 . 913 0 . 380 ∗∗∗ 0 . 397 ∗∗∗ ( 0 . 0680 ) ( 0 . 103 ) ( 0 . 0755 ) I R EU 0 . 942 0 . 313 ∗∗∗ 0 . 345 ∗∗∗ ( 0 . 0712 ) ( 0 . 0849 ) ( 0 . 0669 ) I R AA 0 . 746 ∗∗∗ 0 . 229 ∗∗∗ 0 . 191 ∗∗∗ ( 0 . 0567 ) ( 0 . 0651 ) ( 0 . 0386 ) I R NA × I 2014 + 1 . 074 ∗ 1 . 007 1 . 012 ( 0 . 0352 ) ( 0 . 0186 ) ( 0 . 0218 ) I R EU × I 2014 + 0 . 955 1 . 038 ∗ 0 . 938 ∗∗ ( 0 . 0313 ) ( 0 . 0186 ) ( 0 . 0192 ) I R AA × I 2014 + 1 . 111 ∗∗ 0 . 898 ∗∗∗ 0 . 950 ∗ N 602599 602599 207281 602599 602599 207281 Pseudo R 2 0 . 0725 0 . 1945 0 . 3081 0 . 0735 0 . 2045 0 . 3191 Exponentiated coefficients ; Standard errors in parentheses ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 22 TABLE S2 : Conditional definition of X p – identifying “Neighboring” or shorter - distance cross - domain combinations . Article - level analysis implemented using the logit model . The dependent variable is a binary indicator variable taking the value 1 if the article features cross - domain combinations ( represented by X Neighboring , SA , p or X Neighboring , CIP , p or X Neighboring , SA & CIP , p ) and 0 otherwise . Publication data included : articles published in period y p ∈ [ 1970 , 2018 ] with k p ≥ 2 and w p ≥ 2 . Robust standard errors are shown in parenthesis below each point estimate . Reported are odds ratios , exp ( β ) . ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) X Neighboring , SA X Neighboring , CIP X Neighboring , SA & CIP X Neighboring , SA X Neighboring , CIP X Neighboring , SA & CIP y 1 . 030 ∗∗∗ 1 . 002 1 . 025 ∗∗∗ 1 . 028 ∗∗∗ 1 . 012 ∗∗ 1 . 036 ∗∗∗ ( 0 . 00243 ) ( 0 . 00267 ) ( 0 . 00284 ) ( 0 . 00294 ) ( 0 . 00378 ) ( 0 . 00513 ) z j 1 . 488 ∗∗∗ 1 . 344 ∗∗∗ 1 . 765 ∗∗∗ 1 . 428 ∗∗∗ 1 . 266 ∗∗∗ 1 . 646 ∗∗∗ ( 0 . 0855 ) ( 0 . 0929 ) ( 0 . 122 ) ( 0 . 0746 ) ( 0 . 0757 ) ( 0 . 0954 ) ln k 0 . 530 ∗∗∗ 1 . 755 ∗∗∗ 1 . 132 0 . 543 ∗∗∗ 1 . 832 ∗∗∗ 1 . 188 ( 0 . 0362 ) ( 0 . 131 ) ( 0 . 145 ) ( 0 . 0346 ) ( 0 . 133 ) ( 0 . 154 ) ln w 1 . 756 ∗∗∗ 0 . 889 ∗∗∗ 1 . 816 ∗∗∗ 1 . 788 ∗∗∗ 0 . 889 ∗∗∗ 1 . 799 ∗∗∗ ( 0 . 0992 ) ( 0 . 0304 ) ( 0 . 106 ) ( 0 . 0828 ) ( 0 . 0237 ) ( 0 . 0888 ) N R 1 . 763 ∗∗∗ 6 . 297 ∗∗∗ 8 . 424 ∗∗∗ 1 . 853 ∗∗∗ 2 . 617 ∗∗ 4 . 071 ∗∗∗ ( 0 . 181 ) ( 0 . 845 ) ( 1 . 485 ) ( 0 . 268 ) ( 0 . 867 ) ( 1 . 679 ) N CIP 1 . 429 ∗∗ 1 . 415 ∗∗ ( 0 . 172 ) ( 0 . 171 ) N SA 1 . 230 ∗∗∗ 1 . 215 ∗∗∗ ( 0 . 0526 ) ( 0 . 0529 ) I 2014 + 1 . 029 0 . 770 ∗∗∗ 0 . 795 ∗∗ ( 0 . 0560 ) ( 0 . 0478 ) ( 0 . 0653 ) I R NA 1 . 122 0 . 405 ∗ 0 . 511 ( 0 . 182 ) ( 0 . 151 ) ( 0 . 225 ) I R EU 1 . 192 0 . 327 ∗∗ 0 . 404 ∗ ( 0 . 202 ) ( 0 . 121 ) ( 0 . 179 ) I R AA 0 . 626 ∗∗ 0 . 172 ∗∗∗ 0 . 156 ∗∗∗ ( 0 . 110 ) ( 0 . 0651 ) ( 0 . 0700 ) I R NA × I 2014 + 1 . 053 1 . 040 ∗ 1 . 009 ( 0 . 0481 ) ( 0 . 0187 ) ( 0 . 0418 ) I R EU × I 2014 + 1 . 044 1 . 114 ∗∗∗ 1 . 035 ( 0 . 0481 ) ( 0 . 0163 ) ( 0 . 0429 ) I R AA × I 2014 + 1 . 274 ∗∗∗ 1 . 081 ∗∗∗ 1 . 210 ∗∗∗ ( 0 . 0578 ) ( 0 . 0187 ) ( 0 . 0475 ) N 602599 602599 430801 602599 602599 430801 Pseudo R 2 0 . 0496 0 . 1716 0 . 1919 0 . 0554 0 . 1837 0 . 2041 Exponentiated coefficients ; Standard errors in parentheses ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 23 TABLE S3 : Conditional definition of X p – identifying “Distant” or longer - distance cross - domain combinations . Article - level analysis im - plemented using the logit model . The dependent variable is a binary indicator variable taking the value 1 if the article features cross - domain combinations ( represented by X Distant , SA , p or X Distant , CIP , p or X Distant , SA & CIP , p ) and 0 otherwise . Publication data included : articles pub - lished in period y p ∈ [ 1970 , 2018 ] with k p ≥ 2 and w p ≥ 2 . Robust standard errors are shown in parenthesis below each point estimate . Reported are odds ratios , exp ( β ) . ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) X Distant , SA X Distant , CIP X Distant , SA & CIP X Distant , SA X Distant , CIP X Distant , SA & CIP y 1 . 033 ∗∗∗ 1 . 017 ∗∗∗ 1 . 043 ∗∗∗ 1 . 036 ∗∗∗ 1 . 035 ∗∗∗ 1 . 071 ∗∗∗ ( 0 . 000769 ) ( 0 . 00445 ) ( 0 . 00451 ) ( 0 . 00174 ) ( 0 . 0101 ) ( 0 . 00955 ) z j 0 . 635 ∗∗∗ 1 . 210 0 . 838 0 . 624 ∗∗∗ 1 . 127 0 . 750 ∗∗ ( 0 . 0253 ) ( 0 . 141 ) ( 0 . 0837 ) ( 0 . 0202 ) ( 0 . 119 ) ( 0 . 0659 ) ln k 0 . 867 ∗∗∗ 1 . 740 ∗∗∗ 1 . 289 ∗ 0 . 879 ∗∗∗ 1 . 861 ∗∗∗ 1 . 403 ∗ ( 0 . 0331 ) ( 0 . 166 ) ( 0 . 166 ) ( 0 . 0318 ) ( 0 . 178 ) ( 0 . 185 ) ln w 2 . 258 ∗∗∗ 0 . 918 2 . 584 ∗∗∗ 2 . 261 ∗∗∗ 0 . 894 2 . 496 ∗∗∗ ( 0 . 114 ) ( 0 . 111 ) ( 0 . 211 ) ( 0 . 115 ) ( 0 . 105 ) ( 0 . 167 ) N R 1 . 107 4 . 594 ∗∗∗ 5 . 094 ∗∗∗ 0 . 986 1 . 652 ∗ 1 . 924 ∗∗ ( 0 . 0627 ) ( 1 . 235 ) ( 1 . 476 ) ( 0 . 0549 ) ( 0 . 340 ) ( 0 . 432 ) N CIP 1 . 181 ∗∗∗ 1 . 169 ∗∗∗ ( 0 . 0204 ) ( 0 . 0204 ) N SA 1 . 183 1 . 171 ( 0 . 114 ) ( 0 . 115 ) I 2014 + 0 . 871 ∗∗∗ 0 . 735 ∗ 0 . 648 ∗∗ ( 0 . 0130 ) ( 0 . 105 ) ( 0 . 0991 ) I R NA 0 . 872 ∗ 0 . 378 ∗∗∗ 0 . 450 ∗∗∗ ( 0 . 0494 ) ( 0 . 0957 ) ( 0 . 100 ) I R EU 0 . 894 0 . 123 ∗∗∗ 0 . 132 ∗∗∗ ( 0 . 0537 ) ( 0 . 0306 ) ( 0 . 0298 ) I R AA 0 . 725 ∗∗∗ 0 . 188 ∗∗∗ 0 . 130 ∗∗∗ ( 0 . 0460 ) ( 0 . 0505 ) ( 0 . 0305 ) I R NA × I 2014 + 1 . 063 ∗∗∗ 0 . 860 0 . 842 ( 0 . 0106 ) ( 0 . 0900 ) ( 0 . 120 ) I R EU × I 2014 + 1 . 031 ∗∗ 1 . 228 ∗ 1 . 039 ( 0 . 0104 ) ( 0 . 125 ) ( 0 . 144 ) I R AA × I 2014 + 1 . 118 ∗∗∗ 0 . 646 ∗∗∗ 0 . 711 ∗ ( 0 . 0106 ) ( 0 . 0643 ) ( 0 . 0968 ) N 602599 602599 396471 602599 602599 396471 Pseudo R 2 0 . 0375 0 . 1492 0 . 1474 0 . 0496 0 . 1716 0 . 1919 Exponentiated coefficients ; Standard errors in parentheses ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 24 TABLE S4 : Career - level analysis using panel model with individual researcher fixed effects . Publication data included : articles published in period y p ∈ [ 1970 , 2018 ] with k p ≥ 2 and w p ≥ 2 ; only includes researchers with N a ≥ 10 articles satisfying these criteria . Robust standard errors are shown in parenthesis below each point estimate . Y indicates additional fixed effects included in the regression model . ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) ( 6 ) z p z p z p z p z p z p ln k 0 . 415 ∗∗∗ 0 . 416 ∗∗∗ 0 . 421 ∗∗∗ 0 . 438 ∗∗∗ 0 . 424 ∗∗∗ 0 . 406 ∗∗∗ ( 0 . 00470 ) ( 0 . 00468 ) ( 0 . 00469 ) ( 0 . 00551 ) ( 0 . 00547 ) ( 0 . 00527 ) ln w 0 . 0320 ∗∗∗ 0 . 0376 ∗∗∗ 0 . 0379 ∗∗∗ 0 . 0244 ∗∗∗ 0 . 0544 ∗∗∗ 0 . 0385 ∗∗∗ ( 0 . 00468 ) ( 0 . 00465 ) ( 0 . 00466 ) ( 0 . 00615 ) ( 0 . 00528 ) ( 0 . 00536 ) τ - 0 . 0107 ∗∗∗ - 0 . 0106 ∗∗∗ - 0 . 0105 ∗∗∗ - 0 . 0115 ∗∗∗ - 0 . 00987 ∗∗∗ - 0 . 0101 ∗∗∗ ( 0 . 00148 ) ( 0 . 00148 ) ( 0 . 00149 ) ( 0 . 00229 ) ( 0 . 00170 ) ( 0 . 00157 ) I X SA 0 . 0480 ∗∗∗ ( 0 . 00367 ) I X CIP 0 . 0691 ∗∗∗ ( 0 . 00466 ) I X Neighboring , SA 0 . 0878 ∗∗∗ ( 0 . 00461 ) I X Neighboring , CIP 0 . 0675 ∗∗∗ ( 0 . 00496 ) I X Distant , SA - 0 . 00993 ∗∗ ( 0 . 00376 ) I X Distant , CIP 0 . 0205 ∗ ( 0 . 0102 ) I X SA & CIP 0 . 132 ∗∗∗ ( 0 . 00728 ) I X Neighboring , SA & CIP 0 . 132 ∗∗∗ ( 0 . 00886 ) I X Distant , SA & CIP 0 . 0424 ∗∗ ( 0 . 0158 ) constant - 0 . 719 ∗∗∗ - 0 . 719 ∗∗∗ - 0 . 736 ∗∗∗ - 0 . 677 ∗∗∗ - 0 . 781 ∗∗∗ - 0 . 711 ∗∗∗ ( 0 . 0541 ) ( 0 . 0540 ) ( 0 . 0543 ) ( 0 . 0770 ) ( 0 . 0547 ) ( 0 . 0583 ) Year ( y ) dummy Y Y Y Y Y Y Topic category ( −→ SA ) dummy Y Y Y Y Y Y Department category ( −−→ CIP ) dummy Y Y Y Y Y Y Region ( −→ R ) dummy Y Y Y Y Y Y N 825147 825147 825147 358237 552254 527347 adj . R 2 0 . 102 0 . 102 0 . 101 0 . 131 0 . 090 0 . 093 F 265 . 7 262 . 0 251 . 4 231 . 3 198 . 5 193 . 5 # researcher profiles ( df r + 1 ) 8448 8448 8448 8422 8435 8441 Standard errors in parentheses ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 25 TABLE S5 : Flagship Project effect : Career - level analysis using panel model with researcher fixed effects . Publication data included : articles published in period y p ∈ [ 1970 , 2018 ] with k p ≥ 2 and w p ≥ 2 ; only includes researchers with N a ≥ 10 articles satisfying these criteria . Robust standard errors are shown in parenthesis below each point estimate . Y indicates additional fixed effects included in the regression model . ( 1 ) ( 2 ) ( 3 ) z p z p z p ln k 0 . 438 ∗∗∗ 0 . 425 ∗∗∗ 0 . 406 ∗∗∗ ( 0 . 00551 ) ( 0 . 00547 ) ( 0 . 00528 ) ln w 0 . 0250 ∗∗∗ 0 . 0546 ∗∗∗ 0 . 0385 ∗∗∗ ( 0 . 00616 ) ( 0 . 00528 ) ( 0 . 00536 ) τ - 0 . 0108 ∗∗∗ - 0 . 00965 ∗∗∗ - 0 . 00878 ∗∗∗ ( 0 . 00255 ) ( 0 . 00189 ) ( 0 . 00175 ) I 2014 + 0 . 0137 0 . 00818 - 0 . 0571 ∗∗∗ ( 0 . 0160 ) ( 0 . 0133 ) ( 0 . 0111 ) I X SA & CIP 0 . 155 ∗∗∗ ( 0 . 00796 ) I X SA & CIP × I 2014 + - 0 . 0884 ∗∗∗ ( 0 . 00985 ) I X Neighboring , SA & CIP 0 . 182 ∗∗∗ ( 0 . 00973 ) I X Neighboring , SA & CIP × I 2014 + - 0 . 160 ∗∗∗ ( 0 . 0103 ) I X Distant , SA & CIP 0 . 0276 ( 0 . 0180 ) I X Distant , SA & CIP × I 2014 + 0 . 0432 ∗ ( 0 . 0180 ) constant - 0 . 666 ∗∗∗ - 0 . 777 ∗∗∗ - 0 . 690 ∗∗∗ ( 0 . 0729 ) ( 0 . 0515 ) ( 0 . 0559 ) Year ( y ) dummy Y Y Y Topic category ( −→ SA ) dummy Y Y Y Department category ( −−→ CIP ) dummy Y Y Y Region ( −→ R ) dummy Y Y Y N 358237 552254 527347 adj . R 2 0 . 131 0 . 091 0 . 093 F 229 . 0 198 . 6 191 . 3 # researcher profiles ( df r + 1 ) 8422 8435 8441 Standard errors in parentheses ∗ p < 0 . 05 , ∗∗ p < 0 . 01 , ∗∗∗ p < 0 . 001 \ No newline at end of file diff --git a/silver_data/ac17a6b27853121149064a811b56e88e43d4e463.pdf.txt b/silver_data/ac17a6b27853121149064a811b56e88e43d4e463.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..32beb70615b182a62bcfd6a80531eab406f806ed --- /dev/null +++ b/silver_data/ac17a6b27853121149064a811b56e88e43d4e463.pdf.txt @@ -0,0 +1 @@ +Twitter Democracy : Policy versus identity politics in three emerging African democracies Michael L . Best Sam Nunn School of International Affairs and School of Interactive Computing Georgia Institute of Technology Atlanta , GA USA mikeb @ cc . gatech . edu Amanda Meng Sam Nunn School of International Affairs Georgia Institute of Technology Atlanta , GA USA a . meng @ gatech . edu ABSTRACT Social media offers new ways for citizens to discuss and debate politics and engage in the democratic process . These online systems could be places for rich policy relevant debate , which is favored by scholars of deliberative democracy . Alternatively social media might be a platform for an identity driven form of political discourse that is routinely scorned by scholars of democracy . To examine these two possibilities , we analyzed tweets sent during three national elections , the defining participatory process of democracy . Our dataset includes over 760 , 000 tweets gathered during national elections in Nigeria , Ghana and Kenya from 2011 to 2013 . In order to analyze the degree to which Twitter was being used for policy relevant discussion we developed policy term sets through a text analysis of the major political party platforms . To examine the amount of discourse focused on identity issues we created identity term sets based upon national religious , tribal , and regional differences . In Nigeria , where divisive identity politics feed violence and electoral misconduct , discussion of tribe , region , and religion dominate mentions of platform policies . In contrast Ghanaians , who enjoy the most robust democracy of the three countries , were seven times more likely to discuss policy issues rather than identity . Kenyan democracy is still undergoing consolidation , and tweets again reflect this , with almost as many tweets devoted to tribal identity as campaign policy . These findings suggest that social media discussions may echo the state of democratic deepening found in a country during its national elections . Keywords Democracy , social media , Twitter , Africa , elections , text analysis . 1 . 1 . INTRODUCTION Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee pr ovided that copies are not made o r distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for components of this work owned by others than ACM must be honored . Abstracting with credit is permitted . To cop y other wise , or republish , to post on servers or to redistribute to lists , requires prior specific permission and / or a fee . Request permissions from permissions @ acm . org ICTD’15 , May 15 – 18 , 2015 , Singapore , Singapore . Copyright 2015 ACM 978 - 1 - 4503 - 3163 - 0 / 15 / 05 … $ 15 . 00 . http : / / dx . doi . org / 10 . 1145 / 2737856 . 2738017 During the 2013 National Elections in Kenya , one Kenyan tweeted a question to fellow citizens : “ The big question is when will ethnic - based politics end in Kenya ? When issues will guide voters and not just ethnicity . # kenyadecides ” . Kenya , like many emerging African democracies , can count the number of recent democratic elections on one hand . And as common with other ( young ) democracies , Kenyans are working to create robust political discourse among its electorate . According to most democratic theories , prior to marking ink on a ballot , successful elections require rich discourse among citizens and the articulation of a party vision and policy agenda communicated through a candidate ’ s campaign platform . However , as the tweet above from Kenya suggests , discussion of policy is often overwhelmed by the politics of identity . So while some Kenyans cry out for substantive democratic discourse , others concentrate instead on identity . In fact , another Kenyan on Twitter declares : “ I ' ll never ever trust kikuyu … fuck you kikuyuz fuck you . ” This massively retweeted post demonstrates the depth of the mistrust in the Kikuyu tribe among some Kenyans and positions discrimination and suppression as a leading component of political expression . Elections provide a unique opportunity to contrast policy oriented democratic discourse with identity based politics . By evaluating social media collected around election days , this paper attempts to investigate this discourse in three emerging democracies in Africa : the Nigeria 2011 Presidential Election , Ghana 2012 National Election , and Kenya 2013 National Election . Theories ( e . g . Saco 2002 ) suggest that the relative intensities of discussion in these three countries over social media offer an indication of whether citizens are prioritizing identity or policy discourse in their electoral processes . Are citizens engaged in a substantive discussion of policies and party platforms over social media characteristic of deliberative democracy ? Or do identity politics of tribe , religion , or region dominate online discussion ? In this paper we report an analysis of Twitter data from these three elections . Using a text analysis system , we queried each dataset for policy relevant material which we initially had mined from each country ’ s major political party platform . Similarly , we searched the corpus for identity keywords which we acquired by listing the names of major ethno - linguistic groups , religions , and regional territories . We find variation between these three countries in the prevalence of policy versus identity based discourse over Twitter and this variation aligns with the level of political development in each county as determined by scholars of democracy . The next section outlines theories of identity politics versus deliberative democracy . The third section situates this paper in related works and theories . The fourth section gives background on the three countries ’ political landscape prior to the tracked elections . Section five describes the dataset . The analysis methods and tools are outlined in section six . The seventh section reports findings and offers our conclusions . 1 . 2 . DELIBERATIVE DEMOCRACY AND IDENTITY POLITICS How individuals and groups voice political issues and effect governance within a democracy related to the normative values of citizenship , inclusion , and participation . The concept that democracy is most legitimate when citizens engage in debate and reflection on policy is known as deliberative democracy ( Bohman and Rehg 1996 ) . Contemporary normative views of democracy draw from the works of scholars including Cohen ( 1996 ) , Habermas ( 1996 ) , and Christiano ( 2008 ) . Cohen ( 1996 ) evokes rational choice theory to explain how public reasoning allows decision makers to overcome information asymmetry by collective intelligence and thus bring reason and legitimacy to policy . Similarly , Habermas ( 1996 ) contends that democratic authority rests in “ communicative reason , ” or the public contemplation and preparation of political decisions . But Habermas brings a perhaps more radical notion ; he describes a civil society , autonomous from democratic processes , that can resist , organize , and influence democratic legislation . Christiano ( 2008 ) introduces the notion of social justice as an outcome of deliberation . He advocates deliberative democracy through public democratic decision making as a way to achieve social justice . Bringing this normative view of democracy to the developing world , Patrick Heller ( 2000 ) asserts that an effective democracy has two interrelated characteristics : a robust civil society and a capable state . Heller ’ s robust civil society echoes the Habermasian civil society . According to Heller , civil society is made up of individuals , organizations , and social movements that hold the state accountable through ongoing expression of voice , feedback , and negotiation . When democratic institutions and social processes are mutually reinforcing , democratic deepening occurs . Identity politics incorporates individuals into political action based on a constructed identity that could be rooted in religion , ethnicity , or gender ( Heyes 2012 ) . While identity can be beneficially integrated into deliberative democratic practices , political theorist John Dryzek ( 2005 ) has shown that identity politics will actually weaken effective deliberation and democratic deepening when it is validated or constituted as suppression of one identity by another . Deliberative democracy is made more elusive when political elites bring citizens into the political process through clientelism premised upon identity ( Crawford & Lynch 2012 ) . The top - down structure of clientelism limits citizens ’ power to negotiate ( Powell 1970 ) . Accordingly , political elites who gain support and votes through handouts and identity are often uninterested in democratic consultation or deliberation of policy with citizens . As political parties claim subordinate classes through clientelism based on identity politics , society fails to achieve democratic deepening through expansion of social citizenship ( Heller 2000 ) . Voters receive favors , not voice . With new networked technologies , social media offers a space for institutions and civil society to dynamically interact . Citizens can use the 140 - character tweet to reason through campaign platforms and advocate for certain candidates and their policies . They may hashtag a political party , tweet at a candidate , or advocate for particular policy outcomes . Alternatively , social media platforms can be used to amplify identity based politics that focus on race , tribe , religion , and region . This paper uses elections , the foremost democratic institution , and the microblogging platform , Twitter , as an opportunity and artifact to evaluate how public discourse over social media reflects practice of democratic deliberation or divisive identity politics in Nigeria , Ghana , and Kenya . 1 . 3 . RELATED WORKS AND THEORY Since the rise of social media , many scholars model and debate the use of online platforms as public spaces for political discourse during election time ( Gayo - Avello et al . 2011 ; Jungherr et al . 2012 ; Boutet et al . 2012 ; Stieglitz and Dang - Xuan 2012 ) . Tumasjan et al . ( 2010 ) find evidence that Twitter is a space for political deliberation in a dataset from the month leading up to the 2009 German national elections . The authors probe a set of 104 , 003 political tweets using a linguistic inquiry and word count ( LIWC ) software . Results show that despite a limited user group , the tweets reflect German political realities in terms of election outcomes , emerging political coalitions , voter sentiment and voter policy preference . Echoing these finding , Stieglitz and Dang - Xuan ( 2012 ) use the LIWC software and regression analysis to model Twitter discussions and sentiment during German state parliament elections . Results indicate political discussions do occur over Twitter , and articulated sentiment has an impact on whether a statement is retweeted . Stieglitz and Dang - Xuan ( 2012 ) also find tweets are used for information sharing , direct communication , and found election discourse over Twitter to be dominated by a smaller number of influential users . Despite this presence of a smaller number of highly active users , political tweets were still reflective of election outcomes . Many studies have questioned the use of Twitter to describe political outcomes during elections . Junglier et al ( 2012 ) find fault with the method of data selection and analysis used by Tumasjan et al . Their evaluation questions how tweets were collected , the exclusion of a key political party , and the arbitrary time frame of analysis . Including a Pirate Party query and expanding the time frame by even one day would significantly alter the conclusions reached by Tumasjan et al . In “ Limits of Electoral Predictions Using Twitter , ” Gayo - Avello et al . ( 2011 ) further elaborate the debate on how natural language processing of tweets reveals political election realities . The authors use two datasets from the United States ’ congressional elections and two textual analysis methods , frequency count and sentiment analysis , to evaluate the claim that Twitter can be used to predict elections . Results find both methods to be unreliable . Frequency count only correctly predicts winners half of the time . Sentiment analysis only slightly outperforms a random classifier in predicting voter preferences . Moving beyond election predictions , Boutet et al . ( 2012 ) , in a case study of the United Kingdom 2010 general election , substantiate a commonly held belief that Twitter is an echo chamber . Users were more likely to reference their own political party and retweets were highly segregated by political affiliation . Their paper also finds that certain users show preference for news sources based on political association . In addition to describing Twitter behaviors during election time , the authors test and compare classification methods , finding the Bayesian - Volume classifier to out - perform a Bayesian - Volume classifier , Bayesian - Retweet classifier , and a Support Vector Machine classifier in accuracy . In Cybering Democracy ( 2002 ) , Diana Saco theorizes what new forms of democratic sociality are made possible by new technologies . Using seminal theories of Lefebvre , Ardent , Barber , Habermas , etc . , Saco identifies the touch points of technology and civics in the practice of democracy . She argues that cyberspace is not like all spaces , but it is an “other space” where mediated and meaningful social interaction can occur ( xxv ) . Saco would extend Lefebvre ’ s definition of ( social ) space as a ( social ) production to the Twitter cyber space . As micro - bloggers tweet their experiences and expectations on election day , Twitter becomes a lived space that reflects social and political realities . Saco also reminds readers of the technical definition of virtual . She finds evidence that the virtual space of Twitter is a simulation of the democratic process ; therefore , election tweets should represent the social and political systems of identity and policy on election day in three emerging African democracies . While she does not argue that any reciprocal duties of communicative and deliberative democratic practice are evident over the Twitter dataset , her theory would support the claim that tweets do offer a cross - section of evidence towards democratic deepening . 1 . 4 . COUNTRY POLITICAL BACKGROUND Nigeria , Ghana , and Kenya are recently developing democracies . For Nigerians , the 2011 election was their third election since a democratic transition in 1999 . Unhappily , these Nigerian elections have been plagued by violence and electoral misconduct designed to influence voting ( Arowolo and Aluko 2012 ) . Indeed , for many scholars , Nigeria is not considered a full democracy as election outcomes are often a product of electoral fraud , violence , and elite negotiation ( Moller and Skaaning 2013 ) . During the presidential elections in April , 2011 , violence erupted in the North in response to the victory of the presidential incumbent , Goodluck Jonathan . Jonathan of the People ’ s Democratic Party ( PDP ) , a Christian from the South of the country , had as his major challenger Buhammadu Buhari , of the Congress of Progressive Change ( CPC ) and a Muslim from the North . The practice of closed power negotiation , as well as intermittent violence and coups , are rooted in the post - colonial elites ’ inability to construct inclusive citizenship ( Abubakar 2000 ) . Regional identity , derived from both religion and ethnicity , plays a dominant role in the Nigerian political sphere . Ghana is considered the most stable democracy among these three country comparators . The 2012 National Election was the fifth time Ghanaians went to the polls to elect a president and parliamentary representatives . Historically , tribe , colonial legacy and uneven development have shaped and given power to identity in Ghana . But as Paul Nugent ( 2001 ) has explained , unlike Nigeria or Kenya , Ghana does not have the presence of large or dominant super tribes . ⁠ 1 As early as the 2000 elections , Ghanaians began to move away from identity politics to vote for a candidate with political commitments to change , particularly with anti - corruption platforms . For the 2012 election , political analysts forecasted a close race between incumbent John Mahama of the National Democratic Congress ( NDC ) and Nana Akufo - Addo of the National People ’ s Party ( NPP ) . Even given Ghana ’ s recent history , a close and high - stakes election could lead to candidates exhibiting opportunistic strategies of clientelism based on identity and , accordingly , discussion of identity politics could emerge . Ethnicity has played a central role in Kenyan politics since independence . Daniel arap Moi dominated Kenyan politics as president from 1978 to 2002 . While Kenyans went to the polls to vote during his twenty - four year reign , violence and electoral misconduct kept Moi in power ( Bratton and Kimenyi 2008 ) . In particular , during the nineties elections erupted in violence as political elites mobilized Kenyans to vote along ethnic lines . The 2002 election was noted as the most free and fair election in the nation ’ s history and similar to Ghana , Kenyans demonstrated a move towards democratic deepening as opposed to identity politics when Mwai Kibaki ’ s coalition government , made up of many ethnic groups , won ( The Carter Center 2003 ) . Five years later , in the 2007 elections , the Kenyan Human Rights Commission ( 2011 ) reported that post - election civil unrest and violence resulted in the deaths of at least 1 , 300 Kenyans . According to the Commission , this violence was fueled by inter - ethnic cleavages and the identity politics of the two rival presidential candidates . Leading up to the 2013 elections , communities began arming themselves out of concerns of ethnic based violence most notably between supporters of the two leading candidates : Uhuru Kenyatta , a Kikuyu of the JUBILEE coalition and Raila Odinga , a Luo with the CORD coalition . The three elections examined in this study represent the first time these countries have had national elections in the presence of substantial use of social media platforms . We hypothesize that the historical realities of these three new democracies , overviewed above , will inform their citizens ’ use of social media during the elections under consideration . Table 1 : Election Data Summary Country Election Date Data Acquisition Time Span Twitter Reports Nigeria Presidential Apr 16 , 2011 April 15 7 : 00 AM - April 19 7 : 00 AM 192 , 142 Ghana General Dec 7 , 2012 Dec 6 7 : 00 AM - Dec 10 7 : 00 AM 320 , 789 Kenya General Mar 4 , 2013 March 3 7 : 00 AM - March 7 7 : 00 AM 254 , 216 Totals 767 , 147 5 . 5 . DATA Data from Twitter was accumulated during national elections in Nigeria , Ghana , and Kenya . The dates of this data acquisition , along with other details of the data , are summarized in Table 1 . The time spans for data acquisition start one day prior to the election , and includes election day and two days following . The data was accumulated using a social media aggregator tool , named Aggie , developed by the Technologies and International Development ( TID ) Lab at Georgia Tech . Aggie is designed to accumulate reports across multiple social media platforms ( e . g . Facebook , Twitter , Google + ) , as well as RSS syndicated sites , and support real - time analysis , credibility checking , and response . Aggie has been successfully deployed in multiple elections including the three detailed in this paper . In order to collect Twitter content focused on the election at hand , subject matter experts from each country developed a list of specific queries , listed in Table 2 , that were then used to accumulate the tweet dataset via the public Twitter API . Subject matter experts , who were members of collaborating civil society groups with extensive knowledge in social media and Nigerian elections , received training in how Aggie collects social media reports and exercised autonomy in choosing queries . For this study , data originating from sources other than Twitter was removed , leaving just over 765 , 000 tweets across the three elections . Table 2 : Queries Used to Gather Election Relevant Discussion over Twitter Country Queries Nigeria voting materials , ballot box , nigeria , corpers , inec , jega , nigeriadecides , nigeriaelection , nigeria vote , ballot box snatch , ballot box thugs , stuffing ballot box , voters register , violence lga , bomb nigeria , abuja , anambra , enugu , akwa ibom , adamawa , bauchi , bayelsa , benue , borno , cross river , ebonyi , ekiti , gombe , jigawa , kaduna , kano , katsina , kebbi , kogi , kwara , lagos , nasarawa , niger , ogun , ondo , osun , oyo state , plateau state , rivers state , sokoto , taraba , yobe , zamfara , polling unit , plessyahand , pressyahand , buhari Ghana ghana , ghanadecides , ghanaelection , NPP , NDC , ghana ballot snatching , ghana hate speech , ghana bloodshed , ghana police brutality , ghana minors voting , ghana war mongering , Kumasi , Manhyia , Bawku Central , Odododiodoo , Ashaiman , Okaikoi Central , Akwatia , Atiwa , western region , brong ahafo , tamale ghana , CPP , # PPP , CODEO , all die be die , mahama , nadaa , Akufo Addo , nduom , ghana EC , afari - gyan , ghana polling agent , ghana poll , ghana poll closed , ghana spoiled ballot , ghana spoilt ballot , ghana count , ghana registered , ghana not registered , ghana rejected , thumbprint , ede bee keke , ghana long queues , ballot stuffing , delayed voting , machomen , cutlass , double voting , ink ballot , verification vote , verification ghana , biometric verification , late closing , light off ghana , police headquarters , npp office , ndc office , presiding officer , zongo , machete ghana , machete elections , returning officer , election fighting , fax results , @ JDMahama , @ Nadaa2012 , @ joyonlineghana , @ spyghana , @ pkndoum , polling agents , Ghana rig , Ghana thieves , Ghana steal , Ghana stole , cheating Ghana , only in Ghana , save this country , preventing Ghana , ballot attack Ghana , do or die Ghana , looting Ghana , riot Ghana , ghana motorbikes , Ketu , voting materials , ghana mp , ghana death , ashanti region , eastern region , ghana results , ghana counts , blackout ghana , black out ghana , black out count , blackout count , petrol bomb kumasi , recount ghana , recount elections , credible results , credible result , credible election , disputed result , certified result , mensah Kenya kenyaelection , KenyaDecides , kenyadecides2013 , kenyadecides13 , kenyaelections , kenyaelections2013 , kenyaelections13 , uchaguzi , uchaguzi254 , uchaguzibora , uchaguzisivita , uchaguzi2013 , elections2013 , Ke2013 , KeDecides13 , KeElections2013 , KeElections13 , nipeukweli , choice2013 , decision2013 , Odinga , Kenyatta , Ruto , Karua , Dida , Muite , Kiyiapi , Mudavadi , Maamuzi2013 Twitter is being used in these three countries by an expanding subset of people . While theories suggest that Twitter discussions will echo the general political discourses , it is clear that social media users in Africa represent a relative elite subset and so it is possible that Twitter data will be similarly biased . This paper does not try to generalize outside of the set of Twitter users and their discourses virtual and physical . However as social media use continues to grow , Twitter discourse will increasingly reflect most voices . Estimates of Twitter use range from 1 % ( 1 . 8 million ) in Nigeria ( Schoonderwoerd , 2013 ) to 3 – 4 . 5 % ( up to 2 . 1 million ) in Kenya ( Kemibaro , 2014 ) . Estimates are not available for Ghana . 6 . 6 . METHODS After each field deployment of the Aggie system , data ( summarized in Table 1 ) was gathered and organized for offline access within a standard SQL database . We then performed text analysis , transformation , clustering , and visualization of the data using the Luminoso system . Luminoso is an analytics and information visualization software for natural language processing developed at the MIT Media Lab ( Speer , Havasi , Treadway & Liberman 2010 ) . Going beyond standard text analysis , Luminoso augments the textual dataset with common sense knowledge embedded in the ConceptNet semantic network . It then reduces the extraordinarily high dimensionality of this data space via singular value decomposition . Documents can then be visualized and manipulated as points within this dimensionality - reduced space . The two - step process , adding in a semantic network and then the reduction of dimensionality , has the effect of surfacing top concepts salient to the corpus . A concept might be a commonly repeated word ( e . g . “ vote ” or “ constituencies ” ) , or a set of co - occurring words ( e . g . “ provisional results ” or “ let voters decide ” ) , or it might even be a set of words that do not directly co - occur in the study corpus but instead hold strong connections available through the semantic network . Luminoso also allows users to study the corpus through queries of one or more words combined via disjunction or conjunction . These queries are not dissimilar to an advanced Google search . Luminoso reports a percentage of the documents in the corpus that are both exact and conceptual ( via the semantic network ) matches to the query . For example , we might query the corpus around religion by searching for the word “ Christian ” or the word “ Muslim . ” Documents returned may contain exact matches of the word “ Christian ” or “ Muslim ” as well as tweets with words conceptually related through the semantic network such as “ church , ” or “ mosque . ” We use this query tool to extract from the dataset Twitter content indicative of deliberative democracy or of identity - focused discourse . Political parties in Africa outline their campaign promises and policies in published documents called a manifesto ; party manifestos are the most uniform and accessible material describing a candidate ’ s campaign platform . For example , while a manifesto for each candidate in each election could be found and downloaded , media coverage or speeches made by candidates was not uniformly available . Party manifestos therefore served as a surrogate for the overall candidate platforms for the elections we studied . We acquired the party manifestos of the top two candidates running in each election . To systematically determine manifesto key terms , we removed stop words and generated bi - grams using the online platform , Lextutor . Stop words are extremely common words ( such as “ the ” or “ but ” ) that offer little semantic value . A bi - gram is a string of two immediately co - located words in a text . Table 3 . Manifest Bi - Gram and Identity Topic Term Queries Country Candidate , Party Manifesto Bi - Gram Topic Terms Nigeria Goodluck Jonathan , PDP iron steel , party government , human rights , public office , foreign policy , traditional rulers , nigerian culture , civil servants , encourage private , rule law , sustainable development , private entrepreneurs , police force , water transportation , rural areas , natural mineral , scientific technology , local production , primary education , participatory democracy Buhamma du Buhari , CPC private sector , amend constitution , science technology Ghana John Mahama , NDC national development , private sector , oil gas , basic education , social protection , job creation , creative industry , infrastructure development , long term , social compact , natural resources , technical vocational , security services , water resources , economic development , health care , youth sports , shea nut , traditional medicine , school children Nana Akufo - Addo , NPP private sector , national development , economic growth , rural areas , transformation programme , good governance , quality education , road network , armed forces , long term , transformation agenda , fight corruption , raw materials , human rights , science technology , national youth , anti - corruption , development planning , natural resources Kenya Uhuru Kenyatta , JUBILEE economic growth , county governments , coalition government , county level , renewable energy , coalition partners , young people , private sector , natural resources , community land , economic development , water supply , secondary schools , power supply , subsistence farming , vision 2030 , tax incentives , primary school , transformation leadership , security forces Raila Odinga , CORD private sector , county governments , health care , infrastructure development Country Identity Topic Terms Nigeria tribe , hausa , fulani , yoruba , igbo , ibo , south , north , east , west , Christian , Muslim , Boko Haram Ghana tribe , akan , mole - dagbon , ewe , ga - dangme , gurma , Ashanti , Brong Ahafo , Central , Easter , Greater Accra , Northern , Upper East , Upper West , Volta , Western , Christian , Muslim Kenya kikuyu , luhya , luo , kalenjin , kamba , central , coast , masai , eastern , nairobi , north - eastern , nyanza , rift valley , Christian , Muslim With stop words removed , bi - gram analysis returned a list of the most frequently mentioned word pairs which we found generally related to policy issues . The top of Table 3 provides a list of the ( at most ) twenty common bi - grams for each manifesto . We include all bi - grams mentioned at least three times in the manifesto and due to the short length of some manifestos , not all documents yielded twenty top bi - grams . In our analysis , these top word pairs from the party manifesto , or our policy term sets , are used as surrogate indicators of policy relevant content among the tweets . To complement our policy relevant term sets , we also need to develop a collection of queries that will indicate identity focused discussions . We developed these identity term sets by simply listing the terms associated with the relevant tribal or ethnic groups , religion , and geopolitical region specific to each of the three nations ( Table 3 bottom ) . The five largest tribal groups listed in the CIA World Fact Book constituted the ethnic identity terms . Christian and Muslim were included as religious identity terms . Geopolitical regions were sourced from the countries ’ administrative boundary terms . Using Luminoso , we extracted discussion of policy and identity in the Twitter dataset by applying the policy and identity term sets as queries . These queries return two valuable statistics , a count of exact and conceptual ( semantic network ) matches among the tweets along with the percentage of the tweets that contain the exact and conceptual matches . Luminoso also provides a sample of fifty tweets that match the query . Upon inspection we found that conceptual matches ( via the semantic network ) were not always relevant . For this reason , the samples of fifty tweets were hand tested for relevancy . For example , the bi - gram “ quality education ” was mentioned seven times in the Ghanaian National People ’ s Party manifesto and thus this word pair was one of our policy term sets . A query for “ quality education ” returned exact matches as well as conceptual matches of discussion on education policy ; discussion of free tuition to secondary school , or Senior High School was very prevalent . These tweets may not include the exact term “ quality education , ” but instead “ SHS ” or “ 4yrs , ” which are terms often tweeted by individuals discussing education policy , specifically the NPP ’ s promise of four years of free Senior High School . In this case , Luminoso properly identified conceptual matches to a policy query . In contrast , the policy query “ iron steel , ” a bi - gram repeated seven times in the Nigerian People ’ s Democratic Party manifesto , resulted in exact and conceptual matches of irrelevant mentioning of metals and minerals of no pertinence to the election . For policy queries , matches were hand - marked as relevant if they pertained to a discussion of policy , campaign platforms , or change expected with the election . We classified each of the fifty tweets as relevant or irrelevant and in Table 4 ( top ) we report the percentage of the hand tested sample deemed relevant to policy discourse . Identity queries were classified as relevant if they pertained to a specific ethnic , religious or regional groups ’ voting or a certain party or candidate aligning with a certain ethnic , religious , or regional association . Even a tweet advocating against identity politics such as voting or political participation based on religious or ethnic association was considered relevant because it still indicates discussion of identity as opposed to policy . Only tweets with an exact match are reported for ethnic and geopolitical queries because our hand inspection revealed that Luminoso returned consistently unrelated conceptual matches , particularly those related to tribal names . If more than half of the sample returned by a particular query can be classified as relevant , we report it as a policy or identity based discussed by citizens during the election . Finally , we compute an estimated projection of the total number of relevant tweets per topic by multiplying the relevance percentage score times the number of exact and conceptual matches . 7 . 7 . FINDINGS 1 . 7 . 1 . Nigerians on Twitter Only two of twenty - three policy queries returned any tweets in the Nigeria dataset . The two policy queries that did return tweets ( “ participatory democracy ” and “ primary education ” ) were only barely relevant , with fifty and fifty - seven percent of the hand tested sample coded as relevant to the policy terms . “ Participatory democracy , ” which came from Goodluck Jonathan ’ s manifesto , did reveal an intense discussion around democracy . This democratic fervor was not specifically related to Goodluck Jonathan , but instead discussed support for good governance generally post - election and what one tweeter referred to as “ real democracy . ” The second policy topic , “ primary education , ” also came from the party manifesto of Goodluck Jonathan . Compared to Ghana and Kenya , the Nigeria Twitter set returned the least number of tweets related to their political party manifestos . In contrast to policy discussions , the number of tweets returned based on identity queries show that Nigerian citizens engaged in considerable discussions around issues of identity . Indeed , overall the projected number of tweets discussing identity is double the number of tweets of policy queries ( 4014 versus 8323 ) . Accordingly , Nigerians on Twitter were twice as likely to discuss identity over policy during election time . 1 . 7 . 2 . Kenyans on Twitter The Kenyan dataset showed a slightly greater intensity of relevant policy discussion than the Nigerian data set . Compared to the two policy queries that returned relevant tweets from the Nigeria data set , three policy queries returned relevant discussion in the Kenyan tweets . The three relevant policy term sets for Kenya are “ economic growth , ” “ transformational leadership , ” and “ vision 2030 . ” All three of these bi - grams are from the Jubilee Alliance or TNA party manifesto , whose presidential candidate was Uhuru Kenyatta , who went on to win the election . Of these three , Kenyans on Twitter primarily discussed the policy term sets “ economic growth ” and “ transformational leadership . ” Upon inspection of the relevant tweets , we noticed mostly a general desire for future economic growth and stronger political leadership . These general calls for economic or political change are similar to tweets from the Nigerian election . In Kenya , ethnic identity queries , not religious or geopolitical , were the only identity based queries to return relevant results . Similar to the Ghanaian results discussed below , the geopolitical queries only returned tweets and re - tweets of voting tabulation results by region and some tweets indicated party strongholds or a clear expectation of which candidates would carry which regions . In comparison to the policy topics , Kenyan identity based discussions are much quieter and much less intense than Nigeria ’ s . 1 . 7 . 3 . Ghanaians on Twitter In the Ghanaian Twitter dataset , six policy term sets derived from candidate manifestos returned relevant tweets . The six relevant policy queries are , “ quality education , ” “ fight corruption , ” “ infrastructure development , ” “ school children , ” “ economic development , ” and “ good governance . ” The NPP actively promoted education , specifically free tuition for Senior High School , as a major priority of their campaign platform . Policy relevant discussion of educational policy over Twitter often focused on this issue , e . g . “ Think abt other people . . . the fact dat u rich doesnt mean free shs [ Senior High School ] isnt necessary . Those kids u see outta sch [ ool ] need to go back so vote npp . ” We estimate that the Ghana dataset has over twenty - nine thousand tweets discussing educational issues . Just this one policy query has five times more estimated tweets associated with it compared to all of the policy discussion found in the Kenyan dataset . In contrast , a very small portion of the Ghanaian tweets show discussion of tribal identity . The Ewe and the Ashanti speaking Akan tribes were most commonly mentioned . Most of the geopolitical queries returned matches of election tabulation results and re - tweets of vote counts by region . However , a search for the Volta Region alone returned a largely re - tweeted statement that made a joke of how the NPP would have to change their party symbol in order to gain votes in the region . ⁠ 2 Religious identity does not seem to play a prominent role in the election oriented Twitter - sphere in Ghana , as religious terms did not return election relevant tweets . Overall , in Ghana policy discussions were much more prevalent than identity politics . 6 . 8 . DISCUSSION AND CONCLUSIONS Table 5 below summarizes the relative degree of policy and identity discourse occurring over twitter for the three national elections . We estimate that 13 % of the Ghanaian dataset is policy relevant compared with just 2 % of Nigeria and Kenya . And while Ghana and Kenya had roughly similar percentages of identity tweets in their datasets ( 2 % ) , Nigeria had twice as much identity - focused discourse ( 4 % ) . Scholars of democracies , Moller and Skaaning ( 2013 ) , have developed a dataset , which categorizes regime types during the recent wave of state democratization . According to their work , Nigeria and Kenya are classified as multiparty autocracies . Table 4 : Policy and Identity Query Results Count ry Policy Term Set Hand Coded Releva nce Numbe r of Exact Matche s Number of Concept ual Matches Percent of Total Country Data Projected Number of Relevant Tweets Nigeri a Participatory democracy 50 % 728 4 , 744 2 . 90 % 2 , 736 Primary education 57 % 421 1 , 821 1 . 20 % 1 , 278 total 4 , 014 Ghana Quality education 100 % 191 22 , 992 7 . 20 % 23 , 183 Fight corruption 100 % 1 , 407 297 0 . 50 % 1 , 704 Infrastructure development 77 % 441 1 , 493 0 . 60 % 1 , 489 School children 71 % 2 , 165 4 , 854 2 . 20 % 4 , 983 Economic development 65 % 31 3 , 244 1 . 30 % 2 , 129 Good governance 55 % 9 , 364 5 , 141 4 . 5 % 7 , 978 total 41 , 466 Kenya Economic growth 88 % 31 3 , 244 1 . 30 % 2 , 882 Transformational leadership 84 % 252 2 , 034 0 . 90 % 1 , 920 Vision 2030 63 % 83 1 , 706 0 . 70 % 1 , 127 total 5 , 929 Count ry Identity Term Set Nigeri a tribe , hausa , fulani , yoruba , igbo , ibo , ijaw , kanuri , ibibio , tiv 90 % 1 , 198 NA 0 . 63 % 1 , 078 South , North 100 % 4 , 861 NA 2 . 56 % 4 , 861 Christian , Muslim 100 % 1 , 229 1 , 155 1 . 30 % 2 , 384 total 8 , 323 Ghana tribe , Akan , mole - dagbon , ewe , ga - dangme , gurma , grusi , mande - busanga 96 % 200 NA 0 . 06 % 192 Ashanti , Brong Ahafo , Central , Easter , Greater Accra , Northern , Upper East , Upper West , Volta , Western 29 % 2 , 669 NA 0 . 83 % 774 Volta alone 100 % 4 , 606 NA 1 . 40 % 4 , 606 Christian , Muslim 57 % 140 793 0 . 30 % 532 total Kenya kikuyu , luhya , luo , kalenjin , kamba , kisii , meru 94 % 1 , 404 NA 0 . 55 % 1 , 320 Central , Coast , Masai , Eastern , Nairobi , North - eastern , Nyanza , Rift Valley 35 % 7 , 786 NA 3 . 06 % 2 , 725 Christian , Muslim 29 % 31 1 , 097 0 . 40 % 327 total 4 , 327 Table 5 . Country Comparison of Political Discussion versus Identity Discussion Country Projected Number of Tweets Relevant to Policy Terms Policy Percentage of Dataset Projected Number of Tweets Relevant to Identity Terms Identity Percentage of Dataset Nigeria 4 , 014 2 . 09 % 8 , 323 4 . 33 % Ghana 41 , 466 12 . 93 % 6 , 104 1 . 90 % Kenya 5 , 929 2 . 33 % 4 , 372 1 . 72 % Moller and Skaaning describe a multiparty autocracy as a regime with weak civil liberties and political rights indicators but with more than one party represented in government . And while elections are held , they are not considered truly competitive . This may help explain why policy discourse is less intense in the Nigerian and Kenyan Twitter dataset . Elections may appear to be free and fair , but outcomes are known before ballots are cast . Citizens are perhaps less likely to debate and defend a policy stance when elections feel rigged . Nigerians on Twitter seem to be aware of falling short of full democracy and indicate fervor for democratic deepening . Instead of discussing specific policies , Nigerians share beliefs on the quality of democracy and governance . For example , one Nigerian tweets , “ We can pretend bad governance does not affect us , we are cool people . But as long as u r alive , bad governance affects u ! # NigeriaDecides . ” Another tweet states , “ Jonathan in high spirit . Nigeria cannot afford to impose on herself a non - democratic person when her citizens are hungry for food & freedom . ” The following tweet demonstrates an excitement for the democratic process , “ In my family , everyone . Is voting different candidates ! Now that ' s democracy . . . : D # Nigeriadecides . ” Despite these and many other examples of calls for democratic deepening , discussion of democracy in Nigeria and Kenya is dominated by discussion of ethnic , geopolitical , and religious identity . Ghanaians on Twitter are much more engaged in discussions around campaign platforms . This finding is perhaps expected due to Ghana ’ s higher ranking of democracy . Moller and Skaaning ( 2013 ) classify Ghanaian democracy as a polyarchy , a democracy with legitimate elections where citizens also enjoy political rights and civil liberties . The relatively greater intensity of political discussion may be due to active Ghanaian political parties . Many researchers highlight the role of political parties in supporting a robust democracy ( Key 1958 , Stokes 1999 , Aldrich 1995 , Linz and Stepan 1996 ) . During election time , parties do the important work of translating the public ’ s demands and priorities into a policy agenda . The Ghanaian ’ s desire for access to education , revealed on Twitter , is indeed reflected in the promise of free tuition , which was a position of the NPP . And as was also true for Kenya and Nigeria , the top two most popular policy queries were both term sets from the winning party ’ s platform . Perhaps not coincidence , rich and robust discussion around a party ’ s particular policy position may indeed translate to votes at the polls . Turning now to identity , in Nigeria division between a predominantly Muslim Northern region and Christian South dates back to pre - colonial era , and was only intensified by the English ( Lenshie and Abel 2012 ) . This legacy continues to dominate conversations of national identity in the public sphere ( Alubo 2011 ) . In the Twitter data , Nigerians discuss identity , particularly geopolitical and religious identities , with greater intensity than they discuss policy . Examples of tweets focused on identity include , “ it seems the country is voting along religious and ethnic sentiments . . . . sad . . . # nigeriadecides ” or the commonly retweeted joke “ Mayb wht Nigeria nids is a prsidnt who ' s a half Igbo - half Hausa Muslim ; Brn n raised in the Niger Delta with a Yoruba Wife . ” While Kenya ’ s discussion of identity on Twitter takes different form and is significantly quieter than Nigeria , political scientist Ogude ( 2002 ) notes that Kenyan politicians use ethnic identity to foster political loyalty . Politics are charged with tribal identity , particularly affiliation with the Kikuyu and Luo tribes . For the 2013 election , a major debate was over land holdings , which have fueled ethnic tension ( Flood 2013 ) . While scholars claim that tribal politics no longer play a prominent role in Ghanaian elections , discussion of tribes and regions is present at about the same level as within the Kenyan dataset . The most commonly discussed identity relates to the Akan tribe as well as the Ashanti and Volta region , where tribes do indeed tend to vote along long standing ties with the two major Ghanaian political parties . Religion was not a prominent source of identity politics in Ghana or Kenya . This is most likely due to a large majority Christian population in both countries . With seventy - four percent Christian in Ghana and eighty - two percent Christian in Kenya religious identity is less likely to be source of political division . Comparatively , fifty percent of Nigerians are Muslim and forty percent are Christian ( CIA World Factbook ) . Returning to Table 5 , the relative prevalence of discussions of policy material versus identity politics demonstrates that citizens in Ghana , the highest ranked democracy of the three , tweeted more frequently about the campaign manifestos of Presidential candidates . This suggests that as democratic deepening occurs , citizens do engage in more discussion of policy on election day , and they are using Twitter to do so . In Nigeria , where identity is fiercely present and observed , discussion of North versus South overwhelms the policy debate ; Twitter seems to clearly reflect this quality of the sociopolitical landscape . While some consider social media as a politically useless echo chamber , lacking reasonable discourse , political parties and candidates should take note that in all three countries , the most discussed policy material was of the winning candidate ’ s party , even in highly contested elections . Most scholars are in agreement that social media has yet to cause a power shift in politics . Emerging research on the political impact of ICT tools in developing countries find intermediaries to play an important role in engaging with citizens using new technologies . The existing political structure of parties and elections may be viewed as an important intermediary in developing social media as a space for power shift through social engagement . In each election , the most discussed manifesto topics were from winning candidates ’ campaign platforms . This finding may indicate that citizens on Twitter respond positively when political parties correctly envision the public ’ s desires in their policy platforms . To engage in a more deliberative process , political parties can use social media to identify voters ’ priorities and open up the ideation phase of a party policy , using new , networked technologies as tools for substantive democracy . This identifies political parties as key intermediaries in realizing political development through the use of ICTs . 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M . “ Predicting Elections with Twitter : What 140 Characters Reveal about Political Sentiment . ” Proceedings of the Fourth International AAAI Conference on Weblogs and Social Media . 2010 50 . [ 50 ] Twi Teacher . Twitter Post . December 8 , 2012 , 9 : 31 a . m . http : / / Twitter . com / Twi _ teacher 51 . [ 51 ] Waithaka , Francis . Twitter Post , March 4 , 2013 , 10 : 51 a . m . http : / / Twitter . com / waithash . 1 While the Akans make up a large ethnic group , the sub - groups of the Akan , including the Asante , Fantis , Akyems , and Brongs do not align themselves politically ( Nugent 2003 ) . 2 “ NPP are considering changing its symbol from an ELEPHANT to a CAT so they can win some votes in the Volta Region ” \ No newline at end of file diff --git a/silver_data/b04115ce453c118e5414ac1912c19c30c1f59c9a.pdf.txt b/silver_data/b04115ce453c118e5414ac1912c19c30c1f59c9a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..c2b0008792cb052f8dd63cde88fa347dccca89cd --- /dev/null +++ b/silver_data/b04115ce453c118e5414ac1912c19c30c1f59c9a.pdf.txt @@ -0,0 +1 @@ +Journal of Immune Based Therapies , Vaccines and Antimicrobials , 2013 , 2 , 44 - 48 http : / / dx . doi . org / 10 . 4236 / jibtva . 2013 . 24006 Published Online October 2013 ( http : / / www . scirp . org / journal / jibtva ) PKC Is a Target to Modulate the Expression of Receptor Mediated Endocytosis ( RME ) Mice Macrophages BALB / c for Optimizing the Phagocytosis toward Candida albicans Adi Prayitno 1 * , Elyana Asnar 2 , Okid Parama Astirin 3 , Anief Nur Artanti 4 , Meutia Srikandi Fitria 3 , Eva Agustina Perwitasari 3 , Suhartono Taat Putra 2 1 Department of Dental and Oral Disease , Faculty of Medicine , University of Sebelas Maret , Surakarta , Indonesia 2 Departement of Pathobiology , Faculty of Medicine , University of Airlangga , Surabaya , Indonesia 3 Department of Biology , Faculty of Mathematics and Natural Science , University of Sebelas Maret , Surakarta , Indonesia 4 Department of Pharmacy , Faculty of Mathematics and Natural Science , University of Sebelas Maret , Surakarta , Indonesia Email : * drgadiprayitno @ yahoo . com Received August 2 , 2013 ; revised September 3 , 2013 ; accepted September 11 , 2013 Copyright © 2013 Adi Prayitno et al . This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use , distribution , and reproduction in any medium , provided the original work is properly cited . ABSTRACT Introduction : The existence of receptor - mediated endocytosis ( RME ) means that selectivity and selectivity occurs in capturing macromolecules . Protein kinase C ( PKC ) which can be expressed by almost all cells are proteins important in signal transduction groove that plays a role in a number of cell activity , e . g . phagocytosis . Aims : The purpose of this study is to determine the expression of RME after modulating the PKC which is characterized by the number of Can - dida albicans cells attached to the surface of macrophages . Methods : Peritoneal macrophages cultured BALB / c mice are treated with PMA and / or bisindolylmaleimides of providing levels of 5 ng / ml to 100 ng / ml for 10 minutes . Then immediately insert Candida albicans and observe every 30 minutes for 120 minutes . The research design used the same subject . Data collected in the form of number of Candida albicans cells attached to the surface of macrophages are analyzed with ANOVA statistical test ( one way ) to show the differences between treatments . Results : The test shows statistically significant difference in the number of Candida albicans cells attached to the surface of macrophages after administration of various levels of PMA ( p < 0 . 001 ) . The higher level of PMA is given , the more active the PKC is , the more RME are formed , the more Candida albicans cells attached to the surface of macrophages . Another result shows statistically significant difference in the number of Candida albicans cells attached to the surface of macrophages after administration of various levels of bisindolylmaleimides ( p < 0 . 001 ) . The higher level of bisindolylmaleimide is given , the less active PKC is , and the less RME are formed , the less Candida albicans cells attached to the surface of macro - phages . Conclusion : Research shows that activator PKC ( PMA ) can increase the expression of RME on macrophages . Another research shows that inhibitor PKC ( bisindolylmaleimides ) can decrease the expression of RME on macrophages . Keywords : PMA ; Bisindolylmaleimides ; RME ; PKC ; Phagocytosis 1 . Introduction Phorbol esters are polycyclic alcohol that is derived from croton oil . Phorbol ester is highly carcinogenic and known as tumor promoters [ 1 - 3 ] . How phorbol ester activates PKC is identical to how diacylglycerol ( DAG ) activates PKC . Phorbol ester activation is persistent , because it is very similar to the DAG and not immediately degraded [ 2 , 3 ] . Bisindolylmaleimides is a potent and selective in - hibitor of Protein kinase C which provides evidence for the potential use of PKC inhibitor as therapeutic immu - nomodulators [ 4 ] . Protein kinase C ( PKC ) family is a he - terogeneous family of phospholipid - dependent kinases [ 5 ] . Protein kinase C has a number of important roles in cellular growth and differentiation , cellular metabolism , and transcriptional activation , most of which are not well understood [ 2 , 3 , 6 - 8 ] . Protein kinase C as allosteric en - zyme can be modulated so it can make a pharmacological target [ 4 , 9 , 10 ] . * Corresponding author . Copyright © 2013 SciRes . JIBTVA A . PRAYITNO ET AL . 45 Candida albicans is the most common organism to be associated with superficial candidosis . Oral colonization may originate early in infancy , although its incidence is increased by a number of factors , including hospitaliza - tion and bottle feeding . Some factors are systemic , and others are related to local conditions [ 11 ] . Little is known about the precise mechanisms involved in immunity to fungal infection . Macrophages and T cells immunity may be implicated in resistance to fungal infection [ 5 , 12 - 14 ] . The role of PKC , expression of receptor mediated en - docytosis and phagocytosis toward Candida albicans may be explained by pathobiology examination [ 15 , 16 ] . This concept gives a chance to explain how to modulate immune response toward Candida albicans . The existence of receptor - mediated endocytosis ( RME ) means for selectivity and effectiveness of arresting mac - romolecules that may be in very low concentrations in the extracellular fluid , e . g . controlled by a cell receptor to capture various types of substrates , including hormones , growth factors , enzymes - enzymes , and plasma proteins [ 2 , 17 , 18 ] . Then that macromolecules outside the cell have been collected and attached to the cell surface or plasma membrane into the cell to form a basin which means that RME has become the receptor binding in the area at the plasma membrane , known as coated pits [ 2 , 19 ] . Some re - ceptors are concentrated in coated pits for a long time with a fixed concentration in the plasma membrane [ 2 , 20 - 22 ] . 2 . Method Macrophages obtained from the peritoneal cavity of mice BALB / c done in a way as is done by Colligan , et al . with some modifications [ 23 ] . Macrophages obtained by using a 10 ml syringe and hypodermic needle 25 G . The results collected in sizes 25 ml centrifuge tube and stored on ice . Macrophage cultures performed in culture dishes with a diameter of 20 mm . Macrophages were distributed into each well of the plate so that the culture wells containing an average of 1000 cells . For ease of painting it on the basis of prior pitting the cover glass is placed before the cell is inserted . Further culture medium Roswell Park Memorial Institute ( RPMI ) 1640 ( Sigma Chem . Co . . St . Louis , USA ) inserted into the wells as much as 10 ml . Medium replacement done once every 24 hours and in - cubated at room temperature . Before treated culture medium were taken and cultured cells were washed with PBS - 10F ( PD : 137 nM - NaCl - KCl 3 nM , 7 nM - Phosphate Buffer , pH 7 . 4 ) and subse - quently given the PMA ( Sigma Chem . Co . . , St . Louis , USA ) for 10 minutes in the levels of 5 ng / ml to 100 ng / ml at room temperature . After the AMF in the exhaust and cleaned with PBS - 10F , added C . albicans approxi - mately 200 cells per well and were observed every 30 min for 120 minutes [ 24 , 25 ] . After the observation period finish the glass cover at the base of the existing wells cell culture was taken and carried the painting with Giemsa ( MERCK ) . Nikkon microscope equipped with a photo tool used to document the results with 100 multiple magnification , every 30 mi - nutes for 120 minutes [ 2 ] . 3 . Result The number of Candida albicans cells attached to the surface of macrophages can be seen in Figure 1 below , The test shows statistically significant difference in the number of Candida albicans cells attached to the surface of macrophages after administration of various levels of PMA ( p < 0 . 001 ) . The higher level of PMA is given , the more active PKC , the more RME are formed , the more Candida albicans cells attached to the surface of macro - phages ( Table 1 ) . Another result showed statistically significant differ - ence in the number of Candida albicans cells attached to the surface of macrophages after administration of vari - ous levels of bisindolylmaleimides ( p < 0 . 001 ) . The high - er level of bisindolylmaleimide is given , the less active PKC , the less RME are formed , less Candida albicans cells attached to the surface of macrophages ( Table 2 ) . 4 . Discussion Phorbol Ester said can activate PKC . Polyciclic alcohol that derivate from croton oil is very carcinogenic , so it known as tumor promoter . Phorbol ester activate PKC because they similarity with diacylglycerol . The activity is persistent , because phorbol ester is similar with dia - cylglycerol which can’t quickly degradated . If PKCs are going active can increases the signal transduction activity for cells activity . Phorbol ester also can increase the macropinicytosis toward Lucifer Yellow [ 3 , 26 , 27 ] . Dia - cylglycerol had two important signal pathways . First , they are break further for release the arachidonic acid , its ( a ) ( b ) ( c ) Figure 1 . ( a ) : Culture of phagocytozed macrophage toward Candida albicans before treated ; ( b ) : Culture of phagocy - tozed macrophage toward Candida albicans after treatment . We can look the the number of Candida albicans traped into complete phagosomes after treated with 100 ng / ml PMA ; ( c ) : Culture of phagocytozed macrophage toward Candida albi - cans after treatment . We can looks the the number of Can - dida albicans traped into complete phagosomes after treated with 100 ng / ml GF 109203x , and . Copyright © 2013 SciRes . JIBTVA A . PRAYITNO ET AL . 46 Table 1 . Phagocytic index of macrophage phagocytozised activity toward C . albicans after administration with many concentrations of PMA in 30 minutes and 120 minutes . The higher level of PMA is given , the more PKCs are active , the more RMEs are formed , the more Candida albicans cells attached to the surface of macrophages ( % at least one fun - gi attached , collom 2 ) . phagocytic per positive cell # index Concentration of PMA % 30’ 120’ 30’ 120’ 0 ng / ml 95 2 10 19 . 0 92 . 0 5 ng / ml 95 2 6 19 . 0 57 . 0 25 ng / ml 90 4 6 36 . 0 54 . 0 50 ng / ml 98 3 4 29 . 4 39 . 2 100 ng / ml 93 0 1 0 . 0 9 . 3 Note : ng / ml : nanogram per millilittre ; percentages ( % ) : containing mean number of fungi at least one fungi ; phagocytic index ( PI ) : percentages con - taining at least one fungi X mean number of fungi per positive cell . A hun - dred cells macrophages againt 1000 cells Candida albicans per well . # total number of cell Candida albicans which is attached to the surface of the cell membrane of macrophages . Table 2 . Phagocytic index of macrophage phagocytozised activity toward Candida albicans after administration with many concentrations of GF 109203x in 30 minutes and 120 minutes . The higher level of GF 109203x is given , the less PKCs are active , the less RMEs are formed , the less Can - dida albicans cells attached to the surface of macrophages ( % at least one fungi attached , collom 2 ) . phagocytic per positive cell index Concentration of GF 109203x % 30’ 120’ 30’ 120’ 0 ng / ml 92 1 10 9 . 2 92 . 0 5 ng / ml 90 0 10 0 . 0 90 . 0 25 ng / ml 92 0 6 0 . 0 55 . 2 50 ng / ml 80 0 4 0 . 0 32 . 0 100 ng / ml 60 0 1 0 . 0 6 . 0 Note : ng / ml : nanogram per millilittre ; percentages ( % ) containing mean number of fungi at least one fungi ; phagocytic index ( PI ) = percentages containing at least one fungi X mean number of fungi per positive cell . A hundred cells macrophages againt 1000 cells C . albicans per well . In this moment the phagosomes was completed . # total number of cell Candida albicans which is attached to the surface of the cell membrane of macro - phages . mean the can part as the best courier or used in eico - sanoids sintesis . Second , it more important , they can ac - tivate kinase serine / threonine protein kinase which selec - tively to phosphorelate in target cells . The effect of dia - cylglycerol can change with phorbol ester , that in plant production in the dependency with kinase C . The activity is direct . This reagents seen that this pathway always im - ages the cellular response . More type cells can stimu - late the proliferation of cells culture if administrated with kinase C activator [ 7 , 28 ] . Inhibitors of PKC could interact with the substrate - binding site or with regulatory site of PKC . The struc - tural similarity between bisindolylmaleimides , chelery - thine and staurospoorine suggested that bisindolylmalei - mides may be a competitive inhibitor with respect to ATP . The inhibition of PKC by bisindolylmaleimides was demonstrated to be highly dependent on the ATP concentration [ 4 , 29 ] . GF 109203X inhibited collagen - and alpha - thrombin - induced platelet aggregation as well as collagen - triggered ATP secretion . However , ADP - dependent reversible aggregation was not modified . In Swiss 3T3 fibroblasts , GF 109203X reversed the inhibi - tion of epidermal growth factor binding induced by phor - bol 12 , 13 - dibutyrate and prevented [ 3H ] thymidine in - corporation into DNA , only when this was elicited by growth promoting agents which activate PKC [ 4 ] . Cell signalling includes : 1 ) Recognation of the stimu - lus by a specific receptor embedded wthin the membrane . 2 ) Transfer of a signal to its cytoplasmic surface . 3 ) Transmission of the signal to specific receptor molecules within the cytoplasm that trigger the cell’s response 4 ) Cessation of the response as a result of the destruction or inactivation of the signaling molecule . Evidence from re - cent studies indicates are complex , for examples : 1 ) Sig - nal from variety of unrelated growth factors , each bind - ing to its own receptor , can convergence to activate a common effector . 2 ) Signal from same ligand , can diver - ge to activate a variety of different effectors , leading to cellular response . 3 ) Signal can be passed back and forth between different pathways , a phenomenon known as crosstalk [ 3 , 30 ] . Some cells have a mechanism for internalism material needs from the environment outside the cell to inside the cytoplasmic through bubble dent derived from the plas - ma membrane . With the RME then macromolecules bind to specific RME projected on the outer surface of the plasma membrane [ Stites and Terr , 1991 ] . Pinocytosis and macropinocytosis associated with the influx of fluids and soluble molecules into cells are characterized by in - creased metabolic activity of the cells . Phagocytosis pro - cess includes chemotaxis , catch - consuming macromole - cules ( by forming phagosome ) , digest ( by forming secon - dary lysosomes and ongoing enzymatic degradation ) and release it back that has been in the form of small particles particle non - antigenic [ 5 , 31 ] . PKC has the amino - therminal regulatory domain ( 20 - 70 kDa ) which is the binding site for phorbol ester . This domain is divided into carboxyl - terminal catalytic do - main ( approximately 45 kDa ) consisting of a binder Adenosine Tri Phosphate ( ATP ) and substrate binding site where the two are connected by a flexible hinge re - gion [ 32 , 33 ] . PKC conventional dependent calcium and Copyright © 2013 SciRes . JIBTVA A . PRAYITNO ET AL . 47 DAG or phorbol ester as a cofactor , while PKC Novel depends only on DAG or phorbol ester . Atypical PKC is not dependent calcium nor DAG for maximal activity [ 3 ] . PKC can modulate for anti inflammatory [ 34 - 36 ] , diabe - tes complication [ 37 , 38 ] , regulating of actin cytoskeleton [ 39 ] , and apoptosis [ 40 ] . 5 . Conclusion Research shows that activator PKC ( PMA ) can increase the expression of RME on macrophages . Another re - search shows that inhibitor PKC ( bisindolylmaleimides ) can decrease the expression of RME on macrophages . 6 . Acknowledgements We thank to Higher Education Competitive Research Project Ministry of Education and Culture Republic of Indonesia for Grand Featured Research Universities 2013 , LPPT of Gajah Mada University and special thanks to acknowledge , Prof . Dr . Rafik Karsidi , MSc as a rector of Sebelas Maret University Surakarta Indonesia , Prof . Dr . Ir . Darsono , M . Si as a Chairman of the Institute for Re - search and Community Service , Prof . Dr . Zainal Arifin Adnan , SpPD - KR . , FINASIM as a dean of Faculty of Medicine of Sebelas Maret University Surakarta Indone - sia and special thanks to acknowledge Prof . Wihaskoro Sosroseno , DDS . , Ph . D . for much inspirations to write this article . REFERENCES [ 1 ] P . E . Driedger and P . M . 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King , “Activation of Protein Kinase C Isoforms and Its Impact on Diabetic Complications , ” Circulation Research , Vol . 106 , No . 8 , 2010 . pp . 1319 - 1331 . http : / / dx . doi . org / 10 . 1161 / CIRCRESAHA . 110 . 217117 [ 38 ] A . Gonelli , C . Mischiati , R . Guerrini , R . Voltan , S . Sal - vadori and G . Zauli , “Perspectives of Protein Kinase C ( PKC ) Inhibitors as Anti - Cancer Agents , ” Mini Reviews in Medical Chemistry , Vol . 9 , No . 4 , 2009 , pp . 498 - 509 . http : / / dx . doi . org / 10 . 2174 / 138955709787847967 [ 39 ] C . Larsson , “Protein Kinase C and the Regulation of the Actin Cytoskeleton , ” Cellular Signalling , Vol . 18 , No . 3 , 2006 , pp . 276 - 284 . http : / / dx . doi . org / 10 . 1016 / j . cellsig . 2005 . 07 . 010 [ 40 ] A . M . Gonzales - Guerrico , J . Meshki , L . Xiao , F . Benavides , C . J . Conti and M . G . kazanietz , “Molecular Mechanisms of Protein Kinase C - Induced Apoptosis in Prostate Cancer Cells , ” Journal of Biochemistry and Mo - lecular Biology , Vol . 38 , No . 6 , 2005 , pp . 639 - 645 . http : / / dx . doi . org / 10 . 5483 / BMBRep . 2005 . 38 . 6 . 639 \ No newline at end of file diff --git a/silver_data/b0a3192b12d66f3ab093494e27a5f29fb78abba3.pdf.txt b/silver_data/b0a3192b12d66f3ab093494e27a5f29fb78abba3.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..29e690569d6e507f7b5cc6433e704e2491bfec32 --- /dev/null +++ b/silver_data/b0a3192b12d66f3ab093494e27a5f29fb78abba3.pdf.txt @@ -0,0 +1 @@ +This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 DESIGN : ONE , BUT IN DIFFERENT FORMS Willemien Visser INRIA Paris - Rocquencourt ; CNRS LTCI ; TELECOM ParisTech 46 , rue Barrault 75634 Paris Cedex 13 France tel : + 33 ( 0 ) 1 45 81 83 19 fax : + 33 ( 0 ) 1 45 65 95 15 email : Willemien . Visser @ Telecom - ParisTech . fr Abstract . This overview paper defends an augmented cognitively oriented generic - design hypothesis : there are both significant similarities between the design activities implemented in different situations and crucial differences between these and other cognitive activities ; yet , characteristics of a design situation ( related to the design process , the designers , and the artefact ) introduce specificities in the corresponding cognitive activities and structures that are used , and in the resulting designs . We thus augment the classical generic - design hypothesis with that of different forms of designing . We review the data available in the cognitive design research literature and propose a series of candidates underlying such forms of design , outlining a number of directions requiring further elaboration . Keywords . cognitive design research ; generic design ; psychology of design ; design activity ; design cognition This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 This paper is a first step in an endeavour to assert an augmented generic - design hypothesis ( which concluded our book , The Cognitive Artifacts of Designing , Visser , 2006b ) : analysed from a cognitive viewpoint , design has specific characteristics that distinguish it from other cognitive activities , but also takes on different forms depending on the main dimensions of the design situation . Examination of this hypothesis , which is the object of this paper , may have consequences for both theory and practice in the domain of design . Support for the hypothesis may have consequences for design environments , assistance , and education . It may , for example , guide the development of modalities for supporting designers when they are involved in the construction of representations or in the management of constraints and criteria . Given the mostly dispersed and anecdotal discussion of the different components that make up the hypothesis , our aim here is to articulate them in an overview paper . Reviewing various empirical studies of activities " as diverse as software design , architectural design , naming and letter - writing , " Thomas and Carroll ( 1979 / 1984 ) stated that these different design activities " appear to have much in common " ( p . 234 ) . A number of authors have defended that , compared to other professionals , designers have specific forms of knowledge ( e . g . , Cross , 2001b ; 2002b ) . Combining the positions underlying these two claims , Goel and Pirolli ( 1989 ; 1992 ) proposed the notion " generic design . " Still other studies focus on the differences between design in different domains , examining a third aspect in this analysis concerning the nature of design ( e . g . , Akin , 2001 ; Purcell & Gero , 1996 ) . In this paper we review and discuss these different aspects of design , focussing on the third one , whose discussion seems the least organised in the design literature . The position defended in this paper is the following : there are both ( 1 ) significant similarities between the design activities implemented in different situations and ( 2 ) significant differences between design and other cognitive activities ; yet , ( 3 ) characteristics of a design situation ( i . e . , characteristics related to the design process , the designers , and the artefact ) introduce specificities in the corresponding cognitive activities and structures that are used , and in the resulting designs . We thus augment the generic - design hypothesis ( 1 and 2 ) with that of different forms of designing ( 3 ) . The augmented generic - design hypothesis thus connects three different positions with respect to design and nondesign activities that have been espoused , more or less explicitly , by different authors in the domain This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 of cognitive design research . Given the implicitness to this respect that is present in many papers on design , the review and discussion proposed in this paper seem useful . Generally , papers are concerned with only one position , sometimes two ( the generic - design hypothesis ) , but the three have rarely been articulated together ( but see Akin , 2001 , discussed below ) . In addition , corroboration of the generic - design hypothesis is nearly exclusively grounded in Goel and Pirolli ' s ( 1989 ; 1992 ) work . Except for these authors ' research , this double - sided hypothesis has received little substantiation through comparative cognitive analyses . We qualify our hypothesis as " cognitive , " because in the literature the term " generic design " most often is used in other than cognitive acceptations . The notion is used in the domains of software engineering ( cf . Gamma ' s design patterns ) , AI and knowledge acquisition ( e . g . , KADS and successor work ) , based , for example , on Chandrasekaran ' s ( 1983 ) " generic tasks , " or notions such as " generic design methods " and other " generic design agents " ( see , e . g . , Warfield , 1994 ) . All these references are normatively based approaches to design , not concerned with the cognitive validity of the proposed units , be they design patterns , tasks , or methods . The present text focuses on cognitively oriented analyses of design activities . Outline of the paper . This introduction presents our augmented generic - design hypothesis in the context of Goel and Pirolli ' s ( 1992 ) generic - design hypothesis and the view of design ' s domain independence defended by several other authors . Sections 1 and 2 briefly discuss the two constituents of the generic - design hypothesis , that is , the existence of commonalities between designing in different situations ( section 1 ) and differences between designing and nondesigning ( section 2 ) . In the main section of this paper , that is , section 3 , we discuss the third constituent of our hypothesis ( that is , design takes different forms depending on characteristics of the design situation ) ; we do so through an examination of candidate variables underlying such forms of design , outlining a number of directions for further elaboration . In section 4 , the Conclusion , we will discuss the augmented generic - design hypothesis and complete it with a fourth constituent . The generic - design hypothesis . Goel and Pirolli ( 1992 ) formulated their " intuitions about generic design " as a hypothesis that combined two assumptions : " problem spaces exhibit major invariants across design problem - solving situations and major variants across design and nondesign problem - solving situations " ( p . 399 ) . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 According to Goel ( 1994 ) , the authors aim to " motivate the notion of generic design within information - processing theory " ( p . 53 ) , that is , within the symbolic information processing framework that Newell and Simon ( 1972 ) developed in order to analyse problem solving from a cognitive viewpoint . Goel and Pirolli ( 1989 ; 1992 ) seem to have made a strong case for the generic - design hypothesis . Their study may , however , be criticised on two points . On the one hand , certain flaws in the choice of nondesign tasks considerably weaken the authors ' characterisation of design—which mainly depends on its contrast to nondesign ( design is qualified as " X " by contrast to nondesign being " not X " ) . First , the nondesign tasks were brief , artificial games ( that took 15 to 40 minutes ) . Second—something , moreover , noticed by the authors themselves—the study " purposefully took two points ( ill - structured design tasks and well - structured game tasks ) at the extremities of the spectrum of problem types " ( Goel , 1994 , p . 71 ) . The author considers that , " given that [ Goel and Pirolli ] have found interesting differences , it would be instructive to… explore the intermittent points in the space " ( Goel , 1994 , p . 71 ) , but this examination has not been conducted , as far as we know . On the other hand , the design tasks were examined in artificially restricted laboratory situations . The participants in Goel and Pirolli ' s ( 1989 ; 1992 ) study were professionals , but the design sessions , varying from 2 to 3 hours , " simulated the ' design sketch ' exercises which are an integral part of the training program of many design disciplines " ( Goel , 1994 , p . 54 ) —tasks from which generalisation to design is not immediate , in our opinion . Such an approach is typical for the classical cognitive - psychology research on " problem solving " tasks . Since the beginning of our cognitive design research , we have been questioning the representativeness of such studies for professional design projects ( in Visser , 1987b , for example , we identified specificities of professional design that are not observed in limited , artificially constructed design situations ) . In addition , we have come to question the appropriateness of the " problem solving " paradigm for the cognitive analysis of design ( Visser , 2006b ) . The present paper focuses on cognitive design studies performed in real , professional work situations—even if we also review data from other experimental , less ecological research that is related to our topic . We will use , however , the notion " problem " and " problem solving " as authors use them , not always again questioning this view here . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 The domain independence of design . Zimring and Craig ( 2001 ) present the " domain independence " of design as a notion similar to that of generic design . One may interpret domain independence , however , as only referring to design being invariant across domains , not necessarily to design differing significantly from other cognitive activities . It is in this more restricted sense that many design researchers and practitioners defend the idea of a domain - independent theory of design . Certain authors indeed defend such a position because of similarities observed between two or more domains of design . Zimring and Craig ( 2001 ) consider that " common descriptions of design—that designing involves abductive reasoning , construction , ill - defined problem solving skills—…are not always sharp enough to both distinguish design from other types of problem solving and unite design across different design - related disciplines " ( p . 125 ) . The authors consider that the analysis and description of design in terms of " mid - level constructs " " may be more profitable in scaling research across disciplines " ( p . 126 ) . As examples of mid - level processes or types of reasoning , the authors present mental simulation , decision - making , and analogical reasoning . During the 1995 " Design Dialogues : one " meeting , entitled " Universal Theory of Design : is a domain independent theory of design possible ? " 1 the participants explored " the reasons for the apparent lack of progress in design research over [ the decade 1985 - 1995 ] and in particular whether the search for an atemporal , acultural , domain independent theory of design [ was ] a reasonable or realistic goal . " During this meeting , Cross stated , " a primary goal of the Design Research Society since its founding in the 1960s [ had ] been a domain independent theory of design within the context of a science of design " ( from the Meeting report , see Note 1 ) . In her Meeting report , McDonnell wrote , " on the question of whether theories , of whatever kind , can be domain independent , there was a… diversity of views . … some participants believing that some form of universal theory is possible ranged against those who argued for the incommensurability of different views of design or that the elimination of context necessary for a universal theory would result in an activity unrecognisable as design . " 1 " Design Dialogues : one " was " the first in an occasional series of discussion meetings on design theory sponsored by the Design Research Society , " organised by McDonnell and Logan at University College London , on May 17 , 1995 . We quote McDonnell ' s meeting report , which until recently could be retrieved via internet , but is no longer accessible . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 There has been much discussion in the design - research community around the relations between design and science ( Sargent , 1994 ) , some authors considering that a design science is to be developed ( Hubka & Eder , 1987 ) , others , such as Cross ( 2001b ; 2002b ) , judging that the two are to be clearly distinguished . For Hubka and Eder ( 1987 ) , " design science addresses the problem of determining and categorizing all regular phenomena of the systems to be designed , and of the design process " ( p . 124 ) . Cross ( 2002b ) wishes to develop " ' design as a discipline ' , based upon a ' science of design ' , not a ' design science ' " ( cf . also Simon , 1969 / 1999 , characterising the " science of design " in his Sciences of the Artificial ) . For Cross ( 2002b ) , " design science implies an explicitly organised , rational and wholly systematic approach to design ; not just the utilisation of scientific knowledge of artefacts , but design in some sense [ as ] a scientific activity itself . … Science of design refers to that body of work which attempts to improve our understanding of design through ' scientific ' ( i . e . , systematic , reliable ) methods of investigation . Let us be clear that a ' science of design ' is not the same as a ' design science ' . The study of design leaves open the interpretation of the nature of design . " ( see also Cross , 2001b ) " Domain " in the context of " domain independence " is generally equated with a " discipline ( of practice ) , " such as engineering , architecture , computer science , or product design 2 . It may be used in a wider acceptation . Discussing domain - generality versus domain - specificity in cognition , Frensch and Buchner ( 1999 , p . 142 , quoted in Zimring & Craig , 2001 , p . 126 ) define a domain as " anything that a given constraint can potentially be generalized to and from . " In this paper , we will be concerned with design " situations " that can be characterised on three main dimensions , that is , the design process , the designers , and the artefact . The augmented generic - design hypothesis translates our claim that , if we do not " eliminate the context " of design ( cf . McDonnell quoted previously ) , we may observe different forms of design in different design situations . 1 . Design is one : commonalities between designing in different situations From the early 1980s on , authors in the domain of design research have started to characterise design as a cognitive activity , highlighting the differences from design as it had been represented until then in 2 We use the term " product design " where many authors use " industrial design , " because we reserve " industrial design " in a more general acception , that is , for design perfomed in a professional , industrial situation . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 prescriptive models underlying design methods ( e . g . , Pahl & Beitz , 1977 / 1996 ) . An important reference for this new , more cognitively oriented approach to design has been Simon ' s ( 1969 / 1999 ) analysis of design in The Sciences of the Artificial . At the end of the 1990s , the following characterisation of design was prevailing in the domain of cognitive design research—even if authors may differ regarding certain characteristics ( see hereunder xi . Design activity is mostly opportunistically organised ) . Concerning only two qualities , authors generally continue to adhere strictly to Simon ' s ( 1969 / 1999 ) position and analysis of design ( see hereunder i and v ) . For most characteristics , authors have elaborated on Simon ' s characterisation , not so much contradicting him , as extending generally his analysis ( see hereunder , especially , points iii , vi , vii , viii , ix , and x ) . For a last series , they have revised considerably Simon ' s position ( see hereunder , especially , points ii , iv , and xi ) ( for a more detailed and more critical discussion of Simon ' s , 1969 / 1999 , positions , see Visser , 2006b ) . ( i ) Design is a type of cognitive activity rather than a professional status . In 1969 , Simon ( 1969 / 1999 ) states that " design " is not restricted to engineers , who are not the only professional designers . " Everyone designs who devises courses of action aimed at changing existing situations into preferred ones . " ( p . 111 ) ( ii ) Design is a problem - solving activity . This is one of Simon ' s central stances with respect to design , based on the symbolic information - processing framework developed in Newell and Simon ( 1972 ) . In addition , Simon qualifies design as an " ordinary " problem - solving activity , that is , a problem - solving activity for which no new and hitherto unknown problem - solving concepts or techniques are necessary . According to his " nothing special " position ( presented for scientific thinking in Klahr & Simon , 2001 , p . 76 ) , " no qualitatively new components " need to be introduced in the classic general problem - solving mechanisms , in order to be able to handle design problems ( Simon , 1973 / 1984 , p . 197 ) . No " special logic " is necessary ( Simon , 1969 / 1999 , p . 115 ) —even if Simon " admits " that standard logic is to be adapted to the search for alternative solution elements ( p . 124 ) . In recent years , we have started to amend Simon ' s ( 1969 / 1999 ) position : we have developed the idea that designing is more appropriately qualified as the construction of representations ( Visser , 2006b , 2006c ) . From a formal viewpoint , design is certainly a " problem solving " activity : based on the design specifications , designers are rarely able to evoke from memory a pre - existing problem - solving procedure . Numerous studies have shown that , for This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 many components of a design task , designers need to construct procedures in order to formulate a solution . However , qualifying design " simply " as problem solving is not very informative . In this paper , we cannot further detail these ideas ( see Visser , 2006b ) . ( iii ) Design problems are considered ill - defined ( or " ill - structured " in Simon ' s , 1973 / 1984 , terms ) : this design feature , noticed from the earliest cognitive design studies on ( Eastman , 1969 , 1970 ; Reitman , 1964 ; Thomas & Carroll , 1979 / 1984 ; Voss & Post , 1988 ) , has been substantiated in many different kinds of studies since then , and continues to be considered as a specific characteristic of design ( Akin , 2001 ; Michalek & Papalambros , 2002 ; Ormerod , 2005 ) . Rittel and Webber ( 1973 / 1984 ) speak of " wicked " problems , which have no definitive formulation : each formulation corresponds to at least one solution ( Buckingham Shum , 1997 ; Conklin , 2006 ) ( for a discussion of the distinction between ill - defined and wicked problems , see Visser , 2006b , p . 142 ) . ( iv ) In his problem - solving approach to design , Simon ( 1969 / 1999 ; 1973 / 1984 ) distinguishes two stages in problem solving : problem structuring and problem solving . Analysis , synthesis , and evaluation are examples of another decomposition of design proposed by authors adopting , with more or less profound modifications , Simon ' s approach to design—or , more generally , Newell and Simon ' s ( 1972 ) approach ( Akin , 1986a , 1986b ; Baykan , 1996 ; Goel , 1994 ; Goel & Pirolli , 1992 ; Hamel , 1995 ; Lebahar , 1983 ) . However , such stages can be distinguished only in theory as distinct activities : problem analysis and solution elaboration progress in parallel , rather than in separate , consecutive stages . Furthermore , designers constantly generate new task goals and redefine task constraints . Even if they are cognisant of prescriptive models distinguishing analysis and synthesis , designers do not follow them systematically ( Akin , 1979 / 1984 ; Carroll & Rosson , 1985 ; Cross , 1984 ; Dasgupta , 1989 ; Visser , 1987a ) . Authors who analyse design problem solving in terms of " problem space " and " solution space " have proposed the notion of " co - evolution " of these two spaces ( Dorst & Cross , 2001 ; Maher , Poon , & Boulanger , 1996 ; cf . also our idea of problem / solution pairs , Visser , 1991 ) . ( v ) Design is a " satisficing " activity : rather than to optimize , that is , to calculate the optimum value , or to choose the best solution among all possible solutions , designers " settle for the good enough " ( Simon , 1971 / 1975 , p . 1 ) , accepting a satisfactory solution ( Simon , 1987 / 1995 , p . 246 ) . As they have to decide This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 without complete information , they have no other choice . This characteristic has been observed by various authors ( Akin , 2001 ; Ball , Lambell , Reed , & Reid , 2001 ) . According to Akin ( 2001 ) , however , designers from different disciplines vary on this point : while architects indeed proceed to satisficing , engineering designers adopt more objective methods in their selection among possibilities and may proceed to optimisation . ( vi ) Design generally involves complex problems that are rarely decomposable into independent subproblems . Of course , designers proceed to decomposition , in order to make their problems more manageable and easier to solve . In our view , Simon and many design researchers who follow him overestimate , however , the role of systematic problem decomposition , especially through balanced , stepwise refinement . In other than relatively routine design projects , designers rarely decompose in a systematic way ( cf . our critique of Simon ' s " overestimating the role of systematic problem decomposition , " Visser , 2006b , pp . 68 - 70 ) . Moreover , one and the same design component often can be decomposed in different ways ( Reitman , 1964 , p . 296 ) . Simon himself notes that the interdependencies among the subproblems resulting from problem decomposition " are likely to be neglected or underemphasized . " " Such unwanted side effects accompany all design processes that are as complex as the architectural ones " that he considers in his text ( Simon , 1973 / 1984 , p . 191 ) . According to Akin ( 2001 ) , architects use idiosyncratic strategies to decompose a problem into subproblems and to integrate their solutions into a global solution afterwards , whereas in electronic hardware or mechanical design , the interaction between the parts are " theoretically determined . " Notice that Simon ( 1973 / 1984 , pp . 200 - 201 ) analyses complex systems such as social systems as " nearly decomposable " and that Goel ( 1995 ) considers the modules resulting from decomposition as " leaky . " ( vii ) Designers often tend to generate , at the very start of a project , a few simple objectives in order to create an initial solution kernel to which they then are sticking in what is going to become their global design solution . Such an initial solution kernel , which Darke ( 1979 / 1984 ) qualified as " primary generator , " has been identified by many other authors and has received labels such as " kernel idea , " " central concept , " " early solution conjecture , " " primary position , " and " guiding theme " ( Cross , 2001a , 2004b ; Guindon , Krasner , & Curtis , 1987 ; Kant , 1985 ; Lawson , 1994 ; Rowe , 1987 ; Ullman , Dietterich , & Staufer , 1988 ) . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 The ensuing process has been qualified as " position - driven " design , " early fixation , " " premature commitment , " " early crystallisation , " or " solution fixation " ( Ball , Evans , & Dennis , 1994 ; Cross , 2001a ; Goel , 1995 ) ( we will come back upon this characteristic , arguing that it requires inspection ) . ( viii ) Rather than one solution , which would be " the " " correct " solution , design problems have several , acceptable solutions , which are more or less satisfying . This characteristic of design problems , related to their ill definedness and the satisficing character of designing , has been observed in many studies and domains , for example , architecture ( Akin , 2001 ; Eastman , 1970 ) , mechanical design ( Frankenberger & Badke - Schaub , 1999 ) , software design ( Malhotra , Thomas , Carroll , & Miller , 1980 ) , and traffic - signal setting ( Bisseret , Figeac - Letang , & Falzon , 1988 ) . ( ix ) Design problems and solutions lack pre - existing , objective evaluation criteria ( Bonnardel , 1991 ; Ullman et al . , 1988 ) . As evaluative references are forms of knowledge , designers ' expertise in a domain influences how they use them ( D ' Astous , Détienne , Visser , & Robillard , 2004 ) . Given that , in a collaborative design setting , designers may have different representations of their project , solution proposals are evaluated not only based on purely technical , " objective " evaluative criteria ; they are also the object of negotiation , and the final agreement concerning a solution often results from compromises between designers ( Martin , Détienne , & Lavigne , 2001 ) . In addition , not only solution proposals , but also the evaluation criteria and procedures themselves undergo evaluation ( D ' Astous et al . , 2004 ) . ( x ) Reuse of knowledge ( from specific previous design projects ) through analogical reasoning has been observed in many cognitive design studies as a central approach in design ( Ball & Christensen , 2007 ; Ball , Ormerod , & Morley , 2004 ; Bhatta & Goel , 1997 ; Burkhardt , Détienne , & Wiedenbeck , 1997 ; Casakin & Goldschmidt , 1999 ; Détienne , 2002 ; Maiden , 1991 ; Sutcliffe & Maiden , 1991 ; Visser , 1995 , 1996 ) . Of course , this use of specific knowledge is combined with that of generic knowledge ( especially , from design methodology , the application domain , and the technical domains that underlie the design project ) . Most examples of reuse concern software design ( Détienne , 2002 ; Visser , 1987b ) , but we also observed it on product design ( in the Delft study , Visser , 1995 ) . ( xi ) Design activity is mostly opportunistically organised : designers proceed in a non - systematic , multidirectional way ( at moments top - down , at others bottom - up , at moments in breadth , at others in This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 depth ) , formulating plans that are more or less local , at both high , abstract and low , concrete levels . The basis for such organisation is designers taking into consideration the data that they have at the time : specifically , the state of their design in progress , their representation of this design , the information at their disposal , and their knowledge ( cf . the qualification of design as " situated " ) . This last point needs some discussion , because not all researchers share our conclusion that design is opportunistically organised ( for a detailed discussion , see ch . 21 . 4 The opportunistic organization of design : Decomposition and planning , and especially its section " Discussion of our opportunistic - organization position , " in Visser , 2006b , pp . 163 - 177 ) . Especially Davies ( 1991 ) and Ball and Ormerod ( 1995 ) adopt other positions . According to Davies ( 1991 ) , " expert programmers adopt a broadly top - down approach to the programming task , at least during its initial stages " ( p . 186 ; see our discussion of expertise below in Section 3 . 2 ) . Ball and Ormerod ( 1995 ) claim , " much of what has been described as opportunistic design behavior appears to reflect a mix of breadth - first and depth - first modes of solution development " ( p . 131 ) , even if design is also " subject to potentially diverging influences such as serendipitous events and design failures " ( p . 145 ) . Obviously , designers may proceed top - down and depth - first , or top - down and breadth - first—or bottom - up combined with depth - first or breadth - first . What we wish to emphasise is that ( 1 ) they often do so occasionally and locally , rather than systematically throughout the entire design process ; ( 2 ) a top - down - bottom - up mix can take different forms , and even if a mix pattern has several occurrences—and thus gets a systematic character—these will generally be interspersed with other ways of proceeding , so that " top - down " and " bottom - up " are inappropriate as general qualifications of designers ' activity ; and , especially , ( 3 ) an occasional , local top - down - bottom - up and or breadth - first - depth - first mix are just some of the various forms in which opportunism can reveal itself in design . As regards the " broadly top - down with opportunistic local episodes " ( Davies , 1991 ) versus " opportunistic , with hierarchical episodes " ( Visser , 1994a ) issue , we follow Hayes - Roth and Hayes - Roth ( 1979 , p . 307 ) . These authors have proposed that the systematic refinement model be considered as a special case of the opportunistic model , which allows various organisational structures of an activity— rather than only one , or a mix of two structures . An opportunistically organised activity may have This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 hierarchical episodes at a local level , but its global organisation is not hierarchical ( Visser , 1994a ) . With respect to the structured character of design organisation , opportunism proponents ( Guindon et al . , 1987 ; Kant , 1985 ; Ullman et al . , 1988 ; Visser , 1987a ; Voss , Greene , Post , & Penner , 1983 ) question the systematic implementation both of a depth - first ( or breadth - first ) and of a top - down refinement ( or bottom - up ) approach . Notice that Goel ( 1995 ) , who presents design as a quite systematic process , also remarks that " designers differ substantially in the path they take through [ the design problem ] space and how quickly or slowly they traverse its various phases " ( p . 123 ) . In addition , he notices that " problem structuring " ( in his model , the first phase of design development ) " occurs at the beginning of the task , … but may also recur periodically as needed " ( p . 114 ) . These 11 qualifications , based on studies in different application domains , have contributed much to the development of the position that there are important commonalities between the implementations of design in different domains , that is , one of the two components of the generic - design hypothesis . Despite the more or less implicit adherence to this hypothesis in the design literature , there has been little systematic empirical research , however , to corroborate it—that is , apart from Goel and Pirolli ' s ( 1989 ; 1992 ) work . In the rest of this section , we present some rare studies concluding to the existence of more or less similar features between designing in different situations . There is a series of early cognitive design studies conducted by Carroll and various colleagues in different design disciplines . In their review of this work , Thomas and Carroll ( 1979 / 1984 ) conclude that software design , architectural design , naming , and letter writing have many commonalities ( as noticed in our introduction ) . Bringing together observations gathered on product design ( in the Delft study , Visser , 1995 ) and on software design ( Visser , 1987b ) , we concluded that designers from these two disciplines proceeded to reuse . Reymen et al . ( 2006 ) have performed empirical case studies in three design disciplines—architectural , software and mechanical design—in order to develop domain - independent design knowledge . The authors This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 conclude that the supposed " important differences " between these design disciplines " concern mainly differences in terminology " ( p . 151 ) . One may notice , however , that the authors did not observe designers at work . They conducted interviews with designers concerning particular projects and analysed the documentation of these projects . In his paper How is a piece of software like a building ? Toward general design theory and methods , Gross ( 2003 ) advances the thesis that these two types of artefacts are alike on several dimensions : their size , level of complexity , lifetime , and degree to which their components are subject to change , the proportion of reusable components in their structure , the sanitary risks and safety concerns that particular uses or states of these artefacts may introduce , the type of their use or user , and the differences between their client and user . However , Gross ( 2003 ) does not refer to empirical work . We will come back to most of these dimensions below . Notice that in the abovementioned studies , presented in order to show commonalities between designing in different situations , these different " situations " were always different domains of discipline—never different conditions of age , sex , expertise , or working conditions ( e . g . , process variables ) , for example . 2 . Design is different from nondesign The idea that design significantly differs from nondesign activities is stated explicitly less often than its counterpart in the generic - design hypothesis ( that is , that design activities implemented in different situations are significantly similar ) . Cross ( 2001b ; 2002b ) contends—as the underlying axiom of the design discipline he defends— that there are " forms of knowledge special to the competencies and abilities of a designer . " Yet , it is not trivial to indicate what makes design specific . Expertise : design versus nondesign . In his overview paper on design expertise , Cross ( 2004b ) concludes that " expertise in design has some aspects that are significantly different from expertise in other fields " ( p . 427 ) . Referring to results from several empirical studies , he observes that the classical depth - first ( novices ) - breadth - first ( experts ) ( or top - down - bottom - up ) difference is not as systematically displayed in design as in This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 other problem - solving tasks . The other main characteristics identified by Cross ( 2004b ) are the following . A key feature of design expertise is " problem framing , " that is , structuring and formulating the problem . Expert designers are solution - focused rather than problem - focused , especially in their particular domain of expertise ( cf . also Lawson ' s , 1979 / 1984 , results differentiating students from architecture and science on the solution - focused - problem - focused dimension ) . Expert designers frequently switch between different types of cognitive activity . Contrary to what is considered " good practice " in design methodology , they readily commit to an early solution concept that they elaborate , at all costs , patching it , if necessary : they do so instead of generating and examining a large number of alternative solution concepts , and of abandoning their solution concept when confronted with problems in its development ( cf . point vii in Section 1 ) . Advocating that , from a cognitive viewpoint , design is definitely different from other activities , Goel and Pirolli ( in Goel , 1994 ; Goel & Pirolli , 1989 ) quoted the examples of chess and medical diagnosis . These were , however , not the nondesign tasks examined by the authors in order to corroborate their generic - design hypothesis . To this aim , they compared protocols ( collected by Newell & Simon , 1972 , and published in their Human Problem Solving ) concerning cryptarithmetic and the Moore - Anderson logic task with protocols the authors themselves had collected in three design disciplines , architecture , mechanical engineering , and instructional design ( Goel , 1994 ; Goel & Pirolli , 1989 ) . In their conclusion , the authors notice that , now that results have been obtained with such extreme well - structured tasks ( cryptarithmetic and the Moore - Anderson logic task are two perfectly defined play problems ) , other activities need to be examined . In The Sciences of the Artificial , Simon ( 1969 / 1999 ) presents as different the cognitive activities implemented in economics and in design . Analysing economic theories , he is very sensitive to the way in which economists idealise human rationality and neglect its limits . With respect to design , however , Simon seems to underestimate human cognitive limitations—something we illustrated in some detail in The Cognitive Artifacts of Designing ( Visser , 2006b ) . Several authors discuss differences between design and science ( cf . also our introductory section ) . According to Archer ( 1979 ) " there exists a designerly way of thinking and communicating that is both This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 different from scientific and scholarly ways of thinking and communicating , and as powerful as scientific and scholarly methods of enquiry when applied to its own kinds of problems " ( p . 18 ) . This idea of " designerly ways of knowing " is further developed by Cross ( 1982 ) ( see also Cross , 2006 ) . Lawson ( 1979 / 1984 ) compared students from architecture and science ( using artificial tasks supposed to represent architectural - design activities ) . He observed that the science students analysed their problems in order to discover their structure , whereas the design students generated " a sequence of high scoring solutions until one proved acceptable " ( p . 218 ) . Lawson ' s ( 1979 / 1984 ) conclusion has constituted one of the bases for distinguishing architects from scientists , encountered in papers qualifying architects as " solution - focused " and scientists as " problem - focused " ( see also Kruger & Cross , 2006 ) . Other studies seem to show , however , that a solution - focused approach is related to one ' s experience ( Cross , 2004b ; Lloyd & Scott , 1994 ) . In addition , various authors perceive elements of similitude between scientific and design activities . In his analysis of the structure of design processes , Dasgupta ( 1989 ) , for example , considers design problem solving a special instance of scientific discovery . In The Sciences of the Artificial , Simon ( 1969 / 1999 ) establishes a correspondence between social design ( social planning ) and scientific discovery : they share a type of search , that is , a " search guided by only the most general heuristics of ' interestingness ' or novelty " ( p . 162 ) . Cagan , Kotovsky and Simon ( 2001 ) point out the cognitive and computational similarities between the " seemingly disparate activities " of scientific discovery and inventive engineering design ( p . 442 ) . They notice that highly creative design activities are often labelled invention . The major conclusion of the authors ' comparison is that , " at a deep level , the cognitive and computational processes that accomplish [ design and discovery ] are virtually identical " ( Cagan et al . , 2001 , p . 463 ) . These underlying cognitive activities are based on " problem solving , pattern recognition , analogical reasoning , and other cognitive knowledge retrieval mechanisms " ( pp . 452 - 453 ) . The authors thus defend , with respect to scientific - discovery and inventive engineering - design problems , their " nothing special position " ( see point ii in Section 1 ) . They establish , however , a " fundamental difference " between the two : " the goal of the process : Scientific explanation versus creation of a new artifact . . . . Design starts with a desired function and tries to synthesize a device that produces that function . Science starts with an existing function and tries to synthesize a mechanism that can plausibly accomplish or This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 account for that function " ( Cagan et al . , 2001 , p . 455 ) . We do not share this reserve concerning the goals of the two activities : in our view , ultimately science ( be it discovery or invention ) is a design activity , the artefact aimed here being a theory . In short , except with respect to expertise , there is little direct evidence for design differing significantly from nondesign . Indirect evidence might come from the previous section . Implicitly , our stance is that the characteristics presented in Section 1 are specific to design and make design differ from nondesign . However , we cannot refer to empirical studies that show this . 3 . Design is one , but takes different forms The idea that there may be different forms of design has been hinted at in informal discussions , generally without empirical or theoretical evidence ( Löwgren , 1995 ; Ullman et al . , 1988 ) . Without any such underpinning , for example , the engineering - design methodologists Hubka and Eder ( 1987 ) assert that " the object of a design activity , what is being designed… substantially influences the design process . " This assertion expresses a rather generally—more or less implicitly—accepted idea , that is , that the artefact product , characterising the design discipline ( architecture , mechanical , or software design ) is the variable underlying the differences we are examining here . In " Variants in design cognition , " Akin ( 2001 ) states that " in different fields of design , cognitive processes have both similarities and differences " ( p . 105 ) . The author focuses on architectural design , generally contrasting it with engineering design in his paper . Compared to other designers , architects are more inclined to use ( i ) " rich representations , " ( ii ) creative , inventive strategies , ( iii ) non - standard problem decomposition schemata , ( iv ) complexity management strategies , and ( v ) search for alternative solutions . ( i ) On the basis of his extensive research on architects , Akin ( 2001 ) affirms that these designers use many forms of both analog and symbolic , " naïve , " everyday and physical , technical , and domain - specific representations . ( ii ) Confronting both informal observations and experimental work ( Akin , 1986a ) on architects and experimental results concerning electrical engineering designers obtained by Ball et al . ( 1997 , quoted in Akin , 2001 ) , Akin ( 2001 ) considers as plausible that " architects tend towards creative design strategies while engineers tend to routine design . " His comparison between the architects in his Akin ( 1986a ) study This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 producing a richness in novel solutions to constrained , closed problems , and the engineers observed by Ball et al . ( 1997 , quoted in Akin , 2001 ) generating remarkably low numbers of solutions , leads him to suppose that " these engineers tend to apply routine - design strategies even when the problem calls for a novel solution . " ( iii ) Akin ( 2001 ) opposes the conclusion reached by himself and by colleagues that architects adopt individual decomposition schemata , to that formulated by research colleagues ( Frankenberger , 1997 , and Dörner , 1997 , quoted in Akin , 2001 ) that mechanical engineers and industrial designers use standardised schemata . This holds for both the decomposition of the global design process into design phases and that of a larger problem into smaller ones . ( iv ) For Akin ( 2001 ) , the way in which designers recompose a comprehensive design solution from partial ones as an indicator of the way in which they manage complexity . Based on a study he conducted in 1994 ( referred to in his chapter ) , Akin ( 2001 ) explains how architects use ad hoc strategies to integrate partial solutions into global ones . He opposes this approach to the predetermined procedures that electronic or mechanical designers use to handle the interaction between the parts of a VLSI circuit or a mechanical assembly . ( v ) Akin ( 1986a ) observed that architects continue their search for alternative solutions even if they have already formulated a satisfactory concept . They do not commit themselves prematurely to an early selected kernel idea , something that is often considered a general characteristic of designers ( cf . characteristic iv in Section 1 ) . Concerning the second point : various authors have observed that other designers than architects—for example , product designers ( Dorst & Cross , 2001 ; Rodgers , Green , & McGown , 2000 ; Van der Lugt , 2002 ) — may also act in creative , flexible ways ( something distinctive for architects , according to Akin ) . Concerning the last point , we have requested more inspection of the basis for the conclusions about premature commitment that are generally advanced in the cognitive design research literature ( Visser , 2006b ) . There are experimental results supporting Ball and Ormerod ' s ( 2000 ) hypothesis that design induces early fixation on a kernel idea when designers are working individually and / or in artificially restricted situations , and that professional designers collaborating in " natural , " real work situations may come up with more alternative solutions ( Visser , 1993a ) . There are , however , also studies ( 1 ) on teams working in de - contextualised situations that show designers willing to reconsider early concepts ( Smith and Tjandra , 1998 , referred to in Cross , 2001a ) , and ( 2 ) on individually working designers who come up with several solution ideas , ( 2a ) while they are working in artificially restricted conditions ( Eastman , 1969 : bathroom design ; This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Fricke , 1999 : engineering design ; Whitefield , 1989 : mechanical design ) , but also ( 2b ) in natural situations ( Reitman , 1964 : musical composition ) ( see also Atman , Chimka , Bursic , & Nachtmann , 1999 , quoted in Cross , 2004b ) . The apparent contradiction between these observations might be removed in at least two ways . ( i ) Designers may aspire—or simply think or declare—to refrain from premature commitment , but in fact not put these ideas in practice ( see also Malhotra et al . , 1980 ; cf . our observation that designers ' accounts about their activity often do not coincide with their actual activity , Visser , 1990 ) . ( ii ) Early on in the design process , working at a conceptual level , designers may select a kernel idea , but afterwards they may refrain from premature commitment at a more concrete or detailed level , for example , by not fixing all values for its variables . Purcell and Gero ( 1996 ) have observed a difference between mechanical and product designers as regards their susceptibility to use features of example designs . In certain situations , mechanical designers showed " [ design ] fixation in the traditional sense of reproducing the characteristics of [ an example ] design , including incorrect features " ( p . 381 ) . They did so when " the example shown embodied principles that were typical of the knowledge base of the discipline " ( p . 381 ) . However , when they received innovative design examples , they seemed to " identify [ the core innovative principle involved in the example ] and then explore how this could be used in the particular design situation " ( p . 381 ) , leaving out of the designs they produced many of the specific aspects of the example . With the product designers , the fixation effect was completely absent . However , to produce innovative designs , these designers did not use the innovative examples either . Maybe they became " fixated " " on being different " ( p . 381 ) ; maybe their search was for difference rather than for innovation ( p . 380 ) . The authors suggest two sources for the observed differences between the designers from the two disciplines . First , education : that of product designers may emphasize creativity and the search for many different ideas . Second , the more or less varying and / or articulated character of knowledge in a domain : " the areas of knowledge that make up industrial design are more diverse than those studied in mechanical engineering " ( p . 374 ) and many of them " are associated with less well articulated bodies of knowledge than those that make up the knowledge base of mechanical engineering . For example , aesthetics plays a prominent role in industrial design education… , while it plays little formal role in mechanical engineering " ( pp . 374 - 375 ) . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Through several studies of designers from different domains working in their daily professional situation ( software and various types of mechanical design ) , we have been able to identify differences between such professionals working on industrial or other commercial design projects and design - knowledgeable participants ( generally students ) solving design problems in artificially restricted situations ( generally laboratory experiments ) ( Visser , 1995 , 2006b , 2006c ) . Three notable differences are the following . ( 1 ) In software , mechanical , or other professional design projects , designers organise their activity in an opportunistic way , whereas in simpler , more restricted situations , designers are often able to follow systematic decompositional approaches ( as generally prescribed by normative methods , e . g . top - down , breadth - first ; cf . our discussion above in Section 1 ) . ( 2 ) Reuse of elements from previous projects seems a specific professional design approach—even if it has also been observed in experimental research ( Détienne , 2002 ) . ( 3 ) In our study of a professional software designer ( Visser , 1987b ) , we noticed that user considerations were among his guiding principles ( leading him , for example , to adopt certain variable - naming strategies ) , an observation that has not been mentioned by researchers studying design in artificially restricted situations . These observations , which do not seem specific to a particular domain of design , may point to the influence that is exerted on the activity of design by ( 1 ) design education , ( 2 ) the complexity of a design project , and ( 3 ) the design setting . In the cognitive design research literature , one frequently encounters allusions to , or implicit testimonies of the specific character of software design compared to other types of design ( see Visser , 2006b ; 2006c for a discussion of these attitudes ) . Even if design of HCI is much less the object of discussion in this context , researchers studying software design or HCI have themselves also contrasted their domain of research with other domains . The responsible variables remain , however , unexplored . In their bibliographic cocitation analysis , Atwood , McCain , and Williams ( 2002 ) found that a set of authors representing Software Engineering design methodologies was " essentially unconnected with the remainder of the author set " ( p . 129 ) . They noticed , " software design has its own design literature " ( p . 132 ) . On the other hand , generic design journals , such as Design Studies , Design Issues , or The Journal of Design Research rarely publish papers on software or HCI design . The separation between software and HCI , and other types of design holds for scientific events as well . Conferences in the domain of design research concern either software design and / or HCI ( i . e . , treated either together or singly ) , or other types of design . Of course , there are specialised This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 conferences in many domains of design , but when they announce , as their object , " design " without any further specification , conferences generally do not expect papers on software or HCI ( for a list of example references , see Visser , 2006b , Section . 22 . 1 ) . Refining our analysis initiated in Visser ( 2006b ; 2006c ) and continued in Visser ( 2006a ) , this paper proposes three dimensions that we suppose underlie differences between forms of design : the design process , the designer , and the artefact . Under each of these dimensions , we propose several variables . 3 . 1 PROCESS Various process - related variables may affect designers ' cognitive structures and activities , and the designs they produce . We identified the organisation of the design process , the tools used , and the place of the user in the design process , possibly specified into two or more variables that are more specific . 3 . 1 . 1 The organisation of the design process The way designers plan to organise their task or the process they are involved in is liable to influence their activity . Be the organisation imposed by one ' s hierarchy , or devised by oneself , it works as other tools : it not only structures , but also guides people ' s activity , through immaterial and material means , such as design methods and other tools , be they representational , or calculation and simulation aids ( cf . subsection 3 . 1 . 2 , Tools in use ) . The time scale of the design process . Design is considered an off - line activity . One might thus naively suppose that designers , contrary to , for example , controllers of dynamic situations , have all their time to think over their projects , to analyse and change views , to discuss and confront their opinions with colleagues . The reality is different . First , most industrial , or other professional design projects generally take place under temporal constraints . Their stringency , however , may differ depending on external organisational ( due to the workshop or the client ) , artefactual , and other factors . Second , planning—both as a design activity in itself and as a component of other design activities ( Visser , 1994b ) —is obviously subject to temporal variables . Several early empirical studies have examined the role of temporal constraints in the context of planning , for This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 example , the famous study on route - plan design by Hayes - Roth and Hayes - Roth ( 1979 ) . We will come back to temporal constraints in our discussion of " designing in space versus designing in time " ( section 3 . 3 . 3 ) . Individual versus collective design . Certain artefacts are designed generally by an individual designer , others are usually the work of a team . Complexity and size of the artefact ( two dimensions mentioned by Gross , 2003 ) may play a role in this association , but are certainly not the only variables . Product design is often performed by individual designers , whereas many engineering design projects are conducted collectively— but , of course , these are only tendencies . We have defended elsewhere that there is no reason to suppose that cooperation modifies the nature of the basic cognitive activities and operations implemented in design ( i . e . , generation , transformation , and evaluation of representations ) ( Visser , 1993a ) . Because cooperation proceeds through interaction , it introduces , however , specific activities and influences designers ' representational structures ( both on sociocognitive and emotional levels ) . Some examples of such activities are coordination , operative synchronisation , construction of interdesigner compatible representations , conflict resolution , and management of representations that differ between design partners through confrontation , articulation , and integration . Activities involving argumentation—that is , in our view , activities aiming to modify the representations held by one ' s interlocutors—obviously play a particularly important role . The construction of interdesigner compatible representations ( Visser , 2006b , 2006c ) , their existence beside designers ' private representations , and their management introduce factors that may add complexity to collective design situations compared to individual design . 3 . 1 . 2 Tools in use Given our view of design as the construction of representations , we privilege representational tools in this discussion , especially concerned with external representations and the means to produce them . Designers ' internal ( mental ) representations evidently also play a crucial role in their activity , but these representations are mainly dependent on other components of the situation and on individual factors . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Design methods . By definition , designers proceed differently depending on the method they follow . In the domain of software design , several authors have compared the use of different design paradigms ( in the sense of design methods ) and observed differences , both with respect to activities and to resulting designs . Lee and Pennington ( 1994 ) , for example , have shown these two types of differences between software design using an object - oriented and using a procedural paradigm . With respect to the activity , the differences concerned the domain and solution spaces developed , the duration of problem domain analysis and of solution evaluation . As also observed by other authors ( see references in Lee & Pennington , 1994 ) , the resulting designs " [ reflected ] fundamentally different models of the solution . Procedural design methodologies result in designs in which the modules represent procedures that complete subparts of the task , whereas object - oriented methodologies result in modules that represent objects in the environment " ( p . 581 ; for other differences , see the paper ) . Kim and Lerch ( 1992 ) , in their comparison between object - oriented ( OOD ) and " traditional functional decomposition " ( TFD ) methodologies , expected " OOD to radically change the cognitive processes in logical design " ( p . 491 ) . Based on the preliminary results obtained in a pilot study , the authors noted that " OOD may achieve substantial time savings over TFD in logical design . … 1 ) by simplifying rule induction processes used in functional decomposition ; 2 ) by guiding designers on how to build more effective problem spaces ; and 3 ) by allowing designers to run mental simulation more efficiently and more effectively " ( p . 489 ) . The maturity of a domain may influence the availability—and thus the use—of tools . In 2004 , the NSF launched a " Science of Design " program aiming to " develop a set of scientific principles to guide the design of software - intensive systems " ( Science of Design , 2004 ) . An underlying idea was that " in fields more mature than computer science [ such as architecture and other engineering disciplines , for example , civil or chemical engineering ] , design methodology has traditionally relied heavily on constructs such as languages and notational conventions , modularity principles , composition rules , methodical decision procedures and handbooks of codified experience . … However , the design of software - intensive systems is more often done using rough guidelines , intuition and experiential knowledge . " This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 As noticed above , research in the domain of software design has shown that design methodologies may have an influence on the activity and the resulting design . One may suppose that being familiar with the constructs and other tools that have been developed in a domain may influence , probably facilitate , a designer ' s activity—even if cognitive design research has shown the difficulty of designers ' effectively working according to design methodology prescriptions ( Carroll & Rosson , 1985 ; Visser , 2003 ; Visser & Hoc , 1990 ) . One may notice that related to the idea that underlies the present variable and that is only touched upon here , is the question of well - defined versus ill - defined problems and the implications for the nature of the activities involving these problems ( see Visser , 2006b , 2006c ) . From a cognitive - activity viewpoint , most or all ill - defined problems might be analysed as design problems ( Visser , 1993b ) . Going one step further , Falzon ( 2004 ) proposes to adopt design as a paradigm for analysing all problem - solving activities . Eventually , Falzon posits , each design problem becomes a state - transformation problem ( the type of problem typically examined in classical cognitive - psychology laboratory research ) , because of people ' s acquisition of expertise and habits , and of technological evolution . Falzon nevertheless also notes the possibility that there will always remain multiple situations in which people refuse themselves to refer to procedures and routines . As an example , he refers to a study by Lebahar concerning painters who try to establish conditions that rule out the possibility to refer to routines . External representations . According to Zhang and Norman ( 1994 ) , external and internal representations differentially activate perceptual and cognitive processes . With Scaife and Rogers ( 1996 ) , we presume that things are less systematic , and more complex . Nevertheless , we suppose that the use of internal and that of external representations involve processing differences . Therefore , designing may differ between situations depending on the importance of certain types of representations . One may suppose that , for example , design of physical artefacts ( e . g . , architectural or mechanical design ) differs from design of symbolic artefacts ( e . g . , procedures or organisations ) . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Indeed , one of the factors underlying the differences between software and other types of design that are often stressed may be due to the different types of representations primarily used . A classical result in cognitive psychology research on external representations concerns the influence of representation " formats " on problem solving . The standard approach to show this effect has been to compare the way in which isomorphic problems were solved ( for example , the Tower of Hanoi and the Tea Ceremony ) . Another difference in format is that between alphanumeric and figurative representations . The possibilities provided by sketches and other types of drawings compared to those offered by purely alphanumeric representations , for example , with respect to the ease of visualisation and manipulation and their corollaries may facilitate simulation and other forms of evaluation of what are going to become physical artefacts . This observation surely does not only apply to classical ( i . e . , nonvisual ) forms of software design . It probably also holds for other symbolic artefacts , such as other types of procedures , plans , and organisational structures . According to Akin ( 2001 ) , architects differ from designers in other domains with respect to their relatively more frequent use of ( 1 ) analogue compared to symbolic representations and ( 2 ) varying representations . The author attributes this greater representational variety to architectural design ' s situated and user - dependent character . Akin also points to the lack of universally accepted representational standards in architecture ( cf . other elements of Akin ' s position presented in the introduction of Section 3 ) . We already put into perspective Akin ' s view that architectural designers are particularly resourceful and flexible . However , the lack of standards , be they universally accepted or not , may indeed be of influence ( see also our remark concerning The maturity of a domain , Section 3 . 1 . 2 ) . According to Zimring and Craig ( 2001 ) , disciplines of practice differ with respect to the ease or even the possibility for users to understand the intermediate and final representations of the artefact generally used in the domain . They assert that , for example , architectural drawings of layout or appearance of the building to be designed " are at least theoretically understandable by end - users… , resulting in patterns of collaboration , testing and accountability that differ significantly from those associated with more ' invisible ' design This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 processes . An engineer , for example , working on a car engine is not likely to collaborate with end users directly , given the difficulty the average car driver has in understanding the mechanics of engines . " ( p . 128 ) With respect to the role of representation , we wish to state explicitly that the importance of its role undeniably also depends on the designer ( discussed in Section 3 . 2 ) . Possible means for evaluation . Domains differ in the methods and other tools that may be used in order to evaluate design proposals ( Malhotra et al . , 1980 , pp . 129 - 130 ) . In engineering , more or less " objective " measures and other criteria for future artefacts ' performance can be used and different proposals can be ranked rather objectively . One can calculate whether a particular design ( e . g . , a bridge ) meets particular functional requirements , such as accommodation and maximum load . The results of qualitative evaluation used in other domains , based on subjective criteria such as aesthetics , for example , may be more difficult to translate into a " score , " and thus to compare . In between the extremes of completely objective and entirely subjective evaluation , exist different types of simulation , physical and mental . 3 . 1 . 3 The user in the design process Designers design for other people , the " users " of the artefact product . In each domain of design , users are central—even if not always for the designers and even if the use of artefacts may ( seem to ) be more or less direct ( cf . also The artefact ' s impact in Section 3 . 3 . 2 ) . Naïvely , one might think , for example , that industrial design products ( such as , pens , chairs , household boxes ) are in " direct " use by their users , whereas the relation between the product of a city - planning project and its users is much less direct . Yet , domains differ with respect to their common practices regarding the way in which designers usually take into account the potential , future users and their use of the artefact product . Integration of user data into the design . In HCI , for example , there is a tradition and , correspondingly , considerable effort towards developing methods to integrate user data into the design . This has varied from such data being introduced into the design by design participants who " know " the users but are not users This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 themselves , to approaches such as participatory design in which the users have themselves a voice in the design process ( Carroll , 2006 ) . It seems likely that the number and variety of participants who take part in a design process influence this process , probably more its socio - organisational than its cognitive aspects ( see also Section 3 . 1 . 1 , Individual versus collective design ) . Yet , on a cognitive level , the difficulty of integration may increase with the number of different representations to be integrated—thus with the number of types of participants . In addition , the participation of " nontechnical " design participants ( what users generally are ) may introduce a specific difficulty , both for the nontechnical participants themselves and for their professional design colleagues . Gross ( 2003 ) mentions two specific user - related variables on which a piece of software is like a building : the difference or equivalence between client and user , and the type of use or user , which may change more or less , or may remain constant . These two variables get a particular weight in the context of the abovementioned influence that number and variety of participants may have on the design process . 3 . 2 DESIGNER : INTERINDIVIDUAL DIFFERENCES Differences between designers may affect both their activities and their representations . This influence may occur by way of one or more of the variables proposed hereafter . The use of certain types of representations or other tools may influence design thinking , but a particular designer may be more inclined to adopt a particular type of representation , or feel more at ease with its use . 3 . 2 . 1 Design expertise A classical cognitive - psychology result confirmed in cognitive design research is that experts and novices in a domain differ as to their representations and activities . Experts , for example , " recognise underlying principles , rather than focusing on surface features of problems " ( Cross , 2004b , p . 432 ) . Expertise has been examined in many experimental studies on design ( Chi , Glaser , & Farr , 1988 ; Cross , 2004a , 2004c ; " Expertise in Design , " 2004 ; Glaser , 1986 ; Glaser & Chi , 1988 ; Reimann & Chi , 1989 ) . In Section 2 , we saw that besides novice This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 designers differing from expert designers , design expertise in itself differs from that in other fields ( Cross , 2004b ) . We have proposed to distinguish , in addition to levels of expertise , types of expertise ( Falzon & Visser , 1989 ; see also Visser & Morais , 1991 ) . We have indeed observed how designers who were " experts " in the same domain , but had different prior experience in that domain ( workshop vs . laboratory ) , exhibited ( 1 ) different types of knowledge and ( 2 ) different organisations of their knowledge—a result comparable to that regarding levels of expertise . 3 . 2 . 2 Routine character of a task The routine character of a task is not an objective characteristic of the task , but depends on the representation that people construct of it . This task characteristic is thus dependent on interindividual differences . Most design projects comprise both routine and nonroutine tasks . In a comparative analysis of three of our empirical design studies , we have established a link between the more or less routine character of a design project and the way in which analogies are used ( at the action - execution and at the action - management levels ) ( Visser , 1996 ) . This , in turn , influences , at least in part , the possibilities a designer has for reuse in a design project . 3 . 2 . 3 Idiosyncrasies " The reality of professional design practice seems to be that individual designers have differing design abilities—some designers just seem to be better than others , and some are outstandingly good . " ( Cross , 2002a , p . 14 ) In addition to studies comparing experts and novices , there are clinical studies on experts that have led researchers to identify specific characteristics of particular experts ( Cross , 2001c , 2002a ) . It seems , for example , that , contrary to most architects , Frank Lloyd Wright could imagine and develop a design entirely without using external representations , not sketching or drawing , be it until an advanced stage of the project ( Tafel , 1979 , quoted in Ball & Christensen , 2007 ) , or even until the very end of the design process ( Weisberg , 1993 , quoted in Bilda & Gero , 2005 ) . Cross ( 2002a ) , who mentions several studies on exceptional designers , presents three case studies he has performed himself on creative design in engineering and product design . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Concerning the three designers whom he considers " exceptional , " Cross ( 2002a ) notices that " there appear to be striking similarities in their design strategies , which suggest that general models might be constructed of design expertise and creative processes in professional design practice " ( p . 14 ) . 3 . 2 . 4 Different " types of people " An idea often encountered , especially among architects themselves , is that , rather than possessing idiosyncratic characteristics , architects ( as members of the architectural profession ) are of a " special kind , " a different " type of people " than other designers , especially , software designers or engineers . Architects would have a different " personality " : they would be , for example , more creative and more aesthetics - oriented . Several researchers , architects themselves or not , also defend such an idea . Akin ( 2001 ) , for example , considers that architects attribute another value to creative and unique designs than other designers do . Besides advancing more tangible differences presented above ( see the introduction of Section 3 ) , Akin ( 2001 ) claims that " the profession of architecture rewards the heart while engineering rewards the brain " ( p . 105 ) . He notes explicitly , however , that the specificities of architects are not biological or physiological , " or even fundamental intelligence related . " Akin ( 2001 ) believes that " it is a matter of ethos and culture fostered in a given profession , its educational philosophy and the predisposition of its participants . " " Personality " is a familiar notion in psychology ( nearly every theoretical tradition in psychology has its own personality theory ) , which , as many notions from the social sciences and humanities , is widely used outside of this discipline . The personality - related difference between architects and other designers , however , does have no scientific basis : we are unaware of any empirical study concerning possible " personality differences " between designers working in different domains . 3 . 3 ARTEFACT We have identified three artefact variables : social embeddedness , type of artefact ( instantiated by structures versus processes ) , and artefacts ' evolution . Gross ( 2003 ) proposes the proportion of reusable components in the artefact ' s structure as one of the factors that make " a piece of software like a building , " but we do not have any data or hypotheses concerning such a variable . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 3 . 3 . 1 Social embeddedness Referring to Rittel and Webber ( 1973 / 1984 ) , Zimring and Craig ( 2001 ) point to the social embeddedness of planning and design problems . In their analysis of societal planning problems as " wicked , " Rittel and Webber ( 1973 / 1984 ) indeed attribute the major part of this wickedness to the social embeddedness of these problems . Neither Rittel and Webber ( 1973 / 1984 ) , nor Zimring and Craig ( 2001 ) define the notion . Our definition is based on Edmond ( 1999 ) s ' constructivist approach : social embeddedness refers to the extent to which an appropriate characterisation of an agent ' s activities and representations requires that one include the agent ' s social environment ( " the society of agents " in Edmond ' s terms ) as a whole in the characterisation . We consider that it is not an externally defined task that is more or less socially embedded , but people ' s representations involved in dealing with it . Qualifying a design project as socially embedded is then shorthand for qualifying as such the designer ' s representations of that project ( an analogous remark holds for " a project ' s ill definedness " as shorthand for " the ill definedness of the representations that the designer has constructed of the project " ) . A project can become socially embedded because the designers or other stakeholders consider necessary to take into account the insertion and future position of the artefact in its environment ( characterised by its users and / or the global society ) . In apparent opposition with what we advanced above concerning user involvement in design , one might think that a product design project is less socially embedded than an urban project ; that development of HCI involves more social embeddedness than traffic signal design , for example . Given our view that the view held by the designers or other stakeholders makes a project socially embedded , this quality is not limited to what are generally considered " social " or " societal " problems . Even if societal planning problems generally will be socially embedded , this characteristic is not specific to planning problems : for example , planning one ' s route through a city ( Chalmé , Visser , & Denis , 2004 ; Hayes - Roth & Hayes - Roth , 1979 ) or planning a meal ( Byrne , 1977 ) are not necessarily typical instances of socially embedded problems . Yet , planning a meal can become socially embedded depending on the meal " designers " ' view of their guests and of the consequences This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 occasioned by the meal ' s more or less greater " success " —and as such , designers ' activity will be influenced by their view . The influence of social embeddedness on a designer ' s activities is probably similar to that of ill definedness . For example , socially embedded problems probably have various , different solutions . These solutions may be considered more or less appropriate , more or less acceptable , depending on the criteria adopted by the person who judges them . A related issue in which societal questions play an important role is the consideration of an artefact ' s user in the design of the artefact ( see Section 3 . 1 . 3 , The user in the design process ) . Winograd ( 1996 ) considers that its user - oriented character makes software design comparable to architectural and graphic design , and different from engineering design . He considers , however , that the design of interactive software is completely different from other software design ( Winograd , 1997 ) . Among the arguments advanced for these claims , none is based on cognitive analyses of the activity . Simon ( 1969 / 1999 ) , in Sciences of the Artificial , implicitly establishes a radical distinction between design activities in engineering and in social design . In his discussion of social planning , Simon ( 1969 / 1999 ) states that " representation problems take on new dimensions " in this form of design ( and , maybe , also in inventive engineering design , see Visser , 2006b , 2006c ) compared with the " relatively well - structured , middle - sized tasks " of engineering and architectural design ( p . 141 ) , which he presents—implicitly—as the prototypes of design . For " real - world problems of [ the ] complexity " of social planning , Simon considers that designers may refer to " weaker " criteria than in the case of standard design . Processes such as " search guided by only the most general heuristics of ' interestingness ' or novelty " may provide " the most suitable model of the social design process " ( Simon , 1969 / 1999 , p . 162 ) . With respect to the sources of social design problems ' greater " complexity , " Simon suggests that differences of at least three types may be involved : problems ' degree of structuredness ( here qualified as " definedness " ) , their size , and the nature of their object ( see our detailed discussion of these differences in Visser , 2006b , 2006c ) . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 It seems that only when he discusses social problems ( and perhaps scientific discovery problems , see the introduction to Section 2 Design is different from nondesign ) that Simon seriously considers human bounded rationality and takes into account the role of what he calls " representations without numbers , " generative constraints such as " interestingness " or " novelty , " and critical constraints such as the " defensibility " of a decision . The hypothesis that we have formulated in order to explain—at least , in part—this view of design adopted by Simon is that he considers ( 1 ) ( routine ) engineering and architectural design as standard design , and ( 2 ) social planning as radically different from standard design . In ever more domains , people become convinced of the societal aspects of their action . One may suppose that this evolution will have its influence on design . " The common thread in the new approach to traffic engineering is a recognition that the way you build a road affects far more than the movement of vehicles . It determines how drivers behave on it , whether pedestrians feel safe to walk alongside it , what kinds of businesses and housing spring up along it . " ( McNichol , 2004 ) ( see also tendencies such as ecodesign , ecological design , and sustainable design , see , e . g . , Méhier , 2005 ) ( cf . the next subsection , where we discuss the influence of users ' interaction with artefacts on designers ' activities—especially those related to the anticipation of the artefact ' s behaviour over time ) . 3 . 3 . 2 Artefacts ' evolution " Interactive systems are designed to have a certain behavior over time , whereas houses typically are not , " according to Löwgren ( 1995 , p . 94 ) . Even if this assertion is questionable with respect to " behaviour " in general , behaviour over time is a variable on which artefacts differ—and the types of behaviour of different artefact products are quite diverse . An artefact ' s behaviour over time may be related to its impact on people ( the " transformative " nature of artefacts , see Carroll , Rosson , Chin , & Koenemann , 1998 ) , through the interaction that people engage in , and to its use by people who are not necessarily transformed by this use . It may also be due to its deterioration , dependently or independently of people . Two variables introduced by Gross ( 2003 ) are the degree to which components of the artefact may be subject to change or renewal , and the more or less extended lifetime of the artefact . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 All artefacts change over time . Houses may not display " behaviour " over time , but they change . Systems such as organisations or interactive systems are subject to specific types of change . Designers are supposed to anticipate the transformation that their artefact products undergo—be it of deterioration or another evolution type . The possibility of anticipation may vary between situations ( domains ) , not necessarily depending on the degree of impact . It depends , among others things , on the possibility to simulate the artefact , or to test it in another way . For interactive artefacts , anticipation may be performed through simulation . The future behaviour of certain technical artefacts may be anticipated based on calculations . The artefact ' s impact on people ' s activity and the possibility to anticipate it . Predicting people ' s future use of an artefact product and further anticipating the impact of the product on human activity , is one of the " characteristic and difficult properties " of designing ( Carroll , 2000 , p . 39 ) . Indeed , " design has broad impacts on people . Design problems lead to transformations in the world that alter possibilities for human activity and experience , often in ways that transcend the boundaries of the original design reasoning " ( Carroll , 2000 , p . 21 ) . Gross ( 2003 ) mentions sanitary risks and safety concerns that particular uses or states of an artefact may introduce . Even if all design has impact on people , certain domains seem more sensitive than others are . HCI , with which Carroll ( 2000 ) is especially concerned in his discussion quoted above , is an example of a domain in which design has particularly broad impacts on people . Yet , this holds for all design with societal implications . Distance between intermediary representations and final product . The design of an artefact is a different activity than its implementation ( Visser , 2006b , 2006c ) . For certain types of artefacts , however , there seems to be a relatively fluid , steady transition between the different forms that the design concept may take and the final artefact product—what may be qualified as a shorter " distance " between the two . Symbolic artefacts , such as software , are an example . This might elucidate somewhat our observation that software designers find it particularly difficult to separate design from coding ( Visser , 1987b ) . It does not imply , however , that design and implementation are not distinct for symbolic artefacts . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 It is with respect to the distance between the design concept and the final artefact product that Löwgren ( 1995 , p . 94 ) opposes architectural and engineering design to " external " software design ( " design of the external behavior and appearance of the product , the services it offers to users and its place in the organization " ) . Delay of implementation . Design is by definition concerned with artefact products that do not yet exist . A central aspect of designing is thus , once again , anticipation . The bases of this anticipation may vary depending on other variables ( users ' taking part and designer ' s knowledge , experience , and activities , such as simulation ) , but anyhow the conditions of existence , the behaviour , and the use of the artefact products will be more or less different from those anticipated : the world changes without possibility of being completely controlled . The implementation of certain types of artefacts is much longer in coming than that of others—and not because of laziness or indifference of the workshop or the client , or due to lack of resources . Voss et al . ( 1983 ) have noticed that the solving of social - science problems is particularly difficult because of the " delay from the time a solution is proposed and accepted to when it is fully implemented " ( p . 169 ) . Such a delay clearly complicates the anticipation of the artefact ' s evolution and other matters involved in its evaluation ( through simulation or other means ) . Even if this observation is particularly applicable to social - science problems , it may also hold for other types of design . 3 . 3 . 3 Type of artefact " Type of artefact " may seem an evident explanatory variable for the existence of different forms of design . As noticed already , however , few elements are available concerning underlying variables . An example is software design—often considered as " essentially different " from design in other domains , but without discussion or examination of the responsible variables . One candidate variable could be the difference between structures and processes . Data concerning the influence of this variable may come from results obtained in studies concerning what may be considered particular instances of structures and processes , that is , spatial and temporal entities . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Designing in space versus designing in time . Studies comparing problems governed by temporal and problems governed by spatial constraints have shown that designers deal differently with these constraints ( Chalmé et al . , 2004 ; detailed in Visser , 2006b ) . An example of design that preferentially implements temporal constraints is planning ( meal planning , see Byrne , 1977 ; route planning , see Chalmé et al . , 2004 ; Hayes - Roth & Hayes - Roth , 1979 ) . Research , however , has not yet settled clearly the specificity of the relative ease and difficulty involved in the corresponding types of design—it has even less identified the underlying factors . Structures ( which may correspond to states ) are not necessarily spatially constrained , but processes have systematically temporal characteristics . By analogy to the differences between the cognitive treatment of spatial and temporal constraints , one may expect that structures and processes are represented differently ( especially mentally , but also externally ) , thus processed differently , and therefore lead to different design activities ( cf . Clancey ' s , 1985 , distinction between configuration and planning ) . 4 . Conclusion In this section , we will first briefly review the generic - design hypothesis and then focus on the component of our augmented generic - design hypothesis that has been central in this text , that is , design also takes different forms . The validity of the generic - design hypothesis . Given the scarcity of empirical evidence , the generic - design hypothesis needs more research . We formulated above several points of reserve concerning the only systematic study ( Goel & Pirolli , 1989 ; 1992 ) , but we have presented , in Sections 1 and 2 , various elements of support for the hypothesis ' two components . The hypothesis requires , however , more research , especially more systematic research . The validity of the hypothesis that design also takes different forms . If because of the rare and disseminated empirical evidence , the generic - design hypothesis needs more research , this holds a fortiori for our hypothesis that design also takes different forms . Most variables proposed were based on a broad knowledge and analysis This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 of the results of some 25 years of cognitive design research ; they would need comparative studies about the hypothesised differences between design situations . For some of them , we were able to present precise empirical data : for differences between architectural design and engineering design ( Akin , 2001 ) , and between mechanical and product design ( Purcell & Gero , 1996 ) , between design in a professional project and design in an artificially restricted situation ( Visser , 1995 , 2006b , 2006c ) , between design activities and resulting designs in projects adopting different design methods ( Kim & Lerch , 1992 ; Lee & Pennington , 1994 ) ; for differences due to designers ' expertise ( for levels of expertise , see especially Cross ' work ; for types of expertise , see Falzon & Visser , 1989 ) ; for the influence of design projects ' routine character on the way in which designers use analogy ( Visser , 1996 ) , and the influence of idiosyncratic differences between designers ( shown for certain experts , see several references in the text ) . Therefore , in the analysis presented in this paper , we have introduced material that still requires further analysis , and indicated a number of directions—to be followed , modified , completed , and developed , in other research . It is conceivable that not all variables proposed have the same degree of influence . Given our view of design as the construction of representations , we suppose that variables related to representational structures and activities are particularly influential . Referring to classical research on the influence of representation formats on problem solving , we formulated the hypothesis that sketches and other types of drawings may facilitate certain activities ( such as simulation and other forms of evaluation ) , due to the augmented ease of visualisation and manipulation offered by such figurative external representations . Yet , variables may also depend on other underlying factors and their influence on the activity may exert itself by way of representational structures and activities . The variables and the characteristics of the different forms of activities and cognitive structures—if their influence were to be confirmed—may have implications for design support . Given the centrality of representation in designing , the development of appropriate support modalities for representational activities and structures already suggests itself—be such modalities technological ( generally , computerised ) or This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 methodological . However , according to the role of representation , and the type of representation preferentially used in specific design tasks and / or in specific design situations , the development of specific support modalities may be worthwhile . Research on these questions may take advantage of the progress already obtained in other domains , for example , those of software and HCI design . In those domains , there has been considerable research on visualisation and other visual tools , for example , on diagrammatic reasoning ( see Blackwell , 1997 , and the Diagrammatic reasoning site ; see also the research on multiple —external— representations , e . g . by Van Someren , Reimann , Boshuizen , & De Jong , 1988 ) . A question that might be asked after the presentation of all these—possibly—different forms of design is : if there are so many differences between the implementations of design thinking in different situations , then what about the idea that design is a " generic " activity ? In order to answer this question—and counter the underlying opposition to the generic - design hypothesis—we now come up with the fourth member of our augmented cognitively oriented generic - design hypothesis . In its complete form , we see this hypothesis as the following . ( 1 ) Design thinking has distinctive characteristics from other cognitive activities . ( 2 ) There are commonalities between the implementations of design thinking in different design situations . ( 3 ) There are also differences between these implementations of design thinking in different situations . ( 4 ) However , the commonalities between all the different forms of design thinking are sufficiently distinctive from the characteristics of other cognitive activities , to consider design a specific , generic cognitive activity . Given the hypothetical character of the third member , which was examined here , we did not mention this fourth member before . If one defends the idea of design as a generic cognitive activity , it is , however , the counterpart of the third member . At the end of this paper , this fourth member remains completely hypothetical This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 and requires new empirical research comparable to Goel and Pirolli ' s ( 1989 ) work—but preferably , in our opinion , performed in real , i . e . professional design situations . References Akin , Ö . ( 1979 / 1984 ) . An exploration of the design process . In N . Cross ( Ed . ) , Developments in design methodology ( pp . 189 - 207 ) . Chichester , England : Wiley ( Originally published in Design Methods and Theories , 1979 , 13 ( 3 / 4 ) , 115 - 119 ) . Akin , Ö . ( 1986a ) . 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In J . - M . Hoc , T . Green , R . Samurçay & D . Gilmore ( Eds . ) , Psychology of programming ( pp . 235 - 250 ) . London : Academic Press . Visser , W . , & Morais , A . ( 1991 ) . Concurrent use of different expertise elicitation methods applied to the study of the programming activity . In M . J . Tauber & D . Ackermann ( Eds . ) , Mental models and human - computer interaction ( Vol . 2 , pp . 59 - 79 ) . Amsterdam : Elsevier . Voss , J . F . , Greene , T . R . , Post , T . A . , & Penner , B . C . ( 1983 ) . Problem - solving skill in the social sciences . In G . Bower ( Ed . ) , The psychology of learning and motivation ( Vol . 17 , pp . 165 - 213 ) . New York : Academic Press . Voss , J . F . , & Post , T . A . ( 1988 ) . On the solving of ill - structured problems . In M . T . H . Chi , R . Glaser & M . J . Farr ( Eds . ) , The nature of expertise ( pp . 261 - 285 ) . Hillsdale , NJ : Lawrence Erlbaum Associates . Warfield , J . N . ( 1994 ) . A science of Generic Design : Managing complexity through systems design . Ames , IA : Iowa State Press . Whitefield , A . ( 1989 ) . Constructing appropriate models of computer users : The case of engineering designers . In J . Long & A . Whitefield ( Eds . ) , Cognitive ergonomics and human - computer interaction ( pp . 66 - 94 ) . Cambridge , England : Cambridge University Press . Winograd , T . ( 1997 ) . From computing machinery to interaction design . Retrieved July 21 , 2008 , from http : / / hci . stanford . edu / winograd / acm97 . html Winograd , T . ( Ed . ) . ( 1996 ) . Bringing design to software . New York : ACM Press . Zhang , J . , & Norman , D . A . ( 1994 ) . Representations in distributed cognitive tasks . Cognitive Science , 18 , 87 - 122 . This text is a Post - print of : Visser , W . ( 2009 ) . Design : one , but in different forms . Design Studies , 30 ( 3 ) , 187 - 223 . see linkinghub . elsevier . com / retrieve / pii / S0142694X08001051 doi : 10 . 1016 / j . destud . 2008 . 11 . 004 Zimring , C . M . , & Craig , D . L . ( 2001 ) . Defining design between domains : An argument for design research à la carte . In C . Eastman , M . McCracken & W . Newstetter ( Eds . ) , Design knowing and learning : Cognition in design education ( pp . 125 - 146 ) . Amsterdam : Elsevier Science . \ No newline at end of file diff --git a/silver_data/b1ba3c8a98b59a2e02b96f6d1dcf285ea0068d78.pdf.txt b/silver_data/b1ba3c8a98b59a2e02b96f6d1dcf285ea0068d78.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..c5b93e668ec0d0585cac7e5554dc17ad133d3a3a --- /dev/null +++ b/silver_data/b1ba3c8a98b59a2e02b96f6d1dcf285ea0068d78.pdf.txt @@ -0,0 +1 @@ +T r a n s l a t i o n a l r e s e a r c h Affective neuroimaging in generalized anxiety disorder : an integrated review Gregory A . Fonzo , PhD ; Amit Etkin , MD , PhD Introduction G eneralized anxiety disorder ( GAD ) is a preva - lent and debilitating anxiety disorder that is associated with significant distress , functional impairment , and hu - man and economic burden . 1 , 2 The cardinal symptom feature of GAD is uncontrollable , pervasive worry and anxiety occurring more days than not for at least 6 months , occurring alongside somatic and emotional symptoms , such as restlessness , feeling keyed up or on edge , being easily fatigued , difficulty concentrating , ir - ritability , muscle tension , and sleep disturbance . In recent years , significant resources have been devoted to understanding the neurobiological mecha - nisms of the disorder . These efforts have profoundly shaped scientific understanding through noninvasive imaging methods , such as functional magnetic reso - nance imaging ( fMRI ) , which utilize intensity changes in a type of magnetic resonance ( MR ) signal to track hemodynamic changes in the brain . These hemody - namic changes ( known as the blood oxygenation – level dependent or BOLD response ) can be utilized as proxy measures for neuronal function , allowing researchers to infer what brain regions in GAD are activated dur - ing a given behavior , the degree to which these activa - Copyright © 2017 AICH – Servier Research Group . All rights reserved 169 www . dialogues - cns . org Keywords : amygdala ; emotion ; emotion regulation ; fMRI ; generalized anxiety ; neuroimaging ; prefrontal cortex ; worry Author affiliations : Department of Psychiatry and Behavioral Sciences , Stanford University School of Medicine , Stanford , California , USA ; Stan - ford Neurosciences Institute , Stanford University , Stanford , California , USA ; Veterans Affairs Palo Alto Healthcare System and the Sierra - Pacific Mental Illness Research , Education , and Clinical Center ( MIRECC ) , Palo Alto , California , USA Address for correspondence : Gregory A . Fonzo , PhD , 401 Quarry Road , MC 5722 , Stanford , CA 94305 , USA ( email : gfonzo @ stanford . edu ) Affective neuroimaging has contributed to our knowl - edge of generalized anxiety disorder ( GAD ) through measurement of blood oxygenation level – dependent ( BOLD ) responses , which facilitate inference on neural responses to emotional stimuli during task - based func - tional magnetic resonance imaging ( fMRI ) . In this article , the authors provide an integrated review of the task - based affective fMRI literature in GAD . Studies provide evidence for variable presence and directionality of BOLD abnormalities in limbic and prefrontal regions during re - activity to , regulation of , and learning from emotional cues . We conclude that understanding the sources of this variability is key to accelerating progress in this area . We propose that the cardinal symptom of GAD—worry—pre - dominantly reflects stimulus - independent mental pro - cesses that impose abnormal , inflexible functional brain configurations , ie , the overall pattern of information transfer among behaviorally relevant neural circuits at a given point in time . These configurations that are inflex - ible to change from the incoming flux of environmental stimuli may underlie inconsistent task - based findings . © 2017 , AICH – Servier Research Group Dialogues Clin Neurosci . 2017 ; 19 : 169 - 179 . T r a n s l a t i o n a l r e s e a r c h tions may be abnormal , and how the synchrony of brain function across two or more regions may be perturbed . In aggregate , these tools have provided much - needed insight regarding the functional brain abnormalities observed in the GAD phenotype . In this paper , the authors provide an integrated review of the emotional task - based fMRI literature in GAD . Resting state , cog - nitive studies , and pre / post treatment comparisons have been excluded from the scope of this review due to space constraints . The goal of this effort is threefold : ( i ) to synthesize and integrate the body of literature for the purposes of clearly disseminating key patterns of find - ings to the interested reader ; ( ii ) to highlight relevant trends over time in the design , conduction , and findings of GAD imaging studies ; and ( iii ) to provide future di - rections and general guidelines for continued research in this area . Methods In line with the scope of the review , we focused on iden - tifying studies that investigated emotional task - based BOLD fMRI responses in individuals with the diagno - sis of GAD . The following criteria were instituted : ( i ) primary publication in English ; ( ii ) primary comparison of a diagnostically defined GAD sample ( or subsample from larger studies investigating anxiety transdiagnos - tically ) against at least one healthy comparison group and / or other patient group ; ( iii ) use of BOLD fMRI during an affective task as a primary outcome measure ; and ( iv ) sample size within each cell of at least eight participants ( case studies were excluded ) . To identify studies , the authors conducted a literature search in PubMed using broadly defined search terms : [ “gener - alized anxiety” OR “generalised anxiety” OR “GAD” ] AND [ “imaging” OR “fMRI” OR “neuroimaging” ] . The scope of articles was further shortened through re - view of search results and assessment of whether each study met the above criteria . Results Using the search terms listed above , a total of 608 ar - ticles were initially identified . Individual review of each narrowed this pool down to 30 studies ( Table I ) . 3 - 32 We review the results below , subdivided by functional do - main assessed . Facial affect processing Numerous BOLD imaging studies in GAD have fo - cused on the assessment of brain responses to facial expressions of emotion , particularly fear . Fear faces ro - bustly activate limbic circuitry , such as the insula and amygdala , 33 which are implicated as being hyperactive in various anxiety manifestations . 26 , 34 The amygdala is crucial for the induction of a fear response to an ex - ternal stimulus 35 and is critically involved in fear learn - ing 36 and emotion perception . 37 The insula is implicated in representing the physiological state of the body , a process known as interoception , 38 which provides infor - mation regarding changes in internal body signals upon which subjective emotional states are based . 39 Evidence for hyperactive amygdala and insula function in anxi - ety disorders , such as social anxiety and specific phobia are robust , 34 , 40 but results in GAD have been mixed . In adults with GAD , some studies have observed hy - peractive amygdala responses to processing fearful vs happy faces 26 and processing 10 of and adaptation to face - word emotional conflict , 12 whereas others have found decreased amygdala activation to gender identi - fication of fearful vs neutral faces 6 and viewing of nega - tive pictures , 15 and yet others have found no differences in amygdala activation during fearful face processing , 5 facial affect processing of angry , fearful , or neutral faces , 30 or viewing of aversive pictures . 8 , 29 At least one study observed a hyperactive anterior insula response to threatening vs neutral picture viewing in the absence of amygdala abnormalities . 29 Findings in adolescent and child samples have also been mixed , with amygdala hy - peractivity detected in an adolescent GAD sample only when they were attending to their own subjective feel - ings of fear in response to a fearful face but not when attending to the affect of the face itself . 4 Other studies have used a dot - probe paradigm with angry and neutral faces to probe amygdala reactivity . The dot - probe utilizes a rapid presentation of a pair of stimuli ( usually an emotional and neutral face ) fol - 170 Selected abbreviations and acronyms BOLD blood oxygenation – level dependent dmPFC dorsomedial prefrontal cortex fMRI functional magnetic resonance imaging GAD generalized anxiety disorder vACC ventral anterior cingulate cortex vmPFC ventromedial prefrontal cortex Affective neuroimaging in GAD - Fonzo and Etkin Dialogues in Clinical Neuroscience - Vol 19 . No . 2 . 2017 171 Table I . Affective neuroimaging studies of generalized anxiety disorder . 3 - 32 Study Sample Design Outcome Monk et al , 3 2006 18 GAD , 15 HC ( adolescents ) Dot - probe task with angry and neutral face pairs BOLD activation McClure et al , 4 2007 15 GAD , 20 HC ( adolescents ) Viewing of fearful , angry , neutral , and happy faces with various rating conditions ( own distress , face emotion , nose width ) BOLD activation and connectivity Whalen et al , 5 2008 15 GAD , 15 HC Passive viewing of fearful , neutral , and happy faces BOLD activation Blair et al , 6 2008 17 GAD , 17 SAD , 17 HC Gender identification of neutral , fearful , and angry faces BOLD activation Monk et al , 7 2008 17 GAD , 12 HC ( youth ) Dot - probe task with masked angry / happy and neu - tral face pairs BOLD activation and connectivity Nitschke et al , 8 2009 14 GAD , 12 HC Anticipation of negative and neutral pictures BOLD activation Paulesu et al , 9 2010 8 GAD , 12 HC Mood / worry induction with spoken sentences and sad faces BOLD activation Etkin et al , 10 2010 17 GAD , 24 HC Face - word emotional conflict paradigm with fear - ful and happy faces BOLD activation and connectivity Maslowsky et al , 11 2010 14 GAD , 10 HC ( youth ) Dot - probe task with angry , happy , and neutral faces BOLD activation Etkin and Schatzberg , 12 2011 18 GAD , 14 MDD , 25 GAD and MDD , 32 HC Face - word emotional conflict paradigm with fear - ful and happy faces BOLD activation and connectivity Palm et al , 13 2011 15 GAD , 16 HC ( adult women ) Gender judgment of neutral , sad , disgust , happy , fearful , and angry faces BOLD activation Price et al , 14 2011 16 GAD , 12 HC ( older adults ) Emotional Stroop task BOLD activation Blair et al , 15 2012 17 GAD , 19 SAD , 18 HC Reappraisal and upregulation of emotion to nega - tive and positive pictures ; Top - down attentional control task BOLD activation Guyer et al , 16 2012 18 GAD , 14 SAD , 26 HC ( adolescents ) Monetary incentive delay task BOLD activation Strawn et al , 17 2012 10 GAD , 10 HC ( adolescents ) Continuous processing task with emotional and neutral pictures BOLD activation and connectivity Yassa et al , 18 2012 15 GAD , 15 HC Decision - making task with high and low uncer - tainty BOLD activation Ball et al , 19 2013 23 GAD , 18 PD , 22 HC Reappraisal and maintenance of emotion to nega - tive pictures BOLD activation Greenberg et al , 20 2013 32 GAD , 25 HC ( adult women ) Fear generalization paradigm BOLD activation and connectivity Holzel et al , 21 2013 26 GAD , 26 HC Affect labeling of angry and neutral faces BOLD activation and connectivity Cha et al , 22 2014 32 GAD , 25 HC ( adult women ) Fear generalization paradigm BOLD activation and connectivity Fonzo et al , 23 2014 21 GAD , 11 HC Facial affect matching paradigm with fearful , an - gry , and happy faces BOLD activation and connectivity Robinson et al , 24 2014 15 GAD , 7 SAD , 23 HC Facial affect identification of fearful and happy faces BOLD connectivity Andreescu et al , 25 2015 28 GAD , 31 HC ( older adults ) Worry induction and reappraisal paradigm BOLD connectivity T r a n s l a t i o n a l r e s e a r c h lowed by a probe in the location of one of the preced - ing stimuli . The participant signals the location of the probe as quickly as possible , which facilitates inference on attentional bias , ie , the extent to which the partici - pant’s attention was drawn to the location of the emo - tional cue and the level of difficulty disengaging from the emotional cue . These studies observed hyperactivity of amygdala responses in youth with GAD only when the angry faces were masked from conscious awareness through rapid replacement of the emotional probe with a neutral one . 7 When faces were consciously processed , the amygdala response was normal , but the ventrolat - eral prefrontal cortex ( vlPFC ) was hyperactive . 3 Anxi - ety symptoms were inversely correlated with degree of vlPFC activation , suggesting a potential compensatory function . This finding converges with a study in adult GAD that observed a hyperactive lateral prefrontal response to consciously processed angry faces without any amygdalar differences . 6 Although the dot - probe has not yet been examined in adult GAD with imag - ing , these studies preliminarily suggest that amygdala abnormalities may be modulated by the focus or level of attention given to an emotional cue during the task . Conflicting results may also be accounted for by abnormal amygdala responses in GAD to facial stim - uli other than fearful faces , which are often used as baseline or comparator conditions in affective imaging tasks . For example , one study in adult GAD observed diminished amygdala and insula responses to a happy face – processing comparator condition that was con - trasted with fearful and angry face processing , resulting in an exaggerated contrast magnitude with differences driven by the baseline condition only . 23 Another study observed similarly increased magnitude of amygdala responses to neutral faces , but not angry faces , in adults with GAD . 21 Similarly , amygdala interactions with other brain structures may influence variability , often investigated through context - dependent functional connectivity , ie , the degree of synchrony of BOLD responses in two regions in interaction with task conditions . A higher degree of synchrony is thought to indicate that two re - gions show greater connectivity during one task condi - tion relative to another . One study observed elevated amygdala - insula connectivity in adults with GAD dur - ing processing of fearful and angry vs happy facial ex - pressions , 23 consistent with the role of these regions in emotion processing 41 and in anxiety 34 more generally . Findings for abnormal amygdala - prefrontal connectiv - ity in GAD are also abundant . A recent study observed increased connectivity between the amygdala and dor - somedial prefrontal cortex ( dmPFC ) and dorsal ante - rior cingulate ( dACC ) during the processing of fearful vs happy faces in a transdiagnostic anxiety sample com - posed of individuals with GAD and social anxiety ( this finding was also present in the GAD sample alone ) , and higher levels of anxiety symptoms were related to greater connectivity of the amygdala with the dACC / dmPFC . 24 Similarly , another study observed increased connectivity during the processing of threatening vs 172 Table I . Continued Fonzo et al , 26 2015 15 GAD , 15 PD , 14 SAD , 15 HC Facial affect matching paradigm with fearful , an - gry , and happy faces BOLD activation Makovac et al , 27 2015 19 GAD , 21 HC Perseverative cognition induction paradigm BOLD connectivity Mohlman et al , 28 2015 20 GAD , 16 HC ( older adults ) Worry induction paradigm BOLD activation and connectivity Buff et al , 29 2016 21 GAD , 21 PD , 21 SAD , 21 HC Viewing of threatening and neutral pictures BOLD activation and connectivity Karim et al , 30 2016 17 GAD , 20 HC ( older adults ) Facial affect matching paradigm with fearful , an - gry , and neutral faces BOLD activation Ottaviani et al , 31 2016 19 GAD , 20 HC Perseverative cognition induction paradigm BOLD activation White et al , 32 2016 46 GAD , 32 HC Passive avoidance task BOLD activation Unless otherwise stated , “Sample” column indicates number of adults with GAD or other diagnoses and healthy comparison par - ticipants included in each study . BOLD , blood oxygenation level - dependent response ; GAD , generalized anxiety disorder ; HC , healthy control ; PD , panic disorder ; SAD , social anxiety disorder . Affective neuroimaging in GAD - Fonzo and Etkin Dialogues in Clinical Neuroscience - Vol 19 . No . 2 . 2017 neutral pictures between the amygdala and the dorsal mid - cingulate cortex , which is anatomically and func - tionally related to the dACC , 42 as well as increased con - nectivity between the anterior insula and dorsal mid - cingulate . 29 In contrast , other findings demonstrate deficient amygdala - ventral anterior cingulate ( vACC ) connec - tivity in GAD , which supports a specific type of implicit emotional regulatory activity—emotional conflict ad - aptation . In the first of two studies , 10 GAD participants displayed a reduced dmPFC response to face - word emotional conflict ( participants identified fearful or happy facial expressions overlaid with either the con - gruent or incongruent emotional word ) , as well as de - ficient vACC connectivity with the amygdala , which displayed a hyperactive response across all trial types . In healthy individuals , greater vACC - amygdala connec - tivity was related to the ability to adapt ( ie , reduce re - action time slowdown ) to the interference arising from an incongruent face - word emotional conflict trial when that trial was preceded by another incongruent trial vs when that trial was preceded by a congruent trial . De - ficient dmPFC activation and exaggerated amygdala reactivity to emotional conflict trials is consistent with a study in late - life GAD that observed deficient dlPFC recruitment and exaggerated amygdala reactivity dur - ing an emotional Stroop task , 14 a conceptually related paradigm in which participants identify font color dur - ing presentation of negative and neutral words . The second study using the face - word emotional conflict paradigm in GAD observed deficient vACC activation , exaggerated amygdala reactivity , and deficient vACC - amygdala connectivity during emotional conflict adap - tation , which was common across both GAD and major depression . 12 Thus , GAD is associated with selectively diminished connectivity between the amygdala and the vACC / ventromedial prefrontal cortex ( vmPFC ) during the implicit regulation of emotional reactivity , but in - creased amygdala connectivity with the dACC / dmPFC during reactivity itself . This dorsal / ventral distinction in amygdala connectivity abnormalities in GAD parallels research that implicates the vACC / vmPFC in inhibition of fear and fear extinction , whereas the dACC / dmPFC and dorsal – mid - cingulate have been implicated in fear expression / generation 43 and aversive amplification . 24 The PFC has also demonstrated abnormalities dur - ing emotional reactivity paradigms in GAD , such as hy - peractive dACC / dmPFC and vlPFC responses in youth with GAD during assessment of their own emotional response to fear faces , 4 viewing of emotional vs neu - tral images during a continuous processing task , 17 and hyperactive vlPFC responses during a dot - probe para - digm with angry and neutral faces . 3 In adults with GAD , one study observed attenuated medial and lateral pre - frontal responses to fearful , angry , sad , and happy faces ( vs neutral ) , 13 but others observed exaggerated lateral prefrontal responses to angry faces 6 and exaggerated dorsal mid - cingulate and dorsolateral prefrontal ( dlP - FC ) activation to threatening vs neutral pictures , which was specific to GAD relative to healthy individuals and participants with social anxiety ( SAD ) or panic disor - der ( PD ) . 29 In summary , GAD demonstrates a variable pattern of prefrontal and limbic activation and connectivity ab - normalities during the processing of facial affect , which is in stark contrast to other anxiety disorders that dem - onstrate more consistent patterns of hyperactivity in regions such as the amygdala and insula . 34 , 40 The source of this variability is currently unclear , but it may be con - sequent to factors varying across studies , such as depth of processing and attentional engagement necessitated by the task , as well as age - related changes in amygda - la - frontal dynamics during emotion perception . 44 Sec - ondarily , the varying directionality and presence of ab - normalities during facial affect processing may reflect shifting functional configurations of brain regions in - volved in emotional reactivity and emotion regulation due to variation in internal / affective contexts , such as degree of worry or physiological arousal , which may moderate effective inhibition of limbic activation . Affective learning and regulation Beyond emotional reactivity , GAD also manifests ab - normalities in downstream processes , such as learning to associate cues with affective outcomes , 45 and in delib - erately regulating emotional responses to such cues . 46 , 47 Two studies have observed abnormal prefrontal re - sponses during the explicit downregulation of emo - tion in response to affective images . During cognitive reappraisal of aversive images , individuals with GAD displayed deficient activation in dlPFC and dmPFC regions 19 that are crucial for supporting this emotion regulation technique , 48 a phenotype that was also ob - served in individuals with PD . Another study similarly observed reduced dACC recruitment across individu - 173 T r a n s l a t i o n a l r e s e a r c h als with GAD , SAD , or comorbid GAD / SAD during completion of a cognitive reappraisal paradigm , as well as a second paradigm assessing top - down attentional control . 15 Thus , these findings are consistent with an emotional dysregulatory perspective on GAD , 47 pro - viding evidence for deficient recruitment of brain re - gions during explicit emotion regulation in GAD that are consistently implicated in healthy individuals as supporting this psychological process . 48 This may also be a characteristic shared across different anxiety mani - festations . 15 , 19 Other studies have investigated affective learning in individuals with GAD , abnormalities of which may un - derlie processes thought to be involved in the etiology of the disorder , eg , threat generalization and avoidance learning . 45 One study in women observed that GAD was associated with a reduced capacity for vmPFC dis - crimination of a fear - conditioned stimulus from stimuli of similar perceptual characteristics , such that those with GAD displayed a flatter slope of vmPFC activa - tion change as a function of stimulus similarity , ie , vmP - FC activation was not as high for stimuli most different from the fear - conditioned stimulus . 20 This is consistent with the role of the vmPFC in fear inhibition and safety learning 49 and suggests that GAD is associated with a deficient ability of the brain to signal safety in contexts that resemble those associated with threat but that have no threat potential . This same female GAD sample also displayed an abnormally heightened response of the ventral teg - mental area ( VTA ) to stimuli that resembled the fear conditioned stimulus but were never actually paired with shock . 22 This latter finding is quite novel and im - plicates dysfunction of a key node of the mesolimbic dopaminergic system in GAD , which has classically been associated with reward and approach behavior but is also increasingly recognized as being involved in anticipation and response to aversive stimuli . 50 This system , of which the VTA is a crucial part , is critically implicated in learning stimulus - outcome associations and modifying the predictive value of a stimulus for a particular outcome . 51 This process has been increasingly examined using computational models of operant rein - forcement learning in probabilistic decision - making tasks combined with imaging , known as model - based fMRI . 52 With this method , researchers are able to de - rive from behavioral data individual computational model parameters of expected value ( the degree to which a stimulus signals a potential future positive out - come ) and prediction error ( an adjustment signal that is used to update the expected value of a stimulus via trial and error learning ) in paradigms that invoke decision making to obtain rewards or avoid punishments . These individual parameters are then convolved with the he - modynamic response function on a trial - by - trial basis to identify areas of the brain displaying BOLD signal dynamics that conform to this pattern of information processing . A recent study in adults observed that GAD was associated with deficient prediction error signal - ing in key regions implicated in decision making and affective learning regardless of trial outcome . 32 These regions included the vmPFC , the dACC / dmPFC , an - terior insula , posterior cingulate , and ventral striatum . Moreover , when trial outcomes were punishing , the GAD group displayed deficient modulation by punish - ment prediction errors in the bilateral putamen , globus pallidus , and caudate . This valence - specific striatal ab - normality during an incentivized learning task in GAD is partially consistent with a study in pediatric anxiety that observed a valence - specific putamen abnormality in children with GAD during anticipation of increasing magnitudes of monetary gains , but not losses , 16 suggest - ing some continuity of dysfunctional incentive learning circuitry across development . In aggregate , these findings point toward abnormal neural and behavioral processes supporting not only reactivity to emotional cues , but also the downstream processes that underlie the regulation of emotional re - sponses to these salient stimuli and the learning and behaviors that follow . Notably , these abnormalities en - compass regions of the dorsal and ventral prefrontal cortex and basal ganglia , which is consistent with the role of these circuits in supporting goal - directed behav - ior , 53 associative learning , 54 and in regulating emotional state implicitly 10 and explicitly . 48 Thus , the GAD neu - rophenotype manifests a disturbance of behaviorally relevant neural circuits across contexts , rather than a more circumscribed and focal abnormality of structures underlying the fear response and anxiety per se . 34 Worry and perseverative cognition As the cardinal symptom feature of GAD 55 and the center point of its clinical conceptual models , 56 , 57 early imaging studies focused on assessing worry and its neu - ral correlates . An imaging study using worry - inducing 174 Affective neuroimaging in GAD - Fonzo and Etkin Dialogues in Clinical Neuroscience - Vol 19 . No . 2 . 2017 and neutral spoken sentences demonstrated similar lev - els of activation in the dACC and dmPFC across both individuals with GAD and healthy comparison subjects , but only in those with GAD did this elevated regional activity persist into a post worry - induction resting state scan . 9 Furthermore , higher elevations in dACC / dmPFC post - worry resting state activity were correlated with higher self - reported levels of worry . Similarly , another study in elderly GAD participants observed that worry induction elicited robust activation in the amygdala and insula across those with GAD and healthy comparison subjects , but the pattern of activation was more prom - inently frontal in those with GAD . 28 A third study in elderly GAD participants during worry induction and reappraisal observed reduced connectivity between the dmPFC and the dlPFC during worry reappraisal , suggesting an inability of the dmPFC to couple with lateral prefrontal regions for regulation of worry en - gagement . 25 These findings highlight a pattern of frontal predominance and inflexibility in GAD during states of worry , which is broadly consistent with recent findings that have investigated patterns of connectivity before and after induction of perseverative cognition , ie , wor - ry and rumination . One study investigating amygdala connectivity at rest before and after induction of perse - verative cognition observed that individuals with GAD displayed decreased connectivity between the amyg - dala and the dACC / dmPFC before induction of per - severative cognition that was as prominent an abnor - mality as the increased connectivity observed between these regions after the induction . 27 This latter finding converges with other resting state studies that have observed reduced connectivity between the amygdala and dACC / dmPFC in adults with GAD . 58 Furthermore , another study in a largely overlapping sample of these participants also observed that before perseverative cognition induction , individuals with GAD displayed less task - induced deactivation of the posterior cingu - late and precuneus , the posterior nodes of the resting state default - mode network that display frequent deac - tivation in response to task demands and elevated ac - tivity at rest . 59 In aggregate , these findings support the contention that the brain in GAD is characterized by a striking level of inflexibility in response to changing en - vironmental / behavioral demands , 60 which is consistent with the clinical observation that persistent worry and negative beliefs are generally unamenable to change from contradictory evidence in the environment . 61 Discussion These findings highlight limbic and prefontal abnor - malities in GAD across behavioral processes and de - velopmental stages . Perhaps more so than any other anxiety disorder , the presence and directionality of these abnormalities varies greatly across studies . Given the significant heterogeneity of findings observed , what can be gleaned from this body of work that will inform future studies and lead to a deeper and more nuanced understanding of the GAD neurophenotype ? We suggest that understanding the sources of this variability is key to progress in this area . Of the anxi - ety disorders , GAD is unique in that the cardinal , dis - order - defining feature is by its very nature potentially independent of environmental input—that is , worry is persistent , inflexible , future - oriented , and does not necessitate any perceptual stimulus or cue in order to arise in the individual’s subjective experience . We do not mean that worry does not arise as a consequence of a cue or stimulus , but rather that it has a larger poten - tial for stimulus independence relative to other anxiety symptoms such as avoidance , emotional reactivity , and other cued fear states . Thus , the stimulus - independent potential for worry to arise and persist suggests two critical points : ( i ) the neurocircuitry orchestrating the worry cascade is probably heavily dependent on top - down medial prefrontal systems implicated in stimulus - independent mental activity , ie , the anterior default mode network , consistent with the prominent GAD mPFC abnormalities reviewed in this paper , as well as imaging studies of worry in healthy individuals 62 ; and ( ii ) predominant stimulus - independent brain states in GAD that form the foundation for worry are more resistant to change from the perpetual flux of environ - mental input and more likely to persist in a chronic , inflexible manner . This deduction is consistent with re - cent clinical theoretical developments that posit worry is not a way to avoid negative emotion in GAD 56 but actually serves to induce a mild , persistent state of neg - ative affect that facilitates an attenuated magnitude of affective shifts arising from stimuli in the environment ( known as the contrast avoidance theory 63 ) . The idea of a stimulus - independent , maladaptive medial prefrontal “hyperorganization” of the internal milieu is consis - tent with findings for a reduced capacity of the brain in GAD to discriminate among stimuli signaling safety vs threat , 20 reduced modulation of brain activation by en - 175 T r a n s l a t i o n a l r e s e a r c h vironmental feedback , 32 and a decreased ability to shift brain and physiological states in response to environ - mental demands . 9 , 27 , 31 If accurate , the wide variability in task - based acti - vation and connectivity abnormalities in GAD would therefore be expected . Specifically , task - based manipula - tions are less likely to influence regional brain configura - tions in a reliable way in GAD , and these configurations are more likely to be dictated by stimulus - independent factors that are resistant to change by experimental in - put . Thus , depending on the stimulus - independent fac - tors influencing the functional configurations of limbic 176 Configuration 1 • Amygdala activation • dmPFC deactivation Configuration 2 • No amygdala activation • dmPFC activation Configuration 4 • Amygdala hypoactivation • dmPFC hyperactivation Stimulus time series Amygdala BOLD dmPFC BOLD Configuration 3 • Amygdala hyperactivation • dmPFC deactivation Amygdala and dmPFC inflexibility Healthy GAD Figure 1 . Variability of task - induced blood oxygenation – level dependent activity in generalized anxiety disorder : a proposed model . The figure illustrates hypothetical inversely - correlated BOLD signal time courses for the dorsomedial prefrontal cortex ( green line ) , amygdala ( red line ) , and the stimulus ( fearful face ) during an affective imaging task in healthy controls ( graph on top ) and individuals with generalized anxiety disorder ( graph on bottom ) . The black brackets indicate periods of the time course that refer to distinct “configurations” of conjoint activity between the dorsomedial prefrontal cortex and amygdala . In healthy individuals , only the first two configurations are represented in the regional time courses . In the individuals with generalized anxiety disorder , however , there are several other distinct configurations of conjoint activity that are represented over the course of the task . Dotted lines indicate periods of the time course that reflect periods of inflexibility , or a failure to change configurations of conjoint brain activity , which may be secondary to brain func - tion being more heavily influenced in individuals with generalized anxiety disorder by stimulus - independent factors . A brain surface is depicted in the lower left hand corner displaying the regions of interest depicted in the hypothetical graphs . BOLD , blood oxygenation level – dependent response ; dmPFC , dorsomedial prefrontal cortex ; GAD , generalized anxiety disorder . Affective neuroimaging in GAD - Fonzo and Etkin Dialogues in Clinical Neuroscience - Vol 19 . No . 2 . 2017 and prefrontal regions in a given GAD sample , abnor - malities may be assessed as increased activation , de - creased activation , or null activation differences . We il - lustrate this potential dynamic in Figure 1 , wherein the range of potential brain states in GAD is proposed to be broader than that of healthy individuals and individuals with other anxiety disorders , but the shifts among these various states are proposed to be less frequent and more invariant to experimental manipulations . If variability is the norm rather than the exception , how can task - based imaging be used to better under - stand the brain dynamics in GAD ? First , it will be cru - cially important to bring other convergent measures to bear on imaging findings in order to better contextu - alize their significance . The field has already begun to move in this direction with the recent incorporation of physiological measures such as heart rate variability 27 , 31 as well as computational methods that allow for infer - ence concerning the information processed by dysfunc - tional substrates . 32 Second , the variability in activation values across the task itself may in fact be extremely in - formative to explore in regards to GAD . Characteriza - tion of the amplitudes of low frequency fluctuations in GAD at rest have already yielded potentially valuable insights in this area , 64 , 65 but to the authors’ knowledge no such investigations have been undertaken in a task context . Of particular importance would be to under - stand how variability in stimulus - cued activation chang - es over the course of time and is influenced by other stimulus - independent factors such as mood , arousal level , predominance of worry or rumination , and physi - ological state . Third , a careful consideration and assess - ment of the baseline comparator condition in imaging paradigms will be crucial , as existing findings already in - dicate abnormal responses to stimuli often used as the subtractive condition for a contrast . 21 , 23 Thus , multiple baseline or comparator conditions might be warranted , as well as careful theoretical considerations of which stimuli are most suitable for this purpose . Furthermore , understanding how spontaneous fluctuations in BOLD signal might contribute differently to variations in task - evoked activation 66 in GAD might be an important area for focus , particularly given the prominent resting state abnormalities observed in the disorder 58 , 64 and the vari - ability in task - based findings . Finally , consistent with the recent explosion of “big data” analytic techniques and their application to neuroscience , 67 the time is ripe for the application of these principles to the study of GAD . More specifically , the overwhelming majority of imaging studies in this area have relied exclusively on univariate statistical techniques to characterize brain dynamics , which are well - characterized methods with an illustri - ous history of application to brain mapping . 68 However , multivariate statistical methods such as multivariate pattern analysis , representational similarity analysis , and machine learning algorithms are being increasingly applied to the study of mental disorders 69 and neurosci - ence more broadly . 70 Such multivariate tools could be eminently useful for characterizing the functional neu - roanatomy of GAD , in which a wide degree of variabili - ty in observed abnormalities may derive , at least in part , from the confluence of multiple classifiable abnormal brain states across subjects in a sample , or even within the same subject at different points in time . Moreover , the ability to understand how similar observed levels of activation in a given region , eg , the amygdala , may signify very different information - processing states de - pending upon the concomitant activation level in other relevant brain structures will be paramount to better understanding the significance of regional brain config - urations and how differential access to and assumption of these configurations may reveal unique information regarding worry and GAD . o Acknowledgments / Conflict of Interest : Gregory A . Fonzo was partially supported by a grant from the National Institute of Mental Health T32 MH019938 . All authors report no conflicts of interest . 177 REFERENCES 1 . Wittchen HU . Generalized anxiety disorder : prevalence , burden , and cost to society . Depress Anxiety . 2002 ; 16 ( 4 ) : 162 - 171 . 2 . Hoffman DL , Dukes EM , Wittchen HU . Human and economic burden of generalized anxiety disorder . Depress Anxiety . 2008 ; 25 ( 1 ) : 72 - 90 . 3 . 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Toward an integration of deep learning and neuroscience . Front Comput Neurosci . 2016 ; 10 : 94 . 179 Neuroimágenes de las emociones en el trastorno de ansiedad generalizada : una revisión integradora Las neuroimágenes de las emociones han contribuido al conocimiento del trastorno de ansiedad generaliza - da ( TAG ) a través de la medición de las respuestas de la señal BOLD , la cual refleja el nivel de oxigenación de la sangre del cerebro ; esto facilita la inferencia de respues - tas neuronales a los estímulos emocionales durante la imaginería funcional por resonancia magnética ( RNMf ) con una tarea determinada . En este artículo , los auto - res revisan de manera integradora la literatura sobre la RNMf emocional en base a una tarea en el TAG . Los resultados de estudios aportan evidencia sobre la varia - bilidad de la presencia y dirección de las anormalidades de la señal BOLD en las regiones límbicas y prefrontales durante la reactividad a claves emocionales , en la re - gulación y en el aprendizaje que se hace de éstas . Se concluye que la comprensión de los orígenes de esta variabilidad es clave para acelerar el progreso en esta área . Se propone que el síntoma cardinal del TAG - la preocupación excesiva - es reflejo principalmente de los procesos mentales independientes de estímulos que im - ponen configuraciones cerebrales funcionales anorma - les y rígidas ; como por ejemplo , el esquema global de transferencia de información entre los circuitos neurales conductualmente relevantes en un momento dado . Es - tas configuraciones que no se modifican por el flujo de entrada de los estímulos ambientales , pueden estar a la base de resultados contradictorios luego de una tarea determinada . Neuro - imagerie des émotions et anxiété généralisée : une analyse globale La neuro - imagerie des émotions a enrichi notre connais - sance de l’anxiété généralisée ( AG ) en mesurant les réponses par le signal BOLD qui reflète le taux d’oxy - génation du sang dans le cerveau , ce qui facilite l’in - férence des réponses neuronales aux stimuli émotion - nels pendant l’imagerie fonctionnelle par résonance magnétique ( IRMf ) lors d’une tâche donnée . Dans cet article , les auteurs analysent de façon globale la litté - rature sur l’IRMf émotionnelle lors d’une tâche donnée dans l’AG . Des études ont montré une présence et une direction variables des anomalies du signal BOLD dans les régions limbiques et préfrontales lors de la réactivité à certains indices émotionnels , lors de leur régulation et de l’apprentissage qui en est fait . Nous en concluons que la compréhension des origines de cette variabilité est un élément clé d’une progression accélérée dans ce domaine . Selon nous , le symptôme cardinal de l’AG , l’in - quiétude excessive ou l’appréhension , reflète de façon prédominante les processus mentaux indépendants du stimulus qui imposent des configurations anormales et rigides du cerveau fonctionnel , par exemple , le schéma global du transfert de l’information au sein des circuits neuronaux comportementalement pertinents à un ins - tant donné . Ces configurations , qui ne se modifient pas lors du flux entrant des stimuli environnementaux , peuvent être à la base de résultats contradictoires lors d’une tâche donnée . \ No newline at end of file diff --git a/silver_data/b61ed1aa8992aa844f2b8d3b60f12007cd27d2b1.pdf.txt b/silver_data/b61ed1aa8992aa844f2b8d3b60f12007cd27d2b1.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..3d983b657f7ead77370f429a3e5d54f7d273b8f4 --- /dev/null +++ b/silver_data/b61ed1aa8992aa844f2b8d3b60f12007cd27d2b1.pdf.txt @@ -0,0 +1 @@ +BlackBox Toolkit : Intelligent Assistance to UI Design Vinoth Pandian Sermuga Pandian Fraunhofer FIT - UCC Sankt Augustin , Germany pandian @ fit . fraunhofer . de Sarah Suleri Fraunhofer FIT - UCC Sankt Augustin , Germany suleri @ fit . fraunhofer . de Permission to make digital or hard copies of part or all of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page . Copyrights for third - party components of this work must be honored . For all other uses , contact the owner / author ( s ) . Copyright held by the owner / author ( s ) . CHI’20 , Workshop on Artificial Intelligence for HCI : A Modern Approach , April 25 – 30 , 2020 , Honolulu , HI , USA . https : / / sites . google . com / view / ai4hci / accepted - papers Abstract User Interface ( UI ) design is an creative process that in - volves considerable reiteration and rework . Designers go through multiple iterations of different prototyping fidelities to create a UI design . In this research , we propose to mod - ify the UI design process by assisting it with artificial intel - ligence ( AI ) . We propose to enable AI to perform repetitive tasks for the designer while allowing the designer to take command of the creative process . This approach makes the machine act as a black box that intelligently assists the designers in creating UI design . We believe this approach would greatly benefit designers in co - creating design solu - tions with AI . Author Keywords UI Element Dataset , Neural Networks , Deep Learning , Sketch Detection , Sketch Recognition , Blueprints , UI Lay - out , Prototyping CCS Concepts • Computing methodologies → Object detection ; • Human - centered computing → Graphical user interfaces ; User interface design ; Interface design prototyping ; User centered design ; a r X i v : 2004 . 01949v2 [ c s . H C ] 7 A p r 2020 Introduction The user interface ( UI ) acts as the bridge between a human and a machine . It acts as a translator mediating between " Just as the In - dustrial Revolution freed up a lot of humanity from physical drudgery I think AI has the potential to free up humanity from a lot of the mental drudgery . " – Andrew Ng two worlds : one disorderly , irrational , but adept at noticing patterns ; another structured , analytical , however inept in pattern - finding ( as of now ) . A UI designer is an architect who designs this bridge between man and machine . The most important job of a UI designer is not to find a balance between both worlds ; instead , reduce the mental load of the one with issues and emotions and try to fit it into the confinements and restrictions placed by the other . This task of designing such interfaces is strenuous . Among the numerous ways of creating user interfaces to satisfy both the worlds involved , the most commonly used technique is user - centered design . In user - centered design , users are kept at the heart of de - sign , and designers attempt to satisfy their needs by an - alyzing the usage context , user needs , and requirements before starting the design process . Then during the design process , designers go through multiple fidelities of proto - types . Starting from low - fidelity ( lo - fi ) freehand sketches to medium - fidelity ( me - fi ) digital images and finally to high - fidelity ( hi - fi ) interactive screens or code . The different fideli - ties have their strengths and weaknesses . For example , lo - fi is cheap and supports ideating different designs quickly ; however , it does not do justice to the final look - and - feel of the system . Similarly , me - fi contains the most necessary information and is quicker to create than hi - fi ; however , it is hard to create multiple design variations compared to lo - fi . Hi - fi resembles the final product , but the workload of cre - ating such a system is humongous , and it is hard to create multiple design modifications to test the system . Several researchers attempt to solve the issues in this de - sign process . After the advancements of AI , one solution is to automate this whole process - the designers sketch a UI , and then the machine analyses the sketch and generates the hi - fi code . This interesting approach , however , has one major flaw . The whole system acts like a transformer to the designer who enters their sketch and gets a correspond - ing code with no control over tweaking the intricate design details . As a solution , we approach the same problem do - main and propose two solutions . Our goal is to enable the machine to perform repetitive tasks for the designer while allowing the designer to take command of the creative pro - cess . This approach makes the machine act as a black box that intelligently assists the designers in UI design . The machine’s role in our proposed design process is no differ - ent from the apprentices of renaissance art maestros . The apprentice’s task to assist the artist in preparing materials and executing the less critical and quite tedious decora - tive parts of frescoes or statues . We believe this approach would greatly benefit designers where AI and human co - create creative solutions . Research Focus Our focus in this research is on how to move designers as the drivers of creativity and let AI assist designers in the UI design process . Our primary research question in this research is , " How can we automate the UI design process while allowing UI designers to control the design details . " We address this question by using artificial intelligence to automate the transformation of different fidelities of UI de - sign . In the following sections , we expand on each of our proposed solutions , the challenges we faced with imple - mentation and the benefit of that solution . MetaMorph MetaMorph is a UI element detector , created with a Deep Neural Network ( DNN ) object detection model [ 5 ] . Meta - Morph detects constituent UI element categories and their position from a lo - fi sketch using a fine - tuned RetinaNet object detection model trained with a dataset of 125 , 000 synthetically generated lo - fi sketches . Challenge & Solution The major challenge in creating MetaMorph was the dataset . We required a large scale lo - fi sketch dataset , which was non - existent . Therefore , we collected UI element sketches from 350 participants using paper and digital question - naires . Then we processed this data and labeled it to cre - ate the UISketch dataset 1 [ 4 ] . This dataset contains 5 , 917 sketches of 19 UI elements . However , this labeled dataset is only useful for classifying UI elements ; but , a UI element detector would need ground truth of both the identity and location of UI elements in a lo - fi sketch . Figure 1 : Sample generated synthetic data As a solution , we created Syn 2 , a large - scale synthetic dataset containing 125 , 000 synthetically generated lo - fi sketches [ 4 ] . To create Syn , we randomly chose UI ele - ments from the labeled UISketch dataset and stitched them in random locations with random scaling ( Figure 1 ) . This random allocation of elements in an image is similar to the pre - processing and data augmentation techniques used in improving detection metrics of object detection models . We used Syn to train the MetaMorph UI element detector . We then collected 200 lo - fi sketches to evaluate Meta - Morph . The evaluation results indicate that MetaMorph detects UI elements from lo - fi sketches with 63 . 5 % mAP . MetaMorph 3 is available as an open web API 4 . We have also open - sourced the UISketch dataset and Syn . Figure 2 : Usage of MetaMorph to detect lo - fi elements and transforming them to me - fi in Eve 1 https : / / www . kaggle . com / vinothpandian / uisketch 2 https : / / www . kaggle . com / vinothpandian / syn - dataset 3 https : / / metamorph . designwitheve . com / 4 http : / / api . metamorph . designwitheve . com / Benefits By detecting the UI elements present in a lo - fi sketch , the lo - fi prototype can be converted to me - fi or hi - fi instead of fully automating the process ( Figure 2 ) . MetaMorph en - abled us to create Eve , a prototyping workbench [ 5 ] where a designer can sketch or upload a lo - fi , which will be con - verted to me - fi and later to hi - fi by means of UI element detection . Eve enables the designer to control the styling of the UI in me - fi and progress it to hi - fi android XML code . Blu Blu is a tool that generates UI layout information from UI screenshots . With the detected information , it enables con - version of UI screenshots to blueprints and editable vector graphics [ 3 ] . Herring et al . demonstrate the benefits and role of design examples in different aspects of the UI design process [ 2 ] . In this research , we expand on this idea , and from a UI design example screenshot , we detect UI element categories , positions , grouping information , and layout us - ing deep neural networks . Challenge & Solution We faced two significant challenges in implementing Blu : dataset and layout detection . Fortunately , as a solution to dataset issue , Deka et al . col - lected a large scale android UI screenshot dataset , RICO [ 1 ] . RICO contains 72k UI screenshots with UI element hi - erarchy and semantic annotations . However , RICO was annotated using an automated approach ; therefore , the an - notations are sometimes mislabelled . If RICO is directly used as training data of a DNN , these mistakes propagate and provide inadequate results . Therefore , we had to re - annotate the elements and layout information for a subset of RICO to train Blu . Another challenge in creating Blu is identifying the layout in - formation . A UI designer by education and practice groups and aligns UI elements while creating a me - fi prototype . They group UI elements mostly based on gestalt laws . This layout information further helps the front - end developers to create the hi - fi with the constraints placed on them by programming languages and development environments . However , there is no algorithmic way to automate the layout of UI elements using gestalt law yet . To solve this issue , we are attempting to automate the alignment process algorith - mically based on gestalt laws . Figure 3 : UI screen ( left ) and its respective blueprint ( middle ) and UI layout tree ( right ) created using Blu . Benefits Blu reduces the rework of UI designers for starting a de - sign from scratch [ 3 ] . It also assists designers to generate blueprints of UI design and convey the design information to developers . To demonstrate Blu , we created a web applica - tion 5 that utilizes the annotations from RICO and generates a blueprint ( Figure 3 ) . This web app helps to convey the design and layout information of UI screen . Summary & Future work In this paper , we presented our research on utilizing AI to assist designers in the UI designing process . We introduce the first two of our tools ( MetaMorph and Blu ) from our pro - posed solution , BlackBox toolkit . This research is an ex - ploration of applying bleeding - edge AI research in human - computer interaction domain . This ongoing research is at its incipient phase with two tools . Further , we are planning to ideate and implement similar tools , such as generating UIs and automatic detection of accessibility issues in UIs . By this research , we are looking forward to a future where humans and AI collaborate in creative tasks similar to the analytical tasks . 5 https : / / blu . blackbox - toolkit . com / REFERENCES [ 1 ] B Deka , Z Huang , C Franzen , J Hibschman , D Afergan , Y Li , J Nichols , and R Kumar . 2017 . Rico : A Mobile App Dataset for Building Data - Driven Design Applications . In Proceedings of the 30th Annual ACM Symposium on UIST ( UIST ’17 ) . ACM , New York , NY , USA , 845 – 854 . DOI : http : / / dx . doi . org / 10 . 1145 / 3126594 . 3126651 [ 2 ] S R . Herring , C C Chang , J Krantzler , and B P . Bailey . 2009 . Getting Inspired ! Understanding How and Why Examples Are Used in Creative Design Practice . In Proceedings of the SIGCHI Conference on Human Factors in Computing Systems ( CHI ’09 ) . ACM , New York , NY , USA , 87 – 96 . DOI : http : / / dx . doi . org / 10 . 1145 / 1518701 . 1518717 [ 3 ] VPS Pandian , S Suleri , and M Jarke . 2020a . Blu : What GUIs Are Made Of . In Proceedings of the 25th International Conference on IUI Companion ( IUI ’20 ) . ACM , New York , NY , USA , 81 – 82 . DOI : http : / / dx . doi . org / 10 . 1145 / 3379336 . 3381497 [ 4 ] VPS Pandian , S Suleri , and M Jarke . 2020b . Syn : Synthetic Dataset for Training UI Element Detector From Lo - Fi Sketches . In Proceedings of the 25th International Conference on IUI Companion ( IUI ’20 ) . ACM , New York , NY , USA , 79 – 80 . DOI : http : / / dx . doi . org / 10 . 1145 / 3379336 . 3381498 [ 5 ] S Suleri , VPS Pandian , S Shishkovets , and M Jarke . 2019 . Eve : A Sketch - Based Software Prototyping Workbench . In Extended Abstracts of the 2019 CHI Conference on Human Factors in Computing Systems ( CHI EA ’19 ) . ACM , New York , NY , USA , Article Paper LBW1410 , 6 pages . DOI : http : / / dx . doi . org / 10 . 1145 / 3290607 . 3312994 \ No newline at end of file diff --git a/silver_data/b72831776dfb0f9d9e17f754e1f61340c4d4032b.pdf.txt b/silver_data/b72831776dfb0f9d9e17f754e1f61340c4d4032b.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..3b53588a13ccc80206b2a9443e10405a88ac7f8b --- /dev/null +++ b/silver_data/b72831776dfb0f9d9e17f754e1f61340c4d4032b.pdf.txt @@ -0,0 +1 @@ +3061 Research Article Introduction Precise control of cell - matrix adhesion is necessary for cell migration to occur . Fibroblasts lacking paxillin ( PXN ) or focal adhesion kinase ( FAK ) , two core focal - adhesion components , display aberrant adhesion and decreased cell motility in vitro . Embryos deficient in genes that encode these proteins also lack proper mesoderm formation leading to embryonic lethality ( Ilic et al . , 1995 ; Hagel et al . , 2002 ) . In many types of cancer cell , focal adhesion proteins display either modulated protein expression or inappropriate regulation . For example , FAK is overexpressed in many types of cancer cells and is responsible for hyper - phosphorylation of other focal - adhesion proteins ( Mitra and Schlaepfer , 2006 ) . Conversely , targeted disruption of the FAK gene in breast cancer models show that FAK is required for carcinoma formation and metastasis ( Lahlou et al . , 2007 ) . Other focal - adhesion proteins have altered expression profiles in cancer models as well . Recent data suggest that the EGF - induced switch from expression of tensin - 3 to the expression of the anti - adhesive molecule cten leads to increased metastasis of mammary tumor cells ( Katz et al . , 2007 ) . These observations indicate that proper focal - adhesion dynamics are crucial for morphogenesis and play a significant role in cancer progression . Coronins are highly conserved F - actin binding proteins that are important for cell motility and actin dynamics ( de Hostos et al . , 1993 ; Cai et al . , 2007 ; Foger et al . , 2006 ) . Mammalian genomes contain at least six coronin genes that can be separated into three types : type I ( coronin 1A , 1B and 1C ) , type II ( coronin 2A and 2B ) , and type III ( coronin 7 , also known as POD ) ( Uetrecht and Bear , 2006 ) . Each of the coronins displays different tissue expression patterns with at least one type I coronin and POD expressed in all tissues and cell types . Type II coronins show a more restricted expression pattern , with strong enrichment in tissues containing epithelial and neuronal cell types ( Cai et al . , 2005 ) . The subcellular localization of each of the coronin types is also quite distinct . Type I coronins localize primarily to the lamellipodia and some vesicular structures ( Cai et al . , 2005 ; Rosentreter et al . , 2007 ) , type II coronins localize to stress fibers and focal adhesions ( Nakamura et al . , 1999 ) , and POD localizes to the Golgi apparatus ( Rybakin et al . , 2004 ) . Type I coronins have a clear role in regulating cell motility , whereas the function of type II coronins remains unknown . Type I coronins such as coronin 1B coordinately regulate Arp2 / 3 and cofilin activities in lamellipodia ( Cai et al . , 2007 ) . Coronin 1B targets Arp2 / 3 - containing branches and replaces Arp2 / 3 at branches , leading to network remodeling and disassembly ( Cai et al . , 2008 ) . In addition , coronin 1B is required for proper targeting of Slingshot - 1L , an activating phosphatase for the ADF / cofilin family of actin - binding proteins to the rear of lamellipodia ( Cai et al . , 2007 ) . It is unclear whether type II coronins execute similar functions at other cellular locations . ADF / cofilin proteins ( hereafter referred to as cofilin ) promote actin dynamics by several mechanisms , including severing actin filaments ( Bamburg and Bernstein , 2008 ) . Depletion of cofilin by RNAi increases the thickness of stress fibers , decreases F - actin retrograde flow , and impairs whole - cell motility ( Hotulainen et al . , 2005 ; Sidani et al . , 2007 ) . Several pathways have been identified that regulate cofilin activity . Cofilin is inactivated by interacting with phosphatidylinositol ( 4 , 5 ) - bisphosphate on membranes ( van Rheenen et al . , 2007 ) , by conformational changes induced by intracellular pH shifts ( Frantz et al . , 2008 ) , or via phosphorylation of serine 3 by LIM kinase ( LIMK ) or TES kinase ( TESK ) ( Yang et al . , 1998 ; Toshima et al . , 2001 ) . All of these events prevent cofilin from binding to F - actin . Activation of cofilin can be achieved by Coronins are conserved F - actin - binding proteins that are important for motility and actin dynamics . Unlike type I coronins , coronin 2A localizes to stress fibers and some focal adhesions , and is excluded from the leading edge . Depletion of coronin 2A in MTLn3 cells decreases cell motility and turnover of focal adhesions . Surprisingly , none of the pathways known to regulate focal - adhesion turnover are affected by depletion of coronin 2A . Depletion of coronin 2A does , however , increase phospho - cofilin , suggesting that misregulation of cofilin might affect adhesion dynamics . Slingshot - 1L , a cofilin - activating phosphatase , localizes to focal adhesions and interacts with coronin 2A . Depletion of coronin 2A reduces cofilin activity at focal adhesions , as measured by barbed - end density and actin FRAP . In both fixed cells and live cells , cofilin localizes to the proximal end of some focal adhesions . Although expression of wild - type cofilin in coronin - 2A - depleted cells has no major effect on focal - adhesion dynamics , expression of an active mutant of cofilin bypasses the defects in cell motility and focal - adhesion disassembly . These results implicate both coronin 2A and cofilin as factors that can regulate a subset of focal - adhesion - turnover events . Supplementary material available online at http : / / jcs . biologists . org / cgi / content / full / 122 / 17 / 3061 / DC1 Key words : Coronin , Cofilin , Slingshot Summary Coronin 2A regulates a subset of focal - adhesion - turnover events through the cofilin pathway Thomas W . Marshall , Heather L . Aloor and James E . Bear * Lineberger Comprehensive Cancer Center and Department of Cell and Developmental Biology , University of North Carolina - Chapel Hill , Chapel Hill , NC 27599 , USA * Author for correspondence ( jbear @ email . unc . edu ) Accepted 27 May 2009 Journal of Cell Science 122 , 3061 - 3069 Published by The Company of Biologists 2009 doi : 10 . 1242 / jcs . 051482 J ou r na l o f C e ll S c i en c e JCS ePress online publication date 4 August 2009 3062 the dephosphorylation of serine 3 by the Slingshot and Chronophin phosphatases ( Niwa et al . , 2002 ; Gohla et al . , 2005 ) . Interestingly , both LIMK and Slingshot - 1L share a common localization to focal adhesions ( Foletta et al . , 2004 ; Soosairajah et al . , 2005 ) . Despite the localization of these cofilin regulatory proteins to focal adhesions , cofilin activity has never been directly implicated in focal - adhesion dynamics . Several mechanisms are known to regulate the disassembly of focal adhesions . These include microtubule targeting of focal adhesions ( Kaverina et al . , 1999 ) , talin cleavage by calpain ( Franco et al . , 2004 ) , changes in myosin - II - generated tension ( Webb et al . , 2004 ) , and changes in FAK phosphorylation and interactions with dynamin ( Ezratty et al . , 2005 ) . However , the inter - relationship among these mechanisms is poorly understood . An unexplored mechanism could involve cofilin - mediated actin - filament - severing activity at focal adhesions . Here , we have investigated the role of coronin 2A in regulating cofilin activity at focal adhesions and the subsequent effects on whole - cell motility . Results Coronin 2A localizes to stress fibers and focal adhesions , but not lamellipodia To investigate the function of coronin 2A ( also known as IR10 , ClipinB , WDR2 , coronin 4 and CRN5 ) , we expressed coronin 2A tagged with GFP ( Coro2A - GFP ) in MTLn3 cells , a mammary adenocarcinoma cell line . Coro2A - GFP co - localizes along F - actin stress fibers and with vinculin at some focal adhesions ( Fig . 1A ) . Interestingly , coronin 2A did not co - localize with coronin 1B ( Fig . 1A ) or coronin 1C ( data not shown ) at the leading edge of cells and was excluded from the lamellipodial region . These observations , along with the absence of type I coronins from focal adhesions ( data not shown ) , indicate that type I and type II coronins have distinct properties that determine protein localization . Endogenous coronin 2A had the same localization pattern as Coro2A - GFP ( Fig . 1B ) . Interestingly , coronin 2A only localizes to some of the internal focal adhesions that have stress fibers attached , but does not localize to focal complexes and adhesions found in the periphery of cells . Other focal - adhesion markers , such as talin and FAK , show similar partial co - localization with coronin 2A at internal focal adhesions ( data not shown ) . Coronin - 2A - depleted cells display decreased cell speed , but show no change in lamellipodial dynamics To address the functional role of coronin 2A , we developed an shRNA against rat coronin 2A ( shCoro2A ) , delivered by lentivirus , to deplete this gene product . To control for the effects of lentiviral infection , we also used a non - specific shRNA ( shNS ) . To verify that any effects were not due to off - target silencing , we developed a rescue construct that encodes both shCoro2A and an RNAi - resistant human coronin 2A tagged with GFP . Immunoblotting indicated that shCoro2A - expressing cells have nearly undetectable amounts of coronin 2A protein ( Fig . 2A ) . This result was confirmed by qRT - PCR analysis ( supplemental material Fig . S1A ) . Although cells expressing the rescue construct appeared to have levels of coronin 2A protein that were higher than endogenous levels , separate pilot experiments showed that this antibody recognizes human coronin 2A more strongly than rat coronin 2A ( supplemental material Fig . S1B ) , indicating that the level of rescue protein expression was in the physiological range . Since coronin 2A localizes to F - actin stress fibers and focal adhesions , we examined the motility of cells that were depleted of coronin 2A . Single - cell tracking indicated that coronin - 2A - depleted cells have substantially reduced cell speed relative to control cells . Re - expression of human Coro2A - GFP restored cell speed to the level of control cells , indicating that the effects caused by the expression of shCoro2A are due to the specific depletion of coronin 2A ( Fig . 2B ) . The cells depleted of coronin 2A displayed protrusion and retraction at the leading edge , but did not display much overall translocation . We used kymography to determine whether there were any differences in lamellipodial dynamics caused by the depletion of coronin 2A ( example in Fig . 2C ) . Protrusion rate , protrusion distance , and persistence ( Fig . 2D ) were all unaffected by depletion of coronin 2A , suggesting that the decrease in cell motility is not due to alterations in lamellipodial dynamics . Journal of Cell Science 122 ( 17 ) Fig . 1 . Coronin 2A localizes to F - actin stress fibers and some focal adhesions , but is excluded from the leading edge . ( A ) MTLn3 cells expressing Coro2A - EGFP were stained with Alexa 568 phalloidin ( top panel ) or with antibodies against either vinculin ( middle panel ) or coronin 1B ( bottom panel ) . ( B ) Immunofluorescence of endogenous coronin 2A ( green ) with either Alexa 568 phalloidin ( top panel ) or a vinculin antibody ( middle panel ) . In the bottom panel , magnified images ( 6 (cid:2) ) of the insets in the middle panels show that coronin 2A localizes to focal adhesions . Scale bars : 5 μ m . J ou r na l o f C e ll S c i en c e 3063 Coro2A affects focal adhesions via cofilin Cells depleted of coronin 2A have defects in focal - adhesion turnover Since there are defects in cell motility in the coronin - 2A - depleted cells and coronin 2A localizes to focal adhesions , the size and number of these structures were quantified . Quantification of focal adhesions in cells expressing either shNS or shCoro2A indicated that depletion of coronin 2A increases focal - adhesion size and decreases the total number of focal adhesions per cell ( Fig . 3A , B , example images in C ) . To measure cell spreading , we used the ACEA RT - CES system that measures changes of impedance caused by cells interacting with a microelectrode within the surface of the dish . Coronin - 2A - depleted cells showed a slight , but not statistically significant ( P = 0 . 1726 for MTLn3 versus shCoro2A , P = 0 . 0661 for shNS versus shCoro2A ) increase in cell spreading compared to control cells ( Fig . 3D ) . This indicates that adhesion formation and cell spreading appear to be normal in coronin - 2A - depleted cells . Larger focal adhesions in the coronin - 2A - depleted cells indicated that focal - adhesion dynamics might be affected . To investigate possible changes in focal - adhesion turnover , coronin - 2A - depleted cells that co - express GFP - PXN were used to monitor focal - adhesion assembly and disassembly ( Webb et al . , 2004 ) . Since coronin 2A is localized to a subset of focal adhesions in the internal region of the cell ( see Fig . 1 ) , we selected these adhesions for analysis . Compared to control cells , focal - adhesion assembly was not affected in the coronin - 2A - depleted cells ( Fig . 3E ) , but the rate of focal - adhesion disassembly decreased by half ( Fig . 3F ; supplementary material Movie 2 ) . Together , these data indicate that coronin 2A plays a significant role in controlling disassembly of focal adhesions located at internal regions of the cell . Adhesion defects caused by the depletion of coronin 2A are mediated through the cofilin pathway To determine the cause of focal - adhesion turnover defects in the coronin - 2A - depleted cells , we examined various cellular pathways and processes that regulate focal - adhesion turnover . There were no significant differences in talin cleavage by calpain , FAK phosphorylation at Y397 or phospho - MLC ( P - MLC ) levels ( Fig . 4A ) . The frequency of microtubule targeting to focal adhesions was decreased in coronin - 2A - depleted cells , but this appeared to be caused by slightly decreased microtubule growth rates Fig . 2 . Depletion of coronin 2A impairs cell motility , but does not affect lamellipodial dynamics . ( A ) MTLn3 cells were infected with lentivirus that expressed shRNAs against a non - specific sequence ( shNS ) , coronin 2A ( shCoro2A ) or shCoro2A that also co - expressed human Coro2A - GFP ( Rescue ) . Lysates were blotted for coronin 2A and for tubulin as a loading control . ( B ) Time - lapse microscopy of MTLn3 , shNS , shCoro2A and Rescue cell lines was used to determine single - cell speed , depicted in graph . Error bars represent 95 % confidence intervals . * P < 0 . 0001 by Student’s t - test . See supplementary material Movie 1 . ( C ) Representative kymograph of a protruding MTLn3 cell . ( D ) Protrusion rate , protrusion distance and persistence were calculated from the time ( y - axis ) and protrusion distance ( x - axis ) measured from kymographs . Error bars represent 95 % confidence intervals . Fig . 3 . Depletion of coronin 2A increases focal - adhesion size , decreases focal - adhesion number and decreases focal - adhesion disassembly . Focal - adhesion size ( A ) and number ( B ) were measured in MTLn3 cells expressing shNS or shCoro2A . Vinculin - positive focal adhesions were used for the quantifications . Focal - adhesion size was normalized against neighboring uninfected cells on the same coverslip . * P = 0 . 0424 for focal - adhesion size and * P = 0 . 0286 for focal - adhesion number , by Student’s t - test . ( C ) Representative vinculin immunofluorescence for MTLn3 cells expressing shNS or shCoro2A used for quantifications in A and B . Scale bar : 5 μ m . ( D ) Cell spreading as measured by change of impedance with the ACEA RT - CES system . Equal numbers of MTLn3 , shNS and shCoro2A cells were plated in triplicate . P = 0 . 1726 for MTLn3 versus shCoro2A and P = 0 . 0661 for shNS versus shCoro2A , by Student’s t - tests . ( E , F ) Average rates of focal - adhesion assembly ( E ) or disassembly ( F ) visualized by GFP - PXN in cells expressing either shNS or shCoro2A . Cells were imaged once a minute for 30 minutes ( see supplementary material Movie 2 ) . Changes of fluorescent intensity were used to determine focal - adhesion assembly and disassembly rates . * P < 0 . 001 by Student’s t - test . Error bars represent 95 % confidence intervals . J ou r na l o f C e ll S c i en c e 3064 ( supplemental material Fig . S2A , B ) and not to be due to microtubule stability or inability to properly target focal adhesions ( supplemental material Fig . S2C , D ) . These observations suggest coronin 2A might control focal - adhesion turnover through a different pathway . Since coronin 1B regulates cofilin activity at the leading edge of the cell ( Cai et al . , 2007 ) , we investigated whether coronin 2A has a similar role in regulating cofilin phosphorylation . Depletion of coronin 2A led to roughly a 1 . 5 - fold increase in phosphorylated cofilin ( P - cofilin ) by both western blots ( Fig . 4A , B ) and ratiometric immunofluorescence ( Fig . 4C , D ) . ADF phosphorylation was also increased in the coronin - 2A - depleted cells ( supplemental material Fig . S3 ) . To determine whether the increased phosphorylation of cofilin was due to increased LIMK activity in coronin - 2A - depleted cells , we blotted for phosphorylated ( active ) LIMK and observed no change upon depletion of coronin 2A ( Fig . 4A ) . Consistent with a failure to activate LIMK , depletion of coronin 2A did not cause a global change in F - actin content , as measured by changes in phalloidin intensity relative to surrounding control cells ( supplemental material Fig . S4 ) . The cofilin - activating phosphatase Slingshot - 1L has been shown to localize to the leading edge of cells and , in agreement with other reports ( Soosairajah et al . , 2005 ) , we observed Slingshot - 1L – GFP ( SSH1L - GFP ) localizing to focal adhesions ( Fig . 5A ) . Coronin 2A and Slingshot - 1L also co - localized at some , but not all , focal adhesions ( Fig . 5B ) . Immunoprecipitations of Coro2A - GFP or endogenous coronin 2A with an antibody against coronin 2A were able to co - immunoprecipitate Slingshot - 1L ( Fig . 5C , D ) . This interaction is independent of the phosphatase activity of Slingshot because co - immunoprecipitation occurred with a phosphatase - inactive mutant of Slingshot ( CS ) equally as well as with wild - type Slingshot ( Fig . 5C ) . Furthermore , endogenous coronin 2A can co - immunoprecipitate with SSH1L - GFP , indicating that this interaction is not mediated by GFP dimerization or by an interaction with GFP ( Fig . 5D and control blot in supplemental material Fig . S5 ) . One obvious mechanism by which coronin 2A might affect the cofilin pathway at focal adhesions is to serve as a targeting subunit for Slingshot - 1L at this location . However , SSH1L - GFP targeted focal - adhesion structures equally well in control and coronin - 2A - depleted cells ( Fig . 6A ) . Because the depletion of coronin 2A did not appear to affect Slingshot - 1L localization to focal adhesions , we examined the dynamics of SSH1L - GFP at focal adhesions in cells expressing either shNS or shCoro2A . The rate of internal focal - adhesion disassembly , as marked by SSH1L - GFP , was similar to Journal of Cell Science 122 ( 17 ) Fig . 4 . Depletion of coronin 2A increases P - cofilin levels , but has no effect on other focal - adhesion - turnover protein components . ( A ) Representative immunoblots of MTLn3 , shNS , shCoro2A and Rescue cell lysates for talin , P - FAK , FAK , P - MLC , MLC , P - cofilin , cofilin , P - LIMK , LIMK and tubulin ( loading control ) . Asterisk indicates calpain - induced talin cleavage product . ( B ) Quantification of P - cofilin versus total cofilin blots . Error bars represent 95 % confidence intervals . ( C ) Representative ratiometric images of P - cofilin ( P - Cof ) and cofilin ( Cof ) for shNS and shCoro2A cells . Images were normalized to neighboring uninfected cells . Scale bars : 5 μ m . ( D ) Quantification of ratiometric images in C . * P = 0 . 0001 by Student’s t - test . Error bars represent 95 % confidence intervals . Fig . 5 . Coronin 2A interacts with Slingshot - 1L and co - localizes at a subset of focal adhesions . ( A ) SSH1L - GFP localizes to focal adhesions . MTLn3 cells transfected with SSH1L - GFP were fixed and stained with vinculin antibodies . Scale bar : 5 μ m . ( B ) MTLn3 cells expressing Coro2A - GFP and SSH1L - mRFP1 co - localize at focal adhesions . In the lower panel , magnifications ( 3 (cid:2) ) of insets show positive focal adhesions ( arrowheads ) . Some SSH1L - mRFP - positive focal adhesions contain little to no Coro2A - GFP ( arrows ) . ( C ) Lysates from 293FT cells with enforced expression of Coro2A - GFP and SSH1L - GFP or SSH1L ( CS ) - GFP were immunoprecipitated with a coronin 2A antibody and immunoblotted for GFP . ( D ) Co - immunoprecipitation of endogenous coronin 2A with SSH1L - GFP . Immunoprecipitation was performed as for C . Immunoblots were probed with coronin 2A and GFP antibodies . Asterisk indicates antibody immunoglobulin reacting with the secondary antibody . J ou r na l o f C e ll S c i en c e 3065 Coro2A affects focal adhesions via cofilin the rate observed with GFP - PXN and displayed the same ~ twofold decrease in coronin - 2A - depleted cells compared to control cells ( Fig . 6C ) . Another possibility is that coronin 2A regulates Slingshot - 1L activity at focal adhesions . Although Slingshot - 1L regulation is incompletely understood , phosphorylation of two serine residues in its C - terminus ( S937 and S978 ) and subsequent 14 - 3 - 3 binding have been shown to inhibit its activity ( Eiseler et al . , 2009 ; Kligys et al . , 2009 ; Nagata - Ohashi et al . , 2004 ) . We examined whether SSH1L - S937A , S978A ( abbreviated as SSH1L - 2SA ) , a non - phosphorylatable mutant with higher phosphatase activity , had an effect on focal - adhesion disassembly . Similarly to wild - type Slingshot - 1L , SSH1L - 2SA – GFP localized to focal adhesions in both control and coronin - 2A - depleted cells ( Fig . 6B ) . Expression of the active SSH1L - 2SA mutant increased focal - adhesion disassembly rates by ~ twofold . Interestingly , depletion of coronin 2A in the cells expressing SSH1L - 2SA produced an intermediate result . The rate of disassembly was lower than that with the expression of the mutant alone , but higher than with depletion of coronin 2A in the presence of wild - type Slingshot - 1L ( Fig . 6C ) . This result suggests that increased Slingshot activity can compensate for depletion of coronin 2A , but that coronin 2A might also have an effect on focal - adhesion dynamics downstream of Slingshot - 1L . Cofilin localizes to the proximal end of some focal adhesions Although proteins that regulate cofilin activity , like Slingshot , LIM kinase and TESK , localize to focal adhesions , it remains unclear whether cofilin also localizes to these structures . Cells that were permeabilized with saponin prior to fixation displayed punctate spots of cofilin near the proximal end of some FAK - positive focal adhesions ( Fig . 7A ) , suggesting that endogenous cofilin is stably anchored near some focal adhesions . To circumvent the problem of fixation - induced artifacts , we imaged MTLn3 cells expressing GFP - PXN and cofilin - TagRFP . Live - cell TIRF microscopy showed that spots of cofilin - TagRFP localized to the proximal end of the focal adhesion ( Fig . 7B ; supplementary material Movie 3 ) . In some cases , the appearance of cofilin at the base of a focal adhesion was followed by focal - adhesion disassembly ( Fig . 7B , middle and bottom panels ) . Although these results suggest that cofilin transiently localizes to some focal adhesions , the precise molecular interactions of cofilin at these structures will require further elucidation in future studies . Active cofilin can bypass focal - adhesion and motility defects caused by the depletion of coronin 2A Because cofilin localizes to some focal adhesions , assays were performed to determine whether the severing activity of cofilin occurs at focal adhesions . We used two approaches to evaluate the activity status of cofilin in situ after depletion of coronin 2A . The actin - filament - severing activity of cofilin generates free barbed ends that can be detected by the incorporation of labeled G - actin into permeabilized cells ( Chan et al . , 2000 ) . Thus , the presence of free barbed ends can be used as a surrogate marker for cofilin activity , although barbed ends at focal adhesions might arise from other mechanisms , such as anti - capping protein activity ( e . g . VASP ) . To measure the density of actin filament barbed ends at focal adhesions , we labeled free barbed ends and visualized them relative to the focal - adhesion marker vinculin . The relative density of free barbed ends at vinculin - positive focal adhesions ( reported as the ratio of barbed ends to vinculin staining at focal adhesions ) was significantly diminished in coronin - 2A - depleted cells ( Fig . 8A , B ) . In addition , fluorescence recovery after photobleaching ( FRAP ) of TagRFP - actin was performed to monitor any changes in actin dynamics near focal adhesions . Coronin - 2A - depleted cells displayed significantly decreased recovery rates of labeled actin at focal adhesions compared to control cells ( Fig . 8C ) . Along with the increased phosphorylation of cofilin in coronin - 2A - depleted cells , reduced density of barbed ends at focal adhesions , and the interaction of coronin 2A and Slingshot - 1L , these data suggest that coronin 2A enhances disassembly of a subset of internal focal adhesions through the cofilin pathway . To directly determine whether decreased cofilin activity is responsible for the decreased cell motility in the coronin - 2A - depleted cells , we tested whether an active mutant of cofilin ( cofilin S3A ) could bypass these defects . Expression of cofilin S3A in coronin - 2A - depleted cells restored whole - cell motility to that of control cells ( Fig . 9A ) . To test whether the defect in focal - adhesion Fig . 6 . Active mutants of Slingshot increase focal - adhesion disassembly and partially bypass decreases in focal - adhesion disassembly caused by depletion of coronin 2A . ( A ) SSH1L - GFP localizes to focal adhesions in cells expressing shNS or shCoro2A . Cells were co - stained with vinculin antibodies . Scale bars : 5 μ m . ( B ) SSH1L - 2SA - GFP localizes to focal adhesions in cells expressing shNS or shCoro2A . Cells were co - stained with vinculin antibodies . ( C ) Focal - adhesion disassembly rates , carried out as for Fig . 3 , visualized by SSH1L - GFP or SSH1L - 2SA - GFP ( active ) in cells expressing either shNS or shCoro2A . Error bars represent 95 % confidence intervals . J ou r na l o f C e ll S c i en c e 3066 turnover present in the coronin - 2A - depleted cells was due to insufficient cofilin activity , we expressed either wild - type or cofilin S3A in the coronin - 2A - depleted cells . Expression of the wild - type cofilin was unable to rescue the defect in internal focal - adhesion disassembly observed with depletion of coronin 2A ( Fig . 9B ) . However , expression of cofilin S3A in coronin - 2A - depleted cells restored internal focal - adhesion disassembly rates to the rate of control cells ( Fig . 9B ) . Expression of cofilin S3A alone had no effect on whole - cell motility or focal - adhesion turnover , suggesting that the bypass effect of this mutant is specific to depletion of coronin 2A . Together , these data indicate that coronin 2A affects focal - adhesion disassembly through the activation of cofilin . Discussion Considerable evidence points to the important role that coronins play in cell motility , but the function of type II coronins , such as coronin 2A , has never been addressed . Unlike the more well - characterized type I coronins , coronin 2A localizes to stress fibers and some focal adhesions , and is excluded from the leading edge . Our data indicate that coronin 2A plays a key role in regulating whole - cell motility and internal focal - adhesion turnover . Depletion of this gene leads to insufficient cofilin activity at focal adhesions Journal of Cell Science 122 ( 17 ) Fig . 7 . Cofilin localizes to the proximal end of some focal adhesions in fixed and live cells . ( A ) Immunofluorescent images of saponin - permeabilized MTLn3 cells stained for cofilin and FAK . Lower panel shows magnifications ( 7 . 5 (cid:2) ) of insets . Scale bar : 5 μ m . ( B ) Montage of live - cell images of cell expressing shNS - GFP - PXN and cofilin - TagRFP . Images were taken every minute ( see supplementary material Movie 3 ) . Scale bar : 5 μ m . Cofilin - TagRFP localizes to the base of GFP - PXN - positive focal adhesions , leading to focal - adhesion disassembly . Middle panel gives enlarged ( 2 . 2 (cid:2) ) view of white box inset . Bottom panel gives enlarged ( 4 . 3 (cid:2) ) view of black box inset . Fig . 8 . Depletion of coronin 2A leads to reduced barbed - end density and actin turnover at internal focal adhesions . ( A ) Representative images of Oregon green actin conjugate incorporated into free barbed ends at vinculin - stained focal adhesions in MTLn3 cells expressing shNS - TagRFP - actin or shCoro2A - TagRFP - actin . Scale bars : 5 μ m . ( B ) Graph depicting the ratio of barbed ends to focal adhesions ( BE / FA ) , as measured by average fluorescence intensities . These results were normalized to the intensity values of uninfected MTLn3 cells on the same coverslip . Error bars indicate 95 % confidence intervals . * P < 0 . 001 by Student’s t - test . ( C ) Graph of fluorescence recovery after photobleaching ( FRAP ) of TagRFP - actin at focal adhesions in MTLn3 cells expressing either shNS or shCoro2A . T 1 / 2 values indicate time required for 50 % recovery of fluorescence . Values from nine cells were used to calculate the recovery rate . J ou r na l o f C e ll S c i en c e 3067 Coro2A affects focal adhesions via cofilin and reduced adhesion disassembly in the interior region of the cell . This effect might be mediated in part through a coronin - 2A – Slingshot - 1L interaction that regulates Slingshot ( and therefore cofilin ) activity . Consistent with a role of cofilin activation in these processes , defects in motility and internal focal - adhesion turnover induced by depletion of coronin 2A can be bypassed by an active mutant of cofilin . Our data highlight the striking diversity of localization and function in the mammalian coronin family of proteins . Previous data from our laboratory indicate that the primary function of type I coronins ( e . g . coronin 1B ) is to interact with the Arp2 / 3 complex and Slingshot - 1L at the rear of the lamellipodia and remodel the dendritic meshwork ( Cai et al . , 2007 ; Cai et al . , 2008 ) . Like coronin 1B , coronin 2A interacts with Slingshot - 1L . This indicates that cofilin regulatory activity is conserved between type I and II coronins . Despite this similarity , type I coronins and coronin 2A have distinct spatial distributions . Coronin 2A localizes to stress fibers and focal adhesions in the central region of the cell and is excluded from the leading edge , whereas type I coronins are concentrated in this compartment . It remains unclear what causes these differences in localization patterns , and elucidating this will require further investigation . A significant conclusion from these studies is that activation of the severing activity of cofilin is an important factor in a subset of focal - adhesion - disassembly events . Previous studies on TESK1 have shown a requirement of cofilin phosphorylation for proper spreading to occur , but did not address the role of cofilin in regulating focal - adhesion dynamics ( LaLonde et al . , 2005 ) . Although many studies have linked cofilin activity to actin turnover at specific structures , such as the dendritic meshwork , stress fibers and myofibril arrays ( Ono , 2007 ) , our data are the first to directly implicate cofilin activity in the turnover of focal adhesions . One simple model for the direct involvement of cofilin in this process is that actin filaments in stress fibers connected to focal adhesions must be severed to initiate , propagate or complete the turnover of the adhesion . Data from others has already indicated that the linkage between stress fibers and focal - adhesion components , such as PXN , is capable of slipping in a clutch - like fashion ( Hu et al . , 2007 ) . Thus , severing by cofilin might reflect an alternate pathway by which the linkage between stress fibers and focal adhesions can be regulated . Since some studies have indicated that cofilin poorly severs actin filaments in bundled structures ( Michelot et al . , 2007 ) , there might be a requirement for bundle remodeling near the point of cofilin action in order for this mechanism to function efficiently . One intriguing issue that arises from these studies is that of how coronin 2A modulates cofilin activity at focal adhesions . On the basis of the increased levels of P - cofilin observed with coronin 2A depletion and the selective ability of cofilin S3A ( but not wild - type ) to bypass the motility and adhesion turnover defects , it seems probable that coronin 2A affects cofilin activity , at least in part , via its phosphorylation status . The interaction between coronin 2A and Slingshot - 1L supports this notion , but it is important to point out that coronin 2A does not simply target Slingshot - 1L to focal adhesions . Furthermore , coronin 2A is unlikely to exclusively regulate Slingshot - 1L activity through controlling the phosphorylation of the C - terminal serine residues because the enhanced focal - adhesion disassembly caused by expression of the activated Slingshot - 1L 2SA mutant would have been completely unaffected by depletion of coronin 2A in this case . Thus , coronin 2A might control Slingshot - 1L activity by an unknown mechanism . Alternately , coronin 2A might affect cofilin by a Slingshot - 1L - independent mechanism , such as through an effect on the structure of actin filaments in the focal adhesion , or through the direct enhancement of cofilin activity , as described recently for coronin 1A in an in vitro system ( Kueh et al . , 2008 ) . These and other possibilities will need to be addressed in future experiments . Previous studies uncovered multiple mechanisms for focal - adhesion turnover and it is worth considering how cofilin - based severing might be integrated with some of these other mechanisms . One significant mechanism of turnover is the calpain - dependent cleavage of talin ( Huttenlocher et al . , 1997 ; Bhatt et al . , 2002 ) . This event permanently breaks a key linkage between integrin receptors and other components in the focal - adhesion plaque . Cofilin - based severing of actin filaments might serve a similar purpose at the proximal end of the focal adhesion and might synergistically accelerate adhesion turnover . Another important mechanism of adhesion assembly and disassembly is myosin - based contractility . The isometric tension generated by this mechanism is important for the maturation of nascent contacts near the leading edge and is also important for detachment of adhesions at the trailing edge ( Rottner et al . , 1999 ; Webb et al . , 2004 ; Gupton and Waterman - Storer , 2006 ) . Cofilin - based severing could directly impact myosin - based contractility by modifying actin filament structure near the adhesion . It is unclear how these mechanisms and others such as microtubule contact and FAK - dynamin interactions function together to drive focal - adhesion turnover . In addition to the complication of multiple interrelated mechanisms of focal - adhesion turnover , it is clear that different mechanisms might dominate in particular cellular compartments and in different cell types . Cells must balance the formation and dissolution of adhesions in an asymmetric manner to promote efficient cell migration . Thus , adhesion turnover at the front of the Fig . 9 . Expression of cofilin S3A rescues cell motility and focal - adhesion disassembly defects caused by depletion of coronin 2A . ( A ) Graph of average cell speeds of MTLn3 , shNS , shCoro2A , shNS - cofilinS3A and shCoro2A - cofilinS3A expressing cells , as done in Fig . 2 . Error bars indicate 95 % confidence intervals . Asterisk indicates P < 0 . 001 . ( B ) Graph of average rates of focal adhesion ( FA ) disassembly visualized by GFP - PXN in cells expressing either shNS or shCoro2A along with wild - type ( wt ) or S3A cofilin - mRFP1 , as done in Fig . 3 . Error bars indicate 95 % confidence intervals . * P < 0 . 001 by Student’s t - test . J ou r na l o f C e ll S c i en c e 3068 cells might utilize a distinct set of turnover mechanisms from those at the trailing edge of the cells . Because coronin 2A is excluded from the periphery of the cell , it seems likely that it would participate only in turnover of centrally located adhesions . Finally , it is important to note that expression of coronin 2A is not detectable in fibroblasts and thus its role in focal - adhesion turnover might be specific for certain cell types such as epithelial cells . However , various Slingshot and LIMK isoforms are ubiquitously expressed , so regulated cofilin activation might be an intrinsic part of focal - adhesion turnover in all cells . Considering the profound effect that focal - adhesion - turnover kinetics has on overall cell migration , it will be crucial to elucidate this process in detail in order to fully understand migration - dependent processes such as the immune response and cancer metastasis . Materials and Methods Molecular cloning pML2 ( human Coro2A - GFP ) has been described previously ( Cai et al . , 2005 ) . Rat coronin 2A was amplified from cDNA made from MTLn3 cells . pLL5 . 0 base vector and pLL5 . 0 - shNS - GFP / mCherry have been described ( Cai et al . , 2007 ) . shRNA sequences were designed to target rat , but not human , coronin 2A ( shCoro2A ) . Oligonucleotides were annealed and ligated in HpaI and XhoI sites in pLL5 . 0 . The coronin 2A shRNA sequence is 5 (cid:3) - GGAACGTCTTGGACATCAT - 3 (cid:3) . A rescue construct was made by PCR amplification of human coronin 2A and cloning it into EcoRI and BamHI sites in pLL5 . 0 - shCoro2A . The following cDNAs were PCR amplified and inserted in pLL5 . 0 - shNS - GFP and pLL5 . 0 - shCoro2A - GFP with the sites indicated : paxillin ( GFP - PXN ; double blunt ligation into RI / SbfI site ) , Slingshot - 1L ( wt or CS ) ( SSH1L - GFP ; MfeI / BglII into EcoRI / BamHI in pLL5 . 0 ) , cofilin A ( EcoRI / BamHI ) and TagRFP - actin ( EcoRI / SbfI ) . cDNAs for SSH1L and cofilin S3A were PCR amplified , digested with SalI / SacII or EcoRI / BamHI , respectively , and ligated into pML2 - mRFP1 . SSH1L - 2SA - GFP was made by site - directed mutation of serine 937 and serine 978 to alanines . PCR primers sequences are available upon request . Antibodies and reagents GST - coronin 2A short tail ( human , amino acids 493 - 526 ) was produced in Escherichia coli , purified using glutathione - Agarose beads , and used to inject rabbits for the production of polyclonal antibodies for coronin 2A ( Covance ) . Purified antibodies were isolated by applying serum to MBP - coronin 2A short tail that was immobilized on an UltraLink Biosupport ( Pierce ) . Antibodies were eluted from the column with both high and low pH solutions and tested for recognition of coronin 2A protein by immunoblotting ( WB ) , immunofluorescence ( IF ) and immunoprecipitation ( IP ) . The following antibodies were purchased and used at the dilutions as indicated : α - tubulin clone DM1A ( Sigma ; WB 1 : 1000 ) , GFP clone JL - 8 ( Clontech ; WB 1 : 5000 ) or ( Roche ; IP 1 μ l ) , talin ( Sigma ; WB 1 : 5000 ) , FAK [ Millipore ; WB 1 : 1000 , IF 1 : 200 , or BC3 ( a gift from Mike Schaller , University of West Virginia , Morgantown , WV ) , IF 1 : 200 ] , P - FAK ( Biosource ; WB 1 : 1000 ) , cofilin ( Cytoskeleton ; WB 1 : 500 ) or [ Mab22 ( a gift from Jim Bamburg , Colorado State University , Fort Collins , CO ) IF 1 : 100 ] , P - cofilin [ Biosource , WB 1 : 1000 , or 4321 ( a gift from Jim Bamburg ) , IF 1 : 70 ) ] , LIMK ( Cell Signaling ; WB 1 : 1000 ) , P - LIMK ( ECM Biosciences ; WB 1 : 1000 ) , MLC II ( Cell Signaling ; WB 1 : 1000 ) , and P - MLC ( Cell Signaling ; WB 1 : 1000 ) . Coronin 1B rabbit polyclonal antibody is described in the literature ( Cai et al . , 2005 ) . Cy2 , Cy5 , rhodamine red - X , and HRP conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories . Immobilon - P PVDF was from Millipore . Recombinant Rat EGF was purchased from Sigma . Rat tail collagen was purchased from BD Biosciences . 100 (cid:2) Pen Strep glutamine ( PSG ) , α - MEM , DMEM , Alexa Fluor 488 , 568 and 647 labeled with phalloidin , Oregon green and Alexa Fluor 647 actin conjugates were purchased from Invitrogen . Fetal bovine serum ( FBS ) was purchased from Hyclone . Cell Culture and shRNA lentiviral production MTLn3 cells ( a gift from John Condeelis , Albert Einstein College of Medicine , Bronx , NY ) , a rat mammary adenocarcinoma cell line described by Chan et al . ( Chan et al . , 1998 ) , were grown in α - MEM containing 5 % FBS and 1 (cid:2) PSG . 293FT cells were grown in DMEM containing 10 % FBS and 1 (cid:2) PSG . Retroviral and lentiviral production was preformed as previously described ( Cai et al . , 2007 ) . MTLn3 cells were infected with retrovirus or lentivirus for 4 hours and then the media was changed . Effects of lentiviral infections were examined by western blot after 3 - 4 days . ACEA RT - CES experiments After background measurements were taken ( 100 μ l of media ) , 5000 cells in 100 μ l was added to each well ( three experiments , done in triplicate ) . Impedance measurements were taken every 2 minutes for 3 hours and quantified in Prism ( Graph Pad ) Single cell tracking MTLn3 cells were either uninfected or infected with lentiviruses encoding shNS , shCoro2A , rescue , shNS – cofilin - S3A – GFP or shCoro2A – cofilin - S3A – GFP . These cells were plated on 50 μ g / ml rat tail collagen - coated Bioptechs Delta T dishes for 16 - 18 hours . Time - lapse microscopy was performed on an Olympus IX - 81 inverted microscope ( 10 (cid:2) objective ) with a Hamamatsu CCD camera ( model c4742 - 80 - 12AG ) . Cell speed was measured with Tracking software ( Andor Bioimaging ) or Slidebook software ( Intelligent Imaging Innovations ) using manual tracking mode . Graphs displayed were made in Prism ( Graph - Pad ) . Immunoprecipitations 293FT cells were transfected with plasmids as indicated in the figures . An 80 - 90 % confluent 6 - cm dish of cells was lysed in 1 ml of 1 % Triton X - 100 in PBS . Lysates were spun at 16 , 873 g at 4°C for 5 minutes . Approximately 1 μ g of GFP or coronin 2A antibodies were used for immunoprecipitation in combination with 20 μ l of protein A or protein - G Sepharose beads ( Pierce or GE Lifesciences , respectively ) . Immunoprecipitated proteins were separated by SDS - PAGE , transferred to PVDF ( Millipore ) , and immunoblotted for coronin 2A or GFP . Focal - adhesion - assembly and - disassembly experiments MTLn3 cells infected with shNS - GFP - PXN , shCoro2A - GFP - PXN , shNS - SSH1L - GFP , shCoro2A - SSH1L - GFP , shNS - SSH1L - 2SA - GFP , or shCoro2A - SSH1L - 2SA - GFP were plated as in single - cell tracking experiments . Cells were imaged on a Nipkow - type spinning disk confocal scan head ( Yokogawa CSU - 10 ) with a 60 (cid:2) 1 . 45 NA objective . Images were taken 1 frame every minute for 40 minutes . In four cells over two experiments , at least 12 focal adhesions per cell were analyzed with ImageJ software . These adhesions fit the criteria that they were not ( 1 ) localized to the edge of a protruding lamellipodia , and ( 2 ) localized to either the tail or internal region of the cell . The intensity of GFP in each frame was used to determine rates of focal - adhesion assembly and disassembly as described ( Webb et al . , 2004 ) . All images were corrected for photobleaching . Focal - adhesion and cofilin live - cell imaging MTLn3 cells infected with shNS - GFP - PXN and cofilin - TagRFP were plated as in focal - adhesion - assembly and - disassembly conditions . Total internal reflectance microscopy ( Olympus ) was used to illuminate fluorescent proteins in close proximity to the coverslip . Images of cofilin and PXN were taken every minute for 30 minutes . Immunofluorescence MTLn3 cells were plated on acid - washed coverslips coated with 50 μ g / ml rat tail collagen . Cells were fixed with 4 % PFA , or pre - permeabilized in permeabilization buffer ( 20 mM Hepes at pH 7 . 5 , 138 mM KCl , 4 mM MgCl2 , 3 mM EGTA , 0 . 2 mg / ml saponin , 1 mM ATP , 1 % BSA ) for 30 seconds followed by 4 % PFA fixation for 10 minutes . After three washes with PBS , cells were permeabilized with 0 . 1 % Triton X - 100 in PBS for 5 minutes . Cells were blocked for 15 minutes in PBS containing 5 % normal goat serum ( Jackson Laboratories ) and 5 % fatty - acid - free BSA . Primary antibodies were applied to cells in PBS containing 1 % BSA for 1 hour . If needed , diluted primary antibody solutions also contained Alexa Fluor 488 , 568 , or 647 phalloidin ( Invitrogen ) . Cells were washed three times in PBS . Cy2 , Cy5 and rhodamine red - X conjugated secondary antibodies were diluted to 1 : 400 in 1 % BSA in PBS and applied to the coverslips for 1 hour . After three washes in PBS , the coverslips were mounted onto slides with Fluoromount G ( Electron Microscopy Sciences ) . Barbed - end experiments were carried out as described ( Chan et al . , 2000 ) . Briefly , cells were permeabilized in permeabilization buffer containing 2 μ M Oregon green or Alexa Fluor 647 actin conjugates ( Invitrogen ) for 20 seconds . Cells were then fixed and processed as above . Fluorescence recovery after photobleaching MTLn3 cells infected with shNS - TagRFP - actin or shCoro2A - TagRFP - actin were photobleached at focal adhesions with a 405 - nm laser for 30 milliseconds . TagRFP fluorescence intensities were monitored every 0 . 784 seconds on an Olympus FV1000 microscope with a 60 (cid:2) 1 . 2NA Olympus objective . Nine cells were analyzed for each condition . All images were corrected for overall photobleaching . Images were analyzed in ImageJ . Ratiometric imaging of P - cofilin and cofilin MTLn3 cells were fixed with 4 % PFA and processed as in the immunofluorescence experiments . Fields containing both an infected cell expressing either shNS or shCoro2A - mCherry and an uninfected cell were imaged in the linear range of the camera . Fluorescent images of P - cofilin and cofilin were collected and processed using the ImageJ plug - in Ratio plus . Relative intensities were obtained by normalizing the values to the uninfected neighboring cells for 30 cells per treatment . Journal of Cell Science 122 ( 17 ) J ou r na l o f C e ll S c i en c e 3069 Coro2A affects focal adhesions via cofilin EB1 - GFP experiments MTLn3 cells expressing either shNS - EB1 - GFP or shCoro2A - EB1 - GFP with or without TagRFP - actin were imaged either every second for EB1 - GFP dynamics and microtubule growth rate studies , or every 3 seconds for focal - adhesion targeting . Focal - adhesion targeting was determined by EB1 - GFP tracking down an actin stress fiber and terminating at the base of the stress fiber ( considered to be a targeting event ) . EB1 - GFP velocity ( rate of microtubule growth ) was determined by tracking the centroid of the EB1 - GFP spot over time . The distance divided by time was used to determine EB1 velocity . Growth phase was determined to be the amount of time an EB1 - GFP spot is observed over the course of a movie . 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Yang , N . , Higuchi , O . , Ohashi , K . , Nagata , K . , Wada , A . , Kangawa , K . , Nishida , E . and Mizuno , K . ( 1998 ) . Cofilin phosphorylation by LIM - kinase 1 and its role in Rac - mediated actin reorganization . Nature 393 , 809 - 812 . J ou r na l o f C e ll S c i en c e \ No newline at end of file diff --git a/silver_data/b94be41d283190bb23aaf6937eac23f3f972d94a.pdf.txt b/silver_data/b94be41d283190bb23aaf6937eac23f3f972d94a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..308dcfa38b5230fe862024b350182d5cc58c01b2 --- /dev/null +++ b/silver_data/b94be41d283190bb23aaf6937eac23f3f972d94a.pdf.txt @@ -0,0 +1 @@ +Caveolin Moves from Caveolae to the Golgi Apparatus in Response to Cholesterol Oxidation Eric J . Smart , Yun - Shu Ying , Patricia A . Conrad , and Richard G . W . Anderson Department of Cell Biology and Neuroscience , University of Texas Southwestern Medical Center , Dallas , Texas 75235 Abstract . Caveolae are a membrane specialization used to internalize molecules by potocytosis . Caveolin , an integral membrane protein , is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane . We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi appara - tus markers . After the enzyme is removed from the medium , caveolin returns to caveolae . When untreated cells are gently homogenized , caveolin on the plasma membrane is accessible to both anti - caveolin IgG and trypsin . After cholesterol oxidase treatment , however , Golgi - associated caveolin is inaccessible to both of these molecules . Brefeldin A , which inhibits ER to Golgi trafficking , blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane . Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma mem - brane and becomes associated with endoplasmic retic - ulum and Golgi compartments . Surprisingly , the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae . C AVEOLAE are a membrane specialization found on the surface of many different cells ( 23 , 36 ) . There is considerable evidence that they are an endocytic organelle that internalizes substances by forming plasma - lemmal vesicles ( 24 ) . In some cases these vesicles transport molecules across the cell ( 32 ) while at other times they de - liver low molecular weight molecules and ions to the cyto - plasm by a process called potocytosis ( 1 ) . The cytoplasmic surface of each caveola is decorated with a characteristic coat material that appears to be composed of integral mem - brane components ( 28 ) . One of the proteins associated with this coat is caveolin ( 28 ) . The caveolae coat may play a role in controlling membrane invagination . Potocytosis was discovered by studying receptor - coupled transport of folate in MA104 cells ( 15 ) . At physiological con - centrations of 5 - methyltetrahydrofolate , the efficient delivery of the vitamin to the cytoplasm of cells is dependent on a high affinity receptor that is anchored to the membrane by glycosylphosphatidylinositol ( GPI ) 1 . The GPI - anchor causes the receptor to cluster in caveolae ( 30 ) where it becomes se - questered from the extracellular space each time a caveolae closes . Sequestered folate rapidly dissociates from the re - ceptor and crosses the membrane by an anion carrier ( 16 ) . Address all correspondence to Eric J . Smart , Dept . of Cell Biology and Neuroscience , University of Texas Southwestern Medical Center , Dallas , Texas 75235 . 1 . Abbreviations used in this paper : DNE dinitrophenol ; GPI , glycosyl - phosphatidylinositol ; PKC , protein kinase C . Caveolae then open and another round of potocytosis begins . Recently we showed ( 34 ) that activators of protein kinase C ( PKC ) inhibit potocytosis by preventing the invagination of caveolae . Caveolin was originally identified as a 22 - kD tyrosine phosphoprotein in chick embryo fibroblasts infected with the Rous sarcoma virus ( 11 ) . Immunogold cytochemistry showed that over 90 % of the caveolin in the plasma membrane is as - sociated with caveolae and that small amounts of caveolin are also found in the Golgi apparatus ( 28 ) . Independently , Kurz - chalia et al . ( 17 ) showed that a protein of the same molecular weight as caveolin ( designated VIP21 ) is prominent in Golgi - derived vesicles destined for the apical cell surface . Cloning of the cDNA , first for VIP21 ( 17 ) and then for caveolin ( 10 ) , revealed that they are the same protein . The sequence pre - dicts that it is an integral membrane protein synthesized without a leader peptide . The exact orientation of the protein in the plasma membrane is still not clear . Some evidence suggests that the protein forms a hairpin loop in the mem - brane such that both ends are in the cytoplasm ( 9 ) . Other evi - dence ( 21 ) favors the view that it is a type II transmembrane protein . The function of caveolin is not known . The association of this protein with the caveolar coat suggested that it might have a structural role in maintaining the shape of the or - ganelle ( 28 ) . In polarized epithelial cells , caveolin first as - sociates with clusters of GPI - anchored membrane proteins in the Golgi apparatus ( 6 , 21 ) before moving to the cell sur - face in transport vesicles ( 17 ) . Therefore , caveolin may target GPI - anchored proteins and associated glycolipids to the api - © The Rockefeller University Press , 0021 - 9525 / 94 / 12 / 1185 / 13 $ 2 . 00 The Journal of Cell Biology , Volume 127 , Number 5 , December 1994 1185 - 1197 1185 cal cell surface ( 17 , 21 ) . There is not any evidence , however , that plasmalemmal vesicles move from the cell surface to the Golgi apparatus as part of an internalization cycle . Both the structure ( 28 ) and the function ( 7 ) of caveolae are absolutely dependent on membrane cholesterol . Exposure of membranes to sterol - binding drugs such as filipin cause the caveolar coat to disassemble , invaginated caveolae to be - come flat , and the GPI - anchored proteins to uncluster ( 28 ) . Likewise , cells that are depleted of membrane cholesterol have unclustered GPI - anchored proteins , a reduced number of visible caveolae and potocytosis is impaired ( 7 ) . These results are in agreement with the observation that caveolae have a much higher concentration of cholesterol than the sur - rounding plasma membrane ( 33 ) . Oxidation of cholesterol has been implicated as a risk fac - tor in the development of atherosclerosis ( 14 , 25 ) . The es - sential role of cholesterol in caveola function suggests that it may be a plasma membrane domain that is especially sen - sitive to oxidative damage . To investigate this possibility , we used cholesterol oxidase to enzymatically oxidize fibroblast membrane cholesterol in situ . Surprisingly , this treatment caused caveolin to migrate to the Golgi region of the cell without there being a change in the number of caveolae . Cav - eolin recycles back to the plasma membrane after cholesterol oxidase is removed from the media and the level of oxidized cholesterol in the membrane declines . Materials and Methods Materials Dulbecco ' s modified Eagle ' s medium , glutamine , trypsin - EDTA , and peni - cillin / streptomycin were from Gibco Laboratories ( Grand Island , NY ) . Fe - tal calf serum was from Hazleton Research Products , Inc . ( Lenexa , KS ) . The analytical silica gel thin - layer chromatography plates and the following solvents were from J . T . Baker , Inc . ( Philllpsburg , NJ ) : heptane , petroleum ether , ethyl ether , acetic acid , and 2 - propanoL 3Hacetate was obtained from DuPont ( Wilmington , DE ) with a specific activity of approximately 4 . 13 Ci / mmol . Trans - 35S - label was from ICN ( Costa Mesa , CA ) . Cho - lesterol oxidase ( Nocardia erythropolis ) , cholesterol and cholestenone were from Boehrinser Mannheim Biochemicals ( Indianapolis , IN ) . The sulfuric - dichromate spray was from Supelco Inc . ( Bellefonte , PA ) . Protein A - Seph - arose ( CL - 4B ) was from Pharmacla ( Alameda , CA ) . Antibodies were ob - tained from the following sources : anti - caveolln IgG ( mAb 2234 ) was a gift from Dr . John Glenney ( Glentech , Inc . , Lexington , ICO ; rabbit anti - BiP IgG was from David Bole ( University of Michigan , Ann Arbor , MI ) ( 4 ) ; rabbit anti - galactosyltransferase IgG was from E . G . Berger ( Physiolo - gisches Institute Universitat Zurich , Switzerland ) ( 3 ) ; goat anti - mouse IgG conjugated to peroxidase was from Organon Tekuika Corp ( West Chester , PA ) ; goat anti - rabbit IgG conjugated to gold ( 10 rim ) was from Biocell ( Ted Pella , Redding , CA ) ; mouse anti - dinitrophenol IgG was from Oxford Bio - medical ( Oxford , MI ) ; goat anti - mouse IgG conjugated to gold ( 10 nm ) was from Energy Beam Sciences ( A _ gawam , MA ) . All other reagents were from Sigma Chem . Co . ( St . Louis , MO ) . Me ~ o ~ Cell Culture . Cultured fibroblasts were derived from a skin biopsy obtained from a normal patient . Cells were grown in a monolayer and set up accord - ing to a standard format ( 12 ) . On day zero , 2 . 5 x 105 cells were seeded into 100 - ram Petri dishes with 3 ml of Dulbecco ' s modified Eagle ' s medium supplemented with penicillin , streptomycin , 10 % ( vol / vol ) human lipopro - rein deficient serum . Cells were fed with fresh medium on day 3 and day 5 . Cells were labeled with 75 # Ci of 3H - acetate on day six and used on day 7 . Thin - layer Chromatography . The thin - layer chromatography experi - ments were carried out as previously described ( 22 ) . Three confluent 100 - mm dishes of fibroblasts were used for each sample . The labeled cells were washed extensively in PBS and then placed in 3 ml of buffer ( DMEM , 20 mM Hepes , pH 7 . 4 ) before the indicated treatments . The caveolae then were isolated on sucrose gradients ( see below ) . Each gradient fraction was ad - justed to 1 ml final volume and treated with 30 % tanrodeoxycbolate , 2 ml Dole reagent ( 2 - propanol , 78 % : beptane , 20 % : water , 2 % ) , and I rni of heptane . The samples were vortexed and spun in a table top centrifuge for 10 min ( 3000 g ) . The heptane phase ( upper ) contained lipids and was used for thin - layer chromatography . The aqueous phase ( bottom ) contained pro - teins and was used for immunoblots . The heptane phase was dried under N2 and suspended in 50 t ~ l of the solvent system ( 80 : 20 : 1 ; petroleum ether / ethyl ether / acetic acid ) . Pure cholesterol and pure cholestenone were dissolved in the solvent system and used as standards ( 5 / tg / spot ) . The lipids were spotted onto a silica gel G plate and developed in the solvent system . Lipids were visualized by charring with sulfuric acid - dichromate and heat - ing at 180°C for 10 min . Unlabeled cholesterol and cholestenone were added to each fraction to facilitate visualization . The appropriate spots were scraped and the amount of radiation quantified by liquid scintillation counting . Caveolae Isolation . Caveolae were isolated as described by Sargiacomo and colleagues ( 31 ) . Three confluent , 100 - ram dishes of human fibroblasts ( 10 nag protein ) were used for each sample . All of the solutions contained a cocktail of protease inhibitors consisting of : ieupeptin ( 10 / ~ M ) , benzami - dine ( 0 . 5 raM ) , soybean trypsin inhibitor ( 10 / ~ g / ml ) , pepstatin A ( 1 / ~ g / ml ) , and PMSF ( 0 . 2 mg / ml ) . Each dish was washed briefly with MBS ( 25 mM MES , pH 6 . 5 ; NaCI , 0 . 15 M ) at room temperature . MBS plus 0 . 02 % ED ' I ' A was added to each dish for 10 min on ice . The cells were pelleted in a table top centrifuge for 5 min , 1400 g , 4 °C . Cells were suspended with a pipette tip in 1 . 0 ml MBS containing 1 % Triton X - 100 . The samples were incubated on ice for 20 min and then dounced 20x in a I ml tight dounce . The samples were diluted with an equal volume of 85 % sucrose . The mixed material was placed at the bottom of a centrifuge tube ( TH641 ; Sorvaii Instruments , Newton , CT ) and a 10 - 30 % linear sucrose gradient was layered on top . The samples were centrifuged at 143 , 200 g for 4 h at 4°C . After centrifugation the gradient was fractionated in 0 . 65 - ml fractions before analysis . Triton Solubility . Three 100 - ram dishes of confluent fibroblasts were used for each sample . Cells were grown 22 h in methionine - free media that contained 300 ~ tCi of 35Smethionine ( sp act 1132 Ci / mmol ) , washed three times with DMEM and then incubated for 5 h in the presence of 50 ftg / ml of cycloheximide . The cells were washed extensively in PBS and sub - jected to the indicated treatments , all in the presence of cyclobeximide . Cells were released from the dish by incubating in MBS plus 0 . 02 % EDTA for 10 rain on ice . The cells were collected by centrifuging for 5 min , 1400 g at 4°C . The pellets were suspended in 1 . 0 ml of MBS plus 1 % Triton X - 100 and incubated on ice for 20 rain . The samples were dounced 20 times and spun in a mierofuge for 5 rain , 16000 g at 4°C . The supernatant fraction was collected and designated as the Triton - soluble fraction . The pellet was suspended in MBS plus 1 % Triton and designated the Triton - insoluble frac - tion . Caveolin was immunoprecipitated from each of these fractions as de - scribed below . Immunoprecipitation of Caveolin . Protein A - scpbarose beads were first blocked by incubating them for 4 h at 4oc with human fibroblast cell lysate ( 200 / tg / ml ) plus 30 mg / ml of BSA in MBS buffer that contained 1 % Triton X - 100 and 60 mM octylgiucoside ( MBSD buffer ) . Blocked beads were used to pre - clear the Triton X - 100 soluble and insoluble fractions that had been suspended in MBSD . Pre - cleared fractious were then incubated for 19 h at 4°C with a 1 : 400 dilution of mAb anti - caveolin before adding blocked , Protein A - scpharose beads and incubating an additional 2 h at 4°C . Beads were removed by centrifugation , dissolved in sample buffer ( 18 ) , and proteins separated by electrophoresis . The autoradiograph was exposed for 48 h . Protease Protection . Two 100 - mm dishes of confluent fibroblasts were used for each sample . Cells that had been preincubated for 5 h in the pres - ence of 50 / ~ g / ml of cycloheximide were washed extensively in PBS and sub - jected to the indicated treatments , all in the presence of cycloheximide . At the end of the treatments , cells were removed from the dish with Trypsin - EDTA . Cells were collected by centrifugation at 1400 g for 5 rain and then washed in 5 ml of lysis buffer ( 1 mM NaHCO3 ; 1 ram MgC12 ; 10 mM KC1 ; 5 mM Hepes , pH 7 . 5 ; 0 . 3 M sucrose ) . Cells were pelleted at 1400 g for 5 rain and then suspended in 0 . 5 ml of ice cold lysis buffer . Cells were broken by douncing 20 times with a 1 ml type A dounce homogenizer . The appropriate samples were incubated with 300 / ~ g of trypsin for 30 win on ice . Soybean trypsin inhibitor ( 300 ~ g ) was then added and the sample was centrifuged for 1 rain at 600 g to remove unbroken cells . The remaining suspension was centrifuged at 100 , 000 g for 60 min at 4°C . The pellet was suspended in lysis buffer and analyzed by immunoblotting . Electrophoresis and Immunoblots . Protein concentrations were deter - The Journal of Cell Biology , Volume 127 , 1994 1186 mined by the BioRad Bradford assay ( 5 ) or the BioRad D . C . assay ( Bio - Rad Labs , Richmond , CA ) . Proteins were concentrated by trichloroacetic acid precipitation . Pellets were dissolved in sample buffer and heated at 95°C for 3 min before being loaded onto gels . Proteins were separated in a 12 . 5 % SDS - polyacrylamide gel using the method of Laemmli ( 18 ) . Separated pro - teins were then transferred to nylon by the method of Towbin ( 35 ) . The ny - lon was blocked in TBST ( 20 mM Tris , pH 7 . 6 ; 137 mM NaC1 ; 0 . 5 % Tween - 20 ) plus 3 % dry milk for 1 h at room temperature . Anti - caveolin mAb 2234 diluted 1 / 500 in TBST + 1 % dry milk was added for 1 h at room temperature . After incubating with the primary antibody , the nylon was washed four times , 10 win each in TBST plus 1 % dry milk . The secondary antibody ( goat anti - mouse conjugated to horseradish peroxidase ) was di - luted 1 / 30 , 000 and added for 1 h at room temperature . The nylon was then washed and bands visualized using enhanced chemiluminescence . Indirect Immunofluorescence . Cells grown on glass coverslips were washed three times with PBS . After the indicated treatment , the cells were washed in PBS and fixed for 45 min at room temperature either with 3 % ( wt / vol ) paraformaldehyde in PBS or with a fixative that contained 3 . 7 % paraformaldehyde , 70 mM Lysine - HCl , and 10 mM NaIO4 in 20 mM MES buffered saline ( pH 7 . 0 ) . After fixation , the cells were rinsed with PBS and incubated in 100 mM NI - I4CI - PBS for 10 min at room temperature . Cells were permeabilized with 0 . 1 % Triton X - 100 for 7 min on ice and washed extensively in PBS . Cells were then incubated sequentially with PBS plus 0 . 5 % BSA for 1 h at room temperature , anti - caveolin mAb 2234 diluted 1 / 350 in PBS plus 0 . 2 % BSA for 1 h at room temperature and finally with 20 / xg / ml F1TC - goat anti - mouse IgG plus 0 . 2 % BSA for 1 h at room temperature . Cells were washed three times with PBS after each incubation . To colocalize caveolin and galaetosyltransferase , the primary antibody incu - bation contained a mixture of mAb 2234 and polyclonal anti - galactosyl - transferase ( 1 : 350 ) . The secondary antibody incubation contained a mixture of RITC - goat anti - mouse IgG and FITC - goat anti - rabbit IgG . The concen - tration of the individual antibodies in the mixtures was the same as when they were used alone . Cells were then washed briefly in distilled water and mounted in a 2 . 5 % solution of 1 , 4 - diazabicyclo - ( 2 . 2 . 2 ) oetane . Cells were photographed using a Zeiss Photomicroscope HI . Iramunoelectron Microscopy . Cells were washed quickly with EM buffer ( 100 mM sodium phosphate , pH 7 . 6 , 3 mM KC1 , 3 mM MgCi2 ) and then fixed with 3 % ( wt / vol ) paraformaldehyde plus 3 mM trinitrophenol in EM buffer for 45 rain at room temperature . The cells were rinsed with EM buffer for 10 rain and then incubated for 20 min with 100 mM NI - I4CI in EM buffer at 4°C . Cells were rinsed with EM buffer for 10 min and then incubated with EM buffer that contained 0 . 15 % bovine serum albumin and 0 . 01 % saponln for 30 min at 4°C . Samples were incubated either with anti - caveolin mAb 2234 ( 1 / 100 ) or control mAb 2001 ( 20 t ~ g / ml ) , rinsed , and then incubated with 20 ~ tg / ml goat anti - mouse IgG conjugated to dinitro - phenol ( DNP ) ; all with EM buffer plus 0 . 15 % BSA and 0 . 01 % saponin . All antibody incubations were 1 h at 4°C . Cells were fixed with 2 % glutaralde - hyde in EM buffer and incubated with 100 mM NI - IaCI in EM buffer before post - fixation with 1 % OsO4 containing 1 . 5 % potassium ferrieyanide in 100 mM sodium phosphate ( pH 7 . 6 ) . Samples were then dehydrated , embedded in Eponate , sectioned and processed to localize DNP groups by immuno - gold labeling as previously described ( 26 ) . Immunogold quantification was carried out using a modification of a previously described method ( 30 ) . Briefly , electron microscopic negatives were obtained by randomly photo - graphing 60 regions from 40 different cells in each experimental treatment . The negatives were enlarged using a hand held magnifier and the surface length of the cell , the number of caveolae and the number of gold particles were measured directly . Results Cholesterol oxidase converts plasma membrane cholesterol to cholestenone without penetrating the lipid bilayer and en - tering the cell ( 19 ) . To determine how much of the surface cholesterol in a living human fibroblast was accessible to the enzyme , we first metabolically labeled the cholesterol with tritium by growing the cells for 16 h in the presence of 36 / zM 3Hacetate ( 4 . 13 Ci / mmol ) . We then incubated the cells in the presence or absence of 0 . 5 U / ml cholesterol oxi - dase for 1 h at 37°C . The radiolabeled lipids were extracted from the cell with organic solvents , separated by TLC , and measured with a liquid scintillation counter . Cholesterol oxi - dase reproducibly converted 5 - 7 % of the total cellular cho - lesterol to cholestenone . 1 - 2 h after removal of the enzyme from the culture medium , all of the radiolabeled choleste - none disappeared from the cell . Caveolae are easily isolated from cultured cells because they are insoluble in Triton X - 100 and , as a consequence , can be separated from other membranes by flotation on a sucrose gradient ( 31 ) . We used this procedure to measure the amount of caveolin , 3H - cholesterol and 3H - cholestenone that was in caveolae before and after exposure of the cells to cho - lesterol oxidase . Untreated ceils that had been homogenized in detergent were loaded on the bottom of a 10 to 30 % su - crose gradient and centrifuged at 143 , 200 g for 4 h ( Fig . 1 A ) . Immunoblots ( Fig . 1 A , Immunoblot ) showed that the majority of the caveolin was in fraction 10 , which is well separated from the starting material in fraction 14 . Frac - tion 10 contained only 6 % of the total 3H - cholesterol ( Fig . 1 A , 1 ) , with the remainder in fractions 12 - 14 . 3H - cholestenone was not present in any of the fractions ( Fig . 1 A , < 3 ) . A dramatically different profile was seen after cho - lesterol oxidase treatment ( Fig . 1 B ) . Virtually all of the 3H - cholesterol in fraction 10 ( Fig . 1 B , l ) had been replaced by 3H - cholestenone ( Fig . 1 B , o ) . This was the exclusive location of oxidized cholesterol in these cells . More remarkably , caveolin was no longer in fraction 10 ( Fig . 1 B , Immunoblot ) but remained in the starting material ( Fig . 1 B , Fraction 14 ) . The quantitative conversion of the choles - terol in fraction 10 to cholestenone suggests that all of the oxidase - sensitive cholesterol in a living fibroblast is in the caveolae . The caveolin may have remained in the starting material after cholesterol oxidase treatment because oxidized sterols render the protein soluble in Triton X - 100 ( Fig . 2 ) . To test this idea , we first grew cells overnight in 35Smethionine to metabolically label caveolin . Protein synthesis was stopped by adding cycloheximide to the media for 5 h and the cells were incubated in the presence or absence of 0 . 5 U / ml cho - lesterol oxidase for 1 h at 37°C . We then homogenized the cells in Triton X - 100 , separated the soluble ( Fig . 2 , Triton Soluble ) and insoluble ( Fig . 2 , Triton Insoluble ) material by centrifugation , and immunoprecipitated each fraction with mAb anti - caveolin IgG . All of the caveolin in untreated cells was in the insoluble fraction ( Fig . 2 , NT ) . After 60 min of cholesterol oxidase treatment , however , all of the caveolin was in the soluble fraction ( Fig . 2 , CO ) . Removal of the cho - lesterol oxidase from the culture medium caused a progres - sive return of caveolin to the insoluble fraction ( Fig . 2 , chase ) . 90 min after removal of oxidase , all of the caveolin was Triton insoluble ( Fig . 2 , 90 min ) . Separate experiments showed that cholestenone disappeared from the cells within 60 min after cholesterol oxidase was removed from the medium ( data not shown ) . We observed a quantitative ( Fig . 2 , compare band intensities ) return of the Triton soluble cav - colin to the insoluble fraction after cholesterol oxidase was removed from the media even though protein synthesis was blocked during this time . Caveolin Reversibly Moves to the Golgi Apparatus These results suggested that cholesterol oxidase caused the caveolin to leave the caveolae without changing the physical integrity of the caveolar membrane . Indirect immunofluores - Smart et al . Caveolin Traffic to Golgi Region 1187 Figure 1 . Distribution of cho - lesterol , cholestenone and caveolin in caveolae isolated from either untreated ( A ) or cholesterol oxidase - treated ( B ) ceils . Human fibroblasts were incubated for 1 h at 37°C in the presence of either medium alone ( A ) or medium that contained 0 . 5 U / ml of cholesterol oxidase ( B ) . Both sets of cells were solubilized in 1 % Triton X - 100 and caveo - lae isolated by flotation in a sucrose gradient . Fourteen fractions from the gradient were analyzed for the pres - ence of caveolin ( Immuno - blot ) , cholesterol ( I ) and cholestenone ( o ) as described in Materials and Methods . The arrow indicates the caveo - lae fraction . cence ( Fig . 3 , A , C , and E ) and immunogold electron mi - croscopy ( Fig . 3 , B , D , and F ) confirmed this interpretation . The anti - caveolin IgG immunofluorescence staining pattern of untreated cells is shown in Fig . 3 A . The caveolin was pri - marily on the surface , concentrated in patches along the edge and over the body of the cell ( Fig . 3 A ) . Immunogold labeling showed that nearly all of the caveolin was associated with in - Figure 2 . Effects of cholesterol oxidase on the Triton X - 100 solubil - ity of caveolin . Human fibroblasts were incubated in the presence of trans - [ 35S ] - label overnight , washed and further incubated in cold media that contained 50 # g / ml of cycloheximide for 5 h . They were then incubated for 1 h in the presence of either medium alone ( ART ) or medium that contained 0 . 5 U / ml of cholesterol oxidase ( CO and chase ) . One set of cholesterol oxidase treated cells ( chase ) was incubated further for the indicated time in medium that did not contain any enzyme . Cycloheximide was present during all incuba - tions . After each treatment , cells were removed from the dish with 1 % Triton X - IO0 and the soluble ( Triton soluble ) and insoluble ( Triton insoluble ) fractions separated by centrifugation . Caveolin was immunoprecipitated from each fraction as described in Ma - terials and Methods . The Journal of Cell Biology , Volume 127 , 1994 1188 Figure 3 . Immunoltuorescence ( A , C , and E ) and immunogold ( B , D , and F ) localization of caveolin in untreated ( A and B ) and cholesterol oxidase - treated ( C - F ) cells . Human fibroblasts were incubated fo 1 h at 37°C in either medium alone ( A and B ) or medium that contained 0 . 5 U / ml of cholesterol oxidase ( C - F ) . One set of cholesterol oxidase - treated cells ( E and F ) was incubated an additional 1 h in medium that did not contain any enzyme . At the end of the incubations , cells were processed to localize caveolin using either immunofluorescence ( A , C , and E ) or immunogold ( B , D , and F ) immunocytochemistry . Bar , ( a , c , and e ) 5 . 7 # m ; ( b , d , and f ) 0 . 4 # m . vaginated caveolae at the cell surface ( Fig . 3 B , bottom ) and that very little was present in the Golgi apparatus ( Fig . 3 B , top ) . A 1 - h treatment with cholesterol oxidase at 37°C dra - matically changed this distribution . The caveolin now ap - peared in numerous vesicles that were concentrated near the nucleus of the cell ( Fig . 3 C ) . Examination of treated cells with the electron microscope ( Fig . 3 D ) showed that in re - sponse to oxidation of cholesterol , cells accumulated numer - ous vesicles that were associated with the Golgi apparatus . The lumen of many of these vesicles was heavily labeled with anti - caveolin IgG - gold ( Fig . 3 D , top [ * ] ) . There also was a dramatic reduction in the amount of gold in caveolae ( Fig . 3 D , bottom ) although caveolar morphology was unchanged . Quantification of the gold particles ( Table I A ) showed that cholesterol oxidase caused a , , o60 % decrease in the number of gold particles associated with caveolae . By contrast , there was only a 10 % decline in the number of caveolae . Most of the caveolin returned to the cell surface ( Fig . 3 E ) after removal of cholesterol oxidase for 1 h at 37°C . The Golgi re - gion returned to a more normal appearance , although some Smart et al . Caveolin Tra ~ c to Golgi Region 1189 Table L Distribution of Caveolin After Cholesterol Oxidase Treatment A . Cholesterol Oxidase at 37 " C % Labeled Total Gold / labeled Treatment caveolae Total gold caveolae caveola Caveolae / I0 t ~ m Control 47 1519 635 4 . 4 Cholesterol oxidase 29 632 632 3 . 6 B . Cholesterol Oxidase at 18 " C then Warmed ( ~ ) to 370C % Labeled Total Gold / labeled Treatment caveolae Total gold caveolae caveola 1 . 65 1 . 47 Control @ 18°C 65 5710 473 4 . 15 CO @ 18°C 41 4489 421 4 . 17 co @ 18 " c ; 48 3394 560 3 . 92 37°c 30 min CO @ 18 " C 4 31 1927 525 3 . 22 37°C 90 min Human fibroblasts were subjected to the same conditions as described in the legend to Fig . 3 ( for A ) or Fig . 7 ( for B ) and then processed for quantification of anti - caveolin mAb gold as described . of the trans - most cisternae appeared to be dilated ( Fig . 3 F , top * ) . Very little immunogold was present in these cister - nae . The quantity of caveolae - associated gold returned to normal levels ( Fig . 3 F , bottom ) . The return of caveolin to caveolae paralleled the decline in membrane cholestenone . Indirect immunofluorescence was used to determine if caveolin moved to the Golgi apparatus under conditions where protein synthesis was inhibited ( Fig . 4 ) . Cyclohexi - mide was added to cells 5 h before the experiment and kept present throughout the incubations . Cells were either not treated ( Fig . 4 , A and B ) , incubated in the presence of cho - lesterol oxidase ( Fig , 4 , C and D ) , or exposed to cholesterol oxidase and then further incubated 1 h in the absence of the enzyme ( Fig . 4 , E and F ) . We then used immunofluores - cence to colocalize caveolin ( Fig . 4 , A , C , and E ) and the Golgi marker ( 3 ) , galactosyltransferase ( Fig . 4 , B , D , and F ) . Normal surface membrane and Golgi apparatus staining was observed for the two markers in untreated cells ( Fig . 4 , A and B ) . After incubation in the presence of cholesterol oxi - dase , however , anti - caveolin IgG and anti - galactosyltrans - ferase IgG staining were superimposed , indicating that cav - colin had migrated to the Golgi apparatus ( Fig . 4 , C and D ) . Just as we observed with the electron microscope ( Fig . 3 ) , the Golgi apparatus appeared to be enlarged after cholesterol oxidation ( compare Fig . 4 , B with D ) . When the cholesterol oxidase was removed from the media and the cells incubated 1 h , anti - caveolin IgG staining was normal ( Fig . 4 , E and F ) , which indicates that caveolin had returned to caveolae . There - fore , cholesterol oxidase causes caveolin to move to the Golgi apparatus but the same molecules can return once the enzyme is removed . The appearance of anti - caveolin IgG within the lumen of the Golgi cisternae after cholesterol oxidase treatment sug - gested that this integral membrane protein could move from the inside surface of the plasma membrane across the Golgi membrane . Therefore , we used two other standard methods to assess the topology of caveolin : antibody accessibility and protease protection . At the proper temperature and concentration , digitonin will permeabilize the plasma membrane but leave the ER and Golgi membranes intact ( 2 ) . We used indirect immunofluo - rescence with an antibody against the resident ER protein BiP ( immunoglobulin heavy chain binding protein , 4 ) to determine the conditions for selective permeabilization of human fibroblasts ( Fig . 5 , D - F ) . We found that 15 # g / ml of digitonin did not allow the antibody access to the BiP ( Fig . 5 E ) but treatment with 0 . 1 % Triton X - 100 gave a typical anti - BiP IgG staining pattern ( Fig . 5 D ) . We then incubated cells in the presence of cholesterol oxidase and sequentially treated them with 15 t ~ g / ml of digitonin and Triton X - 100 . These cells displayed a typical anti - BiP staining pattern ( compare Fig . 5 , F with D ) . When untreated cells were per - meabilized with 15 # g / ml of digitonin and stained with anti - caveolin IgG , the staining pattern was normal ( Fig . 5 A ) . By contrast , when we incubated the cells in the presence of cho - lesterol oxidase and then permeabilized them with digitonin , anti - caveolin IgG did not bind ( Fig . 5 B ) . Antibody accessi - bility was restored after the cholesterol oxidase treated cells were sequentially treated with digitonin and Triton X - 100 ( Fig . 5 C ) . Protease protection experiments also indicated that cave , o - lin was crossing the ER - Golgi membrane system in choles - terol oxidase - treated cells ( Fig . 6 ) . Cells grown in cyclohexi - mide to prevent protein synthesis were subjected to the different treatments and then gently homogenized in an iso - tonic buffer to preserve the integrity of intracellular or - ganelles . We added either 300 ftg / ml of trypsin or buffer alone directly to the homogenate and incubated the mixture for 30 min on ice . The proteins were separated by elec - trophoresis and immunoblotted with anti - caveolin IgG ( Fig . 6 ) . Untreated cells that were not exposed to the protease showed a distinct caveolin band ( Fig . 6 , NT ) . The addition of trypsin to untreated cell homogenates caused this band to disappear ( Fig . 6 , NT + T ) . Cholesterol oxidase - treated cells that were not exposed to trypsin also had a single im - munoreactive band ( Fig . 6 , CO ) . In contrast to untreated The Journal of Cell Biology , Volume 127 , 1994 1190 Figure 4 . Reversible movement of caveolin to the Golgi apparatus in response to cholesterol oxidase . Human fibroblasts were incubated for 5 h in the presence of 50 tzg / ml of cycloheximide . Ceils were then either not treated ( A and B ) , incubated in the presence of 0 . 5 U / ml of cholesterol oxidase for 1 h at 37°C ( C and D ) , or incubated in the presence of cholesterol oxidase followed by incubation in the absence of the enzyme for 1 h at 37°C ( E and F ) . Cycloheximide was present during all of the incubations . At the end of the incubations , the cells were fixed and processed to co - localize eaveolin ( A , C , and E ) and the Golgi apparatus marker , galactosyltransferase ( B , D , and F ) by indirect immunofluorescence . All ceils were fixed with periodate - lysine - paraformaldehyde fixative . Bar , 5 . 0 ~ m . cells , this band was only slightly reduced in intensity when trypsin was added to the homogenate ( Fig . 6 , CO + T ) . The protected portion of caveolin was degraded when Triton X - 100 was added to permeabilize membranes ( Fig . 6 , CO + T + Triton ) . Caveolin became completely protease sensi - tive after the cholesterol oxidase was removed from the medium and the cells incubated an additional 1 h ( Fig . 6 , R + T ) even though protein synthesis was inhibited . Low Temperatures and BFA Block Movement of Caveolin We next carried out a series of experiments to identify the route that caveolin takes to get to the Golgi membrane . Since Golgi membrane and endosomal membrane traffic are both blocked at 18°C ( 8 , 13 ) , we incubated cells in the presence of cholesterol oxidase at this temperature and looked at the distribution of caveolin . Immunofluorescence showed ( Fig . Smart ¢t al . Caveolin Tragic to Golgi Region 1191 Figure 5 . Effect of cholesterol oxidase on accessibility of caveolin to anti - caveolin IgG after digitonin permeabilization . Cells were incubated in the presence ( B , C , E , and F ) or absence ( A and D ) of cholesterol oxidase as described in Fig . 3 . Cells stained with anti - BiP IgG ( D - F ) were permeabilized either with Triton X - 100 ( D ) , digitonin ( E ) , or digitonin plus Triton X - 100 ( F ) . Cells stained with anti - caveolin IgG were permeabilized either with digitonin ( A and B ) or digitonin followed by Triton X - 100 ( C ) . All cells were fixed with periodate - lysine - paraformaldehyde fixative . Bar , 4 . 8 # m . 7 ) that most of the caveolin remained associated with the cell surface ( compare Fig . 7 , A with B ) . This temperature had no effect on the conversion of cholesterol to cholestenone ( 6 % ) . When we shifted the 18°C - treated cells to 37°C for 30 min , almost all of the caveolin staining was in the Golgi apparatus ( Fig . 7 C ) . 90 min after the shift , the caveolin was predominantly in vesicles near the cell center and in small vesicles distributed throughout the cell ( Fig . 7 D ) . We also found that once caveolin had migrated to the Golgi region it would not return to the cell surface at 18°C ( Fig . 7 ) . Caveolin was induced to move by treating the cells with cholesterol oxidase at 37°C . The cells were washed to re - move the enzyme and further incubated 1 h at 18°C . Fig . 7 E shows that the caveolin remained in the Golgi region . Cel - lular cholestenone levels also remained constant during the incubation . By contrast , caveolin promptly returned to the cell surface when cells were incubated at 37°C for 1 h ( Fig . 7 F ) . We took advantage of the 18°C block and used im - munogold labeling to follow caveolin from the cell surface to the Golgi region after shifting the temperature to 37°C ( Fig . 8 ) . Incubating cells at 18°C in the absence of choles - terol oxidase had no eff - eet on the amount of anti - cave , olin specific immunogold in either the Golgi apparatus ( Fig . 8 A ) The Journal of Cell Biology , Volume 127 , 1994 1192 Figure 6 . Effect of cholesterol oxidase on trypsin sensitivity of caveolin . Human fibro - blasts that had been preincu - bated in the presence of 50 # g / ml of cycloheximide for 5 h were incubated either ; in the absence of cholesterol oxidase ( NT ) , in the presence of 0 . 5 U / ml of cholesterol oxidase ( CO ) for 1 h , or in the presence of choles - terol oxidase for I h followed by an additional 1 h in medium alone ( R ) . Cells were homogenized as described in Materials and Meth - ods and treated with 300 / ~ g / ml of trypsin ( NT + T , CO + T , R + T , and CO + T + Triton ) for 30 min on ice . One set ofhomogenates from cholesterol oxidase treated cells was mixed with 0 . 1 % Triton X - 100 ( CO + T + Triton ) to allow trypsin access to the sequestered protein . After each treatment , the presence of caveolin was deter - mined by immunoblot as described in Materials and Methods . or the ER compartments ( Fig . 8 B ) . With cholesterol oxi - dase present at this temperature , however , there was a sub - stantial amount of immunogold associated with the rough ER ( Fig . 8 D [ * ] ) while the amount of gold label in the Golgi region remained unchanged ( Fig . 8 C ) . Cells shifted to 37°C for 30 min had an increased amount of immunogold in the ER ( Fig . 8 F ) with a pronounced shift in distribution towards the smooth ER ( Fig . 8 F [ * ] ) . Smooth ER could be recog - nized because often it was in continuity with the rough ER ( Fig . 8 , F and H ; arrows ) and the two ER compartments both contained a similar flocculent material in the lumen . Heavily labeled , dilated smooth ER was also found associated with the Golgi apparatus ( Fig . 8 E [ * ] ) . After 90 min at 37°C , rough ER labeling declined ( Fig . 8 , G and H ) but there was still good labeling of both smooth ER ( Fig . 8 H ) and Golgi - associated compartments ( Fig . 8 G ) . Figure 7 . Effect of temperature on the distribution of caveolin in cholesterol oxidase treated cells . Human fibroblasts were incubated under the following conditions : ( A ) no cholesterol oxidase at 18°C ; ( B ) 0 . 5 U / ml cholesterol oxidase at 18°C ; ( C ) cholesterol oxidase at 18°C followed by cholesterol oxidase at 37°C for 30 min ; ( D ) cholesterol oxidase at 18°C followed by cholesterol oxidase at 37°C for 90 rain ; ( E ) cholesterol oxidase at 37°C followed by medium alone at 18°C ; ( F ) cholesterol oxidase at 37°C followed by medium alone at 37°C . At the end of the incubations , the cells were fixed and processed to localize caveolin by indirect immunofluorescence . Bar , 5 . 7 # m . Smart et al . Caveolin Tmffw to Golgi Region I l°B Figure 8 . Immunogold localization of caveolin during its migration to the Golgi region of the cell in cholesterol oxidase treated cells . Human fibroblasts were either incubated in the presence ( C and D ) or absence ( A and B ) of 0 . 5 U / mi of cholesterol oxidase at 18°C or they were incubated in the presence of cholesterol oxidase at 18°C and then warmed to 37°C in the presence of cholesterol oxidase for the indicated time ( E - H ) . At the end of the incubations , cells were fixed and processed for immunogold localization of caveolin as described . Representa - tive areas of the Golgi region ( A , C , E , and G ) and ER ( B , D , F , and H ) compartments are shown for each treatment . Bar , 0 . 4 pro . The Journal of Cell Biology , Volume 127 , 1994 1194 Quantitative electron microscopy confirmed these visual results ( Table I B ) . Incubation in the presence of cholesterol oxidase at 18°C alone caused a 12 % decline in the amount of immunogold that was associated with caveolae . After the temperature was shifted to 37°C , there was a progressive loss of caveolin from caveolae until at 90 min 70 % had migrated away from this compartment . The dispersed arrangement of the ER in fibroblasts made it impossible to determine quan - titatively if all of the caveolin had moved to the ER - Golgi compartment . These results suggest that the rough ER may be involved in the movement of caveolin to the Golgi region of the cell . If this is the pathway , then brefeldin A ( BFA ) should block the accumulation of caveolin in the Golgi area because this drug disrupts membrane traffic between ER and Golgi com - partments ( 20 ) . Incubation of human fibroblasts in the pres - ence of BFA had no effect on the distribution of caveolin ( Fig . 9 A ) . When we switched these cells to medium that con - tained cholesterol oxidase and BFA ( Fig . 9 B ) , anti - caveolin IgG labeling at the cell surface markedly declined . Instead , the staining pattern appeared as small , thin tubules that were diffusely distributed throughout the cell . When the BFA was removed in the presence of cholesterol oxidase , the caveolin migrated to the Golgi apparatus ( Fig . 9 C ) . Caveolin returned to the cell surface after 1 h of incubation in the ab - sence of both BFA and cholesterol oxidase ( Fig . 9 D ) . Discussion Previous immunogold localization studies showed that cav - eolin was a component of the spiral coat that decorates the inside surface of the plasma membrane ( 28 ) . The coat be - haves as if it is constructed from integral membrane proteins ( 28 ) , and the amino acid sequence of caveolin predicts that it belongs to this class of molecules ( 10 , 17 ) . The migratory behavior of caveolin now suggests that it is not a structural molecule of caveolae because the morphology of caveolae appears normal after most of the caveolin moves to the Golgi region . Therefore , caveolae can apparently exist in the ab - sence of caveolin . This raises the possibility that there are tissue cells that have functional caveolae but no caveolin . Caveolin and Membrane Cholesterol The integrity of caveolae appears to depend on cholesterol ( 7 ) . Previous electron microscopic studies have shown that caveolae are enriched in this sterol ( 33 ) . We have also shown that depletion of intracellular cholesterol in MA104 cells causes a 10 - fold reduction in the number of invaginated cav - eolae ( 29 ) and a significant inhibition of potocytosis ( 7 ) . In addition , treatment of cells with cholesterol - binding drugs such as filipin causes invaginated caveolae to become fiat ( 29 ) . These results suggested that potocytosis and , conse - quently the opening and closing of the caveolae , are con - trolled by the lipid phase of the caveolar membrane . Cholesterol oxidase has long been used to distinguish be - tween plasma membrane and intraceUular cholesterol pools ( 19 ) . We can use the conversion of cholesterol to choleste - none by cholesterol oxidase to calculate that (cid:127)6 - 7 % of the total cellular cholesterol is in caveolae . If90 % of the choles - terol is in the plasma membrane ( 19 ) and caveolae occupy 1 - 2 % of this surface ( 30 , 37 ) , then caveolae membranes have Figure 9 . BFA prevents cholesterol oxidase - induced migration of caveolin to the Golgi region of the cell . Human fibroblasts were subjected to the following treatments at 37°C : ( A ) 1 h in 18 # M ( BFA ) ( B ) 1 h pretreatment with BFA followed by 1 h in 0 . 5 U / ml cholesterol oxidase plus BFA ; ( C ) 1 h in BFA plus cholesterol oxidase followed by cholesterol oxidase alone for 1 h ; ( D ) BFA plus cholesterol oxidase for 1 h followed by 1 h in medium alone . At the end of the incubations , the cells were fixed and processed to localize caveolin by indirect immunofluorescence . Bar , 5 . 7 # m . Smart et al . Caveolin Tragic to Golgi Region 1195 four - to eightfold more cholesterol than the surrounding plasma membrane . Our results suggest that caveolin may play a role in main - taining the proper cholesterol environment in the caveolae . Upon oxidation of caveolar cholesterol the caveolin leaves the plasma membrane and moves to the Golgi region of the cell . Ordinarily caveolin is not seen in the Golgi region be - cause it spends most of the time on the cell surface . Oxida - tion of cholesterol perturbs this balance so that caveolin spends more time in the Golgi apparatus , which allows de - tection by indirect immunofluorescence . The cholestenone remains in the caveolae but the caveolae regain the normal amount of cholesterol as the caveolin returns to the plasma membrane . The fate of the cholestenone is not known . In polarized epithelial cells caveolin associates with GPI - anchored proteins , glycolipids , and cholesterol in the Golgi apparatus ( 6 , 21 ) . The caveolin - lipid complex then moves to the cell surface in Golgi - derived transport vesicles ( 17 ) . The cycling of caveolin between the plasma membrane and the Golgi apparatus may allow the cell to specifically control the level of cholesterol in the caveolae and consequently their function . Caveolin is not the only protein that migrates to the Golgi apparatus in response to oxysterols . The cytoplasmic oxy - sterol - binding protein ( OSBP ) also moves to the Golgi ap - paratus ( 27 ) when complexed with 25 - hydroxycholesterol . OSBP is normally diffuse in the cytoplasm but after cells are incubated in the presence of 25 - hydroxycholesterol it be - comes associated with the Golgi apparatus . Our finding that caveolin moves to the Golgi apparatus suggests that the Golgi apparatus may have a general role to play in cholesterol homeostasis . Caveolin Can Move from the Cell Surface to the Golgi Apparatus The most remarkable observation in this study was the finding that caveolin , which is an integral membrane protein , can move to the Golgi apparatus region of the cell . The num - ber of caveolae on the surface of the cell changed very little after cholesterol oxidase treatment , which may mean that the caveolin is not moving to the Golgi apparatus in plasmalem - rnal vesicles that have formed from caveolae . We also were unable to detect caveolin in any type of transport vesicle . While there are other explanations for the observations we have presented , two of the experimental results suggest that the ER has some role to play in the movement of caveolin to the Golgi apparatus : ( a ) oxidation of cholesterol at 18°C caused immunogold - detectable caveolln to first appear as - sociated with the rough ER and this was followed by the progressive appearance of gold labeling in the Gelgi appara - tus after the cells were warmed to 370C for various times ( Fig . 8 ) ; ( b ) brefeldin A , which inhibits membrane traffic be - tween the ER and Golgi apparatus ( 20 ) , did not prevent the initial movement of caveolln into the cell but did prevent it from reaching the Golgi apparatus ( Fig . 9 ) . Further work will be required to determine exactly how the endoplasmic reticulum participates in caveolin transport to the Golgi ap - paratus . The movement of caveolin to the Golgi region was revers - ible . Within one hour after cholesterol oxidase was removed from the medium , caveolin returned to the Triton X - 100 - insoluble fraction ( Fig . 2 ) and to the cell surface ( Fig . 4 ) even when protein synthesis was blocked with cyclohexi - mide . This means that the same molecule of eaveolin can travel between these compartments in a cycle . We do not know how caveolin returns to caveolae . The identification of a novel cycling system for caveolin that is revealed by the oxidation of caveolar cholesterol sup - ports previous findings that cholesterol is critical for the nor - mal function of this membrane specialization ( 7 , 29 ) . The potential existence of a new pathway between the caveolae and the Golgi apparatus has broad implications for under - standing the function of normal and diseased cells . We would like to thank Mr . William Donzell for his important contribution in carrying out the immunofluorescence experiments . We are also grateful to Ms . Grace Liao for technical assistance . This work was supported by grants from the National Institutes of Health , HL 20948 , GM 43169 , and GM 15631 and the Perot Family Foun - dation . Received for publication 15 August 1994 and in revised form 7 September 1994 . References 1 . Anderson , R . G . W . , B . A . Kamen , K . ' G . Rothberg , and S . W . Lacey . 1992 . Potocytosis : sequestration and tran ~ rt of small molecules by cav - eolae . Science ( Wash . DC ) . 255 : 410 - 411 . 2 . Balch , W . E . , W . G . Dunphy , W . A . Braell , and J . E . Rothnmn . 1984 . Reconstitotion of the transport of protein between successive compart - ments of the Golgi measured by the coupled incorporation of N - acetylgin - cosamine . Cell . 39 : 405 - 416 . 3 . Berger , E . G . , U . Muller , E . Aegerter , and G . J . Strous . 1987 . Biology of galactosyltransferase : recent developments . Biochera . Soc . Trans . 15 : 610 - 613 . 4 . Bole , D . G . , L . M . Hendershot , andJ . F . 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Ying , Y . - S . , R . G . W . Anderson , andK . G . Rothberg . 1992 . Each caveola contains multiple glycosyl - phosphatidylinositol anchored membrane pro - teins . Cold Spring Harbor Symp . Quant . Biol . 57 : 593 - 604 . Smart et al . Caveolin Tragic to Golgi Region 1197 \ No newline at end of file diff --git a/silver_data/ba05405b435bbdfd214279b7fb6e0d5dad41a80d.pdf.txt b/silver_data/ba05405b435bbdfd214279b7fb6e0d5dad41a80d.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..96f24a578e01c895a2477b26468214328cbc3ff1 --- /dev/null +++ b/silver_data/ba05405b435bbdfd214279b7fb6e0d5dad41a80d.pdf.txt @@ -0,0 +1 @@ +Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 https : / / doi . org / 10 . 1186 / s12879 - 022 - 07461 - 9 RESEARCH Seroprevalence and characteristics of Coronavirus Disease ( COVID - 19 ) in workers with non - specific disease symptoms Wajiha Javed , Syed Hussain Baqar Abidi * and Jaffer Bin Baqar Abstract Background : The population - based serosurveys are essential for estimating Coronavirus Disease - 19 ( COVID - 19 ) burden and monitoring the progression of this pandemic . We aimed to assess the seroprevalence of SARS - CoV - 2 antibodies and potential predictors of seropositivity in the Pakistani population . Methodology : This population - based seroprevalence study includes consenting subjects from the workplaces ( factories , corporates , restaurants , media houses , schools , banks , and hospitals ) located in the urban areas of Karachi , Lahore , Multan , Peshawar , and Quetta . We analyzed each subject’s serum sample for SARS - CoV - 2 - IgM and / or IgG anti - bodies using UNIPER IgG / IgM Rapid COVID - 19 Testing Kit . The subject’s demographics , exposure history , and symp - toms experienced ( in last 7 days ) were also obtained . The collected data was analyzed using SPSS version 22 . 0 . Results : The overall seroprevalence of SARS - CoV - 2 antibodies was 16 . 0 % ( 2810 / 17 , 764 ) . The total antibody seroposi - tivity was higher in males than females ( OR 1 . 22 , 95 % CI 1 . 110 – 1 . 340 ) . The symptomatic subjects had 2 . 18 times higher odds of IgG seropositivity while 1 . 2 times for IgM seropositivity than the asymptomatic subjects . The multivariable logistic regression model showed that the odds of SARS - CoV - 2 total antibody seroprevalence were affected by the number of dependents ( OR = 1 . 077 ; 95 % CI 1 . 054 – 1 . 099 ) , apparent symptomology ( OR = 1 . 288 ; 95 % CI 1 . 011 – 1 . 643 ) , close unprotected contact with a confirmed or probable case of COVID - 19 ( OR 2 . 470 ; 95 % CI 2 . 164 – 2 . 819 ) , traveling history ( last 14 days ) ( OR = 1 . 537 ; 95 % CI 1 . 234 – 1 . 914 ) and proximity with someone who traveled ( OR = 1 . 534 ; 95 % CI 1 . 241 – 1 . 896 ) . Conclusion : We found a reasonable seroprevalence of SARS - CoV - 2 antibodies in the studied population . Several factors like the number of dependents , apparent symptoms , close unprotected contact with a confirmed or probable case of COVID - 19 , traveling history , and proximity with someone who traveled are associated with increased odds of SARS - CoV - 2 antibody seropositivity . Keywords : SARS - CoV - 2 , COVID - 19 , Seroprevalence , IgM , IgG , Risk Factors , Potential Predictors © The Author ( s ) 2022 . Open Access This article is licensed under a Creative Commons Attribution 4 . 0 International License , which permits use , sharing , adaptation , distribution and reproduction in any medium or format , as long as you give appropriate credit to the original author ( s ) and the source , provide a link to the Creative Commons licence , and indicate if changes were made . The images or other third party material in this article are included in the article’s Creative Commons licence , unless indicated otherwise in a credit line to the material . If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use , you will need to obtain permission directly from the copyright holder . To view a copy of this licence , visit http : / / creat iveco mmons . org / licen ses / by / 4 . 0 / . The Creative Commons Public Domain Dedication waiver ( http : / / creat iveco mmons . org / publi cdoma in / zero / 1 . 0 / ) applies to the data made available in this article , unless otherwise stated in a credit line to the data . Introduction After emerging from Wuhan , the novel Coronavirus has rapidly transmitted throughout the world . It is now regarded as a pandemic [ 1 ] , accompanied by a varying range of mild symptoms including cough , fever , cold and body ache and even severe , like Acute Severe Respira - tory Distress ( ASRD ) become the leading cause of death among these patients [ 2 – 4 ] . According to the World Health Organization ( WHO ) reports , we now have more than 296 million confirmed COVID - 19 cases and more than 5 million deaths worldwide [ 5 ] . Locally in Pakistan , the first two cases were reported in February 2020 , hav - ing a travel history from Iran . Although all preventive Open Access * Correspondence : hussain . baqar @ getzpharma . com Getz Pharma ( PVT ) Limited , Karachi , Pakistan Page 2 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 measures were taken to contain the disease , unfortu - nately , around 1 , 301 , 141 cases have been identified since then , accounting for 28 , 961 deaths [ 6 ] . But the concern regarding disease control , morbidity , and mortality in Pakistan remains ; it is claimed that 415 , 352 cases have successfully recovered [ 6 ] , possibly due to low testing rates in Pakistan compared to the rest of the world [ 7 ] . One of the significant reasons for limited testing facilities is the restricted healthcare budget and resources [ 7 ] . As COVID - 19 has become a global threat , early detec - tion and prevention of viral spread have become cru - cial . Reverse transcription - polymerase chain reaction ( RT - PCR ) is recognized as the gold standard diagnostic technique as it helps in the early recognition of COVID - 19 [ 8 ] . Although , due to compromised test sensitivity in association with inadequate sample collection , the time between sample collection , the onset of symptoms , and the fluctuation in viral load [ 9 ] linger the duration . However , an easy , sensitive , and inexpensive alternate , i . e . , Antibody Test , is now being used to identify SARS - COV - 2 . Antibody screening through serological assays helps determine the actual frequency and seroprevalence of the virus [ 10 ] . The antibodies IgG and IgM can be detected within 1 – 3 weeks after exposure to Coronavi - rus . A rapid increase in the antibody level and the sero - conversion rate is observed during the first two weeks [ 11 ] . Presently the seroconversion rate is being widely studied in order to understand its dynamics , specifically from acute to convalescent phases [ 12 ] . It has been found that the IgM and IgA growth after exposure to SARS - CoV - 2 is relatively slow compared to other acute viral infections contributing to its heterogeneous pathogenic - ity [ 13 ] . Old age , pre - existing comorbid conditions , low CD3 + CD8 + T - cells levels , high troponin I and d - dimer levels have been recognized as the major risk factors associated with seropositivity and mortality among the infected patients [ 14 – 17 ] . More recently , studies con - ducted in Shenzhen , including 1286 close contacts ( 98 COVID positive cases ) and Guangzhou including 2098 close contacts ( 134 COVID positive cases ) , discovered that significant risk factors for COVID - 19 infection were among older age and traveling outside or inside the coun - try , etc [ 18 , 19 ] . Additionally , a Taiwanese study identified exposure to a confirmed or probable case of COVID - 19 as a risk factor [ 20 ] . In the current study , we employed a large dataset , including 17 , 764 participants from Karachi , Lahore , Mul - tan , Peshawar , and Quetta , to estimate the seroprevalence of SARS - CoV - 2 antibodies , characteristics , and potential predictors of seropositivity . This serological investigation intends to elaborate Pakistan’s COVID infection statistics under the worldwide pandemic . Methodology Study design & participants A population - based study was conducted to investigate the seroprevalence of COVID - 19 infection in Pakistan . Sample size was calculated on standard assumptions , which are , 95 % confidence interval with 5 % margin of error and considering 50 % estimated prevalence of COVID - 19 during the first wave of pandemic . With these assumptions we had sample size n = 384 for a sin - gle center . We aimed to cover approx . 40 centers across Pakistan and achieved a well representative sample size n = 17 , 764 for final analysis . Participants between 18 to 65 years of age were recruited during April - September 2020 from the workplaces , including factories , corpo - rates , restaurants , media houses , schools , banks , and hos - pitals , located in Karachi , Lahore , Multan Peshawar , and Quetta through non - probability convenience sampling technique . While non - consenting individuals , PCR - pos - itive at the time of screening , having past 14 - days history of COVID - 19 and COVID - 19 vaccine trials’ participants were excluded . Primary & secondary endpoints The primary endpoint was to assess the seroprevalence of SARS - CoV - 2 antibodies ( IgM and IgG ) among the studied individuals . And the secondary endpoint was to determine the risk factors associated with the seroposi - tivity of these antibodies . Procedures & laboratory analysis For this population - based seroprevalence study , the patient’s baseline demographics , medical history , expo - sure information and details regarding the symptom profile of last 7 - days , seropositive rates and the factors associated with SARS - CoV - 2 antibodies were investi - gated at the time of sample collection and noted using a structured questionnaire . The samples were drawn for SARS - CoV - 2 rapid antibody testing in a temperature controlled environment by a healthcare professional . IgM and / or IgG antibodies were assessed from human serum using Unite Diagnostic Strip for N Coronavirus ( 2019 - nCoV ) IgG / IgM Antibody ( Colloidal Gold Immuno - chromatography Assay ) by Uniper Medical Technology Limited , approved by U . S . Food and Drug Administra - tion ( US FDA ) EUA and certified by European Certified ( C . E . ) . Statistical analysis The data were statistically analyzed using SPSS version 22 . 0 . Categorical variables , including gender , age groups , household size , medical history , symptom profile , contact history , etc . , were summarized as frequencies and per - centages . Moreover , mean and standard deviation were Page 3 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 used for continuous variables like age , weight , and height . The seropositivity rate was calculated by the proportion of test - positive among those tested . Chi - square or Fisher exact test was used to compute the symptomatic associa - tion with antibody seropositivity . The Fitting Information Model was used to test the general fit of the model . More - over , the Multinomial logistic regression was applied to see the association between symptoms and antibodies . A multivariate logistic regression was performed , the IgM and IgG antibody’s significant predictors from Univariate analysis were entered into the final model . The forward Wald stepwise approach was used and the significant variables were determined as the independent predictors of seropositivity . A p - value < 0 . 05 was considered statisti - cally significant . Ethical considerations The study protocol was approved by the Ethical Review Board of AEIRC ( Reference no . MU / ECA / 20 / 17820 ; Dated 2nd April 2020 ) . All the procedures were per - formed in accordance with the Declaration of Helsinki , and the patient’s confidentiality was maintained through - out the investigation . The included patients were well - informed about the study objective and written informed consent was taken from them before inclusion . Results Participant demographics and exposures A total of 17 , 764 individuals were included in the study , with a mean age of 35 . 68 ± 14 . 17 years ( Range : 18 to 65 years ) , and of them , 13 , 003 were males , and 4 , 761 were females ( Table 1 ) . Prevalence of SARS‑CoV‑2 antibodies Out of 17 , 764 subjects , only 2 , 810 ( 16 . 0 % ) cases had either or both IgM and IgG positive antibodies . 951 ( 5 . 4 % ) tested positive for IgG antibody , 982 ( 5 . 5 % ) tested positive for IgM antibody , while the remaining had both antibodies ( n = 877 ) . Symptomatology among study individual The data regarding COVID - 19 symptomatology for last 7 - days was collected before antibody testing . About 8 . 2 % of the study individuals experienced flu - like symptoms , fever ( 7 . 5 % ) , and cough ( 7 . 4 % ) . While among the less common were sore throat , headache , and fatigue ( Fig . 1 ) . Of the IgM seropositive patients , the most com - monly reported symptoms were fever ( 26 . 35 % ) , cough ( 23 . 39 % ) , and flu ( 23 . 02 % ) as shown in Table 2 . Whereas flu ( 56 . 29 % ) , cough ( 48 . 85 % ) , headache ( 40 . 37 % ) and sore throat ( 37 . 14 % ) were the most frequently reported symp - toms among IgG - positive cases . Table 1 Baseline demographic characteristics of the study subjects Variables ( n = 17 , 764 ) Age ; Mean ± S . D . ( years ) 35 . 68 ± 14 . 71 Weight ; Mean ± S . D 68 . 81 ± 15 . 09 Height ; Mean ± S . D 63 . 01 ± 11 . 51 Gender n ( % ) Male 13 , 003 ( 73 . 2 ) Female 4761 ( 26 . 8 ) Household size < 5 3524 ( 19 . 8 ) ≥ 5 12 , 542 ( 70 . 6 ) Not Reported * 1698 ( 9 . 6 ) Median ( IQR ) 6 ( 5 – 8 ) No of dependents < 5 9946 ( 56 ) ≥ 5 4870 ( 27 . 4 ) Not Reported * 2948 ( 16 . 6 ) Median ( IQR ) 4 ( 1 – 5 ) Smoker No 15 , 652 ( 88 . 1 ) Yes 2112 ( 11 . 9 ) Comorbid conditions No 16 , 678 ( 93 . 3 ) Yes 872 ( 4 . 9 ) Not reported * 214 ( 1 . 2 ) Marital status Single 6165 ( 34 . 7 ) Married 11 , 599 ( 65 . 3 ) Monthly income ( PKR ) < 15 , 000 1433 ( 8 . 1 ) 15 , 000 – 30 , 000 1930 ( 10 . 9 ) 31 , 000 – 50 , 000 1143 ( 6 . 4 ) 51 , 000 – 75 , 000 1100 ( 6 . 2 ) 76 , 000 – 150 , 000 1764 ( 9 . 9 ) > 150 , 000 2538 ( 14 . 3 ) Not Reported * 7856 ( 44 . 2 ) Recent malaria history No 17 , 381 ( 97 . 8 ) Yes 164 ( 0 . 9 ) Not reported * 219 ( 1 . 2 ) BCG vaccine status No 3210 ( 18 . 1 ) Yes 14 , 188 ( 79 . 9 ) Not reported * 366 ( 2 . 1 ) Close unprotected contact with a confirmed or probable case of COVID - 19 No 14 , 604 ( 82 . 2 ) Yes 3024 ( 17 ) Not reported * 136 ( 0 . 8 ) Travelled outside or inside the country in the last 14 days No 16 , 840 ( 94 . 8 ) Page 4 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 The presence of symptoms showed higher odds of IgG seropositivity ( OR = 2 . 180 ; CI 1 . 65 – 2 . 88 ) as compared to that for IgM antibody ( OR = 1 . 190 ; CI 0 . 96 – 1 . 48 ) ( Table 3 ) . Factors associated with anti‑SARS‑CoV‑2 antibodies positivity in the studied population The odds of having IgM seropositivity were higher among males as compared to females ( 1 . 142 , 95 % CI 1 . 022 – 1 . 276 ) . While the OR = 1 . 006 ( 95 % CI 0 . 902 – 1 . 122 ) showed that the chances of IgG seropositivity were simi - lar in the two genders ( Table 4 ) . The odds of IgM and IgG seropositivity were similar among all age groups . In asso - ciation with the SARS - CoV - 2 total antibody , the model showed that close unprotected contact with a confirmed or probable case of COVID - 19 ( OR = 2 . 437 ; 95 % CI 2 . 221 – 2 . 675 ) , reporting previous symptoms ( OR = 1 . 391 ; 95 % CI 1 . 152 – 1 . 680 ) and proximity with someone who traveled ( OR = 2 . 270 ; 95 % CI 1 . 955 – 2 . 636 ) gave increased odds of for positive antibodies . Age , gender , number of dependents , marital status , monthly income , comorbid conditions , close unprotected contact with a confirmed or probable case of COVID - 19 travelling history ( outside or inside the country ) , and proximity with someone who traveled ( outside or inside the country in the last 14 days ) , were entered into the final model . In result , number of dependents , marital sta - tus , close unprotected contact with a confirmed or prob - able case of COVID - 19 and proximity with someone who traveled were found to be independent significant predic - tors of IgM seropositivity . The final model for IgG seropositivity included age , the number of dependents , smoker , monthly income , comorbid disease , BCG vaccine status , close unprotected contact with a confirmed or probable case of COVID - 19 , travelling history ( outside or inside the country in last 14 days ) , and proximity with someone who travelled ( outside or inside the country in last 14 days ) ( Table 6 ) . No of dependent , smokers , BCG vaccine status , symp - tomatology , close unprotected contact with a confirmed or probable case of COVID - 19 , travelling history , and proximity with someone who travelled were the inde - pendent significant predictors of IgG seropositivity . * Not Reported = Not available ( Missing data ) Table 1 ( continued ) Variables ( n = 17 , 764 ) Yes 791 ( 4 . 5 ) Not reported * 133 ( 0 . 7 ) Proximity with someone who travelled outside or inside the country in the last 14 days No 16 , 717 ( 94 . 1 ) Yes 916 ( 5 . 2 ) Not reported * 131 ( 0 . 7 ) Fig . 1 Symptom profile of the study subjects Page 5 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 The multivariate regression analysis showed that the potential predictors for total antibody positivity were number of dependents , symptomatology , close unpro - tected contact with a confirmed or probable case of COVID - 19 , traveled outside or inside the country in the last 14 days , and proximity with someone who traveled outside or inside the country in the last 14 days ( Table 7 ) . Discussion This is the first large - scale serological investigation of 2019 - nCoV in Pakistan to the best of our knowledge . As previously published , local studies targeted a spe - cific population or had a small sample size . A serosur - vey from Lahore included 154 policemen [ 21 ] ; similarly , the National Institute of Blood Diseases and Bone Mar - row Transplantation from Karachi published the data on SARS - CoV - 2 seroprevalence among 380 healthy blood donors [ 22 ] , and another included 1 , 675 health - care workers ( HCWs ) [ 23 ] . Moreover , the preprint data also represent seroprevalence in a limited sample size [ 24 , 25 ] . The current findings revealed that the overall seroprevalence of anti - SARS - CoV - 2 antibodies ( IgG and / or IgM ) was 16 . 0 % , i . e . , 2 , 842 out of 17 , 764 inhab - itants had developed SARS - CoV - 2 antibodies . This fig - ure is significantly higher than that reported in Italy ( 11 . 0 % ) , i . e . , 6 , 30 , 000 confirmed SARS - CoV - 2 cases [ 26 ] . This high seroprevalence is consistent with other studies [ 27 – 31 ] , 9 . 7 % reported among 2 , 766 subjects from Switzerland ( 2020 ) [ 27 ] , 13 . 8 % among 674 from Spain ( 2021 ) [ 28 ] , 21 . 4 % of 380 healthy blood donors from Pakistan ( 2020 ) [ 24 ] and 23 % of 390 subjects from Italy ( 2020 ) [ 31 ] . The seroprevalence of anti - SARS - CoV - 2 total anti - body was significantly higher among males ( OR 1 . 220 , 95 % CI 1 . 110 – 1 . 340 ) ( Table 4 ) . In contrast , no signifi - cant differences in the seroprevalence of COVID anti - bodies have been reported among males than females ( OR 1 . 2 , 95 % CI 0 . 8 – 1 . 8 ) , which is also evidently proven by the existing literature [ 26 , 32 , 33 ] . Further - more , we found no significant increase in the odds of SARS - CoV - 2 antibody seropositivity with respect to age ( OR 1 . 003 , 95 % CI 1 . 000 – 1 . 006 ) ( Table 4 ) . In con - trast , a study reported that the risk of seropositivity was lower among individuals ≤ 20 years of age than those aged > 60 years ( OR : 0 . 72 , 95 % CI : 0 . 60 – 0 . 87 ) [ 34 ] . In addition to age and gender , close unprotected con - tact with a confirmed or probable COVID - 19 case was also identified as a potential factor affecting the odds of seropositivity ( OR 2 . 437 , 95 % CI 2 . 221 – 2 . 675 ) ( Table 4 ) and also confirmed by the forward Wald stepwise logis - tic regression analysis , including the significant predic - tors of positive antibody in univariate analysis ( Tables 5 and 6 ) . As per the final observed model , number of dependents , symptomatology , closed unprotected con - tact with the COVID - 19 probable or confirmed case , travelling history , and proximity with someone who travelled were the independent significant predictors of seropositivity ( Table 7 ) . A study conducted by Rudberg et al . reported that the seropositivity was significantly affected by the patient - related work , i . e . , 21 % of the 1 , 764 subjects in patient contact vs . 9 % of the 305 sub - jects without patient contact ( OR 2 . 9 , 95 % CI 1 . 9 – 4 . 5 ) [ 35 ] . Moreover , the subjects with confirmed COVID Table 2 Seropositivity by COVID - 19 compatible symptoms Symptoms IgM seropositivity n ( % ) IgG seropositivity n ( % ) Flu 428 ( 23 . 02 ) 1029 ( 56 . 29 ) Fever 490 ( 26 . 35 ) 876 ( 7 . 92 ) Cough 435 ( 23 . 39 ) 893 ( 48 . 85 ) Dyspnea 102 ( 5 . 48 ) 145 ( 7 . 93 ) Anosmia 109 ( 5 . 86 ) 133 ( 7 . 27 ) Fatigue 343 ( 18 . 45 ) 646 ( 35 . 33 ) Myalgia 178 ( 9 . 57 ) 403 ( 22 . 04 ) Anorexia 112 ( 6 . 02 ) 152 ( 8 . 31 ) Expectoration 94 ( 5 . 05 ) 247 ( 13 . 51 ) Sore Throat 348 ( 18 . 71 ) 679 ( 37 . 14 ) Conjunctivitis 99 ( 5 . 32 ) 99 ( 5 . 41 ) GI Symptoms 160 ( 8 . 60 ) 480 ( 26 . 25 ) Confusion 86 ( 4 . 62 ) 111 ( 6 . 07 ) Dizziness 110 ( 5 . 91 ) 131 ( 7 . 16 ) Headache 266 ( 14 . 30 ) 738 ( 40 . 37 ) Hemoptysis 23 ( 1 . 23 ) 29 ( 1 . 58 ) Rhinorrhea 104 ( 5 . 59 ) 147 ( 8 . 04 ) Chest Pain 117 ( 6 . 29 ) 123 ( 6 . 72 ) Other Symptoms – 11 ( 0 . 60 ) Table 3 Associations between symptoms and seropositivity of SARS - CoV 2 antibodies * p - value < 0 . 05 is considered statistically significant Symptomatic IgM n ( % ) OR ( 95 % CI ) p‑value IgG n ( % ) OR ( 95 % CI ) p‑value Positive Negative Positive Negative No 96 ( 9 ) 971 ( 91 ) Ref . 0 . 107 55 ( 5 . 2 ) 1012 ( 94 . 8 ) Ref . 0 . 001 * Yes 1763 ( 10 . 6 ) 14 , 934 ( 89 . 4 ) 1 . 190 ( 0 . 96 – 1 . 48 ) 1773 ( 10 . 6 ) 14 , 924 ( 89 . 4 ) 2 . 180 ( 1 . 65 – 2 . 88 ) Page 6 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 patient contact had 1 . 4 times higher seropositivity odds than those who had non - COVID patient contact ( p < 0 . 05 ) [ 35 ] . As for clinical symptoms , we found that symptomatic participants had 2 . 18 times higher odds of IgG seroposi - tivity and 1 . 2 times higher IgM seropositivity than the asymptomatic participants ( Table 3 ) . The presence of symptoms was significantly associated with seropositiv - ity of IgG antibody and insignificant for the IgM anti - body . Interestingly , 9 % of the asymptomatic subjects showed IgM antibodies , and 5 . 2 % had IgG antibodies . Also supported by multiple other studies [ 24 , 26 ] , Vena et al . reported seropositivity among 8 . 6 % of the asymp - tomatic subjects , and a similar local study reported that 53 . 2 % of COVID positive HCWs were completely asymp - tomatic [ 26 ] . These findings indicated that non - apparent infections are common among healthy , active individuals . However , the seroprevalence of SARS - CoV - 2 antibod - ies was reportedly high among symptomatic individuals ( p < 0 . 05 ) . The most commonly reported symptoms of the IgM seropositive patients were fever , cough and flu . Whereas headache , flu , cough and sore throat were the most frequently reported symptoms among IgG - positive cases ( Table 2 ) . In contrast , a study described the strong - est association of symptoms like anosmia , ageusia , and fever with seropositivity of SARS - CoV - 2 antibody [ 35 ] . The present study’s findings have several implica - tions for pandemic management since a large propor - tion of COVID infected subjects remain asymptomatic , also supported by the current study findings . Hence , the actual numbers of confirmed SARS - CoV - 2 cases might be higher than those reported . Thus , we need to employ stringent screening strategies targeting the infectious spread beyond the current symptom - driven approach [ 36 ] . Furthermore , the study limitations must also be dis - cussed ; we analyzed the serum samples of only those who voluntarily decided to be tested ( workers from selective Table 4 Univariate analysis of the factors associated with SARS - CoV - 2 Antibodies * Significant at p ≤ 0 . 05 * * Reference category Predictors IgM antibody IgG antibody Total antibody OR 95 % CI p‑value OR 95 % CI p‑value OR 95 % CI p‑value Age ( years ) 1 . 010 1 . 006 – 1 . 012 0 . 001 * 1 . 004 1 . 001 – 1 . 007 0 . 011 * 1 . 003 1 . 000 – 1 . 006 0 . 024 * Gender ( Male ) 1 . 142 1 . 022 – 1 . 276 0 . 019 * 1 . 006 0 . 902 – 1 . 122 0 . 914 1 . 220 1 . 110 – 1 . 340 0 . 001 * Household size 1 . 006 0 . 999 – 1 . 013 0 . 105 1 . 000 0 . 993 – 1 . 008 0 . 909 1 . 001 0 . 995 – 1 . 008 0 . 735 No of dependents 1 . 073 1 . 053 – 1 . 093 0 . 001 * 1 . 065 1 . 045 – 1 . 085 0 . 001 * 1 . 063 1 . 047 – 1 . 080 0 . 001 * Smoker 1 . 129 0 . 979 – 1 . 303 0 . 096 0 . 739 0 . 627 – 0 . 871 0 . 001 * 1 . 069 0 . 946 – 1 . 209 0 . 283 Married 1 . 348 1 . 213 – 1 . 498 0 . 001 * 1 . 101 0 . 994 – 1 . 221 0 . 066 0 . 895 0 . 821 – 0 . 976 0 . 012 * Monthly income ( < 15 , 000 ) * * 15 , 000 – 30 , 000 1 . 278 1 . 003 – 1 . 629 0 . 047 * 0 . 956 0 . 740 – 1 . 235 0 . 73 1 . 208 0 . 980 – 1 . 489 0 . 077 31 , 000 – 50 , 000 1 . 097 0 . 828 – 1 . 453 0 . 518 0 . 867 0 . 643 – 1 . 169 0 . 35 0 . 991 0 . 991 – 0 . 776 0 . 944 51 , 000 – 75 , 000 1 . 404 1 . 072 – 1 . 838 0 . 014 * 1 . 246 0 . 944 – 1 . 644 0 . 121 1 . 355 1 . 074 – 1 . 711 0 . 011 * 76 , 000 – 150 , 000 1 . 471 1 . 155 – 1 . 873 0 . 002 * 1 . 443 1 . 132 – 1 . 841 0 . 003 * 1 . 514 1 . 232 – 1 . 861 0 . 001 * > 150 , 000 1 . 604 1 . 280 – 2 . 009 0 . 001 * 1 . 466 1 . 167 – 1 . 842 0 . 001 * 1 . 484 1 . 221 – 1 . 802 0 . 001 * Comorbid disease 1 . 444 1 . 186 – 1 . 757 0 . 001 * 1 . 244 1 . 011 – 1 . 531 0 . 039 * 1 . 226 1 . 029 – 1 . 462 0 . 023 * Recent history of malaria 1 . 465 0 . 947 – 2 . 265 0 . 086 0 . 955 0 . 569 – 1 . 603 0 . 861 1 . 253 0 . 845 – 1 . 856 0 . 262 BCG vaccine 0 . 928 0 . 820 – 1 . 049 0 . 231 0 . 778 0 . 690 – 0 . 877 0 . 001 * 0 . 907 0 . 818 – 1 . 006 0 . 064 Symptomatic 1 . 194 0 . 963 – 1 . 481 0 . 107 2 . 186 1 . 659 – 2 . 880 0 . 001 * 1 . 391 1 . 152 – 1 . 680 0 . 001 * Close unprotected contact with a confirmed or prob - able case of COVID - 19 2 . 835 2 . 550 – 3 . 151 0 . 001 * 2 . 693 2 . 419 – 2 . 997 0 . 001 * 2 . 437 2 . 221 – 2 . 675 0 . 001 * Traveled outside or inside the country in the last 14 days 1 . 871 1 . 547 – 2 . 263 0 . 001 * 1 . 956 1 . 618 – 2 . 364 0 . 001 * 1 . 890 1 . 602 – 2 . 230 0 . 001 * Proximity with someone who traveled 2 . 597 2 . 204 – 3 . 061 0 . 001 * 2 . 270 1 . 913 – 2 . 693 0 . 001 * 2 . 270 1 . 955 – 2 . 636 0 . 001 * Table 5 Multivariate Hierarchical Models for the association of predictors with IgM Seropositivity AOR Adjusted Odd Ratio * p - value < 0 . 05 is considered statistically significant Final model AOR 95 % CI p‑value No of dependents 1 . 075 1 . 050 – 1 . 101 0 . 001 * Married 1 . 227 1 . 030 – 1 . 460 0 . 022 * Close unprotected contact with a confirmed or probable case of COVID - 19 2 . 387 2 . 060 – 2 . 765 0 . 001 * Proximity with someone who traveled outside or inside the country in the last 14 days 1 . 895 1 . 531 – 2 . 346 0 . 001 * Page 7 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 organizations ) , suggesting selection bias . Moreover , the prevalence estimates might have varied due to false serol - ogy test results and among elderly or immunosuppressed subjects . Hence , the data cannot be generalized to whole population and the objective was to determine the sig - nificant characteristics which are still difficult to predict due to varying and non - specific nature of Coronavirus disease . Conclusion In conclusion , the overall SARS - CoV - 2 antibodies ( IgG and / or IgM ) seroprevalence was 16 . 0 % . The multivari - ate model predicted that number of dependents , symp - tomatology , close unprotected contact with a confirmed or probable case of COVID - 19 , travelling history and proximity with someone who traveled are the significant independent predictors of total antibody seropositiv - ity in the studied population . More efforts concerning public health investigations for COVID - 19 on a regional and national level should be made to identify the par - ticular risks and outcomes related to SARS - CoV - 2 seroprevalence . Abbreviations COVID - 19 : Coronavirus Disease 2019 ; SARS - CoV - 2 : Severe acute respiratory syndrome coronavirus 2 ; ASRD : Acute Severe Respiratory Distress ; WHO : World Health Organization ; RT - PCR : Reverse transcription - polymerase chain reaction ; US FDA : U . S . Food and Drug Administration ; CE : European Certified ; EUA : Emergency Use Authorization ; SPSS : Statistical Package for the Social Sciences ; GI : Gastrointestinal . Acknowledgements The authors would like to acknowledge the support of the Advance Edu - cational Institute & Research Center ( AEIRC ) for their technical support and assistance in the publication process . Special thanks to Wajiha Farooq for her contribution to the review and editing of the manuscript . Author contributions WJ conceived the idea and wrote the protocol , data collection , performed data analysis , manuscript writing , review , and finalization . SHBA contributed to drafting protocol , data collection , manuscript formatting , and review . JBB con - tributed to drafting protocol , data collection , statistical analysis , manuscript editing , and review . All authors read and approved the final manuscript . Funding None . Availability of data and materials The datasets generated and / or analyzed during the current study are available in the Mendeley Data repository , https : / / data . mende ley . com / datas ets / nyd3v j48kf / 1 . Declarations Ethics approval and consent to participate The study protocol was approved by the Ethical Review Board of AEIRC ( Refer - ence no . MU / ECA / 20 / 17820 ; Dated 2 nd April 2020 ) . All the procedures were performed in accordance with the Declaration of Helsinki , and the patient’s confidentiality was maintained throughout the investigation . The included Table 6 Multivariate Hierarchical Models for the association of predictors with IgG Seropositivity AOR Adjusted Odd Ratio * p - value < 0 . 05 is considered statistically significant Final model AOR 95 % CI p‑value No of dependent 1 . 086 1 . 059 – 1 . 114 0 . 001 * Smoker 0 . 550 0 . 424 – 0 . 712 0 . 001 * BCG vaccine status 0 . 739 0 . 619 – 0 . 882 0 . 001 * Symptomatic 2 . 792 1 . 835 – 4 . 249 0 . 001 * Close unprotected contact with a confirmed or probable case of COVID - 19 2 . 762 2 . 362 – 3 . 230 0 . 001 * Traveled outside or inside the country in the last 14 days 1 . 717 1 . 338 – 2 . 203 0 . 001 * Proximity with someone who traveled outside or inside the country in the last 14 days 1 . 396 1 . 092 – 1 . 783 0 . 008 * Table 7 Multivariate Hierarchical Models for the association of predictors with total antibodies AOR Adjusted Odd Ratio * p - value < 0 . 05 is considered statistically significant . Final model AOR 95 % CI p‑value No of dependents 1 . 077 1 . 054 – 1 . 099 0 . 001 * Symptomatic 1 . 288 1 . 011 – 1 . 643 0 . 041 * Close unprotected contact with a confirmed or probable case of COVID - 19 2 . 470 2 . 164 – 2 . 819 0 . 001 * Traveled outside or inside the country in the last 14 days 1 . 537 1 . 234 – 1 . 914 0 . 001 * Proximity with someone who traveled outside or inside the country in the last 14 days 1 . 534 1 . 241 – 1 . 896 0 . 001 * Page 8 of 8 Javed et al . BMC Infectious Diseases ( 2022 ) 22 : 481 patients were well - informed about the study objective and written informed consent was taken from them before inclusion in the study . Consent for publication Not Applicable . Competing interests The author ( s ) declare no competing interest . Received : 24 May 2021 Accepted : 5 May 2022 References 1 . Chang L , Hou W , Zhao L , Zhang Y , Wang Y , Wu L . The prevalence of antibodies to SARS - CoV - 2 among blood donors in China . 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A comparison study of SARS - CoV - 2 IgG antibody between male and female COVID - 19 patients : a possible reason underlying different outcome between sex . J Med Virol . 2020 ; 92 ( 10 ) : 2050 – 4 . 34 . Haq M , Rehman A , Ahmad J , Zafar U , Ahmed S , Khan MA , Naveed A , Rajab H , Muhammad F , Naushad W , Aman M . SARS - CoV - 2 : big sero - prevalence data from Pakistan—is herd immunity at hand ? Infection . 2021 ; 49 ( 5 ) : 983 – 8 . 35 . Rudberg AS , Havervall S , Månberg A , Falk AJ , Aguilera K , Ng H , et al . SARS - CoV - 2 exposure , symptoms and seroprevalence in healthcare workers in Sweden . Nat Commun . 2020 ; 11 ( 1 ) : 1 – 8 . 36 . Tadj A , Lahbib SS . Our overall current knowledge of COVID 19 : an over - view . Micro Infec Chemotherapy . 2021 ; 1 : e1262 . Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub - lished maps and institutional affiliations . \ No newline at end of file diff --git a/silver_data/ba57e74c940e60ea84a993e94ccb4bc94d15fb66.pdf.txt b/silver_data/ba57e74c940e60ea84a993e94ccb4bc94d15fb66.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..85a7489ad04158b992916f9c1eec1546fb23a49b --- /dev/null +++ b/silver_data/ba57e74c940e60ea84a993e94ccb4bc94d15fb66.pdf.txt @@ -0,0 +1 @@ +Gary Laevsky and David A . Knecht University of Connecticut , Storrs , CT , USA ABSTRACT Under - agarose chemotaxis has been used previously to assess the ability of neu - trophils to respond to gradients of chemoat - tractant . We have adapted this assay to the chemotactic movement of Dictyostelium amoebae in response to folic acid . Troughs are used instead of wells to increase the area along which the cells can be visualized and to create a uniform front of moving cells . Imaging the transition zone where the cells first encounter the agarose , we find that the cells move perpendicular to the gradient and periodically manage to squeeze under the agarose and move up the gradient . As cells exit the troughs , their cross - sectional area increases as the cells become flattened . Three - dimensional re - construction of confocal optical sections through GFP - labeled cells demonstrates that the increase in cross - sectional area is due to the flattening of the cells . Since the cells locally deform the agarose and be - come deformed by it , the concentration of the agarose , and therefore its stiffness , should affect the ability of the cells to mi - grate . Consistent with this hypothesis , cells in 0 . 5 % agarose move faster and are less flat than cells under 2 % agarose . Cells do not exit the troughs and move under 3 % agarose at all . Therefore , this assay can be used to compare and quantify the ability of different cell types or mutant cell lines to move in a restrictive environment . INTRODUCTION Dictyostelium discoideum is a eu - karyotic amoeba that normally inhabits the soil . During its life cycle , the hap - loid cells undergo two distinct types of chemotactic movement . In the vegeta - tive phase , the amoebae are attracted to folic acid , which is released by their bacterial food source , and detected by cell surface folate receptors ( 6 , 13 ) . As the bacterial food source is depleted , D . discoideum enters the developmental stage of its life cycle . The number of folate receptors decreases during the first 7 – 9 h of development ( 11 ) , and the cells become responsive to cAMP re - leased by other amoebae ( 8 , 13 ) . The number of cAMP receptors ( cARs ) be - gins to increase immediately after the initiation of starvation , and cAR1 is maximally expressed on the surface 3 – 4 h into development ( 7 ) . Cell motility is most often studied in a laboratory by observing cells crawl - ing on a glass cover slip in liquid medi - um . Under these conditions , there is lit - tle to resist the movement of the cells except their own adhesion to the sub - strate . However , cells in natural envi - ronments , such as amoebae moving in the soil or neutrophils extravasating through the endothelium of a capillary , are presumed to move under more re - strictive conditions . In addition , move - ment in 3 - D environments has the added complexity that cells do not have a clearly defined dorsal and ventral sur - face because they can interact with the substrate on all sides . The molecular mechanisms underlying motility in 3 - D environments are as yet poorly defined . We sought to develop an assay in which cells attempt to move in a restric - tive environment , but where high - reso - lution imaging is possible . Under - agarose assays have been used to show the chemotactic and invasive abilities of neutrophils and macrophages ( 2 , 12 ) . Dictyostelium cells had previously been shown to be able to move under agarose by Yumura and co - workers ( 18 ) as a way to image moving cells while limit - ing their three - dimensionality . Here , we have modified the under - agarose che - motaxis assay by altering the well con - figurations and varying the agarose con - centration to investigate the chemotactic motility of Dictyostelium cells in differ - ent types of restrictive environments . Using time - lapse imaging and confocal microscopy , we show that cells deform the agarose but at the same time become flattened as they move under the aga - rose . The assay is reproducible and movement is quantifiable , allowing de - tailed analysis of the motility of individ - ual cells or populations of cells . MATERIALS AND METHODS Cell Culture NC4A2 cells were transfected with vectors that express cytosolic GFP ( pB15 - GFP ) ( 15 ) , GFP - ABD120 ( 14 ) to visualize F - actin , and myosin II heavy chain - GFP ( mhcA = GFP ) ( 10 ) to visualize myosin II localization . All cells were cultured in HL - 5 medium [ 5 g Bacto (cid:210) Proteose Peptone no . 2 ( Dig - co , Detroit , MI , USA ) , 5 g BBL Thiotone E , 10 g glucose , 5 g yeast ex - Research Report 1140BioTechniques Vol . 31 , No . 5 ( 2001 ) Under - Agarose Folate Chemotaxis of Dictyostelium discoideum Amoebae in Permissive and Mechanically Inhibited Conditions BioTechniques 31 : 1140 - 1149 ( November 2001 ) tract , 0 . 35 g Na 2 HPO 4 , 0 . 35 g KH 2 P O 4 , and double - distilled water to 1 L , pH 6 . 7 ] . Under - Agarose Chemotaxis Assay To prepare plates for the under - agarose assay , SeaKem ® GTG agarose ( BMA , Rockland , ME , USA ) was melt - ed at concentrations as indicated in SM medium [ 10 g Bacto - Peptone ( Difco ) , 10 g glucose , 1 g yeast extract , 1 . 9 g KH 2 PO 4 , 0 . 6 g K 2 HPO 4 , and 0 . 43 g MgSO 4 to 1 L , pH 6 . 5 ] . Similar results were obtained with other types of agarose [ ultraPURE (cid:212) LMP ( Invitrogen , Carlsbad , CA , USA ) , NuSieve ® GTG ( BMA ) , and Ultra Low Gelling ( Fisher - Biotech ) . Motility was generally higher when SM was used to prepare the agarose instead of HL - 5 . The agarose mixture was prepared fresh each day by mixing sterile SM ( previously auto - claved ) with the agarose powder and au - toclaving for 5 min , slow exhausting , and then plating as soon as possible . Agarose solution ( 4 mL ) was added to each 60 - mm plastic petri dish and al - lowed to harden for 1 h . Three troughs were then cut in the agarose using a sin - gle - edge razor blade . The troughs were 2 mm wide , the length of the blade 39 mm long , and spaced 5 mm apart ( Figure 1 ) . The agarose was removed to form the troughs using light suction from a Pasteur pipet applied to the “end” of the agarose plug to be removed . Care was taken at each step not to disturb the agarose interface at the edge of the trough . A solution of 100 mM folic acid ( Research Organics , Cleveland , OH , USA ) was prepared by dissolving 0 . 44 g folic acid in approximately 200 m L 10 M NaOH . This was brought to a final volume of 10 mL , adjusted to pH 7 . 0 , and stored in the dark at 4°C . A 0 . 1 - mM folic acid solution was pre - pared from the stock solution , and then 40 m L were added to the center trough ( Figure 1 ) and the gradient was allowed to develop for 1 h before adding cells . While the gradient developed , cells were prepared for addition to the troughs . It was found that cells were re - duced in their chemotactic response if grown to stationary phase or if cen - trifuged to concentrate and wash the cells . To maximize responsiveness , cells were plated at 4 · 10 5 cells / mL in 10 mL HL - 5 in 100 - mm petri dishes and grown for 2 days to a titer of approxi - mately 1 · 10 6 / mL . The overlying me - dia was aspirated , and the cells were re - suspended in fresh HL5 . For individual cell analysis , the cells were resuspended in 10 mL HL5 , counted with a hemocy - tometer , and the density was adjusted to 1 · 10 6 / mL . For population analysis , the cells were harvested in 1 mL HL5 and adjusted to 1 · 10 7 cells / mL . Sev - enty microliters of cells were then added to the peripheral troughs . For fluorescence imaging experi - ments , 0 . 75 mL agarose was added to a Rose chamber ( 21 ) or a 60 - mm glass bottom petri dish ( Willco Wells , Ams - terdam , The Netherlands ) so that cells could be imaged through a 0 . 17 mm thick glass cover slip . The troughs in the Rose chamber were cut with a shortened razor blade to a length of 10 mm , and the amount of cells and folate was decreased proportionally . Vol . 31 , No . 5 ( 2001 ) BioTechniques 1141 Figure 2 . Under - agarose chemotaxis of Dictyostelium cells to folic acid . Images were taken of cells at three times during movement under 0 . 5 % agarose toward the folate source . Chemotaxis to 0 . 1 mM fo - late was imaged under 0 . 5 % agarose . The images were taken at a fixed location about 1500 m m from the edge of the cell trough , showing the movement of the front of cells across the plate . The 240 ¢ time point is equivalent to 4 h after the addition of cells to the trough . Arrows indicate the origin of gradient and the di - rection of cell movement . A QuickTime movie showing the chemotactic movement of cells can be ac - cessed at http : / / www . BioTechniques . com / Movies / Nov01 / KnechtFig2 . mov . Figure 1 . Experimental setup . ( A ) The under - agarose assay plate . A 60 - mm petri dish is filled with agarose , and three troughs are then cut with a razor blade . The center trough is filled with chemoattractant , and 1 h later cells are added to the outside troughs . ( B ) Side view of the plate . Cells can only move out of the troughs by squeezing between the deformable agarose and the non - deformable plastic or glass surface . Analysis of Chemotaxis Images were taken of the cell popu - lations using a Zeiss (cid:210) inverted micro - scope ( Carl Zeiss , Oberkochen , Ger - many ) , Paultek Imaging CCD camera ( Advanced Imaging Concepts , Prince - ton , NJ , USA ) , Scion LG3 frame grab - ber ( MVI , Avon , MA , USA ) , and NIH Image software ( NIH , Bethesda , MD , USA ) . Cell population speed was deter - mined by measuring the distance the leading cells had moved away from the trough edge at a particular time point . Data from 10 cells were used to calcu - late the average distance the front - most cells had migrated . The distance moved was determined by imaging a stage mi - crometer and superimposing it on the images . The time it took the cell front to travel to successive time points was used to determine cell front speed . Indi - vidual cell speed and direction change were determined using DIAS (cid:210) soft - ware ( Solltech , Oakdale , IA , USA ) . Speed was calculated from the dis - placement of the centroid from frame to frame . Direction change was measured as the absolute value of the difference in the direction of movement of the cen - troid from frame to frame , measured in degrees . Cross - sectional area measure - ments were made using NIH Image software . The cross - sectional area is measured as the area of the image of a cell seen using phase microscopy . Con - focal imaging of GFP - labeled cells was performed using an MRC 600 ( Bio - Rad Laboratories , Hercules , CA , USA ) equipped with a 25 - mW krypton - argon laser and COMOS software . Three - di - mensional reconstruction was done us - ing Viewpoint3 and Volrend software written by Dr . Eric Shelden ( University of Michigan ) . Links to QuickTime (cid:212) movies are shown in the legends to Figures 2 , 3 , and 6 . RESULTS AND DISCUSSION Development of the Under - Agarose Assay The traditional under - agarose che - motaxis assay of macrophages and polymorphonuclear leukocytes ( PMNs ) utilizes small round wells cut in agarose into which cells or chemoattractant are loaded ( 2 , 12 ) . This format provides a small area where the cells are able to move in the direction of the gradient , making imaging of the transition zone difficult . We have modified this assay to make it more suitable to the visualiza - tion of a population of moving cells , al - lowing for quantification of both popu - lation and individual cell behavior . By cutting linear troughs instead of circular wells , movement along the entire front of the trough could be visualized and Research Report 1142BioTechniques Vol . 31 , No . 5 ( 2001 ) Figure 4 . The cross - sectional area of cells under agarose is increased . Images of cells under 0 . 5 % and 2 % agarose at the leading front , 1500 m m from trough edge . The arrow indicates the direction of move - ment . The inset shows the quantification of the cross - sectional area of the cells for the indicated agarose concentrations . Note the increased area of the cells under 2 % agarose . Figure 3 . Movement of cells at the transition zone . The large arrow points toward the source of folate and the direction of cell movement . The dashed line indicates the edge of the cell trough . Cells above the dashed line have moved underneath the 2 % agarose sheet . Note the increased cross - sectional surface area of the cells . The time points indicated are time elapsed after application of cells to trough . The black arrowhead in - dicates a cell that has moved under the agarose during this sequence . A QuickTime movie of cells exiting the trough can be accessed at http : / / www . BioTechniques . com / Movies / Nov01 / KnechtFig3 . mov . quantified . In preliminary experiments , it became clear that , if care was not tak - en in the cutting of the troughs , the inter - face could be disturbed such that gaps were created that allowed cells under the agarose . Even when care was taken , a few small areas where cells exited the trough much more rapidly were some - times observed . These areas were ig - nored in analysis , as they represented a small proportion of the behavior along the entire trough front . When the agarose was lifted and rotated within the petri dish to purposely disturb the inter - face , cell migration was less inhibited at all agarose concentrations . We believe this indicates an interaction between the agarose and the plastic substrate that must normally be broken or disturbed by the cells moving out of the trough . Using this assay , Dictyostelium cells were visualized over time after place - ment in the trough in the presence of a folate gradient . Cells exited the trough and moved up the folate gradient for at least 10 h ( Figure 2 ) . In the first hour af - ter application of cells to the trough , ap - proximately 10 times as many cells moved out of the trough toward the chemoattractant than away ( data not shown ) . After 4 h , the differential in movement approaches 1000 - fold . When cells were assayed using plates in which no folate was added , the results were similar to the results from the dis - tal side of the chemotaxis assay . Thus , randomly moving cells do not signifi - cantly exit the troughs , and it is the at - traction of the folate gradient that leads to cells moving under the agarose . Transition and Cortical Deformation Chemotaxis was initially character - ized using troughs cut in either 0 . 5 % agarose ( the minimum mechanically workable concentration ) or 2 % agarose . Before the arrival of cells , the agarose is in direct contact with the plastic surface of the petri dish . At the trough edge in - terface , the cells might move by climb - ing up and over the agarose , traveling through the agarose , moving under the agarose , or stopping at the edge of the trough . Microscopic analysis confirms that the cells do not climb over or through the agarose . Many but not all cells move out of the trough and under the agarose in the time frame of the as - say . Cells were not immediately able to make the transition to movement under the agarose when they reached the edge of the trough . Time - lapse movies of the trough edge show cells moving back and forth along the trough edge , and only occasionally does a cell actually extend a pseudopod under the agarose and move up the gradient . This behav - ior is particularly evident in the 2 % agarose plates . There is a continuous exit of cells from the trough throughout the assay . Some cells are left behind and do not exit the trough regardless of the concentration of the agarose . These cells are primarily in the middle or dis - tal portion of the trough . This cellular behavior suggests that a stable gradient does not extend very far into the trough . For cells to move out of the trough , they have to flatten themselves to a thickness that will fit between the agarose and the plastic , deform the agarose upwards while maintaining shape , or a combination of the two such that they deform the agarose upwards as they flatten themselves . As cells leave the trough and move under the agarose , there was a dramatic increase in their 2 - D cross - sectional area , indicating a flat - tening of the cells ( Figure 3 ) . The aver - age cross - sectional area of cells in the trough was 32 ± 4 m m 2 , while cells 1500 m m from the trough edge had an average area of 130 ± 4 m m 2 under 0 . 5 % agarose . If the stiffness of the agarose were causing the flattening of the cells , then , as the concentration of agarose is increased , the cells should become flatter . Consistent with this hy - pothesis , cells under 2 % agarose con - centration had a cross - sectional area of 301 ± 25 m m 2 ( Figure 4 and inset ) . Another approach used to assess changes in cell morphology was to use confocal imaging to determine the 3 - D shape of the cells under agarose . Cy - tosolic GFP was expressed in transfect - ed Dictyostelium cells . The chemotaxis assay was set up in a Rose chamber on a cover slip to allow high - resolution 3 - D optical sectioning with the confocal microscope . Reconstruction ( Figure 5 ) of the optical sections showed a clear flattening of the cells under agarose as compared to cells remaining in the trough . Cells in the trough that had not yet moved under the agarose were vari - able in height , ranging as high as 12 m m . Cells that had crawled under the 0 . 5 % agarose were 8 – 10 m m thick , and Research Report 1144BioTechniques Vol . 31 , No . 5 ( 2001 ) Figure 5 . Volume reconstruction of cells . Cells expressing cytosolic GFP were imaged in the trough , under 0 . 5 % or 2 % agarose . “Front” cells images were captured 1500 m m from trough edge . Confocal op - tical sections were collected through each cell and then reconstructed into a 3 - D volume . Each volume is shown in a top down and side view . cells under 2 % agarose were uniformly 4 m m thick . Another approach to understanding the interaction of the cells with the agarose is to determine the extent to which the agarose is deformed upward by the cells moving underneath . Since the cells have a thickness of 4 m m under the 2 % agarose , it is likely that the agarose is locally deformed or moved upward as the cell passes underneath . To visualize this deformation , 0 . 5 m m fluorescent beads were added to the agarose before hardening . Confocal im - ages with a Z - section approximately 1 m m thick were collected with the focal plane fixed 4 – 5 m m above the substrate , near the top of the cells . Beads could clearly be seen to move into and out of the plane of focus as the cells moved underneath ( Figure 6 ) . Some beads ( Figure 6 , lower left ) are visible before the cell arrives and then move out of the plane of focus as the cell passes . Beads at the bottom ( Figure 6 , upper right ) of the agarose were frequently pulled out of the agarose and phagocytosed by the cells . The beads that remained in the agarose after the cells passed were usu - ally in a slightly higher focal plane , in - dicating that the agarose is lifted slight - ly by the passing of cells . Thus , cells trailing the front will be exposed to a slightly different environment . If the imaging plane was set higher in the agarose , then no movement of the beads was observed . This indicates that the entire sheet of agarose is not raised but rather that the agarose is locally de - formed above the cell . In addition , the beads did not move until just before ( approximately 2 m m ) the advancing cell reached it and returned to their po - sitions just after the cell had passed , in - dicating that the deformation of the agarose is quite local . Effect of Agarose Concentration on Cell Movement After moving out of the trough , the cells form a distinct front that progress - es toward the chemoattractant well ( Fig - ure 4 ) . Time - lapse imaging and comput - er analysis were used to visualize and quantify the movement of cells up the folate gradient . The speed of the front was calculated by determining the posi - tion of the leading cells over time . Under 0 . 5 % agarose , the cell front moved at 6 . 4 m m / min ( Figure 7 and inset ) . The rate of movement of the cells was surprisingly consistent throughout the assay . Since the cells deform the agarose away from the surface to move , we hy - pothesized that , by increasing the con - centration of the agarose , the cells would have to work harder to move . Consistent with this idea , the speed of the front decreased as the agarose con - centration increased ( Figure 7 and in - set ) . At 2 % agarose , the cells moved at 3 . 1 m m / min ; however , at 3 % agarose , the cells can no longer move out of the trough , indicating that the cells are un - able to deform this stiff matrix suffi - ciently to squeeze underneath . These data may provide a clue as to why many previous attempts to use un - der - agarose chemotaxis with mam - malian cell lines have been unsuccess - ful . Cells clearly undergo a flattening as they begin to protrude under the agarose . Even Dictyostelium amoebae that move robustly once under the agarose have a difficult time making the transition to flatten out and squeeze under the agarose ( Figure 3 ) . The edge of the agarose trough seems to present a restrictive barrier that cells must over - come to move up the chemoattractant gradient . Cells must have sufficient ad - hesion to the surface to apply a forward force and deform the agarose as they move , and they must have sufficient cortical tension not to be crushed by the downward force of the overlying ma - trix . Many cell types may be unable to meet both of these criteria to move un - der agarose . By varying the substrate composition and reducing the stiffness of the overlay , it may be possible to im - prove the movement of mammalian cell lines . In fact , Mollison et al . ( 9 ) have shown that varying the concentration of agarose affects the under agarose mi - gration distance of PMNs . Research Report 1146BioTechniques Vol . 31 , No . 5 ( 2001 ) Figure 6 . Agarose deformation assay using flu - orescent beads . Fluorescent beads ( FITC , 0 . 5 m m diameter ) were added to 2 % agarose before pour - ing the plates . Images were collected using the confocal microscope as cells moved through the field of view . The plane of focus was just above the height of the cells so that some beads are ini - tially below the plane of focus and some are in the plane . As the cell moves by , beads below the plane are moved through the plane and so become temporarily visible . Beads initially in the plane move out of the plane , becoming invisible , and then reappear after the cell passes by . A Quick - Time movie showing the movement of the beads can be accessed at http : / / www . BioTechniques . com / Movies / Nov01 / KnechtFig6 . mov . Figure 7 . Movement of the leading edge of cells under various agarose concentrations . NC4A2 cells applied to troughs at the 0 time point . The movement of the front of cells over time is plot - ted . The cells at the front move at a constant speed throughout the assay . The inset shows the average speed of the front calculated from the slope of the 2 – 10 h time points . Characterization of Chemotactic and Chemokinetic Motility Cell motility was also analyzed by computerized motion analysis of indi - vidual cell paths over time . Cells at the leading front of 0 . 5 % agarose had a mean velocity of 6 . 9 ± 0 . 1 m m / min and a very low rate of directional change , indicating that they were moving per - sistently up the folate gradient ( Figure 8 and inset ) . This is also evident from the paths of individual cells , which are quite linear . The fact that the cell speed and the population speed are similar also indicates that cells are tracking Vol . 31 , No . 5 ( 2001 ) BioTechniques 1147 Figure 8 . DIAS analysis of leading - edge and lagging cells . The paths of representative cells were mea - sured using DIAS motion analysis software . The arrow points toward the chemoattractant trough and the direction of cell movement . Lagging cells are approximately 450 m m behind the leading cells . The inset table shows the quantification of speed and direction of leading and lagging cells as well as cells exposed to no folate or a uniform concentration of folate throughout the agarose . The speed and direction changes are expressed as the mean value of at least 50 cells monitored for at least 30 min in two independent ex - periments ( mean ± SEM ) . Figure 9 . GFP - labeled cells chemotaxing under agarose . ABD120 and myosin II heavy chain - GFP ( mhcA - GFP ) fusion proteins were expressed in cells to visualize F - actin and myosin II in cells . The top panel shows a mhcA - GFP - transformed cell , and the bottom panel shows a GFP - ABD120 - transformed cell . Cells are imaged under 2 % agarose using confocal microscopy as described . Cells are moving in the direction of the arrows . consistently up the gradient . The cells behind the leading front were slower ( 5 . 0 ± 0 . 7 m m / min ) but had twice the rate of directional change . The more frequent turns are evident in the paths of the trailing cells ( Figure 8 ) . This be - havior is most likely due to the distur - bance of the gradient by the leading cells . Dictyostelium cells have both cell surface and secreted folate deaminases that will alter the local gradient as the leading cell passes . Chemotactic agents can have both chemotactic ( directional ) and / or chemokinetic ( speed ) effects on cells . To determine the effects of folate on D . discoideum cells , we compared the movement of cells in the standard chemotactic chamber to movements in a chamber in which the folate was pre - sent uniformly throughout the agarose and the troughs , or a chamber that con - tained no folate . Cells in a uniform con - centration of folate moved as fast as cells exposed to a gradient ( Figure 8 and inset ) , but the cells made 50 % more direction changes . There was also a 50 % decrease ( data not shown ) in the number of cells migrating out of the troughs compared to the chemotactic conditions . Unlike the chemotactic as - say , in the chemokinetic assay an equal number of cells moved out in both di - rections . When no folate was present , very few cells moved out of the troughs ( data not shown ) . Thus , folate exerts both chemotactic and chemokinetic in - fluences on cells . This is to our knowl - edge the first demonstration of a chemokinetic effect of folate . The evi - dence for chemokinesis is not definitive because the ability of Dictyostelium to bind folate and the presence of extra - cellular and cell - surface folate deami - nases ( 3 , 4 ) may convert a uniform fo - late concentration into a local gradient ( 1 ) . It may be informative to perform this experiment using a non - hydrolyz - able folate analog such as methotrexate to confirm this observation . Localization of GFP - Labeled Proteins One of the benefits of this assay is the ability to image cells that are active - ly undergoing chemotaxis in a mechani - cally inhibited environment . Confocal microscopy of cells expressing GFP - fu - sion proteins was used to examine the localization of several cytoskeletal pro - teins during chemotaxis . It has been shown previously that myosin II moves to the rear of the cell when cells are overlaid with a thin agarose sheet ( 2 ) . The same phenomenon was observed when cells labeled with GFP - myosin II moved from the trough out under the agarose ( Figure 9 ) . The myosin II that Research Report was previously throughout the cortex , rapidly relocalized to the rear of the cell . The F - actin cytoskeleton was also visualized using an actin binding do - main - GFP fusion ( 18 ) . Surprisingly , the bulk of the F - actin co - localized with myosin II at the rear of the cell rather than in the leading lamella ( Figure 9 ) . The under - agarose chemotaxis assay has numerous potential applications that we will explore in the future . The assay has been combined with an electric cell impedance sensing ( ECIS ) system to al - low the arrival of the cells at a specified location to be recorded without visual - ization ( see following paper ) . This as - say opens the way for a high - throughput screen for agonists and antagonists of chemotaxis . Also , a variety of Dic - tyostelium mutants are available with alterations in cytoskeletal and motility - related genes . It is likely that mutations that affect cortical integrity will have adverse affects on motility in this re - strictive environment . Preliminary ex - amination of cells lacking myosin II heavy chain indicates significant defects in movement under agarose ( Laevsky unpublished observations and refer - ences . Furthermore , the visualization of GFP - labeled proteins in directionally moving cells will allow us to assess the localization of proteins in relation to the motility process and how the machinery changes between mechanically inhibit - ed and uninhibited conditions . Last , it should be possible to adapt this assay to isolate the cells left in the trough as a way to screen for mutants deficient in force production or chemotaxis . ACKNOWLEDGMENTS We would like to thank Dr . Sally Zigmond , Dr . Andrew Masselli , Dr . Michael Lynes , Dr . Eunkyung Lee , and Catherine Green for encouragement , thoughtful discussions , and careful reading of the manuscript . This work was funded by the National Institutes of Health grant no . GM40599 to D . A . K . REFERENCES 1 . Condeelis , J . , A . Bresnick , M . Demma , S . Dharmawardhane , R . Eddy , A . L . Hall , R . Sauterer , and V . Warren . 1990 . Mechanisms of amoeboid chemotaxis : an evaluation of the cortical expansion model . Dev . Genet . 11 : 333 - 340 . 2 . Cutler , J . E . and J . J . Munoz . 1974 . A simple in vitro method for studies on chemotaxis . Proc . Soc . Exp . Biol . Med . 147 : 471 - 474 . 3 . Darmon , M . , J . Barra , and P . Brachet . 1978 . The role of phosphodiesterase in aggregation of Dictyostelium discoideum . J . Cell Sci . 31 : 233 - 243 . 4 . Fukui , Y . and S . Inoue . 1991 . Cell division in Dictyostelium with special emphasis on acto - myosin organization in cytokinesis . Cell Motil . Cytoskeleton 18 : 41 - 54 . 5 . Fukui , Y . and S . Inoue . 1997 . Amoeboid movement anchored by eupodia , new actin - rich knobby feet in Dictyostelium . Cell Motil . Cytoskeleton 36 : 339 - 354 . 6 . Fukui , Y . , T . J . Lynch , H . Brzeska , and E . D . Korn . 1989 . Myosin I is located at the leading edges of locomoting Dictyostelium amoebae . Nature 341 : 328 - 331 . 4 . Greiner , R . A . , D . Jacobs - Krahnen , R . Mutzel , D . Malchow , and B . Wurster . 1992 . A 138 - kDa glycoprotein from Dictyostelium membranes with folate deaminase and folate binding activity . J . Biol . Chem . 267 : 5096 - 5101 . 5 . Hadjout , N . G . Laevsky , D . A . Knecht , and M . A . Lynes . 2001 . Automated real - time mea - surement of chemotactic cell motilitg . Bio - Techniques 31 : 1130 - 1138 . 6 . Jones , T . H . and G . M . Brown . 1967 . The biosynthesis of folic acid . VII . Enzymatic syn - thesis of pteridines from guanosine triphos - phate . J . Biol . Chem . 242 : 3989 - 3997 . 7 . Klein , P . S . , T . J . Sun , C . L . d . Saxe , A . R . Kim - mel , R . L . Johnson , and P . N . Devreotes . 1988 . A chemoattractant receptor controls develop - ment in Dictyostelium discoideum . Science 241 : 1467 - 1472 . 8 . Konijn , T . M . , J . G . Van De Meene , J . T . Bon - ner , and D . S . Barkley . 1967 . The acrasin ac - tivity of adenosine - 3 ¢ , 5 ¢ - cyclic phosphate . Proc . Natl . Acad . Sci . USA 58 : 1152 - 1154 . 9 . Mollison , K . W . , G . W . Carter , and R . A . Krause . 1981 . Chemotaxis of human polymor - phonuclear leukocytes under agarose : lack of requirement for media protein and differential effects of buffer and agarose type on locomo - tion . Proc . Soc . Exp . Biol . Med . 167 : 419 - 427 . 10 . Moores , S . L . , J . H . Sabry , and J . A . Spudich . 1996 . Myosin dynamics in live Dictyostelium cells . Proc . Natl . Acad . Sci . USA 93 : 443 - 446 . 11 . Nandini - Kishore , S . G . and W . A . Frazier . 1981 . [ 3 H ] Methotrexate as a ligand for the fo - late receptor of Dictyostelium discoideum . Proc . Natl . Acad . Sci . USA 78 : 7299 - 7303 . 12 . Nelson , R . D . , P . G . Quie , and R . L . Simmons . 1975 . Chemotaxis under agarose : a new and simple method for measuring chemotaxis and spontaneous migration of human polymor - phonuclear leukocytes and monocytes . J . Im - munol . 115 : 1650 - 1656 . 13 . Pan , P . , E . M . Hall , and J . T . Bonner . 1972 . Folic acid as second chemotactic substance in the cellular slime moulds . Nat . New Biol . 237 : 181 - 182 . 14 . Pang , K . M . , E . Lee , and D . A . Knecht . 1998 . Use of a fusion protein between GFP and an actin - binding domain to visualize transient fila - mentous - actin structures . Curr . Biol . 8 : 405 - 408 . 15 . Rietdorf , J . , F . Siegert , and C . J . Weijer . 1996 . Analysis of optical density wave propa - gation and cell movement during mound for - mation in Dictyostelium discoideum . Dev . Biol . 177 : 427 - 438 . 16 . Rose , G . G . , C . M . Pomerat , T . O . Shindler , and J . B . Trunnell . 1958 . A cellophane - strip technique for culturing tissue in multipurpose culture chambers . Biophysic . Biochem . Cytol . 17 . Sussman , R . and M . Sussman . 1967 . Cultiva - tion of Dictyostelium discoideum in axenic medium . Biochem . Biophys . Res . Commun . 29 : 53 - 55 . 18 . Yumura , S . , H . Mori , and Y . Fukui . 1984 . Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence . J . Cell Biol . 99 : 894 - 899 . Received 31 January 2001 ; accepted 15 May 2001 . Address correspondence to : Dr . David Knecht University of Connecticut 75 N . Eagleville Rd . Dept . of MCB / U - 125 Storrs , CT 06269 , USA e - mail : knecht @ uconn . edu Vol . 31 , No . 5 ( 2001 ) BioTechniques 1149 For reprints of this or any other article , contact Reprints @ BioTechniques . com \ No newline at end of file diff --git a/silver_data/bd2a511ced47fd322ba1e14ccf97a5af1bec01e8.pdf.txt b/silver_data/bd2a511ced47fd322ba1e14ccf97a5af1bec01e8.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..a716128be7664599ddf7b40b6f6137801fda3014 --- /dev/null +++ b/silver_data/bd2a511ced47fd322ba1e14ccf97a5af1bec01e8.pdf.txt @@ -0,0 +1 @@ +J _ ID : za5 Customer A _ ID : 09 - 035 . R1 Cadmus Art : CPLX20332 — 2011 / 3 / 3 — page 22 — # 1 The Micromechanics of Three - Dimensional Collagen - I Gels ANDREW M . STEIN , 1 DAVID A . VADER , 2 DAVID A . WEITZ , 2 AND LEONARD M . SANDER 3 1 Modeling and Simulation Group , Novartis Institute for BioMedical Research , Cambridge , Massachusetts 02139 ; 2 School of Engineering and Applied Sciences , Harvard University , Cambridge , Massachusetts 02138 ; and 3 Michigan Center for Theoretical Physics , Department of Physics , University of Michigan , Ann Arbor , Michigan 48109 Received 20 December 2009 ; revised 15 May 2010 ; accepted 9 June 2010 We study the micromechanics of collagen - I gel with the goal of bridging the gap between theory and experiment in the study of biopolymer networks . Three - dimensional images of fluorescently labeled collagen are obtained by confocal microscopy , and the network geometry is extracted using a 3D network skeletonization algorithm . Each fiber is modeled as an elastic beam that resists stretching and bending , and each crosslink is modeled as torsional spring . The stress – strain curves of networks at three different densities are compared with rheology measurements . Themodelshowsgoodagreementwithexperiment , confirmingthatstrainstiffeningofcollagencan beexplainedentirelybygeometricrealignmentofthenetwork , asopposedtoentropicstiffeningofindividualfibers . The model also suggests that at small strains , crosslink deformation is the main contributer to network stiffness , whereas at large strains , fiber stretching dominates . As this modeling effort uses networks with realistic geometries , this analysis can ultimately serve as a tool for understanding how the mechanics of fibers and crosslinks at the microscopic level produce the macroscopic properties of the network . © 2010 Wiley Periodicals , Inc . Complexity 16 : 22 – 28 , 2011 Key Words : biopolymer ; collagen ; strain stiffening 1 . INTRODUCTION C ollagen is the most abundant animal protein [ 1 ] and its mechanics have been studied in great detail [ 2 ] . It takes on many morphologies , including skin , tendons , Correspondence to : Andrew M . Stein , Modeling and Sim - ulation Group , Novartis Institute for BioMedical Research , Cambridge , MA 02139 ( e - mail : astein @ ima . umn . edu ) ligaments , individual fibers , and gels . Of particular interest is the mechanics of collagen - I gels , shown in Figure 1 ( a ) . These gels provide a relatively simple structure that can be nonin - vasively observed by confocal microscopy [ 3 , 4 ] and used as a scaffold for growing artificial tissues [ 5 ] and as a 3D environ - ment for studying cell motility [ 6 ] and tumor invasion [ 7 , 8 ] . A critical first step in understanding these systems is to develop amodelforthecollagengelalone . Inthisarticle , wegiveasuc - cessful theoretical model of the micromechanics of realistic networks . 22 C O M P L E X I T Y © 2010 Wiley Periodicals , Inc . , Vol . 16 , No . 4 DOI 10 . 1002 / cplx . 20332 Published online 17 August 2010 in Wiley Online Library ( wileyonlinelibrary . com ) J _ ID : za5 Customer A _ ID : 09 - 035 . R1 Cadmus Art : CPLX20332 — 2011 / 3 / 3 — page 23 — # 2 FIGURE 1 A typical gel ( 1 . 2mg / mL , 25 . 6 μ m × 25 . 6 μ m × 25 . 6 μ m ) . ( a ) Maximal intensity projection along the axis that is perpendicular to the focal plane of the microscope . ( b ) Projection of 3D network extracted by FIRE . ( c ) Reduced network , where elements that do not contribute to the network stiffness have been removed for improved computational efficiency . ( d ) Deformationafter50 % tension . ( e ) Deformationafter50 % shear . ( f ) Comparison of the stress – strain response between the model and experiment . Collagen - I gels belong to a class of materials known as biopolymers . Other examples include actin , found in the cytoskeleton , and fibrin , a component of blood clots . Com - mon mechanical features of biopolymers are negative nor - mal stresses under shear [ 9 ] , significant contraction and alignment as the gel is axially stretched [ 10 , 11 ] , and strain stiffening by 2 – 3 orders of magnitude at large strains , as shown in Figure 1 ( f ) . The cause of this strain stiffening is not well understood . Storm et al . [ 12 ] attributed strain stiff - ening in all biopolymer networks exclusively to the pulling out of entropic modes of individual filaments . Their calcu - lation required the assumption that deformations are affine . On the other hand , Heussinger et al . [ 13 , 14 ] showed how one could deconstruct the network deformation into a set of nonaffine floppy modes . They concluded that accounting for the nonaffinity was necessary in describing the elastic properties of the network . They hypothesize that the non - linearity comes from a transition from a bending regime to a stretching regime . Onck and coworkers [ 15 , 16 ] have pro - posed the alternative hypothesis that strain stiffening is due to the rearrangement of the fibers as the network is strained . Resolving this debate has been difficult as almost all theo - retical analysis has been on artificially generated networks in 2D . Whether such artificially generated networks can be used to accurately describe real biopolymer gels has not yet been confirmed . In particular , key results of the above theories rest on assumptions on how the crosslink spacing scales with density . Although a direct relationship has been obtained in two dimensions for random stick networks [ 17 , 18 ] , in three dimensions , the scaling relationship will also depend on how thegelpolymerizes . Althoughthenatureofcollagenpolymer - ization is currently the subject of active research [ 19 ] , it is still not well understood . Moreover , the few examples of quanti - tative comparisons to experiment in the literature [ 12 , 20 , 21 ] arenotabletoquantitativelyfitthefullstress – strainresponse of the gel at varying densities using a single set of parameters . The goal of this research is to develop a model for colla - gen gel based on three - dimensional images of fluorescently labeled collagen gels at different densities . The images are obtained by confocal microscopy [ Figure 1 ( a ) ] , and the net - work geometry is extracted using a custom FIbeR Extraction ( FIRE ) algorithm [ Figure 1 ( b ) ] [ 3 ] . The gel is modeled as a ran - dom network of crosslinked fibers , as described below , and thestress – strainresponseiscomparedwiththatmeasuredby an AR - G2 rheometer . Good agreement between model and experiment is obtained by fitting a single parameter , that is , the crosslink stiffness . 2 . EXPERIMENT The experiments were defined in more detail previously [ 3 ] . Briefly , we labeled bovine collagen type I with the fluores - cent molecule TAMRA . Collagen solution at final concentra - tions of 0 . 5 , 1 . 0 , and 1 . 5mg / mL were prepared , and a Leica SP5 resonant confocal microscope with a 63X 1 . 2 - NA water immersion objective was used to image the collagen sam - ples . Four networks at each density were extracted using the FIRE skeletonization algorithm [ 3 ] , as shown in Figure 1 ( b ) . After applying FIRE , the collagen density was re - estimated in two different ways : by counting the number of bright pixels in the image and by the product of the average fiber cross - sectionalarea ( A = 2800nm withanassumedradiusof30nm [ 3 , 22 ] ) and total fiber length in the extracted networks . The re - estimated densities of the three networks were 0 . 5 , 1 . 2 , and 1 . 4mg / mL . The final step before simulation is to remove dangling portions of the network that do not contribute to its stiffness [ Figure 1 ( c ) ] . The shear response of the gel was mea - suredusinganAR - G2rheometerwitha4 ◦ , 40 - mmcone - plate © 2010 Wiley Periodicals , Inc . DOI 10 . 1002 / cplx C O M P L E X I T Y 23 J _ ID : za5 Customer A _ ID : 09 - 035 . R1 Cadmus Art : CPLX20332 — 2011 / 3 / 3 — page 24 — # 3 geometry with a 109 - μ m gap . Approximately 1 . 2mL of colla - gen solution was pipetted onto the 37 ◦ C preheated bottom plate of the rheometer , and the cone was lowered onto the sample . A solvent trap was used to prevent the sample from drying during the measurement . 3 . MODEL In the model for the collagen gel , each fiber is treated as an elastic beam that resists stretching and bending and each crosslink is treated as a torsional spring , making it more stiff than a freely rotating pin joint but less stiff than a welded joint of fixed angle . The stretching modulus of an individual fiber is given by K s = EA , where E is theYoung’s modulus and A is the cross - sectional area . The Young’s modulus of a fiber in aqueous conditions has been estimated to be between 30 and 800MPa [ 23 – 25 ] , and we use a modulus of 50 MPa , which fits the data well and is also close to the value chosen by Stylianopoulous and Barocas ( i . e . , 79MPa ) to fit their model [ 21 ] . Ithasbeenshownthatasinglefiberwillstiffenbyafactor of 2 – 4 when strained [ 25 ] . We choose here to use a constant E both to reduce the number of parameters in the model and to see if geometric reorientation of the network is enough to explainstrainstiffening . StylianopoulosandBarocas [ 21 ] also explored the bilinear and exponential constitutive relations for the individual fibers and observed only minor effects on the macroscopic network behavior . The radius of each fiber is r = 30nm [ 3 , 22 ] . The bending modulus of the fiber is given by K b = EI = 32pN - μ m 2 , where I = π r 4 / 4 [ 26 ] . No crosslinking agent has been added to the gel , and very little is known about the nature of the naturally formed collagen crosslinks . We find that we can fit all the data by setting the torsional spring stiffness to K x = 300pN - μ m . To compare K b to K x , we consider K b / l c , where the mean crosslink spacing is given by l c ∼ 2 μ m [ 3 ] . Thus , we find that K x ∼ 20 K b / l c . One possible reason for an increase in stiffness at the crosslinks could be an increase in fiber radius near the crosslinks by a factorof2 , asbendingstiffnessscalesby r 4 . However , much is still unknown about the internal structural of the fibrils and the interfiril and intrafibril bonding mechanics , and there - fore , there is necessarily no reason to expect K x and K b / l c to be equivalent . We assume that in the undeformed state of the network , there are no internal stresses . Thus , the fibers have an innate curvature and the crosslinks have an equilibrium angle equal to that in their initial configuration . We ignore entropic con - tributions to the fiber mechanics . While the geometric per - sistence length of these fibers has been measured to be 20 μ m [ 3 ] , the thermal persistence length is much longer l p = K b / kT ∼ 1cm . Furthermore , in the case that the strain stiffening is dominated by thermal compliance , one would expect to see a decrease in the yield strain with increasing concentration [ 27 ] . Collagen gels , however , have been shown to have a constant yield strain of about 60 % for a wide range of concentrations [ 28 ] . The total energy in the network for a given configuration is given below . U = N s (cid:2) i = 1 K s L i ( (cid:3) L i ) 2 2 + N b (cid:2) i = 1 K b L i (cid:3) (cid:3)θ i b (cid:4) 2 2 + N x (cid:2) k = 1 K x (cid:3) (cid:3)θ i x (cid:4) 2 2 ( 1 ) Here N a is the number of elements of type a ∈ { s , b , x } , which denotes stretching , bending , and crosslink , L i is the length of stretching element , θ i b and θ i x are the bending and crosslink angles , respectively , and (cid:3) indicates the difference between the deformed and undeformed state . To calculate the stress – strain relationship of our model network , we performed a series of 18 incremental strain steps by imposing a small deformation on one face , F 1 , while hold - ing the opposite face , F 0 , fixed . We impose two types of deformations : tension [ Figure 1 ( d ) ] and shear [ Figure 1 ( e ) ] . In a tensile deformation , we allow the vertices on F 0 and F 1 to move freely in directions perpendicular to the imposed strain to allow for perpendicular contraction , which is seen to occur in these experiments [ 28 , 29 ] . In experiments of this type , the distancebetween F 0 and F 1 isontheorderofcentimeters , and thesimulatednetworkrepresentsasmallregionnearthecen - ter of a sample . In shear , we do not allow the boundary nodes on F 0 and F 1 to move freely . We compare the shear results to cone - plate rheometer experiments , where the shear faces are boundtotherheometer . Here , thedistancebetween F 0 and F 1 is 109 μ m , and the simulated network is one fourth the length of the experimental sample between the boundaries . In both deformations , all other nodes , including those on the four remaining faces of the network , are free to move . The mini - mum energy state of the network at an imposed strain U ( (cid:5) ij ) is found using a conjugate gradient method developed by HagerandZhang [ 30 ] . Thestressrequiredtoholdthenetwork in its current configuration is given by σ ij ( (cid:5) ij ) = ( dU / d (cid:5) ij ) / A , where A denotes the area of F 0 . 4 . RESULTS The results are averaged over four extracted networks and over all six shear deformations (cid:5) ij | i (cid:5) = j in the sheared network and all three principal tensile directions (cid:5) ii in the stretched network . The stress – strain relationship for a single gel is shown in Figure 1 ( f ) , and the results for all gels are summa - r ized in Figure 2 . Below ∼ 5 % strain amplitude , the mater ial deforms primarily in an elastic fashion , but above ∼ 5 % , the material deforms irreversibly . Although the gel can recover some of its initial material properties given sufficient time to relax , it never completely recovers . In Figure 3 ( a ) , we plot the small strain modulus from the samples by calculating the slope of the stress – strain curve at small strains ( 0 . 2 – 2 % ) . In Figure 3 ( b ) , we compare the previously reported tensile mod - ulus of large samples that are centimeters in length [ 28 ] to the simulation . Here , in both simulation and experiment , the largestrainmodulusiscomputedbyfittinglinestothestress – strain curve in the regime of 20 – 40 % strain . At small strains , 24 C O M P L E X I T Y © 2010 Wiley Periodicals , Inc . DOI 10 . 1002 / cplx J _ ID : za5 Customer A _ ID : 09 - 035 . R1 Cadmus Art : CPLX20332 — 2011 / 3 / 3 — page 25 — # 4 FIGURE 2 Stress – strainsweepsofthegel , from0 . 5to100 % strain . Intheartwork , E denotes experiment , W denotes the elastic beam model , S denotes the spring model where we set K x = K b = 0 , and the number denotes the collagen density in mg / mL . ( a ) Unscaled results . Note the good agreement between model and data at small and large strains . ( b ) When the curves are scaled by ρ 2 . 68 , relatively good data collapse is achieved . We denote lines of slope m = 1 and m = 3 to guide the eye . At large strains , scaling breaks down and the low - density curves overtake the high - density curves , because at large strains , stiffness scales linearly with density . Thus , this rescaling mainly serves as a visualization tool and does not represent a true data collapse . the stress – strain response is linear , as expected , and at larger shear strains , the stress – strain response stiffens considerably . In Figure 3 ( a ) , we show that the small strain modulus scales by σ 12 ∼ ρ 2 . 68 (cid:5) 12 , where ρ is the collagen density . At this time , it is not possible to verify the power law scaling in the model as only densities of 0 . 5 , 1 . 2 , and 1 . 4 were observed . The flu - orescent labeling of the network changes the polymerization propertiesofthenetwork , causingittoclumpathigherdensi - ties . We use this scaling relationship to collapse the curves in Figure 2 ( b ) . The close agreement between model and exper - iment indicates that strain stiffening due to the geometric rearrangement of the collagen fibers is enough to explain the strain stiffening seen in experiments . At large strains ( ∼ 50 % in tension and ∼ 200 % in shear ) , the stress – strain curve of the model becomes linear again , although with a much steeper slope . In Figure 3 ( b ) , we com - pare the large strain tensile behavior of the model to the experiments of Roeder et al . [ 28 ] . Although our model under - estimates their experimental measurement by a factor of 2 . 5 , we find this to be reasonable as the two experiments used dif - ferent collagen protocols . In particular , different buffers were used . In Figure 2 , we also explore the case where K x = K b = 0 , such that we have only a network of springs connected at freely rotating pin joints . At low strains , the network can be deformed without exerting any stress , but at strains higher FIGURE 3 ( a ) The small strain shear modulus is compared with the cone – plate rheometer experiments , and a scaling law of G (cid:6) ∼ ρ 2 . 68 is observed for the experiment . ( b ) The large strain tensile modulus is from the model and from the experiments of Roeder et al . [ 28 ] . Results differ by a factor of 2 . 5 , which is reasonable as the two experimental protocols were different [ 28 ] . © 2010 Wiley Periodicals , Inc . DOI 10 . 1002 / cplx C O M P L E X I T Y 25 J _ ID : za5 Customer A _ ID : 09 - 035 . R1 Cadmus Art : CPLX20332 — 2011 / 3 / 3 — page 26 — # 5 FIGURE 4 Average negative normal stresses for the 1 . 2mg / ml gel in response to shear . than25 % , weseethatthissimplificationadequatelydescribes the gel . A topic of investigation explored by many is the validity of the assumption that these networks deform affinely [ 14 , 15 ] . For brevity , we only state that the deformations are highly nonaffine at small strains where the majority of the energy is stored in bending rather than stretching . This is best seen in Figure 2 . Affine deformations have the property that the majority of the energy stored in the network is in stretching the fibers , but here , fiber stretching makes no contribution to the network stiffness until strains are greater than 10 % . In response to shear stress , the simulated networks also exhibited negative normal stresses that grow quadratically with strain ( Figure 4 ) , as observed in the experiments of Janmey et al . [ 9 ] . We also examined the perpendicular con - traction of the gel in response to axial strain in comparison with the experiments of Vader et al . [ 11 ] and Roeder et al . [ 4 ] , as shown in Figure 5 . The perpendicular strain was taken to be the perpendicular displacement divided by the origi - nal perpendicular distance from the origin averaged over all the nodes . Here , the model does not match the experiments well . In particular , in both experiments , the z - compression is significantly greater than the y - compression . By contrast , in the simulation , the z - and y - compression are almost overlap - ping . Furthermore , both experiments exhibited considerably more perpendicular compression than seen in the simula - tion . One possible explanation is that the geometry of the experiments done here ( sheared cone - plate rheometer ) is considerably different from that in the other experiments ( stretched disk and stretched 3D I - beam ) . Another possi - bility is that the FIRE network extraction algorithm over - estimates the number of crosslinks of the network . It may also be that some of the crosslinks in the network slip , thus allowing the network to further compress in response to axial tension . To give the same strain - stiffening behavior , this would require stiffer crosslinks . Further exploration , how - ever , was beyond the scope of this article . Despite the lack of quantitative agreement , we note that nonlinearly increasing perpendicular strain is qualitatively similar to that observed inVader et al . [ 11 ] . 5 . SUMMARY AND DISCUSSION In summary , we have presented a microstructural model of a 3D biopolymer gel using a network geometry that is based on the true network architecture . It differs from pre - vious work in that we use realistic network architectures that have been extracted using the FIRE algorithm . We specifi - cally focus on the mechanics of collagen - I networks , but we emphasize that this modeling approach is generalizable to other biopolymer networks . The model has three parame - ters : { E , r , K x } . The fiber radius and tensile modulus can be measured experimentally , and the model uses realistic para - meters . The crosslink torsional spring constant must be fit to thedataandweused K x ∼ 20 K b / l c . Fittingthissingleparame - ter gives the right strain - stiffening behavior for networks at three different densities at strains that vary from 0 . 5 to 50 % . This result lends support to the hypothesis put forward by Onck et al . [ 15 ] that strain stiffening in polymer networks and particularly collagen - I gels is governed by rearrangement of the gel . Here , we note that the strain - stiffening mechanisms for collagen are likely different than for other gels . Because of itslongpersistencelength ( 1cm ) fibers , theeffectsofentropic stiffening in these gels is known to be minimal . Therefore , the entropic stiffening hypothesis [ 31 ] is not applicable here . However , for other biopolymer gels such as actin , where the FIGURE 5 Perpendicularstrainsforthe1 . 2mg / mLgelinresponsetotensionwhen compared with the experiments from Vader et al . [ 11 ] and Roeder et al . [ 4 ] . 26 C O M P L E X I T Y © 2010 Wiley Periodicals , Inc . DOI 10 . 1002 / cplx J _ ID : za5 Customer A _ ID : 09 - 035 . R1 Cadmus Art : CPLX20332 — 2011 / 3 / 3 — page 27 — # 6 persistence length of the filaments is in the order of the mesh size , entropic stiffening of individual fibers is more likely to be an important component that contributes to strain stiff - ening . The stiffening in fibrin clots may depend not only on geometric rearrangements but also on the unfolding of the fibrin protein [ 32 ] . Anotherfindingofthemodelisthatatshearstrainsgreater than 25 % , the stiffness of the gel is governed almost entirely by stretching of the fibers . This result is relevant for collagen because cells embedded in these gels are seen to produce deformations of this order of magnitude [ 33 ] . In modeling large systems of this type where the strains are large , it may be sufficient to treat each fiber as a spring rather than an elastic beam to reduce the computation time . This work also demonstrates that an understand of the crosslink mechanics in these systems is critical to understand their mechanical properties , as has been seen previously [ 34 ] . In much of the theoretical work that has been done on random stick net - works , the crosslinks are treated either as freely rotating pin joints or welded joints of fixed angle [ 14 , 15 , 27 ] . Although these are sensible simplifying assumptions in develop - ing a theory , they are not adequate for describing actual networks . The mechanical properties of an individual fiber depends on the fiber microstructure and ranges from relatively linear elastic behavior even at large strains [ 24 ] to moderate stiffen - ing [ 35 ] . Here , wenotethatthesimulatedlargestrainmodulus underestimated the actual large strain modulus by a factor of two . This difference could potentially be explained by a stiff - eningoftheindividualfibersbyafactoroftwoatlargestrains , butconfirmationwouldrequireadditionalexperimentsmea - suringthestress – strainpropertiesofindividualfibersinthese collagen gels . We note that this model has been designed to capture the intermediate time scale behavior of the network ( minutes to hours ) , where the network behaves as an elastic solid . The viscous modulus is known to be a factor of 10 less than the elastic modulus within the range of 0 . 1 – 100Hz [ 36 , 37 ] , but at even faster time scales , the viscosity of the fluid will play a critical role . At slower time scales , the assumption that the crosslinks remain relatively fixed [ 36 ] breaks down as plastic deformation and creep occur . This simplified model provides astartingpointinthedevelopmentofamorecompletemodel of collagen gel . Ultimately , a more sophisticated approach , such as that taken by Rodney et al . [ 38 ] will be necessary to capture the full dynamic behavior of the gel , where crosslinks are allowed to slip and break . ACKNOWLEDGEMENTS The authors thankV . Barocas , T . Stylianopoulos , E . A . Sander , E . Tuzel , H . Zhang , H . Othmer , T . Jackson , P . Smereka , R . Krasny , F . MacKintosh , A . Kabla , R . Lee , M . Dewberry , L . Kaufman , and L . Jawerth , for their discussions . This work was supported by NIH Bioengineering Research Partnership ( grantR01CA085139 - 01A2 ) andtheInstituteforMathematics and its Applications . REFERENCES 1 . Lodish , H . ; Berk , A . ; Zipursky , S . L . ; Matsudaira , P . ; Baltimore , D . ; Darnell , J . 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C . Mechanical characterization of collagen fibers and scaffolds for tissue engineering . Biomaterials 2003 , 24 , 3805 – 3813 . 36 . Barocas , V . H . ; Moon , A . G . ; Tranquillo , R . T . The fibroblast - populated microsphere assay of cell traction force - part2 . measurement of the cell traction coefficient . J Biomech Eng 1995 , 117 , 161 – 170 . 37 . Velegol , D . ; Lanni , F . Cell traction forces on soft biomaterials . I . microrheology of type i collagen gels . Biophys J 2001 , 81 , 1786 – 1792 . 38 . Rodney , D . ; Fivel , M . ; Dendievel , R . Discrete modeling of the mechanics of entangled materials . Phys Rev Lett 2005 , 95 , 108004 . 28 C O M P L E X I T Y © 2010 Wiley Periodicals , Inc . DOI 10 . 1002 / cplx \ No newline at end of file diff --git a/silver_data/cbc05a44f18a489c3df237d50d201d8f6e8735f4.pdf.txt b/silver_data/cbc05a44f18a489c3df237d50d201d8f6e8735f4.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..db6d42c630560c45c5571a10b424a9463e91676e --- /dev/null +++ b/silver_data/cbc05a44f18a489c3df237d50d201d8f6e8735f4.pdf.txt @@ -0,0 +1 @@ +Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions The Harvard community has made this article openly available . Please share how this access benefits you . Your story matters Citation Dixon , Jesse R . , Siddarth Selvaraj , Feng Yue , Audrey Kim , Yan Li , Yin Shen , Ming Hu , Jun S . Liu , and Bing Ren . 2012 . Topological domains in mammalian genomes identified by analysis of chromatin interactions . Nature 485 ( 7398 ) : 376 - 380 . Published Version doi : 10 . 1038 / nature11082 Citable link http : / / nrs . harvard . edu / urn - 3 : HUL . InstRepos : 14169378 Terms of Use This article was downloaded from Harvard University’s DASH repository , and is made available under the terms and conditions applicable to Other Posted Material , as set forth at http : / / nrs . harvard . edu / urn - 3 : HUL . InstRepos : dash . current . terms - of - use # LAA Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions Jesse R . Dixon 1 , 3 , 4 , Siddarth Selvaraj 1 , 5 , Feng Yue 1 , Audrey Kim 1 , Yan Li 1 , Yin Shen 1 , Ming Hu 6 , Jun S . Liu 6 , and Bing Ren 1 , 2 , * 1 Ludwig Institute for Cancer Research 2 University of California , San Diego School of Medicine , Department of Cellular and Molecular Medicine , Institute of Genomic Medicine , 9500 Gilman Drive , La Jolla , CA 92093 3 Medical Scientist Training Program , University of California , San Diego , La Jolla CA 92093 4 Biomedical Sciences Graduate Program , University of California , San Diego , La Jolla CA 92093 5 Bioinformatics and Systems Biology Graduate Program , University of California , San Diego , La Jolla CA 92093 6 Department of Statistics , Harvard University , 1 Oxford Street , Cambridge , MA 02138 Abstract The spatial organization of the genome is intimately linked to its biological function , yet our understanding of higher order genomic structure is coarse , fragmented and incomplete . In the nucleus of eukaryotic cells , interphase chromosomes occupy distinct chromosome territories ( CT ) , and numerous models have been proposed for how chromosomes fold within CTs 1 . These models , however , provide only few mechanistic details about the relationship between higher order chromatin structure and genome function . Recent advances in genomic technologies have led to rapid revolutions in the study of 3D genome organization . In particular , Hi - C has been introduced as a method for identifying higher order chromatin interactions genome wide 2 . In the present study , we investigated the 3D organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution . We identify large , megabase - sized local chromatin interaction domains , which we term “topological domains” , as a pervasive structural feature of the genome organization . These domains correlate with regions of the genome that constrain the spread of heterochromatin . The domains are stable across different cell types and highly conserved across species , suggesting that topological domains are an inherent property of mammalian genomes . Lastly , we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF , housekeeping genes , tRNAs , and SINE retrotransposons , suggesting that these factors may play a role in establishing the topological domain structure of the genome . * To whom correspondence should be addressed : biren @ ucsd . edu . Supplementary Information is linked to the online version of the paper at www . nature . com / nature Author Information : All Hi - C data described in this study have been deposited to GEO under the accession number GSE35156 . We have developed a web based Java tool to visualize the high resolution Hi - C data at a genomic region of interest that is available at http : / / chromosome . sdsc . edu / mouse / hi - c / database . html . Reprints and permissions information is available at www . nature . com / reprints . The authors declare no competing financial interests . Author Contributions : JD and BR designed the studies . JD , AK , YL , YS conducted the Hi - C experiments ; JD , SS and FY carried out the data analysis ; JL and MH provided insight for analysis ; FY built the supporting website ; JD and BR prepared the manuscript . NIH Public Access Author Manuscript Nature . Author manuscript ; available in PMC 2012 November 17 . Published in final edited form as : Nature . ; 485 ( 7398 ) : 376 – 380 . doi : 10 . 1038 / nature11082 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t To study chromatin structure in mammalian cells , we performed the Hi - C experiment 2 in mouse embryonic stem cells ( mESCs ) , human embryonic stem cells ( hESCs ) , and human IMR90 fibroblasts . Together with Hi - C data for the mouse cortex generated in a separate study 3 , we analyzed over 1 . 7 billion read pairs of Hi - C data corresponding to pluripotent and differentiated cells ( Supplemental Table 1 ) . We normalized the Hi - C interactions for biases in the data ( Supplemental Figure 1 and 2 ) 4 . To validate the quality of our Hi - C data , we compared the data with previous 5C , 3C , and FISH results 5 – 7 . Our IMR90 Hi - C data shows a high degree of similarity when compared to a previously generated 5C dataset from lung fibroblasts ( Supplementary Figure 4 ) . In addition , our mESC Hi - C data correctly recovered a previously described cell - type specific interaction at the Phc1 gene 6 ( Supplementary Figure 5 ) . Furthermore , the Hi - C interaction frequencies in mESCs are well - correlated with the mean spatial distance separating six loci as measured by 2D - FISH 7 ( Supplemental Figure 6 ) , demonstrating that the normalized Hi - C data can accurately reproduce the expected nuclear distance using an independent method . These results demonstrate that our Hi - C data is of high quality and accurately captures the higher order chromatin structures in mammalian cells . We next visualized 2D - interaction matrices using a variety of bin sizes to identify interaction patterns revealed as a result of our high sequencing depth ( Supplemental Figure 7 ) . We noticed that at bin sizes less than 100kb , highly self - interacting regions begin to emerge ( Figure 1a , Supplemental Figure 7 , seen as “triangles” on the heatmap ) . These regions , which we term “topological domains , ” are bounded by narrow segments where the chromatin interactions appear to end abruptly . We hypothesized that these abrupt transitions may represent boundary regions in the genome that separate topological domains . To systematically identify all such topological domains in the genome , we devised a simple statistic termed the “directionality index” ( DI ) to quantify the degree of upstream or downstream interaction bias for a genomic region , which varies considerably at the periphery of the topological domains ( Figure 1b , see supplemental methods for details ) . The DI was reproducible ( Supplemental Table 2 ) and pervasive , with 52 % of the genome having a DI that was not expected by random chance ( Figure 1c , FDR = 1 % ) . We then used a Hidden Markov model ( HMM ) based on the DI to identify biased “states” and therefore infer the locations of topological domains in the genome ( Figure 1a , see supplemental methods for details ) . The domains defined by HMM were reproducible between replicates ( Supplemental Figure 8 ) . Therefore , we combined the data from the HindIII replicates and identified 2 , 200 topological domains in mESCs with a median size of 880kb that occupy ~ 91 % of the genome ( Supplemental Figure 9 ) . As expected , the frequency of intra - domain interactions is higher than inter - domain interactions ( Figure 1d , e ) . Similarly , FISH probes 7 in the same topological domain ( Figure 1f ) are closer in nuclear space than probes in different topological domains ( Figure 1g ) , despite similar genomic distances between probe pairs ( Figure 1h , i ) . These findings are best explained by a model of the organization of genomic DNA into spatial modules linked by short chromatin segments . We define the genomic regions between topological domains as either “topological boundary regions” or “unorganized chromatin” , depending on their sizes ( Supplemental Figure 9 ) . We next investigated the relationship between the topological domains and the transcriptional control process . The HoxA locus is separated into two compartments by an experimentally validated insulator 5 , 8 , 9 , which we observed corresponds to a topological domain boundary in both mouse ( Figure 1a ) and human ( Figure 2a ) . Therefore , we hypothesized that the boundaries of the topological domains might correspond to insulator or barrier elements . Dixon et al . Page 2 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t Many known insulator or barrier elements are bound by the zinc - finger containing protein CTCF 10 – 12 . We see a strong enrichment of CTCF at the topological boundary regions ( Figure 2b , Supplemental Figure 10 ) , indicating that topological boundary regions share this feature of classical insulators . A classical boundary element is also known to stop the spread of heterochromatin . Therefore , we examined the distribution of the heterochromatin mark H3K9me3 in humans and mice in relation to the topological domains 13 , 14 . Indeed , we observe a clear segregation of H3K9me3 at the boundary regions that occurs predominately in differentiated cells ( Figure 2d , e , Supplemental Figure 11 ) . Since the boundaries we analyzed in Figure 2d are present in both pluripotent cells and their differentiated progeny , the topological domains and boundaries appear to “pre - mark” the end points of heterochromatic spreading . Therefore , the domains do not appear to be a consequence of the formation of heterochromatin . Taken together , the above observations strongly suggest that the topological domain boundaries correlate with regions of the genome displaying classical insulator and barrier element activity , thus revealing a potential link between the topological domains and transcriptional control in the mammalian genome . We compared the topological domains with previously described domain - like organizations of the genome , specifically with the A and B compartments described by Lieberman - Aiden et al . , 2 with Lamina - Associated Domains ( LADs ) 11 , 15 , replication time zones , 16 , 17 and Large Organized Chromatin K9 - modification ( LOCK ) domains 18 . In all cases , we can see that topological domains are related to , but independent from , each of these previously described domain - like structures ( Supplemental Figures 12 – 15 ) . Notably , a subset of the domain boundaries we identify appear to mark the transition between either LAD and non - LAD regions of the genome ( Figure 2f , Supplemental Figure 12 ) , the A and B compartments ( Supplemental Figure 13 , 14 ) , and early and late replicating chromatin ( Supplemental Figure 14 ) . Lastly , we can also confirm the previously reported similarities between the A and B compartments and early and late replication time zone ( Supplemental Figure 16 ) 17 . We next compared the locations of topological boundaries identified in both replicates of mESCs and cortex , or between both replicates of hESCs and IMR90 cells . In both human and mouse , the majority of the boundary regions are shared between cell types ( Figure 3a , Supplemental Figure 17a ) , suggesting that the overall domain structure between cell types is largely unchanged . At the boundaries called in only one cell type , we noticed that trend of upstream and downstream bias in the DI is still readily apparent and highly reproducible between replicates ( Supplemental figure 17b , c ) . We cannot determine if the differences in domain calls between cell types is due to noise in the data or due to biological phenomena , such as a change in the strength of the boundary region between cell types 19 . Regardless , these results suggest that the domain boundaries are largely invariant between cell types . Lastly , only a small fraction of the boundaries show clear differences between two cell types , suggesting that a relatively rare subset of boundaries may actually differ between cell types ( Supplemental Figure 18 ) . The stability of the domains between cell types is surprising given previous evidence showing cell type specific chromatin interactions and conformations 6 , 8 . To reconcile these results , we identified cell - type specific chromatin interactions between mouse ES cell and mouse cortex . We identified 9 , 888 dynamic interacting regions in the mouse genome based on 20kb binning using a binomial test with an empirical false discover rate of < 1 % based on random permutation of the replicate data . These dynamic interacting regions are enriched for differentially expressed genes , ( Figure 3b – d , Supplemental Figure 19 , Supplemental Table 5 ) . In fact , 20 % of all genes that undergo a 4 - fold change in gene expression are found at dynamic interacting loci . This is likely an underestimate , because by binning the genome at 20kb , any dynamic regulatory interaction less than 20kb will be missed . Lastly , > 96 % of dynamic interacting regions occur in the same domain ( Figure 3e ) . Therefore , we favor a Dixon et al . Page 3 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t model where the domain organization is stable between cell types , but the regions within each domain may be dynamic , potentially taking part in cell - type specific regulatory events . The stability of the domains between cell types prompted us to investigate if the domain structure is also conserved across evolution . To address this , we compared the domain boundaries between mouse ES cells and human ES cells using the UCSC liftover tool . The majority of boundaries appear to be shared across evolution ( 53 . 8 % of human boundaries are boundaries in mouse and 75 . 9 % of mouse boundaries are boundaries in humans , compared to 21 . 0 % and 29 . 0 % at random , p - value < 2 . 2×10 −16 , Fisher’s Exact Test ) ( Figure 3f ) . The syntenic regions in mouse and human in particular share a high degree of similarity in their higher order chromatin structure ( Figure 3g , h ) , indicating that there is conservation of genomic structure beyond the primary sequence of DNA . We explored what factors may contribute to the formation of topological boundary regions in the genome . While most topological boundaries are enriched for the binding of CTCF , only 15 % of CTCF binding sites are located within boundary regions ( Figure 2c ) . Thus , CTCF binding alone is insufficient to demarcate domain boundaries . We reasoned that additional factors might be associated with topological boundary regions . By examining the enrichment of a variety of histone modifications , chromatin binding proteins , transcription factors , around topological boundary regions in mESC , we observed that factors associated with active promoters and gene bodies are enriched at boundaries in both mouse and humans ( Figure 4a and Supplemental Figures 20 – 23 ) 20 , 21 . In contrast , non - promoter associated marks , such as H3K4me1 ( associated with enhancers ) and H3K9me3 , were not enriched or were specifically depleted at boundary regions ( Figure 4a ) . Furthermore , transcription start sites ( TSS ) and global run on sequencing ( GRO - Seq ) 22 signal were also enriched around topological boundaries ( Figure 4a ) . We found that “housekeeping genes” were particularly strongly enriched near topological boundary regions ( Figure 4b – d , See Supplemental Table 7 for complete GO terms enrichment ) . Additionally , the tRNA genes , which have the potential to function as boundary elements 23 , 24 , are also enriched at boundaries ( p - value < 0 . 05 , Fisher’s exact test ( Figure 4b ) . These results suggest that high levels of transcription activity may also contribute to boundary formation . In support of this , we can see examples of dynamic changes in H3K4me3 at or near some cell - type specific boundaries that are cell type - specific ( Supplemental Figure 24 ) . Indeed , boundaries associated with both CTCF and a housekeeping gene account for nearly a third of all topological boundaries in the genome ( Figure 4e , Supplemental Figure 24 ) Lastly , we analyzed the enrichment of repeat classes around boundary elements . We observed Alu / B1 and B2 SINE elements in mouse and Alu SINE elements in humans are enriched at boundary regions ( Figure 4a , Supplemental Figures 24 , 25 ) . In light of recent reports indicating that a SINE B2 element functions as a boundary in mice 25 , and SINE element retrotransposition may alter CTCF binding sites during evolution 26 , we believe this contributes to a growing body of evidence suggesting a role for SINE elements in the organization of the genome . In summary , we show that the mammalian chromosomes are segmented into megabase - sized topological domains , consistent with some previous models of the higher order chromatin structure 1 , 27 , 28 . Such spatial organization appears to be a general property of the genome : it is pervasive throughout the genome , stable across different cell types and highly conserved between mice and humans . We have identified multiple factors that are associated with the boundary regions separating topological domains , including the insulator binding factor CTCF , housekeeping genes , SINE elements . The association of housekeeping genes with boundary regions extends Dixon et al . Page 4 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t previous studies in yeast , insects and lower vertebrates and suggests that non - CTCF factors may be also involved in insulator / barrier functions in mammalian cells 29 . The topological domains we identified are well conserved between mice and humans . This suggests that the sequence elements and mechanisms that are responsible for establishing higher order structures in the genome may be relatively ancient in evolution . A similar partitioning of the genome into physical domains has also been observed in Drosophila embryos 30 and in high - resolution studies of the X - inactivation center in mice ( termed Topologically Associated Domains or TADs ) 31 , suggesting that topological domains may be a fundamental organizing principle of metazoan genomes . Method Summary Cell Culture and Hi - C Experiments J1 mouse embryonic stem cells were grown on gamma - irradiated mouse embryonic fibroblasts cells under standard conditions ( 85 % High Glucose DMEM , 15 % HyClone FBS , 0 . 1mM non - essential amino acids , 0 . 1mM β - mercaptoethanol , 1mM Glutamine , LIF 500U / mL , + P / S ) . Before harvesting for Hi - C , J1 mESCs were passaged onto feeder free 0 . 2 % gelatin coated plates for at least 2 passages to rid the culture of feeder cells . H1 Human embryonic stem cells and IMR90 fibroblasts were grown as previously described 14 . Harvesting the cells for Hi - C was performed as previously described , with the only modification being that the adherent cell cultures were dissociated with trypsin prior to fixation . Sequencing and Mapping of Data Hi - C analysis and paired end libraries were prepared as previously described 2 and sequenced on the Illumina Hi - Seq2000 platform . Reads were mapped to reference human ( hg18 ) or mouse genomes ( mm9 ) , and non - mapping reads and PCR duplicates were removed . 2 - dimensional heat - maps were generated as previously described 2 . Data Analysis For detailed descriptions of the data analysis , including descriptions of the directionality index , hidden Markov models , dynamic interactions identification , and boundary overlap between cells and across species , see supplemental methods . Supplementary Material Refer to Web version on PubMed Central for supplementary material . Acknowledgments We are grateful for the valuable comments from and discussions with Drs . Zhaohui Qin ( Emory University ) , Arshad Desai ( LICR / UCSD ) , and members of the Ren lab during the course of the present study . We also thank Drs . Wendy Bickmore and Ragnhild Eskeland for sharing the FISH data generated in mouse ES cells . This work was supported by funding from the Ludwig Institute for Cancer Research , California Institute for Regenerative Medicine ( CIRM , RN2 - 00905 - 1 ) ( to B . R . ) , and NIH ( B . R . R01GH003991 ) . JD is funded by a pre - doctoral training grant from CIRM . YS is supported by a postdoctoral fellowship from the Rett Syndrome Research Foundation . References 1 . Cremer T , Cremer M . Chromosome territories . Cold Spring Harb Perspect Biol . 2 : a003889 . [ PubMed : 20300217 ] 2 . Lieberman - Aiden E , et al . Comprehensive mapping of long - range interactions reveals folding principles of the human genome . Science . 2009 ; 326 : 289 – 93 . [ PubMed : 19815776 ] Dixon et al . Page 5 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t 3 . Shen Y , et al . A Map of cis - Regulatory Sequences in the Mouse Genome . 2012 in submission . 4 . Yaffe E , Tanay A . Probabilistic modeling of Hi - C contact maps eliminates systematic biases to characterize global chromosomal architecture . Nat Genet . 43 : 1059 – 65 . [ PubMed : 22001755 ] 5 . Wang KC , et al . A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression . Nature . 472 : 120 – 4 . [ PubMed : 21423168 ] 6 . Kagey MH , et al . Mediator and cohesin connect gene expression and chromatin architecture . Nature . 467 : 430 – 5 . [ PubMed : 20720539 ] 7 . Eskeland R , et al . Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination . Mol Cell . 38 : 452 – 64 . [ PubMed : 20471950 ] 8 . Noordermeer D , et al . The dynamic architecture of Hox gene clusters . Science . 334 : 222 – 5 . [ PubMed : 21998387 ] 9 . Kim YJ , Cecchini KR , Kim TH . Conserved , developmentally regulated mechanism couples chromosomal looping and heterochromatin barrier activity at the homeobox gene A locus . Proc Natl Acad Sci U S A . 108 : 7391 – 6 . [ PubMed : 21502535 ] 10 . Phillips JE , Corces VG . CTCF : master weaver of the genome . Cell . 2009 ; 137 : 1194 – 211 . [ PubMed : 19563753 ] 11 . Guelen L , et al . Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions . Nature . 2008 ; 453 : 948 – 51 . [ PubMed : 18463634 ] 12 . Handoko L , et al . CTCF - mediated functional chromatin interactome in pluripotent cells . Nat Genet . 43 : 630 – 8 . [ PubMed : 21685913 ] 13 . Xie W , et al . Base - Resolution Analyses of Sequence and Parent - of - Origin Dependent DNA Methylation in the Mouse Genome . Cell . 148 : 816 – 31 . [ PubMed : 22341451 ] 14 . Hawkins RD , et al . Distinct epigenomic landscapes of pluripotent and lineage - committed human cells . Cell Stem Cell . 6 : 479 – 91 . [ PubMed : 20452322 ] 15 . Peric - Hupkes D , et al . Molecular maps of the reorganization of genome - nuclear lamina interactions during differentiation . Mol Cell . 38 : 603 – 13 . [ PubMed : 20513434 ] 16 . Hiratani I , et al . Genome - wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis . Genome Res . 20 : 155 – 69 . [ PubMed : 19952138 ] 17 . Ryba T , et al . Evolutionarily conserved replication timing profiles predict long - range chromatin interactions and distinguish closely related cell types . Genome Res . 20 : 761 – 70 . [ PubMed : 20430782 ] 18 . Wen B , Wu H , Shinkai Y , Irizarry RA , Feinberg AP . Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells . Nat Genet . 2009 ; 41 : 246 – 50 . [ PubMed : 19151716 ] 19 . Scott KC , Taubman AD , Geyer PK . Enhancer blocking by the Drosophila gypsy insulator depends upon insulator anatomy and enhancer strength . Genetics . 1999 ; 153 : 787 – 98 . [ PubMed : 10511558 ] 20 . Bilodeau S , Kagey MH , Frampton GM , Rahl PB , Young RA . SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state . Genes Dev . 2009 ; 23 : 2484 – 9 . [ PubMed : 19884255 ] 21 . Marson A , et al . Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells . Cell . 2008 ; 134 : 521 – 33 . [ PubMed : 18692474 ] 22 . Min IM , et al . Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells . Genes Dev . 25 : 742 – 54 . [ PubMed : 21460038 ] 23 . Donze D , Kamakaka RT . RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae . EMBO J . 2001 ; 20 : 520 – 31 . [ PubMed : 11157758 ] 24 . Ebersole T , et al . tRNA genes protect a reporter gene from epigenetic silencing in mouse cells . Cell Cycle . 10 : 2779 – 91 . [ PubMed : 21822054 ] 25 . Lunyak VV , et al . Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis . Science . 2007 ; 317 : 248 – 51 . [ PubMed : 17626886 ] 26 . Schmidt D , et al . Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages . Cell . Dixon et al . Page 6 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t 27 . Jhunjhunwala S , et al . The 3D structure of the immunoglobulin heavy - chain locus : implications for long - range genomic interactions . Cell . 2008 ; 133 : 265 – 79 . [ PubMed : 18423198 ] 28 . Capelson M , Corces VG . Boundary elements and nuclear organization . Biol Cell . 2004 ; 96 : 617 – 29 . [ PubMed : 15519696 ] 29 . Amouyal M . Gene insulation . Part I : natural strategies in yeast and Drosophila . Biochem Cell Biol . 88 : 875 – 84 . [ PubMed : 21102650 ] 30 . Sexton T , et al . Three - dimensional folding and functional organization principles of the Drosophila genome . Cell . 148 : 458 – 72 . [ PubMed : 22265598 ] 31 . Nora EP , et al . Spatial partitioning of the regulatory landscape of the X - inactivation center . 2012 In submission . Dixon et al . Page 7 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t Figure 1 . Topological Domains in the Mouse ES cell Genome a , Normalized Hi - C interaction frequencies displayed as a 2D heatmap overlayed on ChIP - Seq data ( from ref . 3 ) , DI , HMM Bias State Calls , and domains . For both DI and HMM State calls , downstream bias ( red ) and upstream bias ( green ) are indicated . b , Schematic illustrating topological domains and resulting directional bias . c , Distribution of the DI ( absolute value , in blue ) compared to random ( red ) . d , Mean interaction frequencies at all genomic distances between 40kb to 2Mb . Above 40kb , the intra - versus inter - domain interaction frequencies are significantly different ( p < 0 . 005 , Wilcoxan test ) . e , Box plot of all interaction frequencies at 80kb distance . Intra - domain interactions are enriched for high - frequency interactions . f – i , Diagram of “Intra - domain” ( f ) and “Inter - domain” FISH probes ( g ) and the genomic distance between pairs ( h ) . i , Bar chart of the squared interprobe distance ( from ref . 7 ) FISH probe pairs . Error bars indicate standard error ( n = 100 for each probe pair ) . Dixon et al . Page 8 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t Figure 2 . Topological Boundaries Demonstrate Classical Insulator or Barrier Elements Features a , 2D heatmap surrounding the HoxA locus and CS5 insulator in IMR90 cells . b , Enrichment of CTCF at boundary regions . c , The portion of CTCF binding sites that are considered “associated” with a boundary ( within + / − 20kb window is used as the expected uncertainty due to 40kb binning ) . d , Heat maps of H3K9me3 at boundary sites in human and mouse . e , UCSC Genome Browser shot showing heterochromatin spreading in the human ES cells and IMR90 cells . The 2D heat map shows the interaction frequency in hES cells . f , Heat map of LADs ( from ref . 15 ) surrounding the boundary regions . Dixon et al . Page 9 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t Figure 3 . Boundaries are shared across cell types and conserved in evolution a , Overlap of boundaries between cell types . b , Genome browser shot of a cortex enriched dynamic interacting region that overlaps with the Foxg1 gene . c , Foxg1 expression in mouse ES cells and cortex as measured by RNA - seq . d , Heat map of the gene expression ratio between mouse ES cell and cortex of genes at dynamic interactions . e , Pie chart of inter - and intra - domain dynamic interactions . f , Overlap of boundaries between syntenic mouse and human sequences ( p - value < 2 . 2 * 10 −16 compared to random , Fisher’s exact test ) . g and h , Genome browser shots showing domain structure over a syntenic region in the mouse ( g ) and human ES cells ( h ) . Note : the region in humans has been inverted from its normal UCSC coordinates for proper display purposes . Dixon et al . Page 10 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t Figure 4 . Boundary regions are enriched for housekeeping genes a , Chromatin modifications , TSS , GRO - Seq , and SINE elements surrounding boundary regions in mESCs or IMR90 . b , Boundaries associated with a CTCF binding site , housekeeping gene , or tRNA gene ( purple ) compared to expected at random ( grey ) . c , Gene Ontology p - value chart . d , Enrichment of housekeeping genes ( gold ) and tissue specific genes ( blue ) as defined by on Shannon entropy scores near boundaries normalized for the number of genes in each class ( TSS / 10kb / total TSS ) . e , Percentage of boundaries with a given mark within 20kb of the boundaries . Dixon et al . Page 11 Nature . Author manuscript ; available in PMC 2012 November 17 . N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t N I H - PA A u t ho r M anu sc r i p t \ No newline at end of file diff --git a/silver_data/d124ff435367f314852cd82ca0d1984921bdfa07.pdf.txt b/silver_data/d124ff435367f314852cd82ca0d1984921bdfa07.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..df42d94eb4cd7fc3ca731033438b729629ce55e9 --- /dev/null +++ b/silver_data/d124ff435367f314852cd82ca0d1984921bdfa07.pdf.txt @@ -0,0 +1 @@ +JCB : Article The Rockefeller University Press $ 30 . 00 J . Cell Biol . www . jcb . org / cgi / doi / 10 . 1083 / jcb . 200910136 Cite by DOI : 10 . 1083 / jcb . 200910136 JCB 1 of 15 G . A . Quinones and J . Jin contributed equally to this paper . Correspondence to Anthony E . Oro : oro @ stanford . edu Abbreviations used in this paper : BAR , Bin / Amphiphysin / Rvs domain ; CD2AP , CD2 - associated protein ; GMA , GFP fused to the actin - binding domain of moesin ; IRS , insulin receptor substrate ; MEF , mouse embryonic fibroblast ; MIM , missing - in - metastasis ; PGC , primordial germ cell ; pTyr , phosphotyrosine . Introduction Directed cellular migration facilitates the coordinated move - ment of individual cells , cell clusters , and sheets of cells during development and regeneration . Directed cell migration is im - portant for individual cells as well as groups of cells ; there are key differences between these types of migrating cells . Individ - ual cells migrating , either on a plastic dish or in a living tissue matrix , must correctly sense or respond to migratory cues . These cells then undergo changes in their cytoskeletal structure in order to project their cell bodies forward , put down new ad - hesion complexes , and remove older adhesion complexes at the trailing end of the cell ( Harden , 2002 ; Friedl et al . , 2004 ; Blaser et al . , 2005 ; Montell , 2006 ; Rørth , 2007 ) . Although cells in a cluster or sheet must also respond to directional cues , they must also maintain the correct cell – cell adhesions and spatial aware - ness in order to maintain their structure . Directed cluster migra - tion forms the foundation for organ morphogenesis and , when abnormal , has been implicated in disease states such as mental retardation , birth defects , and cancer ( Friedl et al . , 2004 ; Kopfstein and Christofori , 2006 ) . Insights into directed cell migration of groups of cells have come from the studies of relatively small clusters of cells , such as border cell migration during Drosophila oogenesis ( Rørth , 2002 ; Montell , 2006 ) or the rearrangement of larger sheets , as is the case during vertebrate gastrulation ( Keller , 2005 ) . In Drosophila border cell migration , cell clusters initially become autonomously motile , elaborating nondirec - tional actin - based cellular extensions with little net cellular dis - placement . Border cell migration is mediated in response to local migratory cues emanating from the ovary via guidance receptors , the Drosophila epidermal growth factor receptor ( DER ) and the PDGF / VEGF - like ( PVR ) receptor , on the clus - ter surface . Signaling through the receptors allows border cell membranes to become polarized to form actin - based mem - brane extensions and migrate along the growth factor gradient ( Bianco et al . , 2007 ; Prasad and Montell , 2007 ) . Genetic studies overexpressing DER in border cells indicate that it is not the total levels of receptor , but the location of activated receptors that determines directional migration ( Jékely and Rørth , 2003 ) . Despite its importance , our understanding of signaling mecha - nisms downstream of the guidance receptors that operate in the context of developing organisms remains primitive . Previous work points to a central role for guidance receptor endocytosis in interpreting local migratory cues to the A lthough directed cellular migration facilitates the coordinated movement of cells during devel - opment and repair , the mechanisms regulating such migration remain poorly understood . Missing - in - metastasis ( MIM ) is a defining member of the inverse Bin / Amphiphysin / Rvs domain ( I - BAR ) subfamily of lipid binding , cytoskeletal regulators whose levels are altered in a number of cancers . Here , we provide the first genetic evidence that an I - BAR protein regulates directed cell migration in vivo . Drosophila MIM ( dmim ) is involved in Drosophila border cell migration , with loss of dmim function resulting in a lack of directional movement by the border cell cluster . In vivo endocytosis assays com - bined with genetic analyses demonstrate that the dmim product regulates directed cell movement by inhibiting endocytosis and antagonizing the activities of the CD2 - associated protein / cortactin complex in these cells . These studies demonstrate that DMIM antagonizes pro - endocytic components to facilitate polarity and localized guidance cue sensing during directional cell migration . I - BAR protein antagonism of endocytosis mediates directional sensing during guided cell migration Gabriel A . Quinones , Janet Jin , and Anthony E . Oro Program in Epithelial Biology and Cancer Biology Graduate Program , Stanford University School of Medicine , Stanford , CA 94305 © 2010 Quinones et al . This article is distributed under the terms of an Attribution – Noncommercial – Share Alike – No Mirror Sites license for the first six months after the publication date ( see http : / / www . rupress . org / terms ) . After six months it is available under a Creative Commons License ( Attribution – Noncommercial – Share Alike 3 . 0 Unported license , as described at http : / / creativecommons . org / licenses / by - nc - sa / 3 . 0 / ) . T H E J O U R N A L O F C E L L B I O L O G Y on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 JCB 2 of 15 Here , we provide the first in vivo genetic evidence for the involvement of an I - BAR family member in regulating direc - tional migration . We show that MIM and cortactin antagonism underlies a novel molecular steering mechanism . Results Vertebrate MIM inhibits endocytosis Increasing attention has been directed toward the role of the BAR family proteins in endocytosis and vesicle trafficking . Because each subfamily of BAR proteins has been shown to positively regulate endocytosis , we sought to test whether I - BAR proteins function in a similar manner , or because they are struc - turally distinct from the other families , do they act to antagonize endocytosis . We asked whether MIM , a founding member of the I - BAR family , regulates cell migration and endocytosis using well - characterized , receptor - mediated endocytosis assays in vertebrate cells . Using transferrin endocytosis assays , we found the uptake of HRP - labeled transferrin within 5 min of cell warming to be increased where MIM protein levels are reduced ( Fig . 1 A ) . Similar effects were seen when the kinetics of 125 I - labeled EGF uptake were observed after siRNA knock - down of MIM ( Fig . 1 B ) . This effect was not due to altered ini - tial levels of receptor ( Fig . 1 , F and H ) . We also observed an increase in recycling levels of both transferrin and EGF ( Fig . 1 , C and D ) , consistent with the increase in 125 I - EGF uptake . These results indicate that a reduction in MIM protein levels increases endocytosis and recycling , but not receptor degradation , leading us to examine if MIM knockdown alters signaling downstream of the receptor . We tested this by assaying levels of phospho - ERK1 / 2 ( Fig . 1 , G and I ) . In MIM knockdown cells , peak levels of pERK1 / 2 immunoreactivity remained unchanged , but the du - ration of the signal persisted twice as long as in control cells . In our short - term cell - based assays we noted no differences in proliferation , adhesion , or overall morphology compared with control - treated cells ( not depicted ) . We also found a marked abnormality in the ability of cells to directionally migrate in response to EGF ( Fig . 1 E ) . From these data we conclude that MIM functions to inhibit receptor - mediated endocytosis , resulting in altered downstream signaling and reduced direc - tional cell migration in vitro . DMIM is a novel Drosophila I - BAR protein Although MIM is one of the founding members of the I - BAR subfamily of lipid - binding BAR domain proteins , MIM func - tions have not yet been delineated in vivo , although studies have been conducted with IRSp53 knockout mice ( Kim et al . , 2009 ; Sawallisch et al . , 2009 ) . Because the BAR subfamilies are rep - resented in Dipterans , we chose to use Drosophila to better under - stand the in vivo roles of these important proteins . Drosophila MIM ( DMIM , GenBank / EMBL / DDBJ accession no . CG33558 ) shares strong identity in the I - BAR and WH2 domains with its vertebrate counterpart and ABBA , and less similarity with the I - BAR family member IRS53 ( Fig . S1 ; Scita et al . , 2008 ) . Previous structural studies have demonstrated that MIM binds and bends phospholipid - containing membranes ( Mattila et al . , 2007 ) . To determine whether DMIM retains similar properties , underlying cytoskeleton . In cultured mammalian cells , localized receptor - mediated endocytosis and receptor recycling amplifies the guidance signal to focally activate key regulators of the cyto - skeleton such as the GTPase Rac1 ( Palamidessi et al . , 2008 ) . Similar mechanisms appear to control Drosophila border cell migration ( Jékely et al . , 2005 ; Minina et al . , 2007 ) . Forward genetic screens for migration mutants have identified cue - specific components such as the nonreceptor tyrosine kinase Src , com - ponents of the endocytic machinery ( Jékely et al . , 2005 ; Palamidessi et al . , 2008 ) , and the CD2 - associated protein ( CD2AP ) / cortactin complex ( Lynch et al . , 2003 ; Somogyi and Rørth , 2004 ) . Each of these components has been shown to regulate endocytosis and cell migration , but little information exists about how they function to regulate directionality during migration . Increasing attention has been directed toward the Bin / Amphiphysin / Rvs ( BAR ) superfamily of proteins and their role in endocytosis and vesicle trafficking ( Dawson et al . , 2006 ; Frost et al . , 2007 ) . Each of the BAR domain subfamilies of cur - vature - dependent molecular scaffolds are thought to bring to - gether effector complexes to distinct lipid surface in order to regulate actin cytoskeletal remodeling near vesicles . For exam - ple , the BAR protein endophilin is critical for early vesicle scis - sion and EGF receptor signaling ( Kaneko et al . , 2005 ) , and isoforms have been associated with both tumor suppression and oncogenesis . Endophilin is recruited in a receptor - dependent manner through the formation of complexes with the Cbl - associated proteins CIN85 and CD2AP . The endophilin / CD2AP complex in turn mediates vesicle scission through the recruit - ment of cortactin and the actin - polymerization machinery ( Dikic , 2002 ; Lynch et al . , 2003 ; Kaksonen et al . , 2006 ) . The newest family of BAR domain proteins is the inverse or IMD BAR ( I - BAR ) family . IMD proteins are defined by the proteins Missing - In - Metastasis ( MIM ) and the insulin receptor substrate 53 ( IRSp53 ) cytoskeletal regulators ( Miki et al . , 2000 ; Lee et al . , 2002 ) . MIM was originally identified as a gene whose expression is down - regulated in a variety of urogenital meta - static cancers ( Lee et al . , 2002 ; Wang et al . , 2007 ) , but other studies have also demonstrated elevated MIM levels in many hedgehog - dependent tumors ( Callahan et al . , 2004 ) and meta - static endodermal tumors such as hepatocellular carcinomas ( Ma et al . , 2007 ) . Like many BAR family proteins , MIM contains several protein – protein interaction modules that sug - gest it functions to scaffold protein complexes at membranes ( Mattila et al . , 2003 ; Woodings et al . , 2003 ; Gonzalez - Quevedo et al . , 2005 ; Lin et al . , 2005 ) . Crystal structure analysis indi - cates that the shape of the IMD dimer is the most convex of the family members thus far , suggesting that the I - BAR family senses a very distinct class of membranes ( Lee et al . , 2007 ) . I - BAR family members have also been well studied as membrane - deforming proteins with the capacity to cause membrane tubu - lation and projections ( Suetsugu et al . , 2006 ; Mattila et al . , 2007 ; Saarikangas et al . , 2008 , 2009 ; Yang et al . , 2009 ) . Because each of the other BAR family members has roles in posi - tively regulating endocytosis , the convex shape of the I - BAR proteins is proposed to be involved in antagonizing endocytosis , although this supposition has not been directly tested ( Dawson et al . , 2006 ; Lee et al . , 2007 ) . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 3 of 15 Missing - in - metastasis regulates endocytosis during cell migration • Quinones et al . Figure 1 . Vertebrate MIM loss of function results in increased endocytosis and reduced directional migration . ( A and B ) Quantification of the ratio of internalized to surface - bound Transferrin ( A ) of EGF ( B ) in ptc  /  MEFs treated with indicated siRNAs . Data are represented as the mean ± SEM from three separate experiments ( * , P < 0 . 01 ; t test ) . ( C and D ) Quantification of the amount of recycled Transferrin ( C ) or EGF ( D ) in the medium of ptc  /  MEFs treated with indicated siRNAs . Data are represented as the mean ± SEM from three separate experiments . Inset in C shows immunoblots of protein level knockdown after treatment with indicated siRNAs . ( E ) MIM knockdown alters EGF - induced directional cell migration . Transwell migration assays showing alterations in the migration of ptc  /  MEFs through a permeable membrane in response to an EGF or PDGF gradient over the course of 8 h . Data are represented as the mean ± SEM from three separate experiments ( * , P < 0 . 01 ; t test ) . ( F ) EGFR levels in siRNA - treated cells show that si - MIM – treated cells do not display enhanced degradation of the EGF receptor over time . ( G ) Phospho - ERK1 and 2 levels in cells treated with control or MIM siRNA . MIM knockdown results in prolonged levels of pERK1 / 2 after the addition of EGF to the cell medium . ( H and I ) Quantification of immunoblots used for F and G . Data are represented as the mean ± SEM from three separate experiments ( * , P < 0 . 01 ; t test ) . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 JCB  of 15 partial female sterility ( Fig . S2 , F and G ) . dmim mutant adults appear phenotypically normal , but display defects in locomotor activity ( Fig . S2 , H and I ) . We also observed that dmim mutants display marked abnormalities in guided cell migration . Two cell types requiring precise cellular movement during development are the ovarian border cells and the primordial germ cells ( PGCs ) in the embryo . In the early embryo , PGCs respond to both maternal and zygotic cues from the mesoderm to trans - migrate across an epithelial layer toward the genital mesoderm ( Kunwar et al . , 2006 ) . dmim mutant PGCs fail to arrive at the mesodermal target despite apparently normal mesoderm as measured by Clift immunoreactivity ( unpublished data ) . Approximately 10 % of the PGCs arrest outside the embryo after germ band extension . Of those that enter , all are capable of transmigration , but only 20 % appropriately migrate toward the gonadal mesodermal attractants . The remaining dmim PGCs demonstrate a metastatic phenotype , with PGCs scattered throughout the posterior embryo ( Fig . 3 , A and B ) . In the ovary , border cells also perform a well - defined directional migration in response to oocyte - derived growth factor migratory cues ( Rørth , 2002 ; Montell , 2006 ) . The border cell cluster first migrates toward the posterior end of the egg cham - ber , and then dorsally as the migratory cue changes position . dmim mutant border cell clusters have normal numbers and we compared the lipid - binding properties of purified fly and human MIM I - BAR domains ( Fig . 2 , B and C ) . Both human and DMIM proteins bound to PI ( 4 , 5 ) P2 - containing vesicles , whereas the DMIM mutant I - BAR domain failed to bind to these vesicles . The DMIM I - BAR domain was also able to bind to PI ( 3 , 4 , 5 ) P3 - containing vesicles , whereas the human I - BAR was not . This binding was quite specific as mutation of the con - served lipid - binding motifs completely abrogated lipid binding ( Mattila et al . , 2007 ) . Further , fly or human MIM overexpres - sion in Drosophila S2 cells results in the same dramatic cyto - skeletal reorientation and extensions ( Fig . 2 D ) , indicating that DMIM functions similarly to its vertebrate orthologue . Migration defects in dmim mutants To understand the role of DMIM and other I - BAR family mem - bers , we generated a dmim - null mutant through homologous re - combination ( Fig . 2 A ) . We confirmed that the dmim mutant lacked the conserved I - BAR domain and much of the scaffold - ing domain ( Fig . S2 A ) and dmim / dmim phenotypes equaled those from dmim / deficiency mothers , arguing the dmim mutant is a strong loss - of - function mutant . The dmim locus produces a maternal transcript in the ovary , which is then distributed into the germ cells during early embryogenesis ( Fig . S2 , B – E ) . Consistent with this maternal expression , dmim mutants display Figure 2 . DMIM is the Drosophila homologue of human MIM . ( A ) The genomic structure of the dmim locus on chromosome 2R , 42C - 42E , showing the transcriptional start ( arrow ) and exons encoding the I - BAR , scaffolding , and WH2 domains . The black box above indicates the exons ( 3 – 10 ) deleted by homologous recombination to make dmim - null flies . ( B ) Native gel of hMIM , DMIM , or DMIM mutant ( 4KA ) I - BAR domains binding with lipids . Only when the I - BAR domain ( s ) bind to lipids will they run down the native gel . ( C ) hMIM , DMIM , or DMIM mutant ( 4KA ) I - BAR domain cosedimented with PI ( 4 , 5 ) P2 - or PI ( 3 , 4 , 5 ) P3 - rich ( 30 % ) large multilamellar vesicles . Human and fly MIM display similar specificity in binding for specific lipids . ( D ) Overexpressing human or Drosophila MIM in Drosophila S2 cells results in similar changes in the actin cytoskeleton . Overexpression of either protein results in the formation of actin - based protrusions from the cell membrane . Bar = 20 µm . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 5 of 15 Missing - in - metastasis regulates endocytosis during cell migration • Quinones et al . Figure 3 . dmim - null flies display abnormal cell migration . ( A ) Immunohistochemistry of Drosophila embryos at different developmental stages stained with anti - VASA antibody to highlight the progress of germ cell migration . dmim mutants display retained germ cells outside of the embryo at stage 9 and mislocalized germ cells in the mesoderm at stage 15 . Red arrowheads indicate the position of the primordial germ cells throughout each stage . Bar = 50 µm . ( B ) Quantification of germ cell migration from two different developmental stages where dmim mutants display an aberrant migration pattern . ( C ) dmim mutants are delayed in migration of the border cells in stage 10 egg chambers . The egg chambers are stained with phalloidin ( F - actin , red ) . Bar = 50 µm . ( D ) Expression of a UAS - DMIM - Myc transgene under the 306 - Gal4 driver shows expression of the construct in the border cells in both the dmim mutant and wild - type backgrounds . White arrowheads indicate the border cell cluster . Bar = 50 µm . ( E ) Quantification of border cell migration using the scale described in F ; more than 100 egg chambers were examined per genotype . ( F ) Schematic of a stage 10 egg chamber and scale used to score border cell migration defects . Anterior is to the left . ( G ) Border cell stained for pTyr ( green ) and F - actin ( red ) indicating the portion of the cell membrane where active signaling is occurring . Bar = 5 µm . ( H ) Quantification of the ratio of posterior to anterior pixel staining with the pTyr antibody . Initial polarized pTyr has been shown to be crucial for proper border cell migration ( Jékely et al . , 2005 ) . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 JCB 6 of 15 Figure 4 . dmim mutants show border cell migration defects and altered endocytosis . ( A ) Time - lapse series of confocal micrographs of early stage 9 border cell clusters expressing UAS - GMA under 306 - Gal4 in wild - type , dmim mutant , and dmim mutants expressing UAS - DMIM - Myc transgene in the border cells showing the migration of the border cell clusters over  3 h . White arrowheads indicate the border cell cluster and the dashed line indicates the anterior end of the oocyte ( the end point of border cell migration ) . Bar = 50 µm . ( B ) Quantification of the directionality index ( DI ) of the border cell cluster . The DI is calculated as ( A  B ) / ( A + B ) , where A is the number of forward extensions and B is the number of rearward extensions . Forward and rearward extensions are defined by which direction they point away from a vertical line on the dorsal ventral axis of the egg chamber . ( C ) Quantification of the migration speed of the border cell cluster , independent of the direction of migration . ( D ) Quantification of the average lifetime of each extension from the border cell cluster . All analyses were conducted using five separate movies for each genotype . ( * * , P < 0 . 001 ; * * * , P < 0 . 0001 ; t test ) . ( E ) Time - lapse on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010  of 15 Missing - in - metastasis regulates endocytosis during cell migration • Quinones et al . modes of border cell membrane dynamics : initial random ex - tensions independent of cue direction , posterior - directed move - ment characterized by long extensions toward the guidance cue source , and as the cluster comes closer to the guidance cue source , slower dorsal migration toward the oocyte nucleus . Border cells with constitutively active guidance receptors fail to detach from the anterior chamber and lack cell protrusions , whereas border cells with a lack of guidance receptor activity ( i . e . , those expressing dominant - negative receptors ) have nor - mal cytoplasmic extensions , but lack directional migration ( Prasad and Montell , 2007 ) . To examine how the dmim product effects border cell mi - gration , we imaged dmim mutant border cells expressing the actin - binding domain of moesin tagged with GFP ( GMA ) com - pared with isogenic wild - type controls ( Fig . 4 A ; Video 1 [ wt ] , Video 2 [ dmim mutant ] , Video 3 [ dmim ; DMIM + rescue ] ) . Using a directional migration index ( Prasad and Montell , 2007 ) , we found that wild - type border cells displayed marked direc - tionality toward the cue source , with a directionality index close to 1 . 0 . These data emphasize the marked asymmetry of exten - sions accompanying normal cluster migration . DMIM depletion impairs the directionality of the border cell cluster ( Fig . 4 B ) , and phenocopies border cell clusters lacking guidance receptors ( Prasad and Montell , 2007 ) . Further , we ascertained the dura - tion of the cytoplasmic extensions to determine whether dmim had affected the actin cytoskeleton itself . Measurement of the time to maximal extension or retraction in several independent movies clearly indicated no difference in extension lifetime , suggesting that dmim is not required for general actin dynamics in these cells in vivo ( Fig . 4 D ) . Because the I - BAR proteins have been shown , when overexpressed , to promote formation of small filopodial - like plasma membrane protrusions in migrating cells , we assayed the number of small cellular projections on the cell surface of individual border cells over time ( Fig . 4 E ) . Similar to our results with larger extensions , we observed no difference in the number of these fine projections ( Fig . 4 F ) . The dmim mutants also displayed reduced migration speeds inde - pendent of their direction , with dmim mutant border cells mov - ing much slower . The average speed for dmim mutants was 0 . 35 µm / min as compared with 0 . 65 µm / min displayed by wild - type clusters ( Fig . 4 C ) . Expression of DMIM back into the dmim mutant border cells ( dmim ; DMIM + ) rescues both the directionality index and migration speed phenotypes . Live - cell imaging data in vivo lead us to conclude that DMIM plays a role in properly sensing and responding to directional migratory cues in an otherwise normal migratory environment . DMIM inhibits endocytosis in border cells Our in vitro data suggest that MIM inhibition of endocytosis under - lies the inability to directionally sense guidance cues . To confirm size ( Fig . S3 C ) , but are significantly delayed in the initial phase of polarized movement compared with their wild - type counter - parts . By stage 10 , nearly half of the mutant border cells are still delayed , whereas greater than 95 % of wild - type clusters have traversed the egg chamber to the oocyte ( Fig . 3 , C and E ) . We focused further on dmim function in the border cells because of the ease of genetic manipulation and live imaging in this system . To determine whether the dmim product functions in migrating cells or in the surrounding environment , we ana - lyzed the structure of the dmim mutant ovary . The polarity , membrane , and cytoskeletal structure appeared to be unaffected as detailed by Fas2 , oskar , armadillo , DE - cadherin , and phalloi - din immunoreactivity ( Fig . S3 , A and B ) . Moreover , expression levels of the migratory cues such as gurken exhibited immuno - reactivity comparable to wild type , arguing that dmim mutants retain a normal migratory environment . Because the dmim locus sits immediately adjacent to recombination sites on the second chromosome , the generation of dmim mutants specifically in border cells was not possible . Rather , we aimed to demonstrate cell autonomy by rescuing the dmim phenotype through expres - sion of wild - type DMIM in the border cells . Expression of DMIM in border cells resulted in uniform distribution of DMIM protein at both the leading and lagging edges of the migrating border cells ( Fig . S3 D ) . This uniformly distributed expression restored the ability of the cluster to respond to its normal migra - tory cues ( Fig . 3 E ) . Further , we tested whether higher levels of DMIM expression altered border cell migration using two dif - ferent lines , the slbo - Gal4 and 306 - Gal4 drivers ( Fig . S3 D ) . Gain - of - function dmim border cells expressing elevated levels of DMIM protein displayed normal cell shape and border cell migration ( Fig . 3 D ) . Previous studies indicate that membrane response to directional guidance cues are polarized , as measured by phosphotyrosine ( pTyr ) staining at the leading edge . ( Rørth , 2002 , 2007 ; Jékely et al . , 2005 ) . dmim mutant border cells fail to correctly polarize , as assayed by nonlocalized pTyr staining around the border cell membrane ( Fig . 3 , G and H ) . These data suggest that DMIM acts in migrating border and PGC cells to facilitate the initial membrane polarity as the border cells begin to migrate . dmim mutant border cell migration phenocopies loss of guidance receptors The study of cell dynamics during migration both in vitro and in vivo has been hampered by the lack of a natural environment to study cell movement or the necessity for fixation to observe the migratory cells . Using recent technical developments that allow live imaging of migrating border cell clusters , we examined the role for dmim in border cell motility in a native environment ( Bianco et al . , 2007 ; Prasad and Montell , 2007 ; Tekotte et al . , 2007 ) . In vivo cell motility studies have observed three distinct series of confocal micrographs of individual border cells expressing UAS - GMA under 306 - Gal4 . Red arrowheads indicate tracking of a single cellular projection over time . Images were taken every 5 min , and the area shown is a magnification of the leading edge of a single border cell within a migrating cluster . ( F ) Quantification of the number of small surface projections in E . Data are represented as the mean ± SEM from 10 separate time series for each genotype . ( G ) Quantification of the amount of FM4 - 64 dye uptake over time for each indicated genotype . Data are represented as the mean ± SEM from five separate time series for each genotype . ( H ) Live confocal imaging of border cell clusters stained with the membrane - selective dye FM4 - 64 . Each time series shows the gradual uptake and increase of the dye at the membrane of the border cells . Bar = 5 µm . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 JCB 8 of 15 Figure 5 . Loss of dcortactin or cindr rescues dmim border cell migration defects . ( A ) GST cosedimentation assay using candidate GST proteins and S2 cell - derived DMIM protein . DMIM binds to itself , human MIM , and DCortactin . ( B ) Immunoprecipitation assay using an antibody to endogenous DCortac - tin and lysates from Myc - tagged DMIM constructs . FL ( full length DMIM ) ;  I - BAR ( lacking the IBAR domain ) ;  PRD ( lacking the polyproline - rich domain ) . ( C ) MIM or Cortactin knockdown alters EGF - induced directional cell migration . Transwell migration assays showing alterations in the migration of ptc  /  MEFs through a permeable membrane in response to an EGF or PDGF gradient over the course of 8 h . Data are represented as the mean ± SEM from three separate experiments ( * , P < 0 . 01 ; * * , P < 0 . 001 ; t test ) . ( D ) Schematic of a stage 10 egg chamber and scale used to score border cell migration defects . Anterior is to the left . ( E ) Confocal immunofluorescence images of egg chambers from wild - type , dmim , dcortactin , dmim ; dcortactin , cindr RNAi , and dmim / cindr RNAi double - mutant egg chambers stained with phalloidin ( F - actin , red ) . White arrowheads indicate the border cell cluster . Bar = 50 µm . ( F and H ) Quantification of stage 10 border cell migration for the indicated genotypes ; more than 100 egg chambers were examined per genotype . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 9 of 15 Missing - in - metastasis regulates endocytosis during cell migration • Quinones et al . We determined the functional relationship between DMIM and Dcortactin through the examination of border cell phe - notypes in double mutants . Previous analysis of dcortactin mutants identified a mild border cell migration phenotype ( Somogyi and Rørth , 2004 ) , a phenotype we confirmed ( Fig . 5 , E and F ) . Surprisingly , we found that dmim ; dcortactin double mutant border cells do not enhance the mutant phenotype , but rather display a wild - type phenotype . This suggests that DMIM acts antagonistically to Dcortactin to regulate directional cell migration in border cells . Because cortactin is recruited to en - dophilin via the adapter CD2AP ( Dikic , 2002 ; Soubeyran et al . , 2002 ; Lynch et al . , 2003 ) , if the CD2AP / cortactin complex functions antagonistically to MIM in guided migration , then CD2AP mutant border cells should phenocopy dcortactin mutants , and also rescue the dmim mutant border cell cluster migration phenotype . We assayed the effects of mutations in the single fly CD2AP gene , cindr , on border cell migration . Previ - ous studies have indicated that two cindr i lines have strong effects on photoreceptor cell morphology ( Johnson et al . , 2008 ) . We expressed these two independent cindr i lines in border cells , where they demonstrated a moderate border cell migration phe - notype , mimicking that of dcortactin mutants ( Fig . 5 , E and H ) . Like dcortactin , cindr i expression in a dmim mutant background partially restores the ability of border cell clusters to migrate , increasing the fraction of clusters that have migrated more than half the distance to the oocyte . We also found that the loss of dcortactin or cindr results in a mild reduction in the uptake of the FM4 - 64 dye in border cells ( Fig . 5 , G and I ) . As predicted , knockdown of dmim in combination with either dcortactin or cindr results in a partial rescue of the dmim increase in dye uptake . From these data , we conclude that the cindr / Dcortactin complex acts to promote endocytosis in opposition to that of DMIM . Our genetic data suggest guided cell migration is regu - lated in part from competition for cortactin between the pro - endocytic BAR domain complex endophilin / CD2AP , and the anti - endocytic BAR MIM . To confirm this model , we examined MIM antagonism of cortactin and other endocytic regulators in cultured vertebrate cells to determine whether double mutants would also restore normal receptor uptake in vitro . As predicted , whereas treatment with siRNA against cortactin decreases the levels of EGF receptor endocytosis , siRNA - mediated knock - down of both MIM and cortactin restores receptor uptake levels back to those in the control - treated cells ( Fig . 6 A ) . Consistent with our genetic data , combination knockdowns of MIM with CD2AP also restore EGF uptake levels to control values . We further tested whether MIM antagonism extended to other endocytic regulators . Knockdown of endophilin leads to a reduction in endocytosis with the combined knockdown of en - dophilin and MIM demonstrating a reproducible trend in eleva - tion of endocytosis over endophilin alone . Knockdown of Cbl , clathrin , or dynamin results in decreased endocytosis and is not our in vitro results , we examined border cell membrane dynamics using a live - cell lipophilic dye uptake assay , previously used to measure clathrin - dependent endocytosis rates during Drosophila synaptic reuptake ( Verstreken et al . , 2008 ) . We confirmed the dynamin dependence of dye uptake by showing dramatic reduc - tion of dye uptake in clusters expressing dominant - negative dynamin ( slbo - Gal4 ; shi K44A ) , or those treated with the dyna - min inhibitor Dynasore ( Georgiou et al . , 2008 ; Kirchhausen et al . , 2008 ; Fig . 4 , G and H ) . If MIM inhibits receptor - mediated endocytosis , we would expect increased dye uptake in border cells lacking DMIM . During live imaging the dye is taken up throughout the entire egg chamber , including the border cells , and continues to increase after dye addition . However , in dmim mutants , the rate of dye uptake compared with wild type was dramatically increased ( Fig . 4 G ) . This effect was not simply due to an increase in general membrane uptake or permeability , as the increased uptake was dynamin dependent and expression of the wild - type DMIM protein in mutant border cells partially rescued the uptake phenotype . The live - cell imaging data , in con - junction with dye uptake studies , support the idea that DMIM regulates guided cell migration through its ability to inhibit receptor - mediated endocytosis . MIM competes with endophilin / CD2AP for binding to cortactin BAR domain proteins function through regulating the assem - bly of protein complexes at membrane surfaces ( Ren et al . , 2006 ; Machesky and Johnston , 2007 ) . To understand mecha - nistically how dmim regulates border cell guidance sensing , we sought to discover which proteins , previously known to regulate border cell migration , interact with the scaffolding portion of DMIM . In GST pull - downs and coimmunoprecipi - tation assays , DMIM formed protein complexes with Dro - sophila cortactin ( Dcortactin ) , but not with Rac1 , Cdc42 , several RPTPs , and the BAR domain protein amphiphysin ( Fig . 5 A ) . Dcortactin is a major Src phosphorylation target downstream of growth factor signaling and is part of the pro - endocytic complex comprised of the BAR domain protein endophilin and its binding partner CD2AP . The endophilin / CD2AP / cortactin complex helps to provide force for endo - cytosis and scission of the early endosome by inducing local actin polymerization . Like their vertebrate counterparts , DMIM interacts with Dcortactin through the proline - rich domain ( Fig . 5 B ) . Both MIM and cortactin also affect the directional migration of vertebrate cells in culture . Cells treated with siRNA against either MIM or cortactin display a reduction in cell motility in response of EGF ( Fig . 5 C ) . Simultaneous knockdown of both MIM and cortactin results in normal cell motility , suggesting that these two proteins work antagonisti - cally . The loss of both proteins resulting in a wild - type pheno - type also suggests that there is redundancy with another set of proteins with a similar function . ( G and I ) Quantification of the amount of FM4 - 64 dye uptake over time for each indicated genotype . Data are represented as the mean ± SEM from three separate time series for each genotype . Knockdown of dmim in combination with either dcortactin or cindr is able to partially rescue the increase in dye uptake seen in the dmim mutant border cells . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 JCB 10 of 15 rescued in combination knockdowns with MIM , suggesting that MIM acts uniquely through CD2AP / cortactin ( Fig . 6 A ) . These results , along with previous studies , suggest that endophilin could be interacting with additional proteins when CD2AP and cortactin are unavailable during EGFR endocytosis ( Soubeyran et al . , 2002 ; Schmidt et al . , 2003 ; Kaneko et al . , 2005 ; Gareus et al . , 2006 ; Uezu et al . , 2007 ) . Because circular dorsal ruffles ( CDRs ) and cortactin have been shown to be an alternative endocytosis pathway for the in - ternalization of growth factor receptors , we investigated MIM and cortactin’s role in CDR formation ( Lynch et al . , 2003 ; Bompard et al . , 2005 ; Orth et al . , 2006 ; Machesky and Johnston , 2007 ; Teodorof et al . , 2009 ) . In vivo , we found no evidence for CDR formation in migrating border cells . In cultured cells ( Fig . S4 ) , we observed that the loss of MIM resulted in a reduction of the CDR formation in response to both PDGF - BB and EGF . However , in contrast to border cells , EGF endocytosis in our MIM and cortactin double mutant cells failed to rescue CDR formation . These results suggest that the effects seen in border cell migration are not a result of increased internaliza - tion of the growth factor receptors due to dorsal ruffle forma - tion ( Fig . S4 C ) . We examined the kinetics of cortactin association with guidance cue addition by performing cortactin immunoprecipi - tations upon the addition of EGF ligand . Under serum - free conditions , we find that cortactin associates with both endophilin / CD2AP and MIM complexes , but within 5 min after EGF addition , cortactin begins to disassociate from the CD2AP / endophilin complex ( Fig . 6 C ; Lynch et al . , 2003 ) . In the absence of MIM , cortactin fails to dissociate from CD2AP after EGF Figure 6 . MIM regulates endocytosis by competing with CD2AP for cortactin binding . ( A ) Quantification of the ratio of internalized to surface - bound EGF at 0 and 15 min in ptc  /  MEFs treated with the indicated siRNAs . MIM knockdown shows an increase in EGF uptake , whereas cortactin or CD2AP knockdown shows a decrease in EGF uptake after 15 min . The combination of siRNAs against both MIM and cortactin or MIM and CD2AP restores the phenotype to wild - type levels at 15 min . The phenotype is not restored by simultaneous knockdown with clathrin heavy chain , dynamin , cbl , or endophilin . Data are represented as the mean ± SEM from three separate experiments ( * , P < 0 . 01 ; t test ) . ( B ) Immunoblots indicating the level of protein knockdown after treatment with siRNAs for A . ( C ) Coimmunoprecipitations of CD2AP and endophilin with cortactin in si - GFP – or si - MIMtreated ptc  /  MEF lysate showing a decrease in the association of both proteins with cortactin after EGF stimulation . In the si - MIM – treated cells , a prolonged association of CD2AP and endophilin with cortactin is seen when compared with si - GFP – treated cells . ( D ) Direct competition between in vitro – translated CD2AP and bacterially expressed MIM for the SH3 domain of cortactin . MIM lacking the region that binds cortactin ( MIM 1 – 277 ) is unable to compete with CD2AP for cortactin binding . ( E ) Coimmunoprecipitations of GFP - tagged endophilin or GFP - tagged MIM with cortactin in NIH 3T3 cells treated with increasing concentrations of EGF ligand . The interaction between endophilin and cortactin quickly increases within 5 min after stimulation with EGF , and subsequently dissociates within 30 min . The interaction between MIM and cortactin increases within 5 min , but persists strongly even 30 min after stimulation when compared with endophilin . on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 11 of 15 Missing - in - metastasis regulates endocytosis during cell migration • Quinones et al . but dmim mutants display a wild - type number of border cells . This discrepancy could be explained in part due to the obser - vation that MIM associates with vertebrate Suppressor of Fused ( Callahan et al . , 2004 ) , which is redundant in flies . The data presented here uncover migratory defects in PGCs , border cells , and vertebrate cultured fibroblasts , all responding to different migratory cues . This suggests that although cells use different cues and receptors for migration in a variety of systems , the regulation of this process at the level of endocytosis appears to be shared . Our studies identify DMIM as novel I - BAR protein , and one of the first negative regulators of endocytosis with a role in guided cell migration . Genetic , cell biological , and biochemical data support the model that DMIM and CD2AP compete for cortactin in regulating receptor - mediated endocytosis . The observation that removing both MIM and cindr / cortactin results in wild - type migration suggests that MIM and cortactin consti - tute one of several redundant regulatory systems to control the directional migration of the border cells . Because removal of both proteins restores normal border cell migration but disrup - tion of clathrin and dynamin function does not , we speculate that other combinations of pro - and anti - endocytosis complexes downstream of dynamin must be operating to balance this pro - cess in migrating cells . Although we see a trend in increased en - docytosis with MIM / endophilin double knockdown , the lack of complete rescue further suggests endophilin possesses MIM - independent endocytosis functions . Current studies are ongoing to identify these additional signaling pathways in the sensi - tized dmim ; dcortactin background . More importantly , our data show the dramatic effects on migration when components of the steering mechanism are missing or out of balance . Similar effects have also been seen with gross overexpression of cortac - tin ( Timpson et al . , 2005 ) and may explain the relatively high frequency of cortactin and MIM alterations in late - stage cancers ( Patel et al . , 1996 ; Lee et al . , 2002 ; Timpson et al . , 2007 ; Hofman et al . , 2008 ) . These results provide a new mechanistic understanding of BAR domain function by showing that directional sensing comes in part from protein complexes competing for common effector proteins during endocytosis . These data support the notion that MIM acts to dampen guidance receptor signaling at a variety of ligand concentrations by sequestering cortactin . Guidance cue binding assembles the N - BAR subfamily mem - ber endophilin and its adapter CD2AP , which binds cortactin , shifting it away from MIM sequestration . We postulate that in - creased endocytosis and MIM’s persistent binding of cortactin prevent the cell from improperly sensing guidance cues and misinterpreting directional differences . Previous studies sug - gest that phosphorylation of cortactin modulates its interaction with a number of proteins ( Zhu et al . , 2007 ) ; however , we do not detect such alteration using phosphospecific cortactin anti - bodies in our system ( unpublished data ) . Consistent with this data are the lack of localized bulk MIM protein at the leading edge of cultured cells or rescued dmim ; DMIM + border cells . Altogether , these data suggest that there is a novel MIM - dependent steering mechanism that guides cell migration through interactions with other protein complexes . Jékely et al . ( 2005 ) addition , resulting in an increase of the amount and duration of endophilin / CD2AP in complex with cortactin ( Fig . 6 C ) . The duration of cortactin association mirrors that of the persistent pERK1 / 2 immunoreactivity ( Fig . 1 , H and J ) . Our work and other studies indicate that both CD2AP and MIM associate through their proline - rich domains with cortactin’s SH3 domain ( Fig . 5 B ; Lynch et al . , 2003 ; Lin et al . , 2005 ) . Indeed , in vitro – translated CD2AP readily associates with the purified SH3 do - main of cortactin . Increasing concentrations of purified MIM abrogate the binding of cortactin to CD2AP ( Fig . 6 D ) , support - ing a direct competition between MIM and CD2AP for cortac - tin binding . MIM lacking its proline - rich domain ( MIM 1 – 277 ) fails to compete with CD2AP , providing additional support for the idea that MIM antagonizes the ability of cortactin to associ - ate with the pro - endocytic CD2AP / endophilin complex . We then used stable GFP - tagged MIM and endophilin lines to ex - amine cortactin association with each of the proteins at different EGF ligand concentrations over time . The antagonism between MIM and CD2AP / endophilin for cortactin binding could be ex - plained by the prolonged association between cortactin and MIM , which persists longer after stimulation with EGF ligand than the association between endophilin and cortactin , even at different ligand concentrations ( Fig . 6 E ) . Discussion We have shown through genetic interaction and live - cell imag - ing that migrating cells use a MIM - dependent steering mecha - nism to interpret local migratory signals . MIM’s role appears to be general , as both border cell and PGC migration are affected in dmim mutants and involve different cell types responding to different guidance cues . Our data indicate that MIM inhibits guidance receptor endocytosis by competing directly with CD2AP for cortactin , resulting in dampened guidance receptor signaling . This study provides the first genetic and biochemical evidence for the function of a member of the I - BAR family of proteins in directed cell migration , and provides a mechanistic link between MIM and cell migration . Directional cell migration is a complex process requiring dynamic rearrangements of the cytoskeleton and precise direc - tional sensing of local migratory cues ( Prasad and Montell , 2007 ) . Our live - cell imaging data suggest that DMIM is involved in directing cell migration through the inhibition of endocytosis . Although previous studies demonstrate that MIM is an actin cytoskeletal remodeling protein ( Gonzalez - Quevedo et al . , 2005 ; Lee et al . , 2007 ; Mattila et al . , 2007 ) , our imaging studies argue against a major , direct role for MIM in general actin polymerization . Consistent with this notion is the lack of apparent defects in adherens junctions , the actin cytoskeleton , or anteroposterior polarity in dmim mutant egg chambers . This is not to say that MIM does not affect the actin cytoskeleton in other cases of cell migration , just that in the case of Drosophila border cell migration the function of DMIM is not required for actin cytoskeletal dynamics . Previous studies have also implicated MIM in regulating Sonic Hedgehog signaling ( Callahan et al . , 2004 ) . Mutations in the hedgehog pathway component costal2 result in aberrant border cell numbers ( Liu and Montell , 1999 ) , on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 JCB 12 of 15 North Carolina , Chapel Hill , NC ) . The primers used were , 5  - CCC - GGGAGCGAGTGAGGACGCGCAAATGG - 3  and 5  - GGTACCGAC - CAGTTCCTGGGCCACAGAC - 3  to amplify the 5  region and 5  - AGATC - TAGTCAGGTGAGAGTAGGAAATC - 3  and 5  - GAATTCCTGCCACAC - GCTCCCATTCCATATCC - 3  to amplify the 3  region . Mutant chromosomes were out crossed to yw flies for five generations to minimize linked enhanc - ers and suppressors . DMIM rescue lines were made by cloning the dmim cDNA into the pUAST or pUASp vectors with a myc - his epitope tag on the C terminus . Transgenic lines were generated using standard methods . Transgenic lines were crossed into the 306 - Gal4 ( Bloomington Drosophila Stock Center , Bloomington , IN ) for expression in the border cells . The slbo - Gal4 and shi K44A lines were obtained from the Bloomington Drosophila Stock Center . UAS - cindr - IR 2 . 21A + 2 . 23A ( cindr IR 2 ) targets the long and inter - mediate cindr transcripts and UAS - cindr - IR 3 . 73B + 3 . 81A ( cindr IR 3 ) targets all three transcripts , as described previously ( Johnson et al . , 2008 ) . The Cortactin M7 line has been described previously ( Somogyi and Rørth , 2004 ) . In vitro migration assays For directional cell migration assays in vitro , cells were seeded into the upper compartment of a Transwell 12 - well plate ( Costar 3403 ; Corning ) in serum - free medium after 16 h of serum starvation . The lower compartment of each well contained serum - free medium with or without the indicated growth factor . Cells were allowed to migrate for 8 h , then the medium was removed and the cells were fixed with 4 % paraformaldehyde . Fixed cells were stained with 0 . 5 % crystal violet and the cells in the upper compart - ment were scraped off of the insert membrane . The well was then exten - sively washed with water and allowed to air dry . Only cells that migrated down through the porous membrane remained . The crystal violet remaining in these cells was then extracted with 30 % acetic acid and assessed by measuring the absorbance at 590 nm . Lipid vesicle cosedimentation assays 6XHis fusion proteins of DMIM I - BAR ( aa 1 – 477 ) , hMIM I - BAR ( aa 1 – 255 ) , and DMIM 4KA I - BAR were cloned by standard techniques in pQE80L vector , expressed in bacteria and purified on Superflow Nickel Beads ( QIAGEN ) . DMIM 4KA contained mutations K139A , K140A , K149A , and K150A gen - erated by QuikChange Mutagenesis ( Stratagene ) . PI ( 4 , 5 ) P 2 ( l -  - phosphati - dylinositol - 4 , 5 - bisphosphate ; porcine brain , tri - ammonium salt ) , PI ( 3 , 4 , 5 ) P 3 ( 1 , 2 - dileoyl - sn - glycero - 3 - [ phosphoinositol - 3 , 4 , 5 - trisphosphate ] , tetra - ammonium salt ) , phosphatidylserine ( PS ; brain ) , phosphatidylcholine ( PC ; brain ) , and phosphoethanolamine ( PE ) were purchased from Avanti Polar Lipids , Inc . Fusion proteins were diluted to desired concentrations in F - buffer ( 10 mM im - idazole , pH 7 . 5 , 0 . 2 mM CaCl 2 , 0 . 2 mM ATP , 1 mM DTT , 100 mM KCl , 2 mM MgCl 2 , and 1 mM EGTA ) . For vesicle cosedimentation assays the following mixtures were prepared : 5 % PE , 20 % PS , 45 – 75 % PC , and 0 – 30 % PI ( 4 , 5 ) P 2 or PI ( 3 , 4 , 5 ) P 3 . Samples were vacuum dried under N 2 and hydrated for a minimum of 4 h in 0 . 2 mM Hepes - KOH , pH 7 . 5 , and 100 mM NaCl to a 1 - mM total lipid concentration . Large multilamellar vesicles were formed by repeated freeze / thaw cycles between liquid nitrogen and a hot water bath . Vesicles were also vortexed before each experiment . I - BAR constructs were diluted in desired concentrations in F - buffer and sedimented at 360 , 000 g for 30 min at room temperature . For native gel electropho - resis , fusion proteins were diluted to desired concentration in F - buffer . Lipid / protein reactions were incubated at room temperature for 1 h , run on 12 % native PAGE gels , stained with Coomassie , and scanned . Drosophila egg chamber immunofluorescence For confocal immunofluorescence , ovaries from 1 – 2 - d - old females were dissected in PBS , fixed , and permeabilized in 4 % paraformaldehyde and PBSX ( 0 . 1 % Triton X - 100 ) for 20 min at room temperature and subsequently washed in PBSX , blocked in 5 % normal horse serum ( Vector Laboratories ) , and then incubated overnight in primary antibody at 4°C . The primary antibodies used were : mouse anti - armadillo ( 1 : 200 ; Developmental Studies Hybridoma Bank [ DSHB ] , Iowa City , IA ) , mouse anti - Myc ( 1 : 100 ; Sigma - Aldrich ) , phalloidin - AF633 ( 1 : 100 ; Invitrogen ) , mouse anti - DE cadherin ( 1 : 250 ; DSHB ) , mouse anti - oskar ( 1 : 250 ; DSHB ) , mouse anti - lectin FITC ( 1 : 250 ; Vector Laboratories ) , mouse anti - FasIII ( 1 : 500 ; DSHB ) , mouse anti - gurken ( 1 : 100 ; DSHB ) , rabbit anti - DCortactin ( 1 : 250 ; a gift from Dr . Togashi , Tohoku University , Sendai , Japan ) , mouse anti - GFP ( 1 : 250 ; Sigma - Aldrich ) , and phalloidin - TRITC ( 1 : 100 ; Sigma - Aldrich ) . Alexa Fluor – conjugated anti - bodies ( Invitrogen ) were used as secondaries . Drosophila egg chamber live imaging For live confocal imaging , egg chambers from ovaries were prepared ac - cording to previous protocols ( Bianco et al . , 2007 ; Prasad and Montell , 2007 ; Tekotte et al . , 2007 ) with some modification . Egg chambers were demonstrated the importance for regulating both polarity and a localized response to external stimuli during the migration of the border cell cluster . Their studies focused on regulation of receptor tyrosine kinase signaling through the action of key pro - teins involved in the endocytosis of the receptor . We have fo - cused our studies on a negative regulator of endocytosis , which in the Drosophila border cell cluster regulates both polarity and a local response to guidance cues as a means of mediating direc - tional migration . Materials and methods Transferrin internalization and recycling Ptc1 - null mouse embryonic fibroblasts ( MEFs ; Taipale et al . , 2000 ) were used throughout these studies due to the increased levels of MIM protein expressed when compared with NIH 3T3 cells , as well as the ease of MIM knockdown at the protein level in these cells . Ptc1 - null MEFs were nucleo - fected with the appropriate siRNA 2 d before the endocytosis assays , and were serum starved overnight before the assays . Endocytosis was mea - sured using HRP - conjugated mouse Transferrin ( Tfn ; Sigma - Aldrich ) and detected using a Sensolyte ADHP HRP ELISA Assay kit ( AnaSpec ) . To mea - sure the amount of internalized HRP - Tfn , cells were serum starved over - night , the HRP - Tfn ( 100 µg / ml ) in serum - free DME was added to each well , and the plate was placed at 37°C for the indicated time ( s ) . Endocytosis was stopped by placing the plate on ice , washing with ice - cold PBS , pH 5 . 0 , for 2 min each , and then 4 times with PBS , pH 7 . 0 . The cells were then lysed in 20 mM Hepes , pH 7 . 9 , 150 mM NaCl , and 1 % NP - 40 for 10 min on ice . The amount of HRP - Tfn in the lysate was then measured following the Sensolyte kit protocol . To measure recycling of HRP - Tfn , cells were treated as per the above protocol . After stripping the unbound ligand from the cell surface , fresh serum - free medium was placed on the cells . Medium was then collected from the wells at the indicated time points and assayed for HRP - Tfn content following the Sensolyte kit protocol . EGF stimulation , internalization , and recycling For the EGF stimulation , Ptc1 - null MEFs were grown for 2 d after nucleofec - tion and then serum starved overnight . Cells were stimulated with EGF at 100 ng / ml ( Sigma - Aldrich ) in serum - free DME for the indicated times , washed twice with PBS , and lysed . Lysates were loaded onto SDS - PAGE gels for Western blot analysis . The primary antibodies used were : rabbit anti - cortactin ( 1 : 1 , 000 ; Millipore ) , mouse anti –  - actin ( 1 : 10 , 000 ) , rabbit anti - MIM ( 1 : 500 ) , mouse anti - CD2AP ( 1 : 500 ; Santa Cruz Biotechnology , Inc . ) , mouse anti - endophilin ( 1 : 1 , 000 ; Abcam ) , rabbit anti - dynamin ( 1 : 1 , 000 ; Abcam ) , rabbit anti - clathrin heavy chain ( 1 : 1 , 000 ; Abcam ) , and goat anti - CBL ( 1 : 1 , 000 ; Abcam ) . For 125 I - EGF assays , ptc1 - null cells were grown for 2 d after nucleo - fection and then serum starved overnight . After serum starvation , the cells were stimulated with EGF ( 100 ng / ml ) in serum - free DME for the indicated times . Radiolabeled EGF at 1 ng / ml , ( NEX1600 ; PerkinElmer ) was com - bined with unlabeled EGF to a final concentration of 100 ng / ml . For inter - nalization assays , unbound ligand was removed by washing three times with ice - cold buffer containing : 20 mM Hepes , 130 mM NaCl , 5 mM KCl , 0 . 5 mM MgCl 2 , 1 mM CaCl 2 , and 1 mg / ml polyvinylpyrolidione , pH 7 . 4 . Surface - bound ligand was then collected in ice - cold acid strip buffer ( 50 mM glycine - HCl , 100 mM NaCl , and 1 mg / ml polyvinylpyrolidione , pH 3 . 0 ) for 2 min . Internalized ligand was then released with 5 N NaOH for 30 min , added to reducing sample buffer , and analyzed with a scintillation counter . For recycling assays , unbound ligand was removed as described above , then fresh serum - free medium was added to the cells . The cells were placed at 37°C for the indicated times . At each time point , medium was removed from the well and assayed for EGF content . Results were normalized to the initial amount of EGF in the medium at time 0 . All siRNAs were ordered from Thermo Fisher Scientific ; si - MIM : 5  - GCAAGGCACUCAUCGAA - GAUU - 3  , ON - TARGETplus SMARTpool siRNAs were used for : Cbl , CD2AP , clathrin , cortactin , dynamin , and endophilin . Fly genetics The dmim mutant was made using a modified version of “ends - out” homol - ogous recombination ( Gong and Golic , 2003 ) . The donor vector was con - structed by cloning PCR products of the homologous regions in the dmim genomic region upstream and downstream of the deleted exons ( exons 3 – 10 ) into the pP [ EndsOut2 ] vector ( a gift from Dr . Jeff Sekelsky , University of on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 13 of 15 Missing - in - metastasis regulates endocytosis during cell migration • Quinones et al . ( Promega ) . GST beads containing the SH3 domain of cortactin were incu - bated with equal amounts of CD2AP and increasing amounts of bacterially expressed full - length 6X - His MIM protein or 6X - His MIM lacking the C - terminal proline - rich domain . The beads were washed with PBST and run on SDS - PAGE gels for Western blot analysis using rabbit anti - CD2AP ( 1 : 500 ; Cell Signaling Technology ) . Online supplemental material Fig . S1 shows ClustalW2 alignment of the I - BAR and WH2 domain of Drosophila , human , and mouse MIM as well as mouse ABBA and mouse IRSp53 . Fig . S2 shows eclosion and hatching frequency deficiencies in dmim mutants as well as locomotor defects in the adults . Fig . S3 shows a comparison of staining patterns of cell – cell junctions , membranes , growth factor gradients , and the actin cytoskeleton between wild - type and dmim mutant egg chambers . Fig . S4 shows the effect of MIM knockdown on the formation of circular dorsal ruffles in vertebrate cell culture . Video 1 shows wild - type border cells migrating normally toward the oocyte ( right ) . Video 2 shows dmim mutant border cells fail to migrate toward the oocyte ( right ) . Video 3 shows dmim / DMIM + border cells migrating normally toward the oocyte ( right ) . Online supplemental material is available at http : / / www . jcb . org / cgi / content / full / jcb . 200910136 / DC1 . We wish to thank P . Rorth , J . Sekelsky , and D . Kiehart for flies and plasmids ; R . Johnson and R . Cagan for the cindr RNAi fly lines ; P . Khavari , H . Chang , and S . Pfeffer for comments on the work ; M . Bershteyn for MIM siRNA sequences ; and L . Edgington and A . Bailey for technical support . This work was supported by NSF predoctoral fellowships to G . A . Quinones and J . Jin , and an NIH / NIAMS grant to A . E . Oro ( R01 AR052785 ) . Submitted : 23 October 2009 Accepted : 19 March 2010 References Bianco , A . , M . Poukkula , A . Cliffe , J . Mathieu , C . M . Luque , T . A . 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Differentiation . 70 : 181 – 203 . doi : 10 . 1046 / j . 1432 - 0436 . 2002 . 700408 . x placed in Petri dishes with glass coverslip bottoms ( MatTek ) , surrounded by halocarbon oil 700 ( Sigma - Aldrich ) and covered by the gas - permeable membrane from a Petriperm plate ( Grenier Bio ) . Females were taken 1 – 3 d after eclosion and fed fresh yeast 1 d before dissection . Dissections were performed in Schneider’s Insect medium ( Invitrogen ) supplemented with 15 % fetal bovine serum ( Sigma - Aldrich ) , 0 . 20 mg / ml  1 insulin ( Sigma - Aldrich ) , 0 . 6x penicillin / streptomycin ( Invitrogen ) , and 3 mM FM4 - 64 ( Invitrogen ) . Ovaries were removed , washed in dissection medium , and individual ovarioles were removed from the muscle sheath using fine dis - section forceps . Imaging was performed using a microscope ( LSM - 510 Meta ; Carl Zeiss , Inc . ) with a 340 1 . 3 NA Plan NeoFluor oil immersion objective . Two channels were acquired simultaneously : GFP ( 488 - nm laser and 500 – 550 - nm band - pass filter ) and FM4 - 64 ( 488 - nm laser and 560 - nm long - pass filter ) . 7 sections were taken 5 µm apart with 5 min between stacks . The three to four sections covering the migrating cluster were merged for each time point using the Volocity 4 imaging software package ( PerkinElmer ) . Videos were made using Volocity 4 , and exported as Quick - Time ( . mov ) files at 5 frames per second . Egg chambers were chosen in which border cells had clearly delaminated or were already migrating . For dye uptake assays , egg chambers were incubated in 9 µM FM4 - 64 dye in imaging medium for exactly 30 min before the first image was taken . For Dynasore ( Sigma - Aldrich ) inhibition of dynamin in the egg chambers , dis - sected egg chambers were incubated in imaging medium containing 80 µM Dynasore for 1 h before the addition of FM4 - 64 . Live imaging calculations The directionality index ( DI ) for large cellular extensions emanating from a border cell is calculated as ( A  B ) / ( A + B ) , where A is the number of for - ward extensions and B is the number of rearward extensions . Forward and rearward extensions are defined by which direction they point away from a vertical line on the dorsal ventral axis of the egg chamber . For the pur - poses of this study , cell extensions are defined as transient , large cellular processes that are greater than one quarter of a border cell diameter in width at their base , and extend outward from the cell surface . Cellular filopodial - like projections are defined as cellular processes that are transient , less than one quarter of a cell diameter in width at the base , and extend from the cell surface . Tracking of cellular extensions / projections was per - formed using the tracking module of the Volocity program . Each individual process was manually marked and tracked throughout each frame to cal - culate the amount of time each process persisted . The number of projec - tions was calculated by counting the number of projections along a 10 - µm length of cell surface along a border cell . We are limited in the types of projection studies as our analyses cannot account for projections that form and dissociate faster than the 5 - min time points collected . For FM4 - 64 dye uptake assays , images were taken over time of the border cell cluster and analyzed using ImageJ . A line drawn following the outside of the mem - brane of a single border cell was used to select the area from which the in - tensity measurements were calculated . The intensity measurement was then divided by the area selected to control for normal variations in border cell shapes and sizes . These analyses were all normalized to the overall inten - sity of the entire egg chamber to account for differences in uptake through - out the egg chamber itself . Measurements from single border cells within the same cluster were combined with measurements from cells of a different cluster for both technical and biological replicates within the analyses . A mutant dynamin ( shi K44A ) and a small molecule inhibitor of dynamin were included as negative controls to show that dye uptake is dependent on clathrin - mediated endocytosis , and not just accumulation of the dye in the membrane over time . GST pull - downs and coimmunoprecipitations For GST pull - downs , cells were lysed in PBS with 1 % NP - 40 , protease inhibitors , 1 mM Na 3 VO 4 , and 5 mM NaF for 30 min at 4°C . For coim - munoprecipitations , cell lysates were generated using RIPA buffer ( 50 mM Tris - HCl , pH 8 . 0 , 150 mM NaCl , 5 mM EDTA , 1 % NP - 40 , 0 . 1 % SDS , 10 % glycerol , 0 . 5 % Na deoxycholate , 1 mM Na 3 VO 4 , and protease inhibitors ) and precleared with Protein G PLUS - Agarose beads ( Santa Cruz Biotechnology , Inc . ) for 30 min at 4°C . The primary antibodies used were : rabbit anti - cortactin ( 1 : 1 , 000 ; Millipore ) , rabbit anti - MIM ( 1 : 500 ) , mouse anti - CD2AP ( 1 : 500 ; Santa Cruz Biotechnology , Inc . ) , goat anti - endophilin I ( 1 : 500 ; Santa Cruz Biotechnology , Inc . ) , mouse anti - Myc ( 1 : 500 ; Sigma - Aldrich ) , and rabbit anti - Myc ( 1 : 1 , 000 ; Santa Cruz Bio - technology , Inc . ) . Direct binding and competition assays The coding sequence of CD2AP was cloned into pcDNA3 . 1 MHA . 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M701997200 on A p r il 5 , 2017 D o w n l oaded f r o m Published April 12 , 2010 \ No newline at end of file diff --git a/silver_data/d25c013ba4cccfd2fd27a44fe3d2dd21749450b8.pdf.txt b/silver_data/d25c013ba4cccfd2fd27a44fe3d2dd21749450b8.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..262232e4604c9c31c2034f927f56ac71efa405d3 --- /dev/null +++ b/silver_data/d25c013ba4cccfd2fd27a44fe3d2dd21749450b8.pdf.txt @@ -0,0 +1 @@ +UC Berkeley UC Berkeley Previously Published Works Title Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos Permalink https : / / escholarship . org / uc / item / 09n4v2k6 Journal Proceedings of the National Academy of Sciences of the United States of America , 111 ( 29 ) ISSN 0027 - 8424 Authors Bothma , JP Garcia , HG Esposito , E et al . Publication Date 2014 - 07 - 22 DOI 10 . 1073 / pnas . 1410022111 Peer reviewed eScholarship . org Powered by the California Digital Library University of California Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos Jacques P . Bothma a , 1 , Hernan G . Garcia b , 1 , Emilia Esposito a , Gavin Schlissel a , Thomas Gregor b , c , 2 , and Michael Levine a , 2 a Department of Molecular and Cell Biology , University of California , Berkeley , CA 94720 ; and b Joseph Henry Laboratories of Physics and c Lewis - Sigler Institute for Integrative Genomics , Princeton University , Princeton , NJ 08544 Contributed by Michael Levine , May 30 , 2014 ( sent for review April 9 , 2014 ; reviewed by William McGinnis and Scott Barolo ) We present the use of recently developed live imaging methods to examine the dynamic regulation of even - skipped ( eve ) stripe 2 expression in the precellular Drosophila embryo . Nascent tran - scripts were visualized via MS2 RNA stem loops . The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcrip - tion , beginning with a broad domain of expression during nuclear cycle 12 ( nc12 ) , and progressive refinement during nc13 and nc14 . The mature stripe 2 pattern is surprisingly transient , constituting just ∼ 15 min of the ∼ 90 - min period of expression . Nonetheless , this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods . Analysis of individual transcription foci reveals intermit - tent bursts of de novo transcription , with duration cycles of 4 – 10 min . We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development . T he Drosophila even - skipped ( eve ) stripe 2 enhancer is one of the best - characterized cis - regulatory DNAs in animal development ( 1 ) . A combination of genetic analyses , DNA binding assays , and site - directed mutagenesis led to a detailed model for the regulation of stripe 2 , whereby the maternal Bicoid gradient , in concert with zygotic Hunchback protein , defines a broad domain of activation in the anterior half of the embryo ( 2 – 5 ) . Localized gap repressors , Giant in anterior regions and Kruppel in central regions , establish the anterior and posterior stripe borders , respectively ( summarized in Fig . 1 A ) . Most of our information regarding the regulation of the stripe 2 expression pattern is derived from the analysis of fixed prep - arations of staged embryos ( 2 , 5 , 6 ) . Here , we use a newly developed live - imaging technique ( 7 , 8 ) to explore the detailed temporal dynamics of the eve stripe 2 expression pattern in living embryos . Multiple copies of an MS2 stem loop sequence were inserted into the 5 ′ - UTR of a yellow reporter transgene ( Fig . 1 B ) . The loops form upon transcription by RNA polymerase II ( Pol II ) and are bound by a maternally provided MS2 coat protein fused to GFP ( MCP - GFP ) ( 9 – 14 ) . As a result , fluorescence signals are detected at sites of Pol II elongation and de novo transcription , and the strength of the signals are proportional to the number of elongating Pol II complexes ( 7 ) . This method was recently used to examine the activation of the proximal hunchback enhancer by the Bicoid gradient in the anterior half of the precellular embryo ( 7 , 8 ) . Diminishing levels of Bicoid were shown to cause stochastic on / off transcription of the hunchback > MS2 transgene at the posterior limits of the Hunchback expression pattern . This observation suggests that the Bicoid activator not only augments the levels of transcription but also increases the probability that a given cell within a pop - ulation will initiate expression ( 15 ) . The regulation of the hunchback > MS2 transgene is rather static . Once activated by Bicoid during nuclear division cycle 10 ( nc10 ) ( 16 ) , the spatial features of the pattern remain essentially constant for the next hour until transcription is lost at the mid - point of nc14 ( 7 , 17 ) . In contrast , the eve stripe 2 pattern is highly dynamic , with broad activation during nc11 and nc12 , followed by progressive refinement during nc13 and nc14 ( 4 ) . These regulatory dynamics are nicely captured by the MS2 detection system and reveal surprisingly transient expression of the mature stripe ( Fig . 1 D and Movie S1 ) . We also present evidence for the occurrence of sporadic transcriptional bursts , with fluctuation cycles of 4 – 10 min . We discuss the possibility that these dis - continuities in de novo transcription facilitate the dynamic reg - ulation of eve stripe 2 expression by the localized Giant and Kruppel repressors . Results The first 1 . 7 kb of the eve 5 ′ flanking region was attached to a yellow reporter gene containing 24 tandem repeats of the 60 - to 70 - bp MS2 stem loop motif [ summarized in Fig . 1 B ( 18 ) ] . The eve > MS2 fusion gene contains the “ full - length ” 720 - bp eve stripe 2 enhancer , located between – 1 . 5 kb and – 800 bp upstream of the eve transcription start ( 4 , 19 ) . It also contains dispersed regulatory sequences that mediate weak expression within the limits of stripe 7 ( Fig . 1 C ) . Conventional in situ hybridization assays identify authentic stripe 2 and stripe 7 expression patterns , as seen for similar eve reporter genes lacking MS2 stem loop sequences ( e . g . , refs . 20 and 21 ) , confirming that the presence of the stem loops does not significantly affect the output pattern of expression . Dynamics of Stripe Formation . The eve > MS2 transgene was introduced into embryos containing a maternally expressed MCP - GFP fusion protein , as described in ref . 7 . Sites of de novo transcription were imaged by sampling a series of confocal z Significance There is considerable information about the spatial regula - tion of gene expression during pattern formation in animal development . Significantly less is known about temporal con - trol , in part due to our inability to analyze gene activity in real time . Using a recently developed approach for the visualization of gene expression in living Drosophila embryos , we examined the well - known even - skipped stripe 2 expression pattern . Sur - prisingly , we observe that this classic pattern is quite transient and generated by discontinuous surges of transcriptional ac - tivity in individual cells . These results challenge a purely static framework for dissecting developmental programs and emphasize the importance of the dynamic features of pattern formation . Author contributions : J . P . B . , H . G . G . , E . E . , G . S . , T . G . , and M . L . designed research , per - formed research , contributed new reagents / analytic tools , analyzed data , and wrote the paper . Reviewers : W . M . , University of California , San Diego ; and S . B . , University of Michigan Medical School . The authors declare no conflict of interest . Freely available online through the PNAS open access option . 1 J . P . B . and H . G . G . contributed equally to this work . 2 To whom correspondence may be addressed . Email : mlevine @ berkeley . edu or tg2 @ princeton . edu . Thisarticlecontainssupportinginformationonlineatwww . pnas . org / lookup / suppl / doi : 10 . 1073 / pnas . 1410022111 / - / DCSupplemental . 10598 – 10603 | PNAS | July 22 , 2014 | vol . 111 | no . 29 www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1410022111 sections through the entirety of cortical nuclei . The fluorescence intensities of these expression puncta is a proxy for the number of Pol II molecules actively transcribing the reporter gene and hence is an instantaneous measure of activity ( 7 ) . A broad spatial domain of transcriptional activity is detected during nc11 , nc12 , and nc13 ( e . g . , Fig . 1 D , a – c and Movies S1 – S3 ) . The stripe is gradually refined during nc14 ( Fig . 1 D , d and e ) , and ultimately disappears before the onset of gastrulation ( Fig . 1 D , f ; see below ) . The dynamics of the stripe 2 expression pattern , broad activation followed by localized repression , is consis - tent with previous studies of fixed embryos ( e . g . , ref . 4 ) . As observed in classical studies ( e . g . , ref . 22 ) , there is no evi - dence of transcription during mitosis ( e . g . , Fig . 1 D , a ) . There is a marked restriction in the posterior limits of the reactivated expression pattern following the mitosis at nc13 / nc14 ( Movies S2 and S3 ) . During nc13 , de novo transcription is observed throughout most of the length of the embryo , spanning 10 – 70 % along the anterior - posterior ( AP ) limits of the embryo . However , at the onset of nc14 , the transgene is reactivated within tighter spatial limits , from 20 % to 50 % across the AP axis . This initial nc14 pattern is broader than the final limits of the mature stripe , but considerably more restricted than the pre - nc14 pat - tern ( Movies S2 and S3 ) . The progressive refinement of the mature stripe 2 expression pattern ( Fig . 2 A – C ) is revealed by quantifying the instantaneous fraction of nuclei exhibiting fluorescent signals of de novo transcription . There is relatively uniform activation of eve > MS2 transcription within a broad domain centered at 40 % embryo length of nc13 embryos , This pattern is refined into the mature stripe during a 20 - min window of nc14 ( Fig . 2 D , dashed line ) , but quickly disappears before the onset of gastrulation ( Fig . 2 E ) . The mature stripe 2 pattern is far more transient than the picture formed from conventional in situ hybridization assays using fixed embryos ( e . g . , refs . 20 and 21 ) . Dynamic Regulation Predicts the Mature Expression Pattern . Image analysis methods were used to quantify the signal intensities of individual transcription foci in an effort to understand how the dynamic de novo transcription profile produces a steady - state stripe of expression ( 7 ) . Signal intensities were tracked for individual nuclei during the entirety of nc14 development . Some of these nuclei reside within the limits of the mature stripe , whereas others are located just beyond the anterior and posterior borders of the stripe ( Fig . 3 A ) . Interestingly , there is an overall reduction of signal intensities within the stripe 2 domain at the midpoint of nc14 ( arrow , Fig . 3 A ) . This transient reduction might reflect a change in the regulatory landscape , for example , due to the accumulation of Giant and Kruppel repressors as the mature stripe is formed . A similar reduction in expression was observed for the hunchback > MS2 transgene at ∼ 12 min after onset of nc14 ( 7 ) . By integrating fluorescent traces over time , we calculated the spatial profile of mRNAs produced per nucleus at each time point during nc14 ( Fig . 3 B – D , SI Text , and Movie S4 ) ( 7 ) . The mature stripe of steady - state mRNAs becomes apparent at ∼ 30 min into nc14 , when each nucleus produces ∼ 130 mRNAs ( SI Text ) . To systematically quantify the width of the stripe , a Gaussian curve was fitted to the mRNA accumulation profiles at the end of nc14 ( Fig . 3 D ) . This analysis reveals a mature stripe that is centered at 41 . 0 ± 0 . 1 % egg length with half - maximum at full - width limits of 5 . 3 ± 0 . 1 % egg length . The stripe position is entirely consistent with previous reports based on in situ hy - bridization assays and antibody staining of fixed embryos ( lo - cated between 39 % and 43 % egg length ; see ref . 23 ) . As mentioned above , the de novo transcription profiles pre - dict the final limits of the mature stripe 2 pattern ( Figs . 2 C and 3 C ) as visualized by conventional methods ( Fig . 1 C ) . Thus , posttranscriptional mechanisms , such as differential mRNA stability , are unnecessary to account for the dynamics of the stripe 2 expression pattern . However , mRNA half - life does affect the total number of steady - state mRNAs and proteins present within the stripe . When we assume a half - life of ∼ 7 min , as measured for ftz mRNAs ( 24 ) , the position and width of the stripe remains unchanged , but there is a reduction in the total A B C D Fig . 1 . Live imaging of e ve stripe 2 transcriptional activity . ( A ) Schematic representation of eve stripe 2 regulation ( data from refs . 38 and 39 ) . The stripe is the result of the combined activation of Bicoid and Hunchback , which define a broad activation domain in the anterior part of the em - bryo , and repressors Giant and Kruppel , which restrict expression anterior and posterior of this domain , respectively ( 4 ) . ( B ) Structure of the reporter construct : the eve stripe 2 DNA region ( − 1 . 7 kbp , + 50 bp ) was placed upstream of 24 repeats of the MS2 stem loops and a yellow reporter gene . The MCP - GFP protein that binds to these stem loops is present in the unfertilized egg and in the early embryo . ( C ) Confocal image of a trans - genic embryo carrying the eve > MS2 transgene labeled via in situ hy - bridization with full - length probes for the yellow reporter gene ( plasmid transgene ) and endogenous eve RNAs in the same embryo during nc14 . ( D ) Projected confocal stack of a live Drosophila embryo at six time points ( a – f ) centered at ∼ 40 % embryo length , expressing the eve > MS2 trans - gene , histone RFP ( red ) , and MCP - GFP ( green ) . Each image is 77 μ m × 77 μ m . ( a ) At metaphase of nc11 , no foci of transcription are detectable . ( b ) Same embryo 10 . 3 min later than a during nc12 interphase . There are clear fluorescent foci indicating sites of nascent transcript formation . ( c ) Em - bryo in nc13 interphase showing broad expression of the transgene . ( d ) At the onset of nc14 , the stripe pattern has started to refine . ( e ) Refined stripe by late nc14 . ( f ) Embryo just before gastrulation when the transgene expression has diminished significantly . Bothma et al . PNAS | July 22 , 2014 | vol . 111 | no . 29 | 10599 D E V E L O P M E N T A L B I O L O G Y number of steady - state mRNAs ( Fig . 3 E and Movie S5 ) . Furthermore , assuming a protein translation rate of one protein per mRNA per min ( 25 ) and a protein half - life of 6 – 40 min ( 26 ) , we predict an average of ∼ 1 , 200 Eve proteins per nucleus within the mature stripe 2 domain . It should be possible to test these predictions using quantitative in situ detection methods . Bursts of Transcriptional Activity in Individual Nuclei . The preceding analysis reveals a highly dynamic pattern of de novo transcrip - tion . In an effort to gain insights into the underlying mechanisms , we measured the fluorescence intensities of individual foci dur - ing the entirety of nc14 . There are significant oscillatory fluctu - ations in fluorescence signal intensities during nc14 ( Fig . 4 A and Fig . S2 ) . A typical nucleus within the definitive stripe 2 domain displays reactivation of de novo transcription within ∼ 10 min after the onset of nc14 . There are variable reductions in the levels of transcription followed by surges or bursts of expression . These fluctuations are evocative of transcriptional bursts repor - ted in a number of other systems subject to live - image analysis , including bacteria , yeast , Dictyostelium , and cultured mammalian cells ( 18 ) . Moreover , previous analysis based on fixed Drosophila embryos found evidence of transcriptional bursting at the Hox gene Scr ( 27 ) and in gap gene expression ( 17 ) . Transcriptional “ bursts ” have been associated with promoters that switch between ON and OFF states ( 28 , 29 ) . In this simple “ two - state model , ” transcription occurs only when the promoter is in the ON state and no transcription is permitted when the promoter is in the OFF state ( summarized in Fig . 4 B ) . To determine whether this simple model can describe the fluctua - tions of eve transcription , we calculated the dynamics of Pol II loading based on the fluorescence signal intensities at individual sites of de novo transcription . These signals are a proxy for the number of Pol II molecules actively transcribing the gene ( Fig . 4 A and C ) . Changes in signal intensities can be directly related to the rate of Pol II loading at the promoter ( Fig . 4 D and E ) using a previously described model ( 7 ) ( SI Text ) . We observed highly variable burst cycles of 4 – 10 min , and the production of 20 – 100 mRNAs per typical burst ( Fig . 4 A and E , andFig . S2 ) . Both the time of persistence and the number of mRNAs produced per burst are comparable to those observed in other systems using similar live - imaging methods as well as fixed tissue techniques ( 10 , 11 , 28 , 30 ) . Surprisingly , however , these bursts A B C D E Fig . 2 . Formation and refinement of stripe 2 expression domain . ( A – C ) Snapshots of a Drosophila embryo expressing the eve > MS2 reporter at different times in nc14 centered at ∼ 37 % embryo length . Nuclei that show foci of active transcription have been false - colored yellow . ( D ) Instantaneous fraction of active nuclei as a function of position in nc13 and at different times during nc14 . The expression domain is defined as the area within the full width at half - maximum of a Gaussian fit to the profile at each time point . ( E ) Expression domain width and fraction of active nuclei within the domain as a function of time obtained from Gaussian fits as shown in D . After entry into nc14 , the width of the domain refines and the fraction of active nuclei within it increases . The mature stripe is stable for 15 min and decays rapidly as gastrulation approaches . The temporal progression of the spatial profile of the fraction of active nuclei is also shown in Movie S5 . ( All data were obtained by averaging over four embryos ; error bars correspond to SEMs . ) 10600 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1410022111 Bothma et al . do not present a single characteristic rate of Pol II loading but correspond to discrete values ranging from a peak of 14 elon - gating Pol II complexes per min to a minimum of 4 Pol II per min . The occurrence of multiple rates of Pol II loading argues against a simple two - state model of transcription ( Fig . 4 B ) . Instead , the data are consistent with a “ multistate model , ” with promoter switching between several discrete transcriptional states ( Fig . 4 B and Fig . S2 ) . For both the multistate model and the simpler two - state model , the molecular mechanisms underlying these multiple transcriptional states are uncertain ( see below ) . Discussion During the past 30 years , we have obtained a comprehensive picture of the spatial patterning processes underlying the segmentation of the Drosophila embryo [ e . g . , reviewed by Levine ( 1 ) ] . How - ever , considerably less is known about the temporal dynamics of this process . Here , we applied recently developed live - imaging methods to monitor the transcriptional activity of an eve stripe 2 fusion gene in living Drosophila embryos . We found that the mature eve stipe 2 expression pattern is surprisingly short - lived and persists for only ∼ 15 min after it is fully formed ( Fig . 2 ) . Nonetheless , the temporal dynamics of de novo transcription accurately account for the steady - state expression of eve stripe 2 seen with conventional in situ detection methods ( Fig . 3 ) . A critical observation of this study is the occurrence of transcrip - tional bursts underlying the dynamic eve expression pattern . These bursts are highly variable in duration and in mRNA out - put , and a simple two - state model cannot explain them ( Fig . 4 ) . The ephemeral nature of the stripe 2 expression pattern highlights our ignorance of the temporal dynamics of the segmentation gene network , despite extensive insights into the spatial control of expression ( e . g . , ref . 1 ) . Timing is just as important for developmental fate decisions as the control of the spatial limits , and it is now possible to measure the temporal control of gene expression using newly developed live - imaging methods ( 7 , 8 ) . Indeed , recent evidence suggests that promoters with poised Pol II exhibit more rapid activation dynamics than those lacking poised Pol II , and subtle differences in the timing of expression can influence the coordination of cell invagination events during gastrulation ( 31 ) . Previous live - imaging studies have reported transcription bursts , whereby promoters switch between ON and OFF states ( 10 , 11 , 28 , 30 ) ( Fig . 4 B ) . Such bursts have also been inferred A D E B C Fig . 3 . Dynamics of eve stripe 2 mRNA distribution . ( A ) Mean spot fluorescence , indicating transcriptional activity , as a function of time for different positions along the AP axis of the embryo . The arrow indicates a reduction in the average fluorescence that consistently occurs at about 28 min into nc14 in all embryos observed . ( B and C ) By integrating the total fluorescence as a function of time and assuming no mRNA degradation , it is possible to predict the amount of accumulated mRNA ( SI Text ) . The intensity of the yellow false - color label is proportional to the amount of mRNA produced in each nucleus . ( D ) Total amount of mRNA produced per nucleus , assuming no degradation as a function of position along the AP axis at different time points during nc14 . The absolute number of mRNA molecules should be seen as an estimate ( SI Text ) . ( E ) Number of mRNA and protein molecules per nucleus assuming an eve mRNA half - life of 7 min ( 24 ) , a protein translation rate of one protein per mRNA per min ( 25 ) and a protein half - life of 6 – 40 min ( 26 ) . The exact half - life of eve mRNA that is used for this model has little influence on the qualitative appearance of the stripe ( Fig . S1 and Movie S4 ) . The temporal progression of all parameters is shown in Movie S5 . ( All data were obtained by averaging over four embryos ; error bars correspond to SEMs . ) Bothma et al . PNAS | July 22 , 2014 | vol . 111 | no . 29 | 10601 D E V E L O P M E N T A L B I O L O G Y from fixed embryo data ( 17 , 27 ) . However , our analysis of eve stripe 2 regulation suggests a more nuanced picture of the transcription dynamics . We find evidence for multiple ON states , with each state exhibiting a distinct rate of Pol II loading and release from the eve promoter . Because the nc14 interphase occurs after DNA replication , there are two copies of each allele on adjoining sister chromatids . Independent burst cycles from each copy could contribute to the observed multistate complexity . Additional molecular mechanisms underlying pro - moter switching include occupancy of transcription factor binding sites , nucleosome remodeling , disassembly of the preinitia - tion complex , and stochastic enhancer - promoter looping events ( 32 – 35 ) . It is possible that transcriptional bursting could contribute to the dynamic regulation of the eve stripe 2 expression pattern . In the critical region of refinement , there are overlapping dis - tributions of activators ( Bicoid and Hunchback ) and repressors ( Giant and Kruppel ) . At the beginning of nc14 , the activators have the upper hand and it is only as the concentration of Giant and Kruppel increase that the pattern becomes refined ( 36 ) . Perhaps the “ OFF phase ” of the eve bursts is particularly sus - ceptible to repression during this increase in the levels of Giant and Kruppel . The OFF phase might reflect the uncoupling of the stripe 2 enhancer and transcriptional machinery , thereby ren - dering the enhancer DNA more accessible to the newly synthesized repressors ( e . g . , ref . 37 ) . The hunchback > MS2 transgene exhibits a relatively static pattern of expression ( 7 , 8 ) , and it is currently unclear whether individual nuclei exhibit transcrip - tional bursting behaviors . It remains to be seen whether the transcriptional bursts or surges identified in this study are a general property of gene expression in the Drosophila embryo , or a property of dynami - cally regulated genes such as eve . It is striking that eve tran - scription is stochastic and discontinuous because the Drosophila syncytium exhibits the most rapid regulatory dynamics known in animal development . Future studies will explore the possibility that previously described mechanisms of transcriptional pre - cision , e . g . , paused Pol II and shadow enhancers ( e . g . , ref . 37 ) , somehow suppress transcriptional bursts to produce more uni - form rates of mRNA synthesis . A B C D E Fig . 4 . Transcriptional bursting in eve stripe 2 activity . ( A ) Fluorescence intensity of an individual spot within the stripe ( black ) and manual fits consistent with the simple model put forth in B – D ( red ) ; error bars are imaging errors as in Garcia et al . ( 7 ) . Inset ( 51 μ m × 51 μ m ) shows the nuclear location corresponding to the spot using false coloring as in Fig . 3 C . ( B ) The widespread two - state model of transcription posits that promoters can be in an OFF or ON state . Transcription factors can then regulate the rates of interconversion between these two states or the rate of transcriptional initiation in the ON state . In a more general multistate model of transcription , the promoter can be found in the OFF state as well as several ON states , each one of which has a characteristic rate of transcription initiation , i . e . , polymerase loading rate . ( C ) The strength of eve > MS2 fluorescent foci are proportionate to the number of elongating Pol II complexes across the gene template . ( D ) The rate of Pol II loading is related to the spot fluorescence intensity through the time Pol II molecules spend bound to the gene during transcript elongation . Two example time traces of the rate of Pol II loading and their corresponding fluorescence dynamics are shown . In the example for the two - state model , Pol II molecules are loaded onto the gene at a rate r starting at a time t 1 after mitosis resulting in a linear increase of fluorescence . Once the first Pol II molecule reaches the end of the gene and falls off [ ∼ 4 . 2 ± 0 . 4 min ; see Garcia et al . ( 7 ) ] , the number of Pol II molecules on the gene will reach steady state , resulting in a constant fluo - rescence value . At time t 2 , the promoter is switched OFF and the fluorescence intensity will decline as Pol II molecules terminate transcription . ( E ) Estimated rate of Pol II loading resulting from the manual fits in A . The estimated number of mRNA molecules produced per state and their duration are shown . 10602 | www . pnas . org / cgi / doi / 10 . 1073 / pnas . 1410022111 Bothma et al . Methods Female virgins maternally expressing MCP - GFP and Histone - RFP from ref . 7 were crossed with males of the eve > MS2 - yellow reporter line . Collected embryos were imaged using either two - photon or confocal microscopy . At each time point , a stackof at least 10 images separated by 0 . 5 μ m ( confocal ) or 1 μ m ( two - photon ) was acquired . 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Blackwood EM , Kadonaga JT ( 1998 ) Going the distance : A current view of enhancer action . Science 281 ( 5373 ) : 60 – 63 . 16 . PisarevA , PoustelnikovaE , SamsonovaM , ReinitzJ ( 2009 ) FlyEx , thequantitativeatlas on segmentation gene expression at cellular resolution . Nucleic Acids Res 37 ( Database issue ) : D560 – D566 . 17 . Little SC , Tikhonov M , Gregor T ( 2013 ) Precise developmental gene expression arises from globally stochastic transcriptional activity . Cell 154 ( 4 ) : 789 – 800 . 18 . Lionnet T , Singer RH ( 2012 ) Transcription goes digital . EMBO Rep 13 ( 4 ) : 313 – 321 . 19 . Ludwig MZ , Manu , Kittler R , White KP , Kreitman M ( 2011 ) Consequences of eukaryotic enhancer architecture for gene expression dynamics , development , and fitness . PLoS Genet 7 ( 11 ) : e1002364 . 20 . HardingK , HoeyT , WarriorR , LevineM ( 1989 ) Autoregulatoryandgapgeneresponse elements of the even - skipped promoter of Drosophila . EMBO J 8 ( 4 ) : 1205 – 1212 . 21 . 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Kellerman KA , Mattson DM , Duncan I ( 1990 ) Mutations affecting the stability of the fushi tarazu protein of Drosophila . Genes Dev 4 ( 11 ) : 1936 – 1950 . 27 . Paré A , et al . ( 2009 ) Visualization of individual Scr mRNAs during Drosophila embryogenesis yields evidence for transcriptional bursting . Curr Biol 19 ( 23 ) : 2037 – 2042 . 28 . Sanchez A , Golding I ( 2013 ) Genetic determinants and cellular constraints in noisy gene expression . Science 342 ( 6163 ) : 1188 – 1193 . 29 . Munsky B , Neuert G , van Oudenaarden A ( 2012 ) Using gene expression noise to understand gene regulation . Science 336 ( 6078 ) : 183 – 187 . 30 . Chubb JR , Trcek T , Shenoy SM , Singer RH ( 2006 ) Transcriptional pulsing of a developmental gene . Curr Biol 16 ( 10 ) : 1018 – 1025 . 31 . Lagha M , et al . ( 2013 ) Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo . Cell 153 ( 5 ) : 976 – 987 . 32 . 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Proc Natl Acad Sci USA 110 ( 17 ) : 6724 – 6729 . 39 . Dubuis JO , Samanta R , Gregor T ( 2013 ) Accurate measurements of dynamics and reproducibility in small genetic networks . Mol Syst Biol 9 : 639 . Bothma et al . PNAS | July 22 , 2014 | vol . 111 | no . 29 | 10603 D E V E L O P M E N T A L B I O L O G Y Supporting Information Bothma et al . 10 . 1073 / pnas . 1410022111 SI Text SI Methods Cloning and Transgenesis . A plasmid construct containing the even - skipped ( eve ) enhancer and promoter region ( − 1 . 7 kb , + 50 bp ) was built using the pbPHi backbone vector containing yellow reporter gene ( 1 , 2 ) . The yellow reporter gene ( 6 . 4 kb ) was used instead of lacZ ( 5 . 3 kb ) ( 3 ) to increase the signal strength , which is proportional to the length of the reporter . This is because the number of transcripts associated with the template is directly proportional to the length of the reporter when the MS2 repeats are placed in the 5 ′ position . Primers used for building the construct are atttgcggccgcCAAGAAGGCTTGCATGTGGG and cgggatccAACGAAGGCAGTTAGTTGTTGACTG . Copies of the MS2 stem loops were extracted from plasmid pCR4 - 24XMS2SL - stable ( Addgene ; 31865 ) by digesting it with BamHI and BglII restriction enzymes . This fragment was ligated into eve - yellow pbPHI vector linearized with BamHI . The eve2 - MS2 - yellow plasmid was integrated on chromosome 3 ( Vk33 ) . In Situ Hybridization and Fluorescence Microscopy . Fluorescent in situ hybridization was performed as described previously using hapten - tagged complementary mRNA probes ( 4 , 5 ) . Embryos were imaged on a Zeiss 700 laser - scanning microscope in z stacks through the nuclear layer at 0 . 5 - μ m intervals using a Plan - Apochromat 20 × / 0 . 8 air lens . Live Imaging Sample Preparation and Data Acquisition . Female vir - gins of line yw ; Histone - RFP ; MCP - NoNLS - GFP ( 3 ) were crossed with males of the reporter line ( eve2 - MS2 - yellow ) . Collected embryos were dechorinated with bleach and mounted between a semipermeable membrane ( Biofolie ; In Vitro Systems & Services ) and a coverslip and embedded in Halocarbon 27 oil ( Sigma ) . Excess oil was removed with absorbent paper from the sides to flatten the embryos slightly . The flattening of the embryos makes it possible to image more nuclei in the same focal plane without causing any detectable change to early development processes ( 6 ) . Embryos were either imaged using a custom - built two - photon microscope ( 7 ) at Princeton and a Zeiss LSM 780 confocal microscope at University of California , Berkeley . On the two - photon microscope , imaging conditions were as described by Garcia et al . ( 3 ) : average laser power at the specimen was 10 mW , a pixel size is 220 nm , and image resolution is 512 × 256 pixels . At each time point , a stack of 10 images separated by 1 μ m was acquired , resulting in a final time resolution of 37 s . Confocal imaging on the Zeiss LSM 780 was performed using a Plan - Apochromat 40 × / 1 . 4 N . A . oil immersion objective . The MCP - GFP and Histone - RFP were excited with a laser wave - length of 488 and 561 nm , respectively . Fluorescence was detected with two separate photomultiplier tubes using the Zeiss QUASAR detection unit ( gallium – arsenide – phosphide photo - multiplier was used for the GFP signal , whereas the conventional detector was used for the RFP ) . Pixel size is 198 nm , and images were captured at 512 × 512 pixel resolution with the pinhole set to a diameter of 116 μ m . At each time point , a stack of 22 images separated by 0 . 5 μ m were captured , spanning the nuclear layer . The final time resolution is 32 s . Live - Imaging Data Analysis . Analysis was performed as described in ref . 3 . Histone - RFP slices were maximum projected for each time point . Nuclei were segmented using a blob detection approach based on the Laplacian of Gaussian filter kernel . The segmented nuclei were then tracked over multiple nuclear cycles . Initially , each time frame of the MCP - GFP channel is treated independently . Spots are detected in 3D using raw images and assigned to their respectively closest nucleus . When multiple spots are detected in the vicinity of the nucleus ( due to segre - gating sister chromatids ) , only the brightest one is kept . When single traces are shown , the automated tracking of both nuclei and spots was checked manually frame by frame using custom analysis code . Spot intensity determination requires an estimate of the local fluorescent background for each particle . Two - dimensional Gaussian fits to the peak plane of each particle column determines an offset , which is used as background esti - mator . The intensity is calculated by integrating the particle fluorescence over a circle with a radius of 6 pixels and sub - tracting the estimated background . Imaging error is dominated by the error made in the fluorescent background estimation ( 3 ) . In ref . 3 , it was possible to measure the average fluorescence per polymerase molecule for the hunchback > MS2 transgene with 24 MS2 repeats . The quantitative imaging for the eve > MS2 trans - gene was conducted under the exact same imaging conditions on the same microscope . The eve > MS2 transgene also possess 24 MS2 repeats . However , the specific sequence of the stem loops is slightly different as these repeats have been further optimized to facilitate molecular biology work with them ( 8 ) . Assuming that the MS2 sites are similarly saturated in both cases , we can then use the average fluorescence per polymerase molecule calculated for the hunchback > MS2 transgene to calibrate the eve > MS2 fluorescent traces in terms of the absolute number of transcribing polymerases per fluorescent spot ( Fig . 4 ) . It is im - portant , however , to point out that this is an estimate and that a direct calibration between fluorescence and MS2 - eve tran - scripts will be necessary for further confidence . We quantified expression domain refinement dynamics by fitting a Gaussian curve to the profile of the fraction of active nuclei as a function of anterior - posterior ( AP ) position at each time point in nuclear cycle 14 ( nc14 ) ( Fig . 2 D , dashed line ) . The fits define an expression domain width over which we determined the instantaneous fraction of active nuclei . The width of the expression domain as well as the fraction of active nuclei refine into the mature stripe pattern during the initial 20 min of expression ( Fig . 2 E ) . Determining the Amount of mRNA Accumulated in the Presence of Degradation . In the experiments reported here , the quantity that is measured is the observed fluorescence in foci of transcription , which is proportional to the number of nascent mRNA molecules associated with a locus at a specific time . This is related to , but not exactly equal to , the rate of mRNA production . One quantity that is of particular interest is how much mRNA has accumulated in individual cells at a specific time point . To connect the measured fluorescence to the amount of mRNA produced by a cell up to a given time , we have to obtain the rate of mRNA production from the fluorescence traces and then account for the corre - sponding mRNA degradation . In the following sections , we give details on how this magnitude is calculated . The first section describes how to connect the measured fluorescence with the rate of mRNA production , and the second describes how we to use the obtained mRNA production rate to estimate the amount of accumulated mRNA in the presence of degradation . Bothma et al . www . pnas . org / cgi / content / short / 1410022111 1 of 7 Relating Measured Fluorescence to mRNA Production Rate . The ob - served fluorescence in foci of transcription as a function of time is given by F ð t Þ . This quantity is linearly related to the number of mRNA molecules associated with the DNA template at a given instant . In our model , mRNA molecules remain associated with the DNA template for as long as it takes the transcribing poly - merase to traverse the length of the gene , E t . Hence , after a time ( E t + t ) , all of the mRNA associated with the active locus at a time t have been released from the template . Thus , F ð t Þ = N p ð t + E t Þ − N p ð t Þ ; where N p ð t Þ is the number of mRNAs that have been produced up to time point t , properly scaled by the average fluorescence intensity for a single mRNA molecule . Now , we can expand N p ð t + E t Þ around E t = 2 , which results in the following : N p ð t + E t Þ = N p (cid:1) t + E t 2 + E t 2 (cid:3) = X ∞ n = 0 N ð n Þ p (cid:1) t + E t 2 (cid:3) n ! (cid:1) E t 2 (cid:3) n : Similarly , we can expand N p ð t Þ , obtaining the following : N p ð t Þ = N p (cid:1) t + E t 2 − E t 2 (cid:3) = X ∞ n = 0 N ð n Þ p (cid:1) t + E t 2 (cid:3) n ! (cid:1) − E t 2 (cid:3) n : As a result , N p ð t + E t Þ − N p ð t Þ = X ∞ n = 0 N ð 2 n + 1 Þ p (cid:1) t + E t 2 (cid:3) ð 2 n + 1 Þ ! (cid:1) E t 2 (cid:3) 2 n + 1 = dN p dt (cid:1) t + E t 2 (cid:3) × (cid:1) E t 2 (cid:3) + h : o : t ; where “ h . o . t ” indicates higher - order terms . This implies that F ð t Þ ≈ dN p dt (cid:1) t + E t 2 (cid:3) × (cid:1) E t 2 (cid:3) and dN p ð t Þ dt ≈ 2 E t × F (cid:1) t − E t 2 (cid:3) : As a result , by integrating the measured fluorescence one can ob - tain , up to a multiplicative constant , the amount of mRNA pro - duced as a function of time . The details on how this is done is detailed in the next section . Relating mRNA Production Rate to Amount of mRNA Accumulated . In the previous section , we derived a connection between the measured fluorescence and the rate of mRNA production . In the absence of mRNA degradation and neglecting diffusion of mRNA between cells , the total amount of mRNA associated with a given nucleus at a given time is as follows : N mRNA ð t Þ = Z t 0 dN p ð τ Þ dt d τ : If we have to allow for mRNA degradation according to first - order kinetics , this simply becomes the following : N mRNA ð t Þ = Z t 0 (cid:1) dN p ð τ Þ dt − λ × N mRNA ð τ Þ (cid:3) d τ : Hence , given some initial conditions and the rate of mRNA pro - duction estimated from our measurements , we can integrate the above equation to obtain the accumulated amount of mRNA present in a cell at a given time . SI Discussion Using the methods explained in the previous sections , it is pos - sible to relate the measured fluorescence ( Fig . S1 A ) to the amount of mRNA accumulated ( Fig . S1 B ) . Fig . S1 B shows how the amount of accumulated mRNA changes with time when one assumes different half - lives . When the mRNA half - life is short , the accumulated amount of mRNA plateaus relatively soon into cell cycle 14 while it steadily increases with time for longer half - lives . The overall levels of mRNA accumulated also scales with the mRNA half - life . One of the things we wanted to get a sense of is how different half - lives of mRNA affect the qualitative profile of the stipe . To look at this , we determined the mRNA accumulation profile for a given embryo assuming different half - lives ( Fig . S1 C and D , and Movie S5 ) . Although there appears to be a modest increase in the width of the stripe as the mRNA half - life is increased , it is not a striking difference . Fig . S1 E shows that increasing the mRNA half - life from 5 to 60 min only leads to a small increase in the number of cells on the boundary of the stripe even though the half - life is varied by more than an order of magnitude . Movie S5 does show how the persistence of mRNA increases as the mRNA half - life is increased . It is not clear how biologically relevant the persistence of the mRNA driven by stripe 2 enhancer is later because there is an autor - egulatory enhancer that takes over from the eve 2 enhancer to drive expression later ( 9 ) . In this study , we have looked at the dynamics of mRNA accumulation . How this will connect to the dynamics of the protein distribution will depend strongly on the half - life of the eve protein in the early embryo . Although this has not been measured for eve , the half - lives of the ftz and engrailed proteins have been measured and both found to be short , < 10 min ( 10 , 11 ) . Hence it is likely that the eve protein will have a lifetime on this order [ also consistent with estimates from modeling work ( 12 ) ] . With such a short half - life , the evolution of the protein pattern will be closely coupled to that of the accumulated mRNA pattern . We relate the fluorescence dynamics to the rate of RNA polymerase II ( Pol II ) loading at the promoter by invoking a simple model previously used to analyze the mean transcrip - tional activity of multiple MS2 spots ( 3 ) . In the following section , we describe the general idea of the models , how they are used to fit the data and extract the single - cell dynamics of transcriptional initiation . A cartoon depicting one possible outcome in the context of a two - state promoter is shown in Fig . 4 B – D . Here , at time t 1 after mitosis Pol II molecules are loaded at a constant rate r . Because at this point in time the gene is devoid of Pol II mol - ecules , the fluorescence will increase as more polymerases escape the promoter and are loaded onto the gene . If the tran - scriptionally active state persists for a time longer than the time required for the first Pol II molecule to reach the end of the gene [ 6 . 4 kbp , which take 4 . 2 ± 0 . 4 min to transcribe ( 3 ) ] , the number of Pol II molecules on the gene will reach steady state , resulting in a constant fluorescence signal . At time t 2 , the promoter switches back to the OFF state and Pol II molecules are not further loaded at the promoter while those that have finished elongation fall off the gene , resulting in a steady decrease of fluorescence intensity . This whole process can be repeated with Bothma et al . www . pnas . org / cgi / content / short / 1410022111 2 of 7 different characteristic times resulting in the modulation of the burst size and frequency . In a “ multistate ” transcription model ( Fig . 4 B – D ) , the promoter can load Pol II molecules onto the gene a varying discrete rates . The result is more complex fluorescence dynamics , but the connection between the fluorescent signal and the rate of tran - scriptional initiation remains the same as in the case of the “ two - state ” transcription model . Fits of the model to the fluorescence data to extract the dynamics of transcriptional initiation were performed manu - ally using custom - written code in Matlab . It is important to point out that these fits should only be seen as an estimate of the dynamics . An unbiased algorithm to fit the data in an automated way is currently under development and will allow for the systematic characterization of these in vivo single - molecule data . 1 . Venken KJT , He Y , Hoskins RA , Bellen HJ ( 2006 ) P [ acman ] : A BAC transgenic platform for targeted insertion of large DNA fragments in D . melanogaster . Science 314 ( 5806 ) : 1747 – 1751 . 2 . Perry MW , Boettiger AN , Bothma JP , Levine M ( 2010 ) Shadow enhancers foster robustness of Drosophila gastrulation . Curr Biol 20 ( 17 ) : 1562 – 1567 . 3 . Garcia HG , Tikhonov M , Lin A , Gregor T ( 2013 ) Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning . Curr Biol 23 ( 21 ) : 2140 – 2145 . 4 . Bothma JP , Magliocco J , Levine M ( 2011 ) The snail repressor inhibits release , not elongation , of paused Pol II in the Drosophila embryo . Curr Biol 21 ( 18 ) : 1571 – 1577 . 5 . KosmanD , etal . ( 2004 ) MultiplexdetectionofRNAexpressionin Drosophila embryos . Science 305 ( 5685 ) : 846 . 6 . Di Talia S , Wieschaus EF ( 2012 ) Short - term integration of Cdc25 dynamics controls mitotic entry during Drosophila gastrulation . Dev Cell 22 ( 4 ) : 763 – 774 . 7 . Liu F , Morrison AH , Gregor T ( 2013 ) Dynamic interpretation of maternal inputs by the Drosophila segmentation gene network . Proc Natl Acad Sci USA 110 ( 17 ) : 6724 – 6729 . 8 . HocineS , RaymondP , ZenklusenD , ChaoJA , SingerRH ( 2013 ) Single - moleculeanalysis of gene expression using two - color RNA labeling in live yeast . Nat Methods 10 ( 2 ) : 119 – 121 . 9 . HardingK , HoeyT , WarriorR , LevineM ( 1989 ) Autoregulatoryandgapgeneresponse elements of the even - skipped promoter of Drosophila . EMBO J 8 ( 4 ) : 1205 – 1212 . 10 . Edgar BA , Odell GM , Schubiger G ( 1987 ) Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm . Genes Dev 1 ( 10 ) : 1226 – 1237 . 11 . Kellerman KA , Mattson DM , Duncan I ( 1990 ) Mutations affecting the stability of the fushi tarazu protein of Drosophila . Genes Dev 4 ( 11 ) : 1936 – 1950 . 12 . Jaeger J , et al . ( 2004 ) Dynamic control of positional information in the early Drosophila embryo . Nature 430 ( 6997 ) : 368 – 371 . Fig . S1 . Role of mRNA half - life on mRNA accumulation and profile of the stripe domain . ( A ) Fluorescence intensity as a function of time for an individual spot in the center of the eve expression pattern . ( B ) Calculated accumulated mRNA as a function of time for different mRNA half - lives . ( C and D ) Profile of the accumulated mRNA in an embryo when different half - lives are assumed , the false coloring of nuclei indicating the amount of mRNA produced . Each panel has been rescaled according to the maximum amount of mRNA accumulated during the course of the movie . ( E ) False - colored nuclei showing the stripe domain for 5 - and 60 - min half - lives . Nuclei were chosen to be within the stripe domain if they had an accumulated mRNA amount that was at least 25 % of the maximum amount accumulated . [ A , Error bars are imaging errors as in Garcia et al . ( 3 ) ; B , error bars are propagated from A ; C , error bars correspond to the SEM over four embryos . ] Bothma et al . www . pnas . org / cgi / content / short / 1410022111 3 of 7 Fig . S2 . Further evidence of transcriptional bursting in the eve stripe 2 domain . False coloring of nuclei indicating the amount of mRNA produced ( Fig . 2 D – F ) . ( B – D ) Fluorescence intensity as a function of time for individual spots corresponding to the nuclei shown in A and estimated rate of Pol II loading resulting from manual fits as in Fig . 4 . Note that transitions between multiple discrete states of transcription can be seen both inside and outside the mature stipe . The estimated number of mRNA molecules produced per state and their duration are shown . [ Error bars are imaging errors as in Garcia et al . ( 3 ) . ] Bothma et al . www . pnas . org / cgi / content / short / 1410022111 4 of 7 Movie S1 . Dynamics of eve stripe 2 expression . Maximum projection of eve > MS2 transgene from nc11 to nc14 over 112 μ m × 112 - μ m region centered on the stripe . Anterior is to the Left . The pattern is initially broad and gets refined during successive cell cycles to later on disappear at the onset of gastrulation . The snapshots from Fig . 1 are taken from this movie . Movie S1 Movie S2 . Whole - embryo dynamics of eve stripe 2 expression . Maximum projection of eve > MS2 transgene from end of nc10 to gastrulation for a whole embryo . Anterior is to the Left . Movie S2 Bothma et al . www . pnas . org / cgi / content / short / 1410022111 5 of 7 Movie S3 . Further example of dynamics of eve stripe 2 formation . Maximum projection of eve > MS2 transgene from nc12 to nc14 over 112 μ m × 112 - μ m region centered on the stripe . Anterior is to the Left . The pattern is initially broad and then gets refined during successive cell cycles and then the pattern eventually disappears . Movie S3 Movie S4 . Effect of mRNA degradation on stripe formation . mRNA accumulation in the presence of degradation . The accumulated amount of mRNA per cell is shown as a function of time assuming different mRNA half - lives . The amount of accumulated mRNA is proportional to the degree of yellow false coloring . Because the absolute amount of mRNA accumulated is very different for different half - lives , each panel has been scaled according to the maximum accu - mulation reached during the course of each movie . Movie S4 Bothma et al . www . pnas . org / cgi / content / short / 1410022111 6 of 7 Movie S5 . Quantitative dynamics of stripe formation and refinement . The mean spot fluorescence , instantaneous fraction of active nuclei , and accumulated amount of mRNA produced as a function of AP position are shown for different time points as in Fig . 3 . The time stamp indicates time since the beginning of nc14 . The mRNA produced us shown in the case of infinite half - life ( mRNA accumulation ) and of a half - life of 7 min . All data are obtained by averaging four different embryos . Error bars are SEM . Movie S5 Bothma et al . www . pnas . org / cgi / content / short / 1410022111 7 of 7 \ No newline at end of file diff --git a/silver_data/d758bbd598725f72b210db3c7a6eff7ad07846bf.pdf.txt b/silver_data/d758bbd598725f72b210db3c7a6eff7ad07846bf.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..776a933b5b83ff45d154c56a87ccf58fee7723d9 --- /dev/null +++ b/silver_data/d758bbd598725f72b210db3c7a6eff7ad07846bf.pdf.txt @@ -0,0 +1 @@ +Tumor and Stem Cell Biology A Systematic Analysis Reveals Heterogeneous Changes in the Endocytic Activities of Cancer Cells Sarah R . Elkin 1 , Nawal Bendris 1 , Carlos R . Reis 1 , Yunyun Zhou 2 , Yang Xie 2 , Kenneth E . Huffman 3 , John D . Minna 3 , 4 , and Sandra L . Schmid 1 Abstract Metastasis is a multistep process requiring cancer cell signaling , invasion , migration , survival , and proliferation . These processes require dynamic modulation of cell surface proteins by endocy - tosis . Given this functional connection , it has been suggested that endocytosis is dysregulated in cancer . To test this , we developed In - Cell ELISA assays to measure three different endocytic path - ways : clathrin - mediated endocytosis , caveolae - mediated endocy - tosis , and clathrin - independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene - transformed human lung epithelial cells . We found thatallendocyticactivitieswerereducedinthetransformedversus normal counterparts . However , when we screened 29 indepen - dently isolated non – small cell lung cancer ( NSCLC ) cell lines to determine whether these changes were systematic , we observed signi fi cant heterogeneity . Nonetheless , using hierarchical cluster - ing based on their combined endocytic properties , we identi fi ed two phenotypically distinct clusters of NSCLCs . One co - clustered with mutations in KRAS , a mesenchymal phenotype , increased invasion through collagen and decreased growth in soft agar , whereas the second was enriched in cells with an epithelial phenotype . Interestingly , the two clusters also differed signi fi - cantly in clathrin - independent internalization and surface expres - sion of CD44 and CD59 . Taken together , our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a signi fi cant in fl uence on cancer - relevant phenotypes , with potential implications for inter - ventions to control cancer by modulating endocytic dynamics . Cancer Res ; 75 ( 21 ) ; 4640 – 50 . (cid:1) 2015 AACR . Introduction Tumor cell growth and metastasis involve changes in cell – cell and cell – matrix interactions , survival and proliferative signaling , and nutrient uptake , all of which depend on plasma membrane receptors and transporters ( 1 , 2 ) . Signaling from the cell surface and the interactions of cells with each other and their environment are dynamically regulated by the endo - cytosis of signaling , adhesion , and nutrient receptors . Conse - quently , it has been suggested that endocytosis is dysregulated in cancer cells ( 3 – 5 ) . Indeed , there are numerous examples of cancer - speci fi c mutations in components of the endocytic machinery and / or changes in their levels of expression ( 6 – 10 ) . It has also been reported that endocytic traf fi cking can be perturbed downstream of oncogenes such as p53 and Ras ( 11 , 12 ) . Clathrin - mediated endocytosis ( CME ) and caveolae - mediat - ed endocytosis ( CavME ) remain the best - characterized endo - cytic pathways , although other more recently discovered and mechanistically distinct pathways have been shown to mediate the uptake of different subsets of signaling , adhesion , and nutrient receptors , as well as regulate the surface expression of membrane transporters ( 13 – 15 ) . These alternate pathways , generally referred to as clathrin - independent endocytosis ( CIE ) , include the recently discovered clathrin - and dynamin - 2 ( Dyn2 ) - independent uptake into so - called clathrin - indepen - dent carriers ( CLIC ) , which involve the small GTPases Rac1 , Cdc42 , and Arf6 ( 14 – 18 ) . To what extent these CIE pathways contribute to the endocytic capacity of the cell remains unclear , as some studies suggest they are the major pathway for bulk uptake ( 17 ) , whereas a more recent study suggests that CME can account for virtually all bulk uptake ( 19 ) . Past studies of endocytosis in cancer cells have focused pri - marily on CME and CavME , and these have been studied , indi - vidually , in only a few cancer cell lines . Hence , it is unknown whether endocytic activities are selectively or randomly altered in cancers . Moreover , few studies have correlated the activities of speci fi cendocyticpathwayswithchangesincellularbehaviorsuch as migration , adhesiveness , or proliferation . To address these issues , we have systematically and quantitatively analyzed mul - tiple endocytic activities across a clinically diverse and molecu - larly characterized panel of non – small cell lung cancer ( NSCLC ) cell lines ( 20 , 21 ) . Our studies reveal signi fi cant heterogeneity across cell lines and endocytic pathways , which we utilize to test for correlations between speci fi c endocytic activities and altera - tions in cellular processes related to cancer , including prolifera - tion , adhesion , and migration . 1 Department of Cell Biology , UT Southwestern Medical Center , Dallas , Texas . 2 Department of Clinical Science and Quantitative Biomedical Research Center ( QBRC ) , UT Southwestern Medical Center , Dallas , Texas . 3 The Hamon Center for Therapeutic Oncology Research , UT Southwestern Medical Center , Dallas , Texas . 4 Departments of Internal MedicineandPharmacology , UTSouthwesternMedicalCenter , Dallas , Texas . Note : Supplementary data for this article are available at Cancer Research Online ( http : / / cancerres . aacrjournals . org / ) . Corresponding Author : Sandra L . Schmid , UT Southwestern Medical Center , 6000 Harry Hines Blvd . , Dallas , TX 75390 . Phone : 214 - 648 - 3941 ; Fax : 214 - 648 - 7491 ; Email : Sandra . Schmid @ utsouthwestern . edu doi : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 (cid:1) 2015 American Association for Cancer Research . CancerResearch Cancer Res ; 75 ( 21 ) November 1 , 2015 4640 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 Materials and Methods Cell lines and culture HBEC30KT and the NSCLC cancer cell lines were generated as previously described ( 20 ) . HBEC3KT and their oncogene - trans - formed derivatives were developed by the Minna lab ( 22 ) . All NSCLC lines used in this study were obtained from the Hamon Cancer Center Collection ( UT Southwestern Medical Center ) and maintained in RPMI - 1640 ( Life Technologies ) supplemented with 5 % FCS at 37 (cid:1) C in a humidi fi ed atmosphere containing 5 % CO2 and 95 % air . All cell lines have been DNA fi ngerprinted using the PowerPlex 1 . 2 Kit ( Promega ) and are mycoplasma free using the e - Myco Kit ( Boca Scienti fi c ) . Culture media were pur - chased from Life Technologies . Human bronchial epithelial cell ( HBEC ) , NSCLC , and Human retinal epithelia ARPE - 19 cell lines were obtained from the ATCC and cultivated in complete KSF medium , RPMI - 5 % FBS ( Sigma ) , or in DMEM / F12 - 10 % FBS , respectively . Antibodies and reagents Anti - TfnR ( HTR - D65 ) monoclonal antibody was produced in hybridoma as in ref . 23 . Anti - CHC ( sc - 12734 ) and anti – Dyn - 2 ( sc - 64000 ) antibodies were purchased from Santa Cruz Biotech - nology . FITC - conjugated anti - CD44 ( G44 - 26 ) and anti - CD59 ( p282 - H19 ) monoclonal antibodies were obtained from BD Pharmingen . Horseradish peroxidase ( HRP ) – and AlexaFluor - conjugated antibodies were purchased from Life Technologies . Biotinylated albumin ( # A8549 ) , OPD ( # P1536 ) , nystatin ( # N6261 ) , poly - l - Lysine ( # P1536 ) , fi bronectin ( # F1141 ) , lami - nin ( # L2020 ) , and hyaluronic acid ( # H5388 ) were obtained from Sigma - Aldrich . Rat - tail collagen ( # 354236 ) and streptavidin - POD were purchased from BD Biosciences and from Roche , respectively . Fluoromount G and paraformaldehyde ( PFA ) were purchased from Electron Microscopy Sciences . Transferrin receptor , albumin , CD44 , and CD59 internalization TfnR , CD44 , or CD59 internalizations were performed using receptor - speci fi cmAbs . Weusedbiotinylatedalbumintomeasure Cav - ME . Cells were seeded at a density of 2 . 8 (cid:3) 10 4 cells / well on collagen - coated 96 - well plates and grown overnight . For assays , cells were washed ( 3 (cid:3) PBS ) and incubated with40 m L PBS 4 þ ( PBS supplementedwith1mmol / LMgCl 2 , 1mmol / LCaCl 2 , 5mmol / L glucose , and 0 . 2 % BSA ) containing 4 m g / mL mAb or 30 m g / mL biotinylated albumin at 37 (cid:1) C for the indicated time points before being immediately cooled to 4 (cid:1) C to arrest internalization and washed to remove unbound ligand ( 3 (cid:3) PBS ) . The remaining surface - bound ligand was removed by acid washes ( 4 (cid:3) 1 minute 0 . 2 mol / L acetic acid , 0 . 2 mol / L NaCl , pH 2 ) . Cells were washed withPBSandthen fi xedin4 % PFA ( ElectronMicroscopySciences ) inPBSfor10minutesat4 (cid:1) Cand10minutesatroomtemperature . Cells were then permeabilized with 0 . 1 % Triton - X100 / PBS for 5 minutes , washed , and then blocked with 5 % BSA / casein for 1 hour . Internalized albumin was assessed using streptavidin - POD ( Roche ) . Internalized D65 , CD44 , and CD59 mAbs were assessed using a goat anti - mouse HRP - conjugated antibody and further developed with 200 m L OPD . The reaction was stopped by addition of 50 m L 5 mol / L H 2 SO 4 . The absorbance was read at 490 nm ( Biotek Synergy H1 Hybrid Reader ) . Well - to - well vari - ability in cell number was accounted for by normalizing the reading at 490 nm with a BCA readout at 560 nm ( Biotek Synergy H1 Hybrid Reader ) . The fraction of internalized ligand was calculated relative to the initial total surface - bound ligand at 4 (cid:1) C ( without the acid wash step ) measured in parallel for all the assays . Data represent mean (cid:4) SD from four independent experi - ments , each performed in triplicate . To visualize internalization by fl uorescence microscopy , HBEC30KT and HCC4017 cells were seeded in 8 - well chambers . The same procedure as for the In Cell ELISA was used to generate " Surface - bound " and " Internalized " ligands except that after permeabilization the cells were incubated with suitable Alexa - Fluor - labeled secondary antibodies and then mounted with Fluoromount G . Images were captured using a 60 (cid:3) objective mounted on a Ti - Eclipse inverted microscope equipped with a CoolSNAP HQ2 monochrome CCD camera ( Photometrics ) . For siRNA - mediated inhibition of endocytosis , RNAiMAX trans - fection reagent ( Life Technologies ) was used to deliver siRNA targeting Dyn2 ( 1 : 1 mixture of Dyn2 _ 1 : 5 0 - CCGAAUCAAUCG - CAUCUUCUU - 3 0 and Dyn2 _ 2 : 5 0 - GACAUGAUCCUGCAGUU - CAUU - 3 0 ) or clathrin heavy chain ( CHC ; ref . 24 ) to H1299 cells , following the manufacturer ' s instructions . We used All Star Neg - ative siRNA as a control . Cells were used 72 hours after siRNA transfection . For inhibition by nystatin , H1299 cells were pre - incubated in the absence ( control ) or presence of 25 m g / mL nystatin for 30 minutes at 37 (cid:1) C before internalization assays were performed as described above except the PBS 4 þ contained nystatin . Proliferation , adhesion assays , and 3D migration assays The following microscopy - based assays were performed in 96 - well black plates with clear bottom ( PerkinElmer # 600525 ) . Proliferation . A total of 2 , 000 cells / well of each cell line were dispensed , in triplicate . Five hours after plating , cells from the fi rst plate — " Day 0 " plate — were fi xed with 4 % PFA and stored at 4 (cid:1) C . The same operation was repeated on days 1 through 5 . Cell nuclei were stained for 30 minutes with 10 m g / mL Hoechst / PBS solution . Cell proliferation was expressed as the ratio of cell counts on " Day 5 " / " Day 0 " plates . Adhesion . 96 - well plates were incubated with 40 m L / well for 1 hour containing 10 m g / mL BSA , 0 . 01 % poly - l - Lysine , 50 m g / mL rat tail collagen , 10 m g / mL fi bronectin , 50 m g / mL laminin or incubated overnight with 1 mg / mL hyaluronic acid . After plates were washed with PBS , 15 , 000 cells / well were plated and allowed to adhere for 30 minutes at 37 (cid:1) C . Floating cells were then removed by three washes with PBS . Cells were fi xed with 4 % PFA and nuclei were stained for 30 minutes with 10 m g / mL Hoechst / PBS solution . Cell adhesion represents the number of cells per condition . 3Dmigration assay . Assayswereperformed using1mg / mLbovine collagen and 2 . 5 % FBS as chemoattractant , following the exper - imental procedure described in ref . 25 . Microscopy images of nuclear staining were taken at 50 m m steps from 0 to 150 m m into the collagen plug . Invasion index was calculated as the sum of cell counts at 50 , 100 , and 150 m m over cell counts at 0 m m . Cell nuclei were visualized using a 20 (cid:3) ( migration assays ) or 10 (cid:3) ( proliferation and adhesion assays ) air objective mounted on a T i - Eclipse inverted microscope ( Nikon ) driven by NIS Ele - ments V4 . 13 software and quanti fi ed using the " Object Count " feature of NIS Elements software . Altered Endocytic Activity in Cancer Cells www . aacrjournals . org Cancer Res ; 75 ( 21 ) November 1 , 2015 4641 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 Growth in soft agar . Anchorage - independent growth in soft agar was performed by seeding cells in 0 . 4 % noble agar on top of a 1 % layer . After 3 weeks , cell foci were stained with a Crystal Violet solution and extensively washed with water to remove excess stain . Foci were then counted manually using the Cell Counter tool on ImageJ software . Statistical analysis and hierarchical clustering A hierarchical clustering algorithm ( 26 ) was used to group the 29 NSCLC cell lines based on their collective endocytic activities . For this purpose , we took the averaged endocytic activity across replicates for each cell line and each pathway ( Supplementary Table S1 ) , and then used these values to calculate Pearson cor - relation distance metrics , average linkages ( 26 ) , and then gener - ated the hierarchical clustering results using GENE - E software from GenePattern ( 27 ) with default settings . The hierarchical tree identi fi ed two clusters . We tested the signi fi cance of the clustering result using the R package sigclust by using the 2 - means clustering index as a statistic to test the null hypothesis that a group of samples is from a single Gaussian distribution ( 28 ) . Migration and growth in soft - agar enrichment analysis for two clusters were measured by the Fisher exact test to examine whether the two kinds of classi fi cation from categorical data are associated , which is best suited for small sample sizes and a 2 (cid:3) 2 contingency table . We tested association of the two clusters with endocytic activities and the total surface protein expression using a Wilcoxon rank sum test , which compares the sums of ranks and is more robust to the presence of outliers . The statistical correlations between each endocytic activity and cell proliferation were calculated by the Spearman rank correlation test , which is less sensitive to outliers and queries the extent of dependence of one ranked variable on another . Correlations between KRAS mutations and endocytic activities were also measured by the Wilcoxon rank sum test . Results Choice of pathway - speci fi c and lung cancer – related endocytic markers CME and CavME have been extensively studied , and speci fi c markers allowing the quanti fi cation of these endocytic routes are well described . We chose transferrin receptor ( TfnR ) for CME ( 29 ) and albumin , which binds to gp60 and is internalized by CavME ( 30 ) . Although markers for clathrin - and caveolae - independent endocytotic pathways are less well characterized , glycophospha - tidylinositol - anchored proteins ( GPI - AP ) are reportedly internal - ized by these routes ( 15 , 17 , 18 ) . For our studies , we chose complement lysis restricting factor / CD59 ( 31 ) , a GPI - AP that is involved in cellular processes critical to lung cancer cell survival and internalized via CIE ( 32 , 33 ) . Recent studies have identi fi ed the transmembrane glycoprotein , CD44 , which is a hyaluronic acid receptor , as a cargo molecule speci fi cally internalized by CLICs and recycled via GPI - enriched endocytic compartments ( GEEC ; refs . 15 , 34 ) . CD44 becomes more concentrated in fl o - tillin - low or non - raft fractions , presumably corresponding to CLICs , in migrating breast cancer cells ( 35 ) . Interestingly , CD44 is expressed in NSCLC , but not in small cell lung cancer ( 36 ) . ElevatedexpressionlevelsofCD44combinedwithlowexpression of CD24 ( CD44 high / CD24 low ) is an emergent prognosis tool in the clinic as it is associated with " stemness " features of cancer cells in vivo and in vitro ( 36 – 38 ) . Together , these markers allowed us to directly measure the CME , CavME , and CIE activities in NSCLC cells . Development of pathway - speci fi c 96 - well endocytosis assays To ef fi ciently and systematically measure multiple endocytic pathways using several NSCLC cell lines in parallel , we devel - oped a new sensitive and quantitative method using 96 - well plates , referred to as an In - Cell ELISA ( see Materials and Methods ) . As ARPE - 19 cells have been routinely used to study CME , we used this cell line to set up and validate the high - throughput assay . Upon incubation of ARPE - 19 cells at 37 (cid:1) C with the anti - transferrin receptor antibody HTR . D65 ( D65 ; ref . 39 ) , we could detect the time - and temperature - dependent accumulation ( expressed as the fraction internalized at 37 (cid:1) C over the total surface bound at 4 (cid:1) C ) of intracellular D65 ( Supplementary Fig . S1A , left , quanti fi cation in B ) . The use of the bivalent anti - TfnR mAb , rather than transferrin , as ligand reduces rapid recycling ( 40 ) and enables us to focus our measurements on initial rates of internalization . As a negative control for internalization of surface - bound versus soluble mAb , we incubated these cells with a speci fi c antibody against the T - cell marker CD8 , which is not expressed on epithelial cells . As expected , we did not detect surface binding or any internalization of anti - CD8 in APRE - 19 cells ( Supplementary Fig . S1A , right , quanti fi cation in B ) . This assay was adapted to measure CavME using biotiny - lated - albumin as ligand , and CIE using anti - CD59 or anti - CD44 mAbs as markers , and validated in the NSCLC line , H1299 . We fi rst con fi rmed that these different surface receptors utilized different pathways by siRNA knockdown of CHC , which is speci fi cally required for CME and dynamin 2 ( Dyn2 ) , which is required for both CME and CavME ( Fig . 1A ) . We also treated cells with the antifungal drug nystatin that disrupts the plasma membrane cholesterol and selectively inhibits CavME , but not CME . As expected , CME of TfnR was inhibited upon RNAi - mediated knockdown of either Dyn2 or CHC ( Fig . 1B ) , whereas nystatin showed no effect . Also , as expected , uptake of albumin by CavME was inhibited upon RNAi knockdown of Dyn2 and treatment with nystatin ( Fig . 1C ) , but was unaffected by CHC knockdown . Finally , uptake of CD44 and CD59 was not affected by either of these perturbations ( Fig . 1D and E ) , con fi rming that these markers are internalized by a clathrin - , Dyn2 - , and caveolae - independent mechanism . These data establish our ability to independently and selectively measure at least three distinct endocytic pathways . Altered endocytic activity across pathways in isogenic normal and cancer cell lines We next compared the endocytic activities in an NSCLC cell line ( HCC4017 ) with those in a normal , nontumorigenic HBEC line , HBEC30KT , derived from the same patient . The HBEC30KT cells were immortalized by serial introduction of retroviral expression vectors encoding cyclin - dependent kinase 4 ( Cdk4 ) , which prevents premature growth arrest , and human telomerase reverse transcriptase ( hTERT ) , which bypasses telo - mere - dependent senescence ( 41 ) . The internalization rates of all four receptors , expressed as the fraction internalized after 5 minutes at 37 (cid:1) C over the total surface bound at 4 (cid:1) C , were reduced in the NSCLC cells as compared with their normal HBEC30KT counterpart ( Fig . 2A ) . This was con fi rmed by fl uorescence microscopy ( Fig . 2B – E ) , which also illustrates the uniformity in uptake among the cells within each line . These results suggest that endocytosis may indeed be altered in cancer cells . Elkin et al . Cancer Res ; 75 ( 21 ) November 1 , 2015 Cancer Research 4642 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 Malignant transformation of HBEC alters endocytosis To determine when changes in endocytosis might occur during the transformation process , we obtained normal HBEC3KT cell lines along with their derivatives with the following oncogenetic modi fi cations common to lung cancer : ( i ) p53 de fi ciency trig - geredbystableshRNA - mediatedknockdownofp53 ( sh - p53 ) , ( ii ) overexpression of activated KRAS ( KRAS V12 ) , ( iii ) both sh - p53 and KRAS V12 , or ( iv ) sh - p53 , KRAS V12 , and c - myc overexpression . It had been previously shown that all three oncogenic changes were necessary and suf fi cient for full oncogenic transformation of HBEC3KT cells as assessed by in vitro anchorage - independent growth and in vivo tumor formation after subcutaneous injection into NOD / SCID mice ( 22 ) . Consistent with this , the transfected cells were morphologically indistinguishable from the parent HBEC3KT cells until all three oncogenic mutations were present ( Fig . 3A ) , at which point they adopted a more elongated and irregularly shaped mesenchymal morphology similar to some NSCLC cell lines . We measured the various endocytic activities in these cells , and consistent with the need for all three changes to induce tumor formation in mice , only the HBEC3KT sh - p53 / KRASV12 / c - myc cell line exhibited decreased rates of uptake of all four markers ( Fig . 3B ) . Interestingly , the different endocytic pathways were differentially affected during the transformation process . For example , whereas the rate of CavME did not signi fi cantly decrease until all three oncogenic changes were introduced , CME decreased linearly with each oncogenic modi fi cation . CD44 and CD59 uptake , both thought to report CIE , also respond differently to the oncogenic changes . Introduction of the KRAS V12 mutant was suf fi cient to decreaseCD59 - CIEactivitytoitslowestlevels , whereasCD44 - CIE activity was unaffected by expression of KRAS V12 alone , but was progressively reduced with the addition of sh - p53 and myc . Together , these data provide additional support that our assays measure distinct endocytic processes and establish that the dif - ferent endocytic pathways can be differentially sensitive to onco - genic changes . Importantly , as we had observed in comparing patient - matched normal HBEK30KT cells with HCC4017 NSCLC tumor - derived cells ( Fig . 2 ) , we again found that full oncogenic transformation of otherwise syngeneic HBEK3KT cells resulted in decreased rates of all endocytic activities measured . Systematic analysis of CME , CavME , and CIE in 29 NSCLC cell lines To determine whether there are indeed systematic changes in endocyticactivitiesassociatedwithNSCLC , wemeasuredtherates of uptake for all four endocytic pathways in NSCLC cell lines derived from 29 patients . These cell lines were chosen for their diverse molecular and clinical status ( e . g . , isolated from the primary tumor vs . metastases ) . All assays were performed in quadruplicate over multiple days with H1299 used as an internal control for each experiment ( see Materials and Methods ) . The data , which are summarized in Supplementary Table S1 ( avg . (cid:4) SD , n ¼ 4 for each pathway in each cell line ) and presented in Fig . 4A , show that the four endocytic activities varied signi fi - cantly across all NSCLC lines . This variability cannot be attributed to differences in surface expression of the four molecular markers , as the rates of uptake did not correlate with changes in surface expression of receptors ( Supplementary Fig . S2 ) . For comparison , the rates of uptake determined in normal HBEC30KT and HBEC3KT cells were plotted as solid squares and triangles , respec - tively ( Fig . 4 ) . As shown with theisogenic pairs ( Figs . 2 and 3 ) , the majority of NSCLC cell lines have lower rates of uptake across all four pathways compared with the nontransformed HBEC lines ; however , several NSCLC cell lines had equal or higher rates of endocytosisespecially intheCD44 - CIE pathway , whichexhibited the greatest cell - to - cell variability . Thus , although our analyses of isogenic normal and NSCLC lines showed a consistent and signi fi cant decrease in activity across all endocytic pathways , we fi nd considerable heterogeneity in endocytic activities across a diverse panel of NSCLC cell lines . Given this heterogeneity , we asked whether changes in inter - nalization rates occurred randomly across the different endocytic routes , or if they positively or negatively correlated with one or more pathways . For this comparison , we ordered the 29 NSCLC celllinesfromlowtohigh , based ontheirratesofTfnRuptakeand compared these rates to those measured for other pathways ( Fig . 4B ) . We were unable to detect any correlation between CME or CavME and the other endocytic pathways . As expected , a similar patternemergedwhencomparingtherateofuptakeofthetwoCIE Figure 1 . MeasuringmechanisticallydistinctendocyticpathwaysinNSCLCH1299cells . H1299NSCLCcellsgrownin96 - wellplateswereincubatedwiththeindicated antibodies or ligands , and the mechanistically distinct endocytic activities were measured using an In - Cell Elisa assay ( see Materials and Methods ) . A , representative Western blot showing ef fi ciency of siRNA - mediated knockdownofCHCordynamin2 ( Dyn2 ) measured72hoursaftertransfection of H1299 cells . GAPDH was used as a loading control . CME of D65 anti - TfnR mAb ( B ) , CavME of biotinylated albumin ( C ) , CIE of anti - CD44 mAb ( D ) , or anti - CD59 mAb ( E ) were measured 72 hours after transfection with control ( open circle ) , CHC ( closed circle ) , or Dyn2 ( closed square ) siRNAs , or 30 minutes after incubation with nystatin ( closed triangle ) . Data are expressed as the fraction of total surface - bound ligand internalized after incubation for the indicated time at 37 (cid:1) C ( n ¼ 3 independent experiments , each performed in triplicate (cid:4) SD ) . Two - tailed Student t tests were used to assess statistical signi fi cance . (cid:5) , P < 0 . 05 ; (cid:5)(cid:5) , P < 0 . 005 ; (cid:5)(cid:5)(cid:5) , P < 0 . 0005 . Altered Endocytic Activity in Cancer Cells www . aacrjournals . org Cancer Res ; 75 ( 21 ) November 1 , 2015 4643 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 markers , CD44 and CD59 . However , consistent with their differ - ential response to the introduction of oncogenic changes ( Fig . 3B ) , clear , quantitative differences were also detected . Thus , these four pathways appear to be differentially regulated in cancer cells . Correlation of endocytic activities with cancer - related properties of NSCLC cell lines Giventhe differential effects of lossof p53incombination with overexpression of KRAS V12 and / or c - myc on endocytosis in HBEC3KT cells and their derivatives ( Fig . 3B ) , we next extracted information regarding the status of these three oncogenes for the 29 NSCLC cell lines from existing databases ( Fig . 5A ; refs . 42 – 44 ) . We wondered whether these oncogenic changes might correlate with their measured endocytic activities . The majority of the cell lines had functional mutations in p53 , whereas only about half had functional mutations in KRAS , and only 5 had increased expression of c - myc . In contrast with the results obtained with syngeneic cell lines , the three NSCLC lines exhibiting all three oncogenic changes ( H1792 , Hcc44 , and H2122 ) did not exhibit the lowest levels of endocytic activity for any of the pathways ( see Supplementary Table S1 ) . Indeed , there was no correlation between changes in activity of any of the endocytic pathways with p53 mutations or c - myc overexpression ( Supplementary Fig . S3A and S3B ) . We could detect a small , but signi fi cant ( P < 0 . 1 , Figure 2 . Differential internalization of endocytic markers in matched patient - derived normal HBEC30KT and NSCLC ( HCC4017 ) cells . A , comparison of the extent of internalization after 5 minutes at 37 (cid:1) C via the indicated endocytic pathways in nontumorigenic - HBEC30KT ( black bars ) and the NSCLC line ( HCC4017 , gray bars ) derived from the same patient . Two - tailed Student t tests were used to assess statistical signi fi cance . (cid:5)(cid:5)(cid:5) , P < 0 . 0005 . B – E , representative fl uorescence images of total surface - bound and internalized ( after 5 minutes at 37 (cid:1) C ) ligands in HBEC30KT and HCC4017 cell lines assayed for CME of D65 anti - TfnR mAb ( B ) , CavME of biotinylated - albumin ( C ) , CIE of anti - CD44 mAb ( D ) , or CIE of anti - CD59 mAb ( E ) . Elkin et al . Cancer Res ; 75 ( 21 ) November 1 , 2015 Cancer Research 4644 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 Wilcoxon rank sum test ) correlation between KRAS mutations and a decrease in endocytic activity of both CIE pathways ( Fig . 5B ) ; however , there was no signi fi cant correlation between KRAS mutations and CME or CavME . Thus , we were unable to detect systematic changes in endocytic activity corresponding to speci fi c oncogenic changes in our panel of NSCLC cell lines . Several studies on individual cell lines have suggested roles for CME , CavME , and CIE in cancer progression ( 4 , 6 – 9 , 45 , 46 ) . Therefore , we next asked whether the heterogeneity observed in pathway - speci fi c internalization rates might relate to differential activities of the 29 cell lines assessed in a panel of in vitro cancer - relevant assays . For this purpose , we established 96 - well assays to measure the following cellular processes ( see Materials and Methods ) : ( i ) 3D migration through a collagen matrix ; ( ii ) adhe - sion to different substrates ( collagen , fi bronectin , laminin , and hyaluronic acid ) ; ( iii ) proliferation ; and ( iv ) growth in soft agar . Once again we observed signi fi cant heterogeneity in these activ - ities across the different NSCLC lines ( Supplementary Table S2 ) . We applied the Spearman rank correlation test to determine whether differences in individual endocytic activities correlated with changes in any of the above properties . We detected a signi fi cant negative correlation between proliferation and CIE pathway activity that was not seen for CME or CavME ( Supple - mentary Fig . S4 ) . However , we saw no signi fi cant correlations between 3D migration , adhesion or growth in soft agar , and changes in either of the four individual endocytic activities ( Sup - plementary Figs . S5 and S6A ) . However , adhesion on one sub - strate was correlated with adhesion on another by Pairwise Pearson correlation comparison ( Supplementary Fig . S6B ) , pro - viding evidence for the validity of our measurements and dem - onstrating that the adhesion properties of these cells are not substrate speci fi c . Hierarchical clustering based on endocytic activities re fl ects cancer - related properties Previous studies have used hierarchical clustering analyses to classify NSCLCs based on histologic ( 47 ) , transcriptional ( 48 ) , and genetic differences ( 49 ) . To gain more insight into cancer cell properties that might be linked to their endocytic activities , we took a similar approach and conducted unsupervised hier - archical clustering analysis ( 26 ) of all 29 cell lines based on Pearson correlation distance metrics and average linkage of their measured endocytic activities using the raw data presented in Supplementary Table S1 ( see Materials and Methods ) . Hier - archical clustering based on the functional criteria of their collective endocytic behaviors identi fi ed two clusters ( Fig . 6A ) . The signi fi cance of the clustering result ( P ¼ 0 . 0037 ) was tested using the R package sigclust ( 50 ) . Whereas individual endocytic activities did not appear to correlate with other cancer cell properties , we were able to detect signi fi cant Figure 3 . Changes in endocytic activity accompany progressive oncogenic transformation of HBEC3KT cells . A , phase contrast images of nontumorigenic HBEC3KT cells and the same cells transformed by sh - p53 knockdown and / or mutant KRAS V12 and c - myc overexpression , as indicated . Morphologic changes relative totheparentHBEC3KTcellsareonlyapparentwhenallthreeoncogenictransformationshavebeenintroduced . B , comparisonofinternalizationviaCME , CavME , and CIE in nontumorigenic HBEC3KT , HBEC3KT sh - p53 , HBEC3KT KRASV12 , HBEC3KT sh - p53 / KRASV12 , and HBECK3KT sh - p53 / KRASV12 / c - myc cells . Rates of internalization in HCC4017 NSCLC cells , from Fig . 2 , are also shown for comparison . Data shown are average values for the fraction of surface - bound ligand internalized after 5 minutes at 37 (cid:1) C for three independent experiments , each performed in triplicate . Two - tailed Student t tests were used to assess statistical signi fi cance compared with the parent HBEC3KT ( ns , not signi fi cant ; (cid:5) , P < 0 . 05 ; (cid:5)(cid:5) , P < 0 . 005 ; (cid:5)(cid:5)(cid:5) , P < 0 . 0005 ) . Altered Endocytic Activity in Cancer Cells www . aacrjournals . org Cancer Res ; 75 ( 21 ) November 1 , 2015 4645 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 differences in cancer cell properties between the two clusters derived from analysis of their collective endocytic activities . Cluster 1 was enriched in NSCLC cell lines that expressed mutant KRAS ( Fig . 6B ) and mesenchymal cell lines based on a published 76 gene signature ( 51 ) , whereas cluster 2 was enriched in NSCLC cell lines classi fi ed , based on their gene signature , as epithelial ( P ¼ 0 . 02 , Fisher exact test ) . NSCLC cells in the two clusters also differed in their ability to migrate through collagen and their growth in soft agar . Cluster 1 exhibited a greater ability to migrate through collagen as com - pared with cluster 2 ( Fig . 6C , P ¼ 0 . 0641 , Fisher exact test as assessed by categorical correlation for migratory or nonmigratory behavior of the two clusters ) . However , the majority of NSCLC cell lines ( 54 % of cluster 1 and 81 % of cluster 2 ) failed to migrate undertheseconditions ; thus , itispossiblethatotherassaysforcell migrationmight revealmore signi fi cant differences . Cluster1 also appeared to less effectively grow in soft agar as compared with cluster2 ( Fig . 6D , P ¼ 0 . 117 ) . Thatthisapparentdifferencedidnot reach statistical signi fi cance may be due to the qualitative nature of the assay and its saturation at (cid:6) 300 colonies / well . Consistent with this , only 31 % of cluster 1 cell lines compared with 62 % of cluster 2 cell lines exhibited high growth ( i . e . , at > 300 colonies / well ) in soft agar ( P ¼ . 079 , two - sided Student t test ) . Together , these studies suggest that clustering of NSCLC cells based on functional criteria may be informative as to cancer cell properties . Clearly higher numbers of cell lines will need to be examined to con fi rm these associations . A recent study has suggested a link between CD44 expression and KRAS - driven lung adenocarcinomas ( 52 ) . Given our fi nding ofthesmallbutsigni fi cantcorrelationbetweenCD44endocytosis and KRAS mutations ( Fig . 5 ) , the negative correlation between CD44 endocytosis and proliferation in 2D ( Supplementary Fig . S4 ) , and the apparent differences in proliferation on soft agar between clusters 1 and 2 , we more closely examined uptake and surface expression of CD44 in the two clusters . We found that cluster 1 had signi fi cantly lower clathrin - independent endocytic activity and correspondingly higher surface expression of both CD44 ( Fig . 6E and F ) and CD59 ( Supplementary Fig . S7A ) . In contrast , we did not detect systematic or signi fi cant differences in the other endocytic activities or changes in surface expression of their markers ( Supplementary Fig . S7B and S7C ) between the two clusters . Together , these data suggest that selective changes in endocytic activities , in particular clathrin - independent endocyto - sis , can dynamically alter surface expression of cancer - related molecules and affect cellular processes that contribute to cancer aggressiveness . However , such changes do not appear to occur systematically across endocytic pathways or in all NSCLC cells . Furtherstudieswillbeneededtodeterminewhetherthesechangesaffectclinicaloutcomes . Discussion Endocytosis of cell surface receptors can potentially control many activities related to cancer cell proliferation and migration , including nutrient acquisition , cell – cell and cell – matrix adhe - sion , receptor tyrosine kinase , and G - protein – coupled receptor signaling . Moreover , many components of the endocytic machin - ery are mutated or have altered expression in a number of cancers ( 4 , 5 , 53 ) . Thus , it is generally assumed that endocytosis is somehow altered in cancer cells to enhance their proliferative and metastatic potential . Evidence in support of this concept derives primarily from analysis of the effects of perturbing speci fi c endocytic pathways on signaling , proliferation , and / or migration incancercells ( 6 , 7 , 9 , 45 ) . Here , we soughtadditional support for this hypothesis through a more systematic and quantitative analysis of the endocytic activities of a large panel of NSCLC cell lines to determine whether there were consistent alterations in oneormoreendocyticpathwaythatmightbelinkedtochangesin cancer cell proliferation , including anchorage - independency , adhesion , or migration through a collagen matrix . We measured the uptake of four ligands via mechanistically distinct endocytic pathways in 29 independently isolated NSCLC cell lines and discovered a large degree of heterogeneity in these activities . Our results emphasize the inherent complexity and heterogeneity in cancer cell biology that can preclude drawing general conclusions from acute perturbation studies in single cell lines . Nonetheless , based on their overall endocytic properties , we could identify two phenotypically distinct clusters of NSCLC cell lines , which dif - fered in their preponderance of KRAS mutations , epithelial or mesenchymal gene signatures , 3D migration , and anchorage - independent growth . These data suggest that changes in overall Figure 4 . Summary of data for the activities of four distinct endocytic pathways in 29 NSCLC cell lines . A , mechanistically distinct endocytic activities were measured in 29 NSCLC cell lines , as described in Fig . 1 . Each data point represents the average endocytic activity ( n ¼ 4 independent experiments , each performed in triplicate ) for a different cell line . The closed square and triangle represent the internalization rate of normal , HBEC30KT , and HBEK3KTcells , respectively . B , the29celllineswereorderedfromlowtohigh CMEofTfn ( top ) , andtheotherendocyticactivitiesforeachcelllineareshown in the three bottom plots . Although the extent of CIE of CD44 and CD59 was related , neither CIE nor CavME covaried with CME activity . Elkin et al . Cancer Res ; 75 ( 21 ) November 1 , 2015 Cancer Research 4646 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 endocytic capacity might indeed in fl uence cancer cell behavior and / or vice versa . Heterogeneity in NSCLC cell lines con fi rms diversity of endocytic pathways The functions and activities of different endocytic pathways are frequently measured after selective perturbation of one or other pathway . However , the effects of these perturbations can partially overlap making it dif fi cult to distinguish endocytic pathways , especiallyinthecaseofthelesswell - de fi nedclathrin - independent mechanisms . Indeed , the relationship between CLIC - mediated uptake of CD44 and GPI - anchored proteins and the arf6 - depen - dent uptake of similar markers remains somewhat controversial ( 6 , 15 , 32 , 54 , 55 ) . Adding to this complexity , it has been suggested that there might be cross - talk between endocytic path - ways , such that inhibition of one might lead to upregulation of another . Such a reciprocal relationship has been recently sug - gested for CavME and CD44 uptake ( 56 ) . Finally , a recent study suggested that CME accounts for > 95 % of all bulk endocytosis , including that of the GPI - AP , CD59 ( 19 ) , and brought into question the physiologic relevance of alternate endocytic path - ways . Our fi nding that at least three distinct endocytic pathways can be differentially up - or downregulated in an otherwise non - perturbed panel of NSCLC lines con fi rms that these pathways , including two poorly de fi ned clathrin - independent pathways mediating the uptake of CD59 and CD44 , respectively , can be differentially regulated and hence must be , at least in part , mechanistically independent . Endocytosis and cancer Unexpectedly , weobservedadecreaseinallendocyticpathways measured when we compared syngeneic normal HBEC cell lines with their tumorigenic counterparts . Having lower rates of endo - cytosis might bene fi t the NSCLC cell lines by maintaining higher surface levels of important plasma membrane molecules , includ - ing those for signaling and adhesion . However , this pattern did not hold across all NSCLC lines , highlighting the genetic and mechanistic diversity of cancer . Moreover , it is likely that endo - cytic activities of cancer cells will be in fl uenced by different signaling environments in vitro and in vivo . Previous work using siRNA knockdown of components of the endocytic machinery had suggested a link between CME and Figure 5 . Relationship between endocytic activity and genetic alteration associated with cancer cell oncogenic transformation . A , the mutation status of p53 and KRAS and overexpression statusofMYCinthe29NSCLCcelllines , as derived from ref . 42 . B , correlation between KRAS mutation status and endocytic activity in all four pathways . Wilcoxon rank sum tests were used to assess statistical signi fi cance , as indicated . Altered Endocytic Activity in Cancer Cells www . aacrjournals . org Cancer Res ; 75 ( 21 ) November 1 , 2015 4647 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 CavME and the ability of cancer cells to proliferate , adhere , and migrate . CME has been implicated in integrin ( 45 ) and receptor tyrosine kinases ( 57 ) endocytosis . In order for a cell to sustain directed forward migration , integrins and receptor tyrosine kinases , such as the EGFR , must be constantly internalized by CME and recycled to the leading edge of the cell . CavME has also been found to be important for the internalization of membrane - type 1 matrix metalloproteinase , which helps to degrade the ECM at the leading edge of a cell , ( 58 ) , as well as integrins ( 59 ) contributing to the disassembly of focal adhesions . Correspond - ingly , knockdown of Dyn2 in prostate cancer cells prevented cell invasion in 3D and in vivo ( 9 ) , and siRNA knockdown of Cav1 in NCI - H460 lung cancer cells causes an increased ability of these cells to migrate and invade in vitro ( 7 ) . Thus , we were surprised to see no correlation between CME or CavME endocytic activity and these cancer cell processes . This difference likely re fl ects the effects of strong perturbation of these pathways as compared with the more subtle regulation and variations we observe in our panel of NSCLCcelllines . Itisalsopossiblethatratherthanglobalchanges in CME and CavME , changes in the endocytosis of speci fi c cargo molecules not used here to measure these pathways could be selectively altered to affect migration , proliferation , and / or Figure 6 . Endocytic activities identify two phenotypically distinct clusters of NSCLC cell lines . A , the 29 NSCLC cell lines clustered into two distinct groups after unsupervised learning based on endocytic activity in all four pathways . B . NSCLC cell lines in cluster 1 were enriched in those bearing KRAS mutations ( þ ) and exhibiting a mesenchymal phenotype ( M ) , whereas cells in cluster 2 were enriched in those exhibiting an epithelial phenotype ( E ) . C , invasion ratio for each cell line was measured as the fraction of cells migrating up through a collagen matrix toward serum ( see Materials and Methods ) . D , rate of growth in soft agar reported as colonies / well ( see Materials and Methods ) . Each bar represents the average activity ( n ¼ 3 independent experiments , each performed in triplicate ) for each cell line indicated and clustered in A . E and F , comparison of the extent of internalization of CD44 through CIE after 5 minutes at 37 (cid:1) C ( E ) and total surface binding of CD44 measured at 4 (cid:1) C in cluster 1 and cluster 2 ( F ) . Wilcoxon rank sum tests were used to assess statistical signi fi cance , as indicated . Elkin et al . Cancer Res ; 75 ( 21 ) November 1 , 2015 Cancer Research 4648 on July 25 , 2017 . © 2015 American Association for Cancer Research . cancerres . aacrjournals . org Downloaded from Published OnlineFirst September 10 , 2015 ; DOI : 10 . 1158 / 0008 - 5472 . CAN - 15 - 0939 adhesion . For example , we detected an inverse relationship betweenCIEofCD44andproliferationin2Dthatwaslessevident when measuring CIE of CD59 . Better classi fi cation of NSCLCs would facilitate the diagnosis and treatment plans for individual patients . Numerous studies have applied unsupervised clustering methods to classify NSCLC cell lines based on their diverse patterns of mRNA or protein expression , epigenetic modi fi cations , etc . ( 47 – 49 ) . Here , for the fi rst time , we apply this analysis to their measured diversity in a cell biological activity , endocytosis . Interestingly , despite our inability to correlate differences in individual endocytic activities with other cancer - related cellular properties ( e . g . , migration , adhesion , proliferation ) , hierarchical clustering based on the diversity of their collective endocytic activities identi fi ed two distinct NSCLC cell line clusters that co - cluster with other can - cer - related properties . Cluster 1 appeared enriched in cell lines bearing KRAS mutations , a mesenchymal phenotype , an enhanced ability to migrate in collagen , reduced ability to grow in soft agar , and reduced CIE , in particular of CD44 , leading its increased expression on the cell surface . Consistent with these fi ndings , recent studies reported a role for CD44 in mediating NSCLC proliferation downstream of KRAS ( 52 ) . It will be impor - tant to extend these analyses to other NCSLC cells to test the validity of these linkages and also to determine their functional and / or clinical relevance . Moreover , applying this approach to other NCSLC lines will enhance our statistical power and allow us to assess the functional signi fi cance of the " outliers " we detect among this initial sampling of 29 cell lines . Further work is necessary to directly explore the role and regulation of endocytosis in cancer progression . That many com - ponents of the endocytic machinery are dysregulated or mutated in cancer suggests a functional link . Our results suggest that dynamic regulation of the surface expression of important cancer molecules through endocytosis may , in some cases , contribute to the malignant properties of cancer cells . However , they also highlight cancer cell heterogeneity and reveal that the functional relationship between endocytic activities and cell migration , adhesion , and proliferation may be more complex than suggested by perturbation analyses of single cell lines . Disclosure of Potential Con fl icts of Interest No potential con fl icts of interest were disclosed . Authors ' Contributions Conception and design : S . R . Elkin , N . Bendris , J . D . Minna , S . L . Schmid Development of methodology : S . R . Elkin , N . Bendris , C . R . Reis Acquisition of data ( provided animals , acquired and managed patients , provided facilities , etc . ) : S . R . Elkin , N . Bendris , K . E . Huffman , J . D . Minna Analysis and interpretation of data ( e . g . , statistical analysis , biostatistics , computational analysis ) : S . R . Elkin , C . R . Reis , Y . Zhou , Y . Xie , S . L . Schmid Writing , review , and / or revision of the manuscript : S . R . Elkin , N . Bendris , C . R . Reis , Y . Zhou , J . D . Minna , S . L . Schmid Administrative , technical , or material support ( i . e . , reporting or organizing data , constructing databases ) : S . R . Elkin , S . L . Schmid Study supervision : S . L . Schmid Acknowledgments The authors thank Jerry Shay for helpful discussions and Luc Girard for generously providing access to the mRNA expression database for the NSCLC cell lines . They also thank members of the Schmid lab for thoughtful discussions . Grant Support ThisworkwassupportedbyNIHgrantsR01GM042455andR01GM073165 ( S . L . Schmid ) , NIH grant RO1CA15230 and Cancer Prevention Research Institute of Texas Award RP101251 ( Y . Xie ) , as well as Lung Cancer SPORE P50CA70907 and NASA NSCOR NNX11AC54G ( J . D . Minna ) . S . R . Elkin was supported by an HHMI Med into Grad Grant ( Grant # 56006776 ) to the Mechanisms of Disease and Translational Science Track supported by NIH T32 ( Grant # 1T32GM10977601 ) and the Pharmacological Sciences Training Grant ( GM00706240 ) . The costs of publication of this article were defrayed in part by the payment of page charges . This article must therefore be hereby marked advertisement in accordance with 18 U . S . C . 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CAN - 15 - 0939 \ No newline at end of file diff --git a/silver_data/d790034b8c664f58c892156d562f018a0c99002e.pdf.txt b/silver_data/d790034b8c664f58c892156d562f018a0c99002e.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..9c42e0e7fc3a196767043c5946155901b1b8c892 --- /dev/null +++ b/silver_data/d790034b8c664f58c892156d562f018a0c99002e.pdf.txt @@ -0,0 +1 @@ +© 2011 Society for Laboratory Automation and Screening www . slas . org 1153 IntroductIon M embers of the silent information regulator family ( sirt or sirtuins ) are evolutionarily conserved naD - dependent protein deacetylases and adenosine diphos - phate ( aDP ) – ribosylases . there are seven known isoforms ( sirt1 – 7 ) that differ in their subcellular localization , substrate specificities , and functions ( table 1 ) . 1 – 3 an initial report indi - cated that sirt1 , 6 , and 7 are predominantly nuclear , sirt2 is cytoplasmic , and sirt3 – 5 are localized in the mitochondria . 4 however , it has subsequently been shown that movement of sirt1 and sirt2 between cytoplasmic and nuclear compart - ments may occur , 5 , 6 perhaps indicative of substrate localization . sirtuins catalyze the deacetylation of lysine residues on histones and various proteins , resulting in a deacetylated product , nicoti - namide , and o - acetyl - aDP - ribose . 7 for at least sirt1 , kinetic analysis indicates that lysine deacetylation activity follows a sequential mechanism , whereby the acetylated substrate and naD ( + ) must both bind prior to initiating catalysis . 8 it should be pointed out that although all seven sirtuins are classified as dea - cetylases ( largely based on structural homology ) , the catalytic activity of sirt4 – 7 has been associated primarily with aDP - ribosylation . indeed , little or no lysine deacetylase activity has been found with purified sirt4 or sirt6 enzymes . 9 , 10 it is pos - sible that the biological functions of these isoforms are more closely related to aDP - ribosylation than with lysine acetylation or recognize a specific lysine - containing motif in a protein sub - strate yet to be determined . additional less well - characterized posttranslational modifications have been associated with sirtuin activity , such as depropionylation , 11 but their biological impact has yet to be demonstrated . sirtuins have garnered a great deal of interest in the short time since their discovery . as such , a treatise on the entire sir - tuin family could fill volumes . the founding member of this class of deacetylases , sirt1 ( homolog of yeast silent informa - tion regulator , sir2 ) , is the most widely studied of the sirtuins . early reports in yeast described sir2 / sirt1 as a promoter of genomic stability and enhancer of viability / lifetime . 12 , 13 sirt1 has consequently been associated with aging processes , neural plasticity , and memory , as well as a variety of human disease conditions such as metabolic syndrome , inflammation , neuro - degeneration , and cancer . 3 , 14 because of these pleiotropic func - tions , sirt1 has gained considerable interest by the pharmaceutical industry as a prospective drug target . however , it is unclear whether an activator inhibitor approach will pro - duce the desired therapeutic outcome in the absence of ( or minimal ) unwanted side effects . a thorough understanding of sirtuin biology is needed to establish physiologically relevant enzymatic and cellular assays to screen for these pharmaco - logical modulators . the complexities of sirt1 biology and screening assays have revealed the potential for artifacts and / or misinterpretation . this review is devoted to summarizing the 1 singapore institute for Clinical sciences , agency for science , technology and research ( a * star ) , singapore . 2 lilly research laboratories , eli lilly & Company , indianapolis , indiana . received Jun 17 , 2011 , and in revised form Jul 26 , 2011 . accepted for publica - tion aug 8 , 2011 . Journal of biomolecular screening 16 ( 10 ) ; 2011 Doi : 10 . 1177 / 1087057111422103 Sirtuin 1 ( SIrt1 ) : the Misunderstood HdAc WALter StünkeL 1 and robert M . cAMpbeLL 2 the sirtuin family of naD - dependent histone deacetylases ( hDaCs ) consists of seven mammalian proteins , sirt1 – 7 . many of the sirtuin isoforms also deacetylate nonhistone substrates , such as p53 ( sirt1 ) and α - tubulin ( sirt2 ) . the sirtuin litera - ture focuses on pharmacological activators of sirt1 ( e . g . , resveratrol , srt1720 ) , proposed as therapeutics for diabetes , neurodegeneration , inflammation , and others . however , many of the sirt1 activator results may have been due to artifacts in the assay methodology ( i . e . , use of fluorescently tagged substrates ) . a biological role for sirt1 in cancer has been given less scrutiny but is no less equivocal . although proposed initially as an oncogene , we present herein compelling data sug - gesting that sirt1 is indeed a context - specific tumor suppressor . for oncology , sirt1 inhibitors ( dual sirt1 / 2 ) are indi - cated as potential therapeutics . a number of sirtuin inhibitors have been developed but with mixed results in cellular systems and animal models . it is unclear whether this has been due to poorly understood model systems , signalling redundancy , and / or inadequately potent and selective tool compounds . this review provides an overview of recent developments in the field of sirt1 function . While focusing on oncology , it aims to shed light on new concepts of expanding the selectivity spectrum , including other sirtuins such as sirt2 . ( Journal of Biomolecular Screening 2011 : 1153 - 1169 ) key words : epigenetics , metabolic diseases , cancer and cancer drugs , immune system diseases , Cns and Pns diseases Stünkel and campbell 1154 www . slas . org Journal of biomolecular Screening 16 ( 10 ) ; 2011 table 1 . the sirtuin family of Protein Deacetylases / aDP - ribosyltransferases : Known Crystal structures , subcellular localization , substrates , and Phenotypes from Knockout / Deficient mice SIRT Isoform Crystal Structure Localization Substrates Knockout Mouse Phenotype sirt1 human ( none ) , yeast ( hst2 ) , 169 - 171 Escherichia coli ( cobb ) , 172 Thermotoga maritima ( sir2 - tm ) , 173 - 175 Archaeoglobus fulgidus ( sir2 - af1 / 2 ) 176 - 178 nucleus ( can shuttle to cytoplasm ) h3K9 , h4K16 , 15 , 16 p53 , 24 , 25 p73 , 26 foX01 , foX03a , foX04 , 27 - 30 Pten , 31 niCD , 32 mef2 , 33 hif - 1α , hif - 2α , 34 - 36 taf ( i ) 68 , 37 srebP - 1c , 38 β - catenin , 39 rela / p65 , 40 , 41 PgC1α , 42 bmal1 , Per2 , 43 , 44 Ku70 , 45 XPa , 46 smaD7 , 47 cortactin , 48 irs - 2 , 49 , 50 aPe1 , 52 PCaf , 53 tiP60 , 54 p300 , 20 , 28 , 55 , 56 suV39h1 , 57 aceCs1 , 58 , 59 PParγ , 60 erα , 61 ar , 62 lXr 63 reduced life span , tumor suppressor , or oncogene depending on mouse genetic background , genomic instability , developmental defects , autoimmune syndrome 17 , 23 , 39 , 100 - 102 , 117 , 119 , 179 sirt2 human 180 Cytoplasm ( can shuttle to nucleus ) h4K16 , 181 h3K56 , 20 α - tubulin 182 none reported sirt3 human 183 mitochondria ( can shuttle to nucleus ) h4K16 , 184 h3K56 , 20 Ku70 , 185 iDh2 , 186 , 187 hmgCs2 , 188 gDh , 187 aceCs , 58 , 189 sdha , 190 soD2 , 191 , 192 lCaD 193 normal , increased reactive oxygen species ( ros ) levels , decreased adenosine triphosphate ( atP ) levels 194 , 195 sirt4 none reported mitochondria glutamate dehydrogenase ( gDh ) ( aDP - ribosylation ) 196 normal , gDh activation 197 sirt5 human 198 mitochondria Cytochrome C , 187 CPs1 199 urea cycle dysfunction , hyperammonemia following fasting 199 sirt6 human 10 nucleus h3K9 , 200 , 201 h3K56 202 lethal around 4 weeks , lymphopenia , decreased subcutaneous fat , bone loss , decreased igf1 , metabolic defects 203 sirt7 none reported nucleus ( nucleolus ) rna Pol i ( ? ) , 204 p53 159 reduced life span , kyphosis , decreased subcutaneous fat , degenerative cardiac hypertrophy 159 no deacetylation activity has been found with sirt4 thus far . it is unclear whether rna polymerase i is a direct target of sirt7 . 204 although deacetylation activity has been reported for sirt6 , other reports suggest it is primarily an aDP ribosyltransferase . 10 biochemical / biological activities of sirt1 , status of chemical modulators ( activators , inhibitors ) , assay considerations , and suggestions for paths forward in sirtuin drug discovery . SIRT1 substrates—histones Yeast and mammalian sir2 / sirt1 will deacetylate a vari - ety of histones at specific lysine residues : h1 ( K26 ) , h3 ( K9 , K14 ) , and h4 ( K16 ) in vitro . 15 , 16 of these mark changes observed biochemically , elevation in h3K9 and h4K16 acety - lation is seen in sirt – / – mef cells . 17 loss of h4K16 acetyla - tion and h4K20 trimethylation are commonly observed in human cancers . 18 in yeast , there is an age - associated increase in h4K16 acetylation and concomitant decrease in sir2 ( sirt1 ) protein , causing reduced transcriptional silencing . 19 these early observations were key drivers of investigations into the role of sirt1 in cancer and aging . recently , sirt1 was found to suppress p300 - mediated h3K56 acetylation 20 , 21 at the bclaf1 promoter , thereby reducing t cell activation . 21 sirt1 knockout mice develop an autoimmune syndrome , with increased t cell activation and disruption of CD4 + t cell tol - erance . 22 , 23 given this evidence , sirt1 , perhaps via h3K56 deacetylation , may play a role in innate and adaptive immune function . SIRT1 substrates—nonhistones sirt1 can deacetylate a number of nonhistone substrates , suggesting a broader role than just epigenetic silencing . one of Sirtuin 1 ( SIrt1 ) Journal of biomolecular Screening 16 ( 10 ) ; 2011 www . slas . org 1155 the first substrates of sirt1 identified was p53 ( p53K382 ) , a well - known tumor suppressor , which resulted in repression of p53 - dependent apoptosis in response to Dna damage and oxi - dative stress . 24 , 25 sirt1 deacetylates quite a number of other nonhistone proteins , including other tumor suppressors , tran - scription factors , signaling proteins , enzymes , and nuclear hor - mone receptors : p73 ( putative tumor suppressor involved in cell cycle and apoptosis ) 26 ; foX01 , foX03a , and foX04 ( forkhead transcription factors ) 27 – 30 ; Pten ( tumor suppressor regulating Pi3K / aKt pathway ) 31 ; niCD ( notch1 intracellular domain ) 32 ; myocyte enhancer factor , mef2 ( transcription factor ) 33 ; hypoxia - inducible factors hif - 1α , - 2α 34 – 36 ; taf ( i ) 68 ( Pol i transcription ) 37 ; srebP - 1c ( transcription factor modulating hepatic lipogenesis ) 38 ; β - catenin ( component of Wnt signaling pathway ) 39 ; rela / p65 ( nfκb signaling ; immune function ) 40 , 41 ; PgC1α ( nuclear coactivator involved in control of gluconeo - genic genes ) 42 ; bmal1 and Per2 ( circadian rhythm / clock pro - teins ) 43 , 44 ; Ku70 ( Dna repair protein ) 45 ; XPa ( nucleotide excision repair ) 46 ; smaD7 ( tgfβ - induced signaling protein ) 47 ; cortactin ( promotes cell migration ) 48 ; irs - 2 ( neuroprotec - tion ) 49 , 50 ; tau ( neurodegeneration ) 51 ; aPe1 ( apurinic / apyrimi - dinic endonuclease - 1 , base - excision repair pathway ) 52 ; PCaf 53 ; tiP60 54 ; p300 histone acetyltransferases ( inflammation , Dna damage response ) 20 , 28 , 55 , 56 ; suV39h1 ( histone methyltrans - ferase ) 57 ; aceCs1 ( acetyl Coa synthetase ) 58 , 59 ; and PParγ , 60 erα , 61 ar , 62 and lXr 63 nuclear hormone receptors . given the multitude of potential substrates and the lack of a defined consensus sequence , 64 it is possible that sirt1 sub - strate selectivity may be defined by cellular localization in the presence of sirt1 chaperones / partners in a given complex . sirt1 has been reported to shuttle between the cytoplasm and the nucleus upon various stimuli and has “compartment - dependent” functions . 6 , 65 , 66 SIrt1 And MetAboLIc FunctIonS sirt1 can be considered a master regulator of cellular metabolic pathways . 67 sirt1 plays a direct role in transcrip - tional silencing and , via direct interaction with specific co - repressors , exerts profound effects on metabolic pathways such as lipogenesis and gluconeogenesis . Data suggest that sirt1 is involved in the central regulation of food intake 68 and regula - tion of the circadian clock . 69 its major function in metabolism may be to act as a “nutrient sensor . ” sirt1 has been shown to affect lipogenesis through the regulation of mitochondrial acetyl - Coa synthetase 58 and PParγ . 70 interestingly enough , PParγ directly interacts with sirt1 and inhibits sirt1 activ - ity , forming a highly regulated negative feedback loop . 60 sirt1 has also been implicated in control of metabolic glucose . the deacetylation of lXr by sirt1 suggests a role in cholesterol and lipid homeostasis . 63 sirt1 promotes gluconeogenic gene induction and fatty acid oxidation at least in part via PgC1α . 71 , 72 sirt1 overexpression in the liver attenuates insulin resist - ance . 73 similarly , sirt1 has been downregulated in insulin - resistant C2C12 cells and muscle tissues , and sirt1 knockdown has produced insulin resistance . 74 this effect was thought to be mediated by PtP1b silencing at the chromatin level . insulin secretion may also be regulated by sirt1 repression of uCP2 in pancreatic cells . 75 subsequently , there have been a multitude of reports linking sirt1 activation to enhanced fatty acid oxi - dation , gluconeogenesis , insulin secretion , insulin sensitivity , and decreased lipogenesis ( reviewed in haigis and guarente 76 and liang et al . 77 ) . therefore , it is not surprising to find great excitement surrounding the development of sirt1 activators for metabolic diseases . SIrt1 And neurodegenerAtIon sirt1 has been postulated to serve a neuroprotective func - tion , suggesting that sirt1 activators may be effective thera - peutics for treating neurodegeneration diseases such as alzheimer disease . alzheimer disease is characterized by dep - osition of β - amyloid ( or amyloid β , aβ ) plaques and tau pro - tein – containing tangles . it is believed that these proteins may be causative in the neuronal and functional loss , which eventu - ally results in death . tau protein is deacetylated by sirt1 , albeit with a modest effect in vitro . 51 tau acetylation prevents degradation of phosphorylated tau ( p - tau ) , so deacetylation ( by sirt1 ) may be neuroprotective . there is compelling evidence linking sirt1 to aβ - peptide production . for example , in mixed cortical cultures , sirt1 overexpression inhibits aβ - induced nfκb activation and cytotoxicity , presumably via the deacetylation of rela / p65 ( K310 ) . 40 , 78 neuronal cells from sirt1 transgenic mice display higher α - secretase activity and attenuated aβ peptide ( 1 – 40 and 1 – 42 ) production . 79 introducing a sirt1 transgene to a mouse model of alzheimer disease ( aPPswe / Psen1de9 ) similarly causes an activation of α - secretase and subsequent reduction in alzheimer disease – related aβ peptides . 80 Conversely , reduction of sirt1 via expression of a dominant negative sirt1 construct augments the accumulation of aβ peptides by neuronal cells in vitro . 81 it is conceivable that sirt1 activators may attenuate aβ peptide and tau production and thereby have utility in alzheimer dis - ease therapy . indeed , sirt1 lentiviral insertion in the hip - pocampus was neuroprotective in an inducible p25 transgenic mouse model of alzheimer disease . 82 as mentioned , the activity of sirt1 may be regulated by its nuclear / cytoplasmic localization . upon exposure to neuronal cell precursor differentiation conditions , sirt1 quickly trans - locates from the cytoplasm to the nucleus and later moves back to the cytoplasm . 65 overexpression of sirt1 in these cells promoted differentiation , but this was not seen with a cytoplas - mically restricted sirt1 mutant . 65 similarly , cytoplasmic sirt1 ( but not nuclear ) increased ngf - induced neurite Stünkel and campbell 1156 www . slas . org Journal of biomolecular Screening 16 ( 10 ) ; 2011 outgrowth of PC12 cells . 66 both studies suggest that sirt1 localization was critical to activity . SIrt1 And tHe IMMune SySteM sirt1 is a potent negative regulator of nfκb transcription , at least in part due to direct rela / p65 deacetylation . 40 Data from both overexpression and knockout / down models are con - sistent with an anti - inflammatory role for sirt1 . in mouse macrophages overexpressing sirt1 , nfκb activity is decreased relative to Wt , whereas this effect is not seen with knock - in of a catalytically inactive sirt1 [ h355a ] mutant . 83 mice with myeloid - specific sirt1 knockout display hypera - cetylated nfκb , resulting in elevated transcriptional activation of proinflammatory macrophage target genes . 41 smokers and patients with chronic obstructive pulmonary disease ( CoPD ) have decreased lung sirt1 levels ( especially in alveolar mac - rophages , airway epithelium , and alveolar epithelium ) . 84 Cigarette smoke reduces sirt1 levels in a human monocyte - macrophage cell line ( monomac6 ) , leading to acetylation of the rela / p65 subunit of nfκb and elevation in levels of inter - leukin ( il ) – 8 . it was postulated that reduction of sirt1 and resulting nfκb hyperactivation could possibly be the basis of the chronic inflammatory response , such as in CoPD . 84 in pri - mary mouse peritoneal macrophages , sirt1 suppresses aP - 1 transcriptional activity and CoX - 2 expression . 85 it was also found that raW264 . 7 macrophage - like cells transfected with sirt1 demonstrated improved phagocytic function . in the neonatal cardiomyocyte , sirt1 may depress the inflammatory response , as adenoviral overexpression of sirt1 in these cells blocked phenylephrine - stimulated mCP1 mrna ( monocyte chemoattractant protein – 1 ) and acetylation of rela / p65 and PgC1α . 86 sirt1 may modulate nfκb as a pivotal point in the regulation of endotoxin tolerance , shepherding the transition from an inflammatory to an adaptive state . following lPs stimulation of thP - 1 monocytic cells or infusion in vivo , sirt1 protein transiently decreases ( 8 – 12 h ) and later returns to 87 or exceeds baseline . 88 sirt1 binds to the promoters of tumor necrosis factor α ( tnfα ) and il - 1β in the thP1 cell , deacetylates rela / p65 ( presumably when in complex at the promoter ) , and represses transcription of these proinflamma - tory cytokines . 88 it is believed that , over time , expression of namPt ( nicotinamide phosphoribosyltransferase , a key enzyme in naD biosynthesis ) and thus naD + is increased , stimulating relb expression and recruitment to sites of tran - scription . this “feed - forward” loop resulting in sustained epi - genetic silencing may be the mechanism that produces endotoxin tolerance . 88 as mentioned previously , sirt1 - null mice display autoim - mune - like symptoms , such as the accumulation of immune com - plexes in liver and kidney , as well as the presence of antinuclear antibodies . 23 it is thought that hiV - 1 may act to interfere with sirt1 function and decrease immune surveillance . hiV - 1 tat protein binds to the deacetylase domain of sirt1 and inhibits sirt1 - mediated deacetylation of rela / p65 . 55 , 89 Consequently , nfκb transactivation is greatly potentiated , causing t cell hyperactivation , increased viral replication , and eventually the depletion of CD4 + t cells . targeted deletion of sirt1 in specific t cell populations reveals a contrasting view to global knockout ( i . e . , where data suggest that sirt1 dampens the inflammatory response and suppresses immune response ) . however , when sirt1 was deleted specifically in CD4 + t cells and foxp3 + treg cells , activation , proliferation , and cytokine production were unal - tered relative to wild type . 90 also seen was an increase in foxp3 expression , a protein associated with an immunosup - pressive phenotype , and enhancement of allograft survival . as these results were recapitulated by sirt1 inhibitors , eX527 and splitomicin , 90 one could certainly make a case for the use of sirt1 inhibitors in preventing allograft rejection . dIcHotoMIc FunctIonS oF SIrt1 In cAncer studies of sirt1 function in cancer are littered with seem - ingly contradictory data , with some suggesting sirt1 is a tumor suppressor and others indicating that sirt1 may be tumorigenic . expression data are equivocal , as sirt1 is over - expressed in certain tumors ( prostate , hepatocellular carci - noma , breast ovarian epithelial , gastric carcinoma , colorectal , melanoma ) 91 – 98 while reduced in several others ( bladder , colon carcinoma , glioblastoma , ovary , and prostate ) . 17 , 99 in vivo , the presence or absence of other mutations in key tumor survival pathways may serve to determine the fate of sirt1 function . another thought to consider is that sirt1 may normally pro - tect the cell from oncogenic transformation , but upon or after transformation , sirt1 signaling is co - opted to promote malig - nant growth ( i . e . , those pathways that serve to protect normal cells may prevent the death of cancer cells ) . the various lines of evidence are presented herein with the suggestion that sirt1 function is context dependent . SIRT1 as a tumor suppressor sirt1 knockout and transgenic mouse studies led to the speculation that under certain circumstances , sirt1 may act as a tumor suppressor . 100 in the presence of aPC min / + mutation , sirt1 overexpression reduced polyp formation in mice , a potential precursor to colorectal cancer . 39 however , when using aPC min / + , sirt – / – mice , the presence of intestinal polyps was unchanged relative to aPC min / + , sirt + / + counterparts ( i . e . , the absence of sirt1 did not yield a greater number of polyps ) . 101 additional studies support the concept of sirt1 as a tumor suppressor . for example , sirt1 + / – , p53 + / – mice develop tumors Sirtuin 1 ( SIrt1 ) Journal of biomolecular Screening 16 ( 10 ) ; 2011 www . slas . org 1157 in multiple organs . 17 in another model , homozygous deletion of sirt1 in mice led to the development of prostatic intraepithe - lial neoplasia , which was thought to be mechanistically linked with decreased autophagy . 102 in contrast , skin papilloma devel - opment was unchanged by sirt status ( sirt1 – / – or sirt1 + / – mice ) in a model of chemically induced skin carcinogenesis . 101 nucleotide excision repair ( ner ) is conducted by the care - ful orchestration of many proteins , including aPe1 , nbs1 , XPa , and XPC , to remove and , where possible , fix damaged Dna . apurinic / apyrimidinic endonuclease - 1 , aPe1 , is essen - tial for the repair of single - strand Dna lesions . nijmegen breakage syndrome protein , nbs1 , is part of the larger mre11 - raD50 - nbs1 nuclease complex involved in the detection and repair of damaged Dna . XPa and XPC ( xeroderma pigmento - sum proteins a or C ) are also responsible for sensing the Dna breaks and recruiting repair proteins to the site of damage . it was recently reported that sirt1 could deacetylate and acti - vate each of these proteins ( aPe1 , nbs1 , XPa , XPC ) directly , enhancing ner and cell survival . 46 , 52 , 103 , 104 sirt1 knockdown causes cells to become more sensitive to killing by a variety of genotoxic stresses—for example , uV , h2o2 , and mms ( aba - sic Dna damaging agent ) —potentially due to hyperacetylation of these ner factors . sirt1 – / – mefs and keratinocytes with sirt1 knockdown show dramatically reduced XPC levels , increased aKt ser473 phosphorylation , and a diminished ability to excise base lesions ( cyclobutane pyrimidine dimers ) caused by uV irradiation . 103 the effect on aKt phosphorylation is consistent with previous data showing that sirt1 deacetylates Pten , counteracting acetylation - induced repression of its phosphatase activity , thereby activating aKt . 31 given the well - known oncogenic role of aKt , these data would strongly suggest a tumor - sup - pressing role for sirt1 under these conditions . Downstream of aKt , sirt1 has been shown to interact with tsC2 , a member of the mammalian target of rapamycin complex . 105 sirt1 negatively regulates mtor signaling , implying that targeting sirt1 would enhance mtor activity , which is contraindi - cated in cancer therapy . to complicate the situation further , sirt1 is phosphorylated ( ser47 ) by mtor , resulting in the inhibition of sirt1 catalytic activity . 106 more recently , it was shown that the aKt / mtor pathway is activated in brCa1 - mutated cancers mediating tumorigenesis . 107 brCa1 mutant - dependent breast cancers were found to have decreased sirt1 and increased survivin expression compared to controls . 108 overexpression of sirt1 hindered brCa1 - dependent cancer formation in vitro . survivin and other parts of the Wnt signaling pathway have been shown to foster the proliferative state of stem cells . 109 sirt1 was reported to deacetylate β - catenin , leading to inhibition of proliferation of colon cancer cells . 39 the non - oncogenic cytosolic form of β - catenin was found to be enhanced by sirt1 , coinciding with co - localization of nuclear sirt1 and the oncogenic form of β - catenin in a cohort of 81 human colon tumors . 39 however , in another study , sirt1 shrna caused a reduction in Dishevelled proteins DVl - 2 and DVl - 3 in t47D and heK - 293 cells , suggesting multiple regulation points in the Wnt signaling by sirt1 . 110 stem cell function highly depends on the transcription factor environment . for example , c - myc is part of a transcription factor network that can reverse the differentiated phenotype back to pluripotent and proliferative stem cells ( reviewed in Papp and Plath 111 ) . in the same way described for the p53 - hiC1 - sirt1 axis and its function on the sirt1 promoter , the oncogenic transcription factor c - myc constitutes a negative feedback loop with sirt1 leading to downregulation of c - myc activity , another potential antitumor effect . 112 in light of the putative stem cell function of c - myc , sirt1 - mediated c - myc downregulation in cancer stem cells may lead to differentiation , a trait highly aligned with a potent tumor suppressor function . SIRT1 as tumor promoter / oncogene there is evidence that sirt1 may play an oncogenic role by inactivation of tumor suppressors and / or activation of onco - genic proteins . hiC1 ( hypermethylated in cancer 1 ) is a tumor suppressor that can be either epigenetically silenced or deleted in various cancers . 113 , 114 hiC1 constitutes a circular regulatory feedback loop with p53 and sirt1 , such that inactivation / absence of hiC1 derepresses sirt1 , triggering sirt1 activa - tion and p53 inactivation via deacetylation . 115 , 116 hic1 + / – , apc + / Δ716 double - heterozygous mice have increased numbers of intestinal polyps , leading to hyperplasia . 117 if indeed the hiC1 - sirt1 - p53 loop were in effect , this would provide indirect evidence for sirt1 as a tumor promoter ( i . e . , in the context of hiC1 and aPC ) . sirt1 may also promote oncogenesis via the stabilization of n - myc . n - myc induces sirt1 transcription in neuroblastoma , which then enhances n - myc stability in a positive feedback loop . 118 this is thought to be accomplished by sirt1 - induced repression of mKP3 phosphatase , leading to increased erK phosphorylation ( i . e . , decreased de - phosphoryl - ation ) and then n - myc ser62 phosphorylation , which protects n - myc from degradation . 118 these observations are seemingly contradictory to the tumor suppressor activities of sirt1 men - tioned previously . one could speculate that specific genetic disruptions ( i . e . , such as seen with hic1 ) effect a molecular switch , turning sirt1 from a tumor suppressor to an oncogene . for n - myc , the specific mutation effecting this switch has yet to be elucidated . some of the most intriguing animal data for sirt1 as an oncogene come from the recent 2011 american association for Cancer research ( aaCr ) meeting , where serrano 119 reported that sirt1 transgenic crossed with Pten - null mice produced aggressive thyroid and prostate carcinomas . these thyroid tumors were enriched in cmyc and cmyc targets ( but not p53 , Stünkel and campbell 1158 www . slas . org Journal of biomolecular Screening 16 ( 10 ) ; 2011 p65 , or β - catenin ) and displayed elevated p - aKt levels . more studies will be required to understand the interaction of the Pi3k / mtor pathway , sirt1 , and myc . however , this is the first evidence clearly demonstrating an oncogenic role for sirt1 in vivo . an alternative oncogenic hypothesis is that cancer cells develop addictive mechanisms by which sirt1 overexpres - sion is coupled with aberrant silencing of tumor suppres - sors . 120 addition of transforming growth factor β ( tgfβ ) , known to stimulate epithelial - to - mesenchymal transition ( emt ) , to immortalized human mammary epithelial cells ( htert - hmes ) produces a profound elevation in sirt1 and n - cadherin , as well as a substantial reduction in e - cadherin via direct epigenetic silencing . 121 e - cadherin levels were restored by sirt1 depletion in vitro . anchorage - independent growth and migration of htert - hme cells are inhibited by either sirt1 shrna or mir - 200a . sirt1 and mir - 200a dis - play an inverse relationship , as sirt1 is overexpressed whereas mir - 200a is depressed in breast cancer biopsies . 121 these authors suggest that mir - 200a functions as a tumor suppressor in breast cancer and that reduction in mir - 200a removes the repression of sirt1 , producing sirt1 overex - pression , thus supporting induction of emt . other ways sirt1 could influence tumor suppressor function is via the interaction with other oncogenes such as ski . ski interacts with sirt1 , increasing the binding of p53 to sirt1 and sta - bilizing the p53 - sirt1 interaction . Deacetylation of p53 ( by sirt1 ) is enhanced , which suppresses p53 - dependent tran - scriptional activation . 122 as ski facilitates the p53 - sirt1 interaction , the authors speculated that ski may convey sirt1 substrate selectivity . given the multiplicity of activities attrib - uted to sirt1 and subcellular localization , it seems quite plausible that adapter proteins might exist to associate sirt1 with specific substrates and effector functions . this role for sirt1 , however , is only relevant in those tumors that have intact / competent p53 . sirt1 silencing is reported to cause apoptosis of many tumor lines ( colorectal , breast , cervical ) but not noncancerous lines ( epithelial origin : arPe - 19 , htb - 125 ; and normal fibro - blasts [ nDf ] ) via a p53 - , bcl - 2 - and bax - independent mecha - nism . 123 in the hCt116 colorectal tumor line , co - silencing of foxo4 and sirt1 , but not foxo3 , rescues the cells from apoptosis 123 ( i . e . , suggesting foXo4 is required for the proap - optotic effect in the absence of sirt1 ) . it is quite possible then that the differential sensitivity to sirt1 sirna ( cancer vs . normal ) has a genetic basis , but the enabling molecular target ( s ) are as yet undetermined . in pancreatic cancer , sirt1 levels are inversely correlated with patient survival . sirt1 knockdown in pancreatic tumor lines induces g1 growth arrest and apopto - sis ( miaPaCa , PanC1 ) . 124 sirt1 interference also decreases viability and clonogenic survival of human melanoma tumor lines ( a375 , hs294t , g361 ) 97 and inhibits proliferation of breast ( mCf7 ) and nsClC ( h1299 , p53 - deficient ) cells . 125 , 126 it should be noted that knockdown of both sirt1 and sirt2 was required to induce cell cycle arrest and kill mCf - 7 cells in vitro , 125 suggesting that dual sirt1 / 2 inhibitors might be required for maximal efficacy in breast cancer . sirt1 has been linked to mechanisms of Dna repair , puta - tively serving to protect normal cells against potential carcino - genic agents and / or environmental stresses . another possibility is that during malignant growth , sirt1 provides an antiapop - totic stimulus to maintain the aberrant growth and lifetime of the transformed cells . o’hagan et al . 127 have postulated that chronic exposure to inflammation and / or carcinogens incurring Dna damage could initiate epigenetic silencing of tumor sup - pressor genes . using their exogenous gene promoter model ( containing the Cpg island segment of e - cadherin ) , these investigators found that both sirt1 and eZh2 were transiently recruited to the Dna break site , as well as Dnmt1 and Dnmt3b . Dna methylation and engagement of Dnmt3b were dependent on sirt1 . 127 albeit infrequent in occurrence , gene silencing and Dna methylation were observed in the Cpg island region . these authors provide a compelling argu - ment for sirt1 involvement in aberrant Cpg island Dna methylation and gene silencing contributing to the initiation and / or maintenance of cancer . this may explain why sirt1 and eZh2 proteins are more abundant in cancerous ( Nkx3 . 1 – / – ; Pten – / – mouse model ) versus normal prostate tissue 128 and per - haps also the association of sirt1 overexpression with CimP - high ( Cpg island methylator phenotype ) and microsatellite instability in colorectal cancer . 96 upon Dna damage , the cell triggers a number of key proteins that begin the process of Dna repair and maintain genomic integ - rity , including Ku70 , tiP60 , and e2f1 . Ku70 , a protein that binds to the ends of Dna double - strand breaks and initiates nonhomolo - gous end - joining , is deacetylated by sirt1 . 45 in a model system of radiation - induced Dna damage ( luciferase reporter gene ) , sirt1 expression enhanced annealing of Dna breaks in vitro ; this was not seen using dominant - negative , catalytically inactive sirt1 cells . 45 as such , sirt1 could be acting to enhance Dna repair and cell survival by deacetylation of Ku70 . tiP60 , tat - interacting protein 60 kD , is a histone acetyltransferase required for double - stranded Dna break ( DsDb ) repair following radia - tion . 129 , 130 tiP60 and the mre11 - raD50 - nbs1 ( mrn ) complex act cooperatively to activate atm kinase following Dna damage . mrn is thought to direct atm and tiP60 to the site of DsDbs , where tiP60 and atm become activated . 129 h2aX is then phos - phorylated by atm ( ser139 ) and acetylated ( lys5 ) by tiP60 , 54 , 131 causing cell cycle arrest and apoptosis . tiP60 also acetylates p53 ( K120 ) , enhancing the binding of p53 to proapoptotic genes . 54 sirt1 acts as a negative regulator of tiP60 , directly deacetylating tiP60 , inhibiting tiP60 activation , and promoting tiP60 degrada - tion . 54 , 131 as such , one could envision sirt1 acting to dampen the apoptotic response and promote tumor survival . Sirtuin 1 ( SIrt1 ) Journal of biomolecular Screening 16 ( 10 ) ; 2011 www . slas . org 1159 a common recurring theme in sirt1 and cancer is that of promoting cellular resistance toward damaging conditions . sirt1 has been associated with drug resistance 120 or oxidative stress . 25 , 27 , 29 the interaction between e2f1 ( i . e . , a “proapop - totic” factor induced by Dna damage ) and sirt1 perhaps illustrates the conditional nature of sirt1 activity . sirt1 is elevated following etoposide - induced Dna damage , and this is thought to be mediated by e2f1 ( stabilized by atm phospho - rylation ) . 132 e2f1 is also deacetylated by sirt1 and may even recruit sirt1 to its own promoter . this suggests that a feed - back loop between sirt1 and e2f1 may exist to finely control the Dna damage response . in u2os cells co - transfected with sirt1 and e2f , sirt1 was found to inhibit the apoptotic activity of e2f1 . 132 this effect was also seen with hCt116 p53 – / – cells indicating a p53 - independent mechanism . in h1299 non – small cell lung carcinoma , sirt1 knockdown or addition of a nonselective sirt inhibitor , nicotinamide , sensitized cells to etoposide - induced apoptosis . 132 sirt1 may also function to cultivate a microenvironment conducive to tumor growth and metastasis . sirt1 is highly expressed in the nuclei of vascular endothelium . 133 rna silencing of sirt1 , but not sirt - 2 , - 3 , or - 5 , abolishes endothelial cell migration and sprout formation in vitro . the deacetylase activity of sirt1 is required for these activities as a dominant negative mutant [ h363Y ] mimics the effects of sirt1 sirna . 133 as one might expect from the in vitro obser - vations , knockout of sirt1 in mice ( endothelial cell specific : Tie2Cre tg ; SIRT1 flox / – ) or zebrafish ( antisense ) produced a blunted angiogenic phenotype . subsequent data from this laboratory revealed that sirt1 , via deacetylation of nCiD , acts as a negative regulator of notch signaling in endothelial cells . 32 notch is ubiquinated and degraded under deacetylation conditions , so sirt1 could conceivably control the levels of notch . as notch coordinates various aspects of blood vessel growth , the authors speculated that sirt1 may act as “a rheo - stat” to finely tune notch responses in endothelial cells . 32 Cortactin is an f - actin binding protein and src substrate that , in its acetylated form , attenuates cell migration . Cortactin has been shown to potentiate metastasis in breast and esophageal cancer models . 134 , 135 sirt1 deacetylates cortactin and pro - motes cell migration in vitro , 48 suggesting a role for sirt1 in metastasis . pHArMAcoLogIcAL ModuLAtIon oF SIrt1 Enzymatic assays there are now many assay formats to screen sirt1 enzyme for both activators and inhibitors . these include fluores - cence , 136 – 139 time - resolved fluorescence , chemiluminescence , 140 microfluidic mobility shift , 140 fluorescence polarization , 141 mass spectrometry , 141 , 142 capillary electrophoresis , 143 high - per - formance liquid chromatography ( hPlC ) , 7 , 144 and radioac - tive 145 – 147 formats . these are described in more detail within this issue . 142 SIRT1 activators sirt activators are being pursued in hopes of providing benefit to patients with neurodegenerative , inflammatory / autoimmune , and metabolic disease ( perhaps also cancer in certain tumor types ) . however , the most widely published sirt1 activators have caused a great deal of controversy . it is questioned whether many of these compounds are de facto sirt1 activators or assay artifacts . this debate has arisen from a combination of issues associated with enzymatic assay format and compound selectivity . one of the earliest assays used for sirt1 screening , commercialized by biomol ( Plymouth meeting , Pa ) as fluor de lys , 148 contained a flu - orescent - labeled peptide ( p53 - amC ) for a substrate . the first sirt1 activators identified with this assay were poly - phenolic plant - derived compounds : resveratrol , butein , quercetin , piceatannol , and myrcetin ( table 2 ) . 148 , 149 subsequently , much more potent and efficacious sirt1 acti - vators were reported as potential therapeutics for the treat - ment of diabetes ( e . g . , srt1720 , srt2183 , and srt1460 ) , 141 using a fluorescently labeled substrate in a fluorescence polarization assay . it was realized quickly that sirt1 bio - chemical activation by resveratrol could only be demon - strated when the substrate was fluorescently tagged . 150 – 152 using a variety of nonfluorometric assay formats , it was found that all of these four compounds ( resveratrol , srt1720 , srt2183 , and srt1460 ) did not activate sirt1 when using native peptide or protein substrates . 152 , 153 furthermore , srt1720 and srt2183 decreased acetylated p53 levels in sirt1 - deficient ( sirna ) and null ( sirt – / – mefs ) cells , again suggesting sirt1 was not the direct target . one unconfirmed report claimed that srt1720 and srt2183 , but not resveratrol , were actually p300 hat inhibitors , account - ing for their cellular effect on p53 acetylation in the absence of enzymatic modulation . 153 the value of resveratrol , srt1720 , srt2183 , and srt1460 as sirt1 activators becomes even more dubious when taking into account that they inhibit quite a number of nonepigenetic targets ( e . g . , g - protein - coupled receptors [ gPCrs ] , enzymes , and trans - porters ) . 152 the selectivity of piceatannol for sirt1 is also questionable as it inhibits tyrosine kinases such as lck 154 and mitochondrial atP synthase ( as do other polyphenols ) . 155 it is evident that more highly selective sirt1 activators , which show correlative activity in both enzymatic and cellular sirt1 - dependent assays , are still needed to understand sirt1 biology and therapeutic potential . Stünkel and campbell 1160 www . slas . org Journal of biomolecular Screening 16 ( 10 ) ; 2011 table 2 . small - molecule activators of sirt1 SRT1720 RESVERATROL SRT2183 SRT1460 MYRICETIN BUTEIN QUERCETIN PICEATANNOL C C C OH OH O OH OH O OH OH OH OH O OH OH OH OH OH OH O OH OH OH O OH OH OH OH OH N N O NH N S N N NH O CH 3 O C H 3 O NH N S N N NH O CH 3 O NH N S N N OH SRT1720 RESVERATROL SRT2183 SRT1460 MYRICETIN BUTEIN PICEATANNOL C C C OH OH O OH OH O OH OH OH OH O OH OH OH OH OH OH O OH OH OH O OH OH OH OH OH N N O NH N S N N NH O CH 3 O C H 3 O NH N S N N NH O CH 3 O NH N S N N OH Fold Rate Increase @ 100 µM [ refs . 148 , 149 ] EC 1 . 5 ( % Max ) * [ ref . 142 ] butein 8 . 53 myrcetin 3 . 19 Piceatannol 7 . 90 Quercetin 4 . 59 , 2 . 15 resveratrol 13 . 4 , 4 . 66 46 . 2 µm ( 201 % ) srt1720 0 . 16 µm ( 781 % ) srt2183 0 . 36 µm ( 296 % ) srt1460 2 . 90 µm ( 447 % ) * Concentration of compound required to increase enzyme activity by 50 % . SIRT1 inhibitors a selective ( vs . other hDaCs ) sirt1 inhibitor , eX527 ( aka sen - 196 ) , was initially reported with an iC 50 ≈ 100 nm in enzymatic assays . 156 eX527 ( 1 µm ) was later shown to increase p53 [ K382 ] acetylation in the presence of etoposide ( but not in the absence ) and had no effect on the expression of p53 target genes , cell viability , or proliferation in various tumor lines . 157 this result was quite unexpected as both sirt1 knockdown and overexpression of catalytically inac - tive sirt1 mutants induce proliferation , apoptosis , and / or Dna damage repair repression in cancer cell lines . 24 , 45 , 97 , 123 , 125 , 126 one possible explanation is that the antitumor effects observed with knockdown are independent of catalytic activity and that the point mutation inactivating the enzyme also disrupts the protein – protein interactions that are indeed responsible for sirt1 activity . p53 is a protein harboring multiple acetyla - tion sites , 158 and it is worthwhile noting that the same lysine residue in p53 ( K382 ) is also targeted by sirt7 , another member of the sirt family . 159 this may reflect a functional redundancy , although the role and activity of sirt7 are poorly established . a new selectivity concept for sirtuin inhibitors in oncology has emerged , based on the observation that multi - isoform sirt inhibitors such as cambinol , 160 sirtinol , 126 salermide , 161 Jgb1741 , 162 suramin analogs , 163 and the tenovins 164 are effective in inducing cancer cell death ( table 3 ) . these observations were recently strengthened by simultaneous rnai - mediated gene silencing of both sirt1 and sirt2 , which potently induced apoptosis in mCf - 7 breast cancer cells , whereas the individual knockdowns were ineffective . 125 in addition , both dual sirt1 / 2 inhibitors sirtinol and salermide , but not eX527 , induced p53 [ K382 ] acetylation and stabilization in mCf7 cells . Pharmacological inhibition of sirt1 and sirt2 is also effica - cious in xenograft models , as exemplified by tenovin - 6 ( arn - 8 melanoma ) 164 and cambinol ( Daudi burkitt lymphoma ) . 160 this lends credence to the belief that dual sirt1 / 2 inhibition may provide therapeutic benefit in cancer . there are an increasing number of publications reporting the discovery of novel dual sirt1 / 2 inhibitors 165 – 167 as well as inhibitors of sirt1 – 3 . 168 it remains to be seen whether dual - or broad - spectrum sirtuin inhibitors may prove to be efficacious and safe in the clinic . concLuSIon it is evident that a great deal has been learned about the biology of sirtuins since their original discovery in yeast . although classified as type iii histone deacetylases , the sir - tuins are both deacetylases and aDP - ribosyltransferases , with substrates that include nonhistone proteins . there is evidence that several sirtuins translocate between subcellular Sirtuin 1 ( SIrt1 ) Journal of biomolecular Screening 16 ( 10 ) ; 2011 www . slas . org 1161 table 3 . small - molecule inhibitors of sirt1 JGB - 1741 CAMBINOL SALERMIDE TENOVIN - 6 SPLITOMYCIN EX527 NICOTINAMIDE SIRTINOL Cl NH O NH 2 NH 2 O N O NH N OH O O O NH N OH NH S NH NH O N O OH NH NH S O OH N S NH O JGB - 1741 CAMBINOL SALERMIDE TENOVIN - 6 SPLITOMYCIN EX527 NICOTINAMIDE SIRTINOL Cl NH O NH 2 NH 2 O N O NH N OH O O O NH N OH NH S NH NH O N O OH NH NH S O OH N S NH O SIRT1 IC 50 ( µM ) SIRT2 IC 50 ( µM ) naDh 17 000 205 ( sir2 ) 11 000 205 ( human ) nicotinamide 95 . 1 , 125 130 205 ( hst2 ) 1 . 16 , 125 32 . 3 206 eX527 0 . 16 , 207 0 . 38 , 125 0 . 098 156 48 . 5 , 207 32 . 6 , 125 19 . 6 156 splitomycin ≈100 , 162 * 60 , 146 * 74 208 salermide 76 . 2 125 45 125 sirtinol 131 , 209 37 . 6 125 57 . 7 , 209 103 . 4 125 Cambinol 40 . 7 , 167 57 . 9 , 207 56 160 47 . 9 , 167 40 . 7 , 207 59 160 Jgb1741 15 162 > 100 162 tenovin - 6 21 164 10 164 references listed as superscripts above the iC 50 value . * Yeast sir2 enzyme used ; all others human sirt enzyme . compartments depending on stimuli and form various com - plexes that can be highly regulated by both positive and negative feedback circuits . sirt1 is to date the best studied of the sirtuins . sirt1 is involved in the regulation of meta - bolic , neuronal , immune , and tumor function ( Figs . 1 , 2 ) . however , despite the multitude of data , both molecular and pharmacological , the outcome of selective sirt1 modulation in vivo is still not completely clear . Data from various for - mats explored for both sirt1 enzymatic and cell - based assays suggest great caution should be exercised in assay construction and interpretation of the data . in the case of sirt1 activator assays , the presence of a fluorophore - tagged substrate in certain assays has led to misleading results . it is difficult to reconcile the lack of biological activity for the sole potent and selective sirt1 inhibitor , eX527 , with the opposing results of sirt1 catalytically inactive mutants . moreover , other published inhibitors exert promiscuity with equivocal evidence for inhibition of sirt1 - dependent dea - cetylase activity as the sole mechanism behind the pheno - typic response observed in cancer cells . Perhaps other sirt1 - selective and / or multi - isoform selective compounds will shed light on this situation . advances in structural biol - ogy for the sirtuin family will be quite impactful in guiding further development of isoform - selective and / or broad - spec - trum sirt inhibitors . Co - crystallization with a chemical probe compound ( i . e . , not just apo - protein ) may better aid in understanding the potential binding modes of activators / inhibitors and facilitate medicinal chemistry strategies . it should be noted that although the structures for sirt1 from several lower species have been solved ( table 1 ) , a crystal structure for human sirt1 is yet to be reported . Known sir - tuin isoform crystal structures are outlined and referenced in table 1 . in drug discovery , sirt1 cell - based assays are potentially complicated by the multitude of substrates—that is , which substrate ( s ) is driving the biology / phenotype in question in that specific cell type ? Do they differ between cell types and stimuli ? is there redundancy between sirtuin isoforms ? Comparative data with specific activators / inhibitors and / or molecular tools ( over - expression , knockdown , catalytically inactive mutants ) will be helpful in this regard . having the appropriate animal models will also be critical to understanding sirt1 biology . for oncology , using xenografts in sirt1 - deficient mice with different genetic backgrounds may produce different results ( e . g . , those with inac - tive hiC1 or Pten ; see table 1 ) . animal model selection may affect patient tailoring strategies in oncology if indeed a preferred genetic background lends itself to sirtuin modulation . in summary , the existing data suggest that sirt1 activators may be useful for the treatment of diabetes / metabolic disease , inflammation , and / or neurodegeneration . however , for cancer , the burden of evidence indicates that broad - spectrum sirt1 / 2 inhibitors may provide therapeutic benefit in select tumor types . these opposing approaches bring up many questions , such as whether sirt1 activators will promote cancer ( no evi - dence thus far ) or whether sirt1 ( and sirt2 ) inhibitors will exacerbate metabolic disease , inflammation , and / or neurode - generation ( data equivocal at best ) . more potent and selective compounds will need to be tested in vivo to unequivocally answer these questions . the preponderance of data is indeed compelling enough to justify drug discovery efforts for both sirt1 activators and sirt1 / 2 inhibitors . Stünkel and campbell 1162 www . slas . org Journal of biomolecular Screening 16 ( 10 ) ; 2011 NCoR PPARy LXR AcCoA - S PGC1α Metabolism UCP2 Lipogenesis Insulin - Sensitivity Neuronal Function Immune Function IRS - 2 α - Secretase Aβ Peptide Neuroprotection Neurite Outgrowth NFκB IL - 8 HIV / Tat AP - 1 Cox2 Inflammation Immunity PTP1B SIRT1 FIg . 1 . Diverse roles of sirt1 in biological pathways . sirt1 is a central regulator with impact on metabolic , neuronal , and immunological functions . sirt1 enhances insulin sensitivity , and aberrant function is associated with the etiology of type 2 diabetes mellitus . in addition , sirt1 has been shown to be neuroprotective and anti - inflammatory , both via deacetylation of nfκb . arrows with diamond - shaped ends show inhibitory action via deacetylation . Tumor Suppressor Function Tumor Promoter Function SIRT1 β - Catenin APC min / + Intestinal Polyps Cell Proliferation BRCA1 mutant AKT mTOR PTEN p53 Cell Survival cMyc CellProliferation HIC1 DBC1 FOXO1 Angiogenesis TIP60 Cell Survival H2AX ~ Ac FIg . 2 . sirt1’s dual roles in cancer . sirt1 functions as a context - dependent tumor suppressor and oncogene . sirt1 has direct repressive activity on a number of cancer - relevant pathways involving mtor , mutated brCa1 , and aPC . however , in the context of mutations ( e . g . , negative regulators such as hiC1 and DbC1 ) , sirt1 activity is enhanced , tipping the balance toward increased cancer cell survival , angiogen - esis , and cell cycle progression . arrows with diamond shaped ends show an inhibitory activity . Sirtuin 1 ( SIrt1 ) Journal of biomolecular Screening 16 ( 10 ) ; 2011 www . slas . org 1163 reFerenceS 1 . horio , Y . ; hayashi , t . ; Kuno , a . ; Kunimoto , r . 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Campbell Eli Lilly & Company Lilly Corporate Center , dc0424 , Indianapolis , IN 46285 E - mail : campbell _ robert _ morris @ lilly . com \ No newline at end of file diff --git a/silver_data/db7390ae8daad5e1eb029d1cc1cfc65a6866178b.pdf.txt b/silver_data/db7390ae8daad5e1eb029d1cc1cfc65a6866178b.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..0d370e79db2231d95686dab365d51c5e8ebe757e --- /dev/null +++ b/silver_data/db7390ae8daad5e1eb029d1cc1cfc65a6866178b.pdf.txt @@ -0,0 +1 @@ +Customizing Lotus Notes to Build Software Engineering Tools Jun Ma , Holger M . Kienle , and Piotr Kaminski University of Victoria Victoria , Canada { majun , kienle , pkaminsk } @ cs . uvic . ca Anke Weber ExperEdge Technology Partners Victoria , Canada weber @ experedge . com Marin Litoiu IBM Toronto Labs Toronto , Canada marin @ ca . ibm . com Abstract Many software engineering research tools are stand - alone applications that have trouble in - teroperating with other development tools and do not fit well into the software developers’ es - tablished work processes . Our main hypothesis is that in order for new tools to be adopted ef - fectively , they must be compatible with both existing users and existing tools . Typically , software engineering teams in an organization share a set of common appli - cations for their development activities that are a permanent part of each developer’s ev - eryday workflow . Among these applications are shrink - wrapped office tools such as Lotus Notes , which are used for , among other tasks , email , scheduling , and project reports and pre - sentations . These office tools , are highly inte - grated and offer a mature , well - tested working environment , which can be customized to an extent that allows to provide support for ad - vanced software engineering tasks . This paper describes RENotes , a reverse engineering tool built by customizing Lotus Notes . RENotes targets software developers who use Notes as part of their work environ - ment . We describe Notes’ features and how they can be leveraged to layer new reverse engineering functionality on top . Keywords : Lotus Notes , customization , end - user programmable systems , tool adop - tion , collaboration , Rigi 1 Introduction It takes a lot of effort to go from the con - ceptual design for a new software engineer - ing technique to the development of tools that supports the technique and finally adoption of the tool in industry . Researchers that strive to have their tools adopted struggle to de - velop tools that satisfy the requirements that software engineers in industry place on them . Common examples of adoption hurdles include difficult installation , lack of documentation , unpolished / awkward user interfaces , and poor interoperability with existing tool infrastruc - ture . As a result , most research tools require a significant learning curve while disrupting the established work process of the software engi - neer . Tool development is a significant investment and should focus on the novel features of the tool . Unfortunately , the development of a base - line environment , albeit often trivial to ac - complish , requires significant effort before tool - specific functionality can be tackled . This is especially true for GUI - based tools that use a visual manipulation paradigm . In this paper , we outline a software devel - opment approach that leverages a widely - used , shrink - wrapped office tool—Lotus Notes— by building a software reengineering tool , RENotes , on top of it . Notes is the host ap - plication that provides the baseline environ - ment , including features such as document - based database , collaboration , search , and se - 1 curity . RENotes leverages Notes to provide software reengineering functionality such as ar - tifact filtering and graph manipulation . We believe that tool implementations that follow this approach have desirable features from the user and developer point of view . Spinellis draws a similar conclusion for the field of visual programming tools [ 21 ] : “As many visual programming en - vironments are research - oriented , proof - of - concept projects , they cannot easily compete with the commercially - developed , polished , and supported commercial IDEs . In contrast , visual programming based on the reuse of proven existing components in a widely adopted IDE levels the playing field and allows research to focus on program representations and methodologies that can increase productivity rather than supporting infrastructure . ” The reminder of the paper is organized as follows . Section 2 gives more background in - formation that guides our research . RENotes , our case study , implements part of the func - tionality of the Rigi reverse engineering tool . Therefore , Section 3 gives a brief introduction of Rigi and its functionality . In Section 4 we further introduce Notes as the host application for the RENotes case study . We first analyze the baseline environment that Notes provides and then discuss in Section 5 in detail how we customized Notes to build RENotes . Sec - tion 6 reviews related work . Finally , Section 7 summarizes our development experiences and RENotes’ potential of improved adoption . 2 Background This section discusses what we mean by tool customization and categorizes both the so - called host applications and the targeted users in more detail in order to provide a better un - derstanding of the requirements of our research . 2 . 1 Tool Customization A prerequisite for our proposed tool develop - ment method is that the host tool offers sophis - ticated customization mechanisms . ( Such tools have been also referred to as user - tailorable computer systems [ 14 ] . ) Support for customization can be divided into non - programmatic and programmatic cus - tomization mechanisms . Non - programmatic customization is accomplished , for example , by editing parameters in startup and configura - tion files or with direct manipulation at the GUI level . Programmatic customization in - volves some form of scripting or programming language that allows the modification and ex - tension of the application’s behavior . Program - matic customization is more powerful , but re - quires a significant effort . There is an initial cost in acquiring the necessary customization skills , followed by development and mainte - nance costs of the customization . It is an interesting question to what extent users customize their applications . Page et al . studied the customization changes that users made to the WordPerfect word processor [ 18 ] . A surprising 92 percent of users in their study did some form of customization , with 63 per - cent using macros . They summarize their find - ings with “users who most heavily used their systems have the highest levels of customiza - tion” and “customization features that were simple to adapt ( like the Button Bar ) tended to have higher incidences of tailoring . ” The above study suggests that tool builders should expect that users want to customize their software ; thus , tools should offer extensive customization support while making it simple and fast to use . An early commercial product that allowed users to create customized applications was Hy - perCard , thus making “everybody a program - mer” [ 8 ] . The Emacs text editor can be cus - tomized both by parameter setting and by pro - gramming in Emacs Lisp . Similarly , the Auto - CAD system can be customized with AutoLisp . An example of a highly customizable research tool is Rigi [ 24 ] [ 23 ] . The Rigi graph editor al - lows customization by exposing its graph model and user interface with a Tcl / Tk API . Further - more , Rigi graphs have a generic model that can be customized to different domains . Spec - ification files describe the kinds of nodes and arcs that constitute a certain domain . Tool builders have recognized the impor - tance of making their software customizable 2 and by now many popular tools ( such as Mi - crosoft Office , Lotus Notes , Adobe Acrobat , and Macromedia Dreamweaver ) offer scripting support and APIs to accomplish customization . This paper focuses on the customization of Notes . The feasibility of customization of Notes has been demonstrated by a number of large - scale projects . One such effort reports the following experiences [ 12 ] : “Users and developers have been pos - itive about Notes . Advantages cited include the relative ease of learning how to develop basic applications , the relatively short development cycle— from a few days to a couple of weeks per applications—and the fact that users’ suggestions and feedback can be incorporated into the applications with relative ease and can be done on a continuous basis . ” 2 . 2 Host Applications The development approach that we describe grafts domain - specific functionality on top of highly customizable tool foundations—we call these tools host applications . There is a broad range of candidates for host applications . In our current research , we focus on office suites ( e . g . , Microsoft Office , Lotus SmartSuite , and OpenOffice ) and productivity tools ( e . g . , Lotus Notes and Microsoft Project ) . From the programmer’s point of view , these tools have commercial - of - the - self ( COTS ) char - acteristics . Other promising candidates are ex - tensible IDEs ( e . g . , Eclipse ) . We classify systems that use COTS compo - nents based on the scheme proposed by Carney [ 2 ] : turnkey : These systems use a single COTS component on which they heavily depend . Typically , customization is quite limited and non - programmatic . intermediate : These systems are also built on a single COTS component , but also “have a number of customized elements specific to the given application . ” The amount of customization can vary , but does not fun - damentally change the nature of the em - ployed COTS component and results in a moderate amount of newly developed cus - tomization code . mixed : These systems contain several ( het - erogeneous ) COTS components to provide large - scale functionality that is otherwise not available . They have a significant amount of glue code and are often difficult to develop and maintain . In our current research , we target intermedi - ate systems ( i . e . , only a single host application is selected and customization is done program - matically ) . 2 . 3 Target Users Another important consideration are the users that the application targets . Karsten [ 11 ] reviewed 18 case studies of organizations’ use of Notes and splits them into three groups : exploratory , expanding , and extensive use of Notes . Organizations with extensive use of Notes had the following commonalities [ 11 ] : “In all these cases , there were several applications that were tied directly into established work practices . Ap - plication development was conducted as a careful process , with prototypes and user involvement . ” Thus , in the best case , the host application is a fundamental part of the users’ work pro - cesses . Evidence of such a mission - critical tool are local developers ( “gardeners” ) that work on customizations for their group . This has been observed , for example , for CAD systems [ 6 ] . An other example is the IBM Toronto Lab , which has a dedicated development group to customize Notes . Host applications that are based on familiar office tools provide a number of potential ben - efits to users : a familiar GUI : The user interacts with a fa - miliar environment and paradigm . Appli - cation knowledge ( a . k . a . cognitive support [ 26 ] ) has been typically built up by the user over years . Since the users are already familiar with the standard functionality , they can concentrate on learning the ad - ditional , domain - specific functionality ( in - crementally ) . 3 tool interoperability : Office tools interoper - ate among each other via cut - and - paste and ( file - based ) import / export facilities . tool support : Popular tools come with a large infrastructure that provides useful information to the user . For example , ( on - line ) publications discuss how to use a tool most effectively . Mailing lists and discus - sion forums help troubleshoot users’ prob - lems . Stand - alone research tools are typically found lacking in all of the areas outlined above . 2 . 4 Selection of Host Applica - tions Selection of a host application is a trade - off de - cision . When deciding on a suitable host ap - plication , one typically has to choose among several possible candidates . A host applica - tion has to satisfy two main criteria : it has to ( 1 ) provide a suitable baseline environment for extension , and ( 2 ) be familiar to its tar - get users . The former criterion shortens the development life cycle while the latter can ac - celerate the adoption . Sometimes , these two criteria may conflict with each other . Our host application for RENotes is Lotus Notes . The decision to select Notes was based on its large user base in companies and its customization flexibility . Although we did not formally eval - uate the trade - offs , our observations and dis - cussions with developer at the IBM Toronto Lab hold up our assumptions . Notes appears to be the most pervasive application within the IBM Toronto Lab . Besides its use as a group and peer - to - peer communication infras - tructure , Notes is the host application for tens of customized applications used by the Lab em - ployees . About seven developers in the Lab are currently involved in development and mainte - nance of these applications . 3 The Rigi Reverse Engi - neering Tool In order to gain experiences and validate our tool development approach , we decided to build a reverse engineering tool on top of Notes that is similar to Rigi as a case study . Rigi has been under development for over a decade in our re - search group at the University of Victoria . Be - cause of our previous tool building experience with Rigi , we are already familiar with the ap - plication domain and can focus on understand - ing customizations with Notes . Figure 1 : The Rigi reverse engineering tool . Rigi is an interactive , visual tool designed for program understanding and software re - documentation . Figure 1 shows a snapshot of Rigi . The core of Rigi is a generic graph editor enhanced with domain - specific functionality for reverse engineering tasks . Rigi uses typed , di - rected graphs to convey information about the target software system . Nodes of the graph represent artifacts of the system ( e . g . , func - tions , variables , and types ) and directed arcs represent artifact relationships ( e . g . , functions calls , assignments to variables , and type decla - rations ) . A simple example of a Rigi view is the subject system’s call - graph . Nodes in the call - graph represent functions of the program and arcs represent calls between functions . Differ - ent kind of nodes and arcs are visualized with different colors . Rigi offers operations to select and filter the arcs and nodes in a graph and al - lows to apply several graph layout algorithms . Even though Rigi can be customized with Tcl scripting , it is a stand - alone application that is not easy to integrate with other tools . Most no - tably , short of taking screenshots , Rigi graphs cannot be exported to other applications . This 4 is a severe drawback , because Rigi graphs are the main work products of the reverse engineer - ing activity , usually becoming part of the sys - tem documentation [ 27 ] . Leveraging office tools to build reverse en - gineering functionality seems a promising ap - proach because the reverse engineering process for a larger legacy system is both document - intensive and collaborative . An important problem is the task of organizing and archiv - ing of obtained reverse engineering results , so that they are available for future system evo - lution . As described in Section 4 , Notes has features that address these problems . 4 Lotus Notes / Domino Lotus Notes / Domino is a popular groupware product that is used by many organizations [ 10 ] . It is important to realize that Notes has more to offer than email support . In fact , it is often mentioned as the main software for sup - porting collaboration and its use is extensively studied by researchers in the area of computer supported collaborative work [ 11 ] . Based on the client / server model , Lotus Notes / Domino is often used to construct an in - ternal information platform for an organization [ 4 ] . In this platform , all information is rep - resented as documents , which are stored and organized in databases . Domino acts as the server , providing database access ; Notes hosts the client applications that access the database . In Notes , shared databases are the backbone that enables collaboration among members in an organization . For many organizations Notes is a critical part of their IT infrastructure . An example is Statoil , a Norwegian state - owned oil com - pany [ 17 ] . Statoil has a full company license making it one of the world’s largest users of Notes . Five years after its introduction in 1992 , Notes had diffused to most of the company’s 17000 employees , and its use was made manda - tory . Statoil’s geographical distribution ( 40 - 50 sites ) makes collaboration - based features at - tractive . The following collaborative features of Notes are leveraged : email , document manage - ment , workflow , electronic archive , group cal - endar , news and bulletin boards , and discussion databases . IBM itself is another example of a company that uses Notes extensively . From our obser - vations at the IBM Toronto Labs , Notes has been customized for a wide variety of applica - tion , ranging from a simple carpool database to a project application that allows a devel - opment team to organize the work - products of their project . With the Domino Web server , Notes applications can be published on the Web . When an HTTP request accesses a database , Domino translates the document to HTML . Thus , certain applications can be made accessible with both Notes and a standard Web browser . At the Toronto Lab , Domino servers within the intranet allow Web access to appli - cations . 4 . 1 Features Before deciding on a host application , it is im - portant to understand its key features . The host’s baseline functionality and customization mechanisms determines its suitability for build - ing domain - specific functionality on top of it . Figure 2 : Lotus Notes mail application . The user leverages the following features of Notes’ baseline environment when using RENotes : User interface : Notes has a mature ( if id - iosyncratic ) user interface that has been consecutively refined over six major re - leases . Figure 2 shows a snapshot of the 5 latest release , Lotus Notes 6 . The user in - terface includes a menu bar , a bookmark bar , a tool bar , a status bar and a set of window tabs . Tabs make it easy for user to switch to different applications and databases . Most of the GUI elements can be customized . Furthermore , each tab has its own set of task - specific menu items and buttons . The mail tab , for example , has a button to compose a new email ( “New Memo” ) and a pull down menu with sev - eral options on how to reply to the cur - rently selected email ( “Reply” ) . Document - based database ( DBD ) : The documents in each database are organized with views and folders . In Notes , every document is a basic data unit that con - tains a set of data fields . In this respect , a document is similar to a record in a relational database ; however , a document in Notes can be composed of an arbitrary number of fields . Since all documents adhere to this schema , it is possible to access , manage , and manipulate diverse documents in a uniform manner . Notes databases support many data types , including text , pictures , sound , video , file attachments , embedded objects , and applets . Script automation : Users often take advan - tage of customization to automate work . A study about the customization behavior of 51 users in a Unix environment found that users “were most likely to customize when they discovered that they were do - ing something repeatedly and chose to au - tomate the process” [ 13 ] . Notes provides users agent and actions to accomplish automation . Actions are inte - grated into the GUI and activated by users ( e . g . , by pressing a button ) . Agents , which run on the server , can be triggered by cer - tain events ( e . g . , expiration of a timer ) . Collaboration : Typical Notes applications are message exchange , information shar - ing , and workflow . By sending and receiv - ing emails , members exchange messages in an organization . By accessing document in shared databases on servers , distributed information exchange , retrieval , storage , and consolidation is facilitated . Workflow applications guide users through certain tasks that they have to perform as part of their work . Such guidance can reduce overhead and mistakes , thus speeding up processes . Search : Notes has automatic search capabili - ties as well as full - text indexing support . Users can give keywords in the search bar to retrieve matching documents sorted by significance . For example , users can search a certain folder in the mailbox database . Security : In many large organizations , access to information in databases needs fine - grained access control as well as secu - rity mechanisms for authentication . Ev - ery Notes user has a digital ID and access can be granted at different level , from the server down to individual document fields . 5 RENotes Case Study RENotes is our reverse engineering application that we built as a case study to gain experiences with our approach to tool - building . It lever - ages Notes features wherever possible , supple - menting them with custom functionality where needed . In this section , we describe RENotes’ architecture and implementation , list the sup - posed benefits to its adoption , and relate our experiences with implementing and using the application . 5 . 1 Architecture and Implemen - tation RENotes has been implemented with standard three - layer architecture . Its major components are shown in Figure 3 . RENotes represents the structure of the system under examination with a typed , directed , attributed graph , similar to the one used in Rigi ( see Section 3 ) . The graph is initially produced using existing source code parsers ( e . g . , Rigi’s cparse for C , or the CPPX fact extractor for C + + [ 3 ] ) and saved in Graph Exchange Language ( GXL ) format [ 9 ] . GXL is a XML - based exchange format popular in the reverse engineering community . Its syntax is 6 defined with an XML Document Type Defini - tion ( DTD ) . Figure 3 : RENotes’ layered architecture . The generated GXL file can be imported into RENotes as a Notes database through a Java agent . In order to perform the transformation , the GXL file is parsed into an XML Document Object Model ( DOM ) tree . The XML DOM tree is then traversed and elements are con - verted into the Domino Object Model . Notes exposes its internal state with the Domino Object Model [ 25 ] . This model al - lows programmatic access to and manipulation of the databases and application services . It has been implemented for a broad range of lan - guages , including the Formula language , Lo - tus Script , Visual Basic , JavaScript , and Java . Each object defines a set of properties and methods . For example , the NotesDocument ob - ject represents a document in a database and has the method AppendItemValue to add a new field to the document . The graph to database mapping is simple : each node and arc is mapped to a separate new document with an automatically gener - ated unique identifier . The type and other attributes of each graph element are saved in the corresponding document’s fields . Figure 4 shows a database with a small graph of 14 nodes . During the import , the graph is also checked against a source code language - specific domain schema , encoded in XML and held in a separate Notes document . This ensures that the graph is well - formed and meaningful so that other tools can use it safely . Once the data has been imported , users can manipulate the documents with all the Figure 4 : Nodes in a sample RENotes database . usual Notes tools . They can search for spe - cific nodes or arcs by keyword ( cf . Figure 5 ) , or create filtered , sorted views of the ( auto - matically indexed ) database based on complex queries . The documents can also be accessed through Notes’ standard automation features , allowing users to write ad - hoc scripts to per - form more complex operations such as bulk at - tribute changes or transitive closures on the system’s directed relationships . The user can also select from a set of predefined scripts that perform typical reverse engineering tasks such as to find all callers of a function or accessors of a field . These existing scripts provide use - ful templates as a starting - point for users that want to write their own scripts . Access to the RENotes databases is con - trolled by Notes’ security features ; RENotes defines some common user roles with various degrees of privilege , restricting users’ actions with fine granularity . All this functionality is leveraged unchanged from Notes and should be familiar to its users . 5 . 2 Visualization The generic textual list views provided by Notes are often not optimal for exploring the structure of a system , so RENotes provides a custom - built graphical visualization . The user 7 Figure 5 : Keyword search of a RENotes database . first selects a subset 1 of nodes to visualize . Ref - erences to the nodes are gathered into a per - spective document from which the user can pop up the RENotes graph browser ( cf . Figure 6 ) . Figure 6 : Visualization of a RENotes database . The browser—written in Java using the open - source , Swing - based JGraph [ 1 ] graph editing toolkit and embedded in the RENotes database—provides a visual representation of the nodes and all relationships between them . All graphical elements are connected to the un - derlying Notes documents , using the domain 1 The subset may , in fact , be the whole graph . schema to map their properties to visual at - tributes . The browser offers basic navigation , manipulation ( most notably filtering of nodes and arcs ) , and layout controls to help the user investigate the system’s structure . When the user exits the graph browser , its state is saved in the corresponding perspective document , encoded as XML . This allows the developer to resume exploration of the graph in a subsequent session . Each perspective docu - ment thus represents a separate persistent view on the system graph ; the same node may be in - dependently present in many perspectives . The graph visualization can also be saved in Scal - able Vector Graphics ( SVG ) format [ 5 ] , so that it can be embedded into other documents or shared with people who are not using RENotes or do not have the access privileges necessary to open a perspective document . 6 Related Work There are other office tools besides Notes that are promising candidates for host applications . In related projects in our group we extend Mi - crosoft Visio and PowerPoint to visualize and manipulate Rigi graphs [ 16 ] . Other research , discussed in the following , has leveraged office tools as well to build software engineering func - tionality . Desert is an open tool environment con - sisting of several loosely coupled components [ 19 ] . One of these components is a spe - cialized editor for source code and architec - ture documentation . This editor is based on Adobe FrameMaker and uses a wide variety of FrameMaker’s functionality , such as syntax highlighting with fonts and colors , graphic in - sets , and hypertext links . FrameMaker is ex - tended via the Frame Developer’s Kit API . Riva and Yang have developed a software documentation process that uses Rigi to visu - alize software artifacts [ 20 ] . They used Rigi’s scripting capabilities to export this information to Visio as a UML model . The authors take also advantage of Visio’s ability to export Vi - sio UML drawings as HTML to Web - enable the documentation . The Visual Design Editor ( VDE ) is a domain - specific graph editor implemented with 8 VisualBasic on top of PowerPoint [ 7 ] . VDE personalizes PowerPoint with new pull - down menus and icons . The authors state : “Power - Point offers a highly functional GUI for inter - actively designing presentation graphics . Vir - tually every part of that GUI is useful , without modification , as part of our design editor . ” Tilley and Huang report on their experiences with an industrial client in implementing a soft - ware visualization and documentation system in Visio [ 22 ] . Visio was selected after evalu - ating the visualization capabilities of several candidate tools . The authors were constrained in their technology choices by the client’s poli - cies . For example , ”the company reasonably requested that professional support be avail - able for whichever tools were selected . This requirement immediately ruled out almost all academic and research tools . ” Among the iden - tified benefits of Visio was that the client al - ready employed Visio in their development pro - cess and had a set of custom - developed stencils to represent their software artifacts . 7 Conclusions Even at this early stage , the RENotes project has generated some interesting insights and po - tential benefits to adoption , though more work is necessary to evaluate the effectiveness of our approach . 7 . 1 Development Experience Building our application in Lotus Notes rather than stand - alone has greatly reduced our de - velopment effort . The benefits of reusing Notes functionality more than offset the overhead of adapting to a new development environment , reducing lines of code by an ( estimated ) order of magnitude . This also allowed us to build sev - eral prototypes to verify and test the feasibility of our ideas . The JGraph framework proved also very helpful : the graph browser application weighs in at less than 4000 lines of code . By com - parison , Rigi has about 30000 lines of C / C + + code , though it provides a richer feature set than RENotes does . Overall , the majority of the effort was directed at leveraging the po - tential the Notes environment provides , as op - posed to writing new code from scratch . As with any framework , there are also some limitations . Notes does not let Java clients control its user interface , preventing the graph browser from tightly binding visualized node selection to a Notes document view . While other APIs ( for example , the Notes C API ) may afford more control , we did not consider these languages suitable for rapid development of re - liable software . There are also potential issues with scaling RENotes to handle larger systems . While we did not try large - scale experiments , we are cautiously optimistic on this count since Notes’ database kernel has been refined and op - timized over the last decade . 7 . 2 Benefits to Adoption RENotes made substantial strides towards im - proved adoption by building on top of Notes . Notes’ relatively large user base ( as compared to research prototypes ) means that support is plentiful and basic training easy to obtain . Since RENotes reuses many of Notes’ features , the learning curve is significantly lowered for existing users , and even new users benefit from Notes’ support network . Notes also has a ma - ture , coherent user interface and many con - venient tools , such as search and filters . To - gether with Notes’ popularity , this should make RENotes more appealing to domain experts with no reverse engineering experience . Notes support for ad - hoc end - user pro - grammability is also critical to reverse engi - neering efforts . Many reverse engineering tasks are tedious to perform manually and , if so ac - complished , cannot be recorded and reused . Notes is fully scriptable and even offers users a choice of programming languages that cover the spectrum of formality , letting them pick a tool appropriate for the task at hand . The abun - dance of scripting options also makes it more likely that a user already knows at least one of them , further lowering the barrier to adoption , and comparing favorably with research proto - types that normally support at most one script - ing method . Building on Notes also lets us leverage its support for collaboration , which should become more important as reverse engineering projects 9 grow more complex . RENotes databases also integrate well into the Notes workspace and can be linked to other databases employed by the user . By providing GXL import and SVG ex - port capabilities , RENotes also integrates well with applications outside Lotus Notes , making it easier to fit into an existing workflow . 7 . 3 Future Work In future work , we will add more of the Rigi functionality and reverse engineering capabili - ties to RENotes . Furthermore , we would like to conduct a user study with software engineers at the IBM Toronto Lab to gain a better under - stand if , and how , RENotes lowers the adop - tion barrier . Furthermore , we want to observe how effectively Notes’ baseline environment is utilized by RENotes users and how users will integrate RENotes into their work environment and processes . The work on RENotes described in this pa - per is part of ACRE V1 . 0 [ 16 ] , the first ver - sion of the software evolution environment un - der development at the University of Victoria as part of our Adoption - Centric Reverse Engi - neering ( ACRE ) project [ 15 ] . In addition to RENotes it consists of several other software visualization engines on top of various office products ( e . g . , Microsoft Excel , PowerPoint , and Visio ) . We plan to compare and evaluate our experiences with these approaches and use the implementations for user studies targeted to the question of how to further ( industrial ) adoption of research tools . Acknowledgments This work has been supported by the Natu - ral Sciences and Engineering Research Council of Canada ( NSERC ) , the Consortium for Soft - ware Engineering ( CSER ) , and the Center for Advanced Studies ( CAS ) , IBM Canada Ltd . About the Authors Jun Ma is a Master student in Computer Sci - ence at the University of Victoria , Canada . He received a B . E . from the Harbin Institute of Technology in China . His research interests include software engineering , XML technology , and CSCW . Holger M . Kienle is a Ph . D . student in Computer Science at the University of Victoria , Canada . He received a Master of Science degree in Computer Science from University of Mas - sachusetts Dartmouth and a Diploma in Infor - matics from University of Stuttgart , Germany . His interests include software reverse engineer - ing , exchange formats for re - engineering , pro - gram analyses , and domain - specific languages . Piotr Kaminski is a Ph . D . student in Com - puter Science at the University of Victoria , Canada . He received a Master of Science de - gree in Computer Science from the University of Victoria , and a Bachelor of Mathematics from the University of Waterloo , Canada . His interests include software engineering , aspect - oriented programming , the semantic web and human - computer interaction . Anke Weber is a principal of ExperEdge Technology Partners , an IT consulting com - pany based in Victoria , Canada . Prior to co - founding ExperEdge , she has gained a variety of experiences in the IT field in positions as a co - ordinator for a European Union - funded project at the Paderborn Center for Parallel Computing , as a technical editor and writer at dSPACE Inc . , an international supplier of tools for developing and testing new mecha - tronic control systems , and most recently as a Software Engineering Research Associate in the Department of Computer Science at the Uni - versity of Victoria , Canada . She received her Master’s Degree in Computer Science from the University of Dortmund in Germany . Among here many roles , she appreciates most to work as a designer , editor , and writer in both the technical and non - technical worlds . Her cur - rent research interests include adoption - centric software engineering , web site evolution , and “live” systems documentation . Dr . Marin Litoiu is member of the Centre for Advanced Studies at the IBM Toronto Lab - oratory where he initiates and manages joint re - search projects between IBM and Universities across the globe in the area of Application De - velopment Tools . Prior to joining IBM ( 1997 ) , he was a faculty member with the Department of Computers and Control Systems at the Uni - versity Politechnica of Bucharest and held re - 10 search visiting positions with Polytechnic of Turin , Italy ( 1994 and 1995 ) , and Polytechnic University of Catalunia ( Spain ) , and the Euro - pean Center for Parallelism ( 1995 ) . Dr . Litoiu’s other research interests include distributed ob - jects ; high performance software design ; perfor - mance modeling , performance evaluation and capacity planning for distributed and real time systems . References [ 1 ] Gaudenz Alder . JGraph home page . http : / / jgraph . sourceforge . net / . [ 2 ] David Carney . 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Improving adoptabil - ity by preserving , leveraging , and adding cognitive support to existing tools and en - vironments . 3rd International Workshop on Adoption - Centric Software Engineer - ing ( ACSE 2003 ) , pages 36 – 41 , May 2003 . [ 27 ] Kenny Wong , Scott R . Tilley , Hausi A . M¨uller , and Margaret - Anne D . Storey . Structural redocumentation : A case study . IEEE Software , 12 ( 1 ) : 46 – 54 , January 1995 . 12 \ No newline at end of file diff --git a/silver_data/de6d6f330b0741252e942bf69c583b2a7f07d676.pdf.txt b/silver_data/de6d6f330b0741252e942bf69c583b2a7f07d676.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..4bfd2e930709cf5dbc5015a54d91e22156d1b499 --- /dev/null +++ b/silver_data/de6d6f330b0741252e942bf69c583b2a7f07d676.pdf.txt @@ -0,0 +1 @@ +Crusade for iron : iron uptake in unicellular eukaryotes and its significance for virulence Robert Sutak 1 , Emmanuel Lesuisse 2 , Jan Tachezy 3 and Des R . Richardson 1 1 Department of Pathology and Bosch Institute , Blackburn Building ( D06 ) , University of Sydney , Sydney , NSW 2006 , Australia 2 Laboratoire d’Inge´nie´rie des Prote´ines et Controˆle Me´tabolique , De´partement de Biologie des Ge´nomes , Institut Jacques Monod , Unite´ Mixte de Recherche 7592 CNRS - Universite´s Paris 6 and 7 , Tour 43 , 2 place Jussieu , F - 75251 Paris cedex 05 , France 3 Department of Parasitology , Charles University , Vinicna 7 , 128 44 Prague 2 , Czech Republic The effective acquisition of iron is a pre - requisite for survival of all organisms , especially parasites that have a high iron requirement . In mammals , iron homeostasis is meticulously regulated ; extracellular free iron is essen - tially unavailable and host iron availability has a crucial role in the host – pathogen relationship . Therefore , patho - gens use specialized and effective mechanisms to acquire iron . In this review , we summarize the iron - uptake systems in eukaryotic unicellular organisms with particular focus on the pathogenic species : Candida albicans , Tritrichomonas foetus , Trypanosoma brucei and Leishmania spp . We describe the diversity of their iron - uptake mechanisms and highlight the importance of the process for virulence . The crusade for iron Iron is an essential nutrient for almost all living organ - isms . Its importance originates from the flexibility of redox potentials available to iron by its varied interactions with coordinating ligands and from its capacity to undergo electron transfer and acid – base reactions [ 1 ] . Iron is a co - factor of a variety of proteins that can be classified according to the coordination chemistry of the metal : ( i ) haemoproteins , which function as O 2 carriers , O 2 activa - tors or electron - transfer proteins ; ( ii ) iron – sulfur cluster ( ISC ) proteins , involved mainly in electron transfer ; and ( iii ) non - haem , non - iron – sulfur , iron - containing proteins , including enzymes and proteins involved in iron transport and storage [ 1 ] . Host - iron availability has a crucial role in the host – pathogen relationship . As a general anti - microbial defence mechanism , mammals possess an elaborate iron - withhold - ing system that effectively reduces the amount of iron accessible to pathogenic microorganisms . Iron is predomi - nantly intracellular , and the limited amount of extracellu - lar iron is tightly bound to proteins such as transferrin and lactoferrin [ 2 ] . Therefore , invading pathogens use special - ized and effective mechanisms to acquire iron , and iron availability is often found to have an important role in virulence . Some potential invaders that have impaired iron - acquisition ability cause disease primarily in iron - loaded hosts [ 3 ] . Unlike that in pathogenic bacteria , little is known about iron uptake and its intracellular metabolism in unicellular eukaryotic pathogens . Here , we summarize the available knowledge on iron uptake in : Candida albicans , a pathogenic organism related to Saccharomyces cerevisiae ; Tritrichomonas foe - tus , a parasitic protist with a high requirement for iron ; Leishmania spp . , another group of parasites with both intra - and extra - cellular parts of their life cycles ; and , finally , Trypanosoma brucei , a flagellate parasite that dis - plays unique expression of transferrin receptor genes . Although they are not evolutionarily closely related , these organisms share several features regarding iron metab - olism : ( i ) they are unicellular , taking up iron from their environment and not re - distributing iron between cells ; ( ii ) they have no classical ferritin for iron storage ; ( iii ) they are eukaryotic , with iron being delivered and / or stored in various organelles ; and ( iv ) they are pathogens and need to ‘fight’ for nutrients in a harsh , iron - limited host environ - ment . Moreover , they often occupy diverse niches during their life cycle , such as different tissues or even different organisms during transmission between the host and vec - tor . Hence , these pathogens require multiple uptake sys - tems for a variety of iron sources . These particular organisms were deliberately chosen as examples because they represent some of the most well characterized uni - cellular eukaryotic pathogens in terms of iron - assimilation mechanisms . Metabolism of iron in mammalian cells To ensure iron availability and to eliminate the toxicity of free iron in addition to its accessibility for invading patho - gens , mammals have evolved a strictly regulated system for iron homeostasis ( for review , see Ref . [ 2 ] ) . Free iron is essentially unavailable in blood and extracellular fluids . In plasma , circulating iron is bound to transferrin , whereas , in external secretions such as colostrum , iron is bound to lactoferrin [ 2 ] . The uptake of diferric transferrin is mediated by transferrin receptor - 1 ( TfR1 ) , which facilitates the internalization of the transferrin – TfR1 complex through endocytosis [ 2 ] . Iron is released in early endosomes that are acidified by a proton pump , which reduces the pH to (cid:2) 5 . 5 , resulting in dissociation of iron from transferrin [ 2 ] . The metal ion is then transported through the endosomal membrane via the divalent metal ion transporter 1 ( DMT1 ) Review Corresponding author : Richardson , D . R . ( d . richardson @ pathology . usyd . edu . au ) . 0966 - 842X / $ – see front matter (cid:2) 2008 Elsevier Ltd . All rights reserved . doi : 10 . 1016 / j . tim . 2008 . 03 . 005 261 [ 4 ] . Once within the cytosol , excess iron is stored within ferritin , a polymer of 24 subunits that forms a cage - like structure that is capable of containing up to 4500 iron atoms [ 5 ] . More recently , a mitochondrial ferritin has been ident - ified [ 6 ] , although its biological role is unclear . The only cellular iron exporter that has been identified under phys - iological conditions is ferroportin - 1 [ 7 ] , a transporter at the plasma membrane that transports iron to the extracellular space . Iron metabolism is meticulously regulated and this is ensured , in part , by two iron regulatory proteins [ 8 ] . These molecules bind to iron - responsive elements ( IREs ) of mRNAs that encode proteins involved in iron uptake ( TfR1 and DMT1 ) , utilization ( erythroid d - aminolevulinic acid synthase ) , storage ( ferritin ) and export ( ferroportin - 1 ) . Another key player in the regulation of systemic iron homeostasis is the circulating peptide hormone hepcidin [ 9 ] . Hepcidin regulates cellular iron efflux by binding to ferroportin - 1 and inducing its internalization [ 2 ] . Pathogenic microorganisms use two general strategies for iron acquisition from the host environment . The first depends on direct contact between the pathogen and the host iron source and , mostly , iron is released from the protein extracellularly [ 10 , 11 ] . The second strategy uses secretion and subsequent uptake of small iron - binding molecules called siderophores ( Figure 1 ) , which are able to remove iron from host protein – iron complexes because of their high - affinity for the metal [ 10 , 11 ] . The competition for iron between the host and the pathogen is an exciting and complex phenomenon , which includes a variety of different tactics on both sides . Indeed , this can be seen from the fact that , although lactoferrin has anti - microbial activity , it serves as an iron source for many microbes [ 12 ] . In response to poor iron availability in the host , pathogens produce siderophores to take the element from host proteins . In response , the mammalian protein lipocalin - 2 binds to several kinds of siderophores , prevent - ing the pathogen from accessing siderophore - bound iron [ 13 ] . However , to evade this strategy , some bacteria pro - duce a glycosylated form of the siderophore enterobactin , preventing its sequestration by lipocalin - 2 [ 14 ] . The pathogenic yeast Candida albicans A wide variety of unicellular ( yeasts ) and filamentous fungi ( Box 1 ) have developed sophisticated mechanisms of iron uptake [ 10 ] . Among these organisms , C . albicans is the object of numerous studies because it is an important opportunistic human pathogen . The non - pathogenic yeast , S . cerevisiae , has long been used as a model to study eukaryotic iron metabolism ( Figure 2 ) and several exten - sive reviews are devoted to iron metabolism in this organ - ism ( e . g . see Refs [ 15 – 17 ] ) . Studies based mainly on the search for S . cerevisiae homologues and functional comple - mentation of the corresponding S . cerevisiae mutants led to the identification of two main strategies of iron uptake in C . albicans : reductive and non - reductive ( siderophore - mediated ) [ 18 – 20 ] . A third mechanism of iron import and haem uptake has been identified in C . albicans but not S . cerevisiae [ 21 , 22 ] . These three mechanisms are finely regulated at the transcriptional level , according to iron availability . As in most fungi ( but not in the model yeast S . cerevisiae ) , transcriptional regulation of the ‘iron regulon’ in C . albicans is mediated by a GATA - type repression factor ( Sfu1 ) [ 23 , 24 ] . Another molecule , Tup1 , a global regulator of morphology and metabolism , also has a major role in iron - dependent gene regulation and acts differently on genes involved in reductive and in non - reductive iron uptake [ 19 , 25 ] . Considering the role of Tup1 , it can be hypothesized that different iron - uptake systems might be Figure 1 . Hydroxamate - type siderophores . Low - molecular weight ( < 1500 Da ) ferric - specific chelates produced by microorganisms under iron limitation are called siderophores . Siderophores can be divided into different classes depending on the chemical nature of the oxygen ligand moieties responsible for Fe ( III ) coordination . These are catecholates , hydroxamates or hydroxycarboxylates . The siderophores synthesized and / or used by most fungi are mainly hydroxamates [ 68 ] . Box 1 . Iron acquisition by fungi Among fungi , species with yeast and filamentous forms are found in different phyla . Several species are pathogenic in humans , espe - cially for individuals with impaired immunity . These organisms live in a broad range of ecological niches , and often exhibit life cycles whereby unicellular forms alternate with mycelius ( or filamentous ) forms . Iron - acquisition strategies of fungi essentially consist of siderophore production and / or use , reductive iron - uptake and haem - uptake systems [ 16 ] . Some species are siderophore - generat - ing ( i . e . Aspergillus , Neurospora , Schizosaccharomyces , Rhodotor - ula and Ustilago ) , whereas others are not ( i . e . Saccharomyce s , Candida and Pichia ) [ 33 ] . It has long been thought that only siderophore - generating fungi are able to take up iron in the form of their own native siderophores . However , it is now recognized that many species , including those that cannot produce any side - rophores , can take up xenosiderophores , that is , siderophores produced by other species ( bacteria and fungi ) [ 33 ] . Siderophore production is not directly related to pathogenicity . Some non - pathogenic fungi can produce siderophores ( e . g . Schi - zosaccharomyces pombe ) [ 73 ] and some pathogenic species cannot produce siderophores ( e . g . Candida albicans ) [ 33 ] . However , when a siderophore - producing species is pathogenic , the synthesis of these chelators is often required for virulence ( e . g . Aspergillus fumigatus ) [ 66 ] . The reductive iron - uptake system ( see description in the section ‘the pathogenic yeast Candida albicans ’ in the main text ) was first described in Saccharomyce s cerevisiae as an alternative mechanism to the siderophore - uptake strategy [ 74 ] . Further studies showed that this uptake system is present in most fungi and can be required for virulence ( e . g . in C . albicans and Cryptococcus neofor - mans ) [ 18 ] . This is a well - conserved copper - dependent iron - uptake system , which is also found in higher eukaryotes and enables cells to acquire iron from almost all extracellular ferric complexes , except haem . Haem uptake is probably a widespread mechanism of iron acquisition in pathogenic fungi , but it is still poorly documented . Iron use from haem requires the porphyrin ring to be opened via a reaction catalysed by haem oxygenase [ 75 ] because iron cannot be removed from this molecule by reduction . Review Trends in Microbiology Vol . 16 No . 6 262 used according to the morphological form of C . albicans ( yeast or filamentous form ) . Reductive iron uptake by yeasts : coupling specific permeation to non - specific reduction of iron S . cerevisiae employs two systems of iron acquisition , namely a low - and high - affinity iron - uptake mechanism [ 15 ] ( Figure 2 ) . The utilization of each system is dependent on the availability of iron in the environment [ 26 ] . For both systems , the first step involves reduction of iron by ferrir - eductases ( Fre1 and Fre2 proteins ) that are plasma mem - brane flavo - haemo - proteins capable of reducing a wide variety of ferric complexes , including siderophores [ 15 , 27 ] . This is a low - specificity and low - affinity step , which enables cells to convert structurally diverse extra - cellular ferric complexes into the common ferrous state [ 16 ] . When extracellular iron is in excess , a non - ATP - de - pendent trans - membrane transporter ( Fet4 ) is the main component of the low - affinity transport mechanism [ 28 ] ( Figure 2 ) . The Fet4 - dependent low - affinity transport sys - tem found in S . cerevisiae does not seem to be present in C . albicans , probably because the metabolism of the latter is based upon respiration ( and not fermentation ) , whereas Fet4 is mainly required under anaerobic conditions [ 29 ] . By contrast , when iron is limiting , the high - affinity system is used , which involves an oxidase – permease complex composed of the Fet3 and Ftr1 proteins in the plasma membrane [ 10 ] . The Fet3 component of the Fet3 – Ftr1 complex is a multi - copper ferroxidase that converts ferrous to ferric iron [ 26 ] , and Ftr1 is a permease necessary for high - affinity iron uptake [ 30 ] ( Figure 2 ) . Biogenesis of Fet3 in the late - or post - Golgi compartment requires efficient copper uptake by cells ( mediated by the Fre reductases and the cuprous permease , Ctr1 ) and the activity of an intracellular cupric transporter ( Ccc2 ) [ 21 , 31 ] . Therefore , mutants lacking Ctr1 or Ccc2 copper transport activity are defective in high - affinity reductive iron import [ 21 , 32 ] . Figure 2 . Iron - uptake mechanisms in the model yeast Saccharomyces cerevisiae . During reductive uptake , extracellular ferric complexes are reduced by the ferrireductases ( Fre1 and Fre2 ) and the ferrous iron that is released is taken up via the low - affinity divalent metal permease ( Fet4 ) or via the high - affinity oxidase – permease system ( Fet3 – Ftr1 ) , which requires copper as a co - factorof Fet3 . Extracellular cupricions ( Cu 2 + ) are reduced by the same reductases ( Fre1 or 2 ) and the cuprous ions enter the cell via Ctr1 . Intracellular copper participates in the biogenesis of Fet3 in a post - Golgi compartment . Intracellular iron is stored in the vacuole , from which it can be mobilized by a similar reductive mechanism involving homologous proteins ( i . e . Fre6 , Fet5 and Fth1 ) . ‘Ligands’ are chemical moieties that bind to ferric iron to form coordination complexes , which can be labilized by iron reduction ( with the notable exception of haem , for which the ligands have nearly the same affinity for ferric and for ferrous iron ) . Blue arrows represent the fate of proteins ; green arrows represent the fate of elements . Abbreviations : HA , high - affinity iron uptake ; LA , low - affinity iron uptake . During non - reductive uptake , siderophore transporters ( Sit1 , Arn1 , Arn2 and Enb1 ) mediate the uptake of various siderophores ( FOB , ferrioxamine B ; FCH , ferrichrome ; TAF , triacetylfusarinine ; ENB , enterobactin ) . Some siderophore transporters ( e . g . Sit1 and Arn1 ) cycle from endosomes to the vacuole , where they are degraded , whereas other transporters become targeted to the plasma membrane if their specific substrates are sensed by the cell . Review Trends in Microbiology Vol . 16 No . 6 263 In the reductive strategy of iron uptake , an apparent paradox is that iron has to be reduced ( via Fre ) before being re - oxidized ( via Fet3 ) to enter the cell ( via Ftr1 ) [ 15 , 30 ] . However , this reduction and oxidation of iron does not result in a futile cycle . Ferroxidation – permeation is a well conserved process involved in iron transport across mem - branes [ 10 , 15 ] . The role of the reduction step is most probably to release iron from its ferric ligands in such a way that the cells can acquire iron from a broad variety of ferric compounds with only one high - affinity uptake sys - tem [ 15 ] . Non - reductive uptake of siderophores by yeasts : an opportunistic strategy C . albicans has a specific transporter for ferrichrome - type siderophores , Arn1 ( also known as Sit1 ) ( Figure 2 ) , but it is probably unable to produce its own siderophores because it lacks the genes required for hydroxamate ligand biosyn - thesis [ 33 ] . Similarly to S . cerevisiae , C . albicans can use siderophores produced by other microorganisms [ 34 ] . This opportunistic strategy of iron uptake might be important in some ecological niches where several microorganisms co - exist . Siderophore transporters in fungi are well - conserved integral membrane proteins ( with 13 or 14 potential mem - brane - spanning regions ) that bind to specific siderophores and mediate their import in a high - affinity uptake process [ 10 ] ( Figure 2 ) . However , the mechanism of siderophore import is still poorly understood . It is unclear whether the siderophore transporter functions as a classical permease or as a siderophore receptor that enters the cell by endo - cytosis after binding to its specific substrate [ 35 , 36 ] . In S . cerevisiae , some siderophore transporters accumulate in post - Golgi compartments and are targeted to the plasma membrane in response to the presence of their specific substrate in the extracellular medium [ 17 ] . By contrast , other siderophore transporters are constitutively targeted to the plasma membrane [ 17 ] . This latter scenario seems to prevail for the Arn1 transporter of C . albicans [ 35 ] . The intracellular fate of siderophores is largely unknown and remains an important area of research . Intracellular iron release from the siderophores probably occurs by reduction in an acidic environment , which makes the vacuole a good candidate for this process . Iron uptake and virulence of yeasts During systemic infection , possible iron sources for C . albicans are haem and transferrin . This pathogen was shown to produce haemolytic factors [ 37 ] and is able to use haem as an iron source [ 21 , 22 ] . The enzyme , haem oxygenase ( Hmx1 ) , which is regulated by haemoglobin and iron , is required for iron assimilation from haemin [ 22 , 38 ] . Moreover , surface haem - binding proteins involved in haemin and haemoglobin - iron utilization have been identified [ 39 ] . The role of haem uptake in virulence and the mechanism of haem import remain poorly understood . Transferrin has also been shown to be an iron source for C . albicans [ 40 ] . In fact , iron uptake from transferrin was shown to occur via the reductive pathway , but nothing is known about the mechanism of how this protein is recognized by Candida and how it crosses the cell wall [ 40 ] . Although the iron - uptake systems in Candida seem somewhat redundant ( at least three high - affinity iron - uptake systems ) , particular components of at least two of them might be essential for successful development of infection in the host [ 18 , 41 ] . While deletion of the high - affinity iron permease gene ( FTR1 ) abolished the capacity of Candida to establish systemic infection in mice [ 18 ] , siderophore uptake mediated by the Arn1 transporter was required for colonizing epithelial layers , but not in the infection of the bloodstream [ 41 ] . It is probable that the three different iron - uptake systems help Candida to cope with the variability of iron abundance and form in different environments during infection . Tritrichomonas foetus T . foetus is a bovine parasitic protist that is the causative agent of bovine trichomoniasis , which is a sexually trans - mitted disease leading to abortion and eventually to per - manent infertility [ 42 ] . This organism has high requirements for extracellular iron ( 50 – 100 m M ) [ 43 ] , prob - ably owing to the importance of ISC proteins such as ferredoxin , pyruvate : ferredoxin oxidoreductase and hydro - genase in pyruvate metabolism [ 44 ] . The core metabolic pathway in which ISC proteins are involved takes place in the hydrogenosome , an ATP - producing double - membrane - bound organelle . Iron availability markedly influences carbohydrate metabolism of T . foetus . Under iron - restricted conditions , hydrogenosomal metabolism dramatically decreases and the dominant product of glu - cose breakdown becomes ethanol [ 44 ] . The cells then become sensitive to omeprazole , an inhibitor of pyruvate decarboxylase [ 45 ] . Pyruvate decarboxylase is the rate - limiting enzyme in the production of ethanol , which is overexpressed in iron - restricted T . foetus cells [ 45 ] . Involvement of iron in the virulence of T . foetus has been examined in an experimental mouse infection by the moderately virulent KV - 1 strain of this organism ( (cid:2) 5 % mortality rate ) [ 46 ] . Administration of ferric ammonium citrate to infected mice increased the mortality rate up to a level determined for the highly virulent LUB - 1MIP strain ( (cid:2) 80 % mortality rate ) [ 46 ] . Consistent with this obser - vation , the strain with lower virulence clearly showed lower efficiency in accumulating iron [ 46 ] . T . foetus is able to take up and use iron from a wide range of sources ( Figure 3 ) . In an initial study , it was shown that the cells bind both lactoferrin and transferrin , but only the binding of lactoferrin to the cell surface is inhibited by an excess of unlabelled protein , indicating a receptor - mediated process [ 47 ] . Further investigations demonstrated that iron uptake from these proteins is mediated by two different systems . Iron uptake from lac - toferrin is mediated by endocytosis , whereas iron acqui - sition from transferrin occurs via extracellular release , probably by acidification of the cell micro - environment [ 43 ] ( Figure 3 ) . The dynamics and properties of iron uptake from transferrin are similar to those from the low molecu - lar weight chelate , iron – nitrilotriacetic acid [ 48 ] . Iron from low molecular weight complexes is first reduced to divalent iron at the plasma membrane or in the reducing environ - ment of the cell , and is then taken up by a transport system with a dissociation constant of 2 . 7 m M [ 48 ] ( Figure 3 ) . Review Trends in Microbiology Vol . 16 No . 6 264 Interestingly , this uptake mechanism was shown not to be iron - regulated . Moreover , it was demonstrated that T . foetus also acquires iron from various siderophores by a non - specific and non - saturable mechanism , which prob - ably involves pinocytosis and removal of iron from side - rophores in acidic intracellular vesicles [ 49 ] ( Figure 3 ) . T . foetus is also able to use iron from haem , probably in a process involving haem oxygenase because traces of bili - verdin ( a by - product of haem breakdown ) are found in the growth medium when haemin is the iron source [ 49 ] . Similar sources of iron can be used by the related human parasite , Trichomonas vaginalis , which causes one of the most frequent sexually transmitted diseases worldwide , trichomoniasis [ 42 ] . The growth of the parasite has been shown to be supported by low molecular weight iron , lactoferrin , haemoglobin , cytochrome c and ferritin , but not transferrin [ 50 ] . Specific receptor - mediated binding was suggested for lactoferrin and haemoglobin [ 50 , 51 ] . However , the mechanisms of iron release from the host proteins and its uptake has not been studied in detail . In conclusion , the metabolism and virulence of T . foetus strongly depends on iron availability and , paradoxically , this parasite seems to lack an iron - regulated high - affinity system for iron uptake . This is probably compensated by the ability of the pathogen to use a large variety of iron sources in its environment . Trypanosoma brucei T . brucei is a parasite responsible for the fatal disease known as sleeping sickness in humans and Nagana in domestic livestock in Africa [ 42 ] . These flagellated protists are transmitted between mammalian hosts by the tsetse fly , Glossina spp . [ 42 ] . After transmission , they reside first in the bloodstream , lymphatic system and interstitial spaces and then , during the latter stages of infection , in the central nervous system [ 42 ] . African trypanosomes obtain iron from host transferrin [ 52 ] . The uptake occurs in the flagellar pocket and is mediated by a hetero - dimeric glycosylphosphatidylinosi - tol - anchored receptor consisting of subunits encoded by expression site - associated genes ESAG - 6 and ESAG - 7 [ 52 ] . These genes are located in poly - cistronic transcription units called variant surface glycoprotein ( VSG ) expression sites and are co - transcribed with a gene encoding the VSG protein , forming the surface coat of the parasite [ 52 ] . During chronic infection , T . brucei evades the mammalian immune response by continuously changing a homo - geneous VSG coat . T . brucei has (cid:2) 20 VSG expression sites , only one of which is fully active at a time , and each expression site seems to contain functional copies of the ESAG - 6 and ESAG - 7 genes [ 53 , 54 ] . The transferrin recep - tors encoded by different expression sites vary in their affinities for transferrin from different host species , and switching between expression sites probably helps trypa - nosomes cope with the diversity of transferrins in the hosts they colonize . Indeed , cultivation of trypanosomes expres - sing a receptor with low - affinity for canine transferrin in canine serum results in selection of trypanosomes expres - sing transferrin receptor with higher affinity for canine transferrin [ 55 ] . However , it is not clear whether trypano - somes with low - affinity transferrin receptors need to switch to another expression site encoding a high - affinity receptor to take up sufficient iron for growth in vivo . When dissociation constant values of transferrin recep - tors and the concentration of serum transferrin are com - pared and considered in conjunction with the iron requirements of the trypanosome , it seems unlikely that the bloodstream form of this parasite would be iron - deprived [ 56 ] . It can be hypothesized that high - affinity transferrin receptors are important to enable sufficient iron uptake in the presence of antibodies against transfer - rin receptors formed in a chronic trypanosome infection . However , a recent study examining the host immune response during chronic murine trypanosomiasis showed that anti - transferrin receptor antibody titres are probably too low to prevent sufficient iron uptake by the parasites [ 57 ] . Nevertheless , sensing iron levels is effective in try - panosomes . Within 3 h of transferrin starvation ( i . e . within half a division time ) , an increase in the synthesis Figure 3 . Proposed mechanism of iron uptake by Tritrichomonas foetus from various sources . Iron from transferrin ( Tf ) and non - siderophore , non - haem , low - molecular weight complexes ( L – M – W ) are released in the reducing , acidic environment of the cells and free iron is taken up by an unknown transport system ( T ) . Iron from lactoferrin ( Lf ) is acquired via endocytosis mediated by a specific binding protein ( BP ) . Siderophore - bound iron is non - specifically pinocytosed . Both lactoferrin - and siderophore - bound iron is released within acidic intracellular vesicles . Review Trends in Microbiology Vol . 16 No . 6 265 of transferrin receptor is detectable [ 58 ] . Surprisingly , there is no information about iron uptake in the host vector , although it is likely that trypanosomes have to adapt to different iron conditions when they switch from the mammalian to insect host . Leishmania Leishmania are trypanosomatid parasites that cause leish - maniasis , a complex of diseases that afflict > 12 million people [ 42 ] . Leishmania spp . are digenetic parasites requiring a sand fly vector and a mammalian host to complete their life cycle [ 42 ] . Amastigotes ( non - motile intracellular forms ) live inside the phago - lysosomal system of mammalian macrophages , whereas promastigotes ( motile extracellular forms ) multiply in the sand fly gut [ 42 ] . As expected from the diversity of the environments Leishmania encounters during its life cycle , this parasite is able to use iron from several sources , such as transferrin , lactoferrin or haemin [ 59 ] . Investigation of iron uptake in Leishmania chagasi promastigotes showed non - specific binding of transferrin and lactoferrin to the organism [ 60 ] . Promastigotes preferentially acquire iron in a reduced , rather than an oxidized , form [ 60 ] . In addition , NADPH - dependent iron reductase activity has been detected in Leishmania , indicating reductase - dependent iron uptake from transferrin and lactoferrin [ 60 ] . In contrast to transferrin and lactoferrin , haemoglobin binds to L . donovani promastigotes in a saturable manner via a specific receptor , the binding being independent of the presence of transferrin , haemocyanin or myoglobin [ 61 ] . Furthermore , free globin , haemin and haem - containing a - and b - chains of haemoglobin do not compete with the binding , indicating that the receptor recognizes holo - haemoglobin only [ 61 ] . Following initial binding to the sites localized within the flagellar pocket , haemoglobin is rapidly internalized [ 61 ] . The endocytosis of haemo - globin is regulated by small GTP - binding proteins of the Rab family [ 62 ] . Furthermore , the fusion of early endo - somes containing haemoglobin with late endosomes requires a signal mediated through the cytoplasmic tail of the haemoglobin receptor [ 62 ] . Besides haemoglobin binding , there is evidence for the existence of a receptor on Leishmania mexicana that facilitates binding of haem or other metalloporphyrin compounds to promastigote cells [ 63 ] . The presence of such systems in Leishmania is consistent with the fact that this parasite lacks a com - plete haem biosynthetic pathway and must acquire this essential nutrient from exogenous sources . Duringthe intracellular stageofits lifecycle , Leishmania has to deal with the harsh environment of the mammalian phagolysosome . Divalent metal ions are thought to be removed from the phagolysosome by the natural resist - ance - associated macrophage protein 1 ( Nramp1 ) transpor - ter [ 64 ] . This protein influences the susceptibility of mammalian cells to intracellular pathogens [ 64 ] . In such an iron - restricted environment , it can be hypothesized that Leishmania amastigotes possess a highly effective iron - uptake system . Indeed , in a recent study , the Leishmania iron transporter ( LIT ) member of the ZIP ( Zrt , Irt - like protein ) family of ferrous iron transporters LIT1 was described in Leishmania amazonensis [ 65 ] . Thistransporter is detectable only in amastigotes that replicate intracellu - larly and is essential for the development of pathogenic lesions in mice , but dispensable for growth and differen - tiation in axenic culture [ 65 ] . Concluding remarks and future perspectives Our knowledge of iron - uptake mechanisms by fungi has improved markedly in the past few years and this is largely owing to the use of the yeast S . cerevisiae as a tool . This easy to manipulate microorganism has proven to be a good model for both reductive and non - reductive iron uptake . Certainly , a better understanding of the molecular mech - anisms involved in siderophore uptake by pathogenic fungi should lead to the development of new anti - fungal agents . These could be based on the inhibition of siderophore synthesis [ 66 ] or on the ‘trojan horse approach’ , which consists of coupling anti - fungal drugs with siderophores , enhancing drug accumulation within pathogenic cells [ 67 ] . This therapeutic strategy was recently shown to be unsuc - cessful using S . cerevisiae [ 35 ] , probably because this yeast tightly compartmentalizes intracellular siderophores within the vacuole , preventing the siderophore – drug con - jugate reaching its target . However , this same approach could be promising considering the many fungi that seem to use intracellular siderophores as cytosolic iron - storage depots [ 68 ] . Far less is known about iron uptake in parasitic protists , although substantial progress has been achieved in the understanding of these mechanisms ; for example , in Leish - mania , for which iron - transport systems have been described for both the intra - and extra - cellular forms of the organism . However , despite the importance of effective iron uptake for virulence , surprisingly little is known about iron acquisition in other unicellular eukaryotic pathogens , especially those with serious medical implications . For instance , there is very limited information available on iron uptake by Trypanosoma cruzi . This flagellate parasite is a causative agent of Chagas disease , which has an estimated infection prevalence of 13 million [ 42 ] . Here , we have not described the mechanisms of iron assimilation of Plasmodium , the most important human parasitic protist , because the subject remains controversial and requires a separate detailed analysis that is beyond the scope of this review . In fact , the exact source of iron for Plasmodium growth remains unclear , although the importance of iron for its survival is evident because several iron chelators have anti - malarial activity [ 69 ] . The abundance of haemoglobin in erythrocytes , where Plasmodium spends the major part of its life cycle , makes this protein the most probable iron source , but this still remains unclear . In addition , the presence of haem oxyge - nase , which is required for the effective release of iron from haem , has never been confirmed in this parasite . Moreover , the stable end - product of haem catabolism in Plasmodium , hemozoin , does not seem to be metabolically available because it is an insoluble complex of haem units [ 70 ] . Another mystery of unicellular eukaryote iron metab - olism is iron storage . Once the cell takes up iron , it must be safely sequestered because it can participate in the gener - ation of reactive oxygen species . In most organisms , this is Review Trends in Microbiology Vol . 16 No . 6 266 ensured by iron - storage proteins , mainly ferritin [ 5 ] . The majority of unicellular eukaryotes , such as yeast and parasitic protists , do not possess ferritin , thus the mech - anism of intracellular iron storage in these organisms is not clear . In yeast , the vacuole seems to have the function of an iron - storage compartment [ 71 ] . However , in the case of T . foetus , the majority of iron incorporated from the iron – nitrilotriacetate complex remains in the cytosol [ 72 ] . The lack of a major iron - binding protein within these organ - isms reveals another question : what form does iron take between its uptake and incorporation into target proteins or distribution between different compartments ? It is important to complete our knowledge of the mech - anisms of iron metabolism in unicellular eukaryotic patho - gens because this might lead to the development of novel chemotherapeutic strategies . Without effective mechan - isms for acquisition and utilization of iron , no organism can survive , especially parasites that have intense iron requirements . Acknowledgements We thank David Lovejoy , Rosei Siafakas , Danuta Kalinowski , Erika Becker and Jan Slapeta for their comments on the article before submission , and Zaklina Kovacevic for her additional proof - reading . D . R . R . thanks the NHMRC for fellowship and grant support . R . S . acknowledges a Postdoctoral Fellowship from the University of Sydney . J . 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Biochemistry 45 , 14772 – 14780 AGORA initiative provides free agriculture journals to developing countries The Health Internetwork Access to Research Initiative ( HINARI ) of the WHO has launched a new community scheme with the UN Food and Agriculture Organization . As part of this enterprise , Elsevier has given hundreds of journals to Access to Global Online Research in Agriculture ( AGORA ) . More than 100 institutions are now registered for the scheme , which aims to provide developing countries with free access to vital research that will ultimately help increase crop yields and encourage agricultural self - sufficiency . According to the Africa University in Zimbabwe , AGORA has been welcomed by both students and staff . ‘‘It has brought a wealth of information to our fingertips’’ , says Vimbai Hungwe . ‘‘The information made available goes a long way in helping the learning , teaching and research activities within the University . Given the economic hardships we are going through , it couldn’t have come at a better time . ’’ For more information , visit www . aginternetwork . org Review Trends in Microbiology Vol . 16 No . 6 268 \ No newline at end of file diff --git a/silver_data/e1b357565f362b6ec31a55a638379214b1d8424f.pdf.txt b/silver_data/e1b357565f362b6ec31a55a638379214b1d8424f.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..3a215936c47527e97e51e818ba2f2c877803adfa --- /dev/null +++ b/silver_data/e1b357565f362b6ec31a55a638379214b1d8424f.pdf.txt @@ -0,0 +1 @@ +Testing for the Bradley - Terry - Luce Model Anuran Makur ∗ and Japneet Singh † ∗ Department of Computer Science and ∗ , † School of Electrical and Computer Engineering , Purdue University , West Lafayette , IN 47907 Email : { amakur , sing1041 } @ purdue . edu Abstract —The Bradley - Terry - Luce ( BTL ) model is one of the most widely used models for ranking a set of items given data about pairwise comparisons among them . While several studies in the literature have attempted to empirically test how accurately a BTL model can model some given pairwise comparison data , this work aims to develop a formal , computationally efficient hypothesis test to determine whether the BTL model accurately represents the data . Specifically , we first propose such a formal hypothesis test , establish an upper bound on the critical radius of the proposed test , and then provide a complementary lower bound on the critical radius . Our bounds prove the minimax optimality of the scaling of the critical radius with respect to the number of items ( up to constant factors ) . Finally , we also take the first step towards characterizing the stability of rankings under the BTL model when there is a small model mismatch . I . I NTRODUCTION Many applications , such as sports tournaments , consumer preference surveys , and political voting , generate data in the form of pairwise comparisons between a set of items or agents ( e . g . , choices , teams ) . These datasets are useful for performing various data analysis tasks , such as ranking the items , analyzing the skill level of a particular team over time , and examining market or sports competitiveness , cf . [ 1 ] – [ 15 ] . A popular modeling assumption to perform such learning and inference tasks is the Bradley - Terry - Luce ( BTL ) model [ 1 ] – [ 6 ] . The BTL model assigns a latent skill score α i > 0 to each item i , representing its relative merit compared to other items , and posits that the likelihood of i being preferred over an item j in a pairwise comparison is given by P ( i is preferred over j ) = α i α i + α j . ( 1 ) The BTL model is a natural consequence of the assump - tion of independence of irrelevant alternatives ( IIA ) , which is widely used in economics and social choice theory [ 2 ] . Despite its widespread popularity , it is known that the IIA assumption does not hold well for various real - world datasets [ 16 ] – [ 18 ] . For example , the BTL model is oblivious to the “home - advantage effect” in sports , which refers to a home team’s possible advantage when playing against a visiting team ( see , e . g . , cricket [ 19 ] , soccer [ 20 ] ) . Hence , several other models of pairwise comparisons have been proposed in the literature , e . g . , modifications of the BTL model to incorporate home - advantage effect [ 21 , Chapter 10 ] , Thurstonian models [ 3 ] , other generalizations of the BTL model [ 7 ] , models of The author ordering is alphabetical . rankings based on Borda scores [ 22 ] , [ 23 ] , and other non - parametric stochastically transitive models [ 24 ] , [ 25 ] . Nevertheless , primarily because of its simplicity and in - terpretability , the BTL framework remains one of the most widely - used models . A large fraction of the associated results in the literature focuses on estimation of the skill score parameters of the BTL model . Some popular approaches include maximum likelihood estimation [ 6 ] , [ 7 ] , rank central - ity ( or Markov - chain - based ) methods [ 10 ] , [ 26 ] , least - squares methods [ 13 ] , and non - parametric methods [ 12 ] , [ 24 ] ( also see [ 8 ] , [ 9 ] for Bayesian inference for BTL models ) . Once the parameters are estimated , they are then used for inference tasks , such as ranking items and learning skill distributions [ 14 ] , [ 15 ] . An inherent assumption for such statistical analysis to be meaningful in real - world scenarios is that the BTL model accurately represents the given pairwise comparison data . Hence , it is important to understand precisely when the BTL assumption holds in a systematic manner . A . Main Contributions In contrast to the above works , we focus on the problem of testing whether the BTL assumption accurately models data generated from an underlying pairwise comparison model . First , we devise a notion of “distance” that allows us to quantify the deviation of a general pairwise comparison model from BTL models . We then use this distance to formally construct a hypothesis test to determine whether a BTL model accurately models the underlying data . We establish an upper bound on the minimax critical radius for this test . Furthermore , we also prove an information theoretic lower bound on the critical radius for this problem , thereby demonstrating the minimax optimality of the critical radius . Finally , we utilize the notion of distance mentioned above to analyze the stability of BTL assumptions in the context of rankings . More specifically , we investigate the deviation from the BTL condition that is sufficient for the ranking produced under the BTL assumption to differ from the classical Borda count ranking [ 27 ] . B . Related Literature This work lies at the confluence of two fields of study : hypothesis testing and preference learning . The analysis of preference data , such as pairwise comparisons , has a rich history starting from the seminal works [ 1 ] – [ 6 ] . As mentioned earlier , the BTL model is one of the most well - studied models for pairwise comparison data [ 1 ] . It was first proposed by [ 6 ] as a method for estimating participants’ skill levels in chess 2023 IEEE International Symposium on Information Theory ( ISIT ) 978 - 1 - 6654 - 7554 - 9 / 23 / $ 31 . 00 ©2023 IEEE 1390 2023 I EEE I n t e r n a ti on a l S y m po s i u m on I n f o r m a ti on T h e o r y ( I S I T ) | 978 - 1 - 6654 - 7554 - 9 / 23 / $ 31 . 00 © 2023 I EEE | DO I : 10 . 1109 / I S I T 54713 . 2023 . 10206450 Authorized licensed use limited to the terms of the applicable license agreement with IEEE . Restrictions apply . tournaments . Moreover , it is a special case of the Plackett - Luce model [ 2 ] , [ 4 ] , initially developed in mathematical psychology . We refer readers to [ 28 ] , [ 29 ] for a comprehensive overview of different models of rankings . In the literature , many studies have focused on estimating parameters for the BTL model and characterizing the error bounds , cf . [ 7 ] , [ 10 ] , [ 11 ] , [ 25 ] , [ 26 ] , [ 30 ] – [ 32 ] . For example , [ 11 ] presents non - asymptotic bounds for relative ℓ ∞ and ℓ 2 - norm estimation errors of normalized vectors of skill scores . We use some of these bounds in our arguments , although we need to make alterations since the proofs do not apply to general pairwise comparison models . Hypothesis testing also has a rich history in statistics , ranging from Pearson’s χ 2 - test [ 33 ] to non - parametric tests [ 34 ] . Yet , to the best of our knowledge , no study has developed rigorous hypothesis tests to determine the validity of the BTL assumption in the literature . The minimax perspective of hypothesis testing , which we specialize in our setting , was initially proposed by [ 35 ] . It is worth mentioning that recently , [ 36 ] analyzed two - sample testing on pairwise comparison data , and [ 37 ] derived lower bounds for testing the IIA assumption given general preference data . For the special case of pairwise comparisons , the lower bounds in [ 37 ] agree with ours in terms of the high - level scaling law of the critical radius . However , our hypothesis testing problem is formulated differently to [ 37 ] and [ 37 ] does not provide upper bounds . Furthermore , investigating the stability of the BTL assump - tion is another interesting question in the literature . For exam - ple , [ 22 ] provided empirical evidence that the BTL assumption is not very robust to changes in the pairwise comparison matrix . So , in this work , we also take the first steps towards rigorously characterizing the stability of rankings under the BTL model . II . F ORMAL S ETUP AND D ECISION R ULE A . Notational Preliminaries We briefly collect some notation here that is used throughout this work . Let 1 n ∈ R n be the column vector with all entries equal to 1 , where we drop the subscript when it is clear from context , and [ n ] ≜ { 1 , . . . , n } . Furthermore , for any vector x ∈ R n , diag ( x ) ∈ R n × n is the diagonal matrix with x along its principal diagonal . For a vector x ∈ R n , ∥ x ∥ 2 denotes its ℓ 2 - norm and x p denotes the entry - wise p th power of x , i . e . , x p = [ x p 1 , x p 2 , · · · , x pn ] T . For any matrix A ∈ R n × n , ∥ A ∥ 2 and ∥ A ∥ F denote the operator norm and Frobenious norm of A , respectively . For a strictly positive vector π ∈ R n , we define a Hilbert space on R n with inner product ⟨ x , y ⟩ π = (cid:80) ni = 1 π i x i y i and the corresponding vector and matrix norms ∥ x ∥ π = (cid:112) ⟨ x , x ⟩ π and ∥ A ∥ π = sup ∥ x ∥ π = 1 ∥ Ax ∥ π and ∥ A ∥ π , F = (cid:0) (cid:80) i (cid:80) j π j A 2 ij (cid:1) 1 / 2 . B . Formal Model and Goal We begin by introducing general pairwise comparison mod - els . Consider a set of n agents , indexed by [ n ] with n ∈ N \ { 1 } , that engage in a tournament consisting of several pairwise comparisons . This scenario is ubiquitous in many real - world applications . For example , in a sports tournament , [ n ] rep - resents the teams or players that play pairwise games with each other , and in discrete choice models from economics , [ n ] represents alternatives that an individual may choose from . Several probabilistic models exist in the literature to capture such pairwise comparison settings , e . g . , BTL model [ 1 ] , [ 2 ] , [ 5 ] , Thurstonian models [ 3 ] , and non - parametric models [ 24 ] , [ 25 ] , and all of them turn out to be specializations of the following general pairwise comparison model . Definition 1 ( Pairwise Comparison Model ) : For any pair of agents i , j ∈ [ n ] , i ̸ = j , let p ij ∈ ( 0 , 1 ) denote the probability that agent j beats agent i in a “i vs . j” pairwise comparison . We refer to the collection of n ( n − 1 ) parameters { p ij : i , j ∈ [ n ] , i ̸ = j } as a pairwise comparison model . Specifically , we consider an asymmetric setting where an “ i vs . j ” comparison may have a different meaning to a “ j vs . i ” comparison . Such asymmetric settings are commonly observed in sports like cricket , football , etc . [ 19 ] . Hence , such a model can be aptly summarized by a matrix P ∈ R n × n with P ij = (cid:40) p ij , i ̸ = j , 12 , i = j , ( 2 ) where we have set P ii = 12 for notational convenience . In our analysis , we will find it convenient to assign a time - homogenous Markov chain ( or row stochastic matrix ) on the finite state space [ n ] to any pairwise comparison model . This canonical assignment is defined next . Definition 2 ( Canonical Markov Chain ) : For any pairwise comparison model { p ij ∈ ( 0 , 1 ) : i , j ∈ [ n ] , i ̸ = j } with matrix P ∈ R n × n , its canonical Markov chain is given by the row stochastic matrix S ∈ R n × n , where S ij =   p ij n , i ̸ = j , 1 − 1 n (cid:88) k ∈ [ n ] \ { i } p ik , i = j . As noted earlier , the most well - known specialization of the pairwise comparison model in Definition 1 is the BTL model defined below [ 1 ] , [ 2 ] , [ 5 ] . Definition 3 ( BTL Model [ 1 ] , [ 2 ] , [ 5 ] ) : A pairwise com - parison model { p ij ∈ ( 0 , 1 ) : i , j ∈ [ n ] , i ̸ = j } is known as a BTL ( or multinomial logit ) model if there exist skill score parameters α i > 0 for every agent i ∈ [ n ] such that : ∀ i , j ∈ [ n ] , i ̸ = j , p ij = α j α i + α j . Hence , we can describe a BTL model entirely using the collection of its n skill score parameters { α i : i ∈ [ n ] } . We next describe how a pairwise comparison model char - acterizes the likelihood of a tournament between n agents . To this end , fix any pairwise comparison model { p ij ∈ ( 0 , 1 ) : i , j ∈ [ n ] , i ̸ = j } . For any i ̸ = j , define the outcome of the m th i vs j pairwise comparison between them as the Bernoulli random variable Z m ij ≜ (cid:40) 1 , if j beats i ( with probability p ij ) , 0 , if i beats j ( with probability 1 − p ij ) , ( 3 ) 2023 IEEE International Symposium on Information Theory ( ISIT ) 1391 Authorized licensed use limited to the terms of the applicable license agreement with IEEE . Restrictions apply . for m ∈ [ k ij ] , where k ij denotes the number of i vs j com - parisons . We assume throughout that the observation random variables Z ≜ { Z m ij : i , j ∈ [ n ] , i ̸ = j , m ∈ [ k i , j ] } are mutually independent . Let Z ij ≜ (cid:80) k ij m = 1 Z m ij . Clearly , it follows that for any i ̸ = j , Z ij is a binomial random variable , i . e . , Z ij ∼ Bin ( k ij , p ij ) , and for simplicity , we set Z ii = 0 . We also make the following assumption on the pairwise comparison model . Assumption 1 ( Dynamic Range ) : We assume that there is a constant δ ∈ ( 0 , 1 ) such that for all i , j ∈ [ n ] , δ 1 + δ ≤ p ij ≤ 1 1 + δ . ( 4 ) Goal . Given the observations Z of a tournament as defined above , our objective is to determine whether the underlying pairwise comparison model is a BTL model . This corresponds to solving a composite hypothesis testing problem : H 0 : Z ∼ BTL model for some α 1 , . . . , α n > 0 , H 1 : Z ∼ pairwise comparison model that is not BTL , ( 5 ) where the null hypothesis H 0 states that Z is distributed according to a BTL model , and the alternative hypothesis H 1 states that Z is distributed according to a general non - BTL pairwise comparison model . To pose this hypothesis testing problem more rigorously , we demonstrate an interesting relation between a BTL model and its canonical Markov chain . Recall that a Markov chain on the state space [ n ] , defined by the row stochastic matrix W ∈ R n × n , is said to be reversible if it satisfies the detailed balance conditions [ 38 , Section 1 . 6 ] : ∀ i , j ∈ [ n ] , i ̸ = j , π i W ij = π j W ij , ( 6 ) where W ij denotes the probability of transitioning from state i to state j , and π = ( π ( 1 ) , . . . , π ( n ) ) denotes the invariant distribution of the Markov chain ( which always exists ) . Equiv - alently , the Markov chain W is reversible if and only if diag ( π ) W = W T diag ( π ) . ( 7 ) It turns out that there is a tight connection between reversible Markov chains and the BTL model . This is elucidated in the ensuing proposition , cf . [ 39 , Lemma 6 ] , [ 10 ] . Proposition 1 ( BTL Model and Reversibility ) : A pairwise comparison model { p ij ∈ ( 0 , 1 ) : i , j ∈ [ n ] , i ̸ = j } is a BTL model if and only if its canonical Markov chain S ∈ R n × n is reversible and p ij + p ji = 1 for all i , j ∈ [ n ] . Proof : We provide a proof for completeness . If the pairwise comparison model is BTL , it implies that for some weight vector α ∈ R n + , the pairwise comparison matrix P is given by p ij = α j α i + α j for i ̸ = j . It is easy verify that π ≜ (cid:0) (cid:80) ni = 1 α i (cid:1) − 1 [ α 1 · · · α n (cid:3) T is the stationary distribution of canonical Markov chain matrix S corresponding to P . Moreover , S is reversible as π i S ij = α i (cid:80) ni = 1 α i × α j n ( α i + α j ) = π j S ij . For the converse , since p ij > 0 for all i , j ∈ [ n ] , S is irreducible and has a unique stationary distribution π . By reversibility of S , we have for all i ̸ = j , π i S ij = π j S ij = ⇒ π i p ij = π j p ji = ⇒ p ij = π j π i + π j , where last step follows from the fact that p ij + p ji = 1 . Thus , P corresponds to a BTL model with weight vector π . □ Let π be the stationary distribution of the canonical Markov chain matrix S corresponding to a valid pairwise comparison matrix P . By Proposition 1 , any pairwise comparison matrix P is BTL if and only if it satisfies the reversibility condition Π P = P T Π , where Π = diag ( π ) , and translated skew - symmetry P + P T = 1 n 1 T n . It turns out that both conditions are elegantly captured by the matrix Π P + P Π − 1 n π T as illustrated in the Proposition 2 , and we will later use the norm of this matrix to quantify the deviation of a pairwise comparison matrix from being BTL . Proposition 2 ( Orthogonal Decomposition ) : For any pair - wise comparison matrix P ∈ R n × n and vector π ∈ R n with strictly positive entries , we have (cid:13)(cid:13) Π P + P Π − 1 n π T (cid:13)(cid:13) 2 π − 1 , F = (cid:13)(cid:13) Π P − P T Π (cid:13)(cid:13) 2 π − 1 , F + (cid:13)(cid:13) P + P T − 1 n 1 T n (cid:13)(cid:13) 2 π , F , where Π = diag ( π ) . The proof can be found in the extended version of this paper [ 40 ] . It is important to note here that Assumption 1 and the Perron - Frobenius theorem [ 41 , Chapter 8 ] imply that π i > 0 for all i ∈ [ n ] . Hence , the norm ∥ · ∥ π − 1 , F is always well - defined . Hypothesis testing problem . For a given tolerance param - eter ϵ > 0 , we can formulate the hypothesis testing problem in ( 5 ) as : H 0 : Π P + P Π = 1 n π T , H 1 : 1 n ∥ Π P + P Π − 1 n π T ∥ F ≥ ϵ ∥ π ∥ ∞ , ( 8 ) where Π = diag ( π ) and π is the stationary distribution of the canonical Markov chain matrix S corresponding to P . Proposition 3 , which is proved in [ 40 ] , verifies that the null hypothesis indeed captures BTL models . Proposition 3 ( BTL Model Characterization ) : The pairwise comparison matrix P defined in Section II - B corresponds to a BTL model if and only if the hypothesis H 0 in ( 8 ) is true . C . Minimax Risk and Decision Rule Let ϕ denote a hypothesis test ( or decision rule ) that maps the consolidated observations { Z ij , k ij } i , j ∈ [ n ] to { 0 , 1 } , where 0 represents the null hypothesis and 1 represents the alternative hypothesis . Let P H 0 and P H 1 denote the probability distribu - tions of the input variables under H 0 and H 1 , respectively . Let M 0 and M ( ϵ ) denote the sets of matrices P that satisfy the null and alternative hypotheses in ( 8 ) , respectively . Now , define the minimax risk as R m ≜ inf ϕ (cid:40) sup P ∈M 0 P H 0 ( ϕ = 1 ) + sup P ∈M ( ϵ ) P H 1 ( ϕ = 0 ) (cid:41) , ( 9 ) 2023 IEEE International Symposium on Information Theory ( ISIT ) 1392 Authorized licensed use limited to the terms of the applicable license agreement with IEEE . Restrictions apply . where the infimum is taken over all { 0 , 1 } - valued tests ϕ . Finally , we can define the critical threshold of the hypothesis testing problem in ( 8 ) as the smallest value of ϵ for which the minimax risk is bounded by 13 : ε c = inf (cid:26) ϵ > 0 : R m ≤ 1 3 (cid:27) . ( 10 ) The constant 13 is arbitrary and can be replaced by any constant in ( 0 , 1 ) . Formally , our objective is to characterize the scaling of the critical radius with respect to n . To this end , we consider a hypothesis test which takes the consolidated observations { Z ij , k ij } i , j ∈ [ n ] as input and evaluates the following expres - sion : T = n (cid:88) i = 1 n (cid:88) j = 1 ( ˆ π i + ˆ π j ) 2 Z ij ( Z ij − 1 ) k ij ( k ij − 1 ) + ˆ π 2 j − 2ˆ π j ( ˆ π i + ˆ π j ) Z ij k ij ( 11 ) where ˆ π denotes the stationary distribution ( choosing one arbitrarily if there are several ) of the empirical Markov chain matrix ˆ S ∈ R n × n defined via ˆ S ij ≜ (cid:40) Z ij k ij n , i ̸ = j , 1 − 1 n (cid:80) u : u ̸ = i Z iu k iu , i = j . ( 12 ) The alternative hypothesis is selected if T > γ / n for some appropriately chosen constant γ independent of n . The test has constructed such that if ˆ π = π then E [ T ] = ∥ Π P + P Π − 1 n π T ∥ 2F , i . e . , we “plug - in " ˆ π in an unbiased estimator of ∥ Π P + P Π − 1 n π T ∥ 2F . III . U PPER B OUND ON C RITICAL T HRESHOLD The ensuing theorem establishes an upper bound on the critical radius of the hypothesis testing problem for the BTL model . For simplicity of analysis , we will assume that k ij = k ji = k for all i , j ∈ [ n ] throughout the sequel . Theorem 1 ( Upper Bound on ε c ) : Consider the hypothesis testing problem in ( 8 ) , and suppose the number of comparisons per pair of agents satisfies k ≥ max (cid:8) 2 , 36 C 2 log ( n ) nδ 4 (cid:9) for some constant C > 0 . Then , there exists another constant c > 0 such that for any ϵ > 0 with ϵ 2 ≥ c n , we have R m ≤ 13 . Hence , we obtain the bound ε 2 c ≤ c n . The proof is provided in [ 40 ] . IV . L OWER B OUND ON C RITICAL T HRESHOLD We now prove an information theoretic lower bound on the critical radius for the BTL hypothesis testing problem , thus proving the minimax optimality of the scaling provided in the upper bound ( up to constant factors ) . Theorem 2 ( Lower Bound on ε c ) : Consider the hypothesis testing problem in ( 8 ) . Then , there exists a constant c > 0 such that the critical radius ε c is lower bounded as ε 2 c ≥ c kn . Proof : We will use the Ingster - Suslina method for construct - ing a lower bound on the critical radius [ 42 ] . The method is similar to the well - known Le Cam’s method , but it establishes a minimax lower bound by considering a point and a mixture on the parameter space instead of just two points . ( Although Le Cam’s method could also be used for this proof in principle , the Ingster - Suslina method greatly simplifies the calculations to bound total variation distance in our setting . ) Under the null hypothesis , we assume that the pairwise comparison matrix P is fixed to be an all 1 / 2 matrix , i . e . , H 0 : P = P 0 ≜ 1 2 1 n 1 T n . ( 13 ) We will denote the distribution corresponding to the pairwise comparison matrix P 0 by P 0 . Moreover , note that under H 0 , the stationary distribution of the canonical Markov chain matrix S is uniform , i . e . , π = 1 n 1 n . Under the alternative hypothesis , we assume that the pairwise comparison matrix P θ is generated by sampling the parameter θ uniformly from the set Θ , i . e . , H 1 : P = P θ and θ ∼ Unif ( Θ ) , ( 14 ) and for some θ ∈ Θ , P θ is given by P θ = (cid:34) 12 1 n / 2 1 T n / 2 12 1 n / 2 1 T n / 2 + ηQ θ 12 1 n / 2 1 T n / 2 − ηQ θ 12 1 n / 2 1 T n / 2 (cid:35) , ( 15 ) where Θ is set of all permuation matrices , and Q θ is the n / 2 × n / 2 permutation matrix corresponding to the permutation θ . Let P Θ denote the distribution corresponding to the pairwise comparison matrix P θ . The construction of this mixture was inspired by [ 36 ] . However , there are two notable differences . Firstly , the problem in [ 36 ] is distinguishing whether two sets of data samples consisting of pairwise comparisons are coming from the same underlying distribution or two different distributions described by a pairwise comparison model . In contrast , our work tests whether or not a single dataset is sampled from a BTL model . Secondly , the manner in which a notion of distance is used to define the deviation of the given data from the null hypothesis is very different in the two works . Let S θ denote the canonical Markov chain matrix cor - responding to P θ . It is straightforward to verify that the stationary distribution of S θ is independent of the permutation θ . Let π denote the stationary distribution of S θ . By the symmetry of S θ , the set of first n / 2 elements , and respectively , last n / 2 elements , of π are equal , i . e . , π 1 = · · · = π n / 2 ≜ x and π ( n / 2 ) + 1 = · · · = π n ≜ y . Now x and y can be determined by solving the set of linear equations : π T = π T S θ and n (cid:88) i = 1 π i = 1 . Solving these equations gives x = 1 n (cid:18) 1 − 4 η n (cid:19) and y = 1 n (cid:18) 1 + 4 η n (cid:19) . 2023 IEEE International Symposium on Information Theory ( ISIT ) 1393 Authorized licensed use limited to the terms of the applicable license agreement with IEEE . Restrictions apply . It is also easy to verify that the deviation from BTL ∥ Π P θ + P θ Π − 1 n π T ∥ F is independent of the permutation θ and is given by ∥ Π P θ + P T θ Π − 1 n π T ∥ 2F = n 2 (cid:18) ( x + y ) (cid:18) 1 2 + η (cid:19) − y (cid:19) 2 + n 2 (cid:18) ( x + y ) (cid:18) 1 2 − η (cid:19) − x (cid:19) 2 + n 2 (cid:16) n 2 − 1 (cid:17)(cid:18) x + y 2 − y (cid:19) 2 + n 2 (cid:16) n 2 − 1 (cid:17)(cid:18) x + y 2 − x (cid:19) 2 = 2 η 2 n (cid:18) 1 − 2 n (cid:19) 2 + 2 η 2 n 2 (cid:18) 1 − 2 n (cid:19) . ( 16 ) Let ϵ = ∥ Π P θ + P θ Π − 1 n π T ∥ F / ( n ∥ π ∥ ∞ ) to ensure that P θ ’s satisfy the condition of the alternative hypothesis in ( 8 ) . Substituting the values of ∥ π ∥ ∞ = y and ∥ Π P θ + P T θ Π − 1 n π T ∥ F implies that ϵ ≤ Cη / √ n for some constant C > 0 . Now , the Ingster - Suslina method [ 42 ] states that R m ≥ 1 2 (cid:16) 1 − (cid:112) χ 2 ( P 0 | | P Θ ) (cid:17) , where χ 2 ( · | | · ) denotes the χ 2 - divergence . Similar to the anal - ysis in [ 36 , Theorem 3 ] , for the distributions P 0 and P Θ , if we have η 2 ≤ ck for some constant c independent of n , k , then we can upper bound the χ 2 ( P 0 | | P Θ ) term as χ 2 ( P 0 | | P Θ ) ≤ 19 . Hence , we have shown that there exists a constant c > 0 , such that if nϵ 2 ≤ c / k , then χ 2 ( P 0 | | P Θ ) ≤ 19 , which implies that the minimax risk R m ≥ 13 . Hence , ε 2 c ≥ c / ( kn ) as desired . □ We defer readers to [ 40 ] for a more detailed proof . V . S TABILITY OF THE BTL A SSUMPTION In this section , we analyze the stability of the BTL as - sumption in the context of rankings . The BTL ranking orders agents based on the stationary distribution π of the canonical Markov - chain matrix . Meanwhile , the Borda count ranking is more general , as it doesn’t rely on the BTL assumption , and instead is based on Borda scores [ 27 ] ( defined below ) . If the BTL assumption holds , then Borda ranking equals the BTL ranking . Our goal is to determine the size of the deviation from the BTL condition in a pairwise comparison model that is sufficient to produce a discrepancy between the BTL and Borda count rankings . For this section , we will consider the symmetric setting in which the pairwise comparison matrix P satisfies , p ij + p ji = 1 . Define the Borda count τ i ( P ) of an agent i ∈ [ n ] as the ( scaled ) probability that i beats any other agent selected uniformly at random [ 27 ] : τ i ( P ) ≜ n (cid:88) j = 1 ( 1 − p ij ) . ( 17 ) Now we show that the stability of the BTL assumption decreases as n grows meaning that even smaller deviations from the BTL condition can lead to a reversed ranking . Proposition 4 ( Stability of BTL Assumption ) : There exists a pairwise comparison matrices P ∈ R n × n such that two rows i , j ∈ [ n ] having constant ∆ τ ≜ τ i ( P ) − τ j ( P ) > 0 , but has an 32 40 48 56 64 72 80 88 96 104 112 120 128 Number of Teams n 0 . 32 0 . 3070 . 2930 . 28 0 . 2670 . 2530 . 24 0 . 2270 . 2130 . 2 0 . 1870 . 1730 . 16 P e r t uba t i on 1 2 3 4 5 10 - 3 Fig . 1 . Plot of empirical average of T under hypothesis H 1 . 32 40 48 56 64 72 80 88 96 104 112 120 128 Number of Teams n 0 . 32 0 . 3070 . 2930 . 28 0 . 2670 . 2530 . 24 0 . 2270 . 2130 . 2 0 . 1870 . 1730 . 16 P e r t u r ba t i on 0 0 . 2 0 . 4 0 . 6 0 . 8 1 Fig . 2 . Plot of 1 R m > 4 / 5 . opposite ranking BTL ranking , i . e . , π i < π j . Let Π = diag ( π ) . Moreover , the deviation of P from BTL condition decays as (cid:13)(cid:13) Π P + P Π − 1 n π T (cid:13)(cid:13) F ≤ c √ n for some constant c > 0 . The proof is provided in [ 40 ] . Proposition 4 highlights that the BTL assumption may potentially give a wrong ranking when the underlying pairwise matrix is O ( 1 / √ n ) “distance” away from the BTL condition . Interestingly , this O ( 1 / √ n ) deviation coincides with the critical threshold for the BTL testing problem ( up to constant factors ) . It would be interesting to further explore the stability of the BTL assumption in the context of rankings . VI . N UMERICAL S IMULATIONS In this section , we will empirically analyze the behavior of the minimax risk and the deviation ∥ Π P + P Π − 1 π T ∥ F via a synthetic experiment . We will use the same construction for the pairwise comparison matrix P that we utilized in ( 2 ) under the null and alternate hypothesis which are presented in ( 13 ) and ( 14 ) . We set the number of pairwise comparisons per pair of agents k = 12 , the number of agents n is linearly increased from 32 to 128 , and the perturbation η in ( 15 ) is increased from 0 . 16 to 0 . 32 . Simulations are performed for each value of η and n , and the corresponding value of expected values of test T under hypothesis H 1 and minimax risk R m is estimated . The threshold used for the decision rule is set to η 2 / n . Fig . 2 plots the empirical average of T under H 1 and 1 R m > 4 / 5 for different values of η and n . Note that the behavior of empirical average of T ( under H 1 ) is consistent with ( 16 ) and moreover for a fixed value of η the behavior of R m is independent of n . VII . C ONCLUSION In this work , we studied the problem of testing whether a BTL model accurately represents the data generated from an underlying pairwise comparison model . 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Restrictions apply . \ No newline at end of file diff --git a/silver_data/e3884ebaac0a11eda0218e7032ce861026ef51d9.pdf.txt b/silver_data/e3884ebaac0a11eda0218e7032ce861026ef51d9.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..1cb61324de325f769aad663417f81d6cc57f1495 --- /dev/null +++ b/silver_data/e3884ebaac0a11eda0218e7032ce861026ef51d9.pdf.txt @@ -0,0 +1 @@ +REVIEW Open Access Bone regeneration : current concepts and future directions Rozalia Dimitriou 1 , 2 † , Elena Jones 3 † , Dennis McGonagle 3 † and Peter V Giannoudis 1 , 2 * † Abstract Bone regeneration is a complex , well - orchestrated physiological process of bone formation , which can be seen during normal fracture healing , and is involved in continuous remodelling throughout adult life . However , there are complex clinical conditions in which bone regeneration is required in large quantity , such as for skeletal reconstruction of large bone defects created by trauma , infection , tumour resection and skeletal abnormalities , or cases in which the regenerative process is compromised , including avascular necrosis , atrophic non - unions and osteoporosis . Currently , there is a plethora of different strategies to augment the impaired or ‘ insufficient ’ bone - regeneration process , including the ‘ gold standard ’ autologous bone graft , free fibula vascularised graft , allograft implantation , and use of growth factors , osteoconductive scaffolds , osteoprogenitor cells and distraction osteogenesis . Improved ‘ local ’ strategies in terms of tissue engineering and gene therapy , or even ‘ systemic ’ enhancement of bone repair , are under intense investigation , in an effort to overcome the limitations of the current methods , to produce bone - graft substitutes with biomechanical properties that are as identical to normal bone as possible , to accelerate the overall regeneration process , or even to address systemic conditions , such as skeletal disorders and osteoporosis . Introduction Bone possesses the intrinsic capacity for regeneration as part of the repair process in response to injury , as well as during skeletal development or continuous remodelling throughout adult life [ 1 , 2 ] . Bone regeneration is com - prised of a well - orchestrated series of biological events of bone induction and conduction , involving a number of cell types and intracellular and extracellular molecular - signalling pathways , with a definable temporal and spatial sequence , in an effort to optimise skeletal repair and restore skeletal function [ 2 , 3 ] . In the clinical setting , the most common form of bone regeneration is fracture healing , during which the pathway of normal fetal skele - togenesis , including intramembranous and endochondral ossification , is recapitulated [ 4 ] . Unlike in other tissues , the majority of bony injuries ( fractures ) heal without the formation of scar tissue , and bone is regenerated with its pre - existing properties largely restored , and with the newly formed bone being eventually indistinguishable from the adjacent uninjured bone [ 2 ] . However , there are cases of fracture healing in which bone regeneration is impaired , with , for example , up to 13 % of fractures occurring in the tibia being associated with delayed union or fracture non - union [ 5 ] . In addition , there are other conditions in orthopaedic surgery and in oral and maxillofacial surgery in which bone regeneration is required in large quantity ( beyond the normal potential for self - healing ) , such as for skeletal reconstruction of large bone defects created by trauma , infection , tumour resection and skeletal abnormalities , or cases in which the regenerative process is compromised , including avas - cular necrosis and osteoporosis . Current clinical approaches to enhance bone regeneration For all the aforementioned cases in which the normal process of bone regeneration is either impaired or simply insufficient , there are currently a number of treatment methods available in the surgeon ’ s armamentarium , which can be used either alone or in combination for the enhancement or management of these complex clinical situations , which can often be recalcitrant to treatment , representing a medical and socioeconomic challenge . Standard approaches widely used in clinical practice to stimulate or augment bone regeneration include distrac - tion osteogenesis and bone transport [ 6 , 7 ] , and the use of * Correspondence : pgiannoudi @ aol . com † Contributed equally 1 Department of Trauma and Orthopaedics , Academic Unit , Clarendon Wing , Leeds Teaching Hospitals NHS Trust , Great George Street , Leeds LS1 3EX , UK Full list of author information is available at the end of the article Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 © 2011 Dimitriou et al ; licensee BioMed Central Ltd . This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http : / / creativecommons . org / licenses / by / 2 . 0 ) , which permits unrestricted use , distribution , and reproduction in any medium , provided the original work is properly cited . a number of different bone - grafting methods , such as autologous bone grafts , allografts , and bone - graft substi - tutes or growth factors [ 8 , 9 ] . An alternative method for bone regeneration and reconstruction of long - bone defects is a two - stage procedure , known as the Masquelet technique . It is based on the concept of a “ biological ” membrane , which is induced after application of a cement spacer at the first stage and acts as a ‘ chamber ’ for the insertion of non - vascularised autograft at the sec - ond stage [ 10 ] . There are even non - invasive methods of biophysical stimulation , such as low - intensity pulsed ultrasound ( LIPUS ) and pulsed electromagnetic fields ( PEMF ) [ 11 - 13 ] , which are used as adjuncts to enhance bone regeneration . During distraction osteogenesis and bone transport , bone regeneration is induced between the gradually dis - tracted osseous surfaces . A variety of methods are cur - rently used to treat bone loss or limb - length discrepancies and deformities , including external fixators and the Ilizarov technique [ 6 , 7 ] , combined unreamed intramedullary nails with external monorail distraction devices [ 14 ] , or intramedullary lengthening devices [ 15 ] . However , these methods are technically demanding and have several disadvantages , including associated compli - cations , requirement for lengthy treatment for both the distraction ( 1 mm per day ) and the consolidation period ( usually twice the distraction phase ) , and effects on the patient ’ s psychology and well - being [ 6 , 7 ] . Bone grafting is a commonly performed surgical proce - dure to augment bone regeneration in a variety of ortho - paedic and maxillofacial procedures , with autologous bone being considered as the ‘ gold standard ’ bone - grafting material , as it combines all properties required in a bone - graft material : osteoinduction ( bone morphogenetic pro - teins ( BMPs ) and other growth factors ) , osteogenesis ( osteoprogenitor cells ) and osteoconduction ( scaffold ) [ 16 ] . It can also be harvested as a tricortical graft for struc - tural support [ 16 ] , or as a vascularised bone graft for restoration of large bone defects [ 17 ] or avascular necrosis [ 18 ] . A variety of sites can be used for bone - graft harvest - ing , with the anterior and posterior iliac crests of the pelvis being the commonly used donor sites . Recently , with the development of a new reaming system , the reamer - irriga - tor - aspirator ( RIA ) , initially developed to minimise the adverse effects of reaming during nailing of long - bone fractures , the intramedullary canal of long bones has been used as an alternative harvesting site , providing a large volume of autologous bone graft [ 19 ] . Furthermore , because it is the patient ’ s own tissue , autologous bone is histocompatible and non - immunogenic , reducing to a minimum the likelihood of immunoreactions and trans - mission of infections . Nevertheless , harvesting requires an additional surgical procedure , with well - documented com - plications and discomfort for the patient , and has the additional disadvantages of quantity restrictions and sub - stantial costs [ 20 - 22 ] . An alternative is allogeneic bone grafting , obtained from human cadavers or living donors , which bypasses the pro - blems associated with harvesting and quantity of graft material . Allogeneic bone is available in many prepara - tions , including demineralised bone matrix ( DBM ) , mor - cellised and cancellous chips , corticocancellous and cortical grafts , and osteochondral and whole - bone seg - ments , depending on the recipient site requirements . Their biological properties vary , but overall , they possess reduced osteoinductive properties and no cellular compo - nent , because donor grafts are devitalised via irradiation or freeze - drying processing [ 23 ] . There are issues of immu - nogenicity and rejection reactions , possibility of infection transmission , and cost [ 8 , 23 ] . Bone - graft substitutes have also been developed as alter - natives to autologous or allogeneic bone grafts . They con - sist of scaffolds made of synthetic or natural biomaterials that promote the migration , proliferation and differentia - tion of bone cells for bone regeneration . A wide range of biomaterials and synthetic bone substitutes are currently used as scaffolds , including collagen , hydroxyapatite ( HA ) , b - tricalcium phosphate ( b - TCP ) and calcium - phosphate cements , and glass ceramics [ 8 , 23 ] , and the research into this field is ongoing . Especially for reconstruction of large bone defects , for which there is a need for a substantial structural scaffold , an alternative to massive cortical auto - or allografts is the use of cylindrical metallic or titanium mesh cages as a scaffold combined with cancellous bone allograft , DBM or autologous bone [ 24 , 25 ] . Limitations of current strategies to enhance bone regeneration Most of the current strategies for bone regeneration exhi - bit relatively satisfactory results . However , there are asso - ciated drawbacks and limitations to their use and availability , and even controversial reports about their effi - cacy and cost - effectiveness . Furthermore , at present there are no heterologous or synthetic bone substitutes available that have superior or even the same biological or mechani - cal properties compared with bone . Therefore , there is a necessity to develop novel treatments as alternatives or adjuncts to the standard methods used for bone regenera - tion , in an effort to overcome these limitations , which has been a goal for many decades . Even back in the 1950s , Professor Sir Charnley , a pioneer British orthopaedic sur - geon , stated that ‘ practically all classical operations of sur - gery have now been explored , and unless some revolutionary discovery is made which will put the control of osteogenesis in the surgeon ’ s power , no great advance is likely to come from modification of their detail ’ [ 26 ] . Since then , our understanding of bone regeneration at the cellular and molecular level has advanced enormously , Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 2 of 10 and is still ongoing . New methods for studying this pro - cess , such as quantitative three - dimensional microcom - puted tomography analyses , finite element modelling , and nanotechnology have been developed to further evaluate the mechanical properties of bone regenerate at the micro - scopic level . In addition , advances made in cellular and molecular biology have allowed detailed histological ana - lyses , in vitro and in vivo characterisation of bone - forming cells , identification of transcriptional and translational pro - files of the genes and proteins involved in the process of bone regeneration and fracture repair , and development of transgenic animals to explore the role of a number of genes expressed during bone repair , and their temporal and tissue - specific expression patterns [ 27 ] . With the ongoing research in all related fields , novel therapies have been used as adjuncts or alternatives to traditional bone - regeneration methods . Nevertheless , the basic concept for managing all clinical situations requiring bone regenera - tion , particularly the complex and recalcitrant cases , remains the same , and must be applied . Treatment strate - gies should aim to address all ( or those that require enhancement ) prerequisites for optimal bone healing , including osteoconductive matrices , osteoinductive factors , osteogenic cells and mechanical stability , following the ‘ diamond concept ’ suggested for fracture healing ( Figure 1 ) [ 28 ] . BMPs and other growth factors With improved understanding of fracture healing and bone regeneration at the molecular level [ 29 ] , a number of key molecules that regulate this complex physiologi - cal process have been identified , and are already in clini - cal use or under investigation to enhance bone repair . Of these molecules , BMPs have been the most exten - sively studied , as they are potent osteoinductive factors . They induce the mitogenesis of mesenchymal stem cells ( MSCs ) and other osteoprogenitors , and their differen - tiation towards osteoblasts . Since the discovery of BMPs , a number of experimental and clinical trials have sup - ported the safety and efficacy of their use as osteoinduc - tive bone - graft substitutes for bone regeneration . With the use of recombinant DNA technology , BMP - 2 and BMP - 7 have been licensed for clinical use since 2002 and 2001 respectively [ 30 ] . These two molecules have been used in a variety of clinical conditions including non - union , open fractures , joint fusions , aseptic bone necrosis and critical bone defects [ 9 ] . Extensive research is ongoing to develop injectable formulations for mini - mally invasive application , and / or novel carriers for pro - longed and targeted local delivery [ 31 ] . Other growth factors besides BMPs that have been implicated during bone regeneration , with different functions in terms of cell proliferation , chemotaxis and angiogenesis , are also being investigated or are currently being used to augment bone repair [ 32 , 33 ] , including platelet - derived growth factor , transforming growth fac - tor - b , insulin - like growth factor - 1 , vascular endothelial growth factor and fibroblast growth factor , among others [ 29 ] . These have been used either alone or in combinations in a number of in vitro and in vivo stu - dies , with controversial results [ 32 , 33 ] . One current approach to enhance bone regeneration and soft - tissue healing by local application of growth factors is the use of platelet - rich plasma , a volume of the plasma fraction of autologous blood with platelet concentrations above baseline , which is rich in many of the aforementioned molecules [ 34 ] . ’ Orthobiologics ’ and the overall concept to stimulate the local ‘ biology ’ by applying growth factors ( especially BMPs , because these are the most potent osteoinductive molecules ) could be advantageous for bone regeneration or even for acceleration of normal bone healing to reduce the length of fracture treatment . Their clinical use , either alone or combined with bone grafts , is con - stantly increasing . However , there are several issues about their use , including safety ( because of the supra - physiological concentrations of growth factors needed to obtain the desired osteoinductive effects ) , the high cost of treatment , and more importantly , the potential for ectopic bone formation [ 35 ] . Currently BMPs are also being used in bone - tissue engineering , but several issues need to be further exam - ined , such as optimum dosage and provision of a sus - tained , biologically appropriate concentration at the site of bone regeneration , and the use of a ‘ cocktail ’ of other growth factors that have shown significant promising results in preclinical and early clinical investigation [ 32 ] or even the use of inhibitory molecules in an effort to mimic the endogenous ‘ normal ’ growth - factor produc - tion . Nanoparticle technology seems to be a promising approach for optimum growth - factor delivery in the future of bone - tissue engineering [ 36 ] . Nevertheless , owing to gaps in the current understanding of these fac - tors , it has not been possible to reproduce in vivo bone regeneration in the laboratory . MSCs An adequate supply of cells ( MSCs and osteoprogeni - tors ) is important for efficient bone regeneration . The current approach of delivering osteogenic cells directly to the regeneration site includes use of bone - marrow aspirate from the iliac crest , which also contains growth factors . It is a minimally invasive procedure to enhance bone repair , and produces satisfactory results [ 37 ] . How - ever , the concentration and quality of MSCs may vary significantly , depending on the individual ( especially in older people ) [ 38 , 39 ] , the aspiration sites and techniques used [ 39 ] , and whether further concentration of the Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 3 of 10 bone marrow has been performed [ 37 ] , as bone - marrow aspiration concentrate ( BMAC ) is considered to be an effective product to augment bone grafting and support bone regeneration [ 40 , 41 ] . Overall , however , there are significant ongoing issues with quality control with respect to delivering the requisite number of MSCs / osteoprogenitors to effect adequate repair responses [ 40 ] . Issues of quantity and alternative sources of MSCs are being extensively investigated . Novel approaches in terms of cell harvesting , in vitro expansion and subsequent implantation are promising [ 42 - 44 ] , because in vitro expansion can generate a large number of progenitor cells . However , such techniques add substantial cost and risks ( such as contamination with bacteria or viruses ) , may reduce the proliferative capacity of the cells and are time - consuming requiring two - stage surgery [ 45 ] . This strategy is already applied for cartilage regeneration [ 46 ] . Alterna - tive sources of cells , which are less invasive , such as per - ipheral blood [ 47 ] and mesenchymal progenitor cells from fat [ 48 ] , muscle , or even traumatised muscle tissue after debridement [ 49 ] , are also under extensive research . How - ever , the utility of fat - derived MSCs for bone - regeneration applications is debatable , with some studies showing them to be inferior to bone - marrow - derived MSCs in animal models [ 50 , 51 ] , and the evidence for a clinically relevant Figure 1 Male patient 19 years of age with infected non - union after intramedullary nailing of an open tibial fracture . ( A ) . Anteroposterior ( AP ) and lateral X - rays of the tibia illustrating osteolysis ( white arrow ) secondary to infection . The patient underwent removal of the nail , extensive debridement and a two - staged reconstruction of the bone defect , using the induced membrane technique for bone regeneration ( the Masquelet technique ) . ( B ) Intraoperative pictures showing : ( 1 ) a 60 mm defect of the tibia ( black arrow ) at the second stage of the procedure ; ( 2 ) adequate mechanical stability was provided with internal fixation ( locking plate ) bridging the defect , while the length was maintained ( black arrow ) ; ( 3 ) maximum biological stimulation was provided using autologous bone graft harvested from the femoral canal ( black arrow , right ) , bone - marrow mesenchymal stem cells ( broken arrow , left ) and the osteoinductive factor bone morphogenetic protein - 7 ( centre ) ; ( 4 ) the graft was placed to fill the bone defect ( black arrow ) . ( C ) Intraoperative fluoroscopic images showing the bone defect after fixation . ( D ) Postoperative AP and lateral X - rays at 3 months , showing the evolution of the bone regeneration process with satisfactory incorporation and mineralisation of the graft ( photographs courtesy of PVG ) . Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 4 of 10 or meaningful population of circulating MSCs also remains very contentious [ 52 ] . It is fair to say that the role of MSCs in fracture repair is still in its infancy , largely due to a lack of studies into the biology of MSCs in vivo in the fracture environment . This to a large extent relates to the historical perceived rarity of ‘ in vivo MSCs ’ and also to a lack of knowledge about in vivo phenotypes . The in vivo phenotype of bone - marrow MSCs has been recently reported [ 53 ] and , even more recently , it has been shown that this population was actually fairly abundant in vivo in nor - mal and pathological bone [ 54 ] . This knowledge opens up novel approaches for the characterisation and mole - cular profiling of MSCs in vivo in the fracture environ - ment . This could be used to ultimately improve outcomes of fracture non - union based on the biology of these key MSC reparative cells . Scaffolds and bone substitutes Although they lack osteoinductive or osteogenic proper - ties , synthetic bone substitutes and biomaterials are already widely used in clinical practice for osteoconduc - tion . DBM and collagen are biomaterials , used mainly as bone - graft extenders , as they provide minimal structural support [ 8 ] . A large number of synthetic bone substitutes are currently available , such as HA , b - TCP and calcium - phosphate cements , and glass ceramics [ 8 , 23 ] . These are being used as adjuncts or alternatives to autologous bone grafts , as they promote the migration , proliferation and differentiation of bone cells for bone regeneration . Espe - cially for regeneration of large bone defects , where the requirements for grafting material are substantial , these synthetics can be used in combination with autologous bone graft , growth factors or cells [ 8 ] . Furthermore , there are also non - biological osteoconductive substrates , such as fabricated biocompatible metals ( for example , porous tantalum ) that offer the potential for absolute control of the final structure without any immunogenicity [ 8 ] . Research is ongoing to improve the mechanical prop - erties and biocompatibility of scaffolds , to promote osteoblast adhesion , growth and differentiation , and t0 allow vascular ingrowth and bone - tissue formation . Improved biodegradable and bioactive three - dimensional porous scaffolds [ 55 ] are being investigated , as well as novel approaches using nanotechnology , such as mag - netic biohybrid porous scaffolds acting as a crosslinking agent for collagen for bone regeneration guided by an external magnetic field [ 56 ] , or injectable scaffolds for easier application [ 57 ] . Tissue engineering The tissue - engineering approach is a promising strategy added in the field of bone regenerative medicine , which aims to generate new , cell - driven , functional tissues , rather than just to implant non - living scaffolds [ 58 ] . This alternative treatment of conditions requiring bone regeneration could overcome the limitations of current therapies , by combining the principles of orthopaedic surgery with knowledge from biology , physics , materials science and engineering , and its clinical application offers great potential [ 58 , 59 ] . In essence , bone - tissue engineering combines progenitor cells , such as MSCs ( native or expanded ) or mature cells ( for osteogenesis ) seeded in biocompatible scaffolds and ideally in three - dimensional tissue - like structures ( for osteoconduction and vascular ingrowth ) , with appropriate growth factors ( for osteoinduction ) , in order to generate and maintain bone [ 60 ] . The need for such improved composite grafts is obvious , especially for the management of large bone defects , for which the requirements for grafting material are substantial [ 8 ] . At present , composite grafts that are available include bone synthetic or bioabsorbable scaf - folds seeded with bone - marrow aspirate or growth fac - tors ( BMPs ) , providing a competitive alternative to autologous bone graft [ 8 ] . Several major technical advances have been achieved in the field of bone - tissue engineering during the past dec - ade , especially with the increased understanding of bone healing at the molecular and cellular level , allowing the conduction of numerous animal studies and of the first pilot clinical studies using tissue - engineered constructs for local bone regeneration . To date , seven human studies have been conducted using culture - expanded , non - geneti - cally modified MSCs for regeneration of bone defects : two studies reporting on long bones and five on maxillofacial conditions [ 61 ] . Even though they are rather heteroge - neous studies and it is difficult to draw conclusive evi - dence from them , bone apposition by the grafted MSCs was seen , but it was not sufficient to bridge large bone defects . Furthermore , the tissue - engineering approach has been used to accelerate the fracture - healing process or to augment the bone - prosthesis interface and prevent aseptic loosening in total joint arthroplasty , with promising results regarding its efficacy and safety [ 62 , 63 ] . Recently , an animal study has shown the potential for prefabrication of vascularised bioartificial bone grafts in vivo using b - TCP scaffolds intraoperatively filled with autogenous bone marrow for cell loading , and implanted into the latissimus dorsi muscle for potential application at a later stage for segmental bone reconstruction , intro - ducing the principles of bone transplantation with mini - mal donor - site morbidity and no quantity restrictions [ 64 ] . Overall , bone - tissue engineering is in its infancy , and there are many issues of efficacy , safety and cost to be addressed before general clinical application can be achieved . Cultured - expanded cells may have mutations or epigenetic changes that could confer a tumour - forming potential [ 44 ] . However , in vitro and in vivo evidence Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 5 of 10 suggests that the risk of tumour formation is minimal [ 65 ] . No cases of tumour transformation were reported in 41 patients ( 45 joints ) after autologous bone - marrow - derived MSC transplantation for cartilage repair , who were fol - lowed for up to 11 years and 5 months [ 46 ] . Another important issue is the difficulty of achieving an effective and high - density cell population within three - dimensional biodegradable scaffolds [ 66 ] . Consequently , numerous bioreactor technologies have been investigated , and many others should be developed [ 67 ] . Their degradation prop - erties should also preserve the health of local tissues and the continuous remodelling of bone [ 44 ] . Three - dimen - sional porous scaffolds with specific architectures at the nano , micro and macro scale for optimum surface porosity and chemistry , which enhance cellular attachment , migra - tion , proliferation and differentiation , are undergoing a continuous evaluation process . Gene therapy Another promising method of growth - factor delivery in the field of bone - tissue engineering is the application of gene therapy [ 68 , 69 ] . This involves the transfer of genetic material into the genome of the target cell , allowing expression of bioactive factors from the cells themselves for a prolonged time . Gene transfer can be performed using a viral ( transfection ) or a non - viral ( transduction ) vector , and by either an in vivo or ex vivo gene - transfer strategy . With the in vivo method , which is technically relatively easier , the genetic material is transferred directly into the host ; however , there are safety concerns with this approach . The indirect ex vivo technique requires the collection of cells by tissue harvest , and their genetic modification in vitro before transfer back into the host . Although technically more demanding , it is a safer method , allowing testing of the cells for any abnormal behaviour before reimplantation , and selection of those with the highest gene expression [ 69 ] . Besides the issues of cost , efficacy and biological safety that need to be answered before this strategy of genetic manipulation is applied in humans , delivery of growth factors , particularly BMPs , using gene therapy for bone regeneration has already produced promising results in animal studies [ 70 , 71 ] . Mechanical stability and the role of mechanical stimulation in bone regeneration In addition to the intrinsic potential of bone to regener - ate and to the aforementioned methods used to enhance bone regeneration , adequate mechanical stability by var - ious means of stabilisation and use of fixation devices is also an important element for optimal bone repair , espe - cially in challenging cases involving large bone defects or impaired bone healing . The mechanical environment constitutes the fourth factor of the ‘ diamond concept ’ of fracture healing , along with osteoconductive scaffolds , growth factors and osteogenic cells , interacting during the repair process [ 28 ] . During bone regeneration , intermediate tissues , such as fibrous connective tissue , cartilage and woven bone , pre - cede final bone formation , providing initial mechanical stability and a scaffold for tissue differentiation . The mechanical loading affects the regeneration process , with different stress distribution favouring or inhibiting differ - entiation of particular tissue phenotypes [ 72 ] . High shear strain and fluid flows are thought to stimulate formation of fibrous connective tissue , whereas lower levels stimu - late formation of cartilage , and even lower levels favour ossification [ 72 ] . The interfragmentary strain concept of Perren has been used to describe the different patterns of bone repair ( pri - mary or secondary fracture healing ) , suggesting that the strain that causes healthy bone to fail is the upper limit that can be tolerated for the regenerating tissue [ 73 ] . Since then , extensive research on this field has further refined the effects of mechanical stability and mechanical stimula - tion on bone regeneration and fracture healing [ 74 ] . Numerous in vivo studies have shown contradictory results regarding the contribution of strain and mechanical stimulation , in terms of compression or distraction , in bone formation during fracture healing . In early fracture healing , mechanical stimulation seems to enhance callus formation , but the amount of callus formation does not correspond to stiffness [ 74 ] . During the initial stages of bone healing , a less rigid mechanical environment resulted in a prolonged chondral bone regeneration phase , whereas the process of intramembranous ossification appeared to be independent of mechanical stability [ 75 ] . By contrast , a more rigid mechanical environment resulted in a smaller callus and a reduced fibrous - tissue component [ 76 ] . For later stages of bone regeneration , lower mechanical stabi - lity was found to inhibit callus bridging and stiffness [ 74 ] . Finally , in vitro studies have also shown the role of the mechanical environment on different cell types involved in bone regeneration . It has been demonstrated using cell - culture systems that the different cellular responses in terms of proliferation and differentiation after mechanical stimulation depend on the strain magnitude and the cell phenotype [ 74 ] . Mechanical stability is also important for local vascu - larisation and angiogenesis during bone regeneration . In an i n vivo study , it was shown that smaller interfragmen - tary movements led to the formation of a greater number of vessels within the callus , particularly in areas close to the periosteum , compared with larger movements [ 77 ] , whereas increased interfragmentary shear was associated with reduced vascularisation with a higher amount of fibrous - tissue formation and a lower percentage of mineralised bone during early bone healing [ 78 ] . Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 6 of 10 Finally , the presence of a mechanically stable environ - ment throughout the bone - regeneration process is also essential when additional methods are being used to enhance bone repair [ 28 , 79 ] . Optimal instrumentation with minimal disruption of the local blood supply is required to supplement and protect the mechanical prop - erties of the implanted grafts or scaffolds to allow incor - poration , vascularisation and subsequent remodelling [ 79 ] . Systemic enhancement of bone regeneration As an alternative to local augmentation of the bone - regen - eration process , the use of systemic agents , including growth hormone ( GH ) [ 80 ] and parathyroid hormone ( PTH ) [ 81 ] is also under extensive research . Current evi - dence suggests a positive role for GH in fracture healing , but there are issues about its safety profile and optimal dose , when systemically administered to enhance bone repair [ 80 ] . There are also numerous animal studies and clinical trials showing that intermittent PTH administra - tion induces both cancellous and cortical bone regenera - tion , enhances bone mass , and increases mechanical bone strength and bone - mineral density , with a relatively satis - factory safety profile [ 81 , 82 ] . Currently , two PTH analo - gues , PTH 1 - 34 ( or teriparitide ) and PTH 1 - 84 , are already used in clinical practice as anabolic agents for the treat - ment of osteoporosis [ 81 , 83 ] , and research is being carried out into their off - label use as bone - forming agents in com - plex conditions requiring enhancement of bone repair , such as complicated fractures and non - unions . In addition to the anabolic agents for bone regeneration , current antiresorptive therapies that are already in clinical use for the management of osteoporosis could be used to increase bone - mineral density during bone regeneration and remodelling by reducing bone resorption . Biphospho - nates , known to reduce the recruitment and activity of osteoclasts and increase their apoptosis , and strontium ranelate , known to inhibit bone resorption and stimulate bone formation , could be beneficial adjuncts to bone repair by altering bone turnover [ 84 ] . In addition , a new pharmaceutical agent called denosumab , which is a fully human monoclonal antibody designed to target receptor activator of nuclear factor - (cid:1) B ligand ( RANKL ) , a protein that selectively inhibits osteoclastogenesis , might not only decrease bone turnover and increase bone - mineral density in osteoporosis , but also indirectly improve bone regenera - tion in other conditions requiring enhancement [ 85 ] . Recently , another signalling pathway , the Wnt path - way , was found to play a role in bone regeneration [ 86 ] . Impaired Wnt signalling is associated with osteogenic pathologies , such as osteoporosis and osteopenia . Thus , novel strategies that systemically induce the Wnt signal - ling pathway or inhibit its antagonists , such as scleros - tin , can improve bone regeneration . However , there are concerns about carcinogenesis [ 87 ] . Another approach for systemic enhancement of bone regeneration is the use of agonists of the prostaglandin receptors EP2 and EP4 , which were found to be skele - tally anabolic at cortical and cancellous sites . Promising results have been seen in animal models , without adverse effects , and therefore these receptors may repre - sent novel anabolic agents for the treatment of osteo - porosis and for augmentation of bone healing [ 27 ] . Finally , new treatments for systemic augmentation of bone regeneration may come to light while researchers are trying to elucidate the alterations seen at the cellular and molecular level in conditions with increased bone forma - tion capacity . Fibrodysplasia ossificans progressiva , a rare genetic disorder , is an example of how an abnormal meta - bolic condition can be viewed as evidence for systemic regeneration of large amounts of bone secondary to altera - tions within the BMP signalling pathway [ 88 ] , and may indicate unique treatment potentials . Conclusions There are several clinical conditions that require enhance - ment of bone regeneration either locally or systemically , and various methods are currently used to augment or accelerate bone repair , depending on the healing potential and the specific requirements of each case . Knowledge of bone biology has vastly expanded with the increased understanding at the molecular level , resulting in develop - ment of many new treatment methods , with many others ( or improvements to current ones ) anticipated in the years to come . However , there are still gaps ; in particular , there is still surprisingly little information available about the cellular basis for MSC - mediated fracture repair and bone regeneration in viv o in humans . Further understanding in this area could be the key to an improved and integrated strategy for skeletal repair . In the future , control of bone regeneration with strate - gies that mimic the normal cascade of bone formation will offer successful management of conditions requiring enhancement of bone regeneration , and reduce their morbidity and cost in the long term . Research is ongoing within all relevant fields , and it is hoped that many bone - disease processes secondary to trauma , bone resection due to ablative surgery , ageing , and metabolic or genetic skeletal disorders will be successfully treated with novel bone - regeneration protocols that may address both local and systemic enhancement to optimise outcome . Acknowledgements None . Author details 1 Department of Trauma and Orthopaedics , Academic Unit , Clarendon Wing , Leeds Teaching Hospitals NHS Trust , Great George Street , Leeds LS1 3EX , UK . 2 UK Leeds NIHR Biomedical Research Unit , Leeds Institute of Molecular Medicine , Beckett Street , Leeds , LS9 7TF , UK . 3 Section of Musculoskeletal Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 7 of 10 Disease , Leeds Institute of Molecular Medicine , University of Leeds and Chapel Allerton Hospital , Chapeltown Road , Leeds , UK . Authors ’ contributions RD contributed to the literature review and writing . EJ , DMcG and PVG contributed to the writing of specific sections of the manuscript within their main scientific interest , and critically revised the manuscript for important intellectual content . All authors read and have given final approval of the final manuscript . Competing interests The authors declare that they have no competing interests . Received : 15 February 2011 Accepted : 31 May 2011 Published : 31 May 2011 References 1 . Bates P , Ramachandran M : Bone injury , healing and grafting . In Basic Orthopaedic Sciences . The Stanmore Guide . Edited by : Ramachandran M . London : Hodder Arnold ; 2007 : 123 - 134 . 2 . Einhorn TA : The cell and molecular biology of fracture healing . Clin Orthop Relat Res 1998 , 355 ( Suppl ) : S7 - 21 . 3 . 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Submit your next manuscript to BioMed Central and take full advantage of : • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed , CAS , Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www . biomedcentral . com / submit Dimitriou et al . BMC Medicine 2011 , 9 : 66 http : / / www . biomedcentral . com / 1741 - 7015 / 9 / 66 Page 10 of 10 \ No newline at end of file diff --git a/silver_data/e65f6c15825baacfeea0cb7a3090e9027127a4e4.pdf.txt b/silver_data/e65f6c15825baacfeea0cb7a3090e9027127a4e4.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..674604e0cefdc6a707f5dd8650e836e0fe666c1a --- /dev/null +++ b/silver_data/e65f6c15825baacfeea0cb7a3090e9027127a4e4.pdf.txt @@ -0,0 +1 @@ +Cyclic Stretching of Soft Substrates Induces Spreading and Growth 1 Yidan Cui , Feroz M . Hameed , Bo Yang , Kyunghee Lee , Catherine Qiurong Pan , Sungsu Park , Michael Sheetz 2 Supplementary Information 3 4 2 Supplementary Figure 1 . Design and fabrication . ( a ) Design of the stretching device . ( b ) Cross - sectional 1 design of each stretching unit . Each stretching unit consists of a 3 - mm - wide culture chamber with a PDMS 2 ( polydimethylsiloxane ) membrane layer underneath and a post loading layer ( 2 - mm wide and 100 - µm 3 high ) of PDMS suspended over the third actuation cavity layer . Positive pressure was applied to this cavity 4 to push the loading post upwards , which deforms the flexible PDMS membrane . ( c ) Fabrication process of 5 the stretching device . 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 3 1 Supplementary Figure 2 . Device characterization . ( a ) Changes of center to center distance ( 2 µm pillar 2 arrays ) by 1 % , 5 % and 15 % stretch . Scale bar = 5 um . ( b ) Changes of center - to - center distance after 5 % 3 cyclic stretching for 6 hrs ( n > 300 pillars ) . Surface of the pillar array was contact printed with fluorescent 4 fibronectin - Cy3 . Scale bar = 5 um . Characterization of strains on device surfaces . Circumferential strain 5 parameters were plotted ( Left ) and consistency of strains over time under either cyclic or static stretching 6 pressure was also measured ( Right ) . 7 8 9 4 1 Supplementary Figure 3 . Test the effect of cyclic stretching on cell spreading and stress fiber formation 2 of another cell type . Representative images of primary human umbilical vein endothelial cells ( HUVECs ) 3 on soft pillars made of PDMS with and without stretching . After being seeded onto substrate , cells were 4 incubated with and without stretching for 6 hrs before being stained with fluorescein isothiocyanate 5 labeled phalloidin , anti - paxillin antibody and 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar = 20 µm . 6 Experiments were repeated three times and showed similar results . 7 8 9 10 11 12 13 5 1 Supplementary Figure 4 . Effect of large magnitude ( 15 % ) of cyclic stretching on cell spreading and stress 2 fiber formation on a flat PDMS surface . Percentage of PMEFs with long stable edges and long stress fibers 3 and cell area and cell aspect ratio on each substrate : flat PDMS ( n = 153 cells ) ; soft pillars with 15 % cyclic 4 stretching at 0 . 1 Hz ( n = 68 ) . Cells were fixed after 6 hrs of stretching and then stained with rhodamine - 5 phalloidin and DAPI . Scale bar = 30 µm . Error bars , s . e . m . * * P < 0 . 01 ; Student’s t - test . Experiments were 6 repeated at least three times . 7 8 9 10 11 12 6 1 2 Supplementary Figure 5 . Effect of total duration of cyclic stretching and subsequent relaxation time on 3 cell spreading and stress fiber formation on soft pillars . Percentage of spread PMEFs and cell area and 4 cell aspect ratio of PMEFs . Cyclic stretching was stopped 1 hr ( n = 96 cells ) , 2 hrs ( n = 102 ) and 4 hrs ( n = 5 93 ) after seeding and cells were then statically cultured for 18 hrs before being stained with rhodamine - 6 phalloidin and DAPI . Scale bar = 30 µm . Error bars : s . e . m . * * P < 0 . 01 , * * * P < 0 . 001 ; Student’s t - test . 7 Experiments were repeated at least three times . 8 9 10 7 1 Supplementary Figure 6 . Persistence of spreading . Cells were cultured without further cyclic stretching 2 for 24 hrs ( n = 81 ) or 48 hrs ( n = 79 ) after the initial cyclic stretching at 0 . 1 Hz for 4 hrs . Then cells were 3 fixed and stained with rhodamine - phalloidin and DAPI . Scale bar = 30 µm . Error bars : s . e . m . * * P < 0 . 01 , 4 * * * P < 0 . 001 ; Student’s t - test . Experiments were repeated three times and showed similar results . 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 8 1 Supplementary Figure 7 . Displacement of pillar displacements near the edge of a single cell upon both 2 stretch and relaxation . The orange line represents the approximate cell boundary . The green arrow 3 shown at the bottom of the image corresponds to a 100 nm displacement . 4 5 6 7 8 9 10 11 12 13 14 15 16 9 1 Supplementary Figure 8 . Time - dependent translocation of GFP - MRTF - A ( green fluorescent protein 2 tagged Myocardin - related transcription factor - A ) to the nucleus by cyclic stretch ( 5 % , 0 . 1 Hz ) . Plasmids 3 were transiently transfected into PMEFs through electroporation at least 24 hrs before imaging . 4 Stretching was applied 2 hrs after the seeding of cells in the chamber . Over 20 cells were analyzed . 5 Experiments were repeated three times and showed similar results . Scale bar = 30 µm . 6 7 8 10 1 11 Supplementary Figure 9 . Knockdown experiments . ( a ) Immunoblotting from PMEF cells transfected with 1 the indicated siRNAs . ( b ) Immunofluorescence images of YAP ( Yes - associated protein ) , MRTF - A , DAPI and 2 actin in PMEF cells transfected with the indicated siRNAs ( control indicates no siRNA treatment ) . For 3 immunostaining , cells were trypsinized and seeded either on stretch device or glass coverslips coated with 4 fibronectin . Cells were stretched ( 5 % at 0 . 1 Hz ) for 6 hours ( stretching was applied 2 hrs after seeding of 5 cells in the chamber ) on stretch device , or static cultured for 8 hours on glass coverslips . Then cells were 6 fixed and stained with MRTF - A and YAP antibodies , rhodamine - phalloidin and DAPI . Experiments were 7 repeated three times and showed similar results . Scale bar = 30 µm . 8 12 1 13 Supplementary Figure 10 . Time dependent imaging of knockdown cells . ( a ) Time - dependent 1 fluorescence images of PMEF cells co - transfected with the MRTF - A siRNA and YAP - GFP ( green fluorescent 2 protein tagged Yes - associated protein ) plasmid . Yellow circles in the figure indicate cell nuclear . ( b ) Time - 3 dependent fluorescence images of PMEF cells co - transfected with the YAP siRNA and MRTF - A - GFP 4 plasmid . ( c ) Time - dependent fluorescence images of PMEF cells co - transfected with the control siRNA and 5 MRTF - A - GFP plasmid . Stretching was applied 2 hrs after seeding of cells in the chamber . At least 10 cells 6 were analyzed for each condition . Experiments were repeated three times and showed similar results . 7 Scale bar = 20 µm . 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 14 Supplementary Methods . 1 Cell culture and staining for fluorescent microscopy . HUVECs were cultured in phenol red - free Medium 2 200 ( Medium 200PRF ; M - 200PRF - 500 , Life Technologies ) supplemented with Low Serum Growth 3 Supplement ( LSGS ; S - 003 - 10 , Life Technologies ) and maintained in an incubator at 37 °C with 5 % CO 2 . The 4 surface of the stretching device was functionalized with 10 µg / ml of fibronectin overnight at 4 °C . 5 Stretching was applied right after the seeding of cells in the chamber . For actin and paxillin staining , cells 6 were washed with PBS ( phosphate - buffered saline ) , fixed in 4 % paraformaldehyde for 20 min and 7 permeabilized in 0 . 5 % Triton X - 100 for 10 min , followed by blocking with 3 % bovine serum albumin for 30 8 min in room temperature , all in PBS . Cells were incubated with Anti - Paxillin antibody ( Mouse , ab3127 , 9 Abcam ) diluted at 1 : 500 in PBS for overnight at 4 °C . Then cells were washed three times with 0 . 05 % 10 Triton X - 100 in PBS and incubated with Alexa Fluor 594 Goat Anti - Mouse IgG Antibody ( A - 11005 , 11 Molecular Probes ) and fluorescein isothiocyanate labeled phalloidin ( P5282 ; Sigma - Aldrich Co ) diluted at 12 1 : 1000 in PBS for 30 min . DNA was stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; 0 . 2 mg / ml in PBS , 13 D9542 , Sigma - Chemical Co . ) at room temperature for 5 min . Images were acquired on a Delta Vision 14 System ( Applied Precision ) centered on an Olympus IX70 microscope and equipped with a CoolSNAP HQ 2 15 CCD camera ( Photometrics , Tucson , AZ ) . 16 siRNA transfections . siRNA transfections were done with DharmaFECT™ ( Dharmacon™ ) in antibiotics - free 17 DMEM according to manufacturer’s instructions . The siRNA sequences of the SMARTpool® ( Dharmacon™ ) 18 mouse YAP are 5’ - CCGAAAUCUUGGACGUGGA - 3’ , 5’ - GAAUAAAGGAUGGCGUCUU - 3’ , 5’ - 19 UCUUAAAUCACAACGAUCA - 3’ and 5’ - AAGGAGAGACUGCGGUUGA - 3’ . The siRNA sequences of the 20 SMARTpool® mouse MRTF - A are 5’ - GGACCGAGGACUAUUUGAA - 3’ , 5’ - GCUGCGUCCUGCUGUCUAA - 3’ , 21 5’ - GGUCAGCUCUUGUAACAGC - 3’ and 5’ - GCACAUGGAUGAUCUGUUU - 3’ . 22 Western blotting . Cells were lysed with RIPA buffer [ 150 mM NaCl , 50 mM Tris - HCl pH 7 . 3 , 0 . 25 mM EDTA , 23 1 % ( w / v ) sodium deoxycholate , 1 % ( v / v ) Triton X - 100 , 50 mM NaF , 5 mM sodium orthovanadate , protease 24 15 inhibitors ( Roche Applied Science ) ] . Samples were run in SDS - PAGE gels followed by western blot analyses 1 using the Pierce Pico ECL ( Thermo Scientific ) . Polyclonal anti - YAP ( Rabbit , provided by Prof . Marius Sudol , 2 National University of Singapore ) , polyclonal goat anti - MRTF - A ( Santa Gruz Biotechnology ) and 3 monoclonal anti - α tubulin ( Sigma - Aldrich Co ) were used . 4 Immunofluorescence imaging of knockdown cells . Cells were washed with PBS ( phosphate - buffered 5 saline ) , fixed in 4 % paraformaldehyde for 20 min and permeabilized in 0 . 5 % Triton X - 100 for 10 min , 6 followed by blocking with 3 % bovine serum albumin for 30 min in room temperature , all in PBS . Cells were 7 incubated with Anti - YAP ( Rabbit , provided by Prof . Marius Sudol , National University of Singapore ) 8 antibody diluted at 1 : 1000 in PBS and Anti - MRTF - A ( Goat , sc - 21558 , Santa Gruz Biotechnology ) antibody 9 diluted at 1 : 100 in PBS for overnight at 4 °C . Then cells were washed three times with 0 . 05 % Triton X - 100 10 in PBS and incubated with Alexa Fluor 488 Donkey Anti - Rabbit IgG Antibody ( A - 21206 , Molecular Probes ) , 11 Alexa Fluor 647 Chicken Anti - Goat IgG Antibody ( A - 21469 , Molecular Probes ) and rhodamine – phalloidin 12 ( 200 units in DMSO , R415 , Molecular Probes ) diluted at 1 : 1000 in PBS for 30 min . DNA was stained with 4 ' , 13 6 - diamidino - 2 - phenylindole ( DAPI ; 0 . 2 mg / ml in PBS , D9542 , Sigma - Chemical Co . ) at room temperature 14 for 5 min . Images were acquired on a Delta Vision System ( Applied Precision ) centered on an Olympus 15 IX70 microscope and equipped with a CoolSNAP HQ 2 CCD camera ( Photometrics , Tucson , AZ ) . 16 17 18 19 20 21 22 23 \ No newline at end of file diff --git a/silver_data/e77bb1c86f6d3468ae0c47ce4cb55561ee89d130.pdf.txt b/silver_data/e77bb1c86f6d3468ae0c47ce4cb55561ee89d130.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..ccb17b85b9e8a90dda602ede06701e3334048c00 --- /dev/null +++ b/silver_data/e77bb1c86f6d3468ae0c47ce4cb55561ee89d130.pdf.txt @@ -0,0 +1 @@ +( Un ) making AI Magic : a Design Taxonomy Maria Luce Lupetti m . l . lupetti @ tudelft . nl Delft University of Technology Delft , The Netherlands Dave Murray - Rust d . s . murray - rust @ tudelft . nl Delft University of Technology Delft , The Netherlands Figure 1 : ( un ) making magic . ABSTRACT This paper examines the role that enchantment plays in the design of AI things by constructing a taxonomy of design approaches that increase or decrease the perception of magic and enchantment . We start from the design discourse surrounding recent developments in AI technologies , highlighting speci fi c interaction qualities such as algorithmic uncertainties and errors and articulating relations to the rhetoric of magic and supernatural thinking . Through analyzing and re fl ecting upon 52 students’ design projects from two editions of a Master course in design and AI , we identify seven design principles and unpack the e ff ects of each in terms of enchantment and disenchantment . We conclude by articulating ways in which this taxonomy can be approached and appropriated by design / HCI practitioners , especially to support exploration and re fl exivity . CCS CONCEPTS • Human - centered computing → Interaction design theory , concepts and paradigms ; Interface design prototyping ; • Com - puting methodologies → Arti fi cial intelligence . KEYWORDS arti fi cial intelligence , critical design , research through design , criti - cal computing , magic ACM Reference Format : Maria Luce Lupetti and Dave Murray - Rust . 2024 . ( Un ) making AI Magic : a Design Taxonomy . In Proceedings of the CHI Conference on Human Factors Permission to make digital or hard copies of part or all of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for pro fi t or commercial advantage and that copies bear this notice and the full citation on the fi rst page . Copyrights for third - party components of this work must be honored . For all other uses , contact the owner / author ( s ) . CHI’24 , May 11 – 16 , 2024 , Honolulu , USA © 2024 Copyright held by the owner / author ( s ) . ACM ISBN 979 - 8 - 4007 - 0330 - 0 / 24 / 05 . https : / / doi . org / 10 . 1145 / 3613904 . 3641954 in Computing Systems ( CHI ’24 ) , May 11 – 16 , 2024 , Honolulu , HI , USA . ACM , New York , NY , USA , 21 pages . https : / / doi . org / 10 . 1145 / 3613904 . 3641954 1 INTRODUCTION “The new AI can write and talk ! It can draw , do fake photos and even make video ! It even has AI folklore . Authentic little myths . Legendry . ” [ 114 ] As the science fi ction writer Bruce Sterling emphasizes , there is a discourse of great excitement and wonder about all the novel arti fi cial intelligence ( AI ) algorithms and applications that have been launched in the last years – a Mardi Gras of AI [ 114 ] . After the last winter , the current spring [ 43 ] of AI tools is now being leveraged to speed up and enhance a multitude of works , especially in creative practices where applications range from idea explo - ration [ 22 , 66 , 68 ] and ideation [ 128 ] to project documentation [ 21 ] , creative partnership [ 71 , 86 ] , and so on . AI has always been a metaphor - driven fi eld [ 2 ] , and the search for useful metaphors that articulate the possibilities of technology has a hand in shaping the fi eld [ 7 , 65 ] . As these tools are adopted , along with wonder and excitement , we see a metaphorical vocabulary that stems from the world of the supernatural and emphasizes the ‘magical and enchanting’ nature of these technologies . As an illustration , Derczynski [ 31 ] reports on a series of recurring terms used by a variety of professionals that show this metaphorical link to magic , such as spellcasting for providing or developing an input to a prompted language model ; alchemy , to describe a practice of constructing model inputs to discover the di ff erent model outputs they lead to ; invoking , to change the characteristics of a model output ; and more . Such a tendency to use the language of magic is not new – “it is often the case that new technologies are presented as magical” [ 18 ] . However , around AI tendencies towards magical thinking are emphasized as computer programs are seen to carry out tasks that were once unimaginable for computers to do [ 108 ] ; to have CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust skills that were once thought to be exclusive of humans [ 87 ] , e . g . , to converse [ 57 ] and express creativity [ 38 , 76 , 88 ] . The term ar - ti fi cial intelligence – particularly in contrast to alternatives such as ‘complex information processing systems’ [ 61 ] – invites us to attribute typically human properties such as “thought , imagination , memory , will , sensation , perception , belief , desire , intention , or feel - ings” to AI systems and products in which they are embedded [ 24 ] . Such emphasis on replication of human intelligence , and the ease with which we over - attribute abilities to computational systems , means that AI has now “come to have overtones of trickery” [ 125 ] as we discuss for example the gap between being able to produce sentences and actually understanding language [ 9 ] . Here , we work with the idea of enchantment : “the experience of being caught up and carried away” , where our attention is captured , we are “charmed and disturbed” , and our “background sense of order has fl own out the door” [ 12 ] . Contemporary AI algorithms , especially deep learning , have a particular power to enchant . They embody a level of complexity that is hard for experts – let alone the general public – to grasp [ 18 ] . This complexity renders AI algo - rithms inherently opaque [ 5 , 74 , 96 ] , in their structure , their internal patterns , and their links to the world [ 39 ] : “when a computer learns and consequently builds its own representation of a classi fi cation decision , it does so without regard for human comprehension” [ 16 ] . This gap of comprehension is the space through which magic fl ows [ 4 ] , opening space for ambiguity and unexpectedness , and leading to truly enchanting experiences [ 85 ] . The language of magic and superhuman abilities is purposefully embraced by tech industries to build a public imaginary of AI as a silver bullet [ 73 , 122 ] for solving all problems . As some examples : Future Tools [ 118 ] “collects & organizes all the best AI tools so you too can become superhuman ! ” ; Google’s new text feature for the Chrome browser is called Magical [ 81 ] ; Figma’s AI toolset is called Magician [ 42 ] : “Every little thing it does is magic . A magical design tool for Figma powered by AI . ” ; and Runway AI [ 106 ] introduces its AI tools with the header : “AI Magic Tools . Dozens of creative tools to ideate , generate and edit content like never before” . However , as well as generating “interest in the fi eld , spur [ ing ] fi nancial invest - ment , and trigger [ ing ] research and development” , this language also contributes to obfuscating the actual practices involved in the ‘making’ of AI systems [ 36 ] ; it disproportionately emphasizes the possibilities and opportunities o ff ered by AI rather than its actual functionalities , blurring the line between the fantasy and the real - ity of these technologies [ 36 ] . Designing and communicating AI things as supernatural – enchanted – products , in fact , shapes the social perceptions of these systems , taking them out of the realm of mere technical tools to be regarded as socially capable agents [ 113 ] and / or socially valuable applications [ 18 ] . Building on Bennett’s development of enchantment [ 12 ] , Campolo and Crawford [ 18 ] de - fi ne this phenomenon as enchanted determinism : “a discourse that presents AI , and deep learning techniques especially , as magical , thus outside the scope of present scienti fi c knowledge , yet also de - terministic , in the sense that AI algorithms can nonetheless detect patterns that give unprecedented access to people’s identities , emo - tions , and social character” . Working in this magical domain allows designers to focus on mastery of the illusions that they create [ 113 ] while minimizing concern for the consequences they cause [ 18 , 36 ] . This paper investigates the ways in which design can contribute to increasing and decreasing the sense of enchantment in experi - ences with AI products , supporting an understanding of the dy - namics and e ff ects of magical thinking in the design of AI things . We do this by building a taxonomy of design principles that en - gage with magic and AI in the creation of interactive products . The taxonomy is based on a combination of theoretical development and critical re fl ection on two years of the Interactive Technology Design Master’s course held at Delft University of Technology ( TU Delft ) between the years 2021 and 2023 . While student work may not represent the full landscape and richness of AI things we can design , it gives a unique window into the conceptual development of designers who are engaging with AI . We re fl ected upon students’ projects following the notions of enchantment and disenchantment , and used these to iterate on our taxonomy , developing a set of archetypal principles that contribute to creating AI - based products and services . We expand the discussion of these by articulating how designers can approach and appropriate the taxonomy in their practice . 2 MAGICAL THINKING IN THE DESIGN OF TECHNOLOGY Figure 2 : AI products are inherently enchanting things as they hold a magical aura that is fed by AI uncertainty and errors . We de fi ne magical aura ( the circles around the cube ) in section 4 , building on the work by Gell [ 51 ] “Any su ffi ciently advanced technology is indistinguish - able from magic” [ 26 ] This famous quote summarizes a complex entanglement of phe - nomena at play when it comes to the perception of technology as a product of magic . Gell [ 51 ] , committed to sca ff olding the value and workings of art , provides an extensive explanation of how creative practices such as painting , poetry , and fi ction can contribute to creating a sense of enchantment toward man - made things . But as Gell [ 51 ] further clari fi es : “enchantment is immanent in all kinds of technical activity” and stems from the uncertainty we experience when encountering novel technologies about which we only have partial knowledge . Magical thinking and language have a long his - tory in the design of technology and human - computer interactions ( HCI ) . Designers and makers of technology are often regarded as the “magicians of the twenty - fi rst century” [ 27 ] . Computational technologies are increasingly used to generate novel product con fi g - urations whose functionings cannot be fully understood [ 69 , 104 ] ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA generating a sense of wonder . These blend the familiarity of ex - isting products with the unexpectedness and ambiguity of novel technologies [ 69 , 104 ] . As Landin [ 69 ] explains , technological prod - ucts can become magical as they bring unexpectedness and surprise to the interaction , while maintaining recognizable features . In this section , we review two distinct approaches at the inter - section of technology , HCI , design , and enchantment . 2 . 1 Designing for Enchantment Figure 3 : Main actions identi fi ed in literature regarding the design of AI things that contribute to emphasizing AI’s en - chanting potential : ’Apply Magic Metaphors’ and ’Apply Stage Magic Principles’ “Magical technology consists of representing the techni - cal domain in enchanted form” [ 51 ] Over the last decades , several authors have been exploring the parallels between HCI design and stage magic , such as “a need for consistency , the use of metaphors , and smoothness throughout the interactions” [ 4 , 117 ] . This is particularly evident in those applica - tions of computing where entertainment and engagement of the audience are key to a successful user experience , such as interactive performances , installations , and games [ 83 ] . Some metaphors from the world of magic have even gained so much popularity in HCI that they became emblems of interaction paradigms . The concept of a magic wand , and the related metaphor of spell - casting , is regu - larly used for embodying handled navigation systems that allow to control of smart environments [ 25 ] , while the idea of magic mirror has become the recurring metaphorical reference for materializing the ‘on - user’ augmented reality paradigm [ 37 , 80 ] . Magic , however , often materializes in HCI in a much more subtle way . When enhanced through the use of emerging technologies , familiar products become “more useful , more delightful , more infor - mative , more sensate , more connected , more engaging” [ 104 ] . But together with becoming extraordinary , these products also become less easy for people to understand their inner functioning , creating a sense of wonder and enchantment . Di ff erent views do exist on what an object with the power to enchant is and does . Bennet [ 12 ] looks at enchantment more as a state in perception rather than as a quality of objects . Conversely , Rose [ 104 ] believes that an en - chanted object is something that starts as an everyday object that gains ‘magical powers’ by the addition of technology , which makes the user both comfortable and captivating . Fisher ( as in [ 84 ] ) , in - stead , also has a focus on enchantment as a quality of an object , yet this “does not remind us of anything we know and we fi nd ourselves delaying in its presence for a time in which the mind does not move on by association to something else” . Despite the di ff erent views , enchantment can be understood as a relational quality between user perception and the object’s performativity . In particular , objects’ ambiguity disorients but also engages , and enchants by o ff ering the potential for unexpectedness and discovery [ 85 ] . This more implicit way of embedding magic in HCI , the design for enchant - ment , is actually very popular in a variety of HCI domains , such as the Internet of Things ( IoT ) and the human - robot interaction ( HRI ) fi elds where products are intentionally designed for encouraging people to “attribute properties such as thought , imagination , mem - ory , will , sensation , perception , belief , desire , intention , or feelings to these products” [ 24 ] . Leveraging enchanting mechanisms ( e . g . , deceptive behaviors as in [ 79 ] ) products are conceptualized and experienced as social agents rather than tools [ 24 , 93 , 113 ] . In this regard , Watson and colleagues [ 123 ] identi fi ed thirteen principles of stage magic that can be applied to user experience design , such as vanishing , transformation , and prediction . Designing for enchantment , then , allows the achievement of seamless and engaging interactions with arti fi cial agents [ 102 ] , while also encouraging consideration of “aspects of interaction that often remain underdeveloped in HCI , such as cultural aspects of interaction” [ 105 ] . As Ylipulli and colleagues [ 129 ] argue “We deem that magic as a concept can mobilize thinking that helps to come up with idealized and alternative realities , and related ideas” . The use of enchantment and ‘make - believe’ in technology design , however , is a double - edged sword . Many examples show how computational products easily turn from friendly enchanted things to spooky presences that surround us [ 32 , 104 ] . Targeted advertisements on personal devices , for instance , are highly associated with feelings of creepiness [ 130 ] , especially due to the partial inexplicability of how the systems work and the people’s impression of devices being always listening [ 17 ] . 2 . 2 Perils of Enchantment The perceptual mechanism of enchantment itself – the gap in under - standing that disorients and captivates – often plays a signi fi cant role in the key ethical challenges associated with digital products , especially when powered by AI . On the one hand , people have higher chances of improperly calibrating trust in arti fi cial agents [ 30 ] if the capabilities of this are enhanced through the language of magic . As Wolf and colleagues [ 126 ] argue : “Users ‘enchanted’ by deceptive machines are likely to make inappropriate decisions based on these deceptions” . Building on a classical quote from Plato [ 98 ] , in fact , Turkle [ 116 ] argues that every deceiving thing can be also considered a form of enchantment and vice versa : every - thing that enchants also deceives . Deception , whether intentional or not , is inherently risky as it can give a false sense of mutuality in human - agent interaction ( e . g . , in sensitive care settings ) ) , and it can be used to conceal non - anthropomorphic capacities ( e . g . , to hide a user monitoring mechanism present in a robot ) making people less alert towards possible dangers , thus vulnerable [ 29 ] . On the other hand , the problem of enchanted technologies extends far beyond the issue of users’ misunderstanding of product capabili - ties . Notions of magic and mechanisms of enchantments hinder the CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust way for people to properly grasp the complexity of the ecosystems populated by technological products [ 82 ] and their risks [ 18 ] . “The dominant narrative of technology as enchantment helps maintaining a cloak of impenetrability as to how technologies actually work , exploiting the genuine dif - fi culty in grasping machine operations that run in the background , unseen , unheard , unknown , and incom - mensurable to human scale” [ 18 ] With technological products becoming increasingly more com - plex and appearing as “magical unknowns” [ 4 ] , enchantment also becomes a mechanism for masking exploitative work ethics [ 27 , 36 ] , power struggles [ 111 ] , and dramatic environmental impacts [ 82 , 112 ] . This calls for more cautious approaches to the design and communication of advanced technologies , especially AI for which it has become harder and harder to separate actual capabili - ties from industry and media promises [ 125 ] . This contributes to a phenomenon that Campolo and Crawford [ 18 ] de fi ne as enchanted determinism : “a discourse that presents AI , and deep learning tech - niques especially , as magical , thus outside the scope of present scienti fi c knowledge , yet also deterministic , in the sense that AI algorithms can nonetheless detect patterns that give unprecedented access to people’s identities , emotions , and social character” . 2 . 3 Designing for Disenchantment Figure 4 : Design actions that contribute to reducing AI prod - ucts’ magical aura and diminishing their enchanting poten - tial The more AI products take part in our everyday practices , the more it becomes of crucial importance to “distinguish research , pouring from companies and laboratories , from speculation , fan - tasy , and fi ction” [ 125 ] and to address social and ecological implica - tions along with technical discourses [ 101 ] . Research in the area of explainable AI and AI auditing implicitly sets out to answer these needs . As Campolo and Crawford [ 18 ] explain , experts in the area of AI interpretability in machine learning are developing ways to provide rational explanations to classi fi cations and predictions that enchanted discourses of AI withhold . AI auditing , instead , looks at AI algorithms documentation and related operations to account for and ensure the AI system’s trustworthiness [ 67 ] as opposed to the dominant claims of progress and liberation . These approaches represent important strategies for experts in various domains , from technical to legal , to disenchant AI , as they allow for “dissecting and inspecting” algorithms and the systems surrounding them . How - ever , their e ff ect on the way AI products are shaped , communicated , and perceived by the public is indirect : it is up to designers and developers to take on the responsibility for the rhetorics that AI is presented with and the semantics of how it is experienced . Conversely , a distinctively designerly perspective comes from the fi eld of Critical and Speculative design in which provocative designs are used to bring awareness to the myths and beliefs we be - stow on AI things and technology more broadly [ 33 , 77 ] as well as to manifest often obscure mechanisms of technology . For instance , allegorical steering wheels have been used to challenge the myth of the autonomous car and its related narratives [ 77 ] , and custom PCBs satirically intended to predict luck and harmony , have been designed to actually make us re fl ect on the beliefs and assumptions of superhuman capabilities and objectivity that we project on AI [ 41 ] . With complex technological infrastructures , a range of design practices needs to be brought together to articulate the multiple mechanisms at play [ 89 ] . Critical and Speculative projects , how - ever , often remain marginal practices , and their e ff ects are limited to public debate , rather than getting embedded in actual product development processes [ 127 ] . 3 TOWARDS A TAXONOMY OF MAGIC IN AI DESIGN As critical design and HCI researchers , we share the view of the many authors [ 18 , 36 , 113 , 125 ] that problematize the use of the magic rhetoric concerning novel technologies , and especially AI systems . We agree with Sharkey and Sharkey’s [ 113 ] position that it is a moral imperative for designers of AI systems to be truthful about the actual capabilities of the technologies they are devel - oping , along with being honest about the intent of creating the illusion of intelligence . Fanciful construction of AI as magic , as holding higher - than - human capabilities , while disregarding its ac - tual feasibility would be a great mistake [ 92 ] : being swept up in enchantment can blind one from the actual implications of AI , and prevent the development of strategic methodological sensitivities that critically ground AI analysis and claims [ 36 ] . However , as pre - viously discussed ( Section 2 . 1 ) , we also see the pull of enchantment around new technologies , and argue that rather than dismissing it altogether , we should build a deeper understanding of its dynam - ics and how to skillfully and honestly leverage the possibilities of enchantment in the design of future technological products . More ontological resources are needed to navigate this intricate space [ 23 , 75 ] , and theoretical tools such as taxonomies hold great potential . In this case , we set out to create a taxonomy that can not only help structure the body of knowledge in the area of magic and AI and predict its future developments [ 54 ] but also provide de - sign / HCI practitioners , educators , and researchers with a resource for informing future designs and for practicing re fl exivity on their processes . In what follows , we illustrate the process and results of our ( Un ) making AI Magic taxonomy development . We focus speci fi cally on AI technologies not only because these are considered the most important of the fourth industrial revolution [ 53 ] but also and foremost because of the strong connection between AI and the narrative of technology as magic [ 72 , 92 , 107 ] . We aim to tease out the mechanisms for enchantment – and disenchantment – that designers can draw on around AI systems . ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Figure 5 : Taxonomy building process : grounding , applying , and articulating . This does not mean eliminating the sense of enchantment en - tirely : as Sharkey and Sharkey [ 113 ] discuss , just as cartoon anima - tors do not need to sell their characters as truly thinking beings , AI designers can leverage the active participation of people and their willing suspension of disbelief to maintain a sense of enchantment without deception . Our taxonomy builds on Gell’s “halo - e ff ect” of technology – the inherent property of the technological product to enchant . This halo is intrinsically bound to the beholder’s mental model of the technology in use , as it responds to the uncertainty we experience when encountering novel technologies about which we only have partial knowledge [ 51 ] . We focus on strategies that designers can employ to a ff ect the halo – the perceived magical aura of technological artifacts . 3 . 1 Taxonomy Building Approach Following [ 94 , 95 ] , we employed a “conceptual – to – empirical” ap - proach to building the taxonomy . This involves setting out the characteristics of the taxonomy , de fi ning a stopping condition , and then a process of iteration where an initial conceptual version of the taxonomy is grounded in a fi xed set of examples . We fi rst set our area of concern , or meta - characteristic , as the di ff erent strategies that can be employed to modulate the magical aura of interactive AI products : that is each dimension in our taxonomy will be something that can be done to a ff ect the kind of enchantment that a product engages in . Our example set is a collection of 52 student projects ( see Section 3 . 2 ) . The stopping condition is that i ) objectively , all projects are associated with at least one strategy , and ii ) subjec - tively , all strategies of interest seen in the projects are accounted for . After setting these conditions , the process involved three main steps , expanded in Sections 4 – 6 : • Grounding ( Section 4 ) . An initial deductive approach allowed us to derive the fi rst set of categories from a combination of literature and personal re fl exivity . This process started from intuition , theory , or conceptualization and identi fi es dimensions and characteristics in the taxonomy by a log - ical process derived from sound conceptual or theoretical foundations [ 95 ] . • Applying and revising ( Section 5 ) . Next , we empirically artic - ulated the taxonomy through observations and re fl ections on the collection of 52 design student projects . Building on design academic traditions , here the empirical work was ap - proached as an annotative form of knowledge production [ 50 ] . As with annotated portfolios [ 49 ] , which are ’descriptive ( of past occurrences ) and intended to be generative - inspirational ( of future possibility ) ’ [ 15 ] , we used projects’ curation and critique to fi nd patterns and to explicate how particular arti - facts and their features embody elements and dynamics of a theoretical space [ 50 ] . This was carried out by the fi rst au - thor’s examination of the projects through the classi fi cation identi fi ed in step 1 with the addition of missing categories to account for new discoveries . • Iteration , agreement and articulation ( Section 6 ) . Through iteration and discussion with the second author , we veri fi ed the fi rst author’s classi fi cation and fi nalized the taxonomy . 3 . 2 Taxonomy Corpus To develop the taxonomy , we re fl ected upon a collection of 52 design student projects , illustrated in Figures 7 and 8 that were the outcomes of a 20 - week interaction prototyping course from two years ( 2022 / 23 ) of the Interactive Technology Design master course held at TU Delft . Students were explicitly focused on understanding and using novel AI tools in interaction design projects , developed through an iterative prototyping process . They were provided with tutorials and dedicated technical support for working with AI tools such as Teachable Machine , Edge Impulse , Voice Flow , and more , as well as with brief and provocative lectures on conceptual aspects of interaction design and AI . This represents a large body of work by developing designers , giving a window into their conceptual development . It illustrates how designers may be inclined to think about AI in magical terms and whether and how this may translate into speci fi c product features and design dynamics . Design education is often a testbed for the exploration of theories and methodologies in design [ 52 , 90 ] . For instance , the notion of en - chanted objects by Rose [ 104 ] was explored and articulated through his educational work at the Massachusetts Institute of Technology ( MIT ) where he was teaching the Tangible Media graduate course . CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Figure 6 : Initial version of the ( Un ) making AI Magic taxonomy , based on literature , showing two approaches to designing for enchantment , through applying magic metaphors or stage magic principles , and two strategies for disenchantment : manifesting mechanisms and materializing beliefs . However , we do acknowledge that students’ work may not rep - resent the full landscape and richness of design in relation to magic and AI , and address this further in the discussion . 3 . 2 . 1 Ethical approval and students’ consent . Students were asked to provide consent for using their educational outputs in research under university ethics board cases 2850 ( 2023 ) and 2251 ( 2022 ) . In all cases , students were free to opt - out , and the consent was gath - ered by someone who was not part of the core teaching / marking team to avoid power imbalances . They were asked whether their concept cards ( project images with descriptions ) - which are the basis of this work - could be interpreted . We intentionally do not mention student names in the paper to avoid a sense of public critique of their work . 4 GROUNDING THE TAXONOMY Starting from the focus on the magical aura of AI technology and building on the literature discussed in Section 2 , we identi fi ed two broad categories of action between design and the magical aura of an AI product : enhancement and diminishment . Design for enchantment covers actions that enhance this aura , making products feel more magical , building on Section 2 . 1 , Figure 3 . Concretely , Borrowing magic metaphors is an explicit approach , engaging with the language and imaginary of magic , such as when naming products ( see many examples in Section 2 . 1 ) . Applying stage magic principles works implicitly , to create seamless , fasci - nating , and engaging product experiences . For example , the NEST smart thermostat uses anticipation [ 62 ] to create a sense of magic functioning . Design for disenchantment conversely focuses on reducing the magical aura ( Section 2 . 3 , Figure 4 ) . Materializing beliefs involves using provocative designs to bring awareness on the myths and assumptions we bestow to AI [ 59 ] or highlighting myths of AI objectivity about non - quanti fi able things like luck [ 35 ] . Manifesting mechanisms and properties of AI products , such as the heavy hunger for data that AI systems come with [ 1 ] , reduce the aura by making the mechanics more apparent to the end user . This led to the initial structure seen in Figure 6 . 5 APPLYING AND REVISING THE TAXONOMY The students’ projects ( Figures 7 and 8 ) , summarised through pic - tures and descriptions , were clustered and annotated by the fi rst author following the four main design principles identi fi ed in the fi rst version of the taxonomy ( Figure 6 ) : apply magic metaphors ( Section 6 . 2 ) , apply stage magic principles ( 6 . 1 ) , manifest mecha - nisms ( 6 . 5 ) , and materialize beliefs ( 6 . 4 ) . To properly understand the projects , and especially the way AI was used and conceptualized , the analysis made use of various project materials that captured the students’ process and outcomes , such as pictures , illustrations , videos , descriptions , and written re fl ections . As a result of this activity , we found that a great number of projects embodied principles of stage magic ( 18 ) , while some lever - aged magic metaphors ( 4 ) and manifested mechanisms ( 3 ) , and only one seemed to represent the principle of materializing beliefs . As a great number of projects ( N 28 ) did not fi t within our four initial categories , we iteratively revised the taxonomy ( Figure 9 ) and identi - fi ed three additional categories : presume AI ( 4 ) , dispel enchantment through play with AI ( 1 ) , and summon AI as supernatural entity ( 15 ) . Finally , we excluded 5 projects from our classi fi cation and analysis , ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Figure 7 : Overview of the 28 projects resulting from the 2022 edition of the Interactive Technology Design Course CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Figure 8 : Overview of the 24 projects resulting from the 2023 edition of the Interactive Technology Design Course as these did not engage with AI , either in the conceptualization or prototyping . We note that the vast majority of projects operated within the space of enchantment , rather than disenchantment . This could be interpreted as a partial lack of critical perspectives in the design of AI things , or a result of the framing of the course . Rather than quantifying , however , our interest here is to learn what possibilities and dynamics each of the identi fi ed design principles a ff ords and how . In the following , we unpack these discussing a selection of representative examples . ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Figure 9 : The revised version of the ( Un ) making AI Magic taxonomy from Figure 6 after the analysis of the projects uncovered the additional enchanting principle of summoning supernatural AI , possibilities around playing with AI , and a new category of projects that take AI as a given without directly engaging with it . 6 ARTICULATING THE TAXONOMY Here we present our fi nal taxonomy ( Figure 9 ) , through images and descriptions of representative projects . 6 . 1 Apply stage magic principles Several projects ( 18 ) manifested an approach to design character - ized by the use of stage magic principles to create engaging and seamless interactions . Miron , an iron that physically resists and op - poses user intentions when ironing clothes , lets people experience the agency of products . Summit of the Objects takes this further to allow users to negotiate with objects , so that opening a tap for wa - ter begins an animated debate about how resources should be used , and which product should operate at a certain time . Projects in this category also create enhanced experiences of private and public environments : the Fordy car manifests its ability to anticipate needs by opening the rear door when a user approaches with shopping bags ; StairsOverElevatorUse uses computer vision to understand when to nudge users to use the stairs instead of the elevator or not ( e . g . when carrying luggage ) . In contrast to other categories , projects that apply stage magic principles present a relatively ad - vanced implementation of technology , in a way that is so seamless that the technological mechanisms are not perceivable by the user . They create smooth and intuitive experiences that ‘feel like magic’ . The Under the Loop project ( Figure 10 ) , for instance , consists of a device that resembles a magnifying glass . This allows citizens to provide input on future city developments , thus enabling adminis - trations to learn about the thoughts and concerns of the public . The device is fi tted with sensors ( e . g . , for measuring air quality ) as well CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Figure 10 : Under the Loop : citizen participation in placemak - ing using a magic magnifying glass to collect data and opin - ions as a microphone , used to collect spoken comments about problem - atic situations that a citizen may encounter in their surrounding . A speech - to - text algorithm is used to translate the comments into data and , together with the sensor data , place these on a heat map of the city . Of all this technical functioning , the user only perceives the presence of the microphone and interacts with a button . The interaction with the device is an act of transformation : from a per - sonal experience of space and place , spoken opinion is brought into a publicly accessible map of issues and contestation . As such , the designers engaged with key principles of stage magic : the changing state of the matter at hand [ 123 ] and the misdirection of attention [ 117 ] . Projects in this category , then , focus on promoting engage - ment and seamlessness [ 124 ] in use , and AI here mostly represents a tool for achieving unprecedented user experiences . 6 . 2 Applying Magic Metaphors This category includes a few ( 4 ) projects , yet they are powerful . This category makes use of magic metaphors to describe and shape projects , and enhance the perception of the designed artifacts as magical . Our initial conception of this category was that the magic metaphors work as communication devices , aimed at supporting the end users’ mental image of the AI product . However , through the re fl exive activity of engaging with the projects , it became clear that the borrowing of magic metaphors is also a way for the designers themselves to unpack the features and interaction dynamics that an AI product may embed . The LUMI project ( Figure 11 ) is designed to sensitize hotel guests about the scarcity of energy and promote sustainable behaviors . It very explicitly builds on Harry Potter’s concept of deluminator : a magical lighter - like device that is used to absorb as well as return light from any light source to provide cover to the user . LUMI works under a similar principle : it is a lantern - like device , a single light point that holds light or sends it to di ff erent objects in a hotel room . In this case , while the metaphor may be known or not to the audience , it was strongly present in the imagination of all the students in the team who , through this , managed to agree on an overarching working principle , to envision the interaction architecture and components , and to build a coherent and understandable interactional aesthetics . In a way , then , magic metaphors can help consciously guide the design of AI products as enchanted things , to bring focus on how magic principles may be embedded . These projects often leverage stage magic principles ( Section 6 . 1 ) : LUMI uses an optical illusion to give the impression that the same light ‘jumps’ from object to object . Figure 11 : LUMI : embodying light energy through the magic metaphor of an enchanted lantern . However , by explicitly using magic metaphors these projects emphasize and build on the supernatural conceptualization of the designed artifacts . This is a signi fi cant di ff erence for the phenome - non of enchantment : as Tognazzini [ 117 ] explains , with stage magic spectators engage with an illusion and suspend their disbelief , go - ing along with the idea that the magician is magical , even if only within the boundaries of the performance . AI products leveraging magic metaphors invite users to suspend their disbelief , making explicit the staging nature of the magic at play but centering the magic on the object itself , which both enchants and disenchants . 6 . 3 Summon AI as supernatural entity The most prominent category that we identi fi ed in the attempt to understand the projects that did not fi t with our initial classi fi cation is one that manifests a view of AI as a supernatural entity . This view is coherent with our general understanding of how the magical aura of AI in fl uences designers’ understanding of the technology they are starting to engage with . Through the analysis of projects , we further realized that this vision can also translate into an approach to the design of AI things themselves . A great number of projects ( 15 ) , in fact , engages with AI in a fashion that sits in between the use of magic metaphors and the application of stage magic principles , yet has very distinct implications . AI is ‘summoned’ as an entity with supernatural powers , such as the ability to interpret and interfere with complex human experiences . In the Reframe your Thought project ( Figure 12 ) , AI is described as a tool that adds a visual dimension to therapy , understands conversations between therapists and clients , and generates positive images as alternatives to the ones representing the struggles of the person ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Figure 12 : Reframe your Thought : generative AI techniques that create the illusion of a superintelligent being assisting in therapy . going through therapy . The underlying assumption is that AI is capable of understanding what human struggles are and what better alternatives there could be . The narrative , in these projects , is the one of AI as a possible superintelligence [ 14 ] . This is a controversial subject , and it is not uncommon for projects in this group to reveal a lack of thorough and critical engagement with the nature and qualities of AI . Despite the technical accomplishments of Reframe your Thought , in many projects seen here , storytelling and Wizard of Oz techniques are extensively leveraged to materialize the design concepts , often at the expense of technical development . The aesthetics of the projects also contribute to conveying this vision : AI things are shaped as abstract and symbolic objects , in which it is hard to determine where technology sits and how it works . An AI system may remind an Egyptian temple ( Reframe your Thought ) , a shell ( The Shell ) , a totem ( Connective Senses ) , and even a sand garden ( Sand me Awareness ) . Designers are caught themselves falling for the enchanting power of AI and ‘dragging’ the audience with them through the staging of the ‘supernatural AI’ . 6 . 4 Materialize Beliefs This category covers projects where provocative artifacts are used to bring awareness to the myths and assumptions we bestow to AI . Somewhat surprisingly it includes only 1 project from the course , despite being a strong staple of critical practice ( e . g . [ 28 , 64 ] ) . While examples in the category summoning AI as supernatural entity ( Sec - tion 6 . 3 ) can also be considered as implicitly materializing the belief that AI holds superior abilities , projects in this category take a crit - ical stand to the beliefs articulated , exposing them for scrutiny . As we learned from the literature , critical and speculative designs can be used to address matters of concern regarding AI beliefs and in - tentionally promote discussion about these . In the Colored Realities project ( Figure 13 ) this approach was translated into a generative newspaper , presented on a screen and controlled through a dial placed on a pedestal . The dial allows people to select a writing style or a political standpoint they would like to read their article with . Figure 13 : Colored Realities : a dynamic digital newspaper gen - erated by large language models that allow users to control the political leanings and writing style of the text of any given news item . When turning the dial , the text in the article instantly changes to re fl ect the desired style . The project makes evident how using AI capabilities for generating personalized content can easily turn into an instrument of power . The generated articles are based on the same facts but leave out details in order to follow a speci fi c style and set of values , obscuring other viewpoints and hindering people from getting the complete picture of events . In contrast , while the project Reframe your Thought is grounded on the assumption that AI is capable of understanding human struggles and acting to promote positive change , this capability is not emphasized but rather quietly embedded into a ‘gentle’ product that mediates between a therapist and a client’s conversations . A project designed for materializing beliefs [ 28 , 59 , 64 ] , instead intentionally puts the spotlight on typically hidden features of the technology . These beliefs are translated into the product features - such as the dial in the Colored Realities project - inviting the audience to try out , indulge in experimentation , and see and feel what happens if we engage with a given belief . By prominently materializing beliefs , exaggerating and confronting them , this type of design disenchants . 6 . 5 Manifest mechanisms This category covers projects that manifest the mechanisms of AI , as a way to disenchant . This is close to the approach of materializing beliefs ( Section 6 . 4 ) , but distinct in that here it is the mechanisms of action rather than beliefs and possibilities that surround them that are highlighted . The projects in this category ( 3 ) built seamless products and interactions that engage with popular concepts asso - ciated with AI , such as personalization ( Ready for your tea ? ) , social connectivity ( Own Faces ) , and behavior change ( A Closer Look ) . To - gether with providing an apparently clear and smooth interaction , the projects in this category also all present a ‘backside’ intended to put a spotlight on the hidden mechanisms at play and their poten - tial intricacies . A Closer Look ( Figure 14 ) is a smart health system in the form of a mirror that allows users to do daily checkups at CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Figure 14 : A Closer Look : a critical exploration of AI data collection and personal wellbeing , manifesting surveillance capitalism through a magic mirror , that invites visitors to play the role of either a client or the invisible data architec - tures that supply them with pharmaceuticals . home , as well as to get medications . The panel is equipped with several knobs and sliders that allow the user to select preferences and set parameters . However , together with the interface in the front , the mirror has knobs and sliders on the back too . As the piece is intended to be presented as an exhibition artifact , two people can experience the e ff ect of shifting control between choices from the primary user in the front and the ones from the person hidden in the back . The last embodies the hidden mechanisms at play when we interact with AI - powered products that heavily leverage user pro fi ling and provide info and recommendations based on hidden parameters . This design approach takes a distinctively critical stand and ex - plicitly operates to disenchant the audience , in line with the idea from the literature that when we look inside the enchanted object and understand the inner workings we see that no magic exists [ 104 ] . Our re fl exive analysis of the student projects allowed us to add a speci fi c design principle that seems to be intuitive and e ff ec - tive for engaging with inner workings : the backside interface . The backside panel of A Closer Look project has some similarities with the backside screen display of the Ready for your tea ? project in which user data is shown together with the overview of ongoing nudging activities , based on industry interests . In this project , in fact , the focus is on the intricacies of having smart products using nudging behaviors as they represent proxies for industries having at heart clear commercial interests , rather than the well - being of clients . In the Own Faces project , instead , the concept of a backside interface translated into a much more experiential design feature . The project , which revolves around the importance of image own - ership when interacting with social media , invites the user to take a sel fi e in a photo booth and then post it online . In the moment the photo is taken frontally , other fl ashes appear from the back and sides , and multiple pictures end up being posted on social me - dia . Through this , the experience translates the hidden working of image appropriation and manipulation that can happen online and makes it experiential and confronting . The concept of backside interface , then , is a useful way of manifesting mechanisms that can translate into diverse forms of design . 6 . 6 Play with AI Figure 15 : Future Dialogue : playful control of small home appliances through training on personal vocal languages . The category play with AI includes a single project , Future Dia - logue , yet we believe it brings an important approach to the design of AI things , that is distinctively designerly . It also has a unique status in the taxonomy , as it involves both disenchanting and re - enchanting . The characteristic element of this approach is the focus on the playful - and to some extent purposeless - engagement with AI . With play , we describe interactions that engage the user in intrin - sically motivating and curiosity - driven experiences of AI . Playful approaches to technology are seen as a valuable way to let people explore possibilities , see how new technologies function , and imag - ine what they can be used for [ 48 ] . When allowed to purposelessly interact with AI and ‘see what happens’ , users can experience many of the characterizing elements of play , such as anticipation , surprise , pleasure , and understanding [ 34 ] . By playing with technology , the experience of AI products favors the occurrence of ‘a - ha’ moments that help the user understand the technology , as well as to keep the engagement high . The Future Dialogue project ( Figure 15 ) focuses on a novel mode of interaction with small home appliances . Devices can be con - trolled only through personalized sounds , and each object fi rst needs to be trained by the user . The underlying speculative narra - tive is that AI will enable an unprecedented level of personalization in product experiences , including hyper - personalized product in - terfaces that leverage secret languages de fi ned by users . To engage with this vision , the appliances in the project ( a co ff eemaker , a toothbrush , and a blender ) are provided with components that materialize the key conceptual elements of a machine learning al - gorithm for classi fi cation : a rotary encoder and LEDs are used as an interface for selecting a training category , a button is used to activate the recording of sounds for training , and a switch allows to shift from a training modality to play with the trained objects . This moves the activity of training AI models from an engineering ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA practice to an end - user engagement activity , developed by making use of the MLTK01 board [ 78 ] . Through this interface , the objects tell the story of a possible future but also open the space for playing and testing the limits of the AI . This disenchants the users who grasp the hidden workings of AI , yet , at the same time also invites the user to suspend their disbelief and “play along” [ 99 ] with the AI products . This allows for re fl ective cycles of enchantment and disenchantment as play uncovers new qualities of the underlying mechanisms which are quickly deployed by the end user as new interactions , where the mechanism disappears again . This design approach , then , playfully engages with distinctive AI characteristics , such as unpredictability and uncertainties [ 19 ] , that when encountered in interfaces could disenchant the user [ 99 ] , and intentionally makes them part of the experience . This creates new spaces for enchantment where we don’t trick ourselves and the audience into believing in the magical and supernatural powers of AI , but rather invite them and us to suspend disbelief and play along with the pretense that AI things work as if they were magical . 6 . 7 Presume AI Figure 16 : Reflection through Collection : data rituals for surfers engaged in citizen science This category includes projects ( 4 ) that do not engage explicitly with AI , but design products that live within the data - driven world of AI and implicitly provide anchors for imagining potential connec - tions with AI and ML capabilities . The Re fl ection through Collection project ( Figure 16 ) , for instance , focuses on the development of a leash for surfers that has sensors embedded for collecting water quality data , such as temperature , PH levels , salt quantities , and more . The whole concept revolves around the ritual of wearing and then washing the leash as a way to fi rst collect and then release data . The act of ‘releasing’ data implicitly suggests the presence of an intelligent processing element in the system that does something with that data , yet this remains unaddressed and something for the audience to wonder about . In this category of projects , we see design presuming AI as a background condition for the project to exist , although this does not come directly into the interactions with the products and services envisioned . In contrast to other categories , here design does not explicitly operate on a speci fi c direction ( dis ) enchantment . A product can either be enchanting or disenchanting and simultaneously use other principles , such as magic metaphors or the manifestation of mechanisms . In Re fl ection through Collection , for instance , there is no explicit AI enchantment at play – although one could argue for the project drawing on Summoning of AI as supernatural entity – but the ritual of washing out data from the leash clearly sets out to create an enchanting experience of the wearable technology . 7 DISCUSSION The descriptions of each category in the ( un ) making AI magic taxon - omy provided here illustrate di ff erent strategies by which designers can act within the space of AI and magic . In this discussion , we look at the relations and dynamics between the strategies and the design projects , discuss the limitations of this work , and then discuss the implications of the taxonomy for both design / HCI research and practice . 7 . 1 Taxonomy dynamics The taxonomy unpacked here is not designed to precisely and uniquely de fi ne the position of particular projects , but rather to help navigate the space of AI magic by articulating various principles and their e ff ects , to support the responsibility and agency of designers who deploy them . Here , we draw out some key relations between projects , designers , and the principles we have derived . First of all , each project can embody multiple principles . For in - stance , the artifact in Under the Loop project ( Figure 17 ) applies magicmetaphors ofenchantedmagnifyingglasses ( e . g . [ 100 ] ) , declar - ing the intent of scrutinizing space for invisible signals . At the same time , the description of the functioning also reveals that the designers presumed a layer of connectivity and algorithmic pro - cessing ( presume AI ) and that there is a potentially disenchanting role to play as the mechanisms are manifest and ’citizens might be prompted to provide input on speci fi c locations’ . All in all , however , the smoothness of the designed artifact and related interaction , together with the lack of technical details in the description of the project manifests an emphasis on the enchanting power of the project , an interest in creating a seamless experience , for which technical mechanisms remain hidden . The Colored Realities project , which we used to exemplify the principle of materializing beliefs , may also be seen as a reinterpre - tation of a magical thing or a sophisticated transposition of stage magic principles . The dynamically changing con fi guration of the news piece in the project is reminiscent of the ‘live newspaper’ from the world of Harry Potter in which pictures become alive when the reader is watching , but also represents a seamless embedding of the stage magic principle of transformation that feels enchanting to the user who experiences it . Similarly , the A Closer Look project , which we used to describe the principle of manifesting mechanisms , clearly also borrows the metaphor of the magic mirror and a ff ords play with the AI algorithms , by providing buttons and sliders that instantly let the user a ff ect the product behavior . However , the overall emphasis is put on the intricacies of the mechanisms at play and reveals an explicitly critical view about AI . The e ff ects of combined principles in a single project tend to either enhance or diminish the perception of the magical aura of AI . CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Figure 17 : Analysis of Under the Loop project . The example shows how a single project and its description can map onto di ff erent principles . However , one principle and an e ff ect ( enchantment or disenchantment ) tend to prevail . ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA While multiple principles can coexist in a single project , in most of the projects one dominates over the others . This means that if a designer wishes to increase or decrease enchantment , the taxonomy o ff ers multiple complimentary routes to do so : the principles give inspiration for how to design enchanting or disenchanting AI prod - ucts , and consciously direct the e ff ects that those products will have on user’s perception . The exception to this is play with AI , which asks for a continual iteration of enchantment and disenchantment . The taxonomy can be used as a re fl exive tool to understand one’s attitude and standing and to shape the choice of whether critical or a ffi rmative principles are manifested most prominently . However , when enchanting and disenchanting principles are both present the e ff ects of disenchanting principles tend to dominate . In the projects here , materializing beliefs and manifesting mechanisms take over , as we “open up the unfamiliar device to see what makes it tick [ and ] our reductionist curiosity and microanalysis kill the enchantment” [ 104 ] . These principles a ff ect the designers as well as the intended end users . Through our analysis , it was clear that the magical aura of AI a ff ects not only the user but also , and foremost the designer . The principles of summoning AI as magical entity , manifesting mecha - nisms and materializing beliefs in particular , put the spotlight on the designers themselves , and their fascination or fears about AI . The designers could hold multiple standpoints through their projects ; for example , the manifesting mechanisms and materializing beliefs principles declare a critical view of AI , yet , this does not prevent the designers from deeply engaging with the technology . In contrast , the projects embodying the summoning AI as magical entity prin - ciple often did not deeply engage with the AI tools and technical components , instead , AI is rather ‘talked about’ and staged through Wizard of Oz techniques . This may sound counter - intuitive as one would think that fascination for AI may lead to a rich exploration of it , while skepticism may discourage engagement . However , this tends to have to do with the actual level of engagement and making with AI , and the corresponding disenchantment on the part of the designers . The play with AI principle is again an exception as it works both to disenchant and re - enchant and invites the user to play along with the designer . Users can participate in a continuous recon fi guration of AI that brings awareness on the functionings and limits of the technology while feeling the magical aura when this performs as desired . Finally , a rich engagement with AI is key to developing a con - scious approach to the design of AI products . Designers in this survey who had a strong engagement with AI were able to leverage the dynamics of magical thinking and doing somewhat purposefully , to develop their positionality . This does not mean that designers should necessarily express a critical stand towards AI and only design for disenchantment but rather be aware of the position they take and be responsible for the e ff ects they might generate on users through their designs . In contrast , the designers who engaged less strongly with the workings of the AI technology they were using , found it harder to construct an articulated position , whether critical or a ffi rmative . 7 . 2 The Taxonomy as a Nascent Design Theory of AI Magic To further appreciate the value of the ( un ) Making AI Magic taxon - omy , we should consider how this responds to the HCI need for more ontological resources [ 23 , 75 ] , and speci fi cally around the space of AI perception and design [ 31 ] . The taxonomy , we argue , can be considered as a form of theory in the broad sense [ 55 ] , what Forlizzi terms nascent theory [ 45 ] : it suggests a lens through which we can understand phenomena and dynamics around the design of AI things and stimulate questions by introducing new constructs and proposing relations between these and established concepts . The design principles composing our taxonomy ( illustrated in Section 6 ) and the related dynamics ( Section 7 . 1 ) introduce new constructs . This lens allows us to look at the existing research landscape around AI , in particular connecting creative research endeavors with more technical approaches . For instance , Manifesting Mecha - nisms , a disenchanting principle , has strong analogues in technical approaches such as algorithmic auditing [ 67 ] and explainable AI approaches [ 5 ] , but also connects to the critical and speculative explorations we saw in students’ projects . Apply Magic Metaphors , instead , intuitively connects to artistic and designerly research en - deavors in the space of metaphors and product semantics , such Benjamin et al . ’s Entoptic Field Camera [ 11 ] that looks at how a perceptual concept can help us understand AI , or Murray - Rust et al . [ 91 ] who explored how metaphors can help designers in thinking about the creation of AI systems . However , the framing in magic metaphors also encourages investigations into matters of human cognition , and mental models in particular . The ( un ) Making AI Magic taxonomy , then , provides anchors for di ff erent HCI research identities to engage with , and opportunities for cross - pollination . Nevertheless , we argue for this to be primarily a Design theory ( as opposed to a theory for design ) [ 103 ] since it foregrounds making and experiential exploration as a primary way for understanding AI and magic . It speci fi cally centers around the exploration of multiple ways there can be to make AI and the impli - cations of these possibilities in terms of enchantment’s intentions and e ff ects . Due to this centering on making as a way of learning about AI , in what follows , we unpack potential implications of the taxonomy for both design education and wider practice . 7 . 3 How to ( un ) Make AI Magic Taxonomies are powerful theoretical instruments that can strongly impact future research , as demonstrated by popular examples , such as the Taxonomy for Autonomous Agents [ 46 ] which is now a foun - dation for thinking about arti fi cial agents and multi - agent systems , or the Socially Interactive Robots taxonomy [ 44 ] , that has become a key resource for human - robot interaction designers . Taxonomies are useful for several purposes , from the classi fi cation of exist - ing knowledge to the identi fi cation of knowledge gaps , from the identi fi cation of objects and characteristics to the positioning of a research output , and more [ 110 ] . Despite successful examples , how - ever , the HCI fi eld historically su ff ers from a gap between research and professional design practices , and even more , between theory and speci fi c design instances [ 120 ] . CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Table 1 : Translating principles into ’what if’ questions to use the taxonomy for design exploration Principle What if . . . Presume AI . . . product X would implicitly leverage an AI infrastructure ? Summon Supernatural AI . . . product X would have supernatural AI capabilities ? Apply Magic Metaphors . . . product X would embed AI looking and behaving as Y ? Apply Stage Magic Principle . . . product X would use AI as a magic trick ? Manifest Mechanism . . . product X would declare the AI mechanisms embedded into it ? Materialize Beliefs . . . product X would manifest designers’ or users’ beliefs about AI ? Play with AI . . . product X would invite users to play with AI ? In this vein , our ambition here is to create a taxonomy that can be applied and appropriated in many situations . We suggest two particular ways to make use of the ( un ) Making AI Magic taxonomy for design / HCI practice : exploration and re fl exivity . 7 . 3 . 1 ’What if ? ’ – using enchantment for design exploration . The seven principles composing the taxonomy can be explicitly used for inspiring and guiding design interventions by translating each into a ‘what if’ question ( see Table 1 ) to support exploratory design processes [ 109 ] . This simple act of translation from principles to what if questions is highly generative as it automatically opens the way to a variety of practical questions . In Figure 18 we illustrate this using the project Glow ( project 24 , year 2 ) , through an exploration of the questions from Table 1 . A What if the product leverages an AI infrastructure ? In this initial framing , the artifact does not embed any AI capability but clearly lives in a data - driven world presuming hypothetical AI capabilities to function , warming up when the user needs it based on contextual and user data . B What if it has supernatural AI capabilities ? This begs the question what kind of superior ability related to heating and sensing the environment would be su ffi ciently sophisticated to make it perceived as ‘supernatural’ . Based on existing possi - bilities , one could imagine a product able to ‘see’ through walls and eventually turn on . D What if the product uses AI as a magic trick ? invites us to consider what spaces for unexpectedness there can be . What would be a surprising and counterintuitive modality of interaction ? The answers can be many . One could be functional anticipation of the user’s state and need , e . g . , the heater predicting that the user would feel cold after working on a laptop for a while and heating up preventively . Another could be interactional , where AI is leveraged to detect users’ gestures for activation . C What if the heater was a magical creature ? Multiple metaphors can be used , but only some can ‘talk’ about both the functional scope of the product as well as the capabilities of AI embedded into it . The magical fi gure of a phoenix , for instance , could be used as a metaphor for designing a heater that uses AI capabilities to ‘see’ user need for heat ( as in Summon Supernatural AI ) , but that would completely erase – or burn – any data and knowledge about the user any time it ‘dies’ – or f - switches o ff – and is reborn as a way to preserve privacy . E what if the heater would declare the AI mechanisms embedded into it ? asks foremost to re fl ect on what the heater’s mechanisms and their implications are , drawing on explainable AI techniques to either aid user understanding or highlightprivacyconcernsasaresultofhyper - personalization . For the latter , one could design a heather that shows how a user is being tracked and provoke a re fl ection on how we grant smart products access to personal data . F What if the heater manifests beliefs about AI heaters ? One could assume a user’s belief of AI as objective and infal - lible , and hence better suited to making decisions regarding sustainable heating behaviors . The heater could then be de - signed to switch on exclusively when ‘objectively needed’ with no possibilities for the user to control , resulting in a frustrating and confrontational AI heating interface . G What if the heater would invite users to play with AI ? This could potentially touch on many of the design concepts mentioned above , such as gesture detection , environment data collection , the manifestation of user monitoring , and more , but with the explicit intent of enabling exploration playfully . Here the emerging questions would be about how a heater can be an interface for interacting with AI and what a ff ordances can we design to enable play . As this brief thought exercise shows , the taxonomy principles when turned into what if questions open a generative design space . These allow us to consider a wide spectrum of possibilities and o ff er a framing for the confrontation of design ideas that can also help detect possible issues in the conceptualization of AI products , such as design fi xation [ 63 ] and normativity [ 47 ] . 7 . 3 . 2 ’Why ? ’ - investigating enchantment for reflexivity in design . The taxonomy can also be used to encourage designers to re fl ect on their intentions and positionality . What if questions come with e ff ects in terms of enchantment , but Why ? questions have impli - cations in terms of crticiality in design practices [ 6 ] . As Fallman [ 40 ] argues , through exploration , design can show alternatives and examples , but it also becomes a statement of what is possible , what would be desirable or ideal . The principles in the taxonomy can pull in di ff erent directions : Apply Stage Magic Principles enchants through seamless interaction while Manifest Mechanisms disen - chants through confrontation the second . ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Figure 18 : Examples of ’what if questions in use : What if a heater would . . . These potentially con fl icting viewpoints can serve as a frame for re fl exivity and scrutiny if looked at as a ‘map’ for designers to re fl ect on their positionality . As background , there are many exam - ples from professional design work that materialize the principles included in the taxonomy . AI products integrate metaphors from the world of magic , such as the The Mirror Home Gym that uses the metaphor of the magic mirror , or the Bosch Series 8 Accent Line Sensor Oven [ 13 ] that uses the metaphor of the crystal ball . Others use stage magic principles to create smooth and enchanting user experiences , such as the family of Amazon products where Alexa manifests the principle of teleportation by giving the impression of ‘jumping’ from object to object , or the NEST smart thermostat that learns and estimates house heating behaviors [ 62 ] to predict user needs and anticipate actions . In these examples , there is hardly any engagement with matters of values , assumptions , and power which , instead , are core to re fl exive practices [ 97 ] . In contrast , examples from critical and speculative practices [ 127 ] inherently come with a more explicit commitment to the critique of norms [ 6 ] . For instance , the design studio Super fl ux confronts us with the beliefs we imbue technology with , such as Our Friends Electric [ 115 ] which explores the assumptions we hold regarding our relationship with voice - activated AI assistants . The Paragraphica camera [ 121 ] uses location data and AI to visu - alize a “photo” of a speci fi c place and moment – an explicit re fl ection on aspects of data processing and decision - making around what is deemed relevant and , thus , ‘captured’ from the environment : a clear CHI’24 , May 11 – 16 , 2024 , Honolulu , USA Lupe tt i & Murray - Rust Table 2 : Translating principles into ’why’ questions to use the taxonomy for design re fl exivity Principle Why . . . Presume AI . . . would a product X implicitly leverage an AI infrastructure ? Summon Supernatural AI . . . would a product X have supernatural AI capabilities ? Apply Magic Metaphors . . . would a product X embed AI looking and behaving as Y ? Apply Stage Magic Principle . . . would a product X use AI as a magic trick ? Manifest Mechanism . . . would a product X declare the AI mechanisms embedded into it ? Materialize Beliefs . . . would a product X manifest designers’ or users’ beliefs about AI ? Play with AI . . . would a product X invite users to play with AI ? example of the principle of Manifesting Mechanisms . Re fl exivity in these cases usually revolves around the development of a single standing , the materialization of a personal view , rather than the exploration of a spectrum of possibilities . To facilitate the use of the taxonomy for re fl exive purposes , we suggest another simple act of translation : from principles to ‘Why ? ’ questions ( Table 2 ) . In asking why , we automatically engage with matters of intention , e ff ects , and values that an AI artifact can carry . Looking back at the example of the smart desk heater , we could then ask why a desk heater would be able to see through walls . An - swering this would invite us to engage with matters of control and agency , as such capabilities enable the product to make decisions and act autonomously . Or one could ask why a desk heater would behave as a phoenix , leading us to consider matters of data protec - tion and privacy . Such ‘Why ? ’ questions should be engaged not only for re fl ecting on what is being designed but also and foremost for considering how else AI things could be shaped . Asking why then helps us consider what are the underlying values and narratives we are embedding into AI products , and what alternative stories we could wave . The taxonomy provides a platform for diverging from either norms or personal views , opening up a spectrum of alternatives . 7 . 4 Limitations and Future Work Our work , of course , is not free from limitations . In particular , one could question how relevant a taxonomy is for professional design practices when it’s built out of re fl ections on students’ design work . Several are the di ff erences in the way students approach design as compared to professionals . For instance , experienced designers – in contrast to students – have tendencies to refer to past designs , to question if a project is worth pursuing , and to consider possible issues [ 3 ] . Furthermore , students’ work is highly in fl uenced by the perspectives and practices brought into the course by the educators . In our collection , for instance , we see a connection between our own critical and experimental approach to design research and education , and the students’ projects . Design theories , however , are often built by re fl ecting on educational work ( see more in Section 5 ) . Students’ work is valuable not because of its potential capacity to emulate professional design practices , but rather because of its dialogical relationship between the educators’ conceptual under - standing of a theoretical space surrounding design / HCI practice and its manifestations . It is exactly in this dialogical space between the educators’ standing and the students’ interpretation that we fi nd a powerful ground for confrontation and re fl ection , where we ’retroactively’ look at our education practice to understand how the - oretical principles can translate and be appropriated into practice [ 103 ] . We acknowledge , however , that as for most design theories , our taxonomy is generative and suggestive , rather than veri fi ed [ 50 ] . Further empirical evidence could be gained both in the class - room and in professional environments by using the taxonomy as a guide for AI product development or as a re fl exive tool for self - scrutinizing processes and then running comparative studies . Future empirical investigations could look for unpacking the dy - namics of enchantment and disenchantment : How does a designer’s experience a ff ect the way they use the principles ? How does the context of a project a ff ect the success of applying any of these tech - niques ? How does prolonged exposure for designers or end users a ff ect the perception of the magical aura ? For all of these dynam - ics , we found hints in our work , yet they open up a broad space that could not be addressed in this single analysis . Nevertheless , we as design / HCI researchers should be mindful of the role of the di ff erent ways of knowing that exist in the HCI landscape and that design theory , compared to theory for design , is hardly generaliz - able and veri fi able , but nonetheless holds great value in its capacity to inspire future work and drive research agendas [ 50 , 103 ] . Thus , the taxonomy presented here is open - ended and represents a fi rst – high - level – layer classi fi cation in the space of ( un ) making AI magic which could be seen as a limitation , but we look at this optimistically : as Nickerson and colleagues [ 95 ] argue “a useful taxonomy should allow for the inclusion of additional dimensions and new characteristics” . For each design principle , additional char - acterizations can be found and nuances in the related dynamics can be understood . For instance , the general principle of using magic metaphors can be further investigated to understand how di ff erent magic categories , whether creatures , objects , or ‘fait’ types [ 17 ] , can bring distinct implications for how a product is perceived and approached . Similarly , one could develop speci fi c practical tech - niques for working with each of the principles , for instance by delving into the distinct e ff ects and implications of diverse stage magic techniques . We hope that this initial framing gives a fertile structure for building such future work and that more design / HCI researchers will feel encouraged to engage in theory - making work . 8 CONCLUSIONS In this paper , we illustrated and problematized the fuzzy design space of magic and enchantment in AI product development . De - signing enchanting products contributes to developing seamless ( Un ) making AI Magic : a Design Taxonomy CHI’24 , May 11 – 16 , 2024 , Honolulu , USA and engaging experiences , but can also encourage users’ misunder - standing of products’ capabilities and hide the complexity and risks of ecosystems populated by AI products . Designers need to navigate between the ambition to be critical and conscious about the use of AI while also being caught up in the magic rhetorics that drive the ‘economy of appearances’ [ 119 ] shaping public engagements . En - chantment is not just designed but is inherently ingrained in novel technologies as it is tightly bound to the di ffi culty of grasping the mechanisms at play : roads and railways once enchanted with their stories of speed and freedom [ 58 ] , now AI technologies enchant with their dreams of agency and care . Designers hold a responsibil - ity to approach AI technologies carefully and skillfully . For this , it is of primary importance to build literacy around AI not only in its technical terms but also in its socio - cultural components . Hence , it is crucial to understand the more or less subconscious principles of magic and supernatural thinking that heavily characterize current AI development , in order to shape the societal and cultural impact of these technologies . As the fi eld still lacks an understanding of the dynamics and ef - fects of magical thinking in the design of AI things , we developed a taxonomy of the di ff erent con fi gurations of magic present in the de - sign of AI things . By re fl ecting primarily on student design projects but also connecting professional design works , we identi fi ed and unpacked the implications of 7 design principles that can distinc - tively characterize AI products’ development : applying stage magic principles , applying magic metaphors , summoning AI as supernatural entity , materializing beliefs , manifesting mechanisms , play with AI , and presuming AI . These , we believe , provide an initial overview of di ff erent ways design can operate in relation to AI and emphasize or diminish its enchanting power . We do not see this taxonomy as closed , and hope that the ‘un fi n - ished’ nature of our taxonomy serves as an invite for the community to engage and contribute . It is a framework to look at AI magic not as a marginal and disconnected topic but as a lens to re fl ect upon and understand many of the critical perspectives that are currently being investigated in the design and HCI fi eld to make sense of AI , such as metaphors [ 8 , 11 , 70 , 91 ] , narratives [ 20 , 56 , 60 , 77 ] , and expressive implications of AI uncertainties and errors [ 10 , 19 ] . To conclude , we invite designers and researchers fi nding them - selves engaged in AI products’ development to re fl ect upon their thinking and whether there is some ‘magic at play’ . For this , we o ff er at our taxonomy as a re fl exive tool that allows critical scrutiny of the things we design and the expectations we bring in our ac - tions . As such , we believe our work will contribute to building a disciplinary sensitivity for the role of rhetorics and ‘the irrational’ in the design of AI things , and technology more broadly . ACKNOWLEDGMENTS This work owes a great deal of gratitude to all the students who pas - sionately engaged with the Interactive Technology Design course at TU Delft . Their ways of appropriating the technologies and the AI discourse were an inspiration and a motivation for us as educators to grow awareness about aspects of AI perception and rhetoric . Especially , we would like to thank Lenny Martinez who relentlessly contributed to this work as a design student fi rst , and as a teaching assistant for the course later . REFERENCES [ 1 ] AminaAdadi . 2021 . Asurveyondata - e ffi cientalgorithmsinbigdataera . Journal of Big Data 8 , 1 ( 2021 ) , 24 . [ 2 ] Philip Agre . 1997 . Computation and human experience . Cambridge University Press . [ 3 ] Saeema Ahmed , Ken M Wallace , and Lucienne T Blessing . 2003 . Understanding the di ff erences between how novice and experienced designers approach design tasks . Research in engineering design 14 ( 2003 ) , 1 – 11 . 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On Creative Metaphors in Technology Design : Case " Magic " . In Proceedings of the 8th International Conference on Communities and Technologies . 280 – 289 . [ 130 ] Hui Zhang , Munmun De Choudhury , and Jonathan Grudin . 2014 . Creepy but inevitable ? The evolution of social networking . In Proceedings of the 17th ACM conferenceonComputersupportedcooperativework & socialcomputing . 368 – 378 . \ No newline at end of file diff --git a/silver_data/e9751ee3d3f05dc99e0d4c277affc93ddaf0673a.pdf.txt b/silver_data/e9751ee3d3f05dc99e0d4c277affc93ddaf0673a.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..45ddae3ddee46f9f874b746b069e5460aff9d50c --- /dev/null +++ b/silver_data/e9751ee3d3f05dc99e0d4c277affc93ddaf0673a.pdf.txt @@ -0,0 +1 @@ +Structural Basis of Membrane Bending by the N - BAR Protein Endophilin Carsten Mim , 1 , 2 Haosheng Cui , 3 Joseph A . Gawronski - Salerno , 1 , 2 Adam Frost , 5 Edward Lyman , 4 Gregory A . Voth , 3 and Vinzenz M . Unger 1 , 2 , * 1 Department of Molecular Biosciences , Northwestern University , 2205 Campus Drive , Evanston , IL 60208 , USA 2 Chemistry of Life Processes Institute , Northwestern University , 2170 Campus Drive , Evanston , IL 60208 , USA 3 Department of Chemistry , Institute for Biophysical Dynamics , James Franck Institute , and Computation Institute , University of Chicago , 5735 South Ellis Avenue , Chicago , IL 60637 , USA 4 Department of Physics and Astronomy and Department of Chemistry and Biochemistry , University of Delaware , Newark , DE 19716 , USA 5 Department of Biochemistry , University of Utah , 15 North Medical Drive East , Salt Lake City , UT 84112 , USA * Correspondence : v - unger @ northwestern . edu DOI 10 . 1016 / j . cell . 2012 . 01 . 048 SUMMARY Functioning as key players in cellular regulation of membrane curvature , BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms . Using electron cryo - microscopy , we present reconstructions of full - length endophilin and its N - terminal N - BAR domain in their membrane - bound state . Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin’s amphipathic N - terminal helices . Coarse - grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N - terminal helices are required for formation of a stable and regular scaffold . Furthermore , endophi - lin accommodates different curvatures through a quantized addition or removal of endophilin dimers , which in some cases causes dimerization of endo - philin’s SH3 domains , suggesting that the spatial presentation of SH3 domains , rather than affinity , governs the recruitment of downstream interaction partners . INTRODUCTION The cell membrane is a dynamic barrier whose shape is constantly remodeled with high spatial and temporal accuracy during essential cellular processes ranging from cell motility and signaling , to maintenance and generation of organelles ( Gallop and McMahon , 2005 ; Hurley et al . , 2010 ) . Though critical for normal cell function , the mechanisms by which cells control membrane remodeling are only beginning to come into focus ( Dawson et al . , 2006 ) . At the molecular level , proteins of the Bin / Amphiphysin / Rvs ( BAR ) domain superfamily have emerged as major players in membrane remodeling ( Farsad et al . , 2001 ; Frost et al . , 2009 ) . Divided into four distinctive subfamilies , N - terminal - BAR ( N - BAR ) , extended - FCH - BAR ( F - BAR ) , inverse BAR ( I - BAR ) ( Ren et al . , 2006 ) , and Pinkbar proteins ( Pyka¨la¨inen et al . , 2011 ) , the majority of BAR domain proteins are scaffolding proteins that combine a membrane - remodeling BAR domain with additional protein : protein interaction or catalytic domains , which are responsible for the biological coupling of specific processes to distinct membrane curvature states . At the struc - tural level , BAR domains are dimers of an antiparallel helix bundle ( Peter et al . , 2004 ; Shimada et al . , 2007 ; Weissenhorn , 2005 ) that display various degrees of intrinsic curvature . The simple design of these modules allows cells to generate a large range of different curvature states , including membrane invagi - nations , like tubules , or extrusions like filopodia ( Guerrier et al . , 2009 ; Lee et al . , 2002 ) . At the mechanistic level , high - resolution structures and spectroscopic studies have inspired two major models for how BAR domains accomplish changes in membrane curvature . One model holds that membranes are remodeled through a pure scaffolding mechanism in which a ‘‘banana - shaped’’ BAR domain dimer binds the bilayer through electro - static interactions and imposes its intrinsic curvature on the substrate bilayer . In the second model , curvature sensing and generation are thought to critically depend on the membrane insertion of amphipathic wedges like the N - terminal helix 0 ( H0 ) that are found in N - BAR proteins such as endophilin and amphi - physin ( Chernomordik and Kozlov , 2003 ; Farsad et al . , 2001 ; Johannes and Mayor , 2010 ) . There is good experimental evidence in support of both models , yet mechanistic details remain largely unknown , in part because direct visualization of BAR domain proteins in membrane - bound states has proven to be challenging . The structure of the membrane - bound CIP4 F - BAR domain demonstrated that bending of the membrane can be realized solely through scaffolding ( Frost et al . , 2008 ) . However , the same study also revealed a second physiologically relevant binding mode in which the F - BAR domains engage planar bila - yers through an alternate , flat binding interface . This suggested that the repertoire and mechanistic complexity of BAR : mem - brane interactions likely is larger than was previously anticipated . In contrast , how insertion of amphipathic sequences into the bilayer promotes curvature sensing and generation remains less clear . A recent study showed that N - BAR domains of Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . 137 endophilin are able to form ordered surface lattices on membranes . However , the study focused on tubular structures with very narrow diameter . Individual N - BAR domain dimers were not resolved , and details about how the N - BAR domains interact with the membrane were not provided ( Mizuno et al . , 2010 ) . Here we present reconstructions of full - length endophilin and its N - terminal N - BAR domain bound on tubules whose diameters are in a range that is relevant for action by down - stream interaction partners , such as the GTPase dynamin ( Chappie et al . , 2011 ; Faelber et al . , 2011 ; Roux et al . , 2010 ; Sundborger et al . , 2011 ) . Our key findings are that at their struc - tural level N - BAR lattices are fundamentally different from F - BAR scaffolds , that highly promiscuous and generic interactions between N - terminal H0 helices are essential for the formation of ordered scaffold structures , and that the geometry of the N - BAR endophilin lattice in some cases results in dimerization of the SH3 domain . Combined with our observation that the concentration of SH3 domains close to the scaffold surface is independent of curvature , our study suggests that recruitment of downstream partners is driven by steric selection rather than thermodynamic affinity . RESULTS N - BAR Lattices Expose Large Membrane Areas To directly visualize the design principles of membrane - bound N - BAR lattices , we used electron cryomicroscopic imaging , maximum likelihood classification and an iterative helical real space reconstruction approach ( Egelman , 2007 ; Sorzano et al . , 2004 ) to determine class averages and the 3D structures of tubules decorated with either the endophilin N - BAR domain or full - length endophilin ( Figure 1 and Figure S1 and Table S1 avail - able online ) . Starting from data sets of (cid:1) 75 , 000 overlapping segments for endophilin , and (cid:1) 35 , 000 segments for tubes coated with the endophilin N - BAR domain , we were able to determine structures of tubules of different diameters for each sample ( see Extended Experimental Procedures ) . In addition , we also obtained a class average for another N - BAR protein , amphiphysin ( (cid:1) 2 , 000 overlapping segments [ Figure S1A ] ) to test whether the smaller number of amphipathic wedges in amphiphysin ( two versus four in endophilin ) resulted in a change in overall scaffold design . Class averages obtained from our data sets showed a distinc - tive pattern where dark and light regions alternated along the vertical axis of the membrane tubule with a period of (cid:1) 50 A˚ ( Figures 1 , S1A , and S1B ) . Moreover , the pattern was highly conserved regardless of differences in the number of amphi - pathic wedges ( endophilin versus amphiphysin ) , the presence / absence of domains other than the N - BAR domain ( full - length endophilin / amphiphysin versus endophilin N - BAR domain ) , or different curvatures of the underlying membrane ( Figure S1C ) . This suggested that N - BAR lattices were highly pliable , which was further supported by the observation that tube diameters readily and frequently changed , even along a single tube . To better understand the lattice design , we generated 3D reconstructions of full - length endophilin ( Figure 1 ) and endo - philin’s N - BAR domain ( Figure S1E ) . In the reconstructions , indi - vidual N - BAR domains were easily recognizable ( Figure 1 ) . Inspection of the structures revealed that tip - to - tip interactions betweenconsecutive N - BAR dimers didnotseem toplay amajor Figure 1 . Endophilin Scaffold Structure ( A ) Class averages ( top ) and reconstructed volumes ( bottom ) of membrane tubules decorated withfull - lengthendophilin . Thesizeofthescalebar is the same in corresponding reconstructions and class averages . The bilayer is yellow and the protein green . Apparent holes seen in the bilayer of the 32 nm tube reconstruction are dueto the thresholding level , which was chosen to emphasize the molecular envelope of the protein component . ( B ) Modelofendophilinlattice , viewedfromthetop ( left ) andincross - sectionperpendiculartothetube axis ( right ) . The high - resolution crystal structure of rat endophilin A1 ( pdb : 1ZWW , orange ) was fitted into the reconstructions of the 28 nm full - length endophilin tubule . Only one full dimer of the BAR domaincoreisshownforclarity . Thedisposition of H0 ( magenta ) and insert ( cyan ) helices is indicated by cylinders . The molecular envelope ( gray mesh ) suggests that H0 helices from adjacent BAR domaindimerspairinanantiparallelfashion . Atthe same contouring threshold , one of the insert helices is not fully accounted for , reflecting the partially disordered state of this element in our reconstructions . Thelengthof thevertical thinblue lines ( right ) is 30 A˚ and marks the approximate positioning of the hydrophobic core of the bilayer . At the chosen contouring threshold we did not observe density for the leaflet that is in direct contact with the BAR domain . This indicates that the bilayer is highly disordered and stressed . See also Figure S1 and Table S1 . 138 Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . role in stabilizing the lattice and also seemed to switch between two modes . In the 25 nm and 32 nm tubes , the N - BAR domains were oriented perpendicular to the tube’s long axis and appeared to have little to no direct tip - to - tip contact , whereas in the 28 nm tubes , direct tip - to - tip interactions seemed to link consecutive dimers that were inclined by (cid:1) 10 (cid:3) with respect to the tube’s long axis . Cross - sections of these tubes revealed that the distinct orientations coincided with lattices that differed by an integral number of N - BAR dimers around the circumfer - ence of the tube ( data not shown ) . Moreover , N - BAR dimers in adjacent rows of the lattice did not show direct lateral interac - tions between the coiled - coil core domains of N - BAR dimers , which had been identified as a defining structural feature in scaf - folds formed by the F - BAR protein CIP4 ( Frost et al . , 2008 ) . This marked difference in lattice design exposed large areas of continuous membrane surface between adjacent lattice ridges ( e . g . , (cid:1) 350 nm 2 from one notch to the next in 28 nm tubules ) that , independent of curvature , were spaced (cid:1) 50 A˚ apart from each other . Endophilin’s Amphipathic H0 Helix Provides Key Contacts in the N - BAR Scaffold The absence of lateral interactions between the N - BAR cores , and the apparently weak or absent tip - to - tip interactions between consecutive N - BAR dimers suggested that interactions between the amphipathic helices of endophilin played an impor - tant role in lattice formation and stability . To better understand the role of these structural elements , we sought to identify their positioning in our reconstructions . For this purpose , reconstruc - tions were thresholded to tightly fit the backbone of endophilin’s high - resolution N - BAR crystal structure to emphasize the molec - ular envelope of the protein components ( Protein Data Bank [ pdb ] : 1ZWW ) . At this contouring level , additional densities bridged adjacent windings of the surface lattice and likely repre - sented contributions from the amphipathic helices ( Figure 1B ) . Based on the envelope , the reconstructions suggested that , in the 28 nm tubes , H0 helices from adjacent rows of the N - BAR lattice interacted in an antiparallel fashion , placing the helix pair roughly parallel to both the membrane surface and the long axis of the tube ( Figure 1B ) . In contrast to the H0 helices , posi - tioning of the insert helices was less certain because the volume elements accounting for this helix were less well defined than the envelope for enclosing H0 ( Figure 1B ) . Whether this poorer defi - nition was due to flexible linkers that connected the insert helix to the BAR domain core or was caused by a spread of orientations of the insert helix could not be resolved at the resolution of our reconstructions . However , the molecular envelopes suggested that the insert helix was tilted toward the bilayer core and in the 28 nm tubules did not make direct contacts with insert helices from the adjacent N - BAR domain dimer ( Figure 1B ) . Interactions between H0 Helices Are Essential for Lattice Stability The conclusion that interactions between the H0 helices from neighboring N - BAR dimers were essential to lattice formation and stability was independently established by coarse - grained molecular dynamics simulations ( Figure 2A ; see Extended Experimental Procedures and Figure S2 for details ) . The simula - tions revealed that the oligomer structure was retained during the course of simulation for the N - BAR system ( Figure 2A , ‘‘H0 + BAR’’ ) , whereas the oligomer rapidly became scrambled for the system where H0 had been deleted ( Figure 2A , ‘‘BAR’’ ) . Notably , even in the presence of H0 , the lattices had order parameters of only (cid:1) 0 . 8 ( Figure S2B ) . This further emphasized that endophilin lattices were very dynamic and explained why segment sorting and classification were necessary to obtain reli - able class averages for reconstruction of the volumes . To further test the importance of H0 : H0 interactions for the formation of the lattices , mutant endophilins with serial deletions lacking 3 , 6 , 9 , 12 , 16 , or 20 residues along H0 were tested for their ability to tubulate liposomes . Of these mutants the D 2 – D 17 and D 2 – D 21 mutants did not tubulate liposomes ( data not shown ) . However , the shorter partial deletion mutants were still functional in this assay , but displayed a lower efficiency than wild - type protein if the amounts of endophilin were lowered to the smallest amount necessary to yield robust tubulation ( data not shown ) . Moreover , cryoEM data collected from tubes formed by the D 2 – D 10 and D 2 – D 13 mutants lacked the characteristic striation pattern that was observed for tubes formed by wild - type endophilin ( Figure 2C ) . This suggested a higher degree of lattice disorder in the tubes formed by the mutant endophilins and further supported the idea that antiparallel interactions between H0 helices were necessary for the formation of a well - defined lattice structure . The Interactions between H0 Helices Are Degenerate and Dynamic To further characterize the interactions between H0 helices , we generated 12 single Cys mutants along H0 in a Cys - less endo - philin background and determined their ability to spontaneously form crosslinked dimers in their membrane - bound state ( Figures 3A and S3 ) . Surprisingly , all mutants were able to crosslink , and the efficiency increased toward the C - terminal end of H0 , except in the case of the Q9C mutant , which for unknown reasons crosslinked less well than other mutants in this region of H0 . At the same time , all mutants retained their ability to tubulate lipo - somes ( Figure S3D for examples ) . This indicated that the mutant endophilins were functional and that the observed crosslinks occurred in the context of a scaffold . The latter implied that the helices were able to interact in many different registers , including pairings on either side of the helix . To acknowledge this observa - tion , H0 helices are represented by generic cylinders in Figure 1B to indicate that the observed densities represent an ensemble average over all possible lateral alignments of H0 pairs . Consis - tent with the crosslinking experiments , all - atom molecular dynamics calculations revealed a very smooth energy landscape for H0 : H0 interactions , with the exception of two configurations that were slightly more stable , and a more general trend that small overlaps toward the N - terminal ends of the helices were energetically slightly less favorable than more extensive overlaps ( Figure 2B ) . The hypothesis that lattice formation depended on lateral interactions between H0 helices implicitly made the prediction that both tubulation and crosslinking of the various mutants would be sensitive to the presence of chemically synthesized H0 peptide . As shown in Figure 3B , this condition was met , as Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . 139 two different H0 peptides ( wild - type and a T14D mutant peptide that mimicked a naturally phosphorylated H0 variant [ Kaneko et al . , 2005 ] ) significantly reduced crosslinking for all but the most N - terminal mutants of H0 . The latter observation not only served as an internal negative control , but was also consistent with the slightly less favorable interactions that were observed in the molecular dynamics simulations ( Figure 2B ) . In further agreement with the crosslinking results , tubulation was reduced in the presence of submillimolar concentrations of the peptide and almost absent at nominal peptide concentrations of 2 mM that were used to carry out the competitive crosslinking experi - ments ( Figure 3B ) . Moreover , wild - type endophilin H0 peptide also blocked membrane tubulation by another N - BAR protein , an isoform of amphiphysin 2 , with the same concentration dependence , but had a much less inhibitory effect on membrane tubulation by the F - BAR domain of FBP17 , which does not employ amphipathic wedges to induce membrane curvature ( Figures 3B and S3E for titration results of N - BAR proteins ) . This suggested that the presence of the free peptide in the membrane did not interfere with membrane bending per se but acted by disrupting the cooperative assembly of a coherent lattice that required a permissive spatial positioning of H0 helices . A rough estimate revealed that in fully assembled scaf - folds , H0 is present at a concentration of (cid:1) 14 mM within the leaflet that accommodates the wedging element . With this in mind , the inhibition we observed at a nominal peptide concentra - tion of 2 mM was deemed significant . At this concentration , the peptide alone did not cause tubulation or disruption of the bilayer ( data not shown ) . However , our reconstruction revealed that insertion of the much higher concentrations of amphipathic wedges in the actual scaffolds caused significant stress on the bilayer . This stress manifested itself in the absence of density Figure 2 . The N - Terminal Helix H0 Plays a Crucial Role in Lattice Formation ( A ) Starting ( top ) and final ( bottom ) snapshots of coarse - grained molecular dynamics simulations ofendophilinoligomers ( N - BARontheleftandH0 - deleted BAR on the right ) . Green represents the coiled - coil BAR scaffold , blue represents the H0 helix , and white represents the insert helix . ( B ) Free energy profile for antiparallel H0 pairs as afunctionofdisplacementagainsteachother . The helix pair is shoulder - to - shoulder when the offset is 0 A˚ . Error bars represent the SEM . The free energywascalculatedfromtheumbrellasampling data by the weighted - histogram analysis method and the error bar was calculated by the bootstrap method . The orange line is for reference purposes only and shows that the energy is slightly higher in the direction of positive displacement . The label ‘‘a’’ ( at (cid:4) 13 A ˚ ) refers to one of the configurations shown in C . ( C ) Two stable configurations for the helix pair . The pair labeled ‘‘a’’ corresponds to the same label in B . ( D ) CryoEM images ( top ) and single - particle class averages ( bottom ) of endophilin wild - type ( wt ) and partial H0 - deletion mutants of endophilin ( Δ 2 – D 10 and Δ 2 – D 13 ) . For the D 2 – D 10 mutant , 294 out of 1 , 641 segments overlapping segments in the data set contributed to the average shown . For the D 2 – D 13 mutant , 156 out of 561 overlapping segments in the data set contributed to the average shown . The small number of usable segments was caused by the small number of well - preserved tubes formed by this mutant en - dophilin . The scale bar in the cryoEM images is 25 nm . The scale bar in the single - particle class averagesis5nm . Tubulesofendophilin ( wt ) exhibit acharacteristic ‘‘studded’’appearance , indicating a coherent protein coat with a repeat distance of (cid:1) 5 nm . This spacing is not detectable in tubules formed by either of the H0 - truncation mutants of endophilin ( Δ 2 – D 10 and Δ 2 – D 13 ) , suggesting a loss of coherence within the protein coat . See also Figure S2 . 140 Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . for the bilayer leaflet closest to the N - BAR domain if the recon - structions were thresholded to emphasize the protein compo - nent of the ensemble ( Figure 1B ) . It should be pointed out that the missing leaflet was visible at a less significant thresholding level . However , at this level the definition of the BAR domains was lowered to the point where individual dimers were no longer visible . Thus , the reconstructions shown in Figure 1 were the best overall compromise for representing the structures . Some , but Not All , Lattice Geometries Induce Dimerization of Endophilin’s SH3 Domains As is the case in many BAR domain proteins , endophilin employs src3 - homology domains for the recruitment of its two down - stream interaction partners , dynamin and synaptojanin ( Chang - Ileto et al . , 2011 ; Llobet et al . , 2011 ; Ringstad et al . , 1997 ; Takei et al . , 1999 ) . How each of these partners is selectively recruited is unknown , which prompted us to compare reconstructions of membrane - bound full - length endophilin with reconstructions of membrane - bound N - BAR domain alone to determine whether the SH3 domains adopted well - defined spatial patterns above Figure 3 . Interactions between H0 Helices Are Nonspecific and Degenerate ( A ) Crosslinking efficiency for 12 unique Cys substitutions alongH0 . Alldataarepresentedasthemeanofthe " dimer / total endophilin ratio " + SEM ( triplicates ) . ( B ) Impact of synthetic H0 peptides on crosslinking of unique Cys - substituted endophilin mutants . Shown are crosslinking efficiencies in the absence of the peptide ( striped bars ) , and in the presence of 22 - residue wild - type endophilin H0 peptide ( gray bars ) and phosphomimetic mutant peptide ( T14D ) ( black bars ) . All but one mutant ( S2C ) showed a significant reduction in crosslinking effi - ciency in the presence of either H0 peptide . In these experiments , peptideswereusedat2mMconcentrations . All data are presented as mean + SEM ( triplicate ) . Signifi - cance levels : * p > 0 . 05 , * * p > 0 . 01 , with Student’s t test . The error bars represent the SEM . ( C ) Negative - stain EM images of tubulation reactions with liposomes in the absence ( left ) and presence ( right ) of 2 mM of endophilin H0 peptide ( residues 1 – 22 ) . Concen - trations of 2 mM effectively inhibited the ability of endo - philin to bend membranes . This block of tubule formation was also observed with amphiphysin 2 , an N - BAR protein similar to endophilin . In contrast , tubulation by the F - BAR protein FBP17 was less affected , indicating that the inhi - bition of N - BAR - dependent curvature generation was caused by a disruption of H0 : H0 interactions between N - BAR dimers . Scale bar = 2 m m . See also Figure S3 . the scaffold surface . Overlay of the reconstruc - tions of the 25 nm tubes for both proteins failed to show any significant differences . This indicated that the SH3 domains were highly mobile above the scaffold surface , which caused their density to be averaged out during the reconstruction . In contrast , overlay of the 28 nm tubes revealed additional densities in the case of the full - length endophilin lattice ( Figures 4 and S1E for data of N - BAR - only tubes ) . The additional densities were large enough to accommo - date the backbone of the high - resolution structure of an endophilin SH3 domain dimer ( pdb : 3IQL [ Trempe et al . , 2009 ] ) . Interestingly , the putative SH3 domain dimers were located above the region where the tips of two consecutive en - dophilin dimers come close to each other and thus provided additional lattice contacts in this case ( Figure 4 ) . Based on this model , placement of Cys residues into the putative dimerization interface should allow the spontaneous formation of disulfide bridges . To test this prediction , we generated a Thr320Cys mutant in an otherwise Cys - less background . Consistent with our model , this mutant efficiently formed crosslinks when bound to bilayers , which set it apart from wild - type endophilin A1 , in which the nearby Cys294 in the SH3 domain failed to form signif - icant amounts of disulfide - linked dimers . The fact that the cross - linking was less than 100 % was likely due to the fact that only a fraction of the tubes in any given population had a diameter that would allow dimerization to occur ( Figure S1D ) . Neverthe - less , the pronounced difference in the behavior of the T320C mutant and wild - type endophilin A1 was consistent with the Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . 141 idea that SH3 dimers can form above the surface of some , but not all , endophilin lattices . DISCUSSION N - BAR Scaffolds Are Fundamentally Different Than F - BAR Scaffolds Based on their structure , BAR domains are very similar to each other overall ( Frost et al . , 2009 ) . Once assembled into scaffolds , however , lattices of F - BAR ( Frost et al . , 2008 ) and N - BAR domains are remarkably different . The extensive lateral contacts between the coiled - coil regions of the F - BAR protein CIP4 , a defining hallmark of the coats it forms on membranes , were entirely absent from any of the N - BAR lattices we reconstructed . Similarly , tip - to - tip interactions between consecutive F - BAR dimers made a significant contribution to controlling the curva - ture state of the bilayer ( Frost et al . , 2008 ) , yet corresponding N - BAR dimer interactions did not seem to make notable contri - butions to the formation of N - BAR lattices . These fundamental differences in lattice design both led to the same conclusion , that antiparallel interactions between H0 helices from N - BAR domains in adjacent rows of the lattice are essential for scaffold assembly and stabilization of membrane curvature because the limited or absent tip - to - tip interactions between consecutive N - BAR domain dimers would be too weak on their own to main - tain the highly curved membrane tubules ( Lyman et al . , 2010 ) . As a direct consequence , the lateral association of H0 helices along the tube axis and between adjacent ridges of the lattice exposed large areas of membrane surface , which may be critical to allowing access for proteins like the GTPase dynamin or the inositol 5 - phosphatase synaptojanin that in endocytosis act downstream of N - BAR proteins ( Ringstad et al . , 1997 ; Sund - borger et al . , 2011 ; Takei et al . , 1999 ) ( Figure 5 ) . Also very different from the F - BAR domain of CIP4 , cross - sections of endophilin - covered tubes revealed that they differ by an integral number of dimers around the circumference of the tube ( data not shown ) . This suggested that the N - BAR domains accommodate different curvatures primarily through a ‘‘quantized’’ addition / removal of dimers , which contrasts with CIP4 scaffolds , in which different curvatures are accommodated by a combination of lattice reorientation and noninteger changes in F - BAR dimers around the circumference of the tubule ( Frost et al . , 2008 ) . H0 : H0 Interactions Are Generic and Degenerate One of the most surprising and counterintuitive findings of our work was that interactions between the H0 helices appeared to be extremely pliable and nonspecific . Fortifying the crosslinking data , the observation that the endophilin H0 peptide was able to poison lattice formation by another N - BAR protein , amphiphysin 2 , was perhaps the most striking illustration of the nonspecific nature of H0 interactions because the sequences of the H0 helices from the two proteins do not share high homology . The notion that these types of amphipathic wedges provide a generic , low - affinity amphiphilic hook seemed at odds with their indisputable importance for establishing ordered arrays . Specifically , our findings raise the question of how such generic interactions allow for proper biological function . In part , an answer may arise from the observation that N - BAR decorated tubes are very flexible in vitro and readily accommodate changes in diameter even within a given lattice . If flexibility were a func - tional requirement , then pliable interactions between the main lattice contact points would be necessary , and such interactions could not be accomplished with a scaffold design , as seen in F - BAR lattices of CIP4 , where extensive lateral interactions between F - BAR coiled - coil domains create a very rigid cast ( Frost et al . , 2008 ) . Similarly , a low - affinity interaction facilitates lattice disassembly , which may be necessary after a given process has run to completion . Contemplating possible reasons Figure 4 . Disposition of SH3 Domains ( A ) Partial overlay of the envelopes for reconstructions of full - length endophilin ( black ) and the endophilin N - BAR domain only ( red ) . Shown are the overlays for tubes of 28 nm ( left ) and 25 nm ( right ) diameter . An additional volume element was observed in the full - length endophilin reconstruction of the 28 nm tube . ( B ) The volume of the additional density was large enough to accommodate the high - resolution crystal structure of theratendophilinA1SH3domaindimer ( pdb : 3IQL ) , which was fitted without any modifications to the coordinates . ( C ) The positions of Cys294 ( pink ) and Thr320 ( yellow ) are marked in the structure ( left ) . The black scale bar above the dimer is 10 A˚ . Shown to the right are Coomassie - stained , nonreducing SDS - PAGE gels of wild - type endo - philin and a Thr320Cys mutant after tubulation and crosslinking ; M , molecular weight marker . In the crystal structure of the SH3 domain dimer , the pair of Cys294 residues isjustover 10A ˚ apart , slightlytoofar toefficiently crosslink . However , substituting Thr320 for Cys should , anddid , allowefficientcrosslinking of endophilin based on the appearance of the (cid:1) 75 kD endophilin dimer . This is consistent with the idea that in some cases , SH3 domains dimerize above the BAR domain scaffold . 142 Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . other than lattice dynamics , a highly specific recognition between amphipathic wedges may also not be necessary , as the concentration of unrelated segments is likely to be small in the vicinity of the site where membrane remodeling occurs . Though without doubt , a final explanation for the perplexing lack of specificity still needs to be found , it seems that part of the answer may lie in a cell’s need to mount a versatile , yet robust , response in cases for which assembly of N - BAR lattices is required . Reorientation of the Insert Helix May Be Associated with Curvature Generation Although the resolution of our reconstructions did not allow the assignment of a clear position for endophilin’s insert helix , a reasonable fit for this helix within the molecular envelope would require it to be tilted toward the membrane core ( Figure 1B ) . Interestingly , the direction of tilt would be the opposite of that observed in the only crystal structure in which the insert helix was resolved ( pdb : 2Z0V ) and also would contrast with recent spectroscopic evidence showing that a membrane - bound insert helix bound at an angle that made it slope away from the hydro - phobic core ( Jao et al . , 2010 ) . Notably , these latter studies had to be performed on liposomes that were too small to support tubulation because tubulation would hinder interpretation of the spectra . In an attempt to reconcile these disparate observa - tions , we suggest that in order to efficiently induce curvature at a macroscopic scale , the bilayer environment must be permissive for lateral H0 : H0 interactions to occur and to allow the insert helix to undergo a conformational change that would cause it to tip toward the hydrophobic core of the bilayer . How these two components individually contribute to curvature sensing , stabilization , and generation is a question that remains unanswered . Scaffold Structure Suggests a Steric Selection Mechanism for Interacting Proteins By nature , most BAR domain proteins are scaffolding compo - nents that drive curvature - dependent biological processes through the recruitment of other proteins . Selection of interaction partners in many cases depends on SH3 domains , which often are able to interact with multiple different proteins ( Ferguson et al . , 2009 ; Solomaha et al . , 2005 ) . This promiscuity raises the important question of how BAR - dependent scaffolds accom - plish specificity in recruiting only those components that are required to drive any given biological process . The question becomes more pressing in light of the observation , based on our reconstructions , that endophilin coats generate effective SH3 domain concentrations of 3 – 5 mM within a 100 A˚ annulus above the scaffold surface . Notably , this concentration is inde - pendent of curvature , as the same number is obtained for tubes of different width . Even more unsettling , calculations for the scaf - folds formed by the F - BAR domain of CIP4 ( Frost et al . , 2008 ) yield the same result . Considering that proline - rich target peptides typically bind SH3 domains with nanomolar to low - micromolar affinities ( Demers and Mittermaier , 2009 ) , our reconstructions reveal that selection of downstream interaction partners cannot be based on binding affinities because the high local concentration of SH3 domains would cause nonselective recruitment , even of low - affinity binding partners , to any type of BAR domain lattice , and in a curvature - independent manner . Our finding that SH3 domains dimerize above the surface of some , but not all , lattices offers a potential solution to this vexing problem , as it suggests that unique spatial presentation signatures , rather than binding affinities , identify the type of BAR - scaffold and communicate the curvature state of an underlying membrane to potential inter - action partners . For instance , the 28 nm tubes present SH3 domains as dimers . This spatial arrangement may be particularly useful for recruitment of the GTPase dynamin , which has two spatially close proline - rich segments within its proline - rich domain . Incidentally , a diameter of 28 nm of the endophilin - covered tube is also in the middle of the range of diameters sug - gested to be appropriate for dynamin - dependent membrane fission ( Faelber et al . , 2011 ) . Consequently , the 28 nm tubes not only may recruit dynamin more readily than tubes of different diameters but also may aid membrane scission by matching the curvature state of the target membrane with what is required for dynamin action . EXPERIMENTAL PROCEDURES Protein Purification cDNA fragments encoding rat endophilin A1 , rat endophilin A1 N - BAR domain ( 1 – 247 ) , and rat amphiphysins 1 and 2 were subcloned into pGEX6P - 1 ( GE Healthcare , Piscataway , NJ ) via polymerase chain reaction . Cysteine mutants were generated by site - directed mutagenesis ( QuikChange , StrataGene , Santa Clara , CA ) . cDNA for rat endophilin A1 , rat amphiphysin 1 , and rat amphiphysin 2 were kindly provided from P . DeCamilli , Yale University , New Haven , CT . Fusion proteins were bacterially expressed and purified first on a GST - glutathione affinity column ( GE Healthcare ) . The GST tag was cleaved Figure 5 . N - BAR Scaffolds Compartmentalize the Membrane Surface for Interactions with Downstream Effectors In contrast to F - BAR lattices , endophilin lattices expose large membrane surface areas . This figure shows how the size of these areas is matched to the size of membrane - binding domains from downstream effectors that are re - cruitedbyendophilin . Specifically , thePHdomainofdynamin ( pdb : 2DYN ) and the inositol polyphosphate phosphatase catalytic domain ( IPP5C ) of syn - aptojanin from S . pombe ( pdb : 1I9Z ) are perfectly accommodated within the constraints of the endophilin lattice . Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . 143 by PreScission protease followed by gel filtration chromatography ( see Supplemental Information for composition ) . Endophilin H0 - truncation mutants were cloned and generated in pET24 ( Novagen / EMD Chemicals , Gibbstown , NJ ) to improve yield . H0 - truncation mutants were generated by site - directed mutagenesis ( QuikChange , StrataGene ) . The fusion proteins were purified on a Talon - metal - affinity column ( Clonetech , Mountain View , CA ) . Aliquots of 4mg / ml ( endophilinNBAR ) protein , 10mg / ml ( endophilin ) , 25mg / ml ( endophi - lin H0 - truncation mutants ) , and 2 mg / ml ( amphiphysin 1 and amphiphysin 2 ) were stored at (cid:4) 80 (cid:3) C . Liposome Preparation and Tubulation In Vitro For all experiments , synthetic lipids were used ( Avanti , Alabaster , AL ) . For the imaged sample we prepared lipids with the compositions ( w / w ) 50 % DOPS , 45 % DOPE , and 5 % cholesterol ( endophilin ) , and 75 % DOPS , 25 % DOPE , and 5 % cholesterol ( amphiphysin , endophilin N - BAR ) . These mixtures were driedunderastreamofdryargonwithgentlevortexinginglassvials , dissolved inabsolutehexane , driedwithargonagain , anddesiccatedunderhighvacuum for 1 hr . Lipids were then hydrated with buffer A ( 50 mM K - aspartate , 10 mM Tris / HCl , and 1mM EGTA , pH 7 . 5 ) , sonicated , and used immediatelyor stored in aliquots at (cid:4) 80 (cid:3) C . The in vitro tubulation was performed with liposomes ( 0 . 1 – 0 . 25 mg / ml ) equilibrated at room temperature before adding the protein at a lipid / protein ratio ( w / w ) of 1 . 4 : 1 ( endophilin N - BAR ) or 1 : 1 ( amphiphysin 1 N - BAR ) . Cysteine Crosslinking of Lipid - Bound Endophilin Endophilin cysteine mutants were reduced in buffer A containing 1 mM tris ( 2 - carboxyethyl ) phosphine ( TCEP ) before adding liposomes . After an additional incubation period , TCEP was removed by repeated centrifugation and resus - pensionofthepelletwithTCEP - freeandEGTA - freebufferA . Theresuspended pellet wasincubated inbuffer A containing 1mMCu - O - phenanthroline for 1hr and the reaction was stopped with 1 mM EDTA . The pellet and supernatant were analyzed by nonreducing SDS - PAGE and stained with Coomassie Blue . An aliquot of the pellet of all tested mutants was assessed by electron microscopy . For competition experiments with H0 peptides , nominal peptide concentrations of 2 mM were used . Electron Microscopy Imaging The tubulation reaction was screened using 1 % uranyl - acetate - stained samples and a Tecnai 12 microscope ( Philips , FEI , Eindhoven , The Netherlands ) operatingat120kV . Imagesofunstainedsampleswereacquired at a sample temperature of (cid:4) 170 (cid:3) C on a Tecnai F20 Twin transmission elec - tron microscope operating at 120 kV , and recorded with a Tietz F415 4k 3 4k pixel CCD camera using the Leginon data collection software ( Suloway et al . , 2005 ) at nominal magnifications of 29k 3 , and defocus values of (cid:4) 1 . 5 m mto (cid:4) 2 m m . Electroncryomicrographsusedinthefigureswerecontrast enhanced to increase visibility of fine molecular features . Detailed methods can be found in the Extended Experimental Procedures . Helical Image Processing Fourier - Bessel reconstruction proved to be impossible due to the lack of high - resolution features in the power spectrum from these tubules . Moreover , variations in the diameter over the length of even a single tubule precluded reciprocal space averaging . We therefore decided to sort overlapping segments of the tubules into defined classes using maximum - likelihood methods ( XMIPP ) ( Sorzano et al . , 2004 ) before initiating reconstruction by an iterative helical real - space reconstruction ( IHRSR ) single - particle algorithm asimplementedinSPIDER ( Egelman , 2007 ; Franketal . , 1996 ) . Theresolutions of our reconstructions ( Table S1 ) were calculated with the program RMEAS - URE ( Sousa and Grigorieff , 2007 ) . See Extended Experimental Procedures for a detailed description of the reconstruction strategy . ACCESSION NUMBERS Volume files for the reconstructions have been deposited at EMDB under the following accession numbers : N - BAR - only ( 25 nm ) EMD - 5365 , N - BAR - only ( 28 nm ) EMD - 2007 , endophilin ( 25 nm ) EMD - 5366 , endophilin ( 28 nm ) EMD - 5367 , and endophilin ( 32 nm ) EMD - 5368 . SUPPLEMENTAL INFORMATION Supplemental Information includes Extended Experimental Procedures , three figures , and one table and can be found with this article online at doi : 10 . 1016 / j . cell . 2012 . 01 . 048 . ACKNOWLEDGMENTS We thank Pietro De Camilli and members of his lab for critical discussions and feedback . The majority of data for this work were collected at the National Resource for Automated Molecular Microscopy , which is supported by the National Institutes of Health through the National Center for Research Resources’ P41 program ( RR017573 ) . We are also indebted to David Morgan for letting us use the cryoEM facility at Indiana University . This work was funded by Deutsche Forschungsgemeinschaft ( C . M . ) and the Cancer Research Institute ( C . M . ) , as well as PHS grants DA24101 ( V . M . U . ) , GM094479 ( V . M . U . ) , and GM063796 ( G . A . V . ) from the National Institutes of Health . 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Cell 149 , 137 – 145 , March 30 , 2012 ª 2012 Elsevier Inc . 145 \ No newline at end of file diff --git a/silver_data/f3b94893a6c96192021b923540945b222c05e602.pdf.txt b/silver_data/f3b94893a6c96192021b923540945b222c05e602.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..70aca35480c91e4fcd85e6fb19fed3ebd12f51bd --- /dev/null +++ b/silver_data/f3b94893a6c96192021b923540945b222c05e602.pdf.txt @@ -0,0 +1 @@ +1 Tripartite Inhibition of SRC - WNT - PKC Signalling Consolidates Human 1 Naïve Pluripotency 2 3 Jonathan Bayerl 1 * , Muneef Ayyash 1 * , Tom Shani 1 * , Yair Manor 1 * , Ohad Gafni 1 , Yael Kalma 2 , Alejandro 4 Aguilera - Castrejon 1 , Mirie Zerbib 1 , Hadar Amir 2 , Daoud Sheban 1 , Shay Geula 1 , Nofar Mor 1 , Leehee 5 Weinberger 1 , Vladislav Krupalnik 1 , Bernardo Oldak 1 , Nir Livnat 1 , Shadi Tarazi 1 , Shadi Tawil 1 , Lior 6 Lasman 1 , Suhair Hanna 3 , Noa Novershtern 1 , Dalit Ben - Yosef 2 @ # , Sergey Viukov 1 # @ and Jacob H . Hanna 1 @ # . 7 1 The Department of Molecular Genetics , Weizmann Institute of Science , Rehovot 7610001 , Israel . 2 Wolfe PGD 8 Stem Cell Lab , Racine IVF Unit at Lis Maternity Hospital Tel Aviv Sourasky Medical Center , Tel Aviv University , 9 Tel Aviv , Israel . 3 Department of Pediatrics , Rambam Hospital , Haifa , Israel . 10 11 @ Correspondence should be addressed to Jacob H . Hanna ( jacob . hanna @ weizmann . ac . il ) , Sergey Viukov 12 ( Sergey . viukov @ weizmann . ac . il ) & Dalit Ben - Yosef ( dalitb @ tlvmc . gov . il ) . 13 * Co - first authors . # Co - senior authors . 14 15 16 Abstract 17 Different conditions have been devised to isolate MEK / ERK signalling independent human 18 naïve pluripotent stem cells ( PSCs ) that are distinct from conventional primed PSCs and better 19 correspond to pre - implantation developmental stages . While the naïve conditions described thus 20 far endow human PSCs with different extents of naivety features , isolating human pluripotent cells 21 that retain characteristics of ground state pluripotency while maintaining differentiation potential 22 and genetic integrity , remains a major challenge . Here we engineer reporter systems that allow 23 functional screening for conditions that can endow both the molecular and functional features 24 expected from human naive pluripotency . We establish that simultaneous inhibition of SRC - NF κ B , 25 WNT / ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma 26 competent fully naïve human PSCs in defined or xeno - free conditions . Divergent signalling and 27 transcriptional requirements for maintaining naïve pluripotency were found between mouse and 28 human . Finally , we establish alternative naïve conditions in which MEK / ERK inhibition is 29 substituted with inhibition for NOTCH / RBPj signalling , which allow obtaining alternative human 30 naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA 31 hypomethylation . Our findings set a framework for the signalling foundations of human naïve 32 pluripotency and may advance its utilization in future translational applications . 33 34 35 36 37 38 39 40 2 Highlights of key findings : 1 2 • Combined inhibition of SRC , WNT and PKC signaling consolidates human naïve pluripotency 3 • Stable expansion of DNA / RNA methylation - independent and TGF / ACTIVIN - independent 4 human naïve PSCs 5 • Opposing roles for ACTIVIN and WNT / ßCATENIN signaling on mouse vs . human naive 6 pluripotency 7 • 2i and MEK / ERKi independent alternative human naïve PSC conditions via inhibiting 8 NOTCH / RBPj signaling 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 3 Introduction 1 A continuum of pluripotent configurations represent changes occurring during in vivo transition 2 of naïve pre - implantation pluripotency toward that of primed post - implantation pluripotent state , can be 3 captured in vitro to various extents ( ( Brons et al . , 2007 ; Hackett and Surani , 2014 ; Nichols and Smith , 4 2009 ; Tesar et al . , 2007 ; Weinberger et al . , 2016 ) . Many naïve and primed pluripotency properties can be 5 individually characterized and attributed to pluripotent stem cells ( PSCs ) expanded in distinct conditions . 6 In mice , defined serum free 2i / LIF conditions ( 2 inhibitors of MEK and GSK3 supplemented with LIF ) 7 have been extensively characterized where many naïve molecular and functional properties are endowed 8 by this combination ( Ying et al . , 2008a ) . The latter include global DNA hypomethylation , loss of 9 bivalency over developmental genes ( Marks et al . , 2012 ) , exclusive nuclear localization of TFE3 10 transcription factor ( Betschinger et al . , 2013 ) , tolerance for lack of exogenous L - glutamine ( Carey et al . , 11 2015a ) , tolerance for loss of repressors like DNMT1 , METTL3 and DGCR8 / DICER ( Geula et al . , 2015 ) . 12 Mouse ESCs expanded in Fetal Bovine Serum ( FBS ) / Lif conditions are also considered naïve and possess 13 features such as retention of pre - X inactivation state , ability to tolerate lack of repressors like Mettl3 and 14 Dnmt1 ( Geula et al . , 2015 . However , they do not retain a global hypomethylated epigenome and acquire 15 H3K27me3 over developmental genes ( Weinberger et al . , 2016a ) , and thus are considered relatively less 16 naïve than 2i / LIF grown mouse PSCs ( Marks et al . , 2012 ) . Rodent EpiSCs expanded in Fgf2 / Activin A 17 conditions show further consolidation and acquisition of their milieu of primed pluripotency 18 characteristics ( Brons et al . , 2007 ; Tesar et al . , 2007 ) . EpiSC lines are heterogeneous in their epigenetic 19 and transcriptional patterns ( Kojima et al . , 2014 ) , and while they are pluripotent and give rise to 20 differentiated cells from all three germ layers , they are epigenetically restricted as evident for example in 21 their reduced ability , after long - term maintenance in FGF2 / ACTIVIN A conditions , to differentiate into 22 primordial germ cells ( PGCs ) ( Hayashi et al . , 2011 ) or contribute to chimera formation when injected in 23 the pre - implantation ICM ( Wu et al . , 2015a ) . 24 25 While conventional human embryonic stem cells ( hESCs ) and iPSCs ( hiPSCs ) growth 26 conditions entailed FGF / TGF as typical for murine EpiSC , these two cell types are not identical , and 27 hESC share several molecular features with naïve mESCs including expression of E - CADHERIN ( rather 28 than N - CADHERIN ) ( Weinberger et al . , 2016 ) . Further , conventional human ESCs express high levels of 29 PRDM14 and NANOG as murine naïve ESCs , and they are functionally dependent on their expression 30 4 ( Chia et al . , 2010 ) . Still however , hESCs retain a variety of epigenetic properties that are consistent with 1 possessing a primed pluripotent state . This includes inability to tolerate MEK / ERK signaling inhibition 2 ( Weinberger et al . , 2016 ) , predominant utilization of the proximal enhancer element to maintain OCT4 3 expression , tendency for initiation of X chromosome inactivation ( XCI ) in most female ESC lines 4 ( Mekhoubad et al . , 2012 ) , high levels of DNA methylation , prominent deposition of H3K27me3 and 5 bivalency acquisition on lineage commitment regulators ( Gafni et al . , 2013 ) . 6 7 The proof of concept for the metastability between naïve and primed state in rodents ( Hanna et 8 al . , 2009 ) , have raised the possibility that the human genetic background is more “stringent” in regards to 9 requirement for exogenous factors provided in allowing preservation of ground state - naïve pluripotency 10 in comparison to rodents . Indeed , several groups have previously established condition to derive naïve 11 MEK / ERK signaling independent genetically unmodified human PSCs . For example , NHSM conditions 12 do not require the use of exogenous transgenes or feeder cells , maintain teratoma formation competence 13 and entail the following components : 2iLIF , P38i / JNKi , PKCi , ROCKi , ACTIVIN and FGF2 . NHSM 14 conditions endow human PSCs with variety of naïve features including maintaining pluripotency while 15 MEK / ERK signaling is inhibited , predominant TFE3 nuclear localization , resolution of bivalent domains 16 over developmental regulators , in vitro reconstitution of human PGCLC and a mild reduction in DNA 17 methylation ( Gafni et al . , 2013 ; Irie et al . , 2015 ) . The latter effect is profoundly weaker than that seen in 18 mouse pluripotent cells , suggesting sub - optimal human naïve pluripotency growth conditions . 19 20 Exciting alternative conditions that generate MEK independent human naïve cells and retain a 21 more compelling milieu of transcriptional markers expressed in the human ICM were developed . Several 22 components found in NHSM conditions ( 2i , ROCK inhibitor , ACTIVIN , FGF2 ) were supplemented with 23 BRAF inhibitors , to generate MEF dependent naïve cell lines ( termed : 5iLA - , 5iLAF - , 6i / LA - and 4i / LA - 24 MEF ) ( Theunissen et al . , 2014 ) . Globally these conditions generated more pronounced downregulation in 25 DNA methylation and upregulation of naïve pluripotent cell markers . However , the hypomethylation in 26 these conditions was accompanied by immediate and global loss of imprinting over all imprinted loci 27 ( Theunissen , 2016 ) ( Bar et al . , 2017 ; Pastor et al . , 2016 ) and obligatory confounding chromosomal 28 abnormalities in nearly 100 % of the lines generated within 10 passages only ( Liu et al . , 2017 ) . Derivation 29 of human naïve ESC in t2iL - Go conditions has been reported with and without the use of exogenous 30 5 transgenes induction . In both cases , the generated cell lines do not form teratomas in vivo and can only 1 differentiate in vitro after an extended 3 - week transfer to primed - like conditions , thus questioning their 2 pluripotent functionality and stability ( Guo et al . , 2016 ; Liu et al . , 2017 ; Takashima et al . , 2014 ) . The 3 latter is in striking difference from rodent ground state naïve PSCs , which are fully pluripotent and can 4 initiate differentiation in vivo following autonomous induction of the needed priming ( capacitation ) 5 signals toward differentiation ( Ko et al . , 2009 ; Müller et al . , 2010 ; Takahashi et al . , 2003 ; Ying et al . , 6 2008 ) . 7 8 Therefore , these prior studies collectively suggest that none of the conditions devised so far are 9 optimal or enable closing the gap between mouse and human naive pluripotency characteristics . In this 10 study , we set out to devise enhanced defined conditions that enable the stabilization of hPSCs with 11 stringent functional and molecular properties , previously attributed to mouse ground state naïve ESCs and 12 to human preimplantation stages , while preserving cell - autonomous teratoma formation competence and 13 decipher defining features of their signaling and regulatory circuitry . We also aimed to establish 14 principles for capturing alternative human naïve PSCs without inhibiting MEK / ERK pathway that tends 15 to compromise DNA imprinting stability in both mouse and human naive PSCs ( Choi et al . , 2017 ; Di 16 Stefano et al . , 2018 ; Yagi et al . , 2017 ) . 17 18 Results 19 Defining human naïve pluripotency conditions that endow tolerance for depletion of RNA / DNA 20 methylation 21 22 A major challenge for capturing human naïve PSC conditions has been the lack of stringent 23 functional assays that allow screening for conditions endowing functionally naïve human PSCs ( De Los 24 Angeles et al . , 2015a ) . Recent studies have defined the functional ability of mouse naïve 2i / LIF 25 conditions to maintain their pluripotency upon knocking - out epigenetic repressors like Mettl3 and Dnmt1 26 ( m 6 A mRNA and DNA methyltransferase enzymes , respectively ) , while EpiSCs differentiate and undergo 27 apoptosis upon complete ablation of the same factors ( Geula et al . , 2015 , Liao et al . , 2015 ) . Further , 28 DNMT1 ablation leads to cell death and differentiation of human primed ESCs further underscoring this 29 primed aspect of their molecular identity ( Liao et al . , 2015 ) . As such , we hypothesized that the latter 30 6 functional attributes might be utilized to screen and define conditions endowing this stringent naïve 1 pluripotency feature ( i . e . maintenance of pluripotency in the absence of defined epigenetic repressors ) . 2 We engineered human WIBR3 hESC lines with conditional inducible ablation of the expression of 3 METTL3 ( Fig . 1a ) . This was obtained via introducing an exogenous METTL3 transgene under the 4 regulation of Tet - OFF promoter ( Liao et al . , 2015 ) , followed by CRISPR / Cas9 mediated ablation of both 5 endogenous human METTL3 alleles ( Fig . 1a ) . Two resultant clones were validated for METTL3 6 expression only from the exogenous allele , which can be shut off by addition of DOX to the media ( Tet - 7 OFF - METTL3 lines ) ( Fig . 1b ) . 8 9 Primed Tet - OFF - METTL3 hESCs expanded in primed TeSR or KSR / FGF2 conditions could 10 not be sustained in the presence of DOX more than four passages ( both on MEF or Geltrex coated dishes ) 11 and resulted in massive cell death and differentiation ( Fig . 1c ) , analogous to the result previously reported 12 for mouse EpiSCs ( Geula et al . , 2015 ) . The latter is also consistent with lack of reports showing 13 generation of complete KO METTL3 cells in human primed cells ( Bertero et al . , 2018 ) . In the presence 14 of MEFs , Tet - OFF - METTL3 could be poorly maintained in previously described human naive NHSM 15 conditions and with a very slow proliferation rate , but not in 4iLA - MEF , 5iLAF - MEF , 5iLA - MEF , 6iLA - 16 MEF , TESR / 3iL - MEF previously described naïve conditions ( Fig . 1c , S1a ) . However , in the absence of 17 MEFs , also NHSM conditions could not support maintenance of pluripotency when METTL3 was 18 completely ablated , suggesting that NHSM conditions can possibly be enhanced to endow the cells with 19 such ability ( Fig . S1a ) . We thus set out to test candidate molecules that may enrich NHSM condition and 20 allow to maintain Tet - OFF - METTL3 on Geltrex coated plates in the presence of DOX . 21 22 We tested whether supplementing NHSM conditions with individual small molecules that have 23 been previously implicated in boosting mouse or human naïve pluripotency formation or iPSC derivation 24 efficiency might achieve this goal ( Fig . 1d ) . Remarkably , supplementing NHSM conditions with the 25 Tankyrase inhibitor named IWR1 , but not any of the other 15 compounds tested , enabled expanding Tet - 26 OFF - METTL3 on DOX with great homogeneity ( Fig . 1e ) . IWR1 is a WNT inhibitor ( WNTi ) small 27 molecule that stabilizes AXIN protein in the cytoplasm by inhibiting Tankyrase enzyme ( abbreviated 28 herein as TNK inhibitor – TNKi ) ( Huang et al . , 2009 ) . It was included serendipitously among our 29 compounds of interest since when combined with GSK3 inhibitors on mouse and human primed cells , 30 7 this effect lead to enriching β Catenin only in the cytoplasm and such primed cells could be expanded 1 without exogenous FGF2 ( Kim et al . , 2013a ) . Using an independent specific TNKi , XAV939 , yielded a 2 similar effect , while using exo - IWR1 an inactive modified version of IWR1 failed to do so , supporting 3 specific inhibition of Tankyrase as the target yielding stability of these pluripotent cells ( Fig . 1f ) . Notably , 4 a number of recent publications have included TNKi among other ingredients for both naïve , extended 5 potential or primed hPSCs , however none of these different conditions that employ TNKi ( Bredenkamp et 6 al . , 2019a ; Smith et al . , 2012 ; Yang et al . , 2017a , 2017b ; Zimmerlin et al . , 2016a ) allow to maintain 7 OCT4 + in the absence of METTL3 or DNMT1 , likely due to the absence of other key ingredients 8 synergistically used in NHSM conditions but not in these previously described conditions ( i . e . 9 combination of ACTIVIN , PKCi , P38i / JNKi together with TNKi combination ) ( Fig . S1b ) . 10 11 We used previously described WIBR3 hESC line carrying knock - in Δ PE - OCT4 - GFP reporter 12 ( Theunissen et al . , 2014 ) ( Fig . S1c ) and found that supplementation of TNKi to NHSM conditions 13 yielded a dramatic increase in GFP signal when compared to primed , NHSM or 4i - LA conditions ( Fig . 14 1g , Fig . S1c ) . Consistent with studies conducted in mice ( Kim et al . , 2013a ) , including TNKi rendered 15 exogenous supplementation of FGF2 dispensable even in feeder free conditions ( Fig . 1g ) . Further , as 16 XAV939 inhibits WNT signaling , we validated that including GSK3 inhibitor is dispensable and , in fact , 17 compromises the intensity of Δ PE - OCT4 - GFP signal ( Fig . S1d ) . JNK / P38 inhibition boosted naïve 18 pluripotency marker expression and cell viability , therefore were maintained in the media used herein 19 ( Fig . S1e ) . Importantly , after optimizing NHSM conditions , we retried to substitute TNKi with other 20 components included in the screen , to exclude the possibility that the latter optimizations may facilitate a 21 different screening result . However , none of them allowed expanding METTL3 depleted cells in vitro as 22 seen with supplementing TNKi ( including VPA , BRAFi , Forskolin , Kenopaullone , SHHi , DOT1Li , 23 LSD1i , TGFRi , ERK5i ) ( Fig . S1f ) . 24 25 Defining human naïve pluripotency conditions independent of TGF / ACTIVIN signaling 26 27 Following the latter modifications human ESCs maintained uniformly high Δ PE - OCT4 - GFP 28 levels only in the presence of exogenous ACTIVIN A , and consistently differentiated when TGFR 29 inhibitor was provided ( Fig . 1h - i , Fig . S1g ) . We next tested whether a second targeted screening strategy 30 8 would allow us to identify a small molecule whose supplementation will render human PSCs that are 1 independent of exogenous ACTIVIN / TGF supplementation . It should be noted that none of the 2 previously described human naïve conditions have been able to maintain teratoma competent pluripotent 3 cells that can be maintained long term and validated for their naïve identity after prolonged specific 4 inhibition of ACTIVIN / NODAL signaling ( Liu et al . , 2017 ) . To do this , the latter TNKi supplemented 5 NHSM conditions were used in the absence of ACTIVIN A , and candidate molecules were added to 6 screen for allowing expanding OCT4 + PSCs independent of METTL3 expression ( on DOX ) and the 7 absence of exogenous ACTIVIN A ( Fig . 2a ) . While in most conditions OCT4 + cell fraction rapidly 8 deteriorated , we noted that a validated SRC inhibitor ( SRCi = CGP77675 ) dramatically maintained the 9 stability of dome like cells that were uniformly OCT4 + ( Fig . 2b ) . The latter led us to assemble a defined 10 FGF / TGF / ACTIVIN / MEF independent and serum free conditions which we term Human Enhanced 11 Naïve Stem cell Medium – HENSM ( Fig . 2c - d ) , which we systematically characterize and validate 12 herein . We also note that supplementation of SRCi in ACTIVIN A containing conditions , although was 13 not essential to maintain Δ PE - OCT4 - GFP + when ACTIVIN A was provided , supported consistency in 14 domed like morphology among naïve cells ( Fig . S1h ) ( we term this conditions HENSM - ACT and is 15 highlighted herein when used ) . 16 17 Both with and without METTL3 depletion in HENSM conditions , WIBR3 cells maintained their 18 typical domed like morphology and uniformly expressed pluripotency markers including KLF17 that is 19 specific to the human naïve state ( Fig . S2a - b ) . Measurement of m 6 A on mRNA showed over 90 % 20 depletion of total levels after DOX addition ( Fig . 2e ) , comparable to those seen upon knockout of the 21 Mettl3 / 14 in mouse naïve cells . ESCs maintained in the absence or presence of DOX for over 30 passages 22 remained pluripotent and , as it is typically standardized for murine ESCs and iPSCs , the human cells were 23 capable of generating mature teratomas in vivo without the need to passage them first for a period of time 24 in primed conditions or capacitation via unique conditions ( Rostovskaya et al . , 2019 ) prior to their 25 injection in immune - compromised mice ( Fig . 2f ) . The latter validate maintenance of naïve pluripotency 26 in human PSCs expanded in HENSM conditions , including when METTL3 protein and m 6 A levels on 27 mRNA were ablated . 28 29 9 To extend the previous findings to another repressor machinery , Δ PE - OCT4 - GFP - WIBR3 1 reporter human ESCs were targeted by CRISPR / Cas9 to generate DGCR8 null cells ( Fig . S2d - e ) . While 2 conducting such targeting on primed cells did not yield any null clones ( Fig . S2d ) , consistent with 3 inability to sustain mouse primed and human ESCs without DGCR8 due to its essentiality in this state 4 ( Geula et al . , 2015 ) , DGCR8 null cells could be obtained when the targeted cells were expanded in 5 HENSM conditions ( Fig . S2e - f ) . DGCR8 KO cells expanded in HENSM retained human naïve 6 pluripotency specific traits despite lacking microRNA expression equivalent to results obtained in mouse 7 naïve PSCs ( Fig . S2g ) . To test which of the naïve conditions enable expanding human naïve PSCs in the 8 absence of DNMT1 cells , a similar approach to that applied for making Tet - OFF - METTL3 herein , has 9 been recently used to generate TET - OFF DNMT1 in HUES64 ESC line ( Fig . 2g ) ( Liao et al . , 2015 ) . Cells 10 expanded in previously described naïve conditions including NHSM , 5i / LA . t2iL - GO ( or PXGL ) and 11 TESR / 3iL - MEF could not be maintained in the presence of DOX for more than 3 passages ( Fig . 2h - i , 12 S2h ) . HENSM and HENSM - ACT conditions allowed stable and unlimited expansion of DNMT1 13 depleted human naïve ESCs both in feeder and feeder free conditions ( Fig . 2i , S2h ) . Whole Genome 14 Bisulfite Sequencing ( WGBS ) confirmed global loss of DNA methylation in naïve DNMT1 depleted cells 15 expanded in HENSM conditions ( Fig . S2i ) that also maintained expression of all pluripotency markers 16 ( Fig . S2j ) . Removal either of ERKi , PKCi , SRCi or TNKi in HENSM conditions resulted in loss of 17 pluripotency when DNMT1 or METTL3 were depleted ( Fig . S2k ) . Collectively these results demonstrate 18 that HENSM conditions closely mimic stringent characteristics of mouse naïve ground state ESCs and , 19 for the first time , enable generation of human PSCs ablated for epigenetic repressors ( both in feeder and 20 feeder free conditions ) and that are independent from ACTIVIN / TGF signaling . 21 22 Tolerance for absence of exogenous L - Glutamine in HENSM conditions 23 24 Murine naïve ESCs retain bivalent metabolic capability utilizing both oxidative phosphorylation 25 ( OXPHOS ) and glycolytic metabolism , while upon priming they become dependent only on glycolytic 26 metabolism . As shown previously , naïve hPSCs in NHSM , 5i - LA and transgene induced t2iL - Go 27 conditions increase OXPHOS activity leading to retention of a bivalent metabolic profile ( Sperber et al . , 28 2015 ) . HENSM conditions were similarly tested herein . Measured basal oxygen consumption rate ( OCR ) 29 was substantially higher in HENSM conditions than in primed PSC ( Fig . S2l ) . Higher electron transport 30 10 chain activity in HENSM was evidenced by a greater OCR increase in response to the mitochondrial 1 uncoupler FCCP ( Fig . S2l ) . Both NHSM , HENSM and transgene dependent t2iL - Go reset conditions , but 2 not primed or 5iLA conditions , PSCs maintained pluripotency when glucose uptake was blocked with 3 1mM 2 - Deoxy - D - Glucose ( DG ) , similar to what was observed with mouse naïve PSCs in 2i / LIF ( Fig . 4 S2m ) . 5 More stringently , a newly identified metabolic feature in naive ESCs in 2i or 2i / LIF is that they 6 can endogenously synthesize glutamine at sufficient levels to maintain adequate alpha - ketogluterate 7 ( α KG ) levels ( Carey et al . , 2015a ) . While they benefit from exogenous L - Glutamine supplementation , it 8 is not essential for their stability or pluripotency as they can metabolically synthesize it endogenously as 9 part of their altered metabolic configuration ( Carey et al . , 2015a ) . FBS / LIF naïve murine ESCs or primed 10 EpiSCs cannot be maintained in the absence of exogenous L - Glutamine for an extended period of time 11 ( Carey et al . , 2015a ) . To compare the latter observation and apply them on distinct human pluripotent 12 states , WIBR3 - OCT4 - GFP knock - in ESC line , Δ PE - WIBR3 - OCT4 - GFP knock - in ESC line and H9 - 13 NANOG - GFP ESC lines were then tested for ability to maintain pluripotency in the presence and absence 14 of exogenously added L - Glutamine ( Fig . 2j ) . Importantly , we failed to maintain primed PSCs or other 15 previously described naïve PSCs in the absence of L - Glutamine ( in NHSM , 4i / LA - MEF , 5i / LAF - MEF , 16 6iLA - MEF , TESR / 3iL - MEF , t2iLGo even in feeder cells presence for more than 10 days ( Fig . 2j ) . 17 However , Δ PE - WIBR3 - OCT4 - GFP expanded in HENSM was not compromised when L - glutamine was 18 not included in HENSM conditions ( Fig . S3a ) and GFP signal was positive for H9 - NANOG - GFP ESC 19 both in the presence and absence of L - Glutamine ( both on feeder and feeder free conditions ) ( Fig . S3a ) . 20 Cells expressed general and naïve specific pluripotency markers in HENSM with and without exogenous 21 L - Glutamine and generated differentiated teratomas without the need for in vitro passaging in primed 22 conditions before subcutaneous in vivo injection ( Fig . S3b - c ) . Other previously published combination 23 that entailed TNKi among other ingredients for both naïve and primed PSCs ( Bredenkamp et al . , 2019 ; 24 Wu et al . , 2015 ; Yang et al . , 2017 , Zimmerlin et al . , 2016 ) , failed to preserve pluripotency in the absence 25 of exogenous L - Glutamine as seen in HENSM based conditions described herein ( Fig . 2j ) . The latter is 26 due to the absence of other key ingredients synergistically used in NHSM conditions contributing to naive 27 pluripotency that are used herein but not in these previously described conditions ( i . e . the unique 28 combination of SRCi , PKCi together with TNKi combination in HENSM ) ( Fig . 2j ) . Collectively , these 29 11 results validate that HENSM conditions can maintain naïve pluripotency characteristics and endow the 1 cells with ability to be expanded in the absence of exogenous L - Glutamine as previously described for 2 murine 2i / LIF naïve PSCs ( Carey et al . , 2015a ) . 3 4 Transcriptional characterization of human PSCs in HENSM conditions 5 6 We next aimed to revert previously established primed PSCs lines and to derive new lines 7 directly in HENSM - ACT and HENSM conditions from the ICM of human blastocysts . Human 8 blastocysts were plated on mouse embryonic fibroblast ( MEF ) coated plates and medium successfully 9 generated domed cell outgrowths following 6 - 8 days of plating . ICM derived outgrowths were then 10 trypsinized and passaged . Subsequently , we were able to establish and characterize 5 newly derived stem 11 cell lines termed LIS36 , LIS42 and LIS46 in HENSM - ACT and LIS41 and LIS49 ESCs in HENSM 12 conditions ( Fig . S4a ) . Multiple conventional ( hereafter will be named “primed” ) hESC lines ( WIBR1 , 13 WIBR2 , WIBR3 , HUES64 , H1 , H9 ) were plated on Geltrex coated dishes in HENSM medium ( Fig . 14 S4b ) . Within 4 - 8 days of applying this protocol , dome - shaped colonies with packed round cell 15 morphology , typical of mESCs , could be readily isolated and further expanded ( Fig . S4b ) . Adult human 16 dermal fibroblast cells or peripheral blood cells were reprogrammed to iPSCs in HENSM conditions 17 following either lentiviral transduction with DOX inducible OKSM factors ( BF1 hiPSC ) or by non - 18 integrating sendai viruses ( JH1and MECP5 hiPSC ) ( Fig . S4c ) . All polyclonal and subcloned hESC and 19 iPSC lines expanded in HENSM conditions were uniformly positive for pluripotent markers alkaline 20 phosphatase ( AP ) , OCT4 , NANOG , SSEA4 , TRA1 - 60 and TRA1 - 81 ( representative images in Fig . S5 ) 21 and robustly formed mature teratomas in vivo without the need for short - or long - term exposure to primed 22 growth conditions prior to their injection into host mice , and as typically observed with rodent ground 23 state naïve PSCs ( Fig . S6a ) . Naïve lines were passaged with TryplE every 3 - 5 days and had single cell 24 cloning efficiency up to 40 % , while primed cell single cell cloning increased only up to 10 % even when 25 ROCK inhibition was used . Human naïve pluripotent lines maintained normal karyotype after extended 26 passaging in HENSM - ACT or HENSM in most lines of tested ( Fig . S7a ) . In some cultures ( < 10 % ) and 27 after over 30 passages , minor aneuploidy cells were observed however we did not observe a recurrent 28 abnormality between any of these lines as determined by G - banding of metaphase chromosomes ( Fig . 29 S7a ) . The latter results were corroborated by performing e - karyotyping that is based on RNA - seq 30 12 expression of HENSM derived lines ( Fig . S6b ) , thus indicating that epigenetic resetting in HENSM does 1 not cause obligatory chromosomal abnormalities nor select for pre - existing abnormal variants , as has been 2 seeing for other naïve conditions like 5iLA - MEF or t2iL - Go conditions that induce 96 - 100 % 3 chromosomal abnormality already by passage 10 ( Liu et al . , 2017 ) . 4 5 We compared global gene expression patterns between naïve and primed hESCs and hiPSCs , 6 many of which were genetically matched . Unbiased clustering of genome - wide expression profiles 7 demonstrated that naïve hESC and hiPSCs possess a distinct gene expression pattern and clustered 8 separately from conventional / primed hESCs and hiPSCs ( Fig . 3a - b ) . Transcripts associated with naïve 9 pluripotency were significantly upregulated in naïve cells ( Blakeley et al . , 2015a ) . The later included 10 NANOG , TFCP2L1 , KLF17 , KLF4 , STELLA ( DPPA3 ) , DPPA5 , DNMT3L , KHDC1L and ARGFX 11 shown in two independent datasets ( Fig . S8a and Table S1 - S2 ) . RT - PCR analysis validated the dramatic 12 upregulation in naïve PSCs expanded both in HENSM and HENSM - ACT conditions ( Fig . 3c ) . When 13 including previously and independently published naïve datasets generated in 5iLA - MEF , 4i - LAF and 14 t2i - LIF - GO - NK2 , we note that cells generated in HENSM and HENSM - ACT conditions clustered with 15 all the latter naïve conditions and not with primed samples ( Fig . 3a - b , Fig . S8b - f ) . The same 16 transcriptional qualities were detected in human PSCs expanded in HENSM Xeno - Free ( HENSM - XF 17 conditions ) on BioLaminin511 ( Fig . 3a ) . FACS analysis confirmed upregulation of previously identified 18 human naïve pluripotency markers Gb3 / CD77 and IL6ST / CD130 in HENSM conditions , and primed 19 pluripotency marker CD24 was downregulated in HENSM conditions ( Fig . S8g ) ( Collier et al . , 2017 ) . 20 SUSD2 surface marker was also expressed on all naïve cells lines tested , however was meaningfully 21 expressed on nearly 50 % human primed cell lines tested ( Fig . S9e ) , which provides a cautionary note 22 regarding recent claims ( Bredenkamp et al . , 2019 ) that it is an exclusive and decisive marker to exclude 23 the human primed state in vitro . Importantly , naïve pluripotent cells had profoundly down regulated 24 transcripts associated with lineage commitment genes including T , ZIC2 and VIM1 that are expressed in 25 primed hESCs ( Fig . S8a , S9a - b ) . By comparing the transcriptome to that described during early human 26 pre - implantation development , cells expanded in HENSM conditions , but not primed cells , showed 27 specific enrichment to profile of late morula - early blastocysts specific signature ( Fig . 3d ) . We 28 additionally introduced STELLA - CFP knock - in allele via CRISPR / Cas9 ( Fig . S9c - d ) , to monitor 29 pluripotency maintenance in the different tested conditions , and STELLA - CFP was upregulated in both 30 13 HENSM and 5iLA conditions ( Fig . S9d ) . Similar to its upregulation and importance in maintaining 1 human naïve pluripotency in 5i / LA conditions ( Pastor et al . , 2018 ) , TFAP2C KO lines ( Fig . S10a - d ) 2 showed that it is essential for deriving and maintaining human naïve PSCs in both HENSM and HENSM - 3 ACT conditions ( Fig . S10e ) . ESRRB is not detectable in human genetically unmodified cells expanded in 4 HENSM conditions ( Fig . S10f - j ) , consistent with its negative expression in the human ICM ( Blakeley et 5 al . , 2015b ; Petropoulos et al . , 2016 ) . The latter results confirm that HENSM conditions attain consensus 6 transcriptional features observed in other previously published naïve hPSCs studies or in vivo measured 7 human embryo transcriptional data , while uniquely retaining the critical ability to generate teratomas after 8 being directly injected as naïve cells in immune - compromised mice . 9 10 We profiled and compared Transposable Element ( TE ) - derived transcripts in conventional and 11 naïve human PSCS expanded in HENSM conditions ( Theunissen et al . , 2016 ) . The top 5 , 000 TEs with 12 largest SD separated naïve and primed samples both in hierarchical clustering ( Fig . S11a , Table S3 ) and 13 in PCA based analysis ( Fig . S11b ) . Members of the SINE - VTR - Alu ( SVA ) family of TEs and HERVK - 14 associated LTR were transcribed almost exclusively in HENSM conditions similar to previously obtained 15 in 5i / LA and transgene dependent t2iLGo conditions ( Fig . S12 ) ( Theunissen et al . , 2016 ) . We used TE 16 profiling to measure the degree to which HENSM and primed conditions resemble pluripotent cells in 17 early human embryos in vivo . HENSM naïve , but not primed cells , demonstrated the most significant 18 overlap with the human morula and epiblast stages when looking at TEs ( Fig . S11c ) , as was similarly 19 shown for coding genes ( Fig . 3d ) . 20 21 Epigenetic characterization of human PSCs in HENSM conditions 22 23 To further delve into the patterns which distinguish the naïve condition from its primed 24 counterpart in hESC and converge with its in vivo equivalents , we explored change in chromatin 25 accessibility of each condition by ATAC - seq . By comparing unique chromatin accessibility loci found in 26 different pre - implantation stages ( i . e . 2 - cell , 8 - cell and ICM ) we compared how much of these loci show 27 an increase or decrease in chromatin accessibility of naïve compared to primed hESCs . Like in datasets 28 generated in 5iLA - MEF and t2iL - Go - MEF , HENSM hESCs chromatin accessibility corresponds more to 29 the ICM than earlier stages ( Fig . 3e ) . These results support the endowment of late pre - implantation like 30 14 transposon expression and chromatin accessibility profile in PSCs expanded in HENSM conditions in 1 vitro . 2 3 We next moved to test whether HENSM conditions endow human naive PSCs with a pre - X 4 chromosome configuration . We used previously generated primed human WIBR2 hESCs carrying 5 knock - in MECP2 - tdTomato and MECP2 - GFP alleles ( Fig . 4a ) ( Theunissen et al . , 2016 ) . Correctly 6 targeted clone # 29 - 9 expresses only the red allele , however upon transferring the cells into HENSM 7 conditions > 99 % of cells expressed both fluorescent markers consistent with reactivation of both x 8 chromosome alleles . Transferring the cells into primed media allowed inactivation of X chromosome as 9 evident by obtaining GFP - / tdTomato + pattern > 95 % of the reprimed cells ( Fig . 4a ) . We carried out FISH 10 analysis for ATRX in female cells as this locus is expressed from one copy even in human primed 11 WIBR3 hESCs that have undergone erosion of X chromosome ( Xe ) . Indeed , two ATRX foci could be 12 uniformly found in naïve , but not primed , human female PSCs supporting reactivation of X chromosome 13 ( Fig . 4b ) . SNP based analysis of X chromosome allele expression as detected in RNA - seq datasets 14 showed biallelic expression of X chromosome encoded genes in HENSM but not primed conditions ( Fig . 15 4c ) , consistent with functional reactivation of X chromosome in female naïve PSCS induced in HENSM 16 ( Bar et al . , 2019 ) . 17 18 We sampled human naïve PSCs in HENSM and HENSM - ACT for DNA methylation status by 19 Whole genome Bisulfite Sequencing ( WGBS ) . Lines tested displayed profound downregulation of global 20 methylation levels ( Fig . 4d - e ) from 80 % in primed hPSCs to 50 % in HENSM - ACT and to 65 % in 21 HENSM expanded human hPSCs . It has been previously shown in mouse ground state naive conditions 22 that rapid loss of global DNA methylation level result from partial decrease in UHRF1 protein expression 23 levels while maintaining DNMT1 levels ( von Meyenn et al . , 2016 ) . Indeed , in human cells expanded in 24 HENSM , DNMT1 methyltransferase protein expression is maintained , while UHRF1 protein is partially 25 ( 50 - 60 % ) depleted ( Fig . 4f ) which may underlie the global downregulation in DNA methylation in 26 HENSM conditions . It is important to note that supplementing HENSM with BRAF inhibitor used in 27 5i / LAF conditions leads to dramatic downregulation also in DNMT1 ( Fig . 2h ) . This pattern is also 28 observed in 5i / LAF condition which might explain the immediate and global loss of imprinting ( loss of 29 imprinting of > 95 % of imprinted loci within 5 passages ) . As DNMT1 levels are maintained in HENSM 30 15 conditions and UHRF1 protein downregulation was partial , we did not observe immediate global loss of 1 all imprinted genes in HENSM or HENSM - ACT conditions ( Fig . S13 ) as has been previously seen in 2 5iLA or 2iL - Go based conditions ( Pastor et al . , 2016b ; Theunissen , 2016b ) . However , loss of imprinting 3 in HENSM occurred sporadically only at some individual loci , at lower frequencies and only after 4 prolonged cell passaging ( > 15 - 20 passages , Fig . S13 ) , which constitutes an improvement over previously 5 reported hypomethylated human naïve PSCs and comparable frequency to those observed in human 6 primed cells ( Bar et al . , 2017 ) . Collectively , the above findings indicate that HENSM and HENSM - ACT 7 conditions consolidate human naïve pluripotency identity and endow them with nearly all known naïve 8 pluripotency and differentiation features that have been attributed to human ICM in vivo , previously 9 derived human naïve cells and murine naïve ground state cells . 10 11 Differentiation competence of naive hiPSC following interspecies chimaerism in mice 12 13 Given that the naïve hPSCs described above maintained genetic integrity and were capable of 14 forming teratoma without the need for exogenous priming or capacitation prior to injection , we next 15 moved to evaluate whether human naïve iPSCs can integrate and differentiate following microinjection 16 into host mouse blastocyst ( Gafni et al . , 2013b ; Wu and Izpisúa Belmonte , 2016 ) . Microinjection of 17 human GFP labeled naïve iPSCs ( maintained for at least 3 passages in HENSM based conditions ) into 18 mouse E2 . 5 morulas showed robust integration in mouse in vitro developed ICMs at E3 . 5 . Blastocysts 19 obtained after micro - injection with human naïve iPSCs were implanted in pseudo - pregnant female mice 20 and their survival and integration was assayed throughout next 14 days using various imaging techniques . 21 HENSM - derived hiPSCs are able to colonize mouse embryos up to E17 . 5 at various anatomic regions of 22 different embryonic germ layers ( Fig . S14 - S15 ) . Specific marker staining for human nuclei excluded any 23 contamination by mouse PSC lines ( Fig . S14 ) . 24 25 Our results indicate that the contribution levels of human GFP + cells in mouse embryos , although 26 meaningful and reproducible , are relatively low ( ~ 3 % of mice are chimeric with naïve hiPSC derived 27 cells ) . Thus , we sought to develop technical platforms that can enhance such integration and also allow us 28 to better visualize integrating human cells in the developing mouse embryo . Recently , mouse ESC 29 depleted for p53 outcompete host pluripotent cells after blastocyst microinjection by cell competition 30 phenomenon that has been recently documented in mice ( Dejosez et al . , 2013 ) , and can generate very 31 16 high contribution chimeras by blocking their tendency for apoptosis . Based on the latter , we tested 1 whether manipulating these pathways might similarly endow injected naïve hiPSCs with competitive 2 advantage and increase human cell chimerism in developing mouse embryos . GFP hiPSC lines were 3 targeted with CRISPR / Cas9 to generate P53 - / - cell lines ( Fig . S16 , S17 ) . Remarkably , after microinjection 4 into mouse morulas or aggregation with 2 - cell embryos ( Fig . S18 ) , a 4 - fold increase in the number of 5 chimeric animals was obtained ( Fig . 4g , Fig . S18 - S25 ) and a dramatic increase in the extent of chimerism 6 per embryo ( up to 20 % cells per embryo Fig . S25c ) . Furthermore , differentiation was validated in 7 chimaeric embryos for major developmental markers covering all three embryonic germ layers 8 overlapping extensively with human - derived GFP signal ( Fig . 4h , Fig . S18 - S25 ) . Taken together , these 9 results substantiate generation of advanced human - mouse interspecies chimaera with various engraftment 10 in many defined lineages up to E17 . 5 from HENSM based conditions . 11 WNT / ß - CATENIN and SRC / NFkB signaling are major priming pathways compromising human 12 naïve pluripotency 13 14 The results above indicate that functional naïve pluripotency in HENSM composition not only 15 relies on inhibition of ERK and PKC pathways , but also on inhibition of TNK and SRC . Depletion of any 16 of the inhibitors for these 4 pathways compromised naïve pluripotency as exemplified by X chromosome 17 inactivation in reporter female hESCs ( Fig . 5a ) . Combined depletion of TNKi and SRCi pushed human 18 PSCs toward complete loss of pluripotency within a number of passages ( Fig . 5b , S26a ) , underlining the 19 conclusions that ERKi together with simultaneous tripartite inhibition of PKCi - SRCi - TNKi are essential 20 for functional defined conditions for human naïve pluripotency . We next aimed to define the signaling 21 pathway downstream of Tankyrase inhibition that facilitate human naïve PSCs stabilization . WNT 22 signaling is a well - established promoter of rodent naive pluripotency where it leads to ß - CATENIN 23 ( ßCAT ) nuclear localization which in turn neutralizes the repression by TCF3 effector and derepresses 24 naïve pluripotency targets ( ten Berge et al . , 2011 ; Wray et al . , 2011 ) . Although some studies have 25 indicated that titrating WNT stimulation level is needed when working with human naïve cells via using 26 lower doses of GSK3 inhibitor or by adding Tankyrase inhibitor together with GSK3i ( leading to high 27 cytoplasmic ß - CAT ) ( Theunissen et al . , 2014 ; Zimmerlin et al . , 2016 ) , they concluded that WNT 28 stimulation is helpful for promoting human naïve pluripotency exactly as seen in mouse PSCs . Other 29 studies , indicated that WNT inhibition is beneficial for initial reversion of primed to naïve pluripotency , 30 17 however its continued application compromised human naive pluripotency and that 2i with titrated WNT 1 stimulation is the optimal pattern for maintaining human naive PSCs ( Bredenkamp et al . , 2019a , 2 Bredenkamp et al . , 2019b ) , and that WNT inhibition , rather than stimulation , was needed for priming and 3 capacitation of human naïve pluripotency ( Bredenkamp et al . , 2019a ) . 4 5 Given that prior studies did not rely on exacting reporter and knockout models for WNT signaling 6 components and did not use teratoma competent cell lines , we turned to generate ßCAT KO cells on naïve 7 pluripotency reporter cell lines to give a definitive answer for this fundamental question ( Fig . S27 ) . 8 ßCAT - KO hESCs maintained high levels of Δ PE - OCT4 - GFP in HENSM condition , and upon removal of 9 XAV939 , GFP level was not decreased in BCAT - KO , but only in WT ESCs ( Fig . 5c ) . Supplementing 10 naïve cells with WNT stimulator , like CHIR99021 , or depleting BCAT inhibitor ( TNKi ) , activated WNT 11 signaling in human PSCs ( Fig . 5d ) compromised Δ PE - OCT4 - GFP levels ( Fig . 5c ) , compromised their 12 domed shape like morphology ( Fig . 5e ) and their naïve - like transcriptional profile ( Fig . 3a ) . We 13 introduced tamoxifen induced ßCAT - ERT transgene into ßCAT - KO hESCs , and we noted that in human 14 Δ PE - OCT4 - GFP signal , naïve transcription profile and domed morphology were compromised upon 15 4OHT stimulation ( Fig . 5f , Fig S26b - d ) . This is in striking contrast to mouse Δ PE - Oct4 - GFP ESCs 16 expanded in N2B27 LIF conditions that upon tamoxifen treatment induced Δ PE - Oct4 - GFP reporter and 17 naive characteristic domed like morphology ( Fig . 5f ) . Similarly , while KO of Tcf3 boosts mouse naïve 18 pluripotency and alleviates the need for WNT stimulation ( Wray et al . , 2011b ) , TCF3 KO human ESCs 19 ( Fig . S28a - b ) still required WNTi to maintain their naïve identity in HENSM conditions highlighting a 20 major difference between mouse and human naïve pluripotency ( Fig . S28c - d ) . Finally , supplementing 21 HENSM conditions with CHIR compromised their ability to maintain pluripotency upon depletion of 22 DNA and RNA methylation or omitting L - Glutamine from the culture conditions ( Fig . S28e ) . Cross - 23 species comparison of in vivo mouse and human RNA - seq data of pre - implantation blastocysts shows a 24 strong signature for AXIN1 / 2 upregulation in human but not mouse ICM ( Fig . S28f ) . Collectively , these 25 findings clearly establish WNT - ßCAT signaling as a major priming agent for human , but not mouse , 26 naïve pluripotency and establish that ablation of ßCATENIN can substitute for the need for Tankyrase or 27 WNT pathway inhibition . 28 29 18 SRC inhibition has been shown previously to deplete activation of downstream effectors 1 including ERK , PKC and NFkB signaling ( Lee et al . , 2007 ; Lluis et al . , 2007 ) . Given that SRCi was 2 needed in HENSM conditions despite continued direct blocking of ERK and PKC pathways , this has led 3 us to focus on NFkB as a potential effector mediating the beneficial effect of the use of SRCi ( Torres and 4 Watt , 2008 ) . Luciferase reporter assay activity showed pronounced activity for NFkB signaling in primed 5 conditions in comparison to HENSM naïve expanded cells ( Fig . 5g ) , and activity of NFkB became 6 prominently induced upon omission of SRC in HENSM conditions ( Fig . 5g , Fig . S29a ) . The transfection 7 of dominant negative IkB (cid:0) subunit , which blocks NfKB signalling , in ßCAT - KO ; Δ PE - OCT4 - GFP 8 hESCs allowed maintenance of Δ PE - OCT4 - GFP not only in HENSM without TNKi , but also without 9 SRCi ( Fig . 5h ) . These results establish that WNT - BCAT and SRC - NFkB pathways compromise human 10 naïve pluripotency . Further , the results on Δ PE - OCT4 - GFP , RT - PCR and DNA hypomethylation 11 establish that ACTIVIN A , rather than BMP4 , supports human naïve pluripotency ( Fig . 1h , 3c and 4d - 12 e ) . The latter can be consistent with the observations that single cell - RNA - seq data on mouse and human 13 ICMs show that human ICM retains high NODAL expression but not that of BMP4 which is highly 14 expressed only in the mouse ICM ( Blakeley et al . , 2015a ; Weinberger et al . , 2015 ) . 15 16 In mouse ground state naïve conditions , LIF / Stat3 has been shown to be a booster for naïve 17 marker expression however they can be omitted without entire collapse of the naïve PSC circuit ( Ying et 18 al . , 2008b ) . By omitting LIF from HENSM conditions and by generating STAT3 KO human naïve PSCs , 19 we show that LIF can slightly boost the purity of undifferentiated cells in culture ( Fig . 5a ) and naïve 20 marker expression by RT - PCR ( Fig . S29a ) however it is dispensable and human naive PSCs can maintain 21 their naïve identity even in the absence of LIF / STAT3 signaling ( Fig . S29b - d ) as has been previously 22 shown for rodent ground state naïve PSCs in 3i conditions ( 3 inhibitors of ERK , GSK3 and FGFR ) ( Ying 23 et al . , 2008b ) . 24 25 KLF17 is functionally essential for human , but not mouse , naïve pluripotency 26 27 Analysis of chromatin accessibility revealed differences in accessibility of DNA binding proteins 28 and protein families , unique for each pluripotent configuration . Binding motifs of different TFs like 29 KLF17 and KLF4 were found to be significantly enriched ( corrected p - value ~ 0 ) in naïve chromatin 30 19 accessible loci whereas GATA family are more prone to be found in the primed condition ( Fig . 6a , Table 1 S4 ) . We initially focused on KLF17 gene , which is specifically expressed in human naïve conditions and 2 has been shown to promote ERV expression pattern in humans ( Pontis et al . , 2019 ) however the 3 essentiality of its functional importance for maintaining human naïve circuitry remains to be established . 4 We generated single and double KO ESC lines for KLF4 and KLF17 TFs ( Fig . S30 ) . Remarkably KO of 5 KLF17 led to collapse of naïve pluripotency in HENSM conditions , while cells maintain their 6 pluripotency in HENSM - ACT as measured by OCT4 - GFP reporter ( Fig . 6b ) . The latter also support the 7 notion how ACTIVIN A supplementation promotes naïve pluripotency in humans . However , double KO 8 naïve ESCs for KLF4 / KLF17 deteriorated toward differentiation and most cells lost OCT4 - GFP 9 expression in both HENSM and HENSM - ACT conditions ( Fig . 6b ) . In mice , Klf17 is not expressed in 10 the mouse ICM or ESCs , but rather only until the 4 Cell stage in vivo ( Fig . S31a ) . Newly generated Klf17 11 KO mice were viable and pups were obtained at the expected mendelian ratios and were fertile ( Fig . 12 S31b - f ) . Unlike the strong phenotype seen in human KLF17 KO ESCs in HENSM conditions , mouse 13 Klf17 KO ESCs were indistinguishable from WT cells and did not show any compromise in their naïve 14 pluripotency marker expression ( Fig 6c , Fig . S31g ) . These results establish a great dependency of human , 15 but not mouse , naïve pluripotency on KLF17 that greatly stabilizes the TF circuitry . 16 17 To better understand the role of naïve specific pluripotency factors like KLF4 and KLF17 versus 18 regulators of both naïve and primed cells like OCT4 and SOX2 , we examined the DNA binding profile of 19 the master pluripotent TF regulators OCT4 and SOX2 and naïve promoting TFs like KLF17 , KLF4 and 20 TFAP2C using ChIP - seq . KLF17 binds 12197 genes , mostly in their promoter ( 59 % in < = 1kb from TSS , 21 Fig . 6d ) , unlike OCT4 / SOX2 that bind typically to enhancers ( 72 % and 85 % in Distal intergenic regions 22 and introns , respectively ) . KLF17 binds more substantially to naïve - specific open regions ( Fig . 6e ) . 23 Interestingly , OCT4 and SOX2 , known to work as a complex ( Merino et al . , 2014 ) , show high correlation 24 between their binding targets , yet share significantly less reciprocal binding sites with other naïve specific 25 KLF17 and KLF4 ( Fig . 6f ) . The latter explain why despite the presence of OCT4 and SOX2 in human 26 naïve PSCs , they could not compensate for depletion of KLF4 or KLF17 as the latter constitute major 27 regulators of pluripotency specific genes and that are not bound by OCT4 or SOX2 ( e . g . KLF4 , DPPA3 , 28 NODAL , HORMAD1 , Fig . 6g , Table S5 ) . The latter also provides wider basis for the importance of 29 KLF17 in human naïve pluripotency . 30 20 1 2 Inhibition of NOTCH / RBPj pathway facilitates maintenance of human naïve pluripotency without 3 ERK inhibition 4 5 As has been previously shown in mice ( Choi et al . , 2017 ; Yagi et al . , 2017 ) , the use of MEK / ERK 6 inhibition is the major mediator for inducing global DNA hypomethylation which in turns leads to 7 sporadic erosion of imprinting that gets more severe with extended passaging . In mice , using alternative 8 naïve conditions that do not employ ERK inhibitor or titrating ERKi , allows isolating murine PSCs with 9 all features of naivety except from global hypomethylation ( Choi et al . , 2017 ; Yagi et al . , 2017 ) . The 10 latter murine cells are naïve by nearly all features and are capable of generate all - iPS mice with 11 contribution to the germline , and thus provide a safer route for exploiting mouse naïve PSCs . 12 13 Although erosion of imprinting was slow and sporadic on few loci in HENSM conditions and 14 only after extended passaging ( Fig . S13 ) , this may complicate the use of naïve cells in future clinical 15 applications . We thus aimed to define alternative HENSM ( a HENSM ) conditions that allow naïve hPSC 16 isolation and maintenance but without global DNA hypomethylation . Notably , recent studies showing 17 titration of ERKi in 5i / LA conditions slightly improved the chromosomal aberrancies frequency per line 18 in hESCs , still all lines had at least one genetic abnormality , and titrating ERKi levels in these conditions 19 did not abrogate global hypomethylation and global loss of imprinting at all loci ( Di Stefano et al . , 2018 ) . 20 The latter was likely because these conditions still had BRAF inhibitor which is upstream to MEK / ERK 21 pathway and contributes to the rapid and radical DNA hypomethylation induced in these cells ( Pastor et 22 al . , 2016 ) . 23 24 Withdrawal or partial ( 33 % ) titration of ERKi from HENSM compromised the naivety of human 25 ESC as evident by a decrease in Δ PE - OCT4 - GFP levels and loss of X - reactivation state in most of the 26 cells within the expanded population ( Fig . 7a - b ) . Supplementing ACTIVIN A upon omission of ERKi 27 from HENSM conditions did not block loss of X reactivation in female cell lines ( Fig . 7d – lower 28 panels ) . Thus , we set out to screen for added compounds that would enable maintenance of pre - x 29 inactivation upon omitting or titrating MEKi / ERKi ( Fig . 7c ) . Remarkably , we noted that adding of 30 21 gamma secretase inhibitor DBZ , which blocks NOTCH pathway ( Ichida et al . , 2014 ) , allowed robust and 1 feeder free maintenance of human naïve cells when ERKi was omitted ( 0 HENSM conditions ) or titrated 2 down from 1 μ M to 0 . 33 μ M termed ( t HENSM conditions ) ( Fig . 7d - e ) 3 4 Human PSCs expanded in aHENSM conditions ( 0HENSM and tHENSM ) maintain Δ PE - OCT4 - 5 GFP signal equivalent to HENSM conditions ( Fig . 7f ) , and maintained pre - X inactivation state in female 6 cell lines ( Fig . 7d ) . RT - PCR analysis showed that the lines expressed naïve pluripotency markers ( Fig . 7 7h ) , although levels were less induced in comparison to when ERKi was used . Global gene expression 8 analysis showed that the cells clustered with HENSM naïve PSCs rather than primed PSCs ( Fig . 7i , Fig 9 S32a ) . At the functional levels , the cells showed no enhanced tendency for obtaining chromosomal 10 abnormalities and were competent in differentiating into PGCLCs in vitro ( Fig . S32e ) and making 11 teratomas without any need for priming in vitro before injections ( Fig . S6a ) . Cells could be maintained 12 upon depletion of DNMT1 , METTL3 or exogenous L - glutamine and these qualities depended on the 13 presence of DBZ ( Fig . S32b - d ) . WGBS analysis showed that these alternative 0HENSM and tHENSM 14 conditions did not upregulate DNMT3L enzyme and did not show trends of global hypomethylation that 15 were seen in HENSM or HENSM - ACT even after extended passaging ( Fig . 4d - e , Fig . S33a ) and 16 consistently did not show accelerated loss of imprinting compared to parental primed cells Fig . S33b - c ) . 17 Generation of RBPj KO hESCs ( Fig . S34 ) allowed expanding human naïve PSCs in tHENSM and 18 0HENSM without adding DBZ , proving that NOTCH / RBPj ( Castel et al . , 2013 ) is the main mediator of 19 dismantling naïve pluripotency in human cells when ERKi is depleted ( Fig . 7j - k ) . These results show 20 that , equivalent to what was obtained in rodent in vitro PSCs with t2iL and a2iL mouse conditions that 21 endow naïve features in murine PSCs without compromising global DNA methylation and imprinting 22 regulation ( Choi et al . , 2017 ; Yagi et al . , 2017 ) , this can now be obtained also with human naïve - like 23 PSCs . 24 25 Discussion 26 27 The findings reported herein consolidate the previously raised notion that different genetic 28 backgrounds assume distinct states of pluripotency in vitro , the stability of which is regulated by 29 endogenous genetic determinants that remain unknown , and can be modified by defined exogenous 30 22 factors that support the naïve ground state of pluripotency ( Reik and Surani , 2015 ) . The stringency in 1 requirement for these exogenous factors appears to be different among distinct species , as exemplified 2 by the requirement for simultaneous inhibition of SRC , PKC , NOTCH and multiple MAPK kinase 3 pathways in human naïve cells . However , certain pathway like WNT and ACTIVIN signaling are 4 divergent between mice and humans , as only in humans WNT inhibition and ACTIVIN stimulation 5 promote naïve pluripotency and contribute to the unique molecular and epigenetic configuration of 6 naïve pluripotency described herein . The molecular basis for the latter findings remains to be 7 mechanistically defined . Our results also suggest that epigenetic priming of human pluripotent cells is 8 predominantly caused by supplementation of WNT and FGF . 9 10 The above results on the requirement for integrated WNT - SRC - PKCi to maintain human naive 11 pluripotency can explain why previous described conditions that applied only some of these three 12 essential components had compromised and unstable naïve pluripotency features . For example , Zambidis 13 and colleagues indicated Tankyrase to promote human PSCs in ERKi containing conditions , however did 14 not include PKCi , SRCi and still used WNT stimulation ( Zimmerlin et al . , 2016 ) , which yields cells with 15 low Δ PE - OCT4 - GFP signal and lack of X reactivation . Similarly , conditions describing extended 16 potential cells in mouse or humans did not utilize PKCi or SRCi and still used WNT activation together 17 with TANKYRASE inhibition ( Yang et al . , 2017 ) , explaining again why these cells have unique 18 characteristics , they fail to contain naïve pluripotency defining features and cannot sustain stable in vitro 19 naive pluripotency independent of repressive DNA and RNA methylation marks . 5iLA conditions did not 20 include PKCi and did not actively inhibit WNT pathway ( Theunissen et al . , 2014 ) . 21 22 Equivalent to what has been previously established for rodent ESCs in 3i conditions ( Ying et al . , 23 2008 ) , we now establish the ability to expand transgene independent human naïve PSCs independent of 24 any exogenous cytokine supplementation ( i . e . without LIF or ACTIVIN ) and by only inhibiting human 25 PSC autologous driven signaling pathways with small molecule inhibitors . We can also expand bona 26 fide human naïve pluripotent cells by omitting MEK / ERK inhibitors . It will be important to decipher 27 the downstream regulators of pluripotency influenced by NOTCH / RBPj , SRC - NFkB and PKC 28 pathways . We cannot exclude that alternative growth conditions may be devised to capture human 29 naïve pluripotent cells with features similar to those described herein , or that HENSM conditions might 30 23 be modified for improving and consolidating the extent of naïve features in human pluripotent cells . 1 Indeed , X reactivation in the naïve conditions described here in still cannot yield random inactivation 2 upon differentiation of human PSCs and this is similar to previously generated conditions ( Sahakyan et 3 al . , 2016 ) . 4 5 The fact that murine naïve cells , rather than primed cells , are tolerant to ablation of epigenetic 6 repressors supports the concept of naïve pluripotency as a synthesis with a relatively minimal 7 requirement for epigenetic repression ( in comparison to primed pluripotent and somatic cells ) ( Geula et 8 al . , 2015 ; De Los Angeles et al . , 2015 ) . In this study , conditional mutants for METTL3 and DNMT1 9 were used for optimizing more stringent human naïve pluripotency growth conditions . Further , our 10 results in mice and human indicate that the ability to tolerate complete and permanent ablation of such 11 repressors may be one of the key features of naïve pluripotency across different species and might 12 prove an efficient method for defining ground state of pluripotency from other mammalian species in 13 vitro ( e . g . monkeys , bovine , swine ) . 14 15 16 17 Acknowledgements 18 19 This work was funded by Pascal and Ilana Mantoux ; Nella and Leon Benoziyo Center for 20 Neurological Diseases ; David and Fela Shapell Family Center for Genetic Disorders Research ; Kekst 21 Family Institute for Medical Genetics ; Helen and Martin Kimmel Institute for Stem Cell Research ; Flight 22 Attendant Medical Research Council ( FAMRI ) ; Helen and Martin Kimmel Award for Innovative 23 Investigation ; Dr . Beth Rom - Rymer Stem Cell Research Fund ; Edmond de Rothschild Foundations ; 24 Zantker Charitable Foundation ; Estate of Zvia Zeroni ; European Research Council ( ERC - CoG ) ; Israel 25 Science Foundation ( ISF ) ; Minerva ; Israel Cancer Research Fund ( ICRF ) and BSF . We thank Dr . Rada 26 Massarwa for help in setting up roller culture incubators . The experimental setting established here can be 27 purchased from Arad Technologies Ltd . , Ashdod , Israel . We thank D . Trono and J . Pontis for TE 28 analysis , N . Benvenisty and S . Bar for RNA - seq based karyotyping and SNP analysis for LOI , R . 29 Jaenisch for sharing WIBR2 X chromosome reporter cell lines , A . Meissner for sharing HUES64 with 30 24 DNMT1 TET - OFF reporter cell line and constructs , A . Smith for H9 - NK2 reset cell line , R . Massarwa for 1 technical advice on embryo imaging , Q . 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Development138982 - 78 . 32 33 34 35 36 37 38 39 40 41 42 43 44 45 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 METHODS 25 HENSM conditions 26 The following serum free conditions , termed HENSM ( H uman E nhanced N aïve S tem cell 27 M edium ) were used to isolate , generate , derive and stabilize naïve human pluripotent stem cells ( iPSCs 28 and ESCs ) with the unique biological properties described in this study . HENSM medium was generated 29 by including : 240 ml Neurobasal ( ThermoFisher 21103049 ) and 240ml of DMEM - F12 without HEPES 30 ( ThermoFisher 21331020 ) , 5 ml N2 supplement ( ThermoFisher 17502048 or in - house prepared ) , 5mL 31 GlutaMAX ( ThermoFisher 35050061 ) , 1 % nonessential amino acids ( BI 01 - 340 - 1B ) , 1 % Penicillin - 32 Streptomycin ( BI 03 - 031 - 1B ) , 10ml B27 supplement ( Invitrogen 17504 - 044 or in - house made or 33 Xenofree B27 Invitrogen A1486701 ( for XF conditions ) ) , 0 . 8mM Dimethyl 2 - oxoglutarate ( aKG ) ( add 34 60µL from purchased solution from Sigma 349631 ) , Geltrex ( Invitrogen A1413202 / A1413302 - add 1ml 35 rapidly in media to obtain 0 . 2 % final conc ) , 50µg / ml Vitamin C ( L - Ascorbic acid 2 - phosphate 36 sesquimagnesium salt hydrate - Sigma A8950 ) , 20 ng / ml recombinant human LIF ( Peprotech 300 - 05 ) and 37 the following small molecule inhibitors : MEKi / ERKi ( PD0325901 1 µM - Axon Medchem 1408 ) ; 38 31 WNTi / TNKi ( IWR1 - endo 5µM - Axon Medchem 2510 or XAV939 2µM - Axon Medchem 1527 ) ; 1 P38i / JNKi ( BIRB0796 0 . 8µM - Axon Medchem 1358 ) , PKCi ( Go6983 2µM - Axon Medchem 2466 ) , 2 ROCKi Y27632 ( 1 . 2µM - Axon Medchem 1683 ) , SRCi ( CGP77675 1 . 2µM – Axon Medchem 2097 ) . For 3 HENSM - ACT , the latter HENSM conditions were supplemented with 4 - 20 ng / ml recombinant human 4 ACTIVIN A ( Peprotech 120 - 14E ) . We avoided using β - mercaptoethanol or adding additional BSA . Cells 5 in HENSM were grown on 1 % Growth factor reduced Matrigel ( BD ) or 1 % GELTREX ( ThermoFisher 6 A1413202 or A1413302 ) or Biolaminin - 511 ( Biolamina Inc ; including for HENSM - XF conditions ) 7 coated plates for at least 1 hour in 37 o C . Notably , coated plates with 0 . 2 % gelatin and irradiated ICR - DR4 8 MEFs can also be used to maintain human naïve cells in HENSM conditions . Enhanced single cell 9 cloning efficiency can be obtained with HENSM supplementation with additional 5µM of Y - 27632 10 ROCKi for 24 hours after cell passaging . TrypLE ( ThermoFisher 12604013 or 12604054 ) ( diluted into 11 0 . 5X - EDTA 0 . 75mM ) is optimal for single cell passaging ( 2mL per 10cm dish , 0 . 75ml per 6 well - for 5 12 min at 37°C ) . After removal of TrypLE and air - drying at RT for 3 min . , cells are resuspended in PBS . 13 Rapid 0 . 05 % trypsinization ( 0 . 5 - 1 min at 37C ) is optimal for passaging as small clumps . Longer 0 . 05 % 14 trypsinization for 3 - 5 minutes at 37°C is optimal for microinjection into as the cells come out less sticky . 15 Harvested cells should be centrifuged for 4 min x 1300RPM . Cells were expanded in 5 % O 2 and 5 % CO 2 16 conditions and passaged every 4 - 5 days . Medium was freshly replaced every 24 hours . Assembled media 17 can be used for up to 7 days after preparation if kept at 4C . Passage numbers of naïve - hiPSC / hESCs 18 indicate number of passages counted after induction or stabilization of the naïve state ( and do not include 19 previous passages when the cells were established and maintained in other conditions ) . For transfection of 20 mouse and human naïve iPSCs / ESCs , 10 million cells were harvested with 0 . 05 % trypsin - EDTA solution 21 ( Invitrogen ) , and cells resuspended in PBS + / + were transfected with 100 - 150 μ g DNA constructs ( Gene 22 Pulser Xcell System ; Bio - Rad ; 220V , 25 μ F , square wave , 20 ms , 4 mm cuvettes ) . Cell lines were 23 monthly checked for Mycoplasma contaminations ( LONZA – MYCOALERT KIT ) , and all samples 24 analyzed in this study were not contaminated . Instead of adding 5ml of commercially available N2 25 supplements , individual components can be added to 500 ml media bottle at the indicated final 26 concentrations : 1 ) Recombinant Human Insulin ( Sigma I - 1882 ) – 12 . 5 µg / ml final concentration ; 2 ) Apo - 27 Transferrin ( Sigma T - 1147 ) – 500 µg / ml final concentration ; 3 ) Progesterone ( Sigma P8783 ) – 0 . 02 28 µg / ml final concentration ; 4 ) Putrescine ( Sigma P5780 ) – 16 µg / ml final concentration ; 5 ) Sodium 29 Selenite ( Sigma S5261 ) - add 5 µL of 3 mM stock solution per 500ml HENSM media . For 0HENSM 30 32 conditions – PD0325901 was omitted and replaced with DBZ ( 0 . 15 - 0 . 3 µM – TOCRIS 4489 ) . For 1 tHENSM conditions titrate concentration of ERKI were used ( 0 . 33microM PD0325901 - Axon Medchem 2 1408 ) together with DBZ ( 0 . 15 - 0 . 3 µM – TOCRIS 4489 ) . Previously described NHSM conditions ( Gafni 3 et al . , 2013a ) were assembled as follows : 240 ml Neurobasal ( ThermoFisher 21103049 ) and 240ml of 4 DMEM - F12 without HEPES ( ThermoFisher 21331020 ) , 5 ml N2 supplement ( ThermoFisher 17502048 5 or in - house prepared ) , 5mL GlutaMAX ( ThermoFisher 35050061 ) , 1 % nonessential amino acids ( BI 01 - 6 340 - 1B ) , 1 % Penicillin - Streptomycin ( BI 03 - 031 - 1B ) , 10ml B27 supplement ( Invitrogen 17504 - 044 ) , 7 50µg / ml Vitamin C ( L - Ascorbic acid 2 - phosphate sesquimagnesium salt hydrate - Sigma A8950 ) , 20 8 ng / ml recombinant human LIF ( Peprotech 300 - 05 ) and the following small molecule inhibitors : 9 MEKi / ERKi ( PD0325901 1 µM - Axon Medchem 1408 ) ; CHIR99021 ( 0 . 3 - 1µM - Axon Medchem 2510 ) ; 10 P38i / JNKi ( BIRB0796 2µM - Axon Medchem 1358 ) , PKCi ( Go6983 2µM - Axon Medchem 2466 ) , 11 ROCKi Y27632 ( 1 . 2µM - Axon Medchem 1683 ) , 20 ng / ml recombinant human ACTIVIN A ( Peprotech 12 120 - 14E ) and 8ng / ml bFGF ( Peprotech ) . 13 14 Small molecule compounds and cytokines used in small scale screens 15 Small molecules and cytokines were supplemented as indicated in screening related figure legends : 16 recombinant human BMP4 ( 10ng / ml , Peprotech ) , BMPRi ( LDN193189 0 . 3µM , Axon Medchem 1509 ) , 17 recombinant human IGF1 ( 10ng / ml , Peprotech ) , Forskolin ( FK , 10µM , TOCRIS ) , FGFR inhibitor 18 PD173074 ( 0 . 1µM , TOCRIS ) , TGFRßi / ALK inhibitor A83 - 01 ( 0 . 3µM , Axon Medchem 1421 ) , 19 Kenopaullone ( KP , 5µM , Sigma Aldrich ) , DOT1L inhibitor ( SGC0946 0 . 5µM , SigmaAldrich SML1107 ) , 20 recombinant human SCF ( 10ng / ml , Peprotech ) , SRCi CGP77675 ( CGP77675 1µM – Axon Medchem 21 2097 ) , TNKi ( IWR1 - endo 4µM - Axon Medchem 2510 ) , Gamma Secretase / NOTCH pathway inhibitor 22 ( DBZ 0 . 35µM – Axon Medchem 1488 ) , BRAFi ( SB590885 , 0 . 5µM , Axon Medchem 2504 ) , ERK5i 23 ( BIX02189 5µM , Axon Medchem 1809 ) , SHHi ( RU - SKI43 3µM , Axon Medchem 2035 ) , G9a inhibitor 24 ( BIX01294 0 . 5µM – Axon Medchem 1692 ) , Medium with inhibitors was replaced every 24 hours . 25 Doxycycline ( DOX - SigmaAldrich D9891 ) was used at a final concentration of 2µg / ml and refreshed 26 every 48 hours upon media exchange . For exogenous L - GLUTAMINE withdrawal assay ( “no GLUT 27 assay” ) ( Carey et al . , 2015b ) , media indicated were used without addition of non - essential amino acids , 28 without L - Glutamax , without added aKG , and without Matrigel / Geltrex as some of these components can 29 introduce L - Glut precursors as well . Laminin511 or Gelatin & irradiated feeders were used as coating 30 33 material for no - GLUT assay . 1 2 ESC derivation from human blastocysts in HENSM conditions 3 The use of human preimplantation embryos for ESC derivation was performed in compliance 4 with protocols approved by LIS hospital ESCRO committee , Lis hospital Institutional review committee 5 and Israeli National Ethics Committee ( 7 / 04 - 043 ) and following the acceptance of a written informed 6 consent . The couples ' participation in the study was voluntary after signing informed consent forms and 7 there was no monetary compensation for their embryo donation . Inner cell masses ( ICMs ) were isolated 8 mechanically by laser - assisted micromanipulation from spare IVF embryos , at day 6 - 7 following 9 fertilization . The intact ICM clumps were placed on a feeder cell layer of irradiation treated DR4 mouse 10 embryonic fibroblasts and cultured in HENSM or HENSM - ACT media . Initial outgrowths of 11 proliferating ESCs were evident by days 4 - 8 , and were trypsinized into single cells , 6 - 10 days following 12 ICM plating . The newly established cell lines were further propagated by 0 . 05 % trypsin and then either 13 frozen or used for further analysis . 14 15 Culture of conventional / primed human ESCs and iPSCs 16 The following already established conventional human ESCs and iPSC lines were used 17 ( indicated passage number of the cell line taken for conversion into naïve pluripotency is indicated in 18 parentheses ) : Human induced pluripotent stem cells C1 ( P25 ) hiPSC lines and the human embryonic 19 stem cell ( hESC ) lines H1 ( P42 ) , H9 ( P39 ) , WIBR1 ( P51 ) , WIBR2 ( P44 ) , WIBR3 ( P42 ) , HUES64 , LIS1 , 20 WIS1 , LIS2 hESCs were maintained in 5 % O2 conditions ( unless indicated otherwise ) on irradiated 21 mouse embryonic fibroblast ( MEF ) feeder layers or Matrigel coated plates as indicated . Two conventional 22 and established conditions were used as indicated throughout the figures and manuscript : TeSR 23 conditions on Matrigel coated plates ( Stem Cell Technologies ) or FGF / KSR conditions on MEF substrate : 24 DMEM - F12 ( Invitrogen 10829 ) supplemented with 15 % Knockout Serum Replacement ( Invitrogen 25 10828 - 028 ) , 1mM GlutaMAX ( Invitrogen ) , 1 % nonessential amino acids ( Invitrogen ) , 1 % Sodium - 26 pyruvate ( BI 03 - 042 - 1B ) , 1 % Penicillin - Streptomycin ( BI 03 - 031 - 1B ) , 0 . 1mM β - mercaptoethanol 27 ( Sigma ) , and 8ng / mL bFGF ( Peprotech ) and 2ng / ml recombinant human TGF β 1 ( Peprotech ) . Cultures 28 34 were passaged every 5 – 7 days either manually or by trypsinization ( 24 hours pre and 24 hours after 1 addition of ROCK inhibitor at 5 - 10µM concentration ) . For transfection of hiPSC and hESC lines , cells 2 were cultured in Rho kinase ( ROCK ) inhibitor ( Y27632 ) 24 h before electroporation . Primed human ESC 3 and iPSC cells were harvested with 0 . 25 % trypsin - EDTA solution ( Invitrogen ) , and cells resuspended in 4 PBS were transfected with 75 μ g DNA constructs ( Gene Pulser Xcell System ; Bio - Rad ; 250 V , 500 μ F , 5 0 . 4 - cm cuvettes ) . Cells were subsequently plated on MEF feeder layers ( DR4 MEFs for puromycin or 6 neomycin selection ) in hESC medium supplemented with ROCK inhibitor for the first 24 hours , and then 7 antibiotic selection was applied . Previously described 5iLA or 4iLA were assembled and applied as 8 previously described ( Theunissen et al . , 2016 ) . T2iL - Go reset and PXGL conditions were used and 9 assembled as previously described ( Brendenkamp et al . , 2019a ; Takashima et al . , 2014b ) . Media for 10 extended potential stem cells or region specific primed cells were also devised where indicated as 11 originally reported ( Wu et al . , 2015 ; Yang et al . , 2017 ) . 12 13 Mouse naïve and primed stem cell lines and cultivation 14 Murine naïve V6 . 5 ESCs ( C57B6 / 129sJae ) pluripotent cells were maintained and expanded in 15 serum - free chemically defined N2B27 - based media : 240 ml Neurobasal ( ThermoFisher 21103049 ) and 16 240ml of DMEM - F12 with HEPES ( SIGMA D6421 ) , 5ml N2 supplement ( Invitrogen ; 17502048 ) , 5ml 17 B27 supplement ( Invitrogen ; 17504044 ) , 2mM glutamine ( Invitrogen ) , 1 % nonessential amino acids 18 ( Invitrogen ) , 0 . 1mM β - mercaptoethanol ( Sigma ) , 1 % penicillin - streptomycin ( Invitrogen ) , 5mg / ml BSA 19 ( Sigma ) . Naïve 2i / LIF conditions for murine PSCs included 20 ng / ml recombinant human LIF ( in - house 20 made ) . Where indicated 2i was added : small - molecule inhibitors CHIR99021 ( CH , 3 μ M - Axon 21 Medchem ) and PD0325901 ( PD , 1 μ M – Axon Medchem 1408 ) . Murine naïve ESCs and iPSCs were 22 expanded on gelatin - coated plates , unless indicated otherwise . Primed 129Jae EpiSC line ( derived from 23 E6 . 5 embryos ) or C57BL6 / 129sJae EpiSC line ( derived following in vitro priming of V6 . 5 mouse ESCs ) 24 were expanded in N2B27 with 12ng / ml recombinant human bFGF ( Peprotech ) and 20ng / ml recombinant 25 Human ACTIVIN A ( Peprotech ) . Murine EpiSC were expanded on feeder free Matrigel coated plates . 26 Cell lines were routinely checked for Mycoplasma contaminations every month ( LONZA – 27 MYCOALERT KIT ) , and all samples analyzed in this study were not contaminated . 28 29 35 Reprogramming of somatic cells and cell infection 1 Virus - containing supernatants of the different reprogramming viruses : STEMCCA - OKSM 2 polycistronic vector ( DOX inducible and constitutive expression ) was supplemented with the FUW - lox - 3 M2rtTA virus ( when necessary ) and an equal volume of fresh culture medium for infection . For 4 derivation of iPSCs directly from fibroblasts ( and not from already established iPSC lines ) , fibroblasts 5 were grown in the presence of DOX in HENSM conditions on Matrigel - coated plates until initial iPSC 6 colonies were observed and subcloned . Generation of JH1 hiPSCs from human PBMCs was conducted by 7 OSKM Sendai virus Cyto - Tune - iPS2 . 0 Kit according to manufacturer’s instructions but in HENSM 8 conditions applied starting from day 4 of the reprogramming process . 9 Sources of cell lines and plasmids 10 WIBR3 - OCT4 - GFP knock in reporter ESCs and Δ PE - OCT4 - GFP WIBR3 hESCs were 11 previously described ( Theunissen et al . , 2016 ) . 29 - 8 WIBR2 and 29 - 9 WIBR2 hESCs reporters for X 12 chromosome reactivation were previously described ( Theunissen et al . , 2016 ) . Targeting strategy and 13 CRIPS / Cas9 used for targeting of human ESCs are delineated in supplementary figures . Correct targeting 14 was validated by southern blot analysis and normal karyotype was revalidated before use of all generated 15 new lines or subclones . Klf17 - / - mouse ESCs ( and its littermate control WT ESC ) were derived from 16 blastocysts obtained following mating of Klf17 - / - adult mice . ßCAT - ERT transgenic construct and ßCat - 17 KO mESCs were previously described and validated ( Kim et al . , 2013 ) . pBabe - Puro - IKBalpha - mut 18 ( super - repressor ) ( dominant negative mutant ) ( Addgene 15291 ) and its control pBabe - Puro - IKBalpha - 19 WT ( Addgene 15290 ) were used to generate transgenic lines analyzed in Fig . 5h . TET - OFF DNMT1 20 HUES64 hESC line was previously described and validated ( Liao et al . , 2015 ) . H9 hNanog - pGZ hESC 21 that contains NANOG - GFP reporter was obtained from WiCell . 22 23 Mouse embryo micromanipulation and imaging 24 Human naïve pluripotent iPSCs were treated with 20 μ M ROCKi the night before injection and 25 trypsinized and microinjected into E2 . 5 BDF2 diploid morulas or aggregated with 2 - Cell BDF2 embryos , 26 and were subsequently allowed to develop in vitro until E3 . 5 ( in KSOM medium ) . Microinjection into 27 E2 . 5 morulas placed in M16 medium under mineral oil was done by a flat - tip microinjection pipette . A 28 36 controlled number of 4 - 8 cells were injected into the morulas . After injection , morulas were returned to 1 KSOM media ( Invitrogen ) supplemented with 10 - 20 um ROCKi for 4h , then transferred to KSOM 2 without ROCKi and placed at 37 o C until transferred to recipient females at E3 . 5 . Ten to fifteen injected 3 blastocysts were transferred to each uterine horn of 2 . 5 days post coitum pseudo - pregnant females . 120 - 4 150 embryos were obtained and transferred for each injection batch . Embryos were dissected at the 5 indicated time points and imaged for GFP + cell localization . The latter experiments were approved by 6 Weizmann Institute IACUC ( 00330111 - 2 ) and by the Weizmann Institute Bioethics and ESCRO 7 committee . In Toto confocal imaging of chimeric mouse embryos was conducted after that the embryos 8 were removed from the uterus into Tyrode’s solution , the decidua removed and the embryo carefully 9 isolated with yolk sac intact . Embryos were moved into a droplet of culture media on a paper filter 10 attached to a coverslip with vacuum grease . Starting farthest away from the embryo , the yolk sac was 11 gently peeled open and lightly pressed onto the paper filter , which has adherence qualities , to anchor the 12 embryo to the filter . To expose the tissue of interest , the embryo can be stretched and lightly pressed onto 13 the filter . After mounting the embryos , a glass - bottomed dish was placed over the embryos using vacuum 14 grease drops for spacing . The dish was inverted , filled with culture media and placed in the imaging 15 chamber on an inverted Zeiss confocal LSM700 microscope . No blinding or randomization was 16 conducted when testing outcome of microinjection experiments . Mounted embryos were placed in a heat - 17 and humidity - controlled chamber ( 37°C ; 5 % O2 , 5 % CO2 and 90 % N2 gas mixture ) on a motorized stage 18 of an inverted Zeiss LSM700 confocal microscope . Images were acquired with a 20x / 0 . 8 M27 lens with 19 0 . 5 - 0 . 6x digital zoom - out or 10x / 0 . 3 Ph1 M27 lens with 0 . 5 - 0 . 6x digital zoom - out . For GFP detection , a 20 448 - nm laser ( 30 - 50 % power ) was used . Orange - Tracker ( Molecular Probes , cat # C2927 . Used at 20 21 μ M ) was excited by a 555 - nm laser ( 30 % power ) . Hoechst ( Sigma , cat # B2261 , used at 10 μ g / ml ) was 22 excited by a 405 - nm laser ( 40 % power ) . RRX dyes were excited by 594 laser ( 20 % power ) and far - red 23 dyes were excited by 647 laser ( 30 % power ) . Images were acquired at 1024x1024 resolution . The 24 thickness of z - slices appears in the relevant figure legends . 25 26 Imaging , quantifications , and statistical analysis 27 Images were acquired with D1 inverted microscope ( Carl Zeiss , Germany ) equipped with DP73 28 camera ( Olympus , Japan ) or with Zeiss LSM 700 inverted confocal microscope ( Carl Zeiss , Germany ) 29 equipped with 405nm , 488nm , 555nm and 635 solid state lasers , using a 20x Plan - Apochromat objective 30 37 ( NA 0 . 8 ) . All images were acquired in sequential mode . For comparative analysis , all parameters during 1 image acquisition were kept constant throughout each experiment . Images were processed with Zen blue 2 2011 software ( Carl Zeiss , Germany ) , and Adobe Photoshop CS4 . 3 4 Immunohistochemistry analysis of mouse embryos 5 Mouse embryos were harvested at the indicate time points , washed once in 1xPBS and fixated 6 overnight in 4 % PFA . Thereafter , embryos were washed three times in 1xPBS and dehydrated in 7 sequential Ethanol dilutions . After standard paraffinization and embedding , tissue blocks were sectioned 8 on a microtome and mounted onto Superfrost plus slides ( Thermo Scientific , Menzel - Glaser ) , dried at 38º 9 C for 4 hours and stored at 4º C until further processing . Tissue sections were stained for following 10 indicated antibodies : goat anti - GFP ( Abcam ab6673 ) , chicken anti - GFP ( Abcam ab13970 ) , rabbit anti - 11 Numa ( Abcam ab84680 ) , mouse anti - human nuclei ( Millipore MAB1281 ) , mouse anti - TUJ1 ( Covance 12 MMS 435P ) , goat anti - SOX2 ( R & D AF2018 ) , goat anti - SOX17 ( R & D AF1924 ) . All secondary 13 antibodies were used from Jackson ImmunoResearch including donkey anti - rabbit Biotin and 14 Streptavidin - Cy3 for signal enhancement . For PFE - staining , sections were rehydrated , antigen retrieved in 15 Sodium citrate buffer and pressure cooker , rinsed in PBS and treated with 0 . 3 % H2O2 to reduce 16 background staining . After permeabilization in 0 . 1 % Triton X - 100 in PBS three times for 2 min . , samples 17 were blocked in 10 % normal donkey serum in PBS in humidified chamber for 20 min . at RT . Slides were 18 then incubated in the appropriate primary antibody diluted in 1 % BSA in 0 . 1 % Triton X - 100 at 4 °C 19 overnight . Sections were then washed three times ( 5 min each ) in washing buffer ( 0 . 1 % Triton X - 100 in 20 PBS ) incubated with appropriate fluorochrome - conjugated secondary antibodies diluted in 1 % BSA in 21 0 . 1 % Triton X - 100 at RT for 1 h in the dark . For human cell specific NUMA staining , signal 22 enhancement was performed by usage of Biotin - SP conjugated 2nd antibody for 30 min and subsequent 23 incubation with Streptavidin - Cy3 antibody for 30 min . All sections were washed three times in washing 24 buffer for 10 min each , counter stained with DAPI for 20 min , rinsed twice in washing buffer and 25 mounted with Shandon Immuno - Mount ( Thermo Scientific , 9990412 ) . For OCT - staining , embryos were 26 fixated o / n in 4 % PFA at 4°C , washed three times in PBS for 10 min each and submerged first in 15 % 27 Sucrose / PBS and then 30 % Sucrose o / n at 4°C . The day after , samples were subjected to increasing 28 gradient of OCT concentration in Sucrose / PBS followed by embedding in OCT on dry ice and stored at - 29 80°C until further processing . Cryoblocks were cut with LEICA CM1950 and washed once with 1xPBS 30 38 and incubated with 0 . 3 % H 2 O 2 for 20 min . After permeabilization with 0 . 1 % Triton X - 100 in PBS for 10 1 min . , slides were again washed three times with 1xPBS for 2 min . each and blocked with blocked in 10 % 2 normal donkey serum in PBS in humidified chamber for 20 min . at RT . Antibody incubations were 3 carried out analogously to PFE stainings . All images were collected on a Zeiss ( Oberkochen , Germany ) 4 LSM700 confocal microscope and processed with Zeiss ZenDesk and Adobe Photoshop CS4 ( Adobe 5 Systems , San Jose , CA ) . For FACS analysis of mouse embryos , they were harvested and washed once in 6 PBS . Thereafter , they were trypsinized in 0 . 05 % trypsin / EDTA for 5 min and filtered through a cell 7 strainer in MEF - medium before subjected to FACS analysis using FACS ARIA III analyzer and sorter 8 system . Non - injected mouse embryos were used for proper gating and exclusion of auto - fluorescence . 9 10 Luciferase activity assays 11 WNT TOP - Flash reporter constructs ( ten Berge et al . , 2011 ) were used to determine the 12 regulation pattern of WNT signalling activity and were electroporated into 0 . 5 – 3 × 10−6 cells along 13 with the pRL - TK ( Renilla ) vector for normalization . Assays were performed 48 h later using the 14 Dual - Glo Luciferase Assay System ( Promega ) . The basal activity of the empty luciferase vector was 15 set as 1 . 0 . Luciferase assay for NF - kB Reporter activity was measured with NF - kB Reporter kit 16 ( BPS Bioscience # 60614 ) according to manufacturer instructions and following using 17 Lipofectamine Stem Transfection reagent ( ThermoFisher Scientific ) . 18 RT - PCR analysis 19 Total RNA was isolated using Trizol ( Ambion life technologies ) and phenol - chloroform 20 extraction . 1 μ g of total RNA was reverse transcribed using a High - Capacity Reverse Transcription Kit 21 ( Applied Biosystems ) and ultimately re - suspended in 200 µl of water . Quantitative PCR analysis was 22 performed in triplicate using 1 / 100 of the reverse transcription reaction in a Viia7 platform ( Applied 23 Biosystems ) . Error bars indicate standard deviation of triplicate measurements for each measurement . RT - 24 PCR primers used herein are : XIST - Forward : GGGTTGTTGCACTCTCTGGA ; XIST - Reverse : 25 TCATTCTCTGCCAAAGCGGT ; OCT4 - Forward : GCTCGAGAAGGATGTGGTCC ; OCT4 - Reverse : 26 CGTTGTGCATAGTCGCTGCT ; SOX1 - Forward : GGGAAAACGGGCAAAATAAT ; SOX1 - Reverse : 27 TTTTGCGTTCACATCGGTTA ; PAX6 - Forward : TGTCCAACGGATGTGTGAGT ; PAX6 - Reverse : 28 39 TTTCCCAAGCAAAGATGGAC ; ZIC2 - Forward : TGCCTCATAAAAAGGAACAC ; ZIC2 - Reverse : 1 TGTCCATTTGTAAAACTCCG ; NANOG - Forward : GCAGAAGGCCTCAGCACCTA ; NANOG - 2 Reverse : AGGTTCCCAGTCGGGTTCA ; DNMT3L - Forward : TGAACAAGGAAGACCTGGACG ; 3 DNMT3L - Reverse : CAGTGCCTGCTCCTTATGGCT ; KHDC1L - Forward : 4 ACGAGTGCTCTCAGCAAGGA ; KHDC1L - Reverse : GTACGTGTCATCAAGTCCGAAGA ; ARGFX - 5 Forward : CCGGAGTCAACAGTAAAGGTTTG ; ARGFX - Reverse : GGTGGGCACATTCTTCTTGGA ; 6 KLF17 - Forward : AGCAAGAGATGACGATTTTC ; KLF17 - Reverse : GTGGGACATTATTGGGATTC ; 7 TFCP2L1 - Forward : TCCTTCTTTAGAGGAGAAGC ; TFCP2L1 - Reverse : 8 ACCAACGTTGACTGTAATTC ; KLF4 - Forward : CGCTCCATTACCAAGAGCTCAT ; KLF4 - Reverse : 9 CACGATCGTCTTCCCCTCTTT ; STELLA - Forward : CGCATGAAAGAAGACCAACAAACAA ; 10 STELLA - Reverse : TTAGACACGCAGAAACTGCAGGGA ; DPPA5 - Forward : 11 TGCTGAAAGCCATTTTCG ; DPPA5 - Reverse : GAGCTTGTACAAATAGGAGC ; OTX2 - Forward : 12 CATCTGATCAAAGTTCCGAG ; OTX2 - Reverse : TTAAGCAGATTGGTTTGTCC ; PRDM14 - Forward : 13 CCTGACACTTTAATTCCACC ; PRDM14 - Reverse : AAGGTAATAACTGGGAGGTC ; FGF4 - Forward : 14 AGAATGGGAAGACCAAGAAG ; FGF4 - Reverse : TGCATTAAACTCTTCATCCG ; REX1 - Forward : 15 GGAATGTGGGAAAGCGTTCGT ; REX1 - Reverse : CCGTGTGGATGCGCACGT ; ACTIN - Forward : 16 CCACGAAACTACCTTCAACTCC ; ACTIN - Reverse : GTGATCTCCTTCTGCATCCTGT ; GAPDH - 17 Forward : CTCCTGCACCACCAACTGCT ; GAPDH - Reverse : GGGCCATCCACAGTCTTCTG ; 18 VIMENTIN - Forward : TGTCCAAATCGATGTGGATGTTTC ; VIMENTIN - Reverse : 19 TTGTACCATTCTTCTGCCTCCTG ; ESRRB - Forward : TCTCACCCAGCACTAGGACACCAG ; 20 ESRRB - Reverse : ACACCCACTTGCTCCAAGCCA ; ESRRB - Taqman : Hs01584024 _ m1 21 22 Detection of miRNA expression in human ESCs / iPSCs 23 RNA harvest was performed with miRNeasy Micro Kit ( Qiagen ) and cDNA synthesized with 24 miScript II RT kit ( Qiagen ) using miScript HiFlex buffer . Real - time quantitative RT - PCR was performed 25 using the miScript SYBR Green PCR kit ( Qiagen ) and a ViiA 7 Real - Time PCR System ( Applied 26 Biosystems ) . The primer concentration used was 1 µM in a final reaction volume of 10µl . Each reaction 27 was performed in triplicate . The thermal cycling parameters were according to manufacturer orders 28 ( miScript SYBR green PCR kit ) . The following primers were used : miR302b : 29 40 TAAGTGCTTCCATGTTTTAGTAG ; miR200c : TAATACTGCCGGGTAATGATGGA ; U6 : 1 CGATACAAGGCTGTTAGAGAG 2 3 Human mitochondrial DNA PCR 4 DNA was harvested with home - made lysis buffer ( 100 mM Tris pH8 . 0 , 0 . 5 mM EDTA , 0 . 2 % 5 SDS , 200 mM NaCl , 200 ug / ml Proteinase K ) , precipitated with isopropanol and washed 1x with 70 % 6 Ethanol and finally eluted in 10 mM Tris - HCL pH 8 , 0 . 1 mM EDTA . 50 ng template was used per 7 reaction with SYBR Green Mastermix in ViiA 7 Real - Time PCR System ( Applied Biosystems ) . As 8 endogenous control , UCNE expression was used and compared to amplification of human mitochondrial 9 element DNA . Quantification was done with delta / delta ct - method . The following primers were used : 10 UCNE - forward : AACAATGGGTTCAGCTGCTT ; UCNE - reverse : CCCAGGCGTATTTTTGTTCT ; 11 hMIT - forward : AATATTAAACACAAACTACCACCTACC ; hMIT - reverse : 12 TGGTTCTCAGGGTTTGTTATAA . 13 14 Immunofluorescence staining 15 Cells were grown for two days on glass cover slips ( 13mm 1 . 5H ; Marienfeld , 0117530 ) fixed 16 with 4 % paraformaldehyde / phosphate buffer for 10 min at room temperature , washed three times with 17 PBS , and permeabilized in PBS / 0 . 1 % Triton for 10 min . Cells were blocked with blocking solution ( 2 % 18 normal goat serum , 0 . 1 % BSA in PBS / 0 . 05 % Tween ) for 1h at RT and incubated with primary antibody 19 diluted in blocking solution overnight at 4°C ( Antibodies in this study have all been validated in the 20 literature and by ourselves ) . Cells were then washed three times with PBS / 0 . 05 % Tween , incubated with 21 secondary antibodies for 1 hour at room temperature , washed in PBS / 0 . 05 % Tween , counterstained with 22 DAPI ( 1 μ g / ml ) , washed again three times with PBS / Tween 0 . 05 % and mounted with Shandon Immu - 23 Mount ( Thermo Scientific , 9990412 ) , and imaged . All comparative experiments were done 24 simultaneously . The following antibodies were use at the indicated dilutions : mouse anti - TRA - 1 - 60 25 ( Abcam ab16288 , 1 : 500 ) , mouse anti - TRA - 1 - 81 ( Abcam ab16289 , 1 : 100 ) , mouse anti - SSEA1 ( Abcam 26 ab16285 , 1 : 100 ) , mouse anti - SSEA4 ( Abcam ab16287 , 1 : 100 ) , rat anti - SSEA3 ( Abcam ab16286 , 1 : 100 ) , 27 goat anti - NANOG ( R / D AF1997 , 1 : 50 ) , rabbit anti - OCT3 / 4 ( Santa Cruz SC9081 , 1 : 400 ) , mouse anti - 28 OCT3 / 4 ( Santa Cruz SC5279 , 1 : 100 ) , rabbit anti - KLF4 ( Santa Cruz SC20691 ) , mouse anti - ECAD 29 ( Abcam ab1416 , 1 : 100 ) , rabbit anti - NCAD ( Abcam ab12221 , 1 : 800 ) , mouse anti - NR3B2 ( R & D 30 41 PPH670500 , 1 : 1000 ) , rabbit anti - TFE3 ( SIGMA HPA023881 , 1 : 500 ) , rabbit anti - H3K27me3 ( Abcam 1 AB5603 ) , goat anti - TFCP2L1 ( R / D AF5726 , 1 : 200 ) , rabbit anti - NUMA ( Abcam ab84680 , 1 : 200 ) , 2 chicken anti - GFP ( Abcam ab13970 , 1 : 1000 ) , rabbit anti - KLF17 ( SIGMA HPA024629 , 1 : 100 ) , anti - 3 TFAP2C ( CSTH2320 , 1 : 100 , Cell Signaling ) , anti - STELLA ( Santa Cruz , sc - 376862 , 1 : 100 ) , anti - 4 DNMT3L ( Novus Biologicals , NPB2 - 27098 , 1 : 200 ) . All secondary antibodies were used from Jackson 5 ImmunoResearch . 6 7 W estern blotting analysis 8 Whole - cell protein extracts were isolated from human cells with conventional RIPA lysis buffer 9 and protein concentration assayed by BCA kit ( Thermo Scientific ) . Blocking was carried out in 10 % skim 10 milk in PBST for 1 h . Blots were incubated with the following antibodies in 5 % BSA in PBST : KLF4 11 ( AF3158 ; 1 : 200 ; R & D ) , OCT4 ( H - 134 ; 1 : 1 , 000 ; Santa Cruz ) , NAANOG ( 397A ; 1 : 1 , 000 ; Bethyl ) , 12 METTL3 ( A301 - 567A , 1 : 2000 , Bethyl ) , HSP90beta ( ab32568 , 1 : 10000 , Abcam ) , DNMT1 ( ab87654 , 13 1 : 1000 , Abcam ) , GAPDH ( ab181602 , 1 : 10000 , Abcam ) , ACTIN ( ab6276 , 1 : 10000 , Abcam ) , UHRF1 ( sc - 14 373750 , 1 : 1000 , Santa Cruz ) , DGCR8 ( 10996 - 1 - AP , 1 : 1000 , Proteintech ) , P53 ( D - 01 , courtesy from 15 Varda Roter’s lab ) , β - catenin ( sc - 7963 , 1 : 1000 , Santa Cruz ) , TCF3 ( CST2883 , 1 : 1000 , Cell Signaling ) , 16 STAT3 ( sc - 482 , 1 : 1000 , Santa Cruz ) , KLF4 ( sc - 20691 , 1 : 1000 , Santa Cruz ) , RBPJ ( C5 5313 , 1 : 1000 , 17 Cell Signaling ) , TFAP2C ( CSTH2320 , 1 : 1000 , Cell Signaling ) , STAT3 ( sc - 7993 , 1 : 1000 , Santa Cruz ) . 18 Secondary antibodies were HRP - linked goat anti - mouse , goat anti - rabbit and rabbit anti - goat ( 1 : 10 , 000 ; 19 Jackson ) . Blots were developed using SuperSignal West Pico Chemiluminescent Substrate ( Thermo 20 Scientific 34580 ) . 21 22 Whole - Genome Bisulfite Sequencing ( WGBS ) Library preparation 23 DNA was isolated from cells using the Quick - gDNA miniprep kit ( Zymo ) . DNA ( 50ng ) was then 24 converted by bisulfite using the EZ DNA Methylation - Gold kit ( Zymo ) . Libraries were prepared using the 25 TruSeq kit ( Illumina ) and length distribution of each library was measured using the Bioanalyzer and 26 product concentration was measured using Qubit Fluorometric Quantitation . For sequencing , the 27 libraries , NextSeq 500 / 550 High Output v2 kit ( 150 cycles ) was used . The following secure token has 28 42 been created to allow review of record GSE125555 while it remains in private status : 1 https : / / www . ncbi . nlm . nih . gov / geo / query / acc . cgi ? acc = GSE125555 2 3 Whole - Genome Bisulfite Sequencing ( WGBS ) analysis 4 The sequencing reads were aligned to the human hg19 reference genome ( UCSC , 2009 ) , using a 5 proprietary script based on Bowtie2 . In cases where the two reads were not aligned in a concordant 6 manner , the reads were discarded . Methylation levels of CpGs calculated by WGBS were unified . Mean 7 methylation was calculated for each CpG that was covered by at least 5 distinct reads ( X5 ) . Average 8 methylation level was calculating by taking the average over all covered X5 covered CpG sites in that 9 genome . 10 11 ChIP - seq library preparation 12 Cells were crosslinked in formaldehyde ( 1 % final concentration , 10 (cid:3) min at room temperature ) or 13 double crosslinked in DSG / formaldehyde ( 2 mM DSG , 30 min at room temperature ) , then formaldehyde 14 1 % final concentration , 10 (cid:3) min at room temperature ) as detail bellow per antibody , and then quenched 15 with glycine ( 2 . 5M 5 (cid:3) min at room temperature ) . Cells were lysed in 50 (cid:3) mM HEPES KOH pH (cid:3) 7 . 5 , 16 140 (cid:3) mM NaCl , 1 (cid:3) mM EDTA , 10 % glycerol , 0 . 5 % NP - 40 alternative , 0 . 25 % Triton supplemented with 17 protease inhibitor at 4 (cid:3) °C ( Roche , 04693159001 ) for 10 min , and later centrifuged at 950 g for 10 (cid:3) min . 18 Supernatant was discarded and pellet was resuspended in RIPA - 1 ( 0 . 2 % SDS , 1 (cid:3) mM EDTA , 0 . 1 % DOC , 19 140 (cid:3) mM NaCl and 10 mM Tris - HCl ) with protease inhibitor . Cells were then fragmented with a Branson 20 Sonifier ( model S - 450D ) at −4 (cid:3) °C to size ranges between 200 and 800 (cid:3) bp and centrifugation at max 21 speed for 10 min . Supernatant lysate was extracted and diluted with RIPA 2 - 3 - fold ( 0 . 1 % SDS , 1 (cid:3) mM 22 EDTA , 0 . 1 % DOC , Triton 1 % , 140 (cid:3) mM NaCl and 10 mM Tris - HCl ) . Small amount of lysate were saved 23 for whole cell extract at this point . Antibody was pre - bound by incubating with Protein - G Dynabeads 24 ( Invitrogen 10004D ) in blocking buffer ( PBS supplemented with 0 . 5 % TWEEN and 0 . 5 % BSA ) for 1 (cid:3) h 25 at room temperature . Washed beads were added to the lysate for incubation as detail per antibody . 26 Samples were washed five times with RIPA buffer , twice with RIPA buffer supplemented with 500 (cid:3) mM 27 NaCl , twice with LiCl buffer ( 10 (cid:3) mM TE , 250mM LiCl , 0 . 5 % NP - 40 , 0 . 5 % DOC ) , once with TE 28 ( 10Mm Tris - HCl pH 8 . 0 , 1mM EDTA ) , and then eluted in 0 . 5 % SDS , 300 (cid:3) mM NaCl , 5 (cid:3) mM EDTA , 29 10 (cid:3) mM Tris HCl pH (cid:3) 8 . 0 . Eluate was incubated treated sequentially with RNaseA ( Roche , 30 43 11119915001 ) for 30 (cid:3) min in 37°C and proteinase K ( NEB , P8102S ) for 2 (cid:3) h in 37°C and de - crosslinked 1 in 65 (cid:3) °C for 8 (cid:3) h . DNA was purified with Agencourt AMPure XP system ( Beckman Coulter Genomics , 2 A63881 ) . Libraries of cross - reversed ChIP DNA samples were prepared according to a modified version 3 of the Illumina Genomic DNA protocol , as described previously ( Rais et al . , 2013 ) . Treatment per 4 antibody : KLF17 – double crosslinked , cell amount : 30 million , antibody : 6ug HPA024629 - atlas 5 antibodies , incubation time : overnight . KLF4 – crosslinked , cell amount : 30 million , antibody : 10ug 6 AF3158 - R & D systems , incubation time : overnight , comment : This is a mouse Klf4 antibody that was 7 verified to work on human using IF staining . NANOG – crosslinked , cell amount : 30 million , antibody : 8 6ug AF1997 - R & D systems , incubation time : overnight . OCT4 – crosslinked , cell amount : 30 million , 9 antibody : 10ug SC8628 Santa - Cruz , incubation time : overnight . SOX2 – crosslinked , cell amount : 30 10 million , antibody : 10ug AF2018 - R & D systems , incubation time : overnight . TFAP2c – crosslinked , cell 11 amount : 30 million , antibody : 5ug sc - 8977 Santa - Cruz , incubation time : overnight . H3K27ac – 12 crosslinked , cell amount : 5 million , antibody : 5ug ab4729 Abcam , incubation time : 6 hours . 13 14 ChIP - seq analysis 15 We analyzed Chip - seq data of the following DNA - binding proteins : NANOG , SOX2 , OCT4 , 16 KLF4 , KLF17 , TFAP2C , H3K27AC , in HENSM or primed conditions , with replicates or triplicates for 17 each antibody per single condition . For alignment and peak detection , we used bowtie2 software to align 18 reads to human hg19 reference genome ( UCSC , 2009 ) , with default parameters . We identified enriched 19 intervals of all measured proteins using MACS version 1 . 4 . 2 - 1 . We used sequencing of whole - cell extract 20 as control to define a background model . Duplicate reads aligned to the exact same location are excluded 21 by MACS default configuration . The number of peaks in Primed conditions were in - par with previous 22 publications ( e . g . PMID 25409831 , PMID 19829295 , Supplementary Table S5 ) . Peaks that were 23 observed in at least two replicates were kept for further analysis . Peaks were assigned to genes using 24 Homer software ( Supplementary Table S5 ) . Motif enrichment analysis was performed using Homers 25 findmotifsgenome . pl , with parameter - size 50 ( Supplementary Table S4 ) . Significance of overlap 26 between TF targets was calculated using Fisher exact test ( Figure 6h ) . 27 28 RNA - seq library preparation 29 44 Total RNA was isolated from indicated cell lines and extracted from Trizol pellets by chloroform - 1 phenol extraction protocol , then cleaned using Ribo - Zero ( Illumina Ribo - Zero Plus rRNA Depletion Kit , 2 ID - 20037135 ) or Poly - A ( Dynabeads mRNA DIRECT Kit ( Invitrogen ) , ID - 61012 ) depletion Kits . 3 Cleaned RNA was next utilized for RNA - Seq by ScriptSeq Preparation Kit v2 ( Illumina ) according to 4 manufacturer’s instruction . 5 6 RNA - seq analysis 7 hESCs grown in naïve and primed conditions , from different cell lines ( LIS41 , LIS49 , WIBR2 , 8 WIBR3 , H1 , H9 , HUES64 ) were used for RNA - seq analysis . Overall 36 samples were sequenced using 9 poly - A single - end strategy , and 19 samples were sequenced using ribo - zero paired - end strategy . STAR 10 software version 2 . 5 . 2b was used to align reads to human GRCh38 reference genome ( 2013 ) , using the 11 following flags : - - outFilterMultimapNmax 1 - - outReadsUnmapped Fastx - - twopassMode Basic - - 12 outSAMstrandField intronMotif . Read count values were estimated with HTSeq V0 . 7 . 2 software over 13 all aligned reads using GRCh38 general feature format ( GFF ) , with the following flags : - a 10 - s no - t 14 exon - i gene _ id . Mitochondrial and ribosomal genes were filtered out . Genes with a sum count across 15 all samples < 10 , were filtered out as well . The filtering was done independently in each analysis , 16 therefore the number of genes included may change , as it is dependent on the samples that were included 17 for that analysis . Count values were normalized using R DESeq2 V1 . 26 software , and corrected for batch 18 effects using R Limma V3 . 42 . Variance stabilizing transformation was performed to all datasets as well . 19 Dataset analysis and plotting was performed by R V3 . 6 . Sample correlation was performed using 20 Spearman method . Hierarchical clustering was carried out using “complete” or “Ward . D2” methods . 21 Heatmap plots were first Z - scored by sample to better emphasize gene separation . Differentially 22 expressed genes between Naïve and Primed samples were performed by the following parameters : 23 log2 ( Fold change ) > 2 or log2 ( Fold change ) < - 2 , and adjusted p - value < 0 . 1 . RNA - Seq data are deposited 24 under GEO no . GSE125555 . 25 26 ATAC - seq library preparation 27 Cells were trypsinized and counted , 50 , 000 cells were centrifuged at 500 g for 3 min , followed by 28 a wash using 50 μ l of cold PBS and centrifugation at 500 g for 3 min . Cells were lysed using cold lysis 29 buffer ( 10 mM Tris - HCl , pH 7 . 4 , 10 mM NaCl , 3 mM MgCl 2 and 0 . 1 % IGEPAL CA - 630 ) . Immediately 30 45 after lysis , nuclei were spun at 500 g for 10 min using a refrigerated centrifuge . Next , the pellet was 1 resuspended in the transposase reaction mix ( 25 μ l 2× TD buffer , 2 . 5 μ l transposase ( Illumina ) and 22 . 5 2 μ l nuclease - free water ) . The transposition reaction was carried out for 30 min at 37 °C and immediately 3 put on ice . Directly afterwards , the sample was purified using a Qiagen MinElute kit . Following 4 purification , the library fragments were amplified using custom Nextera PCR primers 1 and 2 for a total 5 of 12 cycles . Following PCR amplification , the libraries were purified using a QiagenMinElute Kit and 6 sequenced . 7 8 ATAC - seq analysis 9 Reads were aligned to hg19 human genome using Bowtie2 with the parameter - X2000 ( allowing 10 fragments up to 2 kb to align ) . Duplicated aligned reads were removed using Picard MarkDuplicates tool 11 with the command REMOVE _ DUPLICATES = true . To identify chromatin accessibility signal we 12 considered only short reads ( ≤ 120bp ) that correspond to nucleosome free region . To detect and separate 13 accessible loci in each sample , we used MACS version 1 . 4 . 2 - 1 with - - call - subpeaks flag ( PeakSplitter 14 version 1 . 0 ) . To generate the final ATAC loci of naïve and primed conditions , we selected peaks that 15 appear in the majority of the replicates of each condition , i . e . 3 out of 4 naïve samples , and 2 out of 3 16 primed samples . 17 18 Enhancer Identification 19 H3K27ac peaks were detected using MACS version 1 . 4 . 2 - 1 and merged for each condition 20 ( naïve and primed ) using bedtools merge command . All ATAC peaks were filtered to include only peaks 21 which co - localized with the merged H3K27ac peaks in at least one condition . Finally , peaks that co - 22 localized with promoter or exon regions based on hg19 assembly ( UCSC , 2009 ) were filtered out . Finally , 23 we were left with defined genomic intervals which we annotated as enhancers . 24 25 SNP Score Calculation from WGS 26 SNP score calculation was done as previously described ( Bar et al . , 2017 ) . Briefly , we 27 downloaded WGS data for H1 and H9 cell lines from the SRA database 28 ( http : / / www . ncbi . nlm . nih . gov / sra ) . The files were extracted using SRAtools and aligned to the hg19 29 reference genome using BWA . SAMtools was used to convert from SAM to BAM . We then detected 30 46 polymorphisms with the same pipeline as described previously for RNA - seq ( without filtering for 1 FPKM ) . In order to test if genomic SNPs are properly detected in the RNA - seq analysis , we extracted 2 SNPs from exons of control pluripotent genes and calculated the average percentage of overlapping SNPs 3 between WGS and RNA - seq of the appropriate H1 and H9 samples . In order to test if there is a difference 4 in the existence of genomic SNPs between imprinted genes governed by maternal and paternal gDMR , we 5 extracted the SNPs located at exons of imprinted genes from WGS of H1 and H9 cell lines and then 6 calculated the percentage of genes with genomic SNPs in each group . We used a chi - square test , which 7 confirmed that the bias was not significant . 8 9 Quantifying X : A allelic ratio 10 X : A allelic ration was done as previously described ( Bar et al . , 2019 ) . Briefly , to generate a 11 quantifiable score of allelic expression across the X chromosome , we developed the X : Autosomes ( X : A ) 12 allelic ratio . For this we first counted the number of biallelic SNPs in each chromosome . Since our 13 analysis is based on expressed SNPs , this sum is expected to be dependent on the number of genes in the 14 chromosome . Therefore , to overcome this bias we generated a normalized SNP sum by dividing the SNP 15 sum with the number of known genes in each chromosome . We validated that the average of this 16 normalized SNP sum was similar across all autosomes . Next , for each sample we calculated the ratio of 17 normalized SNP sum of X chromosome and the average normalized SNP sum of autosomes : 18 19 NS ( normalized sum ) = Sum ( biallelic SNPs ) / No . of genes in chromosome 20 X : A allelic ratio = X chromosome - NS / Average ( Autosome - NS ) 21 22 By comparing the normalized sum of X and autosomes within each sample , we avoid potential 23 biases which are due to coverage and batch issues . Samples with very low normalized SNP sums in 24 autosomes ( for example haploid samples or their diploid genetic match which are completely 25 homozygous ) , were discarded from this analysis . 26 27 Transposon expression profiling 28 TE profiling was performed as previously described ( Theunissen et al . , 2016 ) . For repetitive 29 sequences an D . Trono lab curated version of the Repbase database was used ( fragmented LTR and 30 47 internal segments belonging to a single integrant were merged ) . TEs counts were generated using the 1 multiBamCov tool from the bedtools software . TEs which did not have at least one sample with 20 reads 2 ( after normalizing for sequencing depth ) were discarded from the analysis . TEs overlapping exons were 3 also removed from the analysis . Normalisation for sequencing depth and differential gene expression 4 analysis was performed using Voom ( Law et al . , 2014 ) as it has been implemented in the limma package 5 of Bioconductor ( Gentleman et al . , 2004 ) . A gene ( or TE ) was considered to be differentially expressed 6 when the fold change between groups was bigger than 2 and the p - value was smaller than 0 . 05 . A 7 moderated paired t - test ( as implemented in the limma package of R ) was used to test significance . P - 8 values were corrected for multiple testing using the Benjamini - Hochberg’s method ( Benjamini , 1995 ) . 9 Single cell RNA - Seq data was downloaded from GEO ( Edgar et al . , 2002 ) ( GSE36552 ) . Data was 10 mapped and processed as explained above . When merging with in - house RNA - Seq , only TEs which were 11 expressed in both datasets were used . To be considered expressed , the TE needed to have at least as many 12 reads across all samples as samples existed in each dataset . Correspondence between naive / primed ESCs 13 and single cell expression data from human embryonic stages w ` s also done as described before 14 ( Theunissen et al . , 2016 ) . For every cell state we perform a statistical test to find the genes ( or TEs ) that 15 have a different expression level compared to the other cell states ( Yan et al . , 2013 ) . For this we use a 16 moderated F - test ( comparing the interest group against every other ) as implemented in the limma package 17 of Bioconductor . For a gene ( or TE ) to be selected as expressed in a specific cell state it needs to have a 18 significant p - value ( < 0 . 05 after adjusting for multiple testing with the Benjamini and Hochberg method ) 19 and an average fold change respective to the other cell states greater than 10 . Note that with this approach 20 a gene ( or TE ) can be marked as expressed in more than one cell state . Once we have the genes ( or TEs ) 21 that are expressed in a specific cell state we ask if those genes are more expressed in primed or in naive . 22 For that we see how many of the genes are up ( or down ) regulated in the primed / naive pairwise 23 comparison . For a gene to be considered differentially expressed it needs to have a p - value ( after multiple 24 testing correction with the Benjamini and Hochberg method ) lower than 0 . 05 and a fold change greater 25 than 2 . 26 27 28 Figure Legends 29 48 1 Figure 1 . Reporter systems for functional screening for enhanced human naive pluripotency 2 conditions . 3 a . Strategy for generating human ESCs with TET - OFF regulated expression of METTL3 m 6 A 4 methyltransferase enzyme . b . Western blot analysis for correctly engineered human ESCs with TET - OFF 5 METTL3 regulation , before and after DOX treatment . c . Representative images for human METTL3 6 TET - OFF engineered cells in different conditions with or without DOX addition . White arrow indicated 7 viable PSC at P + 2 . Previously described primed and naïve conditions do not maintain their pluripotency 8 and viability when DOX is added ( METTL3 is depleted ) . d . Scheme depicting strategy for conducting a 9 screen for identifying a small molecule or cytokine additive to previously described human naïve NHSM 10 conditions that allow maintaining pluripotency in TET - OFF METTL3 human stem cells after adding 11 DOX . e . W3G4 cells were maintained in the presence of DOX for up to 4 passages in different conditions 12 and stained for OCT4 to quantify percentage of cells that retained their pluripotency . Graph shows that 13 supplementing NHSM conditions with an inhibitor for Tankyrase ( TNKi ) allows maintaining 14 pluripotency in majority of cells expanded ( > 75 % positive OCT4 staining ) . f . OCT4 + pluripotency 15 maintenance in NHSM conditions supplemented with various TNK inhibitors and DOX to repress 16 METTL3 expression . g . Quantification of Δ PE - OCT4 - GFP knock in naïve pluripotency reporter , in 17 variety of primed ( red ) and naïve conditions ( blue ) . Mean fluorescence intensity values ( MFI ) are 18 indicated . h . Quantification of Δ PE - OCT4 - GFP knock in naïve pluripotency reporter in variety of 19 conditions with various concentrations of ACTIVIN A recombinant cytokine . Figure shows that in 20 NHSM + TNKi conditions the naivety of human ESCs is still dependent on ACTIVIN A supplementation . 21 i . Representative phase contrast images in human ESCs expanded in NHSM + TNKi conditions showing 22 their maintenance of pluripotent domed - like morphology even in the presence of FGFRi . However , upon 23 blocking of TGF / ACTIVIIN A signaling ( with A83 - 01 designated as TGFRi ) , the cells in NHSM + TNKi 24 lose their pluripotent dome - like shaped morphology and differentiate . 25 26 Figure 2 . Defining enhanced human naive conditions compatible with blocking TGF / ACTIVIN 27 signalling . 28 a . Scheme depicting strategy for conducting a screen for identifying a small molecule or cytokine additive 29 to optimized NHSM conditions after addition of TNKi , allow to maintain pluripotency in TET - OFF 30 49 METTL3 human stem cells ( clone W3G4 ) and without supplementing exogenous TGF or ACTIVIN . b . 1 OCT4 + pluripotency maintenance in optimized naive conditions without TGF / ACTIVIN , indicated that 2 supplementing SRCi CGP77675 allows maintaining OCT4 + cells in optimized conditions and without 3 METTL3 expression ( n = 3 per condition ) . c . Summary of small molecules and their concentrations used in 4 the optimized HENSM conditions used herein . d . Representative phase contrast images showing naïve 5 domed - like morphology of human ESCs expanded in HENSM conditions , that is maintained even when 6 TGFRi small molecule A83 - 01 is supplemented . e . Mass spectrometry - based quantification of m 6 A on 7 isolated mRNA from the indicated cell lines and conditions . Depletion of m 6 A in human cells was 8 validated in HENSM + DOX conditions . f . Mature teratoma obtained following injection of METTL3 9 TET - OFF human ESCs expanded for 15 passages ( P15 ) in HENSM + DOX conditions . Please note that 10 no in vitro priming or media other than HENSM + DOX was used before the cells were injected into the 11 mice to test for teratoma formation . g . Strategy for generating human ESCs with TET - OFF regulated 12 expression of DNA methyltransferase enzyme , DNMT1 . h . Western blot analysis for DNMT1 expression 13 in HENSM conditions supplemented with either DOX of BRAF inhibitor ( SB590885 – 0 . 25µM ) . Please 14 note that DOX ablates DNMT1 expression and that BRAFi depletes DNMT1 expression to much lower 15 levels than seen in HENSM conditions ( without DOX ) , yet still they retain residual DNMT1 expression 16 that is necessary for their survival and viability when BRAFi is added to HENSM conditions . i . DNMT1 17 TET - OFF ESC clone was maintained in the presence of DOX for up to 4 passages in different conditions 18 and stained for OCT4 to quantify percentage of cells that retained their pluripotency . Graph shows that 19 only HENSM and HENSM - ACT conditions ( with and without MEFs ) maintain robust expansion of 20 dome - like undifferentiated human PSCs in vitro . Omitting TNKi and SRCi ( in other words WNTi and 21 SRCi ) form HENSM leads to loss of ability to maintain DNMT1 depleted human naïve PSCs , as evident 22 from loss of OCT4 + cells . j . Mouse or human ESCs carrying NANOG - GFP pluripotency reporter were 23 expanded in the indicated naïve and primed conditions in the absence of exogenous L - Glutamine 24 supplementation for 4 passages . Percentage of pluripotent cells was quantified based on GFP expression 25 levels . Graph shows that only HENSM and HENSM - ACT conditions ( with and without irradiated feeder 26 cells – MEFs ) maintain expansion and stability of human NANOG + pluripotent cells when exogenous L - 27 Glutamine is omitted , and that this is similar to 2iL conditions on mouse naïve ESCs . Omitting TNKi and 28 SRCi form HENSM leads to loss of ability to maintain human naïve PSCs without exogenous L - 29 Glutamine supplementation , as evident from loss of NANOG - GFP + cells . 30 50 Figure 3 . HENSM endows human PSCS with canonical naïve - like transcriptional features . 1 a . Unbiased hierarchical clustering was performed on RNA - seq measurement obtained from different 2 human ESC and iPSCs expanded in HENSM , HENSM - ACT naïve and TeSR primed conditions . The data 3 was also clustered with previous independently generate RNA - seq on human PSCs expanded in 5iLA 4 conditions ( Naïve 5iLA * , Theunissen et al . Cell Stem Cell 2016 ) , Reset conditions ( Naïve Reset * , 5 Takashima et al . Cell 2014 – composed of NANOG - KLF2 transgenes and 2iLGo ) or Primed conditions 6 ( Primed * ) . Figure shows that HENSM and HENSM - ACT conditions ( Dark blue ) resemble 5iLA and 7 Reset conditions ( light blue ) and cluster separately from primed cells ( Red and orange ) . b . PCA analysis 8 of samples represented in a , showing that HENSM and HENSM - ACT conditions ( Dark blue ) resemble 9 5iLA and Reset conditions ( light blue ) and cluster separately from primed cells ( Red ) . c . RT - PCR 10 analysis for naïve pluripotency markers . Values were normalized to ACTIN and GAPDH . Primed 11 expression levels were set as 1 . * t - test p Value < 0 . 01 . Please note that HENSM - ACT show higher 12 expression of naïve pluripotency markers than HENSM , consistent with the notion that ACTIVIN A 13 supports naïve pluripotency in humans . d . Correspondence between gene expression in naïve / primed 14 ESCs and single - cell human embryonic stages ( Yan et al . 2013 ) . For every stage of human embryonic 15 development , a statistical test was performed to find the genes that have a different expression level 16 compared to other stages . The proportions of developmental stage - specific genes that are upregulated ( p 17 < 0 . 05 , 2 - fold change ) in naïve or primed cells are indicated in orange and blue , respectively . e . Uniquely 18 accessible regions were recognized in 2 / 8 - cell and ICM of human early development . The percentage of 19 up - regulated regions ( FC > 2 ) in naïve ( Blue ) or primed ( Red ) are indicated . Analysis was done for 3 20 distinct naïve systems : HENSM , t2iL / Gö and 5iL / A ( previously mapped in Wu et al ) . Accessible regions 21 in naïve tend to have a higher overlap with ICM regions than with 2 - or 8 - cell regions . 22 23 Figure 4 . Chromosome X reactivation status and DNA methylation profile in HENSM based 24 conditions . 25 a . Schematic and FACS results following using WIBR2 ( female 46XX ) 29 - 9 hESC line that carries GFP 26 and tdTomato on each of the X chromosomes in the MECP2 locus . Parental 29 - 9 clone has the X 27 chromosome carrying mCherry allele in the active state , and thus is positive only for tdTomato and 28 negative for GFP in the primed state . Upon transfer to HENSM conditions , all cells turn on both X 29 chromosomes and thus become double positive for both fluorescent markers ( GFP and tdTomato ) . After 30 51 transfer into primed conditions ( i . e . repriming ) , cells start to inactivate the X chromosome again . b . RNA - 1 FISH analysis for ATRX transcription in primed and HENSM WIBR3 cells . Note that ATRX is active on 2 both X chromosomes only in HENSM conditions . c . X : Autosome allelic ratios calculated for primed and 3 naïve PSCs ( value of each single RNA - seq sample ) . d . Average methylation as calculated from primed 4 samples , and naïve samples that were maintained in various HENSM conditions , along with previously 5 published Reset - naïve samples ( Takashima et al ) , and human ICM samples ( Guo et al , Nature 2014 ) . 6 DNMT1 - / - ( from TET - OFF lines ) samples were used as negative control for methylation . e . Global 7 methylation histogram measured on the same samples as in ( d ) . Dark blue - percentage of highly 8 methylated CpGs ( > 0 . 9 methylation level ) , light blue – percentage of lowly methylated CpGs ( < 0 . 1 9 methylation level ) . f . Western blot analysis for DNA methylation regulators , DNMT1 and UHRF1 10 enzymes . DNMT1 protein levels are maintained in all conditions . UHRF1 is partially depleted in 11 HENSM conditions , and this decrease is more enhanced when ERKi concentration is increased . g . 12 Representative images of whole - mount in - toto imaged mouse embryos , after microinjection of the 13 indicated human naïve iPSCs , are shown in comparison to non - injected wild - type embryos . White squares 14 in tiles outline zoomed - in regions in subsequent panels . GFP staining was used to trace hiPSC - derived 15 progeny and CellTracker and Hoechst as counter staining . h . Representative images of IHC for TUJ1 and 16 GFP of injected ( upper panels ) and non - injected E15 . 5 mouse embryos ( lower panels ) for spinal cord 17 region are shown . GFP served as human cell tracer and TUJ1 as neural marker . GFP , TUJ1 , overlap as 18 well as merged constitute zoomed - in regions of tissues depicted in red squares in the tiles . White 19 arrowheads in insets depict co - localization of GFP and TUJ1 . TUJ1 , neuron - specific class III beta - 20 tubulin ; GFP , green fluorescent protein ; p53 , tumor protein p53 ; iPSC , induced pluripotent stem cell . Tile 21 scale bar 200 and 100µm . Zoomed - in scale bar 50µm . 22 23 Figure 5 . Signalling foundations for human naïve pluripotency . 24 a . FACS results following using WIBR2 ( female 46XX ) 29 - 8 hESC line that carries GFP and tdTomato 25 on each of the X chromosomes in the MECP2 locus . Parental 29 - 8 clone has the X chromosome carrying 26 GFP allele in the active state , and thus is positive only for GFP and negative for tdTomato in the primed 27 state . Upon transfer to HENSM conditions , all cells turn on both X chromosomes and thus become double 28 positive for both fluorescent markers ( GFP and mCherry ) . Indicated components from HENSM 29 conditions were individually depleted or added , and cells were subjected to FACS analysis after 10 days . 30 52 Withdrawal of PKCi , SRCi or WNTi compromises XaXa state and cells start inactivating one of the X 1 chromosomes , indicating loss of naïve state identity . Figure shows that upon LIF omission , cells do not 2 become primed and cells maintain XaXa state . b . Quantification of Δ PE - OCT4 - GFP knock in naïve 3 pluripotency reporter in variety of conditions after 2 passage transfer from HENSM starting conditions . 4 * t - test p Value < 0 . 01 . c . Quantification of Δ PE - OCT4 - GFP knock in naïve pluripotency reporter in 5 β CAT + / + and isogenic β CAT - / - cells in variety of conditions after 3 passage transfer from HENSM 6 starting conditions . d . Luciferase assay for measuring WNT activity via luciferase reporter assay . Values 7 were normalized to HENSM no TNKI conditions ( defined as 1 ; n = 3 per condition ) . e . Morphological 8 changes in β CAT + / + and isogenic β CAT - / - cells after 2 passages from TNK withdrawal . Knock in naïve 9 pluripotency reporter in variety of conditions after 2 passage transfer from HENSM starting conditions f . 10 Mouse and human β CAT - / - ESCs were rendered transgenic with a validated Tamoxifen inducible β CAT - 11 ERT transgene . Acquisition or loss of naïve domed like morphology other parameters were assayed after 12 2 passages of the indicated conditions . g . Luciferase assay for measuring NFkB activity via luciferase 13 reporter assay . Values were normalized to HENSM no SRCi conditions ( defined as 1 ; n = 3 per condition ) . 14 * t - test p Value < 0 . 01 ; NS - not significant . h . 29 - 8 hESC line that carries GFP and tdTomato on each of 15 the X chromosomes in the MECP2 locus , were rendered transgenic with IKB (cid:0) WT or dominant negative 16 mutant allele ( IKB (cid:0) - DNmut ) , and cells were FACS analyzed following 10 - day expansion in the 17 indicated conditions . in the indicated conditions . 18 19 Figure 6 . KLF17 is a master regulator of human naïve pluripotency . 20 a . Motif enrichment in HENSM - specific and primed - specific accessible chromatin regions ( n = 20642 and 21 b = 36927 , respectively ) . Motif families are indicated at the right . Shades represent enrichment fold - 22 change . b . WT and KO hESCs for KLF4 and / or KLF17 were expanded in HENSM and HENSM - ACT 23 conditions , imaged and assayed for OCT4 - GFP by FACS analysis ( values indicated per frame ) . c . WT 24 and KO mESCs for Klf17 were expanded in 2iL , phase imaged and assayed for OCT4 - GFP by FACS 25 analysis ( values indicated per frame ) . d . Genomic annotation of binding sites of KLF17 , KLF4 , TFAP2C , 26 NANOG , OCT4 and SOX2 measured in HENSM naïve or primed conditions , showing the preference of 27 KLF17 to bind promoters , compared to all TFs that prefer to bind enhancers ( Distal intergenic + Introns 28 annotations ) . e . KLF17 binding pattern ( average z - score + STD ) in naïve - specific regions ( n = 20642 , 29 blue ) , primed - specific regions ( n = 36927 , red ) , or in all promoters ( n = 43463 , grey ) . f . Overlap between 30 53 target genes of the indicated transcription factors , showing highest overlap between Sox2 and Oct4 target 1 genes ( p - value ~ 0 ) , and a relative lower overlap with KLF17 and KLF4 targets ( p - value < 10 - 21 ) . Scaled p - 2 value is presented ( Methods ) g . ChIP - seq profile of KLF17 , OCT4 and SOX2 in selected regions and 3 different conditions in human PSCs , showing that in some gene regions such as KLF4 , DPPA3 , 4 HORMAD1 and NODAL ; KLF17 is solely bound . IGV range common to all tracks is indicated . 5 6 Figure 7 . NOTCH / RBPj inhibition allows maintaining alternative human naïve cells without using 7 MEK / ERKi . 8 a . FACS analysis showing status of X activation in female 29 - 9 cells following decreasing concentrations 9 of ERKi in HENSM conditions . Note that the fraction of cells inactivating X chromosome increases with 10 depleting ERKi concentrations . b . FACS analysis for Δ PE - OCT4 - GFP knock in naïve pluripotency 11 reporter upon reducing ERKi in HENSM conditions . c . Schematic showing screen strategy for finding 12 small molecule supplements that could allow maintaining GFP + / tdTomato + cells in HENSM conditions 13 in which ERKi is completely omitted ( 0HENSM ) or partially depleted ( tHENSM ) . d . FACS analysis 14 following supplementing 0HENSM or tHENSM with ACTIVIN A or DBZ , a gamma secretase small 15 molecule inhibitor that blocks NOTCH signaling ( NOTCHi ) . e . Summary of small molecules and their 16 concentrations used in the optimized tHENSM and 0HENSM conditions used herein . f . Δ PE - OCT4 - GFP 17 knock in naïve pluripotency reporter in HENSM , tHENSM and 0HENSM conditions . g . Phase images of 18 WIBR1 hESCs in different MEF supplemented or feeder free naïve pluripotency conditions . h . RT - PCR 19 analysis for naïve pluripotency markers in different naïve and primed conditions . Values were normalized 20 to ACTIN and GAPDH . Primed expression levels were set as 1 . i . PCA of gene expression profiles of 21 cells grown in Primed condition ( red ) , or in HENSM , HENSM - ACT , tHENSM or 0HENSM conditions 22 ( blue shades ) . j . Phase images of RBPj + / + and RBPj - / - isogenic WIBR3 hESCs upon removal of DBZ from 23 OHENSM conditions . Arrows indicate flatted and loss of domed colony morphology . k . RT - PCR 24 analysis for changes in naïve pluripotency marker expression in RBPj + / + and RBPj - / - isogenic WIBR3 25 hESCs upon removal of DBZ from OHENSM conditions . Values were normalized to ACTIN and 26 GAPDH . RBPj + / + HENSM sample expression levels were set as 1 . * t - test p Value < 0 . 01 ; NS - not 27 significant . 28 29 30 54 1 Supplementary Figure Legends 2 3 Figure S1 . Reporter systems for screening for enhanced human naive pluripotency conditions . 4 a . W3G4 METTL3 TET - OFF cells were maintained in the presence of DOX for up to 4 passages in 5 different conditions and stained for OCT4 to quantify percentage of cells that retained their pluripotency . 6 Graph shows that NHSM conditions without feeder cells and other previously described naïve and primed 7 conditions for human ESCs / iPSCs failed to maintain pluripotency in majority of cells expanded in the 8 presence of Dox . b . METTL3 TET - OFF and DNMT1 TET - OFF cells were maintained in the presence of 9 DOX for up to 4 passages in different previously published naïve and primed conditions and stained for 10 OCT4 to quantify percentage of cells that retained their pluripotency . c . Quantification of Δ PE - OCT4 - 11 GFP knock in naïve pluripotency reporter , in variety of primed ( red ) and naïve conditions ( blue ) . Mean 12 fluorescence intensity values ( MFI ) are indicated . Figure shows that supplementing NHSM conditions 13 with TNKi like IWR1 small molecules boosts expression of GFP suggesting enhancement of naivety 14 characteristics . d . Quantification of Δ PE - OCT4 - GFP knock in naïve pluripotency reporter , in optimized 15 naïve conditions and various concentrations of GSK3 inhibitor that leads to WNT activation ( CHIR99021 16 is used as GSK3 inhibitor and is abbreviated as CHIR ) . Figure indicates that CHIR addition negatively 17 influences human naive pluripotency as determined by Δ PE - OCT4 - GFP intensity . e . RT - PCR analysis for 18 naïve pluripotency markers in HENSM conditions with and without P38i / JNKi ( BIRB0796 ) . Values were 19 normalized to ACTIN and values in Primed conditions were set as 1 . * t - test p Value < 0 . 01 . Figure 20 indicates that use of BIRB0796 as P38i / JNKi boosts expression of naïve pluripotency markers . f . W3G4 21 METTL3 TET - OFF cells were maintained in the presence of DOX for up to 4 passages in the optimized 22 HENSM conditions but without TNKi , in order to see if other molecules can substitute for TNKi after all 23 optimizations were applies ( e . g . adding of Geltrex , concentration optimization ) . g . FACS analysis for 24 OCT4 - GFP pluripotency reporter expression following addition of TGFRi . In optimized NHSM 25 conditions that still lack SRCi , pluripotency is entirely and rapidly lost upon inhibition of TGFRi . h . 26 Phase images showing how supplementation of 0 . 2 % Geltrex ( Life Technologies ) in the growth media 27 and SRCi additively enhance domed like morphology of human naïve PSCs in HENSM conditions 28 optimized herein . 29 30 55 Figure S2 . Functional engineered systems for evaluating HENSM conditions . 1 a . Immunostaining for W3G4 before and after DOX validating METTL3 protein downregulation after 2 DOX addition in HENSM . b . Immunostaining of W3G4 cells in HENSM conditions with and without 3 DOX . Cells expressed canonical OCT4 and naïve pluripotency specific markers like KLF17 in both 4 conditions . c . Phase images of WIBR3 esc in NHSM and HENSM conditions , showing more domed and 5 uniform morphology in HENSM conditions . d . Efficiency of generating KO and targeting DGCR8 in 6 human primed ( TeSR ) and HENSM naïve conditions . 2 replicates for targeting were done for each growth 7 conditions . Only in HENSM conditions we recovered DGRC8 KO clones based on genotyping and 8 western blot analysis . e . CRISPR targeting strategy for DGCR8 locus , followed by western blot and 9 sequencing validation of KO clones . f . RT - PCR analysis for the indicated microRNA in DGCR8 WT and 10 KO human ESCs . Figure shows loss of microRNA expression in DGCR8 KO human naïve HENSM 11 conditions as expected following ablation of DGCR8 . * t - test p Value < 0 . 01 . Error bars indicate SD from 12 mean . g . FACS analysis showing preservation of Δ PE - OCT4 - GFP naïve marker expression in both WT 13 and DGCR8 KO human ESCS expanded in HENSM conditions . h . Representative images for human 14 DNMT1 TET - OFF engineered cells in different conditions with or without DOX addition . Previously 15 described primed and naïve conditions do not support maintain their pluripotency and viability when 16 DOX is added ( DNMT1 is depleted ) . Only HENSM and HENSM - ACT conditions ( with and without 17 irradiated feeder cells – MEFs ) maintain robust expansion of dome - like undifferentiated human PSCs in 18 vitro even after extended passaging in the presence of DOX . i . WGBS validates global reduction in CG 19 methylation in HENSM conditions following DOX addition ( shutdown of DNMT1 expression ) . j . 20 Immunostaining validating OCT4 + expressed in DNMT1 TET - OFF cells expanded in HENSM + DOX 21 conditions at P10 . k . METTL3 TET - OFF and DNMT1 TET - OFF cells were maintained in the presence of 22 DOX for up to 4 passages in different HENSM based conditions upon depletion of the indicated key 23 inhibitors and stained for OCT4 to quantify percentage of cells that retained their pluripotency . l . Oxygen 24 consumptions rate ( OCR ) measurement in different conditions . m . OCT4 + colony formation in 2 - 25 deoxyglucose ( 2DG ) . 1mM 2DG was added to the indicated conditions . Error bars indicate SD . 26 27 Figure S3 . Optimized HENSM conditions enable naïve pluripotency maintenance in the absence of 28 L - Glutamine . 29 56 a . FACS based validation of pluripotency maintenance in human stem cells expanded in HENSM 1 conditions with and without exogenous L - Glutamine . Percentage of positive cells and intensity of naïve 2 marker expression were not compromised upon omitting GLUT in HENSM based conditions . b . 3 Representative phase contrast and fluorescent images of human cells expanded in HENSM conditions 4 with and without exogenous L - Glutamine ( GLUT ) . TeSR primed human ESCs were used as controls . c . 5 human ESCs expanded for 10 passages in HENSM conditions without exogenous GLUT robustly formed 6 teratomas ( without any need for exogenously induced priming before they were injected ) . 7 8 Figure S4 . Derivation of new human ESC lines in HENSM conditions . 9 a . 5 new lines were derived in HENSM or HENSM - ACT conditions directly from human blastocysts as 10 indicated . At P6 - 8 , a small portion of the cells was taken and expanded in primed conditions ( and thus are 11 labeled as “primed cells” . b . Representative images showing previously established human 12 primed / conventional ESCS were transferred to HENSM conditions , and after at least 5 passages a small 13 portion of them were transferred back into primed conditions ( thus are referred to as “reprimed” cells ) . c . 14 Newly derived iPSC lines from dermal fibroblasts or peripheral blood mononuclear cells ( PBMCs ) were 15 obtained following OSKM transduction and cell culturing in HENSM conditions . Phase images show 16 initial iPSC colony appearance before they were picked for further expansion . 17 18 Figure S5 . Pluripotency marker expression and characterization in HENSM conditions . 19 Representative immunostaining for pluripotency markers in HENSM conditions are shown for LIS49 20 hESC line . Primed cells expanded in TeSR conditions are used as controls . Note that KLF17 , DNMT3L , 21 STELLA and TFCP2L1 are naïve pluripotency specific markers and are expressed in HENSM conditions 22 and not in primed cells . 23 24 Figure S6 . HENSM conditions maintain teratoma formation competence of PSCs . 25 a . Mature teratoma images are shown following their derivation from the indicated cell lines expanded in 26 the different indicated conditions . Please note that without exception , all teratomas were formed 27 following direct subcutaneous injections after being expanded only in the indicated media condition and 28 without the need for any expansion in other primed conditions in vitro before injection . b . Results of e - 29 karyotyping based on RNA - seq data for the indicated lines are shown . 30 57 1 Figure S7 . Chromosomal stability following long term expansion in HENSM based conditions . 2 Metaphase chromosomal spreads are shown from the indicated human ESC and iPSC lines expanded in 3 HENSM ( a ) , tHENSM ( b ) and OHENSM ( c ) based conditions . Passage numbers are indicated 4 throughout . 5 6 Figure S8 . Differentially expressed genes between human naïve and primed states highlights 7 regulatory candidates . 8 a . Volcano plot comparing change in expression of all genes ( log2 ( Naïve HENSM / primed Fold - Change ) 9 in x - axis ) , to their statistic ( - log10 ( q - value ) in y - axis ) . Differentially expressed genes ( Fold - 10 change > 2 ( < 0 . 5 ) , p - adjusted < 0 . 1 ) are marked in red . Extreme genes are highlighted . b . Spearman 11 correlation matrix of naïve HENSM and primed samples , along with previously published naïve and 12 primed samples ( Takashima et al , 2014 ) . c . Clustered expression profile of differentially expressed genes 13 ( Fold change > 2 ( < 0 . 5 ) , p - adjusted < 0 . 1 , n = 2987 ) in naïve and primed samples , along with Takashima et al 14 samples . ( d - f ) Same as a - c , done over an independent RNA - seq dataset . Number of differentially 15 expressed genes here is 7087 . g . FACS analysis for expression levels of the indicated surface markers . 16 CD130 and CD77 are induced in HENSM conditions consistent with their previous designation as 17 markers of human naïve pluripotency ( Collier et al . Cell Stem Cell 2017 ) . CD24 is depleted in HENSM 18 naïve conditions as expected . 19 20 Figure S9 . Expression profile of selected sets of genes in HENSM naïve and primed conditions . 21 a . Expression profile of selected sets of genes in naïve and primed conditions . b . RT - PCR validation of 22 expression of primed pluripotency markers ZIC2 and OTX2 . Both were significantly depleted in HENSM 23 based naïve conditions . * t - test p Value < 0 . 001 . c . Strategy for generating human STELLA - CFP knock in 24 reporter cell line . Both HENSM and HENSM - ACT conditions upregulated STELLA expression in 25 comparison to primed cells and consistent with transcriptome data . d . FACS analysis for STELLA - CFP 26 knock - in reporter expression levels in the indicated primed and naïve conditions . e . FACS staining results 27 on different primed ( red colors ) and HENSM conditions for SUSD2 expression . MFI values are indicated 28 and compared to matched unstained negative controls . 29 30 58 Figure S10 . Generation of TFAP2C KO human ESCS with reporter for human naive pluripotent 1 state . 2 a . Strategy for generating TFAP2C human KO in WIBR2 human ESC line carrying GFP / tdTomato 3 reporters on each of the X chromosomes respectively . b . Immunostaining analysis for TFAP2C ( also 4 known as AP2gamma ) expression in WT and KO human ESC clones ( WIBR2 - 29 - 8 hESC line ) . OCT4 5 expression was not affected in primed KO cells in comparison to WT primed control cells . c . Strategy for 6 generating TFAP2C human KO for TFAP2C via simultaneous targeting of both alleles in WIBR3 Δ PE - 7 O4G hESCs . d . Western blot analysis for validation of TFAP2C KO generation in primed human 8 WIBR3 - Δ PE - O4G hESCs . e . Phase images showing loss of pluripotent cells expansion within 2 passages 9 in both HENSM and HENSM - ACT conditions . f . RT - PCR analysis in naïve HENSM and primed 10 conditions for naïve pluripotency genes and ESRRB . ( UD - Undetectable ) . g . Targeting strategy for 11 generating ESRRB - mCherry knock - in reporter human WIBR3 hESC line . h . PCR and southern blot 12 analysis for detecting and selecting correctly targeted clones . i . FACS analysis for detecting ESRRB - 13 mCherry expression in human PSCs . ( Tg = Transgene ) . ESRRB - mCherry signal was detected only upon 14 ectopic expression of ESRRB and KLF2 exogenous transgenes in addition to applying naïve HENSM 15 conditions . 16 17 Figure S11 . Human PSCs in HENSM conditions have a transposon element ( TE ) transcription 18 signature of the human pre - implantation embryo . 19 a . Heatmap of RNA - seq expression data form primed human ESCs and naïve cells expanded in HENSM 20 and HENSM - ACT conditions . Data shown include 10000 TEs with the highest standard deviation 21 between samples . Figure shows clear separation between naïve HENSM and primed datasets in TE 22 expression and profile . b . Principal component analysis ( PCA ) of primed ( in TeSR or KSR / FGF2 23 conditions ) or HENSM naïve conditions based on the differential expression of transposable elements 24 ( TEs ) . c . Correspondence between TE expression in HENSM naïve vs . primed ESCs and single - cell 25 human embryonic stages ( Yan et al . , 2013 ) . For every stage of human embryonic development , a 26 statistical test was performed to find the TEs that have a different expression level compared to the other 27 stages . The proportions of developmental stage - specific TEs that are upregulated ( p < 0 . 05 , 2 - fold 28 change ) in naive or primed cells are indicated in orange and blue , respectively , while TEs that did not 29 59 change expression are indicated in gray . The samples include all HENSM based naive cells that we 1 examined in the PCA analyses in b . 2 3 Figure S12 . Rewiring TE elements in naïve HENSM conditions . 4 Changes in distinct TE families are with indications whether they were downregulated or upregulated 5 between HENSM naïve and primed conditions . 6 7 Figure S13 . Full analysis of LOI in HENSM and HENSM - ACT PSCs . 8 Heatmap of biallelic expression including the complete list of imprinted genes in hPSC samples . Genes 9 are arranged according to their genomic proximity . NE - not expressed ( FPKM < 0 . 2 ) . 10 11 Figure S14 . HENSM - derived human naïve pluripotent stem cells are competent for interspecies 12 chimaera . 13 ( a ) Representative images of whole mount in toto microscopy of chimaeric embryos over several 14 developmental stages ( e11 . 5 till e13 . 5 ) . Panels on the right are zoomed - in regions of tiles on the left side . 15 Hoechst was used as counterstain and anti - GFP staining used to trace human iPSC - derived descendants . 16 ( b ) Frozen tissue sections of E17 . 5 chimaeric embryos were stained for GFP and Human - Nuclei to 17 confirm human origin identity . Non - injected embryos served as negative control . GFP , Human - Nuclei , 18 overlap as well as merged are zoomed - in regions of lung tissue depicted in red squares in the tiles . White 19 arrowheads in insets point out co - localization between GFP and Human - Nuclei . GFP , green fluorescent 20 protein ; WT , wild type ; iPSC , induced pluripotent stem cell . Tile scale bar 500 um . Zoomed - in scale bar 21 100 um . 22 23 Figure S15 . HENSM - derived human naïve pluripotent stem cells integrate successfully into mouse 24 embryos and acquire respective tissue identity . 25 Representative images of frozen tissue sections of E17 . 5 chimaeric embryos were stained for GFP and 26 Pro - Spc for lung - specific alveolar - surfactant secreting cells . Non - injected embryos served as negative 27 control . a . GFP , Pro - Spc , overlap as well as merged are zoomed - in regions of lung tissue depicted in red 28 squares in the tiles . White arrowheads in insets point out co - localization between GFP and Pro - Spc . GFP , 29 green fluorescent protein ; WT , wild type ; Pro - Spc , prosurfactant Protein C ; iPSC , induced pluripotent 30 60 stem cell . Tile scale bar 1000 um . Zoomed - in scale bar 50 um . b - c . AS above but for Aqp5 ( aquaporin 5 ) 1 and CC10 ( Clara - cell 10 ) lung cell markers . 2 3 Figure S16 . P53 ( TP53 ) targeting in human iPSCs . 4 a . Design of CRISPR / Cas9 targeting Exon 4 of hTP53 to generate knock - out with the guide RNA in red 5 and the PAM sequence in green . B . Western blot analysis showing complete depletion of TP53 protein in 6 various chosen clones . DNA sequence alignment showing out - of - frame insertions / deletions in clone C2 7 and a point mutation in clone E7 . c - d . Staining and karyotyping showed normal pluripotency marker 8 expression and karyotype in representative clones . e . NUMA staining of human vs mouse teratoma to 9 confirm human specificity of the NUMA antibody used in this study to trace GFP - labelled human iPSCs 10 after transplantation . NUMA , nuclear mitotic apparatus protein . Tile scale bar 1000 um . Zoomed - in 11 region scale bar 100 um . 12 13 Figure S17 . Integration of P53 null naïve hiPSCs into mouse blastocysts . 14 a . Phase and GFP image of P53 null naïve hiPSCs C8 clone expanded in HENSM - ACT . b . Phase and 15 GFP images of mouse blastocysts 24h after microinjection into mouse morulas were conducted . c . 16 Representative immunostaining images of mouse blastocysts 24h after GFP labeled human naïve P53 null 17 hiPSCs were microinjected into mouse morulas . d . Representative immunostaining images of mouse 18 blastocysts 48h after GFP labeled human naïve P53 null hiPSCs were micro - aggregated with mouse 2 - 19 Cell embryos . 20 21 Figure S18 . Boost in cell chimerism contribution by depleting P53 endows in hiPSCs before 22 microinjection . 23 a . Representative images depicting integration of P53KO GFP - labelled hiPSCs into different locations 24 within developing E9 . 5 / E10 . 5 mouse embryo in comparison to WT GFP + - cells and non - injected embryos 25 used as negative controls . Red squares in the first column represent zoomed - in areas shown in the 26 following images 1 and 2 . Tile scale bar 200 um . Inset scale bar 50 um . b . Abnormally developed e10 . 5 27 chimaeric embryo with abundant GFP contribution in comparison to non - injected WT embryo lacking 28 any GFP signature . Hoechst and CellTracker were used for counterstaining . Scale bar 50 um . c . 29 Representative images of whole - mount in toto imaged e15 . 5 mouse embryos are shown in comparison to 30 61 non - injected wild - type embryos . White squares in tiles outline zoomed - in regions in subsequent panels . 1 GFP staining was used to trace hiPSC - derived progeny and CellTracker and Hoechst as counterstaining . 2 Scale bar 1 mm . GFP , green fluorescent protein ; p53 , tumor protein p53 ; iPSC , induced pluripotent stem 3 cell ; WT , wild type . 4 5 Figure S19 . Naïve hiPSCs contribution to chimaeric mouse embryos . 6 a . FACS analysis for GFP detection in mouse embryos . Non - injected embryos and a parental GFP + 7 hiPSC line were used as controls . b . Human mitochondrial specific DNA detection assay for dilutions 8 between human and mouse cells , chimaeric embryos , non - injected embryos as well as for different pure 9 mouse tissues . Dotted line represents threshold level set in this PCR . Green arrows highlight mouse 10 chimeric embryos with high levels of human DNA detection . c - e . IHC staining of various regions of 11 chimaeric embryos for NUMA to trace GFP + human P53KO hIPSC derived cells . GFP , NUMA , overlap 12 as well as merged are zoomed - in regions of lung tissue depicted in red squares in the tiles . White 13 arrowheads in insets point out co - localization between GFP and NUMA . Non - injected embryos served as 14 negative control . Tile scale bar 2000µm . Zoomed - in region scale bar 100µm . 15 16 Figure S20 . Naïve hiPSC - derived cells colonize various anatomical regions throughout whole mouse 17 embryo . 18 a - d . IHC staining of various regions of chimaeric embryos for NUMA to trace P53KO hIPSC derived 19 GFP + human cells . GFP , NUMA , overlap as well as merged are zoomed - in regions of lung tissue 20 depicted in red squares in the tiles . White arrowheads in insets point out co - localization between GFP and 21 NUMA . Non - injected embryos served as negative control . Tile scale bar 1000 um and 2000 um . Zoomed - 22 in region scale bar 100µm . 23 24 Figure S21 . Contribution of GFP + hiPSC - derived progeny to ectodermal - neural lineages within 25 chimaeric mouse embryos . 26 a - b . Representative images of IHC staining of injected ( upper panels ) and non - injected E15 . 5 mouse 27 embryos ( lower panels ) for ( a ) brain TUJ1 and ( b ) cochlea SOX2 respectively are shown . GFP served as 28 human cell tracer of microinjected P53KO hiPSC , and TUJ1 and SOX2 as neural progenitor signature . 29 GFP , TUJ1 or SOX2 , overlap as well as merged constitute zoomed - in regions of tissues depicted in red 30 6 2 squares in the tiles . White arrowheads in insets depict co - localization of GFP and TUJ1 . GFP , green 1 fluorescent protein ; TUJ1 , neuron - specific class III beta - tubulin ; SOX2 , sex determining region Y - box ; 2 P53KO , knock - out of tumor protein p53 . Tile scale bar 2000 um . Non - injected scale bar 1 mm . Zoomed - 3 in region scale bar 100µm . 4 5 Figure S22 . Contribution of GFP + hiPSC - derived progeny to ectodermal - neural lineages within 6 chimaeric mouse embryos . 7 a - c . Representative images of IHC for TUJ1 and GFP of injected ( upper panels ) and non - injected E15 . 5 8 mouse embryos ( lower panels ) for each tissue type ( a , b , c ) respectively are shown . GFP served as human 9 cell tracer of microinjected P53KO hiPSC . GFP , TUJ1 , overlap as well as merged constitute zoomed - in 10 regions of tissues depicted in red squares in the tiles . White arrowheads in insets depict co - localization of 11 GFP and TUJ1 . GFP , green fluorescent protein ; TUJ1 , neuron - specific class III beta - tubulin ; P53KO , 12 knock - out of tumor protein p53 . Tile scale bar 2000µm . Zoomed - in region scale bar 100µm . 13 14 Figure S23 . Contribution of GFP + hiPSC - derived progeny to ectodermal - neural lineages within 15 chimaeric mouse embryos . 16 a - d . Representative images of IHC for SOX2 of injected ( upper panels ) and non - injected E15 . 5 mouse 17 embryos ( lower panels ) for each tissue type respectively are shown . GFP served as human cell tracer of 18 microinjected P53KO hiPSC , and SOX2 as neural progenitor signature . GFP , SOX2 , overlap as well as 19 merged constitute zoomed - in regions of tissues depicted in red squares in the tiles . White arrowheads in 20 insets depict co - localization of GFP and SOX2 . GFP , green fluorescent protein ; SOX2 , sex determining 21 region Y - box ; P53KO , knock - out of tumor protein p53 . Tile scale bar 1000µm . Zoomed - in region scale 22 bar 100µm . 23 24 Figure S24 . Contribution of GFP + hiPSC - derived progeny to endoderm and mesoderm lineages 25 within chimaeric mouse embryos . 26 a - d . Representative images of IHC for SOX17 of injected ( upper panels ) and non - injected E15 . 5 mouse 27 embryos ( lower panels ) for each tissue type respectively are shown . GFP served as human cell tracer of 28 microinjected P53KO hiPSC and SOX17 as endoderm progenitor and mesoderm - progeny tissue marker . 29 GFP , SOX17 , overlap as well as merged constitute zoomed - in regions of tissues depicted in red squares 30 63 in the tiles . White arrowheads in insets depict co - localization of GFP and SOX17 . GFP , green fluorescent 1 protein ; SOX17 , SRY - related HMG - box 17 ; P53KO , knock - out of tumor protein p53 . Tile scale bar 2 2000µm . Zoomed - in region scale bar 100µm . 3 4 Figure S25 . Contribution of GFP + hiPSC - derived progeny to endoderm and mesodermal lineages 5 within chimaeric mouse embryos . 6 a - b . Representative images of IHC for SOX17 of injected ( upper panels ) and non - injected E15 . 5 mouse 7 embryos ( lower panels ) for each tissue type respectively are shown . GFP served as human cell tracer of 8 microinjected P53KO hiPSC and SOX17 as endoderm progenitor and mesoderm - progeny tissue marker . 9 GFP , SOX17 , overlap as well as merged constitute zoomed - in regions of tissues depicted in red squares 10 in the tiles . White arrowheads in insets depict co - localization of GFP and SOX17 . GFP , green fluorescent 11 protein ; SOX17 , SRY - related HMG - box 17 ; P53KO , knock - out of tumor protein p53 . Tile scale bar 2000 12 um . Zoomed - in region scale bar 100 um . c . Summarizing table for outcome of injections of different 13 naïve and primed hiPSCs into mouse embryos . 14 15 Figure S26 . Nuclear β CATENIN signaling induces priming of human PSCs . 16 a . FACS analysis for OCT4 - GFP reporter in HENSM conditions before and after depletion of indicated 17 media components . b . Mouse and human β CATENIN null ESCs were made transgenic for an exogenous 18 validated tamoxifen inducible β CATENIN transgene ( βCAT KO - βC ateninERT - Tg ) . Immunostaining 19 confirms increase in nuclear β CATENIN upon tamoxifen addition ( 4OHT ) in mouse and human validated 20 clones used for analysis . c . RT - PCR analysis for naïve pluripotency maker expression in Human WIBR3 - 21 △ PE - βCAT KO ; β CateninERT - Tg line before and after Tamoxifen addition for 48 hours . Values were 22 normalized to ACTIN and GAPDH . Primed expression levels were set as 1 . * t - test p Value < 0 . 01 . Naïve 23 pluripotency marker expression where significantly downregulating upon induction of nuclear βCATENIN 24 signaling in human PSCs . 25 26 Figure S27 . Generation of β CATENIN knockout hESCs with pluripotency reporters . 27 a . Scheme depicts CRISPR / Cas9 based strategy for generating β CATENIN KO in WIBR3 - OCT4 - GFP 28 and WIBR3 - Δ PE - OCT4 - GFP primed human ESCs . b . Sequencing results and c . western blot validation 29 64 for β CATENIN in correctly targeted WIBR3 - OCT4 - GFP clones . d . Sequencing results and e . western 1 blot validation for β CATENIN in correctly targeted WIBR3 - Δ PE - OCT4 - GFP clones . 2 3 Figure S28 . Ablating TCF3 does not support human naïve pluripotency . 4 a . Scheme depicts strategy for TCF3 knockout induction in human WIBR3 - OCT4 - GFP ESC . b . Western 5 blot analysis validates TCF3 protein deletion in candidate correctly targeted clones . c . Phase contrast 6 images of TCF3 - / - ESCs before and after TNKi removal from HENSM conditions . d . RT - PCR analysis 7 for naïve pluripotency maker expression in the indicated conditions , with and without TNKi , in TCF3 WT 8 and KO hESCs . Values were normalized to ACTIN and GAPDH . * t - test p Value < 0 . 01 . 9 TCF3 + / + HENSM expression levels were set as 1 . e . Percentage of OCT4 + cells at P4 from TET - OFF - 10 DNMT1 , DNMT1 - TET - OFF cell lines and OCT4 - GFP cells in the absence of exogenous L - Glut in 11 primed , naïve HENSM , and naïve HENSM supplemented with 1 . 5µM CHIR99021 . f . Normalized 12 expression pattern of TCF3 and AXIN1 / 2 in mouse and human , as reported in previous single - cell RNA - 13 seq measurement in mouse and human embryos . 14 15 Figure S29 . LIF - STAT3 signaling supports human naïve pluripotency but overall can be 16 dispensable . a . RT - PCR analysis for naïve pluripotency maker expression in the indicated naïve and 17 primed conditions , with and without LIF . Values were normalized to ACTIN and GAPDH . Primed 18 expression levels were set as 1 . * t - test p Value < 0 . 01 ; NS - not significant . b . Scheme depicts strategy for 19 generating STAT3 knockout WIBR3 - OCT4 - GFP hESCs . c . Western blot validation for loss of STAT3 20 protein in correctly targeted clones that were validated by southern blot analysis . d . RT - PCR analysis for 21 naïve pluripotency maker expression in the indicated lines and conditions . Values were normalized to 22 ACTIN and GAPDH . Primed expression levels were set as 1 . * t - test p Value < 0 . 01 ; NS - not significant . 23 Naïve pluripotency marker remain highly expressed in STAT3 KO ESCs in HENSM conditions , when 24 compared to primed cells that do not express at all these specific markers . 25 26 Figure S30 . Generation of KLF4 and / or KLF17 knockout human ESCs . 27 a . Scheme depicts strategy for generating KLF17 null WIBR3 - OCT4 - GFP hESCs . b . Southern blot 28 analysis validating correctly targeted clones for KLF17 KO . c . Immunostaining validation for loss of 29 KLF17 protein in early passage KO clone expanded for 2 passages in HENMS - ACT . d . Scheme depicts 30 65 strategy for generating KLF4 knockout WIBR3 - OCT4 - GFP hESCs . e . Southern blot analysis validating 1 correctly targeted clones for KLF4 KO . f . Immunostaining validation for loss of KLF4 protein in early 2 passage KO clone expanded for 2 passages in HENMS - ACT . Same strategy for making KLF4 knockout 3 ( d - f ) was applied on KLF17 - / - cells to generate double knockout validated clones . 4 5 Figure S31 . Klf17 is dispensable for mouse naïve pluripotency in vivo and in vitro . 6 a . KLF4 and KLF17 relative abundance in pre - implantation stages as measured in vivo by scRNA - seq . 7 Please note that KLF17 is not expressed in the mouse ICM ( but rather a 2 Cell stage in the mouse ) , while 8 in humans it is rather upregulated at the morula - ICM stages . b . Scheme depicts strategy for generating 9 Klf17 null allele in V6 . 5 mouse ESCs . c . PCR analysis for checking correctly targeted clones . d . Southern 10 blot analysis validating correctly targeted clones . e . PCR analysis for confirming correct flipping out of 11 antibiotic resistance cassette prior to ESC microinjection . f . Germ line transmission from Klf17 targeted 12 mouse ESCs as confirmed by obtaining agouti colored pups . g . RT - PCR analysis for naïve pluripotency 13 markers in WT and Klf17 - / - mouse ESCs expanded in 2i / LIF conditions . Values per each gene were 14 normalized in WT ESCs as 1 . Klf17 - / - did not show any significant change in naïve pluripotency marker 15 expression . ( NS – not significant ) . 16 17 Figure S32 . Alternative HENSM conditions for human naïve PSCs without using ERKi . 18 a . Spearman correlation matrix of primed samples , along HENSM , HENSM - ACT , tHENSM and 19 0HENSM samples , as well as previously published naïve and primed samples ( Takashima et al , 2014 ) . b - 20 d . Percentage of OCT4 - GFP + cells at P4 from WIBR3 - OCT4 - GFP cells in HENSM , tHENSM and 21 0HENSM conditions without METTL3 ( b ) , DNMT1 ( c ) or exogenously added L - Glutamine ( d ) . Note 22 that removal of DBZ from tHENSM or 0HENSM results in loss of maintenance of pluripotency when 23 these factors are depleted . e . Competence for human naïve PSCs for differentiating into PGCLCs in vitro 24 was assays for NANOS3 - mCherry knock in reporter line expanded for at least 3 passages in HENSM , 25 tHENSM or 0HENSM conditions . Percentage of NANOS3 + cells detected by FACS analysis are shown . 26 27 Figure S33 . Pattern of DNA methylation regulators in alternative tHENSM and OHENSM 28 conditions . 29 66 a . Immunostaining for DNMT3L in primed and different naïve conditions are shown . DNMT3L levels are 1 reduced in tHENSM and 0HENSM conditions as seen in primed conditions . b . Global methylation 2 histogram calculated from primed samples , and naïve samples that were maintained in various conditions , 3 including titrated ERKi supplementation , along with previously published Reset - naïve * and primed * 4 samples ( Takashima et al . Cell 2014 ) , and human ICM samples . DNMT1 - / - ( from TET - OFF lines ) samples 5 were used as negative control for methylation . Dark blue - percentage of highly methylated CpGs ( > 0 . 9 6 methylation level ) , light blue – percentage of lowly methylated CpGs ( < 0 . 1 methylation level ) . Yellow 7 dots – sample methylation average . c . Average methylation as calculated from primed samples , and naïve 8 samples that were maintained in various conditions , including titrated ERKi ( tHENSM and 0HENSM ) 9 supplementation . 10 11 Figure S34 . Generation and validation of RBPj knockout human ESCs . a . Scheme depicts targeting 12 strategy for generating knockout human WIBR3 hESCs for RBPj . b . Correctly targeted clones validated 13 by southern blot analysis were confirmed for loss of RBPj protein by western blot analysis . 14 15 Supplementary Table Legends 16 17 Table S1 . Diferentially expressed genes between naive and primed samples ( Fold change > 2 , adjusted p - 18 value < 0 . 1 ) in dataset 1 , along with normalized counts of all genes . Related to Fig . 3 & S8 . 19 20 Table S2 . Normalized counts of all genes in Dataset 2 ( HENSM naive , primed , tHENSM naïve , 21 0HENSM naïve samples ) . Related to Fig . 7 , S8 , S9 & S32 . 22 23 Table S3 . Differentially expressed transposable elements , based on dataset 1 . Related to Fig . S11 - S12 . 24 25 Table S4 . Significant binding motifs identified by Homer software , calculated from the targets of 26 KLF4 , KLF17 , TFAP2C , SOX2 , OCT4 , NANOG . Related to Fig . 6 . 27 28 Table S5 . Gene targets and sample statistics of ChIP - seq experiments of KLF4 , KLF17 , TFAP2C , SOX2 , 29 OCT4 , NANOG in HENSM and Primed conditions . Related to Fig . 6 . 30 67 1 2 \ No newline at end of file diff --git a/silver_data/f3e59cff414dfcff98b48eb745be7fb95b1bc8ca.pdf.txt b/silver_data/f3e59cff414dfcff98b48eb745be7fb95b1bc8ca.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..caed2dddd9d20782a2fc76eafe68a41592cc0032 --- /dev/null +++ b/silver_data/f3e59cff414dfcff98b48eb745be7fb95b1bc8ca.pdf.txt @@ -0,0 +1 @@ +City , University of London Institutional Repository Citation : Maiden , N . ORCID : 0000 - 0001 - 6233 - 8320 , Brock , G . , Zachos , K . ORCID : 0000 - 0003 - 1977 - 7090 and Brown , A . ( 2018 ) . Making the News : Digital Creativity Support for Journalists . In : CHI ' 18 Proceedings of the 2018 CHI Conference on Human Factors in Computing Systems . ( 475 . ) . New York , USA : ACM . ISBN 978 - 1 - 4503 - 5620 - 6 This is the accepted version of the paper . This version of the publication may differ from the final published version . Permanent repository link : http : / / openaccess . city . ac . uk / 19351 / Link to published version : http : / / dx . doi . org / 10 . 1145 / 3173574 . 3174049 . Copyright and reuse : City Research Online aims to make research outputs of City , University of London available to a wider audience . Copyright and Moral Rights remain with the author ( s ) and / or copyright holders . URLs from City Research Online may be freely distributed and linked to . City Research Online : http : / / openaccess . city . ac . uk / publications @ city . ac . uk City Research Online Making the News : Digital Creativity Support for Journalists Neil Maiden , Konstantinos Zachos , Amanda Brown , George Brock City , University of London 106 Bunhill Row , London EC1Y 8TZ UK N . A . M . Maiden @ city . ac . uk Lars Nyre , Aleksander Nygård Tonheim University of Bergen , P . O . Box 7800 5020 Bergen Norway , Lars . Nyre @ uib . no Dimitris Apostolou Institute Communica - tion and Computer Systems , 15780 Zo - grafou , Greece DApost @ mail . ntua . gr Jeremy Evans Explaain Ltd Urban Innovation Centre , 1 Sekforde Street , London EC1R0BE UK jeremy @ explaain . com ABSTRACT This paper reports the design and first evaluations of new digital support for journalists to discover and examine crea - tive angles on news stories under development . The support integrated creative news search algorithms , interactive crea - tive sparks and reusable concept cards into one daily work tool of journalists . The first evaluations of INJECT by jour - nalists in their places of work to write published news sto - ries revealed that the journalists generated new angles on existing stories rather than new stories , changed their writ - ing behaviour , and reported evidence that INJECT use had the potential to increase the objectivity and the boldness of journalism methods used . Author Keywords Creativity support ; journalism ; Google Docs Add - on ACM Classification Keywords D . 5 . 2 [ User Interfaces ] : User - centered design , voice I / O General Terms Design , human factors INTRODUCTION Journalism involves the search for [ 3 ] and critical analysis of information [ 21 ] to create news stories and reports . How journalists discover and select sources of this information is important , both to avoid bias and to be credible and trusted . However , discovering and examining information sources takes time – time that journalists increasingly lack as news and media organizations are squeezed by reducing circula - tions , revenues and staff numbers [ 29 ] . As a consequence , many journalists use subsets of available and familiar in - formation sources to create stories in the limited time avail - able . However , this behaviour that can reduce the number and diversity of angles used to report news , as well as weaken the role the journalism in democracies [ 3 ] because citizens lack enough information to hold the powerful to account . Journalism is one of the creative industries . Most journalists exhibit professional - level creativity because their news sto - ries generate income and provide a living [ 14 ] . However , support for journalist creative thinking is limited . Most con - tent management systems and search engines only have keyword search functions to discover information sources [ 18 ] , and require journalists already to know and describe the creative angles to investigate . Interactive creativity sup - port tools exist for different creative industries ( e . g . [ 1 , 13 ) ] , but newsrooms lack the digital support to discover possible creative angles on news stories , i . e . stories that are novel and have value to their organisations , their readers and their democracies [ 3 ] . This paper reports new digital creativity support for use by journalists . The tool , called INJECT , integrated natural lan - guage processing , creativity search algorithms and interac - tive creative sparks . To support use by journalists , INJECT was implemented as an Add - on sidebar for the Google Docs text editor often used by journalists , so that its creativ - ity support was visible next to the story being written . The support had the potential to both relieve journalists of a demanding process that is difficult to do well under time pressure , as well as to expand possibilities open to journal - ists . The next 3 sections summarize the creativity challeng - es that newsrooms face , report digital creativity support generated in different creative industries , and define crea - tivity in newsroom journalism . The paper then describes INJECT’s interactive creativity support and software archi - tecture , and how the creativity support was designed within the Add - on sidebar’s constraints . It then reports results from first evaluations of the effect of INJECT use on how journalists discovered news stories . It ends with threats to the validity of the early evaluation results , limitations and conclusions , and how INJECT might be deployed more effectively in newsrooms . RELATED WORK Surprisingly few studies to inform the design of new digital support for journalists have been reported . Exceptions were the design implications for future tools to discover local news information sources reported in [ 9 ] , and the Maater system that corrected news misinformation using high - ranking crowd - sourced entries [ 16 ] . To workaround the lack of digital tools to support creativity , some journalists have adopted general - purpose ones such as import . io and www . social - searcher . com that keyword - search multiple social media channels but not news information sources , and present comparative results . However , these tools were neither tailored to support journalist tasks nor provided ex - plicit support for discovering creative angles on stories . Other digital journalism tools have implemented artificial intelligence techniques . For example , the Alchemy API was developed to support journalists to make sense of unstruc - tured natural language data and generate human insights using text analysis and visualization mechanisms . Likewise the NewsReader tool implemented text analysis and artifi - cial intelligence mechanisms to build structured event in - dexes of large volumes of financial and economic data for decision making from news content [ 23 ] . However , none of these tools had capabilities to search information sources to discover possible creative angles on future stories , or to support human creative thinking to examine these angles . Unlike in journalism , digital creativity support has been implemented for professionals in other creative industries such as the performing arts , music , and film and television [ 1 , 13 ] . Examples of the digital support include StoryCrate , a collaborative editing tool developed to drive users’ crea - tive workflows within a location - based television produc - tion environment [ 4 ] and Trigger Shift , which appropriated information technologies into performance art in theatre [ 25 ] . More recent studies have revealed the need to inte - grate this digital creativity support into daily work practic - es . Not only does this avoid imposing additional cognitive load on professionals , but it also supports pain - free experi - mentation [ 10 ] . It can also deliver simultaneous productivi - ty and creativity benefits to these professionals [ 17 ] . TODAY’S CRISIS IN JOURNALISM The digitalization of the production , distribution and con - sumption of news has led many news businesses to become uncompetitive [ 6 ] . The result has been a decade - long crisis in journalism . Even though this crisis has required news businesses to operate more competitively [ 24 ] , many work practices have remained unchanged , due in part to the con - servative attitudes of journalists [ 7 ] . Newsrooms have not adopted many new digital tools , even though the need to discover and examine information from multiple sources had been recognized widely ( e . g . [ 30 ] ) . That said , case studies reveal that journalists are still moti - vated by opportunities to develop new skills , including new creative skills [ 19 ] . Indeed , investigative and visual journal - ism demand new forms of creative search and association . Computational exploration in journalism increasingly in - volves creative thinking at the intersection of journalism and data technology . This creative thinking transcends geo - graphical , disciplinary and linguistic boundaries [ 11 ] . Mul - ti - skilled journalists have more control over their work and are more creative [ 33 ] , although the high demands of daily production mean that many have struggled to develop these creative capabilities to their full potential . Amabile identified that people who are not expert in crea - tivity work need task motivation , domain - specific skills and creativity skills to produce creative outcomes [ 2 ] . Whilst most journalists are motivated and have journalism skills , most journalists also lack the creativity skills that are need - ed [ 19 ] . Therefore , inspired and informed by the challenge to design new digital tools for journalists that exploit oppor - tunities to support their development and use of creative thinking skills , a new interdisciplinary collaboration be - tween journalists and computer scientists formed . The new collaboration developed digital creativity support for newsroom journalists to : R1 ) discover angles on news stories that are more creative than at present , from more information sources , in order to be more credible and trust - ed ; R2 ) discover new angles more productively than at pre - sent , to be creative with less resources in order to enable their newsrooms to operate more competitively [ 24 ] ; R3 ) use the creativity support in their everyday work tools , to encourage more journalists to adopt it , and ; R4 ) learn good creative practices of more experienced journalists , because journalists seek new creative thinking skills [ 19 ] . The focus was on support for rather than automation of the work of journalists – journalists using the digital support were still expected to browse news information , discover angles on stories and write stories with these angles . This paper re - ports the research to achieve requirements R1 , R2 and R3 , and first evaluations of the new digital creativity support by journalists to investigate 2 research questions . REFRAMING JOURNALISM AS LITTLE - C CREATIVITY To design the digital support needed by journalists , the col - laboration developed a definition of creativity that was spe - cialized to newsroom journalism . Although many journal - ists engage in professional - level creativity [ 14 ] , much of their daily work writing news stories can be characterized as little - c creativity – daily activities not perceived to be creative [ 14 ] , and undertaken by people who are not expert with creativity skills . Therefore , building on established definitions that creativity produces outcomes that are novel and appropriate to the task [ 27 ] , little - c creativity in news - room journalism was defined as activities to write news stories that are novel to the journalist and appropriate for publication . Furthermore , to distinguish these activities from straightforward news reporting , the journalist uses his or her creativity skills to generate the knowledge incorpo - rated into the news stories and / or use in the activities to write the news stories , to render the stories novel . This con - trasts with traditional news reporting , in which the journal - ist acts primarily as a stenographer who reports facts as accurately as possible , and does not introduce new knowledge that s / he generates . For example , a journalist on a local newspaper who reports important facts from a town hall meeting that might be new to the journalist , such as a planning decision , is not engaged in little - c creativity because no creativity skills are used and no new knowledge is generated . In contrast , if the journalist uses creativity skills to generate new knowledge about , for example , a connection between this decision and the busi - ness interests of a politician , and this new angle is appropri - ate for publication , then the journalist exhibits little - c crea - tivity in newsroom journalism . Likewise , if the journalist interviews the politician to acquire more information based on the knowledge generated , then s / he is also exhibiting little - c creativity in newsroom journalism . However , as newsrooms lack the resources [ 6 ] to train jour - nalists to acquire creativity skills using traditional methods , a new digital tool was investigated to support newsroom journalists to use simple creativity skills interactively to discover creative angles on news stories more productively using their everyday work tools . INJECT’S DIGITAL CREATIVITY SUPPORT INJECT is a new digital support tool for individual journal - ists to discover and examine creative angles on news stories under development . It was built to support human - centred creative cognition [ 15 ] , in which idea generation about new angles took place concurrently with information search . INJECT searched news information with creative strategies that codified the expertise of experienced journalists . It supported idea generation by presenting the retrieved news information and interactive sparks that codified creativity heuristics . The presented news information and sparks were designed to encourage individual journalists to use and learn simple creativity skills . INJECT was made available to an individual journalist as a single sidebar , so that the journalist could start news information searches and gener - ate ideas about new angles next to a new story being writ - ten . As such , INJECT did not automate most of the journal - ist’s work . The journalist still needed to understand and select between recommendations to discover angles , as well as examine , interpret , style and present [ 26 ] new articles . INJECT’s User - Centred Design The interaction design of INJECT [ 18 ] involved journalists at all stages . After semi - structured interviews with journal - ism experts to discover the digital tools that journalists cur - rently use , a decision was made to implement INJECT with Google Docs . Many journalists use Google Docs instead of bespoke news content management systems for early story development because of its familiarity , flexibility and high - er number of useful features . To kick - start the user - centred design , different paper - based then digital wireframes of the sidebar were developed and presented to professional jour - nalists . Over the next 8 months , new releases of the work - ing INJECT software were tested for their usability and effectiveness , first with journalism students who had no direct relationship to the authors [ 18 ] , then with profession - al journalists working in UK magazines , regional Norwe - gian newspapers and networks of freelance journalists in the Netherlands . INJECT’s Sidebar : A Google Docs Add - on To support journalists to undertake the pain - free explora - tion of information and ideas that was associated with crea - tive thinking [ 10 ] , INJECT’s digital creativity support was integrated into the Google Docs editor . Google supported a research sidebar of the editor window called the Google Docs Add - on that allowed a user to start searches and browse results [ 31 ] . The team decided to deliver INJECT’s creativity support through this Add - on , referred to simply as the sidebar in this paper . The sidebar was the pre - defined Google Docs component that appeared to the right of the editor . However , the side - bar had a fixed width ( 300px ) and poor performance with server - side libraries . Therefore , design challenges included embedding usable and effective creativity support that called different server - side services into the sidebar . To meet the challenges , INJECT was implemented with : ( A ) features to generate candidate search terms directly from text already written in the larger editor window ; ( B ) small icons with mouse hover - box descriptions , to control the sidebar ; ( C ) mouse hover - boxes to present additional in - formation quickly in context ; ( D ) overlays to present more information in the sidebar space , and ; ( E ) server - side ser - vices that preloaded news information to overcome the sidebar’s performance limitations . The designs and imple - mentations of these 5 features were improved incrementally during the journalist - led prototyping , for example by merg - ing control icons and reducing the number of required in - teractions , so that , for example , the journalist received crea - tivity support in as little as 2 clicks . Figure 1 shows the final design of the INJECT sidebar . The left side shows the launch setup and 4 functional areas . The first area was the news topic space – a journalist could enter terms or import them directly from the text editor to de - scribe topics of the current news story . The second area was the strategy space – a journalist could invoke different crea - tive search strategies using the described topics . The third , which occupied over 80 % of the sidebar , was the infor - mation space . A journalist could scroll , mouse hover - over and click retrieved news information and creative sparks to discover and generate new ideas for news angles . The fourth space , fixed at the bottom of the sidebar , was the control space . The journalist could access different tabs to manage tool settings , collaborate with online communities and follow a tutorial . An example of this tutorial is shown on the right side of Figure 1 . A small number of INJECT features were also implemented in a separate dialog com - ponent that would appear over the editor window . However , due to the limited functionality of that component , most features were implemented in the sidebar , so that journalists could simultaneously use it and write new articles . INJECT’s Creativity Support Imagine a fictional journalist who used the INJECT Google Docs sidebar to discover and examine creative angles for a story related to the 2017 Italian migration crisis . At any time s / he could highlight text written in the editor – in this example : Europe’s annual summer migration crisis impacts one country more than any other – Italy – then click the insert button on the sidebar . INJECT parsed the highlighted text to extract stemmed nouns , verbs and proper names as candidate topics . The journalist could then edit these nouns , verbs and proper nouns before instructing INJECT to dis - cover possible creative angles using topics such as migra - tion , summer , crisis and Italy , shown in the news topic space at the top of the sidebar in Figure 2 . 1 2 3 4 Figure 1 . The INJECT sidebar’s 4 functional areas and an example of the tutorial support available to journalists Figure 2 . The INJECT sidebar on the right side of the Docs editor , showing creativity support with the people strategy The journalist could then instruct INJECT to discover pos - sible creative angles using 6 pre - defined types of angle by clicking icons in the strategy space . Each of the icons in - voked a different creative strategy that searched news in - formation sources associated with a different type of crea - tive angle : A . Quantifiable : discover and examine quantified infor - mation associated with the news story ; B . People : discover and examine information about people associated with the news story ; C . Causal : discover and examine information about events associated with the background of the news story ; D . Quirky : discover and examine comical information as - sociated with the news story ; E . Ramifications : discover and examine information asso - ciated with future consequences of the news story ; F . Data visualizations : discover and examine data sets and visualizations associated with the news story . Each of these 6 strategies codified the expertise that experi - enced journalists had reported to discover creative angles on news stories . To uncover and describe candidate strate - gies , semi - structured interviews with experienced journal - ists and digital experts in journalism had been held . The candidate strategies were then validated with other experi - enced journalists , and extended and refined [ 18 ] . Each strategy operationalized the elicited journalism expertise so that INJECT users could exploit it to discover possible creative angles on news stories . So for example , if the fictional journalist clicked the people icon , INJECT used the selected terms to search and retrieve news information sources , extract the names of individuals from these source and present the extracted people’s names in the sidebar , with basic information about the individual and the original news item . Figure 2 also depicts the side - bar’s information space , in which one individual , Pope Francis , is presented above the information source in which he is named . The journalist could scroll the sidebar to search the information about Pope Francis and generate new ideas for story angles from it . Other individuals pre - sented for this search were politicians Lulzim Basha and Vince Cable and philanthropist George Soros . Then , to ex - amine a selected individual , the journalist clicked the indi - vidual’s name , and INJECT opened the dialog window with a photograph and description of the person extracted from Wikipedia sources , to stimulate more idea generation about new angles . If needed , the journalist could also request more people using the refresh button . If the journalist clicked the causal icon , INJECT searched for and retrieved explainer - style , long - read news articles . It would present each article’s title , source and first line in the sidebar . Figure 3 depicts 2 examples of this sidebar , with an Independent newspaper article on an Amnesty International report on EU naval operations on the left side . Again , the journalist could scroll the sidebar to search presented in - formation , generate new ideas for story angle from it , and request more information by clicking the refresh button . If s / he then clicked the article title , INJECT would open the article in the dialog window . This window then enabled the journalist to search each article with further keywords and view the original article online – features designed to sup - port idea generation – and to quick - reference the article and paste highlighted text from it into the Google Docs editor – features to support more productive work . Figure 3 . The INJECT sidebar showing support using the causal strategy , with mouse hover - over creative sparks gener - ated for news entities ( on left ) and stories ( on right ) Clicking on ramifications would have triggered INJECT to use the topic terms to provide similar support based on arti - cles that describe possible future implications . Moreover , for each news article in the sidebar , INJECT also presented a set of entities that were extracted automatically from the article , in colored rectangles – green for people , red for events , brown for places and blue for organizations . These entities summarized each article’s content . When the journalist positioned the mouse over each rectangle , INJECT presented a pop - up creative spark generated for that entity that codified creativity heuristics specialized to the journalism task . The creative sparks were designed to encourage the journalism to use and learn simple creativity skills . The left side of Figure 3 shows one example of these sparks : Explore recent data that is available about Syria , and build on interesting patterns in it . Other sparks in this sidebar included : Explore the future implications for Italy and how might its people be impacted , and Explore the characteristics of human rights that enhance the emotional impact . Creative sparks were also displayed if the journalist placed the cursor over an article title or individual name in the sidebar , see the right side of Figure 3 . The mouse hover - over feature enabled the journalist to explore multiple piec - es of creative advice generated from retrieved news infor - mation quickly , consistent with design advice in [ 10 ] . Furthermore , if the journalist clicked any colored rectangle , the sidebar displayed an interactive concept card containing a text description of the entity . The journalist could learn from and incorporate the description into his / her article . The journalist could also edit the card’s content to maintain a more personalized set of concept descriptions with which to write future articles more productively . Figure 4 shows the sidebar when the journalist clicked on the Open Europe entity . The concept card was presented in the sidebar , over the original greyed - out news content that the journalist could return to with one click . Figure 4 . An INJECT concept card with the sidebar On the other hand , if the journalist clicked quirky , INJECT searched for and retrieved political cartoons with matching names and captions , and presented each retrieved cartoon as a thumbnail image and caption and creative spark . Clicking a cartoon thumbnail opened it in the dialog window to sup - port further idea generation , as shown in Figure 5 . If the journalist had clicked data visualizations , INJECT would provide similar support using data and information visuali - zations extracted from retrieved news sources . Figure 5 . Use of INJECT’s quirky strategy , showing digital cartoons in the sidebar and dialog window In this example , our fictional journalist might have used the retrieved news information and creative sparks to generate a news story about the moral and legal conflicts between the roles played by the Papal State and Italian Republic . The story might also include the complexities arising from the intervention of non - governmental organizations in the Med - iterranean Sea . INJECT’s Architecture To deliver the described interactive digital creativity sup - port , the INJECT tool had 3 architecture layers : − A user interaction layer that enabled different interfaces , such as the sidebar plug - in for Google Docs ; − A data layer of 1 . 6 million tagged news published news stories discovered using RSS feeds from 150 news sources . The sources were selected by INJECT’s jour - nalist team to represent political perspectives and reduce the risk of echo chambers , and a database of over 40 , 000 political cartoons . INJECT also accessed infor - mation from Wikipedia but did not search it , so it was not part of the data layer ; − An application layer of software services that supported journalists to generate news stories more creatively and productively : ( i ) the creative search service manipulated topic descriptions from the editor to generate queries then implemented the different creative search strate - gies ; ( ii ) the news extraction service collected and in - dexed information from the 150 news sources prior to creative search ; ( iii ) the creative sparks service generat - ed creative sparks that were tailored to entities extracted from news information ; ( iv ) the concept card service that allowed individual journalists to edit and maintain personalised sets of concept cards , and ; ( v ) the persis - tence service that provided search session storage and retrieval capabilities . The news extraction and creative sparks services pre - generated news information content for the sidebar , to reduce the impact of the Add - on’s performance constraints . The news extraction service collected and indexed news information using public RSS feeds to the 150 news sources and tailored machine learning and natural language pro - cessing algorithms . It uploaded this information from the feeds every 30 minutes and stored it in a PostgreSQL data - base as metadata , with raw article data text as strings and a URL link to the source . It removed non - news content such as navigation links and adverts . It detected and extracted the people , location organization and event entities . It applied advanced natural language parsing to determine noun and verb phrases . And it uploaded each processed news article into an external Elasticsearch Cluster . At run - time , in response to a journalist entering topic terms and clicking one of the creative angle icons , the creative search service retrieved relevant news information , in two stages : 1 . It automatically disambiguated each topic term by dis - covering its correct sense according to the online lexi - con at WordNet using context knowledge from other terms in the query ( e . g . that migration is a group of peo - ple migrating together in some given time period rather than a periodic passage of groups of animals from one region to another for feeding or breeding ) [ 20 , 28 ] . It then expanded each term with other terms that have sim - ilar meanings ( e . g . the term migration with the above sense is synonymous with terms such as relocation and exodus ) and included these terms in the query . The ser - vice returned an unordered set of news articles or digital cartoons that achieved a threshold match score with the expanded search terms ; 2 . It filtered retrieved news articles and information using the strategy associated with the clicked icon . For exam - ple , for people , the service extracted from articles the name of each individual with a Wikipedia entry . For c ausal , it filtered to retain matched articles with more than 500 words and a minimum threshold of keywords indicative of causal articles – terms such as cause , im - pact and studies – from sources such as the Economist and the New York Times . And for quantifiable , it filtered to retain articles with a minimum threshold of quantity , measure and value keywords , for example Sterling , population and actual numbers . The creative sparks service generated the pop - up sparks for each retrieved article and entity extracted from each article . An individual creative spark associated 1 extracted entity or news article to 1 creative instruction . The sets of instruc - tions had been manually generated from websites and blogs that teach journalists to uncover new angles on stories . One set of instructions was generated for each of the 4 types of entity that were extracted – people , events , places and or - ganizations . Examples included : Unpick what the relevance of [ Place ] , as opposed to somewhere else , might have on the story and ; Explore the history and background of [ Or - ganization ] to obtain a new perspective on your story . A total of 34 creative instructions were implemented . One set of instructions was also generated for news articles re - trieved with each of the 6 creative strategies – people , causal , quirky , quantifiable , ramifications and data visuali - zations , and a total of 41 such instructions were implement - ed . Examples included : Use data types reported in this sto - ry , to generate a new angle , and : Make your angle more similar to the causal angle in this story . When invoked , the service used a randomizing function to attribute one instruc - tion string to one entity string of the same type , then con - catenated the strings to generate the spark . So , for the ex - tracted organization Open Europe from Figure 4 , INJECT might have presented a spark such as : Explore the history and background of Open Europe to obtain a new perspec - tive on your story . To retrieve and manage the concept cards , INJECT incor - porated Explaain , a service that organized news information in forms other than in articles [ 8 ] . Each concept card nor - mally stored one small chunk of news - related information , often similar to the length of a tweet . Phrases in each card were linked to other cards , similar to hyperlinks . INJECT also implemented other features that were request - ed by journalists during the prototyping to : - Discover news information and provide creative guid - ance in the languages that journalists work in , for exam - ple in Dutch and Norwegian as well as English ; - Discover information from newspaper archive sources , for example the digital archives of participating regional Norwegian newspapers . Although the design of INJECT had involved journalists , there had been no testing of the tool in newsrooms to ex - plore its impact on journalist work . The next section reports first evaluations of the impact of INJECT on journalist’s work using an established journalism framework . EVALUATING INJECT IN NEWSROOMS A version of INJECT that implemented the people , causal , quantitative and quirky strategies and all features reported in previous sections was used by 5 journalists to develop published news stories . Data collected after this use was analyzed to answer the following 2 research questions : RQ1 : Did each journalist use INJECT to discover new and useful angles on news stories ? RQ2 : Did each journalist change how s / he developed news stories as a result of using INJECT ? Evaluation Methods The INJECT version was evaluated by 5 journalists . Three of the journalists worked for 3 different regional Norwegian newspapers from the INJECT consortium and were selected by their editors . The 3 journalists were : an all - round jour - nalist responsible for content in the online version of the newspaper , with 4 years of journalism experience ; a photo - journalist who produced video reports , with 2 years experi - ence , and ; a news journalist who decided the content of all newspaper versions , with 13 years experience . One of the 3 journalists had limited involvement in the earlier co - design of INJECT . The other 2 journalists who evaluated INJECT worked at the same UK newspaper that specialized in digi - tal journalism , and were previously unconnected to the pro - ject . One was an editor with 3 years experience , and other a senior reporter with 2 years of experience . Other journalists also used INJECT , but the lack of time and resources in newsrooms that INJECT sought to alleviate also reduced the opportunities to provide evaluation feedback . Each journalist agreed to use INJECT in his / her work and to be interviewed afterwards about this use . No other incen - tives were offered . After agreeing to participate , each jour - nalist received an email with instructions to download and use the INJECT sidebar . Each was also sent links to an INJECT help website and was able to ask questions to the research team , to become familiar and competent with the tool’s features . The 3 Norwegian reporters used a Norwe - gian - language version of INJECT that also searched local newspaper archives , a screenshot of which is shown in Fig - ure 6 . All other INJECT capabilities were the same . When each journalist was familiar with the tool , his or her indi - vidual evaluation began . Two methods were used to collect data , depending on the amount of time that each journalist was able to commit to the evaluation . Figure 6 . INJECT’s creativity support , in Norwegian The first method was simpler and used with the 3 Norwe - gian journalists . One semi - structured interview with each journalist was held in Norwegian after up to 4 weeks of INJECT use . The interviews asked how the journalist per - ceived and used the tool , and its advantages and disad - vantages for discovering new news angles . The second method was more complex and used with the 2 journalists able to commit more time . It collected two types of data from each journalist over iterations of tool use : ( 1 ) the news stories developed with INJECT’s support , and ; ( 2 ) responses to structured interview questions . The researcher prepared for each interview with each journalist by first reading the news stories and adapting the questions , which were derived from Shapiro’s assessment framework for the practice of journalism [ 26 ] . The framework defined 5 facul - ties of good journalism : discovery , examination , interpreta - tion , style and presentation . INJECT was intended to sup - port the discovery and examination faculties . Discovering was the faculty of deciding what to say , and uncovering the news angle from information sources . Examining was the faculty of testing facts from information sources for their verifiability and coherence . The interview questions were derived from quality - related attributes associated with these 2 faculties . To elicit evidence about discovering , the inter - viewer asked questions about INJECT’s influence to : ( a ) select the stance to investigate the topic ; ( b ) overcome jour - nalist topic biases and interests , in order to achieve prag - matic objectivity ; ( c ) ensure social importance , to leave society in a better place than before with more socially im - portant stories , and ; ( d ) be ambitious in the methods used by the journalist [ 26 ] . To elicit evidence about examining , the interviewer asked questions about : ( e ) use of rigorous efforts to ensure accuracy , and how INJECT use might have increased rigor and accuracy , and ; ( f ) being undaunted in the research to shed new light on complex subjects [ 26 ] . First Evaluation Results The one - off semi - structured interviews with the 3 journal - ists at the 3 Norwegian newspapers revealed that INJECT was used to support the development of 5 published news stories – 3 by 1 journalist and 1 each by the other 2 . All 3 journalists used INJECT to develop stories that were as - signed to them after editorial meetings . All also reported minor usability problems , such as unclear meaning of the sidebar’s 6 creative search icons , and the unnecessary clicks needed to change INJECT’s creative search from strict to relaxed . However , none of these problems impeded the journalist’s use of INJECT . All 3 journalists reported that INJECT use supported them to discover new news angles , at the time that stories needed to be written , and to include more background information in their stories using the quantifiable strategy . INJECT use was reported to be more effective for discovering new an - gles on longer feature stories that required evidence , num - bers and facts , rather than on shorter news stories that re - ported facts . This decision to use INJECT for occasional feature stories explained the low number of articles that were written with INJECT’s support . In addition the 3 jour - nalists also reported that INJECT use supported the discov - ery of information from their own newspaper archives – information that each already should have remembered . And all 3 reported that writing stories more effectively with angles that were generated by themselves , which INJECT supported them to do , rather than with automatically gener - ated angles that the tool recommended to them . The new ideas and angles on the news stories were discov - ered by all 3 journalists using INJECT quickly , often in less than 3 minutes for each story . The journalists then switched to other tools , such as Google search , to retrieve more de - tailed information with which to complete the story . No direct disadvantages of INJECT use were reported , but the journalists expressed reservations about some of its de - sign . The journalists requested more explanation of the cre - ative search service algorithms and news information that they retrieved . None claimed to use the creative sparks , and 1 journalist asked for the sparks to be presented in different forms , to standout from other information in the sidebar . One journalist also asked for digital reminders of other es - tablished journalism angles , such as to think about the eco - nomic angle on a story . Other reported new requirements were integration into the newspaper’s content management systems , and a critical mass of 4 - 5 journalists who would use INJECT regularly in each newsroom , to maintain en - thusiasm for and training and support to use the tool . To summarize , the semi - structured interviews revealed that journalists did use INJECT to discover new angles on news features before these features were written , often quickly , before using other digital tools to complete the stories . A critical mass of journalists using INJECT in newsrooms was requested to support ongoing use of the tool . However , the interviews revealed little about how the individual jour - nalists changed their behaviour as a result of using INJECT . Second Evaluation Results A total of 4 interviews took place . The editor and senior reported that 4 published news stories were written with support from INJECT , and other stories had been prepared with use of the tool . The senior reporter claimed that the permanent presence of INJECT’s sidebar changed how she wrote the news stories . This presence led her to use its fea - tures after writing each paragraph rather than the end of the story – a change that she believed decreased the chances of omissions from stories . She also reported that she perceived INJECT’s strategies as alternative forms of journalism’s 5 Ws ( who , what , where , when and why ) that constituted a formula for developing a complete news story on a subject , and that INJECT’s quirky and ramifications strategies ex - tended this formula in new directions . The editor reported using the creative search icons from left - to - right on the sidebar , in order to structure her exploration of new angles with INJECT . The timing of INJECT use impacted on its effectiveness . At first , the editor and senior reporter used INJECT only after having developed the structure and some of the text of their stories , equivalent to the senior reporter’s use of Google search , and both were disappointed by the wide range of the articles presented by INJECT . Later in the evaluation peri - od , both used INJECT earlier : the editor used it during the research stage of a new article on copyright law , to investi - gate different topics entered into the sidebar . She reported that the information was still “ vague . . gave me articles that were not quite what she was looking for ” , although while this information did not change the overall direction of the article , it did guide her to incorporate new voices into it . The senior reporter used INJECT to research of a new story about news fact - checking , and made use of its quantifiable and causal strategies to explore new angles . In both interviews , the editor reported that INJECT use led her to discover articles that informed her stories from be - yond her usual news sources . Examples of these new news sources were Wired magazine , Guardian newspaper , blogs , digital cartoons and other newspapers about the topic but from 4 - 5 months earlier . INJECT’s use appeared to over - come some of her biases arising from sources used . In par - ticular she reported that news information retrieved with the people strategy led her to introduce new voices from alter - native sources into the article about copyright law . She also reported that one creative spark – think about the opponents of [ Person ] , and how the story evolves from their perspec - tive – directed her research in that direction and to generate new content to the article . On reflection , the senior reporter claimed that INJECT’s less directed searches had the poten - tial to develop new stories with greater social importance , saying : “ it is a very good tool for inspiration ” . To this end she used the people strategy to develop one story from the perspective of people who were impacted by fake news , rather than from the data sources that she usually used . She highlighted the potential of the ramifications strategy to encourage her to explore more about the consequences of events , rather than just report the events themselves . The senior reporter stated that INJECT had the potential to increase the boldness of her journalism methods by recom - mending new angles on her own already - implemented sto - ries . These new angles guided her to re - purpose and publish new versions of these stories , which was a new direction for her journalism . The editor reported that INJECT also had the potential to increase the boldness of her journalism , in part due to the authority that she attributed to news stories retrieved by the INJECT tool compared with her searches of social media sources . She also claimed that the creative sparks had the potential to increase the social importance of her stories , because some of the sparks led her to generate new ideas about other socially relevant organizations . INJECT’s creative search strategies were reported to have changed the behaviour of both journalists , albeit less direct - ly than had been designed for . For example , the existence of the people and quirky strategies made the editor more aware of these types of angle : “ it made me search a bit on the in - dividual and the quirky side . Cos I think that I wouldn’t have thought to look for cartoons . . ” . And the senior reporter claimed that the presence of INJECT’s quantifiable and ramifications strategies reminded her to think more about the accuracy and the future implications of her stories , both with and without INJECT . The editor reported that , on reflection , she understood better INJECT’s purpose and potential benefits to her work . These benefits included greater support for writing feature stories , use of creative sparks to direct her to think of more people , and to be more ambitious in both her stories and investiga - tive methods . About the creative sparks , the editor said : “ some of them are very , very specific , and some are a bit more broad , so in that sense it could potentially give you a bit of a push . So if you are writing a story and you’re not sure on the angle , and if you should include this or this per - son in it , then if you go to the prompts , then some of them might resonate , so yes . . ” . Neither journalist reported strong evidence that INJECT supported examining news information that was retrieved , but the senior reporter said that her use of the quantifiable strategy did provide her with some data and evidence that supported the examining faculty . However , the editor stated that INJECT was not an effective tool for fact checking . CONCLUSION , DISCUSSION AND FUTURE WORK This paper reports the design and first evaluations of new digital support for journalists to discover creative angles on news stories . The digital support integrated natural lan - guage processing , creative search and interactive recom - mendation technologies . It codified the expertise of experi - enced journalists to retrieve news information and direct idea generation , consistent with human - centred creative cognition [ 15 ] . The support was delivered to journalists as a visible sidebar of a text editor . Data was collected from 5 journalists to answer 2 research questions : RQ1 : All of the journalists discovered new and useful an - gles on stories written with INJECT’s support , often quickly , however many of these angles extended ex - isting storylines rather than developed new ones ; RQ2 : Some changed how they developed news stories as a result of using INJECT , and the presence and struc - ture of the INJECT sidebar in the editor window im - pacted on the behaviour of at least some of the jour - nalists . As such , journalist use of INJECT was effective , but not to the degree that the tool was designed to support . Journalist use of INJECT also provided more effective support for writing feature stories rather than news stories , although this finding might have been influenced by INJECT’s data layer , which was only composed of past news rather than current social media information . However , writing feature stories will still require journalists to exhibit more creative thinking than during the evaluations , by applying more cre - ativity skills when using INJECT than was reported . Results revealed that use of INJECT offered more support to journalists when discovering rather than examining [ 26 ] . It appeared to contribute to journalists being more ambi - tious and to overcome biases by retrieving topic infor - mation from alternative news sources and discovering new stances from which to investigate news topics . However , this support for discovering also led journalists to compare INJECT , sometimes unfavorably , to Google search , a com - parison reinforced by some of the journalists using Google search after INJECT to seek more information on new an - gles . At least some of the journalists might have expected INJECT to discover the information that was needed , rather than just to offer creative support with this information , as INJECT was designed to do . Although subsequent versions of INJECT now embed Google search to support journal - ists , to help to distinguish between discovering a new angle with INJECT capabilities and retrieving more information afterwards , journalists will still need to apply more creativi - ty skills to exploit INJECT’s capabilities effectively . The reported uses of INJECT did not appear to deliver and / or encourage sufficient use of these skills . Results also revealed that use of INJECT supported some of the journalists to recognize known news information at the time that news stories are being written . Indeed , different news organizations have identified the potential of INJECT to unlock their own news archives for creative use , to en - hance productivity as well as creativity , but only if INJECT can support journalists to recognize [ 5 ] related news quick - ly when writing stories . New recommendation features will be needed to support such recognition . Of course , many threats to the validity of these first evalua - tion results exist . Although more journalists used INJECT , only 5 provided structured feedback for the evaluation , and their verbal reports were not triangulated with INJECT us - age log data . The journalists worked for regional and spe - cialist newspapers rather than other types , and did not use INJECT support for most of their news stories . The regional newspapers were members of the project consortium and their journalist’s responses might have biased , due to pres - sure from management and / or loyalty . And there has been no systematic evaluation of the creativity , i . e . novelty and value of the news stories generated with INJECT’s support . Therefore , the results need to be interpreted cautiously , and more systematic evaluations of INJECT with larger num - bers of journalists for longer periods will be needed to draw firmer conclusions about the tool’s effectiveness . Journalists will need to demonstrate more creativity skills to exploit INJECT effectively . Some of the journalists in the evaluation expressed a preference to write stories based on ideas generated themselves rather than ones recommended by INJECT – a preference can be interpreted as motivation to be more creative and to develop and apply creativity skills . However , development of the skills remains a chal - lenge in newsrooms with conservative attitudes [ 7 ] , espe - cially in the face of new fears over automation that INJECT itself might exacerbate . For example , the tool could be ex - tended to collect data about the dates and times that readers access stories generated with different INJECT angles , to automate the writing of stories with different angles to be read at different times . Therefore , although future versions of INJECT will include more directed creativity support , digital tools alone will not provide the missing creativity skills . One alternative to explore with newsrooms is upfront journalist training in creativity techniques that align with INJECT’s capabilities . One example of these techniques is SCAMPER [ 22 ] , which guides problem solvers to examine solutions from different perspectives such as combining , eliminating and reversing , and is a simplified form of INJECT’s creative sparks . Another example is Hall of Fame [ 22 ] , a technique that guides problem solvers to ex - amine solutions from the perspective of different well - known people – who could be iconic journalists . We hope to report on the use of these techniques and INJECT togeth - er in the near future . Another possible way to develop and apply creativity skills is to develop a critical mass of journalists using INJECT in each newsroom , as some of the journalists in the evalua - tions reported . It is long established that during idea genera - tion and evaluation activities , cooperation can lead to more creative ideas and better solutions ( e . g . [ 32 ] ) , and coopera - tion between 4 - 5 journalists has the potential to support the development and use of different creativity skills . When journalists used INJECT also appeared to influence the ef - fectiveness of the tool . Therefore , to encourage earlier use of INJECT and the new creativity skills of journalists , we will seek to embed this use into newsroom workflows , for example for short periods after editorial meetings and prior to writing news stories . Another emerging challenge was to design complex interac - tions that inputted to and received outputs from sophisticat - ed algorithms that produced unpredictable outcomes . Arti - ficial intelligence has been framed as a new design material that required interactions that were transparent , opaque and offer shared control [ 12 ] , and journalists both in the evalua - tions and elsewhere requested transparent creative search algorithms to explain the retrieved news information . How - ever , INJECT’s designers also believed that these algo - rithms still needed to be opaque , in order to retrieve infor - mation that was surprising and hence creative to users . Therefore , future work will refine INJECT’s interactions to offer different degrees of support related to the user’s famil - iarity with the tool , for example to offer greater transparen - cy and shared control to less experienced users , and to in - crease the opaqueness for more experienced ones . Other new INJECT features have already been implement - ed . These features include more news and new social media information sources added to the data layer , contextualized explanations of information retrieved by INJECT , new uses of the creative sparks , and new versions of INJECT for the InCopy TinyMCE text editors . Future uses of these new INJECT versions by critical numbers of journalists in news - rooms will enable the research team to investigate broader research questions about changing journalism practices . For example , future versions of INJECT will invoke fact - checkers to detect possible fake news information . Howev - er , if journalists do develop and use new creativity skills with INJECT , the resulting creative thinking might also increase their reflexivity with which to detect fake news sources . More broadly , use of new digital tools such as INJECT have the potential to increase conflicts in news - rooms [ 7 ] , with journalists who resist the adoption of these new tools , and with managers who impose the new tools in order to increase newsroom creativity and productivity . Moreover , as newsroom resources reduce further , uses of more intelligent tools such as INJECT risk substituting ra - ther than supplementing key journalism tasks such as face - to - face interviewing . Therefore , the use of new digital tools to increase journalist creativity cannot be separated from wider socio - political challenges that newsrooms face . The rollout of INJECT in new newsrooms will be a vehicle to investigate emerging challenges and trade - offs that will we anticipate will arise . ACKNOWLEDGEMENTS The research reported in this paper is supported by the EU - funded H2020 723328 INJECT innovation action . REFERENCES 1 . Sara F . 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Bregtje Van Der Haak , Michael Parks and Manuel Castells . 2012 . The Future of Journalism : Networked Journalism . Inter - national Journalism of Communication 6 : 2923 - 2938 . 31 . Google Docs introduces new sidebar research tool . Retrieved December 28 , 2017 from https : / / thenextweb . com / apps / 2012 / 05 / 15 / useful - google - docs - introduces - new - sidebar - research - tool 32 . Andrew Warr and Eamon O ʼ Neill , 2005 . Understanding de - sign as a social creative process . In Proceedings of the ACM Creativity and Cognition Conference ( C & C’05 ) , 118 - 127 . http : / / doi . acm . org / 10 . 1145 / 1056224 . 1056242 33 . Tamara Witschge and Gunnar Nygren . 2009 . Journalism : a profession under pressure ? Journal of Media Business Studies 6 , 1 : 37 - 59 . \ No newline at end of file diff --git a/silver_data/f581848bbfb6cdcac16f3ca2c474feb79d6f2e49.pdf.txt b/silver_data/f581848bbfb6cdcac16f3ca2c474feb79d6f2e49.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..aa9af594e8403f2007429e4eb5387e7cea7dfefc --- /dev/null +++ b/silver_data/f581848bbfb6cdcac16f3ca2c474feb79d6f2e49.pdf.txt @@ -0,0 +1 @@ +1 In Proc . Advanced Visual Interfaces , AVI 2000 , Palermo , Italy , May 2000 . http : / / www . daimi . au . dk / ~ mbl / AVI2000 Reification , Polymorphism and Reuse : Three Principles for Designing Visual Interfaces Michel Beaudouin - Lafon and Wendy E . Mackay University of Aarhus , Department of Computer Science Aabogade 34 DK - 8200 Aarhus N - Denmark + 45 89 42 56 44 / + 45 89 42 56 22 { mbl , mackay } @ daimi . au . dk ABSTRACT This paper presents three design principles to support the development of large - scale applications and take advantage of recent research in new interaction techniques : Reification turns concepts into first class objects , polymorphism permits commands to be applied to objects of different types , and reuse makes both user input and system output accessible for later use . We show that the power of these principles lies in their combination . Reification creates new objects that can be acted upon by a small set of polymorphic commands , creating more opportunities for reuse . The result is a simpler yet more powerful interface . To validate these principles , we describe their application in the redesign of a complex interface for editing and simulating Coloured Petri Nets . The cpn2000 interface integrates floating palettes , toolglasses and marking menus in a consistent manner with a new metaphor for managing the workspace . It challenges traditional ideas about user interfaces , getting rid of pull - down menus , scrollbars , and even selection , while providing the same or greater functionality . Preliminary tests with users show that they find the new system both easier to use and more efficient . Keywords Design principles , reification , polymorphism , reuse , direct manipulation , instrumental interaction , interaction model . 1 . INTRODUCTION Today ' s visual interfaces suffer from an overabundance of functionality : each successive version is marketed based on the number of new functions , with little regard to the corresponding increase in the cost of use . Simple things keep getting harder , as users spend more and more time deciding among an increasing variety of rarely or never - used options . Some users are at a breaking point and are less and less able to cope with new software releases [ 21 ] . Others have begun to actively reject software upgrades and cling to older versions of products such as Microsoft Word ( survey of Microsoft users , Business Week , 5 July , 1999 ) . New interaction techniques , such as toolglasses [ 4 ] and marking menus [ 17 ] , have been proposed to reduce this trade - off between power and ease - of - use . Yet such interaction techniques tend to be developed in isolation , as the focus of a particular research project . While this is a critical first step , it is also important to understand how these techniques scale when combined with other techniques and are placed in the context of complex real - world applications . We also need to develop new interaction models that explain how these and other techniques can increase the functionality available to users without creating a corresponding increase in the cost of use . This paper describes how three design principles , reification , polymorphism and reuse , have provided a framework for redesigning a complex tool for editing and simulating Coloured Petri Nets . Developed in the late 1980 ' s , the Design / CPN tool used a then state - of - the - art WIMP ( windows , icons , menus , pointing ) user interface . The new tool , cpn2000 , is the result of a participatory design process , in which users and designers have collaborated to recreate a tool that supports " Petri - Nets - In - Use " . The goal is to provide Coloured Petri Nets developers with greater functionality through an interface that is more intuitive , efficient and pleasant to use ; one that allows them to think in terms of Petri nets and not the mechanics of the interface . We begin by describing the principles of reification , polymorphism and reuse and then describe the interface to cpn2000 . We explain how these principles have influenced the design of the user interface and discuss how combining them helps address the trade - off between power and ease - of - use . We conclude with directions for future research . 2 . DESIGN PRINCIPLES Graphical user interfaces can be broadly defined as consisting of graphical objects and commands . Graphical objects are represented on the screen and commands can be applied to create , edit and delete them . Visualization techniques describe how to represent these objects while interaction techniques describe how to apply commands to them . Over time , users develop individual patterns of use that depend upon the available objects and commands , the particular application domain and the current context of use . The perceived " ease - of - use " of an interface depends upon many factors , including the effectiveness of the visual representation , the completeness of the command set and the support for efficient patterns of use . 2 We have developed three principles that address the issues surrounding objects , commands and patterns of use : (cid:127) Reification extends the notion of what constitutes an object ; (cid:127) Polymorphism extends the power of commands with respect to these objects ; and (cid:127) Reuse provides a way of capturing and reusing patterns of use . 2 . 1 Reification Reification is the process by which concepts are turned into objects . For example , in a graphical editing tool , the concept of a circle is represented as an image of a circle in a tool palette . Reification creates new objects that can be manipulated by the user , thus increasing the set of objects of interest . Instrumental Interaction [ 1 ] extends the principles of Direct Manipulation [ 26 ] by reifying commands into interaction instruments . An interaction instrument is a mediator between the user and objects of interest : the user acts on the instrument , which in turn acts on the objects . This reflects the fact that , in the physical world , our interaction with everyday objects is mediated by tools and instruments such as pens , hammers or handles . The menu items , tool buttons , manipulation handles and scrollbars seen in today ' s user interfaces are examples of interaction instruments . A scrollbar , for example , is both a visible object on the screen that can be manipulated by the user and also a command the user manipulates to scroll the document . Turning commands into objects provides potentially infinite regression . Since instruments are objects , they can be operated upon by ( meta ) - instruments , which are themselves objects , etc . In real life , we see limited chains of regression , as we move our focus from pencils , to pencil sharpeners that sharpen pencils to screwdrivers that fix pencil sharpeners . In some user interfaces , menus and toolbar buttons can be reconfigured to tailor the interface : they become instrument objects that can be manipulated by meta - instruments . Another example of reification is the notion of style : In a text editor such as Microsoft Word , a style is a collection of attributes describing the look of a text in a paragraph , e . g . , the font and margins . The user can create and edit styles and apply them to paragraphs . Styles thus become objects of interest for the user . Many graphical editors also reify a collection of objects into the notion of a group . Since a group is itself an object , it can be added to a group , giving way to arbitrarily large structures such as trees and DAGs . These structuring mechanisms can be found in a wide variety of interfaces . 2 . 2 Polymorphism Polymorphism is the property that enables a single command to be applicable to objects of different types . Polymorphism allows us to maintain a small number of commands , even as reification increases the number of object types . This property is essential if we want to keep the interface simple while increasing its power . Most interfaces include the polymorphic commands cut , copy and delete , which can be applied to a wide variety of object types , such as text , graphics , files or spreadsheet cells . Undo and redo can also be considered polymorphic to the extent that they can be applied to different commands . Applying a command to a group of objects involves polymorphism at two levels . First , any command that can be applied to an object can also be applied to a group of objects of the same type by applying it to each object in the group . Second , any command can be applied to a heterogeneous group of objects , i . e . objects of different types , as long as the command has meaning for each of the individual object types . 2 . 3 Reuse Reuse can involve previous input , previous output or both . Input reuse makes previously - provided user input available for reuse in the current context . For example , the redo command lets users repeat complex input strings without having to retype them . Output reuse makes the results of previous user commands available for reuse . For example , duplicate and copy - paste let users avoid re - creating complex objects they have just created . Polymorphism facilitates input reuse because a sequence of actions can be applied in a wider range of contexts if it involves polymorphic commands . Prototyping environments such as Self and its Morphic user interface framework [ 22 ] , which are based on cloning and delegation , support and even encourage a high level of input reuse . Reification facilitates output reuse by creating more first - class objects in the interface which are then available for reuse . Thus , for example a Microsoft Word user can create a new style object by reifying the style of an existing paragraph or by duplicating an existing style object , modifying the copy and reapplying it . A more elaborate form of reuse obtains when new styles are created through inheritance from an existing style , which allows changes made in the reused object to be propagated to the edited copies . Macros , such as those found in Microsoft Excel , illustrate the power of combining these three design principles . The user begins by telling the system to " watch " as a sequence of commands is performed . Reification enables the user to capture the particular pattern of use as a sequence of commands that can be applied as a single new command to a new set of objects . A more advanced form of reification turns each component command into an object that can itself be edited , thus changing the pattern of use to accommodate different contexts . The next section briefly describes the cpn2000 interface , which provides a testbed for exploring these three principles . 3 . THE CPN2000 INTERFACE The current cpn2000 interface was created over a period of ten months by a group of ten people . We followed a highly participatory design process beginning with observation of users of an existing system , Design / CPN , in various work settings . We developed scenarios to capture and articulate their work practices and engaged in a variety of video brainstorming and video prototyping exercises to develop the new interface . These activities involved a multidisciplinary group of user interface researchers , programmers and Coloured Petri Nets developers . The first version of cpn2000 was presented at the CPN International Workshop in October 1999 . We also took advantage of the CPN Workshop and an earlier retreat for the University of Aarhus CPN group to conduct more formal studies using CPN developers who were not involved in the development of the new tool . The following sections introduce the basic concepts and vocabulary of Coloured Petri Nets ( CPN ) , the basic interaction techniques we selected and the overall design of the interface . 3 Fig . 1 : A simple Petri net with three places , one transition and four arcs . Two places have a token . Fig . 2 : The Petri net from Fig . 1 after the transition has been fired . Fig . 3 : Design / CPN , the current tool used by CPN designers . 3 . 1 Coloured Petri Nets Both Design / CPN and its successor , cpn2000 , address the application domain of editing and simulating Coloured Petri Nets [ 14 ] . Petri nets are a graphical formalism with a strong underlying mathematical model that extends the power of simple finite state automata . Petri nets are particularly suited for the modeling and analysis of parallel systems such as communication protocols and resource allocation systems . The graphical representation of Petri nets ( Fig . 1 ) is a bipartite graph where the nodes are called places ( depicted as circles or ellipses ) and transitions ( depicted as rectangles ) . Edges of the graph are called arcs and can only connect places to transitions and transitions to places . Each place typically represents a possible state or resource of the system . Places hold tokens , which represent the fact that the system is in a given state or the number of resources that can be allocated . The rules for simulating the net are very simple : a transition is enabled if all the places connected to it by an input arc have a token . Firing an enabled transition consists of removing a token from each input place and adding a token to each output place of the transition ( Fig . 2 ) . Mathematically , a Petri net can be represented by a matrix and simulation of the net is equivalent to a set of linear algebra operations . Properties of the net can be proven , such as the fact that the net has a bounded number of tokens or that there are no deadlocks . A number of higher - level Petri net formalisms have been developed to model complex systems . Most of these formalisms are equivalent in power to a simple Petri net , but are much more concise . One such extension is Coloured Petri Nets [ 14 ] . In this model , the tokens belong to a color set equivalent to a data type in a conventional programming language . Arcs are labeled with pattern - matching expressions that describe which tokens are used when a transition is fired . Typically , colors allow a conventional Petri net to be " folded " onto itself , making models much smaller . In addition Coloured Petri Nets can be hierarchical . A transition can be described by a subnet , equivalent to macro - substitution in a textual language . Hierarchical nets make it possible to structure a complex net into smaller units that can be developed and tested separately . Over the past decade , the CPN group at the University of Aarhus has been developing an editor and simulator for Coloured Petri Nets , called Design / CPN ( Fig . 3 ) . This tool is freely available to the CPN community and is currently in use by over 600 organizations both in industry and academia . Design / CPN users have created models with as many as 100 modules and have run simulations lasting several days . The tool has been used far beyond the expectations of the designers and has reached its limits in terms of usability and complexity of implementation . The goal of cpn2000 is to reimplement the basic functionality of Design / CPN while improving the user interface and adding new editing and simulation capabilities . The project is a joint effort of the CPN , HCI and Beta groups at the University of Aarhus and is funded by the Danish Center for IT Research , Hewlett - Packard and Microsoft . 3 . 2 Interaction techniques We began with two key decisions that have influenced many aspects of the design . First , we decided to explicitly support two - handed input , with a mouse for the dominant hand and a trackball for the non - dominant hand . The keyboard is used only to input text and to navigate within and across text objects . The design of the bi - manual interaction follows Guiard ' s Kinematic Chain theory [ 10 ] in which the non - dominant hand manipulates the context ( container objects such as windows and toolglasses ) while the dominant hand manipulates objects within that context . The exception is direct interaction for zooming and resizing , which , according to Casalta et al . [ 6 ] , should give both hands symmetrical roles . Second , we decided to incorporate a combination of new interaction techniques , rather than using a standard WIMP interface . Our goal is to provide cpn2000 users with easier yet more powerful tools and support more effective patterns of use . Users should be able to spend more time on developing Petri nets and less time on the mechanics of the interface . The current version of cpn2000 incorporates four primary interaction techniques : direct interaction , marking menus [ 17 ] , floating palettes , and toolglasses [ 4 ] . Direct interaction involves pointing directly at objects and either clicking on or dragging them . A direct bi - manual interaction , used for resizing and zooming , involves depressing a trackball button with the non - dominant hand and dragging the mouse with the dominant hand , as if stretching a piece of rubber . 4 Marking menus are radial , contextual menus that appear when clicking the right button of the mouse . Marking menus offer faster selection than traditional linear menus for two reasons . First , it is easier for the human hand to move the cursor in a given direction than to reach a target at a given distance . Second , the menu does not appear when the selection gesture is executed quickly , which supports a smooth transition between novice and expert use . Kurtenbach and Buxton [ 17 ] have shown that selection times can be more than three times faster than with traditional menus . Hierarchical marking menus involve more complex gestures but are still much more efficient than their linear counterparts . Floating palettes contain tools represented by buttons . Clicking a tool with the mouse activates this tool , i . e . the user conceptually holds the tool in his or her hand . Clicking on an object with the tool in hand applies the tool to that object . In many current interfaces , after a tool is used ( especially a creation tool ) , the system automatically activates a " select " tool . This supports a frequent pattern of use in which the user wants to move or resize an object immediately after it has been created but causes problems when the user wants to create additional objects of the same type . cpn2000 avoids this automatic changing of the current tool by getting rid of the notion of selection ( see below ) while ensuring that the user can always move an object , even when a tool is active , with a long click ( 200ms ) of the mouse . This mimics the situation in which one continues holding a physical pen while moving an object out of the way in order to write . Toolglasses , like floating palettes , contain a set of tools represented by buttons . Unlike floating palettes , they are semi - transparent and are moved with the non - dominant hand . A tool is applied to an object with a click - through action : The tool is positioned over the object of interest and the user clicks through the tool onto the object . The toolglass disappears when the tool requires a drag interaction , e . g . , when creating an arc . This prevents the toolglass from getting in the way and makes it easier to pan the document with the non - dominant hand when the target position is not visible . This is a case where the two hands operate simultaneously but independently . Since floating palettes and toolglasses both contain tools , it is possible to turn a floating palette into a toolglass and vice versa , using the right button of the trackball . Clicking this button when a toolglass is active drops it , turning it into a floating palette . Clicking this same button on a floating palette picks it up , turning it into a toolglass . None of the above interaction techniques requires the concept of selection . All are contextual , i . e . the object of interest is specified as part of the interaction . This greatly simplifies the application ' s conceptual model and , one hopes , the users ' mental models . However , this also creates a problem . Traditional interfaces use multiple selection to apply a command to a set of objects . We solve the problem by reifying multiple selection into objects called groups ( see below ) . We considered several other interaction techniques including gesture input [ 25 ] , zoomable interfaces [ 2 ] and dropable tools [ 3 ] . We selected the above set partly due to the participatory nature of our design process , which led us to select the techniques most appealing and natural for our particular set of users . However , the techniques we chose also cover each of the different possible syntaxes for specifying commands : (cid:127) object - then - command : point at the object of interest , then select the command from a contextual marking menu ; (cid:127) command - then - object : select a command by clicking a tool in a floating palette , then apply the tool to one or more objects of interest ; (cid:127) command - and - object : select the command and the object simultaneously by clicking through a toolglass or moving it directly . Preliminary results from our user studies [ 13 ] make it clear that none of these techniques is always better or worse . Rather , each emphasizes a different , but common , pattern of use . Marking menus work well when applying multiple commands to a single object . Floating palettes work well when applying the same command to different objects . Toolglasses work well when the work is driven by the structure of the application objects , such as working around a cycle in a Petri net . 3 . 3 Workspace manager Coloured Petri Nets frequently contain a large number of modules . In the existing Design / CPN tool , each module is presented in a separate window and users spend time switching among them . Early in the project , it became clear that we had to design our own window manager to improve this situation : the Workspace Manager . The workspace occupies the whole screen ( Fig . 4 ) and contains window - like objects called folders . Folders contain pages , each equivalent to a window in a traditional environment . Each page has a tab similar to those found in tabbed dialogs . Clicking the tab brings that page to the front of the folder . A page can be dragged to a different folder with either hand by dragging its tab . Dragging a page to the background creates a new folder for it . Dragging the last page out of a folder removes the folder from the screen . Folders reduce the number of windows on the screen and the time spent organizing them . Folders also help users organize their work by grouping related pages together and reducing the time spent looking for hidden windows . Cpn2000 also supports multiple views , allowing several pages to contain a representation of the same data . For example , the upper - left page in Fig . 4 shows a module with simulation information , while the upper - right page shows the same module without simulation information but at a larger scale . The left part of the workspace is called the index and contains a hierarchical list of objects that can be dragged into the workspace with either hand . Objects in the index include toolglasses , floating palettes and Petri net modules . Dragging an entry out of the index creates a view on its contents , i . e . a toolglass , a floating palette or a page holding a CPN module . Pages and folders do not have scrollbars . If the contents of a page is larger that its size , it can be panned with the left button of the trackball , even while the dominant hand is using the mouse to , for example , move an object or invoke a command from a marking menu . Getting rid of scrollbars saves valuable space but makes it harder to tell which part of the document is currently visible . A future version will display relative position information on the borders of the page during the panning operation in a non - intrusive and space - saving way . Resizing a folder and zooming the contents of a page involves direct bi - manual interaction ( as described above ) . Unlike traditional window management techniques , using two hands makes it possible to simultaneously resize and move a folder , or pan and zoom the contents of a page at the same time . Clicking the mouse on the page tab or on the folder pops up a contextual marking menu with additional commands , such as close , duplicate , collapse and expand . 5 Fig . 4 : The cpn2000 interface . The index appears in the left column . The upper - left folder contains a page with the simulation layer active . The upper - right folder contains a view of the same page , at a different scale . The lower folder contains three pages : the top page shows a horizontal and a vertical magnetic guideline . The VCR - like controls to the left belong to the simulation floating palette . The toolglass in the center is positioned over objects on the page and is ready to apply any of the attributes shown . To the right , a marking menu has been popped up on a folder and is ready to accept a gesture to invoke one of the commands displayed . 3 . 4 Creating and Laying out Objects Creation tools are accessible via any of the three interaction techniques . The user may select the appropriate object from the floating palette , move to the desired position and click , or use the non - dominant hand to move the toolglass to the desired position and click - through with the dominant hand , or move to the desired location and make the appropriate gesture from the marking menu . Users of the current Design / CPN spend a great deal of time creating and maintaining the layout of their Petri net diagrams . The primary technique is a set of align commands , similar to those found in other drawing tools . The limitation is that they align the objects at the time the command is invoked , but do not remember that those objects have been aligned , unless the user groups them together into a new object . Aligning groups creates other problems , since the elements of the group cannot be edited further without ungrouping them , which is cumbersome and risks disturbing the alignment . Groups are also strictly hierarchical : an object cannot be a member of two independent groups . This makes it impossible to , for example , place an object both in a horizontal and vertical alignment group . We also observed that most current users use the same pattern to move an aligned object : They manually select all objects aligned to the object of interest and move them as a group . This dramatically slows down the interaction . Many of the ideas from our early brainstorming sessions involved trying to make layout less cumbersome . We began by reifying the alignment command into alignment objects called guidelines . Graphic designers often use guidelines to define the structure of the layout and to position objects relative to this structure . In the current version , we use horizontal and vertical guidelines . Guidelines are first - class objects that are created in the same way as the elements of the Petri net model , i . e . with tools found in a palette / toolglass or in a marking menu . Guidelines are displayed as dashed lines ( Fig . 4 ) and are magnetic . Moving an object near a guideline causes the object to snap to the guideline . Objects can be removed from a guideline by clicking and dragging them away from the guideline . Moving the guideline moves all the objects that are snapped to it , thus maintaining the alignment . An object can be snapped to both a horizontal and a vertical guideline . We have designed , but not yet incorporated , additional types of guidelines . For example , circular or elliptical guidelines would make it easier to layout the cycles commonly found in Petri nets . We also plan to support spreading or distributing objects 6 over an interval within a line segment , since this is a common layout that current users must implement by hand . Adding these new types of guidelines may create conflicts when an object is snapped to several guidelines . One solution is to assign weights to the guidelines and satisfy the alignment constraints of the guidelines with heaviest weight first . Such conflicts do not exist in the current system because only horizontal and vertical guidelines are available . 3 . 5 Editing Attributes The tools to edit the graphical attributes of the CPN elements are grouped in a palette / toolglass that contains three rows : a row of color swatches , a row of lines with different thicknesses , and a row for user - defined styles ( Fig . 4 ) . The first two rows are fairly standard and are not described further here . Tools in the last row correspond to the reification of groups of graphical attributes into styles . Initially , each tool in this row is a style picker . Applying this tool to an object copies the object ' s color and thickness into the tool and transforms the tool into a style dropper . Applying a style dropper to an object assigns the tool ' s color and thickness to that object . Applying a style dropper to the background of the page empties it and turns it into a style picker . If this is done by mistake , the undo command restores its previous state . In practice , style pickers and style droppers make it very easy and efficient for users to define the styles they use most often and apply them to objects in the diagram . 3 . 6 Simulation tools Once a CPN model has been created , the developer runs simulations to validate it . Simulations correspond to testing traditional programs by running them on different data sets . The simulation of a Petri net is visual : tokens are put into places and go from place to place according to the rules described in the text inscriptions . CPN developers debug Petri nets by running simulations step - by - step or in larger chunks , similar to debugging traditional programs . Cpn2000 displays simulation information in a simulation layer that can be added to any page via any of the three interaction techniques . When the simulation layer is active ( Fig . 4 ) , the background color of the page changes , the number of tokens are displayed as small red disks , the value of the tokens are displayed as yellow text annotations , and enabled transitions are displayed with a green halo . Each of these types of feedback can be toggled by the user using the tools in the simulation palette or toolglass . Cpn2000 uses a VCR metaphor to control the simulation . Next frame lets the user select which transition to fire . Play randomly fires enabled transitions until a deadlock is reached or the user hits the stop button . Fast - forward runs the simulation for a maximum number of steps set by the user . Rewind resets the net to its initial state . The Next frame command is polymorphic : If applied to an enabled transition , it fires that transition . If applied to a page , it fires a randomly - selected transition within the page . If applied to a folder or to the workspace , it fires a randomly - selected transition within the pages of the folder or the whole model , respectively . Our user studies showed that users are either interested in the results of the simulation , and thus do not want to change the underlying diagram , or they are interested in editing the diagram and usually do not need the results of the simulation . Therefore , in our design , a diagram cannot be edited in a page while the simulation layer is active , which makes it easier to adjust the location of the simulation feedback . The user can always edit the underlying diagram in a different page with the simulation layer turned off ( Fig . 4 ) . 4 . USING THE DESIGN PRINCIPLES Many factors have influenced our design , including observations of users , brainstorming ideas , selection of interaction techniques , knowledge of other systems and , of course , the three design principles . The design principles played two key roles . First , they helped us find a way to combine the interaction techniques , preserving and even increasing ease - of - use . Second , they helped generate new ideas that solved problems or increased the power of the system . The design principles served to define a design space that helped us both evaluate and generate potential solutions , which in turn helped us manage the trade - off between power and simplicity . 4 . 1 Grouping The concept of a group is very powerful , resulting from the combination of reification and polymorphism . Groups encourage reuse , since creating a group implies that the user plans to work with that set of objects at a later time . Several aspects of the cpn2000 interface take advantage of the notion of group . Folders represent groups of pages , magnetic guidelines represent groups of objects with layout constraints and styles represent groups of objects that share graphical attributes . Through polymorphism , commands applied to the group operate on its members , with semantics that depend upon the command . Thus , activating the simulation layer on a folder means activating the simulation layer in each page of the folder , while firing a transition in a folder means firing one transition picked from among the set of enabled transitions in the pages of the folder , rather than firing a transition in each page . Similarly , applying a style to a guideline applies it to each object snapped to the guideline . Groups allow users to work at a higher level of abstraction , which increases the power of the interface . Yet using groups is no more complex than using individual objects , since the same interaction methods apply to both . 4 . 2 Decomposition Commands in traditional user interfaces tend to be coarse grained . In order to keep the number of commands reasonably low , designers often use dialog boxes to specify the ever - increasing number of arguments for each command . For example , specifying an alignment or editing a style usually requires a separate dialog box to specify the characteristics of the alignment or style . Our approach leads to a more fine - grained set of commands , as illustrated by our solutions for alignments and styles . Instead of an overall align command , the user creates a guideline , then adds objects to it . Different types of alignment correspond to different types of guidelines , e . g . , horizontal or vertical . Similarly , different parts of objects , e . g . , object centers or sides , can be snapped to the guideline . This decomposition of the alignment command makes it possible to dynamically add and remove objects to the alignment , unlike traditional align commands . New types of alignment , such as alignment around a circle or distribution along a line segment , can be added easily , without increasing the complexity of the interface . 7 Instead of creating styles explicitly , with a set of dedicated commands , styles are extracted from existing objects with the style picker tool . Applying a style to an object involves the same interaction as changing an object ' s attribute . Reification results in an increase of power through the notion of style , while polymorphism keeps the interface simple . In addition , fine - grained commands encourage a wider variety of patterns of use and therefore creates more opportunities for reuse and the opportunity to better meet the specific needs of each user . 4 . 3 Layers User interface modes are often criticized in the HCI literature , since they make interfaces harder to use and less direct . At the same time , modes appear necessary in complex systems , helping to structure the interface according to the different activities of users . For example , when working with Petri nets , users make a clear distinction between editing the net and simulating it . Yet , in the earlier Design / CPN tool , users complained about having to switch explicitly between the two modes . In order to address these issues , we reify the notion of mode into layers : activating a layer in a page gives the user access to the objects and commands relevant to a specific activity . For example , activating the simulation layer displays tokens and enabled transitions . Magnetic guidelines and text inscriptions are also accessible via separate layers . Users have the ability to define for themselves the complexity or simplicity of the visual interface , based on the number of activities they want to engage in simultaneously . Enabling several layers makes more objects directly accessible but may clutter the display while enabling a single layer allows the user to focus on a single activity . Since most activities are naturally separated through the context of the work , users are likely to display only the layers that are directly relevant to the problem at hand . Users thus control for themselves the trade - off between power for simplicity . 5 . RELATED WORK The work presented in this paper builds upon previous work in graphical user interfaces . The Xerox Star [ 15 ] and the Apple Lisa [ 23 ] have led to the desktop metaphor and the now - standard WIMP interfaces . Alternative models of the workspace have been proposed , including Rooms [ 12 ] , the Information Visualizer [ 5 ] , Translucent Patches [ 16 ] and Lifestreams [ 9 ] . Our design clearly draws from existing techniques . For example , the index is similar to the list of files and folders found in many file managers ; Dragging pages into and out of folders resembles the manipulation of the palette tabs in Adobe Photoshop . A number of systems have explored the reification of concepts into user interface objects , including ARK , the Alternate Reality Kit [ 27 ] and Self ' s Morphic user interface [ 22 ] . Dropable Tools [ 3 ] and the Raisamo and Räihä ' s Ruler [ 24 ] also fit into this design approach . Interaction models such as Direct Manipulation [ 26 ] , Direct Combination [ 11 ] and Instrumental Interaction [ 1 ] have also strongly influenced this work . Some of the interaction techniques described here have been applied to real - world applications . Marking menus and bi - manual zooming and resizing are used in the T3 prototype and in Alias | Wavefront ' s Studiopaint [ 18 ] and a variation of toolglasses is used in Maya [ 19 ] . Layers have been used as an architecture model [ 8 ] and in TicTacToon [ 7 ] , a professional animation system . 6 . SUMMARY AND CONCLUSIONS This paper articulates three design principles , reification , polymorphism and reuse , and demonstrates how they , in conjunction with Instrumental Interaction [ 1 ] , have affected the re - design of cpn2000 , a tool for editing and simulating Coloured Petri Nets . One of our goals was to incorporate the latest research in interaction techniques . The design principles led us to the insight that we did not need the concept of selection , which in turn enabled us to combine and integrate four interaction techniques : direct interaction , marking menus , floating palettes , and toolglasses . These specific interaction techniques are of particular interest because they support each of the different patterns of use we observed during our studies of CPN developers at work . Marking menus emphasize the object of interest , floating palettes emphasize the command and toolglasses permit rapid switching between objects and commands as the focus of interest changes , such as working around a cycle in a Petri net . Some users in our studies found that the new interaction techniques , marking menus and toolglasses , took a little bit of time to get used to . But everyone was able to successfully use all three tools after ten minutes of practice and all were more efficient than with the previous pull - down menus . Our approach accommodates individual preferences , allowing users to adapt the tools to their needs rather than having to adapt to the tool . Our preliminary experimental results show that different work contexts change the preferred patterns of use , even when the tasks are identical , which produces a corresponding difference in preference for tools . Thinking about these design principles has also led us to re - examine a number of existing interaction features , extending or redefining them . The resulting interface has no menu bars , title bars or scrollbars , dramatically reducing visual complexity and allocating the extra space to users ' objects of interest . Reification is a property of Instrumental Interaction that allows the creation of first - class objects from a variety of concepts . When applied systematically , reification helps us to avoid some annoying aspects of existing interfaces , such as dialog boxes that seize control and block interaction with the rest of the interface until the dialog is complete . Polymorphism , which applies a single command to objects of different types , helps avoid the potential proliferation of commands that result from extensive use of reification . Explicitly considering reification and polymorphism together enables us to make specific design choices in the former that aid in the smooth execution of the latter . Reuse allows us to capture patterns of use in the form of objects and commands and apply them in different settings . Simple reuse of reified objects and polymorphic commands directly improves the efficiency of the interface . More complex types of reuse provide subtle ways for users to capture and reflect upon their own work patterns , and subsequently tailor both the tool and their future work practices to meet the changing needs of the work and the work context . Tailoring choices need not involve choosing from long lists of difficult - to - interpret system options , but can be implicit records of work practices . As suggested in Mackay [ 20 ] , reified patterns of use can be shared , enabling newcomers to quickly adopt the work style of the group and innovators to share their new strategies for accomplishing work . Throughout our design process , these principles have helped us achieve our most basic goal , which is to provide users with 8 additional power and functionality , without simultaneously increasing the cost of use . We have just begun the development of the next version of cpn2000 , which will provide most of the functionality of the earlier Design / CPN tool . We will need to incorporate many more features , which will further test our claim that these principles make it easier to scale a prototype into a full - scale real - world application . 7 . ACKNOWLEDGMENTS Cpn2000 is a team effort : our thanks to Kurt Jensen , Søren Christensen , Peter Andersen , Paul Janecek , Mads Jensen , Michael Lassen , Kasper Lund , Kjeld Mortensen , Stephanie Munck , Katrine Ravn and Anne Ratzer for their work on the design and implementation of the system as well as extensive discussions of the ideas presented here . We are also indebted to the members of the CPN group at the University of Aarhus for their participation in the design activities . 8 . REFERENCES [ 1 ] Beaudouin - Lafon , M . Instrumental Interaction : An Interaction Model for Designing Post - WIMP User Interfaces . In Proc . Human Factors in Computing Systems , CHI ' 2000 , ACM Press , 2000 . [ 2 ] Bederson , B . and Hollan , J . Pad + + : A Zooming Graphical Interface for Exploring Alternate Interface Physics . In Proc . ACM Symposium on User Interface Software and Technology , UIST’94 , ACM Press , 1994 , p . 17 - 26 . [ 3 ] Bederson , B . , Hollan , J . , Druin , A . , Stewart , J . , Rogers , D . , Proft , D . Local Tools : an Alternative to Tool Palettes . In Proc . ACM Symposium on User Interface Software and Technology , UIST’94 , ACM Press , 1994 , p . 169 - 170 . [ 4 ] Bier , E . , Stone , M . , Pier , K . , Buxton , W . , De Rose , T . Toolglass and Magic Lenses : the See - Through Interface . In Proc . ACM SIGGRAPH , ACM Press , 1993 , p . 73 - 80 . [ 5 ] Card , S . , Robertson , G . , Mackinlay , J . The Information Visualizer , an Information Workspace . In Proc . ACM Human Factors in Computing Systems , CHI ' 91 , ACM Press , 1991 , p . 181 - 187 . [ 6 ] Casalta , D . , Guiard , Y . and Beaudouin - Lafon , M . Evaluating Two - Handed Input Techniques : Rectangle Editing and Navigation . ACM Human Factors In Computing Systems , CHI ' 99 , Extended Abstracts , 1999 , p . 236 - 237 . [ 7 ] Fekete , J - D . TicTacToon : A Paperless System for Professional 2D Animation . Proc . ACM SIGGRAPH , ACM Press , 1995 , p 79 - 90 , 1995 . [ 8 ] Fekete , J - D . & Beaudouin - Lafon , M . Using the Multi - layer Model for Building Interactive Graphical Applications . In Proc . ACM Symposium on User Interface Software and Technology , UIST ' 96 , ACM Press , p . 109 - 118 . [ 9 ] Fertig , S . , Freeman , E . and Gelernter , D . LifeStreams : An Alternative to the Desktop Metaphor . Video , ACM Human Factors in Computing Systems Adjunct Proceedings , p 410 - 411 , 1996 . [ 10 ] Guiard , Y . Asymmetric division of labor in human skilled bimanual action : The kinematic chain as a model . Journal of Motor Behavior , 19 : 486 - 517 , 1987 . [ 11 ] Holland , S . & Oppenheim , D . Direct Combination . In Proc . ACM Human Factors in Computing Systems , CHI ' 99 , ACM Press , p . 262 - 269 , 1999 . [ 12 ] Henderson , D . A . & Card , S . K . Rooms : The Use of Multiple Workspaces to Reduce Space Contention in a Window - Based Graphical User Interface . A C M Transactions on Graphics 5 ( 3 ) : 211 - 243 , 1986 . [ 13 ] Janecek , P . , Ratzer , A . , and Mackay , W . Petri - Nets - In - Use . In Proc . International Workshop on Coloured Petri Nets , Aarhus , Denmark , 1999 . [ 14 ] Jensen , K . Coloured Petri Nets : Basic Concepts ( Vol . 1 , 1992 ) , Analysis Methods ( Vol . 2 , 1994 ) , Practical Use ( Vol . 3 , 1997 ) . Monographs in Theoretical Computer Science . Springer - Verlag , 1992 - 97 . [ 15 ] Johnson , J . , Roberts , T . L . , Verplank , W . , Smith , D . C . , Irby , C . , Beard , M . and Mackey , K . The Xerox " Star " : A Retrospective . IEEE Computer 22 ( 9 ) : 11 - 29 , 1989 . [ 16 ] Kramer , A . Translucent Patches : Dissolving Windows . In Proc . ACM Symposium on User Interface Software and Technology , UIST ' 96 , ACM Press , 1996 , p . 121 - 130 . [ 17 ] Kurtenbach , G . & Buxton , W . User Learning and Performance with Marking Menus . In Proc . ACM Human Factors in Computing Systems , CHI ' 94 , ACM Press , 1994 , p . 258 - 264 . [ 18 ] Kurtenbach , G . , Fitzmaurice , G . , Baudel , T . , Buxton . W . The Design of a GUI Paradigm based on Tablets , Two - hands , and Transparency . In Proc . ACM Human Factors in Computing Systems , CHI ' 97 , ACM Press , 1997 , p . 35 - 42 . [ 19 ] Kurtenbach , G . , Fitzmaurice , G . W . , Owen , R . N . , Baudel , T . The Hotbox : efficient access to a large number of menu - items . In Proc . ACM Human Factors in Computing Systems , CHI ' 99 , ACM Press , 1999 , p . 231 - 237 . [ 20 ] Mackay , W . E . Users and Customizable Software : A Co - Adaptive Phenomenon . Ph . D . Dissertation , Massachusetts Instititute of Technology , 1990 . [ 21 ] Mackay , W . E . Triggers and barriers to customizing software . In Proc . ACM Human Factors in Computing Systems , CHI ' 91 , ACM Press , 1991 , p . 153 - 160 . [ 22 ] Maloney , J . H . & Smith , R . B . Directness and Liveness in the Morphic User Intertface Construction Environment . In Proc . ACM Symposium on User Interface Software and Technology , UIST ' 95 , ACM Press , 1995 , p . 21 - 28 . [ 23 ] Perkins , R . , Keller , D . S . and Ludolph , F . . Inventing the Lisa User Interface . ACM Interactions 4 ( 1 ) : 40 - 53 , 1997 . [ 24 ] Raisamo , R . & Räihä , K - J . A New Direct Manipulation Technique for Aligning Objects in Drawing Programs . In Proc . ACM Symposium on User Interface Software and Technology , UIST ' 96 , ACM Press , 1996 , p . 157 - 164 . [ 25 ] Rubine , D . Specifying Gestures by Example . In Proc . ACM SIGGRAPH , ACM Press , 1991 , p 329 - 337 . [ 26 ] Shneiderman , B . Direct Manipulation : a Step Beyond Programming Languages . IEEE Computer 16 ( 8 ) : 57 - 69 , 1983 . [ 27 ] Smith , R . B . Experiences with the Alternate Reality Kit : An Example of the Tension between Literalism and Magic . In Proc ACM Human Factors in Computing Systems , CHI + GI ' 87 , ACM Press , 1987 , p 61 - 67 . \ No newline at end of file diff --git a/silver_data/f81053d5347f77be070ed7bb14663242b54cfbe9.pdf.txt b/silver_data/f81053d5347f77be070ed7bb14663242b54cfbe9.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..f218237263be4ea10e75381403082effb6cb5ff2 --- /dev/null +++ b/silver_data/f81053d5347f77be070ed7bb14663242b54cfbe9.pdf.txt @@ -0,0 +1 @@ +Epithelial Splicing Regulatory Proteins 1 ( ESRP1 ) and 2 ( ESRP2 ) Suppress Cancer Cell Motility via Different Mechanisms * Receivedforpublication , June12 , 2014 , andinrevisedform , August5 , 2014 Published , JBCPapersinPress , August20 , 2014 , DOI10 . 1074 / jbc . M114 . 589432 Hiroki Ishii ‡ § , Masao Saitoh ‡ , Kei Sakamoto ¶ , Tetsuo Kondo (cid:2) , Ryohei Katoh (cid:2) , Shota Tanaka § , Mitsuyoshi Motizuki ‡ , Keisuke Masuyama § , and Keiji Miyazawa ‡1 From the Departments of ‡ Biochemistry , § Otolaryngology , Head and Neck Surgery , and (cid:2) Human Pathology , Interdisciplinary Graduate School of Medicine and Engineering , University of Yamanashi , Yamanashi 409 - 3898 and the ¶ Section of Oral Pathology , Graduate School of Medical and Dental Sciences , Tokyo Medical and Dental University , Bunkyo - ku , Tokyo 113 - 0034 , Japan Background : The roles of ESRP1 and ESRP2 during carcinogenesis remain unknown . Results : ESRPs are up - regulated during carcinogenesis but down - regulated in invasive fronts . ESRP1 suppresses expression of the Rac1b isoform , whereas ESRP2 represses epithelial - mesenchymal transition - inducing transcription factors . Conclusion : ESRP1 and ESRP2 suppress cell motility through distinct transcriptional and / or post - transcriptional mechanisms . Significance : Our findings reveal a novel molecular network that regulates cancer cell motility . ESRP1 ( epithelial splicing regulatory protein 1 ) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells , and both are down - regulated during the epithelial - mesenchymal transition . However , little is known about their expression and functions during carcinogenesis . In thisstudy , wefoundthatexpressionofbothESRP1andESRP2is plastic : during oral squamous cell carcinogenesis , these proteins areup - regulatedrelativetotheirlevelsinnormalepitheliumbut down - regulated in invasive fronts . Importantly , ESRP1 and ESRP2 are re - expressed in the lymph nodes , where carcinoma cells metastasize and colonize . In head and neck carcinoma cell lines , ESRP1 and ESRP2 suppress cancer cell motility through distinctmechanisms : knockdownofESRP1affectsthedynamicsof theactincytoskeletonthroughinductionofRac1b , whereasknock - down of ESRP2 attenuates cell - cell adhesion through increased expression of epithelial - mesenchymal transition - associated tran - scription factors . Down - regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells . The epithelial - mesenchymal transition ( EMT ) 2 is essential for embryonic organogenesis as well as tissue regeneration and wound healing ( 1 ) , but it is also involved in fibrotic diseases and cancer progression ( 2 , 3 ) . During EMT , individual cells convert from epithelial to mesenchymal phenotypes . EMT is principally characterized by changes in cell morphology , attenuation of cell - cell interaction , and loss of cell polarity , often leading to increased cell motility . These characteristics also contribute to acquisition of malignant phenotypes by cancer cells , including invasion into stromal tissues and distant metastasis ( 4 ) . The EMT process is controlled by four primary regulatory systems : transcriptional control , alternative splicing , noncoding RNA , and post - translational control ( 5 ) . Among these , transcrip - tional control has been explored most extensively . EMT is directly or indirectly controlled by several transcription factors , including (cid:2) EF1 / ZEB1 ( zinc finger E - box - binding homeobox 1 ) , SIP1 ( Smad - interacting protein 1 ) , Snail , and Twist , which are regarded as the most important regulatory components involved in initiating EMT and / or maintaining mesenchymal phenotypes . These EMT - associated transcription factors repress E - cadherin and up - regulate expression of mesenchymal markers ( N - cad - herin , vimentin , and fibronectin , etc . ) ( 4 , 6 , 7 ) . Recent work has revealed the crucial roles played by RNA splicing events during EMT . The RNA - binding proteins ESRP1 and ESRP2 and the RNA - binding protein FOX2 homologue RBFOX2 have been identified as key regulators of splicing events related to EMT ( 8 ) . ESRP1 and ESRP2 , also called RBM35A and RBM35B , respectively , are involved in maintenance of epithelial cell - specific isoforms , whereas RBFOX2 is required for mainte - nanceofmesenchymalphenotypes ( 8 ) . Werecentlydemonstrated that ESRP1 and ESRP2 are down - regulated by (cid:2) EF1 and / or SIP1 during TGF - (cid:3) - induced EMT ( 9 ) . Importantly , this down - regula - tion is essential for EMT progression . Although both ESRP1 and ESRP2 prefer to bind to the UG - rich motif , ESRP2 appears to be less active as a splicing factor ( 10 ) . ESRPs play multiple roles in tumor progression . Down - reg - ulation of ESRPs during TGF - (cid:3) - induced EMT triggers isoform switching of FGF receptors , which sensitizes cells to FGF - 2 ; cooperation of TGF - (cid:3) with FGF - 2 then promotes enhanced EMT with more aggressive phenotypes ( 11 , 12 ) . Furthermore , ectopic expression of ESRP1 / RBM35A suppresses malignant phenotypes of colon and breast cancer cells ( 9 , 13 ) , suggesting that ESRPs are tumor suppressors . In contrast , Yae et al . ( 14 ) reported that ESRP1 is associated with a lower survival rate in * This work was supported by the Vehicle Racing Commemorative Founda - tion ; Japan Society for the Promotion of Science ( JSPS ) KAKENHI Grants 24791764and24592592 ; theresearchprogramoftheProjectforDevelop - mentofInnovativeResearchonCancerTherapeutics ( P - Direct ) oftheMin - istry of Education , Culture , Sports , Science and Technology of Japan ; and the JSPS Core - to - Core Program Cooperative International Framework in TGF - (cid:3) Family Signaling . 1 To whom correspondence should be addressed . Tel . and Fax : 81 - 55 - 273 - 6784 ; E - mail : kmiyazawa @ yamanashi . ac . jp . 2 The abbreviations used are : EMT , epithelial - mesenchymal transition ; HNSCC , head and neck squamous cell carcinoma ; OSCC , oral squamous cell carcinoma ; miRNA / miR , microRNA . THEJOURNALOFBIOLOGICALCHEMISTRY VOL . 289 , NO . 40 , pp . 27386 – 27399 , October3 , 2014 ©2014byTheAmericanSocietyforBiochemistryandMolecularBiology , Inc . PublishedintheU . S . A . 27386 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 40• OCTOBER 3 , 2014 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m breast cancer patients because it enhances the cysteine / glu - tamic acid transporter ( xCT ) - dependent defense against reac - tive oxygen species in cancer cells , thereby promoting coloni - zation of the lung . Thus , whether ESRPs play positive or negative roles during tumor progression remains controversial . Moreover , although genome - wide determinations of ESRP - regulated exons have predicted that they regulate a large num - ber of splicing events in various genes ( 10 , 15 ) , most of the isoform - specific functions have not been elucidated , except in the cases of CD44 , MENA , and Exo70 ( 14 , 16 , 17 ) . In this study , we examined the expression profiles of ESRP1 and ESRP2 in human normal and tumor tissues . The expression levels of both ESRP1 and ESRP2 were low in normal epithelium but up - regulated in precancerous lesions and carcinoma in situ . Expression was maintained in advanced cancer cells but down - regulated in invasive fronts . We also found that knockdown of ESRP1 and / or ESRP2 promoted cell motility in head and neck cancer cell lines that express these proteins . Knockdown of ESRP1 derepressed Rac1b , a self - activating isoform of Rac1 , whereas knockdown of ESRP2 resulted in repression of E - cad - herin through induction of (cid:2) EF1 / SIP1 . We thus conclude that ESRP1 and ESRP2 suppress cell motility through distinct tran - scriptional and / or post - transcriptional mechanisms . EXPERIMENTAL PROCEDURES Cell Lines— Seven human head and neck squamous cell car - cinoma ( HNSCC ) cell lines ( SAS , HSC2 , HSC3 , HSC4 , Ca9 - 22 , Kuma - 1 , and Gun - 1 ) , a human uterine cervix squamous cell carcinoma cell line ( HeLa ) , and a human breast adenocarci - noma cell line ( T47D ) were used in this study . SAS , HSC2 , HSC3 , and HSC4 cells , established from human oral squamous cell carcinomas , were purchased from the Japanese Collection of Research Bioresources Cell Bank ( Osaka , Japan ) . Ca9 - 22 cells , established from a human gingival squamous cell carci - noma , were purchased from the Japanese Cancer Research Resources Bank ( Tokyo , Japan ) . Kuma - 1 cells , established from a human maxillary sinus squamous cell carcinoma , and Gun - 1 cells , established from a human hypopharyngeal squamous cell carcinoma ( 18 ) , were kindly supplied by Dr . Kazuaki Chikamatsu ( Gunma University , Gunma , Japan ) . These cells were cultured in DMEM ( Nacalai Tesque ) supplemented with 10 % heat - inacti - vated FBS , 500 units / ml penicillin , and 500 (cid:4) g / ml streptomycin at 37 °C in a humidified atmosphere containing 5 % CO 2 . Immunohistochemistry— Formalin - fixed , paraffin - embedded tissue blocks were sectioned ( 2 (cid:4) m thick ) and mounted on sil - ane - coated glass slides . These slides were then deparaffinized with xylene , followed by rehydration through graded alcohols to water . For antigen retrieval , deparaffinized sections were placed in plastic Coplin jars and heated in an autoclave at 120 °C for 15 – 20 min in 10 m M citrate buffer solution ( pH 6 ) or target retrieval solution ( pH 9 ) ( Dako S2367 ) . Next , endogenous per - oxidase was quenched with 3 % ( v / v ) H 2 O 2 for 5 min . After blocking with 1 % BSA at room temperature for 2 h , the sections were incubated with a primary antibody diluted with 1 % BSA in PBS . Slides were incubated overnight at 4 °C with primary monoclonal antibodies . Primary antibodies and working dilu - tion rates were as follows : anti - ESRP1 , 1 : 100 ( Sigma - Aldrich HPA023720 ) ; anti - ESRP2 , 1 : 100 ( Abcam ab113486 ) ; anti - E - cadherin , 1 : 100 ( BD Biosciences 610181 ) ; and anti - Rac1b , 1 : 50 ( Millipore 09 - 271 ) . Slides were then incubated with HRP using a ChemMate EnVision kit ( Dako ) for 2 h . After the slides were washed twice with PBS , immunoreactivity was visualized with 0 . 6 n M 3 – 3 (cid:2) - diaminobenzidine tetrahydrochloride ( Dojindo ) , and the slides were counterstained with hematoxylin . Images were acquired with an Olympus BX53 microscope and DP72 camera and analyzed using Olympus cellSens software . All studies were conducted using protocols approved by the ethics committee of the University of Yamanashi and the Tokyo Med - ical and Dental University . Immunofluorescence and Phalloidin Staining— Cells were grown on 24 (cid:3) 24 - mm microscope cover glass ( Matsunami ) , fixed in acetone / methanol for 10 min , and permeabilized for 10 min in 0 . 3 % Triton X - 100 in PBS . After blocking with Blocking One Histo ( Nacalai Tesque ) for 15 min , cells were incubated overnight at 4 °C with anti - E - cadherin primary monoclonal antibody diluted in Blocking One Histo . The cells were incu - bated with Alexa Fluor 633 - labeled goat anti - mouse IgG ( Invit - rogen A - 21052 ) as a secondary antibody for 2 h and then with DAPI for 15 min at room temperature . Fluorescence was exam - inedusinganOlympusFV1000confocalmicroscopeandanalyzedwithOlympusFV10 - ASW imaging software . Phalloidin staining wasperformedasdescribedpreviously ( 19 ) . Imageswereacquired with an Olympus BX50 microscope and analyzed using View - Finder Lite software ( Better Light , Inc . ) . Filopodial length was measured at eight random fields on photographs . Immunoblot Analysis— Immunoblot analysis was performed as described previously ( 20 ) . Cells were lysed in lysis buffer con - taining 20 m M Tris - HCl ( pH 7 . 4 ) , 5 m M EDTA , 150 m M NaCl , 1 % Nonidet P - 40 , 1 % aprotinin , and 1 m M PMSF on ice . The protein concentration was measured using the BCA protein assay reagent ( Thermo Fisher Scientific ) . Proteins were sepa - rated by SDS - PAGE , followed by electrophoretic transfer onto polyvinylidene difluoride membranes and blocking with 50 m M Tris - HCl ( pH 7 . 4 ) , 150 m M NaCl , and 0 . 1 % Tween 20 contain - ing 5 % skim milk . The following primary antibodies were used : anti - ESRP1 ( Sigma - Aldrich HPA023719 ) , anti - ESRP2 ( Abcam ab113486 ) , anti - E - cadherin ( BD Biosciences 610181 ) , anti - Rac1 ( Millipore 05 - 389 ) , anti - Rac1b ( Millipore 09 - 271 ) , and anti - (cid:5) - tubulin ( Sigma - Aldrich DM1A ) . The blots were incu - bated overnight at 4 °C . After incubation with HRP - conjugated mouse or rabbit IgG ( Jackson ImmunoResearch Laboratories ) for 1 h , proteins were visualized using Amersham Biosciences ECL Western blotting detection reagent ( GE Healthcare ) . All images were acquired with a Fujifilm LAS - 4000 mini imager and analyzed with Image Reader LAS - 4000 software . This soft - ware was also used to quantify protein bands on immunoblots . RNA Interference— Transfection of siRNAs into cells ( 5 (cid:3) 10 5 ) was performed using Lipofectamine RNAiMAX transfec - tion reagent ( Invitrogen ) . The Stealth RNAi siRNA sequences used in this study were as follow : human ESRP1 ( 262 ) , 5 (cid:2) - GAGAAGGAGUUGAUCCUGCUGUUCU - 3 (cid:2) ; human ESRP1 ( 981 ) , 5 (cid:2) - CGGAGAAGCUCUGGUUAGGUUUGUA - 3 (cid:2) ; human ESRP2 ( 862 ) , 5 (cid:2) - CCGAGGUGAUAAAGCAGAAA - UACGA - (cid:2) ; human ESRP2 ( 1126 ) , 5 (cid:2) - GCGUCCGCUAUAUU - GAGGUGUAUAA - 3 ; human Rac1b 5 (cid:2) - UGGAGAAACGUA - CGGUAAGGAUAUA - 3 ; human (cid:2) EF1 , 5 (cid:2) - GACCAGAACAG - ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27387 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m UGUUCCAUGCUUAA - 3 (cid:2) ; and human SIP1 , 5 (cid:2) - CCACCAC - AGUGUUACGAAUUGUGAU - 3 (cid:2) . The final concentration of the siRNAs used was 5 n M , except for the SIP1 siRNA , which was used at 10 n M . Unless noted otherwise , we used ESRP1 ( 981 ) and ESRP2 ( 862 ) siRNAs . Conventional PCR and Quantitative Real - time PCR— Total RNA was extracted from each cell line using the RNeasy mini kit ( Qiagen ) . Two (cid:4) g of total RNA were then reverse - tran - scribed into cDNA using the SuperScript VILO cDNA synthe - sis kit ( Invitrogen ) . Conventional PCR was performed with LA Taq polymerase ( TaKaRa ) . The primers used in conventional PCR are as follows : human CD44 , 5 (cid:2) - GCACTTCAGGAGGT - TACATC - 3 (cid:2) ( sense ) and5 (cid:2) - ACTGCAATGCAAACTGCAAG - 3 (cid:2) ( antisense ) ; human Rac1 , 5 (cid:2) - GGATCCTTTGACAATTATTCT - GCCAATG - 3 (cid:2) ( sense ) and 5 (cid:2) - CGGACATTTTCAAATGATG - CAGG - 3 (cid:2) ( antisense ) ; human MENA , 5 (cid:2) - GCTGGAATGGGA - GAGAGAGCGCAGAATATC - 3 (cid:2) ( sense ) and 5 (cid:2) - GTCAAGTC - CTTCCGTCTGGACTCCATTGGC - 3 (cid:2) ( antisense ) ; and human (cid:3) - actin , 5 (cid:2) - GGCATCCTCACCCTGAAGTA - 3 (cid:2) ( sense ) and 5 (cid:2) - GGGGTGTTGAAGGTCTCAAA - 3 (cid:2) ( antisense ) . All PCR conditions included an initial denaturation for 2 min at 95 °C . Amplification reactions were performed for 30 cycles under the following conditions : 95 °C for 1 min , 98 °C for 20 s , and 60 °C for 30 s , followed by an extension of 1 min at 72 °C . PCR products were separated on 1 . 5 % agarose gels , stained with ethidium bromide , and visualized using a Printgraph AE - 6932 gel detection system ( ATTO Corp . ) . The gene encoding (cid:3) - actin was used as an internal control in conventional PCR . Quantita - tive RT - PCR using SYBR Green was performed on an ABI 7300 Fast real - time PCR system ( Applied Biosystems ) as described previously ( 21 ) . mRNA levels were normalized to the level of the mRNA encoding GAPDH in the same sample . The relative expression levels of target genes were determined by the 2 (cid:4) ( (cid:5)(cid:5) Ct ) method . The primers used were as follows : human ESRP1 , 5 (cid:2) - CAATATTGCCAAGGGAGGTG - 3 (cid:2) ( sense ) and 5 (cid:2) - GTCCCCATGTGATGTTTGTG - 3 (cid:2) ( antisense ) ; human ESRP2 , 5 (cid:2) - TGCCACAGAGGATGACTTTG - 3 (cid:2) ( sense ) and 5 (cid:2) - ATTGACTGCTGGGCTCTTTG - 3 (cid:2) ( antisense ) ; human E - cadherin , 5 (cid:2) - TGCACCAACCCTCATGAGTG - 3 (cid:2) ( sense ) and 5 (cid:2) - GTCAGTATCAGCCGCTTTCAG - 3 (cid:2) ( antisense ) ; human (cid:2) EF1 , 5 (cid:2) - CAATGATCAGCCTCAATCTGCA - 3 (cid:2) ( sense ) and 5 (cid:2) - GTCAGTATCAGCCGCTTTCAG - 3 (cid:2) ( antisense ) ; human SIP1 , 5 (cid:2) - AAGCCCCATCAACCCATACAAG - 3 (cid:2) ( sense ) and 5 (cid:2) - AAATTCCTGAGGAAGGCCCA - 3 (cid:2) ( antisense ) ; human Snail , 5 (cid:2) - TTCTCACTGCCATGGAATTCC - 3 (cid:2) ( sense ) and 5 (cid:2) - GCAGAGGACACAGAACCAGAAA - 3 (cid:2) ( antisense ) ; human Slug , 5 (cid:2) - GCCTCCAAAAGCCAAACTACA - 3 (cid:2) ( sense ) and 5 (cid:2) - GAGGATCTCTGGTTGTGGTATGACA - 3 (cid:2) ( antisense ) ; human Twist , 5 (cid:2) - GGCCGGAGACCTAGATGTCATT - 3 (cid:2) ( sense ) and 5 (cid:2) - CCACGCCCTGTTTCTTTGAAT - 3 (cid:2) ( anti - sense ) ; and human GAPDH , 5 (cid:2) - GCACCGTCAAGGCTGAG - AAC - 3 (cid:2) ( sense ) and 5 (cid:2) - ATGGTGGTGAAGACGCCAGT - 3 (cid:2) ( antisense ) . GST - PAK Fusion Protein Binding Assay— Cells ( 5 (cid:3) 10 5 ) were inoculated , cultured for 48 h , and harvested . Rac1b acti - vation was assessed by GST pulldown assay using the Rac1 / Cdc42 activation assay kit ( Millipore 17 - 441 ) . Wound Healing Assay— Cells were inoculated in 6 - well plates . After cells reached confluence , they were scratched with a 10 - (cid:4) l disposable pipette tip . Migration of wound edges was measured at 10 random points on photographs acquired using a Nikon Eclipse TS100 microscope , and then cell migration dis - tance after 12 h was compared with the distance at 0 h . A pre - vious protocol ( 19 ) was used for data analysis . Proliferation Assay— Cells ( 1 (cid:3) 10 5 ) were inoculated in 6 - well plates and cultured for 24 , 72 , 120 , and 168 h . Cells were then trypsinized and counted using hemocytometer . Statistical Analyses— All statistical analyses were performed using StatMate IV software ( ATMS Co . ) . RESULTS ESRP1 and ESRP2 Are Up - regulated during Human Oral Squamous Cell Carcinoma Carcinogenesis— We first assessed ESRP1 and ESRP2 expression during the progression of carci - nogenesis from normal epithelium to dysplasia and invasive carcinoma . To this end , we examined human dysplasia and oral squamous cell carcinoma ( OSCC ) samples by immunohisto - chemical staining . In normal human squamous epithelium , ESRP1 and ESRP2 were detected only weakly . Most of the ESRP - positive cells were located in the basal layer , whereas only a few were present in the cornified layer ( Fig . 1 A ) . To the extent that they were expressed , they were principally localized in the nucleus ( Fig . 1 ) . The expression levels of ESRP1 and ESRP2 were higher in dysplastic lesions than in normal epithelium ( Fig . 1 B ) . Epithelial dysplasia can be divided into three catego - ries : mild , moderate , and severe . As the degree of cellular atypia rose , the proportion of ESRP1 - positive cells increased ( Fig . 1 C ) . In carcinoma in situ and advanced OSCC lesions , which can be classified into well or poorly differentiated types , ESRP1 expres - sion in cancer cells was also significantly elevated . The patterns and intensities of ESRP1 expression in each histological type of advanced OSCC were similar to those in dysplastic lesions ( Fig . 1 D ) . A similar expression pattern was observed for ESRP2 in dysplasia ( data not shown ) and advanced OSCC samples ( Fig . 1 E ) . In addition , we evaluated the intensities of ESRP1 and ESRP2 immunoreactivity in 49 human OSCC samples . The reactivities for ESRP1 and ESRP2 were elevated relative to those for normal epithelium in 46 of 49 and 40 of 49 OSCC samples , respectively . These results clearly indicate that human ESRP1 and ESRP2 are expressed in both normal epithelium and OSCC and are up - regulated during OSCC carcinogenesis . Reduction of ESRP1 and ESRP2 Expression in Invasive OSCC— ESRPs are down - regulated in basal - like breast cancer cell lines , which are invasive ( 9 ) . To determine whether ESRP expression levels are altered in cancer cells during invasion into surrounding stromal tissues , we next analyzed expression of ESRP1 in carcinoma in situ and OSCC with invasive pheno - types . In cancer cells that penetrated through the basement membrane to invade stromal tissues , ESRP1 expression was sig - nificantly reduced ( Fig . 2 , A and B ) . We also examined ESRP1 and ESRP2 expression in cancer cells that invaded the stroma to deeper locations , as well as in cells that metastasized to a lymph node . In both locations , ESRP1 was re - expressed in the cancer nest ( Fig . 2 , C , left ; D ; and E , left ) but repressed in the invasive fronts ( Fig . 2 , C , right ; and E , right ) . These results suggest that ESRP1 and ESRP2 Differently Suppress Cell Motility 27388 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 40• OCTOBER 3 , 2014 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m expression of ESRPs is plastic during invasion and metastasis of cancer cells . We previously reported a positive correlation between expres - sionofESRPsandE - cadherininhumanbreastcancercelllines ( 9 ) . Consistent with our earlier finding , in invasive fronts , where ESRP1 and ESRP2 levels were reduced , we observed loss or inter - nalization of junctional E - cadherin ( Fig . 2 F ) . Thus , ESRP1 and ESRP2 may suppress invasion or cell motility in HNSCC . Expression of ESRPs in HNSCC Cell Lines— To explore the possible suppressive effects of ESRPs on cell motility , we per - formed in vitro experiments using human HNSCC cell lines . We first examined ESRP mRNA expression in seven HNSCC cell lines , using HeLa cells for comparison . ESRP1 gene expres - sion was higher in all seven HNSCC cells than in HeLa cells , whereas there was no striking difference in ESRP2 expres - sion between HNSCC cell lines and HeLa cells ( Fig . 3 A ) . We also examined endogenous ESRP1 and ESRP2 proteins by immunoblotting ( Fig . 3 B ) ; the protein expression profiles were similar . Isoform switching of CD44 mRNA is regulated by ESRPs ( 9 , 14 , 22 ) . Therefore , to assess ESRP activities in HNSCC cell lines , we analyzed splicing isoforms of CD44 mRNA by conventional PCR ( Fig . 3 C ) . In HNSCC cell lines expressing higher levels of ESRP1 ( SAS , HSC4 , and Ca9 - 22 ) , CD44 variant isoforms , including CD44v2 - 10 , CD44v6 - 10 , and CD44v8 - 10 , were exclusively expressed . In contrast , only the CD44 standard iso - form was expressed in HeLa cells lacking ESRP1 . We then designed two siRNAs against ESRP1 and ESRP2 and transfected these siRNAs into SAS and HSC4 cells , in which ESRP1 and ESRP2 are highly expressed ( Fig . 3 , D and E ) . Silencing of ESRP1 resulted in switching from the CD44 variant isoforms to the CD44 standard isoform ( Fig . 3 F ) , indicating that ESRP1 is active in these cell lines . In contrast , silencing of ESRP2 did not affect CD44 isoform switching ( Fig . 3 G ) . We next analyzed splicing FIGURE1 . ESRPsareup - regulatedduringhumanOSCCcarcinogenesis . ExpressionofhumanESRP1andESRP2wasinvestigatedbyimmunohistochemical analysis . A and B , ESRP1 ( left ) andESRP2 ( right ) expression ( brown ) innormalepithelium ( A ) andtheborderbetweennormalepitheliumandthedysplasticlesion ( B ) . C , ESRP1 expression ( brown ) in different stages of dysplasia : mild ( left ) , moderate ( middle ) , and severe ( right ) . D , ESRP1 expression ( brown ) in well ( left ) and poorly differentiated ( right ) OSCC tissues . E , ESRP1 or ESRP2 expression ( brown ) in advanced OSCC . Serial sections were stained with hematoxylin and eosin ( H - E ; left ) , anti - ESRP1 antibody ( middle ) , and anti - ESRP2 antibody ( right ) . Scale bars (cid:6) 100 (cid:4) m . ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27389 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m isoforms of MENA mRNA , also identified as an ESRP target . There are three isoforms of MENA transcripts , MENA , MENA 11a ( (cid:7) ) , and MENA (cid:5) v6 ; MENA 11a ( (cid:7) ) and MENA (cid:5) v6 are the epithelial and mesenchymal types , respectively ( 16 ) . In con - trol cells , MENA 11a ( (cid:7) ) was most abundantly expressed . Knock - down of ESRP1 resulted in down - regulation of MENA 11a ( (cid:7) ) and up - regulation of MENA and MENA (cid:5) v6 . Knockdown of ESRP2 increased the MENA isoform , although it did not induce FIGURE2 . ESRPsaredown - regulatedininvasivefronts . ExpressionofhumanESRP1andESRP2wasinvestigatedbyimmunohistochemicalanalysis . A , ESRP1 expression ( brown ) in the border between carcinoma in situ ( CIS ) and the invasive lesion . B , ESRP1 expression ( brown ) in the basal layer where the basement membrane ( BM ) was intact ( left ) or where cancer cells penetrated through the basement membrane ( right ) . C , ESRP1 ( left ) or ESRP2 ( right ) expression ( brown ) in the invasive fronts of advanced OSCC . Arrows indicate the direction of tumor invasion . Each high - power field is shown in the panels surrounded by dashed lines . ThegradientexpressionofESRP1orESRP2isschematicallyrepresentedasa blackslope . D and E , ESRP1 ( left ) orESRP2 ( right ) expression ( brown ) inalymph node with metastasis : cancer nests ( D , arrows ) or those with invasive fronts in the lymphatic tissue ( E , arrows ) . Each high - power field is shown in the panels surrounded by dashed lines . The gradient expression of ESRP1 or ESRP2 is schematically represented as a black slope . F , representative image of the invasive fronts in advanced OSCC . Serial sections were stained with hematoxylin and eosin ( H - E ) , anti - ESRP1 antibody , anti - ESRP2 antibody , and anti - E - cadherin antibody ( brown ) . High - powerfieldsofthenoninvasiveregion ( panelsa , c , and e ) andthetipoftheinvasivefront ( panelsb , d , and f ) areshown . Scalebars (cid:6) 100 (cid:4) m . ESRP1 and ESRP2 Differently Suppress Cell Motility 27390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 40• OCTOBER 3 , 2014 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m the MENA (cid:5) v6 isoform ( Fig . 3 H ) . These results indicate that ESRP2 is also active in these cell lines . Knockdown of ESRPs Promotes HNSCC Cell Migration— Because we observed down - regulation of ESRPs at invasive fronts , we next examined the phenotypic effects of silencing endogenous ESRP1 and / or ESRP2 in HNSCC cell lines . First , we confirmed knockdown of ESRP1 and / or ESRP2 , as shown in Fig . 4 A . Next , we examined changes in cell morphology . Knock - down of ESRP1 and / or ESRP2 induced cell scattering , and knockdown of ESRP2 appeared to be more effective in SAS cells ( Fig . 4 B ) . Although knockdown of ESRPs did not affect cell proliferation in either of these cell types ( Fig . 4 C ) , wound clo - sure was faster in knockdown cells than in control cells ( Fig . 4 D ) . These findings indicate that knockdown of either ESRP1 or ESRP2 increases cell motility , although double knockdown does not increase motility further . Mechanisms of Enhanced Cell Motility after ESRP Knock - down— To further elucidate the mechanisms underlying enhanced cell motility in ESRP - silenced cells , we examined E - cadherin expression and actin dynamics . E - cadherin protein expression was decreased by knockdown of ESRP2 , but not ESRP1 ( Fig . 4 A ) . Furthermore , knockdown of ESRP2 down - FIGURE 3 . Expression profiles and activities of ESRPs in HNSCC cell lines . A , mRNA levels of ESRP1 and ESRP2 in HNSCC cell lines as determined by quantitativeRT - PCR . ExpressionlevelsarepresentedrelativetothoseinT47Dbreastcarcinomacells , whichexpressbothESRP1andESRP2 , andeachvaluewas normalizedtoexpressionofGAPDHinthesamesample . HeLacellswereusedasacontrol . Valuesaremeans (cid:8) S . D . oftriplicatemeasurements . B , proteinlevels ofESRP1andESRP2inHNSCCcelllinesasdeterminedbyimmunoblotanalysis . (cid:5) - Tubulinwasusedasaloadingcontrol . Bandsforeachproteinwereconfirmed by siRNAs against ESRP1 ( siESRP1 ) and ESRP2 ( siESRP2 ) in SAS cells . Arrows indicate ESRP1 ( upper ) and ESRP2 ( lower ) . Asterisks denote nonspecific bands . C , profiles of CD44 variant ( CD44v ) and standard ( CD44s ) isoforms in HNSCC cell lines examined by conventional RT - PCR . (cid:3) - actin was used as an internal control . D and E , knockdown of ESRP1 ( D ) and ESRP2 ( E ) in SAS and HSC4 cells . Cells were treated with control siRNA ( siControl ) , ESRP1 siRNA , or ESRP2 siRNA for 48 h . The expression levels of mRNAs and proteins were examined by quantitative RT - PCR ( upper ) and immunoblotting ( lower ) , respectively . mRNA levels are presented relative to those in cells treated with control siRNA after normalization to the level of GAPDH mRNA ( upper ) . Values are means (cid:8) S . D . of triplicate measurements . (cid:5) - Tubulin was used as a loading control ( lower ) . In E , the arrow and asterisk denote ESRP2 protein and nonspecific bands , respectively . F , expression of CD44 isoforms was examined by conventional RT - PCR in SAS and HSC4 cells after knockdown of ESRP1 . (cid:3) - Actin was used as an internal control . G , expression of CD44 isoforms was examined by conventional RT - PCR after knockdown of ESRP2 in SAS and HSC4 cells . mRNAs extracted from ESRP1 knockdown in SAS and HSC4 cells were used as positive controls . (cid:3) - Actin was used as an internal control . H , expression of MENA isoforms was examined by conventional PCR in SAS and HSC4 cells after knockdown of ESRP1 or ESRP2 . (cid:3) - Actin was used as an internal control . ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27391 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m regulated E - cadherin at the transcriptional level ( Fig . 5 A ) ; knockdown of ESRP1 had a similar but more modest effect . Localization of E - cadherin was not affected by knockdown of ESRP1 alone ( Fig . 5 B ) , indicating that ESRP1 affects the E - cad - herin status only minimally . We also examined the status of the actin cytoskeleton in knock - down cells . Remarkably , knockdown of ESRP1 dramatically pro - motedformationoflongfilopodiarelativetocontrolcells ( Fig . 5 , C and D ) , whereas knockdown of ESRP2 had no detectable effect on the actin cytoskeleton . These results indicate that ESRPs play sup - pressive roles in the control of cell motility in HNSCC : ESRP1 is involved in the regulation of actin dynamics , whereas ESRP2 prin - cipally maintains E - cadherin expression . ESRP1 Regulates Splicing of Rac1 Isoforms— We next exam - ined the mechanism underlying formation of long filopodia after knockdown of ESRP1 . Actin dynamics are regulated by the Rho family of small G - proteins , Rho , Cdc42 , and Rac1 . Rac1 has a self - activating splice variant , Rac1b , which is characterized by inclusion of exon 3b ( Fig . 6 A ) ( 23 ) . Exon 3b encodes 19 amino acid residues , the insertion of which induces a conformational change in the switch I and II regions in Rac1b , leading to acti - vation by GDP / GTP exchange independent of guanine nucleo - tide exchange factors , as well as impaired hydrolysis of GTP ( 24 ) . A previous report showed that Rac1b promotes cell motil - ity ( 25 ) . In light of these observations , we assessed the alterna - tive splicing of Rac1 mRNA in HNSCC cell lines . ESRP1 knockdown increased the level of the Rac1b isoform in SAS and HSC4 cells , whereas ESRP2 knockdown did not ( Fig . 6 B ) . Rac1b protein was also significantly up - regulated in ESRP1 knockdown cells ( Fig . 6 C ) ; the protein was active , as assessed by FIGURE4 . KnockdownofESRP1and / orESRP2resultsinincreasedcancercellmotility . Cellsweretreatedwithcontrol ( siControl ) , ESRP1 ( siESRP1 ) , orESRP2 ( siESRP2 ) siRNA48hbeforethefollowingassays . A , proteinexpressionofESRP1 , ESRP2 , andE - cadherininSASandHSC4cellsinwhichESRP1and / orESRP2were knocked down as determined by immunoblotting . (cid:5) - Tubulin was used as a loading control . B , phase - contrast images of morphological changes in SAS cells afterknockdownofESRP1and / orESRP2 . Scalebars (cid:6) 10 (cid:4) m . C , proliferationofESRP1 - orESRP2 - silencedSAS ( left ) andHSC4 ( right ) cellswasexamined . Values are means (cid:8) S . D . of triplicate measurements . D , wound healing assay of SAS and HSC4 cells after knockdown of ESRP1 and / or ESRP2 . The results were quantitatedasshown ( lower ) . Migrationofthewoundedgewasmeasuredat10randomlychosenpointsinthephotograph . Cellmigrationdistancesafter12h at 10 randomly chosen points were compared with the distances at 0 h . Values are means (cid:8) S . D . of triplicate measurements . Similar results were obtained in three independent experiments . p values were determined by Student’s t test . * , p (cid:9) 0 . 01 ; n . s . , not significant . ESRP1 and ESRP2 Differently Suppress Cell Motility 27392 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 40• OCTOBER 3 , 2014 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m binding to the GST - PAK fusion protein ( Fig . 6 D ) . Thus , ESRP1 appears to repress expression of the Rac1b isoform by exclud - ing variant exon 3b from splicing of Rac1 mRNA . Intriguingly , we noted that an ESRP - binding motif ( UGGUGG ) ( 10 ) is pres - ent in the intron upstream of exon 3b ( Fig . 6 A ) , suggesting that ESRP1 directly regulates the alternative splicing of Rac1 mRNA . Next , we examined expression of Rac1b in human OSCC tissues by immunohistochemical staining . Consistent with our in vitro findings , the expression levels of Rac1b were increased in invasive fronts ( Fig . 6 E ) . ESRP1 and Rac1b are thus reciprocally expressed in OSCC tissues . We next investigated the role of Rac1b in the promotion of cell motility by ESRP1 knockdown . First , we knocked down Rac1b in ESRP1 - silenced HNSCC cell lines by transfecting siRNA against the Rac1b - specific exon 3b ( Fig . 7 , A and B ) . Knockdown of Rac1b significantly suppressed the elevated cell motility induced by silencing of ESRP1 ( Fig . 7 C ) . In addition , formation of long filopodia was significantly attenuated after FIGURE5 . MechanismsofenhancedcancercellmotilitybyESRPknockdown . A , expressionofE - cadherininESRP1 - and / orESRP2 - silencedSASandHSC4cellsas determinedbyquantitativeRT - PCRanalysis . mRNAlevelsarepresentedrelativetothoseincellstreatedwithcontrolsiRNAafternormalizationtothelevelofGAPDH mRNA . Valuesaremeans (cid:8) S . D . oftriplicatemeasurements . Similarresultswereobtainedinthreeindependentexperiments . p valuesweredeterminedbyStudent’s t test . * , p (cid:9) 0 . 05 ; * * * , p (cid:9) 0 . 001 . siESRP1 , ESRP1siRNA ; siESRP2 , ESRP2siRNA . B , subcellularlocalizationofE - cadherin ( white ) inESRP1and / orESRP2knockdownSASand HSC4 cells as visualized by immunofluorescence . Nuclei were stained with DAPI ( blue ) . Scale bars (cid:6) 2 . 5 (cid:4) m . siControl , control siRNA . C , phalloidin staining of ESRP1 and / orESRP2knockdownSASandHSC4cells . High - powerfieldsareshown ( lower ) . Scalebars (cid:6) 1 . 0 (cid:4) m . D , quantificationoffilopodiallength . Each number belowthe graphsindicatesthenumberofmeasuredfilopodia . p valuesweredeterminedbythemediantest . * , p (cid:9) 0 . 001 ; n . s . , notsignificant . ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27393 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m knockdown of Rac1b in ESRP1 - silenced cells ( Fig . 7 , D and E ) . These results demonstrate that ESRP1 suppresses cancer cell motility by repressing the Rac1b isoform . Knockdown of ESRP2 Increases EMT - associated Transcrip - tion Factors in HNSCC— Finally , we sought to clarify the mech - anism underlying down - regulation of E - cadherin after knock - down of ESRP2 . Because E - cadherin expression is directly regulated by EMT - associated transcription factors , including (cid:2) EF1 , SIP1 , Snail , Slug , and Twist , we examined the expression of these factors after knockdown of ESRP2 . (cid:2) EF1 and SIP1 levels were significantly higher in ESRP2 - silenced SAS cells , whereas knockdown of ESRP2 in HSC4 cells induced only SIP1 ( Fig . 8 A ) . Together , these observations suggest that E - cadherin expres - sion is repressed by (cid:2) EF1 and / or SIP1 upon knockdown of ESRP2 . We then knocked down both (cid:2) EF1 and SIP1 in ESRP2 - silenced SAS cells and SIP1 alone in ESRP2 - silenced HSC4 cells ( Fig . 8 B , upper ) . Knockdown of (cid:2) EF1 and SIP1 was not com - plete , but it was sufficient to compensate for the increase in expression due to knockdown of ESRP2 . E - cadherin mRNA and protein levels were significantly restored by knockdown of (cid:2) EF1 and SIP1 ( Fig . 8 , B , lower ; and C ) . As shown by immunofluores - cence staining ( Fig . 8 D ) , E - cadherin re - accumulated , and cell - cell adhesion partially recovered after silencing of (cid:2) EF1 and SIP1 in ESRP2 - silenced SAS cells . Similarly , E - cadherin expres - sion was restored by knockdown of SIP1 in ESRP2 - silenced HSC4 cells ( data not shown ) . These results indicate that ESRP2 represses (cid:2) EF1 and SIP1 to maintain E - cadherin expression in HNSCC cells . In addition , we examined the motility of cells in which (cid:2) EF1 , SIP1 , and ESRP2 were silenced . As described above , knockdown of ESRP2 promoted cell motility ( Fig . 4 D ) . Additional knockdown of both (cid:2) EF1 and SIP1 or SIP1 alone canceled the effect of ESRP2 in SAS or HSC4 cells , respectively ( Fig . 8 E ) . Thus , we conclude that ESRP2 inhibits cancer cell motility by suppressing EMT - related transcription factors . DISCUSSION ESRP1 and ESRP2 , which belong to the RBM family of RNA - binding proteins , were originally identified as epithelium - spe - cific splicing regulators in a genome - wide cDNA expression screen aimed at finding key factors that enable alternative splic - ing of FGF receptor - 2 mRNA in epithelial cells ( 26 ) . They have molecular masses of (cid:10) 75 kDa and consist of three conserved tandem RNA recognition motifs ( 26 ) . Although several recent in vitro studies explored the functions of ESRPs as regulators of EMT - associated alternative splicing , the roles of these proteins in cancer progression remain to be elucidated . Ueda et al . ( 27 ) FIGURE 6 . ESRP1 is associated with regulation of alternative splicing of Rac1 mRNA . A , schematic presentation of alternative splicing of Rac1 mRNA . Insertion of exon 3b ( 57 bp ) , which yields the Rac1b isoform , was examined . Primers were designed in exons 3 and 4 . An ESRP - binding consensus sequence ( UGGUGGGUG ) islocated274bpupstreamofexon3b ( shownbythe asterisk ) . Fw , forward ; Rv , reverse . B and C , changesinRac1isoformsinESRP1 - , ESRP2 - , and ESRP1 / ESRP2 - silenced HNSCC cells as determined by conventional PCR ( B ) or immunoblotting ( C ) . siESRP1 , ESRP1 siRNA ; siESRP2 , ESRP2 siRNA . In C , (cid:5) - tubulin wasusedasaloadingcontrol . ThequantificationofRac1bproteinisshown ( right ) . ThedensityofRac1bproteinbandsonWesternblotimageswasmeasured andquantified . ProteinlevelsarepresentedrelativetothoseincellstreatedwithcontrolsiRNA ( siControl ) afternormalizationtotheproteinlevelof (cid:5) - tubulin . Values are means (cid:8) S . D . of triplicate measurements . Similar results were obtained in three independent experiments . p values were determined by Student’s t test . * , p (cid:9) 0 . 05 ; n . s . , notsignificant . D , detectionoftheactiveRac1bisoforminESRP1 - silencedHNSCCcellsbyapulldownassayusingGST - PAKfusionprotein . Rac1b ( brown ) was visualized using a specific monoclonal antibody raised against the amino acid sequence encoded by exon 3b . (cid:5) - Tubulin was used as an input control . E , immunohistochemical detection of Rac1b in human advanced OSCC . The arrow indicates the direction of tumor invasion . The gradient expression of Rac1b is shown schematically as a black slope . ESRP1 and ESRP2 Differently Suppress Cell Motility 27394 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 40• OCTOBER 3 , 2014 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m recently reported that ESRP1 is weakly expressed in normal pancreatic ductal cells . Using in situ RNA hybridization , Revil and Jerome - Majewska ( 28 ) demonstrated that ESRP1 is expressed in the head region of developing mouse embryos . ESRP1 is also expressed in human breast and pancreatic can - cers ( 9 , 27 ) . In contrast , in vivo expression of ESRP2 has not been reported . Moreover , it remains unclear how ESRP1 and ESRP2 expression is regulated in the process of carcinogenesis . In this study , we examined the expression profiles of ESRP1 and ESRP2 during carcinogenesis using 49 samples of human HNSCC tissue . The process of squamous cell carcinogenesis consists of several developmental steps , including dysplasia , FIGURE 7 . Knockdown of ESRP1 increases cell motility through up - regulating Rac1b . Shown is the knockdown of the Rac1b isoform in SAS or HSC4 cells . Cellsweretreatedwithcontrol ( siControl ) , ESRP1 ( siESRP1 ) , orRac1b ( siRac1b ) siRNA . After48h , RNAsorproteinswereextractedandsubjectedtoconventional RT - PCR ( A ) orimmunoblotting ( B ) usinganti - Rac1antibody . In A , (cid:3) - actinwasusedasaninternalcontrol . In B , the asterisk denotesRac1 , and (cid:5) - tubulinwasused as a loading control . C , wound healing assay of SAS and HSC4 cells in which ESRP1 alone or with Rac1b were knocked down . The results were quantitated as shown ( lower ) . Migrationofthewoundedgewasmeasuredat10randomlychosenpointsinthephotograph . Cellmigrationdistancesafter12hat10randomly chosen points were compared with the distances at 0 h . Values are means (cid:8) S . D . of triplicate measurements . Similar results were obtained in three indepen - dentexperiments . p valuesweredeterminedbyStudent’s t test . * , p (cid:9) 0 . 01 . D , phalloidinstainingofSASandHSC4cellsinwhichESRP1andRac1bwereknocked down . Scale bars (cid:6) 1 . 0 (cid:4) m . E , quantification of filopodial length . Each number below the graphs indicates the number of measured filopodia . p values were determined by the median test . * , p (cid:9) 0 . 001 ; n . s . , not significant . ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27395 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m FIGURE 8 . ESRP2 maintains E - cadherin expression by repressing the (cid:2) EF1 family of transcription factors . A , expression of EMT - associated transcription factors in SAS and HSC4 cells in which ESRP2 or both ESRP1 and ESRP2 were knocked down . After incubation for 48 h , gene expression levels were measured byquantitativeRT - PCR . mRNAlevelsarepresentedrelativetothoseincellstreatedwithcontrolsiRNAafternormalizationtothelevelofGAPDHmRNA . Values aremeans (cid:8) S . D . oftriplicatemeasurements . Similarresultswereobtainedinthreeindependentexperiments . p valuesweredeterminedbyStudent’s t test . * * , p (cid:9) 0 . 01 ; * * * , p (cid:9) 0 . 001 ; n . s . , not significant . B – E , SAS cells were transfected with control siRNA ( siControl ) , with ESRP2 siRNA ( siESRP2 ) , or with ESRP2 plus (cid:2) EF1 ( si (cid:2) EF1 ) and SIP1 ( siSIP1 ) siRNAs . HSC4 cells were transfected with control siRNA , with ESRP2 siRNA , or with ESRP2 plus SIP1 siRNAs . After incubation for 48 h , cells were used for the following assays . B , mRNA expression of ESRP2 ( upper ) , (cid:2) EF1 / SIP1 ( middle ) , and E - cadherin ( lower ) examined by quantitative real - time PCR . mRNA levels are presented relative to those in cells treated with control siRNA after normalization to the level of GAPDH mRNA . Values are means (cid:8) S . D . of triplicate measurements . Similar results were obtained in three independent experiments . p values were determined by Student’s t test . * , p (cid:9) 0 . 05 ; * * , p (cid:9) 0 . 01 ; * * * , p (cid:9) 0 . 001 . C , protein levels of E - cadherin and ESRP2 as determined by immunoblotting . (cid:5) - Tubulin was used as a loading control . D , subcellular localization of E - cadherin ( white ) in SAS cells in which ESRP2 or both ESRP2 and (cid:2) EF1 / SIP1 were knocked down as visualized by immunofluorescence . Nuclei were stained with DAPI ( blue ) . Scale bars (cid:6) 2 . 5 (cid:4) m . E , wound healing assay of SAS and HSC4 cells in which ESRP2 or both ESRP2 and (cid:2) EF1 / SIP1 were knocked down . The results were quantitated as shown ( lower ) . Migration of the wound edge was measured at 10 randomly chosen points in the photograph . Cell migration distances after 12 h at 10 randomly chosen points were compared with the distances at 0 h . Values are means (cid:8) S . D . of triplicate measurements . Similar results were obtained in three independent experiments . p values were determined by Student’s t test . * * , p (cid:9) 0 . 01 . ESRP1 and ESRP2 Differently Suppress Cell Motility 27396 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 40• OCTOBER 3 , 2014 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m carcinoma in situ , and invasive carcinoma . HNSCC samples are favorable for evaluating expression of ESRP1 and ESRP2 during carcinogenesis because multiple steps associated with HNSCC carcinogenesis can be observed in the same specimens . We found that ESRP1 and ESRP2 were only weakly expressed in normal oral epithelium , where both could be detected in the basal layers . The expression levels of both proteins in dysplasia and carcinoma in situ were higher than in normal epithelium . Moreover , they were also highly expressed in advanced OSCC and cancer nests in metastatic lymph nodes . These findings indicate that expression of ESRPs is elevated during HNSCC carcinogenesis . In contrast , ESRP1 and ESRP2 were repressed in cancer cells that penetrated through the basement mem - brane into the stroma and those invading from cancer nests into stromal tissues . In light of these findings , we asked how altered expression of ESRPs affects cellular phenotypes . ESRPs regulate alternative splicing events in a number of genes associated with reorgani - zation of the actin cytoskeleton and maintenance of cell - cell adhesion , tight junctions , and cell polarity during EMT ( 29 ) . However , these splicing products are often uncharacterized . In our study , we demonstrated that ESRP1 and ESRP2 suppress cell motility through distinct mechanisms , although involve - ment of ESRPs in cell motility regulation has been reported previously ( 10 , 27 ) . First , we found that knockdown of ESRP1 resulted in inclu - sion of variant exon 3b in alternative splicing of Rac1 mRNA , thereby increasing expression of the Rac1b isoform . In ESRP1 knockdown cells , Rac1b modulated actin dynamics to induce formation of long filopodia and augment cell motility . Variant products of other ESRP1 - targeted genes ( MENA and Exo70 , etc . ) are also associated with actin dynamics ( 16 , 17 ) . Taken together , these data indicate that ESRP1 principally inhibits cell motility via regulation of actin dynamics . Second , we found that knockdown of ESRP2 effectively repressed E - cadherin expression in HNSCC cell lines at both the mRNA and protein levels . Several reports have shown that ectopic expression or knockdown of ESRP1 does not affect E - cadherin expression ( 8 , 10 , 22 , 30 ) . We previously found that overexpression of ESRP1 and ESRP2 in basal - like breast cancer cells results in up - regulation of E - cadherin expression ( 9 ) . These observations are consistent with our present finding that ESRP2 contributes to maintenance of E - cadherin expression . Furthermore , we found that among EMT - associated transcrip - tion factors that repress E - cadherin expression , the levels of (cid:2) EF1 and SIP1 were significantly elevated in ESRP2 - silenced cells . (cid:2) EF1 and SIP1 bind directly to the E - cadherin promoter ( 7 , 31 , 32 ) . Down - regulation of E - cadherin by (cid:2) EF1 and SIP1 is associated with acquisition of a migratory / invasive phenotype by epithelial tumor cells ( 32 – 35 ) . In addition , (cid:2) EF1 and SIP1 are also direct suppressors of ESRP expression in cancer cells , indi - cating that there is a double - negative feedback loop between ESRP2 and (cid:2) EF1 / SIP1 . It remains to be elucidated how ESRP2 represses (cid:2) EF1 and SIP1 . Among the many systems involved in the regulation of EMT , microRNAs ( miRNAs ) have been specifically implicated in the regulation of EMT - associated transcription factors . In particu - lar , a double - negative feedback loop is formed between miR - 34 , the miR - 200 family , and Snail or (cid:2) EF1 / SIP1 ( 6 , 36 ) . It is thus possible that ESRP2 suppresses (cid:2) EF1 and SIP1 by regulating miRNA expression . Melamed et al . ( 37 ) recently reported that the alternative splicing machinery competes with the miRNA processing machinery when a pre - miRNA sequence overlaps with active splice sites . However , EMT - associated pre - miRNA sequences ( miR - 34 , miR - 200 family , and miR - 9 ) do not overlap exon - intron junctions , ruling out such a mechanism . Wu et al . ( 38 ) reported that ASF / SF2 ( SRSF1 ) has a splicing - independent function in miRNA processing . Previous reports ( 8 , 10 ) and our own data ( this study ) have shown that ESRP2 is a less potent regulator of splicing than ESRP1 . Therefore , ESRP2 may influ - ence (cid:2) EF1 and SIP1 expression by regulating miRNA process - ing via splicing - independent mechanisms . Elucidation of such a mechanism might lead to identification of a novel mechanism of regulation of cancer progression . One of the important findings of our study is the plasticity of ESRP expression during cancer cell invasion and metastasis ( Fig . 9 ) . ESRP1 and ESRP2 were re - expressed in cancer cells that reached the deep stroma and the lymph nodes , whereas cells invading from cancer nests again lost expression of ESRPs . These in vivo data suggest that down - regulation of ESRP1 and ESRP2 is restricted to cells that acquire a motile phenotype during cancer invasion . As noted above , whether ESRP expres - sion has a positive or negative impact on survival rates of cancer patients remains controversial . Ueda et al . ( 27 ) reported that in a cohort of pancreatic cancer patients , the overall survival rate of an ESRP1 - high group was significantly higher than that of an ESRP1 - low group . In contrast , Yae et al . ( 14 ) showed that a high level of ESRP1 expression is significantly associated with a lower rate of overall survival in breast cancer patients , suggest - ing that increased ESRP1 expression is related to the malignant phenotypes of human breast cancer . In this study , we observed plasticity of ESRP1 and ESRP2 expression during malignant progression . Therefore , it would be difficult to draw a definitive conclusion regarding the relationship between ESRP expression andcancerprognosis . Consistentwiththis , analysisofinformation on HNSCC patients in public databases ( PrognoScan ) revealed no difference in prognosis between ESRP1 / ESRP2 - positive and ESRP1 / ESRP2 - negative groups . It is important to elucidate the mechanisms underlying the plasticity of ESRP expression . Previous reports showed that EMT - associated transcription factors ( (cid:2) EF1 , SIP1 , Snail , Slug , and Twist ) down - regulate ESRP1 or ESRP2 ( 9 , 26 , 39 , 40 ) . In contrast , the factors involved in up - regulation of ESRP1 and ESRP2 are relatively unknown . Recently , the Grainyhead tran - scription factor Grhl2 was shown to induce ESRP1 expression in breast cancer cells ( 41 ) . Ras signaling is also implicated in up - reg - ulation of ESRP1 and ESRP2 during carcinogenesis because Ha - Ras - transformed MCF10A cells express higher levels of ESRP1 and ESRP2 compared with parental MCF10A cells . 3 It remains to be determined why normal epithelial cells with low expression of ESRP1 have low motility . In this regard , it is noteworthy that Rac1b was first identified as a tumor - associ - ated splicing isoform in breast and colon cancers ( 42 , 43 ) . A 3 H . Ishii and K . Miyazawa , unpublished data . ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27397 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m previous report demonstrated that ASF / SF2 , a member of the serine / arginine - rich protein family , acts as an inducer of the endogenous Rac1b isoform by promoting inclusion of exon 3b ( 23 ) . In contrast , we found that ESRP1 represses Rac1b expres - sion by causing the variant exon to be skipped . Similar to the pattern for ESRPs , ASF / SF2 expression is also weak in normal epithelium but increases significantly during lung carcinogen - esis ( 44 ) . Therefore , even though ESRP1 is expressed weakly in normal epithelium , Rac1b is not expressed due to low expres - sion of ASF / SF2 . In cancer tissues , ESRP1 appears to be func - tionally competitive with ASF / SF2 in alternative splicing of Rac1 mRNA . 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PLoS ONE 7 , e46539 ESRP1 and ESRP2 Differently Suppress Cell Motility OCTOBER 3 , 2014• VOLUME 289•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27399 a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m Keiji Miyazawa Mitsuyoshi Motizuki , Keisuke Masuyama and Tetsuo Kondo , Ryohei Katoh , Shota Tanaka , Hiroki Ishii , Masao Saitoh , Kei Sakamoto , Cell Motility via Different Mechanisms ( ESRP1 ) and 2 ( ESRP2 ) Suppress Cancer Epithelial Splicing Regulatory Proteins 1 Cell Biology : doi : 10 . 1074 / jbc . M114 . 589432 originally published online August 20 , 2014 2014 , 289 : 27386 - 27399 . J . Biol . Chem . 10 . 1074 / jbc . M114 . 589432 Access the most updated version of this article at doi : . JBC Affinity Sites Find articles , minireviews , Reflections and Classics on similar topics on the Alerts : When a correction for this article is posted • When this article is cited • to choose from all of JBC ' s e - mail alerts Click here http : / / www . jbc . org / content / 289 / 40 / 27386 . full . html # ref - list - 1 This article cites 44 references , 12 of which can be accessed free at a t NO R T H E R N I LL I NO I S UN I V on J a nu a r y 27 , 2015 h tt p : / / www . j b c . o r g / D o w n l o a d e d fr o m \ No newline at end of file diff --git a/silver_data/fa25610fb8586c2b50a3654edc5bb42fa7fc4729.pdf.txt b/silver_data/fa25610fb8586c2b50a3654edc5bb42fa7fc4729.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..76bd16d7c7cf29f668cf176d71da2f8064f7aba9 --- /dev/null +++ b/silver_data/fa25610fb8586c2b50a3654edc5bb42fa7fc4729.pdf.txt @@ -0,0 +1 @@ +EECS 940 Theoretic Foundation of Data Science Instructor : Name : Dr . Luke Huan Meeting Hours : Wednesday 8 : 30 - 11 : 00 at ITTC 250 Office Hour : Wednesday 1 : 00 - 2 : 00 Eaton 2134 Phone : 864 - 5072 Email : jhuan @ ku . edu Class Web Page : http : / / people . eecs . ku . edu / ~ jhuan / EECS940 _ S14 Class Objectives : We will review statistical and mathematical principles that are utilized in machine learning and data mining research . Covered topics include asymptotic analysis of parameter estimation , sufficient statistics , model selection , information geometry , function approximation and Hilbert spaces . Prerequisite : EECS 738 , EECS 837 , EECS 844 or equivalent . Text Book : Recommended ( not required ) : 1 . All of Statistics , by Larry Wasserman , Springer , ISBN - 10 : 0387402721 ISBN - 13 : 978 - 0387402727 2 . The Elements of Statistical Learning : Data Mining , Inference , and Prediction , by Trevor Hastie , Robert Tibshirani , Jerome Friedman , Springer , ISBN - 13 : 978 - 0387952840 . 3 . Pattern Recognition and Machine Learning , By Christopher Bishop , Springer , 2nd printing edition , 2007 , ISBN - 10 : 0387310738 , ISBN - 13 : 978 - 0387310732 4 . Essential of Statistical Inference , Young & Smith , Cambridge University Press , 2010 , ISBN - 13 : 978 - 0521548663 5 . Model selection and model averaging , Claeskens & Hjort , Cambridge University Press , 2008 , ISBN - 13 : 978 - 0521852258 6 . Numerical Optimization , by Jorge Nocedal and Stephen Wright , Springer ; 2nd edition , 2006 , ISBN - 10 : 0387303030 , ISBN - 13 : 978 - 0387303031 7 . Scaling up machine learning , edited by Bekkerman , Bilenko , Langford , Cambridge University Press , 2012 , ISBN - 13 : 978 - 0521192248 Grading : Homework assignments 25pts One in - class presentation 25pts Final Project : 25pts In - class discussions 25pts Total : 100pts We will use the following scale to assign final grades ( tentative and curving will be used ) : A : over 90 % B : 80 % - 89 % C : 70 % - 79 % D : 60 % - 69 % F : below 60 % Extra Credit : Extra credits will be given to creativity and / or additional efforts shown in the team project and exams . Details will be given in the related assignments . Attendance : I expect you to come to lectures on a regular basis and will generally be unwilling to answer questions about material covered in a class you missed ( unless you were sick or had another legitimate excuse ) . You are responsible for all announcements made in class . Participation is encouraged ; please feel free to stop me if you do not understand something that has been said . Academic Misconduct : The department , school and university have very strict guidelines regarding academic misconduct . Obviously , copying is not allowed on exams . Students are expected to submit their own work on individual programming projects . Lending or borrowing all or part of a program from another student is not allowed . Students ARE allowed to borrow and modify any code on this class web site in their labs or programming projects . Instances of cheating will result in a loss on one letter grade in the course and referral to the department chairman and the dean of engineering . If a second case of academic misconduct is reported in any class , a dismissal hearing may be initiated by the dean of engineering . Topics Covered ( subject to change during the course ) : 1 . Measure , σ - algebra , random variables 2 . Law of large numbers 3 . Statistical decision theory , subjective Bayesian 4 . Maximum likelihood , Bayesian , Minimax 5 . Linear regression 6 . Linear classification 7 . Bayesian regression 8 . SVM 9 . Decision tree , Boosting , Boosted Decision Tree 10 . Gaussian processes 11 . EM 12 . Statistical Learning Theory 13 . Approximate inference in Bayesian 14 . Probabilistic graphical models 15 . Bayesian nonparametrics 16 . Big data 17 . Other topics related to Machine learning and data mining \ No newline at end of file diff --git a/silver_data/fd495af30283dda92fb68cf99f2e4ccb7d55b249.pdf.txt b/silver_data/fd495af30283dda92fb68cf99f2e4ccb7d55b249.pdf.txt new file mode 100644 index 0000000000000000000000000000000000000000..03d08a525307436ff8fab501bbf046050ac295a3 --- /dev/null +++ b/silver_data/fd495af30283dda92fb68cf99f2e4ccb7d55b249.pdf.txt @@ -0,0 +1 @@ +ED 306 093 AUTHOR TITLE INSTITUTION SPONS AGENCY REPORT NO PUB DATE CONTRACT NOTE PUB TYPE EDRS PRICE DESCRIPTORS IDENTIFIERS ABSTRACT DOCUMENT RESUME SE 050 487 Hall Rogers ; And Others Exploring the Episodic Structure of Algebra Story Problem Solving . Revised . California Univ . , Irvine . Dept . of Information and Computer Science . Office of Naval Research , Arlington , Va . Personnel and Training Research Programs Office . TR - 86 - 24 12 Dec 88 N00014 - 85 - K - 0373 95p . ; Small print may not reproduce well . Reports - Research / Technical ( 143 ) MF01 / PC04 Plus Postage . * Algebra ; Mathematical Applications ; Mathematical Concepts ; Mathematical Logic ; * Mathematical Models ; Mathematics Achievement ; * Mathematics Instruction ; * Problem Solving ; Secondary Education ; * Secondary School Mathematics ; * Word Problems ( Mathematics ) * Mathematics Education Research This paper analyzes the quantitative and situational structure of algebra story problems , uses these materials to propose an interpretive framework for written problem soving protocols , and then presents an exploratory study of the episodic structure of algebra story problem solving in a sizable group of mathematically competent subjects . Analyses of written protocols compare the strategic . tactical , and conceptual content of solution attempts , looking within these attempts at the interplay between problem comprehension and solution . Comprehension and solution of algebra story problems are complimentary activities , giving rise to a succession of problem solving episodes . While direct algebraic problem solving is sometimes effective , results suggest that the algebraic formalism may be of little help in comprehending the quantitative constraints posed in a problem text . Instead , competent problem solvers often reason within the situational context present by a story problem , using various forms of " model - based reasoning " to identify , pursue , and verify quantitative constraints required for solution . The paper concludes by discussing the implications of these findings for acquiring mathematical concepts ( e . g . , related linear functions ) and for supporting their acquisition through instruction . ( Author ) * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Reproductions supplied by EDRS are the best that can be made * from the original document . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * I ii Information and Computer Science Exploring the Episodic Structure of Algebra Story Problem Solving * Rogers Hall Dennis Kibler Etienne Wenger Chris Truxaw Irvine Computational Intelligence Project Department of Information and Computer Science University of California TECHNICAL REPORT - 5 r U . S . DEPARTMENT Or EDUCATION Once of Educational Research and Improvement EDUCATIONAL RESOURCES INFORMATION CENTER IERIC ) XThis document has been reproduced as received horn the person or organization originating rt O Minor changes have been made to improve reproduction quality o ' , wits of view or oprnions stated in ttusdocu - nrent do not necessarily represent official OERI pcsrfion or policy UNIVERSITY OF CALIFORNIA IRVINE 2 Exploring the Episodic Structure of Algebra Story Problem Solving * Rogers Hall Dennis Kibler Etienne Wenger Chris Truxaw Irvine Computational Intelligence Project Department of Information and Computer Science University of California Irvine , CA 92717 Technical Report 86 - 24 Revised : December 12 , 1988 To appear in Cognition el Instruction " Supported by Personnel Training and Research Division of the Office of Naval Re - search , contract N00014 - 85 - K - 0373 . 1 3 Unclassified SECURITY CLASSIFICATION OF THIS PAGE ( When Data Entered ) REPORT DOCUMENTATION PAGE READ INSTRUCTIONS BEFORE COMPLETING FORM 1 . REPORT NUMBER Technical Report No . 7 2 , GOVT ACCESSION NO , 3 , RECIPIENT ' S CATALOG NUMBER 4 . TITLE ( and Subtitle ) Exploring the episodic structure of algebra story problem solving 5 , TYPE OF REPORT & PERIOD COVERED Research Report 12 / 31 / 88 6 PERFORMING ORG . REPORT NUMBER UCI - ICS Tech Rep 86 - 24 ( revised ) 7 . AJTHOR ( s ) Rogers Hall , Dennis Kibler , Etienne Wenger , Chris Truxaw 8 . CONTRACT OR GRANT NUMBER ( s ) N00014 - 85K - 0373 9 . PERFORMING ORGANIZATION NAME AND ADDRESS Department of Information & Computer Science University of California , Irvine , CA 92717 10 . PROGRAM ELEMENT , PROJECT , TASK - - AREA & WORK UNIT NUMBERS 11 . CONTROLLING OFFICE NAME AND ADDRESS Cognitive Science Program Office of Naval Research Arlington , Virginia 22217 12 . REPORT DATE January 1 , 1989 13 . NUMBER OF PAGES 90 14 . MONITORING AGENCY NAME & ADDRESS ( if different from Controlling Office ) 15 . SECURITY CLASS . ( of this report ) Unclassified 15a . DECLASSIFICATION / DOWNGRADING SCHEDULE 16 . DISTRIBUTION STATEMENT ( of this Report ) Approved for public release ; distribution unlimited 17 . DISTRIBUTION STATEMENT ( of the abstract entered in Block 20 . if different from Report ) 18 . SUPPLEMENTARY NOTES To appear in Cognition 6 Instruction , 1989 . 19 . KEY WORDS ( Continue on reverse side if necessary , and identify by block number ) mathematical problem solving algebra story problems modelbased reasoning analogical problem solving teaching representations 20 . ABSTRACT ( Continue on reverse side if necessary and identify by block number ) OVER DD 1 FJOARNM73 1473 EDITION OF 1 NOV 6515 OBSOLETE Unclassified SECURITY CLASSIFICATION OF THIS PAGE ( When Data Entered ) Unclassified SECURITY CLASSIFICATION OF THIS PAGE ( When Data Entered 20 . ABSTRACT This paper analyz - s the quantitative and situational structure of algebra story problems , uses these materials to propose an interpretive framework for written problem - solving protocols , and then presents an exploratory study of the episodic structure of algebra story problem solving in a sizable group of mathematically competent subjects . Analyses of written protocols compare the strategic , tactical , and conceptual content of solution attempts , looking within these attempts at the interplay between problem comprehension and solution . Comprehension and solution of algebra story problems are complimentary activities , giving rise to a succession of problem solving episodes . While direct algebraic problem solving is sometimes effective , results suggest that the algebraic formalism may be of little help in comprehending the quantitative constraints posed in a problem text . Instead , competent problem solvers often reason within the situational context presented by a story problem , using various forms of " model - based reasoning " to identify , pur - sue , and verify quantitative constraints required for solution . The paper concludes by discussing the implications of these findings for acquiring mathematical concepts ( e . g . , related linear functions ) and for supporting their acquisition through instruction . Unclassified SECURITY CLASSIFICATION OF THIS PAGE ( When Data Entered ) 2 . Exploring the Episodic Stntcture of Algebra Story Problem Solving Rogers Hali , Dennis Kibler , Etienne Wenger and Chris ` D . uxaw Department of Information and Computer Science University of California at irutne Send proofs to : Ro 5ers Hall Department of Information and Computer Science University of California , Irvine , CA 92717 6 ABSTRACT This paper analyzcs the quantitative and situational structure of algebra story problems . uses these materials to propose an interpretive framework For written problem solving pro tarots , and then presents an exploratory study of the episodic structure of algebra story problem solving in a sizable group of mathematically competent subjects Analyses of writ ten protocols compare the strategic , tactical , and conceptual content of solution attempts , looking within these attempts at the interplay between problem comprehension and solo tion . Comprehension and solution of algebra story problems are complimentary activities , giving rise to a succession of problem solving epiisodes . While direct algebraic problem solv ing is sometimes effective , results suggest that the algebrak formalism may be of little help in comprehending the quantitative constraints posed in a problem text . Instead , competent problem solvers often reason within the situational context presented by a story problem , using various forms of " model - based reasoning " to identify , pursue , and verify quantitative constraints required for solution . The paper concludes by discussing the implications of these findings for acquiring mathematical concepts ( e . g . , related linear functions ) and fur supporting their acquisition through instruction . 2 . 1 . I . I CZ : . Confronted with an algebra story problem , a student faces a fundamental sort of " ill structured problem " ( Newell , 1969 ; Simon , 1973 ) . The problem text gives information about initial and goal states , but state transition operators taking the text into a quanti . tative solution are hardly well defined . Even assuming the student has an adequate grasp of mathematical principles and operators within the formalisms of arithmetic and algebra ( e . g . , the distributive property of multiplication over addition or using algebraic substi . tution ) , a solution to the presented problem is often obvious only in retrospect . Rather than searching for a solution path in a well - defined apace of representational states , the problem solver is more likely to be searching among a space of alternative representations in an attempt to make the problem routine or familiar . Omitted or incorrectly introduced constraints within the problem representation can lead to prolonged and often meaningless calculations , and may encourage otherwise sophisticated problem solvers to give up entirely . Information - processing models of ill - structured problem solving remain elusive . This state of affairs might be puzzling but acceptable if algebra story problems were transient disturbances in the secondary school curriculum . However , these problems recur as a general task throughout the mathematics curriculum and are even found in the quan . titative sections of entrance examinations for professional schools . If prevalence alone is an insufficient basis for study , the unique role of these problems in bringing mathematical formalisms into contact with everyday experience recommends them highly . Viewed from within the classroom , story or " applied " problems provide students with an opportunity to validate acquired mathematical abstractions in more familiar domains ( e . g . , traveling or shopping ) . Viewed in a wider context , these problems may provide a curricular microcosm of a central pedagogical problem : transfer of training from the algebra classroom to StIV dents ' later educational or life experiences . Interpretations derived from either vantage are controversial . For example , we have anecdotal evidence that these problems arc avoided by some teachers as being too difficult for both students and teachers . On the other hand , S studies of mathematics in practice suggest that " real world " curricular materials may have little correspondence with mathematical problems or their solution in " real life " ( Lave , 1986 , 1988 ) . For psychologists and educationalists alike , the problem iv to determine how applied problems are solved by competent problem solvers and how acquisition of that competence might be supported . ' Insert Table I about here . j Algebra story problems of the sort shown in Table 1 have heen studied extensively by cognitive and educational psychologists , both as a representative task for mathematical problem solving ( e . g . , Ilinsley , Hayes , & Simon , 1977 ; Kilpatrick , 1967 ; Mayer , Larein , Kadane , 1984 ; and Paige & Simon , 1968 ) and as experimental materials for studies of trans far ( e . g . , Dellarosa , 1985 ; Reed , 1987 ; Reed , Dempster , & Ettinger , 1985 , and Silver , 1979 , 1981 ) . Many studies treat problem solving as an opaque process with an inspet table output ( i . e . , correct or incorrect ) and duration . Maniculations in problem toutent or presentation ; are introduced , performance data are collected , and inferences are drawn concerning hypo thetical problem - solving mechanisms . In contrast , much as in Kilpatrick ' scatty work ( 1967 ) and subsequent studies of mathematical problem solving by ! Aral ( 1979 ) amid Schoenfeld ( 1985 ) , we have chosen instead to present subjects with representative problems and then to observe and analyze their uninterrupted responses in some detail This approach trades experimental control over the problem solving setting for a richer ( albeit interpretive ) view of problem - solving activities . In addition to finding whether or not a subject has gotten a problem " right , " we are at least partially able to explore the solution strategies that sub jests select and their tactical course in achieving solutions , right or wrong We find this a Woeful approach to characterizing what competent problem 6 , , Ivers actually do when solving these problems ( i . e . , a succession of strategic and tactical efforts ) . This characterization is a necessary first step towards finding methods for supporting aquisition of competent problem - solving behaviors . 4 When describing models of algebra story problem solving , we will distinguish between the ocormtive and predictive capacity ' of models ( computational or otherwise ) as successive approximations to a robust instructional theory . A model with generative capacity uses an expressive language for describing problems and their solutions : o produce descriptions of problem solving activity that obey certain constraints . For example , given a language that is adequate for expressing arbitrary algebraic expressions , we might like to generate only those expressions , liat reflect mathematical relations stated directly in a story problem tttt . For various instructional purposes . this is an improvement over generating all syntactically vermlsOble algebraic expressions , but it sails well short 41 addressing typical instructional problems e . g . , why or how has a student generated some particular algebraic expres shins ? This sort of predictive capacity will require considerable extensions to the expressive language ( e . g . , a notation for intermediate representational slates ) and to constraints tc . st restrict the process for generating algebraic expressions ( e . g . , a vocabulary of justifications for a subject ' s choices among alternative problem solving activities ) . Given a sufficiently expressive la7cguage and a , appropriate set of constraints , a model may generate & scrip , lions that correspond closely with students ' activities . When this correspondence is of high fidelity i . e . , the model answers questions of why or how in a psychologically plausible fashion it can be used to support a variety of important instructional tasks . For example , a predictive model of algebra story problem solving might be used to diagnose students ' errors , to plan tutorial activities , or even to provide basic instructional materials . Work reported in this paper approaches a predictive model by presenting descriptive languages for problem - solving activities , examining constraints that arise from interactions between these languages , and then exploring problem - solving behaviors observed in a siz able group of competent problem solvers . In the first section of the paper we examine some ' We are not arguing foe explanatory ulequery in the wise nasally reserved loo linguistic theories Whom shy . 1965 ) ' fie models discussed in this paper approul descriptive adequacy but do sot yet piopose mow ( constraints on moulting problem solving competence 5 10 basic materials out of which algebra story problems and their solutions c an be constructed Out working hypothesis Is that ID Order to generate a MA1111011 enabling representation of 4 problem , reasoners must a . ssemble quantitative constraints under the guidance of their tin derstanding of the situarimud rmacrt preseid ell by the story problem context serves not only as a vehicle for the quantitative problem , but also as a framework for justifying the existence of quantitative constraints and their interrelationships to cordinglv . we examine the quantitative and situational structure or typical algebra story prohlems , and then use representative problems in the exploratory study In later sections of the paper we analyze the written protocols of 85 upper division computer science undergraduates who were instru , tech widow their work when solving four , representative algebra story problems . An interpretive framework is des eloped in which a written solution attempt is divided into a series of coherent problem solving episodes Each of these episodes is coded along a set of categories tenor ling strategic and tactical role , conceptual content , manipulative or conceptual errors , and relationship tic : oil : rounding episodes . Exploratory analyses of the scored protocols provide evidenc e for the frequency with which various problem solving behaviors occur within subjects ' solution attempts , the content and outcome of the " final episodes " during which subjects ( MON & their errOrth , and the role that " model based reasoning " plays in solution attempts One of our ceutral findings is that competent problem solvers frequently engage in problem solving activities " outside " of the traditional algebraic formalism These activities , based on an analysis of protocol results , often take the form of constructive and elaborative problem solving inferences within the situational context presented by an algebra story problem These findings are interpreted as evidence for a model of quantitative problem solving in which mathematical formalisms ( e . g algebraic expressions ) provide a sometimes useful tool for comprehending quantitative constraints In the discussion section , ae relate this model to existing accounts of mathematical problem solving , and then consider the implications of 6 these findings for acquiring mathematical concepts ( c . . g . , related linear functi , ns ) and for supporting their acquisition through instruction . PROBLEM STRUCTURE Before presenting our exploratory study , we ertamine the domain of algebra story prob Ions at two levels of abstraction : the quantitative structure of related mathematical entities and the situational structure of related physicat entities within a problem . The central ac tivity in our model of problem solving is to find convergent constraints through constructer ( elaboration of a problem representation . Structural abstractions examined in this section give two basic materials for such a constructive process . Ultimately , these and other levels of analysis may provide a relatively complete domain " ontology " ( Greeno , 1983 ) for algebra story problems aed other situations that give rise to mathematical problem solving . For the purposes of this paper , we want to identify materials that can provide a descriptive vocabulary for behavioral observations presented in later sections and can assist our ulna . itions in framing a model of problem solving , learning , and teaching within this domain . These materials can play several roles : as a description of the task of solving algebra story problems , as a hypothetical account of the representations held by subjects during the so . tuition process , and as an illustrative medium for teaching . This section focuses or . task and represent nticmal issues ; the utility of quantitative and situational structures in education is examined in the discussion section . Quantitative structure Hy the quantitative structure of algebra story problems , we mean the mathematical enti ties and relationships presented or implied in the problem text . A particular problem I s a " structure " at this level of analysis to the extent that the relationships between mathemat - ical entities take a distinguishable fon u when compared with other algebra story problems . Of course , there might be many ways of characterizing the quantitative structure of an 7 12 arbitrary problem or group of ostensibly related problems e g . , as algebraic equations or as matrices of coefficients . llohrow ( 1968 ) uses algebraic equations as a canonical [ Menial representation of meaning for story problem texts , while Reed et al . ( 1985 ) use equations to define the a priori similarity of problems and their solution procedures . The language of algebraic equations may be sslicient for analysing the task of algebra ! ' inampulation , hut it is less useful when the analysis us to include what students actually understand and use while learning to solve algebra story problems . A network language of quantitative entities . We start with a conceptual frame work originally proposed by Quintero ( 1981 , Quintero do Schwartz , 1981 ) and later extended by Sbalin te Bee ( 1985 ) and ammo ( 1985 , 1987 , Greeno , Brown , RI . , Shales , Ike , Lewis , Vitolo , 1986 ) . The framework serves all three roles mentioned above . as au analysis of task structure , as x hypothetical account of subjects ' representations of algebra problems , and as an instructional medium . Our interest in this work iv twofold Furst , we will use the framework as a means for describing constraints essential for problem solution . although several additions to the framework would be necessary for it to serve as a representatinmil hypothesis . Second , we will employ some aspects of the framework to describe how an arbitrary pair of problems might lie considered similar for problem sok mg purposes Shalin lc Bee ( 1985 ) describe the mathematical structiire of an algebra story problem as a network consisting of quantitative elements , relations over those elements , dud ioni positions of these relations Quantitative elements are divided olio four them types . an extensive eletnent denotes a primary quantity ( e g . , S0111e number of nob a hours ) , an intensive element denotes a map between two eeensiveS ( e . g . , a IWO II / II rate relates tune and distance ) ; a difference element poses an additive contrast of two extensive , ( e g . . one time interval is 2 hours longer than another ) ; and a factor element gives a nodUphra live comparison of two extensive % ( e . g . . one distance Is twice . 1111 / i her ) ' omposing these elements according to their type yields quantitative relations A quantitative relation us 8 13 defined as an arithmetic operation ( i . e . . addition . suhtraction , multiplication , or division ) relating three quantities . For example , the fact that a train traveling 100 km / h for 5 . 5 hours covers a distance of 550 km can he expressed as a relational triad over two extensives ( 550 kilometers and 5 . 5 hours ) and a single intensive ( 100 kne / h ) as shown in Figure 1 Each element is presented grae ' irally as a box containing several expressions . The shape at the top of the box designates element type - e . g . , a reztangular top designates an extensive , a triangular top an intensive . ! Insert Figure 1 aboitt As an additional level of structure , relational triads can be composed by sharing various quantities to yield " problem structures . " These are quantitative networks describing typed quantities and constraints among them . As shown with solid lines2 in Figure 2 ( a ) , a single quantitative network can be used to graphically represent the problem of trains traveling in opposite directions ( problem MOD from Table 1 ) . Sharing a common time , two rates combine through multiplicative triads to yield parts of the total distance . These parts are conihined in an additive triad to give a single extensive quantity representing the total distance . Figure 2 ( b ) shows a quantitative network corresponding to the round trip ( MR7 ) problem . In both networks , the term " output " serves as a generalization over distance and work . taken together , quantities , relations and structures provide a language for descrihing the quantitative form of particular algebra story problems . While a variety of equiva - lent graphical languages might be used ( e . g . , parse trees for arithmetic expressions ) , this language gives explicit representational status to mathematical entities , associates a quanti - tative type with each , and incorporates a metaphorical sense of storage for holding semantic information ( e . g . , textual phrases ) and intermeuit . te calculations . Constraints on the arith metic composition of typed quantitative entities restrict the space of possible quantitative 21 ' ottions or the network in dashed Imes will be discussed shortly rel . ttions ( Green ( ) et al . , 1986 ) For example , the multiplicative ( mimosa lllll of intensive And extensive quantities ( rate and time ) in Figure I is allowed , while an additive composition of the same quantities would be disallowed . Greene ( 1987 ) points out that constraints are also availahle Irani compositional restrictions on the units of measurement for quantities3 , although the network language does not presently incorporate these constraints . Finally , the interconnectivity of a quantitative network supports a form of written algebraic cairn lus . Expressions can be propagated through the network with the goal of finding convergent constraints on the given unknown . ! Insert Figure 2 about here . ) Quantitative networks provide a visually accessible notation for comparing the structure of different algebra story problems . However , the notation and compositional constraints do not specify which of the permissible quantitative structures a subject might generate when solving an algebra story problem . For example , the quantitative network shown with solid lines in Figure 2 ( a ) describes the opposite direction problem after several crucial inferences have occurred : component distances have been inferred within the total of 880 kilometers , and a single extensive quantity for travel time has been correct ' ) inserted in the network . For the same prohlem , consider elahorating the quantitative network to include network components shown with dashed lines in Figure 2 ( a ) We might imagn a subject inferring that the given rates can be adoed . The resulting combined rate ( 160 km / h ) , when multiplied by the unknown time , gives the total distance without adding constituent distances . During empirical studies with this and similar problems , we find considerable variety in the solution approaches taken by different subjet ts as well as by individual subjects within a single prohlein solving effort . The r . tworks shown in Figure 2 are idealized graphical representations of prohlem ' An instructional tool developed by Sehwait : ( 1932 ) enforces unit constraints to lido users avoid met evant calculations , particularly when using intensive quaniaties Thompson ( 1988 ) conilnne , imaniitative networks and unit constraints in another tool named " Wok ' Problem Aseiatant 10 structure as they might be constructed by problem solvers who understand the quantitative network language and are able to use the language to comprehend and solve algebra story problems . These networks give a particular quantitative representation , but their content is largely the result of inferential processes that draw on other knowledge sources . These processes may include : recognizing quantitative entities directly contained in or implied by the problem text , composing these entities into local relational structures , composing relational substructures into larger problem structures , recognizing familiar substructural arrangements , and detecting when constraints are sufficient for solution . The results of each action lie within the quantitative formalism for which Shalin & Bee ' s ( 1985 ) framework provides a functional description . However , the enablement conditions for these actions or the knowledge sources that support them lie partly outside the formalism . These issues are explored further when we consider the situational structure of problems . Quantitative networks as problem classes . Quantitative networks provide an analytic tool for examining aspects of quantitativesimilarity . At the level of entire problems , this approach gives a stronger basis for mathematical similarity than simply noting common equations . At a more fine - grained level , there may be significant areas of substructural isomorphism in globally dissimilar problems . The problems from ' Fable I can be grouped into structurally similar pairs as follows : MOD / WT and AIRT / WC . Each problem in a pair is a " quantitative isomorph " of the other , as shown graphically in Figure 2 . In the MOD / WT pair , extensives for kilometers travzlerl correspond with those for parts of a job completed ( output I and output2 ) . In the AflIT / IVC pair , a round trip travel extensive corresponds with an extensive for boxes filled and then checked ( output ) . Comparing prohlems within each pair , extensive and intensive quantities play identical roles in the surrounding network structure . However , when cone paring problems across pairs , structural roles of similar quantitative entities change or are even reversed . For example , the additive extensive relation for combined distance ( or work II output ) in Figure 2 ( a ) is locally similar to the additive extensive Melton for combined time in Figure 2 ( h ) . but these relations play very different roles in their overall imantita live structures . In general , A specific quantitative network de fines an equivalence class of algebraic problems , each of which may hue a different situational imtaiittetion Of being directly similar in form does not mean that problems must be solved in the same way . Figure 2 presents the quantitative structure of problem materials required for a quantitative solution . We could as well depict the quantitative structure of intermediate representational states in subjects ' solutions , an exercise that sumetimes leads to a surprising sequence of graphical images as various conceptual error : . are introduced or repaired . Turning to a finer grained level of comparison , we can identify classes of problems that are similar to each other by sharing particular quantitative substructures . A substructure is a subgraph within a larger quantitative network consisting of stated quantities , inferred quantities , and relationships among these quantities . For example , " current " problems are similar at a quantitative level because they contain an additive relationship between . he rate of the vehicle ( steamer , canoe , etc . ) and the rate of the medium in which it travels ( current , tide , etc . ) . While other aspects of the quantitative structure for a pair of cur rent problems can be dissimilar , such a shared substructure may contrilmte to subjects ' estimates of problem similarity . As in the results of llinsley et ra . ( 1979 ) , similarity judg ments at the level of " river " problems may appear an educational failure : problem solvers acquire content - specific categorizations when the true pedagogiral goz1 is to 1 ; , . . . . : ; ate their learning of mathematical forms . / mother interpretation is that quantitative suktrurtures are learned through instruction and problem solving experience and this form pert of the underlying competence in this domain . Since particular substructures are correlated with problem types , the resulting categorizations appear overly content specific . However , there may be a functional or pragmatic hasis for learning these problem classes : despite dissim ilarity of ovzrall mathematical structure , shared quantitative substructures require similar 12 7 partial solution strategies . Situational structure The quantitative network formalism does not attempt to account for the problem struc - tures that subjects actually generate during problem solving , although some constraints are placed on combining quantitative types into relational triads . In this section , we examine another level of abstraction - - the situational structure of a story problem as a source of additional constraint when subjects construct a solution - enabling representation of an algebra story problem . Our view of the situational structure of an algehra story problem is not synonymous with what other researchers have called " surface content . " Although surface materials like trains , buses , or letters are important problem constituents , and may be particularly so for novice problem solvers , we will not focus on these materials . Instead , we present a language for describing the situational structure of " compound " algebra story problems involving related linear functions , and use the language in a detailed examination of probles s involving motion or work4 ( see Table 1 ) . As with the quantitative network formalism , our language fur describing the situational structure of problems can play several roles : as an analysis of problem structure , as a hypothetical cognitive represen tation , or as an educational medium . Here we develop a relational language for describing problems , argue for its utility in generating key problem - solving inferences , and then use the language to piesent a viewpoint on the space of possible algebra story problems that is complementary to problem classes based on quantitative structure . In later sections of the paper , we also use the language to help interpret various activities observed in an exploratory st . . . / ; of algebra story problem solving and then to consider the educational implications of these findings . A relational language of situational contexts . We present the basic terms of our ' Motion and work are frequently used u the setting for story problems in algebra texts , comprising 70 % of an extensive sampling by Mayer ( MM . 13 8 relational language fin followed by an example of their use shown III Figure 3 Onnpound motion and work problems are assembled around related events e . g . , traveling in opposite directions , working together , riding a bus and walking , or filling envelopes and checking them . In each event , an agent engages in activity that produces some output ( distance or work ) over a period of tune . Hence , output and time are the basic dimensions that organize story activities . These activities start and stop with particular times , los atioes , or work products that can be modeled as places , dung the appropriate dimension . Places that bound an activity define particular segments of output or time , and these segments i an he placed in relation to each other within a common dimension ' . Rates of motion or work give a systematic correspondence between dimensions of output and time , and using rates in the solution of a quantitative problem requires a strategy for integrating these Arranging output and time dimensions orthogonally gives a rectilinear framework in which rate is a two - dimensional entity . We model these rate entities as inclines that associate particular output and time segments . Relational descriptions involving typed dimensions , places , segments , and inclines provide a language for expressing the situational context of an individual problem . ( Insert Figure 3 ahout here . ' The situational context of problem MOD ( from Table I ) is shown in Figure 3 . Parts ( a ) and ( b ) of the figure show place and segment representations for output ( distance ) and time , while part ( c ) of the figure shows an orthogonal integration of these dimensions with time on the vertical axis . In part ( a ) of the figure , trains traveling in opposite directions from the same station provide two spatial segments ( ) ustance 1 and Distance 2 ) sharing a place of origin ( S ) but with unknown places for destinations . These segments are eellinear and oriented in opposite directions . Since trains leave from the same place of origin , these distance segments are also adjacent and can be arranged within the horizontal dimension ' Segment relations within a dimension ate minds , to Allen ' s Mgt , 19641 relational descripttons of temporal intervals , 11 9 20 shown in part ( c ) of the figure . In part ( b ) of the figure , trains leave at the same time ( t0 ) and are separated by 880 kilometers at some later time , providing time segments ( Time 1 and Time 2 ) that are congruent ( i . e . , coinciding at all points ) when arranged within the vertical dimension . We assume collinearity and the same directional orientation for all time segments . Representing rates of travel as two - dimensional inclines , part ( c ) of the figure puts particular instances of output and time in correspondence ( e . g . , 60 versus 100 kilometers in the rirst hour of travel ) . Inclines can either represent a concrete situation , as shown here , or an invariant relation between output and time dimensions . Treating rate as an invariant relation approaches the algebraic sense of rate as a linear function . Each interpretation enables different problem - solving activities , discussed below . Problem - solving inferences based on situational contexts . Before using this re - latinnal language to describe a space of situational contexts for algebra story problems , we briefly consider its utility as a representation for problem solving . First , we describe how a representation of situational context like that shown in Figure 3 could be constructed ; sec . and , we consider how this relational description might be useful for problem comprehension and solution . Both are ongoing research questions that touch on the role of our situational language as a representational hypothesis and an instructional medium . On the issue of how these representations might be constructed ( either spontaneously or as an educational exercise ) , we propose a series of constructive inferences that operate nn a case frame representation° of the events described in the text of a cm , ud Algebra stnty problem . These inferences build a situational model of the problem by assembling a relational description of a particular situational context . Assuming the case frames contain roles that specify typed places and segments ( e . g . , the starting location versus the starting time ) , we can model these roles as situational places and segments within output and time dimensions . From these initial situatinnal entities , a series of elaborative inferences F . ' t Badman ( 1919 ) for a review of related representation schemes and Kintsch tit Creeno ( 1985 ) for sr ' rumple of a cafe frame representation for the tent of word arithmetic problems 15 identify places and segments implicit in the problem statement and relations over segments within ear , dimension . What results is a relational description of situational context as in Figure 3 . Constructive inferences that assemble a relational description of situational context are similar to the comprehension strategies tha . Kintsch k Green° ( 1985 ) use to take propositional encodings of arithmetic word problems into a net based representation . On the issue of utility , we suspect that segment relations within situatinnal dunes SiOnfi support the construction of quantitative representations like the rotworks of Shalin fit Bee ( 1985 ) . For example , knowing that spatial segments are collinear and adjacent while times are congruent supports two useful problem - solving inferences in problem MOD : con stituent distances can be added to yield a tntal distance , and the rates of each train can be added to give a combined rate . The first inference is a necessary quantitative constraint for solution , while the second inference effectively compresses the compound problem into a simpler problem which can be solved without extended algebraic manipulation . These are precisely the inferences about problem structu ; e that were not accounted for in our examination of quantitative structure . For enample , the netwnrk components shown with duhed lines in Figure 2 ( a ) would result if a student decided to add motion or working rates . Hence , in addition to constructive inferences that build a situatinnal context , there are also constraint - generating inferences that take descriptions of situational strut tore into quantitative relations . Each inference about a quantitative constraint , supported by red evant situational relations , gives a substructural component in a larger set of constraints that may enable a solution . It is also possible to use dimensions , places , segments , and inclines directly in a solution attempt by treating these representational entities as a model of the problem situation We will develop a general account of model based rrasomng as a prnblem solving tat tic here . Following sections introduce operational categories fnr interpreting thus tactic within the structure of written protocnis and give an empirical a , count of its use and consent : mice in 16 21 algebra story problem solving . ( Insert Figure 4 about here . ) Placed within a single dimension to model time or output , segments provide an explicit spatial representation that enables a variety of problem - solving operations like " copying , " " stacking , " " comparing , " or " decomposing " their one - die . ensional extent . Similarly , us . ing inclines as models of rate enables operations like " joining " or " scaling " their two - dimensional extent . Joining , shown in part ( a ) of Figure 4 , places copies of the concrete incline along the diagonal in an iterative fashion . Scaling , In put ( b ) of the figure , treats the incline as an invariant relation by estimating the extent of a segment In one dimension And then projecting that value through the incline to generate an associated extent in the other dimension . Each operation Is based on a different interpretation of rate as a relation across dimensions , and each coon4inates operations on associated segments within single dimensions . Both join and scale operations enable problem solving by model - based reasoning with . out requiring algebraic representation . Figure 5 shows solution attempts using join and scale operations on the opposite direction motion problem ( MOD ) . Treating indines as concrete entities in part ( a ) of the figure , the join operator enables an iterative simulation over five successive one hour increments in the time dimension . These correspond to in . termediate states in a two - dimensional model of the problem , successively constructed and tested against the given constraint or being 880 kilometers apart after a common interval of time . Treating inclines as invariant relations in p . tzt ( b ) of the figure , the scale operator enables a heuristic estimate of the problem ' s final state by choosing five hours as the time at which the trains will be 880 kilometers apart and projecting this choice of a common time through each incline to find associated distance segments . In both solution attempts , spatial relations within the twodimensional model support and organize relatively simple quanti - tative operations like addition , multiplication , and value comparison . Thus , even without 17 22 utilizing the metric qualities that such a model might afford ( e g . . testing whether adjacent distance segments precisely " fill " the composed 880 kilometer segment ) . 1111 ) 1114 based sea sorting can lead to a solution without explicitly constructing an algebraic representation of the problem . ( Insert Figure 5 about here . ( While entities and operations in model based reasoning can support solution attempts directly , they also provide a vocabulary of problem solving activit es that could be used to construct an algebraic representation , For example , introducing a variable , 1 , as a label on the unknown common time in part ( b ) of Figure 5 , we can use the scale operator to project that variable into expressions for labels on each distance segment in the horizontal dimension . Since these segments are adjacent and must fill the given combined distance of 880 kilometers , addition of label expressions in the horizontal dimension gives an A : bran expression for the combined distance , 100t + 601 = 880 . Thus , model based reasoning operations can also participate in constraint mauling infervices described earlier In general , inferences in model based reasoning correspond to relatively opaque oper ations in the algebraic formalism ( e . g . , distribution of a product ) Their spatial character and granularity may provide an accessible problem solving medium for subjects who are newcomers to the algebraic formalism . In addition , the results al these operations could jus Wy more abstract activities in an algebraic or quantitative network representation , allowing problem solvers to verify quantitative constraints or results about which they are uncertain Evidence for these hypothetical roles of tootlel based reasoning , even in competent problem solvers , is presented in the sections that follow . Situational contexts as problem classes . Ileyond their role as a representational hypothesis or an instructional medium , situational contexts provide a viewpoint on the space of possible compound algebra story problems that is complementary to the problem classes provided by quantitative structure Even if we restrict analysis to compound ma 18 23 lion problems in which movement must be collinear and directed , a variety of situational contexts are possible . Taking two collinear distance segments we can select from a set of spatial relationships ( e . g . , congruent or adjacent ) and combine this selection with direc tional orientation ( e . g . , same or opposite ) to yield a distinct spatial situation . Also selecting a relation between time segments ( e . g . , congruent or adjacent ) , we can combine segment relations for distance and time dimensions to yield a particular situational context for a compound motion problem . For example , problem MOD has adjacent distance segments oriented in opposite directions and has congruent time segments , yielding the situational context used in Figure 5 . A similar approach is possible with compound cork problems . Work outputs can also be modeled as collinear segments , although their direttional orientation is less directly interpretable . In the present analysis , we exclude a sense of direction for work outputs , Working " together " can be modeled as adjacent output segments and " competitive " work as congruent output segments . For example the work together ( W7 ) problem has adjacent output segments that add to yield a single job and congruent time segments that , in concert with additive output , allow addition of working rates . This corresponds directly with the situational context of problem MOD , without direttiona ! orientation of output segments . The competitive work problem ( WC ) can be modeled In a similar fashion . Since Randy and Jo each work on the same set of boxes , we choose congruent segments to model the same output . Adjacent time segments are associated with the completion of each output , leading to a direct situational correspondence with the round trip problem ( MN ) . ( Insert Figure 6 about here . ) Figure 6 shows a matrix of situational contexts formed by crossing segment relations from output and time dimensions . Compound motion and work problem : in each cell have a common situational structure ( e . g . , problems MOD and WTia the upper right cell ) , and oft diagonal cells contain pairs of problems that reverse segment relations for time and output 19 r24 For example , reversing ndjarcnt distances and mignont times in problem MOD produces problem AfRT , provided that opposite directions are retained in both problems . Problem structures in diagonal cells of the figure ( shaded ) are not used in this study but also provide the basis for particular algebra story problems . For example . the lower right cell of Figure 6 contains what Mayer ( 1981 ) calls " speed change " problems . This constructive approach to situational contexts can be extended to larger eelational vocabularies for output and time ( e . g . , including overlap , disjoint , etc . ) , yielding a sizable space of situational contexts that provide the dimensional basis for algebra " stories " about motion and work . These examples show that our language of dimensions , places , segments , and inclines can be used to model compound motion and work problems . We have also examined the coverage of this language over different classes of Algebra story problems , like those in eluded in Mayer ' s exhaustive taxonomy ( 1981 ) . Useful models of situational contest can be constructed f - 4 most of these classes , including current , mixture , simple interest , cost , and coin problems . Some extensions of the language appear necessary to model relational con strainta involving additive and multiplicative comparisons ( e . g . , " 12 stone than " or ' twits as fast u " ) . In general , however , models of situational context are possible for any problem In which related linear functions can sensibly be shown within two dimensions Although arbitrarily complex quantitative relations can be graphed in a Cartesian plane , the pro Odor that their dimensions be " sensible " restricts our modeling language to situations where onedimensional relations like adjacent and two dimensional operatnrs like " joining " or " scaling " have meaning . Thus . dimensional models of situational context may be ap plicable beyond textbook algebra story problems and include everyday situations revolving related linear functions . Comparison of situational and qunntletlive structure . Isomorphism within cells and reversed structure across cells of the matrix in Figure 6 partition the space of ( ( impound algebra story problems In a way that is complementary to the problem clAssea deotthell 20 in the preceding section on quantitative structure . In fact , the problems paired in each cell also have an isomorphic quantitative structure , and problems from off - diagonal cells reverse quantitative relations . For example , an additive triad over distance extensives in problem MOD contrasts with a shared extensive for distance in problem AVM . In our view , this complementarity arises precisely because the quantitative substructures serve as a mathematical abstraction for describing situational contexts . In turn , our relational language of situational contexts provides an abstraction for describing ( or modeling ) events within particular , , problems . Thus , choosing segment relations for output and time gives rise to an organized space of situational contexts for compound motion and work problems , each with a corresponding quantitative structure . While quantitative and situational viewpoints on algebra story problems are comple - mentary , they are not identical . The quantitative network formalism models conceptual entities of time , output , and rate at abstractions that preserve quantitative type ( e . g . , ex - tensive " versus intensive ' ) and value , either as a number o ; an algebraic expression . In contrast , situational segments and inclines model these same entities as individuals that preserve semantic type ( e . g . , time versus output ) , dimensional order ( i . e . , segments versus inclines ) , quantitative value , a physical sense of extent ( i . e . , the length of a segment or the slope of an incline ) , and local " spatial " relations between individual instances of extent ( e . g . , the 60 and 100 kilometer segments after the first hour of travel are adjacent ) . Pre - serving physical extent and relations of locality allows a problem solver to utilize spatial knowledge when identifying or verifying quantitative constraints . For example , when a total distance can be decomposed into component distances which exactly fit within the total , there is a direct physical justification for their addition . " Joining " or " scaling " inclines us ing a two - dimensional model of rate promises a similar physical justification for operations on intensive quantities . Whether students actually use su . h a vocabulary for justification is an interesting issue , not directly addressed in the present study , that we are exploring 21 26 I i further ( Hall , 1987 ) . We suspect that shared situational strut sire , in addition to quanti tative structure , contributes to subjects ' judgments of similarity between an arbitrary pair of algebra story problems . Quantitative and situational structure are not the only materiels in the domain of algebra story problems that are important for problem solving , learning , and teaching . Neither can we tacitly assume that these structures , as described above , are actually held by subjects during problem solving . However , these structural abstractions may help to understand what subjects actually do when confronted with a problem to he solved , and to hypothesize what must be learned for competent problem solving to be achieved . Knowl edge sources that guide the generation of quantitative representations , and the manner in which they are manifested during problem solving , comprise an important part of compe , tent performance . By grounding quantitative structure in conceptual understar . ding , these knowledge sources may allow a problem solver to effectively assemble and validate refire sentational structures and operators in the algebraic formalism . Having described some aspects of the underlying situational and quantitative structure of algebra story problems , we now turn to an exploratory study of problem solving . METHOD The primary goal of this study is to characterize the activities of " competent " problem solvers on representative algebra story problems . When compared with the activities of beginning algebra subjects , the contrast should give a rough image of the terrain over which a learner must travel to become a skilled problem solver . We chose to study subjects who have clearly mastered the algebra curriculum up to existing institutional standards , but who were not recent recipients of algebra based instruction , Thus we are attempting to describe a primary target of traditional instruction in algebra : a problem solver who has mastered the tools of the algebraic formalism , has practiced these skills during instruction , and should be able to apply these skills in novel settings , The study In - dyes minimal 22 27 experimental intervention , and our interpretation and analysis of problem - solving protocols are primarily descriptive . Subjects Subjects in this study were 85 undergraduate computer science majors in their junior and senior years . They were enrolled in an introductory course in artificial intelligence , and par . ticipated in the study as part of their classroom activities . These subjects could be viewed as " experts " in algebra story problem solving since they must have successfully completed courses in algebra during secondary schooling . In addition , prerequisites to the artificial intelligence course include three university - level courses in calculus and completion or cur rent enrollment in courses covering discrete mathematics . Thus the level of mathematical sophistication in this sample of problem solvers should be high . Alternately , one might argue that these subjects were expert algebra story problem solvers at one time but that their skills have in some sense been " retired " with the passage of time . As will be clear shortly , the solutions offered by many members of this sample do not fit an image of smooth execution of a practiced " skill . " Materials Subjects were asked to solve the four algebra story problems shown in Table 1 . Problems MOD , MRT and WT were taken directly front Mayer ' s ( 1981 ) sample of algebra story problems , with minor alterations in their number set and phrasing . These alterations were intended to free students from unwieldy calculations during problem solving and to make wording between selected pairs of problems more similar . Problem WC was constructed to be isomorphic to the MRT problem at the level of quantitative structure . These problems were selected for two reasons . First , r ith the possible exception of WC , they are highly typical of problems found in secondary school texts . Out of an exhaustive set of 1097 algebra story problems drawn from 10 texts , Mayer found that problems like MOD , 23 MRT , and WT accounted for 7 . 8 % of all observed problems . Second , different pairings of there problems allow us to present subjects with opportunities for positive or negative transfer across contiguously presented problems . Specifically , problem pairings MOD / WT and MRT / WC are isomorphic in their quanti tative structure ( see Figure 2 for a graphical representation of these pairs ) and have similar situational contexts . In the MOD / WT pair , output dimensions are adjacent , heing collinear and sharing a starting point , while time dimensions are congruent , overlapping completely by sharing both starting and ending times . In the MRT / WC ' pair , outputs are congruent while time segments are adjacent and of different value ( see Figure 3 ) . Should subjects recognize this similarity , they may exhibit some form of positive transfer . Alternately . problem pairings MOD / MRT and WT / WCare similar at a more superficial level , sharing types of surface materials ( e . g . , distance traveled or parts of a job completed ) while having quite dissimilar quantitative and situational structures . In fact , relations over output and time dimensions are exactly reversed , as described in the preceding section on quantitative structure . In the MOD / MRT pair for example , outputs in MOD are adjacent and times are congruent , while outputs in MRT are congruent and times are adjacent . When presented contiguouely , these problem pairs may induce fairly specific forms of negative transfer ( e . g . , adding rates in the MRT problem after correctly solving the MOD problem ) . Procedure Problem materials were distributed so that subjects with adjacent seating during data collection would be in different groups . Group membership was not randomly determined but should reflect no systematic bias . Subjects were allowed eight minutes to solve each problem , and all subjects worked through the prohlems at the same time Those finishing early on an individual problem waited until the eight minute time limit expired before proceeding to the next problem . Before solving any problems , subjects were asked to " show all of your work " in a written form , to " work from top to hottom , writing new material 24 below previous material , " and not to erase after making a mistake . Instead , they were asked to mark through any mistake with a single line . Finally , subjects were instructed to " . . . draw a box around your answer . " After solving all four problems , subjects were given 20 minutes to explain their solutions in writing on facing pages of the text booklet without changing their original work . Problem ordering . The first group of subjects ( group M , n = 46 ) saw problems in the following order : MOD , WT , WC , MRT . The second group ( W , n = 39 ) saw the following order : WT , MOD , MRT , WC . Thus , each group solved pairs of problems that were isomorphic at quantitative and situational levels ( MOD / WT or WC / MRT ) and also solved pairs of problems that were superficially similar but had reversed relations in quantitative and situational structure ( WT / WC or MOD / MRT ) . Data collection . The " behaviors " reported here , and all interpretations of them , are based entirely on subjects ' written protocols . Relying solely on written protocols has several obvious disadvantages . There is no timing information . While students were allowed eight minutes to solve each problem , we can neither determine how long a subject works on any single prob . lent , nor how long any particular written episode lasts e . g . , performing algebraic manipulation . Written material may be a lean or even distorting window on a subject ' s cognitive processing . A subject may omit materials that seem unimportant or potentially em barrassing ; alternately the subject may give written evidence of processes or strategies that bear little relation to what she actually does . Since this study is exploratory in nature , we present our results as a heuristic tool for generating hypotheses , and leave more manipulative procedures for confirmatory studies Scoring . Written protocols were scored in committee by the authors , using majority rule for categorization of troublesome cases . A scoring system was constructed around the 25 analysis of problem structure described earlier , using an iterative process with subsets from the total pool of protocols . Scoring categories were added , refined , or dropped from the final system when scorers had persistent difficulty reaching consensus . THE EPISODIC STRUCTURE OF WRITTEN PROTOCOLS This section describes a qualitative framework for interpreting written problem solving protocols , showing representative protocols as examples of scored categories within the framework . We point out connections between several of these categories and hypothetical representations and inferences described earlier , although these connections are open to many interpretations . Our framework resembles Schoenfeld ' s ( 1985 ) analysis of mathemat lea ] problem solving by concentrating on coherent episodes of problem solving behavior ( see Ericsson k Simon ( 1984 ) for a review of aggregation techniques ) . We also explicitly score the transition between problem solving episodes . A subject ' s written protocol for a given problem is interpreted in two stages . First the protocol is divided into a sequence of coherent problem - solving episodes . and then each episode is scored individually with re ect to its content , its correctness and its function in the overall sequence . In nearly all c s , the following definition of a problem solving episode allowed scorers to reach consensus : Strategic coherence . The subject is pursuing the same overall goal . Tactical coherence . The subject is using the same method for attaining this goal . Conceptual coherence . The subject is exhibiting the sante conceptualization of the problem . Although episodes divide problem solving into coherent chunks , the context created by earlier episodes is assumed to he inherited by later ones , unless there is evidence that a reconceptualization has occurred . Our definition of an episode will he sharpened in the 26 following paragraphs as we specify in detail the scoring categories used to describe episodic coulee ! . After dividing the written protocol into coherent problem - solving episodes , each episode is examined to determine its general content . Content categories include : strategic purpose , tactical content , conceptual content , the presence of conceptual or manipulative errors , and finally the status of the episode in the overall solution attempt . The latter covers relative correctness and the reason for transition to a new episode . With the exception of conceptual content . each of these categories is further differentiated into alternative subcategories , as shown in Table 2 . In some cases only one subcategory is selected as best describing the more general category ( e . g . , simulation as a type of model - based reasoning under tactical content ) ; in other cases , each subcategory can occur within a single episode ( e . g . , various kinds of conceptual and manipulative errors ) . ( Insert Table 2 about here . ' The remainder of this section takes up each of these interpretive categories in detail , showing representative written protocols as examples of their use in scoring the episodic structure of subjects ' solution attempts . For example , subject m20 in Figure 7 goes through three error - free episodes , each with a specific purpose , tactic , content , and transition . In the protocols shown in figures as illustrations of various categories , episodes are separated by dashed lines , and their sequence is shown with circled numbers . Several protocol excerpts are presented directly in the text without accompanying figures . ( Insert Figure 7 about here . ' Strategic purpose The strategic purpose of an episode is its relation to the ultimate goal of finding a solution . Judgments of a problem solver ' s " purpose " are clearly a matter of our own interpretation , although we present scoring criteria that maim these judgments operatioral across individ - ual ratings . In this regard , our scoring distinguishes between three abstract problem - solving 27 F 32 modes . Comprehension . The subject is not directly seeking a final solution , but is construct ing a representation of the problem by incorporating various constraints . In episode I of Figure 7 , the subject finds a way to express working rates by considering their outputs after one hour . Solution atP . empt . The subject is attempting a series of operations that work directly toward a solution ( Figure 7 , episode 2 ) . Verification . The subject has already produced a solution to the problem and is now seeking confirmatory evidence for it . for instance by rederiving the solution with another method or by inserting the answer in some intermediate equations ( Figure . 7 . episode 3 ) . Tactical content The tactical content of an episode is the method used by a subject to achieve some strategic . purpose . Our operational criteria refer primarily to the protocol material for the current episode , but , in a few cases information contained directly in the protocol was insufficient to make an operational category judgment . In these cases , surrounding episodes and post hoe written explanations supplied by the subject were used to assist scoring . ( Insert Figure 8 about here . ] Annotation . These episodes usually occur early in the protocol when subjects are collecting information about the problem . Three cases are covered . Problem elements . The subject is recording elements of the problem text ( e . g . , VA = 60kmIhr , Figure 8 , episode 2 ) . Retrieval of formulas . The subject is remembering and writing down memorized formulas which seem relevant , ( e . g . , v = I , Figure 8 , episode 4 ) . Diagram . The subject draws a pictorial representation of the problem situation ( e . g . , Figure 8 , episode I ) . 28 33 Algebra . An episode is algebraic if it makes use of one or more equations placing constraints on the value of one or more variables . However , simple assignments are not treated as equations . Thus neither 100 + 60 = 160 nor d = 880 are considered equations , while d = 100 x t is considered an equation . As ehown unusually clearly in the protocol of Figure 9 , the tactical approach of the typical algebraist is to express constraints as a system of one or more equations ( or proportions ) and to solve for the appropriate unknown . We have also found cases of subjects trying equations in a generate - and - test fashion until , as one subject explained , an equation " looks good . " ( Insert Figure 9 about here . ) Model - based reasoning , This category is scored when a ribiets " executes " a model of the problem situation along the dimension defined by an unknown quantity such as time , distance or work . Subcategories of model - based reasoning relate to constructive problem - solving inferences des : ribed in the preceding section on situational structure . Simulation ' . The subject selects a . base unit for the chosen dimension and " runs " the model for each successive unit increment as illustrated in episode 3 of Figure 8 . Consistent with our earlier development of situational structure , a simulation episode could be interpreted as an iterative " joining " of concrete individual inclines . Simula . tion can also be partial ( just one or two increments ) in that it is not used to reach a solution , but to examine relations between quantities and to enable some other solu tion method . In both episode I of Figure 7 and episode 5 of Figure 13 , a simulation for one hour establishes the quantitative combination of entities from distinct events . heuristic . The base quantity " jumps " by variable increments whose magnitude is determined at each point by estimations of closeness to the solution . A heuristic ' Our use of ' simulation " in somewhat different from its use in computational studies of common - sense reasoning , for example , de Kleer ' s ( 1977 , 1979 ) envisionment " uses quantitative calculation to resolve east . ' Wive ambiguity , whiteout sense of M mutation uses physical construction to help disambiguate quantitative constraints . 29 model - based reasoning episode could be interpreted as " scaling " inclines that repre sent invariant relations , as described earlier The progression of this generate and test approach can be monotonic , as iii episode 2 of Figure 10 , or follow some form of interpolation search . After each generation of a value , the state of the problem situation being modeled is reconstructed and evaluated . Pima Figure 10 about here . ) Ratio . This subcategory covers a number of tactics by which relations of proportion ality between quantities are used , sometimes providing clever " shortcuts " to a solution . These tactics clearly utilize a representation of quantity ( e . g . , intensive quantities . as de scribed earlier ) , but the manner in which related quantities are Integrated may depend upon constructive inferences within the situational context ( e . g . , composing segments or inclines ) . ( Insert Figure II about here . ) Whole / port . The subject vilws a part as fitting sonic number of times unto a whole quantity , as in episode 6 o ' Figure 13 . Part / whole and part / part . These two types of ratios compare portions of entities . Ilse of the part / whole ratio is illustrated in episodes 2 - 4 of Figure 11 , where the subject considers parts of the total jobs . A version of the part / part ratio appears in episode 2 of Figure 12 , involving the respective rates of bus and foot travel . Proportion . Non - algebraic proportions cover reasoning of the type exhibited by sub , jest m05 on the work - together ( WT ) problem : " . . . they ' ve done a ( of a job ) in 2 hrs , so hr more would do for ( the job ) left to he done . . " ' Although this protocol Illustrates the category fleetly , it is probable that successful hoe of thin ratio veu somewhat fortuitous on the pot of this student , since a general iustifii shun fur its validity IP rather complex , 30 Sealing . The subject solves a related version of the problem or reaches an unexpected answer , and simply scales the answer to fit the quantities given in the problem . This may relate to our earlier description of " scaling " rates as invariant two dimensional inclines . In episodes 3 4 of Figure 12 , for example , the subject solves an easier problem by heuristic model - based reasoning and then scales her answer to " fit " the MT problem . ( Insert Figure 12 about here . ( Unit . In a few cases , a subject reasons purely in terms of units of measurement given in the problem . For instance , on the work competitive problem ( WC ) , subject ni44 examines alternative rate forms with the following manipulations : box min ( ules ) rain = min - wz - box = min Procedure . This subcategory is scored when there is evidence that a subject is execute lug mine stored sequence of actions or operations other than routine algebraic or arithmetic manipulation . For example , on the work together problem ( WT ) subject m21 appears to use a simple averaging tactic for combining quantities , writing " total = 1 ( 5 + 4 ) = = 41hrs . " Conceptual content ' The conceptual content of an episode reflects the subject ' s conceptualization of the problem situation and the resulting set of constraints between problem entities . There is a subtle but crucial distinction between situational understanding and the quantitative constraints that are implied by it , as suggested in previous sections . Without further subcategorization , our scoring of conceptual content simply contains the constraints that the subject clearly recognizes and uses in the episode . For instance , subject nr39 in Figure 9 manifests an understanding of all necessary constraints : equal distances , additive composition of times , and the distance - rate - time relation . 35 31 ( Insert Figure 11 about heaI Errors Within each problem solving episode , we consider two broad classes of errors Conceptual errors . These are stored when a subject either unladen a constianit that is inappropriate for the problem or excludes a constraint that is a critical requirement for the current episode . Errors of commission . These errors are incorrect constraints that the subject intro duces during an episode , either by incorrectly representing the situational ( 1 / 11i1 . % t of the problem or by making erroneous quantitative inferences . For example , in episodes 4 6 of Figure 13 the subject subtracts distances because she thinks that the trams are going in the same direction . Errors of omission . These errors are overlooked constraints To be scored as an error of omission , an overlooked constraint has to be critical to the solution method applied by the subject . This usually means that two entities are explicitly used while the relation between them is ignored . In Figure 14 , episode : 1 , the subject lugs overlooked that working times represented as z and y are equal . ' Insert Figure 14 about here . ( Manipulation errors . Since written protocols usually display algebraic or arithmetic manipulations clearly , our scoring identifies manipulative errors of three types . Algebrmi ctrues . For example , on the MOD problem , subject w39 writes " WM = 1 . ? " followed by " 7 ' = tE . Variable e - rors . We observed two types of errors related to the concept of VAttablv hi " switch errors , " the meaning of a variable changes in the enuroe of problem solving In label errors , " subjects are using variables as labels ( or quantities . ha Umlaute , in : 12 the round trip problem ( MR7 ) , subject m10 writes the equation " ID . 4 8W 7 . 1 Wirt . " and explains that for every 1 hour on the bus , it takes $ hours to get back . " Mit / melte errors . For example , on the opposite direction motion problem ( MOD ) subject m20 writes = lit . " After detecting this arithmetic error in a verification episode , the subject recovers by using the ratio scaling tactic mentioned earlier . Status of episode within solution attempt Categories listed so far deal with internal characteristics of an episode . The two aspects of the storing scheme described here , consistency and transition , concentrate on the relation of an individual episode to the overall problem - solving effort . Consistency . This category assesses the correctness of an episode in the context of the problem - solving sequence and is scored correct or incorrect for three facets . Before . This subcategory reflects the correctness 4 the context inherited by the episode . For example , errors may be generated in former episodes and passed into the current episode , as with the conceptual error of commission ( same direction ) passed between episodes 4 and 5 of Figure 13 . During . This scores the correctness of the current episode with respect to the inherited context . An episode producing an incorrect result can be internally correct if it is consistent with an incorrect context . For example , episodes 5 and 6 of Figure 13 are internally consistent with the conceptual error of commission introduced in episode 4 . After This subcategory assesses the absolute correctness of the outcome of the current episode If a solution is presented , the scoring reflects its correctness , otherwise scoring assesses whether or not the subject is on a possible right track . 33 Transition , The intent here is to determine the reason why a subject passes from one episode to the next . Unlike comistency , which reflects the scorers ' judgment of correctness . this aspect attempts to captute the subject ' s point of view . Sutgoot . The subject accomplishes an intermediate goal . bringing the episode to an end ( Figure 7 , episodes I and 3 ) . Information identified when achieving a sub goal ( e . g . . changing the form of a working rate ) is generally carried into subsequent episodes . Wrong . The subject decides that she is on the wrong track and abandons the current approach , usually by marking through the work ( Figure 13 , episode 3 ) This transition is often the result of an explicit verification episode . Impasse . The subject reaches a point where she cannot continue with the current method . A good example of impasse is shown in episode 3 of Figure it , where the subject correctly applies simulation by hourly increments , overshoots the non integer solution , and then switches to an algebra / 0 tactic after adding rates , Lost , The subject reaches a point where she cannot determine how to proceed . as in episode 2 of Figure 14 . Final solution . The subject reaches a result and presents it as a solution to the problem . Found so / triton wrong . The subject realizes or believes that the solution Neu - Mist is incorrect . This presentation of our framework fur interpreting wn . ten proton . . gives An overly linear picture of its use in scoring subjects ' solution attempts In fact , categorising the episodic structure of a written protocol within this framework was usually dime quickly ( from S to 20 minutes per Protocol ) and with little Aukcequent disagreement AtimiiK the 3l scorers . By design , each category was rated with at least 75 % agreement over four scorers ; most categories approached unanimous agreement . In addition to determining whether or not a subject has managed to find the " cor - rect " solution to an algebra story problem , this framework for interpreting problem - solving episodes allows us to describe the internal structure of the subject ' s solution attempt . Our interpretation of episodic structure supports more fine - grained explorations of the strate - gic and tactical course of problem solving . In the quantitative results section that follows , we form composite analytic categories by identifying episodic patterns among the atomic category judgments described above . Thus we will be able to speak of subjects reaching a " final episode " with some particular tactic and content or to examine a series of contiguous episodes during which model - based reasoning is used . Beyond the results presented here , we expect the set of scored protocols to provide a rich dataset for continuing analysis . QUANTITATIVE ANALYSIS OF PROBLEM - SOLVING EPISODES In the section on problem structure , we argued that competent problem solving pro - ceeds as an elaborative , interdependent exploration of two distinct problem spaces : the situational context of a story problem and the quantitative constraints given explicitly or implied in the prolem statement . Results presented in this section provide evidence for this interdependency at a global level of problem - solving activity and at a more detailed level of episodic content . Our aralysis distinguishes betwee Jubjects ' problem - solving attempts and the episodic structure of those attempts . By problem - solving attempt , we mean all of the activities evident in the written protocol , which may include several distinct episodes . By episodic structure we mean the alternation of problem - solving episodes of various types , and the constraints or errors that are contained within and across those episodes . First we examine the tactical content , strategic purpose , transitional status , and er - rors present in subjects ' solution attempts . These analyses pool episodes within solution attempts to show the prevalence of different interpretive categories , and so they provide 35 only a coarse image of competent problem solving Second , we look within individual so lution attempts and examine two episodic patterns in detail . Aii analysis of the episode during which a final solution is offered provides a liner image of problem solving ouicome , describing relations between solution outcomes and other interpretive categories within the episode . We also identify individual episodes of model based reasomny to permit a closer examination of problem - solving activity outside of the traditional algebraic formalism . fly considering the content of surrounding problem solving episodes , we can begin to examine subjects ' reasons for using model - based reasoning and to assess its effectiveness for making correct problem - solving inferences or recovering from existing errors . The section ends with summary of major qzontit alive findings . Problem - solving attempts Since many of our rated categories represent hypotheses about problem solving processes , we present their frequency of occurrence within s jects ' problem solving attempts . ' fa ble 3 shows the percentage of subjects having one or more episodes in which various rated categories were observed . Percentages are shown separately for each problem ( MOD , MRT . WT , WC ) but are collapsed over groups ( M , W ) since none of these contrasts were statis - tically reliable . Most findings are as expected , while several are surprising . ( Insert Table 3 about here . ) Tactical content of scored episodes . While most subjects use algebra in their solution attempts ( 63 . 5 to 85 . 9 % across problems ) , reasoning within the situational context presented by the problem is surprisingly common . Looking within individual problems , at least one model based episode is used by 22 . 4 % to 47 . 1 % of subjects , depending on the problem . A separate aralysis pooling across problems shows that 72 . 9 % of subjects have one or more episodes of model based reasoning in their written protocols . These episodes are explored more fully later . 36 41 . Use of ratios is the next most prevalent non - algebraic tactic ( 14 . 1 % to 42 . 4 % across problems ) and may depend upon a variety of factors : the complexity of the constraints presented by a problem ' s quantitative structure , the accessibility of situational justi - fications for those constraints , and the manner in which the constraints are presented in the problem text . Few solution attempts contain episodes using a " procedure " or reasoning with " units . " Most subjects using a procedure on the WT problem chose to take an average over working rates , a strategy that violated the situational meaning of " working together " in that problem and generally led to an incorrect solution . Annotations , in the form of diagrams or notations about problem elements , were ei - ther scarce or common , depending upon the situational and surface content of the story problem . Motion problems ( MOD , MRT ) showed few notations ( 7 . 1 % , 15 . 3 % ) but more frequent diagrams ( 69 . 4 % , 36 . 5 % ) , while work problems showed frequent notations ( 21 . 2 % , 29 . 4 % ) but fewer diagrams ( 8 . 2 % , 9 . 4 % ) . Although it is likely that the spatial content of motion problems makes them more accessible to diagram - matic representation , some subjects are able to construct effective diagrams for work problems ( e . g . , see Figure 11 , episode 3 ) . Strategic purpose of scored episodes . Most subjects show explicit attempts at comprehension in their written protocols ( 57 . 6 % to 84 . 7 % across problems ) , typically through diagrams , notations or model - based reasoning . While all subjects make some attempt to solve the problem , only a minority give evidence of attempting to verify the results or their work ( 7 . 1 % to 28 . 2 % across problems ) . 37 42 Transitions out of scored episodes . Most subjects find and explicitly present a solution ( either correct or incorrect ) as part of their problem - solving attempt , although problems MR ' / ' and WT appear more difficult than their quantitative isomorphs in this regard ( WC and MOD ) A more detailed analysis of solution outcomes follows shortly . Likewise , the three transitions without sol in ' oil ( i . e . , impasse , lost , or wrong ) are most common in the more difficult problems ( MRT and WT ) . Errors in scored episodes . Conceptual errors of omission and commission increase for the more difficult prob - lems ( MRT and WT ) , and appear much more frequently than manipulative errors ( arithmetic , algebraic , or variable errors ) on all problems . Several interesting patterns emerge in these findings . First , subjects ' written protocols are not composed solely of material generated while performing algebraic transformations . Instead , many subjects appear to use various forms of direct situational reasoning , which we have termed model - Lased reasoning , conducted within their understanding of the context posed by & story problem text . Second , although most subjects du present a solution in some form , their efforts do not appear as a smooth progression toward a quantitative solution . Rather , their problem - solving efforts are often interrupted by varied conceptual difficulties that must be repaired before asolution is found . Third , manipulation errors within algebraic and arithmetic formalisms do occur , but these are overshadowed by conceptual errors of omission or commission as a primary source of problem - solving difficulty . Consistent with our earlier treatment of problem structure , we interpret these findings to mean that students form an understanding of the problem at the level of its situational context and then use this understanding to introduce quantitative constraints . As a result , many of the activities 38 43 present in an episodic analysis of algebra story problem solving fall outside the traditional algebraic formalism . Final episodes : outcome , tactical content , and errors Examination of the written protocols c . . sarly shows that subjects undertake a variety of problem - solving activities when attempting to solve these problems , particularly when they encounter difficulties in reaching a solution . However , the previous findings speak only to the presencr of various conditions in subject ' s problem - solving efforts . By our scoring , subjects averaged approximately 2 . 5 scored episodes per problem - solving effort , with some protocols presenting evidence for as many as 10 distinct episodes . In the following analyses , we look within individual protocols for more finely - detailed episodic structure . Within an individual ' s efforts on any given problem , we extract a ` final episode " for a first level of detailed analysis . This episode seed not be the subject ' s last effort in a solution attempt , but it is final in one of three senses : it Is the last episode during which a subject presents a solution that is correct , the last episode during which they present & solution that is incorrect , or the last episode of a problem - solvingeffort in which no solution is presented . " Incorrect " mesas the subject presents an incorrect final solution without detecting any errors . The " no solution " category includes subjects who present an incorrect solution but realize they have done so during a subsequent attempt at verification , without being able to recover . Thus , the final episode may be either correct , incorrect , or present 110 solution . ( Insert Table 4 about here . ) Performance outcomes across groups . Table 4 shows the final outcomes for each problem , broken out to show anticipated effects of problem ordering . For example , on oroblem MOD group W should perform better than group M ( shown as M < W in the taLte ) , since subjects in group W are exposed to an isomorphic problem ( WT ) just before seeing problem MOD . If positive transfer occurs , subjects in group M should be at a relative disadi Wage , having seen no prior problem . None of the group contrasts were statistically 39 44 significant , even taking into account whether subjects were correct or incorrect on preceding problems . Thus . the problem ordering manipulation introduced to provide opportuniti : s for positive and negative transfer appears to have had little effect on subjects ' performance at the level of solution correctness . We consider this finding at a more detailed level in the discussion section . Clearly , problems MRT and WT were most difficult , with percentages of subjects reaching a correct solution on these problems ( 51 . 8 % and 61 . 2 % ) falling well below those reaching correct solutions on problems MOD and WC ( 90 6 % and 91 . 8 % ) . [ Insert Table 5 about here . ) Relation between solution outcome and tactical content . Tables shows tactical content and error categories for final problem solving episodes . For tactical content , subject receives a sinzle category score , so cell frequencies sum to give appropriate column totals . A few protocols contain insufficiert information to score tactical content in the final episode . For errors , a subject may achieve a correct solution in the final episode but still demonstrate an error , et they may have several types of errors . As a result , cell entries for errors do not always add up to coincide with column totals . The prevalence of tactical content and error categories in the final episode is generally consistent with findings for overall solution attempts . However . by looking within these attempts we can focus more closely on relations between tactic and outcome Even within the final episode , not all solutions ( correct or incorrect ) are found using algebra . Excluding those with no solution or with contents that were not scorable , between 22 . 0 % and 44 . 0 % of subjects ( across problems ) ised other tactics to find their final solution . Use of ratios is the most prevalent form of non algebraic reasoning in final episodes . with the exception of an incorrect averaging procedure on problem WT . Model based reasoning is the next most prevalent tactic . Algebra , model - based reasoning , and ratio tactics are about equally effective in the 40 45 final episode . Pooling across problems , algebra is slightly more successful ( number cor . rest / total observed ) and slightly less error - prone ( number incorrect / total observed ) than either of the non - algebraic tactics . Thus , even within the final episode where a solution might be found , a normative account of problem solving consisting of successive algebraic transformations would be disconfirmed by these data . Instead , subjects find solutions through a variety of reasoning strategies that , in some taste , involve relatively little formal algebra . In a moment , we examine the episodic structure of model - based reasoning tactics more closely . Relations between solution outcome and errors . Errors observed during final episodes are also interesting although more difficult to interpret since individual subjects can have multiple errors . We distinguish between " conceptual errors , " which arise through omission or ccatmission of specific quantitative constraints , and " manipulative errors , " which arise through improper use of arithmetic , algebraic operations , or variables . These error categories are shown in the lower panel of Table 5 . With the exception of problem MOD , conceptual errors are more prevalent than manipulation errors . This is particularly true of the more difficult problems ( MRT and Wn . Subjects who achieve a correct solution have fewer conceptual errors than those with an incorrect solution or no solution ( 1 : 6 , 0 : 30 , 1 : 37 and 1 : 4 across problems ) . In the few cases where a solution is found despite conceptual errors , offsetting manipulative MOTs fortuitously " correct " these conceptual errors . Although manipulative errors are found among subjects who do not reach a correct solution , they are also observed among subjects giving a correct solution . These errors are repaired within the final episode to allow for a correct solution . 41 46 . A niong subjects who reach an incorrect solution , the t ttttt iber with manipulative errors could not account for more than a third of these failures ( 2 / 6 . 5 / 15 , 7 / 21 . and 1 / 5 across problems ) . Alternately , at least two thirds of the incorrect solutions muss be based oil conceptual errors . One interpretation of these results is that manipulative errors are less frequent and more recoverable than conceptual errors . That is , subjects who make an error during a problem - solving episode are more likely to recover from that error lit stems from arithmetic or algebraic manipulation than if it is a result of misunderstanding or misencoding the structure of the problem . Since errors may persist across episodes , this conclusion cannot be unambiguously supported . Nonetheless , the most serious errors among this group of relatively competent problem solvers are conceptual rather than manipulative . Episodic structure of model - based reasoning One of the most intriguing findings in these data are subjects ' use of what we 4 all ' model based reasoning . " In these episodes , subjects depart from the algebraic formalism and reason directly within the situational context presented by the story problem . In this section , we examine the functional role that model based reasoning plays within the overall solution effort . We are interested in determining under what rtreninstances this form of reasoning occurs , what purpose it serves within a particular solution attript . and what outcomes are likely when subjects reason iii this fashion . As with the analysis of final episodes , we identify specific episodes within subjects ' solution attempts where model based reasoning occurs . We also extract the preceding problem - solving episode in the hopes of identifying enabling conditions for model based reasoning . Since some subjects ' only use of model based reasoning occurs during their first scored episode , they will have no preceding episode . ( Insert Table 6 about here . ) 42 47 Precursors to modelbased reasoning . A first task for describing the role of model based reasoning in subjects ' solution attempts is to determine their reasons for using this method . We will contrast the correctness and transition out of an immediately preceding episode with the purpose ( as we have rated it ) for using model - based reasoning . Table 6 shows the number of subjects who use model - based reasoning for some purpose ( scored as comprehension , solution attempt , or verification ) subsequent to various condi . Lions in the preceding episode . A subject may either have no preceding episode , have a preceding episode without errors , or five a preceding episode with one or more scored errors ( i . e . , an error of commission , omission , or manipulation from which the subject does not recover in that episode ) . From 26 . 3 % ( 5 of 19 on MRT ) to 70 . 0 % ( 21 of 30 on WT ) of model - based reasoning episodes occur as the first episode in a solution attempt . Of these initial model - based episodes , the majority ( except for problem MRT ) are undertaken for the apparent purpose of comprehending some aspect of the presented problem . The remaining initial episodes are scored as solution attempts . For subjects having a preceding episode , their transition out of this episode is scored as achieving a subgoal , finding a solution , reaching an impasse , or deciding they are wrong . Of the model - based reasoning episodes following an error - free episode , there are two essentially different conditions . In the first , a subject ' s preceding episode ends with achieving a subgoal or finding a solution . This subject could be considered " on track " in her solution attempt . In the second condition , subjects " abandon " the preceding episode after reaching an impasse ( also after getting lost , as described earlier ) or deciding that their efforts are wrong . These subjects are technically on track since their preceding episodes are free of errors , but they encounter sufficient difficulty that they abandon a previous line of reasoning in favor of model based reasoning 43 48 Amost all subjects who are " on track " in a preceding episode either attempt a solution or continue attempts at comprehension during the model based reasoning crignd . . Only a few subjects are " on track " and undertake model based reasoning for the purpose of verification . On problem WC these verification episodes follow finding a solution ; the single verification attempt on problem IV7 ' comes from a subject who verifies a recalled formula using a simplification of the original problem . Subjects " abandon " ( i . e . , lost , impasse or wrong ) a prior , error free episode infre quently and subsequently use model - based reasoning for comprehension or to attempt a solution . Model - based reasoning episodes following an episode with errors are less frequent than those discussed above , but fall into similar categories . Relatively few subjects have preced ing errors , are unaware of those errors , and proceer : as if " on star k " ( sebum . . . a subgoal or find a solution ) . Subjects who are aware of their preceding error nearly always deride that they are wrong and " abandon " the preceding episode . Among those who " abandon " a preceding episode with errors , subsequent model based reasoning is used either for comprehension or as an attempt to find a solution . Although based on a sunset ssf all s ' . ojecte studied , these findings support an inter . pretation in which model - based reasoning plays four basic roles in problem solving : as a pmparnfory comprehension strategy when the model based episode is either the first problem - solving activity attempted or follows other comprehension episodes , as a solution strategy when subjects feel they are on track , a . an evidence gathering strategy " hen a so . lution has been found previously ( this is infrequent ) , or as a creamery strategy when subjects suspect that their comprehension or solution efforts te . . ) , be " off track " These interpret tions are consistent with our earlier argument that reasoning within the situational context 44 49 of a problem supports the generation of quantitative constraints , can be used directly as a solution method , or can he used to verify that these constraints are appropriate . ( Insert Table 7 about here . ) Effectiveness of model - based reasoning . As well as inferring subjects ' reasons for undertaking model - based reasoning , we would like to characterize the effectiveness of this reasoning strategy . To assess efficacy , we examine the occurrence of any errors within successive episodes . Table 7 shows the relationship between errors during a preceding episode ( when there is one ) and errors within the model - based tenoning episode . When model - based reasoning is the subject ' s first evident activity , as indicated by " No episode " in Table 7 , errors are not often encountered within that episode . The two errors shown for problem MRT are mis - conceptualizations in which subjects assume that round trip times are equal . The error in problem WT comes from a subject who assumes that Mary and Jane do equal amounts of work . When a previous episode contains errors , the subsequent model - based episode is usually error - free . Thus , existing errors may be " repaired " during model - based rea - soning . Following an error - free episode , only one subject introduces a new error with model - based reasoning by omitting the constraint that distances are equal on problem MRT . While these findings are not conclusive , they are again consistent with the four hypotheti - cal roles for model - based reasoning described in the analysis of final episodes . Preparatory comprehension promotes an error - free conceptualization of the problem situation , enabling subjects to correctly assemble the quantitative structure of the problem during later rea soning episodes . Subjects also attempt to find solutions directly through model - based reasoning , generally without introducing errors . Alternately , after encountering an error during previous problem - solving activities , subjects may be able to recover through the 45 5Q use of model - based reasoning . Finally , model based reasoning can play a confirmatory role when subjects have identified imp . rtant problem constraints or a possible solution . Summary of quantitative findings As part of our effort to explore the episodic structure of algebra story problem solv ing , this section presents three levels of quantitative analysis : the prevalence of different interpretive categories in subjects ' overall solution attempts , relations between outcomes , tactical content , and errors in subjects ' final episodes of problem solving , and the role and effectiveness of model - based reasoning episodes within the wider problem - solving context . Each successive . . 4 of analysis tightens the focus on findings at coarser levels . A global view of solution attempts reveals significant non ' algebraic reasoning as a preva - lent and somewhat unexpected constituent of competent problem solving . Most prevalent among these tactics is model - based reasoning . Among observed errors , conceptual omis sions or commissions are more frequent than manipulative errors within arithmetic or alge . braic formalisms . An examination of final episodes , the " bottom line " in a very lean view of these problems , corroborates this global image of significant non algebraic reasoning on non - routine problems . Looking more closely at errors , we find that manipulative errors are both less frequent and less damaging than conceptual morn , since subjects are more likely to recover from errors of manipulation within the final episode . Finally , we examine the episodic structure of model - based reasoning and propose four roles for this tactic : as preparatory comprehension , as a solution method , as evidence - gathering for a candidate solution , or as a recovery method for errors generated earlier in the solution attempt . Chew quantitative analyses of problem solving agree with out earlier description of the interplay between the quantitative and situational structure of algebra story problems DISCUSSION Interpreted as a series of problem solving episodes , the written protocols described 46 51 above provide an opportunity to look within individual solution attempts for evidence of strategic and tactical upproach . We have also been able to look across a relatively large sample of mathematically sophisticated subjects in an effort to describe ' typical " problem - solving behaviors . In this section , we compare the results of our study with other research on mathematical problem solving and discuss the Implications of these findings for conceptions of mathematical " knowledge and instruction . Competent problem solving Our findinge are offered as a preliminary exploration of " competent " algebra story problem solving . By choosing the term competent , we hope to contrast the problem - solving behay . iota we have observed against images of " expertise " in problem solving as they are often portrayed in the literature . Po example , Mosley et aL ( 1977 ) and Mayer et aL ( 1984 ) report that experienced problem solvers use problem - solving schemata to categorize prob . lems by type and then represent these problems using familiar quantitative constraints . While this account corresponds with some of our protocols , many subjects in our sample appear to eon : inlet solutions to algebra story problems . Rather than a smooth execution of a highly practiced Al , these constructions often proceed with some difficulty and include reasoning : divide . only partly connected to algebraic or arithmetic formalisms . As noted earlier , subjects In this study should be considered mathematically sophis . Reeled . Nonetheless , judging from the varied behaviors we have observed , the algebra story problems we presented to subjects are not routine problems . On problems MRTand WT , for example , many subjects fail to reach a correct solution , and those who do sue coed often experience considerable difficulty . Analyses of errors encountered by subjects when attempting solutions suggest that conceptual errors of omission and commission are both more prevalent and more damaging than manipulative errors In algebra or arithmetic . These results support a model of algebra story problem solving in which problem compre hension and solution are complimentary processes . Integrating dual representations of a 47 problem at situational and quantitative levels is a central aspect of competence . These intermediary structures provide a representational bridge between the text of a problem and a quantitative solution . Reasoning about the situational context of a problem can serve as a justification foe assembling quantitative constraints that may eventually lead to a correct solution . Thus , a substantial portion of a subject ' s activity is devoted to reach . ing an understanding of the problem that is sufficient for applying the routine of formal manipulation . Despite their mathematical backgrounds , perhaps our subjects have yet to achieve corn . petent algebra story problem solving , well beyond the curricula : setting designed to teach it . Alternately , they may have been " experts " during and shortly after algebra instruction , but with the passage of time have lost the facile performance demonstrated by Ilinsley et al . ( 1977 ) . Whichever explanation is chosen , the iuue remains how to characterize os . tensility competent problem solving in a population for whom the algebra curriculum is designed . Recent studies of mathematical problem solving in " practice ( Carraher , Calm her , Sc Schliemann , 1987 ; Carraher & Schliemann , 1987 ; and de . It Rothe , 1986 ) present similar images of competent quantitative reasoning ; problem solvers organize their quan titative knowledge around the demands of the situational context presented by the task , art . " using the problem situation ( or knowledge of it ) to assemble or verify quantitative constraints . If an image of competent problem rolving in this domain is to inform teaching efforts i . e . , it is to have some predictive capacity as described in the introduction of this paper then activities like these are a legitimate topic of study . We return to issues of competence and acceptable transitional performance in a moment Transfer effects Aside from their use as representative wildcat solving tasks , algebra story ptohlents of ten serve as materials for studies of analogical transfer . ( inert a target Nowell , to wise . subjects exhibit positive transfer when they can use the solution method from a ; nevi 48 53 ously encountered source problem to help solve the target problem . Alternately , subjects exhibit negative transfer when they access and use the solution from an inappropriately related source problem . Studies of analogical transfer with algebra story problems have produced mixed results , but show that both positive and negative transfer sometimes oc - cur . Positive transfer has been more likely when subjects are alerted to the experimental manipulation ( Reed , 1987 ; Reed , Dempster , & Ettinger , 1985 ) or are high in mathematical achievement ( Novick , 1987 ) . Transfer effects related to higher achievement have been at . tributed to subjects ' improved attention to aspects of quantitative structure ( Novick , 1987 ; Silver , 1979 ) and better memory for previous solution methods ( Silver , 1981 ) . Negative transfer in subjects with lower achievement ( Novick , 1987 ) has been attributed to a re . Hance on inappropriate problem features and an inability to reject misleading analogical sources . Finally , Dellarosa ( 1985 ) has experimentally manipulated subjects ' use of analog . ical and schematic problem comparisons to produce improvements in their categorization and solution of related problem : . In the present study , we did not alert subjects to the comparability of problems , nor did we encourage them to look back over their prior solutions as they worked through the problems . Their backgrounds insare high mathematical achievement , and entrance requirements for academic majors in computer science and engineering preselect for high quantitative abilities . There is no performance - level evidence of positive or negative transfer within the problem - solving session , despite ozir manipulation of ptobiem structure and presentation order to elicit these effects . At the aggregate level , our suhjects appear to take the " school math " task we present them at face value : each problem , presented individually on a blank sheet of paper , is treated as a sail - contained exercise , rather like vch at a student might face during examinations in a course on algebra . However , en inspection of individual protocols and explanatory remarks we find that several subjects give evidence for some form of negative transfer . 49 54 In some cases , transferred material directly violates the quantitative and situational structure of the target problem . For example , subject w08 incorrectly attempts to add working rates on problem WC , first writing 1 / 5 bores + 1 / 2 bores = 56 , followed by 7 / 10 bores = 56 . In explanatory remarks , w08 states that ' Ile mistake 1 made was that I assumed it was like problem 1 where they work together . " In the preceding solution to WI ' , this subject had written " Together = 1 / 5 + 1 / 4 in one hour = 9 / 20 " and then correctly divided one job by the combined rate . Adding working rates in problem WT is justified since Mary and Jane work together at the same time . However , situational and quantitative relations are exactly reversed in problem WC ( see Figures 6 and 2 ( 1 ) ) ) . Since times are added together ( adjacent ) and work is performed on the same boxes ( congruent ) , the addition of working rates ( i . e . , output over time ) cannot be similarly justified . In other cases , subjects recognize an appropriate source problem , but then fail to transfer information at the correct level of abstraction . For example , on problem MOD subject wet correctly attempts to add motion rates , but uses an algebraic expression of the form : 1 / 60 + 1 / 100 = z / 880 . On the previous ( WT ) problem , the subject manages a correct solution using an expression of the form , 1 / 5 + 1 / 4 = 1 / z , and remarks that this " . . . is a formula used to find a total of time they work together . " Although the addition of rates can be justified in both problems , it appears that the rate form in the retrieved formula is reversed ( i . e . , time over output ) when used in a solution attempt on the MOD problem . Thus in a situation where we anticipate that the subject will benefit by transfer of a solution approach , their failure to justify transferred material actually produces a negative effect . it may be that the problem - solving context , completing a test booklet in a proctored examination setting , as well as our decision not to alert subjects to the comparability of problems , prevented them from recognizing and elaborating effecti ve analogical comparisons between problems . In more detailed verbal protocol studies where subjects are encouraged to make problem comparisons ( Hall , 1987 , 1988 ) , attempts at analogical inferences between algebra story problems are quite common . These comparisons are usually lengthy and can introduce misconceptions , but also frequently lead to fruitful explorations of problem structure , both quantitative and situational . In addition , comparisons need not encompass the entire problem structure , but often instead make effective use of relevant substructural similarities . These alternative findings are largely consistent with other verbal protocol studies of learning from worked examples ( Pirolli & Anderson , 1985 ; Singley , 1986 ; Chi , Bassok , Lewis , Reiman , & Glaser , 1987 ) , and suggest that analogical comparison may be a common problem - solving and learning strategy in settings where subjects have some control over their work . Model - based reasoning We are not the only researchers to note the prevalence of model - based reasoning during mathematical problem solving . A number of psychological studies have found similar ev - idence , although interpretations of this activity vary . Paige & Simon ( 1966 ) , comparing human protocols with Bobrow ' s ( 1964 ) computational model of translating algebra story problems into equations , found that subjects with varied mathematical backgrounds used " auxiliary representations " of the physical setting of a problem . These representations allowed some subjects to detect impossible problems or to assemble relevant quantitative constraints ( e . g . , additivity in part - whole relations ) . Using verbal protocols to study the prevalence of Pclya ' s ( 1945 ) heuristics for mathematical problem solving , Kilpatrick ( 1967 ) reported that 60 % of an above - average group of eighth graders usc . , " successive approxima tion " while attempting to solve word problems . These trial - and - error approaches were often successful and were sometimes combined effectively with more deductive solution strate - gies . Silver ( 1979 ) found similar successful approximation strategies in students who had yet to receive formal algebraic training . Studying geometry problems , Schoenfeld ( 105 ) found that students used a trial - and - error approach to generate hypotheses about geomet tic relations and then evaluated these hypotheses by physical construction . Ile argued that 51 56 these exploratory episodes of " naive empiricism " were usually poorly organized and often interfered with forms of deductive verification that students knew how tc use . Finally , Kintsch & Gismo ( 1985 ) described a process model of solving arithmetic word problems in which quantitative strategies were triggered by information contains , ' in a " situation model " of the problem . The situation model was constructed during text comprehension and contained a set - based representation of typed quantities and their interrelationships ( e . g . , part - whole ) . Follow - on studies ( Kintsch , 1986 ) have shown that the construction of a situation model is important for recall , inference , and learning from text . Looking over this evidence , we find that studies of mathematical problem solving con sistently encounter activities similar to what we call model - based reasoning : subjects con . struct some form of situation model , take inferences within the model to help comprehend and sometimes to solve a quantitative prohlem , and use the model in a supportive role for assembling or verifying quantitative constraints . Beyond model - based reasoning in math - ematical problem solving , similar evidence is available across a wide range of cognitive activities . For example , Johnson - Laird ( 1983 ) argues for a model driven arc of syllo - gistic reasoning that underlies common - sense inference . Given a pair of prem < . like , AU the artists are beekeepers / Alt the beekeepers are chemists , Johnson Laird ' s subjects appear to build successively more elaborate models of the situation described by the premises when searching for valid inferences . The validity of each inference , rather than being logically deduced by sound rules of inference , is evaluated with respect to these concrete models of the premises . Errors occur when subjects are unable to build sufficient models of the premises and thus overlook or fail to eliminate various inferences . Relatively concrete forms of reasoning outside traditional ( i . e . , schooled ) formalisms have also been observed for de - cision making under uncertainty ( Tversky & Kahneman , 1974 ) , various forms of statistical reasoning ( Nisbett , Fong , Lehman , & Cheng , 1987 ) , and explanations of physical processes ( Clement , 1983 ; McCloskey , 1983 ) . 52 57 In general , these studies raise questions about the relationship between what students bring to an educational setting i . e . , their " preconceptions " about a subject matter and materials that the curriculum explicitly presents . In the domain of mathematical prob - lem solving , students ' " preconceptions " and associated activities are often pushed to the background of legitimate practice and inquiry . At best they are " auxiliary " to quantitative reasoning , while at worst they interfere with preferred problem - solving activities and pro - duce ' lost opportunities , unfocused work , and wasted effort " ( Schoenfeld , 1985 , p . 308 ) . In their stead , the manipulation of symbolic representations of quantity , quite apart from the situations that give rise to these quantities , is held in the foreground . Our findings on model - based reasoning , in concert with other studies reviewed briefly above , suggest that this foreground / background conception of quantitative problem solving may need to be reconsidered . In our sample of " competent ' subjects , a routine problem is one in which the use of familiar algebraic operations will provide a precise value for an unknown entity . This is the power of the algebraic formalism : it is perfectly general , sound , and often simple to apply . However , quantitative precision is of little value when the subject is uncertain about the problem ' s structure . Our characterization of overall episodic activity , the frequency and consequence of conceptual versus manipul dive errors during those episodes , and the role of model - based reasoning show that routhe activities within the algebraic formalism make up only a portion of competent problem - solving . For many of our subjects , algebra story problems are not routine exercises . Instetd , much of their problem - solving activity is devoted to assembling a sensible set of constra . nts on a desired quantity , an effort that uu covers the problem ' s structure . When algebraic constraints are unclear , subjects sometimes attempt solutions using model - based reasoning ( e . g . , Figure 8 ) , a tactic that approximates a certain value for an unknown = t1ty . The value is certain when quantitative constraints that determine its derivation are grounded in a representation of problem structure that is 53 58 familiar to the subject . The strategic significance of this activity is consistent with varying explanations . On one hand , enacting a set of physical constrai . ay provide otherwise skilled quantitative problem solvers with an efficient means of estimating quantitative solutions . Under this interpretation , the model - based episode shown in Figure 8 may result simply from the subject ' s preference for repeated additions over a more complicated thoisian . ( 1981 ) makes a similar argument when interpreting results of a developmer . tal study on the relationship between velocity , time , and distance . In contrast , we argue that episodes of model - based reasoning serve as problem solving strategies in their own right , and are used when more " ( nemal " activities ( e . g . , algebraic substitution ) are unreachable given the cur - rent problem representation . Under this interpretation , the subject in Figure 8 undertakes model - based reasoning because her representation of the problem cannot justify a division of the total distance by a combined rate . Enacting motion and time constraints over suc - cessive hours of travel makes the quantitative structure of the problem more certain . The results of model - based reasoning support a conceptualization of quantitative constraints in which the total distance can be divided by a combined rate to give a precise account of the elapsed time . Further constraints are introduced by establishing that the correct quantitative solution falls between the fifth and sixth hours of travel . Interpreting model - based reasoning as an alignment of certain and precise represen tations of problem structure leads to deeper questions about a competent understanding of mathematical concepts , in this case related linear functions . One point of view takes mathematical concepts as objects of knowledge in and of themselves , quite apart from their physical embodiment in a situational context . Hence the story in an algebra story problem serves only as a vehicle for carrying a mathematical structure . An alternative point of view takes mathematical concepts as tools for modeling physical situati - s , in this case related motion or work events as presented in problem texts . The question is how far vehicles 54 59 60 will travel or how long it will take to complete a job , and mathematical concepts serve as sometimes - useful tools for answering these questions . We suspect that these points of view are not incompatible . In fact , the latter view may provide an educational bridge to mathematical concepts as self - containel sources of knowledge . That is , a competent mathematical conception of related linear functions is based on and extended through a physical understanding of the situational context that the " story " of an applied problem presents . An activity like iterative simulation " joins " concrete inclines , allowing the subject to successively construct a systematic relationship between rata and providing an introduction to related linear functions that tan be directly supported within a familiar context . Over time , the mathematical concept reflects a history of use as a tool for modeling physical tituations . The concept of rate changes as its modeling role is extended over a wider range of situational contexts , perhaps leading to heuristic estimates or algebraic construction based on " scaling " inclines as invariant relations . The fault could eventually resemble a relatively context - free mathematical abstraction . Of course , this account of the acquisition of mathematical concepts is highly speculative and not a focus of our study . However , judging from the problem - solving behavior observed in this study , even ostensibly " competent " mathematical problem solvers continue tc base their quantitative efforts within the situational context of presented problems . Educational implications Wo have interpreted the relative prevalence and consequence of conceptual versus manipula tive errors as evidettee that subjects have difficulty is assembling the quantitative structure of algebra story problems , long after they have mastered the algebraic formalism . Likewise , the prevalence and functional role of model - based reasoning are interpreted as evidence that even mathematically - sophisticated problem solvers explore the situational context of these problems in an attempt to construct or repair a representation that will , ipport a solution . Rased on these findings and their interpretation , we examine several implications 55 I for teaching mathematical problem solving . The primacy of conceptual errors and use of model based reasoning , in some cases to recover from these errors , suggest that instruction based solely within the mathematical formalism may be inadequate for solving non - routine problems . Textbook instruction in algebra story problem solving typically addresses this issue by providing some suggestions for " . . . translating from words to appropriate algebraic forms " ( Kolman & Shapiro , 1981 , p . 64 ) . These range from direct translation rules taking textual phrases into equations ( e g . , rewrite " twice " as 2x ) to the construction of tables that organize quantitative entities and their interrelationships around known formulas . The desired result is a set of simultaneous linear equations amenable to algebraic operations . While these suggestions provide a sort of organizational strategy for the student ' s problem - solving activity , they fall well short of specifying how quantitative relations , particularly those that are only implied by the problem text , can be identified , arranged as entries in a table , or effectively used . Instead , the results of our study point to persistent problem - solving difficulties that the traditional algebra curriculum addresses weakly if at all . How might these components of competent problem solving be taught more effectively ? We argue that the situational context of an algebra story problem , and in particular the correspondence ber . veen situational relations and quantitative constraints , should be a le gitimate object of teaching in the algebra curriculum . This is clearly appreciated in other problem - solving curricula . For example , consider the utility of forte diagrams for solving statics problems in physics . Students who ignore or incorrectly construct force diagrams can be expected to manipulate equations or formulas without visible signs of progress . This is quite similar to Paige & Simon ' s ( 1966 ) finding that " auxiliary representations " helped subjects to detect impossible algebra story problems , sometimes before writing any equations at all . Our quest : on , then , is whether there might not be a similar organizing representation for algebra story problem solving ? There are some suggestive precedents : 56 61 11 Gould & Finzer ( 1982 ) describe an animated computational environment that allows stu - dents to make guesses in a one - dimensional world of notion ; Greeno ( 1983 ) describes an effective instructional technique in which students use an electric train set to help calculate solutions to compound motion problems . As one possibility among many , we present a representation that drawa directly from the analysis of situational structure presented earlier and consider under what circumstances it could provide a useful instructional model for constructive problem solving . As with any model used in teaching , there are problems of registration : the model may cover some aspects of the target domain well but cover other aspects poorly . Our proposal addresses relations and operations possible within a representation of the situational structure of compound algebra story problems , and the correspondence of these aspects to relations and operations possible with a representation of quantitative structure . We expect that in combination with a quantitative model like that proposed by Greeno et at ( 1986 ) , their joint contribution could prove more effective than either used alone . ( Insert Figure 15 about here . ) Figure 15 shows paired graphical representations of situational and quantitative struc . ture for the AMT problem . At the top of the figure , a dimensional frame displays orthogo - nal output ( in this case , distance ) and time dimensions , with entities arranged along those dimensions by their respective situational relations : times are adjacent and distances con - gruent . At the bottom of the figure , a quantitative network ( Shalin & Bee , 1985 ) shows the common distance found by applying motion rates to component times . Esch represents - tional device provides a directly accessible illustration for important aspects of competence in this problem - solving domain . In contrast with translation rules or tabular arrangements , the illustroive medium of dimensional frames provides a spatial abstraction for compound rate problems that promotes a physical justification for essential quantitative constraints . Time segments 57 62 add because they are adjacent within the vertical dimension , while distance segments are equal because they are congruent within the horizontal dimension . As noted in our earlier discussion of quantitative structure , substructures corresponding to these constraints must be constructed before using the quantitative network to find a solution e . g . , the additive triad over time extenaives that centers the quantitative network in Figure 15 . The ability to appropriately select and place these quantitative substructures appears to require a substantial investment in training time ( Greeno et at , 1986 ) . We expect chat a well designed illustrations around the idea of dimensional frames could effectively support the acquisition and use of a quantitative network illustration In contrast with a set of algebraic equations , quantitative networks provide a spatial abstraction for variables and equivalence relations that makes the global structure of what would otherwise be a linear encoding more apparent . Rather than writing a set of equa - tions with repeated variable names or constants , a notation that can obscure the role of quantitative entities and make the applicability of certain algebraic operations difficult to recognize , the quantitative network directly captures the notion of shared variables or con . stants and multiple ways of reaching a particular unknown . The network provides a visually inspectable form of algebraic calculus , essentially constraint propagation , that may prove easier for students to learn than more traditional instructional methods ( i . e . , algebraic op - erations on linear equations ) . Thus , the two illustrative media are collaborative III that they provide interdependent representational stages intermediate between a problem text and a correctly manipulated set of algebraic constraints . Returning to Figure 15 , we give a more detailed treatment of this collaborative inter - dependence . As a compound motion problem , MRT involves two events , each contributing entities modeled as segments on output and time dimensions . Across events , segments on each dimension are related in manner that determines their quantitative composition . . 01ilasoa ( ix press ) give * prescriptive methodology for co aaaaa coax mteractore tfluarohons u well as a particular illsatratioa , called " Rectsagle World , for the ratio state of rational lumbers 58 63 Adjacent time segments can be composed to yield a single segment whose extent along the vertical dimension corresponds directly to the value of total time traveled , thus implying an additive relation over extensive ' in the quantitative network . Similarly , congruent segments in the distance dimension have an identical extent , implying the same ( and same - valued ) extensive in the quantitative network . Within each event , the rate provides a comparative mapping between dimensions , modeled as Individual inclines in the figure . Placed at the top of the dimensional frame , walking covers 3 miles In one hour , and after transformation to reflect a common output ( discussed in a moment ) , the bus is shown to cover the same 3 miles in hours at the bottom of the frame . la addition to sanctioning relations among quantitative entities , more direct problem - solving inferences using model - bated reasoning are also possible within the dimensional frame . Treated as invariant relations across dimensions , motion inclines can be " scaled " to give heuristic estimates of common distance and composed times , as shown with dashed lines in Figure 15 . Alternately , treating rates as concrete associations , inclines could be " joined " together during an iterative simulation of compound motion . In eath case , a model - based solution is reached when a common distance is found that precisely requires six hours for round trip traversal . Both forms of model - based solution attempts are consistent with observed protocols . For example , subject m31 uses a form of " scaling " to make heuristic estimates of 24 , 12 , and 15 miles for a common distance , checking the combined time required for each estimate against the given six hours . After the third estimate , she notices that " each mile takes . . . A hours " and later uses this constraint to construct an algebraic expression in a single unknown , - A x X = 6 . " In contrast , subject m18 uses a form of " joining " by choosing 3 miles as a concrete distance segment , determining that the bus takes 7 . 5 minutes to cover this distance ( shown as hours in Figure 15 ) , and then extending these concrete relations in a simulation of successive three - mile return trips . Both subjects alter the form in which motion rates are expressed ( i . e . , output over time ) during their model - 59 P 64 ; . 5 based solution attempts , and subject mil finds a way of conihining rates for a " return trip mile . " In each case , activities within model based reasoning episodes observed in written protocols directly sanction multiplicative relations between rates ( intensives ) and timer , ( extensive ' ) shown in the quantitative network of Figure 15 . An appropriate combination of these representations could be a helpful artifact for in . struction in algebra story problem solving . First , representational choices in the dimensional frame can serve as justifications for more abstract relations or operations in the quantita live network . As argued above , a justification for adding times within the quantitative formalism is that their composed spatial extent is tensible within the situational context of the story . As a more complex example , subject m3I ' s decision to transform and then add motion rates in this problem cleverly restructures the dimensional frame to have single segments on both time and output diroensions e . g . , A hours for each " return trip " mile . The corresponding quantitative network would require only three entities : a time extensive ( 6 hours , given ) results from multiplying the combined rate intensive ( A hours per mile , inferred ) by an unknown extensive for round trip distance . This is a sensible change in representation only because the time segment given in the " goal state " of the problem is presented as a composed whole ( i . e . , " . . . he was gone for 6 hours " in the text of problem ART ) , and round trip distance segments are congruent . Thus , representational choices in the dimensional frame provide justification for construction of a simplified quantitative network . Second , problem - solving activity ( e . g . , iterative simulation ) within the dimensional framework can actually help to recover from prior conceptual errors . For example , con . eider a subject who first attempts a solution within the algebraic formalism and omits the constraint that distances are the same ( i . e . , the same variable ) . Finding two simultaneous Ham equations in three variables , this subject reaches an impasse . ( looting model based reasoning for the purpose of comprehension in the next episode , the subject immediately 60 faces a representational decision in the distance dimension : should positionally distinct or identical spatial segments be chosen ? Certainly , the possibility of an incorrect choice remains , but when mailing this choke In the algebraic formalism of the prior episode , the consequences of an Incorrect representational decision were less apparent . Correctly choosing congruent distance segments in the dimensional frame could allow this subject to achieve a solution within the model - based reasoning episode , or to return to the algebraic formalism with a more complete representation . in summary , choosing an apt combination of situational and quantitative models for instructional purposes is SSW hating problem . Our suggestion for the dimensional frame as an illustrative mechanism would require further refinement to achieve effective integration with an algebraic Illustration , as discussed above . Nonetheless , we feel this approach is Interesting in several respect , . First , our proposal is consistent with an empirical picture of episodic problem - solving behavior in mathematically sophisticated subjects . Taking these findings as evidence for competent ( if not expert ) problem solving , we are interested in supporting what problem solvers actually do during their attempts to solve non - routine problems . Our instructional proposal is based on a characteritarion of these attempts and an analysis of common problem - solving difficulties . Second , although the solution of a particular class of problems may become routine with practice , the ability to construct an algebraic representation will continue to be important for novel problems or problems that have become unfamiliar with the passage of time . Being able to construct a representation in the algebraic formalism , based on the constraint - generating Inference : A we have des : ribed AS one role for model - based reasoning , may never become entirely routine . Last , combined Illustrative media may be of some practical value in delivering instruction on algebra story problem solving , whether provided through computer - based instruction or a traditional algebra curriculum . CI 66 I ACKNOWLEDGEMENTS This paper is an expanded version of au earlier report on the episodic structure of algebra story problem solving . The research was primarily supported by the Cognitive Science Program of the Office of Naval Research . contract N00014 RS K 0 . 173 Rogers Hall was also supported by a University of California Regents ' Dissertation Fellowship . Etienne tVenger was also supported by the Institute for Research on Learning ; and Chris ' Pruxaw was also supported by a graduate fellowship from the Office of Naval Research and the American Society for Engineering Education , We wish to thank Jun Greene , . I , auren itesnirk . and Karen Wieckert for valuable editorial comments . 62 87 REFERENCES Allen , J . F . ( 1983 ) . Maintaining knowledge about temporal intervals . Communications of the Association for Computing Machinery , 26 ( 11 ) , 832 - 843 . Allen , J . F . ( 1984 ) . Towards a general theory of acting and time . Artificial Intelligence , 25 ( 2 ) , 123 - 154 . Bobrow , D . C . ( 1968 ) . Natural language input for a computer problem - solving system . In M . 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Publishers . McCloskey , M . ( 1983 ) . Naive theories of motion . In D Gentner & A . L . Stevens ( Eds . ) Mental models ( pp . 325 - 341 ) . Hillsdale , New Jersey . Lawrence Erlbaum Associates , Publishers . Ne II , A . ( 1969 ) . Heuristic programming : Ill structured problems . In . 1 S Aronofsky ( Ed . ) , Progress in Operations Research , 3 , 360 . 114 . Nisbett , R E . , Fong , G . T . , 1 . ehman , 1 ) 11 . , & Cheng , P . W . ( 1987 ) leaching reasoning Science , 30 , 625 - 631 . Novick , L R . ( 1987 ) . Problem similarity , analogical transfer , and individual differences University of California at Los Angeles , Unpublished manuscript , Ohlsson , S ( in press ) Sense and reference in the design of interactive ellustrstions for rational numbers . In It . Lawler & M Yazdani ( Eds . ) , Artificial intelligence and education . Norwood , New Jersey Aides . fife 71 l ' aige , J . M . , & Simon , II . A . ( 1966 ) . Cognitive processes in solving algebra word problems . In B . Kleinmuntz ( Ed . ) Problem solving : Research , method , and theory ( pp . 51 119 ) . New York : John Wiley . P . 1 . . , & Anderson , J . R . ( 1985 ) . The role of learning from examples in the acquisi tion of recursive programming skills . Canadian Journal of Psychology 32 ( 2 ) , 240 - 272 . Polya , G . ( 1945 ) . How to solve it . Princeton , New Jersey : Princeton University Press . Quintero , A . H . ( 1981 ) . The role of semantic understanding in solving multiplication word problems : Two step it x E problems . In Schwartz , J . L . The role of semantic un - derstanding in solving multiplication and division word problems . Final report to the National Institute of Education , NIE - G - 80 - 0144 . Quintero , A . H . , & Schwartz , J . L . ( 1981 ) . The development of the t . oncept of ratio in children . In Schwartz , J . L . The risk of semantic understanding in solving multiplica - tion and division word problems . Final report to the National Institute of Education , NIEG . 80 - 0144 . Reed , S . K . , Dempster , A . , & Ettinger , M . ( 1985 ) . Usefulness of analogous solutions for solving algebra word problems . Journal of Experimental Psychology : Learning , Mem - ory , and Cognition , 11 ( 1 ) , 106 - 125 . Reed , S . K . ( 1987 ) . A structure - mapping model for word problems . Journal of Experi - mental Psychology ; Learning , Memory , and Cognition , 15 ( I ) , 124 - 139 . Riley , M . S . , Green° , J . G . , & Heiler , 3 . 1 . ( 1983 ) . Development of children ' s problem - solving ability in arithmetic . In H . P . Ginsburg ( Ed . ) The development of mathematical thinking ( pp . 153 - 196 ) . New York : Academic Press . Schoenfeld , A . II . ( 1985 ) . Mathematical problem solving . New York : Academic Press , Inc . PP 72 67 Schwartz , J . L . ( 1982 ) . The semantic calculator . Classroom Computer News 2 , 22 24 . Shalin , V . , & Bee , N . V . ( 1985 ) . Analysis of the semantic structure of a domain of word problems . Technical Report No . APS 2e . University of Pittsburgh Learning Research and Development Center . Silver , E . A . ( 1979 ) . Student prceptions of relatedness among mathematical verbal prob ! ems . Journal for Research in Mathematics Education , 10 , 195 210 . Silver , E . A . ( 1981 ) . Recall of mathematical problem information . solving related prob ! ems . Journal for research in mathematics education , 12 ( 1 ) , 54 . 64 . Simon , II . A . ( 1973 ) . The structure of ill - structured problems . Artificial hitelhgcnce . 4 , 181 - 201 . Singley , M . K . ( 1986 ) . beveloping models of skill acquisition in the context of intelligent tutoring systems . Ph . D . Thesis , Carnegie - Mellon University . Thompson , P . W . ( 1988 ) . Artificial intelligence , advanced technology , and learning and teaching algebra . To appear in C . Kieran & S . Wagner ( Eds . 1 , Research woes to the learning and teachms of algebra . Reston , VA : National Council of Teachers of Mathematics . Tveteky , A . & Kahneman , D . ( 1974 ) . Judgment under uncertainty . Heuristics and biases . Science , 185 , 1124 - 1131 . Wilkening , F . ( 1981 ) . Integrating velocity , time , and distance information . A develop mental study . Cognitive Psychology . 13 , 231 247 . 68 r ; Table 1 : Representative algebra story problems . Motion : Opposite direction ( MOD ) . Two trains leave the same station at the same time . They travel in opposite directions . One train travels 60 km / h and the other 100 km / h . In how many hours will they be 880 kin apart ? Motion : Round trip ( MRT ) . George rode out of town on the bus at an average speed of 24 miles per hour and walked back at an average speed of 3 miles per hour . How far did he go if he was gone for six hours ? Work : Together absolute ( WT ) . Mary can do a job in 5 hours and Jane can do the job in 4 hours . If they work together , how long will it take to do the job ? Work : Competitive ( WC ) . Handy can fill a box with stamped envelopes in 5 minutes . His boss , Jo , can check a box of stamped envelopes in 2 minutes . Randy works filing boxes . When A is done , Jo starts checking his work . How many boxes were filled and checked if the entire project took 56 minutes ? 69 Table 2 : Categories for interpreting the purpose , content , errors , and relative statue . of problem - solving episodes . Strategic purpose Comprehension Solution attempt Verification Tactical : ontent Annotation Problem elements Retrieval of formulas Diagram Algebra Model - based reasoning Simulation Heuristic Ratio Whole / part Part / whole , part / part Proportion Scaling Unit Procedure Conceptual content Erro , ptual errors Errors of commission Errors of omission Manipulation errors Algebraic errors Variable errors Arithmetic errors Status of episode within solution attempt Conwaency Before During After Transition Subgoal Wrong Impasse Lost Final solution Found solution wrong 70 Table 3 : Percentage of subjects with a scored category during their solution attempts . Problem HOD MRT WT WC Tactical content Algebra 82 . 4 85 . 9 71 . 8 63 . 5 Model 30 . 6 22 . 4 35 . 3 47 . 1 Ratio 17 . 6 14 . 1 15 . 3 42 . 4 Pncedure 0 . 0 1 . 2 21 . 2 0 . 0 Units 3 . 5 1 . 2 1 . 2 1 . 2 Notations 7 . 1 15 . 3 21 . 2 29 . 4 Diagram 69 . 4 36 . 5 8 . 2 9 . 4 Strategic purpose Comprehension 84 . 7 64 . 7 57 . 6 60 . 0 Soluttoz attempt 100 . 0 100 . 0 100 . 0 100 . 0 Verification 28 . 2 20 . 0 7 . 1 20 . 0 Episode transitions Solution 97 . 6 75 . 3 85 . 9 97 . 6 Impasse 9 . 4 10 . 6 7 . 1 4 . 7 Lost 4 . 7 21 . 2 15 . 3 3 . 5 Wrong 16 . 5 38 . 8 25 . 9 16 . 5 Errors . Omission 7 . 1 21 . 2 23 . 5 11 . 8 Commission 17 . 6 49 . 4 42 . 4 14 . 1 Arithmetic 9 . 4 4 . 7 3 . 5 2 . 4 Algebra 5 . 9 8 . 2 8 . 2 0 . 0 Variable 1 . 2 5 . 9 14 . 1 2 . 4 71 76 Table 4 : Final episodes : percentage correct by subject groupings . Problem MOD MRT WT WC Group contrast ' M < W M > W M > W M < W Correct 89 . 1 92 . 3 47 . 8 56 . 4 58 . 7 64 . 1 93 . 5 89 . 7 Incorrect 6 . 5 7 . 7 19 . 6 15 . 4 28 . 3 20 . 5 6 5 5 . 1 Nosolution 4 . 3 0 . 0 32 . 6 28 . 2 13 . 0 15 . 4 0 . 0 5 . 1 * M sees MOD . WT . WC . MRT : W sees WT . MOD . Af RT . WC 72 7 Table 5 : Final episodes : tactical content and errors uy correctness . Problem MOD MKT WT WC Outcome ' ` C I NCIINC IN n 77 6 2 44 15 1 26 52 21 12 78 5 2 Tactical content Algebra 58 6 0 36 8 20 43 5 7 44 2 I Model 3 0 0 4 2 6 2 1 2 12 1 0 Ratio 13 0 2 4 3 0 5 3 2 22 1 1 Procedure 0 0 0 0 0 0 1 11 1 0 0 0 Units 2 0 0 0 0 0 0 0 0 0 0 0 Not scored 1 0 0 0 2 0 I 1 0 0 1 0 Errors 1 Conceptual 1 6 0 0 14 16 1 27 I0 1 4 0 Manipulative 7 2 0 1 5 2 4 7 1 II 2 1 0 ` C = correct ; I = incorrect ; N = no soIution 7 8 73 Table 6 : Errors and transitional status of a previous episode compared with the impost - of a model - based reasoning episode . Problem MOD ( MET I WT WC n 26 I 19 I 30 I 40 V Purpose EjEguirm S pc C S VIC s No preceding episode 7 1 0 1 4 0 17 4 0 10 2 0 No errors in preceding episode r On track Abandon 3 1 9 0 0 0 0 I 6 I 0 0 2 0 2 2 I 0 10 0 11 0 2 0 U 0 Errors in preceding episode On track Abandon 1 2 0 2 0 0 4 ( 1 1 0 5 0 0 0 0 0 2 0 0 1 1 1 2 ate _ . . . - 74 7g Table 7 : Errors before and during model - based reasoning . Problem MOD MRT WT WC n 26 19 30 40 Model episode Errors None Errors None Errors None Errors None Previous episode No episode Errors No errors 0 1 0 8 4 13 2 2 4 0 2 r I 12 4 23 IR distance 100 5 . 5 550 kilometers A . rate 100 kph III time 5 . 5 hours Figure 1 : A multiplicative relation involving two extenSIVeN and a tilligle 11111 . 11SIVi 81 14 Y 8C , 75 76 ( a ) output ratel ( total time2 ) = rate2 , time2 unknown l ' igure 2 : The quantitative structure of two problem classes : ( a ) contains problems MOD , 11 ' 7 ' while ( b ) contains MRT , WC . 77 82 ( a ) Collinear A Opposite direction ( b ) Colt : near Distance t Distance 2 ' Time 1 ' rmie 2 0 0 0 C . ) 0 ( C ) Congruent 60 k 100k A stpse e Id Figure 3 A situational context for motion in opposite directions ( a ) ant ( b ) show places and segments for output and time , while ( c ) shows inclines for rates wh . . 41 these dimensions are arranged orthogonally . 78 83 4 I ( a ( b 4 . 1 Figure 4 : Operations based on different interpretations of two - dimensional inclines : ( a ) shows a concrete situation successively " foiled " to give an iterative simulation of states within the problem model ; ( b ) shows an invariant relation " scaled " to give a heuristic estimate of a final state in the model . 79 ff \ ? 81 I i 0 . i . . : . , : / : O ki o A t r I U U i i t . . ° . a i l \ ii I I t . . . . - . 1011 - > a Ite 880 kilometers ( b ) 144 880 kilometers Figure 5 : Solution attempts using model - based reasoning on problem MOO : ( a ) loins " successive concrete inclines in an iterative simulation ; ( b ) " scales " inclines as an invariant multiplicative relation in a heuristic estimation . NO 85 C T o I M g , r " u e R E L 3 A d T j 1 a O c N e S n t OUTPUT RELATIONS Congruent Adjacent Figure 6 : A matrix of situational contexts for pairs of isomorphic motion and work problems . Mary can do job in 5 hours and Jane can do the job in 4 hours . If they work together , how long will it take to do the job ? Nal doe / . jeud 1 - 4 . 1011 . voNs . olmr . alm 1 IRMO dovece CU Q - ) . 1111 , gRea Figure 7 : Protocol of subject m20 on the WT problem . 82 7 Two trains leave the same station at the same time . They travel opposite directions . One train travel . 60 km / h and the other 100 km / h . In haw many hours will they be 880 km apart ? C . ) A \ far . - _ - . 14 - / 14 . . . v , C . ) 1 P I z - < 1 5 womm . ago . . = qzp f A . . 4 1 ; 441 6 49 17O , / , . . I ; tV \ e 1 . . / , . ) . 2J ) ' ) o ' 1 - c 99 ft / - . / 1 S - t ? 3r1 . 2 i 44 ( tr . - , d 4 V 4 r° L170 . , 5 1 . 2 1 ( if - 700 - 17 - AP , Cpl . 3 ( 0 U O - - ) J - roma 111 . ono * mm0 d 4 - - - a - 3 iv° Figure 8 : Protocol of subject w06 on the MOD problem . 83 88 George rode out of town on the bus at an average speed of 24 miles per hour and walked back at an average spe - , : d of 3 miles per . lur . How far did he go if he was gone for six hours ? ais - fmeice 4 Ar ) ( x how ' s ) to0 . 165 oils - ft : no r ( 3 r4 , 1es / kr ) ( so als4ance uutttkvt3 aviance & % 4 miles - ( & r ) ( X Lan ) = ( 3 mi lerA , ) ( , ayx - t8 - 3x 0 27x . IS X 3 1100 ' bus dis4toice = ( tk rA1esAr ) t Ft ) tikrs ) 1 ) ( kS 151 - rtnce I 2 $ rni \ eS walking Ais4ance 1 > te way = Ilo miles gou . na - trif Ba Figure 9 : Protocol of subject m39 on the MRT problem . 84 8 Two trains leave the same station at the same time . They travel in opposite directions . One train travels 60 km / h and the other 100 km / h . In how many hours will they be 880 km apart ? 0 r . t1 / 4 / 00 lc / It / it 60 em / H ieo Km / h . . . 1 = 110 mgeeee NINIIM AMINO alMa ffer / 80 01 : 100 tn : lik ' S 300 , r / t7 5 . 00 330 kin 350 ktn Figure 10 : Protocol of subject m03 on the MOD problem . 35 90 Mary can do a job in 5 hours and Jane can do the job in 4 hours . If they work together , how long will it take to do the job ? 111a4 - 4 . 0 hrs His Sark - 4 r 5A1 cc : 10 ) i 0 fn : : HA Di - 1017 aAk - Cti : 9 ' 1 T . 4 / 1 1C4 old . 4 - o Job u . ) . 4 It - bk . t . rm . 241 hrs , o e ( i . ko CD Figure 11 : Protocol of subject m32 on the WT problem . 86 9 1 TrINVIPMBRINCEIMMIZOCIEIMIELSOD - Aarenter , George rode out of town on the bus at an average speed of 24 miles per hour and walked back at as average speed of 3 miles per hour . How far did he go if he was gone for six hours ? gct 24 mbj : ® 3 , niiir = q , 4 - ca5tir t4 . 4o1 64 - y e f3us + ravels 24 - mile ; - cc ( one Ivor , 67639c - frAileI6 back 2it wiles 6 Vscor6 remlb _ n _ 45 - . _ 3 hoar ; 47 : ADJ . . 304 - L6e wan - 4 - ( 0 hours u . hTick vvi 2 - - raii 3 1 ( . 0 mites Figure 12 : Protocol of subject w17 on the MRT problem . . . , . . . . D . . . RT - - - > 1 - - - - - - x - 0 C ) . . . . . . . . . . . , . . . . Two trains leave the same station at the same time . They travel in opposite directiono . One cram travels 60 km / h and the other 100 km / h . In bow many hours will they ' oe 880 km apart ? . 1E 1 - . 1 - Sn - 1 ( con O O r4t - ONISE TrAvs ( s I It 11 1 , . : 5 j eySJA3 . 5fr . 0 1 f t i v A / O . NA A s 0 . rv . MA iS Zt1 ovi 0 2 . 122 S 4 . 0 3 i , A _ _ _ . . - - L - - staat 641 , . . . . . . . . . . . . . . . . . . . . . . . . . . . vity4 . L - , . . r k , : - . 2 . : v 4 , Li , 4 . C 4 . " ^ - , Ai , A ) AAA4 - 0A - k ) ea ( 01 , - 6 , 4 - e . S tj bi . . 45 , SO e . . % 4 cm - , c , , ti 0 . r C < 60 vi 40 / h / KO MN . 411111 , MEM . = OM = ONO Kok t Figure 13 : Protocol of subject m19 on the MOD problem . 88 3 Mary can do a job ln 3 hours and Jane can do the job in 4 hours . If they work together , how long will it take to do the job ? Asti 1144 " Ta . , / t4 . 4 . 4 = . . ti g 1 - 01 = 5 - vx P YI - 1 v9 . 1 4 11 " 74 = 2 ' 0 . = Wm / 0 O ammo mew . . , 6114 ' Id kv4 - 1 . " ( . ( 4 d JT71 4 : ( ) ( A . . Figure 14 : Protocol of subject w23 on the WT problem . \ No newline at end of file diff --git a/task1-test.json b/task1-test.json new file mode 100644 index 0000000000000000000000000000000000000000..edaaf9a8886a9dc5c01e3fb66d401ca98f53d68a --- /dev/null +++ b/task1-test.json @@ -0,0 +1,668 @@ +[ + { + "id": "akamatsulab-3YkqDzf-k", + "claim": "\"Disruption of actin with Latrunculin A or Jasplakinolide permited tubule formation as shown by Alexa555-CTxB, whereas no membrane tubules were observed with pretreatment of 16uM Nocodozole prior to LatA or Jasplak treatment.\"", + "citekey": "day2015microtubule", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab--Oe1lYDk9", + "claim": "\"DNA-PAINT resolution enhancement by sequential imaging (RESI) of DNA origami resolved DNA bases 0.7 nm apart\"", + "citekey": "reinhardt2023angstromresolution", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-SL11GKMhj", + "claim": "\"Membrane Vacuole Like Dilations (VLDs) with 2um diameter and 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networks\"", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-jjW0_d3ak", + "claim": "\"Disruption of microtubule network with high dose (16uM) nocodozole eliminated CTxB+ tubules, whereas low dose (150nM) nocodozole had little effect\"", + "citekey": "day2015microtubule", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ebn-UtwZK", + "claim": "\"Optical tweezers revealed limited force propagation when only the membrane was pulled\"", + "citekey": "debelly2023cell", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-xY-GewW3E", + "claim": "\"Truncated GRAF1 missing GAP and SH3 domains (GRAF1-BAR-PH) displayed long tubules that persisted for >10minutes when incubated with Dil dye\"", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-P87cNqwZr", + "claim": "\"Epsin triple knockout mouse embryonic fibroblasts exhibited increased actin accumulation at sites of clathrin-mediated endocytosis as assayed by expressed utrophin calponin homology domain and TIRF microscopy\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-A1HdO-GrO", + "claim": "\"Membrane Vacuole Like Dilations (VLDs) were observed in MEFs seeded on PDMS upon return to iso-osmotic conditions after hypo-osmotically induced swelling, and showed no colocalization between pEYFP-Mem structures and actin.\"", + "citekey": "kosmalska2015physical", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-hGyVfnHYL", + "claim": "\"Epsin triple knockout mouse embryonic fibroblasts exhibited an increased number of shallow and U shape clathrin-coated vesicles than wild type based on electron microscopy\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-VLnm3aII7", + "claim": "\"mCherry-Arp3 reached peak accumulation at SecGFP-GPI endocytic sites at 6 seconds prior to scission in hAGS cells as shown by TIRF microscopy\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-COBrjEXGF", + "claim": "\"Myc-tagged GRAF1 colocalized with GFP-GPI in Caveolin1-knockout mouse embryonic fibroblasts based on immunofluorescence\"", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-Cmy4D8bhz", + "claim": "\"Dynamin pulled down with GST-GRAF1-SH3 domain based on Coomassie staining in a pulldown assay of rat brain cytosol with GST-GRAF1-SH3-bound beads\"", + "citekey": "doherty2011endocytic", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-i2NXezq0Q", + "claim": "\"TagRFPt-PICK1 reached peak intensity accumulation at CG endocytic sites at 12 seconds prior to scission (-12sec), dropping until just prior to scission and then increasing, in hAGS cells as shown by colocalization with SecGFP-GPI spots using TIRF microscopy\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-scjz_DMJY", + "claim": "\"Deletion of ICAP-1 led to recovery of traction forces in beta integrin KOs.\"", + "citekey": "kyumurkov2023force", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-EowpPsowf", + "claim": "\"Membrane reservoirs with 500nm diameter and ~1um height were observed in pEYFP-mem-transfected MEFs upon release of 6% stretch for 3min.\"", + "citekey": "kosmalska2015physical", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-MlLsP5aAR", + "claim": "\"Hip1R expression increased by ~75% in epsin triple knockout mouse embryonic fibroblasts\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-M3NOdLhAe", + "claim": "\"Alexa488-STxB and Alexa488-CTxB accumulated in tubular invaginations in ATP-depleted COS-7 cells, however, ATP-depletion inhibited uptake of Dextran.\"", + "citekey": "day2015microtubule", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-27gEPZJHs", + "claim": "\"\u00b5-track performed better that other tracking pipelines in datasets with high particle density with regards to precision and accuracy.\"", + "citekey": "roudot2023utrack3d", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ihaqh3iX6", + "claim": "\"Under 150 mOsm hypotonic conditions, CK-666 increased clathrin-RFP lifetime by 60% in SK-MEL-2 cells\"", + "citekey": "kaplan2022load", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-EIEiB15oW", + "claim": "\"Based on STORM imaging, actin marked by phalloidin was asymmetrically distributed around endocytic sites, with an average distance of 71 nm from the center of the clathrin coat\"", + "citekey": "jin2022branched", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-RuSMj5Tja", + "claim": "\"Using COS-7, monkey Kidney cells, pre- and post-labeling of microtubules exhibited increased spatial resolution compared to pre-labeling, in Ex-SMLM\"", + "citekey": "zwettler2020molecular", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-HRNgiqUaO", + "claim": "\"After treatment with 30uM Dyngo4a (a dynamin inhibitor) CLICs were identified using electron microscopy as the remaining CTxB-containing structures, contributing to 90% of the total CTxB-containing endocytic volume after 15sec of CTxB uptake, 86% after 1min, and 74% after 2min in Cav-/- MEFs as compared to cells untreated with Dyngo4a. \"", + "citekey": "howes2010clathrinindependent", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-BNMAVkp2E", + "claim": "\"Snx33-/- dHL60 cells display higher levels of actin than WT dHL60 cells upon fMLP chemoattractant addition as shown by phalloidin staining detetected by flow cytometry.\"", + "citekey": "sitarska2023sensing", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-6B_aybd1z", + "claim": "\"mEmerald-p21, an Arp2/3 complex subunit, colocalized with mCherry-IRSp53 at filopodia in fixed AGS cells as shown by TIRF microscopy.\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ca1lLfdLf", + "claim": "\"Using a worm-like chain model to fit the force dependence of actin filament bending, the persistence length of actin was determined to be 10.7 $$\\pm$$ 1 $$\\mu$$m.\"", + "citekey": "bibeau2023twist", + "dataset": "akamatsulab" + }, + { + "id": "megacoglab-9z_zo-lGV", + "claim": "\"social innovation concept proposals that cited inspirations with greater maximum conceptual distance from their problem domain were less likely to be shortlisted as creative by a panel of experts\"", + "citekey": "chanBestDesignIdeas2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xwrgZU20w", + "claim": "\"participants who received automatically labeled clusters of ideas as inspiration generated a higher quantity of ideas compared to receiving no external stimulation\"", + "citekey": "chanComparingDifferentSensemaking2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-HPVx3ziPZ", + "claim": "\"project ideas that cited a diverse set of inspiration sources were not more likely to be marked as creative by an expert panel\"", + "citekey": "chanImportanceIterationCreative2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-0DNrdxdn6", + "claim": "\"participants who saw examples in a list for an exploratory creativity task were more likely to do initial hill-climbing-like local exploration compared to participants who saw examples contextualized in the search space or in a dropdown\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-PrtoB_uNu", + "claim": "\"participants who received automatically labeled clusters of ideas as inspiration generated more diverse ideas compared to receiving no external stimulation\"", + "citekey": "chanComparingDifferentSensemaking2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-R8NAx8AqR", + "claim": "\"participants generated more alternative uses for everyday objects in a large room compared to participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-nZ8moeasL", + "claim": "\"skip-gram model with vanilla BOW contexts achieved 0.3 correlation with human judgments of verb similarities on SimLex999, about 50 percent worse than correlations with human judgments of nouns (0.5 correlation) or adjectives (0.6 correlation)\"", + "citekey": "schwartzSymmetricPatternsCoordinations2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xVGvCQlr4", + "claim": "\"social innovation concept proposals that cited inspirations with greater mean conceptual distance from their problem domain were less likely to be shortlisted as creative by a panel of experts\"", + "citekey": "chanBestDesignIdeas2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-fiT1U1BNY", + "claim": "\"the most novel alternative uses for everyday objects that were generated by undergraduate psychology students in a large room were marginally (but not statistically significantly) more novel than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-qK4L2OmZ8", + "claim": "\"constantly recommending examples that were semantically distant from current ideas --- regardless of cognitive state --- led to lower maximum novelty of ideas compared to receiving no stimuli\"", + "citekey": "chanSemanticallyFarInspirations2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-SgCPC8G1k", + "claim": "\"forced colocation increased the likelihood that previously non-colocated French labs would collaborate by 3.5 times, primarily amongst lab pairs that had previously semantically distant topics and cited references\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-XZ-GAXsTK", + "claim": "\"constantly recommending examples that were semantically distant from current ideas -- regardless of cognitive state -- led to longer time intervals between ideas compared to receiving no stimuli\"", + "citekey": "chanSemanticallyFarInspirations2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-8NLbZ1oEO", + "claim": "\"Getting pairs of scientists in a breakout room to discuss their work increased the likelihood they would collaborate on a grant by about 75%\"", + "citekey": "boudreauFieldExperimentSearch2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-ydBOxMKiQ", + "claim": "\"after being forcibly colocated, French lab pairs grew increasingly similar in topics and literature cited\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-t3LvtWlNM", + "claim": "\"Crowd workers generated the most practical ideas when given analogical inspirations found from domain-independent problem descriptions with concrete constraints, compared to abstract constraints\"", + "citekey": "yuDistributedAnalogicalIdea2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-fOKN4sbBt", + "claim": "\"word2vec model with skip-gram architecture and 300 dimensions trained on a subset of the Google News corpus for three days achieved 50 percent accuracy on semantic four-term analogy word problems, better than a set of NNLM and RNNLM models\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-31i8gG7Kn", + "claim": "\"alternative uses for everyday objects that were generated by participants in a large room were judged as less practical on average than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-ayJPkOD1i", + "claim": "\"the most novel alternative uses for everyday objects that were generated by participants in a large room were more novel than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-SInNihzph", + "claim": "\"scientist pairs who were randomly assigned to talk to each other (and did so, face-to-face) at an interdisciplinary medical imaging symposium used about 6 new MeSH keywords from their partners in future publications when the pair had moderate overlap in intellectual interests, compared to pairs who did not interact at the symposium\"", + "citekey": "laneEngineeringSerendipityWhen", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Oryy_ukuK", + "claim": "\"engineering students generated ideas with lower mean quality when they received analogical examples that were distant from their problem domain (vs. close to their domain)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-AX2AnkiDh", + "claim": "\"participants who saw examples contextualized in the search space for an exploratory creativity task were more likely to self-report using examples to model the problem compared to participants who saw examples in a list or in a dropdown\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-XwVHuT02v", + "claim": "\"word2vec model with CBOW architecture and 600 dimensions trained on a subset of the Google News corpus achieved 50 percent accuracy at completing semantic and syntactic four-term analogy word problems\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-lpcaTGd-U", + "claim": "\"word2vec model with skip-gram architecture and 640 dimensions trained on LDC corpora achieved 55 percent accuracy on semantic four-term analogy word problems, better than an NNLM model (23 percent accuracy), and RNNLM model (9 percent accuracy), trained on the same data\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-murOlQXGZ", + "claim": "\"positive effect of analogical paper recommendations (vs. keyword-based recommendations) on inducing creative adaptation ideas was statistically mediated by analogical recommendations' likelihood of being partial (vs. full) purpose matches\"", + "citekey": "kangAugmentingScientificCreativity2022", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-2Ey6CRR--", + "claim": "\"the occurrence of planning disagreements in an interdisciplinary science team was associated with subsequent increases in expressed uncertainty in their conversations\"", + "citekey": "paletzUncoveringUncertaintyDisagreement2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-tD_biL9dm", + "claim": "\"participants who saw diverse sets of examples had higher maximum task scores on an exploratory creativity task compared to participants who saw non-diverse sets of examples\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Wvp7Jsu6k", + "claim": "\"the occurrence of science-related disagreements in an interdisciplinary science team was associated with subsequent increases in expressed uncertainty in their conversations early -- but not late -- in their mission\"", + "citekey": "paletzUncoveringUncertaintyDisagreement2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-rrA3LVjvk", + "claim": "\"crowd workers were more likely to generate creative product ideas when given descriptions of types of experts recommended by other workers who were given schematized abstract problem descriptions, compared to workers who were given descriptions of types of experts recommended by workers who were given the original concrete problem description, experts recommended from an irrelevant problem, or allowed to search for their own inspirations\"", + "citekey": "yuEncouragingOutsideBox2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-M1fSi8xQv", + "claim": "\"participants who received automatically labeled clusters of ideas as inspiration generated lower quality ideas compared to receiving no external stimulation\"", + "citekey": "chanComparingDifferentSensemaking2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-HwTluMGQh", + "claim": "\"participants who saw examples in a list for an exploratory creativity task were more likely to self-report selecting individual examples as starting points, rather than using them to model the problem or ignoring the examples\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-bf1_85feE", + "claim": "\"undergraduates spontaneously transferred a solution from an analogous story they had just read about 70-100 percent of the time with a hint that the story was useful for their problem\"", + "citekey": "gickAnalogicalProblemSolving1980", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-31S7Tx1Y9", + "claim": "\"undergraduate mechanical engineering students generated ideas with the same level of variety when given examples --- either surface dissimilar in the form of biological examples, or surface similar to their problem --- compared to students who received no examples\"", + "citekey": "wilsonEffectsBiologicalExamples2010", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-kPgwfWLY2", + "claim": "\"project ideas that cited other projects that cited a diverse set of inspiration sources were more likely to be marked as creative by an expert panel\"", + "citekey": "chanImportanceIterationCreative2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-ofmkVMxwh", + "claim": "\"undergraduates spontaneously transferred a solution from an analogous story they had just read about 40 percent of the time without a hint that the story was useful for their problem; about the same amount transferred the solution after a hint\"", + "citekey": "gickAnalogicalProblemSolving1980", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-tqn-PBdXQ", + "claim": "\"after forcible separation, previously colocated labs' cited references became less similar over time\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-sMBayPMZl", + "claim": "\"undergraduate mechanical engineering students generated marginally more novel ideas when given examples that were surface dissimilar from their problem (in the form of biological examples) compared to students who received examples that were surface similar to their problem\"", + "citekey": "wilsonEffectsBiologicalExamples2010", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-eLMnvM_CG", + "claim": "\"analogical paper recommendations were judged as more novel by engineering PhD students compared to keyword-based paper recommendations\"", + "citekey": "kangAugmentingScientificCreativity2022", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-v5Gb8mkfK", + "claim": "\"scientist pairs who were randomly assigned to talk to each other (and did so, face-to-face) at an interdisciplinary medical imaging symposium and had pre-symposium moderate similarity in clinical fields were more likely to co-author new publications in the 5 years following the symposium, compared to pairs who did not interact with each other at the symposium\"", + "citekey": "laneEngineeringSerendipityWhen", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-TP1cdR2tl", + "claim": "\"undergraduate mechanical engineering students generated more novel ideas when given examples --- either surface dissimilar in the form of biological examples, or surface similar to their problem --- compared to students who received no examples\"", + "citekey": "wilsonEffectsBiologicalExamples2010", + "dataset": "megacoglab" + }, + { + "id": "megacoglab--JkuVF9as", + "claim": "\"constantly recommending examples that were semantically distant from current ideas --- regardless of cognitive state --- led to ideas that were on average less similar to their immediately preceding idea compared to receiving no stimuli\"", + "citekey": "chanSemanticallyFarInspirations2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-AyfbosEHF", + "claim": "\"skip-gram model with symmetric pattern contexts achieved 0.46 correlation with human judgments of verb similarities on SimLex999, comparable to correlations with human judgments of nouns (0.42 correlation) or adjectives (0.65 correlation) and outperforming skip-gram models with other contexts, such as dependency parses and BOW\"", + "citekey": "schwartzSymmetricPatternsCoordinations2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-XyiUTIRAT", + "claim": "\"chemists and chemical engineers in self-reported high-scatter fields were about 2x more likely to self-report spending more than 5 hrs a week on keeping up with their field, compared to chemists and chemical engineers in low-scatter fields\"", + "citekey": "packerImportanceSDICurrent1979", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-rc7fs4Cwg", + "claim": "\"for two out of four design problems, concepts generated by engineering graduate students who received stimulus verbs that were antonyms of the core functions of the problem generated ideas that were judged to be more useful, and cohesive than students who receive stimulus verbs that were synonyms of the core functions of the problem\"", + "citekey": "chiuInvestigatingEffectsOppositely2012", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-SUT_hq92I", + "claim": "\"Crowd workers generated the most original ideas when given analogical inspirations found from domain-independent problem descriptions with concrete constraints, compared to abstract constraints\"", + "citekey": "yuDistributedAnalogicalIdea2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-MDvKeTZZN", + "claim": "\"word2vec model with CBOW architecture trained on LDC corpora and 640 dimensions achieved 24 percent accuracy on semantic four-term analogy word problems, about the same as NNLM model (23 percent accuracy), and better than an RNNLM model (9 percent accuracy), trained on the same data\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Eas3Av6eX", + "claim": "\"crowd workers who were given a more abstract schematic description of a problem recommended more unique types of experts who might provide useful perspectives on the problem, compared to workers who were given the original concrete problem description\"", + "citekey": "yuEncouragingOutsideBox2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-VH37eM1Ba", + "claim": "\"engineering students generated ideas with the highest mean novelty when they received analogical examples that were both distant from their problem domain and unfamiliar to them (vs. all other types of examples or no examples)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-fD5LD5jZs", + "claim": "\"chemists and chemical engineers in self-reported high-scatter fields were less likely to self-report a high success-to-time-spent ratio in keeping up with their field, compared to chemists and chemical engineers in low-scatter fields\"", + "citekey": "packerImportanceSDICurrent1979", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xdDrUY5pK", + "claim": "\"engineering students generated ideas with higher mean novelty when they received analogical examples that were distant from their problem domain (vs. close to their domain)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-TjSBH1e8i", + "claim": "\"forced separation did not negatively affect collaboration between previously colocated French labs\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-WB32ukGUI", + "claim": "\"engineering PhD students were more likely to generate creative adaptation vs. direct application ideas for their research when given analogical vs. keyword-based paper recommendations\"", + "citekey": "kangAugmentingScientificCreativity2022", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-VcP5d-4Lp", + "claim": "\"chemists and chemical engineers in self-reported low-scatter fields who subscribed to selective dissemination of information (SDI) services were more likely to self-report low efficiency in keeping up with their field compared to non-subscribers \"", + "citekey": "packerImportanceSDICurrent1979", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-D8dmJsaRz", + "claim": "\"word2vec model with skip-gram architecture trained on LDC corpora and 640 dimensions achieved 59 percent accuracy on syntactic four-term analogy word problems, better than RNNLM and NNLM models trained on the same data (which achieved 36 and 53 percent accuracy)\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-04yh2Agp6", + "claim": "\"engineering students generated ideas with higher variability in quality when they received analogical examples that were distant from their problem domain (vs. close to their domain or no examples)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-tZtKHmnD7", + "claim": "\"alternative uses for everyday objects that were generated by undergraduate psychology students in a large room were judged as less practical on average than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-IMWGvZ2_G", + "claim": "\"crowd workers who were given a more abstract schematic description of a problem recommended experts who might provide useful perspectives on the problem that were more semantically distant from the problem, compared to workers who were given the original concrete problem description\"", + "citekey": "yuEncouragingOutsideBox2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Hn7gmJnZU", + "claim": "\"participants who saw examples in a list had lower maximum task scores on an exploratory creativity task compared to participants who saw examples contextualized in the search space or in a dropdown\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-4rDvdG2-8", + "claim": "\"for one out of four design problems, concepts generated by engineering graduate students who received stimulus verbs that were antonyms of the core functions of the problem generated ideas that were judged to be more novel than students who receive stimulus verbs that were synonyms of the core functions of the problem\"", + "citekey": "chiuInvestigatingEffectsOppositely2012", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xwEsF3MFC", + "claim": "\"engineering students' ideas were more likely to contain solution elements from analogical examples when examples were distant from their problem domain (vs. close to their domain)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-0Jkc9NJV2", + "claim": "\"word2vec model with CBOW architecture trained on LDC corpora and 640 dimensions achieved 64 percent accuracy on syntactic four-term analogy word problems, better than RNNLM and NNLM models trained on the same data (which achieved 36 and 53 percent accuracy)\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + } +] \ No newline at end of file diff --git a/task1-train-dev-2024-04-25-update.json b/task1-train-dev-2024-04-25-update.json new file mode 100644 index 0000000000000000000000000000000000000000..2b5bb8711bd8021db76844b7cbafe6a43d9906c5 --- /dev/null +++ b/task1-train-dev-2024-04-25-update.json @@ -0,0 +1,4473 @@ +[ + { + "id": "akamatsulab-1YXRXmgXU", + "claim": "Fluid uptake as measured by fluorescent 10kDa Dextran increased upon decreased membrane tension as a result of deadhering CHO cells during trypsinization.", + "citekey": "thottacherry2018mechanochemical", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-9E0jVhav_", + "claim": "Swip1 bound to actin via Swip1 EF1 domain as shown by GFP-pulldown assay in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B" + ] + }, + { + "id": "akamatsulab-QMQIJuzAI", + "claim": "Knocking down \u03b1v\u03b25 integrin expression abolished clathrin plaque assembly in HeLa cells", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C", + "FIG 2A" + ] + }, + { + "id": "akamatsulab-WJvOy9Exn", + "claim": "The density of free barbed ends increased as a function of growth stress", + "citekey": "li2022molecular", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-ZR6_FyY3a", + "claim": "Overexpressed CRIB domain of N-WASP localized to Cdc42-containing spots on the plasma membrane in FRaTB cells", + "citekey": "chadda2007cholesterolsensitive", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D", + "FIG 6E" + ] + }, + { + "id": "akamatsulab-cRFz4n0Pc", + "claim": "Coronin and cofilin-1 intensity peaked at about 4-5 seconds after ABP1-GFP maximal intensity at endocytic patches in budding yeast.", + "citekey": "lin2010overlapping", + "dataset": "akamatsulab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "akamatsulab-TBLuywz6G", + "claim": "Endogenous Swip1 localized to Rab21+ and B1-integrin-containing early endosomes.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C", + "FIG 2A" + ] + }, + { + "id": "akamatsulab-WQY-tljFm", + "claim": "A computational assessment of theoretical conformational changes upon binding of cofilin monomers to an actin filament suggest a minimum of 3 cofilactin interfaces, or two adjacent bound cofilin monomers, induces severing of an actin filament.", + "citekey": "tanaka2018structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 7" + ] + }, + { + "id": "akamatsulab-rPE_IW2JJ", + "claim": "Endothelial cells grown on stiffer cells had more actin stress fibers and were themselves stiffer than cells grown on more compliant substrates.", + "citekey": "byfield2009endothelial", + "dataset": "akamatsulab", + "findings": [ + "FIG 3A", + "FIG 3B", + "FIG 3C", + "FIG 3D", + "FIG 3E", + "FIG 3F", + "FIG 1A", + "FIG 1B", + "FIG 1C" + ] + }, + { + "id": "akamatsulab-GqpzNJuEZ", + "claim": "siRNA-A1 silenced Hip1R showed Arp3 and F-actin 'tails' in HeLa cells.", + "citekey": "engqvistgoldstein2004rnaimediated", + "dataset": "akamatsulab", + "findings": [ + "FIG 4C" + ] + }, + { + "id": "akamatsulab-A63ow10iT", + "claim": "Mechano-sensing clathrin plaques formed independent of actin contractility", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "akamatsulab-yQg2vbfkG", + "claim": "Cofilin peaked in concentration about 10 seconds after endocytic scission in mouse 3T3 fibroblasts.", + "citekey": "taylor2011high", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-2-bJaFjob", + "claim": "About 30 DNM2 molecules were required for forming the encytosis in SK-MEL-2 cells.", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 3G" + ] + }, + { + "id": "akamatsulab-QLP8zq7GU", + "claim": "Platinum-replica electron micrographs of unroofed B16F1 mouse melanoma cells showed actin networks in collar-like arrangements around clathrin coat necks and at small lateral patches at the periphery of clathrin coats", + "citekey": "collins2011structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "akamatsulab-u95Z_9Xq7", + "claim": "Total bending energy of actin filaments increased as a function of membrane tension in endocytic simulations", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 7H" + ] + }, + { + "id": "akamatsulab-kZckUXf1w", + "claim": "Treatment of H9 cells with CK-666 resulted in a~50% decrease in the formation of apical membrane\u00a0initiation sites (AMIS) en route to lumen formation", + "citekey": "taniguchi2015lumen", + "dataset": "akamatsulab", + "findings": [ + "FIG 4G" + ] + }, + { + "id": "akamatsulab-kimWcz_h4", + "claim": "Endocytic protein ABP1-GFP lifetime increased by 2-3x in budding yeast cells with cofilin mutants", + "citekey": "lin2010overlapping", + "dataset": "akamatsulab", + "findings": [ + "FIG 8" + ] + }, + { + "id": "akamatsulab-kayrJnALo", + "claim": "Endocytic adaptor protein End4p-mGFP accumulated more slowly and to a greater number of molecules in fission yeast cells with mutant cofilin allele", + "citekey": "chen2013actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-kIupe544T", + "claim": "The surface area overlap of actin over clathrin increased from ~5% to ~10% coverage from flat to curved pits in four cell types by platinum-replica electron microscopy", + "citekey": "yang2022actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 2N" + ] + }, + { + "id": "akamatsulab-rYe33dsbF", + "claim": "Knockdown of either \u03b1v or \u03b25 integrins in HeLa cells using specific siRNAs resulted in a complete loss of large and static CCSs", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 2F", + "FIG 2E", + "FIG 2D" + ] + }, + { + "id": "akamatsulab-QsJF6msql", + "claim": "Intensity over time traces of hiPSCs endogenously expressing AP2-RFP and Dynamin2-GFP peaked sequentially", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 2G" + ] + }, + { + "id": "akamatsulab-BX3Cqqj90", + "claim": "Purified swip1 bound human platelet actin with an affinity of 1.9 \u00b1 0.8 uM", + "citekey": "kwon2013swiprosin1", + "dataset": "akamatsulab", + "findings": [ + "FIG 3A" + ] + }, + { + "id": "akamatsulab-vmbAp09g2", + "claim": "MiTAB, a inhibitor of DNM2 PH domain, led to increased DNM2 recruitment in SK-MEL-2 cells", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S3F" + ] + }, + { + "id": "akamatsulab-Dd1n6KIBV", + "claim": "siRNA silenced Swip1 reduced GFP-Rab21+ (CG-associated) endosome trafficking in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6G" + ] + }, + { + "id": "akamatsulab-WbWLJVWcF", + "claim": "Knockout of __Dictyostelium__ WASP (__wasA-__) led to bipolar localization of WAVE rather than WT localization of WAVE to the leading edge.", + "citekey": "amato2019wasp", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A", + "FIG 1B" + ] + }, + { + "id": "akamatsulab-taeyp3oH-", + "claim": "Membrane dye (DiI dye) or Dextran uptake for 1 or 5 minutes in HeLa cells colocalize with endogenous GRAF1 staining.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B", + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-LlehpEErX", + "claim": "Immunofluorescence of folate-binding protein showed fluorescence in the periphery of KB cells", + "citekey": "numasawa2020fluorescent", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S1" + ] + }, + { + "id": "akamatsulab-wLCgd8nNI", + "claim": "Switching media from isotonic to hypotonic (75m0sm) media for 2-20 minutes increased the membrane tension from 33.0 \u00b1 7.4 pN to 48.0 \u00b1 17.1 pN, as measured by AFM.", + "citekey": "kaplan2022load", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A", + "FIG 2B" + ] + }, + { + "id": "akamatsulab-dFhjWUgD7", + "claim": "TIRF revealed that Cortactin, Hip1R, and Clathrin were simultaneously recruited to endocytic sites in 3T3 cell.", + "citekey": "leclainche2007hip1r", + "dataset": "akamatsulab", + "findings": [ + "FIG 4E" + ] + }, + { + "id": "akamatsulab-boQ6UJJMP", + "claim": "Decreased clathrin coat dynamics were measured under increased membrane tension using micropipette aspiration", + "citekey": "ferguson2017mechanoregulation", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "akamatsulab-5i2BACcCn", + "claim": "Pyrene-actin binding assays between human myo1e and actin plateaued at a dissociation rate of 440 \u00b1 9 s-1 for ATP concentrations > 2 \u00b5M", + "citekey": "mezgueldi2002kinetic", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A" + ] + }, + { + "id": "akamatsulab-3-az1CxcL", + "claim": "Cofilin seems to have different effects on actin filaments, such as severing and nucleation, based on its concentration.", + "citekey": "andrianantoandro2006mechanism", + "dataset": "akamatsulab", + "findings": [ + "FIG 5", + "SUPP FIG S5" + ] + }, + { + "id": "akamatsulab-chLz-7JK6", + "claim": "The rate constant of cofilin binding to actin filaments was measured to be k+\u00a0= 0.013 \u03bcM\u22121s\u22121", + "citekey": "andrianantoandro2006mechanism", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C" + ] + }, + { + "id": "akamatsulab-W8Zi3Y46u", + "claim": "Overexpression of GFP-GRAF1 in HeLa cells showed long (1-5um) tubules that turn over completely within 10 minutes based on fluorescence microscopy", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 1E" + ] + }, + { + "id": "akamatsulab-JJ6CSK6w3", + "claim": "Depleting actin polymerization with latrunculin A led to increased STxB-induced tubular membrane invaginations in HeLa cells", + "citekey": "romer2010actin", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S1A", + "SUPP FIG S1B" + ] + }, + { + "id": "akamatsulab-MIpYGNa-U", + "claim": "WAVE2 and IRSp53 both localized at ruffles and cell-cell junctions in A431 cells.", + "citekey": "suetsugu2006optimization", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-6wkebK9Rb", + "claim": "Depletion of \u03b21 or \u03b23 integrins by siRNA in HeLa cells showed no difference in the percentage of dynamic or static CCSs as compared to siCTRL cells.", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 4E", + "FIG 4F" + ] + }, + { + "id": "akamatsulab-HiUvvjFf0", + "claim": "More actin filaments accumulated at sites of simulated endocytosis as a function of membrane tension", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 7G" + ] + }, + { + "id": "akamatsulab-k90itT5sI", + "claim": "After washout of ATP-reducing agents, lifeact-GFP increased photobleaching recovery and preceded scission of the membrane tubule a few minutes later", + "citekey": "romer2010actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D", + "FIG 5E" + ] + }, + { + "id": "akamatsulab-gfaXg2-bh", + "claim": "Endogenous Swip1 associated with Rab21 in Rab21-containing early endosomes in MDA-MB-231 cells as shown by proximity ligation assay (PLA).", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "akamatsulab-yGIijdoa_", + "claim": "Endocytic track lengths of SKMEL-2 cells endogenously expressing Clathrin-RFP and Dynamin-GFP were distributed with peak value around 20-30 seconds", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "akamatsulab-3IOlxyzLb", + "claim": "Applying force to clathrin lattices on carbon-coated films caused a larger change in height when clathrin light chain was also present", + "citekey": "dannhauser2015effect", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D" + ] + }, + { + "id": "akamatsulab-5BnB27O-f", + "claim": "Endocytic adaptor protein Sla1-GFP accumulated more slowly to sites of endocytosis in a cof1-22 mutant in budding yeast", + "citekey": "okreglak2007cofilin", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-QigGJImJz", + "claim": "CortF, withtriple-mutant in tyrosine residues, increased fillopodia retraction frequency", + "citekey": "he2015src", + "dataset": "akamatsulab", + "findings": [ + "FIG 4D" + ] + }, + { + "id": "akamatsulab-NU1hryH_8", + "claim": "GRAF1-depletion by siRNA knockdown in HeLa cells has no effect on transferrin uptake", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 4D" + ] + }, + { + "id": "akamatsulab-nC0geNc63", + "claim": "Depleting Cdc42 with siRNA led to the buildup of GRAF1-GFP in tubular structures", + "citekey": "francis2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 3C" + ] + }, + { + "id": "akamatsulab-FxTUmxjh2", + "claim": "Simulated fission yeast endocytic actin networks produced ~3000 pN of force, while simulated budding yeast endocytic actin networks produced ~1000 pN of force", + "citekey": "nickaeen2022model", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "akamatsulab-txXrNK-wA", + "claim": "At fission yeast endocytic sites, scission protein (spHob1) first detected ~2 seconds before initiation of membrane invagination", + "citekey": "sun2019direct", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-45WDQVJkn", + "claim": "ABI2 formed a complex with GRAF1 based on immunoprecipitation of HeLa cell lysates, in a manner dependent on GRAF1 phosphorylation and treatment with mitochondrial damaging agent oligomycin-A and antimycin-A", + "citekey": "zhu2023graf1", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "akamatsulab-u3tkdx52k", + "claim": "Fluorescently labeled cortactin assembled coincident with Arp3 and Abp1 in NIH3T3 cells", + "citekey": "taylor2011high", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "akamatsulab-q0LJEXQsw", + "claim": "Resistance to internalization (spring constant) was proportional to membrane tension in continuum membrane mechanics endocytosis simulations", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-tZwLhk7sM", + "claim": "Cryo-electron tomograms of flat clathrin lattices in HeLa cells showed a wide variety of angles for clathrin vertices (120.0 \u00b1 13.3 degrees)", + "citekey": "sochacki2021structure", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D" + ] + }, + { + "id": "akamatsulab-cixbubehW", + "claim": "Swiprosin-1 inhibited cofilin-mediated actin depolymerization in vitro.", + "citekey": "huh2013swiprosin1", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D" + ] + }, + { + "id": "akamatsulab-kIHG6-Ila", + "claim": "IRSp53 coimmunoprecipitated with WAVE in NIH3T3 cell lysates.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-lNL2WkukA", + "claim": "Cryo-EM analysis of actin filaments showed that filaments bound to ADP-Pi were stiffer than filaments bound to ADP", + "citekey": "reynolds2022bending", + "dataset": "akamatsulab", + "findings": [ + "FIG 4F" + ] + }, + { + "id": "akamatsulab-1q7uo-zzw", + "claim": "Efficient lamellipodia actin filament severing requires a cofilin cluster longer than one-half of a helix", + "citekey": "ngo2015cofilininduced", + "dataset": "akamatsulab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "akamatsulab-p-Fy1XHf_", + "claim": "GRAF1-depleted HeLa cells (via siRNA treatment) demonstrate ~50% reduction in fluid phase uptake as compared to control cells, shown by FITC-dextran uptake.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 4B", + "FIG 4C" + ] + }, + { + "id": "akamatsulab-6yP4l1xIu", + "claim": "Swip1 preferentially bound WT-Rab21 and constitutively active Rab21 over the closely related Rab5 GTPase.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 1E" + ] + }, + { + "id": "akamatsulab-JvW-piCf8", + "claim": "Cortactin mutants lacking phosphorylatable Tyrosine site could not rescue the Knockdown of endogenous cortactin in Hela Cell", + "citekey": "zhu2007receptormediated", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A", + "FIG 2B" + ] + }, + { + "id": "akamatsulab-hmfv-Bz4r", + "claim": "Incubation of STxB with Arp2-depleted cells for 5 min at 37C led to the appearance of STxB-containing tubular structures in HeLa cells", + "citekey": "romer2010actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A", + "SUPP FIG S1" + ] + }, + { + "id": "akamatsulab-AP8l66U1L", + "claim": "Focal adhesion area decreased in A549 cells grown on softer PDMS substrates", + "citekey": "lee2022substrate", + "dataset": "akamatsulab", + "findings": [ + "FIG 5", + "TAB 1" + ] + }, + { + "id": "akamatsulab-Qo0PhBepV", + "claim": "NWASP was found around clusters of clathrin heavy chain by TIRF microscopy + super resolution microscopy", + "citekey": "leytonpuig2017flat", + "dataset": "akamatsulab", + "findings": [ + "FIG 1E" + ] + }, + { + "id": "akamatsulab-gW__kcqup", + "claim": "Membrane tether pulling on one part of a HeLa cell did not affect membrane tether force on another part of the cell", + "citekey": "shi2018cell", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-841E070Cm", + "claim": "Knockdown of DNM2 via siRNA showed a reduction of 488-Choleratoxin internalization in differentiated neuronal CAD cells", + "citekey": "mcfarland2008rna", + "dataset": "akamatsulab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "akamatsulab-IBn59ZWXY", + "claim": "Membrane mechanics simulations showed that 15 pN of force orthogonal to the plasma membrane base was sufficient to reshape a U shaped pit into a pinched shape", + "citekey": "hassinger2017design", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D" + ] + }, + { + "id": "akamatsulab-1-C-g0qCW", + "claim": "The curvature of the clathrin coat increased as a function of angle to the plasma membrane in SK-MEL2 cells fixed in plastic", + "citekey": "avinoam2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D" + ] + }, + { + "id": "akamatsulab-NbFDiQaUh", + "claim": "Swip1 colocalized with actin at lamellipodia of CHO-K1 cells transfected with GFP-Swip1.", + "citekey": "kwon2013swiprosin1", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "akamatsulab-TndUNP272", + "claim": "Measurements by super resolution microscopy showed a ~20 nm radial distance between Las17 (WASP) and MYO1 in budding yeast", + "citekey": "mund2018systematic", + "dataset": "akamatsulab", + "findings": [ + "FIG 2F" + ] + }, + { + "id": 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"FIG 5B" + ] + }, + { + "id": "akamatsulab-PECll5ZFw", + "claim": "B3 integrin lifetime increased by ~50% in the absense of B1 integrin and ICAP-1", + "citekey": "kyumurkov2023force", + "dataset": "akamatsulab", + "findings": [ + "FIG 3E" + ] + }, + { + "id": "akamatsulab-UlZY0Mnwt", + "claim": "hiPSCs at the colony edges showed large, paxillin-positive focal adhesions with thick actin stress fibers parallel to the colony edge.", + "citekey": "narva2017strong", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-iLicutBoX", + "claim": "Fluid uptake as measured by fluorescent 10kDa Dextran increased upon relaxation after mechanically induced stretch by vacuum-based equi-bi-axial stretching device in CHO cells.", + "citekey": "thottacherry2018mechanochemical", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "akamatsulab-0-2tkuu4l", + "claim": "Using the TNT T7-coupled reticulocyte lysate systems kit, translated WT-IRSp53 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"akamatsulab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "akamatsulab-_N-gQJ0eV", + "claim": "Expressing WT GFP-WASP in __wasA-__ mutants rescues confinement of active Rac to the leading edge of __Dictyostelium__ cells.", + "citekey": "amato2019wasp", + "dataset": "akamatsulab", + "findings": [ + "FIG 5F" + ] + }, + { + "id": "akamatsulab-nK9okhwH0", + "claim": "In H1299 cells, Knockdown of DNM2 with siRNA decreased the filopodial formation, and filopodia length", + "citekey": "yamada2016actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "akamatsulab-4UYkBZdjG", + "claim": "hiPSCs at the edge of the colony had larger focal adhesions than cells in the hiPSC colony center", + "citekey": "narva2017strong", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-nwh8sv1mV", + "claim": "Cells grown on curved quartz substrates coated with fibronectin or vitronectin showed preferential localization of integrin B5 to curved 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"lachowski2022substrate", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D", + "FIG 6E" + ] + }, + { + "id": "akamatsulab-Vmwk-ddxa", + "claim": "FRaTb-1 cells transfected with dominant negative dnm2-K44A had similar or slightly increased levels of folate receptor antibody internalization", + "citekey": "sabharanjak2002gpianchored", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-92Xr7KIW0", + "claim": "Treatment of SK-MEL2 cells with Jasplakinolide resulted in slower assembly and disassembly rates of dynamin2-GFP at sites of endocytosis", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5F", + "FIG 5G" + ] + }, + { + "id": "akamatsulab-L3a7k3HOw", + "claim": "The folate probe FolateRSense showed fluorescence patterns in internal spots and at the periphery of KB cells", + "citekey": "numasawa2020fluorescent", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S2" + ] + }, + { + "id": "akamatsulab-5QnBT-MFL", + "claim": "Endogenously RFP-tagged clathrin light chain A recovered from photobleaching with a half life of 2 seconds at sites of endocytosis in SKMEL-2 cells", + "citekey": "avinoam2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-c__rWg__T", + "claim": "IRSp53-/- MEFs or LG186-treated WT MEFs showed significant reduction in the number of CLICs per field, but no effect on clathrin or caveolae-derived structures.", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D" + ] + }, + { + "id": "akamatsulab-CIyKP3Kxe", + "claim": "Steady-state ATPase rate of human myo1e motor and IQ domain pleateaued at 1.2 \u00b1 0.3/s", + "citekey": "mezgueldi2002kinetic", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-oG3x2904t", + "claim": "CLIC/GEEC endosomes internalize GPI-anchored proteins.", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B", + "FIG 1A" + ] + }, + { + "id": "akamatsulab-Tz9iPR6Hy", + "claim": "GFP-cofilin associated with actin patches between 3 and 4.2 s after initial association of actin/Abp1p -", + "citekey": "okreglak2007cofilin", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-C8_EKRIsh", + "claim": "GFP-tagged ARPC3/Arc18 at C terminus did not affect __in vitro__ actin nucleation rates", + "citekey": "egile2005mechanism", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-Ed-cn4hbd", + "claim": "In Cav-/- MEFs, a dominant negative Dynamin mutant (DynK44A) showed an extensive tubular network of internalizing cholera toxin binding subunit (CTB), but inhibited trafficking of CTB to the Golgi.", + "citekey": "kirkham2005ultrastructural", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S4B", + "FIG 3A", + "SUPP FIG S4C" + ] + }, + { + "id": "akamatsulab-RniLbP0dj", + "claim": "Angle between membrane and clathrin pit increased from 0 to 90 (early phase/U-shaped) and 90 to 180(neck construction & scission) -", + "citekey": "avinoam2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-L1nUdRWr2", + "claim": "GST-WAVE1 in presence of IRSp53 and GST-WAVE2 VCA demonstrate high rates of Arp2/3-mediated actin polymerization in vitro.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 5C" + ] + }, + { + "id": "akamatsulab-ht_gHxaFG", + "claim": "The peak number of molecules of twinfilin (Twf1p) per endocytic patch in fission yeast was measured to be 210(+/- 40) by fluorescence microscopy", + "citekey": "sirotkin2010quantitative", + "dataset": "akamatsulab", + "findings": [ + "FIG 6F", + "FIG 6D" + ] + }, + { + "id": "akamatsulab-7VUX5lkA_", + "claim": "Fluorescence microscopy and image analysis of fluorescently tagged clathrin, dynamin, and arp2/3 complex in hiPSCs showed ~30% of CME events completing in the absence of detectable actin assembly", + "citekey": "jin2022branched", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S4B" + ] + }, + { + "id": "akamatsulab-ctop9ejQ4", + "claim": "Endocytic proteins Clathrin light chain A-RFP and dynamin2-GFP increased in lifetimes as a function of concentration of Arp2/3 complex inhibitor CK666 in SK-MEL-2 cells", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "akamatsulab-HAM7DEdlD", + "claim": "The GRAF1 inhibitor LG186 showed significantly reduced fluid phase uptake in WT MEFs that was comparable to the fluid phase uptake in IRSp53-/- MEFS.", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 3C" + ] + }, + { + "id": "akamatsulab-yspWhgcnu", + "claim": "Association rate constant of cofilin to actin by presteady state kinetics was 0.008/uM/s", + "citekey": "cao2006energetics", + "dataset": "akamatsulab", + "findings": [ + "FIG 3", + "TAB 2" + ] + }, + { + "id": "akamatsulab-gFbw239AO", + "claim": "Mouse embryonic fibroblasts (MEFs) from IRSp53-/- mice showed a significantly reduced fluid phase uptake as compared to WT-addback MEFs without affecting transferrin receptor internalization .", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "akamatsulab-rS-hEJjYE", + "claim": "Purified Arp2/3 complex activation by GST-WAVE2 and GST-WAVE2 VCA was stimulated in the presence of purified IRSp53 using pyrene-labelled and free monomeric actin in an actin polymerization kinetics study.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "akamatsulab-ZWw9A8p4C", + "claim": "Between hPSC colonies plated on glass/polystyrene and on softer VTN-funtionalized hydrogels, no significant change in hPSC colony organizeation and no or modest change in FA number was observed.", + "citekey": "narva2017strong", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S2D", + "FIG 2", + "SUPP FIG S2" + ] + }, + { + "id": "akamatsulab-ee1ItmKqR", + "claim": "The minimum number of yeast cofilin molecules required for severing is 23 +/- 11 molecules.", + "citekey": "gressin2015architecture", + "dataset": "akamatsulab", + "findings": [ + "FIG 7", + "FIG 3" + ] + }, + { + "id": "akamatsulab-9CPBwF3cc", + "claim": "The persistence length of non-stabilized actin in physiological ionic conditions was found to be $$9 \\pm 0.5 \\mu m$$", + "citekey": "isambert1995flexibility", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-nDfNWJdL-", + "claim": "Co-sedimentation assay of purified Swip1/EFhd2 and actin showed actin binds to EFhd2 in vitro.", + "citekey": "park2016structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "BIOL403-ZsSZ8qIYT", + "claim": "Mean AP-2 fluorescence lifetime increased for VSV particles compared to DI-T particles and for other pits.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3B", + "FIG 3C" + ] + }, + { + "id": "BIOL403-2oMaUVZb3", + "claim": "Camostat, a TMPRSS2 inhibitor, had a significantly greater % inhibition for Delta pseudotyped virus (PV) than for Omicron PV when infecting A549 cells overexpressing ACE2 and TMPRSS2.", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3F" + ] + }, + { + "id": "BIOL403-65HQLSARh", + "claim": "Both the mean LCa and Clathrin fluorescence lifetime increased for VSV particles compared to pit and DI-T.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3D", + "FIG 3E" + ] + }, + { + "id": "BIOL403-kibez2rYZ", + "claim": "Inhibiting polymerization of actin with latB reduced the internalization efficiency of VSV but not DI-T.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "BIOL403-LfApmzbHq", + "claim": "AP-2 fluorescence intensity increased after DI-T attachment to the BSC1 cell as the clathrin-coated pit assembled", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "BIOL403-2HHW-wfqd", + "claim": "Electron microscopy showed that with increasing exposure to heat (37C), the number of HCoV-229E viruses localized with or near the orifices (neck) of caveolae increased, where \"cross-bridge structures of high electron density were frequently observed between virus particles and the plasma membrane.\"", + "citekey": "nomura2004human", + "dataset": "BIOL403", + "findings": [ + "FIG 4A", + "FIG 4B" + ] + }, + { + "id": "BIOL403-yLpL38PuF", + "claim": "The geometries and clathrin coverage of endocytic carries for internalization of DI-T and VSV particles were different when visualized using electron microscopy", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3A", + "FIG 3B" + ] + }, + { + "id": "BIOL403-qrc8wp2qc", + "claim": "DI-T particles did not alter the process of clathrin-coated vesicle formation.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3A", + "FIG 3B", + "FIG 3C", + "FIG 3D" + ] + }, + { + "id": "BIOL403-on60mfeqE", + "claim": "\"Binding of HCoV-229E and incubation at 37\u00b0C caused clustering of CD13 and its colocalization with caveolin-1.\"", + "citekey": "nomura2004human", + "dataset": "BIOL403", + "findings": [ + "FIG 3" + ] + }, + { + "id": "BIOL403-W6_BwdDw8", + "claim": "AP-2 fluorescence in VSV particles is 2x higher and ~2x longer-lived than that of DI-T particles, which are comparable to non-viral endocytic pits; after AP-2 fluorescence peaks at 45s for VSV particles, it drops to 0 a.u. by 60s.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "BIOL403-LgTH0yTQO", + "claim": "Pre-treatment of cells with the clathrin inhibitor Pitstop or Dynamin inhibitor MiTMAB reduced the number of internalized HCoV-NL63 particles in LLC-Mk2 cells", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D" + ] + }, + { + "id": "BIOL403-NwFQlJbzD", + "claim": "A strong inhibition of virus internalization in HAE cultures preincubated with clathrin or dynamin inhibitors (Pitstop and MitMAB) compared to DMSO-treated control cells was observed, using confocal microscopy.", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 7" + ] + }, + { + "id": "BIOL403-UAYD3rA57", + "claim": "DI-T particles internalized in BSC1 cells through canonical, circular clathrin coated pits, while VSV particles internalized into Vero cells in non-canonical, oblong pits as shown by electron microscopy", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 4A", + "FIG 4B" + ] + }, + { + "id": "BIOL403-oH_7_-Ju7", + "claim": "Compared to the control with DMSO, there was significant inhibition of HCoV-NL63 infection in LLC-Mk2 cells", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 1A", + "FIG 1B", + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "BIOL403-fZjAAn6XP", + "claim": "Electron microscopy showed a complete clathrin coat around DI-T particles, whereas VSV endocytic carriers seemed to be incompletely coated with clathrin.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "BIOL403-fsXgzlA-a", + "claim": "Treatment with pitstop or MiTMAB reduced the number of infected LLC-Mk2 cells following incubation with the HCoV-NL63 virus.", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 9" + ] + }, + { + "id": "BIOL403-uns4_1odd", + "claim": "During coated pit formation of DI-T, there was minimal cortactin recruitment (fluorescence), which peaked before full clathrin assembly", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "BIOL403-DRplh7Vza", + "claim": "Cortactin intensity spiked at late timepoints during VSV internalization.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5C" + ] + }, + { + "id": "BIOL403-bPmO00UbS", + "claim": "Di-T and VSV spots colocalized with AP-2 and LCa over similar periods of time", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3B", + "FIG 3C", + "FIG 3D", + "FIG 3E" + ] + }, + { + "id": "BIOL403-vKrivrfAQ", + "claim": "inhibition of actin polymerization drastically increases the the time of VSV internalization but not DI-T, as seen with increased time of AP-2 fluorescence.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 6B", + "FIG 6D" + ] + }, + { + "id": "BIOL403-QK-IPLtB0", + "claim": "When human fibroblasts were treated with anti-CD13 antibody and anti-caveolin-1 antibody for 60 minutes at 37C, an increase in fluorescence of CD13 and colocalization of caveolin-1 was observed.", + "citekey": "nomura2004human", + "dataset": "BIOL403", + "findings": [ + "FIG 2" + ] + }, + { + "id": "BIOL403-o-DGibStS", + "claim": "LLC-MK2 cells showed no significant differences in replication of HCoV-NL63 under a controlled environment versus a camostat environment", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 11A", + "FIG 11B" + ] + }, + { + "id": "BIOL403-1bD48wYTX", + "claim": "The creation of clathrin-coated pits in BSC1 cells showed minimal cortactin recruitment based on time lapse fluorescence assays", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "BIOL403-8jYihJXs1", + "claim": "Cortactin intesity spikes during VSV internalization", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5B", + "FIG 5C", + "FIG 5D" + ] + }, + { + "id": "BIOL403-C0vEc-VbK", + "claim": "Depletion of TMPRSS2 afftected the entry of WT and Delta PV to a greater extent than that of the Omicron PV, in a manner dependent on ACE2 expression levels.", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3C", + "FIG 3D" + ] + }, + { + "id": "BIOL403-JEZVJFX83", + "claim": "E64d, a cathepsin inhibitor, had a significantly greater % inhibition for Omicron pseudotyped virus (PV) than for Delta PV.", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3F" + ] + }, + { + "id": "BIOL403-kPjXx4aUP", + "claim": "VSV and DI-T particles are different lengths", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "BIOL403-8-zvLzqhz", + "claim": "There was a decrease in internalized viral particles when treated with classical clathrin-mediated endocytosis inhibitors Pitstop 2 and MiTMAB.", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D" + ] + }, + { + "id": "BIOL403-jUOkXBg6J", + "claim": "Using A549-ACE2/TMPRSS2 cells a noticeable, but not statistically significant, difference between live viral forms of omicron and delta, when treated with either TPRSS2 or cathepsin inhibitors", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3G" + ] + }, + { + "id": "dg-social-media-polarization-NAv6HKLye", + "claim": "Respondents were not very polarized in function of their party alignment in the US, with fewer than one in five scoring high on the polarization scale.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization--eEnETf9u", + "claim": "Heavy Facebook users were more likely to consume information from untrustworthy websites, which was often immediately preceded by a visit to Facebook.", + "citekey": "guessExposureUntrustworthyWebsites2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-dOZ6zAzVl", + "claim": "SEM modeling of social media use showed an indirect yet positive effect on polarization for moderate liberals via increased polarization", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-wg-tL_YH6", + "claim": "According to a conditional latent growth curve model (LGM), American adults' online political expression increased support for their candidate.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-PKXjaKH1E", + "claim": "Strong pro-refugee sentiment amongst Germans was associated with ~2x higher rates of higher education compared to strong anti-refugee sentiment", + "citekey": "arltBiasWantedExamining2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4" + ] + }, + { + "id": "dg-social-media-polarization-RV0XA6D3B", + "claim": "The majority of Germans fall between extreme positions regarding refugees, with only a small minority expressing extreme pro-refugee or anti-refugee attitudes", + "citekey": "arltBiasWantedExamining2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-LP0AEgKvL", + "claim": "Polarized users in likewise polarized neighborhoods distributed their likes across their community pages in a similar way, both in science and conspiracy communities.", + "citekey": "brugnoliRecursivePatternsOnline2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-1OX4Ja3Bs", + "claim": "The interaction between dual national identity, identifying as both Hong Kongese and Chinese, and political use of social media was negatively associated with attitudinal polarization on position issues.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-GJX02PUU3", + "claim": "Self-reporting a dual nationality identity, identifying as both Hong Kongese and Chinese, was negatively associated with attitudinal polarization on valence issues in Hong Kong.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-rwVKYrlHN", + "claim": "Substantial selective exposure was heavily concentrated among a subset of Americans with conservative information diets.", + "citekey": "guessExposureUntrustworthyWebsites2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1", + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-BGpeJrpUb", + "claim": "According to a repeated-measures ANOVA, and latent growth curve modeling (LGM, unconditional model), individuals' candidate preferences grew more stable as Election Day approached.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-w5xrqKLI_", + "claim": "According to analyses of Facebook data, people's egonets misrepresented how many friends other people have.", + "citekey": "feldEgonetsSystematicallyBiased2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-SVzr-eboO", + "claim": "Authors on the liberal sites were much more favorable toward left-leaning people and groups than authors on the conservative sites. In addition, those on the left- and right-leaning sites were more extreme than those at the moderate blog on this variable", + "citekey": "suhayForgingBondsBurning2015", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-CfQF8Cm-z", + "claim": "Fifty-five percent of issue-related conversations involved interactions between users from both sides of union debate.", + "citekey": "balcellsCrossingLinesTwitter2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-NwBCcwVBq", + "claim": "Extreme users strongly prioritized cognitive congruence in news sharing, in both pro- and anti-Bolsonaro communities. Ideological congruence become less important towards the network center.", + "citekey": "arugueteNewsSharingGatekeeping2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 7" + ] + }, + { + "id": "dg-social-media-polarization-TMN0eQB8C", + "claim": "For respondents in Hong Kong with a single identity, either only Hong Kongese or only Chinese, model estimates of affective polarization increased from 30\u00b0 to 40\u00b0 with frequent social media use, while for those with a dual identity, both Hong Kongese and Chinese, it decreased from 30\u00b0 to 12\u00b0 with frequent social media use.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-NTikknFZ7", + "claim": "Both Republican and Democrat users reported change in self-reported political ideology towards their own ideological extreme when exposed to opposing views on Twitter, yet only Republicans' effects were statistically significant", + "citekey": "bailExposureOpposingViews2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-gyOhM4gnR", + "claim": "Confidence in the president and Congress was positively associated with levels of selective exposure to social media in the US.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-G6Dt9EUlZ", + "claim": "AccoA Random Forest algorithm was able to predict the polarization of Twitter users with high accuracy and recall.", + "citekey": "marozzoAnalyzingPolarizationSocial2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 7" + ] + }, + { + "id": "dg-social-media-polarization-i61FjD7N_", + "claim": "Deactivating facebook reduced various measures of political polarization, particularly exposure to news that improve understanding of their own political party (vs. other party), and issue polarization, but not affective polarization.", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-r-1ymhoVX", + "claim": "Enthusiasm among US respondents toward the out-party presidential candidate was positively associated with disagreement expression on social media.", + "citekey": "choiEnthusiasmOtherSide2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-VlBq9h_85", + "claim": "According to SEM modeling, social media use was negatively associated with a shift towards conservatism for political neutrals, and positively associated with a shift towards liberalism", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-FnfG3r2bC", + "claim": "Facebook users who reported higher differences between feeling-thermometer measures for their own party vs. the other party registered more Facebook use which lead to over-time depolarization", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-ttY5ZRIo8", + "claim": "The fragmentation of the audiences of Russian media outlets on the Vkontakte platform occurred mainly along political lines", + "citekey": "urmanNewsConsumptionRussian2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-GMnFJ3he4", + "claim": "SEM modeling showed that, for moderate conservatives, there was no direct or indirect effect of social media use for adopting extreme conservatism", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-Wtxp_oJDN", + "claim": "Most of the users who retweeted rumor rebuttal tweets displayed the agree attitude. However, the users who commented on rumor rebuttal tweets had more diversified attitudes under various topics, with the majority of individuals holding query or unknown attitudes", + "citekey": "wangEchoChamberEffect2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3", + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-Zo4cVaD34", + "claim": "Longer lifetime and higher levels of activity of Facebook users corresponded with a lesser number of Facebook pages being consumed.", + "citekey": "schmidtPolarizationVaccinationDebate2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-sV9j_ssrE", + "claim": "All of the derogatory words used to talk about Remoaners on Twitter contain negative tribal associations.", + "citekey": "northBattleBritainAnalyzing2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "dg-social-media-polarization-AQy-nbEwf", + "claim": "27 out of 38 events of the Brexit timelines were associated with unusual spikes in the volume of tribal keywords outside of the normal range.", + "citekey": "northBattleBritainAnalyzing2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-85I3FMzkf", + "claim": "Smaller Japanese media outlets Twitter accounts had ideologically distinctive followers and were mainly isolated in the network.", + "citekey": "kobayashiNewsAudienceFragmentation2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-TEas5VTq3", + "claim": "More reputable outlets had greater incentives to adjust editorial lines. Less reputable outlets were already sending messages congruent to their preferred user.", + "citekey": "arugueteNewsSharingGatekeeping2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-YnZuD0iG2", + "claim": "Republican users exposed to opposing views on Twitter had an increase in conservatism between 0.11 and 0.59 standard deviations", + "citekey": "bailExposureOpposingViews2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-5xJkG_dQv", + "claim": "The majority of Facebook users were active either in the pro-vaccines or anti-vaccines community, not both.", + "citekey": "schmidtPolarizationVaccinationDebate2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-1JvBH0nCz", + "claim": "Only at the first wave of data, Facebook users who reported higher differences between feeling-thermometer measures for their own party vs. the other party were more likely to view news on Facebook", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-DL7lKY_N6", + "claim": "Exposure to counter-attitudinal content on Facebook modestly decreased the affective polarization index compared to the pro-attitudinal treatment", + "citekey": "levySocialMediaNews2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-sffZqWHW4", + "claim": "Dual identifying Hong Kong residents were 14.8% more likely to have a weak partisan selectivity than single identifiers.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-PjQJXsOzF", + "claim": "Anger and anxiety among US respondents toward the out-party presidential candidate wass not associated with disagreement expression on social media.", + "citekey": "choiEnthusiasmOtherSide2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-tQy4sEF_R", + "claim": "Perceived polarization increased as a function of time spent reading a tweet, but only for Republican users", + "citekey": "banksPolarizedFeedsThreeExperiments2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 6", + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-2L5_w1IYK", + "claim": "Deactivating Facebook boosted participants' perception of its role in improving news consumption and increased agreement on the potential impact of deactivating one's account.", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-aJngFbteo", + "claim": "deactivating Facebook resulted in improvements on measurements of subjective well-being", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 5" + ] + }, + { + "id": "dg-social-media-polarization-5OWXeEZWn", + "claim": "Selective exposure and selective avoidance predict polarization, but they are weaker indicators than strength of party ties.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-eIcyC6cqo", + "claim": "The level of encountered disagreement on social medi predicted the likelihood of expressing disagreement for US respondents, even when accounted for the initial level of expressed disagreement.", + "citekey": "choiEnthusiasmOtherSide2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-qyujJigSk", + "claim": "The retweeting and commenting user networks of rumor rebuttal under different topics showed highly modular structures; however, the large clusters in retweeting user networks showed high homophily, while the large clusters in commenting user networks had mixed attitudes", + "citekey": "wangEchoChamberEffect2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-PSA7511kK", + "claim": "According the a polarization index, Italian news sites are strongly polarizated on the issue of the Italian constitutional referendum.", + "citekey": "marozzoAnalyzingPolarizationSocial2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-7joN8S4Cb", + "claim": "Pro-attitudinal news uses were not found to mediate the relationship between Facebook use at wave 1 and higher differences between feeling-thermometer measures for users' own party vs. the other party at wave 3", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-DH7kbqJd1", + "claim": "According to regression analysis, heterogeneous conversations were longer when they involved users who are placed on the more moderate and central positions of the debate, and were probably more disposed to hear the other side.", + "citekey": "balcellsCrossingLinesTwitter2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4" + ] + }, + { + "id": "dg-social-media-polarization-V9kqzF5Vc", + "claim": "The variance of response about EU issues between those who got most of the news from social networks or other types of media was very small for participants who had positive predispositions toward the EU.", + "citekey": "nguyenTestingPopularNews2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-4pRj56pL2", + "claim": "Regression analyses of American adults showed that the relationship between political party and political opinions increased as people expressed themselves politically.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-fjmFnfZxi", + "claim": "Germans across the ideological spectrum perceived the media to have biases favoring their opposing ideological group on the topic of refugees", + "citekey": "arltBiasWantedExamining2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4" + ] + }, + { + "id": "dg-social-media-polarization-l-GuCGg0p", + "claim": "According to regression analysis, heterogeneity increased the level of depth of conversations.", + "citekey": "balcellsCrossingLinesTwitter2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4", + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-KwHHkPC99", + "claim": "SEM modeling showed that, among neutrals, social media use was positively associated with political engagement, which was also positively associated with political polarization toward liberalism.", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-tad5MxAmU", + "claim": "According to a survey, educated Ethiopians believed that social media helps and hurts democracy in Ethiopia.", + "citekey": "worknehSocialMediaProtest2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 5" + ] + }, + { + "id": "dg-social-media-polarization-F6qxFkTUx", + "claim": "Facebook news use at wave 1 resulted in depolarization at wave 3 via exposure to counter-attitudinal news", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-00d1hdyKn", + "claim": "According to regression analyses, individuals on WhatsApp and Twitter became more embedded in homophilic interaction networks than Facebook users.", + "citekey": "yarchiPoliticalPolarizationDigital2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-CYdetKaa4", + "claim": "Deactivating Facebook resulted in significant declines in both self-reported news attention and objectively measured news knowledge.", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-EEv4t_Zmw", + "claim": "Engaging in polarizing content increased the probability to have friends with similar characteristics.", + "citekey": "brugnoliRecursivePatternsOnline2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 5" + ] + }, + { + "id": "dg-social-media-polarization-D6WhqzXF4", + "claim": "The out-party feed increased affective polarization compared to the control group, but the difference was not statistically significant", + "citekey": "shmargadSortingNewsHow2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-fjnZM77gZ", + "claim": "The in-party feed slightly decreased affective polarization compared to the control group, but this difference was not statistically significant", + "citekey": "shmargadSortingNewsHow2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-J8B-ui82c", + "claim": "Most Japanese media accounts were followed on Twitter by both liberal and conservative users, and the followers of major media accounts overlapped to a significant extent.", + "citekey": "kobayashiNewsAudienceFragmentation2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-Oij-q4JsF", + "claim": "Republicans were more likely than Democrats to share extreme *pro*-Republican news sources and less likely to share extreme *pro*-Democratic news sources, and vice versa.", + "citekey": "osmundsenPartisanPolarizationPrimary2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-Q2jgcTrzF", + "claim": "The effect of being offered subscriptions to news outlets from the opposite ideological position on a political opinions index was small and not statistically significant.", + "citekey": "levySocialMediaNews2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-FhiobBveu", + "claim": "SEM modeling showed that social media use by liberals was positively associated with political engagement, and in turn, was positively associated with political polarization toward strong liberalism", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-rW9j9BLFE", + "claim": "Estimating the conditional effects of the data for American adults showed that party identification predicted political opinions at any value of political expression, but the relationship was stronger when political expression was high.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-Dnlq8A7OQ", + "claim": "The cognitive reflection test was weakly associated with sharing real news sources.", + "citekey": "osmundsenPartisanPolarizationPrimary2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-pz9Ofmmt_", + "claim": "The majority (80-90%)of networks of tweets about the Italian referendum shared links had the same model-estimated stance towards the referendum as the users who shared them. Retweet and quote networks had almost 100% of within-stance.", + "citekey": "laiStancePolarityPolitical2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 10" + ] + }, + { + "id": "dg-social-media-polarization-OzPq5b9Bu", + "claim": "Derogatory Brextremist discourse on Twitter included many terms that suggested lack of sanity (e.g., nutters, loonies) and references to fear and hatred of foreigners and destruction of Britain.", + "citekey": "northBattleBritainAnalyzing2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "dg-social-media-polarization-rw_F8t-BK", + "claim": "The variance of response about EU issues between those who got most of the news from social networks or other types of media was very small for participants who had negative predispositions toward the EU.", + "citekey": "nguyenTestingPopularNews2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-wF7z2zjF7", + "claim": "Party identification predicts political opinions at any value of political expression but the relationship is stronger when political expression is high.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-tsKBuSu4g", + "claim": "US respondents who self-reported selectively using social networking sites with political content that aligned with their own views were more likely to have a stronger difference in favorability ratings between Republican vs. Democratic parties.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-URbbXnWk0", + "claim": "According to regression analysis, social network heterogeneity positively influenced ethnic, ideological, and party polarization.", + "citekey": "kibetSociallyNetworkedHeterogeneity2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-b1nT-0nmZ", + "claim": "As shown by comparing percentages between Facebook, Twitter, and WhatsApp users, homophily was higher on Twitter and WhatsApp than Facebook.", + "citekey": "yarchiPoliticalPolarizationDigital2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-fZx8-Rfca", + "claim": "US survey respondents self-reported greater ideological distance between Donald Trump and Hillary Clinton after seeing negative campaign tweets from either candidate, compared to seeing no tweets", + "citekey": "banksPolarizedFeedsThreeExperiments2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3", + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-xkB9Y4f6Z", + "claim": "Users polarized towards a narrative tended to consume nearly exclusively content adhering to their system of beliefs.", + "citekey": "brugnoliRecursivePatternsOnline2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-nHQRpPGyv", + "claim": "Multiple mediation analyses showed that posting news in WhatsApp groups was associated with exposure to diverse perspectives.", + "citekey": "kibetSociallyNetworkedHeterogeneity2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-F0emNfngi", + "claim": "There were no striking divisions between users exposing different stances in the reply-to Twitter network of the Italian referendum.", + "citekey": "laiStancePolarityPolitical2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-LSw8SXuEz", + "claim": "According to Poisson regression, women were underrepresented among top Instagram degrees, indicative of a glass ceiling effect (p.3).", + "citekey": "stoicaAlgorithmicGlassCeiling2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-zwlHilzun", + "claim": "Students provided expert instruction(A teacher with a P.H.D in physics) performed as well as groups given the same instruction by a different teacher and provided analogical instruction", + "citekey": "jacobsonSchemaAbstractionProductive2020", + "dataset": "megacoglab", + "findings": [ + "TAB 8" + ] + }, + { + "id": "megacoglab-xMEXh8Glg", + "claim": "papers with high median conventionality and high tail atypical combinations of journals they cited were 2x more likely than average to be in top 5 percent of citation distribution", + "citekey": "uzziAtypicalCombinationsScientific2013", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-LJtUs-1OU", + "claim": "statistically significant but small differences in bibliometric measures of consensus across papers in physical, hard and soft biological, and social sciences", + "citekey": "fanelliBibliometricEvidenceHierarchy2013", + "dataset": "megacoglab", + "findings": [ + "FIG 4", + "TAB 2" + ] + }, + { + "id": "megacoglab-3jTc6a_9h", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventors who was new to the patent's field tended to receive fewer forward citations, but less so when they collaborated with an expert in the new field", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 7" + ] + }, + { + "id": "megacoglab-g8wge1Vez", + "claim": "Final advertisements from designers who prototyped designs in parallel performed better on advertising analytics compared to ads from designers who prototype designs serially", + "citekey": "dowParallelPrototypingLeads2010", + "dataset": "megacoglab", + "findings": [ + "FIG 7", + "TAB 1" + ] + }, + { + "id": "megacoglab-uwJCgdA2o", + "claim": "undergraduates used more complex integration approaches (vs. property transfer and hybridization approaches) when generating definitions for noun phrases from dissimilar vs. similar noun pairs", + "citekey": "wilkenfeldSimilarityEmergenceConceptual2001", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-AZ-LvsLz_", + "claim": "students scored higher on biology post-test learning quizzes when given instruction with complex instructional analogies compared to no analogies", + "citekey": "bakerComplexInstructionalAnalogies2001", + "dataset": "megacoglab", + "findings": [ + "TAB 1", + "TAB 2" + ] + }, + { + "id": "megacoglab-BV-n51_DU", + "claim": "There was an average of 33% improvement in forecasting accuracy (3 out of 4 debate questions answered more accurately.)", + "citekey": "irwinForecastingArgumentationFrameworks2022", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-inEj6W9YH", + "claim": "engineering students used more between-domain keywords used to search for patents in interactive visualization with VISION (functional component representations of patents compared to a standard Google Patents interface)", + "citekey": "songDesignbyAnalogyEffectsExplorationBased2022", + "dataset": "megacoglab", + "findings": [ + "FIG 12" + ] + }, + { + "id": "megacoglab-TAgkEfWtq", + "claim": "Students given analogical instruction with two models performed better on far domain problems than students provided only a single model to study", + "citekey": "jacobsonSchemaAbstractionProductive2020", + "dataset": "megacoglab", + "findings": [ + "TAB 7", + "FIG 3" + ] + }, + { + "id": "megacoglab-TkvbwvSxI", + "claim": "users were more likely to select clusters when an ACT-IF model's prediction of cluster profitability was equal to the expected rate of information gain", + "citekey": "pirolliInformationForaging1999", + "dataset": "megacoglab", + "findings": [ + "FIG 18" + ] + }, + { + "id": "megacoglab-rjVivNG51", + "claim": "a substantial number of IDEO-designed products incorporated technological solutions from other industries", + "citekey": "hargadonTechnologyBrokeringInnovation1997", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-6ddZeIyjB", + "claim": "Subjective ratings of information scent model derived from the task environment with ACT-IF tightly correlated with users' judgments of information relevance on a Scatter-Gather task", + "citekey": "pirolliInformationForaging1999", + "dataset": "megacoglab", + "findings": [ + "FIG 16" + ] + }, + { + "id": "megacoglab-e5Os8ZONI", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventors who were new to the patent's field tended to be more novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 7" + ] + }, + { + "id": "megacoglab-r9OyxsAJe", + "claim": "patenting teams with a greater number of prior patenting fields were more likely to generate patents that were technological outliers", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-RTVDpn3Mv", + "claim": "A patent's odds of being an outlier increases by 1.20 times for each doubling of other patents cited", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-tYJs3SOGO", + "claim": "inventors' prior experience with knowledge in focal and citing US patents subclasses intensified the U-shaped relationship between technological complexity and exaptation", + "citekey": "mastrogiorgioInnovationExaptationIts2016", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-QgrzIjqGl", + "claim": "amongst 9 major medical question-answering 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"claim": "similar secondary attack rate for household contacts 18yo or less (compared to other age groups) in Midwestern US", + "citekey": "yousafProspectiveCohortStudy2020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-3rIy1jtKe", + "claim": "teams were more likely than single or pair-authored papers to cite atypical combinations of journals", + "citekey": "uzziAtypicalCombinationsScientific2013", + "dataset": "megacoglab", + "findings": [ + "FIG 3A" + ] + }, + { + "id": "megacoglab-inz0nxyak", + "claim": "undergrad 1980s textbooks in physics and chemistry cited far fewer texts compare to sociology textbooks", + "citekey": "coleHierarchySciences1983", + "dataset": "megacoglab", + "findings": [ + "TAB 10" + ] + }, + { + "id": "megacoglab-_FWs0NXx5", + "claim": "Experts employed more schema-driven analogizing than case-driven analogizing; novices showed the reverse pattern", + "citekey": "ballSpontaneousAnalogisingEngineering2004", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-UIVkpC3D_", + "claim": "viewers in Twitch chat engaged in less bad behaviors after a user was banned by a moderator for that bad behavior", + "citekey": "seeringShapingProAntiSocial2017", + "dataset": "megacoglab", + "findings": [ + "TAB 8" + ] + }, + { + "id": "megacoglab-FpvCvjJU9", + "claim": "interdisciplinary humanities scholars' work required a variety of information sources across different stages of exploration and translation work", + "citekey": "palmerInformationWorkInterdisciplinary2002", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-GxyCjeS8g", + "claim": "No analogical transfer amongst undergraduates between insight problems without hints about analogical mapping", + "citekey": "reedRoleAnalogyTransfer1974", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-L5D-9bBxY", + "claim": "approximately 2x lower prevalence of secondary 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This process unfolded over the course of years.", + "citekey": "okadaAnalogicalModificationCreation2009", + "dataset": "megacoglab", + "findings": [ + "FIG 10", + "FIG 3" + ] + }, + { + "id": "megacoglab-C1uFTRfBX", + "claim": "less than 1% of pubpeer comments were positive reviews", + "citekey": "ortega2022classification", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 3" + ] + }, + { + "id": "megacoglab-W3sdOb60i", + "claim": "US patents filed by inventors who were new to the patent's field tended to be more novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-zHpJGF1Ih", + "claim": "undergraduates generated novel word combination noun phrases with more emergent features when noun pairs were dissimilar vs. similar", + "citekey": "wilkenfeldSimilarityEmergenceConceptual2001", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-lJcUil9G6", + "claim": "higher conceptual flexibility to classify organisms based on taxonomic and ecological relations if children were older, from more rural areas, and engaged in more unstructured interactions with organisms", + "citekey": "betzDevelopmentConceptualFlexibility2020", + "dataset": "megacoglab", + "findings": [ + "TAB 9" + ] + }, + { + "id": "megacoglab-yMCU6h2YU", + "claim": "in an interview and survey, data scientists reported major (unsupported) pain points around sharing and collaborating on notebooks in data science teams", + "citekey": "chattopadhyayWhatWrongComputational2020", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 2" + ] + }, + { + "id": "megacoglab-6e7Wu6JTF", + "claim": "overall marginally significant negative effect of analogically distant cases on creativity of campaign ad ideas, mostly for students with high creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 2" + ] + }, + { + "id": "megacoglab-2yEG0_6MI", + "claim": "engineering students who prototyped designs in parallel generated significantly more diverse designs compared to students who did iterative prototyping", + "citekey": "murphyComparingParallelIterative2022", + "dataset": "megacoglab", + "findings": [ + "FIG 5", + "TAB 2" + ] + }, + { + "id": "megacoglab-zWGskgjzd", + "claim": "When provided visual analogies 4th graders performed better than the students given just the target concept. However, This was not observed in 5th graders.", + "citekey": "matlenEnhancingComprehensionScience", + "dataset": "megacoglab", + "findings": [ + "FIG 4", + "FIG 3" + ] + }, + { + "id": "megacoglab-4oupzhYHs", + "claim": "No secondary cases amongst preschoolers in a COVID cluster in Singapore with a student as index case", + "citekey": "yungNovelCoronavirus20192020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-fdgQruzE4", + "claim": "higher levels of interdisciplinarity was significantly associated with fewer publications, particularly in fields with less interdisciplinarity", + "citekey": "leaheyProminentLessProductive2017", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-ledAQMl6P", + "claim": "highest self-rated map quality, as measured by overall quality, and helpfulness to others, when creating map based on previous map that was iterated on by multiple people, compared to creating from scratch, and 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+ "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-d77x3B_Ui", + "claim": "case-based reasoning system loaded with diverse cases increased overall usefulness and creativity of ad campaign ideas, especially for people with lower creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 1" + ] + }, + { + "id": "megacoglab-mPZM8-hmC", + "claim": "when given a (far) analogy for numerical representations, students did no better (and possibly worse) than on a post-test than not having it", + "citekey": "vamvakoussiBridgingGapDense2012", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-GL4yvLPha", + "claim": "low prevalence of EBPR references in state statutes and regulations pertaining to behavioral health care; 20 states had a total of 33 mandates that referenced an EBPR", + "citekey": "lee2022references", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-3sChuA1j8", + "claim": "No secondary cases amongst preschoolers in a COVID cluster in Singapore with adults as index cases", + "citekey": "yungNovelCoronavirus20192020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-0dCO_io-p", + "claim": "increased word ambiguity in abstracts was associated with slightly lower modularity of citation networks for those abstracts", + "citekey": "mcmahanAmbiguityEngagement2018", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-t_AiQeKkK", + "claim": "evaluators gave lower scores to biomed grant proposals that were very novel; but higher scores to proposals that were somewhat novel", + "citekey": "boudreauLookingLookingKnowledge2016", + "dataset": "megacoglab", + "findings": [ + "TAB 5", + "FIG 3" + ] + }, + { + "id": "megacoglab-xRWgPZXX9", + "claim": "Better model fit to Wuhan case data with age-dependent susceptibility, with children ~2x lower than adults", + "citekey": "cmmidcovid19workinggroupAgedependentEffectsTransmission2020", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "FIG 4" + ] + }, + { + "id": "megacoglab-eEjsNkFV1", + "claim": "Members in a data science team mainly use meetings and documentations to develop shared understanding on a project", + "citekey": "wangHowDataScientists2019", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-iG1V6dHgy", + "claim": "Far analogies' effects on MechE students' ideation fluency and diversity were different depending on timing: more ideas (but with more functional repeats) before ideation began, vs. more functionally distinct designs after problem solving began (during a break), both compared to seeing nothing", + "citekey": "tsengRoleTimingAnalogical2008", + "dataset": "megacoglab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "megacoglab-PDfEGZaLe", + "claim": "\ud83d\udd11Outlier Patents receive more citations on average than \ud83d\udd11Adjacent Patents", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-MLSP020ju", + "claim": "Generating (but not evaluating) solutions to far analogy problems positively predicted performance on a subsequent analogy task (compared to generating or evaluating near analogy problems)", + "citekey": "vendettiFarOutThinkingGenerating2014", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-ML0rEDL6_", + "claim": "higher salience of ecological relations for organizing living things for children from rural vs. urban communities", + "citekey": "betzDevelopmentConceptualFlexibility2020", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-1A-vRkLMT", + "claim": "highly novel papers had higher variance in their citation outcomes over a 15-year window, biased towards the higher impact tail of the distribution", + "citekey": "wangBiasNoveltyScience2017", + "dataset": "megacoglab", + "findings": [ + "FIG 1A", + "TAB 2" + ] + }, + { + "id": "megacoglab-5gJjNdjMH", + "claim": "US patents filed by inventor teams that included an expert in the patent's field tended to be less novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-9ic6uJY1D", + "claim": "Women were more likely than men to submit winning solutions for open innovation contests", + "citekey": "jeppesenMarginalityProblemsolvingEffectiveness2010", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-Ig-q6lfVE", + "claim": "Only small business owners with high divergent thinking ability generated more ideas with diverse (vs. constrained) stimuli", + "citekey": "gielnikCreativityOpportunityIdentification2011", + "dataset": "megacoglab", + "findings": [ + "TAB 4", + "FIG 2" + ] + }, + { + "id": "megacoglab-2C17QaTLH", + "claim": "science students made more planning errors and fewer structural errors on a spatial block design task; architecture students made fewer planning errors and more structural errors", + "citekey": "lawsonCognitiveStrategiesArchitectural1979", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-2mTPyT8sj", + "claim": "Limited increases in transfer between analogous insight problems when explicit hint (mapping + solution) was given", + "citekey": "reedRoleAnalogyTransfer1974", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-hZB-PgxZX", + "claim": "~60 percent of InstructGPT-generated analogical explanations for scientific concepts were rated by crowd workers as containing a meaningful analogy, comparable to human-generated analogies", + "citekey": "bhavyaAnalogyGenerationPrompting2022", + "dataset": "megacoglab", + "findings": [ + "TAB 10" + ] + }, + { + "id": "megacoglab-mtKPi-Abu", + "claim": "no overall effect of analogical distance of cases on novelty or usefulness of campaign ad ideas", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-u2Rw1hwtY", + "claim": "College students imagined novel alien creatures with more atypical features vs. Earth creatures when they were instructed to consider survival-critical features of life forms, vs. building on specific Earth animal exemplars", + "citekey": "wardRoleSpecificityAbstraction2004", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-I5ElqeQBh", + "claim": "prevalence of COVID (excluding first-reported case) increased with age in New York State, ~2x lower for children and youth compared to adults", + "citekey": "rosenbergCOVID19TestingEpidemic2020", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-PNHl7tkly", + "claim": "10 scholars self-reported that they read specific fragments and sections, such as findings and background and references, in patterns that varied by tasks such as keeping track, finding new ideas, and writing articles or new projects", + "citekey": "ribaupierreExtractingDiscourseElements2017", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-JszLLa3Q4", + "claim": "Experienced facilitator participants created inspirations for crowd brainstormers with strategies like crafting simulation prompts or inquiries, in contrast to novice facilitators, who relied more heavily on simply passing on examples without further processing", + "citekey": "chanImprovingCrowdInnovation2016", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-5ypAq6Csv", + "claim": "A patent's odds of being an outlier increases by 1.05 times for each doubling of scientific articles cited", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-_QOSgyUEe", + "claim": "~half of InstructGPT-generated analogies for a small benchmark science analogy dataset matched the reference set; ~70 percent were valid", + "citekey": "bhavyaAnalogyGenerationPrompting2022", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-J9X-JZ5b5", + "claim": "Prior patenting experience is negatively related to the likelihood of being granted \ud83d\udd11Outlier Patents", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-N3idE-TeS", + "claim": "Papers citing papers that refute the Hawthorne effect predominantly cited those papers to (incorrectly) affirm the Hawthorne effect", + "citekey": "letrudAffirmativeCitationBias2019", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "FIG 2", + "TAB 1", + "TAB 2", + "FIG 3", + "TAB 3" + ] + }, + { + "id": "megacoglab-H-9bped9d", + "claim": "the sensemaking translucence interface significantly improved clue finding and crime solving performance", + "citekey": "goyalEffectsSensemakingTranslucence2016", + "dataset": "megacoglab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "megacoglab-GvDDTwcsJ", + "claim": "In general, MechE students' ideas when given near analogies were at least as novel, and sometimes moreso, then ideas generated with far analogies, or seeing nothing", + "citekey": "tsengRoleTimingAnalogical2008", + "dataset": "megacoglab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "megacoglab-Qt5iwJecI", + "claim": "after noticing a key element of a problem representation for the mutilated checkerboard problem (namely the lack of parity between the squares on the checkerboard), solutions to the problem came almost immediately after", + "citekey": "kaplanSearchInsight1990", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-kvQUSZy5J", + "claim": "higher estimated helpfulness and clarity of map from others for their own sensemaking for an everyday sensemaking task when the map was iterated on by multiple people vs. created by one person", + "citekey": "fisherDistributedSensemakingImproving2012", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-3BGDkcK-w", + "claim": "Only small business owners with high divergent thinking ability generated more original ideas with diverse (vs. constrained) stimuli", + "citekey": "gielnikCreativityOpportunityIdentification2011", + "dataset": "megacoglab", + "findings": [ + "TAB 4", + "FIG 3" + ] + }, + { + "id": "megacoglab-Smh3nQpqn", + "claim": "Participants who marked up their own papers with a formal semantic scheme self-reported that formally marking rhetorical relations between claims seemed less clear in terms of payoff compared to formally making claims", + "citekey": "grozaSALTWeavingClaim2007", + "dataset": "megacoglab", + "findings": [ + "FIG 8B" + ] + }, + { + "id": "megacoglab-X9jkreduk", + "claim": "direct comparison of dissimilar analogs approximately doubled rates of spontaneous far analogical transfer for the radiation problem", + "citekey": "gickSchemaInductionAnalogical1983", + "dataset": "megacoglab", + "findings": [ + "TAB 5" + ] + }, + { + "id": "megacoglab-TE2krdmGY", + "claim": "Lower secondary attack rate in households in China for children 4-18 compared to other age groups", + "citekey": "wuHouseholdTransmissionSARSCoV22020", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-C5L_KQ5fy", + "claim": "higher levels of interdisciplinarity associated with higher variance in their interdisciplinary papers' citation outcomes", + "citekey": "leaheyProminentLessProductive2017", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-4cjTc4Vju", + "claim": "Universities are 1.36 times more likely to file \ud83d\udd11Outlier Patents than non-universities", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-lMdlmzR0o", + "claim": "most undergrad 1980s textbooks in physics and chemistry cited work published before 1960; sociology texts mostly cited work published after 1960", + "citekey": "coleHierarchySciences1983", + "dataset": "megacoglab", + "findings": [ + "TAB 10" + ] + }, + { + "id": "megacoglab--wTYRJFK3", + "claim": "when placed in the same semantic space, computationally selected near and far analogical stimuli were farther away from human-selected near and far analogical stimuli for the same problem", + "citekey": "fuMeaningFarImpact2013", + "dataset": "megacoglab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "akamatsulab-vcvO9153l", + "claim": "Simulated endocytic filaments were exponentially distributed in length with a median length of 90 \u00ac\u00a8\u00ac\u00b1 80 nm", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 3S1E" + ] + }, + { + "id": "akamatsulab-zqrfjcGub", + "claim": "Only \u201a\u00c4\u00f6\u221a\u00a2\u00ac\u00df 2.5 filaments were growing at a given time point in a simulation of endocytic actin networks", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 3S1C" + ] + }, + { + "id": "akamatsulab-geHT15jC4", + "claim": "Internalization of simulated endocytic pits did not change as a function of filament stall force", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 1S1D" + ] + }, + { + "id": "akamatsulab-Q78XQ9h0X", + "claim": "Simulated endocytic internalization efficiency increased from 0.1% to 1.5% as a function of increasing membrane tension", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 7S1B" + ] + }, + { + "id": "akamatsulab-n6lZLQULG", + "claim": "Endocytic pit internalization was robust for actin confinement strengths >= 100 pN/\u00ac\u00a8\u00ac\u00b5m", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 1S1K" + ] + }, + { + "id": "akamatsulab-Whvj4Tjxl", + "claim": "Knocking down \u2248\u00ed\u201a\u00e2\u00a75-integrin resulted in loss of flat clathrin lattice expansion upon EGF treatment in HSC3-EGFR-GFP cells", + "citekey": "alfonzomendez2022dual", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A" + ] + }, + { + "id": "akamatsulab-iAzvRq7UW", + "claim": "FCHSD2 localization at sites of endocytosis formed a partial ring around the base of the endocytic site by STED microscopy", + "citekey": "almeidasouza2018flat", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-x0oKAeneB", + "claim": "WASP knockout (__wasA-__) __Dictyostelium__ cells displayed bipolar localization of active Rac, rather than localization to the leading edge.", + "citekey": "amato2019wasp", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "BIOL403-S-EATBLOE", + "claim": "[[EVD]] - Eps8 accumulates at bleb membrane sites ~5s before actin accumulation, and is followed by myosin light chain accumulation about 15s after actin in DLD1 cells. - @aoki2016rhoa", + "citekey": "aoki2016rhoa", + "dataset": "BIOL403", + "findings": [ + "FIG 1" + ] + }, + { + "id": "BIOL403-3e7GhrMiE", + "claim": "[[EVD]] - In actively blebbing DLD1 cells, LifeAct-RFP-labeled actin polymerized at the base of blebs and at many individual sites around the bleb membrane, increasing in intensity as blebs fully retracted. - @aoki2016rhoa", + "citekey": "aoki2016rhoa", + "dataset": "BIOL403", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "BIOL403-EaXaNtpwZ", + "claim": "Ezrin knockout DLD1 cells (transfected with LifeAct-RFP and GFP-tagged Eps8) showed slower bleb retraction as compared to WT DLD1 cells as assessed by microscopy.", + "citekey": "aoki2016rhoa", + "dataset": "BIOL403", + "findings": [ + "FIG 3F", + "FIG 3J" + ] + }, + { + "id": "BIOL403-7D_RuzN1q", + "claim": "[[EVD]] - DLD1 cells showed a decreased bleb retraction velocity as compared to migratory blebs or early stage apoptotic blebs. - @aoki2020coordinated", + "citekey": "aoki2020coordinated", + "dataset": "BIOL403", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "BIOL403-KXw0hotNz", + "claim": "local particle shape at the point of initial contact with macrophage not the overall size is shown to influence phagocytic fate", + "citekey": "champion2006role", + "dataset": "BIOL403", + "findings": [ + "FIG 2A", + "FIG 2B" + ] + }, + { + "id": "BIOL403-Aje0TewwL", + "claim": "\u201a\u00c4\u00f6\u221a\u00d1\u221a\u222bFigure 4 shows the number of phagocytosed 3 \u2248\u00ed\u00ac\u222bm IgG-adsorbed spheres and worms after 22 h of incubation at a concentration of 30 particles per cell. Worms still exhibited very little internalization compared to spheres\u201a\u00c4\u00f6\u221a\u00d1\u221a\u03c0", + "citekey": "champion2009shape", + "dataset": "BIOL403", + "findings": [ + "FIG 4" + ] + }, + { + "id": "BIOL403-XcS5PUVkD", + "claim": "Significantly lower excitatory postsynaptic currents (EPSCs) were measured in mutated and the knock-out experiment than in those with normal synaptotagmin-1 function and the rescue experiment.", + "citekey": "chang2018synaptotagmin1", + "dataset": "BIOL403", + "findings": [ + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "BIOL403-5pwU4KU5w", + "claim": "[[EVD]] - Once bleb expansion stops, ezrin was recruited to the bleb membrane and retraction of bleb began in DLD1 cells. - @charras2006reassembly", + "citekey": "charras2006reassembly", + "dataset": "BIOL403", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "BIOL403-WvSjanXYR", + "claim": "[[EVD]] - RhoA was present at all stages of blebbing in HeLa cells. - @charras2006reassembly", + "citekey": "charras2006reassembly", + "dataset": "BIOL403", + "findings": [ + "FIG 1" + ] + }, + { + "id": "BIOL403-AyWDrvoLN", + "claim": "It was observed that miniature synaptic release was not significantly affected by blocking known components of the evoked release in Drosophila neuromuscular junctions when treated with UAS-TeTxLC and Plectreurys toxin II, separately, and that there was reduced synaptic area in vglutMN mutants.", + "citekey": "choi2014miniature", + "dataset": "BIOL403", + "findings": [ + "FIG 1O", + "FIG 1P" + ] + }, + { + "id": "megacoglab-xRWgPZXX9", + "claim": "Better model fit to Wuhan case data with age-dependent susceptibility, with children ~2x lower than adults", + "citekey": "cmmidcovid19workinggroupAgedependentEffectsTransmission2020", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "FIG S4" + ] + }, + { + "id": "akamatsulab-tfg0w7qid", + "claim": "Platinum replica EM of mouse B16F1 cells showed substantial networks of branched actin persisting around clathrin-coated pits after 30 min treatment of 5 \u00ac\u00a8\u00ac\u00b5M Latrunculin B", + "citekey": "collins2011structural", + "dataset": "akamatsulab", + "findings": [ + "FIG S4" + ] + }, + { + "id": "akamatsulab-b3xP1Gqc0", + "claim": "The clathrin lattice height change caused by applied force is reversible.", + "citekey": "dannhauser2015effect", + "dataset": "akamatsulab", + "findings": [ + "FIG S4" + ] + }, + { + "id": "akamatsulab-01PrcWW71", + "claim": "Polyacrylamide gel substrates were fabricated with stiffnesses from 6 to 165 kPa.", + "citekey": "denisin2016tuning", + "dataset": "akamatsulab", + "findings": [ + "TAB S4" + ] + }, + { + "id": "akamatsulab-h0B5oi6H4", + "claim": "Gel formulation (total polymer content, crosslinker concentration, initiator and accelerators), gelation temperature and time, and storage conditions affected the stiffness heterogeneity of polyacrylamide substrates.", + "citekey": "denisin2016tuning", + "dataset": "akamatsulab", + "findings": [ + "TAB S4", + "FIG NA" + ] + }, + { + "id": "akamatsulab-NlLFa2dlZ", + "claim": "Both clathrin-coated structures and integrins accumulated along collagen fibers in 3D cell culture", + "citekey": "elkhatib2017tubular", + "dataset": "akamatsulab", + "findings": [ + "FIG S5A", + "FIG 1A" + ] + }, + { + "id": "akamatsulab-GqpzNJuEZ", + "claim": "siRNA-A1 silenced Hip1R showed Arp3 and F-actin 'tails' in HeLa cells.", + "citekey": "engqvistgoldstein2004rnaimediated", + "dataset": "akamatsulab", + "findings": [ + "FIG 4C" + ] + }, + { + "id": "BIOL403-MntJXxDIB", + "claim": "Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons.", + "citekey": "evans2020degradation", + "dataset": "BIOL403", + "findings": [ + "FIG 6D", + "FIG 6E" + ] + }, + { + "id": "BIOL403-f1y2v5Ne5", + "claim": "Using a quantitative based model of TCR triggering, as-well-as in vivo TIRF microscopy receptor dwell time at phosphatase-depleted close contacts facilitates TCR activation and immunosignaling -", + "citekey": "fernandes2019cell", + "dataset": "BIOL403", + "findings": [ + "FIG 3D", + "FIG 3E", + "FIG 5B", + "FIG 5C" + ] + }, + { + "id": "akamatsulab-ka1IiK3zJ", + "claim": "Latrunculin A and cytochalasin D increased GRAF1 assembly on tubules", + "citekey": "francis2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG S2" + ] + }, + { + "id": "akamatsulab-j0UGLJ3e6", + "claim": "Cells treated with the WASP-inhibitor wiskostatin showed a time-dependent increase of GFP\u201a\u00c4\u00f6\u221a\u00d1\u221a\u00a8GRAF1-decorated tubes at the cell surface", + "citekey": "francis2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D", + "FIG 3E" + ] + }, + { + "id": "akamatsulab-D9FYGZNmk", + "claim": "0.021 \u00ac\u00a8\u00ac\u00b1 0.05 MPa turgor pressure was measured for budding yeast by relating measurements of cellular stiffness and radius of the cell", + "citekey": "goldenbogen2016dynamics", + "dataset": "akamatsulab", + "findings": [ + "FIG S8" + ] + }, + { + "id": "akamatsulab-1B3Jax3yY", + "claim": "Without clathrin-mediated endocytosis, flat-clathrin lattices and reticular adhesions could not form.", + "citekey": "hakanpaa2023reticular", + "dataset": "akamatsulab", + "findings": [ + "FIG 2", + "FIG 4", + "FIG S2", + "FIG 3" + ] + }, + { + "id": "BIOL403-UakdwE_zW", + "claim": "The N-terminal fragment, but not the C-terminal fragment or full length GSDMD, caused cell death (visualized by propidium iodide staining) following transient transfection in HEK-29T cells.", + "citekey": "he2015gasdermin", + "dataset": "BIOL403", + "findings": [ + "FIG 3E" + ] + }, + { + "id": "BIOL403-nZABR2spA", + "claim": "Stimulating Pam3CSK4-primed bone-marrow derived macrophages (BMDM's) with inflammasome activators leading to extracellular IL-1beta secretion.", + "citekey": "heilig2018gasdermind", + "dataset": "BIOL403", + "findings": [ + "FIG 1A", + "FIG 1B", + "FIG 1C" + ] + }, + { + "id": "BIOL403-vagWoh8Ar", + "claim": "\u201a\u00c4\u00f6\u221a\u00d1\u221a\u222bThe ATP/ADP translocase drives mitophagy independent of nucleotide exchange\u201a\u00c4\u00f6\u221a\u00d1\u221a\u03c0", + "citekey": "hoshino2019adp", + "dataset": "BIOL403", + "findings": [ + "FIG 2A", + "FIG 2B" + ] + }, + { + "id": "dg-social-media-polarization-sikUtfWyf", + "claim": "An SVM classifier trained on vocabulary from 40 model-estimated topics over subreddits was able to predict subreddits' political bias with 85% accuracy.", + "citekey": "kaneCommunitiesWeChoose2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-S3Jz8Rq0j", + "claim": "LDA modeling showed that overall political bias varied significantly across subreddits .", + "citekey": "kaneCommunitiesWeChoose2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "BIOL403-Oduoj3hLG", + "claim": "While eIF3j+ is able to bind to 40s on its own, eIF3j- was observed to bind to the 40s ribosomal subunit in certain conditions detailed in the figure (but notably in the presence of other transcription factors)", + "citekey": "kolupaeva2005binding", + "dataset": "BIOL403", + "findings": [ + "FIG 1D", + "FIG 1E", + "FIG 1C", + "FIG 1A", + "FIG 1B", + "FIG 1F" + ] + }, + { + "id": "BIOL403-ziiwRmbbx", + "claim": "\u201a\u00c4\u00f6\u221a\u00d1\u221a\u222bUsing traction force microscopy, we show that cells exert significant strain on the underlying substrate during the contraction phase but little strain during the spreading phase, demonstrating that phagocytes actively constrict during late-stage phagocytosis\"", + "citekey": "kovari2016frustrated", + "dataset": "BIOL403", + "findings": [ + "FIG 3F" + ] + }, + { + "id": "BIOL403-1uvjnCEpK", + "claim": "\u201a\u00c4\u00f6\u221a\u00d1\u221a\u222bIn each PBS core basal cylinder, four phycobilin chromophores in corresponding APC trimers (Fig. 2) are close to the bottom surface of the whole PBS [6, 21], and we will refer to them as \u201a\u00c4\u00f6\u221a\u00d1\u221a\u2264bottom\u201a\u00c4\u00f6\u221a\u00d1\u221a\u00a5 phycobilins. These phycobilins appear to be the closest to the cytoplasmic side of PSII and its chlorophylls. The resulting vicinity of the chromophores creates an opportunity for the energy transfer from PBS to PSII.\"", + "citekey": "krasilnikov2020rates", + "dataset": "BIOL403", + "findings": [ + "FIG 2" + ] + }, + { + "id": "BIOL403-pRP48JdtW", + "claim": "\u201a\u00c4\u00f6\u221a\u00d1\u221a\u222bThe obtained absorption spectrum of PSII has typical peak position at 675 nm (Fig. 1B). Comparing it with the fluorescence emission spectra of the APC680 and APC660 demonstrates, in both cases, the high degree of their overlap that is one of the basic prerequisites of successful energy transfer. \u201a\u00c4\u00f6\u221a\u00d1\u221a\u222b", + "citekey": "krasilnikov2020rates", + "dataset": "BIOL403", + "findings": [ + "FIG 1A", + "FIG 1B" + ] + }, + { + "id": "akamatsulab-DG74gc3CJ", + "claim": "Barbed end capping proteins inhibit actin bursting", + "citekey": "Kueh2008", + "dataset": "akamatsulab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "akamatsulab-Qo0PhBepV", + "claim": "NWASP was found around clusters of clathrin heavy chain by TIRF microscopy + super resolution microscopy", + "citekey": "leytonpuig2017flat", + "dataset": "akamatsulab", + "findings": [ + "FIG 1E" + ] + }, + { + "id": "akamatsulab-WJvOy9Exn", + "claim": "The density of free barbed ends increased as a function of growth stress", + "citekey": "li2022molecular", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-LtD05YJZT", + "claim": "[[EVD]] - In budding yeast cof1[delta] cells transformed with pCOF1-RFP CEN plasmid, there were 930 molecules of cofilin per patch which normalized to about 530 molecules per patch when accounting for overexpression and comparison to WT cells - @lin2010overlapping", + "citekey": "lin2010overlapping", + "dataset": "akamatsulab", + "findings": [ + "FIG S1D" + ] + }, + { + "id": "BIOL403-iLMDqSoqF", + "claim": "Using a force prove 2D single-bond lifetimes under a range of constant forces applied via a pMHC engaged to a TCR on a naive T cell from OT1 transgenic mice, with fluorescently tagged Ca2+ release used as a proxy for TCR triggering, TCRs were found to form catch bonds with agonists and slip bonds with antagonists. This differential bond formation, based off pressure, impacted the association time with pMHC, and the resulting TCR triggering.", + "citekey": "liuAccumulationDynamicCatch2014a", + "dataset": "BIOL403", + "findings": [ + "FIG 3A", + "FIG 3B", + "FIG 2B", + "FIG 2C" + ] + }, + { + "id": "akamatsulab-g8zQj06en", + "claim": "The SH3 domain of GRAF1 bound to the polyproline domain of dynamin1 with an apparent dissociation constant of 106 \u00ac\u00a8\u00ac\u00b5M via isothermal titration calorimetry", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG S2D" + ] + }, + { + "id": "akamatsulab-_HN8EB6zZ", + "claim": "GRAF1 tubules were disrupted in HeLa cells when fixed with 4% PFA at 4degC, but intact when fixed with 4% PFA at 37degC.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG S1C" + ] + }, + { + "id": "akamatsulab-amE_0Wcnm", + "claim": "Actin polymerization assay showed that cortactin phosphorylation by ERK increased the WASP function", + "citekey": "martinezquiles2004erk", + "dataset": "akamatsulab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "BIOL403-qB6i0BV-n", + "claim": "Under photostimulation of Th IRES-Cre LC-BLA ChR2 there are changes in firing frequency.", + "citekey": "mccall2017locus", + "dataset": "BIOL403", + "findings": [ + "FIG 3C", + "FIG 3D" + ] + }, + { + "id": "BIOL403-5Y-UQGr52", + "claim": "[[EVD]] - \"CD13 clustering by activating but not inactive antibodies can be visualized by confocal imaging of U-937 cells treated with fluorescently labeled antibodies.\" - [[[[@mina-osorio2008cd13]]]]", + "citekey": "mina-osorio2008cd13", + "dataset": "BIOL403", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "akamatsulab-Hcxv0n6n7", + "claim": "Internalisation of Transferrin (Tf), a marker of clathrin mediated endocytosis, was reduced 10 minutes, but not 1 hour after incubation when REF52 fribroblast cells were cultured on \"soft\" hydrogels, as opposed to \"intermediate\" or \"stiff\" hydrogels.", + "citekey": "missirlis2014effect", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B", + "FIG 1A", + "FIG 1D", + "FIG 1C" + ] + }, + { + "id": "akamatsulab-w0rQV7oyI", + "claim": "Deletion of Swip1-EF1 actin-binding region abolished Swip1 ability to facilitate integrin endocytosis.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "akamatsulab-gfaXg2-bh", + "claim": "Endogenous Swip1 associated with Rab21 in Rab21-containing early endosomes in MDA-MB-231 cells as shown by proximity ligation assay (PLA).", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "akamatsulab-TBLuywz6G", + "claim": "Endogenous Swip1 localized to Rab21+ and B1-integrin-containing early endosomes.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A", + "FIG 2C" + ] + }, + { + "id": "akamatsulab-c4Ecoobt4", + "claim": "Endogenous Swip1 preferentially bound WT-Rab21 and constitutively active Rab21 over the closely related Rab5 GTPase in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG S1E", + "FIG 1B", + "FIG 1C" + ] + }, + { + "id": "akamatsulab-9E0jVhav_", + "claim": "Swip1 bound to actin via Swip1 EF1 domain as shown by GFP-pulldown assay in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B" + ] + }, + { + "id": "akamatsulab-6yP4l1xIu", + "claim": "Swip1 preferentially bound WT-Rab21 and constitutively active Rab21 over the closely related Rab5 GTPase.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG S1E" + ] + }, + { + "id": "akamatsulab-24El5w68I", + "claim": "siRNA silenced IRSp53 significantly reduced internalized 10kDa TMR Dextran, while siRNA silenced Swip1 did not in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 5B" + ] + }, + { + "id": "akamatsulab-Dd1n6KIBV", + "claim": "siRNA silenced Swip1 reduced GFP-Rab21+ (CG-associated) endosome trafficking in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6G" + ] + }, + { + "id": "akamatsulab-6-XC7noR7", + "claim": "siRNA silenced Swip1 showed reduced internalization of B1-integrin in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "BIOL403-7nz9HilB1", + "claim": "Using digitalized EM tomograms Actin Filament angle distribution became more ordered at +/- 35 degrees relative to membrane closer to the leading edge.", + "citekey": "mueller2017load", + "dataset": "BIOL403", + "findings": [ + "FIG 3E", + "FIG 3F" + ] + }, + { + "id": "BIOL403-QdjQnpn48", + "claim": "Following transient global cerebral ischemia (tGCI) induced by bilateral common carotid artery occlusion, immunoprecipitation of rat hippocampal CA1 subregion lysates revealed the association of active CASP2 with PIDDosome components.", + "citekey": "niizuma2008piddosome", + "dataset": "BIOL403", + "findings": [ + "FIG 3A" + ] + }, + { + "id": "BIOL403-PIb_pytWu", + "claim": "\u201a\u00c4\u00f6\u221a\u00d1\u221a\u222bWe found that 90% of the spherical targets (irrespective of size or material) are engulfed within 10 min of making contact with the cell, whereas only 10% of the ellipsoidal targets are engulfed during this period\u201a\u00c4\u00f6\u221a\u00d1\u221a\u03c0", + "citekey": "paul2013phagocytosis", + "dataset": "BIOL403", + "findings": [ + "FIG 4" + ] + }, + { + "id": "BIOL403--VO-_WYxs", + "claim": "No correlation was noted about the time at which spontaneous and evoked neurotransmissions occur at a synapse during an 8 minute recording during which the neuron was stimulated every 30 seconds.", + "citekey": "reese2016single", + "dataset": "BIOL403", + "findings": [ + "FIG 2C" + ] + }, + { + "id": "BIOL403-J3a8rpdM0", + "claim": "Immunoblotting with antibodies against CD13 in monocytes showed CD13 content in the low density raft (detergent-resistant) fractions .", + "citekey": "santos2000aminopeptidase", + "dataset": "BIOL403", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-Pu_eipQdq", + "claim": "Matrigel stiffness can be tuned between ~10 Pa to ~300 Pa", + "citekey": "slater2018tuning", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "BIOL403-F1br3itAE", + "claim": "No reduction of eIF3j affinity of the 43s preinitiation complex was observed when eIF4A was not complexed to eIF4G", + "citekey": "sokabe2017helicaseindependent", + "dataset": "BIOL403", + "findings": [ + "FIG 4A", + "FIG 4B" + ] + }, + { + "id": "BIOL403-r2XHRduO2", + "claim": "[[EVD]] - eIF4A activity reduces eIF3j affinity of the 43s preinitiation complex - [[Spokabe and Fraser 2017]]", + "citekey": "sokabe2017helicaseindependent", + "dataset": "BIOL403", + "findings": [ + "FIG 2" + ] + }, + { + "id": "BIOL403-XluxTD7Ik", + "claim": "[[EVD]] - The addition of HB27 following H014-S binding exhibits cooperative allosteric binding - [Sun, 2021] -", + "citekey": "sun2021structurebased", + "dataset": "BIOL403", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "akamatsulab-Gn6iewvYF", + "claim": "[[EVD]] - Presence of Cofilin, Coronin and Aip1 resulted in rapid loss of actin filaments over time based on electron microscopy - [[@tang2020bursting]]", + "citekey": "tang2020bursting", + "dataset": "akamatsulab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "BIOL403-VZoKTW0Yr", + "claim": "eIF3 NIP1 subunit was observed to have attenuated association with eIF1 and eIF5 when a mutating residues in in the NIP1-NTD", + "citekey": "valasek2004interactions", + "dataset": "BIOL403", + "findings": [ + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-rYN6VK3QX", + "claim": "[[EVD]] - Measurement of severing rates of actin filaments by eGFP-cofilin - @wioland2017adf", + "citekey": "wioland2017adf", + "dataset": "akamatsulab", + "findings": [ + "FIG 2E" + ] + }, + { + "id": "akamatsulab-QXPFaZd9j", + "claim": "Simulated actin filament polymerization in the presence of cofilin", + "citekey": "wioland2017adf", + "dataset": "akamatsulab", + "findings": [ + "FIG 6E" + ] + }, + { + "id": "akamatsulab-2wT9Yv_cF", + "claim": "Appearance rate constant of eGFP-cofilin-1 domains onto unlabeled cytoplasmic actin was ~2.15x10^-4/ \u00ac\u00a8\u00ac\u00b5M / sec", + "citekey": "wioland2017adf", + "dataset": "akamatsulab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "akamatsulab-_xYEUv42O", + "claim": "Summary of the ADF/cofilin assembly mechanism and corresponding rates", + "citekey": "wioland2017adf", + "dataset": "akamatsulab", + "findings": [ + "FIG 1H", + "FIG 1I", + "FIG 1J" + ] + }, + { + "id": "akamatsulab-9gFfjaS5s", + "claim": "In the presence of calcium, beads in motility assays with Drosophila Myosin-ID had shorter actin tails", + "citekey": "xu2024myosini", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-5LQe2yX0I", + "claim": "Rhodamine-actin density decreased by~20% in the presence of Drosophila Myosin-ID in branched actin bead motility assays", + "citekey": "xu2024myosini", + "dataset": "akamatsulab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "akamatsulab-tvBYlW_Kf", + "claim": "Rigor delayed the growth of actin comet tails at 50 nM Cp", + "citekey": "xu2024myosini", + "dataset": "akamatsulab", + "findings": [ + "FIG 5C", + "FIG 2A" + ] + }, + { + "id": "akamatsulab-m1Yc-GGvJ", + "claim": "SNAP-Arp2/3 complex was ~50% as intense in the presence of Drosophila Myosin-ID in bead motility assays", + "citekey": "xu2024myosini", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A", + "FIG 4C" + ] + }, + { + "id": "akamatsulab-lFsPSfH_z", + "claim": "The actin:Arp2/3 fluorescence ratio in bead tails was 50% higher in the presence of drosophila Myosin-ID", + "citekey": "xu2024myosini", + "dataset": "akamatsulab", + "findings": [ + "FIG 4D" + ] + }, + { + "id": "akamatsulab-82vjreeE4", + "claim": "WAVE2-ABI1 complex formation via immunoprecipitation was decreased in HeLa cells with GRAF1 knocked down by siRNA", + "citekey": "zhu2023graf1", + "dataset": "akamatsulab", + "findings": [ + "FIG 4B" + ] + } +] \ No newline at end of file diff --git a/task1-train-dev.json b/task1-train-dev.json new file mode 100644 index 0000000000000000000000000000000000000000..992b15ea1030901fc9f9dd7e515be99b8349eb8b --- /dev/null +++ b/task1-train-dev.json @@ -0,0 +1,3701 @@ +[ + { + "id": "akamatsulab-1YXRXmgXU", + "claim": "Fluid uptake as measured by fluorescent 10kDa Dextran increased upon decreased membrane tension as a result of deadhering CHO cells during trypsinization.", + "citekey": "thottacherry2018mechanochemical", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-9E0jVhav_", + "claim": "Swip1 bound to actin via Swip1 EF1 domain as shown by GFP-pulldown assay in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B" + ] + }, + { + "id": "akamatsulab-QMQIJuzAI", + "claim": "Knocking down \u03b1v\u03b25 integrin expression abolished clathrin plaque assembly in HeLa cells", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C", + "FIG 2A" + ] + }, + { + "id": "akamatsulab-WJvOy9Exn", + "claim": "The density of free barbed ends increased as a function of growth stress", + "citekey": "li2022molecular", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-ZR6_FyY3a", + "claim": "Overexpressed CRIB domain of N-WASP localized to Cdc42-containing spots on the plasma membrane in FRaTB cells", + "citekey": "chadda2007cholesterolsensitive", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D", + "FIG 6E" + ] + }, + { + "id": "akamatsulab-cRFz4n0Pc", + "claim": "Coronin and cofilin-1 intensity peaked at about 4-5 seconds after ABP1-GFP maximal intensity at endocytic patches in budding yeast.", + "citekey": "lin2010overlapping", + "dataset": "akamatsulab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "akamatsulab-TBLuywz6G", + "claim": "Endogenous Swip1 localized to Rab21+ and B1-integrin-containing early endosomes.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C", + "FIG 2A" + ] + }, + { + "id": "akamatsulab-WQY-tljFm", + "claim": "A computational assessment of theoretical conformational changes upon binding of cofilin monomers to an actin filament suggest a minimum of 3 cofilactin interfaces, or two adjacent bound cofilin monomers, induces severing of an actin filament.", + "citekey": "tanaka2018structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 7" + ] + }, + { + "id": "akamatsulab-rPE_IW2JJ", + "claim": "Endothelial cells grown on stiffer cells had more actin stress fibers and were themselves stiffer than cells grown on more compliant substrates.", + "citekey": "byfield2009endothelial", + "dataset": "akamatsulab", + "findings": [ + "FIG 3A", + "FIG 3B", + "FIG 3C", + "FIG 3D", + "FIG 3E", + "FIG 3F", + "FIG 1A", + "FIG 1B", + "FIG 1C" + ] + }, + { + "id": "akamatsulab-GqpzNJuEZ", + "claim": "siRNA-A1 silenced Hip1R showed Arp3 and F-actin 'tails' in HeLa cells.", + "citekey": "engqvistgoldstein2004rnaimediated", + "dataset": "akamatsulab", + "findings": [ + "FIG 4C" + ] + }, + { + "id": "akamatsulab-A63ow10iT", + "claim": "Mechano-sensing clathrin plaques formed independent of actin contractility", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "akamatsulab-yQg2vbfkG", + "claim": "Cofilin peaked in concentration about 10 seconds after endocytic scission in mouse 3T3 fibroblasts.", + "citekey": "taylor2011high", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-2-bJaFjob", + "claim": "About 30 DNM2 molecules were required for forming the encytosis in SK-MEL-2 cells.", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 3G" + ] + }, + { + "id": "akamatsulab-QLP8zq7GU", + "claim": "Platinum-replica electron micrographs of unroofed B16F1 mouse melanoma cells showed actin networks in collar-like arrangements around clathrin coat necks and at small lateral patches at the periphery of clathrin coats", + "citekey": "collins2011structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "akamatsulab-u95Z_9Xq7", + "claim": "Total bending energy of actin filaments increased as a function of membrane tension in endocytic simulations", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 7H" + ] + }, + { + "id": "akamatsulab-kZckUXf1w", + "claim": "Treatment of H9 cells with CK-666 resulted in a~50% decrease in the formation of apical membrane\u00a0initiation sites (AMIS) en route to lumen formation", + "citekey": "taniguchi2015lumen", + "dataset": "akamatsulab", + "findings": [ + "FIG 4G" + ] + }, + { + "id": "akamatsulab-kimWcz_h4", + "claim": "Endocytic protein ABP1-GFP lifetime increased by 2-3x in budding yeast cells with cofilin mutants", + "citekey": "lin2010overlapping", + "dataset": "akamatsulab", + "findings": [ + "FIG 8" + ] + }, + { + "id": "akamatsulab-kayrJnALo", + "claim": "Endocytic adaptor protein End4p-mGFP accumulated more slowly and to a greater number of molecules in fission yeast cells with mutant cofilin allele", + "citekey": "chen2013actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-kIupe544T", + "claim": "The surface area overlap of actin over clathrin increased from ~5% to ~10% coverage from flat to curved pits in four cell types by platinum-replica electron microscopy", + "citekey": "yang2022actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 2N" + ] + }, + { + "id": "akamatsulab-rYe33dsbF", + "claim": "Knockdown of either \u03b1v or \u03b25 integrins in HeLa cells using specific siRNAs resulted in a complete loss of large and static CCSs", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 2F", + "FIG 2E", + "FIG 2D" + ] + }, + { + "id": "akamatsulab-QsJF6msql", + "claim": "Intensity over time traces of hiPSCs endogenously expressing AP2-RFP and Dynamin2-GFP peaked sequentially", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 2G" + ] + }, + { + "id": "akamatsulab-BX3Cqqj90", + "claim": "Purified swip1 bound human platelet actin with an affinity of 1.9 \u00b1 0.8 uM", + "citekey": "kwon2013swiprosin1", + "dataset": "akamatsulab", + "findings": [ + "FIG 3A" + ] + }, + { + "id": "akamatsulab-vmbAp09g2", + "claim": "MiTAB, a inhibitor of DNM2 PH domain, led to increased DNM2 recruitment in SK-MEL-2 cells", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S3F" + ] + }, + { + "id": "akamatsulab-Dd1n6KIBV", + "claim": "siRNA silenced Swip1 reduced GFP-Rab21+ (CG-associated) endosome trafficking in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6G" + ] + }, + { + "id": "akamatsulab-WbWLJVWcF", + "claim": "Knockout of __Dictyostelium__ WASP (__wasA-__) led to bipolar localization of WAVE rather than WT localization of WAVE to the leading edge.", + "citekey": "amato2019wasp", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A", + "FIG 1B" + ] + }, + { + "id": "akamatsulab-taeyp3oH-", + "claim": "Membrane dye (DiI dye) or Dextran uptake for 1 or 5 minutes in HeLa cells colocalize with endogenous GRAF1 staining.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B", + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-LlehpEErX", + "claim": "Immunofluorescence of folate-binding protein showed fluorescence in the periphery of KB cells", + "citekey": "numasawa2020fluorescent", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S1" + ] + }, + { + "id": "akamatsulab-wLCgd8nNI", + "claim": "Switching media from isotonic to hypotonic (75m0sm) media for 2-20 minutes increased the membrane tension from 33.0 \u00b1 7.4 pN to 48.0 \u00b1 17.1 pN, as measured by AFM.", + "citekey": "kaplan2022load", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A", + "FIG 2B" + ] + }, + { + "id": "akamatsulab-dFhjWUgD7", + "claim": "TIRF revealed that Cortactin, Hip1R, and Clathrin were simultaneously recruited to endocytic sites in 3T3 cell.", + "citekey": "leclainche2007hip1r", + "dataset": "akamatsulab", + "findings": [ + "FIG 4E" + ] + }, + { + "id": "akamatsulab-boQ6UJJMP", + "claim": "Decreased clathrin coat dynamics were measured under increased membrane tension using micropipette aspiration", + "citekey": "ferguson2017mechanoregulation", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "akamatsulab-5i2BACcCn", + "claim": "Pyrene-actin binding assays between human myo1e and actin plateaued at a dissociation rate of 440 \u00b1 9 s-1 for ATP concentrations > 2 \u00b5M", + "citekey": "mezgueldi2002kinetic", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A" + ] + }, + { + "id": "akamatsulab-3-az1CxcL", + "claim": "Cofilin seems to have different effects on actin filaments, such as severing and nucleation, based on its concentration.", + "citekey": "andrianantoandro2006mechanism", + "dataset": "akamatsulab", + "findings": [ + "FIG 5", + "SUPP FIG S5" + ] + }, + { + "id": "akamatsulab-chLz-7JK6", + "claim": "The rate constant of cofilin 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+ "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-6wkebK9Rb", + "claim": "Depletion of \u03b21 or \u03b23 integrins by siRNA in HeLa cells showed no difference in the percentage of dynamic or static CCSs as compared to siCTRL cells.", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 4E", + "FIG 4F" + ] + }, + { + "id": "akamatsulab-HiUvvjFf0", + "claim": "More actin filaments accumulated at sites of simulated endocytosis as a function of membrane tension", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 7G" + ] + }, + { + "id": "akamatsulab-k90itT5sI", + "claim": "After washout of ATP-reducing agents, lifeact-GFP increased photobleaching recovery and preceded scission of the membrane tubule a few minutes later", + "citekey": "romer2010actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D", + "FIG 5E" + ] + }, + { + "id": "akamatsulab-gfaXg2-bh", + "claim": "Endogenous Swip1 associated 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"okreglak2007cofilin", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-QigGJImJz", + "claim": "CortF, withtriple-mutant in tyrosine residues, increased fillopodia retraction frequency", + "citekey": "he2015src", + "dataset": "akamatsulab", + "findings": [ + "FIG 4D" + ] + }, + { + "id": "akamatsulab-NU1hryH_8", + "claim": "GRAF1-depletion by siRNA knockdown in HeLa cells has no effect on transferrin uptake", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 4D" + ] + }, + { + "id": "akamatsulab-nC0geNc63", + "claim": "Depleting Cdc42 with siRNA led to the buildup of GRAF1-GFP in tubular structures", + "citekey": "francis2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 3C" + ] + }, + { + "id": "akamatsulab-FxTUmxjh2", + "claim": "Simulated fission yeast endocytic actin networks produced ~3000 pN of force, while simulated budding yeast endocytic actin networks produced ~1000 pN of force", + "citekey": "nickaeen2022model", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "akamatsulab-txXrNK-wA", + "claim": "At fission yeast endocytic sites, scission protein (spHob1) first detected ~2 seconds before initiation of membrane invagination", + "citekey": "sun2019direct", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-45WDQVJkn", + "claim": "ABI2 formed a complex with GRAF1 based on immunoprecipitation of HeLa cell lysates, in a manner dependent on GRAF1 phosphorylation and treatment with mitochondrial damaging agent oligomycin-A and antimycin-A", + "citekey": "zhu2023graf1", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "akamatsulab-u3tkdx52k", + "claim": "Fluorescently labeled cortactin assembled coincident with Arp3 and Abp1 in NIH3T3 cells", + "citekey": "taylor2011high", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": 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[ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-lNL2WkukA", + "claim": "Cryo-EM analysis of actin filaments showed that filaments bound to ADP-Pi were stiffer than filaments bound to ADP", + "citekey": "reynolds2022bending", + "dataset": "akamatsulab", + "findings": [ + "FIG 4F" + ] + }, + { + "id": "akamatsulab-1q7uo-zzw", + "claim": "Efficient lamellipodia actin filament severing requires a cofilin cluster longer than one-half of a helix", + "citekey": "ngo2015cofilininduced", + "dataset": "akamatsulab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "akamatsulab-p-Fy1XHf_", + "claim": "GRAF1-depleted HeLa cells (via siRNA treatment) demonstrate ~50% reduction in fluid phase uptake as compared to control cells, shown by FITC-dextran uptake.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 4B", + "FIG 4C" + ] + }, + { + "id": "akamatsulab-6yP4l1xIu", + "claim": "Swip1 preferentially bound WT-Rab21 and constitutively active Rab21 over the 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"citekey": "hassinger2017design", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D" + ] + }, + { + "id": "akamatsulab-1-C-g0qCW", + "claim": "The curvature of the clathrin coat increased as a function of angle to the plasma membrane in SK-MEL2 cells fixed in plastic", + "citekey": "avinoam2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D" + ] + }, + { + "id": "akamatsulab-NbFDiQaUh", + "claim": "Swip1 colocalized with actin at lamellipodia of CHO-K1 cells transfected with GFP-Swip1.", + "citekey": "kwon2013swiprosin1", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "akamatsulab-TndUNP272", + "claim": "Measurements by super resolution microscopy showed a ~20 nm radial distance between Las17 (WASP) and MYO1 in budding yeast", + "citekey": "mund2018systematic", + "dataset": "akamatsulab", + "findings": [ + "FIG 2F" + ] + }, + { + "id": "akamatsulab-0AnyJYOE1", + "claim": "Overexpressed NWASP-GFP colocalized with fluorescent dextran spots in FRaTb cells", + "citekey": "chadda2007cholesterolsensitive", + "dataset": "akamatsulab", + "findings": [ + "FIG 4C", + "FIG 4D", + "FIG 4G", + "FIG 4H" + ] + }, + { + "id": "akamatsulab-Hcxv0n6n7", + "claim": "Internalisation of Transferrin (Tf), a marker of clathrin mediated endocytosis, was reduced 10 minutes, but not 1 hour after incubation when REF52 fribroblast cells were cultured on \"soft\" hydrogels, as opposed to \"intermediate\" or \"stiff\" hydrogels.", + "citekey": "missirlis2014effect", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A", + "FIG 1B", + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-Ga8ORIY8v", + "claim": "Higher rates of purified actin polymerization are seen with purified WAVE2 in the presence of purified IRSp53 as compared to WAVE2 alone in vitro.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "akamatsulab-PECll5ZFw", + 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IRSp53(DELTA)SH3 did not pull down N-WASP.", + "citekey": "lim2008cdc42", + "dataset": "akamatsulab", + "findings": [ + "FIG 3A" + ] + }, + { + "id": "akamatsulab-lljpG3FsT", + "claim": "More actin accumulated at sites of clathrin-mediated endocytosis in SK-MEL-2 cells treated with hypo-osmotic shock to elevate membrane tension", + "citekey": "kaplan2022load", + "dataset": "akamatsulab", + "findings": [ + "FIG 4C" + ] + }, + { + "id": "akamatsulab-Whvj4Tjxl", + "claim": "Knocking down \u03b25-integrin resulted in loss of flat clathrin lattice expansion upon EGF treatment in HSC3-EGFR-GFP cells", + "citekey": "alfonzomendez2022dual", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A", + "FIG 2B", + "FIG 2C", + "FIG 2D", + "FIG 2E", + "FIG 2F", + "FIG 2G" + ] + }, + { + "id": "akamatsulab-1Orp-fkjH", + "claim": "Cells injected with anti-cortactin antibodies decreased the degree of transferrin internalization into clone 9 cells", + "citekey": "cao2003cortactin", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-1B3Jax3yY", + "claim": "Without clathrin-mediated endocytosis, flat-clathrin lattices and reticular adhesions could not form.", + "citekey": "hakanpaa2023reticular", + "dataset": "akamatsulab", + "findings": [ + "FIG 4B" + ] + }, + { + "id": "akamatsulab-iyjRL_mO4", + "claim": "Increased membrane tension, achieved by compression between a polymer cushion, resulted in increased clathrin coat lifetimes.", + "citekey": "ferguson2017mechanoregulation", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C" + ] + }, + { + "id": "akamatsulab-de4YJna7K", + "claim": "hPSCs were found to exert strong inward-directed mechanical forces on the ECM at specific locations coinciding with VSFs at the colony edge.", + "citekey": "narva2017strong", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D" + ] + }, + { + "id": "akamatsulab-32Cg5C3il", + "claim": "Branched actin filament barbed ends were densely oriented towards the clathrin coated vesicle in B16F1 Cells", + "citekey": "collins2011structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-41SA-aHd6", + "claim": "As bending energy decreased in simulated endocytic actin filaments, the pit internalization increased proportionally", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 5G", + "FIG 5H", + "FIG 5I" + ] + }, + { + "id": "akamatsulab-pHdxFkkDB", + "claim": "eGFP-60mer and 120mer nanocages expressed in Arabodopsis with CAAX membrane binding showed a linear relationship between fluorescence intensity and molecule number", + "citekey": "alamos2021quantitative", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C", + "FIG 2D" + ] + }, + { + "id": "akamatsulab-4u5hI2-Ys", + "claim": "The diameter of GRAF1-positive tubules by electron microscopy found in HeLa cells was ~40nm.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 1G" + ] + }, + { + "id": "akamatsulab-6-XC7noR7", + "claim": "siRNA silenced Swip1 showed reduced internalization of B1-integrin in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "akamatsulab-MOrPymM0p", + "claim": "At zero seconds, 7000 actin proteins associated with endocytic invagination", + "citekey": "sirotkin2010quantitative", + "dataset": "akamatsulab", + "findings": [ + "FIG 7A", + "FIG 7B" + ] + }, + { + "id": "akamatsulab-B7UPmuzgg", + "claim": "N-WASP failed to recruit to forming CG endocytic sites.", + "citekey": "sathe2018understanding", + "dataset": "akamatsulab", + "findings": [ + "FIG 5.4A" + ] + }, + { + "id": "akamatsulab-jjw60hnsi", + "claim": "Clathrin-coated plaques formed on stiff substrates (<31 kPa) more than on softer substrates", + "citekey": "baschieri2018frustrated", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B", + "FIG 2A", + "FIG 1F", + "FIG 1A", 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"akamatsulab-gOeTvBjuK", + "claim": "2 mins after addition, ~70% of a fluorescent monoclonal folate receptor-binding antibody was localized to internal spots nonoverlapping with spots marked by fluorescent transferrin", + "citekey": "sabharanjak2002gpianchored", + "dataset": "akamatsulab", + "findings": [ + "FIG 2A", + "FIG 2B", + "FIG 2C", + "FIG 2D", + "FIG 2E", + "FIG 2F" + ] + }, + { + "id": "akamatsulab-0LXSOKdht", + "claim": "Integrin \u03b1V\u03b25 correlated with flat clathrin lattices in human keratinocytes", + "citekey": "zuidema2018mechanisms", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-Gv-IeF8Wl", + "claim": "The per-filament capping rate of __in vitro__ loaded actin filament networks decreased exponentially as a function of applied force", + "citekey": "li2022molecular", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C" + ] + }, + { + "id": "akamatsulab-up45yF1mO", + "claim": "In loaded in vitro networks, VCA preferentially bound the free barbed ends of actin filaments rather than soluble actin monomers", + "citekey": "li2022molecular", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A", + "FIG 4B", + "FIG 4C", + "FIG 4D" + ] + }, + { + "id": "akamatsulab-iNPTKMfD8", + "claim": "Of 10 genes investigated, the top 10 candidate off-target sites were not mutated based on sanger sequencing after Cas9 RNP electroporation", + "citekey": "roberts2017systematic", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S3", + "FIG 1B", + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-6r_duPu8G", + "claim": "Blocking integrin \u03b21 led to reversal of the inhibitory effect of fibronectin on flat formations lattice formation.", + "citekey": "hakanpaa2023reticular", + "dataset": "akamatsulab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "akamatsulab-DHGP0FRjV", + "claim": "87 \u00b1 1.6% of all dynamin containing endocytic events displayed RFP beta actin recruitment", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 4F" + ] + }, + { + "id": "akamatsulab-YYYVszFr6", + "claim": "Pulldown assay of NIH3T3 lysates demonstrated a physical linkage between WAVE and Rac by IRSp53.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "akamatsulab-aKH5q105S", + "claim": "Lumenoids formed over 3 days in hESCs", + "citekey": "taniguchi2015lumen", + "dataset": "akamatsulab", + "findings": [ + "FIG 1F" + ] + }, + { + "id": "akamatsulab-F0N83vxEe", + "claim": "Knockdown of Dyn2 decreased the length of filopodia in H1299 cells", + "citekey": "yamada2016actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 3C" + ] + }, + { + "id": "akamatsulab-9_9FlVy1D", + "claim": "Simulated endocytosis decreased as a function of membrane tension", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 7" + ] + }, + { + "id": "akamatsulab-ZblBen2ee", + "claim": "GFP-cdc42L61 (dominant-active) -expressing HeLa cells demonstrate co-localization with Myc-GRAF1 as shown by confocal microscopy images.", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D" + ] + }, + { + "id": "akamatsulab--Sz9IbUra", + "claim": "Plasma membrane tension and stiffness increased in hepatic stellate cells as a function of substrate stiffness", + "citekey": "lachowski2022substrate", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-A6dSILE8L", + "claim": "OVCAR3 and HT1080 cells showed minimal folate receptor expression by immunofluorescence", + "citekey": "numasawa2020fluorescent", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S1" + ] + }, + { + "id": "akamatsulab-iAzvRq7UW", + "claim": "FCHSD2 localization at sites of endocytosis formed a partial ring around the base of the endocytic site by STED microscopy", + "citekey": "almeidasouza2018flat", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-Ke0qFhkQL", + "claim": "Cortactin Knockdown by siRNA decreased the degree of transferrin endocytosis in MDA-MB-231 cells", + "citekey": "chen2006roles", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "akamatsulab-A61nf3QNl", + "claim": "Fibronectin inhibited the formation of flat clathrin lattices and associated reticular adhesions", + "citekey": "hakanpaa2023reticular", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D" + ] + }, + { + "id": "akamatsulab-MvZiM_zgO", + "claim": "U2O cells plated on Col IV and LN111 produced fibronectin which assembled into elongated fibrils.", + "citekey": "hakanpaa2023reticular", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "akamatsulab-0JSkTOnvs", + "claim": "2000 pN of force produced by simulated fission yeast endocytic actin networks", + "citekey": "nickaeen2019actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A" + ] + }, + { + "id": "akamatsulab-qOwFV9hTg", + "claim": "The majority of bending energy from simulated endocytic actin networks came from capped filaments", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "akamatsulab-phR4apt3P", + "claim": "Actin polymerization kinetics assay showed that Hip1R-cortactin complex reduced actin assembly based on pyrene fluorescence", + "citekey": "leclainche2007hip1r", + "dataset": "akamatsulab", + "findings": [ + "FIG 5B" + ] + }, + { + "id": "akamatsulab-bZQte2Eb-", + "claim": "Association rate constant of Alexa-488-cofilin to rhodamine-actin filaments was 0.05-0.07 s^-1 uM^-1 for concentrations of cofilin less than 3 uM", + "citekey": "hayakawa2014singlemolecule", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C" + ] + }, + { + "id": "akamatsulab-1SinPyIpx", + "claim": "Cosedimentation assays showed a binding affinity of 0.9 \u00b1 0.1 \u00b5M between Swip1 and actin in the presence of calcium", + "citekey": "lehne2022calcium", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D" + ] + }, + { + "id": "akamatsulab-ZeSbzUTLA", + "claim": "Knockdown of integrin \u03b23 or \u03b15 in cancer associated fibroblasts (CAF's) reduced fibrillar fibronectin (but not secreted fibronectin) and migration of CT26 cancer cell spheroids.", + "citekey": "attieh2017cancerassociated", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D", + "FIG 4C", + "FIG 4B", + "FIG 4A" + ] + }, + { + "id": "akamatsulab-FmR6Quoxu", + "claim": "__in vitro__ branched actin networks decreased in capping rate as a function of growth stress", + "citekey": "li2022molecular", + "dataset": "akamatsulab", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "akamatsulab-OT8BRD36j", + "claim": "Clathrin Light chains gives the clathrin lattice more stronger bending power.", + "citekey": "dannhauser2015effect", + "dataset": "akamatsulab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "akamatsulab-_N-gQJ0eV", + "claim": "Expressing WT GFP-WASP in __wasA-__ mutants rescues confinement of active Rac to the leading edge of __Dictyostelium__ cells.", + "citekey": "amato2019wasp", + "dataset": "akamatsulab", + "findings": [ + "FIG 5F" + ] + }, + { + "id": "akamatsulab-nK9okhwH0", + "claim": "In H1299 cells, Knockdown of DNM2 with siRNA decreased the filopodial formation, and filopodia length", + "citekey": "yamada2016actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "akamatsulab-4UYkBZdjG", + "claim": "hiPSCs at the edge of the colony had larger focal adhesions than cells in the hiPSC colony center", + "citekey": "narva2017strong", + "dataset": "akamatsulab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "akamatsulab-nwh8sv1mV", + "claim": "Cells grown on curved quartz substrates coated with fibronectin or vitronectin showed preferential localization of integrin B5 to curved parts of the substrate", + "citekey": "zhang2023curved", + "dataset": "akamatsulab", + "findings": [ + "FIG 2I" + ] + }, + { + "id": "akamatsulab-DSc19RzyF", + "claim": "Knockdown of DNM2 increases the acetylated microtubule, inducing the increased adenovirus trafficking in primary keratocyte", + "citekey": "lee2019impact", + "dataset": "akamatsulab", + "findings": [ + "FIG 2C" + ] + }, + { + "id": "akamatsulab-eryKP0bHY", + "claim": "Pulldown assay with Swiss3T3 lysates showed that GST-bound IRSp53 strongly associated with WAVE, but did not precipitate N-WASP.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "akamatsulab-y7NvExQj1", + "claim": "Nanocages ranging from 12-120 tagGFP2 molecules expressed in human induced pluripotent stem cells showed a linear relationship between intensity and number of molecules", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D" + ] + }, + { + "id": "akamatsulab-x0oKAeneB", + "claim": "WASP knockout (__wasA-__) __Dictyostelium__ cells displayed bipolar localization of active Rac, rather than localization to the leading edge.", + "citekey": "amato2019wasp", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "akamatsulab-fxWgfx7yr", + "claim": "The immunoelectron microscopy revealed that Cortactin localized on flat or dome shaped-clathrin lattice, and actin filament of COS7 cells", + "citekey": "cao2003cortactin", + "dataset": "akamatsulab", + "findings": [ + "FIG 2D", + "FIG 2E", + "FIG 2F" + ] + }, + { + "id": "akamatsulab-ZMI7u4SyM", + "claim": "Increased membrane tension due to hypotonic swelling resulted in increased clathrin coat lifetimes and reduction in standard deviation of clathrin growth rates.", + "citekey": "ferguson2017mechanoregulation", + "dataset": "akamatsulab", + "findings": [ + "FIG 3A", + "FIG 3B", + "FIG 3C", + "FIG 3D", + "FIG 3E" + ] + }, + { + "id": "akamatsulab-XG3wvRdRY", + "claim": "Overexpression of a cortactin mutant lacking 3 tyrosine residues reduced CME transferrin uptake by nearly 40%", + "citekey": "zhu2007receptormediated", + "dataset": "akamatsulab", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "akamatsulab-iO13PSTbc", + "claim": "Knockdown of DNM2 reduced the transferrin internalization in neuronal differentiated CAD cells", + "citekey": "mcfarland2008rna", + "dataset": "akamatsulab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "akamatsulab-x7qaygcfw", + "claim": "Membrane invagination occurred ~6 seconds after scission protein (spHob1-GFP) was detected in budding yeast cells", + "citekey": "sun2019direct", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-bpzU9tgBb", + "claim": "Nanodissected clathrin lattices in unroofed PTK2 cells relaxed to higher curvature, lower energy configurations", + "citekey": "tagiltsev2021nanodissected", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-c4Ecoobt4", + "claim": "Endogenous Swip1 preferentially bound WT-Rab21 and constitutively active Rab21 over the closely related Rab5 GTPase in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 1E", + "FIG 1B" + ] + }, + { + "id": "akamatsulab-eglh75iLo", + "claim": "Inhibition of CG pathway in CHO cells by LG186 resulted in no increase in fluid uptake as indicated by fluorescent 10kDa Dextran upon relaxation of membrane tension following stretch.", + "citekey": "thottacherry2018mechanochemical", + "dataset": "akamatsulab", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "akamatsulab-uY8-oBWwy", + "claim": "Overexpression of the CRIB domain of N-WASP inhibited dextran and folate receptor but not transferrin internalization in FRaTb cells", + "citekey": "chadda2007cholesterolsensitive", + "dataset": "akamatsulab", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "akamatsulab-Y0BOVsMrA", + "claim": "Fluorescent actin label generally localized to STxB-induced membrane tubules in HeLa cells", + "citekey": "romer2010actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5B" + ] + }, + { + "id": "akamatsulab-bDbdRTc20", + "claim": "The folate probe FolateRSense showed fluoresecence patterns in internal spots in OVCAR-3 cells", + "citekey": "numasawa2020fluorescent", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S2" + ] + }, + { + "id": "akamatsulab-w0rQV7oyI", + "claim": "Deletion of Swip1-EF1 actin-binding region abolished Swip1 ability to facilitate integrin endocytosis.", + "citekey": "morenolayseca2021cargospecific", + "dataset": "akamatsulab", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "akamatsulab-Q5F4o4kDA", + "claim": "Hepatic stellate cells grown in a 3D environment showed the same responses to high matrix stiffness as cells grown in 2D culture (TIMP-1 and Caveolin-1 overexpression)", + "citekey": "lachowski2022substrate", + "dataset": "akamatsulab", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D", + "FIG 6E" + ] + }, + { + "id": "akamatsulab-Vmwk-ddxa", + "claim": "FRaTb-1 cells transfected with dominant negative dnm2-K44A had similar or slightly increased levels of folate receptor antibody internalization", + "citekey": "sabharanjak2002gpianchored", + "dataset": "akamatsulab", + "findings": [ + "FIG 5D" + ] + }, + { + "id": "akamatsulab-92Xr7KIW0", + "claim": "Treatment of SK-MEL2 cells with Jasplakinolide resulted in slower assembly and disassembly rates of dynamin2-GFP at sites of endocytosis", + "citekey": "grassart2014actin", + "dataset": "akamatsulab", + "findings": [ + "FIG 5F", + "FIG 5G" + ] + }, + { + "id": "akamatsulab-L3a7k3HOw", + "claim": "The folate probe FolateRSense showed fluorescence patterns in internal spots and at the periphery of KB cells", + "citekey": "numasawa2020fluorescent", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S2" + ] + }, + { + "id": "akamatsulab-5QnBT-MFL", + "claim": "Endogenously RFP-tagged clathrin light chain A recovered from photobleaching with a half life of 2 seconds at sites of endocytosis in SKMEL-2 cells", + "citekey": "avinoam2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "akamatsulab-c__rWg__T", + "claim": "IRSp53-/- MEFs or LG186-treated WT MEFs showed significant reduction in the number of CLICs per field, but no effect on clathrin or caveolae-derived structures.", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 3D" + ] + }, + { + "id": "akamatsulab-CIyKP3Kxe", + "claim": "Steady-state ATPase rate of human myo1e motor and IQ domain pleateaued at 1.2 \u00b1 0.3/s", + "citekey": "mezgueldi2002kinetic", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-oG3x2904t", + "claim": "CLIC/GEEC endosomes internalize GPI-anchored proteins.", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 1B", + "FIG 1A" + ] + }, + { + "id": "akamatsulab-Tz9iPR6Hy", + "claim": "GFP-cofilin associated with actin patches between 3 and 4.2 s after initial association of actin/Abp1p -", + "citekey": "okreglak2007cofilin", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-C8_EKRIsh", + "claim": "GFP-tagged ARPC3/Arc18 at C terminus did not affect __in vitro__ actin nucleation rates", + "citekey": "egile2005mechanism", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-Ed-cn4hbd", + "claim": "In Cav-/- MEFs, a dominant negative Dynamin mutant (DynK44A) showed an extensive tubular network of internalizing cholera toxin binding subunit (CTB), but inhibited trafficking of CTB to the Golgi.", + "citekey": "kirkham2005ultrastructural", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S4B", + "FIG 3A", + "SUPP FIG S4C" + ] + }, + { + "id": "akamatsulab-RniLbP0dj", + "claim": "Angle between membrane and clathrin pit increased from 0 to 90 (early phase/U-shaped) and 90 to 180(neck construction & scission) -", + "citekey": "avinoam2015endocytic", + "dataset": "akamatsulab", + "findings": [ + "FIG 1A" + ] + }, + { + "id": "akamatsulab-L1nUdRWr2", + "claim": "GST-WAVE1 in presence of IRSp53 and GST-WAVE2 VCA demonstrate high rates of Arp2/3-mediated actin polymerization in vitro.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 5C" + ] + }, + { + "id": "akamatsulab-ht_gHxaFG", + "claim": "The peak number of molecules of twinfilin (Twf1p) per endocytic patch in fission yeast was measured to be 210(+/- 40) by fluorescence microscopy", + "citekey": "sirotkin2010quantitative", + "dataset": "akamatsulab", + "findings": [ + "FIG 6F", + "FIG 6D" + ] + }, + { + "id": "akamatsulab-7VUX5lkA_", + "claim": "Fluorescence microscopy and image analysis of fluorescently tagged clathrin, dynamin, and arp2/3 complex in hiPSCs showed ~30% of CME events completing in the absence of detectable actin assembly", + "citekey": "jin2022branched", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S4B" + ] + }, + { + "id": "akamatsulab-ctop9ejQ4", + "claim": "Endocytic proteins Clathrin light chain A-RFP and dynamin2-GFP increased in lifetimes as a function of concentration of Arp2/3 complex inhibitor CK666 in SK-MEL-2 cells", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "akamatsulab-HAM7DEdlD", + "claim": "The GRAF1 inhibitor LG186 showed significantly reduced fluid phase uptake in WT MEFs that was comparable to the fluid phase uptake in IRSp53-/- MEFS.", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 3C" + ] + }, + { + "id": "akamatsulab-yspWhgcnu", + "claim": "Association rate constant of cofilin to actin by presteady state kinetics was 0.008/uM/s", + "citekey": "cao2006energetics", + "dataset": "akamatsulab", + "findings": [ + "FIG 3", + "TAB 2" + ] + }, + { + "id": "akamatsulab-gFbw239AO", + "claim": "Mouse embryonic fibroblasts (MEFs) from IRSp53-/- mice showed a significantly reduced fluid phase uptake as compared to WT-addback MEFs without affecting transferrin receptor internalization .", + "citekey": "sathe2018small", + "dataset": "akamatsulab", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "akamatsulab-rS-hEJjYE", + "claim": "Purified Arp2/3 complex activation by GST-WAVE2 and GST-WAVE2 VCA was stimulated in the presence of purified IRSp53 using pyrene-labelled and free monomeric actin in an actin polymerization kinetics study.", + "citekey": "miki2000irsp53", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "akamatsulab-ZWw9A8p4C", + "claim": "Between hPSC colonies plated on glass/polystyrene and on softer VTN-funtionalized hydrogels, no significant change in hPSC colony organizeation and no or modest change in FA number was observed.", + "citekey": "narva2017strong", + "dataset": "akamatsulab", + "findings": [ + "SUPP FIG S2D", + "FIG 2", + "SUPP FIG S2" + ] + }, + { + "id": "akamatsulab-ee1ItmKqR", + "claim": "The minimum number of yeast cofilin molecules required for severing is 23 +/- 11 molecules.", + "citekey": "gressin2015architecture", + "dataset": "akamatsulab", + "findings": [ + "FIG 7", + "FIG 3" + ] + }, + { + "id": "akamatsulab-9CPBwF3cc", + "claim": "The persistence length of non-stabilized actin in physiological ionic conditions was found to be $$9 \\pm 0.5 \\mu m$$", + "citekey": "isambert1995flexibility", + "dataset": "akamatsulab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "akamatsulab-nDfNWJdL-", + "claim": "Co-sedimentation assay of purified Swip1/EFhd2 and actin showed actin binds to EFhd2 in vitro.", + "citekey": "park2016structural", + "dataset": "akamatsulab", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "BIOL403-ZsSZ8qIYT", + "claim": "Mean AP-2 fluorescence lifetime increased for VSV particles compared to DI-T particles and for other pits.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3B", + "FIG 3C" + ] + }, + { + "id": "BIOL403-2oMaUVZb3", + "claim": "Camostat, a TMPRSS2 inhibitor, had a significantly greater % inhibition for Delta pseudotyped virus (PV) than for Omicron PV when infecting A549 cells overexpressing ACE2 and TMPRSS2.", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3F" + ] + }, + { + "id": "BIOL403-65HQLSARh", + "claim": "Both the mean LCa and Clathrin fluorescence lifetime increased for VSV particles compared to pit and DI-T.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3D", + "FIG 3E" + ] + }, + { + "id": "BIOL403-kibez2rYZ", + "claim": "Inhibiting polymerization of actin with latB reduced the internalization efficiency of VSV but not DI-T.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 6C" + ] + }, + { + "id": "BIOL403-LfApmzbHq", + "claim": "AP-2 fluorescence intensity increased after DI-T attachment to the BSC1 cell as the clathrin-coated pit assembled", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 2B" + ] + }, + { + "id": "BIOL403-2HHW-wfqd", + "claim": "Electron microscopy showed that with increasing exposure to heat (37C), the number of HCoV-229E viruses localized with or near the orifices (neck) of caveolae increased, where \"cross-bridge structures of high electron density were frequently observed between virus particles and the plasma membrane.\"", + "citekey": "nomura2004human", + "dataset": "BIOL403", + "findings": [ + "FIG 4A", + "FIG 4B" + ] + }, + { + "id": "BIOL403-yLpL38PuF", + "claim": "The geometries and clathrin coverage of endocytic carries for internalization of DI-T and VSV particles were different when visualized using electron microscopy", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3A", + "FIG 3B" + ] + }, + { + "id": "BIOL403-qrc8wp2qc", + "claim": "DI-T particles did not alter the process of clathrin-coated vesicle formation.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3A", + "FIG 3B", + "FIG 3C", + "FIG 3D" + ] + }, + { + "id": "BIOL403-on60mfeqE", + "claim": "\"Binding of HCoV-229E and incubation at 37\u00b0C caused clustering of CD13 and its colocalization with caveolin-1.\"", + "citekey": "nomura2004human", + "dataset": "BIOL403", + "findings": [ + "FIG 3" + ] + }, + { + "id": "BIOL403-W6_BwdDw8", + "claim": "AP-2 fluorescence in VSV particles is 2x higher and ~2x longer-lived than that of DI-T particles, which are comparable to non-viral endocytic pits; after AP-2 fluorescence peaks at 45s for VSV particles, it drops to 0 a.u. by 60s.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3B" + ] + }, + { + "id": "BIOL403-LgTH0yTQO", + "claim": "Pre-treatment of cells with the clathrin inhibitor Pitstop or Dynamin inhibitor MiTMAB reduced the number of internalized HCoV-NL63 particles in LLC-Mk2 cells", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D" + ] + }, + { + "id": "BIOL403-NwFQlJbzD", + "claim": "A strong inhibition of virus internalization in HAE cultures preincubated with clathrin or dynamin inhibitors (Pitstop and MitMAB) compared to DMSO-treated control cells was observed, using confocal microscopy.", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 7" + ] + }, + { + "id": "BIOL403-UAYD3rA57", + "claim": "DI-T particles internalized in BSC1 cells through canonical, circular clathrin coated pits, while VSV particles internalized into Vero cells in non-canonical, oblong pits as shown by electron microscopy", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 4A", + "FIG 4B" + ] + }, + { + "id": "BIOL403-oH_7_-Ju7", + "claim": "Compared to the control with DMSO, there was significant inhibition of HCoV-NL63 infection in LLC-Mk2 cells", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 1A", + "FIG 1B", + "FIG 1C", + "FIG 1D" + ] + }, + { + "id": "BIOL403-fZjAAn6XP", + "claim": "Electron microscopy showed a complete clathrin coat around DI-T particles, whereas VSV endocytic carriers seemed to be incompletely coated with clathrin.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 4A" + ] + }, + { + "id": "BIOL403-fsXgzlA-a", + "claim": "Treatment with pitstop or MiTMAB reduced the number of infected LLC-Mk2 cells following incubation with the HCoV-NL63 virus.", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 9" + ] + }, + { + "id": "BIOL403-uns4_1odd", + "claim": "During coated pit formation of DI-T, there was minimal cortactin recruitment (fluorescence), which peaked before full clathrin assembly", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "BIOL403-DRplh7Vza", + "claim": "Cortactin intensity spiked at late timepoints during VSV internalization.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5C" + ] + }, + { + "id": "BIOL403-bPmO00UbS", + "claim": "Di-T and VSV spots colocalized with AP-2 and LCa over similar periods of time", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 3B", + "FIG 3C", + "FIG 3D", + "FIG 3E" + ] + }, + { + "id": "BIOL403-vKrivrfAQ", + "claim": "inhibition of actin polymerization drastically increases the the time of VSV internalization but not DI-T, as seen with increased time of AP-2 fluorescence.", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 6B", + "FIG 6D" + ] + }, + { + "id": "BIOL403-QK-IPLtB0", + "claim": "When human fibroblasts were treated with anti-CD13 antibody and anti-caveolin-1 antibody for 60 minutes at 37C, an increase in fluorescence of CD13 and colocalization of caveolin-1 was observed.", + "citekey": "nomura2004human", + "dataset": "BIOL403", + "findings": [ + "FIG 2" + ] + }, + { + "id": "BIOL403-o-DGibStS", + "claim": "LLC-MK2 cells showed no significant differences in replication of HCoV-NL63 under a controlled environment versus a camostat environment", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 11A", + "FIG 11B" + ] + }, + { + "id": "BIOL403-1bD48wYTX", + "claim": "The creation of clathrin-coated pits in BSC1 cells showed minimal cortactin recruitment based on time lapse fluorescence assays", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5A", + "FIG 5B" + ] + }, + { + "id": "BIOL403-8jYihJXs1", + "claim": "Cortactin intesity spikes during VSV internalization", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 5B", + "FIG 5C", + "FIG 5D" + ] + }, + { + "id": "BIOL403-C0vEc-VbK", + "claim": "Depletion of TMPRSS2 afftected the entry of WT and Delta PV to a greater extent than that of the Omicron PV, in a manner dependent on ACE2 expression levels.", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3C", + "FIG 3D" + ] + }, + { + "id": "BIOL403-JEZVJFX83", + "claim": "E64d, a cathepsin inhibitor, had a significantly greater % inhibition for Omicron pseudotyped virus (PV) than for Delta PV.", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3F" + ] + }, + { + "id": "BIOL403-kPjXx4aUP", + "claim": "VSV and DI-T particles are different lengths", + "citekey": "cureton2010length", + "dataset": "BIOL403", + "findings": [ + "FIG 1C" + ] + }, + { + "id": "BIOL403-8-zvLzqhz", + "claim": "There was a decrease in internalized viral particles when treated with classical clathrin-mediated endocytosis inhibitors Pitstop 2 and MiTMAB.", + "citekey": "milewska2018entry", + "dataset": "BIOL403", + "findings": [ + "FIG 6A", + "FIG 6B", + "FIG 6C", + "FIG 6D" + ] + }, + { + "id": "BIOL403-jUOkXBg6J", + "claim": "Using A549-ACE2/TMPRSS2 cells a noticeable, but not statistically significant, difference between live viral forms of omicron and delta, when treated with either TPRSS2 or cathepsin inhibitors", + "citekey": "meng2022altered", + "dataset": "BIOL403", + "findings": [ + "FIG 3G" + ] + }, + { + "id": "dg-social-media-polarization-NAv6HKLye", + "claim": "Respondents were not very polarized in function of their party alignment in the US, with fewer than one in five scoring high on the polarization scale.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization--eEnETf9u", + "claim": "Heavy Facebook users were more likely to consume information from untrustworthy websites, which was often immediately preceded by a visit to Facebook.", + "citekey": "guessExposureUntrustworthyWebsites2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-dOZ6zAzVl", + "claim": "SEM modeling of social media use showed an indirect yet positive effect on polarization for moderate liberals via increased polarization", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-wg-tL_YH6", + "claim": "According to a conditional latent growth curve model (LGM), American adults' online political expression increased support for their candidate.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-PKXjaKH1E", + "claim": "Strong pro-refugee sentiment amongst Germans was associated with ~2x higher rates of higher education compared to strong anti-refugee sentiment", + "citekey": "arltBiasWantedExamining2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4" + ] + }, + { + "id": "dg-social-media-polarization-RV0XA6D3B", + "claim": "The majority of Germans fall between extreme positions regarding refugees, with only a small minority expressing extreme pro-refugee or anti-refugee attitudes", + "citekey": "arltBiasWantedExamining2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-LP0AEgKvL", + "claim": "Polarized users in likewise polarized neighborhoods distributed their likes across their community pages in a similar way, both in science and conspiracy communities.", + "citekey": "brugnoliRecursivePatternsOnline2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-1OX4Ja3Bs", + "claim": "The interaction between dual national identity, identifying as both Hong Kongese and Chinese, and political use of social media was negatively associated with attitudinal polarization on position issues.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-GJX02PUU3", + "claim": "Self-reporting a dual nationality identity, identifying as both Hong Kongese and Chinese, was negatively associated with attitudinal polarization on valence issues in Hong Kong.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-rwVKYrlHN", + "claim": "Substantial selective exposure was heavily concentrated among a subset of Americans with conservative information diets.", + "citekey": "guessExposureUntrustworthyWebsites2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1", + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-BGpeJrpUb", + "claim": "According to a repeated-measures ANOVA, and latent growth curve modeling (LGM, unconditional model), individuals' candidate preferences grew more stable as Election Day approached.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-w5xrqKLI_", + "claim": "According to analyses of Facebook data, people's egonets misrepresented how many friends other people have.", + "citekey": "feldEgonetsSystematicallyBiased2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-SVzr-eboO", + "claim": "Authors on the liberal sites were much more favorable toward left-leaning people and groups than authors on the conservative sites. In addition, those on the left- and right-leaning sites were more extreme than those at the moderate blog on this variable", + "citekey": "suhayForgingBondsBurning2015", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-CfQF8Cm-z", + "claim": "Fifty-five percent of issue-related conversations involved interactions between users from both sides of union debate.", + "citekey": "balcellsCrossingLinesTwitter2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-NwBCcwVBq", + "claim": "Extreme users strongly prioritized cognitive congruence in news sharing, in both pro- and anti-Bolsonaro communities. Ideological congruence become less important towards the network center.", + "citekey": "arugueteNewsSharingGatekeeping2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 7" + ] + }, + { + "id": "dg-social-media-polarization-TMN0eQB8C", + "claim": "For respondents in Hong Kong with a single identity, either only Hong Kongese or only Chinese, model estimates of affective polarization increased from 30\u00b0 to 40\u00b0 with frequent social media use, while for those with a dual identity, both Hong Kongese and Chinese, it decreased from 30\u00b0 to 12\u00b0 with frequent social media use.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-NTikknFZ7", + "claim": "Both Republican and Democrat users reported change in self-reported political ideology towards their own ideological extreme when exposed to opposing views on Twitter, yet only Republicans' effects were statistically significant", + "citekey": "bailExposureOpposingViews2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-gyOhM4gnR", + "claim": "Confidence in the president and Congress was positively associated with levels of selective exposure to social media in the US.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-G6Dt9EUlZ", + "claim": "AccoA Random Forest algorithm was able to predict the polarization of Twitter users with high accuracy and recall.", + "citekey": "marozzoAnalyzingPolarizationSocial2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 7" + ] + }, + { + "id": "dg-social-media-polarization-i61FjD7N_", + "claim": "Deactivating facebook reduced various measures of political polarization, particularly exposure to news that improve understanding of their own political party (vs. other party), and issue polarization, but not affective polarization.", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-r-1ymhoVX", + "claim": "Enthusiasm among US respondents toward the out-party presidential candidate was positively associated with disagreement expression on social media.", + "citekey": "choiEnthusiasmOtherSide2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-VlBq9h_85", + "claim": "According to SEM modeling, social media use was negatively associated with a shift towards conservatism for political neutrals, and positively associated with a shift towards liberalism", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-FnfG3r2bC", + "claim": "Facebook users who reported higher differences between feeling-thermometer measures for their own party vs. the other party registered more Facebook use which lead to over-time depolarization", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-ttY5ZRIo8", + "claim": "The fragmentation of the audiences of Russian media outlets on the Vkontakte platform occurred mainly along political lines", + "citekey": "urmanNewsConsumptionRussian2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-GMnFJ3he4", + "claim": "SEM modeling showed that, for moderate conservatives, there was no direct or indirect effect of social media use for adopting extreme conservatism", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-Wtxp_oJDN", + "claim": "Most of the users who retweeted rumor rebuttal tweets displayed the agree attitude. However, the users who commented on rumor rebuttal tweets had more diversified attitudes under various topics, with the majority of individuals holding query or unknown attitudes", + "citekey": "wangEchoChamberEffect2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3", + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-Zo4cVaD34", + "claim": "Longer lifetime and higher levels of activity of Facebook users corresponded with a lesser number of Facebook pages being consumed.", + "citekey": "schmidtPolarizationVaccinationDebate2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-sV9j_ssrE", + "claim": "All of the derogatory words used to talk about Remoaners on Twitter contain negative tribal associations.", + "citekey": "northBattleBritainAnalyzing2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1B" + ] + }, + { + "id": "dg-social-media-polarization-AQy-nbEwf", + "claim": "27 out of 38 events of the Brexit timelines were associated with unusual spikes in the volume of tribal keywords outside of the normal range.", + "citekey": "northBattleBritainAnalyzing2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-85I3FMzkf", + "claim": "Smaller Japanese media outlets Twitter accounts had ideologically distinctive followers and were mainly isolated in the network.", + "citekey": "kobayashiNewsAudienceFragmentation2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-TEas5VTq3", + "claim": "More reputable outlets had greater incentives to adjust editorial lines. Less reputable outlets were already sending messages congruent to their preferred user.", + "citekey": "arugueteNewsSharingGatekeeping2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-YnZuD0iG2", + "claim": "Republican users exposed to opposing views on Twitter had an increase in conservatism between 0.11 and 0.59 standard deviations", + "citekey": "bailExposureOpposingViews2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-5xJkG_dQv", + "claim": "The majority of Facebook users were active either in the pro-vaccines or anti-vaccines community, not both.", + "citekey": "schmidtPolarizationVaccinationDebate2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-1JvBH0nCz", + "claim": "Only at the first wave of data, Facebook users who reported higher differences between feeling-thermometer measures for their own party vs. the other party were more likely to view news on Facebook", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-DL7lKY_N6", + "claim": "Exposure to counter-attitudinal content on Facebook modestly decreased the affective polarization index compared to the pro-attitudinal treatment", + "citekey": "levySocialMediaNews2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-sffZqWHW4", + "claim": "Dual identifying Hong Kong residents were 14.8% more likely to have a weak partisan selectivity than single identifiers.", + "citekey": "kobayashiDepolarizationSocialMedia2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-PjQJXsOzF", + "claim": "Anger and anxiety among US respondents toward the out-party presidential candidate wass not associated with disagreement expression on social media.", + "citekey": "choiEnthusiasmOtherSide2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-tQy4sEF_R", + "claim": "Perceived polarization increased as a function of time spent reading a tweet, but only for Republican users", + "citekey": "banksPolarizedFeedsThreeExperiments2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 6", + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-2L5_w1IYK", + "claim": "Deactivating Facebook boosted participants' perception of its role in improving news consumption and increased agreement on the potential impact of deactivating one's account.", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-aJngFbteo", + "claim": "deactivating Facebook resulted in improvements on measurements of subjective well-being", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 5" + ] + }, + { + "id": "dg-social-media-polarization-5OWXeEZWn", + "claim": "Selective exposure and selective avoidance predict polarization, but they are weaker indicators than strength of party ties.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-eIcyC6cqo", + "claim": "The level of encountered disagreement on social medi predicted the likelihood of expressing disagreement for US respondents, even when accounted for the initial level of expressed disagreement.", + "citekey": "choiEnthusiasmOtherSide2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-qyujJigSk", + "claim": "The retweeting and commenting user networks of rumor rebuttal under different topics showed highly modular structures; however, the large clusters in retweeting user networks showed high homophily, while the large clusters in commenting user networks had mixed attitudes", + "citekey": "wangEchoChamberEffect2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-PSA7511kK", + "claim": "According the a polarization index, Italian news sites are strongly polarizated on the issue of the Italian constitutional referendum.", + "citekey": "marozzoAnalyzingPolarizationSocial2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-7joN8S4Cb", + "claim": "Pro-attitudinal news uses were not found to mediate the relationship between Facebook use at wave 1 and higher differences between feeling-thermometer measures for users' own party vs. the other party at wave 3", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-DH7kbqJd1", + "claim": "According to regression analysis, heterogeneous conversations were longer when they involved users who are placed on the more moderate and central positions of the debate, and were probably more disposed to hear the other side.", + "citekey": "balcellsCrossingLinesTwitter2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4" + ] + }, + { + "id": "dg-social-media-polarization-V9kqzF5Vc", + "claim": "The variance of response about EU issues between those who got most of the news from social networks or other types of media was very small for participants who had positive predispositions toward the EU.", + "citekey": "nguyenTestingPopularNews2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-4pRj56pL2", + "claim": "Regression analyses of American adults showed that the relationship between political party and political opinions increased as people expressed themselves politically.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-fjmFnfZxi", + "claim": "Germans across the ideological spectrum perceived the media to have biases favoring their opposing ideological group on the topic of refugees", + "citekey": "arltBiasWantedExamining2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4" + ] + }, + { + "id": "dg-social-media-polarization-l-GuCGg0p", + "claim": "According to regression analysis, heterogeneity increased the level of depth of conversations.", + "citekey": "balcellsCrossingLinesTwitter2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 4", + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-KwHHkPC99", + "claim": "SEM modeling showed that, among neutrals, social media use was positively associated with political engagement, which was also positively associated with political polarization toward liberalism.", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-tad5MxAmU", + "claim": "According to a survey, educated Ethiopians believed that social media helps and hurts democracy in Ethiopia.", + "citekey": "worknehSocialMediaProtest2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 5" + ] + }, + { + "id": "dg-social-media-polarization-F6qxFkTUx", + "claim": "Facebook news use at wave 1 resulted in depolarization at wave 3 via exposure to counter-attitudinal news", + "citekey": "beamFacebookNewsPolarization2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-00d1hdyKn", + "claim": "According to regression analyses, individuals on WhatsApp and Twitter became more embedded in homophilic interaction networks than Facebook users.", + "citekey": "yarchiPoliticalPolarizationDigital2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-CYdetKaa4", + "claim": "Deactivating Facebook resulted in significant declines in both self-reported news attention and objectively measured news knowledge.", + "citekey": "allcottWelfareEffectsSocial2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-EEv4t_Zmw", + "claim": "Engaging in polarizing content increased the probability to have friends with similar characteristics.", + "citekey": "brugnoliRecursivePatternsOnline2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 5" + ] + }, + { + "id": "dg-social-media-polarization-D6WhqzXF4", + "claim": "The out-party feed increased affective polarization compared to the control group, but the difference was not statistically significant", + "citekey": "shmargadSortingNewsHow2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-fjnZM77gZ", + "claim": "The in-party feed slightly decreased affective polarization compared to the control group, but this difference was not statistically significant", + "citekey": "shmargadSortingNewsHow2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 8" + ] + }, + { + "id": "dg-social-media-polarization-J8B-ui82c", + "claim": "Most Japanese media accounts were followed on Twitter by both liberal and conservative users, and the followers of major media accounts overlapped to a significant extent.", + "citekey": "kobayashiNewsAudienceFragmentation2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3" + ] + }, + { + "id": "dg-social-media-polarization-Oij-q4JsF", + "claim": "Republicans were more likely than Democrats to share extreme *pro*-Republican news sources and less likely to share extreme *pro*-Democratic news sources, and vice versa.", + "citekey": "osmundsenPartisanPolarizationPrimary2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-Q2jgcTrzF", + "claim": "The effect of being offered subscriptions to news outlets from the opposite ideological position on a political opinions index was small and not statistically significant.", + "citekey": "levySocialMediaNews2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-FhiobBveu", + "claim": "SEM modeling showed that social media use by liberals was positively associated with political engagement, and in turn, was positively associated with political polarization toward strong liberalism", + "citekey": "leeDoesSocialMedia2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-rW9j9BLFE", + "claim": "Estimating the conditional effects of the data for American adults showed that party identification predicted political opinions at any value of political expression, but the relationship was stronger when political expression was high.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-Dnlq8A7OQ", + "claim": "The cognitive reflection test was weakly associated with sharing real news sources.", + "citekey": "osmundsenPartisanPolarizationPrimary2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-pz9Ofmmt_", + "claim": "The majority (80-90%)of networks of tweets about the Italian referendum shared links had the same model-estimated stance towards the referendum as the users who shared them. Retweet and quote networks had almost 100% of within-stance.", + "citekey": "laiStancePolarityPolitical2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 10" + ] + }, + { + "id": "dg-social-media-polarization-OzPq5b9Bu", + "claim": "Derogatory Brextremist discourse on Twitter included many terms that suggested lack of sanity (e.g., nutters, loonies) and references to fear and hatred of foreigners and destruction of Britain.", + "citekey": "northBattleBritainAnalyzing2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "dg-social-media-polarization-rw_F8t-BK", + "claim": "The variance of response about EU issues between those who got most of the news from social networks or other types of media was very small for participants who had negative predispositions toward the EU.", + "citekey": "nguyenTestingPopularNews2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-wF7z2zjF7", + "claim": "Party identification predicts political opinions at any value of political expression but the relationship is stronger when political expression is high.", + "citekey": "choInfluencingMyselfSelfReinforcement2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-tsKBuSu4g", + "claim": "US respondents who self-reported selectively using social networking sites with political content that aligned with their own views were more likely to have a stronger difference in favorability ratings between Republican vs. Democratic parties.", + "citekey": "johnsonBlindedSpitePath2017", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 2" + ] + }, + { + "id": "dg-social-media-polarization-URbbXnWk0", + "claim": "According to regression analysis, social network heterogeneity positively influenced ethnic, ideological, and party polarization.", + "citekey": "kibetSociallyNetworkedHeterogeneity2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 3" + ] + }, + { + "id": "dg-social-media-polarization-b1nT-0nmZ", + "claim": "As shown by comparing percentages between Facebook, Twitter, and WhatsApp users, homophily was higher on Twitter and WhatsApp than Facebook.", + "citekey": "yarchiPoliticalPolarizationDigital2020", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 1" + ] + }, + { + "id": "dg-social-media-polarization-fZx8-Rfca", + "claim": "US survey respondents self-reported greater ideological distance between Donald Trump and Hillary Clinton after seeing negative campaign tweets from either candidate, compared to seeing no tweets", + "citekey": "banksPolarizedFeedsThreeExperiments2021", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 3", + "TAB 1" + ] + }, + { + "id": "dg-social-media-polarization-xkB9Y4f6Z", + "claim": "Users polarized towards a narrative tended to consume nearly exclusively content adhering to their system of beliefs.", + "citekey": "brugnoliRecursivePatternsOnline2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 4" + ] + }, + { + "id": "dg-social-media-polarization-nHQRpPGyv", + "claim": "Multiple mediation analyses showed that posting news in WhatsApp groups was associated with exposure to diverse perspectives.", + "citekey": "kibetSociallyNetworkedHeterogeneity2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 2" + ] + }, + { + "id": "dg-social-media-polarization-F0emNfngi", + "claim": "There were no striking divisions between users exposing different stances in the reply-to Twitter network of the Italian referendum.", + "citekey": "laiStancePolarityPolitical2019", + "dataset": "dg-social-media-polarization", + "findings": [ + "FIG 9" + ] + }, + { + "id": "dg-social-media-polarization-LSw8SXuEz", + "claim": "According to Poisson regression, women were underrepresented among top Instagram degrees, indicative of a glass ceiling effect (p.3).", + "citekey": "stoicaAlgorithmicGlassCeiling2018", + "dataset": "dg-social-media-polarization", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-zwlHilzun", + "claim": "Students provided expert instruction(A teacher with a P.H.D in physics) performed as well as groups given the same instruction by a different teacher and provided analogical instruction", + "citekey": "jacobsonSchemaAbstractionProductive2020", + "dataset": "megacoglab", + "findings": [ + "TAB 8" + ] + }, + { + "id": "megacoglab-xMEXh8Glg", + "claim": "papers with high median conventionality and high tail atypical combinations of journals they cited were 2x more likely than average to be in top 5 percent of citation distribution", + "citekey": "uzziAtypicalCombinationsScientific2013", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-LJtUs-1OU", + "claim": "statistically significant but small differences in bibliometric measures of consensus across papers in physical, hard and soft biological, and social sciences", + "citekey": "fanelliBibliometricEvidenceHierarchy2013", + "dataset": "megacoglab", + "findings": [ + "FIG 4", + "TAB 2" + ] + }, + { + "id": "megacoglab-3jTc6a_9h", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventors who was new to the patent's field tended to receive fewer forward citations, but less so when they collaborated with an expert in the new field", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 7" + ] + }, + { + "id": "megacoglab-g8wge1Vez", + "claim": "Final advertisements from designers who prototyped designs in parallel performed better on advertising analytics compared to ads from designers who prototype designs serially", + "citekey": "dowParallelPrototypingLeads2010", + "dataset": "megacoglab", + "findings": [ + "FIG 7", + "TAB 1" + ] + }, + { + "id": "megacoglab-uwJCgdA2o", + "claim": "undergraduates used more complex integration approaches (vs. property transfer and hybridization approaches) when generating definitions for noun phrases from dissimilar vs. similar noun pairs", + "citekey": "wilkenfeldSimilarityEmergenceConceptual2001", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-AZ-LvsLz_", + "claim": "students scored higher on biology post-test learning quizzes when given instruction with complex instructional analogies compared to no analogies", + "citekey": "bakerComplexInstructionalAnalogies2001", + "dataset": "megacoglab", + "findings": [ + "TAB 1", + "TAB 2" + ] + }, + { + "id": "megacoglab-BV-n51_DU", + "claim": "There was an average of 33% improvement in forecasting accuracy (3 out of 4 debate questions answered more accurately.)", + "citekey": "irwinForecastingArgumentationFrameworks2022", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-inEj6W9YH", + "claim": "engineering students used more between-domain keywords used to search for patents in interactive visualization with VISION (functional component representations of patents compared to a standard Google Patents interface)", + "citekey": "songDesignbyAnalogyEffectsExplorationBased2022", + "dataset": "megacoglab", + "findings": [ + "FIG 12" + ] + }, + { + "id": "megacoglab-TAgkEfWtq", + "claim": "Students given analogical instruction with two models performed better on far domain problems than students provided only a single model to study", + "citekey": "jacobsonSchemaAbstractionProductive2020", + "dataset": "megacoglab", + "findings": [ + "TAB 7", + "FIG 3" + ] + }, + { + "id": "megacoglab-TkvbwvSxI", + "claim": "users were more likely to select clusters when an ACT-IF model's prediction of cluster profitability was equal to the expected rate of information gain", + "citekey": "pirolliInformationForaging1999", + "dataset": "megacoglab", + "findings": [ + "FIG 18" + ] + }, + { + "id": "megacoglab-rjVivNG51", + "claim": "a substantial number of IDEO-designed products incorporated technological solutions from other industries", + "citekey": "hargadonTechnologyBrokeringInnovation1997", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-6ddZeIyjB", + "claim": "Subjective ratings of information scent model derived from the task environment with ACT-IF tightly correlated with users' judgments of information relevance on a Scatter-Gather task", + "citekey": "pirolliInformationForaging1999", + "dataset": "megacoglab", + "findings": [ + "FIG 16" + ] + }, + { + "id": "megacoglab-e5Os8ZONI", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventors who were new to the patent's field tended to be more novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 7" + ] + }, + { + "id": "megacoglab-r9OyxsAJe", + "claim": "patenting teams with a greater number of prior patenting fields were more likely to generate patents that were technological outliers", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-RTVDpn3Mv", + "claim": "A patent's odds of being an outlier increases by 1.20 times for each doubling of other patents cited", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-tYJs3SOGO", + "claim": "inventors' prior experience with knowledge in focal and citing US patents subclasses intensified the U-shaped relationship between technological complexity and exaptation", + "citekey": "mastrogiorgioInnovationExaptationIts2016", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-QgrzIjqGl", + "claim": "amongst 9 major medical question-answering systems, none addressed conflicting evidence or communicate uncertainty to clinicians", + "citekey": "kellWhatWouldIt2021", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-nSzMQuhLB", + "claim": "case-based reasoning system loaded with diverse cases increased overall novelty of ad campaign ideas, but only for people with lower creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-nFbTK1YKO", + "claim": "Experts generated more analogies per minute than novices", + "citekey": "ballSpontaneousAnalogisingEngineering2004", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-LB6bYkRJj", + "claim": "higher levels of interdisciplinary associated with higher visibility", + "citekey": "leaheyProminentLessProductive2017", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-FLl3xFSpU", + "claim": "similar secondary attack rate for household contacts 18yo or less (compared to other age groups) in Midwestern US", + "citekey": "yousafProspectiveCohortStudy2020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-3rIy1jtKe", + "claim": "teams were more likely than single or pair-authored papers to cite atypical combinations of journals", + "citekey": "uzziAtypicalCombinationsScientific2013", + "dataset": "megacoglab", + "findings": [ + "FIG 3A" + ] + }, + { + "id": "megacoglab-inz0nxyak", + "claim": "undergrad 1980s textbooks in physics and chemistry cited far fewer texts compare to sociology textbooks", + "citekey": "coleHierarchySciences1983", + "dataset": "megacoglab", + "findings": [ + "TAB 10" + ] + }, + { + "id": "megacoglab-_FWs0NXx5", + "claim": "Experts employed more schema-driven analogizing than case-driven analogizing; novices showed the reverse pattern", + "citekey": "ballSpontaneousAnalogisingEngineering2004", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-UIVkpC3D_", + "claim": "viewers in Twitch chat engaged in less bad behaviors after a user was banned by a moderator for that bad behavior", + "citekey": "seeringShapingProAntiSocial2017", + "dataset": "megacoglab", + "findings": [ + "TAB 8" + ] + }, + { + "id": "megacoglab-FpvCvjJU9", + "claim": "interdisciplinary humanities scholars' work required a variety of information sources across different stages of exploration and translation work", + "citekey": "palmerInformationWorkInterdisciplinary2002", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-GxyCjeS8g", + "claim": "No analogical transfer amongst undergraduates between insight problems without hints about analogical mapping", + "citekey": "reedRoleAnalogyTransfer1974", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-L5D-9bBxY", + "claim": "approximately 2x lower prevalence of secondary cases for children vs. adults in households in Wuhan", + "citekey": "wangHouseholdTransmissionSARSCoV22020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-nQ-MNtexz", + "claim": "researchers were far more likely to self-report communicating frequently with their collaborators in the planning and writing stages of the research process if they were physically proximate, especially next door offices", + "citekey": "krautPatternsContactCommunication1988", + "dataset": "megacoglab", + "findings": [ + "FIG 1D" + ] + }, + { + "id": "megacoglab-GOqzI-lPp", + "claim": "engineering students more likely to generate ideas that directly copy (mostly within-domain) analogous patents when using Google Patents to retrieve patents, vs. VISION (interactive visualization with functional component representations of patents)", + "citekey": "songDesignbyAnalogyEffectsExplorationBased2022", + "dataset": "megacoglab", + "findings": [ + "FIG 9" + ] + }, + { + "id": "megacoglab-RqfcscSpW", + "claim": "No secondary cases amongst secondary school students in a COVID cluster in Singapore with adults as index cases", + "citekey": "yungNovelCoronavirus20192020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-udEyztUZu", + "claim": "majority of sample of 70 paper recommendation approaches were evaluated with offline approaches; only 7% were evaluated in real usage scenarios", + "citekey": "beelResearchPaperRecommender2013", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-pyNvD2Acy", + "claim": "three commercial facial recognition classifiers performed better on lighter vs. darker skinned subjects in the pilot parliaments benchmark dataset", + "citekey": "buolamwiniGenderShadesIntersectional2018", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-rllJ6VS1E", + "claim": "participants spent more time looking at content vs. organization in maps created by one other person, but equally at content and organization for maps that were iterated on by multiple people", + "citekey": "fisherDistributedSensemakingImproving2012", + "dataset": "megacoglab", + "findings": [ + "FIG 3", + "TAB 2" + ] + }, + { + "id": "megacoglab-7GVGV9S2j", + "claim": "undergraduate students' ideas were more likely to include critical features of examples they had seen (relative to ideas of students who hadn't seen the examples)", + "citekey": "smithConstrainingEffectsExamples1993", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-KQ6aLkCGV", + "claim": "Molecular biologists with a reputation for innovation rarely used very far analogies in their lab meetings while generating novel scientific concepts; instead, they relied mainly on analogies to the same or other biological organisms", + "citekey": "dunbarHowScientistsThink1997", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-b8pGDFeTc", + "claim": "US patents filed by inventors who were new to the patent's field tended to receive fewer forward citations, but less so when they collaborated with an expert in the new field", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-qbG7q-Tp_", + "claim": "Participants who marked up their own papers with a formal semantic scheme perceived the task of formally marking claims in the text to be intuitive, in contrast to formally marking rhetorical relations between claims", + "citekey": "grozaSALTWeavingClaim2007", + "dataset": "megacoglab", + "findings": [ + "FIG 7" + ] + }, + { + "id": "megacoglab-R--E3kMiR", + "claim": "Two artists used individual artworks to shape not just their ideas, but used a process of \"analogical modification\" to search for and modify higher-order concepts, and their creative vision. This process unfolded over the course of years.", + "citekey": "okadaAnalogicalModificationCreation2009", + "dataset": "megacoglab", + "findings": [ + "FIG 10", + "FIG 3" + ] + }, + { + "id": "megacoglab-C1uFTRfBX", + "claim": "less than 1% of pubpeer comments were positive reviews", + "citekey": "ortega2022classification", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 3" + ] + }, + { + "id": "megacoglab-W3sdOb60i", + "claim": "US patents filed by inventors who were new to the patent's field tended to be more novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-zHpJGF1Ih", + "claim": "undergraduates generated novel word combination noun phrases with more emergent features when noun pairs were dissimilar vs. similar", + "citekey": "wilkenfeldSimilarityEmergenceConceptual2001", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-lJcUil9G6", + "claim": "higher conceptual flexibility to classify organisms based on taxonomic and ecological relations if children were older, from more rural areas, and engaged in more unstructured interactions with organisms", + "citekey": "betzDevelopmentConceptualFlexibility2020", + "dataset": "megacoglab", + "findings": [ + "TAB 9" + ] + }, + { + "id": "megacoglab-yMCU6h2YU", + "claim": "in an interview and survey, data scientists reported major (unsupported) pain points around sharing and collaborating on notebooks in data science teams", + "citekey": "chattopadhyayWhatWrongComputational2020", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 2" + ] + }, + { + "id": "megacoglab-6e7Wu6JTF", + "claim": "overall marginally significant negative effect of analogically distant cases on creativity of campaign ad ideas, mostly for students with high creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 2" + ] + }, + { + "id": "megacoglab-2yEG0_6MI", + "claim": "engineering students who prototyped designs in parallel generated significantly more diverse designs compared to students who did iterative prototyping", + "citekey": "murphyComparingParallelIterative2022", + "dataset": "megacoglab", + "findings": [ + "FIG 5", + "TAB 2" + ] + }, + { + "id": "megacoglab-zWGskgjzd", + "claim": "When provided visual analogies 4th graders performed better than the students given just the target concept. However, This was not observed in 5th graders.", + "citekey": "matlenEnhancingComprehensionScience", + "dataset": "megacoglab", + "findings": [ + "FIG 4", + "FIG 3" + ] + }, + { + "id": "megacoglab-4oupzhYHs", + "claim": "No secondary cases amongst preschoolers in a COVID cluster in Singapore with a student as index case", + "citekey": "yungNovelCoronavirus20192020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-fdgQruzE4", + "claim": "higher levels of interdisciplinarity was significantly associated with fewer publications, particularly in fields with less interdisciplinarity", + "citekey": "leaheyProminentLessProductive2017", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-ledAQMl6P", + "claim": "highest self-rated map quality, as measured by overall quality, and helpfulness to others, when creating map based on previous map that was iterated on by multiple people, compared to creating from scratch, and basing on a map created by one other person, for the same everyday sensemaking topic", + "citekey": "fisherDistributedSensemakingImproving2012", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-KxZbYojmj", + "claim": "Similar secondary attack rate for children vs. adults in Shenzen", + "citekey": "biEpidemiologyTransmissionCOVID192020", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-W-DsHoVoj", + "claim": "evaluators gave higher scores to biomed grant proposals that were farther from their domain of expertise", + "citekey": "boudreauLookingLookingKnowledge2016", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-IFtWTd_4B", + "claim": "Students were able to alter their conceptions after being provided with bridging analogies", + "citekey": "bryceEncouragingConceptualChange2005", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-v22_1oEQw", + "claim": "5x lower secondary attack rate for children compared to adults in households in China", + "citekey": "liCharacteristicsHouseholdTransmission2020", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-JZU-T5AFL", + "claim": "Scientists more often nonlinearly read subsections of papers for findings, hypotheses or other information, rather than linearly reading whole documents", + "citekey": "ribaupierreExtractingDiscourseElements2017", + "dataset": "megacoglab", + "findings": [ + "FIG 3", + "FIG 2" + ] + }, + { + "id": "megacoglab-AyXYy7QOO", + "claim": "overall salience of taxonomic relations for organizing living things for 6-12 year old children during an initial card sort of the organisms", + "citekey": "betzDevelopmentConceptualFlexibility2020", + "dataset": "megacoglab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "megacoglab-Vpd-mdiMN", + "claim": "working in groups with others who share your initial bias towards a hypothesis intensified that bias over time, compared to working alone or with other students who preferred different hypotheses", + "citekey": "convertinoCACHEStudyGroup2008", + "dataset": "megacoglab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "megacoglab-NTnlzmqwG", + "claim": "an ACT-IF model's ranking of actions to do next on a Scatter-Gather task was a better fit to users' actual actions compared to a random model", + "citekey": "pirolliInformationForaging1999", + "dataset": "megacoglab", + "findings": [ + "FIG 17" + ] + }, + { + "id": "megacoglab-sBhdunCTd", + "claim": "In a design team, concepts tended to be more similar to their immediately preceding concepts after far analogy use compared to using near or no analogies", + "citekey": "chanImpactAnalogiesCreative2015", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "TAB 11" + ] + }, + { + "id": "megacoglab-SYNSopX8w", + "claim": "Participants who searched through clumpier distributions in space behaved as if words were more densely clumped in the Scrabble task", + "citekey": "hillsSearchExternalInternal2008", + "dataset": "megacoglab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "megacoglab-k3fuaSbE1", + "claim": "users from subreddits banned for hate speech did not engage in hate speech in new forums they joined", + "citekey": "chandrasekharanYouCanStay2017", + "dataset": "megacoglab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "megacoglab-wMIhhzUaI", + "claim": "participants who used ART - Voyager 2 explored ~25% more of the space of possible visualizations compared to a baseline system without wildcard and related views for recommended visualizations", + "citekey": "wongsuphasawatVoyagerAugmentingVisual2017", + "dataset": "megacoglab", + "findings": [ + "FIG 9" + ] + }, + { + "id": "megacoglab-9c5iLH-3V", + "claim": "Far analogies' effects on MechE students' novelty of ideas were different depending on timing: more novel ideas when seeing them after problem solving began vs. before", + "citekey": "tsengRoleTimingAnalogical2008", + "dataset": "megacoglab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "megacoglab-p2hCTLXaE", + "claim": "Comparable secondary attack rate in households in China for children under 3 compared to other age groups", + "citekey": "wuHouseholdTransmissionSARSCoV22020", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-jScHaY6Fl", + "claim": "estimated semantic networks of animal concepts from montessori-educated children were more interconnected, with shorter paths between concepts and fewer subcommunities, compared to networks from traditional-schooled but comparable children", + "citekey": "denervaudEducationShapesStructure2021", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "FIG 2" + ] + }, + { + "id": "megacoglab-fCGl7qdb0", + "claim": "researchers were more likely to collaborate with researchers whose offices were close by (e.g., same corridor or floor vs. different floor or building); pattern is robust to whether researchers were from the same department or not", + "citekey": "krautPatternsContactCommunication1988", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-k6yG1FHks", + "claim": "household secondary attack rate for children of index cases in Singapore was about 6 percent, and increased with age", + "citekey": "yungHouseholdTransmissionSevere2020", + "dataset": "megacoglab", + "findings": [ + "TAB NA" + ] + }, + { + "id": "megacoglab-ZkiOwUfXJ", + "claim": "No significant correlation between analogical distance of cross-industry inspirations and degree of innovation outcome (incremental, market or breakthrough, and radical)", + "citekey": "enkelCreativeImitationExploring2010", + "dataset": "megacoglab", + "findings": [ + "TAB 5" + ] + }, + { + "id": "megacoglab-zGQwQiY6L", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventor teams that included an expert in the patent's field tended to be less novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 7" + ] + }, + { + "id": "megacoglab-7Xw3gepU9", + "claim": "An additional 10 fields of experience doubles the probability of an inventor with average fields of experience being an outlier", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-pTruDj61B", + "claim": "Far analogies were rare and never used in psychology lab group meetings for reasoning (vs. mere mentions); in contrast, far analogies were frequently used in colloquia", + "citekey": "sanerAnalogiesOutBlue1999", + "dataset": "megacoglab", + "findings": [ + "TAB 3", + "TAB 2" + ] + }, + { + "id": "megacoglab-kWpt95CTL", + "claim": "crowd workers were at chance accuracy in distinguishing human-written from GPT-3-written stories, news stories, and recipes", + "citekey": "clarkAllThatHuman2021", + "dataset": "megacoglab", + "findings": [ + "TAB 1", + "TAB 2" + ] + }, + { + "id": "megacoglab-rGxlquhjc", + "claim": "teachers in countries with high math and science achievement scores also displayed more cognitive supports for analogies, such as bridging analogies, and visuospatial scaffolds for comparison, compared to teachers in a country with lower math and science achievement", + "citekey": "richlandCognitiveSupportsAnalogies2007", + "dataset": "megacoglab", + "findings": [ + "FIG NA" + ] + }, + { + "id": "megacoglab-YLzHxxCDY", + "claim": "People were ~2x worse at solving matchstick arithmetic problems when the elements were harder to decompose into chunks", + "citekey": "knoblichConstraintRelaxationChunk1999", + "dataset": "megacoglab", + "findings": [ + "FIG 3" + ] + }, + { + "id": "megacoglab-uVqwN3vFt", + "claim": "groups generated more ideas within categories when individuals were given high-related (vs. low-related) categories", + "citekey": "baruahCategoryAssignmentRelatedness2011", + "dataset": "megacoglab", + "findings": [ + "FIG 1" + ] + }, + { + "id": "megacoglab-BN8Vqws1Y", + "claim": "people who received inspirations on-demand transferred more structural features from examples compared to participants who received examples on a fixed schedule", + "citekey": "siangliulueProvidingTimelyExamples2015", + "dataset": "megacoglab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "megacoglab-02o3rSVIc", + "claim": "highly novel papers were more likely to be published in lower impact journals", + "citekey": "wangBiasNoveltyScience2017", + "dataset": "megacoglab", + "findings": [ + "FIG 3", + "TAB 6" + ] + }, + { + "id": "megacoglab-FCz9hmMW4", + "claim": "When provided scaffolding for a similar problem students performed similarly compared to their peers given little scaffolding, however, when asked to solve problems with low similarity the group provided scaffolding performed significantly better.", + "citekey": "richlandReducingCognitiveLoad2013", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 3" + ] + }, + { + "id": "megacoglab-lbRNxo-nM", + "claim": "groups given low-related categories of solutions ended up exploring more categories in their solutions compared to high-related category groups", + "citekey": "baruahCategoryAssignmentRelatedness2011", + "dataset": "megacoglab", + "findings": [ + "TAB 4", + "TAB 3" + ] + }, + { + "id": "megacoglab-AD1_6mXtQ", + "claim": "working in groups with others who have different initial preferred hypotheses facilitated convergence away from the bias at similar rates to working alone", + "citekey": "convertinoCACHEStudyGroup2008", + "dataset": "megacoglab", + "findings": [ + "FIG 6" + ] + }, + { + "id": "megacoglab-wlMJq0BJy", + "claim": "Self-rated technical distance from open innovation contest problems was slightly positively correlated with submitting winning solutions", + "citekey": "jeppesenMarginalityProblemsolvingEffectiveness2010", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-d77x3B_Ui", + "claim": "case-based reasoning system loaded with diverse cases increased overall usefulness and creativity of ad campaign ideas, especially for people with lower creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "FIG 2", + "TAB 1" + ] + }, + { + "id": "megacoglab-mPZM8-hmC", + "claim": "when given a (far) analogy for numerical representations, students did no better (and possibly worse) than on a post-test than not having it", + "citekey": "vamvakoussiBridgingGapDense2012", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-GL4yvLPha", + "claim": "low prevalence of EBPR references in state statutes and regulations pertaining to behavioral health care; 20 states had a total of 33 mandates that referenced an EBPR", + "citekey": "lee2022references", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-3sChuA1j8", + "claim": "No secondary cases amongst preschoolers in a COVID cluster in Singapore with adults as index cases", + "citekey": "yungNovelCoronavirus20192020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-0dCO_io-p", + "claim": "increased word ambiguity in abstracts was associated with slightly lower modularity of citation networks for those abstracts", + "citekey": "mcmahanAmbiguityEngagement2018", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-t_AiQeKkK", + "claim": "evaluators gave lower scores to biomed grant proposals that were very novel; but higher scores to proposals that were somewhat novel", + "citekey": "boudreauLookingLookingKnowledge2016", + "dataset": "megacoglab", + "findings": [ + "TAB 5", + "FIG 3" + ] + }, + { + "id": "megacoglab-xRWgPZXX9", + "claim": "Better model fit to Wuhan case data with age-dependent susceptibility, with children ~2x lower than adults", + "citekey": "cmmidcovid19workinggroupAgedependentEffectsTransmission2020", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "FIG 4" + ] + }, + { + "id": "megacoglab-eEjsNkFV1", + "claim": "Members in a data science team mainly use meetings and documentations to develop shared understanding on a project", + "citekey": "wangHowDataScientists2019", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-iG1V6dHgy", + "claim": "Far analogies' effects on MechE students' ideation fluency and diversity were different depending on timing: more ideas (but with more functional repeats) before ideation began, vs. more functionally distinct designs after problem solving began (during a break), both compared to seeing nothing", + "citekey": "tsengRoleTimingAnalogical2008", + "dataset": "megacoglab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "megacoglab-PDfEGZaLe", + "claim": "\ud83d\udd11Outlier Patents receive more citations on average than \ud83d\udd11Adjacent Patents", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-MLSP020ju", + "claim": "Generating (but not evaluating) solutions to far analogy problems positively predicted performance on a subsequent analogy task (compared to generating or evaluating near analogy problems)", + "citekey": "vendettiFarOutThinkingGenerating2014", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-ML0rEDL6_", + "claim": "higher salience of ecological relations for organizing living things for children from rural vs. urban communities", + "citekey": "betzDevelopmentConceptualFlexibility2020", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-1A-vRkLMT", + "claim": "highly novel papers had higher variance in their citation outcomes over a 15-year window, biased towards the higher impact tail of the distribution", + "citekey": "wangBiasNoveltyScience2017", + "dataset": "megacoglab", + "findings": [ + "FIG 1A", + "TAB 2" + ] + }, + { + "id": "megacoglab-5gJjNdjMH", + "claim": "US patents filed by inventor teams that included an expert in the patent's field tended to be less novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-9ic6uJY1D", + "claim": "Women were more likely than men to submit winning solutions for open innovation contests", + "citekey": "jeppesenMarginalityProblemsolvingEffectiveness2010", + "dataset": "megacoglab", + "findings": [ + "TAB 4" + ] + }, + { + "id": "megacoglab-Ig-q6lfVE", + "claim": "Only small business owners with high divergent thinking ability generated more ideas with diverse (vs. constrained) stimuli", + "citekey": "gielnikCreativityOpportunityIdentification2011", + "dataset": "megacoglab", + "findings": [ + "TAB 4", + "FIG 2" + ] + }, + { + "id": "megacoglab-2C17QaTLH", + "claim": "science students made more planning errors and fewer structural errors on a spatial block design task; architecture students made fewer planning errors and more structural errors", + "citekey": "lawsonCognitiveStrategiesArchitectural1979", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-2mTPyT8sj", + "claim": "Limited increases in transfer between analogous insight problems when explicit hint (mapping + solution) was given", + "citekey": "reedRoleAnalogyTransfer1974", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-hZB-PgxZX", + "claim": "~60 percent of InstructGPT-generated analogical explanations for scientific concepts were rated by crowd workers as containing a meaningful analogy, comparable to human-generated analogies", + "citekey": "bhavyaAnalogyGenerationPrompting2022", + "dataset": "megacoglab", + "findings": [ + "TAB 10" + ] + }, + { + "id": "megacoglab-mtKPi-Abu", + "claim": "no overall effect of analogical distance of cases on novelty or usefulness of campaign ad ideas", + "citekey": "althuizenSupportingCreativeProblem2014", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-u2Rw1hwtY", + "claim": "College students imagined novel alien creatures with more atypical features vs. Earth creatures when they were instructed to consider survival-critical features of life forms, vs. building on specific Earth animal exemplars", + "citekey": "wardRoleSpecificityAbstraction2004", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-I5ElqeQBh", + "claim": "prevalence of COVID (excluding first-reported case) increased with age in New York State, ~2x lower for children and youth compared to adults", + "citekey": "rosenbergCOVID19TestingEpidemic2020", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-PNHl7tkly", + "claim": "10 scholars self-reported that they read specific fragments and sections, such as findings and background and references, in patterns that varied by tasks such as keeping track, finding new ideas, and writing articles or new projects", + "citekey": "ribaupierreExtractingDiscourseElements2017", + "dataset": "megacoglab", + "findings": [ + "FIG 2" + ] + }, + { + "id": "megacoglab-JszLLa3Q4", + "claim": "Experienced facilitator participants created inspirations for crowd brainstormers with strategies like crafting simulation prompts or inquiries, in contrast to novice facilitators, who relied more heavily on simply passing on examples without further processing", + "citekey": "chanImprovingCrowdInnovation2016", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-5ypAq6Csv", + "claim": "A patent's odds of being an outlier increases by 1.05 times for each doubling of scientific articles cited", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-_QOSgyUEe", + "claim": "~half of InstructGPT-generated analogies for a small benchmark science analogy dataset matched the reference set; ~70 percent were valid", + "citekey": "bhavyaAnalogyGenerationPrompting2022", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-J9X-JZ5b5", + "claim": "Prior patenting experience is negatively related to the likelihood of being granted \ud83d\udd11Outlier Patents", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-N3idE-TeS", + "claim": "Papers citing papers that refute the Hawthorne effect predominantly cited those papers to (incorrectly) affirm the Hawthorne effect", + "citekey": "letrudAffirmativeCitationBias2019", + "dataset": "megacoglab", + "findings": [ + "FIG 1", + "FIG 2", + "TAB 1", + "TAB 2", + "FIG 3", + "TAB 3" + ] + }, + { + "id": "megacoglab-H-9bped9d", + "claim": "the sensemaking translucence interface significantly improved clue finding and crime solving performance", + "citekey": "goyalEffectsSensemakingTranslucence2016", + "dataset": "megacoglab", + "findings": [ + "FIG 4" + ] + }, + { + "id": "megacoglab-GvDDTwcsJ", + "claim": "In general, MechE students' ideas when given near analogies were at least as novel, and sometimes moreso, then ideas generated with far analogies, or seeing nothing", + "citekey": "tsengRoleTimingAnalogical2008", + "dataset": "megacoglab", + "findings": [ + "FIG 5" + ] + }, + { + "id": "megacoglab-Qt5iwJecI", + "claim": "after noticing a key element of a problem representation for the mutilated checkerboard problem (namely the lack of parity between the squares on the checkerboard), solutions to the problem came almost immediately after", + "citekey": "kaplanSearchInsight1990", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-kvQUSZy5J", + "claim": "higher estimated helpfulness and clarity of map from others for their own sensemaking for an everyday sensemaking task when the map was iterated on by multiple people vs. created by one person", + "citekey": "fisherDistributedSensemakingImproving2012", + "dataset": "megacoglab", + "findings": [ + "TAB 1" + ] + }, + { + "id": "megacoglab-3BGDkcK-w", + "claim": "Only small business owners with high divergent thinking ability generated more original ideas with diverse (vs. constrained) stimuli", + "citekey": "gielnikCreativityOpportunityIdentification2011", + "dataset": "megacoglab", + "findings": [ + "TAB 4", + "FIG 3" + ] + }, + { + "id": "megacoglab-Smh3nQpqn", + "claim": "Participants who marked up their own papers with a formal semantic scheme self-reported that formally marking rhetorical relations between claims seemed less clear in terms of payoff compared to formally making claims", + "citekey": "grozaSALTWeavingClaim2007", + "dataset": "megacoglab", + "findings": [ + "FIG 8B" + ] + }, + { + "id": "megacoglab-X9jkreduk", + "claim": "direct comparison of dissimilar analogs approximately doubled rates of spontaneous far analogical transfer for the radiation problem", + "citekey": "gickSchemaInductionAnalogical1983", + "dataset": "megacoglab", + "findings": [ + "TAB 5" + ] + }, + { + "id": "megacoglab-TE2krdmGY", + "claim": "Lower secondary attack rate in households in China for children 4-18 compared to other age groups", + "citekey": "wuHouseholdTransmissionSARSCoV22020", + "dataset": "megacoglab", + "findings": [ + "TAB 2" + ] + }, + { + "id": "megacoglab-C5L_KQ5fy", + "claim": "higher levels of interdisciplinarity associated with higher variance in their interdisciplinary papers' citation outcomes", + "citekey": "leaheyProminentLessProductive2017", + "dataset": "megacoglab", + "findings": [ + "TAB 3" + ] + }, + { + "id": "megacoglab-4cjTc4Vju", + "claim": "Universities are 1.36 times more likely to file \ud83d\udd11Outlier Patents than non-universities", + "citekey": "kneelandExploringUnchartedTerritory2020", + "dataset": "megacoglab", + "findings": [ + "TAB 6" + ] + }, + { + "id": "megacoglab-lMdlmzR0o", + "claim": "most undergrad 1980s textbooks in physics and chemistry cited work published before 1960; sociology texts mostly cited work published after 1960", + "citekey": "coleHierarchySciences1983", + "dataset": "megacoglab", + "findings": [ + "TAB 10" + ] + }, + { + "id": "megacoglab--wTYRJFK3", + "claim": "when placed in the same semantic space, computationally selected near and far analogical stimuli were farther away from human-selected near and far analogical stimuli for the same problem", + "citekey": "fuMeaningFarImpact2013", + "dataset": "megacoglab", + "findings": [ + "FIG 6" + ] + } +] \ No newline at end of file diff --git a/task2-test.json b/task2-test.json new file mode 100644 index 0000000000000000000000000000000000000000..9df5ae618cb6bc57f0190b016c84ef502b394a60 --- /dev/null +++ b/task2-test.json @@ -0,0 +1,656 @@ +[ + { + "id": "akamatsulab-3YkqDzf-k", + "claim": "\"Disruption of actin with Latrunculin A or Jasplakinolide permited tubule formation as shown by Alexa555-CTxB, whereas no membrane tubules were observed with pretreatment of 16uM Nocodozole prior to LatA or Jasplak treatment.\"", + "citekey": "day2015microtubule", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab--Oe1lYDk9", + "claim": "\"DNA-PAINT resolution enhancement by sequential imaging (RESI) of DNA origami resolved DNA bases 0.7 nm apart\"", + "citekey": "reinhardt2023angstromresolution", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-SL11GKMhj", + "claim": "\"Membrane Vacuole Like Dilations (VLDs) with 2um diameter and ~2um height were observed in pEYFP-mem-transfected MEFs upon return to iso-osmotic conditions after exposure to 50% hypo-osmotic medium for 3min.\"", + "citekey": "kosmalska2015physical", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-F08lbYl1E", + "claim": "\"DNA-PAINT resolution enhancement by sequential imaging (RESI) showed increased packing (decreased spacing between receptors) of C20 receptors in CHO cells in the presence of RTX antibody \"", + "citekey": "reinhardt2023angstromresolution", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-hUo7JOHVF", + "claim": "\"Inhibition of GBF1 with LG186 greatly reduced fluid uptake and the number of CLICs while CME-derived vesicles and uptake were unaffected in AGS cells.\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-dRrpuL9Ns", + "claim": "\"Clathrin coated pits increased in lifetime by 14% under 100 \u00b5M CK666 in isotonic conditions based on live cell fluorescence imaging of SK-MEL-2 cells\"", + "citekey": "kaplan2022load", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-8WVpbNX8F", + "claim": "\"Actin filaments were less resistant to fragmentation due to twisting in the presence of cofilin.\"", + "citekey": "bibeau2023twist", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-E5mC5JCYA", + "claim": "\"The single-molecule and expansion microscopy protocol achieved highly uniform expansion of fission yeast cells at both the macro and micro scales.\"", + "citekey": "vojnovic2024combining", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ttWXeJjs6", + "claim": "\"WT NIH3T3 fibroblast cells (untreated with Dyngo4a) showed comparable total CTxB-containing structures after 2min incubation with CTxB-HRP that aligned with CLIC characteristics to WT and Cav-/- MEFs as evaluated by electron microscopy (total = 47% of cytoplasm, where 38% cytoplasm was comprised of tubules, and 9% vesicles).\"", + "citekey": "howes2010clathrinindependent", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-H8YLpgxgm", + "claim": "\"By directly binding a capping protein, Twinfilin inhibited CARMIL-mediated uncapping of capped actin filament barbed ends\"", + "citekey": "johnston2018novel", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-QWPyIbSxE", + "claim": "\"Combined cryo-EM, subtomogram averaging using RELION4, and unbiased matches with AlphaFold2 identified new protein Tekt5 in mouse sperm flagellum.\"", + "citekey": "chen2023novo", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-LjXvWKMsS", + "claim": "\"GTP-yS-Cdc42 and Pip2-containing vesicles increased the rate of NWASP-mediated branched actin elongation based on pyrene-actin fluorescence\"", + "citekey": "rohatgi1999interaction", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-zspgTfk4Y", + "claim": "\"Endocytic actin filaments had branch angles of 68 \u00b1 9\u02da based on cryo-electron tomography of SK-MEL-2 cells \"", + "citekey": "serwas2022mechanistic", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-34Sse-JI-", + "claim": "\"Arc5p-mEOS3.2 did not internalize in wsp1\u2206 fission yeast cells based on live-cell FPALM microscopy\"", + "citekey": "arasada2018highspeed", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-hDBBP4KpR", + "claim": "\"K560 truncated kinesin 1-Halo with JF646 expressed in live U20S cells showed 16 nm processive directional stepping by MINFLUX microscopy\"", + "citekey": "deguchi2023direct", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-nzQ0RZigK", + "claim": "\"GRAF1 coimmunoprecipitated with Dynamin from rat brain cytosol as identified by mass spectrometry and confirmed with antibodies to both GRAF1 and Dynamin.\"", + "citekey": "doherty2011endocytic", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-mfOuQgaJI", + "claim": "\"When twisted, actin filaments formed a secondary loop (\\\"plectoneme\\\") that was independent of twisting direction under low tension conditions.\"", + "citekey": "bibeau2023twist", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-8Lx-KOlQv", + "claim": "\"In Epsin triple knockout mouse embryonic fibroblasts, Hip1R did not localize to punctate structures characteristic of endocytic sites based on immunofluorescence\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-APVr-0tNG", + "claim": "\"Arc5p-mEOS3.2 did not internalize in myo1\u2206 fission yeast cells based on live-cell FPALM microscopy\"", + "citekey": "arasada2018highspeed", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-K_PZU2gX3", + "claim": "\"In 150 mOsm hypotonic media, CK-666 did not affect the height of clathrin coated pits based on STORM imaging\"", + "citekey": "kaplan2022load", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-x2wgeai5k", + "claim": "\"Membrane mechanics simulations showed a linear relationship between endocytic pit internalization and force, for a variety of values of membrane tension\"", + "citekey": "akamatsu2020principles", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-yirRYe_uR", + "claim": "\"3D MINFLUX imaging allowed for 3D tracking of kinesin-1-Halo added to fixed U20S cells \"", + "citekey": "deguchi2023direct", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-lRBhBNler", + "claim": "\"mMaple-3-Wsp1 internalized over the course of several seconds based on live-cell FPALM microscopy in fission yeast\"", + "citekey": "arasada2018highspeed", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-3MRyHuIf9", + "claim": "\"The 2 terminal subunits at the free barbed end of f-actin filament adopted a \\\"flat\\\" conformation similar to the conformation of subunits in the middle of the filament\"", + "citekey": "carman2023structures", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-rj_RtRmpv", + "claim": "\"The fluorescence intensity of ArpC3 peaked concomitantly with dynamin2 based on live cell imaging of gene edited hiPSCs\"", + "citekey": "jin2022branched", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-z4XGgZPP8", + "claim": "\"100 \u00b5M CK-666 increased clathrin coat height by 8% based on STORM imaging\"", + "citekey": "kaplan2022load", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-YyF8q_zk6", + "claim": "\"3D DNA-PAINT resolution enhancement by sequential imaging (RESI) resolved nuclear pore complexes 5 nm apart in U2OS cells\"", + "citekey": "reinhardt2023angstromresolution", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-jjW0_d3ak", + "claim": "\"Disruption of microtubule network with high dose (16uM) nocodozole eliminated CTxB+ tubules, whereas low dose (150nM) nocodozole had little effect\"", + "citekey": "day2015microtubule", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ebn-UtwZK", + "claim": "\"Optical tweezers revealed limited force propagation when only the membrane was pulled\"", + "citekey": "debelly2023cell", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-xY-GewW3E", + "claim": "\"Truncated GRAF1 missing GAP and SH3 domains (GRAF1-BAR-PH) displayed long tubules that persisted for >10minutes when incubated with Dil dye\"", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-P87cNqwZr", + "claim": "\"Epsin triple knockout mouse embryonic fibroblasts exhibited increased actin accumulation at sites of clathrin-mediated endocytosis as assayed by expressed utrophin calponin homology domain and TIRF microscopy\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-A1HdO-GrO", + "claim": "\"Membrane Vacuole Like Dilations (VLDs) were observed in MEFs seeded on PDMS upon return to iso-osmotic conditions after hypo-osmotically induced swelling, and showed no colocalization between pEYFP-Mem structures and actin.\"", + "citekey": "kosmalska2015physical", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-hGyVfnHYL", + "claim": "\"Epsin triple knockout mouse embryonic fibroblasts exhibited an increased number of shallow and U shape clathrin-coated vesicles than wild type based on electron microscopy\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-VLnm3aII7", + "claim": "\"mCherry-Arp3 reached peak accumulation at SecGFP-GPI endocytic sites at 6 seconds prior to scission in hAGS cells as shown by TIRF microscopy\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-COBrjEXGF", + "claim": "\"Myc-tagged GRAF1 colocalized with GFP-GPI in Caveolin1-knockout mouse embryonic fibroblasts based on immunofluorescence\"", + "citekey": "lundmark2008gtpaseactivating", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-Cmy4D8bhz", + "claim": "\"Dynamin pulled down with GST-GRAF1-SH3 domain based on Coomassie staining in a pulldown assay of rat brain cytosol with GST-GRAF1-SH3-bound beads\"", + "citekey": "doherty2011endocytic", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-i2NXezq0Q", + "claim": "\"TagRFPt-PICK1 reached peak intensity accumulation at CG endocytic sites at 12 seconds prior to scission (-12sec), dropping until just prior to scission and then increasing, in hAGS cells as shown by colocalization with SecGFP-GPI spots using TIRF microscopy\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-scjz_DMJY", + "claim": "\"Deletion of ICAP-1 led to recovery of traction forces in beta integrin KOs.\"", + "citekey": "kyumurkov2023force", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-EowpPsowf", + "claim": "\"Membrane reservoirs with 500nm diameter and ~1um height were observed in pEYFP-mem-transfected MEFs upon release of 6% stretch for 3min.\"", + "citekey": "kosmalska2015physical", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-MlLsP5aAR", + "claim": "\"Hip1R expression increased by ~75% in epsin triple knockout mouse embryonic fibroblasts\"", + "citekey": "messa2014epsin", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-M3NOdLhAe", + "claim": "\"Alexa488-STxB and Alexa488-CTxB accumulated in tubular invaginations in ATP-depleted COS-7 cells, however, ATP-depletion inhibited uptake of Dextran.\"", + "citekey": "day2015microtubule", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-27gEPZJHs", + "claim": "\"\u00b5-track performed better that other tracking pipelines in datasets with high particle density with regards to precision and accuracy.\"", + "citekey": "roudot2023utrack3d", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ihaqh3iX6", + "claim": "\"Under 150 mOsm hypotonic conditions, CK-666 increased clathrin-RFP lifetime by 60% in SK-MEL-2 cells\"", + "citekey": "kaplan2022load", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-edRoDBRsn", + "claim": "\"The use of an optimized mixture of CapZ, tropomodulin, tropomyosin, and actin produced shorter actin filaments and more ends per micrograph compared to actin alone\"", + "citekey": "carman2023structures", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-EIEiB15oW", + "claim": "\"Based on STORM imaging, actin marked by phalloidin was asymmetrically distributed around endocytic sites, with an average distance of 71 nm from the center of the clathrin coat\"", + "citekey": "jin2022branched", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-HRNgiqUaO", + "claim": "\"After treatment with 30uM Dyngo4a (a dynamin inhibitor) CLICs were identified using electron microscopy as the remaining CTxB-containing structures, contributing to 90% of the total CTxB-containing endocytic volume after 15sec of CTxB uptake, 86% after 1min, and 74% after 2min in Cav-/- MEFs as compared to cells untreated with Dyngo4a. \"", + "citekey": "howes2010clathrinindependent", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-BNMAVkp2E", + "claim": "\"Snx33-/- dHL60 cells display higher levels of actin than WT dHL60 cells upon fMLP chemoattractant addition as shown by phalloidin staining detetected by flow cytometry.\"", + "citekey": "sitarska2023sensing", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-6B_aybd1z", + "claim": "\"mEmerald-p21, an Arp2/3 complex subunit, colocalized with mCherry-IRSp53 at filopodia in fixed AGS cells as shown by TIRF microscopy.\"", + "citekey": "sathe2018small", + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ca1lLfdLf", + "claim": "\"Using a worm-like chain model to fit the force dependence of actin filament bending, the persistence length of actin was determined to be 10.7 $$\\pm$$ 1 $$\\mu$$m.\"", + "citekey": "bibeau2023twist", + "dataset": "akamatsulab" + }, + { + "id": "megacoglab-9z_zo-lGV", + "claim": "\"social innovation concept proposals that cited inspirations with greater maximum conceptual distance from their problem domain were less likely to be shortlisted as creative by a panel of experts\"", + "citekey": "chanBestDesignIdeas2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xwrgZU20w", + "claim": "\"participants who received automatically labeled clusters of ideas as inspiration generated a higher quantity of ideas compared to receiving no external stimulation\"", + "citekey": "chanComparingDifferentSensemaking2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-HPVx3ziPZ", + "claim": "\"project ideas that cited a diverse set of inspiration sources were not more likely to be marked as creative by an expert panel\"", + "citekey": "chanImportanceIterationCreative2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-0DNrdxdn6", + "claim": "\"participants who saw examples in a list for an exploratory creativity task were more likely to do initial hill-climbing-like local exploration compared to participants who saw examples contextualized in the search space or in a dropdown\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-PrtoB_uNu", + "claim": "\"participants who received automatically labeled clusters of ideas as inspiration generated more diverse ideas compared to receiving no external stimulation\"", + "citekey": "chanComparingDifferentSensemaking2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-R8NAx8AqR", + "claim": "\"participants generated more alternative uses for everyday objects in a large room compared to participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-nZ8moeasL", + "claim": "\"skip-gram model with vanilla BOW contexts achieved 0.3 correlation with human judgments of verb similarities on SimLex999, about 50 percent worse than correlations with human judgments of nouns (0.5 correlation) or adjectives (0.6 correlation)\"", + "citekey": "schwartzSymmetricPatternsCoordinations2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xVGvCQlr4", + "claim": "\"social innovation concept proposals that cited inspirations with greater mean conceptual distance from their problem domain were less likely to be shortlisted as creative by a panel of experts\"", + "citekey": "chanBestDesignIdeas2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-fiT1U1BNY", + "claim": "\"the most novel alternative uses for everyday objects that were generated by undergraduate psychology students in a large room were marginally (but not statistically significantly) more novel than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-qK4L2OmZ8", + "claim": "\"constantly recommending examples that were semantically distant from current ideas --- regardless of cognitive state --- led to lower maximum novelty of ideas compared to receiving no stimuli\"", + "citekey": "chanSemanticallyFarInspirations2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-SgCPC8G1k", + "claim": "\"forced colocation increased the likelihood that previously non-colocated French labs would collaborate by 3.5 times, primarily amongst lab pairs that had previously semantically distant topics and cited references\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-XZ-GAXsTK", + "claim": "\"constantly recommending examples that were semantically distant from current ideas -- regardless of cognitive state -- led to longer time intervals between ideas compared to receiving no stimuli\"", + "citekey": "chanSemanticallyFarInspirations2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-8NLbZ1oEO", + "claim": "\"Getting pairs of scientists in a breakout room to discuss their work increased the likelihood they would collaborate on a grant by about 75%\"", + "citekey": "boudreauFieldExperimentSearch2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-ydBOxMKiQ", + "claim": "\"after being forcibly colocated, French lab pairs grew increasingly similar in topics and literature cited\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-t3LvtWlNM", + "claim": "\"Crowd workers generated the most practical ideas when given analogical inspirations found from domain-independent problem descriptions with concrete constraints, compared to abstract constraints\"", + "citekey": "yuDistributedAnalogicalIdea2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-fOKN4sbBt", + "claim": "\"word2vec model with skip-gram architecture and 300 dimensions trained on a subset of the Google News corpus for three days achieved 50 percent accuracy on semantic four-term analogy word problems, better than a set of NNLM and RNNLM models\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-31i8gG7Kn", + "claim": "\"alternative uses for everyday objects that were generated by participants in a large room were judged as less practical on average than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-ayJPkOD1i", + "claim": "\"the most novel alternative uses for everyday objects that were generated by participants in a large room were more novel than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-SInNihzph", + "claim": "\"scientist pairs who were randomly assigned to talk to each other (and did so, face-to-face) at an interdisciplinary medical imaging symposium used about 6 new MeSH keywords from their partners in future publications when the pair had moderate overlap in intellectual interests, compared to pairs who did not interact at the symposium\"", + "citekey": "laneEngineeringSerendipityWhen", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Oryy_ukuK", + "claim": "\"engineering students generated ideas with lower mean quality when they received analogical examples that were distant from their problem domain (vs. close to their domain)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-AX2AnkiDh", + "claim": "\"participants who saw examples contextualized in the search space for an exploratory creativity task were more likely to self-report using examples to model the problem compared to participants who saw examples in a list or in a dropdown\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-XwVHuT02v", + "claim": "\"word2vec model with CBOW architecture and 600 dimensions trained on a subset of the Google News corpus achieved 50 percent accuracy at completing semantic and syntactic four-term analogy word problems\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-lpcaTGd-U", + "claim": "\"word2vec model with skip-gram architecture and 640 dimensions trained on LDC corpora achieved 55 percent accuracy on semantic four-term analogy word problems, better than an NNLM model (23 percent accuracy), and RNNLM model (9 percent accuracy), trained on the same data\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-murOlQXGZ", + "claim": "\"positive effect of analogical paper recommendations (vs. keyword-based recommendations) on inducing creative adaptation ideas was statistically mediated by analogical recommendations' likelihood of being partial (vs. full) purpose matches\"", + "citekey": "kangAugmentingScientificCreativity2022", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-2Ey6CRR--", + "claim": "\"the occurrence of planning disagreements in an interdisciplinary science team was associated with subsequent increases in expressed uncertainty in their conversations\"", + "citekey": "paletzUncoveringUncertaintyDisagreement2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-tD_biL9dm", + "claim": "\"participants who saw diverse sets of examples had higher maximum task scores on an exploratory creativity task compared to participants who saw non-diverse sets of examples\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Wvp7Jsu6k", + "claim": "\"the occurrence of science-related disagreements in an interdisciplinary science team was associated with subsequent increases in expressed uncertainty in their conversations early -- but not late -- in their mission\"", + "citekey": "paletzUncoveringUncertaintyDisagreement2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-rrA3LVjvk", + "claim": "\"crowd workers were more likely to generate creative product ideas when given descriptions of types of experts recommended by other workers who were given schematized abstract problem descriptions, compared to workers who were given descriptions of types of experts recommended by workers who were given the original concrete problem description, experts recommended from an irrelevant problem, or allowed to search for their own inspirations\"", + "citekey": "yuEncouragingOutsideBox2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-M1fSi8xQv", + "claim": "\"participants who received automatically labeled clusters of ideas as inspiration generated lower quality ideas compared to receiving no external stimulation\"", + "citekey": "chanComparingDifferentSensemaking2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-HwTluMGQh", + "claim": "\"participants who saw examples in a list for an exploratory creativity task were more likely to self-report selecting individual examples as starting points, rather than using them to model the problem or ignoring the examples\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-bf1_85feE", + "claim": "\"undergraduates spontaneously transferred a solution from an analogous story they had just read about 70-100 percent of the time with a hint that the story was useful for their problem\"", + "citekey": "gickAnalogicalProblemSolving1980", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-31S7Tx1Y9", + "claim": "\"undergraduate mechanical engineering students generated ideas with the same level of variety when given examples --- either surface dissimilar in the form of biological examples, or surface similar to their problem --- compared to students who received no examples\"", + "citekey": "wilsonEffectsBiologicalExamples2010", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-kPgwfWLY2", + "claim": "\"project ideas that cited other projects that cited a diverse set of inspiration sources were more likely to be marked as creative by an expert panel\"", + "citekey": "chanImportanceIterationCreative2015", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-ofmkVMxwh", + "claim": "\"undergraduates spontaneously transferred a solution from an analogous story they had just read about 40 percent of the time without a hint that the story was useful for their problem; about the same amount transferred the solution after a hint\"", + "citekey": "gickAnalogicalProblemSolving1980", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-tqn-PBdXQ", + "claim": "\"after forcible separation, previously colocated labs' cited references became less similar over time\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-sMBayPMZl", + "claim": "\"undergraduate mechanical engineering students generated marginally more novel ideas when given examples that were surface dissimilar from their problem (in the form of biological examples) compared to students who received examples that were surface similar to their problem\"", + "citekey": "wilsonEffectsBiologicalExamples2010", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-eLMnvM_CG", + "claim": "\"analogical paper recommendations were judged as more novel by engineering PhD students compared to keyword-based paper recommendations\"", + "citekey": "kangAugmentingScientificCreativity2022", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-v5Gb8mkfK", + "claim": "\"scientist pairs who were randomly assigned to talk to each other (and did so, face-to-face) at an interdisciplinary medical imaging symposium and had pre-symposium moderate similarity in clinical fields were more likely to co-author new publications in the 5 years following the symposium, compared to pairs who did not interact with each other at the symposium\"", + "citekey": "laneEngineeringSerendipityWhen", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-TP1cdR2tl", + "claim": "\"undergraduate mechanical engineering students generated more novel ideas when given examples --- either surface dissimilar in the form of biological examples, or surface similar to their problem --- compared to students who received no examples\"", + "citekey": "wilsonEffectsBiologicalExamples2010", + "dataset": "megacoglab" + }, + { + "id": "megacoglab--JkuVF9as", + "claim": "\"constantly recommending examples that were semantically distant from current ideas --- regardless of cognitive state --- led to ideas that were on average less similar to their immediately preceding idea compared to receiving no stimuli\"", + "citekey": "chanSemanticallyFarInspirations2017", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-AyfbosEHF", + "claim": "\"skip-gram model with symmetric pattern contexts achieved 0.46 correlation with human judgments of verb similarities on SimLex999, comparable to correlations with human judgments of nouns (0.42 correlation) or adjectives (0.65 correlation) and outperforming skip-gram models with other contexts, such as dependency parses and BOW\"", + "citekey": "schwartzSymmetricPatternsCoordinations2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-XyiUTIRAT", + "claim": "\"chemists and chemical engineers in self-reported high-scatter fields were about 2x more likely to self-report spending more than 5 hrs a week on keeping up with their field, compared to chemists and chemical engineers in low-scatter fields\"", + "citekey": "packerImportanceSDICurrent1979", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-rc7fs4Cwg", + "claim": "\"for two out of four design problems, concepts generated by engineering graduate students who received stimulus verbs that were antonyms of the core functions of the problem generated ideas that were judged to be more useful, and cohesive than students who receive stimulus verbs that were synonyms of the core functions of the problem\"", + "citekey": "chiuInvestigatingEffectsOppositely2012", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-SUT_hq92I", + "claim": "\"Crowd workers generated the most original ideas when given analogical inspirations found from domain-independent problem descriptions with concrete constraints, compared to abstract constraints\"", + "citekey": "yuDistributedAnalogicalIdea2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-MDvKeTZZN", + "claim": "\"word2vec model with CBOW architecture trained on LDC corpora and 640 dimensions achieved 24 percent accuracy on semantic four-term analogy word problems, about the same as NNLM model (23 percent accuracy), and better than an RNNLM model (9 percent accuracy), trained on the same data\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Eas3Av6eX", + "claim": "\"crowd workers who were given a more abstract schematic description of a problem recommended more unique types of experts who might provide useful perspectives on the problem, compared to workers who were given the original concrete problem description\"", + "citekey": "yuEncouragingOutsideBox2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-VH37eM1Ba", + "claim": "\"engineering students generated ideas with the highest mean novelty when they received analogical examples that were both distant from their problem domain and unfamiliar to them (vs. all other types of examples or no examples)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-fD5LD5jZs", + "claim": "\"chemists and chemical engineers in self-reported high-scatter fields were less likely to self-report a high success-to-time-spent ratio in keeping up with their field, compared to chemists and chemical engineers in low-scatter fields\"", + "citekey": "packerImportanceSDICurrent1979", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xdDrUY5pK", + "claim": "\"engineering students generated ideas with higher mean novelty when they received analogical examples that were distant from their problem domain (vs. close to their domain)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-TjSBH1e8i", + "claim": "\"forced separation did not negatively affect collaboration between previously colocated French labs\"", + "citekey": "cataliniMicrogeographyDirectionInventive2018", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-WB32ukGUI", + "claim": "\"engineering PhD students were more likely to generate creative adaptation vs. direct application ideas for their research when given analogical vs. keyword-based paper recommendations\"", + "citekey": "kangAugmentingScientificCreativity2022", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-VcP5d-4Lp", + "claim": "\"chemists and chemical engineers in self-reported low-scatter fields who subscribed to selective dissemination of information (SDI) services were more likely to self-report low efficiency in keeping up with their field compared to non-subscribers \"", + "citekey": "packerImportanceSDICurrent1979", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-D8dmJsaRz", + "claim": "\"word2vec model with skip-gram architecture trained on LDC corpora and 640 dimensions achieved 59 percent accuracy on syntactic four-term analogy word problems, better than RNNLM and NNLM models trained on the same data (which achieved 36 and 53 percent accuracy)\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-04yh2Agp6", + "claim": "\"engineering students generated ideas with higher variability in quality when they received analogical examples that were distant from their problem domain (vs. close to their domain or no examples)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-tZtKHmnD7", + "claim": "\"alternative uses for everyday objects that were generated by undergraduate psychology students in a large room were judged as less practical on average than uses generated by participants in a small room\"", + "citekey": "chanSituativeCreativityLarger2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-IMWGvZ2_G", + "claim": "\"crowd workers who were given a more abstract schematic description of a problem recommended experts who might provide useful perspectives on the problem that were more semantically distant from the problem, compared to workers who were given the original concrete problem description\"", + "citekey": "yuEncouragingOutsideBox2016", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-Hn7gmJnZU", + "claim": "\"participants who saw examples in a list had lower maximum task scores on an exploratory creativity task compared to participants who saw examples contextualized in the search space or in a dropdown\"", + "citekey": "chanFormulatingFixatingEffects2024", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-4rDvdG2-8", + "claim": "\"for one out of four design problems, concepts generated by engineering graduate students who received stimulus verbs that were antonyms of the core functions of the problem generated ideas that were judged to be more novel than students who receive stimulus verbs that were synonyms of the core functions of the problem\"", + "citekey": "chiuInvestigatingEffectsOppositely2012", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-xwEsF3MFC", + "claim": "\"engineering students' ideas were more likely to contain solution elements from analogical examples when examples were distant from their problem domain (vs. close to their domain)\"", + "citekey": "chanBenefitsPitfallsAnalogies2011", + "dataset": "megacoglab" + }, + { + "id": "megacoglab-0Jkc9NJV2", + "claim": "\"word2vec model with CBOW architecture trained on LDC corpora and 640 dimensions achieved 64 percent accuracy on syntactic four-term analogy word problems, better than RNNLM and NNLM models trained on the same data (which achieved 36 and 53 percent accuracy)\"", + "citekey": "mikolovEfficientEstimationWord2013", + "dataset": "megacoglab" + } +] \ No newline at end of file diff --git a/task2-train-dev.json b/task2-train-dev.json new file mode 100644 index 0000000000000000000000000000000000000000..8173f8f8559824413d8320e87530124ba7e41397 --- /dev/null +++ b/task2-train-dev.json @@ -0,0 +1,548 @@ +[ + { + "id": "akamatsulab-WbWLJVWcF", + "claim": "Knockout of __Dictyostelium__ WASP (__wasA-__) led to bipolar localization of WAVE rather than WT localization of WAVE to the leading edge.", + "citekey": "amato2019wasp", + "context": [ + "Super-resolution microscopy was adopted to investigate the ability of WASP CRIB mutants to localize to clathrin-coated pits and to recruit the Arp2/3 complex. Images were acquires using a Zeiss LSM880 equipped with a 63x/1.40 NA objective. GFP was excited at 488 nm, RFP at 561 nm. Images were acquired using the ZEN imaging software every 1 or 2 s. TIRF microscopy was utilized to monitor the dynamics of clathrin, WASPs, Arp2/3 complex and actin on the ventral surface of the cells. Images were acquired using a modified Nikon Eclipse TE 2000-U microscope equipped with a photometrics Evolve 512 camera and a DualView DV2 emission splitter. GFP and RFP were excited using 473 nm and 561 nm wavelengths respectively. A 100x/1.40 NA TIRF objective was used. Images were acquired every 1 or 2 s using the MetaMorph software. (p. 18)", + "Dictyostelium wild-type, wasA knockout, and inducible double null cells were grown at 22\u000eC on Petri dishes in HL5 supplemented with vitamins and micro-elements (Formedium). (p. 17)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-dFhjWUgD7", + "claim": "TIRF revealed that Cortactin, Hip1R, and Clathrin were simultaneously recruited to endocytic sites in 3T3 cell.", + "citekey": "leclainche2007hip1r", + "context": [ + "We show that the complex contributes to the regulation of actin assembly in HeLa cells and we establish the relative timing of recruitment of these proteins to endocytic sites. Specifically, we show that the Hip1R\u2013cortactin complex inhibits actin assembly by capping filament barbed ends.", + "Real-time imaging of Hip1R and cortactin at CCPs", + "To determine when this Hip1R\u2013cortactin complex might function during endocytosis, we quantitatively analyzed the temporal appearance of Hip1R and cortactin at endocytic sites. Previous immuno-EM studies revealed that both proteins are present during all stages of CCP formation (Engqvist-Goldstein et al, 2001; Cao et al, 2003). In addition, Hip1R appeared to link actin filaments to CCPs, whereas cortactin was found on actin filaments and actin filament branches throughout the plasma membrane (EngqvistGoldstein et al, 2001; Cao et al, 2003). Total internal reflection fluorescence microscopy (TIR-FM) was performed to image Hip1R-GFP or cortactin-GFP on the surfaces of cells stably expressing DsRed-clathrin (Figure 4). Consistent with previous reports, Hip1R appeared at essentially all of the clathrin patches, whereas cortactin appeared at about onethird of the clathrin patches (Engqvist-Goldstein et al, 2004; Merrifield et al, 2005). We found that Hip1R recruitment follows the course of CCP growth, albeit with a small delay compared to clathrin (Figure 4A, C and E). When a CCP started to disappear, Hip1R behavior was essentially the same as that of clathrin. These observations are consistent with previous observations, but with greater time resolution (Engqvist-Goldstein et al, 2001; Keyel et al, 2004). In contrast, cortactin recruitment was more precipitous, occurring close to the time of CCV formation, and cortactin fluorescence lingered slightly longer after clathrin disappeared (Figure 4B, D and E).", + "Live cell imaging TIR-FM was performed using an Olympus IX81 microscope equipped with a \b 60/NA1.45. lens and 488 nm argon ion laser (Melles Griot). The temperature was maintained at 27.51C using a Bioptechs chamber. The 488 nm laser was used to excite both GFP and DsRed. Simultaneous two-color imaging was performed using an image splitter (Optical Insight) to separate the red and green emission signals to two sides of the camera sensor using a 565 nm dichroic mirror, and 530/30 and 630/50 nm emission filters. No bleed-through between the red and the green channel was detected under our conditions. Swiss 3T3 cells that stably express DsRedmLCa (mouse clathrin light chain a) were transiently transfected with Hip1R-GFP or cortactin-GFP using Lipofectamine reagent (Invitrogen) 48 h before imaging. Each cell was imaged every 2 s for 200\u2013300 frames. After each experiment, images of immobilized microbeads that fluoresce at both green and red wavelengths were captured. These images were used to align the cell images.", + "(C) Average fluorescence of clathrin (dark green) and Hip1R (red) plotted against time from 30 CCPs in eight cells. The error bars represent the s.d. from 30 events. Time 0 corresponds to the moment at which the clathrin signal started to dim. All data were normalized (see Materials and methods) before averaging. (D) Average fluorescence for clathrin (light green) and cortactin (blue) plotted against time from 30 CCPs in 14 cells. (E) Summary of panels C and D without the error bars. Time 0 is marked by a yellow line." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-5i2BACcCn", + "claim": "Pyrene-actin binding assays between human myo1e and actin plateaued at a dissociation rate of 440 \u00b1 9 s-1 for ATP concentrations > 2 \u00b5M", + "citekey": "mezgueldi2002kinetic", + "context": [ + "Myosin-I Expression and Purification\u2014The cDNA for human myosin-IC (accession NM 004998), kindly provided by W. M. Bement (University of Wisconsin), was truncated at Glu720, generating a construct containing the motor domain and the only IQ motif (referred to as myo1eIQ throughout the paper). A FLAG peptide sequence was inserted at the C terminus and subcloned into the baculovirus transfer vector pVL1392 (Invitrogen). Recombinant baculovirus was generated using standard procedures. Myo1eIQ with bound calmodulin was purified from Sf9 cells that were coinfected with virus containing recombinant myo1eIQ and CaM (Fig. 1). Four liters of log phase cells (2 & 106 cells/ml) were infected and incubated at 27 \u00b0C for 60 h with shaking. Cells were harvested by centrifugation; suspended in lysis buffer (10 mM Tris, pH 7.0, 200 mM NaCl, 2 mM MgCl2,5mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin), 2 mM MgATP, and 0.05% Igepal at 4 \u00b0C; and homogenized with five strokes in a Dounce homogenizer. Cell extract was centrifuged at 100,000 & g for 1 h. The supernatant was loaded onto anti-FLAG antibody columns (Sigma). Columns were washed with 5 column volumes of lysis buffer ' 2mM MgATP and 5 column volumes of lysis buffer. Myo1eIQ was eluted with 10 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT, 5 \"M CaM, 0.2 mg/ml FLAG peptide (Sigma), 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin. Eluted protein was loaded directly on to an 8-ml Mono Q column (Amersham Biosciences) equilibrated in column buffer (10 mM Tris, pH 8.0, 25 mM KCl, 1mM DTT) and eluted with a linear 25 mM\u20131 M KCl gradient. The Mono Q column separated myo1eIQ from FLAG peptide, ADP, ATP, and free CaM. Fractions containing myo1eIQ were dialyzed versus storage buffer (10 mM Tris, pH 7.5, 100 mM KCl, 1 mM EGTA, 1 mM DTT, 50% glycerol), which concentrated the protein and allowed for storage at $20 \u00b0C. Quantitative densitometry showed that myo1eIQ was (95% pure, and the CaM:myo1eIQ ratio was 1:1. Approximately 2 mg of pure myo1e protein was obtained from 4 liters of cells. (p. 2)", + "Stopped Flow, Quenched Flow, and Kinetic Modeling\u2014Transient kinetic measurements were made at 25 \u00b0C with an Applied Photophysics (Surrey, UK) SX.18MV stopped flow having a 1.2-ms dead time. Tryptophan fluorescence (#ex # 295 nm) was measured using a 320 nm WG long pass emission filter (Oriel). A 400 nm long pass filter (Oriel) was used to monitor pyrene (#ex # 365 nm), and mantADP and mantATP (#ex # 295 nm) fluorescence. Usually three to five transients were averaged before nonlinear least square fitting. The time courses presented in the figures show the average of one to four individual traces. Transients were fitted to exponential functions using the software supplied with the stopped flow. (p. 2)", + "Rabbit skeletal muscle actin was prepared and gel filtered (22). Actin concentrations were determined by absorbance at 290 nm, !259 # 26,600 M$1 cm$1 (23). Actin was labeled with pyrenyl iodoacetamide (pyreneactin) and gel filtered (24). All actin was stabilized with a molar equivalent of phalloidin (Sigma). Calmodulin (CaM) was expressed in bacteria and purified as described (25). (Pp. 1-2)", + "Tryptophan fluorescence (#ex # 295 nm) was measured using a 320 nm WG long pass emission filter (Oriel). (p. 2)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-W8Zi3Y46u", + "claim": "Overexpression of GFP-GRAF1 in HeLa cells showed long (1-5um) tubules that turn over completely within 10 minutes based on fluorescence microscopy", + "citekey": "lundmark2008gtpaseactivating", + "context": [ + "HeLa cells were grown in RPMI 1640 or MEM media (GIBCO) supplemented with LGlutamine, 10% foetal bovine serum, non-essential amino acids (for MEM), and transfected using Genejuice (Novagen) for transient protein expression. For primary cultures, rat hippocampal neurons/astrocytes were prepared by trypsin digestion and mechanical trituration from E18 or P1 Sprague-Dawley rats and plated onto poly L-lysine coated coverslips. Cells were cultured in B27-supplemented Neurobasal media. (Supplemental materials p. 2)", + "For real time microscopy of the dynamics of GRAF1- and GRAF1 BAR+PH- positive tubules, transfected cells on glass-bottom Petri dishes (WillCo Wells BV, Amsterdam) were washed with buffer (125mM NaCl, 5mM KCl, 10mM Dglucose, 1mM MgCl2, 2mM CaCl2 and 25mM HEPES) and images were taken using a 5-live scanning microscope (Zeiss) or spinning disc confocal system (Improvision) with subsequent analysis in LSM Image Browser (Zeiss), ImageJ (freeware) or Volocity (Improvision) (Supplemental materials p.3)", + "We found GRAF1-positive tubular structures to be spectacularly dynamic (Figure 1E and Movie S1)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-MIpYGNa-U", + "claim": "WAVE2 and IRSp53 both localized at ruffles and cell-cell junctions in A431 cells.", + "citekey": "suetsugu2006optimization", + "context": [ + "**Cells and purification of the WAVE2 complex** A431 cells were transfected with FLAG-tagged WAVE2 in pCMV-Tag 2 (Stratagene) or with vector alone. FLAG-tagged WAVE2 is functional because the transfection of FLAG-tagged WAVE2 into WAVE2 knockout cells eliminated WAVE2 deficits. After selection with G418, cells with stable FLAG-WAVE2 expression were grown. These cells did not show any significant change in growth or appearance compared with vector-transfected cells or parental cells. The amount of FLAG-WAVE2 expression was similar to the amount of endogenous WAVE2. 106 cells were plated onto 15-cm dishes and cultured for 5 d. After serum starvation overnight, some cultures were stimulated with EGF. To purify the WAVE2 complex from the cytosol (cytosol fraction), cells were harvested in buffer A containing 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 10 \u03bcg/ml aprotinin, and 10 \u03bcg/ml leupeptin.", + "Figure 1. **Localization of WAVE2 and IRSp53 in A431 cells.** (A) Localization of WAVE2 and IRSp53 in EGF-stimulated A431 cells treated with control, IRSp53, or IRTKS stealth RNAi was examined by immunofluorescence with anti-WAVE2 antibody or anti-IRSp53 antibody (M051-3). Actin filaments stained with phalloidin are also shown. WAVE2 and IRSp53 were localized at ruffles (arrowheads) and cell\u2013cell junctions (arrows).", + "All fluorescent images were taken through a microscope (Eclipse E600; Nikon) with a confocal microscopy system (Radiance 2000; Bio-Rad Laboratories) at room temperature. Fluorochromes used include AlexaFluor488, 546, and 647 and rhodamine (all purchased from Invitrogen). A 60× NA 1.40 oil immersion objective (Nikon) was used. Images were assembled with Adobe Photoshop. In each plate, photographs were cropped, and each fluorochrome was adjusted identically for brightness and contrast to represent the observed images. Time-lapse images were taken through a phase-contrast microscope (Axiovert S100; Carl Zeiss MicroImaging, Inc.) with a camera (CCD-782-Y/HS; Princeton Instruments). A 40× NA 1.30 FLUAR oil immersion objective (Carl Zeiss MicroImaging, Inc.) was used." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-6wkebK9Rb", + "claim": "Depletion of \u03b21 or \u03b23 integrins by siRNA in HeLa cells showed no difference in the percentage of dynamic or static CCSs as compared to siCTRL cells.", + "citekey": "baschieri2018frustrated", + "context": [ + "Spinning disk microscopy of live cells. Cells were imaged at 5 s intervals for the indicated time using a spinning disk microscope (Andor) based on a CSU-W1 Yokogawa head mounted on the lateral port of an inverted IX-83 Olympus microscope equipped with a \u00d760 1.35NA UPLSAPO objective lens and a laser combiner system, which included 491 and 561 nm 100 mW DPSS lasers (Andor). Images were acquired with a Zyla sCMOS camera (Andor). The system was steered by IQ3 software (Andor). For CCS dynamics quantification, we measured the lifetime of CCSs using the TrackMate plugin of ImageJ47. Tracks below 5 s of duration (detected on only 1 frame) were discarded. Measured individual lifetimes were pooled into two groups: the \u201cdynamic\u201d group corresponding to structures with a lifetime below the duration of the movie (5 min) and the \u201cstatic\u201d group with a lifetime of 5 min. Of note, the relative percentage of dynamic versus static structures depends on the duration of the movie because static structures are only counted once while dynamic structures continuously nucleate and disappear during the movie. For this reason, all quantifications of CCS dynamics represent the relative number of static or dynamic events detectable at the plasma membrane at a given time point. At least 1000 CCSs from at least five cells per conditions and per experiments were tracked in 3\u20135 independent experiments. Data are expressed as mean \u00b1 SD. (p. 11)", + "HeLa cells (a gift from P. Chavrier, Institut Curie, Paris, France; ATCC CCL-2), genome-edited HeLa cells engineered to expressed an endogenous GFP-tagged or mCherry-tagged \u03bc2 subunit, HepG2 cells (ATCC HB8065), Caco-2 cells (ATCC HTB-37), MDA-MB-231 cells (a gift from P. Chavrier, Institut Curie, Paris, France; ATCC HTB-26), or genome-edited MDA-MB-231 cells engineered to expressed an endogenous GFP-tagged \u03bc2 subunit (a gift from D. Drubin, University of California-Berkeley, California, USA) were grown in DMEM Glutamax supplemented with 10% foetal calf serum at 37 \u00b0C in 5% CO2. All cell lines have been tested for mycoplasma contaminations. For most experiments, cells were grown on substrates coated with a 50 \u03bcg/ml solution of collagen-I (Thermo Fisher Scientific\u2014Cat. Nr. A10483-01) unless otherwise stated. (p. 10)", + "For siRNA depletion, 200,000 cells were plated in six well plates. After 24 h, cells were treated with the indicated siRNA (30 nM) using RNAimax (Invitrogen, Carlsbad, CA) according to the manufacturer\u2019s instruction. Protein depletion was maximal after 72 h of siRNA treatment as shown by immunoblotting analysis with specific antibodies. To deplete CHC or \u03bc2-adaptin, cells were transfected once as described above and then a second time, 48 h later, with the same siRNAs. In this case, cells were analyzed 96 h after the first transfection. The following siRNAs were used: \u03b25-1, 5\u2032-GCUCGCAGGUCUCAACA UA-3\u2032; \u03b25-2, 5\u2032-GGUCUAAAGUGGAGUUGUC-3\u2032; \u03bc2-adaptin, 5\u2032-AAGUGGA UGCCUUUCGGGUCA-3\u2032; Clathrin heavy chain (CHC), 5\u2032-GCUGGGAA AACUCUUCAGATT-3\u2032; \u03b1v-1, 5\u2032-CCUCUGACAUUGAUUGUUA-3\u2032; \u03b1v-2, 5\u2032-C CGAAACAAUGAAGCCUUA-3\u2032; DAB2, 5\u2032-GAGCAUGAACAUCCAGAU AATT-3\u2032; Numb, 5\u2032-GAUAGUCGUUGGUUCAUCATT-3\u2032; Integrin \u03b21 ON-TARGET plus SMART pool (Dharmacon L-004506-00), Integrin \u03b23 siGENOME Human ITGB3 siRNA (Dharmacon M-004124-02); Talin1, 5\u2032-AC AAGAUGGAUGAAUCAAUUUU-3\u2032; non-targeting siRNAs (siControl), ONTARGET plus Non-Targeting SMART pool siRNAs (Dharmacon D-001810-01) (p. 11)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-NU1hryH_8", + "claim": "GRAF1-depletion by siRNA knockdown in HeLa cells has no effect on transferrin uptake", + "citekey": "lundmark2008gtpaseactivating", + "context": [ + "For immunofluorescence trafficking assays, biotinylated holo-transferrin, (Sigma Aldrich), Alexa Fluor 647- conjugated transferrin (Invitrogen), Alexa Fluor 546/555- conjugated CTxB (Invitrogen), DiI (Invitrogen), FITCdextran (10kDa MW, used for fluorimetric uptake assay, Invitrogen), and biotinylated dextran (10kDa MW, used for immunofluorescent uptake assays, Invitrogen), were diluted in pre-warmed media, added to cells and incubated for time periods and temperatures as described in figure legends. After washing, cells were fixed and subjected to immunofluorescence analysis as described", + "GRAF1-depleted cells were assessed for their ability to endocytose dextran (allowing assessment of total endocytic capacity), both by epifluorescence microscopy and by a quantitative fluorimetric assay (Figures 4B and 4C).", + "HeLa cells were grown in RPMI 1640 or MEM media (GIBCO) supplemented with LGlutamine, 10% foetal bovine serum, non-essential amino acids (for MEM), and transfected using Genejuice (Novagen) for transient protein expression. For primary cultures, rat hippocampal neurons/astrocytes were prepared by trypsin digestion and mechanical trituration from E18 or P1 Sprague-Dawley rats and plated onto poly L-lysine coated coverslips. Cells were cultured in B27-supplemented Neurobasal media. For GRAF1 depletion, HeLa cells were transfected with Stealth siRNAs specific against human GRAF1 using Lipofectamine 2000 (Invitrogen) according to manufacturers instructions. The Invitrogen siRNA duplex sequences used were siRNAa (UUA UCU CCC AUU CAG CAC AGA UAU C/ GAU AUC UGU GCU GAA UGG GAG AUA A), and siRNAb (UUU GAA ACU GGU ACA UCA UGA GUG G/CCA CUC AUG AUG UAC CAG UUU CAA A). Cells were cultured for an additional 48 hours for efficient silencing of the GRAF1 expression. Stealth Block-it siRNA (Invitrogen) was used as a control. AP2 siRNA was used as previously described [3]. (Supplemental materials p. 2)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-FxTUmxjh2", + "claim": "Simulated fission yeast endocytic actin networks produced ~3000 pN of force, while simulated budding yeast endocytic actin networks produced ~1000 pN of force", + "citekey": "nickaeen2022model", + "context": [ + "To determine the time-dependent driving force fdrive (t), we integrate over the surface of the invagination, for each t, the active and viscous forces exerted on the invagination by actin flow and then project the resultant force on the invagination axis [see Supplemental Material, Eq (S4)].", + "Our model, described in detail in Nickaeen et al. (2019), couples kinetics of actin nucleation, polymerization, and turnover constrained by counts of each participating protein over time (Berro et al., 2010), with the mechanics of the assembling filamentous meshwork approximated as that of a visco-active gel (Kruse et al. 2005; Prost et al., 2015). Mathematically, the model consists of advection-reaction equations governing densities of proteins involved in patch assembly (see Eq (S1) in the Supplemental Material), and a force-balance equation yielding actin velocities [Eq (S2) of the Supplemental Material].", + "The coupled system of the reaction-transport equations [Eqs (S1), (S1*), and (S1**)], force-balance equation [Eq (S2)], and Eq (1) was solved using a moving-mesh solver of COMSOL Multiphysics, a software package for solving spatial multiphysics problems on finite element meshes (COMSOL Multiphysics, 2015).", + "Figure 4 compares results for budding and fission yeast obtained with the optimized parameter sets. Graphs in panels A and B illustrate how turgor pressure affects fdrive (t) and fnet(t) = fdrive(t) \u2013 fresist(t)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-45WDQVJkn", + "claim": "ABI2 formed a complex with GRAF1 based on immunoprecipitation of HeLa cell lysates, in a manner dependent on GRAF1 phosphorylation and treatment with mitochondrial damaging agent oligomycin-A and antimycin-A", + "citekey": "zhu2023graf1", + "context": [ + "GRAF1b isoform full length or various GRAF1 domains were cloned into pGEX-6P-1 vector and expressed as glutathione-s-transferase (GST) fusion proteins in BL21 E. coli. The expressed GST-fusion proteins were purified with Glutathione Sepharose 4B (Cytiva). For GST pull-down assays, Hela/YFP-Parkin cells were collected in lysis buffer (20 mM Tris HCl, 150 mM NaCl, 10% glycerol, 0.5 % Triton X100 pH7.4 with 1x HALT phosphatase & protease inhibitor cocktail). Cell lysates were incubated on ice for 15 min and centrifuged at 20,000xg for 15 min. The supernatants were incubated with GST-fusion protein-loaded beads for 1 hr end-over-end rotation at 4 \u00b0C. Beads were washed 5 times with cell lysis buffer and stored 4 \u00b0C until use or up to 7 days.", + "a Co-IP of endogenous ABI2 with Flag-GRAF1 WT or phosphor-deficient AAA variant in GRAF1 KO Hela/YFP-Parkin cells. EV: empty vector.", + "For western blot analysis, Hela cells or NRVCMs in 6 well plates with indicated treatment were lysed in Triton X-100 lysis buffer (25 mM Tris-HCL pH7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100 with 1x HALT phosphatase & protease inhibitor cocktail). Lysate concentration was measured by Pierce BCA protein assay kit. Approximately 25 ~ 50 \u03bcg protein per sample was separated on SDS-PAGE and then wet transferred in Tris-glycine transfer buffer to 0.2 \u03bcm pore size nitrocellulose membranes (Bio-Rad). HRP-conjugated secondary antibodies and ECL chemiluminescent substrate (GE Healthcare) were used to detect target proteins in blots.", + "To perform co-immunoprecipitation of target proteins from COS7 cell lysates, COS7 cells were transfected with the specified plasmids and expressed the target proteins for a duration of 20 h. Subsequently, transfected COS7 cells were treated with a combination of Oligomycin A (2.5 \u03bcM) and Antimycin A (250 nM) for a period of 6 h to induce mitophagy. Cell lysates were prepared in lysis buffer (50 mM HEPES-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.8% Chaps and 1x HALT phosphatase & protease inhibitor cocktail). For co-immunoprecipitation of target proteins from Hela cells, cell lysates were prepared in lysis buffer (20 mM Tris HCl, 150 mM NaCl, 10% glycerol, 0.5 % Triton X100 pH7.4 with 1x HALT phosphatase & protease inhibitor cocktail). Cell lysates were incubated on ice for 15 min and centrifuged at 20,000 rcf for 15 min. Protein concentration was measured by Pierce BCA protein assay kit (Thermo Fisher). Protein lysates (500 ~ 800 \u03bcg) were incubated with mouse anti-Flag M2 (F1804,Sigma) or rabbit anti-GFP (A11122, Thermo Fisher) bound with Dynabeads\u00ae Protein G (Thermo Fisher) with end-over-end rotation for 3 h at 4 \u00b0C. Magnetic beads were washed 4 times with lysis buffer and were transferred to a clean tube for one more wash. Co-IPd protein complexes were eluted by boiling in Laemmli buffer plus \u03b2-mercaptoethanol for 5 min." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-JvW-piCf8", + "claim": "Cortactin mutants lacking phosphorylatable Tyrosine site could not rescue the Knockdown of endogenous cortactin in Hela Cell", + "citekey": "zhu2007receptormediated", + "context": [ + "FIGURE 2. Cortactin mutant deficient in tyrosine phosphorylation fails to rescue uptake of Tfn in cortactin knockdown cells. A, MDA-MB-231 cells were infected with retroviruses carrying GFP or GFP-tagged murine cortactin variants (cortactin-GFP, Cort-F-GFP, and Cort\u0003SH3-GFP). The infected cells were treated with either human cortactin (C) or HS1 (H) siRNA for 2 days. The total cell lysates were prepared and analyzed for cortactin expression by immunoblotting using monoclonal cortactin antibody. The samples were also immunoblotted by actin antibody. B, siRNA-treated cells were subjected to Tfn uptake analysis after 2 days of treatment as described under \u201cExperimental Procedures.\u201d The data shown are the mean \b S.D. of three independent experiments. The p values of Student\u2019s t test refer to the difference between cortactin- and HS1 siRNA-treated cells (*) and the difference between cortactin-siRNA-treated cells expressing cortactin-GFP and those expressing CortF-GFP or Cort\u0003SH3-GFP (**).", + "To study the role of cortactin tyrosine phosphorylation in receptor-mediated endocytosis, HeLa cells were infected with a retroviral vector, MGIN (39), encoding a murine cortactin construct tagged by GFP at the C terminus (cortactinGFP), a mutant of Y421F/Y466F/Y482F (Cort-F-GFP), and a mutation with deletion of the SH3 domain (Cort\u0003SH3-GFP) (Fig. 1A).", + "Analysis of the Effect of Tyrosine Phosphorylation on the Affinity of Cortactin for Dynamin-2\u2014Cortactin was phosphorylated by Src kinase (Upstate) in a reaction buffer containing 100 mM Tris, pH 7.4, 125 mM MgCl2,25mM MnCl2,2mM EGTA, 1 mM ATP, and 0.2 mM Na3VO4 at 30 \u00b0C for 30 min, the modified condition recommended by the manufacture. To analyze the affinity of cortactin for dynamin, 18 pmol of cortactin, either phosphorylated or nonphosphorylated, was incubated with different amounts of GST-dynamin-2 immobilized on glutathione-Sepharose in buffer", + "As shown in Fig. 2A, the human cortactin siRNA inhibited cortactin expression by \u000560 \u2013 80% as compared with that of the cells treated with the HS1 siRNA. When biotinTfn uptake was analyzed, a nearly 70% reduction in endocytosis was found in the cells treated with cortactin siRNA (Fig. 2B, left), which is consistent with our previous report (22) and a report from others (23). To determine whether the decrease in Tfn endocytosis was due to the specific loss of cortactin in the siRNA-treated cells, we treated the cells expressing murine cortactin-GFP with HS1 and cortactin siRNAs, respectively. In addition to the endogenous cortactin, the human cortactin siRNA also showed a slight inhibition of the exogenous murine cortactin (Fig. 2B, right), presumably because of the 95% identity between human and mouse mRNAs in the region targeted by the siRNA. Nevertheless, the overall level of exogenous cortactin proteins was greater than or similar to that of the endogenous cortactin of the control cells. At this level cortactin siRNA failed to inhibit Tfn uptake in the cortactinGFP-expressing cells, indicating that cortactin-GFP protein maintains its endocytic function similar to the endogenous cortactin. However, the same cortactin siRNA retained inhibitory activity for Tfn uptake in the cells expressing either mutant Cort-F-GFP or Cort\u0003SH3GFP (Fig. 2B)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-UlZY0Mnwt", + "claim": "hiPSCs at the colony edges showed large, paxillin-positive focal adhesions with thick actin stress fibers parallel to the colony edge.", + "citekey": "narva2017strong", + "context": [ + "Cells were plated on VTNcoated glass plates and fixed after 24 hr with 4% paraformaldehyde for 15 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 15 min, and stained with the appropriate antibodies, prepared in 30% (v/v) horse serum (Gibco), in addition to DAPI and attophalloidin-647 (Sigma) staining.", + "HiPSC lines HEL24.3, HEL11.4, HEL24.3-SOX2-ntdT reporter, hESC line H9, and parental fibroblasts were obtained from the University of Helsinki. Cells were grown on MG (Corning) or on 5 mg/mL VTN (Life Technologies) in Essential 8 medium (Life Technologies). HiPSCs were differentiated in 10 mM RA (Sigma).", + "Surprisingly, hiPSCs displayed large, PAXILLIN-positive FAs at the edges ofthe colonies (\u2018\u2018cornerstone\u2019\u2019 FAs) in stark contrast to the" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-iLicutBoX", + "claim": "Fluid uptake as measured by fluorescent 10kDa Dextran increased upon relaxation after mechanically induced stretch by vacuum-based equi-bi-axial stretching device in CHO cells.", + "citekey": "thottacherry2018mechanochemical", + "context": [ + "For the stretch\u2013relax experiments, cells plated on PDMS membrane were loaded on the cell stretcher system (Fig. 1b) within a temperature-controlled chamber at 37 \u00b0C. Vacuum was applied beneath the ring containing the PDMS sheet, deforming the membrane and stretching the cells plated on the PDMS. The setup was calibrated to stretch cells equi-biaxially to cause 6% strain for 90 s.", + "The quantification of endocytic uptake for a population is done by imaging on 20\u00d7, 0.75 NA on a Nikon TE300 widefield inverted microscope. For the stretch experiments, an upright microscope (Nikon eclipse Ni\u2013U) was used with a water immersion objective (60\u00d7, 1.0 NA). For endosome size calculation, spinning disk confocal microscope (100\u00d7, 1.4 NA) was used with ANDOR iQ software followed by analysis using 3D object counter plugin in Fiji61. The images were analyzed using MetaMorph\u00ae or MicroManager software and were processed for presentation using Adobe Illustrator. All images displayed are equally scaled for intensity unless otherwise mentioned. The integrated intensities, spread area and thus average uptake per cell were determined by drawing regions around each cell using the region measurement option in Fiji61. For plotting endocytic uptake, all values are normalized to the mean value of the control and plotted as a box plot using Origin software (OriginLab, Northampton, MA). Box plot shows uptake per cell (each data point) and it also shows median (middle line), standard deviation (whiskers), 25th percentile (lower line of box) and 75th percentile (upper line of box) value. The box plot contains points pooled from two separate experiments with technical duplicates in each and normalized to their respective controls. The total number of cells in each condition (pooled from all experiments) is mentioned in the legends. Statistical significance was tested using the Mann\u2013Whitney test and p values used to determine significance are reported in the legends. The scale bar is 10 \u03bcm, unless otherwise mentioned.", + "CHO (Chinese Hamster Ovary) cells stably expressing FR-GPI and human transferrin receptor TfR (IA2.2 cells)8, HeLa9, MEF (Mouse embryonic fibroblasts) cells10, Caveolin null MEF10, conditional null dynamin triple knockout MEF11, vinculin null MEF12, FR-AGS (Human adenogastric carcinoma cells stably transfected with FR-GPI)13,14, IRSp53 null MEFs14 and IRSp53 null MEFs stably expressing WT-IRSp5314 were used for the assays. HF12 media (HiMEDIA, Mumbai, India) and DMEM (Invitrogen) supplemented with NaHCO3 and L-Glutamine/Penicillin/Streptomycin solution (Sigma Aldrich) were used for growing CHO/FR-AGS cells and the different MEF lines respectively. (supplemental materials p. 22)", + "Fluid uptake (90 s) in CHO cells at steady state (steady state), immediately on relaxing the stretch (stretch\u2013relax), or after a waiting time of 90 s on relaxing the stretch (stretch\u2013relax\u2013wait) (n = control (316), stretch\u2013relax (257), stretch\u2013relax\u2013wait (277))." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-1Orp-fkjH", + "claim": "Cells injected with anti-cortactin antibodies decreased the degree of transferrin internalization into clone 9 cells", + "citekey": "cao2003cortactin", + "context": [ + "both the cortactin AB3 and C-Tyr antibodies exhibited a profound inhibitory effect on ligand uptake (Fig. 4b and c). In over 80% (AB3-injected) and 70% (C-Tyr-injected) of the cells counted, transferrin internalization appeared completely ablated (Fig. 4e)", + "For immunofluorescence microscopy and confocal microscopy, cells were grown on coverslips for 1 to 2 days and prepared as described previously (2). Clone 9 cells were double stained with anti-cortactin (AB3) polyclonal antibody or anti-cortactin (C-Tyr) polyclonal antibody with clathrin (X-22) monoclonal antibody. Transiently transfected cortactin-RFP-expressing cells were also costained with clathrin (X-22). The anticortactin and anti-SH3 antibodies were used to identify the Cort SH3-pCR3.1expressing cells. For the transferrin uptake experiments, the cells were permeabilized with 0.01% digitonin (Fluka, Ronkonkoma, N.Y.). Cells were viewed with an Axiovert 35 epifluorescence microscope (Carl Zeiss, Inc., Thornwood, N.Y.) equipped with a 100-W mercury lamp and a 100\u0006 objective lens (Zeiss Plan-Neofluar 1.30) and a confocal microscope (LSM-310 or 510; Carl Zeiss, Inc.) equipped with an argon\u2013krypton laser and a 100\u0006 objective lens.", + "Clone 9 cells, an epithelial cell line isolated from normal rat liver (ATCC CRL-1439), were maintained in Ham\u2019s F-12K medium supplemented with 10% fetal bovine serum (Invitrogen), 100 U of penicillin/ml, and 100 \u0005g of streptomycin/ml in 5% CO2\u201395% air at 37\u00b0C. Cells were cultured in T-75 flasks (Fisher Scientific, Pittsburgh, Pa.) or on 22- by 22-mm glass coverslips for transfections and immunocytochemistry, respectively.", + "Images for quantitation were acquired by using a cooled charge-coupled device camera (Photometrics SenSys, Tuscon, Ariz.) driven by Metamorph (Universal Imaging, West Chester, Pa.). All images of microinjected cells were taken at full resolution (1,024 by 1,024 pixels) with the same acquisition settings (exposure time, 5 s; 12-bit grayscale). The percentage of cells demonstrating an inhibition of transferrin or dextran uptake was determined by visual inspection. The mean percentage of cells showing reduced internalization was determined from three separate experiments and at least 150 cells for each condition. Fluorescence intensity quantitation was performed with IPLab (Scanalytics, Inc., Fairfax, Va.). The area occupied by each cell was traced, and the mean fluorescence intensity per unit area was determined for at least 100 cells in each injection condition." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-1B3Jax3yY", + "claim": "Without clathrin-mediated endocytosis, flat-clathrin lattices and reticular adhesions could not form.", + "citekey": "hakanpaa2023reticular", + "context": [ + "Cell culture and reagents U2Os, U2Os-AP2-GFP, U2Os-AP2-halo, U2Os-AP2-GFP-ITGB5mScarlet, HeLa-AP2-GFP, and HeLa-AP2-halo were cultured in MEM supplemented with 10% FBS (Gibco) and penicillinstreptomycin (100 U/ml, Thermo Fisher Scientific). HDF-AP2halo and Caco2-AP2-halo were cultured in DMEM supplemented with 10% FBS (Gibco) and penicillin\u2013streptomycin (100 U/ml, Thermo Fisher Scientific). MCF7-AP2-halo was cultured in DMEM supplemented with 10% FBS (Gibco) and penicillinstreptomycin (100 U/ml, Thermo Fisher Scientific), 2 mM glutamine, 5 \u03bcg/ml human insulin, and 1 \u03bcM sodium pyruvate. hMEC-AP2-GFP was cultured in MEGM complete medium (Lonza).", + "B) Analysis of RA coverage from samples in A. N (images): control = 12, shAP2A1#1 = 8, shAPA1#2 = 11, shAPS1#1 = 10, shAPS1#2 = 19, shAPS1#3 = 10. Data were obtained from two individual experiments and similar results were observed in five independent experiments. One-way ANOVA with Tukey\u2019s multiple comparison, F(5, 64) = 43.11, P < 0.001.", + "These experiments were performed using U2Os cells with an endogenously GFP-tagged \u03b1 adaptin 2 Sigma-Aldrich subunit (AP2S1, hereafter referred to simply as AP2). AP2 is a widely used CME marker that faithfully mirrors clathrin dynamics (Almeida-Souza et al., 2018; Ehrlich et al., 2004; Rappoport and Simon, 2008). We used endogenously tagged cell lines throughout this study as the expression level of the AP2 complex was shown to modulate the amount of FCLs (Dambournet et al., 2018).", + "Manipulation of CME machinery to study RA formation Clathrin assembly at the cell membrane was reduced by silencing two subdomains of AP2 or by overexpressing AP180 ct, which acts as a dominant negative for AP2. U2Os AP2-GFP cells silenced for AP2A1 shRNA #1 and #2 or AP2S1 shRNA #1, #2, and #3, and control shRNA, and plated on non-coated imaging dishes were fixed and stained for integrin \u03b1v\u03b25 and FA marker p-Pax. Alternatively, AP180 ct was overexpressed in U2Os AP2-GFP cells, and cells were plated on non-coated imaging dishes, fixed the next day, and stained for integrin \u03b1v\u03b25 and p-Pax. RA formation (integrin \u03b1v\u03b25 adhesions without FA marker) and FAs (integrin \u03b1v\u03b25 colocalizing with FA marker) were imaged using TIRF microscopy.", + "While all FCLs colocalize to RAs, FCL-free areas of larger RAs are rather common (e.g., Fig. 2 A; Fig. 3, A\u2013C;andFig. S2 D), which may give the impression that FCLs are formed on preexisting RAs. Nevertheless, the fact that both structures are inhibited independently by FN suggests a deeper relationship and led us to ask if RAs can exist without the CME machinery. To answer this question, we quantified the RA coverage in U2OsAP2-GFP cells silenced for the clathrin adaptor AP2 complex subunits \u03b11 (AP2A1) or Sigma-Aldrich 1 (AP2S1) in cells plated on non-coated dishes, a condition where we observe large RAs. Consistent with an important role played by the CME machinery in RA formation, AP2A1- or AP2S1-silenced cells (easily recognizable as cells with little to no AP2-GFP signal) did not display RAs. Instead, integrin \u03b1v\u03b25 localized to FAs (Fig. 4, A and B)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-6-XC7noR7", + "claim": "siRNA silenced Swip1 showed reduced internalization of B1-integrin in MDA-MB-231 cells.", + "citekey": "morenolayseca2021cargospecific", + "context": [ + "Fig. 5 | Swip1 is a cargo adaptor for the CG pathway. ... b, Representative micrographs (top), and levels of dextran\u2013TMR (bottom right) and \u03b21-integrin\u2013AF488 (bottom left) internalization in control-, Swip1- and IRSp53-silenced MDA-MB-231 at 15 min. Swip1 siRNA1, n = 157 cells; Swip1 siRNA2 and siCTRL, n = 160 cells; siCTRL, n = 121 cells; IRSp53 siRNA1, n = 177 cells; and IRSp53 siRNA2, n = 121 cells. ***P = 0.0001 and **P = 0.0004.", + "Screening for Rab21 interaction partners by GFP-pulldown. Each mass-spectrometry experiment consisted of a mixture of a GFP-pulldown in active GFP\u2013Rab21-expressing MDA-MB-231 cells (WT or active Rab21 Q76L mutant) cultured in heavy medium and a GFP-pulldown in control cells (expressing GFP or the inactive GFP\u2013Rab21 T33N mutant) cultured in light medium (forward experiment). In the reverse experiment, the heavy and light media were exchanged (label-swap experiment). Briefly, co-immunoprecipitation samples were prepared as follows. Cells (two 15 cm dishes) were cultured until they reached 60\u201380% confluence, washed with ice-cold PBSM (PBS + 5 mM MgCl2), harvested in PBSM, pooled and washed again. The cell pellets were resuspended in 600 \u03bcl lysis buffer LB (50 mM Tris, pH 7.5, 5 mM MgCl2, 150 mM KCl, 1.3% n-beta-octyl-d-glucopyranoside, 10% glycerol, protease and phosphatase inhibitors, and 500 \u03bcM GppNHp for GFP-active Rab21-expressing cells or 500 \u03bcM GDP for GFP-inactive Rab21-expressing cells) and lysed by douncing 40\u00d7 in a tissue grinder (dounce homogenizer) and incubating on ice for 20 min. The insoluble fraction was removed by centrifugation at 18,000g and the supernatant was incubated with 20 \u03bcl GFP-Trap agarose beads (Chromotek) for 60 min at 4 \u00b0C by overhead rotation. The beads were then washed three times with 500\u03bcl washing buffer WB (50 mM Tris, pH 7.5, 5 mM MgCl2, 300 mM KCl and 10% glycerol), combining the heavy, GFP-active Rab21 immunoprecipitation with the light, GFP-inactive Rab21 immunoprecipitation (forward experiment) during the second wash step. Proteins were eluted from the beads in 100 \u03bcl of U/T buffer (6 M urea + 2 M thiourea in 10mM HEPES, pH 8.0) for 15 min with shaking in a bacterial shaker at room temperature and an agitation rate of 1,400 r.p.m. The eluted proteins were collected and the process was repeated to maximize the protein yield. The eluted proteins were precipitated by adding 70 \u03bcl of 2.5 M Na-acetate, pH 5.0, 1 \u03bcl GlycoBlue (Thermo Fisher) and 1,700 \u03bcl ethanol to the pooled elution fractions (200 \u03bcl) in a 2 ml tube. After an overnight incubation at 4 \u00b0C, the precipitation mixture was centrifuged for 50 min at 20,000g and the resulting pellet was dried for 15\u201320 min at 60\u201370 \u00b0C.", + "Here we identify Swiprosin-1 (Swip1, EFHD2) as an interactor of Rab21 and a cargo-specific adaptor for CG-endocytosis.", + "To assess whether Swip1 is a cargo adaptor or an integral member of the CG pathway, we investigated whether it regulates the endocytosis of other CG cargos. Swip1 silencing had no effect on the uptake of 10 kDa dextran or major histocompatibility complex I (MHCI; Fig. 5b,c).", + "For visualization purposes, all of the identified proteins from each experiment were plotted (Fig. 1a and Extended Data Fig. 1a\u2013d), where each spot corresponds to a protein identified by mass spectrometry. Each plot is representative of two independent experiments (forward and reverse, x and y axis), where every experiment consists of two independent immunoprecipitations. The mean log2-transformed fold-change values from both experiments were plotted against the absolute protein intensities (intensity-based absolute quantification, iBAQ) and significance B was calculated according to Cox and Mann52. The error function was estimated using the erfc as is implemented in the pracma package53 (R package version 2.2.9). Abundance bins were defined by including 100 proteins in a subsequent order.", + "To identify Rab21-interacting proteins, we performed proteomic analyses by stable isotope labelling with amino acids in cell culture (SILAC) of cells expressing wild-type (WT) Rab21, the constitutively active Rab21Q76L mutant (CA-Rab21) or the Rab21T31N inactive mutant16,17." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-Pu_eipQdq", + "claim": "Matrigel stiffness can be tuned between ~10 Pa to ~300 Pa", + "citekey": "slater2018tuning", + "context": [ + "Collagen I Preparation Corning Collagen I High Concentration, rat tail (Collagen HC rat tail, Corning Cat. No. 354249) gelation is pH- and temperature-dependent, and the product must be neutralized in order to polymerize. Collagen HC rat tail was diluted using the alternate gelation procedure included in the lot-specific Certificate of Analysis30,31. All dilutions were prepared on ice in pre-chilled glass tubes. To prepare the desired volume and protein concentration of Collagen I solutions, 0.1X the final volume of 10X Dulbecco\u2019s Phosphate Buffered Saline (10X DPBS, Thermo Fisher), 0.023 mL of 1N Sodium Hydroxide (JT Baker) per mL of Collagen I were added and the final volume was adjusted using ice-cold DI-water. The contents of the tube were mixed by vortexing and the required volume of Collagen I was added to the solution using a positive displacement pipet, mixed by vortexing gently and held on ice. All diluted samples were mixed by inversion immediately before loading the test samples onto the rheometer using a positive displacement pipet (0.680 mL). Each protein concentration was measured in triplicate.", + "To investigate whether varying the protein concentration alters the elastic modulus of the Matrigel matrix gels, Corning Matrigel High Concentration, Growth Factor Reduced (Matrigel HC GFR; at a concentration of 19.1 mg/mL) was diluted using ice-cold DMEM and liquid samples were directly added to the rotational rheometer.", + "Over a range of concentrations from 3 to 19.1 mg/mL for Matrigel HC GFR, the elastic component of the shear modulus increased from 9.1 \u00b1 0.3 Pa to 288.2 \u00b1 9 Pa.", + "Figure 1. Increase of elastic modulus as a function of Corning Matrigel HC GFR concentration. (B) Plateau values of G' (in Pa) shown as a function of Matrigel matrix concentration (n = 3).", + "The temperature of the bottom plate (Peltier-controlled) was ramped from 5\u00b0C to 37\u00b0C and then held at 37\u00b0C, which is the recommended gelation temperature for Matrigel matrix. The evolution of the elastic component (G') and the viscous component (G\") of the complex shear modulus were observed over time and the sample was allowed to reach a state of equilibrium." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-7ioUQ5iO3", + "claim": "The percentage clathrin-coated structures with visible actin overlap decreased from ~50% to ~20% from flat to curved pits in four cell types by platinum-replica electron microscopy", + "citekey": "yang2022actin", + "context": [ + "The shape of CCSs in most cases was obvious in 2D PREM images. In uncertain cases, the degree of CCS invagination was determined using images tilted at \u00b110\u201320 degrees. Based on the degree of invagination, CCSs were classified into three categories: (i) flat CCSs with no obvious invagination; (ii) dome-shaped CCSs that had a hemispherical or less invaginated shape with visible edges of the clathrin lattice; and (iii) spherical CCSs that had a round shape with the clathrin lattice edges not visible in 2D projection images. Dome-shaped subdomains of flat CCSs were quantified separately from the contiguous flat CCSs. The CCS area in PREM images was measured using ImageJ software.", + "Branched actin networks in PREM samples were identified by the presence of two or more of the following characteristics: the presence of at least several Y-shaped configurations, short filament lengths, a wide range of filament orientations, and an abundance of actin filament ends. CCSs were considered to be actin-positive if branched actin networks were located no farther than 20 nm from the edge of the clathrin lattice. (p. 16)", + "Fraction of the actin-positive CCSs with any amount of branched actin extending over their lattice in 2D projection images in the indicated cell types. (p. 4)", + "Genome-edited human osteosarcoma U2OS cells and human cervical cancer HeLa cells, both endogenously expressing RFP-tagged clathrin light chain A (CLTA) and EGFP-tagged dynamin2 (DNM2), were a kind gift from Dr. David Drubin, University of California-Berkeley, California, USA41,93. Conditional epsin DKO MEFs with deleted epsin2 and epsin3 genes and floxed epsin1 gene were a kind gift of Dr. Pietro De Camilli, Yale University, Connecticut, USA33. U2OS, HeLa, potoroo epithelial kidney PtK2 cells and DKO MEFs were cultured at 37 \u00b0C with 5% CO2 in Dulbecco\u2019smodified Eagle\u2019s medium (DMEM) supplemented with GlutaMAX (#10569010, Thermo Fisher Scientific), 10% fetal bovine serum (FBS) (#F2442, Sigma-Aldrich) and 1% penicillin\u2212streptomycin. Mouse melanoma B16F1 cells were cultured in DMEM/F12 (#11330-032, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin\u2212streptomycin at 37 \u00b0C and 5% CO2 (p. 16)", + "For unroofing experiments, B16F1 cells were seeded on coverslips coated with laminin (25 \u03bcg/ml, #L2020, Sigma-Aldrich), and Ptk2, U2OS and Hela cells were seeded on coverslips coated with poly-Dlysine (0.2 mg/ml, #P6407, Sigma-Aldrich) or HistoGrip (1:50 dilution, #008050, Thermo Fisher Scientific)94,95.EpsinDKOandTKOMEFs were cultured on uncoated glass coverslips. B16F1 cells were unroofed as described previously18. For unroofing of other cells, coverslips with cells were quickly transferred into ice-cold PEM buffer (100 mM PIPES \u2212 KOH, pH 6.9, 1 mM MgCl2 and 1 mM EGTA) containing 2 \u03bcM unlabeled phalloidin (#P2141, Sigma) and, in some experiments, 10 \u03bcM taxol (#T7402, Sigma-Aldrich) and unroofed by a brief (1 s) ultrasonic burst from a 1/8-inch microprobe positioned at ~45\u00b0 angle ~3 mm above the coverslip and operated by Misonix XL2020 Ultrasonic Processor at 17\u201320% of output power. After sonication, the coverslips were immediately fixed with 2% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.3 for at least 20 min at room temperature. Sample processing for PREM was performed as described previously96,97. In brief, glutaraldehyde-fixed cells were post-fixed by sequential treatment with 0.1% tannic acid and 0.2% uranyl acetate in water, critical-point dried, coated with platinum and carbon, and transferred onto EM grids for observation. PREM samples were examined using JEM 1011 transmission electron microscope (JEOL USA, Peabody, MA) operated at 100 kV. Images were acquired by an ORIUS 832.10 W CCD camera driven by Gatan Digital Micrograph 1.8.4 software (Gatan, Warrendale, PA) and presented in inverted contrast (p. 16)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-F0N83vxEe", + "claim": "Knockdown of Dyn2 decreased the length of filopodia in H1299 cells", + "citekey": "yamada2016actin", + "context": [ + "Filopodial formation. H1299 cells were serum-starved for 16 h. Thereafter, the cells were transfected with dynamin 2 siRNAs, cortactin siRNAs, or the control siRNA, followed by incubation with DMEM supplemented with 10% FBS for 45 min. For the rescue experiments, cortactin was silenced in H1299 cells with oligo 3, and the cells were cultured for 24 h. The cells (1x105/coverslip) were then transfected with rat wild-type cortactin or cortactin W525K (0.25 \u03bcg each) cloned into the pIRES2-AcGFP1 expression vector (Clontech Laboratories, Santa Clara, CA, USA). Thereafter, the cells were stimulated with serum for 45 min, fixed, and stained with Alexa Fluor 488 or rhodamine-labeled phalloidin for visualization of filopodia.", + "The human non-small cell lung carcinoma cell line H1299 (Cat. no. ATCC CRL-5803; American Type Culture Collection, Manassas, VA, USA) was cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) at 37 \u030aC in an atmosphere of 5% CO2.", + "Compared with the length of filopodia in serum-stimulated control cells (10.2\u00b10.5 \u03bcm), dynamin 2 knockdown decreased filopodial extension in silenced cells (4.7\u00b10.6 \u03bcm) (Fig. 3B and C)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-j0UGLJ3e6", + "claim": "Cells treated with the WASP-inhibitor wiskostatin showed a time-dependent increase of GFP\u2013GRAF1-decorated tubes at the cell surface", + "citekey": "francis2015endocytic", + "context": [ + "Estimations of the fraction of fixed cells with GFPGRAF1 proteins in abundant or tubular structures were performed by epifluorescent visual assessment. (p. 11)", + "HeLa cells (ATCC-CRM-CCL-2) were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM; low glucose, L-glutamine, sodium pyruvate, HEPES and Phenol Red), supplemented with 10% fetal bovine serum (FBS; Gibco). Flp-In T-REx HeLa cell lines with tetracycline-inducible expression of GRAF1, GRAF1\u2013GFP, GFP\u2013GRAF1 and GFP\u2013GRAF1-R412D were generated as previously described (Mohan et al., 2015). Established cultures were grown in the mentioned DMEM, exceptions being during 800CW-SS-cargo internalisation assays and live-cell acquisitions when basic medium containing 4.5 g/l glucose was used (Gibco). The culturing media were further supplemented with 100 \u03bcg/ml hygromycin B and 5 \u03bcg/ml blasticidin S HCl (Gibco) for plasmid selection, and recombinant protein expression was induced by incubation in doxycycline hyclate (Sigma-Aldrich) for 20\u00b14 h. (p. 11)", + "The Imaris Software V7.5 (Bitplane) was utilised to analyse micrographs captured from fixed and live-cell samples as specified in Table S3. (p. 11)", + "The effects of actin polymerisation drugs on GRAF1 structures were analysed by fluorescence microscopy of fixed cells after treatment with 2 \u03bcM cytochalasin D, 2 \u03bcM latrunculin A (Sigma-Aldrich), 5 \u03bcM wiskostatin (Calbiochem) or DMSO.\" (p. 11)", + "Cells were fixed and prepared for immunofluorescence analysis as previously described (Lundmark et al., 2008). Images of fixed cell samples were captured using an epifluorescence Axioimager Z1 system (AxioCam MRm camera) (Zeiss) with the ZEN Software and a 63\u00d7 lens (Plan-Apochromat 1.40 Oil DIC 0.17) or an A1 R Laser Scanning Confocal Microscope system (ANDOR iXon EMCCD camera) (Nikon Instruments) under control of the NIS-Elements Microscope Imaging Software and a 60\u00d7 lens (Apochromat 1.40 Oil \u03bbS 0.17 WD 0.14, Nikon), at the appropriate excitation and emission wavelengths. Live-cell confocal movies were recorded using the 63\u00d7 lens in the Nikon system or the 63\u00d7 lens (Plan-Apochromat 1.40 Oil DIC M27) in a Cell Observer Spinning Disc Confocal Microscope system (ANDOR iXon Ultra) (Zeiss) controlled by the ZEN Software. Real-time TIRF acquisitions were captured by employing the 100\u00d7 lens in respective system [Apochromat 1.49 Oil 0.13-0.20 DIC N2 (Nikon) or Plan-Apochromat 1.46 Oil DIC M27 (Zeiss)]. Micrographs and the acquired movies were prepared (cropped, rotated, linearly adjusted for intensity and converted) using Adobe Photoshop or ImageJ. (p. 11)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-Ke0qFhkQL", + "claim": "Cortactin Knockdown by siRNA decreased the degree of transferrin endocytosis in MDA-MB-231 cells", + "citekey": "chen2006roles", + "context": [ + "While GFP knock-down as a mock treatment had no effect on transferrin uptake, knock-down of cortactin substantially attenuated the ligand uptake [Fig. 1(B,C)]", + "MDA-MB-231, 3T3-L1 cells and NIH/3T3 cells were obtained from ATCC (Maryland, USA). Cells were cotransfected with Dyn-GFP (or Dyn-PRD-GFP) and RFP-Cort (or RFP-Cort-SH3, RFP-Cort\u2206SH3) using the Lipofectamine reagent according to the manufacturer\u2019s instructions. Cells for further experiment", + "Cells were incubated with 10 \u03bcg/ml Oregon green labeled transferrin. The percentages of cells showing normal levels of internalized transferrin were determined by visual inspection in three independent experiments. Cells treated with BioPORTER (Sigma-Aldrich, St. Louis, USA) to introduce immunoreagents were subjected to ELISA-based endocytosis assay. The BioPORTER introduction of immunoreagents was performed according to the manufacturer\u2019s protocol. Capture-ELISA assay to measure endocytic transferrin was performed exactly as described [20,27]. Biotinylated human transferrin (10 \u03bcg/ml) was used in this assay. Each assay point was performed in duplicate.", + "For immunofluorescence microscopy, cells were either permeabilized with P-buffer (0.02% Triton X-100, 0.1 M Mes, pH 7.4, 1 mM MgCl2, 1 mM EGTA and 4% PEG8000) for 10 s or directly fixed with 4% paraformaldehyde for 20 min at room temperature followed by permeabilization with 0.2% Triton X-100, then blocked with 2.5% bovine serum albumin-phosphate-buffered saline (BSA-PBS). After immunostaining, cells were viewed using an inverted Nikon ECLIPSE TE2000-U with a digital camera controlled by Nikon ACT-1 V.2.51 software (Nikon, Tokyo, Japan)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-OT8BRD36j", + "claim": "Clathrin Light chains gives the clathrin lattice more stronger bending power.", + "citekey": "dannhauser2015effect", + "context": [ + "H6-tagged epsin 1 lacking the ENTH-domain (H6-epsin144\u2013575)and His-tagged AP180 lacking the ANTH-domain (H6-AP180328\u2013896)from rat brain were expressed and purified as previously described (44).", + "Liposomes from Brain polar lipid extract (BPLE; Avanti) containing 5% (w/w) Ni2+-NTA-DOGS lipid (Avanti) were prepared and coated with H6-epsin144\u2013575 and clathrin as described before (18).", + "In a two-stage binding assay, first H6-epsin144\u2013575 at 1.8 \u03bcM was attached to the liposomes at 4\u2218C in buffer G. Unbound protein was removed by centrifugation at 6.000 g and 4\u2218C, and in the second stage the resuspended liposomes were incubated with either clathrin (0.15 \u03bcM) or CHC triskelia (0.15 \u03bcM)for30minat15or25\u2218C, respectively, in a thermocycler. For ultrathin sectioning, liposomes were pelleted at 6000 g and 4\u2218C, fixed with 3% glutaraldehyde and further processed for embedding in Epon and staining as described previously (50). For the quantification of clathrin- or CHC-coated profiles images from ultrathin sections of plastic, embedded liposomes were analyzed using NIH ImageJ. Membrane buds were identified as coated outlines of liposomes with an inner diameter \u2264150 nm. The analysis of membrane budding at 15\u2218Cwas based on three independent experiments. Four ultrathin sections from pelleted membranes were examined in detail, and the percentile of coated membrane buds was determined from a total of 608.216-nm coated profiles. A membrane pellet from the 25\u2218Cincubationwasanalyzed in the same way. From three sections, 513.096-nm coated membrane profiles were quantitatively evaluated.", + "at 15\u2218C a marked inhibition of budding by the light-chain-free clathrin supervened (Figure 3).", + "After fixation of the protein on the electron microscope grids, the grids wererinsed,firstwithbufferG,thenwith100mM MES, 1 mM EGTA, 0.5 mM MgCl2, pH 6.4, blotted and then incubated for 1 min with 5% uranylacetateinH2O. Excess staining solution was carefully removed with moist filter paper. Clathrin assemblies on glass were visualized after critical point drying and PT/C-shadowing as described elsewhere (52). The specimens were examined with a Tecnai\u2122 G2 (FEI) electron microscope at an acceleration voltage of 200 kV or with a Morgagni (FEI) electron microscope at 80 kV. Digital images were generated with the TIA-Software (FEI), processed with the program NIH ImageJ and Adobe Photoshop CS.X." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-_N-gQJ0eV", + "claim": "Expressing WT GFP-WASP in __wasA-__ mutants rescues confinement of active Rac to the leading edge of __Dictyostelium__ cells.", + "citekey": "amato2019wasp", + "context": [ + "At a given time, a subset of puncta in WASP CRIB mutants is also enriched in active Rac. WASP-CRIB-mutants and active Rac appear to have distinct dynamics: active Rac appears to remain at the plasma membrane for longer than CRIB-mutated WASPs. Therefore, although some active Rac-positive structures are not enriched in CRIB-mutated WASPs, we cannot exclude that they were enriched in CRIB-mutated WASP at some stage. Representative plots showing active Rac marker localization from front to rear are shown in Figure 5E. Quantification (Figure 5F) reveals that cells expressing CRIB-mutated WASPs accumulate active Rac at the rear to the same degree as wasA\u0001 cells, while wild-type WASP rescues the spatial confinement of active Rac at the leading edge. In line with previous reports from wasA\u0001 cells [6], cells expressing either WASP CRIB mutant are significantly slower than those expressing wild-type WASP", + "Dictyostelium cells lacking Wiskott-Aldrich syndrome protein (WASP)", + "(F) Frequency of active Rac accumulation at the back of wasA\u0001 cells and wasA\u0001 cells expressing wild-type WASP or WASP CRIB mutants. wasA\u0001 (n = 11 cells): 88.2% \u00b1 3.5%; wasA\u0001/GFP-WASP (n = 28 cells): 12.7% \u00b1 1.0%; wasA\u0001/GFP-WASPDCRIB (n = 24 cells): 78.1% \u00b1 2.0%; wasA\u0001/GFPWASP*CRIB (n = 28 cells): 76.7% \u00b1 2.2%; means \u00b1 SEM. One-way ANOVA; wasA\u0001/GFP-WASP versus wasA\u0001/GFP-WASPDCRIB: p < 0.0001; wasA\u0001/GFPWASP versus wasA\u0001/GFP-WASP*CRIB: p < 0.0001.", + "Dictyostelium wild-type, wasA knockout, and inducible double null cells were grown at 22\u000eC on Petri dishes in HL5 supplemented with vitamins and micro-elements (Formedium)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-eryKP0bHY", + "claim": "Pulldown assay with Swiss3T3 lysates showed that GST-bound IRSp53 strongly associated with WAVE, but did not precipitate N-WASP.", + "citekey": "miki2000irsp53", + "context": [ + "As proline-rich regions are known to be involved in protein\u00b1protein interactions, we carried out a two hybrid screening using the proline-rich region of WAVE1 as `bait', which yielded several positive clones. DNA sequencing analysis revealed that they encode three identified proteins (IRSp53, nArgBP2 and profilin) and two unidentified ones. As IRSp53 gave the strongest positive signal on b-galactosidase assay (data not shown), we analysed it further.", + "Pull down assay GST\u00b1fusion proteins (10\u00b150 mg) were first immobilized on 20 ml of glutathionesepharose beads and then mixed with 400 ml protein samples such as cell lysates and purified proteins. After 2 h, the beads were washed with lysis buffer five times and 20 mlof SDS sample buffer was added. Samples (5 ml) were separated by SDS\u00b1PAGE, followed by immunoblotting and Coomassie staining. Cell lysates of Swiss3T3, COS7 and NIH3T3 cells were obtained by lysing half-confluent cells in a dish (diameter, 150 mm) with 1 ml lysis buffer. In the case of Sf9 cells infected with baculoviruses, cells were collected by centrifugation and then lysed in 1/20 volume of lysis buffer.", + "Figure 1 The specific and direct interaction of IRSp53 and WAVE2. ... b, Various GST\u00b1fusion SH3 domain-containing proteins immobilized on beads were mixed with Swiss3T3 cell lysates treated with or without PDGF (WAVE mobility-shift occurs by PDGF-treatment23). The bound proteins were analysed by immunoblotting.", + "We then examined whether the Src-homology (SH)-3 domain, located at the C terminus of IRSp53, associates with WAVE by a pull-down assay. The SH3 domain in IRSp53 strongly associated with WAVE, but N-WASP was not precipitated (Fig. 1b). This is comparable to Grb2/Ash and Fyn, which associate strongly with N-WASP, but less significantly with WAVE (Fig. 1b)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-YsCMystnc", + "claim": "CLIC markers CTxB, CD44, and Thy1 concentrated at the leading edge of mouse embryonic fibroblasts during wound healing", + "citekey": "howes2010clathrinindependent", + "context": [ + "(A) Confluent monolayers of WT MEFs or NIH3T3s were scratched and cells were allowed to migrate for 8\u201312 h. CTxB-555 and Tf-647 were pulsed into migrating cells for 2 min in the presence of anti-CD44 (WT MEF), anti-GFP (GFP-GPI expressing WT MEF), or anti-Thy-1 (NIH3T3) antibodies or cells were labeled for Cav-1 (WT MEF). Dotted lines indicate leading edges. Arrows show colocalization between anti-CD44 or anti\u2013Thy-1 and CTxB-555 but not Tf-647 at the leading edge. Bar, 20 \u03bcm. (p. 12)", + "For immunocytochemistry, cells were permeabilized in freshly made 0.1% saponin for 10 min and blocked in 0.2% fish skin gelatin/0.2% BSA in PBS. Coverslips were incubated with primary antibodies for 1 h at room temperature, followed by three times 10-min wash in PBS before incubation with secondary Alexa Fluor\u2013conjugated antibodies at 1:400 for 1 h (p. 14)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-XG3wvRdRY", + "claim": "Overexpression of a cortactin mutant lacking 3 tyrosine residues reduced CME transferrin uptake by nearly 40%", + "citekey": "zhu2007receptormediated", + "context": [ + "overexpression of a cortactin mutant, Cort-F, which lacks three tyrosine residues targeted by Src, reduced clathrin-mediated Tfn uptake by \u000540%.", + "As shown in Fig. 1C, the cells expressing cortactinGFP internalized Tfn at an efficiency similar to the cells infected with the viral GFP vector, indicating no significant effect of overexpression of wild-type cortactin on endocytosis. However, Tfn uptake in the cells expressing Cort-F-GFP was nearly 40% less than in cells expressing the vector alone (p \u0004 0.05) (Fig. 1C).", + "To study the role of cortactin tyrosine phosphorylation in receptor-mediated endocytosis, HeLa cells were infected with a retroviral vector, MGIN (39), encoding a murine cortactin construct tagged by GFP at the C terminus (cortactinGFP), a mutant of Y421F/Y466F/Y482F (Cort-F-GFP), and a mutation with deletion of the SH3 domain (Cort\u0003SH3-GFP)", + "FIGURE 1. Analysis of the activity of wild-type and mutant cortactin proteins for internalization of Tfn and formation of clathrin-coated vesicles. ... C, cells expressing cortactin variants were grown in serum-free medium at 37 \u00b0C for 60 min and incubated with biotin-Tfn for 30 min on ice. Endocytosis was initiated at 37 \u00b0C for 5 min and terminated on ice. The treated cells were subjected to enzyme-linked immunosorbent assay-based biotin-Tfn uptake as described under \u201cExperimental Procedures.\u201d The p value calculated by Student\u2019s t test refers to the difference between PP2- and non-PP2-treated cells.", + "As shown in Fig. 1C, the cells expressing cortactinGFP internalized Tfn at an efficiency similar to the cells infected with the viral GFP vector, indicating no significant effect of overexpression of wild-type cortactin on endocytosis. However, Tfn uptake in the cells expressing Cort-F-GFP was nearly 40% less than in cells expressing the vector alone (p \u0004 0.05) (Fig. 1C). Similarly, cells overexpressing Cort\u0003SH3GFP showed an impaired Tfn uptake at an efficiency 43% less than the control cells.", + "Capture Enzyme-linked Immunosorbent Assay\u2014Cells expressing cortactin-GFP or Cort-F-GFP were incubated in serum-free Dulbecco\u2019s modified Eagle\u2019s medium containing 1% bovine serum albumin and 20 mM HEPES at 37 \u00b0C for 30 min and incubated with 10 \u0004g/ml bio-Tfn for 30 min on ice. Endocytosis was initiated by incubating the mixture at 37 \u00b0C for 5 min and terminated by placing on ice. To measure internalization of bio-Tfn, total cell lysates were added to a 96-well ELISA (enzyme-linked immunosorbent assay) plate coated with an anti-Tfn antibody. After 12 h of incubation at 4 \u00b0C, each well was added with streptavidin-horseradish peroxidase and Roche BM blue substrate, respectively. Absorption at 450 nm was determined by a microplate reader (Anthos Labtec) and was plotted as a function of time." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-C8_EKRIsh", + "claim": "GFP-tagged ARPC3/Arc18 at C terminus did not affect __in vitro__ actin nucleation rates", + "citekey": "egile2005mechanism", + "context": [ + "Actin was purified from rabbit muscle and isolated as Ca2\u00fe-ATP-G-actin in G buffer (5 mM Tris-Cl [pH 7.8], 0.1 mM CaCl2, 0.2 mM ATP, and 1 mM DTT) according to Pardee and Spudich [15] and pyrenyl labeled. The yeast Arc40/ARPC1-YFP Arp2/3 complex was isolated from a strain expressing an Arp3-CaMBM-tev-ProtA subunit (RLY1945) as previously described [16]. The unlabeled control complex (Arp3MH, which has a [Myc]5His6 tag on Arp3 [16]) as well as the Arp3-, Arp2-, and Arc18/ARPC3-GFP-His10-labeled complexes were isolated as follows. Yeast cells were grown to mid log phase in YPD medium (OD600 2\u00014) washed in U buffer (50 mM HEPES [pH 7.5], 100 mM KCl, 3 mM MgCl2, and 1 mM EGTA) and stored at \u000180 8C until use. A 50- to 100-g cell pellet was resuspended in five volumes of cold U buffer supplemented with 0.5% Triton X-100, 0.2 mM ATP, 1 mM DTT, and protease inhibitor mix (0.5 lg/ml antipain, leupeptin, pepstatin A, chymostatin, and aprotinin, and 1 mM PMSF) and passed through a microfludizer (Microfluidics, Newton, Massachusetts, United States) until 70% lysis was obtained. The cell extracts were cleared by centrifugation at 100,000 3 g for 1 h and filtered through a 0.45-lm filter. A 60% ammonium sulfate precipitation of cell extracts was performed, and the pellet was dialyzed into NaP buffer (100 mM phosphate [pH 7.8], 100 mM KCl, and 20 mM imidazole). This fraction was cleared by centrifugation and incubated with Ni-NTA agarose beads (Qiagen, Valencia, California, United States). Beads were washed with NaP buffer, NaP buffer plus 0.5 M KCl, and NaP buffer plus 0.5% Triton X-100, and the complex was eluted with 250 mM imidazole. The complex was further purified through a HiTrapS column (Amersham Biosciences, Little Chalfont, United Kingdom) in 50 mM MES (pH 6.5), 25 mM NaCl; a UnoQ1 column (Bio-Rad, Hercules, California, United States) in U buffer; and a Superose 12 gel filtration column (Amersham Biosciences) in U buffer on a BioLogic chromatography system (Bio-Rad). (p 5)", + "(C) Pyrenyl-actin polymerization kinetics obtained with actin alone (black), control complex (light blue), Arp3-GFP complex (red), Arp2-GFP complex (purple), Arc40/ARPC1-YFP complex (green), and Arc18/ARPC3-GFP complex (dark blue). (p. 3)", + "(B) Purified yeast Arp2/3 complexes visualized by SDS-PAGE and Coomassie blue staining; unlabeled (control), and GFP- or YFP-labeled Arp3, Arp2, Arc40, or Arc18 complexes. The labeled subunits are marked by arrowheads. The Arc40 subunit in the labeled Arc40/ARPC1-YFP complex ran as 30-kDa and high-molecular-weight species (previously confirmed by immunoblotting and peptide sequencing), owing to an unusual electrophoretic mobility [16]. The Arp3 subunit of the unlabeled complex is denoted by an asterisk, and the Arp3 subunit of labeled Arc40/ARPC1-YFP complex is denoted by two asterisks. (p. 3)", + "Pyrene-actin polymerization assays were performed at Harvard Medical School and repeated at the Burnham Institute. Typically, G-actin was clarified at 436,000 3 g for 30 min. Reactions were performed by mixing 2 lMMg2\u00fe-ATP-Gactin (10% pyrene labeled) with Arp2/3 complex and the appropriate NPF, and actin polymerization was initiated in 13 KMEI buffer (50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.2 mM DTT, 0.1 mM ATP, 0.02% azide, and 2 mM imidazole [pH 7.0]). Polymerization was followed using a fluorescence spectrophotometer (Cary Eclipse Varian at Harvard Medical School and an MOS-250 spectrofluorometer [BioLogic, Claix, France] equipped with BioKine 32 software at the Burnham Institute), using 365 nm as the excitation wavelength and 407 nm as the emission wavelength. All of the GFP-labeled complexes were used at 50 to 200 nM concentration with 100 to 200 nM GST-NWASp WA. When the reaction reached the plateau, 2 lM phalloidin was added to stabilize the branches, and the reaction was diluted as required in F buffer (5 mM Tris-Cl [pH 7.8], 50 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 0.2 mM ATP, and 1 mM DTT). The slight reduction in the polymerization rate with Arc18/ARPC3-GFP complex was due to frozen storage of the complex. This reduction was not observed with fresh complex (data not shown), which was used to obtain the actin branches studied using EM. (p. 6)" + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-RniLbP0dj", + "claim": "Angle between membrane and clathrin pit increased from 0 to 90 (early phase/U-shaped) and 90 to 180(neck construction & scission) -", + "citekey": "avinoam2015endocytic", + "context": [ + "We further determined the maximal angle (q) between the adjacent plasma membrane (PM) and the invaginating segment of the membrane (Fig. 2B). Independently of invagination size and which model is correct, q will increase during invagination from 0\u00b0 to 90\u00b0 in a U-shaped invagination before tending to 180\u00b0 at neck constriction and scission (13) (Figs. 1A and 2B and movie S6).", + "To study clathrin-coated pit (CCP) maturation, we used well-characterized genome-edited human cell lines expressing fluorescently tagged clathrin light chain A at endogenous concentrations (8, 9) (hCLTAEN) (Fig. 1, B and C). To distinguish late stages of CME, we used cells that coexpressed endogenously tagged dynamin-2 (9) (hCLTAEN/hDNM2EN) (Fig. 1B). To label CCPs that recruit cargo, we incubated cells with fluorescently labeled transferrin (TF), a constitutive cargo of CME (hCLTAEN/TF) (Fig. 1C). CME events imaged by total internal reflection fluorescence (TIRF) microscopy showed a buildup of clathrin fluorescence that reached a plateau before a burst of dynamin preceded the site\u2019s disappearance, as previously described (8, 10)(Fig.1,Band C, and movies S1 and S2).", + "Analysis of Clathrin Coated Pits (CCPs) and Vesicles (CCVs) The membrane and coat profiles of invaginations and vesicles were extracted by clicking points along the cytoplasmic leaflet of the membrane and the outer surface of the", + "Two alternative models describe the transition from planar membrane to clathrin-coated vesicle (CCV). The first\u2014derived from electron microscopy images showing both relatively flat and invaginated clathrin lattices in cells\u2014suggests that clathrin assembles as a planar lattice that subsequently bends as the membrane invaginates (6) (Fig. 1A). For this to happen, complex rearrangements within the clathrin network must occur during budding. The second model avoids this difficulty by proposing that large, flat clathrin lattices are not precursors of CME and that, at sites of CME, clathrin directly assembles to produce the curved coat as the membrane invaginates (7)(Fig.1A)." + ], + "dataset": "akamatsulab" + }, + { + "id": "akamatsulab-ctop9ejQ4", + "claim": "Endocytic proteins Clathrin light chain A-RFP and dynamin2-GFP increased in lifetimes as a function of concentration of Arp2/3 complex inhibitor CK666 in SK-MEL-2 cells", + "citekey": "akamatsu2020principles", + "context": [ + "Histograms of endocytic lifetime in SK-MEL-2 cells endogenously expressing clathrin light chain CLTAtagRFP-T and dynamin2-eGFP and treated with CK-666. n = 368 tracks from 10 cells. (p. 14)", + "Cells were imaged on either an Olympus 1X-81 or Nikon Ti-2 inverted microscope fitted with TIRFoptics. The IX-81 microscope used a 60 x 1.49 NA objective (Olympus) and an Orca Flash 4.0sCMOS camera (Hamamatsu). Cells were illuminated with solid-state lasers (Melles Griot) with simul-taneous acquisition by a DV-2 image splitter (MAG Biosystems). The microscope was maintained at37 \u00b0C with a WeatherStation chamber and temperature controller (Precision Control) and imageswere acquired using Metamorph software. The Nikon Ti2 microscope was equipped with a motor-ized stage (Nikon), automated Z focus control, LU-N4 integrated four-wavelength solid state lasersetup, TIRF illuminator (Nikon), quad wavelength filter cube, NI-DAQ triggering acquisition (NationalInstruments), an Orca Flash 4.0 sCMOS camera (Hamamatsu), and triggerable filter wheel (FingerLakes Intstrumentation) with 525/50 and 600/50 wavelength emission filters. Cells were seeded onautoclaved 25 mm #1.5 round coverslips coated with 1 mL matrigel (80 ug/ mL) or recombinantVitronectin-N diluted in PBS (Thermo Fisher). Cells were maintained at 37 \u00b0C with a stage top incuba-tor (OKO Lab) and images were acquired with Nikon Elements. (p. 31)", + "SK-MEL-2 cells endogenously expressing clathrin light chain CLTA-RFP and dynamin2-eGFP (Doyon et al., 2011) were cultured in DMEM/F12 (Thermo Fisher) supplemented with 10% FBS(HyClone) and Penicillin/Streptomycin (Thermo Fisher). A day before imaging, the cells were seeded on 25 mm diameter glass coverslips (Fisher Scientific). (p. 30)", + "Time-lapse fluorescence quantification: We made modifications to automated MATLAB-based tracking software (Aguet et al., 2013; Hong et al., 2015) to track and analyze fluorescence-intensity time lapse data of genome-edited cells. The core tracking program (based on the software package m-track) automatically identifies fluorescent spots and connects them as tracks by minimizing the linear assignment problem (Jaqaman et al., 2008). We used stringent tracking parameters with gap size 0 and search radius 0\u20132.3 pixels (248 nm). GFP and RFP tracks with high variability in the intensity/time profile were automatically rejected (Ferguson et al., 2017) as well as tracks \u0014 3 s in duration (Dambournet et al., 2018) and the remaining tracks were associated spatiotemporally according to a cost matrix (Hong et al., 2015). We used two track rejection schemes. In the first, users were presented with fluorescence montages and XY coordinates of the tracks to assess the fidelity of tracking for each event (Hong et al., 2015). In the second, tracks were automatically rejected based signal-to-noise ratio (>1.05) and proximity to neighboring tracks (>525 nm) (Hong et al., 2015). We checked that the manual and automatic track rejection schemes yielded similar results (lifetime distributions and intensity versus time plots) as well as to manual, kymographbased quantification of lifetimes (below). From the above workflow (Dambournet et al., 2018) we increased throughput by connecting all steps into an automated tracking pipeline requiring minimal user input. For SK-MEL-2 cells expressing CLTA-RFP and DNM2-GFP, we tracked regions that were not near the nucleus (which has a concentration of Golgi-derived clathrin budding) and that did not have large, bright, persistent structures containing invariant RFP and GFP signals (\u2018plaques,\u2019 which are likely sites of adhesion). (p. 33)" + ], + "dataset": "akamatsulab" + }, + { + "id": "megacoglab-3jTc6a_9h", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventors who was new to the patent's field tended to receive fewer forward citations, but less so when they collaborated with an expert in the new field", + "citekey": "artsParadiseNoveltyLoss2018a", + "context": [ + "we select all U.S. inventors who patented in Michigan or in another nonenforcing state before the passing of MARA in 1985, including Alaska, California, Connecticut, Minnesota, Montana, Nevada, North Dakota, Oklahoma, Washington, and West Virginia (Malsberger 1996; Marx et al. 2009, Marx et al. 2015). Inventors who did not patent in a nonenforcing state or only did so after the passing of MARA are excluded to ensure that MARA did not affect sample selection. We track all subsequent patents linked to this set of inventors and assigned to a \ufb01rm and identify each move between \ufb01rms (again, the design relies upon inventors patenting at least twice). Only intrastate moves are taken into account because inventors can emigrate from Michigan to a nonenforcing state to avoid a lawsuit (Marx et al. 2015). Finally, we restrict the analysis to the 1975\u20131995 period, that is, 10 years before and after the passing of MARA. Notice that we collect all patents, both before and after MARA and in and out of Michigan. This results in the full sample of patents by inventors at risk for moving between \ufb01rms both in Michigan and elsewhere before and after MARA. The resulting data set spans 21 years and consists of 29,956 inventors and 162,586 patents of which 13,723 patents represent a move, that is, the \ufb01rst subsequent patent of an inventor after moving to a new \ufb01rm. The \ufb01nal analysis is restricted to these mobile inventors because the pressure to explore new \ufb01elds because of MARA only affects inventors who move between \ufb01rms.", + "In 1985, MARA was passed with the intention of harmonizing state law with the uniform state antitrust act (Bullard 1985). However, while passing MARA, legislators unintentionally revoked statute 445.761, which prohibited the enforcement of noncompete agreements in Michigan (Alterman 1985). After the passing of MARA, employers in Michigan suddenly obtained the legal means to prevent their ex-employees from working in the same \ufb01eld at a different \ufb01rm (Marx et al. 2009). As such, the Michigan experiment provided an exogenous pressure on inventors to explore new \ufb01elds after they left their former employer.", + "To estimate the effect of exploring new \ufb01elds on the novelty and value of invention, we use a two-stage least square model (2SLS). Because the endogenous variable is binary, we use the approach suggested by Angrist (2001) and Angrist and Pischke (2008). For a recent application of the approach, see Galasso and Schankerman (2015). First, we estimate the likelihood of exploring new fields with a logit model in a difference-in-differences configuration. We include the interaction between Michigan and postmara as an exogenous variable capturing the effect of the policy reversal. Using logit instead of OLS in the \ufb01rst stage results in a better \ufb01t. Second, we calculate the \ufb01tted probabilities of exploring new fields and use these nonlinear fitted values bound between zero and one as an instrument for exploring new fjelds in the 2SLS models. Using nonlinear fitted values as an instrument is the same as plugging in \ufb01tted values when the first stage is estimated by OLS, but the advantage is a better predictor of exploring new fields in the first stage (Angrist and Pischke 2008). The 2SLS model uses a single instrument, resulting in just-identified estimates. Standard errors are clustered at the inventor level to control for repeated observations. (p. 12)", + "For each inventor-patent observation, we retrieve the three-digit technology classes of all prior patents of the focal inventor and identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked o all prior patents of the same inventor. We rely on all classes assigned to a patent rather than just the primary class. Exploring new fields is a binary indicator that equals one in the absence of any overlapping class between all prior patents and the focal patent. (p. 6)", + "For each inventor-patent observation, we identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked to all prior patents of the coinventors on the patent (excluding the focal inventor). Expert team is a binary indicator that equals one if at least one of the coinventors has a prior patent in the same class(es) as the focal patent. The measure is used a moderator to test whether collaboration with experts reduces the negative effect of exploring new fields on value. (p. 6)", + "To assess patent value, we calculate forward citations (ln) as the logarithmic trans-formation of one plus the number of times a patent is referenced as prior art within 10 years. (p. 6)", + "Our identi\ufb01cation relies on the fact that only mobile inventors residing in Michigan after the passing of MARA are affected; Michigan inventors before MARA and inventors from other states before and after MARA are not affected by the policy change. Therefore, we can combine differences in exploring new \ufb01elds between inventors from different states (Michigan versus nonMichigan) with differences between cohorts induced by the timing of MARA (pre- versus post-MARA). The interaction between binary indicators for Michigan residence and post-MARA is expected to have a positive signi\ufb01cant effect on the likelihood of exploring new \ufb01elds. Thus, this difference-in-differences (DD) can be used as an instrument for exploring new \ufb01elds (Du\ufb02o 2001). The DD speci\ufb01cation controls for overall time trends in exploring new \ufb01elds (across all states) and for time-invariant unobserved differences between Michigan and non-Michigan inventors (Angrist and Pischke 2008). Furthermore, regression DD allows us to include additional inventor and \ufb01eld characteristics affecting the likelihood of exploring new \ufb01elds as controls. The main assumption is that, in the absence of MARA, the trend in exploring new \ufb01elds would not have been systematically different between Michigan and nonMichigan mobile inventors. (p. 12)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-e5Os8ZONI", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventors who were new to the patent's field tended to be more novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "context": [ + "we select all U.S. inventors who patented in Michigan or in another nonenforcing state before the passing of MARA in 1985, including Alaska, California, Connecticut, Minnesota, Montana, Nevada, North Dakota, Oklahoma, Washington, and West Virginia (Malsberger 1996; Marx et al. 2009, Marx et al. 2015). Inventors who did not patent in a nonenforcing state or only did so after the passing of MARA are excluded to ensure that MARA did not affect sample selection. We track all subsequent patents linked to this set of inventors and assigned to a \ufb01rm and identify each move between \ufb01rms (again, the design relies upon inventors patenting at least twice). Only intrastate moves are taken into account because inventors can emigrate from Michigan to a nonenforcing state to avoid a lawsuit (Marx et al. 2015). Finally, we restrict the analysis to the 1975\u20131995 period, that is, 10 years before and after the passing of MARA. Notice that we collect all patents, both before and after MARA and in and out of Michigan. This results in the full sample of patents by inventors at risk for moving between \ufb01rms both in Michigan and elsewhere before and after MARA. The resulting data set spans 21 years and consists of 29,956 inventors and 162,586 patents of which 13,723 patents represent a move, that is, the \ufb01rst subsequent patent of an inventor after moving to a new \ufb01rm. The \ufb01nal analysis is restricted to these mobile inventors because the pressure to explore new \ufb01elds because of MARA only affects inventors who move between \ufb01rms.", + "To assess patent novelty, we calculate new combinations (ln) as the logarithmic transformation of one plus the number of pairwise subclass combinations of a patent that appear for the first time in the US. patent database (Fleming et al. 2007, Jung and Jeongsik 2016). To do so, each pairwise combination of subclasses is compared with all pairwise combinations of all prior U.S. patents. (p. 5)", + "In 1985, MARA was passed with the intention of harmonizing state law with the uniform state antitrust act (Bullard 1985). However, while passing MARA, legislators unintentionally revoked statute 445.761, which prohibited the enforcement of noncompete agreements in Michigan (Alterman 1985). After the passing of MARA, employers in Michigan suddenly obtained the legal means to prevent their ex-employees from working in the same \ufb01eld at a different \ufb01rm (Marx et al. 2009). As such, the Michigan experiment provided an exogenous pressure on inventors to explore new \ufb01elds after they left their former employer.", + "To estimate the effect of exploring new \ufb01elds on the novelty and value of invention, we use a two-stage least square model (2SLS). Because the endogenous variable is binary, we use the approach suggested by Angrist (2001) and Angrist and Pischke (2008). For a recent application of the approach, see Galasso and Schankerman (2015). First, we estimate the likelihood of exploring new fields with a logit model in a difference-in-differences configuration. We include the interaction between Michigan and postmara as an exogenous variable capturing the effect of the policy reversal. Using logit instead of OLS in the \ufb01rst stage results in a better \ufb01t. Second, we calculate the \ufb01tted probabilities of exploring new fields and use these nonlinear fitted values bound between zero and one as an instrument for exploring new fjelds in the 2SLS models. Using nonlinear fitted values as an instrument is the same as plugging in \ufb01tted values when the first stage is estimated by OLS, but the advantage is a better predictor of exploring new fields in the first stage (Angrist and Pischke 2008). The 2SLS model uses a single instrument, resulting in just-identified estimates. Standard errors are clustered at the inventor level to control for repeated observations. (p. 12)", + "For each inventor-patent observation, we retrieve the three-digit technology classes of all prior patents of the focal inventor and identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked o all prior patents of the same inventor. We rely on all classes assigned to a patent rather than just the primary class. Exploring new fields is a binary indicator that equals one in the absence of any overlapping class between all prior patents and the focal patent. (p. 6)", + "Our identi\ufb01cation relies on the fact that only mobile inventors residing in Michigan after the passing of MARA are affected; Michigan inventors before MARA and inventors from other states before and after MARA are not affected by the policy change. Therefore, we can combine differences in exploring new \ufb01elds between inventors from different states (Michigan versus nonMichigan) with differences between cohorts induced by the timing of MARA (pre- versus post-MARA). The interaction between binary indicators for Michigan residence and post-MARA is expected to have a positive signi\ufb01cant effect on the likelihood of exploring new \ufb01elds. Thus, this difference-in-differences (DD) can be used as an instrument for exploring new \ufb01elds (Du\ufb02o 2001). The DD speci\ufb01cation controls for overall time trends in exploring new \ufb01elds (across all states) and for time-invariant unobserved differences between Michigan and non-Michigan inventors (Angrist and Pischke 2008). Furthermore, regression DD allows us to include additional inventor and \ufb01eld characteristics affecting the likelihood of exploring new \ufb01elds as controls. The main assumption is that, in the absence of MARA, the trend in exploring new \ufb01elds would not have been systematically different between Michigan and nonMichigan mobile inventors. (p. 12)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-nSzMQuhLB", + "claim": "case-based reasoning system loaded with diverse cases increased overall novelty of ad campaign ideas, but only for people with lower creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "context": [ + "Following Amabile [5], we asked three experts to rate independently all of the campaign proposals. two of the experts were creative directors of renowned Dutch marketing agencies. the other expert was the beer brewery\u2019s senior marketing manager in charge of the original campaign. two of the experts had also served as chairmen of the annual Dutch Sales Promotion Campaign Awards. the experts were blind to the study design. All the proposals were given an identical layout and were sent to the experts in randomized batches of 20 proposals. there were no order or batch effects. It took about four hours to rate all 40 proposals. (pp. 9-10)", + "Study 2 was identical to Study 1, except for the experimental treatments. Of the CBR system with 100 diverse cases used in Study 1, 50 cases were classified as \u201cclose\u201d and 50 as \u201cremote\u201d based on the product category (see [16]). Campaigns for consumer-packaged goods (cpg), such as beer and soft drinks, are quite different from campaigns for consumer durables or services (non-cpg), such as cars or insurance products. Since beer is a consumer-packaged good, we classified all cpg cases as \u201cclose\u201d and all non-cpg cases as \u201cremote.\u201d To test H2, we devised a between-subjects study in which one group of participants (n = 20) was given a CBR system with remote cases only, while the other group (n = 20) received a CBR system with close cases only. (p. 13)", + "Forty marketing students from the same university as in Study 1 were randomly assigned to one of the two groups. gender and education level were again found to be proportionally distributed over the two groups (gender: 73 percent male; education level: 70 percent master\u2019s degree, 30 percent bachelor\u2019s degree). (p. 13)", + "the search structure of the CBR system consisted of the following components: (1) general information about the case; (2) problem situation, including market situation, campaign objectives, and constraints; (3) solution, including campaign design and campaign execution; and (4) outcomes. this search structure contained 60 different variables. users could search for cases by specifying the parameters they deemed relevant given the problem situation. the search values were mostly qualitative in nature, such as \u201cintroduction,\u201d \u201cgrowth,\u201d \u201cmaturity,\u201d and \u201cdecline\u201d for the variable \u201cproduct life cycle phase.\u201d users could also attach importance weights to the search variables (ranging from \u201cvery low\u201d to \u201cvery high\u201d). the importance weights could be inferred from the campaign brief. to prevent information overload, the CBR system displayed only the ten most similar past cases. the similarity between the search query and the cases in the CBR system was calculated using a distance-based measure (ranging from 0 = \u201cno similarity\u201d to 1 = \u201cperfect similarity\u201d). users were free to run multiple search queries. (p. 8)", + "to measure the creative ability of the participants, we used the Abbreviated torrance test for Adults (ATTA) developed and validated by Goff and Torrance [35] (see also [4]). this test is a brief 15-minute version of the Torrance tests of Creative thinking and measures the following cognitive subskills of creative thinking: fluency (number of ideas), flexibility (diversity of ideas), originality (rarity of ideas), and elaboration (embellishment of ideas with details), together with 15 other creativity indicators, such as \u201crichness and colorfulness of imagery\u201d and \u201chumor: conceptual incongruity.\u201d the ATTA test consists of one verbal and two figural response tasks. two independent, trained raters scored the responses to the tasks. With raters considered in agreement whenever their scores differed no more than one point on a seven-point scale (1 = \u201cminimal\u201d to 7 = \u201csubstantial\u201d) (see [23, 63]), there was agreement in 100 percent of the cases. We also calculated the r wg coefficient as an index of within-group interrater agreement [41]. the mean r wg for the creative ability scores was 0.97 (range: 0.85\u20131.00), indicating strong agreement [52]. 5 hence, we combined the scores of the raters. (p. 9)", + "For measuring the novelty of the solution, we selected three items (i.e., originality, surprise, and uniqueness) from Besemer and O\u2019Quin\u2019s [9] Creative Product Semantic Scale, which is a well-known instrument for evaluating creative products [60]. (p. 9)", + "to fill the case-base of the CBR system, we collected descriptions of 100 award-winning campaigns in Europe from the 1980s and the first half of the 1990s. the cases were taken from two books: Scoren [56] and European Sales Promotion: Great Campaigns in Action [75]. Examples of how these cases were stored in the CBR system, which we called LEAPS, 3 are given in Appendix A (close case) and Appendix B (remote case). (p. 8)", + "A Dutch beer brewery gave permission to use one of their campaign briefs in our study. the participant\u2019s task was to write a proposal for a campaign that had to boost brand loyalty and enhance the image of the brand. the campaign had to be targeted at the \u201cheavy-user\u201d segment with \u201clive music\u201d as the main theme. the brief included a number of practical constraints: (1) the promotion had to apply to crates (containing 30 cl [centiliter] or 45 cl bottles), (2) promotional items should not be attached to the sides of the crates, and (3) the value of the promotional offer should not exceed 5 euros per crate. the campaign proposal had to contain a brief description of (1) the basic idea, (2) the sales promotion technique, (3) supporting activities, and (4) a rough cost estimation. the task thus involved all the phases of the creative process, including a divergent (generating ideas) and a convergent phase (selecting and working out the best idea) [5]. the participants were instructed not to copy one of the brand\u2019s previous campaigns (see [67]). they were also told that experts would rate their campaign proposal on novelty and usefulness (see [10]). (p. 8)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-b8pGDFeTc", + "claim": "US patents filed by inventors who were new to the patent's field tended to receive fewer forward citations, but less so when they collaborated with an expert in the new field", + "citekey": "artsParadiseNoveltyLoss2018a", + "context": [ + "we begin with the full population of inventors and collect all patents assigned to \ufb01rms but, by design, must restrict the sample to inventors who have at least two patents assigned to the same \ufb01rm. The advantage of this panel setup is that we can use inventor\u2013firm fixed effect models to control for unobserved heterogeneity among inventors and firms, which arguably have a strong effect on the novelty and value of creative output. This approach basically uses repeated patents of the same inventor within the same firm to identify whether the inventor creates more or less novel\u2014and more or less valuable\u2014patents when any subsequent patent is categorized in a new \ufb01eld. The sample includes 2,705,431 patent\u2013inventor observations assigned to 396,336 unique inventors and 46,880 unique firms, accounting for 473,419 unique inventor\u2013firm pairs. (p. 5)", + "For each inventor-patent observation, we retrieve the three-digit technology classes of all prior patents of the focal inventor and identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked o all prior patents of the same inventor. We rely on all classes assigned to a patent rather than just the primary class. Exploring new fields is a binary indicator that equals one in the absence of any overlapping class between all prior patents and the focal patent. (p. 6)", + "we can use inventor\u2013\ufb01rm \ufb01xed effect models to control for unobserved heterogeneity among inventors and \ufb01rms, which arguably have a strong effect on the novelty and value of creative output (p. 5)", + "For each inventor-patent observation, we identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked to all prior patents of the coinventors on the patent (excluding the focal inventor). Expert team is a binary indicator that equals one if at least one of the coinventors has a prior patent in the same class(es) as the focal patent. The measure is used a moderator to test whether collaboration with experts reduces the negative effect of exploring new fields on value. (p. 6)", + "To assess patent value, we calculate forward citations (ln) as the logarithmic trans-formation of one plus the number of times a patent is referenced as prior art within 10 years. (p. 6)", + "we select the full population of inventors with U.S. patents assigned to \ufb01rms for 1975\u20132002 (p. 3)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-W3sdOb60i", + "claim": "US patents filed by inventors who were new to the patent's field tended to be more novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "context": [ + "To assess patent novelty, we calculate new combinations (ln) as the logarithmic transformation of one plus the number of pairwise subclass combinations of a patent that appear for the first time in the US. patent database (Fleming et al. 2007, Jung and Jeongsik 2016). To do so, each pairwise combination of subclasses is compared with all pairwise combinations of all prior U.S. patents. (p. 5)", + "we begin with the full population of inventors and collect all patents assigned to \ufb01rms but, by design, must restrict the sample to inventors who have at least two patents assigned to the same \ufb01rm. The advantage of this panel setup is that we can use inventor\u2013firm fixed effect models to control for unobserved heterogeneity among inventors and firms, which arguably have a strong effect on the novelty and value of creative output. This approach basically uses repeated patents of the same inventor within the same firm to identify whether the inventor creates more or less novel\u2014and more or less valuable\u2014patents when any subsequent patent is categorized in a new \ufb01eld. The sample includes 2,705,431 patent\u2013inventor observations assigned to 396,336 unique inventors and 46,880 unique firms, accounting for 473,419 unique inventor\u2013firm pairs. (p. 5)", + "For each inventor-patent observation, we retrieve the three-digit technology classes of all prior patents of the focal inventor and identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked o all prior patents of the same inventor. We rely on all classes assigned to a patent rather than just the primary class. Exploring new fields is a binary indicator that equals one in the absence of any overlapping class between all prior patents and the focal patent. (p. 6)", + "we can use inventor\u2013\ufb01rm \ufb01xed effect models to control for unobserved heterogeneity among inventors and \ufb01rms, which arguably have a strong effect on the novelty and value of creative output (p. 5)", + "we select the full population of inventors with U.S. patents assigned to \ufb01rms for 1975\u20132002 (p. 3)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-6e7Wu6JTF", + "claim": "overall marginally significant negative effect of analogically distant cases on creativity of campaign ad ideas, mostly for students with high creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "context": [ + "Following Amabile [5], we asked three experts to rate independently all of the campaign proposals. two of the experts were creative directors of renowned Dutch marketing agencies. the other expert was the beer brewery\u2019s senior marketing manager in charge of the original campaign. two of the experts had also served as chairmen of the annual Dutch Sales Promotion Campaign Awards. the experts were blind to the study design. All the proposals were given an identical layout and were sent to the experts in randomized batches of 20 proposals. there were no order or batch effects. It took about four hours to rate all 40 proposals. (pp. 9-10)", + "Study 2 was identical to Study 1, except for the experimental treatments. Of the CBR system with 100 diverse cases used in Study 1, 50 cases were classified as \u201cclose\u201d and 50 as \u201cremote\u201d based on the product category (see [16]). Campaigns for consumer-packaged goods (cpg), such as beer and soft drinks, are quite different from campaigns for consumer durables or services (non-cpg), such as cars or insurance products. Since beer is a consumer-packaged good, we classified all cpg cases as \u201cclose\u201d and all non-cpg cases as \u201cremote.\u201d To test H2, we devised a between-subjects study in which one group of participants (n = 20) was given a CBR system with remote cases only, while the other group (n = 20) received a CBR system with close cases only. (p. 13)", + "Forty marketing students from the same university as in Study 1 were randomly assigned to one of the two groups. gender and education level were again found to be proportionally distributed over the two groups (gender: 73 percent male; education level: 70 percent master\u2019s degree, 30 percent bachelor\u2019s degree). (p. 13)", + "the search structure of the CBR system consisted of the following components: (1) general information about the case; (2) problem situation, including market situation, campaign objectives, and constraints; (3) solution, including campaign design and campaign execution; and (4) outcomes. this search structure contained 60 different variables. users could search for cases by specifying the parameters they deemed relevant given the problem situation. the search values were mostly qualitative in nature, such as \u201cintroduction,\u201d \u201cgrowth,\u201d \u201cmaturity,\u201d and \u201cdecline\u201d for the variable \u201cproduct life cycle phase.\u201d users could also attach importance weights to the search variables (ranging from \u201cvery low\u201d to \u201cvery high\u201d). the importance weights could be inferred from the campaign brief. to prevent information overload, the CBR system displayed only the ten most similar past cases. the similarity between the search query and the cases in the CBR system was calculated using a distance-based measure (ranging from 0 = \u201cno similarity\u201d to 1 = \u201cperfect similarity\u201d). users were free to run multiple search queries. (p. 8)", + "to measure the creative ability of the participants, we used the Abbreviated torrance test for Adults (ATTA) developed and validated by Goff and Torrance [35] (see also [4]). this test is a brief 15-minute version of the Torrance tests of Creative thinking and measures the following cognitive subskills of creative thinking: fluency (number of ideas), flexibility (diversity of ideas), originality (rarity of ideas), and elaboration (embellishment of ideas with details), together with 15 other creativity indicators, such as \u201crichness and colorfulness of imagery\u201d and \u201chumor: conceptual incongruity.\u201d the ATTA test consists of one verbal and two figural response tasks. two independent, trained raters scored the responses to the tasks. With raters considered in agreement whenever their scores differed no more than one point on a seven-point scale (1 = \u201cminimal\u201d to 7 = \u201csubstantial\u201d) (see [23, 63]), there was agreement in 100 percent of the cases. We also calculated the r wg coefficient as an index of within-group interrater agreement [41]. the mean r wg for the creative ability scores was 0.97 (range: 0.85\u20131.00), indicating strong agreement [52]. 5 hence, we combined the scores of the raters. (p. 9)", + "For measuring the novelty of the solution, we selected three items (i.e., originality, surprise, and uniqueness) from Besemer and O\u2019Quin\u2019s [9] Creative Product Semantic Scale, which is a well-known instrument for evaluating creative products [60]. (p. 9)", + "For measuring usefulness, we selected four items from the same scale. We slightly adapted the items to fit the objectives stated in the campaign brief. the four items were effectiveness for increasing the loyalty of heavy users, fit with the brand values, attractiveness for heavy users, and the overall usefulness of the campaign. All seven items were measured on a scale from 0 (very poor) to 10 (excellent). (p. 9)", + "to fill the case-base of the CBR system, we collected descriptions of 100 award-winning campaigns in Europe from the 1980s and the first half of the 1990s. the cases were taken from two books: Scoren [56] and European Sales Promotion: Great Campaigns in Action [75]. Examples of how these cases were stored in the CBR system, which we called LEAPS, 3 are given in Appendix A (close case) and Appendix B (remote case). (p. 8)", + "A Dutch beer brewery gave permission to use one of their campaign briefs in our study. the participant\u2019s task was to write a proposal for a campaign that had to boost brand loyalty and enhance the image of the brand. the campaign had to be targeted at the \u201cheavy-user\u201d segment with \u201clive music\u201d as the main theme. the brief included a number of practical constraints: (1) the promotion had to apply to crates (containing 30 cl [centiliter] or 45 cl bottles), (2) promotional items should not be attached to the sides of the crates, and (3) the value of the promotional offer should not exceed 5 euros per crate. the campaign proposal had to contain a brief description of (1) the basic idea, (2) the sales promotion technique, (3) supporting activities, and (4) a rough cost estimation. the task thus involved all the phases of the creative process, including a divergent (generating ideas) and a convergent phase (selecting and working out the best idea) [5]. the participants were instructed not to copy one of the brand\u2019s previous campaigns (see [67]). they were also told that experts would rate their campaign proposal on novelty and usefulness (see [10]). (p. 8)", + "the novelty items and the usefulness items showed high internal consistency for all experts (Cronbach \u03b1s > 0.86). thus, we averaged the individual item scores to obtain a single \u201cnovelty\u201d score and a single \u201cusefulness\u201d score per expert. to obtain an \u201coverall creativity\u201d score for each expert, we averaged these novelty and usefulness scores (see [13, 55]). (p. 10)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-jJF_B28kC", + "claim": "An insurance benefits help desk employee struggled to reuse a database record to assist a caller because it was missing contextual information such as the history of changes and authorship, and some details about the caller's employment", + "citekey": "ackermanOrganizationalMemoryObjects2004", + "context": [ + "The \ufb01rst task in a distributed cognition analysis is to identify how a functional system works, good and bad (Rogers, 1992). There are low-level functional systems often embedded in larger functional systems that contribute to the overall activity being observed. Functional operation is decomposed into smaller units of analysis that make sense with respect to the particular system. While appearing in some cases to be straightforward task decomposition, it is more essentially an event driven segmentation. (pp. 9-10)", + "This study is based on field observations of a telephone hotline group (called HLG here) at a well-established company, CyberCorp, headquartered inSilicon Valley. HLG answers human resource questions for CyberCorp, primarily about benefits and personnel policies for the company\u2019s thousands of employees. In general, telephone hotlines are of interest in the study of organizational memory, largely because they are so information intensive. HLG agents have to start forming their answer within 45-60 s while simultaneously listening to the caller\u2019s elaborations and information. Many answers came directly from the hotline member\u2019s memory; hotline questions tend to be repetitive. There is also a great need for additional information sources: Facts must be double-checked, new questions arise, and answers become obsolete with new conditions. (p. 11)", + "The \ufb01eld study took place over a period of 18 months. A variety of data collection methods were used, including direct observation, video, semistructured interviews, and social network analyses. (p. 11)", + "Once analyzed into its component representational states and processes, the analyst uses that information to reconstruct the functioning of the system. This allows an analysis with respect to the context of use within an organization. By extension one can speculate about how changes in technologies might a\ufb00ect future operations. We believe that looking at the phenomena of organizational memory is well supported by taking this essentially cognitive view of a system, and in our interpretation, giving it a certain social twist. (p. 10)", + "Because of privacy reasons, only one side of the conversations was taped. Joan, however, described each call fully to the camera. (Moreover, we are concerned here with Joan\u2019s actions, rather than the intent of the caller.) (p. 11)", + "As with other cognitive theories, distributed cognition identi\ufb01es the observed informational inputs entering, as well as those outputs leaving, a system. The functional de\ufb01nition of the unit of analysis helps with this identi\ufb01cation. Stepping down in grain size, inside the system the focus is about how information is represented, and how these representations are transformed, combined, and propagated through that system in order to produce the system\u2019s observable behavior (Simon, 1990). It is the detailing of representational states and processes that helps the analyst to understand much of the system processing as it involves transitions between humans and artifacts. One records the representational state, the material media on which it is instantiated, as well as the processes that transform it. Video recording is particularly helpful here because of the ability to review the record repetitively and glean details that are observable but di\ufb03cult to catch in just \u2018\u2018one-pass\u2019\u2019. (p. 10)", + "Here we describe and analyze the responses to two calls captured on video. (p. 11)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-x62kMph4u", + "claim": "healthcare workers struggled to specify contextual details on patient interactions in a healthcare records system, leading to downstream problems reusing those records, such as in administration", + "citekey": "andersonDataBaseMent2008", + "context": [ + "Bigcity2 Primary Care Trust (BPCT) has some 7,500 members of staff", + "At present, PiMS is used for data entry, retrieval, and analysis by a variety of professional groups, each of which constitutes different communities of knowledge and practice and has different interests. PiMS is used to record patient details at registration and after (such demographic details as contact address and phone number, GP address and phone number); referrals of the patient to BPCT, either from external sources (such as GPs, self-referral), or from elsewhere within the Trust (between practitioners); planned/booked patient appointments; details of each contact the patient has with Trust Mental Health practitioners; details of patient assessments carried out by Trust practitioners. Currently, data entry is performed within the Trust\u2019s multi-disciplinary teams largely by secretarial, nursing, and related staff, such as occupational therapists. Very few medical consultants or junior doctors use the system themselves, preferring to handwrite records, and delegating computer data entry to their team secretaries.", + "The data comprise field notes and transcriptions of interviews with staff about their everyday work, their use of PiMS and other information systems, both manual and computer-based, their training on PiMS, and also their use of the Trust\u2019s previous computer systems. (p. 8)", + "The veracity of findings was established using a number of techniques. These included triangulation through the collection of data by multiple methods and sources. Verification was performed through discussions of findings with clinical and nonclinical BPCT staff; in feedback sessions with the clinical staff interviewed at their weekly team meetings; and in a Trust-wide workshop for feedback and discussion on the PiMS implementation project. As fieldwork progressed, comparisons were also be drawn between the activities of workers over time and between members of different occupational groups. (p. 8)", + "Nonclinical settings in which fieldwork was conducted included regular meetings of the PiMS Implementation Group (weekly) and the PiMS User Group (monthly). These involved members of the Project Team responsible for implementing PiMS, and staff from the Information Services (IS) and Clinical Governance (CG) Teams. The latter two groups were users of PiMS data from an administrative perspective, analyzing data from the system in order to compile mental healthcare statistics for decision making within and beyond BPCT. All had an interest in ensuring the completeness, accuracy, and timeliness of data entry, including the use of the Contact Purpose menu.", + "The investigation reported here is based on 12 months of ethnographic fieldwork carried out in BPCT. Data collection methods included participant observation and semistructured interviews with a range of personnel across a number of sites within the Trust, in both clinical and nonclinical departments or settings. The first round of data collection in BPCT took place between autumn 2001 and spring 2002, after which an initial report of fieldwork findings on the implementation process to date, together with some recommendations for action, were presented to Trust managers (Hardstone 2002) (p. 7).", + "The sites of clinical practice were chosen in order to compare PiMS implementation in a locality-based General Adult Mental Health Service (SW BigCity) with that in one of the larger specialist services, the Alcohol Problems Service. Both of these services were comprised of more than one team: SW Area having three (Outreach, Day Hospital, and Community Psychiatric Nurses, or CPNs) and Alcohol Problems Service having two (Resource and CPNs). Each team covered a different aspect of its service\u2019s mental health provision and had its own caseloads, and therefore had different work patterns and practices influencing its use of PiMS. While CPN Teams were occupationally homogeneous, others had a broader membership, including consultants, nurses, CPNs, occupational and other therapists, and social workers, as well as medical secretaries. In the context of this paper, each team had a different array of purposes for contact with patients. (p. 7)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-jScHaY6Fl", + "claim": "estimated semantic networks of animal concepts from montessori-educated children were more interconnected, with shorter paths between concepts and fewer subcommunities, compared to networks from traditional-schooled but comparable children", + "citekey": "denervaudEducationShapesStructure2021", + "context": [ + "To assess creative thinking, children completed divergent and convergent creativity tasks from the Evaluation of Potential Creativity. Divergent thinking reflects the ability to think of ideas that differ from one another; convergent thinking reflects the ability to think of a single creative solution. Performance on such creative thinking tasks has been shown to predict both academic*' and creative achievement. In the divergent thinking task, the child was asked to draw as many different drawings as possible from one imposed abstract form (i.e, incomplete shape), within 10 min. The final score was the sum of all valid drawings. In the convergent thinking task, the child had to select three different abstract forms out of eight to create an original drawing that combined them, within 15 min. Three blind judges scored the drawings for originality following the EPoC scoring manual (inter-rater agreement; Krippendorff's alpha = 0.905). Independent t tests were computed on each creativity score (divergent and convergent) to test for between-group differences. Pearson\u2019s correlations were computed between each creativity score and the verbal fluency metrics to test whether creative thinking relates to the quantity and quality of words retrieved from semantic memory. (p. 5)", + "Children completed verbal fluency task. Category verbal fluency tasks have been widely used to efficiently assess semantic network organization. Consistent with traditional task administration, each child had 60s to name as many animals as he/she could. Based on previous work in children, we targeted the animal category. Children spoke their responses out loud, which were recorded (and later transcribed) by an experimenter. For each child, fluency data was preprocessed using the SemNA pipeline in R. Repetitions or variation on roots were converged and non-category members were excluded from the final analysis. Number of responses per participant were summed (total number of responses). (p. 5)", + "Finally, using these word association matrices, we applied the triangulated maximally filtered graph TMFGY; to minimize noise and potential spurious associations. The TMFG method filters the word association matrices to capture only the most relevant information (i.e, removal of spurious associations and retaining the largest associations) within the original network. This approach retains the same number of edges between groups (i.e, 3n-6, where n equals the number of responses), which avoids the confound of difference network structures being due to a different number of edges, This resulted in a 68 nodes network with 198 edges for both groups. (p. 5)", + "Next, we computed a word association matrix for each group using the cosine similarity. The cosine similarity is commonly used in related to Pearson\u2019s correlation, which can be considered as the cosine between two normalized vectors. With the cosine similarity measure, all values are positive ranging from 0 (two responses do not co-occur) to 1 (two responses always co-occur). For both groups, each element in the word association matrix, A;, represents the cosine similarity or the co-occurrence between response i and j. (p. 5)", + "A total of 67 children participated in the current study (Mage =9.31, SD=2.23, 47.8% girls) through the University Hospital of Lausanne researchpool as part of a broader research project on education and neurocognitive development. Children were compensated with a ~30 USD gift voucher for completion of the study. Inclusion criteria were schoolings ystem (participants had to be enrolled in Montessori or in traditional classes from the early years on, in the case of the youngest children, or for at least 3 years), age (5-14 years of age); exclusion criteria were parental report of learning disabilities or sensory impairment. To account for variability in our measures due to nonverbal intelligence, or socioeconomic background, we controlled for between-group homogeneity in nonverbal intelligence (black and white short version of the Progressive Matrices\u00ae) and family socioeconomic status (both parents\u2019 education levels (score from 1 to 5) and current job (score from 1 to 4); scores were summed and averaged between both parents (max 9), with higher scores denoting higher SES). (p. 4)", + "The processed data were transferred into a binary response matrix, where columns represent the unique exemplars given by the sample and rows represent participants; the response matrix is filled out by 1 (if an exemplar was generated by that participant) and 0 (if that exemplar was not generated). To control for confounding factors (such as different nodes or edges in both groups), as in previous studies, the binary response matrices only include responses that are given by at least two participants in each group. Then, to avoid the two groups including a different number of nodes, which may bias comparison of network parameters, responses in the binary matrices were equated, so that the networks of both groups in each sample are compared using the same nodes. (p. 5)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-zGQwQiY6L", + "claim": "natural experiment with inventors affected by the 1985 Michigan Antitrust Reform Act showed that patents filed by inventor teams that included an expert in the patent's field tended to be less novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "context": [ + "we select all U.S. inventors who patented in Michigan or in another nonenforcing state before the passing of MARA in 1985, including Alaska, California, Connecticut, Minnesota, Montana, Nevada, North Dakota, Oklahoma, Washington, and West Virginia (Malsberger 1996; Marx et al. 2009, Marx et al. 2015). Inventors who did not patent in a nonenforcing state or only did so after the passing of MARA are excluded to ensure that MARA did not affect sample selection. We track all subsequent patents linked to this set of inventors and assigned to a \ufb01rm and identify each move between \ufb01rms (again, the design relies upon inventors patenting at least twice). Only intrastate moves are taken into account because inventors can emigrate from Michigan to a nonenforcing state to avoid a lawsuit (Marx et al. 2015). Finally, we restrict the analysis to the 1975\u20131995 period, that is, 10 years before and after the passing of MARA. Notice that we collect all patents, both before and after MARA and in and out of Michigan. This results in the full sample of patents by inventors at risk for moving between \ufb01rms both in Michigan and elsewhere before and after MARA. The resulting data set spans 21 years and consists of 29,956 inventors and 162,586 patents of which 13,723 patents represent a move, that is, the \ufb01rst subsequent patent of an inventor after moving to a new \ufb01rm. The \ufb01nal analysis is restricted to these mobile inventors because the pressure to explore new \ufb01elds because of MARA only affects inventors who move between \ufb01rms.", + "In 1985, MARA was passed with the intention of harmonizing state law with the uniform state antitrust act (Bullard 1985). However, while passing MARA, legislators unintentionally revoked statute 445.761, which prohibited the enforcement of noncompete agreements in Michigan (Alterman 1985). After the passing of MARA, employers in Michigan suddenly obtained the legal means to prevent their ex-employees from working in the same \ufb01eld at a different \ufb01rm (Marx et al. 2009). As such, the Michigan experiment provided an exogenous pressure on inventors to explore new \ufb01elds after they left their former employer.", + "To estimate the effect of exploring new \ufb01elds on the novelty and value of invention, we use a two-stage least square model (2SLS). Because the endogenous variable is binary, we use the approach suggested by Angrist (2001) and Angrist and Pischke (2008). For a recent application of the approach, see Galasso and Schankerman (2015). First, we estimate the likelihood of exploring new fields with a logit model in a difference-in-differences configuration. We include the interaction between Michigan and postmara as an exogenous variable capturing the effect of the policy reversal. Using logit instead of OLS in the \ufb01rst stage results in a better \ufb01t. Second, we calculate the \ufb01tted probabilities of exploring new fields and use these nonlinear fitted values bound between zero and one as an instrument for exploring new fjelds in the 2SLS models. Using nonlinear fitted values as an instrument is the same as plugging in \ufb01tted values when the first stage is estimated by OLS, but the advantage is a better predictor of exploring new fields in the first stage (Angrist and Pischke 2008). The 2SLS model uses a single instrument, resulting in just-identified estimates. Standard errors are clustered at the inventor level to control for repeated observations. (p. 12)", + "For each inventor-patent observation, we identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked to all prior patents of the coinventors on the patent (excluding the focal inventor). Expert team is a binary indicator that equals one if at least one of the coinventors has a prior patent in the same class(es) as the focal patent. The measure is used a moderator to test whether collaboration with experts reduces the negative effect of exploring new fields on value. (p. 6)", + "To assess patent value, we calculate forward citations (ln) as the logarithmic trans-formation of one plus the number of times a patent is referenced as prior art within 10 years. (p. 6)", + "Our identi\ufb01cation relies on the fact that only mobile inventors residing in Michigan after the passing of MARA are affected; Michigan inventors before MARA and inventors from other states before and after MARA are not affected by the policy change. Therefore, we can combine differences in exploring new \ufb01elds between inventors from different states (Michigan versus nonMichigan) with differences between cohorts induced by the timing of MARA (pre- versus post-MARA). The interaction between binary indicators for Michigan residence and post-MARA is expected to have a positive signi\ufb01cant effect on the likelihood of exploring new \ufb01elds. Thus, this difference-in-differences (DD) can be used as an instrument for exploring new \ufb01elds (Du\ufb02o 2001). The DD speci\ufb01cation controls for overall time trends in exploring new \ufb01elds (across all states) and for time-invariant unobserved differences between Michigan and non-Michigan inventors (Angrist and Pischke 2008). Furthermore, regression DD allows us to include additional inventor and \ufb01eld characteristics affecting the likelihood of exploring new \ufb01elds as controls. The main assumption is that, in the absence of MARA, the trend in exploring new \ufb01elds would not have been systematically different between Michigan and nonMichigan mobile inventors. (p. 12)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-IUA1p7faR", + "claim": "US patents in the early 90's were more likely to be exapted by inventors who had previous patent experience in the focal and-or citing patent subclasses", + "citekey": "mastrogiorgioInnovationExaptationIts2016", + "context": [ + "The ability of inventors to draw inventive analogies and exapt existing technologies across different domains is a function of their stock of knowledge spanning these domains. We introduced a novel measure of inventors\u2019 analogical abilities that quantifies their multi-domain skills: In order to build the measure, we considered the inventor (or team) belonging to the first citing patent. We then extracted, from the Patent Network Dataverse (Lai et al, 2011),the inventor\u2019s previous patents that belong to the 1975-1990 pre-sample window. We then counted the number of previous patents whose OR class was equal to the OR class of either the focal patent or the citing patent. (p. 7)", + "In order to test our hypotheses, we studied exaptation at the invention level; we identified an invention with a patent and considered a cross-section of U.S. patents. Raw patent data were obtained from the USPTO and/NBER databases (Hall et al., 2001),and the main measures were built after merging these databases to the Patent Network Dataverse (Lai et al, 2011). (p. 5)", + "a forward citation is cross-class if both the OX and XR classes of the focal patent differ from the OR class of the citing patent (p. 6)", + "As our dependent variable is a proportion, we adopted the fractional logit estimation procedure proposed by Papke and Wooldridge (1996). (p. 10)", + "Our empirical framework consisted of the following steps: (1) we considered a random cross-section of US. patents granted between January and June 1991 (both theJanuary-June interval and the year 1991 were chosen randomly); (2) for each patent we used a 1991-1999 window of forward citations to calculate exaptation; (3) for each patent we then considered a 1975-1990 pre-sample window in order to calculate technological complexity, inventor's analogical ability, and other controls. The estimation sample consists of 19076 patents. (p. 5)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-d77x3B_Ui", + "claim": "case-based reasoning system loaded with diverse cases increased overall usefulness and creativity of ad campaign ideas, especially for people with lower creative ability", + "citekey": "althuizenSupportingCreativeProblem2014", + "context": [ + "Participants in the \u201cno CBR system\u201d control condition received only a sales promotion technique inventory on paper. It should be noted that this paper document was also made available to the participants in the CBR system condition. the inventory contained a basic description of sales promotion techniques (such as sampling or organizing a contest) together with the most salient pros and cons of each technique (such as aptitude for inducing trial, implementation times, and predictability of the costs). the descriptions were taken from textbooks and verified by experts (who also verified the CBR system\u2019s search structure).", + "Following Amabile [5], we asked three experts to rate independently all of the campaign proposals. two of the experts were creative directors of renowned Dutch marketing agencies. the other expert was the beer brewery\u2019s senior marketing manager in charge of the original campaign. two of the experts had also served as chairmen of the annual Dutch Sales Promotion Campaign Awards. the experts were blind to the study design. All the proposals were given an identical layout and were sent to the experts in randomized batches of 20 proposals. there were no order or batch effects. It took about four hours to rate all 40 proposals. (pp. 9-10)", + "the search structure of the CBR system consisted of the following components: (1) general information about the case; (2) problem situation, including market situation, campaign objectives, and constraints; (3) solution, including campaign design and campaign execution; and (4) outcomes. this search structure contained 60 different variables. users could search for cases by specifying the parameters they deemed relevant given the problem situation. the search values were mostly qualitative in nature, such as \u201cintroduction,\u201d \u201cgrowth,\u201d \u201cmaturity,\u201d and \u201cdecline\u201d for the variable \u201cproduct life cycle phase.\u201d users could also attach importance weights to the search variables (ranging from \u201cvery low\u201d to \u201cvery high\u201d). the importance weights could be inferred from the campaign brief. to prevent information overload, the CBR system displayed only the ten most similar past cases. the similarity between the search query and the cases in the CBR system was calculated using a distance-based measure (ranging from 0 = \u201cno similarity\u201d to 1 = \u201cperfect similarity\u201d). users were free to run multiple search queries. (p. 8)", + "to measure the creative ability of the participants, we used the Abbreviated torrance test for Adults (ATTA) developed and validated by Goff and Torrance [35] (see also [4]). this test is a brief 15-minute version of the Torrance tests of Creative thinking and measures the following cognitive subskills of creative thinking: fluency (number of ideas), flexibility (diversity of ideas), originality (rarity of ideas), and elaboration (embellishment of ideas with details), together with 15 other creativity indicators, such as \u201crichness and colorfulness of imagery\u201d and \u201chumor: conceptual incongruity.\u201d the ATTA test consists of one verbal and two figural response tasks. two independent, trained raters scored the responses to the tasks. With raters considered in agreement whenever their scores differed no more than one point on a seven-point scale (1 = \u201cminimal\u201d to 7 = \u201csubstantial\u201d) (see [23, 63]), there was agreement in 100 percent of the cases. We also calculated the r wg coefficient as an index of within-group interrater agreement [41]. the mean r wg for the creative ability scores was 0.97 (range: 0.85\u20131.00), indicating strong agreement [52]. 5 hence, we combined the scores of the raters. (p. 9)", + "To test H1, We devised a between-subjects experiment in which one group of participants (n = 20) was given a CBR system filled with a diverse set of cases, plus a sales promotion technique inventory on paper (more details are given in the treatments subsection). the other group of participants (n = 20) received only the sales promotion technique inventory on paper. (p. 7)", + "Forty marketing students from a Dutch university participated in this study. 1 the participants were randomly assigned to one of the two groups. gender and education level were found to be proportionally distributed over the two groups (gender: 65 percent male; education level: 70 percent master\u2019s degree and 30 percent bachelor\u2019s degree). (p. 7)", + "For measuring the novelty of the solution, we selected three items (i.e., originality, surprise, and uniqueness) from Besemer and O\u2019Quin\u2019s [9] Creative Product Semantic Scale, which is a well-known instrument for evaluating creative products [60]. (p. 9)", + "For measuring usefulness, we selected four items from the same scale. We slightly adapted the items to fit the objectives stated in the campaign brief. the four items were effectiveness for increasing the loyalty of heavy users, fit with the brand values, attractiveness for heavy users, and the overall usefulness of the campaign. All seven items were measured on a scale from 0 (very poor) to 10 (excellent). (p. 9)", + "to fill the case-base of the CBR system, we collected descriptions of 100 award-winning campaigns in Europe from the 1980s and the first half of the 1990s. the cases were taken from two books: Scoren [56] and European Sales Promotion: Great Campaigns in Action [75]. Examples of how these cases were stored in the CBR system, which we called LEAPS, 3 are given in Appendix A (close case) and Appendix B (remote case). (p. 8)", + "A Dutch beer brewery gave permission to use one of their campaign briefs in our study. the participant\u2019s task was to write a proposal for a campaign that had to boost brand loyalty and enhance the image of the brand. the campaign had to be targeted at the \u201cheavy-user\u201d segment with \u201clive music\u201d as the main theme. the brief included a number of practical constraints: (1) the promotion had to apply to crates (containing 30 cl [centiliter] or 45 cl bottles), (2) promotional items should not be attached to the sides of the crates, and (3) the value of the promotional offer should not exceed 5 euros per crate. the campaign proposal had to contain a brief description of (1) the basic idea, (2) the sales promotion technique, (3) supporting activities, and (4) a rough cost estimation. the task thus involved all the phases of the creative process, including a divergent (generating ideas) and a convergent phase (selecting and working out the best idea) [5]. the participants were instructed not to copy one of the brand\u2019s previous campaigns (see [67]). they were also told that experts would rate their campaign proposal on novelty and usefulness (see [10]). (p. 8)", + "the novelty items and the usefulness items showed high internal consistency for all experts (Cronbach \u03b1s > 0.86). thus, we averaged the individual item scores to obtain a single \u201cnovelty\u201d score and a single \u201cusefulness\u201d score per expert. to obtain an \u201coverall creativity\u201d score for each expert, we averaged these novelty and usefulness scores (see [13, 55]). (p. 10)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-5gJjNdjMH", + "claim": "US patents filed by inventor teams that included an expert in the patent's field tended to be less novel", + "citekey": "artsParadiseNoveltyLoss2018a", + "context": [ + "To assess patent novelty, we calculate new combinations (ln) as the logarithmic transformation of one plus the number of pairwise subclass combinations of a patent that appear for the first time in the US. patent database (Fleming et al. 2007, Jung and Jeongsik 2016). To do so, each pairwise combination of subclasses is compared with all pairwise combinations of all prior U.S. patents. (p. 5)", + "we begin with the full population of inventors and collect all patents assigned to \ufb01rms but, by design, must restrict the sample to inventors who have at least two patents assigned to the same \ufb01rm. The advantage of this panel setup is that we can use inventor\u2013firm fixed effect models to control for unobserved heterogeneity among inventors and firms, which arguably have a strong effect on the novelty and value of creative output. This approach basically uses repeated patents of the same inventor within the same firm to identify whether the inventor creates more or less novel\u2014and more or less valuable\u2014patents when any subsequent patent is categorized in a new \ufb01eld. The sample includes 2,705,431 patent\u2013inventor observations assigned to 396,336 unique inventors and 46,880 unique firms, accounting for 473,419 unique inventor\u2013firm pairs. (p. 5)", + "we can use inventor\u2013\ufb01rm \ufb01xed effect models to control for unobserved heterogeneity among inventors and \ufb01rms, which arguably have a strong effect on the novelty and value of creative output (p. 5)", + "For each inventor-patent observation, we identify whether there is any overlap between the three-digit technology classes of the focal patent and the three-digit technology classes linked to all prior patents of the coinventors on the patent (excluding the focal inventor). Expert team is a binary indicator that equals one if at least one of the coinventors has a prior patent in the same class(es) as the focal patent. The measure is used a moderator to test whether collaboration with experts reduces the negative effect of exploring new fields on value. (p. 6)", + "we select the full population of inventors with U.S. patents assigned to \ufb01rms for 1975\u20132002 (p. 3)" + ], + "dataset": "megacoglab" + }, + { + "id": "megacoglab-mtKPi-Abu", + "claim": "no overall effect of analogical distance of cases on novelty or usefulness of campaign ad ideas", + "citekey": "althuizenSupportingCreativeProblem2014", + "context": [ + "Following Amabile [5], we asked three experts to rate independently all of the campaign proposals. two of the experts were creative directors of renowned Dutch marketing agencies. the other expert was the beer brewery\u2019s senior marketing manager in charge of the original campaign. two of the experts had also served as chairmen of the annual Dutch Sales Promotion Campaign Awards. the experts were blind to the study design. All the proposals were given an identical layout and were sent to the experts in randomized batches of 20 proposals. there were no order or batch effects. It took about four hours to rate all 40 proposals. (pp. 9-10)", + "Study 2 was identical to Study 1, except for the experimental treatments. Of the CBR system with 100 diverse cases used in Study 1, 50 cases were classified as \u201cclose\u201d and 50 as \u201cremote\u201d based on the product category (see [16]). Campaigns for consumer-packaged goods (cpg), such as beer and soft drinks, are quite different from campaigns for consumer durables or services (non-cpg), such as cars or insurance products. Since beer is a consumer-packaged good, we classified all cpg cases as \u201cclose\u201d and all non-cpg cases as \u201cremote.\u201d To test H2, we devised a between-subjects study in which one group of participants (n = 20) was given a CBR system with remote cases only, while the other group (n = 20) received a CBR system with close cases only. (p. 13)", + "Forty marketing students from the same university as in Study 1 were randomly assigned to one of the two groups. gender and education level were again found to be proportionally distributed over the two groups (gender: 73 percent male; education level: 70 percent master\u2019s degree, 30 percent bachelor\u2019s degree). (p. 13)", + "the search structure of the CBR system consisted of the following components: (1) general information about the case; (2) problem situation, including market situation, campaign objectives, and constraints; (3) solution, including campaign design and campaign execution; and (4) outcomes. this search structure contained 60 different variables. users could search for cases by specifying the parameters they deemed relevant given the problem situation. the search values were mostly qualitative in nature, such as \u201cintroduction,\u201d \u201cgrowth,\u201d \u201cmaturity,\u201d and \u201cdecline\u201d for the variable \u201cproduct life cycle phase.\u201d users could also attach importance weights to the search variables (ranging from \u201cvery low\u201d to \u201cvery high\u201d). the importance weights could be inferred from the campaign brief. to prevent information overload, the CBR system displayed only the ten most similar past cases. the similarity between the search query and the cases in the CBR system was calculated using a distance-based measure (ranging from 0 = \u201cno similarity\u201d to 1 = \u201cperfect similarity\u201d). users were free to run multiple search queries. (p. 8)", + "to measure the creative ability of the participants, we used the Abbreviated torrance test for Adults (ATTA) developed and validated by Goff and Torrance [35] (see also [4]). this test is a brief 15-minute version of the Torrance tests of Creative thinking and measures the following cognitive subskills of creative thinking: fluency (number of ideas), flexibility (diversity of ideas), originality (rarity of ideas), and elaboration (embellishment of ideas with details), together with 15 other creativity indicators, such as \u201crichness and colorfulness of imagery\u201d and \u201chumor: conceptual incongruity.\u201d the ATTA test consists of one verbal and two figural response tasks. two independent, trained raters scored the responses to the tasks. With raters considered in agreement whenever their scores differed no more than one point on a seven-point scale (1 = \u201cminimal\u201d to 7 = \u201csubstantial\u201d) (see [23, 63]), there was agreement in 100 percent of the cases. We also calculated the r wg coefficient as an index of within-group interrater agreement [41]. the mean r wg for the creative ability scores was 0.97 (range: 0.85\u20131.00), indicating strong agreement [52]. 5 hence, we combined the scores of the raters. (p. 9)", + "For measuring the novelty of the solution, we selected three items (i.e., originality, surprise, and uniqueness) from Besemer and O\u2019Quin\u2019s [9] Creative Product Semantic Scale, which is a well-known instrument for evaluating creative products [60]. (p. 9)", + "For measuring usefulness, we selected four items from the same scale. We slightly adapted the items to fit the objectives stated in the campaign brief. the four items were effectiveness for increasing the loyalty of heavy users, fit with the brand values, attractiveness for heavy users, and the overall usefulness of the campaign. All seven items were measured on a scale from 0 (very poor) to 10 (excellent). (p. 9)", + "to fill the case-base of the CBR system, we collected descriptions of 100 award-winning campaigns in Europe from the 1980s and the first half of the 1990s. the cases were taken from two books: Scoren [56] and European Sales Promotion: Great Campaigns in Action [75]. Examples of how these cases were stored in the CBR system, which we called LEAPS, 3 are given in Appendix A (close case) and Appendix B (remote case). (p. 8)", + "A Dutch beer brewery gave permission to use one of their campaign briefs in our study. the participant\u2019s task was to write a proposal for a campaign that had to boost brand loyalty and enhance the image of the brand. the campaign had to be targeted at the \u201cheavy-user\u201d segment with \u201clive music\u201d as the main theme. the brief included a number of practical constraints: (1) the promotion had to apply to crates (containing 30 cl [centiliter] or 45 cl bottles), (2) promotional items should not be attached to the sides of the crates, and (3) the value of the promotional offer should not exceed 5 euros per crate. the campaign proposal had to contain a brief description of (1) the basic idea, (2) the sales promotion technique, (3) supporting activities, and (4) a rough cost estimation. the task thus involved all the phases of the creative process, including a divergent (generating ideas) and a convergent phase (selecting and working out the best idea) [5]. the participants were instructed not to copy one of the brand\u2019s previous campaigns (see [67]). they were also told that experts would rate their campaign proposal on novelty and usefulness (see [10]). (p. 8)" + ], + "dataset": "megacoglab" + } +] \ No newline at end of file diff --git a/test_extracted_captions/akamatsu2020principles.json b/test_extracted_captions/akamatsu2020principles.json new file mode 100644 index 0000000000000000000000000000000000000000..17f106e5689a4b004d7e8262bd3f2c33fbf51039 --- /dev/null +++ b/test_extracted_captions/akamatsu2020principles.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "'\\n\\n**Figure supplement 1.** Relationship between endocytic outcome and active Arp2/3 complex surface density or mother filament nucleating protein surface density at the base of the pit.\\n\\n**Figure supplement 2.** A collar of active Arp2/3 complex near the neck the pit does not affect endocytic outcome.\\n\\n**Figure supplement 3.** Internalization as a function of the number of Hip1R molecules and mechanism of self-organization of endocytic actin filaments.\\n\\n**Figure 4-video 1.** Simulations in which the coverage of linker Hip1R around the pit was varied from 1% to 80% of a sphere.\\n\\n**Figure 4.** Spatial distribution of actin/Hip1R attachments strongly affects actin self-organization and pit internalization. **(A)** Schematic of spatial boundary conditions from endocytic actin-binding proteins. Positions of active Arp2/3 complex (blue) and actin/pit attachments via linker proteins such as Hip1R (purple). **(B)** Initial positions of Hip1R around increasingly large pit surface area, from 1% to 80% of a sphere. The top ~20% of the sphere is occluded by the neck. **(C)** Snapshots of a series of simulations for different values of Hip1R coverage showing actin distribution at t = 13 s. **(D-G)** Changes in the endocytic actin network over time as a function of Hip1R coverage (colors). \\\\(n\\\\) = 96 simulations. **(D)** Internalization; **(E)** Number of barbed ends near the base of the pit (within 7.5 nm); **(F)** Number of Arp2/3 complexes bound in the endocytic network; **(G)** Number of actin filaments bound in the endocytic network. Scale bar: 50 nm.\\n\\nThe online version of this article includes the following video and figure supplement(s) for figure 4:\\n\\n**Figure supplement 1.** Relationship between endocytic outcome and active Arp2/3 complex surface density or mother filament nucleating protein surface density at the base of the pit.\\n\\n**Figure supplement 2.** A collar of active Arp2/3 complex near the neck the pit does not affect endocytic outcome.\\n\\n**Figure supplement 3.** Internalization as a function of the number of Hip1R molecules and mechanism of self-organization of endocytic actin filaments.\\n\\n**Figure 4-video 1.** Simulations in which the coverage of linker Hip1R around the pit was varied from 1% to 80% of a sphere.\\n\\n'", "CAPTION FIG5-1.png": "'Figure 5: Bending of endocytic actin filaments stores elastic energy for pit internalization. **(A)** Snapshot of simulation showing filaments bent between the attachment site in the coat and the base of the pit. Also see _Figure_ 1F. Yellow arrowheads point to a bent actin filament. **(B)** Tomographic slice of cryo-electron monogram of an SK-MEL2 cell. Long actin filaments (yellow arrowheads) bend along the clathrin-coated pit between the coat and the base of the pit. **(C)** Snapshot of membrane mechanics simulation under an internalization force with 60 nm internalization. **(D)** Slice of the same nomogram as shown in **B** at a different Z-level (+37 nm) in which the coated plasma membrane (white arrowheads) is visible. Scale bar for A-D: 50 nm. **(E)** Heat map of the bending angle and free filament length of endocytic actin filaments in simulations. Color code is number of filaments (summed for all time points, average of 24 simulations). Lines demarcate the magnitude of energy stored in these filaments, based on the theory of elastic beam rigidity for filaments of persistence length 10 \u03bcm (Materials and methods), in units of k_M_T_ (4.1 pN-nm). Purple lines: filament conformations expected from thermal fluctuations (passive bending): White lines: filament bending greater than from thermal fluctuations (active bending). Magenta lines: lower.\\n\\n'", "CAPTION FIG5-2.png": "'limit for bending energy expected to sever filaments (De La Cruz et al., 2015b). (F) Total elastic energy stored in bent capped (red) or growing (green) endocytic actin filaments during simulation over time compared to mean energy necessary for internalization (black) (n = 96 simulations). (G) Schematic of an in silico experiment to test the mechanical function of bent endocytic actin filaments. At t = 10 s, the membrane tension was reduced to zero, and the filaments were capped. (H) Internalization (green) after spring cut and filament capping, compared to simulation with no change in tension (black, same data as Figure 3D). n = 48 simulations. (I) Bending energy of endocytic actin filaments with barbed ends near base of pit over time. Release of tension and filament capping at t = 10 s (green) compared to no change in tension (black).\\n\\nThe online version of this article includes the following video and figure supplement(s) for figure S:\\n\\n**Figure supplement 1.** Hexagonal and pentagonal lattices in tomogram of clathrin-coated pit.\\n\\n**Figure supplement 2.** Energetics of endocytic actin network.\\n\\n**Figure 5-video 1.** Cryo-electron tomogram of SK-MEL-2 cell grown on holey carbon grid and vitrified, related to Figure 5.\\n\\n**Figure 5-video 2.** Simulation of actin in endocytosis in which, at t = 10 s, filaments were all capped and the membrane tension was reduced to 0 pN/m.\\n\\nhttps://ellfescances.org/articles/49840Wfig5video2'", "CAPTION FIG6.png": "'Figure 6: Irhibiting Arp2/3 complex nucleation activity stalls endocytosis. (**A**) Schematic of the model parameter corresponding to Arp2/3 nucleation activity, and the step inhibited by the small molecule CK_666_. (**B**) Internalization as a function of Arp2/3 complex nucleation rate. Orange region highlights parameter sensitivity, and green region highlights parameter insensitivity. n = 96 simulations. Reducing Arp2/3 nucleation rate reduces internalization as seen in the orange region. (**C**) Histograms of endocytic lifetime in SK-MEL-2 cells endogenously expressing clathrin light chain CLTatagRFP-T and dynamin2-aGFP and treated with CK_666_. \\\\(n\\\\) = 368 tracks from 10 cells. (**D**) Fluorescence intensity over time for endocytic tracks marked by clathrin-RFP and dynamin2-GFP in SK-MEL-2 cells treated with 0.1% DMSO (\\\\(\\\\gtrsim\\\\) mM) or the indicated concentration of CK_666_ for 45 min. Fluorescence events were tracked automatically (Materials and methods). Tracks in which GFP and RFP colocalized are shown. Each track was normalized to its maximum intensity and then all tracks were averaged and aligned to the time of the disappearance of the clathrin-RFP signal. The lifetimes of these events are plotted in **D**. Shaded bars are standard deviations.\\n\\nThe online version of this article includes the following figure supplements(s) for figure 6:'", "CAPTION FIG7.png": "'Figure 7: Adaptation of endocytic actin network to changes in membrane tension. **(A)** Schematic depicting possible adaptation of the actin network to membrane tension via self-organization and bending **(B\u2013D)** Snapshots of simulations from the same time point (14 s) for **(B)** low membrane tension (0.015 pN/nm); **(C)** medium membrane tension (0.15 pN/nm); **(D)** high membrane tension (1 pN/nm). Scale bar is 50 nm. **(E\u2013H)** Changes in the endocytic actin network as a function of membrane tension. **(E)** Internalization; **(F)** Number of barbed ends near base of pH; **(G)** Number of actin filaments in lipid IR-bound network; **(H)** Bending energy for filaments with barbed ends near base of pH. Mean \\\\(\\\\pm\\\\) standard deviation of time points in the last 5 s of simulations. Dashed line in **(E)** is expected internalization based on constant energy usage with 0.01 pN/nm condition as reference (see **Methods**).\\n\\n'", "CAPTION TABNA.png": "'\\n\\n**Figure Captions**\\n\\nFig. 1. The \\\\(\\\\pi^{0}\\\\'", "CAPTION FIG8.png": "'Figure 8: App2/3 complex activity and HipIR/actin attachments are critical for allowing actin filaments to drive endocytic put internalization and adapt to changing tension. **(A)** Schematic of App2/3 complex activity and HipIR coverage along with membrane tension. **(B)** Phase diagram of endocytic internalization as a function of membrane tension and App2/3 complex nucleation rate shown on a log-log plot. Dotted lines are values taken from the literature (**Beltamer and Pollard**, _2008_, **Diz-Munoz et al.**, _2016_, **(C\u2013G)** Changes in the endocytic actin network as a function of HipIR coverage for different values of membrane tension. Low tension = 0.015 pN/nm; medium tension = 0.15 pN/nm; high tension = 1 pN/nm. \\\\(n\\\\) = 288 simulations. **(C)** Internalization; **(D)** Number of barbed ends near base of pit; **(E)** Number of App2/3 complexes bound in network; **(F)** Number of actin filaments bound in network; **(G)** Bending energy of filaments with barbed ends near the base of the pit. Mean + standard deviation of time points in the last 5 s of simulations. **(H)** Summary of load-dependent adaptation of self-organizing endocytic actin network due to spatial segregation of active Arp2/3 complex at the base and HipIR in a broad distribution within the clathrin coat.\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: Multiscale modeling shows that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. (**A**) Schematic of a section of the cell\u2019s plasma membrane being internalized during mammalian endocytosis depicts plasma membrane deformation against membrane tension [purple arrows] countered by the clathrin coat (yellow) and the actin cytoskeleton (red). (**B**) Shape of the membrane and pit internalization from continuum mechanics simulations of the endocytic pit experiencing axial (\\\\(Z\\\\)) forces corresponding to simplified actin forces. To begin with, the plasma membrane (yellow) is deformed by a coat with preferred curvature that expands in area until the pit stalls. A net Figure 1 continued on next page\\n\\n'", "CAPTION FIG2-2.png": "'\\n\\n**Figure supplement 1.** Optimization and validation of fluorescence calibration method.\\n\\n**Figure supplement 2.** Generation of genome-edited human induced pluripotent stem cell lines endogenously expressing AP2-RFP and ArpC3-GFP.\\n\\n**Figure 2-video 1.** Time lapse images of human induced pluripotent stem cells transiently expressing FKBP-60mer-GFP and treated with 0.5 nM AP21967.\\n\\nhttps://elifesciences.org/articles/49840ffig2video1\\n\\n**Figure 2-video 2.** Time-lapse TIRF microscopy image of a human induced pluripotent stem cell endogenously expressing ArpC3-GFP and AP2-RFP.\\n\\nhttps://elifesciences.org/articles/49840ffig2video2'", "CAPTION FIG3.png": "'Figure 3: Self-organization of actin filaments into a radial dendritic network drives endocytic internalization. (**A**) (Left) Schematic depicting actin barbed (plus) or pointed (minus) ends. (Right) Heat maps of the positions of barbed ends (red) or pointed ends (blue) relative to the endocytic pit. Color code represents the relative number of ends. Each graph is averaged across 96 simulations and 1 s of simulation time. (**B**) Simulation output of endocytic actin filaments color-coded for axial (**Z**) orientation. Blue filaments orient toward the base of the pit (+90) and green filaments orient parallel to the base of the pit (**D**). (**C**) Axial orientation of barbed ends. (Left) Schematic of axes. R is radial position of barbed end. (Right) Heat map of axial orientation of barbed ends as a function of R and Z position. Average of 96 simulations. (**D**) Pit internalization over time (\\\\(n\\\\) = 96 simulations). (**E**) Simulation output of endocytic actin filaments color-coded for radial orientation. (**F**) Radially oriented endocytic actin filaments. (Left) Schematic of axes Radial orientation is defined such that +1 = barbed end oriented away from the center of the pit, and -1 = barbed end oriented toward the center of the pit. (Right) Heat map of radial orientation of barbed ends as a function of X and Y position (\\\\(n\\\\) = 96 simulations). Barbed ends radiate outward. (**G**) Radial orientation of barbed ends over time for 96 simulations. Gray curve is negative control of randomly oriented filaments (\\\\(n\\\\) = 50 filaments in one simulation). (**H**) Concentration of barbed ends near the base of the endocytic pit. (Left) Schematic of positions of the neck and base of the pit. (Right) Number of barbed ends near base (green) or neck (blue) of pit, defined as within 7.5 nm of each surface. (**J**) The majority of forces are directed orthogonal to the base of the pit based on positions of barbed ends in simulations. Shaded bars are standard deviations. The online version of this article includes the following video and figure supplements for figure 3:\\n\\nFigure 1: Assembly and self-organization of endocytic actin network. Figure 3\u2014video 1. Simulation of actin in endocytosis with actin filaments color coded for axial orientation.\\n\\n'", "CAPTION FIG1-2.png": "'force (red arrows) is applied downward from the coat and upward into the base of the endocytic pit (red dotted lines). In this simulation, membrane tension was 0.2 pH/nm, and the coated area was rigid (2400 pH nm). (**C**) Force versus pit internalization relationships for different values of membrane tension. Internalization is defined as the pit displacement in \\\\(Z\\\\). Shading delineates linear force-internalization regime (blue); \"transition point\" from U to omega shape (orange); \"omega-shaped\" regime where the neck is narrower than the pit diameter and the force required for internalization is lower than at the transition point (for tensions > 0.1 pN/rem) [yellow]. Color matches the three snapshots in B. Parameters are given in Supplementary files 1 and 2. (**D**) Resistance of pit to internalization versus membrane tension. Resistance (spring constant) is defined as absolute value of slope in C for the \\'U-shaped\\' region. Each curve is calculated for a different value of membrane rigidity (where \\\\(\\\\text{Ix}=320\\\\) pH nm, the rigidity of the uncoated plasma membrane). (**E**) Computational model of branched actin filament polymerization coupled to endocytic pit internalization. An internalizing endocytic pit is modeled as a sphere with a neck attached to a flat surface by a spring. Active Arp2/3 complex (blue) is distributed in a ring around the base of the pit. An actin nucleation protein (pink) generates an actin filament (white), which polymerizes, stalls under load, and is stochastically capped [red]. Arp2/3 complexes bind to the sides of actin filaments and nucleate new filaments at a 77-degree angle, creating new branches. Linker Hip1R (purple) is embedded in the pit and binds to actin filaments. Model parameters are given in Supplementary file 3. (**F**) Graphical output of the simulations from Cytosim (_Nedelec_ and _Foethke_, 2007) at 2 s intervals. Scale bar: 100 rpm. (**G**) Pit internalization over simulated time as a function of the number of available molecules of Arp2/3 complex. Average of 16 simulations per condition. Shaded bars are standard deviations. The online version of this article includes the following video and figure supplement(s) for figure 1:\\n\\n**Figure supplement 1.** Effect of different actin- and simulation-related parameters on pit internalization dynamics.\\n\\n**Figure supplement 2.** Initiation from a pool of diffusing cytoplasmic actin filaments leads to variable timing of internalization.\\n\\n**Figure 1-video 1.** Simulations of continuum membrane mechanics model.\\n\\n**Figure 1-video 2.** Simulation of actin in endocytosis using Cytosim.\\n\\n**Figure 2-video 3.** Simulation of actin in endocytosis using Cytosim.\\n\\n'", "CAPTION FIGSCHEME1.png": "'\\n\\n**Scheme 1.** Flow chart of multiscale modeling and experimental strategy combining membrane mechanics, actin spatiotemporal dynamics, molecule counting, and cryo-electron tomography.\\n\\n'", "CAPTION FIG2-1.png": "'\\nFigure 2: Molecule counting of endogenously GFP-tagged Arp2/3 complex in live human induced pluripotent stem cells. (**A-D**) Development of a calibration curve relating fluorescence intensity to numbers of molecules in live cells. (**A**) Cartoon of intracellular GFP-tagged 60mer nanocage with inducible plasma membrane tether. Each subunit (blue) is tagged with GFP (green) and FKBP (orange). FRB (T2098L) (Purple) is targeted to the plasma membrane by a palmitoylation and myrioylation sequence and dimerizes with FKBP in the presence of rapamycin analog AP21967. Cartoon showing one of 60 tagged subunits is based on PDB structures: Ssp, 2J0a, and 4di. Scale bar 10 nm. (**B**) Inverse contrast fluorescence intensity images of human induced pluripotent stem cells expressing GFP-tagged plasma membrane-bound nanocages. Sum projection of nine 300 nm confocal images. Scale bar: 2 \u03bcm. (**C**) Histograms of fluorescence intensity per spot for the four calibration constructs showing mean \u00b1 standard deviation. Images were corrected for uneven illumination and intensity was background-corrected. Data from 305 spots in 15 cells over three experiments. (**D**) Calibration curve relating fluorescence intensity to numbers of molecules in mammalian cells. Line is a linear fit through zero. Error bars are standard deviations. (**E**) Cartoon drawn to scale of Arp2/3 complex tagged with GFP at the flexible C-terminus of Arp2/3. Known binding and activation sites are distal to this site. Based on PDB 2p9f. (**F**) Montage of CME event marked by AP2-tagRFP-T and ArpC3-tagGFP2 from TIRF imaging. Montage shows 4s intervals from a movie taken at 2 s intervals. (**G**) Relative fluorescence intensity over time of AP2-tagRFP-T and ArpC3-tagGFP2 in endocytic events imaged by TIRF microscopy. Traces were normalized to maximum intensity and averaged 121 traces from 8 cells in four experiments. Shading is \u00b11 s.d. (**H**) Fluorescence micrographs of (left) 60mer-tagGFP2, (left-center) 120mer-tagGFP2, (right-center) ArpC3-tagGFP2, and (right) ArpC3-tagGFP2 and AP2-tagRFP-T. White arrows mark spots in which ArpC3-tagGFP2 and AP2-tagRFP-T colocalize. Scale bar 2 \u03bcm. (**0**) Numbers of molecules of ArpC3 over time.\\n\\n'", "CAPTION FIG5.png": "'* [16] A.\\n\\n'", "CAPTION FIG1.png": "''", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/arasada2018highspeed.json b/test_extracted_captions/arasada2018highspeed.json new file mode 100644 index 0000000000000000000000000000000000000000..e5b98edeab29d34944d9d747dff0c4cbfba51456 --- /dev/null +++ b/test_extracted_captions/arasada2018highspeed.json @@ -0,0 +1 @@ +{"CAPTION FIG3.png": "'Figure 3: Localization of actin filaments in two zones in endoscopic actin patches in wild-type S, ensemble cells. A (B) Time series of fluorescence micrographs at 14 intervals of representative actin patches in cells expressing proteins that had actin filaments. The images are regions of interest of 500 \\\\(\\\\times\\\\) 500 \\\\(\\\\times\\\\) ground one actin patch for each protein. Scale bars are 250 min. (Arg CPG CMD and \\\\(\\\\sigma\\\\)) for (mGS2.32 and (B);m-PEG2 and (B);m-PEG2 and (B);m-PEG2-PEG2. Top panels, maximum intensity projections of the confocal _z_-locus in the X-plane of individual actin patches from cells expressing GFP-CPG and _m_-PEG2.666 map, _m_-PEG2.\\n\\n'", "CAPTION FIG2.png": "'FIGURE 2.: Localization of resolution promoting feature and \\\\(\\\\mu\\\\)-pixel samples in actin patches by PHARM in wild type cells. (30 time series of PHARM images could be the density of the descriptors at the location; 30, triangles drip-ring, middle '", "CAPTION FIG1.png": "'Figure 1: FPALM improves the spatial resolution of actin patches over conventional fluorescence microscope. (A) Two-zone model based on quantitative confocal fluorescence microscopy. The cartoon represents an actin patch at -2 s when the membrane tubule elongates and at 0 s when the vesicle patches away from the plasma membrane. The plasma membrane is a black line, the clathrin coat is gray, nucleation-promoting factors Wsp1p and Myo1p are yellow, and zones with actin filaments are blue. Growth of the branched filaments from two distinct zones of NPFs is postulated to push the tip of the invaginating tubule away from the cell surface contributing to the elongation of the tubule and scission of the coated vesicle. (B, C) Fluorescence micrographs of an S. pombe cell expressing capping protein Acp2p-mECS3.2 with focusing in the middle plane of the cell. We used continuous epifluorescence illumination to photocomwart mECS3.2 with 40S- and 564-rm lasers to excite the photocomwart mECS3.2 through the entire cell. Top panels, wide-field epifluorescence images reconstructed from the total fluorescence emission. Middle panels, raw FPALM images constructed from the localizatores of single molecules. Bottom panels, each localized emitter in the raw data set was convolved with a Gaussian kernel (\\\\(\\\\alpha=1.5\\\\) pixel) and color coded for density in a heat map. (B) Whole cell during a 1-s interval. Scale bar is 1 \\\\(\\\\mu\\\\)m. (C) Time series of images of one actin patch at 1-s intervals each reconstructed from 200 sequential frames. Top panel, inverted contrast wide-field epifluorescence images. Scale bar is 250 nm.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: Localization of actin filaments in two zones in endocytic actin patches in wild-type \\\\(S\\\\). pombe calls. (A, B) Time series of fluorescence micrographs at 1-s intervals of representative actin patches in cells expressing proteins that bind actin filaments. The images are regions of interest of -S00 \\\\(\\\\times\\\\) 500 nm around one actin patch for each protein. Scale bars are 2S0 nm. (A) GFP-CHD and mECS3.2-CHD and (B) Fin1p-GFP and Frm1p-mECS3.2. Top panels, maximum intensity projections of five confocal \\\\(z\\\\)-slices in the \\\\(XY\\\\)-plane of individual actin patches from cells expressing GFP-CHD or Frm1p-GFP. Bottom panels, FPLAM images of cells expressing mECS3.2-CHD or Frm1p-mECS3.2. Each localized emitter was convolved with a Gaussian kernel (\\\\(\\\\sigma=1.5\\\\) pixel) and color coded for density in a heat map. Yellow dashed lines show the location of the\\n\\n'", "CAPTION FIG3-2.png": "'gradients membrane [PM]. (C-F) Spatial distributions of single molecule localization of mEOS3.2-CHD and Fim1p-mEOS3.2 in FPALM images of actin patches. FWMM is full-width of the distributions at half-maximums. (C) Width distribution and (E) length distribution of mEOS3.2-CHD in 20 actin patches. (D) Width distribution and (F) length distribution of Fim1p-mEOS3.2 in 14 actin patches prepared as in Figure 2. (G, H) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches. The micrographs are aligned to time 0 s when the vesicle pinches off from the plasma membrane. Scale bars are 200 nm. The blue and red lines indicate the membrane-proximal and membrane-distal regions. (G) Twenty-area seconds from a cell expressing mEoS3.2-CHD. (H) Seventeen seconds from a cell expressing Fim1p-mEoS3.2. (H, J) Time courses of the average number of localizations of (H) CHD-mEOS3.2 and (J) Fim1p-mEOS.2 detected in (blue line) the membrane proximal zone [200 x 250-nm region next to the call edge] and (red line) the membrane distal zone (350 x 250-nm region 200 nm away from the cell edge). Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations. Time zero seconds indicates vesicle sosion. Mean total localization were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches. Error bars are SDs. Every third error bar is shown.\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: Localization of nucleation promoting factors and Arp2/3 complex in actin patches by FPALM in wild-type cells. (A) Time series of FPALM images color coded for the density of localizations: top, mMaple3-Myo1p; middle, mMaple3-Wsp1p; and bottom, Arc5p-mMaple3 a subunit of Arp2/3 complex. The micrographs were aligned to time 0 s when the vesicle pirches away from the plasma membrane. For mMaple3-Myo1p the peak localizations were aligned to the peak localizations of mMaple3-Wsp1p. Yellow dashed lines show the position of the plasma membrane. Scale bar is 100 nm. (B) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches: left, 18 s from a cell expressing mMaple3-Myo1p; center, 16 s from a cell expressing mMaple3-Wsp1p; and right,\\n\\n'", "CAPTION FIG2-2.png": "'21 \\\\(\\\\leq\\\\) from a cell expressing ArcSp-mMaple3 a subunit of Arp2/3 complex. The blue and red lines indicate the membrane-proximal and membrane-distal regions. Scale bar is 200 nm. (C, D) Spatial distributions of single molecule localizations in FPALM images of actin patches. FW-HM is full-width of the distributions at half-maximums. (C) Width distributions and (D) length distributions of localizations of left, mMaple3-Myo1p in 20 actin patches; center, mMaple3-Wsp1p in 12 actin patches; and right, ArcSp-mMaple3 in 18 actin patches. The data from separate patches were aligned to their peaks and averaged. Full-widths at half-maximum were calculated from the distributions of the localizations along the \\\\(x\\\\)-axis. The lengths of the patches are given as the distances that include 90% of the localizations from the cell edge. The gray area in the graphs represents the localizations detected outside the cell. (E) Time courses of the average number of localizations detected in (blue line) the membrane proximal zone (200 \\\\(\\\\times\\\\) 250-nm region next to the cell edge) and (red line) the membrane distal zone (350 \\\\(\\\\times\\\\) 250-nm region 200 nm away from the cell edge): left, mMaple3-Myo1p; middle, mMaple3-Wsp1p; and right, ArcSp-mMaple3. Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations. Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches. Time 0 s indicates vesicle scission. Error bars are SDs. Every third arrow bar is shown.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Actin patch assembly in cells lacking nucleation promoting factors Myo1p or Wsp1p. {A, B} Time series of FPALM images color coded for localization density showing the (A) ArcSp-mMaple3 a subunit of Arp2/3 complex or {B} mEOS3.2-CHD to visualize actin filaments. Top panels, wild-type cells; middle panels, _Amyo1_ cells; and bottom panels, _Awsp1_ cells. Yellow dotted lines indicate the cell membrane. The micrographs are aligned to time 0 s when the vesicle pinches off from the plasma membrane. Scale bars are 250 nm. (C, D) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches. The blue and red lines indicate the membrane-proximal and membrane distal regions. (C) Cells expressing ArcSp-mMaple3: left, 16 s of an actin patch in a wild-type cell; center, 18 s of an actin patch in a _Amyo1_ cell; and right, 18 s of an actin patch in a _Am_wsp1_ cell. {D} Cells expressing mEOS3.2-CHD: left, 15 s of an actin patch in a wild-type cell; center, 27 s of an actin patch in a _Am_yo1_ cell; and right, 28 s of an actin patch in a _Am_wsp1_ cells. {E, F} Time courses of the average number of localizations of (E) ArcSp-mMaple3 and {F} mEOS.2-CHD detected in actin patches in (blue line) the membrane proximal zone (200 \u00d7 250-nm region next to the cell edge) and (red line) the membrane distal zone (350 \u00d7 250-nm region 200 nm away from the cell edge): left, wild-type cells; middle, _Am_y_o1 cells; and right, _Awsp1_ cells. Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations. Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches. Time 0 s indicates the vesicle scission. Error bars are SDs. Every third error bar is shown.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Hypothesis for the contributions of the nucleation promoting factors Myo1p and Wsp1p to endocytosis. Different stages of endocytosis with time zero defined as vesicle scission from the plasma membrane. The plasma membrane is a black line, clathrin is gray, nucleation promoting factors Wsp1p and Myo1p are blue, and actin filaments are red, green, and yellow. Clathrin is recruited 2 min prior to invagination. Nucleation promoting factors are recruited beginning at -6 s and peak prior to patch movement at -3 s. Myo1p and Wsp1p both stimulate actin polymerization at the base of the invagination near the cell surface. As the actin zones around Myo1p and Wsp1p grow, they repel each other, driving membrane invagination and redistribution of Wsp1p away from the base along the membrane invagination creating new sites of actin polymerization at the invagination tip. Myo1p remains associated with the plasma membrane at the base of the invagination. The expansion of the two zones triggers vesicle scission between the two zones of actin at time 0 s.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/ayyar2023clic.json b/test_extracted_captions/ayyar2023clic.json new file mode 100644 index 0000000000000000000000000000000000000000..084e34c65dcfa8953350de53dac091a346b8bfb2 --- /dev/null +++ b/test_extracted_captions/ayyar2023clic.json @@ -0,0 +1 @@ +{"CAPTION FIG6.png": "'and membrane recycling processes regulated by **Rabli** and **Rabli** result in membrane reorganization and receptor clustering leading to (6) tubular carrier formation due to multiple interactions of virus with host factors causing membrane bending and (7) endocytosis regulated by Cdc42 and cholesterol. (8) GIL4 entry into the cell results in V-ATPase regulated endosomal acidification causing (9) conformational changes in the virus capsid and release of the viral genome from the endosomal compartment.\\n\\nFig. 6: **GIL4 uses a complex entry mechanism involving cellular wound repair mechanisms and CILC-mediated endocytosis.** (1) Binding of GIL4 with its glycan receptor (HEGAs and possibly with a still unidentified co-receptor) on the cell surface (2) induces plasma membrane wounding (3) triggering signaling responses that direct multiple membrane repair cellular components to the injury site. (4) ASM translocation to the plasma membrane surface results in conversion of sphingomyelin (SM) to ceramide. (5) ceramide formation along with other membrane repair processes involving gal3 (glyceran damage sensor), ALK (Cl2+ sensor)'", "CAPTION TAB2.png": "'\\n\\n**Table 2 | List of inhibitors, additives and recombinant proteins**'", "CAPTION TAB1.png": "'\\n\\n**Table 1** | Macrohrus MLP specifications'", "CAPTION FIG1.png": "'Fig. 1: **GIL4 capsid protein elicits acidification and endocytosis in HIEs.**a Viral replication at 24 h (black dots) compared to bound virus at 1h (blue dots) and inhibition of replication in the presence of VLPs compared to untreated at 24 h. Replication was quantified using \\\\(n\\\\) = 2 independent HIE replicates for the 1 h and \\\\(n\\\\) = 3 independent HIE replicates for 24 h with 2 technical replicates/sample. **b** LysoTracker staining of acidic compartments in the presence of an anti-GIL4 polyclonal antibody (pAb), GIL4 virus, and pAb mixed with GIL4 virus at 37 \u00b0C. Right panel: Mean fluorescence intensity was quantified from different regions of interest (I00Is) for pAb (blue bar, ROIs = 32), GIL4 stool (red bar, ROIs = 33), GIL4 stool +pAb (green bar, ROIs = 32). **c** LysoTracker staining of acidic compartments induced by GIL3 VLP (green, ROIs = 100), GCDCA (purple, ROIs = 100), GIL4 virus (cyan, ROIs = 101), and GIL4 VLP (red, ROIs = 97) compared to media (black, ROIs = 140).\\n\\n'", "CAPTION FIG2.png": "\"kinase (RTK) inhibitor genistein at 1h (black) and 24 h (blue) at 37degC. **g** GIL4 replication in the presence of nocodazole, a microtubule disrupting agent, at 1 h (black) and 24 h (pink) at 37degC. **h** Tubulin staining (red) in the presence of genistein and nocodazole. All the experiments were repeated independently three times within similar results. In **b**, **d**-**g** will GEs were quantified using \\\\(n\\\\) = 2 independent HIE replicates for the 1 h and \\\\(n\\\\) = 3 independent HIE replicates for 24 h with two technical replicates/sample. In **b**, viral GEs were quantified using \\\\(n\\\\) = 3 HIE replicates. In **b**-**g**, error bars represent mean \\\\(n\\\\) SD with significance relative to untreated control, calculated using one-way ANOVA, Dumeret's multiple comparisons test. Source data are provided as a Source Data file.\\n\\nFig. 2: **GIL4 entry is dynamic-independent but depends on cholesterol and actin for infection.****a** Schematic of major endocytosis pathways, key regulators and their inhibitors. **b** GIL4 replication (at 37\u00b0C) was assessed in the presence of dynamin inhibitors, dynasore and mtmrab. Viral RNA replication was quantified at 1 (black) and 24 h (green) by RT-qPCR. **c** Validation of dynamin inhibitor activity by thiol-complexed low density lipoprotein (EH4LDL) uptake (red). Right panel: Quantization of pH-LDL uptake in untreated (80 BOLD, dynasore-treated (38 ROI) and mtmrab-treated (S5 ROI) HEs. **d** GIL4 replication in the presence of cholesterol sequestrants, M6CD and filipin, at 1 h (black) and 24 h (red) at 37\u00b0C. **c** GIL4 replication in the presence of actin depolymerizing agent cytochalasin D (Cyto D) at 1 h (black) and 24 h (green). **f** GIL4 replication in the presence of receptor tyrosine kinase (RTK) inhibitor genistein at 1h (black) and 24 h (blue) at 37\u00b0C. **g** GIL4 replication in the presence of nocodazole, a microtubule disrupting agent, at 1 h (black) and 24 h (pink) at 37\u00b0C. **h** Tubulin staining (red) in the presence of genistein and nocodazole. All the experiments were repeated independently three times within similar results. In **b**, **d**-**g** will GEs were quantified using \\\\(n\\\\) = 2 independent HIE replicates for the 1 h and \\\\(n\\\\) = 3 independent HIE replicates for 24 h with two technical replicates/sample. In **b**, viral GEs were quantified using \\\\(n\\\\) = 3 HIE replicates. In **b**-**g**, error bars represent mean \\\\(n\\\\) SD with significance relative to untreated control, calculated using one-way ANOVA, Dumeret\u2019s multiple comparisons test. Source data are provided as a Source Data file.\\n\\n\"", "CAPTION FIG3.png": "'treatment using anti-gal-3 and Gp Syt-pAb (n = 3 HIE replicates). **g** effect of blocking GLR4 virus-selectin-3 (gal-3) surface interaction using anti-gal-3 antibody on GIL4 replication at 1 h (black) and 24 h (red). **h** Doc blot analysis investigating GIL4 VIP interaction with purified gal-3.1 Probing CILC carriers utilized in HIEs for endocytosis with Alexa Fluor \"S94 conjugated cheler toxin B (CTR) (red) and GIL4 VIPs (green) with similar cargoes marked by white arrows (n = 2 HIE replicates). Inset: Co-occurrence of CTRb and GIL4 VIPs in similar cargoes, scale = 5 um. All the experiments were repeated independently three times with similar results. **h**, **c**, and **g** viral GEs were quantified using \\\\(n\\\\) = 2 independent HIE replicates for the 1 h and \\\\(n\\\\) = 3 independent HIE replicates for 24 h with two technical replicates) sample. The error bars represent mean +-SD with _P_values calculated using one-way ANOVA, Dunnett\\'s multiple comparisons test with comparisons at 24 h relative to untreated control. Source data are provided as a Source Data file.\\n\\nFig. 3: **GIL4 infection in HIEs is sensitive to effectors of GL4 pathway.** **A** Schematic of macropinocytosis and the Clathrin Independent Carrier (CLK) pathways, their effectors and inhibitors. **b** GL4 replication in the presence of HPA (Na*/H) exchanger inhibitor, M141 (CdCl2 inhibitor), Mukostatin (N-WASP inhibitor), NCS23766 (RAC1 inhibitor), CT04 (RbRi inhibitor), Hibbstibrain (myosin inhibitor), and LY29402 (PIX inhibitor) at 1 h (gray) and 24 h (orange). **c** GL4 replication in the presence of AHI Inhibitor Golglide A (GCA at 1 h (black) and 24 h (blue). **d** GL4-induced tubular carriers at 1 h (37 \u00b0C) detected using guinea pig anti-Sydney VIP polyclonal antibody (Gp Syt-pAb) for viral capsid (green) and phalloidin for actin (red). Images were taken using a ZESS laser Scanning Microscope LSM 980 with Airyscan 2. **c** Electron microscopy to identify CILC structures in GL4 VIP-treated HIEs. (1\u20137 structures/cell) compared to, mock-treated cells (no structures in \\\\(n\\\\) = 25 cells). **f** Confocal microscopy to detect GL4 VPI capsid (green) colocalization with gal-3 (red) on the cell surface at 10 min and 1 h (37 \u00b0C) after VLP treatment using anti-gal-3 and Gp Syt-pAb (n = 3 HIE replicates). **g** effect of blocking GIL4 virus-selectin-3 (gal-3) surface interaction using anti-gal-3 antibody on GIL4 replication at 1 h (black) and 24 h (red). **h** Doc blot analysis investigating GIL4 VIP interaction with purified gal-3.1 Probing CILC carriers utilized in HIEs for endocytosis with Alexa Fluor \"S94 conjugated cheler toxin B (CTR) (red) and GIL4 VIPs (green) with similar cargoes marked by white arrows (n = 2 HIE replicates). Inset: Co-occurrence of CTRb and GIL4 VIPs in similar cargoes, scale = 5 \u03bcm. All the experiments were repeated independently three times with similar results. **h**, **c**, and **g** viral GEs were quantified using \\\\(n\\\\) = 2 independent HIE replicates for the 1 h and \\\\(n\\\\) = 3 independent HIE replicates for 24 h with two technical replicates) sample. The error bars represent mean +-SD with _P_values calculated using one-way ANOVA, Dunnett\u2019s multiple comparisons test with comparisons at 24 h relative to untreated control. Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG5.png": "\"LAMP-1 (red) and VPI (green) colocalization was detected using mouse anti-LAMP-1 mAb and Cp Syd-pAb. (\\\\(n=3\\\\) HE replicates). f VPI, ALX and Gal-3 colocalization in VIP-treated and media-treated cells (1 h at 37degC) using confocal microscopy. VP1 (green), ALX (red) and gal-3 (white) were detected using Cp Syd-pAb, rabbit anti-ALXB pAb and rat signals. Right panel. Graph showing colocalization between VPI, gal-3 and ALX as estimated by Pearson Correlation Coefficient using ExCo-localization (ROI = 16, black for VLP- and ROI = 17, blue for media-treated HIEs). Error bars indicate mean \u00b1 SD and \\\\(P\\\\) values were calculated using two-way ANOVA, SiddiK\u2019s multiple comparisons test. All the experiments were repeated independently three times with similar results. Error bars for \\\\(a\\\\) = indicate mean \u00b1 SD calculated using 2 HE replicates for 1 h and 3 HE replicates for 24 h with two technical replicates/sample. \\\\(P\\\\) values were calculated using one-way ANOVA, Dunnett's multiple comparisons test by comparing replication at 24 h to untreated control. Source data are provided as a Source Data file.\\n\\nFig. 5: **GIL4 entry is sensitive to factors controlling endo-lysosomal homeostasis and induces membrane wounding and subsequent wound repair mechanisms.**a. GL4 replication in the presence of PikS/ive inhibitor YM2D1636 at 1 h (black) and 24 h (red) at 37\u00b0C. **b. GL4 replication in the presence of vaccumol-1 (a lysosomal exocytosis inhibitor) at 1 h (gray) and 24 h (purple). **c. GL4 replication in the presence of acid sphingomyelinase (ASM, amtriptyline) and neutral sphingomyelinase (NSM, GW4869) inhibitors at 1 h (black) and 24 h (blue). **d. Cell injury** determination using propidium iodide (PI) uptake assay. Right panel: Graph quantitative membrane injury calculated by counting number of PI spots (red) counterstained with DAPI (blue) when HIEs were incubated with VLPs (ROI = 10), VLP + \\\\(C\\\\)2\u00b0 (ROI = 12) and media (ROI = 10). Significance was calculated using two-way ANOVA, Tukey\u2019s multiple comparisons test. **e** Immunofluorescence staining showing GL4-induced lysosomal exocytosis represented by the presence of LAMP-1 on the apical cell surface in media-treated cells compared to VLP-treated cells. LAMP-1 (red) and VPI (green) colocalization was detected using mouse anti-LAMP-1 mAb and Cp Syd-pAb. (\\\\(n=3\\\\) HE replicates). f VPI, ALX and Gal-3 colocalization in VIP-treated and media-treated cells (1 h at 37\u00b0C) using confocal microscopy. VP1 (green), ALX (red) and gal-3 (white) were detected using Cp Syd-pAb, rabbit anti-ALXB pAb and rat signals. Right panel. Graph showing colocalization between VPI, gal-3 and ALX as estimated by Pearson Correlation Coefficient using ExCo-localization (ROI = 16, black for VLP- and ROI = 17, blue for media-treated HIEs). Error bars indicate mean \u00b1 SD and \\\\(P\\\\) values were calculated using two-way ANOVA, SiddiK\u2019s multiple comparisons test. All the experiments were repeated independently three times with similar results. Error bars for \\\\(a\\\\) = indicate mean \u00b1 SD calculated using 2 HE replicates for 1 h and 3 HE replicates for 24 h with two technical replicates/sample. \\\\(P\\\\) values were calculated using one-way ANOVA, Dunnett\u2019s multiple comparisons test by comparing replication at 24 h to untreated control. Source data are provided as a Source Data file.\\n\\n\"", "CAPTION FIG4.png": "\"induced by wild type GIL4 VLP and GIL4 VLP lacking the ALK-binding motif (\\\\(\\\\Delta\\\\) VLP) using FMI-4JX in HEs at 37degC (\\\\(n\\\\) = 3 kHz replicates). **h** viral replication in the presence of GIL4 WT VLPs (pink bars) and \\\\(\\\\Delta\\\\) VLPs (blue bars) was compared to untreated (black bar) at 24 h (blue does). 1 h (gray dots) represents bound virus. Error bars indicate mean \\\\(\\\\pm\\\\) SD calculated using 2 hIE replicates for 1 h and 3 hIE replicates for 24 h (each condition with two technical replicates). GIL4 replication in WT VL2 and \\\\(\\\\Delta\\\\)V2=\\\\(\\\\Delta\\\\)HHB monolayers indicated by percent fold change in viral RNA using the 2 - \\\\(\\\\Delta\\\\)CT method. Error bars indicate mean \\\\(\\\\pm\\\\) SD calculated using 3 hIE replicates for each condition with two technical replicates. All the experiments were repeated independently three times with similar results. \\\\(P\\\\) values for **d**, **h**, and **i**, relative to untreated control were calculated using one-way ANOVA, Dummet's multiple comparisons test. Source data are provided as a Source Data file.\\n\\nFig. 4: **GIL4 interacts with ALX and TSG01, with ALK being critical for virus entry.**a. GIL4 VLP, 5 and 6 domain binding to immobilized ALK by ELISA. Error bars indicate mean \\\\(\\\\pm\\\\) SD with three replicates. **b** GIL4 VLP, 5 and 7 domain binding to immobilized TSG01 by ELISA. Error bars indicate mean \\\\(\\\\pm\\\\) SD with three replicates. **c** Bio-Layer Interferometry (BL) to determine the binding affinity of GIL4 5 and 7 domains to biotinylated ALK and TSG01 (BALK and IPTG01). **d** Effect of blocking virus interaction with ALK and TSG01 on viral replication using specific mAbs at 1 h (gray) and 24 h (pink). Error bars indicate mean \\\\(\\\\pm\\\\) SD calculated using 2 HHE replicates for 1 h and 4 hIE replicates for 24 h (with two technical replicates/sample). **c** Confocal microscopy to probe GIL4-ALK colocalization on the cell surface by detecting VPI (green) and ALK (red) using _C_P_ Syd-pab and rabbit anti-ALK yb. **f** ELISA to evaluate binding of GIL4 5 and mutant \\\\(\\\\Delta\\\\)S-domains to immobilized ALK. Error bars indicate mean \\\\(\\\\pm\\\\) SD with 3 replicates. **g** Endocytosis induced by wild type GIL4 VLP and GIL4 VLP lacking the ALK-binding motif (\\\\(\\\\Delta\\\\) VLP) using FMI-4JX in HEs at 37\u00b0C (\\\\(n\\\\) = 3 kHz replicates). **h** viral replication in the presence of GIL4 WT VLPs (pink bars) and \\\\(\\\\Delta\\\\) VLPs (blue bars) was compared to untreated (black bar) at 24 h (blue does). 1 h (gray dots) represents bound virus. Error bars indicate mean \\\\(\\\\pm\\\\) SD calculated using 2 HHE replicates for 1 h and 3 hIE replicates for 24 h (each condition with two technical replicates). GIL4 replication in WT VL2 and \\\\(\\\\Delta\\\\)V2=\\\\(\\\\Delta\\\\)HHB monolayers indicated by percent fold change in viral RNA using the 2 - \\\\(\\\\Delta\\\\)CT method. Error bars indicate mean \\\\(\\\\pm\\\\) SD calculated using 3 HHE replicates for each condition with two technical replicates. All the experiments were repeated independently three times with similar results. \\\\(P\\\\) values for **d**, **h**, and **i**, relative to untreated control were calculated using one-way ANOVA, Dummet's multiple comparisons test. Source data are provided as a Source Data file.\\n\\n\""} \ No newline at end of file diff --git a/test_extracted_captions/bernsteinNetworkCentralizationCollective2023.json b/test_extracted_captions/bernsteinNetworkCentralizationCollective2023.json new file mode 100644 index 0000000000000000000000000000000000000000..679fd32d882c25399d3c568be45ea392c3226e6a --- /dev/null +++ b/test_extracted_captions/bernsteinNetworkCentralizationCollective2023.json @@ -0,0 +1 @@ +{"CAPTION FIG7.png": "'Figure 7: (Color online) Visualization of \u201cCommon Language\u201d Effect Size: Choosing One Random Trial of a Centralized Network (Left Panel: Hub-and-Spoke, Right Panel: Core-Periphery) and One Random Trial of a Control Network Structure, What Are the Chances That the Treatment Will Be Better Than, Equal to, or Worse Than the Control in Terms of the Number of Participants Correct at the End of the Trial?\\n\\n'", "CAPTION FIG11-1.png": "'Figure 11: (Color online) Empirical Cumulative Distribution Functions of the Number of Individuals Correct per Trial.\\n\\n'", "CAPTION FIG11-2.png": "''", "CAPTION FIG10.png": "'Figure 10: (Color online) Illustration of the Time Course of Final Correct Answers for Hub-and-Spoke Network Trials\\n\\n'", "CAPTION TABD2.png": "'\\n\\n**Table D.2**.: False-Positive Rates for Networks with Same True Mean'", "CAPTION TABD3.png": "'\\n\\\\begin{table}\\n\\\\begin{tabular}{c'", "CAPTION TABC1.png": "'\\n## Chapter 6 Conclusion\\n\\nIn this thesis, we have developed a new method for the calculation of the energy of the energy of the system. We have developed a method for calculating the energy of the system. We have developed a method for calculating the energy of the system.\\n\\n'", "CAPTION TABD1.png": "'\\n## Appendix D Additional Results\\n'", "CAPTION TABD4.png": "'\\n\\n**Table D.4.** False-Negative Rates for Trials with Different True Means'", "CAPTION FIGE1.png": "'Figure E.1. Illustration of the Four Quadrants (Submatrices) of a Blockmodel of an Adjacency Matrix Representation of a Core-Periphery Structure\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION FIG2.png": "'Figure 2: (Color online) Diagram of Mechanisms Underlying Performance in Adaptation Tasks\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: Relative Amounts of Conformity Pressure to Shift to a Competing and Mutually Exclusive Solution to a Problem Vary by Number and Share of Adopting Peers\\n\\n'", "CAPTION FIG1-2.png": "''", "CAPTION FIG6.png": "''", "CAPTION FIG11.png": "''", "CAPTION FIG1.png": "'* [16]'", "CAPTION FIG5.png": "'\\n\\n**Figure S.** Experimental Treatments'", "CAPTION FIG3.png": "'Figure 3. (Color online) Screenshot of the Experimental Interface\\n\\n'", "CAPTION FIG6-1.png": "'Figure 6: (Color online) Empirical Cumulative Distribution Functions of the Number of Individuals Correct per Trial.\\n\\n'", "CAPTION FIG6-2.png": "'\\n### _Results_\\n\\nWe first consider the case of a single cell in which the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell, and the cell is located in the cell. The cell is located in the cell,'", "CAPTION TAB3.png": "'\\n\\n**Table 3.** Descriptive Statistics by Network Treatment'", "CAPTION TAB4.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1979) The'", "CAPTION FIG8.png": "'Figure 8: Probability of Having the Same Answer as Neighbors by Position During Phase 3 of the Experiment.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: (Color online) Core Nodes See Diverse Answers, Including Correct Answers, Even When They Are Wrong, Themselves\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/bibeau2023twist.json b/test_extracted_captions/bibeau2023twist.json new file mode 100644 index 0000000000000000000000000000000000000000..3f0771299925324dca1f318f6cecc36077068b02 --- /dev/null +++ b/test_extracted_captions/bibeau2023twist.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'\\nFig. 2: Actin filament force-extension response. (_A_) Representative fluorescent images of Alexa 647-labeled actin filaments (magenta) conjugated to an paramagnetic bead (cyan) under fluid flow. No magnetic field is applied. (_B_) Force-extension curves for actin filaments of varying lengths (colored points) within the global best fit to Eq. 1 (colored lines) with pulling forces at \\\\(a\\\\) < \\\\(r\\\\) (_Methods_). (_C_) Average force-extension curves, normalized to filament lengths, for 7 actin filaments and corresponding theory with the global best fit persistence length of 10.7 (\u00b11) \u03bcm (solid red line) [Eq. 1]. Theoretical force-extension curves [Eq. 1], in descending order with \\\\(L_{0}\\\\) = 100, $\u20dd, 1, and 0.1 \u03bcm (dashed red lines). Uncertainty bars indicate standard error of the mean (SEM).\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\nFig. 5: Torsional persistence length of actin filaments determined by electron cryocrinroscopy. (_A_) Cartoon schematic of measured filament subunit twisting. fluctuations. The top cartoon depicts an actin filament (gray) with the average, intrinsic twist (\\\\(\\\\Delta y_{\\\\text{microwave}}\\\\)) between adjacent subunits / and + illustrated as a red curved arrow. The observed twist deviates from the intrinsic twist, either over or under, because of thermal fluctuations. The middle and bottom cartoon illustrates an undertwisted filament (light gray) overlaying a canonical filament with an intrinsic twist (dark gray). Blue arrows illustrate the observed twist between subunits / and +1 (Middle) or / and +3 (Bottom), which differs from the intrinsic twist by \\\\(\\\\Delta y_{\\\\text{microwave}}\\\\) (illustrated by black arrows). (_B_) Histograms of actin filament subunit twist fluctuations (\\\\(\\\\Delta y_{\\\\text{microwave}}\\\\) in degrees) estimated from cryo-EM alignment parameters (reference volume of 5 subunits) for \\\\(n=1\\\\), 10, and 50 subunits. Red lines represent fits to a normal distribution with mean zero and variance (\\\\(\\\\sigma_{\\\\text{dark},n}^{2}\\\\)). (_C_) \\\\(n\\\\) dependence of the twist variance (\\\\(\\\\sigma_{\\\\text{dark},n}^{2}\\\\)). The solid red lines represent the best fit to _S_Appendix_, Eq. **S21**.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Twisted cofilacion filaments fragment more easily than twisted bare actin filaments. (_A_) Representative images of twist-induced fragmentation for undertwisted (\\\\(\\\\Gamma\\\\)D) bare, overtwisted (\\\\(\\\\Gamma\\\\)D) bare, undertwisted cofilin saturated, and overtwisted cofilin saturated filaments. (Scale bar, 4 \u03bcm). (_B_) Survival analysis from the experiments in (_A_) at a pulling force of 0.03 pN. Log-rank test comparing UT bare to UT cofilin (\\\\(p<0.0001\\\\)) and OT bare and OT cofilin (\\\\(p=0.0094\\\\)). Both log-rank tests and Gehan-Breslow-Wilcoxon tests yielded similar \\\\(P\\\\) values, which conclude that the observed twisting response of bare and cofilin-decorated filaments is statistically different.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Modeling twist-induced fragmentation of actin filaments. _(a)_ Experimental actin filament survival curves for undertwisted (blue) and overtwisted (black) bare actin filaments at 0.25 pN pulling force (\\\\(\\\\bar{\\\\alpha}\\\\) mL min\\\\({}^{-1}\\\\) flow rate) and a twisting rate of \\\\(\\\\bar{\\\\alpha}\\\\) = 0.3 rot \\\\(\\\\varepsilon^{-1}\\\\). Data include instances where fragmentation occurs close to the bead or surface interfaces. For comparison, the plot includes simulations of filament survival curves as a function of twist density (\\\\(\\\\bar{S}\\\\)/\\\\(\\\\Delta\\\\)pender, Eq. **531**) at the same twist rate of 0.3 rot \\\\(\\\\varepsilon^{-1}\\\\) with a length of \\\\(\\\\bar{\\\\alpha}\\\\) = 15 \\\\(\\\\mu\\\\)m (red trace), as that typical in our twist-extension experiments in this study, and a twist rate of \\\\(\\\\bar{\\\\alpha}\\\\) = 2.2 rat \\\\(\\\\varepsilon^{-1}\\\\) with a length \\\\(\\\\bar{\\\\alpha}\\\\) = 0.1 \\\\(\\\\mu\\\\)m (gray trace). Onset image is an example of twist-induced fragmentation. Inset graph is the model-predicted filament torque (\\\\(\\\\bar{S}\\\\)/\\\\(\\\\Delta\\\\)pender, Eq. **53**). (\\\\(\\\\bar{B}\\\\)) Model-predicted twisting strain energy per subunit (left y-axis, \\\\(\\\\bar{S}\\\\)/\\\\(\\\\Delta\\\\)pender, Eq. **54**) and the relative increase in fragmentation rate constants of strained relative to relaxed, native filaments (right y-axis, \\\\(\\\\bar{S}\\\\)/\\\\(\\\\Delta\\\\)pender, Eq. **528**). Dashed lines indicate the model-predicted twisting strain energy for the twist density imposed at boundaries of human cofilm clusters (blue) and by singly isolated bound human cofilm (red).\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Direct visualization of actin filament twisting fluctuations. (A) Cartoon schematic of the experimental setup. A cylindrical magnet with a center hole was positioned 5 mm above the coverslip surface-anchored actin filament (i.e., in the \\\\(z\\\\) direction perpendicular to the surface). A DGG-coated marker bead was added to the paramagnetic bead to track rotations. No flow was applied during experiments. Filament length and bending are not to scale, (B) (_a_) Assay is set up by identifying a filament attached to a paramagnetic bead (large dm head) with identifiable marker beads (small bright bead) under fluid flow. (B) Fluid flow is turned off. (C) cylindrical magnet is lowered into position, (_b_) Filament is pulled out of the focal plane, (_c_) Focal plane is adjusted to observe the rotational fluctuations of both beads. Images were taken every 5 s. (C) Example images of the angular fluctuations of the filament visualized by the absolute angle of a line connecting the two beads to the \\\\(x\\\\) direction. (D) The mean-subtracted absolute angle (_S_) _Appendix_, Eq. **525** of the marker beads over time. Black trace indicates the angular fluctuations from the filament tracked in (C). Gray traces represent four other sample traces from different experiments performed at different times. Histogram represents the distribution of absolute angles from the black trace. The actin filament torsional persistence length of 12.9 (\u00b12.4) _mm_ is an average (_n_ = 5) of separate measurements, each was determined from the value of the variance at long times (see Panel _E_) and the filament length according to Eq. **4**. (_E_) Time-dependent variance of the traces in \\\\(D\\\\). It demonstrates that the measurement time of whole filament angular fluctuation has to be long enough for the variance to reach equilibrium such that experiencing all poss_b_-flites.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1.** Actin filament bending and torsional persistence lengths} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Actin filament bending and torsional persistence lengths'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\nFig. 3: Actin filament twist-extension response and supercoiling. (A) Representative fluorescent images of actin filament twist-extension with 0.01 (Top), 0.03 (Middle), and 0.25 (Bottom) pN pulling force (SJ Appendix, Eq. S1 with \\\\(d=\\\\gamma\\\\)). (Scale bar, 5 \u03bcm). (SJ Revt-extension curves for actin filaments under 0.01 (white circles), 0.03(gray circles) and 0.25 (black circles) pN pulling forces with the global best fit to Eq. SJ (red lines). Solid and dashed lines differentiate model before and after plectoconcentration, respectively. Rotations along the positive x-axis indicate filament overtwisting, and rotations along the negative x-axis indicate undertwisting. The complete dataset represents 50 filaments and n > 3 for each experimental condition. Uncertainty bars represent SEM. The asymmetric look of red dashed fitting lines for over- and undertwists under the same pulling force is due to the different fixed parameters \\\\(\\\\left(\\\\frac{1}{\\\\epsilon}\\\\right)\\\\) in the fitting (Methods) and not because of differences in response to applied over- and undertwist.\\n\\n'", "CAPTION FIG1.png": "'\\nFig. 1: Texisting actin filaments with magnetic tweezers. (A) Cartoon schematic of the experimental setup. Alexa 488-labeled actin filament seeds (green) were attached to a Biotin-PEG-Silane surface through biotin (pelloxie circles) and neuratvidin (black diamonds) irter actions and elongated from the barbed ends with Alexa 647-labeled actin (red). Flammets were further elongated from their barbed ends with digoxigenin (DNG)-labeled actin (purple). Paramagnetic beads (2.8 \\\\(\\\\mu\\\\)m in diameter; gray) coated with DIG antibodies (purple) were attached to filaments at or near their barbed ends. Filament-attached beads can be rotated by a permanent magnet (blue and red rectangles) and pulled by buffer flow(black arrow). Relative filament and bead sizes are not drawn to scale. Twisting clockwise or counterclockwise corresponds to under- or bursting, respectively. (B) Rotation of a phalloidin-decorated filament attached to two paramagnetic beads. (C) Cosine of the rotational angle of the second paramagnetic bead (black traces) and the magnet (red trace) indicates that the bead and magnet rotation are in phase. (D) Rotation of an Alexa 647-labeled actin filament with visible attachment to paramagnetic bead indicates that the bead and filament rotation are in phase.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/boudreauFieldExperimentSearch2017.json b/test_extracted_captions/boudreauFieldExperimentSearch2017.json new file mode 100644 index 0000000000000000000000000000000000000000..013d4d6c6ff31a54e7ff795f611935200ad14733 --- /dev/null +++ b/test_extracted_captions/boudreauFieldExperimentSearch2017.json @@ -0,0 +1 @@ +{"CAPTION TAB5.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB3.png": "'\\n\\n**Table 3.-Calculating Drans by Thematic Stat.**'", "CAPTION TAB1.png": "'\\n\\n## Chapter 1 Summary'", "CAPTION TAB2.png": "'\\n\\n**Figure 1.** The \\\\(\\\\pi\\\\)-\\\\(\\\\pi\\\\)-\\\\(\\\\pi\\\\) transition at \\\\(\\\\pi=0\\\\). The transition at \\\\(\\\\pi=0\\\\).\\n\\n'", "CAPTION TAB4.png": "'\\n\\n**Tane 4.--**John Eptet a Treatment on Cellular Automata**'"} \ No newline at end of file diff --git a/test_extracted_captions/carman2023structures.json b/test_extracted_captions/carman2023structures.json new file mode 100644 index 0000000000000000000000000000000000000000..1f42bcc64f6fa2aa475e04372582dcc796ce0247 --- /dev/null +++ b/test_extracted_captions/carman2023structures.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**(b)** Fit to the cryo-EM map of the W loop of the terminal subunit (blue) versus a subunit from the middle of the filament (gray). **(E)** In the middle of F-actin, the W loop and the C terminus of actin form a pinch-like structure that grasps the D loop of the subunit below (gray and cyan, respectively). This interaction is absent for subunits at the barbed end (blue), resulting in the W loop adapting a G-actin conformation and the C terminus moving by 2.0 A (red arrows). Single-letter abbreviations for amino acids referenced throughout are as follows: R. Agi, K. Lysi, L. Ile, P. Pro, F. Phe, Q. Qin, G. Gly, E. Glic, H. Hia; L. Leu: N. Asri, A. Alis; T. Thr\\n\\nFig. 1: **Free barbed end.** (**A**) Schematic representation of the G- to F-actin transition. A crosswhite -20\\\\({}^{\\\\circ}\\\\) rotation of the outer domain (subdomains 1 and 2) is definite to the inner domain (subdomains 3 and 4) produces a flatter conformation of subunits in F-actin. **(B)** Cryo-EM map of the free barbed end at 3.30-A resolution. The terminal and perultimate subunits are shown in two different shades of blue. **(C)** The terminal and penultimate subunits (blue) adopt the classical F-actin conformation of subunits in the middle of F-actin (gray). **(S)** Stimulus were superimposed based on the inner domain (surface representation) to highlight differences in orientation of the outer domain (ribbon representation).\\n\\n'", "CAPTION FIG2.png": "\"(**E**) Comparison of the filament-bound (pink, magenta) and unbound (\\\\(Z\\\\)Z) (gray, PDB code 3AA7) structures of CapZ. The superimposition, based on CapZa, highlights a -15\\\\({}^{\\\\circ}\\\\) rotation of CapZb (red arrow) that flattens the mushroom head. (**F**) The actin-binding surface of CapZ consists mostly of two antiparallel helices and the a and b tetracles. Comparison of the filament-bound (pink, magenta) and unbound (gray) structures shows that the helices contain n bulges that charge confirmation between these two states (red arrows). The tetracles, which are disordered in the unbound structure, project out in the filament-bound structure to engage the two barbed-end subunits. (**G**) Close-up view of CapZ's interaction with the barbed end, with the binding interface colored pink (CapZa) and magenta (CapZb). CapZ residues participating in the interaction are shown and labeled in fig. S9C.\\n\\nFig. 2: **CapZ-capped barbed end.** (**A**) Domain diagram of CapZ subunits \\\\(\\\\alpha\\\\) and \\\\(\\\\beta\\\\). (**B**) Macro-free structure of CapZ and transition between filament-bound and unbound states. In filament-bound CapZ, the mushroom head flattens the tetractes engage the hydrophobic elicits of barbed-end subunits. (**C**) Cry-EM trap of the CapZ-capped barbed end at 2.79-A resolution, showing the terminal and penultimate actin subunits is two states of blue and CapZa and CapZb in pink and magenta, respectively. (**D**) Solving at the short-pitch bar formed by the terminal and penultimate subunits (blue) as compared to a short-pitch pair from the middle of F-actin (gray). The short-pitch pairs were superimposed based on the penultimate subunit to highlight the playing of the terminal subunit, showing a maximum displacement of 1.5 \u00c5 resulting from a rotation of -2\\\\({}^{\\\\circ}\\\\) (red arrow). Solving is likely favored by missing contacts of the interstrand plug at the barbed end compared to the middle of F-actin (dashed red curves).\\n\\n\"", "CAPTION FIG3.png": "'Figure 3: **Free pointed end.** (**A**) Cryo-EM map of the free pointed end at 2.84-A resolution, showing the first and second subunits in two different shades of green. The D long of subunits at the pointed end is disordered. (**B**) The first (left) and second (right) subunits at the pointed end adopt a G-actin conformation, where the outer domain is rotated -20\\\\({}^{\\\\circ}\\\\) (red arrow) compared with subunits in the middle of F-actin (gray). Subunits were superimposed based on their inner domains (surface representation) to highlight differences in the orientation of their outer domains (ribeon representation).\\n\\n'", "CAPTION FIG4.png": "'by the b-strand to a-helix loops of the LRR domain of ABS2. Timod residues involved in the interaction are shown and labeled in fig. SBD. (**C**) The first actin subunit (left) adopts a G-actin conformation with the outer domain rotated by -20\\\\({}^{\\\\circ}\\\\) (red arrow) compared with subunits in the middle of F-actin (gray). The second actin subunit (right) adopts an F-actin conformation. Subunits were superimposed based on their inner domains (surface representation) to highlight differences in orientation of their outer domains (ribleon representation).\\n\\nFig. 4: **Tmod-capped pointed end.** (**A**) Domain diagram of Timod, comprising alternating tropomyosin (TMSSI and TMSS2, disordered in the structure) and fraction (ABS1 and ABS2) binding sites. (**B**) Cryo-EM map of the Tmod-capped pointed end at 3.26-A resolution, showing the first and second subunits in two shades of green and Timod in orange. The view on the right is approximately taken the longitudinal axis of F-actin and shows a ribbon representation of Timod. ABS1 caps the first actin subunit, whereas ABS2 wedges into a cleft formed by the first three actin subunits. Most of the interactions with actin are mediated by the b-strand to a-helix loops of the LRR domain of ABS2. Timod residues involved in the interaction are shown and labeled in fig. SBD. (**C**) The first actin subunit (left) adopts a G-actin conformation with the outer domain rotated by -20\\\\({}^{\\\\circ}\\\\) (red arrow) compared with subunits in the middle of F-actin (gray). The second actin subunit (right) adopts an F-actin conformation. Subunits were superimposed based on their inner domains (surface representation) to highlight differences in orientation of their outer domains (ribleon representation).\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5. Model of subunit association and dissociation at the free and capped ends of F-actin.** Structures described here show that subunits in F-actin have different conformations depending on whether they are in the middle or at the ends of F-actin, and are different from those of G-actin. Notably, these are not nucleotide-dependent conformational differences, which are relatively minor in both G- and F-actin (see text). The conformational differences at the ends of F-actin correlate with the association and dissociation constants of subunits at the ends of F-actin. Only the main pathway at equilibrium is depicted, with ATP-actin preferentially adding to the barbed end and ADP-actin dissociating from the parifed end [see (_I_) for other possible reactions]. Structural differences explain the asymmetric association of ATP-actin monomers to the barbed and parifed ends of F-actin, with ATP-bound monomers more likely to undergo the G- to F-actin transition required for preferential binding to the barbed end than ADP-bound monomers. CapZ and Trzed inhibit subunit exchange at the barbed and pointed ends, respectively, by structural mechanisms revealed in this study.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/cataliniMicrogeographyDirectionInventive2018.json b/test_extracted_captions/cataliniMicrogeographyDirectionInventive2018.json new file mode 100644 index 0000000000000000000000000000000000000000..6927687219b237e26c8a6656879c45c7dd546b66 --- /dev/null +++ b/test_extracted_captions/cataliniMicrogeographyDirectionInventive2018.json @@ -0,0 +1 @@ +{"CAPTION TAB5-1.png": "'\\n\\n**Table 5.** Increase in Collaboration and Collaboration'", "CAPTION TAB5-2.png": "'Notes. The dependent variable in column (1) is a dummy equal to 1 of the lab pair collaborates in the focal year. The dependent variable in columns (2) and [3] is the number of collaborations. After Colocation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. The Potsson specification with fixed effects drops all lab pairs where collaboration is never observed, hence the smaller number of observations. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\\\\({}^{+}n\\\\leq 0.08\\\\); \\\\({}^{+}n\\\\leq 0.01\\\\)'", "CAPTION FIG1-1.png": "'Figure 1. (Color online) Probability of Collaboration and Colocation\\n\\n'", "CAPTION FIG1-2.png": "'Nuts. Estimated coefficient for years before and after the move. The dependent variable is collaboration (1/0). Regression includes lab-pair fixed effects and year fixed effects. Error bars represent 95\\\\(\\\\%\\\\) confidence intervals based on robust standard errors clustered at the lab-pair level.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3. (Color online) Similarity in Cited References Space\\n\\n'", "CAPTION FIG3-2.png": "'Nuts. Estimated coefficient for years before and after the move. The dependent variable is the cosine similarity (based on cited references) between the two labs. The higher line in the post-period is for collocation, and the lower line is for separation. Regression includes lab-pair fixed effects and year fixed effects. Error bars represent 95% confidence intervals based on robust standard errors clustered at the lab-pair level.\\n\\n'", "CAPTION TAB7-2.png": "'Nuts. The dependent variable in all columns is a dummy equal to 1 if the lab pair collaborates in the focal year. In columns (1) and (3), only pairs with above the median cosine similarity (low search costs) are included. In columns (2) and (4), only pairs with below the median similarity (high search costs) are included. The cosine similarity measures are based on the vectors of keywords used by each lab in columns (1) and (2), and on the vectors of cited references in columns (3) and (4). In all cases, the measures are calculated before the moves start and do not include direct collaborations between the focal labs (which would share mechanically all keywords and cited references). _After Calculation_ is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. _After Separation_ is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\\\\({}^{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{ \\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{\\\\text{ \\\\text{\\\\text{\\\\text{ \\\\text{ \\\\text{ \\\\text{ } \\\\text{ }}}}}}}}}}}}}}}}}}}p <0.1\\\\).\\n\\n'", "CAPTION TAB7-1.png": "'\\n\\n**Table 7.** Probability of Collaboration and Search Costs'", "CAPTION TAB8-1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB8-2.png": "'NotesThe dependent variable in all columns is the number of publications by the lab pair in the focal year within the given quartile of the citation distribution. Citation data is obtained from Scopus in 2016, and quartiles for the citation distribution are built by year using a large sample of articles in the relevant fields of science. The first quartile represents papers with the lowest level of citations, the fourth the highest. Results are conditional on collaboration in the focal year. _After_ _Calocariton_ is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. _After_ _Separation_ is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\\'_p_ < 0.1, \"_p_ < 0.05.\\n\\n'", "CAPTION TAB9-1.png": "'\\n\\n**Table 9.** Colocation, Separation and Max Citations'", "CAPTION TAB9-2.png": "'Notes. The dependent variable in all columns is the maximum number of citations received by the publications of the lab pair in the focal year. Citation data is obtained from Scopus in 2016. In column (1), only pairs with above the median create similarity in keywords (low search costs) are included. In column (2), only pairs with below the median similarity in keywords (high search costs) are included. Results are conditional on collaboration in focal year. After Colocation is equal to \\\\(1\\\\) when a lab pair becomes colocated because of the moves and \\\\(0\\\\) otherwise. After Separation is equal to \\\\(1\\\\) when a previously colocated pair is separated because of the moves and \\\\(0\\\\) otherwise. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\"\\\\(p<0.01\\\\).\\n\\n'", "CAPTION TAB2-1.png": "'\\n\\n**Table 2.** Model-Level Chuckenics'", "CAPTION TAB2-2.png": "''", "CAPTION TAB3-1.png": "'\\n\\n**Table 3. Lab Level--Overall Collaboration Portfolio**'", "CAPTION TAB3-2.png": "'* [19]'", "CAPTION TAB1-1.png": "'\\n\\n**Table 1. Summary Statistics for the Main Sample**'", "CAPTION TAB1-2.png": "'* [18] A.\\n\\n'", "CAPTION TAB4-1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'", "CAPTION TAB4-2.png": "''", "CAPTION FIG2.png": "'Figure 2: (Color online) Probability of Collaboration and Separation\\n\\nFigure 2: (Color online) Probability of Collaboration and Notation\\n\\n'", "CAPTION TAB6.png": "'Netes. The dependent variable in column (1) is a dummy equal to 1 if the lab pair collaborates in the focal year. The dependent variable in columns (2) and (3) is the number of collaborations. _After Colocation_ is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. _After Separation_ is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. The Poisson specification with fixed effects drops all lab pairs where collaboration is never observed, hence the smaller number of observations. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\\\\({}^{-}p<0.05;^{-}p<0.01\\\\).\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c} \\\\hline \\\\hline Netes & The dependent variable in column (1) is a dummy equal to 1 if the lab pair collaborates in the focal year. The dependent variable in columns (2) and (3) is the number of collaborations. _After Colocation_ is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. _After Separation_ is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. The Poisson specification with fixed effects drops all lab pairs where collaboration is never observed, hence the smaller number of observations. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\\\\({}^{-}p<0.05;^{-}p<0.01\\\\). \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 6: Colocation and Separation'", "CAPTION TAB5.png": "'* [16] M. C. C. (1999). The \\\\(\\\\alpha\\\\)-function of the \\\\(\\\\alpha\\\\)-function'", "CAPTION FIG1.png": "'Figure 1: (Color online) Probability of Collaboration and Colocation\\n\\n'", "CAPTION FIG3.png": "'Figure 3: (Color online) Similarity in Cited References Space\\n\\n'", "CAPTION TAB7.png": "''", "CAPTION TAB8.png": "''", "CAPTION TAB9.png": "'* _Nuts._ The dependent variable in all columns is the maximum number of citations received by the publications of the lab pair in the focal year. Citation data is obtained from Scopus on 2016. In column (1), only pairs with above the median cosine similarity in keywords (low search costs) are included. In column (2), only pairs with below the median similarity in keywords (high search costs) are included. Results are conditional on collaboration in focal year. _After Calculation_ is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. _After_ _Spinn_ is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. Robust standard errors clustered at the lab-pair level in parentheses.\\n* \\\\(p\\\\) < 0.01.\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c c} \\\\hline \\\\hline _Nuts._ The dependent variable in all columns is the maximum number of citations received by the publications of the lab pair in the focal year. Citation data is obtained from Scopus on 2016. In column (1), only pairs with above the median cosine similarity in keywords (low search costs) are included. In column (2), only pairs with below the median similarity in keywords (high search costs) are included. Results are conditional on collaboration in focal year. _After Calculation_ is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. _After_ _Spinn_ is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. Robust standard errors clustered at the lab-pair level in parentheses. \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 9: Collection, Separation and Max Citations'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Notes._ _Editorial Period is a dummy equal to 1 for all of the years during which a specific lab is moved from its original location because of the asbestos removal. Shares are calculated based on the Scopus author affiliations data available for each paper and then aggregated at the lab level on a yearly basis. \u201cShare French\u201d does not include the Jussieu campus and affiliated labs. Robust standard errors clustered at the lab level in parentheses. \\\\({}^{\\\\star}\\\\)\\\\(\\\\approx 0.1\\\\) \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB4.png": "'* [19]'", "CAPTION FIG2-1.png": "'Figure 2. (Color online) Probability of Collaboration and Separation\\n\\n'", "CAPTION FIG2-2.png": "'Nates. Estimated coefficient for years before and after the move. The dependent variable is collaboration (1/0). Regression includes lab-pair fixed effects and year fixed effects. Error bars represent \\\\(95^{\\\\circ}\\\\) confidence intervals based on robust standard errors clustered at the lab-pair level.\\n\\n'", "CAPTION TAB6-1.png": "'\\n\\n**Table 6. Calculation and Separation**'", "CAPTION TAB6-2.png": "'Notes: The dependent variable in column (1) is a dummy equal to 1 if the lab pair collaborates in the focal year. The dependent variable in column (2) and (3) is the number of collaborations. After Cofacation is equal to 1 when a lab pair becomes colocated because of the moves and 0 otherwise. After Separation is equal to 1 when a previously colocated pair is separated because of the moves and 0 otherwise. The Poisson specification with fixed effects drops all lab pairs where collaboration is never observed, hence the smaller number of observations. Robust standard errors clustered at the lab-pair level in parentheses.\\n\\n\"\\\\(p<0.05\\\\); \"\\\\(p<0.01\\\\).\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chanBenefitsPitfallsAnalogies2011.json b/test_extracted_captions/chanBenefitsPitfallsAnalogies2011.json new file mode 100644 index 0000000000000000000000000000000000000000..d460256d1ade558d3ccb01a81118f37e4e707a5e --- /dev/null +++ b/test_extracted_captions/chanBenefitsPitfallsAnalogies2011.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## Table 2: Patent for each candidate'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2** Example participant solution concepts'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG3.png": "'* [19]'", "CAPTION FIG6.png": "'* [19]'", "CAPTION FIG7.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chanBestDesignIdeas2015.json b/test_extracted_captions/chanBestDesignIdeas2015.json new file mode 100644 index 0000000000000000000000000000000000000000..26100903da2f978d15d94181a7f1e2e14524fc47 --- /dev/null +++ b/test_extracted_captions/chanBestDesignIdeas2015.json @@ -0,0 +1 @@ +{"CAPTION TAB4.png": "'Table 4: Model estimates and fit statistics for cross-classified multilevel logistic regressions of Pr(shortlist) on DIS\\\\(\\\\Gamma_{34A\\\\times 3}\\\\), with comparison to baseline model (controls only)\\n\\n'", "CAPTION TAB5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The \\\\(\\\\beta\\\\)-function of the \\\\(\\\\beta'", "CAPTION TAB2.png": "'\\n\\n## Chapter 4 Machine Learning and Machine Learning'", "CAPTION TAB3.png": "'\\n\\n## Appendix A Machine Learning'", "CAPTION FIG7 (NOISY).png": "'\\n\\n## Chapter 7 Multi-dimensional lattice\\n\\nIn this chapter we will discuss the lattice model in the following chapters.\\n\\n### 7.1 Introduction\\n\\nThe lattice model is a lattice model with a lattice spacing \\\\(a\\\\) and a lattice spacing \\\\(a\\\\).\\n\\n'", "CAPTION FIG4 (NOISY).png": "'Figure 4 (A) Scatterplot of LDA curves vs. averaged human continuous similarity judgments for inspiration in the e-near challenge. (B) Mean cosine against the challenge belief for within- vs. between-domain approximations\\n\\n'", "CAPTION FIG5 (NOISY).png": "'Figure 5: Tests found by L.D.A. within examples of new and far superiorities for the \\\\(e\\\\)-wave turbulence.\\n\\n'", "CAPTION FIG6 (NOISY).png": "'Figure 6: Illustration error-dunified structure of the data\\n\\n'", "CAPTION FIG8 (NOISY).png": "'\\n\\n## Chapter 4 Overall and the\\n\\nIn the first part of the thesis, we have presented a new method for calculating the entropy of the system. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method, and the method is based on the idea of the method. The method is based on the idea of the method, and the method is based on the idea of the method, and the method is based on the idea of the method. The method is based on'", "CAPTION FIG9 (NOISY).png": "'\\n\\n## Chapter 6 Meta-Ghird rule-\\n\\nMeta-Ghird rule-\\n'", "CAPTION FIG10 (NOISY).png": "'\\n\\n## Chapter 4 Conclusion and Future Work\\n\\nIn this thesis we have developed a new method for the calculation of the \\\\(\\\\Delta\\\\)-function in the \\\\(\\\\Delta\\\\)-function. The method is based on the \\\\(\\\\Delta\\\\)-function, which is a function of the form\\n\\n\\\\[\\\\Delta(\\\\Delta)=\\\\frac{1}{2}\\\\sum_{i=1}^{n}\\\\frac{1}{2}\\\\sum_{j=1}^{n}\\\\frac{1}{2} \\\\sum_{i=1}^{n}\\\\frac{1}{2}\\\\sum_{j=1}^{n}\\\\frac{1}{2}\\\\sum_{i=1}^{n}\\\\frac{1}{2} \\\\sum_{j=1}^{n}\\\\frac{1}{2}\\\\sum_{i=1}^{n}\\\\frac{1}{2}\\\\sum_{j=1}^{n}\\\\frac{1}{2} \\\\sum_{i=1}^{n}\\\\frac{1}{2}\\\\sum_{j=1}^{n}\\\\frac{1}{2}\\\\sum_{i=1}^{n}\\\\frac{1}{2} \\\\sum_{j=1}^{n}\\\\frac{1}{2}\\\\sum_{i=1}^{n}\\\\frac{1}{2}\\\\sum_{j=1}^{n}\\\\frac{1}{2} \\\\sum_{j=1}^{n}\\\\frac{1}{2}\\\\sum_{j=1}^{n}\\\\frac{1}{2}\\\\'", "CAPTION FIG11 (NOISY).png": "'Figure 11: Mean for (\\\\(\\\\pm\\\\)1 SE) at. Ismour mid-infrared for LB4 points by level of K.\\n\\n'", "CAPTION FIG3 (NOISY).png": "'Figure 3: Full-first of database brief from the OpenDB0 challenge.\\n\\n'", "CAPTION FIG1 (NOISY).png": "'Figure 1: Example number illustrating the typical amount of detail per concept\\n\\n'", "CAPTION FIG2 (NOISY).png": "\"Figure 2: Depiction of OpenIDEO elution workflow. When porting concepts/adaptrations, users are prompted to cite concepts/adaptrations they should upon' by abrogating how/marked concepts/imprimatives (middle panel) to the citation area (left panel). Users can also search for related concepts/imprimatives at this step (middle panel). These third sources show any an metadata for the concept/imprimatives (right panel).\\n\\n\"", "CAPTION TAB1.png": "'Table 1: Descriptions and number of tests for OpenIDEO challenges in final analysis samples\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chanComparingDifferentSensemaking2016.json b/test_extracted_captions/chanComparingDifferentSensemaking2016.json new file mode 100644 index 0000000000000000000000000000000000000000..c004bbfb2c7f81a05d41b1fcc3c1a018b5012602 --- /dev/null +++ b/test_extracted_captions/chanComparingDifferentSensemaking2016.json @@ -0,0 +1 @@ +{"CAPTION TAB5.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3.** **Participants generate similar numbers of good ideas across conditions, adjusted for baseline fluency.**'", "CAPTION TAB3.png": "'Table 3. Machine-generated paths increase number of **idens relative to control condition. Means are adjusted for baseline fluency**.\\n\\n'", "CAPTION TAB4.png": "'\\n\\n**Table 4. External stimulation increases breadth of search by relative to control condition, for both problems.**'", "CAPTION FIG1.png": "'Figure 1. Screenshot of human labeling task (left) physical setup of human cluster-label task (right)\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Screenshot of ideation interface. Actual inspirations from the machine-machine condition are shown. Participants enter ideas on the left, and view inspirations on the right. Participants can star inspirations they find useful.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1. Example solution paths extract by each method for both problems. For comparability, we select examples that are close in semantic meaning.**'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'"} \ No newline at end of file diff --git a/test_extracted_captions/chanFormulatingFixatingEffects2024.json b/test_extracted_captions/chanFormulatingFixatingEffects2024.json new file mode 100644 index 0000000000000000000000000000000000000000..59da804d753a73ce455460c595e8eea1692c184b --- /dev/null +++ b/test_extracted_captions/chanFormulatingFixatingEffects2024.json @@ -0,0 +1 @@ +{"CAPTION FIG7.png": "'\\n\\n## References\\n\\nFig. 7: Example exploration graphs used for coding hill-climbing strategies: not \u201call hill-climbing\u201d (left) and \u201call hill-climbing\u201d (right), where each transition between participant moves is plotted on the x-axis, and the Euclidean distance between each move and its immediately preceding move is plotted on the y-axis. In the not \u201call hill-climbing\u201d example, the first 10 moves (the 0th-10th moves), where there are substantial variations in Dropdown move distances across the sequences, would be coded as non-hillclimbing (N), while the 10th-20th moves and the 20th-30th moves, where Dropdown move distances are consistently low, would be coded as hillclimbing (H). In the \u201call hill-climbing\u201d example, all first 30 moves would be coded as hillclimbing (H).\\n\\n'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG4.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\nFig. 5: Maximum score at \\\\(n\\\\)-th move for each participant: left) HD; right) LD. We observe (1) cumulative disadvantages for the List condition, as well as (2) early advantages for the In-Context interface, especially with LD examples.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG8.png": "'\\n\\n## References\\n\\nFig. 8: Raw proportion of participants with a predominantly hillclimbing strategy in the first 30 moves, across interface conditions (a) with both HD and LD examples (b). More \u201cList\u201d participants did hillclimbing in the first 30 moves with both high and low diverse example sets than \u201cIn-Context\u201d participants. More \u201cList\u201d participants did hillclimbing for the first 30 moves with LD examples than other combinations of the presentation and example sets.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## References\\n\\nFig. 6: Raw proportion of participants expressed \u201cnot using (examples)\u201d, \u201csimulation-based\u201d or \u201cmodel-based\u201d in their answer to \u201cHow did you use initial examples (the values of ten points given to you)?\u201d. Error bars are standard error of proportion. More participants, self-reported using a model-based strategy in the In-Context condition compared to other conditions.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The \\\\(\\\\alpha\\\\)-function of the \\\\(\\\\alpha'", "CAPTION FIG2.png": "'Figure 2: Screenshot of experimental interface, shown for the List condition: the 100x100 grid, which constituted the search environment for the task, was shown on the left panel: participants explored the space by clicking anywhere on the 100x100 grid. The 10 initial examples, moves remaining, the score of current move, the current max score and score legend were shown on the right panel. In the Dropdown condition, the dropdown menu as seen in Figure 3 was shown in the same position as the list of examples in the List condition. In the In-Context condition, examples were instead overlaid as points, with corresponding values, on the search grid, as shown in Figure 3.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chanImportanceIterationCreative2015.json b/test_extracted_captions/chanImportanceIterationCreative2015.json new file mode 100644 index 0000000000000000000000000000000000000000..fba33581615d659b5727e351b422ce4816da6e25 --- /dev/null +++ b/test_extracted_captions/chanImportanceIterationCreative2015.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Example inspiration (left) and concept (right) for an OpenIDEO problem about managing electronic waste.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Illustrated example of \u201cindirect\u201d sources as sources in levels 2\u20134 of a specific target concept\u2019s genealogy. Teal circles denote concepts; maroon circles denote inspirations. Note that some indirect sources in this example serve as direct sources for the earlier concepts in this genealogy.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{Model estimates and fit statistics for cross-classified multilevel logistic regressions of Pr(shortlist) on COMB-DIST\\\\({}_{\\\\text{obs}}\\\\), with comparison to baseline model (controls).} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Model estimates and fit statistics for cross-classified multilevel logistic regressions of Pr(shortlist) on COMB-DIST\\\\({}_{\\\\text{obs}}\\\\), with comparison to baseline model (controls).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Model-fitted relationship between combination distance and \\\\(\\\\text{Pr}\\\\)(shortlist). Fetted values evaluated at mean values of FEEDBACK, SOURCEQUAL, and SP-DIST. Creyed \\\\(\\\\text{L}\\\\)=es are fitted from pesticide mode estimates of the slope of COMB-DIST\\\\({}_{\\\\text{Ca}}\\\\) for each problem.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Example near (left) and far (right) combinations according to \\\\(\\\\mathsf{COMB-DIST_{\\\\Omega\\\\pi}}\\\\).\\n\\n'", "CAPTION TAB3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 4**} \\\\\\\\ \\\\multicolumn{1}{c}{**Bivariate correlations for indirect combination distance measures**} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Bivariate correlations for indirect combination distance measures'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG6.png": "'Figure 6: Model-fitted relationship between _z_ombination distance of indirect sources and Pr(shortlist). Fitted values evaluated at mean values of FEEDBACK, _SOLWCEQUAL_, and _SP_-DST. Greyed lines are fitted from posterior mode estimates of the slope of \\\\(\\\\text{COMB-DIST}_{\\\\text{ROI}}\\\\) for each problem.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chanSOLVENTMixedInitiative2018.json b/test_extracted_captions/chanSOLVENTMixedInitiative2018.json new file mode 100644 index 0000000000000000000000000000000000000000..9a5a075a879fa9771bab425c48398094df477af4 --- /dev/null +++ b/test_extracted_captions/chanSOLVENTMixedInitiative2018.json @@ -0,0 +1 @@ +{"CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB5.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB6.png": "'\\n\\n'", "CAPTION FIG1.png": "'Fig. 1. A screenshot of the crowd-facing interface for annotating the abstracts. Workers annotate aspects by first selecting the annotation type (an in-line description is provided as a reminder) and then using an intuitive click-and-drag highlighting interaction to annotate one or more words.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB2.png": "'\\n\\n'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/test_extracted_captions/chanSemanticallyFarInspirations2017.json b/test_extracted_captions/chanSemanticallyFarInspirations2017.json new file mode 100644 index 0000000000000000000000000000000000000000..d1e4abe8216eaf9cd53a12548ee38a590e2166ae --- /dev/null +++ b/test_extracted_captions/chanSemanticallyFarInspirations2017.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Participants in our pilot study showed higher levels of cognitive effort during system-inferred impasses (green bars) compared to system-inferred productive ideation (red bars), as indicated by lower levels of \\\\(\\\\Delta\\\\)HlbR (in micromolars, \\\\(\\\\blacklozenge\\\\)**M**) in regions of the prefrontal cortex (measured using functional near-infrared spectroscopy).\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG2.png": "'Figure 2. Participants generated ideas more slowly just before and impasse compared to other points in their ideation session.\\n\\n'", "CAPTION FIG3.png": "\"\\n\\n**Figure 3. Summary of model-adjusted mean contrasts across dependent measures for each inspiration condition against the non-stimuli baseline condition (vertical dashed lines). Mean contrasts are reported on the original scale of the dependent measure. Error bars are 95% confidence intervals. Significant contrasts from the no-stimuli baseline (by Dunnett's t-test) are shown in orange. Contrasts that support a theory's prediction are marked green; contrasts that contradict (i.e., go in the opposite direction of) a theory's prediction are marked red. Here, SIAM's predictions for the always-far (for _inter-idea interval_, _transition similarity_), always-near (for _inter-idea interval_, _transition similarity_, _fluency_), and mismatch-state conditions (for _inter-idea interval_) are supported, while the associationist theory's predictions for always-near is contradicted (for _novelty_).**\"", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chanSituativeCreativityLarger2016.json b/test_extracted_captions/chanSituativeCreativityLarger2016.json new file mode 100644 index 0000000000000000000000000000000000000000..83ded127bfbafc88cb5f761e59c2024bb098a814 --- /dev/null +++ b/test_extracted_captions/chanSituativeCreativityLarger2016.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Figure 1**\\n\\n**Figure of large room (left) and small room (right) in Experiment 1.**'", "CAPTION FIG2.png": "'\\n\\n## Figure 2 Shapes for Invention task in Experiment 1.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Figure 4**\\n\\nNovelty and practicality of alternative uses by room size in Experiment 1. Error bars are \\\\(\\\\pm\\\\)1 SE.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## Figure 5 Summary of effects in Experiment 1. Error bars are 95% CIs.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Example of low and high novelty/practicality inventions. The low and high novelty inventions are a \u201cfunnel\u201d and a device that \u201cslows elevators with centripetal force.\u201dThe low and high practicality inventions are an \u201cunstable martini glass\u201d and a \u201ctool to catch water to measure the rain.\u201dNote that the shapes used for invention are a cone, sphere, and tube.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## Table 1\\n\\nDescriptive statistics for Experiment 1.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n## Figure 7 Shapes for Invention task in Experiment 2.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## Figure 6 Picture of new large room in Experiment 2.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1979) The role of the \\\\(\\\\beta\\\\)-decay of the \\\\'", "CAPTION FIG10.png": "'\\n\\n**Figure 10**\\n\\nNovelty and practicality of alternative uses by room size, pooled across experiments. Error bars are \\\\(\\\\pm\\\\)1 SE.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n## Table 3\\n\\nDescriptive statistics for Experiment 2.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Figure 8**\\n\\nNovelty and practicality of alternative uses by room size in Experiment 2. Error bars are \\\\(\\\\pm 1\\\\) SE.\\n\\n'", "CAPTION FIG9.png": "'\\n\\n## Chapter 9 Summary of effects in Experiment 2. Error bars are 95% CIs.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chen2023novo.json b/test_extracted_captions/chen2023novo.json new file mode 100644 index 0000000000000000000000000000000000000000..585ac9ee838136957beeba07bcb3a6e095b30d16 --- /dev/null +++ b/test_extracted_captions/chen2023novo.json @@ -0,0 +1 @@ +{"CAPTION FIG6.png": "'Figure 6: Colled-coil interfaces are suitable to withstand mechanical stress from orthogonal directions (A) A model of how 3-helix bundles would be able to bear mechanical stress differently compared with the microtubules. The bending curvatures and gaps are exaggerated forustration purposes.\\n\\n(B) A schematic of wild-type and mutant sperm doublet structures highlighting the Takitin 5 bundles and the partial redundancy.\\n\\n'", "CAPTION FIGS1.png": "'Figure S1. Workflow of data processing, related to Figures 1, 2, and 3 and STAR Methods (A) Tift series composed of 25 2D projections were recorded [scale bar: 100 nm]. The image shows the midpiece of sperm flagella that contains mitochondria around the axoneme. Gold beads on the tilt images are indicated (green arrowhead). The fiducial gold beads were used to align the tilt images. (B) 3D micrograms were reconstructed, and subroutines were picked along the microtubules (scale bar: 100 nm). (C) 3D elastifica and refinement were performed using and average the subutemogram for the 96 nm-propagating structures of the mouse sperm doublets. Four views of the 96 nm-repeating structure of doublets from EHNA-treated sperm are shown for the 3D reconstruction generated using RELION3 as reported previously.10 Gold-standard Fourier shaft correlation (FSC) curve calculated between half maps of mouse sperm doublets is shown. The resolution was estimated as 26 A (FSC = 0.143). (D) Two slices of the 96 nm-repeating structure of doublets looking along and perpendicular to the filament axis. Note the red line in the top panel indicates the plane of the bottom slice, and periodic structures are observed inside the microtubules. The coordinates were recartered on the 48-nm repeats and imported into RELION4. In the top panel, note that the features further away from the microtubules are blunier, suggesting that there are conformational heterogenities and that they are resolved at lower resolutions. (E) The initial father3D job of the 48 nm-repeating structures was performed using RELION4.21\\n\\n(F) The 3D reconstructions were matched to the 2D projections of individual particles in the raw tilt images, and this step refined both the geometric and optical parameters of the tilt series (-H). (G) Another round of sub-tetromogram averaging was performed based on refined tilt series. No additional improvement was observed after 3 rounds of refinement and Refine3D as shown in (F)-(G).\\n\\n'", "CAPTION FIGS4.png": "'Figure S4.\u2014 Rigid-body fitting of 29 identified MIPs from bovine trachea cilia into the density map of mouse sperm doublet, related to Figures 2 and 3 and STAR Methods (A-F) Models of 28 known MIPs from bovine trachea cilia (PDB: 7MRC)\\\\({}^{13}\\\\) are fitted into the density map of mouse sperm doublet. The viewing angles for all panels are shown. For proteins that have multiple \u03b1-helices (CFAP161, RIBC2, CFAP53, MNS1, CFAP21, NME7, CFAP141, EFHC1, EFHC2, ENKUR, CFAP210, EFCABE, GFAP45, PACRB, and TEKIN 1\u20134), the arrangement of secondary structures matches densities in sperm doublets. The overall shapes of \u03b2-sheets in \u03b2-sheet-rich proteins (CFAPS2 and CFAP20) match the densities, and these proteins are highly conserved in axoremes. For the proteins that contain random coils, we did observe matching features in the maps and were able to trace some of the main chairs at the current resolution (CFAP96, SPAG8, CFAP107, FAM166B, Pierce1, Pierce2, CFAP128, CFAP276, and TEKIFIP1).\\n\\n'", "CAPTION FIGS5.png": "'Figure S5: Characterization of the 16 nm-repeating structures of doublets from mouse sperm, related to Figure 2 (A and B) Gold-standard Fourier shell correlation (FSC) curves were calculated using half maps of 16 nm-repeating structures of A-bLuMe and B-bLuMe. The resolution was estimated as 6.0 and 7.7 \\\\(\\\\AA\\\\), respectively (FSC = 0.143). The Nyquist limit is 5.26 \\\\(\\\\AA\\\\).\\n\\n(C-E) The local resolution map was calculated from the two half maps of 16 nm-repeating structures of A-bLuMe using RELION4. The viewing angles for (G) and (E) are shown in (C) (black arrow). These viewing angles are similar to Figures 1A, 1D, and 1E, respectively.\\n\\n(F-H) The local resolution map was calculated using the two half maps of 16 nm-repeating structures of B-bLuMe using RELION4. The viewing angles for (G) and (G) are shown in (F) (black arrow). The viewing angles of (F) and (G) are similar to Figures 1A and 1F, respectively.\\n\\n'", "CAPTION FIGS6.png": "'Figure S6.: Tektin 5 and CCDC105 likely form sperm-specific 3-helix bundles associated with the A-tubule, related to Figures 2 and 3 (A) After unbiased matching, Tektin 5 was scored as the 45 h of the predicted structures out of 21,615 proteins from the mouse proteome, marked by cross-correlation scores (the top 10 are shown). Tektin 1-4 were ranked at 47-10 due to their similar tertiary structures.\\n\\n(B) Typical elastic positions (#1-4 and e/b) from the same search. Usually, these are proteins with long single helices that do not match the gaps observed in the map. Also, they do not explain the 3-helix bundles. The fitting of Tektin 5 into the same densities is shown for comparison.\\n\\n(C) The structure of CCDC105 directly predicted by AlphaFold2 (left) is compared with the predicted complex formed by two CCDC105 molecules (right). The half-length CCDC105 molecule in the complex is colored based on the per-residue confidence scores (predicted local distance difference test (pLDDT), red: high confidence) from the AlphaFold2 prediction. The three P-loops have medium confidence scores (green), suggesting the exact conformations of these loops may not be accurately predicted. However, the presence of these structured loops is conclusively confident based on the conserved proline residues (see the sequence alignment in D) and the matching protrusion densities observed in our maps (Figure 2G). Note that the conformations of the three proline-rich loops differ in these two predictions. These differences could be caused by the presence of neighboring molecules during AlphaFold2 analyses.[7]\\n\\n(D) The sequence alignment of CCDC105 from the mammals (H. sapiens, _M. musculus, B. taurus, S. scrota, and F. catus), zebrafish (D. rerio), and sea urchins (S. roupurakis). The three proline-rich loops are marked above the sequences.\\n\\n(E) The models of CCDC105 and Tektin 5 are fitted into the densities of the 3-helix bundle at their ribbon, where the former model explains the extra protrusions and orientation/lengths of helices of the densities, but the latter does not.\\n\\n'", "CAPTION FIGS8.png": "'Figure S8: Biochemical extractions of proteins from mouse sperm, related to Figure 3 (A) SDS-PAGE analyses of protein extractions from mouse sperm using 0.1% Triton in PBS (E1), 0.6 M NaCl in PBS (E2), 0.6 M KClN in PBS (E3), 8 M urea (E4), and 10% SDS (E5).\\n\\n(B) Western blot analyses of protein extractions from mouse sperm using an antibody against a-tubules. Note that strong bands were detected only in E3 and E4, suggesting the microtubule structures were stable in Triton and high NaCl buffers and dissonhol completely in KClN/urea solutions.\\n\\n(C) Bar chart of the number of proteins identified by MS (protein control in each fraction (E1-ES) and biological replicate. We identified a total of 1,677 mouse proteins, with a range of 772 to 1,326 proteins identified in each individual fraction and replicate.\\n\\n(D) Hathamp of proteins with significant changes between any two fractions (absolute log2 fold change [log2-FC] > 1, adjusted p value < 0.05), listed by fractions (E1-ES) and biological replicate, and clustered by correlation of intensity profile. Proteins are colored by the log2,FC in protein intensity normalized to the row median (red, increased intensity; blue, decreased intensity; gray, not detected). Cluster identification _narrows_ (cluster ID) are labeled (err).\\n\\n(E) Hathamp of gene ontology (GC) enrichments among the significantly changing proteins identified in each cluster from (D) that to right: cluster ID 1\u20136, as labeled in ID. GO terms were curated from the top 4 enrichment terms per cluster, and non-redundant terms were selected by an automated clustering procedure (see STAR Methods). Increased shading reflects increased significance of the enrichment term. The number of proteins per enrichment term is shown in white IR significant (adjusted p value < 0.05) and gray if not significant (adjusted p value > 0.05). A bar chart plotting the number of total genes in each cluster ID is included.18\\n\\n(F) Log2 protein intensities (y axis) for eight mouse proteins as quantified by MS in each fraction (E1\u2013ES) and biological replicate (colored dots; maximum n = 3).\\n\\n'", "CAPTION FIGS2.png": "'Figure S2: Characterization of the 48 nm-repeating structure of doublets from mouse sperm, related to Figures 1, 2, and 3\\n\\n(A) Gold-standard Fourier shell correlation (FSC) curves were calculated between half maps of mouse sperm doublets. The resolutions were reported as FSC = 0.148. Note the FSC curves resulting from the iterative frame alignment and CTF refinement between the second and third Refrea3D [abs were not shown for the activity of the figure. Further refinement after the third Refrea3D did not improve the resolution or the quality of the map.\\n\\n(B) The local resolution map of mouse sperm doublets was calculated by RELLOU4. The ribbon region has the highest resolutions. Densities in the A-tubule have higher resolutions than the ones from the B-tubule.\\n\\n(C) Equivalent longitudinal cross-section views of doublets from mouse sperm and bovine trachea cilia (EMDE: EMD-24664) are shown. [(ii)] The latter was low-pass filtered to 7.5 \u00c5, and comparable details of the secondary and tertiary structures of the MPAs are observed.\\n\\n(D) The reconstruction of mouse sperm doublet (gray) is overlaid with the bovine trachea doublets (yellow). The mouse sperm-specific densities are highlighted (dashed axis). The broken helical bundles and the curved helical bundles inside the A-tubule of mouse sperm doublets along the microtubule axis are shown. The discontinuous parts of the broken helical bundles are indicated (dashed rectangle). Note that the curved bundles have one straight and two curved groups of densities in every 48-nm repeat (outlined using dashed shapes).\\n\\n'", "CAPTION FIG1.png": "'Figure 1: The 3D reconstructions of mouse and human sperm doublets revealed novel MIPs (A and B) Transverse cross-section views of the doublets of mouse (A) and human (B) sperm. Conserved sperm MIP densities are highlighted (pink, blue, and green), and the corresponding viewing angles of (C)\u2013(E) are indicated (colored arrowheads). The 3-helix densities in A-tubule shared with bovine trachea doublets (EMD-24664) are colored (yellow).\\\\({}^{13}\\\\) Divergent sperm densities are also indicated (red dashed shapes). Individual protofilaments of the doublets are labeled as A1\u2013A13 and B1\u2013B10. (C\u2013E) Zoom-in views of the conserved sperm MIP densities along the longitudinal axis. In (C), mouse sperm-specific densities are indicated and labeled (red dashed shapes; see more in Figures S2 and S3). In (E), although the situations are 8 nm apart from one another, the overall periodicity is 48 nm. See also Figures S1, S2, and S3.\\n\\n'", "CAPTION FIGS3.png": "'Figure S3. Characterization of the 48 nm-repeating structure of doublets from human sperm, related to Figure 1 (a). The gold-standard Fourier shell correlation (PSC) curve was calculated between half maps of mouse sperm doublets. The resolution was estimated as 10.3 A (FSC = 0.143).\\n\\n(B) The local resolution map of human sperm doublets was calculated by RELION4. The ribbon region has the highest resolutions. Densities in the A-tubule have higher resolutions than the ones from the B-tubule.\\n\\n(C) Equivalent views of doublets from human sperm and bovine trachea cilia (EMOE: EMD-24664) are shown. [13] The latter was low-pass filtered to 10 A, and comparable details of the secondary and tertiary structures of the MMPs are observed.\\n\\n(D) The reconstruction of human sperm doublet (blue) is overlaid with the bovine trachea doublets (yellow) at low and high thresholds.\\n\\n(E) The two broken bundles inside the A-tubule in human sperm are shown at a low threshold (see the corresponding mouse densities in Figure S2D).\\n\\n(F) The curved helical bundles contain one straight and two curved groups of densities inside the A-tubule of human sperm. Human sperm-specific densities were observed to connect one curved bundle to the lumen of A-tubule.\\n\\n(G) The human sperm doublets overlaid with mouse sperm doublets are shown. The inconsistent densities are outlined (dashed line) (also see Figures S2D and S2F).\\n\\n'", "CAPTION FIGS7.png": "'Figure S7. DUSP proteins in the A-tubule, related to Figure 3 (A) at a lower threshold compared with Figure 3C, densities connecting the N-terminal residues of the slanted Takin Ss (magenta models) and the DUSPs (blue models) are observed.\\n\\n(B) The DUSPs is fitted into the globular domain, and three orthogonal views are shown. Other homologous DUSP proteins fit well into the density because of similar tertiary structures [DUSP 3, 13, 14, 18, 21, and 29].\\n\\n'", "CAPTION FIG2.png": "'Figure 2: _De novo protein identification of sperm MIPs assisted by AlphaFold2_\\n\\n(A) Conserved densities in mouse and human sperm were segmented from the averages of 16-nm repeats of mouse sperm doublets and searched in the AlphaFold2 library of the mouse proteasome (21,615 proteins).\\n\\n(B) The predicted structure of Takith 5 based on AlphaFold2 was fitted into the continuous 3-helix bundle.\\n\\n(C) Modeling of a complex formed by a full-length Takith 5 and a truncated one (N-Tait 5: aa 1-149) using CableFold.23\\n\\n(D) Fitting and modeling of Takith 56 into the 3-helix bundle identifies in the A-t-'", "CAPTION FIG5.png": "'Figure 6: Characterization of mutant TeMFS -/- sperm\\n\\n(**A**) The percentages of motile sperm from wild-type and TeMFS knockout mice (>200 cells were counted for each mouse, three knockout -/- mice and two wild-type mice were analyzed, and the pool percentage and 95% confidence intervals Wilson/Brown method were shown).\\n\\n(**B**) The percentages of bent sperm from wild-type and TeMFS knockout mice (>200 cells were counted for each mouse, three knockout -/- mice and two wild-type mice were analyzed, and the pool percentage and 95% confidence intervals by Wilson/Brown method were shown). Two examples of bent sperm are shown.\\n\\n(**C**) An overlay of wild-type models with the densities of TeMFS -/- sperm around the slanted bundles. The continuous 3-helix bundle assigned as TeMFS (high occupancies) and slanted helical bundles (low occupancies) are shown. The densities corresponding to the DUSP proteins are barely resolved. Note that there are substantially less densities for these models compared with Figure 3C.\\n\\n(**D**) An overlay of wild-type models with the densities of TeMFS -/- sperm around the curved bundles. The occupancies of the curved bundles are lower than the other MIPs and tubulins. Note that there are substantially less densities for these models compared with Figure 3D.\\n\\n(**E**) The two broken 3-helix bundles have lower occupancies compared with the surrounding MIPs and tubulins. Note that there are substantially less densities for these models compared with Figure 3A.\\n\\n'", "CAPTION TABNA.png": "'\\n\\n**Acknowledgments**'", "CAPTION FIG4.png": "'Figure 4. Sperm doublet are compared at microtubules and extensive cact-cell bundles\\n\\nFig 5. The pus and rhinus arcade of Takin 5 were named in a fixed C terminal of the protein.\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'Figure 3.: **Conformational plasticities of Tektin 5**(A) The two broken 3-helix bundles could be explained by two complete and a third partial copies of Tektin 5 (dashed rectangles) per 48-nm reagent, instead of three Tektin in the continuous 3-helix bundle.\\n\\n(B) The AlphaFold2 medial of mouse Taktin 5 was fitted into the slanted helical damilies. Sequence alignment of Tektin 5 from _M. musculus_, _H. sapiens_, _B. fauvus_, and _F. cada_ is shown from G133 to F151 (the numbering of ambe acids is based on _M. musculus_ Tektin 5). The conserved Gly137, Gly143, and Gly150 are near the turning point of the bent \u03b1-helix.\\n\\n(C) The fitting of Tektin 5 and DUSF3 protein (its homologs are also possible candidates) into the 16 nm-repeating features; see the same view of the map in Figure 1C.\\n\\n(D) Three modified Taktin 5 were fitted into the densities of the curved bundle in the mouse sperm doublet (as indicated in Figure 1A). The intact intermolecular interaction strength reference, N termini of the Taktin 5s, and curved 2-helix segments are indicated (arrows). Nearby MIPs shared between mouse sperm flagella and bovine trachea cilia are also colored and labeled [MMET, CFAP161, and SPAGB].\\n\\n(E) The cross-section schematic is shown. The highlighted models of (A)\u2013(D) are indicated using arrows. See also Figures 51, 52, 54, 56, 57, and 58.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/chiuInvestigatingEffectsOppositely2012.json b/test_extracted_captions/chiuInvestigatingEffectsOppositely2012.json new file mode 100644 index 0000000000000000000000000000000000000000..cd818fc393bae87ca52fbed3b392773612494700 --- /dev/null +++ b/test_extracted_captions/chiuInvestigatingEffectsOppositely2012.json @@ -0,0 +1 @@ +{"CAPTION FIG3.png": "'Figure 3: Aggregated frequency of stimulus word-use with no unknown uses of stimulus words.\\n\\n'", "CAPTION TAB7.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB9.png": "\"\\n\\n**Table 9.** Kendall's W for Experiment 2 concepts.\\n\\n\"", "CAPTION TAB10.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB8.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Coal language results comparing effects of PQS and Stimulus Type on estimated marginal means of NWP.\\n\\n'", "CAPTION TAB11.png": "'\\n\\n**Table 11.** **Bedging problem contrast results.**'", "CAPTION FIG4.png": "'Figure 4: Graphs of concept novelty, usefulness and cohesiveness ratings. Note the lines in the interaction graph do not signify a relationship, but assist in visualising variable effects and interactions. Reprinted with permission. Copyright \u00a9 ASME 2010.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Bushing language results comparing effects of POS and Stimulus Type on estimated marginal means of NWP.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Show language results comparing effects of POS and Stimulus Type on estimated marginal means of NWP3\\n\\n'", "CAPTION TAB14.png": "'Table 14. Task comparison between control and stimulus participants\\n\\n'", "CAPTION TAB15.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB16.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Explanatory model of opposite-verb NWP introduction and its effect on concept creativity.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG1.png": "'Figure 1: Rating scale for novelty. Reprinted with permission. Copyright ASME 2010.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB3.png": "'\\\\(\\\\frac{1}{2}\\\\).\\n\\n'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l l l} \\\\hline \\\\hline & & & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Kendall\u2019s W for aggregated results of Experiment 1 concepts.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 5: Example concepts and creativity scores for the Sunflower problem.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Graph of novelty, usefulness and cohesiveness results for the Sunflower and Egg problems.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/cureton2010length.json b/test_extracted_captions/cureton2010length.json new file mode 100644 index 0000000000000000000000000000000000000000..c041cd52b83e3eb8cbc80bbf44508562e062c58d --- /dev/null +++ b/test_extracted_captions/cureton2010length.json @@ -0,0 +1 @@ +{"CAPTION FIG5.png": "'Figure 5.: **Clathrin structures containing VSV recruit more cortactin that pits that internalize Di-T.** (A) Cortactin recruitment duringcoated pit formation. Left, snapshot showing the surface of a BSC cell transiently expressing cortactin-EGFP (green) and mCherry-LCa (red) at 18 h post-transfection. Time-lapse images were acquired at 3 s intervals, and frame B3 is shown. Middle, split channel lumpfangsuits of coated pit formation in the cell at left. White arrowheads highlight pits in which cortactin recruitment is clearly visible above the local background. Right, example plot of coctactin and clathrin fluorescence intensity over time during the formation of a single clathrin-coated pit in the cell shown at left (Video S5). (B and C) Examples of cortactin recruitment during Di-T (B) and VSV (C) uptake. BSC1 cells transiently expressing mCherry-LCa (red) and cortactin-EGFP (green) were inoculated with Alexa Fluor 647-labeled Di-T or VSV, and images were acquired as in A. Left, split-channel kymograph views of protein and virion (blue) fluorescence intensity over time (Wideos S6, S7). Images in the right-hand panels show a snapshot of the maximal cortactin on clathrin fluorescence, and white arrowheads highlight the peak cortactin signal. Right, plots of the cortactin and clathrin fluorescence intensity over time for each internalization event. (D) Relative peak fluorescence intensity of cortactin in cells co-expressing mCherry-LCa and cortactin-EGFP. At its best-transfection, samples were separately inoculated with Di-T or VSV particles, and images were acquired as in A. For each cell that was imaged, the maximum cortactin fluorescence associated with \\\\(-\\\\)50 pits lacking virus particles (pits, black) and all pits that internalized a DI-T (left, blue, 3 cells) or VSV (right, red, 5 cells) particle was measured. The data are plotted as described in the legend of Figure 3C, and the number of events is shown above each plot. Numerical values and statistical analyses are provided in Table 1.**\\n\\n'", "CAPTION FIG6.png": "'Figure 6: **Actin polymerization is not required for DI-T internalization.** (_k_) The endocytic fate of virus particles after inhibition of actin polymerization. BSC1 cells stably expressing \u03b12-EGFP (green) were treated with 6.3 \u03bcM IntB for 12 min. and inoculated with DI-T and VSV particles in the continued presence of IntB. Time-lapse images of a single cell were acquired at 4 s intervals for 692 s, and an 8.8 \u00d75.0 \u03bcM2 area of the cell surface is shown. The upper panels show the complete internalization of a DI-T particle (blue, blue arrowheads), where +0 s indicates the first frame of the time-lapse series. The lower panels show the subsequent capture but failed internalization of 2 VSV (red, red arrowheads) particles on the same area of cell membrane (time scale continued from above) (Video S8). (_k_) AP-2 fluorescence intensity for the events shown in A. Note that the adaptor fluorescence associated with the DI-T particle (blue) and a conventional coated pit (black) that formed within the same membrane area peak and disappear normally, while the adaptor signal associated with the upper-most VSV particle (red) does not, signifying failed internalization. (_C_) Effect of latB on the efficiency of virus capture and internalization. BSC1 cells stably expressing \u03b12-EGFP were treated and imaged as described in A. Left, the percentage of virus particles that were captured by a clathrin structure after attachment. Right, the percentage of captured virus particles that were successfully internalized within 300 s of capture (see D. for details). Cumulative data are from 5 cells. (_D_) Effect of latB on the lifetime and peak fluorescence intensity of AP-2 in clathrin structures. Data were acquired as described in A. and displayed as in the legend of Figure 3C. Left, relative lifetime of AP-2 in structures that lack (pits, black) or capture a virus particle. Inset shows a rescaled distribution of the pit and DI-T internalization events. Right, maximum fluorescence intensity of AP-2 in the events at left. Data are from 4 of the 5 cells analyzed in C, as thermal drift during imaging prevented accurate fluorescence intensity measurements in one cell. The number of events in each category is shown above the corresponding plots at right. DI-T (blue) data consists only of productive internalization events. VSV events are categorized as productive internalizations (VSV entry, red) or non-productive captures (trapped VSV, red). A non-productive capture is defined as a stable colocalization between a spot of AP-2 and a VSV particle that began at least 300 s before the last captured image and did not result in virus uptake before cessation of imaging. The 300 s cutoff was chosen because a majority (22/24) of productive internalizations occurred within 300 s of capture. Captures in which the final image frame was acquired before 300 s elapsed were excluded from the analysis, as the eventual endocytic fate of the particle cannot be predicted.\\n\\n'", "CAPTION TAB1-1.png": "'\\n\\n**Table 1.** Summary of kinetic and fluorescence intensity data'", "CAPTION TAB2-1.png": "\"Kinetic and fluorescence values are provided as the main +/- SD for all events in a given context. The number of experiments and cells analyzed is indicated. The absolute lifetime are expressed in seconds (d). The % lifetime and % maximum fluorescence intensity values were calculated as described in the legend of Figure 3C. The % maximum fluorescence intensity of correlation is provided for events analyzed in cells co-expressing mCherry-1Ca and correlation-6FP. A two-tailed Student's +-test was used to determine whether data from 2 categories differ in a statistically significantly manner.\\n\\n(f) denotes a statistical difference with a p-value <0.005 and >0.00005 when comparing data in a given category to data for pick lacking virus in the same category. (g) similarly denotes a p-value <0.00005. All values for _V_SD are significantly different (p<0.005 for % max AP-2 fluorescence; p<0.00005 for all other categories) from those measured for DT-T particles in the same context. ND, not determined.\\n\\ndoi:10.1371/journal.ppat.1001127.1001\"", "CAPTION FIG7.png": "'\\n\\n**Figure 7.** **Model of DI-T and VSV entry.** DI-T (above] and VSV (below) particles engage host cells through interactions between the viral surface glycoproteins and unknown cellular receptor moieties. Following attachment, both particle types undergo slow diffusion diffusion coefficient \\\\(-5\\\\times 10^{-11}\\\\) cm\\\\({}^{2}\\\\)\\\\(\\\\varepsilon^{-1}\\\\); an the cell surface for an average of \\\\(-2\\\\) min before being captured by a clathrin-coated pit. For DI-T, continued clathrin assembly drives complete particle envelopment by the plasma membrane and leads to virus endocytosis. In contrast, the presence of a VSV particle in a coated pit physically prevents complete membrane constriction by the clathrin machinery and causes clathrin assembly to halt prematurely. The force provided by actin polymerization then further remodels the plasma membrane and thereby encloses the virus particle in a partially clathrin-coated vesicle. doi:10.1371/journal.ppa.1001127.g007'", "CAPTION FIG2-1.png": "'\\n\\n**Figure 2.** **Clathrin structures capture VSV and Di-T particles with similar kinetics.** (A) Schematic of clathrin-dependent virus internalization. It. A particle (blue) attaches to receptor moieties: (orange) on the cell surface (black horizontal line), and the virus-receptor complex diffuses in the plane of the membrane. 2. The virus particle is captured by the clathrin endocytic machinery (AP-2, green).\\n\\n'", "CAPTION FIG2-2.png": "\"clathrin, red) after diffusion into an existing clathrin structure (e.g. Genague virus) or entrapment within a clathrin structure that initiates in the close proximity to the virion (e.g. VSW and influenza A virus). 3. Clathrin assembly completes, and the virus-containing pit is screwed from the cell surface in a dynamic-dependent process. Internalization of VSW also requires local actin assembly. Clathrin is rapidly removed from the nascent vesicle, and the vesicle is actively transported further into cell. (B) Example of a complete DI-T internalization event. A single DI-T particle (red) attaches to a BSC1 cell expressing c2-eGFP (green) and diffuses on the cell surface. A dim spot of AP-2 appears beneath the virion, signifying capture of the particle. The AP-2 fluorescence intensity increases as the clathrin coat assembles, and the virus disappears into the cell shortly after the AP-2 signal reaches a maximum (Video S1). Munbered stages correspond to the events described in \\\\(A\\\\). The path of particle motion is depicted as a linear, color-coded trace that progresses with time from blue to red. (C) VSW and DI-T particle capture by clathrin structures in the same cell. BSC1 cells stably expressing c2-eGFP (green) were inoculated with Alexa Fluor 647-labeled DI-T (blue, blue arrowheads) and Alexa Fluor 568-labeled VSW (red, red arrowheads). Time-lapse images were acquired at 4 s intervals using a spinning disk confocal microscope. Left, snapshot of a cell depicting coated pits lacking (white arrowheads) or containing (blue/red arrowheads) virus particles. Right, expanded split-channel views of the region within the dashed box at left. (D) Kinetics of virus capture. BSC1 cells stably expressing c2-eGFP (_T_ cells) or eGFP-LCa (12 cells) were inoculated with VSW and DI-T particles. Images were acquired at 3-4 s intervals as in C, and the time interval between virus attachment and detection of AP-2 or LCa boresath a virion was quantified for productive internalization events. The distribution of capture times is shown for DI-T (blue) and VSW (red) particles, and the mean time to capture \\\\(\\\\langle + \\\\)/\\\\(-\\\\) SD/ for each particle population is provided at right. The kinetics of VSW and DI-T capture are not significantly different (Student's t-test p value = 0.3).\\n\\n\"", "CAPTION FIG1.png": "\"Figure 1.: **Biological properties of DI-T particles.** (A) Structure of VSV and DI-T genomic RNAs. The single-stranded negative-sense RNA genomes are shown in a 3'-5\u2032 orientation. The five viral genes are colored as follows: red nucleocapsid (M) protein gene; orange, phosphoprotein (P) gene; yellow, matrix (M) protein gene; green, glycoprotein (G) gene; blue, large (L) polymerase protein gene. The noncoding genomic terminal leader (Le) and trailer (Tr) regions, which serve as promoters for RNA synthesis and genomic RNA encapsidation, are shown in gray. The DI-T genome comprises 2,208 nts. The 5\u2032 terminal 2,163 nts derive from the 5\u2032 terminus of the parental VSV genome (11,161 nts), and the 3\u2032terminus contains a 45 nts inverted complement of the wild-type Tr (TrC). (B) Electron micrographs of purified VSV and DI-T particles negatively stained with PTA. Middle panel, inset shows an expanded view of virions from the boxed region to facilitate visualization of the viral glycoprotein spikes. Inset scale bar, 100 nm. (C) Vision geometry. The dimensions of individual DI-T (blue) and VSV (red) particles measured from electron micrographs of negatively stained particles. Each open circle represents the measurement for a single particle. Horizontal red lines denote the mean value of each population, and the numerical means (+/- SD) are provided above each plot. (D) Protein composition of purified VSV and DI-T particles. Viral proteins (L, G, P, N, and M) were separated by SDS-PAGE and visualized with Coomassie blue staining. The ratio of N protein to G protein was quantified using ImageJ and is displayed below the gel as a comparative measure of average virion surface glycoprotein density in each particle population. (E) Fluorescence intensity of virus particles labeled with Alexa Fluor dye molecules. Purified DI-T or VSV particles were labeled with Alexa Fluor 647 or 568 and imaged on separate glass coverslips using a spinning disk confocal microscope. The fluorescence intensity of individual spots in a single field of view was quantified, and the distribution of intensity values (in arbitrary units, a.u.) for DI-T (blue) and VSV (red) particles is shown.\\n\\n\"", "CAPTION FIG3.png": "'Figure 3: **Cells internalize Di-T particles using conventional clathrin-coated vesicles.** (A) internalization of VSV and Di-T by the same cell. BSC1 cells stably expressing \\\\(\\\\sigma\\\\)2-eGFP (green) were inoculated with VSV (red) and Di-T (blue) particles, and confocal images were captured at 4 s intervals. The images show the sequential internalization of single VSV (red, red arrowheads) and Di-T (blue, blue arrowheads) particles, followed by the formation of a canonical coated vesicle (white arrowhead) within a 3.5\\\\(\\\\times\\\\)3.5 \u03bcm\\\\({}^{2}\\\\) area of the plasma membrane (Video S2). The first acquired frame of the time-lapse series is designated +0 s, and the capture time of the subsequent images is shown. (B) AP-2 recruitment during the uptake events shown in A. Left, image quadrants depicting AP-2 accumulation over time (left panels) and at the time of maximum AP-2 signal during each event (right panels). Upper panels in each quadrant show the AP-2 channel alone, and the lower panels show overlays of the virus and AP-2 channels. The highlighted pit from A. is indicated with a white arrowhead, and virus particles are colored as in A. Right, fluorescence intensity (in arbitrary units, a.u.t) of AP-2 over time for the events shown at left and in A. The time of AP-2 detection above the local background was set to +-0 s for each event. (C) Kinetics and AP-2 content of endocytic structures. The plots show the relative lifetime (left) and maximum fluorescence intensity (right) of AP-2 during the uptake of coated pits lacking virus (pits, black) or structures that internalized Di-T (blue) or VSV (red) particles (from 4 cells). Values are expressed as percentages to facilitate comparison of viral and nonviral uptake events across multiple cells. Approximately 50 pits lacking virus were analyzed in each cell, and the mean of the measured values was calculated for each parameter. The values for each nonviral and viral uptake event were divided by the mean for pits lacking virus in the same cell, and the resulting values were multiplied by 100. Each open circle represents a single uptake event, and horizontal red lines demark the mean of the compiled population. The number of events is provided above each plot. Numerical values and statistical analyses are provided in Table 1. (D) Clathrin recruitment during virus entry. Left, kymograph views of internalization events from a single BSC1 cell transiently expressing GFP-LCa (Videos 53, 54). Images were captured as in A. and displayed as described in R. Right, fluorescence intensity of eGFP-LCa over time for the events shown at left. (E) Kinetics and clathrin content of endocytic structures. The plots show the relative lifetime (left) and maximum fluorescence intensity (right) of clathrin during the uptake of coated pits lacking virus (pits, black) or structures that internalized Di-T (blue) or VSV (red) particles (from 3 cells). Data were calculated and plotted as described in C. Numerical values and statistical analyses are provided in Table 1.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Figure 4. Electron microscopic images of DI-T and VSV particles in clathrin-coated pits.** (A) Electron micrographs depicting DI-T particles at early (left) or late (right) stages of clathrin-dependent endocytosis. BSC1 cells were incubated with \\\\(-1000\\\\) particles per cell for 10 min. at 37\\\\({}^{\\\\circ}\\\\)C. Cells were then fixed, and samples were processed for ultra thin sectioning and viewed as described in the materials and methods. (B) Electron micrographs of VSV particles at sequential (left to right, top to bottom) stages of clathrin-dependent endocytosis. VSV cells were inoculated with VSV at an _W_OI of \\\\(\\\\xi\\\\), and samples were prepared for analysis at 6 h post-infection.\\n\\ndoi:10.1371/journal.pga.1001127.g004.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1.** Summary of kinetic and fluorescence intensity data.} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Summary of kinetic and fluorescence intensity data.\\n\\n'", "CAPTION TAB2.png": "'* [18] B.\\n\\n'", "CAPTION FIG2.png": "'Figure 2.: **Cistarius structures capture VSV and D-T particles with similar kinetics.** (U) Schematic of clarity-dependent virus internalization. 1. A particle label attaches to receptor moieties (orange) on the cell surface block horizontal line, and the virus-receptor complex diffuse in the plane of the membrane. 2. The virus particle is captured by the clathrin endocytic machinery (AP-2, green, clathrin, red) after diffusion into an existing clathrin structure to-go. Danger twist of entrapment within a clathrin structure that maintains a force primarily to the vitan tip, 95% and membrane A. 3. Glutrich assembly completes, and the virus-containing tip is screened from the cell surface in a dynamic dependent process. Intercalation of VSV also requires local actin assembly. Glutrich is rapidly removed from the nascent vesicle, and the vesicle is actively perturbed further into cells. (II) Example of a complete D-T internalization event. A single D-T particle (and attach) is a 18-cell experiment 20-24-(95%) (green) and 60% on the cell surface. A final spot of 40-24\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/day2015microtubule.json b/test_extracted_captions/day2015microtubule.json new file mode 100644 index 0000000000000000000000000000000000000000..9db428bf5639515e97c4b13c008b0505a4c1d4de --- /dev/null +++ b/test_extracted_captions/day2015microtubule.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "'Figure 4: **An intact microtubule network is required for the formation of tubular invaginations.** A and C) Microtubule disruption with high dose nocodazole prevents the formation of tubular invaginations in ATP-depleted cells (mean \u00b1 SD, \\\\(N\\\\) = 74 cells,) \\\\({}^{**}p<0.01\\\\), chi-squared test. B and D) Inhibition of microtubule dynamics with low dose nocodazole has no effect on the formation of tubular invaginations in ATP-depleted cells (mean \u00b1 SD, \\\\(N\\\\) = 135\\\\(-\\\\)169 cells), _n.s._\\\\(p>0.05\\\\), chi-squared test. E and F) Microtubule disruption prior to Dynasore treatment blocks the formation of branched tubules in cells labeled with either wild type CTxB (E) or monovalent CTx (F). Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3: Tubular invaginations align along microtubules and undergo complex motions including bidirectional motility and branching events.** A) Microtubules persist in RFP-\\\\(\\\\alpha\\\\)-tubulin expressing HeLa cells following ATP depletion. B) CTxB positive invaginations (green) align with taxol-stabilized microtubules (red) in stably expressing RFP-\\\\(\\\\alpha\\\\)-tubulin HeLa cells under ATP depletion. C) Percentage of CTxB-positive tubules that align with microtubules (black), partially align with microtubules (gray), or do not colocalize with microtubules (light gray) in ATP-depleted HeLa cells expressing RFP-\\\\(\\\\alpha\\\\)-tubulin. \\\\(N\\\\) = 84 cells. D\\\\(-\\\\)J) CTxB-enriched tubules exhibit complex motions in live cells as illustrated by representative frames from time series and corresponding kymographs. Time stamps are in minutes:seconds. D\\\\(-\\\\)F) CTxB-positive invaginations often grow in fluid directed motions. G\\\\(-\\\\)I) CTxB-positive invaginations also extend, retract and regrow along the same axis. _J_) Occasionally, the CTxB positive tubules undergo branching events. Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: The ATPase activity of dynein and an intact dynactin complex are required for the formation of tubular invaginations. A and B) Inhibition of dynein ATPase activity with dilobrevin-A (Cilio-A) significantly reduces the percent of cells displaying tubular invaginations (mean +- SD from 95\\\\(-\\\\)122 cells). \\\\({}^{**}p<0.01\\\\), chi-squared test. C) Expression of GFP-p50 reduced the prevalence of cells with invaginations as compared to untransfected cells (-) or cells expressing EGFP. (mean +- SD from 42\\\\(-\\\\)101 cells). _n.s._, _p_\\\\(>0.05\\\\); \\\\({}^{**}p<0.01\\\\), chi-squared test. D and E) Expression of CCl-dsRed (red) significantly reduces the percent of cells with CTXb positive invaginations (green) as compared to untransfected cells (-) or control cells expressing mCherry (red). (mean +- SD from 40\\\\(-\\\\)64 cells). _n.s._, _p_\\\\(>0.05\\\\); \\\\({}^{**}p<0.01\\\\), chi-squared test. Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG7.png": "\"Figure 7: Bulk endocytosis of CTxB is largely unaffected by disruption of microtubules or the dynactin complex. A and B) Endocytosis of dextran, transferrin and CTxB in cells expressing GFP-p50 (gray bars) compared to cells expressing GFP (black bars). B shows mean +- SD for 37\\\\(-\\\\)278 cells. _n.s._, \\\\(p\\\\) > 0.05, Student's t-test. C and D) Effect of microtubule disruption with 5 mg/mL nocodazole (gray bars) on the uptake of dextran, transferrin and CTxB. Control cells were treated with DMSO (black bars). D shows mean +- SD for 78\\\\(-\\\\)358 cells. _n.s._, \\\\(p\\\\) > 0.05, *, \\\\(p\\\\) < 0.05, Student's t-test. Bars, 10 \u03bcm. E) Representative time course of toxin-induced chloride secretion in T84 cells in response to treatment with 20 nM wt CTx. Cells were either pretreated with NZ (closed symbols) on the DMSO (open symbols) prior to toxin addition to either the apical (blue) or basolateral (red) surface at \\\\(t\\\\) = 30 min as described in the _Materials and Methods_. Forskolin was added to control monolayers (green or gray diamonds) at 90 min in order to demonstrate equivalency of the secretory response and monolayer viability. The error bars indicate the variance calculated as the standard deviation (_n_ = 3). Data are representative of the results of two independent experiments.\\n\\n\"", "CAPTION FIG5.png": "'\\n\\n**Figure 5: Motor-based motions persist in ATP-depleted cells.** A\u2013C) A subset of lysosomes labeled with mCherry-LAMP1 display long range directed motions in ATP-depleted cells. B) Time lapse of zoomed in region of the cell in panel A. An example of a lysosome undergoing long-range directed motion is marked with the arrowhead. C) Tracings of lysosome movement in the cell shown in A. Each track is indicated by a different color. D and E) Frequent long-range directed motions are observed under control conditions. F and G) mCherry-LAMP1 positive lysosomes are immobile in PFA fixed cells. Elapsed time in F, H and J = 470 s. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG7-1.png": "'Figure 7: Learned on next page.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Working model microtubules, dynactin and cytoplasmic dynein facilitate plasma membrane tubulation. Dynein and dynactin provide attachment sites or generate tugging forces on the plasma membrane, leading to microtubule-dependent tubulation. Glycolipid-binding toxins may further sense, induce or stabilize membrane curvature, enabling their efficient sorting into tubular structures.\\n\\n'", "CAPTION FIG1-2.png": "'Figure 1: **No more than one functional GM1 binding site is required to target cholera toxin to plasma membrane invaginations.** A\\\\(-\\\\)E) CTxB accumulates in either linear extended tubules (A\\\\(-\\\\)D) or branched tubules (E) under conditions that block scission. Bar, 10 \\\\(\\\\mu\\\\)m. (F\\\\(-\\\\)M) Cholera toxin binding mutants accumulate in tubular invaginations. F) Cy3-CTx chimera labels tubules in ATP depleted COS-7 cells. G\\\\(-\\\\)I) Quantification of invaginations in ATP-depleted cells labeled with Cy3-CTx chimera or Alexa555-CTxB. G) Percentage of cells displaying invaginations (mean \\\\(\\\\pm\\\\) SD from 117\\\\(-\\\\)119 cells). *, \\\\(p\\\\) < 0.05, chi-squared test. H) Average number of invaginations per cell (mean \\\\(\\\\pm\\\\) SD of 42\\\\(-\\\\)46 cells). _n.s._, \\\\(p\\\\) > 0.05; Student _t_-test. I) Length of invaginations (mean \\\\(\\\\pm\\\\) SD for 219\\\\(-\\\\)332 invaginations). _n.s._, \\\\(p\\\\) > 0.05; Student _t_-test. J\\\\(-\\\\)M) Both wild type CTxB and monovalent CTx accumulate in tubular invaginations in cells subjected to lasplakinolide pretreatment prior to ATP depletion. L) Percentage of cells displaying invaginations. (mean \\\\(\\\\pm\\\\) SD of 59\\\\(-\\\\)63 cells). _n.s._, \\\\(p\\\\) > 0.05; chi-squared test. M) Average number of invaginations per cell. (mean \\\\(\\\\pm\\\\) SD of 59\\\\(-\\\\)63 cells). _n.s._, \\\\(p\\\\) > 0.05; Student _t_-test. N and O) Similar to wild type CTxB, monovalent CTx accumulates in branched tubules in Dynasore-treated cells. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Legend on next page. Figure 1: **No more than one functional GM, binding site is required to target cholera toxin to plasma membrane invaginations.** A\u2013E) CTB accumulates in either linear extended tubules (A\u2013D) or branched tubules (E) under conditions that block incision. Bar, 10 \u03bcm. (F\u2013M) Cholera toxin binding mutants accumulate in tubular invaginations. F) Cy3-CTx chimera labels tubules in ATP depleted C05-7 cells. G\u2013D Quantitation of invaginations in AH-depleted cells labeled with Cy3-CTx chimera or Alexa55-CTB. G) Percentage of cells displaying fragments (mean \u00b150 from 117\u2013119 cells). \\\\(y\\\\) = 0.05, cis-squared test. H) Average number of invaginations per cell (mean \u00b150 of 42\u201346 cells). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. [1] length of invaginations (mean \u00b150 for 219\u2013332 imagination). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. [1\u2013M] Both wild type CTB and monovalent CTx accumulate in tubular invaginations in cells subjected to laqxihizide pretreatment prior to ATP depletion. L) Percentage of cells displaying invaginations. (mean \u00b150 of 59\u201363 cells). _n.s._, \\\\(p\\\\) > 0.05; chi-squared test. H) Average number of invaginations per cell. (mean \u00b150 of 59\u201363 cells). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. H) Average number of invaginations per cell. (mean \u00b150 of 49\u201363 cells). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. [1\u2013M] Both wild type CTB and monovalent CTx accumulate in branched tubules in DNase-treated cells. Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: Lateral on next page.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Toxin binding is not necessary for tubular invaginations to form.** A,B) EGFP-HRas (green) is found in plasma membrane invaginations in ATP-depleted cells in both the presence (A) and absence (B) of Alexa555-CTxB (red). C\u2013F) Similar results were obtained for GFP-HRas in cells subjected to actin disruption (C and D) or actin stabilization (E and F). G and H) A construct containing only the C-terminal 10 amino acids of HRas, EGFP-HRas-tail (green), also localized to tubules in both the presence and absence of CTxB. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/day2015microtubule_supplement1.json b/test_extracted_captions/day2015microtubule_supplement1.json new file mode 100644 index 0000000000000000000000000000000000000000..73a855ebd63bb09e450b64134136ddb96f209d97 --- /dev/null +++ b/test_extracted_captions/day2015microtubule_supplement1.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS1.png": "\"\\n\\n**Supplemental Figure 1. (Related to Figure 1) This figure shows a series of control experiments that characterize the properties of the tubular invaginations generated in response to ATP depletion. It also documents the efficacy of the ATP depletion treatment.**\\n\\n**(A)** Alexa488-STxB and Alexa488-CTxB accumulate in tubular invaginations in ATP depleted COS-7 cells. To facilitate STxB labeling, cells were transfected with Gb3 synthase. **(B, C)** As reported previously (78), fluorescent CTxB labels both plasma membrane invaginations (arrowheads) and plasma membrane protrusions (arrows) in ATP depleted cells. B and C are single frames from a confocal z-stack. Dashes mark the position of the xz-sections shown below. **(D)** Alexa488-STxB and Alexa555-CTxB colocalize in invaginations in ATP depleted COS-7 cells expressing Gb3 synthase. **(E)** Tubules prelabeled with Alexa555-CTxB filled at approximately the same rate as newly added Alexa488-CTxB accumulates at the plasma membrane, indicating that the tubules are open to the fluid phase in ATP depleted cells. Time stamps are in minutes:seconds. **(F-H)** ATP depletion inhibits uptake of dextran and internalization of transferrin (Tfn) (Mean +- SD, N = 325-417 cells). ***, p < 0.001, Student's t-test. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n\""} \ No newline at end of file diff --git a/test_extracted_captions/day2015microtubule_supplement3.json b/test_extracted_captions/day2015microtubule_supplement3.json new file mode 100644 index 0000000000000000000000000000000000000000..fd331c190b2314f76d582db7ce48c79f7a8613d7 --- /dev/null +++ b/test_extracted_captions/day2015microtubule_supplement3.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS3.png": "'Supplemental Figure 3.\\n\\n**Supplementary Figure 3. (Related to Figures 4 and 6) This figure provides additional evidence that intact microtubules and an intact dynactin complex are required for tubule elongation. It also shows the results of control experiments determining the concentration of ciliobrevin A required to inhibit delivery of CTxB to perinuclear compartments.**\\n\\n**(A, B) Pretreatment of cells with 16.6 M nocodazole prior to LatA (A) or Jasplakinolide treatment (B) blocks tubule formation. (C, D) Pretreatment with ciliobrevin A (Cilio-A) disrupts delivery of CTxB to perinuclear compartments in ATP replete cells in a dose-dependent manner. n = 36-108 cells; error bars = SD. (E, F) Expression of GFP-p50 (E), but not GFP alone (F) blocked the formation of long branched tubules in Dynasore-treated cells. Similar results were obtained for both wild type CTxB or monovalent CTx. Bars, 10 m.**'"} \ No newline at end of file diff --git a/test_extracted_captions/debelly2023cell.json b/test_extracted_captions/debelly2023cell.json new file mode 100644 index 0000000000000000000000000000000000000000..286d4fa3d137066e081bf0297984c82e775dd992 --- /dev/null +++ b/test_extracted_captions/debelly2023cell.json @@ -0,0 +1 @@ +{"CAPTION FIG3-2.png": "'(3) Relative force changes (\\\\(\\\\beta\\\\) axis) for membrane tension monitored on the static tether as a function of the extending tether length (\\\\(\\\\nu\\\\) axis) upon continuous pulling. In the case of bibels or cells with heavily disassembled actin cortex (light and dark green), the tension on static tether increases as the extending tether lengthens; the however, there are no perceptible tension changes on the static tether tension from the cell body (pink, intact central) even when the other tether has extended by more than 60 \\\\(\\\\mu\\\\)m \\\\(\\\\left\\\\{ \\\\text{h} > 14,\\\\text{h} > 3\\\\right\\\\}\\\\). Graphical data represent means \\\\(\\\\approx\\\\) SDs. See also Figure S3 and Video S2.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Actin-driven protrusions stimulate global, nearly undampened membrane tension propagation (A) A dual-tether pulling assay to simultaneously monitor membrane tension on the far end (left, trap 1 at 180\u00b0) and on the side of the cell (top, trap 2 at 90\u00b0) during light-activated protrusion. (B) Representative time traces of dual trap forces over successive cycles of light-activated protrusion show coinciding tension increases on both membrane features adjacent to (trap 2) and at the opposite cell surface from (trap 1) protrusion; light: 90 s on (shaded area), 180 s off. (C) Correlation between trap forces at the two tether positions during activation (blue) remains rocky transit from first activation cycle to the next; for comparison, minimal correlation is seen between the two tethers before osteogenic activation (gray). Dashed line: Inner progression. (D) (L-left) Time delay measured between tension rise on membrane tethers adjacent to (trap 2 at 90\u00b0, black) and opposite from trap 1 at 180\u00b0, red) cell protrusion. (Right) In most cells, the traps detect membrane tension increase on both tethers within a second or less of one another, indicating a rapid propagation of tension across the cell. (E) Averaged traces of dual trap forces before, during (light), and after activation (yreams \\\\(\\\\pm\\\\) SD; \\\\(n\\\\) > 25, N = 4). (F) Pearson correlation coefficient between dual trap forces measured at steady state, during light activation, and recovery afterward (70 s post light). Error bar: means \\\\(\\\\pm\\\\) SD; p values from Welch\u2019s unpaired Student\u2019s test in \\\\(>\\\\) 10, N \\\\(\\\\times\\\\) 4). See also Figure S2 and Video S2.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Long-range tension propagation is accompanied by directed membrane and actin flows toward the protrusion (A) Confocal images of opta-PI3K ceils expressing membrane marker (CAX-HaIaTaj): before and during light-activated protrusion. Scale bars: 5 \u03bcm. (B) Kymographs of membrane fluorescence along the normalized cell circumference (a axis) show that over time (x axis) membrane accumulates toward the protruding cell front and is depleted from the back (n > 50, N = 6; Figure S4; see STAR Methods). (C) Flow of membrane and actin during protrusion are calculated assuming optimal transport (see STAR Methods). (D) Membrane flow field referred using optimal transport from kymograph intensity changes over time: shortly after activation begins (f = 70 s, dark text traces), the magnitude of membrane flow speed increases (red dashed arrows), with positive speed for clockwise flow along the cell upper hat and negative speed for counter-clockwise flow along the bottom hat (G), all moving toward the cell protruding front (x). During recovery (f = 170 s, light green traces), the direction of membrane flow reverses (black dashed arrows). (E) Membrane flow around the cell before, during, and after (f = 30, 70, and 170 s) right-side protrusion; the flow magnitude is denoted by the arrow size (red; forward flow, blue; backward). Membrane flows toward the protrusion in the protruding phase and away from the protrusion during the recovery phase. (F) Attracting membrane diffusion assay in which we bleach the membrane marker CellMask across a wide section of the cell (sparing a small section of the membrane mask), opto-activate a portion of the cell angled 90\u00b0 from the unbleached area (or use no light as control), and monitor the diffusion pattern of the unbleached area over time. (G) (Top) Example kymograph of unbiased diffusion in a control cell (no activating light). (Bottom) Same as top but in a protruding cell, showing biased diffusion and bulk flow of the unbleached membrane signal toward the protrusion. Heatmap similar as in (B). (H) Sample fits of individual timepoints of kymograph data (points colored by respective time points) with a gaussian equation (thick curves, colored by respective time points). Shifts in the means of the gaussian fits, quantified bulk membrane flow, are shown as vertical lines (colored by respective timepoints). (I) Quantification of mean shifts fit by linear regression to assay membrane flow rate in control cells (gray, no apparent flow, u = 3.34 nm/x) and protruding cells (red, biased flow toward side of protrusion, u = 35.51 nm/x) (N = 3, n = 3). See also Figure S4 and Video S3.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: Membrane tension does not propagate upon direct mechanical pulling on the cell membrane (A) A dual-iether assay to detect tension propagation (static tether, left) while a nearby force is exerted through the use of an optically trapped bead to pull on the membrane -2-\\\\(\\\\mu\\\\)m away (moving tether, right).\\n\\n(B) An example time trace of trap force for dual membrane tension measurements, in which one moving trap (T2, gray) dynamicaly pulls on the cell membrane by continuously pulling and extending the membrane tether, whereas the other trap controls a second static membrane tether (T1, black) to monitor nearby changes in membrane tension. The increase in the length of the actadge tether from the cell body is plotted in gray along the right y axis.\\n\\n(C) Correlation plots of normalized trap forces between the moving and static tethers. Five representative measurements from different cells are shown: dashed lines: linear regression.\\n\\n(D-F) Similar to (A\u2013C), but probing tension in bibels (membrane detached from actin cortex generated by using altrunculin B treatment to weaken the actin cortex), a high correlation is observed between static and moving tethers.\\n\\n(A\u2013B) Similar to (A\u2013C), but probing tension in cells where the actin cortex has been significantly disassembled using a combination of altrunculin B treatment and osmotic shock; a high correlation is observed between static and moving tethers even at a significant distance from one another (here, 90\\\\({}^{\\\\circ}\\\\), but in Figures S3H-S31, 180\\\\({}^{\\\\circ}\\\\)).\\n\\n(J) Pearson correlation coefficient between dual trap forces measured before perturbations (none; light gray), upon light-activated protrusions (purple; Figure 2), during cell membrane pulling (pink; A\u2013C), during membrane pulling on a bleb (light green; D\u2013F), and during cell membrane pulling in cells with nearly ds-assembled actin cortex (dark green; G\u2013I). Error bar: means +- SD; p values from Welch\u2019s unpaired Student\u2019s t test (\\\\(\\\\eta\\\\sim 15\\\\), N \\\\(>3\\\\)).\\n\\n'", "CAPTION FIG5.png": "'Figure 5.\u2014 **Optogenetically induced actomyosin contractions generate rapid long-range membrane tension propagation and membrane flows (A) Optogenetic approach for light-induced activation of isulamia-associated Rho guanine nucleotide exchange factor (LARG), resinEng in Rho GTPase activation to initiate actomyosin-driven cell contraction (see STAR Methods).**\\n\\n(B) Time-lapse confocal images of a neutrophil-like HL-60 cell expressing opto-corastruct (Opto-LARG) and membrane marker (CellMask), showing localizad membrane contraction and cell flattening upon light activation.\\n\\n(C) After light-activated contraction on one side of the cell (top), changes in membrane tension on the opposite side (bottom) are measured via a membrane tether held by an optical trap.\\n\\n(D) Aupward image trace of trap force before (steady state), during (light), and after activating cell contraction (means = SD; n > 55, N = 7).\\n\\n(E) Aupward trap force before (steady state) and during actuation. Box and whiskers: median and min to max; p values from Wilcoxon paired Student\u2019s t test.\\n\\n(F) A dual-tether pulling assay to simultaneously monitor membrane tension on the far end (left, trap 1 at 180\\\\({}^{\\\\circ}\\\\)) and on the side of the cell (top, trap 2 at 90\\\\({}^{\\\\circ}\\\\)) during light-activated contraction.\\n\\n(G) Average traces of dual trap forces before, during (left), and after activation showing coinciding tension increases on both membrane tethers adjacent to (trap 2) and at the opposite cell surface from (trap 1) contraction (means = SD; n = 25, N = 4).\\n\\n(H) Pearson correlation coefficient between dual trap forces measured at steady state and during light activation. Error bar: means = SD; p values from Welch\u2019s unpaired Student\u2019s t test (n > 20, N > 4).\\n\\n(I) Confocal images of opto-LARG cells stained with membrane marker (CellMask) before and during light-activated contraction.\\n\\n(J) Kipographs of membrane fluorescence along the normalized cell circumference (_y_ axis) show that over time (_x_ axis) membrane accumulates toward the contracting cell front and is depleted from the back (_n_ = 40, N = 3; see STAR Methods).\\n\\n(B) Membrane flow field infrared using optimal transport from lymphograph intensity changes over time: shortly after activation begins (_t_ = 120 s, basal traces), the magnitude of membrane flow speed increases (red dashed arrows), with positive speed for clockwise flow along the cell upper half and negative speed for counter-clockwise flow along the bottom half, at moving toward the site of cell contraction (_n_). During recovery (_t_ = 200 s, light green traces), the direction of membrane flow reverses (blue dashed arrows).\\n\\n(L) Membrane flow around the cell before, during, and after (_t_ = 30, 120, and 240 s) right-side contraction; the flow magnitude is denoted by the arrow size (red forward flow, blue-backward). Membrane flows toward the contraction in the contracting phase and away from the contraction during the recovery phase. Scale bars: 5 \u03bcm. See also Figure SS and Video S4.\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIGS6.png": "'Figure S6. Mechanical perturbations applied on both membrane and cortex lead to rapid tension propagation across the cell, related to Figure 6\\n\\n(A) Tether pulling assay in which tethers are pulled at constant speed until they break. Maximum tether length is used as a proxy for local membrane reservoirs [16]. (B) Maximum tether length comparison of 3T3s fibroblasts versus H-6ths cells. In red are cells for which the maximum pulling length was reached on our setup, without tether breaking occurring, suggested high local membrane reservoir availability. Error bar: means +- SD; \\\\(n\\\\) > 15, N > 3. (C) Average trap force of different opto-cells (OptoT1M-based portation induction and OptoT4G-based actomyosin contractility), before and after light in the absence or presence of the Earn inhibit MSC668394 (25 mM). These data show that lowering MCA or alkyl sticky attractive membrane tension increase in protruding cells but severely impedes membrane tension increases in recontracting cells. Error bar: means +- SD; \\\\(n\\\\) values from Welch\u2019s unpaired Student\u2019s t test (n > 25, N > 30, (D) A dual-stufher pulling assay to simultaneously monitor membrane tension on the far end (bottom, trap 1 at 180-) and on the side of the cell (right, trap 2 at 30-) during micropipette aspiration (top, ~4-5 mm in tip diameter), which mechanically pulls on both the membrane and actin cortex underneath. (E) Representative time traces of dual trap forces over successive cycles of aspiration (shaded area) and relaxation; the magnitude of aspiration progressively increased in the last two cycles (+ and +; the first three cycles were also shown in Figure S6, The nearby superimposable tension rise and tail on the two membrane tethers show that membrane tension propagates rapidly across the cell upon mechanical perturbations exerted to both the cortex and membrane. Note that the profiles of tension rise upon aspiration and of tension drop during relaxation resemble those observed with light-activated actin-driven protrusions (Figure 2B). (F) Zoom-in on the first aspiration event shows that the trap force for membrane tension on the tether closer (pink) to the aspiration site started increasing slightly earlier and ended up slightly higher compared with that measured on the tether opposite from the aspiration (purple). (G) An example trace of tether tension response monitored on the opposite side of micropipette aspiration (trap 1 at 180-). Here, the recording lasted for six rounds of aspiration and relaxation. (H) Another example of dual-stufher membrane tension measurement upon micropipette aspiration; the tether in trap 2 broke (?) shortly after the aspiration stopped. (I) An example time trace of trap force for cell membrane tension exhibits robust responses over three aspiration cycles using a micropipette of slightly smaller diameter (~2 mm). (J) (Latin) Time delay measured between tension rise on membrane tethers adjacent to (trap 2 at 90-, pink) and opposite from (trap 1 at 180-, purple) cell aspiration using micropipettes. (Right) In most cells, the traps detect membrane tension increase on both tethers within a second or less of one another, indicating a rapid propagation of tension across the cell. (D) Pearson correlation coefficient between dual trap forces measured before any perturbations (steady state) and during mechanical pulling upon micropipette aspiration. Error bar: means +- SD; \\\\(n\\\\) values from Welch\u2019s unpaired Student\u2019s t test (n > 15, N > 3). (L) Correlation plots of normalized trap forces between the two tethers during micropipette aspiration. Five representative measurements from different cells are shown; dashed lines: linear regression. (M) Comparing membrane flows of light-induced protrusions at the mid and ventral plane of the cell. (N) Confocal images at a cell membrane (visualized using CAXX-HialTag) before and during protrusion at two different \\\\(z\\\\) planes (mid-section and ventral plane of the cell, Scale bar: 5 \u03bcm. (Q) Average zymograph of relative membrane fluorescence intensity along the normalized cell circumference (y axis) at the ventral and mid-plane of the cell over time (x axis) showing a decreased membrane flow at the ventral side of the cell, likely due to friction between the cell and the substrate (n > 30, N = 3). (P) Normalized membrane fluorescence intensity across the blue dotted line in (Q).\\n\\n'", "CAPTION FIGS2.png": "'Figure S2: Membrane tension propagates within seconds across the cell during actin-driven protrusion, related to Figure 2 (A) Rad and blue: averaged time traces of trap force for dual membrane tension measurements before (steady state), during (B)H, and after (recovery) activating cell protrusion. A nearly coinciding tension increase is observed between the membrane tether adjacent to (trap 2, blue) and opposite from (trap 1, red) cell protrusion. Gray: as a control, averaged trace from cells treated with actin inhibitor (10 \u03bcM latrunculin B) shows no membrane tension change upon activation (means \\\\(\\\\pm\\\\) SD; n \\\\(>\\\\) 15, 18 \u03bcM \\\\(\\\\div\\\\) 4).\\n\\n(B) Zcom-in on traces in (A): increases in membrane tension emerging on both tethers within the first 5\u201310 s of light activation.\\n\\n(C) Three example time traces of trap force for dual membrane tension measurements before, during, and after light-induced cell protrusion. At steady state, durations from the two tethers show little correlation, but they become highly correlated upon light activation (purple shaded area). During the recovery phase, we often observe a lag in time between the two tethers\u2019 tension drop, with the tether opposite from the protrusion she recovering more slowly (red).\\n\\n(D) Three example time traces of trap force for dual membrane tension measurements with cells treated with actin inhibitor (10 \u03bcM latrunculin B) before, during (purple shaded area), and after light activation of cell protrusion.\\n\\n'", "CAPTION FIGS4.png": "'Figure S4: Long-range tension propagation coincides with directed membrane flows toward the protrusion, related to Figure 4 (A and B) Apparent membrane thickness is measured based on the width of fluorescence intensity profile across the cell contour, a.g., on the side of cell protrusion (black line). At steady state (pre-activation), the cell membrane contour appears tagged (top image) and thick in width (light green curve in B). **(B)**, **(K)** due to the presence of membrane reservoirs. As the cell protrudes, the membrane intensity outside of the protruding region drops (bottom image) and becomes thinner in width (purple curve in B). **(C)** Kymograph of averaged apparent membrane thickness along the normalized cell circumference (\\\\(y\\\\) axis) over time (\\\\(x\\\\) axis): before, during, and after localized light-activated protrusion (box in white dashed line). Apart from the protruding site, apparent membrane thickness reduces on average throughout the cell, **(K)** reflecting a decrease in membrane reservoirs and a redistribution of extra membranes toward the protrusion site. **(D)** Representative confocal images of an opto-PI3K cell stained with plasma membrane (a) (CellMask) before light activation or during protrusion. **(E)** Kymograph of membrane fluorescence intensity (from cells stained with CellMask) along the normalized cell circumference (\\\\(y\\\\) axis) over time (\\\\(x\\\\) axis): before, during, and after localized light-activated protrusion (box in white dashed line; \\\\(n>25\\\\), \\\\(N=4\\\\)). **(F)** Confocal images of opto-PI3K cells expressing actin marker (actin-HaloTap: before and during light-activated protrusion. **(G)** Kymographs of actin fluorescence along the normalized cell circumference (\\\\(y\\\\) axis) show that over time (\\\\(x\\\\) axis) actin accumulates toward the protruding cell front and is depleted from the back (\\\\(n>30\\\\), \\\\(N=6\\\\); see STAR Methods). **(J)** Left, evolution of the total membrane intensity across the cell contour (means = SD; \\\\(n>30\\\\), \\\\(N=6\\\\)). Except for a small intensity decrease due to the bleaching of the fluorophore, the membrane quantity is conserved. Right, evolution of the total actin density across the cell contour (means = SD; \\\\(n>50\\\\), \\\\(N=6\\\\)). **(B)**.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Mechanical forces acting on the actin cortex drive rapid long-range membrane tension propagation in cells (A) A-3-tier composite model for membrane tension propagation in cells: membrane displacements (_k_) as a readout for tension propagation upon cortical flows (_k_), depend on the membrane elasticity (_k_) and the membrane-cortex friction \\\\(m\\\\) imposed through the adhesive linkers. (B) Model predictions of membrane tension response at moderate membrane-cortex friction (see Methods S1-f) only actin-based puffing leads to tension increase and propagation (red rectangles); external pulfing on the membrane alone is inefficient (blue circles). (C) Predicted membrane tension transmission as a function of membrane-cortex friction (_k_ axis) for different targets of force application: plasma membrane only (blue) and actin cortex only (red). (D) Membrane tension measurements during light-induced protrusions in cells with decreased MCA by using 25 uM of Ezrin inhibitor NSC66394. (E) Red: averaged time trace of trap force before steady state), during (light) and after activating cell protrusion in control cells (same data as Figure 1F). Orange: averaged trace from cells with decreased MCA by using 25 uM of Ezrin inhibitor NSC66394, showing slight defects in membrane tension propagation during light-activated protrusions (_m_ <= <= <= > = >\\n\\n'", "CAPTION FIGS3-1.png": "'Figure S3. Mechanical perturbations affecting only the plasma membrane do not result in measurable membrane tension propagation in cells but do in blebs detached from actin cortex, related to Figure 3 (A). An example time trace of trap force for dual membrane tension measurements, where one moving trap (T2, blue) mechanically perturbs on the cell membrane by continuously pulling and extending the membrane tether, and the other trap remains static (T1, red) to monitor changes and propagation in tension to a nearby membrane tether. The increase in length of the astending tether from the cell body is plotted in gray along the right y axis. \u201c\u201c annotations when the astending tether broke. Note that a sudden tension release upon breakage of the astending tether (blue, at 1~50 s) does not lead to changes in tension on the static tether (red, which is in close proximity to the astending tether (<=2 \u03bcm). This observation shows that mechanical perturbations affecting only the plasma membrane in cells are locally constrained and inadequate to generate measurable tension propagation between the two tethers. (B) Similar operations as (A) but monitoring tension propagation between two membrane tethers on cellular blebs (i.e., a vesicle-like, small section of membrane detached from actin cortex upon intravenous B treatment). The tension readouts between the extending and the static tethers on blebs appear highly correlated, (_\\n\\n'", "CAPTION FIGS5.png": "'Figure S5: Optogenetically induced actomyosin contractions generate rapid long-range membrane tension propagation and actin flows, related to Figure 5 (A and B) Representative time traces of trap force (a direct readout of cell membrane tension charge) during light-induced actomyosin contraction. Revealing robust increase in membrane tension during light-activated contractions on the opposite end of the cell; light: 90 s on (shaded area). (C) Averaged time trace of trap force before (steady state), during (light), and after activating call contraction, measured at the side (90\u00b0) of the contraction (means \u00b1 SD; n > 30, N = 7). (D) Left: Time delay measured between tension rise on membrane tethers adjacent to (trap 2 at 90\u00b0, blue) and opposite from (trap 1 at 180\u00b0, red) call contraction. (Right) In most cells, the traps detect membrane tension increase on both tethers within a second or less of one another, indicating a rapid propagation of tension across the cell. Error bar: means \u00b1 SD. (E) Confocal images of opto-LARG cells stained with actin marker (SPV650-FastAct); before and during light-activated contraction. (F) Average kymograph of relative actin fluorescence intensity along the normalized call circumference (y axis) show that over time (x axis). Actin accumulates toward the contracting call front (_n_ > 25, N = 3; see STAM Methods). (G) Actin flow field traces using optimal transport from kymograph intensity changes over time: shortly after activation begins (_t_ = 120 s, tail traces), the magnitude of membrane flow speed increases (red dashed arrows), with positive speed for clockwise flow along the cell upper half and negative speed for counter-clockwise flow along the bottom half, at moving toward the cell contracting front (_n_). During recovery (_t_ = 230 s, light yellow traces), the direction of membrane flow reverses (blue dashed arrows). (H) Actin flow around the cell before, during, and after (_t_ = 30, 80, and 230 s) right-side contraction; the flow magnitude is denoted by the arrow size (red: forward flow, blue: backward). Membrane flows toward the contraction in the contracting phase and away from the protrusion during the recovery phase. (I) Two examples of actin speckle tracking during light-induced cell contraction. Tracked actin patches are circled in red and their trajectory is represented by lines of different colors. Scale bars: 5 \u03bcm.\\n\\n'", "CAPTION TABNA.png": "'\\n\\n**Acknowledgments**'", "CAPTION FIG1.png": "'Figure 1: Local cell protrusions elicit a sharp increase in membrane tension on the opposite side of the cell within seconds (A) Optogenetic control for light-induced activation of phosphatidylinositol 3-kinase (PI3R) via localized recruitment of inter SH2 domain (ISH2), resulting in Rac GTPase activation that initiates acsin-driven cell protrusions (see STAR Methods).\\n\\n'", "CAPTION FIGS1.png": "'Figure S1: Optogenetic control of PI3K leads to local Rac activation, which triggers localized actin-driven cell protrusion and rapid membrane tension increase, related to Figure 1 (A) Membrane-anchored optogenetic control for light-induced activation of phosphoinositide 3-kinase (PI3K): upon localized 488-nm excitation, the membrane-anchored protein (LiRL-BFP-CAAX) undergoes a conformational change, which results in the binding of inter SH2 domain (SH2) to the illuminated region. ISB2 proceeds to recruit PI3K, whose lipid product (PIP3) induces the activation of Rac GTPase (Rac), Active Rac than triggers actin polymerization leading to localized membrane protrusion. By imaging the mCherry-labeled Rac biosensor (Pak-PBD-mCherry), which recognizes and binds the active GTP-bound Rac, we can monitor Rac activation during light-induced protrusions (see STAR Methods). (B) Time-lapse confocal images of HL-60 cells expressing opto-coreshrt (Opto-PI3K), membrane marker (CAAX-HaToq. imaged on top), and Rac biosensors (Pak-PBD-mCherry, imaged on bottom). Middle and right: localized recruitments of active Rac is confirmed at the site of right extinction for cell protrusion (box h). (C) Time-lapse brightfield (top) and confocal images (bottom) of an opto-PI3K cell during light activation. The specific recruitment of PI3K activator, (SH2-EGFP) to the illuminated area (box in white dashed line) is monitored upon 488-nm excitation. Within 2 s [between the first two frames], SH2 has redistributed from the cytoplasm to the plasma membrane. Scale bar: 1 \u03bcm. (D) Fluorescence intensity line scans (along the white dashed line in (C) show the enrichment of opto-construct (ISH2-EGFP) at the cell protruding site over time. (E) Kymograph of the above line scan (white dashed line in (C) shows that after SH2 is required to the membrane, the cell contour (j.e., its membrane) rapidly expands outward. (F) In red, average time trace of cells before and during light-induced protrusion. In green, apparent cell diameter (long axis) over time as proxy of cell shape change and increases in apparent surface area during protrusion. Trap force and shape change are correlated during the initial phase of the protrusion (giving phase) but when are decoupled as the cell access its membrane reservoirs limiting further increases in membrane tension (platanu phase) even as the protrusion continues to extend (means a SD; m = 15, N = 5). (G) Representative time trace of trap force measured from the tether pulling assay with a cell at steady state: membrane tension remains stable with low magnitude of stochastic fluctuations. (H) As a control, we light activate the wild-type (WT) cells, which lack opto-constructs, and use the same tether pulling assay described above to monitor membrane tension response before, during, and after 488-nm illumination (purple shaded area). Representative time trace of trap force for cell membrane tension recorded from WT cells with light activation. The activation light alone does not elicit any changes in cell morphology or membrane tension responses. (I) In another control, we light activate cells lacking the membrane anchor protein for opto-control (LiRL-BFP-CAAX) and monitor their membrane tension response upon 488-nm illumination (purple shaded area). No perceptible changes in cell morphology or membrane tension were observed. (J) Averaged time trace of trap force (red) for cell membrane tension recorded before (steady state), during (activation), and after (recovery and return to steady state) light-induced protrusion on the opposite side of the cell (see Figure 1C). Individual data traces are shown in light gray (same data as in Figure 1F, n > 60, N = 3.). (S).\\n\\n'", "CAPTION FIGS3-2.png": "'Unlike those on cell body in (A), Specifically, during the \"step-wise pulling\" to extend tether in trap 2 (blue), the static tether held in trap 1 (red) exhibits immediate stability rises in transfer, mirroring the pattern in trap 2. When a smooth increase is exerted on the extending tether by trap 2 (blue, at \\\\(\\\\sim\\\\) 13 s), the tension increase on static tether (red) accordingly becomes gradual. Furthermore, the sudden drop in tension back to initial level on the static tether (red, t \\\\(\\\\sim\\\\) 26 s)--in response to the sudden tether breakage (\\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\)) and thus tension release of the ascending tether (blue)--reflects a direct tension transmission and rapid propagation (see E) within a membrane bleb detached from the constraining actin cortex.\\n\\n(C) Average time trace of relative distance between head and cell in untreated cells and cells treated with 10 uM of the actin inhibitor lattunculin B. After tether pulling measurements, the trapping laser is turned off and the elastic recoil of the bead toward the cell is observed to confirm the absence of cytoskeleton in the tether. Similar tether recoil is observed between untreated and lntunculin-treated cells (means \\\\(\\\\pm\\\\) SD; n \\\\(>\\\\) 13, N \\\\(>\\\\) 3).\\n\\n(D) Similar to (A) but we alternate which buffer is pulling and which tether is static. Trap forces (readout of membrane tension) from static tether is concentrated to that of moving tether ?e., little to no change in tension on the static tether during pulling of the moving tether.\\n\\n(E) Similar to (C) but probing tension in blobs (membrane detached from actin cortex); here, a high correlation is observed between static and moving fathers. (F) Example zoom-in traces of dual trap forces (raw data at 78 kHz) showing the time difference between a sudden tension release upon breakage (\\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\)) of the extending tether (blue) and the subsequent reduction (\\\\(\\\\pm\\\\)) in tension on the static tether (red; traces slightly offset in \\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\) axis for illustration clarity). Typically, this time delay observed is \\\\(\\\\leq\\\\) 100 ms (measured between the inflection points, \\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\) and \\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\), on each trace), which is right around the temporal resolution of our optical trapping instrument (limited by the core frequency of a 2-\\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\)-unhead held by a trap with stiffness of \\\\(\\\\sim\\\\)0.2 pN/nm), indicating that the actual timescale of tension propagation on cellular blobs is less long to fast to be resolved in our experiments.\\n\\n(G) Representative confocal images of actin in cells using actin dye SIR-actin, comparing untreated cells as control with cells treated with either 10 uM of lntunculin B or with a combination of 10 uM of lattunculin B and in hypotonic media (+60% H2O). Scale bar: 10 uM.\\n\\n(H) Borghetti made of dual-tather pulling from opposite sides of a cell treated with a combination of 10 uM of lattunculin B and hypotonic shock.\\n\\n(H) Representative force traces of at cell treated with a combination of 10 uM of lattunculin B and a hypotonic shock showing long-range membrane tension propagation in cells with heavy depolymerated cytoskeleton.\\n\\n(J) Two example time traces of distance between head and cell in cells treated with 10 uM of the actin inhibitor lattunculin B and with an hypotonic camote shock to heavily depolymerize the actin cytoskeleton. After tether pulling measurements, the trapping laser is turned off and the elastic recoil of the bead toward the cell is observed to confirm the absence of cytoskeleton in the tether. We observe similar tether recoil as with untreated and lattunculin-treated cells.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/deguchi2023direct.json b/test_extracted_captions/deguchi2023direct.json new file mode 100644 index 0000000000000000000000000000000000000000..b89d077d6e3d374d642075c4223c8ae5dd0f8a71 --- /dev/null +++ b/test_extracted_captions/deguchi2023direct.json @@ -0,0 +1 @@ +{"CAPTION FIG2-1.png": "'\\nFig. 2: **MINFLUX tracking of kinesin in live cells.** Panels (**A**) to (**D**) show tracking of full-length kinesin labeled N-terminally with a HaloTag bound to JF645 in live U20S cells. (**A**) Confocal images of GFP-a-tubulin in untreated live U20S cells and overlaid full-length human kinesin trajectories. (**B**) Knesin'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG1.png": "'vertical kinesin trajectories with color-coded walking directions. (**F**) Traads as indicated in (E) show side stepping. (**G**) Traads as done as 12 nm are clearly resolved and display lineshitching inter-family between neighboring microtubules or pretrellaments (movie S2). (**H**) Representative track and the corresponding time versus position pick at saturating ATP concentrations (>1 mM, here 6 mM) showing 8-nm making steps. (**I**) Histogram of step size at saturating ATP concentrations from seven experiments, (**A**) tracks, and 995 steps with a Gaussian fit (73 nm +- 2.7 SD +- 0.09 nm SEM red line). (**J**) Dual time histogram and H with a contribution of the experimental functions (average dwell time of 30.8 ms reci red line). (**K** and **L**) Representative track at low ATP concentrations (10 mM) and a corresponding time versus position, raw data (gray), and 20-ms running mean (black) clearly showing 8-nm walking steps (see movie S3). (**M**) Representative track showing a zigzag trajectory, indicating an asymmetric arrangement of the label within a kinesin molecule (see fig. S2, A and E, and movie S4; see fig. S3 for additional examples). Scale bars: (**L**) and (**M**), 10 nm (**F**) to (0), 100 nm; (**D**) and (**E**), 1 nm.\\n\\nFig. 1: **IMFLU tracking of kinesin in fixed cells. (A) Kiresin walls on microtubules in a hard-over-hard manner. The apparent step size is 8 nm when the label is attached to the C-terminal tail domain and 16 nm when it is attached to the N-terminal motor domain. (B) 20 MIFLUX tracking of a single molecule. A draw beam probes seven positions around a fluorophore to determine its location with nanometer precision. The scan pattern is iteratively centered on the fluorophore during tracking. (C) Micro-PMNT approach to track, kinesin in fixed cells. Cells are first permeabilized to extract cell contents and then gently feed to pressure microtubules. Purified fluorescently labeled kinesin (DmKHC (0-42L))-SNAP-tag-to-offel are added and tracked as they mask toward the plus ends of the microtubules (move S1). Parts (D) to (M) show MNFLUX motor-PMNT in fixed cells. (**D**) Confocal images of a neuron and overlaid kinesin trajectories in four neurites. Most of the neurites show kinesin trajectories in both directions, i.e., toward (magenta) and away (cyan) from the soma, as expected for dendrites. (**E**) Confocal microscopy images of green fluorescent protein (GFP)\u2013a-tubulin in U2OS cells showing what appears to be a centrosome and overlaid kinesin trajectories with color-coded walking directions. (**F**) Traads as indicated in (E) show side stepping. (**G**) Traads as done as 12 nm are clearly resolved and display lineshitching inter-family between neighboring microtubules or pretrellaments (movie S2). (**H**) Representative track and the corresponding time versus position pick at saturating ATP concentrations (>1 mM, here 6 mM) showing 8-nm making steps. (**I**) Histogram of step size at saturating ATP concentrations from seven experiments, (**A**) tracks, and 995 steps with a Gaussian fit (73 nm +- 2.7 SD +- 0.09 nm SEM red line). (**J**) Dual time histogram and H with a contribution of the experimental functions (average dwell time of 30.8 ms reci red line). (**K** and **L**) Representative track at low ATP concentrations (10 mM) and a corresponding time versus position, raw data (gray), and 20-ms running mean (black) clearly showing 8-nm walking steps (see movie S3). (**M**) Representative track showing a zigzag trajectory, indicating an asymmetric arrangement of the label within a kinesin molecule (see fig. S2, A and E, and movie S4; see fig. S3 for additional examples). Scale bars: (**L**) and (**M**), 10 nm (F**) to (0), 100 nm; (**D**) and (**E**), 1 nm.\\n\\n'", "CAPTION FIG2-2.png": "'track in which the localizations are rendered as a super-resolution image in the region indicated in (A). (**E**) Line plot connecting each localization. (**D**) Time versus position plot of the highlighted portion of the track in (C) showing steps of 16 nm. Panels (E) to (J) show tracking of truncated kinesin (HaloTag-K560) in Taxol-treated live U2OS cells. (**E**) Confocal images of GFP-a-tubulin and overlaid kinesin tracks. (**F** and **G**) The tracks indicated in (E) rendered as a super-resolution image (F) and line plots connecting each localization (G) (see movie S9) showing clear walking steps (localization precisier. 2 nm, temporal resolution 1 ms). (**H**) Time versus position picks of representative kinesin tracks as indicated in (E) showing clear 16 nm stepwise movements. (**J**) Step-sse histogram [6] experiments, 330 tracks, and 2887 steps) and a Gaussian fit (16.2 +- 3.8 SD +- 0.07 SEM rms). (**J**) Dual-time histogram, fit with a convolution of four exponential functions (average dwell time of 27.5 ms; red line). Panels (K) to (M) show tracking of kinesin (HaloTag-K560) in untreated live primary mouse cortical neurons. (**K**) Confocal images of GFP-a-tubulin and overlaid kinesin tracks. (**L** and **M**) Representative tracks corresponding to those indicated in (K) as line plots (L) and time versus position plots (M) showing 16-nm stepwise movements (see fig. S2, C and G, for step size and dwell time histograms). Scale bars: (B), (C), (F), (G), and (L), 100 nm; (A), (E), and (K), 1 nm.\\n\\n'", "CAPTION FIG3.png": "'switch microtubules (arrows). Panels (E) and (F) show 3D tracking in live cells. (**E**) Representative kinesin tracks in live U2DS cells in top and side views, showing stepwise movements both in the \\\\(x\\\\)-\\\\(y\\\\) plane and along the \\\\(z\\\\) axis (see fig. S2, D and H, for histograms and fig. S3 for confocal overview images; also see movie S10). (**F**) Position versus time plots of the tracks from (E) showing 16-nm steps. Scale bars: (E), 100 nm; (C) and (D), 200 nm.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/doherty2011endocytic.json b/test_extracted_captions/doherty2011endocytic.json new file mode 100644 index 0000000000000000000000000000000000000000..dd9c347bbc1bd6ce574fc684049fd59009a9ccec --- /dev/null +++ b/test_extracted_captions/doherty2011endocytic.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "\"FIGURE 2: GRAF1 is not a general component of focal adhesions but localizes to PLAs. (A) Fluorescence micrograph of HeLa cells coexpressing myc-tagged GRAF1 and GFP-tagged GIT1 and costained for myc and vinculin. Insets show magnifications of the areas indicated by yellow squares. (B) Bar graph showing the percentage of cells in which myc-GRAF1 or myc-GRAF1 R412D was found localized with vinculin in PLAs following the indicated treatments or overexpression of GFP-GIT1. Cells expressing myc-GRAF1 were treated with Y-27632, an inhibitor of rhoA kinase (5 min), or blebbistatin, an inhibitor of nonmuscle myosin III (10 min), and the number of cells where GRAF1 colocalized with vinculin was counted. Bars and error bars correspond to mean and SEM calculated from three independent experiments (\\\\(\\\\alpha\\\\) > 250; \\\\(\\\\alpha\\\\) = 0.05; two-tailed Fisher's exact test, \\\\({}^{\\\\ast\\\\ast\\\\ast}\\\\), p < 0.001). (C) Fluorescence micrograph of cells coexpressing myc-tagged GRAF1 and GFP-cdc42-DA and costained for myc and vinculin. (D) Merged confocal micrograph of a cell expressing src YS27F and GFP-GRAF1 and costained for vinculin. Inset below shows three-dimensional view of the section indicated by the dotted line. Bar graph depicting the percentage of cells with untagged or GFP-tagged GRAF1 localized to vinculin-defined, src-induced podosomes. Bars and error bars correspond to mean and SEM calculated from six independent experiments, each including 30 cells. (E) Fluorescence micrographs of cells treated with the ROCK inhibitor Y-27632 for 15 min or untreated before fixation and staining for vinculin and overexpressed myc-GRAF1. Insets in the top panel show magnification and three-dimensional rotation of the area indicated in the vinculin panel. Arrows indicate basal structures in which GRAF1 and vinculin colocalize. Scale bars: 10 \u03bcm.\\n\\n\"", "CAPTION FIG1.png": "'FIGURE 1: GRAF1 interacts with proteins involved in membrane remodeling and cell adhesion. (A) Domain model of GRAF1. (B) Immunoprecipitation of GRAF1 from rat brain cytosol showing that GRAF1 interacts with dynamin, GIT1, and FAK as identified by mass spectrometry and confirmed by Western blotting with the indicated antibodies. (C) Immunoprecipitates of GIT1, FAK, dynamin, and pFAK from rat brain cytosol were analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. (D) Coomassie-sucanoyl-stained gel of pulldown experiments against brain lysates with beads bound to GST or GST-tagged GRAF1 SH3 domain (GST-SH3). Indicated proteins were identified by mass spectrometry. (E) Confocal micrograph of a HeLa cell expressing GFP-GIT1 and myc-GRAF1, contained for dynamin. Merged image shows section views (as indicated by yellow lines). Note that structures with all three proteins colocalizing are found at the basal surface, while dynamin is also found at the top surface. Scale bar: (5 mm. (F) Schematic representation of the GRAF1 interactome, showing the interactions that link cell adhesion, small G-protein regulation, and GRAF1-mediated membrane trafficking (Hildebrand et al., 1996; Zamir and Geiger, 2001a, 2001b; Hoefen and Berk, 2006; Lundmark et al., 2008). Dotted lines show interactions known to be directly activating (arrowheads) or inhibiting (no arrowheads) the active state of depicted small G protein.\\n\\n'", "CAPTION FIG3.png": "'FIGURE 3: Adhesion reorganization results in clustering of CTxB at the cell surface in GRAF1-positive structures. (A) Fluorescence micrographs of myc-GRAF1-expressing HeLa cells incubated with Y-27632 together with CTxB-Alexa555 for 5 min as indicated before washing, fixation, and costaining for vinculin. Insets are magnifications of the area marked by a rectangle in panel 1. (B) Vinculin- and myc-GRAF1-positive PLA areas were selected from four cells from two different experiments, and the percent colocalization of CTxB was measured as described in the text. The mean is indicated by a red line and the error bars represent standard deviation (SD) above and below the mean. (C) Confocal micrograph of cell expressing src YS27F and GFP-GRAF1 and incubated together with CTxB-Alexa555 for 5 min before washing, fixation, and costaining for vinculin. The merged image is rotated to highlight the basal localization of GRAF1-positive structures. Scale bars; 10 um.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Membrane remodelling by GRAF1 and uptake of CTxB are stimulated at the leading edge of migrating cells. (A) Fluorescence micrographs of myc-GRAF1-expressing HeLa cells incubated with CTxB-Alexa555 for 5 min before washing, fixation, and costairing for vinculin. (B) Confluent wild-type MEF monolayers were wounded by scratching, and cells were allowed to migrate into the wound for 4\u20136 h. CTxB-555 and Tfn-647 were then added to migrating cells for 2 min of uptake at 37\\\\({}^{\\\\circ}\\\\)C. Cells were acid-stripped and fixed and were then labeled for endogenous pxillin. Arrows indicate colocalization between pxillin and CTxB but not Tfn. (C) Twenty-four cells across three independent experiments were treated as in (B), and the percentage of pxillin (green) pixels that colocalized with either CTxB (red) or Tfn (blue) pixels was calculated using Volocity version 3.0. Bars and error bars correspond to mean and SEM (n = 12-15; a = 0.05; Student\u2019s \\\\(t\\\\) test, **, p < 0.01). (D) Fluorescence micrographs of HeLa cells expressing myc-GRAF1 and DA rac1 (GFP-Rac-DA; left panels) and incubated with CTxB-Alexa555 for 5 min (right panels). The length of GRAF1 tubules was measured in fluorescence micrographs of seven different cells (n = 193) as described. The mean length is indicated by a red line and the error bars represent standard deviation (SD) above and below the mean. (E) Fluorescence micrographs of cells expressing myc-GRAF1 and DA rhoA (GFP-rhoA-DA) and contained for vinculin. GRAF1-positive tubules were found in 14.3 +- 3.7%, as determined from three independent experiments (n = 124; error values represent SEM). Length of GRAF1 tubules in \u03bcm was measured from nine different cells (n = 108) and depicted as in Figure 4D. Scale bars: 10 \u03bcm.\\n\\n'", "CAPTION FIG5.png": "\"Figure 5: GRAF1 is necessary for cell spreading and migration. (A) Immunoprecipitation andimmunodetection of GRAF1 from HeLa cells treated with either a control siRNA or siRNAs against GRAF1. Tubulin was detected in the cell lysates as an immunoprecipitation control. GRAF1 expression following siRNA treatment (GRAF1 expe. [%]) was quantified as described from three independent experiments. Values correspond to normalized means +-SD. (B) Micrographs of cells treated with either a control siRNA or siRNA against GRAF1 for 72 h before fixation and imaging. Inset 1 (left panel) shows magnification of the marked square. Inset 1 (right panel) exemplifies cell area indicated in blue, and length and width indicated in red and green, respectively. (C) Bar graphs showing quantification of the cell area and length:width ratios, as described in the text, of cells treated with control siRNA or siRNAs against GRAF1, as in (B). In the left panel, bars and error bars correspond to mean and SEM calculated from five or six independent experiments (n = 50\u201360; a = 0.05; Student's t test, ***, p < 0.001). In the right panel, bars and error bars correspond to mean and SEM calculated from two independent experiments (n > 100; a = 0.05; Student's t test, ***, p < 0.001). Length:width ratio was scored as the ratio between length (longest straight line within a cell (red line in (B)) and width (the\\n\\n\""} \ No newline at end of file diff --git a/test_extracted_captions/ezzatSpecificityAbstractionExamples2020.json b/test_extracted_captions/ezzatSpecificityAbstractionExamples2020.json new file mode 100644 index 0000000000000000000000000000000000000000..a4ad632a6fd3a41b37ef8eb249f010270f7dd2b0 --- /dev/null +++ b/test_extracted_captions/ezzatSpecificityAbstractionExamples2020.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: (A) Mean number of responses according to the experimental condition (Specificity/Control/Abstract) and the type of solution (Fixation/Expansion). (B) Mean originality scores according to the experimental condition (Specificity/Control/Abstract).\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l c c} \\\\hline \\\\hline \\\\multicolumn{1}{c}{TABLE 1} & Categories of Solutions of the Egg\u2019s Task (Agogu\u00e9, Kazakci, et al., 2014) \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Categories of Solutions of the Egg\u2019s Task (Agogu\u00e9, Kazakci, et al., 2014)'"} \ No newline at end of file diff --git a/test_extracted_captions/francis2015endocytic.json b/test_extracted_captions/francis2015endocytic.json new file mode 100644 index 0000000000000000000000000000000000000000..1621ee3c7db2170c3ed07f1aa24efac9d7353d76 --- /dev/null +++ b/test_extracted_captions/francis2015endocytic.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'\\n\\n**Fig. 2. Dynamic assembly of GRAF1 at the leading edge.** (A) Immunobists detecting endogenous and recombinant GRAF1 in Flp-fn T-REx HeLa GFP-GRAF1, GRAF1 and GRAF1-GFP cell lysates after induction with the eluted doxycycline (Deoxy) concentrations for 24 h. A deoxycycline concentration of 1 ng/ml was chosen for all subsequent experiments. (B) Fluorescence micrograph detecting GRAF1-GFP, corresponding to the last frame of a live-cell spinning disc, confocal microscopy acquisition. As the leading edge, tracks of structure movement and duration over three are illustrated as colour-coded lines. Raprecardation frames from the same acquisition are presented as a time series. Scale bar: 10 um. (C) Duration time of GRAF1-GFP structure tracks derived from acquisitions exemplified in B. 19 cells from three independent experiments were analyzed. The bar graph depicts the distribution of the means-d values derived from each evaluated cell (D) Duration time of GRAF1-GFP and GFP-GRAF1 structure tracks derived from live-cell TIRF microscopy acquisitions. Bar graphs depicts the distribution of the mean+s.d. values derived from each cell included in the respective analysis. Three independent experiments were analyzed (Mann-Whitney U terz, r=11-12 cells, a=0.08, ris, not significant). (E) Fluorescent micrograph showing GRAF1-GFP and mCherry-tagged clathrin light chain (CLC) at a protrusion, corresponding to core frame from a live-cell TIRF microscopy acquisition. Traces visualise the detected structure movement in the respective channel. The edge of the cell is outlined in the upper panels. ArroMethods highlight GRAF1 assemblies. Scale bar: 2 um.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3.**Inhibition of **G**dc42 function and **ac**ln polymerization effects **G**RAF1 carrier processing. (A,B) Relative normalization of aminodextrin-S-G-C/V**400 and transferrin-S-S-C/V**400 after 30 min uptake in Fip-ln T-R**2x HeLa GFP-G**RAF1 cells depleted at AP-2, Cdc42 or G**RAF1 by siRNA transfection (A), as confirmed by the immunodetection of the respective protein in camparan to clathrin heavy chain (CHC) in the cell lysates (B). The cargo uptake was normalized to the corresponding control _a_-RNA-transfected sample and the significance in relation to this sample was determined by analyses of at least three independent experiments (means,d; Mann-Whitney U tests, _n_=3-4 samples, a=0.0%, ns, not significant; \"pe0.0%). (C) Fluorescent micrographs depicting G**RAF1-GFP** in control cells and cells depleted of Cdc42. The ratio of cells with G**RAF1** in at least one tubular structure (i.e. a structure with a length >2 mm) was quantified on the basis of three independent experiments (means,d - Chi-square test, _n_>480 cells, a=0.0%, +++p<0.0001). (D) Ratio of cells with GFP-G**RAF1** in tubular structures after treatment with DMSO (Vehice) or withostatin (Vhisk), for the indicated time interval. Cells from three independent experiments were included in the analysis (means,d.; Chi-square tests, a=0.0%, +++p<0.0001). (E) Cordocal stack at a GFP-G**RAF1-expressing cell after 20 min dissociation treatment, with presented top and slice views positioned as indicated (white arrow). Scale bar. 10 mm.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **GRAF1 recruitment to Cdc42** microdomains coincides with a local decreasing plasma membrane association of the GTPase. \\\\(\\\\langle\\\\Delta\\\\rangle\\\\) Time series from a live-cell spinning disc confocal microscopy acquisition, visualizing a protrusion from a Fip-In T-REx HeLa GRAF1-GFP call co-expressing mCherry-Cdc42. White arrows mark detected GRAF1 assemblies. (B) Intensity profiles of the green and red channels, calculated over time as described in the Materials and Methods, for the first-appearing GRAF1 assembly in A. The grey area highlights the peak duration for the green channel. The insert visualizes the ROI used to derive the plotted fluorescence intensity curves. (C-F) Parameters calculated for single GRAF1 assemblies on the basis of the corresponding intensity peak recorded within the intensity profiles derived from live-cell acceptance, as exemplified in A and B. The three points for the intensity peak start, maximum and end were defined for each structure and channel as described in the Materials and Methods. 34\u201336 GRAF1 structures from three cells were included in the analysis and results are meaning.d.\\n\\n'", "CAPTION FIG7-2.png": "'\\n\\n**Fig. 7. Cdc42 GTPase deficiency disrupts the maturation of internalized GRAF1 carriers.** (A) Fluorescence micrograph representing the first frame of a live-cell confocal acquisition detecting dextran-Alexa-Flucr-555 and GFP\\\\({}_{\\\\infty}\\\\) = GRAF1 in Fip-In T-REx HaLa cells co-expressing Myc-Cdc42-Q61L (upper pannels). The acquisition was started after a 5-min incubation with the cargo followed by medium exchange. Analysis on the basis of four captured cells revealed dextran enrichment in 31\\\\(\\\\pm\\\\)16% (means.e.m.) of the identified GRAF1 structures. Areas 1 and 2 from the acquisition are presented as time series (lower panels). Red stars indicate cells affected by Myc-Cdc42-Q61L Scale bar: 10 mm. (B) Epifluorescence micrograph of GFP-GRAF1 and dextran structures after a 5-min uptake in two cells, one defined as phenotypically unaffected (control) and the other defined as affected by Myc-Cdc42-Q61L (red star). Scale bar: 10 mm. (C,D) Quantification of the total dextran uptake and descriptive parameters for dextran-positive compartments for comparison of GFP-GRAF1 cells unaffected and affected by the Cdc42-Q61L transfection, as defined in B, to circumvent inter-sample variation of cargo fluorescence. Error bars represent the s.e.m. from three independent experiments (Mann-Whitney U tests, n=12-14 cells, a=0.05; ns, not significant; \"P<=0.01). (E) Relative internalisation of aminodextrin-S-S-CW800 and transferrin-S-S-CN800 after a 30-min uptake in Fip-In T-REx HaLa GFP-GRAF1-R412D cells. The cargo uptake of the mutant-expressing cells collected from at least three independent experiments was normalised to the corresponding wild-type sample and the significance in relation to this sample was assessed (means.d.; Mann-Whitney U tests, _n_=3-4, a=0.05; ns, not significant). (F) EEA1 intensity per endosome and number of EEA1-positive endosomes per GFP-GRAF1 cell transfected with mCherry-tagged Cdc42 or Cdc42-Q61L. Three independent experiments were analyzed [mean +-s.d.; left diagram, Student\\'s +-test, _n_>13,500 EEA1-stained endosomes (from 50 cells), a=0.05; ns, not significant; right diagram, Mann-Whitney U test, _n_=3 cells, a=0.05; ns, not significant). Bars in the left graph depict the distribution of the mean values derived from each experiment. (G) Confocal fluorescence micrographs of GFP-GRAF1 cells transfected with mCherry-Cdc42-Q61L and immunostained with anti-EEA1 antibody. Scale bar: 5 mm. (H) Ratio of dextran-filled Rab6 compartments in BFP-Rab5-transfected GFP-GRAF1 cells without or with co-expressed Myc-Cdc42-Q61L after a 5-min cargo uptake. Cells from three independent experiments were included in the analysis [means.d.; Mann-Whitney U test, _n_>5 captured frames (at least 5 cells), a=0.05; \"P<=0.05). (I) Representative time series from a live-cell spinning disc confocal microscopy acquisition visualising the dynamics of dextran-filled internal compartments in GFP-GRAF1 cells co-expressing BFP-Rab5 and Myc-Cdc42-Q61L. The yellow arrow marks a Rab6 vesicle devoid of dextran, adjacent to a cargo-filled GRAF1 compartment pointed out by the magenta arrow. The turquoise arrow marks a second GRAF1-labeled structure devoid of dextran, which over the depicted time range fuses with the first GRAF1 compartment (specifically note the redistribution of cargo between these two compartments). Scale bar: 2 mm.\\n\\n'", "CAPTION FIG7.png": "\"\\n\\n**Fig. 7.** **Cdc42 GTPase deficiency disrupts the maturation of internalized **GRAF1 carriers**. (A) Fluorescence micrograph representing the first frame of a live-cell confocal acquisition detecting dextran-Alexa-Fluco-565 and GFP-GRAF1 in Fip-In T-REk HeLa cells co-expressing Myc-Cdcd42-Q6 IL (upper panels). The acquisition was started after a 5-min incubation with the cargo followed by medium exchange. Analysis on the basis of four captured cells revealed dextran enrichment in 31+-16% (means.s.m.) of the identified GRAF1 structures. Areas 1 and 2 from the acquisition are presented as time series (lower panels). Red stars indicate cells affected by Myc-Cdcd42-Q6 IL. Scale bar: 10 um. (B) Erythroscence micrograph of GFP-GRAF1 and dextran structures after a 5-min uptake in two cells, one defined as phenotypically unaffected (control) and the other defined as affected by Myc-Cdcd42-Q6IL (red star). Scale bar: 10 um. (C,D) Quantification of the total dextran uptake and descriptive parameters for dextran-positive compartments for comparison of GFP-GRAF1 cells unaffected and affected by the Cdc42-Q6IL transfection, as defined in B, to circumvent inter-sample variation of cargo fluorescence. Error bars represent the s.e.m. from three independent experiments (Mann-Whitney U tests, _n_=12-14 cells, _a_=0.05; ns, not significant; *P<0.01). (E) Relative internalisation of aminod dextran-S-S-CWN800 and transferrin-S-CWN800 after a 30-min uptake in Fip-In T-REk HeLa GFP-GRAF1-R412 cells. The cargo uptake of the mutant-expressing cells collected from at least three independent experiments was normalised to the corresponding wild-type sample and the significance in relation to this sample was assessed (means.s.d.; Mann-Whitney U tests, _n_=3-4, _a_=0.05; ns, not significant). (F) EEA1 intensity per endosome and number of EEA1: positive endosomes per GFP-GRAF1 cell unaffected with mCherry-tagged Cdc42 or Cdc42-Q6IL. Three independent experiments were analysed [means +s.d.; left diagram, Student's +Rest, _n_=13,500 EEA1-stained endosomes (from 60 cells), _a_=0.05; ns, not significant; right diagram, Mann-Whitney U test, _n_=5 cells, _a_=0.05; ns, not significantly. Bars in the left graph depict the distribution of the mean values derived from each experiment. (G) Confocal fluorescence micrographs of GFP-GRAF1 cells transfected with mCherry-Cdcd42-Q6 IL and immunostained with anti-EEA1 antibody. Scale bar: 5 um. (H) Ratio of dextran-filed Rab5 compartments in BFP-Rab5-transfected GFP-GRAF1 cells without or with co-expressed Myc-Cdcd42-Q6IL after a 5-min cargo uptake. Cells from three independent experiments were included in the analysis [means.d.; Mann-Whitney U test, _n_=5 captured frames (at least 5 cells), _c_=0.05; *P<0.05). (I) Representative time series from a live-cell spinning disk confocal microscopy acquisition visualizing the dynamics of dextran-filed internal compartments in GFP-GRAF1 cells co-expressing BFP-Rab5 and Myc-Cdcd42-Q6IL. The yellow arrow marks a Rab5 vesicle devoid of dextran, adjacent to a cargo-filled GRAF1 compartment pointed out by the magenta arrow. The turquoise arrow marks a second GRAF1-labelled structure devoid of dextran, which over the depicted time range fuses with the first GRAF1 compartment (specifically note the redistribution of cargo between these two compartments). Scale bar: 2 um.\\n\\n\"", "CAPTION FIG5.png": "'\\nFig. 5: **Interaction with transition state Cdc42 enforces membrane assembly of GRAF+.** (A) Ratio of Fig-In T-Rea HeLa cells showing a phenotype of abundant (>15) GFP-GRAF+1 or GFP-GRAF-1.R412D structures in the absence (-) or presence of co-expressed Coral/Hue-tagged small GTPases and mutants thereof. For statistical analysis, cells from at least three independent experiments were included. The results were compared to the GRAF1 samples expressing wild-type Cdc42, or in the case of GRAF1-R412D cells, were related to the corresponding GRAF1 samples (means.d.; Chi-square tests, _n_>=0.05, ns, not significant; \"\"PcO.0001). (B) Maximum intensity projections of cordocal stacks detecting GFP-GRAF1 in cells co-expressing the indicated _Coral/Hue-_tagged Cdc42 proteins. (C) Cocomas-stained SDS-PAGE gel from a platform experiment using purified GST GST or a GST-Tagged GRAF1 GAP-domain (GAP) as bait, and purified Cdc42 or Cdc42-Q61L loaded with GTP-S or GDP in the presence of AIF, (GDP/AIF, as grey). The bound (B) and unbound GTPases (J) were detected. (D) Maximum intensity projections of cordocal stacks detecting GFP-GRAF1 or indicated truncates (the BAR-PH-GAP and BAR-PH domains) and _Coral/Hue\u2013_C_dc42-Q61L in transfected HeLa cells. (E) Percentage volume of GFP\u2013GRAF1 proteins colocalized with Coral/Hue\u2013_C_dc42-Q61L, as visualised in D. Three independent experiments were analysed (means.d.; Kruskal-Wallis test, Dunn\u2019s post test, _n_=15 cells, _\u03b1_=0.05; ns, not significant: \"\"PcO.0001). Scale bars: 10 \u03bcm.\\n\\n'", "CAPTION FIG7-1.png": "'\\n\\n**File T:** See text page for text.\\n\\n'", "CAPTION FIG6.png": "\"Figure 6: **Cdc42 GTPase deficiency affects GRAF1 carrier dynamics after their formation**. (A) Fluorescence micrographs depicting the start frames from the-cell TIRF microscopy acquisitions of GFP-_GRAF1 structures in Fb-in T-REx HeLa cells transfected with the indicated _Coral/Hue-tagged Cdc42 constructs (upper panels). The corresponding structure tracks over time, derived from the acquisitions, are represented as colour-coded lines (lower panels). Structures detected over the entire basal cell surface were included in the track analysis. (B\u2013E) Quantification of the number and dynamic parameters of GFP-_GRAF1_ structure tracks from acquisitions exemplified in A. Three independent experiments were analysed [Student's +-tests on log10-transformed data, \\\\(n\\\\)2-136 structure tracks (from four cells), _\u03b1_=0.05; ns, not significant; ***P>0.0001]. Bar graphs in C\u2013E depict the distribution of the means.d, values derived for each cell included in the respective analysis. (F) Fluorescence micrograph showing the first frame from a live-cell spinning disc confocal microscopy acquisition of GFP-_GRAF1_ structures in cells transfected with the indicated _mCherry_-tagged Cdc42-Q61L. Speeds measured over the detected GRAF1 tracks are represented as colour-coded lines. Magnifications of areas 1 and 2 are visualised as a time series to highlight the mobile membrane structures positive for both proteins. The white arrow marks a structure at the cell surface, the yellow arrow follows the lateral movement of an internal structure and the red arrow points out the fission of a tubular structure from the cell surface. (G) Quantification of duration of colocalisation between GFP-_GRAF1_ membrane assemblies and respective indicated _mCherry_-tagged Cdc42 protein, based upon track analyses performed on live-cell spinning disc confocal microscopy acquisitions like that exemplified in F. Cells from two independent experiments were analysed and the means.d. is indicated [Student's +-test on log10-transformed data, \\\\(n\\\\)2-29 structure tracks (from three cells), _\u03b1_=0.05; ***P>0.0001]. Scale bars: 10 \u03bcm.\\n\\n\""} \ No newline at end of file diff --git a/test_extracted_captions/fuExpertRepresentationDesign2013.json b/test_extracted_captions/fuExpertRepresentationDesign2013.json new file mode 100644 index 0000000000000000000000000000000000000000..142cba07712a8f594e43e951d9a6883973fd2ae1 --- /dev/null +++ b/test_extracted_captions/fuExpertRepresentationDesign2013.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n**Table 3 Algorithm structure nodes evaluated by experts during interview**'", "CAPTION FIG4.png": "'\\n\\n## Chapter 4 Algorithm\\n\\n### 4.1 Algorithm\\n\\nThe algorithm is a simple algorithm for the computation of the \\\\(n\\\\)-th order polynomial in \\\\(n\\\\) variables. The algorithm is based on the following algorithm:\\n\\n1. [label=()]\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Correlations between participant and algorithm-generated structures of patents'", "CAPTION TAB6.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB4.png": "'\\n\\n**Table 4 Mean extent reaches to each of the algorithm nodes.**'", "CAPTION TAB5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The \\\\(\\\\chi\\\\)-function of the \\\\(\\\\chi'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG5.png": "\"\\n\\n## Chapter 4 Hurwitz and Euler's\\n\\n\"", "CAPTION FIG6.png": "'\\n\\n## Appendix A Proof of Theorem 1\\n\\n### Proof of Theorem 1\\n\\nProof of Theorem 1\\n'", "CAPTION FIGAPPB.png": "'* [16] A.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n## Appendix A'", "CAPTION TAB APPA.png": "'\\n\\n## Appendix A\\n\\n45 Patents used in algorithm generated structure'", "CAPTION FIG1.png": "'Figure 1: Planning chain of possible outcomes of study and main possible inference.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/gickAnalogicalProblemSolving1980.json b/test_extracted_captions/gickAnalogicalProblemSolving1980.json new file mode 100644 index 0000000000000000000000000000000000000000..e6751dda66e5213e52771ec9b9ac530c1607238a --- /dev/null +++ b/test_extracted_captions/gickAnalogicalProblemSolving1980.json @@ -0,0 +1 @@ +{"CAPTION TAB4.png": "'TABLE 4\\n\\nMean Ratings of Likelihood that Stories Would Help to Think of Various Radiatione Solutions\"'", "CAPTION TAB2.png": "\"TABLE 2\\n\\nSchematic Outline of Duncker's Radiation Problem Showing\\n\\nCorrespondences with Analogous Stories\"", "CAPTION TAB3.png": "'TABLE 3\\n\\nPercentage of Subjects in Each Condition of Experiment 1 Who Fuguese Various Solutions to the Radiation Problem'", "CAPTION TAB5.png": "'TABLE 5\\n\\n**Fortion of Protocol for S15 (Attack-Dispersion Condition)**'", "CAPTION TAB7.png": "'TABLE 7\\n\\nPortion of Proceed for $19 (Open Supply Route Condition)'", "CAPTION TAB6.png": "'TABLE 6\\n\\n**Fortion of Protocol for S38 (Attack-Dispersion Condition)**'", "CAPTION TAB9.png": "'TABLE 9\\n\\nSchematic Outline of Paradox-Disperation Story'", "CAPTION TAB8.png": "'\\n\\n**TABLE 8**\\n\\n**[1.5ex]**\\n\\n**[1.5ex]**\\n\\n**[1.5ex]**\\n\\n**[1.5ex]**\\n\\n**[1.5ex]**'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{TABLE 10} \\\\\\\\ Percentage of Subjects in Each Condition of Experiment II Who Proposed \\\\\\\\ \\\\multicolumn{1}{c}{the Dispersion Solution to the Radiation Problem} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 10: Percentage of Subjects in Each Condition of Experiment II Who Proposed \\\\\\\\ \\\\multicolumn{1}{c}{the Dispersion Solution to the Radiation Problem}\\n\\n'", "CAPTION TAB11.png": "'\\n\\n**Figure Captions**\\n\\n* **Figure 1**: The \\\\(\\\\pi^{+}\\\\) and \\\\(\\\\pi^{-}\\\\)'", "CAPTION TAB12.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{TABLE 12} \\\\\\\\ Percentage of Subjects in Experiment V Who Produced Dispersion Solution at Each Step of the Procedure \\\\\\\\ \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 12: Percentage of Subjects in Experiment V Who Produced Dispersion Solution at Each Step of the Procedure'", "CAPTION TAB1.png": "'TABLE 1\\n\\nA Summary of Attack-Dispersion Story and of Corresponding Solution to Radiation Problem (See Fig. 1)'", "CAPTION FIG1.png": "'Figure 1: Analogical correspondences between the Attack-Dispersion story and the radiation problem.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/gilonAnalogyMiningSpecific2018.json b/test_extracted_captions/gilonAnalogyMiningSpecific2018.json new file mode 100644 index 0000000000000000000000000000000000000000..31ca676590add0f5059655470ca18d236529398a --- /dev/null +++ b/test_extracted_captions/gilonAnalogyMiningSpecific2018.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: An example product description from our dataset: Soapy, slider\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Illustration of the process of abstracting the corpus based on a given focus-abstracted query. All terms in each document that match the designer-selected abstracted _properties_ (shown in monospace font) are replaced by the matching properties. This brings documents that might be different in domain (e.g., about \u201cknives\u201d) but are nevertheless similar at the desired level of abstraction (e.g., PersonalProduct) closer to the focus-abstracted query.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Illustration of the process of expressing focus-abstracted queries in our system. Designers express a focus-abstracted query by (1) selecting important sentences in a product description that relate to an intended focus, (2) ignoring irrelevant terms, and (3) replacing important terms (where desired) with appropriate abstracted properties, yielding a sequence of important terms and abstracted properties.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Illustrative matches from each method for the scenario: \u201cmake the dish compatible with different sizes of soap bars\u201d. The abstraction of \u201csoap\u201d seems to allow the FocusAbstracted method to ignore the domain difference of knives vs. soap. OverallPurpMech finds a match from a different domain that is analogous in terms of its overall purpose of keeping something clean/dry, but misses the core purpose of adapting to different sizes. In contrast, both OverallGioVe and FocusOnly find a highly relevant match from the same domain.**\\n\\n'", "CAPTION FIG5.png": "'Figure 5: FocusAbstracted matches achieve comparable relevance to FocusOnly and GloVe baselines, and more relevance than OverallPurpMech (left panel) while being more domain distant than FocusOnly and GloVe baseline matches, and equivalently domain distant as OverallPurpMech matches (right panel)\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1. Seed product descriptions and associated redesign scenarios**} \\\\\\\\ & used for our evaluation experiment. Descriptions shortened. \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Seed product descriptions and associated redesign scenarios used for our evaluation experiment. Descriptions shortened.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/hopeAcceleratingInnovationAnalogy2017.json b/test_extracted_captions/hopeAcceleratingInnovationAnalogy2017.json new file mode 100644 index 0000000000000000000000000000000000000000..5683747f84f8a0d5c1293d696923da3f889af937 --- /dev/null +++ b/test_extracted_captions/hopeAcceleratingInnovationAnalogy2017.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "'Figure 4: Showing proportion estimates by our random-effect logistic regression, for \\\\(k=2\\\\) (left) and \\\\(k=3\\\\) (right). Participants are significantly more likely to generate good ideas for the redesign ideation task when given inspirations from our analogy approach compared to baseline-surface and baseline-random approaches\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG3.png": "'\\n\\n**Figure 3: Overview and excerpts of the ideation experiment. Top-Seed product. Workers were asked to solve the same problem in a different way. Middle: Top 3 inspirations for each of the conditions. Note that the TF-IDF baseline returns results from the same domain, while our method returns a broader range of products. Bottom: Ideas generated by users exposed to the different conditions.**'", "CAPTION FIG2.png": "'\\n\\n**Figure 2:** **Screenshot of analogy search interface**'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG1.png": "'\\n\\n**Figure 1: Collecting purpose and mechanism annotations from the crowd.**'"} \ No newline at end of file diff --git a/test_extracted_captions/hopeScalingCreativeInspiration2022.json b/test_extracted_captions/hopeScalingCreativeInspiration2022.json new file mode 100644 index 0000000000000000000000000000000000000000..e6ac34b569ab6b518a3d3b67445120b167ca2d38 --- /dev/null +++ b/test_extracted_captions/hopeScalingCreativeInspiration2022.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "\"Figure 4: Comparing our GCN model predictions (right) to human annotations (left). Interestingly, our model managed to correct some annotator errors (\u201cit's\u201d, \u201cheated\u201d, \u201ccoffee warm\u201d, \u201cbeverages\u201d). Purposes in pink, mechanisms in green.\\n\\n\"", "CAPTION FIG3.png": "'Figure 3: Left: Precision\\\\(0\\\\)K results for the best performing model (GCN + self-training). Right: Raw extraction accuracy evaluation. All approaches use CRF loss. GCN with syntactic edges outperforms baselines. Self-training further improves results. Random-label achieves only \\\\(16.01\\\\)\\\\(\\\\mathbf{F}_{1}\\\\).\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Extracting fine-grained purposes and mechanisms at scale enables mapping the landscape of ideas. Suppose an inventor is looking for a way to wash clothes without water. On the left we see a snippet from the graph of purposes. Each node in the graph represents a cluster of similar purpose spans extracted from documents (labels are manually generated for illustration purposes). Edges reflect abstract structural similarity, capturing co-occurrence patterns of spans in the corpus (see Section 5.1 and Figure 9). On the right we see example documents containing purposes from the four clusters (corresponding cluster numbers appear in boxes). Purpose/mechanism spans in documents are shown in pink/green, respectively. One could find direct matches to the query \u2013 i.e., documents with purpose from cluster 1 and mechanism not \u201cwater\u201d (e.g., waterless washing machine using dry ice), or explore neighboring purpose clusters, reformulating the problem as removing odor, removing dirt, or getting rid of the dirty clothes, each resulting in a different set of inspirations.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Crowdsourcing interface for fine-grained purposes and mechanisms.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: **Inspiration user study results. Left: Proportion of inspirations selected by at least \\\\(2\\\\) raters, per condition. Right: Proportion of boxes (clusters) with at least \\\\(2\\\\) spans marked by \\\\(\\\\geq 2\\\\) raters.**\\n\\n'", "CAPTION FIG11.png": "'\\n\\n**Figure 11:** Schematic of our GCN model'", "CAPTION FIG5.png": "'Figure 5: Applications for light where light is not in the purpose. **Left: Interface. Right: Two of the results and their automatic annotations (purposes in pink, mechanisms in green).**\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Results for search evaluation test case. Mean average precision (MAP) and Normalized Discounted Cumulative Gain (NDCG) by method, averaged across queries. Methods in bold use our model.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{|c|c|} \\\\hline \\\\hline \\\\multicolumn'", "CAPTION FIG9.png": "'Figure 9: Excerpt from our Functional Concept Graph. Nodes represent concepts (clusters of purposes). To give intuition for how edges were created, they are annotated with example products containing spans from both concepts. All nodes and edges in this figure were automatically constructed and used to create the user-fixing inspirations shown in Figure 8. This figure provides a graphic illustration (not shown to users) explaining how the boxes in Figure 8 are generated, with node names provided here by us for readability. The problem of \"medicine morning reminder\" is mapped (via embedding) to the _Alert/remind_ concept cluster (as named by us), which is linked to the concepts of medical monitoring and making hot drinks through products such as \"smart medicine injector\" and \"coffee machine alarm\" (among others, not displayed in the figure). These links serve as the source for inspirations in our study, as seen in Figure 8.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Scenarios and example results retrieved by our FineGrained-AVG method. Queries reflect non-trivial uses of mechanisms (e.g., using water not for drinking/cleaning, retrieving a lighter running on hydrogen from water and sunlight).\\n\\n'", "CAPTION FIG7.png": "'Figure 7: An example of our learned functional concept graph extracted from texts. Mechanism in green, purpose in pink. Titles are tags nearest to cluster centroids (redicted to fit).\\n\\n'", "CAPTION FIG8.png": "'Figure 8: A snippet from our ideation interface for \\\\({}^{4}\\\\)morning medicine reminder\u201d. Users see inspirations grouped into boxes. Each box is supposed to represent a concept \u2013 a cluster of related spans as found by our method or by the baselines (see \u00a75.1). Users indicate which inspirations were useful, and what ideas they inspired. For example, seeing \"real time health checker\" inspired one user to suggest a monitoring device for finding the best time for reminding to take the medicine.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/howes2010clathrinindependent.json b/test_extracted_captions/howes2010clathrinindependent.json new file mode 100644 index 0000000000000000000000000000000000000000..849edee206e45231337fc664d184b9eacae4822d --- /dev/null +++ b/test_extracted_captions/howes2010clathrinindependent.json @@ -0,0 +1 @@ +{"CAPTION FIG3.png": "'Figure 3: **3D morphology of CUCs.** [_A_]_ Conv1/- _M_EFs were incubated with CTxB-HRP for 20 min on ice, before internalization for 15 s. The DAB reaction was performed and calls were processed for electron tomography [see Materials and methods]. A single section of the original tomogram is shown [left]. Various rotations of a 3D convoluted electron density render were generated [middle]. Enlarged sections selected from the tomogram [right] show internal vesicles [arrows] and a complete connection around the circumference of the structure (arrowhead). (B) V/T MFFs grown on sapphire discs were incubated with CTxB-HRP for 15 s before DAB reaction and high-pressure freezing. Tubular extensions [large arrows] are seen emanating from vascular bubbles (arrowheads) in the characteristic ring-shaped CUC morphology. CCPs without CTxB label [double arrowheads] indicate that they are still surface connected. Bars, 200 nm.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Biochemical enrichment of CICs.** [A] 3T3-GPI calls were subjected to density fractionation as described in Materials and methods. Western blots of membrane markers are shown. Within the first gradient [left] anti-GFP-, Cov1-, and Gpp78-positive membranes are present in Fraction 1.2. highlighted by outline. CHC, GMI 30., and TR-positive membranes are below the detection limit within this fraction. Within the second gradient, anti-GFP-positive membranes concentrate within Fraction 2.8. highlighted by outline. This represents a yield of 9.6 \\\\(\\\\pm\\\\) 0.2% of anti-GFP-positive membranes. Cav1- concentrates in Fraction 2.6 and Gpp78 in Fraction 2.10. (B) Fraction 2.8 was fixed and visualized by EM. Structures within Fraction 2.8 share similar profiles to CUCs scan within intact. Bar, 200 nm. [C] High resolution electron micrographs of Fraction 2.8 structures, providing examples of vesiculation [arrows] and 3 spherical bulb connected to lobular extension [arrowheads]. Bar, 100 nm. [D] NH4T33 cells were incubated with CIC8-HRP fractionation and the DAB reaction was performed on Fraction 2.8. Bar, 200 nm. (E) NH4T33 cells transfected with CIC42-WT or CD-N were incubated with CTx6 buffer fractionation. Western blots were probed with anti-CTx6. 3T3-GPI cells were treated or not with mipCD before incubation with anti-GFP antibodies. Western blots of fractions were probed with anti-Rb+HRP. (F) Western blots of fractions from cells transfected with Hat1-HA, GRAF1-myc, or left untransfected. Fractions were probed with anti-HA, _myc_-, _dynamin_, _F_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_-_G_-_-_G_-\\n\\n'", "CAPTION FIG1.png": "'Figure 1: **CIA** **does not affect** **CUC** **adocytosis**. (**A**) **Coy1**\u2013/\u2013 **MEFs** were incubated with HRP in the presence or absence of CIA/R for 1.5 s. Cells were cooled and DAB reaction performed in the presence of ascorbic acid (AA) before fixation. Bars, 200 nm. (**B**) Quantification of the number of HRP-filled carriers per cell across 10\u201312 cells treated as in A. CILCs were defined by their characteristic ring-shaped morphology, CCVs were defined as coated vesicular carriers. Error bars show SEM. [**C**] **Coy1**\u2013/\u2013 **MEFs** were grown in normal media (plus energy), media containing 2-deoxyglucose (no energy), or 2-deoxyglucose (no energy), or 2-deoxyglucose (no energy) for 1 h followed by a 1-h washout in normal media (recovery) before CTaR-R/R internalization and DAB reaction. Labeled structures were counted across 10 cells. Error bars show SEM. (**D**) and [**E**] **Coy1**\u2013/\u2013 **MEFs** were incubated with CTaR-R/R for 5 min before the DAB reaction for either 5 min at 37degC or 10 min at 4degC, followed by fixation at 37degC. All CTaR-R/R-positive structures were counted in 10\u201312 cells across two independent experiments. Error bars show SEM. Bar, 200 nm. (**F**) NIH3T3s were treated for 20 min with vehicle (Influotated) or Dyngoda before internalization of CTaR-555 (left panels) and Tf647 (right panels) for 5 min. Cells were placed on ice and acid stripped to remove surface labeling. Cells were then bound with CTaR-488 (middle panels). Bar, 10 \u03bcm. (**S**) NIH3T3s cells were left untreated or were treated with Dyngoda before internalization of CTaR-HRP, followed by DAB reaction. Arrows show CTaR-HRP\u2013Loden CILCs, double arrowheads show internalized, labeled CCVs, arrowheads show surface connected unlabeled CCVs. Bars, 200 nm.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Verification of novel CUC cargo.** (A) NIH3T3 cells were incubated with either anti-Thy-1 or anti-CD44 antibodies and CTb-555 and Tb6.47 for 2 min. Myoferlin was detected after fixation, showing steady-state localization. Arrows indicate colocalization. Bar, 10 \u03bcm. (B) 3T3-GPI cells were left untreated or were treated with 100 nM wortmannin before internalization of anti-GFP for 10 min. Arrows indicate colocalization. Bar, 10 \u03bcm. (C) Quantification of \\\\(b\\\\) from 12\u201315 cells in three independent experiments. (D) Quantification of colocalization between internalization and CTb44 antibodies and Tb6.47, CTb-555, or anti-type antibodies for GFP-dyoferlin-myc-expressing cells after 2, 10, and 40 min. Error bars show SEM. (E) Anti-CD44.H8P was internalized into WT MEFs before DAB reaction. Arrows show anti-CD44-HRP-positive carriers with morphology of CUCs. Arrowheads show large, tubular ring-shaped anti-CD44-HRP-positive compartment. Bars, 200 nm. (F) Anti-CD44-HRP or THHR was pulsed into Cav1\u2212/- MEFs for 15 s, 1 min, or 2 min. Cells were fixed and processed for vertical sections. Arrows show HRP-labeled carriers. Bar, 200 nm. (G) Strategy measurements were captured across 20\u201325 cells in three independent areas as treated in F. Error bars show SEM.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG7.png": "'Figure 7. **Model of CUC endocytosis.** [_A_] [_1_] CUC cargo, such as Thy-1, CD44, and myofarlin are concentrated within Fluillin-1 and cholesterol-enriched microdomains. [_2_] Actin and G&AF-1 drive the inside formation of the carriers, within 15 s. [_3_] Recruitment of dynamin, Sab11, and Rab5/2 EEA-1 complexes within 2 min provides the ability for these carriers to facilitate bulk membrane flow to early endosomes (4a) and fast plasma membrane recycling [_4b_]. [_3_] After abrasion to the PM an influx of Ca2+ activates the fusogenic C2 domains of dyafarlin/myofarlin, resulting in free-praferential recycling of the CUC pathway.\\n\\n'", "CAPTION FIG6-1.png": "'Figure 6: **CICs become polarized during and are necessary for efficient cellular migration.** [A] Corbitant monolayers of WT MEFs or NH3T3s were scratched and cells were allowed to migrate for 8\u201312 h. CTxB-555 and Tf-647 were pulsed into migrating cells for 2 min in the presence of anti-CD44 (WT MEFI), anti-GFP (GFP-GPI expressing WT MEF), or anti-Thy-1 (NH3T3) antibodies or calls were labeled for Cox-1 [WT MEFI]. Dotted lines indicate leading edges. Arrows show colocalization between anti-CD44 or anti-Thy-1 and CTxB-555 but not Tf-647 at the leading edge. Bor, 20 \u03bcm. [B] Questions of average pixel intensity from the leading to trailing edges for CTxB-555, Cox-1, and Tf-647. Heart shows the concentration of tubular, Cox-1, and Tf-negative CTxB-labeled CULCs at the leading edge. A rectangular area, outlined, was used to calculate the average pixel intensity (along the y axis) across the leading to trailing edge [along the x axis] for each endocytic marker. Plot profiles identify a concentration of CTxB in the leading edge and Cox-1 in the trailing edge whereas Tf shows uniform intensity across the cell. Bar, 10 \u03bcm. [C] Electron micrographs of a migrating WT MEFI. Large arrow indicates direction of migration. Magnifications from the leading edge and the trailing edge show representative images of CTxB-HRP-labeled CULCs\\n\\n'", "CAPTION FIG6-2.png": "'[1, 2] and surface-connected caveolae [3, 4]. Bar, 500 nm. [D] Quantitation of the number of endocytic structures at the leading and trailing edge of cells treated as in C. Budded caveolae and CCVs are positive for CTxB-HRP label, whereas caveolae and CCPs are not. Error bars show SEM. [E] WT MEFs were incubated with or without CTxB+HRP for 2 min. The DAB reaction was performed on live cells for 5 min. Cells were washed and allowed to grow for a further 4 h. CLbS-555, anti-CD44 antibodies, and Tf-647 were added directly to cells for 2 min of uptake before acid stripping and fixation. Bar, 10 \u03bcm. [E] 12\u20131.5 cells treated as in E were quantitated for orange fluorescence intensity of CTxB-555, Tf46.47, or goat anti-mouse-488 for mouse-anti-CD44. [G] Confluent monolayers of WT MEFs were scratched and cells were allowed to migrate into the space for 1 h. Cells were incubated with serum alone or serum with 10 ug ml-1 CTxB+HRP for 2 min and the DAB reaction was performed as in E. After 4 h of migration, cells were fixed and distance migrated was determined by measuring the distance of the gap between both sides of the wound at time zero and after 4 h of incubation. Error bars show SEM from three independent experiments.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Quantification of CIIC endocytosis.** [A] Cav1\u2212/- or wildtype [WT] MEFs were left untreated or were treated with Dyngo4a. CTRh-HRP was internalized for 15 s, 1 min, or 2 min. Examples of CTxRh-labeled structures after 15 s of upticks are shown [inset, left panel]. Substratum indicated by large arrowhead, grid sizes are 2,000 or 200 nm, examples of intersections shown by arrows. [B] 20\u201325 cells treated as in A were used to calculate the volume fraction [V(g)]. Error bars show SEM. [C] NIH3T3 cells not treated with inhibitor were processed as in A and counted as in B. [V] was calculated for both tubular and vesicular structures. Error bars show SEM. [D] Cav1\u2212/- MEFs were incubated with HRP for 15 s, and in an ion and HRP-laden carriers counted as in B. Error bars show SEM. [E] CTRh was conjugated with NH3T5S-biotin and added to untreated Cav1\u2212/- MEFs or Cav1\u2212/- MEFs treated with Dyngo4a for 15 s or 2 min or was bound to untreated cells on ice for 10 min [Surface + MesNa]. Cells were placed on ice and residual surface biotin cleaved with MesNa. Western blots of cell lysates were probed with streptavidin-HRP. Chart represents the average intensity of streptavidin-HRP across three independent experiments. Residue luminescence in Surface + MesNa samples indicates level of uncarboxylic bisin. Error bars show SEM. [F] Absolute volume of CIICs was estimated from the volume fraction, [V(g)], multiplied by the average volume of a Cav1\u2212/\u2212 MEF, 2,3.47 \\\\(\\\\mu\\\\)m2. Surface density [S(g)] was calculated from high resolution images of labeled structures using a cycloid grid as described in Materials and methods and multiplied by the absolute volume to give absolute surface area. Volume of a single carrier was calculated as described in Materials and methods. Number of CIIC budding events per minute per cell was calculated based on the absolute volume internalized by all CIICs divided by the volume of a single carrier. Volume adjustments for overprojection effects are in brackets (see Materials and methods).\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/jeeRelationalScaffoldingEnhances2019.json b/test_extracted_captions/jeeRelationalScaffoldingEnhances2019.json new file mode 100644 index 0000000000000000000000000000000000000000..08d4943e8e1696d374045e403a1c12b972bf83af --- /dev/null +++ b/test_extracted_captions/jeeRelationalScaffoldingEnhances2019.json @@ -0,0 +1 @@ +{"CAPTION TAB4.png": "'\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Posttest and delayed-posttest day/night-cycle-understanding scores for participants who scored below and above the median on the pretest. Results are shown separately for sequential scaffolding (SS) and relational scaffolding (RS). Individual participant scores are shown in gray; means are in orange. Error bars show standard errors. Individual scores were jittered to produce separation on the \\\\(x\\\\)-axis. SS = sequential scaffolding; RS = relational scaffolding.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5.** Overview of procedure for Experiments 1 and 2.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB3.png": "'\\n\\n**Table 3.** Means for Demographic and Cognitive Variables for Experiment 2'", "CAPTION FIG2.png": "'Figure 2: Screen captures of relational-scaffolding video footage from an embodied simulation (top row) and a 3-D model simulation (bottom row). In the embodied simulation, the student enacted Earth\u2019s rotation in relation to the yellow ball representing the sun (top left). In the 3-D model simulation, the student watched from a third-person perspective as the model of Earth rotated on its axis (bottom left). Images on the right were captured by a GoPro camera mounted on the participant\u2019s head (top right) and on model Earth (bottom right). Arrows indicate paths of motion or apparent motion.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table 1.** Means for Demographic and Cognitive Variables for Experiment 1.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB2.png": "'\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The mathematical theory of the mathematical theory'"} \ No newline at end of file diff --git a/test_extracted_captions/jin2022branched.json b/test_extracted_captions/jin2022branched.json new file mode 100644 index 0000000000000000000000000000000000000000..f98e84fdc5676cb758a28ac68ab4c361411d75fa --- /dev/null +++ b/test_extracted_captions/jin2022branched.json @@ -0,0 +1 @@ +{"CAPTION FIG5.png": "'\\nFig. 5: **Action positive CME sites show distinct dynamics.****a** Histograms of ARPC3 negative (blue) and positive (orange) CME lifetimes. CME lifetime is measured from the first frame of the AP2 signal to the presumed scission time (the peak of DNM2 signal). ARPC3 positive CME events have longer lifetimes. p-value from two-sided Welch\u2019s t-test: 2.1e-76. **b** Averaged intensity vs time plots of ARPC3 negative (top) and positive (bottom) CME sites in ADA cells. Events were aligned to the frames showing the maximum DNM2 intensity. Error bar: **%** standard deviation. **c** Lateral motility of ARPC3 negative (blue) and positive (yellow) CME sites before (solid line) and after (dashed line) vesicle excision. ARPC3 positive CME sites move slower than ARPC3 negative ones. p-value from two-sided Kolmograv-Smirnov test: ARPC3 + before vs ARPC3- before: 1.27e-14, ARPC3 + after vs ARPC3- after: 5.18e-08. **d** Straightness-index of ARPC3 negative (blue) and positive (yellow) CME sites before (solid line) and after (dashed line) excision. The straightness-index is defined by the ratio between the sum of frame-to-frame distances to the end-to-end distance of a single event\u2019s trajectory, where a perfectly straight-lined trajectory would have an index of 1. ARPC3 positive CME sites move with a more straight trajectory. p-value from two-sided Kolmogorov-Smirnov test: ARPC3 + before vs ARPC3- before: 1.07e-11, ARPC3 + after vs ARPC3- after: 2.06e-6. **a\u2013d** ARPC3 + \\\\(N\\\\)= 840, ARPC3 + \\\\(N\\\\)= 1,385. **a, c, d** Source data are provided in the Source Data file.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n## References\\n\\nFig. 7: **An updated schematic model of actin-negative and actin-positive clathrin-coated pits in human cells.** Actin assembly is induced at stalled CME sites, where asymmetric forces pull, bend and possibly twist the plasma membrane against membrane tension to drive membrane invaginations and vesicle scission.\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: **Airscan live-cell imaging reveals asymmetric actin organization at CME sites.****a** A representative single time frame image of an Airscan movie (Supplementary Movie 3) of DNM2-tagGP72 (red) and JFG35 ligand-conjugated ARPC3-HaloTag (cyan) on the ventral plasma membrane surface of ADAM cells. The highlighted region is boxed by a dashed line. Scale bar: 1\\\\(\\\\mu\\\\)m. **b** Histogram of distance between the center of mass of DNM2 and ARPC3 at frame corresponding to scission. Mean and standard deviation are shown on the graph. Data were analyzed using Prism 9. Source data are provided in the Source Data file. **c** Montage of average intensity projection of representative ARPC3 positive CME sites. Frame corresponding to scission was determined by maximum DNM2 intensity. \\\\(33\\\\times 33\\\\) pixels square region centered at DNM2 maximum intensity pixel was cropped for six continuous frames ending in scission. Cropped frame series were rotated by 90 or 180 degrees as needed to align the ARPC3 signal to the left of the center of the image at the frame corresponding to scission. *: Averaged image of the frame corresponding to scission (maximum DNM2 intensity) is marked to show the displacement between the CME neck (DNM2) and branched actin (ARPC3). Scale bar: 1\\\\(\\\\mu\\\\)m. Intervals: 0.2\\\\(\\\\times\\\\). \\\\(N-72\\\\). **d** The line scan function in ImageJ software was used to measure the fluorescence intensity along a one pixel wide, 33 pixel long horizontal line drawn across the center of the averaged intensity image of the frame corresponding to scission* in **c**. The signals were normalized to the minimum signal along the line, and intensity was calculated as a ratio to the maximum signal along the line. Source data are provided in the Source Data file.\\n\\n'", "CAPTION FIG6.png": "'\\nFig. 6: **Asymmetric N-WASP recruitment to stalled CME sites.****a** A representative single time frame image of a TIRF movie (Supplementary Movie S) of AP2MI-tagRFP-T (magenta), DNM2-tagGFP2 (green) and JF635 ligand-conjugated HaloTag-N-WASP (cyan) in ADW cells. The highlighted region is boxed by a dashed line. Scale bar: 5 \\\\(\\\\mu\\\\)m. **b** A representative kymograph of CME sites in ADW cells over a 4 min movie. Scale bar: 5 \\\\(\\\\mu\\\\)m. **c** Averaged intensity (solid line) and distance (dashed line) vs time plots of N-WASP positive CME sites in ADW cells. Events are aligned to the frames showing the maximum DNM2 intensity. Intensity is scaled to 1 at peaks for each channel. Error bar: 3\\\\(\\\\mu\\\\) standard deviation. **d** N-WASP positive CME events have longer lifetimes. p-value from two-sided Welch\u2019s t-test: 9.06t-20. **e** Intensity vs time plots of averaged N-WASP negative (top) and positive (bottom) CME sites in ADW cells. Events were aligned to the frames showing the maximum DNM2 intensity. Error bar: 3\\\\(\\\\mu\\\\) standard deviation. Source data are provided in the Source Data file. **c**, **d** N-WASP negative CME sites: \\\\(N=299\\\\), N-WASP positive CME sites: \\\\(N=199\\\\).\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Fig. 1 Two-color, 3D stochastic optical reconstruction microscopy (STORM) shows that actin structures are off-centered with respect to clathrin coats.****a**, **b** Two-color 3D STORM image of bottom membrane of ADA cells immunostained with clathrin light chain antibody (clathrin, CF-680, magenta) and phalloidin (actin, AF647, rainbow). **c** **A** histogram of distances between centroids of clathrin and actin signals. Mean and standard deviation are reported on the graph. Data were analyzed using Prism 9. Source data are provided in the Source Data file. **d**, **e** Two color 3D STORM image of the bottom membrane of ADA cells immunostained with clathrin light chain antibody (clathrin, AF647, magenta) and HaloTag antibody (ARPC3-HaloTag, CF-680, rainbow). Dotted lines label lamellipodia. **b**, **e** The highlighted CME sites, which are labeled by white arrows in **a**, **d**, are rotated and shown in magnified top and side view projections. Color bar shows the \\\\(z\\\\) position of ARPC3-HaloTag. **f** A histogram of distances between centroids of clathrin and ARPC3 signals. Mean and standard deviation are reported on the graph. Data were analyzed using Prism 9. Source data are provided in the Source Data file. Scale bars: **a**, **d** 2 \u03bcm, **b**, **e**: 100 nm.\\n\\n'", "CAPTION FIG2.png": "'\\nFig. 2: **Triple-genome-edited iPSCs reveal dynamic actin organization at CME sites.****a** Models of branched actin assembly at invaginating CME sites.****a** Model 1: Asymmetric actin assembly at CME sites results in separated actin-coat and actin-neck signals. Model 2: Symmetric actin assembly at tilted CME sites results in separated actin-coat signals but overlapped actin-neck signals. Model 3: Symmetric actin assembly at perpendicularly inaginating CME sites will result overlapped actin, coat and neck signals. **b** A representative single time frame image of a TRF movie (Supplementary Movie 2) of AP2Multi-tagRFP-T (magenta), DNM2-tagGFP2 (green) and JF635 ligand\\\\({}^{\\\\text{61}}\\\\)-conjugated ARPC3-HaloTag (cyan) in ADA cells. The highlighted region is boxed by a dashed line. Scale bar: 5 \u03bcm. **c** A representative kymograph of AP2M1-tagRFP-T (magenta), DNM2-tagGFP2 (green) and JF635 ligand-conjugated ARPC3-HaloTag (cyan) at CME sites in ADA cells. Scale bar: 5 \u03bcm. **d** Montage of a representative ARPC3 positive CME site in ADA cells. Individual channels and pair-wise merges are shown. *: Images from one frame before scission (maximum DNM2 intensity) are marked to show the displacement between the CME coat (AP2)-ARPC3 and CME neck (DNM2)-ARPC3. Size of field of view: 2 \u03bcm \u00d7 2 \u03bcm. intervals: 1 s.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n## 3 Results\\n\\n### Results\\n\\nWe first present the results of the _'"} \ No newline at end of file diff --git a/test_extracted_captions/jin2022branched_supp.json b/test_extracted_captions/jin2022branched_supp.json new file mode 100644 index 0000000000000000000000000000000000000000..028db18ef71f0d27268b7924a6d57906d61057fc --- /dev/null +++ b/test_extracted_captions/jin2022branched_supp.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS1-1.png": "'\\n\\n**Supplementary Fig. 1: Genome-edited iPSCs show dynamic CME sites.**\\n\\n**a** Schematic model of CME. Mammalian CME proteins can be grouped into several modules, including the coat, WASP and Myosin / actin nucleation promoting factor (NPF), actin and scission modules. Actin networks provide pulling forces to invaginate the membrane against membrane tension. **b** Immunoblot analysis of cell extracts from the WT (WTC) and genome-edited (AP2M1-tagRFP-T/DNM2-tagGFP2/ARPC3-HaloTag; ADA) human iPSCs. The labeled proteins were detected with tag(CGY)FP, HaloTag, and GAPDH (loading control) antisera respectively. N=1. Uncropped and unprocessed scans of the blots are provided in the Source Data file. **c** Kymograph of representative CME sites of double-edited (AP2M1-tagRFP'", "SUPP CAPTION FIGS1-2.png": "'T/DNM2-tagGFP2; AD) and triple-edited (AP2M1-tagRFP-T/DNM2-tagGFP2/ARPC3-HaloTag; ADA) cells. Scale bar: 5um.\\n\\n'", "SUPP CAPTION FIGS4.png": "'\\n\\n**Supplementary Fig. 4: Computational analysis reveals actin network assembly at the late stage of CME.**\\n\\n**a** We generated a randomized data set by pairing ARPC3 images with AP2 and DNM2 images from an unrelated movie. **b** The fraction of CME sites without actin assembly (Negative), with early actin assembly (ARPC3 signal disappears before DNM2 signal peak), and with late actin assembly (ARPC3 signal overlaps with DNM2 signal peak) were calculated for real data movies and randomized control movies (**a**). In the randomized dataset, we detected early \"assembly\" of actin in a similar fraction of CME events as in the real data set, indicating that presence of actin detected early in CME is very likely an artificial association caused when nearby actin structures overlap with CME sites by chance. Mean and standard deviation are shown on the graph. Source data are provided in the Source Data file.\\n\\n'", "SUPP CAPTION FIGS2.png": "'\\n\\n**Supplementary Fig. 2: Actin assembles at different types of CME sites.**\\n\\n**a** Montage of a representative ARPC3 positive CME plaque from a TIRF movie of triple-edited (AP2M1-tagRFP-T/DNM2-tagGFP2/ARPC3-HaloTag; ADA) human iPSCs (Supplementary Movie 2). **b** Montage of a representative ARPC3 positive splitting CME site from a TIRF movie of triple-edited (AP2M1-tagRFP-T/DNM2-tagGFP2/ARPC3-HaloTag; ADA) human iPSCs (Supplementary Movie 2).\\n\\n'", "SUPP CAPTION FIGS7.png": "'\\n\\n**Supplementary Fig. 7: Dynamics of N-WASP at CME sites.**\\n\\n**a** Immunoblot analysis of cell extracts from the control and genome-edited (AP2M1-tagRFP-T/DNM2-tagGFP2/HaloTag-WASL; ADW) human iPSCs. The labeled proteins were detected with HaloTag and GAPDH (loading control) antisera respectively. N=1. Uncropped and unprocessed scans of the blots are provided in the Source Data file. **b** Histogram of N-WASP lifetime at CME sites. The lifetime is measured from the first frame of the N-WASP signal to the presumed scission time (the peak of DNM2 signal). **c** Intensity vs time plots of cohorts of N-WASP positive CME sites in ADW cells. Events are grouped into cohorts by the lifetimes of AP2 and aligned to the frames showing the maximum DNM2 intensity. Data are presented as mean values +/- 1/4 standard deviation. N=1,199. Source data are provided in the Source Data file.\\n\\n'", "SUPP CAPTION FIGS6.png": "'\\n\\n**Supplementary Fig. 6: Arp2/3-mediated actin assembly facilitates CME.**\\n\\n**a** Intensity vs time plots of averaged ARPC3 negative (top) and positive (bottom) CME sites in ADA cells after 1-5min treatment with 2% DMSO (solid lines) or 100 mM CK666 (dashed lines). Events were aligned to the frames showing the maximum DNM2 intensity. Error bar: 1/4 standard deviation. ARPC negative CME sites: DMSO: N=402, CK666: N=261, ARPC3 positive CME sites: DMSO: N=1,006, CK666: N=580. **b** CK666-treated cells have longer CME lifetimes than DMSO-treated control cells. p-value from two-sided Kolmogorov-Smirnov test: 1.83e-3. Source data are provided in the Source Data file.\\n\\n'", "SUPP CAPTION FIGS3.png": "'\\n\\n**Supplementary Fig. 3: Filtering methods for selection of CME sites.**\\n\\n**a** 2-D histogram of the first two principal components (PCs) of AP2 and DNM2 dynamic features. Of the 121,349 total tracks detected by CMEanalysis, \\\\(\\\\sim\\\\)49% were valids (as defined previously[30]), \\\\(\\\\sim\\\\)31% had detection gaps, \\\\(\\\\sim\\\\)15% were persistent events, and \\\\(\\\\sim\\\\)5% were split/merges. Only valid tracks (N=59,239), which appear and disappear during the course of movie acquisition, were used to generate filtering methods and for subsequent analysis. The shaded underlay represents simulated data points in principal component space and their individual probabilities of belonging to the nearest cluster center. Cluster 0 shows data points in the DNM2-positive cluster, which contains 10.02% of total valid tracks. Clusters 1-4 represented 24.60%, 10.43%, 17.03%, and 37.91% of valid tracks, respectively. **b** Cohort plots of the shortest AP2 events (\\\\(<\\\\)40 seconds) from each cluster. Cluster 0 represents DNM2-positive events where a strong DNM2 signal is detected. Data are presented as mean values \\\\(+\\\\)/- 1/4 standard deviation. **c** DNM2-positive events are sorted by the number of DNM2 peaks using a peak-detection scheme. Representative intensity vs time plots of a single-peaked event (top), non-peak event (bottom left) and a multi-peaked event (bottom right). Percentage of the number of the events in each class is shown next to the plot. **d** Single-peaked DNM2 events (N=2,538), hereon named CME sites, are grouped into lifetime cohorts and aligned to the peak of the DNM2 channel. Percentage of the number of the CME sites in each cohort is shown next to the plot. Data are presented as mean values \\\\(+\\\\)/- 1/4 standard deviation.\\n\\n'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Supplementary Fig. 5: AP2-ARPC3 separation is not due to imaging artifacts.**\\n\\n**a** Montage from a TIRF movie of a multi-fluorescence bead (Supplementary Movie 4). Size of field of view: 2\\\\(\\\\upmu\\\\)m x 2\\\\(\\\\upmu\\\\)m. Intervals: 1sec. **b** A boxplot of inter-channel distances (note: y-axis is base-10 log scale) between centroids of either CME sites or beads to highlight that the observed separation between marked proteins exceeds the measured chromatic aberration. Beads were compared between the same pairs of channels as used to image the tagged proteins. Box plot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Source data are provided in the Source Data file. **c** A heat map graph of distance between AP2 and ARPC3 signals before scission, and average AP2 frame to frame displacement within 6 seconds before scission. Over 95% of the CME events present larger AP2-ARPC3 separation than AP2 displacement. N= 1,385.\\n\\n'", "SUPP CAPTION FIGS1.png": "'\\n## References\\n\\n* [1]\\n* [2]**Supplementary Fig. 1: Counter-coded PICC same dynamic CEM data.**\\n* [3]**: A schematic model of CEM. Materials CEM points can be grouped into several modules, including the core, WAY and layouts (with modifications requiring core), and modules including the core, WAY and layouts (with modifications requiring core), and modules include module, Multi-core concepts, multiple design concepts to highlight the mechanism, pipeline mechanism training, Simulation analysis and cell detection from the WT (VTC) and geometric center (V2/31-tag/1-2008-tag/2007-2008-tag'"} \ No newline at end of file diff --git a/test_extracted_captions/jingHouseholdSecondaryAttack2020.json b/test_extracted_captions/jingHouseholdSecondaryAttack2020.json new file mode 100644 index 0000000000000000000000000000000000000000..c992422f40218b32e56755d5d68a4cba9c1e875a --- /dev/null +++ b/test_extracted_captions/jingHouseholdSecondaryAttack2020.json @@ -0,0 +1 @@ +{"CAPTION FIG2 (NOISY).png": "'\\n\\n**Figure 2:** Epidemic curve based on symptom onset dates of COVID-19 crisis in Guangzhou from Jan &, 2020, to Feb 18, 2020\\n\\n**Estimated \\\\(R\\\\), for those scenarios: scenario 1, all imported cities (who traveled to or resided in Huisei province 14 days before symptom onset) considered as primary cities, and all secondary cities were infected by primary cases in the same case district; scenario 2, which is identical to scenario 1, with the additional assumption that local primary cases might have been infected by earlier cases in other clusters; and scenario 3, which is identical to scenario 2, with the additional assumption that imported secondary cases were considered as infected by primary cases in the same cluster. \\\\(R\\\\)=effective reproductive number.**'", "CAPTION TAB2 (NOISY).png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG1 (NOISY).png": "'Figure 1: **Spatial distribution of COVID-19 case clusters on the basis of contact tracing data from Guangzhou, China, from Jan 7, 2020, to Feb 18, 2020**\\n\\nOverall distribution of COVID-19 case clusters in Guangzhou (A), and distribution in the submissions defined in panel A (B-G). Individuals were considered as primary cases if their symptom onset dates were the earliest or 1 day (14) days for an imported case) after the earliest in the cluster and as secondary cases otherwise. Non-infected contacts are not shown. The displayed location of such case is randomly perturbed away from the actual residential address.\\n\\n'", "CAPTION TAB4 (NOISY).png": "'Data are 00 [95% CI]. Initration were repeated for selected settings of the natural history of disease (in, mean incubation and maximum infectious periods). The model was adjusted for age groups, epidemic phase, and household size. (Q1=odds ratio).\\n\\n'", "CAPTION TAB3 (NOISY).png": "'Data are estimating (95% C). Estimation of the daily probability of infection from an external source and the GHR for the relative infectivity during the disease versus irradiation period are also provided. Extrusion as reported using two different definitions of household contact (dose relation, or only individual sharing the same residential address) and for selected setting of the natural history of disease (no mean incubation and maximum infectious period). This model was not adjusted for age group, epidemic phase, or household use. D6-odds ratio.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/johnston2018novel.json b/test_extracted_captions/johnston2018novel.json new file mode 100644 index 0000000000000000000000000000000000000000..10fc134b6b504290a7fa89ec236c7bb87ea551bf --- /dev/null +++ b/test_extracted_captions/johnston2018novel.json @@ -0,0 +1 @@ +{"CAPTION FIG6-1.png": "\"Figure 6: mTwf1 and Capping Protein colocalize and have similar phenotypes in B16F10 melanoma cells. **(A)** Representative images from immunoblotting staining showing colocalization of endogenous mTwf1 (yellow) and Capping Protein (magenta). Scale bar, 20 \u03bcm. Close ups of boxed regions shown in Zooms; scale bar, 4 \u03bcm. **(B)** Mander's correlation coefficient **(M1 and M2)** values of overlap between mTwf1 and Capping Protein (CP) measured from cells (\\\\(n\\\\) = 47 cells) as in **(A)** Error bars, s.e.m. **(C)** Comparison of the relative abundance of mTwf1 and Capping Protein (CP) in B16F10 cells measured from western blot analysis. Data averaged from four separate experiments. Error bars, s.d. n.s. p>0.05 by t-test. **(D,E)** Representative western blots and quantification of cellular levels of mTwf1 **(D)** and CP **(E)** in B16F10 cells treated with siRNA against mTwf1 **(si-Twf1)** or Figure 6_continued on next page_\\n\\n\"", "CAPTION FIG7.png": "'Figure 7: Overexpression of Tsurfilin suppresses morphological defects caused by CARMIL hyperactivity. (**A** Representative images of F-actin staining in untreated B16F10 cells [control], and cells transfected with Flag-CARMIL1, full-length (FL)-myc-mTurf1, or both. Scale bar, 20 mm. (**B** Average Microspike density in cells treated as in (**A**). Box and whisker plots show mean, first and third quartile, and the maximum and minimum values. Data averaged from two experiments (_n_ = 19-25 cells per condition). Data averaged from two experiments. From left to right: \\\\(n\\\\) = 19, 25, 20, and 25; mean microspike density 0.75, 1.13, 0.62, 0.58 filopodia per 10 mm of cell cortex. Error bars, s.a.m. ***p<=0.001, n.s. p>0.05 by one-way ANOVA with Tukey post hoc test. (**C** Earlier model for CP regulatory cycle, adapted from Fujiwara and colleagues (Fujiwara et al., 2014). Proposed steps in model: (1) V-1 globally inhibits Capping Protein (CP) in the cytoplasm, (2) membrane-associated CARMIL [at the protruding cell edge] catalyzes dissociation of V-1 from CP, (3) the resulting CARMIL-CP complex is partially active, binding weakly to free barbed ends to provide capping function, (4) an unknown factor or mechanism promotes dissociation of CARMIL from CP, allowing V-1 to rebind CP and complete the cycle. (**D** Our revised working model for the CP regulatory cycle. We propose that V-1 functions to maintain a cytosolic reservoir of inactive CP, from which Twinfilin and CARMIL activate CP, generating two distinct forms of active CP in cells: Twinfilin-CP complexes and CARMIL-CP complexes. Twinfilin-CP complexes are fully active and support stable capping of barbed ends. In contrast, CARMIL-CP complexes have ~100 fold reduced affinity for barbed ends, and may therefore more transiently cap barbed ends, permitting restricted network growth at the cell membrane where CARMIL localizes. CARMIL and Twinfilin directly compete with each other for binding CP (shown in close up of Transition state), which may result in the displacement of CP from Twinfilin. This would disease Twinfilin at the barbed end to catalyze depolymerization, or alternatively return filaments back to the original state of assembly.\\n\\nDOI: https://doi.org/10.7554/elte.41313013\\n\\nThe following figure supplement is available for figure 7:\\n\\nFigure 1: Structural model for a ternary complex formed by Twinfilin, Capping Protein and the barbed end of an actin filament. Figure 7 continued on next page\\n\\n'", "CAPTION FIG6-2.png": "'Figure 6 continued\\n\\nCP (si-CP) or negative control (Control). Band intensity for control cells was set to 1.0. Data averaged from at least three separate experiments, error bars, s.d. (**F**) Representative images showing F-actin immunofluorescence in B16F10 cells treated with siRNA against mTwf1 (si-Twf1) or CP (pi-CP) or negative control (Control); siRNA treated cells (si-Twf1 or si-CP) were also rescued using plasmids expressing si-resistant FL-myc-mTwf1 (MT or mTwf1-1). Scale bar, 20 mm. Close ups of boxed regions shown in Zooms; scale bar, 4 mm. (**G**) Microspike density in cells treated as in (**D**). Box and whisker plots show mean, first and third quartile, and the maximum and minimum values. Data averaged from two experiments. From Left to right: \\\\(n\\\\) = 45, 53, 51, 24, 24, and 20 and mean microspike density 0.69, 1.34, 1.77, 0.59, 1.24, and 1.01 filopodia per 10 mm of cell cortex. Error bars, s.e.m. ***p\\\\(\\\\leq\\\\)0.0001, *p\\\\(\\\\leq\\\\)0.05, n.s. p\\\\(>\\\\)0.05 by one-way ANOVA with Tukey post hoc test.\\n\\nDOI: https://doi.org/10.7554/eLife.41313.011\\n\\nFigure supplement 1. Supporting data for Figure 6.\\n\\nDOI: https://doi.org/10.7554/eLife.41313.012'", "CAPTION FIG2.png": "'Figure 2: Twartlin is a Capping Protein Interaction (CPI)-motif protein that competes with CARML for binding Capping Protein. (**A**) CARML domain organization: PH, plackstrin-homology domain; L, linker; N-cap (**N**), LRR, leucine-rich repeat domain; C, C-cap; HD, helical dimerization domain; CBR, Capping Protein binding domain, consisting of CPI, Capping Protein interaction domain, and CSI, CARML-specific interaction sequence; MBD, membrane binding domain; PRD, proline-rich domain. Alignment between the Capping Protein Interaction (CPI) motif consensus sequence, and the CPI regions of H-1 sequences (H-1,C-AMM11 (UnProttKB CSV20; 1), CARML12 (UnProttKB CSV26; 2), CARML13 (UnProttKB OSND23), CCPI UniProttKB O53610.2), CD2AP (CB1 NP_Q6262.1), WASHCAP (Fam21) (UnProttKB GP7451.3), CapZIP (CB1 NP_A43094.3), CINRS (UniProttKB O96897.2), and the tail sequences of Twartlin homologs from D. melanogaster (Dm), C. lectalurius (C1.), S. cerevisiae (S.c.), O. Taurus (D.t), S. libra (S.l), D. rerio (Dr.), H. sapiens (H-s.), and M. musculus (Mm.). Twartlin isoforms (Dm. Twaf1 UniProttKB NP_60338. C. Twaf1 UniProttKB XP_Q14258437.1, S.c. Twf1 GenBank SAK68393.1, C.t. Twf1 XP_Q22917989.1, S.l. Twf1 XP_Q22816377.1, D.r. Twf1 AAH+67368.1, _H-s. Twf1 UniProttKB NO_001229326.1, and M.m. Twf1 GenBank. GenBank: AAH+15081.1). Amino acid color coding illustrates side chain chemistry similarities. The asterisk marks the residue we mutated in mTwf1 in panel. (**C**) The alignments were generated using the MAFFT algorithm in the DNASTAR Lasergene Suite/MegAlign Pro application (MegAlign Pro. Version 15.0, DNASTAR. Madison, WI.1(**B**) Fluorescence anisotropy measurement of 60 nM Hylyt4488-labeled mTwf1 tail peptide mixed with increasing concentrations of the indicated CP construct. (**C**) Fluorescence anisotropy measurement of 40 nM TAMRA-labeled mTwf1 tail peptide incubated with 1 mM CP and different concentrations of wild type and mutant mTwf1 tail peptides. (**D**) Fluorescence anisotropy measurement of 60 nM Hylyt488-labeled mTwf1 tail peptide incubated in the presence of 240 nM CP and increasing concentrations of the indicated CARML fragment (CBR, CSI, or CPI). CSI failed to compete with Hyltyte 488-mTwf1 tail peptide at the concentrations tested. Anisotropy values for each condition were averaged from three independent experiments.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Barbed end capping by Capping Protein inhibits Twinfilin1-mediated depolymerization. **(A)** Mouse Twinfilin-1 {mTwTtl} domain organization: ADF-H, actin depolymerization factor homology domain; L, linker; T, tail. Sequence alignment of tail regions of Twinfilin isoforms from different species with boxed region highlighting conservation of residues critical for binding to Capping Protein (CP), mTwT-11 carries a mutation in the tail region (XR332,333AA) that disrupts binding to CP. **(B)** Fluorescence anisotropy measurement of 100 nM Hylte488-labeled mTwTtl tail peptide mixed with increasing concentrations of the indicated CP construct. **(C)** Fluorescence anisotropy measurement of 100 nM Hylte488-labeled mTwTtl tail peptide incubated in the presence 1 uM CP and increasing concentrations of either mTwTtl or mTwTtl-11. Anisotropy values for each condition averaged from three independent experiments. **(D,E)** Rates of barbed end depolymerization (subunits s\u22121) induced by 1 uM of the indicated mouse Twinfilin, in the **(D)** absence or **(E)** presence of 10 nM CP, determined from TIRF assays. Rates for each condition averaged from at least five filaments in each of two independent experiments. From left to right: **(D)**\\\\(n\\\\) = 19, 26, and 15 and mean depolymerization rates 1.13, 2.784 and 2.81 subunits s\u22121; **(E)**\\\\(n\\\\) = 13, 15, and 20 and mean depolymerization rates 1.13, 2.784 and 2.81 subunits s\u22121. **(F)** Rates of barbed end depolymerization [subunits s\u22121) induced by 1 uM that TNF, in the absence or presence of 1 uM of the indicated CP construct, determined from TIRF assays. Rates for each condition averaged from at least five filaments from at least one experiment. From left to right \\\\(n\\\\) = 21, 25, 6, and 10, mean depolymerization rates 1.45, 2.991, 0.11, and 3.58 subunits s\u22121. **(G)** Summary of barbed end depolymerization activity of mTwTtl constructs in combination with different CP constructs determined from TIRF assays (as in D,E,F). Error bars, s.o.m. ****ps0.0001, n.s. p>0.05 by one-way ANOVA with Tukey post hoc test.\\n\\nFigure 2: The following video is available for figure 1:\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Direct interactions of Tworillin with Capping Protein attenuate CARML-mediated uncapping. **(A)** Bulk fluorescence assays comparing the rates of actin assembly in the presence of 25 nM muscle Capping Protein (CPer[B]) and increasing concentrations of mTwf1. To initiate uncapping, 250 nM CBR fragment of CARML (see schematic, Figure 2A) was spiked into the reaction at 400 s. Data shown are representative curves from experiments repeated three independent times. **(B)** Representative time-lapse images from TIRF microscopy assays monitoring the displacement of labeled CP from barbed ends. Filaments were first polymerized and tethered using 1 uM actin (10% OG-labeled, 0.5% biotin-actin), then capped at their barbed ends by flowing in SNAP-649-CP (100% labeled). Next, 50 nM CBR fragment of CARML and different concentrations of mTwf1 were flowed in, and CP dissociation was monitored over time. Scale bar, 5 \u03bcm. **(C)** Quantification of the percentage of filaments retaining CP at the barbed ends in the presence of 50 nM CBR fragment of CARML and variable concentrations of mTwf1, determined from TIRF reactions as in **(B)**. Control curve, buffer alone (no CBR or mTwf1). \\\\(n>45\\\\) events measured from at least two independent experiments. **(D)** Representative time-lapse images from TIRF microscopy assays monitoring CP displacement from barbed ends, analyzed as in **(B)**, except using 1 uM mTwf1-1 instead of mTwf1. \\\\(n>45\\\\) events measured from at least two independent experiments. **(E)** Quantification of the percentage of filaments retaining CP at the barbed end in the presence of 50 nM CBR fragment of CARML and different concentrations of mTwf1-11, determined from TIRF assays as in **(D)**. \\\\(n>45\\\\) events measured from at least two independent experiments.\\n\\n'", "CAPTION FIG5.png": "'Figure 5.: HDX-MS analysis of Twinfilin reveals effects on Capping Protein structure near the CPI motif-binding site. (**A**) A cartoon representation of a crystal structure of CP, based on PDB 3AAA (Takeda et al., 2010). Differences in deuterium uptake induced by mTwf1 binding to CP are displayed as a color gradient (see scale at bottom of panel (B) CPI domain of CARMIL overlaid on to its binding site on CP (around the stalk). Representative comparisons of deuterium uptake curves for free CP (black) with mTwf1 bound CP (red) for CP alpha subunit (upper panels) and CP beta subunit (lower panels). Error bars representing the results of t-tests between samples are shown above each time point to illustrate statistical significance. When error bars are not shown explicitly, the error is within the radius of the symbol. Data shown are representative curves from experiments repeated two independent times. (**B**) A cartoon representation of a crystal structure of CP, showing the differences in deuterium uptake induced by CBR domain of CARMIL binding to CP are displayed as a color gradient (see scale at the bottom). CPI domain of CARMIL overlaid on to its binding site on CP (around the stalk), V-I is overlaid on its binding site on CP (barbed and binding surface) for comparison.\\n\\nDOI: https://doi.org/10.7554/eLife.4131.006'", "CAPTION FIG4.png": "\"Figure 4: Tsurflin's direct binding to Capping Protein accelerates the disassociation of V-1 to promote capping of filaments. **(A, B)** Seeded elongation assays comparing the rates of actin assembly from spectrin-F-actin seeds (grey) in the presence of 0.5 mM actin (10% pyrene-labeled), 25 nM muscle Capping Protein (CapZi, 500 nM V-1, and variable concentrations of mTwf1 **(A)** or mTwf1-11 **(B)** as indicated. Data shown are representative curves from experiments performed three independent times. **(C)** Fluorescence anisotropy measurement of 100 nM Hilyto488-labeled mTwf1 tail peptide mixed with 1 mM mouse Capping Protein (CP) and variable concentrations of CBR fragment of CARMill or V-1. Rates for each condition averaged from three independent experiments. **(D)** Stopped-flow fluorescence assays measuring the kinetics of dissociation of 50 nM TAMRA-V-1 from 1 mM CP upon addition at time zero of 2.5 mM unlabeled V-1 and variable concentrations of mTwf1 as indicated. Apparent dissociation rates are listed for each condition. **(E)** Apparent dissociation rates of TAMRA-V-1 for different concentrations of mTwf1 are from **(D)**; and for 12 mM mTwf1-11 = 1.0 +- 0.003 s\u22121. Anisotropy values for each condition were averaged from five independent experiments. **(D)** Only: https://doi.org/10.7554/eLife.41313.007\\n\\n\"", "CAPTION FIG6.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kamrahHowDiverseInitial2023.json b/test_extracted_captions/kamrahHowDiverseInitial2023.json new file mode 100644 index 0000000000000000000000000000000000000000..409c556b57cc4603d91ff19441406b07ec509bfb --- /dev/null +++ b/test_extracted_captions/kamrahHowDiverseInitial2023.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Fig. 1 Correlation matrix showing the relative correlation between two gammas by comparing the way our DPP approach ranks 10,000 sampled sets of cardinality, \\\\(k\\\\!=\\\\!10\\\\). The gammas values in both axes here are logarithmic values with base 10.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\n* [1] A. A. Barabasi, A.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Scatter plots showing randomly chosen sets with \\\\(k=5\\\\) high-diversity and low-diversity samples with their diversity score on top of each of the chosen set\\n\\n'", "CAPTION FIG4.png": "'\\n\\n## 4 Conclusion\\n\\nFigure 4: Experiment 1: optimality gap grid plot showing the difference in current optimality gap between optimizers initialized with 5th versus 95th percentile diverse sample (y-axis) as a function of optimization iteration (x-axis). The different factors in the factor grid plot the effects of diversity as the noise amplitude and smoothness are varied in the range [0.2, 0.8]. Each plot also has text indicating the net cumulative optimality gap (NCOG), a positive value corresponds to a better performance by high-diversity samples compared to the low-diversity samples. The plot shows that BO benefits from diversity in some cases but not others. There is no obvious trends in how the NCOG values change in the grid. The results are further discussed in Sec. 3.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\nFig. 5: Experiment 2: box plot showing distribution of \u201clengthscale\u201d hyper-parameter learned by BO when initiated with diverse (right-side) and non-diverse samples (left-side) for 16 different families of wildcat wells functions of the same parameters but 100 different seeds. The optimal hyper-parameter for each of the 100 wildcat wells instances from each family is also plotted as horizontal lines\u2014in many but not all cases these overlap. Each cell in the plot also has the 95th percentile confidence bound on MAE for both diverse and non-diverse samples. The results show that MAE confidence bounds for non-diverse samples are smaller compared to diverse samples for all the families of wildcat wells function. Thus, indicating a presence of model building advantage for non-diverse initial samples. The results of this figure are further discussed in Sec. 4.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## 4 Conclusion\\n\\nFigure 6: Experiment 3: optimality gap plot showing effects of diversity when the optimizer is not allowed to fit the hyper-parameters for the Gaussian process and the hyper-parameters are instead fixed to the values found in experiment 2. The results from this plot show positive NCOG values for all families of wildcat wells function, showing that once the model building advantage is taken away, the diverse samples outperform non-diverse samples. Further discussion on this plot can be read in Sec. 5.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kangAugmentingScientificCreativity2022.json b/test_extracted_captions/kangAugmentingScientificCreativity2022.json new file mode 100644 index 0000000000000000000000000000000000000000..405024cf71f0f1707950fb38d9e91dc0b07d698c --- /dev/null +++ b/test_extracted_captions/kangAugmentingScientificCreativity2022.json @@ -0,0 +1 @@ +{"CAPTION FIG5.png": "'Figure 5: (Left) Participants judged analogy articles significantly more novel. The mean response to the question _\u201cHave you seen this article before?\u201d_ was significantly higher in Analogy: 2.7 (SD: 0.48) than in Keyword: 2.3 (SD: 0.55). (Right) There were significantly more overlapping words between search query terms provided by participants and the title and abstract text of articles: Keyword: 4.1 (SD: 1.74) vs. Analogy: 1.6 (SD: 1.42).\\n\\n'", "CAPTION FIG4.png": "'Fig. 4. The front-end interfaces. (Left) Human reviewers used this filtering interface to input search queries received from the participants and remove articles with obviously irrelevant purposes. To assist the reviewers\\' filtering process, model predicted purpose (e.g., the noise reduction and time, highlighted in red at the bottom of the filtering interface) and mechanism (highlighted in green) tokens were also provided along with the title and the abstract text. The background color turned green when the \"Similar\" button is clicked and red when the \"Dissimilar\" button is clicked. (Right) The ideation task interface was populated with a list of human filtered articles for review by the participants in Study 1 (the order of articles was randomized).\\n\\n'", "CAPTION TAB3-1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3-2.png": "\"Each participant's research problem is described in the Problem column. While the topics of research problems vary, Creative Adaptation ideas are more distant in terms of content compared to the source problem than Direct Application ideas are, and may be characterized by the use of different sets of verbs (|design, invent|) in Creative Adaptation ideas versus |apply, adopt| in Direct Application ideas).\\n\\n\"", "CAPTION FIG6.png": "'Figure 6: Frequency of the ideation outcome types by condition. Darker colors represent higher rates. Creative adaptation is 5.3 times more frequent among analogy articles (\\\\(53\\\\) in Analogy vs. \\\\(10\\\\) in Keyword), while most of direct application is from keyword articles (\\\\(3\\\\) in Analogy vs. \\\\(16\\\\) in Keyword). The distributions differed significantly (chi-squared test, \\\\(\\\\chi^{2}(3)=52.12,p<1.0\\\\times 10^{-10}\\\\) overall and \\\\(\\\\chi^{2}(1)=28.41,p=9.84\\\\times 10^{-8}\\\\) for the contrast between the rates of creative adaptation and direct application ideas).\\n\\n'", "CAPTION TAB4-1.png": "'\\\\(\\\\frac{1}{2}\\\\).\\n\\n## 4 Example of Different Purpose-match Types\\n\\n### 4.1 Example of Different Purpose-match Types\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{\\\\(\\\\frac{1}{2}\\\\)} & \\\\multicolumn{1}{c}{\\\\(\\\\frac{1}{2}\\\\)} \\\\\\\\ \\\\hline \\\\hline \\\\(\\\\frac{1}{2}\\\\) & \\\\(\\\\frac{1}{2}\\n\\n'", "CAPTION TAB4-2.png": "\"Purpose-Match shows the level of purpose-match between a recommended article and each participant's research problem (see Table 3 for descriptions of research problems). Fully matching purposes are those that match at both high- (more abstract) and low-levels (specific details). Partial matches only match at the high-level abstraction and differ in details. The Participant Comment column shows relevant excerpts from the participant.\\n\\n\"", "CAPTION TAB5-1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}\\n\\n'", "CAPTION TAB5-2.png": "'Purpose-match was the only significant mediator between Condition and Creative Adaptation (indirect effect = \\\\(-0.09\\\\), significant using a bootstrapping method [91] with 1,000 iterations, \\\\(p<2\\\\times 10^{-16}\\\\)).\\n\\n'", "CAPTION FIG1.png": "'Figure 1: A diagram of two different yet analogical approaches (dashed arrow) for building lighter and more compact solar arrays, and their representations in purposes and mechanisms.\\n\\n'", "CAPTION TAB6-1.png": "'Table 6. F1 Scores of Different Models, Sorted by the Overall F1 Score of Purpose (PP) and Mechanism (MN) Detection\\n\\n'", "CAPTION TAB6-2.png": "\"The span-based Model 2 gave the best overall F1 score (blue). In comparison, the average agreement (\\\\(\\\\overline{\\\\kappa}\\\\)) between two experts' and crowdworkers' annotations was 0.68 (PP) and 0.72 (dNN) [21]. We used AllenNLP [41] to implement the baseline models 1-5.\\n\\n\"", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}\\n\\n'", "CAPTION TAB1.png": "'\\n\\n'", "CAPTION FIG3.png": "'Fig. 3. Example articles for the purpose of facilitating heat transfer heat in semiconductors. (Top) A Direct Application article involves directly applicable ideas and techniques for manipulating the interface material and structure to control thermal conductance. (Bottom) A Creative Adaptation example involves transferring a distant idea (fin-based design for heat sinks) and creatively adapting it into the target problem context (designing nano-scale fins that could absorb heat and convert it to useful energy). Figure credits: contact configurations and interface resistance from [116], fin-based heat sink from [104], nano-fins from [94].\\n\\n'", "CAPTION TAB3.png": "''", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l c'", "CAPTION TAB5.png": "'* [19]'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c} & Table 6: F1 Scores of Different Models, Sorted by the Overall F1 Score of Purpose (PP) and Mechanism (MN) Detection \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 6: F1 Scores of Different Models, Sorted by the Overall F1 Score of Purpose (PP) and Mechanism (MN) Detection'", "CAPTION FIG9.png": "'Fig. 9. Mean ranks of human-judged high and low purpose match articles from the span-based pipeline Low matches were ranked significantly lower (the rank number was higher, or average at 465th (SD) 2&1.92) than high matches at 343th (SD) 2&1.948).\\n\\n'", "CAPTION FIG7.png": "\"Fig. 1. Preparation of creative adaptation ideas among the partial purpose-match articles. Creative Adaptation was significantly more frequent among the analogy articles (area) than keyword articles (area) (which's new-tailed t-test, \\\\(\\\\rho=90\\\\times 10^{-4}\\\\).\\n\\n\"", "CAPTION FIG8.png": "'Figure 8: The rate of ideation outcome types in full and partial purpose matches. Among the keyword articles as the purpose mismatch increases, the rate of creative adaptation also increases from \\\\(0\\\\%\\\\) to \\\\(21\\\\%\\\\) (middle). However, this rate is significantly higher among the analogy articles (\\\\(47\\\\%\\\\)) than the keyword articles (\\\\(21\\\\%\\\\)). Note that while purpose mismatches led to more creative adaptation among analogy articles, a large portion of them also resulted in no ideation outcome (\\\\(38\\\\%\\\\)).\\n\\n'", "CAPTION FIG11.png": "\"Fig. 11. The distribution of mean purpose match scores over different conditions (mappings Name \\\\(\\\\mapsto 0\\\\), Part \\\\(\\\\mapsto 1\\\\), and Full \\\\(\\\\mapsto 2\\\\)). The mean purpose-match score of the system backed by Model 2 (0.63, SD 0.53) is significantly higher than that of the system used in Study 1 without the human-in-the-loop (BiLSTM, \\\\(\\\\mu=0.45\\\\), SD 0.58) (Welch's two-tailed t-test, \\\\(\\\\tau(237.87)=2.49,p=0.0135\\\\)), similar to that of the system with the human-in-the-loop (BiLSTM with filtering, \\\\(\\\\mu=0.62\\\\), SD 0.52) (\\\\(\\\\tau(244.65)=0.25,p=0.80\\\\)), and significantly lower than that of the keyword-based search (Keyward, \\\\(\\\\mu=1.04\\\\), SD 0.65) (\\\\(\\\\tau(159.38)=-4.57,p=0\\\\)).\\n\\n\"", "CAPTION FIG12.png": "'Figure 12: The search interface used for case studies featured an input for query reformulation which participants used to iteratively reformulate their queries.\\n\\n'", "CAPTION FIG10.png": "'\\n\\n## 4 Conclusion\\n\\nIn this paper, we have proposed a novel method for generating the full-width and full-width and full-width and full-width and full-width, and full-width, respectively. We have shown that the full-width and full-width are preserved in the full-width and full-width, respectively. We have shown that the full-width and full-width are preserved in the full-width and full-width, respectively.\\n\\n'", "CAPTION TAB7.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG13.png": "'Fig. 13. Diagram showing different abstraction levels of purposes and their relations. Node \\\\(\\\\bar{\\\\mathfrak{A}}\\\\) corresponds to a more specific query than its higher-level representation, denoted as \\\\(\\\\bar{\\\\mathfrak{B}}\\\\). Similarly, node \\\\(\\\\bar{\\\\mathfrak{C}}\\\\) represents a more specific purpose representation of \\\\(\\\\bar{\\\\mathfrak{A}}\\\\), accessible via the \\\\(\\\\bar{\\\\mathfrak{A}}\\\\underset{\\\\text{abstraction}}{\\\\rightarrow}\\\\bar{\\\\mathfrak{B}} \\\\underset{\\\\text{specification}}{\\\\rightarrow}\\\\bar{\\\\mathfrak{C}}\\\\) path.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Components of our system design that address the three core challenges. 1 Purpose and mechanism tokens are extracted from article abstracts at scale. We develop Seq2Seq classifiers to classify tokens into purpose, mechanism, or neither, going beyond previous approaches that worked on sentences or relied on crowds. 2 We embed the extracted purpose texts using a pre-trained language model (Google\u2019s USE [20]) and train a tree-based index of vectors to place high semantic similarity vectors in close neighborhoods for efficient lookup. 3 When the user query arrives at the system, it is first embedded with USE. This query embedding is then used to lookup the pre-computed tree indices for high similarity purpose embeddings. Article abstracts for the corresponding purpose embeddings are retrieved from Google Datastore. In the first system, additional human filtering is performed to remove obviously irrelevant results that may have been included due to model errors. Finally, a set of articles with similar purposes to the query but different mechanisms are returned to the users for ideation.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kaplan2022load.json b/test_extracted_captions/kaplan2022load.json new file mode 100644 index 0000000000000000000000000000000000000000..64091ecac69fa68193e9589da8e435b597c36141 --- /dev/null +++ b/test_extracted_captions/kaplan2022load.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: 2c-3D STORM resolves clathrin structures highly connected to actin networks at different stages of endocytosis. (A) STORM image of the ventral surface of an SK-MEL-2 cell immunoblotabeled with the CF-680 antibody (clathrin coats in red) and phhaloidin-AF647 (actin in cyan). Orange squares are areas shown in panel B. Color bar shows the \\\\(\\\\mathrm{z}\\\\) position of actin. Scale bar: 5 \u03bcm. (B) Magnification of highlighted areas 1 and 2 in panel A. Magenta squares are shown in panel C. Scale bars: 250 nm. (C) X-Z projections of the regions highlighted in panel B. Scale bars: 100 nm. (D) Illustration of binning clathrin coats (red) into three geometric stages based on their aspect ratio (shape index S|). Shallow: S! 0.7; U-shape: 0.7 < S! 0.9 and \\\\(\\\\mathrm{\\\\Omega}\\\\): S! 0.9. (E) X-Z projections of representative STORM images showing clathrin coats (red) with different actin (cyan) coverages around clathrin. Calculated S! of shallow CCSs from left to right image: 0.56, 0.53, 0.51, 0.55; for U-shaped CCPs from left image to right image: 0.87, 0.89, 0.86, 0.82; for \\\\(\\\\mathrm{\\\\Omega}\\\\)-shaped CCPs from left image to right image: 1.31, 1.06, 1.31, 1.52. Scale bars: 100 nm. (F) Graph of endocytic coat SI as a function of actin coverage for shallow (black dots), U-shaped (blue dots), and \\\\(\\\\mathrm{\\\\Omega}\\\\)-shaped (gray dots) pits. Categories of shape indices are chosen similar to E. Pits with actin coverage >5% are shown. R = -0.04, \\\\(n\\\\) = 719. Events accumulated from six cells. (G) Cartoon depicting the clathrin coat with actin either at the tip of the coat (top), covering the clathrin coat completely (middle), or at the base of the clathrin coat (bottom). Dashed black lines indicate the average \\\\(Z\\\\) position of actin and clathrin. \\\\(\\\\mathrm{D}_{x}\\\\) is the difference between average actin and clathrin \\\\(\\\\mathrm{Z}\\\\) positions. \\\\(\\\\mathrm{D}_{x}\\\\) < 0 is defined as the average actin \\\\(Z\\\\) position nearer the base of the pit. Schematic is a hypothetical plot of \\\\(\\\\mathrm{D}_{x}\\\\) vs. actin coverage for scenarios in which actin grows from the tip of the coat (red line) or the base of the pit (black line). (H) \\\\(\\\\mathrm{D}_{x}\\\\) as a function of actin coverage (for actin coverage >5%, R = 0.66, \\\\(n\\\\) = 719, \\\\(N\\\\) = 6 cells). (I) Cartoon of actin (blue) growing from the base of the pit (black lines) to cover clathrin coat (red) from a shallow membrane invagination to a fully formed membrane vesicle. X-Z projection (side profile) is shown. Dashed arrows indicate that growth of the actin network is not tightly coupled to the endocytic coat geometry and is variable in extent.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Quantitative analysis of CME mechanosensitivity under elevated membrane tension. (A) Schematic of cells in isotonic media (top) or hypotonic media (bottom), which causes water influx and stretches the cell membrane. In this figure, hypotonic treatment is 75 mOsm. (B) Mean membrane tether force values measured by AFM of cells in isotonic media (\\\\(n=18\\\\)) or in hypotonic media (\\\\(n=17\\\\)). Mean values were obtained by pulling at least three tethers (three independent experiments). In hypotonic treatment, circles are mean tether values from 2 to 10 min after hypotonic media exchange and triangles are mean tether values obtained between 10 and 16 min after hypotonic media exchange. Bars are mean +- SD. \\\\(p\\\\) = 0.002 by two-tailed Mann-Whitney test. (C) Kymographs of TIRF micrographs of live SK-MEL-2 cells endogenously expressing CLTA-TagRFP-TEN (magenta) and DNIM2-eGFPEN (green). Time is on the \\\\(X\\\\) axis. Kymographs are 4.8 min long. Cells were imaged in isotonic media (top) or hypotonic media for 2 min (middle) or 10 min (bottom). (D) Cumulative distribution plot of clathrin lifetimes marked by CLTA-TagRFP-TEN in isotonic media (red), hypotonic media for 2 min (violet), and hypotonic media for 10 min (orange). These tracks were associated with DNIM2-eGFPEN. (E) Cumulative distribution plot of dynamin2 lifetime marked by DNIM2-eGFPEN in isotonic media (light green), hypotonic media for 2 min (blue), and hypotonic media for 10 min (dark green). These tracks were associated with CLTA-TagRFP-TEN. \\\\(n\\\\) = 5831 tracks in 17 cells across four experiments for D-H. (D, E) Detailed statistics in Supplemental Table S1. (F) Plot of endocytic initiation rate for the three conditions. \\\\(p\\\\) < 0.05 by two-tailed Mann-Whitney test for both comparisons. (G) Endocytic completion rate in the three conditions. \\\\(p\\\\) < 0.05 by two-tailed Mann-Whitney for both comparisons. (H) Percentage of persistent tracks (defined as tracks lasting the entirety of the image acquisition) for the three conditions. \\\\(p\\\\) < 0.05 by two-tailed Mann-Whitney for both comparisons. (F-H) Barplots show mean +- SD. Statistics to plots in Supplemental Figure S7H.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: The actin network at CME sites increases in size in response to elevated membrane tension. In this figure, hypotonic refers to 75 mOsm media. (A) Schematic of cells in hypotonic media, which increases plasma membrane tension. The response of the actin network (blue) to elevated plasma membrane tension (purple) was previously unknown. (B) Representative STORM images of clathrin (red) and actin (cyan) in \\\\(\\\\times\\\\)z projections for cells fixed after treatment in the hypotonic media for 5 min (bottom). Coated pits are classified as shallow, U-shaped, or \\\\(\\\\Omega\\\\)-shaped based on the aspect ratio of the coat. Scale bars: 100 nm. (C) Plots of actin \\\\(Z\\\\) height at CCPs from calls in the isotonic (\\\\(n=736\\\\)) and hypotonic (\\\\(n=527\\\\)) media measured from STORM \\\\(\\\\times\\\\)z projections. Lines are median \\\\(\\\\pm\\\\) interquartile range. \\\\(p<0.0001\\\\) determined by Mann-Whitney test. (D) Plots of actin coverage over the clathrin coat in pits found in STORM \\\\(\\\\times\\\\)z projection images in isotonic (\\\\(n=719\\\\)) and hypotonic (\\\\(n=509\\\\)) conditions. Pits with actin coverage \\\\(>\\\\)1% are plotted. Lines are median \\\\(\\\\pm\\\\) interquartile range. \\\\(p<0.0001\\\\) determined by Mann-Whitney test. (E) Actin \\\\(Z\\\\) height as a function of coconut shape in isotonic (gray, \\\\(n=736\\\\)) and hypotonic (purple, \\\\(n=527\\\\)) conditions. (F) Actin \\\\(Z\\\\) height as a function of actin coverage over the clathrin coat in isotonic (gray, \\\\(n=719\\\\)) and hypotonic (purple, \\\\(n=509\\\\)) conditions. The data for isotonic conditions were also used to generate the plots in Figure 1. Three independent STORM experiments with \\\\(N_{-}=6\\\\) cells in isotonic and \\\\(N_{-}=7\\\\) cells in hypotonic media. (G) Cartoon depicting an adaptive actin force-generating mechanism that counteracts elevated membrane tension to ensure robust CME progression. This schematic describes three scenarios in which membrane tension is low, intermediate, or high and how CME is accomplished efficiently by adaptive actin network organization. Under low tension (bottom), the clathrin coat provides sufficient force to advance CME. At intermediate tension (middle), actin polymerization is required for the transition from U to \\\\(\\\\Omega\\\\) shape. At high tension (top), endocytic progression slows. More pits stall at the shallow conformation. In response to increased resistance, the actin network grows to envelop the coat and provide additional force against high membrane tension.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Importance of Arp2/3 complex-mediated actin polymerization during CME increases under elevated membrane tension. In this figure, CK666 (Arp2/3 complex inhibitor) treatment is 100 \u03bcM and hypotonic shock is 150 mOsm. \\\\(\\\\langle\\\\)A\\\\(\\\\rangle\\\\) Kymographs of cells expressing CLTA-TagRFP-TEN (magenta) and DNIM2-eGFPEN (green) imaged by TIRF. Cells were imaged in isotonic media. The media was then exchanged to include CK666 (bottom panel) and imaged after 4 min. \\\\(\\\\langle\\\\)B\\\\(\\\\rangle\\\\) Cumulative distribution plots of clathrin lifetimes in control, DMSO-treated conditions [orange, \\\\(n=4219\\\\)], and CK666-treated (red, \\\\(n=3124\\\\)) conditions. \\\\(\\\\langle\\\\)C\\\\(\\\\rangle\\\\) Cumulative distribution plots for control, DMSO-treated (dark green, \\\\(n=4219\\\\)), and CK666-treated (light green, \\\\(n=3124\\\\)) dynamin2 lifetimes associated with clathrin lifetimes in B. \\\\(\\\\langle\\\\)BC\\\\(\\\\rangle\\\\) Control, \\\\(N=10\\\\) cells, and CK666 treatment, \\\\(N=10\\\\) cells, measured in three independent experiments. Complete statistics in Supplemental Table S3. \\\\(\\\\langle\\\\)D\\\\(\\\\rangle\\\\) Kymographs of cells in hypotonic media. In the top panel, cells were placed in hypotonic media and imaged after 4 min. In the bottom panel, cells were treated with CK666 in hypotonic media and imaged after 4 min. \\\\(\\\\langle\\\\)E\\\\(\\\\rangle\\\\) Cumulative distribution plots of clathrin lifetimes for control, DMSO-treated conditions (magenta, \\\\(n=1405\\\\)), and CK666-treated conditions (blue, \\\\(n=2783\\\\)) in hypotonic media. \\\\(\\\\langle\\\\)F\\\\(\\\\rangle\\\\) Cumulative distribution plots of DMSO-treated (black, \\\\(n=1405\\\\)) and CK666-treated (blue, \\\\(n=2783\\\\)) dynamin2 lifetimes in hypotonic media associated with clathrin lifetimes in E. \\\\(\\\\langle\\\\)E\\\\(\\\\rangle\\\\) Control, \\\\(N=9\\\\) cells, and CK666 treatment, \\\\(N=10\\\\) cells, measured in three independent experiments. Complete statistics in Supplemental Table S3. \\\\(\\\\langle\\\\)G\\\\(\\\\rangle\\\\) Representative STORM images of immunolabeled clathrin-coated structures in control cells arranged by coat height. Top panel shows the \\\\(x\\\\)-\\\\(y\\\\) projections and bottom panel the corresponding \\\\(x\\\\)-\\\\(z\\\\) projections. The white square in the \\\\(x\\\\)-\\\\(y\\\\) projections shows the area that was cropped to generate the \\\\(x\\\\)-\\\\(z\\\\) projections. The heights of clathrin coats in the \\\\(x\\\\)-\\\\(y\\\\) projection from left to right image are 61, 96, 124, 158, and 177 nm. Scale bars: 100 nm. \\\\(\\\\langle\\\\)H\\\\(\\\\rangle\\\\) Clathrin coat heights when cells were treated with DMSO (\\\\(n=154\\\\)) or CK666 (\\\\(n=158\\\\)) in isotonic media or CK666 in hypotonic media (\\\\(n=159\\\\)). Clathrin coat images for quantitative analysis were collected from at least three different cells for each condition from a single experiment. Statistics are given in Supplemental Figure S8F. \\\\(p<0.05\\\\) in both comparisons by Mann-Whitney test.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kordeAlternatingIndividualGroup2016.json b/test_extracted_captions/kordeAlternatingIndividualGroup2016.json new file mode 100644 index 0000000000000000000000000000000000000000..00904241e745e30642909aab76221d544baa1c23 --- /dev/null +++ b/test_extracted_captions/kordeAlternatingIndividualGroup2016.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Number of unique of least generated in the different conditions across phases in Experiment 1.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The structure of the surface of a sphere. _Phys. Rev. E_, 71:036002, 2005.\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Effect of conditions on novelty using the dual-pathway model. The model shows the effect of the hybrid and group-conditions as compared to the alone condition.\\n\\n'", "CAPTION FIG6.png": "'\\nFigure 6: Effect of condition on novelty using the dual-pathway model with direct paths. The model shows the effect of the hybrid and gauge conditions as compared to the data-candErane.\\n\\n'", "CAPTION TAB3.png": "'* [16] A.\\n\\n'", "CAPTION TAB4.png": "'* [16] A.\\n\\n'", "CAPTION TAB5.png": "'* [16] A.\\n\\n'", "CAPTION TAB2.png": "'* [16] A.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Number of unique of ideas generated in the different conditions across phases in Experiment 2.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kosmalska2015physical.json b/test_extracted_captions/kosmalska2015physical.json new file mode 100644 index 0000000000000000000000000000000000000000..8535d96a3d747a8215baeef02019b30a77b0764f --- /dev/null +++ b/test_extracted_captions/kosmalska2015physical.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: **Membrane response to stretch and osmotic changes.****(a)** Cells transfected with pETYP-memb before (top panel) and after (middle and bottom panels) applying different magnitudes of constant stretch. Yellow arrow indicates a membrane rutile flattened by stretch. (**b**) Cells transfected with DEYFP-memb before and after reducing medium osmolarity to either 50 or 0% of original medium. Cells submitted to 0% osmolarity (de-ionized water) for 3 min sometimes rounded and flattened membrane ruffles (middle panel) and sometimes underwent membrane lysis (right panel). Yellow arrows indicate membrane ruffles, which either remain or flatten after applying 50 or 0% hypo-osmotic medium, respectively. (**c**) Confocal slice showing a cell before (green) and after (red) application of medium with 50% osmolarity for 3 min. (**d**) Corresponding quantification of the increase in cell volume and required membrane area (\\\\(n=5\\\\) cells). (**e**) % of cells showing membrane tearing after 3 min of constant stretch application (\\\\(n=70\\\\) cells). (**f**) % of cells showing membrane lysis after 3 min of application of medium with different osmolarity (\\\\(n=50\\\\) cells). Scale bars, 20 \u03bcm. Error bars are mean \\\\(\\\\pm 5\\\\) s.m.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Membrane remodelling can be described through pathways along the surface/volume phase diagram. (a-d) Different pathways tested in the phase diagram by applying stretch and hypo-osmotic shocks (red arrows, numbers refer to the corresponding image in the panel below). (e-h) Corresponding response of pEYFP-mem-transfected cells after applying stretch and somatic shocks as indicated to allow the pathways. Time flows from top to bottom. In all cases, the first application of stretch/hypo-osmotic shock (second row of cells) lasted 3 min. Subsequent steps were carried out as quickly as possible to evaluate membrane response before cells had time to actively eliminate structures. (f) In cells submitted to hypo-osmotic shock, co-localization of membrane structures formed after first restoring iso-osmotic medium (red) and then applying stretch (green). (j) Confocal vertical slices showing VLD shape before (top) and after (bottom) stretch release. Zoomed image to the right shows the superimposed shape prediction from the theoretical model in red. (k) Quantification of VLD fluorescence for cells under hypo-osmotic medium after either restoring iso-osmotic medium (blue symbols) or restoring iso-osmotic medium and then releasing stretch application (pink symbols, arrow indicates moment of stretch release). (N = 100/50 structures from 10/5 cells. (l) In cells submitted to both hypo-osmotic shock and stretch, co-localization of membrane structures formed after first releasing stretch (red) and then restring iso-osmotic medium (green). (m) Quantification of VLD diameter (_n_ = 100/50/70 structures from 10/3/3 cells, \u204e\\\\(\\\\rightarrow\\\\)P < 0.001, analysis of variance (ANOVA)) and density (_n_ = 50/30/30 regions from 8/3/3 cells, \u204e\\\\(\\\\rightarrow\\\\)P < 0.001, ANOVA/A) in cells submitted only to semantic shocks or also to de-stretch (stretch release) before or after restoring iso-osmolarity). Scale bars are 5 min in j and 20 min elsewhere. Insets show zoomed views (\\\\(10\\\\times 10\\\\,\\\\mu\\\\)m\\\\({}^{2}\\\\)) of membrane structures. Error bars are mean \u00b1 s.e.m.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: **VLD formation is driven by the confinement of liquid flows at the cell-substrate interface.** Response of pEGFP-mem-transfected cells seeded on either poly-acrylamide (PA) gels or soft silicone elastomers to: (**a**) the application of 50% hypo-osmotic medium for 3 min, (**b**) the application of 6% strain for 3 min and (**c**) a fast 6% strain pulse. Insets show zoomed views (0x10 \\\\(\\\\mu\\\\)m\\\\({}^{2}\\\\)) of membrane structures. Scale bars, 2.0 \u03bcm. No significant differences were observed between any of the cases either in the diameter of reservoirs (n = 150 reservoirs from 3 cells) or in their density (\\\\(n=30\\\\) cells) regions from 3 cells. (**d**) Time sequence of VLD formation and resorption in pEGFP-mem-transfected cells exposed to dextran-labelled iso-osmotic media after 3 min incubation with 50% unlabelled hypo-osmotic media. (**e**) Zoomed insets (\\\\(20\\\\times 20\\\\) \u03bcm\\\\({}^{2}\\\\)) corresponding to red square in **d** showing the evolution of membrane and dextran fluorescence, and merged images. (**f**) Corresponding quantification of pEGFP-mem and dextran relative fluorescence levels.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Cell membranes use different strategies to readapt to normal surface and volume.****(a)** pETFP-mem-transfected cells before, during and after constant stretch application during 3 min. **(b)** Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, \\\\(\\\\overline{\\\\alpha}\\\\): background). \\\\(n=100\\\\) reservoirs from 10 cells. **(c)** Confocal vertical slice from a pEYFP-mem-transfected cell before (top) and after (bottom) application of 6% stretch for 3 min. **(d)** Staining images of cells fixed immediately after stretch release showing the membrane (pEYFP-mem transfection), pixillin and actin. **(f)** Co-localization images are shown to the right. **(e)** pEYFP-mem-transfected cells before, during and after application of 50% hypo-osmotic medium during 3 min. **(f)** Quantification of VLD fluorescence after re-application of iso-osmotic medium (\\\\(\\\\overline{\\\\alpha}\\\\): initial fluorescence, \\\\(\\\\overline{\\\\alpha}\\\\): background). \\\\(n=100\\\\) VLDs from 10 cells. **(g)** Confocal images of a pEYFP-mem-transfected cell before (top) and after (bottom) application of 50% hypo-osmotic medium for 3 min. **(h)** Staining images of cells fixed immediately after re-application of iso-osmotic medium showing the membrane (pEYFP-mem transfection), pixillin and actin. **(f)** Garaged co-localization images are shown to the right. **(f)** Quantification of mean diameter of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). \\\\(n=250/100\\\\) structures from 8/10 cells. **(f)** Quantification of mean density of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). \\\\(n=30/50\\\\) regions from 5/8 cells. **(k)** Quantification of mean height of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). \\\\(n=80/50\\\\) structures from 6/4 cells (**P\\\\(<\\\\)0.001, two-tailed Student\u2019s t-test). We note that reservoir heights are close to the axial resolution of our confocal microscope (0.9 \u03bcm) and thus represent upper estimates rather than accurate measurements. Scale bars are 5 \u03bcm in **c.g** and 20 \u03bcm in **d.h**. In all cases, zoomed insets (\\\\(10\\\\times 6\\\\,\\\\mu\\\\)m\\\\({}^{2}\\\\) in **c.g** and \\\\(10\\\\times 10\\\\,\\\\mu\\\\)m\\\\({}^{2}\\\\) in **d.h**) show a magnification of the area marked in the main image. Error bars are mean \\\\(\\\\pm\\\\) s.e.m.\\n\\n'", "CAPTION FIG6.png": "\"Figure 6: **I Membrane mechanical adaptation is explained by minimization of the strain and adhesion energies required to generate surface and volume containers.** (**a**) Phase diagram showing the predicted structures that require minimal energy to deform the membrane and detach it from the substrate in order to accommodate membrane surface area (upon stretch release) and liquid volume at the cell-substrate interface (upon an increase in osmolarity)[38]. Surface storage is achieved optimally with increasingly long tubules, whereas volume storage leads to the formation of spherical caps (VLDs). When both volume and surface storage are required, spherical caps 'bud' and become more invagulated. (**b**) Left: time-course sequences of cell membrane regions showing the formation of either reservoirs or increasingly long tubules after releasing different stretch magnitudes. Right: Mean reservoir/tubule length (black dots, experimental data, red line, theoretical prediction) as a function of de-stretch magnitude (for increasing stretch, \\\\(n=80/50/50\\\\) structures from 6/3/3 cells). (**c**) Left: images showing the formation of increasingly large VLDs after restoring iso-osmotic medium in cells previously exposed to different magritudes of hypo-osmotic shocks for 3 min. Right: mean VLD diameter (black dots, experimental data, red line, theoretical prediction) as a function of hypo-osmotic shock magnitude (for increasing osmotic shock, 60/100/100/50 structures from 5/5/10/3 cells). Scale bars, 5 \u03bcm. Error bars are mean \\\\(\\\\pm\\\\) s.e.m.\\n\\n\"", "CAPTION FIG4.png": "'Figure 4: **Effect of stimulus application rate on membrane structure formation.** (**a**) Cell submitted to two successive steps of 6% stretch for 3min, in which the first is released immediately and the second slowly (ISs). (**b**) Cell submitted to two successive applications of 50% hypo-osmotic media, in which iso-osmotic medium is restored first immediately and then slowly (1min). Scale bars, 20 \u03bcm. Insets show zoomed views (\\\\(10\\\\times 10\\\\,\\\\mathrm{\\\\SIUnitSymbolMicro m}^{2}\\\\)) of membrane structures.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: _Membrane mechanical adaptation is a passive process followed by active recovery._ (**a**) Examples of control and ATP-depleted pYFP-mem-transfected cells before, during and after application of transfected cells before, during and after application of two 3-min pulses. (**f**) Quantification of VLD fluorescence after the first re-application of iso-osmotic medium and during application and release of the second pulse (\\\\(n=35/40\\\\) VLDs from 3/3 cells). The effect of ATP depletion was significant (P \\\\(<\\\\) 0.001, two-tailed Student\u2019s t-test). (**g**) Quantification of reservoir size in control and ATP-depleted cells (\\\\(n=150/200\\\\) reservoirs from 3/3 cells). No significant differences were observed, two-tailed Student\u2019s t-test. (**d**) Quantification of reservoir density in control and ATP-depleted cells (\\\\(n=50/50\\\\) regions from 5/5 cells). No significant differences were observed. (**e**) Examples of control and ATP-depleted pYFP-mem-transfected cells before, during and after application of 50% hypo-osmotic medium in two 3-min pulses. (**f**) Quantification of VLD fluorescence after the first re-application of iso-osmotic medium and during application and release of the second pulse (\\\\(n=35/40\\\\) VLDs from 3/3 cells). The effect of ATP depletion was significant (P \\\\(<\\\\) 0.001, two-tailed Student\u2019s t-test). (**g**) Quantification of VLD size in control and ATP-depleted cells (\\\\(n=30/35\\\\) VLDs from 3/3 cells). No significant differences were observed. (**h**) Quantification of VLD density in control and ATP-depleted cells (\\\\(n=20/25\\\\) regions from 3/3 cells). No significant differences were observed. Scale bars, \\\\(20\\\\,\\\\mathrm{\\\\SIUnitSymbolMicro m}\\\\). Insets show zoomed views (\\\\(10\\\\times 10\\\\,\\\\mathrm{\\\\SIUnitSymbolMicro m}^{2}\\\\)) of membrane structures.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kosmalska2015physical_supplement.json b/test_extracted_captions/kosmalska2015physical_supplement.json new file mode 100644 index 0000000000000000000000000000000000000000..4f61af2efb05deef30ab09e6dc6b55c2092c544d --- /dev/null +++ b/test_extracted_captions/kosmalska2015physical_supplement.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS6-1.png": "'\\n\\n**Supplementary Figure 6. Reservoirs and VLDs are unrelated to caveolae**. **a**, Time sequence of pEYFP-mem and Cav1-mcherry transfected cells before, during, and after application of 6% stretch for three minutes. Image to the right shows the lack of co-localization between pEYFP-mem (green) and Cav1-mcherry (red). **b**, pEYFP-mem transfected cell (green in merged image) fixed right after restoring iso-osmotic media and stained for caveolin 1 (red in merged image). No co-localization was observed between caveolin and VLDs. **c**, Time sequence of pEYFP-mem and'", "SUPP CAPTION FIGS1.png": "'\\n\\n**Supplementary Figure 1. Stretch system.****a**, Schematic of stretch system. A ring clamping a PDMS membrane (shown in red) is placed on a support containing a central loading circular post and an external ring, with an opening in between. Imaging objectives can then be placed either below (inverted microscope) or above (upright microscope). Cells are then cultured on the membrane after coating with fibronectin. **b**, Once vacuum is applied through the opening, it deforms and stretches the membrane. In some cases, cells were seeded on polyacrylamide and soft silicone gels previously attached to the membrane (see methods). **c**, Level of strain in the X and Y axis obtained after applying different vacuum suction pressures. Stretch was biaxial.\\n\\n'", "SUPP CAPTION FIGS2.png": "'\\n\\n**Supplementary Figure 2. Response of cells to 2% strain.** Response of a peYFP-mem transfected cell to the application of 2% strain for three minutes. No visible effects were observed either upon stretch application or upon stretch release. Insets display magnifications of the areas in red, showing that membrane ruffles were not flattened upon stretch application, and reservoirs were not formed upon stretch release. Scale bar is 20 mm.\\n\\n'", "SUPP CAPTION FIGS9.png": "'\\n\\n**Supplementary Figure 9. Adhesion dependence of reservoirs and VLDs.****a**, Time sequence of pEYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes for control cells and cells treated with 10 mg mL-1a5b1 antibody. **b**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background) (n=100/150 reservoirs from 10/6 cells). **c**, Corresponding quantification of mean reservoir diameter (n=250/200 reservoirs from 8/6 cells). **d**, Corresponding quantification of mean reservoir density (n=30/60 regions from 5/5 cells). **e**, Time sequence of pEYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes for control cells and cells treated with 10 mg mL-1a5b1 antibody. **f**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=100/40 VLDs from 10/5 cells. **g**, Corresponding quantification of mean VLD diameter (n=100/150 VLDs from 10/7 cells). **h**, Corresponding quantification of mean VLD density (n=50/70 regions from 8/7 cells). Scale bars indicate 20 mm. N.s., non-significant, *, p<0.05, ***, p<0.001. Scale bars are 20 mm. In all cases, Zoomed insets (10x10 mm) show the formation and evolution of membrane structures.\\n\\n'", "SUPP CAPTION FIGS10.png": "'\\n\\n**Supplementary Figure 10.** Model predictions. **a**, Model prediction for reservoir length upon stretch release as a function of applied stretch. **b**, model prediction for VLD diameters formed after restoring iso-osmotic medium from osmotic shocks of different magnitude. In a and b, red line corresponds to the bending modulus used throughout the work, dashed black line shows the prediction for a 5-fold increase in bending modulus. **c**, VLD shape after restoring osmolarity from hypo-osmotic shocks of different magnitude and **d**, VLD shape after restoring osmolarity from a 50% hypo-osmotic shock in a pre-stretched membrane (top) and then de-stretching the membrane by 6% (top).\\n\\n'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Supplementary Figure 5. Poroelasticity of polyacrylamide gels.****a**, graph showing the height of polyacrylamide gels during and after application of 6% stretch for three minutes. Values are shown normalized to the height before stretch application. As gels are stretched, they first decrease their height to maintain volume, but they progressively incorporate water and swell. Once stretch is released, the progressive reduction in thickness is indicative of water expulsion flow. **b**, As a control, gels submitted to a 50% decrease in media osmolarity did not modify their height. (n=5 gels)'", "SUPP CAPTION FIGS7.png": "'\\n\\n**Supplementary Text:**_Abb_ and **inversion** (**Supplementary Text:**_Abb_, **Abb'", "SUPP CAPTION FIGS6.png": "'\\n## References\\n\\n* [1] A. B.\\n\\n'", "SUPP CAPTION FIGS3.png": "'\\n\\n**Supplementary Figure 3. Dynamic formation and resorption of membrane structures.****a,** Quantification of VLD fluorescence after re-application of iso-osmotic medium (1:maximum fluorescence, 0: background). n= 20 VLDs from 2 cells. Imaging was carried out using an inverted microscope (60x objective) which allowed to visualize the initial VLD formation period. **b**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background). n= 100/80/30 reservoirs from 10/3/3 cells. Imaging was carried out using an upright microscope (60x objective). Both decreasing temperature (pink symbols) and increasing stretch magnitude (black symbols) slowed the process of reservoir formation. Images to the right show corresponding examples of membrane structures at the times indicated in the graph. **c**, Quantification of VLD fluorescence after re-application of iso-osmotic medium (1: initial fluorescence, 0: background). n= 100/50/60 VLDs from 10/5/4 cells. Imaging was carried out using an upright microscope (60x objective). Both decreasing temperature (pink symbols) and increasing the magnitude of the hyposomic shock (black symbols) slowed the process of VLD formation. Images to the right show corresponding examples of membrane structures at the times indicated in the graph. All insets have a size of 10x10 um.\\n\\n'", "SUPP CAPTION FIGS7-2.png": "'(n=100/100 reservoirs from 10/4 cells). **c**, Corresponding quantification of mean reservoir diameter (n=250/100 reservoirs from 8/4 cells). No significant differences were observed. **d**, Corresponding quantification of mean reservoir density (n=30/30 regions from 5 cells). **e**, Time sequence of peYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes for control cells and cells treated with 0.5 mM cytochalasin D. Zoomed insets show the formation and evolution of membrane VLDs. **f**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=100/100 VLDs from 10/5 cells. **g**, Corresponding quantification of mean VLD diameter (n=100/60 VLDs from 10/3 cells). **h**, Corresponding quantification of mean VLD density (n=50/30 regions from 8/3 cells). **i**, Time sequence of peYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes for control cells at 37(r) and cells at 26(r). Zoomed insets show the formation and evolution of membrane reservoirs. **j**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background) n=100/60 reservoirs from 10/3 cells. **k**, Corresponding quantification of mean reservoir diameter (n=250/140 reservoirs from 8/3 cells). No significant differences were observed. **l**, Corresponding quantification of mean reservoir density (n=30/30 zones from 5/3cells). No significant differences were observed. **m**, Time sequence of peYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes for control cells at 37(r) and cells at 26(r). Zoomed insets show the formation and evolution of membrane VLDs. **n**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=100/24 VLDs from 10/3 cells. **o**, Corresponding quantification of mean VLD diameter (n=100/45 VLDs from 10/3 cells ). No significant differences were observed. **p**, Corresponding quantification of mean VLD density (n=50/25 zones from 8/3 cells). No significant differences were observed. Scale bars indicate 20 mm. N.s., non-significant, *, p<0.05, ***, p<0.001. Scale bars are 20 mm.\\n\\n'", "SUPP CAPTION FIGS6-2.png": "'Cav1-mcherry transfected cells before, during, and after application of 50% hypo-osmotic media for three minutes. Image to the right shows the lack of co-localization between peYFP-mem (green) and Cav1-mcherry (red). **d**, peYFP-mem transfected cell (green in merged image) fixed right after releasing 6% stretch and stained for caveolin 1 (red in merged image). No co-localization was observed between caveolin and reservoirs. **e**, Time sequence of peYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes. Zoomed insets show the formation and evolution of VLDs. Caveolin **1** knock-out cells were reconstituted either with caveolin 1-GFP or an empty control vector (IRES-GFP). **f**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=50/25 VLDs from 5/3 cells. **g**-**h**, Corresponding quantification of mean VLD diameter (**g**) (n=100/60 VLDs from 5/3 cells) and density (**h**) (n=30/20 regions from 5/3 cells).No significant differences were observed. **i**, Time sequence of peYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes. Zoomed insets show the formation and evolution of membrane reservoirs. Caveolin **1** knock-out cells were reconstituted either with caveolin 1-GFP or an empty control vector (IRES-GFP). **j**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background) (n=30/60 reservoirs from 3/4 cells) . **k**, Corresponding quantification of mean reservoir diameter (n=250/100 reservoirs from 3/3 cells). No significant differences were observed. **l**, Corresponding quantification of mean reservoir density (n=40/20 regions from 3/3 cells). No significant differences were observed. **S**cale bars are 20\\\\(\\\\mu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS7-1.png": "'\\n\\n**Supplementary Figure 7. Actin and temperature dependence of reservoirs and VLDs.****a**, Time sequence of peYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes for control cells and cells treated with 0.5 mM cytochalasin D. Zoomed insets show the formation and evolution of membrane reservoirs. **b**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background)'", "SUPP CAPTION FIGS4.png": "'\\n\\n**Supplementary Figure 4. Co-localization of actin and membrane structures before, during and after stretch and hypo-osmotic shocks.****a**, cells transfected with pEYFP-mem and Lifeact-Ruby before, during, and after application of 6% stretch for 3 minutes. **b**, equivalent images obtained before, during, and after application of a 50% hypo-osmotic shock for 3 minutes. Insets (10x10 \\\\(\\\\upmu\\\\)m) show zoomed views of membrane and actin structures. Scale bars are 20\\\\(\\\\upmu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS8.png": "'\\n\\n**Supplementary Figure 8. Membrane reservoirs and VLDs are observed across cell types and species.** Images of Chinese Hamster Ovary (CHO) cells, A431 human squamous carcinoma cells, and human keratinocytes (HaCaT) transfected with pEYFP-mem before and after being submitted to biaxial stretch (a) or hypo-osmotic shock (b). All cell types consistently showed the formation of reservoirs or VLDs, respectively. Zoomed insets (10x10 \\\\(\\\\upmu\\\\)m) show a magnification of both membrane structures. Scale bar indicates 20 \\\\(\\\\upmu\\\\)m.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/kyumurkov2023force.json b/test_extracted_captions/kyumurkov2023force.json new file mode 100644 index 0000000000000000000000000000000000000000..0ccab7ed23fc3e49fa00337b64c7856afd3270e6 --- /dev/null +++ b/test_extracted_captions/kyumurkov2023force.json @@ -0,0 +1 @@ +{"CAPTION FIGS3.png": "'Figure 53: **Efficiency of clathrin silencing in osteoblast cell lines.** Clathrin and actin are visualized by Western blot after treatment with siRNA against clathrin as compared to Scramble conditions. Source data are available for this figure: SourceData F53.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: **Size and dynamic of b3 integrin FAs are dependent on b1 integrins and ICAP-1.****(A)** Staining area of b3 integrins was carried out on osteoblast cells spread on fibronectin-coated coverglass for 4 h using LucAS antibody. Scale bar, 20 \u03bcm. **(B)** Quantification of the adhesive area of b3 integrins FA.\\n\\n'", "CAPTION FIGS4.png": "'Figure S4: **NME/B3 integrin interaction occurs at a-adaptin-marked CCPs and does not need b1 integrin in osteoblasts.****(A)** PLA signal using NME antibody in combination with ICAP-1 antibody (4d1d6) or ICAP-1 antibody (9b10) or b3 integrin antibody or B1 integrin antibody in WT osteoblasts. Immunoblotting of a-adaptin was performed after PLA. Insets are higher magnification of boxed regions. Insets show PLA signals (NME/ICAP-1 or NME/B1 integrin or NME/B3 integrin) colocalizing with a-adaptin-positive CCPs (arrowheads). PLA signal indicates a dose proximity, possibly in situ interaction of NME with ICAP-1, BL, and B3 integrins at CCPs. Scale bar, 10 \u03bcm. Inset scale bar, 5 \u03bcm. (**B and C)** Representative images of PLAs (\u03b2) and PLA assay quantification (C) of the number of PLA spots per cell performed with antibodies against NME and B3 integrin. Red dots denote regions of signal amplification consistent with NME/B3 integrin proximity in ostechbast cells deficient or not in B1 integrin. PLA performed on ICAP-1 and B1 integrin deficient cell line is used as control. Scale bar, 20 \u03bcm. Error bars represent SD. \\\\(R\\\\) = 15 cells/condition from three independent experiments. ns, adjusted \\\\(P\\\\) value > 0.05.\\n\\n'", "CAPTION FIGS1.png": "'Figure S1: **[B3 integrin expression level is unchanged upon loss of b1 integrin. (A-C) Western blot of total cell lysate (A) and quantification (B and C) confirmed the deletion of b1 integrin or IXAP-1 in osteoblast cells. Tubulin is used as loading control. (B) The deletion of b1 integrin and IXAP-1 do not affect the expression of b3 integrin. Actin is used as loading control. (D) The expression of b3 integrin mRNA is not changed upon deletion of b1 integrin or IXAP-1. Luminescence signal is normalized to the b1 integrin/+- _x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_x_-_-x_-_x_-_x_-_x_-_-x_-_x_-_x_-_-x_-_x_-x_-_-x_-_x_-x_-_-x_-x_-_-x_-x_-_-x_-x_-x_-_-x_-\\n\\n'", "CAPTION FIG2-2.png": "'and B3 integrin (LucA.5, green) was performed and analyzed by fluorescent confocal microscopy. Silencing the expression of B3 integrin leads to the complete ablishment of the ppMLC-decorated stress fibers in the B12+/-/-/- and shrinkage of the cell area. Scale bar, 20 mm. **(b)** Quantification of the ppMLC area normalized to the cellular surface area after treatment with b3 integrin siRNA (si B3, clear colors) or with scramble siRNA (si Scr, dark colors). Customized particle analysis script from ImageJ was used after application of Unshappen mask and Despede filters. The error bars represent SD. **(f)** Western blot showing the p value > 0.05, + \\\\(P\\\\) value < 0.05, + \\\\(P\\\\) value < 0.01, ***, P value < 0.0001. **(e and D)** Representative fraction forces map. (TF/H) from _\u03b2_/-/- and _\u03b2_ integrin/-/--/- cells treated with b3 integrin siRNA (siB3, bottom panels) or with scramble siRNA (si Scr, upper panels) and quantification (D) of the total force applied (nN) on fibronectin-coated polyacrylamide gel with a defined rigidity of S kPa. The additional silencing of b3 integrins in the B1-/-/-/- cells declines the traction forces, confirming that, in absence of b1 integrin and ICAP-1, generation of strong cellular contractility at adhesion sites is dependent on b3 integrins. Scale bar, 10 mm. Error bars represent SD. **(**)**, P value < 0.0001. **(e)** FACS analysis of median fluorescence intensity of b3 integrin on cell surface in B1 integrin KD osteoblast cell lines. Error bars represent SD. **(f)** Western blot showing the efficiency of b3 integrin silencing in _\u03b2_ integrin/-/-/- and _\u03b2_ integrin/-/-/-/- cell lines. Source data are available for this figure: SourceData F2.\\n\\n'", "CAPTION FIG4-1.png": "'Figure 4: **The \\\\(\\\\beta\\\\)3 integrin dependent contractility is associated with the defect of \\\\(\\\\beta\\\\)3 integrin endocytosis.****(A)** The \\\\(\\\\beta\\\\)3 integrin uptake was measured using \\\\(\\\\beta\\\\)3 integrin specific antibody (Luck.S), coupled with pH-Rhado. The volume of the endocytotic vesicles per cell volume was measured at 37 and 4\u00b0C. Note the decrease of \\\\(\\\\beta\\\\)3 integrin endocytosis in cell lines deleted in ICAP-L. The quantification was performed after 45 min incubation. Error bars represent SD. \\\\(N\\\\) = 127 cells. ns, adjusted P value > 0.05; *, P value < 0.05; **, P value < 0.01; ***, P value < 0.00L; ***, P value < 0.0001. ** Representative images of PLA.\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: **ppMyssin area and traction force are dependent on b3 integrin and ICAP-1 expression.****(A)**\u03b21 integrin KO osteoblast cell lines were treated with b3 integrin siRNA or scramble siRNA for 48 h and then let spread on FN coated glass for 24 h. Staining of myosin phosphorylation (cpMLC antibody, red)\\n\\n'", "CAPTION FIGS5.png": "\"Figure S5: **Requirement of NNE in COP upstream of dynamin and auxillin steps to allow optimal clathrin-coated vesicle budding.****(A)** Still image and kymographs of simultaneous two-color TIRF-M time series of mock- or siRNA-treated BSC-1 cells overexpressing mRFP-LCa and GFP-auxulin (1 image/s). In mock-treated cells, vertical arrowhead points to a burst of GFP-auxulin coincident with mRFP-LCa signal disappearing. In siRNA-treated cells, arrowhead points to mRFP-LCa signal disappearing without associating GFP-auxulin. **(b)** Percentage of disappearing mRFP-LCa CCP's ending with a burst of GFP-auxulin in mock- and siRNA-treated BSC-1 cells. At least 300 events were analyzed for each condition.\\n\\n\"", "CAPTION FIG3-2.png": "'normalized to the cellular surface area. Error bars represent SD. \\\\(N\\\\) = 208 cells. ns, adjusted P value > 0.05, *, P value < 0.05, **, P value < 0.01, ***, P value < 0.001, ***, P value < 0.001, ***, P value < 0.001. (**) Quantification of the mean of b3 integrin FAs area. The deletion of ICAP-1 in b1 deficient cell line (grey box) drives massive leap of b3 integrin stained FAs size. Error bars represent SD. \\\\(N\\\\) = 208 cells. ns, adjusted P value > 0.05, *, P value < 0.05, **, P value < 0.01, ***, P value < 0.001. (**) Representative time series of the lifetime of eGFP-b3 integrin FAs of the four osteoblastic cell lines. Asteric points out typical eGFP-b3 integrin FAs in the cells with typical disassembly lifetime. Note the translocation of FAs to the cell center in b1 integrin-/-1/2-1/- cells. Scale bar 2, _\u03bc_m. **[**]** The lifetime analysis on the eGFP-b3 integrin FAs. The lifetime of the eGFP-b3 integrin containing FAs is increased in b2 integrin-/-1/- cells. Spinning disk videos of eGFP-b3 integrin were taken for the duration of 2 h. The adhesion lifetime was analyzed by Focal Adhesion Analysis Server (FAAS; Berginski and Gomez, 2013) and verified visually. Error bars represent SD. \\\\(N\\\\) = 50 focal adhesions. ns, adjusted P value > 0.05, *, P value < 0.05, **, P value < 0.01, ***, P value < 0.001, ***, P value <= 0.0001. (**) FRAP analysis on the eGFP-b3 integrin FAs reveal that the b3 integrin mobility at the plasma membrane is halted in the absence of ICAP-1 and b1 integrin. At least six FAs per cell lines were bleached and their recovery was monitored for 5 min. Error bars represent SD. ns, adjusted P value > 0.05, *, P value < 0.05, **, P value < 0.01, ***, P value < 0.001. ***, P value < 0.0001.\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: **Osteoblasts are able to exert traction force on fibronectin-coated substrate in the absence of \u03b21 integrin and ICAP-1. (A and B) Representative traction forces maps (A) and quantification (B) of the total force applied on fibronectin-coated polyacrylamide gel with a defined rigidity of 5 kPa.**\\n\\n'", "CAPTION FIGS2.png": "'Figure S2: **ICAP-1 is required for cells to adapt cell velocity as function of substrate stiffness independently on the presence of B1 integrin and is involved in\u03b23 integrin FA translocation to the cell center. (A)** Osteoblast cells were spread on FN-coated PAA gels of different rightiles (3, 15, and 60 kPa). Cell migration was monitored for 5 h using time-lapse microscopy. The cell velocity was determined by individually tracking 200-300 cells in three independent experiments. ICAP-1 deficient cells displayed a constant migration velocity whatever the substrate rigidity as compared to the b1 integrin/*: kap-1/* cells, independently of the presence of B1 integrin highlighting the crucial role of ICAP-1 in rigidity sensing. Error bars represent SD. \\\\(N\\\\) >= 199 cells. *, **, ** < 0.05; **, **, ** < 0.005; **, **, ** **, ** **, ** **, ** **, ** **, ** **,\\n\\n'", "CAPTION FIG5-2.png": "'colors) or scramble siRNA (sis Scr, dark colors) impedes the turnover of scfP-B3 integrins at the plasma membrane in B1 integrin-/-/facp-1/+ cell line. Six FAs per cell were bleached for each experiment. scfP-B3 integrin recovery was monitored for 5 min. 19 cells \\\\(\\\\leq N\\\\leq\\\\) 107 cells. Error bars represent SD. ns, adjusted P value > 0.05 *, P value \\\\(\\\\leq\\\\) 0.05 *, P value \\\\(\\\\leq\\\\) 0.05 *, P value \\\\(\\\\leq\\\\) 0.01 ***, P value \\\\(\\\\leq\\\\) 0.001 ***, P value \\\\(\\\\leq\\\\) 0.0001 **, P value \\\\(\\\\leq\\\\) 0.0001 **, P value \\\\(\\\\leq\\\\) 0.0001 **, (C and D)** Representative images of PLAs (C) and PLA assay quantitation (D) of the number of PLA spots per cell performed with antibodies against NME and ICAP-1 or AP2 or Dynamic 2. Red dots denote regions of signal amplification consistent with NME/ICAP-1 interaction, NME/AP2 interaction, and NME/dynamic 2 interaction in B1 integrin-/-/-scap-1/+ osteoblastic cells (left). PLA performed on ICAP-1 def experiment cell line is used as control. The deletion of ICAP-1 induces a decrease of red dots in all three cases indicating the crucial role of ICAP-1 for keeping NME in clathrin endocytosis machinery (right). Nuclei are stained in blue with DAPI. Scale bar, 20 mm. Error bars represent SD. ns, adjusted P value > 0.05 *, P value \\\\(\\\\leq\\\\) 0.05 *, P value \\\\(\\\\leq\\\\) 0.01 ***, P value \\\\(\\\\leq\\\\) 0.001 ***, P value \\\\(\\\\leq\\\\) 0.0001 **, P value \\\\(\\\\leq\\\\) 0.0001 **, P value \\\\(\\\\leq\\\\) 0.0001 ** (E and F)** Representative images (E) of PLAs with quantification (F) performed with antibodies against NME and B1 integrin or b3 integrin or transferrin receptor. Red dots denote regions of signal amplification consistent with NME/integrin proximity in B1 integrin/-/-icap-1/+ osteoblast cells (left). The deletion of ICAP-1 induces a decrease of the number of red dots indicating the crucial role of ICAP-1 for keeping NME and integrin vicinity (right). PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. Scale bar, 20 mm. Error bars represent SD. \\\\(N\\\\) = 25 cells/condition from three independent experiments. ns, adjusted P value > 0.05 *, P value \\\\(\\\\leq\\\\) 0.05 *, P value \\\\(\\\\leq\\\\) 0.01 ***, P value \\\\(\\\\leq\\\\) 0.001 ***, P value \\\\(\\\\leq\\\\) 0.0001. Source data are available for this figure: SourceData FS.\\n\\n'", "CAPTION FIG5-1.png": "'Figure 5: **ICAP-1 is required for NME recruitment in clathrin-coated pits and for keeping the proximity between integrin and NME.****(A)** Western blots analysis showing the efficiency of SNAME1/2 in osteoblast cells. **(B)** TIRF/FRAP analysis shows that deletion of NME1/2 complex by siRNAs (sRNAs (sRNAs (sRNAs 1/2, light))).\\n\\n'", "CAPTION FIG6-1.png": "'Figure 6: **Requirement of ICAP-1 for NME function in CCP upstream of dynamin and auxillin steps to allow optimal clathrin-coated vesicle budding in the vicinity of focal adhesion.****(A)** Highly inclined illumination fluorescence microscopy analysis was performed at the basal face of adherent cell on FN coated.\\n\\n'", "CAPTION FIG6-2.png": "'coverslide. Left panel shows cells immunostained for vinculin (green) and b1integrin/NME diudink signal (PLA event, red), and the right panel shows cells stained for vinculin (green) and b3 integrin/NME diudink signal (PLA event, red). Scale bar, 5 mm. **(b)** Boxplot representation of the fraction of the hits estimated for b1 integrin/NME-vinculin and for b3 integrin/NME-vinculin and for randomized images (rand.). **(c and D)** Representative images of PLA with quantification performed with antibodies against NME and b3 integrin. Red dots dentate regions of signal amplification revealing (GAP-1/b3 integrin proximity in b1 integrin deficient cells. PLA performed on KAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. **In** \\\\(x\\\\) 2 cells/condition from three independent experiments. Scale bar, 20 mm. **(e and e)** Spinning disk highly magnified video micrographs (every 4 s) of an FA, (vinculin - green) and acuillic bursts in red (indicated by yellow arrow) in b1 integrin/~1-GAP-1+ cells. The quantification in F shows that ICAP-1 deletion slows down the auxillin bursts/FA by 50%. At least 35 FAs and 3 squares in at least 4 cells per condition were imaged in 3 independent experiments. Error bars represent SD. ns, adjusted P value > 0.05, *, P value s 0.05, **, P value s 0.01, ***, P value s 0.001; ***, P value s 0.0001.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Recruitment of NME by ICAP-1 to regulate cellular force by promoting inactive integrin internalization.** Scheme representing the sequence of molecular events during clathrin-mediated integrin endocytosis occurring at the edge of FA. ICAP-1 acts as an adaptor protein in integrin endocytic process. ICAP-1 mediates cargo selection by positioning NME close to integrin (1), AP2, and dynamic (2) in order to fuel dynamic at the clathrin-coated pks (3) allowing execution of subsequent membrane deformation and vesicle fission (4) to ensure the turnover of integrins.\\n\\n'", "CAPTION FIG1-2.png": "'B1 integrin KO cells exert less force than the other osteoblasts mutants. The additional deletion of KAP-1 led to generation of traction forces revealing a novel pathway independent of B1 integrins to generate traction forces on fibronectin. Scale bar, 10 \\\\(\\\\mu\\\\)m. Error bars represent SD. \\\\(N\\\\) \\\\(\\\\times\\\\) 60 cells. _ns_, adjusted P value \\\\(>\\\\)0.05; *, P value \\\\(\\\\approx\\\\) 0.05; **, P value \\\\(\\\\approx\\\\) 0.01; ***, P value \\\\(\\\\approx\\\\) 0.001; ***, P value \\\\(\\\\approx\\\\) 0.0001. **(C)** Immunofluorescence staining of the ppMoxin (ppMMC antibody, red) and F-actin (phalloidin, green) in the four osteoblasts cell lines showed that deletion of KAP-1 alone does not change the organization of astro-myc cytoskeleton but increases slightly the intensity and the thickness of the stress fibers (see quantification of the ppMMC area in E). Deletion of B1 integrin leads to a decrease of the cell area (D) and disorganization and decrease of thickness and number of the ppMMC decorated stress fibers. Scale bar, 20 \\\\(\\\\mu\\\\)m. **(D)** Quantified cell spreading area from staining of fluorescent F-actin. Error bars represent SD. \\\\(N\\\\) \\\\(\\\\times\\\\) 429 cells. _ns_, adjusted P value \\\\(>\\\\) 0.05; *, P value \\\\(\\\\approx\\\\) 0.05; **, P value \\\\(\\\\approx\\\\) 0.01; ***, P value \\\\(\\\\approx\\\\) 0.0001. **(C)** Quantified epMMC staining, normalized to cell area. Error bars represent SD. \\\\(N\\\\) \\\\(\\\\times\\\\) 211 cells. _ns_, adjusted P value \\\\(>\\\\) 0.05; *, P value \\\\(\\\\approx\\\\) 0.05; *, P value \\\\(\\\\approx\\\\) 0.01; ***, P value \\\\(\\\\approx\\\\) 0.001. **(F)** Osteoblasts cells were spread in serum-free medium on uncoated glass for 24 h. Immunofluorescence staining of the extracellular fibronectin (cellular fibronectin antibody, red). F-actin (phalloidin, blue) and B3 integrins (Luc \\\\(K\\\\) antibody, green) in the four osteoblasts cell lines were analyzed by fluorescent confocal microscopy. The \\\\(\\\\beta\\\\)1-/- cells increase rather FN fibrillogenesis events and \\\\(\\\\beta\\\\)1 integrin-/-'", "CAPTION FIG4-2.png": "'performed with antibodies against ICAP-1 and b3 integrin. Red dots denote regions of signal amplification indicating the close proximity between ICAP-1 and b3 integrin. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. **(c)** Representative images of proximity ligation assays performed with antibodies against ICAP-1 and AP2 or Dynamic 2. Red dots denote regions of signal amplification indicating that ICAP-1 belongs to clathrin endocytosis machinery. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. Scale bar, 20 mm. **(d)** PLA performed with antibodies against AP2 and b1 integrin or b3 integrin and quantitation of the number of PLA spots per cell shows that deficiency in ICAP-1 leads to increase of the association of b1 and b3 integrins with AP2. \\\\(N\\\\) > 25 cells/condition from three independent experiments. Scale bars, 20 mm. Error bars represent SD. \\\\(n\\\\) value > 0.05; *, \\\\(p\\\\) value < 0.05; **, \\\\(p\\\\) value < 0.01; ***, \\\\(p\\\\) value < 0.001; ***, \\\\(p\\\\) value < 0.0001. **)** Immunofluorescence staining of the phospho-myosin (p6uDIC antibody, red) and b3 integrin (Luca5 antibody, green). Representative micrographs of the four osteoblast cell lines treated with clathrin siRNA (si Cir, right) or scramble siRNA (si Cir, left). Inhibition of clathrin expression in b1 integrin/~/~/rap-1/+ cell line rescues cell spreading through b3 integrin-mediated FA and development of at-a-myosin cytoskeleton (see also graphs F-H). Scale bar, 20 mm. **(f)** Quantification of cell spreading area of the four osteoblast cell lines treated with clathrin sRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. \\\\(N\\\\) > 27 cells. _n_s, adjusted P value > 0.05; *, \\\\(p\\\\) value < 0.05; ***, \\\\(p\\\\) value < 0.01; ***, \\\\(p\\\\) value < 0.0001. **(g)** Quantification of area of b3 integrin containing FAs, normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA (si Citrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. \\\\(N\\\\) > 27 cells. _n_s, adjusted P value > 0.05; *, \\\\(p\\\\) value < 0.05; **, \\\\(p\\\\) value < 0.01; ***, \\\\(p\\\\) value < 0.001. ***, \\\\(p\\\\) value < 0.0001. **)** Quantification of phospho-myosin staining area normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin sRNA (si Citrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. \\\\(N\\\\) > 27 cells. _n_s, adjusted P value > 0.05; *, \\\\(p\\\\) value < 0.05; *, \\\\(p\\\\) value < 0.05; **, \\\\(p\\\\) value < 0.001. ***, \\\\(p\\\\) value < 0.0001. **, \\\\(p\\\\) value < 0.0001. **, \\\\(p\\\\) value < 0.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG4.png": "'* [11]'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG6.png": "'* [16] A.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/laneEngineeringSerendipityWhen.json b/test_extracted_captions/laneEngineeringSerendipityWhen.json new file mode 100644 index 0000000000000000000000000000000000000000..5f58e292d5ece4f4f6255e963ee3d8d27daaa121 --- /dev/null +++ b/test_extracted_captions/laneEngineeringSerendipityWhen.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'Figure 2: Yearly trend in copublication (left) and forward citation (right) rates between same room (treatment) and different room (control) pairs with 95% CIs\\n\\n'", "CAPTION TAB2.png": "''", "CAPTION TAB4-2.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system of coupled equations. We show that the two-dimensional case is equivalent to the two-dimensional case. We show that the two-dimensional case is equivalent to the two-dimensional case.\\n\\n'", "CAPTION TAB3-1.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional and the two-dimensional Gaussian distributions of the two-'", "CAPTION TAB1.png": "'\\n\\n## Appendix A'", "CAPTION TAB4-1.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system of coupled equations. We show that the two-dimensional case is equivalent to the two-dimensional case. We show that the two-dimensional case is equivalent to the two-dimensional case.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**FIGURE 3** Change in (a) knowledge transfer, (b) knowledge creation, and (c) knowledge diffusion and knowledge similarity for communicating and noncommunicating pairs'", "CAPTION TAB3-2.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system. We study the relationship between the two-dimensional and the two-dimensional case of a two-dimensional system. We study the relationship between the two-dimensional and two-dimensional case of a two-dimensional system.\\n\\n'", "CAPTION TAB5-1.png": "'\\n\\n**Abstract**\\n\\nThe \\\\(\\\\beta\\\\)-function of the \\\\'", "CAPTION FIG1.png": "'\\n\\n**FIGURE 1** Randomization of participants by night, room, group, and poster locations'", "CAPTION TAB5-2.png": "'\\n\\n**Figure Captions**\\n\\nFig. 1. The \\\\(\\\\chi^{2}\\\\) dependence of the \\\\(\\\\chi^{2}\\\\) dependence'", "CAPTION TAB4.png": "'* [10] A.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n**Abstract**\\n\\nThe _Semi-supervised learning_ of a class of supervised learning is a powerful method for classifying the classes of objects in a given class. The supervised learning method is a powerful method for classifying objects in a given class.\\n\\n'", "CAPTION TAB5.png": "'* [10] J. M. C. Collins, \"The \\\\(\\\\pi^{+}\\\\) and \\\\(\\\\pi^{-}\\\\) production cross sections in the \\\\(\\\\pi^{+}\\\\'"} \ No newline at end of file diff --git a/test_extracted_captions/linEffectivenessTeachingScience1996.json b/test_extracted_captions/linEffectivenessTeachingScience1996.json new file mode 100644 index 0000000000000000000000000000000000000000..0636313cebd4390f08f47ce5daf7418c87d13157 --- /dev/null +++ b/test_extracted_captions/linEffectivenessTeachingScience1996.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "'Figure 4: Pictorial analogy and demonstration of water pressure.\\n\\n'", "CAPTION FIG1.png": "'Figure 1., Electrical analogy of density.\\n\\n'", "CAPTION TAB1.png": "'Table 1\\n\\n_Means and Standard Deviations of the Conceptual Problem Solving Test for Experimental and Control Groups_'", "CAPTION FIGA2.png": "'As shown in the following figure, an empty flask is sealed with a rubber stopper which includes a glass tube (at the end of the glass tube, there is a drop of mercury).\\n\\n(a) Can you predict and explain the movement of the mercury if the flask is immersed in the beaker filled with water of 3degC?\\n\\n(b) Can you predict and explain the movement of the mercury if the flask is immersed in the beaker filled with water of 80degC?\\n\\n(c) Can you predict and explain the movement of the mercury if the flask (not including the beaker) is put inside the refrigerator of 5degC?'", "CAPTION FIGA3.png": "'3. As shown in the following figure, a plastic bottle with a small hole on the side is filled with water. The water flows out from the small hole. As soon as you put your hand on the top of the bottle, the water stops flowing. Why does the water stop flowing?'", "CAPTION FIGA4.png": "'In a jewelry store, there are two pieces of golden metal with the same size, shape, and appearance. Knowing that the density of pure gold is 19.3gcm\\\\({}^{-3}\\\\), how can you use the following equipment to identify which piece is the real gold?'", "CAPTION TAB2.png": "'\\n\\n'", "CAPTION FIGA1.png": "'1. As shown in the following figure, a test tube is filled with \\\\({}^{1}\\\\!f_{3}\\\\) volume of water and heated to boiling point (100degC). The test tube is then removed from the burner and a rubber stopper (equipped with a thermometer) is inserted in the end of the test tube. Why does the water boil again if a sack of ice is put around the upper end of the test tube (the reading of the thermometer is below 100degC)?'", "CAPTION FIG2.png": "'Figure 2: Potential analogy of pressure.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Torricelli\u2019s experiment, pictorial analogy, and demonstration of atmospheric pressure.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1979) The \\\\(\\\\chi^{2}\\\\) of the \\\\(\\\\chi^{2}\\\\)'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Summary of Students\u2019 Alternative Conceptions and Difficulties About Density, Atmospheric Pressure, and Pressure_} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Summary of Students\u2019 Alternative Conceptions and Difficulties About Density, Atmospheric Pressure, and Pressure'", "CAPTION FIG5.png": "'Figure 5: The learning progress pattern of the control and the experimental group.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/linseyStudyDesignFixation2010.json b/test_extracted_captions/linseyStudyDesignFixation2010.json new file mode 100644 index 0000000000000000000000000000000000000000..9024e48afc1c7df4bee01920d517a03f532f0798 --- /dev/null +++ b/test_extracted_captions/linseyStudyDesignFixation2010.json @@ -0,0 +1 @@ +{"CAPTION FIG13.png": "'Figure 13: Participants believe they were influenced by the provided example solution\\n\\n'", "CAPTION FIG14.png": "'Figure 14: Participants in the fixation and the defixation groups felt the example solution had a positive influence or at least were unsure that the influence was positive or not\\n\\n'", "CAPTION FIG15.png": "'Fig. 18 Participants were undecided if the example solution negatively influenced them\\n\\nFig. 19: Participants were undecided if the example solution negatively influenced them '", "CAPTION TAB3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: The defixation group used, on average, more energy, sources in total than participants in the other two groups. Each error bar is \\\\(\\\\pm 1\\\\) standard error.\\n\\n'", "CAPTION FIG12.png": "'Fig. 12: All groups used analogies in developing solutions to the design problem. The deflization group, on average, employed slightly more analogies during ideation. The error bars are \\\\(\\\\pm 1\\\\) standard error.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Abstract**\\n\\nWe study the relationship between the two-dimensional (non-)equilibrium dynamics of the system and the two-dimensional (non-)equilibrium dynamics of the system. We show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system. We show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system. We show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system. We also show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system. We also show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system. We also show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system. We also show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system. We also show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system. We also show that the system is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, is a (non-equilibrium) system, and that the system is a (non-equilibrium) system,'", "CAPTION FIG4.png": "'* [19] A.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Fig. 1 Design gradient description**'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2 Example solution provided to the participants in the fixation group**'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8** The fixation group repeated, on average, features from the example solution more often than the other two groups. Each error bar is \\\\(\\\\pm 1\\\\) standard error.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: The fixation group used, on average, a higher percentage of the features from the example solution in their concepts. Each error bar is \\\\(\\\\pm 1\\\\) standard error.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: The defixation group produced more designs powered by a gas engine than individuals in the other conditions. The error bars are \\\\(\\\\pm 1\\\\) standsrd error.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The \\\\(\\\\beta\\\\)-function of the \\\\(\\\\beta'", "CAPTION FIG6.png": "'Figure 6: A set of solutions showing a high degree of fixation on the provided example solution\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## Table 2 Sample size for each condition'", "CAPTION FIG7.png": "'Fig. 7 The fixation group produced fewer ideas, on average, than either the control group or the deflization group. Each error bar is \\\\(\\\\pm 1\\\\) standard error.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## Table 1 Energy source categories'"} \ No newline at end of file diff --git a/test_extracted_captions/lundmark2008gtpaseactivating.json b/test_extracted_captions/lundmark2008gtpaseactivating.json new file mode 100644 index 0000000000000000000000000000000000000000..33e06c5ab0d2d5c8d0028343e7f5ecfa83ee0a05 --- /dev/null +++ b/test_extracted_captions/lundmark2008gtpaseactivating.json @@ -0,0 +1 @@ +{"CAPTION FIG1-1.png": "'Figure 2: The BAR and FH Domaire Localize GRAF1 to Highly Curved, Plains(4,5)P2-Zeninched Membranes, and the SH3 Domain Binds Dynamic (A) Coomassie-stained gel of liposome cosedimentation assay showing the preference of GST-tagged GRAF1 BAR+PH protein for binding to smaller-sized liposomes derived from total brain lipids (the average diameters of liposomes are shown). Pellet (P) and supernatant (S) fractions were separated by ultra-centrifugation. The graph shows quantifications of total band intensities for each condition normalized to binding of the non-curvature-sensitive protein Dah2 (as a way of controlling for total lipid in each experiment). The error bars show 95% confidence intervals (calculated by t) tests for each condition. (B) Liposome cosedimentation assay as performed in (A) but with 0.8-\u03bcm-diameter liposomes enriched with varying phospholipids. (C) Electron micrographs of negatively stained liposomes incubated in the presence or absence of GST-tagged GRAF1 BAR+PH protein. Note the protein-dependent presence of tubular structures. The scale bar represents 200 nm. (D) Immunoprecipitation of GRAF1 from rat brain cytosol (via Ab2) reveals a GRAF1-Dynamim1 complex as identified by mass spectrometry and confirmed by immunoblot. (E) Coomassie-stained gels of pull-down experiments with purified Dynamim and either bead-bound GST-tagged GRAF1/Amphighysin SH3 domain or GRAF1 BAR+PH protein. P = pellet fraction, S = supernatant fraction. (F and G) Epifluorescence micrographs of HeLa cell overexpressing Myc-tagged GRAF1 and incubated with CT&B for 5 min before fixation and staining. (G) Epifluorescence micrographs of a HeLa cellincubated with CT&B for 5 min before fixation and staining for endogenous GRAF1. Scale bars represent 10 \u03bcm.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: GRAF1 Is Indispensable for Ciaithrin-Independent Fluid-Phase Uptake in Fibroblasts (A) Immunoblots on HeLa cell lysates transfected with a control siRNA or either of two siRNAs directed against GRAF1 mRNA (siRNA a and b). Detection of GRAF1 and tubulin (loading control) was performed with specific antibodies on lysates obtained 48 hr after transfection. (B) GRAF1-depleted cells show a major reduction in fluid-phase endocytosis as shown by the decrease in the uptake of FITC-labeled dextran (control siRNA \\\\(n\\\\) = 5), siRNA (\\\\(n\\\\) = 8), or APE siRNA (\\\\(n\\\\) = 3). The error bars show the standard deviation of the mean. (C and D) HeLa cells depleted of GRAF1 were incubated with dextran (C) or transferrin (D) for 15 min before fixation and staining. Scale bars represent 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG1-2.png": "'\\n\\n**(G) Electron micrographs prepared as described and immunolabeled for GRAF1 (10 nm gold particles). Note the GRAF1-positive tubular structures. Distance between arrowhead tips = 40 nm. Scale bars represent 10 \u03bcm.**'", "CAPTION FIG3.png": "'Figure 3: GRAF1 Regulates the CLIC Endocylic Pathway (A) Representative electron micrographs of 65 nm ultrathin cryosections of HeLa cells transiently transfected with GFP-GPI alone or with both GFP-GPI and Myc-tagged GRAF1 FL. Cells were fixed in 2% PFA with 0.2% glutaraldehyde and labeled with anti-GFP and anti-_Myc_ antibodies. Protein A 10 nm gold was used for revealing the GFP in the single labeling (upper image). As shown, GFP-GPI was found in uncoated vesicles and tubules within the cell. Double labeling of GRAF1 (Protein A gold 10 nm) and GFP-GPI (Protein A gold 15 nm) is shown in the bottom image, where colocalization of GRAF1 and GFP-GPI is seen in an intracellular tubule. Arrowheads point to 10 nm gold particles. Due to limitations of the \\n\\n'", "CAPTION FIG1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/lundmark2008gtpaseactivating_supplement.json b/test_extracted_captions/lundmark2008gtpaseactivating_supplement.json new file mode 100644 index 0000000000000000000000000000000000000000..d6787f6a800236284b25af4c17939861c8488d50 --- /dev/null +++ b/test_extracted_captions/lundmark2008gtpaseactivating_supplement.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS1.png": "'\\n\\n**Figure S1 \\\\(|\\\\) Localisation of GRAF1 to tubular membrane structures is temperature sensitive and dependent on the BAR and PH domains.****A,** Western blots showing the different forms of GRAF1 detected in adult rat brain and their differential presence/absence in cultured SH-SY5Y (human neuroblastoma), HeLa (human fibroblast), K562 (human Chronic Myeloid Leukaemia), and MEF (mouse embryonic fibroblast), cells. Western blots of purified GRAF1 and myc-tagged GRAF1 (from lysates of HeLa cells overexpressing this protein) are shown for comparison. **B,** Confocal micrograph of an NIH 3T3 cell stained for endogenous GRAF1 distribution. **C,** Confocal micrographs of HeLa cells fixed either at 4degC or 37degC for 10 minutes in 4% paraformaldehyde and then stained for endogenous GRAF1. Note the absence of GRAF1-positive tubules in the 4degC fixation image. **D**, Confocal micrographs showing the tubular localization of overexpressed myc-tagged GRAF1 BAR+PH protein in HeLa cells and the cytoplasmic localization of a similarly overexpressed protein with a BAR domain mutation (KK131/132EE). **E,** Confocal micrograph showing the cytoplasmic and punctate localization of overexpressed myc-tagged GRAF1 BAR protein in HeLa cells. **Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Figure S5 \\\\(|\\\\) Nature of GRAF1-positive endocytic structures.****A and B,** Confocal micrographs of HeLa cells overexpressing GFP-tagged flotillin1 (A) or GFP-tagged caveolin1 (B) and co-stained for endogenous GRAF1. Note the lack of colocalization. **C and D,** Confocal micrographs of HeLa cells overexpressing myc-tagged GRAF1 BAR+PH and flotillin1 (E) or caveolin1 (F) incubated with CTxB for 5 minutes. Note the colocalization of GRAF1 BAR+PH and flotillin1 in CTxB-positive tubular structures. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "SUPP CAPTION FIGS4.png": "'\\n\\n**Figure S4 \\\\(|\\\\)****GRAF1 BAR+PH overexpression affects CTxB uptake but not transferrin uptake.****A and B,** Confocal micrographs of HeLa cells transfected with myc-tagged GRAF1 BAR+PH and incubated with CTxB or transferrin for 15 minutes before fixation and staining. **C,** The graph shows the quantification of images such as depicted in (A). Cells were scored for expression of GRAF1 BAR+PH (over a threshold corresponding to maximum autofluorescence) and CTXB internalization (over an arbitrarily-set threshold above background). Note the reduction of the number of transfected cells internalising CTxB compared with controls. **D,** Live cell microscopy of NIH 3T3 cells expressing GFP-tagged GRAF1 and incubated with CTXB and transferrin (Tfn) at 4degC before chasing their internalization from the time of warming to 37degC (time=00:00). Note the internalising GRAF1-positive tubule containing CTxB. Note also the lack of colocalization of GRAF1-positive tubules with internalized transferrin. Time is given as minutes:seconds. This sequence is taken from Movie S3. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "SUPP CAPTION FIGS6.png": "'\\n\\n**Figure S6 \\\\(|\\\\)****GRAF1 BAR+PH overexpression does not affect the uptake of MHC Class I which enter GRAF1-negative compartments.****A and B,** Epifluorescence micrographs of HeLa cells, transiently transfected with GFP-tagged GRAF1 FL (A) or GRAF BAR+PH (B) were pulsed with anti-MHC Class I antibody for 5 (A) or 15 minutes (B) at 37\\\\({}^{\\\\circ}\\\\)C followed by a brief acid wash to remove surface-bound antibody. MHC Class I was visualized using Alexa568-conjugated secondary antibodies. **C,** Quantitation of surface GFP-GPI levels in cells overexpressing this protein with myc-tagged GRAF1 FL or GRAF1 BAR+PH. **D,** Confocal micrographs of HeLa cells treated with siRNA against GRAF1 or a control siRNA and stained for endogenous GRAF1. **Scale bars = 10\\\\({}_{\\\\mu}\\\\)m.**'", "SUPP CAPTION FIGS2.png": "'Figure S.A.: **Supplementary behavioral data in the binding between GAU1 and Dyes1.**\\n\\n'", "SUPP CAPTION FIGS2-1.png": "\"\\n\\n**Figure S2 A-D\\\\(|\\\\) Supplementary biochemical data on the binding between GRAF1 and Dynamin.****A,** Coomassie-stained gel and confirmatory Western blots of coimmunoprecipitation experiments in rat brain cytosol performed with either control pre-immunization serum (pre-serum) or the Ab3 antibody. Bands in the Coomassie-stained gel were identified by mass spectrometry as described. **B and C,** Coomassie-stained gel and Western blots of pull-down experiments from mouse brain lysate (B) or HeLa cell cytosol (C) with beads bound to GST (control) or GST-tagged GRAF1 BAR+PH, or SH3 proteins. The bands in the Coomassie-stained gel were identified by mass spectrometry as described. Note the major band of Dynamin present in the SH3 lanes, which is not present in the control or BAR+PH condition. 'cyt' marks the HeLa cell lysate (positive control) lane. **D,** The upper panel shows a raw trace from isothermal titration calorimetry performed as described. The lower panel shows the fitting of this data to a one-site binding model from which the affinity (shown) can be calculated. GRAF1 SH3 domain and peptide concentrations, as well as injection volumes and times are shown.\\n\\n\"", "SUPP CAPTION FIGS3.png": "'\\n\\n**Figure S3 \\\\(|\\\\) GRAF1-positive endocytic structures are Clathrin-independent and exclude transferrin A-C, Confocal fluorescent micrographs of HeLa cells stained for endogenous GRAF1 and Clathrin (A), transferrin (B) or transferrin receptor (B). The depicted images are used to show some of the different morphologies of GRAF1-positive structures that are observed in these cells. Scale bars = 10\\\\(\\\\mu\\\\)m.**'", "SUPP CAPTION FIGS2-2.png": "'\\n\\n**Figure S2 E \\\\(\\\\mid\\\\) Dynasore inhibits the uptake of dextran and affects the localization of GRAF1. E, Confocal micrographs (maximum projections) of HeLa cells treated with either DMSO (vehicle) or 100\\\\(\\\\mu\\\\)M dynasore for 1 hour before addition of dextran for 15 minutes, fixation, and immunostaining for dextran and the focal adhesion marker paxillin. Scale bars = 10\\\\(\\\\mu\\\\)m.**'"} \ No newline at end of file diff --git a/test_extracted_captions/mcfarland2008rna.json b/test_extracted_captions/mcfarland2008rna.json new file mode 100644 index 0000000000000000000000000000000000000000..ed19b22bc05f4fa9c77b0ce2eab3e5411cdf6f96 --- /dev/null +++ b/test_extracted_captions/mcfarland2008rna.json @@ -0,0 +1 @@ +{"CAPTION FIG5.png": "\"\\n\\n**Fig. 5.** Effect of RNAi knockdown of dynamin 2 and the g2 subunit of the AP2 complex on the internalization of 488-Tf in dCAD cells. Seventy-two hours after transfection with either mock, g2 subunit, or dyananin 2 siRNA, dCAD cells were incubated with 488-Tf for 10 min and imaged by confocal microscopy. Both g2 subunit- and dyananin 2 siRNA-transfected cells showed a reduction of 488-Tf internalization (indicated by arrows) compared with mock-transfected dCAD cells. Images were obtained using confocal microscopy as described under Materials and Methods. The internal fluorescence intensity of the intracellular space of all cells in the field of view was quantified using MetaMorph software (Molecular Devices, Sunnyvala, CA) (bottom right). Fifteen to 20 cells per experiment were analyzed. Data are representative of three separate experiments. Asterisks denote a significant difference in intracellular mean internal fluorescence intensity compared with mock-transfected cells (analysis of variance and Dunnett's multiple comparison test; \\\\(\\\\rho<0.01\\\\)).\\n\\n\"", "CAPTION FIG6.png": "\"Figure 5: Effect of RNAi knockdown of dynamin 2 and the g2 subunit of the AP2 complex on the internalization of 488-CT in dCAD cells. Seventy-two hours after transfection with either mock, g2 subunit, or dyananin 2 siRNA, dCAD cells were incubated with 488-CT for 10 min and imaged by confocal microscopy. Dynamin 2 siRNA-transfected cells showed a reduction of 488-CT internalization (indicated by arrows) compared with neck-transfected dCAD cells. g2 subunit siRNA-transfected cells did not display any difference in 488-CT internalization compared with neck-transfected dCAD cells, confirming the specificity of the treatments. Images were obtained using confocal microscopy as described under Materials and Methods. The internal fluorescence intensity of the intracellular space of all cells in the field of view was quantified using MetaMorph software (bottom right). Fifteen to 20 cells per experiment were analyzed. Data are representative of three separate experiments. Asterisks denote a significant difference in intracellular mean fluorescence intensity compared with neck-transfected cells (analysis of variance and Dannett's multiple comparison test; \\\\(p<0.05\\\\)).\\n\\n\"", "CAPTION FIG7.png": "'Figure 7: SKM 4-45-1 internalization by dCAD cells after siRNA transfection. dCAD cells were treated with SKM 4-45-1 (35 \\\\(\\\\mu\\\\)M) for 10 min at 72 h after transfection with mock, b2 subunit, or dynamin 2 siRNA. Knockdown of the b2 subunit of the AP2 complex had no effect on the SKM 4-45-1-derived fluorescence signal compared with mock. Knockdown of dynamin 2 in dCAD cells significantly decreased the accumulation of SKM 4-45-1-derived fluorescence. Images were obtained using confocal microscopy as described under Materials and Methods. Phase contrast images are shown below each corresponding fluorescence image. Data are representative of three separate experiments.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Expression of FAAH in CAD cells. A, eCAD and dCAD cells express FAAH. Western blots were performed as described under Materials and Methods. Data are representative of three separate experiments. B, cCAD and dCAD cells were homogenized and then incubated at \\\\(\\\\pm\\\\)7\u00b0C for 10 min. Cell homogenates were then incubated with 5 nM [3H]AEA in the presence or absence of 600 nM MAFP. After the incubation, the reaction was terminated, and the aqueous and organic phases were separated. Bars represent AEA-derived [3H]ethanalanine present in the aqueous phase (i.e., metabolized NEA) after the termination of the reaction as a direct measure of FAAH activity. Data shown are means \\\\(\\\\pm\\\\) S.D. of three separate experiments performed in duplicate.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 1.** Effect of lipid raft disruption on \\\\(\\\\Lambda\\\\)EA uptake in dCAD cells. dCAD cells were pretreated for 30 min with 25 mg/ml nystatin and 10 mg/ml progesterone (NP treatment). After NP treatment, AEA uptake assays were performed in 1\\\\(\\\\times\\\\) KRH at 37degC in the presence or absence of 100 mM AM404 to define nonspecific transport. Transport assays were performed for 5 min with 1 nM [\\\\(\\\\Gamma\\\\)H]\\\\(\\\\Lambda\\\\)EA as described under Materials and Methods. Data are means \\\\(\\\\pm\\\\) S.E.M. combined from three separate experiments performed in triplicate. The cpm values for control cells were total = 5755 \\\\(\\\\pm\\\\) 118 and nonspecific = 502 \\\\(\\\\pm\\\\) 41. The cpm values for NP-treated cells were total = 3117 \\\\(\\\\pm\\\\) 40 and nonspecific = 729 \\\\(\\\\pm\\\\) 61. Inset, offset of NP treatment on membrane cholesterol content. Cholesterol content in CAD cell membranes was determined as described under Materials and Methods. Data are means \\\\(\\\\pm\\\\) S.E.M. from two separate experiments performed in triplicate. *, \\\\(p\\\\)\\\\(<\\\\) 0.05.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: RNA-mediated knockdown of the g2 subunit of the AP2 complex and dynamin 2. dCAD cells were transfected with Stealth RNAi (Invitrogen) using Lipofectamine 2000 (Invitrogen) and then incubated at 37\u00b0C in serum-fros media for 72 h to allow for protein degradation. The expression of both the g2 subunit of the AP2 complex and the protein dynamin 2 was decreased by >90% compared with mock siRNA (response sequence) transfection. siRNA oligonucleotides had no effect on the level of control protein \u03b2-actin (data not shown). To control for protein loading, equal amounts of total protein (20 mg) from the cell lysates were added to each well for Western blot analysis as described under Materials and Methods. Both proteins resolved at the expected molecular weights. Data are representative of three separate experiments.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: \\\\({}^{3}\\\\)H\\\\(\\\\|\\\\)MEA uptake by dCAD cells after siRNA transfection. Seventy-two hours after transfection with mock, dynamic 2, or g2 subunit siRNA, \\\\({}^{3}\\\\)H\\\\(\\\\|\\\\)MEA uptake assays were performed in 1\\\\(\\\\times\\\\) KRH at 37\\\\({}^{\\\\circ}\\\\)C as described under Materials and Methods. AM404 (100 pM) was used to define nonspecific transport. Data represent maan \\\\(\\\\pm\\\\) S.E.M. for three separate experiments performed in triplicate. The epm values for mock cells were total = 2887 \\\\(\\\\pm\\\\) 136 and nonspecific = 1126 \\\\(\\\\pm\\\\) 128. The epm values for dynamin 2 knockdown cells were total = 2973 \\\\(\\\\pm\\\\) 89 and nonspecific = 505 \\\\(\\\\pm\\\\) 83. The epm values for g2 subunit knockdown cells were total = 2638 \\\\(\\\\pm\\\\) 104 and nonspecific = 717 \\\\(\\\\pm\\\\) 27.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Metabolism of [\\\\({}^{3}\\\\)H]\\\\(\\\\lambda\\\\)EA in dynamic 2 siRNA-transfected dCAD cells. \\\\(\\\\Lambda\\\\), 72 h after transfection with either neck or dynamic 2 siRNA, dCAD cells were treated with [\\\\({}^{3}\\\\)H]\\\\(\\\\lambda\\\\)EA for 5 min and then lysed. Cellular lipids were extracted from the cell lysates using chloroform/methanol and analyzed by TLC for intact [\\\\({}^{3}\\\\)H]\\\\(\\\\lambda\\\\)EA. Noaspecific uptake was determined using 100 \\\\(\\\\mu\\\\)M \\\\(\\\\Delta\\\\)M404. The graph represents data from three separate experiments. Statistical analysis was performed using a one-sample \\\\(t\\\\) test comparing with the value of 100 (neck control). \\\\({}^{\\\\ast}\\\\), \\\\(p<0.05\\\\). The cpm values from the TLC analysis for mock cells were total = 2986 \\\\(\\\\pm\\\\) 795 and nonspecific = 2243 \\\\(\\\\pm\\\\) 407. The cpm values from the TLC analysis for dynamic 2 knockdown cells were total = 3257 \\\\(\\\\pm\\\\) 545 and nonspecific = 1799 \\\\(\\\\pm\\\\) 198. B, PAAH expression after RNAi-mediated knockdown of dynamin 2 Knockdown of dynamin 2 in cCAD cells was performed as described under Materials and Methods except siLentPect Lipad Reagent (Bio-Rad) was used as the transfection reagent for 48 h with 10 nM siRNA oligo. Western blots were performed using the polyclonal dynamin 2 antibody, monoclonal PAAH antibody (1:1000; Aknova Corporation, Taipei City, Taiwan), monoclonal actin antibody (1:2000; Sigma-Aldrich, St. Louis, MO). C, PAAH activity after RNAi-mediated knockdown of dynamin 2. PAAH activity assays were performed on whole cell lysates as described under Materials and Methods. Data shown represent means \\\\(\\\\pm\\\\) 5.D. from two experiments performed in quadruplicate.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Dose-dependent inhibition of AEA uptake and SKM 4-45-1 internalization by AM404. Both dCAD (\\\\(\\\\blacksquare\\\\)) and dCAD (eiref) cells were treated with increasing concentrations of AM404 for 10 min at 37\u00b0C. A, after the incubation with AM404, [H]AEA was added at a final concentration of 1 nM as described under _Materials and Methods_. Data shown are means \\\\(\\\\pm\\\\) S.E.M. and are representative of four separate experiments performed in triplicate. _K_i values (means \\\\(\\\\pm\\\\) S.E.M., \\\\(n\\\\) = 3) were 850 \\\\(\\\\pm\\\\) 600 nM (dCAD) and 930 \\\\(\\\\pm\\\\) 150 nM (dCAD). The cpm values for cCAD cells were total = 6440 \\\\(\\\\pm\\\\) 914 and nonspecific = 850 \\\\(\\\\pm\\\\) 86. The cpm values for dCAD cells were total = 5124 \\\\(\\\\pm\\\\) 1456 and nonspecific = 794 \\\\(\\\\pm\\\\) 35. B, cells were treated with SKM 4-45-1 (25 mM) and increasing concentrations of AM404 for 10 min, and fluorescence was determined on a FUSION microplate reader (PerkinElmer Life and Analytical Sciences). IC30 values were converted to _K_i values using the Cheng-Prusoff equation (_K_i value for SKM 4-45-1 = 16 mM). _K_i values (means \\\\(\\\\pm\\\\) S.E.M.; \\\\(n\\\\) = 31) were 24 \\\\(\\\\pm\\\\) 12 mM (cCAD) and 16 \\\\(\\\\pm\\\\) 10 mM (dCAD). Data shown are representative of three separate experiments performed in duplicate. Arbitrary fluorescence unit values for dCAD cells were total = 13,858 \\\\(\\\\pm\\\\) 1856, nonspecific = 7321 \\\\(\\\\pm\\\\) 573, and background = 4313 \\\\(\\\\pm\\\\) 625. Arbitrary fluorescence unit values for dCAD cells were total = 23,137 \\\\(\\\\pm\\\\) 6590, nonspecific = 11,309 \\\\(\\\\pm\\\\) 4024, and background = 2006 \\\\(\\\\pm\\\\) 74.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/messa2014epsin.json b/test_extracted_captions/messa2014epsin.json new file mode 100644 index 0000000000000000000000000000000000000000..b73534b19625e1c9df5a14e89210349861de5ba1 --- /dev/null +++ b/test_extracted_captions/messa2014epsin.json @@ -0,0 +1 @@ +{"CAPTION FIG6-1.png": "'Figure 6: Epsin directly binds synaptobrevin 2/VAMP2. {**A**} Interaction of purified epsin ENTH\u2013His6 (\\\\(\\\\delta\\\\)00 ng) with increasing amounts of GST-cytosolic portion of synaptobrevin2 (\\\\(\\\\mathrm{S}\\\\)/\\\\(\\\\mathrm{b}\\\\)2) as revealed by anti-His immunoblotting of bound material in a GST pull-down (top). Coomassie blue stained gel of the baits (bottom). {**B**} The interaction of epsin ENTH\u2013His6 with equal amounts of GST\u2013Syb2 (\\\\(\\\\mathrm{S}\\\\)/\\\\(\\\\mathrm{b}\\\\)2) is enhanced by the presence of soluble diC8-PI(4,5)P2 (PI(4,5)P3), IP2 or IP4 (final concentration S0 \u03bcM). Top: anti-His immunoblotting. Bottom: Coomassie blue stained Figure 6. Continued on next page\\n\\n'", "CAPTION FIG6-2.png": "'Figure 6: Continued\\n\\nFigel of the baits. EB: empty beads. (**C**) The interaction of apsin ENTH-His6 with equal amounts of GST-Syb2 is lost in a construct missing the N-terminal helix zero (**A**-**H**a**k**0) irrespective of the presence of \\\\(\\\\text{IP}_{k}\\\\) (final concentration 50 mM). EB: empty beads. (**D**) Quantitative analysis of the binding of ENTH-His6 to increasing amounts of GST-Syb2 in the presence or absence of \\\\(\\\\text{IP}_{k}\\\\) as revealed by densitometry of anti-His immunoreactivity in western blots of bound material. \\\\(\\\\text{K}_{\\\\text{6}}\\\\)s are also indicated. (**E**) Schematic representation of constructs used for (**F**\u2013**I**). SNARE motif and transmembrane region (TM) are denoted by dotted lines. (**F** and **G**) Pull-down of ENTH-His6 by GST fusions of different cytosolic fragments of Syb2. Anti-His immunoblotting (**F**) shows binding only to SNARE motif containing fragments (top). Coomassie blue stained gel of the baits (Bottom). Quantitative analysis of the results is shown in (**G**). (**H** and **I**) ENTH-His6 domain binding to SNARE motif fragments (**H**) and corresponding quantification (**I**). Anti-His- immunoblotting detects ENTH interaction with the N-terminal portion of the SNARE motif. (**J**) Purified yeast His-Snct binds a GST fusion of the ENTH domain of Ent1 (yeast apsin 1) in a pull down assay (top). Coomassie blue stained gel of the baits (Bottom). Corresponding quantification is shown. Data are represented as mean +- SEM. See also **Figure 6\u2014figure supplement 1**.\\n\\nFigel of the baits. EB: empty beads. (**C**) The interaction of apsin ENTH-His6 with equal amounts of GST-Syb2 is lost in a construct missing the N-terminal helix zero (**A**-**H**a**k**0) irrespective of the presence of \\\\(\\\\text{IP}_{k}\\\\) (final concentration 50 mM). EB: empty beads. (**D**) Quantitative analysis of the binding of ENTH-His6 to increasing amounts of GST-Syb2 in the presence or absence of \\\\(\\\\text{IP}_{k}\\\\) as revealed by densitometry of anti-His immunoreactivity in western blots of bound material. \\\\(\\\\text{K}_{\\\\text{6}}\\\\)s are also indicated. (**E**) Schematic representation of constructs used for (**F**\u2013**I**). SNARE motif and transmembrane region (TM) are denoted by dotted lines. (**F** and **G**) Pull-down of ENTH-His6 by GST fusions of different cytosolic fragments of Syb2. Anti-His immunoblotting (**F**) shows binding only to SNARE motif containing fragments (top). Coomassie blue stained gel of the baits (Bottom). Quantitative analysis of the results is shown in (**G**). (**H** and **I**) ENTH-His6 domain binding to SNARE motif fragments (**H**) and corresponding quantification (**I**). Anti-His- immunoblotting detects ENTH interaction with the N-terminal portion of the SNARE motif. (**J**) Purified yeast His-Snct binds a GST fusion of the ENTH domain of Ent1 (yeast apsin 1) in a pull down assay (top). Coomassie blue stained gel of the baits (Bottom). Corresponding quantification is shown. Data are represented as mean +- SEM. See also **Figure 6\u2014figure supplement 1**.\\n\\nFigel of the baits. EB: empty beads. (**C**) The interaction of apsin ENTH-His6 with equal amounts of GST-Syb2 is lost in a construct missing the N-terminal helix zero (**A**-**H**a**k**0) irrespective of the presence of \\\\(\\\\text{IP}_{k}\\\\) (final concentration 50 mM). EB: empty beads. (**D**) Quantitative analysis of the binding of ENTH-His6 to increasing amounts of GST-Syb2 in the presence or absence of \\\\(\\\\text{IP}_{k}\\\\) as revealed by densitometry of anti-His immunoreactivity in western blots of bound material. \\\\(\\\\text{K}_{\\\\text{6}}\\\\)s are also indicated. (**E**) Schematic representation of constructs used for (**F**\u2013**I**). SNARE motif and transmembrane region (TM) are denoted by dotted lines. (**F** and **G**) Pull-down of ENTH-His6 by GST fusions of different cytosolic fragments of Syb2. Anti-His immunoblotting (**F**) shows binding only to SNARE motif containing fragments (top). Coomassie blue stained gel of the baits (Bottom). Quantitative analysis of the results is shown in (**G**). (**H** and **I**) ENTH-His6 domain binding to SNARE motif fragments (**H**) and corresponding quantification (**I**). Anti-His- immunoblotting detects ENTH interaction with the N-terminal portion of the SNARE motif. (**J**) Purified yeast His-Snct binds a GST fusion of the ENTH domain of Ent1 (yeast apsin 1) in a pull down assay (top). Coomassie blue stained gel of the baits (Bottom). Corresponding quantification is shown. Data are represented as mean +- SEM. See also **Figure 6\u2014figure supplement 1**.\\n\\nFigel of the baits. EB: empty beads. (**C**) The interaction of apsin ENTH-His6 with equal amounts of GST-Syb2 is lost in a construct missing the N-terminal helix zero (**A**-**H**a**k**0) irrespective of the presence of \\\\(\\\\text{IP}_{k}\\\\) (final concentration 50 mM). EB: empty beads. (**D**) Quantitative analysis of the binding of ENTH-His6 to increasing amounts of GST-Syb2 in the presence or absence of \\\\(\\\\text{IP}_{k}\\\\) as revealed by densitometry of anti-His immunoreactivity in western blots of bound material. \\\\(\\\\text{K}_{\\\\text{6}}\\\\)s are also indicated. (**E**) Schematic representation of constructs used for (**F**\u2013**I**). SNARE motif and transmembrane region (TM) are denoted by dotted lines. (**F** and **G**) Pull-down of ENTH-His6 by GST fusions of different cytosolic fragments of Syb2. Anti-His immunoblotting (**F**) shows binding only to SNARE motif containing fragments (top). Coomassie blue stained gel of the baits (Bottom). Quantitative analysis of the results is shown in (**G**). (**H** and **I**) ENTH-His6 domain binding to SNARE motif fragments (**H**) and corresponding quantification (**I**). Anti-His-phospho blotting detects ENTH interaction with the N-terminal portion of the SNARE motif. (**J**) Purified yeast His-Snct binds a GST fusion of the ENTH domain of Ent1 (yeast apsin 1) in a pull down assay (top). Coomassie blue stained gel of the baits (Bottom). Corresponding quantification is shown. Data are represented as mean +- SEM. See also **Figure 6\u2014figure supplement 1**.\\n\\nFigel of the baits. EB: empty beads. (**C**) The interaction of apsin ENTH-His6 with equal amounts of GST-Syb2 is lost in a construct missing the N-terminal helix zero (**A**-**H**a**k**0) irrespective of the presence of \\\\(\\\\text{IP}_{k}\\\\) (final concentration 50 mM). EB: empty beads. (**D**) Quantitative analysis of the binding of ENTH-His6 to increasing amounts of GST-Syb2 in the presence or absence of \\\\(\\\\text{IP}_{k}\\\\) as revealed by densitometry of anti-His immunoreactivity in western '", "CAPTION FIG7.png": "\"\\n\\n**Figure 7.** Functions of apsin in clathrin-mediated endocytosis. (**A**) Schematic representation of the role of apsin in the coupling of the endocytic clathrin coat to actin in cooperation with Hip1R (left). This coupling helps invaginate the pit. Only apsin localized at the equator of the bud is shown to emphasize its actin-related function in pit invagination, but apsin is not restricted to the equator. Higher magnification representation of the interactions of apsin (night): the N-terminal ENTH domain binds (1) the PI[4,5]P\\\\({}_{2}\\\\)-rich membrane bilayer of the plasma membrane (and partially penetrates it), [2] the ANTH domain of Hip1R, and (3) synaptobrevin2/NAMP2; its tail binds (1) ubiquitinated cargo proteins (via UIMs), (2) AP-2 and clathrin heavy chain (via DPF/DPW motifs and 'clathrin boxes', respectively), and (3) the EH domain region of Eps15 (via NPF motifs). Epsin's tail also binds F-actin and co-operates with the THATCH domain of Hip1R in the coupling of the actin cytoskeleton to the clathrin-coated pit. In both fields Hip1R is depicted as a monomer but functions as a dimer, or heterodimer with Hip1. (**B**) Schematic representation of the multiple functions of apsin in clathrin-mediated endocytosis. Epsin participates in the early stages of the reaction as a coat nucleator, curvature inducer/sensor and also helps coupling bud formation to SNARE incorporation. As the coat matures, Lbequitinated cargo is recruited. Additionally, the link between apsin/Hip1R and actin is required for the deep invagination of the pit, and this is the process at which the action of apsin becomes essential. Subsequently, the force produced by actin and the bilayer destabilizing properties of apsin's ENTH domain may co-operate with dynamin in fission.\\n\\n**Figure 8.** The binding of the actin cytoskeleton to the actin cytoskeleton to the actin cytoskeleton to the actin cytoskeleton. The binding of the actin cytoskeleton to the actin cytoskeleton is shown to be a monomer with Hip1R.\\n\\n\"", "CAPTION FIG1-2.png": "'used as a control. (**C**) Anti-opsin 1 immunofluorescence shows that the typical punctate eosin 1 signal of WT cells (top) was completely absent in TKO cells (bottom). The perimeter of the TKO cell is indicated by a dotted white line and demonstrates a very large size relative to WT cells. The insets show higher magnification of the boxed regions. (**D**) DAPI staining showing single nuclei in WT and multiple nuclei in a TKO cell. The inset of the WT field shows the accumulation of AuroraB kinase immunoreactivity at the mid-body during cytokinesis. (**E**) The increase in cell number during a 3-day incubation is lower in TKO cells. Cells were counted at day 7 and 9 after addition of OHT (\"+p < 0.01, Student\\'s t test, \\\\(n\\\\) = 3 experiments). (**F**) As shown by a morphometric analysis, cytokinesis events are only rarely observed in TKO cells. Scale bar represents 10 mm. Data are represented as mean +- SEM. See also Figure 1**--figure supplement 1**.\\n\\nDOI: 10.7554/eLife.03311.003\\n\\nThe following figure supplement is available for figure 1:\\n\\n**Figure supplement 1**. The generation of triple KO cells, nestin Cre brain specific triple KO mice, and the observations of nuclear defect in TKO cells.\\n\\nDOI: 10.7554/eLife.03311.004'", "CAPTION FIG2-1.png": "'Figure 2: Absence of epain stalls endocytic clathrin-coated pit maturation at an early stage. Confocal microscopy (**A\u2013D**) Immunofluorescence staining for a-adsptin (**A** and **B**) and clathrin light chain (**C** and **D**) indicates an increase of clathrin-coated pits (CCPs) number in cells that lack all three epins. In Figure 2, Continued on next page\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: Mitotic defects in apsin TKO fibroblasts. (**A**) Anti-opsin 1 immunoblot shows the disappearance of apsin 1 from Epn1(a,b,f(a,b); Epn2(a,b); Epn3(b,c); Cre-ER(a,d) cells after 7-day treatment with 4-hydroxy-tamoxifen (OHT). Tubulin was used as a loading central (top). Densitometric analysis of the apsin 1 bundle during OHT-treatment (bottom). (**B**) Anti-epsin immunoblots with isoform-specific antibodies showing OHT-treatment for 7 days results in the loss of apsin 1 in apsin 2/3 double knock-out (DKO) thus generating triple KO (TKO) cells. Wild type (MT) cell lysate was Figure 1. Continued on next page\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Abnormal actin distribution in epsilon deficient cells. **(A-C)** Confocal microscopy images. Phalloidin staining of WT and organ TKO cells shows a major loss of stress fibers with a corresponding accumulation of elongated F-actin foci (see inset) in TKO cells. These changes were rescued by the expression of epsilon1-GFP **(C). **(D)** Quantification of the actin foci shown in **(A-C, ***p \\\\(<\\\\) 0.001, \\\\(\\\\mu\\\\) = 8 cells/conditions, one-way ANOVA). **(E)** Phalloidin staining and Arp 2/3 immunoreactivity in a WT and a TKO cell showing the co-localization of Arp 2/3 with the actin foci, as visualized by confocal microscopy. **(F)** TIRF microscopy of WT and TKO cells expressing clathrin light chain-GFP (CLC-GFP) and the F-actin binding protein utrophin-mCherry (CH4-mCh). Note the increase in F-actin (CH4-mCh signal) typically surrounding the clathrin-coated pits, in the cortical region (TIRF phase) of the TKO cell. Virtually all pits are positive for CH4-mCh. Scale bars: 10 \u03bcm for **(A-C)**, 5 \u03bcm for **(E and F). Data are represented as mean \u00b1 SEM. DOI: 10.7554/eLife.03311.007\\n\\n'", "CAPTION FIG2-2.png": "\"TKO cells, clathrin-coated pits generally occur in small clusters. Insets show the boxed regions at high magnification. Note the large size of TKO cells relative to control. (**E**) CCP number in WT and TKO cells as assessed by **a**-adaptin immunofluorescence (**P** < 0.001, Student's t test, \\\\(n\\\\) = 10 cells/genotype). (**F**) Kymographs from a time series of WT and TKO cell expressing **u**2-adaptin-GFP Each line represents a single **u**2-GFP spot. Note the short length of the line for WT, reflecting the turnover of the pits and the continuous lines in TKO cells, reflecting an arrest of the pit maturation. (**G**) Clatin-coated pit turnover (appearance and disappearance events) as analyzed by spinning-disk confocal imaging of **u**2-adaptin-GFP fluorescence in WT and TKO cells (_n_ = 5 cells/genotype). (**M** and **I**) impaired uptake of pre-bound Alexa594-transferrin (Tf) in TKO cells during a 15-min incubation. In WT, the bulk of Tf was internalized, while in TKO Tf remained at the call surface. (**J** and **K**) Transferrin receptor (TfR-GFP predominantly localizes in intracellular vesicles in WT but at the call surface in TKO cells. (**L**) A surface biotinylation assay reveals elevated amounts of endogenously expressed TfR at the plasma membrane of TKO cells relative to WT, as assessed by anti-TfR immunoblotting of streptavidin affinity-purified maternal. (**M**-**P**) Increased surface localization of stably expressed Syb2-HA in TKO fibroblasts as shown by total (**M**-**O**) and surface-only (**N**-**P**) immunofluorescence. (**O** and **N** surface biotinylation performed as a (**U**) demonstrating an increased fraction of cell surface exposed Syb2-HA in TKO cells relative to WT (**T***P** < 0.01, Student's t test, \\\\(n\\\\) = 4 experiments, Surf. surface, Int. internal. (**S** and **T**) Representative electron microscopy images of different stage endocytic clathrin-coated intermediates in TKO cells (**S**) and quantification of the corresponding stages (**T**, ***P** < 0.01, ***P** < 0.001, \\\\(n\\\\) = 33 cells/genotype, one-way ANOVA). (**U**) Comparative analysis of the localization of clathrin immunoreactivity (CLC) with the localization of dynamin 2, endophilin 2, and myosin 1E immunoreactivity. In WT cells, these three proteins co-localize with a subset of clathrin-coated pits (examples are indicated by small white arrows), which represent late-stage pits. In TKO cells, where more numerous clathrin-coated pits are observed, the punctate localization of dynamin 2, endophilin 2, end myosin 1E is completely lost. Scale bars: 10 _\u03bc_m for (**A**-**D**, **H**-**K**), 20 _\u03bc_m for (**M**-**P**), 5 _\u03bc_m for (**U**), and 200 _\u03bc_m for (**S**). In **E**, **G**, **R**, and **T** black bars indicate WT and red bars open TRCO. See also **Figure 2**--figure supplement**1.\\n\\nThe following figure supplement is available for figure 2:\\n\\nFigure 2: _Continued_\\n\\n\"", "CAPTION FIG6.png": "'* [19] J.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG2.png": "'* [16]'", "CAPTION FIG4.png": "'Figure 4: Epsin is required for the recruitment of Hip1R at the endocytic clathrin-coated pits. (**A**) Immunoblot analysis of Hip1R (left) shows an increase (as quantified in the right) of its levels in \\\\(\\\\left( \\\\mathbf{B} \\\\right)\\\\) Full down experiments from rat brain homogenate, using ENTH-GST or GST alone as bait, shows the affinity-purification of Hip1R in the presence of dI(4,5)P, \\\\(\\\\left[ \\\\mathbf{P}\\\\left[ \\\\mathbf{V}\\\\left[ \\\\mathbf{A} \\\\right] \\\\right\\\\rbrack_{\\\\mathbf{P}} \\\\right]_{\\\\mathbf{Q}}\\\\), \\\\(\\\\left[ \\\\mathbf{c} \\\\right\\\\rbrack_{\\\\mathbf{P}}\\\\), \\\\(\\\\left[ \\\\mathbf{c} \\\\right\\\\rbrack_{\\\\mathbf{Q}}\\\\), \\\\(\\\\left[ \\\\mathbf{c} \\\\right\\\\rbrack_{\\\\mathbf{P}}\\\\), \\\\(\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Coupling of clathrin-dependent budding to actin dynamics in a cell-free assay is perturbed by the absence of open. Plasma membrane sheets of PTX2 cells expressing PM-anchored GFP (PM-GFP) were incubated in the presence of WT, open TKO or Hip1/Hip1R double KD (DKO) brain cytosol, and nucleotides as described in _Wu et al._ (2010), fixed, immunostained, and observed by confocal microscopy. Views orthogonal to the substrate are shown. (**A**) Immunofluorescence staining for clathrin light chain and open 1. The GFP-positive columns represent narrow tubular invaginations of the plasma membrane capped by clathrin-coated pits, as revealed by the presence of clathrin and open immunoreactivity (_Wu et al._, 2010), in the control preparation (_Jeff_) tubules are straight and perpendicular to the substrate, while in the preparation incubated with TKO cytosol (right, note the absence of open immunoreactivity) they have a more disordered orientation. (**B**) Phalloidin staining of the membrane sheets revealing a well-organized actin scaffold around the tubules after incubation with WT cytosol (left) and an exaggerated and disorganized F-actin network in sheets incubated with TKO cytosol (right). (**C**) Myosin TE immunoreactivity locales at the bottom of tubular invaginations in sheets incubated with WT cytosol (left), and at the tip of the invaginations after incubation with TKO cytosol (right). Details of the sheets are shown at high magnification below the main panels. (**D**) Hip1R immunoreactivity is present at the tip of tubular invaginations in the control samples (left), but is absent in sheets incubated with open TKO cytosol (right). (**C**) Quantification of F-actin polymerization upon incubation with the cytosol of the three tested genotypes (MT, open TKO, and Hip1/Hip1R RDC). The average phalloidin fluorescence per unit area of membrane sheets was calculated (_n_ = 10 sheets/conditions, ****P < 0.0001, one-way ANOVA). (**F**) Phalloidin staining of sheets incubated with Hip1/Hip1R double KD (right) cytosol reveals an exaggerated and disorganized F-actin network relative to WT (left), but not as prominent as that observed in preparations incubated with open TKO cytosol. (**G**) Immunofluorescence staining for open 1 and Hip1R shows that the presence of open 1 at the tips of the invagination is not strongly modified by the absence of Hip1 and Hip1R. (**H**) The disordered C-terminal tail of open binds F-actin. GST fused again 1 fragments (ENTH domain, DPW, NPF, and DPW-NPF containing regions) were incubated (5 mM final concentration) with previously polymerized F-actin (15 mM) and then subjected to ultracentrifugation followed by SDS-PAGE and anti-GST immunoblotting of the supernatant (S) and pellet (P) materials. Scale bars: 5 \u03bcm. Data are represented as mean \u00b1 SEM. See also _Figure_**5_\u2013figure supplement_ 1.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/mikolovEfficientEstimationWord2013.json b/test_extracted_captions/mikolovEfficientEstimationWord2013.json new file mode 100644 index 0000000000000000000000000000000000000000..f13612c3639a63a8cfe0cf8598541dd1bb7526c4 --- /dev/null +++ b/test_extracted_captions/mikolovEfficientEstimationWord2013.json @@ -0,0 +1 @@ +{"CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}\\n\\n'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Comparison of models trained for three epochs on the same data and models trained for one epoch. Accuracy is reported on the full Semantic-Syntactic data set._} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 5: _Comparison of models trained for three epochs on the same data and models trained for one epoch. Accuracy is reported on the full Semantic-Syntactic data set._'", "CAPTION FIG1.png": "'Figure 1: New model architectures. The CBOW architecture predicts the current word based on the context, and the Skip-gram predicts surrounding words given the current word.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Accuracy on subset of the Semantic-Syntactic Word Relationship test set, using word vectors from the CBOW architecture with limited vocabulary. Only questions containing words from the most frequent 30k words are used._} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: _Accuracy on subset of the Semantic-Syntactic Word Relationship test set, using word vectors from the CBOW architecture with limited vocabulary. Only questions containing words from the most frequent 30k words are used._'", "CAPTION TAB3 (NOISY).png": "'\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}\\n\\n'", "CAPTION TAB8 (NOISY).png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Examples of the word pair relationships, using the best word vectors from Table \\\\(\\\\cline{2-3}\\\\hskip-4.0pt\\\\)_\\\\(\\\\sharp\\\\)_(_Skip-gram model trained on 783M words with 300 dimensionality_)._} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 8: _Examples of the word pair relationships, using the best word vectors from Table \\\\(\\\\cline{2-3}\\\\hskip-4.0pt\\\\)_\\\\(\\\\sharp\\\\)_(_Skip-gram model trained on 783M words with 300 dimensionality_)._'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{_Comparison of models trained using the DistBelief distributed framework. Note that training of NNLM with 1000-dimensional vectors would take too long to complete._} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 6: _Comparison of models trained using the DistBelief distributed framework. Note that training of NNLM with 1000-dimensional vectors would take too long to complete._'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'"} \ No newline at end of file diff --git a/test_extracted_captions/milewska2018entry.json b/test_extracted_captions/milewska2018entry.json new file mode 100644 index 0000000000000000000000000000000000000000..022619b6691986741cff74798e7a535407397414 --- /dev/null +++ b/test_extracted_captions/milewska2018entry.json @@ -0,0 +1 @@ +{"CAPTION FIG12.png": "'Figure 12: Actin is important for HCov-NL63 entry. LLC-MK2 cells were incubated with DMSO (\\\\(\\\\Delta\\\\)V, 10 \\\\(\\\\mu\\\\)M) cytochalasin D (B and E), 1.5 \\\\(\\\\mu\\\\)M japakirzolide (C and F), or 400 nM nocodazole (D and G) for 1 h at 37\\\\({}^{\\\\circ}\\\\)C and then inoculated with purified HCov-NL63 and incubated at 32\\\\({}^{\\\\circ}\\\\)C for 1 h. Actin and virus localization was verified with confocal microscopy; fixed cells were immunostained for HCov-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: TIPRSS2 is required for entry into HAE calls but does not enable virus-cell fusion on the call surface. (A) HC_av_-NL63 replication in LLC-Mik2 cells and HAE cultures in the presence of camostat or control DMSO was analyzed using RT-qPCR. Cultures were incubated with 100 \\\\(\\\\mu\\\\)M camostat or DMSO for 1 h at 37\u00b0C and inoculated with HC_av_-NL63 (TCID\\\\({}_{\\\\text{1.0}}\\\\) = 400/ml). After 2 h of incubation at 32\u00b0C, virions that were not internalized were inactivated with acidic buffer [pH 3], and cells were incubated for 5 days at 32\u00b0C. The data are presented as log reduction value [LRV] compared to the control sample. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(P\\\\) values of <0.05 were considered significant and are denoted with asterisks. (B) HAE cultures were incubated with control DMSO or 100 \\\\(\\\\mu\\\\)M camostat for 1 h at 37\u00b0C. Further, cells were inoculated with purified HC_av_-NL63 and incubated at 32\u00b0C for 2 h. Subsequently, cells were fixed and immunostained for HC_av_-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 5 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: HC\\\\(\\\\omega\\\\)-NLR\\\\(\\\\delta\\\\) binding to ACE2 triggers clathrin-mediated endocytosis. Precooled LLC-Mk2 cells were incubated with gradient-purified HC\\\\(\\\\omega\\\\)-NLR\\\\(\\\\delta\\\\) for 40 min at 4\u00b0C following 0 min (A and B) or 5 min (C) of incubation at 32\u00b0C. Colocalization of the virus (green) and ACE2 (red) was analyzed using confocal microscopy (A). No colocalization with clathrin was observed after 0 min of incubation [B]. Triple colocalization of virus with ACE2 and clathrin (blue) is visible in panel C. Images on the right side are zoomed-in regions indicated by white rectangles on the left-side slides. A representative image is shown. Scale bars = 10 \u03bcm.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Importance of endosomal entry for HCoV-NL63 infection. (A) Inhibition of HCoV-NL63 infection in LLC-Mik2 cells and HAE cultures by the lysosomotropic agents ammonium chloride (NH4Cl) (50 mM) and bafilmomycin A (BaF A) (100 mM), as determined by RT-qPCR. Data on the \\\\(y\\\\) axis represent LRVs. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(P\\\\) values of \\\\(<\\\\)0.05 were considered significant and are denoted with an asterisk. [B] The cytotoxicity of the tested inhibitors was measured with an XTT assay. Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(\\\\times\\\\) and \\\\(\\\\rm D\\\\) Confocal images showing colocalization of HCoV-NL63 virions with the early endosomal marker EEA1 on LLC-Mik2 cells (C) and HAE cultures [D]. Scale bars = 5 \\\\(\\\\mu\\\\)m. Green, HCoV-NL63; red, EEA1.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: HC_av_-NL63 colocalizes with daltrin but root caveolin. (A and B) LLC-Wide2 cells were incubated with gradient-purified HC_av_-NL63 for 40 min at 4\u00b0C following 5 min (A) or 20 min (B) of incubation at 32\u00b0C. HAE cultures were incubated with gradient-purified HC_av_-NL63 for 40 min at 4\u00b0C following 120 min of incubation at 32\u00b0C. HC_av_-NL63 colocalization with daltrin (A) or caveolin (B) was analyzed with confocal microscopy (HC_av_-NL63, green; clathrin and caveolin, red; nuclei, blue). (C) Cells mock incubated and stained with isotypic antibodies were used as a control. Scale bars = 5 \u03bcm.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Clathrin and dynamic inhibitors prevent HCOV-NL63 from entering the cell in the HAE model. HAE cultures were incubated with central DMSO (A), 10 \\\\(\\\\mu\\\\)M Pistrop 2 [B], or 10 \\\\(\\\\mu\\\\)M MITMAB (C) for 1 h at 37\u00b0C. Cells were then inoculated with purified HCOV-NL63 and incubated at 32\u00b0C for 2 h. Subsequently, cells were fixed and immunostained for HCOV-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 5 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Cytotoxicity of Pitstop 2 and MitTMAS on HAE cultures. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with control DMSO, 10 \u03bcM Pittsburgh 2, or 10 \u03bcM MITMAB for 2 h at 37\u00b0C. Data on the \\\\(y\\\\) axis represent the viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG4.png": "'\\nFigure 4: Clathrin and dynamic inhibitors hamper internalization of HC_ON_-ML63. (A to C) In order to verify the effectiveness of inhibitors, LLC-Mk2 cells were incubated with control DMSO (A), 10 \u03bcM Pitstop 2 (B), or 10 \u03bcM MITMAB (C) for 30 min at 37\u00b0C and inoculated with Alexa Fluor 488-labeled transferrin. Following incubation (45 min, 37\u00b0C), cells were fixed and stained for actin (red). Transferrin entry was evaluated with confocal microscopy. (E to G) LLC-Mk2 cells were incubated with control DMSO (E), 10 \u03bcM Pitstop 2 (F), or 10 \u03bcM MITMAB (G) for 30 min at 37\u00b0C. Cells were inoculated with purified HC_ON_-ML63 and incubated at 32\u00b0C for 1 h. Subsequently, cells were fixed and immunostained for HC_ON_-ML63 particles (green) and actin (red). [D/] Mock-infected cells were used as a control. Scale bars = 10 \u03bcM.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Cytotoxicity of Pristop 2 and MitMAB on LLC-Mk2 cells. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with control DMSO, 10 \\\\(\\\\mu\\\\)M Pristop 2, or 10 \\\\(\\\\mu\\\\)M MitMAB for 2 h at 37\u00b0C Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: Clathrin and dynamic inhibitors limit the number of LLC-Mk2 infected cells. (A to D) LLC-Mk2 cells were incubated with control DMSO (A), 5 mg/ml nystatin [B], 10 mM MITMAB [C], or 10 \u03bcM Pittstop 2 (D) for 1 h at 37\u00b0C and inoculated with HC6N-ML63 (TGD3 = 100/ml). After 2 h of incubation at 32\u00b0C, virions that were not internalized were inactivated with acidic buffer [pH 3], and cells were incubated for 4 days at 32\u00b0C in the presence of the tested inhibitors or control DMSO. (E and F) The identical procedure was applied to cells, but in these MITMAB (E) and Pittstop 2 (F) were applied after the acid wash. Fixed cells were immunostained with anti-NL63 nucleocapsid protein (green) and nuclei (blue), and confocal images were collected. Scale bar = 200 \u03bcM.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Numerical image analysis: clathrin and dynamic inhibitors block HCON-NL63 entry. (S to D) LLC-MK2 cells were incubated with DMSO (B), 10 \\\\(\\\\mu\\\\)M MITMAB (C), or 10 \\\\(\\\\mu\\\\)M Pitstop 2 (D) for 30 min at 37\u00b0C and subsequently inoculated with purified HCoV-NL63 and incubated for 45 min at 32\u00b0C. Confocal images were digitalized, and the localization of each virus particle relative to the cellular membrane was assessed. (A) Number of internalized virus particles relative to number of virions on the cell surface (_y_ acids) for cells treated with DMSO (control, Pitstop 2, or MITMAB. In panels B, C, and D, raw data for cells treated with DMSO, Pitstop 2, or MITMAB, respectively, are presented. Histograms show the average number of virus particles (_y_ acids) versus the distance from the cell surface (_x_ axis). Values of <0 on the \\\\(x\\\\) axis indicate that the virus is inside the cell, while for extracellular virions, the \\\\(x\\\\) value is =0.\\n\\n'", "CAPTION FIG10.png": "'Figure 10: Clathrin and dynamin inhibitors hamper replication of HCov-NL63 in LLC-MXB cells. [A] HCov-NL63 replication in LLC-MXB cells and HAE cultures in the presence of entry inhibitors or control DMSO was analyzed using RT-qPCR. Cultures were incubated with 10 \u03bcM Pitstop 2, 10 \u03bcM MMTMAB, 5 \u03bcM mg/ml mystatin, or DMSO for 1 h at 37\u00b0C and inoculated with HCov-NL63 (TCIDho = 400/ml). After 2 h of incubation at 32\u00b0C, virions that were not internalized were inactivated with acidic buffer [pH 3], and cells were incubated for 5 days at 32\u00b0C. The data are presented as log reduction value [LEW] compared to the control sample. The assay was performed in triplicate, and average values with standard errors are presented. \\\\(P\\\\) values of <0.05 were considered significant and are denoted with an asterisk. [B] The cytotoxicity of the inhibitors was tested with an XTT assay. Cells were incubated with 10 \u03bcM Pitstop 2, 10 \u03bcM MMTMAB, 5 \u03bcM mg/ml mystatin, or DMSO for 5 days at 32\u00b0C. Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG13.png": "'Figure 13: Cytotoxicity of the cytoskeleton-modifying compounds. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with DMSO, 10 \\\\(\\\\mu\\\\)M cytochalasin D, 1.5 \\\\(\\\\mu\\\\)M jasplakinolide, or 400 nM nocodazole for 2 h at 37\u00b0C. Data on the \\\\(y\\\\) axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.\\n\\n'", "CAPTION FIG14.png": "'\\n\\n**FIG 14 Early event during HCR-ML83 infection.**'"} \ No newline at end of file diff --git a/test_extracted_captions/mueller2017load.json b/test_extracted_captions/mueller2017load.json new file mode 100644 index 0000000000000000000000000000000000000000..61107475a47d137efd53dc7cd7e8b8c43fa7507a --- /dev/null +++ b/test_extracted_captions/mueller2017load.json @@ -0,0 +1 @@ +{"CAPTION FIG6.png": "'Figure 6: **Stochastic Model Receightulating Light and Electron Microscopy Results**\\n\\n(A) The critical angle at which filaments are not able to keep up with the advancing membrane, \\\\(\\\\varphi_{c}\\\\), depends on the maximal polymerization rate, set here as 450 nm/s, and the angle of the filament toward the membrane, \\\\(\\\\varphi\\\\) (black line). A drop in external force leads to a transient increase in protrusion speed leading to a decrease in \\\\(\\\\varphi_{c}\\\\) and a subsequent, angle-dependent decrease in filaments (red line). Force increase causes a decrease in protrusion speed, leading to an increase in \\\\(\\\\varphi_{c}\\\\) and filament density (blue line).\\n\\n(B) Scheme of filaments growing at steady state at intermediate load (middle, gray arrow). Following an increase in load (top, blue arrow) filament density increases and the angle distribution broadens, whereas at a decrease in load bottom, red arrow) filaments at a higher angle \\\\(\\\\varphi_{c}\\\\) are preferentially capped and density decreases.\\n\\n(C) Visualization of model results with filaments shown in green, barred ends in red, and pointed ends in blue. Visualization of model results in a regime with force increase (top) and decrease (bottom), \\\\(\\\\Delta F\\\\), at around 900 nm. Subsequent panels are aligned next to the respective force regimes.\\n\\n(D) Flament numbers, barred, and pointed and density quantified for the three conditions like in Figures 3, 4, and 5. In the unperturbed situation with fixed external force, \\\\(F\\\\), the network assumes a steady state with balanced filament density, branching, and capping. After a decrease in \\\\(F\\\\), capping increases, nucleation decreases, and the network is thread out, whereas force increase causes a nucleation peak and an increase in filament density.\\n\\nFor all graphs, mean and SD for an average of 20 runs are shown.\\n\\nSee also Figure S7, Tables S1 and S2, Movie S5, and the STAR Methods.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: The Stochastic Model Recapitulates Cell Migration Parameters of Fish Keratocytes (A) Example of Temporal Machistators in Heact:GFP intensity, area, and protrusion speed for the cell shown in Figure 1 compared to the migration parameters produced by a simulation run with the projected cell area as scaled transic input.\\n\\n(B) Average of temporal cross-correlation functions of the simulations obtained with the projected cell area of the 21 migrating keratocytes shown in Figure 1. Temporal correlation peaks at time lag zero between Heact:GFP intensity, projected cell area, and protrusion match the ones observed in vivo. Mean and SEM are shown.\\n\\n(C) Histogram of the order parameters obtained for the 21 steady-state migrating keratocytes. The stochastic model predicts cells migrating with a predominantly negative order parameter, with only occasional switches to positive order parameter values as cells undergo rapid shrinkage and a transient burst in protrusion speed.\\n\\nSee also Figure S7.\\n\\n'", "CAPTION FIGS1-1.png": "'Figure S1. Details of Quantification of Live Cell Imaging Parameters, Related to Figure 1 (A) Temporal fluctuations of fluctaGFP signal, projected cell area and protrusion speed of the cell shown in Figure 1 are shown normalized from the maximum value to zero. The fractalGFP signal multiplied by the instantaneous speed is shown normalized from the maximum to zero as well.\\n\\n'", "CAPTION FIGS1-2.png": "\"(B) Temporal crosscorrelation functions of Area - Heart:GFP interaibly, Area-protrusion speed and Heart:GFP intensity - Protrusion speed for 21 individual cells are shown in black. Averaged crosscorrelation functions from a spline fit to the combined single-cell crosscorrelation functions are shown in purple (/rea - Heart:GFP intensity), green (/area - Protrusion speed) and yellow (/fluct:GFP intensity - Protrusion speed).\\n\\n(C) Confocal images of a migrating keratocyte expressing Heart:GFP were analyzed using consecutive 1.09-mm wide regions spanning the lamellipodium.\\n\\n(D) Resulting fibact:GFP intensity maps are plotted against time.\\n\\n(E) Temporal crosscorrelation functions of region '1' at the very front of the cell to consecutive regions are plotted as a function of time lag.\\n\\n(F) Time lag between peaks of the curves shown in H multiplied by cell velocity and resulting distances are plotted against distance between analyzed regions. Measured mean and s.d. of twelve cells are shown together with a linear regression (Pearson constant = 0.9997, p = 0.0003).\\n\\n\"", "CAPTION FIGS3.png": "'Figure S3. Membrane Tension Measurement by Tether Pulling and Membrane Tension Manipulation by Changing Osmotic Pressure, Related to Figure 2 (A) A migrating keratocyte was imaged with bright field microscopy and simultaneously a coated bead controlled by a laser tweezer was used to pull a membrane tether. (B) The measured tether force decrease correlated with rapid decreases in projected cell area. (C) A Hexact:GFP expressing keratocyte was imaged by confocal microscopy and subjected to rapid changes in osmotic pressure of the medium by alternating additions of 0.2M sucrose and pure water. (D) Hexact:GFP signal is negatively correlated with the osmolarity of the medium. When the pressure increases upon addition of 0.2M sucrose, Hexact:GFP signal drops, which is reversible upon restoring a lower osmotic pressure by addition of pure water.\\n\\n'", "CAPTION FIGS5.png": "'Figure S5. Changes in Filament Parameters at a Decrease in Filament Density, Related to Figure 5 (A and B) Network structure analysis of seven lamellipodia tomograms in three correlated cells revealed consistent features at the point, where the actin density decreased due to the manipulation by the micropipette. Overview electron micrographs and 5.5nm tomogram slices are shown for all regions. (C) Averages of filament density, barbed and pointed ends and median angle from the cell edge showed the consistent changes at the density step. (D) Median angles of at analyzed regions are plotted for 300nm space bins before and after the decrease in actin filament density light. (Pared tost, p = 0.0249). (E) Normalized ratio of filaments after density step to filaments before density step is plotted against the filament angle from the cell edge together with a linear regression curve. (Pearson constant, \\\\(r=-0.996\\\\)) Mean and s.o.m. are shown on all graphs.\\n\\n'", "CAPTION FIGS2.png": "'Figure S2. Live Cell Imaging Controls with a Membrane Marker and actin:GFP, Related to Figure 1 (A) Example images of migrating keratocytes incubated with the membrane dye CalMask. (B and C) Temporal fluctuations at the resulting intensity maps are shown normalized as in Figure 1D (B) and as in Figure S1A (C). Neither an amplification of area. Instructions for a correlation could be observed. (D) Resulting temporal cross correlation functions for eight individual cells are shown in black and averaged cross correlation in dark blue. The averaged cross correlation as mean with s.a.m. is shown in dark blue on the right. (E) Zebrafish keratocytes transfected with actin:GFP were analyzed in the same way as shown before for IfReact:GFP. (F) Temporal cross correlation analysis area - GFP signal intensity showing peaks at time lag zero for transfected cells expressing actin:GFP and IfReact:GFP. Additionally cells generated from actin:GFP microinjected Zebrafish embryos were analyzed and showed a positive cross correlation coefficient at time lag zero. Plot shows averaged cross correlation and s.a.m. for seven cells for both actin:GFP transfection and mRNA injection.\\n\\n'", "CAPTION FIGS4.png": "'Figure S4: **Actin Network Parameters for Individual Tomogram Montages of Keratocyte Lamellipodia, Related to Figures 3 and 4**(A)** Filament density, barbad and pointed ends for individual cells shown in Figure 3. Data for individual lamellipodia are shown in black together with average. (B) Flament densities growing at the indicated angle from the callmembrane in 212nm distance bins of the steady state lamellipodia shown in Figure 3. Mean and a.m. are shown. (C) Flament densities growing at the indicated angle from the cell membrane in 212nm distance bins of the correlated tomogram shown in Figure 4. The densities are shown as in Figure 4 begirting from toward the rear of the cell on the left until the leading edge on the right. (D) 5.5nm slice of a negatively stained electron tomogram mortag\\n\\n'", "CAPTION FIGS6.png": "'Figure S6: **Quantification of Correlated Live Microscopy-Electron Tomography, Related to Figure 5** (A) A 5.5rm slice of the tomogram mortg used for Figures 5 (left) is shown together with automated tracking results (middle and right). (B) Close-up of region containing the drop in Hamart density and change in actin-architecture with Siaments shown in green, barbed ends in red and pointed ends in blue. Mainually tracked filaments, barbed and pointed ends of a region including the density step are shown. (C) Hamart density quantified by manual and automated tracking, lastk image analysis and the fltact:GFP signal of the correlated cell shown in Figure 5. (D) Filament densities in 212nm distance bins growing at different angles toward the membrane. The densities are shown as in Figure 5 beginning from toward the rear of the cell on the left until the leading edge on the right. (E) 5.5rm slice of negatively stained electron tomogram montage showing region marked with red box in Figure 5A (left) and corresponding automatically tracked filaments shown color-coded according to their angle from the leading edge (right). Additionally, the spatial bins used for quantification of network parameters in Figures 5 and S6D are displayed on the left. The cell edge is seen on the right side.\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: Cytoskeletal Response to Manipulations of Membrane Tension (A) L/Heact:GFP Imaging following microplights aspiration. (1) Migrating keratocyte before contact with the micromanipulator, (2) aspiration of membrane, and (3) release of vacuum. (B) Temporal fluctuations of fillout:GFP intensity at the leading edge defined as in Figure 1B, area, and protrusion speed. The area between the dotted lines shows, where the cell was aspirated. (C) Kymograph along dashed yellow line in (A). (D) Change in Ifaact:GFP signal following aspiration with four different vacuum levels. (E) Change in cell edge protrusion for the same cells as in (B). Mean and SEM are shown for 28 aspiration events in 13 individual cells in (D) and (E).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Correlated Live Microscopy-Electron Tomography of Ultrastructural Changes in Networks with Decreasing Filament Density** (A) Migrating keratocyte manipulated with a microneade to induce a rapid decrease in projected cell area with an accompanying decrease in Ifacet:GFP signal, fixed within = 3 s, and prepared for electron microscopy. A rapid decrease in the Ifacet:GFP signal is preserved in the fixed Ifacet:GFP sample and the low-magnification electron micrograph. (B) 5.5-nm tomogram since showing the region marked with a red box in (A). The cell edge is seen on the right side, and the region of lower density is distinguishable toward the middle of the micrograph. Region of steady-state network density and decreased density are marked in black and red throughout the whole figure. (C) Flament tracks of the lamellipodium shown in (B), with actin filaments shown in green, barbed ends in red, and pointed ends in blue. (D) Flament numbers and densities of barbed and pointed ends in 106-nm-wide spatial bins throughout the lamellipodium shown in (B) and (C). (E) Histogram showing filament densities growing at the indicated angle from the membrane in 212-nm distance bins. An order parameter (blue) in 212 nm is defined like in Figure 3. See also Figures 5S and 5S and Movie S4.\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1989) The mathematical theory of the mathematical theory'", "CAPTION FIGS7.png": "'Figure S7: **Details of Stochastic Simulation, Related to Figures 6 and 7**\\n\\nThe graphs in A-C are aligned as in Figure 6.\\n\\n(A) The temporal changes in filaments in three different angle bins are shown. The number of filaments in the indicated angle bins is constant at growth at constant external force. In the force decrease scenario filaments growing at higher angles are capped preferentially and the network thus out. When force increase is used as an input, filaments in all angle bins increase.\\n\\n(B) Histograms of filament density (black) and order parameter as defined in Figure 3 (black). Without perturbation the network architecture is dominated by a 35\u00b0 peaks and the order parameter is consistently negative. Histograms of filament density before (black) and after (red) decrease in \\\\(F\\\\) and order parameter (blue) show biased elimination of filaments growing at higher angle. This leads to a change from the \\\\(\\\\pm\\\\) 35\u00b0 architecture to one dominated by straight, 0\u00b0, filaments and a transitionly positive order parameter. Filaments of all angles increase upon increase in force, which is also reflected in a negative order parameter.\\n\\n(C) The migration parameters force (the input parameter), actin density and protrusion speed fluctuate around steady state values during the unperturbed simulation. The force decrease causes an increase in protrusion speed and a decrease in actin density, with the reciprocal situation observed for the force increase.\\n\\n(D) Capging, elongation and branching rates used for the stochastic model.\\n\\n(E) Underlying lamellipodial feedback loop: Force, \\\\(F\\\\), is set as an external parameter and decreases velocity, \\\\(v\\\\), which is in turn increased by increased filament density, \\\\(D\\\\), as suggested by the elastic ratchet model of actin polymerization. Actin branching is modeled as a zeroth order process and therefore actin density decreases branching rate, \\\\(\\\\beta\\\\). The parts of the feedback loop linking velocity, \\\\(v\\\\), to the filament density, \\\\(D\\\\), shown in this study are marked in red: Velocity, \\\\(v\\\\), increases capping rate, \\\\(v\\\\), in an angle-dependent manner and leads to a decrease in filament density, \\\\(D\\\\). For all graphs mean and s.d. for an average of 20 runs are shown, for details see STAR Methods.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: **Electron Tomography of Migrating Wild-Type Keratocytes** (A) Overview electron micrograph of migrating keratocyte with acquired tomogram mortagam marker in red. (B) 5.5-nm slice of a negatively stained tomogram of the actin network behind the loading edge. (C) Automated tracking results of the same region with filaments shown in green, barbed ends in red, and pointed ends in blue. (D) Normalized densities of filaments, barbed ends, and pointed ends in 105-nm-wide bins of four averaged tomogram mortagam. Graph shows mean and SEM. (E) Scheme showing the filament angle bins used for calculating the global order parameter. (F) Histogram of combined filament length growing at indicated angle toward the cell membrane (black) is shown together with a global order parameter (blue) in 212-nm distance bins defined as (Filtraments \\\\(0^{-}\\\\)-\\\\(20^{\\\\circ}\\\\))-(Filtraments \\\\(30^{-}\\\\)-\\\\(50^{\\\\circ}\\\\))/Filtraments \\\\(0^{-}\\\\)-\\\\(20^{\\\\circ}\\\\))+Filtraments \\\\(30^{-}\\\\)-\\\\(50^{\\\\circ}\\\\)). See also Figures S4A and S4B.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Correlated Live Microscopy-Electron Tomography of Ultrastructural Changes in Networks with Increasing Filament Density (A) A migrating karyotype expressing ileact:GFP was aspirated at the rear with a micropipette, fixed within ~ 3 s, and prepared for electron microscopy. The cell shifted out of focus because of the manipulating procedure. A transient increase in actin density can be seen in the low-magnification electron micrograph on the right.**\\n\\n**(B) 5.5-nm tomogram slice of the region marked by a red box in (A). The rear of the cell is toward the left side of the picture, and the cell front is seen on the right.**\\n\\n**Fig. 5:** Schematic of the model for the intracellular transport '", "CAPTION FIG2-2.png": "'(F) Lifact:GFP signal following fast cell shrinkage. (i) Karatocyte exhibiting stretched morphology, while its trailing edge is tethered to the substrate. (2) The trailing edge is cut with a pulsed laser (yellow arrowhead). (3) Temporal fluctuations of leading edge fractal:GFP intensity, area, and protrusion speed. The rapid retraction is marked with a dotted line. (4) Kymograph along dashed yellow line in (F). (See also Figure S3 and Movie S2.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: **Correlative Analysis of Actin, Projected Gell Area, and Protrusions in Migrating Zebrafish Keratocytes (A) Corcoral imaging of Heact:GFP-expressing keratocyte moving on a glass covarlin.**\\n\\n**(B) Time frame from (A) showing the 1.09-\\\\(\\\\mu\\\\)m wide area of Heact:GFP intensity measurements (left), a binary mask used for quantifying the area (middle), and an pseudo-colored Horn-Schunck optical flow analysis image of the leading edge (right).**\\n\\n**(C) Fluorescence intensity and leading edge velocity maps for the time lapse of the cell shown in (A).**\\n\\n**(D) Temporal fluctuations of cell area, Heact:GFP intensity, and protrusion speed averaged across the analyzed region shown in red in (B).**\\n\\n**(E) Average of temporal cross-correlation functions of 21 migrating keratocytes. Mean and SEM are shown.**\\n\\nSee also Figures S1 and S2 and Movie S1.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/mund2023clathrin.json b/test_extracted_captions/mund2023clathrin.json new file mode 100644 index 0000000000000000000000000000000000000000..79edf9698780e4c408a39ecfa4e904ee93222dba --- /dev/null +++ b/test_extracted_captions/mund2023clathrin.json @@ -0,0 +1 @@ +{"CAPTION TAB1-1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1**} & \\\\multicolumn{1}{c}{**Summary of clathrin coat growth model fits in SK-NEL-2**} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Summary of clathrin coat growth model fits in SK-NEL-2'", "CAPTION TAB1-2.png": "'* Fitted parameter values for the constant area model (CA/M), the constant curvature model (CCM) and the cooperative curvature model (CoopCM) when fitting curvature \\\\(H[\\\\delta(\\\\theta)]\\\\), surface area \\\\(A[\\\\delta(\\\\theta)]\\\\), edge length \\\\(\\\\delta(\\\\theta)\\\\), and projected area \\\\(A_{\\\\gamma}[\\\\delta(\\\\theta)]\\\\). A: Surface area fitted with CAM; R: Radius fitted with CCM; \\\\(y\\\\): Constant rate of curvature increase fitted with CoopCM; H\\\\({}_{2}\\\\): Preferred curvature of the clathrin coat fitted with CoopCM; R\\\\({}_{2}\\\\): preferred radius of the clathrin coat fitted with CoopCM; A\\\\({}_{5}\\\\): Fraction of surface area growing as a flat lattice before curvature initiation, defined as \\\\(A_{0}\\\\sim A[\\\\theta\\\\sim 0.01]/A[\\\\theta=180^{\\\\circ}]\\\\); k\\\\({}_{\\\\text{act}}\\\\): local growth rate obtained from \\\\(\\\\delta(\\\\theta)\\\\) fitted with CoopCM and measured in nm per pseudentate units \\\\(\\\\xi^{2}\\\\). \\\\(N=13\\\\) cells; \\\\(n=1,645\\\\) sites.\\n\\n'", "CAPTION TAB2-1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB2-2.png": "'* Fitted parameter values for the constant area model [CAM], the constant curvature model [CCM], and the cooperative curvature model [CoopCM] when fitting curvature _H_[_d_], surface area _A_[_d_], edge length _E_[_d_], and projected area _A_[_d_]. A: Surface area fitted with CAM; R: Radius fitted with CCM; _y:_ Constant rate of curvature increase fitted with CoopCM; H2: Preferred curvature of the clathrin coat fitted with CoopCM; R3: preferred radius of the clathrin coat fitted with CoopCM; A5: Fraction of surface area growing as a flat lattice before curvature initiation, defined as \\\\(A\\\\)0 ~ A(_th_ = 0.01)/_A_[_th_ = 180%); kax: local growth rate obtained from _d_[_d_] fitted with CoopCM and measured in nm per pseudotime units _g_-1. \\\\(N\\\\) = 3 cells; \\\\(n\\\\) = 241 slices.\\n\\n'", "CAPTION FIGS4.png": "'Figure S4. **Clathrin coat remodeling in three different cell lines.****(A-C)** Results of LocMeFit analysis for clathrin structures. Different growth models are fitted to curvature H over \\\\(\\\\theta\\\\). The resulting fitting parameters are then used to map the same models also over the surface area, edge length, and projected area (left to right). Purple. Completely flat sites with \\\\(\\\\mu\\\\) = 0 mm\\\\({}^{-1}\\\\) and \\\\(\\\\theta\\\\) = 0. Red. Disconnected sites that were excluded from the fitting. Black line: Rolling median (window width = 5% of total number of sites; A) U2OS (\\\\(N\\\\) = 3 cell, \\\\(n_{\\\\text{gray}}\\\\) = 241 sites, \\\\(n_{\\\\text{nat}}\\\\) = 53 disconnected sites, \\\\(n_{\\\\text{purple}}\\\\) = 1 completely flat sites), (\\\\(\\\\theta\\\\)) 3T3 mouse fibroblasts (\\\\(N\\\\) = 7 cells, \\\\(n_{\\\\text{grey}}\\\\) = 688 sites, \\\\(n_{\\\\text{red}}\\\\) = 51 disconnected sites, \\\\(n_{\\\\text{purple}}\\\\) = 8 completely flat sites), and (C) SK-MEL-2 cells (\\\\(N\\\\) = 13 cells, \\\\(n_{\\\\text{grey}}\\\\) = 1,631 sites, \\\\(n_{\\\\text{red}}\\\\) = 153 disconnected sites, \\\\(n_{\\\\text{purple}}\\\\) = 14 completely flat sites). **(D-F)** Temporal reconstruction of clathrin coat remodeling. (\\\\(\\\\langle\\\\rangle\\\\)) Distribution of \\\\(\\\\theta\\\\) slightly differs between cell lines, especially in the earlier states. Median \\\\(\\\\theta\\\\) shown as dotted lines correspond to 99.6\\\\({}^{\\\\circ}\\\\) for U2OS; I21.4\\\\({}^{\\\\circ}\\\\) for 3T3; and 108.5\\\\({}^{\\\\circ}\\\\) for SK-MEL-2 cells. (\\\\(\\\\langle\\\\rangle\\\\)) The cooperative curvature model (red line) highlights the square-root dependence between \\\\(\\\\theta\\\\) and pseudoutine. (\\\\(\\\\langle\\\\rangle\\\\)) The cooperative curvature model is used to describe the curvature H propagation over pseudoutine. Resulting fitting parameters are then used to map the same model to surface area \\\\(A_{c}\\\\), edge length \\\\(\\\\theta\\\\), and projected area \\\\(A_{c}\\\\). A rolling median is plotted \\\\(\\\\theta\\\\) black (window width = 5% of total number of sites).\\n\\n'", "CAPTION FIGS2.png": "'Figure S2: **Simulations.****(A and B)** Estimation error of the closing angle \\\\(\\\\theta\\\\) (\\\\(N\\\\) = 1,800 sites). **(A)** The comparison of fitted \\\\(\\\\theta\\\\) of simulated clathrin coat structures for corresponding ground-truth \\\\(\\\\theta\\\\) shows no systematic bias and a narrow spread of the error across the ground truth although the spread increases when \\\\(\\\\theta\\\\) < 20\\\\({}^{\\\\circ}\\\\) or \\\\(\\\\theta\\\\) > 160\\\\({}^{\\\\circ}\\\\). We reasoned that in the earlier range, where the structures are flat, the slightly increased error corresponds to the insensitivity of \\\\(\\\\theta\\\\) to flat structures. In the later range, where the vesicles are almost closed, the error was caused by indistinguishable tiny holes corresponding to the real vesicle openings and unlabeled clathrin in the coat. The fitted structures were simulated to have similar quality as the experimental data and to distribute evenly across \\\\(\\\\theta\\\\). **(B)** The distribution of \\\\(\\\\theta\\\\) shows no significant error compared to the expectation corresponding to the evenly distributed \\\\(\\\\theta\\\\), except for the small potential underestimation of entirely closed coats. **(C-G)** Averaging preserves U-shapes in simulated endocytic sites. **(C)** A ground-truth structure was used for smithating U-shaped clathrin coats. The model for simulation was built by combining a hemisphere with a radius of R = 97 nm and a cylinder with a height D = 0.5 R. We choose the radius value according to the median radius of bin 6 in Fig. 4. G. This bin has a closing angle slightly larger than 90\\\\({}^{\\\\circ}\\\\). 20-rm-thick cross-sections of the averages (\\\\(N\\\\) = 100 sites) registered based on (D) the ground truth and (E) the spherical fit are shown with the ground-truth U-shaped model (dotted line). Histograms of normalized estimation errors are shown for the parameters (F) surface area and (G) radius, with mean values of \\\\(-2.6\\\\) and \\\\(2.5\\\\%\\\\) respectively. The scale bar is 100 nm.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: **3D single-molecule localization microscopy (SMLM) of clathrin-coated structures.****(A)** 3D SMLM image of clathrin immunoblotted with AF647 at the bottom membrane of a SK-MEL-2 cell. **(B)** Enlarged view of the region indicated in A. **(C)** All structures found in B are shown in top view (gy) and as S0 nm-thick z-slices (x2), and orientation of slice is indicated by a dotted line. Scale bars are 10 \u03bcm (A); 1 \u03bcm (B), and 100 nm (C). **(D)** Geometric analysis pipeline. All clathrin coats are segmented, and their localizations are directly fitted with a spherical cap model using the fitting framework LortMoFit (Wu et al., 2023). **(E)** In LortMoFit, individual clathrin coats are parametrized by their size (radius \\\\(R\\\\)), closing angle (\\\\(\\\\theta_{\\\\text{d}}\\\\)), position (\\\\(\\\\theta_{\\\\text{x}}\\\\),\\\\(\\\\phi_{\\\\text{y}}\\\\),\\\\(\\\\phi_{\\\\text{z}}\\\\)), and 3D orientation. **(F)** Using \\\\(\\\\theta\\\\) as a proxy for endocytic progression, the relative endocytic time point for each structure can be determined.\\n\\n'", "CAPTION TAB3-1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 3.** Summary of clathrin coat growth model fits in 3T3 mouse fibroblasts.} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 3: **Summary of clathrin coat growth model fits in 3T3 mouse fibroblasts.**'", "CAPTION TAB3-2.png": "'* Fitted parameter values for the constant area model [CAM], the constant curvature model [CCM], and the cooperative curvature model [CoopCM] when fitting curvature _H_[_d_], surface area _A_[_d_], edge length _E_[_d_], and projected area _A_[_d_]. A: Surface area fitted with CAM; R: Radius fitted with CCM; _y:_ Constant rate of curvature increase fitted with CoopCM; H2: Preferred curvature of the clathrin coat fitted with CoopCM; R3: preferred radius of the clathrin coat fitted with CoopCM; A5: Fraction of surface area growing as a flat lattice before curvature initiation, defined as \\\\(A\\\\)0 = _A_(_th_ = 0.01)/_A_(_th_ = 180%); kax: local growth rate obtained from _d_(_t_) fitted with CoopCM and measured in nm per pseudotime units \\\\(\\\\frac{5}{2}\\\\). \\\\(N\\\\) = 7 cells; \\\\(n\\\\) = 688 sites.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Temporal reconstruction of clathrin coat remodeling.****(A)** Endocytic sites are sorted by \\\\(\\\\theta\\\\) to reconstruct pseudotime. Enriched \\\\(\\\\theta\\\\) states, for example the peak at 135\u00b0, represent long-lived states that remain for extended periods in pseudotime. _Color_ code represents the same number of sites in both histograms. **(b)** The square-root dependence between \\\\(\\\\theta\\\\) and pseudotime approximated by the cooperative curvature model (red line). **(c)** Curvature generation over pseudotime is approximated by the cooperative curvature model. **(d\u2013f)** Fit results in \\\\(C\\\\) were used to describe (D) edge length, (f) surface area, and (f) projected area change over pseudotime. A rolling median (window of 82 sites) is plotted alongside (black line). **(c)** Superresolution averages for distinct endocytic stages, resulting from all collected snapshots. Each bin contains the same number of snapshots of clathrin-coated structures sorted along their pseudotime (\\\\(n\\\\) = 163 per bin), so that all bins represent equally long pseudotime intervals. Individual sites were rescaled to the median bin radius and aligned by their center coordinates as well as rotation angles. Scale bar is 100 nm. (\\\\(n\\\\) = 1,645 sites, \\\\(N\\\\) = 13 cells).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Quantitative analysis of clathrin-coated structures in SK-MEL-2 cells.****(A)** Clathrin coat geometry is quantified using LotMoFit. The fitted spherical cap model is drawn as a circle with its outer radius (long row, xy), as cross-sectional profile (50 nm xz slices, middle row), and as surface with the corresponding closing angle \\\\(\\\\theta\\\\) (bottom row). Scale bar, 100 nm. **(B)** Distributions of closing angle \\\\(\\\\theta\\\\) (median \\\\(\\\\sim\\\\) 108\\\\({}^{\\\\circ}\\\\)), curvature H (median = 0.011 nm\\\\({}^{-1}\\\\)), and surface area \\\\(\\\\hat{A}\\\\) (median = 54,000 m\\\\({}^{2}\\\\)) of endocytic sites in SK-MEL-2 cells (\\\\(n\\\\) = 1,798 sites, \\\\(N\\\\) = 13 cells) as determined from the model fit. **(C)** Two previously proposed mechanistic models for clathrin coat assembly during endocytosis (for details, see text). In both scenarios **(**(**)** increases monotonically) and is thus a proxy for endocytic progression. **(D)** Development of curvature, surface area, and projected area of the clathrin coat during endocytosis. _Color_ indicates point density.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: **Model for clathrin coat growth.****(A)** Schematic of the cooperative curvature model, where clathrin lattices grow by the addition of trisikelia to the edge at a constant growth rate \\\\(k_{av}\\\\). Curvature generation is driven toward a preferred curvature, ultimately creating a spherical vesicle. **(B)** Distinct clathrin growth models and rolling median (window width = 82 sites) fitted to curvature over \\\\(\\\\partial\\\\). **(C and D)** The resulting fitting parameters are then used to map the same models also over **(C)** surface area and **(D)** edge length. _(n =_ 1,645 sites, _N =_ 13 cells).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Temporal reconstruction of clathrin coat remodeling across three different cell lines.****(A and B)** Superresolution averages for distinct endocytic stages, resulting from all collected snapshots for (A) U2O5 (\\\\(n\\\\) = 241 sites, \\\\(N\\\\) = 3 cells) and (B) 373 (\\\\(n\\\\) = 688 sites, \\\\(N\\\\) = 7 cells). Each bin contains the same number of snapshots of clathrin-coated structures sorted along their pseudotime (\\\\(n_{\\\\text{U2O5}}\\\\) = 24 per bin, \\\\(n_{\\\\text{3T3}}\\\\) = 68) and represent equally long pseudotime intervals.\\n\\n'", "CAPTION TAB1.png": "'* [16] J. S. B. (1981). The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)), the constant-law model (\\\\(\\\\alpha\\\\)) and the temperature-law model (\\\\(\\\\alpha\\\\)). The constant-law model (\\\\(\\\\alpha\\\\)) and the temperature-law model (\\\\(\\\\alpha\\\\)) are shown in Figure 10. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) are shown in Figure 11. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 12. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 13. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 14. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 15. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 16. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 17. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 18. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 19. The power-law index for the constant-law model (\\\\(\\\\alpha\\\\)) is shown in Figure 19.\\n\\n'", "CAPTION TAB2.png": "'* [1]_Summary of deficits out growth model fits to 1995.\\n* [2]_Summary of deficits out growth model fits to 1996.\\n* [3]_Summary of deficits out growth model fits to 1996.\\n* [4]_Summary of deficits out growth model fits to 1996.\\n* [5]_Final parameter values to be consistent results (Table 1), to construct consistent results (Table 2), to construct consistent results (Table 3), to construct consistent results (Table 4), to construct consistent results (Table 5), to construct consistent results (Table 6), to construct consistent results (Table 7), to construct consistent results (Table 8), to construct consistent results (Table 9), to construct consistent results (Table 10), to construct consistent results (Table 11), to construct consistent results (Table 12), to construct consistent results (Table 13), to construct consistent results (Table 14), to construct consistent results (Table 15), to construct consistent results (Table 16), to construct consistent results (Table 17), to construct consistent results (Table 18), to construct consistent results (Table 19), to construct consistent results (Table 11), to construct consistent results (Table 12), to construct consistent results (Table 13), to construct consistent results (Table 14), to construct consistent results (Table 15), to construct consistent results (Table 16), to construct consistent results (Table 17), to construct consistent results (Table 18), to construct consistent results (Table 19), to construct consistent results (Table 11), to construct consistent results (Table 12), to construct consistent results (Table 13), to construct consistent results (Table 14), to construct consistent results (Table 15), to construct consistent results (Table 16), to construct consistent results (Table 17), to construct consistent results (Table 18), to construct consistent results (Table 19), to construct consistent results (Table\\n'", "CAPTION TAB3.png": "''", "CAPTION FIGS1.png": "'Figure S1: **Examples of diverse clathrin coat structures.****(A)** Large clusters of clathrin molecules excluded from further analysis. Shown in top view \\\\(\\\\langle\\\\langle xy\\\\rangle\\\\) with the dotted line indicating a 50 nm-thick z-slice \\\\(\\\\langle xz\\\\rangle\\\\) shown below. **(B)** Vesicular structures are sometimes fitted with a lower \\\\(\\\\hat{\\\\theta}\\\\) than expected. **(C)** While a spherical model describes the structure of most endocytic clathrin coats faithfully, there are few cases, as exemplified here, where the elliptical and irregular shape of an assembling coat is difficult to approximate with a simple geometric model. Two orthogonal 50-nm-thick z-slices are shown here in xz and yz, and the respective spherical model fit is plotted as dotted line. **(D)** Non-continuous labeling of clathrin manifests itself as holes in the coat, indicated with a blue arrow. All scale bars are 100 nm.\\n\\n'", "CAPTION FIGS3.png": "'Figure 53: **Non-endocytic clathrin structures.** Results of _LocMe_Et analysis of clathrin structures in SK-MEL-2 cells (_N_ = 13 cells, \\\\(n\\\\) = 1,798 sites). **(A)** A strong correlation between curvature and \\\\(\\\\delta\\\\) can be observed for most structures (_n_ = 1,645 sites, black). A disconnected point cloud (_n_ = 153 sites, 8.5% red) indicates the presence of endocytosis-unrelated clathrin structures. **(B\u2013D)** The same distinct population of data points can be observed for the (B) surface area. **(C)** edge length, and **(D)** projected area. **(B)** Example structures from the disconnected population of sites in top view (_y_) and 50 non-thick _z_-slices (_xz_) and their respective fitted \\\\(\\\\delta\\\\) values. Scale bar, 100 nm. **(C\u2013F)** Analysis of clathrin casts not following general trajectory of curvature generation. **(C)** 3T3 cell transiently overexpressing AP2-GFP. Left: Single-molecule localization microscopy image of immunolabeled clathrin. Middle: Diffraction-limited image of the AP2-GFP signal. Right: Overlay of the two targets (scale bar, 10 \u03bcm), **(D)** Enlarged image of the section indicated in **C** (scale bar, 1 \u03bcm). **(E)** example sites indicated in **B** (scale bar, 100 nm). **1**: Example for a structure annotated as \u201cGFP positive.\u201d 2: Example for an \u201cinconclusive\u201d GFP signal. 3 and 4: Example of \u201cGFP negative\u201d structures.* **(F)** Analysis results when estimating \\\\(\\\\delta\\\\) and curvature from clathrin structures and annotating them depending on their AP2 signal (_N_ = 3 cells and \\\\(n\\\\) = 277 sites). No AP2-GFP positive structures are found in the disconnected population of sites, suggesting that they are most likely not generated via clathrin-mediated endocytosis.\\n\\n'", "CAPTION FIGS5.png": "'Figure S5: **Linkage error investigation and number of localizations per clathrin coat.****(a-1)** Impact of linkage error on geometric model fit. Indirect immunolabeling displaces the label from the target molecule by two antibodies. This generates a so-called linkage error of an average \\\\(\\\\pm\\\\) 10 nm, and resulting localizations might not accurately represent the underlying structure of interest (Fruh et al., 2021). **(a)** An ideal case of no linkage error, where the model (dotted black line) with radius \\\\(R_{\\\\text{M}}\\\\) accurately represents the underlying clathrin coat (red) with a radius \\\\(R_{\\\\text{c}}\\\\). **(b)** Histogram of quantified curvature with a median of 0.011 nm\\\\({}^{-2}\\\\). **(c)** Models fitted to curvature propagation over \\\\(\\\\delta\\\\). **(b)** Uniform displacement of localizations (light red, \\\\(\\\\pm\\\\) 10 nm) due to unbiased labeling by the antibodies. The radius \\\\(R_{\\\\text{M}}\\\\) still accurately represents the true radius \\\\(R_{\\\\text{c}}\\\\). **(e)** Histogram of the curvature, assuming no needed correction of \\\\(R_{\\\\text{M}}\\\\). Median: 0.011 nm\\\\({}^{-2}\\\\). **(f)** Models fitted to curvature propagation over \\\\(\\\\delta\\\\) remain the same as for **(c)** Biased labeling of the antibodies (light red, \\\\(\\\\pm\\\\) 10 nm) could result in an overestimation of \\\\(R_{\\\\text{M}}\\\\) by 10 nm. **(f)** Histogram of the curvature corrected by subtracting 10 nm from quantified \\\\(R_{\\\\text{M}}\\\\). Median: 0.013 nm\\\\({}^{-1}\\\\). **(f)** Models fitted to corrected curvature propagation over \\\\(\\\\delta\\\\). **(f)** Biased labeling could result in an underestimation of \\\\(R_{\\\\text{M}}\\\\) by 10 nm. **(f)** Microgram of the curvature corrected for the overestimation in radius by adding 10 nm from quantified \\\\(R_{\\\\text{M}}\\\\). Median: 0.010 nm\\\\({}^{-2}\\\\). **(i)** Models fitted to corrected curvature propagation over \\\\(\\\\delta\\\\). **(c, F, I, and I)** Fitting parameters are \\\\(A_{\\\\text{c}}\\\\): Fraction of surface area growing as a flat lattice before curvature irritation, defined as \\\\(A_{0}\\\\) = \\\\(A\\\\)(\\\\(\\\\delta\\\\) = 0.01)/\\\\(A\\\\)(\\\\(\\\\delta\\\\) = \\\\(\\\\pi\\\\)); \\\\(R_{\\\\text{c}}\\\\): Preferred radius of the clathrin coat fitted with CoopCM. \\\\(A_{\\\\text{c}}\\\\): Preferred curvature of the clathrin coat fitted with CoopCM. Analysis of \\\\(N\\\\) = 13 SK-MEL-2 cells, \\\\(n\\\\) = 1,645 sites. While the fitting parameters scale with the error in radius estimation, the relationships among the parameters and thus our mechanistic interpretation by the cooperative curvature model still holds true. **(M\u20130)** Number of localizations versus surface area. For \\\\(N\\\\) = 6 SK-MEL-2 cells, \\\\(n\\\\) = 700 sites the number of localizations found in one clathrin-coated structure was extracted. This is plotted against the quantified surface area determined for each coat.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/mwangangi2021structure.json b/test_extracted_captions/mwangangi2021structure.json new file mode 100644 index 0000000000000000000000000000000000000000..2e046b36cf96bae135d93304b45122664b1e0950 --- /dev/null +++ b/test_extracted_captions/mwangangi2021structure.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\nFigure 1: **The twinfilin/CP/actin complex.** (**A**) Front view of the permanent complex in cartoon representation. CP consists of two subunits, a-subunit (CPa) and b-subunit (CPb). Twinfilin comprises two ABC + domains (D1 and D2), a linker between D1 and D2 that includes a helix (residues 151 to 165), and a C-terminal tail (Taaf; residues 316 to 342, the last eight amino acids are disordered). Actin subunit 1 is bound to twinfilin D1, and subunit 2 to twinfilin D2. The twinfilin secondary structure elements are colored differentially, helices in red, strands in cyan, and loops and extended regions in orange, with the Taaf in lime green. (**B**) Front view (same as in (**D1**), in which the actin subunits and twinfilin are represented as surfaces. Twinfilin is shown entirely in orange. (**C** and **D**) Back and side views, respectively.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2. Binary interactions in the twinfilin/CP/actin complex.****(A)** Front and back views of actin bound to twinfilin D1 or twinfilin D2. (B) Two views of the CP interactions with actin, in which actin 1 and actin 2 are shown in similar orientations. (C) Three orientations of the CP/twinfilin interaction. Examples of the electron density of key features are shown in fig. 55 (H to K).\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Comparison of the CP-binding mode to Arp1 in the dynamic complex (Arp 1/CP) with the binding mode to actin in actin/twinfilin/CP complex.** (**A** and **B**) Focus on the \\\\(\\\\beta\\\\)- and \\\\(\\\\omega\\\\)-entacles, respectively, which are highlighted by black circles. The \\\\(\\\\beta\\\\)-entacle is disordered and unbound in the actin/twinfilin/CP complex, and the \\\\(\\\\omega\\\\)-entacle is partially bound and ordered, relative to the dynaxton complex. Enlargements of the tentacle regions show the superimpositions of the CP \\\\(\\\\beta\\\\)- and \\\\(\\\\beta\\\\)-subunits colored red and blue, respectively) from the dynaxton complex on to actin (green and teal) and twinfilin (yellow) from the actin/twinfilin/CP complex. (**A**) Enlargement, the twinfilin linker (yellow) binds to the \\\\(\\\\beta\\\\)-entacle-binding site on actin 2 (green). (**B**) Enlargement, the N terminus of twinfilin D1 (yellow) occupies half of the \\\\(\\\\alpha\\\\)-teractle-binding site on actin 1 (teal).\\n\\n'", "CAPTION FIG6.png": "'\\nFig. 6: **Models of the filament uncapping.** (**A** Cartoon comparison of CP and CP/twufflin association at the batbed end of a filament. (**B**) Twufflin-sided uncapping is a result of twufflin ADF+ domains inducing G-actin-like conformations in the terminal two actin protomers, weakening actin-actin interactions in the filament, leading to the dissociation of the complex. (**C**) Twinflin-V-1-aided uncapping requires space for V-1 to reach its binding site on CP via a wobble state of the twufflin-bound complex. Once V-1 is bound, CP is unable to reassociate with actin. In vitro effects of twufflin alone on actin filaments and comparisons of uncapping models in the absence of twinflin are shown in fig. 59.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: V-1 uncaps filament barbed ants in the presence of twinfilin. Pyrene-actin polymerization assay showing uncapping of F-actin seeds by V-1 in the presence of twinfilin. Unlabeled F-actin seeds (0.5 mM) were capped with 100 mM CP and subsequently polymerized in either 2 mM actin (10% pyrene)/28 mM grofilm (red) or 2 mM actin (10% pyrene)/28 mM grofilm supplemented with either 1 mM twinfilin (yellow), 5 mM V-1 [purple], or a mixture of 1 mM twinfilin and 5 mM V-1 (blue). As a control F-actin seeds were polymerized in the absence of CP (black). As shown in the profile, the presence of both twinfilin and V-1 induces accelerated polymerization of pre-capped F-actin seeds (blue) to almost the same level as the positive control (black). By contrast, twinfilin alone (yellow) induces very low level of polymerization, similar to CP alone (red), while V-1 alone (purple) only induces minimal polymerization.\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: **Conformations of CP.** (**A**) Comparison of the twinfilin tail (yellow) binding site with CAMRIL (cyan) on CP. Both CP-binding peptides run in the same direction, and N terminus of the twinfilin tail is labeled N. (**B**) The actin-binding site on the CP \\\\(\\\\alpha\\\\)-tetracle is distant from the twinfilin tail-binding site. (**C** and **D**) Structural superimposition of the \\\\(\\\\beta\\\\)-subunits of CP reveals that the conformation of CP in the twinfilin/CP/actin complex is different to the CARML-bound and unbound conformations of CP. (**E**) Superimposition reveals that the _coordination_ of CP in the twinfilin/CP/actin complex is similar to the V-1-bound conformation of CP. \\\\(\\\\alpha\\\\)- and \\\\(\\\\beta\\\\)-subunits of CP are colored light green and dark green, respectively, for the CARML, unbound, and V-1 complexes. Black arrows indicate conformational changes in CP in adopting the twinfilin/CP/actin complex structure.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/numasawa2020fluorescent.json b/test_extracted_captions/numasawa2020fluorescent.json new file mode 100644 index 0000000000000000000000000000000000000000..0830159039a5125f56e346a37b0ddcf3e5248a8b --- /dev/null +++ b/test_extracted_captions/numasawa2020fluorescent.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1. a) Molecular design of fluorescent probes for detection of folate receptors. The structures of FolateSiR-1 and FolateSiR-2 are also shown. b) Absorption and emission spectra of 1 um FolateSiR-1 in 100 mm sodium phosphate buffer at pH 7.4, \\\\(\\\\lambda_{\\\\rm in}=652\\\\) nm. c) Absorption and emission spectra of 1 um FolabeSR-2 in 100 mm sodium phosphate buffer at pH 7.4, \\\\(\\\\lambda_{\\\\rm in}=656\\\\) nm.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: a) The ovarian cancer tissue microarray was incubated with 5 um FolateSiR-1 and 2.9 um D4PI (nuclear stain) in phosphate-buffered saline containing 0.05 % Tween 20 (PBST) for 2 h. There, the tissue microarray was washed with PBST three times and the fluorescence image was obtained. The microarray contains 37 tumor tissues and 3 normal tissues. Fluorescence images of the 3 normal tissues and 3 representative tumor tissues are shown, scale bar = 2 mm. b) immunostaining of folate receptors in the tissue microarray. The immunostained 3 normal tissues and 3 tumor tissues correspond to those in (a), scale bar = 2 mm.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: a) Fluorescence images at different time points of KB tumor-bearing mouse injected with 100 \\\\(\\\\mu\\\\)m **FolateSR-1** in 100 \\\\(\\\\mu\\\\)L saline containing 1% DMSO as a cosolvent. Fluorescence and white light images were obtained before and 0, 0.5, 1, 2, 3 and 6 h after the probe injection; \\\\(\\\\lambda_{in}=\\\\) 661 (641\u2013681) nm, \\\\(\\\\lambda_{in}=\\\\) 700\u2013800 nm. \\\\(T=\\\\) tumor; \\\\(M=\\\\) muscle. Fluorescence intensity scale: gray scale 0 to 233. b) Time-dependent change of fluorescence intensity in tumor and non-tumor (muscle) areas of three mice, including the mouse in (a). Error bar shows S.E. c) Fluorescence images at different time points of KB tumor-bearing mouse injected with 100 \\\\(\\\\mu\\\\)m **FolateSR-2** in 100 \\\\(\\\\mu\\\\)L saline containing 1% DMSO as a cosolvent. Fluorescence and white light images were obtained before and 0, 0.5, 1, 2, 3 and 6 h after the probe injection; \\\\(\\\\lambda_{in}=\\\\) 661 (641\u2013681) nm, \\\\(\\\\lambda_{out}=\\\\) 700\u2013800 nm. \\\\(T=\\\\) tumor; \\\\(M=\\\\) muscle. Fluorescence intensity scale: gray scale 0 to 233. d) Time-dependent change of fluorescence intensity in tumor and non-tumor (muscle) areas of three mice, including the mouse in (c). Error bar shows S.E.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: a) Bright-field (left) and fluorescence (right) images of KB cells incubated with \\\\(\\\\delta\\\\) um **FolateSIR-1** or **FolateSIR-2** in the presence or absence of 1 mm folic acid and \\\\(0.5\\\\,\\\\%\\\\) DMSO as a cosolvent. White arrows indicate bright dots inside cells; \\\\(\\\\lambda_{\\\\text{res}}=650\\\\) nm, \\\\(\\\\lambda_{\\\\text{res}}=670\\\\)-\\\\(750\\\\) nm. Scale bars = \\\\(20\\\\) um. b) Bright-field (left) and fluorescence (right) images of OVCAR-3 cells incubated with \\\\(5\\\\) um **FolateSIR-1** or **FolateSIR-2** and \\\\(0.5\\\\,\\\\%\\\\) DMSO as a cosolvent. White arrows indicate bright dots inside cells, \\\\(\\\\lambda_{\\\\text{res}}=650\\\\) nm, \\\\(\\\\lambda_{\\\\text{res}}=670\\\\)-\\\\(750\\\\) nm. Scale bars = \\\\(20\\\\) \u03bcm.\\n\\n'", "CAPTION FIG3.png": "'Figure 3. a) Schematic of folate receptor (\\\\(-\\\\)Folbp 1) expression in mouse embryo: regions reported to show folate receptor expression are shown in gray. b) Fluorescence image of mouse embryo incubated with 20 um FolateSiR-1. Locations stained with FolateSiR-1 are indicated by white arrowheads. c) Fluorescence image of mouse embryo incubated with 20 um FolateSiR-2.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/numasawa2020fluorescent_supp.json b/test_extracted_captions/numasawa2020fluorescent_supp.json new file mode 100644 index 0000000000000000000000000000000000000000..a94d91e422397074b15c8e957f5d1a136eba2913 --- /dev/null +++ b/test_extracted_captions/numasawa2020fluorescent_supp.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS6.png": "'and fluorescence images of extracted organs of the mouse in Figure 4a. (b) White-light and fluorescence images of extracted organs of the mouse in Figure 4c.\\n\\n'", "SUPP CAPTION FIGS3-1.png": "'\\n\\n**Figure S3.** (a) Fluorescence images of KB cells (left) and OVCAR-3 cells (right) incubated with fluorophore-labeled folates. Emission: 490 nm (**Fluorescein Folate**, **Alexa488 Folate**), 555 nm (**TAMRA Folate**) and 595 nm (**2-Me DCTM Folate**, **2-COOH DCTM Folate**). Excitation: 510-540 nm (**Fluorescein Folate**, **Alexa488 Folate**), 575-600 nm (**TAMRA Folate**) and 610-650 nm (**2-Me DCTM Folate**, **2-COOH DCTM Folate**). Scale bars: 20 \\\\(\\\\mu\\\\)m. (b) Photophysical properties of fluorophore-labeled folates. \\\\({}^{a}\\\\)Photophysical properties were measured in 0.1 N NaOH aq. with fluorescein in 0.1 N NaOH aq. (\\\\(\\\\Phi_{\\\\text{fl}}\\\\)\\\\(=0.85\\\\)) as a standard. \\\\({}^{b}\\\\)Photophysical properties were measured in 100 mM sodium phosphate buffer (pH 7.4) with fluorescein in 0.1 N NaOH aq. (\\\\(\\\\Phi_{\\\\text{fl}}\\\\)\\\\(=0.85\\\\)) as a standard. \\\\({}^{b}\\\\)Photophysical properties were measured in 100 mM sodium phosphate buffer (pH 7.\\n\\n'", "SUPP CAPTION FIGS3-2.png": "'0.85) as a standard. \\\\({}^{c}\\\\) Photophysical properties were measured in 100 mM sodium phosphate buffer (pH 7.4) with Rhodamine B in EtOH (\\\\(\\\\Phi_{\\\\mathrm{fl}}\\\\) = 0.65) as a standard. \\\\({}^{d}\\\\) Photophysical properties were measured in 100 mM sodium phosphate buffer (pH 9.0) with 2-Me TokyoMagenta in 100 mM sodium phosphate buffer (pH 9.0) (\\\\(\\\\Phi_{\\\\mathrm{fl}}\\\\) = 0.42) as a standard.\\n\\n'", "SUPP CAPTION FIGS4-1.png": "'\\n\\n**Figure S4.** (a) KB cells were incubated with **FolateSiR-1**, then fixed with 4% formaldehyde, and bright-field and fluorescence images were obtained. (b) Fluorescence image of mouse embryo incubated with 10 mM **FolateSiR-1**'", "SUPP CAPTION FIGS4-2.png": "'in DMEM containing 10% rat IC-serum. Then, the mouse embryo was fixed with 4% formaldehyde. Locations stained with **FolateSiR-1** are indicated by white arrowheads. (c) Fluorescence image of mouse embryo incubated with 10 mM **FolateSiR-1** in the presence of 1 mM folic acid, and then fixed with 4% formaldehyde.\\n\\n'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Figure S5.** (a) Fluorescence images of mouse embryos incubated with 10 \\\\(\\\\upmu\\\\)**M****FolateSiR-1** and fixed with 4% formaldehyde. The right images are magnifications of the indicated portions of the left images. The regions stained with **FolateSiR-1** are indicated by white arrowheads. The cells indicated by yellow arrows appear to be dead cells. Scale bars: 50 \\\\(\\\\upmu\\\\)m. (b,c) Bright-field (left) and fluorescence (right) images of mouse embryos fixed with 4% formaldehyde in the absence (b) or presence (c) of 1 mM folic acid. These data indicate that the autofluorescence of the fixed embryos with or without 1 mM folic acid is sufficiently low compared with those in (a). Scale bars: 50 \\\\(\\\\upmu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS1.png": "'\\n\\n**Figure S1.** Immunostaining images of KB cells (a), OVCAR-3 cells (b), and HT1080 cells (c). Cells were fixed with 4% PFA/PBS for 20 min, labeled with human anti-folate binding protein antibody for 1 hr, washed, and labeled with second antibody conjugated with Alexa Fluor(r) 488 for 1 hr. Ex. 488 nm; Em. 500-535 nm. Scale bars: 20 \\\\(\\\\upmu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS2.png": "'\\n\\n**Figure S2.** Fluorescence images of KB cells (FR+) (a) and OVCAR-3 cells (FR-) (b) obtained with a commercially available near-infrared fluorescent probe, **FolateRSense** (PerkinElmer Inc.). White arrows indicate bright dots inside cells. Ex. 650 nm, Em. 670-750 nm. Scale bars: 20 \\\\(\\\\mu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS8.png": "'\\n\\n**Figure S8.**_In vivo_ fluorescence imaging of HT1080 tumor-bearing mice injected with 100 \\\\(\\\\upmu\\\\)M **FolateSiR-2** in 100 \\\\(\\\\upmu\\\\)L saline (n = 3). (a) Time-lapse white-light (top) and fluorescence (bottom) images of a mouse. The images were obtained before the probe injection and 0, 0.5, 1, 2, 3 and 6 h after the probe injection. Ex./Em. = 661/700-800 nm. T: Tumor; M: Muscle. Fluorescence intensity scale: gray scale 0 to 255. (b) The time-dependent fluorescence intensity changes in tumor and non-tumor (muscle) areas of three mice. Error bar shows S.E. (c) White-light (top) and fluorescence (bottom) images of extracted mouse organs.\\n\\n'", "SUPP CAPTION FIGS9.png": "'\\n\\n**Figure S9.**_In vivo_ fluorescence imaging of KB tumor-bearing mice injected with 6 mM folic acid in 100 mL saline, then with 100 mM **FolateSiR-1** in 100 mL saline (n = 3). (a) Time-lapse white-light (top) and fluorescence (bottom) images of a mouse. The images were obtained before the probe injection and 0, 0.5, 1, 2, 3 and 6 h after the probe injection. Ex./Em. = 661/700-800 nm. T: Tumor; M: Muscle. Fluorescence intensity scale: gray scale 0 to 255. (b) The time-dependent fluorescence intensity changes of tumor and non-tumor (muscle) areas of three mice. Error bar shows S.E. (c) White-light (top) and fluorescence (bottom) images of extracted mouse organs.\\n\\n'", "SUPP CAPTION S13.png": "'fluorescence intensities were weak in C3 and A5 specimens. (b) The magnified folate receptor-immunostaining images of C4, B8, C3 and A5 in Figure S12 are shown. All specimens were immunostained, though the sample condition of B8, C3 and A5 were not good. (c) The magnified fluorescence image (left) and the magnified immunostaining image (right) of C4 specimen in (a) and (b) are shown. Arrows indicate the boundary between tumor tissues and non-tumor tissues (vascular tissues and fibrous tissues). The tumor tissues are selectively stained by the fluorescent probe. **FalterSIR-I**\\n\\nfluorescence intensities were weak in C3 and A5 specimens. (b) The magnified folate receptor-immunostaining images of C4, B8, C3 and A5 in Figure S12 are shown. All specimens were immunostained, though the sample condition of B8, C3 and A5 were not good. (c) The magnified fluorescence image (left) and the magnified immunostaining image (right) of C4 specimen in (a) and (b) are shown. Arrows indicate the boundary between tumor tissues and non-tumor tissues (vascular tissues and fibrous tissues). The tumor tissues are selectively stained by the fluorescent probe. **FalterSIR-I**'", "SUPP CAPTION FIGS13.png": "'Figure S13. (a) The magnified fluorescence images of C4, B8, C3 and A5 specimens in Figure S11 are shown. Red color indicates the fluorescence of **FlatestBl-4**. All specimens showed the fluorescence signal, though the color indicates the fluorescence of **FlatestBl-4**. All specimens showed the fluorescence signal, though the color indicates the fluorescence of **FlatestBl-4**.\\n\\n'", "SUPP CAPTION FIGS3.png": "'Figure 3: (a) The \\\\(\\\\Delta\\\\)-\\\\(\\\\Delta\\\\) relationship, slope of \\\\(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\Delta\\\\)(\\\\(\\n\\n'", "SUPP CAPTION FIGS4.png": "'Figure 10. (a)(b)(c)(d) shows the normalized (a) normalized (b) normalized (b) normalized (c) normalized (b) normalized (c) normalized (d) normalized (d) normalized (e) normalized (f\\n\\n'", "SUPP CAPTION S13-2.png": "'fluorescence intensities were weak in C3 and A5 specimens. (b) The magnified folate receptor-immunostaining images of C4, B8, C3 and A5 in Figure S12 are shown. All specimens were immunostained, though the sample condition of B8, C3 and A5 were not good. (c) The magnified fluorescence image (left) and the magnified immunostaining image (right) of C4 specimen in (a) and (b) are shown. Arrows indicate the boundary between tumor tissues and non-tumor tissues (vascular tissues and fibrous tissues). The tumor tissues are selectively stained by the fluorescent probe, **FolateSiR-1**.\\n\\n'", "SUPP CAPTION FIGS14.png": "'\\n\\n**Figure S14.** Absorption spectra of 1 \\\\(\\\\mu\\\\)M 2-COOH SiR650 in 100 mM sodium phosphate buffer at pH 7.4 with or without 10% fetal bovine serum (FBS).\\n\\n'", "SUPP CAPTION FIGS15.png": "'\\n\\n**Figure S15.** Absorbance spectra of 1 \\\\(\\\\upmu\\\\)**M **FolateSiR-1** in phosphate buffered saline (PBS) containing 0.1% DMSO with or without 10% FBS (Biowest, Cat. No.: S1810-500), or in mouse serum (FUJIFILM Wako Pure Chemical Corp., Cat. No.: 146-06551).\\n\\n'", "SUPP CAPTION FIGS12.png": "'\\n\\n**Figure S12.** Immunostaining of folate receptors in the tissue microarray described in Figure S8. Scale bar represents 2 mm.\\n\\n'", "SUPP CAPTION FIGS13-1.png": "'\\n\\n**Figure S13.** (a) The magnified fluorescence images of C4, B8, C3 and A5 specimens in Figure S11 are shown. Red color indicates the fluorescence of **FolateSiR-1**. All specimens showed the fluorescence signal, though the'", "SUPP CAPTION FIGS10.png": "'\\n\\n**Figure S10.** Components of the frozen human ovary tumor tissue array (Catalog No.: T6235183-5; Lot No.3 B705061).\\n\\n'", "SUPP CAPTION FIGS11.png": "'\\n\\n**Figure S11.** Fluorescence staining of the tissue microarray described in Figure S8 with **FolateSiR-1** (pink) and DAPI (Blue). Scale bar represents 2 mm.\\n\\n'", "SUPP CAPTION FIGS7.png": "'\\n\\n**Figure S7.**_In vivo_ fluorescence imaging of HT1080 tumor-bearing mice injected with 100 \\\\(\\\\upmu\\\\)M **FolateSiR-1** in 100 \\\\(\\\\upmu\\\\)L saline (n = 3). (a) Time-lapse white-light (top) and fluorescence (bottom) images of a mouse. The images were obtained before the probe injection and 0, 0.5, 1, 2, 3 and 6 h after the probe injection. Ex./Em. = 661/700-800 nm. T: Tumor; M: Muscle. Fluorescence intensity scale: gray scale 0 to 255. (b) The time-dependent fluorescence intensity changes of tumor and non-tumor (muscle) areas of three mice. Error bar shows S.E. (c) White-light (top) and fluorescence (bottom) images of extracted mouse organs.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/obashi2023conformational.json b/test_extracted_captions/obashi2023conformational.json new file mode 100644 index 0000000000000000000000000000000000000000..d0ca92c70481f2fa2e307bc361a4c2b2e4c12c55 --- /dev/null +++ b/test_extracted_captions/obashi2023conformational.json @@ -0,0 +1 @@ +{"CAPTION FIG3.png": "\"of fluorescence lifetime changes similar. Mean fluorescence lifetimes from single CCs were analyzed by categorizing them according to lattice structures and compared to average values of flat structures. \\\\(n\\\\) = 6 cells from 4 experiments for each condition. Each dot is from one cell experiment and errors are SE. One-way ANOVA, then Tukey's test. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. FA proposed structural model of CLC conformational changes predicted from both FRET-CLEM with EGFP-ShadowW and EGFP-DPA. The N-terminus of CLC moves away from both the CLC-terminus (triskelion vertex) and the plane of the plasma membrane as clathrin hitclics curve. The structural model is based on PDB 3LVG. Source data are provided as a Source Data file.\\n\\nFig. 3: **CLC N-terminal position perpendicular to the plane of the plasma membrane.** A dipicylumclime (DPA) is a nonfluorescent hydrophobic anion that incorporates into membranes. DPA quenches EGFP in a distance dependent manner by FRET. PM is the plasma membrane. **b** FLIM images of unrooted membranes of HeLa cells expressing CLC-EGFP (left), EGFP-CLEM (center), or EGFP-CLC (right) without (top) or with 20 \u03bcM DPA (bottom). Scale 10 \u03bcM. **c** Mean fluorescence lifetimes with different DPA concentrations. \\\\(n\\\\) = 16 (EGFP-CLC), 17 (EGFP-CLEM), and 15 cells (CLC-EGFP) from 3 experiments. Errors are SE. **d** A structural model of EGFP positions of CLC probes predicted from the EGFP-PAGE FRET experiments. The model is based on PDB 4KW4 and 3LVG. **e** FRET-CLEM on HeLa cells expressing either CLC-EGFP or EGFP-CLC with DPA. DPA concentrations were 3 \u03bcM for CLC-EGFP and 50 \u03bcM for EGFP-CLC to obtain -50% FRET efficiencies to make the degree of fluorescence lifetime changes similar. Mean fluorescence lifetimes from single CCs were analyzed by categorizing them according to lattice structures and compared to average values of flat structures. \\\\(n\\\\) = 6 cells from 4 experiments for each condition. Each dot is from one cell experiment and errors are SE. One-way ANOVA, then Tukey\u2019s test. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. FA proposed structural model of CLC conformational changes predicted from both FRET-CLEM with EGFP-ShadowW and EGFP-DPA. The N-terminus of CLC moves away from both the CLC-terminus (triskelion vertex) and the plane of the plasma membrane as clathrin hitclics curve. The structural model is based on PDB 3LVG. Source data are provided as a Source Data file.\\n\\n\"", "CAPTION FIG1.png": "'Fig. 1: **FRRT-GLEM.****a** A Correlative FLM-FRET and PREM images of unroofed membranes of HeLa cells expressing EGFP-CLC (top) or EGFP-ShadowY-CLC (bottom). FLM images (left; photon counts are represented by brightness and fluorescence lifetimes are represented by pseudo color), PREM images (center), and emerge images (right). \\\\(n\\\\) = 1 cell for each condition. Scale 500 nm. **b** PREM images of ECSs on an unroofed membrane of a HeLa cell expressing EGFP-CLC that were classified as flat, domed, or sphere and corresponding areas from a FLIM image. \\\\(n\\\\) = 1 cell. Scale 200 nm. **c** Fluorescence lifetime decays from single CCSs indicated by arrows in panel **a**. **d** Mean fluorescence lifetimes from single CCSs on an unroofed membrane of HeLa cells expressing EGFP-CLC or EGFP-ShadowY-CLC. \\\\(n\\\\) = 8S CCSs from 1 cell (EGFP-CLC) and 81 CCSs from 1 cell (EGFP-ShadowY-CLC). For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG4.png": "'with PH-miRP-FRB, or PH-miRP-FRB-2 without or with 20 uM DPA. Cells were unrooted after 15 min incubation with AP21967 or ethanol (control). \\\\(n\\\\) = 20 (PH-miRP-FRB, control), 19 (PH-miRP-FRB, AP21967), 20 (PH-miRP-FRB-2, control), and 19 cells (PH-miRP-FRB-2, AP21967) from 3 experiments. **d** Differences in fluorescence lifetimes (shown in panel c) without and with DPA. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. Source data are provided as a Source Data file.\\n\\nFig. 4: **Manipulation of CL N-terminal position using a chemically inducible dimerization system.** A Schematic models of the chemically inducible FKBP/FRB dimerization system. FKBP is attached to the N-terminus of CLC, and two FRBs (FRB-2) are attached to the C-terminus of PH domain from PLC81. A rapamycin analog, AP21967, induces heterodimerization between FKBP and the T2098L mutant of FRB. **b** Potential FRET pairs without or with AP21967 treatment either absence or presence with DPA. **c** Fluorescence lifetime measurements were performed on unrooted membranes of HeLa cells expressing FKBP-EGFP-CLC either with PH-miRP-FRB, or PH-miRP-FRB-2 without or with 20 uM DPA. Cells were unrooted after 15 min incubation with AP21967 or ethanol (control). \\\\(n\\\\) = 20 (PH-miRP-FRB, control), 19 (PH-miRP-FRB, AP21967), 20 (PH-miRP-FRB-2, control), and 19 cells (PH-miRP-FRB-2, AP21967) from 3 experiments. **d** Differences in fluorescence lifetimes (shown in panel c) without and with DPA. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG2.png": "\"the average values of flat structures. \\\\(n=6\\\\) cells from 3 experiments (EGP-CLC) and \\\\(n=6\\\\) cells from 4 experiments (EGP-CLC and CLC-S ShadowWt). **c**RET-CLEM on HeLa cells expressing either EGF-CLC, or EGF-CLCAN and CLC-S ShadowWt. \\\\(n=6\\\\) cells from 5 experiments (EGP-CLC/can) and \\\\(n=6\\\\) cells from 4 experiments (EGP-CL/can). \\\\(n\\\\) FRET-CLEM on HeLa cells expressing either EGF-CLR/can and \\\\(n=6\\\\) cells from 4 experiments (EGP-CL/can) and \\\\(n=6\\\\) cells from 4 experiments (EGP-CL/can). **f**RET-CLEM on HeLa cells expressing either EGF-QQN (QQN mutant of CLC), or EGF-QQN and CLC-S ShadowWt. \\\\(n=6\\\\) cells from 3 experiments for each condition. One-way ANOVA, then Tukey's test. Each dot is from one cell experiment and errors are SE. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of LS. Source data are provided as a Source Data file.\\n\\nFig. 2: **CLC conformational changes in cells.** a schematic models of CLC conformation at different assembly states in vitro or in living cells and their expected FRET efficiencies between the N- and C-terminus of CLCs. **b** A structural model with the assumption that either extended (light blue) or bent conformations (magenta) of CACs assemble into the lattice. The model is based on PDB 3.WC and 6WQ. 3.WC is overlaid with 6WQ. **c** Schematic models of two assembled triskells with extended (left) or bent CLCs (right). A CLC N-terminus position (yellow) and C-terminus positions of surrounding CLCs (orange) are shown. **d** FRET-CLEM was performed on HeLa cells expressing either EGF-CLC, or EGF-CLC and CLC-S ShadowWt. Mean fluorescence lifetimes from single CCSs were analyzed by categorizing them according to lattice structures (flat, domed and sphere) and they were compared to the average values of flat structures. \\\\(n=6\\\\) cells from 3 experiments (EGP-CLC) and \\\\(n=6\\\\) cells from 4 experiments (EGP-CLC and CLC-S ShadowWt). **e** FRET-CLEM on HeLa cells expressing either EGF-CLR/can, or EGF-CLCAN and CLC-S ShadowWt. \\\\(n=6\\\\) cells from 5 experiments (EGP-CL/can) and \\\\(n=6\\\\) cells from 4 experiments (EGP-CL/can) and \\\\(n=6\\\\) cells from 4 experiments (EGP-CL/can). **f** FRET-CLEM on HeLa cells expressing either EGF-QQN (QQN mutant of CLC), or EGF-QQN and CLC-S ShadowWt. \\\\(n=6\\\\) cells from 3 experiments for each condition. One-way ANOVA, then Tukey\u2019s test. Each dot is from one cell experiment and errors are SE. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of LS. Source data are provided as a Source Data file.\\n\\n\"", "CAPTION FIG6.png": "'Figure 6: **Models of conformational switch in CLC in living cells. Schematic models of conformational switch in CLC at the plasma membrane in cells. CLC assumes an extended conformation in unassembled triskelia in the cytoplasm similar to that seen in x-ray crystal structures. Next, when CLC assembles at the membrane as a flat lattice, CLC changes conformations from the extended to a new folded conformation. The N-terminus is then displaced deeper into the cytosol as clathrin lattices curve into vesicles. The extended and bent models are based on PDE 3LVG.**\\n\\n'", "CAPTION FIG5.png": "'(AP21967). A two-sided unpaired t test was used. e Histogram of residence times. f Confocal projection images of Alexa Fluor 647 conjugated transferrin (TF-AF647) uptake in HeLa cells expressing FKBP-EGFP-CLC and PH-mCherry-FRB-2 treated with AP21967 or ethanol (control). Fluorescence intensity of TF-AF647 (arbitrary units) is represented by pseudo color. \\\\(n\\\\) = 3 experiments. Scale 500 \\\\(\\\\mu\\\\)m = Transforme uptake in HeLa cells expressing FKBP and FRB probes treated with AP21967. Fluorescence intensities of incorporated TF-AF647 normalized by non-transfected cells in the same sample were compared between non-transfected and transfected cells. \\\\(n\\\\) = 116-198 cells from 3 experiments for each conditions. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. A two-sided unpaired t test was used. Source data are provided as a Source Data file.\\n\\nFig. 5: **Inamputation of CLC conformation changed lattice structures, dynamics, and endocytosis. a\u2013c Unroofed membranes from cells expressing FKBP-EGFP-CLC and PH-mRFP-FRB-2 treated with AP21967 (AP) or ethanol (control) were imaged with PREM. Two-dimension area of single CCS were manually segmented and measured. Membrane area occupation against the total analyzed membrane area (a), two-dimension projection area (b), and density (c) of flat, diamond, and sphere CCSs were compared. Each dot is from one cell experiment, and errors are SE. \\\\(n\\\\) = 6 cells from 3 experiments for each condition. The average measured area / cell (mean + SE) = 213 \u00b1 34 (control) and 165 \u00b1 10 \u03bcm\\\\({}^{2}\\\\)(AP21967). d Live cell time lapse TIRF imaging on HeLa cells expressing FKBP-EGFP-CLC and PH-mRFP-FRB-2 without or with AP21967 treatment. Tracks with over 205 were analyzed and residence times were compared. \\\\(n\\\\) = 206 spots from 5 cells from 3 experiments (no treatment) and 234 spots from 5 cells from 4 experiments (AP21967). A two-sided unpaired t test was used. e Histogram of residence times. f Confocal projection images of Alexa Fluor 647 conjugated transferrin (TF-AF647) uptake in HeLa cells expressing FKBP-EGFP-CLC and PH-mCherry-FRB-2 treated with AP21967 or ethanol (control). Fluorescence intensity of TF-AF647 (arbitrary units) is represented by pseudo color. \\\\(n\\\\) = 3 experiments. Scale 500 \\\\(\\\\mu\\\\)m = Transforme uptake in HeLa cells expressing FKBP and FRB probes treated with AP21967. Fluorescence intensities of incorporated TF-AF647 normalized by non-transfected cells in the same sample were compared between non-transfected and transfected cells. \\\\(n\\\\) = 116-198 cells from 3 experiments for each conditions. For box plots, box is interquartile range, center line is median, center circle is mean, whiskers are minimum and maximum data points with a coefficient value of 1.5. A two-sided unpaired t test was used. Source data are provided as a Source Data file.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/okrut2015allosteric.json b/test_extracted_captions/okrut2015allosteric.json new file mode 100644 index 0000000000000000000000000000000000000000..27cc250f2864796a2c02d0a1a545e32212b76396 --- /dev/null +++ b/test_extracted_captions/okrut2015allosteric.json @@ -0,0 +1 @@ +{"CAPTION FIG6.png": "'Figure 5: Proposed mechanism of N-WASP activation by _Nck_. In the auto-inhibited _contamination_, the N-WASP C helix binds intramolecularly to the GBD. _Nck_ binds to tyrosine-phosphorylated receptors _can_ the membrane and activates N-WASP by competitively displacing the C helix from the GBD with its inter-SH3AB linker. The released VCA segment is available to activate the Arp2/3 complex. Polyvalent interactions between the SH3 domains of _Nck_ and the proline-rich region (PFR) of N-WASP can lead to higher-order olig-camar formation, increasing the local density of activated N-WASP molecules.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\nFig. 5: NMR analysis reveals direct binding of the Ndix inter-SH3 linker and the N-WASP GBD. (_A_) [\\\\({}^{15}\\\\)N, \\\\({}^{16}\\\\)H]-heteronuclear single-quantum coherence (BSCQNMR spectra of \\\\({}^{15}\\\\)N-labeled N-WASP GBD acquired in the presence and absence of unlabeled Ndix linker peptide (amino acids 61-106). (_B_) NMR titration of \\\\({}^{15}\\\\)N-labeled GBD (ES \\\\(\\\\mu\\\\)M) with the Ndix linker peptide. Chemical shift changes, along with the corresponding curve fit, are shown for a representative peak from the [\\\\({}^{15}\\\\)N, \\\\({}^{16}\\\\)H]-HSQC spectrum. A binding \\\\(K_{\\\\alpha}\\\\) of \\\\(33\\\\pm 8\\\\)\\\\(\\\\mu\\\\)M was determined by fitting titration curves to a total of 17 peaks. (_C_) Sequence alignment of a WASP-GBD C-helix construct (10) and the N-WASP GBD Ndix linker fusion protein. The secondary structure of the WASP-C helix complex (1EJS.pdb) is indicated. (_D_) Secondary structure prediction of the N-WASP Ndix fusion protein by Talos+ is shown. (_E_) Coomassie-strained gel from a competition pull-down experiment (bound fraction). N-WASP GST-GBD was used to pull down Ndix in the presence of increasing N-WASP VCA. (_F_) integrated band intensities from the Coomassie-stained gel showing Hdx and VCA bound to GST-GBD.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n## References\\n\\nFig. 2: Nick is the most effective SH3 adapter in promoting N-WASP-dependent actin assembly, (_A_) integrated fluorescence intensity and representative images of bead-loazlized Alexa558 H-WASP (200 nM) incubated with NTAD-doped lipid-coated beads and the indicated His-SH3 adapters (250 nM). To compensate for differences in N-WASP recruitment efficiency, the NTAD density was increased to 5% for Crk-H, contraction, CIR, and Tks4, whereas 1% NTAD-was used for Nok1 and Grb2. (_B_) Representative phase contrast and fluorescence images of actin assembly reactions under conditions reported in \\\\(A\\\\). (Scale bars: 5 \u03bcm), (_C_) Integrated fluorescence intensity of Alexa488 actin tails and tail lengths (mean \u00b1 SD; \\\\(n\\\\) > 20). Asterisks indicate significant differences between Nok and Grb2: ***_P_ < 0.001.\\n\\n'", "CAPTION FIG3.png": "'\\nFigure 3: An inter-SH3 linker in Nick promotes actin network assembly by N-WASP. (A) Experimental strategy for localization of Nick to membranes containing PY+-nephrin. (B) Nick constructs used in the deletion analysis. (C) Representative images of actin tails formed by Nick deletion constructs. Motility reactions contained 200 nM N-WASP and 100 nM Nick. (Scale bar: 5 \u03bcm). (D) Integrated fluorescence intensity of Alexa88 actin tails. (F) Actin tail length. (F) Integrated fluorescence intensity of bead-localized Alexase58 Nick constructs. (Mean \u00b1 SD; \\\\(n>20\\\\); ***\\\\(p<0.001\\\\).) fil., Full length; PRR, proline-rich region.\\n\\n'", "CAPTION FIG1.png": "'\\nFig. 1: Membrane-associated actin networks assembled by SH3 adapters and N-WASP. (_A_) Domain organization of SH3 adapter proteins used in this study. (_B_) Experimental strategy for localization of His-tagged SH3 adapters using NTAD-doped membranes supported on silica microspheres. (_C_) Phase contrast and fluorescence images of lipid-coated beads (NTAD density: 1%, 2.5%, or 5%) incubated with the indicated SH3 adapter (250 nM), N-WASP (50 nM), Alexa488 actin, and actin regulatory components. After 15 min, reactions were fixed with glutaraldehyde. (Scale bar: 5 \u03bcm.) (_D_) Integrated fluorescence intensity of Alexa488 actin tails. (_E_) Actin tail length. (_F_) Integrated fluorescence intensity of bead-localized Alexa588 SH3 adapters (250 nM). (Mean \u00b1 50; \\\\(n\\\\) > 20.) Asterisks indicate a significant difference between Mck1 and Grb2 at 1% and 5% NTAD: ***_\\\\(<\\\\)_0.01; ***_P_\\\\(<\\\\) 0.001, respectively.\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: The inter-SH3AB linker contains a conserved motif and binds directly to the N-WASP GBD. (A) Nick domain structure and sequence of the inter-SH3AB linker. (B) Sequence alignment comparing the Nick inter-SH3 linker from various organisms and previously known N-WASP GBD ligands. (C) Coomassie-stained gel of GST pull-down samples. Immobilized GST-GBD from N-WASP or PAK1 was used to pull down full-length Nck. (D) N-WASP GST-GBD was used to pull down the indicator Nick deletion constructs. (E) N-WASP GST-GBD was used to pull down Nick amino acids 1-270 WT and mutants. Samples comprising 1.25% of the unbound supernatant (S) and 12.5% of the bound fraction (S) were separated by SDS/PAGE and stained with Comanatic. GS aa 67-105, substitution of the entire SH3AB linker with a Gly-Ser linker; GS aa 67-77, substitution of the hydrophobic motif with a Gly-Ser linker. (F) Representative images of actin tails formed by Nick amino acids 61-377 WT and 4A mutant. Mobility reactions contained 200 nM N-WASP and 100 nM Nck. (Scale bar: 5 \u03bcm.)'"} \ No newline at end of file diff --git a/test_extracted_captions/packerImportanceSDICurrent1979.json b/test_extracted_captions/packerImportanceSDICurrent1979.json new file mode 100644 index 0000000000000000000000000000000000000000..09c33dc97a91f8c9a41be26a08e20dee0b6bf837 --- /dev/null +++ b/test_extracted_captions/packerImportanceSDICurrent1979.json @@ -0,0 +1 @@ +{"CAPTION TAB2.png": "'\\n\\n**CHART 2. Summary of tests on current awareness methods.**'", "CAPTION TAB3.png": "'TABLE 3. Relationship between active and time sent.\\n\\n'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline TABLE 4. Relationship between scatter of information and success in keeping up-to-date.[3] \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table} TABLE 4. Relationship between scatter of information and success in keeping up-to-date.[3]'", "CAPTION FIG2.png": "'* [16]'", "CAPTION FIG3.png": "'* [16] A.M. 3. Success in keglin up-to-date.\\n\\n'", "CAPTION TAB1.png": "''", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION FIG1.png": "'* [14] A. M. K.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Summary of the effect of scatter of information and subscribing to an SDI service on efficiency in keeping up-to-date [Tables 5, 5(a), 5(b), 5(c), 6, 6(a), and 6(b)]. Arrows point to group with higher efficiency; \\\\(\\\\varepsilon\\\\) = significant, \\\\(\\\\iota\\\\) = tendency, \\\\(\\\\iota\\\\) = not even a tendency.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB8.png": "'TABLE 8. Relationship between using manual current awareness tools and efficiency (subscribers to SDI excluded).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Summary of the effect of scatter of information and the use of manual current awareness tools on efficiency in keeping up-to-date [Tables 7, 7(a), 7(b), 7(c), 8, 8(a), and 8(b)]. Arrows point to group with higher efficiency; s = significant, t = tendency; but not significant, nt = not even t tendency.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/paletzDynamicsMicroconflictsUncertainty2017.json b/test_extracted_captions/paletzDynamicsMicroconflictsUncertainty2017.json new file mode 100644 index 0000000000000000000000000000000000000000..312a12382880bce65a4833562a4698a0e9c73ca3 --- /dev/null +++ b/test_extracted_captions/paletzDynamicsMicroconflictsUncertainty2017.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'Table 1: Segmented transcript with uncertainty (italics) and micro-conflict (bold) codes\\n\\n'", "CAPTION FIG2.png": "'\\n\\n## Chapter 4 Printing'", "CAPTION FIG1.png": "'\\n\\n## Chapter 1 Introduction\\n\\nThe _Chandra_ satellite is a _Chandra_ satellite.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1999) The \\\\(\\\\beta\\\\)-function of the \\\\(\\\\beta'", "CAPTION FIG3.png": "'\\n\\n## Appendix A Proof of Theorem 1\\n\\n### Proof of Theorem 1\\n\\nProof of Theorem 1\\n'", "CAPTION TAB3.png": "'\\n\\n**Table 3** **Process micro-conflict and team success on uncertainty**'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/test_extracted_captions/paletzUncoveringUncertaintyDisagreement2016.json b/test_extracted_captions/paletzUncoveringUncertaintyDisagreement2016.json new file mode 100644 index 0000000000000000000000000000000000000000..8f62283c9b8c747ed62f97b792dc6e7fde0c52a5 --- /dev/null +++ b/test_extracted_captions/paletzUncoveringUncertaintyDisagreement2016.json @@ -0,0 +1 @@ +{"CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Examples of uncertainty from Mars Exploration Rover scientists (key words in bold) in conversational contexts, excerpts from Chan et al. (2012)'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION FIG1.png": "'Figure 1: Data structure: blocks nested within clips, and time-lagged analyses of micro-conflicts to uncertainty moderated by temporal context.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 3: Frequency of presence of lagged science, planning, process, quickly resolved, positive, and negative micro-conflicts by block'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION FIG2.png": "'Figure 2. Predicted number of uncertain utterances controlling for covariates by time-lagged (A) process micro-conflicts, (B) prover planning micro-conflicts, and (C) science micro-conflicts for early and later in the first 90 days of the mission\\n\\n'", "CAPTION TAB5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} &'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'"} \ No newline at end of file diff --git a/test_extracted_captions/pandit2020force.json b/test_extracted_captions/pandit2020force.json new file mode 100644 index 0000000000000000000000000000000000000000..b54b92bf26d5cea7f3185153db9df0f3cebecaa5 --- /dev/null +++ b/test_extracted_captions/pandit2020force.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'Figure 2: Effects of mechanical force and nucleotide bound to Arp2/3 complex on the time course of dissociation of actin filament branches. Arp2/3 complex branches were assembled in the flow chamber before applying flow as described in Materials and Methods. In \\\\(A\\\\) and \\\\(C\\\\), the force on each observed branch was calculated from its length and the flow rate, and then binned at the indicated average forces values (see Materials and Methods). (_A_) The effect of force on the time course of dissociation of actin filament branches formed by ATP-actin monomers and ATP-Arp2/3 complex and aged for 30 min, when most branches had ADP bound to Arp2/3 complex. The fraction of branches remaining is plotted. Smooth curves are the best fits of single exponentials to the data. Each trace includes at least 14 branches. (_B_) Dependence of the time course of dissociation of branches formed with ATP-actin monomers and ATP-Arp2/3 complex with different aging times (2.5, 4, 12, and 30 min) and the presence of BaFe. For all time courses, 500 mL min-1 of buffer flow was applied to the branches for discharging, producing a force of ~1 pN for a branch of 1.5 mm. The force on each branch was not calculated for the data shown. Smooth curves are the best global fits of double exponentials to the data for aging branches and yielded two shared rate constants for detachability: slow \\\\(k_{SF}\\\\) = 0.32 (\\\\(\\\\pm\\\\)0.005) min\u20131 and fast \\\\(k_{SF}\\\\) = 6.67 (\\\\(\\\\pm\\\\)0.44) min\u20131. Smooth curves are the best single exponential fits to the data for branches aged for 4 and 30 min with BaFe, with lifetimes of 13.9 (\\\\(\\\\pm\\\\)0.2) min\u20131 for the sample aged for ~4 min and 14.8 (\\\\(\\\\pm\\\\)0.09) min\u20131 for the sample aged for 30 min. Fig. 40 presents the fractional amplitudes obtained from the double exponential fits. (_C_) The effect of force on the time course of the dissociation of actin filament branches formed by ATP-actin monomers and ATP-Arp2/3 complex and aged for ~4 min using the same data collection and analysis methods as in \\\\(A\\\\). The smooth curves are best fits of the data to single (_F_ < 0.5 pN) or double (_F_ > 0.6 pN) exponentials. The fractional amplitudes of the slow phase in the time courses that follow the double exponentials are 0.71 +- 0.05 (_F_ = 0.98 pN), 0.39 +- 0.06 (_F_ = 1.25 pN), 0.81 +- 0.05 (_F_ = 1.60 pN), 0.38 +- 0.02 (_F_ = 1.93 pN), and 0.17 +- 0.02 (_F_ = 2.86 pN). Each trace includes at least 19 branches. (_N_est Time courses of branch dissociation under a range of forces for branches formed from ATP-Arp2/3 complex in the presence of 2 mM BaFe, and aged for ~4 min. Each trace includes at least 13 branches. The smooth curves are single exponential fits to the time course. Branches dissociate slowly under 0.2 pN of force, so the dehydration time course cannot be reliably fitted to obtain the branch lifetime. (_D_) Dependence of branch lifetimes on force for four different conditions: (filled black circles) branches formed from ATP-Arp2/3 complex and aged for 30 min to form branches with ADP-Arp2/3 complex (time courses in _A_); (filled red squares) branches formed from ADP-BaFe-Arp2/3 complex and aged for ~4 min (time courses in \\\\(C\\\\), inset; (filled pink triangles) slow denaturing phase of branches formed from ATP-Arp2/3 complex and aged for ~4 min (ADP-P), branch population; time courses in _C_); and filled blue triangles) fast debraaching phase of branches formed from ATP-Arp2/3 complex and aged for ~4 min (ADP branch population; time courses in _C_). The uncertainty bars for all data represent the SDs from the fits to exponentials shown in \\\\(A\\\\) and \\\\(C\\\\). The smooth black curve is the best single exponential fit (_Eq._1) to the ADP-Arp2/3 complex debraening data points, yielding a half-life (_F_10) of 0.54 (\\\\(\\\\pm\\\\)0.060) pH and branch lifetime in the absence of force (_z_) of 106 (\\\\(\\\\pm\\\\)8) min (observed rate constant = \\\\(\\\\tau_{0}^{-1}\\\\) = 0.01 (\\\\(\\\\pm\\\\)0.007) min\u20131). _Invest_ shows that 11 fast phase lifetimes (blue triangles; 0.3 min to 0.8 min at \\\\(F\\\\) > 1 pN) differ from the slow phase lifetimes (pink triangles; 2 min to 4 min at \\\\(F\\\\) > 1 pN; \\\\(t\\\\) = 6.85, one-tail \\\\(T\\\\)6) was \\\\(\\\\pm\\\\)2.13 and \\\\(P\\\\) = 0.001 by Welch\u2019s unequal variances _t_est); 2) the slow phase lifetimes (pink triangles; 2 min to 4 min at \\\\(F\\\\) > 1 pN) differ from the debraening lifetimes with BaFe, (red squares; 8 min to 9 min at \\\\(F\\\\) > 1 pN; t = 6.65, one-tail \\\\(T\\\\)6) was \\\\(\\\\pm\\\\)2.02 and \\\\(P\\\\) = 0.0006 by Welch\u2019s unequal variances \\\\(t\\\\) test); and 3) the fast phase lifetimes (blue triangles) do not differ significantly from the debraening with ATP aged for 30 min lifetimes (black circles) at \\\\(F\\\\) > 1 pN (_t_ = -0.86, two-tail \\\\(T\\\\)6) was \\\\(\\\\pm\\\\)2.45 and \\\\(P\\\\) = 0.42 by Welch\u2019s unequal variances \\\\(t\\\\) test).\\n\\n'", "CAPTION FIG1.png": "'\\nFig. 1: Microfluidics assay to measure Arp2/3 complex debranching under force. (A) Diagram showing a short segment of an actin filament containing 10% biotinylated and 15% Alexa 58B-labeled (red) actin subunits immobilized on the neutralvidin-coated surface. The surface is passivated with 0.2% Tween illustrated with gray vertical lines). This seed was elongated at its barbed end with 1.5 mM 15% Alexa 647-labeled Mg-ATP-actin (green), and Arp2/3 complex formed a branch with Alexa 647-labeled Mg-ATP-actin. The green filaments fluctuate freely and are subject to viscous drag forces applied by fluid flow. (B) TIRF microscopy images of representative branched filaments under slow flow (2 mL min-1) - 0.004 ph of force for a 1.5-mm branch; Trap) and fast flow (500 mL min-1, -1.02 ph of force for a 1.5-mm branch; Bottom). Branches are aligned in the direction of flow. (Scale bar, 1 mm). (C) The Arp5 subunit of the Arp2/3 complex was labeled with Alexa 488 via snap tag and tracked during debranching. Time-lapse images with the actin filaments represent in red and the Alexa 488-Arp2/3 complex located at the junction of the daughter branch and mother filament represented in green. (Scale bar, 1 mm). (D) Top shows the spatially integrated fluorescence intensity of actin at a branch junction as a function of time, used to determine the observed debranching event time (t = 0). Middle shows a kymograph measured across a branched actin filament. Bottom shows the time course of spatially integrated fluorescence intensity of Arp5 subunit at a branch junction with time aligned to its corresponding actin frame. The fluorescence intensity from Arp2/3 complex reproducibly decreased in a single step for all 12 debranching events observed.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} &'", "CAPTION FIG3.png": "'Figure 3: BaF\\\\({}_{x}\\\\) inhibits deharchung by GMF. [A] Dependence of the time course of dissociation of branches with ADP\u2013BaF\\\\({}_{x}\\\\)\u2013Arp2/3 complex on the concentration of GMF. Brandes with ADP\u2013BaF\\\\({}_{x}\\\\)\u2013Arp2/3 complex were assembled in buffer containing 0.2 mM ATP, 2 mM BaSO\\\\({}_{4}\\\\), and 10 mM NaF and aged for \\\\(\\\\sim\\\\)4 min. Branches with ADP\u2013Arp2/3 complex were assembled in buffer with 0.2 mM ATP and aged for 30 min to allow for ATP hydrolysis and phosphate dissociation (Fig. 2B). Deharchung was initiated by flowing buffer with GMF at 15 \\\\(\\\\mu\\\\)L\\\\(\\\\cdot\\\\)min\\\\({}^{-1}\\\\) and continued throughout the deharchung measurements. The smooth curves are the best fits of single exponentials to the data, yielding the (average) branch lifetimes; \\\\(\\\\alpha=30\\\\) branchas for each trace. A concentration of 1 \\\\(\\\\mu\\\\)M GMF did not dissociate branches with ADP\u2013BaF\\\\({}_{x}\\\\)\u2013Arp2/3 complex assembled from ATP-actin and ATP\u2013Arp2/3 complex with BaF\\\\({}_{x}\\\\) and aged for \\\\(\\\\sim\\\\)4 min. [B] Dependence of the lifetimes of branches with ADP\u2013Arp2/3 complex on the concentration of GMF at a buffer flow rate of 15 \\\\(\\\\mu\\\\)L\\\\(\\\\cdot\\\\)min\\\\({}^{-1}\\\\). The line is the best fit of Eq. 3 to the data, yielding a GMF, binding affinity (\\\\(K_{\\\\alpha,\\\\alpha\\\\alpha}\\\\)) of 40 (\\\\(\\\\pm\\\\)10) nM and a maximum deharchung rate, constant (\\\\(K_{\\\\alpha\\\\alpha\\\\alpha}\\\\)) of 0.31 (\\\\(\\\\pm\\\\)0.05) min\\\\({}^{-1}\\\\). At this low flow rate, branches with ADP\u2013Arp2/3 complex (without GMF) dissociated with a rate constant (\\\\(K_{\\\\alpha\\\\alpha\\\\alpha}\\\\) = 0.014 \\\\(\\\\pm\\\\) 0.0002 min\\\\({}^{-1}\\\\), indicated by an open circle) similar to that under zero force [Fig. 2B and Table 1]. The uncertainty bars are within the data points and represent the SDs of lifetimes in the best single exponential fits of time traces in \\\\(A\\\\).\\n\\n'", "CAPTION FIG4-1.png": "'Figure 4: Model and simulations of the pathways of branch formation, aging, and dabranching. (_A_) Schematic of our hypothesis. Formation of a branch by ATP\\\\(-\\\\)Arp2/3 complex (red) is coupled to hydrolysis of ATP bound to the Argos (5, 7) with rate constant \\\\(k_{\\\\rm force}\\\\), yielding ADP\\\\(-\\\\)Pi\\\\(-\\\\)Arp2/3 complex (orange) in state 1. Irreversible phosphate dissociation with rate constant \\\\(k_{\\\\rm force}\\\\) converts state 1 to ADP\\\\(-\\\\)Arp2/3 complex (blue) in state 2. Brandes dissociate from another filaments with rate constants \\\\(k_{1}\\\\) for state 1 and \\\\(k_{2}\\\\) for state 2, both sensitive to force. (_B_) Simulated time course of the model showing how the popularizers of state 1 branches, state 2 branches, and dissociated branches evolve over time in the absence of force. We assumed that Mg-ATP-actin-memoramers and Mg-ATP\\\\(-\\\\)Arp2/3 complex formed branches for 2.5 min, when the free proteins were removed, and the reactions continued without additional branch formation. The red line represents the best single exponential fit to the observed dabranching (i.e., combined from both states) starting with a normalized value of 1 at the end of branch formation (2.5 min). The experimentally determined or estimated rate constants used in the simulation are \\\\(k_{\\\\rm force}[\\\\Delta\\\\varphi]=0.12\\\\) min\\\\({}^{-1}\\\\), \\\\(k_{\\\\rm case}=0.14\\\\) min\\\\({}^{-1}\\\\), \\\\(k_{1}=0.012\\\\) min\\\\({}^{-1}\\\\), and \\\\(k_{2}=0.01\\\\) min\\\\({}^{-1}\\\\). (C) Aging time dependence of the fractional the slow and fast phase amplitudes in the dabranching time courses under 500 mL\\\\(\\\\cdot\\\\)min\\\\({}^{-1}\\\\) of buffer flow, obtained from double exponential fits to the time courses (Fig. 2B). These data were used for extraction of fundamental rate constants in Table 1. The best global fits of the two-state model (Eq. 2) to the fractional amplitude data gave rate constants for conversion \\\\(k_{\\\\rm force}\\\\) of 0.14 min\\\\({}^{-1}\\\\), state 1, branch dissociation \\\\(k_{1}\\\\) of 0.012 \\\\(\\\\varepsilon^{-1}\\\\), and branch formation \\\\(k_{\\\\rm force}\\\\) of 0.02 \\\\(\\\\mu\\\\)M\\\\({}^{-1}\\\\), \\\\(\\\\varepsilon^{-1}\\\\) without force (Table 1). The one negative fast phase amplitude at 2.5 min results from a net increase in the state 2 branch population during\\n\\n'", "CAPTION FIG4-2.png": "'deharching under force after 2.5 min aging time. The state 2 branch population is the net sum of depletion from deharching (negative contribution to population, exponential decay) and gain from conversion of state 1 branches (positive contribution, exponential rate). For short aging times, little or no state 2 branches exist for deplete, and the conversion from state 1 branches, represented by an exponential rise (negative amplitude), dominates the time course (54, 55). The uncertainty bars are SDs of the fractions of branches from the global double exponential fits of deharching time courses with different aging times in Fig. 2B.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Model and automation of the pathway of branch formation, aging, demonstrating under force after 2.8 min aging time. The state 2 branch and adaptability, (3) demonstrate of our hypothesis. Formation of a branch by rapid adaptation in the net sum of depletion from the glutathione (negative center-ATP-spill) complex both occupied to moderate and high-resolution (3). ATP-spill complex both occupied to moderate and high-resolution (3). ATP-spill complex (orange) is 0.7 with rate constant. Also, feedback from the high resolution, exponential decay and gain from conversion of state 1.7 through single degradation dissociation with rate constant. Zero-passive depletion (red) is 0.5 with rate constant. Zero-passive depletion (red) is 0.8 with rate constant. Zero-passive depletion (red) is 0.8 with rate constant.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/pedersen2023endocytic.json b/test_extracted_captions/pedersen2023endocytic.json new file mode 100644 index 0000000000000000000000000000000000000000..450998360054619a85a9767b82641785bf7b207c --- /dev/null +++ b/test_extracted_captions/pedersen2023endocytic.json @@ -0,0 +1 @@ +{"CAPTION FIG3-2.png": "'measured total displacement of Myo5 was 5.0 nm at 10 \\\\(\\\\mu\\\\)M ATP, with the first _sub_step contributing a 4.8 nm displacement (arrow 1 m G) and the second _sub_ contributing a 0.2-nm displacement (arrow 2 m G). **(F-H)** Left: Forward-averaged ensembles synchronized at the beginning of events. Right: Reverse-averaged ensembles synchronized at the ends of events. Black and gray lines are single exponential fits in the forward and reverse ensembles, respectively. **(I)** Cumulative distributions of attachment durations for Myo5 at 1.10, and 1,0.00 \\\\(\\\\mu\\\\)M ATP. Blue lines show the cumulative frequency of attachment durations at the indicated ATP concentrations, and the red, yellow, and green lines indicate fitted exponential distributions at 1, 10, and 1,0.00 \\\\(\\\\mu\\\\)M ATP, respectively. 1 and 10 \\\\(\\\\mu\\\\)M ATP were fit well to single exponentials, and the 1,0.00 \\\\(\\\\mu\\\\)M ATP data were best described by the sum of two exponentials. **(J)** Summary of rates at 1, 10, and 1,0.00 \\\\(\\\\mu\\\\)M ATP calculated from F-H. Blue boxes are the fitted exponential distributions from I, black diamonds are forward ensemble fits from F-H (left), and gray diamonds are reverse ensemble fits from F-H (right). At lower concentrations of ATP (I and 10 \\\\(\\\\mu\\\\)M), the rate of detachment is limited by ATP association, corresponding to the reverse ensemble fits, while at saturating ATP concentration [L000 \\\\(\\\\mu\\\\)M], the detachment rate is limited by the rate of ADP dissociation, corresponding to the forward ensemble fits. **(K)** Summary of rates determined via single-molecule optical trapping. Errors for detachment rates are 95% confidence intervals. Errors for forward and reverse ensemble fits are standard errors of the fits. \"Detachment rates at 1,0.00 \\\\(\\\\mu\\\\)M ATP were best fit to the sum of two exponents. The major component of the fit (67.8 s-3) comprises 92.1% of the total with the remaining 7.9% having a rate of 11.6 s-3.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Myo5 attachment lifetimes are substantially less force-dependent than other known type I myosins.** An isometric optical force clamp was utilized to determine the force sensitivity of the detachment of _Myo5_ from actin. **(A)** Durations of individual actomyosin attachments as a function of force, plotted on a semi-log scale. **(B)** The solid black line shows the force dependence of the detachment rates determined by MLE fitting of unaveraged points in A. For illustration purposes, attachment durations from A were binned by force at every 10 points, averaged, and converted to rates. Best-fit parameters were determined by MLE fitting and 95% confidence intervals were calculated via bootstrapping. The solid black line is calculated from best-fit parameters (k = 67.6 s-1, d = 114 nm), while the gray shaded region is the 95% confidence interval [k = 62.4-72.9 s-1, d = 1.03-126 nm]. All MLE fitting was performed on unaveraged data and was corrected for instrument deadtime. **(C)** The force dependent detachment rate of Myo5 (from B) plotted alongside the force dependent detachment rates for Myo1b, Myo1c, and B-cardiac muscle myosin, Myh7. **(D)** Power output for the same four myosins is calculated over a range of forces by multiplying the functions from C by the applied force F, and the step size and duty ratios of each myosin.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: **Single molecule, optical trap analysis of Mryo5 step size and kinetics.****(A)** Cartoon schematic of the three-head optical trapping setup. A biotinylated actin filament is tethered between two neutrardin-coated beads that are trapped in a dual-beam optical trap. This bead-actin-bead \"dumbbell\" is lowered onto pedestal beads that have been sparsely coated with \\\\(\\\\text{H}_{\\\\text{ls}}\\\\), antibody to attach Mryo5-motor/lever-AuI-Tev-Hls, **(B\u2013D)** Single Mryo5 displacements for a single bead position and covariance traces, calculated using both beads, showing single molecule interactions acquired in the presence of 1 \\\\(\\\\mu\\\\)M(B) 10 \\\\(\\\\mu\\\\)M (C) and 1,000 \\\\(\\\\mu\\\\)M ATP **(D)**. Blue bars indicate attachment events as identified by covariance (gray) decreases. The threshold of event detection by the covariance traces are indicated by dashed gray lines. **(E)** Schematic of displacement traces depicting the two-step nature of actomyosin displacements in the optical trap. **(F\u2013M)** Binding events were synchronized at their beginnings (left) or ends (right) and averaged forward or backward in time, respectively. The\\n\\n'", "CAPTION FIGS1.png": "'Figure S1: **P21-activated Kinase 1 (PaKi) phosphorylates _My_oS on **5357**. Crude preparations of wild type and S357A MyoS motor/_lever_ constructs were mixed with 250 \u03bcM ATP including 20 \u03bcM of ATPvP32 in kinase assay buffer (5 mM MOPS pH 7.2.5 mM B-glycerophosphate, 5 mM MgCl4, 400 \u03bcM EDTA, 1mM EGTA, and 50 \u03bcM DTT) in either the presence or absence of PaKi. Reactions were incubated at 25degC for 60 min, then quenched by adding an equal volume of 2-3 Tris urea sample buffer (125 mM Tris pH 6.8, 6 M urea, 205 SDS, 0.13 bromophenol blue, 100% B-mercaptoethanol) and resolved on a 10% polyacrylamide gel. The gel was stained with Comosaic, then dried onto Whatman paper and exposed to a storage phosphor screen (Amersham). The Comosaic-stained gel was imaged on a standard photo scanner and the phosphor screen on a Typhoon gel imager (Amersham). Note that there are differences in baseline labeling in the absence of added kinase between the two different protein preps, but the addition of PaKi clearly results in radiolabeling of wild type but not mutant MyoS.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: **Models for the functions of actin assembly and myosin activity during membrane deformation for clathrin-mediated endocytosis.** Cartoon diagram illustrating the organization of actin filaments and Noyos molecules at endocytic sites. Actin filaments are bound by coat proteins at the tip of the growing membrane invagination and oriented with their growing ends toward the plasma membrane, powering membrane invagination. The type I myosin Myos could either anchor the actin network in a favorable orientation (left) or provide an assisting force (right).\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: **In-solution, population biochemical characterization of Myes.****(A)** Comaxise-stained SDS-polyacrylamide gels showing example preparations of the purified Myes0 motor/lewer construct and calmodulin (Cmidi, light chain) used in all experiments. **(B)** The actin concentration dependence of the steady-state ATPase activity of 100 nM unphosphorylated (gray circles) and phosphorylated _Myes_5 (black circles). Each data point represents the average of 6\u20137 time courses, which were 100 s each. The orange line is the best fit of the phosphorylated _Myes_5 data to a rectangular hyperbola. **(C)** Schematic pathway for the Myes ATPase cycle. Blue motors are in tightly bound conformations and green motors are weakly bound/unbound. **(D)** Example of light scattering transients reporting on ATP-induced dissociation of phosphorylated (left, \\\\(k_{obs}\\\\) = 17 S\u22121) and unphosphorylated (right, \\\\(k_{obs}\\\\) = 64.1 S\u22122) act_Myes_05, obtained by mixing 100 nM act_Myes_ (AM) with 94 and 72 \u03bcM ATP, respectively, as shown in the inset schematic. The black line is the fit of a single exponential function to the data. **(E)** ATP\\n\\n'", "CAPTION FIG2-2.png": "'concentration dependence of dissociation of 100 nM unphosphorylated (gray circles) and phosphorylated arctaMy5 (black circles). Each data point represents 3-6 time courses averaged and fit to a single exponential decay function. The orange line is a linear best fit of the phosphorylated Myo5 data. The purple line is the best fit of the unphosphorylated Myo5 data to a rectangular hyperbola. **(f)** Example light scattering transients reporting ATP-induced dissociation of ADP-saturated phosphorylated (left) and unphosphorylated (right) arctaMy5, obtained by preinonubating 200 nM actaMy5 (AM) with 100 \\\\(\\\\mu\\\\)M ADP, then mixing rapidly with 2.5 mM ATP, as shown in the inset schematic. The black line is the fit of a single exponential function to the data. **(c)** Velocity of actin filament gliding, measured at varying surface densities of Phospho-My5 (black circles, orange line) and unphosphorylated Myo5 (gray circles, purple line) in in vitro motility assays. _Myosin_ concentrations indicate the quantity of protein incubated in the flow chamber before washing. Each data point represents the average velocity of 30-60 filaments, and the error bars are standard deviations. Source data are available for this figure. SourceData F2.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K. (1989) The mathematical theory of the mathematical theory'"} \ No newline at end of file diff --git a/test_extracted_captions/purcellDesignOtherTypes1996.json b/test_extracted_captions/purcellDesignOtherTypes1996.json new file mode 100644 index 0000000000000000000000000000000000000000..578933af882105c525cc4c99960f88faad681996 --- /dev/null +++ b/test_extracted_captions/purcellDesignOtherTypes1996.json @@ -0,0 +1 @@ +{"CAPTION TAB5.png": "'\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Illustrations of five bicycle rack designs.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 3: Number of designs and number of solution types produced by the experimental and control groups in two design disciplines for the bath access problem, A utolift fixating example.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Illustrations of the two example designs of devices for use by the blind in measuring quantities for cooking.\\n\\n'", "CAPTION 3.png": "'Figure 3: Illustrations of four devices designed to assist the elderly in entering and leaving a bath.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} \\\\\\\\ \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}\\n\\n'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l l} \\\\hline \\\\hline & & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Frequencies of solution types for the bath access problem produced in the experimental and control groups in two design disciplines, Autolift fixating example.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/reinhardt2023angstromresolution.json b/test_extracted_captions/reinhardt2023angstromresolution.json new file mode 100644 index 0000000000000000000000000000000000000000..0c72bf573aea34d704b0b6c215a93ce585d6f581 --- /dev/null +++ b/test_extracted_captions/reinhardt2023angstromresolution.json @@ -0,0 +1 @@ +{"CAPTION FIGS13.png": "'\\n\\n**Extended Data Fig. 13** **Comparison of Rituximab treated CD20 data simulated CD20 hexamer arrangements.** A. Example of ground truth simulated CD20 hexamers (light blue circles, simulated as triangles of dimers with intra-dimer distances of 13.5 nm as measured experimentally) with random distribution and orientation on a 2D surface at the experimentally determined density. **b.** Label uncertainty and labeling efficiency (black circles indicate labeled molecules) are taken into account in the simulation for a realistic comparison. **c.** Simulated proteins in hexameric arrangements represented as gaussians. **d**, Hexamers after DBSCAN cluster analysis (colors indicate clusters). **e.** RES1 image of CD20 data after RTX treatment. **f.** RES1-localizations of CD20 data after DBSCAN cluster analysis (colors indicate clusters). **g.** Number of molecules per detected cluster for the experimental data and the simulated hexamers. **h.** Circularity metric of experimental data and the simulated hexamers after convex hull analysis of the clusters. We note that the sharp drop at 0.605 stems from the maximum circularity metric for clusters where the convex hulls defined by three molecules. Notably, the absence of a circularity peak at -0.7 in the experimental data suggests that CD20 molecules are not arranged in isolated ring-like hexameric structures.\\n\\n'", "CAPTION TABS1.png": "'\\n\\n**Extended Data Table 1 -- Imaging and RESil parameters**'", "CAPTION FIGS9.png": "'\\n\\n**Extended Data Fig. 9 (Sub-nm RES1 measurements. a, DNA origami featuring six alignment strands (green R4) and six pairs of orthogonal docking strands (red R1, blue R3) spaced one base pair apart. b, RES1 representation with RES1-localizations from round 1 in red and round 2 in blue illustrates excellent alignment. The distances between RES1-localizations from round 1 and 2 are defined as illustrated. c, Overlaying 42 DNA origami and performing a particle average recovers the structure with an alignment uncertainty of 1.2\\\\(\\\\Delta\\\\)CI = [0, 4, 6]A, showing distances between the average positions of the sites at 9.5\\\\(\\\\pm\\\\)2.6A (mean over six distances in the average \\\\(\\\\pm\\\\) mean over the error-propagated uncertainties of the six distances). Same scale applies to all magnification panels. CI describes 68% confidence interval.**'", "CAPTION FIGS14.png": "'\\n\\n**Extended Data Fig. 14** **[Stochastic labeling.****a**, Exemplary simulation of proteins with a Complete Spatial Random (CSR) distribution of a given density. **b**, Histogram of Nearest Neighbor Distances (NNDs). The red line indicates the smallest distance (_d_) that can be resolved by DNA-PAINT for a given set of imaging parameters. The fraction of molecules with a NNbelow this distance threshold (blue, shaded) can be computed for a given density and a given DNA-PAINT resolution. **c**, 2D map of the fraction of non-resolvable molecules as a function of density and resolution. **d**, 1D cuts of a different resolutions (color-coded) can be used as a user guide to estimate the number of multiplexing rounds needed to perform RESF efficiently given a certain target fraction of non-resolvable distances.\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: **ISESI resolves the distance of single DNA base pairs at Angstrom resolution.****a**, DNA origami with docking strands spaced by a single base pair (bp;red and blue strands, with alignment markers in green) provided a platform to demonstrate the highest resolution achievable by RSI. **b**, DNA-PAINT resolved 20 nm spacing but the resolution was insufficient to distinguish individual docking sites, spaced one base apart. **c**, RESI resolves the adjacent docking strands. **d**, **d** Euclidean distance of \\\\(8.5\\\\pm 1.7\\\\) \u00c5 was calculated from individual localizations with an average precision of \\\\(1.2\\\\,\\\\text{\\\\AA}\\\\) (left) for the single-base-pair backbone distance, which is within \\\\(1\\\\,\\\\text{s.d.}\\\\) of the expected value of roughly \\\\(7\\\\,\\\\text{\\\\AA}\\\\) (right). **e**, Experimental localization precision in RESI is in good agreement with \\\\(\\\\frac{\\\\text{\\\\Delta}\\\\text{\\\\Delta}\\\\text{\\\\Delta}\\\\text{\\\\Delta}\\\\text{\\\\Delta}\\\\text{ \\\\Delta}\\\\text{\\\\Delta}\\\\text{\\\\Delta}\\\\'", "CAPTION FIG4.png": "'\\nFig. 4: **RESIs shows CD20 receptor (re)organization at subnanometre precision following drug treatment.****a**, Diffraction-limited and DNA-PAINT overview image of CHO cells expressing mEGFP-CD20 labelled with anti-GFP nanobodies. **b**, Zoomed+in DNA-PAINT image showing apparently randomly distributed CD20 receptors for untreated cells (top) and clustered receptor arrangement for RTK-treated cells (bottom). **c**, Comparison of DNA-PAINT and RESI for both untreated and RTK-treated cells showing sub-10-nm-spaced receptor pairs in the RESIimages, which are unresolvable with DNA-PAINT. **d**, RESI data suggest that CD20 proteins occur in dimers (spaced at \\\\(d_{\\\\text{trans}}\\\\)), which are in turn distributed according to complete spatial randomness (CSR; distances between dimers, \\\\(d_{\\\\text{trans}}\\\\)) in untreated cells. Chains of dimers were observed following administration of RTX. **e**, Whole-cell analysis of first NNDs of CD20 receptors (histograms of distances and kernel density estimation are shown). Only RESI, but not DNA-PAINT, allows the routine detection of sub-10-nm distances between proteins. Whereas DNA-PAINT overestimates dimer distance, RESIs shows allele-limited distance of 13.5 nm (see main text for discussion), **f**, Fitting RESINND data from to a numerical model reveals CD20 dimers and monomers. **g**, CD20 receptors in untreated cells showed second to fourth NNDs consistent with CSR, thus excluding the presence of higher-order protein complexes. **h**, CD20 receptors in RTK-treated cells, however, showed first to fourth NNDs, inconsistent with complete spatial randomness.\\n\\n'", "CAPTION FIGS12.png": "'\\n\\n**Extended Data Fig. 12 RESF reveals higher order arrangement of CD20 dimers in Rituximab-treated CHO cells.****a**, DNA-PAINT imaging of whole mEGFP-CD20-expressing CHO cells, labeled with anti-GFP-nanobodies, shows clustered CD20- molecules in Rituximab-treated cells for three independent experiments. **b**, Zoom-in regions of DNA-PAINT show mEGFP-CD20 clustered into chain-like arrangements. **c**, RESF reveals sub-10-nm spaced receptor pairs within the clusters, unresolvable by DNA-PAINT. **d**, Whole-cell analysis of first nearest neighbor distances (12-NNDS) of CD20 receptors bound to Rituximab-inhibitor diagrams of the distances are displayed. Only RESI, but not DNA-PAINT, allows the routine detection of sub-10-nm distances between proteins. **e**, Routine detection of sub-10-nm distances by RESI recapitulates the first NND measured in the untreated case. Notably the NND peaks measured in the three repeats are consistent, independently of the protein density.\\n\\n'", "CAPTION FIGS10.png": "'\\n\\n**ExtendedDataFig. 10**: **RES1resolves CD20 dimers in untreated CHO cells for different expressionlevels.****a**, DNA-PAINT imaging of whole mEGFP-CD20-expressing CHO cells, labeled with annt-GFP-nanobodies, shows homogeneously distributed molecules for three independent experiments. **b**, Zoom-in regions of DNA-PAINT show cases in which dimers could not be resolved. **c**, RES1reveals sub-10-nmspaced receptor pairs, which are unresolvable in the DNA-PAINT cases. **d**, Whole-cell analysis of first nearest neighbor distances (12+- NNPs) of CD20 receptors (histograms of the distances are displayed). Only RES1, but not DNA-PAINT, allows the routine detection of sub-10-nm distances between proteins. **e**, RES1-localization precision below 1 nm allows for routine detection of sub-10-nm distances, resulting in an accurate assessment of the first NND.\\n\\n'", "CAPTION FIGS8.png": "'\\n\\n**Extended Data Fig. 8 [Sub-nm DNA origami].** Representative DNA origami from across the field of view of the measurement. **a**, Four DNA origami, shown at DNA-PAINT resolution (upper row) and RESI resolution (lower row). The inserts show pairs of directly adjacent docking strands resolved by RESI. **b**, 42 additional DNA origami, shown at DNA-PAINT resolution (upper row) and RESI resolution (lower row).\\n\\n'", "CAPTION FIG1.png": "'\\nFig.1: **REStConcept.****a**, in SM.**M**, \\\\(\\\\alpha_{\\\\text{max}}\\\\)**of a single dye scales with \\\\(\\\\frac{\\\\text{target}}{2\\\\pi}\\\\) ultimately limiting the achievable spatial resolution. **b**, SMIM approaches such as DNA-PAINT feature approximately 10 nm spatial resolution (resolution approximated as full-width at half-maximum = 2.35 \\\\(\\\\alpha_{\\\\text{max}}\\\\)). Whereas targets separated by 20 nm (d); **c** than thus be routinely resolved, objects spaced 2 nm apart (d) are unresolvable because the resulting distributions of localizations overlap. **c**, Using orthogonal DNA sequences (blue and green) and sequential acquisition as in Exchange-PAINT, localizations from targets spaced more closely than the SMIM resolution limit can be unambiguously assigned for each target. **d**, Combining all localizations per target (\\\\(K\\\\)) for each imaging round improves localization precision froms. \\\\(\\\\text{d}\\\\) (\\\\(\\\\alpha_{\\\\text{max}}\\\\)) tos. **e**. **m**, **e**. As super-resolution revolutionized fluorescence microscopy, RESI results in another paradigm shift by reapplying the concept of localization to super-resolution data. **f**, Localization precision in RESI scales with \\\\(\\\\frac{1}{2\\\\pi}\\\\) and thus resolution improvement in RESIs independent of \\\\(\\\\alpha_{\\\\text{max}}\\\\) reaching localization precision on the Angstrom scale.\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n**Extended Data:** **1** **RES1resolution estimation.a**, **A**, **A**grid of defined positions of binding sites is generated (top left), SNLM (DNA-PANT) decalizations are simulated as examples from a gaussian distribution (top right). Localizations for only one binding site were plotted for clarity. For each binding site, subsets of **K** localizations are randomly selected (bottom left) and averaged (bottom right). One exemplary subset and its average is highlighted. **b**, **Resulting**, **RES1-localizations are histogrammed to produce images at different resolutions (\\\\(K\\\\) values). **c**, **RES1-localization** precision \\\\(\\\\alpha_{\\\\text{mvs}}\\\\)K. Analytical dependence on \\\\(\\\\sqrt{K}\\\\) (blue line) and numerical results (black dots). A total of 1200 SNLM localizations per site are simulated. Error bars represent mean +- 1s.d.\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n**Extended Data Fig. 3 12D DNA origami.** Representative DNA origami from across the field of view of the measurement. **a**, Four DNA origami, shown at DNA-PAINT resolution (upper row) and REST resolution (lower row). The insert depicts a pair of docking strands spaced at approx. 5 nm. **b**, 40 additional DNA origami, shown at DNA-PAINT resolution (upper row) and REST resolution (lower rows).\\n\\n'", "CAPTION FIGS2.png": "'\\n\\n**Extended Data Fig. 2**: **2** **(RES11 2D DNA origami. a, DNA origami design featuring six 5 nm-spaced orthogonal docking strand pairs (red R1, blue R3) and six alignment docking strands (green R4). See Methods for sequence details. **b**, DNA-PAINT acquisition parameters were tuned such that 5 nm were not consistently resolvable. **c**, First imaging round conducted with R1 (target) and R4 imagers (alignment, sites circled). **d**, Second imaging round conducted with R3 (target) and alignment imagers (R4, sites circled). The alignment sites were used for translational and rotational alignment between rounds. **e**, RES1 resolves the 5 nm distances. **f**. The distance and orientation between R1 and R3 docking strands are consistent with the design. **g**. An average of 90 DNA origami structures reveals consistent results and excellent alignment performance. The numbers indicate the distance between rounds.\\n\\n'", "CAPTION FIGS7.png": "'\\n\\n**Extended Data Fig. 7.1 Averaging of Nup96 proteins.****a**, Model-free averaging for DNA-PAINT measurements of Nup96 (N = 1045mCs). An angled isometric view is shown. **b**-**d**, DNA-PAINT resolves nucleoplasmic and cytoplasmic rings and recapitulates their eight-fold symmetry, but fails to resolve individual Nup96 proteins. **e**, Side views of all Nup96 pairs in both rings reveal the angled orientation but do not resolve individual Nup96 proteins. **f**, Model-free averaging for RES measurements of Nup96 (N = 1190mNCs). **g**-**i**, RESIC recapitulates nucleoplasmic and cytoplasmic rings as well as their eight-fold symmetry and resolves individual adjacent Nup96 proteins in the majority of cases. **j**, Side views of all eight Nup96 pairs in both rings reveal the angled orientation as well as, in some cases, adjacent in individual Nup96 proteins. **k**, The Cryo-EM structure of the nuclear pore complex indicates that a given Nup96 protein will have neighbors spaced at 11 mm, 39 nm, 71 mm, 93 mm and 101 mm. **l** Performing clustering and nearest neighbor analysis for DNA-PAINT data reveals a peak at approx. 40 nm, corresponding to the distance between two Nup96 pairs, but not below that. RESIC, on the other hand, features a first peak at approx. 15 nm, corresponding to the distance between adjacent Nup96 while taking linkage error (label size) into account. **m**, Analysis of first to tenth nearest neighbor distances for RES1 and DNA-PAINT recapitulates the distances from (**k**), but only RESIC resolves the smallest distance. All scale bars: 20 nm.\\n\\n'", "CAPTION FIGS4.png": "'\\n\\n**Extended Data Fig. 4**: **iESI ln 3D DNA origami.****a**, DNA origami design featuring one pair of orthogonal docking strands (red RL, blue R3) as well as six alignment docking strands (green R4). Docking strands extend from both the top and bottom surface of the DNA origami (insert). **b**, The design ensures that all but the RL/R3 docking strand pair are spaced sufficiently to be resolved by DNA-PAINT. **c**, 3D DNA-PAINT imaging resolves R4 alignment sites, barely resolves R1/R3 axially and does not resolve R1/R3 laterally. **d**, Sequential 3D DNA-PAINT imaging with R4 sites used for alignment. **e**, RESI resolves R1/R3 both axially and laterally. **f**, An overlay of 88 DNA origami reveals overall good alignment despite structural heterogeneity. **g**, Average of 88 DNA origamis. **h**, The particle average recovers the structure with an alignment uncertainty of 0.7 nm CI = [0,1.6] nm, showing a distance between the average R1/R3 positions of IL6 +- 0.8 nm (xy-distance: 2.5 +- 0.4 nm, \\\\(z\\\\) distance: 11.3 +- 0.8 nm), matching the designed distances30. Same scale applies to all magnification panels. CI describes 68% confidence interval.\\n\\n'", "CAPTION FIGS6.png": "'\\n\\n**Extended DataFig. 6 (U2OS Nup96-mEGFP, Representative NPCs from across the field of view of the measurement.****a**, Six NPCs, measured using DNA-PAINT (upper row) and RESI (lower row). The color scale to the right represents the z position of localizations. The measured z coordinates for each NPC have been shifted by a constant such that the lowest localization for a given structure is defined to be at z = 0. This ensures full use of the color range. **b**, 72 additional NPCs, measured using DNA-PAINT (upper rows) and RESI (lower rows). The z positions are colored according to the color scale in panel **a**.\\n\\n'", "CAPTION FIG2.png": "'\\nFig. 2: **NPC proteins in whole cells resolved with Angstrom precision by RESL.**a. Diffraction-limited and DNA-PAINT overview image of Nup96-mEGFP labelled with DNA-conjugated anti-GFP nanodiodes. Zoomed-in view (bottom right) shows high labelling efficiency and image quality for standard DNA-PAINT conditions, recapitulating the eight-fold symmetry of the NPC. **b.** Cryo-EM structure representation of the location of Nup96 proteins (red; C-terminal mEGFP position marked in blue) as part of the Y-complex in nuclear and cytoplasmic rings (NR and CR, respectively). Adapted from PDB JPEG. Nup96is present in 32 copies per NPC. **c.** To enable RESL, Nup96-mEGFP proteins were stochastically labelled with orthogonal DNA sequences by incubation of the sample with anti-GFP nanobodies, each conjugated with one of four orthogonal sequences (represented by blue, yellow, magenta and green dots). **d.** Sequential 3D imaging (colour represents \\\\(z\\\\) position) of the four labels yielded sufficiently spaced localization distributions. The average number of localizations per target is \\\\(K_{\\\\text{nup96}}\\\\) = 38 (background represents cryo-EM structure from \\\\(b\\\\) for context). **e.** Comparison of 3D DNA-PAINT (top left) and 3D RESL (bottom right) for the same NPC illustrating improvement in spatial resolution by RESL. Localizations are rendered as gaussians with \\\\(\\\\sigma_{\\\\text{max}}\\\\) and \\\\(\\\\sigma_{\\\\text{max}}\\\\), respectively. **f.** Localization precision (\\\\(\\\\sigma_{\\\\text{max}}\\\\)) as good as \\\\(S\\\\)A was achieved by combining \\\\(K\\\\)localizations for each target, unambiguously resulting single Nup96 proteins. **g.** The 3D NPC cryo-EM structure was recapitulate using optical microscopy by applying a model-free average of 1,217 NPCs from a single nucleus. **h.** RESL resolved adjacent Nup96 in a structural average by optical microscopy. **l.** Consistent with the cryo-EM structure (taking into account linkage error arising from label size), adjacent Nup96 proteins were spaced \\\\(11.9\\\\pm 1.2\\\\) nm apart laterally (top) and \\\\(5.4\\\\pm 0.4\\\\) nm axially (bottom).\\n\\n'", "CAPTION FIGS5.png": "'\\n\\n**Extended Data Fig. 5**: **3D DNA origami.** Representative 3D DNA origami from across the field of view of the measurement. **a**, Five DNA origami, shown at DNA-PAINT resolution (upper row) and RESI resolution (lower row). The color scale to the right represents the z position of localizations. The measured z coordinates for each DNA origami have been shifted by a constant such that the lowest localization for a given structure is defined to be at z = 0. This ensures full use of the color range. **b**, 32 additional DNA origami, shown at DNA-PAINT resolution (upper rows) and RESI resolution (lower rows). The z positions are colored according to the color scale in panel **a**.\\n\\n'", "CAPTION FIGS11.png": "'\\n\\n**Extended Data Fig. 11**: **RES1resolves the substructure in RTX-induced chain-like arrangements of CD20 receptors with sub-nanometer precision.****a**, DNA-PAINT overview image of mEGFP-CD20 expressing CHO cells treated with RTX. **b**, Labeling with DNA-conjugated anti-GFP-nanobodies and imaging with DNA-PAINT reveals higher-order organization after RTX-treatment. RES1 (insets1-iii) achieves molecular resolution and thereby resolves the molecular arrangement of mEGFP-CD20. **c**, DNA-PAINT imaging shows clustered CD20 molecules. Performing RES1 with sequences R1, R2, R3 and R4 in four separate imaging rounds (color-coded) allows for clustering of localizations originating from a single target. From the clustered localizations, RES1-localizations were calculated, enabling true single-protein resolution.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/rhyscoxDirectedDiversityLeveraging2021a.json b/test_extracted_captions/rhyscoxDirectedDiversityLeveraging2021a.json new file mode 100644 index 0000000000000000000000000000000000000000..2a74000d0b14556498a3c31f3529552a5b45f25d --- /dev/null +++ b/test_extracted_captions/rhyscoxDirectedDiversityLeveraging2021a.json @@ -0,0 +1 @@ +{"CAPTION TAB14.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 14: The rotated factor loading of factor analysis on metrics of perceived quality of the generated messages. Factors explained 80.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\gamma^{2}\\\\)= 5810, p.\\\\(\\\\sim\\\\)0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 14: The rotated factor loading of factor analysis on metrics of perceived quality of the generated messages. Factors explained 80.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\gamma^{2}\\\\)= 5810, p.\\\\(\\\\sim\\\\)0001).\\n\\n'", "CAPTION TAB15.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB16.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 16: The rotated factor loading of factor analysis on metrics of message distinctness. Factors explained 74.8% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1022, p-.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 16: The rotated factor loading of factor analysis on metrics of message distinctness. Factors explained 74.8% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1022, p-.0001).\\n\\n'", "CAPTION FIG12.png": "'Figure 12: Influence of prompt selection technique, prompt size, and prompt count on various distance and diversity metrics. Higher values for all metrics indicate higher diversity. Span and Sparseness results are not shown, but are similar to Mean Distance. Note that we computed the mean of MST edge distances instead of sum, which is independent of number of prompts. Error bars are extremely small, and not shown for simplicity.\\n\\n'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 10: The rotated factor loading of factor analysis on metrics of prompt distance and consistency. Factors explained 73.6\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 5810, p\\\\textless{}0.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 10: The rotated factor loading of factor analysis on metrics of prompt distance and consistency. Factors explained 73.6\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 5810, p\\\\textless{}0.0001).\\n\\n'", "CAPTION TAB11.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 11: The rotated factor loading of factor analysis on metrics of perceived helpfulness of prompts. Factors explained 68.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 2575, p\\\\textless{}.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 11: The rotated factor loading of factor analysis on metrics of perceived helpfulness of prompts. Factors explained 68.9\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 2575, p\\\\textless{}.0001).\\n\\n'", "CAPTION TAB6.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 6: **Independent variables used in the simulation and user studies to manipulate how prompts are shown to ideators.**'", "CAPTION FIG7.png": "'Figure 7: Results of computed individual and collective diversity from ideations for different prompt configurations. See Figure 6 caption for how to interpret charts.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: **Results of diversity in categories and themes derived from thematic analysis of ideations.**\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Pipeline of the overall technical approach to extract, embed, and select phrases to generate diverse prompts. a) Phrase extraction by collecting phrases from online articles and discussion forums (shown as pages), filtering phrases to select a clean subset (shown as the black dash for each phrase); b) Phrase embedding using the Universal Sentence Encoder [14] to compute the embedding vector of each phrase (shown as scatter plot); c) Phrase Selection by constructing the minimal spanning tree to select optimally spaced phrases (see Figure 2 for more details).\\n\\n'", "CAPTION FIG19.png": "'Figure 19: **Ideators are asked to evaluate the message they wrote by providing Likert scale ratings for many different factors along with a short reflection about the message writing process. The screenshot above shows the evaluation screen for Directed(3).**\\n\\n'", "CAPTION FIG20.png": "'\\n\\n**Figure 20: The instruction for individual message rating tasks.**'", "CAPTION FIG17.png": "'Figure 17: The instructions of Ideation User Study for the Random(3) and Directed(3) conditions.\\n\\n'", "CAPTION FIG18.png": "'Figure 18: Random(3) and Directed(3) prompts consist of three phrases per prompt. Note that selected phrases for each trial will be different.\\n\\n'", "CAPTION FIG21.png": "'Figure 21: **Validators rated a randomly selected message on a Likert scale and gave a justification.**\\n\\n'", "CAPTION FIG22.png": "'\\n\\n**Figure 22: The instruction for group message ranking tasks.**'", "CAPTION FIG15.png": "'Figure 15: The instructions of Ideation User Study for the Random(1) and Directed(1) conditions.\\n\\n'", "CAPTION FIG16.png": "'Figure 16: Random(1) and Directed(1) prompts consisted of one phrase per prompt. Note that selected phrase for each trial will be different.\\n\\n'", "CAPTION TAB17.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 17: The rotated factor loading of factor analysis on metrics of ideation effort. Factors explained 59.0\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1008, p\\\\(\\\\sim\\\\)0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 17: The rotated factor loading of factor analysis on metrics of ideation effort. Factors explained 59.0\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1008, p\\\\(\\\\sim\\\\)0001).\\n\\n'", "CAPTION FIG13.png": "'Figure 13: The instructions in the Ideation User Study for the None condition.\\n\\n'", "CAPTION FIG14.png": "'Figure 14: For the None, users are asked to write a message that is at least one to three sentences long.\\n\\n'", "CAPTION FIG23.png": "'Figure 23: **Validators were asked to rank groups of messages for motivation, informativeness and repetitiveness. Note that while we used the word \u201crepetitive\u201d for usability in the survey, we analyzed this dependent variable as \u201cunrepetitive\u201d to be consistent with other diversity metrics.**\\n\\n'", "CAPTION FIG24.png": "'Figure 24: Validators were asked to rate the difference of two messages in a message-pair.\\n\\n'", "CAPTION TAB18.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 18**: Messages generated in our study and the phrase prompt(s) that were shown to ideators.} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 18: Messages generated in our study and the phrase prompt(s) that were shown to ideators.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Demonstration of pairwise embedding angular distances between an example text items (first data row) and neighboring text items. Text items with semantically similar words have smaller distances. For interpretability, we highlighted words to indicate darker color with higher cosine similarity to the first phrase.\\n\\n'", "CAPTION TAB19.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 19: **Themes and categories identified with the qualitative coding of ideated messages.**'", "CAPTION TAB20.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB21.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 21: Statistical analysis and results of mediation effects (RQ2.3) of how prompt configurations (a) and perceived prompt creativity (b) affect ideation diversity. See Table 20 caption to interpret tables. Positive and negative numbers in second column represent estimated model coefficients indicating how much each fixed effect influences the response.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 21: Statistical analysis and results of mediation effects (RQ2.3) of how prompt configurations (a) and perceived prompt creativity (b) affect ideation diversity. See Table 20 caption to interpret tables. Positive and negative numbers in second column represent estimated model coefficients indicating how much each fixed effect influences the response.\\n\\n'", "CAPTION TAB22.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 22: Statistical analysis of how prompt selection influences ideation diversity defined by different metrics (RQ3): a) individual diversity, b) collective diversity, and c) thematic diversity. See Table 20 caption for how to interpret tables.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 22: Statistical analysis of how prompt selection influences ideation diversity defined by different metrics (RQ3): a) individual diversity, b) collective diversity, and c) thematic diversity. See Table 20 caption for how to interpret tables.\\n\\n'", "CAPTION TAB23.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & **Table 23**: **Statistical analysis of how prompt selection influences ideation creativity as validated by different methods (RQ3.1): a) individual rating, b) collective ranking, and c) collective pairwise rating. See Table 20 caption for how to interpret tables.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 23: **Statistical analysis of how prompt selection influences ideation creativity as validated by different methods (RQ3.1): a) individual rating, b) collective ranking, and c) collective pairwise rating. See Table 20 caption for how to interpret tables.**'", "CAPTION FIG25.png": "'Figure 25: **Results of computed individual diversity from ideations for different prompt configurations for (left) prompts that users understood (-0) or did not and (right) ideations that were fast or slow.**\\n\\n'", "CAPTION TAB24.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 24: Statistical analysis of a) how ideators\u2019 understanding of phrases influences ideation diversity and b) how similar. Directed ideations are to their prompts compared to other None and Random Messages.\\n\\n'", "CAPTION FIG9.png": "'Figure 9: **Results of perceived individual and collective creativity from the three validation user studies.**\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Results of ideators\u2019 perceived prompt creativity (Top) and ideation effort (Bottom) for different Prompt Selection technique and Prompt Size. All factors values on a 5-point Likert scale (-2=\u201cStrongly Disagree\u201d to 2=\u201cStrongly Agree\u201d). Dotted lines indicate extremely significant p<.0001 comparisons, otherwise very significant with p-value stated; solid lines indicate no significance at p>.01. Error bars indicate 90% confidence interval.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Example prompts shown to participants in different conditions: None (left, \\\\(g=0\\\\)), Directed(1) (center, \\\\(g=1\\\\)), and Directed(3) (right, \\\\(g=3\\\\)). Phrase texts would be different for Random(1) and Random(3) selection techniques.\\n\\n'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 7: **Metrics of creativity of ideation based on categories and themes derived from a thematic analysis of generated ideas. Metrics are shown for categories, but are the same for themes.**'", "CAPTION TAB8.png": "'\\n\\n**Table 8: Metrics of prompt diversity for all phrases in a single prompt.**'", "CAPTION TAB9.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 9: **Metrics indicating how much of prompt text and concepts are adopted into the ideations.**'", "CAPTION FIG10.png": "'Figure 10: Distribution of pairwise distances between the extracted phrases (N=3,666). The pairwise distances ranged from Min=0.057 to Max=0.586, Median=0.430, inter-quartile range 0.394 to 0.460, SD= 0.047.\\n\\n'", "CAPTION FIG11.png": "'Figure 11: Distribution of pairwise distances between the messages (N\\\\(=\\\\)250) idented in the pilot study with no prompting (None). The pairwise distances ranged from Min\\\\(=\\\\)0.169 to Max=0.549, Median\\\\(=\\\\)0.405, inter-quartile range 0.376 to 0.432, SD\\\\(=\\\\)0.043.\\n\\n'", "CAPTION TAB12.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 12: The rotated factor loading of factor analysis on metrics of prompt adoption. Factors explained 65.1\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1315, p-0.0001).** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 12: The rotated factor loading of factor analysis on metrics of prompt adoption. Factors explained 65.1\\\\% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 1315, p-0.0001).\\n\\n'", "CAPTION TAB13.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 13: The rotated factor loading of factor analysis on diversity metrics of generated messages. Factors explained 75.2% of the total variance. Bartlett\u2019s Test for Sphericity to indicate common factors was significant (\\\\(\\\\chi^{2}\\\\)= 2676, p-.0001).\\n\\n'", "CAPTION FIG26.png": "'Figure 26: Results of prompt-message distance (how dissimilar a prompt is from a message) comparing different messages with respect to Directed(3) prompts.\\n\\n'", "CAPTION TAB25.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 2: Metrics of distances between two points in a multi-dimensional vector space. Each metric can be calculated for an individual text item. These metrics can apply to the embedding of phrases or ideations.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 2: Metrics of distances between two points in a multi-dimensional vector space. Each metric can be calculated for an individual text item. These metrics can apply to the embedding of phrases or ideations.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 3: **Metrics of diversity of phrases or ideation embeddings in a vector space. These capture more characteristics of diversity than average distances in Table 2. Each metric can only be calculated collectively for multiple items.**'", "CAPTION FIG2.png": "'Figure 2: Procedure to direct ideation towards diverse phrases (top) and away from prior or redundant ideas (bottom). To attract ideation with diverse prompts: a) start with embeddings of corpus-extracted phrases; b) construct minimum spanning tree (MST); c) traverse tree to select distant prompts from clusters (most distant points as green dots, in clustered phrases as green ellipses); d) selected prompts are the most diverse. To repel ideation from prior ideas, e) compute embeddings of prior ideas (red hollow dots); f) compute prompt-ideation pairwise distances of all prompts from each prior ideation, exclude phrases (dotted black circles) with pairwise distance less than a user-defined threshold (red bubble), and construct the MST with remaining phrases; g) traverse MST to select a user-defined number of prompts; h) selected prompts are diverse, yet avoids prior ideas.\\n\\n'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Comparison of diversity metrics for canonical examples of distributed points in a 2D space. Points farther apart mean, higher diversity. Here, we calculate Euclidean instead of angular distance, but intuitions are similar.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Diversity prompting evaluation framework to evaluate prompting to support diverse ideation along the ideation chain. We pose research questions (RQ1-3) between each step to validate the ideation diversification process. For each step, we manipulate or measure various experiment constructs to track how well ideators are prompted to generate creative ideas. Except for prompt selection, each construct refers to a statistical factor determined from factor analyses of multiple dependent variables. Constructs are grouped in colored blocks indicating different data collection method (\\\\(\\\\square\\\\) Computed embedding-based metric, \\\\(\\\\square\\\\) ratings from ideators, \\\\(\\\\square\\\\) ratings from validators, \\\\(\\\\square\\\\) thematic coding of ideations).\\n\\n'", "CAPTION FIG4.png": "'Figure 4: 2D UMAP projection showing how diversely selected prompts and resulting ideation messages are distributed based on Directed or Random prompt selection technique and prompt size (number in brackets). Each point represents the embedding of a text item. Light grey points represent all phrases in the extracted corpus, dark grey points represent selected phrases from the simulation study (Section 5.1) and blue dots represent the ideated messages written by crowdworkers in the ideator user study (Section 5.2). Gradient lines connect ideation messages to their stimulus prompts.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/rohatgi1999interaction.json b/test_extracted_captions/rohatgi1999interaction.json new file mode 100644 index 0000000000000000000000000000000000000000..3793d97c6a2f017edd6cfed39553b3c520e11901 --- /dev/null +++ b/test_extracted_captions/rohatgi1999interaction.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'Figure 2: Full-Lang5 M-WASP Cooperative with the Arp23 Complex to Accelerate Actin Polymerization in Vero (A) The co-propagation of the Arp23 complex settled from bovine brain to shown on a 12\\\\(\\\\times\\\\) polyacrylamide gelstained with Galcede Blue (Cosmazole G-250). The thin, unlabeled lines on the right indicate the seven subcuts of the complex. Lested in descending order of molecular weight, the subunits are Arp3, Arp2, p41-ARC, p34-ARC, p21-ARC, p20-ARC, and p16-ARC. (b) The pyrene actin assay was used to monitor the polymerization of 2.5 \\\\(\\\\mu\\\\)M G-actin (D.S mM unlabeled actin + 1 \\\\(\\\\mu\\\\)M pyrenes actin) in the presence of 250 mM Arp23 complex alone, 400 mM K-WASP alone, or both components added together. In all the curves shown here and in the remainder of the figures, polymerization was initiated at time = 0. (C) Comparison of the abilities of wild-type H-WASP (p40) and the seed mutant (p400 mM or 800 mM) to stimulate actin polymerization in the presence of 60 mM Arp23 complex under conditions described in (B) The control curve depicts actin polymerization in the presence of Arp23 alone.\\n\\n'", "CAPTION FIG7.png": "'\\n\\n**Figure 1.** Signal-dependent Action Detection **A**'", "CAPTION FIG6.png": "'Figure 6: Cdc42 and PI(4,5)P\\\\({}_{z}\\\\) Coordinately Activate Full-Length N-WASP in Vitro (A) The effect of GTP-S-Cdc42 (500 nM), PI(4,5)P\\\\({}_{z}\\\\)-containing vesicles (100 \u03bcM, PC:PI:PI)(4,5)P\\\\({}_{z}\\\\), 48:48:41, or both on actin polymerization (2.5 \u03bcM actin and 60 nM Arp2/3 complex) stimulated by N-WASP (200 nM). The solid line shows stimulation of actin polymerization under the same conditions by GST-VCA (200 nM) alone.\\n\\n(B) Dose-response curves showing the maximum elongation rate of actin polymerization (2.5 \u03bcM actin and 60 nM Arp2/3 complex) as a function of the following molecules added: GST-VCA, full-length N-WASP, and full-length N-WASP stimulated with GTP-S-Cdc42 (50 nM) and PI(4,5)P\\\\({}_{z}\\\\)-containing vesicles (100 \u03bcM, PC:PI:PI)(4,5)P\\\\({}_{z}\\\\) 48:48:41. Note that the curve of N-WASP activated by PI(4,5)P\\\\({}_{z}\\\\) and GTP-S-Cdc42 starts to taper off between 8 and 80 nM because the Cdc42 is at a relatively low concentration in the experiment [50 nM]\\\\({}_{z}\\\\) addition of higher concentrations of Cdc42 will drive activation to higher levels approaching those of GST-VCA (Figure 6A).\\n\\n(C) Effects of Cdc42 bound to different nucleotides on N-WASP. Actin polymerization (2.5 \u03bcM actin and 60 nM Arp2/3 complex) was stimulated by N-WASP at the half-handed concentrations in the presence of Cdc42 [50 nM, GTP-S or GDP\\\\(\\\\backslash\\\\)S charged) and PI(4,5)P\\\\({}_{z}\\\\)-containing vesicles (10 \u03bcM, PC:PI:PI)(4,5)P\\\\({}_{z}\\\\),48:48:41.\\n\\n(D) Actin polymerization (2.5 \u03bcM actin and 60 nM Arp2/3 complex) was stimulated by N-WASP (5 nM) in the presence of Cdc42 (50 nM, GTP-S charged) and the indicated concentrations of lipid vesicles containing PC/PI (50:50) or PC/PI/PI(4,5)P\\\\({}_{z}\\\\) (48:48:41).\\n\\n'", "CAPTION FIG1.png": "'Figure 1: M-WASP is Required for Cdc42-Induced Actin Assembly in High-Speed Supernatants of Xenopus Egg Extracts\\n\\n(A) Schematic diagram of N-WASP showing its domain structure. Abbreviations for the domains used throughout the text are indicated inside the boxes. Numbers refer to amino acid residues. The H208D and _X_cof mutants are shown below along with the sites of the mutations.\\n\\n(B) Immunodepletion of N-WASP from Xenopus HSS. Affinity-purified _n_-N-WASP antibody or antibody buffer (mock) was used to immunodeplete N-WASP, and the material left in the supernatant or captured on the beads was analyzed by immunoblotting using the affinity-purified _n_-N-WASP antibody. 0.6% of the input material (HSS), 0.7% of the supernatant material, and 2% of the material captured on beads was loaded on the gel.\\n\\n(C) Comparison of actin assembly stimulated by 250 nM GTP-S-charged GST-Cdc42 in untreated HSS, mock-depleted HSS, _n_-N-WASP depleted HSS, and in depleted HSS reconstituted with 50 nM recombinant N-WASP. Polymerization kinetics were monitored in the HSS using the pyrene actin assay in which fluorescence increase indicates filament formation.\\n\\n(D) Comparison of actin assembly stimulated by 250 nM GTP-S-charged GST-Cdc42 in _n_-WASP depleted HSS to which the indicated concentrations of recombinant N-WASP were added. The initial rate of actin assembly and the maximum level of F-actin attained were quantitated from curves of the type shown in (C).\\n\\n(E) Relative ability of wild-type N-WASP, the H208D mutant, and the _X_cof mutant to restore actin assembly activity to HSS depleted of endogenous N-WASP. In all cases, actin assembly was initiated by the addition of 250 nM GTP-S-charged GST-Cdc42.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Full-Length N-WASP and the GST-VCA Fragment Active Actin Polymerization with Different Potencies (A) Comparison of spontaneous (Cdc42-independent) actin polymerization in Xenopus H5S treated with the indicated concentrations of full-length N-WASP, GST-CA, or GST-VCA. (B) Actin polymerization (2.5 \u03bcM-N actin and 60 nM Arp2/3 complex) stimulated by full-length N-WASP (200 nM) or GST-VCA (0 nM). (C) Dose-response curves showing the variation in the maximum rate of filament elongation as a function of increasing concentrations of either GST-VCA or full-length N-WASP in the presence of 60 nM Arp2/3 complex. The elongation rate was calculated from the linear phase of polymerization curves of the type shown in (B). The main graph depicts the concentrations (abscissa) on a log scale, whereas the inset depicts them on a linear scale. At higher concentrations of GST-VCA, the elongation rate saturates and is no longer a good indicator of activity. Instead, the shortening of the lag phase becomes more dramatic (Figure 3D).\\n\\n'", "CAPTION FIG3.png": "'Figure 3.: N-WASP Binds to and Activates the Arp2/3 Complex via its C Terminus (A) Schematic showing the domain structure of full-length N-WASP and the various C-terminal fragments of N-WASP constructed as GST fusion proteins. All fusion proteins are named according to the C-terminal segments (_N_, C, or A) of N-WASP that they contain. (B) The GST fusion proteins shown in (A) were immobilized on glutathione-Sepharose beads and tested for their abilities to pull down either the Arp2/3 complex or G-actin from solution. The presence of the Arp2/3 complex or G-actin bound to the beads was assayed by immunoblotting with \u03b1-Ar2 or \u03b1-actin, respectively. (C) Actin polymerization (2.5 \u03bcM actin and 60 nM Arp2/3 complex) was followed in the presence of GST-VCA (50 nM), GST-V (200 nM), GST-VCO 200 nM) GST-CA (200 nM), or in the absence of any additions (control). (D) The initial phases of actin polymerization (2.5 \u03bcM actin and 60 nM Arp2/3 complex), including the lag phase, are shown in the presence of indicated concentrations of GST-VCA. (E) Summary of the G-actin binding, Arp2/3 binding, and Arp2/3 activation properties of full-length N-WASP and the fragments shown in (A). Binding of full-length N-WASP to Arp2/3 was determined byimmunoprecipitation rather than GST pull down. Binding of GST-V and GST-VCA was determined by both methods.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: The GST-CA Fragment Can Inhibit the Interaction between N-WASP and the Arp2/3 Complex [A] Stimulation of actin polymerization (2.5 \u03bcMM actin and 60 nM Arp2/3 complex) by N-WASP (200 nM) is inhibited by GST-CA but not GST-V-[B] Stimulation of actin polymerization (2.5 \u03bcMM actin and 60 nM Arp2/3 complex) by GST-VCA (1 nM) is inhibited by GST-CA in a dose-dependent manner.\\n\\n[C] Cdc42-stimulated actin polymerization in Xenopus HSS is inhibited by GST-CA, but not GST-V or GST-VC (200 nM each).\\n\\n[D] Dose-dependent inhibition of Cdc42-stimulated actin polymerization in Xenopus HSS by GST-CA as compared using the initial rate and maximal level of F-actin assembly. The control depicts assembly in the absence of any GST-CA.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/roudot2023utrack3d.json b/test_extracted_captions/roudot2023utrack3d.json new file mode 100644 index 0000000000000000000000000000000000000000..8f0d4cb574355f31123a1aaac48344fb6b8ce25c --- /dev/null +++ b/test_extracted_captions/roudot2023utrack3d.json @@ -0,0 +1 @@ +{"CAPTION TABNA.png": "'\\n\\n## Key Results\\n\\n### Key Results\\n\\nThe Key Results'", "CAPTION FIG1.png": "'Figure 1. **u-traces** **is** a complete pipeline for the measurement, visualization, and creation of large sets of 30 trajectories. The plot is here Ikerated on lattice light-sheet imaging of Hala cells undergoing three-s backend with aGFF-labeled ESS (marking microtubule plus-ends rendered in gray) and mercury-shed experience.\\n\\nFigure 2. **u-traces** **is** a complete pipeline for the measurement, visualization, and creation of large sets of 30 trajectories. The plot is here Ikerated on lattice light-sheet imaging of Hala cells undergoing three-s backend with aGFF-labeled ESS (marking microtubule plus-ends rendered in gray) and mercury-shed experience.\\n\\n'", "CAPTION FIG5.png": "'Figure 6: DynROIs drive the visualization of chromosome capture by microtubules and reveal possible interactions between neighboring kinetochore fibers\\n\\n(A-D) Dual-colored orthogonal MIP of HeLa cells undergoing mitosis labeled with aGFP-labeled EB3 (marking microtubule plus-ends rendered in gray) and mCherry-labeled centromere protein A (marking kinetochores rendered in red). Overlays highlight (A) a dynROU bait around centrosome trajectories, (B) a dynROU bait around kinetochores trajectories, and (C) a plane built to visualize the dynamics of chromosomes relative to the spindle location. (D) View of the dynROIs following description in H-I)\\n\\n(E) Definition of a cortical synROI between a centrosome and a kinetochore.\\n\\n(F) Dual-colored orthogonal MIP of HeLa cells during premetatphase. Overlay highlights the motion of the dynROI.\\n\\n(G) Cumulative overlaps of the detected microtubule plus-and position for three periods of 10 s between 53 and 102 s post nucleus envelope breakage.\\n\\n(H) Plus-ards count function of time and distance from the pole (_n_ = 1 dynROI).\\n\\n'", "CAPTION FIG6.png": "'Figure 6. The trackability score relies on the stochastic footprint of each trajectory to infer tracking accuracy\\n\\n[A] Example of a tracking ambiguity due to three trajectories in close proximity (orange, blue, and red). Dashed lines represent the true motion between track heads at time \\\\(t\\\\)-\\\\(1\\\\) and detections at time \\\\(t\\\\), represented by gray dots. Colored gradients represent the likelihood of each expected particle location at time \\\\(t\\\\), astimated using the history of positions up to time \\\\(t\\\\)-\\\\(1\\\\) and considering multiple motion model hypotheses. The optimal assignment between the expected and detected particle positions at time \\\\(t\\\\) in this case yields an erroneous assignment from the orange track head to detection \\\\(2\\\\) and from the blue track head to detection \\\\(3\\\\) (graph A.I). Resampling of the expected locations results in a new assignment (graph A.II), this time without error.\\n\\n[B] Orthogonal MIP of ES calls expressing aGFP-labeled Sox2 molecules imaged by multilocus microscopy. Overlaid boxes highlight the ROI enlarged in (C)-(E).\\n\\n[C] Orthogonal MIP of ROI. Overlay shows a trajectory where two close detections create assignment ambiguity.\\n\\n[D] Overlay illustrates the stochastic resampling of the predicted particle positions at this time point; blue circles, assignments in agreement with the original solution, red circles, assignments that differ from the original solution.\\n\\n[E] Overlay shows trajectory segments colored according to estimated trackability scores.\\n\\n[G] Examples of simulated trajectories with diffusion coefficients ranging from \\\\(0.1\\\\) to \\\\(1\\\\)\\\\(\\\\mu\\\\)m\\\\({}^{2}\\\\)/s with a fixed particle density of \\\\(0.1\\\\)\\\\(\\\\mu\\\\)m\\\\({}^{-2}\\\\). Visualization is limited to five consecutive frames to reduce clutter.\\n\\n[F] Lifetime of simulated trajectories (the charge in distribution is due to trajectories leaving the field of view as the diffusion coefficient increase).\\n\\n[H] Lifetime distribution measured through tracking shows a loss of the original distributions when the diffusion coefficient exceeds \\\\(0.2\\\\)\\\\(\\\\mu\\\\)m\\\\({}^{2}\\\\)/s.\\n\\n[I] Accuracy measured through the Jaccard index [J.I, blue]; the trackability score (orange, dashed), which is derived without external ground truth, closely follows the J.I up to a diffusion coefficient \\\\(0.5\\\\)\\\\(\\\\mu\\\\)m\\\\({}^{2}\\\\)/s beyond which tracking is random.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n## Chapter 4 Drinking reveal the behavior of molecular adhesions in 3D environments\\n\\n(a) Dual-calared extensional MIF of catacancinana cells egregarding GFP-labeled and non-inflammatory induced in collagen labeled with Alexa Fluor 568. Querley highlights dyred\\n\\n(b) View of the dyred.\\n\\n(c) Detection of adhesion related to a function of the degra of collagen contact and a gradient.\\n\\n(e) Probability density of coagulation for adhesions with high and low degree of contact with collagen fibers (n = 1 cells).\\n\\n'", "CAPTION FIG2-2.png": "\"(C) Probability density of lifetime for the set of trajectories above and below the threshold value T, with and without gap closing [_n_ = 6 cellular layers, pooled trajectories lifetimes].\\n\\n(D) MIP of HLA cells in metaphase imaged with lattice light-sheet microscopy (LLSM) expressing GFP-labeled EBi [orange area is 30 x 32 x 7 mm]. Overlay highlights EBi trajectories.\\n\\n(E) Average microtubule lifetimes, mitochondria growth rate, as well as average number and duration of pause and shrinkage events per trajectory for increasing concentrations of nocodazole [_n_ = 5 par conditions; carrier line, median; box limits, 25 and 75 percentiles; whiskers, extremum].\\n\\n(F) MIP of HLA cells in metaphase imaged with LLSM along with 45'rotation around the vertical axis. Overlay highlights EBi trajectories.\\n\\n(G) Same as (E) measured for cells in metaphase [_n_ = 5 par conditions].\\n\\n(H) MIP of mouse embryonic stem (ES) cell nucleus imaged with LLSM expressing GFP-labeled TFs. Green box is 13 x 13 x 3 mm. Overlay highlights SC0X2 trajectories. (I) MIP of ES cell nucleus imaged with LLSM expressing GFP-labeled TFs. Overlay highlights SC0X2 trajectories tracked after MIP transformation.\\n\\n(J) Probability density of SC0X2 binding time measured in LLSM overlaid with a two-component decay fit (_n_ = 1 cell).\\n\\n(K) Probability density of SC0X2 binding time measured in projected LLSM data overlaid with a two-component decay fit (_n_ = 1 cell).\\n\\n\"", "CAPTION FIG7.png": "'Figure 7: Demonstration of trackability score on experimental data\\n\\n(A) Orthogonal MIP of breast cancer calls imaged with daSLM expressing aGFP-labeled alpha subunit of the AP-2 complex. Boxes show ROIs with quiescent (blue ROI) and slow/fast protrusion-extraction activity (orange and yellow ROIs). Dot overlaps show local level of ambiguity.\\n\\n(B) Ramker of track segments over time for the three ROIs (n = 1 cell).\\n\\n(C) Trackability score over time for the three ROIs (n = 1 cell).\\n\\n(D) Cumulative distribution of the average trackability score of trajectories for both EB3 and kinetochore channels sampling the dynamics of the mitotic spindle shown in Figure 5.\\n\\n(E) Four ROIs (two for each channel) showing trajectories colored according to their mean trackability score. Trajectories were selected near the 10th and 90th percentiles of the cumulative distribution. Yellow dots show surrounding detections.\\n\\n'", "CAPTION TABNA-1.png": "'\\n\\n**Acknowledgments**'", "CAPTION FIG3.png": "'Figure 3: u-track3D performance in comparison to existing methods evaluated on a standard 3D test dataset with high particle density (A) MIP of the simulated virus dynamics overlayed with trajectories reconstructed by u-track3D. Trajectories are colored following a random colormap. (B) Performance metrics for precision (alpha and beta) and accuracy (JSC and JSC) to compare different tracking pipelines.\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: u-track3D supports a variety of imaging and biological scenarios (A) Maximum intensity projections (MIPs) of a rat kidney cell layer imaged with diagonally scarred light-sheet microscopy (diaSLM). Cells are expressing aGPP-labeled alpha subunit of the AP-2 complex. Green box is 160 x 40 \u00d7 12 \u03bcm. Instant shows trajectories of clathrin aggregates classified as clathrin-coated structures or maturing pits. (B) Normalized maximum intensity of each trajectory as a function of lifetime plotted for six cellular layers composed of multiple cells each. The green line denotes the median of the cumulated distribution (value 7).\\n\\n'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/sathe2018small.json b/test_extracted_captions/sathe2018small.json new file mode 100644 index 0000000000000000000000000000000000000000..c4428c162fa86f0ac9bb9d080fd2aa6bf9039e44 --- /dev/null +++ b/test_extracted_captions/sathe2018small.json @@ -0,0 +1 @@ +{"CAPTION TAB2.png": "'\\n\\n**Table 2 Cross-correlation calculated for indicated traces**'", "CAPTION FIG2.png": "'\\nFig. 2: RNA screen reveals BAR domain proteins involved in CG endocytosis. **a** List of _Drosophila_ proteins in the PFAM database that contained one of the following BAR domains, PFAM IDs\\\\(-\\\\)PF06456, PF09325, PF06456, PF0061f and PF08397. The list was filtered to remove duplicates to give 18 genes. **b** The histogram shows normalised 5-min fluid-phase uptake in S2R\\\\({}^{+}\\\\) cells treated with 10 \u03bcg of dsRNA for 4 days as indicated with dsRNA against GBFI (gazr) as positive, and GFP as negative controls. In a single experiment, mean uptake of one of GFP dsRNA coverslip was used to normalize the mean for rest of the coverslips. Data were pooled from three independent experiments and the cell numbers are indicated in the graph. The bars in green are significantly different from GFP dsRNA using two-sample _t_-test (_b_ value <0.05). Version 27 of the PFAM database was used to generate the list '", "CAPTION FIG5.png": "'Figure 5: IRSp3 is reconstructed to forming CG endosomes. **a** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of three different regions [circles, violet, \\\\(t\\\\) = 170 nm and black (_r_ = 250 nm) and annulus, orange (_t_ = 250-420 nm)] for the recruitment of IRSp3-mCherry to the forming SecGFP-GPI endocytic sites and its corresponding random intensity trace (_n_, Table 1). **b** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of TagRFP-CDC42 to the forming SecGFP-GPI endocytic sites and its corresponding random intensity trace to two different regions [circle, black, \\\\(r\\\\) = 250 nm and annulus, orange (_r_ = 250-420 nm)]. Error bars, (**a\u2013b**) represent s.e.m. (_n_, Table 1). The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution (**a**) by Mann-Whitney \\\\(U\\\\) test and the log\\\\({}_{10}\\\\) (_p_) is plotted below each trace [Log\\\\({}_{100}\\\\) (0.05) is \\\\(-1.3\\\\) and log\\\\({}_{10}\\\\) (0.001) is \\\\(-2.5\\\\)]. Representative montages are depicted below the graphs (**a\u2013b**). Arrowheads indicate the newly formed endocytic vesicle. **c** Electron micrographs of AGS cells co-transfected-GFP-IRSp53 and GFP-binding protein coupled to Apex (GBP-Apex). The DAR reaction was performed and the cells were processed for electron tomography. **A** single section of the original tomogram (left) and density-based thresholded of the same plane (middle) reveal electron dense structures containing IRSp3 at membrane surfaces. The whole of PM of the tomographic volume was rendered and different examples of enlarged tubular regions of interest show GFP-IRSp53 recruitment patterns (right). Scale bar, 15 \u03bcm (**a\u2013b**) and 0.5 \u03bcm (**c**), respectively\\n\\n'", "CAPTION FIG7.png": "'\\n\\n## 4 Conclusion\\n\\nIn this paper, we have developed a new method for estimating the surface area of the'", "CAPTION FIG8.png": "'\\n\\n## References\\n\\nFig. 8: Schematic depicting the proposed biphasic mechanism for CG endocytic vesicle formation. **a** Phase I: Characterised by the recruitment of ARF/1 GBRI, PICKI, ARP2/3 and IRSp53 but not the buildup of F-actin and CDC42. Here, IRSp53 may be recruited by its I-BAR domain in the absence of GTP-CDC42, keeping its SH3 domain in an intra-molecular inhibited state. PICKI keeps ARP2/3 in an inactive state. **b** Phase II: Characterised by the recruitment of CDC42 and a sharp increase in ARFI leading to the removal of PICXI. This allows for the activation of ARP2/3 and buildup of F-actin. CDC42 binds to the CRIB domain of IRSp53 thereby activating it. The SH3 domain of IRSp53 can now bind to ARP2/3 activators and create F-actin. **c** Phase II: Characterised by endocytic vesicle formation, the presence of CDC42, ARFI/GBF1 and F-actin'", "CAPTION FIG6.png": "'Figure 6: Arp2/3-based actin machinery is required for CG endocytosis. **a** Histograms (top) show quantification of fluid phase and TR uptake in AGS cells treated with DMSO alone (0 uM) or the indicated concentrations of ARP2/3 inhibitor, CK666, normalised to DMSO-treated controls, along with its representative images (below). Data are pooled from two independent experiments and the number of cells shown indicated the graph. **b\u2013d** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of mCherry-ARP3 (**b**), pRuby-Lifacet (**c**) and mCherry-NWASP (**d**) to the forming SecGFP-GPI endocytic sites, and its corresponding random intensity trace (_n_, Table 1). The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution by Mann-Whitney \\\\(U\\\\) test and the log10 (_p_) [log10 (0.05) is -1.3 and log10 (0.001) is -2.5] is plotted below each trace (**b\u2013d**). Representative montages are depicted below the graphs. Arrowheads indicate the newly formed endocytic vesicle. **e** Histogram (left) shows normalised S-min mean fluid-phase uptake in AGS cells overexpressing plRRS-CA domain, GFP-VCA domain and GFP-N-WASPAVCA from N-WASP compared to un-transfected cells and representative images (right). The transfected cells are outlined. Data were pooled from two independent experiments and the number of cells shown below the graph. Error bars represent s.e.m. (**b\u2013d**) and s.d. (**a**, **e**). p value <0.01 (*), and 0.001(*) by Mann-Whitney \\\\(U\\\\) test (**a**, **e**). Scale bar, 15 \u03bcm (**b\u2013d**), 20 \u03bcm (**a**, **e**)\\n\\n'", "CAPTION FIG3.png": "'\\nFig. 3: IRSp53 is involved in CG endocytosis. Schematic depicting the domain organisation of IRSp53. IRSp53 exists in an inactive dimer state, which upon binding to GTP-CDC42 is activated, allowing SH3 domain to bind to its effectors. **b** Histogram (left) shows 5-min uptake of TFR and fluid phase in IRSp53-WT cells normalised to IRSp53-/- cells, along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated in the figure. **c** Histogram (top) shows normalized 5-min fluid uptake in IRSp53-/- and IRSp53WT cells when treated with LG186 or vehicle (DMSO) along with representative images (bottom). Data were pooled from two independent experiments and the number of cells indicated in the figure. **d** Histogram (top) shows an average number of endocytic structures quantified per field from the electron microscope images (bottom). Data pooled from three independent blocks. Untreated WT MEFs (WT, top row), IRSp53 null MEFs (IRSp53-/-, bottom left) or LG186-treated WT MEFs (LG186, bottom right) were incubated for 5 min at 37 \u00b0C with 10 mg/ml HRP as a fluid-phase marker before processing for electron microscopy. Endocytic structures close to the plasma membrane (PM) are filled with the electron dense peroxidase precipitate. WT cells show a range of endocytic structures including vesicular structures (double arrowheads) and tubular/ring-shaped putative CLIC/GEECs (large arrowheads) but the IRSp53-/- cells and LG186-treated cells only show predominant labelling of vesicular profiles. \\\\(p\\\\) value <0.05 (*), 0.001 (**) Mann-Whitney \\\\(U\\\\) test (**b-c**) and two-sample Student\u2019s test (**d**). Error bars, s.d. (**b-d**). Scale bar, 20 \u03bcm (**b-c**), 1 \u03bcm (**d**), respectively. Schematic (**a**) was adapted with permission from M\u00fcllrio (www.mechanobio.info) Mechanobiology Institute, National University of Singapore'", "CAPTION FIG1.png": "'\\nFig. 1: Identification of newly formed SecGFP-GPI endocytic vesicles using a pH pulse assay. **a** Schematic (top panel) of the pH pulling assay depicting the effect of pH on SecGFP-GPI fluorescence during endocytic vesicle budding. Note the fluorescence of SecGFP-GPI is retained at high pH (top and bottom left panel) or when exposed to low pH if sequestered in a newly formed endocytic vesicle (bottom right panel), and quenched only when exposed to low pH (top right panel). Sequential TIRF images of ACS cell co-expressing SecGFP-GPI and mCherry-ARFI collected at pH 7, pH 5 and in the RFP channels (middle panel). Newly formed endocytic vesicles (inset) (identified as in Supplementary Figure 1c) are used to construct a single frame (yellow rectangle) of the montage depicted (bottom panel). **b** Average of the normalised fluorescence intensities of pH 5 and pH 7 traces at the site of newly formed SecGFP-GPI endocytic vesicles compared to their respective random traces constructed from 120 endocytic events (pH 5 and pH 7) and 3428 random spots, derived from 17 cells pooled from four independent experiments. **c** The graph shows the fold enrichment of fluorescence intensity over the local background of pH 5 vs. pH 7 at the time of formation of the endocytic vesicles (data from 1b). **d\u2013f** Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of TagRFP-CDC42 (**d**), mCherry-ARFI (**e**) and mCherry-GBT (**f**) to the forming SecGFP-GPI endocytic sites, and its corresponding random intensity trace (n, Table 1). The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution by Mann-Whitney \\\\(U\\\\) test, and the log\\\\({}_{10}\\\\) (p) (log\\\\({}_{10}\\\\) (0.05) is \\\\(-\\\\)1.3 and log\\\\({}_{10}\\\\) (0.001) is \\\\(-\\\\)2.5) is plotted below each trace (**d\u2013f**). Representative montages from the respective data sets are depicted below the graphs (**d\u2013f**). Arrowheads indicate the newly formed endocytic vesicle. Error bars, s.e.m. (**b**, **d\u2013f**). Scale bar, 1.5 \u03bcm (**a**, **d\u2013f**)'", "CAPTION TAB1.png": "'Table 1 pH pulsating assay data set\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: CG pathway is abolished in the absence of IRSp53. **a** The box plot shows the number of endosomes per cells (left) for endocytosed GFP-GPI (a-GFP (Fab)), fluid phase and TIR in IRSp53\\\\(-\\\\)/\\\\(-\\\\) and IRSp53 WT cells when pulsed for 2 min along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated below the graph. **b** Plot (left) shows quantification of the fraction of GFP-GPI endocytic/vic vesicles containing fluid phase or TI images (right) show representative single confocal slices of a 2-min pulse of a-GFP Fab (green)/TMR-Dextran (magenta) and a-GFP Fab (green)/A568-Tf (magenta) in IRSp53\\\\(-\\\\)/\\\\(-\\\\) (top row) and IRSp53WT (bottom row) cells. The inset depicts magnified views of the indicated region; single-channel images are in panel **a**. Data were pooled from two independent experiments and the number of cells is indicated below the graph. **c**plot (left) showing quantification of the fraction of \u20181-min fluid-phase endocytof vesicles containing Tf. Images show representative single confocal slices of a \u2020-min pulse of TMR-Dextran (green) and A647-Tf (magenta) in IRSp53\\\\(-\\\\)/\\\\(-\\\\) (top row) and IRSp53WT (bottom row) cells. Inset depicts magnified views of the indicated region. Data were pooled from two independent experiments and the number of cells indicated below the graph. **d** Histogram (left) shows G-min uptake of fluid phase in IRSp53\\\\(-\\\\)/\\\\(-\\\\) MEFs transduced with virus expressing GFP-IRSp53 WT, GFP-IRSp53 4KE, GFP-IRSp53 1266N, GFP-IRSp53 14Q8P and GFP-IRSp53 V522G, normalised to that in IRSp53\\\\(-\\\\)/\\\\(-\\\\) MEFs, along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated in figure except for IRSp53\\\\(-\\\\)/\\\\(-\\\\) (381). \\\\(p\\\\) value <0.01(*) and 0.001(**) by Mann-Whitney U test (**a\u2013d**). Scale bar, 20 \u03bcm (**d**), 5 \u03bcm (**a\u2013d**), respectively. Error bars (**d**) represent s.d.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/sathe2018small_supplement.json b/test_extracted_captions/sathe2018small_supplement.json new file mode 100644 index 0000000000000000000000000000000000000000..17a9459375ae1ec59e7136c81fa18ec57d1aa199 --- /dev/null +++ b/test_extracted_captions/sathe2018small_supplement.json @@ -0,0 +1 @@ +{"SUPP CAPTION FIGS2-1.png": "'\\n\\n**Supplementary Figure 2: Characterization of the pH pulsing assay for visualizing SecGFP-GPI endocytic vesicle formation. (a-b) Graphs show the average normalized'", "SUPP CAPTION FIGS1-2.png": "\"(top) showing quantification of the fraction of GFP-GPI endocytic vesicles containing fluid or Tf. The number of cells is shown below the graph. (**d**) Schematic of pH pulsing analysis, steps (1-3) used for identifying and quantifying the fluorescence spots associated with newly formed endocytic vesicles in the sequential frames of the pH 5 montage. **Step 1** - New spot identification: Each spot (green) in ith frame is compared with the previous frame and is considered new if no nearest neighbour is found by euclidean distance search within 5 pixels (1 pixel = 84nm). Random (red) are generated randomly within the cell mask. **Step 2** - Spot mask (green filled) and background mask (green dashed) considered around the centroid the new spot identified in the step - 1 [also see pH 5 frame in the middle panel of (**Figure 1a**)]. The process is repeated for Random spot mask (red filled) and background mask (red dashed). **Step 3** - schematic of the temporal profile of a new spot (black) and a random spot. Multiple traces pooled from different spots and cells over different days was averaged and shown in rest of the figures. The x-axis represents time and y-axis represents fold change in intensity in the spot over the background. See S.I. for a detailed description. (**e**) AGS cells were pre-treated with either pH 7.3 or pH 5.5 buffer for 5 minutes followed by 5-minute fluid uptake in pH 7.3 buffer. Data was pooled from 2 independent experiments and the number of cells indicated in the graph. (**f**) Untreated AGS (Control, (i)) or LG186-treated AGS (ii) were incubated for 2 minutes at 37degC with 10mg/ml HRP as a fluid phase marker before processing for electron microscopy. Endocytic structures close to the plasma membrane (PM) are filled with the electron dense peroxidase precipitate. Control cells show a range of endocytic structures including vesicular structures (CCP/Cav & EE) (a pair of small arrowheads) and tubular/ring-shaped putative CLIC/GEECs (large arrowhead) but the drug-treated cells show predominant labelling of vesicular profiles. Histogram shows mean endocytic structures quantified per cell (n = 5). CCP/Cav represent vesicles derived from clathrin mediate or caveolar endocytosis and EE represent early endosomes (See S.I.). (**g**) The histogram shows quantification of 5-minute fluid-uptake in AGS cells when treated with indicated concentrations of LG186 or DMSO. Data was pooled from 2 independent experiments and the number of cells indicated in the graph. Scale bar, 5um (**a-b**), 4um (**a-b**, **inset**), 1 um (**f**) & 20um (**e and g**) respectively. Error bars (**c**) represent s.e.m. and (**e-g**) s.d. respectively. p-value < 0.001 (**) 2-sample student's T-test (**f**) and Mann-Whitney U test (**e**).\\n\\n\"", "SUPP CAPTION FIGS2-2.png": "'fluorescence intensity versus time traces for the recruitment of TagRFPt-CDC42 (CDC42 All) to all forming SecGFP-GPI endocytic sites (**a**) or endocytic sites that do not co-detect TagRFPt-CDC42 (CDC42 NoColoc; 44%) and its corresponding random intensity trace (n, Table 1). (**c-g**) Graphs show the average normalized fluorescence intensity versus time trace for the recruitment of mCherry-dynamin to forming SecTfR endocytic sites [**c**; (n = 21 SecTfR and 1448 random spots from 6 cells, 2 experiments), mCherry-clathrin to forming SecGFP-GPI endocytic sites [**d**; n, Table 1], mCherry-dynamin [**e**; n, Table 1] to forming SecGFP-GPI endocytic sites, mCherry-ARF1 to forming SecGFP-GPI endocytic sites [**f**; (n = 228, as in **Figure 1e**); Note the extended time axis], mCherryGBF1 to forming SecGFP-GPI endocytic sites [**g**; (n = 75, as in **Figure 1e**); Note the extended time axis], and their corresponding random intensity trace. A representative montage depicted below (**c-e**). A rowheads indicate the spot. The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution by Mann-Whitney U test and the log10 (p) [log10 (0.05) is -1.3 and log10 (0.001) is -2.5] is plotted below each trace (**a**, & **c-g**). Error bars represent s.e.m. for (**a**, & **c-g**). Scale bar, 1.5um (**c-e**).\\n\\n'", "SUPP CAPTION FIGS6-2.png": "'intensity versus time traces for the recruitment of mCherry-ARF1 NoColoc (**a**), mCherry-GBF1 NoColoc (**b**), mCherry-IRSp53 NoColoc (**c**), mCherry-ARP3 NoColoc (**d**), pRuby-Lifeated NoColoc (**e**), TagRFPt-PICK1 NoColoc (**f**), mCherry-NWA SP NoColoc (**g**) compared to their respective random trace. The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. They represent the fraction of SecGFP-GPI that failed to show a co-detected the X-FP spots. For n see Table 1 and S.I. for further information.\\n\\n'", "SUPP CAPTION FIGS3.png": "'\\n\\n**Supplementary Figure 3: RNA1 screen reveals BAR domain proteins involved in CG endocytosis.** Representative images for data shown in Figure 2b. Scale bar is 20\\\\(\\\\mathrm{\\\\SIUnitSymbolMicro m}\\\\).\\n\\n'", "SUPP CAPTION FIGS4-2.png": "\"treated with 10\\\\(\\\\upmu\\\\)M LG186 for 45 minutes along with the representative images (bottom). Data was pooled from 2 independent experiments and the number of cells is indicated in the figure. (**b**) Histogram (top) shows mean number of endocytic structures per cell from the electron microscope images (below). Data pooled from 3 independent blocks with 5 cells each. Untreated WT MEFs (WT, i), IRSp53 null MEFs (IRSp53-/-, ii) or LG186-treated WT MEFs (LG186, iii) were incubated for 2 minutes at 37\\\\({}^{\\\\circ}\\\\)C with 10mg/ml HRP as a fluid phase marker before processing for electron microscopy. Endocytic structures close to the plasma membrane (PM) are filled with the electron dense peroxidase precipitate. WT cells (left) show a range of endocytic structures including vesicular structures (double arrowheads) and tubular/ring-shaped putative CLIC/GEECs (large arrowheads) but the KO cells (middle) and LG186-treated (right) cells show predominant labelling of vesicular profiles. (**c**) Electron micrographs of AGS cells co-transfected GFP-IRSp53 and GBP-Apex. (**i-ii**) The reaction product is highly patched (arrowheads) on the plasma membrane (PM) along with zoomed regions on the right. (**iii**) Double arrowheads indicate specific labelling within defined microdomains of filopodia. (**d**) Plot (top) showing quantification of co-localizing IRSp53 with p21 (ARP2/3 complex subunit) using ImageJ plugin (Van Steensel's CCF, See Methods.) when compared with its random. Representative (bottom) images of AGS cells co-expressing mEmerald-p21 subunit with mCherry-IRSp53, which were fixed and imaged with TIRFM, with zoomed inset at top left corner. Data was pooled from 2 independent experiments and the cell number is indicated below the graph. (**e**) (1a-c) Wide-field images of cells at 1x (1a), 4x (1b) along with a representative confocal slice of a cell (4x, 1c). (2a-c) Inset from the 4x cell (Supplementary Movie 5) depicting enrichment of IRSp53 (magenta, 2b) within filopodia (CD44, green, 2a) along with the merge (2c). (3-6) Insets of CD44 (green) labelled invaginations showing recruitment of IRSp53 (magenta) at various stages. Each example depicts from left to right CD44, IRSp53 and Merge respectively. Scale bar, 20\\\\(\\\\upmu\\\\)m (**a**), 1\\\\(\\\\upmu\\\\)m (**b-c**), 5\\\\(\\\\upmu\\\\)m (**d**), 20\\\\(\\\\upmu\\\\)m (**e**, **1a-b**), 5\\\\(\\\\upmu\\\\)m (**e**, **1c**), 1\\\\(\\\\upmu\\\\)m (**e**, **2a-c & 3-6**) respectively. p-value < 0.01 (*), and 0.001(**) by Mann-Whitney U Test (**d**) and 2-sample student's T-test (**b**).\\n\\n\"", "SUPP CAPTION FIGS5-2.png": "'fixed and imaged with TIRFM, with zoomed inset at bottom right corner. (**c**) Histogram (left) showing quantification of mean 5-minute pulse fluid-phase in AGS cells overexpressing either plIRES-PICK1 WT or PICK1 KK-EE mutant. Data was pooled from 2 independent experiments and the number of cells is indicated in the figure. Error bar represents s.d. (**a**, & **c**) Scale bar, 20\\\\(\\\\upmu\\\\)m (**a & c**), 5\\\\(\\\\upmu\\\\)m (**b**). p-value < 0.01 (*), and 0.001 (**) by Mann-Whitney U Test (**a**-**c**).\\n\\n'", "SUPP CAPTION FIGS5-1.png": "\"\\n\\n**Supplementary Figure 5:** ARP2/3 is negatively regulated by PICK1 for CG endocytosis independent of N-WASP. (**a**) Histogram (left) showing quantification of \\\\(5\\\\)-minute pulse fluid-phase in AGS cells treated with either DMSO or 10\\\\(\\\\upmu\\\\)M SMIFH2 along with representative images (right). Data was pooled from 2 independent experiments and the cell number is indicated in the figure. (**b**) Plot (left) showing quantification of co-localizing PICK1 with ARP3 or ARF1 using ImageJ plugin (Van Steensel's CCF, See Methods) when compared with its random. Data was pooled from 2 independent experiments and the number the cell number is indicated below the graph. Representative (right) images of AGS cells co-expressing GFP-ARF1 with TaqManRFP-PICK1 or GFP-PICK1 and mCherry-ARP3 which were\"", "SUPP CAPTION FIGS4-1.png": "'\\n\\n**Supplementary Figure 4:** IRSp53 is involved in CG endocytosis. (a) Histogram (top) showing quantification of mean 5-minute normalized TfR uptake in IRSp53 WT cells when'", "SUPP CAPTION FIGS1-1.png": "'\\n\\n**Supplementary Figure 1: Characterization of endocytosis in AGS cells. (a-b)** Endocytic route of SecGFP-GPI in AGS cells. Single confocal plane shows a 3 minute pulse of \\\\(\\\\alpha\\\\)-GFP Fab [**a-b**: magenta in Merge] at 37\\\\({}^{\\\\circ}\\\\)C (upper panel) and 30\\\\({}^{\\\\circ}\\\\)C (lower panel) along with TMR-dextran (**a**: green in Merge) or A 647-Tf (**b**: green in Merge) in AGS cells transiently transfected with SecGFP-GPI. Insets show a magnified view of the marked areas. (c) Plot'", "SUPP CAPTION FIGS6-1.png": "'\\n\\n**Supplementary Figure 6: Fold change over time profiles of SecGFP-GPI spots that did not show a co-detected X-FP spot**. (**a-h**) Graphs show the average normalized fluorescence'", "SUPP CAPTION FIGS1.png": "'\\n**Supplementary Text:** **Characterization of categories in AC/S cells:** (a)-(b)-(c)-(d)-(e)-(f)-(g)-(g)-(h)-(i)-('", "SUPP CAPTION FIGS2.png": "'\\n\\n**Supplementary.****Tests 2.** **Characterization of the pH pH values used for translating SacGr-P/CH electrolyte results (in (a)).** (Origin show the average sensitized\\n\\n\\\\begin{tabular}{c c} &'", "SUPP CAPTION FIGS6.png": "'\\n\\n**Supplementary Figure 6.** Full change over time profiles of SOC-P-P-P type. **full** **full** **inactive** **yout** **time** **from'", "SUPP CAPTION FIGS4.png": "'Instead with 100M L1018 for 0.5 minutes along with the representative images (labeled), then we would form 2 independent experiments and the number of 0th is indicated in the figure. All histogram (only across the number of subjects returning yet from the detection image image shown). Dual pool has 2 independent trials with 5 trials and, unless 100M L1018 was used. (100M L1018 was used for 0.5 minutes), 100M L1018 was used for 1 minutes at 4.7degC with 100M L1018 is used for 2 minutes at 3.7degC with 100M L1018 is used for 1 minutes at 4.7degC with 100M L1018 is used for 2 minutes at 3.7degC with 100M L1018 is not listed below because previous for electron microscopy. Electron microscopy also the plasma membrane (or drift) that will be detected from multiple trials.\\n\\nIn addition to the 2-D slice, the 2-D slice was used for 0.5 minutes and 100M L1018 was used for 10 minutes at 4.\\n\\n'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Supplementary.** **Zheng X.A.Z.Z.Nappler** **Imagingly provided by PICK II (IC) for analysis and imaged with TRIM, with mounted slot-to-light sensor (IC).** **(10 Interoper (40) independent of N-NAB).** **(11 Interoper (40) design configuration of 5-minute plan following injection in ACS cells conveysing through voltage addition of 5-minute plan following injection in ACS cells conveysing through voltage addition of 5-minute plan following injection in ACS cells conveysing through voltage addition.** **(12 Multi-PICK II) for PICK II (IC) for analysis and imaged with TRIM, with mounted slot-to-light sensor (IC).** **(13 Interoper (40) design configuration'"} \ No newline at end of file diff --git a/test_extracted_captions/savinainenUsingBridgingRepresentation2005.json b/test_extracted_captions/savinainenUsingBridgingRepresentation2005.json new file mode 100644 index 0000000000000000000000000000000000000000..dd406e323891454c72b0cef17c3233cfa127586c --- /dev/null +++ b/test_extracted_captions/savinainenUsingBridgingRepresentation2005.json @@ -0,0 +1 @@ +{"CAPTION TAB2.png": "'TABLE 2: Research Instruments Administered to the Study Group'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**Table 3** & **Newton\u2019s Third Law Results for the Pilot Group (\\\\(n=22\\\\)) and Study Group (\\\\(n=23\\\\))** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 3: Newton\u2019s Third Law Results for the Pilot Group (\\\\(n=22\\\\)) and Study Group (\\\\(n=23\\\\))'", "CAPTION FIG7.png": "'Figure 7: Percentage of students exhibiting contextual coherence in the questions addressing Newton\u2019s third law for the pilot and study groups. The tests are in chronological order.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: A free-body diagram representing a block sliding on a horizontal table with constant acceleration. Friction, normal force, and gravitational forces are denoted by the symbols used in Giancoli (1998). Directions of velocity and acceleration are also shown.\\n\\n'", "CAPTION TAB1.png": "'TABLE 1\\n\\nLearning Demand Analysis: The Force Concept'", "CAPTION FIG6.png": "'Figure 6: The interview questions (derived from McDermott et al., 1998, pp. 28\u201329).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: A SRI diagram of a block sliding on a horizontal table. Interaction type is indicated by \\\\(\\\\mathbb{C}\\\\) or \\\\(\\\\mathbb{D}\\\\) (contact or distant interaction).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: The extended response written question used in the study (Ref. 1993b, p. 361).\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Figure 1.** The SRI diagram as a bridging representation.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: A sample question from the survey on Newton\u2019s third law (Bao et al., 2002). This question involves only the contextual feature of velocity. The correct answer is shown in bold point.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/schwartzSymmetricPatternsCoordinations2016.json b/test_extracted_captions/schwartzSymmetricPatternsCoordinations2016.json new file mode 100644 index 0000000000000000000000000000000000000000..1aeff47a881b666342cabb0604ad4734b92c356e --- /dev/null +++ b/test_extracted_captions/schwartzSymmetricPatternsCoordinations2016.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n## 1 Introduction\\n\\nThe \\\\(\\\\nu\\\\)-\\\\(\\\\bar{\\\\nu}\\\\) interaction is a strong interaction between the \\\\(\\\\nu\\\\)-\\\\(\\\\bar{\\\\nu}\\\\) interaction and the \\\\(\\\\bar{\\\\nu}\\\\) interaction. The \\\\(\\\\nu\\\\)-\\\\(\\\\bar{\\\\nu}\\\\) interaction is a strong interaction between the \\\\(\\\\nu\\\\)-\\\\(\\\\bar{\\\\nu}\\\\) interaction and the \\\\(\\\\bar{\\\\nu}\\\\) interaction.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/serwas2022mechanistic.json b/test_extracted_captions/serwas2022mechanistic.json new file mode 100644 index 0000000000000000000000000000000000000000..ea43f36e226ebbd1417ecad921b95f4bfa49b040 --- /dev/null +++ b/test_extracted_captions/serwas2022mechanistic.json @@ -0,0 +1 @@ +{"CAPTION FIG5.png": "\"Figure 5: **Actin filament orientation at CME sites**\\n\\n(**A**) Color code indicating filament orientation relative to the normal (\\\\(\\\\overline{\\\\text{II}}\\\\)') of a reference plane representing the plasma membrane. **At** \\\\(\\\\overline{\\\\text{II}}\\\\)', filament plus ends point toward the membrane.\\n\\n(**B** and **C**) Color-coded filament orientation at two late-stage CME imaginations (green), (**C**) shown a site toward the end of vesicle scission. Note the presence of filaments with their plus ends oriented toward the neck region (red arrowheads).\\n\\n(**D**) Relative filament to reference plane orientation of late CME sites 1 and 2 shown in (**B**) and (**C**), respectively.\\n\\n(**E**) Number of filament plus ends pointing toward base and neck region of CME sites. Mean and SD are shown in (**D**) and (**E**). See also Figure S5.\\n\\n\"", "CAPTION FIG7.png": "'Figure 7: Harnessing actin polymerization force for the pushing, pulling, and squeezing of cellular membranes\\n\\nActin polymerization can generate a pushing force to extend the plasma membrane during the formation of Hypoxia or lamellipodia and to push vesicles and intracellular pathogens through the cytoplasm. Proteins with both actin- and membrane-binding abilities (e.g., Hip1R) can anchor actin filaments to the membrane, converting pushing force into pulling and squeezing forces based on their spatial localization. In addition to the distribution of anchoring points, actin network morphology and polymerization force direction are defined by a second geometrical constraint, the position of actin assembly factors. Note that the precise localization of assembly factors and actin-membrane linkers is not clear for most of the displayed processes.\\n\\n'", "CAPTION TABNA.png": "'\\n\\n**Acknowledgments**'", "CAPTION FIG1.png": "'Figure 1: Native clathrin coat architecture and structure (A) Tomographic slices of a CCV at 2-positions relative to central ice (Z = 0 nm). Top and bottom slice show characteristic cage-like clathrin coat architecture with three log-like actarations emanating from each vertex (yellow arrowheads). Yellow and white circles in central slice indicate vesicle and coated area, respectively. (B) Density map of the clathrin coat determined within intact cells. (C) Resolution determination of the clathrin density map based on Fourier shell correlation (FSC). (D) Structural model of the clathrin hub (PDB: ESCT, in rainbow colors, from Morris et al. (2019)) Ifteadinto subtomogram average isosurface model of cytoplasmic clathrin vertex. Scale bars, 50 nm in (A) and 10 nm in (B) and (D). See also Figure S1.\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: CME-actin networks consist of branched and unbranched filaments (A) Tomographic slices at 2-positions relative to central slice (Z = 0 mm) of CME events at indicated stages. Clathrin coat and individual actin filaments are highlighted with yellow and red arrowheads, respectively.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n## Figure 4. Branched action filaments are organized into clusters\\n\\nSimulation snapshot bridging clusters organization of branched non filaments in mathematical model. Individual clusters are center eroded. The simulation of many cluster number during intra-clusterization.\\n\\nSegmentation model of late CME site from Fig. 2A highlighting clusters organized organized into clusters. Individual clusters are color cached.\\n\\n### Number of clusters and individual CME events. Mean and SD are shown in (\\\\(\\\\beta\\\\)) and (\\\\(\\\\beta\\\\)).\\n\\n### Simulation of the cluster size\\n\\nThe number of clusters in the simulation is \\\\(N_{\\\\rm{max}}=1000\\\\), where \\\\(N_{\\\\rm{max}}\\\\) is the number of clusters in the simulation.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Hip1R-like linkers observed at the neck of CME invaginations increase internalization efficiency (a) Tomographic slices of the regions indicated in the segmentation model of the tomogram shown in Figures 2A and 2B. Slices highlight putative Hip1R dimers (cyan boxes) that are associated with the central membrane of the CME invagination (green arrowsheads) and their positions are highlighted by cyan spharas in the segmentation model composed. Red arrowheads in a slot 1 point at an actin filament associated with a putative Hip1R dimer (b) Supermycositon of the surface model of the density map in (Figure SGB) with previously published atomic models of two actin-binding domains (ABD) (PDB;2,3SW, Girozas et al. (2008)) and two dimerization domains (PDB: 2QCO, Girozas et al. (2008)) of the homologous protein talin. Model of actin filament (PDB: 6D,N, Chou and Poland (2019)) was added as a reference. Mathematical model readout showing filament distribution between CME invagination base and neck in the presence or absence of Hip1R molecules at the neck. (C) Result of simulations showing the effect of Hip1R neck localization on the number of pLis ends at the base (orange and green curves) and neck regions (cyan) and purple curves) of CME invaginations. Mean and SD are shown. (D) Simulated internalization efficiency with and without Hip1R molecules at the neck. Mean and SD are shown. Scale bars, 50 nm in (a) and 5 nm in (b). See also Figure S6.\\n\\n'", "CAPTION FIG2-2.png": "'(B) Segmentation models of the tomograms in [A], bottom row shows branched actin filaments only. Color-coded legend describes elements shown in the models.\\n\\n(C) Filament length distribution of unbranched and branched filaments across all CME events shown in this publication. Median filament length for unbranched and branched actin filaments are highlighted. Scale bars, 50 nm in (A and B). See also Figure S2.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: CME is robust against branch angle variation (A) Tomographic slices of an individual branch junction at Z-positions, left to right, relative to central slice (Z = 0 nm). Positions of mother, daughter filaments, and plus and minus ends are indicated. Arrow points to the arc-like density of the Arp2/3 complex.\\n\\n(B) Branch angle distribution measured in tomograms. Mean value and standard deviation are displayed.\\n\\n(C) Result of simulations showing the effect of branch angle variation on the number of plus ends at the base of CME unvinations [green and orange curves] and the streamalisation rate (cyan and purple curves). Mean and SD are shown. Scale bar, 5 nm in [A]. See also Figure S3.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/sitarska2023sensing.json b/test_extracted_captions/sitarska2023sensing.json new file mode 100644 index 0000000000000000000000000000000000000000..9bc1d97247d6a1ba01f47a9d05d5abc3254a2df2 --- /dev/null +++ b/test_extracted_captions/sitarska2023sensing.json @@ -0,0 +1 @@ +{"CAPTION FIG3.png": "'cell from the 50\\\\({}^{th}\\\\) percentile of the distribution. If the effective rule wavelength increases upon loss of Snx33 (\\\\(p\\\\) = 3.171e-7, Mann-Whitney-U-test, two-sided). \\\\(n\\\\) = 175 (wt), \\\\(n\\\\) = 170 (Smx3-\\\\(j\\\\)). Statistics: r-test or non-parametric Mann-Whitney-U-test. Scale bars = 10 mm, \\\\(p\\\\) < 0.001 (\\\\(\\\\lx@math@degree\\\\)), \\\\(p\\\\) < 0.01 (\\\\(\\\\lx@math@degree\\\\)), \\\\(p\\\\) < 0.05 (\\\\(\\\\lx@math@degree\\\\)). Box-plots: the lower and upper hinges correspond to the 25th and 75th percentile. The upper whisker extends from the hinge to the largest value, but no further than 1.STQR. The lower whisker extends from the hinge to the smallest value, but no lower than 1.STQR of the hinge. Data beyond the whiskers: black dots. Black line: median. Black dot: mean.\\n\\nFig. 3: **IWAVE2 pattern width and rutile wavelength are increased in Snx33 knockout cells.****a** TIRFM images of a wild-type and Snx33/- cell show the distribution of WAVE2, the actin nuclear promoting factor upstream of the Arp2/3 complex in neutrophils. **b** The total area occupied by WAVE2 patches in the leading edge increases upon loss of Snx33 (\\\\(p\\\\) = 0.000408, Mann\u2013Whitney-U- test, two-sided). \\\\(n\\\\) = 82 (wt), \\\\(n\\\\) = 78 (Snx33/-). **c** Segmentation of dynamic WAVE2 patches in a wild-type and snx33/- cell. Time frames (55s) are color-coded. **d** The size of WAVE2 patches increases upon loss of Snx33 (\\\\(p\\\\) = 0.000464, Mann\u2013Whitney-U-test, two-sided). \\\\(n\\\\) = 82 (wt), \\\\(n\\\\) = 78 (Smx33/-). **e** SEM images with ruffle segmentations (red) show the leading edges of a wild-type and Snx33/- cell from the 50\\\\({}^{th}\\\\) percentile of the distribution. If the effective rule wavelength increases upon loss of Snx33 (\\\\(p\\\\) = 3.171e-7, Mann\u2013Whitney-U-test, two-sided). \\\\(n\\\\) = 175 (wt), \\\\(n\\\\) = 170 (Smx33/-). Statistics: r-test or non-parametric Mann\u2013Whitney-U-test. Scale bars = 10 mm, \\\\(p\\\\) < 0.001 (\\\\(\\\\lx@math@degree\\\\)), \\\\(p\\\\) < 0.01 (\\\\(\\\\lx@math@degree\\\\)), \\\\(p\\\\) < 0.05 (\\\\(\\\\lx@math@degree\\\\)). Box-plots: the lower and upper hinges correspond to the 25th and 75th percentile. The upper whisker extends from the hinge to the largest value, but no further than 1.STQR. The lower whisker extends from the hinge to the smallest value, but no lower than 1.STQR of the hinge. Data beyond the whiskers: black dots. Black line: median. Black dot: mean.\\n\\n'", "CAPTION FIG4.png": "'edge of single and collided cells (\\\\(n_{\\\\rm{a}aplus}\\\\) = 6, \\\\(n_{\\\\rm{a}a\\\\&\\\\&\\\\;'", "CAPTION FIG2.png": "'intensity in fixed wt and Snx33/- dtl-60 cells quantified by flow cytometry. Data from 3 independent experiments (_p_00 = 0.184, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.0373, \\\\(t\\\\) = -4.26, df = 2.375, two-sided; \\\\(p\\\\)00 = 0.1298, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.184, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1281, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.164, Mann-Whitney-d-test, two-sided; \\\\(p\\\\)00 = 0.1281, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.1281, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.1289, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J_Schemat for full-length Snx3 with its domains and two Smx33 truncations (_AP_SRPAR and PRNAR). Volcano plots from co-immunoprecipitation experiments comparing Snx33/- with Smx33/- dtl-60 cells; \\\\(p\\\\)00 = 0.1281, \\\\(t\\\\) = -1.9728, df = 3.5035, two-sided; _J'", "CAPTION FIG1.png": "'incomplete crystal structure (PDB ID: 4AKV; gray). **1** Probability histogram of the mean curvature (b) sampled at the center of mass of PX:BAR (green, mean curvature)\\\\({}_{\\\\text{PX:BAR}}=16\\\\,\\\\text{\\\\mu m}^{-5}\\\\) and at a random lipid phosphate position (yellow). red density estimates to smooth the distributions are shown. The mean values are indicated as dashed vertical lines. **1** Pop and side views from snapshots of the coarse-grained simulation of a buckled membrane with the PX-BAR domain. Protein backbone beads (green), phosphate beads (yellow spheres), and lipid tails (gray sticks) are shown. Water and ions are omitted for clarity. **2** Pop and side view are shown. Time points are indicated for the respective frames. **1** G cross-sections (_xz_) of migrating cells using lattice-light-sheet microscopy visualizing eGFP-SmX33 and CAAK:mCherry with a zoom-in on a membrane ruffle (**k**), and on a lamellipodium (**l**), \\\\(n\\\\) = 6, **m** Quantification of the average _z_-position of SmX33 relative to the plasma membrane and off-ruffles (\\\\(\\\\text{p}_{\\\\text{trigon}\\\\text{-}\\\\text{nucleus}}\\\\) = 0.01635, \\\\(r\\\\) = -3.5524, \\\\(r\\\\) = 5, paired, two-sided). \\\\(n\\\\) = 6 cells which data were obtained from two-color 3D modes. Each point represents the average for 1 cell. Scale bars = 10 mm. \\\\(p\\\\) = 0.05 (\\\\({}^{\\\\circ}\\\\)).\\n\\nFig. 1: **Curvature patterning in the lamellipodium and the curvature-sensitive protein SnX3.****a** The leading edge of migrating cells is characterized by intricate curvature patterns. The arrow indicates the direction of cell movement. Lines depict a complex 3D environment. **b** Time-lapse bright-field imaging shows migrating immune-like differentiated HL-60 cells. **c** Scanning electron microscopy (SEM) image of a wild-type cell with zoom-in at the leading edge. \\\\(n\\\\) = 17S. **d** Time-lapse p-polarization of pT-RPM imaging reveals dynamic membrane waves at the leading edge. **c** Up-regulated (orange) and down-regulated (blue) BAM domains genes between differentiated (migratory) and undifferentiated (non-migratory) HL-60 cells. **f** Fluorescently tagged SnX33 (eGFP-SmX33) and membrane marker (CAK-mCherry) in upper (close-to-the-cell-top) and lower (near-surface plane) _z_-planes in a cell. **g** Pearson correlation coefficient of fluorescently tagged SnX33 and membrane marker at different heights acquired by confocal microscopy. \\\\(n\\\\) = 10. Error bars denote the standard error of the mean. **h** Structure of the SmX33 PX-BAR domain as modeled with AlphaFold-multitimer (green) compared with the incomplete crystal structure (PDB ID: 4AKV; gray). **1** Probability histogram of the mean curvature (b) sampled at the center of mass of PX:BAR (green, mean curvature)\\\\({}_{\\\\text{PX:BAR}}=16\\\\,\\\\text{\\\\mu m}^{-5}\\\\) and at a random lipid phosphate position (yellow). red density estimates to smooth the distributions are shown. The mean values are indicated as dashed vertical lines. **1** Pop and side views from snapshots of the coarse-grained simulation of a buckled membrane with the PX-BAR domain. Protein backbone beads (green), phosphate beads (yellow spheres), and lipid tails (gray sticks) are shown. Water and ions are omitted for clarity. **a** Top and side view are shown. Time points are indicated for the respective frames. **k** Cross-sections (_xz_) of migrating cells using lattice-light-sheet microscopy visualizing eGFP-SmX33 and CAAK:mCherry with a zoom-in on a membrane ruffle (**k**), and on a lamellipodium (**l**), \\\\(n\\\\) = 6, **m** Quantification of the average _z_-position of SmX33 relative to the plasma membrane and off-ruffles (\\\\(\\\\text{p}_{\\\\text{trigon}\\\\text{-}\\\\text{nucleus}}\\\\) = 0.01635, \\\\(r\\\\) = -3.5524, \\\\(r\\\\) = 5, paired, two-sided). \\\\(n\\\\) = 6 cells which data were obtained from two-color 3D modes. Each point represents the average for 1 cell. Scale bars = 10 mm. \\\\(p\\\\) = 0.05 (\\\\({}^{\\\\circ}\\\\)).\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/smaldinoNaturalSelectionBad2016.json b/test_extracted_captions/smaldinoNaturalSelectionBad2016.json new file mode 100644 index 0000000000000000000000000000000000000000..9f3be548a20ac4dc5ff076ef8ffb7c88a2ca2402 --- /dev/null +++ b/test_extracted_captions/smaldinoNaturalSelectionBad2016.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Average statistical power from 44 reviews of papers published in journals in the social and behavioural sciences between 1960 and 2011. Data are power to detect small effect sizes (\\\\(d=0.2\\\\)), assuming a false-positive rate of \\\\(\\\\alpha=0.05\\\\), and indicate both very low power (mean = 0.24) but also no increase over time (\\\\(R^{2}=0.0009\\\\)).\\n\\n'", "CAPTION FIG5.png": "'Figure 5. The coordinate of effort and regulation.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: The evolution of effect when zero, 2% or 30% of all studies performed are replications.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Lab pay-offs from the non-evolutionary model. Each graph shows count distributions for high and low effort labs\u2019 total pay-offs after 110 time steps, 100 of which included replication. (\\\\(a-c\\\\)) Total count for each pay-off is totalled from 50 runs for each condition. Panel (\\\\(c\\\\)) includes an inset that displays the same data as the larger graph, but for a narrower range of pay-offs. The punishment for having one\u2019s novel result fail to replicate is orders of magnitude greater than the benefit of publishing, reflected in the discrete peaks in (\\\\(b\\\\)) and (\\\\(c\\\\)).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: The relationship between power and false-positive rate, modified by effort, \\\\(e\\\\). Runs analysed in this paper were initialized with \\\\(\\\\varepsilon_{\\\\rm B}=75\\\\) (shown in orange), such that \\\\(\\\\alpha=0.05\\\\) when power is 0.8.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. Jain, \"The quantum theory of gravity,\" _Phys. Rev. D_**48**, 1988 (1993).\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Power evolves. The evolution of mean power (\\\\(W\\\\)), false-positive rate [\\\\(\\\\alpha\\\\)] and false discovery rate (FDR).\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Effect evolves. The evolution of low mean effort corresponds to evolution of high false-positive and false discovery rates.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/soriano-castell2017rock1.json b/test_extracted_captions/soriano-castell2017rock1.json new file mode 100644 index 0000000000000000000000000000000000000000..72a4382062414bc1d4c0a0e207ddc2d7d07e617e --- /dev/null +++ b/test_extracted_captions/soriano-castell2017rock1.json @@ -0,0 +1 @@ +{"CAPTION FIG7.png": "\"Figure 7: ROCK1 participates in Rac1 induction of actomyosin, and myosin activity is required for Rac1-mediated tubule inhibition. (**a,b,c**) By immunofluorescence, myosin IIA and F-actin were detected in starved Vero cells expressing GFP-Rac1612V-V37A (**a**), GFP-Rac1612V (**b**), or GFP-Rac1612V-V966A (**c**) after treatment with Y27632 (30 min, 25 mM). The magnification insets show GFP-Rac1, phalloidin-A555, and myosin-IIA-A647 in transfected (1) and non-transfected cells (2). In the cells expressing GFP-Rac1612V and GFP-Rac1612V-V966A, the images show the loss of stress fibers (and consequently their staining with myosin II) plus myosin recruitment to cortical F-actin (arrow heads), which is reduced after treatment with Y27632 (bars, 5 mm). (**d**) The percentage of cells presenting tubules was determined in the presence or absence of the myosin inhibitor blebbistatin (30 min, 50 mM). Mean values +- standard error of the mean (SEM) from three independent experiments is shown. Statistical significance between different conditions was determined by Student's _t_-test, *P < 0.05.\\n\\n\"", "CAPTION FIG8.png": "'Figure 8: Molecular machinery implicated in the biogenesis and inhibition of PI(4,5)P\\\\({}_{2}\\\\)-induced endocytic tubulation. An increase of PI(4,5)P\\\\({}_{2}\\\\) in PM ordered domains could induce the recruitment of several PI(4,5) P\\\\({}_{2}\\\\)-binding proteins to generate an incipient membrane deformation (1). When Rac1 activity is low (2), the invagination can be elongated by dyneins toward the center of the cell along microtubules. PI(4,5)P\\\\({}_{2}\\\\) accumulation, as well as the high degree of membrane curvature, could lead to the recruitment of dynamin or BAR-domain proteins, which in turn, could propagate and stabilize the tubule. By contrast, when Rac1 activity is high, tubulation process could be inhibited by either PLC activation (reducing PIP\\\\({}_{1}\\\\) levels) or cytoskeleton regulation (inducing cortical actomyosin at the PM) (3). Rac1 appears to stimulate cortactin PM translocation and ROCK1 activity, thereby triggering cortical actin polymerization and association with myosin (i.e., actomyosin). The resulting over-activation of local actomyosin networks could potentially impede tubulation in one of two ways: (i) by forming a local barrier to increasing PM tension or by causing a steric hindrance that impedes the recruitment of tubulating proteins (4); or (ii) by generating mechanical forces needed to pinch off membrane invaginations more efficiently (5).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Increased levels of PI(4,5)P\\\\({}_{2}\\\\) induces membrane tubules at the PM. (**a**) COS1 cells expressing Cherry-Mem grown on coverslips were incubated with W13 (20 min, 4.5 mg/ml). After fixation (PFA 4%, 15 min at 37 \u00b0C), endogenous PI(4,5)P\\\\({}_{2}\\\\) was detected with a specific antibody and the corresponding anti-mouse Alexa-488 labeled secondary antibody. The images were acquired with a confocal microscope (Leica TCS SP5) and magnification insets show the presence of PI(4,5)P\\\\({}_{2}\\\\) in W13-induced membrane tubules. (**b**) The percentage of cells presenting more than 5 tubules was determined after 20 min of diC8-PI(4,5)P\\\\({}_{2}\\\\) dose-response at the indicated concentrations. (**c**) The percentage of cells with tubules after 20 min incubation with 50 mM diC8-PI(4,5)P\\\\({}_{2}\\\\) or neomycin control. (**d**) Confocal image of cherry-mem depicted tubules in cells treated with diC8-PI(4,5)P\\\\({}_{2}\\\\) as in (**c**) (bars, 10 \u03bcm). (**e**) In Cherry-Mem expressing cells, the percentage of cells presenting tubules and the number of tubules per cell were determined in different experimental conditions: W13 (20 min, 4.5 mg/ml), diC8-PI(4,5)P\\\\({}_{2}\\\\) (20 min, 50 \u03bcm) or GFP-PIP5K expression. Statistical significance between the different conditions and the control (NT, non-treatment) was determined by the unpaired Student\u2019s _t_-test, **p** < 0.01 (n = 100 cells per condition). Representative confocal images acquired through the red channel and insets displaying cells presenting cherry-Mem decorated tubules are shown (bars, 5 \u03bcm). (**f**) In the same experimental conditions as in (**e**), the intracellular levels of PI(4,5)P\\\\({}_{2}\\\\) were determined by immunofluorescence performed as in (**a**). Graph shows the percentage of fluorescence (a.u., arbitrary units) of each condition vs NT, mean values \u00b1 standard error of the mean (SEM) from three independent experiments. Statistical significance between the different conditions and the NT was determined by the paired Student\u2019s _t_-test, *p < 0.05. Representative \u03b1-stacks confocal projection images are shown (bars, 10 \u03bcm).\\n\\n'", "CAPTION FIG1-2.png": "'conditions and the corresponding control was determined by the two-way ANOVA, \"p \\\\(<\\\\) 0.05, ***p \\\\(<\\\\) 0.001. Statistical significance in integrin internalization assay (d) between W13t and W13nt at 5, 10 and 20 minutes were \"p \\\\(<\\\\) 0.05, ***p \\\\(<\\\\) 0.001, ***p \\\\(<\\\\) 0.001, respectively. (**f**) COS1 cells co-transfected with GFP-mem and a specific clathrin heavy chain siRNA or a non-specific siRNA as a control (72 h) were incubated with transferrin-TRITC during 15 minutes at 37oC in the presence or absence of W13 (4.5 mg/ml). After fixation, clathrin was detected with an anti-mouse antibody (clone \\\\(\\\\times\\\\)22) and the corresponding Alexa-647 secondary antibody. Confocal images were acquired with a confocal microscope (Leica TCS SP5) through the corresponding channels (bars, 10 mm). Downregulation of clathrin expression by its specific siRNA is shown by western blotting using a rabbit polyclonal antibody Graph shows the percentage of cells presenting tubules (\\\\(>\\\\)5 tubules/cell) in the indicated conditions. Mean values \\\\(\\\\pm\\\\) standard error of the mean (SEM) from three independent experiments are shown. Statistical significance between W13-treatment and the corresponding control was determined by the paired Student\\'s \\\\(t\\\\)-test, ***p \\\\(<\\\\) 0.01.\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: W13-induced PM tubules provide an internalization pathway for \\\\(\\\\left|\\\\lambda 1\\\\right|\\\\)-integrin. (**a**) COS1 cells grown on coverslips expressing the membrane marker GFP-mem were incubated with an anti-\\\\(\\\\beta 1\\\\)-integrin rat antibody for 30 minutes at 4\\\\({}^{\\\\circ}\\\\)C to avoid endocytosis, followed by incubation for 10 minutes at 37\\\\({}^{\\\\circ}\\\\)C to allow internalization in the presence of W13 (20 min, 4.5 mg/ml). After fixation, \\\\(\\\\left|\\\\lambda 1\\\\right|\\\\)-integrin was detected with an Alexa-555 labeled anti-rat antibody, and images were acquired with a confocal microscope (Leica TCS SP5). The higher magnification insets show \\\\(\\\\left|\\\\lambda 1\\\\right|\\\\)-integrin localization in W13-induced tubules (green arrowheads). (**b**,**c**) Following the same procedure explained as in (**a**), \\\\(\\\\left|\\\\lambda 1\\\\right|\\\\)-integrin was detected with an Alexa-647 labeled anti-rat antibody and EEA1 with a specific antibody and the secondary Alexa-555 anti-mouse in untreated (**b**) or W13-treated cells (**c**). Insets show \\\\(\\\\left|\\\\lambda 1\\\\right|\\\\)-integrin in EEA1-positive endosomes (red arrowheads) (**b**) or in tubules (green arrowheads) (**c**) (bars, 10 \\\\(\\\\upmu\\\\)m). (**d**,**e**) Quantification of internalized \\\\(\\\\left|\\\\lambda 1\\\\right|\\\\)-integrin (**d**) and transferrin (**e**), as explained in the _Materials and Methods_, in COS1 cells expressing GFP-mem or GFP-Rac1\\\\({}^{\\\\mathrm{G11V}}\\\\) for the indicated conditions (W13t, cells presenting tubules; W13nt, cells without tubules). Mean values \\\\(\\\\pm\\\\) standard error of the mean (SEM) from three independent experiments are shown. Statistical significance between different\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Actin cytoskeleton is involved in Rac1-dependent tubule inhibition. (**a**) Vero cells expressing GFP-Rac1G12V, GFP-Rac1G12V-F37A, or GFP-Rac1G12V-W56A were grown on coverslips, and F-actin was visualized by confocal microscopy using phalloidin-TRITC (15 min, 0.04 U/ml) (bars, 10 \u03bcm). (**b**) The percentage of COS1 cells presenting tubules was determined in starved cells expressing GFP-Rac1G12V or GFP-Rac1G12V-W56A and treated with the F-actin depolymerizing agent latrunculin A (LatA; 20 min, 200 nM) in the presence or absence of W13. (**c**,**d**) Percentage of cells with tubules expressing GFP-mem after pre-incubation with nocodazole (10 min, 30 \u03bcm) (**c**) or the dynein inhibitor EHNA (6 hours, 1 mM) (**d**) and incubation with W13 for 20 min. Mean values \u00b1 standard error of the mean (SEM) from three independent experiments are shown in all cases. Statistical significance between W13-treatment and the corresponding control were determined by Student\u2019s _t_-test, *P < 0.05, **p < 0.01. (**e**) In cells expressing Venus-Rac1wt and treated with W13, tubulin was detected by immunofluorescence using a mouse anti-(3-tubulin antibody and a secondary anti-mouse-Alexa594, and F-actin was detected using SiR actin (SC006). Representative confocal images and STED images from the selected areas are shown (bars, 10 \u03bcm).\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Rac1/ROCK1 pathway prevents tubulation without altering PM localization of cortactin. (**a**) Lysates of COS1 cells expressing GFP-Rac1612V, GFP-Rac1612V-937A, GFP-Rac1612V-956A or the empty GFP-C1 vector (EV) were incubated with immobilized GST-ROCK1725\u20133424 in glutathione Sepharose beads, as described in the _Materials and Methods_. GFP-Fusion proteins present in the input lysates and in the bound fraction were detected by western blotting, using an anti-GFP antibody. A representative western blotting is shown (n = 3). (**b**) Vero cells were co-transfected with cherry-Rac1612V and GFP-ROCK1. Confocal image insets show regions with high co-localization. (**c**,**d**) COS1 cells were transfected with GFP-Rac1612V-956A and treated with or without W13 (20 min, 4.5 mg/ml). The percentage of cells presenting tubules was determined after the inhibition of ROCK1 activity with Y27632 (**c**) or after the inhibition of ROCK1 expression by transfection with a specific siRNA (**d**). Downregulation of ROCK1 expression by its specific siRNA is shown by western blotting. (**e**) The percentage of cells presenting tubules was determined in untreated and W13-treated COS1 cells expressing GFP-RhoA718N, GFP-RhoA614V, or GFP-mem as a control. Mean values \u00b1 standard error of the mean (SEM) from three independent experiments are shown in all cases. Statistical significance between different conditions was determined by Student\u2019s _t_-test, *p < 0.05, **p < 0.01, ***p < 0.001. (**f**) Vero cells co-transfected with Cherry-mem and GFP-Rac1 (left panel) or GFP-RhoA614V (right panel) were treated with W13. Images and insets show localization of GFP-Rac1, but not of GFP-RhoA614V, in the cherry-mem tubules. All images were acquired using the Leica TCS SP5 confocal microscope (bars, 10 \u03bcm).\\n\\n'", "CAPTION FIG3.png": "'Figure 3: PI(4,5)P\\\\({}_{2}\\\\)-induced tubulation is dynamin-dependent and its inhibition by active Rac1 involves PLC activity. (**a**,**b**) In the presence or absence of W13 (20 min, 4.5 mg/ml), the percentage of cells with tubules was determined after dynasore treatment (30 min, 150 mM) or dynamin\\\\({}^{\\\\mathrm{K44A}}\\\\) expression (24 h) (**a**) and in cells transfected with a specific dynamin siRNA or a non-targeting siRNA as a control (48 h) (**b**). Downregulation of dynamin expression by its specific siRNA is shown by western blotting. (**c**) The percentage of cells with tubules was determined in cells expressing Cherry-Mem alone or co-expressed with L10-GFP InP54p or L10-GFP InP54p\\\\({}^{\\\\mathrm{Th281A}}\\\\), in the presence or absence of W13 (20 min, 4.5 mg/ml). (**d**) The percentage of cells with tubules was determined in 1-hour starved cells expressing Cherry-Mem or Cherry-Rac1\\\\({}^{\\\\mathrm{G12V}}\\\\) in the presence or absence of PI(4,5)P\\\\({}_{2}\\\\) (20 min, 50 mM), W13 (20 min, 4.5 mg/ml), or GFP-PIP5K expression. (**e**,**f**) The percentage of cells displaying tubules among the starved cells expressing GFP-mem, GFP-Rac1\\\\({}^{\\\\mathrm{G12V}}\\\\), or active Rac1 mutants (F37A and W56A) after the treatment with the PLC-inhibitor U73122 (20 min, 5 mM) (**e**, W13 (20 min, 4.5 mg/ml), or PIP5K overexpression (**f**). Mean values \\\\(\\\\pm\\\\) SEM from three independent experiments are shown in all cases. Statistical significance between different conditions and the corresponding controls were determined by Student\u2019s f-test, *p \\\\(<\\\\) 0.05, **p \\\\(<\\\\) 0.01, ***p \\\\(<\\\\) 0.001.\\n\\n'", "CAPTION FIG5.png": "\"Figure 5: Cortactin is involved in Rac1-dependent tubule inhibition. (**a**) Vero cells grown on coverslips were transfected with GFP-Rac1G12V, GFP-Rac1G22V-83A, or GFP-Rac1G12V-W56A. After fixation, immunofluorescence was performed to detect endogenous cortactin using an antibody and the corresponding Alexa-555 anti-mouse secondary antibody. Magnification insets show the presence or absence of cortactin at the PM (bars, 10 \u03bcm). (**b**) Percentage of COS1 cells presenting tubules, after W13 treatment, in cells transfected with cherry-Rac1G12V-W56A alone or co-transfected with full length cortactin or with a cortactin mutant lacking the PH and SH3 domains, acting as a dominant negative. (**c**) The percentage of cells presenting more than 5 tubules per cell was quantified in untreated (NT) and W13-treated cells co-transfected with GFP-Mem or GFP-Rac1G12V-W56A and the non-specific (NS) or cortactin siRNAs (48 h). Downregulation of cortactin expression by its specific siRNA is shown by western blotting. The W13-induced tubules and cortactin downregulation in GFP-Rac1G12V-W56A and cortactin siRNA-transfected cells is shown by immunofluorescence as in (**a**) (bars, 10 \u03bcm). Mean values \u00b1 standard error of the mean (SEM) from three independent experiments are shown in all cases. Statistical significance between different conditions was determined by Student's _t_-test, *p < 0.05, **p < 0.01.\\n\\n\"", "CAPTION FIG1.png": "'Figure 1. (a): (b): (c): (d): (e): (f): (g): (i): (h):\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/srinivasanAnalogiLeadImprovingSelection2023.json b/test_extracted_captions/srinivasanAnalogiLeadImprovingSelection2023.json new file mode 100644 index 0000000000000000000000000000000000000000..0cc309164e5cfda523b5d37b417e5cd6b0b30057 --- /dev/null +++ b/test_extracted_captions/srinivasanAnalogiLeadImprovingSelection2023.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Our proposed System. The interface is split into three rows. In the first row, the users select _magnets_ (2.1) from the Problem and their corresponding Analogy, mixing them or adding their own in the _Playspace_ (2.2) to generate insightful questions/_recombinations_ (2.3) to facilitate divergent thinking and as a result, aid in recognition of beneficial analogies.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/ulrichs2023multicomponent.json b/test_extracted_captions/ulrichs2023multicomponent.json new file mode 100644 index 0000000000000000000000000000000000000000..90fc272a6077b1a5363e519067835100f5f7fda9 --- /dev/null +++ b/test_extracted_captions/ulrichs2023multicomponent.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Supplementary Movie 3), **f** Survival fraction of formin-bound filaments (BF) as a function of time in the presence of PA supplemented with a range of CP concentrations. Experimental data (symbols) are fitted to a single-exponential decay function (lines) to determine the formin dissociation rate \\\\(k_{x}\\\\)(BF + **g** + **g** + **f**). Number of filaments analyzed for each condition (0 to 10 uM CP): 70, 36, 75, 56, 26, 26, 62, 62, 62 dissociation rate \\\\(k_{x}\\\\) as a function of CP concentration, determined from data in (**f**). Survival fraction of formin-bound filaments (BF) as a function of time in the presence of PA alone (black symbols, \\\\(n\\\\) = 31 filaments) or supplemented with 1 uM mT/t (magenta symbols, \\\\(n\\\\) = 30 filaments), 50 mM CP (yellow symbols, \\\\(n\\\\) = 51 filaments) or 1 uM mT/t, and 50 mM CP together (orange symbols, \\\\(n\\\\) = 50 filaments). Experimental data (symbols) are fitted to a single-exponential decay function (lines). **i** Formin dissociation rate \\\\(k_{x}\\\\) as determined from data in (**f**). Error bars in **g**, **i** indicate 65% confidence intervals based on fits (see Methods). Source data are provided as a Source Data file.\\n\\nFig. 1: **Effect of twinfilin and capping protein (CP) on processivity of formin.****a** Schematic of the experimental strategy. Actin filaments were nucleated from coverdp-anchored formins by a flow containing 1 uM G-actin (15% Alexa-488 labeled) and 0.5 uM profilin. Filaments were then elongated in the presence of 1 uM unlabeled G-actin and 4 uM profilin to ensure insertiual elongation between fluorescent fragments and surface-anchored forms. Filaments were then exposed to a flow containing 0.2 uM unlabeled G-actin and 0.7 uM profilin (PA) (control) with or without a range of concentrations of CP and/or mT/t (alone or together). The survival fraction of formin-bound filaments attached was monitored as a function of time. **b** Representative laryngographs of a formin-anchored filament elongating from 0.2 \u03bcM unlabeled G-actin and 0.7 \u03bcM profilin (PA) (see Supplementary Movie 1). **c** Same conditions as (**b**) but supplemented with 50 nM CP (see Supplementary Movie 2). **d** Same conditions as (**b**) but supplemented with 1 \u03bcM mT/t. **e** Same conditions as (**b**) but supplemented with 50 nM CP and 1 \u03bcM mT/t (see Supplementary Movie 3). **f** Survival fraction of formin-bound filaments (BF) as a function of time in the presence of PA supplemented with a range of CP concentrations. Experimental data (symbols) are fitted to a single-exponential decay function (lines) to determine the formin dissociation rate \\\\(k_{x}\\\\)(BF + **g** + **g** + **f**). Number of filaments analyzed for each condition (0 to 1 \u03bcM CP): 70, 36, 75, 56, 26, 26, 62, 62, 62, 62, 62 association rate \\\\(k_{x}\\\\) as a function of CP concentration, determined from data in (**f**). Survival fraction of formin-bound filaments (BF) as a function of time in the presence of PA alone (black symbols, \\\\(n\\\\) = 31 filaments) or supplemented with 1 uM mT/t (magenta symbols, \\\\(n\\\\) = 30 filaments), 50 nM CP (yellow symbols, \\\\(n\\\\) = 51 filaments) or 1 uM mT/t, and 50 nM CP together (orange symbols, \\\\(n\\\\) = 50 filaments). Experimental data (symbols) are fitted to a single-exponential decay function (lines). **i** Formin dissociation rate \\\\(k_{x}\\\\) as determined from data in (**f**). Error bars in **g**, **i** indicate 65% confidence intervals based on fits (see Methods). Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG2.png": "'exponential function (lines) to determine the rate of PFC disassembly. Number of filaments analyzed per condition (0 to 1M mTwfL): 31, SL, 30, 44, 42, and 50. **d** BFC dissociation rate into BC or BF as a function of mTwf concentration, determined from (**c**). **c** Fraction of BFC (black symbols) filaments transitioning to BF (magenta symbols) or BC (yellow symbols). Experimental data (symbols) are fitted to exponential fits (lines), such that \\\\(k_{\\\\mathit{BFC}}\\\\) = \\\\(K_{\\\\mathit{e}}\\\\) + \\\\(K_{\\\\mathit{c}}\\\\) where \\\\(K_{\\\\mathit{e}}\\\\) is the form dissociation rate from BFC (BFC + BC + F) and \\\\(K_{\\\\mathit{c}}\\\\) is CP dissociation rate from BFC (BFC + BF + C). Conditions - left (0 to 0M mTwfL, 50 filaments), center (50 nM mTwfL, 37 filaments), right (1M mTwfL, 30 filaments). See Supplementary Fig. 2 for the range of mTwf concentrations. **f** Dissociation rate of GP (\\\\(K_{\\\\mathit{c}}\\\\)) or Formin (\\\\(K_{\\\\mathit{d}}\\\\)) from BFC complexes **g** Percentages of BFC complexes in (**e**) transitioning to BC (yellow) or BF (magenta) at different mTwf concentrations. Error bars in (**d**) indicate 65% confidence intervals based on fits (see methods). Source data are provided as a Source Data file.\\n\\nFig. 2: **Effect of twinfilin on capping protein (GP)\u2013formin decision complex.****a** Experimental strategy schematic. Actin filaments were nucleated from coverslip-anchored formins by flowing 1M c-actin (15% Alexa-488 labeled) and 0.5 mM profilin. Filaments were then exposed to flow containing 1M unlabeled G actin, 4 mM profilin, and 1 mM CP for -10 s to convert formin-bound barbed ends (BF) to formin-CP-bound barbed ends (BF + C + BFC). BFC complexes were then exposed to flow containing PA only with a range of mTwfL concentrations. **b** Representative filament kymographs are transitioning to the BFC state upon exposure to 1M ATP. Upon CP removal from solution and exposure to PA (with or without mTwfL), filaments resume elongation following CP dissociation from BFC (top, BFC + BF + C) or detach from formin (bottom, BFC + BC + F). White arrowheads denote BFC complex dissociation. **c** Dissociation fraction of formin-CP-bound filaments (BFC) as a function of time in the presence of PA with/without a range of mTwfL concentrations. Experimental data (symbols) are fitted to a single-exponential function (lines) to determine the rate of PFC disassembly. Number of filaments analyzed per condition (0 to 1M mTwfL): 31, SL, 30, 44, 42, and 50. **d** BFC dissociation rate into BC or BF as a function of mTwfL concentration, determined from (**c**). **c** Fraction of BFC (black symbols) filaments transitioning to BF (magenta symbols) or BC (yellow symbols). Experimental data (symbols) are fitted to exponential fits (lines), such that \\\\(k_{\\\\mathit{BFC}}\\\\) = \\\\(K_{\\\\mathit{e}}\\\\) + \\\\(K_{\\\\mathit{c}}\\\\) where \\\\(K_{\\\\mathit{e}}\\\\) is the formin dissociation rate from BFC (BFC + BC + F) and \\\\(K_{\\\\mathit{c}}\\\\) is CP dissociation rate from BFC (BFC + BF + C). Conditions - left (0 to 0M mTwfL, 50 filaments), center (50 nM mTwfL, 37 filaments), right (1M mTwfL, 30 filaments). See Supplementary Fig. 2 for the range of mTwfL concentrations. **f** Dissociation rate of GP (\\\\(K_{\\\\mathit{c}}\\\\)) or Formin (\\\\(K_{\\\\mathit{d}}\\\\)) from BFC complexes **g** Percentages of BFC complexes in (**e**) transitioning to BC (yellow) or BF (magenta) at different mTwfL concentrations. Error bars in (**d**) indicate 65% confidence intervals based on fits (see methods). Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG4.png": "'recorded until the filament started depolymerizing, i.e., CP dissociated. The green arrow denotes polymerization, and the red arrow denotes depolymerization.\\n\\n**d**: Distr button of residence times of 549-mTwM on unlabeled CP-bound filament barbed ends (_n_ = 510 binding events across 16 filaments). Mean dwell time = 19 +- 0.1 S (sem). The histogram represents 99% of all binding events (remaining 1% outliers are not shown). 100% of binding events were included in calculations of mean dwell time. **e**: Top: time records of 549-mTwM fluorescence intensity at the unlabeled CP-bound barbed end of an actin filament. Intensity is integrated over 5 x 5 pixel square centered around the barbed end of the filament. Bottom: presence (1) or absence (0) of 549-mTwM molecules at the CP-649-bound filament barbed end shown above. Inset: cropped fluorescence images of a 10 x 10-pixel box around the barbed end of the filament showing the arrival, presence, and departure of a single 549-mTwM molecule at the CP-bound barbed end. Source data are provided as a Source Data file.\\n\\nFig. 4: **Visualization and characterization of twinfilin\u2019s interactions with CP-bound barbed ends.****a** Schematic of three-color single-molecule experiments with labeled CP and labeled mTwM. Actin filaments were assembled from 1/1M d-actin (33% Alexa-488 labeled, 1.4% biotin-labeled) and then capped by 10 nM 649-CP. These filaments were then exposed to 10 nM 549-mTwM. The green arrow denotes polymerization. **b** Binding of labeled twinfilin on CP-bound filament barbed ends recorded at 1s time resolution. Kymographs show Alexa-488 actin (top), 649-CP (second from top), 549-mTwM (third from top), and merge (bottom). Magenta bars denote episodes in which a 549-mTwM molecule was present at the 649-CP-bound barbed end of the filament. **c** Schematic of the two-color single-molecule experiments with unlabeled CP and labeled mTwM. Actin filaments were polymerized from 1/1M d-actin (15% Alexa-488 labeled, 1.4% biotin-labeled) and 0.5/M profiling, then capped by unlabeled CP. The capped filaments were then exposed to 15 nM S49-mTwM. Arrival and departure of 549-mTwM molecules at the barbed end were recorded until the filament started depolymerizing, i.e., CP dissociated. The green arrow denotes polymerization, and the red arrow denotes depolymerization. **d**: Distr button of residence times of 549-mTwM on unlabeled CP-bound filament barbed ends (_n_ = 510 binding events across 16 filaments). Mean dwell time = 19 +- 0.1 S (sem). The histogram represents 99% of all binding events (remaining 1% outliers are not shown). 100% of binding events were included in calculations of mean dwell time. **e**: Top: time records of 549-mTwM fluorescence intensity at the unlabeled CP-bound barbed end of an actin filament. Intensity is integrated over 5 x 5 pixel square centered around the barbed end of the filament. Bottom: presence (1) or absence (0) of 549-mTwM molecules at the CP-649-bound filament barbed end shown above. Inset: cropped fluorescence images of a 10 \u00d7 10-pixel box around the barbed end of the filament showing the arrival, presence, and departure of a single 549-mTwM molecule at the CP-bound barbed end. Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG5.png": "'\\nFig. 5: **Twinfilliv\u2019s direct interaction with actin filament is essential for its effects on** **CP** **decision complexes. a** **D**oman diagram of wild-type and mutant mT/W/M constructs used here\\\\({}^{\\\\text{m}}\\\\). \\\\(\\\\times\\\\)\\\\({}^{\\\\text{c}}\\\\) denotes the location of mutations. B Schematic representation of the experimental strategy. Actin filaments were nucleated from coverslip-anchored forms by introducing a flow containing 1uM G-actin (15% Alexa-488 labeled) and 0.5uM profilin. The filaments were then exposed to a flow containing 1uM unlabeled _C_-actin, 4 uM profilin, and 500 nM CP for about 10s to convert formium-bound barbed ends (2%) to formin-CP-bound barbed ends or decision complexes (BF + C + B/FC). These B/C complexes were then exposed to a flow containing PA only or supplemented with wildtype or mutant mT/W/L. **C** Survival fraction of formin-CP-bound filaments (BEC complexes) as a function of time in the presence of PA only (black symbols, 92 filaments), or supplemented with 1uM wild type mT/W/L (black, 36 filaments), mT/W/L (green, 77 filaments), or mT/W/L (left quadrant (blue symbols, 22 filaments). Experimental data (symbols) were fitted to a single-exponential function (lines) to determine B/FC dissociation rate \\\\(k_{\\\\text{-}\\\\text{GC}}\\\\). Error bars indicate 65% confidence intervals based on fits (see methods). **d** B/FC dissociation rate for wild-type and mutant mT/W/L, determined from data in (**c**). Error bars in (**d**) indicate 65% confidence intervals based on fits (see Methods). Source data are provided as a Source Data file.\\n\\n'", "CAPTION FIG6.png": "'into monomers. B Formin (\\\\(\\\\lx@sectionsign\\\\)7) bound barbed ends would get paused by CP to form BFC complexes. Twonfillin\\'s binding to BFC complexes would cause CP\\'s dissociation and renewal of formin-based filament elongation. As a result, the filament would appear to a treadmill, i.e., continue elongating at the barbed end while at the same time being disassembled at the pointed end by CAP-conflin synergy. \"R\" denotes barbed end, \"F\" denotes formin, \"C\" denotes CP, and \"T\" denotes twinfilin.\\n\\nFig. 6: **Working model for regulation of actin dynamics by twinfilin, forming, and CP.** Barbed-end outcomes would depend upon whether barbed ends were free for formin-bound. **a** Free barbed ends (B) would rapidly get capped by CP (C). followed by CP\\'s dissociation by twinfilin (T). This would leave twinfilin alone at the filament end, causing its depolymerization. At the same time, coflin would bind the sides of the aging filament and synergize with cyclase-associated protein (CAP) to initiate the filament\u2019s pointed-end depolymerization. The simultaneous depolymerization at the two ends would result in the complete disassembly of the filament into monomers. B Formin (\\\\(\\\\lx@sectionsign\\\\)7) bound barbed ends would get paused by CP to formin-BFC complexes. Twonfillin\u2019s binding to BFC complexes would cause CP\u2019s dissociation and renewal of formin-based filament elongation. As a result, the filament would appear to a treadmill, i.e., continue elongating at the barbed end while at the same time being disassembled at the pointed end by CAP-conflin synergy. \"R\" denotes barbed end, \"F\" denotes formin, \"C\" denotes CP, and \"T\" denotes twinfilin.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**e** Distribution of lifetimes of 649-mDial:549-CP decision complexes at barbed ends in presence of 0 nM mV/tl (left, \\\\(n\\\\) = 100 EF complexes), 5 nM mV/tl (center, \\\\(n\\\\) = 60 PFC complexes), and 20 nM mV/tl (right, \\\\(n\\\\) = 57 EF complexes). **f** Mean lifetimes (asem) of 649-mDial:549-CP decision complexes at barbed ends as a function of mV/tl concentration, determined from data in (**e**). **g** Fluorescence intensity and length records of a filament with formation and resolution of the decision complex formed in the presence of 100 pM 649-mDial, 10 nM unlabeled CP, and 40 nM 549-mTrvl. The gray shaded box indicates the time duration when both 549-mTrvl, 649-mDial, and unlabeled CP were simultaneously present at the barbed end (BFCT). **h** Cropped fluorescence images of a 13 x 13-pixel box around the barbed end of the filament in (**g**) show the formation and dissociation of a 649-mDi1-unlabeled CP complex in the presence of 549-mTrvl. The formin channel is misperra and twinfilin channel is yellow. Source data are provided as a Source Data file.\\n\\nFig. 3: **Direct visualization of formula, CP, and mV/tl at barbed ends.** Representative time-lapse images of a multicolor single-molecule TIRF experiment (see Supplementary Movie 4). Actin filaments were initiated by incubation of 0.5 mM G-actin (15% Alexa-488 labeled, 0.5% biotin-labeled), 1 \u03bcM profilin, and 50 pM 649-mDial (magenta). 649-mDial bound filaments were then exposed to PA and 20 nM 549-CP (yellow) with or without mV/tl. The white box around each filament and indicates the location of the barbed end. Insets show individual and merged channels localized at the barbed end. Scale bar, 2 \u03bcm. **b** Fluorescence images of a 13 x 13-pixel box around the barbed end of the filament from (**a**) show the formation and dissociation of a mDial\u2013CP complex at the barbed end. The Formin channel is magenta and the CP channel is yellow. **c** Fluorescence intensity and length records of the filament in (**a**) with formation and resolution of the decision complex in the absence of mV/tl. **d** Same as (**c**) but in the presence of 20 nM mV/tl. **d** Same as (**f**) but in the presence of 20 nM mV/tl. **d** Same as (**f**) but in the presence of 20 nM/tl. **e** Distribution of lifetimes of 649-mDial:549-CP decision complexes at barbed ends in presence of 0 nM mV/tl (left, \\\\(n\\\\) = 100 EF complexes), 5 nM mV/tl (center, \\\\(n\\\\) = 60 PFC complexes), and 20 nM mV/tl (right, \\\\(n\\\\) = 57 EF complexes). **f** Mean lifetimes (asem) of 649-mDi1:549-CP decision complexes at barbed ends as a function of mV/tl concentration, determined from data in (**e**). **g** Fluorescence intensity and length records of a filament with formation and resolution of the decision complex formed in the presence of 100 pM 649-mDial, 10 nM unlabeled CP, and 40 nM 549-mTrvl. The gray shaded box indicates the time duration when both 549-mTrvl, 649-mDial, and unlabeled CP were simultaneously present at the barbed end (BFCT). **h** Cropped fluorescence images of a 13 \u00d7 13-pixel box around the barbed end of the filament in (**g**) show the formation and dissociation of a 649-mDi1-unlabeled CP complex in the presence of 549-mTrvl. The formin channel is magenta and twinfilin channel is yellow. Source data are provided as a Source Data file.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/vasconcelosCopyNotCopy2017.json b/test_extracted_captions/vasconcelosCopyNotCopy2017.json new file mode 100644 index 0000000000000000000000000000000000000000..f10eb3ac76e5150b1895bee313d9cc26c9edbcd7 --- /dev/null +++ b/test_extracted_captions/vasconcelosCopyNotCopy2017.json @@ -0,0 +1 @@ +{"CAPTION TAB3.png": "'\\n\\n**Table 3** Summary of modular and non-modular ideas generated across groups.\\n\\n'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION FIG1.png": "'\\n\\n**Fig1.** Example solution provided to participants along with the following description. \"_A modular bike with parts of various sizes that can be connected and swapped to fit people with very different heights. Apart from the socketing parts and expansible/contractible wheels, the angles between tubes can also be modified in specific joints_\". The sketch used is a modification of the ECO 07 Compactable Urban Bicycle [24].\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{} & \\\\multicolumn{1}{c}{} & \\\\multicolumn{'", "CAPTION TAB2.png": "'\\n\\n**Table 2** Summary of bike and non-bike ideas generated across groups'"} \ No newline at end of file diff --git a/test_extracted_captions/vasconcelosEffectExplicitInstructions2018.json b/test_extracted_captions/vasconcelosEffectExplicitInstructions2018.json new file mode 100644 index 0000000000000000000000000000000000000000..8da6df410baa5575701e469ed5f1f4fac23dbc12 --- /dev/null +++ b/test_extracted_captions/vasconcelosEffectExplicitInstructions2018.json @@ -0,0 +1 @@ +{"CAPTION FIG2.png": "'Figure 2: Example solution provided to the participants along with the following description: \"A telescoping blue with parts that can be extended or shortened to fit people with very different heights. Apart from the adjustable tubes and wheels, the angles between tubes can also be modified in specific joints.\" The sketch used is a modification of the Zae-K Ergestamic Bike [Flass, 2010].\\n\\n'", "CAPTION TAB6\n.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 6.** Location metrics used in the study and their computed agreement coefficients and interpretation} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 6: Location metrics used in the study and their computed agreement coefficients and interpretation'", "CAPTION TAB9.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB10.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG3.png": "'Figure 3: Some of the participants\u2019 ideas: [a] bila idea, [b] non-bike idea, [c] modular idea, and [d] helioscopic idea. Note that these examples are not representative of the design work of any particular experimental group.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Bar chart showing the average number of states with features of the example and in the average number of example features per keen across groups (retargets are given per participant, error bars indicate standard deviation).\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB7.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB8.png": "'\\n\\n**Table 8. Summary of blue and non-blue ideas generated across groups**'", "CAPTION FIG1.png": "'\\n\\n**Fig. 1.** Example solution provided to the participants along with the following description: \"A modular bike with parts of various sizes that can be connected and swapped to fit people with very different heights. Apart from the stocking parts and equareible/contractable wheels, the angles between tubes can also be modified in specific joints.\" The sketch used is a modification of the ECO 07 Con repeatable Urban Bigcle [1].\\n\\n'", "CAPTION TAB2.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n**Table 3.** Summary of blue and non-bike ideas generated across groups'", "CAPTION TAB4.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'"} \ No newline at end of file diff --git a/test_extracted_captions/vojnovic2024combining.json b/test_extracted_captions/vojnovic2024combining.json new file mode 100644 index 0000000000000000000000000000000000000000..29509a202fbe3199feba8c5ebf39d8ba9ef0f402 --- /dev/null +++ b/test_extracted_captions/vojnovic2024combining.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'Figure 1: Principle and performance of the SKV method. (_a_) Principle of SKV sample preparation, a correlative ECPAIM protocol for expanding yeast. Shown are cell wall (black), cell membrane (grey), inner and outer nuclear membrane (magenta), target protein (blue), FP marker (red) and the PAA gel mesh (orange). The acrylate group of the protein anchoring agent _ACV_ is highlighted in orange. (_b_) Fixian yeast cells expand approximately fivefold using the SKV protocol. (_d_) SKV provides a macroscale isotropic expansion as determined by two different samples, measuring (_i_) cytosol widths and (_ii_) nuclear diameter (_liu_132) of non-expanded and expanded cells imaged over a total of nine different experiments. The expansion factor was determined to be 4.9 [e.g. 0.2, s.d. 0.8) (cytosel width) and 4.9 [e.g. 0.2, s.d. 0.9] (nuclear diameter), respectively. Note that nuclear widths were estimated from diffraction limited epifluorescence data and cannot be compared directly to cytosolic widths derived from SLIM data. (_d_) Additionally, the isotropic microscale expansion of the SKV protocol was probed investigating cell borders. Here, cells expanded with the SKV protocol show no mfos2 localizations being pulled outside of the cell area and hence an isotropic expansion on the microscale. (_e_) SKV retains 46% (s.e. 3.1%, s.d. 21.8%) of FP signal retention, whereas the original protein retained 22% (s.e. 2.0%, s.d. 12.4%).\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Examples of 5kY microscopy 5kY imaging of high (\\\\(a\\\\)\u2013\\\\(c\\\\)) and low (\\\\(d\\\\)\u2013\\\\(f\\\\)) abundant proteins in fission yeast. Super-resolved targets are marked in italic, targets imaged by conventional epifluorescence in regular script. (\\\\(a\\\\)) mIso2 expressed in the cytosol, (\\\\(b\\\\)) nuclear DNA binding protein chp1, (\\\\(c\\\\)) nuclear protein m16 and DNA (TO-PR0-3 iodide), (\\\\(d\\\\)) nucleus pore protein Map132, (\\\\(e\\\\),\\\\(f\\\\)) centromeric histone protein cmp1 relative to the spindle pole body protein sad1; combined with visualizing DNA (TO-PR0-3 iodide) (\\\\(e\\\\), inset \\\\(i\\\\)).\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Protocol optimizations for SKx7 (_a_) Examples of incomplete cell wall digestion using zymolyase, snail enzyme, a combination of zymolyase and lysing enzyme and btriase. All tested conditions lead to a partial and non-isotropic expansion constrained by remaining cell wall. (_b_) The proteinase activity of proteinase K (red) in comparison to different cell wall digesting enzymes measured by the release of tyrosine during casein digestion. Zymolyase (blue), \u03b2-glucoronidase (green), lysing enzyme (yellow) and Lalzyme _MMIX_ (magenta). Adding proteinase inhibitor PMSF successfully suppressed tyrosine release. (_c_) sad1-mStarlet-I signal shows mean increase of 27% (s.e. 12.8%, s.d. 85%) in FP retention when adding 0.05% glutathione during gelation. Gel rigidity decreases with increasing glutathione concentration. Thus, no gel formation when adding 0.4% glutathione or higher was achieved.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/wilsonEffectsBiologicalExamples2010.json b/test_extracted_captions/wilsonEffectsBiologicalExamples2010.json new file mode 100644 index 0000000000000000000000000000000000000000..39145ff00dcd780bd18e42467e6cbcb5e20c743c --- /dev/null +++ b/test_extracted_captions/wilsonEffectsBiologicalExamples2010.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n**Figure Captions**\\n\\n**Fig. 1.** The \\\\(\\\\pi^{0}\\\\) dependence of the \\\\(\\\\pi^{0}\\\\'", "CAPTION FIG2.png": "'\\n\\n## Chapter 4 Summary and Future Work\\n\\nIn this chapter we have presented a new method for the calculation of the \\\\(\\\\Delta\\\\)-function in the \\\\(\\\\Delta\\\\'", "CAPTION TAB2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIGA2.png": "'Figure 4.2 Statistical diagram example for Study 1\\n\\n'", "CAPTION FIGA3.png": "'Figure 4-3 Haman-retischer-Kine einquple for Study II\\n\\n'", "CAPTION TAB3.png": "'\\n\\n## Table 1.\\n\\n'", "CAPTION FIG3.png": "'Figure J. _Geometry_ tree for the entire set of design produced in the study.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n## Chapter 1 Summary\\n\\nIn this thesis we have developed a new method for the calculation of the \\\\'"} \ No newline at end of file diff --git a/test_extracted_captions/xu2024myosini.json b/test_extracted_captions/xu2024myosini.json new file mode 100644 index 0000000000000000000000000000000000000000..02c6bc84424b3c74d31fe904153f1bd257e7b1c5 --- /dev/null +++ b/test_extracted_captions/xu2024myosini.json @@ -0,0 +1 @@ +{"CAPTION FIGS3.png": "'\\n\\n**Fig. S3. The myosin power-stroke is required for altering network architectures (supplement for Fig. 5 in the main text). (A) Time-series of actin assembly around (top) control- and (bottom) HaloABD-bead at 50 nM CP. HaloABD of \\\\(\\\\alpha\\\\)-actinin heavily delayed the growth of actin comet tail as rigor myosin. Scale bar 5 \\\\(\\\\mu\\\\)m. (B) Quantification of comet tail length generated under different ATP concentrations (0-1 mM) for control-, myosin- and HaloABD-beads (N=2, n>15). Error bars are SD. p values were calculated using unpaired two-tail t test. (C) Network density quantified by total fluorescence intensity per area for control-, myosin-, rigor-myosin and HaloABD-beads. Normalized to the mean value of control-beads (N=2, n>11). Dot plots show mean \\\\(\\\\pm\\\\) SD. p values were calculated using unpaired two-tail t test. (D) Percentages of control-, myosin-, rigor-myosin and HaloABD-beads that performed symmetry breaking or no symmetry breaking at 15nM CP (N=3, n>23). (E) Time collapsed images of actin filaments gliding (with no Ca\\\\({}^{2+}\\\\)) or swirling (with 100 mM free Ca\\\\({}^{2+}\\\\)) on a Myo1d-coated surface. Rainbow bar represents timescale over 61 frames with 5 s frame interval. Scale bar 5 \\\\(\\\\mu\\\\)m. (F) Quantification of actin gliding speed with and without 100 mM free Ca\\\\({}^{2+}\\\\). (G) Total actin fluorescence intensity and (H) Growth efficiency for control- and myosin-beads with and without the presence of 100 \\\\(\\\\mu\\\\)M calcium (N=1, n=15). Efficiency is defined as comet tail length per unit actin fluorescence intensity. Box plots (G, H) show median (center line), interquartile range (box) and min-max values (whiskers). p values were calculated using two-tail paired t test.**'", "CAPTION FIG6-2.png": "'with different extents of shell breaking for control (50 nM CP) (n=182), myosin (n=158), myosin (with 10mM ADP) (n=89), control (100 nM CP) (n=65), N=2. Conditions: 4mM actin (5% Rhodamine labeled), 200 nM Arp2/3, 50 or 100 nM CP. 20mM (5 molar excess) phalloidin and 2 mM ATP were also added to prevent actin depolymerization and preserve myosin motor activity. Actin assembly was arrested 100s after mixing. Scale bar is 5 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Schematic of how myosin-l modulates actin network structure through its power-stroke.\\n\\n'", "CAPTION FIGS2.png": "'\\n\\n**Fig. S2. Myosin-I decreases the actin density in comet tails (supplement for Fig. 3 in the main text).** (A) Time-series of (top) control- and (bottom) 0.43:1 myosin-beads growing comet tails in the presence of 50 nM CP. The actin network growing from the myosin-bead is less dense but has a similar tail length as the control. (**B**) Actin comet tail length as a function of time (**C**) Comet tail fluorescence as a function of time. The fluorescent intensity of control- and myosin-bead experimental pairs are normalized to the average fluorescence level of the control-beads from 1100-1300 s. Large points show the averaged value at binned time interval (every 100 sec). Traces are from individual beads with each myosin-bead acquired with a control-bead in the same field of view (N=5, n=11). Error bars are SD. Conditions: 4 mM actin (5% Rhodamine labeled), 200 nM Arp2/3 complex and 50 nM CP. Scale bar 5 um.\\n\\n'", "CAPTION FIGS1.png": "'\\n\\n**Fig. S1. Phase diagram of actin comet tails assembled under different CP concentrations and myosin densities.** Experimental points demonstrate control- and myosin-bead pairs assembled under each specified conditions. Star - myosin-beads promote symmetry breaking; Triangle- myosin-beads have longer tails than control; Circle - myosin-beads exhibit similar tail length to control; Square - myosin-beads have shorter tails than control; Diamond - myosin-beads shows no comet tail. For extremely low CP concentration (e.g. 6.5 nM, Crossline), both myosin-coated and control-beads displayed aster-like structure. No significant difference was observed between the two types of beads. Conditions: 4 mM G-actin, 200 nM Arp2/3 complex, 0-200 nM of CP as indicated in graph.\\n\\n'", "CAPTION TABS1.png": "'\\n\\n**Table 81. Table of simulation constraints**'", "CAPTION FIGS6.png": "'\\n\\n**Figure S6. Simulated actin intensity, Arp2/3 intensity and Actin:Arp2/3 ratio across the entire actin comet tail for intermediate length condition (intermediate CP concentration) under different debranching thresholds. (A) Actin:Arp2/3 complex ratio (calibrated to number of actin monomers per Arp2/3 complex), average actin intensity, and average Arp2/3 complex intensity from simulated epifluorescent images of comet tails formed at reference debranching conditions with filament length 0.5 um (\\\\(\\\\approx\\\\)187 subunits). Solid circles indicate averages calculated where the Arp2/3 complex is removed upon debranching; empty squares indicate averages calculated where the Arp2/3 complex remains attached to the daughter filament upon debranching. (B) Same as in panel A except under enhanced debranching condition (debranching occurring at \\\\(\\\\pm\\\\)10\\\\({}^{\\\\circ}\\\\) away from 70\\\\({}^{\\\\circ}\\\\) equilibrium branch instead of \\\\(\\\\pm\\\\)25\\\\({}^{\\\\circ}\\\\) as in the reference case in A). The ratio varies with myosin force when Arp2/3 complex dissociates after debranching, but in the opposite manner as compared to the experimental results in Figure 4D.**'", "CAPTION FIGS5.png": "'\\n\\n**Figure S5. Simulated comet tail structure and tensile/compression force distributions as a function of branch length and myosin force \\\\(F_{\\\\text{myo}}\\\\). (A) Simulated epifluorescence images as in Figure 7D, but with filaments grow to a length of 0.3 \\\\(\\\\upmu\\\\)m before capping. The simulations can be compared to high CP concentration experiments of Figure 2A where a comet tail fails to form for myosin-beads. (B) Same as in panel A, but for filament length of 2.0 \\\\(\\\\upmu\\\\)m. The simulations mimic the low CP concentration experiments of Figure 2B where myosin was found to promote symmetry breaking. (C) State diagram for actin comet tails as a function of filament length and myosin force. Images show a cut through the center of the comet tail. Color scale indicates filament tension (red: tensile; blue: compressive).**'", "CAPTION FIG5.png": "'Figure 1. The original system consists in integrated for changing ambient environment. (3) In the presence of a single sensor, (4) in the presence of a single sensor, (5) in the presence of a single sensor, (6) in the presence of a single sensor, (7) in the presence of a single sensor, (8) in the presence of a single sensor, and (9) in the presence of a single sensor, (10) in the presence of a single sensor, (11) in the presence of a single sensor, and (12) in the presence of a single sensor, (13) in the presence of a single sensor, and (14) in the presence of a single sensor, (15) in the presence of a single sensor, and (16) in the presence of a single sensor, (17) in the presence of a single sensor, and (18) in the presence of a single sensor, (19) in the presence of a single sensor, (10) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, (12) in the presence of a single sensor, and (13) in the presence of a single sensor, (14) in the presence of a single sensor, and (15) in the presence of a single 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sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a single sensor, and (11) in the presence of a single sensor, and (11) in the presence of a single sensor, (11) in the presence of a\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Flament level model of actin comet tail recapitulates experimental results.** (**A**) Schematic of model of actin polymerization around the nucleating bead. Model includes filament level nucleation, branching, filament fragmentation, determining, capping, and force exerted by implicit myosin. (**B**) Timespace of symmetry breaking event under intermediate capping condition (branch length = 0.5 \u03bcm) with one myosin. Color scale indicates filament tension (Red tensile, Blue: compressive). (**C**) Tension\\n\\n'", "CAPTION FIG6.png": "\"\\n\\n**Figure 6. The myosin power stroke can fracture the actin shell.** (**A**) Representative actin shells of control-, myosin-beads assembled under 50 nM CP, arrested by adding 20uM (5 molar excess) of Latrunculin B and CK-666 before symmetry breaking; as well as myosin-beads assemble under the same conditions but arrested with the addition of 10mM ADP to inhibit myosin power stroke. Myosin-beads fractured and ejected from the actin shell, while control- and myosin-beads (with 10mM ADP) remained enclosed in the shell. Control-bead assembled under 100 nM CP showing similar network density as the myosin-beads, didn't show shell fracture or bead ejection. Image was captured approximately 40min after arrest. (**B**) The extents of shell breaking was classified by shell-breaking angle \\\\(\\\\theta\\\\): \\\\(\\\\theta\\\\) = 0, no symmetry breaking; 0 < \\\\(\\\\theta\\\\) < 180, shell fracture; \\\\(\\\\theta\\\\)\\\\(\\\\geq\\\\) 180, bead ejected. Scale bar is 5um. (**C**) Percentage of populations\"", "CAPTION FIG7-1.png": "'\\n\\n**Figure 7. Filament level model of actin comet tail recapitulates experimental results.** (**A**) Schematic of model of actin polymerization around the nucleating bead. Model includes filament level nucleation, branching, filament fragmentation, debranching, capping, and force exerted by implicit myosin. (**B**) Timelapse of symmetry breaking event under intermediate capping condition (branch length = 0.5 \\\\(\\\\mu\\\\)m) with no myosin. Color scale indicates filament tension (Red: tensile; Blue: compressive). (**C**) Tension'", "CAPTION FIG5-2.png": "'15-20 min after mixing. (**E**) Length and (**F**) network density quantified by total fluorescence intensity per area for control- and myosin-beads in the absence and presence of 100 mM free Ca\\\\({}^{2+}\\\\) (N=1, n=15). Control- and myosin-bead experimental pairs are normalized to the average fluorescence level of the control-beads. Box plots (**E**, **F**) show median (center line), interquartile range (box) and min-max values (whiskers). p values were calculated using two-tail paired t test. Conditions: 4uM actin (5% Rhodamine labeled), 200 nM. Arp2/3 complex, 15-200 nM CP, 0-1 mM ATP, as indicated. Scale bar 5 um.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3. Myosin-I decreases the actin density of comet tails.** (**A**) Time-series of (top) control- and (bottom) 0.43:1 myosin-beads growing comet tails in the presence of 50 nM CP. The actin network growing from the myosin-bead is less dense but has a similar tail length as the control. Conditions: 4 \\\\(\\\\mu\\\\)M actin (5% Rhodamine labeled), 200 nM Arp2/3 complex and 50 nM CP. Scale bar 5 \\\\(\\\\mu\\\\)m. **(B)** Comparison of the mean fluorescence intensities (Fluor. Int. / Area) of actin comet tails grown from control- and myosin-beads, captured 20 min after mixing. The solid lines connect experimental pairs (N=5, n = 11). **(C)** Actin comet tail length as a function of time and **(D)** tail growth rates derived from the slopes of the time courses in **(C)**. **(E)** Comet tail fluorescence as a function of time. Control- and myosin-bead experimental pairs are normalized'", "CAPTION FIG2.png": "'Figure 2: **Disruption and fracturing of actin shells by myosin-I.** (**A**) Time series of (top) control-beads and (bottom) 0.43:1 myosin-beads acquired in the same imaging field in the presence of 200 nM CP showing the inability of myosin-beads to form a comet tail. (**B**) Time series of (top) control-beads and (bottom) 0.43:1 myosin-beads acquired in the same field in the presence of 25 nM CP showing fracturing of the actin shell and comet tail growth from a myosin-bead but not the control-bead. Conditions: 4 \\\\(\\\\upmu\\\\)M actin (5% Rhodamine labeled), 200 nM Arp2/3 complex, 200 nM or 25 nM CP. Scale bar 5 \\\\(\\\\upmu\\\\)m.\\n\\n'", "CAPTION FIGS8.png": "'\\n\\n**Figure S8. Simulated expulsion of bead after halting actin polymerization/branching.** Simulation snapshots at intermediate filament length (0.5 \\\\(\\\\upmu\\\\)m) (intermediate CP concentration) show a cut through the bead center during the evolution of the actin network after halting actin polymerization and branching. Expulsion of the bead only occurs at intermediate cloud thickness (Growth time = 42.7 s, middle row and Fig. 7J). Thinner shells fall apart (Growth time = 31.5 s), while thicker shells cannot be broken by 0.2 pN myosin alone (Growth time = 59.85 s). Network evolved in the presence of the same myosin force (0.2 pN). Histograms show filament tension distribution at t = 0 s (time of growth arrest); dashed lines indicate region containing 95% of data, centered at zero.\\n\\n'", "CAPTION FIGS7.png": "'\\n\\n**Figure S7. Force acting on beads during comet tail elongation.** Subdivision of forces acting on beads along the direction of bead propulsion due to actin polymerization (light green, gray, dark green continuous; calculated as excluded volume interactions between barbed end segment and bead), actin excluded volume (light green, gray, dark green dashed; calculated as excluded volume interactions on the bead from filament segments other than barbed ends), and myosin forces on the bead (orange line). The bead drag force that balances the sum of these forces is shown in black. Actin forces are subdivided into whether they are located behind the bead (light green), at the side of the bead (gray), or in front of the bead (dark green).\\n\\n'", "CAPTION FIG1.png": "'\\n\\n**Figure 1. Alteration of comet tail morphology by myosin-I. (A) Schematic of (left) control- and (right) myosin-beads coated with NPF and neutravidin, where neutravidin is further conjugated with (control-bead) biotinylated CF640 fluorescent dye or (myosin-bead) biotinylated _Drosophila_ Myo1d. (B) Time-lapse sequence of a (red) myosin-bead walking on a (green) single-actin-filament track. Speed ~8 nm/s. Scale bar 2 um. (C) Time series of the growth of actin comet tail from symmetry breaking to generation of a polarized comet tail from (top) control-bead and (bottom) myosin-bead. Scale bar 5 um. (D) Phase map showing representative examples of 2 um-diameter beads coated with a range of myosin-densities in the presence of \\\\(30-200\\\\) nM CP, 4 uM (5% Rhodamine labeled) actin, and 200 nM Arp2/3 complex. For each myosin density condition, the left column show control-beads that were acquired in the same imaging field as the myosin-beads in the right column. The myosin and NPF densities were determined by SDS-PAGE gel, where the NPF density is the same for group 0.28:1, 0.35:1 and 0.43:1, - 6000 um-2, and - 4000 um-2 for group 0.80:1. Images were acquired 20-35 min after mixing. Brightness and contrast were linearly set to be the same for each control- and myosin-bead pair but different among panels at different conditions for better visualization. Scale bar 5 um.**'", "CAPTION FIG3-2.png": "'to the average fluorescence level of the control-beads from 1100 s -1300 s. (**F**) Rate of fluorescent actin incorporation into comet tails derived from the slopes of the time courses in (**E**). (**C**, **E**) Large points show the averaged value at binned time interval (every 100 sec). Traces are from individual beads with each myosin-bead acquired with a control-bead in the same field of view (N=5, n=11). Error bars are SD. (**G**) Growth efficiency for control and myosin-beads. Efficiency is defined as comet tail length per unit actin fluorescence intensity. Box plots (**B**,**D**, **F**, **G**) show median (center line), interquartile range (box) and min-max values (whiskers). p values were calculated using two-tail paired t test. Each point represents a control- and myosin-bead pair acquired in same field of view. See also Supplementary Fig.\\n\\n'", "CAPTION FIG5-1.png": "'\\n\\n**Figure 5. The myosin power stroke is required for altering network architectures.** (**A**) Time-series of actin assembly around (top) control and (bottom) rigor myosin-beads at 50 nM CP. Rigor myosin heavily delayed the growth of actin comet tails. (**B**) Representative images of actin comet tails grown under different ATP concentrations (top: control beads; bottom: myosin-beads). Adding ATP back rescued comet tail growth. Images were acquired 20-30 min after mixing. (**C**) Representative actin network patterns assembled on control-, myosin-, rigor-myosin, and HaloABD-beads under three different CP concentrations, as indicated. Images were acquired \\\\(20-30\\\\) min after mixing. Brightness and contrast were set to the same values for each panel. (**D**) Representative actin comet tails generated by (top) control- and (bottom) myosin-beads in the absence and presence of 100 uM free Ca\\\\({}^{2+}\\\\) at 50 nM CP. Images were acquired approximately'", "CAPTION FIG4.png": "'\\n\\n**Figure 4.****Comets grown from myosin-beads incorporate less Arp2/3 complex**. (**A**) Representative actin comet tails assembled from (top) control- and (bottom) myosin-beads showing (magenta) actin and (green) SNAP-Arp2/3 complex. Images were acquired approximately 25 - 35 min after mixing. Brightness and contrast were set differently for actin and SNAP-Arp2/3 complex panels for visualization. Conditions: 4 mM actin (5% Rhodamine labeled), 200 nM Arp2/3 complex (80% SNAP-Surface 488 labeled), 40 nM CP. Scale bar is 5 \\\\(\\\\mu\\\\)m. (**B**) Total actin fluorescence intensity, (**C**) total SNAP-Arp2/3 fluorescence intensity and (**D**) actin to SNAP-Arp2/3 fluorescence ratio over the entire comet tail region. (**E**) Total SNAP-Arp2/3 fluorescence intensity on bead surface. Plot shows median (center line), interquartile range (box) and min-max values (whiskers). p values were calculated using two-tail paired t test. Each point represents a pair of control- and myosin-beads (N=2, n=33)'", "CAPTION FIGS4.png": "\"\\n\\n**Fig. S4. Myosin power-stroke alone can break sparse actin shell (w/o Phalloidin). (A-B). Time-lapse series of (top) control- and (bottom) myosin-bead that performed (A) shell fracture or (B) bead ejection. Control- and myosin-beads were mixed with 4 mM actin (5% Rhodamine labeled), 200 nM Arp2/3, and 50 nM CP, incubated for 100s, and then actin assembly was arrested by adding 20\\\\(\\\\upmu\\\\)M (5 molar excess) of Latrunculin B and CK-666. (C) Status of shell breaking approximately 50min after arrest. The extents of shell breaking was classified by shell-breaking angle \\\\(\\\\theta\\\\). (D) Percentage of different populations with different extents of shell breaking. Without Phalloidin: control (n=141), myosin (n=55), myosin with 10mM ADP (n=125); With Phalloidin: control (n=182), myosin (n=158), myosin with 10mM ADP (n=89). Conditions: 4 mM actin (5% Rhodamine labeled), 200 nM Arp2/3 complex, 50 nM or 100 nM CP was incubated for 100 s and arrested by adding 5 molar excess of Latrunculin B, CK-666 and phalloidin (for '+ Phalloidin' experiments) or 10mM ATP (for 'myosin with 10 mM ADP' experiments). Scale bar 5\\\\(\\\\upmu\\\\)m.**\""} \ No newline at end of file diff --git a/test_extracted_captions/yamada2016actin.json b/test_extracted_captions/yamada2016actin.json new file mode 100644 index 0000000000000000000000000000000000000000..83f9bdb2ea7b8f7d5c1a12041414c62ee92b821d --- /dev/null +++ b/test_extracted_captions/yamada2016actin.json @@ -0,0 +1 @@ +{"CAPTION FIG4.png": "'Figure 4: Knockdown of corractin decreases filopodial formation in H1299 cells. (A) Western blotting showing knockdown of cortactin expression by RNA1 in H1299 cells. \u03b2-actin was used as the control. Three micrograms of cell lysate from each sample was analyzed by gel electrophoresis. (B) F-actin was visualized in serum-stimulated H1299 cells by Alexa Fluor 488-phalloidin staining. Boxed areas correspond to enlarged images shown below. Similar to results from dynamin 2-depleted cells, filopodial formation decreased in cortactin-depleted cells (right). Scale bar, 20 \\\\(\\\\mu\\\\)m (upper panels), 5 \\\\(\\\\mu\\\\)m (lower panels). (C) Analysis of filopodial formation in H1299 cells cultured with or without serum. The samples were analyzed by fluorescent confocal microscopy, and the filopodial length was measured. (D) Expression of wild-type cortactin rescues filopodial formation. Cortactin-depleted H1299 cells were transfected with rat wild-type cortactin (left) or cortactin W525K (right) cloned into the plRES2-AcGFP1 expression vector. Boxed areas correspond to enlarged images shown (middle panels). Transfected cells were identified by GFP expression (bottom panels). Scale bar, 20 \\\\(\\\\mu\\\\)m (top and bottom panels), 3.5 \\\\(\\\\mu\\\\)m (middle panels). (E) Analysis of filopodial formation in H1299 cells. The samples were analyzed by fluorescent confocal microscopy, and the filopodial length was measured. Results in (C) and (E) represent the means a SEM from three independent experiments. (F) Rescue of the punctate-like localization of dynamin 2 along F-actin bundles in filopodia by re-expression of cortactin in cortactin-depleted cells. Cortactin-depleted H1299 cells (left panels) were transfected with rat wild-type cortactin or cortactin W525K closed into the plRES2-AcGFP1 expression vector (right panels). The cells were immunostained with an anti-dynamin 2 antibodies. Boxed areas correspond to enlarged images shown below. Transfected cells were identified by GFP expression (right bottom panels). Scale bar, 5 \\\\(\\\\mu\\\\)m (top and right bottom panels), 2.3 \\\\(\\\\mu\\\\)m (left bottom and right middle panels).\\n\\n'", "CAPTION FIG3.png": "'Figure 3: Knockdown of dynamin 2 decreases filopodial formation in H1299 cells. (A) Western blotting showing knockdown of dynamin 2 (Dyn2) expression by RNAi in H1299 cells. (b)-actin served as the control. Three micrograms of cell lysate from each sample was analyzed by gel electrophoresis. (B) F-actin was visualized in H1299 cells by Alexa Fluor 488-phalloidin staining after knockdown of dynamin 2. Extensive filopodial formation was observed in cells after serum stimulation (left). Filopodial formation was inhibited in dynamin 2-silenced cells (right). Boxed areas correspond to enlarged images shown below. Scale bar, 20 \\\\(\\\\mu\\\\)m (upper panels), 5 \\\\(\\\\mu\\\\)m (lower panels). (C) Filopodial length in H1299 cells cultured in the presence or absence of serum. The cells were visualized by fluorescent confocal microscopy, and filopodial length was measured as described in Materials and methods. (D) Inhibition of filopodial formation by dynasore in serum-stimulated H1299 cells. Serum-starved cells were incubated with 240 \\\\(\\\\mu\\\\)M dynasore for 30 min, and then stimulated with 10% FBS for 45 min in the presence of 240 \\\\(\\\\mu\\\\)M dynasore (middle). Thereafter, dynasore was removed, and the cells were incubated in serum-containing medium for 45 min (right). For the negative control, cells were cultured in the presence of 3% DMSO (left). All steps were performed at 37\\\\({}^{\\\\circ}\\\\)C. (E) Analysis of ikopodial formation in the H1299 cells shown in (D). The cells were analyzed by fluorescent confocal microscopy, and filopodial length was measured. Results in (C) and (E) represent the means \\\\(\\\\pm\\\\) SEM from three independent experiments.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Dynamin GTPase inhibitors inhibit the migration of H1299 cells. (A) Representative images acquired by light microscopy showing cell migration in a wound healing assay. Confluent H1299 cells were wounded and then incubated for 8 h in the presence or absence of dynamin GTPase inhibitors at the indicated concentrations. For the negative control (control 0 h), cells were incubated with 1% DMSO. Scale bar, 200 \\\\(\\\\mu\\\\)m. (B) Morphometric analysis of the wound area filled by migrating cells after treatment with inhibitors at the indicated concentrations. The changes were normalized to the control. Results represent the means \\\\(\\\\pm\\\\) SEM of three independent experiments.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Actin bundling by dynamin 2 and cortactin stabilizes F-actin bundles. (A) Long F-actin bundles were formed in the presence of dynamin 2 and cortactin (lower left). Preformed F-actin (3.3 \\\\(\\\\mu\\\\)M) was incubated with or without the indicated proteins (5 \\\\(\\\\mu\\\\)M each). F-actin was visualized with Alexa Fluor 488-balloidin. Scale bar, 30 \\\\(\\\\mu\\\\)m. (B) Representative images acquired by fluorescent microscopy showing the localization of dynamin and cortactin along F-actin bundles. Actin bundles were formed in vitro by including dynamin 2 with wild-type cortactin (left) or dynamin 1 and wild-type cortactin (right). Protein colocalization was performed by double-immunofluorescence. Scale bar, 2 \\\\(\\\\mu\\\\)m. (C) Kinetics of F-actin disassembly induced in 10-fold diluted preformed pyrene-labeled F-actin solution with buffer. F-actin bundles disassembled in the presence of dynamin 2 and cortactin, as well as in the presence of 5 \\\\(\\\\mu\\\\)M a-actinin. The rate of F-actin bundle disassembly was measured by pyrene-fluorescence.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Dynamin 2 colocalizes with cortactin along F-actin bundles in filopodia of serum-stimulated H1299 cells. (A) Colocalization of dynamin 2 (Dyn2, left) and cortactin (Cort, middle) by double-immunofluorescent staining in filopodia of serum-stimulated H1299 cells. Boxed areas correspond to enlarged images shown below. Dynamin 2- and cortactin-positive puncta were present periodically along F-actin bundles in filopodia (arrowheads). Scale bar, 5 \\\\(\\\\mu\\\\)m (upper panels), 1.6 \\\\(\\\\mu\\\\)m (lower panels). (B) In the negative controls, the primary antibodies were omitted for dynamin 2 (left) and cortactin (right). Boxed areas correspond to enlarged images shown below. Bar, 10 \\\\(\\\\mu\\\\)m (top and bottom panels), 2.8 \\\\(\\\\mu\\\\)m (middle panels). (C) Representative images acquired by immunoclectron microscopy showing the localization of dynamin 2 (top three panels) and cortactin (bottom three panels) in filopodia of serum-stimulated H1299 cells. Immunoreactive dynamic 2 and cortactin were present along F-actin bundles (arrowheads). Scale bar, 20 nm. (D) Immunoprecipitation (IP) results demonstrating an _in vivo_ interaction between dynamin 2 and cortactin. H1299 cells were co-transfected with GFP-tagged dynamin 2 (Dyn2-GFP) and neither myc-tagged wild-type cortactin (Cort WT-myc, left) or cortactin ASH3 (Cort ASH3-myc, right). The protein complexes were immunoprecipitated using a polyclonal anti-myc antibody or preimmune IgG (IgG), and then visualized by western blotting (WB) with monoclonal anti-GFP or anti-myc antibodies. Total cell lysates (4, 10 and 20 \\\\(\\\\mu\\\\)g) were also analyzed (input).\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Putative role of the dynamin 2-cortactin complex in cancer cell migration. The dynamin 2-cortactin complex bundles actin filaments, which stabilizes filopodia. In addition, the dynamin 2-cortactin complex participates in the formation of pseudopodia. The regulation of actin by dynamin 2 and cortactin may also be involved in cancer cell invasion and metatasis.\\n\\n'"} \ No newline at end of file diff --git a/test_extracted_captions/yuDistributedAnalogicalIdea2016.json b/test_extracted_captions/yuDistributedAnalogicalIdea2016.json new file mode 100644 index 0000000000000000000000000000000000000000..626013ea72e1ef5fc692f808d67d0ccfaf841cd1 --- /dev/null +++ b/test_extracted_captions/yuDistributedAnalogicalIdea2016.json @@ -0,0 +1 @@ +{"CAPTION TAB2.png": "'\\n\\n## Table 2. Conditions in Experiment 1.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n**Table I.** Workflow for generating constraints.\\n\\n'", "CAPTION TAB5.png": "'\\n\\n**Table 5.** Typical chairs used in judging originality.\\n\\n'", "CAPTION TAB6.png": "'\\n\\n**Table 6 Experiment 2: Design examples**'", "CAPTION TAB7.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline & \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 7: **The effects of abstractness of examples on design quality in Experiment 2.**'", "CAPTION FIG1.png": "'\\n\\n**Figure 1** **Practielity: Interaction between abstraction of the domain and constraints, with 95\\\\(\\\\diamondsuit\\\\) CIs.**'", "CAPTION FIG2.png": "'\\n\\n**Figure 2****Originality: Interaction between abstraction of the domain and constraints, with 95% CIs.**'", "CAPTION TAB3.png": "'\\n\\n**Table 3. Examples of inspirations found when the domains and constraints were presented concretely and abstractly.**'", "CAPTION TAB4.png": "'\\n\\n**Table 4. Distance of inspirations from the kindergarten furniture domain and their diversity in Experiment 1.**'"} \ No newline at end of file diff --git a/test_extracted_captions/yuEncouragingOutsideBox2016.json b/test_extracted_captions/yuEncouragingOutsideBox2016.json new file mode 100644 index 0000000000000000000000000000000000000000..8a08b7727b1ccdfdb53055dec052e87c09f7c100 --- /dev/null +++ b/test_extracted_captions/yuEncouragingOutsideBox2016.json @@ -0,0 +1 @@ +{"CAPTION TAB1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION TAB4.png": "'\\n\\n**Table 4. The effects of inspiration condition on design quality.**'", "CAPTION TAB5.png": "'\\n\\n**Table 5. Examples of inspirations and resulting solutions from participants.**'", "CAPTION TAB2.png": "'\\n\\n**Table 2. Examples of the recommended expert domains from Experiment 1 in the Original and Schematic problem representation conditions.**'", "CAPTION TAB3.png": "'Note on frequency: There were 68 participants in _Original representation_ and 54 participants in _Schematic representation_. Each participant provided three recommendations.\\n\\n**Table 3. Experiment 1: Means and standard errors.**'"} \ No newline at end of file diff --git a/test_extracted_captions/zhu2022mem3dg.json b/test_extracted_captions/zhu2022mem3dg.json new file mode 100644 index 0000000000000000000000000000000000000000..10906b81f55d8d321d0f6739e748d505ad8ca4c5 --- /dev/null +++ b/test_extracted_captions/zhu2022mem3dg.json @@ -0,0 +1 @@ +{"CAPTION FIG6.png": "'Figure 6: Protein aggregation on a realistic dendritic spine geometry. (_A_) Mesh of the dendritic spine and its boundary elements. (_B_) Trajectory of protein evolution and components of system energy. (_C_) Mean curvature of the geometry. The normal component of (_D_) the bending force at \\\\(t=0\\\\), (_E_) the line tension force produced by the equilibrium protein distribution, and (_F_) the difference in the bending force profile produced by final protein distribution as opposed to the initial distribution.\\n\\n'", "CAPTION TAB3.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION TAB4.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline\\n**TABLE 4** & **Parameters used in section \u201cMembrane dynamics with full mechanochemical feedback\u201d for models with full mechanochemical feedback.** \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 4: Parameters used in section \u201cMembrane dynamics with full mechanochemical feedback\u201d for models with full mechanochemical feedback.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: Modeling budding from a vesicle driven by the full mechanochemical feedback of membrane-protein interactions. _(A)_ Validation of the exactness of the discrete forcing with respect to the discrete energy. The terms correspond to forces from bending \\\\(f_{i}\\\\), tension area \\\\(f_{i}\\\\), pressure-volume \\\\(f_{i}\\\\), Dirichlet \\\\(f_{d}\\\\), and protein binding \\\\(f_{i}\\\\). \\\\(\\\\mu_{A}\\\\), \\\\(\\\\mu_{A}\\\\), and \\\\(\\\\mu_{s}\\\\) are the chemical potential of diffusion, bending, and binding respectively. _(B)_ The time trajectory of budding dynamics in hypertonic, isotonic, and hypotonic osmotic conditions. _(C)_ The final snapshot of the system configuration under hypertonic, isotonic, and hypotonic conditions. _(D)_ Similar geometries to those shown in _(C)_ have been observed in experiments by Saleem et al. (59).\\n\\n'", "CAPTION FIGC1.png": "''", "CAPTION FIGE2.png": "'Figure 2: Pointwise directional comparison of continuous and discrete measurements: discrete vertex normal based on (A) volume spacecraft, \\\\(\\\\nabla V_{V}\\\\), mean curvature vector, \\\\(\\\\bar{A}\\\\), and Gaussian curvature vector, \\\\(\\\\bar{A}\\\\), and (B) Schlafli reactor, \\\\(H\\\\bar{S}\\\\).\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Budding dynamics by robust mechanisms of protein scaffolding and interfacial line tension constriction. (_A_-_C_) Control group, (_D_-_F_) bending-driven scaffolding mechanism, and (_G_\u2013_f_) Interfacial line tension assisted budding. (_A_) Spontaneous curvature distribution, \\\\(H\\\\), on initially flat patch. (_D_) Normal projection of the bending force at \\\\(T~{}=~{}15\\\\). (_G_) Normal projection of the line tension force at \\\\(T~{}=~{}7\\\\). (_B_, \\\\(E\\\\), _H_) Shape evolution through time-series snapshots of the Y-Z cross sections of the membrane, corresponding to the vertical dash lines in (_C_), (_F_), (_f_) trajectory plots of system energy and its competing components.\\n\\n'", "CAPTION FIGA1.png": "'FIGURE A.1 A. Symmetric metastable state with two beads instead of a single layer heat in sheared, prior to releasing to the solution shown in Fig. 4 C. high tension.\\n\\n'", "CAPTION FIGA2.png": "\"FIGURE 4.2: Spiner's formula in continuous and discrete geometry. chain of smooth and discrete shape derivative of integrated geometric measurements (144).\\n\\n\"", "CAPTION FIG3.png": "'FIGURE 3: Comparison of discrete quantities with their smooth counterparts on selected shape [4] (Convergence plot of global quantities, including total area, volume, mean curvature (squared), and Gaussian curvature; and [8] (Convergence plot of \\\\(\\\\mathcal{L}\\\\) norm of scalar and vector local quantities, including the mean curvature, Gaussian curvature, and the harmonic term).\\n\\n'", "CAPTION FIGE1.png": "'Figure 1: Pointwise magnitude comparison of continuous and discrete measurements: (\\\\(A\\\\)) scalar mean curvature, (\\\\(B\\\\)) scalar Gaussian curvature, (\\\\(C\\\\)) (scalar) bi-Laplacian term \\\\(\\\\nabla H\\\\) based on the cotan formula, (\\\\(D\\\\)) vector mean curvature, (\\\\(E\\\\)) vector Gaussian curvature, and (\\\\(F\\\\)) (vector) bi-Laplacian term based on Schlafli vector. Note that the result of the cotangent Laplacian approach in (\\\\(C\\\\)) produces a scalar result while our approach using the Schlafli vector in (\\\\(F\\\\)) is a vector result, thus their direct comparison is not meaningful.\\n\\n'", "CAPTION TAB2.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l'", "CAPTION FIG1.png": "'Figure 1: Overview of the DDG framework\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{c c'", "CAPTION FIG2.png": "'Figure 2: Overview of data flow within Mem3DG. The user provides Mem3DG with an initial condition in the form of a triangulated mesh and vertexwise state and kinematic variables (green box). The main loop (black loop) of Mem3DG evaluates the discrete energy and forces and propagates the trajectory, among other supporting steps. Modules in dashed lines are optional depending on whether the system mesh has boundaries and if external forces are specified. User-specified options and possible extensions of Mem3DG to accommodate various physics are highlighted in yellow boxes. Mem3DG automatically exits the simulation when the system converges or the maximum time step is reached.\\n\\n'", "CAPTION FIG4.png": "'FIGURE 4: Receiver typical equilibrium shapes of membranes with homogeneous material properties. (_A_ and _B_) Equilibrium solutions under different centrality (_C_) and agentaneous curvature (_H_) conditions, with initial condition of (_A_) Oblate-shaped'"} \ No newline at end of file diff --git a/test_extracted_captions/zwettler2020molecular.json b/test_extracted_captions/zwettler2020molecular.json new file mode 100644 index 0000000000000000000000000000000000000000..0bc53a115c693757dceecd85952f5e7cb68aafb5 --- /dev/null +++ b/test_extracted_captions/zwettler2020molecular.json @@ -0,0 +1 @@ +{"CAPTION FIG1.png": "'\\n\\n**Fig. 1****Re-embedding enables Ex-distortion.****a** Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm [22]. **b** STORM image of pre-labeled pre-skM expanded and re-embedded Cos-7 cells stained with primary antibodies against a-tubulin and secondary Alexa Fluor 532 conjugated antibodies (AU532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). **c** Zoom in on highlighted region in **(b)**. **d** Averaged cross-sectional profile of nine microtubule segments with a total length of 29.1 mm (segment lengths range from 2.1-4.5 mm) measured in two cells from 1 expanded sample. **e** Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by bi-Gaussian fitting of the data analyzed in **(c)** with an average distance of 137.1 +- 10.1 nm (mean +- sd). The data were obtained from \\\\(n\\\\) = 9 microtubule segments in 2 cells from 1 expanded sample. **f** Unexpanded Cos-7 cells labeled with an anti a-tubulin primary antibody and Alexa Fluor 532 (AU532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded **g** Zoom in of the white boxed region in **(f)**. **h** Average intensity profile of 35 microtubule segments with a length between 11 and 5.8 mm (mean = -2.0 mm) and a total length of 69.6 mm analyzed in 12 dSTORM images. **i** Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in **(h)** with a mean peak-to-peak distance of 36.2 +- 5.4 nm (mean +- sd). The data were obtained from \\\\(n\\\\) = 35 microtubule segments in 12 cells and 3 independent experiments. Scale bars, 2 mm (**b, f**), 500 nm (**c, g**).\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4****Ex-SMLM of U-ExM expanded centrioles.****a-c** 3D dSTORM image of U-ExM expanded and re-embledded _Chamydomonas_ centrioles stained post re-embedding with anti at-tubulin primary antibody and Alexa Fluor 647 conjugated secondary antibodies measured in MEA buffer. **b** Zoom-rin on highlighted region in **(a)** revealing the 9-fold symmetry of the shown overrediticle. **c** side view of two mature centrioles with clearly separated triplets. The inlet shows the cross-sectional profile along the centriole (white box) showing five distinct peaks of microtubule triples (marked with arrow heads). **d** Comparison of the diameters determined from expanded centrioles measured using different protocols (re-embedded and labeled with Alexa Fluor 647, and imaged in MEA photoswitching buffer, labeled with HMSR 647 and imaged in double-deionized water or in pH optimized PBS (1x) buffer with pH 7.4). Mean values are 657 +- 90 nm (mean +- _sd_) for Alexa Fluor 647 in MEA buffer (_n_ = 12 centrioles), 428 +- 74 nm (mean +- sd_) for HMSR in PBS (n = 7 centrioles), and 750 +- 34 nm (mean +- sd_) for HMSR 647 in water (_n_ = 8 centrioles). Data from \\\\(n\\\\) = 2 independent experiments for each condition. Divided by the previously analyzed diameter of a-tubulin labeled centriole expansion factors translates into -3.4x, -2.2x, and -3.9x for expanded centrioles labeled with Alexa Fluor 647 in MEA buffer, HMSR in PBS (1x), and HMSR 647 in water, respectively. Statistical significance was assessed by one-way ANOVA: the mean values of the diameters are significantly different with _p_<0.02 (_F_ = 3.80) (_P_ <= 0.05, \"_P_ <= 0.01). **e-g** 2D dSTORM image of U-ExM expanded centrioles labeled with HMSR 647 imaged in water **(****e-f**) or PBS(1x) (**g**) **f** Zoom-rin on highlighted region in **(e). **h** 3D dSTORM image of unexpanded isolated _Chamydomonas_ centrioles immunostained with antibodies against glutanylated tubulin and Alexa Fluor 647 conjugated secondary antibodies. Scale bars, 1 \u03bcm (**a**, **e**), 500 nm (**b**, **f**, **g**), 1.5 \u03bcm (**c**), 250 nm (**h**).\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3 **3D post-labeling Ex-dSTORM.** SMLM image of re-embedded and post-expansion labeled microtubules. **a** 3D _d_STORM image of re-embedded Cos-7 cells expanded according to the Protein-Retention protocol (_q_preLM)4 pre-labeled with anti _a_- and _b_-tubulin antibodies and additionally post-labeled with anti _a_-tubulin. The secondary antibodies were labeled with Alexa Fluor 532 (_q_preLM A1S32). The small logo in the upper left corner symbolizes the labeling method, e.g. pre- and post-immunolabeling with AIS32 secondary antibodies. **b** (Magnetized view of highlighted region in **(a)**. **c**_xz_-side view cross-sections (white lines)) (\\\\(\\\\hat{\\\\cup}\\\\)) and (\\\\(\\\\hat{\\\\cup}\\\\)) shown in **(b)** revealing the hollow structure of microt_bules. **d** Magnified view of highlighted region (white box) in **(b)**. Since post-labeling dominates the signal, the method is termed _p_c_e_k_M A1S32 (post-labeled). **e** Averaged cross-sectional profile (blue) of 11 analyzed microtubule segments along a total of 28.2 _\u03bc_m_ filament (21-5.5 _\u03bc_m segments) of one expanded sample. The simulated cross-sectional profile for 3.2x proExM expanded pre- and post-labeled microtubule assuming a pre- to post-labeling ratio of 0.1 is shown in red. **f** Histogram of peak-to-peak distances with normalized normal curve (red) of fitted profiles analyzed in **(e)** with an average distance of 79.5 \\\\(\\\\pm\\\\) 6.6 nm (mean 1 \\\\(\\\\pm\\\\) 5d) analyzed along \\\\(n\\\\) = 11 microtubule segments in 2 cells from 1 expanded sample. **g** image projection of the _xz_-axes averaged along two microtubule filaments (_\u03bc_) and (_v_) shown in **(b)** (red dotted lines) using the \"_z_ projection analysis\" of the software \"Line Profiler\". **h** Cross-sectional profile (blue dots) of the _xz_-projection shown in **(g)**. Using a bi-Gaussian fit (red) the peak-to-peak distance is determined to 81.2 _\u03bc_m_. Scale bars, 10 _\u03bc_m_ (**a**), 5 _\u03bc_m_ (**b**), 1 _\u03bc_m_ (**c**), 500 nm (**d**), 100 nm (**g**).\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2 Pre-labeling Ex-dSTORM.****a** Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. **b** dSTORM image of expanded and re-embedded **a**- and **b**-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (AIS32) using the MA-NIH/S/GA method6, i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-AIS32 (GA)). **c** Zoom in of white boxed region in (**b**). **d** Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5-6.4 \u03bcm and 28.6 \u03bcm in total measured in a expanded cells. **e** Histogram of peak-to-peak distance distribution with normalized normal curve (red) of microtubule segments analyzed in (**d**) at **n** = 8 microtubule segments in 4 cells from 1 expansion experiment with a mean distance of 133.8 \u00b1 11.29 mm (mean + sdf). **f** Unexpanded STORM image of ssDNA-C/S secondary antibody hybridized with CyS bearing oligonucleotides pre-expansion (DNA-C/S protocol). **g** Magnified view of white boxed region in (**f**). **h** Average cross-sectional profile of 7 microtubule segments with a length between 1.0-1.8 \u03bcm and 8.7 \u03bcm in total. **i** Histogram of peak-to-peak distances with normalized normal distribution curve (red) of the data analyzed in (**b**) along \\\\(n\\\\) = 7 microtubule segments in 2 cells from 1 experiment with a mean distance of 43.9 \u00b1 3.7 nm (mean + sdf). **j** Expanded dSTORM image of microtubules labeled with a-tubulin and dsDNA (DNA-AIS32) conjugated secondary antibodies exhibiting a metacarpady group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM bifunctional label concept3). **k** Zoom-in of white boxed region in (**f**). **l** Average intensity profile of 26 microtubule segments with a length of 2.4-10.7 \u03bcm and 118.6 \u03bcm in total. **m** Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined from \\\\(n\\\\) = 26 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 226.7 \u00b1 15.3 nm (mean + sdf). **n** dSTORM image of **a**- and _b_-tubulin expanded according to the DNA-C/S protocol strategy with labels at CyS-bearing oligonucleotides introduced post-re-embedding. **e** Zoom in of white boxed region in (**n**). **p** Average intensity profile of 15 microtubule segments with a length between 1.6-25.1 \u03bcm and a total length of 126.0 \u03bcm in 1 expanded sample. **q** Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by fitting the cross-sectional profiles analyzed in (**p**) along \\\\(n\\\\) = 22 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 201.0 \u00b1 12.9 nm (mean + sdf). The small logas in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 \u03bcm (**b**, **f**, **i**, **n**), 500 nm (**c**, **g**, **k**, **o**).\\n\\n'"} \ No newline at end of file diff --git a/test_figures-tables/.DS_Store b/test_figures-tables/.DS_Store new file mode 100644 index 0000000000000000000000000000000000000000..56f00929767fafa9d90bab1d3b8448792caad71e Binary files /dev/null and b/test_figures-tables/.DS_Store differ diff --git a/test_figures-tables/akamatsu2020principles/CAPTION FIG1-1.png b/test_figures-tables/akamatsu2020principles/CAPTION FIG1-1.png new file mode 100644 index 0000000000000000000000000000000000000000..80d7eb11652901dbbfcec677a659c6b3a574ccd8 --- /dev/null +++ b/test_figures-tables/akamatsu2020principles/CAPTION FIG1-1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8cd86985dd04f530cd6c43bb99e0bc417fcc4f5880d08b82ce9ad102ab1f3a01 +size 15343 diff --git a/test_figures-tables/akamatsu2020principles/CAPTION FIG1-2.png b/test_figures-tables/akamatsu2020principles/CAPTION FIG1-2.png new 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